CN115192707A - 一种肿瘤抗原诱捕纳米粒及其制备方法与应用 - Google Patents
一种肿瘤抗原诱捕纳米粒及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种肿瘤抗原诱捕纳米粒及其制备方法与应用。该肿瘤抗原诱捕纳米粒包括核壳结构,所述核为携氧型全氟碳化合物,所述壳为负载有光敏剂和化疗药物的两亲性嵌段共聚物,所述壳的表面连接有铝基佐剂。本发明的肿瘤抗原诱俘纳米粒(PPIAO‑NPs)集光声/超声‑双模态成像和化疗/PTT/PSDT/免疫治疗为一体,实现了全面的肿瘤诊疗一体化,可同时实现肿瘤细胞的杀伤与肿瘤抗原的暴露,为更好地治疗转移和复发肿瘤提供了一种简单有效的个体化肿瘤疫苗策略。
Description
技术领域
本发明涉及纳米制剂技术领域,尤其涉及一种肿瘤抗原诱捕纳米粒及其制备方法与应用。
背景技术
癌症免疫疗法基于人体能够识别和破坏其自身异常细胞的观念,通过激活自身免疫系统清除肿瘤细胞,是抑制肿瘤复发及转移的重要策略。非特异性免疫治疗手段,包括细胞因子疗法、免疫检查点阻断疗法、过继性T细胞转移等,在肿瘤复发及转移防治中取得一定进展。但同时这类方法也面临诸多挑战,如个体反应差异性、低治疗反应性及免疫毒性等。基于肿瘤特异性抗原或肿瘤相关抗原的特异性免疫治疗是肿瘤免疫治疗的理想策略。
目前肿瘤特异性抗原获得方法有:灭活的自体或异体肿瘤组织成分、自体或异体肿瘤组织或细胞提取复合物、肿瘤相关蛋白或多肽、表达肿瘤抗原的基因等。基于自体或异体肿瘤组织制备的肿瘤疫苗需有创获取肿瘤组织,制备过程复杂,同时疗效的发挥受肿瘤异质性和免疫焦点丢失的制约。针对个体的肿瘤组织进行个性化肿瘤疫苗构建则缺乏现实可行性。
肿瘤原位灭活理论上可解决上述问题。通过激活死亡肿瘤的免疫原性而产生个性化原位疫苗的治疗策略备受关注。但原位灭活的肿瘤组织能否产生肿瘤疫苗效应依赖于:肿瘤抗原暴露丰度、抗原提呈细胞(APCs)对肿瘤抗原的加工与提呈、淋巴细胞活化增殖及细胞毒性T淋巴细胞(CTLs)的瘤内浸润。其中,肿瘤抗原的暴露和提呈不足是导致肿瘤免疫原性低下,肿瘤组织内CTLs浸润匮乏的主要原因。研究表明,肿瘤免疫治疗发挥可靠疗效急需突破两大瓶颈:肿瘤组织中细胞毒性T淋巴细胞(CTL)的浸润不足和免疫抑制性肿瘤微环境(ITM)。其实质为肿瘤免疫原性不足和免疫耐受性存在。如何将低免疫原性的“冷肿瘤”转变为高免疫原性的“热肿瘤”是解决肿瘤原位疫苗科学问题的关键。
发明内容
针对现有技术中所存在的不足,本发明提供了一种肿瘤抗原诱捕纳米粒及其制备方法与应用,其解决了现有技术中存在的肿瘤抗原暴露和呈递不足的问题。
本发明一方面,提供一种肿瘤抗原诱捕纳米粒,所述核为携氧型全氟碳化合物,所述壳为负载有光敏剂和化疗药物的两亲性嵌段共聚物,所述壳的表面连接有铝基佐剂。
进一步地,所述纳米粒的流体力学直径为333.13±5.34nm,表面电位为-4.98±0.73mV。
进一步地,所述携氧型全氟碳化合物为携氧全氟正戊烷,所述光敏剂为吲哚菁绿、二氢卟吩、IR-780碘化物中的一种,所述化疗药物为铂类药物或蒽环类药物,所述两亲性嵌段共聚物为聚乳酸羟基乙酸共聚物-聚乙二醇,所述铝基佐剂为纳米氢氧化铝。
本发明实施例中,以吲哚菁绿为光敏剂,奥沙利铂为化疗药物进行具体说明。可以理解的是,本发明中列举的其他光敏剂和化疗药物可以达到同样的效果。
进一步地,所述纳米氢氧化铝的粒径为87.77±4.18nm,表面电位为41.38±1.24mV。
本发明另一方面,提供一种肿瘤抗原诱捕纳米粒的制备方法,包括以下步骤:
S1:将光敏剂、化疗药物、铝基佐剂、携氧型全氟碳化合物加水乳化;
S2:加入两亲性嵌段共聚物继续乳化,得到混合液1;
S3:在混合液1中加入表面活性剂进行乳化,得到混合液2;
S4:在混合液2中加入交联固化剂进行固化。
进一步地,步骤S1中,所述光敏剂、化疗药物、铝基佐剂的质量比为1.5-3.0:1.5-3.0:1-2;
以mg/uL计,所述光敏剂和携氧型全氟碳化合物的质量体积比为1.5-3.0:100-200。
进一步地,步骤S2中,两亲性嵌段共聚物与化疗药物的质量比为50-100:1.5-3.0。
进一步地,步骤S1-S3中,步骤S1-S3中,所述乳化在超声条件下进行,乳化时间为3-5min。
进一步地,步骤S3中,所述表面活性剂为PVA,按w/v计,所述PVA的浓度为3-5%;所述交联固化剂为异丙醇,按w/v计,所述异丙醇的浓度为2-5%。
本发明又一方面,提供一种肿瘤抗原诱捕纳米粒在制备治疗抗肿瘤药物中的应用。
本发明的技术原理为:本发明构建的肿瘤抗原诱俘纳米粒(PPIAO-NPs)在治疗过程中发挥协同效应,包括基于活性氧杀伤机制的光声动力效应、基于热损伤机制的光热效应和化疗药物奥沙利铂的细胞毒性,以及通过提高肿瘤免疫原性促进特异性抗肿瘤免疫效应。同时,超声触发纳米粒产生的空化效应引起的机械损伤也发挥着重要作用。此外,联合治疗后肿瘤细胞释放的其它特异性抗原或肿瘤相关抗原可能在后期APCs的激活与T淋巴细胞活化过程中也发挥着至关重要的作用。
PPIAO-NPs结合光声治疗通过诱导肿瘤细胞的免疫原性死亡来促进更多的肿瘤抗原暴露;通过纳米级氢氧化铝捕获肿瘤细胞暴露和释放的抗原,并形成抗原存储库以促进树突状细胞对抗原的识别、加工和提呈。充分的抗原的暴露和提呈有效诱导体内CD8+T淋巴细胞活化,促进T淋巴细胞的瘤内浸润,从而抑制原发肿瘤和转移瘤的生长。同时,激活的特异性抗肿瘤免疫对体内残留及转移的肿瘤细胞进行围剿,并形成抗肿瘤免疫记忆,以预防肿瘤的复发。
肿瘤细胞释放的抗原在氢氧化铝的抓捕下被更多的储存在原位还促进了免疫细胞向淋巴结的迁移,将更多的抗原暴露于免疫系统,从而使CD8+T淋巴细胞更加准确的对肿瘤细胞展开围剿。同时,PPIAO-NPs中的纳米氢氧化铝通过改变抗原物理性状,延缓抗原降解,延长抗原的潴留时间,刺激APCs,增强APCs对抗原的加工和提呈,为抗原提呈细胞(APCs)摄取抗原创造了有利条件。而纳米氢氧化铝能更高效的促进抗原抗原递呈细胞的相互作用,如促进树突状细胞和巨噬细胞对抗原的吞噬和加工。
相比于现有技术,本发明具有如下有益效果:
(1)本发明的肿瘤抗原诱俘纳米粒(PPIAO-NPs)集光声/超声-双模态成像和化疗/PTT/PSDT/免疫治疗为一体,实现了多模式图像监测、肿瘤治疗和肿瘤疫苗免疫治疗,从而使肿瘤诊疗一体化,为更好地治疗转移和复发肿瘤提供了一种简单有效的个体化肿瘤疫苗策略。
(2)PPIAO-NPs中的纳米氢氧化铝具备预测基因捕获能力,可俘获的肿瘤蛋白中含特异性肽段的主要蛋白2632个。其中已知肿瘤相关蛋白抗原约2.85%,预测基因9个,还包括与肿瘤免疫代谢相关的诸多功能蛋白。相较于PPIO-NPs和和PPIO-NPs+Al,PPIAO-NPs具有更强的肿瘤抑制作用和抗肿瘤免疫激活作用,可通过免疫和非免疫成分之间的相互配合实现抗肿瘤免疫效应,并且更有利于清除体内存在的微小肿瘤转移病灶,更有利于诱导抗肿瘤免疫和形成免疫记忆。
(3)PPIAO-NPs中的纳米氢氧化铝可增强纳米粒的光声成像信号;赋予了纳米粒免疫调控的功能,纳米氢氧化铝粒径为87.77±4.18nm,具有比大颗粒更强的佐剂活性,从而诱导更强的抗原特异性抗体应答。
(4)本发明的PPIAO-NPs的联合治疗适用于多数强侵袭性肿瘤,包括黑色素瘤、三阴性乳腺癌、结直肠癌。
(5)本发明的PPIAO-NPs副作用小,生物安全性高,降低了化疗药物的细胞毒性。
附图说明
图1为本发明实施例2中纳米粒的表征结果图,其中,A为PPIAO-NPs扫描电镜图(标尺2um);B为PPIAO-NPs能谱元素扫描;C为PPIAO-NPs和纳米氢氧化铝的傅里叶红外波谱;D为PPIAO-NPs、PPIO-NPs、游离ICG的紫外吸收光谱;E为PPIAO-NPs、PPIO-NPs、游离ICG的荧光光谱;F为PPIAO-NPs水悬浮液在不同强度808nm激光辐照下的热红外像;G为PPIAO-NP和PPIO-NPs(OXA=15.21ug·mL-1)在相同强度808nm激光(2.0W·cm-2,5min)辐照下温度曲线;H为SOSG检测不同处理组的单线态氧生成。
图2为本发明实施例3-4中纳米粒的双模态成像、生物安全、药代动力学(pK)和生物分布检测的结果图,其中,A为PPIAO-NPs、PPIO-NPs、游离ICG的体外超声成像与光声成像对比;B为超声成像时B-mode平均灰度值(n=3);C为超声成像时CEUS平均灰度值(n=3);D为光声成像时B-mode平均灰度值(n=3);E为光声成像时PA-mode平均光声值(n=3);F为小鼠尾静脉注射PPIAO-NPs后不同时间的肿瘤超声/光声成像;G为健康小鼠注射治疗浓度PPIAO-NPs后不同时间的主要脏器HE染色(OXA=3mg·Kg-1);H为用电感耦合等离子体发射光谱仪(ICP-MS)测定PPIAO-NPs在小鼠体内不同时间的定量生物分布。
图3为本发明实施例5-6中纳米粒的体外细胞摄取、抗肿瘤活性和抗原暴露诱导,其中,A为激光共聚焦显微镜观察PPIAO-NPs在肿瘤小体中的渗透和聚集(蓝色荧光为Hoechst标记活ID8细胞核,红色荧光为DiI标记的PPIAO-NPs);B为流式细胞术检测肿瘤细胞对PPIAO-NPs的吞噬率;C为ID8细胞与PPIAO-NPs(OXA=15.21ug·mL-1)共同孵育2h后,经808nm激光(2Wcm-2,5min)和LIFU(1Wcm-2,5min)处理,胞内活性氧的生成共聚焦图像(绿色荧光显示DCFH-DA染色的ROS阳性),标尺50um;和不同处理组的ID8细胞活/死(CAM/PI)染色,共聚焦图像(绿色荧光显示CAM染色的活细胞,红色荧光显示PI染色的死细胞),标尺100um;D为流式细胞术分析各组细胞死亡和凋亡;E为不同处理组的ID8细胞CRT和HMGB1表达(蓝色荧光为DAPI标记ID8细胞核,绿色荧光为FITC标记CRT,红色荧光为APC标记HMGB1),标尺50um。
图4为实施例7-8中抗原捕获和体外DC刺激的结果图,其中,A为质谱检测纳米氢氧化铝捕获肿瘤蛋白中的已知肿瘤抗原与7个预测基因(根据蛋白质中所含特异性肽段的数量分层统计);B为流式细胞仪分析不同处理组的DCs成熟情况;C为ELISA检测不同处理组IL-12分泌水平(n=5);D为对照组和PPIAO+L.U.组DCs细胞的典型形态(蓝色荧光为Hoechst标记活细胞核,红色荧光为DiI标记ID8细胞,绿色荧光为CFSE标记DC细胞。
图5为实施例9中体内抗肿瘤作用的结果图,其中,A为荷瘤C57BL/6小鼠,静脉注射PPIAO-NPs后6-8小时,808nm激光辐照肿瘤部位的温度检测;B为治疗小鼠的全身热红外成像,仅限肿瘤辐照部位温度升高;C为不同治疗后6组小鼠原发肿瘤体积变化(n=5);D为不同治疗后小鼠体重变化(n=5);E为不同治疗后小鼠肿瘤体积变化(n=5);F为不同治疗后各组小鼠的生存曲线(n=5);G为不同治疗后各组肿瘤切片的H&E/PCNA/TUNEL免疫组织荧光染色。
图6为实施例9-10中体内抗原暴露和抗肿瘤免疫激活的结果图,其中,A为不同治疗后各组肿瘤切片的CRT/HMGB1免疫组织荧光染色;B为流式细胞分析各组瘤内DC细胞成熟;C为流式细胞分析各组瘤内CD4+T淋巴细胞和CD8+T淋巴细胞活化;D为流式细胞分析各组脾脏CD8+T淋巴细胞活化;E-H为ELISA检测各组小鼠治疗后7天血清中IL-6、IL-12、TNF-α、IFN-γ分泌水平。
图7为实施例11-12中PPIAO-NPs改善远位效应和疫苗效应的结果图,其中,A为PPIAO-NPs联合808nm激光/LIFU治疗抑制雌性C57BL/6小鼠远处肿瘤示意图;B为不同治疗后各组小鼠体重变化(n=6);C为不同治疗后各组小鼠原发肿瘤体积变化(n=6);D为不同治疗后各组小鼠转移肿瘤体积变化(n=6);E为不同治疗后各组小鼠的原发瘤与转移瘤切片的CD4+T/CD8+T淋巴细胞免疫组织荧光染色;F为乳酸脱氢酶检测试剂盒检测各组小鼠脾脏淋巴细胞对ID8肿瘤细胞活性抑制;G为ELISA检测各组小鼠脾脏淋巴细胞IFN-γ分泌水平;H为雌性C57BL/6小鼠接种了不同处理的ID8肿瘤小体疫苗后,各组小鼠ID8细胞再挑战的成瘤率;I为流式细胞术分析各组疫苗小鼠脾脏CD8+/CD4+T记忆淋巴细胞活化。
具体实施方式
下面结合附图及实施例对本发明中的技术方案进一步说明。本发明中所用试剂、细胞和小鼠模型如下:聚乳酸羟基乙酸共聚物(Poly(lactic-co-glycolic acid),PLGA,乳酸:乙醇酸=50:50,30,000-60,000Da MW),聚乙二醇(PEG,2000DaMW),聚乙烯醇(polyvinyl alcohol,PVA),吲哚菁绿(indocyanine green,ICG)购自SigmaAldrich(St.Louis,MO,USA)。全氟正戊烷(perfluoropentane,PFP,沸点29℃)购自StremChemicals(MA,USA)。奥沙利铂(OXA)购自MedChemExpress(NJ,USA)。纳米氢氧化铝(Aluminum hydroxide nanoparticles,Al(OH)3)购自瑞禧生物科技(中国西安)。1,1'-二乙基-3,3,3',3'-四甲基吲哚菁碘化物(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate,DiI)购自碧云天。
ID8小鼠卵巢上皮癌细胞由美国堪萨斯大学医学中心提供。ID8细胞复苏后在37℃,5%CO2的培养箱中,用含有10%胎牛血清、50μgL-1链霉素和50μgL-1青霉素的DMEM培养基中培养。当细胞达到80%融合时,用于实验。雌性C57BL/6小鼠(4-6周,体重18-22g)由重庆医科大学实验动物中心提供。所有动物均按照《实验动物的护理和使用指南》进行处理。所有动物实验均经重庆医科大学动物伦理委员会批准。
荷瘤小鼠建立:将悬浮在生理盐水中的ID8细胞(1×106细胞·mL-1)皮下注射到小鼠的右后背部。当肿瘤体积达到约200mm3时,所有荷瘤小鼠都用于治疗实验。
实施例1PPIAO-NPs的制备
1、用超声探头(Sonics&Materials Inc.,USA)将1mLICG水溶液(1.5mg·mL-1)、1mLOXA水溶液(3mg·mL-1)、1mLAl(OH)3水溶液(2mg·mL-1)与200uL携氧PFP充分乳化60s。进而促进药物的充分溶解和Al(OH)3的均匀分散。
2、加入2mLPLGA-PEG2000二氯甲烷溶液(25mg·mL-1),声震乳化3min(超5s,空5s,功率25%)。
3、加入2mLPVA溶液(5%,w/v),声震乳化3min(超5s,空5s,功率25%)。
4、加入10mL异丙醇溶液(2%,w/v)固化纳米粒(NP)壳。将上述乳状溶液低温磁力搅拌12h,以充分去除有机溶剂。收集乳状溶液离心(12000rmp,4℃,5分钟),用去离子水洗涤三次,直到上清液透明,得到PPIAO-NPs。将PPIAO-NPs避光保存在4℃下,并在24h内用于进一步的实验。PPIO-NPs(无Al(OH)3)通过相似的方法合成。在二氯甲烷中加入DiI,合成DiI标记的纳米粒PPIAO-NPs(DiI-NPs)。
实施例2纳米粒PPIAO-NPs的表征
将实施例1制备的抗原诱捕纳米粒(PPIAO-NPs,简称纳米粒)分散到水中,由图1A可知,PPIAO-NPs可在水中均匀分散。通过扫描电镜和透射电镜对PPIAO-NPs的结构和形态进行表征,由图1B可知,PPIAO-NPs为球形结构,表面存在颗粒结构。通过激光动态光散射系统(Malvern Instruments,Malvern,UK)测定PPIAO-NPs、PPIO-NPs和纳米氢氧化铝的平均粒径和zeta电位。结果显示,PPIAO-NPs的流体力学直径为333.13±5.34nm,表面电位为-4.98±0.73mV。PPIO-NPs的表面光滑,平均粒径为313.90±7.28nm,表面电位为-14.60±1.75mV。纳米氢氧化铝分散性好,大小均一,平均粒径87.77±4.18nm,表面电位为41.38±1.24mV。
经能谱扫描分析PPIAO-NPs表面,如图1C可知,主要元素是氧、碳和铝在元素映射中显示出均匀的分布。红外光谱(图1D)数据显示,在564.19和556.59的两个峰对应于特征性的Al-O,在1088.24、950.76、1384.37和1384.32四个峰对应特征性的OH-。经计算,PPIAO-NPs中氢氧化铝的包封率为10.4%,载药量为0.19%,铝含量(0.208ug·mL-1)符合铝佐剂的安全性限度标准。高效液相色谱检测PPIAO-NPs中OXA包封率为19.60%,载药量为1.05%。游离ICG,PPIO-NPs和PPIAO-NPs的紫外吸收光谱(图1E)和荧光光谱(图1F)表明,纳米粒的紫外吸收光谱在785nm处有最大吸收峰,与游离ICG相比红移了约5nm,而游离ICG和纳米粒的荧光光谱均在780nm处有最大荧光发射峰,表明ICG被成功包封进纳米粒中,且其光学性质未发生明显改变。PPIAO-NPs中ICG的包封率为91.95%,载药量为2.49%。
通过红外热像仪监测温度变化,结果显示,在不同强度的808nm激光辐照下,治疗浓度的PPIAO-NPs温度逐渐升高。在2.5W·cm-2的激光照射2分钟时,温度上升至62.4℃(图1G)。肿瘤细胞的致死温度临界点在42.5-43℃,正常细胞则为45℃。温度过高将导致肿瘤细胞内蛋白抗原产生不可逆的变性损坏。因此,2.5W·cm-2作为最佳治疗激光强度(图1H)。3D热成像展现了激光辐照下溶液温度的上升和均匀分布。
通过检测非细胞环境中单线态氧(1O2)的生成,评估PPIAO-NPs的光声动力治疗潜力。图1I显示,808nm激光结合PPIAO-NPs组在525nm处的荧光强度显著高于对照组,原因是大量生成的1O2与SOSG荧光探针结合后形成SOSG-EP复合物。
评估PPIAO-NPs和PPIO-NPs的粒径稳定性和光学稳定性,将合成后的纳米粒分散在DMEM、PBS、ddH2O、10%BSA中,第0、7、14、21和28天测量平均粒径。前两周,两种纳米粒的粒径基本维持稳定;两周之后,粒径缓慢增加,在10%BSA中较明显,但纳米粒的粒径始终小于400nm。第28天,游离ICG的荧光强度下降了43.58%,紫外吸收值下降了60.81%。而PPIAO-NPs中ICG荧光强度下降了20.01%,紫外吸收值下降17.9%。PPIO-NPs中ICG的荧光损失也低于游离ICG。这些结果表明,纳米结构有助于维持ICG的光学稳定性,并改善其易淬灭和半衰期短的缺点。
实施例3双模态成像
用超声诊断仪(MyLab90)的线性探头(5~12MHz)观察808nm激光(2.0W·cm-2,5min)和低强度超声(1W·cm-2,5min)照射前后PBS、游离ICG、PPIAO-NPs(实施例1)和PPIO-NPs(实施例1)悬浮液(ICG浓度为35.67ug·mL-1)的标准B模式和CEUS模式。各组的回声强度(EI)通过图像分析软件进行量化分析。PPIAO-NPs的光声性能由Vevo LAZR光声成像系统评估。并观察不同样本在处理前后的光声强度的变化。在680~970nm(间隔=5nm)不同波长下进行PA成像,检测最大吸光度。然后,使用激发波长为780nm的激光。各组的PA信号强度通过Vevo LAZR软件进行量化分析。在C57BL/6小鼠ID8卵巢癌皮下移植瘤模型中评估PPIAO-NPs的体内双模态成像(n=3)。在静脉注射PPIAO-NPs溶液(ICG7.36mg·Kg-1,OXA3mg·Kg-1)后的不同时间点(Pre,2,4,6,12,24h)采集超声和光声图像,并统计肿瘤区域的PA信号强度。观察静脉注射PPIAO-NPs溶液6h时,肿瘤组织在808nm激光和低强度超声处理后的PA信号值变化。
如图2A所示,在超声成像中,808nm激光辐照后的,与PBS组和游离ICG组比较,PPIAO-NPs组和PPIO-NPs都显示出信号增强(p<0.001)。在B模式中,PPIAO-NPs平均声强(EI)由48.06±1.47dB增加到129.22±2.83dB(图2B);在CEUS模式中,由1.97±0.1dB增加到28.37±0.61dB(图2C)。进一步的低功率超声作用后,PPIAO-NPs组平均声强降低了82.6%(B模式)和68.82%(CEUS模式)。在光声成像中,各组的信号值在激光辐照和超声作用后的变化趋势与超声成像一致。激光辐照后平均声强增强,其中PPIO-NPs组增加了75.6%,PPIAO-NPs组增加了74.51%(图2D)。与PBS组比较,游离ICG组光声值增加不明显,而两个纳米粒PPIAO-NPs组和PPIO-NPs组则显示出光声值的显著增强(p<0.001),PPIO-NPs组由0.29±0.03增至0.98±0.05,PPIAO-NPs组由0.40±0.02增至1.06±0.09(图2E)。经过超声作用后,各组的光声值出现不同程度的降低。并且,在光声成像中,PPIAO-NPs组的光声值始终高于PPIO-NPs组(p<0.05),由此推测这可能是由于金属铝存在的原因。激光辐照后的光热效应加速了纳米粒内核中PFP的相变,即由液相变为气相。这一改变使得纳米造影剂与周围环境的声阻抗差值增大,因此我们观察到超声成像的显著增强。超声作用后,纳米粒组的平均声强和光声值下降,提示相变后的纳米粒在超声作用下发生了破裂,同时这也表明纳米结构对维持ICG的光学稳定性至关重要。
在尾静脉注射纳米粒之前(Pre)和注射后的2、4、6、12、24小时,观察小鼠肿瘤部位的超声和光声图像。结果如图2F所示,在注射后2小时,肿瘤组织中便开始呈现光声信号,随着时间延长,光声信号逐渐增强,在注射后6-8小时达到峰值,随后信号逐渐减弱。这项结果为我们明确了体内治疗和成像研究的最佳时间点。因此,在给药后6-8小时,我们对荷瘤小鼠进行激光辐照和超声处理,并观察干预前后瘤体的光声信号变化。808激光辐照后,瘤内光声信号增强,进一步超声作用后,PA值由0.86±0.03下降到0.68±0.07,表明PPIAO-NPs具备作为PA造影剂的良好性能。
实施例4生物安全、药代动力学(pK)和生物分布检测
通过CCK-8法检测游离OXA的IC50,含不同浓度OXA的纳米粒的细胞毒性,以及PPIAO-NPs结合808nm激光(2.0W·cm-2,5min)和低强度超声(1W·cm-2,5min)处理的IC50。以此确定体外实验的纳米粒浓度。
与游离OXA相比,相同浓度的OXA由于纳米粒的包载而降低了其细胞毒性,这也是纳米载体的突出优势之一。在OXA浓度为16.5ug·mL-1时,游离OXA组的细胞活性为75.47±2.3%,两个纳米粒组的细胞活性依旧高于80%。游离OXA的IC50为42.46ug·mL-1,PPIAO-NPs结合激光和超声处理后,50%细胞存活率时奥沙利铂浓度为15.21ug·mL-1。这明确了我们在这项研究中的药物治疗浓度。
在健康的雌性C57BL/6小鼠体内评估PPIAO-NPs的生物安全性。21只小鼠随机分为7组(n=3):生理盐水组(静脉注射生理盐水),1天组(静脉注射PPIAO-NPs后1天),7天组(静脉注射PPIAO-NPs后7天),14天组(静脉注射PPIAO-NPs后14天),21天组(静脉注射PPIAO-NPs后21天),28天组(静脉注射PPIAO-NPs后28天)。收集各组小鼠的血液和主要脏器(心、肝、脾、肺、肾)。通过HE染色分析主要脏器的组织结构变化。HE染色(图2G)观察小鼠的主要脏器(心肝脾肺肾)病理改变,与生理盐水组一样,纳米粒组的小鼠脏器在28天内未见明显的组织病理学改变。此外,血常规与肝肾功能检测结果也表明治疗浓度的纳米粒未对小鼠造成明显的健康损害。
在C57BL/6小鼠ID8卵巢皮下移植瘤模型中评估PPIAO-NPs的药代动力学(pK)和生物分布。在静脉注射PPIAO-NPs溶液(OXA3mg·Kg-1)后的不同时间点(0.5,1,2,4,6,8,12,24,48h)收集小鼠血液、心脏、肝脏、脾脏、肺、肾脏、肿瘤(每个时间点3只)。各脏器和血液在浓硝酸中消化24h,用电感耦合等离子体质谱(ICP-MS)分析铂(Pt)浓度。奥沙利铂的血药浓度随时间变化符合二室模型。血液循环半衰期2.06±0.11h。生物分布分析表明(图2H),PPIAO-NPs在静脉注射后6-8h,肿瘤摄取峰值达7.37±0.88ug·g-1。这些结果表明PPIAO-NPs可以安全地用作体内成像和治疗的纳米治疗试剂。
实施例5体外细胞摄取
将ID8肿瘤细胞(1×105细胞)接种于共聚焦皿中。待细胞贴壁后,加入DiI标记的PPIAO-NPs(DiI-NPs)(OXA=15.21ug·mL-1)。在细胞与纳米粒共孵育的不同时间(0.5、1.0、1.5、2h),PBS清洗未被吞噬的纳米粒。Hoechst 33342活细胞染色液标记细胞核。通过CLSM的3D成像观察纳米粒在肿瘤小体中的渗透和聚集。六孔板中培养的ID8细胞与DiI-NPs共孵育不同时间后,被胰酶消化收集。流式细胞术检测各组细胞对纳米粒的吞噬率。
共孵育2h后,激光共聚焦扫描显微镜(CLSM)下观察到DiI标记的纳米粒在肿瘤细胞内高效积聚,流式分析吞噬率为96.3%。3D成像图(图3A,B)表明纳米粒可以成功的进入到细胞球内部并稳定聚集。
实施例6体外抗肿瘤活性和抗原暴露
细胞实验包括6组:对照(PBS)组、ICG+光声联合(L.U.)组、游离OXA组、PPIO-NPs+L.U.组、PPIAO-NPs+L.U.组、PPIO-NPs+Al+L.U.组。实验参数:ICG=35.67ug·mL-1,OXA=15.21ug·mL-1,808nm激光(2.0W·cm-2,5min)和低强度超声(1W·cm-2,5min)。将ID8肿瘤细胞(1×105cells)接种于共聚焦皿中。待细胞贴壁后,分别替换含PBS、ICG、游离OXA、PPIO-NPs、PPIAO-NPs或PPIO-NPs+Al(OH)3的新鲜培养基。共孵育2h后,对L.U.组进行808nm激光和低强度超声处理。活性氧荧光探针(DCFH-DA)检测各组胞内活性氧生成。通过钙黄绿素(CAM)/碘化丙啶(PI)双染检测相同处理的ID8活细胞/死细胞。在CLSM图像中,胞内活性氧为绿色荧光标记,活细胞呈绿色,死细胞呈红色。接种在6孔板或24孔板中的ID8细胞经过相同的分组处理后,通过流式细胞术检测细胞的凋亡比例或CCK-8检测细胞存活率。收集处理后的上清液,通过ATPAssay Kit检测ID8细胞的ATP释放。
吞噬了纳米粒(PPIAO-NPs和PPIO-NPs)的肿瘤细胞经过激光和超声的处理后,胞内活性氧显著增加,与2,7-二氯二氢荧光素二乙酸酯(DCFH-DA)探针结合后,表现为CLSM下的强绿色荧光(图3C)。通过活/死细胞染色和流式分析评估各组细胞处理后的死亡情况,结果均表明(图3D)纳米粒结合激光/超声组都诱导了更彻底的细胞凋亡和坏死(PPIO+L.U.95.96%,PPIAO+L.U.95.71%和PPIO+Al+L.U.90.94%)。CCK8检测中得到进一步证明,细胞存活率在PPIO+L.U.组(45.24±3.56%)、PPIAO+L.U组(42.10±1.31%)和PPIO+Al+L.U.组(39.10±1.29%)之间,无显著性差异(p>0.05)。
免疫荧光检测肿瘤抗原暴露:将培养在共聚焦皿中的ID8细胞分组处理后的,置于冰面,PBS清洗3次,每次3min。4%多聚甲醛固定20min后,PBS清洗3次,每次3min。0.5%Triton-100破膜10min后,PBS清洗3次,每次3min。去掉PBS,封闭液(10%胎牛血清)孵育30min。加入一抗(抗钙网蛋白抗体或者抗HMGB1抗体),样本放入湿盒,4℃孵育过夜。然后在PBS清洗后加入荧光(488或者Cy5)标记的羊抗兔IgGH&L(2ug·ml-1),孵育1h。DAPI染色标记细胞核10min。最后,加入抗荧光淬灭剂,CLSM观察ID8细胞CRT的膜反转和HMGB1的分泌。
表现为CLSM下特征性的钙网蛋白(calreticulin,CRT)膜反转,HMGB1的外泌(图3E)和细胞培养上清液中ATP的分泌增加。
流式细胞术检测肿瘤抗原暴露:细胞用80%甲醇固定(5分钟),然后用0.1%PBS-Triton X-100渗透15分钟。然后将细胞在封闭液(10%胎牛血清)孵育,以阻断非特异性蛋白质-蛋白质相互作用,然后一抗(抗钙网蛋白抗体或者抗HMGB1抗体,1μg·ml-1),室温孵育30分钟。Alexa 488或者Cy5标记的二抗,以1:2000稀释度预吸附30分钟。使用与一抗相同浓度和条件的兔IgG同种型对照抗体。流式细胞仪检测分析。
流式检测结果也表明,在纳米粒结合激光和超声处理组中,检测到更多AlexaFlour 488标记的ID8细胞。而在这些组内被Cy5标记的细胞则较少,因为胞内HMGB1的大量外分泌。PPIAO+L.U组ATP分泌水平显著高于其余各组(p<0.0001)。
以上结果表明基于PPIAO-NPs的光声治疗可以同时实现肿瘤细胞的杀伤与肿瘤抗原的暴露。
实施例7抗原捕获
首先,以牛血清白蛋白为标准,通过Bradford法测定PPIAO-NPs和纳米氢氧化铝捕获的蛋白质含量。纳米粒吸附蛋白质量为总蛋白量减去上清液中蛋白量。通过动态光散射法测定PPIAO-NP吸附蛋白前后的流体动力学粒径和Zeta电位改变。透射电镜观察氢氧化铝捕获蛋白前后的形态改变。SDS-PAGE检测和比较裂解肿瘤细胞蛋白和捕获蛋白差异。ID8细胞与含有ICG(35.67ug·ml-1)、OXA(15.21ug·ml-1)的DMEM(无FBS)培养基共孵育2h。808nm激光和低强度超声处理细胞后,收集上清液,离心(200g,5min),以去除所有不溶的细胞碎片。氢氧化铝(5.38ug·ml-1)与上清液孵育4h后,PBS清洗3次,重悬于PBS用于进一步检测。
通过布拉德福实验检测,结果表明包载氢氧化铝纳米粒的PPIAO-NPs蛋白俘获量为551.67ug·mL-1,与未包载纳米铝的PPIO-NPs+L.U.组比较,具有显著性差异(p<0.001),与相同浓度的单独纳米铝比较,差异不显著。纳米粒俘获蛋白抗原后平均粒径增加979.3±13.316nm,表面电位降低-26.63±2.922mV。透射电镜下观察到氢氧化铝纳米粒与BSA孵育后形态发生改变,颗粒表面出现膜状物质,这可能是由于蛋白吸附所致。为了明确捕获蛋白质中是否含有肿瘤相关抗原,在对ID8细胞处理后,分离和鉴定纳米铝结合的蛋白。凝胶电泳结果提示,与细胞裂解蛋白比较,捕获蛋白的蛋白谱(proteinprofile)发生了改变。
通过LC/MS/MS鉴定氢氧化铝捕获的肿瘤蛋白:200ug捕获蛋白溶液首先由10Kd超滤管置换溶液为8MUA溶液,随后加入100uL吲哚乙酸(IAA)(50mM,终浓度不低于20mM)。室温避光孵育30min后,通过烷基化反应以封闭巯基。反应结束后,溶液通过10kd的超滤管置换至50mM NH4HCO3酶切溶液中。酶切反应通过加入4ug胰蛋白酶,在37℃孵育振荡过夜。12h后,酶切肽段由超滤收集。三氟乙酸(TFA)终止酶切。超滤液进行Sep-Pak C18脱盐。除盐后的肽段溶液经离心浓缩仪抽干后,冻存于-20℃待用。0.1FA%复溶后上机检测。肽段样品通过自动进样器吸入后结合至C18捕获柱(3um,75um×20mm,),接着被洗脱至分析柱(50um×150mm,2um粒径,孔径,Thermo)进行分离。利用两个流动相(流动相A:99%H2O,0.1%甲酸和流动相B:80%乙腈(CAN),0.1%甲酸)建立起100分钟的分析梯度(0min in 3%B,0-5min of 3-5%B;5-70min of 5-23%B,70-90min of 23-55%B,90-92min of55-90%B,90%B for 8min)。液相的流速设置为300nL·min-1。质谱DDA模式分析时,每个扫描循环中包含一个MS全扫描(m/z范围是350-1800,离子累积时间200ms),以及随后跟着的40个MS/MS扫描(m/z范围是100-1500,离子累积时间50ms)。MS/MS采集的条件设置为母离子信号大于3e6,电荷数为+2~+5。离子重复采集的排除时间设置为35s。QE产生的质谱数据通过Protein Discover(V2.2)进行检索,采用的数据库检索算法是Percolator。检索使用的数据库是Uniprot中小鼠的蛋白质组参考数据库(UniProt_mouse_20190908.fasta)。检索结果以对PSM的Maximum Delta Cn和Maximum Rank进行卡值≥0.05为标准进行筛选,删去反库中检索的条目和污染蛋白,余下的鉴定/定量信息用作后续分析。结合已知的肿瘤抗原清单和从质谱学数据中获得的捕获蛋白的列表,捕获的肿瘤抗原根据含有特异性肽段的数量呈现。
经质谱检测,筛选出Sum PEP Score≥1.5的蛋白,并删去污染蛋白,俘获蛋白中含特异性肽段的主要蛋白2632个。其中已知肿瘤相关蛋白抗原约2.85%,预测基因9个,还包括与肿瘤免疫代谢相关的诸多功能蛋白(图4A)。经数据库比对检索分析后确认,检索肽段的质量数偏差符合正态分布。
实施例8体外DC刺激
在Trans well小室中共培养ID8(上层)和DCs(下层)。对上层的肿瘤细胞进行上述相同的分组处理后,流式细胞术检测DCs共刺激分子CD11c(FITC标记抗小鼠CD11c抗体)、CD80(APC标记抗小鼠CD80抗体)、CD86(PE标记抗小鼠CD86抗体)的表达情况。酶联免疫法(ELISA)检测小室上清液中IL-12的含量。每组设定5个复孔。分别用CFSE Cell DivisionTracker标记DC细胞,DiI标记ID8细胞,Hoechst 33342活细胞染色液标记细胞核。CLSM观察DC形态变化。此外,为了观察DC和巨噬细胞对捕获抗原的内化,将DiI-NPs捕获FITC标记的OVA(OVA-FITC)后,与DAPI标记的DC和巨噬细胞共孵育2h。PBS清洗后,CLSM观察两种细胞对抗原的摄取。
Transwell实验中,通过对DC细胞的共刺激分子表达,细胞因子分泌和细胞形态改变来综合评估捕获肿瘤抗原的纳米粒能否有效促进DC成熟。流式检测结果(图4B)表明,与对照组比较,ICG+L.U.组(3.04倍)与游离OXA组(2.95倍)中的DC成熟度(CD11c+CD86+CD80+)增加,而纳米粒结合光声处理组则显著增加,其中PPIO+L.U.组3.57倍,PPIAO+L.U.组5.12倍,PPIO+Al+L.U.组5.27倍。体外实验中,纳米铝在治疗体系中的存在形式(包载或游离)未对DC成熟产生明显的影响。IL-12是成熟DC分泌的细胞因子,可以诱导初始T细胞(Th0)分化为Th1细胞,产生Th1型免疫应答。ELISA检测各组处理后上清液中IL-12水平,结果(图4C)表明在纳米粒结合光声处理组都有较高水平的分泌量,PPIAO+L.U.组与其余各组比较,具有显著性差异(p<0.0001)。CLSM下观察PPIAO+L.U.组的DC细胞形态,呈典型的树枝样突起(图4D),系未成熟DC(immature DC)摄取抗原后逐渐成为成熟DC(mature DC)。CLSM观察到两种细胞的胞膜和胞质中除了出现红色荧光和绿色荧光的共定位,还存在单独的绿色荧光,这可能是APCs摄取了溶液中未被DiI染色的吸附了OVA-FITC的氢氧化铝纳米粒所致。
由上可知,PPIAO-NPs结合光声治疗可以实现肿瘤抗原的充分暴露,并在成功捕获蛋白抗原基础上,有效的诱导DC成熟,促进抗原的加工与提呈,从而激活抗肿瘤免疫反应。
实施例9体内抗肿瘤作用及抗原暴露
通过雌性C57BL/6小鼠ID8卵巢癌皮下移植瘤模型,评价PPIAO-NPs介导的联合治疗效果。分组包括对照(PBS)组、ICG+光声(L.U.)组、游离OXA组、PPIO-NPs+L.U.组、PPIAO-NPs+L.U.组、PPIO-NPs+Al+L.U.组。实验参数:ICG=7.36mg·kg-1,OXA=3.0mg·kg-1,808nm激光2.0W·cm-2,5min和低强度超声1W·cm-2,5min。对照组和游离OXA组仅静脉注射PBS或游离OXA。ICG+L.U.组、PPIO-NPs+L.U.组、PPIAO-NPs+L.U.组、PPIO-NPs+Al+L.U.组的小鼠在静脉给药后6-8h,肿瘤部位接受光声联合治疗。所有治疗重复三次,分别在第1天、第4天、第7天。1%戊巴比妥钠用于小鼠麻醉(35mg·kg-1)。热红外成像仪监测治疗过程温度变化。小鼠的体重、肿瘤体积每隔2天记录一次,连续21天。小鼠图像采集需在小鼠麻醉下进行,故每周采集一次。分别在第8天,第11天,第14天收集小鼠的血液、肿瘤和主要脏器(心、肝、脾、肺、肾)。肿瘤组织进行HE染色、增殖细胞核抗原(增殖细胞核抗原)染色、末端脱氧核苷酸转移酶dUTP缺口末端标记(TUNEL)。剩余小鼠用于90天的生存期观察。当肿瘤体积大于1500mm3时,处死小鼠。
肿瘤抗原的暴露通过石蜡切片免疫荧光同源双标法检测:将4%多聚甲醛固定的石蜡包埋肿瘤组织切片。石蜡切片经二甲苯和酒精脱蜡,蒸馏水水化。组织切片置于EDTA抗原修复缓冲液(pH8.0)中,在微波炉内进行抗原修复。PBS洗涤后,进行3%双氧水孵育,以封闭内源性的过氧化物酶。PBS清洗后,血清封闭30min。加入一抗(抗HMGB1抗体),组织切片在湿盒中4℃过夜孵育。加入HRP标记的二抗,室温孵育1h。加入Cy5-TSA,避光孵育10min。脱色摇床洗涤3次后,组织切片置于EDTA抗原修复缓冲液(pH8.0)中,微波炉加热去除抗体-TSA复合物。加入一抗(抗钙网蛋白抗),组织切片在湿盒中4℃过夜孵育。加入对应的Alexa488标记的二抗,室温孵育1h。DAPI复染细胞核10min。PBS洗涤后,自发荧光淬灭剂孵育5min。最后,切片封存于抗荧光淬灭剂中,通过Pannoramic P-MIDI扫描。使用SlideViewer图像分析软件分析图片。
热红外监测表明,治疗过程中肿瘤部位的温度持续稳定在55℃左右(图5A),3D成像显示光声辐照对肿瘤部位进行了精确治疗(图5B)。在给与不同的治疗后,监测各组小鼠肿瘤的生长和体积变化(图5C)。观察期间,各组小鼠的体重均有增加(图5D),但差异不显著(p>0.05)。纳米粒结合光声组的小鼠肿瘤生长得到抑制(抑制率:PPIO+L.U.组45%、PPIAO+L.U.组49%、PPIO+Al+L.U.组44%)。与对照组、ICG+L.U.组和游离OXA组比较,具有显著性差异(p<0.001)(图5E)。对这批治疗小鼠的生存期统计结果也表明,通过联合治疗降低了荷瘤小鼠的肿瘤负荷之后,它们获得了比对照组和单一治疗组更长的存活时间(p<0.001)(图5F)。H&E染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)和增殖细胞核抗原(PCNA)免疫组织荧光、DAPI用于进一步了解治疗后肿瘤组织的组织学特征改变(第8天)。图5G展示的结果中,在PPIAO+L.U.组瘤内出现更大范围的组织破坏和细胞凋亡,肿瘤细胞增殖得到明显抑制。
实施例10抗肿瘤免疫激活
通过组织免疫荧光(Immunofluorescence staining)观察各组小鼠治疗后瘤内细胞ICD相关的分子表达情况。流式细胞术检测上述处理后收集的小鼠脾脏和肿瘤组织中DC成熟与CD4+T、CD8+T淋巴细胞丰度。通过酶解液(1%透明质酸酶,1%胶原酶,0.5%脱氧核糖核酶)获取单细胞悬液。通过ACK红细胞裂解液(ACK Lysis Buffer)去除脾脏组织红细胞。通过活/死细胞染色试剂盒检测鉴定活/死细胞。在细胞染色前,将样品先与FC Block(抗小鼠CD16/32单克隆抗体)冰上孵育5分钟。然后用FITC标记抗小鼠CD4和PC5.5标记抗小鼠CD8a标记CD4+T、CD8+T淋巴细胞。成熟DC表型为CD11c+/CD80+/CD86+,用FITC标记抗小鼠CD11c抗体、APC标记抗小鼠CD80抗体和PE标记抗小鼠CD86抗体标记DC。流式细胞仪使用Becton-Dickinson进行分析,并使用软件进行分析。
如图6A所示,在PPIAO+L.U.组的瘤内细胞几乎全部出现CRT的膜反转(高亮绿色荧光所示),和HMGB1的胞外分泌(红色荧光所示)。这与对照组和单一治疗组呈现的CRT胞质低荧光信号和HMGB1核共定位完全不同。理论上PPIAO+L.U.治疗能够像体外研究一样,诱导其它未能被准确检测的肿瘤抗原暴露,甚至是新抗原的表达。
流式细胞术检测结果如图6B所示,PPIAO+L.U.组的瘤内mDC峰度明显高于其余各组。其机理可能是由于肿瘤组织中存在的纳米氢氧化铝发挥了关键作用,通过有效的抗原吸附和储存,以及将可溶性抗原转变为颗粒抗原。研究表明铝的佐剂性可能与缓慢的抗原释放或增加的细胞募集无关,但与在注射部位的抗原保留以及24小时后成熟迁移DCs对颗粒抗原的摄取增加有关。这些DC前往次级淋巴器官(脾脏、淋巴结),通过MHC-T细胞受体识别和共受体参与与初始CD4+T细胞和CD8+T细胞的相互作用,并活化它们。细胞毒性T淋巴细胞是介导特异性抗肿瘤细胞免疫的主力军。在PPIAO+L.U.组的小鼠脾脏和肿瘤组织中,均检测到了最高峰度的CD8+T细胞(CD3+/CD8+)(图6C,D)。如图6E-H所示,在治疗后7天,接受联合治疗的小鼠血清中出现较高的细胞因子水平(IL-6、IL-12、TNF-α和IFN-γ)。尤其是IFN-γ具备强免疫调节功能,促进细胞免疫。PPIAO+L.U.组的IFN-γ水平显著高于其余各组(p<0.001)。由此可知,免疫和非免疫成分之间的默契配合成功实现了抗肿瘤免疫效应。
实施例11PPIAO-NPs改善远位效应的效果
建立双侧肿瘤模型,其中右侧肿瘤定义为原发肿瘤,左侧肿瘤定义为转移瘤,用于评估免疫细胞对转移肿瘤的反应(图7A)。当原发肿瘤生长至200m3时,分别在第1天、第4天、第7天,对小鼠的原发瘤进行光声治疗,小鼠的双侧肿瘤生长被密切监测。观察期间,对照组的一只小鼠由于肿瘤负荷过大而死亡,其余各组小鼠体重未见明显异常(图7B)。联合治疗组的原发肿瘤生长抑制明显(抑制率:PPIO+L.U.组46%、PPIAO+L.U.组57%、PPIO+Al+L.U.组49%)。与对照组、ICG+L.U.组和游离OXA组比较,具有显著差异(p<0.001)。PPIAO+L.U.组平均肿瘤体积最小,与PPIO+L.U.组和PPIO+Al+L.U.组比较,具有显著差异(p<0.01)(图7C)。这种抑制差异在转移瘤的生长中更加明显(抑制率:PPIO+L.U.组23%、PPIAO+L.U.组50%、PPIO+Al+L.U.组34%)。PPIAO+L.U.组小鼠的转移瘤平均体积显著低于PPIO+L.U.组(p<0.001)和PPIO+Al+L.U.组(p<0.01)(图7D)。在PPIAO+L.U.组中,有5只小鼠的转移瘤在第21天未能触及。在原发肿瘤不同治疗后7天,通过组织免疫荧光染色,观察各组小鼠原发肿瘤和转移肿瘤组织中CD4+T细胞和CD8+T细胞的浸润水平。在PPIAO+L.U.组的原发瘤和转移瘤瘤内,都分布了大量的CD8+T细胞(绿色荧光抗体标记)和CD4+T细胞(红色荧光抗体标记),PPIO+L.U.组和PPIO+Al+L.U.组次之,而在对照组、ICG+L.U.组和游离OXA组分布最少(图7E)。进一步的脾脏淋巴细胞流式检测结果表明,在PPIAO+L.U.组活化的CD8+T淋巴细胞丰度最高。这些结果表明,基于PPIAO+L.U.的联合治疗能有效地诱导T细胞向CD8+T细胞分化,并显著提高肿瘤内CD8+T细胞的浸润。
为了评估基于PPIAO+L.U.联合治疗的全身疗效,检测小鼠脾脏淋巴细胞对ID8肿瘤细胞的体外杀伤活性。获得淋巴细胞单细胞悬液,并对细胞计数。以脾淋巴细胞为效应细胞(effector cells,E),对数生长期的ID细胞为靶细胞(target cells,T)。将50:1比例的效应细胞和靶细胞在37℃,5%CO2培养箱中共孵育4小时。通过乳酸脱氢酶检测试剂盒检测ID8细胞活性。ELISA检测上清液中IFN-γ细胞因子分泌水平。每组11个复孔,实验重复3次。
结果表明PPIAO+L.U.组的细胞活性最低(37.02±4.86%),与其余各组比较,具有显著性差异(p<0.05)(图7F)。ELISA检测PPIAO+L.U.组的细胞上清液中IFN-γ浓度最高(图7G)。体内IFN-γ主要由活化的T细胞和NK细胞产生。这提示PPIAO-NPs结合光声处理能够诱导全身T细胞的激活,这有利于清除体内存在的其它微小肿瘤转移病灶。
实施例12疫苗效应
将等量的ID8细胞(1×106cells mL-1)在低吸附孔板中连续培养7天形成肿瘤小体。用含有PPIO-NPs、PPIAO-NPs或PPIO-NPs+Al(OH)3的无血清培养基更换原培养液。共孵育2h后,在24孔板中进行808nm激光和低强度超声处理。收集各组肿瘤小体及上清液,接种于C57BL/6小鼠左后腿根部。分组:对照(PBS)组、PPIO+L.U.组、PPIAO+L.U.组和PPIO+Al+L.U.组,n=8。每组小鼠都接种两次肿瘤细胞疫苗,间隔7天。在末次接种后一周,各组小鼠右后背部皮下接种同一批次培养的、相同量(1×106cells·mL-1)的ID8肿瘤细胞。随后观察各组小鼠成瘤情况。在各组小鼠接种细胞疫苗后7天,各组随机抽取3只小鼠,收集脾脏,通过流式细胞术检测记忆T淋巴细胞(CD3+/CD8+/CD44+)丰度,流式抗体为:APC anti-mouseCD3 Antibody、PC5.5 anti-mouse CD8aAntibody和ER780 anti-mouse CD44Antibody。细胞挑战实验在接受了三次联合治疗的PPIAO+L.U.组单边荷瘤小鼠身上进行(n=5)。在末次治疗后7天,在所有小鼠的左侧背部接种等量的(1×106cells mL-1)ID8细胞。随后观察小鼠皮下成瘤情况。
21天后,对照组和PPIO+L.U.组的所有小鼠均可视触到瘤体,成瘤率100%。PPIO+Al+L.U.组也有4只(80%),PPIAO+L.U.组仅2只(40%)且瘤体较小(图7H)。40天内,PPIAO+L.U.组成瘤率40%,其余各组为100%。90天的观察期内,PPIAO+L.U.组的剩余3只小鼠未见肿瘤形成。
挑战后的第4周,其中2只小鼠的ID8注射部位(左后背部)观察到粉色肿块形成,代表肿瘤出现。90天的生存观察期内,剩余3只小鼠的注射部位未发现新生肿块。与细胞球相比,在体肿瘤展示了更好的疫苗效果,如推迟肿瘤的发生。
这可能是由于PPIAO-NPs的联合治疗可诱导更多的肿瘤抗原暴露、促进更多成熟DC细胞的抗原提呈、使更多的T淋巴细胞活化并增加其瘤内浸润,从而有利于小鼠体内抗肿瘤免疫的诱导与免疫记忆的形成。
实施例13PPIAO-NPs的制备
1、用超声探头(Sonics&Materials Inc.,USA)将2mL ICG水溶液(1.5mg·mL-1)、0.5mL OXA水溶液(3mg·mL-1)、0.5mLAl(OH)3水溶液(2mg·mL-1)与100uL携氧PFP充分乳化60s。进而促进药物的充分溶解和Al(OH)3的均匀分散。
2、加入4mLPLGA-PEG2000二氯甲烷溶液(25mg·mL-1),声震乳化5min(超5s,空5s,功率25%)。
3、加入2mLPVA溶液(3%,w/v),声震乳化5min(超5s,空5s,功率25%)。
4、加入10mL异丙醇溶液(3%,w/v)固化纳米粒(NP)壳。将上述乳状溶液低温磁力搅拌12h,以充分去除有机溶剂。收集乳状溶液离心(12000rmp,4℃,5分钟),用去离子水洗涤三次,直到上清液透明,得到PPIAO-NPs。
经检测,该实施例制备的纳米粒取得了与实施例1制备的PPIAO-NPs类似的效果。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.一种肿瘤抗原诱捕纳米粒,包括核壳结构,所述核为携氧型全氟碳化合物,所述壳为负载有光敏剂和化疗药物的两亲性嵌段共聚物,所述壳的表面连接有铝基佐剂。
2.如权利要求1所述的一种肿瘤抗原诱捕纳米粒,其特征在于:所述纳米粒的流体力学直径为333.13±5.34nm,表面电位为-4.98±0.73mV。
3.如权利要求1所述的一种肿瘤抗原诱捕纳米粒,其特征在于:所述携氧型全氟碳化合物为携氧全氟正戊烷,所述光敏剂为吲哚菁绿、二氢卟吩、IR-780碘化物中的一种,所述化疗药物为铂类药物或蒽环类药物,所述两亲性嵌段共聚物为聚乳酸羟基乙酸共聚物-聚乙二醇,所述铝基佐剂为纳米氢氧化铝。
4.如权利要求3所述的一种肿瘤抗原诱捕纳米粒,其特征在于:所述纳米氢氧化铝的粒径为87.77±4.18nm,表面电位为41.38±1.24mV。
5.权利要求1-4任一项所述一种肿瘤抗原诱捕纳米粒的制备方法,其特征在于:包括以下步骤:
S1:将光敏剂、化疗药物、铝基佐剂、携氧型全氟碳化合物加水乳化;
S2:加入两亲性嵌段共聚物继续乳化,得到混合液1;
S3:在混合液1中加入表面活性剂进行乳化,得到混合液2;
S4:在混合液2中加入交联固化剂进行固化。
6.如权利要求5所述的一种肿瘤抗原诱捕纳米粒的制备方法,其特征在于:步骤S1中,所述光敏剂、化疗药物、铝基佐剂的质量比为1.5-3.0:1.5-3.0:1-2;
以mg/uL计,所述光敏剂和携氧型全氟碳化合物的质量体积比为1.5-3.0:100-200。
7.如权利要求5所述的一种肿瘤抗原诱捕纳米粒的制备方法,其特征在于:步骤S2中,两亲性嵌段共聚物与化疗药物的质量比为50-100:1.5-3.0。
8.如权利要求5所述的一种肿瘤抗原诱捕纳米粒的制备方法,其特征在于:步骤S1-S3中,所述乳化在超声条件下进行,乳化时间为3-5min。
9.如权利要求5所述的一种肿瘤抗原诱捕纳米粒的制备方法,其特征在于:步骤S3中,所述表面活性剂为PVA,按w/v计,所述PVA的浓度为3-5%;所述交联固化剂为异丙醇,按w/v计,所述异丙醇的浓度为2-5%。
10.权利要求1-4任一项所述的一种肿瘤抗原诱捕纳米粒在制备治疗抗肿瘤药物中的应用。
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