载普鲁士蓝靶向纳米复合物多模态显像及综合治疗裸鼠乳腺癌的实验研究

发布时间：2018-04-27 20:17

本文选题：普鲁士蓝 + 纳米复合物　；参考：《重庆医科大学》2017年博士论文

【摘要】：第一部分载普鲁士蓝靶向纳米复合物的制备及性能检测目的1.制备一种载普鲁士蓝靶向纳米复合物(PLGA-PB-PTX-PEG-FA),并检测其基本性质。2.研究载普鲁士蓝靶向纳米复合物体外寻靶能力和联合激光对细胞活性的影响。方法1.以PEG化连接了叶酸(folic acid,FA)的PLGA作为成膜材料,采用双乳化法,制备装载普鲁士蓝纳米粒子(Prussian blue nanoparticles,PB NPs)和紫杉醇(Paclitaxel,PTX)的PLGA-PB-PTX-PEG-FA靶向纳米复合物,并对其粒径、电位、表面形态等进行检测;用紫外-可见分光光度法、傅里叶红外光谱仪、激光共聚焦显微镜检测其紫外吸收光谱、红外光谱和各组分结构特征;采用0.647 W/cm2,808nm的激光辐照检测其体外光热特性;纳米复合物中紫杉醇的包封率及载药量用高效液相色谱法检测。2.体外培养乳腺癌细胞株MDA-MB-231,激光共聚焦评价PLGA-PB-PTX-PEG-FA靶向纳米复合物体外细胞寻靶能力。采用CCK8法检测所制备的靶向纳米复合物联合激光辐照对细胞的毒性。结果1.透射电镜和扫描电镜观察制得的PLGA-PB-PTX-PEG-FA纳米复合物呈球形,形态规则,大小尚均匀,分散度好。Malvern激光粒径仪检测出PLGA-PB-PTX-PEG-FA纳米复合物粒径为(236.6±55.04)nm,表面电位为(-24.44±1.7)m V。紫外-可见分光光度计检测出在PLGA-PB-PTX-PEG-FA纳米复合物和PB NPs吸收波峰位于702nm附近。傅里叶红外光谱图结果显示,PLGA-PTX-PEG-FA和PB NPs的吸收峰位于1756.23 cm-1 and 2086 cm-1,而PLGA-PB-PTX-PEG-FA纳米复合物同时具有上述两个特征峰。激光共聚焦显微镜显示,PB NPs激发后为绿色荧光,PLGA-PTX-PEG-FA为红色荧光,而PLGA-PB-PTX-PEG-FA纳米复合物为橙黄色荧光。激光辐照下,PLGA-PB-PTX-PEG-FA纳米复合物具有良好的光热特性和光热稳定性,且随着PLGA-PB-PTX-PEG-FA纳米复合物浓度增加或激光能量的增加,纳米复合物的光热转化效能增高。由高效液相色谱法检测PLGA-PB-PTX-PEG-FA纳米复合物中紫杉醇包封率为77.82%,载药量为7.22%,具有良好的缓释特性,在激光辐照下可加快药物的释放。2.体外细胞寻靶实验显示,红色荧光靶向纳米复合物附着于MBA-MD-231乳腺癌细胞表面,而非靶向组,受体封闭组这两个组的细胞周围几乎没有红色的荧光纳米复合物。CCK8法结果显示,PLGA-PB-PTX-PEG-FA联合激光辐照组MBA-MD-231的细胞活性最低,证实其具有体外综合化疗和光热治疗的作用。结论成功制备出球形、形态规则,大小均匀,性质稳定的PLGA-PB-PTX-PEG-FA靶向纳米复合物。该纳米复合物具有良好的光学吸收性能和光热转化特性,载药量较高,在体外对MBA-MD-231乳腺癌细胞有良好的靶向能力,联合激光辐照,在体外具有良好的联合化疗和光热治疗肿瘤细胞的能力。第二部分载普鲁士蓝靶向纳米复合物体内寻靶,光声/磁共振多模态显像实验研究目的观察PLGA-PB-PTX-PEG-FA纳米复合物体外增强磁共振成像(Magnetic resonance imaging,MRI)、光声成像(Photoacoustic imaging,PAI)成像的能力;于体内实验观察PLGA-PB-PTX-PEG-FA纳米复合物对裸鼠MDA-MB-231移植瘤的靶向效果,及PA/MRI多模态成像效果,了解PLGA-PB-PTX-PEG-FA纳米复合物增强PA/MRI的能力及原理。方法1.选取10只MDA-MB-231移植瘤裸鼠进行小动物活体荧光成像,随机分为两组:靶向纳米复合物(DIR/PLGA-PB-PTX-PEG-FA)组和非靶向纳米复合物(Di R/PLGA-PB-PTX-PEG)组,每只裸鼠经尾静脉缓慢注射0.2m L(20mg/m L)纳米复合物。注射成功后分别于1h、2h、4h、6h及24h利用小动物活体荧光成像系统采集图像。对比观察两组肿瘤局部的荧光强度,图像的定量分析利用Living Image软件系统测得。待24h成像完毕后,处死裸鼠,解剖肿瘤、心、肝、脾、肺、肾、脑,行离体荧光成像,并计算各部位离体荧光强度。2.配置不同浓度的PLGA-PB-PTX-PEG-FA纳米复合物和PLGA-PTX-PEG-FA,对照组为双蒸水,使用Vevo?LAZR光声成像系统和3.0T磁共振扫描仪分别采集光声图像和T1*WI图像。应用光声系统自带软件测量各样品的光声值。应用系统软件测定各样本信号强度,计算各样品的信号增强百分比。建立裸鼠MDA-MB-231移植瘤模型,选择15只随机分为三组,经尾静脉分别缓慢注射靶向纳米复合物(PLGA-PB-PTX-PEG-FA)、非靶向纳米复合物(PLGA-PB-PTX-PEG)及生理盐水。注射前及注射后1h、2h、4h、6h及24h利用光声成像仪采集图像。利用仪器自带软件对注射试剂前后的肿瘤感兴趣区的图像进行光声信号定量分析,计算各样品的光声信号比率。同样的分组方法,利用Philips 3.0T超导型磁共振仪和小动物线圈采集肿瘤在不同时间的T1WI图像,测量各时间点增强前后同一层面裸鼠肿瘤及大腿肌肉感兴趣区的信号强度值(Signal intensity,SI)SItumor及SImuscle,计算相对信号强度(Relative signal intensity,SIr)及信号强度增强率(Percentage of signal intensity enhancement,PSIE)结果1.结果显示注射前两组均未见荧光显示,在尾静脉注射靶向纳米复合物后,靶向组肿瘤内部、脾、脑、脊柱可见Di R标记的靶向纳米复合物聚集发出的红色荧光,在注射后1h荧光最强。随时间延长,肿瘤、脊柱、脑部的荧光强度逐渐减低,而肝区的荧光强度逐渐加强。在注射后24h,肿瘤区域、脾和肝区仍可见少量荧光显示。而尾静脉注射非靶向纳米复合物后,非靶向组肿瘤内部没有荧光显示,而肝区见荧光显示,24h后消失。离体活体荧光可见两组的肝、脾、肺均有荧光显示,但只有靶向组的肿瘤有荧光显示,非靶向组的肿瘤没有荧光聚集。Living Image软件系统分析靶向组在体和离体肿瘤及离体脾组织区域荧光强度均明显高于非靶向组,P0.05,差异具有统计学意义。2.体外光声显影结果和体外MRI结果显示,与不含PB NPs的纳米粒(PLGA-PTX-PEG-FA)及双蒸水相比,PLGA-PB-PTX-PEG-FA纳米复合物可明显增强光声显像和MRI T1显像,且随着PLGA-PB-PTX-PEG-FA纳米复合物浓度的增加,光声显像和MRI显像增强,光声值和MRI信号增强百分比(PSIE)也逐渐增强,与PLGA-PTX-PEG-FA组和双蒸水组相比,P0.05,差异有统计学意义。体内部分,靶向组在注射靶向纳米复合物后,肿瘤区域光声信号和MRI信号在1h最明显,然后逐渐下降,但在注射后24h依然还有明显光声信号和MRI信号。而非靶向组和生理盐水组未见明显光声和MRI增强显影。靶向组光声信号比率和信号强度增强率与其他两组比较,P0.05,差异有统计学意义。结论制备的PLGA-PB-PTX-PEG-FA靶向纳米复合物,具有良好的体内靶向能力,可以特异性靶向至肿瘤区域。其具有体外光声和磁共振显影的性能,并且可增强乳腺癌移植瘤的光声及磁共振显像,是一种有效的多模态成像靶向纳米复合物。第三部分载普鲁士蓝靶向纳米复合物综合治疗裸鼠乳腺癌的研究目的研究PLGA-PB-PTX-PEG-FA靶向纳米复合物联合激光促进药物释放对裸鼠MDA-MB-231移植瘤的综合治疗效果。方法选取35只MDA-MB-231移植瘤裸鼠,随机分为7组:PBS对照组(control)、单纯紫杉醇药物组(PTX)、不含药物纳米粒组(PLGA-PB-PEG-FA)、靶向纳米复合物组(PLGA-PB-PTX-PEG-FA)、不含药物纳米粒+激光辐照组(PLGA-PB-PEG-FA+NIR)、靶向纳米复合物+激光辐照组(PLGA-PB-PTX-PEG-FA+NIR)和单纯激光辐照组(NIR)。各组给予尾静脉注射相对应试剂,其中PLGA-PB-PEG-FA+NIR和PLGA-PB-PTX-PEG-FA+NIR组在注射试剂后1h左右,用808nm输出功率0.647 W/cm2的激光辐照肿瘤区域10min。激光辐照组则仅用上述参数激光辐照肿瘤区域10min。用红外成像仪检测上述处理导致的温度变化,每10s记录一次温度。定期测量肿瘤体积大小,计算相对肿瘤体积(The relative tumor volumes,RTV)。于治疗后21d处死裸鼠,剥取肿瘤组织并称取质量,计算肿瘤抑瘤率(Tumor inhibition rate,TIR)。肿瘤细胞的增殖和凋亡分别用免疫组化PCNA法和TUNEL法检测,计算肿瘤细胞增殖指数(Proliferation index,PI)和凋亡指数(Apoptotic index,AI)。HE染色查看各组裸鼠肿瘤坏死情况及PLGA-PB-PTX-PEG-FA+NIR组和对照组裸鼠的心、肝、脾、肺、肾病理切片情况。结果尾静脉注射PLGA-PB-PTX-PEG-FA靶向纳米复合物后1h,激光辐照肿瘤区域后,肿瘤表面的温度迅速上升,上升至52±3.05℃。PLGA-PB-PTX-PEG-FA+NIR组荷瘤裸鼠的肿瘤在处理后3天结痂,处理后12天明显缩小,到处理后第21天,未见明显增长,肿瘤体积与其他各组相比缩小最明显,相对肿瘤生长曲线呈逐渐下降的趋势。其他各组肿瘤均有不同程度的增长。除了NIR组,PLGA-PB-PEG-FA组和对照组,其他各处理组相对肿瘤体积两两比较P0.05,差异具有统计学意义。PLGA-PB-PTX-PEG-FA+NIR组表现出明显的抑瘤效果,质量抑瘤率为98.86%,其他各组抑瘤率依次下降。PCNA和TUNEL结果显示PLGA-PB-PTX-PEG-FA+NIR组PI指数最低,AI指数最高。除了NIR组,PLGA-PB-PEG-FA组和对照组,其他各处理组PI指数及AI指数分别两两比较P0.05,差异具有统计学意义。肿瘤组织HE染色,PLGA-PB-PTX-PEG-FA+NIR组可见大量坏死,镜下呈红色片状无结构区域。PLGA-PB-PTX-PEG-FA+NIR组的心、肝、脾、肺、肾和对照组对比,HE染色未见明显异常。结论制备的PLGA-PB-PTX-PEG-FA靶向纳米复合物能够靶向至肿瘤区域,结合光热和化疗作用,增加肿瘤综合治疗效果。其有能力成为光声、磁共振多模态显影剂及影像介导下结合化疗和光热综合肿瘤治疗的靶向纳米物质。
[Abstract]:The first part of the preparation and performance detection of Prussian blue targeted nanocomposites 1. preparation of a Prussian blue targeting nanocomposite (PLGA-PB-PTX-PEG-FA), and detection of its basic properties.2. study the target ability of Prussian blue target to nano composite objects and the effect of combined light excitation on cell activity. Method 1. PEG connection The PLGA of folic acid (FA) was used as a film forming material, and a double emulsification method was used to prepare the PLGA-PB-PTX-PEG-FA target of Prussian blue nanoparticles (Prussian blue nanoparticles, PB NPs) and paclitaxel (Paclitaxel, PTX), and the particle size, potential and surface morphology were detected, and UV visible spectrophotometry was used. The UV absorption spectrum, infrared spectrum and the structure of each component were detected by laser confocal microscopy, and the photothermal characteristics in vitro were detected by 0.647 W/cm2808nm laser irradiation. The encapsulation efficiency and drug loading of paclitaxel in nanocomposites were detected by high performance liquid chromatography with.2. in vitro culture of breast cancer cell line MDA-MB-2 31, the target ability of the PLGA-PB-PTX-PEG-FA target to the outer cells of nanocomposite objects was evaluated by laser confocal microscopy. The toxicity of the target nanocomposite combined with laser irradiation on the cells was detected by CCK8. Results the PLGA-PB-PTX-PEG-FA nanocomposites obtained by 1. transmission electron microscopy and scanning electron microscopy were spherical, shape rule, size Shang Junyun, The particle size of PLGA-PB-PTX-PEG-FA nano composite was detected by a good dispersion.Malvern laser particle size analyzer (236.6 + 55.04), and the surface potential was (-24.44 + 1.7) m V. UV VIS spectrophotometer. The PLGA-PB-PTX-PEG-FA nanocomposite and PB NPs absorption wave peak were located near the 702nm. The Fourier infrared spectrogram results showed that PLGA-PTX-PEG-FA The absorption peaks of PB NPs and PB NPs are located at 1756.23 cm-1 and 2086 cm-1, while PLGA-PB-PTX-PEG-FA nanocomposites have the above two characteristic peaks. The laser confocal microscope shows that PB NPs is excited by green fluorescence, PLGA-PTX-PEG-FA is red fluorescence, and PLGA-PB-PTX-PEG-FA nanocomplex is orange yellow fluorescence. Under laser irradiation, PLGA-PB-PTX -PEG-FA nanocomposites have good photothermal properties and photothermal stability. The photothermal conversion efficiency of nanocomposites increases with the increase of the concentration of PLGA-PB-PTX-PEG-FA nanocomposites or the increase of laser energy. The encapsulation efficiency of paclitaxel in PLGA-PB-PTX-PEG-FA nanocomposites is 77.82% and the drug loading is 7.2 by HPLC. 2%, with good release characteristics, the release of.2. in vitro could be accelerated by laser irradiation. The target experiment showed that the red fluorescent target was attached to the surface of MBA-MD-231 breast cancer cells, but not in the target group, and the two groups in the receptor closed group had almost no red fluorescent nanocomposite.CCK8 method. The PLGA-PB-PTX-PEG-FA combined laser irradiated group MBA-MD-231 has the lowest cell activity, which proves that it has the effect of comprehensive chemotherapy and photothermal treatment in vitro. Conclusion the spherical, regular, uniform and stable PLGA-PB-PTX-PEG-FA targeted nanocomposites have been successfully prepared. The nanocomposites have good optical absorption properties and light. Heat conversion characteristics, high drug loading, good targeting ability for MBA-MD-231 breast cancer cells in vitro, combined laser irradiation, with good combination of chemotherapy and photothermal treatment of tumor cells in vitro. Second part of Prussian blue targeted nanocomposite objects in the target, photoacoustic / MRI multi-modal imaging experimental research purposes Observe the ability of PLGA-PB-PTX-PEG-FA nanocomposite objects to enhance magnetic resonance imaging (Magnetic resonance imaging, MRI), photoacoustic imaging (Photoacoustic imaging, PAI) imaging, and to observe the target effect of PLGA-PB-PTX-PEG-FA nanocomposites on MDA-MB-231 transplanted tumor in nude mice in vivo, and the effect of PA/MRI multimodal imaging, and to understand PLGA-PB-PTX-P The ability and principle of EG-FA nanocomposites enhanced PA/MRI. Methods 1. selected 10 MDA-MB-231 xenografts in nude mice in vivo fluorescence imaging, randomly divided into two groups: target nanocomposite (DIR/PLGA-PB-PTX-PEG-FA) group and non target nanocomposite (Di R/PLGA-PB-PTX-PEG) group, each nude mice were injected slowly via the tail vein of 0.2m L (20mg/m) L) nanocomposites. After the injection, the images were collected from 1H, 2h, 4h, 6h and 24h using the living fluorescent imaging system of small animals. The fluorescence intensity of the two groups was observed. The quantitative analysis of the images was measured by the Living Image software system. After the 24h imaging was completed, the tumor, the heart, the liver, the spleen, the kidney, the brain, and the brain were dissected. Optical imaging was used to calculate the fluorescence intensity of different parts of each part of.2. with different concentrations of PLGA-PB-PTX-PEG-FA nanocomposites and PLGA-PTX-PEG-FA. The control group was double water. The Vevo? LAZR photoacoustic imaging system and the 3.0T magnetic resonance scanner were used to collect the photoacoustic images and T1*WI images respectively. The photoacoustic values of the samples were measured by the photoacoustic system self band software. The signal intensity of each sample was measured by system software, and the percentage of signal enhancement in each sample was calculated. A model of MDA-MB-231 transplanted tumor in nude mice was established. 15 rats were randomly divided into three groups, and the target nano complex (PLGA-PB-PTX-PEG-FA), non target nanocomposite (PLGA-PB-PTX-PEG) and physiological saline were injected through the tail vein respectively. 1H, 2h, 4h, 6h and 24h are used to collect images using a photoacoustic imaging instrument. The instrument is used to carry out a quantitative analysis of the photoacoustic signal of the tumor in the region of interest before and after the injection of the injection reagent and calculate the ratio of the photoacoustic signal to each sample. The same grouping method uses the Philips 3.0T superconductance MRI and the small animal coils to collect tumors in no T1WI images at the same time were used to measure the signal intensity (Signal intensity, SI) SItumor and SImuscle at the same level of nude mice and thigh muscles before and after each time point enhancement, and the relative signal intensity (Relative signal intensity, SIr) and the enhancement of signal intensity were calculated (Percentage of) results 1. The results showed that there was no fluorescence in the two groups before the injection. After the injection of the target nano complex in the tail vein, the target group of the tumor, the spleen, the brain and the spinal column showed the red fluorescence of the Di R labeled nanocomposite aggregation. The fluorescence intensity of the 1H was strongest after the injection. The fluorescence intensity of the tumor, spinal column and brain gradually decreased with time, and the liver region was gradually reduced. The fluorescence intensity was gradually strengthened. After injection of 24h, the tumor area, the spleen and the liver still showed a small amount of fluorescence. After the injection of the non targeting nanocomposite, the non targeting group had no fluorescent display in the non targeting group, while the liver region showed fluorescence display, and then disappeared after 24h. The fluorescence of two groups of liver, spleen and lung were shown in the isolated living body, but only the target was targeted. The tumor of the group had fluorescent display, the tumor of the non target group had no fluorescence aggregation.Living Image software system analysis, the fluorescence intensity of the target group in the target group and the isolated tumor and the isolated spleen tissue were significantly higher than that of the non targeting group, P0.05, the difference was statistically significant in.2. in vitro photoacoustic development and in vitro MRI results, with the non PB NPs content. Compared with the rice grain (PLGA-PTX-PEG-FA) and double water, the PLGA-PB-PTX-PEG-FA nanocomposites can obviously enhance the photoacoustic imaging and MRI T1 imaging, and with the increase of the PLGA-PB-PTX-PEG-FA nanocomposite concentration, the photoacoustic imaging and MRI imaging are enhanced, and the percentage of the photoacoustic and MRI signal enhancement (PSIE) is also enhanced gradually, with the PLGA-PTX-PEG-FA group and the double steam water. Compared with the group P0.05, the difference was statistically significant. In the body part, the target group was most obvious in the tumor area after injection of the targeted nanocomplex, and the tumor region photoacoustic signal and MRI signal were most obvious in 1H, and then decreased gradually, but there was still obvious photoacoustic signal and MRI signal after the injection, but no obvious photoacoustic and MRI enhanced development was not found in the target group and the normal saline group. Compared with the other two groups, the ratio of the photoacoustic signal to the target group and the enhancement rate of the signal intensity were compared with the other two groups. The difference was statistically significant. Conclusion the prepared PLGA-PB-PTX-PEG-FA targeted nanocomposite has good target ability in vivo and can specifically target to the tumor area. It has the performance of photoacoustic and magnetic resonance imaging in vitro, and can enhance milk. Photoacoustic and magnetic resonance imaging of adenocarcinoma xenograft is an effective multi-modal imaging target nanocomposite. The study of the third part of Prussian blue targeting nanocomposite in the treatment of breast cancer in nude mice: a study of PLGA-PB-PTX-PEG-FA targeted nanocomposite combined with laser induced drug release for nude mice MDA-MB-231 xenografts Methods 35 MDA-MB-231 transplanted nude mice were randomly divided into 7 groups: PBS control group (control), simple paclitaxel group (PTX), no drug nanoparticles group (PLGA-PB-PEG-FA), targeted nanocomposite group (PLGA-PB-PTX-PEG-FA), no drug nanoparticles + laser irradiation group (PLGA-PB-PEG-FA+NIR), and target nanocomposite + excitation Light irradiation group (PLGA-PB-PTX-PEG-FA+NIR) and laser irradiation group (NIR). Each group was given a relative test agent in the tail vein, of which the PLGA-PB-PEG-FA+NIR and PLGA-PB-PTX-PEG-FA+NIR groups were about 1h after the injection of the reagent, and the laser irradiation group of the tumor area 10min. laser irradiated by the laser output power of 0.647 W/cm2 was only used for the above parameters. The tumor area 10min. was used to detect the temperature changes caused by the above treatment by infrared imager. The tumor volume was measured at a time of 10s. The volume of tumor was measured regularly and the relative tumor volume (The relative tumor volumes, RTV) was calculated. After the treatment, 21d was killed in nude mice, the tumor tissue was stripped and the mass was called, and the tumor suppressor rate (rate, T) was calculated. T (Tumor inhibition rate, T) IR). The proliferation and apoptosis of tumor cells were detected by immunohistochemistry PCNA method and TUNEL method respectively. The tumor cell proliferation index (Proliferation index, PI) and apoptosis index (Apoptotic index, AI).HE were calculated to examine the tumor necrosis in nude mice and the heart, liver, spleen, lung and kidney pathology of the mice in the PLGA-PB-PTX-PEG-FA+NIR group and the control group. Results after the PLGA-PB-PTX-PEG-FA targeted nanocomposite was injected into the tail vein 1H, the tumor surface temperature increased rapidly after laser irradiation, and increased to 52 + 3.05 C.PLGA-PB-PTX-PEG-FA+NIR group of tumor bearing nude mice after 3 days after treatment. After treatment, the tumor was markedly reduced, and no significant growth was found on the twenty-first day after treatment. Tumor body had not been increased. The volume of the product decreased most obviously compared with the other groups, and the relative tumor growth curve decreased gradually. Other groups had different degrees of growth. In the group of NIR, group PLGA-PB-PEG-FA and control, the other groups were compared with 22 of the tumor volume, P0.05, and the difference had the significance of the group.PLGA-PB-PTX-PEG-FA+NIR group. The tumor suppressor effect was 98.86%, and the tumor inhibition rate in other groups decreased by.PCNA and TUNEL. The PI index in PLGA-PB-PTX-PEG-FA+NIR group was the lowest, and the AI index was the highest. The PI index and AI index of other treatment groups were 22 compared with the NIR group and the control group, and the difference was statistically significant. HE staining, group PLGA-PB-PTX-PEG-FA+NIR showed a large number of necrosis, under the microscope, the heart, liver, spleen, lung, kidney and the control group were compared with the red flaky unstructured area.PLGA-PB-PTX-PEG-FA+NIR group, and the HE staining was not obvious. Conclusion the prepared PLGA-PB-PTX-PEG-FA targeted nanocomposites can be targeted to the tumor area, combined with the effect of photothermal and chemotherapy. Combined with tumor combined therapy, it has the ability to become a target nanomaterial with photoacoustic, magnetic resonance multimodal developer and imaging mediate combined with chemotherapy and photothermal integrated tumor therapy.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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