Department of Polymer Chemistry and Biomaterials, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, Enschede, The Netherlands.
Biomaterials. 2011 Dec;32(34):9144-53. doi: 10.1016/j.biomaterials.2011.08.036. Epub 2011 Aug 26.
Polymersomes (Ps) based on a biodegradable and biocompatible block copolymer of methoxy poly(ethylene glycol) (mPEG) and poly(D,L-lactide) (PDLLA) in which apeptide sequence, Gly-Phe-Leu-Gly-Phe (GFLGF), was introduced in between the two blocks(mPEG-pep-PDLLA) were developed. The peptide linker is cleavable by the lysosomal enzymecathepsin B (Cath B). Ps containing the peptide linker (Ps(pep)) with an average diameter of about 124 nm were prepared by injecting a THF solution of the block copolymer into DI water. The Ps had a membrane thickness of about 15 nm as determined by transmission electron microscopy (TEM). In order to investigate the enzymatic degradation of the Ps (pep), dynamic light scattering (DLS) measurements of Ps(pep) dispersions with different concentrations of Cath B at pH 5.5 and 7.4 were performed as a function of time. A gradual decrease in kilo counts per second (Kcps) of the Ps (pep) over 7 d was observed after incubation of the Ps (pep) dispersions with 5 units/ml of Cath B at pH 5.5 at 37 °C. The size distribution became also bimodal, indicating that aggregation and precipitation of Ps (pep) occurred by disintegration of the Ps (pep) as a result of cleavage of the peptide. The rate of disintegration of the Ps (pep) was depending on the concentration of Cath Band the pH. No changes by DLS were seen when the dispersions were incubated with the enzyme at pH 7.4. Acridine orange (AO) was encapsulated in Ps (pep)as a model drug and rapid release of AO triggered by Cath B degradation of Ps (pep) was observed at pH 5.5. Anti-epidermal growth factor receptor (anti-EGFR) antibody (abEGFR) was immobilized on the surface of Ps(pep)in order to enhance the cellular uptake of Ps (pep). Fluorescein isothiocyanate labeled dextran (40,000 g/mol) (FD40) was incorporated in the Ps (pep) for the cell study and Ps either without peptide or antibody or without both peptide and antibody were used as negative controls. After 3 d exposure to SKBR3 cells, abEGFR-conjugated Ps (pep) (abEGFR-Ps (pep)) were directly bound to the membrane of the cells and were endocytosed more rapidly as compared to Ps (pep)without abEGFR. Intracellular release of FD40 from Ps (pep) was observed, suggesting that the peptide linker in Ps (pep) was cleaved in the lysosomal compartments of the cells leading to Ps (pep) membrane disruption. An Alexa Fluor(®) 488 labeled fragment of anti-mouse IgG (F(ab')(2)A) was also coupled to Ps (pep). Specific binding of the Ps (pep) coupled IgG (F(ab')(2)A) onto SKBR3 cells treated with primary mouse antibody was observed, whereas no binding was found with SKBR3 cells treated with goat antibody.
基于甲氧基聚乙二醇（mPEG）和聚（D，L-丙交酯）（PDLLA）的可生物降解和生物相容的嵌段共聚物的聚合物囊泡（Ps）被开发出来，其中在两个嵌段（mPEG-pep-PDLLA）之间引入了一个肽序列，甘氨酰-苯丙氨酰-亮氨酰-甘氨酰-苯丙氨酸（GFLGF）。肽接头可被溶酶体酶组织蛋白酶 B（Cath B）切割。通过将嵌段共聚物的四氢呋喃溶液注入去离子水中，制备了含有肽接头（Ps（pep））的平均直径约为 124nm 的 Ps（pep）。通过透射电子显微镜（TEM）测定，Ps 的膜厚度约为 15nm。为了研究 Ps（pep）的酶降解，在 pH5.5 和 7.4 下，用不同浓度的 Cath B 对 Ps（pep）分散体进行了动态光散射（DLS）测量，作为时间的函数。在 37°C 下，将 Ps（pep）分散体与 5 单位/ml 的 Cath B 在 pH5.5 孵育 7 天后，观察到 Ps（pep）的每秒千计数（Kcps）逐渐下降。当分散体在 pH7.4 下与酶孵育时，通过 DLS 没有观察到任何变化。吖啶橙（AO）被包裹在 Ps（pep）中作为模型药物，并且在 pH5.5 下观察到由于 Ps（pep）的肽裂解引发的 AO 的快速释放。为了增强 Ps（pep）的细胞摄取，将抗表皮生长因子受体（anti-EGFR）抗体（abEGFR）固定在 Ps（pep）的表面上。为了细胞研究，将荧光素异硫氰酸酯标记的葡聚糖（40000g/mol）（FD40）掺入 Ps（pep）中，并且 Ps 既没有肽也没有抗体，或者两者都没有肽和抗体用作阴性对照。在将 SKBR3 细胞暴露 3 天后，与 abEGFR 偶联的 Ps（pep）（abEGFR-Ps（pep））直接与细胞的膜结合，并与没有 abEGFR 的 Ps（pep）相比，被更快地内吞。从 Ps（pep）中观察到 FD40 的细胞内释放，表明 Ps（pep）中的肽接头在细胞的溶酶体隔室中被切割，导致 Ps（pep）膜破裂。还将 Alexa Fluor（®）488 标记的抗小鼠 IgG（F（ab'）（2）A）片段偶联到 Ps（pep）上。观察到与用初级小鼠抗体处理的 SKBR3 细胞偶联的 Ps（pep）偶联 IgG（F（ab'）（2）A）的特异性结合，而与用山羊抗体处理的 SKBR3 细胞未发现结合。
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