Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation
Introduction
Type I diabetes mellitus, also known as insulin-dependent diabetes mellitus, is an autoimmune disease wherein pancreatic islets of Langerhans are destroyed. Type I diabetes affects over 15.7 million people in the United States, a number predicted to increase to as many as 25 million by 2010 [1]. Patients with type I diabetes can no longer produce insulin in response to glucose in their diet because insulin is synthesized in the islets. Current therapy for patients with type I diabetes includes insulin injections, dietary constraints, and exercise. However, insulin therapy cannot duplicate a normal physiological response and thus diabetics experience an increased incidence of heart disease, nephropathy, and neuropathy [2]. Severe complications of the disease have prompted other types of treatments to be investigated, including transplantation of the entire pancreas or of purified islet preparations (cell therapy). However, the morbidity of surgery and the chronic immunosuppression that accompanies transplantation must be weighed against the potential benefit of improved glucose metabolism. Usually, this option is not considered unless another transplant is required at the same time (e.g., a simultaneous kidney transplant [3]). The desire to transplant islet tissue without the need for immunosuppression has led to development of immunoisolation devices where islets might be isolated from the host's immune system by barriers or membranes permeable to low molecular weight (MW) species such as glucose and insulin but impermeable to high MW immune proteins such as immunoglobulins M (Ig M) and G (Ig G) as well as other complement cytotoxins.
Although allogenic islet transplantation is clinically effective for type I diabetic patients via immunosuppressive agents, the future for allografts seems unclear because of donor scarcity [4]. As an alternative, xenogeneic pancreatic islets are strong candidates for islet transplantation. In this regard, pigs are an attractive source of islets because they breed rapidly and there exists a long history of porcine insulin use in humans as well as the potential for genetic engineering. Although the potential for infection of recipients with xenogeneic agents and the risk of transmission to the general population, particularly, infection with porcine endogenous retrovirus in human, are major concerns, recent research has shown that natural xenoreactive antibodies can prevent the infection [5], [6]. In addition to it, several immunological obstacles, however, must also be overcome, in particular, the susceptibility of porcine pancreatic islets to destruction by immunological processes and exposure to human blood. It is known that antibodies (or immunoglobulins) and complement system destroy cells via abundant surface antigens on the transplanted cells [7]. These surface antigens are mainly composed of oligosaccharides on glycolipids and glycoproteins (Fig. 1(A)), which are solely responsible for antigen–antibody and complement-mediated reactions [7]. Recent advances in biotechnology suggest that there are some scientific strategies that can prevent the immune response induced by foreign cells or xenogens [8], which include microencapsulation of cells [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22] and surface modifications [23], [24], [25], [26], [27], [28], [29].
Many immunoglobins or antibodies and components of the complement system exist in blood serum. IgG, IgM, IgA, IgD and IgE are the most common immunoglobins [8]. The complement system comprises a group of more than 30 serum and cell surface proteins with MWs in the range between 25 and 750 kDa. Both immunoglobulins and the complement system are responsible for cytotoxicity to transplanted cells and tissues. Islets or tissues can be rejected via action of both antibodies and antibody-activated complement systems [8].
To protect islets from immune-mediated destruction, camouflaging the surface of islets is necessary for immunoisolation and immunoprotection. Two major approaches have been tried so far to prevent immunogenic reactions on the cellular surface. One is microencapsulation of the cells [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22] and the other is surface modification of the cells [23], [24], [25], [26], [27], [28], [29]. The strategy in the former is very similar to those applied in drug delivery. Synthetic poly(vinyl alcohol) [11], [12], poly(lactide-co-glycolide) [13], dimethylaminoethyl methacrylate-methyl methacrylate copolymer [14], natural and biodegradable polymer alginate with or without polylysine [4], [7], [10], [15], [16], [17], [18], [19] and natural agarose [20], [21] have been used for encapsulation of pancreatic islets. Microcapsulation of islets by a polylysine–alginate polymer complex is the most successful example of this technology [9]. The formed semi-permeable membrane permits nutrient flow and oxygen transport but prevents immunogenic reactions. However, pitfalls to microencapsulation include reduced lifespan of the cells due to polymer biodegradation, permeability of the capsules, fragility, limited surface areas, etc. [22].
Poly(ethylene glycol) (PEG) has been successfully used to reduce plasma protein adsorption and platelet adhesion in making blood compatible vessels and devices or surface modification of the blood compatible polymers due to its low interfacial free energy with water, unique solution properties in aqueous solution, high surface mobility, and steric stabilization effects [30], [31].
Surface modification studies of the cells have been given to several cells including red blood cell, white blood cell, islet cell, etc. Most studies have been focused on red blood cells. Modification of the surface of the red cell using PEG originated from the concept that PEG modified proteins and enzymes render them non-immunogenic [32], [33]. Applying the same concept, Jeong and Byun [23] first used a non-immunogenic PEG-cyanuric chloride to modify the surface of red cells and found decreased agglutination and antibody binding after the modification. The morphology of the cells remained intact. Murad et al. [24], [25], Scott and Murad [26] applied the same strategy to anchor PEG-cyanuric chloride onto both red cells and T lymphocytes and likewise found that both morphology and biological function of the cells did not change but their immunogeneicity dramatically dropped. Hortin et al. [27] and Hortin and Huang [28] modified mouse red cells using a 4-arm star-branched PEG succinimidyl propionate (PEG-SPA) combined with albumin and found that the lifespan of the modified cell was almost the same as that of the normal cell. To date there is only one report on islet surface modification and the preliminary results are encouraging [29]. In this study, PEG monoisocyanate was used to modify the rat islet surface and an in vitro glucose release study supported the feasibility of the concept. However, no in vivo long-term immune and complement-mediated cytotoxicity studies were reported. Furthermore, the isocyanate group seems to be an unsuitable candidate for selectively coupling with amino groups on membrane proteins because it is known that isocyanate can rapidly react with water under physiological conditions. In this study, we proposed to use succinimidyl PEG derivatives, including monosuccinimidyl PEG (MSPEG) and disuccinimidyl PEG (DSPEG) plus albumin, to camouflage the surface of the porcine islets for cytoprotection of islet xenotransplantation (Figs. 1(B) and (C)). This exploratory biotechnology may provide a novel route for successful allo- and/or xeno-transplantation in the near future.
The objective of this study was to synthesize and characterize novel succinimidyl PEG derivatives, to use them to modify the surface of porcine islets, and to evaluate the response of the modified cells to in vitro antibody/complement-mediated cytotoxicity in human serum as well as in vitro cell viability. In vitro and in vivo islet functionalities were also evaluated.
Section snippets
Materials
PEG methyl ether (Mn (number average molecular weight)=2000 or 2 kDa and 5000 or 5 kDa), PEG (Mn=3400 or 3 kDa), N-hydroxysuccinimide (NHS) and pyridine were used as received from Aldrich Chemical Co. (Milwaukee, WI) without further purifications. PEG (Mn=6300 or 6 kDa), succinic anhydride (SAn), dicyclohexylcarbodiimide (DCC), diethyl ether, and tetrahydrofuran (THF) were used as received from Acros/Fisher Scientific Inc. (Pittsburgh, PA). Streptozotocin, Hanks’ balanced salt solution, DNAase I,
Characterization of functional PEG derivatives
The FT-IR spectra for PEG3K (3 kDa), SAn, PEG disuccinate (PEGSAn), NHS and DSPEG are shown in Fig. 3. In order to dynamically demonstrate the characteristic peak change, we selectively chose the wavenumber range from 1900 to 1500 cm−1. The spectrum for PEG shows a peak at 1648 cm−1 for methylene bending. The spectrum for SAn shows two strong typical peaks at 1860 and 1784 for anhydride and a peak at 1638 cm−1 for CH2 group. The spectrum for PEGSAn shows peaks at 1731 and 1645 for carbonyl (ester)
Conclusions
We have developed a novel biotechnology to modify the surface of porcine islets by using functional PEG derivatives. All the PEG derivatives used in the study showed a significant in vitro cytoprotection in human serum and in vivo cytoprotection in diabetic SCID mice to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to monosuccinimidyl derivatives. The effects of both MW and concentration of PEG derivatives on cytoprotection were
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