生物可降解交联纳米药物用于肿瘤高效靶向治疗与诊断

发布时间：2018-04-30 19:55

  本文选题：生物可降解 + 可逆自交联纳米药物　； 参考：《苏州大学》2016年博士论文

【摘要】：随着肿瘤发病率及死亡率急速增长,寻求高效、安全的治疗方式刻不容缓。纳米药物由于提高了抗癌药物溶解性、延长体内循环时间、通过EPR效应增强在病灶部位累积量等优点而发展迅速。其中,Doxilò作为首例纳米药物在1995年被FDA批准主要用于卡波济式肉瘤、卵巢癌及多发性骨髓瘤的治疗;Abraxaneò因提高了PTX的溶解度而在2005年上市,用于临床治疗转移性乳腺癌、胰腺癌及非小细胞肺癌。随着韩国三阳公司研制的首个聚合物纳米药物Genexol-PM被批准用于卵巢癌、肺癌和乳腺癌,研究者们继续推动包括NK911和NK105在内的一系列聚合物纳米药物进入临床试验,并取得了阶段性成果。纳米药物的初步成功鼓励着人们探究更多多功能纳米载体用于肿瘤靶向治疗,其中,基于脂肪族聚酯、聚碳酸酯和聚氨基酸的聚合物载体由于优越的生物相容性、生物可降解性、无毒的降解产物而受到广泛青睐。然而,体内稳定性欠缺、难以在肿瘤部位渗透、肿瘤细胞内吞少、细胞内释放缓慢是传统药物提高治疗效率并走向临床需翻越的四座大山。除此之外,探究特异性强、高效率、无毒的基因治疗及诊断治疗一体化多功能纳米平台也是聚合物纳米药物发展的两大趋势。论文第一章对聚合物纳米药物用于肿瘤治疗的发展现状、面临的挑战以及解决方法等作了概述。为此,本论文中我们设计了肿瘤主动靶向、细胞内还原敏感、具有自交联功能的生物可降解聚合物胶束和囊泡纳米药物平台技术,发现其在不同肿瘤模型中的优异表现,对新型抗肿瘤纳米药物的临床试验起到了积极的推动作用。本论文第二章中,我们设计合成了具有自主知识产权的新型二硫戊环三亚甲基碳酸酯(DTC),并基于它制备了一系列肿瘤靶向、可逆自交联的生物可降解聚合物胶束及囊泡纳米药物多功能平台。基于DTC我们合成了表面偶联cRGD的两亲性聚合物聚乙二醇-b-聚二硫戊环三亚甲基碳酸酯(PEG-b-PDTC),制备了载阿霉素的自交联胶束纳米药物,用于主动靶向到α_vβ_3整合蛋白过表达的新生血管及黑色素瘤细胞(B16)。该胶束在生理环境下稳定而在模拟细胞内还原环境下(10 mM GSH),硫硫键断裂解交联,快速释放药物;在小鼠体内消除半衰期t1/2,β为6.19小时(自由dox:0.18小时),6小时后肿瘤积累量为6.13%id/g,最大耐受量(mtd100mg/kg)为自由阿霉素的20倍以上,并对荷b16黑色素瘤小鼠呈现高抗肿瘤活性和低毒副作用,显示了设计的优越性。胶束纳米药物的成功鼓舞我们在第三章研究自交联的聚合物囊泡纳米药物。我们设计合成了基于dtc和三亚甲基碳酸酯(tmc)的两亲性聚合物peg-p(tmc-dtc)和crgd-peg-p(tmc-dtc),其疏水链可调控制以适宜囊泡的制备。通过ph梯度法高效装载dox·hcl并自交联后得到靶向、稳定囊泡纳米药物crgd-ps-dox(载药量为15.4wt.%,120纳米)。crgd-ps-dox被α_vβ_3过表达的人脑胶质瘤细胞u87mg主动靶向摄取,在细胞内和小鼠体内有效抑制肿瘤细胞生长(半致死率ic50为1.77μg/ml)。其呈现小鼠体内循环时间长(t1/2,β为4.49小时)、肿瘤富集多(6.78%id/g)、肿瘤抑制作用强、毒副作用低的特点,拓展了以dtc为基础的自交联囊泡用于抗肿瘤药物的靶向传递,为研究该体系在其他肿瘤模型的治疗应用奠定了基础。针对目前全球范围内发病率及死亡率最高的肺癌,在第四章中基于第三章的自交联囊泡,我们分别探究了三种肺癌主动靶向的囊泡纳米药物crgd-ps-dox、cngq-ps-dox及cngq-ps-dtx,研究了其在a549肺癌皮下瘤和原位瘤模型的靶向治疗效果。三者均对a549皮下瘤体现了高肿瘤富集量、强肿瘤抑制能力、长生存期和低毒性。而在荷a549原位瘤裸鼠中的靶向治疗效果更佳,尤其是crgd-ps-dox和cngq-ps-dox能有效遏制肿瘤增长及转移,治疗16天后肿瘤体积缩小;对小鼠主要脏器无毒副作用;各小鼠在45天实验期间的生存率为100%,而临床使用的聚乙二醇化脂质体阿霉素里葆多lipo-dox虽然也能抑制肿瘤增长但毒副作用大,32天小鼠全部死亡。由此可见,可逆交联囊泡无论装载亲水或疏水药物均不改变表面性质,能有效抑制肺癌皮下和原位肺癌增长,向临床应用前进了一大步。这些令人振奋的实验结果加之囊泡本身和脂质体类似的结构特性,促使我们在第五章将该囊泡纳米药物和里葆多lipo-dox在治疗恶性卵巢癌方面来进行头对头的比较,因后者是卵巢癌临床治疗的一线用药。我们制备了多肽ge11表面修饰的交联囊泡(ge11-ps-dox),其中ge11对卵巢癌细胞表面过表达的egfr受体特异性靶向的。ge11-ps-dox最大耐受剂量为160mg/kg,是lipo-dox至少8倍,大大提高了治疗窗。更令人振奋的是,ge11-ps-dox在60mg/kg单剂量静脉给药治疗荷skov-3卵巢瘤小鼠时,可有效遏制肿瘤增长,减小毒副作用,延长小鼠生存期。故ge11-ps-dox为替代lipo-dox治疗卵巢癌提供了可能性。针对纳米药物应用中肿瘤渗透性低这一难题,我们在第六章设计将具有强肿瘤渗透功能、同时能靶向肿瘤新生血管的多肽irgd偶联到囊泡表面得到irgd-ps-dox,系统研究了其中irgd表面密度对血液循环、肿瘤富集、渗透及治疗的影响。irgd-ps-dox实现了它在血管丰富、α_vβ_3整合蛋白过表达的b16肿瘤模型中高效的渗透性及抗肿瘤活性。体内外实验表明,含50%irgd的irgd50-ps-dox有最长的循环时间(4.19小时)、最高的肿瘤富集量(4.89%id/g)、最佳的渗透及肿瘤抑制效率、最长的生存期,体现了高肿瘤渗透性囊泡对提高纳米药物体内疗效的重要性。生物大分子药物如核酸和蛋白质具有小分子药物无法比拟的优点,包括特异性强和高效无毒等,尤其是小干扰rna(sirna)具有直接的抗肿瘤作用。第七章中我们拓展了囊泡纳米药物技术,设计了囊泡内层为短链带正电荷的支化聚乙烯亚胺pei的囊泡,其pei内壳可高效复合sirna(sirna-cngq/rccps)并助其更好逃逸内涵体,实现了sirna的高效体内递送。该囊泡sirna在体内循环的半衰期为1.78小时,在荷a549原位肺癌裸鼠肺中富集量为4.02%id/g,复合治疗基因siplk1后的囊泡可有效抑制肿瘤增长且小鼠中值为54.0天,远高于其他对照组。该囊泡纳米药物为低毒、高效的生物疗法,尤其是基因疗法靶向治疗肿瘤提供了技术平台。癌症死亡率攀高的重要原因之一就是癌症缺乏安全有效精准的早期诊断,发现时已是中晚期且已转移,给癌症的治疗带来巨大挑战。包括ct在内的非侵入性影像学诊断方法在临床上较常使用,但是需要使用大量的造影剂。在第八章中我们制备了基于生物可降解、含碘聚碳酸酯的囊泡纳米造影剂,用于肿瘤的诊断及诊疗。首先我们设计合成了具有知识产权的含双碘的碳酸酯单体2,2'-碘代甲基-三亚甲基碳酸酯(ic),通过可控开环聚合得到含碘量高达60.4wt.%的两亲性聚合物peg-pic和peg-p(dtc-ic)。制备的peg-pic囊泡ips在相同碘量下在体内外的造影效果均优于小分子造影剂碘海醇,其体内t1/2,β为3.73小时,尾静脉注射入荷u87mg皮下瘤小鼠中发现肿瘤处ct值增加了43.2hu,而在荷a549原位瘤小鼠中ct值增加量为121.3hu,为肿瘤的早期诊断奠定了基础。接着我们探究了表面偶联crgd、可逆自交联囊泡显影剂纳米药物crgd-rcips-dox在荷b16皮下瘤小鼠的诊断与治疗。其呈现优异的体内外稳定性,t1/2,β长达6.84小时;尾静脉注射在荷b16皮下瘤小鼠体内4小时后,发现肿瘤的特异性ct成像,肿瘤的ct增长量为碘海醇的16.4倍。此外,DOX在肿瘤积累量达6.68%ID/g,佐证了其特异性CT成像和高抗肿瘤活性且小毒副作用。cRGD-RCIPs-Dox集特异性CT成像诊断、主动靶向、高效装载抗癌药物、细胞内触发药物释放、高效抑瘤等优点于一身,在肿瘤的早期诊断及靶向治疗上前景广阔。第九章对本论文工作进行了全面总结,并展望了今后可开展的工作。
[Abstract]:With the rapid growth of tumor incidence and mortality, it is urgent to seek efficient and safe treatment. Nanodrugs have developed rapidly because of improving the solubility of anticancer drugs, prolonging the circulation time in the body, and increasing the accumulation of the lesions by the EPR effect. In 1995, Doxil was approved by FDA as the first drug. Mainly used for the treatment of kapoji's sarcoma, ovarian cancer and multiple myeloma; Abraxane was listed in 2005 for improving the solubility of PTX, for clinical treatment of metastatic breast, pancreatic and non-small cell lung cancer. The first polymeric nanomoto drug Genexol-PM was approved for ovarian cancer and lung cancer with the development of South Korea's San Yang company. And breast cancer, the researchers continue to promote a series of polymer nanometers, including NK911 and NK105, to enter clinical trials and achieve stage results. The initial success of nanomedicine encourages people to explore more multifunctional nanoscale for tumor targeting therapy based on aliphatic polyester, polycarbonate and polyamino acids. Polymer carriers are widely favored because of their superior biocompatibility, biodegradability and non-toxic degradation products. However, the lack of stability in the body is difficult to infiltrate in the tumor sites, the tumor cells are swallowed up, and the intracellular release is slow to be the four mountains that traditional drugs increase the therapeutic efficiency and move towards the clinical need. In addition, It is also the two trend of the development of polymer nanomaterials to explore the specific, high efficiency, non-toxic gene therapy and diagnosis and treatment integrated multi-functional nano platform. Chapter 1 of the thesis is a summary of the current development of polymer nanometers for cancer treatment, the challenges faced and the solution method. In this paper, we designed the paper. The tumor active target, the cell reduction sensitivity, the biodegradable polymer micelles and the vesicular nano drug platform technology, which have the self intersection function, found their excellent performance in different tumor models, and played an active role in the clinical trials of new anti-tumor nanoscale drugs. In the second chapter of this paper, we designed and synthesized A new type of two sulfur amyl ring Sanya methyl carbonate (DTC) with independent intellectual property rights was used to prepare a series of tumor targeting, reversible self crosslinked biodegradable polymer micelles and vesicular nano drug multifunction platforms. Based on DTC, we synthesized the two Pro polymer polyethylene glycol two sulphate ring three on the surface of the coupling of cRGD. Methylene carbonate (PEG-b-PDTC), prepared a self crosslinked micellar nano drug carrying doxorubicin, used to actively target the overexpressed alpha _v beta _3 integrin's neovascularization and melanoma cells (B16). The micelles are stable under the physiological environment and in the simulated cell reduction environment (10 mM GSH), sulfur sulfur bond cleavage and crosslinking, and the rapid release of drugs In mice, the half-life t1/2, beta 6.19 hours (free dox:0.18 hours), 6 hours after the tumor accumulation was 6.13%id/g, the maximum tolerance (mtd100mg/kg) was more than 20 times that of free adriamycin, and showed high anti-tumor activity and low toxicity against B16 melanoma mice. It showed the superiority of the design. We have inspired us to study self crosslinked polymer vesicles in the third chapter. We designed and synthesized two amphiphilic polymer peg-p (tmc-dtc) and crgd-peg-p (tmc-dtc) based on DTC and Sanya methyl carbonate (TMC). The hydrophobic chain is adjustable for the preparation of the vesicles. The DOX. HCl and self crosslinking are efficiently loaded by the pH gradient method. Targeting, stable vesicle nanoscale drug crgd-ps-dox (15.4wt.%, 120 nm).Crgd-ps-dox is active targeting of human glioma cells that are overexpressed by alpha _v beta _3, and effectively inhibits the growth of tumor cells in the cells and in mice (the semi lethal rate IC50 is 1.77 mu g/ml). It presents a long cycle time in mice (t1/2, beta 4.49 small). When the tumor is enriched and enriched (6.78%id/g), the tumor suppresses and the side effects are low, the DTC based self crosslinking vesicle is used to target the target delivery of antitumor drugs. It lays the foundation for the study of the system in the treatment of other tumor models. In the four chapter, based on the third chapter, we explored three kinds of lung cancer active targeted vesicle nanometers, crgd-ps-dox, cngq-ps-dox and cngq-ps-dtx, and studied the target therapy effect in A549 lung cancer subcutaneous tumor and in situ tumor model. All of the three were high tumor accumulation, strong tumor suppressor ability and long tumor suppressor ability in A549 subcutaneous tumor. The survival time and low toxicity. But in the nude mice bearing A549 tumor in situ, the target therapy was better, especially the crgd-ps-dox and cngq-ps-dox could effectively inhibit the tumor growth and metastasis. The tumor volume reduced after 16 days of treatment and the nontoxic side effect on the main organs of the mice; the survival rate of each mouse during the 45 day experiment was 100%, and the clinical use of polyb two. The alcohol liposomes, adriamycin lipo-dox, can also inhibit the growth of the tumor but the toxic and side effects are large, and all the mice died in the 32 day. Thus, the reversible cross-linked vesicles, regardless of the hydrophilic or hydrophobic drugs, do not change the surface properties, can effectively inhibit the growth of lung cancer subcutaneous and in situ lung cancer, and make a big step forward to clinical application. The exhilarating experimental results, coupled with the structural characteristics of the vesicle itself and liposomes, prompted us to compare the vesicle nanoscale with the Li Pao lipo-dox in the treatment of malignant ovarian cancer in the fifth chapter, because the latter is the first line of clinical treatment for ovarian cancer. We have prepared the cross-linked sac of the peptide ge11 surface modification. Ge11-ps-dox, in which the maximum tolerance dose of ge11 to the EGFR receptor specific targeting of the ovarian cancer cell surface is 160mg/kg, at least 8 times as much as lipo-dox, which greatly improves the treatment window. It is more exciting that ge11-ps-dox can effectively contain the tumor when the single dose of ge11-ps-dox is administered intravenously to the mice with SKOV-3 ovarian tumor. Increase, reduce toxic side effects and prolong the survival time of mice. Therefore, ge11-ps-dox provides a possibility to replace lipo-dox in the treatment of ovarian cancer. In the sixth chapter, we design a peptide irgd that has strong tumor permeability and can target the neovascularization of tumor to the surface of the vesicle. Irgd-ps-dox, a systematic study of the effects of irgd surface density on blood circulation, tumor enrichment, infiltration and treatment,.Irgd-ps-dox realized its high permeability and anti-tumor activity in the vascular rich, _v beta _3 integrin's overexpressed B16 tumor model. In vivo and in vivo, it was shown that irgd50-ps-dox containing 50%irgd had the longest cycle time. Between 4.19 hours, the highest concentration of tumor (4.89%id/g), the best osmosis and tumor inhibition efficiency, the longest life period, reflects the importance of high tumor osmotic vesicles to improve the efficacy of nanoscale drugs. Biological macromolecules such as nucleic acids and proteins have advantages unmatched by small molecular drugs, including specific and high efficiency. Non-toxic, especially small interference RNA (siRNA) has direct anti-tumor effect. In the seventh chapter, we expanded the vesicle nano drug technology, designed the vesicle of the vesicle with a short chain and positive charge of the branched polyethyleneimine Pei, and its Pei inner shell can efficiently compound siRNA (sirna-cngq /rccps) and help it to escape the endosomes better and realize siRNA The half-life of the vesicle siRNA in the body is 1.78 hours, and the concentration of the lung in nude mice bearing A549 in situ lung cancer is 4.02%id/g. The vesicles after the compound therapy gene siplk1 can effectively inhibit the growth of the tumor and the median value of the mice is 54 days, far higher than that of the other control groups. The vesicle nanoscale drug is low toxic and efficient biotherapy, In particular, gene therapy provides a technical platform for cancer treatment. One of the important reasons for the rise of cancer mortality is the lack of effective and accurate early diagnosis of cancer. It has been found in the middle and late stages and has been transferred to a great challenge for the treatment of cancer. The noninvasive imaging diagnosis, including CT, is more often in clinical practice. Use, but need to use a large number of contrast agents. In the eighth chapter, we have prepared a biodegradable, iodine containing polycarbonate vesicle nano contrast agent for the diagnosis and diagnosis of cancer. First, we designed and synthesized the iodized carbonate monomer 2,2'- iodide methyl Sanya methyl carbonate (IC) with intellectual property. The two Pro polymer peg-pic and peg-p (dtc-ic) with iodine content up to 60.4wt.% were obtained by the controlled open loop polymerization. The effect of peg-pic vesicle IPS in the same iodine amount in vivo and in vivo was better than that of small molecule contrast agent iodiol, and the t1/2, beta was 3.73 hours in its body, and the CT value increased in the tumor at the tail vein injected into the tumor of U87MG subcutaneous tumor in mice. 43.2hu, in A549 in situ tumor bearing mice, the increase of CT value was 121.3hu, which laid the foundation for the early diagnosis of the tumor. Then we explored the diagnosis and treatment of the surface coupled crgd, the reversible self crosslinking vesicle nano drug crgd-rcips-dox in the subcutaneous tumor bearing mice of B16, which showed excellent stability, t1/2, and beta as long as 6.84 small. 4 hours after the injection of the tail vein in the mice bearing B16 subcutaneous tumor, the tumor specific CT imaging was found, and the tumor's CT growth was 16.4 times as much as iodiol. In addition, the accumulation of DOX in the tumor was 6.68%ID/g, which confirmed the specific CT imaging and high antitumor activity and the small toxic side effect.CRGD-RCIPs-Dox set specific CT imaging diagnosis, active targeting The ninth chapters make a comprehensive summary of the work of this paper and look forward to the work that can be carried out in the future.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ460.1
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本文编号：1825973