Nanoscale Properties of Biocompatible materials

INRSScience in ACTION for a World in EVOLUTION
Université du Québec
Institut national de la recherche scientifique
Nanoscale Properties ofNanoscale Properties of
Biocompatible materialsBiocompatible materials
Induction Ceremony, Academia de IngegneriaInduction Ceremony, Academia de Ingegneria
Mexico 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)
rosei@emt.inrs.carosei@emt.inrs.ca
Federico RoseiFederico Rosei
UNESCO Chair in Materials and Technologies for EnergyUNESCO 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 10
9
2 10
9
3 10
9
4 10
9
-50-40-30-20-100
I
ds
(A)
EL(photons/s)
V
ds
(V)
V
gs
= -30
V
gs
= -20
V
gs
= -40
V
gs
= -10
OLETs Chemical
mapping
Molecular Self-assembly
Gatti J Phys Chem C (2014)
MacLeod Langmuir (2015)
Group IV nanostructures
Moutanabbir Phys Rev B (2012)
Multifunctional materials
Nechache Nature Phot (2015)
Li Small (2015)
Zhao Small (2015)
Organic Electronics
Dadvand Angew Chem (2012)
Dadvand J Mater Chem C (2013)
Organic/hybrid Photovoltaics
Dembele J Mater Chem A (2015)
Dynamic Transmission
Electron Microscopy
Nikolova Phys Rev B (2013)
Nikolova J Appl Phys (2014)
Nanostructured catalysts
Chen Adv Func Mater (2012)
Nanostructured Biomaterials
MacLeod Nature Mater (2013)
Cloutier Diam Rel Mater (2014)
Cloutier Trends Biotech (2015)
Surface polymerization
Surface Polymerization
Di Giovannantonio ACS Nano (2013)
Gutzler Nanoscale (2014)
Vasseur Nature Comm (2016)
QD solar cells
Jin Adv. Sci. (2016)
Zhou Adv. En. Mater. (2016)
Emerging
Phenomena
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 μm
Average 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 (uncalcified
bone matrix)
Calcified bone matrix
Cellular interaction
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 μm
Average 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
m
in
30
m
in
1
h
4
h
Overnight
Celldensity
(ControlBase100)
0
200
400
600
6 hours
3 days
Control
5
m
in
30
m
in
1
h
4
h
Overnight
Celldensity
(ControlBase100)
0
100
200
300
400
500
600
6 hours
3 days
Control
5
m
in
30
m
in
1
h
4
h
Overnight
Celldensity
(ControlBase100)
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
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)

Chemical oxidation: general strategy
Ti 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 nm
L. Richert et al., Surf. Sci. 604, 1445 (2010)

Cell spreading
Comparative 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) and
DAPI (nuclei) staining.
(b) Proportions of cells in
stages I-IV at 4 h postplating.
(c) Cell spreading at days 3,
12.

Hindering 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

Covalent Attachment of Bioactive
Molecules to Ti Surfaces

Functionalized nanostructured Ti
AFM images (5x5 μm2
) of Ti
substrates; (a) smooth
surface, clean; (b) smooth
surface, coated with
Dodecylphosphoric acid
(DDPA); (c) nanotextured
surface, clean; (d) nanotextured
surface, coated with DDPA; (e)
height profiles
along 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.
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
exert
differential
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 surfaces
Nosocomial 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 activity
Good mechanical properties & strong
adhesion
Biocompatibility
High 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).
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
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 pending

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 nanotheranostics
Targeted delivery
Combined imaging tracking & therapeutics

Core/Shell structure of
RE3+
co-doped UCNPs
Functional group
Chemotherapeutic drugs
RE based multifunctional nanoplatform
(MFNP)
NIR light
NIR
Imaging(e.g.,optical,MR
Targeting (passive and
UV/VIS
Combination therapy (e.g. Chemotherapy, UC-PDT)
Thin silica shell of SNC
Photodynamic therapy
(PDT) drugs
Singlet oxygen (1
O2)

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.
GNRsUCNPs
UCNPs&GNRs
+
=
GNR@SiO2@UCNPs Nanocomposite
Absorbance[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)

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 Nanoparticles
Photon 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, 4217
L. 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

Strategies 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-Stokes
Narrow emission bandwidth
Long-lived luminescence
High photostability:
Low autofluorescence
Deep 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
F
Cu
Gd
Na
Yb
Au
Si
Yb
Au
Au
Gd Gd Yb
Gd
Yb
Gd
Cu
Gd
Yb
AuYb
Cu
Yb
Au
Er
Er
Er
Er
Er
* 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

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 nm
NIR-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 nm
NIR-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 approach
Excellent colloidal stability
Targeted delivery

Surface 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

Surface functionalization for targeted delivery
+
Folate PEO-bis-NH2
NHS
DCC
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 F127
DMAC
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+
UCNPs
Uniform, 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
Increasing
0.62 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 dish
24h 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 control
Structure/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

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 surface
adsorbed water).

0 50 100 150 200 250
0.000
5.000
10.000
15.000
20.000
25.000
20um
5um
1um
Power (W)
RMSroughness(nm)
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)

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, 3805C. 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 fracturing
Mechanism 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 silk
AFM-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 fibre
C.P. Brown et al., Nanoscale 3, 3805

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

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): nanometres
Globular/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 to
higher 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
Contr
ol
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)

Guiding stem cells
Human 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-textured
samples
70%
Biological Effects:
fibroblasts

100 nm100 nm
100 nm100 nm
Effect of Treatment Time:
Increase in Oxide Layer Thickness and Microtexture
AFM-Depth MeasurementsEllipsometry
FT-IR
Control 30 mins 4 hrs
30 mins:
β-grains (V rich)
preferentially
etched (pitting
starts elsewhere)
4 hrs: the whole
surface is
entirely
Nanotextured
AFM:
Increasing cavity
depth caused by
β-grain
preferential
etchingTiO2 thickness
(Ti-O stretching between
400-1000 cm-1
in IR)
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 nanotexture
above 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
H2SO4
H2O2
pirana
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

Surface Topography by AFM
Before
Oxidation
After Oxidation:
Drastic change in
surface roughness
Evolution of average
surface roughness (Ra)
during treatment by AFM
on 5x5 μm2
(*) and 0.5x0.5
μm2
.

Surface Chemistry of TiO2 by XPS
Before
Oxidation
After
Oxidation

TiAlV: Surface crystallinity
by 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

Contro
l
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
1
G4 → 3
H6
1
G4→3
F4
3
F0→3
F4
1
D2→3
H6
1
D2→3
F4
1
G4 → 3
H6
1
G4→3
F4
Intensity(a.u)
Intensity(a.u)
Intensity(a.u)
Intensity(a.u)
2 F 7/2→2 F 5/2
2 F 7/2→2 F 5/2
4F11/2→4F3/2

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 year
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
Annealin
F. Variola et al., in preparation
Bottom: thermal oxidation (air, 400 °C, 3 hrs)
Rutile Rutile
Top: controls
Etching of
Crystalline TiO2
Is not possible
Raman
Annealing etched
Sample yields
Nanotextured
Crystalline 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
Chemical
linker
Puleo & Nanci, Biomaterials, 20, 2311 (1999)
Stupp & Braun, Science, 277, 1242 (1997)

The atomic concentration of the main cons
dramatically, but suboxides
such as TiO and Ti2O3 were no longer de
main oxide layer after 30 min of etching. T
comprises a mixture of amorphous TiO2, A
O3, and small quantities of V2O5 after tre
is composed of three different layers, nam
with the metal), Ti2O3 (intermediate layer)
layer) (Fig. These findings, coupled
with IR and ellipsometric results, suggest t
process increases mainly TiO2 to a degre
allows detection of the underlying suboxid
organization is not altered. This behavior i
and can be explained by assuming that su
TiO and Ti2O3 are transformed into TiO2
medium of piranha solution [72], and by as
etching solution penetrates the nanopits a
metal. When the solution reaches the subo
further oxidized into TiO2, thereby increas
the dioxide layer in a manner consistent w
measurements. When the underlying meta

TiO2: Surface crystallinity by XRD

Spectroscopic Analysis:
FT-IR and Ellipsometry

Nanoscale Properties of Biocompatible materials

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