氧化还原敏感的紫杉醇—油酸前药自组装纳米药物传递系统的构建和评价

发布时间：2018-05-27 07:22

本文选题：紫杉醇 + 不饱和脂肪酸前药　；参考：《沈阳药科大学》2016年博士论文

【摘要】：恶性肿瘤严重威胁着人类的健康。化疗是治疗恶性肿瘤的一个重要手段,然而,普通制剂的化疗方案存在化疗效率低下和毒副作用严重等问题。随着纳米技术在药物传递领域的广泛应用,这些问题有所改善。但是,目前应用于临床的纳米制剂还存在诸多不足,包括:载药量低(通常低于10%)、稳定性差、载体材料相关的毒副作用和储存过程中药物的结晶和泄漏等问题。因此,设计和构建新型的高效纳米药物传递系统仍然是亟待解决的难题。此外,前药策略也被广泛用于抗癌药物的传递。其中,紫杉醇-不饱和脂肪酸前药的研究取得了很大的进展。例如,紫杉醇-二十二碳六烯酸前药(PTX-DHA)已经进入了临床Ⅲ期研究。然而,最新的临床结果显示,与一线化疗药物相比,PTX-DHA前药的疗效并没有显著优势。在PTX-DHA前药结构中,紫杉醇和二十二碳六烯酸是通过酯键直接相连的,这可能会导致紫杉醇很难从强疏水的脂溶性前药上断裂下来,影响其抗肿瘤效果。与正常细胞相比,肿瘤细胞内的高氧化还原微环境已经被广泛用来设计氧化还原敏感的药物传递系统。针对PTX-DHA的临床效果不佳和传统纳米制剂的不足,本课题分别以单硫醚键、间隔双硫醚键和二硫键作为氧化还原敏感的化学连接臂设计了一系列紫杉醇-油酸前药,并构建了氧化还原敏感的紫杉醇-油酸小分子前药自组装纳米药物传递系统。在此基础上,比较了这些不同的氧化还原敏感的化学连接臂对前药纳米粒的体外释放、细胞毒性、药动学行为、体内分布以及抗肿瘤效果的影响。首先,以单硫醚键、间隔双硫醚键和二硫键作为氧化还原敏感的化学连接臂设计了三个氧化还原敏感的紫杉醇-油酸前药(PTX-S-OA、PTX-2S-OA和PTX-S-S-OA),并以酯键直接相连的非敏感紫杉醇-油酸前药(PTX-OA)作为对照。四个前药化合物均能在乙醇-水混合溶剂体系中自组装形成前药纳米粒(PTX-OA纳米粒、PTX-S-OA纳米粒、PTX-2S-OA纳米粒和PTX-S-S-OA纳米粒),前药纳米粒的粒径均在100nm左右。然而,由于这些前药纳米粒表面的疏水性很强,它们在盐溶液中的胶体稳定性较差,会因"盐析"作用而使得纳米结构被破坏,并析出沉淀。为了应对这一问题并延长前药纳米粒在血液中的循环时间,在前药纳米粒制备过程中加入15%的维生素E聚乙二醇2000琥珀酸酯(TPGS2k)作为PEG修饰剂,制备了PEG化的小分子前药自组装纳米粒(PTX-OA/TPGS2k 纳米粒、PTX-S-OA/TPGS2k 纳米粒、PTX-2S-OA/TPGS2k 纳米粒和 PTX-S-S-OA/TPGS2k纳米粒)。这些PEG修饰的前药纳米粒具有粒径均匀(100nm左右)、载药量高(50%)和稳定性好等优点。以含过氧化氢(H2O2)或二硫苏糖醇(DTT)的磷酸盐缓冲液(PBS,pH7.4)-乙醇混合溶液(70/30,v/v)为释放介质,考察了PEG化的小分子前药自组装纳米粒在氧化条件或还原条件下释放紫杉醇的情况。PTX-OA前药具有很高的稳定性,无论在含氧化还原剂的释放介质还是在空白释放介质中,PTX-OA/TPGS2k纳米粒几乎不释放紫杉醇。相比之下,PTX-S-S-OA具有非常好的还原敏感特性,在含有10mMDTT的释放介质中,PTX-S-S-OA/TPGS2纳米粒在2 h内释放了超过90%的紫杉醇。PTX-S-OA和PTX-2S-OA都具有氧化还原双敏感特性,且PTX-S-OA较PTX-2S-OA具有更高的氧化还原双敏感响应能力。在含有H2O2或DTT的释放介质中,PTX-S-OA/TPGS2k纳米粒均比PTX-2S-OA/TPGS2k纳米粒能更快地释放紫杉醇。体外释放结果表明酯键相连的PTX-OA前药的释药速度非常慢,释药过于缓慢可能是导致临床中PTX-DHA前药药效不佳的原因。本课题设计的两个氧化还原双敏感的前药(PTX-S-OA和PTX-2S-OA)和一个还原敏感的前药(PTX-S-S-OA)能在氧化还原环境中选择性快速释放紫杉醇,有望实现在肿瘤细胞内氧化还原微环境中特异性快速释药,提高紫杉醇的抗肿瘤效果并降低对正常组织的毒副作用。采用MTT法考察了 PEG修饰的小分子前药自组装纳米粒对人鳞状上皮细胞癌细胞(KB-3-1),人大细胞肺癌细胞(H460)和人卵巢癌细胞(OVCAR-8)的细胞毒性。由于前药存在释放母药的过程,因此,前药自组装纳米粒的体外细胞毒性均比Taxol低一些。不同前药自组装纳米粒的细胞毒性大小顺序为PTX-S-S-OA/TPGS2k纳米粒PTX-S-OA/TPGS2k纳米粒PTX-2S-OA/TPGS2k纳米粒PTX-OA/TPGS2k纳米粒。值得注意的是,在设定的药物浓度范围内,PTX-OA/TPGS2k纳米粒没有表现出明显的细胞毒性。细胞毒性结果表明,前药纳米粒的细胞毒性大小与紫杉醇从前药中的释放难易程度相关,化学连接臂对肿瘤细胞内氧化还原环境的敏感性越高,紫杉醇就越容易从前药中被释放出来,其细胞毒性就越大。此外,我们还研究了KB-3-1细胞对香豆素-6标记的PEG化小分子前药自组装纳米粒的细胞摄取情况。香豆素-6溶液剂和香豆素-6标记的小分子前药自组装纳米粒的细胞摄取都具有时间依赖性。与香豆素-6溶液剂相比,香豆素-6标记的小分子前药自组装纳米粒在0.5 h和2 h均具有更高的细胞摄取。以Sprague-Dawley(SD)大鼠为模型动物,比较了Taxol、未修饰的小分子前药自组装纳米粒和PEG修饰的小分子前药自组装纳米粒的体内药动学行为。与未修饰的前药纳米粒和Taxol相比,PEG修饰的前药纳米粒均能显著延长药物在血液中的循环时间。由于化学连接臂的氧化还原敏感性不同,PEG化的各前药纳米粒的药动学也存在一定差异。其中,由于PTX-OA前药酯键的高度稳定性,PTX-OA/TPGS2k纳米粒的AUC0-24h值(前药与母药加和)最大。不同PEG化的前药纳米粒的AUC0-24h值(前药与母药加和)的大小顺序为:PTX-OA/TPGS2k纳米粒PTX-2S-OA/TPGS2k纳米粒PTX-S-S-OA/TPGS2k纳米粒PTX-S-OA/TPGS2k纳米粒。此外,我们以人鳞状上皮细胞癌(KB-3-1)荷瘤裸鼠为研究对象,考察了DiR标记的PEG化小分子前药自组装纳米粒的组织分布情况。DiR溶液剂的荧光主要分布在肺部,在肿瘤组织中的荧光十分微弱。与DiR溶液剂相比,DiR标记的PEG化小分子前药自组装纳米粒在肿瘤组织的荧光强度显著增加。体内分布结果表明,前药纳米粒能显著增加药物在肿瘤部位的积蓄,减少药物在非肿瘤部位的分布。以人鳞状上皮细胞癌(KB-3-1)荷瘤裸鼠为动物模型,考察了 Taxol和PEG修饰的小分子前药自组装纳米粒的体内抗肿瘤效果。PBS和PTX-OA/TPGS2k纳米粒不能有效抑制肿瘤细胞的生长,荷瘤裸鼠的肿瘤体积生长迅速。相比之下,PTX-2S-OA/TPGS2k纳米粒和Taxol能在一定程度上延缓肿瘤的生长,但二者之间的抗肿瘤效果并没有统计学差异。更重要的是,PTX-S-OA/TPGS2k纳米粒和PTX-S-S-OA/TPGS2k纳米粒组展现出很强的抑制肿瘤细胞生长的能力。给药结束后,PTX-S-OA/TPGS2k纳米粒给药组中荷瘤裸鼠的肿瘤体积几乎没有增长。更令人惊喜的是,在PTX-S-S-OA/TPGS2k纳米粒组中荷瘤裸鼠的肿瘤几乎消失不见了。体内抗肿瘤效果与体外释放结果和细胞毒性结果相一致,表明化学连接臂对肿瘤细胞内氧化还原条件的敏感性越高,越容易实现紫杉醇在肿瘤细胞内的特异性快速释放,相应的前药纳米粒的抗肿瘤效果就越好。此外,在整个给药过程中,对照组和给药组的荷瘤裸鼠的体重都没有减轻,也没有发现明显的肝肾毒性。体内药效结果表明PTX-S-OA/TPGS2k纳米粒和PTX-S-S-OA/TPGS2k纳米粒在具有明显的抗肿瘤效果的同时,没有对机体造成显著的非特异性毒性,是安全有效的抗癌药物传递系统。
[Abstract]:Malignant tumor is a serious threat to human health. Chemotherapy is an important means for the treatment of malignant tumors. However, there are some problems in the chemotherapy regimen of general preparation, such as low efficiency of chemotherapy and serious side effects. With the extensive application of nanotechnology in the field of drug delivery, these problems have improved. However, it is currently used in clinical nanoscale. There are many deficiencies in the preparation, including low drug loading (usually less than 10%), poor stability, toxic and side effects associated with carrier materials, and the crystallization and leakage of drugs during storage. Therefore, the design and construction of a new efficient nanoscale delivery system is still a difficult problem to be solved. In addition, the prodrug strategy is also widely used for anticancer. Drug delivery. Among them, paclitaxel - unsaturated fatty acid predrugs have made great progress. For example, paclitaxel - twenty-two - carbon six - acid prodrug (PTX-DHA) has entered clinical stage III studies. However, the latest clinical results show that the efficacy of PTX-DHA prodrugs is not significantly superior to first-line chemotherapeutic drugs. Before PTX-DHA In drug structure, taxol and twenty-two carbon six enoic acids are connected directly through ester bonds, which may cause Taxol to be difficult to break down from strong hydrophobic, fat soluble prodrugs and affect its antitumor effect. Compared with normal cells, high redox microenvironment in tumor cells has been widely used to design redox sensitive drugs. In view of the poor clinical effect of PTX-DHA and the shortage of traditional nanoscale preparation, we designed a series of taxol - oleic acid precursor with monosulfide bond, septal disulfide bond and two sulfur bond as redox sensitive chemical arm, and constructed the self assembly of paclitaxel oleic acid small molecular prodrug with redox sensitivity. On this basis, the effects of these different redox sensitive chemical connections on the release, cytotoxicity, pharmacokinetics, in vivo distribution and antitumor effects of these different redox sensitive chemical connections are compared. First, the mono ether bond, the septal disulfide bond and the two sulfur bond are used as the oxidation-sensitive chemical connections. Three redox sensitive paclitaxel oleic acid prodrugs (PTX-S-OA, PTX-2S-OA and PTX-S-S-OA) were designed and the non sensitive paclitaxel oleic acid prodrug (PTX-OA) was directly linked by ester bonds as a control. The four prodrug compounds could be self assembled to form precursor nanoparticles (PTX-OA nanoparticles, PTX-S-OA nanoparticles, PT) in a mixed solvent system of ethanol water. X-2S-OA nanoparticles and PTX-S-S-OA nanoparticles), the particle size of the precursor nanoparticles is around 100nm. However, because of the strong hydrophobicity of these nanoparticles, the colloids in the salt solution are less stable, and the nanostructures will be destroyed and precipitated because of the "salting out" effect. In the blood circulation time, 15% vitamin E polyethylene glycol 2000 succinate (TPGS2k) was added to the preparation of prodrug nanoparticles as PEG modifier, and the PEG small prodrug self assembled nanoparticles (PTX-OA/TPGS2k nanoparticles, PTX-S-OA/TPGS2k nanoparticles, PTX-2S-OA/TPGS2k nanoparticles and PTX-S-S-OA/TPGS2k nanoparticles) were prepared. The PEG modified prodrug nanoparticles have the advantages of homogeneous particle size (about 100nm), high drug loading (50%) and good stability. The release medium containing the phosphate buffer solution (PBS, pH7.4) ethanol mixture (70/30, v/v) containing hydrogen peroxide (H2O2) or two sulfur sugol (DTT) is used as the release medium, and the PEG small prodrug self assembled nanoparticles in the oxidation strip are investigated. The release of taxol in parts or reduction conditions.PTX-OA prodrug has high stability, PTX-OA/TPGS2k nanoparticles almost do not release taxol in the release medium containing redox agents or in the blank release medium. By contrast, PTX-S-S-OA has a very good and original sensitivity, P in the release medium containing 10mMDTT. TX-S-S-OA/TPGS2 nanoparticles release more than 90% of paclitaxel.PTX-S-OA and PTX-2S-OA in 2 h, both of which have redox dual sensitivity, and PTX-S-OA has a higher redox dual sensitive response ability than PTX-2S-OA. In the release medium containing H2O2 or DTT, PTX-S-OA/TPGS2k nanoparticles can be faster than PTX-2S-OA/TPGS2k nanoparticles. Release of paclitaxel. In vitro release results show that the release rate of the PTX-OA prodrug linked by the ester bond is very slow and the release is too slow may be the cause of the poor efficacy of the clinical PTX-DHA prodrug. Two redox two redox prodrugs (PTX-S-OA and PTX-2S-OA) and a reductive prodrug (PTX-S-S-OA) can be oxidized by this subject. The selective rapid release of taxol in the reduction environment is expected to achieve specific and rapid drug release in the redox microenvironment of tumor cells, improve the antitumor effect of taxol and reduce the toxic and side effects on normal tissues. The MTT method was used to investigate the PEG modified small prodrug self assembled nanoparticles on human squamous cell carcinoma cells (KB- 3-1) cytotoxicity of human lung cancer cells (H460) and human ovarian cancer cells (OVCAR-8). The cytotoxicity of prodrug self assembled nanoparticles in vitro is lower than that of Taxol because of the presence of precursor drugs. The order of cytotoxicity of different prodrug self assembled nanoparticles is PTX-S-S-OA/TPGS2k nanorp PTX-S-OA/TPGS2k Na It is worth noting that PTX-OA/TPGS2k nanoparticles do not exhibit obvious cytotoxicity in the range of drug concentration, and the cytotoxicity results of PTX-OA/TPGS2k nanoparticles show that the cytotoxicity of prodrug nanoparticles is related to the release of paclitaxel in the prodrugs, and the chemical link arm pair The higher the sensitivity of the redox environment in the tumor cells, the more easily the paclitaxel is released in the prodrug, the more cytotoxic it is. In addition, we also studied the cell uptake of the KB-3-1 cells labeled by the coumarin -6 PEG small molecule prodrug self assembled nanoparticles. The small molecules marked by coumarin -6 solution and coumarin -6 The cell uptake of the prodrug self assembled nanoparticles was time dependent. Compared with the coumarin -6 solution, the small molecular prodrug self assembled nanoparticles marked by coumarin -6 had higher cell uptake at 0.5 h and 2 h. Sprague-Dawley (SD) rats were used as model animals to compare Taxol, unmodified small molecular prodrug self assembled nanoparticles and P The pharmacokinetics of EG modified small molecular prodrug self assembled nanoparticles in vivo. Compared with unmodified prodrug nanoparticles and Taxol, PEG modified prodrug nanoparticles can significantly prolong the cycle time of the drug in the blood. The pharmacokinetics of PEG precursor nanoparticles are also certain because of the different redox sensitivity of the chemical arm. Differences. Among them, the AUC0-24h value of PTX-OA/TPGS2k nanoparticles is the largest because of the high stability of the ester bond of the PTX-OA precursor. The AUC0-24h value of the different PEG precursor nanoparticles (prodrugs and mother drugs) is the PTX-OA/TPGS2k nanoparticles PTX-2S-OA/ TPGS2k nanoparticles PTX-S-S-OA/TPGS2k nanoparticles PTX-S-OA/TPGS2k. Nanoparticles. In addition, we studied human squamous cell carcinoma (KB-3-1) tumor bearing nude mice. The tissue distribution of DiR labeled PEG small molecular prodrug self assembled nanoparticles was investigated. The fluorescence of.DiR solution was mainly distributed in the lungs, and the fluorescence in the tumor tissues was very weak. Compared with the DiR solution, the PEG fraction of the DiR marker The fluorescence intensity of the self assembled nanoparticles in the tumor tissues increased significantly. The distribution in vivo showed that the prodrug nanoparticles could significantly increase the accumulation of drugs at the tumor site and reduce the distribution of drugs in the non tumor sites. Human squamous cell carcinoma (KB-3-1) nude mice were used as animal models, and the small molecules modified by Taxol and PEG were investigated. The anti-tumor effects of.PBS and PTX-OA/TPGS2k nanoparticles in the prodrug self assembled nanoparticles do not effectively inhibit the growth of tumor cells, and tumor bearing nude mice grow rapidly. In contrast, PTX-2S-OA/TPGS2k nanoparticles and Taxol can delay the growth of the tumor to a certain extent, but the anti tumor effect between the two is not statistically significant. More importantly, PTX-S-OA/TPGS2k nanoparticles and PTX-S-S-OA/TPGS2k nanoparticles showed a strong ability to inhibit the growth of tumor cells. After the end of the drug, the tumor volume of the tumor bearing nude mice in the PTX-S-OA/TPGS2k nanoparticles group was almost no increase. And, surprisingly, the tumor bearing nude mice in the PTX-S-S-OA/TPGS2k nanoparticles group The tumor is almost disappeared. The antitumor effect in vivo is in accordance with the results of in vitro release and cytotoxicity, which indicates that the more sensitive the chemical connection arm is to the redox conditions in the tumor cells, the more easily the specific rapid release of taxol in the tumor cells, the better the antitumor effect of the corresponding prodrug nanoparticles. In addition, the body weight of the tumor bearing nude mice in the control group and the administration group was not reduced and no obvious liver and kidney toxicity was found in the control group and the administration group. The results showed that the PTX-S-OA/TPGS2k nanoparticles and PTX-S-S-OA/TPGS2k nanoparticles had no significant non specific toxicity to the body while having obvious antitumor effects. It is a safe and effective anti-cancer drug delivery system.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

【相似文献】