Elsevier

Biomaterials

Volume 21, Issue 11, June 2000, Pages 1155-1164
Biomaterials

Treatment of rat pancreatic islets with reactive PEG

https://doi.org/10.1016/S0142-9612(99)00283-5Get rights and content

Abstract

Covalent attachment of polymers to cells and tissues could be used to solve a variety of problems associated with cellular therapies. Insulin-dependent diabetes mellitus is a disease resulting from the autoimmune destruction of the beta cells of the islets of Langerhans in the pancreas. Transplantation of islets into diabetic patients would be an attractive form of treatment, provided that the islets could be protected from the host's immune system in order to prevent graft rejection. If reaction of polyethylene glycol (PEG) segments with the islet surface did not damage function, the immunogenicity and cell binding characteristics of the islet could be altered. To determine if this process damages islets, rat islets have been isolated and treated with protein-reactive PEG-isocyanate (MW 5000) under mild reaction conditions. An assessment of cell viability using a colorimetric mitochrondrial activity assay showed that treatment of the islets with PEG-isocyanate did not reduce cell viability. Insulin release in response to secretagogue challenge was used to evaluate islet function after treatment with the polymer. The insulin response of the PEG-treated islets was not significantly different than untreated islets in a static incubation secretagogue challenge. In addition, PEG-isocyanate-treated islets responded in the same manner as untreated islets in a glucose perifusion assay. Finally, the presence of PEG on the surface of the islets after treatment with the amine-reactive N-hydroxysuccinimide-PEG-biotin (not PEG-isocyanate) was confirmed by indirect fluorescence staining. These results demonstrate the feasibility of treating pancreatic islets with reactive polymeric segments and provide the foundation for further investigation of this novel means of potential immunoisolation.

Introduction

Type I diabetes mellitus, also known as insulin-dependent diabetes mellitus (IDDM) and formerly known as juvenile-onset diabetes, is an autoimmune disease, where the pancreatic islets of Langerhans are destroyed. Insulin is synthesized in the islets; therefore, patients with type I diabetes can no longer produce insulin in response to glucose in their diet. Current therapy for patients with type I diabetes includes insulin injections, dietary constraint, and exercise. However, insulin therapy cannot duplicate a normal physiological response, and diabetics experience an increased incidence of heart disease, nephropathy, and neuropathy [1]. Because of the complications of the disease, other types of treatments are being investigated. One such treatment is transplantation of the entire pancreas or of purified islet preparations; 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 [2]). The desire to transplant islet tissue without the need for immunosuppression has lead to the development of immunoisolation devices where islets might be isolated from the host's immune system by barriers or membranes permeable to low molecular weight species (glucose, insulin) but impermeable to high molecular weight immune proteins.

Encapsulation of individual islets is a commonly explored immunoisolation option. The most popular encapsulation technique involves a coacervation between alginate polyanions and poly-l-lysine polycations, where a polymer lattice results due to the complementary ionic charges [3]. Another encapsulation technique is interfacial precipitation where a polymer, such as poly(HEMA-MMA), dissolved in an organic solvent is precipitated around an islet surface by extraction of the organic solvent in a non-solvent [4]. Islet surfaces have also been isolated with a conformal poly(ethylene glycol) (PEG) coating, a technique in which a polyethylene glycol pre-polymer is photopolymerized around an islet [5]. Many other encapsulation devices can be found in the literature.

In our laboratories, we have an interest in direct covalent modification of the protein surfaces of islets with potentially biocompatible polymers such as PEG [6]. By utilizing linear PEG molecules containing a reactive isocyanate end group, we expect to surface-modify the islet with PEG. We hypothesize that the isocyanate functional group on the PEG molecule will react with amine groups on proteins at the surface of the islet and form urea linkages. Isocyanate end groups react very rapidly with amines under mild conditions [7]. The proteins (and thus lysine residues) that are the target for modification are the extracellular proteins of the islet capsule.

Previously, studies have shown that PEG-diisocyanate can create an effective molecular-scale barrier to cell adhesion when attached to proteinaceous surfaces. PEG-diisocyanate was shown to reduce platelet deposition onto denuded arterial tissue in vitro [8] and in vivo [9]. Denuded placental arteries treated with PEG-diisocyanate in vitro exhibited 87% less platelet deposition than untreated control vessels [8] and a balloon angioplasty damaged rabbit femoral artery treated with PEG-diisocyanate in vivo showed 84% less platelet deposition than the untreated balloon damaged control artery [9]. In addition, treatment of polyethylene, polytetrafluoroethylene, and glass that had been pre-absorbed with fibrinogen and then treated with PEG-diisocyanate showed 96, 97, and 94% less platelet deposition, respectively, than untreated protein absorbed surfaces [10]. Furthermore, platelet deposition onto collagen-coated glass coverslips and pre-clotted Dacron after treatment with PEG-diisocyanate was reduced by 93 and 91%, respectively, versus untreated control surfaces [10]. Since PEG modification of arterial tissue and protein-treated biomaterials can mask thrombogenic protein recognition by platelets, it is an indication that PEG-treated islets may differ from their native precursors in immune cell and protein binding.

The first step in determining the feasibility of PEG-coating islets is to demonstrate that the islets are not intrinsically damaged by treatment with PEG-isocyanate. In the present study, rat islets have been treated with protein-reactive PEG. Islet viability, islet function, and the presence of PEG at the surface of the islet after treatment are investigated.

Section snippets

Islet isolation

Pancreatic islets were isolated from male Sprague-Dawley rats weighing between 300 and 325 g. The animal care and use committee of the University of Pittsburgh approved all procedures. Four to five rats were used per isolation procedure. The animals were anesthetized with 3–5 ml of metafane (Mallinkrodt, Rossville, KS) after which the pancreas of each rat was distended with a 10 ml intraductal injection of 1 mg/ml collagenase (Boerhinger Mannheim, Indianapolis, IN) in Hanks salt solution (Gibco

Islet viability

Fig. 3 shows the results of the MTT assay on the islets in culture 1 day after treatment. There were 51 samples for both untreated and PEG-treated groups of islets. Each sample contained approximately 10 islets. The ratio of the mean absorbance of the PEG-treated samples to the mean absorbance of the untreated islets is displayed on the y-axis. The PEG-isocyanate-treated group remained viable, and the two groups were not found to be significantly different.

Fig. 4 shows the results for rat islets

Discussion

Rat islets have been used in the present study as the model for human islets due to their similarity. Both human and rat islets contain an extracellular matrix membrane capsule [16] and it is the proteins in this capsule that are the target for modification. Cell membrane proteins can also be modified covalently; however, proteins that are a part of the cell membrane have a turnover rate and will be replaced at some time during the cell life with new proteins. These are not the proteins that

Conclusions

Islets were treated with protein-reactive PEG molecules and studies were performed to determine the effects of the treatment. An MTT assay confirmed that the PEG-isocyanate-treated rat islets were still viable and statistical analysis of the data indicated that there was no significant difference between the viability of the PEG-isocyanate treated and untreated groups for 1 and 5 days post-treatment. A small but significant difference in viability was found between the untreated and PEG-treated

Acknowledgements

We are indebted to those people who assisted in many aspects of this project. We would like to thank Ms Darinka Sipula for performing the radioimmunoassays and Mr Sean Alber, Ms Audra Natalio, and Dr Simon Watkins for their assistance with fluorescent staining and confocal microscopy. The NIH Training Grant provided financial support for this project.

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