RGD偶联PAMAM树形分子—金纳米棒探针基础上的恶性黑素瘤在体靶向和光热治疗

发布时间：2018-05-10 21:27

本文选题：黑素瘤 + RGD多肽　；参考：《安徽医科大学》2010年博士论文

【摘要】： 背景早期诊断和治疗是肿瘤研究中的关键科学问题。纳米科技的快速发展为解决这一关键科学问题带来了新机遇。具有独特等离子共振效应的金纳米棒粒子有潜力应用于肿瘤的早期诊断和治疗。目的本研究将利用树形分子取代金纳米棒表面的毒性分子十六烷基三乙基溴化铵(CTAB),然后与精氨酸-甘氨酸-天冬氨酸多肽(RGD)偶联构建生物相容性好的靶向肿瘤细胞和肿瘤血管内皮细胞的生物探针,并解决在病变局部选择性治疗的理论问题,旨在为黑素瘤的早期诊断和治疗提供新的策略。方法首先应用“晶种生长”法合成均一的金纳米棒粒子(GNRs),然后利用巯基改性的G4.0聚酰胺胺(PAMAM)树形分子替代金纳米棒表面的CTAB分子。PAMAM树形分子修饰的纳米粒子与生物配体RGD多肽通过共价键偶联得到具有靶向功能的生物探针(RGD-dGNRs)。利用高分辨率透射电镜、原子力显微镜、核磁共振波谱和紫外-可见吸收光谱等仪器对纳米探针进行表征。选择人脐静脉血管内皮细胞(HUVEC)、黑素瘤A375和乳腺癌MCF-7等细胞系来研究纳米探针的靶向性。应用CCK-8方法评价RGD-dGNRs纳米探针对细胞的毒性作用。利用暗视野显微镜来观察RGD-dGNRs纳米探针与肿瘤细胞结合的敏感性和特异性。同时设立严格的竞争抑制结合实验来推测细胞的结合位点。当肿瘤细胞与RGD-dGNRs纳米探针结合后,应用与金纳米棒纵向等离子共振吸收峰相近的近红外(NIR)808nm激光照射,能量分别采用30 mW,70 mW,110 mW,150 mW四个等级,光斑直径为1.0 mm,照射时间为4分钟。应用Trypan blue(台盼蓝)染色和Calcein-AM染色评价纳米探针吸收激光转化为热能后杀伤肿瘤细胞的效能。将5×106黑素瘤细胞注射到裸鼠右侧大腿皮下,当肿瘤直径达到5mm左右,200μg的RGD-dGNRs纳米探针从尾静脉注入小鼠体内,然后分别在3h, 6h, 9h和12h四个时间点处死3只小鼠,收集血液、肿瘤组织以及心、肝、肾等重要脏器,用电感偶合质谱仪(ICP-Mass)测定各个脏器和肿瘤组织的金纳米棒含量,评价RGD-dGNRs纳米探针在动物体内的分布状况。为推测RGD-dGNRs纳米探针与细胞结合的位点,同时进行竞争抑制结合实验,在注射RGD-dGNRs纳米探针前,给予小鼠过量的RGD多肽(0.5mg)。 30只荷瘤小鼠被随机分为三组,分别为实验组(200μg RGD-dGNRs +NIR激光照射);PBS对照组(PBS+NIR激光照射);空白对照组(未予以任何处理)。纳米探针从小鼠静脉注射6小时后,肿瘤部位予以NIR的808nm激光照射,功率密度24 W/cm2,光斑直径5mm,照射时间5分钟。每周治疗1次,共治疗4次。应用皮肤反射式共聚焦显微镜(RCM)观察肿瘤的组织学和血流变化。通过观察肿瘤体积变化和Kaplan-Meier生存曲线来观察RGD-dGNRs光热治疗对小鼠生存期的影响。结果通过“晶种生长法”制备的金纳米棒(GNRs-CTAB)大小均一,面径比为4.2左右,横向等离子共振吸收峰在520 nm,纵向等离子共振吸收峰在821nm左右。树形分子修饰后的金纳米棒(dGNRs)显示了很好的分散性。RGD-dGNRs纳米探针的纵向等离子共振吸收峰发生了轻度红移(3nm)。RGD-dGNRs纳米探针在生理环境下显示了很好的分散性和稳定性。金纳米棒探针在200ug/ml浓度范围内未见明显的细胞毒性作用,而GNRs-CTAB在浓度在50μg/ml左右即可显示明显的细胞毒性作用。在暗视野显微镜观察下,A375黑素瘤细胞和HUVEC细胞与RGD-dGNRs纳米探针结合后显示明亮的金黄色,不能被水冲洗掉。过量的RGD多肽明显抑制了RGD-dGNRs纳米探针与黑素瘤细胞的结合。另外RGD-dGNRs纳米探针并不与αvβ3整合素阴性乳腺癌细MCF-7胞结合。 A375黑素瘤细胞与RGD-dGNR纳米探针结合30分钟后,在70mW的NIR激光照射下,可见部分细胞被杀死,当激光能量达到110mW时,细胞死亡明显增加。dGNRs纳米粒子孵育或单纯的NIR激光照射并未导致细胞明显死亡。RGD-dGNRs纳米探针与αVβ3整合素阴性的MCF-7乳腺癌细胞孵育,然后在NIR激光照射下,未见明显的细胞死亡。细胞的杀伤情况与RGD-dGNR纳米探针的浓度也密切相关。在110m的激光能量照射下,当RGD-dGNR纳米探针的浓度增加到100ug/ml时,几乎所有的黑素瘤细胞被杀死。因此RGD-dGNRs体外光热治疗的最佳浓度可能为100μg/ml。药代动力学实验显示,随着时间的延长,RGD-dGNRs纳米探针在肿瘤部位的蓄积逐渐增加。6小时后,RGD-dGNRs纳米探针在肿瘤部位可见明显的蓄积,约占金纳米棒总量的17%。过量的RGD多肽几乎完全抑制了RGD-dGNRs纳米探针在肿瘤部位的蓄积。与空白对照组和PBS治疗组相比,实验组小鼠的肿瘤体积在整个观察期间明显变小(P㩳0.01),其中四只小鼠的肿瘤组织几乎完全消失。空白对照组小鼠的平均生存期大约为3周,PBS治疗组的小鼠平均生存期大约为4周,而实验组小鼠的平均生存期大于7周,组间有显著性差异(P =0.006)。Kaplan-Meier曲线也显示RGD-dGNRs纳米探针的光热治疗能明显延长小鼠的生存时间。 RCM实时观察到RGD-dGNRs光热治疗荷瘤小鼠3小时后,肿瘤部位血流明显减少,而对照组小鼠的肿瘤组织血流无明显改变。治疗7周后,RCM观察到RGD-dGNRs纳米探针光热治疗小鼠的肿瘤组织内出现大量的网状或树枝状胶原纤维束,组织学HE染色显示为大量胶原纤维增生,类似疤痕结构。而RCM在对照组小鼠的肿瘤组织内可见大量折射增强的细胞,部分细胞聚集成簇,其对应的HE染色可见大量的黑素瘤细胞。结论本研究成功合成了生物相容好的RGD-dGNRs纳米探针,这些新颖的纳米探针能准确识别活体肿瘤细胞与肿瘤中的新生血管内皮细胞。在NIR激光照射下,纳米探针吸收光能转化为热能将肿瘤血管破坏和肿瘤细胞杀伤。这种纳米复合物探针也适用于其他αvβ3整合素阳性的肿瘤细胞。这些智能化的生物探针在未来的肿瘤分子成像与光热治疗方面具有潜在的应用价值。
[Abstract]:Background Early diagnosis and treatment are key scientific problems in tumor research . The rapid development of nanotechnology brings new opportunities to solve this critical scientific problem . The gold nanorod particles with unique plasma resonance effects have the potential to be applied to early diagnosis and treatment of tumors .

Objective In order to provide a new strategy for the early diagnosis and treatment of melanoma , the cytotoxic molecule cetyl trimethyl ammonium bromide ( CTAB ) of gold nanorod surface was replaced with tree - shaped molecule , and then coupled with the arginine - glycine - aspartic acid polypeptide ( RGD ) to construct a biological probe for targeting tumor cells and tumor vascular endothelial cells .

Methods All gold nanorod particles ( GNRs ) were synthesized by " seed growth " method , and then the CTAB molecules on the surface of gold nanorods were replaced by thiol - modified G4 .

The sensitivity and specificity of RGD - dGNRs nano - probe to tumor cells were studied by using CCK - 8 method . The sensitivity and specificity of RGD - dGNRs probe to tumor cells were evaluated by CCK - 8 method .

5 脳 106 melanoma cells were injected subcutaneously into the right thigh of nude mice . When the diameter of tumor reached 5 mm , 200 渭g of RGD - dGNRs nano - probe was injected from the tail vein into the mice . Then , three mice were sacrificed at 3 h , 6 h , 9 h and 12 h . The distribution of RGD - dGNRs probe was evaluated .

Thirty - six tumor - bearing mice were randomly divided into three groups : experimental group ( 200 渭g RGD - dGNRs + NIR laser irradiation ) , PBS control group ( PBS + NIR laser irradiation ) , PBS control group ( PBS + NIR laser irradiation ) .

Under a dark field microscope , A375 melanoma cells and HUVEC cells were combined with RGD - dGNRs nanoprobes to show bright golden yellow , which could not be washed away by water . Excess RGD polypeptide significantly inhibited the binding of RGD - dGNRs nanoprobes to melanoma cells . The RGD - dGNRs nanoprobes did not bind to 伪v尾3 integrin negative breast cancer cells MCF - 7 .

After 30 minutes of binding of A375 melanoma cells with RGD - dGNR nano - probe , some cells were killed when the laser energy reached 110 mW . The cell death was significantly increased when the laser energy reached 110 mW . The cell killing conditions were also closely related to the concentration of RGD - dGNR nano - probe .

In comparison with the control group and PBS group , the tumor volume of the RGD - dGNRs nano - probe was significantly reduced ( P ? 0.01 ) . The average survival time of the mice in the experimental group was about 4 weeks compared with the blank control group and the PBS treatment group , while the average survival time of the mice in the experimental group was more than 7 weeks , and there was a significant difference between the groups ( P = 0.006 ) . Kaplan - Meier curves also show that the photothermal treatment of RGD - dGNRs nanoprobes can significantly prolong the survival time of mice .

After 7 weeks of treatment , a large number of reticular or dendritic collagen bundles appeared in the tumor tissues of mice with RGD - dGNRs .

Conclusion The biological compatible RGD - dGNRs nanoprobes are successfully synthesized in this study . These novel nano - probes can accurately identify tumor cells and tumor cells in vivo .

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5

【引证文献】