Mesoporous silica nanoparticles for biomedical application

Prasanna Lakshmi Abbaraju

B. Pharm, M. Pharm

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2017

Australian Institute for Bioengineering and Nanotechnology

i

Abstract

Ordered mesoporous silica materials disclosed over the past two decades have

been extensively used in many applications as nanocarriers for delivering various

therapeutic/ diagnostic agents, due to their ease of synthesis and surface

functionalization, tunable pore size, and pore structure, high surface area, pore

volume and controllable morphology. Currently, many research attempts have

focused on the synthesis of mesoporous silica nanoparticles (MSN). Significant

progress has been achieved to date. However, specific biomedical applications, for

example, in the design of tablet formulations, as safe adjuvants in vaccine delivery

and in combined cancer therapy should be developed further to explore the potential

of this family of interesting materials.

The aim of this project is to develop novel and facile approaches to prepare

monodispersed MSN with finely controllable pore size and morphology for 1) to

developing floating tablets, and 2) to gain insight into the role of particle morphology

on hemocompatibility and adjuvanticity, and 3) to study the efficiency of combined

therapy for cancer treatment. The main achievements obtained in this thesis are

listed below.

In the first part, novel floating tablets are designed using MSN for enhancing the drug

delivery performance of both hydrophobic and hydrophilic drugs compared to

conventional floating tablets. The drug released was tested from floating tablet,

where the water soluble drug loaded MSN containing floating tablet show a

sustained release than a conventional tablet. More importantly, dissolution

improvement for the hydrophobic drug was achieved, an issue that was not solved

with a conventional floating tablets.

In the second part, asymmetric MSN with controllable head-tail structures were

successfully synthesised. The tail length (~25-170 nm) and tail coverage on head

particles are adjustable with varying the reaction time and tetraethyl orthosilicate

(TEOS). Furthermore the head particle is tunable (solid silica to porous silica). The

synthesised particles were tested for hemocompatibility. Furthermore, we applied the

smooth solid silica spheres and head-tail MSN for uptake and activation efficiency in

dendritic cells (DCs) and macrophages. Compared to solid head particles or MSN

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with fully covered, dendritic large pores, asymmetrical HTMSN show surprisingly

higher hemocompatibility towards red blood cells. Further, HTMSN were analyzed

for immunoadjuvant activity. Interestingly, we found that asymmetrical HTMSN

exhibit a higher level of uptake and in vitro maturation of antigen presenting cells

compared to spherical head particles.

In the third part, multifunctional core-shell-structured dendritic mesoporous silica

nanoparticles with a fullerene doped silica core and a dendritic silica shell with large

pores have been successfully prepared. The designed dendritic silica spheres with

fullerene core (FD) have a particle size of ~110 nm and pore size of ~25 nm, which

can be easily modified with octadecyl group. The prepared FD with big pore size

after hydrophobic modification (FD-C18) shown good loading efficiency of big

antibody (anti-pAkt). The FD-C18 has high cell internalization compared to FD, which

was confirmed by ICPOES test and confocal and, the particle are less toxicity to

breast cancer cell line (MCF-7) up to 160 ug/mL. The FD-C18 showed significant cell

inhibition of ~ 85 % in combined therapy, while pure antibody shown only negligible

cytotoxicity in breast cancer cell line (MCF-7).

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

Peer-reviewed papers

1. Abbaraju PL, Meka AK, Jambhrunkar S, et al., Floating tablets from mesoporous

silica nanoparticles. Journal of Materials Chemistry B, 2014; 2, 8298-8302.

2. Meka AK, Abbaraju PL, et al., A vesicle supra-Assembly Approach to synthesize

Amine-Functionazed Hollow Dendritic Mesoporous Silica Nanoparticles for Protein

Delivery. Small, 2016, 12, 5169-5177.

3. Yang Y, Lu Y, Abbaraju PL, et al., Multi-shelled Dendritic Mesoporous

Organosilica Hollow Spheres: Roles of Composition and Architecture in Cancer

Immunotherapy. Angewandte Chemie, 2017 (DOI: 10.1002/anie.201701550R1),

accepted on 2017, 4, 3.

Conference proceedings

1. Abbaraju PL, Chengzhong Yu, et al., Floating drug delivery systems using

mesoporous silica nanoparticles.

Nanobio Australia 2014, 6-10 July 2014, Brisbane, Australia.

2. Abbaraju PL, Chengzhong Yu, et al., Dendritic mesoporous silica nanoparticles:

Combined therapeutic protein and fluorescence-image guided photosensitizer for

cancer treatment.

8th International Symposium on Nano and Supramolecular Chemistry (ISNSC-8), 13-

16 July 2016, Mercure Hotel, Australia (Poster presentation).

**Received best poster award

3. Abbaraju PL, Chengzhong Yu, et al., Asymmetric silica nanoparticles as a

vaccine platform: Influence of shape on Immunological responses in vitro and in

vivo.

Emerging Therapeutics Summit 2016, 23-25 November 2016, Melbourne, Australia.

**Received Travel Grant

v

Publications included in this thesis

Abbaraju PL, Meka AK, Jambhrunkar S, et al., Floating tablets from mesoporous

silica nanoparticles. Journal of Material Chemistry B 2014; 2, 8298-8302.-

incorporated as Chapter 4.

Contributor Statement of contribution

Author Abbaraju, PL (Candidate) Experimental design and performance (80%)

Analysis and interpretation of data (75%)

Drafting and writing (75%)

Author Meka, Anand Kumar Analysis and Interpretation of data (20%)

Author Jambhrunkar, Siddharth Analysis and Interpretation of data (5%)

Author Zhang, Jun Experimental performance (3%)

Author Xu, Chun Experimental performance (2%)

Author Popat, Amirali Drafting and Writing (5%)

Author Yu, Changzhong Experimental design and performance (5%)

Analysis and interpretation of data (10%)

Drafting and writing (15%)

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Contributions by others to the thesis

Prof. Chengzhong Yu contributed to the concept and design of the project, and

assisted with the editing of the written work in this thesis.

Dr. Meihua Yu contributed to guidance and interpretation of data in Chapters 6.

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

degree

None.

vii

Acknowledgements

Firstly, I would like to express my sincere gratitude to my advisor Prof. Chengzhong

Yu for his patience, motivation and continuous support during my PhD study and

related research. His guidance helped me in all the time of research and writing of

this thesis. I could not have imagined having better advisor and mentor for my PhD

study. Beside my advisor, I would thank Dr. Amirali Popat for his support in early

stage of PhD study, and Dr. Meihua Yu for her support in my final year of PhD study.

I would specially thank Dr. Anand Kumar for his timely help.

I thank my fellow labmates Dr. Surajit Karmakar, Dr. Siddharth Jambhrunkar, Dr.

Liang Zhou, Dr Jun Zhang, Yannan Yang, Dr. Hongwei Zhang, Dr. Chang Lei, Dr.

Chun Xu, Owen Noonan, Dr. Yusilawati Ahmad Nor, Xiaoran Sun, Hao Song,

Swasmi Purwajanti, Mohammad Kalantari, Manasi Mantri, Trisha Ghosh, Min Zhang

and Yue Wang for the stimulating discussions, and help in the lab. I would like to

thank centre executives Ms. Cheryl Berquist, Ms. Colette for their support in office.

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

Australian Microscopy and Microanalysis Research Facility at the Centre 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 scholarship from the University of Queensland,

PhD Scholarship Top Up from Australian Institute for Bioengineering and

Nanotechnology are greatly appreciated.

Finally, and most importantly, I express my deepest gratitude to my family members.

To my parents, Satyanarayana and Kameswari, I am forever indebted to you for your

support, care and unconditional love to me throughout my life. I thank my partner Dr.

Ashwini Kumar for his unconditional love and support. Many thanks to my brothers

Praveen, Nanda Kishore, who have always believed in me and taken good care of

my parents when I am away from home. I love them so much. Their unconditional

love and care are the best support for me as I face every challenge in my life. I could

not have accomplished my dissertation without the support of all these wonderful

people.

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Keywords

Porous silica nanoparticles, Head tail silica nanoparticles, drug delivery, antibody

and peptide delivery, uptake, Maturation, cell inhibition.

Australian and New Zealand Standard Research Classification (ANZSRC)

ANZSRC code: 111504, Pharmaceutical Sciences, 40 %

ANZSRC code: 100708, Nanomaterials, 40 %

ANZSRC code: 090302, Biomedical Engineering, 20 %

Fields of Research (FoR) Classification

FoR code: 1115 Pharmacology and Pharmaceutical Sciences, 50%

FoR code: 0903, Biomedical Engineering, 50 %

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Table of Contents Abstract .................................................................................................................................................... i

Declaration by author ..............................................................................................................................iii

Publications during candidature ............................................................................................................. iv

Conference proceedings ........................................................................................................................ iv

Publications included in this thesis ......................................................................................................... v

Contributions by others to the thesis ...................................................................................................... vi

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

Acknowledgements ................................................................................................................................vii

Keywords............................................................................................................................................... viii

Australian and New Zealand Standard Research Classification (ANZSRC) ........................................ viii

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

Table of Contents ................................................................................................................................... ix

List of Figures and Tables ......................................................................................................................xii

List of Abbreviations used in the thesis ................................................................................................. xx

Chapter 1 ............................................................................................................................................- 1 -

Introduction .......................................................................................................................................- 1 -

1.1 Significance of the work ............................................................................................................- 1 -

1.2 Research objective and scope ..................................................................................................- 2 -

1.3 Structure of the thesis ...............................................................................................................- 2 -

1.4 References ................................................................................................................................- 3 -

Chapter 2 ............................................................................................................................................- 6 -

Literature Review ..............................................................................................................................- 6 -

2.1 Nanoparticles for application in medicine .................................................................................- 6 -

2.2 Silica nanoparticles in solubility enhancement and controlled drug release .............................- 7 -

2.2.1 Enhancement of drug solubility using traditional method ..................................................- 7 -

2.2.2 Enhancement of drug solubility using various approaches ................................................- 7 -

2.2.3 MSNs as solubility enhancers ............................................................................................- 8 -

2.2.4 Nanomaterial for controlled drug release ........................................................................ - 10 -

2.2.5 Floating tablet preparations............................................................................................. - 11 -

2.3 Silica nanoparticles in cell imaging and photosensitiser carrier ............................................ - 12 -

2.3.1 Silica particles in imaging ................................................................................................ - 12 -

2.3.2 Silica particles in photodynamic therapy ......................................................................... - 14 -

2.4 PDT in Combination therapy for cancer treatment ................................................................ - 17 -

2.4.1 Anti-oxidant agents ......................................................................................................... - 18 -

2.4.2 Chemotherapeutic agents ............................................................................................... - 18 -

2.4.3 Protein therapeutics for cancer therapy .......................................................................... - 20 -

2.5 Hemocompatability of MSNs .................................................................................................. - 22 -

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2.6 Mesoporous silica nanoparticles in vaccine ........................................................................... - 24 -

2.7 Synthesis of mesoporous silica nanoparticles ....................................................................... - 27 -

2.7.1 Synthesis of small pore mesoporous silica nanoparticles .............................................. - 27 -

2.2.1 Synthesis of small pore mesoporous silica nanoparticles .............................................. - 28 -

2.7.2 Synthesis of large pore symmetric and asymmetric silica nanoparticles (SiNPs). ......... - 32 -

2.8 Conclusion ............................................................................................................................. - 39 -

2.9 References ............................................................................................................................. - 40 -

Chapter 3 ......................................................................................................................................... - 58 -

Methodology ................................................................................................................................... - 58 -

3.1 Material synthesis .................................................................................................................. - 58 -

3.1.1 Synthesis of monodispersed MCM-41 and preparation of floating tablets with drug loaded

MCM-41 .................................................................................................................................... - 58 -

3.1.2 Synthesis, functionalization and dye tagging of head-tail nanoparticles ........................ - 59 -

3.1.3 Synthesis of dendritic silica nanoparticle with fullerene core .......................................... - 60 -

3.2 Characterisation ..................................................................................................................... - 60 -

3.2.1 X-ray diffraction ............................................................................................................... - 60 -

3.2.2 Thermogravimetric analysis ............................................................................................ - 61 -

3.2.3 Transmission Electron Microscopy (TEM) ...................................................................... - 61 -

3.2.4 Scanning Electron Microscopy (SEM) ............................................................................ - 61 -

3.2.5 Nitrogen sorption ............................................................................................................. - 61 -

3.2.6 Attenuated Total Reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy ... - 62 -

3.2.7 Dynamic light scattering (DLS)........................................................................................ - 62 -

3.2.8 Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) ...................... - 62 -

3.3 Biological assays .................................................................................................................... - 63 -

3.3.1 MTT assay ...................................................................................................................... - 63 -

3.3.2 Confocal Laser Scanning Microscopy (CLSM) ............................................................... - 63 -

3.3.3 Flow cytometry ................................................................................................................ - 64 -

Chapter 4 ......................................................................................................................................... - 65 -

Floating tablets from mesoporous silica nanoparticles ............................................................ - 65 -

4.1 Introduction ............................................................................................................................ - 66 -

4.2 Results and discussion .......................................................................................................... - 67 -

4.3 Conclusion ............................................................................................................................. - 73 -

4.4 References ............................................................................................................................. - 73 -

Supporting information ................................................................................................................. - 77 -

Supplementary Figures and Tables ............................................................................................. - 81 -

Chapter 5 ......................................................................................................................................... - 93 -

Asymmetric silica nanoparticles with tunable head-tail structures enhances hemocompatibility

and maturation of immune cells ................................................................................................... - 93 -

5.1 Introduction ............................................................................................................................ - 94 -

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5.2 Results and discussion .......................................................................................................... - 94 -

5.3 Conclusion ........................................................................................................................... - 111 -

5.4 References ........................................................................................................................... - 111 -

Supporting information ............................................................................................................... - 118 -

Supplementary Figures and Tables ........................................................................................... - 124 -

Chapter 6 ....................................................................................................................................... - 150 -

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic

therapy and antibody delivery .................................................................................................... - 150 -

6.1 Introduction .......................................................................................................................... - 151 -

6.2 Results and discussion ........................................................................................................ - 152 -

6.3 Conclusion ........................................................................................................................... - 158 -

6.4 References ........................................................................................................................... - 159 -

Supporting information ............................................................................................................... - 163 -

Supporting Figures and Tables .................................................................................................. - 168 -

Chapter 7 ....................................................................................................................................... - 176 -

Conclusion and outlook .............................................................................................................. - 176 -

7.1 Conclusions .......................................................................................................................... - 176 -

7.2 Recommendations for future work ....................................................................................... - 178 -

7.2.1 Active targeting and fullerene solubility ......................................................................... - 178 -

7.2.2 Replacing head with various metal nanoparticles ......................................................... - 178 -

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List of Figures and Tables

Figure 2.1 A typical photodynamic reaction.

Figure 2.2 Delivery of silica nanoparticles based nanovaccines (20-200 nm particle

size) by parental (A) and mucosal (B) routes.

Figure 2.3 The scheme shows the leading nanocarriers for drug delivery and their

general stages of development. The top row shows the representative conventional

nanocarriers such ad liposomes, micelles, dendrimers, and polymers. The bottom

row shows novel inorganic nanocarriers such as carbon nanotubes, quantum dots,

iron oxide, gold, and mesoporous silica nanoparticles.

Figure 2.4 A) Schematic formation mechanism of oil in water system introduced by

Okuyama et al. and B) TEM image of the obtained dendritic MSNs.

Figure 2.5 A) SEM and (B) TEM images of KCC-1.

Figure 2.6 Schematic representation of influence of cyclohexane volume on silica

nanoparticles and respective TEM images.

Figure 2.7 TEM (a,b,c) and SEM (d,e,f) images of three generations of pore

structures.

Figure 2.8 TEM images at low magnification (A) and high magnification (B) and an

ET slice (C) of core cone particles.

Figure 2.9 SEM images at low magnification and high magnification (a, b) and TEM

(c) images and ET slice (d) of A-HDMSN.

Figure 2.10 Synthesis procedure for the dual-compartment Janus mesoporous silica

nanocomposites UCNP@SiO2@ mSiO2&PMO by anisotropic island nucleation and

growth method.

Figure 2.11 TEM images (A-C) of the morphology evolution of the eccentric

nanocomposites. TEM images of Janus UCNPs@SiO2 and PMO and the eccentric

hollow structured nanocomposites.

Scheme 4.1 Schematic representation of floating tablets made from drug loaded

MCM-41, polymer and sodium bicarbonate (A). After blending and compression (B),

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captopril (white coloured) and curcumin (yellow) floating tablets are obtained (C),

which can float in stomach >12 h (D).

Figure 4.1 Cross sectional SEM images of (a) floating tablet with MSNs and (b) after

12 h of dissoution study.

Figure 4.2 The morphology of floating tablet at different time points. (F1) Cur-

MCM-41(1.7nm), (F2) Physical mixture of curcumin and MCM-41, (F3) Pure

curcumin, (F4) Cur-MCM-41(2.1nm). All the formulations contain polymer and

sodium bicarbonate.

Figure 4.3 The release profile of curcumin from floating tablets. (F1) Cur-

MCM-41(1.7nm), (F2) Physical mixture of curcumin and MCM-41, (F3) Pure

curcumin and (F4) Cur-MCM-41(2.1nm). All the formulations contain polymer

and sodium bicarbonate.

Figure 4.4 The release profile of captopril from floating tablet. (F5) Cap- MCM-

41, (F6) Pure captopril. All the formulations contain polymer and sodium

bicarbonate.

Figure 4.S1 XRD patterns (A) of a) Calcined MCM-41 with 2.1 nm b) Calcined MCM-

41 subjected to VVD process with 1.7 nm, N2 adsorption-desorption isotherms (B) of

calcined MCM-41 and MCM-41 subjected to VVD cycle and BJH pore size

distribution plots (C) of calcined MCM-41 and MCM-41 subjected to VVD cycle.

Figure 4.S2 Transmission electron microscope (TEM) image (a) of MCM-41(1.7nm),

(b) MCM-41 (2.1nm); Field-emission scanning electron microscope (FE-SEM) image

(c) of MCM-41(1.7nm), (d) MCM-41 (2.1nm), (e) HPMC K100M, (f) Cross-section

image conventional floating subjected to 12 h of dissolution study.

Figure 4.S3 A) The chemical structure of HPMC polymer. B) The amount of MSNs

remained in floating dosage form after 12 h of dissolution study analysed by TGA.

Figure 4.S4 TGA of Curcumin, MCM-41, Cur-MCM-41(1.7nm), Cur-MCM-41(2.1nm)

Figure 4.S5 (A) XRD pattern of Curcumin, physical mixture of curcumin and MCM-

41, Cur- MCM-41 (1.7nm), Cur-MCM-41 (2.1nm) and MCM-41, and (B) Differential

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Scanning Calorimetry of Curcumin, Cur-MCM-41 (1.7nm), Cur-MCM-41 (2.1nm) and

MCM-41.

Figure 4.S6 (A) Nitrogen adsorption-desorption isotherms of Cur- MCM-41(1.7nm),

Cur-MCM-41(2.1nm) and (B) pore size distribution plot of Cur- MCM-41(1.7nm), Cur-

MCM-41(2.1nm).

Figure 4.S7 (A) Nitrogen adsorption-desorption isotherms of MCM-41, Cap- MCM-

41 and (B) pore size distribution plot of MCM-41, Cap-MCM-41.

Figure 4.S8 TGA analysis of a) Captopril, b) Cap-MCM-41.

Figure 4.S9 FTIR spectra of MCM-41, Captopril, Cap-MCM-41.

Figure 4.S10 A scheme showing the home-made Rosette rise apparatus

Scheme 5.1 Illustration of the synthesis of HTMSN and their advantages in

hemocompatibility and maturation of immune cells.

Figure 5.1 TEM and SEM images of HTMSN prepared at varied TEOS volume V.

(A, D, G) HTMSN0.41-12-60, (B, E, H) HTMSN1.25-12-60, (C, F, I) HTMSN3.75-12-60.

Figure 5.2 TEM images (A-E) of HTMSN1.25-t-60 collected at different reaction time

(t=2, 3, 6, 9 and 24 h, respectively). (F) The variation of tail length (black bar) and

head coverage (grey bar) as a function of t. Bars represent the mean ± SEM (n=20).

Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple

comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, ns (P

≥ 0.05) indicates no significant difference.

Figure 5.3 TEM image (A) and, ET slice (B) of HTDMSN1.25-60-12.

Scheme 5.2 Schematic illustration of (A) an oil-in-water droplet stabilized by Stöber

spheres and (B) the orientation of dendritic tail growth.

Figure 5.4 SEM images of the HTMSN1.25-9-60 emulsion at low (A) and high (B)

magnifications.

Figure 5.5 Hemolytic percentages of mouse RBCs upon incubation with different

nanoparticles at 240 µg/mL. D.I water and PBS was used as positive and negative

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control, respectively. Bars represent the mean ± SEM (n=3). All samples were

compared with HTMSN1.25-12-400.

Figure 5.6 Hemolytic percentages of mouse RBCs upon incubation with different

particles at various concentrations. Bars represent the mean ± SEM (n=3).

Figure 5.7 (A) Extracellular ROS generation of three nanoparticles and SEM images

of RBCs incubated at room temperature for 2 h with (B) HTMSN1.25-12-400, (C) Stöber

spheres and (D) HTMSN1.25-12-60. Scale bar is 10 µm. In (A), bars represent the mean

± SEM (n=3).

Figure 5.8 Flow cytometric analysis showing FITC positive (A) CD11c+ DCs and (B)

F4/80+ macrophages treated with FITC conjugated Stöber spheres HTMSN0.41-12-60,

HTDMSN1.25-12-60, MSN-CC and DMSN. Bars represent mean ± SEM (n=3).

Figure 5.9 Flow cytometry analysis of maturation marker CD40 (A, C) and CD 86 (B,

D) expression levels on CD11c+ DCs (A, B) and F4/80+ macrophages (C, D)

triggered by Stöber spheres, HTMSN0.41-12-60, HTDMSN1.25-12-60, MSN-CC and

DMSN. Bars represent mean ± SEM (n=3).

Figure 5.S1 TEM image of the dense stöber spheres as head with size ~ 200 nm.

Figure 5.S2 Partice size distribution curves of a) HTMSN0.41-12-60, b) HTMSN1.25-12-60,

c) HTMSN3.75-12-60 by DLS measurement.

Figure 5.S3 A TEM image showing the small mesopores in the porous layer fully

covering the head region in HTMSN0.41-12-60. Scale bar: 100 nm.

Figure 5.S4 Tail length and head coverage length of samples HTMSN0.41-12-60,

HTMSN1.25-12-60, and HTMSN3.75-12-60. Statistical analysis was performed using one-

way ANOVA with Dunnett’s multiple comparisons test. Data were considered

significant different at (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, ns

(P ≥ 0.05) indicates no significant difference. Bars represent the mean ± SEM (n=5).

Figure 5.S5 N2 sorption isotherms (A) and the BJH pore size distribution curves

derived from adsorption branch (B) of a) HTMSN0.41-12-60, b) HTMSN1.25-12-60 and, c)

HTMSN3.75-12-60.

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Figure 5.S6 TEM images of A) Stöber spheres; B) HTMSN1.25-2-60; C) HTMSN1.25-3-

60; D) HTMSN1.25-6-60 ; E) HTMSN1.25-9-60; F) HTMSN1.25-24-60.

Figure 5.S7 Particle size distribution of a) HTMSN1.25-0-60; b) HTMSN1.25-2-60; c)

HTMSN1.25-3-60; d) HTMSN1.25-6-60 ; e) HTMSN1.25-9-60; f) HTMSN1.25-24-60 ; g) MSN-CC;

h) HTMSN1.25-12-400.

Figure 5.S8 SEM images of A) Stöber spheres; B) HTMSN1.25-2-60; C) HTMSN1.25-3-

60; D) HTMSN1.25-6-60 ; E) HTMSN1.25-9-60; F) HTMSN1.25-24-60.

Figure 5.S9 N2 sorption isotherms (A) and BJH pore size distribution curves derived

from adsorption branch (B) of a) Stöber spheres; b) HTMSN1.25-2-60; c) HTMSN1.25-3-

60; d) HTMSN1.25-6-60 ; e) HTMSN1.25-9-60; f) HTMSN1.25-24-60.

Figure 5.S10 ET slices of A) HTMSN1.25-3-60 and, B) HTMSN1.25-12-60.

Figure 5.S11 TEM images of A) HTMSN1.25-12-0; B) HTMSN1.25-12-30; C) HTMSN1.25-12-

60; D) HTMSN1.25-12-100; E) HTMSN1.25-12-200; F) HTMSN1.25-12-400, SEM image of G)

HTMSN1.25-12-400.

Figure 5.S12 Digital image of emulsion (A1) after addition of chlorobenzene to

HTMSN1.25-9-60 emulsion, (A2) after addition of water to HTMSN1.25-9-60

emulsion, (B) optical image of emulsion.

Figure 5.S13 TEM image of MSN1.25-60-12 sample prepared at 50 rpm.

Figure 5.S14 TEM image of DMSN

Figure 5.S15 N2 sorption isotherms (A) and the BJH pore size distribution curves

derived from adsorption branch (B) of dendritic particles (a) and HTDMSN1.25-60-12 (b).

Figure 5.S16 Hemolytic percentages of mouse RBCs upon incubation with Stöber

spheres, HTMSN1.25-t-60 (t= 2, 3, 6, 9, 12), and HTMSN1.25-12-400 at different

concentrations. D.I water (+) and PBS (-) were used as controls. Bars represent the

mean ± SEM (n=3). Samples were compared with HTMSN1.25-12-400 under all tested

concentrations.

Figure 5.S17 Hemolysis results. Photographs of RBCs treated with HTMSN at

different concentrations. The released haemoglobin from the damaged cells in the

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supernatant can be seen from the photographs. (-) and (+) controls are the RBCs in

PBS and water, respectively.

Figure 5.S18 Hemolysis results. Photographs of RBCs treated with DMSN,

HTDMSN and MSN-CC at different concentrations. The released haemoglobin from

the damaged cells in the supernatant can be seen from the photographs. (-) and (+)

controls are the RBCs in PBS and water, respectively.

Figure 5.S19 SEM images of (A) free RBCs and (B, C) RBCs incubated with

HTMSN1.25-12-60.

Figure 5.S20 Flow cytometry analysis showing FITC positive RBCs incubation with

Stöber spheres, HTMSN1.25-12-60, and HTMSN1.25-12-400 for 2 h.

Figure 5.S21 Flow cytometer analysis of the uptake of A) Stober spheres, B)

HTMSN1.25-12-60 C) HTMSN0.41-12-60 (C) HTDMSN1.25-12-60 (D) MSN-CC, (E) DMSN

Dendritic cells (left panel) and macrophages (right panel). Q2 represents: % of

dendritic cells positive for silica nanoparticles, Q2-1 represents: % of macrophages

cells positive for silica nanoparticles.

Figure 5.S22 A) Flow cytometry analysis of maturation marker CD40 and CD86

expression levels on CD11c+ DCs and F4/80+ macrophages triggered by HTMSN0.41-

12-60 and Alum, (B) live cell numbers of CD11c+ DCs and F4/80+ macrophages

treated with Alum and HTMSN0.41-12-60.

Figure 5.S23 Flow cytometry analysis of maturation marker CD40 (A, C) and CD86

(B, D) expression levels on CD11c+ DCs (A, B) and F4/80+ macrophages (C, D)

triggered by OVA loaded Stöber spheres, HTMSN0.41-12-60, HTDMSN1.25-12-60, MSN-

CC and DMSN. Bars represent mean ± SEM (n=3).

Scheme 6.1 Schematic illustration of preparation procedure of FD for applications in

imaging and combined protein-/photodynamic therapy.

Figure 6.1 TEM and SEM images of (A,C) Fullerene dendritic (FD), B,D) Fullerene

dendritic-C18 (FD-C18).

xviii

Figure 6.2 Photobleaching of ABDA by 1O2 generated by (A) pure D20, (B) FD, (C)

FD-C18, (D) decay curves of pure D20, FD, FD-C18 at 376 nm as a function of UV

irradiation time.

Figure 6.3 Cell viability of MCF-7 cells treated with FD and FD-C18 under different

UV irradiation times.

Figure 6.4 Treatment of nanoparticles and anti-pAkt at various concentrations in the

presence or absence of UV irradiation for 5 min.

Figure 6.5 Western-blot analysis of Bcl-2 protein in MCF-7 cells after 5 min UV

irradiation along with samples. Blots presented are representative of typical results.

GADPH served as an internal standard.

Figure 6.S1 Particle size distribution curves of FD and FD-C18.

Figure 6.S2 FTIR of a) FD, b) FD-C18. The two extra peaks at 2858 and 2929 cm-1

can be attributed to asymmetric and anti-symmetric –CH2- stretching, respectively,

indicating the successful attachment of octadecyl groups.

Figure 6.S3 N2 adsorption-desorption isotherms of FD and FD-C18 (A),

corresponding pore size distribution curves calculated from adsorption branches

using the BJH method (B), and UV-Vis absorption spectra of the pure silica, FD, and

FD-C18 (C).

Figure 6.S4 Weight loss curves of SD, SD-C18, FD and, FD-C18 using TGA

analysis.

Figure 6.S5 Confocal microscopy images of MCF-7 cells (a) without adding

nanoparticles, with treatment of (b) FD, (C) FD-C18 with 160 µg/ml for 4 h. The

cytoskeleton and nuclei in cells were stained by Alexa Fluor® 488 phalloidin (green)

and DAPI (blue), respectively. Scale bar 50 µm.

Figure 6.S6 Cellular uptake performance of FD and FD-C18 measured by ICPOES.

Figure 6.S7 Cytotoxicity of FD, and FD-C18 as a function of particle concentration in

MCF-7 cells after 24 h.

Table 2.1 Poorly soluble drugs studied on mesoporous materials.

xix

Table 2.2 List of photosensitisers approved for human use.

Table 2.3 Representative drug delivery carriers on the market or in clinical trial.

Table 4.S1 Physicochemical properties of MCM41 (1.7nm), Cur- MCM-41-1.7nm,

MCM-41(2.1nm), Cur-MCM-41-2.1nm, MCM-41, Cap- MCM-41.

Table 4.S2 The compositions of floating tablets with bare MCM-41.

Table 4.S3 Composition of curcumin floating tablet.

Table 4.S4 Composition of captopril floating tablet.

Table 5.S1 Physical properties of samples HTMSN0.41-12-60, HTMSN1.25-12-60,

HTMSN3.75-12-60.

Table 5.S2 Physical properties of stöber, HTMSN1.25-2-60, HTMSN1.25-3-60, HTMSN1.25-

6-60, HTMSN1.25-9-60, HTMSN1.25-24-60.

Table 5.S3 Physical properties of HTMSN1.25-12-60 prepared at a stirring speed of 150

rpm.

Table 5.S4 Physical properties of DMNS and HTDMSN

Table 5.S5 Physical properties of HTMSN1.25-12-400 and MSN-CC

Table 6.S1 Structural properties of FD and FD-C18

Table 6.S2 Fullerene content in FD and FD-C18

xx

List of Abbreviations used in the thesis

MSN: mesoporous silica nanoparticles

MCM-41: mobile crystalline materials

HTMSN: head-tail mesoporous silica nanoparticles

GIT: gastro intestinal tract

VVD: vacuum assisted vapour deposition

RRA: Rosette rise apparatus

HPMC- hydroxyl propyl methyl cellulose

Cur- curcumin

Cap- captopril

TEOS: tetraethyl orthosilicate

CTAB: cetylmethylammonium bromide

CTAC: cetyltrimethylammonium chloride

FITC: fluorescein-5-isothiocyanate

RITC: rhodamine isothiocyanate

CTAC: cetyltrimethylammonium chloride

TEA: triethanolamine

OVA: antigen ovalbumin

APTES: (3-aminopropyl)triethoxysilane

XRD: X-ray Diffraction

TEM: transmission Electron Microscopy

SEM: scanning Electron Microscopy

ET: electron tomography

xxi

BJH: Barrett–Joyner–Halanda

BET: Brunauer–Emmett–Teller

ATR-FTIR: attenuated total reflectance -Fourier transform infrared spectroscopy

DLS: dynamic light scattering

TGA: thermogravimetric analysis

ICP-OES: Inductively Coupled Plasma-Optical Emission Spectroscopy

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

CLSM: confocal laser scanning microscopy

ROS: reactive oxygen species

PSs: photosensitizers

PDT: photodynamic therapy

ABDA: 9,10-anthracenediyl-bis(methylene) dimalonic acid

SSF: solid silica fullerene

F-DMSN: fullerene dendritic mesoporous silica nanoparticles

n-ODMS: n-octadecyltrimethoxysilane

FD: Fullerene dendritic

APCs: Antigen presenting cells

Chapter 1

Introduction

1.1 Significance of the work

From past three decades, traditional polymer based floating tablets have been used

for the delivery of hydrophilic and hydrophobic drugs. [1, 2] However, a conventional

formulation cannot help in enhancement of bioavailability of hydrophobic drugs,

which further hinders clinical applications. Thus, the quest to find an appropriate

method for the formulation of floating tablet with improved drug solubility and

controlled drug release still persists. Recently mesoporous silica nanoparticles

(MSNs) have been extensively studied as one class of the most promising

nanocarriers in biomedical applications, including cell imaging, diagnosis, vaccine,

gene/antibody/drug delivery.[3-6] Increasingly, porous nanomaterials with adjustable

pore sizes have been successfully applied in solubility enhancement[7-9] and

controlled drug release.[10] By understanding the effect of pore size on the drug

release and solubility enhancement a modified floating tablet in combination with

polymer and MSNs can be developed for effective drug release.

On the other hand, with parallel to the pore size, silica nanoparticle shape is also

playing an important role in bio-applications. Recent studies have shown the

advantage of silica nanoparticles shape in tumor cell targeting, [11] bimodal drug

delivery. [12, 13] However, the influence of asymmetric silica nanoparticle shape on

hemocompatibility and adjuvant effect remains unclear. Therefore, systemic

synthesis of series of asymmetric silica nanoparticles and its influence on

hemocompatibility and adjuvant effect should be explored for developing a safe and

potent nanocarrier.

There are tremendous studies in the synthesis and application of large pore silica

nanoparticles in cancer therapy.[14] In addition, combination of chemotherapy and

Chapter 1

Introduction

- 2 -

photodynamic therapy for cancer treatment has also been reported.[15] However,

chemotherapeutics drugs have limitations such as toxicity to normal cells, and

multidrug resistant. In this context, silica nanocarrier with therapeutic proteins and

photosensitizer delivery is required for effective treatment of cancer.

1.2 Research objective and scope

This research aims to develop novel floating tablets with improved solubility of

hydrophobic drug and controlled release for hydrophilic drugs. This thesis does not

only focus on the development of floating tablets, but also provides some guidelines

for the synthesis of symmetric and asymmetric large pore silica nanoparticles. The

synthesised materials were applied in the field of formulation, vaccines, and cancer

therapy. The sentence is not very clear.The objectives of this project are specified as

follows:

a) To fabricate floating tablets from mesoporous silica nanoparticles, and to confirm

the significance of MSN in drug release.

b) To synthesise silica nanoparticles with varied tail lengths and testing the

hemocompatibility and immunoadjuvant property.

c) To synthesise dendritic mesoporous silica nanoparticles for combined therapeutic

protein and photosensitizer for cancer treatment.

1.3 Structure of the thesis

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

The chapters in this thesis are presented in the following sequence:

Chapter 1 Introduction

This chapter includes a brief overview of the background of this research work

including aims and significance of the work.

Chapter 2 Literature review

This chapter presents a detailed review of the contributions made by mesoporous

silica nanoparticles in drug delivery systems. In addition, a detailed review on the

contribution of particle shape and pore size of MSN in biological application.

Chapter 1

Introduction

- 3 -

Chapter 3 Methodology

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

material synthetic methods for small and large pore MSN, and the techniques for

material characterizations and biological evaluations.

Chapter 4 Floating tablets from mesoporous silica nanoparticles

In this chapter we reported the preparation of floating tablets using MCM-41. Floating

tablets were prepared with a specific pore size of MSN which has a significant

solubility enhancement of hydrophobic drug and sustain release of hydrophilic drug.

This chapter was published in Journal of Material Chemistry B.

Chapter 5 Asymmetric silica nanoparticles with tunable head-tail structures

enhance hemocompatibility and maturation of immune cells

In this chapter we reported the synthesis of novel head-tail MSN using emulsion

system. We achieved to synthesize various lengths of tail over head by changing the

parameters like TEOS amount, reaction time and the head amount. The particles

were tested for hemocompatibility and uptake efficiency into APC and inducing the

maturation of APC cells.

Chapter 6 Dendritic mesoporous silica nanoparticles: Combined therapeutic

protein and fluorescence-image guided photosensitizer for cancer treatment

In this chapter we reported the synthesis of novel dendritic particles with big pore

size with a fullerene core and its advantage in cancer treatment. We have shown the

advantage of big pore size dendritic particles with fullerene core in combined protein

and photodynamic therapy in cancer treatment.

Chapter 7 Conclusions and recommendations

Conclusion and major contributions of this work are highlighted and

recommendations for future works are presented.

1.4 References

[1] Jimenez-Martinez, I., T. Quirino-Barreda, and L. Villafuerte-Robles, Sustained

delivery of captopril from floating matrix tablets. International Journal of

Pharmaceutics, 2008. 362(1-2): p. 37-43.

Chapter 1

Introduction

- 4 -

[2] Suwannateep, N., et al., Mucoadhesive curcumin nanospheres: Biological

activity, adhesion to stomach mucosa and release of curcumin into the circulation.

Journal of Controlled Release, 2011. 151(2): p. 176-182.

[3] Zhou, S., et al., Mesoporous silica-coated quantum dots functionalized with folic

acid for lung cancer cell imaging. Analytical Methods, 2015. 7(22): p. 9649-9654.

[4] Trewyn, B.G., et al., Mesoporous silica nanoparticle based controlled release,

drug delivery, and biosensor systems. Chemical Communications, 2007(31): p.

3236-3245.

[5] Xia, T.A., et al., Polyethyleneimine Coating Enhances the Cellular Uptake of

Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA

Constructs. Acs Nano, 2009. 3(10): p. 3273-3286.

[6] Mai, W.X. and H. Meng, Mesoporous silica nanoparticles: A multifunctional nano

therapeutic system. Integrative Biology, 2013. 5(1): p. 19-28.

[7] Zhang, Y., et al., Spherical mesoporous silica nanoparticles for loading and

release of the poorly water-soluble drug telmisartan. J Control Release, 2010.

145(3): p. 257-63.

[8] Jambhrunkar, S., et al., Stepwise pore size reduction of ordered nanoporous

silica materials at angstrom precision. J Am Chem Soc, 2013. 135(23): p. 8444-7.

[9] Zhang, Y., et al., Mesoporous silica nanoparticles for increasing the oral

bioavailability and permeation of poorly water soluble drugs. Mol Pharm, 2012. 9(3):

p. 505-13.

[10] Qu, F., et al., Effective controlled release of captopril by silylation of mesoporous

MCM-41. Chemphyschem, 2006. 7(2): p. 400-6.

[11] Gai, S.L., et al., Fibrous-structured magnetic and mesoporous Fe3O4/silica

microspheres: synthesis and intracellular doxorubicin delivery (vol 21, pg 16420,

2011). Journal of Materials Chemistry, 2011. 21(48): p. 19422-19422.

Chapter 1

Introduction

- 5 -

[12] Yang, Y., et al., Preparation of fluorescent mesoporous hollow silica-fullerene

nanoparticles via selective etching for combined chemotherapy and photodynamic

therapy. Nanoscale, 2015. 7(28): p. 11894-8.

[13] Wang, F., et al., Dual Surface-Functionalized Janus Nanocomposites of

Polystyrene/Fe3O4@SiO2 for Simultaneous Tumor Cell Targeting and Stimulus-

Induced Drug Release. Advanced Materials, 2013. 25(25): p. 3485-3489.

[14] Li, X.M., et al., Anisotropic Encapsulation-Induced Synthesis of Asymmetric

Single-Hole Mesoporous Nanocages. Journal of the American Chemical Society,

2015. 137(18): p. 5903-5906.

[15] Li, X.M., et al., Anisotropic Growth-Induced Synthesis of Dual-Compartment

Janus Mesoporous Silica Nanoparticles for Bimodal Triggered Drugs Delivery.

Journal of the American Chemical Society, 2014. 136(42): p. 15086-15092.

Chapter 2

Literature Review

This chapter reviews the exiting studies on silica based nanoparticles (SiNPs) for

pharmaceutical and biomedical applications. It begins with a brief introduction to

current nanomaterials that have been widely used in medicine and the applications

of silica nanoparticles in formulations, cancer therapy, photodynamic therapy and

vaccines.

2.1 Nanoparticles for application in medicine

Many of the conventional organic drug delivery systems (such as liposomes,

micelles and polymers) have reached the later stages towards clinical applications

(Figure 2.1) and few of them have even received U.S. Food and Drug Administration

(FDA) approval (Table 2.1). The conventional nanocarriers developed so far have

few major drawbacks hindering the clinical translation of laboratory-developed

nanocarriers: 1) inherent toxicity, 2) cost and scalable fabrication. A variety of

inorganic delivery systems with special morphologies and chemophysical properties

are emerging due to the limitations of conventional nanocarriers. Among various

inorganic materials, gold nanoparticles reached the clinical stage which are used in

drug delivery, hyperthermia based treatment and detection.[1] Next to gold, a wide

variety of silica nanoparticles such as solid, mesoporous, hollow silica nanoparticles,

dendritic, asymmetric have been developed for biomedical applications to carry

therapeutic drug/ gene/ protein/ peptide delivery, cell imaging agents. In addition,

silica based nanoparticles with tunable pore structures and particles size, surface

area demonstrated excellent biocompatibility and safety profile at the cellular and

animal level. These unique properties endow them with advantages to load and

deliver various therapeutic agents to the targeted site.[2-4] Recently, the first silica

based diagnostic nanoparticles called C-dots have received the FDA approval for

Chapter 2

Literature Review

- 7 -

stage I clinical trials to use in melanoma patients. This proves the important step

towards real application of silica nanoparticles.

2.2 Silica nanoparticles in solubility enhancement and controlled

drug release

2.2.1 Enhancement of drug solubility using traditional method

Bottom up and top down approaches are basically used for enhancement of drug

solubility. The bottom up approach involves a controlled precipitation process in

which the drug is dissolved in a solvent and this solvent solution is then added to a

non-solvent medium resulting in precipitation of drug into amorphous or crystalline

form. However, the main disadvantage of bottom up process is removal of the harsh

organic solvents used in the process. Hence, this approach is not the preferable

approach in enhancing the drug solubility.[5]

Top-down approach includes techniques such as milling and homogenization.[6, 7]

The most preferable method for milling is wet milling process, where the drug is

dispersed in surfactant solution and subjected to milling process. In homogenization

process drug is broken down to small size by exposing to high energy. [8]The

sample preparation is very similar to wet milling process which involves preparation

of dispersion medium containing drug and stabilizer.[8] The advantage of

homogenization process is reduction in time, microbiological issues and less

contamination then milling process.[9] The major disadvantage of top-down

approach is that it is time, energy consuming, and degradation due to mechanical

stress.[10] In addition, special equipment are required for generating high energy for

milling.[11] Due to the limited success achieved to date by nanotechnology

approach, there is a need for the development of general technique to further

increase the solubility of hydrophobic drugs.

2.2.2 Enhancement of drug solubility using various approaches

The hydrophobic drugs for example, low aqueous solubility hinder the ability of the

drugs to be administered through the intravenous and oral routes. The solubility of

drug is highly important for having high bioavailability while administering the drugs

through oral route. According to the Biopharmaceutical classification system (BCS),

Chapter 2

Literature Review

- 8 -

drugs are classified into four groups based on their solubility in gastrointestinal fluid

and permeability through gastro intestinal tract (GIT). The four classes are: class I-

high soluble and high permeable, Class II- low soluble and high permeable, class III-

high soluble and low permeable, and class IV-low soluble and low permeable. The

poor solubility in aqueous gastrointestinal fluid cause low oral bioavailability.

Especially for class II drugs, low bioavailability is due to its poor solubility and

dissolution in the aqueous solution. The solubility of drugs was enhanced by

converting the pure drug to its salt form by conjugating with chlorides, carbonate,

maleate, citrate etc. Many formulations with drug-salt conjugates have been

commercially available such as Diclofenac sodium, chlorphenaramine maleate,

etc.[12]

Common methods for solubility enhancement are complexation of drug molecules

within the cavity of cyclodextrin (CD). Various kinds of CD are currently available

such as α-CD, β-CD, γ-CD, Hydroxypropyl β-CD, etc. Till date several dosage forms

have been developed using CD to enhance the solubility of drugs such as Piroxicam,

Itraconozole, Hydrocortisone, etc.[13] Co-solvents have also been used for

enhancing the solubility of hydrophobic drugs. The most commonly used co-solvents

are propylene glycol, ethanol, glycerine, polyethylene glycol, and Dimethylsulfoxide

(DMSO), etc.[12] The conventional strategies applied for solubility enhancement face

the problems of precipitation, toxicity and altered pharmacological activity [14, 15]

and also this techniques cannot be applied for all hydrophobic drug molecules.

2.2.3 MSNs as solubility enhancers

Utilizing the inorganic mesoporous materials for enhancing the solubility of poorly

soluble drugs is a rapid growing area in pharmaceutical research. The pore size is

one of the most important factors of MSNs for its application in catalysis and drug

delivery. The pore size of MSN was controlled by changing the synthetic conditions

or by grafting process such as atomic layer deposition [16] and chemical vapour

deposition (CVD)[17] and, vacuum assisted vapour deposition (VVD)[18] process.

The pore size of MCM-41 was controlled by adjusting the pH of the synthesis mixture

from pH 11.5 to 10.0 resulting in pore size variation from 3.8 nm to 5.3 nm. This pH

variation during synthesis causes change in the electrostatic charge distribution

which affects the interaction of micelle head group resulting in pore expansion. The

Chapter 2

Literature Review

- 9 -

MCM-41 obtained by this process has different morphology based on the pH of the

synthesis mixture.[19] Incorporation of drug molecules into pores of MSN will leads

to drug size reduction at nanoscale range leading to high surface area and also

converting from crystalline to non-crystalline form.[20] These effect leads to

enhancement of drug solubility. The enhancement of drug solubility by MSN has

been studied in MCM-41, MCM-48 and SBA-15 materials. The list of hydrophobic

drugs used for solubility enhancement using inorganic nanoparticles is listed in Table

2.1.[21]

Table 2.1 Poorly soluble drugs studied on mesoporous materials. Reported from the

reference.[21]

Drug Solubility

(mg/ml)

BCS class Carrier Drug loading capacity

(wt. %)

Atazanavir 4.0–5.0 II NFM-1, AMS-6,

STA-11

28.2,31.5,32.8

Carvedilol 0.583 II SBA-16, MCM-41,TiO2,

Hydroxycarbonate,

apatite

25.0,25.0,22.8,22.5

Celecoxib 0.003 II Carbon 28.5

Ezetimibe 0.0085 II SBA-15 17.9

Fenofibrate 0.25 II SBA-15, MCM-41 20.0,21.5

Flurbiprofen 0.008 II FSM-16 30.0

Furosemide 0.25 IV TCPSi, TOPSi 41.3

Glibenclamide 0.004 II SBA-15 22.5

Griseofulvin 0.009 II TCPSi, TOPSi 16.5, 17.3

Ibuprofen 0.049 II MCM-41 2.5-37.1

Indomethacin 0.0009 II Syloid 244, MCM-41,

3DOM

25-50

Itraconazole 0.0096 II SBA-15 11.2

Lovastatin 0.0004 II Mesoporous carbon 25.6-36.3

Telmisartan 0.0035 II MCM-41, SBA-15 27.5

Carbamazepine 0.018 II SBA-15 22.5

Cinnarizine 0.75 II SBA-15 20.7

Chapter 2

Literature Review

- 10 -

2.2.4 Nanomaterial for controlled drug release

Since 2001, when MCM-41 was first proposed as drug-delivery system[22-24] silica-

based materials, such as SBA-15[25, 26] or MCM-48[27] and some metal-organic

frameworks have been discussed as drug carriers and controlled-release systems.

Recently several groups have reported drug storage and controlled delivery from

MSNs. The advantages of the relatively large pore diameter and pore channels

allowed easy accessibility as well as interesting release characteristics for the uptake

and release of the drugs.

In 2001, Maria-vellet and co-workers tested the ibuprofen release from MCM-41 with

larger and small pore size. Interestingly, 68% of drug released was achieved from

larger pore size and 55% from small pore size at 24 h.[22] The same group used a

series of periodic mesoporous organosilica with same structural symmetry (p6mm)

and similar morphology but different pore size (from 2.5 to 3.6 nm).[28] An increase

in drug release was observed with relatively increase in pore size. These advances

highlight the considerable potential of nanoporous materials with controlled pore size

for high controlled drug delivery performance.

In addition to the pore size effect, surface chemistry is another key factor affecting

the drug release.[29, 30] Mesoporous silica shows a high density of silanol groups,

which can be used to obtain functionalized surfaces by grafting organic silanes. In

the case of unmodified MSNs, it consists only silanol groups which simply form weak

hydrogen bonds with the drug. Hence, they are not strong enough to hold drug which

leads to burst release. In light of its potential application in the area of sustained drug

delivery, attempts have been made to introduce functional groups on the pore

channel walls. Balas et al., reported amino functionalized MCM-41 and SBA-15

mesoporous silica-based materials containing alendronate for bone repair or

regeneration.[31] After 24 h in an aqueous alendronate solution, the amino-modified

materials showed a drug loading almost 3 times higher than that of the unmodified

materials. The adsorption of alendronate molecules on the amino-modified materials

was 22 and 37% in SBA-15-NH2 and MCM-41-NH2, respectively. These alendronate

loads are significantly larger than those obtained for the modified materials: 8 % in

SBA-15 and 14 % in MCM-41. The work demonstrates that the amount of drug

Chapter 2

Literature Review

- 11 -

adsorption can be modulated through modification of the surface of the pore walls

through organic molecules. The control release of the drug can also be achieved by

modifying the surface with hydrophobic species. The effect of organic modification

on highly water soluble (captopril) drug release was studied. A well-defined,

controlled drug release was achieved by tailoring the surface properties of

mesoporous silica materials by regulating the degree of silylation.[32] Therefore, it

can be concluded that the surface characteristics of the mesoporous carrier system

play an important role in drug delivery profiles.

2.2.5 Floating tablet preparations

Control of placement of drug delivery system (DDS) in a specific region of the GI

tract offers numerous advantages, especially for drugs: 1) exhibiting an absorption

window in the GI tract, 2) drugs which are insoluble or undergoes degradation at

high pH, and 3) disease which need a local effect for effective treatment.[33, 34]

Over the last there decades, various approaches have been pursued to increase the

retention of an oral dosage form in the stomach, including floating systems, swelling

and expanding systems, bio adhesive systems,[35] modified-shape systems, high

density systems, and other delayed gastric emptying devices. Among these floating

delivery system is considered as an effective technique. The polymer and sodium

bicarbonate are the two key ingredients used in the design of low density gastro

retentive drug delivery system. Floating drug delivery systems were first described

by Davis in 1968.[36] Since then several approaches have been used to develop an

ideal floating delivery system. The design of floating technique has been used in

making captopril (highly water soluble drug) tablets by changing the metolose

(polymer) concentration.[37] The drug release decreased as the polymer

concentration increases. However, 100% of drug released at 8th h at the highest

polymer concentration. Mina Ibrahim and co-workers studied the drug release from

ciprofloaxacin floating tablet with polymers HPMC K15M and sodium alginate.[38]

The release profile of the drug was studied systematically by varying the ratios of two

polymers. Tablet containing HPMC K15M (21.42%, W/W), Na alginate (7.14%, w/w)

was showed the satisfactory results with 95% drug release at 12 h. Many

researchers formulated floating tablets for hydrophilic drugs, but very few have

addressed the issue of solubility for hydrophobic drugs to formulate them as a

floating tablet. In the polymer based designs controlling the release of highly water-

Chapter 2

Literature Review

- 12 -

soluble drug is limited. Moreover, drug and polymer are generally physically mixed

which does not aid in enhancing solubility of inherently insoluble drugs.

2.3 Silica nanoparticles in cell imaging and photosensitiser carrier

Molecular imaging is the visualization, characterization, and measurement of

biological processes at the molecular and cellular levels in humans and other living

systems, which is very important to gain better understanding on mystery of life.[39]

The functionalized fluorescent nanoparticles based cell imaging and sensing

technique is of great interest because they are fast responsive, highly stable, in situ

and in real time.[40]

2.3.1 Silica particles in imaging

There are two principal approaches to synthesize dye-doped SiNPs: the Stöber

method and reverse microemulsion method. The Stöber method is simple and

efficient approach to synthesise monodispersed solid SiNPs with a broad range of

diameters from 50 nm to 2µm. Van Blaaderen and coworkers for the first time

synthesised stable dispersions of monodispersed colloidal silica spheres containing

dye or fluorophores according to general procedures and dispersed in polar and

apolar solvents.[41] Wiesner and co-workers at Cornell University further extended

this approach to develop water-soluble, non-toxic fluorescent core-shell silica

nanoparticles (C dots) in a size range of 20-30 nm.[42] The C dots are biological

probes shown enhanced dye quantum efficiencies, brightness and bio stability and

reduced energy transfer effects, because of the restricted rotational mobility of

organic dyes entrapped in the core of the C dots and the protection from molecular

quenchers and solvent effects.[43] Bawendi and Frangioni group have studied the

size effect on effective clearance, and they found that particle diameter between 3-

7nm will have balanced reasonable circulation time and efficient clearance.[44] In

2009, the authors developed poly(ethylene glycol) (PEG) modified C-dots (~7nm)

which showed efficient renal clearance in mice compared with unmodified C

dots.[45] Moreover, the C dots modified with the cyclic arginineglycine-aspartic acid

(cRGD) peptide and radiolabeled with 124I showed its selective tumor targeting and

real-time multimodal imaging in both small and large animal models.[46] The C dots

received FDA investigational new drug approval for a first-in-human clinical trial in

2011. For the trial conducted with C dots, administered under the FDA’s IND

Chapter 2

Literature Review

- 13 -

guidelines showed that particles are safe for human use and leave no trace after

renal excretion. The researchers are organising to test the silica based inorganic

nanoparticle in melanoma patients in developing an approved diagnostic agent.

The second method to prepare dye-doped SiNPs is a water-in-oil reverse

microemulsion method, which involves water, surfactant and oil. The hydrolysis and

condensation of silica precursors and the formation of nanoparticles with dye trapped

inside occur at the interface of surfactant stabilized water droplet inside the oil

phase, to form fluorescent SiNPs. By changing the charge and packing properties of

the surfactant in the micelles, water to surfactant ratio or the amount of free water

and ammonium hydroxide concentration, the diameter of spherical monodispersed

SiNPs can be adjusted from 20 to 500 nm.[47, 48] Monodispersed SiNPs with the

diameters of 30-60 nm was achieved by microemulsion method. However, the

dispersity of SiNPs was not better in the Stöber method. However, the utilization of

large amount of surfactants required extensive washing steps, which limits the

potential of microemulsion method for industrial/mass production. At the beginning,

the reverse microemulsion method can only be used to incorporate inorganic dyes,

but the resultant inorganic-dye-doped SiNPs have limited fluorescence intensity,

because inorganic dyes have lower quantum yields compared with organic

fluorophores.[49-51] Organic dye doped SiNPs are difficult to prepare using reverse

microemulsion method due to hydrophobic properties of the organic dyes compared

with the hydrophilic surface of the SiNPs. In 2004, Tan and co-workers reported a

modified microemulsion method to successfully incorporate the organic dye into

SiNPs.[52] Since then, various single-dye doped SiNPs and multiple-dye

incorporated SiNPs can be generated using this method.[53] Multiple dyes

synchronously encapsulated in SiNP will help in fluorescence resonance energy

transfer (FRET) mediated large stokes shifting fluorescence resonance probes.

Hybrid SiNPs doped with PbSe quantum dots (QDs) have also been reported for cell

imaging using the microemulsion method.[54] The toxicity of QDs was reduced by

single or multiple coating of silica shell, which further enhanced the water-solubility.

Huo et al., developed an hybrid fluorescent SiNPs with uniform particle size of ~ 10

nm by micelle templating approach, with significant improved stability.[55] In

addition, Wang et al., utilized similar approach to obtain an ultra-small, highly stable

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and sensitive dual nanosensor for imaging intracellular oxygen and pH in cytosol.[56]

Furthermore, the dual fluorescent dyes doped SiNPs was developed by Wolfbeis et

al., using same approach. The nanosensors are pH-sensitive which have capacity of

sensing pH value and oxygen in cellular cytosol.[57] Furthermore, hollow

mesoporous silica nanoparticles (HMSNs) with a large cavity inside and mesopores

in the silica walls have also been reported for optical imaging, positron emission

tomography (PET), magnetic resonance imaging [58] and ultrasound imaging in both

in vitro and in vivo models.[59]

2.3.2 Silica particles in photodynamic therapy

Photodynamic therapy is an alternative tumor ablative oncological intervention.

Essentially, it involves the administration of a photosensitizer [58] followed by local

illumination of the tumor with light of a specific wavelength to activate the PS. The

excited PS then transfer its energy to molecular oxygen, thus generating cytotoxic

reactive oxygen species (ROS), such as singlet oxygen (1O2) that can oxidize key

cellular macromoleculer leading to tumour cell ablation (Figure 2.1).[60]

Figure 2.1 A typical photodynamic reaction. Reproduces from reference.[60]

For application in photodynamic therapy Stöber silica nanoparticles and mesoporous

silica nanoparticles are frequently used. These nanoparticles are often used as a

vector for PDT due to its flexible synthesis procedure, chemical inertness,

transparency of the matrix to light absorption and porosity. The effectiveness of PDT

depends on the production of reactive oxygen species (1O2) within the cell. The

efficacy of the photodynamic treatment strongly depends on the type, concentration

and intracellular localization of the photosensitizer. Some of the most common

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photosensitizers that have been approved for use in humans are indicated in Table

2.2.

Table 2.2 List of photosensitisers approved for human use.

Photosensitizer Type of disease Country

(5-ALA) Actinic Keratosis U.S., EU

5-aminolevulinate Basal cell carcinoma

Photofrin Barrett‘s displasia U.S., Canada, EU, UK

Photofrin Cervical cancer Japan

Photofrin Endobronchialcancer Canada, Most EU Countries, Japan, U.S.

Photofrin Esophageal cancer Canada, Most EU Countries, Japan, U.S.

Photofrin Gastric cancer Japan

Photofrin Papillary bladder cancer Canada

Foscan Head and neck cancer EU, Norway, Iceland

Verteporfin Age-related Macular

Degeneration

Canada, Most EU Countries, Japan, U.S.

In 2003, Kopelman first used amino-propyltriethoxysilane reagent for the initiating the

hydrolysis polycondesation of tetramethylorthosilicate for the high efficient

encapsulation of meso meta-tetral(hydroxyphenyl)chlorine (m-THPC) into silica

nanoparticles. To characterise the 1O2 formation, anthracene-9,10-dipropionic acid

(ADPA) was used. The same group published the encapsulation of methylene blue

to vectorize it and protect it from degradation. In 2003, Prasad group prepared

ORMOSIL nanoparticles for the entrapment of 2-devinyl-2-(1-hexyloxyethyl)-

pyropheophorbide (HPPH), a photosensitizer which is in phase I/II clinical trials for

treating esophageal cancer.[61] The entrapped PS was found to generate 1O2

efficiently upon irradiation. In a subsequent study the same group covalently

conjugated PS iodobenzylpyrophenophorbide to ORMOSIL nanoparticles, to avoid

premature release. Another type of PS phthalocyanine 4 Pc4, PpIX [62] and

mTHPC[63] was also encapsulated in ORMOSIL nanoparticles by method

developed by Prasad group, which has improved solubility, stability and delivery into

A-375 cells compared to free drug.[64]

Other type of silica nanoparticles have been used for the encapsulation of PS. Wei

and collaborators[65] prepared hollow SiNP by careful hydrolysis of N-(β-

aminoethyl)-α-aminopropyl triethoxysilane by ammonia for successful encapsulation

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of hypocrelline A into hollow SiNP. These particles were found to have good light

and thermal stability. The same collaborator[66] reported the preparation of

hypocrelline A nanoparticles by reprecipitation followed by encapsulation using N-(β

aminoethyl)-α-aminopropyl triethoxysilane to form 110 nm silica nanovehicles. These

particles have also shown superior light stability and singlet oxygen generation ability

than bare PS. Qian et al. reported the synthesis of ultra small (25 nm) MSN with high

dispersity for encapsulation of PpIX.[67] These nanoparticles induced high cell

death via necrotic pathway (HeLa cell line) upon photoexcitation with 532 nm light for

8 min.

Encapsulation of PS into MSN may leads to leaching from the nanocarrier, leading to

reduced efficiency. Zhang et al., developed multifunctional core shell structured

nanoparticles composed of FITC doped silica core and PS, hematoporphyrin

covalently linked to MSN shell. [68] The particles exhibited excellent photo-oxidation

efficiency and cell imaging ability. Teng et al., synthesised folate conjugated

phospholipid-capped PpIX-loaded and FITC-sensitized MSN for targeted intracellular

delivery of the nanocarriers.[69] The developed nanocarrier has high cell uptake and

efficiency in decreasing cell toxicity than free PpIX. The developed nanocarrier

decreased 65 % of tumour volume in nude mice bearing melanoma tumours after

irradiating the carrier with laser at 630 nm. Another dual delivery using MSN

composite was fabricated by zhao et al., for the combined delivery of carboxy

aluminium phthalocyanine (AlC4Pc) for photodynamic therapy (PDT) and small Pb

nanosheets for photothermal therapy (PTT).[70] While the PS was covalently

conjugated to MSN, the Pb was coated on the surface of MSN via electrostatic

interaction. The cell viability of HeLa cells was dropped to 35 % upon treatment with

functional nanocomposites, which was significantly higher than individual treatment.

Yang et al., precisely synthesised various morphologies of MSN by changing the

amount of organic PS (Ce6) molecules covalently doped in the silica matrix.[71] The

shape of MSN was transformed from spherical to rod shape with increase in Ce6

amount. A chemotherapeutic drug doxorubicin was physically adsorbed into the

mesopores. Among different shape particles, the Ce6 loading efficiency and uptake

into cancer cell was high in rod shape particle with an aspect ratio of 4.6. Moreover,

the synergistic effect of PS/DOX was found to be much higher than single therapy.

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Some nanoscale materials act as a PS due to its ability to generate ROS upon

exposed to lazer light because of its optical absorption properties. Fullerenes are

one class of PS discovered in 1985, with spherical shape and composed of 60 (C60)

or 70 (C70) carbon atoms (Figure). The diameter of fullerene is very small, thus

making it’s less likely to be taken up by the macrophages cells. Fullerenes absorb

light in the ultraviolet [72] or blue region of electromagnetic spectrum to form long-

lived triplet state and generate ROS upon illumination. Fullerenes are photostable

and highly resistant to photo bleaching[73] and undergo both Type I and Type II

reactions in producing both free radicals and 1O2. The major disadvantage of

fullerene is its poor biodegradability, solubility. This short comes have been solved

by PEG modifications, encapsulation in liposomes,[74] micelles[75] or chitosan.[76]

Liu and others developed theranostic hybrid system in which

diethylenetriaminepentaacetic acid (DTPA) was attached to the terminal group of

PEGylated C60 (C60-PEG-DTPA).[77] This complex was conjugated to gadolinium to

form C60-PEG-DTPA-Gd. Upon light irradiation, following intravenous injection of

complex to tumor bearing mice, there was a drastic reduction in tumour volume due

to the PDT effect. In another study, malonic acid C60 derivatives (DMA-C60) modified

with DSPE-PEG2000-maleimide was conjugated to Asn-Gly-Arg peptide, to form a

novel tumor-targeting drug delivery system.[78] The presence of targeted peptide

shown enhanced cell uptake and strong cell inhibition on MCF-7 cell line.

2.4 PDT in Combination therapy for cancer treatment

Combination therapy is a common practice in many medical disciplines. Previously

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

hydrophobic anticancer drug of doxorubicin,[79] hydrophobic drug of tamoxifen[80]

and curcumin.[81, 82] 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 much attention as new therapeutics due

to its advantages.

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2.4.1 Anti-oxidant agents

Buettner and co-workers demonstrated that in the presence of iron, ascorbate

combined with photofrin/ PDT enhanced the production of radicals and decreased

cell survival of various cancer cell lines.[83] Other authors studied the effect of the

combination with benzoporphyrin/PDT in HL60 cells. They concluded that the

addition of ascorbate to cells, followed by photosensitization would strongly enhance

the toxicity of cells.[84] Melnikova et al., demonstrated a remarkable reduction of

tumor after injection of alpha-tocopherol analogue in combination with

mTHPC/PDT.[85]

2.4.2 Chemotherapeutic agents

Chemotherapeutic agents are divided into two categories according to their direct

and indirect effect on DNA. Drugs which directly act on DNA are composed of

alkylating agents, antitumor antibiotics and inhibitors of topoisomerases.

Cisplatin, oxaliplatin and carboplatin are commonly used drugs to treat different

cancers. However, their good clinical efficacy is hindered by severe adverse effects.

Lottner et al.,[86] developed a new approach for the combination of cisplatin and

PDT. The authors have synthesized different hematoporphyrin based platinum

derivatives bearing phototoxic ligands for photodynamic effect. The authors

evaluated the cytotoxicity and phototoxicity of some of these derivatives against

bladder cancer and normal urothelial cells. Carboplatin a less nephrotoxic analogue

of cisplatin, has been employed in combination of 9-hydroxypheophorbide alpha (9-

HPbD)/PDT to treat head and neck cancer cell lines in vitro.[87]

Among the antitumor antibiotics, doxorubicin is commonly used in the treatment of a

wide range of cancer such as haematological malignancies, carcinomas and soft

tissue sarcomas. Casas et al.,[88] evaluated the interaction between 5-ALA/PDT and

doxorubicin in mice bearing transplantable mammary adenocarcinomas. Treatments

with two cytotoxic drugs in common clinical use did not cause any reduction of the

murine tumors tested. Tumor explants of doxorubicin-treated mice were first

subjected to 5-ALA/PDT in vitro and then re-implanted into test animals that showed

strong inhibition of tumour growth in combined treatment. The authors assigned the

observed enhancement of PDT to the weakening of cellular defence mechanisms by

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the pre-treatment involving free radical generation by doxorubicin. Canti et al.,[89]

investigated the combination of PDT, with disulfonated aluminium phthalocyanine

(AlS2Pc) and lazer light, and doxorubicin on mice bearing murine leukemia and

lymphoma. Low chemotherapy doses were ineffective, but the combination of

doxorubicin and AlS2Pc/PDT had improved clinical implications.

Shiah et al., [90] studied the antitumor activity of (N-(2-

hydroxypropyl)methacrylamide (HPMA) copolymer and Adriamycin conjugates in

combination therapy. Combination therapy and PDT of P-ADR and P-Mce6 exhibited

high therapeutic efficacy against human ovarian OVCAR-3 carcinoma xenografts.

The cytotoxic and antitumor effects of doxorubicin in combination with mTHPC)/PDT

have also been verified both in vitro (murine hepatoma cells) and in vivo (murine

liver)[91]. The anticancer efficacy of doxorubicin in combination with methylene

blue/PDT has been investigated in a drug-resistant mouse tumor model [92].

Nanoparticle-mediated combination treatment resulted in enhanced tumor

accumulation of both doxorubicin and methylene blue, significant inhibition of tumor

cell proliferation, increased induction of apoptosis and improved animal survival.

Jianquan et al., [93] developed one strategy delivery system for targeting anticancer

prodrug with both chemo and photodynamic therapeutic actions. The prodrug can

enter folate receptor positive cancer cells and kill the cells via intracellular release of

active drug form. The combined effect of chemotherapy and PDT increased the

therapeutic efficacy of the DOX-fullerene combined prodrug. Guangbao et al., [71]

synthesised mesoporous silica nanorods and spheres, which was intrinsically doped

with photosensitizer, chlorine e6(Ce6) and loaded with DOX. The significance of

combined therapy was successfully realized in both in vitro cellular experiments and

in vivo animal studies. Compared with combination therapy using free drugs, the

nanoparticle formulation had longer retention times in the tumor and thus offered

improved therapeutic outcome. Yannan et al., [94] prepared well dispersed hollow

silica fullerene-DOX nanoparticles with the particle size of ~50 nm. Hollow silica

nanoparticles has shown high drug loading capacity and improved photodynamic

activity compared with solid silica nanoparticles. Moreover, the combined of chemo-

photodynamic therapy demonstrated excellent performance in cancer cells leading to

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high cell apoptosis. Multifunctional hollow mesoporous silica nanocages for cancer

cell detection and the combined chemotherapy and photodynamic therapy was

studied.[95] In vitro cell assays demonstrated that the nanocages could be used as

probes for fluorescence imaging and biocompatible carrier for loading and delivery of

chemotherapeutic drugs DOX and photosensitizer hematoporphyrin.

2.4.3 Protein therapeutics for cancer therapy

Multidrug resistance is the most important impediment for the successful use of

chemotherapeutic drugs. The drawback of multidrug resistance has led to the

development of safe and effective therapeutic system. Among several, protein

therapy has been significantly improved, especially in the last decade. In this part,

the classification of protein therapeutics for cancer therapy will be summarized.

2.4.3.1 Enzymes

The use of enzyme proteins as therapeutics is very promising because of their high

targeted binding affinity and specificity. An enzyme catalytic activity allows for rapid

conversion of multiple target molecules into product. These unique properties make

enzymes promising therapeutic agents [96] and use as a therapeutic agent since the

beginning of twenty century.[97]

Intracellular delivery of proteins with enzymatic activities can help to catalysing a

chemical reaction in cancer cells. One of the examples is ribonuclease A (RNase A),

which is a mature enzyme, as secreted by exocrine cells in the bovine pancreas.[98]

After intracellular delivery of RNase A, it initiates the degradation of mRNA and tRNA

and stops protein synthesis, thus strongly influence cell functions and cause

deleterious effects on cell viability.[99] In addition, type-1 ribosome-inactivating

protein, saporin-S6 (also known as saporin) has also have similar properties of

RNase A. 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. The high

enzymatic activity, stability and resistance to conjugation to blood protease, makes

saporin-S6 a useful tool for cancer treatment.[100] A variety of nanocarriers have

been studied for the delivery of enzymatic protein for cancer therapy. For instance,

hydrophobic modified, negatively charged silica nanoparticles (SiNP) were used to

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immobilize RNase A on the surface and deliver it into human breast cancer cells

(MCF-7) for cell growth inhibition. A dose dependent cytotoxicity was observed for

the SiNP-RNase upon incubation with MCF-7.[101]

2.4.3.2 Targeting proteins

Proteins or peptides can be conjugated to nanoparticles with the help of electrostatic

forces, hydrogen bonds and disulphide bonds, to form 3-dimensional (3-D) rough

and flawy structures, which can help in targeted interaction of proteins with other

molecules.[102] The targeting proteins will specifically interact with molecules inside

or on the surface of cancer cells to block the cell functions. 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 (antipAkt) into cytosols of cancer cells

has been reported.[101] In addition, the delivery of functionalised mesoporous silica

entrapped anti-CTLA4 IgG mAb induced much greater extent of therapeutic

response than the same given alone.[103]. Alternatively, cytotoxic drug conjugated

peptides can also be used for cancer treatment. One example is application of

mRNA for targeting SSTR3 and SSSTR5 receptors (appears to express in some

cancers). However, this lead to some inconclusive and controversial results. AN-238

a targeted cytotoxic SST analogue, which consist of DOX, 2-pyrrolino-DOX was

developed for effective cell growth inhibition in multiple cancer cell lines.[104]

Yuting et al., [105] reported a successful delivery of the anti-pAKt antibody

conjugated to hydrophobic modified rough silica nanoparticles to MCF-7 cell lines.

The rough silica nanoparticles with high loading of anti-pAKt antibody demonstrated

high efficiency of intracellular delivery of therapeutic protein in cancer cells and

caused significant cell growth inhibition. Xin et al., [106] synthesised multifunctional

upconversion nanoplaform with combined PDT and gene therapy for effective cancer

killing. Combining PDT and gene therapy both delivered by upconversion

nanoparticles also served as an imaging tool for real time tracking. Utilizing

multifunctional nano-complex with both SiRNA, photosensitizer (Ce6) an effective

cancer killing was achieved. MSN as a codelivery system has also been designed for

co-delivering of chemotherapeutic drug and SiRNA silencing ABC transporters gene

MDRI,[107] chemotherapeutic drug and SiRNA silencing Bcl-2 gene,[108] and

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chemotherapeutic drug and surfactant chemosensitizer.[109] The co-delivery of Bcl-

2- SiRNA and DOX, has increased the anti cancer activity 132 times higher

compared to pure drug.[108]

2.5 Hemocompatability of MSNs

Mesoporous silica nanoparticles have been put forward as the prominent drug

delivery system which was proven by several studies conducted in the last decade.

However, there are a few concerns regarding its hemocompatability in scientific

community. Silica is reported to have LD50 of 1.5 mg/kg for intravenous

administration in rats while LD50 of 3.16 g/kg for oral administration in rats proposing

its safety. However, considering numerous variants such as particle size, porosity,

shape, surface functionalization and structure within the mesoporous silica family, it

is not surprising to encounter several conflicting results, making the biocompatibility

assessment of these materials a subject of intense debate.[110]

The influence of aminopropyl, mercaptoproplyl, and carboxyethyl-tetheraed modified

SBA-15 on hemolysis was studied by comparing with unmodified SBA-15 and

modified amorphous silica (a-SiO2). a-SiO2 showed high hemolysis than unmodified

SBA-15 at an incubation time of 24 h. While aminopropyl, mercaptoproplyl, and

carboxyethyl-tetheraed modified SBA-15 showed less hemolysis at low

concentration. However, at high concentration of 750 µg/ml aminomodified SBA-15

showed 50 % hemolysis.[111] On the other hand, in the other study it was shown

that the MSN functionalized with organic groups has less or reduced hemolytic

activity. [112]

Impact of shape and pore size of MSN on serum proteins adsorption and RBCs

hemolysis was evaluated, where MSNs with the particle size of 67 and 68 nm and

pore size of 2 and 3 nm were found to be less toxic than porous rod with an aspect

ratio of 3.[113] In the same study they have shown that MSN with the aspect ratio of

4 and 8 displayed lower hemolysis than the MSN with an aspect ratio of 2. Similarly

silica nanoparticle design dependent hemocompatibility effect was studied using

various aspect ratios mesoporous SiO2. Mesoporous SiO2 with high aspect ratio of 4

and 8 demonstrated lower hemolysis activity than spherical or lower aspect ratio (2)

mesoporous SiO2.[114] However, the amine modified SiO2 caused rapid hemolysis

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for all types of nanoparticles irrespective of aspect ratio. In another study the

interaction effect of MSN with different morphologies on RBCs was studied where it

was found that the higher aspect ratio MSN demonstrated lower hemolysis at a

concentration of 250 and 500 µg/ml than spherical and lower aspect ratio MSN

particles.[115]

The influence of MSN size, pore ordering and pore integrity on hemocompatibility

was studied by comparing solid silica spheres with porous MSN of various

sizes.[116] It was found that small silica nanoparticles (SS NP) with the diameter of

24 nm caused high hemolysis compared to SS NP with the diameter of 37, 142 and

263 nm. It was found that the mesoporous silica nanoparticles (MS NP) with the

diameter of 24 nm were caused less hemolysis than the SS NP with the diameter of

24 nm. Overall, compared to SS NP with similar size, MS NP caused reduced

hemolysis activity. Furthermore, the haemolytic activity of MS NP with particle size of

25 and 42 nm increased when the ordered pores was collapsed. However, the

haemolytic activity of MS NP with particle size of 25 and 42 nm was decreased after

polyethylene glycol modification. Additionally, Slowing et al. demonstrated that

haemolytic effect of amorphous silica is higher than MSN.[117] The author further

demonstrated that the haemolytic activity of amorphous silica can be supressed after

amino modification.

As research efforts towards the synthesis of various MSNs and their potential

applications in biological and medicine have intensively continued, concerns

regarding the potential toxicity of the engineered nanomaterials in human health and

on the environment have also been raised. Shami et.al.,[118] investigated the

biological responses of various extracted murine tissues, including lung, liver, kidney,

spleen, and pancreas after exposing to 200 µg/ml calcined SBA-15 and MCM-41

particles for hours. The results showed that the murine tissues did not exhibit any

changes in their microscopic structures and bioenergetics after exposure to both

type of MSNs. The results show that calcined MSNs have very promising

biocompatibility when administered at reasonable dose. Tang and co-workers[119]

investigated the biodistribution and excretion of different MSNs in mice, after

injecting the tail veins of mice with rod-like fluorescein-conjugated MSNs possessing

different aspect ratios of 1.5 and 5. MSNs were found in both urine and feces

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samples, there were detected in lower amounts in the kidney and liver, suggesting

that the MSNs could be more rapidly excreated via renal uptake than hepatic

digestion.

2.6 Mesoporous silica nanoparticles in vaccine

The current vaccine challenges comprise the induction of strong immunoprotective

responses in the host in a safe manner and at lower cost. MSN with a highly ordered

pore structure have been used as an antigen carrier due to the interesting properties

like antigen protection and targeting antigen to antigen presenting cells [120]. Mody

et al., summarized the immunomodulatory properties of different nanoparticles such

as aluminium salts, chitosan, ISCOMs, liposomes, and polymeric and silica

nanoparticles (SPs).[121] Luciana et al., compared the adjuvant effect of SBA-15

with the licenced adjuvant Al(OH)3 and IFA. SBA-15 has high immunogenicity and

improved immune response than the licenced adjuvant. Moreover, SBA-15

stimulated mutually high TH1 and TH2 immune response. In vitro assays with

macrophages demonstrated that SBA-15 has high phagocytic uptake and least cell

integrity.[122] Helen et al.[123] compared the activation of human monocyte derived

dendritic cell after exposing to AMS-6, calcined and cyanopropyl functionalized SBA-

15 (SBA-15 PrCN). SBA-15 at the highest concentration showed similar immune

regulatory effect on MDDCs as AMS-6 for CD86. In contrast, SBA-15 PrCN did not

cause statistically significant changes on MDDCs. In addition, SBA-15 at the highest

concentration also induced a significant increased number of cells producing IL-4

and IL-13 and generated a mixed response of both TH1 and TH2 cytokines. Xiupeng

et al.[124] studied the hollow mesoporous silica (HMS) in generating antitumor

immunity, promoting memory response and stimulating TH1 antitumor immunity in

vivo. HMS nanospheres markedly improved the population of CD4+ and CD8+

effector memory T cells in the bone marrow. Moreover, plain HMS nanospheres

showed greatly inhibited tumor growth compared with alum and without adjuvant. In

addition, plan HMS stimulated both TH1 and TH2 immunity than commercial adjuvant

alum. Jaeyun et al., studied the injectable pore forming scaffolds based on

mesoporous silica rods (MSR), and tested their efficacy in modulation of immune

cells and potentials as a vaccine platform to provoke adaptive immune

response.[125] MSR with the aspect ratio (AR) of 88 X 4.5 µm and 37 X 3.2 µm in

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length and diameter were studied for immune response. In which higher AR particle

has 2.5 fold more host immune cells residing in the structure than lower AR MSRs.

MSR in conjugation with antigen, recruited more dendritic cells and high cell surface

expression of CD86+ and MHCII+, as compared to bare MSR particles. Mice

immunized with full MSR vaccine showed significant proliferation of Thy 1.2+ cells

and generated a high CD4+ CXCR5+ T helper cell clonal expression and TFH

differentiation.[125]

Smith et al., systematically studied the influence of particle size on targeting dendritic

cells (DCs). They have demonstrated that particle size less than 20-30 nm can reach

DCs directly. A scheme below shows the SPs delivery draining into lymph nodes

(Figure 2.2). On the other hand, particle size above 20 nm can be phagocyted by

and uptake by DCs is shown below.[126]

In addition to the particle size, the surface charge of the nanoparticles can also affect

the immune response. A positive charge on the particle surface leads to an effective

macrophage and DC uptake due to the negative charged DCs surface.[128] Foged

et al.,[129] observed that nanoparticles with the particle size of 500 nm has high DCs

uptake, and further concluded that the uptake of nanoparticles with the particles size

> 500 nm can be enhanced with amino modification. Weiwei et al., studied the effect

of surface modification of mesoporous silica micro rod scaffold on immune cell

activation and infilteration.[130] PEG and PEG-RGD modified MSR particles were

tested for immune cell adhesion, cell infiltration and Nlrp3 inflammasome production.

PEG modified MSR induced high CD86 expression than RGD modified MSR.

Moreover, PEG-MSR induced high IL-1b expression than RGD-MSR nanorods.

PEG-RGD modified MSRs displayed decreased inflammatory effect as compared to

PEG-MSR.

The role of other important parameters of particles in modulating the immune

response is not thoroughly understood. Several recent in vitro studies have provided

evidence that particles shape will also plays an important role in modulating

interaction with the APC, and T cells. Kumar et al.[131] conducted a comparative

study of spherical and rod shape particles of different size in modulating an immune

response. It has been reported that spherical particle of 193 nm, rods-376 and

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Figure 2.2 Delivery of silica nanoparticles based nanovaccines (20-200 nm particle

size) by parental (A) and mucosal (B) routes. Reproduces from the reference.[127]

spherical-521 nm produced both Th1 (IgG-2a) and Th2 (IgG-1) type immune

response. However, rods-1530 failed to develop IgG-2a antibody titres. Sphere-193

produced significantly increased level of IFN-γ as compared to other groups.

Bingbing et al., [132] prepared rod shaped aluminium hydroxide nanoparticles with

the length of 810, 592 and 451 nm, and studied the influence of shape and

crystallinity for NLRP3 inflammosome activation, cytokine production, and adaptive

humoral immunity response. Rod shape nanoparticles induced significantly high IL-1

beta than the commercial adjuvant Alum. All lengths of rods generated MHC-II

expression and induced significant expression of costimulatory molecules (CD40,

CD80 and CD86), which was significantly higher than Alum. Wang et al., [133] in

2012 studies the effect of silica nanoparticle pore size, geometry and particle size on

immunological properties by injecting the nanoparticles orally into Balb/C mice using

Bovine serum albumin (BSA) as model adjuvant. The three silica nanoparticles with

the particle size of 130(S1), 430(S2), nm and 1-2(SBA-15) µm was used in the study

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with an average pore size of 4, 60 and 7 nm, respectively. The IgG antibody in the

plasma after oral vaccination of BSA loaded S1, S2, SBA-15 was in the order of

S1>S2>SBA-15.

Kenichi et al., [134] studied the influence of gold nanoparticle size and shape on

immunological responses in vitro and in vivo for the production of antibodies for West

Nile virus (WNV). Where they prepared spherical (20 and 40 nm diameter), rod (40

×10 nm) and cubic (40 ×40 × 40 nm) as adjuvant and coated with WNV protein. The

40 nm spherical particle induced highest level of WNV specific antibodies than rod

and cubic particles. In contrast, uptake of the rod shape nanoparticles was more

efficient than the spherical and cubic particles. Meanwhile, spheres and cube shape

particles induced significantly high inflammatory cytokine production, TNF-alpha, IL-

6, IL-12 than rods. In another study, Zhen et al.[135] studied the shape effect of

glycol-nanoparticles (GNP) on macrophage cellular uptake and immune response. It

was found that spherical GNP was internalized more by RAW 264.7 macrophages

than cylindrical GNP. It was found that all the GNP irrespective of shape stimulated

higher secretion of the inflammatory cytokines compared to the control group.

2.7 Synthesis of mesoporous silica nanoparticles

2.7.1 Synthesis of small pore mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSNs) are inorganic silica nanoparticles composed

of silica with uniform particle size and ordered mesopores. [136] The first MSNs were

discovered by Mobile researchers in 1992 which was named as Mobile Crystalline

Materials (MCM-41) for catalysis.[137] MCM-41 materials were prepared by using

two different surfactants-dodecyltrimethylammonium bromide (C12TAB) and

hexadecyltrimethylammonium bromide (C16TAB) to achieve a pore size of 2.5 nm

and 1.8 nm, respectively. MSN whose attributes include high surface area,

adjustable pore size and particle size, easy surface modification which leads to its

application in adsorption of therapeutic molecules, catalysis activity, sensor and

various other fields.[138-145] The use of MSN in as a drug carrier was first reported

by Maria Vallet-Regi et al., in delivering Ibuprofen.[22] They have demonstrated that

the drug release appeared to be pore size dependent and thus demonstrated the

application of MCM-41 as drug carrier. Szegedi at al., [146] also reported the loading

of ibuprofen into different amounts of 3-aminopropyltriethoxysilane (APTES) modified

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MCM-41. Amino modification resulted in high degree of ibuprofen loading and slow

release into dissolution medium.

2.2.1 Synthesis of small pore mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSNs) are inorganic silica nanoparticles composed

of silica with uniform particle size and ordered mesopores. [5] The first MSNs were

discovered by Mobile researchers in 1992 which was named as Mobile Crystalline

Materials (MCM-41) for catalysis.[6] MCM-41 materials were prepared by using two

different surfactants-dodecyltrimethylammonium bromide (C12TAB) and

hexadecyltrimethylammonium bromide (C16TAB) to achieve a pore size of 2.5 nm

and 1.8 nm, respectively. MSN whose attributes include high surface area,

adjustable pore size and particle size, easy surface modification which leads to its

application in adsorption of therapeutic molecules, catalysis activity, sensor and

various other fields.[7-14] The use of MSN in as a drug carrier was first reported by

Maria Vallet-Regi et al., in delivering Ibuprofen.[15] They have demonstrated that the

drug release appeared to be pore size dependent and thus demonstrated the

application of MCM-41 as drug carrier. Szegedi at al., [16] also reported the loading

of ibuprofen into different amounts of 3-aminopropyltriethoxysilane (APTES) modified

MCM-41. Amino modification resulted in high degree of ibuprofen loading and slow

release into dissolution medium. Tang et al., [22] reported dimethyl silyl (DMS)

modified mesoporous silica for loading ibuprofen by adsorption method. The

adsorption capacity of ibuprofen was dependent on the amount of silanol groups.

Later on, MCM-41 have been used as a nanocarrier for various types of

pharmaceutical drugs such as hydrophobic anticancer drug doxorubicin, [23]

hydrophobic camptothecin [24] and curcumin. [25] Arean et al., [26] investigated

effect of amino and carboxyl functionalized MCM-41 on loading of cisplatin. Their

investigation has shown that modified MCM-41 showed a superior loading efficiency

than unmodified one. Ambrogi et al., [27] reported furosemide loaded drug delivery

system for oral administration. The results demonstrated dissolution enhancement

and complete drug release within 90 min at the absorption region. The advantage of

MCM-41 helped in the invention of variety of inorganic silica nanoparticles such as

SBA-15[28], MSU-1[29], KIT-6[30], FSM-16[30], FDU family [31] and many more are

synthesized till date. MCM-48 is another important type of MSNs which has attracted

Chapter 2

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- 29 -

Figure 2.3 The scheme shows the leading nanocarriers for drug delivery and their

general stages of development. The top row shows the representative conventional

nanocarriers such ad liposomes, micelles, dendrimers, and polymers. The bottom

row shows novel inorganic nanocarriers such as carbon nanotubes, quantum dots,

iron oxide, gold, and mesoporous silica nanoparticles.[3]

much attention in biomedical applications. [32, 33] These materials have hexagonally

arranged tubes, with diameter ranging from 20-100 Å. The discontinuous hexagonal

pore channels are considered to be helpful for fast molecular transport and easy

molecular accessibility.

Tang et al., [151] reported dimethyl silyl (DMS) modified mesoporous silica for

loading ibuprofen by adsorption method. The adsorption capacity of ibuprofen was

Chapter 2

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- 30 -

dependent on the amount of silanol groups. Later on, MCM-41 have been used as a

nanocarrier for various types of pharmaceutical drugs such as hydrophobic

anticancer drug doxorubicin,[152] hydrophobic camptothecin[153] and

curcumin.[154] Arean et al., [155] investigated effect of amino and carboxyl

functionalized MCM-41 on loading of cisplatin. Their investigation has shown that

modified MCM-41 showed a superior loading efficiency than unmodified one.

Ambrogi et al., [156] reported furosemide loaded drug delivery system for oral

administration. The results demonstrated dissolution enhancement and complete

drug release within 90 min at the absorption region. The advantage of MCM-41

helped in the invention of, variety of inorganic silica nanoparticles such as SBA-

15[157], MSU-1[158], KIT-6[159], FSM-16[159], FDU family[160] and many more are

synthesized till date.

MCM-48 is another important type of MSNs which has attracted much attention in

biomedical applications.[161, 162] These materials have hexagonally arranged

tubes, with diameter ranging from 20-100 Å. The discontinuous hexagonal pore

channels are considered to be helpful for fast molecular transport and easy

molecular accessibility. The synthesis of MCM-48 was rather complicated in 1990s,

where cationic-anionic co-surfactants were utilized as templates and high

temperature and long reaction time was involved. Moreover, the particle sizes of

MCM-48 were greater than1µm, which were not suitable for biomedical applications.

In 2010, Kim et al., [163] reported a facile approach to synthesize mono-dispersed

spherical MCM-48 MSN with cubic Ia3d mesostructure by modified Stöber method

Table 2.3 Representative drug delivery carriers on the market or in clinical trial. [3]

Nanocarrier

platform

Delivered

therapeutic

agent

Stage (Date) in

development

Drug name Indications

Liposome Doxorubicin Approved

((November 17,

1995)

Doxil Kaposi's

sarcoma,

recurrent

breast cancer,

and ovarian

Chapter 2

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cancer[17]

Polymeric

micelles

Paclitaxel Phase II clinical

trial

Genexol-PM Non-small- cell

lung cancer[18]

Albumin

(protein-drug

conjugate)

Paclitaxel Approved (January

7th, 2005)

Abraxane Metastic breast

cancer[19]

Gold Recombinant

human

tumor necrosis

factor

(rhTNF)

Phase II clinical

trial

AurImmune

™

Pancreatic

cancer,

melanoma,

soft tissue

sarcoma,

ovarian,

and breast

cancer[20]

Silica cyclic

arginineglycine

aspartic acid

(cRGD)

peptide, and

124I

Phase I clinical

trial

C dots Human

melanoma[2]

utilizing triblock copolymer Pluronic F127 as a particle size designer. The particle

sizes can be controlled within the range of 70-500 nm by adjusting the amount of

F127. Moreover, the pore diameter was also precisely controlled from 2.3 to 3.3 nm

by using different alkyl chain surfactants. The monodispersed spherical MCM-48

MSN was applied for enzyme responsive controlled release and pH responsive

neutraceuticals.[164, 165]

In addition, SBA-15 was also used for biomedical applications. SBA-15 was

synthesised in strong acidic condition using amphiphilic triblock copolymers

EO20PO70EO20 [Pluronic P123, EO refers to poly (ethylene oxide), PO refers to

poly(propylene oxide)] as structure directing agent.[157] The pore size of SBA-15 is

usually 6 nm in diameter, larger than the pore size of MCM-41. The size of SBA-15

was reduced with time, and there was a successful development of different shapes

such as ellipsoid, sphere and rod.[166, 167] Popova et al., [168] conducted a

Chapter 2

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comparative study of sulfadiazine loading into MCM-41 and SBA-15 nanoparticles. In

vitro studies showed slower release rate of sulfadiazine from carboxylic modified

MCM-41 and SBA-15 than parent particles. Most of the SBA-15 is still far away from

ideal candidates for biomedical applications due to its big particle size.

2.7.2 Synthesis of large pore symmetric and asymmetric silica nanoparticles

(SiNPs).

2.7.2.1 Synthesis routes of solid SiNPs and modifications

In 1968, StÖber et al. introduced the synthesis of mono-dispersed solid SiNPs by

hydrolysis and condensation of silicon alkoxides in a mixture of alcohol and water

using ammonia as a catalyst.[169] The silica nanoparticles size was adjusted from

50 nm to 2 µm, by changing the ammonia concentration. Silica nanoparticles

synthesized by StÖber method have been used for various applications. Non-viral

silica nanoparticles mimicking virus surface topology have been synthesised by

inducing surface roughness on the surface of StÖber spheres and tested for gene

efficacy.[170] In addition, non-viral silica nanoparticles were modified with n-ODMS

for therapeutic protein delivery.[105, 171] A modified StÖber method, C dots have

been synthesized for diagnostic applications and have reached stage I human

clinical trials.[42, 46]

2.7.2.2 Synthesis of large pore dendritic symmetric silica nanoparticles

In the recent times, symmetric dendritic silica micro/ nanoparticles with centre-radial

pore structures, a kind of newly created porous materials, have attracted

considerable attention owing to their unique open three dimensional dendritic

superstructures with large pore channels and highly accessible internal surface

areas compared with conventional MSN. Great attention has been paid to this family

of materials due to their unique properties. Different research groups have come with

an innovative different synthesis conditions for the generation of centre radial

structures.

Okuyama et al.[172] introduced oil in water emulsion system to synthesise dendritic

spherical MSNs using octane as the oil phase, styrene monomers and CTAB as a

dynamic templating agent, lysine as a base, TEOS as silica source, 2,2-azobis (2-

methylpropionamide) dihydrochloride as an initiator and water (Figure 2.4A). The

TEM image of calcined sample was shown in Figure 2.4B.

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- 33 -

Figure 2.4 A) Schematic formation mechanism of oil in water system introduced by

Okuyama et al. and B) TEM image of the obtained dendritic MSNs. Reproduced from

the reference. [172]

Polshettiwar et al.[173] reported the synthesis of dendritic silica nanoparticles with

centre radial wrinkle structure through a microemulsion system composed of

cyclohexane as an oil phase, pentanol as a co-solvent, and water as a dispersion

phase, CTAB as a structural directing agent, TEOS as a silicone source and urea as

a base catalyst under microwave-assisted hydrothermal conditions. The internal

surface area of fibrous silica nanospheres (KCC-1) is easily accessible which can be

clearly seen from the TEM image (Figure 2.5B). The proposed mechanism for the

formation of radial wrinkle structure is the arrangement of silicates molecules in the

space available between the self-assembled template molecules to aggregate along

the free radial and restricted tangential directions. The application of KCC-1 was

shown by shili et al., where hydrophobic Fe3O4 was adsorbed on to the fibrous

mesoporous silica nanoparticles (FMSN) structure of KCC-1. Further DOX was

successfully entrapped within the mesopores of Fe3O4-FMSN composite and

investigated the intracellular drug delivery, fluorecsence and therapeutic effect. DOX-

Fe3O4-FMSN composite shown pronounced cytotoxic effect in L929 fibroblast cells

than free DOX at high concentration. [174] Later, Atabaev et al., synthesised

multifunctional core shell mesoporous silica with fibrous morphology with slight

modifications of the procedure reported by Polshettiwar. The multifunctional

Fe3O4@SiO2 composites are spherical morphology with clearly distinguishable

magnetic core and mesoporous silica shell. The size of the Fe3O4@SiO2 NP was

tuned by changing the time, TEOS or urea concentration by keeping the other

Chapter 2

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- 34 -

parameters constant. The Fe3O4@SiO2 composite with the fluorescent property, at

concentration >125 ppm significantly reduced the cell viability of L-929 cells. [175]

Magnetic fibrous monodispersed core-shell Fe3O4/SiO2/KCC-1 structures were

successfully synthesised by surfactant template microemulsion formation process.

The BET surface area and pore volume were 213 m2/g and 0.45 cm3/g, respectively,

which were considerably large. Fe3O4/SiO2/KCC-1 have shown good methylene blue

adsorption capacity.[176]

Figure 2.5 A) SEM and (B) TEM images of KCC-1. Reproduced from the reference.

[173]

Later, Yang’s[177] group and Lee’s[178] group also synthesised KCC-1 type of

particles by employing similar microemulsion systems under more mild experimental

conditions. Lee group systematically investigated and demonstrated that wrinkled

silica nanoparticles were formed in a bicontinuous microemulsion phase of the

winsor III system (Figure 2.6). Moreover, increasing the chain length of the co-

solvents can also alter the phase behaviour by varying the relative solubility of

surfactants in oil and water phases, which results in the increase of interwrinkle

distances in the dendritic particles.

Zhao., at al[179] group developed a new oil-water biphase stratification approach to

achieve 3-D dendritic MSN with tuneable multigenerational and central-radial

mesoporous channels. They used CTAC as surfactant, TEA as base catalyst, TEOS

as silica source, and water in lower phase and upper oil phase includes

cyclohexane, 1-octadecene, decahydronapthalene. The pore size in each generation

was adjusted from 3-13 nm by changing the concentration of hydrophobic solvent

and TEOS in the upper oil phase (Figure 2.7). The particle formation mechanism

was explained by interfacial emulsion funnelling gradient assembly and growth

Chapter 2

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process. They further extended the approach to synthesise functional core-shell

materials with different shapes of cores.

Figure 2.6 Schematic representation of influence of cyclohexane volume on silica

nanoparticles and respective TEM images. Reproduced from reference.[178]

Chun Xu et al.[180] introduced core-cone structures by using chlorobenzene-water

system, in which CTAC as a surfactant, TEA as a base catalyst and TEOS as silica

source. The formed particles are shown in Figure 2.8, which has a pore size ~ 45 nm

and particle size of 175 ± 12 nm (Figure 2.8). Meka et al.[181] synthesised amino-

functionalized hollow dendritic MSN by inducing APTES in the chlorobenzene-water

systems under more diluted CTAC concentration. From the TEM (Figure 2.9d) the

hollow cavity diameter is of 173 ± 29 nm and the large opening pore is of 20 nm.

Figure 2.7 TEM (a,b,c) and SEM (d,e,f) images of three generations of pore

structures. Reproduces from reference. [179]

Chapter 2

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- 36 -

Figure 2.8 A picture of dahlia (A), TEM images at low magnification (B) and high

magnification (A) and an ET slice (D) of core cone particles. Reproduces from

reference.[180]

Figure 2.9 SEM images at low magnification and high magnification (a, b) and TEM

(c) images and ET slice (d) of A-HDMSN. Reproduced from reference. [181]

2.7.2.3 Synthesis of asymmetric silica nanoparticles

Asymmetric nanoparticles with polymeric, inorganic and polymeric-inorganic hybrid

composition have attracted much attention in diverse fields because of their unusual

behaviour that cannot be observed for isotropic particles. Precise design of

asymmetric nanoparticles promises a wide range of application in displays, sensors

and drug delivery systems.

Zhao et al., [182] synthesised multifunctional dual-compartment Janus mesoporous

nanocomposites of upconversion nanoparticles (UCNP) @Sio2@mSio2@PMO

Chapter 2

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containing UCNP@SiO2@mSiO2 nanospheres and periodic mesoporous

organosilica (PMO) single crystal nanocubes using novel anisotropic island

nucleation and growth approach. The asymmetric Janus nanocomposites have a

uniform size of ~300 nm with a unique dual independent mesopores with different

pore sizes of 2.1 and 3.5-5.5 nm (Figure 2.10). The Janus nanocomposite possess

an excellent NIR to UV-vis upconversion optical property from the UCNP and dual

drug loading capacity. A mechanism is proposed to explain the heterogeneous

nucleation process, where the mesostructured CTAB/ silicate micelles can be

assembled and nucleated on the surface of the UCNP@SiO2@mSiO2 through a

heterogeneous nucleation process. Once the nucleation sites are formed the growth

manner of PMO is determined by the hydrolysis environment of 1,2-bis

(triethoxysilyl) ethane (BTEE) and the mesostructure of the obtained PMO. There

observations are consistent with the classical island growth mode of Volmer-Weber.

Figure 2.10 Synthesis procedure for the dual-compartment Janus mesoporous silica

nanocomposites UCNP@SiO2@ mSiO2&PMO by anisotropic island nucleation and

growth method. Reproduced from reference. [182]

The same group synthesised asymmetric single hole nanocages by using dense

SiO2 spherical nanoparticles as a seed. [183] By using CTAB as mesostructure

template and BTEE as a silica source, periodic mesoporous organosilica could be

nucleated and encapsulated on dense SiO2 by novel anisotropic encapsulation

method to form eccentric core@ shell nanocomposite. The unique asymmetric

nanocages have a particle size of 100-240 nm, with uniform mesopores of 3-10 nm

and controllable eccentric hollow cavity of 35-170 nm (Figure 2.11). The obtained

single hole mesoporous nanocages was further functionalized with upconversion

Chapter 2

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nanoparticles (UCNP). The obtained nanocages were used for codelivery of

doxorubicin and bovine serum albumin. The formation of asymmetric nanocages was

because of asymmetric growth of PMO growth on dense SiO2.

Figure 2.11 TEM images (A-C) of the morphology evolution of the eccentric

nanocomposites. TEM images of Janus UCNPs@SiO2 and PMO and the eccentric

hollow structured nanocomposites. Reproduced from reference. [183]

Kuroda et al.,[184] reported the asymmetric capping of mesoporous silica with

phenylsilsesquioxane, where they added phenyltriethoxysilane (PTES) to the

colloidal dispersion of silica and surfactant composite nanoparticles (CMSS). It was

first report on asymmetric modification to produce janus nanoparticles. It was

proposed that PTES acts as an oil phase, and the hydrolysed PTES slowly move

and deposit on the surface of CMSS (located in water phase). Here, it was also

proposed that CMSS can be located at the interface of water and oil droplets as an

emulsifier; hence, nucleation and growth essentially occur on one side of CMSS.

Suteewong et al., synthesised multicompartment mesoporous silica nanoparticles

with branched shape using CTAB as structure directing agent, ammonia as a base

catalyst, and mixed organosilica precursor via one pot synthesis method.

Multicompartnat epitaxial growth was controlled by varying the ethyl acetate

concentration in the reaction system.[185] First, it was shown that increase in

ethylacetate concentration lead to an increase in hydrolysis rate and a pH drop. This

pH drop was drawn to decrease in the acid dissociation constant and was increased

from 91mM to 457mM by keeping ammonium hydroxide and APTES constant.

Novel multifunctional magnetic mesoporous janus particles with controlled aspect

ratio was developed by a simple one step synthesis approach by Zhang et al.[186]

The janus structures with controlled aspect ratios were prepared by varying the

molar ratios of silica source (TEOS) to iron oxide. The formation of ordered

Chapter 2

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- 39 -

mesoporous architecture on iron oxide particles was the self-organization of low-

ordered aggregates on colloidal surface and eventual growth along both axis and

radial direction.

2.8 Conclusion

Mesoporous silica has received considerable attention and emerging as an

alternative carrier system to deliver the therapeutic cargo or diagnostic aids into the

specific disease/ targeting site because of their tuneable pore size, high surface

area, large pore volume, and ease of functionalization. Although there have been

tremendous efforts in the synthesis of mono-dispersed SiNPs for various

applications, more efforts are still needed to develop novel mono-dispersed SiNPs

with desired pore size and morphology. As this Ph.D. thesis is combination of

synthesis and development of silica nanoparticles and its morphology advantage in

biomedical field, chapter 2 mainly focus on the broad picture over synthesis and

applications. Current synthetic approaches for symmetric and asymmetric silica

nanoparticles with small and large pores have been discussed. Later,

physiochemical parameters especially the influence of pore size and morphology

were discussed concerning biomedical applications of nanomaterials, such as

solubility enhancement, controlled drug release, hemocompatibility, combination

therapy, and vaccines.

The novel designed SiNPs are expected to expand their capacity in various

biomedical applications. Tuning the morphological characteristics of particle is an

essential parameter and that helps to load the desired cargo into the silica.

Particularly, conventional floating tablet formulation failed to enhance the solubility of

hydrophobic drugs and control release the hydrophilic drugs. Thus, it is necessary to

develop simple method for the design of floating tablets with improved performance.

For example, SiNPs with desired pore size is expected to have much higher

solubility enhancement of hydrophobic drugs and controlled release of hydrophilic

drugs. Thus chapter 4 of the thesis addresses this issue. In another example, better

understanding on the hemocompatibility of asymmetric SiNP not only allows a better

estimation of the potential risk, but also helps to find a balanced point between

efficacy and safety. Chapter 5 of this thesis investigates the morphology influence of

Chapter 2

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symmetric and asymmetric SiNP on hemocompatibility. Furthermore, large pore

dendritic silica nanoparticles are synthesized using different methods, but the

combination of photosensitiser and large biomolecules in cancer therapy was not

studies. So far all the designed combined therapy is in combination with

chemotherapeutic and photosensitizers. However, the chemotherapeutic caused

various side effects. Thus it is necessary to develop smart combination approach for

cancer treatment. Chapter 6 of the thesis address this issue and demonstrates the

effect of protein and photodynamic therapy in cancer treatment.

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

p. 5949-5955.

[181] Meka, A.K., et al., A Vesicle Supra-Assembly Approach to Synthesize

Amine-Functionalized Hollow Dendritic Mesoporous Silica Nanospheres for

Protein Delivery. Small, 2016. 12(37): p. 5169-5177.

[182] Li, X., et al., Anisotropic encapsulation-induced synthesis of asymmetric

single-hole mesoporous nanocages. J Am Chem Soc, 2015. 137(18): p. 5903-

5906.

[183] Li, X.M., et al., Anisotropic Growth-Induced Synthesis of Dual-

Compartment Janus Mesoporous Silica Nanoparticles for Bimodal Triggered

Drugs Delivery. Journal of the American Chemical Society, 2014. 136(42): p.

15086-15092.

[184] Ujiie, H., A. Shimojima, and K. Kuroda, Synthesis of colloidal Janus

nanoparticles by asymmetric capping of mesoporous silica with

phenylsilsesquioxane. Chemical Communications, 2015. 51(15): p. 3211-

3214.

Chapter 2

Literature Review

- 57 -

[185] Suteewong, T., et al., Multicompartment mesoporous silica

nanoparticles with branched shapes: an epitaxial growth mechanism. Science,

2013. 340(6130): p. 337-341.

[186] Zhang, L., et al., Magnetic-mesoporous Janus nanoparticles. Chemical

Communications, 2011. 47(4): p. 1225-1227.

Chapter 3

Methodology

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

synthetic methods for small pore MCM-41, dual pore head-tail MSN, large pore

dendritic MSN and the techniques for material characterizations and biological

evaluations.

3.1 Material synthesis

Mono-dispersed MSN with various pore size and morphologies are synthesised by

the hydrolysis and condensation of silicon alkoxides in either aqueous solution using

ammonia as a catalysis or in oil in water emulsion system using TEA as a catalyst.

3.1.1 Synthesis of monodispersed MCM-41 and preparation of floating tablets

with drug loaded MCM-41

MCM-41 was synthesized according to an approach reported in our previous work

with slight modifications. [1] In a typical synthesis, 1.0 g of CTAB was dissolved in

480 g of deionized water under stirring at room temperature followed by the addition

of 3.5 mL of NaOH (2 M). The temperature of the solution was raised and kept at 80

°C. To this solution, 6.7 mL of TEOS was added. The mixture was continuously

stirred for additional 2 h. The resultant products were collected by filtration and dried

at room temperature. The templates were removed by calcination at 550 °C for 5 h.

The floating tablets were prepared by a direct compression method. Weights of each

ingredient were taken according to Table S2, Table S3 for each formulation. Micro-

Chapter 3

Methodology

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crystalline cellulose (MCC, bulking agent) was added in order to maintain the same

weight of each tablet in different formulations. All the ingredients were mixed

homogeneously with mortar and pestle for 10 min. Zinc stearate (a lubricant) was

added at last to the mixed powders and then mixed further for 3 min. Finally, the

powder was fed manually into die with spatula of a single punch tablet punching

machine, equipped with concave punches (10 mm diameter). The hardness of the

tablets was adjusted to 50 N by adjusting the punches and the hardness was

measured by using Erweka hardness tester.

3.1.2 Synthesis, functionalization and dye tagging of head-tail nanoparticles

Uniform nonporous silica nanoparticles with a smooth surface were synthesized

using a well-known method developed by Stöber et al. [2] Typically, absolute ethanol

(50 ml) was mixed with deionized water (3.8 ml) and ammonium hydroxide solution

(2 ml, 28%,) at 35 ˚C. Then, TEOS (2.8 ml) 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 was

obtained by drying at 100 ˚C overnight.

In a typical synthesis, 60 mg of silica spheres of ~200 nm size were dispersed in 6

ml of milliQ water, and 0.035 g Triethanolamine (TEA), by sonication for 15 min.

Then, 4 ml of CTAC (25 wt%) solution was added into the solution and kept at 60 ˚C

at a stirring speed of 400 rpm for 1 h. After stirring for 1 h, 8.75 ml of chlorobenzene

and, TEOS was added into the above solution. The reaction was continued at 60 ˚C

for 12 h. Particles were collected by centrifugation at 15,000 rpm, washed with water

and ethanol twice and dried at 50 ˚C overnight. The particles are calcined at 550 ˚C

for 5 h to remove CTAC. We studied the influence of TEOS amount, reaction time

and head amount on the morphology by varying the reaction time, TEOS volume and

head amount.

Amino silane was grafted onto the surface of stöber spheres, HTMSN0.41-12-60,

HTMSN1.25-12-60, HTMSN1.25-12-400, to create positive charge particles. 200 mg of

particles was suspended in 30 ml of toulene, and sonicated for 10 min, and then 0.19

ml APTES was added and the mixture was refluxed at 110 ˚C for 20 h. The product

Chapter 3

Methodology

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were collected by centrifugation and washed twice with ethanol and water and dried

at 50 ˚C for overnight.

50 mg of amino modified particles were suspended in 20 ml of ethanol with

sonication for 10 min. Afterwards, 5 mg of FITC was added and stirred the

suspension for overnight in dark. FITC tagged particles were collected by

centrifugation followed by several times washing with ethanol to remove the free dye.

3.1.3 Synthesis of dendritic silica nanoparticle with fullerene core

Uniform SSF nanoparticles was synthesized according to literature method.[3] In a

typical synthesis, 16 mg of C60 fullerene were well dispersed in toluene using

sonication. The dispersed fullerene solution was mixed and stirred for 30 min, with

microemulsion solution composed of cyclohexane (20 ml), 1-hexanol (8 ml), distilled

water (2 ml), and Triton X-100 (6.8 ml). After 30 min, 400 µl of TEOS solution was

added and stirred for another 30 min before addition of ammonium hydroxide (28 wt

%, 2.4 ml). The mixture was continuously stirred at room temperature for 24 h. 40 ml

of ethanol was added to break the microemulsion and the particles were collected

using 20,000 rpm and washed with ethanol twice.

FD nanoparticles were synthesised according to literature with slight modification. In

a typical synthesis, 60 mg of SSF were dispersed in 6 ml of milliQ water, and 0.035 g

Triethanolamine (TEA), by sonication for 15 min. Then, 4 ml of CTAC (25 wt%)

solution was added into the solution and kept at 60 ˚C at a stirring speed of 400 rpm

for 1 h. After stirring for 1 h, 8.75 ml of chlorobenzene and, TEOS was added into the

above solution. The reaction was continued at 60 ˚C for 12 h. Particles were

collected by centrifugation at 20,000 rpm, washed with water and ethanol twice and

vacuum dried overnight.

3.2 Characterisation

3.2.1 X-ray diffraction

Small-angle XRD patterns of MCM-41 were recorded on a German Bruker D4 X-ray

Diffractometer (40 kV, 30 mA) with Ni-filtered Cu Kα (λ = 0.15418 nm) Radiation at a

scanning rate of 0.2˚ min-1 from 0.5˚ to 5.0˚. The interplanar spacing is calculated

based on the following Bragg equation: 2dhkl sinθ = nλ where dhkl is the interplanar

Chapter 3

Methodology

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distance between a set of parallel planes described by the Miller indices (hkl) (nm), λ

is the wavelength of Cu Kα ray (nm), θ is the angle of incident beam (˚), and n is

integer determined by a given order.

3.2.2 Thermogravimetric analysis

Thermogravimetric analysis (TGA) is used to calculate the loading of curcumin and

captopril in MCM-41 and also the fullerene amount in solid silica spheres. Around 5-

10 mg of drug loaded nanoparticles was loaded in an aluminium pan and heated

from 25 to 900°C at a heating rate of 1.5 °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).

3.2.3 Transmission Electron Microscopy (TEM)

Transmission electron microscopy is an important tool for structure determination

(TEM). In this thesis all the (TEM) images were obtained using JEOL 1010 operated

at 100 kV, JEOL 2100 at 200 kV or FEI Tecnai F30 operated at 300 kV. The samples

for TEM measurements were prepared by dispersing the powder samples in ethanol,

after which they were dispersed and dried on carbon film on a Cu grid.

3.2.4 Scanning Electron Microscopy (SEM)

The morphologies of the samples MMNSs were observed using a JEOL6610

scanning electron microscope (SEM) operated at 5 kV. Samples were dispersed on

a carbon tape.

3.2.5 Nitrogen sorption

Nitrogen adsorption/desorption isotherms were measured at 77 K by using a

Micromeritics ASAP Tristar II 3020 system. The samples were degassed at 473 K

overnight on a vacuum line. The pore size distribution curve was derived from the

adsorption branch of the isotherm using the Barrett– Joyner–Halanda (BJH) method.

The Brunauer–Emmett–Teller (BET) method was utilized to calculate the specific

surface areas. The total pore volume was calculated from the amount adsorbed at a

maximum relative pressure (P/P0) of 0.99.

Chapter 3

Methodology

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3.2.6 Attenuated Total Reflectance (ATR)-Fourier transform infrared (FTIR)

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 Dynamic light scattering (DLS)

Dynamic light scattering (DLS) is a non-invasive, well-established technique for

particle size characterization of proteins, polymers and colloidal dispersions. The

Brownian motion of particles or molecules in suspension causes laser light to be

scattered at different intensities. Analysis of these intensity fluctuations yields the

velocity of the Brownian motion and hence the particle size using the Stokes-Einstein

relationship. The hydrodynamic sizes of particle samples in this PhD project were

measured using DLS measurements on a Malvern NanoZS zetasizer at 25 ◦C in

ethanol or water solutions.

3.2.8 Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES)

Inductively coupled plasma-optical emission spectrometry (ICP-OES) is an analytical

technique used for the detection of trace metals. It uses the inductively coupled

plasma to produce excited atoms and ions that emit electromagnetic radiation at

wavelengths characteristic of a particular element. The intensity of this emission is

indicative of the concentration of the element within the sample. In this PhD project,

ICP-OES technology was utilized to quantify the silicon concentrations in cells after

treatment of nanoparticles with cells. The MCF-7 cells were incubated with the FD,

FD-C18 nanoparticles for 4 hours, then washed with PBS and harvested with trypsin.

After washing with PBS and centrifugation, cell lysis buffer was added to allow

dissolution of cells under sonication process. The supernatants (containing cell

components) were discarded after centrifugation, and the precipitates were washed

with PBS, followed by drying at 50C for 12 h. After 12 h, 1M sodium hydroxide

Chapter 3

Methodology

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solution was added and incubated for 24 h to completely dissolve silica

nanoparticles. The dissolved silica solution was diluted and measured using ICP-

OES with a Vista-PRO instrument (Varian Inc, Australia).

3.3 Biological assays

3.3.1 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 NADH and NADPH. The

resulting intracellular purple formazan can be solubilized by DMSO and quantified by

spectrophotometric means. The cell growth inhibition by delivering nanocarriers was

tested in multiple cell lines, including HeLa, MCF-7, and RAW 264.7 cell lines. One

day before the test, 5000-8000 cells were seeded in each well of 96-well plates. The

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, MTT reagent was added and further incubated for 4h. Finally, DMSO was added

to the wells analysed the plates by setting the absorbance at 540 nm using a

Synergy HT Microplate Reader. Results were compared to cells not exposed to

nanoparticles.

3.3.2 Confocal Laser Scanning Microscopy (CLSM)

Laser scanning confocal microscopy (CLSM) has become an invaluable tool for a

wide range of investigations in the biological and medical sciences for imaging thin

optical sections in living and fixed specimens ranging in thickness up to 100

micrometres. It is a technique to obtain high resolution optical images with selected

depth. CLSM is normally equipped with 3-5 laser systems with a precise control in

wavelength and excitation intensity. These microscopes coupled with

photomultipliers are capable of detecting fluorescence emission ranging from 400 to

750 nm. In this thesis, all the confocal images of fixed cells were observed under a

confocal microscope (LSM Zeiss 710).

Chapter 3

Methodology

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3.3.3 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.

In this thesis, the intracellular uptake of fluorescent silica nanoparticles was

quantified using a FACS LSR II flowcytometer (BD Biosciences).

Chapter 4

Floating tablets from

mesoporous silica nanoparticles

This chapter reports a design of novel floating tablets using mesoporous silica

nanoparticles for enhancing the drug delivery performance of both hydrophobic and

hydrophilic drugs compared to conventional floating tablets. This work has been

published in Journal of Material Chemistry B, 2014; 2, 8298-8302.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 66 -

4.1 Introduction

Since the first report of ordered mesoporous materials in the early 1990s,[1] great

attention has been paid to this family of materials due to their unique properties (e.g.,

easy synthesis, tunable structures, controllable surface chemistry)[2, 3] and a broad

range of applications.[4-6] Recently, mesoporous silica nanoparticles (MSNs) have

attracted enormous interest in drug delivery.[7] The large surface area and high pore

volume of MSNs render a high efficiency to load cargo molecules, while the

adjustable pores with a range of sizes can be used to control the drug release in a

sustainable manner.[8, 9] MSNs have been demonstrated as targeted delivery

systems to tumor cells[10, 11] and as stimuli-responsive systems.[12-14] MSNs have

also been used in developing oral tablets because the oral route is still the preferable

route of drug administration.[15, 16] However, there is no report on using MSNs in

localised drug delivery for the upper region of gastrointestinal tract (GIT), i.e.,

stomach.

High residence time and local drug release in stomach is prerequisite for the drugs

with narrow absorption window in upper GIT, and poor solubility or degradation in

small intestine.[17] Various approaches have been developed for retaining a dosage

form in stomach.[18, 19] Among these floating delivery system is considered as an

effective technique.[20-22] A successful example of floating drug delivery systems is

floating tablets.[23] Traditionally, floating phenomenon in floating tablets is achieved

by using swellable polymers or polymer mixtures and gas generating agents.[17]

However, for polymer based floating systems, controlling the release of highly water-

soluble drug is limited.[24] Moreover, drug and polymer are generally physically

mixed which does not aid in enhancing solubility of inherently insoluble drugs. In light

of the advantages of MSNs in solubility enhancement[25] and controlled drug

release,[26] it is hypothesised that a floating tablet based on MSNs will provide

unprecedented benefits to address the limitations of conventional polymer based

floating tablets.

In this communication, we report the first example of novel floating tablets based on

MSNs for the localised drug release. As shown in Scheme 4.1A, MCM-411 type

MSNs are chosen as drug carriers. Hydroxypropylmethylcellulose (HPMC) is chosen

as a gelling agent due to its non-toxic nature, easy compression and swelling

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 67 -

properties, which is widely used in oral and topical pharmaceutical formulations.

Sodium bicarbonate is used as a gas generating agent. The floating tablet is

prepared by blending drug loaded MSNs, HPMC and sodium bicarbonate (Scheme

4.1B) and compressing into tablet (Scheme 4.1C) using a single punch tablet

compression machine. Curcumin and captopril are chosen as model hydrophobic

and hydrophilic drugs respectively to demonstrate the application of novel MSNs

based floating tablet. The resultant tablets showed extensive floating behaviour (>12

h) with improved dissolution rate and enhanced solubility for curcumin while

extended release in case of captopril (Scheme 4.1D), providing a leap forward

towards new application of mesoporous materials in localized oral drug delivery.

Scheme 4.1 Schematic representation of floating tablets made from drug loaded

MCM-41, polymer and sodium bicarbonate (A). After blending and compression (B),

captopril (white coloured) and curcumin (yellow) floating tablets are obtained (C),

which can float in stomach >12 h (D).

4.2 Results and discussion

MCM-41 type MSNs were used in this study and prepared according to published

methods [27] with slight variations (see ESI†). Calcined MCM-41 with two different

pore sizes (2.1 and 1.7 nm) were used and the pore size adjustment was performed

by a reported vacuum-assisted vapour deposition (VVD) process [25] (ESI†). The X-

ray diffraction (XRD) patterns (Figure 4.S1A, ESI†) of calcined MCM-41 before and

after VVD process both showed three well resolved peaks at 2θ of 2.68, 4.76 and

5.42˚, which can be indexed as 100, 110, and 200 reflections of an ordered two

dimensional hexagonal mesostructure. For MCM-41 after VVD process, the intensity

of 110 and 200 peaks was decreased due to the decreased scattering contrast

between the pore and silica walls. [28] This is evidenced from the N2 adsorption-

desorption analysis. Comparing the isotherms of calcined MCM-41 before and after

VVD process (Figure 4.S1B, ESI†), the capillary condensation step shifted to a lower

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 68 -

relative pressure range after VVD treatment, reflecting a pore size reduction from 2.1

to 1.7 nm (Figure 4.S1C, ESI†), in accordance with our previous results. [25] The

surface areas and pore volumes are summarised in Table 4.S1 (see ESI†).

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

were used to study the nanostructure of MCM-41. SEM images of two MCM-41

materials showed a spherical morphology with particle sizes of ~120 nm (Figure

4.S2c and d, ESI†). TEM images of both materials showed stripe-like or hexagonal

pattern (Figure 4.S2a, b, ESI†), typical of MCM-41 materials.[29] Because MSNs

have not been used in the formulation of floating tablets, screening tests were

conducted to optimise the floating tablet formulation by increasing the amount of

MCM-41. The compositions of floating tablets (F0-FD) are shown in Table 4.S2,

ESI†. F0 is conventional polymer based, while FA-FD has a silica weight ratio of 42,

55, 58, and 62%, respectively. All tablets floated in simulated gastric fluid (SGF, pH

1.2) longer than 12 h. The silica content after 12 h of floating test decreased as the

amount of silica increased (Figure 4.S3B, ESI†). FA with the initial silica ratio of 42%

has the highest retention ratio of 80%. An initially larger MCM-41 content is

beneficial for a higher dosage form, while the silica content remained in the tablet

after floating is important to reduce the unwanted entry of drug into intestine.

Consequently, the silica content of 42% was chosen as the optimised ratio (FA) in

the following studies.

SEM was also used to investigate the morphology of floating tablets. A typical cross-

section SEM images of FA is presented in Figure 4.1a, showing a homogenous

distribution of MCM-41 particles in the tablet. The HPMC (see SEM image in Figure

4.S2e, ESI†) and other ingredients cannot be seen in the tablet, suggesting a

homogenous distribution of all ingredients. After 12 h of floating test, the SEM image

of F0 prepared without silica nanoparticles revealed a porous structure (Figure 4.S2f,

ESI†). The pores are generated by entrapped CO2 produced by the reaction of

sodium bicarbonate with HCl in the gastric juice (pH 1.2). In contrast, the SEM image

of FA prepared in the presence of MSNs after floating for > 12 h still showed

homogeneously distributed MCM-41 nanoparticles in the polymer matrix (Figure

4.1b), suggesting that HPMC play a dual role of gelling agent to entrap both gas and

MSNs throughout the dissolution process.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 69 -

In order to prepare a drug loaded floating tablet, curcumin (Cur) was separately

loaded into two calcined MCM-41 with different pore sizes by a rotary evaporation

method (see ESI†). Curcumin loaded MCM-41 samples were characterised by

thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), XRD and

N2 adsorption-desorption. TGA (Figure 4.S4, and Table 4.S1, ESI†) showed that the

weight percentage of curcumin was 20.0% and 20.9% in Cur-MCM-41(1.7nm) and

Cur-MCM-41(2.1nm) respectively.

Figure 4.1 Cross sectional SEM images of (a) floating tablet with MSNs and (b) after

12 h of dissoution study.

Sharp diffraction peaks were observed in 2θ range of 5-40˚ for both pure curcumin

and the physical mixture of curcumin and MCM-41 as observed from wide-angle

XRD patterns (Figure 4.S5A, ESI†). Pure MCM-41 showed no diffraction peaks in 2θ

range of 5-40˚, indicating its amorphous nature. After loading curcumin into MCM-41,

no obvious diffractions of curcumin were observed, suggesting that the drug has

been successfully loaded as nano-aggregates into the pore channels of MCM-41 in

an amorphous state. This conclusion is further confirmed by DSC analysis. The DSC

curve (Figure 4.S5B, ESI†) of pure curcumin showed a sharp endothermic peak at

172 ˚C corresponding to its melting point. After loading curcumin into MCM-41 this

peak showed very week intensity, suggesting that the crystallinity of curcumin has

been significantly reduced after loading inside the pore channels.

Figure 4.S6A (ESI†) showed the nitrogen adsorption/desorption isotherms of Cur-

MCM-41(1.7nm) and Cur-MCM-41(2.1nm) were typical type IV with clear capillary

condensation steps in the relative pressure (P/P0) range of 0.2-0.4. The details of

surface area, pore volume, and pore size are given in Table 4.S1, ESI†. After

loading curcumin, the surface area and the pore volume decreased. However, the

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 70 -

pore size remained the same for Cur-MCM-41(1.7nm) and Cur-MCM-41(2.1nm)

even after loading curcumin (Figure 4.S6 B, ESI†). It is suggested that due to the

hydrophobic nature of curcumin, the drug molecules tend to repel from the

hydrophilic silanol groups on the surface and exist as isolated nano-aggregates

inside the pore channels, thus the pore size after loading is not changed.

A hydrophilic drug (captopril) was also loaded into MCM-41 with a pore size of 2.1

nm by a wetting impregnation method. The N2 sorption isotherm and pore size

distribution curve of MCM-41 after drug loading was shown in Figure 4.S7, ESI†.

After drug loading the BET surface area decreased from 946 to 534 m2/g, the pore

volume from 0.66 to 0.38 cm3/g, and pore size from 2.1 to 1.7 nm (Table 4.S1,

ESI†). These data suggest that the drug molecules were introduced into the

mesopores. From the TGA analysis (Figure 4.S8, ESI†) it is confirmed that the

weight percentage of captopril loaded is 16.4. Because captopril is hydrophilic in

nature, the drug molecules may locate preferentially adjacent to the hydrophilic

surface silanol, leading to a decrease pore size.

The drug loading is further confirmed by Fourier transform infrared (FTIR) studies

(Figure 4. S9, ESI†). FTIR spectra for the pure captopril showed peaks at 2880,

2955 and 2987 cm-1, corresponding to asymmetrical C-H stretching vibration of

captopril anion, and the characteristic bands at 2569, 1745, 1584 and 1475 cm-1

could be attributed to S-H, C=O of COOH group, C=O of amide and quaternary

carbon atom respectively. A sharp peak at 3745 cm-1 can be observed for MCM-41,

which is attributed to the isolated silanol groups and water molecules. The FT-IR

spectrum of captopril loaded MCM-41 sample shows weak shoulder at 2959 cm-1

which is attributed to symmetrical CH3 stretching mode of captopril and a band at

1749 cm-1 due to the COOH group on captopril.[30] The C=O of amide can be clearly

observed at 1646 cm-1, this shift is attributed to the weak interaction with the silanol

groups on the surface of MCM-41. The signal from isolated silanol disappears for

captopril loaded MCM-41. The corresponding peaks of the pure drug were observed

in the Cap-MCM-41 particles, confirming that the drug was loaded effectively.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 71 -

Figure 4.2 The morphology of floating tablet at different time points. (F1) Cur-MCM-

41(1.7nm), (F2) Physical mixture of curcumin and MCM-41, (F3) Pure curcumin, (F4)

Cur-MCM-41(2.1nm). All the formulations contain polymer and sodium bicarbonate.

The in-vitro drug release profile of floating tablets was performed in SGF, pH 1.2 at

37 ºC using Rosette rise apparatus (RRA) to mimic the environment of stomach

(Figure 4.S10, ESI†).[31] Four different formulations were studied to show the

importance of MSNs and its pore size in the release profile of curcumin (Table 4.S3,

ESI†). The total weight of tablets for four formulations and the amount of curcumin in

each tablet are the same. F1 and F4 are made with polymer and drug loaded MCM-

41 with the pore size of 1.7 nm and 2.1 nm, respectively. F2 is the physical mixture

of MCM-41 (2.1nm), polymer and drug. F3 is made of polymer and drug in the

absence of MSNs. Figure 4.2 showed the morphology of floating tablets (F1-F4) at

different time points of dissolution study. The diffusion of dissolution medium into the

tablets was clearly visible at 1 h as evidenced by the volume change. However, there

was no disintegration of tablets suggesting effective binding ability of HPMC and

MSNs.

The release profiles of curcumin loaded floating tablet F1-F4 are shown in Figure

4.3. The release rate of F4 Cur-MCM-41(2.1nm) was similar to conventional floating

tablet F3. There is no significant difference in the release profile of F2 (floating tablet

made from physical mixture) compared to F3 and F4. The dissolved curcumin from

floating tablets was less than 19% at 20 h in three groups (F2-F4). Remarkably, the

cumulative percentage drug release of F1 Cur-MCM-41(1.7nm) was twice that of

conventional floating tablet at 20 h (42.8%). The solubility of a substance is a

function of its particle size according to the Ostwald-Freundlich equation.[25] After

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 72 -

loading curcumin into MSNs, the size of curcumin is dependent on the pore size of

MSNs. The enhanced solubility of curcumin in F1 can be attributed to the use of

MSNs with a carefully chosen pore size of 1.7 nm, which has been demonstrated

before with the highest solubility enhancement. Additionally, it is noted that when

curcumin was physically mixed with MSNs there was no improvement in release

kinetics, indicating the encapsulation of curcumin into the nanopores is important for

the solubility enhancement.

For captopril floating tablets, two formulations were prepared. F5 was made from

drug loaded MCM-41, while F6 using HPMC polymers in the absence of MCM-41

(Table 4.S4, ESI†). As expected for F6 batch, 60% of captopril was released within

the first 6 h, and almost 100% release was observed at 8 h (Figure 4.4). This

observation indicates that in the case of conventional polymer based floating tablets,

swelling of HPMC allows quick exposure of the drug molecules to dissolution

medium, causing fast release of captopril into the SGF. While for floating tablet F5

made with MCM-41, a sustained drug release of captopril was observed reaching

100% only after 24 h. At all time points the release rate of floating tablet F5 was

slower than that of conventional tablet F6. Because the drug molecules are pre-

loaded inside the nanopores, the dissolution medium has to diffuse through two

barriers: the polymer matrix to induce the gelation of HPMC and the nanopores in

order to dissolve the drug molecules. Consequently, the floating tablet made from

MSNs gives rise to more control over hydrophilic drugs.

Figure 4.3 The release profile of curcumin from floating tablets. (F1) Cur-MCM-

41(1.7nm), (F2) Physical mixture of curcumin and MCM-41, (F3) Pure curcumin and

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 73 -

(F4) Cur-MCM-41(2.1nm). All the formulations contain polymer and sodium

bicarbonate.

Figure 4.4 The release profile of captopril from floating tablet. (F5) Cap- MCM-41,

(F6) Pure captopril. All the formulations contain polymer and sodium bicarbonate.

4.3 Conclusion

In conclusion, we have successfully prepared a new generation of floating tablets

using MSNs as a key formulation ingredient. With this finding, the advantages of

mesoporous materials can be further integrated into floating tablets with fine control

over the sustainable release of water-soluble drugs and, more importantly,

enhancing the solubility of poorly water-soluble drugs, an issue that cannot be solved

by conventional floating tablet technology. Our success has paved the way for

formulation improvement in localised drug release.

We acknowledge the support from the Australian Research Council, the Queensland

Government, the Australian National Fabrication Facility and the Australian

Microscopy and Microanalysis Research Facility at the Centre for Microscopy and

Microanalysis, The University of Queensland. We also thank The Pharmacy

Australia Centre of Excellence for providing access to tablet punching machine.

4.4 References

[1] C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli and J. S. Beck, Ordered

Mesoporous Molecular-Sieves Synthesized by a Liquid-Crystal Template

Mechanism. Nature, 1992, 359, 710-712.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 74 -

[2] V. Mamaeva, C. Sahlgren and M. Linden, Mesoporous silica nanoparticles in

medicine--recent advances. Adv Drug Deliv Rev, 2013, 65, 689-702.

[3] D. Douroumis, I. Onyesom, M. Maniruzzaman and J. Mitchell, Mesoporous silica

nanoparticles in nanotechnology. Crit Rev Biotechnol, 2013, 33, 229-245.

[4] C. Y. Lai, B. G. Trewyn, D. M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija and V. S.

Lin, A mesoporous silica nanosphere-based carrier system with chemically

removable CdS nanoparticle caps for stimuli-responsive controlled release of

neurotransmitters and drug molecules. J Am Chem Soc, 2003, 125, 4451-4459.

[5] K. K. Unger, D. Kumar, M. Grun, G. Buchel, S. Ludtke, T. Adam, K. Schumacher

and S. Renker, Synthesis of spherical porous silicas in the micron and submicron

size range: challenges and opportunities for miniaturized high-resolution

chromatographic and electrokinetic separations. J Chromatogr A, 2000, 892, 47-55.

[6] M. Grun, I. Lauer and K. K. Unger, The synthesis of micrometer- and

submicrometer-size spheres of ordered mesoporous oxide MCM-41. Adv Mater,

1997, 9, 254-&.

[7] S. H. Wu, Y. Hung and C. Y. Mou, Mesoporous silica nanoparticles as

nanocarriers. Chem Commun, 2011, 47, 9972-9985.

[8] X. Li, Q. R. Xie, J. X. Zhang, W. L. Xia and H. C. Gu, The packaging of siRNA

within the mesoporous structure of silica nanoparticles. Biomaterials, 2011, 32,

9546-9556.

[9] Y. Gao, Y. Chen, X. F. Ji, X. Y. He, Q. Yin, Z. W. Zhang, J. L. Shi and Y. P. Li,

Controlled Intracellular Release of Doxorubicin in Multidrug-Resistant Cancer Cells

by Tuning the Shell-Pore Sizes of Mesoporous Silica Nanoparticles. Acs Nano,

2011, 5, 9788-9798.

[10] M. Liong, J. Lu, M. Kovochich, T. Xia, S. G. Ruehm, A. E. Nel, F. Tamanoi and

J. I. Zink, Multifunctional inorganic nanoparticles for imaging, targeting, and drug

delivery.Acs Nano, 2008, 2, 889-896.

[11] D. S. Lin, Q. Cheng, Q. Jiang, Y. Y. Huang, Z. Yang, S. C. Han, Y. N. Zhao, S.

T. Guo, Z. C. Liang and A. J. Dong, Intracellular cleavable poly(2-dimethylaminoethyl

Chapter 4

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methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA

delivery in vitro and in vivo. Nanoscale, 2013, 5, 4291-4301.

[12] S. W. Song, K. Hidajat and S. Kawi, pH-Controllable drug release using

hydrogel encapsulated mesoporous silica. Chem Commun, 2007, 4396-4398.

[13] K. Zhang, W. Wu, K. Guo, J. F. Chen and P. Y. Zhang, Synthesis of

Temperature-Responsive Poly(N-isopropyl acrylamide0/Poly(methyl

methacrylate)/silica Hybrid capsules from Inverse Pickering Emulsion Polymerization

and Their Application in Controlled Drug Release. Langmuir, 2010, 26, 7971-7980.

[14] B. Tzankov, K. Yoncheva, M. Popova, A. Szegedi, G. Momekov, J. Mihaly and

N. Lambov, Indometacin loading and in vitro release properties from novel carbopol

coated spherical mesoporous silica nanoparticles. Micropor Mesopor Mat, 2013,

171, 131-138.

[15] Y. P. Xu, F. Y. Qu, Y. Wang, H. M. Lin, X. A. Wu and Y. X. Jin, Construction of a

novel pH-sensitive drug release system from mesoporous silica tablets coated with

Eudragit. Solid State Sci, 2011, 13, 641-646.

[16] Y. Z. Zhang, J. C. Wang, X. Y. Bai, T. Y. Jiang, Q. Zhang and S. L. Wang,

Mesoporous Silica Nanoparticles for Increasing the Oral Bioavailability and

Permeation of Poorly Water Soluble Drugs. Mol Pharmaceut, 2012, 9, 505-513.

[17] V. D. Prajapati, G. K. Jani, T. A. Khutliwala and B. S. Zala, Raft forming system-

An upcoming approach of gastroretentive drug delivery system. J Control Release,

2013, 168, 151-165.

[18] S. Strubing, H. Metz and K. Mader, Characterization of poly(vinyl acetate) based

floating matrix tablets. J Control Release, 2008, 126, 149-155.

[19] G. P. Andrews, T. P. Laverty and D. S. Jones, Mucoadhesive polymeric

platforms for controlled drug delivery. Eur J Pharm Biopharm, 2009, 71, 505-518.

[20] V. A. Eberle, J. Schoelkopf, P. A. Gane, R. Alles, J. Huwyler and M. Puchkov,

Floating gastroretentive drug delivery systems: Comparison of experimental and

simulated dissolution profiles and floatation behavior. Eur J Pharm Sci, 2014, 58, 34-

43.

Chapter 4

Floating tablets from meoporous silica nanoparticles

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[21] I. Jimenez-Martinez, T. Quirino-Barreda and L. Villafuerte-Robles, Sustained

delivery of captopril from floating matrix tablets. Int J Pharm, 2008, 362, 37-43.

[22] V. K. Pawar, S. Kansal, G. Garg, R. Awasthi, D. Singodia and G. T. Kulkarni,

Gastroretentive dosage forms: A review with special emphasis on floating drug

delivery systems. Drug Deliv, 2011, 18, 97-110.

[23] B. N. Singh and K. H. Kim, Floating drug delivery systems: an approach to oral

controlled drug delivery via gastric retention. J Control Release, 2000, 63, 235-259.

[24] A. O. Nur and J. S. Zhang, Recent progress in sustained/controlled oral delivery

of captopril: an overview. Int J Pharm, 2000, 194, 139-146.

[25] S. Jarnbhrunkar, M. H. Yu, J. Yang, J. Zhang, A. Shrotri, L. Endo-Munoz, J.

Moreau, G. Q. Lu and C. Z. Yu, Stepwise Pore Size Reduction of Ordered

Nanoporous Silica Materials at Angstrom Precision. J Am Chem Soc, 2013, 135,

8444-8447.

[26] Y. Zhang, Z. Zhi, T. Jiang, J. Zhang, Z. Wang and S. Wang, Spherical

mesoporous silica nanoparticles for loading and release of the poorly water-soluble

drug telmisartan. J Control Release, 2010, 145, 257-263.

[27] S. Yang, L. Z. Zhao, C. Z. Yu, X. F. Zhou, J. W. Tang, P. Yuan, D. Y. Chen and

D. Y. Zhao, On the origin of helical mesostructures. J Am Chem Soc, 2006, 128,

10460-10466.

[28] L. Mercier and T. J. Pinnavaia, Heavy metal ion adsorbents formed by the

grafting of a thiol functionality to mesoporous silica molecular sieves: Factors

affecting Hg(II) uptake. Environ Sci Technol, 1998, 32, 2749-2754.

[29] J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D.

Schmitt, C. T. W. Chu, D. H. Olson, E. W. Sheppard, S. B. Mccullen, J. B. Higgins

and J. L. Schlenker, A new family of mesoporous molecular sieves prepared with

liquid crystal templates. J Am Chem Soc, 1992, 114, 10834-10843.

[30] I. F. Alexa, M. Ignat, R. F. Popovici, D. Timpu and E. Popovici, In vitro controlled

release of antihypertensive drugs intercalated into unmodified SBA-15 and MgO

modified SBA-15 matrices. Int J Pharm, 2012, 436, 111-119.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 77 -

[31] P. Arya and K. Pathak, Assessing the viability of microsponges as gastro

retentive drug delivery system of curcumin: Optimization and pharmacokinetics. Int J

Pharm, 2014, 460, 1-12.

Supporting information

Materials and Methods

4.S1. Preparation of MCM-41 particles

MCM-41 material was synthesized using the method reported by Yang et al., with

slight modifications.1In a typical synthesis, 1.0 g of CTAB was dissolved in 480 g of

deionized water with 3.5 ml of NaOH (2 M) under stirring at 80˚C. Then, 6.7 ml of

TEOS was added into the mixture and kept under continuous stirring for an

additional 2 h. The resultant product was collected by filtration and dried at room

temperature. The surfactant templates were removed by calcination at 550˚C for 5 h.

The procedure for step wise pore size reduction of MCM-41 was followed according

to literature.2 MCM-41 material (0.8 g) was held on the porous disc and TEOS was

placed in the bottom chamber. The VVD apparatus is placed in the vacuum oven

(175 Torr) at 60˚C for 24 h. TEOS vapour molecules can only pass through the

porous disc and the mesoporous silica placed on the top of the disc. The vacuum

(175 Torr) helps in continuous contact between mesoporous silica and TEOS vapour

generated from the bottom chamber, so that TEOS molecules can be adsorbed on

the silica surface. After 24 h of the VVD process, the mesoporous silica materials

adsorbed with TEOS was calcined at 550˚C for 5 h.

4.S2. Loading of curcumin and captopril

Curcumin loading

Curcumin was loaded into MCM-41 with two different pore sizes 2.1 and 1.7 nm

using Rotavapor technique. To rotavapor flask (RF) containing 80 mg of silica

material, 20 mg of curcumin was added followed by addition of 5 ml of methanol.

This suspension was sonicated for 2 min in ultrasonic bath and the RF was affixed to

the rotavapor assembly and methanol was slowly evaporated at 50˚C. The solvent

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 78 -

evaporation procedure was continued for another 30 min to ensure complete solvent

removal. The obtained samples were denoted as Cur-MCM-41(1.7nm), Cur-MCM-

41(2.1nm).

Captopril loading

The captopril loading was done by wetting impregnation method. Initially 217.29 mg

of drug was dissolved in 10 ml of water. To the drug solution 200 mg of MCM-41was

added and kept for stirring at room temperature for 24 h. The drug loaded MCM-41

were separated from the solution by filtration and kept in vacuum overnight. The

obtained sample was denoted as Cap-MCM-41.

4.S3. Characterization

TEM images were taken using a JEOL 2100 microscope operated at 200Kv. The

samples were prepared by dispersing MCM-41 in ethanol, and then transferred to a

copper grid with dropper. SEM images were taken using a JEOL 7800 accelerating

voltage operated at 1Kv. SEM samples were prepared by cutting thin cross

sectional layer of the tablet and placed on carbon tape mounted on the stage. X-ray

diffraction (XRD) patterns were recorded on a German Bruker D8 X-ray

diffractometer with Ni-filtered Co Kα radiation (λ=1.79 A). The XRD patterns were

obtained at 40 kV and 30 mA with a step size of 0.01˚. Nitrogen adsorption-

desorption isotherms were measured at -196˚C by using a Micromeritics Tristar II

3020 system. The drug loaded samples were degassed at 50˚C overnight on a

vacuum line and pure MCM-41 is degassed at 180˚C for 6 h. The Brunauer-Emmett-

Teller (BET) method was utilized to calculate the specific surface area. The pore size

distribution curves were derived from the adsorption branches of the isotherms using

the Barrett-Joyner-Halanda (BJH) method. The total pore volume was calculated

from the amount adsorbed at a maximum relative pressure (P/P0).

Thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC)

measurements were performed by TGA/DSC Thermogravimetric Analyser (Mettler-

Toledo Inc) with a heating rate of 2˚C /min in air flow. Erweka hardness tester was

used to measure the hardness of the tablet.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 79 -

4.S4. Preparation of tablets

Preparation of floating tablets without and with curcumin

The floating tablets were prepared by a direct compression method. Weights of each

ingredient were taken according to Table S2, Table S3 for each formulation. Micro-

crystalline cellulose (MCC, bulking agent) was added in order to maintain the same

weight of each tablet in different formulations. All the ingredients were mixed

homogeneously with mortar and pestle for 10 min. Zinc stearate (a lubricant) was

added at last to the mixed powders and then mixed further for 3 min. Finally, the

powder was fed manually into die with spatula of a single punch tablet punching

machine, equipped with concave punches (10 mm diameter). The hardness of the

tablets is adjusted to 50 N by using Erweka hardness tester.

Preparation of captopril tablets

The floating tablets were prepared by direct compression method. Weights of each

ingredient are taken according to Table S4, for each formulation and mixed

homogeneously with mortar and pestle for 10 min. Zinc stearate was added to the

final mixed power and mixed for additional 3 mins. Finally, the powder was fed

manually into die with spatula of a single punch tablet punching machine, equipped

with concave punches (10 mm diameter). The hardness of the tablets is adjusted to

50 N by using Erweka hardness tester.

4.S5. Determination of MSNs content in the tablet

The amount of silica content in tablet after floating for 12 h in dissolution medium

was determined. The floating tablet F0 to FD was used in this test. F0 was prepared

without silica nanoparticles. After 12 h, the water content was removed by freeze

drying overnight. The obtained dry tablet was kept in furnace at 500˚C for 5 h. The

decomposition of all the ingredients except silica was below 500˚C (Melting point of

HPMC K100M-260˚C, Sodium bicarbonate-50˚C, zinc stearate-120-130˚C, Silica-

1600-1725˚C). This was confirmed by the control formulation F0. Thus the weight

after thermal treatment is attributed to the silica retained in the tablet after floating

test.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 80 -

4.S6. Dissolution studies

Dissolution studies for curcumin

The dissolution test was performed in rosette rise apparatus for maintaining sink

condition as reported by Gohel et al.3,4 The tablet was placed in a beaker (100 ml)

modified at the base with inverted v shaped glass tube, containing 70 ml of HCl

(0.1M) containing 0.8% tween 80, maintaining at 37˚C, stirred at 50 rpm. The burette

was mounted on the top of the beaker to deliver the dissolution medium at a flow

rate of 2 ml/min. At the same time 2 ml of medium was drained out of the beaker. At

regular time interval 1 ml of sample was collected from the beaker and replaced with

the same volume of fresh medium. Collected samples were filtered through 0.2 µm

membrane filter and analysed spectrophometrically at 424 nm.

Dissolution studies for captopril

The dissolution test was performed in 200 ml of HCl (0.1M), maintaining at 37˚C,

stirred at 50 rpm.5 At regular time interval 2 ml of sample was collected and replaced

with the same volume of fresh medium. Collected samples were filtered through 0.2

µm membrane filter and analysed spectrophometrically at 216 nm.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 81 -

Supplementary Figures and Tables

Figure 4.S1 XRD patterns (A) of a) Calcined MCM-41 with 2.1 nm b) Calcined MCM-

41 subjected to VVD process with 1.7 nm, N2 adsorption-desorption isotherms (B) of

calcined MCM-41 and MCM-41 subjected to VVD cycle and BJH pore size

distribution plots (C) of calcined MCM-41 and MCM-41 subjected to VVD cycle.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 82 -

Figure 4.S2 Transmission electron microscope (TEM) image (a) of MCM-41(1.7nm),

(b) MCM-41 (2.1nm); Field-emission scanning electron microscope (FE-SEM) image

(c) of MCM-41(1.7nm), (d) MCM-41 (2.1nm), (e) HPMC K100M, (f) Cross-section

image conventional floating subjected to 12 h of dissolution study.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 83 -

Figure 4.S3 A) The chemical structure of HPMC polymer. B) The amount of MSNs

remained in floating dosage form after 12 h of dissolution study analysed by TGA.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 84 -

Figure 4.S4 TGA of Curcumin, MCM-41, Cur-MCM-41(1.7nm), Cur-MCM-41(2.1nm)

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 85 -

Figure 4.S5 (A) XRD pattern of Curcumin, physical mixture of curcumin and MCM-

41, Cur- MCM-41 (1.7nm), Cur-MCM-41 (2.1nm) and MCM-41, and (B) Differential

Scanning Calorimetry of Curcumin, Cur-MCM-41 (1.7nm), Cur-MCM-41 (2.1nm) and

MCM-41.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 86 -

Figure 4.S6 (A) Nitrogen adsorption-desorption isotherms of Cur- MCM-41(1.7nm),

Cur-MCM-41(2.1nm) and (B) pore size distribution plot of Cur- MCM-41(1.7nm), Cur-

MCM-41(2.1nm).

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 87 -

Figure 4.S7 (A) Nitrogen adsorption-desorption isotherms of MCM-41, Cap- MCM-

41 and (B) pore size distribution plot of MCM-41, Cap-MCM-41.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 88 -

Figure 4.S8 TGA analysis of a) Captopril, b) Cap-MCM-41.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 89 -

`

Figure 4.S9 FTIR spectra of MCM-41, Captopril, Cap-MCM-41.

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 90 -

Figure 4.S10 A scheme showing the home-made Rosette rise apparatus.

Table 4.S1 Physicochemical properties of MCM41 (1.7nm), Cur- MCM-41-1.7nm,

MCM-41(2.1nm), Cur-MCM-41-2.1nm, MCM-41, Cap- MCM-41

Sample Surface area (m2/g)

Pore volume (cm3/g)

Pore size (nm)

Drug loading (% TGA)

MCM41(1.7nm) 806 0.57 1.7 Cur-MCM-41(1.7nm) MCM-41(2.1nm)

397 1333

0.24 0.81

1.7 2.1

20

Cur-MCM-41(2.1nm) MCM-41(2.1)

751 946

0.46 0.66

2.1 2.1

20.9

Cap- MCM-41 534 0.38 1.7 16.4

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 91 -

Table 4.S2 The compositions of floating tablets with bare MCM-41

Formulati

on

MCM-41

(mg)

HPMC

K100M

(mg)

Sodium

bicarbonate

(mg)

Zinc

stearate

(mg)

Total weight

of tablet (mg)

F0 0 60 33 4.4 97.4

FA 80 60 33 4.4 187.4

FB 120 60 33 4.4 217.4

FC 140 60 33 4.4 237.4

FD 160 60 33 4.4 257.4

Note: MCM-41 with a pore size of 2.1 nm was used.

Table 4.S3 Composition of curcumin floating tablet

Formu

lation

MCM-

41(mg)

Cur-

MCM-41

(mg)

Curc

umin

(mg)

HPMC

K100M

(mg)

Sodium

bicarbonate

(mg)

MCC

(mg)

Zinc

stearate

(mg)

Total

weight

of tablet

(mg)

F1 0 100 0 60 33 0 4.4 197.4

F2 80 0 20 60 33 0 4.4 197.4

F3 0 0 20 60 33 80 4.4 197.4

F4 0 100 0 60 33 0 4.4 197.4

Note: In F2, F4, MCM-41 with a pore size of 2.1 nm was used. In F1, MCM-41

with a pore size of 1.7 nm was chosen.

Table 4.S4 Composition of captopril floating tablet

Note: MCM-41 with a pore size of 2.1 nm was used.

Formula

tion

Cap-

MCM-41

(mg)

Captopr

il (mg)

HPMC

K100M

(mg)

Sodium

bicarbon

ate (mg)

MCC

(mg)

Zinc

stearate

(mg)

Total weight

of tablet

(mg)

F5 92 0 60 33 0 4.4 189.4

F6 0 15 60 33 77 4.4 189.4

Chapter 4

Floating tablets from meoporous silica nanoparticles

- 92 -

References:

[1] S. Yang, L. Z. Zhao, C. Z. Yu, X. F. Zhou, J. W. Tang, P. Yuan, D. Y. Chen and D. Y.

Zhao, On the origin of helical mesostructures. J Am Chem Soc, 2006, 128, 10460-10466.

[2] S. Jarnbhrunkar, M. H. Yu, J. Yang, J. Zhang, A. Shrotri, L. Endo-Munoz, J. Moreau, G.

Q. Lu and C. Z. Yu, Stepwise Pore Size Reduction of Ordered Nanoporous Silica Materials

at Angstrom Precision. J Am Chem Soc, 2013, 135, 8444-8447.

[3] P. Arya and K. Pathak, Assessing the viability of microsponges as gastro retentive drug

delivery system of curcumin: Optimization and pharmacokinetics. Int J Pharm, 2014, 460, 1-

12.

[4] I. Jimenez-Martinez, T. Quirino-Barreda and L. Villafuerte-Robles, Sustained delivery of

captopril from floating matrix tablets. Int J Pharm, 2008, 362, 37-43.

Chapter 5

Asymmetric silica nanoparticles

with tunable head-tail structures

enhances hemocompatibility and

maturation of immune cells

Asymmetric mesoporous silica nanoparticles (MSN) with controllable head-tail

structures have been successfully synthesized. The head particle type is tunable

(solid or porous) and the tail has dendritic large pores. The tail length and tail

coverage on head particles are adjustable. Compared to spherical silica

nanoparticles with a solid structure (Stöber spheres) or large-pore symmetrical MSN

with fully covered tails, asymmetrical head-tail MSN (HTMSN) show superior

hemocompatibility due to reduced membrane deformation of red blood cells and

decreased level of reactive oxygen species. Moreover, compared to Stöber spheres,

asymmetrical HTMSN exhibit a higher level of uptake and in vitro maturation of

immune cells including dendritic cells and macrophage. This study has provided a

new family of nanocarriers with potential applications in vaccine development and

immunotherapy.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 94 -

5.1 Introduction

Asymmetric nanoparticles with polymeric, [1-3] inorganic, [4-6] and polymeric-

inorganic [7-10] hybrid compositions have attracted much attention in diverse fields.

As an interesting family of porous materials with promising applications in

therapeutic delivery, mesoporous silica-based nanoparticles (MSN) with asymmetric

architectures have emerged only very recently.[10,11] Zhang et al. synthesized

magnetic mesoporous Janus nanoparticles with aspect ratios from 2:1 to 7:1 and

studied their magnetic properties.[5] Multicompartment MSN were synthesized via an

epitaxial growth of hexagonally mesostructured silica branches on cubic mesoporous

silica.[12] Asymmetric single hole mesoporous nanocages have been prepared by

growth of cubic periodic mesoporous organosilica (PMO) on the surface of dense

SiO2 nanoparticles followed by etching of SiO2. The particle was further

functionalized with upconversion nanoparticles (UCNP) and used for a co-delivery

system.[13] Recently, Kuroda and co-workers showed that MSN were asymmetrically

capped by organosilica.[14] The asymmetric MSN reported so far were mainly

prepared in aqueous systems, where relatively simple architectures with limited pore

sizes (< 10 nm) can be obtained. Moreover, the advantage of asymmetric structures

over symmetric ones has rarely been reported.

Recently, dendritic mesoporous silica nanoparticles (DMSN) prepared in water/oil

biphasic systems are emerging as a promising platform for biomedical applications.

[15-19] The unique central-radial pore structures offer vast advantages, such as

large pore size and highly accessible surface areas compared to conventional

MSN.[20-22] To the best of our knowledge, constructing asymmetric MSNs with a

highly controllable dendritic silica compartment with large pores has not been

reported.

5.2 Results and discussion

In this work, head-tail mesoporous silica nanoparticles (HTMSN) with controllable

structures have been successfully prepared for the first time using an oil/water

emulsion system. As shown in Scheme 5.1, spherical silica head particles with either

a solid or porous nature can be used to grow a dendritic tail with large pores (11-28

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 95 -

nm), forming asymmetrical HTMSN. The tail length and tail coverage on head

particles are adjustable. Compared to solid head particles or MSN with fully covered

Scheme 5.1 Illustration of synthesis of HTMSN and their advantages in

hemocompatibility and maturation of immune cells.

dendritic large pores, asymmetrical HTMSN show surprisingly higher

hemocompatibility towards red blood cells. Further, HTMSN were analyzed for

immunoadjuvant activity. Interestingly, we found that asymmetrical HTMSN exhibit a

higher level of uptake and in vitro maturation of antigen presenting cells compared to

spherical head particles. HTMSN were synthesized by dispersing monodispersed

Stöber spheres[23] as head particles (Figure 5.S1) with a uniform size of ~ 200 nm in

a water-chlorobenzene system, in which cetyltrimethylammonium chloride (CTAC)

was used as a structure directing agent, tetraethylorthosilicate (TEOS) as a silica

source and triethanolamine (TEA) as a catalyst (see Supporting Information). The

calcined product is named as HTMSNV-t-H, where V represents the volume of TEOS

(0.41, 1.25 and 3.75 mL), t is the reaction time (2, 3, 6, 9, 12 and 24 h) and H is the

amount of head particles (0, 30 60,100, 200 and 400 mg).

Figure 5.1 shows transmission electron microscope (TEM) and field-emission

scanning electron microscopy (FE-SEM) images of HTMSN prepared with varied

amount of TEOS V at a fixed t of 12 h and H of 60 mg. TEM images of HTMSNV-12-60

(V=0.41, 1.25 and 3.75 mL, Figure 5.1A-C) clearly show a monodispersed

asymmetric head-tail nanostructure with a uniform particle size of 377±5, 399±5, and

427±5 nm, respectively. The particle size and tail coverage of HTMSNV-12-60

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 96 -

Figure 5.1 TEM and SEM images of HTMSN prepared at varied TEOS volume V. (A,

D, G) HTMSN0.41-12-60, (B, E, H) HTMSN1.25-12-60, (C, F, I) HTMSN3.75-12-60.

increased with increased TEOS amount V. Dynamic light scattering (DLS)

measurements were used to further determine the particle size and polydispersity of

HTMSNV-12-60 (Figure 5.S2). The size and polydispersity index (PDI) values of

HTMSNV-12-60 (V=0.41, 1.25, 3.75 mL) were 392, 408, 478 nm and 0.18, 0.12, 0.20,

respectively. The DLS sizes exceeded by 10-20 % of the particle diameters

determined by TEM analysis due to the hydration of particles. The low PDI values

suggest that HTMSNV-12-60 are monodispersed without significant aggregation.

Figure 5.2 TEM images (A-E) of HTMSN1.25-t-60 collected at different reaction time

(t=2, 3, 6, 9 and 24 h, respectively). (F) The variation of tail length (black bar) and

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head coverage (grey bar) as a function of t. Bars represent the mean ± SEM (n=20).

Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple

comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, ns (P ≥

0.05) indicates no significant difference.

The variation in TEOS volume V has a significant impact on the head-tail structure

as shown by TEM images observed at higher magnifications. HTMSN0.41-12-60

synthesized under a low TEOS amount have a tail length of ~ 80 nm (shown by a

solid white line) and a uniform head coverage of porous shell (indicated by dotted

circles) with a thickness of ~ 12 nm (Figure 5.1 D). The tail has a dendritic-like

radially arrayed pore structure with large pores (> 10 nm), which are clearly seen

under SEM observations (Figure 5.1G). The porous layer fully covered on the head

has relatively smaller pores (< 3 nm, Figure 5.S3). When V is increased to 1.25 and

3.75 mL, the length of both tail and head coverage is increased (summarized in

Figure 5.S4). Increases in tail length and coverage also lead to tails with larger sizes,

eventually an umbrella like structure is formed in HTMSN3.75-12-60 where the overall

size of tail is larger than the head. For HTMSN1.25-12-60, radially aligned mesopores

can be observed on the head surface with sizes similar to that in the tail region

(Figure 5.1E). However, for HTMSN3.75-12-60, it appears that the mesopore sizes in

both head and tail regions (Figure 5.1F) are smaller compared to HTMSN1.25-12-60.

The relatively larger pore sizes in the head region can be observed for HTMSN1.25-12-

60 (Figure 5.1H), but not for HTMSN3.75-12-60 (Figure 5.1I).

To characterize the porous nature of HTMSN, N2 sorption analysis was conducted.

The nitrogen adsorption and desorption plots of three HTMSNV-12-60 (V=0.41-3.75)

samples show type IV isotherms (Figure 5.S5A), the pore size distribution curves are

shown in Figure 5.S5B. As summarized in Table 5.S1, all three samples have dual

pores, one set of large pores (10-30 nm) and another set of small pores (1-3 nm).

When V is increased from 0.41 to 1.25 and 3.75 mL, the pore sizes decrease for

both large (28, 16 and 11 nm) and small pores (2.7, 2.1 and 1.8 nm). In contrast,

both the surface area and pore volume increase with increasing V.

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In order to elucidate the evolution of tail on the head, a time-resolved study was

conducted. The collected samples at 2, 3, 6, 9 and 24 h are denoted HTMSN1.25-t-60

(t=2-24 h). TEM images of the samples are shown in Figure 5.2A and Figure 5.S6 (at

low magnification to show the uniform structures). As shown in Figure 5.2A, at 2 h a

small tail with dendritic large pores (> 10 nm) and a length of ~ 20 nm was formed.

When t was extended to 3 h, a thin layer with small mesopores (< 3 nm) uniformly

covered on the head with a thickness of ~ 7 nm. At the same time the tail length

increases to ~ 56 nm. When t was further increased (6-24 h), both the

length/thickness of the tail/porous shell increased continuously (Figure 5.2F). When t

was not longer than 9 h, only small mesoporous (< 3 nm) were observed in the

porous shell covering the head particles (Figure 5.2D). Another dendritic porous

layer with structures similar to that in the tails was formed after 12 h (Figures 5.1E

and 2E).

DLS measurements reveal a mean particle size of 255, 295, 342, 396, 458, and 531

nm for HTMSN1.25-t-60 samples collected at 0, 2, 3, 6, 9 and 24 h (Figure 5.S7),

consistent with both TEM (Figure 5.S6) and SEM (Figure 5.S8) results. N2 sorption

analysis of HTMSN1.25-t-60 samples (Figure 5.S9) shows that large pores (> 10 nm,

derived from the dendritic tail region) appeared at t = 2 h compared to solid silica

head particles, in accordance with the SEM (Figure 5.S8B) and TEM (Figure 5.2A)

observations. However, small sized mesopores (~ 2 nm) attributed to the head cover

were generated at t = 3 h, also in agreement with the TEM results (Figure 5.2B). The

BET surface area and pore volume both increased with increases in the tail length

and thickness of head cover at prolonged t (Table 5.S2).

The pore structures of HTMSN1.25-3-60 and HTMSN1.25-12-60 were studied by electron

tomography (ET).[24-26] As shown by the ET slice, when t is 3 h a dendritic porous

tail with large pores and a mesoporous layer homogeneously covering the head with

smaller pores were observed (Figure 5.S10A). The small-sized mesopores also

appeared in the inner layer of tail region. For HTMSN1.25-12-60 obtained at 12 h, it can

be seen that bimodal mesopores were located on both head and tail nanostructures

(Figure 5.S10B). The thickness of the small-sized mesoporous layer in the head and

tail region is ~ 15 and > 50 nm, respectively. At the outer layer in both the head and

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tail areas, dendritic large mesopores were also observed, in accordance with N2

sorption results.

We further studied the influence of the head amount on the final structures for

sample HTMSN1.25-12-H by varying the head amount H from 0-400 mg in the reaction

system while keeping the other synthesis conditions unchanged. Without adding

dense silica spheres as the head (H=0), mesoporous silica nanoparticles with a

spherical core-cone structure (MSN-CC) [27] were produced with ~ 200 nm in

diameter (Figure 5.S11A). A mixture of MSN-CC and HTMSN particles was observed

for HTMSN1.25-12-30 due to the limited number of head particles in the system for the

production of HTMSN (Figure 5.S11B). The obvious difference in morphology of tail

was observed between the samples prepared with 60 mg of head (HTMSN1.25-12-60,

Figure 5.S11C) and greater than that (HTMSN1.25-12-100, HTMSN1.25-12-200 and

HTMSN1.25-12-400). When the seed amount is larger than 60 mg, the head-tail

morphology is not clear (TEM images Figure 5.S11D-F). HTMSN1.25-12-400 has a

nearly spherical morphology with a fully covered dendritic shell and a mean particle

size of ~300 nm (Figures 5.S7, 5.S11F, G), suggesting that the head particle amount

is another important parameter to produce high yield HTMSN.

In our synthesis system, the silica head particles were negatively charged before

adding into reaction system ( potential: –31.9 mV). When the

cetyltrimethylammonium cations (CTA+) were introduced into the aqueous solution

containing silica nanoparticles, the surface of head particles changed to positively

charged ( potential: +33.5 mV) due to electrostatic attraction. After the addition of oil

(chlorobenzene) containing TEOS into the aqueous solution at a stirring speed of

400 rpm, an oil-in-water emulsion (Figure 5.S12 A) was formed with an average oil

droplet size of ~ 2-5 µm (Figure 5.S12 B). The identification of emulsion type was

conducted by adding either oil or water. The added chlorobenzene was separated

from the emulsion, indicating that oil is the dispersed phase. The emulsion remained

stable after dilution with water, indicating that the continuous phase is water in the

oil-in-water emulsion.

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As reported in literature, [29] silica particles may arrange at the oil/water interface as

an emulsifier to stabilize the emulsion droplet (Scheme 5.2A). Hydrolysis and

condensation of TEOS leads to negatively charged silicate species, which

cooperatively self-assemble with CTA+ as structure-directing agents to form the first

mesostructured layer with relatively small pore sizes.[28] At the same time

nucleation and growth of tails occurs preferentially on one side, possibly near the

water phase, leading to the formation of HTMSN (Scheme 5.2B).

Scheme 5.2 Schematic illustration of (A) an oil-in-water droplet stabilized by Stöber

spheres and (B) the orientation of dendritic tail growth.

To support the proposed mechanism, the emulsion in the HTMSN1.25-9-60 reaction

system was directly treated by vacuum evaporation for 24 h for SEM observation. As

shown in Figure 5.3A, some partially collapsed hollow spherical shapes can be

observed. One clear hollow structure is shown in Figure 5.3B. The size of the hollow

sphere is 2.5 µm, similar to the size of emulsion droplets. Interestingly, the wall of the

hollow sphere is composed of one layer of asymmetric particles. Because the

sample is not washed nor calcined in order to reduce the damage to sample, the tail

structure is not that clear compared to the calcined samples. Nevertheless, the head-

tail morphology can still be distinguished with the spherical head towards the hollow

cavity while the non-spherical tail outwards. The above observations are consistent

with Scheme 5.2: Stöber spheres stabilize the oil-in-water emulsion droplets and the

growth direction of dendritic tail is towards water.

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Figure 5.3 SEM images of the HTMSN1.25-9-60 emulsion at low (A) and high (B)

magnifications.

The observation of MSN-CC without a head-tail asymmetrical morphology in the

absence of head particles (H=0) is consistent with the above mechanism. To test

whether the emulsion system is essential for the formation of head-tail structures, we

also carried out reactions at a slower stirring speed (50 rpm) under which two

phases were maintained. The TEM images of the resulting sample demonstrated a

homogenous growth on the head (Figure 5.S13), suggesting that HTMSN cannot be

prepared under conventional biphasic conditions.

From the time-dependent growth studies, it is proposed that the formation of the

large-pore dendritic tail is similar to what we reported before [27] and can be

predicted by reference to the surfactant packing parameter (g). [28] It is reported that

surfactant CTA+ presented in aqueous solutions forms spherical micelles,[29] thus at

early reaction time (< 2 h) silica coated composite micelles aggregate together to

form mesostructural coating on head region with small pores. The organic cosolvent

(chlorobenzene in our case) diffuses into hydrophobic region of CTA+ micelles with

time, leading to structural transformation to bilayered structures (g≈1).[30] Thus at 2

h, large pore dendritic tail with a pore size of ~ 15 nm was observed (Figure 5.2A).

The diffusion of chlorobenzene into CTA+ micelles is faster near the oil phase

(where the nucleation of tails occurs) than in aqueous solution, therefore only

composite micelles are coated at the head region until 9 h of reaction (Figure 5.2D),

afterwards dendritic shell is also generated in addition to the dendritic tail (Figure

5.2E).

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The proposed mechanism can be used to explain the influence of TEOS amount.

When the reaction time is kept at 12 h, reducing V from 1.25 to 0.41 mL leads to less

silica source available in the system, thus further growth of dendritic shell in the head

region is inhibited. When V is increased to 3.75 mL, HTMSN3.75-12-60 is formed with

increased surface area and pore volume due to increased mesoporous

compartments over dense silica head particles.

Our approach to synthesize asymmetrical HTMSN can be applied to head particles

with other structures. To demonstrate the formation of dendritic tails on porous head

particles, dendritic MSNs (DMSNs) with an average particle size of ~ 150 nm (Figure

5.S14) and a pore size ~ 4 nm (Figure 5.S15, Table 5.S4) were prepared according

to a previous report [31] and used as head particles in our synthesis. Figure 5.4A

clearly shows the formation of head-tail morphology with an asymmetrical

architecture (named as HTDMSN1.25-12-60). The ET slice cutting from the centre of

HTDMSN1.25-12-60 is displayed in Figure 5.4B, showing small mesopores in the head

region and large pores (~ 16 nm) in the tail. The surface area and total pore volume

of HTDMSN1.25-12-60 (303 m2/g and 0.47 cm3/g, Table 5.S4) with a short tail length

were slightly higher than those of HTMSN1.25-6-60 with a solid head by a longer tail

(295 m2/g and 0.35 cm3/g, Table 5.S2). These results suggest that our approach is

versatile to grow dendritic tail on heads with either a solid or porous nature.

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Figure 5.4 TEM image (A) and, ET slice (B) of HTDMSN1.25-60-12.

In order to highlight the unique properties of asymmetrical HTMSN over symmetrical

silica nanoparticles, their hemolytic activity was compared. HTMSN synthesized at

different reaction time (HTMSN1.25-t-60, t=2, 3, 6, 9, 12 h) with tailored tail length

(Figure 5.2) were chosen. For comparison, two symmetrical silica nanoparticles with

only head or tail structure were included: the solid Stöber sphere and HTMSN1.25-12-

400 (fully covered by dendritic pores, Figure 5.S11F, G). All the samples included in

this comparison share a similar surface zeta potential ranging from -30 to -40 mV

(Table 5.S2, S5). A dose dependent hemolysis assay was conducted for all these

samples. As shown in Figures 5.S16 and 5.S17, the hemolysis percentage increases

as the particle concentration increases from 20 to 160 µg/mL for all these samples.

To be noticed, only the symmetrical nanoparticles, Stöber spheres and HTMSN1.25-12-

400, show significantly higher hemolytic activity compared to all asymmetrical

HTMSNs.

By further increasing the particle concentration to 240 µg/mL (Figure 5.5),

HTMSN1.25-12-400 show the highest hemolysis percentage of 92.0±1.7%, followed by

Stöber spheres (26.2±0.7%). The hemolysis percentages of HTMSN1.25-t-60 (t= 2, 3,

6, 9, 12, 24 h) exhibit a tail length dependent behaviour. Once the dendritic large-

pore tail is formed on Stöber spheres as head particles at 2 h (HTMSN1.25-2-60), the

hemolysis percentage decreased rapidly from ~26% to 4.2±0.6%. With increasing

reaction time and tail length the hemolysis percentages decreased (t=3 h, 4.1±0.8%,

t=6 h, 2.9±1.0%), reaching the lowest value of 1.6±0.3% for HTMSN1.25-9-60. When

the reaction time is prolonged to 12 h when the head particles were fully coated with

dendritic structures (HTMSN1.25-12-60), the hemolysis percentage increased to 6.9±

1.1%. The results demonstrate that the asymmetrical HTMSN have advantages over

Stöber spheres by introducing a tail with large pores and reduced hemolysis, and

also over HTMSN1.25-12-400 with large-pore but significantly high hemolysis. [32]

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Figure 5.5 Hemolytic percentages of mouse RBCs upon incubation with different

nanoparticles at 240 µg/milk D.I water and PBS was used as positive and negative

control, respectively. Bars represent the mean ± SEM (n=3). All samples were

compared with HTMSN1.25-12-400.

The hemolytic activity of DMSNs, HTDMSN1.25-12-60 and MSN-CC was also studied.

MSN-CC show a significant increase in hemolysis percentage from 3% at 20 µg/mL

to 90% at 240 µg/mL (Figure 5.6, Figure 5.S18), similar to HTMSN1.25-12-400. For

comparison, only a slight increase in hemolytic activity can be seen in both DMSN

(from 3% to 7%) and HTDMSN1.25-12-60 (from 2 to 5%). The lower hemolytic activity of

small-pore DMSN (4 nm) compared to large-pore MSN-CC (32.8 nm) is consistent

with a literature report. [33] Nevertheless, the asymmetric HTDMSN1.25-12-60 with a

large-pore dendritic tail also display highly improved safety with RBCs compared to

MSN-CC (7% vs 90%). It is concluded that the asymmetric morphology significantly

improves the hemocompatibility, and this concept is applicable in head particles with

both solid and nonporous nature.

The influence of surface charge, [34-36] shape and pore size, [37-38] particle size

[33] and aspect ratio [32] of conventional MSNs on hemolytic activity has been

reported. DMSN with highly accessible and large mesopores have emerged recently

as promising carriers for intracellular delivery of large biomolecules. [20,27]

However, their hemolytic activity has received less attention. Our studies have

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demonstrated their potential risk causing high hemolysis. The fabrication of

unsymmetrical HTMSN with large-pore tails and significantly improved

hemocompatibility provides new candidates and approaches to overcoming this

limitation.

Figure 5.6 Hemolytic percentages of mouse RBCs upon incubation with different

particles at various concentrations.

To understand the underlying reason of the particle symmetry dependent

hemocompatibility, symmetrical particles Stöber spheres, HTMSN1.25-12-400 and one

asymmetrical particle HTMSN1.25-12-60 were chosen to study two possible

contributions: extracellular reactive oxygen species (ROS) generation[39] and the

contact mode of nanoparticles with RBC membrane.[38,40] It has been reported that

Si-O three-membered rings can be formed on amorphous silica nanoparticle surface

after high temperature calcination, and will further cleave and react with water

molecules thus inducing hydroxyl free radicals in solution.[36] ROS generated by

silica nanoparticles could result in peroxidation of cell components of RBCs, causing

cell lysis. Figure 5.7A shows that HTMSN1.25-12-400 with the highest hemolysis

percentages have a significantly higher extracellular ROS level than HTMSN1.25-12-60,

suggesting the ROS level contributes to hemolytic activity of HTMSN1.25-12-400.

However, there is no significant difference in ROS level between Stöber spheres and

HTMSN1.25-12-60. Considering the large difference in hemolytic activity between

Stöber spheres and HTMSN1.25-12-60 (Figure 5.5), we speculate that the low ROS

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level is not the only important parameter for the excellent hemocompatibility of

unsymmetrical HTMSN1.25-12-60.

The interaction between silanol groups on silica surface and RBC membrane is

recognized as one of the main reason for hemolysis. As reported by Slowing et al,

compared to a solid silica sphere, a mesoporous silica nanosphere could have

reduced silanol density contacting with RBCs, thus decreased hemolytic activity.[41]

However, in our study, HTMSN1.25-12-400 with symmetrical coverage of large

mesopores show much higher hemolysis than solid Stöber spheres and HTMSN1.25-

12-60, which indicates that the silanol groups interacting with RBCs may not be the

dominant reason to explain for the hemolysis difference in our particles.

SEM image shows the intact biconcave disks of free RBCs (Figure 5.S19A). A

number of HTMSN1.25-12-400 (Figure 5.7B) were engulfed by RBC membranes,

inducing a strong local membrane deformation. The Stöber spheres (Figure 5.7C)

mainly adhered to RBC membranes with slight local membrane deformation. In the

case of HTMSN1.25-12-60 (Figure 5.7D), the asymmetric particles lie flatly on the

surface of RBC with no obvious membrane deformation (indicated by red arrows).

Although a large number of HTMSN1.25-12-60 were found attached to the RBCs (Figure

5.S19 B,C) similar to Stöber spheres and HTMSN1.25-12-400, the normal biconcave

shape is maintained similar to control RBCs (Figure 5.S19A).

To quantify the cellular uptake performance of Stöber spheres, HTMSN1.25-12-60, and

HTMSN1.25-12-400 in RBCs, fluorescein isothiocyanate (FITC) was conjugated to silica

particles and incubated with RBCs for 2 h for flow cytometry analysis. Almost all

RBCs (98.7%) are positive to FITC-HTMSN1.25-12-400 (Figure 5.S20), suggesting

strong interaction between HTMSN1.25-12-400 and RBCs. In contrast, FITC positive

percentages of RBCs are significantly lower after incubation with FITC-HTMSN1.25-12-

60 (76.8%) and FITC-Stöber spheres (60.9 %). The overall trend in RBC uptake

performance is in good agreement with SEM observations.

It is suggested that the unique head-tail morphology makes it difficult for the particle

to contact the RBC membrane in a vertical manner, either through the head or the

tail region. Instead, the "flat" mode is preferred, leading to reduced contact sites and

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surface area compared to symmetrical particles. Nanoparticle encapsulation by

RBCs is dependent on the binding energy of nanoparticles toward RBC membranes

and bending energy of RBC membranes. [40] The successful particle encapsulation

occurs when the released binding energy conquers the desired bending energy. The

reduced contact sites and surface area of asymmetric HTMSN1.25-12-60 than

symmetric particles (HTMSN1.25-12-400, Stöber spheres and MSN-CC) toward RBC

membrane determines less binding energy, leading to decreased particle penetration

inside RBCs and subsequent cell membrane deformation. It is concluded that both

the ROS level and contact mode with RBC membrane are important for the

hemolytic property of nanoparticles.

In recent years, engineered MSN have been extensively studied as a new class of

adjuvants, stimulating robust antibody [42-45] or cellular immunity responses [46-49]

of antigens with a great potential in disease and cancer treatment. For example, a

recent report revealed that hollow mesoporous silica nanospheres acted as effective

cancer vaccine adjuvants, significantly inhibiting tumour growth compared to the

Figure 5.7 (A) Extracellular ROS generation of three nanoparticles and SEM images

of RBCs incubated at room temperature for 2 h with (B) HTMSN1.25-12-400, (C) Stöber

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spheres and (D) HTMSN1.25-12-60. Scale bar is 10 µm. Scale bar is 10 µm. In (A), bars

represent the mean ± SEM (n=3).

commercial adjuvant Alum. [47] However, it is not clear to date whether the shape of

MSN will have influence on modulating immunological response, even though there

are some reports on the shape effect on adjuvanticity of non-silica nanomaterials.

[50-52]

We compared the uptake level of asymmetrical HTMSN0.41-12-60 and HTDMSN1.25-12-

60, symmetric Stöber spheres, MSN-CC and DMSN, using DCs and macrophages.

Fluorescein isothiocyanate (FITC) was conjugated to silica particles and incubated

with freshly isolated mouse splenocytes in which DCs and macrophages were

marked with CD11c-A600 and F4/80-APC-Cy7 antibodies, respectively. The

resultant cell-associated fluorescence was measured by flow cytometer (Figure

5.S21). As shown in Figure 5.8, the positive percentages of DCs and macrophage

cells for Stöber spheres and DMSN are only ~40-50%. However, up to 92±0.1%,

77±0.4 % DCs and 91±3.2%, 85±1.6% macrophages are positive to HTMSN0.41-12-60

and HTDMSN1.25-12-60. The uptake of MSN-CC (48±2.8% in DCs, and 68±3.3% in

macrophages) was slightly higher than Stöber spheres in macrophages, but also

lower than asymmetric structures.

Figure 5.8 Flow cytometric analysis showing FITC positive (A) CD11c+ DCs and (B)

F4/80+ macrophages treated with FITC conjugated Stöber spheres HTMSN0.41-12-60,

HTDMSN1.25-12-60, MSN-CC and DMSN. Bars represent mean ± SEM (n=3).

The head-tail particles (HTMSN0.41-12-60 and HTDMSN1.25-12-60) significantly enhance

APC uptake compared to conventional symmetrical ones (Stöber spheres, MSN-CC

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and DMSN), possibly because the head-tail morphology provides a geometry

favourable for phagocytosis.[53] Enhanced uptake of non-spherical nanoparticles

than spherical ones was also observed in non-phagocytic human cervical cancer

cells (HeLa).[54] The difference in the uptake of asymmetric structures between

APCs and RBCs may be attributed to their different roles because macrophages and

other APCs are professional scavengers to internalize foreign particles. Our results

have shown that controlling the morphology of nanoparticles is an alternative

approach to achieve targeting towards APCs, even without any targeting ligands,

which is important for applications such as vaccines and immunotherapy.

Next, we studied the maturation of APCs induced by these particles in the presence

or absence of a model antigen (ovalbumin, OVA) to highlight the potential application

of asymmetrical nanoparticles as potent immunoadjuvants. Splenocytes were

incubated with bare particles and OVA loaded particles followed by specific

fluorochrome-coupled antibody staining for analysing the expression of costimulatory

molecules (CD40 and CD86), which are important signals for triggering CD4+ and

CD8+ T lymphocytes. The cell population of DCs and macrophage was determined

by CD11c+ and F4/80+ mean fluorescent intensity (MFI) during FACS analysis,

respectively.

For bare particles, asymmetric HTMSN0.41-12-60 and HTDMSN1.25-12-60 induced

averagely higher levels of CD40 and CD86 expression on DCs and macrophages

than symmetric Stöber spheres, MSN-CC and DMSN, although the difference is not

always significant (Figure 5.9). The provoked APC maturation by asymmetric

particles over symmetric ones can be explained by their enhanced cellular uptake

(Figure 5.8). [51,55]

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Figure 5.9 Flow cytometry analysis of maturation marker CD40 (A, C) and CD86 (B,

D) expression levels on CD11c+ DCs (A, B) and F4/80+ macrophages (C, D)

triggered by Stöber spheres, HTMSN0.41-12-60, HTDMSN1.25-12-60, MSN-CC and

DMSN. Bars represent mean ± SEM (n=3).

For comparison, APC maturation level stimulated by Alum was also studied. As

shown in Figure 5.S22A, Alum shows significantly higher maturation level in both

DCs and macrophages than asymmetric HTMSN0.41-12-60. However, the numbers of

live DCs and macrophages treated with Alum are much lower than those treated with

asymmetric HTMSN0.41-12-60 (Figure 5.S22B), indicating a significantly higher toxicity

of Alum to APC. Our asymmetric HTMSN0.41-12-60 or HTDMSN1.25-12-60 presents a

promising adjuvant with a balanced adjuvant efficiency and safety. Alum has been

widely used as adjuvants in prophylactic vaccines in human but show weaker

therapeutic efficacy in anticancer activity comparing with silica materials.[47] Our

HTMSN with improved safety have a great potential as promising adjuvants in

therapeutic cancer vaccines. We also investigated the capability of OVA (20 µg/mL)

loaded particles (200 µg/mL) in triggering CD40 and CD86 expression on APCs

(Figure 5.S23) and the observation is consistent with the data obtained from the

blank nanoparticles. However, the overall expression level of CD40 and CD86

triggered by OVA loaded particles is much higher than bare particles, which is

consistent with previously reported results.[56] Delivery of encapsulated antigen in

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nanoparticles provides synergistic effect in surface marker upregulation compared to

either soluble antigens or bare nanoparticles.[57] These results suggest that the

asymmetrical particles are more potent adjuvants to trigger the APC maturation than

conventional symmetrical particles.

5.3 Conclusion

In conclusion, asymmetric HTMSNs with a unique head-tail morphology, adjustable

large-pore dendritic tail structures (tail length and tail coverage) and tunable head

structures (solid or dense) have been successfully synthesized for the first time using

an oil/water emulsion system. The HTMSNs showed a tail length dependent

hemolytic activity, which is dramatically lower than conventional symmetric

nanoparticles with solid or dendritic mesoporous structures. The superior

hemocompatibility was attributed to the reduced ROS generation and the “flat”

contact mode to RBCs membrane. As a proof of concept, we further demonstrated

the enhanced uptake of HTMSN into APCs compared to that of Stöber spheres,

which in turn led to higher level of maturation. Our contribution provides a new family

of asymmetric large-pore MSNs with potential applications as efficient

immunoadjuvants and delivery vehicles for antigens or therapeutic biomolecules.

5.4 References

[1] Wang, Y. H.; Zhang, C. L.; Tang, C.; Li, J.; Shen, K.; Liu, J. G.; Qu, X. Z.; Li, J. L.;

Wang, Q.; Yang, Z. Z. Emulsion Interfacial Synthesis of Asymmetric Janus Particles.

Macromolecules 2011, 44, 3787.

[2] Park, J. G.; Forster, J. D.; Dufresne, E. R. High-Yield Synthesis of Monodisperse

Dumbbell-Shaped Polymer Nanoparticles. Journal of the American Chemical Society

2010, 132, 5960.

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Supporting information

Materials and Methods

5.S1. Materials

Chemicals: Cetyltrimethylammonium chloride (CTAC) solution (25 wt% in H2O);

triethanolamine (TEA); chlorobenzene and tetraethylorthosilicate (TEOS, >98%), 3-

aminopropyl triethoxysilane (APTES), fluorescein isothiocyanate (FITC), ammonia

aqueous solution (28 wt%) and ethanol were purchased from Sigma-Aldrich.

PE/CY7-CD11c, BV605-F4/80, FITC-CD40 and, APC-CD86 were from BD

Biosciences. All chemicals were used as received without further purification.

5.S2. Synthesis of dense SiO2 head nanoparticles

Uniform nonporous silica nanoparticles with a smooth surface were synthesized

using a well-known method developed by Stöber et al. [1] Typically, absolute ethanol

(50 mL) was mixed with deionized water (3.8 mL) and ammonium hydroxide solution

(2 ml, 28%) at 308 K. Then, TEOS (2.8 mL) was added to the solution under

vigorous stirring. After 6 h, the as-synthesized nanoparticles were separated by

centrifugation, and washed with ethanol and deionized water. The final product was

obtained by drying at 373 K overnight.

5.S3. Synthesis of dendritic nanoparticles

Dendritic mesoporous silica nanoparticles were synthesised according to literature

with slight modification. [2] At first, 0.18 g of TEA was weighed in 100 ml round

bottom flask. To flask 24 mL of CTAC (25 wt%) solution and 36 Ml of water were

added and stirred slowly at 60 °C for 1 h. After 1 h, 16 mL of cyclohexane and 4 mL

of TEOS was slowly added from sides of the flask and left the reaction under slow

stirring for 24 h. The particles were collected after 24 h by centrifugation and washed

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for several times with ethanol. The surfactant was removed by calcination at 650 °C

for 6 h. The calcined product were named as DMSN.

5.S4. Synthesis of head-tail nanoparticles

In a typical synthesis, 60 mg of Stöber silica spheres of ~200 nm size were

dispersed in 6 mL of milliQ water and 0.035 g TEA by sonication for 15 min. Then, 4

mL of CTAC (25 wt%) solution was added into the solution and kept at 60 °C at a

stirring speed of 400 rpm for 1 h. After stirring for 1 h, 8.75 mL of chlorobenzene and

a pre-determined volume of TEOS (V=0.41, 1.25, 3.75 mL) was added into the

above solution. The reaction was continued at 60 °C for 12 h. Particles were

collected by centrifugation at 15,000 rpm, washed with water and ethanol twice and

dried at 50 °C overnight. The calcined products were named as HTMSNV-t-H, where V

represents the volume of TEOS, t is the reaction time and H is the amount of head

particles. In this synthesis, the final products were labelled as HTMSN0.41-12-60,

HTMSN1.25-12-60, HTMSN3.75-12-60, respectively. We also conducted experiments to

study the influence of reaction time and head amount. The particles synthesized at

reaction time (t=2, 3, 6, 9, 12 and 24) and head amount (H=0, 30, 60, 100, 200 and

400 mg).

The synthesis of HTDMSN1.25-12-60 was similar to that of HTMSN1.25-12-60, where 60

mg of uncalcined DMSN were dispersed in 6 ml of milliQ water and 0.035 g TEA by

sonication for 15 min. Then, 4 mL of CTAC (25 wt%) solution was added into the

solution and kept at 60 °C at a stirring speed of 400 rpm for 1 h. After stirring for 1 h,

8.75 mL of chlorobenzene and a 1.25 mL of TEOS was added into the above

solution. The reaction was continued at 60 °C for 12 h. Particles were collected by

centrifugation at 15,000 rpm, washed with water and ethanol twice and dried at 50

°C overnight. The surfactant was removed by calcination at 650 °C for 6 h. The

calcined product were named as HTDMSN1.25-12-60.

5.S5. Amino group modification of silica nanoparticles

Amino silane was grafted onto the surface of Stöber spheres, HTMSN0.41-12-60,

HTDMSN1.25-12-60, DMSN, and MSN-CC to create positive charge particles. 200 mg

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of particles was suspended in 30 mL of toluene, and sonicated for 10 min, and then

0.19 mL APTES was added and the mixture was refluxed at 110 °C for 20 h. The

product were collected by centrifugation and washed twice with ethanol and water

and dried at 50 ˚C for overnight.

5.S6. Grafting of 5(6)-isothiocyanate dye (FITC)

50 mg of amino modified particles were suspended in 20 mL of ethanol with

sonication for 10 min. Afterwards, 5 mg of FITC was added and stirred the

suspension for overnight in dark. FITC tagged particles were collected by

centrifugation followed by 3 times washing with ethanol to remove the free dye. FITC

is the original fluorescein molecules functionalized with an isothiocyanate reactive

group (-N=C=S). This derivative is reactive towards nucleophiles such as amine and

sulfhydryl groups on protein.

5.S7. Characterization

Nitrogen adsorption-desorption measurements were conducted at 77 K with a

Micromeritcs Tristar ІІ system, before testing the samples were degassed at 453 K

overnight on a vacuum line. The total pore volume was calculated from the amount

adsorbed at a maximum relative pressure (P/P0) of 0.99. The Barrett-Joyner-

Halenda (BJH) method was used to calculate the pore size of samples from the

adsorption branches of the isotherms. The Brunauer-Emmett-Teller (BET) method

was utilized to calculate the specific surface areas.

The SEM images were obtained using JEOL JSM 7800 field-emission scanning

electron microscope (FE-SEM) operated at 1-1.5 kV using gentle bean mode. For

FE-SEM measurements, the samples were prepared by dispersing the powder

samples in ethanol, after which they were dropped to an aluminium foil piece and

attached to conductive carbon film on SEM mount. Then the samples were dried in a

vacuum oven at 60 °C for 12 hours and the samples were observed without any

coating.

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TEM images were obtained using JEOL 1010 and FEI Tecnai F30 operated at 100

kV and 300 kV, respectively. Dynamic light scattering (DLS) were carried out at 298

K using a Zetasizer Nano-ZS from Malvern Instruments.

The ET experiment was performed in bright-field TEM mode on a FEI Tecnai F30

operated at 300 kV. All TEM images were recorded digitally at a given defocus in

bright-field mode to show the mass–thickness contrast. The ET specimens were

prepared by dispersion of the samples in ethanol under ultrasonication for 5 min, and

then deposition directly onto a formvar film supported by a copper grid (3×1 mm slot,

Electron Microscopy Science). Colloidal gold particles (10 nm) were deposited on

both surfaces of the grid as fiducial markers for the subsequent image alignment.

The tomographic tilt series were carried out by tilting the specimen inside the

microscope in dual axis ranging from +65 ° to -65 ° at an even increment of 1°.

Tomograms revealing the fine structures of nanoparticles were obtained by using the

software of IMOD. [3]

For the optical microscopy measurement the samples were prepared by adding

emulsion on to a glass slide mounted with cover slip and the samples were observed

under optical microscopy (Olympus BX51).

5.S8. Uptake of nanoparticles by dendritic and macrophages in in-vitro model

Briefly, mice spleens were passed through a cell strainer to obtain single cell

suspension, and red blood cells were lysed using erylysis buffer (Sigma–Aldrich).

The resulting cells were seeded in a 96-well plate at a density of 2x 105 cells/well in

phenol free IMDM Glutamax medium (Gibco®, Life technologies), supplemented with

10% fetal bovine serum (FBS), 50 µM 2-mercaptoethanol (Gibco®, Life

technologies), 100 U/mL penicillin, and 100 lg/ml streptomycin (Gibco®, Life

technologies). Dextran-FITC (positive control) and samples (Stöber spheres,

HTMSN0.41-12-60, HTDMSN1.25-12-60, MSN-CC and DMSN) labelled with FITC were

added to the wells and incubated for 6 h. The adherent cells were scraped from the

plate and incubated with Fc-block for 20 min at 4°C, centrifuged and resuspended in

a buffer containing CD11c (eBioscience), F4/80 (BioLegend, Pacific Heights Blvd,

San Diego, CA, USA) antibodies for 30 min at 4°C. The cells were then centrifuged

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and resuspended in 0.5 mL of FACS buffer (PBS, 0.02% sodium azide, 0.5% bovine

serum albumin (BSA)) and analyzed using LSR II flowcytometer (BD Biosciences).

The fluorescence intensities of dendritic cells and macrophages treated with PBS

were also measured as a negative control. The actual uptake was calculated as the

percentage of cells double positive for FITC and CD11c and FITC and F4/80.

5.S9. Maturation study of dendritic and macrophages in in-vitro model

Briefly, mice spleens were passed through a cell strainer to obtain single cell

suspension, and red blood cells were lysed using erylysis buffer (Sigma–Aldrich).

The resulting cells were seeded in a 96-well plate at a density of 2x 105 cells/well in

phenol free IMDM Glutamax medium (Gibco®, Life technologies), supplemented with

10% FBS, 50 µM 2-mercaptoethanol (Gibco®, Life technologies), 100 U/mL

penicillin, and 100 lg/ml streptomycin (Gibco®, Life technologies). Stöber spheres,

HTMSN0.41-12-60, HTDMSN1.25-12-60, MSN-CC and DMSN loaded with ovalbumin (20

µg/mL) loaded particles (200 µg/mL) were added to the wells and incubated for 24 h.

The adherent cells were scraped from the plate and incubated with Fc-block for 20

min at 4 °C, centrifuged and resuspended in a buffer containing CD11c

(eBioscience), F4/80, CD40, CD86 (BioLegend, Pacific Heights Blvd, San Diego,

CA, USA) antibodies for 30 min at 4 °C. The cells were then centrifuged and

resuspended in 0.5 mL of FACS buffer (PBS, 0.02% sodium azide, 0.5% BSA) and

analyzed using LSR II flowcytometer (BD Biosciences).

5.S10. Hemolysis Assay

Ethylenediamine tetraacetic acid (EDTA)-stabilized mouse blood was provide by

Australian Institute for Bioengineering and Nanotechnology Animal Facility,

University of Queensland Biological Resources, Australia. Whole blood was

centrifuged at 1600 rpm for 5 min, and the supernatant was discarded. After further

washing RBCs with PBS until the supernatant was clear, 200 µL of packed RBCs

was diluted to 4 mL with PBS (5% hematocrit). The diluted RBCs suspension (0.2

mL) was mixed with 0.8 mL of varied nanoparticles at concentration of 20, 40,

80,120, 160, 240 µg/mL. PBS and deionized water were used as negative and

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positive control, respectively. The mixtures were gently vortexed and incubated at

room temperature for 2 h.

After centrifugation at 1600 rpm for 5 min, the absorbance of the supernatant was

measured at 541 nm using a Synergy HT microplate reader. The percent hemolysis

of RBCs was calculated based on following equation.

Percent hemolysis = ((sample absorbance - negative control absorbance)/(positive

control absorbance - negative control absorbance)) × 100.

5.S11. SEM observation of RBC samples

The diluted RBC suspension (0.2 mL) was mixed with 0.8 mL of varied nanoparticles

at concentration of 240 µg/mL and incubated at room temperature for 2 h. The

samples were fixed by adding 40 µL of glutaraldehyde solution (25 %) into 1 mL of

above solutions. After incubation at 37 °C for 1.5 h, the RBCs samples were washed

with PBS for 3 times, followed by postfix with 1% osmium tetroxide for 1.5 h, and

then washing with PBS for three times. The RBCs were then dehydrated in

increasing concentrations of ethanol (50, 60, 70, 80, 90, 95 and 100%) for 5 min

each. Cell suspensions were dropped onto plastic coverslips, dried, and coated with

Au and then viewed under JEOL 7001F.

5.S12. Extracellular ROS study

The measurement of extracellular ROS was conducted using H2DCF (2,7-

dichlorofluorescein). H2DCF working solution was prepared by dissolving 50 µg of

H2DCF in 17.3 µL of ethanol. 0.01 M sodium hydroxide solution was added to the

stock solution and placed at room temperature for 30 min. Later, 3.5 mL of

phosphate buffer (pH=7.1) was added to neutralize the reaction. 200 µg/mL of

nanoparticle solution was added to H2DCF stock solution and incubated at room

temperature for 3 h. The fluorescence was measured at Ex497/Em527 using

microplate reader.

5.S13. Test to identify the emulsion type

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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2-4 mL of chlorobenzene or water was slowly added to 2-4 mL of emulsion under

static condition.

5.S14. Flow cytometry analysis of RBCs

The diluted RBC suspension (0.2 mL) was mixed with 0.8 mL of FITC conjugated

Stöber spheres, HTMSN1.25-12-60, and HTMSN1.25-12-400 at a concentration of 40µg∙mL-

1 and incubated at room temperature for 2 h. PBS was used as negative control.

After 2 h, RBCs were analysed by accuri C6 flow cytometer.

Supplementary Figures and Tables

Figure 5.S1 TEM image of the dense stöber spheres as head with size ~ 200 nm.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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D ia m e t e r (n m )

Inte

ns

ity

1 0 1 0 0 1 0 0 0

0

5

1 0

1 5

2 0

2 5a

b

c

Figure 5.S2 Partice size distribution curves of a) HTMSN0.41-12-60, b) HTMSN1.25-12-60,

c) HTMSN3.75-12-60 by DLS measurement.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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- 126 -

Figure 5.S3 A TEM image showing the small mesopores in the porous layer fully

covering the head region in HTMSN0.41-12-60. Scale bar: 100 nm.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S4 Tail length and head coverage length of samples HTMSN0.41-12-60,

HTMSN1.25-12-60, and HTMSN3.75-12-60. Statistical analysis was performed using one-

way ANOVA with Dunnett’s multiple comparisons test. Data were considered

significant different at (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, ns

(P ≥ 0.05) indicates no significant difference. Bars represent the mean ± SEM (n=5).

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S5 N2 sorption isotherms (A) and the BJH pore size distribution curves derived

from adsorption branch (B) of a) HTMSN0.41-12-60, b) HTMSN1.25-12-60 and, c)

HTMSN3.75-12-60.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S6 TEM images of A) Stöber spheres; B) HTMSN1.25-2-60; C) HTMSN1.25-3-

60; D) HTMSN1.25-6-60 ; E) HTMSN1.25-9-60; F) HTMSN1.25-24-60.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

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D ia m e te r (n m )

Inte

ns

ity

1 0 1 0 0 1 0 0 0

0

5

1 0

1 5

2 0a

b

c

d

e

f

Figure 5.S7 Particle size distribution of a) HTMSN1.25-0-60; b) HTMSN1.25-2-60; c)

HTMSN1.25-3-60; d) HTMSN1.25-6-60 ; e) HTMSN1.25-9-60; f) HTMSN1.25-24-60.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S8 SEM images of A) Stöber spheres; B) HTMSN1.25-2-60; C) HTMSN1.25-3-

60; D) HTMSN1.25-6-60 ; E) HTMSN1.25-9-60; F) HTMSN1.25-24-60.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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P o r e d ia m e te r (n m )

Po

re v

olu

me

(c

m3

/g

)

1 1 0 1 0 0

0 .0

0 .2

0 .4

0 .6

ba

c

d

e

f

B

Figure 5.S9 N2 sorption isotherms (A) and BJH pore size distribution curves derived

from adsorption branch (B) of a) Stöber spheres; b) HTMSN1.25-2-60; c) HTMSN1.25-3-

60; d) HTMSN1.25-6-60 ; e) HTMSN1.25-9-60; f) HTMSN1.25-24-60.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S10 ET slices of A) HTMSN1.25-3-60 and, B) HTMSN1.25-12-60.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S11 TEM images of A) HTMSN1.25-12-0; B) HTMSN1.25-12-30; C) HTMSN1.25-12-

60; D) HTMSN1.25-12-100; E) HTMSN1.25-12-200; F) HTMSN1.25-12-400, SEM image of G)

HTMSN1.25-12-400.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S12 Digital image of emulsion (A1) after addition of chlorobenzene to

HTMSN1.25-9-60 emulsion, (A2) after addition of water to HTMSN1.25-9-60 emulsion, (B)

optical image of emulsion.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S13 TEM image of MSN1.25-60-12 sample prepared at 50 rpm.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S14 TEM image of DMSN

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S15 N2 sorption isotherms (A) and the BJH pore size distribution curves

derived from adsorption branch (B) of dendritic particles (a) and HTDMSN1.25-60-12 (b).

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 139 -

Figure 5.S16 Hemolytic percentages of mouse RBCs upon incubation with Stöber

spheres, HTMSN1.25-t-60 (t= 2, 3, 6, 9, 12), and HTMSN1.25-12-400 at different

concentrations. D.I water (+) and PBS (-) were used as controls. Bars represent the

mean ± SEM (n=3). Samples were compared with HTMSN1.25-12-400 under all tested

concentrations.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 140 -

Figure 5.S17 Hemolysis results. Photographs of RBCs treated with HTMSN at

different concentrations. The released haemoglobin from the damaged cells in the

supernatant can be seen from the photographs. (-) and (+) controls are the RBCs in

PBS and water, respectively.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 141 -

Figure 5.S18 Hemolysis results. Photographs of RBCs treated with DMSN,

HTDMSN and MSN-CC at different concentrations. The released haemoglobin from

the damaged cells in the supernatant can be seen from the photographs. (-) and (+)

controls are the RBCs in PBS and water, respectively.

Chapter 5

Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 142 -

Figure 5.S19 SEM image of (A) free RBCs and (B, C) RBCs incubated with

HTMSN1.25-12-60.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

- 143 -

Figure 5.S20 Flow cytometry analysis showing FITC positive RBCs incubation with

Stöber spheres, HTMSN1.25-12-60, and HTMSN1.25-12-400 for 2 h.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Figure 5.S21 Flow cytometer analysis of the uptake of A) Stöber spheres and B)

HTMSN0.41-12-60 (C) HTDMSN1.25-12-60 (D) MSN-CC, (E) DMSN in dendritic cells (left

panel) and macrophages (right panel). Q2 represents the percentage of dendritic

cells positive for silica nanoparticles, Q2-1 represents the percentage of

macrophages cells positive for silica nanoparticles.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

hemocompatibility and maturation of immune cells

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Figure 5.S22 A) Flow cytometry analysis of maturation marker CD40 and CD86

expression levels on CD11c+ DCs and F4/80+ macrophages triggered by HTMSN0.41-

12-60 and Alum, (B) live cell numbers of CD11c+ DCs and F4/80+ macrophages

treated with Alum and HTMSN0.41-12-60.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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- 146 -

Figure 5.S23 Flow cytometry analysis of maturation marker CD40 (A, C) and CD86

(B, D) expression levels on CD11c+ DCs (A, B) and F4/80+ macrophages (C, D)

triggered by OVA loaded Stöber spheres, HTMSN0.41-12-60, HTDMSN1.25-12-60, MSN-

CC and DMSN. Bars represent mean ± SEM (n=3).

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Table 5.S1 Physical properties of samples HTMSN0.41-12-60, HTMSN1.25-12-60,

HTMSN3.75-12-60.

Samples S

(m2/g)

V (cm3/g) D

(nm)

PDI Mean

particle

diameter

(nm)

HTMSN0.41-12-60

HTMSN1.25-12-60

HTMSN3.75-12-60

198

369

406

0.40

0.46

0.49

2.7, 28

2.1, 16

1.8, 11

0.183

0.128

0.236

387

408

466

Note: S is BET surface area, V is total pore volume, and D is the pore size.

Table 5.S2 Physical properties of stöber; HTMSN1.25-2-60; HTMSN1.25-3-60;

HTMSN1.25-6-60; HTMSN1.25-9-60; HTMSN1.25-24-60.

Samples S

(m2/g)

V

(cm3/g)

D

(nm)

PDI Mean

particle

diameter

(nm)

-

Potential

(mV)

Stöber spheres

HTMSN1.25-2-60

HTMSN1.25-3-60

HTMSN1.25-6-60

HTMSN1.25-9-60

HTMSN1.25-24-60

15

73

219

295

380

403

0.07

0.17

0.32

0.35

0.38

0.44

0

15

1.8, 18

1.9, 18

2.1, 18

2.1, 13

0.092

0.197

0.153

0.237

0.224

0.125

289

297

347

406

462

540

37.8

-36.4

-35.9

-35.6

-36.1

-40.8

Note: S is BET surface area, V is total pore volume, and D is the pore size.

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Asymmetric silica nanoparticles with tunable head-tail structures enhances

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Table 5.S3 Physical properties of HTMSN1.25-12-60 prepared at a stirring speed of 150

rpm.

Sample S (m2/g) V (cm3/g) D (nm)

HTMSN1.25-12-60 234 0.34 2.5, 16

Note: S is BET surface area, V is total pore volume, and D is the pore size.

Table 5.S4 Physical properties of DMSN and HTDMSN1.25-12-60

Samples S (m2/g) V (cm3/g) D (nm) -Potential

(mV)

DMSN

HTDMSN

162

303

0.40

0.47

4

3, 16

-32.6

-35.45

Note: S is BET surface area, V is total pore volume, and D is the pore size.

Table 5.S5 Physical properties of HTMSN1.25-12-400 and MSN-CC

Sample S(m2/g) V

(cm3/g)

D (nm) PDI Mean

particle

diameter

(nm)

-Potential

(mV)

HTMSN1.25-12-400

MSN-CC

182

612

0.36

1.51

2.1,

16.5

32.8

0.17

0.25

338

293

-33.6

-30.8

Note: S is BET surface area, V is total pore volume, and D is the pore size.

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Reference:

[1] W. Stöber, A. Fink, E. Bohn, Controlled growth of monodispersed silica spheres

in micron size range. J. Colloid Interface Sci. 1968, 26, 62-69.

[2] Shen, D. Yang, J. Li, X, Biphase stratification approach to three-dimensional

dendritic biodegradable mesoporous silica nanospheres. Nano Lett. 2014, 2, 923-32.

[3] Kremer, J. R.; Mastronarde, D. N.; McIntosh, J. R. Computer visualization of

three-dimensional image data using IMOD. Journal of Structural Biology 1996, 116,

71

Chapter 6

Core-shell-structured dendritic

mesoporous silica nanoparticles

for combined photodynamic

therapy and antibody delivery

Multifunctional core-shell-structured dendritic mesoporous silica nanoparticles with a

fullerene doped silica core and a dendritic silica shell with large pores have been

prepared. The combination of photodynamic therapy and antibody therapeutics

significantly inhibits the cancer cell growth by effectively reducing the level of anti-

apoptotic proteins.

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Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

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6.1 Introduction

In the past decades, with the rapid development of nanotechnology, a variety of

nanoparticles such as polyesters and polyacrylamide, [1] liposomes, [2-4]

dendrimers, [5, 6] magnetic [7, 8] and other inorganic nanoparticles [9-11] have been

widely used as carriers for photodynamic therapy (PDT). The combination of chemo

and photodynamic therapy[12] is an effective approach for many cancer treatments

with further enhanced therapeutic efficacy.[13-15] However, conventional

chemotherapy using small molecular weight anti-cancer drugs has caused drug

resistance and this problem exists in combinational therapy.[16] One promising

alternative to chemotherapy for cancer treatment is antibody therapy. The combined

PDT and antibody delivery is discovered as a promising strategy for cancer

treatment by conjugating photosensitizers [17] with monoclonal antibodies (mAbs).

However, there are some intrinsic issues, such as technical difficulty of chemical

coupling and reduced phototoxicity. [18-22] Design of suitable carriers holds promise

for improved combination therapy of photosensitizers and protein / antibody

therapeutics by addressing these problems.

Recently, mesoporous silica nanoparticles (MSN) have attracted attention mainly

due to their easy functionalization, large surface area and high pore volume for high

payload of therapeutic agents and effective cellular delivery.[23,24] Monodispersed

MSN with small pores have applications limited to small drug delivery.[25] The

discovery of dendritic mesoporous silica nanoparticles (DMSN) with large and open

pore channels makes them attractive candidates for delivering large

biomolecules.[26,27] Lin et al. reported the advantage of magnetic DMSN for

doxorubicin hydrochloride (DOX) delivery.[28] Later, Zhao and co-workers reported

the synthesis of 3D-DMSN grown on gold and silver nanoparticles.[29] The

composite DMSN fabricated so far have been used for real time fluorescence

imaging, magnetic resonance imaging,[30] adsorption[31] and protein delivery.[29]

However, to the best of our knowledge, there is no report using DMSN as a

multifunctional platform for both PS with monoclonal antibodies (mAbs).

Herein, we report the synthesis of a multi-functional core-shell structured

nanoparticle consisting of a solid silica-fullerene core and a dendritic silica shell with

Chapter 6

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

- 152 -

large pores (Scheme 6.1). The silica core doped with fullerene (C60) acts as PS and

fluorescent agents for imaging. The large-pore dendritic silica shell after hydrophobic

C18 modification is used to load a therapeutic mAb anti-pAkt. After intracellular

delivery, the doped fullerene in the core generates single oxygen (1O2) and

fluorescence under UV light excitation, whereas anti-pAkt provokes cell inhibition by

blocking pAkt and downstream anti-apoptotic protein Bcl-2. [32] Compared to

antibody treatment itself, the combination of PDT and antibody treatment significantly

promote cell inhibition by reducing the downstream anti-apoptotic protein levels.

Scheme 6.1 Schematic illustration of preparation procedure of FD for applications in

imaging and combined protein-/photodynamic therapy.

6.2 Results and discussion

Solid silica-fullerene nanoparticles were prepared using a literature method [33] and

used as core particles to generate a large-pore dendritic shell in an emulsion system

(Scheme 6.1, see details in Experimental section, ESI). The templates were

removed using solvent extraction and the sample is named FD. Modification with

octadecyl groups resulted in a hydrophobic sample named FD-C18. Both FD and

FD-C18 samples were used for combinational PDT and protein therapy.

Transmission electron microscopy (TEM) images of FD and FD-C18 are shown in

Figure 6.1A, B. Both nanoparticles are highly uniform and monodispersed with a

diameter of 116 ± 2 and 119 ± 1 nm, respectively (n=50). A distinct dendritic

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Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

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structure on fullerene loaded solid silica core can be clearly observed from TEM

images. The large pore openings can be seen directly from scanning electron

microscopy images (Figure 6.1C, D). The dynamic light scattering (DLS)

measurement revealed that FD and FD-C18 has a hydrodynamic diameter of 122

and 121 nm, respectively (Figure 6.S1, Table S1). The particle sizes of FD and FD-

C18 are close to those of TEM measurement. The successful modification of

octadecyl groups is evidenced by two peaks at 2858 and 2929 cm-1 in the Fourier

transform infrared (FTIR) spectra. As shown in Figure 6.1C, D, the pore openings

and spherical morphology is well preserved after C18 modification.

Figure 6.1 TEM and SEM images of (A,C) Fullerene dendritic (FD), B,D) Fullerene

dendritic-C18 (FD-C18).

The nitrogen adsorption/desorption isotherms of FD and FD-C18 are shown in

Figure 6.S3. Both samples show similar type IV isotherms and capillary

condensation steps at relative pressure (P/P0) higher than 0.9, indicative of large

mesopores. From the pore size distribution curves, both FD and FD-C18 show a

broad pore size centered at 28 nm. The Brunauer-Emmett-Teller surface area and

pore volume of FD is 328 m2/g and 1.11 cm3/g (Table 6.S1, SI). After hydrophobic

modification, the surface area and pore volume of FD-C18 decrease to 294 m2/g and

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Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

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1.01 cm3/g due to the grafted layer of silica-octadecyl-groups. The structural

parameters of both FD and FD-18 are summarized in Table 6.S1 for comparison.

The presence of C60 in FD and FD-C18 was studied according to a literature report.

Pure solid silica (SS) nanoparticles without C60 were used as a control group. Figure

6.S3C shows the UV-absorption spectra of SS, FD and FD-C18. FD and FD-C18

show peaks at 234, 217, 308 nm, which indicate the presence of C60. [33] TGA

analysis was further used to quantitatively measure the C60 content in FD and FD-

C18 (Figure 6.S4). Silica dendritic [34] and SD-C18 without C60 were used as

control groups. The weight loss for SD and SD-C18, FD and FD-C18 was 0.6, 7.5,

5.3 and 11.6 %, respectively. The C60 amount of FD and FD-C18 was calculated by

subtracting the weight loss (%) obtained from SD and SD-C18, respectively. The C60

amount in FD and FD-C18 was 4.7 and 4.1 %, respectively (Table 6.S2).

To verify the generation efficiency of singlet oxygen (1O2) produced by FD and FD-

C18, 100 µL of 9,10-anthracenediyl- bis(methylene) dimalonic acid (ABDA, a 1O2

sensor) solution (2 mg/mL) was mixed with FD (1 mg/mL) and FD-C18 (1.136

mg/mL). The C60 concentration was kept the same (65 µg) in two positive groups

and pure ABDA solution was used as a control group. ABDA after chemical reaction

with 1O2 will bleach to endoperoxide, which results in a decrease in optical density

(OD) at 376 nm. The change of OD in the ABDA control group was negligible in the

absence of C60 (Figure 6.2A). For FD (Figure 6.2B), with increase in the UV

exposure time the OD decreased, suggesting an increased amount of 1O2 produced

by the FD over time. A similar trend was observed for the FD-C18 (Figure 6.2C),

suggesting that both FD and FD-C18 have good 1O2 generation efficiency. Figure

6.2D is the normalized OD values shown in Figures 6.2A-C, showing that the

bleaching of ABDA is time dependent under UV illumination in the presence of C60.

However, the 1O2 generation of FD-C18 was slightly less than FD.

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Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

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Figure 6.2 Photobleaching of ABDA by 1O2 generated by (A) pure D20, (B) FD, (C)

FD-C18, (D) decay curves of pure D20, FD, and FD-C18 at 376 nm as a function of

UV irradiation time.

Confocal microscopy was used to investigate the cellular uptake capability of

nanoparticles. As C60 has fluorescent activity, external conjugation of fluorescent

dyes onto nanoparticles for cellular imaging can be eliminated. The fluorescence

images of FD and FD-C18 treated MCF-7 cells were captured after 4 h post

incubation. The cytoskeleton of the cell was marked with Alexa Fluor® 488 phalloidin

(green), while the nucleus is stained with DAPI (blue). As shown in Figure 6.S5, cells

without nanoparticle treatment have no red fluorescence. However, cells treated with

FD and FD-C18 show red fluorescence in the cytoplasmic region, indicating that

nanoparticles were internalized successfully into cells. Inductive coupled plasma

optical emission spectroscopy (ICPOES) was used to quantify the cellular uptake

efficiency of FD and FD-C18 by incubating 50 µg/mL particles with MCF-7 cells for 4

h. As shown in Figure 6.S6, 64 pg FD/cell while 120 pg FD-C18/cell (50% increase)

was detected. The significant improvement in cellular uptake performance of FD-C18

is attributed to the hydrophobic modification, which was similar to the literature

report. [32]

In order to study the in vitro efficacy of our designed particles, the cell toxicity of FD

and FD-C18 as a function of particle concentration was tested in a MCF-7 cell line.

As shown in Figure 6.S7, the cell viability decreased with increasing dose. At low

Chapter 6

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

- 156 -

concentrations of 20, 40 and 80 µg/mL, the cell viability of FD and FD-C18 was

similar (96 vs 96 %, 84 vs 83 %, and 80 vs 79%, respectively). At higher dose of 120

and 180 µg/mL, FD-C18 caused slightly higher toxicity to MCF-7 cells than FD. The

cell viability of FD and FD-C18 was 82 vs 75 % and 69 vs 60 %, respectively. The

toxicity of FD-C18 at the highest concentration is similar to that of C18 modified

dense silica particles reported in literature. [35]

To demonstrate the PDT efficacy of FD and FD-C18, the cancer cell inhibition as a

function of UV irradiation time was tested (Figure 6.3). Under UV light exposure, the

cancer cell inhibition was negligible within 5 min, while 20% cell inhibition was

observed at 10 min irradiation time. The cell viability in the presence of FD (110

µg/mL) and FD-C18 (125 µg/mL) was UV light exposure time dependent. The cell

Figure 6.3 Cell viability of MCF-7 cells treated with FD and FD-C18 under different

UV irradiation time.

viability of FD and FD-C18 after exposing to UV light was 81% and 72% for 2 min, 76

% and 55 % for 5 min, while 47% and 42% for 10 min. The higher cell inhibition (FD-

C18 over FD) is mainly attributed to the higher cellular uptake of FD-C18 (50%

higher than FD), although its 1O2 generation efficacy is slightly lower than FD.

Because the cell toxicity is relatively high caused by a longer UV exposure time such

as 10 min, therefore we selected 5 min as a suitable UV exposure time for

conducting combination therapy studies. It was reported that cooperative, synergistic

therapies using dual or multiple therapy types can produce remarkable therapeutic

efficacy and help various types of therapies effectively play coordinate roles in

cancer therapy. The synergistic effects of PDT and therapeutic mAb anti-pAkt were

Chapter 6

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

- 157 -

investigated using anti-pAkt, FD-C18 + anti-pAkt and FD-C18 + anti-pAkt + UV

groups at different particle and protein concentrations. FD-C18 was chosen for

combined therapy studies because it has high uptake into MCF-7 cell line than FD

and good loading capacity of 12 µg/mg. Efficient cellular uptake performance of

nanoparticles is a prerequisite for successful cellular delivery. As shown in Figure

6.4, free anti-pAkt at various concentrations showed little effect on cell viability. The

cell viability at the highest anti-pAkt concentration of 1.5 µg/mL was 94 %. However,

FD-C18 + anti-pAkt group in the absence of UV light exhibited cell viability of ∼ 85%,

72% and 45% at particle concentrations of 35, 90, and 125 µg/mL, respectively. After

UV light treatment for 5 min, the cell viability was further dropped to ∼ 51%, 33% and

23%. This may be because of efficiency delivery of FD-C18 into the cells.

Figure 6.4 Treatment of nanoparticles and anti-pAkt at various concentrations in the

presence or absence of UV irradiation for 5 min.

To study the knockdown efficiency of downstream Bcl-2 protein in MCF-7 cells after

treatment, western-blot analysis was conducted (Figure 6.5). GAPDH serves as an

internal standard. Bcl-2 protein regulates the cell death or apoptosis.[36] Compared

to the blank control group, anti-pAkt treatment group showed similar levels of Bcl-2,

in accordance with previous results,[37] indicating that anti-pAkt mAb alone cannot

enter into the cells and further showing the significance of nanoparticles in cellular

delivery of anti-pAkt mAb. For FD-C18 + anti-pAkt group, suppression in Bcl-2 level

was observed compared to the control and anti-pAkt groups. The results suggest

that activation of caspases, and inhibition of cancer cell growth by blocking pAkt in

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Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

- 158 -

the cells can be achieved by successful uptake of nanoparticles loaded with anti-

pAkt antibodies. After treatment with FD-C18 + anti-pAkt + UV, the Bcl-2 protein

level was significantly decreased compared to all the other groups. The combination

of protein and photodynamic therapy showed significant reduction of Bcl-2 protein

than single therapy, indicating that cell growth inhibition is associated with the

cytosolic delivery of FD-C18 and efficient blocking of pAkt by PSs and mAbs and

further reducing the level of Bcl-2.

Figure 6.5 Western-blot analysis of Bcl-2 protein in MCF-7 cells after 5 min UV

irradiation in blank control, anti-pAkt, FD-C18 + anti-pAkt and FD-C18 + anti-pAkt +

UV groups. Blots presented are representative of typical results. GADPH served as

an internal standard.

6.3 Conclusion

In conclusion, we have fabricated a new class of core-shell-structured dendritic

mesoporous silica nanoparticles with multifunctions for combined PDT and antibody

therapy. The C60 dispersed in the silica core led to efficient PDT and fluorescence for

intracellular tracking. The dendritic shell with hydrophobic modification is utilized for

intracellular delivery of large therapeutic mAbs. The combined treatment of antibody

/ photodynamic therapy in MCF-7 cancer cells have shown significantly improved

cancer cell inhibition by reducing the level of anti-apoptotic proteins. The

nanoparticles developed in this work have the potential to be used diverse

combination therapies.

Chapter 6

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

- 159 -

6.4 References

[1] J. M. Lu, X. W. Wang, C. Marin-Muller, H. Wang, P. H. Lin, Q. Z. Yao and C. Y.

Chen, Current advances in research and clinical applications of PLGA-based

nanotechnology. Expert Rev Mol Diagn, 2009, 9, 325-341.

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Supporting information

Materials and reagents

6.S1. Materials

Cetyltrimethylammonium chloride (CTAC) solution (25 wt% in H2O); triethanolamine

(TEA); chlorobenzene and tetraethylorthosilicate (TEOS, >98%), C60 fullerene (99%),

cyclohexane, 1-hexanol, Triton X-100, n-octadecyltrimethoxysilane (n-ODMS, 90%),

9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA), (3-4,5-dimethylthiazol-2-

yl]-2,5 diphenyltetrazolium bromide), ammonium hydroxide (28 wt%, 240 mL) were

purchased from Sigma-Aldrich. Toluene was purchased from Merck. Dulbecco’s

Modified Eagles Medium (DMEM), penicillin-streptomycin (10,000 U/mL) and trypsin-

EDTA (0.25 %) were purchased from GIBCO or Invitrogen, Life Sciences, Life

Technologies. 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™) cell line was purchased from American Type Culture Collection (ATCC). All

chemicals were used as received without further purification.

6.S2. Synthesis of SSF and SS nanoparticles

Uniform solid silica fullerene (SSF) nanoparticles were synthesized according to

literature method. [1] In a typical synthesis, 16 mg of C60 fullerene was well

dispersed in toluene by sonication. The dispersed fullerene solution was mixed , with

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microemulsion solution composed of cyclohexane (20 mL), 1-hexanol (8 mL),

distilled water (2 mL), and Triton X-100 (6.8 mL) under stirring for 30min. 400 µL of

TEOS was added into the above mixture and stirred for another 30 min before

addition of ammonium hydroxide (28 wt %, 2.4 mL). The mixture was continuously

stirred at room temperature for 24 h, followed by addition of 40 mL of ethanol to

break the microemulsion. The particles were collected by centrifugation at 20,000

rpm for 10min and then washed with ethanol twice.

Solid silica (SS) nanoparticles without fullerene was prepared using the same

procedure as SSF in the absence of fullerene.

6.S3. Synthesis of FD and SD

In a typical synthesis, 60 mg of SSF was dispersed in 6 mL of milliQ water with

dissolved 0.035 g of TEA by sonication for 15 min. Then, 4 mL of CTAC (25 wt%)

solution was added into the solution and stirred at 60°C with a stirring speed of 400

rpm. After stirring for 1 h, followed by addition of 8.75 mL of chlorobenzene and 1.25

mL of TEOS. The reaction was continued at 60°C for 12 h. Particles were collected

by centrifugation at 20,000 rpm for 10min, washed with water and ethanol twice. The

final fullerene dendritic (FD) nanoparticles were obtained by vacuum drying

overnight. Solid dendritic (SD) nanoparticles were prepared using the same

procedure as FD, except the replacement of SSF with SS.

6.S4. C18 modification of FD and SD

60 mg of FD or SD was suspended in 9 mL of toluene, and sonicated for 10 min.

Then 150 µL of n-ODMS was added into the mixture and refluxed at 110°C for 20 h.

The final products (FD-C18 and SD-C18) were obtained by centrifugation, washing

twice with ethanol and water and vacuum dried overnight.

6.S5. Characterization

Transmission electron microscopy (TEM) images were obtained with JEOL 1010

operated at 100 kV. Scanning electron microscopy (SEM) images were taken using

JEOL 7800 operated at 1kV. Dynamic light scattering (DLS) was carried out in D.I.

water at 298 k using a Zetasizer Nano-ZS from Malvern Instrument. Nitrogen

adsorption-desorption measurements were conducted at 77 K with a Micromeritcs

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Tristar ІІ system, before testing the samples were degassed at 310 K overnight on a

vacuum line. The total pore volume was calculated from the amount adsorbed at a

maximum relative pressure (P/P0) of 0.99. The Barrett-Joyner-Halenda (BJH)

method was used to calculate the pore size of samples from the adsorption branches

of the isotherms. The Brunauer-Emmett-Teller method was utilized to calculate the

specific surface area. Thermogravimetric analysis (TGA) measurements were

performed by TGA/DSC Thermogravimetric Analyser (Mettler-Toledo Inc) with a

heating rate of 2°C /min in air flow. Fourier transform infrared (FTIR) spectra of FD

and FD-C18 were recorded on ThermoNicolet Nexus 6700 FTIR spectrometer

equipped with Diamond ATR (attenuated total reflection) crystal. UV-vis

transmittance spectra were measured with a shimadzu UV-2450 double beam

spectrophotometer.

6.S6. Detection of singlet oxygen

The generation of singlet oxygen was detected using 9,10-anthracenediyl-bis

(methylene) dimalonic acid (ABDA) reagent. In our experiment, ABDA was chosen to

monitor the release of singlet oxygen into solution (deuterium water) by recording the

decrease in absorption of ABDA at 376 nm via UV-vis spectroscopy. 100 µL of

ABDA solution (2 mg/mL) was mixed with FD (1 mg/mL) and FD-C18 (1.136 mg/mL)

and analysed with UV-vis spectroscopy. ABDA dissolved in deuterium water was

used as control sample. The absorption intensity of ABDA at 376 nm was monitored

under different UV exposure times.

6.S7. Cell culture

MCF-7 cells were maintained as monolayer cultures in Dulbecco’s Modified Eagle’s

Medium (DMEM), supplemented with fetal bovine serum (FBS) and 1 % penicillin-

streptomycin in a 5% CO2 incubator at 37°C. The media was changed for every 2-3

days and the cells were trypsinized after reaching 80 % confluence.

6.S8. Cellular uptake assay (Confocal)

MCF-7 cells were seeded onto 24-mm glass coverslip in a 6-well plate with a density

of 1×105 cells / well, one day before the assay. FD or FD-C18 nanoparticles was

added into the cells with a concentration of 160 µg/mL and incubated for 4 h in

serum free media. Afterwards, cells were washed twice with PBS and fixed with 4 %

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formaldehyde solution for 30 min, followed by washing with PBS twice. The

cytoskeleton of cells was stained with Alexa Fluor® 400 phalloidin (Life technologies,

Australia) according to the vendor protocol. The coverslip was washed twice with

PBS and mounted onto glass slides by fluoroshield with DAPI (sigma). The prepared

slides were observed under confocal microscopy (LSM 710, ZEISS).

6.S9. Cellular uptake assay (ICPOES)

The cellular uptake of FD and FD-C18 nanoparticles was measured quantitatively

using inductive coupled plasma optical emission spectrometry (ICPOES). MCF-7

cells were seeded in a 6-well plate at 1×105 cells per well, one day before the assay.

FD and FD-C18 nanoparticles was added into cells at a concentration of 50 µg/mL

and incubated for 4 h in serum free media. The cells were then washed twice with

PBS to remove the remaining particles and dead cells, and harvested with trypsin.

The number of cells was recorded and the cell pellets were washed twice with PBS,

followed by sonication in DI water to break the cells. The pellets were collected by

centrifugation and the supernatant containing cell components were discarded.

Fresh aqueous NaOH solution (1 M) was added into the pellets to dissolve silica

components. The silicon concentration was measured by ICPOES, and the amount

of silica per cell was calculated.

6.S10. Cytotoxicity assay

The MTT assay was used to determine the cell viability of MCF-7 cells treated with

FD and FD-C18 nanoparticles. MCF-7 cells were seeded in 96 well plates at a

density of 8×103 cells / well, one day before the assay. The cells were exposed to FD

and FD-C18 nanoparticles with various concentrations (20, 40, 80, 120, 180 µg/mL)

and incubated in 200 µL of serum containing medium for 24 h at 37 °C in a

humidified atmosphere with 5% CO2. Afterwards, 20 µL of MTT solution (5 mg/mL)

was added into each well and incubated for another 4 h. After removing media, 200

µL of DMSO was added to each well. The absorbance at 540 nm was measured

using a Synergy HT microplate reader. The cells without treatment were used as a

control. All experiments were performed in triplicates for each group. Data was

shown as the mean±SEM.

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6.S11. The combined therapeutic effect of antibody and fullerene

The combined therapeutic effect of anti-pAkt and fullerene delivered by FD-C18 was

evaluated in MCF-7 cells. Cells were seeded in a 96-well plate with a density of

8×103 cells / well one day before the assay. Cells were then treated with different

concentrations of free anti-pAkt antibody, FD-C18 + anti-pAkt antibody for 4 h in

culture medium. After 4 h’s incubation, cell culture media were replaced with PBS

and the cells treated with FD-C18+anti-pAkt antibody were exposed to UV light for 5

minutes. PBS in the UV exposed and non-exposed plates was replaced with culture

media. After 24 h incubation the cell viability was measured using MTT assay as

described above.

6.S12. Western-blot analysis

MCF-7 cells were plated in a 6 well plate at a seeding density of 2×105 cells/ well.

The anti-pAkt antibody was incubated with FD-C18 in PBS at 4°C for 4 h for loading.

The final concentration of FD-C18 was 125 µg/mL and anti-pAkt was 1 µg/mL. The

cells were then treated with free anti-pAkt or anti-pAkt-FD-C18 complex for 4 h.

Media were replaced with PBS and the cells treated with FD-C18+anti-pAkt antibody

were exposed to UV light for 5 min. PBS in the UV exposed and non-exposed plates

was replaced with culture media. After 24h incubation, cells were washed with cool

PBS and lysed. The lysed cells were denatured at 95°C for 5 min followed by

characterization by SDS-PAGE. Obtained 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 internal standard. Bands were visualized using

ChemiDoc MP System (Bio-Rad).

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Supporting Figures and Tables

Figure 6.S1 Particle size distribution curves of FD and FD-C18 in distilled water.

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Figure 6.S2 FTIR spectra of a) FD and, b) FD-C18. The two peaks at 2924 and

2854 cm-1 was attributed to asymmetric and anti-symmetric –CH2- stretching,

respectively, indicating the successful attachment of octadecyl groups.

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Figure 6.S3 N2 adsorption-desorption isotherms of FD and FD-C18 (A),

corresponding pore size distribution curves calculated from adsorption branches

using the BJH method (B), and UV-Vis absorption spectra of the SS, FD and FD-C18

(C).

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Figure 6.S4 Weight loss curves of SD, SD-C18, FD and, FD-C18 using TGA analysis.

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Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

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Figure 6.S5 Confocal microscopy images of MCF-7 cells without treatment (a),

treatment with FD (b), FD-C18 (C) at a concentration of 160 µg/ml for 4 h. The

cytoskeleton and nuclei of cells were stained by Alexa Fluor® 488 phalloidin (green)

and DAPI (blue), respectively. Scale bar 50 µm.

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Figure 6.S6 Cellular uptake performance of FD and FD-C18 measured by ICPOES.

Chapter 6

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

- 174 -

Figure 6.S7 Cytotoxicity of FD and FD-C18 as a function of particle concentration in

MCF-7 cells after 24 h.

Chapter 6

Core-shell-structured dendritic mesoporous silica nanoparticles for combined photodynamic therapy and antibody delivery

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Table 6.S1 Structural properties of FD and FD-C18

Sample SBET

(m2/g)

Pore

volume

(cm3/g)

Pore

size

(nm)

Diameter (nm)

TEM Mean

particl

e

FD 327.5 1.11 28 116±2 122.4

FD-C18 294.3 1.01 28 119±1 121

Table 6.S2 Fullerene content in FD and FD-C18

Sample Weight loss (%) % of fullerene

SD 0.6 -

SD-C18 7.5 -

FD 5.3 4.7

FD-C18 11.6 4.1

References: [1] J. Jeong, M. Cho, Y. T. Lim, N. W. Song and B. H. Chung, Synthesis and

Characterization of a Photoluminescent Nanoparticle Based on Fullerene-Silica

Hybridization. Angew Chem Int Edit, 2009, 48, 5296-5299.

Chapter 7

Conclusion and outlook

The research that I have accomplished during my PhD is presented in this

dissertation provides a useful guide for the formulation and development of solid

dosage forms, and fabrication of new nanocarriers. Currently, there are many

research attempts in the design and modification of nanocarrier for efficient drug

delivery and treating disease. In this thesis MSN based floating tablets with long

retention time in acidic medium was successfully formulated. A series of head-tail

silica nanoparticles, fullerene dendritic MSN has been successfully synthesised and

studied for hemocompatibility, antigen presenting cells uptake, maturation of

dendritic cells for former particles, and demonstrated the advantage of dual therapy

using antibodies and photosensitiser for cancer treatment for later.

7.1 Conclusions

1. A new generation of floating tablets based on MSNs for the localised drug delivery

was developed. Calcined MCM-41 type MSNs with two different pore size (2.1 and

1.7 nm) were used in the preparation of tablets. The loading of Curcumin into MCM-

41 was confirmed by XRD and DSC analysis and the loading percentage is 20.0%

and 20.9 % in MCM-41 with the pore size of 1.7 and 2.1 nm, respectively. In

addition, the loading of captopril into MCM-41 with the pore size of 2.1 nm was

confirmed by TGA with the loading efficiency of 16.4%. Floating tablets with

curcumin or captopril loaded MCM-41 was prepared by tablet compression machine.

The homogenous distributed and retention of MCM-41 in the tablet was even evident

Chapter 7

Conclusion and outlook

- 177 -

after 12 h of dissolution process. The percentage of curcumin release from the

formulation prepared with MCM-41 (1.7nm) was 42.8 % at 20 h, which was

comparatively higher than the formulation prepared with MCM-41 (2.1nm), without

MCM-41 and physical mixture. In addition, captopril release from the floating tablet

prepared with captopril loaded MCM-41, was only 60 % at 6 h and it was 100% at

the same time point for conventional captopril floating tablet. (Relevant content:

Chapter 4)

2. The second major contribution of this work is synthesis of head tail dual pore

mesoporous silica nanoparticles, via emulsion system. Although, there are few

literatures on the synthesis of asymmetric silica nanoparticles, but still there is dearth

for synthesis of safe, dual pore asymmetric silica nanoparticles for biomedical

application. Stöber spheres with a fixed head particle of ~200 nm, with different tail

length and head coverage were fabricated by changing the reaction time, silica

source and amount of head particles. With increase in the reaction time from 2 to 24

h, the tail length was increased from 20 nm to 18 nm, respectively. In addition, with

increase in the silica source the tail length can be tuned from 97 to 176 nm. All head

tail nanoparticles (HTMSN) contain a dual pore with the pore size of ~ 2nm and 28

nm. We further replaced the dense silica head with the porous silica nanoparticle for

generation of porous head tail MSN. To the best of our knowledge this is the first

report on the synthesis of asymmetric silica nanoparticles with tuneable tail lengths.

All head tail nanoparticles has good hemocompatibility with less than 8 % hemolysis

after 4 h of incubation. However, symmetric particles have > 90 % hemolysis at

similar time point. In addition, to hemocompatibility, head tail nanoparticles have high

efficient uptake by APC than stöber spheres. Furthermore, the dendritic cell

expression of CD40 and CD86 induced by stöber spheres was slightly lower than

head-tail silica nanoparticles. (Relevant content: Chapter 5)

3. Utilizing the emulsion system, fullerene core large pore dendritic silica

nanoparticles was synthesised (FD-MSN). The advantage of combined

photodynamic and antibody therapy in treatment of cancer was demonstrated by FD-

C18. The synthesized FD-C18 has a unique feature in terms of its structure, porosity.

The synthesised nanoparticles are about the size of 100-120 nm, with the fullerene

core size of 50 nm and the dendritic shell size of 70 nm. The FD-C18 exhibited a

Chapter 7

Conclusion and outlook

- 178 -

surface area of 327.5 m2/g, pore size of 28 nm and pore volume of 1.11 cm3/g. After

hydrophobic modification the surface area came down to 294.3 m2/g. The

hydrophobic modified particles were successfully loaded with anti-pAKt antibody.

The hydrophobic modified and unmodified particles was efficient in generating

reactive oxygen species under light excitation. The fluorescence generated due to

the dispersed fullerene in the core of FD-C18 was used for intracellular tracking of

particles in MCF-7 cell line. The FD-C18 facilitated the intracellular delivery of

therapeutic protein anti-pAKt antibody, and has good fluorescence generating

activity. The combined antibody/photodynamic therapy were conformed in cancer

cells, leading to cell viability down to ~21 % at the concentration of 1.5 µg/ml of anti-

pAKt antibody. (Relevant content: Chapter 6)

7.2 Recommendations for future work

This work has introduced the improvement in the formulation design of floating

tablets, synthesis of head tail nanoparticles and symmetric dendritic fullerene core

silica nanoparticles and its application in vaccines and cancer therapy. However,

there is still room to develop for improving the efficiency of nanoparticles.

7.2.1 Active targeting and fullerene solubility

In chapter 5, we have shown the advantage of combined therapy in cancer

treatment. However, conjugating targeted peptide can help in uneven accumulation

of nanoparticles in the body. To make current nanoparticles more reliable targeting

moiety like folic acid can help targeting the nanoparticles to the cancer cells. Thus

attachment will greatly help in unwanted side effects and improve cell specificity.

Pegah et.al.,[1] have conjugated folic acid/methionine for active targeting delivery of

docetaxel to tumor. They observed that targeted delivery enhanced the antitumor

efficacy for cancer therapy.

7.2.2 Replacing head with various metal nanoparticles

In chapter 2, we have shown the advantages of head-tail nanoparticles in

hemocompatibility and as an adjuvant. We have also shown that solid head part can

be replaceable with porous nanoparticles. In the similar way solid or porous head

part can be replaced with silica coated gold or iron nanoparticles. The gold or iron

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Conclusion and outlook

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nanoparticle HTMSN not only serves as nanovaccine, it can also be used as

diagnostic agents and in lateral flow immunoassays. Gold nanoparticles have high

surface to volume ratios and can be functionalised to detect specific targets, offering

lower detection limits and higher selectivity than conventional strategies.[2] Mirkin’s

group were pioneers in using gold nanoparticles in molecular detection system. The

product developed was used for warfarin metabolism and F5/F2/MTHFR mutations.

7.2.3 In vivo studies

Finally, in this thesis we have shown the advantages of asymmetric particles in the

uptake and activation of antigen presenting cells. We also tested the in vivo efficacy

of HTMSN loaded with J8 antigen. However, due to the limited time we did not

include the in vivo data in this thesis. We tested the DMSN and HTDMSN

nanoparticle shape influence on generating antibodies response in vivo by using J8

as model antigen. Both the nanoparticles showed a J8 (model antigen) loading

capacity of greater than 800 µg/mg of particles. The effect of particle shape on

generating antibody response was studied, where HTDMSN showed high overall

antibody titre and IgG1 than DMSN. This high antibody titre could be contributed

from high uptake into macrophages. This finding helps in developing promising

candidates in vaccine development. The influence of particle morphology (e.g.,

spheres versus rods) on immune response has been reported in silica,[3] aluminium

oxyhydroxides,[4] and gold nanoparticles,[5] showing that the shape of nanoparticles

is another important parameter on their adjuvanticity.

7.3 References

[1] Khosravian, P., et al., Mesoporous silica nanoparticles functionalized with folic

acid/methionine for active targeted delivery of docetaxel. Oncotargets and Therapy,

2016. 9: p. 7315-7330.

[2] Mieszawska, A.J., et al., Multifunctional Gold Nanoparticles for Diagnosis and

Therapy of Disease. Molecular Pharmaceutics, 2013. 10(3): p. 831-847.

[3] Wang, T.Y., et al., Enhanced mucosal and systemic immune responses obtained

by porous silica nanoparticles used as an oral vaccine adjuvant: Effect of silica

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Conclusion and outlook

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architecture on immunological properties. International Journal of pharmaceutics,

2012. 436(1-2): p. 351-358.

[4] Sun, B.B., et al., Engineering an Effective Immune Adjuvant by Designed Control

of Shape and Crystallinity of Aluminum Oxyhydroxide Nanoparticles. Acs Nano,

2013. 7(12): p. 10834-10849.

[5] Niikura, K., et al., Gold Nanoparticles as a Vaccine Platform: Influence of Size

and Shape on Immunological Responses in Vitro and in Vivo. Acs Nano, 2013. 7(5):

p. 3926-3938.