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Introduction

The advent of biomaterials has significantly influenced the development and rapid growth of various technologies in modern medicine. Biodegradable polymers are mainly used where the transient existence of materials is required and they find applications as scaffolds for tissue regeneration, tissue adhesives and transient barriers for tissue adhesion, as well as drug delivery systems. Each of these applications demands materials with unique physical, chemical, biological, and biomechanical properties to provide efficient therapy. Biodegradable polymers can be produced chemically or recombinantly, each with their own advantages and drawbacks. Chemical synthesis is a quick and versatile method to create polymers with different physico-chemical characteristics, whereas recombinant production of protein-based polymers provides precise control -to a level at present unattainable with chemical polymerization techniques- over the sequence and chain length of these polymers. Furthermore, protein-based polymers can adopt secondary structures thereby increasing the array of supramolecular architectures that can be built. Finally, recombinant engineering can provide biological functionality to the hybrid biomaterials (e.g. ligands, specific protease substrates, adhesion domains).

 

Within the department of Pharmaceutics expertise is available on the design, construction and pre-clinical evaluation of a variety of sustained release- and drug delivery systems, including liposomes, polymeric micelles, microspheres and macroscopic hydrogels [1-6]. Polymer synthesis has been applied to create a variety of drug delivery systems. Recently, the department of Pharmaceutics also started to design and prepare drug carriers making use of protein engineering approaches. This has resulted in the design and synthesis of amphiphilic peptides that can self-assemble into vesicular submicron-sized structures in which water-soluble drugs can be transiently entrapped (van Hell et al., manuscript in preparation). Moreover, in collaboration with the department of Medicinal Chemistry, chemoselective coupling reactions have been used for making new peptide-based materials [7]. The combined expertise on polymer chemistry, chemo-selective conjugation reactions and protein engineering will form a sound basis for constructing novel drug delivery systems based on semi-synthetic biomaterials.

 

Semi-synthetic polymers

 

Aim of the project

The aim of the present project is to construct hybrid biomaterials by combining genetically engineered protein-based polymers with synthetic polymers. The advantages of such hybrid biomaterials are diverse. It allows optimal control over the sequence and length of polymers with the versatility of introducing reactive cross-linking groups. Furthermore by using peptides or protein-based polymers, biological functionality can be introduced into synthetic materials such as hydrogels, microspheres, polymeric vesicles or micelles used for controlled drug release. In hydrogels for tissue engineering applications, peptides containing e.g. RGD sequences can also be used to increase the cell adhesion properties.

 

Work plan:

Hybrid polymers will be generated by conjugating recombinant protein polymers with synthetic polymer domains [8,9]. Different chemoselective conjugation methods will be explored to allow site-specific, reversible and irreversible cross-linking under physiological conditions, including native chemical ligation [10,11], expressed protein ligation [12], Michael addition reactions [13] and metal-catalyzed azide/alkyneclick” reactions [7,14]. Protein-based polymers from naturally derived proteins (keratin, zein, collagen, casein, elastin) or designed de novo will be recombinantly produced in bacteria using the SUMO fusion protein technology currently up and running in our lab [15]. These protein-based polymers will be engineered to contain functional groups (e.g. cysteines and lysines) to which synthetic (biodegradable) polymers can be attached to form hydrogels or micelles for use as targeted and/or controlled drug delivery systems.

 

Hydrogels

 

Polyethylene glycol (PEG)- or hydroxypropyl methacrylamide (HPMA)-based polymers will be cross-linked with functional peptide domains to form a functionalized hydrophilic network. Both covalent and non-covalent approaches will be followed:

 

1.     Chemical covalent cross-linking. In this approach, peptides will be generated (either by solid phase synthesis or recombinant production) that contain N- and C-terminal cysteine residues, allowing them to form covalent cross-links between the synthetic polymers by native chemical ligation as is indicated in the figure below:

 

Cross-linked peptides may serve as substrates for matrix-metalloproteases to obtain in situ degradation of the hydrogel network or may contain cell adhesion sites to facilitate entrapment of cells inside the hydrogel network as illustrated above [13].

 

2.     Enzymatic covalent cross-linking. Synthetic polymers (pHPMA, pHEMA, pEG) can be equipped with two different peptides, one containing a lysine residue and the other containing one or more glutamine residues. These peptides can subsequently be enzymatically cross-linked using transglutaminase [16]. Enzymatic cross-linking is particularly useful for in situ gelling systems and in situations where control over speed of hydrogel formation is required. Peptides containing adhesion sites, such as RGD motifs to allow adhesion of cells within the hydrogel matrix can be used for cross-linking. Alternatively, they may contain substrate recognition sites for matrix-metalloproteases to allow biological degradation of the hydrogel network in vivo.

 

3.     Non-covalent cross-linking. Synthetic polymers described above will be functionalized with peptides or proteins that can dimerize or oligomerize. Examples are coiled coil domains, such as leucine zippers or high-affinity protein-protein interactions, such as the interaction between the subunits of the ferritin protein or the interaction between cohesin and dockerin. The heterodimerization/oligomerization domains will be covalently linked to the synthetic polymeric backbone by using Michael addition or expressed protein ligation (see picture below).

 

Micelles

Hybrid micelles consisting of synthetic hydrophobic polymers with hydrophilic, functional polypeptides for cell-targeting or triggered release

Micelles will be generated in which the hydrophobic domain consist of synthetic, semi-telechelic polymers to which a hydrophilic peptide domain can be attached [17,18]. The peptide domain should enable targeting or triggered release of the micellar systems.

Alternatively, monodisperse micelles may be generated by making recombinant polypeptide chains, such as (Lys)n(Glu)m which will subsequently be chemically modified to create a hydrophobic block (by modifying lysine side chain residues). The advantage of the latter approach is the precise control over hydrophobic block length.

 

 

References

 

1              Carstens, M.G. et al. (2005) Poly(ethylene glycol)--oligolactates with monodisperse hydrophobic blocks: preparation, characterization, and behavior in water. Langmuir 21 (24), 11446-11454

2              Rijcken, C.J. et al. (2005) Novel fast degradable thermosensitive polymeric micelles based on PEG-block-poly(N-(2-hydroxyethyl)methacrylamide-oligolactates). Biomacromolecules 6 (4), 2343-2351

3              Van Tomme, S.R. et al. (2006) Degradation behavior of dextran hydrogels composed of positively and negatively charged microspheres. Biomaterials 27 (22), 4141-4148

4              Vermonden, T. et al. (2006) Rheological studies of thermosensitive triblock copolymer hydrogels. Langmuir 22 (24), 10180-10184

5              Soga, O. et al. (2005) Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery. J Control Release 103 (2), 341-353

6              Lin, C. et al. (2007) Novel bioreducible poly(amido amine)s for highly efficient gene delivery. Bioconjug Chem 18 (1), 138-145

7              Dijk, M. et al. (2007) Synthesis of Peptide-based polymers by microwave-assisted cycloaddition backbone polymerization. Biomacromolecules 8 (2), 327-330

8              Wang, C. et al. (1999) Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains. Nature 397 (6718), 417-420

9              Yang, J. et al. (2006) Hybrid hydrogels self-assembled from HPMA copolymers containing peptide grafts. Macromol Biosci 6 (3), 201-209

10            Otaka, A. et al. (2004) Facile synthesis of membrane-embedded peptides utilizing lipid bilayer-assisted chemical ligation. Chem Commun (Camb) (15), 1722-1723

11            Yeo, D.S. et al. (2004) Expanded utility of the native chemical ligation reaction. Chemistry 10 (19), 4664-4672

12            David, R. et al. (2004) Expressed protein ligation. Method and applications. Eur J Biochem 271 (4), 663-677

13            Mather, B.D. et al. (2006) Michael addition reactions in macromolecular design for emerging technologies. Progress in Polymer Science (Oxford) 31 (5), 487-531

14            Binder, W.H. and Sachsenhofer, R. (2007) 'Click' chemistry in polymer and materials science. Macromolecular Rapid Communications 28 (1), 15-54

15            Butt, T.R. et al. (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43 (1), 1-9

16            Hu, B.H. and Messersmith, P.B. (2003) Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels. J Am Chem Soc 125 (47), 14298-14299

17            Bontempo, D. et al. (2004) Cysteine-reactive polymers synthesized by atom transfer radical polymerization for conjugation to proteins. J Am Chem Soc 126 (47), 15372-15373

18            Bontempo, D. and Maynard, H.D. (2005) Streptavidin as a macroinitiator for polymerization: in situ protein-polymer conjugate formation. J Am Chem Soc 127 (18), 6508-6509

 

 

ProjectsWiki: Recombinant and Semi-Synthetic Drug Delivery Systems