Lipid-based Nanocapsules and Triggered Chemotherapy

该项目的目的和范围细分为三个具体目标，具体如下：具体目标1。放射诱导和靶向化疗（RITCH）。这个概念设想了一种无毒的前药，当静脉内给药时，它将分布在全身。当前药经受局部电磁辐射时，它将在肿瘤部位经历化学转化成细胞毒性化合物。我们已经使用了作为原型RITCH化合物的疏水膜探针碘萘基叠氮化物（INA），它在光照射后经历了与膜蛋白的跨膜部分的共价反应。我们已经研究了RITCH化合物在体外和体内的反应性，使用各种触发模式，包括光，声空化和X-射线辐射。动物研究涉及使用NCI-Frederick的小动物成像设施的药代动力学，生物分布和毒性。我们已经确定了两个新的高能叠氮化物衍生物，响应超声和组织结合。我们优化了超声反应性化合物叠氮化曙红对小鼠肿瘤的递送和保留的方案。我们发现，肿瘤特异性过氧化氢可以诱导曙红-叠氮化物与肿瘤组织结合，因此可以用于将叠氮化合物靶向肿瘤，而无需超声或任何其他能量来源的参与。靶向开发多功能脂质体，成像和药物输送能力我们正在开发基于脂质的纳米颗粒本发明涉及具有靶向和按需药物释放性质的脂质体，用于改善癌症治疗剂的递送。对于光触发的应用，我们设计了脂质体从光聚合的二乙炔磷脂（DC 8，9 PC），基于其独特的分配在脂质体膜。我们的配方还包括可调的水性光敏剂，以促进电磁辐射触发的药物释放。对于生物学应用（局部药物递送），我们使用了不影响细胞活力的可见光源（514 nm激光）。将脂质体/细胞悬浮液预暴露于激光导致多柔比星递送至细胞的效率提高（基于细胞毒性测定）。这些脂质体在小鼠中的生物分布研究表明，肝脏和脾脏摄取，注射后长达三周没有明显的毒性体征。使用鼻咽喉癌模型（KB），我们表明脂质体在肿瘤中积累。通过254 nm（UV）处理在肿瘤组织切片中的离体光触发表明脂质体的破坏和内容物释放到肿瘤中。具体目的3阐明膜活性抗癌设计的β-发夹肽的作用模式。我们发现设计的模型肽在含磷脂酰丝氨酸的脂质体表面折叠，通过形成多聚体孔释放有限大小的包封分子。MD模拟表明，这些肽与Protegrin-1和Alzheimer淀粉样蛋白离子通道具有共同的亚基结构。肽与天然存在的毒性肽共有β折叠形态的事实证明了这种机制的普遍性。因此，我们应用了合成生物学的学科，以了解更多天然存在的有毒肽如何工作。

The purpose and scope of this project is subdivided in three specific aims that are detailed below: Specific Aim 1. Development of Radiation Induced and Targeted Chemotherapy (RITCH). The concept envisions a non-toxic pro-drug that when administered intravenously will distribute throughout the body. When the pro-drug is subjected to localized electromagnetic radiation it will undergo a chemical transformation into a cytotoxic compound at the site of the tumor. We have used as a prototype RITCH compound the hydrophobic membrane probe Iodonaphthyl-azide (INA), which upon light irradiation undergoes a covalent reaction with transmembrane portions of membrane proteins. We have examined the reactivity of RITCH compounds in vitro and in vivo using various modes of triggering that include light, sono-cavitation and X-ray radiation The animal studies involve pharmacokinetics, bio-distribution and toxicity using the small animal imaging facility at NCI-Frederick. We have identified two new energetic azide derivatives that respond to ultrasound and bind to tissues. We optimized the protocol for delivery and retention of the ultrasound reactive compound eosin-azide to tumors in mice. We discovered that tumor specific hydrogen peroxide can induce binding of eosin-azide to tumor tissue and thus can be used to target azido compound to tumors without the involvement of ultrasound or any other source of energy.Specific Aim 2: Develop Multifunctional Liposomes with Targeting, Imaging and Drug Delivery Capabilities We are developing lipid-based nanoparticles (liposomes) bearing targeting and on-demand drug release properties for improved delivery of cancer therapeutics. For light-triggered applications, we have designed liposomes from a photopolymerizable diacetylenic phospholipid (DC8,9PC), based on its unique partitioning in the liposome membrane. Our formulations also include a tunable aqueous photo-sensitizer to promote electromagnetic radiation-triggered drug release. For biological applications (localized drug delivery), we have used a visible light source (514 nm laser) that does not affect cell viability. Pre-exposure of liposome/cell suspensions to the laser results in improved efficiency of doxorubicin delivery to cells (based on cytotoxicity assays). Biodistribution studies of these liposomes in mice indicate uptake by liver and spleen with no apparent signs of toxicity up to three weeks post injection. Using a nasophareangeal carcinoma model (KB), we show that liposomes accumulate in tumors. Ex-vivo phototriggering in tumor tissue sections by 254 nm (UV) treatment indicate disruption of liposomes and release of contents into the tumor. Specific Aim 3 Elucidate mode of action of membrane-active anti-cancer designed beta-hairpin peptides. We found that the designed model peptides fold at the surface of phosphatidyl serine-containing liposomes causing release of encapsulated molecules of limited size by forming multimeric pores.MD simulations indicate that the peptides share a common subunit organization with Protegrin-1 and Alzheimer amyloid ion channels. The fact that the peptides share the beta-sheet morphology in common with naturally occurring toxic peptides lends credence to the universality of this mechanism. Thus we have applied the discipline of synthetic biology in order to learn more how naturally occurring toxic peptides work.