LIPOSOME-MEDIATED INTRACELLULAR DELIVERY IN VITRO

我们建议开发新的方法来优化细胞毒性药物的递送。 药物和其他大分子在体外针对特定肿瘤细胞，通过使用 抗体靶向脂质体。 我们计划采取三管齐下的方法 这涉及新脂质体系统的开发及其 应用于特定选择的肿瘤细胞。 1. pH敏感的融合脂质体：我们计划优化 用于获得最大细胞质的pH敏感性脂质体的行为 内吞作用后释放。 具体而言，我们将实现这一目标 通过：（a）适当的脂肪酸/磷脂酰乙醇胺制剂 (FA/PE）脂质体，其通过避免阻碍相而融合而不泄漏 PE的转变;（B）合成合适的新的pH敏感性脂质 分子结构 将跟踪细胞质释放的程度 通过荧光探针，毒素A片段抑制蛋白质合成 和病毒DNA的传染性。 2.内吞作用和溶酶体加工的新测定法：脂质体将是 用作荧光或荧光分子的载体， 对低pH值或特定水解酶的存在作出反应， 内切酶 为了实现这一点，我们将使用以下类型的探头：（a） 荧光探针显示出容易测量的pH诱导的光谱 移位;（B）显示容易测量pH诱导的荧光探针 光谱位移;（B）附着于脂质体包封的荧光探针 通过pH敏感的共价键连接配体;（c）连接的荧光探针 通过特定酶、弹性蛋白酶或其他蛋白酶可裂解的键。 明智地使用这种分析将提供有价值的定量信息， 内吞作用和噬菌体-溶酶体融合的动力学， 通过脂质体的药物递送，而且对致病性 病毒和细菌。 3.靶向特定的肿瘤细胞：我们选择了模型系统 人（卵巢癌）和鼠（肝转移）肿瘤细胞系 出于与预期的未来治疗应用相关的特定原因 在动物身上，最终在人类身上。 我们计划将细胞毒性最大化 载药脂质体的靶向脂质体， 有效细胞质递送包封的 药

We are proposing to develop new methods for optimal delivery of cytotoxic drugs and other macromolecules to specific tumor cells in vitro, by the use of antibody-targeted liposomes. We have planned a three-pronged approach which involves both the development of new liposome systems and their application to specifically-selected tumor cells. 1. pH-sensitive, fusogenic liposomes: We are planning to optimize the behaviour of pH-sensitive liposomes for obtaining maximal cytoplasmic release following endocytosis. Specifically, we will accomplish this goal by: (a) formulation of appropriate fatty-acid/phosphatidylethanolamine (FA/PE) liposomes that fuse without leakage by avoiding the Hexagonal phase transition of PE; (b) synthesis of new pH-sensitive lipids of appropriate molecular architecture. The extent of cytoplasmic release will be followed by fluorescent probes, inhibition of protein synthesis by toxin A fragments and infectivity of viral DNA. 2. New Assays for endocytosis and lysosomal processing: Liposomes will be used as carriers of fluorescent or fluorogenic molecules capable of responding either to low pH or to the presence of specific hydrolytic enzymes. To achieve this we will use the following types of probes: (a) fluorescent probes that show an easily measurable pH-induced spectral shift; (b) fluorogenic probes that show an easily measurable pH-induced spectral shift; (b) fluorogenic probes attached to liposome-encapsulated ligands via a pH-sensitive covalent bond; (c) fluorogenic probes attached by a bond cleavable by specific enzymes, by elastase, or other proteases. Judicious use of such assays will give valuable quantitative information on the kinetics of endocytosis andphage-lysosome fusion applicable both to drug delivery via liposomes but also to studies on the fate of pathogenic viruses and bacteria. 3. Targeting to specific tumor cells: We have selected as model systems human (ovarian carcinoma) and murine (liver metastatic) tumor cell lines for specific reasons relating to intended future therapeutic applications in animals and ultimately in humans. We plan to maximize the cytotoxicity of drug-loaded liposomes by using targeted liposomes that have the necessary properties for efficient cytoplasmic delivery of the encapsulated drug.