【摘要】：現(xiàn)今,結(jié)直腸癌已經(jīng)成為美國男性和女性除皮膚癌外的第三大被檢出的癌癥,且其成為全美癌癥病人的第二大死因[9]。盡管,市場上有成千上萬的候選藥物,但是它們大多數(shù)都不理想,均產(chǎn)生巨大的毒副作用。其中非常有潛力的藥物為斑蝥素,其為倍半萜化合物,為許多種類的斑蝥蟲所分泌的一種化合物。斑蝥素長期以來被用做中藥,治療一系列的癌癥,包括肝癌,肺癌,胃癌和結(jié)直腸癌等[10]。國際上廣泛認(rèn)同,斑蝥素及其衍生物體現(xiàn)出了對蛋白磷酸酶2A(PP2A)強烈的特異性作用,且其對PP2A的抑制能力與其細(xì)胞的毒性成正比例[11]。近來,斑蝥素也被發(fā)現(xiàn)通過多種途徑對結(jié)直腸癌有作用,主要通過抑制相關(guān)的熱休克蛋白和BCL-2等。然而,斑蝥素會引起強烈的毒副作用,比如消化不良,咯血,排尿困難。因此,通過某種方案降低其毒副作用,并保留其抗癌療效顯得非常必要[12]。納米載藥體系,通常指的是用于載藥的粒子,其粒徑在100nm左右。這類納米載藥體系已經(jīng)引起了世界范圍內(nèi)的極大關(guān)注,因為它能夠克服傳統(tǒng)小分子抗癌藥物的一系列缺陷。選擇正確的藥物載體對于構(gòu)建高效的載藥體系顯得尤為重要[13]。比較好的一個藥物載體為維生素E-聚乙二醇琥珀酸酯(TPGS),其含聚二醇的雙親性表面活性劑分子,能用來包裹、鍵合疏水性藥物并組裝成納米粒子。葉酸為一小分子化合物,其對腫瘤細(xì)胞的增殖和存活具有非常大的意義。相比較正常細(xì)胞,很多腫瘤細(xì)胞表面過表達(dá)葉酸受體,甚至高達(dá)200多倍。癌細(xì)胞表面過表達(dá)的葉酸受體,對癌細(xì)胞攝取葉酸進(jìn)入細(xì)胞具有非常重要的意義[14]。一般來說,葉酸受體在卵巢癌、頭頸癌以及一些兒童癌癥細(xì)胞表面過表達(dá)。因為葉酸受體的這些原因,葉酸已經(jīng)廣泛作為很多載藥體系的靶向基團,其中包裹脂質(zhì)體、高分子膠束、膠囊、納米粒子及碳納米管。葉酸受體也被報道在結(jié)直腸癌細(xì)胞表面過表達(dá),因此在納米粒子表面引入葉酸配體,有可能靶向傳輸藥物到結(jié)直腸癌上[15]。考慮到斑蝥素獨特的抗癌特性和到目前為止藥物傳遞技術(shù)發(fā)展,全球的研究者嘗試了大量的方式直接傳遞斑蝥素及其衍生物等藥物到癌細(xì)胞進(jìn)行癌癥治療。Zhu[16]等人報道了薄膜分散超聲破碎法方法制備具有口服生物利用度的負(fù)載斑蝥素的固體脂質(zhì)納米粒(CA-SLNs)的方法。結(jié)果顯示這種CA-SLNs具有持續(xù)釋放性質(zhì)并且沒有突釋效應(yīng),口服后其生物利用度也高于游離的斑蝥素。隨后,zhu[17]等人報道了一種斑蝥素的環(huán)糊精包結(jié)絡(luò)合物用于藥物傳遞。然而并沒有對其體外和體內(nèi)的藥物效能進(jìn)行研究。最近,去甲斑蝥素,一種具有更低毒性的斑蝥素去甲基衍生物,被shen[18]等人綴合到聚乙烯亞胺(PEI)和6聚賴氨酸(PLL)表面,用于酸傾向性藥物的釋放。盡管這一系統(tǒng)表現(xiàn)出了合理的酸響應(yīng)性,但其藥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認(rèn)可的,很難進(jìn)一步進(jìn)行臨床應(yīng)用[19]。綜合來看,我們在這里展示出了第一個合理的設(shè)計的負(fù)載斑蝥素TPGS納米顆粒實例,用它來靶向傳遞斑蝥素并對結(jié)腸直腸癌進(jìn)行有效治療。由于斑蝥素是一種酐類物質(zhì),很容易和羥基反應(yīng)。斑蝥素可以通過一步簡單開環(huán)反應(yīng)與TPGS的端羥基反應(yīng),即可得到斑蝥素-TPGS共混物(Can-TPGS,Scheme 1a)。由于TPGS具有兩親性的特點,這種共混物在水溶液中可以自組裝成納米顆粒作為一種高效的藥物傳遞系統(tǒng)。通過FA-TPGS和Can-TPGS共同組裝的方法將葉酸鹽引入到了這些納米顆粒中,以進(jìn)一步提高這一系統(tǒng)的效能。我們系統(tǒng)地表征了這種新型的負(fù)載了斑蝥素的納米顆粒,并在兩個系列的癌細(xì)胞上進(jìn)行了體外實驗研究。結(jié)果顯示負(fù)載了斑蝥素的納米顆粒可以增強斑蝥素對葉酸過表達(dá)HT-29細(xì)胞的影響;更重要的是,在納米顆粒中引入葉酸能夠進(jìn)一步提高其效能,而這種明顯的增強作用在MCF-7細(xì)胞中卻觀察不到。目的:通過實驗證實傳統(tǒng)中藥斑蝥素的納米粒子通過引入靶向腫瘤的葉酸基團,實現(xiàn)了納米粒子對結(jié)腸癌的靶向傳輸,降低斑蝥素的毒性,保持并增強其抗癌療效。驗證斑蝥素負(fù)載的納米粒子的靶向能力與治療效果。方法:本論文以小鼠為研究對象建立腫瘤模型,分別以熒光標(biāo)記高分子納米膠束和葉酸高分子納米膠束檢測在荷瘤小鼠體內(nèi)的分布情況;將傳統(tǒng)中藥斑蝥素的納米粒子引入靶向腫瘤的葉酸基團,通過在葉酸受體過表達(dá)的人結(jié)腸癌HT-29細(xì)胞和葉酸低表達(dá)的人乳腺癌MCF-7細(xì)胞,分別進(jìn)行了MTT細(xì)胞毒性評價,細(xì)胞內(nèi)定位和吸收率測定及PP2A抑制試驗。結(jié)果:實驗(一)1.活體成像羅丹明膠束組:給藥后腫瘤組織熒光強度比值不斷增高,12 h達(dá)到最高,熒光強度值為3.07±0.57,腫瘤藥物濃度為正常組織的3倍,隨后比值緩慢下降。2.離體成像羅丹明膠束組:給藥后1h藥物聚集濃度梯度:肝腫瘤腎腦肺心,6h腫瘤藥物濃度略高于肝,12h腫瘤藥物濃度最高,明顯高于其他臟器,24h腫瘤仍存在藥物聚集。實驗(二)無葉酸配體的聚合物膠束P(NIR)在肝、肺有大量聚集,在脾、腎有少量聚集,腫瘤部位聚集很少,心臟沒有聚集。含有葉酸配體的聚合物膠束FA-P(NIR)1,30h時在肝臟有明顯的聚集,腫瘤和腎有少量聚集;48h時在肝臟聚集減弱,腫瘤部位聚集增強。含有葉酸配體的聚合物膠束FA-P(NIR)2在30h時在肝和腫瘤部位聚集較強,腎有少量聚集,心、脾無聚集;48h時在肝臟中聚集減弱,在腫瘤部位聚集增強,腫瘤熒光強度明顯高于肝;以上離體器官成像結(jié)果與活體成像結(jié)果一致。實驗(三)對HT-29細(xì)胞的藥效的按以下順序排列:FA-Can-NPsCan-NPs≈FA-Can-NPs+FA斑螯素,測試了葉酸受體低表達(dá)的MCF-7細(xì)胞,我們發(fā)現(xiàn)藥效排序是FA-Can-NPs≈FA-Can-NPs+FA≈Can-NPs斑螯素。與沒有處理過的細(xì)胞相比,PP2A在用斑螯素、Can-NPs、FA-Can-NPs以及FA-Can-NPs+FA處理過的細(xì)胞上的活性分別為52%、41%、25%和33%。結(jié)論及創(chuàng)新點:實驗證實高分子納米膠束對小鼠腫瘤部位具有明顯增強的通透性和滯留效應(yīng)即EPR效應(yīng)。然而,帶有葉酸配體的高分子納米膠束相對于不帶葉酸配體的納米膠束在小鼠腫瘤部位具有明顯的聚集,并且隨著葉酸含量的增大,聚集效果更明顯。將斑螯素所負(fù)載的納米粒子Can-NPs進(jìn)行藥物傳遞,通過進(jìn)一步引入靶向腫瘤的葉酸基團,我們可以實現(xiàn)斑螯素所負(fù)載的葉酸靶向納米粒子FA-Can-NPs。斑螯素負(fù)載的葉酸靶向納米粒子通過依靠PP2A來殺傷結(jié)直腸癌細(xì)胞。本實驗證實高分子納米膠束是優(yōu)良的被動靶向藥物的優(yōu)良載體,葉酸靶向納米粒子是優(yōu)良的主動靶向藥物載體,而斑螯素所負(fù)載的葉酸靶向納米粒子FA-Can-NPs被證實是有效的針對人結(jié)直腸癌的主動靶向化療藥物,整體效果明顯優(yōu)于單純斑蝥素中藥化療及斑蝥素的被動靶向藥物,針對結(jié)直腸癌病人具有重要的臨床意義。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.34

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