基于纳米泡的前列腺癌超声靶向诊断及治疗的实验研究
发布时间：2018-08-11 19:28
【摘要】：背景目前肿瘤仍然是威胁人类健康的重要疾病,其中前列腺癌在老年男性中为高发的致死性肿瘤疾病之一,其早期诊断和治疗对患者的预后至为重要。在前列腺癌诊断方面,除了常用的物理检查和实验室检查外,影像学方法在早期发现肿瘤中也扮演着越来越重要的作用。特别是近年来多种新技术应用在医学成像领域,使得多角度评价肿瘤生物学行为和患者预后成为可能。但是,多数基于解剖结构的成像技术在发现肿瘤病灶时,患者往往多处于中晚期。而在前列腺癌的治疗方面,手术治疗、放射治疗和药物治疗适合不同适应症的患者,其中化疗作为早期的辅助治疗及晚期的重要治疗方式,发挥着重要的作用。但是,化疗药物由于其具有较强的副作用,如全身毒性反应,因此如何提高化疗药物对病灶的靶向性一直是肿瘤治疗急需解决的问题。超声造影技术是一种通过静脉注射超声造影剂,进而实现增强组织对比度的方法。随着对超声造影剂认识地不断深入,研究者们开发了多种不同种类的超声造影剂。超声纳米泡是一种粒径在1000nm以下的声学造影剂,能够突破传统微米级超声造影剂单纯血池显像的局限,可以用于血管外显像。而通过在纳米泡上搭载针对病灶的特异性配体,特别是针对肿瘤细胞的特异性配体,能够利用肿瘤内皮细胞间隙较大、缺乏基底膜及淋巴循环不良的穿透和滞留增强效应(EPR效应)进入组织间隙进一步与肿瘤细胞结合产生特异性的超声增强信号,实现从分子水平诊断肿瘤的目的。前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)是一种特异性表达于前列腺癌细胞表面的膜蛋白,特别是在雄激素非依赖型前列腺癌和死亡率极高的转移性前列腺癌组织中呈高表达。我们的前期分别将针对PSMA的单克隆抗体、纳米抗体与脂质纳米泡相连,成功构建和制备了针对前列腺癌的靶向纳米泡,并进一步研究证实,与非靶向纳米泡相对比,靶向纳米泡能在体外与前列腺癌细胞特异性结合,在体内超声显像实验中表现为肿瘤显像时间延长、峰值强度增高等特异性显像特征。但是,与已经开发出的多种针对PSMA的配体分子(单克隆抗体、工程化抗体、小分子结合物及我们课题组前期研究的纳米抗体等)不同,分子量更小的适体分子是一种能够与其他物质进行结合的核苷酸或者脱氧核苷酸序列,以其作为靶向纳米泡的配基分子,既能够避免像单克隆抗体、工程化抗体产生的免疫原性,又能避免生产过程中接触到的生物活性物质,保证了其无毒、安全的特点。因此,将A10-3.2适体作为配基搭载在脂质纳米泡上构建出粒径小、性能稳定、安全性好的针对前列腺癌的靶向纳米泡将是我们需要进一步深入研究的内容。超声技术同时在肿瘤的治疗中发挥着重要的作用,如高强度聚焦超声在临床上用于治疗子宫肌瘤取得良好的效果。但是在治疗体内深部肿瘤时,需要考虑到超声能量对声通道上组织的热损伤及其肿瘤周围神经的破坏。而低强度超声下联合超声微泡产生的超声微泡靶向破坏技术,相对来说较为安全,能够在需要作用的靶部位应用超声定点破坏超声造影剂,从而促进辐照区域组织对药物的吸收,该技术目前已经在多个疾病领域(包括心血管、眼和肿瘤等相关疾病)中进行了较为广泛的研究探索。相比于微米尺寸的超声造影剂,粒径更小的纳米泡具有更强的穿透能力、更好的稳定性能等特点,但是超声辐照下纳米泡破裂能否产生空化效应促进阿霉素(Doxorubin,DOX)抑制前列腺癌生长仍需进一步研究和阐述。基于上述研究进展和我们前期的研究基础,本将分别从两部分进行基于纳米泡的前列腺癌超声靶向诊断及治疗的实验研究,首先将A10-3.2适体作为配基搭载在脂质纳米泡上,构建出粒径小、安全性好、性能稳定的靶向纳米泡,研究其在PSMA阳性表达的前列腺癌中靶向显像的能力和效果;其次进一步探索纳米泡在低强度超声爆破技术下辅助广谱、经典的化疗药物-DOX抑制前列腺癌的效果。目的1.研究纳米泡的超声显像能力及其在肿瘤组织中的穿透力,同时制备携载针对PSMA的适体A10-3.2的靶向超声纳米泡,并研究其在诊断前列腺癌中的显像效果,为前列腺癌的靶向超声分子显像提供一种穿透力强、安全、高效的靶向造影剂,也为携载适体的靶向超声纳米泡的相关研究提供方法。2.在基于纳米泡的前列腺癌靶向超声分子显像的基础上,进一步探讨化疗药物DOX在低强度超声靶向破坏纳米泡技术下对前列腺癌生长的抑制作用及其潜在机制,为超声辐照下纳米泡破裂促进阿霉素抑制前列腺癌提供详实的研究基础,也为靶向纳米泡真正实现肿瘤诊疗一体化功能奠定实验基础。方法1.以PSMA适体A10-3.2为导向的脂质纳米泡在前列腺癌诊断中的研究(1)将一定比例的脂质材料在水合液中进行机械震荡,并采用离心漂浮法分离超声纳米泡,在动物体内观察纳米泡注射前后彩色血流信号的变化情况。在光镜及共聚焦显微镜下观察生理盐水灌注后,纳米泡在肿瘤组织及心脏组织中的分布情况。(2)化学法合成氟化的适体A10-3.2,通过细胞免疫荧光及流式细胞技术验证适体对PSMA阳性表达细胞的特异性。随后,通过酰胺反应将A10-3.2适体连接到纳米泡表面构建靶向纳米泡,采用凝胶电泳及免疫荧光验证纳米泡的靶向性。(3)检测靶向纳米泡与非靶向纳米泡的大小及体外超声显像情况,溶血实验验证靶向纳米泡的安全性。在细胞水平,通过流式细胞技术和体外结合实验观察靶向纳米泡与细胞的结合能力。(4)构建C4-2和PC-3前列腺癌移植瘤裸鼠模型,观察靶向纳米泡和非靶向纳米泡的超声造影参数(达峰时间、峰值强度、减半时间及1/2峰值强度曲线下面积)的变化情况,以及通过活体荧光显像观察靶向纳米泡在荷瘤小鼠中的分布情况。2.脂质纳米泡在超声靶向破坏技术下辅助阿霉素抑制前列腺癌生长的实验研究(1)在动物PC-3前列腺癌移植瘤中通过超声造影观察纳米泡在不同强度的超声功率(1W/cm2、1.75W/cm2和2.5W/cm2)下,超声辐照的肿瘤区域纳米泡的破坏情况,从而确定超声参数,包括超声功率、辐照时间和辐照模式。(2)将荷PC-3前列腺癌的裸鼠分为阿霉素组(DOX)、阿霉素加纳米泡组(DOX+NB)、阿霉素加超声组(DOX+US)、超声辐照下阿霉素加纳米泡组(DOX+NB+US)共四组。于处理后6小时处死裸鼠,取心、肝、脾、肺、肾和移植瘤组织匀浆后取上清,通过各自组织的阿霉素标准定量曲线中计算各组药物含量。(3)将PC-3前列腺癌细胞分为DOX、DOX+NB、DOX+US和DOX+NB+US共四组,进行分别干预,采用CCK-8法测定细胞生存率。在上述荷瘤裸鼠动物分组基础上增加生理盐水组(Control)共5组。超声参数保持不变,治疗每隔1天处理1次,共处理9次,并监测裸鼠体重、肿瘤体积变化情况。对治疗结束的裸鼠心肌和肿瘤组织进行HE染色和TUNEL染色分析凋亡情况。(4)按照Control、NB、US和NB+US对PC-3细胞和移植瘤进行分组处理,通过扫描电镜观察细胞表面形态,透射电镜观察眶后静脉注射硝酸镧电子示踪剂后的各组肿瘤内电子示踪剂分布情况。结果1.以PSMA适体A10-3.2为导向的脂质纳米泡在前列腺癌诊断中的研究(1)注射纳米泡后,动物腹部血流信号显示更为敏感清晰;光镜和激光共聚焦技术均发现纳米泡能够进入肿瘤血管间隙,而不能进入心肌组织间隙。(2)免疫组化技术和流式细胞技术证实适体分子A10-3.2对PSMA阳性表达的细胞具有良好的特异性。凝胶电泳和免疫荧光技术证实靶向纳米泡构建成功,溶血实验表明靶向纳米泡对红细胞没有明显的溶血作用。(3)非靶向纳米泡和靶向纳米泡的粒径分别为(519.4±74.6)nm和(576.6±40.2)nm,两者体外显像效果没有明显差别(P0.05),靶向纳米泡能够与PSMA阳性表达的C4-2细胞发生特异性结合,而与PSMA阴性表达的PC-3细胞不能结合。(4)通过在PSMA阳性表达的动物移植瘤中比较靶向纳米泡和非靶向纳米泡的超声造影指标,发现达峰时间没有显著差异(P0.05),而峰值强度、减半时间和1/2峰值强度下曲线面积存在明显差异[分别为(19.48±2.59)vs(16.86±2.64)d B,(862.53±143.83)vs(347.69±74.86)s和(1978.60±370.21)vs(795.60±115.41)d B·s,P0.05];PSMA阴性表达的动物移植瘤中两种纳米泡的四种造影指标均无明显差异(P0.05)。同时,小动物活体荧光显像表明靶向纳米泡在C4-2移植瘤中具有一定的聚集能力。2.脂质纳米泡在超声靶向破坏技术下辅助阿霉素抑制前列腺癌生长的实验研究(1)用于治疗得到的纳米泡粒径为(485.7+33.0)nm,多分散系数为0.026。确定在1W/cm2超声功率下、100Hz的间歇脉冲及手动调节下“5s-on-5s-off”的辐照模式,能够在15min内有效地破坏肿瘤辐照区域内的纳米泡且不会产生明显的热效应。(2)药物分布情况测定时,阿霉素在DOX+NB+US组[(43.71±5.03)ng/g]的移植瘤中分布最多,其次为DOX+US组[(36.91±5.72)ng/g];而心肌组织中药物含量与此相反,在DOX+NB+US组[(13.49±3.14)ng/g]中分布最少。(3)在治疗效果评价的观察中,发现相比于Control组,DOX+NB+US组的前列腺癌细胞生存率[(43.16±2.47)%]和移植瘤体积[(68.16±14.00)mm3]明显受到抑制,超声造影治疗前后的强度变化及病理切片的亦证实DOX+NB+US组坏死情况最为明显。(4)在扫描电镜观察中,发现NB+US组前列腺癌细胞上出现较多孔洞,且细胞表面的褶皱明显增多;而透射电镜亦证实NB+US组有更多的硝酸镧颗粒进入移植瘤的实质细胞和组织间隙内。结论1.纳米泡具有良好的超声显像效果,能够利用肿瘤的EPR效应进入肿瘤组织间隙,为实现肿瘤实质细胞的靶向超声显像奠定了造影剂方面的基础。2.携载针对PSMA的适体A10-3.2的靶向纳米泡能在体外与PSMA阳性表达的前列腺癌细胞靶向结合,能使PSMA阳性表达的前列腺癌移植瘤具有特征性超声分子显像特征,为肿瘤的靶向超声分子诊断提供了一种新的超声造影剂和方法。3.超声辐照下纳米泡靶向破坏技术是一种能有效促进药物作用特定部位的方法,能够有效增加化疗药物在肿瘤区域的含量,减少化疗药物副作用,并发挥有效抑制前列腺癌生长的作用,其背后的潜在机制可能与纳米泡能穿过肿瘤血管、进入肿瘤组织间隙内,在肿瘤细胞周围近距离发生超声空化效应相关。
[Abstract]:Background Nowadays, cancer is still an important disease threatening human health. Prostate cancer is one of the most common fatal cancer diseases in elderly men. Early diagnosis and treatment of prostate cancer is very important to the prognosis of patients. Tumors are also playing an increasingly important role. Especially in recent years, many new techniques have been applied in the field of medical imaging, making it possible to evaluate tumor biological behavior and prognosis from multiple perspectives. Surgical treatment, radiotherapy and drug therapy are suitable for patients with different indications. Chemotherapy, as an important adjuvant therapy in the early and late stages, plays an important role. Targeting has always been an urgent problem in tumor therapy. Contrast-enhanced ultrasound (CEUS) is a method to enhance tissue contrast by intravenous injection of CEUS. With the deepening understanding of CEUS, researchers have developed a variety of CEUS agents. Ultrasound nanobubbles are a kind of ultrasound contrast agents with a diameter of 1000 nm. The following ultrasound contrast agents can break through the limitation of traditional micron-scale ultrasound contrast agent blood pool imaging and can be used for vascular imaging. Poor circulatory penetration and retention enhancement effect (EPR effect) enters the tissue gap and further binds with tumor cells to produce specific enhanced ultrasound signals for molecular diagnosis of cancer. prostate specific membrane antigen (PSMA) is a specific expression on prostate cancer cell surface. Facial membrane proteins are highly expressed, especially in androgen-independent prostate cancer and metastatic prostate cancer with high mortality. We successfully constructed and prepared targeted nanobubbles for prostate cancer by linking monoclonal antibodies against PSMA and nanoantibodies to lipid nanobubbles, respectively, in our previous studies. In contrast to non-targeted nanobubbles, targeted nanobubbles can specifically bind to prostate cancer cells in vitro and exhibit specific imaging characteristics in vivo, such as prolonged tumor imaging time and increased peak intensity. Different from small molecular conjugates and nano-antibodies previously studied by our team, aptamers with smaller molecular weights are nucleotide or deoxynucleotide sequences that can bind to other substances and serve as ligand molecules for targeting nanobubbles, thus avoiding immunogenicity such as monoclonal antibodies and engineered antibodies. Therefore, using aptamer A10-3.2 as ligand to construct lipid nanobubbles with small size, stable performance and good safety will be the content of further research. Surgery also plays an important role in the treatment of tumors, such as high intensity focused ultrasound (HIFU) in the clinical treatment of uterine fibroids achieved good results. But in the treatment of deep tumors in vivo, we need to take into account the ultrasound energy on the sound channel tissue thermal damage and the destruction of the tumor peripheral nerves. Targeted destruction of ultrasound microbubbles produced by ultrasound microbubbles is relatively safe and can be used to destroy ultrasound contrast agents at targeted sites where they are needed, thus promoting the absorption of drugs by irradiated tissue. This technology has been compared in a number of disease areas (including cardiovascular, eye and tumor related diseases). Compared with micron-sized ultrasound contrast agents, the smaller size nanobubbles have stronger penetration ability and better stability. However, whether the cavitation effect of nanobubbles rupture under ultrasound irradiation can promote doxorubin (DOX) inhibiting prostate cancer growth still needs further study and elaboration. In this study, we will carry out ultrasound targeted diagnosis and treatment of prostate cancer based on nanobubbles in two parts. Firstly, aptamer A10-3.2 was loaded on lipid nanobubbles as ligand to construct targeted nanobubbles with small particle size, good safety and stable performance, and its PSMA-positive was studied. Objective 1. To study the ultrasound imaging ability of nanobubbles and their penetrating power in tumor tissues, and to prepare PSMA-loaded nanobubbles for PSMA. Targeted ultrasound nanobubbles of body A10-3.2 were used to study the imaging effect of targeted ultrasound nanobubbles in the diagnosis of prostate cancer. It provides a high-penetrating, safe and efficient targeted contrast agent for targeted ultrasound molecular imaging of prostate cancer. It also provides a method for the study of targeted ultrasound nanobubbles carrying aptamers. 2. Targeting prostate cancer based on nanobubbles. On the basis of ultrasound molecular imaging, the inhibitory effect of chemotherapy drug DOX on the growth of prostate cancer and its potential mechanism under low intensity ultrasound targeted destruction of nanobubbles were further explored, which provided a detailed research basis for promoting the inhibition of prostate cancer by adriamycin by nanobubbles rupture under ultrasound irradiation, and also provided a real tumor targeting nanobubbles. Methods 1. Lipid nanobubbles directed by PSMA aptamer A10-3.2 were used in the diagnosis of prostate cancer. (1) Lipid nanobubbles were separated by centrifugal floatation and mechanical vibration in the hydrate solution. The color hemodynamics before and after injection of nanobubbles were observed in vivo. (2) Fluorinated aptamer A10-3.2 was synthesized by chemical method, and the specificity of aptamer to PSMA-positive cells was verified by immunofluorescence and flow cytometry. Afterwards, the amide reaction was performed. The targeted nanobubbles were constructed by attaching aptamer A10-3.2 to the surface of nanobubbles. The targeting of nanobubbles was verified by gel electrophoresis and immunofluorescence. (3) The size of targeted nanobubbles and non-targeted nanobubbles and ultrasound imaging in vitro were detected, and the safety of targeted nanobubbles was verified by hemolysis test. The binding ability of targeted nanobubbles to cells was observed by external binding experiment. (4) The nude mice models of prostate cancer transplanted with C4-2 and PC-3 were constructed to observe the changes of contrast-enhanced ultrasound parameters (peak time, peak intensity, halving time and area under 1/2 peak intensity curve) of targeted nanobubbles and non-targeted nanobubbles, as well as in vivo fluorescence imaging. Objective To observe the distribution of targeted nanobubbles in tumor-bearing mice.2.Lipid nanobubbles assisted doxorubicin in inhibiting the growth of prostate cancer by ultrasound-targeted destruction technique(1)Contrast-enhanced ultrasound was used to observe the effect of nanobubbles on the growth of prostate cancer in animal PC-3 prostate cancer xenografts under different ultrasound power(1W/cm 2,1.75W/cm 2 and 2.5W/cm 2). (2) Nude mice bearing PC-3 prostate cancer were divided into four groups: doxorubicin group (DOX), doxorubicin plus nanobubbles group (DOX+NB), doxorubicin plus ultrasound group (DOX+US), doxorubicin plus nanobubbles group (DOX+NB+US) and doxorubicin plus nanobubbles group (DOX+NB+US). After 6 hours of treatment, the nude mice were sacrificed, the heart, liver, spleen, lung, kidney and transplanted tumor tissues were taken out, and the supernatant of each group was calculated by the standard quantitative curve of adriamycin. (3) PC-3 prostate cancer cells were divided into four groups: DOX, DOX+NB, DOX+US and DOX+NB+US. The cell survival rate was determined by CCK-8 method. On the basis of the above-mentioned grouping of tumor-bearing nude mice, 5 groups were added to the normal saline group (Control). Ultrasound parameters remained unchanged, treatment was given once every other day for 9 times, and the changes of body weight and tumor volume were monitored. PC-3 cells and transplanted tumor were grouped by NB, US and NB+US. The cell surface morphology was observed by scanning electron microscopy. The distribution of electron tracers in tumor was observed by transmission electron microscopy after intravenous injection of lanthanum nitrate. (2) Immunohistochemistry and flow cytometry demonstrated that aptamer A10-3.2 had good specificity for PSMA positive cells. Gel electrophoresis and immunofluorescence techniques confirmed that the targeted nanobubbles were successfully constructed. Hemolysis experiments showed that the targeted nanobubbles had no obvious hemolysis effect on red blood cells. (3) The diameter of non-targeted nanobubbles and targeted nanobubbles were (519.4 (74.6) nm and (576.6 (40.2) nm, respectively. Bubbles can specifically bind to C4-2 cells which are PSMA-positive, but not to PC-3 cells which are PSMA-negative. (4) By comparing the target nanobubbles and non-target nanobubbles in PSMA-positive animal transplanted tumors, we found that there was no significant difference in peak time (P 0.05), peak strength, halving time and non-target nanobubbles. There were significant differences in the area of curves under 1/2 peak intensity [19.48 (+ 2.59) vs (16.86 (+ 2.64) D B, (862.53 (+ 143.83) vs (347.69 (+ 74.86) s) and (1978.60 (+ 370.21) vs (795.60 (+ 115.41) D B (+) s, P 0.05)]; there were no significant differences in the four angiographic indices of the two kinds of nanobubbles in PSMA-negative animals (P 0.05). Fluorescence imaging showed that the targeted nanobubbles had a certain aggregation ability in C4-2 transplanted tumor. 2. Lipid nanobubbles assisted doxorubicin to inhibit the growth of prostate cancer by ultrasound targeted destructive technology (1) The diameter of the nanobubbles was (485.7 + 33.0) nm, and the polydispersity coefficient was 0.026. (2) When the drug distribution was determined, doxorubicin was most distributed in the transplanted tumors of DOX+NB+US group [(43.71+5.03) ng/g], followed by DOX+US group [(36.91+5.72)]. (3) Compared with the control group, the survival rate of prostate cancer cells in the DOX+NB+US group [(43.16+2.47)%] and the volume of transplanted tumor [(68.16+14.00) mm3] were significantly inhibited before contrast-enhanced ultrasound treatment. The changes of intensity and pathological section after transplantation also confirmed that the necrosis was most obvious in DOX+NB+US group. (4) In scanning electron microscopy, more holes were found in prostate cancer cells of NB+US group, and the wrinkles on the cell surface were significantly increased. Transmission electron microscopy also confirmed that more lanthanum nitrate particles entered the transplanted tumor cells and tissues in NB+US group. Conclusion1. Nanobubbles have a good ultrasound imaging effect, can use the EPR effect of tumor into the tumor tissue gap, to achieve tumour. 2.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25;R445.1
[Abstract]:Background Nowadays, cancer is still an important disease threatening human health. Prostate cancer is one of the most common fatal cancer diseases in elderly men. Early diagnosis and treatment of prostate cancer is very important to the prognosis of patients. Tumors are also playing an increasingly important role. Especially in recent years, many new techniques have been applied in the field of medical imaging, making it possible to evaluate tumor biological behavior and prognosis from multiple perspectives. Surgical treatment, radiotherapy and drug therapy are suitable for patients with different indications. Chemotherapy, as an important adjuvant therapy in the early and late stages, plays an important role. Targeting has always been an urgent problem in tumor therapy. Contrast-enhanced ultrasound (CEUS) is a method to enhance tissue contrast by intravenous injection of CEUS. With the deepening understanding of CEUS, researchers have developed a variety of CEUS agents. Ultrasound nanobubbles are a kind of ultrasound contrast agents with a diameter of 1000 nm. The following ultrasound contrast agents can break through the limitation of traditional micron-scale ultrasound contrast agent blood pool imaging and can be used for vascular imaging. Poor circulatory penetration and retention enhancement effect (EPR effect) enters the tissue gap and further binds with tumor cells to produce specific enhanced ultrasound signals for molecular diagnosis of cancer. prostate specific membrane antigen (PSMA) is a specific expression on prostate cancer cell surface. Facial membrane proteins are highly expressed, especially in androgen-independent prostate cancer and metastatic prostate cancer with high mortality. We successfully constructed and prepared targeted nanobubbles for prostate cancer by linking monoclonal antibodies against PSMA and nanoantibodies to lipid nanobubbles, respectively, in our previous studies. In contrast to non-targeted nanobubbles, targeted nanobubbles can specifically bind to prostate cancer cells in vitro and exhibit specific imaging characteristics in vivo, such as prolonged tumor imaging time and increased peak intensity. Different from small molecular conjugates and nano-antibodies previously studied by our team, aptamers with smaller molecular weights are nucleotide or deoxynucleotide sequences that can bind to other substances and serve as ligand molecules for targeting nanobubbles, thus avoiding immunogenicity such as monoclonal antibodies and engineered antibodies. Therefore, using aptamer A10-3.2 as ligand to construct lipid nanobubbles with small size, stable performance and good safety will be the content of further research. Surgery also plays an important role in the treatment of tumors, such as high intensity focused ultrasound (HIFU) in the clinical treatment of uterine fibroids achieved good results. But in the treatment of deep tumors in vivo, we need to take into account the ultrasound energy on the sound channel tissue thermal damage and the destruction of the tumor peripheral nerves. Targeted destruction of ultrasound microbubbles produced by ultrasound microbubbles is relatively safe and can be used to destroy ultrasound contrast agents at targeted sites where they are needed, thus promoting the absorption of drugs by irradiated tissue. This technology has been compared in a number of disease areas (including cardiovascular, eye and tumor related diseases). Compared with micron-sized ultrasound contrast agents, the smaller size nanobubbles have stronger penetration ability and better stability. However, whether the cavitation effect of nanobubbles rupture under ultrasound irradiation can promote doxorubin (DOX) inhibiting prostate cancer growth still needs further study and elaboration. In this study, we will carry out ultrasound targeted diagnosis and treatment of prostate cancer based on nanobubbles in two parts. Firstly, aptamer A10-3.2 was loaded on lipid nanobubbles as ligand to construct targeted nanobubbles with small particle size, good safety and stable performance, and its PSMA-positive was studied. Objective 1. To study the ultrasound imaging ability of nanobubbles and their penetrating power in tumor tissues, and to prepare PSMA-loaded nanobubbles for PSMA. Targeted ultrasound nanobubbles of body A10-3.2 were used to study the imaging effect of targeted ultrasound nanobubbles in the diagnosis of prostate cancer. It provides a high-penetrating, safe and efficient targeted contrast agent for targeted ultrasound molecular imaging of prostate cancer. It also provides a method for the study of targeted ultrasound nanobubbles carrying aptamers. 2. Targeting prostate cancer based on nanobubbles. On the basis of ultrasound molecular imaging, the inhibitory effect of chemotherapy drug DOX on the growth of prostate cancer and its potential mechanism under low intensity ultrasound targeted destruction of nanobubbles were further explored, which provided a detailed research basis for promoting the inhibition of prostate cancer by adriamycin by nanobubbles rupture under ultrasound irradiation, and also provided a real tumor targeting nanobubbles. Methods 1. Lipid nanobubbles directed by PSMA aptamer A10-3.2 were used in the diagnosis of prostate cancer. (1) Lipid nanobubbles were separated by centrifugal floatation and mechanical vibration in the hydrate solution. The color hemodynamics before and after injection of nanobubbles were observed in vivo. (2) Fluorinated aptamer A10-3.2 was synthesized by chemical method, and the specificity of aptamer to PSMA-positive cells was verified by immunofluorescence and flow cytometry. Afterwards, the amide reaction was performed. The targeted nanobubbles were constructed by attaching aptamer A10-3.2 to the surface of nanobubbles. The targeting of nanobubbles was verified by gel electrophoresis and immunofluorescence. (3) The size of targeted nanobubbles and non-targeted nanobubbles and ultrasound imaging in vitro were detected, and the safety of targeted nanobubbles was verified by hemolysis test. The binding ability of targeted nanobubbles to cells was observed by external binding experiment. (4) The nude mice models of prostate cancer transplanted with C4-2 and PC-3 were constructed to observe the changes of contrast-enhanced ultrasound parameters (peak time, peak intensity, halving time and area under 1/2 peak intensity curve) of targeted nanobubbles and non-targeted nanobubbles, as well as in vivo fluorescence imaging. Objective To observe the distribution of targeted nanobubbles in tumor-bearing mice.2.Lipid nanobubbles assisted doxorubicin in inhibiting the growth of prostate cancer by ultrasound-targeted destruction technique(1)Contrast-enhanced ultrasound was used to observe the effect of nanobubbles on the growth of prostate cancer in animal PC-3 prostate cancer xenografts under different ultrasound power(1W/cm 2,1.75W/cm 2 and 2.5W/cm 2). (2) Nude mice bearing PC-3 prostate cancer were divided into four groups: doxorubicin group (DOX), doxorubicin plus nanobubbles group (DOX+NB), doxorubicin plus ultrasound group (DOX+US), doxorubicin plus nanobubbles group (DOX+NB+US) and doxorubicin plus nanobubbles group (DOX+NB+US). After 6 hours of treatment, the nude mice were sacrificed, the heart, liver, spleen, lung, kidney and transplanted tumor tissues were taken out, and the supernatant of each group was calculated by the standard quantitative curve of adriamycin. (3) PC-3 prostate cancer cells were divided into four groups: DOX, DOX+NB, DOX+US and DOX+NB+US. The cell survival rate was determined by CCK-8 method. On the basis of the above-mentioned grouping of tumor-bearing nude mice, 5 groups were added to the normal saline group (Control). Ultrasound parameters remained unchanged, treatment was given once every other day for 9 times, and the changes of body weight and tumor volume were monitored. PC-3 cells and transplanted tumor were grouped by NB, US and NB+US. The cell surface morphology was observed by scanning electron microscopy. The distribution of electron tracers in tumor was observed by transmission electron microscopy after intravenous injection of lanthanum nitrate. (2) Immunohistochemistry and flow cytometry demonstrated that aptamer A10-3.2 had good specificity for PSMA positive cells. Gel electrophoresis and immunofluorescence techniques confirmed that the targeted nanobubbles were successfully constructed. Hemolysis experiments showed that the targeted nanobubbles had no obvious hemolysis effect on red blood cells. (3) The diameter of non-targeted nanobubbles and targeted nanobubbles were (519.4 (74.6) nm and (576.6 (40.2) nm, respectively. Bubbles can specifically bind to C4-2 cells which are PSMA-positive, but not to PC-3 cells which are PSMA-negative. (4) By comparing the target nanobubbles and non-target nanobubbles in PSMA-positive animal transplanted tumors, we found that there was no significant difference in peak time (P 0.05), peak strength, halving time and non-target nanobubbles. There were significant differences in the area of curves under 1/2 peak intensity [19.48 (+ 2.59) vs (16.86 (+ 2.64) D B, (862.53 (+ 143.83) vs (347.69 (+ 74.86) s) and (1978.60 (+ 370.21) vs (795.60 (+ 115.41) D B (+) s, P 0.05)]; there were no significant differences in the four angiographic indices of the two kinds of nanobubbles in PSMA-negative animals (P 0.05). Fluorescence imaging showed that the targeted nanobubbles had a certain aggregation ability in C4-2 transplanted tumor. 2. Lipid nanobubbles assisted doxorubicin to inhibit the growth of prostate cancer by ultrasound targeted destructive technology (1) The diameter of the nanobubbles was (485.7 + 33.0) nm, and the polydispersity coefficient was 0.026. (2) When the drug distribution was determined, doxorubicin was most distributed in the transplanted tumors of DOX+NB+US group [(43.71+5.03) ng/g], followed by DOX+US group [(36.91+5.72)]. (3) Compared with the control group, the survival rate of prostate cancer cells in the DOX+NB+US group [(43.16+2.47)%] and the volume of transplanted tumor [(68.16+14.00) mm3] were significantly inhibited before contrast-enhanced ultrasound treatment. The changes of intensity and pathological section after transplantation also confirmed that the necrosis was most obvious in DOX+NB+US group. (4) In scanning electron microscopy, more holes were found in prostate cancer cells of NB+US group, and the wrinkles on the cell surface were significantly increased. Transmission electron microscopy also confirmed that more lanthanum nitrate particles entered the transplanted tumor cells and tissues in NB+US group. Conclusion1. Nanobubbles have a good ultrasound imaging effect, can use the EPR effect of tumor into the tumor tissue gap, to achieve tumour. 2.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25;R445.1
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