Development of new polymeric biomaterials for in vitro and in vivo liver reconstruction

Size: px
Start display at page:

Download "Development of new polymeric biomaterials for in vitro and in vivo liver reconstruction"

Transcription

1 SIXTH FRAMEWORK PROGRAMME PRIORITY 3 NMP- Nanotechnology and nano sciences, knowledge-based multifunctional materials and new production processes and devices Development of new polymeric biomaterials for in vitro and in vivo liver reconstruction Loredana De Bartolo Institute on Membrane Technology ITM-CNR

2 Partnership Coordinator: Partners: - Universität Leipzig, Cell Techniques and Applied Stem Cell Biology, Prof. Augustinus Bader - National Research Council of Italy Institute on Membrane Technology ITM- CNR (Italy), Dr. L. De Bartolo - University Ramon Llull of Barcelona, Barcelona Bioengineering Center, Institut Quimic de Saria (Spain) Dr. C. Semino, - University of Bari, Department of Chemistry, (Italy) Prof. P. Favia, - LSMW GmbH Total Life Science Solution, Stuttgart Dr. Hartmut Jank

3 Project Objectives The design and development of new bioactive polymeric membranes and scaffolds to be used for the reconstruction of liver tissue-model in vitro and the development of a biodegradable polymer for the reconstruction of liver tissue in vivo. New polymeric surfaces that are able to activate specific cellular responses in terms of genomic, proteomic and biochemical functions of differentiated tissue analogues.

4 Why bomaterials for in vitro and in vivo liver regeneration Therapy: In Europe, around one thousand people develop acute liver failure each year Acute hepatic failure can only be treated reliably by liver transplantation although % of the affected patients could recover without transplantation. Physiological tissue analogue to study disease pathogenesis and to develop drugs: Drug development in the pharmaceutical industry is facing increasing challenges as developmental strategies in the search for novel therapeutics is under pressure for efficiency, speed, reliability and predictivity for a human situation. Classical animal testing strategies are time consuming (1-2 year) and therefore rather expensive (several million euros). Drug development costs up to million Euros for a pharmaceutical company. Animal models also suffer from shortcomings regarding the predictivity for a human situation as significant species differences in enzyme expression exist between man and animals.

5 Potential Impact The development of new intelligent polymeric materials able to activate specific responses by human liver cells: Substantial breakthrough in their potential application in biomedical field and tissue engineering. Scientific foundation for molecular design of materials for tissue engineering and for in situ tissue regeneration and repair with minimally invasive surgery. Significant clinical applications. Realisation of a highly differentiated human tissues that can be a valuable tool to the pharmaceutical industry for the drug development and vaccines. Chance to improve human predictability and to have alternatives to animal testing through in vitro the development of high throughput screening for detecting toxicity. Major advances in prevention, diagnosis and molecular treatment of disease

6 Project Achievements Development of new polymeric membranes made of modified polyetheretherketone (PEEK-WC) and polyurethane as potential materials to be used for liver cell culture The modification of membrane surface by plasma process and immobilization of functional group and/or biomolecules could control over the adhesion, proliferation and differentiation of liver cells. The development of scaffolds composed purely of self-assembling peptide nanofibers that can be rationally designed to biomimic the architecture and functional activities of collagens and laminins, two main components in the extracellular matrix. Investigation of the ability of the developed biomaterials to activate the expression of genes responsible of cell proliferation and metabolic functions Hepatic bioreactor for in vitro pharmacological screenings

7 Structure of the Project WP1 Preparation of new polymeric membranes WP2 Characterisation of structural and transport properties WP11 Management activities WP8 Human liver cell isolation and culture on bioactive materials WP7 Development of the bioreactor WP9 Characterisation of the genome and proteome of the human hepatocytes WP10 Validation of polymeric membranes and scaffolds (evaluation of gene activation) WP3 Surface modification of materials WP4 Characterisation of the modified surfaces WP5 Synthesis and preparation of peptide scaffold WP6 Characterisation of the bioactive polymers

8 ITM-CNR PEEK-WC-PU membranes Membrane biohybrid system UL Hepatocyte isolation and culture genomic and proteomic analysis Bioreactor UNIBA Modification of membrane surfaces with plasma processes and biomolecule immobilizaton LSMW GmbH Guidelines for quality of materials and products IQS Synthesis of scaffold and peptides Peptide modification of PEEKWC-PU membranes ITM

9 Morphology of PEEK-WC-PU Membrane Surface b) a) SEM s images of the PEEK-WC-PU membrane surface at different magnification L. De Bartolo et al., Biomaterials 2005; 25: ITM

10 Modification of the surface properties of membrane and scaffold by means of plasma processes (deposition/grafting) and immobilization of biomolecules on plasma-processed surfaces Surfaces (coatings/grafted groups) with better properties to stimulate cell adhesion growth Surfaces covalently immobilized with peptides. Surfaces covalently immobilized with saccharide molecules. Achieving a wide set of plasma-modified modified surfaces where liver cells can be tested and materials with the surface modified, suitable for assembling the bioreactor.

11 Characterization of the modified surfaces Optimization of plasma processes and immobilization procedures Characterisation of the surface properties of modified materials and of the distribution of functional specific groups by means of XPS with derivatization techniques, SIMS, and Contact Angle methods) Identification of key parameters of the modified materials to obtain most stabile surfaces, to drive cell behaviour and to obtain the best permeability properties

12 Diclofenac Biotransformation of Human Hepatocytes in OPMB 10 4'-OH-dic 5-OH-dic lactam Diclofenac metabolites [ µm ] Time [days] Time course of 4 -OH-diclofenac and 5-OH-diclofenac lactam metabolite formation by human hepatocytes cultured in the oxygen-permeable membrane bioreactor in presence of 80 µm diclofenac. L. De Bartolo et al., Biomaterials 2006; 27:

13 Hepatocyte Spheroid Morphology After cell isolation (100X) After 2-4h (100X) After 24h (100X) After 24h (200X) Light microscopy images of cell/ml suspension of hepatocytes E. Curcio, S. Salerno, G. Barbieri, L. De Bartolo et al., Biomaterials 2007; 28:

14 Hepatocyte Spheroids in RWMS 100 µm 100 µm 100 µm 100 µm E. Curcio, S. Salerno, G. Barbieri, L. De Bartolo et al., Biomaterials 2007; 28:

15 Microarray Technology (ITM-CNR, UNIBA, IQS) - Spotting of oligos complementary to genes of interest on the surface at defined position of slides - Aminoallyl labeling of purified mrna from samples (mrna arna) - Binding of arnas to corres- ponding oligos immobilized on slides Spotting robot - Detection of bound arnas by fluorescence D20 (30 months): Establishment of genome data bank for hepatocytes es in culture D22 (30 months): Design of gene / proteome chips that characterise the growth and differentiation

16 Control (sandwich culture only) PEEK-WC-PU Membranes (ITM - CNR) next: ITM-CNR,UNIBA, IQS e.g. PEEK-WC-PU 1. Collect the cells 2. Isolation of total mrna 3. Synthesis of Amino allyl modified RNA (arna) arna arna Cy3 dutp Cy5 dutp 4. Dye coupling reaction 5. Mix the labeled arna 6. Array Hybridisation 7. Scanning 8. Data Analysis

17 LSMW part: provide consulting and information about the regulatory requirements (GMP) for selection of biomaterials and design of the bioreactor. Elements subject to GMP requirements

18 Biomaterial Research The goal of the first developed biomaterials was to be inert Today, intelligent biomaterials able to stimulate specific cellular responses at the molecular levels are required in the living tissue-engineered constructs and in the tissue regeneration

19 Multidisciplinary Aspects Life Sciences Engineering Materials The multidiscipliary approaches encompassing Materials Engineering and Life Science could give opportunities for the design and optimization of innovative biomaterials and biohybrid devices ITM