Exploiting Cholesterol Nanodomains to Improve Targeting

描述（由申请人提供）：本提案中描述的工作代表了一种真正的新策略，用于改善针对体内特定部位的脂基运载工具。先前关于静脉给药的研究表明，为了使载药体在靶组织（如肿瘤）中积累，需要延长循环时间。延长循环时间的主要策略是通过聚乙二醇化，但研究表明，这种策略可能会损害输送，这表明开发替代策略是有利的。我们之前的工作已经证明含有高胆固醇含量的脂质纳米颗粒能够避免在血液中聚集和在肿瘤中积聚。此外，我们最近的工作已经证明，胆固醇可以在非常高的浓度下形成纳米结构域，模拟脂质“筏”，赋予生物膜多种功能。提出的研究利用这种脂质混合行为来创建递送载体的区域，该区域可用作靶向配体的锚定位点。这种方法的一个显著优点是胆固醇纳米结构域似乎不含蛋白质，这表明位于纳米结构域内的配体在静脉给药时不会受到蛋白质污染的损害。我们的初步结果与这一假设是一致的，并且证明了位于纳米结构域内的配体在细胞培养和体内显著提高转染率，相比之下，相同的配体被排除在纳米结构域之外。本实验旨在研究脂质组成对纳米结构域形成的影响，以充分利用脂质混合行为来提高靶向性。拟议的研究还将优化配体密度、间隔长度、细胞培养中的摄取和递送，然后在异种移植小鼠模型中测试该策略。此外，实验将评估两种小分子配体（KYT-0353，氨甲酰胺）在体外和体内递送三种不同药物（质粒，siRNA和阿霉素）的能力。因此，本研究将深入研究利用纳米结构域来提高载体靶向性，并评估该方法对不同药物类型的适用性。

DESCRIPTION (provided by applicant): The work described in this proposal represents a truly new strategy for improving the targeting of lipid-based delivery vehicles to specific sites in vivo. Previous studies on intravenous delivery have demonstrated the need to have prolonged circulation times in order for vehicles to accumulate in target tissues (e.g., tumors). The predominant strategy for achieving prolonged circulation times is via PEGylation, but studies have shown that this strategy can compromise delivery, suggesting that it would be advantageous to develop alternative strategies. Our previous work has demonstrated the ability of lipid nanoparticles containing high cholesterol contents to avoid aggregation in blood and accumulate in tumors. In addition, our most recent work has demonstrated that cholesterol can form nanodomains at very high concentrations, mimicking lipid "rafts" that endow biological membranes with multiple functionalities. The proposed studies exploit this lipid mixing behavior to create regions of the delivery vehicle that can be used as anchoring sites for targeting ligands. A significant advantage of this approach is that cholesterol nanodomains appear to remain free of protein, suggesting that ligands located within nanodomains will not be compromised by protein fouling upon intravenous administration. Our preliminary results are consistent with this hypothesis, and demonstrate that ligands located within the nanodomain dramatically improve transfection rates in cell culture and in vivo, in contrast to the identical ligand when it is excluded from the nanodomain. The experiments outlined in this proposal investigate the effects of lipid composition on nanodomain formation in order to fully exploit lipid mixing behavior to improve targeting. The proposed studies will also optimize ligand density, spacer length, uptake and delivery in cell culture, before testing this strategy in a xenograft mouse model. Furthermore, the experiments will assess the ability of two small molecule ligands (KYT-0353, anisamide) to deliver three different drug cargoes (plasmid, siRNA, and doxorubicin) both in vitro and in vivo. Accordingly, the proposed studies will thoroughly investigate the use of nanodomains to improve vehicle targeting, and assess the applicability of this approach to different drug types. PUBLIC HEALTH RELEVANCE: There is a need to target drugs directly to specific sites (e.g., tumors) by developing delivery systems that are able to bind to these tissues in vivo, i.e., a "magic bullet". Previous attempts at utilizing molecules that bind specifically to certain cells have met with limited success, although some critical lessons have been learned. The proposed work takes advantage of the physical properties of lipids to create a delivery system with enhanced binding capacity. This entirely new strategy for targeting drugs to specific sites should be applicable to a wide variety of drugs (e.g., genes, RNA, anti-cancer agents).