LIPOSOME MEDIATED INTRACELLULAR DELIVERY IN VITRO

我们建议最大限度地利用脂质体作为载体 各种大分子的胞质递送。我们计划通过以下方式实现这一目标 引入新的程序，分为三类。1) 内吞作用后脂质体内容物的细胞质释放：这将是 通过构建pH敏感的脂质体与 暴露在低pH环境中的胞内膜 内吞作用，或ii)在内吞作用后，胞质小体的渗透裂解 高渗介质中的脂质体。2)配基特异性的内化 脂质体：我们将脂质体与表面抗体等偶联 识别特定细胞的蛋白质--表面受体。优势将 服用迅速内化的受体，也服用脂质体 识别不止一个感受器的细胞。3)与质膜融合 膜活性物质诱导：我们将优化条件 聚乙二醇、生育酚、PVA、DMSO等融合化合物诱导融合， 同时将它们的细胞毒性作用降至最低。细胞质内释放 脂质体含量之后将有特别选择的荧光探针 (钙黄绿素和荧光葡聚糖)和胶体金。 这些显微探针通常定位在细胞表面 或者在次级溶酶体中。它们的细胞质定位将被用于 作为优化拟议程序的指南。交付的是 功能完整的大分子将随后进行细胞毒性测试 使用甲氨蝶呤-γ-天冬氨酸和白喉毒素 用病毒(SV40)DNA进行感染性分析。这些 生物探针也将作为细胞的有用模型 A)非故意的药理制剂，b)酶和 蛋白质和c)外源基因和其他重组DNA。

We propose to maximize the utility of liposomes as a carrier for the cytoplasmic delivery of various macromolecules. We plan to achieve this by the introduction of new procedures which fall into three categories. 1) Cytoplasmic release of liposome contents after endocytosis: This will be enhanced by constructing pH-sensitive liposomes that fuse with the endocytic membranes when exposed to the low pH environment following endocytosis, or ii) by osmotic lysis of pinosomes after endocytosis of liposomes in hyperosmotic media. 2) Internalization of ligand-specific liposomes: We will conjugate to the liposome surface antibodies and other proteins which recognize specific cells-surface receptors. Advantage will be taken of receptors that are internalized rapidly, and also of liposomes that recognize more than one receptor. 3) Fusion with the plasma membranes induced by membrane-active compounds: We will optimize the conditions for fusion induced by PEG, tocopherol, PVA, DMSO and other fusogenic compounds, while minimizing their cytotoxic effects. The cytoplasmic release of liposome contents will be followed by especially chosen fluorescent probes (calcein and fluorescinated dextran) and encapsulated colloidal gold. These microscopic probes are normally localized either on the cell surface or in the secondary lysosomes. Their cytoplasmic localization will be used as a guide for optimization of the proposed procedures. The delivery of functionally intact macromolecules will be followed by cytotoxicity assays using encapsulated methotrexate-Gamma-aspartate as well as diphtheria toxin A fragment, and by infectivity assays using viral (SV40) DNA. These biological probes will also serve as useful models for cellular incorporation of a) non-permeant pharmacological agents, b) enzymes and proteins and c) foreign genes and other recombinant DNA.