Neutral pressure-sensitive nanoemulsion for localized siRNA delivery

描述(申请人提供)：将小干扰RNA(SiRNA)应用于癌症治疗的最大障碍是将位点特异性siRNA输送到肿瘤部位的可靠方法。病毒载体具有不可接受的毒性水平，阳离子脂质体和聚合物胶束在循环和非特异性递送中受到快速清除的影响。因此，仍然需要长期循环的非病毒载体，能够高效地将siRNA特异性地输送到靶细胞。为了满足这些要求，我们提出了一个中立的 超声波触发的纳米乳剂，可以系统地给药，并以特定部位的方式传递siRNA。纳米乳液由全氟碳纳米液滴组成，包覆有阳离子亲脂肽、辅助脂、聚乙二醇化脂类和磷脂的混合物。在siRNA络合之后，添加乙酰化剂来中和剩余的表面电荷，从而最大限度地减少细胞对络合物的非特异性摄取。液体全氟碳核心可以用聚焦的超声波脉冲蒸发，产生气泡并释放siRNA。额外的声脉冲被用来驱动空化(即气泡振荡)，通过气泡/细胞相互作用暂时增加肿瘤细胞的渗透性，并促进siRNA向癌细胞的输送。使用这种创新的方法，我们绕过了内吞途径，这是siRNA进入细胞质的传统途径。此外，siRNA只被传递到转导焦点区域内的细胞，从而解决了困扰传统非病毒siRNA传递方法的缺乏特异性的问题。在初步研究中，我们已经证实这种方法可以成功地将荧光标记的siRNA传递到培养细胞，从而成为第一个将压敏纳米乳液和超声相结合用于局部siRNA传递的小组。建议的递送方案有可能解决与目前的siRNA递送技术相关的限制，从而使siRNA能够充分发挥其作为癌症治疗试剂的潜力。 与公共健康相关：癌症是美国第二大死因。这项提案中概述的研究将开发局部传递siRNA的技术和方案，用于实体肿瘤的治疗。

DESCRIPTION (provided by applicant): The greatest barrier to the application of small interfering RNA (siRNA) for cancer therapy is a reliable method for site-specific siRNA delivery to the tumor site. Viral vectors have an unacceptable level of toxicity, and cationic liposomes and polymeric micelles suffer from rapid clearance from circulation and non-specific delivery. Thus, there remains a need for long- circulating non-viral carriers that can deliver siRNA efficiently and specifically to targeted cells. To fulfill these requirements, we propose a neutral ultrasound-triggerable nanoemulsion that can be administered systemically and deliver siRNA in a site-specific manner. The nanoemulsion consists of perfluorocarbon nanodroplets coated with a mixture of cationic lipophilic peptides, helper lipids, and PEGylated lipids and phospholipids. After siRNA complexation, an acetylating agent is added to neutralize the residual surface charge, which minimizes non- specific cellular uptake of the complex. The liquid perfluorocarbon core can be vaporized with focused ultrasound pulses, creating bubbles and releasing the siRNA. Additional acoustic pulses are used to drive cavitation (i.e. bubble oscilations), which temporarily increases the permeability of tumor cell through bubble/cell interactions and facilitates the delivery of siRNA.to cancer cells. Using this innovative approach, we bypass the endocytic pathway, which is the conventional pathway for siRNA to gain access to the cell cytoplasm. Furthermore, siRNA is delivered to cells only within the transducer focal zone, thus addressing the lack of specificity that plagues conventional non-viral siRNA delivery methods. In preliminary studies, we have confirmed that this approach can successfully deliver fluorescently-labeled siRNA to cultured cells, thus becoming the first group to combine pressure-sensitive nanoemulsions and ultrasound for localized siRNA delivery. The proposed delivery scheme has potential to resolve the limitations associated with current siRNA delivery techniques, thus allowing siRNA to reach its full potential as an agent for cancer therapy. PUBLIC HEALTH RELEVANCE: Cancer is the second leading cause of death in the United States. The research outlined in this proposal will develop technology and protocols for localized delivery of siRNA for the management of solid tumors.