polymeric nanocapsules as tools to face challenges in … · 2017. 12. 28. · gemma vilar palos...
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Gemma Vilar Palos ([email protected]) Lorena García Fernández ([email protected])
Polymeric Nanocapsules as tools to face challenges in Regenerative Medicine
ETPN RegMed WG 23rd August 2016
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MISSION Create and transfer economic,
social and sustainable value to companies and entities, through research and technology processes.
VISION Be a Technology Partner to companies and Administration, by generating a corporate culture allowing sustained growth and efficient functioning.
PRINCIPLES: We believe in
• Creativity • Innovation • Sustainability • Environmental Awareness • Diversity • Efficiency • Efficacy
CORPORATE CULTURE
VALUES: We act with
•Dynamism • Independency • Commitment • Confidentiality • Market-orientation • Global perspective • Talent
About us
Leitat is the brand of the institution Acondicionamiento Tarrasense, a private and non-profit organisation. It is recognised by the Catalan Government (TECNIO) and by the Spanish Ministry of Science and Innovation.
Since 1906
We develop and bank on development, expanding activities towards the knowledge generation and its transfer to the productive
fabric.
LEITAT
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Who we target?
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MULTISECTORAL ANSWERS to the technology needs of the
companies and institutions
Large companies
SMEs
Public Entities
Sponsors
Multinational companies
Investment Funds
+ PROCESS IMPROVEMENT + PRODUCTS IMPROVEMENT + CHANGE ADAPTATION + INNOVATION CAPACITY
+ COMPETITIVE IMPACT + SOCIAL IMPACT + INTERNATIONALIZATION + ECONOMIC RETURN
LEITAT
Results
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Figures 2013
Our customers’ value: Quality, personal contact, clarity of results Society value: Innovation, sustainability and environmental responsibility, market orientation Level of loyalty (future collaboration and recommendation) > 97%
+ Corporate
Social Responsibility
265 Proposals managed
137 R+D+2i projects being executed*
4 Lead projects
240 Private R+D+2i projects
2.915 Advanced technology solutions
2 Patents
*We participate in European projects
with 839 partners, overall budget of
432M € and collaborating with 28 countries
Figures 2014
LEITAT
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CEA 15 10.043.683,75 €
Fraunhofer 14 8.733.357,25 €
TECNALIA 7 4.698.132,50 €
LEITAT 7 4.518.352,75 €
CNRS 8 4.398.264,25 €
CIDETEC 4 4.306.997,50 €
EUROPE RTO Top list - 2014
LEITAT: 1st as NMP coordinator
58 M€ secured (2014-2015)
LEITAT NMP Results – 2014-15
Materials Safety
Human & Environmental Health & Safety
Group Leader: Socorro Vázquez Campos
Nanomedicine & Nanobiosensors
Nanotoxicology & Risk Assessment
Efficacy & Safety In vitro kits
production
Bioanalytics
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&
Diagnostics: Biofunctionalization of NPs for
electrochemical and colorimetric nanobiosensor platforms
Therapy: Biofunctionalization of NPs and
nanoencapsulation for drug delivery purposes
Nanomedicine Nanobiosensors
Nanoparticles and nanocapsules with size
1-500 nm
Nanomed group
Nanomedicine & Nanobiosensors group
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Regenerative Medicine
Homing and fixing Stem Cells to their desired site for therapy
Stem Cells tracking
Drug delivery Stem Cells
Delivery and retention of complex mixtures of compounds: grow factors (GF) and other pharmacological agents in a specific organ/tissue/cell
Nanotechnology approaches (regenerative pharmacology)
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Regenerative Medicine
Homing and fixing Stem Cells to their desired site for therapy
Stem Cells tracking
Stem Cells
Delivery and retention of complex mixtures of compounds: grow factors (GF) and other pharmacological agents in a specific organ/tissue/cell
Nanotechnology approaches (regenerative pharmacology)
Drug delivery
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What offer nanoencapsulation in curative therapeutics?
Protection of API from pH,
enzymes
Protection of API from
light, chemicals in
the final formulation…
Curative therapeutics
Some APIs produce side effects at high doses
No biological action:
API doesn't arrive to target cell
Some APIs have poor stability in the body
Some APIs have poor stability during storage
Controlled and sustained
release
Targeted nanoparticles
Targeted delivery
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Hydrophobic
Amphyphilic
pH
Enzyme
Temperature Small molecules
Biomolecules
API
Hydrophylic
Polymer Degradation, Swelling…
NP
Nano Toolbox
Co-encapsulation of different active ingredients
Sustained release
Controlled release
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Systems for GF delivery
Sufeng Zhang and Hasan Uludağ. Nanoparticulate Systems for Growth Factor Delivery. Pharmaceutical Research, Vol. 26, No. 7, July 2009.
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Targeted NPs systems
Sufeng Zhang and Hasan Uludağ. Nanoparticulate Systems for Growth Factor Delivery. Pharmaceutical Research, Vol. 26, No. 7, July 2009.
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200 nm 200 nm
0
5
10
15
50 500
Inte
nsi
ty (
%)
Size (nm)
~ 150-200 nm
Sustained release
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200 nm 200 nm
0
5
10
15
50 500
Inte
nsi
ty (
%)
Size (nm)
HPLC AI
tR ~ 8 min
200 nm
Versatile nanocapsules: APIs with different physical and chemical nature
~ 150-200 nm
94%
6%
PLGA + amphihilic drug
ATRA encapsulated ATRA non encapsulated
83%
17%
PLGA + hydrophobic drug
IM encapsulated
33%
67%
PLGA + hydrophilic drug
HQ encapsulated HQ non encapsulated
Sustained release
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pH sensitive nanocapsules Negative polyelectrolyte
Positive polyelectrolyte
API
Biodegradable polymer
Hyd
rod
ynam
ic d
iam
eter
(n
m)
Zeta
-po
ten
tial
(m
V)
0
5
10
15
20
1 10 100 1000 10000
NP-API
NP-API + shell B1
-50
-40
-30
-20
-10
0
500 nm 200 nm NCs NCs+Shell
On switch (smart systems) + sustained release
0
5
10
15
20
1 10 100 1000 10000
NP-API
NP-API + Shell A1
-50
-40
-30
-20
-10
0
10
Hyd
rod
ynam
ic d
iam
eter
(n
m)
Zeta
-po
ten
tial
(m
V)
500 nm 1 mm
Enzyme sensitive nanocapsules
Labile to pH 5.5
Labile to proteases
Encapsulated HQ in PLA Nc showed to be slightly less toxic than free on human dermal fibroblasts (HDF)
0
25
50
75
100
1 10 100
Ce
ll vi
abili
ty (
%)
API concentration (µg/ml)
NCs-amphipilic API cytotoxicity on HDF
ATRA
PLA + ATRA
0
25
50
75
100
10 100 1.000
Ce
ll vi
abili
ty (
%)
API concentration (µg/ml)
NCs-Hydrophobic APIcytotoxicity on HDF
PLA + IM
PLGA + IM
IM 0
25
50
75
100
1 10 100
Ce
ll vi
abili
ty (
%)
API Concentration (µg/ml)
NCs –hydrophilic APIcytotoxicity on HDF
HQ
PLA + HQ
PLGA + HQ
Encapsulated active is less toxic than free on human dermal f ibroblasts (HDF)
NC1_Hydrophobic
NC2_Hydrophobic
Hydrophobic API
NC1_Hydrophilic
NC2_Hydrophilic
Hydrophilic API
NC1_Amphiphilic
Amphiphilic API
Toxicity & Efficacy
NC1
NC2
API
0
40
80
120
160
200
ATRA (4µg/ml)
PLA + ATRA (4µg/ml)
PLGA + ATRA (4µg/ml)
Non-treated cells
Culture medium 10%FBS
Pro
life
rati
on
ind
ex
(%)
Proliferation index of HDF after 24h of
exposure
HUMAN DERMAL FIBROBLASTS NC+PA (120µg/ml) PA(120µg/ml)
PA NC1-PA NC2-PA Non treated cells
CM 10% FBS
Toxicity & Efficacy
The efficacy is increased with nanoencapsulation
Nanoencapsulation offers solubility and
bioavailability of AI
PA NC1-PA NC2-PA NC1 NC2 Non treated cells
CM 10% FBS
2 mg/ml PA 4 mg/ml PA 1 mg/ml PA
Co-encapsulation. Synergic effect
Bone regeneration
Craig A. Simmons, Eben Alsberg, Susan Hsiong, Woo J. Kim, and David J. Mooneya. Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells. Bone 35 (2004) 562– 569
(A) blank, (B) BMP2 only, and (C) TGF-h3 only conditions, the new tissue was primarily fibrous, with little evidence of bone formation. However, in the implants with BMP + TGF (D), there was significant bone formation (pink).
Histomorphometric analysis confirmed the dual growth factor condition had significantly more bone tissue than any other condition analyzed
Tendon and muscle tissue
http://muscletechnetwork.org
GENERATION OF A NEW MODEL OF SKELETAL MUSCLE LESION IN RATS
GENERATION OF A NEW MODEL OF ACHILLES TENDON INJURY IN RATS
Co-encapsulation growth factors Synergic effect
MuscleTech Network Most research network in muscle and tendon
Tendon and muscle tissue
http://muscletechnetwork.org
LESION BY RUPTURE OF MUSCLE SARCOLEMA: NECROSIS-REGENERATION PROCESS
HISTOLOGY, IMMUNOFLUORESCENCE AND MRI
ULTRASOUND INTRAMUSCULAR ANALYSIS
Drug delivery
Stem Cells tracking
Delivery and retention of complex mixtures of compounds: grow factors (GF) and other pharmacological agents in a specific organ/tissue/cell
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Regenerative Medicine
Homing and fixing Stem Cells to their desired site for therapy
Stem Cells
Nanotechnology approaches (regenerative pharmacology)
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Chen, J.; Huang, N.; Ma, B.; Maitz, M.F.; Wang, J.; Li, J.; Li, Q.; Zhao, Y.; Xiong, K.; Liu, X. Guidance of stem cells to a target destination in vivo by magnetic nanoparticles in a magnetic field. ACS Appl. Mater. Interfaces 2013, 5, 5976-5985
Homing and fixing SC SMEInst
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Nanosystems
Encapsulation of inorganic NPs in polymeric NPs
Linkers 1 and 2 Ab
NP functionalization
with mixed monolayer
of ligands by
adsorption
Ab immobilization
by covalent
binding NP
Surface (bio)conjugation of inorganic nanoparticles
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Topics of interest
Topic Title Type of action
1.1 Understanding health, well-being and disease
PM 02 – 2017 New concepts in patient stratification RIA
PM 03 – 2017 Diagnostic characterisation of rare diseases RIA
1.3 Treating and managing diseases
PM 08 – 2017 New therapies for rare diseases RIA
PM 11 – 2016/2017 Clinical research on regenerative medicine RIA
1.4 Active ageing and self-management of health
PM 15 – 2017 Personalised coaching for well-being and care of people as they age RIA
1.5 Methods and data
PM 16 – 2017 In-silico trials for developing and assessing biomedical products RIA
PM 17 – 2017 Personalised computer models and in-silico systems for well-being RIA
ADVANCED MATERIALS AND NANOTECHNOLOGIES FOR HEALTHCARE
NMBP-12-2017 Development of a reliable methodology for better risk management of engineered biomaterials in Advanced Therapy Medicinal Products and/or Medical Devices
RIA
NMBP-13-2017 Cross-cutting KETs for diagnostics at the point-of-care RIA
NMBP-14-2017 Regulatory Science Framework for assessment of risk benefit ratio of Nanomedicines and Biomaterials
RIA
NMBP-15-2017 Nanotechnologies for imaging cellular transplants and regenerative processes in vivo RIA
Drug delivery
Delivery and retention of complex mixtures of compounds: grow factors (GF) and other pharmacological agents in a specific organ/tissue/cell
Homing and fixing Stem Cells to their desired site for therapy
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Regenerative Medicine
Stem Cells
Stem Cells tracking
Nanotechnology approaches (regenerative pharmacology)
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SC tracking
Fluorescent organic dyes
The strongly enhanced surface plasmon resonance of Au NPs optical frequencies makes them excellent scatterers and absorbers of visible light
Fluorescent organic dyes could be either physically entrapped in the polymer interior during the preparation of NPs or covalently bound to the polymer chain
They act as good contrast agents in MRI, enhancing the contrast between different tissues present by inducing a darker area (negative contrast).
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Complete development of nanosystems
Nanomedicine: Complete development of nanosystems
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Other services: Formulation & Stability
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0%
5%
10%
15%
20%
25%
30%
35%
0 50 100 150 200
AI
rele
ase
d
Time (h)
Release IM in water
Release IM in medium
0%
20%
40%
60%
80%
100%
120%
0 10 20 30
AI
rele
ase
d
Time (h)
Release ME in water
Release IM in water
Release PA1 in water
Release studies at 37 °C
Transformation studies: -Aggregation state -NM size and shape -Release kinetics of the AI -Chemical degradation
Final application
Release PA2 in water
Release PA3 in water
Release PA3 in culture medium
Formulation & Stability
Release and stability in final application and storage
Release studies at 4 and 25 °C Transformation studies: -Aggregation state -NM size and shape -Release kinetics of the AI -Chemical degradation
Storage
Formulation & Stability
Release and stability in final application and storage
0,0% 0,2% 0,4% 0,6% 0,8% 1,0% 1,2% 1,4% 1,6%
0 100 200
AI
rele
ase
d
Time (h)
Release IM in cream Parameters affecting stability: T, light, pH, final formula composition
Physical and chemical structure
Initial sample T.a. 1 month 4°C 1 month
Release PA3 in cream
33
Local tolerance, Pharmacokinetics & Efficacy
34
Nanotoxicology
• Viability, proliferation, apoptosis
• Oxidative stress
• Inflammatory responses
• Phagocytic activity
• Phototoxicity
• Genotoxicity/Photogenotoxicity
• Membrane integrity
In-vitro toxicity
In-vitro biokinetics
• Cell uptake and intracellular trafficking
• Intestinal barrier permeability
• ICP-MS, confocal microscopy, fluorescent microscopy, TEM, SEM.
In-vivo toxicity / biokinetics
• Animal models: Rats and mice
• Administration Routes: Oral, dermal, parenteral
• OECD-like evaluations + Nanospecific focus
Kid
neys
Caecum
Sple
en
Liv
er
Epid
idym
is
Sm
all Int
PP
MLN
Lungs
Teste
s
0
1
2
3
4
Ti
(p
pm
)
V e h ic le - N o n fa s te d
V e h ic le - F a s te d
E 1 7 1 - N o n F a s te d
E 1 7 1 - F a s te d
*#
#
* *
* *
*
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Nanotox and Efficacy
Metabolism and Tox
• Hepatotoxicity
• Stability
• Clearance
• Metabolic profiling
• Enzyme mapping
• Panel Screening
• Bio-analytics
ADME
Human Hepatocytes In vitro intestinal models
( brain )
Filter
Endothelial cells
Astrocytes
Apical
Basal
Blood Brain Barrier (BBB) in vitro model
Absorption
• Intestinal
• Kidney
• Brain barriers
• Gastric epithelium
• Cell transport
Safety
• Skin permeation
• Percutaneous absorption
• In vitro skin irritation
• In vitro ocular irritation
• Skin sensitisation
• Skin inmuno and neuro inflammatory responses
• Skin disorders models
Skin biology and topical Nc
Efficacy
• Anti-aging and vitalizing
• Anti-oxidant
• Anti-inflammatory
• Firmess and elasticity
• Moisturizing
• Skin and DNA repair/protection
• Photoprotection
Nanocapsules
Human skin samples
Skin explant culture
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Other Capacities - RegMed
Stem cells Biology •Primary cell cultures from human or animal models •Isolation and characterization of stem cells
Cell Therapy •Stem cell transplantation in vivo. Advanced surgical techniques or ultrasound-guided cell implantation. •Small (mice, rat) and large (rabbit, pig, sheep) animal models
Advanced surgical techniques in experimental in vivo models •Skeletal muscle injuries in vivo models. •Tendon injuries (Achilles and patellar) in vivo models •Surgically-induced congenital malformations. Myelomeningocele or diaphragmatic hernia in fetuses in large animal models.
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Introduction
Scale-up
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2 mm
1 mm
200 nm
Scale up
Spray dryer tecnique
pH-sensitive nanocapsules
Relatively uniform spherical particles
Large production (up to Kg)
Continuous operation and automatic control
Sample purification
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SOME PARTNERS
Thank you for your attention!!
Izabel Alfany, PhD – [email protected] International Project Manager