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，用于改善脂质载体在体内特定部位的靶向。先前关于静脉内递送的研究已经证明需要具有延长的循环时间以使载体在靶组织中积累（例如，肿瘤）。实现延长循环时间的主要策略是通过PEG化，但研究表明，这种策略可能会损害递送，这表明开发替代策略将是有利的。我们以前的工作已经证明了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，我们最近的工作表明，胆固醇可以在非常高的浓度下形成纳米结构域，模仿赋予生物膜多种功能的脂质“筏”。所提出的研究利用这种脂质混合行为来产生可用作靶向配体的锚定位点的递送载体区域。这种方法的显著优点是胆固醇纳米结构域似乎保持不含蛋白质，表明位于纳米结构域内的配体在静脉内施用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，并表明，位于纳米结构域内的配体显着提高细胞培养和体内的转染率，相反，当它被排除在纳米结构域的相同的配体。该提案中概述的实验研究了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改善靶向。在异种移植小鼠模型中测试该策略之前，拟议的研究还将优化配体密度、间隔区长度、细胞培养中的摄取和递送。此外，实验将评估两种小分子配体（KYT-0353，茴香酰胺）在体外和体内递送三种不同药物货物（质粒、siRNA和多柔比星）的能力。因此，拟议的研究将彻底调查纳米结构域的使用，以提高车辆的目标，并评估这种方法对不同药物类型的适用性。 公共卫生相关性：需要将药物直接靶向特定部位（例如，肿瘤），即，一颗“神奇子弹”。以前利用特异性结合某些细胞的分子的尝试取得了有限的成功，尽管已经吸取了一些重要的教训。拟议的工作利用脂质的物理性质来创建具有增强的结合能力的递送系统。这种将药物靶向特定部位的全新策略应该适用于多种药物（例如，基因、RNA、抗癌剂）。

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).