靶向传输维生素E-琥珀酸酯—斑蝥素键合物纳米粒子治疗结直肠癌的实验研究

发布时间：2018-07-29 06:58

【摘要】：现今,结直肠癌已经成为美国男性和女性除皮肤癌外的第三大被检出的癌症,且其成为全美癌症病人的第二大死因[9]。尽管,市场上有成千上万的候选药物,但是它们大多数都不理想,均产生巨大的毒副作用。其中非常有潜力的药物为斑蝥素,其为倍半萜化合物,为许多种类的斑蝥虫所分泌的一种化合物。斑蝥素长期以来被用做中药,治疗一系列的癌症,包括肝癌,肺癌,胃癌和结直肠癌等[10]。国际上广泛认同,斑蝥素及其衍生物体现出了对蛋白磷酸酶2A(PP2A)强烈的特异性作用,且其对PP2A的抑制能力与其细胞的毒性成正比例[11]。近来,斑蝥素也被发现通过多种途径对结直肠癌有作用,主要通过抑制相关的热休克蛋白和BCL-2等。然而,斑蝥素会引起强烈的毒副作用,比如消化不良,咯血,排尿困难。因此,通过某种方案降低其毒副作用,并保留其抗癌疗效显得非常必要[12]。纳米载药体系,通常指的是用于载药的粒子,其粒径在100nm左右。这类纳米载药体系已经引起了世界范围内的极大关注,因为它能够克服传统小分子抗癌药物的一系列缺陷。选择正确的药物载体对于构建高效的载药体系显得尤为重要[13]。比较好的一个药物载体为维生素E-聚乙二醇琥珀酸酯(TPGS),其含聚二醇的双亲性表面活性剂分子,能用来包裹、键合疏水性药物并组装成纳米粒子。叶酸为一小分子化合物,其对肿瘤细胞的增殖和存活具有非常大的意义。相比较正常细胞,很多肿瘤细胞表面过表达叶酸受体,甚至高达200多倍。癌细胞表面过表达的叶酸受体,对癌细胞摄取叶酸进入细胞具有非常重要的意义[14]。一般来说,叶酸受体在卵巢癌、头颈癌以及一些儿童癌症细胞表面过表达。因为叶酸受体的这些原因,叶酸已经广泛作为很多载药体系的靶向基团,其中包裹脂质体、高分子胶束、胶囊、纳米粒子及碳纳米管。叶酸受体也被报道在结直肠癌细胞表面过表达,因此在纳米粒子表面引入叶酸配体,有可能靶向传输药物到结直肠癌上[15]。考虑到斑蝥素独特的抗癌特性和到目前为止药物传递技术发展,全球的研究者尝试了大量的方式直接传递斑蝥素及其衍生物等药物到癌细胞进行癌症治疗。Zhu[16]等人报道了薄膜分散超声破碎法方法制备具有口服生物利用度的负载斑蝥素的固体脂质纳米粒(CA-SLNs)的方法。结果显示这种CA-SLNs具有持续释放性质并且没有突释效应,口服后其生物利用度也高于游离的斑蝥素。随后,zhu[17]等人报道了一种斑蝥素的环糊精包结络合物用于药物传递。然而并没有对其体外和体内的药物效能进行研究。最近,去甲斑蝥素,一种具有更低毒性的斑蝥素去甲基衍生物,被shen[18]等人缀合到聚乙烯亚胺(PEI)和6聚赖氨酸(PLL)表面,用于酸倾向性药物的释放。尽管这一系统表现出了合理的酸响应性,但其药物本身不是更加高效的斑螯素,其使用的高分子材料也不是FDA认可的,很难进一步进行临床应用[19]。综合来看,我们在这里展示出了第一个合理的设计的负载斑蝥素TPGS纳米颗粒实例,用它来靶向传递斑蝥素并对结肠直肠癌进行有效治疗。由于斑蝥素是一种酐类物质,很容易和羟基反应。斑蝥素可以通过一步简单开环反应与TPGS的端羟基反应,即可得到斑蝥素-TPGS共混物(Can-TPGS,Scheme 1a)。由于TPGS具有两亲性的特点,这种共混物在水溶液中可以自组装成纳米颗粒作为一种高效的药物传递系统。通过FA-TPGS和Can-TPGS共同组装的方法将叶酸盐引入到了这些纳米颗粒中,以进一步提高这一系统的效能。我们系统地表征了这种新型的负载了斑蝥素的纳米颗粒,并在两个系列的癌细胞上进行了体外实验研究。结果显示负载了斑蝥素的纳米颗粒可以增强斑蝥素对叶酸过表达HT-29细胞的影响;更重要的是,在纳米颗粒中引入叶酸能够进一步提高其效能,而这种明显的增强作用在MCF-7细胞中却观察不到。目的:通过实验证实传统中药斑蝥素的纳米粒子通过引入靶向肿瘤的叶酸基团,实现了纳米粒子对结肠癌的靶向传输,降低斑蝥素的毒性,保持并增强其抗癌疗效。验证斑蝥素负载的纳米粒子的靶向能力与治疗效果。方法:本论文以小鼠为研究对象建立肿瘤模型,分别以荧光标记高分子纳米胶束和叶酸高分子纳米胶束检测在荷瘤小鼠体内的分布情况;将传统中药斑蝥素的纳米粒子引入靶向肿瘤的叶酸基团,通过在叶酸受体过表达的人结肠癌HT-29细胞和叶酸低表达的人乳腺癌MCF-7细胞,分别进行了MTT细胞毒性评价,细胞内定位和吸收率测定及PP2A抑制试验。结果:实验(一)1.活体成像罗丹明胶束组:给药后肿瘤组织荧光强度比值不断增高,12 h达到最高,荧光强度值为3.07±0.57,肿瘤药物浓度为正常组织的3倍,随后比值缓慢下降。2.离体成像罗丹明胶束组:给药后1h药物聚集浓度梯度:肝肿瘤肾脑肺心,6h肿瘤药物浓度略高于肝,12h肿瘤药物浓度最高,明显高于其他脏器,24h肿瘤仍存在药物聚集。实验(二)无叶酸配体的聚合物胶束P(NIR)在肝、肺有大量聚集,在脾、肾有少量聚集,肿瘤部位聚集很少,心脏没有聚集。含有叶酸配体的聚合物胶束FA-P(NIR)1,30h时在肝脏有明显的聚集,肿瘤和肾有少量聚集;48h时在肝脏聚集减弱,肿瘤部位聚集增强。含有叶酸配体的聚合物胶束FA-P(NIR)2在30h时在肝和肿瘤部位聚集较强,肾有少量聚集,心、脾无聚集;48h时在肝脏中聚集减弱,在肿瘤部位聚集增强,肿瘤荧光强度明显高于肝;以上离体器官成像结果与活体成像结果一致。实验(三)对HT-29细胞的药效的按以下顺序排列:FA-Can-NPsCan-NPs≈FA-Can-NPs+FA斑螯素,测试了叶酸受体低表达的MCF-7细胞,我们发现药效排序是FA-Can-NPs≈FA-Can-NPs+FA≈Can-NPs斑螯素。与没有处理过的细胞相比,PP2A在用斑螯素、Can-NPs、FA-Can-NPs以及FA-Can-NPs+FA处理过的细胞上的活性分别为52%、41%、25%和33%。结论及创新点:实验证实高分子纳米胶束对小鼠肿瘤部位具有明显增强的通透性和滞留效应即EPR效应。然而,带有叶酸配体的高分子纳米胶束相对于不带叶酸配体的纳米胶束在小鼠肿瘤部位具有明显的聚集,并且随着叶酸含量的增大,聚集效果更明显。将斑螯素所负载的纳米粒子Can-NPs进行药物传递,通过进一步引入靶向肿瘤的叶酸基团,我们可以实现斑螯素所负载的叶酸靶向纳米粒子FA-Can-NPs。斑螯素负载的叶酸靶向纳米粒子通过依靠PP2A来杀伤结直肠癌细胞。本实验证实高分子纳米胶束是优良的被动靶向药物的优良载体,叶酸靶向纳米粒子是优良的主动靶向药物载体,而斑螯素所负载的叶酸靶向纳米粒子FA-Can-NPs被证实是有效的针对人结直肠癌的主动靶向化疗药物,整体效果明显优于单纯斑蝥素中药化疗及斑蝥素的被动靶向药物,针对结直肠癌病人具有重要的临床意义。
[Abstract]:Nowadays, colorectal cancer has become the third largest cancer found in American men and women except skin cancer, and it has become the second major cause of death for cancer patients in the United States, [9]. although there are thousands of candidate drugs on the market, but most of them are not ideal and have huge toxic and side effects. Cantharidin, a sesquiterpene compound, is a compound secreted by many species of cantharidis. Cantharidin has long been used as a traditional Chinese medicine to treat a series of cancers, including liver cancer, lung cancer, gastric cancer, and colorectal cancer, which are widely recognized in [10]. international. Cantharidin and its derivatives reflect the strong specificity of the protein phosphatase 2A (PP2A). Sex, and its inhibitory ability to PP2A and its cell toxicity is a positive proportion of [11]. recently, cantharidin has also been found to play a role in colorectal cancer through a variety of pathways, mainly by inhibiting related heat shock proteins and BCL-2. However, cantharidin can cause strong toxic and side effects such as dyspepsia, hemoptysis, and dysuria. It is very necessary for the [12]. nano drug delivery system to reduce its side effects and retain its anticancer effect. It usually refers to the particles used for drug loading, with a particle size of about 100nm. This kind of nano drug delivery system has attracted worldwide attention because it can overcome a series of deficiency of traditional small molecule anticancer drugs. The choice of the correct drug carrier is particularly important for the construction of an efficient drug delivery system. A good [13]. drug carrier is vitamin E- polyethylene glycol succinate (TPGS), a amphiphilic surfactant containing polyglycol, which can be used to encapsulate, bond hydrophobic substances and assemble nanoparticles. Folic acid is a small molecule. It is of great significance for the proliferation and survival of tumor cells. Compared to normal cells, many tumor cells overexpress folic acid receptors, even more than 200 times. The folic acid receptors expressed over the surface of the cancer cells are of great significance for the uptake of folic acid into cells in cancer cells, [14]. generally speaking, the folic acid receptor is in the egg. Nests, head and neck cancer and some children's cancer cells are overexpressed. Because of these reasons, folic acid has been widely used as a target group for many drug loading systems, including liposomes, polymeric micelles, capsules, nanoparticles and carbon nanotubes. With the introduction of folic acid ligands on the surface of the nanoparticles, it is possible to target the transmission of drugs to colorectal cancer and [15]. to take into account the unique anticancer properties of cantharidin and the development of drug delivery technology so far. Researchers around the world have tried a lot of ways to direct cantharidin and its derivatives and other drugs to cancer cells for cancer treatment.Zhu[16] and so on. The method of preparing solid lipid nanoparticles (CA-SLNs) with oral bioavailability of cantharidin with oral bioavailability was reported by the thin film dispersive method. The results showed that the CA-SLNs had continuous release properties and had no sudden release effect, and its bioavailability was higher than that of free cantharidin after oral administration. Subsequently, zhu[17] and others reported that A cantharidin cyclodextrin inclusion complex is used for drug delivery. However, it does not study its drug efficacy in vitro and in vivo. Recently, norcantharidin, a lower toxic cantharidin dimethyl derivative, is conjugated to the surface of polyethyleneimine (PEI) and 6 polylysine (PLL) by shen[18] et al. For use in acid propensity drugs. Although the system has shown a reasonable response to acid, the drug itself is not more efficient, and its polymer material is not recognized by FDA. It is difficult to further carry out the clinical application of [19]. synthesis. Here we show the first reasonable design of the loaded cantharidin TPGS nanoparticles As an example, it is used to target cantharidin and effective treatment for colorectal cancer. As cantharidin is an anhydride, it is easy to react with the hydroxyl group. Cantharidin can get the cantharidin -TPGS blends (Can-TPGS, Scheme 1a) by a simple ring reaction with the hydroxyl terminal of TPGS. Because TPGS has two affinity. The blends can be self assembled into nanoparticles in aqueous solution as an efficient drug delivery system. Folate salts are introduced into these nanoparticles by FA-TPGS and Can-TPGS together to further improve the efficiency of the system. We systematically characterize the new type of cantharidin loaded nanoscale. Rice particles, and in vitro experiments in two series of cancer cells, showed that cantharidin loaded nanoparticles could enhance the effect of cantharidin on folic acid overexpression HT-29 cells; more importantly, the introduction of folic acid in nanoparticles could further improve its efficiency, which was significantly enhanced in MCF-7 cells. Objective: it is proved that the traditional Chinese medicine cantharidin nanoparticles can achieve the target transmission of the colon cancer by introducing the folic acid group targeted to the tumor, reduce the toxicity of cantharidin, maintain and enhance its anticancer efficacy. In this paper, the tumor model was established in mice. The distribution of the tumor bearing mice was detected by fluorescent labeling polymer nanomicelles and folic acid polymer nanomicelles, and the traditional Chinese medicine cantharidin nanoparticles were introduced into the folic acid group targeting the tumor, and the HT-29 cells in human colon cancer cells expressed by folic acid receptor were used. MCF-7 cells with low expression of folic acid were evaluated for MTT cytotoxicity, intracellular localization and absorptivity and PP2A inhibition test. Results: experiment (1) 1. living body imaging Luo Danming micelle group: the fluorescence intensity ratio of tumor tissues increased continuously after administration, the maximum of 12 h, the value of fluorescence intensity was 3.07 + 0.57, and the concentration of tumor drugs 3 times the normal tissue, then the ratio of.2. was slowly decreased in the Luo Danming micelles group: the concentration gradient of 1H drug concentration after administration: liver tumor kidney and brain lung heart, 6h tumor drug concentration is slightly higher than liver, 12h tumor drug concentration is highest, obviously higher than other organs, 24h tumor still exists in drug aggregation. Experiment (two) polymer glue free ligands ligands glue P (NIR) has a large aggregation in the liver and lungs, in the spleen, a small amount of aggregation in the kidney, little aggregation of the tumor site, and no aggregation of the heart. When the polymer micelles FA-P (NIR) 1,30h containing folic acid ligand (NIR) 1,30h has obvious aggregation in the liver and a small amount of aggregation in the tumor and kidney, the aggregation of the liver is weakened at the time of 48h and the aggregation of the tumor is enhanced. A polymer containing folate ligands The aggregation of FA-P (NIR) 2 at the liver and tumor sites was stronger at 30h, with a small amount of aggregation in the kidney and no aggregation in the heart and spleen; the aggregation of the liver in the liver was weakened when 48h was weakened, and the fluorescence intensity of the tumor was obviously higher than that of the liver; the imaging results above in vitro were the same as in vivo imaging. Experiment (three) the following order for the efficacy of HT-29 cells was in the following order Arrangement: FA-Can-NPsCan-NPs FA-Can-NPs+FA plaetin, which tested the MCF-7 cells with low expression of folate receptor, we found that the pharmacodynamic order was FA-Can-NPs FA-Can-NPs+FA Can-NPs plaques. Compared with the untreated cells, the activity of PP2A in the cells treated with chelate, Can-NPs, FA-Can-NPs and FA-Can-NPs+FA was 5, respectively. 2%, 41%, 25% and 33%. conclusions and innovation points: the experiment confirmed the obvious enhanced permeability and retention effect of the polymer nanomicelles on the tumor site of mice. However, the nano micelles with folate ligands have obvious aggregation in the tumor sites of the mice compared with those of the non folate ligands. With the increase of acid content, the aggregation effect is more obvious. By further introducing the Can-NPs nanoparticles loaded by the chelate, we can further introduce the folic acid target of the folic acid targeted to the nanoparticles, FA-Can-NPs., by using the folic acid group, which is loaded by the chelate, to kill the nanoparticles by relying on the PP2A. Colorectal cancer cells. This experiment proved that the polymer nanomicelles are excellent carriers of passive targeting drugs, and folic acid targeted nanoparticles are excellent active targeting drug carriers. The folic acid targeted nanoparticles FA-Can-NPs supported by chelate are proved to be effective targeted chemotherapeutic drugs for human colon cancer. The overall effect is clear. It is superior to cantharidin chemotherapy and cantharidin passive targeting drugs, and has important clinical significance for colorectal cancer patients.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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