本文主要研究内容
作者张妮（2019）在《载aPD1靶向相变型纳米粒对恶性黑色素瘤光热治疗及增效免疫治疗的研究》一文中研究指出：第一部分载aPD1靶向相变型纳米粒的制备及性能检测目的制备一种载程序性死亡受体1抗体(aPD1)、全氟戊烷(perfluoropentane,PFP)和四氧化三铁(Fe3O4)的靶向纳米粒(GOP@aPD1),检测其粒径电位、稳定性、光热性能、相变特性、药物含量及药物释放等性能。方法首先制备粘附多肽(Gly-Arg-Gly-Asp-Ser,GRGDS)靶向的聚乳酸羟基乙酸/聚乙二醇高分子材料(PLGA-PEG-GRGDS),并鉴定其化学结构。采用改良的三步超声乳化法制备GOP@aPD1纳米粒(nanoparticles,NPs)。检测该纳米粒基本的理化性质,包括粒径、电位、形态结构、吸收光谱、傅里叶红外吸收峰及X射线光电子能谱分析吸收峰。于制备后不同时间点(0.5h,1d,3d,7d)测量纳米粒在PBS及含胎牛血清PBS溶液中粒径变化。流式细胞术检测纳米粒与GRGDS抗体连接情况。光镜下观察不同温度下(室温、37℃和45℃)纳米粒的相变情况。在660nm激光辐照下,检测不同浓度条件下该纳米粒的光热特性。观察660nm激光辐照后纳米粒不同时间点(2min,6min,10min,12min)超声增强信号。用酶联免疫吸附(enzyme-linked immuno sorbent assay,ELISA)测定检测GOP@aPD纳米粒内的aPD1药物含量,以及激光辐照触发aPD1药物释放情况。激光共聚焦观察GOP@aPD纳米粒对aPD1载药及释药情况。结果通过氢谱核磁分析对聚合物PLGA-PEG-GRGDS进行了结构确认。用超声乳化法制备的GOP@aPD1纳米乳呈棕褐色,扫描电镜(scanning electron microscopy,SEM)观察到该纳米粒呈球形结构,大小均一,分散度好。透射电镜(transmission electron microscope,TEM)显示点状Fe3O4颗粒均匀分布在该纳米粒内。马尔文粒径仪测得GOP@aPD1 NPs平均粒径218nm,平均电位为-5mV。在PBS及含10%胎牛血清PBS溶液4℃放置7天,GOP@aPD1 NPs粒径无明显改变,证明该纳米粒稳定性良好。流式细胞术测得对比非靶向纳米粒,GOP@aPD1 NPs与GRGDS抗体连接率更高,证明纳米粒表面修饰了GRGDS多肽。原子吸收光谱法测得GOP@aPD1 NPs中Fe3O4的包封率为63%。光学显微镜观察到,在室温及37℃条件下纳米粒基本保持稳定。但是在45℃纳米粒发生液气相变,体积增大,相互融合并破裂。说明GOP@aPD1 NPs相变温度约45℃。光声仪吸收光谱显示GOP@aPD1 NPs在700nm左右有强吸收峰。根据PLGA-PEG-GRGDS浓度,将GOP@aPD1 NPs稀释为1.25-10mg/mL。在660nm激光辐照下,不同浓度GOP@aPD1 NPs均有升温效果,且升高温度与纳米粒浓度正相关,其中5mg/mL组可升温达45℃左右。在660nm激光连续辐照下,纳米粒超声增强信号随时间呈逐渐上升-下降趋势,辐照10min纳米粒超声增强信号最大,说明其液气相变随激光辐照时间逐渐增强10min达高峰。ELISA测的GOP@aPD1 NPs中aPD1药物的载药量为1.56%(w/w),包封率为98.9%。体外药物释放试验显示,GOP@aPD1 NPs内的aPD1药物在激光辐照下2h内快速释放,6h后药物释放趋平缓。激光共聚焦显微镜观察到,在激光辐照前DiI标记GOP@aPD1 NPs的红色荧光与FITC标记aPD1的绿色荧光共定位呈黄色荧光信号,而在激光辐照后红色荧光与绿色荧光两者分离,说明激光辐照产生的光热效应促使GOP@aPD1 NPs释放其内负载的aPD1药物。结论本实验通过乳化法制备了GOP@aPD1纳米粒,形态规则,大小较均匀,性质稳定,负载aPD1及Fe3O4,具有较好的光热能力及液气相变的能力,能够在660nm激光辐照作用下释放aPD1药物。第二部分载aPD1靶向相变型纳米粒对黑色素瘤靶向性及体内分布研究目的观察GOP@aPD1 NPs在体外、体内对黑色素瘤细胞B16F10靶向性,以及该纳米粒的在体内的生物分布、血液中循环时间及体内生物安全性。方法首先制备GOP@aPD1 NPs与非靶向NPs。在体外实验中,激光共聚焦显微镜分别观察Di I标记的两组纳米粒与DAPI染色的B16F10细胞共定位情况,流式细胞术分别测量Di I标记的两组纳米粒与B16F10细胞孵育不同时间后连接率。CCK8法观察不同浓度(2.5mg/ml,5mg/ml,10mg/ml,15mg/ml)GOP@aPD1 NPs与B16F10细胞孵育后细胞活性变化。在体内实验中,首先建立C57B6鼠B16F10-luc黑色素瘤模型,当肿瘤体积大约长至约100mm3时,经C57B6鼠尾静脉分别注射Di R标记GOP@aPD1 NPs与非靶向NPs。在Xenogen IVIS生物发光成像系统中采集荧光图像,分析肿瘤及重要器官的信号强度。C57B6鼠尾静脉分别注射不同浓度(5mg/ml,10mg/ml)GOP@aPD1 NPs,14天后取小鼠血清分析CK、LDH-L、AST、ALT、BUN及CR水平,观察GOP@aPD1 NPs在体内生物安全性。用PerCP/Cy5.5标记的aPD1制备GOP@aPD1 NPs。经荷瘤C57B6鼠尾静脉分别注射PerCP/Cy5.5标记的GOP@aPD1 NPs及PerCP/Cy5.5标记的游离aPD1药物。注射后不同时间点(6h,10h)取肿瘤及重要器官,制备冰冻切片,在激光共聚焦显微镜下观察各组织切片中PerCP/Cy 5.5红色荧光信号,Image-Pro Plus 6.0软件分析信号强度。小鼠尾静脉注射PerCP/Cy5.5标记的GOP@aPD1 NPs后不同时间点(0.5h,1h,2h,4h,6h,8h,12h,24h)采集小鼠血液,进行荧光分光光度计分析。结果激光共聚焦显微镜观察到Di I标记GOP@aPD1 NPs的红色荧光,较Di I标记非靶向NPs的红色荧光更多的定位在B16F10细胞周围。GOP@aPD1 NPs及非靶向NPs与B16F10细胞孵育后2h及10h后,流式细胞术测得GOP@aPD1 NPs与细胞连接率显著高于非靶向NPs(p<0.01)。不同浓度GOP@aPD1 NPs(2.5mg/ml,5mg/ml,10mg/ml,15mg/ml)与B16F10细胞孵育后细胞活性无显著变化。但B16F10细胞与GOP@aPD1 NPs孵育后再接受激光辐照后细胞活性显著降低,且细胞活性随GOP@aPD1 NPs浓度升高而降低。在体内靶向性实验中,GOP@aPD1 NPs通过高通透性和滞留(enhanced permeability and retention,EPR)效应及主动靶向性在肿瘤部位逐渐富集,小动物活体荧光观察到肿瘤部位荧光信号随时间推移而逐渐增强,在给药6h后达到最高峰、24h后减弱。在非靶向NPs组,小鼠肿瘤部位各时间点荧光信号均显著低于GOP@aPD1 NPs组。在C57B6鼠尾静脉分别注射不同浓度GOP@aPD1 NPs(5mg/ml,10mg/ml)14天后,血清CK、LDH-L、AST、ALT、BUN及CR变化无统计学差异。经荷瘤鼠尾静脉分别注射PerCP/Cy5.5标记的GOP@aPD1 NPs 6h及24h后,通过激光共聚焦显微镜均可观察到肿瘤组织中GOP@aPD1NPs红色荧光信号。但在PerCP/Cy5.5标记的aPD1游离药物组,通过激光共聚焦显微镜几乎不能观察到肿瘤组织中红色荧光信号。肿瘤组织中荧光信号强度分析显示,在6h及24h时间点GOP@aPD1 NPs组肿瘤组织中aPD1荧光强度均显著高于aPD1游离药物组。荷瘤鼠尾静脉注射PerCP/Cy5.5标记的GOP@aPD1 NPs 24h后,仍可通过荧光分光光度计检测血液中荧光信号。GOP@aPD1 NPs在体内循环的半衰期约4.39h。结论制备的GOP@aPD1 NPs可通过EPR效应及主动靶向性在肿瘤组织中富集,实现靶向黑色素瘤递送aPD1药物,并具有较长的体内循环时间及良好的生物安全性,为下一步体内治疗实验奠定了基础。第三部分光热介导载aPD1靶向相变型纳米粒治疗恶性黑色瘤及增效免疫治疗机制研究目的研究光热(Photothermal therapy,PTT)介导GOP@aPD1NPs治疗黑色素瘤的效果,及其增效aPD1免疫治疗的相关机制。方法为了观察光热介导GOP@aPD1 NPs在体内光热效应,建立C57B6鼠B16F10-luc黑色素瘤模型,当肿瘤体积约80mm3时开始给予不同处理。把荷瘤小鼠随机分成3组,对照组(不处理),PTT组及GOP@aPD1+PTT组。其中GOP@aPD1+PTT组先经小鼠尾静脉注射GOP@aPD1 NPs(~200μL,5mg/m L),6h后给予肿瘤部位660nm激光辐照(0.1W/cm2,10min)。PTT组仅给予660nm激光辐照(0.1W/cm2,10min)肿瘤组织。用红外热成像仪观察肿瘤部位的升温情况。小鼠经处理后不同时间点(24h,72h,168h)取血清,通过ELISA分析炎症因子IL-6、TNF-α及IFN-γ变化水平。为了观察光热介导GOP@aPD1 NPs在体内对恶性黑色素瘤治疗作用,首先制备GOP@aPD1 NPs,单载Fe3O4 NPs及单载aPD1 NPs。其中单载Fe3O4 NPs不含aPD1,单载aPD1 NPs不含Fe3O4。建立C57B6鼠B16F10-luc黑色素瘤模型,当肿瘤体积大约80 mm3时开始于不同处理。把小鼠随机分成8组,1.对照组(生理盐水),2.游离aPD1组,3.PTT组,4.游离aPD1+PTT组,5.单载Fe3O4 NPs+PTT组,6.单载aPD1NPs+PTT组,7.GOP@aPD1组,8.GOP@aPD1+PTT组。其中2及7组仅分别静脉注射游离aPD1及GOP@aPD1 NPs;3组仅给予肿瘤部位660 nm激光辐照(0.1W/cm2,10min);4、5、6及8组分别先经小鼠尾静脉注射游离aPD1、单载Fe3O4 NPs、单载aPD1 NPs及GOP@aPD1NPs,6h后给予肿瘤部位660nm激光辐照(0.1W/cm2,10min)。经过不同的处理后,观察小鼠的肿瘤体积大小、体重和生存期。在处理后不同时间点(0d、7d、14d)小鼠腹腔注射luc荧光素底物,Xenogen IVIS生物发光成像系统中采集肿瘤荧光信号。在处理7d后,每组随机处死小鼠,收集心、肝、脾、肺、肾和肿瘤组织进行苏木精伊红(hematoxylin-eosin,HE)染色,并对肿瘤组织进行TUNEL免疫荧光染色检测肿瘤细胞的增殖和凋亡。为研究光热介导GOP@aPD1 NPs增效aPD1免疫治疗的机制。实验中小鼠分组及处理同上。在处理7d后处死小鼠,收集肿瘤组织进行流式细胞术及免疫荧光染色检测肿瘤浸润T淋巴细胞数量,CD8+T淋巴细胞及CD4+T淋巴细胞亚群的比例。结果体内光热反应实验发现,PTT组及GOP@aPD1+PTT组给予激光辐照后,肿瘤部位的最高温度分别达到约42℃和45℃,均可产生光热效果。但ELISA分析血清炎症因子IL-6、TNF-α及IFN-γ结果显示,GOP@aPD1+PTT组动物血清炎症因子水平显著高于PTT组。在处理后72h,GOP@aPD1+PTT组血清IFN-γ水平是PTT组3.5倍;在处理后168h后,GOP@aPD1+PTT组血清IFN-γ水平是PTT组3倍。这部分结果证明了光热介导GOP@aPD1 NPs治疗可产生肿瘤光热治疗效应,并有效地激活体内炎症反应水平。在体内黑色素瘤治疗观察中,游离aPD1组、PTT组、单载Fe3O4NPs+PTT组及GOP@aPD1组肿瘤生长未受到明显的抑制作用,和对照组的肿瘤生长无明显差异。在游离aPD1+PTT组及单载aPD1NPs+PTT组中,肿瘤生长受到了一定程度的抑制。然而GOP@aPD1+PTT组肿瘤生长受到了几乎完全抑制。活体荧光实验显示,在治疗14天时GOP@aPD1+PTT组肿瘤荧光强度最弱。GOP@aPD1+PTT组动物生存期均超过35天,而游离aPD1+PTT组及单载aPD1 NPs+PTT组动物平均生存期分别为19天及20天。肿瘤组织HE染色发现,GOP@aPD1+PTT组肿瘤组织可见大量肿瘤细胞坏死,无完整细胞结构,镜下呈红染无结构区,且TUNEL免疫荧光染色结果显示该组肿瘤组织的凋亡荧光最高。各组主要器官的HE染色均未见明显异常,证明了该治疗方式的生物安全性。激光共聚焦显微镜发现对照组肿瘤组织切片中几乎没有肿瘤浸润T淋巴浸润,而GOP@aPD1+PTT组肿瘤组织切片中可见大量CD3+T淋巴细胞,CD8+T淋巴细胞及CD4+T淋巴细胞浸润。流式细胞术分析肿瘤微环境中浸润T淋巴细胞数量及亚群结果显示,GOP@aPD1+PTT组肿瘤浸润CD3+T淋巴细胞数量在各组中最高,且CD8+T淋巴细胞比例也在各组中最高。证明了光热介导的GOP@aPD1NPs治疗有效提高了肿瘤组织微环境中抗肿瘤效应T细胞量,从而增效aPD1免疫治疗疗效。结论光热介导的GOP@aPD1 NPs治疗,提高了体内抗肿瘤炎症反应水平,增加了肿瘤微环境T淋巴细胞量,实现免疫“冷”肿瘤向免疫“热”肿瘤转变,从而增强aPD1的免疫治疗效果,有效地抑制恶性黑色素瘤生长,为恶性黑色素治疗提供一种新途径。
Abstract
di yi bu fen zai aPD1ba xiang xiang bian xing na mi li de zhi bei ji xing neng jian ce mu de zhi bei yi chong zai cheng xu xing si wang shou ti 1kang ti (aPD1)、quan fu wu wan (perfluoropentane,PFP)he si yang hua san tie (Fe3O4)de ba xiang na mi li (GOP@aPD1),jian ce ji li jing dian wei 、wen ding xing 、guang re xing neng 、xiang bian te xing 、yao wu han liang ji yao wu shi fang deng xing neng 。fang fa shou xian zhi bei nian fu duo tai (Gly-Arg-Gly-Asp-Ser,GRGDS)ba xiang de ju ru suan qiang ji yi suan /ju yi er chun gao fen zi cai liao (PLGA-PEG-GRGDS),bing jian ding ji hua xue jie gou 。cai yong gai liang de san bu chao sheng ru hua fa zhi bei GOP@aPD1na mi li (nanoparticles,NPs)。jian ce gai na mi li ji ben de li hua xing zhi ,bao gua li jing 、dian wei 、xing tai jie gou 、xi shou guang pu 、fu li xie gong wai xi shou feng ji Xshe xian guang dian zi neng pu fen xi xi shou feng 。yu zhi bei hou bu tong shi jian dian (0.5h,1d,3d,7d)ce liang na mi li zai PBSji han tai niu xie qing PBSrong ye zhong li jing bian hua 。liu shi xi bao shu jian ce na mi li yu GRGDSkang ti lian jie qing kuang 。guang jing xia guan cha bu tong wen du xia (shi wen 、37℃he 45℃)na mi li de xiang bian qing kuang 。zai 660nmji guang fu zhao xia ,jian ce bu tong nong du tiao jian xia gai na mi li de guang re te xing 。guan cha 660nmji guang fu zhao hou na mi li bu tong shi jian dian (2min,6min,10min,12min)chao sheng zeng jiang xin hao 。yong mei lian mian yi xi fu (enzyme-linked immuno sorbent assay,ELISA)ce ding jian ce GOP@aPDna mi li nei de aPD1yao wu han liang ,yi ji ji guang fu zhao chu fa aPD1yao wu shi fang qing kuang 。ji guang gong ju jiao guan cha GOP@aPDna mi li dui aPD1zai yao ji shi yao qing kuang 。jie guo tong guo qing pu he ci fen xi dui ju ge wu PLGA-PEG-GRGDSjin hang le jie gou que ren 。yong chao sheng ru hua fa zhi bei de GOP@aPD1na mi ru cheng zong he se ,sao miao dian jing (scanning electron microscopy,SEM)guan cha dao gai na mi li cheng qiu xing jie gou ,da xiao jun yi ,fen san du hao 。tou she dian jing (transmission electron microscope,TEM)xian shi dian zhuang Fe3O4ke li jun yun fen bu zai gai na mi li nei 。ma er wen li jing yi ce de GOP@aPD1 NPsping jun li jing 218nm,ping jun dian wei wei -5mV。zai PBSji han 10%tai niu xie qing PBSrong ye 4℃fang zhi 7tian ,GOP@aPD1 NPsli jing mo ming xian gai bian ,zheng ming gai na mi li wen ding xing liang hao 。liu shi xi bao shu ce de dui bi fei ba xiang na mi li ,GOP@aPD1 NPsyu GRGDSkang ti lian jie lv geng gao ,zheng ming na mi li biao mian xiu shi le GRGDSduo tai 。yuan zi xi shou guang pu fa ce de GOP@aPD1 NPszhong Fe3O4de bao feng lv wei 63%。guang xue xian wei jing guan cha dao ,zai shi wen ji 37℃tiao jian xia na mi li ji ben bao chi wen ding 。dan shi zai 45℃na mi li fa sheng ye qi xiang bian ,ti ji zeng da ,xiang hu rong ge bing po lie 。shui ming GOP@aPD1 NPsxiang bian wen du yao 45℃。guang sheng yi xi shou guang pu xian shi GOP@aPD1 NPszai 700nmzuo you you jiang xi shou feng 。gen ju PLGA-PEG-GRGDSnong du ,jiang GOP@aPD1 NPsxi shi wei 1.25-10mg/mL。zai 660nmji guang fu zhao xia ,bu tong nong du GOP@aPD1 NPsjun you sheng wen xiao guo ,ju sheng gao wen du yu na mi li nong du zheng xiang guan ,ji zhong 5mg/mLzu ke sheng wen da 45℃zuo you 。zai 660nmji guang lian xu fu zhao xia ,na mi li chao sheng zeng jiang xin hao sui shi jian cheng zhu jian shang sheng -xia jiang qu shi ,fu zhao 10minna mi li chao sheng zeng jiang xin hao zui da ,shui ming ji ye qi xiang bian sui ji guang fu zhao shi jian zhu jian zeng jiang 10minda gao feng 。ELISAce de GOP@aPD1 NPszhong aPD1yao wu de zai yao liang wei 1.56%(w/w),bao feng lv wei 98.9%。ti wai yao wu shi fang shi yan xian shi ,GOP@aPD1 NPsnei de aPD1yao wu zai ji guang fu zhao xia 2hnei kuai su shi fang ,6hhou yao wu shi fang qu ping huan 。ji guang gong ju jiao xian wei jing guan cha dao ,zai ji guang fu zhao qian DiIbiao ji GOP@aPD1 NPsde gong se ying guang yu FITCbiao ji aPD1de lu se ying guang gong ding wei cheng huang se ying guang xin hao ,er zai ji guang fu zhao hou gong se ying guang yu lu se ying guang liang zhe fen li ,shui ming ji guang fu zhao chan sheng de guang re xiao ying cu shi GOP@aPD1 NPsshi fang ji nei fu zai de aPD1yao wu 。jie lun ben shi yan tong guo ru hua fa zhi bei le GOP@aPD1na mi li ,xing tai gui ze ,da xiao jiao jun yun ,xing zhi wen ding ,fu zai aPD1ji Fe3O4,ju you jiao hao de guang re neng li ji ye qi xiang bian de neng li ,neng gou zai 660nmji guang fu zhao zuo yong xia shi fang aPD1yao wu 。di er bu fen zai aPD1ba xiang xiang bian xing na mi li dui hei se su liu ba xiang xing ji ti nei fen bu yan jiu mu de guan cha GOP@aPD1 NPszai ti wai 、ti nei dui hei se su liu xi bao B16F10ba xiang xing ,yi ji gai na mi li de zai ti nei de sheng wu fen bu 、xie ye zhong xun huan shi jian ji ti nei sheng wu an quan xing 。fang fa shou xian zhi bei GOP@aPD1 NPsyu fei ba xiang NPs。zai ti wai shi yan zhong ,ji guang gong ju jiao xian wei jing fen bie guan cha Di Ibiao ji de liang zu na mi li yu DAPIran se de B16F10xi bao gong ding wei qing kuang ,liu shi xi bao shu fen bie ce liang Di Ibiao ji de liang zu na mi li yu B16F10xi bao fu yo bu tong shi jian hou lian jie lv 。CCK8fa guan cha bu tong nong du (2.5mg/ml,5mg/ml,10mg/ml,15mg/ml)GOP@aPD1 NPsyu B16F10xi bao fu yo hou xi bao huo xing bian hua 。zai ti nei shi yan zhong ,shou xian jian li C57B6shu B16F10-luchei se su liu mo xing ,dang zhong liu ti ji da yao chang zhi yao 100mm3shi ,jing C57B6shu wei jing mai fen bie zhu she Di Rbiao ji GOP@aPD1 NPsyu fei ba xiang NPs。zai Xenogen IVISsheng wu fa guang cheng xiang ji tong zhong cai ji ying guang tu xiang ,fen xi zhong liu ji chong yao qi guan de xin hao jiang du 。C57B6shu wei jing mai fen bie zhu she bu tong nong du (5mg/ml,10mg/ml)GOP@aPD1 NPs,14tian hou qu xiao shu xie qing fen xi CK、LDH-L、AST、ALT、BUNji CRshui ping ,guan cha GOP@aPD1 NPszai ti nei sheng wu an quan xing 。yong PerCP/Cy5.5biao ji de aPD1zhi bei GOP@aPD1 NPs。jing he liu C57B6shu wei jing mai fen bie zhu she PerCP/Cy5.5biao ji de GOP@aPD1 NPsji PerCP/Cy5.5biao ji de you li aPD1yao wu 。zhu she hou bu tong shi jian dian (6h,10h)qu zhong liu ji chong yao qi guan ,zhi bei bing dong qie pian ,zai ji guang gong ju jiao xian wei jing xia guan cha ge zu zhi qie pian zhong PerCP/Cy 5.5gong se ying guang xin hao ,Image-Pro Plus 6.0ruan jian fen xi xin hao jiang du 。xiao shu wei jing mai zhu she PerCP/Cy5.5biao ji de GOP@aPD1 NPshou bu tong shi jian dian (0.5h,1h,2h,4h,6h,8h,12h,24h)cai ji xiao shu xie ye ,jin hang ying guang fen guang guang du ji fen xi 。jie guo ji guang gong ju jiao xian wei jing guan cha dao Di Ibiao ji GOP@aPD1 NPsde gong se ying guang ,jiao Di Ibiao ji fei ba xiang NPsde gong se ying guang geng duo de ding wei zai B16F10xi bao zhou wei 。GOP@aPD1 NPsji fei ba xiang NPsyu B16F10xi bao fu yo hou 2hji 10hhou ,liu shi xi bao shu ce de GOP@aPD1 NPsyu xi bao lian jie lv xian zhe gao yu fei ba xiang NPs(p<0.01)。bu tong nong du GOP@aPD1 NPs(2.5mg/ml,5mg/ml,10mg/ml,15mg/ml)yu B16F10xi bao fu yo hou xi bao huo xing mo xian zhe bian hua 。dan B16F10xi bao yu GOP@aPD1 NPsfu yo hou zai jie shou ji guang fu zhao hou xi bao huo xing xian zhe jiang di ,ju xi bao huo xing sui GOP@aPD1 NPsnong du sheng gao er jiang di 。zai ti nei ba xiang xing shi yan zhong ,GOP@aPD1 NPstong guo gao tong tou xing he zhi liu (enhanced permeability and retention,EPR)xiao ying ji zhu dong ba xiang xing zai zhong liu bu wei zhu jian fu ji ,xiao dong wu huo ti ying guang guan cha dao zhong liu bu wei ying guang xin hao sui shi jian tui yi er zhu jian zeng jiang ,zai gei yao 6hhou da dao zui gao feng 、24hhou jian ruo 。zai fei ba xiang NPszu ,xiao shu zhong liu bu wei ge shi jian dian ying guang xin hao jun xian zhe di yu GOP@aPD1 NPszu 。zai C57B6shu wei jing mai fen bie zhu she bu tong nong du GOP@aPD1 NPs(5mg/ml,10mg/ml)14tian hou ,xie qing CK、LDH-L、AST、ALT、BUNji CRbian hua mo tong ji xue cha yi 。jing he liu shu wei jing mai fen bie zhu she PerCP/Cy5.5biao ji de GOP@aPD1 NPs 6hji 24hhou ,tong guo ji guang gong ju jiao xian wei jing jun ke guan cha dao zhong liu zu zhi zhong GOP@aPD1NPsgong se ying guang xin hao 。dan zai PerCP/Cy5.5biao ji de aPD1you li yao wu zu ,tong guo ji guang gong ju jiao xian wei jing ji hu bu neng guan cha dao zhong liu zu zhi zhong gong se ying guang xin hao 。zhong liu zu zhi zhong ying guang xin hao jiang du fen xi xian shi ,zai 6hji 24hshi jian dian GOP@aPD1 NPszu zhong liu zu zhi zhong aPD1ying guang jiang du jun xian zhe gao yu aPD1you li yao wu zu 。he liu shu wei jing mai zhu she PerCP/Cy5.5biao ji de GOP@aPD1 NPs 24hhou ,reng ke tong guo ying guang fen guang guang du ji jian ce xie ye zhong ying guang xin hao 。GOP@aPD1 NPszai ti nei xun huan de ban cui ji yao 4.39h。jie lun zhi bei de GOP@aPD1 NPske tong guo EPRxiao ying ji zhu dong ba xiang xing zai zhong liu zu zhi zhong fu ji ,shi xian ba xiang hei se su liu di song aPD1yao wu ,bing ju you jiao chang de ti nei xun huan shi jian ji liang hao de sheng wu an quan xing ,wei xia yi bu ti nei zhi liao shi yan dian ding le ji chu 。di san bu fen guang re jie dao zai aPD1ba xiang xiang bian xing na mi li zhi liao e xing hei se liu ji zeng xiao mian yi zhi liao ji zhi yan jiu mu de yan jiu guang re (Photothermal therapy,PTT)jie dao GOP@aPD1NPszhi liao hei se su liu de xiao guo ,ji ji zeng xiao aPD1mian yi zhi liao de xiang guan ji zhi 。fang fa wei le guan cha guang re jie dao GOP@aPD1 NPszai ti nei guang re xiao ying ,jian li C57B6shu B16F10-luchei se su liu mo xing ,dang zhong liu ti ji yao 80mm3shi kai shi gei yu bu tong chu li 。ba he liu xiao shu sui ji fen cheng 3zu ,dui zhao zu (bu chu li ),PTTzu ji GOP@aPD1+PTTzu 。ji zhong GOP@aPD1+PTTzu xian jing xiao shu wei jing mai zhu she GOP@aPD1 NPs(~200μL,5mg/m L),6hhou gei yu zhong liu bu wei 660nmji guang fu zhao (0.1W/cm2,10min)。PTTzu jin gei yu 660nmji guang fu zhao (0.1W/cm2,10min)zhong liu zu zhi 。yong gong wai re cheng xiang yi guan cha zhong liu bu wei de sheng wen qing kuang 。xiao shu jing chu li hou bu tong shi jian dian (24h,72h,168h)qu xie qing ,tong guo ELISAfen xi yan zheng yin zi IL-6、TNF-αji IFN-γbian hua shui ping 。wei le guan cha guang re jie dao GOP@aPD1 NPszai ti nei dui e xing hei se su liu zhi liao zuo yong ,shou xian zhi bei GOP@aPD1 NPs,chan zai Fe3O4 NPsji chan zai aPD1 NPs。ji zhong chan zai Fe3O4 NPsbu han aPD1,chan zai aPD1 NPsbu han Fe3O4。jian li C57B6shu B16F10-luchei se su liu mo xing ,dang zhong liu ti ji da yao 80 mm3shi kai shi yu bu tong chu li 。ba xiao shu sui ji fen cheng 8zu ,1.dui zhao zu (sheng li yan shui ),2.you li aPD1zu ,3.PTTzu ,4.you li aPD1+PTTzu ,5.chan zai Fe3O4 NPs+PTTzu ,6.chan zai aPD1NPs+PTTzu ,7.GOP@aPD1zu ,8.GOP@aPD1+PTTzu 。ji zhong 2ji 7zu jin fen bie jing mai zhu she you li aPD1ji GOP@aPD1 NPs;3zu jin gei yu zhong liu bu wei 660 nmji guang fu zhao (0.1W/cm2,10min);4、5、6ji 8zu fen bie xian jing xiao shu wei jing mai zhu she you li aPD1、chan zai Fe3O4 NPs、chan zai aPD1 NPsji GOP@aPD1NPs,6hhou gei yu zhong liu bu wei 660nmji guang fu zhao (0.1W/cm2,10min)。jing guo bu tong de chu li hou ,guan cha xiao shu de zhong liu ti ji da xiao 、ti chong he sheng cun ji 。zai chu li hou bu tong shi jian dian (0d、7d、14d)xiao shu fu qiang zhu she lucying guang su de wu ,Xenogen IVISsheng wu fa guang cheng xiang ji tong zhong cai ji zhong liu ying guang xin hao 。zai chu li 7dhou ,mei zu sui ji chu si xiao shu ,shou ji xin 、gan 、pi 、fei 、shen he zhong liu zu zhi jin hang su mu jing yi gong (hematoxylin-eosin,HE)ran se ,bing dui zhong liu zu zhi jin hang TUNELmian yi ying guang ran se jian ce zhong liu xi bao de zeng shi he diao wang 。wei yan jiu guang re jie dao GOP@aPD1 NPszeng xiao aPD1mian yi zhi liao de ji zhi 。shi yan zhong xiao shu fen zu ji chu li tong shang 。zai chu li 7dhou chu si xiao shu ,shou ji zhong liu zu zhi jin hang liu shi xi bao shu ji mian yi ying guang ran se jian ce zhong liu jin run Tlin ba xi bao shu liang ,CD8+Tlin ba xi bao ji CD4+Tlin ba xi bao ya qun de bi li 。jie guo ti nei guang re fan ying shi yan fa xian ,PTTzu ji GOP@aPD1+PTTzu gei yu ji guang fu zhao hou ,zhong liu bu wei de zui gao wen du fen bie da dao yao 42℃he 45℃,jun ke chan sheng guang re xiao guo 。dan ELISAfen xi xie qing yan zheng yin zi IL-6、TNF-αji IFN-γjie guo xian shi ,GOP@aPD1+PTTzu dong wu xie qing yan zheng yin zi shui ping xian zhe gao yu PTTzu 。zai chu li hou 72h,GOP@aPD1+PTTzu xie qing IFN-γshui ping shi PTTzu 3.5bei ;zai chu li hou 168hhou ,GOP@aPD1+PTTzu xie qing IFN-γshui ping shi PTTzu 3bei 。zhe bu fen jie guo zheng ming le guang re jie dao GOP@aPD1 NPszhi liao ke chan sheng zhong liu guang re zhi liao xiao ying ,bing you xiao de ji huo ti nei yan zheng fan ying shui ping 。zai ti nei hei se su liu zhi liao guan cha zhong ,you li aPD1zu 、PTTzu 、chan zai Fe3O4NPs+PTTzu ji GOP@aPD1zu zhong liu sheng chang wei shou dao ming xian de yi zhi zuo yong ,he dui zhao zu de zhong liu sheng chang mo ming xian cha yi 。zai you li aPD1+PTTzu ji chan zai aPD1NPs+PTTzu zhong ,zhong liu sheng chang shou dao le yi ding cheng du de yi zhi 。ran er GOP@aPD1+PTTzu zhong liu sheng chang shou dao le ji hu wan quan yi zhi 。huo ti ying guang shi yan xian shi ,zai zhi liao 14tian shi GOP@aPD1+PTTzu zhong liu ying guang jiang du zui ruo 。GOP@aPD1+PTTzu dong wu sheng cun ji jun chao guo 35tian ,er you li aPD1+PTTzu ji chan zai aPD1 NPs+PTTzu dong wu ping jun sheng cun ji fen bie wei 19tian ji 20tian 。zhong liu zu zhi HEran se fa xian ,GOP@aPD1+PTTzu zhong liu zu zhi ke jian da liang zhong liu xi bao huai si ,mo wan zheng xi bao jie gou ,jing xia cheng gong ran mo jie gou ou ,ju TUNELmian yi ying guang ran se jie guo xian shi gai zu zhong liu zu zhi de diao wang ying guang zui gao 。ge zu zhu yao qi guan de HEran se jun wei jian ming xian yi chang ,zheng ming le gai zhi liao fang shi de sheng wu an quan xing 。ji guang gong ju jiao xian wei jing fa xian dui zhao zu zhong liu zu zhi qie pian zhong ji hu mei you zhong liu jin run Tlin ba jin run ,er GOP@aPD1+PTTzu zhong liu zu zhi qie pian zhong ke jian da liang CD3+Tlin ba xi bao ,CD8+Tlin ba xi bao ji CD4+Tlin ba xi bao jin run 。liu shi xi bao shu fen xi zhong liu wei huan jing zhong jin run Tlin ba xi bao shu liang ji ya qun jie guo xian shi ,GOP@aPD1+PTTzu zhong liu jin run CD3+Tlin ba xi bao shu liang zai ge zu zhong zui gao ,ju CD8+Tlin ba xi bao bi li ye zai ge zu zhong zui gao 。zheng ming le guang re jie dao de GOP@aPD1NPszhi liao you xiao di gao le zhong liu zu zhi wei huan jing zhong kang zhong liu xiao ying Txi bao liang ,cong er zeng xiao aPD1mian yi zhi liao liao xiao 。jie lun guang re jie dao de GOP@aPD1 NPszhi liao ,di gao le ti nei kang zhong liu yan zheng fan ying shui ping ,zeng jia le zhong liu wei huan jing Tlin ba xi bao liang ,shi xian mian yi “leng ”zhong liu xiang mian yi “re ”zhong liu zhuai bian ,cong er zeng jiang aPD1de mian yi zhi liao xiao guo ,you xiao de yi zhi e xing hei se su liu sheng chang ,wei e xing hei se su zhi liao di gong yi chong xin tu jing 。
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