Lipid-based Nanocapsules and Triggered Chemotherapy

该项目的目的和范围细分为两个具体目标，具体如下：具体目标1：开发具有靶向，成像和药物递送能力的多功能脂质体靶向。我们正在构建靶向过表达叶酸、HER 2或CD 22受体的癌细胞的脂质体。 通过聚乙二醇（PEG）间隔物（folatePEGDSPE）将叶酸与二硬脂酰磷脂酰乙醇胺（DSPE）连接，并以0.1-1%总脂质掺入脂质体中，用于使用人鼻咽癌（KB）细胞靶向叶酸受体。我们已经启动了成像研究，以确定叶酸靶向荧光脂质体在植入KB异种移植物的小鼠中的生物分布。对于HER 2靶向，我们通过其C-末端半胱氨酸将HER 2特异性亲和体（ZHER 2：342-Cys，8.3 kDa）缀合至脂质体，所述C-末端半胱氨酸与缀合至聚乙二醇化磷脂的马来酰亚胺（MaL-PEG-DSPE）反应。将荧光探针掺入这些脂质体中用于生物物理和/或生物化学分析和/或触发释放测定。亲和体缀合产率在20 μ g/mg的蛋白质/脂质比下为70%，每个亲和体平均200个亲和体分子。亲和体缀合对脂质体的流体动力学尺寸分布或稳定性没有任何显著影响。正在使用基于荧光的测定来检查Affisomes与人乳腺癌细胞（SKBR 3）上的HER 2分子的特异性相互作用。对于CD 22靶向，以类似的方式使用具有增加的可溶性表达的CD 22 scFv（HA 22）的新突变体（mut-HA 22）构建脂质体。mut-HA 22-脂质体与表达CD 22的淋巴瘤细胞系（BJAB）的结合显著大于对照脂质体。mut-HA 22-脂质体在370 ℃而不是40 ℃的细胞内定位表明，我们的靶向脂质体通过受体介导的内吞作用通过能量依赖性过程被摄取。目前正在评价装载有阿霉素的Mut-HA 22-脂质体对人B淋巴瘤细胞的选择性细胞毒性。触发释放。我们设计了一类新型的光致脂质体，其由可光聚合的磷脂DC 8，9 PC（1，2-双（二十三碳-10，1,2-二炔酰基）-sn-甘油-3-磷酸胆碱）和DPPC（1，2-二棕榈酰-sn-甘油-3-磷酸胆碱）制备。在25 ℃下暴露于UV（254 nm）辐射0-45分钟导致这些脂质体中DC 8，9 PC的光聚合和包封的荧光染料（钙黄绿素）的释放。钙黄绿素释放的动力学和程度与脂质体中DC 8，9 PC的mol%相关。DPPC/DC 8，9 PC脂质体的光聚合和钙黄绿素释放仅发生在DPPC/DC 8，9 PC脂质体中，而Egg PC/DC 8，9 PC脂质体则没有发生。这些数据与脂质体双层中的可聚合脂质的相分离和堆积是导致脂质体膜中局部缺陷和/或多孔形成的光活化的主要决定因素的概念一致。这一假设得到了分子动力学模拟的支持，表明固相脂质双层中DC 8，9 PC和DPPC的分层。当适当的可调光敏剂染料包含在脂质体的水性隔室中时，内容物的释放通过用包封染料的波长的激光激发来触发。在氧自由基清除剂存在下的释放的抑制表明，释放的机制涉及与光聚合无关的DC 8，9 PC中的化学变化。DC 8，9 PC中的激光介导的化学修饰正在由SAIC-Frederick先进技术计划的分离技术组通过MS，LC，GC和NMR进行分析。DPPC的物理表征：DC 8，9 PC脂质体包括熔融转变温度（Tm）、溶质装载效率、大小和在血清中的稳定性。我们正在进一步开发这些脂质体，因为它们能够触发释放化疗药物（例如阿霉素），并在体外和体内测试它们的功效。我们还在用超顺磁性氧化铁纳米颗粒（SPION）和药物配制脂质体，用于磁共振成像（MRI）和高血压介导的药物释放的潜在应用。具体目标2。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一个原型的疏水膜探针碘萘基叠氮化物（INA），它在光照射下经历了一个共价反应与膜蛋白的跨膜部分。INA的光活化影响许多细胞受体的信号传导能力并导致细胞死亡。由于INA治疗消除了除其他膜蛋白之外的多药转运蛋白功能，因此该方法对于治疗多药耐药肿瘤是有利的。因此，INA靶向膜蛋白的独特作用机制提供了一种新的有效的化疗方法。最近，我们观察到，交替的辐射方式（如声空化和X射线辐射）可以触发RITCH化合物的反应性。我们计划使用各种触发模式（包括光、声空化和X射线辐射）在体外和体内检查RITCH化合物的功效。动物研究涉及使用NCI-Frederick的小动物成像设备进行的药代动力学、生物分布和毒性。我们正在设计新的RITCH化合物，这些化合物将更适合于各种触发模式。此外，我们正在进行新化合物的化学基础研究，以及导致细胞凋亡和细胞死亡的细胞生物学事件。

The purpose and scope of this project is subdivided in two specific aims that are detailed below: Specific Aim 1: Develop Multifunctional Liposomes with Targeting, Imaging and Drug Delivery Capabilities Targeting. We are constructing liposomes that target to cancer cells that over-express either folate, HER2 or CD22 receptors. Folate tethered to distearoylphosphatidylethanolamine (DSPE) via a polyethylene glycol (PEG) spacer (folatePEGDSPE) is incorporated into liposomes at 0.1-1% total lipid for targeting to folate receptors using human nasopharyngeal carcinoma (KB) cells. We have initiated imaging studies to determine biodistribution of folate-targeted fluorescent liposomes in mouse implanted with KB xenografts. For HER2 targeting we have conjugated HER2-specific Affibody (ZHER2:342-Cys, 8.3 kDa) to liposomes via its C-terminal cysteine that reacts with maleimide conjugated to pegylated phospholipid (MaL-PEG-DSPE). Fluorescent probes were incorporated into these affisomes for biophysical and/or biochemical analysis and/or triggered release assays. Affibody conjugation yields were 70% at a protein/lipid ratio of 20 g/mg with an average number of 200 affibody molecules per Affisome. Affibody conjugation did not have any significant effect on the hydrodynamic size distribution or stability of the liposomes. Affisomes are being examined for their specific interactions with HER2 molecules on human breast cancer cells (SKBR3) using fluorescence-based assays. For CD22 targeting, liposomes were constructed in a similar way using a new mutant of the CD22 scFv (HA22) with increased soluble expression (mut-HA22). The binding of mut-HA22-liposomes to CD22-expressing lymphoma cell lines (BJAB) was significantly greater than to control liposomes. Intracellular localization of mut-HA22-liposomes at 370C but not at 40C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis. Mut-HA22-liposomes loaded with doxorubicin are currently being evaluated for their selective cytotoxicity to human B-lymphoma cells. Triggered Release. We have designed a novel class of light-triggerable liposomes prepared from a photo-polymerizable phospholipid DC8,9PC (1,2- bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine). Exposure to UV (254 nm) radiation for 0-45 minutes at 25 0C resulted in photo-polymerization of DC8,9PC in these liposomes and the release of an encapsulated fluorescent dye (calcein). Kinetics and extents of calcein release correlated with mol% of DC8,9PC in the liposomes. Photopolymerization and calcein release occurred only from DPPC/DC8,9PC but not from Egg PC/DC8,9PC liposomes. These data are consistent with the notion that phase separation and packing of polymerizable lipids in the liposome bilayer are major determinants of photo-activation resulting in the formation of local defects and/or lipidic pores in the liposome membrane. This hypothesis is supported by Molecular Dynamics simulations that indicate de-mixing of DC8,9PC and DPPC in the solid phase lipid bilayer. When an appropriate tunable photo-sensitizer dye is included in the aqueous compartment of liposomes, release of contents is triggered by excitation with a laser at the wavelength of the encapsulated dye. Inhibition of release in the presence of oxygen radical scavengers indicate that the mechanism of release involves chemical changes in DC8,9PC unrelated to photo-polymerization. The laser-mediated chemical modifications in DC8,9PC are being analyzed by MS, LC, GC and NMR by the separations technology group at the advanced technology program, SAIC-Frederick. Physical characterization of DPPC: DC8,9PC liposomes include melting transition temperature (Tm), solute loading efficiency, size, and stability in serum. We are further developing these liposomes for their ability to undergo triggered release of chemotherapeutic agents (e.g. doxorubicin) and are testing their efficacy in vitro and in vivo. We are also formulating liposomes with superparamagnetic iron oxide nanoparticles (SPION) and drugs for potential applications in magnetic resonance imaging (MRI) and hyperthermia-mediated drug release. Specific Aim 2. 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 the hydrophobic membrane probe Iodonaphthyl-azide (INA), which upon light irradiation undergoes a covalent reaction with transmembrane portions of membrane proteins. Photo-activation of INA affects the signaling capabilities of numerous cellular receptors and results in cell death. Since the INA treatment eliminates multidrug transporter function in addition to other membrane proteins, this approach is advantageous for treatment of multidrug resistant tumors. The unique mechanism of action INA targeting membrane proteins thus provides a novel and potent chemotherapeutic approach. Recently we observed that alternate radiation modalities (e.g. sono-cavitation and X-ray radiation) can trigger the reactivity of RITCH compounds. We plan to examine efficacy of our 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 are designing new RITCH compounds that will be more amenable to various triggering modes. In addition we are pursuing basic studies on the chemistry of the new compounds as well on cell biological events that lead to apoptosis and cell death.