Biodegradable nanoparticles
for targeting
Tumor Associated Macrophages:
an innovative cancer treatment
Claudia Cella, PhD candidate
Filarete Foundation, Viale Ortles 22/4 – Milan (Italy)
University of Milan, Via Celoria – Milan (Italy)
European School of Molecular Medicine, via Adamello 16 – Milan (Italy)
Supervisor: Cristina Lenardi, PhD
Internal advisor: Paolo Milani, PhD
External advisor: Simon Richardson, PhD
Immune system and cancer development
Condition such as chronic inflammation can lead to macrophages
polarization toward a M2-like phenotype. The so-formed Tumor Associated
Macrophages (TAMs) support tumor growth, angiogenesis and invasion, while
inhibit natural role of the immune system.
Tumor
proliferation
Angiogenesis
Inflammation
Metastasis
Immune
suppression
Adapted from Nature Medicine,19 (2013) 1423 – 1437
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TAMs and cancer therapy
TAMs are considered a promising target candidate in cancer therapy,
however no specific drugs are available so far, with the except of
trabectedin, that is authorized only in few cancer treatments.
Tumor
proliferation
Angiogenesis
Inflammation
Metastasis
Immune
suppression
Adapted from Nature Medicine ,19 (2013) 1423 – 1437
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Innovative strategies for TAMs targeting
• Curcumin
o
o
o
o
Anti inflammatory
Anti cancer
Able to interact with the immune system
Safe
• Small interfering RNA (siRNA)
o Gene silencing via interaction with the
RNA-induced silencing complex (RISC)
complex
o Potential for TAMs re-education
Both the drugs present low bioavailability due to their low stability in vivo
They need to be vehiculated
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Promising tool for drug delivery
Requirements
Able to protect
drugs until
tumor site reaching
Material
biocompatible,
biodegradable,
FDA approved
curcumin
siRNA
Poly-Lacticco-glicolic
acid (PLGA)
Endosomal escape
properties
Surfactant
TAMs specific targeting
Mannose
Cella Claudia, PhD candidate
Polymeric
Nanoparticles
(NPs)
5
Challenges in NPs mediated drug delivery
1- PLGA NPs interaction
with complex medium
2- Endosomal escape
Complex
medium
components
Nucleus
NPs
Biomolecular
(protein)
corona
Cell cytosol
RISC complex
pH
+
enzymes
Lysosome
Endoplasmatic
pH
Late
reticulum
endosome
NPs
Endocytosis
Cella Claudia, PhD candidate
Sorting
endosome
6
Strategies
1- Modulate NPs surface charge: aminopolyvinyl alcohol (amino-PVA)
2- Modulate compatibility: Calcium Stearate
(CSt)
3- Surface decoration for specific TAMs
targeting
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Outline
1 - PolyVinyl Alcohol (PVA) stabilized PLGA NPs:
synthesis and encapsulation properties
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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Outline
1 - PVA stabilized PLGA NPs:
encapsulation properties
synthesis
and
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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PVA stabilized NPs: parameter investigation
Single emulsion
Polymer molecular weight
Polymer concentration
Freeze-drying
Organic solvent
PLGA
solution
Oil-in-water
emulsion
Purification
Ultrasound
Organic solvent
evaporation
Surfactant
Surfactant
concentration
Nanospheres
Cooling bath
Ultrasound application
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PVA stabilized NPs: parameter investigation
Polymer molecular weight
Double emulsion
Polymer concentration
Freeze-drying
Organic solvent
PLGA
solution
Ultrasound
Water-in-oil
emulsion
Water-in-oil-in-water
emulsion
Ultra-
Purification
sound
Primary
aqueous
phase
Surfactant
Aqueous
phase content
Surfactant
concentration
Cella Claudia, PhD candidate
Organic solvent
evaporation
Core-shell
nanocapsules
Cooling bath
Ultrasound application
11
PVA role
Surfactant is fundamental in modulating NPs size: the more surfactant was
added, the smaller and less polydisperse the NPs were.
Hydrodynamic diameter (nm)
No surfactant added
PVA added at different percentage
Dynamic Light Scattering, DLS
Zetasizer nano ZS90, Malvern Instruments
Cella et al. Polymer International, submitted
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Encapsulation properties
Hydrophobic (curcumin) and hydrophyilic (IgG1 functionalized with
Alexa488®, IgG-488) were actually encapsulated in single or double emulsion
synthetized NPs
PLGA
solution
Curcumin
Single emulsion
Curcumin
encapsulating
PLGA NPs
P0.2-OW-cur
PLGA
solution
IgG-488
aqueous solution
Double emulsion
IgG-488
encapsulating
PLGA NPs
P0.2-IgG-488
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Encapsulation properties: confocal microscopy
P0.2-OW-cur
P0.2-OW-ctr
P0.2-IgG-488
P0.2-ctr
Left: fluorescence channel; Right: contrast phase; Scale bar: 5 µm
Potenza et al. Scientific reports 2015, doi:10.1038/srep18228
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Outline
1 - PVA stabilized PLGA NPs:
encapsulation properties
synthesis
and
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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Degradation studies: DLS results
Due to the decrease in scattered light, DLS was not suitable to follow NPs
degradation for these samples after 24 hours
Derived Count Rate (%)
Particle size (nm)
Degradation
studies
were
performed at 37°C by suspending
NPs in phosphate buffer at
physiological pH (7.4)
Dynamic Light Scattering, DLS
Zetasizer nano ZS90, Malvern Instruments
Potenza et al. Scientific reports 2015, doi:10.1038/srep18228
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SPES as innovative techniques for NPs degradation
Single Particle Extinction and Scattering (SPES) apparatus
NPs flow
lens
A lens
PC
laser
B
A
E
detector
D
F
G
B laser beam
C flow cell
D transmitted light
E scattered light
F interference pattern
C
G photodetector
SPES gives information on particle distribution in size as
well as in refractive index (m)
Potenza et al. Scientific reports 2015, doi:10.1038/srep18228
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SPES: particle size distribution
Particle size distribution gave no information about the PLGA NPs different structures
Potenza et al. Scientific reports 2015, doi:10.1038/srep18228
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SPES: refractive index distribution
Differences in refractive index (m) distribution indicated curcumin encapsulation as well
as the actual formation of a core-shell structure.
m
for
the
reference
sample P0.2-OW-ctr
m for the corresponding
samples
Potenza et al. Scientific reports 2015, doi:10.1038/srep18228
Cella Claudia, PhD candidate
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SPES: degradation studies
NPs degradation after 24 hours was assessed by the variation in the size
distribution profile and by the shift in the refractive index distribution.
SPES analyses after 24 hours degradation
Refractive index distribution
Particle size distribution
m at Time 0
m after 24h incubation
Cella Claudia, PhD candidate
Potenza et al. Scientific reports 2015, doi:10.1038/srep18228
20
Outline
1 - PVA stabilized PLGA NPs:
encapsulation properties
synthesis
and
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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Modulate NPs surface charge: Amino-PVA
By taking advantage of the PVA backbone, a new surfactant with
positive charge was synthesized by radical polymerization and
chemically characterized.
Synthesis by radical
polymerization
PVA backbone
Tertiary
group
amino
Chemical
characterization
Cella et al. Polymer International, submitted
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Amino-PVA vs. PVA
By using the amino-PVA alone, compared with PVA alone, NPs showed
micrometric diameters and a very positive charge (+40.0 mV)
Hydrodynamic diameter (nm)
z-potential (mV)
Zetasizer nano ZS90, Malvern Instruments
Cella et al. Polymer International, submitted
Cella Claudia, PhD candidate
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Mixing amino-PVA and PVA to tailor NPs properties
By properly mixing the two surfactants, size as well as z-potential can be
modulated, until desired parameters were reached (259.3 nm in diameter
and +20.0 mV z-potential).
Hydrodynamic diameter (nm)
z-potential (mV)
Zetasizer nano ZS90, Malvern Instruments
Cella et al. Polymer International, submitted
Cella Claudia, PhD candidate
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Outline
1 - PVA stabilized PLGA NPs:
encapsulation properties
synthesis
and
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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Modulate NPs compatibility: calcium stearate
Calcium Stearate (CSt) was investigated as low molecular weight, costeffective and biocompatible surfactant. Notably, CSt has never been used
for NPs synthesis.
Apolar tails are embedded in the
polymeric matrix, while polar head are
exposed on the NPs surface
Minimum amount of an additional
couple of surfactant was added
NPs
Cella et al. Biomacromolecules, in preparation
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CSt: parameter investigation
Synthetic parameters were carefully studied for both single and double
emulsions synthesis.
No
additional
surfactant
Determining
the P60 / S60
ratio
Reducing
P60 / S60
concentration
Investigating
optimal CSt
concentration
4.8 / 3
mg/mL
3.2 / 2
mg/mL
40.0
mg/mL
Curcumin role
P60
<
S60
1.6 / 1
mg/mL
30.0
mg/mL
P60
=
S60
1.2 / 0.8
mg/mL
20.0
mg/mL
Ratio with
PLGA
1:20
0.8 / 0.5
mg/mL
10.0
mg/mL
Ratio with
PLGA
1:10
P60
>
S60
0.6 / 0.4
mg/mL
5.0
mg/mL
0.4 / 0.3
mg/mL
0
mg/mL
Ratio with
PLGA
1:5
Cella et al. Biomacromolecules, in preparation
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Encapsulation properties
Hydrophobic (curcumin) and hydrophyilic (fibrinogen functionalized with
Alexa647®, fib647) were actually encapsulated in single or double emulsion
synthetized NPs
Single emulsion
Double emulsion
Curcumin
Fib647
C-OW-ctr
C-ctr
C-cur
C-fib
C-fib-cur
PLGA
C-OW-cur
Cella et al. Biomacromolecules, in preparation
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Microscopy characterization
Different model drugs encapsulated in the same
nanoparticles
Microscope Images
Confocal Microscope
Scanning Electron Microscopy
TCS SP5 AOBS (Leica Microsystem)
Curcumin
Fib647
Scale bars: 5 µm
Cella Claudia, PhD candidate
Zeiss Sigma
Cella et al. Biomacromolecules, in preparation
29
CSt: curcumin modified NPs degradation properties
When curcumin was encapsulated, PLGA NPs
degradation and increase in NPs size during time.
Derived Count Rate (%)
showed
fast
Particle size (%)
Zetasizer nano ZS90, Malvern Instruments
Cella et al. Biomacromolecules, in preparation
Cella Claudia, PhD candidate
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Curcumin and fib647 release studies
Release studies were performed at 37°C by suspending NPs in
phosphate buffer at physiological pH (7.4).
Curcumin release
Fib647 release
FluoroMax 4, Horiba, JobinYvon
Cella et al. Biomacromolecules, in preparation
Cella Claudia, PhD candidate
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Outline
1 - PVA stabilized PLGA NPs:
encapsulation properties
synthesis
and
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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Surface functionalization for TAMs targeting
Both amino-PVA and CSt stabilzed PLGA NPs were functionalized by the
formation of a protein coating enriched by mannose, for specific TAMs
targeting.
Fetal Bovin Serum
(FBS, 10% v/v)
components
Incubation
NPs
NPs
Mannose
(20% w/v)
Protein corona
enriched by
mannose
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XPS analysis evidenced mannose presence
In samples treated with mannose, NPs surfaces presented an increased
percentage of -C-OH bond. This led to a different response in X-ray
photoemission spectroscopy (XPS) analysis.
XPS analysis
PLGA NPs
PLGA NPs with mannose
decorated surface
difference
X-ray source Mg K = 1253.6 eV
Pass Energy = 30 eV
Energy resolution 0.7 eV
UHV apparatus Leybold LHS 10/12
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Surface functionalization modified z-potential
For CSt stabilzed PLGA NPs, surface functionalization did not modify the
surface charge. On the contrary, amino-PVA stabilzed NPs showed a switch
from positive to negative surface charge, after incubation with the mixture 10%
v/v FBS and 20% w/v mannose.
Z-potential (mV)
Time 0
After
incubation
with 10% v/v
FBS and 20%
w/v mannose
CSt stabilized
Amino-PVA stabilized
Zetasizer nano ZS90, Malvern Instruments
Cella Claudia, PhD candidate
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Outline
1 - PVA stabilized PLGA NPs:
encapsulation properties
synthesis
and
2 - NPs characterization and degradation studies
with innovative technique (SPES)
3 - Surface properties modulation: amino-PVA
stabilized NPs
4 - Cytocompatibility and cost-effectiveness: CSt
stabilized NPs
5 - Surface functionalization for TAMs targeting
6 - Cytocompatibility towards macrophages
Cella Claudia, PhD candidate
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MTT test for cytocompatibility
MTT is a test for assess cells viability through the investigation of mitochondrial
activity.
Cell lines
1. RAW264.7 macrophages cell line
2. Bone marrow-derived macrophages
from mice
Tested conditions
Surfactant Amino-PVA and CSt stabilized NPs
Medium
10% v/v Fetal Bovine Serum (FBS)
Mannose
20% w/v
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RAW264.7 macrophages – MTT test
Amino-PVA stabilized PLGA NPs were found to slightly reduce cell viability,
while CSt stabilized PLGA NPs were found to be cytocompatible
Amino-PVA stabilzed PLGA NPs
Cella Claudia, PhD candidate
CSt stabilized PLGA NPs
38
Bone-marrow derived macrophages – MTT test
Results were confirmed also for bone marrow-derived macrophages
Amino-PVA stabilzed PLGA NPs
Cella Claudia, PhD candidate
CSt stabilized PLGA NPs
39
Conclusions
PVA stabilized PLGA NPs were characterized with an innovative
techniques (SPES) that allowed to monitor drugs encapsulation
and particles degradation.
PLGA NPs were additionally synthesized with two innovative
surfactants, namely amino-PVA and calcium stearate.
Both of these surfactants presented attractive characteristics for
enabling delivery of curcumin or hydrophilic molecules in the
Tumor Associated Macrophages cytosol.
An effective approach that took advantage on the protein
corona was developed to functionalize the NPs surface with
mannose as specific TAMs target molecule.
Finally, selected formulations showed good cytocompatibility
and looked promising for further in vitro/in vivo investigations.
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Appendix – the nanotox project
A Practical Approach to Assess the
Stability of Isolated Silver
Nanoparticles in Complex Biological
Media
Toxicological impact of silver nanoparticles
Commercial goods
Silver
Nanoparticles
(AgNPs) release
To
determine
the
AgNPs
toxicity, it is of key importance
to fully characterized them in
the tested biological medium
Selected AgNPs
Selected techniques
Surface coating
AgNPs size
Increased size
Altered optical
properties
Citrate
10nm
Dynamic Light Scattering (DLS)
PolyVynilPyrrolidone
(PVP)
40nm
Transmission Electron Microscopy (TEM)
100nm
UV-Visible spectroscopy
Argentiere, Cella et al. Journal of Nanoparticle Research, in preparation
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Focus on TEM analysis
Scale bars: 50 nm; FEI Tecnai G2, Eindhoven
Cella et al. Biomacromolecules, in preparation
Cella Claudia, PhD candidate
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Conclusion
The DLS and TEM techniques present critical points in sample
preparation and characterization of 10 nm-sized AgNPs
The UV-Vis spectroscopy represent a reliable, affordable technique
for the characterization of AgNPs with different sizes
Guidelines for interpreting the UV-vis spectroscopy results have
been established, to ensure complete characterization of AgNPs
under the exposure conditions of in vitro/ in vivo experiments
Argentiere, Cella et al. Journal of Nanoparticle Research, in preparation
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Fundings
These works were possible thanks to:
Fondo per gli Investimenti della Ricerca di Base (FIRB): Inflammation
and
cancer:
nanotechnology-based
innovative
approaches,
protocol RBAP11H2R9, year 2010.
NANOTOX project: Nanoparticles, nanotechnologies and ultrafine
particles Call 2011 CARIPLO FOUNDATION
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Collaborations and list of publication - FIRB
Prof. Marco Potenza, Tiziano Sanvito
Dipartimento di fisica (University of Milan) and EOS s.r.l.
Serena Ghisletti
European Institute of Oncology (Milan), department of Experimental Oncology
Laura Blasi, Marta Madaghiele, Luca Salvatore, Lucia Giampetruzzi
NNL_Institute of Nanoscience CNR – Lecce
Alessandro Sannino
Biomaterials Science Laboratory, Università del Salento, Lecce
Patrizia Rosa
Cimaina, Dipartimento di fisica (University of Milan)
Single particle optical extinction and scattering allows real time quantitative characterization of drug payload and
degradation of polymeric nanoparticles
M. Potenza, T. Sanvito, S. Argentiere, C. Cella, B. Paroli, C. Lenardi, P. Milani
Scientific reports, 2015, 5, Article number: 18228
Amine-modified Poly(vinyl alcohol) as a novel surfactant to modulate size and surface charge of Poly-Lactic-co-Glycolic
Acid nanoparticles
C. Cella, F. Martello, S. Ghisletti, C. Lenardi, P. Milani, S. Argentiere
Polymer international, submitted
Calcium Stearate as an effective alternative to Poly(vinyl alcohol) in Poly-Lactic-co-Glycolic Acid nanoparticles synthesis
C. Cella, I. Gerges, S. Ghisletti, C. Lenardi, P. Milani, S. Argentiere
Biomacromolecules, to be submitted
Embedded poly(lactide-co-glycolide) nanoparticles in a micro-pattern collagen scaffold enhanced neuronal tissue
regeneration
L. Giampetruzzi, L. Blasi, M. Madaghiele, L. Salvatore, S. Argentiere, C. Cella, A. Sannino
To be submitted
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Collaborations and list of publication - Nanotox
Camilla Recordati, Marcella de Maglie, Silvia Bianchessi, Eugenio Scanziani
MapLab (Filarete Foundation), Milan
Cinzia Cagnoli, Lorena Passoni, Matteo Tamborini, Michela Matteoli
Cellular models platform (Filarete Foundation), Milan
Francesco Cubadda, Federica Aureli, Marilena D’Amato, Andrea Raggi
Istituto Superiore di Sanità, Roma
Davide Marchesi, Simona Rodighiero
Imaging Platform (Filarete Foundation), Milan
Maura Cesaria
Dipartimento di Matematica e Fisica, Università del Salento, Lecce
Silver nanoparticles in complex biological media: an effective characterization method before in vitro/in
vivo experiments
S. Argentiere, C. Cella, M. Cesaria, P. Milani, C. Lenardi
Journal of nanoparticles research, to be submitted
Tissue distribution and acute toxicity of silver after single intravenous administration in mice: nano-specific and sizedependent effects
C. Recordati, M. De Maglie, S. Bianchessi, S. Argentiere, C. Cella, S. Mattiello, F. Cubadda, F. Aureli, M. D’Amato, A.
Raggi, C. Lenardi, P. Milani, E. Scanziani
Particle and fiber toxicology, under revision
Investigating biodistribution and toxicity of silver nanoparticles and silver citrate after subacute oral administration in mice
C. Recordati, M. De Maglie, S. Bianchessi, S. Argentiere, C. Cella, S. Mattiello, F. Cubadda, F. Aureli, M. D’Amato, A.
Raggi, C. Lenardi, P. Milani, E. Scanziani
To be submitted
Fill the gap in the nanomaterials legislation: determination of the partition coefficient of silver nanoparticles
C. Cella, S. Argentiere, C. Marotta, P. Milani, C. Lenardi
To be submitted
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Acknowledgements
Advanced Biomaterials platform
(present and former)
Prof. Cristina Lenardi
Simona Argentiere
Maria Vittoria Cavanna
Smbat Gevorgyan
Chiara Marotta
Eleonora Rossi
Martino Alfredo Cappelluti
Laura Morelli
Marco Indrieri
Chantal Speziali
Irini Gerges
Alessandro Tocchio
Federico Martello
Federica Tamion
Chiara Zecconi
Elisa Sogne
Contact:
[email protected]
Internal advisor
Prof. Paolo Milani
External advisor
Prof. Simon Richardson,
University of Greenwich
Thank you all for your attention