Controlled release of proteins from self-assembling hydrogels based on oppositely charged dextran microspheres

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1 Controlled release of proteins from selfassembling hydrogels based on oppositely charged dextran microspheres Sophie Van Tomme a Rene van Nostrum a, Stefaan De Smedt b, Wim Hennink a a Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences Utrecht University, The Netherlands b Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University, Belgium GPEN 2006, 25 th ctober 2006 University of Kansas, Lawrence Utrecht UIPS Institute for Pharmaceutical Sciences

2 utline Hydrogels general features crosslinking methods Aim of the project Approach Results: network characterization Conclusions protein release degradation

3 Introduction Hydrogel 3dimensional hydrophilic network Good biocompatibility minimal tissue irritation low tendency for proteins and cells to adhere Possible applications Drug delivery system Release can be tailored Scaffold for tissue engineering Degradation rate can be tailored Growth factors can be incorporated RGD peptides can be coupled to promote cell adhesion

4 Introduction 1. Chemical crosslinking physical crosslinking Radical polymerization Reaction of complementary groups (e.g. NH 2 /CH) Enzymes (e.g. proteases) Crystallization Hydrogen bonding Ionic interactions Covalent bonds Nonpermanent bonds high mechanical strength toxicity/denaturation selfassembly weaker network 2. Implants injectable matrix

5 Aim of the Project Synthesis, characterization and evaluation of an injectable, selfassembling hydrogel for pharmaceutical and tissue engineering applications

6 Design H H DexHEMA H methacrylic acid Size: 220 µm N dimethylaminoethyl methacrylate

7 Design DexHEMA H methacrylic acid H H Size: 220 µm dexhemamaa microspheres mixing dexhemadmaema microspheres network formation N dimethylaminoethyl methacrylate

8 Design DexHEMA H methacrylic acid H H dimethylaminoethyl methacrylate N Size: 220 µm dexhemamaa microspheres shear mixing dexhemadmaema microspheres network formation

9 Design DexHEMA H methacrylic acid H H dimethylaminoethyl methacrylate N Size: 220 µm dexhemamaa microspheres shear mixing in situ gelling at site of injection dexhemadmaema microspheres network formation

10 Microsphere preparation Radical polymerization of dexhema, emulsified in an aqueous PEG solution initiation by KPS (=potassium peroxodisulphate) ) and TEMED (=tetramethyl ethylene diamine) dexhema PEG dexhema PEG stirring radical polymerization waterin inwater emulsion

11 Network formation Rheology Study of flow and deformation Geometry: 20mm flat plate Gap: 500 µm G = Storage modulus (elasticity( elasticity) G = Loss modulus (viscosity( viscosity) tan δ= = G /G tan δ= = 0 fully elastic tan δ= Newtonian 0< tan δ< < 1 viscoelastic

12 Rheology: controlled strain tan(δ) G' (Pa) G' (Pa) tan(δ) % solid Strength (modulus) of the network can be tailored by the solid content c of the hydrogel

13 Rheology: creep % strain time (s) dexhema HEMAMAA/dexHEMADMAEMA mixture Elastic network

14 Rheology: creep % strain time (s) Newtonian liquid dexhema HEMAMAA MAA dispersion % strain dexhema HEMAMAA/dexHEMADMAEMA mixture Elastic network time (s)

15 Injectability Reversibility of the network? G' and G" (Pa) G' (Pa) G" (Pa) tan (δ) tan (δ) oscillatory stress (Pa) shear Network rebuilds

16 Degradation: influence of charge Swelling ratio S=L t /L 0 L H H swelling ratio neutral time (days)

17 Degradation: influence of charge Swelling ratio S=L t /L 0 L H H swelling ratio /100 neutral time (days)

18 Degradation: influence of charge Swelling ratio S=L t /L 0 L H H swelling ratio /0 0/100 neutral time (days)

19 Degradation: influence of charge Swelling ratio S=L t /L 0 L H H swelling ratio /50 100/0 0/100 neutral time (days) Ratio / degradation time

20 dexhema microspheres (R 1 ) ran H H H H normal situation dexhemamaa microspheres (R 2 ) ran H H repulsion dexhemadmaema microspheres (R 3 ) ran H N H H H attraction stabilization transition state

21 In vitro protein release protein solution dexhemamaa microspheres dexhemadmaema microspheres

22 In vitro protein release protein solution Possible release mechanisms dexhemamaa microspheres dexhemadmaema microspheres

23 In vitro protein release protein solution Possible release mechanisms dexhemamaa microspheres dexhemadmaema microspheres

24 In vitro protein release protein solution Possible release mechanisms dexhemamaa microspheres dexhemadmaema microspheres

25 In vitro protein release Hydrogels composed of 15% microspheres and 85% buffer lysozyme cumulative release (%) lysozyme 20 BSA 10 0 IgG time (days) cumulative release (%) BSA 60 IgG time (days) Quantitative release of lysozyme and BSA Full preservation of enzymatic activity of released lysozyme

26 In vitro protein release diffusioncontrolled release

27 In vitro protein release diffusioncontrolled release degradationcontrolled release

28 In vitro rhodaminebdextran dextran release rhodaminebdextran release (%) time (days) 50 rho/50 50 rho/50

29 In vitro rhodaminebdextran dextran release rhodaminebdextran release (%) time (days) 50 rho/50 50 rho/50 rho 50 rho/50 Combination of release from positive and negative microspheres gives a zero order release

30 Conclusions Mainly elastic hydrogels are formed by mixing oppositely charged dextran microspheres Reversible gelation occurs Continuous diffusioncontrolled release of proteins Degradation behavior can be tailored Hydrogel properties can be tailored for various applications in drug delivery and tissue engineering

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32 Abstract submission deadline 15 th December 2006 Travel grant requests: see website

33 Abstract submission deadline 15 th December