基于介孔二氧化硅的控释靶向性药物投递系统的构建及对B细胞淋巴瘤的治疗研究

发布时间：2018-04-21 14:30

本文选题：介孔二氧化硅纳米粒子 + 利妥昔单抗　；参考：《东南大学》2017年博士论文

【摘要】：目的迄今为止化疗仍然是恶性淋巴瘤全身治疗的主要手段之一,但是化疗过程中不可避免地伴随着严重毒副作用的发生,从而限制了其在临床工作中的有效应用。因此,为了降低抗肿瘤药物的毒副作用,提高药物的主动靶向性,增强药物的抗肿瘤疗效,我们构建了一种以介孔二氧化硅纳米粒子(MSNs)为载体的控释靶向性药物投递系统,即Rituximab修饰的荷载细胞毒性药物盐酸多柔比星(DOX)的MSNs纳米粒子(RDMSNs)。该靶向性药物投递系统既能特异性靶向B细胞淋巴瘤细胞膜受体又能在细胞内的弱酸性环境中释放药物发挥抗肿瘤作用。我们运用RDMSNs靶向性药物投递系统治疗B细胞淋巴瘤,评价其抗肿瘤疗效,探讨其抗肿瘤的作用机制。方法1.RDMSNs靶向性药物投递系统的构建和表征:首先通过共聚法合成了羧基修饰的MSNs纳米粒子,并以其作为药物载体负载抗肿瘤药物DOX,构建细胞内pH值响应性的药物投递系统。在此基础上,为了进一步优化纳米粒子,我们将Amine-PEG2000-Biotin修饰到MSNs纳米粒子母核表面,最后通过生物素-亲和素桥接方法连接B细胞淋巴瘤细胞膜表面高表达的CD20受体靶向的配体Rituximab,构成靶向肿瘤细胞的RDMSNs靶向性纳米粒子。分别通过扫描电子显微镜(SEM)、透射电子显微镜(TEM)、傅里叶变换红外光谱仪、激光粒度分析仪、紫外分光光度计等仪器对所制备的纳米粒子进行物理表征,计算纳米粒子的包封率和载药率,抗体的链接效率;通过体外透析实验了解载药纳米粒在不同pH值的条件下释药特性。2. RDMSNs的细胞靶向性评价及体外细胞毒性研究:分别采用CD20+的Raji细胞和CD20-的Jurkat细胞验证RDMSNs纳米粒子的靶向性性能;并使用流式细胞仪(FCM)、共聚焦激光扫描显微镜(CLSM)和TEM检测和观察细胞内的DOX荧光强度和纳米粒子分布和数量。为了验证RDMSNs纳米粒子的细胞靶向性性能及体外细胞毒性效应,分别以CD20+的Raji和Daudi细胞株及CD20-的Jurkat细胞株为体外研究模型,采用CCK-8法评估载药纳米粒子的细胞毒效应。并且通过CLSM观察不同纳米粒子作用于细胞后细胞凋亡时核的形态学变化,FCM评价不同纳米粒子作用于细胞后的细胞凋亡率。3. RDMSNs对Raji细胞淋巴瘤的治疗疗效及体内荧光成像研究:构建Raji细胞株裸鼠皮下移植瘤模型,当肿瘤体积达到100mm3左右时,将荷瘤裸鼠随机分为4组:Saline组,Free DOX组,DMSNs组和RDMSNs组,每组5只,按照2.0mg/kg尾静脉注射给药,4天给一次药,连续4次,探讨载药纳米粒子的抗肿瘤疗效,并测定肿瘤生长体积及体重,计算抑瘤率,取各组裸鼠心脏、肝脏、脾脏、肺脏、肾脏行HE染色检查观察药物的安全性。采用TUNEL法检测各组肿瘤组织细胞的凋亡情况,免疫组织化学染色后,观察肿瘤细胞凋亡相关蛋白Bax、caspase-3、Bcl-2及增殖细胞相关的核抗原Ki67的表达。此外,还采用近红外活体成像方法评价Cy5.5-DMSNs和Cy5.5-RDMSNs纳米粒子对B细胞淋巴瘤的靶向性性能。结果1.成功构建了结构明确的MSN-COOH纳米粒子,以其作为载体制备RDMSNs靶向性纳米粒子。SEM和TEM显示纳米粒子大小一致和形态规则,傅里叶变换红外光谱仪证实羧基基团被成功连接;RDMSNs靶向性载药纳米粒子的粒径和zeta电位分别是56.3±11.2 nm和-31.5±5.2 mv; RDMSNs靶向性纳米粒子DOX的载药率和包封率分别是(23.5±4.7)%和(45.2±6.2)%;抗体Rituximab的连接率为(66.2±4.1)%;体外药物释放实验显示在pH 5.0的条件下药物释放率明显高于pH 7.4的条件(P0.05),提示RDMSNs靶向性纳米粒子具有pH值响应性的释药特性。2. Raji和Jurkat细胞株分别与RDMSNs靶向性纳米粒子孵育2h后,FCM检测细胞内DOX荧光强度发现Raji细胞株中的荧光强度大约是Jurkat细胞株的三倍(P0.01),同样在CLSM下观察到Raji细胞株中的荧光强度明显高于Jurkat细胞株,通过TEM也观察到类似的结果。在Raji、Daudi和Jurkat细胞株中,RDMSNs靶向性纳米粒子对Raji和Daudi细胞株的细胞毒性效应高于DMSNs组和FreeDOX组(P0.05),且其细胞毒性效应呈现浓度依赖性。而DMSNs和RDMSNs对Jurkat细胞株的细胞毒性效应无差异。在细胞凋亡实验中,CLSM观察到RDMSNs组具有凋亡细胞核形态学变化的细胞株明显多于其他治疗组。FCM检测显示24h对照组,MSNs组,FreeDOX组,DMSNs 组和 RDMSNs 组 Raji 细胞凋亡率分别是(3.0± 0.4) %,(3.9 ±0.6) %,(18.2 ± 1.2) %, (10.1 ± 1.2) % 和(23.3 ± 1.4) %, RDMSNs 组凋亡率明显高于其他组(P0.05)。且RDMSNs组凋亡率呈现浓度依赖性(P0.01)。3.在RDMSNs纳米粒子抗肿瘤疗效评估实验中,Saline组、Free DOX组和DMSNs组的平均肿瘤体积分别是 623.5±156.9mm3, 481.2±55.2mm3 和 335.6±57.3mm3,而RDMSNs处理组的平均肿瘤体积是98.0±51.8 mm3,与其他三组相比差异具有统计学意义(P0.05)。Saline组、RDMSNs组和DMSNs组裸鼠平均体重分别是25.2±0.9g、22.4±0.5g和21.6±0.5g,而Free DOX处理组的平均肿瘤体积是18.1±0.6g,与其他三组相比差异具有统计学意义(P0.01)。Free DOX组,DMSNs组和RDMSNs组的肿瘤抑制率分别为(22.79±4.37)%, (46.22±6.03)%和(84.28±5.92)%,RDMSNs 组的肿瘤抑制率最高,RDMSNs组分别与Free DOX组和DMSNs组相比差异具有统计学意义(P0.01)。这些结果表明RDMSNs组抗肿瘤作用最强且毒副作用较小。Free DOX组裸鼠的心脏HE染色显示心肌纤维排列紊乱,而Saline组,RDMSNs组和DMSNs组裸鼠心脏、肝脏、脾脏、肺脏、肾脏HE染色显示未见明显病理学改变,表明载药纳米粒子在体内具有良好的生物安全性。在TUNEL染色实验中,RDMSNs组肿瘤组织中绿色荧光最多,凋亡细胞数量最多。肿瘤组织免疫组化染色结果显示,与Saline组和Free DOX组相比,Bax和caspase-3蛋白在DMSNs组和RDMSNs组中表达量明显增加,而Bcl-2蛋白和Ki67抗原表达量明显减少,并且在RDMSNs组中Bax和caspase-3蛋白表达最强,Bcl-2蛋白和Ki67抗原表达最弱,表明RDMSNs靶向性纳米粒子具有较强的诱导细胞凋亡能力和抗细胞增殖能力。在近红外活体成像实验中,24h时间点Cy5.5-RDMSNs靶向组荧光信号最强,主要聚集于肿瘤区域,此后,随着时间延长荧光信号逐渐减弱。Cy5.5-DMSNs非靶向组具有类似的活体成像结果,但荧光信号半定量分析发现6h后肿瘤区域的荧光信号强度较Cy5.5-RDMSNs靶向组弱(P0.05)。结论1. RDMSNs靶向性纳米粒子结构完整、形态均一、载药量高,其结构中的PEG成分及表面修饰的Rituximab使载药纳米粒子具备了靶向性给药的性能,并且具有pH值响应性的控释药物性能。2. RDMSNs靶向性纳米粒子能够被淋巴瘤B细胞特异性的内吞,是通过受体介导的内吞作用进入淋巴瘤B细胞内。RDMSNs靶向性纳米粒子在细胞内所形成的溶酶体或核内体的酸性环境中能够促进药物释放,增加了细跑内化疗药物的累积,从而发挥了增强的细胞毒性效应和较高的诱导细胞凋亡能力。3. RDMSNs靶向性纳米粒子具有特异性的靶向性功能和增强的抗肿瘤活性,较低的毒副作用,较强的凋亡诱导能力。控释靶向性药物投递系统RDMSNs有可能成为化疗药物的载体,将药物靶向于淋巴瘤B细胞,提高化疗药物的疗效,减轻化疗药物的毒副反应,为B细胞淋巴瘤的靶向治疗提供了一个崭新平台。
[Abstract]:Objective chemotherapy is still one of the main methods for the systemic treatment of malignant lymphoma, but it is inevitably accompanied by severe toxic and side effects in the course of chemotherapy, which restricts its effective application in clinical work. Therefore, in order to reduce the side effects of antitumor drugs, improve the active targeting of the drug and enhance the drug We have constructed a controlled release targeting drug delivery system based on mesoporous silica nanoparticles (MSNs), the Rituximab modified load cytotoxic drug DOX MSNs nanoparticles (RDMSNs). The targeting drug delivery system can not only specifically target B cell lymphoma cells. Membrane receptors can also release drugs in the weak acid environment of cells. We use RDMSNs targeting drug delivery system to treat B cell lymphoma, evaluate its antitumor effect, and explore the mechanism of its anti-tumor action. Methods the construction and characterization of 1.RDMSNs targeting drug delivery system were synthesized by copolymerization method first. The carboxyl modified MSNs nanoparticles, which are used as drug carriers to load the antitumor drug DOX, construct the intracellular pH responsive drug delivery system. On this basis, in order to further optimize the nanoparticles, we modify the Amine-PEG2000-Biotin to the surface of the MSNs nanoparticle nucleus and finally connect with the biotin avidin bridging method. The CD20 receptor targeting ligand Rituximab, which is highly expressed on the membrane surface of B cell lymphoma cells, constitutes RDMSNs targeted nanoparticles for target tumor cells. By scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectrometer, laser particle size analyzer, ultraviolet spectrophotometer and other instruments The nanoparticles were characterized by physical characterization, and the encapsulation efficiency and drug delivery rate of the nanoparticles were calculated and the efficiency of the antibody was linked. The cell targeting evaluation and in vitro cytotoxicity of the drug loaded nanoparticles under the conditions of different pH values of.2. RDMSNs were investigated by the dialysis experiment in vitro: the Raji cells of CD20+ and the Jurkat cells of CD20- were used separately. To verify the targeting properties of RDMSNs nanoparticles, and use flow cytometry (FCM), confocal laser scanning microscopy (CLSM) and TEM to detect and observe the DOX fluorescence intensity and the distribution and quantity of nanoparticles in the cells. In order to verify the cell targeting and cytotoxic effects of RDMSNs nanoparticles, the Raji and Daudi of CD20+ are used respectively. The cell strain and the Jurkat cell line of CD20- were used as the model in vitro, and the cytotoxic effect of drug loaded nanoparticles was evaluated by CCK-8 method. And the morphological changes of nucleus were observed by different nanoparticles on the apoptosis of cells after cell apoptosis by CLSM. FCM was used to evaluate the apoptosis rate of different nanoparticles after cell apoptosis.3. RDMSNs to Raji cells. The therapeutic effect and fluorescence imaging in vivo: a model of subcutaneous transplantation of Raji cells in nude mice was constructed. When the tumor volume reached about 100mm3, the tumor bearing nude mice were randomly divided into 4 groups: Saline group, Free DOX group, DMSNs group and RDMSNs group, each group was injected in accordance with 2.0mg/kg tail vein, for 4 days for 4 consecutive times. The tumor growth volume and body weight were measured and the tumor suppressor rate was calculated. The safety of the drugs was observed by HE staining in the heart, liver, spleen, lungs and kidney of nude mice. The apoptosis of the tumor tissue cells was detected by TUNEL method. The apoptosis related protein Bax of tumor cells was observed after immunohistochemical staining. Caspase-3, Bcl-2 and the expression of nuclear antigen Ki67 related to proliferating cells. In addition, the targeting properties of Cy5.5-DMSNs and Cy5.5-RDMSNs nanoparticles for B cell lymphoma were evaluated by near infrared imaging methods. Results 1. successfully constructed a clearly structured MSN-COOH nanoparticle, which was used as a carrier to prepare RDMSNs targeted nanoparticles. SEM and TEM showed the uniform size and shape rule of nanoparticles. The Fourier transform infrared spectrometer confirmed that the carboxyl group was successfully connected; the particle size and zeta potential of the RDMSNs targeted drug loaded nanoparticles were 56.3 + 11.2 nm and -31.5 + 5.2 MV, respectively, and the drug loading rate and encapsulation efficiency of RDMSNs targeted nanoparticles were (23.5 + 4.7)% and 45.2 + respectively. 6.2)%, the connection rate of antibody Rituximab was (66.2 + 4.1)%, and the release rate of drug release in vitro showed that the drug release rate was significantly higher than that of pH 7.4 (P0.05) under the condition of pH 5, suggesting that RDMSNs targeted nanoparticles had pH responsive release properties,.2. Raji and Jurkat fine cell lines incubated with RDMSNs targeted nanoparticles respectively. The fluorescence intensity of DOX in the cell was measured to be about three times as high as that of the Jurkat cell line (P0.01). The fluorescence intensity of the Raji cell line was also observed under CLSM. The similar results were observed in the Raji cell lines by TEM. The cytotoxic effect of Audi cell line was higher than that of group DMSNs and FreeDOX group (P0.05), and its cytotoxic effect showed concentration dependence. There was no difference between DMSNs and RDMSNs on the cytotoxicity of Jurkat cell lines. In the apoptosis experiment, CLSM observed that the cell lines with the morphological changes of apoptotic cells in the RDMSNs group were obviously more than other treatments. .FCM test showed that the apoptosis rate of Raji cells in group MSNs, FreeDOX, DMSNs and RDMSNs was (3 + 0.4)%, (3.9 + 0.6)%, (18.2 + 1.2)%, (10.1 + 1.2)% and (23.3 + 1.4)% (23.3 + 1.4)%) in group DMSNs and RDMSNs group, and the apoptotic rate in RDMSNs group was significantly higher than that in other groups (P0.05). The apoptotic rate in RDMSNs group was concentration dependent (P0.01).3. in the The average tumor volume of Saline group, Free DOX group and DMSNs group was 623.5 + 156.9mm3, 481.2 + 55.2mm3 and 335.6 + 57.3mm3 respectively, and the average tumor volume of RDMSNs treatment group was 98 + 51.8 mm3, and the difference between the other three groups was statistically significant (P0.05).Saline group, RDMSNs group and The average weight of the group nude mice was 25.2 0.9g, 22.4 + 0.5g and 21.6 + 0.5g, while the average tumor volume of the Free DOX treatment group was 18.1 + 0.6g, and the difference was statistically significant (P0.01).Free DOX group compared with the other three groups. The tumor inhibition rate of the DMSNs and RDMSNs groups was (22.79 + 4.37)%, (46.22 + 6.03)% and (84.28 + 5.92)%, and the swelling of the RDMSNs group The tumor inhibition rate was the highest in group RDMSNs and group Free DOX and DMSNs group respectively (P0.01). These results showed that group RDMSNs had the strongest anti tumor effect and the heart HE staining of.Free DOX group of.Free DOX group showed the disorder of myocardial fiber arrangement, while the Saline, RDMSNs, and DMSNs group nude mice heart, liver, spleen, lung The HE staining of the kidney showed no obvious pathological changes, which showed that the drug loaded nanoparticles had good biological safety in the body. In the TUNEL staining experiment, the green fluorescence of the tumor tissue of the group RDMSNs was the most and the number of apoptotic cells was the most. The immunohistochemical staining results of the tumor tissue showed, compared with the group Saline and the Free DOX group, Bax and caspase-3. The expression of protein in group DMSNs and RDMSNs group increased obviously, while the expression of Bcl-2 protein and Ki67 antigen decreased obviously, and the expression of Bax and caspase-3 protein was the strongest in RDMSNs group, and the expression of Bcl-2 protein and Ki67 antigen was the weakest. It showed that RDMSNs targeted nanoparticles have strong ability to induce apoptosis and anti cell proliferation. In the infrared imaging experiment, the 24h time point Cy5.5-RDMSNs target group fluorescence signal is the strongest, mainly concentrated in the tumor area. After that, the fluorescence signal gradually weakened and the.Cy5.5-DMSNs non target group has similar living body imaging results, but the fluorescence signal semi quantitative analysis found that the fluorescence signal intensity of the tumor region after 6h is Cy5.5-R. DMSNs target group is weak (P0.05). Conclusion 1. RDMSNs targeted nanoparticles have complete structure, uniform morphology and high drug loading. The PEG components in the structure and the surface modified Rituximab make the drug nanoparticles have the performance of the targeted drug delivery, and the pH value responsiveness of the controlled-release drug.2. RDMSNs target nanoparticles can be lymphatic. The specific endocytosis of tumor B cells is through the receptor mediated endocytosis into the B cells of lymphoma, which can promote the release of drugs in the acid environment of the lysosomes or nuclear bodies formed by the.RDMSNs targeting nanoparticles in the cells, increasing the accumulation of chemotherapeutic drugs in the fine run, and exerts an enhanced cytotoxic effect and higher level of cytotoxicity. .3. RDMSNs targeted nanoparticles have specific targeting function and enhanced anti-tumor activity, lower toxic and side effects and strong apoptosis induction ability. The controlled release targeting drug delivery system RDMSNs may be a carrier of chemotherapeutic drugs, targeting the drug to lymphoma B cells and improving chemotherapeutic drugs. The therapeutic effect can reduce the side effects of chemotherapeutic drugs and provide a new platform for targeted therapy of B cell lymphoma.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.1

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