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.发展辐射诱导和靶向化疗(RITCH)。这一概念设想了一种无毒的亲药物，当静脉给药时，这种药物将在全身分布。当前药物受到局部电磁辐射时，它将在肿瘤部位经历化学转化为细胞毒性化合物。我们使用疏水性膜探针碘-萘叠氮化物(INA)作为Ritch化合物的原型，它在光照射下与膜蛋白的跨膜部分发生共价反应。我们使用各种触发模式，包括光、声空化和X射线辐射，在体外和体内检测了Ritch化合物的反应性。动物研究涉及药代动力学、生物分布和毒性，使用NCI-Frederick的小动物成像设施。我们已经确定了两个新的高能叠氮化物衍生物，它们对超声波有反应，并与组织结合。我们优化了超声反应性化合物曙红叠氮在小鼠肿瘤中的传递和保留方案。我们发现肿瘤特异性过氧化氢可以诱导曙红叠氮与肿瘤组织结合，从而可以在不需要超声或任何其他能源的情况下将叠氮化合物靶向肿瘤。具体目标2：开发具有靶向、成像和药物递送能力的多功能脂质体我们正在开发具有靶向和按需药物释放特性的脂基纳米粒(脂质体)，以改善癌症治疗药物的递送。对于光触发应用，我们设计了由可光聚合的双乙酰磷脂(DC8，9PC)制成的脂质体，基于其在脂质体膜中的独特分配。我们的配方还包括一种可调的水性光敏剂，以促进电磁辐射引发的药物释放。对于生物应用(局部药物输送)，我们使用了不影响细胞活力的可见光光源(514 nm激光)。预先将脂质体/细胞悬浮液暴露在激光下可提高阿霉素对细胞的输送效率(基于细胞毒性分析)。这些脂质体在小鼠体内的生物分布研究表明，在注射后三周内，肝和脾都没有明显的毒性迹象。使用鼻咽癌模型(KB)，我们发现脂质体在肿瘤中积聚。通过254 nm(UV)治疗的肿瘤组织切片的体外光触发表明脂质体被破坏，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-发夹多肽的作用机制。我们发现，所设计的模型多肽折叠在含有磷脂酰丝氨酸的脂质体表面，通过形成多聚体孔来释放有限大小的包裹分子。MD模拟表明，这些多肽与蛋白质-1和阿尔茨海默病淀粉样离子通道具有共同的亚基组织。这些多肽与自然产生的有毒多肽具有相同的β-折叠形态，这一事实证明了这一机制的普遍性。因此，我们应用了合成生物学的学科，以便更多地了解自然产生的有毒多肽是如何起作用的。

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.