bio-inspired virus-mimicking nanoparticles for efficient ...375409/s4256501_phd... · conference...

Bio-inspired Virus-mimicking Nanoparticles for Efficient

Cellular Delivery

Yuting Niu

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2015

Australian Institute for Bioengineering and Nanotechnology

I

Abstract

Delivery of various drugs and biomolecules into cells is crucial in modern medicine, providing

promising potential in the treatment of incurable diseases. Most naked therapeutic biomolecules, for

example, proteins, siRNAs and some free drugs can hardly penetrate into cells, thus various natural

particulates and synthetic vectors have been used as cellular delivery vehicles. The understanding of

structure–function relationships of natural particulates provides a useful guide for the design of new

nanocarriers with better safety and higher delivery efficiency. Currently, many research attempts

have focused on the synthesis of new drug delivery systems by mimicking the advantages of

enveloped viruses, which have evolved sophisticated mechanisms that make use of or shield off

cellular signalling and transport pathways to traffic within host cells and deliver cargos into

appropriate subcellular compartments. However, there are still some parameters of enveloped

viruses requiring intensive study, for example, the contribution of viral surface topography (rough

surface) to intracellular delivery of cargo molecules.

This thesis focuses on the development of a novel drug delivery system with high performance

based on the preparation of silica nanoparticles with virus-mimicking rough morphology and gains

insight into the roles of surface roughness variation and surface functionality (e.g. polyethylenimine

and octadecyl-group) in biomolecule (e.g. siRNAs and therapeutic proteins) delivery performance.

The main achievements obtained in this thesis are listed below.

In the first part, a new and facile approach has been developed to prepare the virus-mimicking silica

nanoparticle (VMSN) with a rough surface. We show that increases in nanoscale surface roughness

promote both binding of biomolecules (e.g., genetic molecules) and cellular uptake; thus, the cargo

delivery efficiency is significantly increased, regardless of surface functionality and cell types.

Finally, gene delivery efficiency was tested, where the biomimetic nanoparticles shows a better cell

growth inhibition performance than the smooth silica nanoparticle and a commercial delivery

reagent.

In the second part, the novel and facile approach for systematically controlling surface roughness of

silica nanoparticles has been developed. Based on our "neck-enhancing" approach, rough silica

nanoparticles (RSNs) with a fixed core particle (211 nm in diameter) and varied shell particles are

obtained. The increase of shell particle s z s rom 13 to 98 nm enlarges interspacing distance

tw n n our n s ll p rt l s rom 7 to 38 nm, where protein molecules will favourably

absorb onto one of RSNs without impacting protein binding ability. Moreover, hydrophobically

modified RSN having the optimized interspacing distance of 38 nm successfully complexes with

II

therapeutic anti-pAkt antibody, and it shows enhanced intracellular delivery efficiency in human

breast cancer (MCF-7) cells, leading to significant cell growth inhibition by blocking pAkt and the

downstream anti-apoptotic protein of Bcl-2.

In the third part, we quantitatively demonstrate both the individual and combined contributions of

surface roughness and hydrophobic modification for the improvement of protein therapeutics. Both

surface roughening and hydrophobic modification enhance the protein adsorption capacity, while

the contribution from surface roughness is more effective. For sustained protein release,

hydrophobic modification has a stronger effect compared to rough surface. Both structural

parameters improve cellular uptake performance; however the contribution difference is cell type-

dependent. It is clear that surface roughness has little contribution to endo/lysosomal escape. Only

surface chemistry, i.e., hydrophobic modification, facilitates the release of nanoparticle/cargo

molecules from endosome/ lysosome entrapment. Collectively, octadecyl-functionalized rough

silica nanoparticle (C18-RSN) shows the best performance in therapeutic protein (RNase A)

delivery, causing significant cell viability inhibition in MCF-7 and SCC-25 cell lines compared to

RSN and smooth silica nanoparticle with (C18-SSN) and without (SSN) C18-modification.

III

Declaration by author

This thesis is composed of my original work, and contains no material previously published or

written by another person except where due reference has been made in the text. I have clearly

stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical

assistance, survey design, data analysis, significant technical procedures, professional editorial

advice, and any other original research work used or reported in my thesis. The content of my thesis

is the result of work I have carried out since the commencement of my research higher degree

candidature and does not include a substantial part of work that has been submitted to qualify for

the award of any other degree or diploma in any university or other tertiary institution. I have

clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and,

subject to the policy and procedures of The University of Queensland, the thesis be made available

for research and study in accordance with the Copyright Act 1968 unless a period of embargo has

been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright

holder(s) of that material. Where appropriate I have obtained copyright permission from the

copyright holder to reproduce material in this thesis.

IV

Publications during candidature

1. Yuting Niu, Amirali Popat, Meihua Yu, Surajit Karmakar, Wenyi Gu and Chengzhong Yu.

Recent advances in the rational design of silica-based nanoparticles for gene therapy. Therapeutic

Delivery, 2012, 3(10), 1217–1237 (Review paper, Yuting and Amirali contribute equally to this

paper)

2. Yuting Niu, Meihua Yu, Sandy B. Hartono, Jie Yang, Hongyi Xu, Hongwei Zhang, Jun Zhang,

Jin Zou, Annette Dexter, Wenyi Gu and Chengzhong Yu. Nanoparticles Mimicking Viral Surface

Topography for Enhanced Cellular Delivery. Adv. Mater. 2013, 25, 6233. (This work has been

highlighted as the Frontispiece)

3. Yuting Niu, Meihua Yu, Jun Zhang, Yannan Yang, Chun Xu, Michael Yeh, Elena Taran, Jeff Jia

Cheng Hou, Peter P. Gray and Chengzhong Yu. Synthesis of Silica Nanoparticles with Controllable

Surface Roughness for Therapeutic Protein Delivery. Journal of Materials Chemistry B, 2015,

2015,3, 8477-8485.

4. Yuting Niu, Meihua Yu, Anand Meka, Yang Liu, Jun Zhang, Yannan Yang, and Chengzhong

Yu. Understanding the Contribution of Surface Roughness and Hydrophobic Modification on Silica

Nanoparticles for Enhanced Therapeutic Protein Delivery. Journal of Materials Chemistry B, 2015,

DOI: 10.1039/C5TB01911G.

5. Yannan Yang, Yuting Niu, Jun Zhang, Anand Kumar Meka, Hongwei Zhang, Chun Xu, Chun

Xiang Cynthia Lin, Meihua Yu and Chengzhong Yu. Biphasic Synthesis of Large-Pore and Well-

Dispersed Benzene Bridged Mesoporous Organosilica Nanoparticles for Intracellular Protein

Delivery. Small, 2015, 11, 2743-2749 (Yannan and Yuting contribute equally to this paper)

6. Meihua Yu, Yuting Niu, Jun Zhang, Siddharth Jambhrunkar, Hongwei Zhang, Wenyi Gu, Peter

Thorn, and Chengzhong Yu. Size-dependent gene delivery efficiency of amine modified

monodisperse Stöber spheres. Nano Research, 2015, DOI: 10.1007/s12274-015-0909-5. (Meihua

and Yuting contribute equally to this paper)

7. Chun Xu, Yuting Niu, Amirali Popat, Siddharth Jambhrunkar, Surajit Karmakar and

Chengzhong Yu. Rod-like mesoporous silica nanoparticles with rough surfaces for enhanced

cellular delivery. Journal of Materials Chemistry B, 2014, 2, 253-256

8. Meihua Yu, Yuting Niu, Yannan Yang, Sandy Budi Hartono, Jie Yang, Xiaodan Huang, Peter

Thorn, and Chengzhong Yu. An approach to prepare polyethyleneimine functionalized silica-based

spheres with small size for siRNA delivery, ACS Applied Materials & Interfaces, 2014, 6, 15626-

15631.

9. Chun Xu, Meihua Yu, Owen Noonan, Jun Zhang, Hao Song, Hongwei Zhang, Chang Lei,

Yuting Niu, Xiaodan Huang, Yannan Yang, Chengzhong Yu. Core-cone Structured Monodispersed

V

Mesoporous Silica Nanoparticles with Ultra-large Cavity for Protein Delivery. Small, 2015, 11(44):

5949–5955.

10. Yusilawati Ahmad Nor, Yuting Niu, Surajit Karmakar, Liang Zhou, Jun Zhang, Meihua Yu,

Donna Mahony, Neena Mitter, Matthew Cooper, Chengzhong Yu. Shaping Nanoparticles with

Hydrophilic Compositions and Hydrophobic Properties as Nano-carriers for Antibiotic Delivery.

ACS Central Science, 2015, 1 (6), 328–334.

Conference proceedings

1. Yuting Niu, Meihua Yu, Sandy B. Hartono, Jie Yang , Hongyi Xu, Hongwei Zhang, Jun Zhang,


Topography for Enhanced Cellular Delivery. 2013 International Conference on BioNano Innovation

(ICBNI), 8th

September 2013, Shanghai, FuDan University, China (Oral presentation).

Publications included in this thesis

1. Yuting Niu, Amirali Popat, Meihua Yu, Surajit Karmakar, Wenyi Gu and Chengzhong Yu.

Recent advances in the rational design of silica-based nanoparticles for gene therapy. Therapeutic

Delivery, 2012, 3(10), 1217–1237 – partially incorporated in Chapter 2.

Contributors Statement of contribution

Author Y. Niu (Candidate)

Draft design (60 %)

Drafting and writing (65 %)

Author A. Popat

Draft design (25 %)


Author M. Yu Drafting and writing (3 %)

Author S. Karmakar Drafting and writing (3 %)

Author W. Gu Drafting and writing (4 %)

Author C. Yu

Draft design (15 %)


VI

2. Yuting Niu, Meihua Yu, Sandy B. Hartono, Jie Yang, Hongyi Xu, Hongwei Zhang, Jun Zhang,


Topography for Enhanced Cellular Delivery. Adv. Mater. 2013, 25,6233.–incorporated as Chapter 4



Experimental design and performance (75 %)

Analysis and interpretation of data (70 %)


Author M. Yu Experimental design and performance (5 %)


Author S. Hartono




Author J. Yang




Author H. Xu




Author H. Zhang Analysis and interpretation of data (1 %)

Author J. Zhang Experimental design and performance (1 %)

Author J. Zou Analysis and interpretation of data (1 %)

Author A. Dexter Analysis and interpretation of data (1 %)

Author W. Gu




Author C. Yu




VII

3. Yuting Niu, Meihua Yu, Jun Zhang, Yannan Yang, Chun Xu, Michael Yeh, Elena Taran, Jeff Jia

Cheng Hou, Peter P. Gray and Chengzhong Yu. Synthesis of Silica Nanoparticles with Controllable

Surface Roughness for Therapeutic Protein Delivery. Journal of Materials Chemistry B, 2015,3,

8477-8485. – incorporated as Chapter 5.






Author M. Yu





Author Y. Yang Experimental design and performance (1 %)

Author C. Xu Experimental design and performance (1 %)

Author M. Yeh




Author E. Taran Analysis and interpretation of data (1 %)


Author J. Hou




Author P. Gray Drafting and writing (1 %)

Author C. Yu




VIII

4. Yuting Niu, Meihua Yu, Anand Meka, Yang Liu, Jun Zhang, Yannan Yang and Chengzhong Yu.

Understanding the Contribution of Surface Roughness and Hydrophobic Modification on Silica

Nanoparticles for Enhanced Therapeutic Protein Delivery. Journal of Materials Chemistry B, 2015,

DOI: 10.1039/C5TB01911G--incorporated as Chapter 6.






Author M. Yu




Author A. Meka Experimental design and performance (3 %)

Author Y. Liu Experimental design and performance (3 %)


Author Y. Yang Experimental design and performance (2 %)

Author C. Yu Experimental design and performance (10 %)



Contributions by others to the thesis

ICPOES analysis was performed by Dr. David Appleton (Chapter 4, 5 & 6).

Statement of parts of the thesis submitted to qualify for the award of another degree

None

IX

Acknowledgements

Most importantly, I would like to show my gratitude and great appreciation to my supervisor Prof.

Chengzhong (Michael) Yu for his supervision, excellent guidance, support and friendship during

my PhD period. Also, I would like to thank my co-supervisors Dr. Wenyi Gu and Dr. Annette

Dexter, for their advice, guidance, kind help and continuous encouragement in my PhD research

work. In addition, I would thank Dr. Meihua Yu for her enormous help during my PhD life, and

appreciate the help from Dr. Jeff Jia cheng Hou, Dr. Michael Yeh, Anand Meka and Yang Liu when

I am in urgent situation.

I would also thank the help and support from kind group members, including Dr. Jie Yang, Dr. Jun

Zhang, Dr. Sandy Budi Hartono, Dr. Surajit Karmakar, Dr. Amirali Popat, Dr. Siddharth

Jambhrunkar, Dr. Liang Zhou, Dr. Kun Qian, Yannan Yang, Hongwei Zhang, Chang Lei, Chun Xu,

Owen Noonan, Yusilawati Ahmad Nor, Prasanna Lakshmi Abbaraju, Xiaoran Sun, Hao Song,

Swasmi Purwajanti, Mohammad Kalantari, Min Zhang and Yue Wang for their direct or indirect

help in my research, and appreciate the help from the staff in the office, including Ms. Celestien

Warnaar-Notschaele, Ms. Cheryl Berquist and Mr. Chaoqing Lu.

Many thanks to the staff in the Australian National Fabrication Facility and the Australian

Microscopy and Microanalysis Research Facility at the Center for Microscopy and Microanalysis,

The University of Queensland, for providing the training and technical analysis help. I would like to

thank Mr. David Appleton for his help in ICPOES analysis from the School of Agriculture and

Food Sciences, the University of Queensland.

The financial support in terms of UQI from the University of Queensland, PhD Scholarship Top Up

from Australian Institute for Bioengineering and Nanotechnology and PhD scholarship from China

Scholarship Council are greatly appreciated.

Last but not least, I would like to express my deepest acknowledgement to my parents and Dr.

Xianyuan Shao for their endless love, support, and encouragement during my PhD study.

X

Keywords

bio-inspired, virus-mimicking, silica nanoparticle, surface roughness, intracellular delivery, gene

therapy, protein therapeutics, surface functionalization

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 100708, Nanomaterials, 40%

ANZSRC code: 090302, Biomedical Engineering, 40%

ANZSRC code: 100712, Nanoscale Characterisation, 20%

Fields of Research (FoR) Classification

FoR code: 0304, Medical & biomolecular chemistry, 70%

FoR code: 0601, Biochemistry & cell biology, 30%

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Table of Contents

Abstract ................................................................................................................................................. I

Declaration by author ......................................................................................................................... III

Publications during candidature ......................................................................................................... IV

Conference proceedings ...................................................................................................................... V

Publications included in this thesis ..................................................................................................... V

Contributions by others to the thesis ............................................................................................... VIII

Statement of parts of the thesis submitted to qualify for the award of another degree ................... VIII

Acknowledgements ............................................................................................................................ IX

Keywords ............................................................................................................................................ X

Australian and New Zealand Standard Research Classifications (ANZSRC) .................................... X

Fields of Research (FoR) Classification ............................................................................................. X

Table of Contents ............................................................................................................................... XI

List of Figures & Tables .................................................................................................................. XV

List of abbreviations used in the thesis ......................................................................................... XXII

Chapter 1 Introduction....................................................................................................................... 1

1.1 Significance of the project .......................................................................................................... 1

1.2 Research objectives and scope ................................................................................................... 2

1.3 Thesis outline ............................................................................................................................. 2

1.4 References .................................................................................................................................. 4

Chapter 2 Literature Review ............................................................................................................. 5

2.1 Modern medications for bio-application .................................................................................... 5

2.1.1 Therapeutic nucleic acids for gene therapy ......................................................................... 5

2.1.1.1 DNA-based therapeutics ............................................................................................... 7

2.1.1.2 RNA-based therapeutics ............................................................................................... 8

2.1.2 Therapeutic proteins for cancer therapy ............................................................................ 10

2.1.2.1 Enzyme........................................................................................................................ 10

2.1.2.2 Targeting protein ......................................................................................................... 11

2.1.2.3 Vaccine........................................................................................................................ 11

2.2 Recent advances in bio-inspired drug delivery carriers ........................................................... 12

2.2.1 Brief introduction of natural particulates as drug delivery vehicles .................................. 12

2.2.2 The advances of bio-inspired nanoparticles in bio-application ......................................... 14

2.2.2.1 Pathogen-mimicking strategies ................................................................................... 14

XII

2.2.2.2 Cell-mimicking strategies ........................................................................................... 15

2.2.2.3 Other strategies ........................................................................................................... 17

2.3 Strategies to engineer silica-based nanoparticles as effective gene and protein carriers ......... 17

2.3.1.1 Surface charge ............................................................................................................. 18

2.3.1.2 Surface hydrophobicity ............................................................................................... 20

2.3.1.3 Influence of serum protein .......................................................................................... 20

2.3.2 Modification with other materials ..................................................................................... 21

2.3.2.1 Organic materials ........................................................................................................ 21

2.3.2.2 Inorganic materials ...................................................................................................... 24

2.3.3 Particle nanotechnology ..................................................................................................... 25

2.3.3.1 Particle size ................................................................................................................. 25

2.3.3.2 Pore size and pore structure ........................................................................................ 26

2.3.3.3 Particle shape and surface morphology ...................................................................... 27

2.4 Conclusions and future perspective .......................................................................................... 28

2.5 References ................................................................................................................................ 29

Chapter 3 Methodology .................................................................................................................... 45

3.1 Materials synthesis ................................................................................................................... 45

3.1.1 Synthesis of monodispersed solid silica nanoparticles as core particle ............................. 45

3.1.2 Synthesis of monodispersed solid silica nanoparticles as shell particles .......................... 45

3.1.3 Synthesis of virus-mimicking silica nanoparticles (VMSNs) ........................................... 46

3.1.4 Functionalization of silica nanoparticles ........................................................................... 46

3.1.4.1 Amine-group modification .......................................................................................... 46

3.1.4.2 Hydrophobic modification .......................................................................................... 46

3.1.4.3 PEI attachment ............................................................................................................ 46

3.2 Characterizations ...................................................................................................................... 47

3.2.1 Transmission electron microscopy .................................................................................... 47

3.2.2 High resolution scanning electron microscopy .................................................................. 47

3.2.3 Dynamic light scattering .................................................................................................... 48

3.2.4 Z t (ζ) pot nt l n lys s .................................................................................................. 48

3.2.5 Nitrogen sorption ............................................................................................................... 48

3.2.6 Attenuated total reflectance-Fourier transform infrared spectroscopy .............................. 49

3.2.7 Elemental analysis ............................................................................................................. 49

3.2.8 Atomic force microscopy measurements ........................................................................... 49

3.2.9 Inductively coupled plasma optical emission spectroscopy .............................................. 50

3.2.10 Surface plasmon resonance measurements ...................................................................... 51

XIII

3.2.11 Thermogravimetric analysis ............................................................................................ 52

3.2.12 X-ray photoelectron spectroscopy ................................................................................... 52

3.3 Biological techniques ............................................................................................................... 52

3.3.1 Agarose gel electrophoresis ............................................................................................... 52

3.3.2 Confocal laser scanning microscopy ................................................................................. 53

3.3.3 MTT assay ......................................................................................................................... 53

3.3.4 Flow cytometry .................................................................................................................. 54

3.3.5 Western-blot analysis......................................................................................................... 54

3.3.6 TEM study on nanoparticle-cell interaction ...................................................................... 55

3.4 References ................................................................................................................................ 55

Chapter 4 Nanoparticles Mimicking Viral Surface Topography for Enhanced Cellular

Delivery ............................................................................................................................................. 57

4.1 Introduction .............................................................................................................................. 58

4.2 Results and discussion .............................................................................................................. 59

4.3 Conclusion ................................................................................................................................ 65

4.4 References ................................................................................................................................ 66

Supplementary Information ............................................................................................................ 68

Material and Methods ................................................................................................................. 68

4.S1. Synthesis of Samples ..................................................................................................... 68

4.S2. Characterizations ........................................................................................................... 69

4.S3. Estimation of shell / core ratio from ET data ................................................................. 70

4.S4. Biological experiments .................................................................................................. 70

Supplementary Figures and Tables ............................................................................................. 74

References ................................................................................................................................... 83

Chapter 5 Synthesis of Silica Nanoparticles with Controllable Surface Roughness for

Therapeutic Protein Delivery .......................................................................................................... 86

5.1 Introduction .............................................................................................................................. 87

5.2 Experimental section ................................................................................................................ 89

5.2.1 Materials ............................................................................................................................ 89

5.2.2 Synthesis of different nanoparticles ................................................................................... 89

5.2.3 Characterizations ............................................................................................................... 91

5.2.4 Biological experiments ...................................................................................................... 92

5.3 Results and Discussion ............................................................................................................. 94

5.4 Conclusion .............................................................................................................................. 103

5.5 References .............................................................................................................................. 103

XIV

Supplementary Figures and Tables .............................................................................................. 106

References .................................................................................................................................... 113

Chapter 6 Understanding the Contribution of Surface Roughness and Hydrophobic

Modification on Silica Nanoparticles for Enhanced Therapeutic Protein Delivery ................ 114

6.1 Introduction ............................................................................................................................ 115

6.2 Experimental .......................................................................................................................... 116

6.2.1 Materials and reagents ..................................................................................................... 116

6.2.2 Synthesis of nanoparticles ............................................................................................... 117

6.2.3 Characterizations ............................................................................................................. 117

6.2.4 Biological experiments .................................................................................................... 119

6.3 Results and discussion ............................................................................................................ 120

6.4 Conclusion .............................................................................................................................. 128

6.5 References .............................................................................................................................. 129

Supplementary Figures and Tables .............................................................................................. 131

References .................................................................................................................................... 138

Chapter 7 Conclusions and outlook .............................................................................................. 139

7.1 Conclusions ............................................................................................................................ 139

7.2 Recommendations for future work ......................................................................................... 141

7.3 References .............................................................................................................................. 141

XV

List of Figures & Tables

Figure 2.1 Various engineering strategies for virus-based vectors, virus-like particles and

virosomes.

Figure 2.2 Preparation and structure of the virus-mimicking nanogel.

Figure 2.3 Mechanism for the assembly of influenza virus-inspired polymer, binding with siRNA

and release of siRNA through a self-catalyzed degradation of PDMAEA.

Figure 2.4 Schematics of the preparation process of the RBC-membrane-coated PLGA

nanoparticles.

Figure 2.5 Endocytic pathways traversed by nonviral carriers. (A) Macropinocytosis of cationic

particles. (B) Nonviral vectors can also be internalized by other pathways such as clathrin-mediated

endocytosis, which is a receptor-mediated pathway. (C) Caveolae-mediated endocytosis proceeds

by oligomerization of caveolin, actin-dependent internalization of caveolae to form cavicles and

merger with the degradative lysosomal compartment, or nondegradative trafficking to the nucleus

via caveosomes. Each pathway relies on microtubules for rapid transport of endocytic vesicles.

HSPG: Heparan sulfate proteoglycans.

Figure 2.6 Steps of multivalent binding and internalization of targeted protocells, followed by

endosomal escape and nuclear localization of protocell encapsulated cargo. (1) DOPC protocells

particles binds to cell surface with high affinity due to attachment of targeting peptides; (2) then

goes through cell memberane via receptor-mediated endocytosis; (3) release drugs into cytosol on

endosome acidification and protonation of H5WYG fusogenic peptide. (4) Finally, NLS-attached

cargo is transported in the nucleus. DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine; NLS:

nuclear localization signal.

Figure 2.7 Nuclear-targeted drug delivery of Tat peptide-conjugated mesoporous silica

nanoparticles. (A) Preparing amino group- and Tat-FITC peptide-conjugated MSNs. (B) Transport

of DOX@MSNs-Tat across the nuclear membrane. (C) Confocal laser scanning microscopy images

of MSNs-Tat with diameters of (1) 25, (2) 50, (3) 67 and (4) 105 nm after incubation with HeLa

cells for (i) 4, ( ) 8 nd ( ) 24 . S l rs: 5 μm. APTES: Am nopropyl tr t oxy s l n ; CTAC:

Hexadecyltrimethylammonium chloride; MSN: Mesoporous silica nanoparticle; TEA: Tetraethyl

amine; TEOS: Tetraethyl orthosilicate.

XVI

Figure 3.1 Schematic illustration of VMSN, followed by amine-group and PEI modification.

Figure 3.2 Illustration of two-stage tomography process with (left) acquisition of an ensemble of

images (projections) about a single tilt axis and (right) the back-projection of these images into 3D

object space.

Figure 3.3 An atomic force microscope probes a molecule adsorption onto a surface, using a carbon

monoxide molecule at the tip for sensitivity.

Figure 3.4 Schematic illustration of a Surface Plasmon Resonance (SPR) system.

Figure 4.1 Illustration of: a) the synthesis procedure, and, b) the comparison of cellular delivery

performance between two nanocarriers. a) Sample 1 represents silica nanoparticles, which can be

further modified with positively charged amine groups or polyethylenimine (PEI). Sample 2

comprises the negatively charged silica nanoparticles with small diameters. Sample 3 was prepared

by using amino-modified 1 as the core and 2 as the shell particles after calcination, which is

modified with amine groups or PEI. b) Compared to smooth nanocarriers, rough ones exhibit both

higher binding ability towards biomolecules (e.g., proteins and genetic molecules) and increased

cellular uptake efficiency, independent of surface functionality.

Figure 4.2 Surface characteristics of 3. a,b) SEM images show the virus-mimicking rough surface.

c) A zero-tilt TEM projection from a tilt series. d) Reconstructed surface rendering of a single

particle. The core particle is shown in blue and the shell spikes in yellow. Scale bar: 100 nm.

Figure 4.3 Virus-mimicking nanoparticles enhance cellular delivery performance in HeLa cells. a,b)

Confocal microscopy images of Cy3-oligoDNA (red color) delivered by (left) 1 -NH2 and (right) 3 -

NH2 . The nuclei are stained in blue (DAPI) and the cell membranes in green (FITC). Scale bar: 20

μ m. ) FACS n lys s o Cy3-oligoDNA delivery, showing stronger Cy3- signals in MFI using 3 -

NH 2 than 1 -NH2. d) Identification of Cy3-oligoDNA binding for 1 -NH2 and 3-NH2. e)

Investigation of biomolecule holding ability calculated from gel retardation assay. f) Comparison of

cellular uptake efficiency of the complexes (1-NH2 / 3-NH2 +Cy3-oligoDNA) measured by

ICPOES. For bar charts, data represent mean ± s.e.m. **indicates p < 0.01,*** p < 0.001, **** p <

0.0001 based on a t -test.

Figure 4.4 Gene delivery performances of virus-mimicking nanoparticles in KHOS cells. a) The

inhibition of cell viability by PLK1-siRNA transfection. S10-siRNA was used as a negative control.

Both 1-PEI and 3-PEI were used as vectors, and a commercial reagent, OligofectamineTM

, was also

applied as a positive control. b) PLK1-siRNA adsorption ability of 1-PEI and 3-PEI. c) Comparison

XVII

of cellular uptake efficiency of the complexes (1-PEI/ 3-PEI+PLK1-siRNA) measured by ICPOES.

Data represent mean ± s.e.m. * indicates p < 0.05, *** p < 0.001 based on a t -test.

Figure 4.S1 TEM images of all samples and size distribution. a, e) sample 1 & 3. b, f) 1-NH2 & 3-

NH2. c, g) 1-PEI & 3-PEI. d) Sample 2 (shell particle). h) Size distribution curves obtained by the

dynamic light scattering (DLS) method.

Figure 4.S2 Surface area tested by Nitrogen adsorption. The surface area of 1 (with a smooth) and

3 (with a rough surface) was measured to be 19 and 24 m2/g, respectively.

Figure 4.S3 Confocal microscopy image shows no Cy3 signals is observed when the same amount

of Cy3-oligoDNA alone is incubated with cells, indicating the inability of genetic molecules

t ms lv s to p n tr t nto lls. S l r: 20 μm.

Figure 4.S4 FACS analysis showing the peak shifts in the MFI from HeLa cells incubated with the

complexes of 1-NH2 (blue colour)/ 3-NH2 (yellow colour) +Cy3- oligoDNA. The Cy3-oligoDNA

in the absence of nano-carriers (red colour) is used as a control.

Figure 4.S5 Comparison of cellular delivery of cargos among different nanoparticles. a, b, c) Very

weak signals were observed in the groups of naked Cy3-oligoDNA, Cy3- oligoDNA coupled with d,

e, f) 2-NH2 (0.9 μ /mL, dosage calculated from ET results) and g, h & i) 1-NH2. j, k & l) Only 3-

NH2 induced evident Cy3 signals across all groups.

Figure 4.S6 Agarose gel analysis. a) The complex of amino-modified nanocarriers with Cy3-

oligoDNA. Lane 0: 25 pmol Cy3-oligoDNA only. Lane 1: 25 pmol Cy3- oligoDNA coupled with

50 μ o 1-NH2. Lane 2: 25 pmol Cy3-ol oDNA oupl d w t 50 μ o 3-NH2. In the case of 1-

NH2, a small amount of released Cy3-oligoDNA was observed. For 3-NH2, nearly no release could

be seen by naked eyes. b, c) the complexes of PEI-modified nano-carriers with PLK1-siRNA. Lane

0: 25 pmol PLK1- siRNA only. Lane 1, 2, 3: 100, 50 and 25 pmol PLK1-siRNA are coupled with

50 μ o 1-PEI and 3-PEI, respectively. In each lane from 1 to 3, the PLK1-siRNA release was

evident in the case of 1-NH2, compared to that of 3-NH2.

Figure 4.S7 Investigation of pure silica nano-carriers in adsorption and cellular uptake. a)

Cytochrome C adsorption. The adsorption amount is 2.71 nmol/mg on 3, and 0.88 nmol/mg on 1. b)

cellular uptake into KHOS cells. 128 pg/cell was internalized for 3, while 62 pg/cell for 1. c)

cellular uptake into HeLa cells. 242 pg/cell was internalized for 3, while 187 pg/cell for 1. Data

represent mean ± s.e.m of three independent experiments. *p<0.05, ***p<0.001,****p<0.0001 (t-

test).

XVIII

Figure 4.S8 Cytotoxicity of nanoparticles. a, b) sample 1 & 3 in HeLa (left) and KHOS (right) cells.

c, d) 1-NH2 & 3-NH2 in HeLa (left) and KHOS (right) cells. e) 1-PEI & 3- PEI in KHOS cells.

Pure and amino-modified silica nanoparticles have relatively mild cytotoxicity to both HeLa and

KHOS cells, and the ones with rough surface are more toxic due to the improved cellular uptake.

However, the attachment of PEI caused a high toxicity to cells, so that the concentrations of 1-PEI

& 3-PEI w ll d r s d to 20 μ /mL in gene silencing experiment. Data represent mean ± s.e.m

of three independent experiments.

Figure 4.S9 Gene delivery performance of virus-mimicking nanoparticles in KHOS cells using

amino-modified nanoparticles. Neither PLK1-siRNA nor S10-siRNA delivered by 1-NH2 and 3-

NH2 induced the decrease of cell viability by gene silencing effect. In the bar chart, data represent

mean ± s.e.m of three independent experiments, and were analyzed using t-test.

Figure 4.S10 TGA of PEI-conjugation. In both a) 1–PEI and b) 3-PEI, the TGA curves of (i)

represent pure silica nanoparticles, (ii) show epoxy-group modified nanoparticles, (iii) are the PEI-

conjugated separately.

Figure 4.S11 Morphology adjustment of virus-mimicking nanoparticles. After fabricating the virus-

mimicking nanoparticles by mixing shell particles with core particles, morphology changes were

traced by TEM images. On adjusting the feed volumes of shell particle solutions from a) 0.68 mL to

b) 1.35 mL, to 2.70 mL (Figure. S1e), to c) 5.40 mL and finally to d) 6.75 mL, the surface

morphologies changed significantly, with the best sample being obtained when the feed volume was

2.70 mL. It is noted that a failure to obtain optimized virus-mimicking morphologies can be

attributed to either insufficient or excessive feed amount of shell particles, where only a proper feed

amount will lead to successful synthesis. Scale bar: 100 nm.

Figure 4.S12 The influence of calcination treatment. TEM image a) shows that without calcination,

shell particles peeled off on ultrasonication. In contrast, b) shows that the calcined sample 3

maintained its morphology after ultrasonication. Scale bar: 100 nm.

Figure 5.1 Schematic illustrations of the synthesis of RSNs using a "neck-enhancing" approach (a)

and a conventional interaction approach (b). Scheme c shows the cellular delivery of therapeutic

anti-pAkt antibody using C18-RSNs and the cell growth inhibition mechanism.

Figure 5.2 TEM (a-d) and HRSEM (e-h) images of RSNs with varied shell particle sizes: a&e)

RSN-211@13, b&f) RSN-211@28, c&g) RSN-211@54, d&h) RSN-211@98 and the red arrows

indicate the formation of bigger “n ks” onn t n s ll nd or p rt l s. S l r: 100nm.

XIX

Figure 5.3 Protein Adsorption profiles ( IgG-A; IgG-F; ▲ yto rom c). Solid lines represent

different protein adsorption onto unmodified rough silica nanoparticles. The dash line also

represents the IgG-A adsorption onto different rough silica nanoparticles, except they are all

modified with C18-groups. Data represent mean ± SD. Specific surface area variations (×) of

different unmodified rough silica nanoparticles are displayed to compare with protein adsorption

trend.

Figure 5.4 Surface topography studies. AFM images of RSN-211@54 before (a) and after (c) the

adsorption of IgG-F. A cross-sectional line is drawn to characterize the height changes of shell

particles on the top region before (b) and after (d) protein adsorption. Scale bar: 100 nm.

Figure 5.5 SPR sensorgrams showing the binding signals of RSN-IgG-F complexes with receptors.

All RSN-IgG-F complexes showed positive values of the binding with ligand.

Figure 5.6 Cell growth inhibition by the delivery of therapeutic protein. a) Cell viability of MCF-7

cells incubated with increasing concentrations of C18-RSN-211@98+anti-pAkt (), C18-RSN-

211@98+non-specific-IgG-A (♦) nd C18-RSN-211@98. Data represent mean ± SD. b) Western

blotting confirming the degradation of downstream anti-apoptotic protein, Bcl-2 in MCF-7 cells.

Blots presented are representative of typical results. GAPDH served as an internal reference.

Figure 5.S1 TEM images (a-g) and particle size distribution curves (f) of shell and core particles.

Shell particles: a&h-i) 13nm, b&h-ii) 28nm, c&h-ii) 54nm, d&h-iv) 98nm, e&h-v) 135nm and f&h-

vi) 175nm. Core particle: g&h-vii) 211nm. Scale bar: 100 nm

Figure 5.S2 The interspacing distance of RSNs. a) RSN-211@13, b) RSN-211@28, c) RSN-

211@54, d) RSN-211@98. The interspaces are measured from SEM images by recording 50 edge-

to-edge interspacing data in each sample.

Figure 5.S3 TEM images showing the synthesis of RSN-211@28 using previous recipe after

washing and drying process (a) and in reaction solution (b). Scale bar: 100 nm

Figure 5.S4 TEM images of failed synthesis of RSNs with much larger shell sizes. a) Core particles

(dotted arrow) mixed with the shell of 135 nm (solid arrow), b) Core particle (dotted arrow) mixed

with the shell of 175 nm (dotted arrow). Scale bar: 100 nm.

Figure 5.S5 Fourier transform infrared (FTIR) spectra of pure liquid n-ODMS (a) and a series of

RSNs with and without hydrophobic modification (b).

XX

Figure 5.S6 TEM images of hydrophobic modified RSNs. a) C18-RSN-211@13, b) C18-RSN-

211@28, c) C18-RSN-211@54 and d) C18-RSN-211@98 (d). Scale bar: 100 nm.

Figure 5.S7 Cell viability of MCF-7 cells incubated with varying concentrations of non-specific

IgG-A () only and anti-pAkt antibody () only, respectively. Data represent mean ± SD.

Figure 5.S8 The comparison of anti-pAkt antibody delivery efficiency (a) and cellular uptake

performance of C18-RSNs measured by ICPOES. (b) Data represent mean ± SD.

Figure 6.1 TEM (a, b, d, e) and HRSEM (c) images and particle size distribution curves (f) of RSN

(a&f-i), C18-RSN (b, c&f-ii), SSN (d&f-iii), C18-SSN (e&f-iv). Scale bar: 100 nm.

Figure 6.2 (a) RNase A adsorption and (b) release profiles of SSN, RSN, C18-SSN and C18-RSN.

Data represent mean ± SD.

Figure 6.3 Cellular uptake performance of RNase A loaded nanoparticles in (a) MCF-7 and (b)

SCC-25 cells, measured by ICPOES. Cells only were used as a control group. Data represent mean

± SD.

Figure 6.4 Typical TEM images of ultra-thin sections of MCF-7 cells incubated with (a) SSN, (b)

RSN, (c) C18-SSN and (d) C18-RSN for 24 h. White arrow indicates cell membrane (CM), black

arrow indicates nanoparticles entrapped in endosome (E) or lysosome (L), black arrowhead shows

locally disrupted membrane of endosomes, black dash arrow indicates nanoparticles distributed in

cytoplasm (C). "N" refers to nucleus. Scale bar: 100 nm.

Figure 6.5 Cell viability of (a) MCF-7 and (b) SCC-25 cells treated with RNase A at a dosage of 2

μ /mL after 24, 48, and 72 h incubation. Data represent mean ± SD.

Figure 6.S1 TEM images (a) OH-core particle, (b) NH2-core particle and (c) shell particle. Scale

bar: 100 nm. (d) Particle size distribution curves.

Figure 6.S2 Fourier transform infrared (FTIR) spectra of (a) pure liquid OTMS, (b) bare

nanoparticles, (c) RNase A and (d) nanoparticles complexed with RNase A.

Figure 6.S3 (a, c, e) AFM images and (b, d, f) void height profiles of (a & b) RSN, (c & d) C18-

RSN and (e & f) C18-RSN + RNase A, generated by drawing a typical cross-sectional line on the

top region, Scale bar: 100 nm. The average height values are measured and calculated by recording

20 data. Data represent mean ± SD.

XXI

Figure 6.S4 Particle size distribution curves tested in PBS. (a) SSN, (b) C18-SSN, (c) RSN and (d)

C18-RSN.

Figure 6.S5 Silica dissolution of different nanoparticles, tested by (a) ICPOES and TEM images of

(b) SSN, (c) C18-SSN, (d) RSN and (e) C18-RSN, after shaking in PBS for 3 days. Scale bar: 100

nm.

Figure 6.S6 RNase A release profiles of SSN, RSN, C18-SSN and C18-RSN at (a) acidic condition

(pH 4.5) and (b) in DMEM under static condition at 37 °C for 4 h. Data represent mean±SD.

Figure 6.S7 The evaluation of toxicity from pure nanoparticles (a,c,e) in MCF-7 cells and (b,d,f) in

SCC-25 cells, at 24 h, 48 h and 72 h, respectively. Data represent mean ± SD.

Table 2.1 Function and features of nucleic acids as therapeutic agents for gene therapy.

Table 2.2. Function and features of different therapeutic proteins for cancer therapy.

Table 4.S1 Physicochemical Properties of nanoparticles

Table 4.S2 Atomic composition (%) of nanoparticles

Table 4.S3 Characterization of PEI-modification by TGA

Table 5.S1 Size and ζ potential characterizations of shell and core particles.

Table 5.S2 Characterizations of RSNs with varied surface topography.

Table 5.S3 Estimation of protein coverage on RSNs/C18-RSNs

Table 5.S4 SPR signal intensity.

Table 6.1 Summary of the contribution of surface roughness and hydrophobic modification

Table 6.S1 Size and ζ pot nt l r t r z t ons o n nop rt l s.

Table 6.S2 DLS size measurements in PBS

Table 6.S3 RNase A adsorption density and C18-modification characterization of nanoparticles

XXII

List of abbreviations used in the thesis

Pc4: silicon phthalocyanine 4

MDR: multiple drug resistance

PDT: photodynamic therapy

DNA: deoxyribonucleic acid

RNA: ribonucleic acid

pDNA: plasmid DNA

PLL: poly-l-lysine

PS: polystyrene

PEI: polyethyleneimine

miRNA: microRNA

siRNA: small interfering RNA

mRNA: messenger RNA

SELEX: systematic evolution of ligands by

exponential enrichment

MSNs: mesoporous silica nanoparticles

RNAi: RNA interference

RISC: RNA-induced silencing complex

MB: molecular beacon

MF: magnetic fluorescence

FDA: food and drug administration

RNase A: ribonuclease A

mAb: monoclonal antibody

TAA: tumor-associated antigen

HPV: human papillomavirus

RBC: red blood cell

RES: reticuloendothelial system

DOX: doxorubicin

BSA: bovine serum albumin

PEG: polyethylene glycol

Tf: transferrin

PAA: poly(acrylic acid)

PLGA: poly(lactic-co-glycolic acid)

PFT: pore-forming toxin

LLV: leuko-like vector

RGD: Arg-Gly-Asp

CREKA: Cys-Arg-Glu-Lys-Ala

HUVEC: human umbilical vein endothelial

cell

CME: clathrin-mediated endocytosis

CvME: caveolae-mediated endocytosis

IEP: isoelectric point

PBS: phosphate buffered saline

AP: 3-aminopropyl

GP: guanidinopropyl

GEGP: 3-[N-(2-guanidinoethyl)-

guanidino]propyl

XXIII

FAP: N-folate-3-aminopropyl

hMSC: human mesenchymal stem cell

ROS: reactive oxygen species

SCC-25: human squamous carcinoma cells

PAMAM: polyamidoamine

R8: octaarginines

FP: fusogenic peptides

NLS: nuclear localization signal

NPC: nuclear pore complex

ER: endoplasmatic reticulum

CM: chloromethyl

MP: mercaptopropyl

Oc: octyl

FCS: fetal calf serum

EPR: enhanced permeation and retention

CTAB: cetyltrimethylammonium bromide

CTAC: hexadecyltrimethyl ammonium

chloride

TEA: tetraethyl amine

HMSN: hollow mesoporous silica

nanoparticle

MOSF: macroporous ordered siliceous foam

PMO: periodic mesoporous organosilica

AR: aspect ratio

TEOS: tetraethyl orthosilicate

APTES: (3-aminopropyl)triethoxy silane

VMSN: virus-mimicking silica nanoparticle

n-ODMS/OTMS: n-octadecyltrimethoxy

silane

3-GPS: 3-glycidoxypropyl trimethoxysilane

TEM: transmission electron microscopy

SEM: scanning electron microscopy

UED: upper electron detector

ET: electron tomography

3D: three-dimensional

DLS: dynamic light scattering

ZP: zeta potential

BET: Brunauer–Emmett–Teller

ATR-FTIR: attenuated total reflectance -

Fourier transform infrared spectroscopy

EA: elemental analysis

AFM: atomic force microscopy

SPM: scanning probe microscope

ICP-OES: inductively coupled plasma-optical

emission spectroscopy

RU: resonance unit

SPR: surface plasmon resonance

EMSA: electrophoretic mobility shift assay

CLSM: confocal laser scanning microscopy

XXIV

MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyl tetrazolium bromide

FITC: fluorescein-5-isothiocyanate

HSV: herpes simplex virus

HIV: human immunodeficiency virus

Cy3: cyanine 3

FACS: fluorescein-activated cell sorting

MFI: fluorescence intensity

PLK1: polo-like kinase 1

XPS: X-ray photoelectron spectroscopy

TGA: thermogravimetric analysis

DMEM: Dulbecco's Modified Eagle's

Medium

DMEM/F12: Dulbecco's modified Eagle's

medium and Ham's F12 medium

DAPI: 4’-6-diamidino-2-phenylindole

TAE: tris-acetate-EDTA

PFA: paraformaldehyde

RSN: rough silica nanoparticle

ATCC: American Type Culture Collection

SDS-PAGE: sodium dodecyl sulphate-

polyacrylamide gel electrophoresis

IgG-A: IgG antibody

IgG-F: IgG fragment

C18-SSN: octadecyl-functionalized smooth

silica nanoparticles

SR: surface roughness

Chapter 1 Introduction

1

Chapter 1

Introduction

1.1 Significance of the project

Effective cellular delivery of various drugs and/or biomolecules is pivotal to satisfy the

requirements of modern medicine.1, 2

Most naked biomolecules and some free drugs are poorly

delivered to cells owing to poor stability, low solubility and/or unwanted toxicity. For these reasons,

various natural particulates and synthetic vectors have been used as cellular delivery vehicles.

Natural particulates, for example, enveloped viruses, have evolved sophisticated mechanisms that

make use of or shield off cellular signalling and transport pathways to traffic within host cells and

deliver cargos into the appropriate subcellular compartment with maximal efficiency.3 However,

several limitations are associated with viral vectors, such as carcinogenesis,4 immunogenicity,

5

broad tropism,6 limited DNA packaging capacity

7 and difficulty of vector production,

8 and an

alternative solution is required. Inspired by viruses, currently, many research attempts have focused

on the synthesis of non-viral delivery systems by mimicking the advantages of viruses, including

the attachment of viral receptors (e.g., Tat9), the imitation of virus core-shell structure10

and the

incorporation of functional polymer to mimic endosomal escape of viruses.11

However, there are

still some parameters of viruses requiring intensive study, for example, the contribution of viral

surface topography to intracellular delivery.

The understanding of structure–function relationships of enveloped viruses provides a useful guide

for the design of new nanocarriers. Recent developments in state-of-the art electron tomography

(ET) v prov d d “n no- olo y” n orm t on or m ny nv loped viruses, for example,

influenza virus,12

herpes simplex virus (HSV),13

and human immunodeficiency virus (HIV),14

all

showing rough surfaces patched by glycoprotein spikes. However, the influence of nanoscale

surface roughness on cellular delivery efficiency remains unclear because it is always associated

with receptor–ligand specific interactions in viral systems. Therefore, a systematic study to explore

the impacts of surface roughness of nano-vectors on the improvement of cargo delivery is required.


2

1.2 Research objectives and scope

This research aims to develop a novel approach to prepare silica-based non-viral vectors by

mimicking the surface topography of enveloped viruses and gain insight into the roles of surface

roughness variation and surface functionality (e.g. polyethylenimine and octadecyl-group) in

biomolecule (e.g. siRNAs and therapeutic proteins) delivery performance. This thesis does not only

focus on the development of facile synthesis of nanoparticles with novel structures, but also

provides some guidelines for the design of highly efficient delivery systems for various biomedical

applications. The objectives of this project are specified as follows:

1) To fabricate silica nanoparticles with virus-mimicking rough morphology and particle size

smaller than 300 nm, and to confirm the significance of VMRM in intracellular delivery of genetic

molecules.

2) To synthesize silica nanoparticles with varied surface roughness for controlled therapeutic

protein adsorption and optimized protein therapeutics.

3) To understand the effects of the surface morphology and functionality of silica nanoparticles on

therapeutic protein adsorption, cellular uptake, cargo release and endosomal escape.

1.3 Thesis outline

This thesis is written according to the guidelines of the University of Queensland. The outcomes of

this PhD thesis are presented in the form of journal publications. The chapters in this thesis are

presented in the following sequence:


This chapter introduces the background of this project and outlines the research objectives

Chapter 2 Literature review

This chapter presents an overview on recent advances in bio-inspired drug delivery systems and the

current strategies to engineer silica-based nanoparticles as effective genetic molecules and

therapeutic protein carriers.

Chapter 3 Methodology

This chapter summarizes the strategies utilized in the whole PhD project, including material

synthetic methods for virus-mimicking silica nanoparticles, and the techniques for material

characterizations and biology experiments.


3

Chapter 4 Nanoparticles Mimicking Viral Surface Topography for Enhanced Cellular

Delivery

This chapter reports the synthesis of novel non-viral nanoparticles mimicking virus surface

topography by attaching small shell particles (~10nm) onto the core particles with large sizes

(~200nm). The increase in nanoscale surface roughness improved both binding of biomolecules

(e.g., genetic molecules) and cellular uptake, regardless of surface functionality and cell types.

Moreover, the delivery efficiency of siRNA was significantly increased, compared to conventional

silica nanoparticles with a smooth surface and a commercial transfection reagent.

Chapter 5 Synthesis of Silica Nanoparticles with Controllable Surface Roughness for

Therapeutic Protein Delivery

This chapter reports a novel "neck-enhancing" approach to synthesize silica nanoparticles with

controlled surface roughness. By roughening the surface of silica core particles (211 nm in

diameter) with smaller shell particles having various sizes, a series of rough silica nanoparticles

(RSNs) with stable structures were obtained. The interspacing distance between neighbouring shell

particles increased from 7 to 38 nm with increasing shell particle sizes from 13 to 98 nm. Protein

loading capacity was dependent on both protein molecule size and interspacing distance, and

protein binding activity was not influenced. Hydrophobically modified RSNs with the interspacing

distance of 38 nm showed effective intracellular delivery of anti-pAkt antibody in breast cancer

MCF-7 cells, leading to significant cell growth inhibition by blocking pAkt and the downstream

anti-apoptotic protein of Bcl-2.

Chapter 6 Understanding the Contribution of Surface Roughness and Hydrophobic

Modification on Silica Nanoparticles for Enhanced Therapeutic Protein Delivery

This chapter quantitatively demonstrates both the individual and combined contributions of surface

roughness and hydrophobic modification for the improvement of protein therapeutics. Both surface

roughening and hydrophobic modification enhance protein (RNase A) adsorption capacity, while

the effect of surface roughness is more dominant. Hydrophobic modification strongly retards RNase

A release. The contribution difference to enhance cellular uptake is cell type-dependent.

Importantly, only surface chemistry, i.e., hydrophobic modification in this work, facilitates the

release of nanoparticle/cargo molecules from endosome/ lysosome entrapment. Collectively,

octadecyl-functionalized rough silica nanoparticle (C18-RSN) shows the best performance in

RNase A delivery, causing significant cell viability inhibition in both human breast cancer (MCF-7)


4

and SCC-25 cell lines, compared to unmodified rough silica nanoparticle and smooth silica

nanoparticles with or without octadecyl-group modification.

Chapter 7 Conclusion and outlook

This chapter presents a general discussion of the work in this thesis and outlook for the future work.

1.4 References

1 Y. Niu, A. Popat, M. Yu, S. Karmakar, W. Gu and C. Yu, Ther. Deliv., 2012, 3, 1217-1237.

2 R. A. Morgan, M. E. Dudley, J. R. Wunderlich, M. S. Hughes, J. C. Yang, R. M. Sherry, R. E.

Royal, S. L. Topalian, U. S. Kammula, N. P. Restifo, Z. L. Zheng, A. Nahvi, C. R. de Vries, L. J.

Rogers-Freezer, S. A. Mavroukakis and S. A. Rosenberg, Science, 2006, 314, 126-129.

3 D. J. Glover, Infect. Disord. Drug Targets, 2012, 12, 68-80.

4 C. Baum, O. Kustikova, U. Modlich, Z. X. Li and B. Fehse, Hum Gene Ther, 2006, 17, 253-263.

5 N. Bessis, F. J. GarciaCozar and M. C. Boissier, Gene Ther, 2004, 11, S10-S17.

6 R. Waehler, S. J. Russell and D. T. Curiel, Nat Rev Genet, 2007, 8, 573-587.

7 C. E. Thomas, A. Ehrhardt and M. A. Kay, Nat Rev Genet, 2003, 4, 346-358.

8 D. Bouard, N. Alazard-Dany and F. L. Cosset, Brit J Pharmacol, 2009, 157, 153-165.

9 L. Pan, Q. He, J. Liu, Y. Chen, M. Ma, L. Zhang and J. Shi, J. Am. Chem. Soc., 2012, 134, 5722-

5725.

10 E. S. Lee, D. Kim, Y. S. Youn, K. T. Oh and Y. H. Bae, Angew. Chem. Int. Edit., 2008, 47,

2418-2421.

11 N. P. Truong, W. Y. Gu, I. Prasadam, Z. F. Jia, R. Crawford, Y. Xiao and M. J. Monteiro, Nat.

Commun., 2013, 4.

12 A. Harris, G. Cardone, D. C. Winkler, J. B. Heymann, M. Brecher, J. M. White and A. C.

Steven, P. Natl. Acad. Sci. USA., 2006, 103, 19123-19127.

13 K. Grunewald, P. Desai, D. C. Winkler, J. B. Heymann, D. M. Belnap, W. Baumeister and A. C.

Steven, Science, 2003, 302, 1396-1398.

14 P. Zhu, J. Liu, J. Bess, E. Chertova, J. D. Lifson, H. Grise, G. A. Ofek, K. A. Taylor and K. H.

Roux, Nature, 2006, 441, 847-852.

Chapter 2 Literature Review

5

Chapter 2

Literature Review

This chapter reviews the advances in the rational design of bio-inspired nanocarriers for drug

delivery. It begins with an introduction of the development of modern medicines in 2.1. Therapeutic

proteins and genetic molecules as promising medications for cancer therapy will be introduced in

details in this section. The recent achievements of bio-inspired drug delivery systems will be

summarized in 2.2. Afterwards in 2.3, the engineering of silica-based nanoparticles (SiNPs) will be

systematically described for controlled cargo delivery. Finally, a summary of current state-of-the-art

technology and future challenges to fabricate bio-inspired SiNPs are proposed in 2.4. Part of the

contents in this literature review has been published as a review paper. (Y. Niu, A. Popat, M. Yu,

S. Karmakar, W. Gu, C. Yu, Therapeutic Delivery. 2012, 3, 1217 – 1237).

2.1 Modern medications for bio-application

Small molecule drugs are the most commonly used for cancer treatment, such as hydrophilic

anticancer drug of doxorubicin,1 hydrophobic drug of tamoxifen

2 and curcumin,

3 as well as

hydrophobic photosensitiser of silicon phthalocyanine 4 (Pc4) for photodynamic therapy (PDT).4

However, pharmaceutical drugs have evident drawbacks of non-specific toxicity to normal cells and

multi-drug resistance (MDR). Therefore, new drugs with better safety and high specificity are

required. Currently, genetic and protein-based biomolecules have attracted increasing attention as

new therapeutics. In the follow part, the classifications of both genetic and protein-based molecules

with therapeutic potential will be summarized.

2.1.1 Therapeutic nucleic acids for gene therapy

With the developments in gene engineering and the understanding of pathogenesis in molecular

level, the efficiency of gene therapy has been significantly improved, especially in the last decade.

In this part, various therapeutic DNA/RNA molecules will be summarized. Table 2.1 summarizes

the features of functional nucleic acids and their application in gene therapy.


6

Table 2.1 Functions and features of nucleic acids as therapeutic agents for gene therapy.

Nu l ds Fun t on D s r pt on Ex mpl s

DNA

pDNA 5-8

Tr ns r n od n

DNA s qu n s

1 to ov r 1,000 k p,

nt rn l z nto lls lon or

om n w t v r l or non-

v r l v tors

Intr tumor l nj t on 6, nt rn l z d y v r l 9 or non- v r l

10

v tors

Sup r o l d

m n - r l

DNA 11

Tr ns r n od n

DNA s qu n s

L ss t n 4k p, d vo d o

t r lly or n t d

mmuno n un-m t yl t d

CpG mot s, mu r

tr ns t on n y

Tr ns r Ad pon t n

DNA s qu n w t

PEI 12

Junk DNA13

D m n s t us o

n od n DNA

D lut n t un t on l DNA

nd n r s n t mount o

t v NPs

D m n s t us o

r port r DNA, w l

s ow t s m t13

RNA

R ozym 14-17

C t lyz RNA or v n

DNA l v Dou l -str nd d RNA

Corr t t mut t d

xpr ss on o α1-AT-

prot n n p tom

lls, us n

ulov rus v tor 18

Ant s ns RNA 19-21

B n ompl m nt ry

to mRNA, n t n

tr nsl t on w t n ll

S n l -str nd d ol o-RNA

Suppr ss t HPV-

r l t d tumor rowt ,

d l v r d y v r l

v tor 22

Apt m r 23-27

T r t n omol ul s

s l nds

RNA or DNA mol ul s o

20–50 nu l ot d s, old n

nto un qu t rt ry

on orm t ons, non-

mmuno n , ood st l ty,

n ty nd sp ty

Att on t NPs o

PLGA- -PEG,

sp lly r o n z

t nt n on prost t

n r ll m m r n 28

s RNA /

m RNA 29-31

Cl v nd d r d

t r t n mRNAs,

n t t tr nsl t on

o t t r t n n

Dou l -str nd d, d s n d

nd r t d n m l

w y, 20-25 nu l ot d s

T r t to t d

r m d s n ,

ul l n kno kdown 32

S n l -str nd d, or n t d

rom ndo nous nom

DNA s qu n s, 20-25

nu l ot d s

D t t nd n t

m RNA un t on n C6

n r lls, r du

ll v l ty 33

Ot r

C m r pl st 34,

35

Corr t nd r p r t

po nt mut t on w t n

DNA mol ul

Cons st n o DNA nd RNA,

ont n n on - s m sm t

w t t t r t DNA

Ful l n t r py o

β –t l ss m 36


7

2.1.1.1 DNA-based therapeutics

Plasmid DNA

A plasmid DNA (pDNA) is a double-stranded and circular DNA molecule, with the size fluctuating

from 1 to over 1,000 kilo base pair (kbp), and it has been widely used in the fields of biology and

medicine, for example, cancer therapy. The pDNAs are usually collected from bacteria, although

some of them are found in eukaryotic organisms (e.g. 2 µm plasmid DNA in Saccharomyces

cerevisiae5). Independent from chromosomal DNA, pDNAs are able to replicate and stably pass to

daughter cells.

The application of pDNA with inserted functional DNA sequences as a framework for gene therapy

has attracted much attention during the last decade. Although some researchers reported similar

transfection and expression efficiency of naked pDNA, compared to equipped ones6, it is reported

only a thousandth of the presented naked pDNAs to cells can successfully arrive at the nucleus and

express,37

because they may be quickly digested by nucleases or cleared from blood circulation.7, 8

Many methods and materials have been selected as pDNA vectors. For instance, by electrostatic

force-driven complexation with pDNA, polymer-based (e.g. poly-l-lysine [PLL]-polystyrene [PS]38

)

DNA vaccine has shown great induction of CD8+ T cell immune response to viruses. In addition,

the expression product of a pDNA, such as GFP39, 40

and luciferase10

, may exhibit fluorescent signal

for diagnosis and simulation of gene therapy.

Minicircle

Minicircle is a newly discovered member of pDNA. This kind of circular DNA molecules with

mini-size (~4 kbp) is able to avoid the disadvantages of conventional pDNA, such as a risk of

uncontrolled dissemination of therapeutic gene or antibiotic resistance gene sequence originating

from bacterial,11

because those genes, together with native regulatory gene sequences, have been

removed, and minicircles will not replicate and be passed to daughter cells. Park et al. performed

the adiponectin gene delivery, using a minicircle-polyethylenimine (PEI) complex to treat diet

induced obese C57BL/6J mice, and it showed higher adiponectin expression than conventional

pDNA in vitro and in vivo.12

Junk DNA

Apart from functional DNA, non-coding DNA molecules, known as junk DNA, have shown some

interesting effects on improving transfection efficiency and, meanwhile, decreasing active DNA

usage. Van Gaal et al. 13

confirmed that by using the same amount of PEI as the vector and the same


8

vector to cargo (DNA) ratio, similar transfer activity was observed between the loading of pure

reporter pDNA and the mixture of reporter pDNA with junk DNA. The results have demonstrated

the total amount of active DNA-containing vectors is more important rather than total DNA cargos.

2.1.1.2 RNA-based therapeutics

RNA is another big family of therapeutic nucleic acids, mainly including ribozymes, aptamers,

microRNAs (miRNAs) and small interfering RNAs (siRNAs). Numerous RNA-based therapeutics

are currently under clinical investigation for diseases ranging from HIV infection to genetic

disorders to various cancers.41

As a result of their flexibility and versatility in structure and

function,42-45

gene therapy protocols could be designed in various ways. However, due to the

instable feature of RNA molecules, vehicles to target cells are required.

Ribozyme

A ribozyme is a type of double-stranded RNA molecules. Due to the unique tertiary structure,14-16

it

possesses the ability to catalyse RNA or even DNA cleavage (e.g., hairpin ribozyme14

or

hammerhead ribozyme15-17

), which has the potential to regulate gene expression. For example,

Ozaki and coworkers reported a hammerhead ribozyme was used to correct the mutated expression

o α1- ntryps n (α1-AT)-protein in hepatoma cells.18

In addition, using baculovirus as a vector,

HIV-1 replication was significantly suppressed by HIV-1 U5 gene-specific ribozyme.46

Antisense RNA

An antisense RNA is a single-stranded oligo-RNA. Being complementary to a messenger RNA

(mRNA), it is able to inhibit translation processes.19, 20

Antisense RNAs are confirmed to exist

widely in nature, and up to 72% of the transcripts are demonstrated to have antisense partners in

human and mouse transcriptomes,21

which show antisense RNAs have promising exploitation in

gene therapy. For example, Zhu et al. reported that PLL-modified SiNPs could successfully

compact and protect the antisense c-myc.47

After transfection, c-myc mRNA levels were

significantly deregulated. In addition, Barnor et al. showed the replication of HIV-1 vif antisense

RNA fragments in MT-4 and H9-infected cells and reduced HIV-1 vif mRNA transcripts.48

Aptamer

To be different from protein-based ligands (e.g. proteins23

and ATP24

), aptamers are very good

candidates for biomolecular targeting. They are RNA/DNA molecules of 20–50 nucleotides in

length, and selected from in vitro experiments (termed SELEX: systematic evolution of ligands by

exponential enrichment).25, 26

They are able to fold into the unique tertiary conformation to bind


9

with viral or cellular proteins with high affinity and specificity.27

Better than common protein-based

ligands, aptamers are non-immunogenic and have evident stability in a wide range of pH (4–9),

temperature, and organic solvents without the loss of activity. Their synthesis is an entirely

chemical process that can decrease batch-to-batch variability when production is scaled up.28

Usually, aptamers are used as ligands for cell recognition. For example, sgc8-aptamer was

covalently connected to mesoporous silica nanoparticles (MSNs)-polyelectrolyte multilayer-

complex for specific cell recognition. 49

Interestingly, some aptamers also have the ability to

specifically block protein functions, such as, transcription factors in E2F family, so that cell

proliferation can be controlled.50-52

siRNA and miRNA

RNA interference (RNAi) is a post-transcriptional level process that suppresses the activity of

specific genes and is referred to as "gene silencing", discovered by Fire and Mello in 1998.31

Two

types of non-coding small RNA molecules – miRNA (single-stranded, originated from endogenous

genome DNA sequences) and siRNA (double-stranded, designed and fabricated in chemical way)

are pivots to RNAi, and both of them have the length of 20-25 nucleotides. By the activation of

RNA-induced silencing complex (RISC), both siRNAs and miRNAs are able to cleave and degrade

targeting mRNAs. Nowadays, they have been effective and popular agents for studying and treating

diseases, such as cancer, respiratory disease, neuronal disease, and autoimmune disease.29, 30

Due to their different origins, siRNAs and miRNAs are exploited in different ways. In cancer cells,

some miRNAs are over expressed, and these will wrongly up- or down-regulate cell activity. In that

case, the deregulation of miRNAs is a fundamental treatment to the pathogenesis of many cancers.53

For instance, Kim et al. conjugated the miRNA-221 molecular beacon (miR-221 MB) on a

magnetic fluorescence NP and transferred the complex into C6 (glial cell) cancer cells,33

and a

significant reduction in cell viability of cancers was observed. On the contrary, siRNAs were often

directly delivered into cells to down-regulate the expression of a specific gene. Ashley et al.

reported a successful gene silencing using large pore MSN-lipid bilayers containing siRNAs in

pores, leading to significant regression of protein expression and apoptosis.54

Chimeraplasty

Besides pure DNA/RNA molecules, chimeric RNA–DNA oligonucleotides (termed as

chimeraplasts) have been designed as therapeutics. First, they will pair with related genomic DNA

sequences, and then correct and repair the point mutation within the sequence. The designed one-

base mismatch between a chimeraplast and the mutated chromosomal DNA will activate endog-


10

enous repair mechanisms, in order to produce a single nucleotide replacement.34, 35

For example, Li

et al. d s n d RNA/DNA ol onu l ot d s or n t r py o β–thalassemia, and nucleotide

mut t on n β-globin gene cluster was successfully corrected.36

However, not every study supports

this mechanism. Tagalakis et al.55

have attempted to create a point mutation in the mouse ApoE

gene by microinjection of chimeraplast, to confirm the potential to design defined mutations.

Despite of successful transfection of chimeraplasts into one-cell mouse eggs, no evidence for

successful conversion was observed.

2.1.2 Therapeutic proteins for cancer therapy

Nowadays, hundreds of proteins and peptides applied as therapeutic agents have been approved for

clinical use by the US Food and Drug Administration (FDA). This indicates that protein-based

therapeutics with variable functions and applications has attracted increasing attention in modern

medicine. In this part, the classification of protein-based therapeutics and their potential for cancer

therapy is summarized (Table 2.2).

2.1.2.1 Enzyme

Enzymatic proteins are superior catalysts that can specifically decompose substrates in

physiological conditions, converting target molecules into products and amplifying following chain

reactions.56

Because of these unique properties, enzymatic proteins have been considered as

therapeutic agents in cancer therapy since the beginning of 20th

century.57

Intracellular delivery of proteins with enzymatic activities can help to terminate protein synthesis in

cancer cells. One of the examples is ribonuclease A (RNase A), which is a predominant form of the

enzyme in the pancreas of Bos Taurus.58

After intracellular delivery of RNase A into cytosols, it

will degrade mRNA and tRNA and inhibit protein synthesis, thus strongly influence cell functions

and cause deleterious effects on cell viability.59

In addition, saporin-S6 (also known as saporin) is

also effective to interfere with cell functions. Saporin belongs to ribosome-inactivating protein

family, classically identified as rRNA N-glycosylase (EC 3.2.2.22). It is widely distributed among

plant genera, and confirmed to specifically remove A4324 adenine residue in 28S rRNA in the 60S

subunit of rat ribosome, so that it can damage ribosomes irreversibly and inhibit protein synthesis.60

Many vectors have been used to deliver enzymatic proteins for cancer therapy. For instance,

enhanced by hydrophobic modification, negatively charged silica nanoparticles were used to

immobilize RNase A on the surface and deliver it into human breast cancer cells (MCF-7) for cell

growth inhibition.61

Saporin was encapsulated in lipid-like nanoparticles, known as lipidoids, and


11

this complex inhibited cell proliferation in vitro and suppresses tumor growth in a murine breast

cancer model.62

2.1.2.2 Targeting protein

With the help of intermolecular forces of electrostatic forces, hydrogen bonds and disulphide bonds,

proteins or peptides can form 3-dimensional (3-D) rough and flawy structures, which are the bases

of targeted interaction of proteins with other molecules.63

The targeting proteins specifically

interacting with molecules inside or on the surface of cancer cells can block the functions of target

molecules for cell destruction. In addition, cytotoxic drugs to cancer cells can be conjugated with

targeting proteins for enhanced cancer therapy. Monoclonal antibodies (mAbs) with high binding

specificity have been widely used in numerous ways, and grown to be the largest class of human

medicine within the top ten best-selling drugs. The delivery of monoclonal antibody to pAkt (anti-

pAkt) into cytosols of cancer cells has been reported to result in apoptosis.61

In addition, the

delivery of mAbs to CTLA4 to the tumor site successfully caused greater and extended inhibition of

the tumor growth than antibody given systematically.64

Some cell targeting peptides have also been

applied for cancer treatment.64

Cytotoxic drug-conjugated peptides can specifically recognize the

target cells expressing its receptors and direct killing effect. One example is the application of a

target cytotoxic somatostatin analogue AN-238, consisting of doxorubicin derivative linked to SST

octapeptide analogue RC-121. AN-238 shows effective cell growth inhibition in multiple cancer

cell lines.65

2.1.2.3 Vaccine

Cancer vaccines have great potential for anti-tumor immunity based on the activation of immune

system. This strategy depends on the selection of tumor-associated antigens (TAAs), which are

mutated protein molecules on tumor cell surface or peptides (with only 8-10 amino acids long),

derived from the mutated proteins, signalling t pr s n o ll t t s om “ or n”.

TAAs can be recognized by immunocytes (e.g., T-cell), which will then be activated and induce an

effective, tumor-specific immune reaction, leading to tumor regression.66, 67

For example, for the

prevention of cervical cancer and genital warts and vulvar and vaginal precancerous lesions, a

recombinant quadrivalent vaccine (commercially GARDASILs) has been prepared from capsid

proteins of human papillomavirus (HPV). It has been approved for commercial use in women aged

9-26 years, without pathogenicity.68

Moreover, peptide vaccines have attracted increasing attention

because of the diversity and smaller size, compared to large protein vaccines. Recently, three people

with melanoma have received personalized treatment with a dendritic cell vaccine (an amino acid


12

substitution peptide) to alert their immune system the threats of mutated proteins in tumours,

providing an important proof of concept for the application of vaccines in cancer therapy.69

Table 2.2. Function and features of different therapeutic proteins for cancer therapy.

Types Function Description Examples

Enzymes

specifically decompose

substrates in physiological

conditions

terminate protein synthesis

in cancer cells RNase A,

61 Saporin

62

Targeting

proteins

specifically interact with

molecules inside or on the

surface of cancer cells

block cell functions or

targeted delivery of

cytotoxic drug for cell

destruction

mAb to pAkt,61

mAb

to CTLA4,64

AN-23865

vaccines activation of immune system

selection of TAAs and

recognition by T-cells,

followed by cell regression

GARDASILs,68

a

dendritic cell vaccine69

In summary, various types of nucleic acids and protein-based molecules have been applied in

modern medicine, especially in cancer therapy. However, because of their difficulties to cross cell

membranes and the fragility in severe conditions, for example, the potential of proteolysis and

degradation when they are localized in endosome/lysosome with acidic surroundings, effective

nanocarriers are required for enhanced biomolecule delivery efficiency.

2.2 Recent advances in bio-inspired drug delivery carriers

2.2.1 Brief introduction of natural particulates as drug delivery vehicles

The intracellular delivery of therapeutic drugs is crucial in modern medicine. Most naked

biomolecules (e.g., siRNAs and therapeutic proteins) and some free drugs are poorly delivered into

cells owing to poor stability, low solubility and/or native toxicity. Natural particulates have evolved

to deliver cargo molecules with maximal efficiency; therefore, provide useful examples and raw

materials for the application as nanocarriers. Until now, plenty of natural particulates have been

engineered accordingly and used for drug delivery. Based on the sources of natural particulates used

for drug delivery, they are classified into two groups, pathogens and mammalian cells.


13

Pathogenic particulates (e.g., bacteria and virus) can effectively invade into target cells, followed by

cargo release and replication of themselves, without rapid clearance by immune system. For

example, engineered lactic acid bacteria can deliver vaccines by the quick recognition and

engulfment by immunocytes (dendritic cells).70

In addition, live or attenuated bacterium can

combine with different nanoparticles for efficient vaccine/drug delivery71

or magneto-optical

applications.72

For virus-based vectors,73

virus-like particles (with74

or without synthetic materials75

)

and virosomes (Figure 2.1),76

they have small sizes of 30-400 nm77

and, more importantly, the

ligands on the spikes of enveloped viruses (e.g., influenza virus78

and herpes simplex virus79

) can

guide specific ligand-receptor interactions with host cells for effective viral entry, followed by viral

genome release in cytosols.80

Apart from pathogen-based drug delivery system, various types of mammalian cells, such as red

blood cells (RBCs),81

dendritic cells82

and macrophages83

have been used as drug delivery carriers.

Although they cannot directly deliver drugs into target cells for disease treatment, they possess

evident advantages of excellent biocompatibility, prolonged circulation time (up to 120 days) and

final clearance from blood by reticuloendothelial system (RES). In that case, free drugs or drug-

loaded nanoparticles can combine with mammalian cells, either inside the cells or attached on the

surface, and can be specifically transported to the disease sites for sustained therapeutic effects.84

Figure 2.1 Engineering strategies for virus-based vectors, virus-like particles and virosomes.85

Although natural particulate-based drug delivery systems have been confirmed effective, the

clinical application is still immature and several issues need to be resolved. One of the top

challenges is the safety concerns of immunogenicity or even pathogenicity of pathogen-based

carriers,73

unless, vaccine delivery is considered as the application. The immunogenicity would be

the key point of pathogen-based carriers to function as adjuvants. In addition, since ex vivo


14

engineering is always required to modify cell-based vehicles, the membrane integrity is highly

possible to be influenced, which will cause even faster clearance of the drug-loaded carriers from

circulation.

2.2.2 The advances of bio-inspired nanoparticles in bio-application

The understanding of structure–function relationships of natural particulates provides a useful guide

for the design of new nanocarriers. Currently, many research attempts have focused on the synthesis

of new drug delivery systems by mimicking the advantages of natural particulates, such as

structures (e.g., shape and inner structure) and surface chemical properties. In the following part,

the current development of bio-inspired nanocarriers is summarized.

2.2.2.1 Pathogen-mimicking strategies

The pursuit of low toxicity and little immunogenicity, but high delivery efficiency has led to the

development of synthetic vectors-mimicking the structure and/or composition of different

pathogens. Lee et al., described a pH-sensitive nanogel system mimicking the core-shell structure of

non-enveloped viruses (Figure 2.2). 86

The core is made of the doxorubicin (DOX)-encapsulated

hydrophobic polymer, while the shell mimicking capsid structure is composed of bovine serum

albumin (BSA), connected by polyethylene glycol (PEG) with the core. The surface of BSA is

further conjugated with folate ligands for targeted delivery to tumor sites. When the surrounding pH

changes from a physiological value (pH 7.4) to endosomal level (pH 6.4), the nanogel will

reversibly swell from 55 nm to 355 nm, facilitating endosomal escape and DOX release for

significant cancer killing effect. Noticeably, the nanogel will move into and kill the neighbouring

cells in the same way, mimicking the cycles of viruses.

Figure 2.2 Preparation and structure of the virus-mimicking nanogel.86


15

Researchers also tried to mimic the features of enveloped viruses for improved drug delivery. A

virus-mimicking liposome-pDNA complex, with an onion-like core of pDNA-lipid complex and

spikes of human transferrin (Tf) coating, has been fabricated.87

Tf-polyplex exhibits improved in

vivo pDNA transfection efficiency and long-term efficacy for systemic p53 gene therapy of human

prostate cancer in combination with conventional radiotherapy. In addition, polymer-based

materials are also used to mimic the features of influenza viruses for enhanced siRNA delivery

(Figure 2.3).88

In the system, siRNAs can compact with the cationic polymer at physiological pH.

After endocytosis and entrapment in endosome/lysosome at acidic condition, this cationic polymer

will degrade into negatively charged and non-toxic poly(acrylic acid) (PAA), releasing siRNAs.

After that, another polymer will be in charge of the fusion with endosome membranes, similar to the

function of fusion peptide (HA2) in influenza viruses, and result in endosomal escape of siRNAs for

gene silencing.

Figure 2.3 Mechanism for the assembly of influenza virus-inspired polymer, binding with siRNA

and release of siRNA through a self-catalyzed degradation of PDMAEA.88

2.2.2.2 Cell-mimicking strategies

Cell-mimicking nanoparticles have gained promising attention to drug delivery because they

possess plenty of excellent properties of the source cells. One of the attractive features of cell-

biomimetic nanoparticles is the long-circulating cargo delivery. Zhang and coworkers successfully

separated erythrocyte membranes, acquiring the membrane lipids associated membrane proteins,

and coated them on biodegradable polymeric nanoparticles of poly(lactic-co-glycolic acid) (PLGA)

(Figure 2.4).89

The erythrocyte membrane-camouflaged PLGA nanoparticles can achieve sustained

circulation in the blood of mice for 72 h following the particle injection. After that, the erythrocyte

membrane-coated PLGA nanoparticles were used for effective removal of pore-forming toxins

(PFTs) in mice.90

Following that, they inserted a model PFT of staphylococcal a-haemolysin (Hla)

into erythrocyte membranes, and used it as a toxoid vaccine. Comparing the vaccination effect with

heat-attenuated PFT, the PFT loaded erythrocyte membrane-coated PLGA nanoparticles showed

superior immunogenicity without toxin-mediated adverse effects.91


16

Figure 2.4 Schematics of the preparation process of RBC-membrane-coated PLGA nanoparticles.89

In addition, the membranes from other cell types have also been applied to coat nanoparticles to

form biomimetic nanocarriers. For example, after isolation of the leukocyte membrane, the

engineered leuko-like vectors (LLV) encapsulating a nanoporous silicon core avoided blood

clearance by mononuclear phagocytic cells, promote active communication with endothelial cells,

and improve the circulation time and accumulation in tumor sites, so that increase drug delivery

efficiency.92

Cancer cell-derived membranes bound with tumor antigens have also been used to

package PLGA nanoparticles for potential application of vaccine and/or drug delivery. The antigens

d r v d rom B16−F10 mous m l nom lls on n no rr r sur lp to v n r

targeted delivery, due to the inherent homotypic binding phenomenon among the tumor cells.

Importantly, the nanocarrier successfully delivers an immunological adjuvant (MPLA), promoting a

tumor-specific immune response for the use in vaccine application.93

Synthetic particles mimicking cell morphology and functions have also been developed. Different

RBC-like particles have been fabricated using hollow polystyrene (PS) microparticle94

or hydrogel-

based microspheres95, 96

as the template, possessing the ability of deformation in capillaries.

Noticeably, PS-based RBC-like particle is able to mimic the functions of natural RBCs to carry

oxygen, and deliver drugs and imaging agents. Platelet-mimicking nanoparticles with the features to

induce additional local clotting have also been developed. This particle possesses a specific binding

affinity for activated platelets, and it is able to adhere and activate platelets at the bleeding site, so

that it can halt bleeding.97

To be different, the iron oxide nanoparticles coated with a homing

peptide, CREKA (Cys-Arg-Glu-Lys-Ala), is applied to simultaneously recognize clotted plasma

proteins at vessel walls and selectively homes to tumors stroma, amplifying tumor imaging, and

potentially enhance drug delivery efficiency.98


17

2.2.2.3 Other strategies

Besides pathogen- and mammalian cell-mimicking strategies, creatures in nature will also inspire

the development of nanoparticles with unique properties. Learning from the functions of jellyfish

with long tentacles in catching prey from surroundings, affinity DNA polymers (DPs) and DNA

polymers with aptamers (PAs) on iron nanoparticles have been synthesized for drug sequestration

and detoxification.99

It is demonstrated the model drugs of DOX and thrombin were effectively

sequestered from solution. More importantly, the toxic effect of DOX to human umbilical vein

endothelial cells (HUVECs) is largely decreased, and the coagulation time is also shortened,

indicating the ability of different nanoparticles in mitigating biological effect of different drugs.

2.3 Strategies to engineer silica-based nanoparticles as effective gene and protein carriers

The key factors for successful gene and protein therapy are effective intracellular delivery of

cargos, overcoming in a number of barriers before the released genetic molecules and therapeutic

proteins exhibit their functions. First, NP-cargo complexes must penetrate across cell membrane,

usually via endocytic pathways (Figure 2.5), which predominantly include phagocytosis, clathrin-

mediated endocytosis (CME), caveolae-mediated endocytosis (CvME) and macropinocytosis.100-102

These cell-penetration processes can be regulated and improved by surface charge adjustment or

ligand conjugation. In addition, engineered surface modification with polymers, peptides or

coupling with some other materials helps the escape of cargos captured in endosomes or lysosomes,

and may even assist to direct nuclear targeting or entry.

Silica-based nanoparticles (SiNPs) are promising materials for genetic molecule or protein delivery.

Through the sol-gel processing route, surface properties, particle size and shape, which may have

different impacts on cell activity and transfection efficiency, can be easily controlled. In addition,

after the additions of surfactants, the nanoparticles with different pore sizes and pore volumes have

been fabricated, providing access to load cargo molecules inside the pores for multifunctional

applications. In this part, the effects of different surface modifications to SiNPs from simple surface

charge adjustments to conjugation with functional molecules, on particle endocytosis efficiency,

endo/lysosomal escape and nuclear targeting will be introduced at first. Then, particle parameters,

with respect to endocytosis efficiency and the interaction with cells, will be discussed.


18

Figure 2.5 Endocytic pathways traversed by nonviral carriers. (A) Macropinocytosis of cationic

particles. (B) Nonviral vectors can also be internalized by other pathways such as clathrin-mediated

endocytosis, which is a receptor-mediated pathway. (C) Caveolae-mediated endocytosis proceeds

by oligomerization of caveolin, actin-dependent internalization of caveolae to form cavicles and

merger with the degradable lysosomal compartment, or non-degradable trafficking to the nucleus

via caveosomes. Each pathway relies on microtubules for rapid transport of endocytic vesicles.

HSPG: Heparan sulfate proteoglycans.102

2.3.1 Surface engineering

Surface properties are an important factor to influence the upload and release of genetic molecules

and proteins into cells on cancer therapy. For gene therapy, negatively charged genetic molecules

are able to complex with amine-modified SiNPs with a positive charge,103

either on the surface or

inside the pores. In addition, the conjugation/hybridation of silica materials with polymers39

or other

inorganic materials104, 105

will acquire some new properties. For protein therapeutics, different

strategies are required to achieve protein loading, due to the variable sizes and isoelectric points

(IEPs) of protein molecules. This section focuses on the development in surface modification of

SiNPs and the impact on cargo delivery and cellular response.

2.3.1.1 Surface charge

Electrostatic force is the most widely applied approach to interact with cargo molecules, NPs and

cell membrane. Due to the negatively charged cell membrane and phosphate backbone of genetic

molecules, NPs with positive charges have been considered more effective to immobilize


19

DNA/RNA and achieve cell uptake than those, which are negatively charged or neutral.30, 39, 106

However, since SiNPs have an intrinsically negative charge on the surface, derived from silanol

groups, surface charge modification is required, for example, by attaching amine-groups on particle

surface. An amino-modified SiNP (45 nm) showed better GFP pDNA adsorption and expression

behavior.103

In addition, cationic polymer-coated SiNPs (e.g., PEI-MSN or PLL-MSN) can also

acquire a positive charge on the surface and provided enhanced cell uptake and transfection

compared to unmodified SiNPs.39, 107, 108

However, the nature of protein molecules with variable

IEPs suggests different surface charge controls. For example, pristine mesoporous silica

nanospheres were first used for delivery of cytochrome c with a IEP of 10.5 into cervical cancer

cells (HeLa),109

where cytochrome c possessed positive charges in physiological buffer of

phosphate buffered saline (PBS, pH 7.4). On the contrary, positively charged surface is required for

the adsorption of therapeutic proteins with low IEP values (e.g., insulin, IEP 5.3).110

Noticeably, by varying the surface charge value, endocytic pathways will change. Slowing et al.

reported that by changing the grafted groups, SiNPs acquired positive charge with zeta potential

(ZP) values.111

In the order from 3-aminopropyl (AP), guanidinopropyl (GP), 3-[N-(2-

guanidinoethyl)-guanidino]propyl (GEGP) to N-folate-3-aminopropyl (FAP) groups, the ZP value

of the original MSNs (-34 mV) increased from -4.68 to +12.81 mV. FAP-MSN and negatively

charged pure MSNs were taken up into cells via a clathrin-pitted mechanism, and AP- and GP-

MSNs were internalized via a caveolae-mediated pathway. However, the pathway for GEAP-MSNs

was unclear, since none of the pathway inhibitors had dramatically slowed down or decreased the

internalization. Strangely, in this research, negatively charged MSNs exhibited better endo-

/lysosomal escape compared with positively charged MSNs. It was explained that the groups of

SiO- on negative charged MSNs can neutralize more H

+, leading to better behavior of the proton

sponge effect. Moreover, surface charge-related endocytosis was reported to be cell type-dependent.

Chung and co-workers claimed that unmodified (negative charged) MSNs were internalized

through a clathrin-mediated and an actin-dependent endocytosis in both murine adipocytes (3T3-

L1) and human mesenchymal stem cells (hMSCs).112

However, when using positive-charged

MSNs, such a pathway was only applied by 3T3-L1 cells, while the pathway for hMSCs have

switched to an unknown one. These results suggested that a threshold of positive surface charge was

responsible for MSN uptake by hMSC, and the surface charge-related MSN uptake was cell type-

specific.

In addition to the effect on endocytosis, surface charge is related to cytotoxicity. Usually, pure

SiNPs show more toxicity than amino-modified ones, and many researchers have given possible

explanations, such as reactive oxygen species (ROS) induced by the hydroxyl groups, denaturation


20

of membrane proteins through electrostatic interactions with silicate and the high affinity of silicate

for binding with the tetra-alkyl ammonium groups that are abundant in the membranes of RBCs.113

Although the exact mechanism is still under investigation, most researchers agree that cytotoxicity

is related to ROS induced by surface silanol groups, causing membrane damage, for example,

hemolysis of RBC.114

Shi et al. confirmed that when SiNPs were treated with alkaline media

(NH4OH), hemolysis was significantly reduced.115

This reduction was possibly related to the shift to

SiO- from Si-OH where hydroxyl groups were neutralized. Moreover, when SiNPs were incubated

with plasma or whole blood, hemolysis and cytotoxicity were also inhibited because of a protective

protein corona layer over the surface of silica particles. This result showed that the interaction of

SINPs with serum proteins was an important event that should be considered in the first place in

future clinical application.

2.3.1.2 Surface hydrophobicity

Hydrophobic interaction has been considered as an important property for enhanced genetic

molecule or therapeutic protein delivery. Nanocarriers with hydrophobic modification are believed

to either improve cell membrane interactions116

or strengthen the interactions between nanoparticle

and cargo molecules by excluding water molecules around hydrophobic moieties.117

Alshamsan et

al. have tried to modify branched PEI with stearic/oleic acid, which has a fatty acid chain of 18

carbon atoms, and the hydrophobically modified PEI increased siRNA delivery up to 3-folds

compared to the parent PEI.116

Bale and co-workers61

developed an octadecyl (C18)-group

functionalized solid silica nanoparticle. They absorbed therapeutic proteins, such as the antibody to

pAkt, on the surface and delivered them into MCF-7 breast cancer cells. The loaded therapeutic

protein resulted in significant cell growth inhibition without extended entrapment in endosomes.

Moreover, Zhang and co-workers confirmed a significant cell growth inhibition to human squamous

carcinoma cells (SCC-25) by the delivery of RNase A using C18-functionalized silica hollow

spheres.118

Therefore, hydrophobic feature is one of the promising factors to control nanoparticle

behaviors for successful drug delivery.

2.3.1.3 Influence of serum protein

Prolonged blood circulation before being cleared via nonspecific binding to serum protein is of

utmost importance to achieve sustained release of cargos, improved retention with cell membrane,

as well as enhanced penetration into targeted cells.119

Surface properties are one of the key influential factors for serum protein adsorption. For example,

the adsorption behavior of BSA at pH 5 was tested on siliceous mesocellular foams (MCF)


21

functionalized with chloromethyl (CM), mercaptopropyl (MP), octyl (Oc) or AP groups.120

The

results showed that positively charged MCF-AP exhibited the highest adsorption capacity with

negatively charged BSA, because of electrostatic interaction. However, hydrophobicity and steric

hindrance of functional groups may significantly decrease serum protein loading amount and

adsorption rate, for example, octyl-group or high amounts of CM-groups. Cell culture media with or

without fetal calf serum (FCS) can have different effects. Related investigation showed that less dis-

tinct particle aggregation was observed in serum-free conditions, while the presence of FCS led to

increased aggregation. Meanwhile, the decreased cytotoxicity in 3T3 fibroblast cells was seen after

interacting with FCS. 121

It is possible that large agglomerates significantly consumed NPs, so only

a small quantity of small agglomerates or primary particles could be internalized into cells.

Therefore, attention should be paid to increase cargo loading efficiency and proper decoration of

NPs in order to reduce aggregation, SiNP dosage and cytotoxicity. For example, PEG, with low

cytotoxicity and good hydrophilicity, has been conjugated to enhance biocompatibility and mitigate

nonspecific adsorption of SiNPs to proteins.122, 123

2.3.2 Modification with other materials

With the purpose to fabricate smart drug carriers, NPs have been used to overcome a series of

obstacles for the successful transfection into planned sites. However, simply modified silica-NP

alone cannot complete the entire work. The complexation of SiNPs with other materials has proven

to be extremely necessary.108

A variety of complexation materials have been utilized in order to

improve the specificity and efficiency of cargo delivery.

2.3.2.1 Organic materials

A wide variety of organic polymers have been coupled with SiNPs for effective genetic molecules

and therapeutic protein delivery, such as cationic polymers (e.g., PEI,39, 114, 124

PLL30, 125, 126

and

polyarginine127, 128

), hydrophilic polymer (e.g., PEG107, 126, 129

) dendrimers (e.g., polyamidoamine

[PAMAM] dendrimers130, 131

), carbohydrate-based polymers (e.g., chitosan132, 133

and dextran134-136

)

and polypeptide.137-139

Although most of the organic materials alone are capable of effectively directing cargo molecules

into cytosols or even nucleus, the combination with SiNPs have shown supplementary advantages,

providing an excellent platform for enhanced cargo delivery performance for multifunctional

applications. For example, after the connection of solid silica NPs with Tat138

or MSN with

octaarginines (R8),139

successful cell internalization was observed. Following cell uptake,

endosomal escape is the second process affecting transfection efficiency, and various


22

polymers/peptides are experienced in overcoming these obstacles. For instance, non-covalent

attachment of 10 kD PEI with pDNA/siRNA showed efficient endocytosis, expression, and gene

silencing effect.39

The PEI-grafted enzymes (superoxide dismutase and catalase) encapsulated in

hollow silica nanoparticles were delivered into cytosols to neutralize the generated superoxide,

protecting cells.124

In addition, fusogenic peptides (FP) are also effective for endosomal escape.

They are classified into three types: anionic amphiphilic, cationic amphiphilic and histidine-rich

peptides. Anionic FPs contain glutamate residues that protonates in low pH conditions (~5) in

endosome and then the formation of alpha-helix causes the disruption of endosome membrane. The

basic residues in cationic FPs are responsible for loading DNA/RNA via electrostatic force. These

basic residues have lytic activity due to the proton-sponge effect and will trigger membrane lysis,

causing related cytotoxicity. Similar to cationic FPs, histidine-rich peptides will also be protonated

at pH values of approximately 5.6–6, so that to achieve endosomal escape.140-142

Ye et al. reported a

successful synergistic application of CPP and fusogenic peptide, and this system exhibited

enhanced cell uptake, pDNA–silica NP complex transfection and gene expression.143

The complexation of silica NPs with a single material may not satisfy all the requirements for

effective transfection. For example, the strong positive charge of polycationic polymers may lead to

aggregation in the presence of serum protein. Extra molecules may also be necessary for special

purposes, such as cell recognition. In addition, polycationic polymers, such as PEI with a large

molecular weight, may cause significant cytotoxicity, due to powerful proton pump activity in

endosome/lysosomes, where excessive hydrogen ions may cause endosomal osmotic-swelling,

rupture and cell death by a mitochondrial dysfunction.5, 37

Hence, proper alleviation or modification

is required. For example, by reducing PEI molecule weight39

or coupling with other polymers, such

as hydrophilic polymer of PEG,107

or the cell-specific ligands of mannose,108

cytotoxicity resulting

from proton pump activity was significantly reduced. Apart from simply reducing cytotoxicity, cell

recognition and enhanced transfection efficiency could be achieved by the connection of cell

specific ligands, for example, mannose.108

Remarkably, a fascinating carrier system has been

synthesized recently, where the composites of SiNPs and QDs were loaded with anticancer-drugs

and siRNA inside the pores, followed by coating with liposomes forming protocells (Figure 2.6).

Then, the protocell surface was conjugated with different functional molecules, including PEG,

targeting peptides (specific to hepatosarcoma cells) and FPs. Results showed a 10,000-fold higher

affinity to hepatosarcoma cells, compared with hepatocytes, endothelial cells or immune cells. More

importantly, it is capable of killing the drug-resistant human hepatocellular carcinoma cells,

representing a 106-fold improvement over comparable liposomes.

144 This research provided a well-


23

designed strategy to synthesize multifunctional SiNPs, so that all the requirements for successful

gene therapy can be met, even comparable to viral carriers.

Figure 2.6 Steps of multivalent binding and internalization of targeted protocells, followed by

endosomal escape and nuclear localization of protocell encapsulated cargo. (1) DOPC protocells

particles binds to cell surface with high affinity due to attachment of targeting peptides; (2) then

goes through cell memberane via receptor-mediated endocytosis; (3) release drugs into cytosol on

endosome acidification and protonation of H5WYG fusogenic peptide. (4) Finally, NLS-attached

cargo is transported in the nucleus. DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine; NLS:

Nuclear localization signal.144

The cargo release behavior is also an important factor for nanocarriers, thus stimuli-responsive and

controlled cargo release has attained considerable eminence. Many physicochemical factors are

responsible for triggering cargo release, for example, light-irradiation,145

cleavage of disulfide,145

enzymatic cleavage,146

temperature147

and pH.148

For example, Sauer et al. reported a successful

release of ATTO633-labeled cysteine into cytoplasm of HuH7 cells by the cleavage of disulfide

from inner surface of MSNs, after endosomal escape.145

For enzymatic cleavage, a co-delivery

system was designed, consisting of PLL-modified hollow MSNs loaded fluorescein inside and CpG

ODN on the surface. The enzyme of alpha-chymotrypsin is used as a model. Fluorescein and CpG

ODN can simultaneously release from the particles stimulated by alpha-chymotrypsin that can

induce PLL polymer degradation.146

Compared with others stimuli, pH-response is the most logical

trigger, because of the intrinsic pH value differences in different cells or cell compartments, for

example, approximately 5 in endosome, 6.8 in tumor tissues and 7.4 in normal tissues. Chen et al.

reported a pH-sensitive drug (ibuprofen) delivery system of chitosan-coated MSNs, where the

release of encapsulated drug could be controlled by the narrow difference between pH 6.8 and

7.4,148

avoiding nonspecific drug leakage in normal cells.


24

After cargo release, genetic molecules (e.g., pDNA) or even some proteins (e.g., saporin60

) should

go into nucleus to show their functions. Usually, free diffusion is a common route for cargo release,

however, the efficiency is not guaranteed. To be different, targeted nuclear delivery will be more

effective for gene/protein therapy, with the help of nuclear localization signal (NLS). Based on

sequence differences, NLS is divided into classical and non-classical groups. After the binding of

NLS w t t mport r ptors o mport n α nd β, nd t ollow n nt r t on w t nu l r por

complex (NPC), cargo translocation will be initiated.149

Shi and co-workers first reported the

connection of NLS with MSNs (MSN–Tat complex) for nuclear targeting and nuclear entry (Figure

2.7).150

Tat showed significant nuclear membrane targeting behavior and the DOX absorbed into

MSNs–Tat complex exhibited cancer cell killing effect. This work opens new avenues of SiNPs for

improved gene therapy.

2.3.2.2 Inorganic materials

Inorganic materials have also coupled with SiNPs, aiming at either improving localization of NPs at

tumor sites, or helping to track NPs in vivo. Magnetic NPs have been extensively studied, because

of their application in efficient magnetic targeting, imaging and bio-separations.105, 151

Using

different protocols, the encapsulation of magnetite (Fe3O4) nanospheres onto or into porous SiNPs

has been reported for the improvement of drug delivery efficiency.105, 151-153

Another type of inorganic material, semi-conductor QDs, has also attracted great research interest in

nanomedicine, because they are effective optical probes for in vivo imaging.154, 155

When silica

materials coat onto QDs, silanol groups on the surface can be accessible to conjugate with other

functional molecules. For example, silica-coated QDs (e.g., CdSe104

) connecting with single-

stranded oligonucleotide hybridizations will work as molecular beacons (MBs), where QDs are

used as fluorophores, and they show fluorescent signals after DNA pairing with a target sequence.

This MB has shown excellent behavior for DNA recognition.156

Although gold and silver NPs themselves have been effective to be used as genetic molecule157

and

protein158

vectors, studies on their combination with silica NPs have been paid increasing attention,

recently. Christen et al. reported that SiO2–Ag-NPs induced an increased endoplasmatic reticulum

(ER)-stress response and reduced cytochrome P4501A activity of human liver cells (Huh7) and

pimephales promelas (fathead minnow) fibroblast cells (FMH).159

SiO2–Au-NPs-oligonucleotide

could also be used as a fast colorimetric DNA.160

In addition, the co-delivery of BSA and pDNA

using gold NP-functionalized MSNs to plant tissues has also been reported.161


25

As a brief conclusion, the strategies to combine inorganic/organic materials with SiNPs hold a

potential to possess all the necessary abilities for highly improved gene/protein delivery, as efficient

as natural particulates. However, other parameters of SiNPs, such as pore size and particle sizes,

need to be further studied.

2.3.3 Particle nanotechnology

2.3.3.1 Particle size

Control over particle size at nanoscale is an important aspect in nanotechnology and has significant

impact on particle endocytosis162-164

and cytotoxicity.165

The size of SiNPs can be adjusted from a

few to hundreds of nanometers.163

There are plenty of evidence showing that cell internalization of

most of non-viral NPs, such as liposomes,166

quantum dots,167

gold nanoparticles162

and SiNPs165

is

particle size-dependent. For example, phagocytosis, performed by immune cells, has been

tr d t on lly sso t d w t t upt k o p rt l s l r r t n 1 μm, r o nizing by specific

receptors. Similarly, macropinocytosis has been exploited for non-specific uptake of large particles,

which is also approxim t ly 1 μm.168

In addition, clathrin- and caveolae-mediated pathways allow

the entry of NPs (e.g., SiNPs) from 30 to 300 nm. 43, 61

In detail, cell uptake efficiency was reported

in the sequence of 50 > 30 > 110 > 280 > 170 nm. The uptake of 50 nm fluorescein-5-

isothiocyanate (FITC)-MSNs was approximately 2.5-times that of 30 nm ones, four-times that of

110 nm ones, 20-times that of 170 nm ones and 11 times that of 280-nm particles.164

Similar trends

have also been found in other materials, such as QDs167

and gold NPs.162, 169

Moreover, particles

with small sizes are easier to target to tumor sites, due to the leaky behaviour of vessels in tumor

tissues compared with healthy tissue, leading to uptake of NPs via enhanced permeation and

retention (EPR) effect.170, 171

On top of the behavior of internalization, particle size is further responsible for systemic

cytotoxicity. Independent research groups have studied the relationship of hemolytic activity or

cytotoxicity with particle size.115, 163, 165

All the results showed that SiNPs caused hemolysis or cell

death (human hepatoma cells) in a size-dependent manner, that is, smaller particles that have larger

surface areas, may cause more cell damage resulting from the ROS. However, Zhao et al. provided

a converse result, where the 600 nm MSNs showed more toxicity than 100 nm MSNs.172

The

authors claimed that membrane wrapping followed by the MSN engulfment is related to particle

curvature rather than particle size. Smaller particle possesses larger curvature, thus more free

energy is required for the membrane bending and binding with MSNs, resulting in

thermodynamically favorable cell uptake of larger MSNs. It is possible that a threshold of particle

size exists for hemocompatible MSNs (below 225 nm).163, 172

Moreover, particle size for nuclear


26

entry should be limited within 20–70 nm, being consistent with the diameter of NPC. Shi and co-

workers (Figure 2.7) firstly confirmed the silica-based nuclear entry of MSN–NLS–Tat complex,

where the threshold size is 50 nm.150

Figure 2.7 Nuclear-targeted drug delivery of Tat peptide-conjugated MSNs. (A) Preparing amino

group- and Tat-FITC peptide-conjugated MSNs. (B) Transport of DOX@MSNs-Tat across nuclear

membrane. (C) CLSM images of MSNs-Tat with diameters of (1) 25, (2) 50, (3) 67 and (4) 105 nm

t r n u t on w t H L lls or ( ) 4, ( ) 8 nd ( ) 24 . S l rs: 5 μm. APTES:

Aminopropyl triethoxy silane; CTAC: Hexadecyltrimethylammonium chloride; MSN: Mesoporous

silica nanoparticle; TEA: Tetraethyl amine; TEOS: Tetraethyl orthosilicate.150

2.3.3.2 Pore size and pore structure

Conventional MSNs have relatively small pore sizes within 2–4 nm173, 174

and only small molecule

drugs can be loaded inside the pores. For example, Bhattarai et al.175

and Meng et al.,176

respectively, reported the co-delivery of siRNAs (luciferase-targeted siRNA or P-Glycoprotein

siRNA) and anticancer drugs (chloroquine or doxorubicin) into different cancer cell lines (mouse

melanoma cell line or epidermoid carcinoma cell line), where the small drugs were loaded into the

pores of MSNs (2–3 nm) and siRNAs were adsorbed on the external surface. It is noted that MSNs

with small pores suffer from disadvantages of low encapsulation efficiency, and large bio-

molecules such as siRNAs, DNAs and proteins can hardly be adsorbed into pores. This limits the

potential advantages of MSNs over solid silica spheres in cargo loading and protection.

SiNPs with different pore sizes and pore structures can be synthesized by varying silica

precursors,177

surfactant types (e.g., hexadecyltrimethylammonium bromide [CTAB],177

Pluronic

F127 [EO106PO70EO106, EO = ethylene oxide, PO = propylene oxide]178

and Pluronic P123


27

[EO20PO70EO20]179

), as well as adding swelling agents (e.g., 1,3,5-trimethylbenzene180

). Related

products with various pore parameters have been fabricated, such as SBA-15 (large-pore 2d

hexagonal),181

MCM-48 (gyroid cubic Ia3d),182

KIT-6 (large-pore gyroid cubic Ia3d),181

FDU-12

(ultra-large-pore, Fm3m type)178, 183, 184

, macroporous (~110nm in diameter) ordered siliceous foams

(MOSF)179

and periodic mesoporous organosilica (PMO).87, 88

It is notable that some surfactants

show cytotoxicity to cells. He and Shi reported as-synthesized MSNs (without removing surfactant)

could kill cancer cells at a much higher efficiency by the release of surfactant molecules than

common anticancer drugs.185

In other words, surfactants must be totally removed prior to bio-

application.

Large pore SiNPs are another type of attractive candidates for genetic molecule or protein delivery.

Due to their high pore volume and much lower density, large amount of therapeutic agents can be

loaded while reducing native cytotoxicity of silica. For example, HMSNs of ~250 nm size were

combined with a chitosan-antibody decoration, and the material enabled a pH-controlled release of

TNF-a, which significantly suppressed the growth of cancer cells and even killed them with high

therapeutic efficacy.185

Although conventional FDU-12 possesses large pores up to 40 nm,174

the

particle size is in micrometer range, which is not suitable for cell uptake. Hartono et al. successfully

prepared a PLL-conjugated FDU-12-type SiNP with particle size of ~100–200 nm and pore size of

~28 nm.30

After the intracellular delivery of siRNA loaded NPs in inner pores, significant decrease

of osteosarcoma cancer cell viability was observed (Figure 2.8). Apart from the good performance

of SiNPs in cargo delivery, the intrinsic cytotoxicity of both porous and nonporous SiNPs has also

been investigated. The damage to cell membrane of RBCs163, 186

and human promyelocytic

leukemia cells (HL-60)186

have been investigated, where porous SiNPs exhibited lower cytotoxicity

than nonporous counterparts of similar size. It is claimed that the external surface area is important

in determining cytotoxicity. Porous SiNPs have smaller external surface areas, thus less silanol

groups on the cell-contactable surface, compared to nonporous SiNPs, resulting in less ROS and

reduced toxicity.113

2.3.3.3 Particle shape and surface morphology

The shapes and surface morphologies of SiNPs can be adjusted by changing synthesis parameters

such as chemical ratios,188, 189

initial temperature,190

stirring rate,190

micelle packing parameter,165,

191 additives,

105, 192, 193, or post-modification,

194, 195. For instance, anisotropic ellipsoid-like MSNs

were fabricated via the organic–inorganic cooperative assembly in the presence of additive agents

(KCl and ethanol105

). In addition, the delivery of Cy3-oligo-DNA into tumor cells showed its

potential in drug/gene delivery.105

Silica nanotubes were made using anodic aluminum oxide


28

membrane as a template. By coating the inner surface of amino-groups, such materials efficiently

loaded pDNA of GFP and expressed in COS-7 cell.196

Endocytosis performance of SiNPs is also influenced by particle shapes. For instance, the

relationship between cell uptake and three rod-like MSNs with similar particle diameter, chemical

composition and surface charge, but with different aspect ratios (ARs, 1, 2 and 4) were investigated.

The results indicated that these different-shaped particles were readily internalized in human

melanoma (A375) cells by non-specific cell uptake. Among them, the particles with larger ARs

were taken up in larger amounts and had faster internalization rates, however, showing a greater

impact on cell proliferation, apoptosis, cytoskeleton formation, adhesion and migration.189

A pos-

sible explanation for this behavior could also be attributed to curvature of SiNPs, since larger AR

means larger contact and interaction area with the cell membrane, causing higher cytotoxicity from

ROS. Evidence showed bio-distribution, clearance and biocompatibility of MSNs will also be

affected by different shapes of MSNs. For example, short-rod MSNs (AR = 1.5) were confirmed to

be easily distributed in the liver, while long-rod MSNs (AR = 5) stayed in the spleen. The main

excretion route was observed by urine and feces. Moreover, short-rod MSNs showed a more rapid

clearance rate. Although in this investigation, hematology, serum biochemistry and histopathology

results indicated that MSNs did not cause significant toxicity in vivo, but systemic side effects may

be caused by the accumulation of MSNs in those organs,188

especially in the long-term.

The surface morphology or topology is another interesting factor for effective modification. By

investigating the features of plenty of surface morphologies, from viruses to plants to animals,

researchers have noticed that morphologies of living creatures in microcosmic scale seldom showed

homogeneousness. For example, the lotus leaves have tiny raspberry-like structures on the surface.

The bubble-like structures between the shells show super-hydrophobicity and hence resistance to

water.197

Nel et al.198

depicted the effect of heterogeneous structures, for example, local protrusions

or depressions with radii smaller than that of the particle, may have the potential to strengthen NP–

cell interactions. Although some silica-based bimodal or heterogeneous structures have been

fabricated, for example, the raspberry-like structure,84, 100, 104

that is, the combination of a large core

and plenty of small shells on the surface, only a few studies have focused on the relationship

between topology and gene/protein delivery efficiency.

2.4 Conclusions and future perspective

Various therapeutic agents with good specificity, such as genetic molecules and therapeutic

proteins, have been applied in modern medicine. However, effective carriers are required for

enhanced intracellular delivery of cargos. Although natural particulates have superior properties for


29

genetic molecules and therapeutic protein delivery, some intrinsic issues prevent their wide

applications, which propels the development of synthetic vectors mimicking the features of natural

particulates. For example, silica-based nanoparticles (SiNPs) have emerged as excellent candidates.

Although several successful delivery systems based on SiNPs have been studied, the investigation

in other parameters, for example, the effect of surface morphology by mimicking enveloped viruses

(objective 1 in this thesis), is required for further improved cargo delivery efficiency. The virus-

mimicking silica nanoparticles (VMSNs) are expected to enhance biomolecule delivery efficiency

in various bio-applications. For instance, the VMSNs are expected to increase siRNA loading

amount and cellular uptake effect, causing significantly enhanced gene silencing efficiency

(objective 1 in this thesis). Following that, the systematic investigation of VMSNs with varied

surface roughness in drug delivery efficiency will be very important, highlighting the roles of

surface topography (objective 2 in this thesis). Furthermore, intensive investigation to one given

VMSN is extremely necessary in understanding the individual contribution of surface roughness

and surface modification to the therapeutic drug delivery processes (objective 3 in this thesis).

The research on bio-inspired nanoparticles for biomedical applications is taking off and has

demonstrated tremendously positive results. The opportunities could be realized by mimicking the

surface topography of enveloped viruses, and combining interdisciplinary knowledge in chemistry,

biology, material science and clinical medicine.

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174 Fenelonov, V. B.; Romannikov, V. N.; Derevyankin, A. L. Mesopore size and surface area

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180 Suteewong, T.; Sai, H.; Cohen, R.; Wang, S. T.; Bradbury, M.; Baird, B.; Gruner, S. M.;

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45

Chapter 3

Methodology

This chapter summarizes the strategies utilized in the whole PhD project, including synthetic

methods for virus-mimicking silica nanoparticles (VMSNs) and the techniques for material

characterizations and biological evaluations.

3.1 Materials synthesis

Mono-dispersed solid SiNPs with different sizes are synthesized by the hydrolysis and condensation

of silicon alkoxides in either aqueous solutions or in alcohol/water mixtures using amino acid and

ammonia as a catalyst, respectively. The silica particle diameter can be well-tuned from 10 nm to

300 nm, simply by varying catalyst and precursor concentrations or reaction temperatures.

3.1.1 Synthesis of monodispersed solid silica nanoparticles as core particle

Uniform nonporous silica nanoparticles with a size of ~200 nm were synthesized using a well-

known method developed by Stöber.1 Typically, absolute ethanol (50 mL) was mixed with

deionized (DI) water (3.8 mL) and ammonium hydroxide solution (2 mL, 28%) at 25°C. Then,

tetraethyl orthosilicate (TEOS, 2.8 mL, 98%) was added to the solution under vigorous stirring.

After 6 hours, as-synthesized nanoparticles were separated by centrifugation, and washed with

ethanol and DI water. The final product was obtained by drying at 100°C overnight.

3.1.2 Synthesis of monodispersed solid silica nanoparticles as shell particles

Shell nanoparticles with the mean sizes of 28, 54, 98, 135 and 175 nm were also fabricated using

Stöber method with the same recipe as the core particle, except for reacting at 70, 60, 50, 40 and 30

°C, respectively. The reactions were first carried out for 20 minutes for the formation of shell

particles (28, 54, 98 and 135 nm). For the shell particle of 175 nm, the reaction time was 2 h. In

addition, a modified Stöber method was used to fabricate shell particle of ~13 nm diameter.2 First,

L-arginine (87 mg) was dissolved in DI water (69.5 mL) containing octane (5.23 mL). Then, TEOS

(0.5 mL) was added under vigorous stirring to react at 60 °C for 3.5 h for the formation of shell

particles.


46

3.1.3 Synthesis of virus-mimicking silica nanoparticles (VMSNs)

Figure 3.1 Schematic illustration of VMSN, followed by amine-group and PEI modification.

The amine-modified core particle (200 mg, modification method can be found in section 3.1.4.1)

was suspended (2 mL) in DI water (for the synthesis of VMSN with small shell particles) or ethanol

(for the synthesis of VMSNs with larger shell particles). The suspensions were added into different

shell particle solution keeping the original volume and condition, reacting for another 2 h. The as-

synthesized VMSNs were washed three times with ethanol and isolated by centrifugation at 4750

rpm for 10 min (shell particles cannot be recovered by centrifugation under these conditions),

followed by drying in a fume cupboard at 25 °C overnight. Finally, VMSNs were obtained after

calcination treatment at 550 °C for 5 h to remove organic components in silica frameworks (Figure

3.1).

3.1.4 Functionalization of silica nanoparticles

3.1.4.1 Amine-group modification

Amine silane was grafted onto the surface of core particle or VMSN to create positively charged

surface. First, core particles or VMSNs (200 mg) were suspended in toluene (30 mL). Then, 3-

aminopropyltriethoxysilane (APTES, 0.19 mL, 0.8 mmol) was added. The mixture was refluxed for

20 hours at 383 K. Then, amine-group modified core particles or VMSNs were obtained by

centrifugation, washing with ethanol and deionized water and drying in a vacuum oven at 25°C

overnight.

3.1.4.2 Hydrophobic modification

Smooth or VMSNs were functionalized with n-octadecyl-trimethoxy silane (n-ODMS, 90%).

Different nanoparticles (200 mg) were suspended in toluene (25 mL) containing 0.5% (v/v) n-

ODMS. Then, the mixture was refluxed for 20 h at 110 °C, followed by centrifugation at 10000

rpm, washing with ethanol and drying in a fume cupboard at 25 °C overnight.

3.1.4.3 PEI attachment

In order to achieve efficient endosomal escape for successful cargo delivery into cytosols, 10 kDa

polymer of PEI was attached to both smooth and VMSNs, through a linker of 3-glycidoxypropyl


47

trimethoxysilane (3-GPS). First, epoxysilane was connected onto the surfaces of smooth and

VMSNs by suspending the silica solid (100 mg) in toluene (30 mL) and stirring for 15 minutes at

343 K. Then, 3- GPS (1.5 mL) was added into the solution, which was further stirred for 24 hours at

the same temperature. The solid products were centrifuged, washed three times with toluene and

methanol and dried. Solid products (50 mg) were mixed with 10 kDa PEI (250 mg) in carbonate

buffer (100 mL, 50 mM, pH 9.5) for 24 hours at 298 K. In this step, PEI is attached to the

nanoparticles via epoxy-groups. The products were washed with NaCl solution (20 mL, 1.0 M),

followed by three washes with deionized water, and were then recovered by centrifugation. At the

final stage, the solid products were re-suspended in ethanolamine (20 mL, 1.0 g/L, pH 9) and stirred

at 25°C for 6 hours to block free epoxy groups. The solids were then washed again with NaCl

solution (20 mL, 1.0 M) and deionized water (20 mL).

3.2 Characterizations

3.2.1 Transmission electron microscopy

Transmission electron microscopy (TEM) images were taken using a JEOL 1010 microscope

operated at 100 kV. TEM specimens were dispersed in ethanol, and transferred to a copper grid.

3.2.2 High resolution scanning electron microscopy

High resolution scanning electron microscopy (HRSEM) images were obtained on a JEOL JSM

7800 FE-SEM equipped with an in-column upper electron detector (UED) and gentle beam

technology. HRSEM was operated at a low accelerating voltage of 0.8-1.5 kV with 20% specimen

bias. HRSEM samples were prepared by dispersing the powder samples in DI water, after which

they were dropped to aluminium foil pieces and attached to conductive carbon film on SEM

mounts. The SEM mounts were dried in a vacuum oven at 70 °C for 8 hours before observations.

3.2.3 Electron tomography

Figure 3.2 Illustration of two-stage tomography process with (left) acquisition of an ensemble of

images (projections) about a single tilt axis and (right) the back-projection of these images into 3D

object space. 3


48

Electron tomography (ET) has been adopted rapidly as an advanced TEM technique for the study of

the morphologies and chemical compositions of nanostructures three-dimensional (3D) views. The

ET processing includes two steps. First, multiple 2-dimentsional projections must be acquired to

reveal quantitative 3D information. Then, in combination with automation and analysis software, 3-

dimentional structure of materials will be reconstructed by back-projection techniques, resulting in

the need for challenging and often lengthy experiments (Figure 3.2).3

ET data was collected with a FEI Tecnai F30 transmission electron microscope operating at 300 kV.

ET specimens were prepared in the same way of the TEM specimens. All TEM images for ET were

recorded at a given defocus in a bright-field mode to show the thickness contrast. The tomographic

tilt series were obtained by tilting the specimen inside the microscope around a single axis under the

electron beam. TEM images were recorded over a tilt range of +57 to -57° at an increment of 1°.

Images of the tilt-series were aligned with respect to a common origin and rotation axis using the

fiducially markers.

3.2.3 Dynamic light scattering

Dynamic Light Scattering (DLS) is used to measure particle and molecule size. This technique

measures the diffusion of particles moving under Brownian motion, and converts this to size and a

size distribution using the Stokes-Einstein relationship. Non-Invasive Back Scatter technology

(NIBS) is incorporated to give the highest sensitivity simultaneously with the highest size and

concentration range.4 DLS measurements were carried out at 25 °C using a Zetasizer Nano-ZS from

Malvern Instruments. The samples were dispersed in deionized water or ethanol by ultra-sonication

before analysis.

3.2.4 Zeta (ζ) potential analysis

The ζ-potential of colloidal dispersions is routinely measured using the technique of micro

electrophoresis. In this technique, a voltage is applied across a pair of electrodes at either end of a

cell containing the particle dispersion. Charged particles are attracted to the oppositely charged

electrode and their velocity is measured and expressed in unit field strength as their mobility.5 ζ -

potential was carried out at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments. The

samples were dispersed in deionized water or PBS by ultra-sonication before analysis.

3.2.5 Nitrogen sorption

Nitrogen sorption isotherms of the samples were obtained at -196 °C using a Micrometrics Tristar II

system. Before the measurements, the samples were degassed at 180 °C overnight in vacuum. The


49

Brunauer–Emmett–Teller specific surface area (SBET) was calculated using experimental points at a

relative pressure of P/P0 = 0.05-0.25.

3.2.6 Attenuated total reflectance-Fourier transform infrared spectroscopy

Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy was utilized to

trace functional group modifications of different SiNPs. The powder of samples was simply added

onto the crystal to obtain optimal contact. The surface functional groups, for example, methyl

groups, were investigated by ATR-FTIR. ATR-FTIR spectra were collected using the Thermo

S nt ™ N ol t™ 6700 FT-IR spectrometers. Each spectrum was obtained using dried powder

against a background measured under the same condition. For each spectrum, 128 scans were

collected at a resolution of 4 cm-1

over the range 400–4000 cm-1

.

3.2.7 Elemental analysis

Elemental analysis (EA) was carried out on a CHNS-O Analyzer (Flash EA1112 Series, Thermo

Electron Corporation) to determine the percentages of carbon (C), according to the dynamic flash

combustion (modified Dumas method). The samples of octadecyl-group modified rough silica

nanoparticles with 10-20 milligrams were tested. Cysteine with 29.99% the element of carbon was

used as the standard control.

When the sample enters the reactor, inserted in the special furnace heated at 950°C, a small volume

of pure Oxygen (250mL/min) is added to the system and helps to burn the material, converting the

sample into elemental (simple) gases. A separation column and TCD detector allows to determine

element concentrations without using a complex splitting system, aliquot dosing device or purge &

trap absorbers.

3.2.8 Atomic force microscopy measurements

Figure 3.3 An atomic force microscope probes a molecule adsorption onto a surface, using a carbon

monoxide molecule at the tip for sensitivity.6


50

Atomic force microscope (AFM) is one kind of scanning probe microscopes (SPM). SPMs are

designed to measure local properties, such as height, friction, magnetism, with a probe. To acquire

an image, the SPM raster-scans the probe over a small area of the sample, measuring the local

property simultaneously (Figure 3.3).6

In the thesis, the surface morphology changes of rough silica nanoparticles before and after

incubating with the protein of IgG-F were investigated. IgG-F was mixed with RSN-211@54 (500

µg) in PBS pH 7.4 for 2 h at 4 °C, at a protein concentration of 1 mg mL-1

. After incubation, the

samples were washed with DI water to remove free and loosely attached proteins and salts in

solution by centrifugation and pipetting. Then, the suspension (10 µL) was placed onto silicon

wafers. The wafer was evaporated at 25 °C before AFM observation. RSN-211@54 without protein

incubation was used as a control. A Cypher S AFM (Asylum Research, an Oxford Instruments

company) was used for all the measurements. The images were obtained by employing the tapping

mode of the AFM in air by using Al-coated silicon probe with tip radius of 2 nm

(NANOSENSORS™, Sw tz rl nd).

3.2.9 Inductively coupled plasma optical emission spectroscopy

Inductively coupled plasma optical emission spectroscopy (ICPOES) is a trace-level, elemental

analysis technique that uses the emission spectra of a sample to identify, and quantify the elements

present. Samples are introduced into the plasma in a process that desolvates, ionises, and excites

them. The constituent elements can be identified by their characteristic emission lines, and

quantified by the intensity of the same lines.7 In this thesis, ICPOES was applied to quantitatively

compare cellular uptake between smooth nanoparticle and VMSNs by testing silicon concentration.

At first, 2×105 cells (including HeLa, KHOS, MCF-7 or SCC-25 cells) were seeded in 6-well plates

on d y or tr ns t on. N nop rt l s (50 μ /mL) alone or nanoparticle-cargo complexes were

incubated with cells under serum free condition for 4 hours. Afterwards, the cells were washed with

PBS three times and harvested with trypsin. After centrifugation, the cell pellets were washed twice

and dried. Cell lysis buffer was added to allow dissolution of the cells with ultrasound. The

supernatants (containing cell components) were removed by centrifugation at 13,000 rpm for 5

minutes, followed by two washes with PBS. Aqueous NaOH solution (1 M) was then added to

allow dissolution of the nanoparticles with ultrasound. The silicon concentrations in the final

solutions were measured by ICPOES using a Vista-PRO instrument (Varian Inc, Australia), which

were then converted to be silica amount.


51

3.2.10 Surface plasmon resonance measurements

Surface plasmon resonance (SPR) is a fast and real-time detection technique used to examine the

interaction between wide ranges of biological targets. Based on detecting small changes in the

refractive index, SPR is able to specifically monitor the interaction between analytes (e.g., antibody

or peptide) and the ligand molecules (e.g., peptide), which have been immobilized onto an inert

surface (e.g., gold surface). "Resonance units" (RU, equal to a critical angle shift of 10−4

deg) is

used to describe binding signals between analytes and ligands (Figure 3.4).8, 9

Figure 3.4 Schematic illustration of a SPR system.9

The interaction between the domain antibody of IgG-F and complementary antigen was monitored

utilizing a SPR-based biosensor (BiacoreTM

T200, GE Healthcare). IgG-F was incubated with one

of VMSNs (500 µg) in PBS pH 7.4 for 2 h at 4 °C, and the final protein concentration was 0.5 mg

mL-1

. Following that, IgG-F-RSNs complexes were washed several times until the supernatant

showed the same UV-vis absorbance at 280 nm as PBS only. Then, IgG-F-RSNs complexes were

suspended in HBS-EP buffer (BiacoreTM

T200, GE Healthcare). Biotinylated peptide was

immobilized via streptavidin capture on a sensor chip CAP (GE Healthcare) pre-immobilized with

ssDNA-streptavidin (Biotin CAPture kit, GE Healthcare) to yield the peptide surface densities in

the range of 2500-5000 R.U. A reference flow cell was generated by omitting only ssDNA-

streptavidin onto the chip surface. Interaction analyses were performed by injecting IgG-F-RSNs

complexes over the reference and peptide surfaces in series for 120 seconds at a flow-r t o 10μl

min-1

. Complex dissociation was monitored for 120 seconds. The binding intensity was determined

at the peak point 128 seconds after sample injection. Surface regeneration was performed at the end

of each analysis cycle by injecting guanidine (8 M) mixed with NaOH (1 M) at the ratio of 3:1,

followed by washing with the mixture of acetonitrile (30%), NaOH (0.25 M) and SDS (0.05%).


52

3.2.11 Thermogravimetric analysis

Thermogravimetric analysis (TGA) is used to monitor the thermal decomposition of epoxy-group

and PEI attachment in air. Around 10-15 mg of epoxy-group/PEI-modifed nanoparticles was loaded

in an aluminum pan and heated from 25 to 900°C at a heating rate of 2 °C min-1

at an air flow of 20

mL min-1

. TGA was conducted using a TGA/DSC 1 Thermogravimetric Analyzer (Mettler-Toledo

Inc).

3.2.12 X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) was used to characterize the amount of Carbon (C),

Nitrogen (N), Oxygen (O) and Silicon (Si) on the external surface of smooth and rough silica

n nop rt l s. XPS w s r ord d on Kr tos Ax s Ultr w t mono rom t Al Kα X-ray

source. Each spectrum was recorded at a survey scan from 0 to 1200 eV with a dwell time of 100

ms, and pass energy of 160 eV at steps of 1 eV with 1 sweep. C1s with a binding energy of 285 eV

was used.10

3.3 Biological techniques

3.3.1 Agarose gel electrophoresis

In the field of nanomedicine, an electrophoretic mobility shift assay (EMSA) is electrophoretic

separation of a nanoparticle–DNA or nanoparticle–RNA mixture on an agarose gel for a short

period. It is also referred as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay,

band shift assay, or gel retardation assay, and is a common affinity electrophoresis technique used

to study nanoparticle–DNA or nanoparticle–RNA interactions. This procedure can determine if a

nanoparticle is capable of binding to a given DNA or RNA sequence. The speed at which different

molecules move through the gel is determined by their size and charge, and to a lesser extent, their

shape (see gel electrophoresis). The control lane (DNA probe without nanoparticle present) will

contain a single band corresponding to the unbound DNA or RNA fragment. Under the correct

experimental conditions, the interaction between the DNA (or RNA) and nanoparticle is stabilized

and the ratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound

probe molecules as the binding reaction enters the gel. By comparison with a set of standard

dilutions of free probe run on the same gel, the number of moles of protein can be calculated.11

In this thesis, gel electrophoresis analysis was performed to examine the holding ability of VMSNs

to genetic molecules. For Cy3-oligoDNA, t omol ul (25 pmol, 100 μM) w s m x d w t

amino-mod d smoot n nop rt l s or VMSNs (50 μ ). M nw l , PLK1-siRNA (100, 50 and


53

25 pmol) were coupled with PEI-mod d smoot n nop rt l s or VMSNs (50 μ ), r sp t v ly.

For control, Cy3-oligoDNA (25 pmol of) or PLK1-siRNA (25 pmol) was prepared in the absence of

nano-carriers. Four hours later, the complexes were loaded into each well of a 2% agarose gel,

which was then run in Tris-acetate-EDTA buffer containing SYBR® Safe DNA gel Stain at 80 V

for 40 min. Genetic molecule bands were visualized using a GelDoc UV illuminator. For

comparison, software of ImageJ was used to calculate the pixel values of each genetic molecule

band in a fixed area. Genetic molecule amount was estimated after comparing with control group.

3.3.2 Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) is a technique for obtaining high-resolution optical

images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-

focus images from selected depths, a process known as optical sectioning. Images are acquired

point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of

topologically complex objects.12

In this thesis, high quality fluorescent images were acquired by CLSM. After seeding 1×105 HeLa

cells onto 24-mm glass coverslips in 6 well-plates, Cy3-oligoDNA-treated (100 nM) and the

complexes of amino-mod d smoot n nop rt l s nd VMSNs (50 μ /mL) +Cy3- oligoDNA

(100 nM)-treated cells were washed with PBS. DNase I solution was added to digest the Cy3-

oligoDNA outside the cells. Cells were washed twice with PBS then incubated at 25°C with

tr tm nt solut on (500 μl), w ont ns 1 mM C Cl2, 0.5 mM MgCl2, 0.1% (w/v) Bovine serum

albumin (BSA, Sigma) and 20 Units of DNase I (Sigma) in PBS. After 30 minutes, the cells were

washed 3 times with PBS and fixed with PFA/PBS (1 mL, 4%) at 4°C for 30 minutes. The liquid

was removed and cells were pre-incubated with PBS containing 1% BSA for 20–30 minutes prior to

dd n t st n n solut on. T n, st n n solut on (200 μl) ont n n 1% (w/v) BSA nd Hum n

CD24 FITC Conju t (5 μl) n PBS w s dd d or 2 ours t 25°C. A t r t t, t ov rsl ps w r

washed three times with PBS and mounted onto glass slides by fluoroshield with DAPI (Sigma).

The slides were viewed using confocal microscopy (LSM ZEISS 710).

3.3.3 MTT assay

The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay

measures cell proliferation rate and conversely, when metabolic events lead to apoptosis or

necrosis, reduction in cell viability. The yellow tetrazolium MTT is reduced by metabolically active

cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as


54

NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified

by spectrophotometric means.13

The cell growth inhibition by delivering therapeutic proteins or siRNAs using VMSNs as

nanocarriers was tested in multiple cell lines, including HeLa, KHOS, MCF-7 and SCC-25 cell

lines. One day before the test, 3000-5000 cells were seeded in each well of 96-well plates. The

complexes of therapeutic drug-nanoparticles were dispersed in culture medium supplemented with

10% FBS and 1% penicillin-streptomycin and added into each well. After the incubation at 37 °C

for 24 to 72 hours, cell viability was measured by adding MTT agent and reading the absorbance at

540 nm using a Synergy HT Microplate Reader. Results were compared to cells not exposed to

nanoparticles.

3.3.4 Flow cytometry

Flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting,

biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing

them by an electronic detection apparatus. It allows simultaneous multi-parametric analysis of the

physical and chemical characteristics of up to thousands of particles per second.14

In this thesis, HeLa cells were seeded in 6 well-plates at 2×105 cells per well one day before the

assay. Cy3-oligoDNA was used as a model for siRNA, and it was pre-mixed with amino-modified

smooth nanoparticles and VMSNs for 4 hours, followed by incubation with cells for another 4

hours, and the con ntr t on o n nop rt l s s 50 μ /mL and Cy3-oligoDNA is 100 nM. To

discriminate between cell-association and actual internalization, extracellular Cy3-oligoDNA was

degraded by addition of a high concentration DNase I solution. For this treatment, the cells were

w s d tw w t PBS t n n u t d t 25°C w t tr tm nt solut on (500 μl), w ont ns 1

mM CaCl2, 0.5 mM MgCl2, 0.1% (w/v) Bovine serum albumin (BSA, Sigma) and 20 Units of

DNase I (Sigma) in PBS. After 30 minutes, the cells were washed 3 times with PBS and harvested

by trypsination. After two more washes with PBS, the cells were re-suspended in paraformaldehyde

(M r k) n PBS (PFA/PBS, 600 μl, 2%) or FACS t st us n BD LSR-II Analyzer (USA). The

results were analysed using FlowJo software.

3.3.5 Western-blot analysis

The western blotting (sometimes called the protein immunoblot) is a widely used analytical

technique used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel

electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of

the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or


55

PVDF), where they are stained with antibodies specific to the target protein. The gel electrophoresis

step is included in western blotting analysis to resolve the issue of the cross-reactivity of

antibodies.15

In this thesis, western blotting was used to define the degradation of pro-apoptotic protein of Bcl-2.

MCF-7 cells were seeded in 6-well plates at a seeding density of 3×105 cells per well. The anti-

pAkt antibody and the non-specific IgG antibody were incubated with hydrophobically modified

VMSNs in PBS pH 7.4 at 4 °C for 2 h. Then, protein-nanoparticle complexes, along with

nanoparticle only group were mixed with serum containing culture medium and incubated with

lls or 24 . T n l on ntr t on o n nop rt l s s 50 μ mL-1

and protein is 1 µg mL-1

. At

the end of incubation, cells were washed with PBS and lysed. The solutions containing cell lysates

were heated at 95 °C for 15 min followed by characterization by SDS-PAGE. The protein bands

were transferred to a PVDF membrane. Targeting protein of Bcl-2 was targeted using Bcl-2

monoclonal antibody mAb as the primary antibody, and HRP-linked anti-rabbit IgG antibody as the

secondary antibody. GAPDH mAb was used as an internal reference. Bands were visualized on a

ChemiDoc MP System (Bio-Rad).

3.3.6 TEM study on nanoparticle-cell interaction

MCF-7 cells were seeded in 3-cm petri-dishes at a cell number of 3×105 for 24 h. Cells were then

incubated with a suspension of nanoparticle (SSN, RSN, C18-SSN and C18-RSN) for 24 hours.

Cells were washed with PBS buffer three times and then fixed with 2.5% glutaraldehyde at 25°C for

30min, before they were postfixed in 1% osmium tetraoxide in microwave condition. After that, the

cells were washed with PBS and embedded into 2% agarose gel cube. The cell cubes were

dehydrated in acetone of increasing concentration (50%, 70%, 90%, 100% and 100%) in a

sequential manner in microwave condition. Then, dehydrated cell cubes were embedded in epon

resin, and solidified in 60°C for 2 days. Microtome (Leica, EM UC6) was then used to cut the

embedded cell-resin cube into thin slices (70–90 nm in thickness). The samples were collected on

copper grids and double stained with the aqueous solution of uranyl acetate (2%) and commercially

available aqueous solutions of lead citrate. The ultra-thin slides were transferred to a form-bar

coated copper grid. TEM images were taken using a JEOL 1010 microscope operated at 80 kV.

3.4 References

1 Stöber, W.; Fink, A.; Bohn, E. Controlled Growth of Monodisperse Silica Spheres in Micron Size

Range. J. Colloid Interface Sci, 1968, 26, (1), 62-69.


56

2 Yokoi, T.; Sakamoto, Y.; Terasaki, O.; Kubota, Y.; Okubo, T.; Tatsumi, T. Periodic arrangement

of silica nanospheres assisted by amino acids. J. Am. Chem. Soc, 2006, 128, (42), 13664-13665.

3 Midgley, P. A.; Dunin-Borkowski, R. E. Electron tomography and holography in materials

science. Nat. Mater., 2009, 8, (4), 271-280.

4 Naiim, M.; Boualem, A.; Ferre, C.; Jabloun, M.; Jalocha, A.; Ravier, P. Multiangle dynamic light

scattering for the improvement of multimodal particle size distribution measurements. Soft Matter,

2015, 11, (1), 28-32.

5 Kaszuba, M.; Corbett, J.; Watson, F. M.; Jones, A. High-concentration zeta potential

measurements using light-scattering techniques. Philos. T. R. Soc. A, 2010, 368, (1927), 4439-

4451.

6 Shibata, M.; Uchihashi, T.; Ando, T.; Yasuda, R. Long-tip high-speed atomic force microscopy

for nanometer-scale imaging in live cells. Sci. Rep-Uk, 2015, 5.

7 Fassel, V. A.; Kniseley, R. N. Inductively Coupled Plasma - Optical Emission-Spectroscopy.

Anal. Chem., 1974, 46, (13), 1110-&.

8 Besenicar, M.; Macek, P.; Lakey, J. H.; Anderluh, G. Surface plasmon resonance in protein-

membrane interactions. Chem. Phys. Lipids, 2006, 141, (1-2), 169-178.

9 Richens, J. L.; Urbanowicz, R. A.; Lunt, E. A. M.; Metcalf, R.; Corne, J.; Fairclough, L.; O'Shea,

P. Systems biology coupled with label-free high-throughput detection as a novel approach for

diagnosis of chronic obstructive pulmonary disease. Resp Res, 2009, 10.

10 Kristensen, E. M. E.; Nederberg, F.; Rensmo, H.; Bowden, T.; Hilborn, J.; Siegbahn, H.

Photoelectron spectroscopy studies of the functionalization of a silicon surface with a

phosphorylcholine-terminated polymer grafted onto (3-aminopropyl)trimethoxysilane. Langmuir,

2006, 22, (23), 9651-9657.

11 Scott, V.; Clark, A.; Docherty, K., The Gel Retardation Assay. In Protocols for Gene Analysis,

Harwood, A., Ed. Humana Press: 1994; Vol. 31, pp 339-347.

12 Claxton, N. S.; Fellers, T. J.; Davidson, M. W., Microscopy, Confocal. In Encyclopedia of

Medical Devices and Instrumentation, John Wiley & Sons, Inc.: 2006.

13 Ferrari, M.; Fornasiero, M. C.; Isetta, A. M. Mtt Colorimetric Assay for Testing Macrophage

Cytotoxic Activity Invitro. J Immunol Methods, 1990, 131, (2), 165-172.

14 Ornatsky, O.; Bandura, D.; Baranov, V.; Nitz, M.; Winnik, M. A.; Tanner, S. Highly

multiparametric analysis by mass cytometry. J. Immunol. Methods, 2010, 361, (1-2), 1-20.

15 Mahmood, T.; Yang, P.-C. Western Blot: Technique, Theory, and Trouble Shooting. N. Am. J.

Med. Sci., 2012, 4, (9), 429-434.

Chapter 4 Nanoparticles Mimicking Viral Surface Topography for Enhanced Cellular Delivery

57

Chapter 4

Nanoparticles Mimicking Viral Surface

Topography for Enhanced Cellular Delivery

This chapter reports the synthesis of novel non-viral nanoparticles mimicking virus surface

topography by attaching small shell particles (~10nm) onto the core particle with a large size

(~200nm). We show that increases in nanoscale surface roughness improve both binding of

biomolecules (e.g., protein or genetic molecules) and cellular uptake; thus, the cargo delivery

efficiency is significantly increased, compared to conventional silica nanoparticles with a smooth

surface. We further demonstrate that the contribution of surface roughness is general, regardless of

surface functionality and cell types. Finally, gene delivery efficiency was tested, and the biomimetic

nanoparticles showed a better performance than a commercial transfection reagent. This work has

been published and highlighted as frontispiece paper in Advanced Materials (2013,

Communication).


58

4.1 Introduction

Delivery of various drug molecules into cells is crucial in modern medicine.1, 2

Most naked

biomolecules and some free drugs are poorly delivered to cells owing to poor stability, low

solubility and/or unwanted toxicity. For these reasons, various natural and synthetic vectors have

been used as cellular delivery vehicles. Compared to viral vectors having a high delivery

efficiency,3, 4

non-viral vectors such as nanoparticles are safer delivery tools,5 but their cellular

delivery efficiency is far from satisfactory.6 It remains an ongoing challenge to develop non-viral

carrier systems having both good safety and high delivery efficiency.

Understanding structure-function relationship in natural systems, such as enveloped viruses,

provides useful guides for the design of new nano-carriers. Enveloped viruses have sizes around 30-

400 nm, which appears to be ideal for cellular uptake.7 Their high infectivity is mainly attributed to

receptor-specific interactions, facilitating subsequent viral fusion or cellular uptake.3, 8

This concept

has been used in the design of nano-carriers.9, 10

Recent developments in state-of-the-art electron

tomo r p y (ET) v prov d d “n no- olo y” n orm t on or m ny nv lop d v rus s, or

example, influenza virus,8 HSV

11 and HIV,

12 all showing rough surfaces patched by glycoprotein

spikes. However, the influence of nanoscale surface roughness on cellular delivery efficiency

remains unclear because it is always associated with receptor-ligand specific interactions in viral

systems.

Synthetic nanoparticles mimicking the surface topography of enveloped viruses may provide useful

tools to study the roles of surface roughness while excluding the influence of receptor-specific

interaction. Cellular delivery efficiency of nano-vectors is dependent on both the binding capability

to cargo molecules and subsequent cellular uptake. It was reported that the saturation uptake of

biomolecules on roughened films increased by up to 70%, higher than the increase of surface area

(20%).13

The enhanced adsorption is attributed to a change in geometrical arrangement and

accumulation of biomolecules in a multi-layered manner on the rough surface.13, 14

Unfortunately,

the impact of surface roughness on biomolecule binding has not been reported for nano-vectors.

Moreover, there exist a small number of literature reports investigating the roles of surface

roughness of polymer nanoparticles in cellular uptake of nanoparticles themselves,15

however, the

contribution from surface roughness fluctuated and may have been interfered by inconsistent

material compositions. A study the impacts of surface roughness of nano-vectors on both binding

ability to cargo molecules and cellular uptake performance has not been reported to our knowledge.


59

Figure 4.1 Illustration of (a) the synthesis procedure and (b) the comparison of cellular delivery

performance between two nano-carriers. a) Sample 1 represents silica nanoparticles, which can be

further modified with positively charged amine groups. Sample 2 comprises the negatively charged

silica nanoparticles with small diameters. Sample 3 was prepared by using amino-modified 1 as the

core and 2 as the shell particles after calcination, which is modified with amine groups. b)

Compared to smooth nano-carriers, rough ones exhibit both higher binding for biomolecules (e.g.

proteins and genetic molecules) and increased cellular uptake efficiency, independent of surface

functionality.

Herein we report the synthesis of novel non-viral nanoparticles mimicking virus surface topography

(Figure 4.1a). We show that increase in nanoscale surface roughness promotes both binding of

biomolecules (e.g. protein or genetic molecules) and cellular uptake, so thst the cargo delivery

efficiency is significantly increased (Figure 4.1b). We further demonstrate that the contribution of

surface roughness is general, hardly dependent on the surface functionality of the silica

nanoparticles and the types of cells used in this work. Finally, gene delivery efficiency was tested,

where the biomimetic nanoparticles showed a better performance than a commercial product.

4.2 Results and discussion

Silica-based nanoparticles were chosen in our study due to their high biocompatibility 16

and ease of

functionalization.17

The synthesis procedure is illustrated in Figure 4.1a. The solid silica

nanoparticle (Sample 1) was prepared by the Stöber method18

(see Supplementary Information,

Materials and Methods). The solid nature of these nanoparticles is important in avoiding any

influences of internal porosity on adsorption behavior, making them ideal for studying the influence

of outer surface roughness. Transmission electron microscopy (TEM) images showed that 1 had a

uniform diameter of 209±20 nm (Figure 4.S1a). After surface modification of negatively charged 1


60

(ζ pot nt l o -24 mV) by amine groups, 1-NH2 was obtained with a similar size (211±19 nm,

Figure 4.S1b) but a positively charged surface (+51 mV). By using a modified Stöber method to

prepare silica nanoparticles,19

2 w s o t n d w t ζ pot nt l o -42 mV and a small diameter of

~13 nm (Figure 4.S1d). The association between 1-NH2 as the core and 2 as the shell particle gave

rise to sample 3 after calcination, which was negatively charged (-36 mV) and showed a rough

surface (Figure 4.S1e).

Figure 4.2 Surface characteristics of 3. a, b) SEM images show the virus-mimicking rough surface.

c) A zero-tilt TEM projection from a tilt series. d) Reconstructed surface rendering of a single

particle. The core particle is shown in blue and the shell spikes in yellow. Scale bar: 100 nm.

Scanning electron microscopy (SEM) images (Figure 4.2a & b) clearly showed that the core

particles (~200 nm) of 3 were studded with shell particles (~10 nm). This surface topography is

similar to that of the envelope-spike structure in some viruses, such as HSV.11

3 was also modified

with amine groups to produce 3-NH2 (224 ±16 nm) with a rough (Figure 4.S1f) and positively

charged surface (+54 mV). Moreover, 1 and 3 were conjugated with polyethylenimine (PEI) with

little change in surface topography (1-PEI: Figure 4.S1c, 220±13 nm, +47mV; 3-PEI: Figure 4.S1g,

226±16 nm, +43mV). The size distribution of all samples measured using the dynamic light

scattering (DLS) method is shown in Figure 4.S1h, indicating that all the samples are monodisperse

(see Table 4.S1 and 4.S2 for the polydispersity index, PDI). The surface area of 1 and 3 was

measured to be 19 and 24 m2/g, respectively (Figure 4.S2). It is noted that 1 and 3 (similarly, 1-NH2

and 3-NH2, 1-PEI and 3-PEI) possess similar sizes, surface charges and chemical compositions,

except their surface topographies.


61

Precision information of the nanoscale surface roughness is important in understanding the impact

o “n no- olo y”.11

Conventional electron microscopy techniques cannot readily be used to

quantitatively determine the surface coverage of shell particles. Therefore, an ET technique was

applied using developed protocols.20

Tilt series were recorded on a 300 kV microscope from virus-

mimicking nanoparticles (3); a bright field TEM image at zero-tilt is shown in Figure 4.2c. The core

diameter was measured to be 168 nm, and the shell particles protruding from the core surface were

clearly visible. The tomographic reconstructed structure of the rough nanoparticle is shown in

Figure 4.2d, reconstructed from 1151 tomogram slices (each 0.15 nm in thickness).

Useful and quantitative information can be obtained from the ET data. By following consecutive ET

slices, a total number of ~320 spikes were found. The size of spikes lay in a broad range of 3.2 to

10.6 nm with an average diameter of 6.4 nm, which we attribute to the calcination treatment in the

preparation of 3. The packing density of the shell particles was variable. The centre-to-centre

distance varies from ~10 to ~40 nm. The structure shown in Figure 4.2d is comparable to the

surface features of some enveloped viruses, such as HSV with a spherical shape (~ 225 nm in

diameter) and 600-750 spikes (~10 nm) in one virion.11

Using the information from ET, the weight

ratio of shell to core particles was estimated to be ~1.8% (see SI). This information will be used in

the evaluation of cellular delivery performance.

The comparison of cellular delivery performance between smooth and rough nano-carriers was first

tested using 1-NH2 and 3-NH2. The cyanine dye (Cy3)-labeled oligoDNA (Cy3-oligoDNA) was

used as a model biomolecule. After coupling 1-NH2 and 3-NH2 (50 µg/mL) with negatively charged

Cy3-oligoDNA (100 nM), the complexes were incubated with HeLa cells for 4 h. Confocal

microscopy showed very weak Cy3 signals (red color) in the sample using 1-NH2 (Figure 4.3a). In

contrast, strong Cy3 signals (Figure 4.3b) localized within the cell borders (green color) were

observed using 3-NH2 as the vector. In addition, no Cy3 signals were observed when the same

amount of Cy3-oligoDNA alone was incubated with cells (Figure 4.S3), indicating the inability of

genetic molecules themselves to penetrate into cells. Fluorescein-activated cell sorting (FACS)

analysis was used to quantitatively evaluate the delivery efficiency of Cy3-oligoDNA (Figure 4.S4).

By comparing the median fluorescence intensity (MFI; Figure 4.3c), it was shown that 3-NH2

delivered ~5.6 times Cy3-OligoDNA into cells compared to 1-NH2, indicating a higher cargo

delivery efficacy of the nano-carrier with a rough surface.

In order to exclude the possibility that the improved cellular delivery performance is attributed to

the unexpected detachment of shell particles, fluorescence microscopy was used. Similar to the


62

results from confocal microscopy, very weak signals were observed in the groups of naked Cy3-

oligoDNA (Figure 4.S5a-c), Cy3-oligoDNA coupled with 2-NH2 (0.9 µg/mL, dosage calculated

from ET results, Figure 4.S5d-f) and 1-NH2 (Figure 4.S5g-i). Only 3-NH2 induced evident Cy3

signals across all groups (Figure 4.S5j-l). These results further confirmed the enhanced delivery is

attributed to the surface roughness.

Figure 4.3 Virus-mimicking nanoparticles enhance cellular delivery performance in HeLa cells. a,

b) Confocal microscopy images of Cy3-oligoDNA (red color) delivery by (left) 1-NH2 & (right) 3-

NH2. The nuclei are stained in blue (DAPI) and the cell membranes in green (FITC). Scale bar: 20

μm. ) FACS n lys s o Cy3-oligoDNA delivery, showing higher increase of Cy3-signals in MFI

using 3-NH2 than 1-NH2. d) Identification of Cy3-oligoDNA binding for 1-NH2 & 3-NH2. e)

Investigation of biomolecule holding ability by gel retardation assay. f) Comparison of cellular

uptake efficiency of the complexes (1-NH2/3-NH2+Cy3-oligoDNA) measured by ICPOES. For bar

charts, data represent mean ± s.e.m.**p<0.01,***p<0.001, ****p<0.0001 (t-test).


63

To understand why surface roughness improved cellular delivery of biomolecules, we separately

investigated the impact of surface roughness on either binding towards cargo molecules or cellular

uptake. Figure 4.3d shows that 3-NH2 absorbed ~85% more Cy3-oligoDNA (1.29 nmol/mg) than 1-

NH2 (0.69 nmol/mg). Moreover, agarose gel electrophoresis was used to evaluate the cargo holding

ability of nano-carriers to mimic the release behaviour. 25 pmol of Cy3-oligoDNA was mixed with

50 µg of nanoparticles, a feed ratio (0.5 nmol/mg) lower than the saturation adsorption amount of

both 1-NH2 and 3-NH2. As shown from Figure 4.S6a, in the absence of nano-carriers (Lane 0), all

naked Cy3-oligoDNA migrated to the positive electrode. In the case of 1-NH2, a small amount of

released Cy3-oligoDNA was observed (Lane 1). For 3-NH2, nearly no release could be seen by

naked eyes (Lane 2). After quantitative analysis (ImageJ, see SI), the released amount of Cy3-

oligoDNA was calculated to be 15.1 and 3.9 pmol for 1-NH2 and 3-NH2, respectively (Figure 4.3e).

To quantitatively study the cellular uptake performance, cells were collected after incubating with

the complexes of 1-NH2 /3-NH2 + Cy3-oligoDNA. Because small oxide suspensions rapidly

agglomerate in biological fluids, and this will influence the transport of nanoparticles to cells in a

time dependent manner,21, 22

we fixed the cell incubation time to be 4 h in all experiments. The mass

of silica internalized per cell was measured using inductively coupled plasma optical emission

spectrometry (ICPOES). As shown in Figure 4.3f, 290 pg/cell was detected for 3-NH2 while 178

pg/cell for 1-NH2 (63% increase). Our results have demonstrated that the nanoscale surface

roughness improves both binding towards biomolecules (enhancing adsorption and reducing release

of bound biomolecules simultaneously) and cellular uptake, all aspects contributing to the highly

increased cellular delivery performance.

In addition to 1-NH2 and 3-NH2, pure silica nano-carriers 1 & 3 with negatively charged surfaces

were also investigated. Using a positively charged protein, Cytochrome C (pI= 9.823

), as the model

molecule, its adsorption amount is 2.71 nmol/mg on 3, nearly 2 times higher than that on 1 (0.88

nmol/mg, Figure 4.S7a). In addition, compared to 1, cellular uptake of 3 increased by 108% in

KHOS cells (an osteosarcoma cell line, Figure 4.S7b) and 29.4% (Figure 4.S7c) in HeLa cells.

Dose-dependent cytotoxicity of nano-carriers (1 and 3, 1-NH2 and 3-NH2) was investigated for

further cellular delivery tests by a standard MTT assay in HeLa and KHOS cells. As shown in

Figure 4.S8a-d, generally the cell viability decreases with increasing dose. It is important to notice

that at each dose, the rough particles caused higher toxicity than the smooth ones, independent of

the surface charge or cell types. For example, the cell viability was 91% for KHOS and 93% for

HeLa after incubating with 1 at the dose of 50 µg/mL, but to 82% and 88% after incubation with 3.

The cytotoxicity of 1-PEI and 3-PEI was also tested in KHOS cells for cellular delivery application,


64

which is higher compared to that of pure and amino-functionalized particles (Figure 4.S8e).

Similarly, 3-PEI was more toxic compared to 1-PEI at all doses. The higher toxicity of rough

particles compared to smooth ones is consistent with the higher cellular uptake induced by the

surface roughness. The above results have shown that the impacts of surface roughness are general

and independent of surface charge, cargos or cell types.

Figure 4.4 Gene delivery performance of virus-mimicking nanoparticles in KHOS cells. a) The

inhibition of cell viability by PLK1-siRNA transfection. S10-siRNA was used as a negative control.

Both 1-PEI and 3-PEI were used as vectors, and a commercial reagent, OligofectamineTM

, was also

applied as a further control. b) PLK1-siRNA adsorption ability of 1-PEI and 3-PEI. c) Comparison

of cellular uptake efficiency of the complexes (1-PEI/3-PEI+PLK1-siRNA) measured by ICPOES.

Data represent mean ± s.e.m.*P<0.05, ***p<0.001 (t-test)

To demonstrate the potential of nanoparticles mimicking virus surface as effective cellular delivery

vehicles, a functional siRNA against polo-like kinase 1 (PLK1) was delivered into KHOS cells.

PLK1 gene is highly expressed in cancer cell lines, such as KHOS (osteosarcoma),16, 22

MCF-7

(breast cancer)24

and HeLa (cervical cancer).25

Silencing PLK1 gene with siRNAs can decrease

PLK1-mRNA and PLK1-protein levels, thus will inhibit cell viability. Due to the inability of


65

amino-modification to achieve endosomal escape (see Figure 4.S9), both smooth and rough

nanoparticles were modified with PEI to induce proton sponge effect to trigger cargo release.2, 16, 17

The characterizations for PEI-modification were shown in Table 4.S2, 4.S3 and Figure 4.S10. An

ineffective siRNA, S10, was chosen as a negative control.

In Figure 4.4a, the negative group using S10-siRNA and the sample using only naked PLK1-siRNA

did not impact cell viability. In contrast, the cell viability was significantly decreased in the groups

using nano-carriers to deliver PLK1-siRNA in a dose-dependent manner (12.5, 25, 50 nM). This

observation is in accordance with literature reports.24

Importantly, 3-PEI promoted stronger

inhibition effects (29%, 43%, 61%) than 1-PEI (21%, 25%, 40%), and the differences were

statistically significant at PLK1-siRNA concentrations of 25 and 50 nM. Notably, the reduction in

cell viability using 3-PEI (61%) was higher compared to that using a commercial non-viral vector,

OligofectamineTM

(50%).

To study the effect of the surface roughness on enhanced gene delivery efficiency, it was measured

that 3-PEI fixed 1.41 nmol/mg PLK1-siRNA while 1-PEI fixed 0.82 nmol/mg (Figure 4.4b).

Moreover, gel electrophoresis results showed that the rough surface of 3-PEI has stronger binding

ability compared to 1-PEI (Figure 4.S6b & c). In addition, the internalized amount was 186 pg/cell

for 3-PEI and 121 pg/cell for 1-PEI (Figure 4.4c). Our results have further confirmed that the

nanoscale surface roughness improves binding towards biomolecules and cellular uptake, leading to

the enhanced gene delivery efficiency.

We have systematically studied the synthesis parameters to obtain and adjust the virus-mimicking

morphology. When the feed volume of sample 2 solution was varied from 0.68 mL (Figure 4.S11a)

to 1.35 mL (Figure 4.S11b), the surface coverage of shell particles was lower compared to sample 3

obtained at a volume of 2.7 mL (Figure 4.S1e). When the feed volume was further increased to 5.4

mL (Figure 4.S11c) and finally to 6.75 mL (Figure 4.S11d), excess unbound shell particles were

observed in addition to the desired morphology. The calcination treatment in the preparation of 3 is

also an important step. TEM images (Figure 4.S12) showed that without calcination, the shell

particles were easily peeled off after ultra-sonication, which is a common treatment in sample

preparation.

4.3 Conclusion

In summary, nanoparticles mimicking virus surface topography have been successfully synthesized.

It is demonstrated that nanoscale surface roughness enhances both binding towards biomolecules


66

(more binding and less release) and cellular uptake efficacy, thus the cellular delivery performance

can be improved. This understanding is important in the rational design of new cellular delivery

vectors. By combining both the nanoscale surface roughness and receptor-specific binding moieties,

or by introducing rough surfaces or core particles having other compositions and structures, a

platform of synthetic vectors with high cellular delivery performance is on the horizon.

4.4 References

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3 D. S. Dimitrov, Nat Rev Microbiol 2004, 2, 109.

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5 C. E. Thomas, A. Ehrhardt, M. A. Kay, Nat. Rev. Genet. 2003, 4, 346.

6 D. A. Jackson, S. Juranek, H. J. Lipps, Mol. Ther. 2006, 14, 613.

7 J. Mercer, M. Schelhaas, A. Helenius, Annu. Rev. Biochem. 2010, 79, 803.

8 A. Harris, G. Cardone, D. C. Winkler, J. B. Heymann, M. Brecher, J. M. White, A. C. Steven,

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9 M. Soliman, R. Nasanit, S. R. Abulateefeh, S. Allen, M. C. Davies, S. S. Briggs, L. W. Seymour,

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Shah, N. Sridevi, A. Prabhune, V. Ramaswamy, Microporous Mesoporous Mater. 2008, 116, 157.

10 K. Grunewald, P. Desai, D. C. Winkler, J. B. Heymann, D. M. Belnap, W. Baumeister, A. C.

Steven, Science 2003, 302, 1396.

11 P. Zhu, J. Liu, J. Bess, E. Chertova, J. D. Lifson, H. Grise, G. A. Ofek, K. A. Taylor, K. H. Roux,

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14 M. Massignani, C. LoPresti, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis, G. Battaglia,

Small 2009, 5, 2424; C. LoPresti, M. Massignani, C. Fernyhough, A. Blanazs, A. J. Ryan, J.

Madsen, N. J. Warren, S. P. Armes, A. L. Lewis, S. Chirasatitsin, A. J. Engler, G. Battaglia, ACS

Nano 2011, 5, 1775.

15 S. B. Hartono, W. Y. Gu, F. Kleitz, J. Liu, L. Z. He, A. P. J. Middelberg, C. Z. Yu, G. Q. Lu, S.

Z. Qiao, ACS Nano 2012, 6, 2104.

16 T. A. Xia, M. Kovochich, M. Liong, H. Meng, S. Kabehie, S. George, J. I. Zink, A. E. Nel, ACS

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17 W. Stöber, A. Fink, E. Bohn, J. Colloid Interface Sci. 1968, 26, 62.

18 T. Yokoi, Y. Sakamoto, O. Terasaki, Y. Kubota, T. Okubo, T. Tatsumi, J. Am. Chem. Soc. 2006,

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19 P. Yuan, J. Sun, H. Xu, L. Zhou, J. Liu, D. Zhang, Y. Wang, K. S. Jack, J. Drennan, D. Zhao, G.

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68

Supplementary Information

Material and Methods

4.S1. Synthesis of Samples

Sample 1. Uniform nonporous silica nanoparticles with a smooth surface were synthesized using a

well-known method developed by Stöber et al.18

Typically, 50 mL of ethanol was mixed with 3.8

mL of deionized water and 2 mL of 28% ammonium hydroxide solution (Sigma-Aldrich) at 308 K.

Then, 2.8 mL of 98% tetraethyl orthosilicate (TEOS, Aldrich) was added to the solution under

vigorous stirring. After 6 hours, the as-synthesized nanoparticles were separated by centrifugation,

and washed with ethanol and deionized water. The final product 1 was obtained by drying at 373 K

overnight.

Sample 1-NH2. Amine silane was grafted onto the surface of 1 to create positively charged smooth

silica nanoparticles. First, 0.2 g of sample 1 was suspended in 30 mL toluene. Then, 0.19 mL of (0.8

mmol) of 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) was added. The mixture was

refluxed for 20 hours at 383 K.10

Then, 1-NH2 was obtained by centrifugation, washing with ethanol

and deionized water and drying in a vacuum oven at 313 K overnight.

Sample 2. Silica nanoparticles with a small diameter were prepared using a modified Stöber

method.19

First, 87 mg of L-arginine (Sigma-Aldrich) was dissolved in a solution containing 69.5

mL of deionized water and 5.23 mL of 98% octane (Sigma-Aldrich) with stirring overnight at 333

K. Then, 0.5 mL of TEOS was added to the mixture and stirred for a further 5 hours. The solution

containing as-synthesized 3 was used immediately in the synthesis of sample 3 (see below).

Sample 3. As-synthesized 2 (2.7 mL solution) was mixed with 50 mg of 1-NH2 dispersed in 30 mL

of deionized water and stirred for 20 hours at 333 K. The as-synthesized 3 was isolated by

centrifugation at 10000 rpm for 10 min (sample 2 cannot be recovered by centrifugation under these

conditions), followed by drying in a vacuum oven at 313 K overnight. Finally, 3 was obtained by

calcination treatment at 823 K for 5 hours.

Sample 3-NH2. The positively charged 3-NH2 was synthesized by amino-modification of 3, using

the procedure described in the preparation of 1-NH2.

Sample 1-PEI & 3-PEI. In order to achieve endosomal escape for successful gene silencing, 10

kDa polymer of PEI (BioScientific Pty Ltd) was attached to 1 and 3, through a linker of 3-

glycidoxypropyl trimethoxysilane (3-GPS, Sigma-Aldrich).16

First, epoxysilane was connected onto


69

the surfaces of sample 1 and 3 by suspending 100 mg of solid in 30 mL of toluene and stirring for

15 minutes at 343 K. Then, 1.5 mL of 3-GPS was added into the solution, which was further stirred

for 24 hours at the same temperature. The solid products were centrifuged, washed three times with

toluene and methanol and dried. Solid products (50 mg) were mixed with 250 mg of 10 kDa PEI in

100 mL of carbonate buffer (50 mM, pH 9.5) for 24 hours at 298 K. In this step, PEI is attached to

the nanoparticles via epoxy-groups. The products were washed with 20 mL of 1.0 M NaCl,

followed by three washes with deionized water, and were then recovered by centrifugation. At the

final stage, the solid products were re-suspended in 20 mL of 1.0 M ethanolamine (pH 9) and stirred

at 298 K for 6 hours to block free epoxy groups. The solids were then washed again with 20 mL of

1.0 M NaCl and 20 mL of deionized water.

4.S2. Characterizations

TEM images were taken using a JEOL 1010 microscope operated at 100 kV. The TEM specimens

were dispersed in ethanol, and then transferred to a copper grid. The HRSEM images were obtained

on a JEOL7800F microscope operated at 1 kV with a through-the-lens system and gentle beam

technology.26

T HRSEM sp m ns w r un o t d or o s rv t on. DLS nd ζ pot nt l

measurements were carried out at 298 K using a Zetasizer Nano-ZS from Malvern Instruments. The

samples were dispersed in deionized water by ultrasonication before analysis. ET data was collected

with a FEI Tecnai F30 transmission electron microscope operating at 300 kV. ET specimens were

prepared in the same way of the TEM specimens. All TEM images for ET were recorded at a given

defocus in a bright-field mode to show the thickness contrast. The tomographic tilt series were

obtained by tilting the specimen inside the microscope around a single axis under the electron beam.

TEM images were recorded over a tilt range of +57 to -57° at an increment of 1°. Images of the tilt-

series were aligned with respect to a common origin and rotation axis using the fiducially markers.

Alignment and tomogram generation were performed with IMOD software.20

Nitrogen sorption

isotherms of the samples were obtained at 77 K using a Quantachrome's Quadrasorb SI analyzer.

Before the measurements, the samples were degassed overnight in vacuo. The BET surface area

was calculated using experimental points at a relative pressure of P/P0 = 0.05-0.25. TGA analysis

was conducted using a TGA/DSC 1 Thermogravimetric Analyzer (Mettler-Toledo Inc) at a heating

rate of 2 K/min. X-ray photoelectron spectroscopy (XPS) were recorded on a Kratos Axis Ultra

w t mono rom t Al Kα X-ray source. Each spectrum was recorded at a survey scan from 0 to

1200 eV with a dwell time of 100 ms, and pass energy of 160 eV at steps of 1 eV with 1 sweep. C1s

with a binding energy of 285 eV was used.


70

4.S3. Estimation of shell / core ratio from ET data

The weight ratio of shell to core particles in 5 can be estimated from the ET data by assuming both

core and shell particles are spheres and that they have the same density. The weight ratio is then the

same as the volume ratio. To calculate the volumes, the diameters of core and shell particles are

measured from ET (Figure 4.2d), giving 168 and 6.45 nm, respectively. The number of shell

particles is measured to 320 from ET. The volume ratio is calculated to be

(320×4/3×3.14×3.2253×10

-21) / (4/3×3.14×83.7

3×10

-21) = 1.83%.

4.S4. Biological experiments

Cell culture: Cell culture reagents were purchased from GIBCO Invitrogen Corporation/Life

Technologies Life Sciences unless otherwise specified. Cell lines used including cervical cancer

cell lines HeLa (ATCC, CCL-2) and osteosarcoma cell line KHOS/NP (CRL-1544) were purchased

from ATCC (American Type Culture Collection). Cells were maintained as monolayer cultures at

310 K and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal

bovine serum (FBS) and 1% penicillin-streptomycin. The final concentration is 100 u/mL for

penicillin, 100 u/mL for streptomycin. Twenty-one-nucleotide (oligo) DNA conjugated with

cyanine dye (Cy-3), fetal bovine serum, paraformaldehyde, antifade fluorescent mounting medium

w t 4’-6-diamidino-2-phenylindole (DAPI) and MTT (3-4,5-dimethylthiazol-2-yl-2,5-

diphenyltetrazolium bromide; thiazolyl blue) were purchased from Sigma-Aldrich. A Cell-Titer-Glo

cell viability assay kit was from Promega. The sequences of human PLK1-siRNA (Ambion at

Applied Biosystems, Foster City, California) are PLK1-S: 5’-CCAUUAACGAGCUGCUUAATT-3’

and PLK1-AS: 5’-UUAAGCAGCUCGUUAAUGGTT-3’. T s qu n s o synt t S10-siRNA

(Proligo, Lismore, Australia) are as follows: S10-S, 5’-GCAACAGUUACUGCGACGUUU-3’ nd

S10-AS, 5’-ACGUCGCAGUAACUGUUGCUU- 3’. T Cy3-oligoDNA has the same sequence as

S10-siRNA, except the base of U is replaced with T.

Biomolecule adsorption assay: The adsorption ability of nanoparticles with biomolecules (Cy3-

oligoDNA, PLK1-siRNA and Cytochrome C) was first evaluated. Final results were given as an

v r o tr pl t m sur m nts. For t dsorpt on o n t mol ul s, 4 μl o Cy3-oligoDNA

(100μM) or PLK1-s RNA PBS solut on (100 μM) w r m x d w t 50 μ o 1-NH2/ 3-NH2 or 1-

PEI/ 3-PEI n 30 μl o p osp t u r d s l n (PBS, pH=7.4), r sp t v ly. In dd t on, 1 μl o

Cytochrome C (5 mg/mL), a positively charged protein, was coupled with 50 μ o 1 or 3 in PBS

(25 μl). In ontrol roup, no n nop rt l s w r dd d. T m xtur s w r d sp rs d y vort x n

for 30 seconds and then incubated at 277 K for 4 hours. After this time, the mixtures were


71

centrifuged at 13000 rpm, and the supernatants were collected for testing. The absorbance of

genetic molecules was determined using a NANODROP 1000 spectrophotometer (Thermo

Scientific), while protein adsorption was tested using a Synergy HT Microplate Reader at 409 nm.

Agarose gel electrophoresis: To examine the holding ability, gel electrophoresis analysis was

performed. For Cy3-oligoDNA, 25 pmol of it was mixed with 50 µg of 1-NH2 or 3-NH2.

Meanwhile, 100, 50 and 25 pmol of PLK1-siRNA were coupled with 50 µg of 1-PEI or 3-PEI,

respectively. For control, 25 pmol of Cy3-oligoDNA or 25 pmol of PLK1-siRNA was prepared in

the absence of nano-carriers. Four hours later, the complexes were loaded into each well of a 2%

agarose gel (UltraPureTM

Agarose, invitrogen), which was then run in Tris-acetate-EDTA (TAE,

UltraPureTM

, invitrogen) buffer containing SYBR® Safe DNA gel Stain (invitrogen) at 80 V for 40

min. Genetic molecule bands were visualized using a GelDoc UV illuminator (Bio-rad

Laboratories). For comparison, software of ImageJ was used to calculate the pixel values of each

genetic molecule band in a fixed area. The genetic molecule amount was estimated after comparing

with the control group.

Cellular delivery assay: HeLa cells were seeded in 6 well-plates at 2×105 cells per well one day

before the assay. Cy3-oligoDNA was used as a model for siRNA, and it was pre-mixed with 1-NH2

and 3-NH2 for 4 hours, followed by incubation with cells for another 4 hours. The final

concentration of nanoparticles is 50 µg/mL and Cy3-oligoDNA is 100 nM. To discriminate between

cell-association and actual internalization, extracellular Cy3-oligoDNA was degraded by addition of

a high concentration DNase I solution. For this treatment, the cells were washed twice with PBS

t n n u t d t 298 K w t 500 μl o tr tm nt solut on, w ont ns 1 mM C Cl2, 0.5 mM

MgCl2, 0.1% (w/v) Bovine serum albumin (BSA, Sigma) and 20 Units of DNase I (Sigma) in PBS.

After 30 minutes, the cells were washed 3 times with PBS and harvested by trypsination. After two

more washes with PBS, the cells were re-susp nd d n 600 μl o 2% p r orm ld yd n PBS

(PFA/PBS) for FACS test using BD LSR-II Analyzer (USA). The results were analysed using

FlowJo software.

Microscopy: For fluorescent imaging, HeLa cells were seeded at 1×105 cells per well in 24 well-

plates one day before the assay. Cy3-oligoDNA alone (100 nM) or the complexes of 50 µg/mL 1-

NH2/ 3-NH2+Cy3 (100 nM), or 1.8 μ o 2-NH2+Cy3 (100 nM) were added to wells containing

serum free medium, and incubated at 310 K for 4 hours. Afterwards, cells were washed twice with

PBS and fixed with 1 mL of 4% PFA/PBS at 277 K for 30 minutes. Subsequently, the cells were

washed again with PBS and t l qu d w s dr n d. A 200 μl port on o nt -fade fluorescent

mounting medium with DAPI was added to stain the nuclei, and the cells were viewed under an


72

Olympus IX51 fluorescent microscope. High quality fluorescent images were acquired by confocal

laser scanning microscopy (CLSM). After seeding 1×105 HeLa cells onto 24-mm glass coverslips

(PST, Australia) in 6 well-plates, Cy3-oligoDNA-treated (100 nM) and the complexes of 50 µg/mL

1-NH2/ 3-NH2+Cy3 (100 nM)-treated cells were washed with PBS and fixed with 1 mL 4%

PFA/PBS at 277 K for 30 minutes. The liquid was removed and cells were pre-incubated with PBS

containing 1% bovine serum albumin (BSA) for 20–30 minutes prior to adding the staining solution.

T n, 200 μl o st n n solut on ont n n 1% (w/v) BSA nd 5 μl o Hum n CD24 FITC

Conjugate in PBS was added for 2 hours at 298 K. After that, the coverslips were washed three

times with PBS and mounted onto glass slides by fluoroshield with DAPI (Sigma). The slides were

viewed using confocal microscopy (LSM ZEISS 710).

Detection of cellular uptake: To quantitatively compare cellular uptake between smooth and virus-

mimicking silica nanoparticles, 2×105

HeLa or KHOS cells were seeded in 6-well plates one day

or tr ns t on. N nop rt l s (50 μ /mL) alone (sample 1 and 3) or complexes (1-NH2/3-

NH2+Cy3-oligoDNA or 1-PEI/3-PEI+PLK1-siRNA) were incubated with cells under serum free

condition for 4 hours. Afterwards, the cells were washed with PBS three times and harvested with

trypsin. After centrifugation, the cell pellets were washed twice and dried. Cell lysis buffer (Cell

Signaling Technology) was added to allow dissolution of the cells with ultrasound. The

supernatants (containing cell components) were removed by centrifugation at 13,000 rpm for 5

minutes, followed by two washes with PBS. Aqueous NaOH solution (1 M) was then added to

allow dissolution of the nanoparticles with ultrasound. The silicon concentrations in the final

solutions were measured by ICPOES using a Vista-PRO instrument (Varian Inc, Australia), which

were then converted to be silica amount.

MTT assay: The cytotoxicity of nanoparticles was tested in both HeLa and KHOS cell lines. One

day before the test, 5000 cells were seeded in each well of 96-well plates. Nanoparticles (1, 1-NH2,

3, 3-NH2) were dispersed in 2-fold series, from 400 μ /mL to 12.5 μ /mL, in culture medium

supplemented with 10% FBS and 1% penicillin-streptomycin. Because PEI attachment brought

relatively higher toxicity, the concentrations for PEI-modified nanoparticles (1-PEI and 3-PEI) were

determined from 50 to 1.56 μ /mL. After 24 hours incubation at 310 K, the liquid was replaced

with fresh medium and the cells were cultured for another 24 hours. Cell viability was measured by

adding MTT agent and reading the absorbance at 540 nm using a Synergy HT Microplate Reader.

Results were compared to cells not exposed to nanoparticles.

Gene silencing assay: Osteosarcoma KHOS cells were seeded in 96-well plates at 5000 cells/well

one day before transfection. PLK1-siRNA or S10-siRNA was pre-mixed with either 1-NH2/3-NH2


73

(50 µg/mL) or 1-PEI/3-PEI (20 µg/mL) in PBS at 277 K for 4 hours. Then, nanoparticle-siRNA

(12.5, 25 or 50 nM) complexes were incubated with cells. After 24-hour incubation, the transfection

medium was replaced with fresh medium, followed by another 24 hours culture. The cell

proliferation was determined using Cell-Titer-Glo assay. The positive controls comprised cells

treated with PBS alone. Both S10-siRNA and PLK1-siRNA (50 nM) were also delivered using a

commercial transfect reagent, OligofectamineTM

, as a separate control.


74

Supplementary Figures and Tables

Figure 4.S1 TEM images of all samples and size distribution. a, e) sample 1 & 3. b, f) 1-NH2 & 3-

NH2. c, g) 1-PEI & 3-PEI. d) sample 2 (shell particle). h) size distribution curves obtained by the

dynamic light scattering (DLS) method.


75

Figure 4.S2 Surface area tested by Nitrogen sorption. The surface area of 1 (with a smooth) and 3

(with a rough surface) was measured to be 19 and 24 m2/g, respectively.

Figure 4.S3 Confocal microscopy image shows no Cy3 signals is observed when the same amount

of Cy3-oligoDNA alone is incubated with cells, indicating the inability of genetic molecules

t ms lv s to p n tr t nto lls. S l r: 20 μm.


76

Figure 4.S4 FACS analysis showing the peak shifts in the MFI from HeLa cells incubated with the

complexes of 1-NH2 (blue colour)/ 3-NH2 (yellow colour) +Cy3-oligoDNA. The Cy3-oligoDNA in

the absence of nano-carriers (red colour) is used as a control.


77

Figure 4.S5 Comparison of cellular delivery of cargos among different nanoparticles. a, b, c) Very

weak signals were observed in the groups of naked Cy3-oligoDNA, Cy3-oligoDNA coupled with d,

e, f) 2-NH2 (0.9 µg/mL, dosage calculated from ET results) and g, h & i) 1-NH2. j, k & l) Only 3-

NH2 induced evident Cy3 signals across all groups.


78

Figure 4.S6 Agarose gel analysis. a) The complex of amino-modified nanocarriers with Cy3-

oligoDNA. Lane 0: 25 pmol Cy3-oligoDNA only. Lane 1: 25 pmol Cy3-oligoDNA coupled with 50

µg of 1-NH2. Lane 2: 25 pmol Cy3-oligoDNA coupled with 50 µg of 3-NH2. In the case of 1-NH2, a

small amount of released Cy3-oligoDNA was observed. For 3-NH2, nearly no release could be seen

by naked eyes. b, c) the complexes of PEI-modified nano-carriers with PLK1-siRNA. Lane 0: 25

pmol PLK1-siRNA only. Lane 1, 2, 3: 100, 50 and 25 pmol PLK1-siRNA are coupled with 50 µg

of 1-PEI and 3-PEI, respectively. In each lane from 1 to 3, the PLK1-siRNA release was evident in

the case of 1-NH2, compared to that of 3-NH2.

Figure 4.S7 Investigation of pure silica nano-carriers in adsorption and cellular uptake. a)

Cytochrome C adsorption. The adsorption amount is 2.71 nmol/mg on 3, and 0.88 nmol/mg on 1. b)

cellular uptake into KHOS cells. 128 pg/cell was internalized for 3, while 62 pg/cell for 1. c)

cellular uptake into HeLa cells. 242 pg/cell was internalized for 3, while 187 pg/cell for 1. Data

represent mean ± s.e.m of three independent experiments. *p<0.05, ***p<0.001,****p<0.0001 (t-

test).


79

Figure 4.S8 Cytotoxicity of nanoparticles. a, b) sample 1 & 3 in HeLa (left) and KHOS (right) cells.

c, d) 1-NH2 & 3-NH2 in HeLa (left) and KHOS (right) cells. e) 1-PEI & 3-PEI in KHOS cells. Pure

and amino-modified silica nanoparticles have relatively mild cytotoxicity to both HeLa and KHOS

cells, and the ones with rough surface are more toxic due to the improved cellular uptake. However,

the attachment of PEI caused a high toxicity to cells, so that the concentrations of 1-PEI & 3-PEI

will be decreased to 20 µg/mL in gene silencing experiment. Data represent mean ± s.e.m of three

independent experiments.


80

Figure 4.S9 Gene delivery performance of virus-mimicking nanoparticles in KHOS cells using

amino-modified nanoparticles. Neither PLK1-siRNA nor S10-siRNA delivered by 1-NH2 and 3-

NH2 induced the decrease of cell viability by gene silencing effect. In the bar chart, data represent

mean ± s.e.m of three independent experiments, and were analyzed using t-test.

Figure 4.S10 TGA of PEI-conjugation. In both a) 1–PEI and b) 3-PEI, the TGA curves of (i)

represent pure silica nanoparticles, (ii) show epoxy-group modified nanoparticles, (iii) are the PEI-

conjugated separately.


81

Figure 4.S11 Morphology adjustment of virus-mimicking nanoparticles. After fabricating the virus-

mimicking nanoparticles by mixing shell particles with core particles, morphology changes were

traced by TEM images. On adjusting the feed volumes of shell particle solutions from a) 0.68 mL to

b) 1.35 mL, to 2.70 mL (Figure 4.S1e), to c) 5.40 mL and finally to d) 6.75 mL, the surface

morphologies changed significantly, with the best sample being obtained when the feed volume was

2.70 mL. It is noted that a failure to obtain optimized virus-mimicking morphologies can be

attributed to either insufficient or excessive feed amount of shell particles, where only a proper feed

amount will lead to successful synthesis. Scale bar: 100 nm.


82

Figure 4.S12 The influence of calcination treatment. TEM image a) shows that without calcination,

shell particles peeled off on ultrasonication. In contrast, b) shows that the calcined sample 3

maintained its morphology after ultrasonication. Scale bar: 100 nm.

Table 4.S1 | Physicochemical Properties of nanoparticles

Sample

ID

Average Particle size (nm) PDI

ζ pot nt l

(mV) TEMa DLS

1 209 ± 20 249 0.006 -24

1-NH2 211 ± 19 291 0.120 +53

1-PEI 220 ± 13 472 0.403 +47

2 13 ± 2 15 0.105 -42

3 226 ± 18 279 0.135 -36

3-NH2 224 ± 16 298 0.159 +54

3-PEI 226 ± 16 473 0.437 +43

a The particle sizes are obtained by measuring 100 particles from TEM images and shown in

mean±SD.


83

Table 4.S2 | Atomic composition (%) of nanoparticles

Sample C Si O N

1 2.61 29.74 67.64 --

1-NH2 24.72 21.24 49.41 4.63

1-PEI 37.86 16.79 37.42 7.92

3 1.65 30.92 67.43 --

3-NH2 23.46 22.38 49.69 4.47

3-PEI 36.89 17.49 38.11 7.51

Uncalcined-2 4.22 29.11 66.67 --

Calcined-2 0.84 29.83 69.33 --

Table 4.S3 | Characterization of PEI-modification by TGA

1-PEI 3-PEI

Weight loss % 1 1-epoxy 1-PEI 3 3-epoxy 3-PEI

1.75 3.17 0.44 2.78 3.24 0.32

References

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Chapter 5 Synthesis of Silica Nanoparticles with Controllable Surface Roughness for Therapeutic

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Chapter 5

Synthesis of Silica Nanoparticles with

Controllable Surface Roughness for

Therapeutic Protein Delivery

This chapter reports a novel "neck-enhancing" approach to synthesize silica nanoparticles with

controllable surface roughness. By roughening the surface of solid silica core particles (~211 nm)

with smaller shell particles having various sizes, a series of rough silica nanoparticles (RSNs) are

obtained, where the shell and core particles are stably connected by bigger "necks", compared to our

previous method. The surface roughness is correlated to the core-to-shell size ratio (from 16.2:1 to

2.2:1), and the interspacing distance between neighboring shell particles increases from 7 to 38 nm

with the increased shell particle sizes from 13 to 98 nm. Protein loading capacity of RSNs is

dependent on the protein size relative to the interspacing distance. The optimal interspacing distance

of RSNs for high protein loading capacity is 7, 21 and 38 nm for cytochrome c (~3nm), IgG-

fragment (IgG-F, ~10nm) and non-specific rabbit IgG antibody (IgG-A, ~20nm), respectively.

Using surface plasmon resonance (SPR), it is demonstrated that the IgG-F maintained efficient

binding function after loading on RSNs. After hydrophobic modification of octadecyl-groups (C18),

C18-RSN with the interspacing distance of 38 nm shows effective intracellular delivery of anti-

pAkt antibody in breast cancer MCF-7 cells, leading to significant cell growth inhibition by

blocking pAkt and downstream anti-apoptotic protein of Bcl-2. This work has been accepted for

publication by Journal of Materials Chemistry B (2015, full paper).


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5.1 Introduction

Protein therapeutics has attracted increasing attention in cancer therapy due to the high

specificity and less interference with normal biological processes.1 By introducing proteins

that specifically recognize and influence target molecules, deactivation or activation of key

signalling pathways within cells can be manipulated, which strongly affect cell functions.2

However, proteins are poorly delivered into cells owing to poor stability and inability to

cross cell membranes.3 It remains an on-going challenge to develop delivery systems to

efficiently compact and deliver therapeutic proteins for enhanced cancer therapy.4

In the past decade, various nano-carriers have been generated to deliver therapeutic proteins

into cells, including liposomes,5-7

polymers,8-11

inorganic nanoparticles12-14

and protein-based

carriers.15

Among them, silica-based nanomaterials are a promising delivery platform for

protein therapeutics. Bale et al.16

reported a successful delivery of the antibody to phospho-

Akt (anti-pAkt) into MCF-7 cells with a significant cell growth inhibition where anti-pAkt

antibody was absorbed on the surface of solid silica nanoparticles modified by n-

octadecyltrimethoxy silane (n-ODMS) . A recent study has demonstrated that there was an

optimized pore size in the shell of silica hollow spheres for high loading and improved

intracellular delivery of a therapeutic protein, Ribonuclease A.17

The size of protein

molecules may vary from several nanometres (e.g. cytochrome c18

) to dozens of nanometres

(e.g. IgG antibodies19

). Therefore, it is highly desired to fabricate silica nanoparticles with

adjustable voids to optimize the loading/release ability toward therapeutic proteins having

various sizes.

Besides the interaction with cargo molecules, efficient cellular uptake performance of silica

nanoparticles is a prerequisite factor for successful cellular delivery. Recently, silica

nanoparticles mimicking the surface topography of enveloped viruses20

were prepared by

attaching shell particles with a smaller size onto core particles with a larger size.21

The

interspaces between neighbouring shell particles provide the void space for the entrapment

of biomolecules (e.g. siRNA). Compared to nanoparticles with smooth surfaces, silica

nanoparticles with rough surfaces exhibited higher loading, sustained release and enhanced

cellular uptake performance, consequently delivering siRNA successfully into cells.

However, systematic control over core-to-shell size ratios of silica nanoparticles and their

protein delivery efficacy has not been reported. If the surface roughness is adjustable on a


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88

nanoparticle, nano-carriers with advantages of both rough surface and controllable void

space would be crucial for exploring the impact of interspacing distance on protein delivery

efficiency. For silica nano-carriers with rough surface, it is also important to investigate the

bioactivity of proteins, for example, the binding activity of antibody, after loading into void

space. Answers to these questions will provide fundamental knowledge to the rational

design of silica nano-carriers for therapeutic protein delivery.

Various approaches have been developed to fabricate rough nanoparticles by attaching small

shell particles on a large core, including the electrostatic interaction22, 23

and covalent

bonding pathways.24

However, the synthesis of inorganic and/or organic rough nanoparticles

with uniform sizes generally have larger particle sizes ( 400 nm), which is not ideal for

cellular delivery.25, 26

Although rough nanoparticles with particle size of 300 nm can be

synthesized,27-29

the surface morphologies are not uniform. More importantly, in all previous

literature reports, the core-to-shell size ratio is larger than 5.6:1. Therefore, it is a challenge

to attach shell particles with relatively large sizes onto core particles.

Herein we report a novel "neck-enhancing" approach to synthesize silica nanoparticles with

controlled surface roughness. By roughening the surface of solid silica core particles (211

nm in diameter) with smaller shell particles (Figure 5.1a) having various sizes, a series of

rough silica nanoparticles (RSNs) are obtained. By forming a big "neck", shell particles with

large sizes can be stably connected to the core particles. The surface roughness is correlated

to the core-to-shell size ratios (from 16.2:1 to 2.2:1), and the interspacing distance between

neighbouring shell particles increases from 7 to 38 nm w t n r s n s ll p rt l s z s

rom 13 to 98 nm. T prot n lo d n p ty o RSNs s d p nd nt on t prot n s z

relative to the interspacing distance. The optimal interspacing distance of RSNs for high

protein loading capacity is 7, 21 and 38 nm for cytochrome c (M.W. 12 kDa), IgG-

fragment (IgG-F, domain antibody, M.W.76 kDa) and non-specific rabbit IgG antibody

(IgG-A, M.W. 150 kDa), respectively. Using surface plasmon resonance (SPR), it is

demonstrated that the IgG-F maintains efficient binding function after loading on RSNs. As

shown in Figure 5.1c, after hydrophobic modification of octadecyl-groups (C18), the C18-

RSN with the interspacing distance o 38 nm s ows t v ntr llul r d l v ry of anti-

pAkt antibody (having a similar structure and animal source with non-specific rabbit IgG-A)

in human breast cancer (MCF-7) cells, leading to significant cell growth inhibition by

blocking pAkt and the downstream anti-apoptotic protein of Bcl-2.


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Figure 5.1 Schematic illustrations of the synthesis of RSNs using a "neck-enhancing" approach (a)

and a conventional interaction approach (b). Scheme c shows the cellular delivery of therapeutic

anti-pAkt antibody using C18-RSNs and the cell growth inhibition mechanism.

5.2 Experimental section

5.2.1 Materials

Ammonium hydroxide solution (28%), L-arginine, octane (98%), 3-

aminopropyltriethoxysilane (APTES), cytochrome c (95%, from bovine heart), fetal bovine

serum (FBS) and trypan blue solution (0.4%) were purchased from Sigma-Aldrich.

Tetraethyl orthosilicate (TEOS, 98%) and n-octadecyl-trimethoxy silane (n-ODMS, 90%)

were purchased from Aldrich. Toluene was purchase from Merck. Dulbecco's Modified

Eagle Medium (DMEM), penicillin-streptomycin (10000 U mL-1

) and trypsin-EDTA

(0.25%) were purchased from GIBCO or Invitrogen, Life Sciences, Life Technologies. The

monoclonal antibodies (rabbit source) to Bcl-2 and GAPDH and HRP-linked anti-rabbit IgG

antibody were purchased from Cell Signalling. MCF-7 (HTB-22™) ll l n w s pur s d

from American Type Culture Collection (ATCC). IgG-F for adsorption and SPR

m sur m nt w s k ndly prov d d rom P t r P Gr y’s roup. All m als were used

without further purifications.

5.2.2 Synthesis of different nanoparticles

Synthesis of core particle

Uniform nonporous silica core particles were synthesized using a well-known method

developed by Stöber et al.30

Typically, absolute ethanol (50 mL) was mixed with deionized

(DI) water (3.8 mL) and ammonium hydroxide solution (2 mL) at 25 °C. Then, TEOS (3


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mL) was added to the solution under vigorous stirring. After 6 h, the as-synthesized

nanoparticles were separated by centrifugation at 20000 rpm, and washed with ethanol. The

final product was obtained by drying at 100 °C overnight. After that, amine-silane was

grafted to create positively charged surface. First, dried samples (200 mg) were suspended in

toluene (30 mL) and APTES (0.19 mL, 0.8 mmol) was added. The mixture was refluxed for

20 h at 110 °C.31

Then, amino-modified nanoparticles were obtained by centrifugation at

20000 rpm, washing with ethanol and drying in fume cupboard at 25 °C overnight.

Synthesis of shell particles

Shell nanoparticles with the mean sizes of 28, 54, 98, 135 and 175 nm were also fabricated

using Stöber method with the same recipe as the core particle, except for reacting at 70, 60,

50, 40 and 30 °C, respectively. The reactions were first carried out for 20 minutes for the

formation of shell particles (28, 54, 98 and 135 nm). For the shell particle of 175 nm, the

reaction time is 2 h. In addition, a modified Stöber method was used to fabricate the shell

particle of 13 nm diameter.32

First, L-arginine (87 mg) was dissolved in deionized water

(69.5 mL) containing octane (5.23 mL). Then, the mixture was sonicated and TEOS (0.5

mL) was added to react at 60 °C for 3.5 h for the formation of shell particles.

Synthesis of RSNs with varied shell sizes

The amino-modified core particle (200 mg) was suspended (2 mL) in DI water (for the

synthesis of RSN-211@13) or ethanol (for the synthesis of other RSNs). The core particle

suspensions were added into different shell particle reaction solutions as described above

(including 69.5 mL of DI water, 87 mg of L-arginine, 5.23 mL of octane and 0.5 mL of

TEOS for the synthesis of RSN-211@13; 50 mL of absolute ethanol, 3.8 mL of DI water, 2

mL of ammonium hydroxide solution and 3mL of TEOS for the synthesis of other RSNs),

reacting for another 2 h at the original temperatures for shell particle synthesis. The as-

synthesized RSNs were washed three times with ethanol and isolated by centrifugation at

4750 rpm for 10 min (shell particles cannot be recovered by centrifugation under these

conditions), followed by drying in a fume cupboard at 25 °C overnight. Finally, RSNs were

obtained after calcination treatment at 550 °C for 5 h to remove organic components in silica

frameworks, enabling all RSNs to have a similar surface composition (amorphous silicon

oxide) and surface property (zeta potential).


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Hydrophobic modification to RSNs

RSNs were functionalized with n-ODMS. Different RSNs (200 mg) were suspended in

toluene (25 mL) containing 0.5% (v/v) n-ODMS. Then, the mixture was refluxed for 20 h at

110 °C, followed by centrifugation at 10000 rpm, washing with ethanol and drying in a fume

cupboard at 25 °C overnight.

5.2.3 Characterizations

Transmission electron microscopy (TEM) images were taken using a JEOL 1010

microscope operated at 100 kV. The TEM specimens were dispersed in ethanol, and then

transferred to a copper grid. The high resolution scanning electron microscopy (HRSEM)

images were obtained on a JEOL JSM 7800 FE-SEM equipped with an in-column upper

electron detector (UED) and gentle beam technology. HRSEM was operated at a low

accelerating voltage of 0.8-1.5 kV with 20% specimen bias.33

For FE-SEM measurements,

the samples were prepared by dispersing the powder samples in DI water, after which they

were dropped to the aluminium foil pieces and attached to conductive carbon film on SEM

mounts. The SEM mounts were dried in a vacuum oven at 70 °C for at least 8 hours before

observations. Dynamic light scattering (DLS) and zeta potential (ZP) measurements were

carried out at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments. The samples

were dispersed in DI water or ethanol by ultra-sonication before analysis. Nitrogen sorption

isotherms of the samples were obtained at -196 °C using a Micrometrics Tristar II system.

Before the measurements, the samples were degassed at 180 °C overnight in vacuum. The

Brunauer–Emmett–Teller specific surface area (SBET) was calculated using experimental

points at a relative pressure of P/P0 = 0.05-0.25. Fourier transform infrared (FTIR) spectra of

rough silica nanoparticles before and after hydrophobic modification were collected using

t T rmo S nt ™ N ol t™ 6700 FT-IR spectrometers. Each spectrum was obtained

using dried powder against a background measured under the same condition. Atomic force

microscopy (AFM) measurement was conducted, where IgG-F was mixed with RSN-

211@54 (500 µg) in PBS pH 7.4 for 2 h at 4 °C, at a protein concentration of 1 mg mL-1

.

After incubation, the samples were washed with DI water to remove free and loosely

attached proteins and salts in solution by centrifugation and pipetting. Then, the suspension

(10 µL) was placed onto silicon wafers. The wafer was evaporated at 25 °C before AFM

observation. RSN-211@54 without protein incubation was used as a control. A Cypher S

AFM (Asylum Research, an Oxford Instruments company) was used for all the

measurements. The images were obtained by employing the tapping mode of the AFM in air


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by using Al- o t d s l on pro w t t p r d us o 2 nm (NANOSENSORS™, Sw tz rl nd).

The interaction between IgG-F and complementary antigen was monitored utilizing a SPR-

based biosensor (BiacoreTM

T200, GE Healthcare). IgG-F was incubated with RSNs (500

µg) in PBS pH 7.4 for 2 h at 4 °C, and the final protein concentration was 1 mg mL-1

.

Following that, IgG-F-RSNs complexes were washed several times until the supernatant

showed the same UV-vis absorbance at 280 nm as PBS only. Then, IgG-F-RSNs complexes

were suspended in HBS-EP buffer (BiacoreTM

T200, GE Healthcare). Biotinylated peptide

was immobilized via streptavidin capture on a sensor chip CAP (GE Healthcare) pre-

immobilized with ssDNA-streptavidin (Biotin CAPture kit, GE Healthcare) to yield the

peptide surface densities in the range of 2500-5000 R.U. A reference flow cell was

generated by omitting only ssDNA-streptavidin onto the chip surface. Interaction analyses

were performed by injecting IgG-F-RSNs complexes over the reference and peptide surfaces

in series for 120 seconds at a flow-r t o 10μl m n-1

. Complex dissociation was monitored

for 120 seconds. The binding intensity was determined at the peak point 128 seconds after

sample injection. Surface regeneration was performed at the end of each analysis cycle by

injecting guanidine (8 M) mixed with NaOH (1 M) at the ratio of 3:1, followed by washing

with the mixture of acetonitrile (30%), NaOH (0.25 M) and SDS (0.05%).

5.2.4 Biological experiments

Protein adsorption assay

The adsorption ability of RSNs with different proteins (cytochrome c, IgG-F and IgG-A)

was evaluated. Different proteins were mixed with RSNs (100 µg) in PBS pH 7.4 for 2 h at

4 °C, and final protein concentration is 1 mg mL-1

. IgG-A was also incubated with C18-

RSNs. After this time, the mixtures were centrifuged at 15000 rpm, and the supernatants

were collected for testing. The adsorption of protein molecules was determined using a

NANODROP 1000 spectrophotometer (Thermo Scientific) at 280 nm.

Protein therapeutics assay

Cells were maintained as monolayer cultures at 37 °C and 5% CO2 in DMEM supplemented

with 10% FBS and 1% penicillin-streptomycin. MCF-7 cells were seeded in 24 well-plates

at 5×104 cells per well and incubated for 4~6 h. The dose-dependent cytotoxicity of C18-

RSN-211@98+anti-pAkt composite was tested. The anti-pAkt antibody (1 mg mL-1

, sigma)

was incubated with C18-RSN-211@98 in PBS pH 7.4 at 4 °C for 2 h. Following this,

protein-nanoparticle complexes were suspended in serum containing culture medium in two-


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93

old d lut ons. T st n nop rt l on ntr t on s 50 μ mL-1

, corresponding to 1 µg

mL-1

of anti-pAkt antibody on the surface of the nanoparticle. Non-specific rabbit IgG-A

and nanoparticle only were used as control. After incubation for 24 h, cells were detached by

incubating with trypsin/EDTA mixture. Detached cells were then suspended in the medium

previously collected from the sample. Cell suspension was diluted with trypan blue solution

in a 1:1 ratio, and live and dead cells were counted on a hemacytometer.

The comparison of protein therapeutics among all the C18-RSNs was also evaluated. The

same amount of C18-RSNs (50 μ ) w s m x d w t nt -pAkt nt ody (1 μ ) n PBS pH

7.4 at 4 °C for 2 h and the complexes were incubated with MCF-7 cells in serum containing

culture medium for 24 h at the nanoparticle concentration of 15 µg mL-1

. Non-specific rabbit

IgG-A and C18-RSNs only were used as control. The cell viability was also determined by

counting live and dead cells.

Detection of cellular uptake performance of C18-RSNs

To quantitatively compare cellular uptake performance of C18-RSNs, 3×105 MCF-7 cells

were seeded in 6-well plates one day before transfection. C18-RSNs (50 μ mL-1

) were

incubated with cells under serum free condition for 4 h. Afterwards, the cells were washed

with PBS three times and harvested with trypsin. Cell number for each sample was recorded.

After centrifugation, the cell pellets were washed twice and dried. DI water was added to

allow dissolution of the cells under ultra-sonication condition. The supernatants (containing

cell components) were removed by centrifugation at 15000 rpm for 5 min. Aqueous NaOH

solution (1 M) was then added to allow dissolution of silica nanoparticles with ultrasound.

The silicon concentrations in the final solutions were measured by inductively coupled

plasma optical emission spectrometry (ICPOES) using a Vista-PRO instrument (Varian Inc,

Australia), which were then converted to be the mass of silica per cell.

Western-blot analysis

MCF-7 cells were seeded in 6-well plates at a seeding density of 3×105 cells per well. The

anti-pAkt antibody and the non-specific IgG-A were incubated with C18-RSN-211@98 in

PBS pH 7.4 at 4 °C for 2 h. Then, protein-nanoparticle complexes, along with C18-RSN-

211@98 only group, were mixed with serum containing culture medium and incubated with

lls or 24 . T n l on ntr t on o n nop rt l s s 50 μ mL-1

and protein is 1 µg mL-

1. At the end of incubation, cells were washed with PBS and lysed. The solutions containing

cell lysates were denatured at 95 °C for 15 min followed by characterization by SDS-PAGE.


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The protein bands were transferred to a PVDF membrane. Bcl-2 bands were targeted using

Bcl-2 monoclonal antibody (mAb) as the primary antibody, and HRP-linked anti-rabbit IgG

antibody as the secondary antibody. GAPDH mAb was used as an internal reference. Bands

were visualized on a ChemiDoc MP System (Bio-Rad).

5.3 Results and Discussion

Preparation of RSNs

Three relatively large silica shell particles (28±3 nm, Figure 5.S1b, ZP -42±8.3 mV; 54±5

nm, Figure 5.S1c, ZP -53±2.6 mV; 98±7 nm, Figure 5.S1d, ZP -53±1.0 mV) were prepared

by the classical Stöber method.30

The smallest silica shell particle (13±2 nm, Figure 5.S1a,

ZP -42±2.4 mV) was synthesized by a modified Stöber method.32

The silica core particle

(diameter of 211±11 nm, Figure 5.S1g, ZP +31±0.2 mV) was also fabricated using the

classical Stöber method, followed by amino-modification to generate positive charges on the

surface. The particle size distribution curves measured by the DLS method are narrow for all

silica particles (Figure 5.S1h), indicating that the nanoparticles are monodispersed in size

(see Table 5.S1 for the polydispersity index, PDI).

To fabricate RSNs with varied shell sizes, the core particles were suspended and added into

the reaction solution of different shell particles. After reaction for 2 h and washing, the final

calcined samples were denoted as RSN-211@13, RSN-211@28, RSN-211@54 and RSN-

211@98, possessing ZP values of -30, -26, -29 and -29 mV, respectively. The numbers

before and after @ refer to the mean size of core particle and shell particles measured from

TEM images (Figure 5.S1). The core-to-shell size is 16.2:1, 7.5:1, 3.9:1 and 2.2:1,

respectively.

TEM images of four RSNs are shown in Figure 5.1. The particle size of RSN-211@13,

RSN-211@28, RSN-211@54 and RSN-211@98 is 254±26, 270±15, 297±20 and 380±39

nm, respectively (Figure 5.2a-d). HRSEM images (Figure 5.2e-h) clearly show that core

particles are uniformly studded by different shell particles, and the interspacing distance

tw n n our n s ll p rt l s nl r s s t s ll p rt l s z n r s rom 13

to 98 nm. The interspacing distance between shell particles is crucial to the accumulation

of cargo molecules. Although the interspacing distances for RSNs are not uniform and

difficult to measure precisely, the values were determined by measuring 50 edge-to-edge

interspacing between adjacent shell particles to allow a semi-quantitative comparison. As

shown in Figure 5.S2 and Table 5.S2, the average interspacing distance of RSN-211@13,


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RSN-211@28, RSN-211@54 and RSN-211@98 is 7±2, 14±4, 21±9 and 38±22 nm,

respectively. The particle sizes of the four samples from DLS measurements are close to that

obtained from TEM measurements and the small PDI values confirm that all RSNs are

uniform and well-dispersed (Table 5.S2).

Figure 5.2 TEM (a-d) and HRSEM (e-h) images of RSNs with varied shell particle sizes:

a&e) RSN-211@13, b&f) RSN-211@28, c&g) RSN-211@54, d&h) RSN-211@98 and the

r d rrows nd t t orm t on o r “n ks” onn t n s ll nd or p rt l s.

Scale bar: 100nm.

The nitrogen sorption analysis was further utilized to measure SBET of RSN-211@13, RSN-

211@28, RSN-211@54 and RSN-211@98, which is 25.4, 26.9, 25.2 and 22.4 m2 g

-1,

respectively (Table 5.S2). For RSN-211@28, RSN-211@54 and RSN-211@98 prepared by

the same protocol, SBET of RSNs decreases with increasing shell particle size. The lower

SBET of RSN-211@13, compared to that of RSN-211@28, can be attributed to the difference

in synthesis methods: a longer reaction time (3.5 h vs. 20 min) of shell particles may lead to

more condensed structures and thus reduced surface area.

Using our previous protocol,21 s ll p rt l s o 13 nm w r su ss ully tt d on t

or p rt l s o 211 nm. How v r, l r r s ll p rt l s, or x mpl , w t t s z o 28

nm, are easily peeled off from core particles during washing and drying processes (Figure

5.S3a), even when core-shell structures were formed in solution (Figure 5.S3 ). In t

urr nt ppro , t l r s ll p rt l s ( 28, 54 nd 98 nm) w r orm d using the

classical Stöber method after reaction of 20 min, as the particle sizes were barely changed


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after about 15 min.30

Afterwards, positively charged core particle suspension was added into

the above shell particle reaction solution. By comparing the difference of current synthesis

strategy to the previous one (shown in Figure 5.1), the pH value of reaction medium (11.0

vs. 9.4) and the weight ratio of shell to core particles (4:1 vs. <1:1) is increased (Figure

5.1a(i) vs. b(i)).

The driving force for the attachment of negatively charged shell particles onto positively

charged core particles is electrostatic interaction21, 22

(Figure 5.1a(ii) and 1b(ii)), however

less attention has been paid to how to stabilize the formed core-shell structure and hence it is

difficult to synthesize rough particles with relatively large shell sizes. The focus of this study

is to address this challenge through silicate chemistry.34

At pH 9.4 the silica solubility is

reduced with both SiO(OH)3- (~50%) and Si(OH)4 (~50%) presenting in solution (Scheme

2b(i)). In contrast, at a stronger basic condition of pH 11, SiO(OH)3- is the predominant

silicate species with a relatively high concentration and thus more negative charges (Figure

5.1a(i)). When shell particles attach to core particles, a space with surfaces of negative

curvature is generated as indicated by a violet arrow in Figure 5.1a(ii). The solubility of a

surface with negative curvature is lower than that with positive curvature (i.e., normal

surface of spheres). Therefore, the net result is silica migration and deposition into the space

near the point of contact between core and shell particles,35

leading to the formation of a

bigger "neck" connecting core and shell particles (indicated by a violet arrow in Figure

5.1a(iii)).

The solubility difference between surfaces with positive and negative curvatures increases

with pH (e.g., 11.0 vs. 9.4), thus a higher pH favours the formation of bigger "necks"35

stabilizing the rough particle morphology. If the neck size is small and not strong enough

(e.g., at pH of 9.4) to hold core and shell particles (especially shell particles with relatively

larger sizes), the shell particles adhered on core particle surface mainly through electrostatic

interaction would easily peel off (iii in Figure 5.1b) during the subsequent treatments

(washing, drying, sonication, etc.). In addition to the increase of pH, the shell particle

concentration is also increased in our synthesis, which is beneficial for the attachment

between core and shell particles and eventually the enhanced neck formation.

The "neck-enhancing" mechanism for core-shell connection is supported by experimental

observations. The formation of neck regions between shell and core particles can be directly

seen using RSN-211@98 as an example (indicated by red arrows in Figure 5.2h).

Noticeably, there is a limitation for our current method. When shell size was further


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increased to 135 nm (Figure 5.S1e, 135±8nm, ZP -33±1.9 mV) and 175nm (Figure 5.S1f,

175±8nm, ZP -32±0.9 mV), the even larger shell particles failed to attach on the surface of

core particles (Figure 5.S4).

Protein adsorption behaviours on RSNs

To investigate the influence of interspacing distance of RSNs on protein adsorption capacity,

three model proteins with various molecular weights, including cytochrome c, IgG-F and a

non-specific rabbit IgG-A were used. Non-specific rabbit IgG-A was chosen because it has a

similar structure and animal source with anti-pAkt antibody, which will be utilized as the

therapeutic protein in the following biological study. Among the three proteins, cytochrome

c has an isoelectric point (IEP) of ~10, a diameter of ~3 nm and the smallest molecular

weight of ~12 kDa.36

IgG-A with the largest molecular weight of 150 kDa has a diameter of

~20 nm19

and the IEP of ~9. The IgG-F is a domain antibody and exists as a dimer, which

has an IEP value of ~8 and a molecular weight of 76 kDa with an estimated diameter of 10

nm. All proteins can be directly loaded onto RSNs at pH 7.4 in a phosphate buffer solution

(PBS) due to the electrostatic interaction between negatively charged silica nanoparticles

(Figure 5.S5) and the positively charged proteins. All RSNs demonstrate higher adsorption

ability in three tested proteins compared to the smooth core particles (data not shown),

which is consistent with previously reported results.21

Figure 5.3 shows protein adsorption capacity of RSNs with various interspacing distances.

RSN-211@98 with the interspacing distance o 38 nm x ts t st dsorpt on

ability (22.6 µg mg-1

) of IgG-A. RSN-211@54 (18.1 µg mg-1

) and RSN-211@28 (17.2 µg

mg-1

) with small interspacing distances show lower adsorption amount. In addition, the IgG-

A adsorption ability of RSN-211@98 is ~78% higher than RSN-211@13 (12.7 µg mg-1

),

which has the smallest interspacing distance. For IgG-F, the adsorption behaviour becomes

different. RSN-211@54 exhibits the highest protein entrapment ability (48.8 µg mg-1

). RSN-

211@98 shows a lower loading capacity of 42.5 µg mg-1

, because the interspacing distance

of RSN-211@98 is too large to hold the relatively small protein molecules in the voids.17

The lower adsorption ability of RSN-211@28 (40.1 µg mg-1

) and RSN-211@13 (36.7 µg

mg-1

) is attributed to the smaller void size compared to RSN-211@54.


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Figure 5.3 Protein adsorption profiles ( IgG-A; IgG-F; ▲ yto rom c). Solid lines

represent different protein adsorption onto unmodified rough silica nanoparticles. The dash

line also represents the IgG-A adsorption onto different rough silica nanoparticles, except

they are all modified with C18-groups. Data represent mean ± SD. Specific surface area

variations (×) of different unmodified rough silica nanoparticles are displayed to compare

with protein adsorption trend.

Noticeably, adsorption trend depends on the void size but not the surface area of RSNs

(Figure 5.3, solid line with cross symbol). As calculated in Table 5.S3, the protein coverage

(adsorption capacity versus surface area) is 63%, 80%, 91% and 127% for IgG-A, 91%,

94%, 122% and 117% for IgG-F on RSN- 211@13, RSN-211@28, RSN-211@54 and RSN-

211@98, respectively. The multilayer deposition behaviour of IgG-A on RSN-211@98 and

IgG-F on both RSN-211@54 and RSN-211@98 suggests that the adsorption occurs in the

interspace of particles with rationally controlled surface roughness.37

To confirm this hypothesis, RSN-211@54 was chosen to explore the surface topography

changes before and after IgG-F adsorption using AFM, due to its highest protein adsorption

capacity among the three cases. As shown in Figure 5.4, at the top region, shell particle

height of pure RSN-211@54 (Figure 5.4a, b) is 34-39 nm. After IgG-F adsorption, the shell

particle height (Figure 5.4c, d) decreases to 16-19 nm, suggesting that protein molecules are

entrapped and accumulated into the shell interspaces of RSN-211@54.

When protein size is much smaller than the interspacing distance, the surface area change

dominates protein adsorption behaviour. For the adsorption of cytochrome c, RSN-211@28

(7.5 µg mg-1

) shows the highest adsorption ability, followed by RSN-211@54 (6.9 µg mg-1

).

The adsorption ability of RSN-211@13 and RSN-211@98 are only 5.2 and 3.6 µg mg-1

,

respectively. This adsorption trend is consistent with SBET variations (Figure 5.3, solid line


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with cross symbol) of different RSNs. In addition, the coverage is calculated to be at most

9% (RSN-211@28, Table 5.S3). These results demonstrate that the interspacing distances of

RSNs are too large to confine very small protein molecules.

Figure 5.4 Surface topography studies. AFM images of RSN-211@54 before (a) and after

(c) the adsorption of IgG-F. A cross-sectional line is drawn to characterize the height

changes of shell particles on the top region before (b) and after (d) protein adsorption. Scale

bar: 100 nm.

Desired surface functionality (e.g., octadecyl-group16

) is of significance for high loading and

efficient delivery of therapeutic proteins into cells. All RSNs were hydrophobically-

modified on the surface, referred to as C18-RSN-211@13, C18-RSN-211@28, C18-RSN-

211@54 and C18-RSN-211@98 with slightly increased ZP values of -23, -18, -25 and -25

mV, respectively. They are used to further investigate the interspacing distance influence of

C18-RSNs on IgG-A adsorption capacity for the following application in therapeutic protein

delivery. FTIR results confirm the successful conjugation of octadecyl-groups for all C18-

RSNs (Figure. 5.S5). No significant topography changes are observed under TEM images

for all samples (Figure. 5.S6).

The IgG-A adsorption ability of C18-RSNs is displayed in Figure 5.3 (dash line). C18-RSNs

show highly improved adsorption capacity, compared to unmodified RSNs. The protein

coverage is calculated to be 179% for C18-RSN-211@13 (37 µg mg-1

), 229% for C18-RSN-

211@28 (49 µg mg-1

), 277% for C18-RSN-211@54 (55 µg mg-1

) and 337% for C18-RSN-

211@98 (60 µg mg-1

). The results are interpreted as IgG-A adsorption in a multi-layered

fashion, both on surface and in voids, where some proteins did not necessarily have strongly

physical contact with the silica surface.19

In addition, the adsorption amount of IgG-A to

C18-RSNs is much higher than the results in literature,16

where IgG-A loading capacity of

octadecyl-group modified smooth solid silica nanoparticles is only 1.25 µg mg-1

.


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The surface chemistry and nanoscale roughness play important roles in the immobilization

of biomolecules. The adsorption of positively charged IgG-A in PBS onto negatively

charged RSNs is mainly attributed to the electrostatic attraction.38

For C18-RSNs, the

hydrophobic modification leads to partially reduced charge density compared to

corresponding RSNs, however the IgG-A adsorption capacity was increased as much as 2-3

times than that of RSNs. For example, C18-RSN-@211@13 shows 2.8 times of IgG

antibody adsorption compared to RSN-211@13 without C18 modification (Figure 5.3),

indicating that the hydrophobic interaction is more important than the electrostatic

interaction in this case.38, 39

However, when comparing the adsorption capacity of either

RSNs or C18-RSNs with the same surface chemistry but tunable surface roughness (Figure

5.3), it shows that the size of surface voids (or core-to-shell ratios) is also important, e.g. the

IgG-A adsorption capacity of C18-RSN-@211@98 is 1.7 times compared to C18-RSN-

@211@13, which is attributed to protein immobilization in the void spaces of rough

surfaces.37

Evaluation of protein binding ability

Protein secondary structures are possibly disturbed after absorbed onto nanoparticle

surface.40

It is very important to maintain the activity of protein molecules loaded into nano-

carriers for therapeutic applications. Therefore, SPR measurements were conducted to

evaluate whether the binding capability of IgG-F with its complementary antigen will be

maintained after contacting rough nanoparticles.

SPR is a fast and real-time detection technique used to examine the interaction between wide

ranges of biological targets. Based on detecting small changes in the refractive index, SPR is

able to specifically monitor the interaction between analytes (e.g., antibody or peptide) and

the ligand molecules (e.g., antigen), which have been immobilized onto an inert surface.

"Resonance units" (RU, equal to a critical angle shift of 10−4

deg) is used to describe binding

signals between analytes and ligands.41

Unmodified RSNs were used to absorb IgG-F in this

test, because IgG-F is immobilized on unmodified RSNs generally in a monolayer manner,

so that the SPR results can be directly compared with the protein binding ability. After

sample injection, the negative signals from the control groups demonstrate that bare RSNs

and PBS did not exhibit any binding events. In comparison, all RSN-IgG-F complexes show

positive signals in binding with the antigen, which has been immobilized to the sensor chip

as the ligand, indicating the binding activity of IgG-F is maintained after complexing with

all RSNs (Figure 5.5 and Table 5.S4). It is noted that the trend of SPR signal intensity


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(peaked at 128 seconds after sample injection) of various RSNs-IgG-F complexes is

consistent with the adsorption trend of IgG-F onto various RSNs (Figure 5.3).

Figure 5.5 SPR sensorgrams showing the binding signals of RSN-IgG-F complexes with

receptors. All RSN-IgG-F complexes showed positive values of the binding with ligand.

Typically, there is a linear relationship between the SPR signal intensity and the surface

concentration of immobilized molecules.41

In our test, equal amount of RSNs was mixed

with excessive amount of IgG-F in PBS solution to achieve a saturated adsorption of IgG-F

on complexes. After removing free IgG-F and re-suspending into the injection buffer, the

SPR test for each sample is generally finished after 10 min of re-suspending the IgG-

F/RSNs complexes to ensure a minimized release of IgG-F from RSNs.42

The IgG-F

coverage of RSN-211@54 is 122%; however, RSN-211@13 and RSN-211@28 with

relatively low IgG-F adsorption are not fully covered. Therefore, higher IgG-F density on

the surface of RSNs favours the binding with ligands on the sensor chip43

and generates

higher SPR signal intensity. As a result, RSN-211@54 shows the highest SPR signal

intensity followed by RSN-211@28, and RSN-211@13 has the lowest SPR signal intensity.

Besides the influence of protein coverage on particle surface, higher shell particle density

provides more voids for accommodating IgG-F. RSN-211@98 with the largest shell particle

size and thus the lowest void density on the surface has less binding sites with ligands,

leading to an even lower SPR intensity, compared to RSN-211@28.

Intracellular delivery of therapeutic protein

It has been reported that pAkt plays an important role in transcriptional activation of

proteins involved in cell growth.44

Delivering anti-pAkt antibody into cytosols inactivates

pAkt and induces the decrease of anti-apoptotic protein (e.g. Bcl-245

), resulting in apoptosis


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in some human cancer cell lines, for example, ovarian cancer, breast cancer and pancreatic

cancer.46-48

Hydrophobic-modified silica nanoparticles have been reported to deliver

therapeutic proteins successfully into cytoplasm.16, 17

In this study, C18-RSN-211@98 was

used to deliver anti-pAkt antibody (Figure 5.1c) into human breast cancer cells (MCF-7) due

to its highest antibody adsorption ability.

Figure 5.6 Cell growth inhibition by the delivery of therapeutic protein. a) Cell viability of

MCF-7 cells incubated with increasing concentrations of C18-RSN-211@98+anti-pAkt (),

C18-RSN-211@98+non-specific-IgG-A (♦) nd C18-RSN-211@98. Data represent mean ±

SD. b) Western blotting confirming the degradation of downstream anti-apoptotic protein,

Bcl-2 in MCF-7 cells. Blots presented are representative of typical results. GAPDH served

as an internal reference.

Dose-dependent cell growth inhibition is observed for the complex of C18-RSN-

211@98+anti-pAkt, and a maximum exposure of 1 µg mL-1

of immobilized anti-pAkt

antibody at 50 µg mL-1

of nanoparticle shows an increase in cell growth inhibition up to

85% (Figure 5.6a). This cell growth inhibition of anti-pAkt antibody is greater than the

literature report, where 80% of cell growth inhibition was induced by using as high as 800

µg mL-1

of a C18-modified silica nanoparticle (15 nm in diameter) to deliver 1 µg of anti-

pAkt antibody. In the absence of silica nano-carrier, free anti-pAkt antibody is unable to


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cause cell growth inhibition, indicating its poor cell internalization ability (Figure 5.S7). In

addition, when cells were treated with C18-RSN-211@98+non-specific rabbit IgG-A or

nanoparticle only, no obvious cell growth inhibition is observed (Figure 5.6a). The anti-pAkt

antibody delivery efficiency was also evaluated using other C18-RSNs (as shown in Figure

5.S8), and C18-RSN-211@98 holds the best performance.

To further test the downstream effects after blocking pAkt, Bcl-2 degradation was evaluated

using western blotting. As shown in Figure 5.6b, Bcl-2 degradation in MCF-7 cells is only

observed following cytosolic delivery of the C18-RSN-211@98+anti-pAkt, compared to cell

only, nanoparticle only and nanoparticle with non-specific IgG-A groups, indicating that cell

growth inhibition is associated with the degradation of Bcl-2 levels in MCF-7 cells.

5.4 Conclusion

In summary, rough silica nanoparticles (RSNs) with varied topographies were successfully

synthesized using a novel "neck-enhancing" approach. Relatively high pH value and shell

particle concentration favour the formation of bigger "necks", which is crucial for the

generation of RSNs with controllable core-to-shell ratios. The increase of shell particle sizes

from 13 to 98 nm while keeping the core particle size at 211 nm enlarges the shell

particle interspacing distance rom 7 to 38 nm, where proteins with comparable sizes are

favourably accumulated without influencing its binding ability. The rough silica

nanoparticles with an optimized loading capacity demonstrate a high efficiency of

intracellular delivery of therapeutic proteins in cancer cells, causing a significant cell growth

inhibition. The "neck-enhancing" approach provides new understanding in the rational

design of cellular delivery vectors with controllable surface roughness for the delivery of

therapeutic proteins.

5.5 References

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Figure 5.S1 TEM images (a-g) and particle size distribution curves (f) of shell and core particles.

Shell particles: a&h-i) 13nm, b&h-ii) 28nm, c&h-ii) 54nm, d&h-iv) 98nm, e&h-v) 135nm and f&h-

vi) 175nm. Core particle: g&h-vii) 211nm. Scale bar: 100 nm.

Figure 5.S2 The interspacing distance of RSNs. a) RSN-211@13, b) RSN-211@28, c) RSN-

211@54, d) RSN-211@98. The interspaces are measured from SEM images by recording 50 edge-

to-edge interspacing data in each sample.


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Figure 5.S3 TEM images showing the synthesis of RSN-211@28 using previous recipe after

washing and drying process (a) and in reaction solution (b). Scale bar: 100 nm.

Figure 5.S4 TEM images of failed synthesis of RSNs with much larger shell sizes. a) Core particles

(dotted arrow) mixed with the shell of 135 nm (solid arrow), b) Core particle (dotted arrow) mixed

with the shell of 175 nm (dotted arrow). Scale bar: 100 nm.


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Figure 5.S5 Fourier transform infrared (FTIR) spectra of pure liquid n-ODMS (a) and a series of

RSNs with and without hydrophobic modification (b).

The RSNs before and after modification of octadecyl group is characterized by FTIR technique.

Figure 5.S5a shows the FTIR spectrum of pure liquid n-ODMS, where the six characteristic peaks

at 809, 1085, 1190, 1465, 2856 and 2926 cm-1

n ttr ut d to ν(S -O), ν(C-O)+ ν(C-C),

ρ(CH3), δ(C-H)1 and symmetric and anti-symmetric -CH2- stretching,

2, 3 respectively. All the

spectra of silica nanoparticles show the same characteristic bands at 802 cm-1

and broad band

centrated at 1058 cm-1

, suggesting –Si-O-Si- bonding.4 In the spectra of all silica nanoparticles after

hydrophobic modifications (Figure 5.S5b i, iii, v, vii), two extra peaks at 2856 and 2926 cm-1

can be

observed, shown as the curves enlarged by 5-folds, which can be attributed to symmetric and anti-

symmetric -CH2- stretching, respectively, indicating the successful modification of octadecyl

groups.5


Protein Delivery

109

Figure 5.S6 TEM images of hydrophobic modified RSNs. a) C18-RSN-211@13, b) C18-RSN-

211@28, c) C18-RSN-211@54 and d) C18-RSN-211@98 (d). Scale bar: 100 nm.

Figure 5.S7 Cell viability of MCF-7 cells incubated with varying concentrations of non-specific

IgG-A () only and anti-pAkt antibody () only, respectively. Data represent mean ± SD.


Protein Delivery

110

Figure 5.S8 The comparison of anti-pAkt antibody delivery efficiency (a) and cellular uptake

performance of C18-RSNs measured by ICPOES (b). Data represent mean ± SD.

C18-RSN-211@13, C18-RSN-211@28 and C18-RSN-211@54 were also used apart from C18-

RSN-211@98 to deliver anti-pAkt antibody into cells. Both C18-RSNs alone or loaded with non-

specific IgG-A did not cause significant cell growth inhibition. Equal amount of C18-RSNs (50 µg)

was mixed with excessive amount of anti-pAkt antibody (1 µg) in PBS solution to ensure a

saturated adsorption. At the nanoparticle concentration of 15 µg mL-1

, cell viability decreases to

50%, 51% and 46% using C18-RSN-211@13, C18-RSN-211@28 and C18-RSN-211@54 as nano-

carriers, respectively, while C18-RSN-211@98+anti-pAkt induces a lower cell survival rate to 31%

(Figure 5.S8a). This difference is attributed to the combined effects of two factors: the cellular

uptake efficiency and protein adsorption ability. As shown in Figure 5.S8b, averagely 127, 139, 121

and 88 pg of silica from C18-RSN-211@13, C18-RSN-211@28, C18-RSN-211@54 and C18-RSN-

211@98 is taken up by each cell, respectively, because nanoparticles with larger sizes have lower

cell penetration.6-8

This trend is opposite to the adsorption trend (Figure 5.3), where C18-RSN-

211@98 shows the highest adsorption ability to large protein (IgG-A). Consequently, the opposite

trends of adsorption and cellular uptake lead to the highest cell growth inhibition of C18-RSN-

211@98+anti-pAkt.


Protein Delivery

111

Table 5.S1 Size and ζ potential characterizations of shell and core particles.

aMean size±SD of shell/core particles by recording 50 data from TEM images.

bNumber mean size

±SD of shell/core particles by DLS method. cPolydispersity index.

Table 5.S2 Characterizations of RSNs with varied surface topography.

Sample ID

Size(nm)

PDIc

SBET

(m2 g

-1) d

Interspace e

(nm) TEMa DLS

b

RSN-211@13 254±26 287±5 0.16±0.07 25.4 7±2

RSN-211@28 270±15 371±13 0.23±0.05 26.9 14±4

RSN-211@54 297±20 398±16 0.13±0 25.2 21±9

RSN-211@98 380±39 480±13 0.22±0.04 22.4 38±22

a Mean size±SD of RSNs by recording 50 data from TEM images.

bNumber mean size ±SD of

RSNs by DLS method. cPolydispersity index of RSNs.

d BET specific surface area of RSNs.

e Mean

interspacing distance ±SD of RSNs by recording 50 data from SEM images.

Sample

size (nm)

PDIc ζ-potential (mV)

TEMa DLS

b

Shells

13±2 10±0.3 0.11±0.01 -42±2.4

28±3 33±0.4 0.03±0.02 -42±8.3

54±5 54±0.7 0.01±0.01 -53±2.6

98±7 96±1.3 0.03±0.01 -53±1.0

135±8 179±3.6 0.02±0.02 -33±1.9

175±8 219±2.4 0.03±0.03 -32±0.9

Core 211±11 241±1.4 0.01±0.01 +31±0.2


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112

Table 5.S3 Estimation of protein coverage on RSNs/C18-RSNs

Protein Sample ID Nmaxa Nads

b Coverage %

c

Cytochrome c

RSN-211@13 3.59×1018

2.55×1017

7.11

RSN-211@28 3.81×1018

3.65×1017

9.59

RSN-211@54 3.56×1018

3.38×1017

9.49

RSN-211@98 3.18×1018

1.77×1017

5.58

IgG-F

RSN-211@13 3.23×1017

2.94×1017

91.07

RSN-211@28 3.43×1017

3.22×1017

94.02

RSN-211@54 3.20×1017

3.92×1017

122.23

RSN-211@98 2.86×1017

3.41×1017

117.65

IgG-A

RSN-211@13 8.08×1016

5.08×1016

62.90

RSN-211@28 8.56×1016

6.88×1016

80.40

RSN-211@54 8.02×1016

7.26×1016

90.59

RSN-211@98 7.15×1016

9.07×1016

126.87

C18-RSN-211@13 8.08×1016

1.44×1017

178.57

C18-RSN-211@28 8.56×1016

1.97×1017

229.49

C18-RSN-211@54 8.02×1016

2.22×1017

276.83

C18-RSN-211@98 7.15×1016

2.41×1017

336.88

aNmax represents the theoretical number of protein molecules that is assumed to fully cover the

surface of 1 gram of RSNs in a single-layered fashion.

(1)

bNads represents the actual number of protein molecules (calculated from adsorption amount, Wads)

that associated on the surface of 1 gram of RSNs/C18-RSNs. NA stands for Avogadro constant, and

MWprotein shows protein molecular weight.

(2)

cCoverage% represents the actual protein coverage percentages, compared to theoretical number of

protein molecules where the surface is assumed to be fully covered in a single-layered manner.

⁄ (3)


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113

Table 5.S4 SPR signal intensity.

Sample ID without IgG-F(RU) with IgG-F(RU)

RSN-211@13 -1.9 462.3

RSN-211@28 -3.8 531.2

RSN-211@54 -4 564

RSN-211@98 -2.6 527.5

PBS -0.4 --

Note: SRP signal intensity was determined at the peak point 128 seconds after sample injection

References

1 M. I. Tejedor-Tejedor, L. Paredes and M. A. Anderson, Chem. Mater, 1998, 10, 3410-3421.

2 Q. Wu, L. Wang and L. Zu, J. Autom. Methods Manage. Chem., 2011, 2011, 7.

3 K. Kailasam and K. Muller, J. Chromatogr. A, 2008, 1191, 125-135.

4 D. B. Mahadik, A. V. Rao, A. P. Rao, P. B. Wagh, S. V. Ingale and S. C. Gupta, J. Colloid Interf

Sci, 2011, 356, 298-302.

5 J. Zhang, S. Karmakar, M. H. Yu, N. Mitter, J. Zou and C. Z. Yu, Small, 2014, 10, 5068-5076.

6 F. Lu, S. H. Wu, Y. Hung and C. Y. Mou, Small, 2009, 5, 1408-1413.

7 J. Zhu, L. Liao, L. N. Zhu, P. Zhang, K. Guo, J. L. Kong, C. Ji and B. H. Liu, Talanta, 2013, 107,

408-415.

8 J. Rejman, V. Oberle, I. S. Zuhorn and D. Hoekstra, Biochem J, 2004, 377, 159-169.

Chapter 6 Understanding the Contribution of Surface Roughness and Hydrophobic Modification on

Silica Nanoparticles for Enhanced Therapeutic Protein Delivery

114

Chapter 6

Understanding the Contribution of Surface

Roughness and Hydrophobic Modification on

Silica Nanoparticles for Enhanced Therapeutic

Protein Delivery

This chapter quantitatively demonstrates both the individual and combined contributions of surface

roughness and hydrophobic modification for the improvement of protein therapeutics. Both surface

roughening and hydrophobic modification enhance protein (RNase A) adsorption capacity, while

the contribution from surface roughness is more dominant. Hydrophobic modification has a

stronger effect to retard RNase A release. The contribution difference to enhance cellular uptake is

cell type-dependent. Importantly, only hydrophobic modification facilitates endo/lysosomal escape.

Collectively, octadecyl-functionalized rough silica nanoparticles (C18-RSNs) thus show the best

performance in RNase A delivery, causing significant cell viability inhibition in both human breast

cancer (MCF-7) and SCC-25 cell lines, compared to unmodified rough silica nanoparticle and

smooth silica nanoparticles with (C18-SSN) or without (SSN) octadecyl-group modification. This

work has been submitted for publication in Journal of Materials Chemistry B (2015, full

paper).



115

6.1 Introduction

Protein-based therapeutics has attracted increasing attention in the field of cancer therapy.1

Compared to small molecule drugs, therapeutic proteins such as enzymes can manipulate

key signalling pathways without influencing normal biological functions.2 However, cell

membrane impermeability to protein molecules limits intracellular delivery of therapeutic

proteins. To address this issue, various synthetic vectors have been developed to compact

therapeutic proteins, for example, liposomes,3, 4

cationic polymers,5 micelles,

6 dendrimers

7

and inorganic nanoparticles (e.g., silica nanoparticles8, 9

and gold nanoparticles10

). Among

these synthetic vectors, silica-based nanomaterials possess excellent biocompatibility,

tuneable nanostructures and the ease of surface modification, making them attractive

candidates for therapeutic protein delivery.11

Pristine mesoporous silica nanospheres were

firstly used for intracellular delivery of cytochrome c into cervical cancer cells (HeLa),12

but

the therapeutic efficacy was not demonstrated.

To achieve successful intracellular delivery of proteins, it is crucial to use nano-carriers with

high protein loading level, effective cellular uptake and sustained protein release in

cytosols13

without proteolysis and degradation in endosome/lysozymes.14, 15

Bale and co-

workers16

illustrated that hydrophobically modified solid silica nanospheres can immobilize

protein molecules on the surface and deliver them into cancer cells without extended

entrapment in endosomes. However, extremely large amount of nanoparticles was required

for significant cell growth inhibition, because solid particles have limited surface area for

protein loading. Recently, Zhang and co-workers17

developed silica vesicles with hollow

cavities to provide sufficient space for enhanced protein loading capacity. After octadecyl-

group (C18) attachment, C18-modified silica vesicles successfully delivered ribonuclease A

(RNase A) to human squamous carcinoma cells (SCC-25), causing a significant cell growth

inhibition. Roughening the surface of nanoparticles has been demonstrated as another

approach to improve biomolecule loading capacity.18

More importantly, surface roughness

was confirmed to enhance cellular uptake efficiency.18, 19

As two independent parameters of

nano-carriers, unfortunately there is no report to compare the individual contribution of

surface roughness and hydrophobic modification for protein delivery. A comparative study

to understand their roles is of fundamental importance in the design of advanced nano-

carriers with enhanced delivery efficacy.



116

Herein, we report a systematic study on the contribution of surface roughness and

hydrophobic modification on silica nanoparticles for therapeutic protein delivery. The

influence of one individual parameter and their combination on protein loading capacity,

release behavior, cellular uptake and endosome escape performance of silica nanoparticles

have been demonstrated. Both surface roughening and hydrophobic modification enhance

protein adsorption capacity, while the contribution from surface roughness is higher. For

sustained protein release, hydrophobic modification has a higher impact compared to rough

surface. Both surface roughness and hydrophobic modification improve cellular uptake

performance; however the contribution difference is cell type-dependent. It is clear that the

surface roughness has little contribution to endo/lysosomal escape. Only the surface

chemistry, i.e., the hydrophobic modification, facilitates the release of nanoparticle/cargo

molecules from endosome/lysosome entrapment. Collectively, octadecyl-functionalized

rough silica nanoparticle (C18-RSN) shows the best performance in therapeutic protein

(RNase A) delivery, causing significant cell viability inhibition in both human breast cancer

(MCF-7) and SCC-25 cell lines among all groups under study.

6.2 Experimental

6.2.1 Materials and reagents

For nanoparticle synthesis: ammonium hydroxide solution (28%), 3-aminopropyltriethoxy

silane (APTES, 98%) and L-arginine (98%), octane (98%) were purchased from Sigma-

Aldrich. Tetraethyl orthosilicate (TEOS, 98%) and trimethoxy(octadecyl) silane (OTMS,

90%) were purchased from Aldrich. Toluene was purchased from Merck. All chemicals

were used without further purifications.

For biology experiments: Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's

modified Eagle's medium and Ham's F12 medium (DMEM/F12), penicillin-streptomycin

(10,000 U/mL) and trypsin-EDTA (0.25%) were purchased from GIBCO or Invitrogen, Life

Sciences, Life Technologies. Fetal bovine serum (FBS) and trypan blue solution (0.4%)

were purchased from Sigma-Aldrich. Ribonuclease A (RNase A) from bovine pancreas was

purchased from ROCHE (Germany). Adenocarcinoma cell line MCF-7 (HTB-22™) nd

human squamous cell carcinoma cell line SCC-25 (ATCC® CRL-1628™) w r pur s d

from ATCC (American Type Culture Collection). MCF-7 cells were maintained as

monolayer cultures in DMEM, supplemented with 10% FBS, 1% penicillin-streptomycin at

37 °C and 5% CO2. SCC-25 cells were maintained as monolayer cultures in DMEM/F12,



117

supplemented with 10% FBS, 1% penicillin-streptomycin, 0.5 mM sodium pyruvate and

400ng/mL hydrocortisone at 37 C and 5% CO2.

6.2.2 Synthesis of nanoparticles

Synthesis of smooth silica nanoparticles (SSN): Un orm sol d s l p rt l w t t s z

o 232 nm w s synt s z d us n St r m t od.20

Typically, 3.8 mL of deionized (DI)

water and 2 mL of ammonium hydroxide solution were added into 50 mL of absolute

ethanol, and the reaction was carried out for 6 hours after adding 3 mL of TEOS at room

temperature under vigorous stirring. Nanoparticles were collected by centrifugation,

washing and drying. Then calcination treatment at 550 °C was conducted to remove organic

components.

Amine-group modification: Uncalcined SSN was functionalized with amine-silane and used

as positively charged core particle. Dried samples (200 mg) were re-suspended in 30 mL of

toluene with 1% (v/v) APTES. The mixture was refluxed for 2 h at 110 °C. Then, the amino-

modified nanoparticle was obtained by centrifugation, washing and drying at room

temperature overnight, and it will be used as core particles.

Synthesis of shell particle: Shell nanoparticle with the size of 20 nm was also fabricated

using the Stöber method. The reaction components were the same as that for SSN, but the

reaction temperature was increased to 70 °C.

Synthesis of RSN: After reacting for 20 min of the shell particle above, core particle (200

mg) suspension in 2 mL of ethanol was added into the shell reaction solution at 70 °C. Then,

RSN was collected 2 h later by washing with ethanol and drying at room temperature

overnight, followed by calcination treatment at 550 °C for 5 h to remove organic

components in silica framework.

Hydrophobic (octadecyl-group) modification: SSN and RSN were functionalized to have

hydrophobic feature. Nanoparticles (200 mg) were re-suspended in 30 mL of toluene

containing 0.5% (v/v) OTMS. Then, C18-SSN and C18-RSN were obtained after refluxing

at 110 C for 20 h, followed by washing and drying at room temperature overnight.

6.2.3 Characterizations

Hydrodynamic size and z t (ζ)-potential (ZP) measurements: Dynamic light scattering

(DLS) size and ZP values were collected at 25 °C using a Zetasizer Nano-ZS from Malvern



118

Instruments. The nanoparticles were suspended in DI water or phosphate buffered saline

(PBS, pH 7.4) by ultra-sonication.

Transmission electron microscopy (TEM) and high resolution scanning electron microscopy

(HRSEM): TEM images were taken using a JEOL 1010 microscope operated at 100 kV for

observing nanoparticles, which were dispersed in ethanol and then transferred to a copper

grid. HRSEM images were obtained on a JEOL JSM 7800 FE-SEM operated at a low

accelerating voltage of 0.8-1.5 kV with 20% specimen bias.21

The microscope was equipped

with an in-column upper electron detector (UED) and gentle beam technology. SEM

samples were prepared by dropping nanoparticle suspensions onto the aluminum foil pieces,

which attach to conductive carbon film on SEM mounts, followed by baking in vacuum at

70 °C for overnight.

Surface area test: Brunauer–Emmett–Teller (BET) specific surface area of nanoparticles

were collected from nitrogen sorption isotherms, carried out at -196 °C using a

Micromeritics Tristar II system. Before the measurements, the samples were degassed at 180

°C for at least 6h in vacuum. BET specific surface area was calculated from experimental

points at a relative pressure of P/P0 = 0.05- 0.25.

Fourier transform infrared (FTIR) measurements: FTIR spectra of OTMS, RNase A and

different nanoparticles before and after RNase A adsorption were collected using the

T rmo S nt ™ N ol t™ 6700 FT-IR spectrometers.

C18-modification determination: Elemental analysis was carried out on a CHNS-O Analyzer

(Flash EA1112 Series, Thermo Electron Corporation) to determine the weight percentages

of carbon (C) element. Different nanoparticles of 10-20 mg were required for the test.

Cystine with 29.99% the element of carbon was used as the standard control.

Atomic force microscopy (AFM) measurements: RSN and C18-RSN were dispersed in DI

water, and then the suspensions (10 µl) were placed onto silicon wafers, evaporating at room

temperature. For the preparation of C18-RSN+RNase A, C18-RSN was first mixed with

RNase A, at nanoparticle concentration of 10mg/ml and RNase A concentration of 1mg/ml.

After that, the complex was washed with DI water to remove salts by centrifugation and

pipetting, followed by mounting on silicon wafer. AFM measurements of RSN, C18-RSN

and C18-RSN+RNase A were processed using the instrument of Cypher S AFM (Asylum

Research, an Oxford Instruments company). AFM images were obtained at the tapping



119

mode of AFM in air using Al-coated silicon probe with tip radius of 2 nm

(NANOSENSORS™, Switzerland).

Stability test of silica nanoparticles: Different nanoparticles were dispersed in PBS pH 7.4 at

the concentration of 1mg/ml, shaking at room temperature for 72h. The suspension (3ml)

was picked up every 24 h and then centrifuged at 20000 rpm for 10 minutes. Silica

concentration of different nanoparticles in supernatant was tested by inductively coupled

plasma optical emission spectrometry (ICPOES) using a Vista-PRO instrument (Varian Inc,

Australia). After 72 h, TEM images of different nanoparticles were taken using a JEOL

1010 microscope operated at 100 kV.

6.2.4 Biological experiments

Loading and Release Profile of RNase A by nanoparticles

Different nanoparticles were mixed with RNase A in PBS for 2 h at room temperature. Final

nanoparticle concentration is 10 mg/ml and RNase A concentration is 1 mg/ml. After this

time, the mixtures were centrifuged, and protein adsorption amount was determined using a

NANODROP 1000 spectrophotometer (Thermo Scientific) at 278 nm, based on the original

and residual RNase A concentrations and volumes. ANOVA analysis is processed. The

precipitates (RNase A loaded nanoparticles) were re-suspended in PBS or acetic buffer (pH

4.5) for protein release, shaking at 1000 rpm at 25 °C using an eppendorf MixMate (Thermo

Fisher Schentific). Certain volume of the mixtures was picked up at different time points and

the same volume of solvent was refilled until 72 h. In addition, RNase A release was also

investigate in DMEM, under static condition at 37 °C for 4h. The released RNase A content

was stained with BCA reagents (Thermal Scientific, Life Sciences, Life Technologies) and

the absorbance was collected at 562 nm using a Synergy HT Microplate Reader.

Detection of cellular uptake performance

To quantitatively compare the cellular uptake of different nanoparticles, 3×105 MCF-7 and

SCC-25 cells were seeded in 6-well plates. RNase A was incubated with nanoparticles for

protein adsorption for 2h, and then the complexes were incubated with cells in serum free

medium for 4h. RNas A on ntr t on s 2 μ /mL, and nanoparticle concentration is 50

μ /mL for each sample. Afterwards, cells were harvested by the digestion of trypsin/EDTA

mixture, and cell number for each sample was recorded. After centrifugation, cell pellets

were washed twice followed by supersonic schizolysis in DI water. Supernatants (containing

cell components) were removed, and aqueous NaOH solution (1 M) was used to allow the



120

dissolution of silica components with ultrasound. The silicon concentrations were detected

by inductively coupled plasma optical emission spectrometry (ICPOES) using a Vista-PRO

instrument (Varian Inc, Australia), and the mass of silica per cell was then calculated.

ANOVA analysis is processed.

Toxicity assay

Both MCF-7 and SCC-25 cells were seeded in 24-well plates at a density of 3×104 cells per

well and incubated for 24 h. To evaluate the cytotoxicity of different pure nanoparticles,

they were made in a 2-fold serial dilution in culture medium, and incubated for 24 h, 48 h

and 72 h. For the measurements of cell growth inhibition caused by RNase A, protein was

first mixed with SSN, C18-SSN and C18-RSN in PBS for 2 h at 4 °C to achieve a saturated

adsorption. Then the complexes were suspended in serum containing culture medium. The

final RNase A on ntr t on s 2 μ /mL, nd n nop rt l on ntr t on s 50 μ /mL. After

24, 48h and 72 h, culture medium from each sample was collected, and cells were harvested

by incubating with trypsin/EDTA mixture. Detached cells were then re-suspended in the

medium previously collected from the samples. Dead cells were marked with trypan blue

reagent in a 1:1 ratio, and cell viability was recorded by counting live and dead cells.

TEM study on nanoparticle-cell interaction

MCF-7 cells were seeded in 3-cm petri-dishes at a cell number of 3×105 for 24 h, and they

were incubated with a suspension of nanoparticle (SSN, RSN, C18-SSN and C18-RSN) at

t on ntr t on o 50 μ /mL for another 24 hours. Then cells were first fixed with 2.5%

glutaraldehyde at room temperature for 30min, and post-fixed in 1% osmium tetraoxide in

microwave condition. After that, cells were embedded into 2% agarose gel cube, followed

by dehydration in acetone of increasing concentration (50%, 70%, 90%, 100% and 100%) in

microwave condition. The dehydrated cell cubes were embedded in Epon resin, and

solidified in 60°C for 2 days. Microtome (Leica, EM UC6) was then used to cut the

embedded cell-resin cube into ultra-thin slices (70–90 nm in thickness). Samples were

mounted on form-bar coated copper grids and double stained with 2% aqueous uranyl

acetate and commercial lead citrate aqueous solution. TEM images were taken using a JEOL

1010 microscope operated at 80 kV.

6.3 Results and discussion

Preparation of nanoparticles



121

RSN (ZP -22±0.7 mV) with a core-shell structure was fabricated according to the reported method

with minor modification.18

A typical TEM image of RSN is shown in Figure 6.1a. The average

particle size is measured to be 296±10.0 nm. C18-modified silica nanospheres have been

demonstrated to successfully deliver therapeutic proteins into cancer cells.15

Therefore, C18-RSN

(299±14.6 nm, Figure 6.1b, ZP -22±0.6 mV) was synthesized by refluxing RSN and

trimethoxy(octadecyl) silane (OTMS) in toluene. HRSEM image (Figure 6.1c) clearly shows that

the core particles are uniformly attached by shell particles. The mean void size between neighboring

shell particles is 14.9±3.6 nm. For control groups, smooth silica nanoparticles (SSN) without

surface roughening (240±9.3 nm, Figure 6.1d, ZP -34±0.4 mV) and C18-SSN (240±17.5 nm, Figure

6.1e, ZP -33±0.5 mV) were also synthesized. FTIR spectra confirm successful C18-modification of

C18-RSN and C18-SSN (Figure 6.S2a&b). AFM images indicate that the surface topography of

RSN (Figure 6.S3a) is not influenced after hydrophobic modification (C18-RSN, Figure 6.S3c).

The void height of C18-RSN (16.7±3.6 nm, Figure 6.S3d) remains similar to that of RSN (16.7±3.3

nm, Figure 6.S3b).

Figure 6.1 TEM (a, b, d, e) and HRSEM (c) images and particle size distribution curves (f)

of RSN (a&f-i), C18-RSN (b, c&f-ii), SSN (d&f-iii), C18-SSN (e&f-iv). Scale bar: 100 nm.

DLS method was further utilized to characterize particle size and dispersity in both DI water

and biological media. As shown in Figure 6.1f and Table 6.S1, the intensity mean size of

SSN, RSN, C18-SSN and C18-RSN in DI water is 324 nm (PDI: 0.30), 630 nm (PDI: 0.23),

277 nm (PDI: 0.03) and 499 nm (PDI: 0.16), respectively, indicating that all nanoparticles

have good dispersibility, while hydrophobically modified ones are more uniform in size. In

PBS, the intensity mean sizes of four samples increase (391 nm for SSN, 512 nm for RSN,

599 nm for C18-SSN, and 683 nm for C18-RSN), and PDI values grow to 0.3-0.4,

suggesting that silica nanoparticles tend to aggregate in some extent (Table 6.S2, Figure



122

6.S4), due to the decrease of electrical double layer thickness under the condition of high

ionic strength.22

Nitrogen sorption analysis was used to measure BET specific surface area (SBET) of SSN,

RSN, C18-SSN and C18-RSN, which is 13.0, 26.5, 11.6 and 21.8 m2/g, respectively (Table

6.1). The shell particles with a smaller size on the core particle surface provide higher SBET

to RSN compared to SSN. The slight decrease in surface area after hydrophobic

modification can be attributed to the grafted layer of silica-octadecyl-groups.17

All

nanoparticles showed excellent stability in physiological solution of PBS after shaking for

three days (Figure 6.S5).

RNase A (13.7 kD) with hydrophobic nature and dimensions of 2.2 nm × 2.8 nm × 3.8 nm

was chosen as a model of therapeutic protein.23

The isoelectric point of RNase A is 9.3, so

that it can be directly immobilized onto negatively charged silica nanoparticles at pH 7.4 in

PBS.24

FTIR data (Figure 6.S2c&d) demonstrate the successful immobilization of RNase A

molecules on four different silica nanoparticles. As shown in Figure 6.2a, both RSN (16.4

µg/mg) and C18-SSN (9.4 µg/mg) have higher RNase A adsorption capacity compared to

SSN (7.3 µg/mg). The increment of RSN over SSN is much higher than that of C18-SSN

(225% vs 129%), suggesting surface roughness is more effective in the improvement of

RNase A loading than hydrophobic modification. By the combination of surface roughness

and hydrophobic modification, C18-RSN shows the highest adsorption capacity of RNase A

(20.7 µg/mg) among the four samples. A decrease in void height from 16.7±3.6 nm (Figure

6.S3c&d) to 14.8±5.3 nm (Figure 6.S3e&f) is detected after RNase A loading under AFM

characterization, suggesting that protein molecules are entrapped and accumulated into the

voids on C18-RSN.

Compared to SSN, the surface area of RSN increased to 204% (p<0.05), which is slightly

lower than the increase of RNase A adsorption (to 225%, see also Table 6.1). RNase A

adsorption capacity per unit surface area (RNase A adsorption density, mg/m2) is calculated

(Table 6.S3), dividing the adsorption capacity by SBET. It can be seen that the rough surface

also enhances protein adsorption density (to 109%), which can be explained by regional

protein aggregation in void spaces of rough surface.25

Therefore, the improvement of RNase

A adsorption caused by surface roughness is resulted from the increase of both surface area

and in-void protein adsorption.



123

Figure 6.2 (a) RNase A adsorption and (b) release profiles of SSN, RSN, C18-SSN and C18-RSN.

Data represent mean ± SD.

Hydrophobic modification improves protein adsorption by the dehydration around C18-

groups, resulting in the reduction of Gibbs energy and the enhancement of spontaneous

RNase A adsorption.24, 26

C18-RSN with 82% surface areas of RSN promotes stronger

RNase A adsorption to 126% (p<0.05). This enhancement is consistent with that of smooth

nanoparticles, where C18-SSN having 89% surface areas of SSN enables RNase A

adsorption to 129% (p<0.05). Therefore, the improvement of RNase A adsorption caused by

hydrophobic modification is limited compared to the role of surface roughness.

It should be noted that C18-RSN increases RNase A loading amount to 220% (p<0.05) and

284% (p<0.05), compared to C18-SSN and SSN, highly beyond their corresponding surface

area enlargement of 188% and 168%, respectively. This can be explained by the

significantly increased RNase A adsorption density of 117% and 170%, due to the densified

C18-groups on C18-RSN surface (2.42 µmol/m2), compared to C18-SSN (1.61 µmol/m

2,

Table 6.S3), respectively, implying stronger interaction with RNase A molecules.

Noticeably, although stronger negative charges on C18-SSN and SSN (Table 6.S1) are

beneficial for RNase A adsorption, C18-SSN and SSN show much lower loading capacity



124

compared to C18-RSN and RSN. It can be concluded that surface roughness and

hydrophobic modification play dominant roles in the enhancement of RNase A adsorption

(Table 6.1).

Both high loading capacity and sustained release are crucial for intracellular delivery of

therapeutic proteins,27

therefore we further investigated the roles of surface roughness and

C18-modification in the release profile of protein molecules. As shown in Figure 6.2b, SSN

has a burst release of 82% of the loaded RNase A within 1 hour (h) in PBS at 25 °C. In

comparison, C18-SSN and RSN shows a relatively slow release with a percentage of 52% &

64% at 1 h, respectively, suggesting that hydrophobic modification is more effective in

retarding protein release, because of strong hydrophobic interaction between RNase A and

C18-SSN (Table 6.1). Nevertheless, up to 90% of the loaded proteins release from both RSN

and C18-SSN at 6 h, and the protein release reaches to plateau (up to 95% of total loaded

RNase A) at 24 h. However, C18-RSN only exhibits a release percentage of 71% at 6 h and

89% at 24 h (Table 6.1). At 48-72 h, it reaches to the release plateau (of 94%). The sustained

release profile of C18-RSN could be explained by the combination of both surface

roughness and the hydrophobic surface with densified C18-groups compared to C18-SSN.

RNase A release profiles from four samples were also evaluated in acidic buffer (pH 4.5),

mimicking the cancer cell compartments (endosome/lysosome),14

and in DMEM at 37 °C

under static condition for 4 h, imitating cell uptake process in the following experiments. A

slower release can be seen in both acidic buffer (Figure 6.S6a, 36%, 34%, 22% and 18% for

SSN, RSN, C18-SSN and C18-RSN at 72 h, respectively) and DMEM medium (Figure

6.S6b, 50%, 27%, 20% and 10% for SSN, RSN, C18-SSN and C18-RSN, respectively),

compared to those values in PBS solution with pH 7.4 (Figure 6.2b). The most sustained

release of RNase A is observed in the sample of C18-RSN (Figure 6.S6a). When solution

pH value decreases from 7.4 to 4.5, RNase A tends to have more positive charges, enabling

improved electrostatic interaction with negatively charged silica nanoparticles and

subsequent retarded release. Static condition in DMEM medium (Figure 6.S6b) is also

beneficial for retarded release, compared with shaking process in PBS solution. These data

suggest C18-RSN is effective to deliver protein molecules into cytosols, avoiding premature

release in cancer cell compartments.

Efficient cellular uptake is another important property for successful protein delivery. We

studied the roles of surface roughness and C18-modification on cellular uptake performance

of the complexes of RNase A and silica nanoparticles in MCF-7 and SCC-25 cells,



125

measured by ICPOES. In both cell lines (Figure 6.3), RSN/ RNase A and C18-SSN/ RNase

A show a higher cellular uptake performance than SSN/ RNase A, suggesting that both

surface roughness and hydrophobic modification could enhance cellular uptake,

independently. Noticeably, in MCF-7 cells, RSN/RNase A (10.8 pg silica/cell) shows better

performance than C18-SSN/RNase A (8.2 pg silica/cell). However, this difference trend is

reversed and C18-SSN/ RNase A (9.0 pg silica/cell) is taken up more than RSN/ RNase A

(7.6 pg silica/cell) in SCC-25 cells. Therefore, the influence of surface roughness and

hydrophobic modification on cellular uptake performance is cell type-dependent. In

addition, C18-RSN/RNase A exhibits the best cellular uptake performance of 14.8 pg

silica/cell in MCF-7 and 10.4 pg silica/cell in SCC-25, which is almost 3 (p<0.05) and 2

(p<0.05) times of SSN/RNase A (5.5 and 5.6 pg silica/cell), respectively. These significant

improvements of C18-RSN in cellular uptake are also attributed to the contribution of both

surface roughness and hydrophobic modification.

Figure 6.3 Cellular uptake performance of RNase A loaded nanoparticles in (a) MCF-7 and

(b) SCC-25 cells, measured by ICPOES. Cells only were used as a control group. Data

represent mean ± SD.

The endocytic pathway is the major cell uptake mechanism of silica nanoparticles with the

sizes from several nanometers to hundreds of nanometers.28

After taken up by cells, the

internalized protein-nanoparticle complexes tend to be entrapped in endosome/lysosomes,

followed by protein degradation by enzymes.14, 29

Therefore, it is very important to facilitate

efficient endo/lysosomal escape (EE/LE) to ensure cytosolic delivery of the therapeutics.

The EE/LE performance of SSN, RSN, C18-SSN and C18-RSN was evaluated by TEM

(Figure 6.4), which show nanoparticle distributions on outer surface of cell membranes

(CM, white arrows), in cytoplasm (C), electron-lucent endosomes (E) or electron-dense

lysosomes (L) of MCF-7 cells after 24 h incubation with nanoparticles. Both SSN (Figure



126

6.4a) and RSN (Figure 6.4b) are generally localized in endosome/lysosomes (black arrow),

indicating inefficient EE/LE. On the contrary, the majority of C18-SSNs (Figure 6.4c) and

C18-RSNs (Figure 6.4c) are found distributed in cytoplasm (black dash arrow). The

disrupted membranes of endosome/lysosomes (Figure 6.4c &d, black arrowhead) indicate

successful release of the enclosed C18-SSNs and C18-RSNs into cytosols, with the help of

hydrophobic modification.16

Therefore, hydrophobic modification plays the dominant role in

EE/LE.

Figure 6.4 Typical TEM images of ultra-thin sections of MCF-7 cells incubated with (a)

SSN, (b) RSN, (c) C18-SSN and (d) C18-RSN for 24 h. White arrow indicates cell

membrane (CM), black arrow indicates nanoparticles entrapped in endosome (E) or

lysosome (L), black arrowhead shows locally disrupted membrane of endosomes, black dash

arrow indicates nanoparticles distributed in cytoplasm (C). "N" refers to nucleus. Scale bar:

100 nm.

The contribution of surface roughness, hydrophobic modification and their combination on

protein loading capacity, sustained release, cellular uptake and endosome escape

performance is summarized in Table 6.1. Both surface roughness and hydrophobic

modification can enhance RNase A adsorption compared to the smooth particle, while the

contribution from surface roughness is more important. The combination of surface

roughening and hydrophobic modification (C18-RSN) thus significantly increase the RNase

A loading capacity compared to SSN (20.7 vs 7.3 mg/g). In terms of protein release, the

hydrophobic modification is more effective compared to rough surface. It is clear only



127

hydrophobic modification can induce effective endo/lysosomal escape, independent of

smooth or rough silica surface. Moreover, different cell types have varied sensitivity to

surface roughness and hydrophobic modification for cellular uptake performance. MCF-7

cells internalized higher amount of the nanoparticles with a rough surface, while more

hydrophobically modified nanoparticles have been taken up into SCC-25 cells. However, it

is evident both functionalization increases the cellular uptake of silica nanoparticles

although their individual contribution is cell type-dependent. Collectively, silica

nanoparticles with both rough surface and hydrophobic modification are desired nano-

carriers for the cellular delivery of proteins.

Table 6.1 Summary of the contribution of surface roughness and hydrophobic modification

SBETa

(m2/g)

Adsb

(µg/mg)

Desc(%) CU (pg /cell)

d

eEE/

LE 1h 24h MCF-7 SCC-25

SSN 13.0 7.3 82 97 5.5±0.5 5.6±0.6 -

RSN 26.5 16.4 64 95 10.8±0.2 7.6±1.9 -

C18-SSN 11.6 9.4 52 93 8.2±0.7 9.0±0.2 +

C18-RSN 21.8 20.7 36 89 14.8±2.1 10.4±0.4 +

Note: a: surface area; b: adsorption capacity of RNase A; c: RNase A desorption percentage;

d: cellular uptake of silica amount per cell; e: qualitative endo/lysosomal escape (EE/LE)

performance, where "-" represents inefficient escape, "+" represents efficient escape.

It has been reported the delivery of RNase A into cytosols will degrade mRNA and tRNA

and inhibit protein synthesis, thus strongly influence cell functions and cause deleterious

effects on cell viability.4 Therefore, we evaluated the cytotoxicity of RNase A delivered by

different silica nanoparticles to MCF-7 and SCC-25 cells. C18-SSN and C18-RSN were

mixed with excessive RNase A to achieve saturated adsorption for intracellular delivery, and

SSN was used as a control group. The total protein concentration in each well is 2 µg/mL

and the nanoparticle concentration is 50 µg/mL, which is much lower than that in the

literature report (800 µg/mL).16

The toxicity of SSN, C18-SSN and C18-RSN is mild to

either MCF-7 or SCC-25 cells at a concentration of 50 µg/mL (Figure 6.S7). As shown in

Figure 6.5, free RNase A cannot significantly impact cell viability in both cell lines at all-

time points of 24, 48, 72 h, because naked protein cannot enter cells. As expected, C18-

SSN/ RNase A and C18-RSN/ RNase A exhibit evident reduction of cell survival rate in a

time-dependent manner. C18-RSN/ RNase A inhibits the viability of MCF-7 and SCC-25



128

cells from 76%/69% at 24 h to 44%/50% at 48 h and finally to 25%/24% at 72 h,

respectively. This effect is stronger than C18-SSN/ RNase A (from 85%/92% at 24 h to

67%/75% at 48 h and finally to 45%/53% at 72 h). In contrast, SSN/RNase A causes little

inhibition to cell viability in both MCF-7 (75%) and SCC-25 (79%) cells even after 72 h

incubation, because pure silica nanospheres (SSN and RSN) cannot achieve effective

endo/lysosomal escape30

as demonstrated in Figure 6.4.

Figure 6.5 Cell viability of (a) MCF-7 and (b) SCC-25 cells treated with RNase A at a dosage of 2

μ /mL after 24, 48, and 72 h incubation. Data represent mean ± SD.

6.4 Conclusion

In summary, we have systematically studied the contribution of surface roughness and

hydrophobic modification on silica nanoparticles for protein (RNase A) delivery in different

aspects of protein loading capacity, release behavior, cellular update and endosome escape

performance. Both surface roughness and hydrophobic modification demonstrate improved

RNase A adsorption, sustained release and cellular uptake performance. However, surface

roughness plays more important role in RNase A adsorption enhancement and hydrophobic

modification has stronger influence on sustained release. The contribution difference of two

structural parameters in enhanced cell uptake efficiency is cell-type dependent. Only



129

hydrophobic modification demonstrates contribution to endo/lysosomal escape. Therefore,

the combination of surface roughening and hydrophobic modification demonstrates

enhanced therapeutic RNase A delivery, leading to significant cell growth inhibition in

cancer cells of MCF-7 and SCC-25. Our work provides understanding of the importance of

surface roughness and modification in the design of promising nano-carriers with enhanced

delivery efficacy.

6.5 References

1 S. D. Putney and P. A. Burke, Nat. Biotechnol., 1998, 16, 153-157.

2 C. H. Lee, T. S. Lin and C. Y. Mou, Nano Today, 2009, 4, 165-179.

3 G. Storm, F. Koppenhagen, A. Heeremans, M. Vingerhoeds, M. C. Woodle and D. J. A.

Crommelin, J. Control. Release, 1995, 36, 19-24.

4 S. Martins, B. Sarmento, D. C. Ferreira and E. B. Souto, Int. J. Nanomed., 2007, 2, 595-607.

5 M. R. Islam, Y. F. Gao, X. Li and M. J. Serpe, J. Mater. Chem. B, 2014, 2, 2444-2451.

6 W. Y. Kim, J. T. Xiao and E. L. Chaikof, Langmuir, 2011, 27, 14329-14334.

7 H. K. Bayele, C. Ramaswamy, A. F. Wilderspin, K. S. Srai, I. Toth and A. T. Florence, J

Pharm Sci-Us, 2006, 95, 1227-1237.

8 S. Martin-Ortigosa, D. J. Peterson, J. S. Valenstein, V. S. Y. Lin, B. G. Trewyn, L. A. Lyznik

and K. Wang, Plant Physiol., 2014, 164, 537-547.

9 J. S. Lim, K. Lee, J. N. Choi, Y. K. Hwang, M. Y. Yun, H. J. Kim, Y. S. Won, S. J. Kim, H.

Kwon and S. Huh, Nanotechnology, 2012, 23, 085101.

10 Y. Z. Huang, F. Q. Yu, Y. S. Park, J. X. Wang, M. C. Shin, H. S. Chung and V. C. Yang,

Biomaterials, 2010, 31, 9086-9091.

11 C. Argyo, V. Weiss, C. Brauchle and T. Bein, Chem Mater, 2014, 26, 435-451.

12 I. I. Slowing, B. G. Trewyn and V. S. Y. Lin, J. Am. Chem. Soc., 2007, 129, 8845-8849.

13 K. E. Sapsford, W. R. Algar, L. Berti, K. B. Gemmill, B. J. Casey, E. Oh, M. H. Stewart

and I. L. Medintz, Chem Rev, 2013, 113, 1904-2074.

14 A. K. Varkouhi, M. Scholte, G. Storm and H. J. Haisma, J. Control. Release, 2011, 151,

220-228.

15 S. Frokjaer and D. E. Otzen, Nat. Rev. Drug. Discov, 2005, 4, 298-306.

16 S. S. Bale, S. J. Kwon, D. A. Shah, A. Banerjee, J. S. Dordick and R. S. Kane, Acs Nano,

2010, 4, 1493-1500.

17 J. Zhang, S. Karmakar, M. H. Yu, N. Mitter, J. Zou and C. Z. Yu, Small, 2014, 10, 5068-

5076.



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18 Y. T. Niu, M. H. Yu, S. B. Hartono, J. Yang, H. Y. Xu, H. W. Zhang, J. Zhang, J. Zou, A.

Dexter, W. Y. Gu and C. Z. Yu, Adv. Mater, 2013, 25, 6233-6237.

19 C. LoPresti, M. Massignani, C. Fernyhough, A. Blanazs, A. J. Ryan, J. Madsen, N. J.

Warren, S. P. Armes, A. L. Lewis, S. Chirasatitsin, A. J. Engler and G. Battaglia, Acs Nano,

2011, 5, 1775-1784.

20 W. Stöber, A. Fink and E. Bohn, J. Colloid Interface. Sci., 1968, 26, 62-69.

21 S. Asahina, S. Uno, M. Suga, S. M. Stevens, M. Klingstedt, Y. Okano, M. Kudo, F. Schüth,

M. W. Anderson, T. Adschiri and O. Terasaki, Micropor. Mesopor. Mat, 2011, 146, 11-17.

22 H. Ohshima, in Electrical Phenomena at Interfaces and Biointerfaces, John Wiley & Sons,

Inc., 2012, pp. 27-34.

23 A. Wlodawer and L. Sjolin, P. Natl. Acad. Sci-Biol, 1981, 78, 2853-2855.

24 W. Norde, Macromol Symp, 1996, 103, 5-18.

25 P. E. Scopelliti, A. Borgonovo, M. Indrieri, L. Giorgetti, G. Bongiorno, R. Carbone, A.

Podesta and P. Milani, Plos One, 2010, 5, e11862.

26 M. Holmberg and X. L. Hou, Langmuir, 2009, 25, 2081-2089.

27 J. P. Yang, F. Zhang, W. Li, D. Gu, D. K. Shen, J. W. Fan, W. X. Zhang and D. Y. Zhao,

Chem Commun, 2014, 50, 713-715.

28 Ling Hu, Zhengwei Mao, Yuying Zhang and C. Gao, J. Nanosci. Lett., 2011, 1, 1-16.

29 M. Dominska and D. M. Dykxhoorn, J. Cell Sci., 2010, 123, 1183-1189.

30 I. Slowing, B. G. Trewyn and V. S. Y. Lin, J. Am. Chem. Soc., 2006, 128, 14792-14793.



131


Figure 6.S1 TEM images (a) OH-core particle, (b) NH2-core particle and (c) shell particle. Scale

bar: 100 nm. (f) Particle size distribution curves measured by DLS.

Figure 6.S2 Fourier transform infrared (FTIR) spectra of (a) pure liquid OTMS, (b) bare

nanoparticles, (c) RNase A and (d) nanoparticles complexed with RNase A.



132

The nanoparticles with and without modification of octadecyl group was characterized by FTIR

technique. Figure S2a shows the FTIR spectrum of pure liquid OTMS, in which the characteristic

peaks at 809, 1085, 1190, 1465, 2856 and 2926 cm-1

n ttr ut d to ν(S -O), ν(C-O)+ ν(C-C),

ρ(CH3), δ(C-H) 1 and symmetric and anti-symmetric -CH2- stretching,2, 3 respectively. The spectra

of SSN (Figure S2b i), C18-SSN (Figure S2b ii), RSN (Figure S2b iii) and C18-RSN (Figure S2b

iv) show the same characteristic bands at 804 cm-1

and broad band peaked at 1050 cm-1

, which

indicate -Si-O-Si- bonding.4 In the spectra of hydrophobic modified silica nanoparticles (Figure S2b

ii & iv), besides the characteristic peaks of silica, two extra peaks at 2858 and 2929 cm-1

can be

observed, which are assigned to symmetric and anti-symmetric -CH2- stretching, respectively,

indicating the successful attachment of octadecyl-groups.5

Figure S2c exhibits the FTIR spectrum of RNase A. The characteristic amide I band centered at

1635 cm-1

is mainly attributed to C꞊O str t n v r t on and the amide II band centered at 1515

cm-1

can be assigned to in-plane N-H bending and C-N stretching.6, 7

After RNase A adsorption

onto silica nanoparticles, the two characteristic peaks of RNase A at 1635 and 1515 cm-1

can be

clearly observed (Figure S2d). SSN (Figure S2d i) and RSN (Figure S2d iii) also show

characteristic peaks of silica, while C18-SSN (Figure S2d ii) and C18-RSN (Figure S2d iv) show

additional characteristic peaks of silica and octadecyl-groups. These results suggest RNase A

molecules have been successfully loaded onto the four samples.



133

Figure 6.S3 (a, c, e) AFM images and (b, d, f) void height profiles of ( & ) RSN, ( & d) C18-

RSN nd ( & ) C18-RSN + RN s A, generated by drawing a typical cross-sectional line on the

top region, Scale bar: 100 nm. The average height values are measured and calculated by recording

20 data. Data represent mean ± SD.



134

Figure 6.S4 Particle size distribution curves of (a) SSN, (b) C18-SSN, (c) RSN and (d) C18-RSN.

Figure 6.S5 Silica dissolution of different nanoparticles, tested by (a) ICPOES and TEM images of

(b) SSN, (c) C18-SSN, (d) RSN and (e) C18-RSN, after shaking in PBS for 3 days. Scale bar: 100

nm.

SSN, RSN, C18-SSN and C18-RSN show good stability in PBS with silica dissolution percentages

less than 10% after 3 days (Figure S5a). Slight increases in the silica dissolution percentage are

observed in four samples as shaking time increases. After 3 days, RSN and C18-RSN exhibit

relatively higher silica dissolution percentages (7.6% and 4.1%), compared to SSN and C18-SSN

(6.4% and 3.1%), respectively. This can be explained by the larger surface area of rough

nanoparticles, which facilitates quicker interaction with medium and subsequent silica dissolution.



135

In addition, the hydrophobic layer has an obvious protection effect to reduce silica dissolution. The

stability of nanoparticles was further confirmed by TEM images, where the surface morphologies of

both smooth (Figure S5b&c) and rough nanoparticles (Figure S5d&e) were not significantly

affected after shaking in PBS for 3 days.

Figure 6.S6 RNase A release profiles of SSN, RSN, C18-SSN and C18-RSN at (a) acidic condition

(pH 4.5) and (b) in DMEM under static condition at 37 °C for 4 h. Data represent mean±SD.



136

Figure 6.S7 The evaluation of toxicity from pure nanoparticles (a,c,e) in MCF-7 cells and (b,d,f) in

SCC-25 cells, at 24 h, 48 h and 72 h, respectively. Data represent mean ± SD.



137

Table 6.S1 S z nd ζ pot nt l r t r z t ons o n nop rt l s.

Sample ID

Size

PDIc ζ-potential

TEMa DLS

b

Shell particle 20±2.4 34±0.6 0.07±0.01 -42±8.3

OH-core particle 232±9.6 281±10.4 0.13±0.03 -24±1.3

NH2-core particle 235±10.9 328±11.5 0.24±0.09 +38±0.9

SSN 240±9.3 324±10.3 0.30±0.07 -34±0.4

C18-SSN 240±17.5 277±7.0 0.03±0.03 -33±0.5

RSN 296±10.0 630±44.3 0.23±0.01 -22±0.7

C18-RSN 299±14.6 499±42.6 0.16±0.01 -22±0.6

aMean size±SD of shell/core particles by recording 50 data from TEM images.

bIntensity mean size

±SD of shell/core particles by DLS method. cPolydispersity index.

Table 6.S2 DLS size measurements in PBS.

Sample ID Sizea PDI

b

SSN 391±68.1 0.40±0.05

C18-SSN 599±111.3 0.31±0.01

RSN 512±158.2 0.43±0.01

C18-RSN 683±12.9 0.30±0.01

aIntensity mean size ±SD of shell/core particles by DLS method.

bPolydispersity index.



138

Table 6.S3 RNase A adsorption density and C18-modification characterization of nanoparticles

aRNase A adsorption density

bCarbon percentage tested by Elemental Analysis.

cOctadecyl-group (C18) -group density: C18-group amount per unit surface area. The molecular

weight of C18 (MWC18) is 236g/mol. SSN nd RSN w r us d s ontrol roup.

C 8 density

(1)

References

1 M. I. Tejedor-Tejedor, L. Paredes and M. A. Anderson, Chem. Mater, 1998, 10, 3410-3421.

2 H. Huang, Y. Ji, Z. Qiao, C. Zhao, J. He and H. Zhang, Microporous Mesoporous Mater, 2010,

2010, 7.

3 K. Kailasam and K. Muller, J. Chromatogr A, 2008, 1191, 125-135.

4 D. B. Mahadik, A. V. Rao, A. P. Rao, P. B. Wagh, S. V. Ingale and S. C. Gupta, J. Colloid Interf

Sci, 2011, 356, 298-302.

5 J. Zhang, S. Karmakar, M. H. Yu, N. Mitter, J. Zou and C. Z. Yu, Small, 2014, 10, 5068-5076.

S mpl ID

DRNase Aa

(m /m2)

C r on (%)

DC18

(µmol/m2)

SSN 0.56 0.011 --

C18-SSN 0.81 0.453 1.61

RSN 0.61 0.025 --

C18-RSN 0.95 1.274 2.42

Chapter 7 Conclusions and Outlook

139

Chapter 7

Conclusions and outlook

7.1 Conclusions

The understanding of structure–function relationship of natural particulates provides a useful guide

for the design of new nanocarriers. Currently, many research attempts have focused on the synthesis

of new drug delivery systems by mimicking the advantages of natural particulates, for example,

enveloped viruses, which have evolved sophisticated mechanisms that make use of or shield off

cellular signalling and transport pathways to traffic within host cells and deliver cargos into the

appropriate subcellular compartment.1 In this thesis, virus-mimicking silica nanoparticles with well-

controlled surface roughness have been synthesized. The understanding of the structure-function

relationship for the significantly enhanced intracellular cargo delivery, including genetic molecules

and therapeutic proteins, has been revealed. In addition, the mechanism for the synthesis of stable

core-shell structure has been investigated, providing guidelines for the design of effective non-viral

carriers. The following conclusions can be drawn based on the studies in this thesis.

1. A virus-mimicking silica nanoparticle (VMSN) for enhanced gene delivery has been developed.

It possesses a core-shell structure, where small shell particles (~13 nm) were studded onto large

core particles (~211 nm), forming a rough morphology. The increase in nanoscale surface

roughness can promote adsorption, while reducing the leakage of genetic molecules, including cy3-

oligoDNAs and siRNAs. Cellular uptake of VMSN was also improved, thus cargo delivery

performance was significantly enhanced. Importantly, the advantages from nanoscale surface

roughness were confirmed generalizable, regardless of surface functionality (amine-groups or PEI-

modification) and cell types (HeLa or KHOS cells). Moreover, using VMSN as the nanocarrier to

deliver PLK1-siRNA into KHOS cells, cell growth inhibition by knocking down PLK1 gene was

significantly enhanced, compared to smooth nanoparticles and a commercial transfection reagent

(OligofectamineTM

). (Relevant content: Chapter 4)

2. Following the intracellular delivery of genetic molecules, in Chapter 5, therapeutic protein

delivery performance using rough silica nanoparticles (RSNs) as the nanocarriers, with

systematically controlled surface morphologies, have been investigated. RSNs with a fixed core


140

particle of ~211 nm and different shell particles, from ~13 nm to ~98 nm, were fabricated by the

"neck-enhancing" approach, and the surface roughness was correlated to the core-to-shell size ratios

from 16.2:1 to 2.2:1. In comparison to the method detailed in Chapter 4 and other reported methods,

rough morphologies of different RSNs can be stably maintained by a bigger "neck" between shell

and core particles, which extended the synthesis limitation of core-to-shell ratio of larger than 5.6:1.

The increase of shell particle size from ~13 nm to ~98 nm, enlarged interspacing distance between

neighbouring shell particles from ~7 to ~38 nm, and protein loading capacity of RSNs was also

found interspacing distance-dependent. Proteins with a suitable molecule size were favourably

absorbed onto one of the RSNs. The optimal interspacing distance of RSNs for high protein loading

capacity was 7, 21 and 38 nm for cytochrome c (~3 nm), IgG-fragment (IgG-F, domain antibody,

~10 nm) and non-specific rabbit IgG antibody (IgG-A, ~20 nm), respectively. Moreover, the

hydrophobically modified RSNs (C18-RSNs) were confirmed effective nanocarriers. The C18-RSN

with an interspacing distance of ~38 nm facilitated the intracellular delivery of therapeutic anti-

pAkt antibody (an IgG type antibody), causing enhanced cell growth inhibition to MCF-7 cells,

compared to literature reports.

3. After understanding the influence of surface morphology on protein adsorption behaviour, we

further quantitatively investigated the individual and combined contribution of surface roughness

and surface modification (e.g., C18-modification) for the improvement of protein therapeutics. To

achieve this goal, four samples, smooth silica nanoparticle (SSN), rough silica nanoparticle (RSN),

C18-SSN and C18-RSN, have been fabricated, where the core size was ~232 nm and shell diameter

was ~20 nm, suitable for the entrapment of ribonuclease A (RNase A, ~3 nm). Both surface

roughening and hydrophobic modification enhance RNase A adsorption capacity, while the

contribution from surface roughness was more dominant. Hydrophobic modification had a stronger

effect to retard RNase A release. The contribution difference to enhance cellular uptake was cell

type-dependent. Importantly, only hydrophobic modification facilitated endo/lysosomal escape.

Collectively, C18-RSN showed the best performance in RNase A delivery, causing significant cell

viability reduction in both human breast cancer (MCF-7) and SCC-25 cell lines, compared to other

nanoparticles in this study. (Relevant content: Chapter 6)

In summary, rough silica nanoparticles bio-inspired by the nanoscale structure of enveloped viruses

are promising nanocarriers for intracellular delivery of either genetic molecules or therapeutic

proteins, regardless of surface chemistry and cell types. The surface roughness allows the

entrapment of biomolecules in the valley or void space between neighbouring shell particles,

improving adsorption capacity and retarding release r. In addition, cellular uptake performance has

also been increased. Taking these advantages, the delivery performance of therapeutic agents has


141

been significantly enhanced, compared to silica spheres with smooth surfaces. Our research

suggests a new parameter for the rational design and synthesis of efficient drug delivery carriers for

highly improved disease therapy.

7.2 Recommendations for future work

The following recommendations are made for the future work.

1. To extend the advantages of rough nanoparticles, porous structures should be included for

enhanced drug loading or co-delivery of therapeutic molecules.

2. To further imitate the features of enveloped viruses with specific infectivity, targeting moieties,

for example, F3 peptide2 or Tat peptide,3 are needed to be conjugated onto the rough surface for

targeted and enhanced delivery.

3. Apart from replacing solid shell or core particle by porous ones, the particles can pre-encapsulate

drug molecules inside for multi-functional applications. For example, the photosensitiser of silicon

phthalocyanine dichloride (SiPCCl2) can be encapsulated in solid silica nanoparticle of 20-50 nm,4

and they can be used as shell particles for combined chemotherapy and photodynamic therapy.

4. In addition to silica materials, other materials can be introduced into the system. Calcium

phosphate nanoparticles (CaP-NPs) are good candidates. For example, the siRNA encapsulated and

lipid-coated CaP-NPs of ~30 nm can be used as shell particles for multifunctional drug delivery.5

7.3 References

1 Glover, D. J. Artificial Viruses: Exploiting Viral Trafficking for Therapeutics. Infect. Disord.

Drug Targets, 2012, 12, (1), 68-80.

2 Christian, S.; Pilch, J.; Akerman, M. E.; Porkka, K.; Laakkonen, P.; Ruoslahti, E. Nucleolin

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bio-inspired virus-mimicking nanoparticles for efficient ...375409/s4256501_phd... · conference...

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