Encapsulation with Alginates. Berit L. Strand, Yrr Mørch and Gudmund Skjåk-Bræk Norwegian University of Science and Technology

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1 Encapsulation with Alginates Berit L. Strand, Yrr Mørch and Gudmund Skjåk-Bræk Norwegian University of Science and Technology

2 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)

3 Microcapsules in cell therapy Immunoisolation: Nutrients and oxygen Immune cells Cell products Insulin Graft rejection + autoimmune disease Cells (Islets) Capsule (Lanza et al. 1999)

4 (chicagodiabetesproject.org)

5 Fibroblast Capsule membrane Cytokines, free radicals reactive oxygen- and nitrogenintermediates Insulin xygen and nutrients Cell Waste products Complement components Y c c Autoimmune antibodies YY YYY Macrophages Secreted proteins Y Y Y v v c Antibodies Natural occurring antibodies Cytotoxic cells Antigenpresenting cells Lymfokines T-cells B-cells Antibody-producing cells Cellular response Humoral response (Adapted and modified from Colton 1996)

6 Important capsule parameters Stability Biocompatibility Permeability Size Cell function after encapsulation

7 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

8 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 Capsules without fibrosis: 0%, n=6 mice 0.05% PLL exp.5min Capsules without fibrosis: 91 ± 5%, n=3 mice without PLL Capsules without fibrosis: 91 ± 6%, n=3 mice (Strand et al. 2001)

9 Effects of PLL on tumor necrosis factor (TNF) production and necrosis in monocytes TNF 80 TNF (pg/ml) Necrosis % necrosis Concentration of PLL (g/ml) (Strand et al. 2001)

10 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

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12 Alginate H H H H H H H H -D-Mannuronic acid (M) H H H H H C- C- H H H H -L-Guluronic acid (G) H G : 1 C 4 M : 4 C 1 C - H C - H -C H H H H C - H H H C - H G 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 )

13 Formation of Ca-alginate gel beads Cells Alginate CaCl 2 (or BaCl 2 ) (Smidsrød and Skjåk-Bræk 1990)

14 Alginate sources and composition Alginate source FG FM FGG FMM FGM FMG FGGG FGGM FMGM NG>1 Algal alginates: Durvillea antarctica Macrocystis pyrifera Laminaria hyperborea, leaf L. hyperborea, stipe Bacterial alginates: FG FGG Pseudomonas aeruginosa 0-0,5 0 Azotobacter vinelandii 0,10-0,85 0,02-0,85 (Smidsrød and Skjåk-Bræk 1990)

15 Alginate properties depend on alginate composition C Ca 2+ H H H G H C G Ca 2+ (Grant et al 1973; Smidsrød 1974)

16 Alginate properties depending on composition GG/GG junctions MG/MG junctions GG/MG junctions (Donati et al, 2005)

17 Alginate properties depending on alginate composition - stability High-G alginate (69% G) High-M alginate (43% G) 50mM CaCl 2 50mM CaCl 2 + 1mM BaCl 2 10mM BaCl 2 50mM SrCl 2 Diameter (µm) Change of NaCl-solution (Mørch et al. 2006)

18 H H H -C Epimerisation of alginate with mannuronan C-5 epimerases H H -C H H -C H H -C H -C H -C H H H H H -C H -C H M M M M M M M M AlgE4 H - C H - C H H H - C H - C H H H - C H - C H H H H - C - C H H M G M G M G M G AlgE1/AlgE6 H - C H - C H H H - C H - C H H H - C - C H H H - C - C H H H - C H M G M G G G G G M (Adapted and modified from Ertesvåg, Valla and Skjåk-Bræk 1996)

19 Swelling of alginate beads - effect of epimerisation High G alginate (69% G) Epimerised polymg alginate (67% G) Epimerised High M alg (56% G) Epimerised polymg alginate (56% G) 1000 Diameter [µm] Change of NaCl-solution (Mørch et al. 2007)

20 Inhomogeneous alginate distribution Intensity 250 Profile 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?)

21 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)

22 Insulin IL-1β TNF-α Transferrin IgG 5.8 kda 17.5 kda 51 kda 80 kda 150 kda Radius of gyration (R G ) for a globular molecule is proportional to the cubic root of the molecular weight (a) : R G M w ⅓ ( a Tanford, C. Physical chemistry of macromolecules. New York, John Wiley & Sons 1961)

23 Permeability (IgG, 150 kda) Permeability of IgG into beads of high-g alginate cpm Positive control Negative control Ca supplemented with Ba (Mørch et al. 2006)

24 Alginate properties depending on alginate composition - permeability Native alginates Epimerised alginates 30 Binding of IgG [%] Negative control Positive control L.hyp, FG = 0.65 M.pyr, FG = 0.42 FG=0.52 FG=0.55 FG=0.61 FG=0.65 (Mørch et al. 2007)

25 Capsules of different permeability TNF 55kDa IgG 150kDa Alg/PDL/pMG Alg/PLL/pMG Alg (Strand et al. 2003)

26 Human pancreatic islets encapsulated in alginate microbeads Day 1: Day 16: Encapsulated human Islet in culture for 141 days: (Strand 2006)

27 Human pancreatic islets from Chicago encapsulated in alginate beads - viability and function after 2x overseas shipments and encapsulation non-encapsulated islets encapsulated islets Viability (%) Stimulation index (SI) Groups non-encapsulated islets encapsulated islets 0 Groups (Qi, Strand et al. 2008)

28 non-fasting blood glucose (mg/dl) Encapsulated human islets to nude mice IE IE IE 1000 IE days (posttransplantation) (Qi, Strand et al. 2008)

29 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

30 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)

31 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 berholzer, 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