polymeric nanocapsules as tools to face challenges in … · 2017. 12. 28. · gemma vilar palos...

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

mailto:[email protected]

mailto:[email protected]

2

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

3

Who we target?

3

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

4

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

5

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

7

&

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

8

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)

9



Stem Cells tracking

Stem Cells



Drug delivery

10

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

11

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

12

Systems for GF delivery

Sufeng Zhang and Hasan Uludağ. Nanoparticulate Systems for Growth Factor Delivery. Pharmaceutical Research, Vol. 26, No. 7, July 2009.

13

Targeted NPs systems

Sufeng Zhang and Hasan Uludağ. Nanoparticulate Systems for Growth Factor Delivery. Pharmaceutical Research, Vol. 26, No. 7, July 2009.

14

200 nm 200 nm

0

5

10

15

50 500

Inte

nsi

ty (

%)

Size (nm)

~ 150-200 nm

Sustained release

15

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

16

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


22



Stem Cells


24

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

25

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

26

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



27


Stem Cells

Stem Cells tracking


28

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).

29

Complete development of nanosystems

Nanomedicine: Complete development of nanosystems

30

Other services: Formulation & Stability

31

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 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

*#

#

* *

* *

*

35

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

36

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.

37

Introduction

Scale-up

38

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

39

SOME PARTNERS

Thank you for your attention!!

Izabel Alfany, PhD – [email protected] International Project Manager

polymeric nanocapsules as tools to face challenges in … · 2017. 12. 28. · gemma vilar palos...

Documents