Department of Chemical and Biological Engineering, College of Engineering, Koç University, Istanbul, Turkey.
Tissue Eng Part A. 2010 Jul;16(7):2217-28. doi: 10.1089/ten.TEA.2009.0640.
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 is an attractive form of treatment, provided that the islets could be protected from the host's immune system to prevent graft rejection, and smaller numbers of islets transplanted in smaller volumes could be sufficient to reverse diabetes. Therefore, a need exists to develop islet encapsulation strategies that minimize transplant volume. In this study, we demonstrate the formation of nano-thin, poly(ethylene glycol) (PEG)-rich functional conformal coatings on individual islets via layer-by-layer assembly technique. The surface of the islets is modified with biotin-PEG-N-hydroxysuccinimide (NHS), and the islets are further covered by streptavidin (SA) and biotin-PEG-peptide conjugates using the layer-by-layer method. An insulinotropic ligand, glucagon-like peptide-1 (GLP-1), is conjugated to biotin-PEG-NHS. The insulinotropic effect of GLP-1 is investigated through layer-by-layer encapsulation of islets using the biotin-PEG-GLP-1 conjugate. The effect of islet surface modification using the biotin-PEG-GLP-1 conjugate on insulin secretion in response to glucose challenge is compared via static incubation and dynamic perifusion assays. The results show that islets coated with the functional PEG conjugate are capable of secreting more insulin in response to high glucose levels compared to control islets. Finally, the presence of SA is confirmed by indirect fluorescent staining with SA-Cy3, and the presence of PEG-peptide on the surface of the islets after treatment with biotin-PEG-GLP-1 is confirmed by indirect fluorescent staining with biotin-PEG-fluorescein isothiocyanate (FITC) and separately with an anti-GLP-1 antibody. This work demonstrates the feasibility of treating pancreatic islets with reactive polymeric segments and provides the foundation for a novel means of potential immunoisolation. With this technique, it may be possible to encapsulate and/or modify islets before portal vein transplantation and reduce transplantation volume significantly, and promote islet viability and insulin secretion due to the presence of insulinotropic peptides on the islet surface. Layer-by-layer self-assembly of PEG-GLP-1 offers a unique approach to islet encapsulation to stimulate insulin secretion in response to high glucose levels.
聚合物与细胞和组织的共价附着可用于解决与细胞治疗相关的各种问题。胰岛素依赖型糖尿病是一种疾病，其起因是胰岛β细胞在胰腺中的朗格汉斯胰岛中发生自身免疫性破坏。胰岛移植是一种有吸引力的治疗形式，只要胰岛可以免受宿主免疫系统的影响以防止移植物排斥，并且可以用较小体积的较小数量的胰岛进行移植以逆转糖尿病即可。因此，需要开发最小化移植体积的胰岛封装策略。在这项研究中，我们通过层层组装技术证明了在单个胰岛上形成纳米薄的聚乙二醇（PEG）丰富的功能共形涂层。通过生物素-PEG-N-羟基琥珀酰亚胺（NHS）修饰胰岛表面，并用链霉亲和素（SA）和生物素-PEG-肽缀合物通过层层法进一步覆盖胰岛。将胰岛素促分泌素配体，胰高血糖素样肽-1（GLP-1）与生物素-PEG-NHS 缀合。通过使用生物素-PEG-GLP-1 缀合物进行胰岛的层层包封来研究 GLP-1 的胰岛素促分泌作用。通过静态孵育和动态灌注测定比较了用生物素-PEG-GLP-1 缀合物对胰岛表面修饰对葡萄糖刺激下胰岛素分泌的影响。结果表明，与对照胰岛相比，用功能化 PEG 缀合物包被的胰岛在响应高葡萄糖水平时能够分泌更多的胰岛素。最后，通过间接荧光染色用 SA-Cy3 证实了 SA 的存在，并且通过间接荧光染色用生物素-PEG-异硫氰酸荧光素（FITC）和单独用抗 GLP-1 抗体证实了在用生物素-PEG-GLP-1 处理后胰岛表面上的 PEG-肽的存在。这项工作证明了用反应性聚合物段处理胰腺胰岛的可行性，并为潜在免疫隔离的新方法提供了基础。通过该技术，可以在门静脉移植前对胰岛进行包封和/或修饰，并且由于胰岛表面上存在胰岛素促分泌肽，可以显著减少移植体积，并提高胰岛的活力和胰岛素分泌。PEG-GLP-1 的层层自组装为刺激胰岛对高葡萄糖水平的胰岛素分泌提供了一种独特的胰岛包封方法。
