pH- SENSITIVE BIS-DETERGENTS FOR MACROMOLECULAR DELIVERY

描述(申请人提供)：大分子，不能轻易穿透细胞膜，通过内吞作用进入细胞。内体的内容物最终会被传送到富含酶的溶酶体，并很可能会被降解。如果治疗性大分子的药理活性只有当它进入胞浆时才能实现，那么必须在递送系统中建立一种机制，使这些药剂从内体逃逸到胞浆中。随着内切体的成熟，内涵体的pH值下降到5.0。这为设计这种胞质递送策略提供了一个独特的机会。 我们设计了一系列酸敏性脂类，可以很容易地并入脂质体药物载体系统中。一旦进入细胞，遇到内体的酸性环境，对酸敏感的脂类就会分解，释放出有效的洗涤剂。目前正在检验的关键假设是：(1)这些洗涤剂的存在启动了脂质体到胶束的转变，触发了包裹在内噬体内的大分子的灾难性释放，(2)胶束然后诱导内体膜的通透性，促进大分子的胞质转移。然而，如果不在脂质体双层中加入临界浓度的洗涤剂，就不可能检验这些假设。超过这个浓度可以发生从囊泡到胶束的转变，这是无法达到的高水平，仅仅是因为在高洗涤剂浓度下，不能制造脂质体。在目前的研究中，我们建议合成“双去污剂”，即惰性双尾脂，它可以在高浓度下嵌入脂质体双层中，并仅在低pH值下分解为“膜活性”去污剂。 一旦实现，这项研究开发的技术不仅将在基础生物医学研究中具有深远的意义，需要在体外进行大分子的胞浆递送，而且在体内的各种治疗应用中也将具有深远的意义，从递送用于诱导细胞介导的免疫的抗原蛋白到用于基因调节的核酸。

DESCRIPTION (provided by applicant): Macromolecules, being unable to penetrate cell membranes readily, enter the cell via endocytosis. The contents of the endosome are eventually routed to enzyme-rich lysosomes and in all likelihood degraded. If pharmacological activity of a therapeutic macromolecule is realized only when it enters the cytosol, then a mechanism must be built within a delivery system by which these agents escape from the endosome into the cytosol. The endosomal pH drops to as low as 5.0 as the endosome matures. This provides a unique opportunity in devising such a strategy for cytosolic delivery. We have designed a series of acid-sensitive lipids that can be readily incorporated into liposomal drug carrier systems. Upon entering the cell and encountering the acidic environment of the endosome, the acid sensitive lipid breaks down to release potent detergents. Critical hypotheses being tested at this point are:(1) presence of these detergents initiates liposome-to-micelle transition that triggers catastrophic release of entrapped macromolecules within endosomes and (2) micelles then induce permeabilization of endosomal membrane to promote cytosolic transfer of macromolecules. However, it is impossible to test these hypotheses without incorporating a critical concentration of detergent into liposomal bilayers. This concentration, above which vesicle-to-micelle transition can take place is unattainably high simply because at high detergent concentrations, liposomes cannot be made. In the present study, we propose to synthesize "bis-detergents", inert double-tailed lipids that can be embedded at high concentrations in liposomal bilayers and break down only at low pH into "membrane-active" detergents. When realized, the technology developed from this study will have profound implication not only in basic biomedical research entailing cytosolic delivery of macromolecules in vitro but also in a variety of therapeutic applications in vivo, ranging from delivery of antigenic proteins for induction of cell-mediated immunity to nucleic acids for gene regulation.