CN112870377A - 用于肿瘤光热和光动力协同治疗的复合纳米粒及制备方法 - Google Patents
用于肿瘤光热和光动力协同治疗的复合纳米粒及制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于肿瘤光热和光动力协同治疗的复合纳米粒及制备方法,所述复合纳米粒包括雷帕霉素,光敏剂,聚多巴胺,聚乙二醇。所述雷帕霉素和光敏剂通过自组装的方式形成纳米内核,在碱性条件下多巴胺自聚在该纳米粒表面包裹聚多巴胺,并修饰聚乙二醇。本发明的复合纳米粒在细胞内可下调HSP70和HIF‑1α,最终增强其引起的光热治疗和光动力治疗,导致肿瘤细胞死亡,可应用于制备恶性肿瘤光疗的靶向纳米药物。
Description
技术领域
本发明涉及一种纳米生物技术领域,具体涉及一种用于肿瘤光热和光动力协同治疗的复合纳米粒及制备方法。
背景技术
光动力和光热协同治疗是一种有前景的联合抗肿瘤策略。然而,由于肿瘤多种耐药机制的存在,其长期治疗效率往往受到限制。例如,由于肿瘤低氧及光动力治疗过程中不断的氧气消耗,造成了低氧诱导因子(HIF-1α)的上调,进而导致了肿瘤细胞增殖和转移及对多种治疗手段的耐受。另外,在光热治疗过程中,肿瘤细胞会上调热休克蛋白(HSP)获得耐热性,从而导致热损伤耐受,肿瘤细胞存活,凋亡抑制,削弱了光热治疗的效率。因此,克服光疗过程中的诸多耐受机制,是一种有效增强肿瘤光疗的手段。
设计多功能复合纳米粒同时下调HIF-1α和HSP 70,从而克服多重耐药可有效增强肿瘤的光疗。然而,多功能纳米制剂设计的复杂性及相关小分子抑制剂递送的困难性提高了其设计难度。因此,亟需一种兼具HIF-1α和HSP 70调控功能的复合纳米粒实现光动力和光热协同治疗,增强光疗效果,从而完全消除肿瘤。
发明内容
为解决上述问题,本发明的目的在于克服现有光动力/光热治疗的缺陷,提供一种光动力和光热协同治疗的复合纳米粒及制备方法,所述复合纳米粒可靶向至肿瘤细胞,并下调HIF-1α和HSP 70,增强其引起的光热治疗和光动力治疗,导致肿瘤细胞死亡。其制备方法简单有效,有效避免了载体的毒性,载药量和修饰率高,可应用于制备肿瘤靶向药物。
为了实现上述目的,本发明首先公开了一种用于肿瘤光热和光动力协同治疗的复合纳米粒,所述复合纳米粒包括雷帕霉素,光敏剂,聚多巴胺和聚乙二醇,所述雷帕霉素和光敏剂通过自组装的方式形成纳米内核,所述聚多巴胺包裹纳米内核,并表面修饰聚乙二醇。
进一步,所述光敏剂为二氢卟吩e6。
进一步,所述聚乙二醇分子量为2000。
进一步,所述雷帕霉素和光敏剂的药载比为2-3∶1,具有良好协同效应。
进一步,所述复合纳米粒粒径为100-200nm,电位为-5~-15mV。
进一步,所述复合纳米粒为球形核壳结构。
基于一个总的发明构思,本发明还提供了上述复合纳米粒的制备方法,包括以下步骤:
S1、将光敏剂和雷帕霉素溶于有机溶剂中,在超声条件下加至超纯水中
S2、碱性条件下将缓冲液和多巴胺加至上述溶液,搅拌自聚;
S3、加入缓冲液、聚乙二醇,搅拌,反应,离心清洗即得复合纳米粒。
基于一个总的发明构思,本发明还提供了一种所述复合纳米粒在制备靶向抗肿瘤药物中的应用。
基于一个总的发明构思,本发明还提供了一种所述复合纳米粒在制备恶性肿瘤光疗的靶向纳米药物的应用。优选的,所说恶性肿瘤为三阴性乳腺癌细胞。
上述的药物,进一步的,所述药物为外用制剂、口服制剂或注射剂。作为优选,所述的外用制剂为外用凝胶剂。所述的口服制剂为包含所述的复合纳米粒的颗粒剂、片剂、口服溶液剂等。所述的注射剂为包含所述的复合纳米粒的静脉注射液。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供了一种复合纳米粒,雷帕霉素和光敏剂通过自组装的方式形成纳米内核,在碱性条件下多巴胺自聚在该纳米粒表面包裹聚多巴胺,并修饰聚乙二醇。该复合纳米粒可同时下调HIF-1α和HSP 70,从而克服多重耐药,增强肿瘤光热和光功力协同光疗,最终完全消除肿瘤。本发明所述的复合纳米粒是由纯药纳米粒,聚多巴胺外壳及聚乙二醇外层组成的复合多功能纳米体,纯药纳米内载药量高,外部修饰了聚乙二醇,稳定性良好,属于一种工艺简单且稳定性高的复合纳米粒的制备方法。
2、本发明提供了一种复合纳米粒,其外层修饰聚乙二醇,该复合纳米粒具有纳米级粒径,可以通过实体瘤的高通透性和滞留效应(EPR效应)被动靶向至肿瘤部位,增加药物在肿瘤部位的累积量,降低药物对正常组织的毒副作用。聚乙二醇可有效屏蔽纳米粒子表面的电性,增加纳米粒的体内循环时间,增强肿瘤蓄积,增加肿瘤细胞的摄取,从而进一步增强其对肿瘤治疗的疗效。
3、本发明提供了一种复合纳米粒的制备方法,其制备过程简单可控,有效避免了载体的毒性,载药量和修饰率高。
4、本发明提供了一种复合纳米粒在制备肿瘤药物中的应用,复合纳米粒可靶向富集于肿瘤病灶部位,通过增强光热和光动力协同治疗导致肿瘤细胞死亡,对心、肝、脾、肾等无损伤,可以对肿瘤治疗提供依据和思路。复合纳米粒可被动富集于所有实体瘤组织(通过公认的EPR效应),复合纳米粒可同时下调HIF-1α和HSP 70,而增强肿瘤的光疗。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1和2中RC,RC@PDA,RC@PP的粒径变化图
图2为本发明实施例1和2中RC,RC@PDA,RC@PP的电位变化图
图3为本发明实施例1和2中RC@PP的透射电镜图
图4为本发明实施例1和2中Ce6,RAP,RC@PP的紫外吸收图谱
图5为本发明实施例1和2中RC@PP的荧光光谱
图6为本发明实施例1和2中RC@PP在不同条件下释放曲线图
图7为本发明实施例3中不同浓度RC@PP的温度动态变化图
图8为本发明实施例3中不同制剂的温度动态变化图
图9为本发明实施例3中离心管的红外热成像图
图10为本发明实施例3中间隔给予激光后的温度变化
图11为本发明实施例3中瘤内注射小鼠的红外热成像图
图12为本发明实施例4中各条件下的ROS变化图
图13为本发明实施例5中RC@PP或Ce6细胞孵育后的细胞摄取图
图14为本发明实施例6中对照组,Ce6及RC@PP处理后细胞内的ROS探针荧光强度
图15为本发明实施例6中各种处理组的HSP 70和HIF-1α水平
图16为本发明实施例6中RC@PP与细胞孵育48h并用不同波长激光照射后,用MTT法测定细胞存活率结果
图17为本发明实施例7中药物在体内的分布研究
图18为本发明实施例8中尾静脉小鼠的红外热成像图
图19为本发明实施例9中分别注射各种制剂并给予激光照射后的肿瘤体积变化曲线
图20为本发明实施例9中分别注射各种制剂并给予激光照射后的肿瘤重量
图21为本发明实施例9中分别注射各种制剂并给予激光照射后的肿瘤照片
图22为本发明实施例9中为各组小鼠肿瘤病理切片分析
图23为本发明实施例10中各组小鼠的体重变化
图24为本发明实施例10中各组小鼠心、肝、脾、肾病理切片分析
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段;若未特别指明,实施例中所用试剂均为市售。
本发明涉及到的百分号“%”,若未特别说明,是指质量百分比;但溶液的百分比,除另有规定外,是指100ml溶液中含有溶质的克数。
本发明所述重量份可以是μg、mg、g、kg等本领域公知的重量单位,也可以是其倍数,如1/10、1/100、10倍、100倍等。
以下实施例中,所采用的仪器及生产厂家的详细信息参见表1:
表1主要仪器名称及生产厂家
以下实施例中,所采用的主要试剂名称及生产厂家参见表2:
表2主要试剂名称及生产厂家
实施例1
本实施例提供了一种纯药纳米粒的制备方法,包括如下步骤:
50μL浓度为20mg/mL的雷帕霉素(RAP)乙醇溶液与50μL浓度为20mg/mL的二氢卟吩e6(Ce6)DMSO溶液依次加至5mL超纯水中,超声搅拌,离心清洗即得纯药纳米粒。
实施例2
本实施例提供一种复合纳米粒的制备方法,包括如下步骤:
(1)50μL浓度为20mg/mL的雷帕霉素(RAP)乙醇溶液与50μL浓度为20mg/mL的二氢卟吩e6(Ce6)DMSO溶液依次加至5mL超纯水中,超声搅拌,离心清洗即得纯药纳米粒RC。
(2)上述纯药纳米粒(5mL),加入2.4mL超纯水,2mLTris缓冲液(pH8.5,100mM),0.6mL多巴胺(10mg/mL),搅拌48小时即得聚多巴胺包裹纯药纳米粒RC@PDA。
(3)上述纳米粒(RC@PDA)10mL,加入2mL2mL Tris缓冲液(pH8.5,100mM),1mL聚乙二醇(10mg/mL),搅拌24小时,离心清洗即得复合纳米粒RC@PP。
(4)2.4mL超纯水中加入2mL Tris缓冲液(pH8.5,100mM),0.6mL多巴胺(10mg/mL),搅拌48小时即得聚多巴胺纳米粒PDA。
实验例一:
对实施例1和实施例2的复合纳米粒进行如下检测:
一、粒径与电位:分别检测RC,RC@PDA,RC@PP的粒径与电位,测量方法为:取样品溶液置于Marlven Nano ZS仪器,采用动态光激光散射法检测粒径,测定池温度设定为25℃,每个样品平行操作3份。图1和图2为RC,RC@PDA,RC@PP的粒径及电位变化图,从图的结果可知:RC粒径为160nm,电位为-43mV,而包裹聚多巴胺(RC@PDA)粒径增至190nm,电位升至-22mV,继续包裹聚乙二醇形成复合纳米粒RC@PP的粒径增至210nm,电位降至-9mV。
二、形态:观察RC@PP的形态,形态的检测方法:样品滴加在覆盖碳膜的400目铜网上,置于干燥器中,待其自然干燥后置于透射电镜Titan G2-F20下观察。图3为RC@PP的透射电镜图,从图中可知:本发明的RC@PP在透射电子显微镜下为球形核壳结构。
三、紫外光谱:分别对Ce6,RAP,RC@PP进行紫外光谱扫描,测定方法为:以蒸馏水为空白对照液,测定Ce6,RAP,RC@PP紫外吸收图谱。图4为Ce6,RAP,RC@PP的紫外吸收图谱,从图中可知:Ce6在404nm及640nm有特征吸收峰,RAP在278nm有特征吸收峰,而RC@PP在278nm,404nm及640nm也有较强的紫外吸收。
四、荧光光谱:对RC@PP进行荧光光谱扫描。图5为RC@PP的荧光光谱。从图中可知随着聚多巴胺外壳的包裹,荧光发生淬灭。
五、释放率检测:将RC@PP取1mL至于截留分子量为3500的透析袋中,将装有纳米粒的透析袋分别置于装有释放介质的50mL离心管中,释放介质分别为:pH 7.4、pH 5.5,pH5.5+激光。分别于1、2、4、8、12,24h取出透析袋的溶液,在640nm处测定吸光度A,以未进行释放的纳米粒的吸光度为A0,累计释放率=(1-A/A0)×100。图6为RC@PP在不同条件下释放曲线图。结果表明酸性或激光折射可以促进RC@PP中的药物释放。
实施例3
考察实施例1和实施例2复合纳米粒光热催化性能:
(1)取不同浓度(0.1,0.2,0.4mg/mL)RC@PP或0.4mg/mL不同制剂(RC,PDA,RC@PDA)置于离心管中,用激光照射6分钟,每10秒测定一次温度,并用红外热成像照相机获取热成像照片。图7为不同浓度RC@PP的温度动态变化图。图8为不同制剂的温度动态变化图。图9为离心管的红外热成像图。图10为间隔给予激光后的温度变化。结果表明RC@PP产生光热具有浓度依赖性,并且与PDA,RC和水相比,具有更高的高热转化效率。红外成像图也表明RC@PP在照射4分钟后温度可达到50℃以上,并且RC@PP具有良好的热转换重复性能。
(2)建立荷瘤裸鼠模型:收集对数生长的M231细胞分散于PBS中,细胞密度为1×107/100μL,等体积与基质胶混合,注射于BALB/c裸鼠(雌性,6周)的腋下部位。雌性BALB/c裸鼠,6周龄,购自常州卡文斯实验动物有限公司。待小鼠肿瘤增长至200mm3时,分别给小鼠瘤内注射游离PBS和RC@PP。24小时后,给予激光照射,并用红外热成像照相机获取热成像照片。图11为小鼠的红外热成像图。结果表明RC@PP可在小鼠肿瘤中产生光热转换,导致局部肿瘤温度的升高。
实施例4
考察实施例1和实施例2的复合纳米粒产生ROS能力:
取RC@PP,Ce6,PDA,加入100μM的过氧化氢,并给予激光照射,用ROS探针(SOSG)检测ROS产生量。图12为各条件下的ROS变化图。结果表明RC@PP在酸性条件下用808nm激光处理后,在630nm激光条件下产生较多的ROS。
实施例5
考察实施例1和实施例2的复合纳米粒对肿瘤的靶向作用:
(1)取对数生长的MDA-MB-231细胞(人源性三阴性乳腺癌细胞MDA-MB-231细胞,购自中南大学湘雅医学实验中心),消化计数,适量DMEM完全培养基稀释至2×105cells/mL的细胞悬液,每孔2mL接种于24孔板,总共接种3个孔。贴壁培养24h后吸弃培基,PBS润洗3次。
(2)将RC@PP用DMEM培养基(无FBS)稀释。
(4)加入2mL RC@PP或Ce6。37℃孵育4h后吸弃培基,PBS润洗3次。
(5)每孔加入1mL多聚甲醛避光固定20min,吸弃上清,PBS洗涤三次。
(6)每孔加入0.5mL 1μg/mL DAPI,避光染核15min,吸弃上清,PBS洗涤3次,激光共聚焦显微镜下观察各孔荧光强弱。
图13为细胞摄取结果。其中,Ce6及RC@PP分别与M231细胞共孵育4h后,激光共聚焦显微镜观察。DAPI通道表明细胞核染为蓝色荧光,Ce6通道表明NPs标记为红色荧光,Merged表明叠加DAPI和Ce6通道。标尺=50μm。
从图中可知:Ce6及RC@PP与M231细胞孵育4h,荧光显微镜下可见细胞内有明显的红色荧光,表明纳米粒被细胞摄取,其中RC@PP孔荧光较Ce6孔强。结果说明复合纳米粒对肿瘤细胞具有靶向作用,制备成纳米粒后能增强肿瘤细胞对Ce6的摄取。
实施例6
考察实施例1和实施例2的复合纳米粒对肿瘤细胞的调控和毒性:
(1)取胰酶消化对数生长的MDA-MB-231细胞,用含10%胎牛血清的DMEM培养基稀释成细胞悬液,以105/孔接种到24孔培养板中。在二氧化碳培养箱内(37℃、5%CO2、饱和湿度)培养24h后移弃培养液。每孔加入液。每孔加入RC@PP或Ce6,孵育2h后用激光照射。加入ROS探针(DCFDA)孵育30分钟,用激光共聚焦显微镜下观察各孔荧光强弱。
图14为对照组,Ce6及RC@PP处理后细胞内的ROS探针荧光强度。结果表明相比于Ce6,RC@PP在激光照射下可产生较多的ROS。
(2)取MDA-MB-231细胞以400000/孔种于6孔板,并进行处理,孵育24h。使用Western裂解液裂解MDA-MB-231细胞,收集细胞中的蛋白样品,测定蛋白样品的蛋白浓度。配制SDS-PAGE凝胶,在收集的蛋白样品中加入适量浓缩的SDS-PAGE蛋白上样缓冲液,100℃或沸水浴加热3-5分钟,以充分变性蛋白。冷却至室温后把蛋白样品直接上样到SDS-PAGE胶加样孔内进行电泳,溴酚蓝到达胶的底端处附近停止电泳。选用PVDF膜进行转膜,使用Bio-Rad的标准湿式转膜装置,转膜完毕后,加入5%脱脂牛奶室温封闭1h。吸尽封闭液,加入稀释好的一抗,室温孵育过夜。回收一抗,加入Western洗涤液,洗涤3次。按照适当比例用Western二抗稀释液稀释辣根过氧化物酶(HRP)标记的二抗。吸尽洗涤液,加入稀释好的二抗,室温孵育1h。洗涤3次。最后使用BeyoECL Plus(P0018)等ECL类试剂检测蛋白。
图15为各种处理组的HSP 70和HIF-1α水平。结果表明与对照组相比,RC@PP可有效下调细胞内的HSP 70和HIF-1α的水平。
(3)取胰酶消化对数生长的MDA-MB-231细胞,用含10%胎牛血清的DMEM培养基稀释成细胞悬液,以5000/孔接种到24孔培养板中。在二氧化碳培养箱内(37℃、5%CO2、饱和湿度)培养24h后移弃培养液。每孔加入不同制剂,同一浓度重复6个复孔,激光照射并孵育48h。每孔加入10μL MTT溶液(5mg/mL),继续孵育4h后终止培养。每孔加入DMSO溶液150μL,置摇床上低速振摇10min使结晶溶解完全,用酶标仪测定570nm波长处的吸光值(OD)。
图16为RC@PP与细胞孵育48h并用不同波长激光照射后,用MTT法测定细胞存活率结果。从图16可以看出,本发明的复合纳米粒RC@PP的细胞存活率均为剂量依赖性,并且在两种激光照射下RC@PP的细胞毒性最强。
实施例7
考察实施例1和实施例2的复合纳米粒在体内的分布研究:
(1)建立荷瘤裸鼠模型:收集对数生长的M231细胞分散于PBS中,细胞密度为1×107/100μL,等体积与基质胶混合,注射于BALB/c裸鼠(雌性,6周)的腋下部位。雌性BALB/c裸鼠,6周龄,购自常州卡文斯实验动物有限公司。
(2)处理:待小鼠肿瘤增长至200mm3时,分别给小鼠尾静脉注射游离Ce6和RC@PP(Ce6,2.5mg/kg)。
(3)检测:分别在注射后1h和24h麻醉小鼠,活体成像系统对小鼠进行成像。24h活体成像后,将小鼠处死,取出心肝脾肺肾和肿瘤,成像系统进行成像。
图17为药物在体内的分布研究。分别给裸鼠尾静脉注射游离Ce6和RC@PP,分别在不同时间点拍摄。分别为尾静脉注射后1h和24h的小鼠体内荧光分布图,及注射24h后,将小鼠处死,心、肝、脾、肺、肾和肿瘤的荧光分布图。
从图17中可知:1h时,注射游离Ce6的小鼠荧光强度强于注射RC@PP的小鼠,24h后两只小鼠荧光强度相反。24h后,注射RC@PP的小鼠肿瘤部位可见明显的纳米粒蓄积,表明其靶向作用。离体肿瘤中,注射RC@PP的小鼠肿瘤荧光强度明显强于注射游离Ce6的荧光强度,进一步表明本发明的复合纳米粒可在肿瘤部位蓄积,对肿瘤具有靶向性。
实施例8
考察实施例1和实施例2的复合纳米粒的体内光热成像:
建立荷瘤裸鼠模型:收集对数生长的M231细胞分散于PBS中,细胞密度为1×107/100μL,等体积与基质胶混合,注射于BALB/c裸鼠(雌性,6周)的腋下部位。雌性BALB/c裸鼠,6周龄,购自常州卡文斯实验动物有限公司。待小鼠肿瘤增长至200mm3时,分别给小鼠尾静脉注射游离PBS和RC@PP。24小时后,给予激光照射,并用红外热成像照相机获取热成像照片。图18为小鼠的红外热成像图。结果表明注射有RC@PP的老鼠可见肿瘤部位明显的温度上升,有较好的光热成像性能。
实施例9
考察实施例1和实施例2的复合纳米粒的体内抗肿瘤活性:
按照实施例6的方法处理小鼠,待荷瘤小鼠肿瘤长至100mm3左右时,将小鼠随机分成5组(n=5),每组在第0天分别注射各种制剂,每两天给小鼠称重并用游标卡尺测量肿瘤的体积至第14天,通过各组肿瘤的相对体积比较各组抗肿瘤效率。肿瘤体积计算公式:V=长×宽2/2。
图19为分别注射各种制剂并给予激光照射后的肿瘤体积变化曲线;图20为分别注射各种制剂并给予激光照射后的肿瘤重量;图21为分别注射各种制剂并给予激光照射后的肿瘤照片;其中G1为PBS,G2为RC@PP,G3为RC@PP+L630,G4为RC@PP+L808,G5为RC@PP+L630+L808。
图22为各组肿瘤组织H&E染色图。其中G1为PBS,G2为RC@PP,G3为RC@PP+L630,G4为RC@PP+L808,G5为RC@PP+L630+L808。
从图19中可知,与PBS组相比,RC@PP组均具有一定的抗肿瘤效果,其中RC@PP在两种激光照射下肿瘤抑制效果最强。图20,图21的肿瘤重量变化和肿瘤照片也反应出同样的趋势。
从图22图中可知,RC@PP+L630+L808组具有最多的肿瘤坏死区域。结果表明,复合纳米粒可实现增强的光疗,具有较强的抗肿瘤疗效,可完全消除肿瘤。
实施例10
考察实施例1和实施例2的复合纳米粒的体内调控作用:
按照实施例6的方法处理小鼠,给药第14天后,处死四组小鼠,取出心、肝、脾、肾,生理盐水洗涤,滤纸吸干水分,4%多聚甲醛固定24h。将组织石蜡包埋、切片、HE染色,利用光学显微镜观察病理改变。
图23为各组小鼠的体重变化,都未出现明显的体重降低。图24为各组小鼠心、肝、脾、肾病理切片分析。标尺=100μm。与PBS组相比,其他三组各器官无明显病理改变。说明纳米复合纳米粒体内安全性良好。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种用于肿瘤光热和光动力协同治疗的复合纳米粒,其特征在于,所述复合纳米粒包括雷帕霉素,光敏剂,聚多巴胺和聚乙二醇,所述雷帕霉素和光敏剂通过自组装的方式形成纳米内核,所述聚多巴胺包裹纳米内核,并表面修饰聚乙二醇。
2.根据权利要求1所述的复合纳米粒,其特征在于,所述光敏剂为二氢卟吩e6。
3.根据权利要求1所述的复合纳米粒,其特征在于,所述聚乙二醇分子量为2000。
4.根据权利要求1所述的复合纳米粒,其特征在于,所述雷帕霉素和光敏剂的药载比为2-3:1。
5.根据权利要求1所述的复合纳米粒,其特征在于,所述复合纳米粒粒径为100-200nm,电位为-5~-15mV。
6.根据权利要求1所述的复合纳米粒,其特征在于,所述复合纳米粒为球形核壳结构。
7.一种权利要求1至6中任一项所述复合纳米粒的制备方法,其特征在于,包括以下步骤:
S1、将光敏剂和雷帕霉素溶于有机溶剂中,在超声条件下加至超纯水中;
S2、碱性条件下将缓冲液和多巴胺加至上述溶液,搅拌自聚;
S3、加入缓冲液、聚乙二醇,搅拌,反应,离心清洗即得复合纳米粒。
8.一种权利要求1至6中任一项所述复合纳米粒在制备靶向抗肿瘤药物中的应用。
9.一种权利要求1至6中任一项所述复合纳米粒在制备恶性肿瘤光疗的靶向纳米药物的应用。
10.根据权利要求9所述的应用,所说恶性肿瘤为三阴性乳腺癌细胞。
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