Lipid-based Nanocapsules and Triggered Chemotherapy

该项目的目的和范围可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。我们正在构建针对过表达叶酸、HER2或CD22受体的癌细胞的脂质体。叶酸通过聚乙二醇（PEG）间隔剂（folatePEGDSPE）连接到二硬脂酰磷脂酰乙醇胺（DSPE），以0.1-1%的总脂质加入脂质体，用于人鼻咽癌（KB）细胞靶向叶酸受体。我们已经启动了影像学研究，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。针对HER2靶向，我们将HER2特异性的粘附体（HER2:342- cys, 8.3 kDa）通过其c端半胱氨酸与偶联聚乙二醇磷脂（MaL-PEG-DSPE）的马来酰亚胺偶联到脂质体上。荧光探针被纳入这些亲和体进行生物物理和/或生化分析和/或触发释放试验。在蛋白/脂质比为20 g/mg，平均编号为&amp；#61566；每个附体有200个附体分子。附着物偶联对脂质体的水动力大小分布和稳定性没有显著影响。目前正在使用基于荧光的检测方法检测亲和体与人乳腺癌细胞（SKBR3）上的HER2分子的特异性相互作用。对于CD22靶向，脂质体以类似的方式构建，使用CD22 scFv （HA22）的新突变体，增加可溶性表达（mut-HA22）。mut- ha22脂质体与表达cd22的淋巴瘤细胞系（BJAB）的结合明显大于与对照脂质体的结合。在370C而不是40C时，mut- ha22脂质体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过程被吸收。目前正在评估装载阿霉素的mut - ha22脂质体对人b淋巴瘤细胞的选择性细胞毒性。触发释放。我们设计了一种新型的光触发脂质体，由光聚合磷脂dc8,9pc（1,2-二（tricosai -10,12-二酰基）-sn-甘油-3-磷酸胆碱）和DPPC（1,2-双棕榈酰基-sn-甘油-3-磷酸胆碱）制备。在25℃的紫外线（254 nm）辐射下暴露0-45分钟，导致DC8、9PC在这些脂质体中光聚合，并释放出包封的荧光染料（钙黄蛋白）。钙黄素释放的动力学和程度与脂质体中DC8、9PC的摩尔百分比相关。仅DPPC/DC8、9PC发生光聚合和钙黄素释放，Egg PC/DC8、9PC脂质体不发生光聚合和钙黄素释放。这些数据与脂质体双层中可聚合脂的相分离和堆积是光活化的主要决定因素，从而导致脂质体膜中局部缺陷和/或脂质孔的形成。这一假设得到了分子动力学模拟的支持，该模拟表明DC8、9PC和DPPC在固相脂质双分子层中发生了脱混。当适当的可调光敏剂染料被包括在脂质体的水室中时，内容物的释放是通过在被封装染料的波长处用激光激发来触发的。氧自由基清除剂对释放的抑制表明，释放机制涉及与光聚合无关的DC8、9PC的化学变化。激光介导的DC8、9PC的化学修饰由上汽弗雷德里克先进技术项目的分离技术小组用质谱、液相色谱、气相色谱和核磁共振进行分析。DPPC: DC8,9PC脂质体的物理特性包括熔融转变温度（Tm）、溶质装载效率、大小和血清稳定性。我们正在进一步开发这些脂质体，使其能够承受化疗药物（如阿霉素）的触发释放，并在体外和体内测试其功效。我们也在配制含有超顺磁性氧化铁纳米颗粒（SPION）和药物的脂质体，用于磁共振成像（MRI）和高温介导的药物释放。具体目标2。放射诱导和靶向化疗（RITCH）的发展。这个概念设想了一种无毒的前药，当静脉注射时，它会分布在全身。当前体药物受到局部电磁辐射时，它将在肿瘤部位发生化学转化为细胞毒性化合物。我们使用了疏水膜探针碘萘叠氮（INA）作为原型，它在光照射下与膜蛋白的跨膜部分发生共价反应。INA的光激活会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治疗除了消除其他膜蛋白外，还消除了多药转运蛋白的功能，因此这种方法有利于治疗多药耐药肿瘤。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（如声纳空化和x射线辐射）可以触发RITCH化合物的反应性。我们计划在体外和体内使用各种触发模式（包括光、声空化和x射线辐射）来检测我们的RITCH化合物的功效。动物研究包括使用NCI-Frederick的小动物成像设备进行药代动力学、生物分布和毒性研究。我们正在设计新的RITCH化合物，它将更适合各种触发模式。此外，我们正在对新化合物的化学性质以及导致细胞凋亡和细胞死亡的细胞生物学事件进行基础研究。

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.