超声产气介导释药的PLGA中空微球诊疗一体化研究

发布时间：2017-12-28 16:13

本文关键词：超声产气介导释药的PLGA中空微球诊疗一体化研究　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：恶性肿瘤严重威胁人类生命健康,传统的化疗药物体内消除快、低靶向性,副作用大,而且肿瘤诊断和治疗是相互分离的两次医疗过程,令患者难以耐受。因此为了实现抗肿瘤药物诊疗一体化,我们构建了靶向肿瘤细胞、在靶部位浓集释药、有效治疗肿瘤的同时具备诊断功能的给药体系,即共载产气剂NaHCO3和抗癌药阿霉素的聚乳酸-羟基乙酸中空微球-PLGA-DOX@NaHCO3 HMs诊疗一体化给药体系。本课题研究内容主要分为三部分:第一部分:构建PLGA-DOX@NaHCO3 HMs给药体系采用复乳溶剂挥发法制备内腔包载阿霉素和NaHCO3的PLGA中空微球。透射电镜可见PLGA-DOX@NaHCO3 HMs中空结构,壳厚约420nm。光学显微镜观察其外观均匀分散,圆整规则。平均粒径分布在1~2μm,电位为(-21.8±1.31)mV,紫外分光光度法法测定其包封率和载药量分别为(39.18±2.20)%和(3.92±0.22)%。包载NaHCO3的制剂-PLGA-DOX@NaHCO3 HMs相比于未包封NaHCO3制剂——PLGA-DOX HMs可显著增强体内外超声显影效果,具备pH响应释药特性和更高的累积释药量,同时在超声作用下累积释药量明显增加。第二部分:研究PLGA-DOX@NaHCO3 HMs给药体系的体外细胞毒性及靶向性以乳腺癌细胞MCF-7为模型细胞考察PLGA-DOX@NaHCO3 HMs的体外细胞毒性和靶向性。MTT结果显示制剂组细胞抑制率均显示良好的浓度及时间依赖性,PLGA-DOX@NaHCO3 HMs相比于PLGA-DOX HMs细胞存活率明显下降,尤其在施加超声治疗后细胞抑制率进一步增加。细胞摄取定性结果显示肿瘤细胞更加倾向于摄取具备pH敏感性能的PLGA-DOX@NaHCO3 HMs制剂组,且其与PLGA-DOX HMs相比DOX在细胞核部位蓄积量明显增多。流式细胞术检测细胞摄取定量实验更加证实了包封NaHCO3的PLGA HMs更加有助于DOX在细胞内蓄积和滞留。细胞凋亡实验结果显示PLGA-DOX@NaHCO3 HMs相比于PLGA-DOX HMs细胞总凋亡率明显增加,在施加超声治疗后超声治疗与化疗双重作用使细胞总凋亡率更高,显示其良好的抗肿瘤细胞效果。第三部分:研究PLGA-DOX@NaHCO3 HMs给药体系的药物代谢动力学研究以昆明种雌性小鼠为模型动物,分别以瘤内原位注射方式给予PLGA-DOX@NaHCO3 HMs,静脉注射方式给予原料药DOX,以高效液相色谱法为定量方法测定阿霉素在血浆中含量的方式考察制剂和原料药中药物在小鼠体内的药物代谢动力学行为。分析结果得到制剂组和原料药组在小鼠体内药物代谢动力学行为差异显著。制剂组相比于原料药DOX,采用瘤内原位注射可将更多药物蓄积于肿瘤部位,减少药物经血渗透入正常组织,降低对正常组织的损伤。其半衰期和平均滞留时间明显增加,提高了生物利用度,延长了DOX作用时间。第四部分:PLGA-DOX@NaHCO3 HMs给药体系的药效学研究采用S180荷瘤昆明小鼠为动物模型,以瘤内注射制剂组和静脉注射原料药组的方式连续给药,以小鼠体重、相对瘤体积、瘤重、肿瘤形态等作为评价指标,考察了PLGA-DOX@NaHCO3 HMs对小鼠生命质量的影响以及抑制肿瘤生长的效果。实验结果得出PLGA-DOX@NaHCO3 HMs相比于未包封产气剂NaHCO3的制剂具有较好的治疗效果,尤其在合并局部超声治疗后,通过超声靶向微泡爆破技术引发气体空化效应使得肿瘤治疗效果更加显著,与原料药DOX治疗效果相当(P0.05)。联合组织病理学切片结果表明DOX对肿瘤生长抑制作用显著,但其存在严重的心肾毒性。制剂组对各组织无明显的毒副作用,同时在合并超声作用后,因化疗与超声治疗的协同作用,肿瘤治疗效果显著提高,使得荷瘤小鼠生命质量得以改善,毒副作用降低,安全性提高。本课题成功构建的PLGA-DOX@NaHCO3 HMs给药体系,经体内外考察得知,该体系具备pH敏感性能,可在肿瘤微酸环境下产生CO2气体用于超声显影定位诊断肿瘤,同时引发空化效应抑制肿瘤生长;在进行超声治疗后,通过超声靶向微泡爆破引发空化效应和声孔效应有效增加靶区药物蓄积浓度,提高肿瘤治疗效果,实现化疗、超声治疗、诊断于一体的的目的。
[Abstract]:Malignant tumor is a serious threat to human life and health. The traditional chemotherapeutic drugs eliminate the fast and low targeting in vivo, and the side effects are large. Moreover, tumor diagnosis and treatment are the two separate medical processes, which make it difficult for patients to tolerate. Therefore, in order to achieve anti-tumor drug treatment of integration, we construct simultaneous targeting of tumor cells, in the target site concentration release, effective treatment of tumors with the diagnosis function of the administration system, namely carrying gas producing agent NaHCO3 and anticancer drug doxorubicin PLGA microspheres -PLGA-DOX@NaHCO3 HMs hollow theranostics administration system. The research contents are divided into three parts: the first part is to build PLGA-DOX@NaHCO3 HMs drug delivery system, and prepare PLGA hollow microspheres loaded with adriamycin and NaHCO3 by emulsion evaporation. PLGA-DOX@NaHCO3 HMs hollow structure was found by transmission electron microscopy, and the thickness of the shell was about 420nm. The optical microscope shows that its appearance is evenly distributed and round the rules. The average particle size distribution was 1~2 mu m and the potential was (-21.8 + 1.31) mV. The encapsulation efficiency and drug loading of UV spectrophotometry were (39.18 + 2.20)% and (3.92 + 0.22)% respectively. The NaHCO3 -PLGA-DOX@NaHCO3 HMs, compared with the unencapsulated NaHCO3 preparation, PLGA-DOX HMs, significantly enhanced the ultrasound imaging effect in vivo and in vitro, and had pH responsive release characteristics and higher cumulative release dose. Meanwhile, the cumulative release amount increased significantly under ultrasound. The second part: We studied the cytotoxicity and targeting of PLGA-DOX@NaHCO3 HMs delivery system in vitro, and investigated the cytotoxicity and targeting of PLGA-DOX@NaHCO3 HMs in vitro with breast cancer cell MCF-7 as a model cell. MTT results showed that the cell inhibition rate in the preparation group showed a good concentration and time dependence. The survival rate of PLGA-DOX@NaHCO3 HMs was significantly lower than that of PLGA-DOX HMs cells, especially after the application of ultrasound treatment, the cell inhibition rate increased further. The qualitative results of cell uptake showed that tumor cells tended to ingest the PLGA-DOX@NaHCO3 HMs preparations with pH sensitive properties, and compared with PLGA-DOX HMs, DOX accumulation in nuclear sites increased significantly. Flow cytometry detection of cell uptake quantitative tests confirmed that the PLGA HMs encapsulated in NaHCO3 was more conducive to the accumulation and retention of DOX in the cells. The apoptosis test results showed that the total apoptosis rate of PLGA-DOX@NaHCO3 HMs increased significantly compared with PLGA-DOX HMs cells. After ultrasound treatment, the dual effect of ultrasound therapy and chemotherapy made the total apoptosis rate of cells higher, showing its good anti-tumor effect. The third part: To study the pharmacokinetics study of drug delivery system PLGA-DOX@NaHCO3 HMs to female Kunming mice as the model animal, respectively by injection of tumor in situ within the given PLGA-DOX@NaHCO3 HMs, intravenous injection of given drug DOX, determined by HPLC on preparations and raw materials in the plasma concentration of adriamycin in drugs pharmacokinetic behavior of mice for quantitative method. The results of the analysis showed that the pharmacokinetics of the drug group and the drug group were significantly different in the mice. In the preparation group, compared with the crude drug DOX, intratumoral injection in situ can accumulate more drugs in the tumor site, reduce the penetration of drugs into normal tissues and reduce the damage to normal tissues. The half-life and the average retention time increased significantly, increasing the bioavailability and prolonging the time of DOX action. The fourth part: the research on the S180 tumor in Kunming mice animal model for the efficacy of PLGA-DOX@NaHCO3 HMs administration system, with intratumoral injection preparation group and intravenous injection of API Group continuously administered to mice weight, relative tumor volume, tumor weight, tumor morphology as the evaluation index, the effects of PLGA-DOX@NaHCO3 HMs on the quality of life of mice and the effect of inhibiting tumor growth. The experimental results show that PLGA-DOX@NaHCO3 HMs compared to the preparation of non encapsulated gas producing agent NaHCO3 has a better therapeutic effect, especially in combination with local ultrasonic treatment, causing gas cavitation effect makes the tumor treatment effect is more significant to the micro bubble blasting technology by ultrasonic target, equivalent treatment medicine raw materials DOX (P0.05). The results of the joint histopathological section showed that DOX had a significant inhibitory effect on tumor growth, but it had serious cardionrenal toxicity. The preparation group had no obvious toxic and side effects on all tissues. Meanwhile, after the combination of ultrasound, the therapeutic effect of tumor was significantly improved due to the synergistic effect of chemotherapy and ultrasound treatment, so that the quality of life of tumor bearing mice was improved, the toxicity and side effects were reduced, and the safety was improved. This study successfully constructed PLGA-DOX@NaHCO3 HMs administration system, the external and internal analysis, the system has pH sensitive performance in tumor acidic environment to produce the CO2 gas for ultrasound imaging diagnosis of tumors, and cause cavitation effect to inhibit tumor growth; in ultrasound after treatment by ultrasound targeted microbubble cavitation caused by blasting the effect of sound hole effect effectively increase the drug target volume concentration, improve the tumor treatment, chemotherapy, ultrasonic treatment, achieve the purpose of diagnosis in one.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

【相似文献】