【作者】 丁宝月；

【导师】 高申； 丁雪鹰；

【作者基本信息】 第二军医大学 ， 药剂学， 2011， 博士

【摘要】 恶性黑素瘤(malignant melanoma,MM),简称恶黑,是一种来源于黑色素细胞的恶性程度很高的肿瘤,易血行转移、死亡率高,而且近年来,恶黑的发病率有明显上升的趋势,包括原先发病率较低的地区也是如此,至今仍缺乏有效地治疗手段。目前,临床上治疗转移性恶性黑色素瘤的主要手段仍然是在术后予以全身辅助的化学治疗,但目前用于MM的首选化疗药物(达卡巴嗪dacarbazine,DTIC)显效率也只有20%。且剂型单一,只有普通注射粉针剂,存在毒副作用大、靶向性差、用药顺应性差等缺点,因此急需对其化疗方法和剂型进行改进。有研究发现黑素瘤细胞表面高表达DR4或DR5,因此将DR4或DR5的激动型抗体DR4或DR5 mAb用于MM的免疫治疗具有良好的应用前景。本课题主要从如何更好地将化疗和免疫治疗相结合,制得高效低毒的主动靶向给药系统用于恶性黑素瘤治疗方面进行相关探索。为了改进恶性黑素瘤的治疗方法,提高疗效,降低毒副作用。在本课题研究中,采用对正常细胞无毒副作用、对MM ( MM细胞表面高表达DR5)具有高特异性和高选择性且自身具有抗肿瘤活性的的抗DR5单克隆抗体(DR5 mAb)为靶头,以达卡巴嗪(DTIC)为模型药物,聚乙二醇修饰的聚乳酸纳米微粒(MPEG-PLA NPs)为递药系统制备兼具化疗和免疫治疗作用的主动靶向免疫纳米微粒,并对其性质及体内外生物性能进行了考察。本文主要从以下几部分进行研究:第一部分,本文以DTIC为模型药物,MPEG-PLA为载体材料,采用超声复乳法制备了DTIC纳米粒(DTIC-NPs),通过单因素考察和正交设计优化制备纳米粒的处方工艺,确定制备纳米粒最优条件。然后以最佳工艺制得DTIC-NPs,并对其形貌、粒径、Zeta电位、表面元素、载药量、包封率、药物体外释放行为进行了考察。粒径电位分析仪及透射电镜测定结果显示,制得的纳米粒外形圆整,表面光滑,纳米粒平均粒径在144.2±7.8 nm左右,粒径分布均匀。其Zeta电位绝对值在-32.1±5.4 mV左右,静电稳定性好。XPS光电能谱仪空载纳米粒表面含有N元素,说明纳米微粒表面包裹有一层HSA,为DTIC-NPs的进一步功能化提供了条件。所制得的DTIC-NPs包封率为71.06±2.63 %、载药量为15.2±0.4μg/mg,DTIC的体外释放可分三个阶段:突释阶段、稳定释放阶段和加速释放阶段。在突释阶段中,纳米释放药量在4h时约为20%,纳米微粒释药在扩散阶段释药保持稳定增长,24h时释药量在40%左右,在72h时可达到81%左右,在最后阶段为纳米微粒MPEG-PLA降解,累积释药量达到91 %左右。第二部分,采用碳二亚胺法,将对正常细胞无毒副作用、将对MM具有高特异性和高选择性且自身具有促肿瘤细胞凋亡活性的DR5 mAb偶联到以上所构建载药纳米粒表面,设计了一种以具生物活性的抗体为靶头的靶向免疫纳米载药系统(DTIC-NPs -DR5 mAb),并对其形貌、粒径分布等进行了表征,DTIC-NPs-DR5 mAb平均粒径为166.0±8.1 nm, Zeta电位值为-36.8±3.2mV。通过双荧光标记法和Micro BCA蛋白含量测定法对单抗靶头是否顺利连接到纳米粒表面进行了定性验证和定量测定,计算得到连接在纳米粒表面的DR5 mAb数量为12.8±2.4μg DR5 mAb/mg NPs,抗体连接效率大约为43%左右。所构建的纳米靶向载药系统DTIC-NPs-DR5 mAb保持原DTIC-NPs缓释药物的特点,具有双重活性功能,即免疫导向和缓释药物,有利于提高肿瘤局部药物浓度。第三部分,采用流式细胞仪和激光共聚焦显微镜考察了靶纳米载药系统对靶细胞人恶性黑素瘤细胞A375的特异性结合、结合效率和摄取效果。用MTT法考察了靶向纳米粒A375细胞的抑制增殖作用和促凋亡作用,还考察了载体系统的安全性。流式测定和激光共聚焦结果表明, DTIC-NPs -DR5 mAb能跟A375细胞特异性结合,在5 min内就已经结合在细胞表面,结合效率较高,30 min后纳米粒已明显进入细胞内,入胞效果良好。MTT实验表明DTIC-NPs -DR5 mAb对A375细胞有良好的抑制增殖作用,其对A375细胞的抑制增殖作用和促凋亡作用显著强于其它对照组,细胞生存率仅为19.3±3.6%,DTIC-NPs-DR5 mAb作用组的A375细胞凋亡率为58.5±5.0%,而DTIC作用组的A375细胞凋亡率只有31.7±3.9%。通过MTT实验,我们发现本实验制得的递释系统MPEG-PLA-NPs和MPEG-PLA-NPs-DR5 mAb对正常细胞的毒性不大,在浓度高达2.2 mg/ml时对于细胞活力均没有受到显著影响,细胞活力均保持在大约80%以上,而且接了靶头的MPEG-PLA-NPs-DR5 mAb跟未接靶头的MPEG-PLA-NPs相比对正常细胞杀伤力并没有明显差异,说明该靶向递释系统(MPEG-PLA-NPs-DR5 mAb)具有良好的安全性,是一种安全的药物载体。第四部分,我们通过人恶性黑素瘤肿瘤移植裸鼠模型的建立,考察了靶向纳米粒的抗肿瘤效果及体内安全性。参比各种治疗因子,考察并评价了DTIC-NPs-DR5 mAb在体内的非特异性毒性情况以及对DR5高表达的黑素瘤的治疗情况。裸鼠的血常规及肝肾功能检测结果均表明,DTIC-NPs-DR5 mAb毒性较小,比起DTIC原药,MPEG-PLA对DTIC的包裹及抗体靶头的导向作用可以有效地减与正常组织细胞对DTIC的非特异性摄取,减小了DTIC在体内的非特异性毒性。在裸鼠的体内治疗试验中,DTIC-NPs-DR5 mAb对高表达DR5的恶性黑素瘤具有明确疗效,对肿瘤的生长产生了明显抑制作用,最终肿瘤平均体积为241.86±84.74 mm3 (PBS空白对照为1308.12±182.73 mm3)。综上所述,本课题成功构建了本身具有促靶细胞凋亡活性的DR5 mAb为靶头的主动靶向给药系统DTIC-NPs-DR5 mAb,可将化疗和免疫治疗有效结合,从内源性和外源性两条途径促进靶细胞凋亡,可起双重和协同治疗作用。体内外实验结果证明其靶向效率高、具有很好的抗肿瘤活性,大大提高了疗效、且毒副作用较低,是一种安全有效的新型抗肿瘤靶向制剂。通过该研究可为MM及其他肿瘤的化疗和靶向生物治疗提供相关的研究方法和思路,也可为今后靶向药物控释系统的设计提供试验资料和理论依据。更多还原

【Abstract】 The malignant melanoma (MM), a most aggressive form of skin cacer , which is easy to hematogenous metastasis and has high mortality. Despite a greater understanding of the risk factors as well as the genetic and epigenetic causes of melanoma, the death rate from melanoma continues to increase fast including original low incidence area in recent years. To date, no agent has promoted a clinically meaningful prolongation of overall survival. The current clinical treatment of patients with metastatic melanoma is mainly by systemic chemotherapy after adequate surgery. Although DTIC is so far the most active single agent for treatment of metastatic melanoma, its efficiency is only 20%, the targeting effect of the chemotherapeutic agents is poor and toxic side effect of it is serious, otherwise, malignant melanoma cell is generally refractory to conventional chemotherapy agents. Thus, novel therapy strategy is still to be proposed and there is a critical need for the development of therapeutic agents that could target aberrant survival pathways in metastatic melanomas to improve the treatment of this disease. As DR4 or DR5 is abundantly expressed on the surface of MM cells, it can be expected that the TRAIL-R1 or TRAIL-R2 monoclonal antibodies (DR4 or DR5 mAb) will be very promising immunotherapy bioactive molecules to MM. The purpose of this thesis is to study how to constructe a active targeting drug delivery system with high therapeutic effect and low-toxic, which can best combine chemotherapy and active speci?c immunotherapy for MM therapy.In our study, to overcome the limitations of the current therapy strategies, we take the DR5 monoclonal antibody (TRAIL-receptor 2 mAb, DR5 mAb) which can efficiently induce the apoptosis of tumor cells without any toxic side effects on normal cells as the targeting functional group, MPEG-PLA nanoparticles and dacarbazine (DTIC) are selected as drug-loaded systems and model drug , respectively, to build a new active targeting drug delivery system (DTIC-NPs-DR5 mAb) that combining of chemotherapy and active specific immunotherapy. This thesis was carried out as follow four parts:On the first part, Dacarbazine (DTIC) loaded methoxy polyethylene glycol– polylactide (MPEG-PLA) nanoparticles (NPs) were prepared by modified w/o/w double emulsion-solvent evaporation method through ultrasonic processor without any additional additives. The prescription and preparation technology of DTIC-NPs were optimizd by single-factor test and orthogonal design. Then the DTIC-NPs was prepared by the optimizd prescription and preparation technology, and its pharmaceutical characterization, such as appearance, strutcture, particle size ,zeta potential, surface element, drug loading, encapsulation efficiency as well as in vitro release characters were evaluated and tested by dynamic light-scattering detector (DLS), transmission electron microscope (TEM), x-ray photoelectron ppectroscopy (XPS) and HPLC. The small sized nanoparticles (NPs) with a particle size of 148.6±9.8 nm in diameter and drug encapsulation ef?ciency of 70.1±2.3% are easy to be dispersed in water. The results showed that the nanoparticles were uniformly spherical and the average diameter of DTIC-NPs-DR5 mAb was 148.6±9.8 nm, DL and EE were 15.2±0.4μg/mg and 71.06±2.63 %, respectively. The behavior of drug release in vitro indicated that nanoparticles took on sustained-release character, within 7 days, up to 91% drug release was observed from DTIC-NPs.On the second part, to obtain the nano targeted drug delivery system DTIC-NPs-DR5 mAb, TRAIL-receptor 2 monoclonal antibody (DR5 mAb) which can efficiently induce the apoptosis of tumor cells without any toxic side effects on normal cells were covalently conjugated to the surface of DTIC-NPs by a two-step carbodiimide method. Then the characterization of the DTIC-NPs-DR5 mAb, such as particle size, zeta potential and morphology, were evaluated by DLS and TEM. The resulting DTIC-NPs-DR5 mAb were sized at 166.0±8.1 nm with the zeta potential of -36.8±3.2 mV. The dual fluorescent marker method and Micro BCA protein content determination method were utilized to determine whether the DR5 mAb conjugated to the DTIC-NPs and the quantity of DR5 mAb on the DTIC-NPs, the conjugation quantity and efficiency were about 12.8±2.4μg DR5 mAb/mg NPs and 43%. The DTIC-NPs-DR5 mAb can keep the activity of DR5 mAb and increase the therapeutic efficiency due to its double activity of antiangiogenesis and local enrichment.On the third part, the cellular uptake and the targeted effect of DTIC-NPs-DR5 mAb on human malignant melanoma cell A375 were investigated by flow cytometry and laser-scanning confocal microscopy. The cytotoxic activity of DTIC-NPs-DR5 mAb against A375 or NIH cells was evaluated by MTT assay, and the in vitro cytotoxicity of blank NPs (MPEG-PLA-NPs and MPEG-PLA-NPs-DR5 mAb which did not encapsulate DTIC) was also tested by MTT assay. The FCM results showed that DTIC-NPs-DR5 mAb could recognize and specificity bind to the targeted cell, the biological activities of DR5 mAb on the NPs were well protected during the preparation process. The binding and internalization of targeted NP were detected on the A375cell membrane only after 5 min incubation and in the cytoplasm of cells after 30 min incubation by LSCM. The MTT tests results indicated that the DTIC-NPs-DR5 mAb was more cytotoxic against A375 (cells viability of 19.3±3.6%) and can induce remarkable quantity of A375 cell apoptosis (58.5±5.0%) when compared with DTIC. The blank NPs were found to be non-toxic at each of the tested concentrations. We did not observe a signi?cant difference in the toxicity of MPEG-PLA NPs and MPEG-PLA-DR5 mAb NPs, although there was a slight reduction in cell viability at higher concentrations (2.2 mg/ml), average cell viability was keeped above 80.1±3.9%, suggesting that MPEG-PLA-NPs-DR5 mAb may be used as a safety target delivery carrier.On the fourth part, we inspected the anti-tumor effect and internal security of targeted nanoparticles though human malignant melanoma tumors xenograft model. Compared with various therapeutic agents, we investigated and evaluated the in vivo nonspecific toxicity and anti-tumor effect of DTIC-NPs-DR5 mAb on malignant melanoma with high expression of DR5. The nude routine blood test、liver and kidney function test results indicate that the toxicity DTIC-NPs-DR5 mAb is much lower than DTIC, DTIC embedding in MPEG-PLA and oriented functions of the target head of antibody can effectively reduce the nonspecific intake of DTIC in normal tissue. The results of in vivo treatment on nude demonstrated that DTIC-NPs-DR5 mAb has a much better therapeutic efficacy in inhibiting tumor growth compared with DTIC and other controls, the final mean tumor volume was 241.86±84.74 mm3 (the final mean tumor volume of PBS blank control group was 1308.12±182.73 mm3).These results suggested that the nano active targeting drug delivery system (DTIC-NPs-DR5 mAb) were successfully, mediated by DR5 mAb which can efficiently induce the apoptosis of tumor cells without any toxic side effects on normal cells. In this approach, antibody is programmed through chemistry to bind a target of biological interest, the programmed antibody maintains advantages characteristic of both antibodies and the targeting agent. It is a combination of chemotherapy and immunotherapy, DTIC and DR5 mAb in the active targeting delivery system (DTIC-NPs-DR5 mAb) induce cancer cell apoptosis through intrinsic pathway and extrinsic pathway, respectively. Such chemoimmunotherapy could be resulted in enhanced anti-tumor immunity and improved therapeutic outcome with decrease side-effect. The in vitro and in vivo experiments demonstrated that DTIC-NPs-DR5 mAb is a safe and effective noval antitumor targeted formulation, which has high targeted efficiency、significant antitumor activity as well as lower side-effect. This study could provid a new research route for the therapy of metastatic malignant melanoma as well as the chemotherapy and targeting immunotherapy for other cancers.更多还原