LIPOSOME-MEDIATED INTRACELLULAR DELIVERY IN VITRO

我们建议开发新的方法来优化细胞毒药物的输送 药物和其他大分子在体外特异性肿瘤细胞，通过使用 以抗体为靶标的脂质体。我们已经计划了一个三管齐下的方法 这涉及到新的脂质体系统的开发和他们的 应用于特定选择的肿瘤细胞。 1.对PH值敏感的融合脂质体：我们计划优化 PH敏感脂质体获得最大细胞质的行为 内吞作用后释放。具体地说，我们将实现这一目标 (A)适当的脂肪酸/磷脂酰乙醇胺配方 (FA/PE)脂质体通过避免六方相而融合而无泄漏 PE的转变；(B)适当pH敏感的新脂质的合成 分子结构。细胞质释放的程度将被跟踪。 通过荧光探针，毒素A片段抑制蛋白质合成 以及病毒DNA的传染性。 2.新的内吞和溶酶体加工检测方法：脂质体将 用作荧光或荧光分子的载体，能够 对低pH或特定水解物的存在作出反应 酵素。为此，我们将使用以下类型的探测器：(A) 显示易于测量的pH诱导光谱的荧光探针 漂移；(B)荧光探头，显示易于测量的pH诱导 光谱漂移；(B)附着在脂质体上的荧光探针 通过pH敏感的共价键形成的配体；(C)附着的荧光探针 通过可被特定的酶、弹性酶或其他蛋白酶裂解的键。 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均适用于 通过脂质体给药，也是对致病原命运的研究 病毒和细菌。 3.靶向特定肿瘤细胞：我们选择了作为模型系统 人(卵巢癌)和小鼠(肝转移)肿瘤细胞系 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。我们计划最大限度地提高细胞毒性 通过使用具有以下特性的靶向脂质体 有效的细胞质输送所需的性质 毒品。

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.