nanoscale properties of biocompatible materials

INRSScience in ACTION for a World in EVOLUTION

Université du Québec

Institut national de la recherche scientifique

Nanoscale Properties of Nanoscale Properties of Biocompatible materialsBiocompatible materials

Induction Ceremony, Academia de IngegneriaInduction Ceremony, Academia de IngegneriaMexico City, Nov 22nd 2017Mexico City, Nov 22nd 2017

Nano–Femto Laboratory (NFL)Nano–Femto Laboratory (NFL)INRS – Énergie, Matériaux et Télécommunications,INRS – Énergie, Matériaux et Télécommunications,

Université du Québec, Varennes (Québec)Université du Québec, Varennes (Québec) [email protected]@emt.inrs.ca

Federico RoseiFederico Rosei

UNESCO Chair in Materials and Technologies for Energy UNESCO Chair in Materials and Technologies for Energy Conversion, Saving and Storage (MATECSS)Conversion, Saving and Storage (MATECSS)




Worldwide Societal Challenges(Broad, General => affect everybody)

• Clean and sustainable energy

• Preserving and protecting the environment

• Improving our health and quality of life

“Our generation will ultimately be defined by how we live up to the energy challenge”

The Future of Energy Supply: Challenges and Opportunities; N. Armaroli, V. Balzani, Angew. Chem. Int. Ed. 2007, 46, 52.




TMA-alcohol assembly

Multi-ferroic BFCO

Template-driven assembly

Biomaterials – TiO2

Nanoscale phenomena

-1,5 10-4

-1 10-4

-5 10-5

0 0

1 109

2 109

3 109

4 109

-50-40-30-20-100

I ds (A)

EL (photons/s)

Vds(V)

Vgs

= -30

Vgs

= -20

Vgs

= -40

Vgs

= -10

OLETs Chemical mapping

Molecular Self-assemblyGatti J Phys Chem C (2014)MacLeod Langmuir (2015)

Group IV nanostructuresMoutanabbir Phys Rev B (2012)

Multifunctional materialsNechache Nature Phot (2015)Li Small (2015)Zhao Small (2015)

Organic ElectronicsDadvand Angew Chem (2012)Dadvand J Mater Chem C (2013)

Organic/hybrid PhotovoltaicsDembele J Mater Chem A (2015)

Dynamic TransmissionElectron MicroscopyNikolova Phys Rev B (2013)Nikolova J Appl Phys (2014)

Nanostructured catalystsChen Adv Func Mater (2012)

Nanostructured BiomaterialsMacLeod Nature Mater (2013)Cloutier Diam Rel Mater (2014)Cloutier Trends Biotech (2015)

Surface polymerization

Surface PolymerizationDi Giovannantonio ACS Nano (2013)Gutzler Nanoscale (2014)Vasseur Nature Comm (2016)

QD solar cellsJin Adv. Sci. (2016)Zhou Adv. En. Mater. (2016)

EmergingPhenomena

Complexity




Guiding Principles

• The role of surfaces & interfaces in materials functionalities (e.g.: catalysis relates to surface structure and properties) & devices

• Structure vs. function in materials: understanding role of morphology & composition in materials properties functionalities => harnessing this knowledge in devices

• Examples in:– Supramolecular host/guest architectures

– Biocompatible materials– Multifunctional materials




• Designing “intelligent” surfaces involves properly managing interactions with surface of, and at interface between, material and host tissue at the nanoscale

• Healing process after surgery: formation of interfacial layer between implant and bone (2–4 months)

Implant

Interface

Biomaterials:Towards Intelligent Surfaces

F. Variola et al., Small 5, 996 (2009)

Average size of a cell: 10 to 15 μmAverage size of a protein: 10 to 15 nm



Institut national de la recherche scientifiqueCellular reactions occur at surfaces/interfaces

Osteogenic cell (osteoblast precursor)

Osteoblast

Osteoid (uncalcif ied bone matrix)

Calcif ied bone matrix

Cellular interaction

Osteocyte

Interfacial interaction!

Deposition of bone matrix by osteoblasts

Cell/substrate interactions result in cellular signaling, which regulates cell attachment, spreading, migration, differentiation, gene expression

What the cell “feels” is in the nanoscale range

Average size of a cell: 10 to 15 μmAverage size of a protein: 10 to 15 nm




Controlled chemical oxidation

Strategy: Nanotechnology

Self-assembly:

Covalent attachment of proteins (growth factors)

New generation of implant surfaces

Improving healing response and tissue integration

Cell cultures (osteogenic cells: critical for successful integration of implants in bone; fibroblasts: formation of fibrous capsules weakens bone/implant interface – complications for permanent implants)

TiO2, Ti alloys: High biocompatibility, resistance to

corrosion, excellent mechanical properties (intrinsic)

F. Variola et al. Biomaterials (2008)L. Richert et al. Adv. Mater. (2008)F. Vetrone et al. NanoLetters (2009)

S. Clair et al. J. Chem. Phys. (2008)L. Richert et al. Surf. Sci. (2010)




Titanium, Titanium alloys

Biocompatibility, resistance to corrosion, excellent mechanical properties (intrinsic)

Improving biocompatibility by nanoscale surface modification

Develop nanotextured surfaces by controlled surface modification of TiO2 / TiAlV using chemical oxidation or plasma based approaches

Surface Modification of Biomaterials



Institut national de la recherche scientifiquePlaying tetris at the nanoscale

General Objective: Control of cell behavior by controlling surface topography and chemistry

Understanding how molecules assemble at surfaces




Before Oxidation

After Oxidation

22.4±7nm

Nanostructured Biomaterials

J.H. Yi et al., Surf. Sci. 600, 4613 (2006)L. Richert et al., Adv. Mater. 20, 1488 (2008)

Titanium, Titanium alloys

Nanotextured surfaces by controlled chemical oxidation of Ti (H2SO4/H2O2)

• Comparative SEM images: primary osteoblasts - 3 days culture on smooth (control, left) & nanotextured (right) portions of Ti6Al4V disk.

• Side-by-side surfaces obtained by treating half the disk for 1 hour.




Control

5 min

30 min 1 h 4 h

Overnight

Ce

ll d

en

sity

(Co

ntr

ol B

ase

10

0)

0

200

400

600

6 hours3 days

Control

5 min

30 min 1 h 4 h

Overnight

Ce

ll d

en

sity

(Co

ntr

ol B

ase

10

0)

0

100

200

300

400

500

600

6 hours3 days

Control

5 min

30 min 1 h 4 h

Overnight

Ce

ll d

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sity

(Co

ntr

ol B

ase

10

0)

0

200

400

600

800

6 hours 3 days

b

a

c

Measure of cell density by SEM after 6 h (black) and 3 days (red) on different etched Ti6Al4V substrates (& control) for different cell lines:

(b) fibroblasts (c) osteoblasts

Selectivity of nanotextured Ti6Al4V

Reduced proliferation of fibroblasts

Enhanced behavior towards osteoblast adhesion and growth

Influence on cell behavior

L. Richert et al., Adv. Mater. 20, 1488 (2008)F. Vetrone et al. NanoLetters 9, 659 (2009)F. Variola et al. Small 5, 996 (2009)L. Richert et al., Surf. Sci. 604, 1445 (2010)O. Seddiki et al., Appl. Surf. Sci. 308, 275 (2014)L. Cardenas et al., Nanoscale 6, 8664 (2014)



Institut national de la recherche scientifiqueChemical oxidation: general strategyTi nanostructured by oxidation:etchant acidity/basicity changed by mixing trifluoromethanesulfonic (triflic) acid (CF3SO3H), sulfuric acid (H2SO4), trifluoroacetic acid (CF3COOH) & ammonium hydroxide (NH4OH). CF3SO3H (>>> more acidic than H2SO4) combined with 30% aqueous H2O2 => spongelike network of nanopores similar to H2SO4/H2O2. CF3COOH (weaker fluorinated acid) with 30% aqueous H2O2 => distinct pattern with patches of nanopores across surface.Concentrated aqueous NH4OH & 30% aqueous H2O2 (basic oxidative etchant) => large, shallower pits (diameter ~50–100 nm) with irregular polygonal shapes. F. Vetrone et al. NanoLetters 9, 659 (2009)F. Variola et al. Small 5, 996 (2009)

scale bar: 100 nmL. Richert et al., Surf. Sci. 604, 1445 (2010)




Cell spreadingComparative cell spreading number & proliferation profile of primary calvaria-derived osteogenic cells on control & nanotextured Ti. (a) Cell adhesion / spreading visualized by epifluorescence of phalloidin (actin cytoskeleton) andDAPI (nuclei) staining.(b) Proportions of cells in stages I-IV at 4 h postplating.(c) Cell spreading at days 3, 12.

F. Vetrone et al. NanoLetters 9, 659 (2009)



Institut national de la recherche scientifiqueHindering cell growth

(a-c) Osteogenic cell growth on control Ti and surfaces etched with NH4OH/H2O2. (Scale bar: 500 μm).(c) 14 days culture: Alizarin red staining for mineral => high calcification on control surface (L); none on treated surfaces (R).(d, e) Fibroblasts growth on control Ti and surfaces etched with NH4OH/H2O2. (d) Evaluation of cell number (MTT viability test) (e) SEM image. (Scale bar: 100 μm).surface features limit growth of osteogenic *and* fibroblastic cells

F. Vetrone et al. NanoLetters 9, 659 (2009)




Covalent Attachment of Bioactive

Molecules to Ti Surfaces




Functionalized nanostructured Ti

AFM images (5x5 μm2) of Ti substrates; (a) smoothsurface, clean; (b) smooth surface, coated with Dodecylphosphoric acid

(DDPA); (c) nanotextured surface, clean; (d) nanotextured surface, coated with DDPA; (e) height profilesalong lines in b, d.

S. Clair et al., J. Chem. Phys. 128, 144795 (2008)





STM images of DDPA covered titanium;(a) and (b) smooth substrate;(c) and (d) nanotextured substrate;(e) height profiles along dashed lines in a, c. Molecular resolution visible in b (0.7 nm pitch)

Functionalized nanostructured Ti – 2




Wettability of functionalized TiO2

Water static contact angle and ellipsometry for dodecylphosphoric acid coated TiO2.On nanotextured surfaces, ellipsometry estimates deposited organic material (not real film thickness)


High hydrophobicity



Institut national de la recherche scientifiqueAging effects

Aging DDPA films on titanium (storage in air or Phosphate Buffered Saline solution)Filled circles: smooth substrate;Open circles: nanotextured substrate.

S. Clair et al., J. Chem. Phys. 128, 144795 (2008)F. Variola et al. in preparation

Perspectives:SAMs on Ti disks with crystalline oxide layer (by annealing). Formation of organic film is delayed => lower water contact angles are found => significant influence of substrate order on molecular self-assembly.




Protein adsorption on nano-Ti• Protein adsorption on control (smooth) & nanotextured Ti

L. Richert, F. Variola, F. Rosei, J. Wuest, A. Nanci, Surf. Sci. 604, 1445 (2010)

SEM images of sputtered titanium before (a) and after (b) treatment with H2SO4/H2O2.

|ΔD/Δf | values of QMC measurements for proteins adsorbed on untreated (Control) & nanopatterned (Nano) surfaces.

surfaces exertdifferential activity on proteins by promoting or limiting adhesion.





Influencing healing speed

Inter-related material/surface (synergistic) factors – understanding cell–surface interactions from a fundamental point of view:

• Surface composition• Surface energy

• Surface roughness• Surface topography• Surface charge distribution• Surface crystallinity

Interfacial interactions - Surface modification

- The next challenge…



Institut national de la recherche scientifiqueNew materials:non-permeable, self-cleaning, anti-septic

Lotus leafLotus leaf (artificial):

nm sized hydrophobic wax size: water rolls (not slides) -> cleans

sol-gel based technique -> on market

Self-cleaning plastic, textiles:Self-cleaning plastic, textiles: CNT stabilized enzymes in polymer

Textiles with ‘Stain Defender’

Air-D-FenseAir-D-Fense (InMat, New Jersey):

nanoclay/butyl thin film: 3000 fold

decreased permeability

- Nanopatterned surfaces promote cell activity (Nanoletters 9, 659 (2009)): What happens to much smaller cells, e.g. bacteria?

M. Cloutier, D. Mantovani, F. Rosei, Trends in Biotechnology 33, 637 (2015)




Influence of surface morphology on bacterial adhesion

Motivation:

- Nanopatterned surfaces promote cell activity(e.g. F. Vetrone et. al, Nanoletters 9, 659 (2009))

- What happens to much smaller cells, e.g. bacteria?




Anti-bacterial surfacesNosocomial infections (Nis): major issue in hospitals, healthcare service units & generally closed/crowded ecosystems. Contamination from instruments & surfaces by pathogenic bacteria => frequent cause of Nis. Addressing this problem requires developing functional coatings:

High antibacterial activityGood mechanical properties & strong adhesionBiocompatibilityHigh deposition rate for large-scale applications

- DLC films excellent biocompatibility, mechanical hardness, wear-resistance & chemical inertness- Ag: antibacterial element; broad-spectrum antibiotic used since ancient times, with low toxicity for humans- nanostructured titanium



Institut national de la recherche scientifiqueSurface preparation

.

Substrates: Ti sheet, cut in 1x1 cm2 pieces

Small scale roughness(1x1 µm2 )

Large scale roughness (50x50 µm2 )

As received 30 nm

500 nm

Polished (mirror) 1-2 nm 30 nm

Piranha treatment, 25˚

5-7 nm 15 nm

Piranha treatment, 80˚

6-10 300 nm

Bacterial adhesion influenced by surface properties: composition, topography & wettability

SEM images of Ti surfaces: (a) as received (untreated), (b) after polishing, (c, d) after treating polished samples for 1 hour in piranha solution at 25 °C (c) & at 80 °C (d).

O. Seddiki et al., Appl. Surf. Sci. 308, 275 (2014)M. Cloutier et al., Diam. Rel. Mater. 48, 65 (2014)



Institut national de la recherche scientifiqueInfluence of surface morphology on bacterial adhesion

- Contrary to primary calvaria-derived osteogenic cells (Vetrone et al, Nanoletters) surfaces with lower roughness significantly inhibit E-coli adhesion.

- Next: study effect of other etchants (e.g. ammonium persulfate) on cell adhesion, to clarify role of oxidative etchant on antibacterial activity

Bacteria tested: E-coli

P T25 T80

O. Seddiki et al., Appl. Surf. Sci. 308, 275 (2014)M. Cloutier et al., Diam. Rel. Mater. 48, 65 (2014)M. Cloutier, D. Mantovani, F. Rosei, Trends in Biotechnology 33, 637 (2015)




Reduced graphene oxide (rGO) on 316L stainless steel

• Stainless steel 316L (SS316L): widely used in implantable devices, coronary/cardiovascular stents, cranial fixation, orthopedic stents & dental implants.

• Challenges: limited resistance to corrosion & wear => material degradation, harmful metallic ions release => clinical complications (thrombus, apoptosis)

• Solution: coating SS316L by direct synthesis of reduced graphene oxide (rGO) => protective layer against corrosion & degradation

• Approach: coronene solution drop cast on electropolished SS316L, followed by annealing (600-800 C, 30 min) in flowing atmosphere of 98% nitrogen + 2% hydrogen in quartz tube, then cooled over 10 min in N2/H2 flow

L. Cardenas et al., Nanoscale 6, 8664 (2014); Patent pendingM. Cloutier, D. Mantovani, F. Rosei, Trends in Biotechnology 33, 637 (2015)




Properties of rGO on SS316L

• (a) Raman spectra of rGO (red), coronene on untreated SS316L (black) & coronene on glass (blue) on same area where optical images were taken for: (b) rGO/SS316L & (c) coronene / untreated SS316L.

• Scale bars: 20 µm




Institut national de la recherche scientifiqueSurface morphology & properties

• Wettability (static water contact angles): Mean static contact angle between rGO/treated SS316L & water: 62±2

• Untreated & treated SS316L used as references (mean contact angles 92± 2 & 52±2)

• => rGO layer improves SS316L wettability due to hydroxyl & carboxylic groups

Untreated SS316L: patterns of well-defined grain boundaries ~ stainless steel. After treatment => smoother surface. rGO coating => steel surface covered by flake multi-layers. (d) flakes completely cover surface (SEM).





Cell viability and cytotoxicity

• HUVEC cell growth on untreated SS316L, treated SS316L & rGO (triple sampling, repeated surveys) based on Alamar blue assay (common to screen adverse effect of nanomaterials in cell culture. Fluorescence signals => proportional to number & metabolic activity of cells)

Cytotoxicity tests on rGO, treated SS & untreated SS. Human Umbilical Vein Endothelial Cells (HUVECs) growth used to quantify cytotoxicity. HUVECs (cells that line inner surface of blood vessels) are sensitive compared to fibroblasts & smooth muscle cells





• Phase-contrast microscopy images (2D cultures): cell morphology & spreading not affected compared to control for all three samples (rGO, untreated SS & treated SS)

Cell viability and cytotoxicity




Institut national de la recherche scientifiqueGiant core-shell QD nanothermometers

The concept

Double PL emission

Color (& lifetime of 650 nm band) changes with temperatureMultiparametric response High sensitivity

H. Zhao et al., Nanoscale 8, 4217 (2016)H. Zhao et al., Small 11, 5741 (2015)G. Sirigu et al., Phys. Rev. B, in press (2017)




Nanotheranostics

Nanotheranostics:drugs & imaging agents combined into single formulation=> targeted therapeutics (e.g. radiation therapy and/or drug delivery) & diagnostics for personalized medicine

Advantages of nanotheranosticsTargeted deliveryCombined imaging tracking & therapeutics




Core/Shell structure of RE3+ co-doped UCNPs

Functional group

Chemotherapeutic drugs

RE based multifunctional nanoplatform (MFNP)

NIR light

NIR

Imag

ing

(e.g., o

ptical, M

RI, an

d C

T scan

)

Targeting (passive and active)

UV/VIS

Combination therapy (e.g. Chemotherapy, UC-PDT)

Thin silica shell of SNC

Photodynamic therapy (PDT) drugs

Singlet oxygen (1O2)




Platform concept

Gold Nanorods (GNRs)

UCNPs

GNRs/UCNPs Nanocomposite

Near infrared light (NIR)

Red emission

Green emission

43ºC




Gold nanorods (GNRs) with tunable optical absorptions at visible & NIR wavelengths

Photophysical processes in GNRs. Light irradiation => excitation of longitudinal plasmon resonance mode => mostly absorption & resonant light scattering

Gold nanorods (GNRs) based platforms for photothermal therapy

Tong et al. 2009 Photochem Photobiol.

PL




GNRs SiO2 NaGdF4: Er3+, Yb3+ UCNPs

Prashant et al. 2008 Acc. Chem. Res.

GNRsUCNPsUCNPs&GNRs

+=

GNR@SiO2@UCNPs Nanocomposite

Ab

sorb

ance

[a.

u.]

Y. Huang et al., J. Phys. Chem. B 120, 4992 (2016)Y. Huang et al., Nanoscale 7, 5178 (2015)




• Nanostructured materials new properties

• Controlling cell–surface interactions:• Nanostructuring Ti/Ti alloys: enhanced

biocompatibility (accelerated formation of calcified

tissue)• Selectivity (osteoblasts vs. fibroblasts)• New concepts for antibacterial coatings:

• Nanotextured surfaces – changes in wettability

• rGO coatings, cytotoxicity

• Giant QDs to measure nanoscale temperature• Nanotheranostics

Conclusions and OutlookConclusions and Outlook




F. Rosei, A. Pignolet, T.W. Johnston, J. Mater. Ed. 31, 65 (2009)F. Rosei and T.W. Johnston, J. Mater. Ed. 31, 293 (2009)F. Rosei and T.W. Johnston, J. Mater. Ed. 32, 163 (2010)F. Rosei and T.W. Johnston, J. Mater. Ed. 33, 161 (2011)F. Rosei and T.W. Johnston, J. Mater. Ed. 34, 197 (2012)F. Rosei and T.W. Johnston, J. Mater. Ed. 35, 127 (2013)




Future Opportunities

3D printing (additive manufacturing) of multifunctional material systems

Combined with

Surface functionalization (altering wettability, controlled drug release)



Institut national de la recherche scientifiqueAcknowledgementsAcknowledgementsGe/Si, Si, Ge nanostructuresGe/Si, Si, Ge nanostructures::• F. Ratto (CNR), D. Riabinina, C. Durand (Univ./CEA Grenoble), K. Dunn, L. Nikolova, J.

Derr (Univ. Paris), M. Chaker (INRS), J. Margot (UdeM)

Nanostencil / functional materialsNanostencil / functional materials::• A. Pignolet, C. Cojocaru (NRC), R. Nechache, S. Li (USTB), A. Vomiero (Lulea), D. Obi, C.

Harnagea (INRS), J. Chakrabartty, S. Barth (TU Wien), G. Chen (Jinan)

Organic molecules: supramolecular structures, 2D polymers, organic electronic devicesOrganic molecules: supramolecular structures, 2D polymers, organic electronic devices• INRS: J. Miwa (UNSW), A. Dadvand (NRC), F. Cicoira (EPM), C. Santato (EPM), J.

MacLeod & J. Lipton-Duffin (QUT), T. Dembele, C. Yan (Souzhou Dresden), G. Galeotti, R. Gutzler (Max Planck), L. Cardenas (CNRS), M. El Garah, K. Moonoosawmy, M. Rybachuk (Griffith), S. Clair (CNRS); D.F. Perepichka (McGill)

• B.J. Eves, G.P. Lopinski (NRC–SIMS, Ottawa)

Nanostructured Biomaterials:• K.G. Nath (Corning Japan), F. Variola (UofO), C. Brown (Oxford), O. Seddiki, A. Vittorini,

F. Vetrone (INRS), L. Richert (CNRS), A. Nanci, J.D. Wuest (UdeM), D. Mantovani (Laval)

Carbon Nanotubes:• S. Miglio, M.A. El Khakani (INRS), P. Castrucci, M. Scarselli, M. De Crescenzi (Roma 2)

AFOSRAFOSR




Upconverting NanoparticlesPhoton upconversion: sequential absorption of two or more photons => emission of light at shorter wavelength than excitation wavelength (anti-Stokes type emission)

Near infrared light (NIR) Activator (Er3+, Ho3+ and Tm3+)

Host

Sensitizer(Yb3+)

Visible light

F. Wang, X Liu. Analyst 2010 (135): 1839




Cell viability of GNR@SiO2@UCNPs

Viability of Hela cells treated with different samples with and without laser irradiation at 980 nm. Standard deviations are shown (n=3).





OFF

OFF

OFF

ON

ON

Drug loading and drug release

Production of singlet oxygen under consumption of ABDA (different samples over time)

Production of singlet oxygen under consumption of ABDA (absence & presence of laser irradiation)





TEM a single core/shellTEM a single core/shell

XRDEDX

Cd:S molar ratio 1:1 Cd:S molar ratio 1:0.8

CdS shell: Zinc Blende (ZB) and Wurtzite (WZ)

Gradient interfacial layer facilitates hole transfer, regulates transition from double- to single-color emission.

Double 5.5 nm

Single 4.9 nm

H. Zhao et al, Nanoscale, 2016, 8, 4217L. Jin et al, Nano Energy, 2016, 30, 531

Mechanism for double emission

Controlling molar ratio of Cd/S to control the interfacial gradient layer

Cation exchangeSILAR




Excitation/emission & interatomic energy transfer process in UCNPs

http://foundry.lbl.gov/schuckgroup/index.html

Upconversion in rare earths




UCNPs for biomedical applications

• Significantly reduced background autofluorescence

• Remarkable penetration depths in vivo & high spatial resolution

• Fluorescence bands lie within “biological window” (650-1350 nm)

• Low cyto- and phototoxicity to biological specimen

Advantages:

Biomedical applications of UCNPs

• Imaging diagnostics

• Photodynamic therapy

• Photothermal therapy

• Drug delivery system

UCNPs injection

▶ UCNPs locating a tumor in a live mouse

Peng et al. Nano Res. 2012 (5): 770




43°C

Laser

Nanoparticle-based photothermal therapy

Photothermal therapy (PTT) is based on laser heating of metal nanoparticles.

Advantages of Au NPs as antitumor photothermal agents:

1)Unique optical properties

2)Photostability

3)Low toxicity

4)Well-known synthesis protocolsDickerson et al. 2011 Chem. Soc. Rev



Institut national de la recherche scientifiqueStrategies to achieve high luminescence efficiency and deep tissue penetration




972 nm

983 nm

GNR@SiO2 Synthesis Procedure

Mix GNRs solution with tetraethyl orthosilicate (TEOS) in methanol and NaOH to form a porous silica

shell

GNRs Synthesis

Seed solution (μL)

CTAB (g)

Ascorbic acid (aq) (μL, mM)

AgNO3 (aq) (mL, mM)

32 0.72 80, 64 0.60, 4

GNRs GNR@SiO2

Synthesis of GNRs and GNR@SiO2




Rare earth (RE) doped nanoparticles (NPs)

Advantages:

Large anti-StokesNarrow emission bandwidth Long-lived luminescence High photostability:Low autofluorescenceDeep tissue penetration

Upconversion emission spectrum of (0.5 mol%) Tm3+ (25 mol%) Yb3+-doped LiYF4 nanocrystals spanning the UV to NIR regions.

Multimodal NPs:

Optical imaging

Magnetic resonance imaging (MRI)

Computed tomography (CT) scans

Therapeutic functionality Mahalingam et al. Adv. Mater. 2009, 21, 4025.




Drug loading and drug release

Drug loading (ZnPc) efficiency: 2.5 wt.%

Upconversion emission spectrum of UCNPs and UV-visible absorption spectra of ZnPc




Cellular uptake of UCNPs and GNR@SiO2@UCNPs

Control UCNPs GNR@SiO2@UCNPs




Lanthanide Trifluoroacetate

Precursors

OA/OD

240ºC

Ligand Exchange

Citric Acid

OA = Oleic Acid OD = Octadecene

Oleate Stabilized NaGdF4:Er3+, Yb3+

(Hydrophobic)Citrate Stabilized NaGdF4:Er3+, Yb3+

(Hydrophilic)

TEM of NaGdF4:Er3+, Yb3+ UCNPs

Synthesis of hydrophobic OA capped UCNP and subsequent hydrophilic ligand exchange

Synthesis of NaGdF4:Er3+, Yb3+ UCNPs

α-NaGdF4 JCPDS: 27-0697




T sensing using NaGdF4:Er3+,Yb3+ UCNPs

Upconversion luminescence spectra of NaGdF4:Er3+, Yb3+ UCNPs at two different temperatures

Temperature dependence of ratio calculated from luminescence spectra. Dots are experimental results, red line is best linear fit




C

O

FCu

GdNa

Yb

AuSiYb

Au

AuGdGdYb

GdYbGd

CuGd

YbAuYbCu

YbAu

Er

Er

Er

ErEr

* Stars indicate typical diffraction peaks of GNRs

* * * *

Synthesis of GNR@SiO2@UCNPs




Luminescence of GNR@SiO2@UCNPs

Thermal change of GNR@SiO2@UCNPs determined using calibration curve of intensity ratio

Upconversion luminescence spectra of UCNPs and GNRs@SiO2@UCNPs




Surface quenching site

RE ion(Sensitizer, e.g. Yb3+ )

RE ion (Activator, e.g. Er3+, Tm3+)

Host

Crystal structures of host, energy transfer process, surface deactivations

High luminescence efficiency => high performance nanotheranostics

Wang, Liu, J. Am. Chem. Soc., 2008, 130, 5642

Boyer et al., Nano Lett., 2007, 7, 847




Krämer et al., Chem. Mater., 2004, 16, 1244

c: Hexagonal (β) and d: Cubic (α) Green plus red emissions of hexagonal

phase are 4.4 times stronger than those of cubic one

Crystal structures of α-NaREF4 and β-NaREF4 built by CERIUS2 software (Http://www.accelrys.com/cerius2). (Thoma et al. Inorg. Chem. 1966, 5, 1222)

Influences of crystal structures on UC efficiency

Low crystal field symmetry Low phonon cut-off energy

http://www.accelrys.com/cerius2




Vetrone et al., Adv. Funct. Mater., 2009, 19, 2924

UC luminescence spectra of colloidal β-NaGdF4: 20%Yb3+, 2%Er3+ UCNPs

Influence on UC efficiency

Suppression of surface deactivation

Modulation of the energy transfer

Core-only Active core/inert shell Active core /active shell

NaGdF4 Yb3+ Er3+

NaGdF4 Yb3+ Er3+

NaGdF4

NaGdF4 Yb3+ Er3+

NaGdF4

Yb3+




NIR-I: 700-950 nmNIR-II: 100-1350 nm NIR-III: 1550-1870 nm

Hemmer et al., Nanoscale Horiz. 2016, 1, 168

Deep tissue penetration: firm requirement for in vivo application




Yb3+ or Nd3+ ?

Wang et al., ACS Nano 2013, 7, 7200




NIR-I: 700-950 nmNIR-II: 100-1350 nm NIR-III: 1550-1870 nm

RF: Eva Hemmer, Antonio Benayas, François Légaré and Fiorenzo Vetrone*, Nanoscale Horiz., 2016, 1, 168—184.

Deep tissue penetration is a firm requirement for in vivo application




Features: •Single step approach•Uniform, monodispersed nanoparticles •More potential to control particle morphology

Schematic illustration of one-step thermolysis

Chen, Chem. Rev. 2014, 114, 5161

Morphology controlled synthesis of RE-doped NPs by thermolysis




The surface engineering of RE-doped NPs is a crucial step for biomedical applications.

Silica based nanocapsules (SNCs)

RE-doped NPs caped by hydrophobic ligands (e.g. oleic acid) are not dispersible in an aqueous solution or physiological buffer.

• Ligand exchange• Ligand oxidation • Ligand removal• Ligand attraction• Surface silanization (e.g.

Silica nanocapsules)

Strategies of surface engineering for hydrophobic RE-doped NPs:

Limitations: poor colloidal stability under physiological conditions

Silica nanocapsules (SNCs) are especially suitable for the application of nanotheranostics.

TEM images of: (a) ‘naked’, and (b) PEO-SiO2 coated MnO nanoparticles.

T1-weighted MRI images of MDA-MB-231 cells incubated with PEOMSNs at various concentrations for 24 h.

RF: B. Y. W. Hsu, M. Wang, Y. Zhang, V. Vijayaragavan, S. Y. Wong, A. Y.-C. Chang, K. K. Bhakoo, X. Li and J. Wang, Nanoscale, 2014, 6, 293-299.




PEOlated silica nanocapsules via interfacial templating condensation

Silica encapsulation

RF: Y. Zhang, M. Wang, Y.-g. Zheng, H. Tan, B. Y.-w. Hsu, Z.-c. Yang, S. Y. Wong, A. Y.-c. Chang, M. Choolani and X. Li, Chem. Mater., 2013, 25, 2976-2985.

F127

Uniqueness:Benign approachExcellent colloidal stabilityTargeted delivery



Institut national de la recherche scientifiqueSurface functionalization for targeted delivery

RF: Fabienne Danhiera, Olivier Feronb, Véronique Préata, Journal of Controlled Release, 2010, 148(2), 135–146.

Size ≥ 8 nm Delivered by enhanced

permeability and retention (EPR) effects

Enhanced the accumulation of drugs in tumor tissue

Delivered by the receptors overexpressed on the targeted cell membrane

Further enhanced the accumulation of drugs in tumor tissue



Institut national de la recherche scientifiqueSurface functionalization for targeted delivery

+

Folate PEO-bis-NH2

NH

S

DC

C

RF: H. Tan, Y. Zhang, M. Wang, Z. Zhang, X. Zhang, A. M. Yong, S. Y. Wong, A. Y.-c. Chang, Z.-K. Chen and X. Li, Biomaterials, 2012, 33, 237-246.

Carboxylic functionalized SNCs Folic acid conjugated SNCs

+

Succinic anhydride F127D

MA

C

DMAC:N,N-dimethylacetamide, NHS:N-Hydroxysuccinimide, DCC:N,N'-Dicyclohexylcarbodiimide, DEC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

PEO–PPO-PEO




Morphology and crystal structure study by transmission electron microscopy (TEM), high resolution TEM (HRTEM), 3 dimension TEM (3DTEM), and powder X-ray diffraction analysis (XRD)

UC and NIR luminescence emission study by photoluminescence spectroscopy

Composition analysis of MFNP by Fourier Transform Infrared (FTIR) Spectroscopy

Loading capacity measurement of UCNPs by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

Stability against physiological aqueous environment by Dynamic Light Scattering (DLS)

Bio-compatability study by cell viability assay

Cellular uptake study by optical confocal microscopy, and MRI

Characterization



Institut national de la recherche scientifiqueMorphologies of β-NaGdF4: 20%Yb3+, 2%Er3+ UCNPsUniform, Monodispersed, Narrow Size Distribution

43.5±2.5x24.7±1.6 (nm) 62.9±3.1x29.8±2.1 (nm)28.85±1.04x17.19±1.05 (nm) 21.2±1.09 (nm) 19.74±1.29x15.36±1.07 (nm)

Diameter: the distance from corner to corner of the surface perpendicular to the c-axis

Height: the vertical distance between the top and bottom surface

Aspect ratio: Diameter/Height

Increasing0.62 2.14



Institut national de la recherche scientifique3DTEM and HRTEM analysis of the hexagonal nanorods

[001]




UC luminescence spectra display differences based on morphology of β-NaGdF4 : 20%Yb3+, 2%Er3+ UCNPs

The UCPL intensity inversely proportional to the surface to volume ratio (S/V) in the logarithmic scale due to the surface quenching effects.

The emission ratio of green to red (fG/R) is related to the aspect ratio of UCNPs: the higher the fG/R is, the closer the aspect ratio to 1.

Sha Liu, Theranostics 2013; 3(4):275-281




Quantitative bacterial adhesion protocol

.

rinse

sonication

TSA petri dish24h incubation

(colony forming unit counting )

1 hr

Bacteria tested: E-coli




Synthesis of LiYF4 based UCNPs co-doped with Yb3+, Tm3+, Nd3+, and Gd3+

Selection of low symmetry lattice host

Suppression of surface related

deactivations by active core/active

shell/inert shell

Engineering energy transfers by

tuning the dopants concentration

Strategies to achieve high emission efficiency:

Gd3+ as T1 contrast agent

Energy transfer of Nd3+→ Yb3+→ Tm3+

LiYF4: Yb3+ Tm3+

Gd3+

LiYF4:

Yb3+ Nd3+

LiYF4:

LiYF4:Yb3+,Tm3+@LiYF4:Yb3+,Nd3+ @Li(Y,Gd)F4




Applications

- Biocompatible materials (implantable):- Cardiovascular stents- Orthopaedic implants

- Tissue engineering- Regenerative medicine- Antibacterial coatings

Approach:Using advanced processing techniques to controlStructure/property relationships in materials




Challenges• Similar to those of any manufacturing area:

– Improve performance

– Reduce costs

– Increase longevity

Effective processing tools-Top down-Bottom up-Chemical (etching, oxidation)-Physical (plasma processing)

Materials of interest:-Titanium, Ti alloys-Cr/Co alloys-Stainless steel




M. Cloutier et al., Diam. Rel. Mater. 48, 65 (2014)

Raman spectroscopy (λ = 488 nm) of DLC films a) deconvoluted peaks & fitted background, b) Pos(G), c) I(D)/I(G) ratio, d) FWHM(G) & e) H content of as-deposited (squares) and aged (triangles) DLC films as a function of deposition power.

Aging of DLC Samples

After aging, Pos(G), I(D)/I(G) & FWHM(G) show same trends as their as-deposited counterparts, with similar values⇒no significant phase change.H concentration increases (18 to 27%) in all samples (attributed to surfaceadsorbed water).




0 50 100 150 200 250

0.000

5.000

10.000

15.000

20.000

25.000

20um

5um

1um

Power (W)

RM

S r

ou

gh

ne

ss

(n

m)

SS316L 150W DLC on SS316L

Roughness (RMS) of SS316L & DLC–SS316L samples.

DLC coatings on stainless steel

0 50 100 150 200 250 300 350

-4.00

-3.50

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

Film stress (Gpa)

Stress (GPa) in DLC coating

Challenges: (i) stress control to prevent delamination; (ii) surface nanotexturing & incorporation of antibacterial elements (Ag,F)

O. Seddiki et al., Appl. Surf. Sci. 308, 275 (2014)M. Cloutier et al., Diam. Rel. Mater. 48, 65 (2014)



Institut national de la recherche scientifiqueStress optimization

Low stress film prepared at 200 W: most resistant to delamination after autoclave test (sterilization under high pressure saturated steam)




Institut national de la recherche scientifiqueIn situ interface treatment Developed in situ interface treatment (in same

PECVD-PVD reactor as DLC deposition) Modified interface (MI): vastly improved adhesion &

minimal delamination after scratch & autoclave tests.

50µm50µm

Endurance in autoclave(2 hour cycle)

Scratch test

DLC/MI/SSDLC/SS

DLC/MI/SSDLC/SS





Spider silk knot (SEM): impressive ductility & toughness under shear, withstands both compressive & tensile stresses=> No damage to inside regions of bends, (large compressive stress), or outer regions of bend (large tensile stress)

“Visions” of silk

C. Brown et al., ACS Nano 6, 1961 (2012)

J. MacLeod, F. Rosei, Nature Mater. 12, 98 (2013)



Institut national de la recherche scientifiqueHierarchical structure of spider silk

S. Keten, M. J. Buehler, Nanostructure and molecular mechanics of dragline spider silk protein assemblies, J. Roy. Soc. Interface 7, 1709–1721 (2010).

AFM of spider silk fibre cross-section (a) two skin layers, with fiber centre towards image bottom-left (b) core region with globular morphology

(A) Hierarchical organisation of spider silk(B) Stress-strain behaviour of wet and dry spider silk.

C.P. Brown et al., Nanoscale 3, 3805 (2011)

C. Brown et al., Nanoscale 3, 870 (2011)




Fibril morphology in spider silk: normal conditions => non-slip fibril kinematics, restricting shearing between fibrils, yet allowing local slipping under high shear stress, dissipating energy without bulk fracturingMechanism could increase fracture resistance in synthetic materials under bending/torsion conditions.

Nanoscale mechanics of spider silk

C. Brown et al., Nanoscale 3, 870 (2011) C. Brown et al., Nanoscale 3, 3805 (2011)



Institut national de la recherche scientifiqueNanoscale mechanics of spider silkAFM-nanoindentation: protein interaction with water dominates energy processing, providing sacrificial bond => ‘plastic’ effect in inner core (black) in dry/ambient conditions. Hydrophobic outer core is elastic under these conditions

Interactions with H20 => stiffness differential across fibre, provides balance between stiffness, strength & toughness under dry/ambient conditions.Wet conditions => balance destroyed as stiff outer core reverts to behaviour of inner core

Basic features of spider silk are known => challenging to reproduce in a wet fibreC.P. Brown et al., Nanoscale 3, 3805 (2011)




SAXS&WAXS: no change in crystal size with increasing hydration (a) Integrated region (to obtain SAXS/WAXS profiles); (200)&(120) peaks indicated with fibre axis direction & location. Inset: entire scattering pattern (b) Integrated average SAXS/WAXS profiles (0–100%)Inset right: enlarged view of WAXS region

SAXS/WAXS insights

C.P. Brown et al., Nanoscale 3, 3805 (2011)




Fibrils interaction: critical at high strains in bending, torsion & combined loading with high shear stress between fibrils.AFM: fibril structure across size ranges (A)–(D): fibrils in spider silk fibres core region, (E): two bundles of interlocking collagen fibrils in fascia, (F): collagen in tendon (A),(B),(E),(F): microns; (C),(D): nanometresGlobular/banding patterns appear in each fibril & interlocking of globules/bands between fibrils.

Fibrils and toughening mechanism

Homogeneous properties: valid for axial tension with fibrils aligned parallel to fiber C. Brown et al., ACS Nano 6, 1961 (2012)




Hierarchical supramolecular structure of spider silk:Network of rubber-like chains reinforced by β–sheet crystals.Increased extensibility in infiltrated fibres: due tohigher proportion of rubber-like amorphous domains & size reduction of β–sheets from water infiltration process

Y. Termonia, Macromolecules 27, 7378 (1994) S.M. Lee, Science 324, 488 (2009)

Hierarchical Supramolecular Structure




F. Variola et al. Biomaterials 29, 1285 (2008)

Morphological Analysis: Statistics

110000 nnmm 110000 nnmm

110000 nnmm 110000 nnmm 110000 nnmm

Control

15 min

30 min

1 h 4 h2 h

Evolution of nanopit diameter vs. etching time in α-phase grains by SEM.Measurements at 15 min refer to β-phase grains (β-phase is preferentially etched)



Institut national de la recherche scientifiqueGuiding stem cellsHuman umbilical cord stem cells grown on control Ti surfaces, nanotextured Ti & control glass coverslips. (a) Day 1: HUC cells spread on all surfaces (elongated shape). Nanostructured Ti: areas of higher cell density. (b, c) Dual nuclear labeling with anti-Ki-67 antibody (red fluorescence) and DAPI (blue fluorescence) at day 3 => 1.6-fold increase of cycling cells compared to control Ti. Phalloidin labeling appears green in (a) and pale white in (b).

Scale bar: 200 μm (a) and 100 μm (b)F. Vetrone et al. NanoLetters 9, 659 (2009)



Institut national de la recherche scientifiqueCompositional/Morphological Analysis by SEM: TiAlV

Back-scattered image of treated (4 h) Ti6Al4V surface

Al (wt%) V (wt%)

Bulk 6.3±0.2 3.5±0.4

α-phase 6.9±0.3 2.7±0.4

β-phase 4±0.8 11.2±1.7


α-phase grains

β-phase grains




Surface Coverage

Controls 95%

Nano-texturedsamples

70%

Biological Effects:

fibroblasts





100 nm100 nm100 nm100 nm

Effect of Treatment Time:Increase in Oxide Layer Thickness and Microtexture

AFM-Depth Measurements

Ellipsometry

FT-IR

Control 30 mins 4 hrs

30 mins:β-grains (V rich) preferentiallyetched (pittingstarts elsewhere)

4 hrs: the wholesurface is entirelyNanotextured

AFM:Increasing cavitydepth caused byβ-grain preferentialetchingTiO2 thickness

(Ti-O stretching between 400-1000 cm-1 in IR)

F. Variola et al. Biomaterials 29, 1285 (2008)F. Variola et al., Appl. Spectroscopy 63, 1187 (2009)




Temperature

100 nm100 nm

100 nm100 nm 100 nm100 nm

100 nm100 nm

Temperature and H2O2 Concentration:Increase Oxide Thickness and Create Sub-µ Texture

% H2O2

F. Variola et al., Adv. Eng. Mater. 11, B227 (2009)F. Variola et al., Appl. Spectroscopy 63, 1187 (2009)

5 °C 25 °C 80 °C Microtexture is superimposed on nanotextureabove 50 °C.

FT-IR

5 °C

25 °C

50 °C

80 °C

H2SO4

H2O2-25%-H2SO4-75%

H2O2-50%-H2SO4-50%

H2O2-75%-H2SO4-25%

H2O2

H2SO4H2O2pirana

1 hr




SEM micrographs of untreated (a, b) polished Ti6Al4V surfaces & surfaces exposed to H2O2/H2SO4 for 1 h (c, d) and 20 h (e, f).

Chemical oxidation induces both micro and nanotexture on TiAlV

Surface Modification: Morphology




Institut national de la recherche scientifiqueSurface Topography by AFM

Before Oxidation

After Oxidation:

Drastic change in surface roughness

J.H. Yi et al., Surf. Sci. 600, 4613 (2006)

Evolution of average surface roughness (Ra) during treatment by AFM on 5x5 μm2 (*) and 0.5x0.5 μm2.

L. Richert et al., Adv. Mater. 20, 1488 (2008)




Surface Chemistry of TiO2 by XPS

Before Oxidation

After Oxidation





TiAlV: Surface crystallinityby Raman and XRD

Raman spectra of an untreated Ti-alloy disk and one exposed to piranha solution (1 h)

Grazing-angle XRD pattern of a treated alloy surface (4 h). Inset: XRD patterns in the 20-30° range





Control

15 min

30 min

1 h

2 h

4 h

AFM topographies (5x5 mm2) of polished Ti-alloy disks


Morphological Analysis: AFM

AFM: increasing cavity depth caused by β-grain preferential etching




UC emission and NIR spectra under excitation of 806 nm

200 nm

Dual upconverting and near-infrared emitting core/shell LiYF4: Yb3+, Tm3+ @LiYF4: Yb3+, Nd3+

3 F0 →

3 F4

1 D2→

3 H6

1 D2 →

3 F4

1G4 → 3H6

1G

4 →

3F

4

3 F0 →

3 F4

1 D2→

3 H6

1 D2 →

3 F4

1G4 → 3H6

1G

4 →

3F

4

Inten

sity (a.u)

Inte

nsity (a.u

)

Inten

sity (a

.u)

Inten

sity (a.u

)

2 F 7/2 → 2 F 5/2

2 F 7/2 → 2 F 5/2

4F

11/2 →

4F

3/2




Biomaterial Implants

• Practical Goals: to design new devices allowing– Controlled healing– Faster healing– More stable implants

• Consequently– Decrease patient morbidity– Decrease health cost– Increase patient happiness! (psychology)

Hip and knee implants: over 300000*

Dental implants: 100 000 to 200000**

per yea

r

only in

the US

* Graves, E. Vital and health statistics, … Hyattsville, MD: National Center for Healt Statistics 1993 **Dunlap, J. Dent Econ, 78, 101 (1988)

Fundamental goal: understanding cell – surface interactions




Amorphous

Nanotexture on Amorphous or Crystalline Ti

An

nealin

g F. Variola et al., in preparationBottom: thermal oxidation (air, 400 °C, 3 hrs)

Rutile Rutile

Top: controls

Etching of Crystalline TiO2

Is not possible

Raman

Annealing etchedSample yieldsNanotexturedCrystalline TiO2




Covalent Immobilization =Surface Science!

Based on silane chemistry:

OH-surf.-- (SinH2n+2) -- biomolecule

Plasma deposition of SAMs

Functional group diversity

Plasma treatments

OH- OH- OH-

Increase surface [OH-]

OH- OH-

OH-

Quantum dots

Different electrical properties

Chemicallinker

Puleo & Nanci, Biomaterials, 20, 2311 (1999)

Stupp & Braun, Science, 277, 1242 (1997)




The atomic concentration of the main constituent, TiO2, did not vary dramatically, but suboxidessuch as TiO and Ti2O3 were no longer detected in themain oxide layer after 30 min of etching. The superficial layercomprises a mixture of amorphous TiO2, Al2O3, and small quantities of V2O5 after treatment. The oxide filmis composed of three different layers, namely TiO (inner layer in contactwith the metal), Ti2O3 (intermediate layer), and TiO2 (outerlayer) (Fig. These findings, coupledwith IR and ellipsometric results, suggest that the oxidationprocess increases mainly TiO2 to a degree that no longerallows detection of the underlying suboxides, but their layerorganization is not altered. This behavior is chemically plausibleand can be explained by assuming that suboxides such asTiO and Ti2O3 are transformed into TiO2 in the oxidativemedium of piranha solution [72], and by assuming that theetching solution penetrates the nanopits and reaches the underlyingmetal. When the solution reaches the suboxides, they arefurther oxidized into TiO2, thereby increasing the thickness ofthe dioxide layer in a manner consistent with ellipsometricmeasurements. When the underlying metal is exposed to theinfiltrating solution, natural passivation conditions are recreated.This re-establishes the initial native layered structure,composed of TiO (in contact with the metal), Ti2O3 (intermediate),and TiO2 (outer layer). However, now it is no longer incontact with the environment but rather with a nanoporousTiO2 layer derived from suboxides transformation (Fig. 16b).




TiO2: Surface crystallinity by XRD





Spectroscopic Analysis:

FT-IR and Ellipsometry
