Encapsulation with Alginates Berit L. Strand, Yrr Mørch and Gudmund Skjåk-Bræk
advertisement
Encapsulation with Alginates Berit L. Strand, Yrr Mørch and Gudmund Skjåk-Bræk Norwegian University of Science and Technology Microcapsules in cell therapy Stem cells for the formation of new tissue Chondrocytes for the formation of new cartilage (Lanza et al. 1999) Endostatic producing cells for hindering re-growth of tumour tissue (gliomas) Microcapsules in cell therapy Immunoisolation: Nutrients and oxygen Immune cells Graft rejection + autoimmune disease Cell products Insulin Cells (Islets) Capsule (Lanza et al. 1999) (chicagodiabetesproject.org) Insulin Oxygen and nutrients Waste products Complement components Fibroblast c c Capsule membrane Y Autoimmune antibodies Cell Cytokines, free radicals reactive oxygen- and nitrogenintermediates Y Y Y YY Y vv Y Y c c Secreted proteins Macrophages Natural occurring antibodies Antibodies Antigenpresenting cells Cytotoxic cells Lymfokines T-cells B-cells Cellular response Antibody-producing cells Humoral response (Adapted and modified from Colton 1996) Important capsule parameters • • • • Stability Biocompatibility Permeability Size • Cell function after encapsulation Biocompatibility • Host cells growing on the capsule surface reduces the amount of nutrients and oxygen that passes through the capsule to the encapsulated cells • Host immune cells may be stimulated by capsule components to secrete elements that are harmful to the encapsulated cells Biocompatibility - depending on exposure of poly-L-lysine (PLL) Empty capsules transplanted to Balb/c mice and explanted after 4 weeks 0.1% PLL exp.10min 0.05% PLL exp.5min without PLL Capsules without fibrosis: 0%, n=6 mice Capsules without fibrosis: 91 ± 5%, n=3 mice Capsules without fibrosis: 91 ± 6%, n=3 mice (Strand et al. 2001) Effects of PLL on tumor necrosis factor (TNF) production and necrosis in monocytes TNF 80 60 1000 40 100 Necrosis % necrosis TNF (pg/ml) 10000 20 10 0 10 100 Concentration of PLL (g/ml) (Strand et al. 2001) Need of new solutions of controlling stability and permeability when omitting the polycation Strategies • Using gelling ions of higher affinity to alginate • Using high-G alginate with a high MW • Formation of inhomogeneous beads • Using enzymatically tailored alginates (epimerased alginates) • Using chemoenzymatic strategy to covalently crosslink the alginate Alginate H COOH H OH OH OH OH H H H COO G: - OH OH O O H -L-Guluronic acid (G) -OOC OH O COO HO O O HO O COO - OH O - O O OH O M : 4C 1 H H -D-Mannuronic acid (M) 1C 4 O OH COOOH OH H O OH OH OH G COO - OH G M M G GM M M MGGGGGGGMGMGMGMGM M M M M MG M - block G - block MG - block M - block (Fisher and Dörfel 1955; Atkins et al. 1970; Haug et al 1964-1967) Formation of Ca-alginate gel beads Cells Alginate CaCl2 (or BaCl2) (Smidsrød and Skjåk-Bræk 1990) Alginate sources and composition Alginate source GM FGG FMM F FMG FGGG FGGM FMGM NG>1 FG FM Durvillea antarctica 0.32 0.68 0.16 0.51 0.17 0.11 0.05 0.12 4 Macrocystis pyrifera 0.42 0.58 0.20 0.37 0.21 0.16 0.04 0.17 6 Laminaria hyperborea, leaf 0.49 0.51 0.31 0.32 0.19 0.25 0.05 0.13 8 L. hyperborea, stipe 0.63 0.37 0.52 0.26 0.11 0.48 0.05 0.07 15 Algal alginates: Bacterial alginates: Pseudomonas aeruginosa Azotobacter vinelandii FG 0 - 0,5 0,10-0,85 FGG 0 0,02-0,85 (Smidsrød and Skjåk-Bræk 1990) Alginate properties depend on alginate composition ¯OOC Ca2+ OH OH O O O OH G O OH ¯OOC G Ca2+ (Grant et al 1973; Smidsrød 1974) Alginate properties depending on composition GG/GG junctions MG/MG junctions GG/MG junctions (Donati et al, 2005) Alginate properties depending on alginate composition - stability High-M alginate (43% G) High-G alginate (69% G) Diameter (µm) 50mM CaCl2 50mM CaCl2 + 1mM BaCl2 10mM BaCl2 1000 1000 900 900 800 800 700 700 600 600 500 500 400 0 1 2 3 4 5 6 7 50mM SrCl2 400 0 1 2 3 4 5 6 7 Change of NaCl-solution (Mørch et al. 2006) Epimerisation of alginate with mannuronan C-5 epimerases OOC - HO O -OOC O OH M OOC - OHO O HO HO O -OOC M OH O HO O OH OOC - O HO O O OH OOC - M M OH O HO OOC - O HO O OOC - M M OH O HO O OH O O M M AlgE4 - O HO O -OOC O OH M OOC OH O HO O -OOC OH G - OOC O - OH O HO O -OOC OH O OH M OOC OH O HO O -OOC OH O OH M G - O OH O O OH O OH M G OOC O G AlgE1/AlgE6 - HO O -OOC OOC O OH O HO O -OOC OH O OH - O O OH OH OOC O G M O OH O OH G OH OOC - OOC G - O O OH O OH O O OH M - OH G - OOC G OOC OH O HO O -OOC OH G O O OH M (Adapted and modified from Ertesvåg, Valla and Skjåk-Bræk 1996) Swelling of alginate beads - effect of epimerisation High G alginate (69% G) Epimerised High M alg (56% G) Epimerised polyMG alginate (67% G) Epimerised polyMG alginate (56% G) Diameter [µm] 1000 900 800 700 600 500 400 0 1 2 3 4 5 6 7 8 Change of NaCl-solution (Mørch et al. 2007) Inhomogeneous alginate distribution Profile Intensity 250 200 150 100 50 0 0 100 200 300 400 500 Microcapsule center An inhomogeneous alginate core • (binds more PLL) • forms a more stable capsule • forms a less permeable capsule • (reduces the risk of protruding islets?) 600 Permeability assay Dynabead coupled with mice antibody (with anti-TNF specificity) 125-I labelled anti-mice IgG (or TNF) Capsule membrane (Kulseng et.al.1997) Insulin IL-1β TNF-α Transferrin IgG 5.8 kDa 17.5 kDa 51 kDa 80 kDa 150 kDa Radius of gyration (RG) for a globular molecule is proportional to the cubic root of the molecular weight(a): RG Mw⅓ (a Tanford, C. Physical chemistry of macromolecules. New York, John Wiley & Sons 1961) Permeability (IgG, 150 kDa) Permeability of IgG into beads of high-G alginate 8000 7000 6000 cpm 5000 4000 3000 2000 1000 0 Positive control Negative control Ca supplemented with Ba (Mørch et al. 2006) Alginate properties depending on alginate composition - permeability 100 Native alginates 80 70 60 50 40 Epimerised alginates 30 20 10 0 N eg at iv e co Po nt si ro tiv l e L. co hy nt p, ro l FG = M 0. .p 65 yr ,F G = 0. 42 FG =0 .5 2 FG =0 .5 5 FG =0 .6 1 FG =0 .6 5 Binding of IgG [%] 90 (Mørch et al. 2007) Capsules of different permeability TNF 55kDa Alg/PDL/pMG IgG 150kDa Alg/PLL/pMG Alg (Strand et al. 2003) Human pancreatic islets encapsulated in alginate microbeads Day 1: Encapsulated human Islet in culture for 141 days: Day 16: (Strand 2006) Human pancreatic islets from Chicago encapsulated in alginate beads - viability and function after 2x overseas shipments and encapsulation 100 non-encapsulated islets Viability (%) 90 encapsulated islets 80 70 60 50 Groups Stimulation index (SI) 7 6 non-encapsulated islets 5 encapsulated islets 4 3 2 1 0 Groups (Qi, Strand et al. 2008) Encapsulated human islets to nude mice 600 600 non-fasting blood glucose (mg/dl) 500 10 000 IE 500 400 400 300 100 0 0 5 16 27 35 52 69 95 109 130 173 208 600 3000 IE 500 500 300 100 0 0 5 16 27 35 52 69 95 109 130 173 208 200 200 400 300 600 5000 IE 400 300 200 100 0 0 5 16 27 35 52 69 95 109 130 173 208 200 100 1000 IE 130 173 208 0 0 5 16 days (posttransplantation) 27 35 52 69 95 109 (Qi, Strand et al. 2008) Conclusions • Polycations provoke immune responses • Alginate capsules without polycation can protect transplanted pancreatic islets in allo- and xenomodels (mice) • Stable alginate capsules can be made by the right selection of alginate and gelling ions • By enzymatic modification, new alginates can be made tailored for their use in encapsulation “A group of highly qualified scientists and their teams who have committed themselves to achieving a functional cure for diabetes as soon as possible” (via transplantation of insulin producing cells) • Finding new sources for insulin producing cells • Transplantation wihtout immuno suppression (encapsulation) www.chicagodiabetesproject.org Acknowledgements Norwegian University of Science and Technology Gudmund Skjåk-Bræk Terje Espevik Yrr A. Mørch Anne Mari Rokstad Liv Ryan Bjørg Steinkjer Wenche Strand The Chicago Diabetes Project, Encapsulation Team: Chicago, Illinois, USA: Jose Oberholzer, Meirigeng Qi Urbana-Champagne, Illinois USA: Kevin Kim, Hyungsoo Choi Bratislava, Slovakia: Igor Lacik Geneva, Switzerland: David Hunkeler Sydney, Australia: Bernie Tuch University of Trieste, Italy: Ivan Donati University of Alberta, Canada: Greg Korbutt Financial Support: Norwegian Research Council The Norwegian Diabetes Association via Extra Funds from the Norwegian Foundation for Health and Rehabilitation The Chicago Diabetes Project
