WO2014160363A1

WO2014160363A1 - Surface modification of porcine islets

Info

Publication number: WO2014160363A1
Application number: PCT/US2014/026394
Authority: WO
Inventors: Robert R. Kane; Bashoo Naziruddin; Jeffrey A. SORELLE; Mazhar A. KANAK
Original assignee: Baylor Research Institute
Priority date: 2013-03-14
Filing date: 2014-03-13
Publication date: 2014-10-02

Abstract

Compositions and methods are provided relating to surface-modified porcine islet cells that may be used to treat prediabetes or diabetes. In particular, a composition comprising surface-modified porcine pancreatic islet cells, wherein a cell surface functional group is covalently attached to one or more biochemically inert compounds (e.g., polyethylene glycol, PEG) that provide a barrier against host immune response is provided.

Description

DESCRIPTION

SURFACE MODIFICATION OF PORCINE ISLETS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/784825, filed on March 13, 2013, the entire contents of which are hereby incorporated by reference without disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0002] The present invention relates generally to the field of medicine. More particularly, it concerns diabetes and treatment for diabetic patients.

2. Description of Related Art

[0003] Transplantation of pancreatic islets into the liver by percutaneous puncture of the hepatic portal vein is a procedure that restores glycemic control to patients suffering from type 1 diabetes (Shapira et al , 2000; Ricordi et al, 2003). The widespread utility and effectiveness of this treatment is compromised by various factors including especially islet graft loss due to both the innate and adaptive immune system. Many islets are lost during the immediate blood-mediated inflammatory reaction known as IBMIR (Bennet et al, 2000; Goto et al, 2008). Additionally, T-cell mediated alloimmune and autoimmune reactions lead to long term graft rejection (Ryan et al, 2005) and islet transplant recipients must be placed on life-long immunosuppressant regimens that lead to increased risk. In many cases, multiple islet infusions are required for a successful outcome (Robertson, 2004; Shapiro et al, 2003), placing significant demand on the limited number of available pancreata and increasing the financial and surgical burden (Frank et al, 2004). Similar cellular therapies may also be useful to treat conditions like Type 2 diabetes and prediabetes (Prentki and Nolan, 2006; Kahn, 2013). Change reference style, where is this sentence from? While significant developments may eventually lead to the widespread use of single donor infusions (Froud et al. , 2005), the limited availability of transplantable islets will remain a limiting factor as this therapy becomes more common. While porcine islets present an attractive alternative source of islets for xenotransplantation, there are significant barriers to this strategy including the acute immune reaction between pig islets and human blood that quickly destroys unprotected islets (Cabric et ah, 2007).

SUMMARY OF THE INVENTION

[0004] Embodiments provided herein are based upon compositions comprising surface-modified porcine pancreatic islet cells. In a first embodiment, a cell surface functional group is covalently attached to one or more biochemically inert compounds that provide a barrier against host immune response. In a further embodiment, an amino acid residue provides the cell surface functional group. In some embodiments the one or more biochemically inert compounds is an organic long chain polymer. In particular embodiments, the long chain polymer is a 1 -5 kDa poly-ethylene glycol.

[0005] In some embodiments of the invention, the biochemically inert compound is covalently attached to the islets through an amide bond. In specific embodiments, a lysine residue is covalently attached to the one or more biochemically inert compounds. In other embodiments, the biochemically inert compound is covalently attached to the islets through a hydrazone bond. In particular embodiments, an oxidized sialic acid residue is covalently attached to the one or more biochemically inert compound. In other embodiments, the biochemically inert compound is covalently attached to the islets through a thioether bond. In some embodiments, the amide bond involves a cysteine residue.

[0006] In some aspects of the invention, the biochemically inert compound comprises biotin. In particular embodiments, the biochemically inert compound comprises biotinylated acyl. In specific embodiments, the biotinylated acyl has the chemical structure:

[0007] In other aspects of the invention, the surface-modified porcme pancreat cells have the structure:

(Π)

wherein n is 1 ,2 ,or 3.

[0008] In some embodiments of the invention, the biochemically inert compound comprises a biotinylated hydrazide. In specific embodiments, the biotinylated hydrazide has the chemical structure:

(III) wherein n is 1 ,2 ,or 3.

[0009] In some embodiments, the surface-modified porcine pancreatic islet cells have the structure:

(IV)

wherein n is 1, 2 or 3.

[0010] In some aspects of the invention, the biochemically inert compound comprises biotinylated-polyethylene glycol imide. In particular aspects of the invention, the biotinylated-polyethylene glycol imide comprises the chemical structure:

(V) wherein n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1.

[0011] In some embodiments, the surface-modified porcine pancreatic islet cells have the structure:

glycol imide

[0012] In some embodiments, the surface-modified porcine pancreatic islet cells are covalently attached to at least two of structures I, III, or V.

[0013] Particular aspects of the invention relate to a method for preparing surface- modified porcine pancreatic islet cells, wherein a cell surface functional group is covalently attached to one or more biochemically inert compounds that provide a barrier against host immune response. In some embodiments, the method comprises reacting porcine islet surface primary amines with a biotinylated-N-hydroxysuccinimide ester reagent. In some embodimennts, the biotinylated-N-hydroxysuccinimide ester reagent has the structure:

wherein n may be 0, 1 , 2 or 3. In some embodiments, the modified pancreatic islet cells have a biocompatible immunoisolation barrier.

[0014] In some embodiments, a method for preparing surface-modified porcine pancreatic islet cells comprises oxidizing islet surface sialic acid residues with sodium metaperiodate, and reacting aldehydes and ketones formed during oxidation with a biotinylated hydrazide reagent, In some embodiments, the biotinylated hydrazide reagent has the structure:

(VIII) wherein n may be 0, 1, 2 or 3. In some embodiments, the modified pancreatic islet cells have a biocompatible immunoisolation barrier.

[0015] In particular embodiments, a method for preparing surface-modified porcine pancreatic islet cells comprises reducing islet surface disulfide bridges with tris-(2- carboxyethyl)-phosphine to free thiols, and reacting the islet surface thiols with a biotinylated-polyethylene glycol maleimide reagent. In specific embodiments, the biotinylated-polyethylene glycol maleimide reagent has the structure:

[0016] In certain aspects of the invention, the modified pancreatic islet cells have a biocompatible immunoisolating barrier.

[0017] In some embodiments of the invention, a method of treating diabetes comprises administering a composition comprising the surface-modified porcine pancreatic islet cells to a patient. In particular embodiments, the patient is a diabetic patient. The diabetic patient may be a Type I or a Type II diabetic. In other embodiments, the patient suffers from prediabetes. In particular embodiments, a method of treating diabetes comprises administering the composition through a catheter into the portal vein of the liver. In particular embodiments, one or more anti- inflammatory, immunosuppressive, anticoagulant, and/or antihyperglycemic agents are administered at the time of islet transplant. In specific embodiments, the anti-inflammatory agent may be a human interleukin-1 antagonist or a Tumor Necrosis Factor (TNF) antagonist. In particular embodiments, the antihyperglycemic agent is a dipeptidyl peptidase-4 inhibitor. In further embodiments, the immunosuppressive agent is thymoglobulin. In yet further embodiments, the anticoagulant is heparin. In other embodiments, human interleukin-1 agonist is anakinra. In other embodiments, TNF antagonist is etanercept. In further embodiments, the dipeptidyl peptidase-4 inhibitor is sitagliptin. In some embodiments, the surface-modified porcine pancreatic islet cells are administered to the patient multiple times. [0018] Further embodiments provided herein are based upon surface-modification of porcine pancreatic islet cells with biologically-active compounds. In some embodiments, a combination of one or more biologically-active compounds are covalently attached to porcine pancreatic islet cells. In further embodiments, a combination of one or more biochemically inert compounds and one or more biologically-active compounds are covalently attached to porcine pancreatic islet cells. In some embodiments, the biologically active compound is thrombomodulin. In other embodiments, the biologically-active compound is urokinase. In further embodiments, the biologically-active compound is heparin. In yet further embodiments, the biologically-active compound is a peptide that inhibits phagocytic clearance of nanoparticles. In a specific embodiment, the peptide is derived from the membrane protein CD47.

[0019] In further embodiments, compositions comprising surface-modified human, bovine, or other mammalian pancreatic islet cells are described. Mammalian is defined as belonging or pertaining to an animal of the class Mammalia. In a first embodiment, a cell surface functional group is covalently attached to one or more biochemically inert compounds that provide a barrier against host immune response. In a further embodiment, an amino acid residue provides the cell surface functional group. In some embodiments the one or more biochemically inert compounds is an organic long chain polymer. In particular embodiments, the long chain polymer is a 1-5 kDa poly-ethylene glycol.

[0020] In other aspects of the invention, the surface-modified human, bovine, or other mammalian pancreatic islet cells have the structure:

(X) wherein n is 1 ,2 ,or 3.

[0021] In some embodiments, the surface-modified human, bovine, or other mammalian pancreatic islet cells have the structure:

(XI) wherein n is 1 ,2 ,or 3.

[0022] In some embodiments, the surface-modified human, bovine, or other mammalian pancreatic islet cells have the structure:

(XII) wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1.

[0023] In some embodiments, the surface-modified human, bovine, or other mammalian pancreatic islet cells are covalently attached to at least two of structures I, III, or V.

[0024] Further embodiments provided herein are based upon surface-modification of human, bovine, or other mammalian pancreatic islet cells with biologically-active compounds. In some embodiments, a combination of one or more biologically-active compounds are covalently attached to human, bovine, or other mammalian pancreatic islet cells. In further embodiments, a combination of one or more biochemically inert compounds and one or more biologically-active compounds are covalently attached to human, bovine, or other mammalian pancreatic islet cells. In some embodiments, the biologically active compound is thrombomodulin. In other embodiments, the biologically-active compound is urokinase. In further embodiments, the biologically-active compound is heparin. In yet further embodiments, the biologically-active compound is a peptide that inhibits phagocytic clearance of nanoparticles. In a specific embodiment, the peptide is derived from the membrane protein CD47. In some embodiments of the invention, islet cells are acquired from a patient. In further embodiments the acquired islet cells are surface-modified by any of the above-mentioned methods, and the autologous cells are re-administered to the patient. In other embodiments, xenologous cells are acquired from a subject, surface-modified, and administered to a patient. In further embodiments of the invention, administration of surface- modified islet cells may be used to treat diseases of the pancreas, including but not limited to, acute pancreatitis, chronic pancreatitis, Type I diabetes, Type II diabetes, exocrine pancreatic insufficiency, cystic fibrosis, or pancreatic pseudocysts.

[0025] "Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, the islet cell compositions of the present invention may be administered to a subject for the purpose of treating or preventing diabetes in a patient who has been identified as being at risk for developing diabetes. The terms "therapeutic benefit," "therapeutically effective," or "effective amount" refer to the promotion or enhancement of the well-being of a subject. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

[0026] "Prevention" and "preventing" are used according to their ordinary and plain meaning. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of preventing or delaying the onset of a disease or health-related condition. For example, one embodiment includes administering the islet cells compositions of the present invention to a subject at risk of developing diabetes for the purpose of preventing diabetes.

[0027] The terms "inhibiting," "reducing," "treating," or any variation of these terms, includes any measurable decrease or complete inhibition to achieve a desired result. Similarly, the term "effective" means adequate to accomplish a desired, expected, or intended result.

[0028] The claims described herein provide a summary of the invention, in addition to what is set forth in the Examples and elsewhere in the application. As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more. [0029] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0030] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0032] FIG. 1 (A-E) - Viability of Human and Mouse Islets. (A) Representative human and mouse islets shown for the control, ACYL, OX, and RED chemical treatments. Hochest 33342 stained all nuclei blue, and propidium iodide stained nuclei of necrotic cells red. (B) Human islet (ACYL) stained for Hochest 33342 and PI overlayed with SA-DyLight 488 indicating location of surface modification. (C) Graph showing the mean viability (±SD) of mouse islets 48 hours after surface modification. (D) Bar graph displaying the mean viability (±SD) of human islets 48 hours after surface modification.

[0033] FIG. 2 (A-C) - Insulin Secretion of Human and Mouse Islets - (A) Graph displaying the mean stimulation index (±SD), calculated as the amount of insulin secreted under high glucose stimulation divided by the insulin secreted under low glucose, for three treatments and control for mouse islets. (B) Graph displaying the mean stimulation index (±SD) for three treatments and control for human islets. (C) Graph displaying the mean stimulation index (±SD) for three treatments and control for pig islets.

[0034] FIG. 3 (A-C) - Fluorescent Quantification of Mouse Islets - (A) Mean fluorescent intensities (MFI) of SA-DyLight 488 (green) labeled mouse islets for three different biotinylation protocols on days 0, 1 , 2, and 7. Asterisks indicate a significant difference between groups as determined by ANOVA, *p<0.05 compared to time-matched control unless indicated otherwise by bars. (B) Fluorescent images of representative islets for each treatment at each time point. (C) Linear fit (slope) of mean surface modification over time for each treatment. [0035] FIG. 4 (A-C) - Fluorescent Quantification of Human Islets -- (A) Mean fluorescent intensities (MFI) of SA-DyLight 488 (green) labeled human islets for three different biotinylation protocols on days 0, 1 , 2, and 7. Asterisks indicate a significant difference between groups as determined by ANOVA, *p<0.05 compared to time-matched control unless indicated otherwise by bars. (B) Fluorescent images of representative islets for each treatment at each time point. (C) Linear fit (slope) of mean surface modification over time for each treatment.

[0036] FIG. 5 (A-C) - Fluorescent Quantification of Porcine Islets ~ (A) Mean fluorescent intensities (MFI) of SA-DyLight 488 (green) labeled pig islets for three different biotinylation protocols on days 0, 1 , 2, and 7. Asterisks indicate a significant difference between groups as determined by ANOVA, *p<0.05 compared to time-matched control unless indicated otherwise by bars. (B) Fluorescent images of representative islets for each treatment at each time point. (C) Linear fit (slope) of mean surface modification over time for each treatment.

[0037] FIG. 6 (A-B) - Species Comparison. Fluorescence intensity at Oh for mouse, human, and porcine islets modified by the ACYL, OX, and RED methods at Oh (top) and over 7 days (bottom).

[0038] FIG. 7 - Uniformity of Human Islets. Flattened representations from confocal 3D images of human islets modified by the ACYL (A), OX (B), and RED (C) techniques on day 2. 3D videos are available in supplementary material. [0039] FIG. 8 — Chemical modification strategies: Top (OX) Conjugation of biotin

(·) via hydrazone bond formation between biotin hydrazide and aldehydes generated through mild sodium metaperiodate (NaI0₄) oxidation of sialic acid residues. Middle (ACYL) Islet biotinylation (·) using NHS ester functionalized reagents to acylate free amines. Bottom (RED) Biotinylated (·) maleimide tethered to islet surface by reaction with free sulfhydryls produced by TCEP reduction of disulfide bonds [0040] FIG. 9 (A-C) - Peptide Conjugation to Islets. (A) Islets are reacted with NHS-phosphine followed by reaction with conjugating peptide. (B) Fluorescent confocal image of modified islet stained with streptavidin-488 to reveal biotinylated human self peptide (Biotin-PEG4-Lys(N₃)-GNYTCEVTELTREGETIIELK, SEQ ID NO: 1). (C) Fluorescent confocal image of modified islet stained with streptavidin-488 to reveal biotinylated murine self peptide (Biotin-PEG4-Lys(N₃)-GNYTCEVTELTREGETVIELK, SEQ ID NO: 2). Lysine(N₃) is a lysine-derivative wherein the side-chain amine is replaced by an azide (-N₃) functional group.

[0041] FIG. 10 (A-B) - Fluorescence Imaging of Sequential Reactions. (A) Fluorescent islet image after sequential maleimide/NHS reactions showing Alexa-Fluor 594 conjugation using maleimide chemistry. (B). Fluorescent image of the same islet showing Alexa-Fluor 405 conjugation via NHS chemistry after maleimide chemistry

[0042] FIG. 11 - Biologically-Inert and Biologically- Active Compound Conjugation to Islets. A multi-step procedure for conjugation of both biologically-active and biologically-inert molecules to islets is illustrated.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. Islet Modification

[0043] Surface modification with a combination of passive barriers and active protective agents improves the effectiveness and availability of islet transplantation as a treatment for diabetes. Acute immune reactions often depend upon recognition of surface antigens, and thus the creation of a biocompatible barrier between the islet and host immune system (immunoisolation) may represent an attractive alternative to systemic immunosuppression (Elliott et al, 2007). Immunoisolation has been primarily focused on macroencapsulation (multiple islets within a capsule) and microencapsulation (individual islets encapsulated) using alginate gels (Elliott et al, 2005; Duvivier-Kali et al , 2001). However these strategies are limited by concomitant increases in islet volume, which can result in hypoxic damage due to poor oxygen and nutrient diffusion, and which may require the use of alternative transplant sites (Lee et al , 2010; Lanza et al , 1996; de Vos et al, 2002). Islet surface modification with a biologically inert, hydrophilic, long-chain polymer - poly-ethylene glycol (PEG) - has emerged as a nanoencapsulation technique that may avoid many of the issues of gel encapsulation while creating a functional nano-scale immunological barrier (Lee et ah , 2010). For example, PEGylation of islet surfaces using N- hydroxysuccimide-functionalized PEG has resulted in long term allogenic graft survival with reduced use of systemic immunosuppressants (Lee et ah , 2006). Thus PEGylation provides an exciting possibility for islet graft protection and long-term survival. [0044] Several approaches to islet PEGylation, have been described. Non-covalent strategies have included the spontaneous incorporation of amphiphilic PEG-lipids into lipid bi-layers (Teramura et ah , 201 1), and the electrostatic interaction of charged PEGs with charged islet surfaces (Wilson et ah , 201 1 ; Veerabadran et ah, 2007; Miura et ah, 2006). The covalent tethering of PEG to islet surface functional groups has been most commonly accomplished by reacting surface proteins on murine islets with N-hydroxysuccimide functionalized PEG (Panza et ah , 2000; Xie et ah, 2005). In another covalent approach, the mild oxidation of carbohydrate moieties has been used to produce aldehydes on the surface of murine islets that were subsequently reacted with hydrazide-functionalized PEGs (Wilson et ah , 2010). [0045] In addition to the installation of passive PEG barriers, these surface modification strategies can also be used to attach molecules that actively protect islets from insult such as thrombomodulin (Ryan et ah , 2001), urokinase (Wilson et ah, 2010), and heparin (Cabric et ah , 2007), to the islet surface in order to provide active protection of the islets from IBMIR. A synthetic stable heparin analog has been recently reported (Nguyen et ah, 2013), and that compound may prove advantageous in the context of islet modification due to its simple structure and the consequential stability and ease of modification.

[0046] A minimal "self peptide has been identified that inhibits phagocytic clearance of nanoparticles (Rodriguez et ah, 2013). This peptide was derived from the membrane protein CD47 which is "marker of self and which impedes phagocytosis. The CD47 extracellular domain is known to interact with CD 172a expressed on phagocytes. Interaction of CD47 and CD 172a has been shown to inhibit mouse macrophage uptake of antibody coated mRBCs as well as human macrophage uptake of both human RBSc and hCD47- coated nanoparticles. It has also been demonstrated that CD47 can inhibit platlet adhesion and activation and neutraphil adhesion (Finley et ah, 2012). [0047] Coating human islets with a CD47 derived peptide protects transplanted islets from macrophage-mediated innate immune response, as has been seen for islets modified with anti-CD154 mAbs (Jung et al., 2012). During the early period of islet transplantation islets are known to elicit IBMIR and infiltration of macrophages and neutrophils is one of the early events during IBMIR (Bennet et al., 2000; Goto et al., 2008). A 21 amino acid long mouse CD47-derived peptide, a 21 amino acid long human CD47-derived peptide, and a control peptide representing a scrambled sequence of amino acids are employed herein as immunoprotective agents for transplanted islets.

[0048] Islet modification studies disclosed herein focus on employing a 'toolbox' of chemical protocols for the production of robustly protected islets fitted with multiple reagents for passive nanoencapsulation and for active protection. With the chemical methods well characterized, the effect of several concurrent orthogonal chemical modifiers on surface coverage, tailored stability, and innovative active protection is described. The concurrent orthogonal chemical modifiers include a CD47-derived 'self peptide, a stable synthetic heparin analog, and a passive PEG coating. The resulting islets' enhanced viability in transplantation models are presented. [0049] .Most islet modification studies have reported the development of chemical methods using rodent islets as a model. Unfortunately, a fundamental understanding of islet surface chemistry, the dynamics of modified islet surfaces, and interspecies differences in these critical properties is lacking. Accordingly, the examples below provide an analysis of islet surface chemistry, directly comparing murine, porcine, and human islets. The efficiency, uniformity, and stability of covalent islet surface modification (biotinylation) using three orthogonal surface modification chemistries was analyzed using fluorescent probes that provided the advantage of high sensitivity with low background (Lichtman et al, 2005), allowing the inventors to readily quantitate the efficiency of the reactions and to approximate the percent surface covered for each islet (via confocal fluorescence microscopy (Teramura et a/. ,201 1 ; Wilson et a/. ,201 1). In addition to the characterization of the modifications, the modified islets were thoroughly analyzed as to their viability and functionality

[0050] As is shown below, three orthogonal covalent islet modification techniques were compared across three species (human, porcine, and murine), using multiple measures to determine effectiveness. All three conjugation chemistries were found to be well tolerated, and the overall efficiency, gross uniformity, and stability of each modification were evaluated in the context of human, porcine, and murine islet surfaces. [0051] Cellular therapy may also be useful to treat conditions like Type 2 diabetes and prediabetes (Prentki and Nolan, 2006; Kahn, 2013).

IV. Examples

[0052] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Quantitative Comparison of Surface Modification Chemistries in Mouse,

Porcine, and Human Islets

[0053] Human Islet isolation — Three human research grade pancreata were obtained through two local organ procurement agencies (LifeGift, Fort Worth, TX and Southwest Transplant Alliance, Dallas, TX) and were obtained by an experienced procurement team of surgeons belonging to the islet isolation team at the Baylor University Medical Center (Matsumoto 201 1). Islet isolation was performed using the modified Ricordi method (Takita 2010; Ricordi 1989). Liberase MTF (Roche Diagnostics GmbH, Penzber, Germany) was used as the collagenase, and an iodixanol based continuous density gradient was used for purification. Islet yield and purity was assessed with dithizone staining (Sigma Chemical Co., St. Louis, MO) (2mg/mL). Human islets used were handpicked to greater than 95% purity. Islets were cultured with human islet culture media (CMRL-based media containing IGF, Niacinamide, and 10% human serum albumin) at 37°C and 5% C0₂ for 24h followed by 22°C and 5% C0_2.

[0054] Mouse islet isolation — Islets were isolated from male inbred B6 mice (C57BL/6N Inbred Mice, Harlan Labs). Collagenase type V (Sigma, C9263) (2mg/mL) was injected through the common bile duct into the pancreas (Matsuoka 2010; Itoh 2012) and the pancreata were digested by incubation at 37°C for 28 minutes. After washing, the islets were purified with a discontinuous Ficoll based gradient (1.085 g/mL, 1.077g/mL, and Cap of DMEM) and centrifugation at 1000 rpm for 10 minutes. Purified islets were further purified in a 65mm dish in DMEM by removing acinar cells to a purity of greater than 95%. Islets were cultured in mouse islet culture media (DMEM supplemented with Kanamycin and 10% fetal calf serum) at 37°C and 5% C0₂.

[0055] Porcine islet isolation ~ Porcine islets were isolated as originally described by Brandhorst et al, 1999. Following exsanguinations of pigs, the pancreas was dissected and distended intraductally with University of Wisconsin (UW) solution containing 0.15%o (w/v) Liberase PI (Roche Biochemical, Basel, Switzerland). Further digestion and collection of dissociated tissue was performed at 28 to 32°C using the automated method (Ricordi 1990). Liberated islets were purified from non-islet tissue by using continuous UW/OptiPrep (Axis- Shield, Dundee, UK) density gradients on a COBE 2991 cell processor. Subsequently, the islets were cultured free-floating in Medium 199 (Sigma, St Louis, MO, USA) supplemented with 10% donor pig serum and ciprofloxacin for 48h. The purity of the isolated islets was assessed by the percentage of dithizone -positive cells and islets used in this study were 80%> pure. Assessment of viability by staining fluorescein diacetate and propidium iodide showed 98%o live cells.

[0056] Surface modification ~ Human islets were cultured 2-7 days to increase islet purity and reduce the proportion of islets damaged by the isolation process. Mouse and porcine islets were used after overnight culture.

[0057] ACYL ~ Acylation of primary amines with N-hydroxysuccinimide esters (NHS) affords stable amide bonds (FIG. 8). For surface modification using the ACYL method, 200-300 islets were handpicked and placed in a 1 .8mL conical centrifuge tube. In order to remove contaminating amine products in culture media islets were washed three times with DPBSG (1 ImM D-glucose) by adding lmL DPBSG, letting islets settle then using a pipette tip attached to a vacuum line to remove the supernatant to 20μΙ_^. The washed islets were reconstituted in DPBSG, and then 500 of ImM sulfo-NHS-LC-biotin (0% DMSO) was added immediately. The mixture was reacted for 1 hour at room temperature with occasional agitation, after which the islets were washed three times with DPBSG and then maintained in culture in species appropriate media.

[0058] OX ~ The reaction of hydrazide reagents with carbonyls (aldehydes and ketones; FIG. 8) forms hydrazone linkages. The OX method of surface modification was accomplished by replacing media with glucose-free DPBS followed by treatment of the islets (200-300) with a mild oxidant (500 μί, ImM NaI0₄ in DPBS) for 15 minutes at room temperature protected from the light, in order to convert vicinal diols on glycoproteins and/or glycolipids to aldehydes. After oxidation, islets were washed three times with DPBSG to limit hypoglycemic stress. Finally, hydrazide-LC-biotin (Pierce/Thermo) was added to the activated islets (500 ΐ. of ImM hydrazide-LC-biotin in DPBSG containing 0.5% DMSO. After incubation for 1 hour at room temperature with occasional agitation, the modified islets were washed with DPBSG and cultured.

[0059] RED ~ Maleimides react with free sulfhydryls to form stable thioether bonds (FIG. 8). The RED method was performed by treating 200-300 islets, prewashed with DPBSG as described above, with the reducing agent TCEP (500 μΤ of a 5mM solution in DPBSG) for 15 minutes at room temperature in order to convert disulfide bridges to free thiol groups. To minimize re-oxidation, the reduced islets were not washed to remove the excess TCEP. Instead, all but 25 μΐ, of the TCEP-containing buffer was removed, followed by the immediate addition of maleimide-PEGn-biotin (500 μΐ, of ImM solution in DPBSG including 0.5% DMSO). The mixture was allowed to react for 1 hour at room temperature with occasional agitation. After one hour, the islets were washed with DPBSG and cultured.

[0060] .CD-47-derived minimal "self peptide -Human mini-CD47 (S I ; Biotin- PEG4-Lys(N₃)-GNYTCEVTELTREGETIIELK, SEQ ID NO: 1) and mouse mini-CD47 (S2; Biotin-PEG4-Lys(N₃)-GNYTCEVTELSREGKTVIELK, SEQ ID NO: 2) peptides were designed and synthesized. Each peptide is fitted with azido-lysine and biotin residues, providing two potential routes for islet conjugation - through the Staudinger ligation (van Berkel et ai, 201 1 ; Stabler et al., 2007) or through avidin-bridging subsequent to islet biotinylation. Conjugation experiments used the Staudinger ligation to attach the peptides to islets that have been acylated with commercially-available linkers. 3.3 mM of azide- modified peptide was dissolved in 200 μΐ_^ DPBS. 2.3 mg of NHS-phosphine was dissolved in 500 μΕ (50% DMSO) to yield a 10 mM solution. Islets were washed 3 times with DPBSG to remove any growth medium. 50 μΕ of 10 mM NHS-Phosphine was added to islets in 450 μΐ, DPBSG. The resulting mixture was incubated at room temperature for 1 hour with gentle agitation. The islets were then rinsed 3 times with 500 μΕ of DPBSG. 350 μΕ of DPBSG was added to the islets followed by incubation at 37 °C for 1 hour with gentle agitation. The islets were then rinsed 3 times with DPBSG. The islets were stained with streptavidin-488 to reveal the attached biotinylated peptide. The peptide-conjugating process is illustrated in FIG. 9A. Fluorescent confocal images of peptide-conjugated islets are illustrated in FIGS. 9B and 9C.

[0061] Stable synthetic heparin mimic - The oligomeric heparin mimic includes a terminal thiol residue that can be used for conjugation. The terminal thiol was reacted with a bis-maleimide crosslinker, and subsequently attached to reduced thiols revealed on the islet surface upon treatment with TCEP. The functionality of the heparin mimic was determined by measuring the production of activated protein C and also via a clotting time assay (Stebler et al, 2007).

[0062] General Sequential Modification Reactions - Solutions employed in sequential islet modification reactions include: 100 mM NaI0₄ (10.7mg Sodium meta— periodate in 500 PBS); 100 mM TCEP (41.3 mg TCEP in 500 xh PBS); 10 mM NHS reagent (in DPBSG, PBS or DMSO); 10 mM hydrazide reagent (in DPBS with no glucose, or DMSO); 10 mM maleimide reagent (in DPBSG, PGS or DMSO). NHS Reaction - Add 25 of 10 mM NHS reagent to -100 islets in 225 μΤ DPBSG. Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG (or DPBS-no glucose if hydrazide is next reaction).

[0063] Hydrazide Reaction - If hydrazide is first reaction, rinse -100 islets 3 times with DPBS (no glucose). Add 3 μΐ, of 100 mM NaI0₄ to -100 islets in 247 μΐ, of DPBS (no glucose). Incubate at room temperature for 15 minutes. Rinse 3 times with DPBSG. Resuspend in 225 μΐ, DPBSG and add 25 μΐ, 10 mM hydrazide reagent. Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG.

[0064] Maleimide Reaction - Add 13 μΤ of TCEP to -100 islets in 237 μΐ, of DPBSG. Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG. Resuspend in 225 μΤ DPBSG and add 25 μΐ_^ 10 mM maleimide reagent. Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG (or DPBS- no glucose if hydrazide is next reaction).

[0065] Sequential Dye Labeling Reactions - Solutions employed in sequential islet modification reactions include: 100 mM NaI0₄ (10.7mg Sodium meta— periodate in 500 μΐ, PBS); 100 mM TCEP (41.3 mg TCEP in 500 μΕ PBS); 10 mM NHS-Alexa-Fluor 405 (1 mg in 97 μΐ, DMSO); 10 mM hydrazide-Alexa-Fluor 488 (1 mg in 175 μΕ DMSO); 10 mM maleimide-Alexa-Fluor 594 (1 mg in 1 10 μΕ DMSO). [0066] NHS reaction - Add 25 μΐ, of Alexa-Fluor 405 ( 10 mM working solution) to -100 islets in 225 μΐ_^ of DPBSG. Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG (or DPBS-no glucose if hydrazide is next reaction).

[0067] Hydrazide reaction - If hydrazide is first reaction, rinse -100 islets 3 times with DPBS (no glucose). Add 3 μΐ, of 100 mM NaI0₄ to -100 islets in 247 \iL of DPBS (no glucose). . Incubate at room temperature for 15 minutes. Rinse 3 times with DPBSG. Resuspend in 225 μΤ of DPBSG and add 25 Alexa Fluor 488 hydrazide (10 mM working solution). Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG. [0068] Maleimide reaction - Add 13 μΐ, of TCEP to -100 islets in 238 μΐ, of

DPBSG. . Incubate at room temperature for 15 minutes. Rinse 3 times with DPBSG. Resuspend in 225 μΤ of DPBSG and add 25 μΐυ Alexa Fluor 594 maleimide (10 mM working solution). Incubate at room temperature for 1 hour with gentle agitation. Rinse 3 times with DPBSG (or DPBS-no glucose if hydrazide is next reaction). [0069] Two or more coupling reactions can be performed in sequence to add multiple reagents, potentially with varying half-lives to islets. Alternatively, two or more of these coupling reactions can be performed in sequence to increase the density of a single modification. As an example, murine islets were modified sequentially using the maleimide reaction (Alexa-Fluor 594 maleimide) followed by the NHS reaction (NHS-Alexa-Fluor 405). The fluorescent images depicted in FIGS. 10A and 10B demonstrate that both the Alexa-Fluor 594 and Alexa-Fluor 405 were successfully attached to murine islets.

[0070] Viability Determination of Hoechst 33342/PI— For in vitro determination of necrosis, islets were incubated with Hoechst 342 (10μg/mL) and propidium iodide (20μg/mL) for 10 minutes at 37°C before imaging via fluorescent microscopy (Itoh 2012). Fluorescent micrographs were merged in Image J (NIH Bethesda, Maryland) and the propidium iodide positive area (necrotic nuclei) was divided by the Hoechst 342 positive area (all nuclei) to provide a calculation of islet viability. At least ten islets per aliquot were used.

[0071] Glucose Stimulated Insulin Secretion -- Islets in each experimental condition were incubated sequentially with low (1.67mM) followed by high (16.7mM) concentrations of glucose solution in Functionality/Viability Medium CMRL1066 (Mediatech, Inc. Manassas, VA) for 1 hour at 37°C (Itoh et al., 2012). Mouse (ultra-sensitive insulin ELISA, Mercodia, Uppsula, Sweden), human (ALPCO Diagnostics, Salem, NH), and porcine (Porcine insulin ELISA, Mercodia, Uppsula, Sweden) insulin levels secreted under low and high glucose conditions were then measured by ELISA. High glucose levels were divided by low glucose levels to obtain the stimulation index (Ryan 2001 ). Experiments were performed with five replicates and 10 islets in each well.

[0072] Fluorescent Quantification -- On days 0, 1 , 2, and 7, aliquots of islets were imaged immediately after staining with streptavidin conjugated DyLight 488 (Thermo SA- Dylight488; 10 g/mL at 37°C for 10 minutes protected from light) to reveal biotin residues. After staining, the islets were washed three times with DPBS, placed on a microscope slide (50μΙ, on a 2cm area outlined with a DA O pen), and imaged with an Olympus DX-72 camera. Images were collected with the following specifications: sensitivity ISO200, exposure time 30.0msec, focus xl OO, filter 515 rrm. The mean fluorescent intensity (MFI) was calculated as the integrated gray pixel value for an islet divided by the area of that islet. Eight and to fifteen islets were measured. Some islets were fixed in formalin before staining; repeated experiments demonstrated that formalin fixation had no effect on streptavidin binding.

[0073] Uniformity Assessment ~ Islets were fluorescently labeled as described above and were visualized with laser scanning confocal microscopy in order to evaluate the uniformity of surface modification. [0074] Statistics ~ Statistical analysis of data sets containing more than two groups was performed by One-way ANOVA with Tukey post-hoc tests. Differences were considered significant when p<0.05.

Results

[0075] Viability and Functionality Determination ~ Exposing islets to chemical modification could be expected to be detrimental to the health of the islets. Accordingly, islet function and viability were tested 48 hours after each conjugation procedure. There was no significant difference in viability of ACYL, OX, or RED treated mouse, pig, or human islets (Fig. 1A-D) as compared to untreated control islets (data analyzed by one-way ANOVA; p>0.05). Notably, any necrosis observed (PI positive area) was not co-localized with areas of surface modification (Fig. I E). In addition to viability, islet function, as reflected by stimulation index, was evaluated (Fig. 2A-C). There was no significant difference in islet potency in any treatment group (human, pig, or mouse, with ACYL, OX, or RED treatments) compared to species-matched control (p>0.05).

[0076] Biotinylation Efficiency and Stability — To compare the extent of biotinylation (modification efficiency) among the different methodologies and between the species specific models, fluorescence was measured on biotin-modified islets after staining with fluorescently-labeled streptavidin (Fig. 3A, B). Mouse islets, which remain the standard model for islet surface conjugation with PEG and other biological molecules (Wilson 2008), were examined first. In this species the ACYL and OX modified islets exhibited the greatest level of surface modification; both treatments were significantly higher than control as well as the RED treated islets (P<0.05) at Oh. The ACYL treated murine islets retained maximal levels of modification through the first three time points (48 hours), with a significant drop in intensity by day 7. The OX treated murine islets lost the covalent label the most quickly, and after 24 hours the detectable biotin was at control levels. Finally, although the RED treated mouse islets began with the lowest levels of surface modification, that chemical modification appeared to be relatively stable over 7 days. Control islets (no biotinylation) consistently exhibited low levels of fluorescence that did not vary over the course of the experiment. An examination of the slope of mean fluorescence vs. time allowed a gross evaluation of the stability of the surface treatments (Fig. 3C). In the murine islet experiments the slope of the OX line reveals a rapid loss of biotin from these islets, while ACYL and RED lines were flatter, suggesting that those treatments afford more stable modifications in the murine model.

[0077] Although rodent islets provide an important experimental model, human islets are used to treat patients with insulin-dependent diabetes and might be expected to exhibit different chemical properties. Accordingly, we repeated the modification experiments using human islets. Immediately after staining, the ACYL and RED treated human islets exhibited similar levels of fluorescence, and were significantly higher than OX treated human islets (Fig. 4A, B, C). The ACYL and RED groups were exceptionally stable and the amount of label did not decrease significantly over the 7 days of measurement. The OX modification, while lower in absolute intensity than ACYL or RED, was also relatively stable, with the only statistically noticeable decrease being between Oh and day 7. As observed for murine islets, untreated human islets exhibited minimal retention of the labeled streptavidin over the course of the experiment. [0078] Looking to the future of islet transplantation, porcine islets provide an attractive alternate source of beta cells, but their use is currently limited by several factors including especially the hyperacute rejection of the xenogenic graft. Porcine islets are structurally and chemically distinct from human and mouse islets, and we therefore explored the surface modification of this potentially important beta cell source. Each chemical treatment (ACYL, OX, and RED) was successful in covalently conjugating biotin to the porcine islet surface (Fig 5A-C), with the ACYL and OX methods affording a higher density of biotin to the surface when compared to the RED method (p<0.05). However, in each case the modifications were rapidly lost in the porcine model, with the ACYL-treated islets appearing especially unstable, returning to control levels at 48h.

[0079] As one clear demonstration the importance of this cross-species study, an analysis of the initial effectiveness of the chemical reactions revealed similar levels of biotinylation for the mouse, porcine and human islets when using the ACYL and OX chemistries (p>0.05; Fig. 6), while the results when using the RED treatment were significantly different between three species (p<0.05; Fig. 6).

[0080] Modification Uniformity ~ Finally, confocal microscopy was used to examine the bulk uniformity of the biotinylation reactions. Three-dimensional models rendered from consecutive confocal images of the ACYL and RED-treated human islets (flattened renderings in Fig. 7A, C) show a relatively uniform surface coating. Surprisingly, inspection of the OX-treated human islets reveals an irregular polygonal net-like pattern (Fig. 7B). These patterns are especially clear in the 3D renderings, which are available as Supplementary Material.

Example 2 - Development of a New Analytical Method and New Surface Modification Method to Optimize Microencapsulation of Human Islets

[0081] Background ~ Immunoisolation of islets by covalent attachment of PEG could protect islets from acute and chronic rejection. Several methods of surface modification have been explored, but evaluating new covalent attachment methods could lead to improved nanoencapsulation. Many studies have evaluated nanoencapsulation of mouse islets, but little has been performed on human islets. Furthermore, no data has been shown on long term retention of attached long-chain moieties. [0082] Methods ~ Islets were isolated from C57BL/6 mice and human donor pancreata. All experiments were performed at 22°C, lh, in DulbuPBSG (l lmM glucose) unless specified otherwise.

[0083] NHS Method: sNHS-LC-biotin reacted with primary amines of islet surface proteins.

[0084] OX Method: Hydrazide-LC-biotin reacted with aledehydes formed from mild oxidation (ImM NaI04, 15min, DPBS) of surface carbohydrates.

[0085] RED Method: Maleimide-PEGl 1-biotin reacted with free sulfhydryls created from the reduction of disulfide bridges of surface proteins (5mM TCEP, 15 min, DPBSG). [0086] Evaluation: PI viability assay, Glucose-stimulated insulin secretion (GSIS) test, PEG quantity (by flluorescent microscopy), PEG uniformity (by confocal microscopy). Stimulation index was calculated as the ratio of insulin released from high-glucose by low- glucose. The statistical significance was determined by one-way ANOVA and Tukey/Kramer post-hoc test. Differences were considered significant when p values were less than 0.05. (* in the figures reveal p<0.05)

[0087] Results ~ Human and mouse islets can be chemically modified without any significant decrease in viability or potency. Surface modification with the OX method slightly increase islet potency in mouse and human islets compared to control. RED and NHS methods in human islets demonstrate similar levels of modification as indicated by quantitative fluorescent microscoopy. The rate of quantitative attrition is much higher in the mouse NHS method compared to the RED treatment. The human islets retain chemically attached molecules much longer than mouse islets, as evidenced by quantitative measurement and uniformity assessment. The RED method on the islet surface showed results comparable to the established NHS method. Confocal microscopy of covalently modified human islets demonstrated dynamic changes over time. As an alternative to the OX, RED and NHS passive-molecule ligation techniques, covalent attachment of molecules that actively protect the islets from insult can be used as a treatment for diabetes. The combined ligation of both passively-protecting and actively-protecting molecules offers added islet protection for diabetes treatment. A scheme for a 3-component treatment is found in FIG. 11. These "triply' protected islets represent a more robust approach for islet protection and diabetes treatment. Alternative routes for each conjugation are readily. [0088] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A composition comprising surface-modified porcine pancreatic islet cells, wherein a cell surface functional group is covalently attached to one or more biochemically inert compounds that provide a barrier against host immune response.

2. The method of claim 1 , wherein an amino acid residue provides the cell surface functional group.

3. The method of claim 1 or 2, wherein the one or more biochemically inert compounds is an organic long chain polymer.

4. The method of claim 3, wherein the long chain polymer is a 1-5 kDa poly-ethylene glycol.

5. The composition of any of claims 1 to 4, wherein the biochemically inert compound is covalently attached to the islets through an amide bond.

6. The composition of any of claims 1 to 5, wherein a lysine residue is covalently attached to the one or more biochemically inert compounds.

7. The composition of claim 1 , wherein the biochemically inert compound is covalently attached to the islets through a hydrazone bond.

8. The composition of any of claims 1 to 2 or 7, wherein an oxidized sialic acid residue is covalently attached to the one or more biochemically inert compound.

9. The composition of claim 1 , wherein the biochemically inert compound is covalently attached to the islets through a thioether bond.

10. The composition of claim 9, wherein the amide bond involves a cysteine residue.

1 1. The composition of any of claims 1 to 9, wherein the biochemically inert compound comprises biotin.

12. The composition of any of claim 1 to 6 or 1 1 , wherein the biochemically inert compound comprises biotinylated acyl.

13. The composition of claim 12, wherein the biotinylated acyl has the chemical structure:

wherein n is 0, 1, 2, or 3.

14. The composition of claim 13, wherein the surface-modified porcine pancreatic islet cells have the structure:

(Π)

wherein n is 1 ,2 ,or 3.

15. The composition of any of claims 1 to 2, 7, 8, or 1 1 wherein the biochemically inert compound comprises a biotinylated hydrazide

16. The composition of claim 15, wherein the biotinylated hydrazide has the chemical structure:

(III) wherein n is 1 ,2 ,or 3.

17. The composition of claim 16, wherein the surface-modified porcine pancreatic islet cells have the structure:

wherein n is 0, 1 , 2, or 3.

18. The composition of any of claims 1 to 5, 9 to 1 1 wherein the biochemically inert compound comprises biotinylated-polyethylene glycol imide.

19. The composition of claim 18, wherein the biotinylated-polyethylene glycol imide comprises the chemical structure:

wherein n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1.

20. The composition of claim 19, wherein the surface-modified porcine pancreatic islet cells have the structure:

wherein n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1.

21. The composition of claim 1, wherein the surface-modified porcine pancreat cells are covalently attached to at least two of structures I, III, or V.

22. A method of preparing the composition of any of claims 2 to 6, 1 1 to 14 or 21 comprising reacting porcine islet surface primary amines with a biotinylated-N- hydroxysuccinimide ester reagent.

23. The method of claim 22, wherein the biotinylated-N-hydroxysuccinimide ester reagent has the structure:

wherein n may be 0, 1, 2, or 3.

24. The method of any of claims 22 or 23, wherein the modified pancreatic islet cells have a biocompatible immunoisolation barrier.

25. A method of preparing the composition of any of claims 1-2, 7-8, 1 1 , 15-17, or 21, comprising oxidizing islet surface sialic acid residues with sodium metaperiodate, and reacting aldehydes and ketones formed during oxidation with a biotinylated hydrazide reagent.

26. The method of claim 25, wherein the biotinylated hydrazide reagent has the structure:

wherein n may be 0, 1 , 2, or 3.

27. The method of any of claims 25 or 26, wherein the modified pancreatic islet cells have a biocompatible immunoisolation barrier.

28. A method of preparing the composition of any of claims 1 to 2, 9 to 1 1 , 18 to 20, or 21 , comprising reducing islet surface disulfide bridges with tris-(2-carboxyethyl)-phosphine to free thiols, and reacting islet surface thiols with a biotinylated-polyethylene glycol maleimide reagent.

29. The method of claim 28, wherein the biotinylated-polyethylene glycol maleimide reagent has the structure:

wherein n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

30. The method of any of claims 28 or 29, wherein the modified pancreatic islet cells have a biocompatible immunoisolating barrier.

31. A method of treating diabetes comprising administering a composition comprising the surface-modified porcine pancreatic islet cells of any of claims 1 to 21 to a diabetic patient.

32. The method of claim 31 , wherein the diabetic patient is a Type I diabetic.

33. The method of claim 31 , wherein the diabetic patient is a Type II diabetic.

34. The method of claim 31 or 32, wherein the composition is administered through a catheter into the portal vein of the liver.

35. The method of any of claims 31 to 34, further comprising administering one or more anti- inflammatory, immunosuppressive, anticoagulant, and/or antihyperglycemic agents at the time of islet transplant.

36. The method of claim 35, wherein the anti-inflammatory agent is a human interleukin- 1 antagonist.

37. The method of claim 35, wherein the anti-inflammatory agent is a Tumor Necrosis Factor (TNF) antagonist.

38. The method of claim 35, wherein the antihyperglycemic agent is a dipeptidyl peptidase-4 inhibitor.

39. The method of claim 35, wherein the immunosuppressive agent is thymoglobulin.

40. The method of claim 35, wherein the anticoagulant is heparin.

41. The method of claim 36, wherein the human interleukin-1 agonist is anakinra.

42. The method of claim 37, wherein the TNF antagonist is etanercept.

43. The method of claim 38, wherein the dipeptidyl peptidase-4 inhibitor is sitagliptin.

44. The method of any of claims 31 to 43, wherein the cells are administered multiple times to the patient.

45. A method of reducing transplanted porcine pancreatic islet graft loss, comprising providing the liver of a subject with any of the surface-modified compositions of claims 1 to 21.

46. A method of treating a patient at risk for diabetes comprising administering a composition comprising the surface-modified porcine pancreatic islet cells of any of claims 1 to 21 to the patient.

47. The method of claim 46, wherein the patient suffers from prediabetes.

48. The method of claim 46, wherein the patient is at risk for Type I diabetes.

49. The method of claim 46 or 47, wherein the patient is at risk for Type II diabetes.