WO2014146677A1 - Peg hydrogels functionalized with glucagon and rgds-tetrapeptide and containing stem cells for islet coating - Google Patents

Peg hydrogels functionalized with glucagon and rgds-tetrapeptide and containing stem cells for islet coating Download PDF

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WO2014146677A1
WO2014146677A1 PCT/EP2013/055543 EP2013055543W WO2014146677A1 WO 2014146677 A1 WO2014146677 A1 WO 2014146677A1 EP 2013055543 W EP2013055543 W EP 2013055543W WO 2014146677 A1 WO2014146677 A1 WO 2014146677A1
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hydrogel
polyethyleneglycol
peg
islets
peptide
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PCT/EP2013/055543
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French (fr)
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Seda Kizilel
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Koc Universitesi
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)

Definitions

  • Diabetes mellitus has a Europe prevalence of approximately 48 million people, including approximately 1 million with Type I diabetes. Despite the availability of exogenous insulin, life expectancy and quality are still diminished by chronic or late complications of the disease. These complications can be mitigated by restoring near-normal levels of glucose. The most effective strategy to do so relies on transplanting islets from donor tissue in the form of a whole pancreas or isolated islets. Islet transplantation has evolved as a meaningful treatment option for Type I diabetes, but its widespread application has been limited by the need for immunosuppression and limited donor tissue supply. Immunoisolation though microencapsulation offers the possibility of addressing both these limiting factors by allowing immunosuppression-free transplantation of allogeneic and possibly xenogeneic tissue.
  • the transplanted tissue e.g. human islet is isolated from the recipient's immune system by a semipermeable membrane.
  • Prior microencapsulation techniques have been based primarily on alginate chemistry. These coatings typically have thicknesses of hundreds of microns.
  • Two critical factors that affect the success of immunoprotective devices are the thickness and permeability of the membrane. These are crucial, because a thick membrane can present a large diffusion barrier to oxygen nutrient, metabolites and/or the therapeutic agent released by the cell, yet a very thin barrier is more likely to have defects that may expose the transplanted cells to the immune system of the host.
  • Encapsulation of islets using either of these techniques is necessary to achieve higher yields to test in vivo function and immunoprotection of islets encapsulated by this method.
  • An understanding of ways to control the thickness and permeability of coating as well as functionality of islets is beneficial not only in advancing the state of knowledge in the islet and diabetes area but also because of the potential impact that this information would have on the development of novel technologies for medical implants and tissue engineering.
  • it can be possible to coat rodent and canine islets in capsules of adequate quantity and consistent quality and the function of coated islets will be equivalent to that of unencapsulated islets in environments where allogeneic and xenogeneic immunologic rejection are not factors, and superior in models in which rejection is a factor.
  • Another object of the invention is allotransplantation and xenotransplantation of islets microencapsulated by surface-initiated polymerization in larger mammals, non-human primates, and preferably humans.
  • Figure 2 shows a process scheme for the production of functionalized PEG hydrogel
  • Figure 3 shows a process scheme of immobilization of photoinitiator eosin-Y onto an islet
  • Figure 4 shows a process scheme for the production of islet encapsulated within functionalized PEG hydrogel.
  • the present invention provides PEG hydrogel coating which is functionalized by GLP-1 (7- 37) (14) and RGDS (16).
  • Functionalized PEG hydrogel (20) of the present invention is used for the encapsulation of different cells, preferably the cells used for the transplantation, more preferably pancreatic islet cells (21 ).
  • the thin and functional PEG coating of the present invention also allows the transplantation of cells through the portal vein that flows into the liver.
  • the functionalized PEG hydrogel (20) is ready for the coating of the cells to be transplanted.
  • the cells to be transplanted are coated with functionalized PEG hydrogel (20) by a photopolymerization method.
  • the surface of the cells to be transplanted e.g. the islets (21 ) was functionalized by eosin-Y (22), which acts as a photoinitiator ( Figure 3).
  • polymerization starts to grow from the surface, unlike many other studies used in the field where they encapsulate 20-30 islets within a gel via bulk polymerization approach. This means that with the current study, transplantation volume of islets (21 ) will be significantly smaller than other approaches and may allow for transplantation of coated islets into the portal vein of the liver.
  • the PEG hydrogels of the invention is used for islet (21 ) transplantation.
  • Pancreatic islets encapsulated within functionalized PEG hydrogel (23) of the present invention are used in the treatment of type I and type II diabetes.
  • the presence of crosslinked functionalized PEG hydrogel (20) protects islets from the large components of the immune system, and the presence of stem cells provides protection against the small components of immune system.
  • Functional hydrogel membranes incorporating the insulinotropic agent, GLP-1 (7-37) around islets increase insulin secretion in a glucose dependent manner, which may reduce the total number of islets required to cure a diabetic patient, and hence decrease transplantation volume which allows flexibility in choosing a transplantation site.
  • Arginine-glycine-aspartic acid-serine (RGDS) cell adhesion sequence achieves the binding of a v 3 3 -integrin located at the outer cell membrane of stem cells to the PEG hydrogel-coating around islets (21 ). This would allow for the attachment of stem cells (19) onto the PEG hydrogel structure.
  • Co-localization of stem cells (19) within PEG hydrogel through RGDS (16) cell adhesion sequences has the following potentials: providing modulation of the immune response, leading to enhanced islet (21 ) function and prolonged graft survival.
  • the most efficient condition for encapsulation is obtained by coupling experiments with mathematical models. Therefore, depending on the diffusion limitations of molecules (oxygen, insulin, waste products or components of immune system) through the membrane, it is possible to tune the properties of the microcapsule in the radial direction.
  • This regulation strategy blocks the passage of immune cells and large molecules like immunoglobulin, however small molecules secreted by activated neutrophils and macrophages, such as reactive oxygen species (ROS) and cytokines can easily diffuse through membranes and induce damage to the islets (21 ).
  • ROS reactive oxygen species
  • cytokines can easily diffuse through membranes and induce damage to the islets (21 ).
  • the strategy used here through incorporation of stem cells (19) to the coating is beneficial to provide protection from small cytotoxic molecules and improve the functionality of the membrane coating.
  • islet coating strategy based on PEG hydrogel coating technique did not compromise islet function, furthermore, islets microencapsulated within GLP-1 (7-37) (14) functionalized PEG hydrogel were found to exhibit higher insulin secretion compared to the islets in the remaining groups in response to high glucose. Interestingly, no such bioactivity was observed when islets (21 ) were microencapsulated within GLP-1 (9-37) functionalized PEG hydrogel, which also confirms the previous studies that the bioactivity of GLP-1 (7-37) peptide sequence would be lost upon its metabolism to GLP-1 (9-37) sequence by its peptidase dipeptidyl peptidase IV.

Abstract

The present invention is related to a method for the production of coated cells to be transplanted; preferably islets encapsulated within functionalized PEG hydrogel coating comprising glucagon-like peptide-1(7-37),arginine-glycine-aspartic acid-serine cell adhesion peptide (RGDS) and stem cells.

Description

DESCRIPTION
PEG HYDROGELS FUNCTIONALIZED WITH GLUCAGON AND RGDS-TETRAPEPTIDE AND CONTAINING STEM CELLS FOR ISLET COATING
Technical Field
The present invention is related to a method for the production of coated cells to be transplanted; preferably islets encapsulated within functionalized polyethyleneglycol (PEG) hydrogel coating comprising glucagon like peptide (GLP) -1 (7-37), arginine-glycine- aspartic acid-serine peptide (cell adhesion peptide) and stem cells.
Prior Art In the United States and Europe, advanced immunoisolation strategies for islet transplantation have been used in order to protect transplanted islets from the immune system, wherein islets are encapsulated within polymeric coatings that exclude the large molecular components of the immune system but still maintain cell viability and permit diffusion of small molecules such as glucose, insulin, oxygen, metabolites and nutrients. This strategy requires optimization of the permeability and thickness of the coatings for diffusive transport of low molecular weight molecules. Tuning of the coat thickness has been particularly difficult, as the current methods, based primarily on droplet generation by extrusion through a needle, make capsules of a constant outer diameter. This proposal will also be the first in islet research area to functionalize membrane capsule with biological ligands and stem cells simultaneously in order to stimulate higher insulin release and to provide local protection from recipient's immune system.
Diabetes mellitus has a Europe prevalence of approximately 48 million people, including approximately 1 million with Type I diabetes. Despite the availability of exogenous insulin, life expectancy and quality are still diminished by chronic or late complications of the disease. These complications can be mitigated by restoring near-normal levels of glucose. The most effective strategy to do so relies on transplanting islets from donor tissue in the form of a whole pancreas or isolated islets. Islet transplantation has evolved as a meaningful treatment option for Type I diabetes, but its widespread application has been limited by the need for immunosuppression and limited donor tissue supply. Immunoisolation though microencapsulation offers the possibility of addressing both these limiting factors by allowing immunosuppression-free transplantation of allogeneic and possibly xenogeneic tissue. Theoretically, this membrane serves as a mechanical barrier isolating the graft from recipient leukocytes and antibodies while continuing to allow the diffusion of glucose, water, insulin, oxygen, nutrients, and cellular waste. Finally, with encapsulation, an opportunity to functionalize the coat to enhance glucose-stimulated insulin secretion by encapsulated islets exists by incorporating pharmacologic agents into the coatings.
In the article titled "Mathematical model for microencapsulation of pancreatic islets within a biofunctional PEG hydrogel", microencapsulation of islets within an insulinotropic peptide functionalized PEG hydrogel is disclosed.
In the article titled as "Glucagon-Like Peptide- 1 Functionalized PEG Hydrogels Promote Survival and Function of Encapsulated Pancreatic β-Cells", encapsulation of a group of pancreatic islets in a semipermeable cyclindiric (5 mm diameter 1 mm thick) PEG hydrogel membrane formed via thiol-acrylate bulk photopolymerization is disclosed. In an example, totally 20 islets are loaded into hydrogel structure.
In microencapsulation process, the transplanted tissue e.g. human islet is isolated from the recipient's immune system by a semipermeable membrane. Prior microencapsulation techniques have been based primarily on alginate chemistry. These coatings typically have thicknesses of hundreds of microns. Two critical factors that affect the success of immunoprotective devices are the thickness and permeability of the membrane. These are crucial, because a thick membrane can present a large diffusion barrier to oxygen nutrient, metabolites and/or the therapeutic agent released by the cell, yet a very thin barrier is more likely to have defects that may expose the transplanted cells to the immune system of the host.
The technique described as surface-initiated polymerization generates much thinner coatings, having a mean thickness of about 100 micrometres. Unfortunately, even with this technique; obstacles remain in the path of developing effective diabetes treatments using microencapsulated pancreatic islet cells. Experimentally a variety of parameters such as the chemical composition of the photopolymerization system, the duration of photopolymerization and the intensity of the excitation light source have been found to effect gel thickness and permeability characteristics of the capsule wall. Kizilel et al have studied a mathematical model based on the Numerical Fractionation technique for hydrogel multilayers formed by interfacial photopolymerization of PEG diacrylate/VP.
Another difficulty that limits the potential for diabetes treatments using microencapsulated pancreatic islet cells is the scarcity of human pancreas or islet sources and the large number of islet cells required for such treatment.
Encapsulation of islets using either of these techniques is necessary to achieve higher yields to test in vivo function and immunoprotection of islets encapsulated by this method. An understanding of ways to control the thickness and permeability of coating as well as functionality of islets is beneficial not only in advancing the state of knowledge in the islet and diabetes area but also because of the potential impact that this information would have on the development of novel technologies for medical implants and tissue engineering. With the present invention, , it can be possible to coat rodent and canine islets in capsules of adequate quantity and consistent quality and the function of coated islets will be equivalent to that of unencapsulated islets in environments where allogeneic and xenogeneic immunologic rejection are not factors, and superior in models in which rejection is a factor.
Brief Description of the Invention
Present invention relates to a method for the production of coated islets encapsulated within PEG hydrogel. Said method comprises the production of functionalized PEG hydrogel with glucagon-like peptide [GLP-1 (7-37)j, cell adhesion peptide arginine-glycine- aspartic acid-serine (RGDS) and stem cells, and the coating of islets with said functionalized PEG hydrogel by a surface initiated photopolymerization method.
For the production of said functionalized PEG hydrogel, firstly PEG conjugates which are acryloyl-polyethyleneglycol-GLP-1 (7-37) (acr-PEG-GLP-1 (7-37)) and acryloyl- polyethyieneglycol-arginine-glycine-asparticacid-serine (acr-PEG-RGDS) are synthesized. Said acr-PEG-GLP-1 (7-37) and acr-PEG-RGDS are incorporated into the PEG prepolymer solution, and finally, stem cells are incorporated into the PEG hydrogel prepolymer comprising acr-PEG-GLP-1 (7-37) and acr-PEG-RGDS conjugates to obtain functionalized PEG hydrogel.
Present invention is also related to coated islet encapsulated within PEG hydrogel which is functionalized with glucagon-like peptide [GLP-1 (7-37)], cell adhesion peptide (RGDS) and stem cells and to the use thereof in the treatment of diabetes via cell transplantation. In the present invention, the requirement of large number of islets is addressed through functionalization of the membrane with an insulinotropic agent, GLP-1 (7-37) through covalent conjugation to PEG.
Object of the Invention
The object of the invention is to provide functionalized PEG hydrogel comprising GLP-1 (7- 37), RGDS and stem cells used as a coating for the cell transplantation.
Another object of the invention is to reduce the transplantation volume of islets and to enhance the transplantation of human islets into the portal vein that flows into the liver by the use of functionalized PEG hydrogel comprising GLP-1 (7-37), RGDS and stem cells.
Another object of the invention is allotransplantation and xenotransplantation of islets microencapsulated by surface-initiated polymerization in larger mammals, non-human primates, and preferably humans.
Description of the Drawings
Figure 1 shows a process scheme for the formation of PEG hydrogel prepolymer comprising acr-PEG-GLP-1 (7-37) and acr-PEG-RGDS conjugates,
Figure 2 shows a process scheme for the production of functionalized PEG hydrogel, Figure 3 shows a process scheme of immobilization of photoinitiator eosin-Y onto an islet, Figure 4 shows a process scheme for the production of islet encapsulated within functionalized PEG hydrogel. The references in the figures may possess following meanings;
PEG prepolymer (13)
GLP-1 (7-37) (14)
Acr-PEG-GLP-1 (7-37) (15)
RGDS cell adhesion sequence (16)
Acr-PEG-RGDS (17)
PEG hydrogel prepolymer comprising acr-PEG-GLP-1 (7-37) (18)
and acr-PEG-RGDS conjugates
Stem cell (19)
Functionalized PEG hydrogel (20)
Islet (21 )
Eosin-Y (22)
Islet encapsulated within functionalized PEG hydrogel (23)
Detailed Description of the Invention
The present invention provides PEG hydrogel coating which is functionalized by GLP-1 (7- 37) (14) and RGDS (16). Functionalized PEG hydrogel (20) of the present invention is used for the encapsulation of different cells, preferably the cells used for the transplantation, more preferably pancreatic islet cells (21 ). The cells which are used for the transplantation in the present invention herein after referred to as cells to be transplanted. The thin and functional PEG coating of the present invention also allows the transplantation of cells through the portal vein that flows into the liver.
Functionalization with GLP-1 (7-37) (14) and RGDS (16) and also immunoprotection with stem cells (19) improve active immunoisolation barriers and survival of transplanted cells.
Functionalized PEG hydrogel (20) of the invention generates a thinner coating to the cells to be transplanted and has enhanced functionality and local immunoprotection of coated cells from immune system components.
Such a coating allows, not only immunosuppression free allotransplantation, but also prevents xenogeneic rejection, an expansion of available donor tissue with the new utility of animals as donors, with a resultant increase in feasibility and applicability of cellular transplantation for diabetes. This interdisciplinary project contributes to standardize islet encapsulation technology and provides an effective long term treatment or cure Type I diabetes.
Functionalized and crosslinked PEG hydrogel of the invention is synthesized in four phases.
The first phase involves the synthesis of the PEG conjugates, which are acr-PEG-GLP- 1 (7-37) (15) and acr-PEG-RGDS (17). In order to functionalize PEG hydrogel coatings, GLP-1 (7-37) (14) is incorporated into the crosslinked network of PEG diacrylate hydrogel (PEGDA). This is achieved by functionalizing the amine terminus of the peptide with an acrylate moiety, which enables the peptide to copolymerize rapidly with PEG diacrylate upon photoinitiation. Acrylated peptide; acr-PEG-GLP-1 (7-37) (15) is synthesized as a result of the reaction of the peptide with acryloyl-PEG-N-hydroxysuccinimide ester. Then, PEG hydrogel is functionalized with RGDS (16) cell adhesion sequence and acr-PEG- RGDS (17) is synthesized. PEG prepolymer (13) solutions comprise PEGDA, triethanolamine and N-Vinylpyrrolidone. In the second phase, obtained conjugates; acr-PEG-GLP-1 (7-37) (15) and acr-PEG- RGDS (17) are incorporated into the PEG prepolymer (13) solution used for the microencapsulation of cells to be transplanted and PEG hydrogel prepolymer comprising acr-PEG-GLP-1 (7-37) and acr-PEG-RGDS conjugate (18) is synthesized (Figure 1 ). Then, stem cells (19) are incorporated into the PEG hydrogel prepolymer comprising acr- PEG-GLP-1 (7-37) (15) and acr-PEG-RGDS (17) conjugates in the third step (Figure 2). Thus, the functionalized PEG hydrogel (20) is ready for the coating of the cells to be transplanted. In the final step, the cells to be transplanted are coated with functionalized PEG hydrogel (20) by a photopolymerization method. In the photopolymerization method, the surface of the cells to be transplanted e.g. the islets (21 ) was functionalized by eosin-Y (22), which acts as a photoinitiator (Figure 3). Next, the cells with photoinitiator on the surface were incubated in PEG prepolymer solution functionalized with or without GLP-1 , which resulted in the formation of a crosslinked PEG hydrogel around said cells with a laser exposure time of 3 min at a visible light wavelength of 514 nm. Since photoinitiator is immobilized on the surface of the cells, PEG hydrogel membrane starts to grow from the surface of the cells to be transplanted (Figure 4). This results in the formation of a PEG hydrogel membrane with gradient in permeability where points closer to cell surface is less permeable compared to the points further from cell surface.
In the present invention, polymerization starts to grow from the surface, unlike many other studies used in the field where they encapsulate 20-30 islets within a gel via bulk polymerization approach. This means that with the current study, transplantation volume of islets (21 ) will be significantly smaller than other approaches and may allow for transplantation of coated islets into the portal vein of the liver.
In a preferred embodiment of the invention, the PEG hydrogels of the invention is used for islet (21 ) transplantation. Pancreatic islets encapsulated within functionalized PEG hydrogel (23) of the present invention are used in the treatment of type I and type II diabetes. The presence of crosslinked functionalized PEG hydrogel (20) protects islets from the large components of the immune system, and the presence of stem cells provides protection against the small components of immune system. Functional hydrogel membranes incorporating the insulinotropic agent, GLP-1 (7-37) around islets increase insulin secretion in a glucose dependent manner, which may reduce the total number of islets required to cure a diabetic patient, and hence decrease transplantation volume which allows flexibility in choosing a transplantation site. Transplantation of human islets into the portal vein that flows into the liver can be enhanced by the administration of PEG hydrogel of the present invention. Introduction at this site enhances the exposure of islets to a nutrient rich medium and also approximates the kinetics and metabolism of normally functioning islets by allowing insulin to first pass through the liver before entering into systemic circulation. The large capsules generated by previous techniques have made problematic transplantation of encapsulated islets into this site. Islets (21 ) which are microencapsulated in functional PEG hydrogel and stem cells (19) simultaneously secrete the higher amounts of insulin and the stem cells (19) protect islets (21 ) from low molecular weight components of the immune system due to the anti- apoptotic properties of stem cells. The higher secretion of insulin as a result of GLP-1 bioactivity helps to reduce the total number of islets to be transplanted required to reverse diabetes, and the presence of stem cells (19) will enhance viability of islets and provide local protect ion for islets. Reduction in total number of islets that needs to be transplanted is an advantage as this would mean less pressure on the liver and higher probability of viability for implanted islets.
The conjugate of acr-PEG-GLP-1 (7-37) (15) is useful to increase the glucose-stimulated insulin secretion by pancreatic islets (21 ). GLP-1 (7-37) (14) is known to be a potent glucose dependent insulinotropic hormone, which has important actions on the stimulation of insulin secretion and of its gene expression, the proliferation and differentiation of islet beta cells. The function of pancreatic islets is enhanced due to the presence of GLP-1 (7- 37) (14) on islet (21 ) surface. The presence of GLP-1 might also induce stem cells (19) towards insulin producing cells.
Arginine-glycine-aspartic acid-serine (RGDS) cell adhesion sequence achieves the binding of av33-integrin located at the outer cell membrane of stem cells to the PEG hydrogel-coating around islets (21 ). This would allow for the attachment of stem cells (19) onto the PEG hydrogel structure.
Co-localization of stem cells (19) within PEG hydrogel through RGDS (16) cell adhesion sequences has the following potentials: providing modulation of the immune response, leading to enhanced islet (21 ) function and prolonged graft survival.
In the present invention, the most efficient condition for encapsulation is obtained by coupling experiments with mathematical models. Therefore, depending on the diffusion limitations of molecules (oxygen, insulin, waste products or components of immune system) through the membrane, it is possible to tune the properties of the microcapsule in the radial direction. This regulation strategy blocks the passage of immune cells and large molecules like immunoglobulin, however small molecules secreted by activated neutrophils and macrophages, such as reactive oxygen species (ROS) and cytokines can easily diffuse through membranes and induce damage to the islets (21 ). The strategy used here through incorporation of stem cells (19) to the coating is beneficial to provide protection from small cytotoxic molecules and improve the functionality of the membrane coating. The mathematical models developed for biofunctional PEG hydrogel are used to obtain ideal thickness and crosslink density values for the capsule around islets (21 ). An optimal experimental condition that gives a thickness of about 50-80 μηη and a crosslink density of -3x10"3 is determined by using the mathematical model. These optimal thickness and crosslink density values were assumed as standard conditions by Kizilel et al leading to the most uniform hydrogel membranes and viable islets. Theoretical predictions of the thickness and crosslink density save significant amount of experimental time to achieve optimal encapsulation conditions leading to the efficient encapsulation and viable islets. PEG is nonreactive with the immune system and has an established history as a material that can be implanted into the human body for a wide range of approved clinical applications. PEG hydrogel formation through surface-initiated photopolymerization involves immobilization of photoinitiator eosin-Y (22) onto a substrate or an islet (21 ) surface through covalent bonding followed by irradiation of monomer precursor solution with visible light (A.=514 nm). Upon photoinitiation, the initiator decomposes to form radical fragments. The radical fragments react with the monomer solution and via propagation form radical chains. Hydrogel formation subsequently occurs when excessive branching and/or crosslinking reactions are present in the polymerization system. We were able to show for the first time that islets (21 ) could individually be coated within biofunctional PEG hydrogel, and that it is possible to modulate islet function within this biofunctional PEG hydrogel bearing bioactive peptides.
The activity of encapsulated islets (21 ) was investigated in vitro by measuring total amount of insulin release in response to a change in glucose concentration for 30 minutes, in both low glucose and high glucose buffers separately, using static incubation assay. For all sets of islets the stimulation index (SI) was greater than 2, while PEG-GLP-
1 (7-37) (14) functionalized hydrogel encapsulated islets had the highest SI value of 16.7.
This proves that islet coating strategy based on PEG hydrogel coating technique did not compromise islet function, furthermore, islets microencapsulated within GLP-1 (7-37) (14) functionalized PEG hydrogel were found to exhibit higher insulin secretion compared to the islets in the remaining groups in response to high glucose. Interestingly, no such bioactivity was observed when islets (21 ) were microencapsulated within GLP-1 (9-37) functionalized PEG hydrogel, which also confirms the previous studies that the bioactivity of GLP-1 (7-37) peptide sequence would be lost upon its metabolism to GLP-1 (9-37) sequence by its peptidase dipeptidyl peptidase IV.
GLP-1 peptide of the present invention comprises lle-Lys-Val-Ala-Val (IKVAV) and RGDS peptide which is affecting the function of islet (21 ). These peptides have synergistic effect for the increase of insulin release. It is originated from placing the peptides to the synthetic environment (encapsulation environment) which mimics the natural environment. Peptide sequences RGDS and IKVAV are present in the structure of laminin and integrin proteins. These sequences allow for the adhesion of stem cells (19) and islets (21 ) into the hydrogel scaffold due to the recognition of these sequences by receptors on stem cell (19) or islet (21 ) surface. GLP-1 is a peptide sequence which is completely released from the body and has a receptor on the islet cells.

Claims

1. A method for the production of coated islets encapsulated within functionalized polyethyleneglycol hydrogel characterized in that said method comprises the steps of;
a) Production of functionalized polyethyleneglycol hydrogel (20) comprising glucagon like peptide-1 (7-37) (14), arginine-glycine-aspartic acid-serine peptide (16) and stem cells (19), and
b) Coating of islets (21 ) with said functionalized polyethyleneglycol hydrogel (20) by a surface initiated photopolymerization method.
2. A method according to claim 1 , characterized in that the production of functionalized polyethyleneglycol hydrogel (20) comprises the following steps: a) Polyethyleneglycol conjugates which are acryloyl-polyethyleneglycol- glucagon like peptide-1 (7-37) (15) and acryloyl-polyethyleneglycol-arginine- glycine-aspartic acid-serine peptide (17) are synthesized,
b) Said acryloyl-polyethyleneglycol-glucagon like peptide-1 (7-37) (15) and acryloyl-polyethyleneglycol-arginine-glycine-aspartic acid-serine peptide (17) are incorporated into the polyethyleneglycol prepolymer (13) solution, and c) Stem cells (19) are incorporated into said polyethyleneglycol hydrogel prepolymer (13) comprising acryloyl-polyethyleneglycol-glucagon like peptide- 1 (7-37) (15) and acryloyl-polyethyleneglycol-arginine-glycine-aspartic acid- serine peptide conjugates to obtain functionalized polyethyleneglycol hydrogel (20).
3. A method according to claim 1 , characterized in that functionalized polyethyleneglycol hydrogel (20) comprising glucagon-like peptide-1 (7-37) (14), arginine-glycine-aspartic acid-serine peptide (16) and stem cells (19) is used for the encapsulation of islets (21 ).
4. Coated islets encapsulated within polyethyleneglycol hydrogel produced by a method of claim 1 characterized in that polyethyleneglycol hydrogel coating is functionalized with glucagon-like peptide-1 (7-37) (14), arginine-glycine-aspartic acid-serine peptide (16) and stem cells (19). Coated islets encapsulated within polyethyleneglycol hydrogel (23) produced by method of claim 1 for use in the treatment of diabetes via cell transplantation.
PCT/EP2013/055543 2013-03-18 2013-03-18 Peg hydrogels functionalized with glucagon and rgds-tetrapeptide and containing stem cells for islet coating WO2014146677A1 (en)

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