CN117298065A - 一种多功能纳米免疫调节制剂及其制备方法与免疫系统重塑的抗肿瘤应用 - Google Patents
一种多功能纳米免疫调节制剂及其制备方法与免疫系统重塑的抗肿瘤应用 Download PDFInfo
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Abstract
本发明公开了一种多功能纳米免疫调节制剂及其制备方法与免疫系统重塑的抗肿瘤应用,该多功能纳米免疫调节制剂由金属有机框架材料包裹化疗药物并负载免疫抑制阻断剂之后,再经巨噬细胞膜包膜而得;本发明构建的mRDZ仿生化多功能纳米免疫调节制剂实现了基于ICD,IDO1和自噬的三通路靶向协同抗肿瘤,mRDZ的多途径激活可提高杀伤性免疫细胞在肿瘤微环境、肿瘤引流淋巴结、脾脏中的浸润及数量,同时降低抑制性免疫细胞的数量,相应细胞因子也表现出与免疫细胞相似的变化趋势,并且激活的抗肿瘤免疫响应表现出显著的免疫记忆性;这种肿瘤组织局部与全身性的免疫系统重塑,使得mRDZ可有效抑制肿瘤生长,并阻碍二次接种的肿瘤生长。
Description
技术领域
本发明属于生物医学材料领域,具体涉及一种多功能纳米免疫调节制剂及其制备方法与免疫系统重塑的抗肿瘤应用。
背景技术
癌症免疫疗法通过对患者自身免疫系统进行重塑来根除癌细胞,已在临床上被证明是一种具有重要应用前景的治疗策略。当前,癌症免疫疗法主要有免疫检查点阻断(ICB)疗法、细胞因子疗法、癌症疫苗和嵌合抗原受体(CAR)T细胞疗法等,然而,这些疗法通常只通过缓解肿瘤免疫抑制微环境或激活细胞免疫反应的单一途径,以抑制肿瘤生长、转移和复发。这种单一免疫治疗效果有限,往往只在一小部分患者中具有治疗效果。
多通路靶向联合治疗,有望弥补单一免疫治疗的缺陷,如基于低剂量化疗可诱导肿瘤免疫原性细胞死亡(ICD)的作用,可开发化疗与免疫协同的治疗方法。然而,该方式会产生过激的免疫反应,导致淋巴耗竭,或增强肿瘤的免疫抑制反应。可见,通过多种途径,一味增强抗肿瘤免疫反应,亦不能达到安全有效的抗肿瘤治疗效果。
免疫检查点抑制剂(ICI)可阻断肿瘤的免疫抑制作用,使免疫系统得以识别并杀伤肿瘤,当前,亦有诸如PD-1,PDL-1,IDO-1,CD47等靶点靶向的免疫检查点抑制剂,被开发用于临床研究。然而,仅阻断肿瘤的免疫抑制作用,亦不能使免疫系统充分发挥其抗肿瘤作用,且随之而来还会产生耐药、自身免疫相关副作用及器官毒性等。
为弥补激活抗肿瘤免疫反应与抑制肿瘤免疫抑制作用这两种治疗方式的缺陷,可开发在激活免疫反应的同时,阻断肿瘤免疫抑制的双重抗肿瘤免疫治疗策略。在该治疗策略中,免疫激活剂(如低剂量的化疗药物、肿瘤疫苗、免疫激动剂等)可刺激免疫系统,产生更多的抗肿瘤免疫细胞,分泌大量抗肿瘤细胞因子;于此同时,免疫抑制阻断剂(如免疫检查点小分子抑制剂、可沉默免疫检查点基因表达的小干扰核酸(siRNA)分子等)则可降低肿瘤对免疫系统的抵御作用,使增强的抗肿瘤免疫系统得以有效识别并杀伤肿瘤细胞。该方法能有效避免对免疫系统的过度激活与抑制,双重调节并平衡免疫系统的抗肿瘤反应,有望真正实现安全有效的抗肿瘤免疫治疗。然而,免疫激活剂与免疫抑制阻断剂要发挥作用,需能有效到达体内特定部位并与特定细胞相互作用,游离药物往往不具有体内分布的靶向性,且在生物体内还存在易降解、易被清除等问题。
纳米制剂因其纳米尺寸效应,具有诸如靶向输送药物到目标作用靶点、延长体内循环时间、缓控式释放药物、提高药物的稳定性和疗效、降低药物毒副作用、提高药物溶解程度和速度、利于储存和运输、可改变给药途径等特性,在多种药物的靶向递送中具有重要应用前景。金属有机框架材料(MOF)是一类由金属结点与有机配体的配位作用连接而成的新型材料,其中,沸石咪唑酯骨架-8(ZIF-8)因其巨大的孔隙率、结构可调、易修饰、pH响应降解和良好的生物相容性,有望在药物的靶向可控递送中发挥重要作用。与载体材料相比,ZIF-8具有以下特点:1)ZIF-8由2-甲基咪唑和Zn2+组成,锌是生物体内第二丰富的过渡金属,而咪唑基团是组氨酸的组成部分,生物相容性高;2)ZIF-8具有特殊的化学和热稳定性,在中性条件下稳定存在,而在酸性条件下容易分解,有利于pH控制载荷药物的释放;3)ZIF-8的合成是一种温和的仿生矿化过程,可用于包埋酶、DNA和蛋白质等多种生物大分子,且不影响其生物活性。此外,ZIF-8载体自身具有其特殊的生物活性(如诱发自噬等),其生物活性可与所装载的药物产生协同作用,从而发挥更强的疾病治疗作用。然而,裸露的ZIF-8纳米颗粒在体内仍不稳定,亦被巨噬吞噬系统清除。
细胞膜“隐身技术”通过在纳米颗粒表面包覆细胞膜,可使纳米颗粒模拟源细胞的表面性质,从而提高其稳定性、靶向性、长循环等性能,如基于巨噬细胞膜包被的ZIF-8纳米颗粒,可被用于多种药物的肿瘤或炎症靶向递送中。然而,当前未有基于巨噬细胞膜仿生化ZIF-8纳米载体用于抗肿瘤免疫激活及阻断免疫抑制药物的靶向共递送中,其递送过程及抗肿瘤免疫应用仍鲜有报道。
发明内容
针对上述问题,本发明提供了一种基于细胞膜仿生化ZIF-8的多功能纳米免疫调节剂系统。通过沸石咪唑啉酸盐骨架-8(ZIF-8)包裹化疗药物DOX,负载靶向IDO-1的siRNA,并且给予该纳米颗粒巨噬细胞膜(RAW 264.7)涂层,最终构建了mRDZ仿生化多功能纳米免疫调节制剂,实现了基于ICD,IDO1和自噬的三通路靶向协同抗肿瘤。
本发明mRDZ的多途径激活,可提高杀伤性免疫细胞在肿瘤微环境、肿瘤引流淋巴结、脾脏中的浸润及数量,同时降低抑制性免疫细胞的数量,相应细胞因子也表现出与免疫细胞相似的变化趋势,更重要的是,激活的抗肿瘤免疫响应表现出显著的免疫记忆性。这种肿瘤组织局部与全身性的免疫系统重塑,使得mRDZ可有效抑制肿瘤生长,并阻碍二次接种的肿瘤生长。
本发明的技术方案如下:
一种多功能纳米免疫调节制剂,由金属有机框架材料包裹化疗药物并负载免疫抑制阻断剂之后,再经巨噬细胞膜包膜而得;
其中,
金属有机框架材料为ZIF-8;
化疗药物为DOX(阿霉素);
免疫抑制阻断剂为siRNA;
巨噬细胞膜为RAW264.7细胞中提取的细胞膜。
本发明所述的多功能纳米免疫调节制剂的制备方法,包括如下步骤:
(1)将六水合硝酸锌溶液和DOX溶液混合,搅拌下加入2-甲基咪唑溶液,继续搅拌0.5~2h,离心,收集沉淀,得到DZ载药纳米颗粒;
优选六水合硝酸锌溶液的浓度为2mg/mL,溶剂为纯水;
优选DOX溶液的浓度为1mg/mL,溶剂为纯水;
优选2-甲基咪唑溶液的浓度为40mg/mL,溶剂为纯水;
优选六水合硝酸锌溶液、DOX溶液、2-甲基咪唑溶液的体积比为4:1:4;
该步中,由于金属离子和有机配体的自组装配位反应,迅速形成中空网状晶体结构,此时DOX被包封在中空网状晶体结构内部,得到包裹药物的DZ纳米颗粒;
(2)将步骤(1)所得DZ载药纳米颗粒与siRNA混合,室温静置,通过静电吸附作用siRNA被吸附到DZ载药纳米颗粒表面,形成RDZ纳米颗粒;
优选DZ载药纳米颗粒包封的DOX与siRNA的质量比为25:1;
优选室温静置的时间为30min;
(3)从RAW264.7细胞中提取细胞膜,将步骤(2)所得RDZ纳米颗粒与提取所得细胞膜混合进行冰浴超声包膜,之后离心,收集得到mRDZ纳米颗粒(即所述的多功能纳米免疫调节制剂);
从RAW264.7细胞中提取细胞膜后,用BCA试剂盒定量,提取效率约为每100万细胞提取1mg膜蛋白量;优选RDZ纳米颗粒与巨噬细胞膜膜蛋白量的质量比为10:1;
优选超声的时间为30min。
本发明所述的多功能纳米免疫调节制剂可用于制备免疫系统重塑的抗肿瘤药物,具体的肿瘤例如结肠癌。
本发明所述的多功能纳米免疫调节制剂的作用机制如下:
本发明基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统,主要包括以下几部分:ICD的诱导,IDO1的沉默和自噬的激活。免疫原性细胞死亡(ICD)作为一种特殊的细胞死亡形式,能够触发针对死亡细胞相关抗原的特异性免疫反应。阿霉素(DOX)等化疗药物不仅可诱导肿瘤细胞损伤,还可诱导ICD效应。ICD依赖于一系列损伤相关分子模式(DAMPs)的协调表达和释放,包括内质网伴侣蛋白(CRT)暴露、三磷酸腺苷(ATP)分泌和高迁移率族蛋白1(HMGB1)释放。不过化疗药物杀死的肿瘤细胞具有的免疫原性非常弱,因为化疗药物例如阿霉素(DOX)诱导ICD的同时,也会通过CTL分泌的干扰素-γ(IFN-γ)导致IDO1上调,从而出现免疫抑制现象;吲哚胺2,3-双加氧酶1(IDO1)是肿瘤免疫微环境中的一种重要免疫抑制因子,其在肿瘤组织中高表达,起着关键的免疫抑制作用。此外,IDO1的高表达可通过激活PI3K/AKT通路来抵抗应激诱导的细胞凋亡并抑制自噬;自噬是一种自我降解的活动,通过膜将部分胞质和细胞内需降解的细胞器、蛋白质等组分包裹起来形成自噬体,然后与溶酶体融合形成自噬溶酶体,最终降解其所包裹的内容物的过程。其被作为ICD的“使能器”,因为它可以促进ATP的分泌和诱导显著的抗肿瘤免疫反应。因此,自噬的激活能够增强ICD效应诱导的免疫应答。
在基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统中,所述ICD的诱导是依靠能诱导ICD的化疗药物(DOX),所述IDO1的沉默是依靠能诱导IDO1基因沉默的功能性siRNA(siRNAIDO1),所述自噬的激活是依靠能激活自噬的纳米材料(ZIF-8)。通过诱导自噬的纳米材料(ZIF-8)包裹诱导ICD的化疗药物(DOX),并且负载上能沉默IDO1基因的功能性siRNA(siRNAIDO1)。此外,为了使其能够逃避单核吞噬系统(MPS)的清除,靶向肿瘤部位,并且进入到肿瘤内部缺氧区,赋予了其巨噬细胞膜涂层。
基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统具有众多优势:1)与未涂层相比,巨噬细胞膜的涂层,使得mRDZ能够逃避MPS清除,靶向肿瘤,并且进入肿瘤内部缺氧区,提高纳米颗粒在体内循环时间,从而产生更好的治疗效果;2)单独的DOX在诱导ICD效应,激活特异性免疫反应的同时,会诱导IDO1高表达,从而出现免疫抑制,减弱特异性免疫反应的问题;而当DOX与siRNAIDO1联合使用时,则避免出现了上述的问题;3)与单一的免疫检查点抑制剂相比,siRNAIDO1的特异性更高,可直接靶向性抑制免疫抑制基因IDO1;4)ZIF-8诱导的自噬会促进ICD效应,同时siRNAIDO1抑制IDO1而促进自噬,从而放大了ICD效应。
本发明的有益效果在于:
1.本发明所述的基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统,其中基础材料ZIF-8由无机锌离子通过2-甲基咪唑盐键连接而成,不仅可以通过简单、低成本的生物矿化过程来包埋酶、DNA和蛋白质等多种生物大分子,可以防止生物大分子的泄露,保持其生物活性。而且在生理条件下稳定,在酸性条件下分解,可用于构建pH敏感的药物递送系统,能够实现对肿瘤细胞内微环境的响应。当位于酸性的肿瘤微环境中时,可响应性释放药物。此外,作为ZIF-8结构组成中的金属离子也有着独特的优势。金属离子可以作为提高抗癌免疫力的元素,发挥清除肿瘤的功能。
2.本发明所述的基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统,通过低张溶解过程排空内部以获得细胞膜,巨噬细胞膜的涂层可以通过挤压或在超声波中实现。巨噬细胞膜的涂层不仅赋予其逃避单核吞噬系统(MPS)清除和靶向肿瘤部位的能力,而且有助于延长纳米颗粒在体内的循环时间,从而进一步提高药物递送效率和治疗效果。
3.本发明所述的基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统,通过简单有效的ZIF-8材料将自噬,ICD和IDO1三者相互协同,这种“三联疗法”相较于传统纳米免疫疗法具有显著的优势。仿生化多功能纳米免疫调节剂mRDZ可激活抗肿瘤免疫反应,重塑肿瘤免疫抑制微环境和重构了肿瘤相关巨噬细胞(TAM),可选择性诱导CD8+T细胞从脾脏迁移到肿瘤,并激发了强大的免疫记忆反应。此外,mRDZ能够有效抑制原发肿瘤的生长,抑制肿瘤转移,并且防止肿瘤的复发。
附图说明
图1是本发明中仿生化多功能纳米免疫调节剂mRDZ的合成路线图。
图2是本发明实施例1中相关纳米颗粒的粒径、电势图和具体实物图;(A)纳米颗粒的粒径和实物图像;(B)纳米颗粒的电势。
图3是本发明实施例2中所述载药纳米颗粒在不同pH条件下的体外释放曲线图和体外溶血率柱状图;(A)mRDZ的体外药物释放曲线;(B)mRDZ的体外血液相容性研究。
图4是本发明实施例3中所述载药纳米颗粒在体外细胞摄取共聚焦图。
图5是本发明实施例3中所述载药颗粒在体外细胞胞内溶酶体逃逸共聚焦图。
图6是本发明实施例3中所述MTT检测结果的柱状图。
图7是本发明实施例4中所述siRNAIDO1在体外细胞中的RT-PCR和WB柱状图;(A)mRDZ处理后的IDO1 mRNA的表达情况;(B)mRDZ处理后的IDO1蛋白表达情况。
图8是本发明实施例4中所述载药颗粒在体外细胞胞内诱导自噬的透射电镜图。
图9是本发明实施例4中所述载药颗粒在体外细胞胞内诱导ATP和HMGB1释放以及细胞表面CRT表达的柱状图;(A)ATP释放量;(B)HMGB1释放量;(C)细胞表面CRT表达量。
图10是本发明实施例4中所述载药颗粒在体外诱导BMDC细胞成熟的流式分析结果;流式检测BMDC与不同处理后的肿瘤细胞共孵育后,其CD80+CD86+成熟细胞的数量。
图11是本发明实施例5中所述不同给药体系在Balb/c小鼠中肿瘤体积变化曲线图。
图12是本发明实施例6中所述不同给药体系在Balb/c小鼠肿瘤组织中激活抗肿瘤免疫水平的相关柱状图;(A-H)肿瘤组织中各类免疫细胞的数量。
图13是本发明实施例6中所述不同给药体系在Balb/c肿瘤模型小鼠淋巴结(TDLN)及脾脏中激活抗肿瘤免疫水平的相关柱状图;在TDLN中,(A)M1型及(B)M2型巨噬细胞数量及(C)两种细胞(M1/M2)数量比;(D)成熟DC数量百分比;在脾脏中,(E)CD4+T,(F)CD8+T,(G)MDSC,(H)Treg,(I)记忆性CD4+T,(J)记忆性CD8+T各类细胞数量。
图14是本发明实施例7中所述不同给药体系在Balb/c小鼠中对侧二次接种肿瘤增长曲线图。
具体实施方式
以下通过实施例的具体实施方式再对本发明的上述内容作进一步的详细说明。但不应当将此理解为本发明上述主题的范围仅限于以下的实例。在不脱离本发明的精神和原则之内做的任何修改,以及根据本领域普通技术知识和惯用手段做出的等同替换或者改进,均应包括在本发明的保护范围内。
本说明书中公开的所有特征,或公开的所有方法或过程中的步骤,除了互相排斥的特征和/或步骤以外,均可以以任何方式组合。
以下实施例中,
六水合硝酸锌购于国药集团化学试剂有限公司(CAS号10196-18-6,规格100g),将10mg六水合硝酸锌溶解于5mL超纯水中,配置成浓度为2mg/mL的六水合硝酸锌溶液。
2-甲基咪唑购于上海迈瑞尔生化科技有限公司(CAS号693-98-1,规格250g),将200mg 2-甲基咪唑溶解于5mL超纯水中,配置成浓度为40mg/mL的2-甲基咪唑溶液。
DOX购于大连美仑生物技术有限公司(货号MB1087-3,规格1g),取10mg的DOX溶于10mL的超纯水中,配置成浓度为1mg/mL的DOX溶液。
siRNA购于苏州吉玛基因股份有限公司(目录号A101003,规格2OD),向2OD的siRNA中加入125μL DEPC水,配置成浓度为20μM的siRNA溶液。
从RAW264.7细胞中提取细胞膜是通过试剂盒(北京英文特生物技术有限公司,货号SM-005)中的方法提取。
实施例1:巨噬细胞膜涂层的仿生化多功能纳米免疫调节剂的制备及其粒径、电势、包封率和载药量
a)将0.2mL 2mg/mL的六水合硝酸锌溶液、0.05mL 1mg/mL的DOX溶液,按体积比4:1在磁力搅拌作用下,室温搅拌5分钟,溶液呈橙色;
b)搅拌下加入0.2mL 40mg/mL的2-甲基咪唑溶液,溶液呈浅紫色,磁力搅拌1h;10000rcf 15min除去未反应完全的原材料,两次离心收集沉淀,得到DZ载药纳米颗粒;
c)将1640μg步骤b)得到包封了42.5μg DOX的DZ和1.7μg siRNA按DOX/siRNA质量比25:1混合,室温静置30min,siRNA通过静电作用吸附到DZ外层,得到RDZ载药纳米颗粒;
d)从RAW264.7细胞中提取细胞膜,并用BCA试剂盒定量(提取效率约为每100万细胞提取1mg膜蛋白量);
e)将1750μg步骤c)得到的RDZ载药纳米颗粒和175μg巨噬细胞膜膜蛋白量按质量比10:1进行冰浴超声包膜,超声30min,包膜后5000rcf 15min离心去除多余细胞膜,收集得到mRDZ载药纳米颗粒。
粒径和电势检测
通过DLS检测载药粒子的尺寸,如图2中(A)所示,ZIF-8溶液颜色呈乳白色,而DZ,RDZ和mRDZ溶液颜色成紫色。粒径结果显示,mRDZ粒径约为201.27±0.39nm。如图2中(B)所示,负载上siRNA后,电势由23.03±0.74mV变为-0.27±0.21mV,而当细胞膜涂层后,电势-0.27±0.21mV变为-22.93±0.12mV。
包封率和载药量测试
将DZ载药纳米颗粒重新溶于水中,由于阿霉素(DOX)自带荧光(激发波长为495nm,发射波长为590nm),利用荧光标准曲线法测定其包封率和载药量。测定结果显示,药物DOX包封率为86.01%±1.04%,载药量为2.60%±0.32%。
该实施例中,基于ICD,IDO1和自噬三者相互协同的“三联疗法”的仿生化多功能纳米免疫调节系统的功能主要包含三部分:ICD的诱导,IDO1的沉默和自噬的激活:
a)仿生化:仿生化是依赖于仿生化多功能纳米免疫调节系统中的巨噬细胞膜涂层。巨噬细胞膜的涂层使得mRDZ能够逃避单核吞噬系统(MPS)的清除,巨噬细胞膜上存在着各种特异性蛋白因子和受体,使mRDZ具有肿瘤靶向性,能够进入肿瘤内部缺氧区。并且有助于延长纳米颗粒在体内的循环时间,从而进一步提高药物递送效率和治疗效果。
b)ICD的诱导:ICD的诱导是依赖于仿生化多功能纳米免疫调节系统中的化疗药物DOX。DOX一方面起化疗作用,另一方面可以诱导免疫原性细胞死亡(ICD)。ICD作为一种特殊的细胞死亡形式,能够触发针对死亡细胞相关抗原的特异性免疫反应。不过化疗药物杀死的肿瘤细胞的免疫原性非常弱,因为化疗药物例如阿霉素(DOX)诱导ICD的同时,也会通过CTL分泌的干扰素-γ(IFN-γ)导致IDO1上调,从而出现免疫抑制现象。因此DOX和siRNAIDO1的联合抗肿瘤治疗可以显著提高免疫治疗效果。
c)IDO1的沉默:IDO1的沉默是依赖于仿生化多功能纳米免疫调节系统中的siRNAIDO1。吲哚胺2,3-双加氧酶1(IDO1)是肿瘤免疫微环境中一个重要的免疫抑制因子,并且在肿瘤免疫微环境中高表达,起着关键的免疫抑制作用。此外,IDO1还可以抑制应激诱导的细胞凋亡和自噬。使用siRNAIDO1可以直接靶向性抑制免疫抑制基因IDO1,解决了化疗药物在诱导ICD的同时诱导IDO1高表达而带来免疫抑制的问题,同时siRNAIDO1可以通过抑制IDO1而促进自噬,从而放大DOX诱导的ICD效应。
d)自噬的激活:自噬的激活是依赖于仿生化多功能纳米免疫调节系统中的基础材料ZIF-8。ZIF-8一直被视为良好的药物递送纳米载体,但是其潜在的生物活性开发甚少。ZIF-8含有金属离子,金属离子可以作为提高抗癌免疫力的元素,发挥清除肿瘤的功能。此外,ZIF-8的一个重要特性是其可以诱导自噬。自噬被视为是ICD的“使能器”,可以促进ATP的释放,促进ICD效应,从而产生特异性免疫反应。
mRDZ的免疫系统激活功能:
本发明所述mRDZ在体内局部肿瘤微环境及TDLN与脾脏中,均可有效激活杀伤性免疫反应(M1,CD4+T,CD8+T,CD80+CD86+DC细胞增多),并削弱抑制性免疫反应(M2,MDSC,Tregs细胞减少),这种局部及全身性的抗肿瘤免疫反应,使得mRDZ具有有效的抑制肿瘤生长功能。此外,肿瘤及脾脏中记忆性CD4+T及记忆性CD8+T细胞的增多,也表明mRDZ可激活局部及全身性免疫记忆,有效抑制二次接种的肿瘤复发。
该双重反应在上述的相互协同作用,赋予了mRDZ多功能免疫调节作用。mRDZ重塑了免疫抑制微环境,激发了强大的免疫记忆反应,能够有效抑制原发肿瘤的生长,抑制肿瘤转移,并且防止肿瘤的复发。
实施例2:药物释放曲线测试和体外血液相容性
体外药物释放实验。将mRDZ载药纳米颗粒重溶于不同pH值(7.5、6.5和5)的磷酸盐缓冲液中,放入透析袋(分子量1000Da)中,并将其浸入具有相应pH值的磷酸盐缓冲液中,然后在37℃的摇床上以500rpm速度摇动36小时。在特定时间间隔(0、0.5、1、2、4、6、8、12、24和36小时)时,取出200ul样品进行DOX荧光分析,使用微孔板检测器进行检测,然后根据DOX荧光标准曲线测量释放的DOX含量。
药物释放曲线结果显示(图3中A),当pH从7.5降至5时,DOX的累积释放量从23.31%±0.79%增加到84.72%±0.84%,并且在12h时,药物释放达到最大量,体现了mRDZ的pH响应性释放。
体外血液相容性实验。取Balb/c小鼠新鲜血液,然后将其室温1000rpm 10min,收集红细胞,并用生理盐水洗涤三次。接着用生理盐水将红细胞稀释成2%红细胞稀释液,并与不同纳米颗粒在37℃下共孵育2小时,用水和生理盐水做阳性对照和阴性对照,将混合物室温12000rpm 5min,取上清200ul测得在545nm处的吸光度,分析红细胞释放的血红蛋白含量。
体外血液相容性检测结果显示(图3中B),mRDZ和RDZ的体外溶血率均低于4%,说明了纳米颗粒的给药安全性。此外,mRDZ的体外溶血水平低于未包膜的RDZ纳米颗粒,说明了细胞膜的包被提高了体外血液相容性和给药安全性。
实施例3:体外抗肿瘤作用
体外细胞摄取实验。将CT26结肠癌细胞以细胞密度为2×104个细胞/皿培养在玻底培养皿中。将指定浓度的mRDZ与细胞共培养不同时间(0.5,4,12,24小时)。在相应的时间,将细胞培养基丢弃,用PBS清洗3次,用4% PFA固定细胞15分钟,然后用DAPI染色10分钟,最后用PBS清洗3次。用共焦显微镜观察纳米颗粒的细胞摄取情况(DOX激发波长为488nm,Cy5激发波长为633nm)。
体外细胞摄取实验结果显示(图4),所制备的mRDZ纳米颗粒可有效地被细胞摄取,并且在4小时达到最大摄取。
体外溶酶体释放实验。将CT26细胞按细胞密度为2×104细胞/皿培养在玻底培养皿中,用75nM的溶酶体红色荧光探针染色1.5小时,染色结束后用PBS清洗三遍。再将mRDZ与细胞共培养不同时间(0.5,4,12和24小时)后,用4% PFA固定15分钟和DAPI染色10分钟,然后用PBS清洗3次,最后通过共聚焦显微镜观察siRNA与溶酶体的共定位情况。
体外溶酶体释放实验观察结果显示(图5),当mRDZ与CT26细胞共孵育0.5小时后,siRNA分子(绿色)与溶酶体(红色)共定位在一起,但在4h及以后,观察到siRNA与溶酶体逐渐分离,说明了mRDZ具有逃逸溶酶体的能力。
体外细胞毒性实验。将CT26细胞以细胞密度为5000细胞/孔接种到96孔板上,在与不同纳米颗粒共培养24小时后,再加入四甲基偶氮唑盐(MTT),在37℃下孵育4小时,最后加入二甲基亚砜(DMSO),震荡15分钟后,测定吸光度(490nm和570nm)。
体外细胞毒性实验结果显示(图6),mRDZ对CT26肿瘤细胞有明显的杀伤能力,并且呈浓度依赖性。
实施例4:体外ICD,自噬及IDO-1的协同抗肿瘤作用
体外IDO1表达。通过RT-qPCR检测IDO1mRNA在CT26细胞中的表达。简而言之,将CT26细胞以细胞密度为3×105细胞/孔培养在六孔板中,将不同纳米颗粒与细胞共培养24小时后,通过RNA提取试剂盒提取细胞总RNA,再逆转录成cDNA。用于基因表达定量的样品通过SYBR Green实时PCR试剂盒定量检测样品的基因表达,并通过实时PCR系统下进行分析。将基因表达水平归一化为GAPDH值,采用相对CT法测定相对表达量。通过免疫印迹法检测IDO1蛋白在CT26细胞中的表达,将不同纳米颗粒与细胞共培养24小时,收集细胞,用RIPA裂解液提取细胞总蛋白。接着用BCA试剂盒来计算蛋白质浓度后,用SDS-PAGE对蛋白质样品进行分离。将分离的蛋白依次转移到PVDF膜上,用封闭液封闭1小时,然后与一抗在4℃下共同孵育一夜。在用TBST洗涤3次后,与用HRP标记的二抗在室温下共孵育2小时,最后获取图像。
体外IDO1表达检测结果显示(图7),DOX会诱导IDO1高表达,但是在mRDZ作用下,会显著降低IDO1表达水平。
体外诱导细胞自噬。通过透射电镜来研究纳米颗粒诱导自噬的能力,将CT26细胞以细胞密度为3×105细胞/孔培养在六孔板中,在不同纳米颗粒与细胞共培养24小时后,收集细胞,加入电镜固定液,室温避光固定30分钟后,通过包埋和制片,最后用透射电镜(TEM)来观察细胞中产生的自噬体的数量。
体外自噬作用电镜实验结果显示(图8),在mRDZ处理下细胞中自噬体的数量明显增多,说明了mRDZ可显著诱导自噬,此外还发现在mRDZ处理下整个细胞呈空泡化,说明mRDZ诱导了肿瘤细胞发生明显的损伤。
体外ICD效应。将CT26细胞以细胞密度为3×105细胞/孔接种于6孔板上,与不同的纳米颗粒共培养24小时后。用ATP和HMGB1检测试剂盒检测不同纳米颗粒处理后CT26细胞上清液中ATP和HMGB1的含量。将CT26细胞接种在6孔板上,与不同的纳米颗粒共培养24小时。细胞用0.1%BSA封闭1小时后,在4℃下与CRT一抗(0.1μg/mL)孵育过夜,用PBS洗涤3次,然后与Alexa FluorTM633偶联的二抗(1μg/mL)在室温下孵育1小时,用PBS清洗三次,用4%PFA对细胞进行固定,最后通过流式细胞仪来检测细胞表面CRT的表达量。按以上方法,处理后的肿瘤细胞与骨髓来源树突状细胞(BMDC)共孵育,24h后,用流式检测BMDC的成熟因子(CD80,CD86)表达量。
体外ICD效应研究实验结果显示(图9),mRDZ可显著诱导ATP(图9中A)和HMGB1(图9中B)的释放,以及细胞表面CRT的表达增加(图9中C),相对其它给药处理,mRDZ处理后的肿瘤细胞,可有效诱导BMDC的成熟(图10),说明了mRDZ可显著诱导ICD效应。
以上结果表明,本发明所述mRDZ兼具诱导ICD、抑制IDO-1、促进自噬的功能,三者协同作用,可激活DC免疫细胞成熟。
实施例5:体内抗肿瘤作用
体内肿瘤抑制作用。在6周大的Balb/c小鼠背部皮下注射CT26细胞悬液(5×105个细胞)。当肿瘤体积增大近50mm3,将小鼠随机分为5组(n=10/group)。小鼠每隔2天尾静脉分别注射生理盐水,DOX,DZ,RDZ和mRDZ,其中DOX剂量为2.5mg/kg,一共注射5次。在预定的时间点监测肿瘤体积,计算肿瘤体积的公式为:V=长度×宽度2/2。
体内肿瘤抑制实验监测结果显示(图11),mRDZ对肿瘤生长的抑制程度最大。
实施例6:体内抗肿瘤免疫反应激活效应
体内局部肿瘤微环境免疫反应激活。监测结束后,取出肿瘤器官,将肿瘤组织剪碎,在组织解离仪中用胶原酶IV(1.0mg/mL)和脱氧核糖核酸酶I(0.1mg/mL)在37℃下解离0.5小时,再用70um的滤网过滤,从而获得单细胞。单细胞与CD16/32抗体共孵育,用于阻断非特异性Fc片段。接着单细胞与带有荧光偶联的抗体共孵育,用流式细胞仪进行检测。
体内免疫反应分析实验检测结果显示(图12),在mRDZ处理下肿瘤组织内CD4+T细胞,MDSCs,Tregs和M2巨噬细胞的数量显著减少,而CD8+T细胞,M1巨噬细胞,记忆性CD4+T细胞和记忆性CD8+T细胞的数量显著增多,说明了mRDZ不仅可激活特异性免疫反应,而且激活了特异性免疫记忆反应。
体内全身性免疫反应激活。监测结束后,取出肿瘤引流淋巴结及脾脏器官,将组织剪碎,在组织解离仪中用胶原酶IV(1.0mg/mL)和脱氧核糖核酸酶I(0.1mg/mL)在37℃下解离0.5小时,再用70um的滤网过滤,从而获得单细胞。单细胞与CD16/32抗体共孵育,用于阻断非特异性Fc片段。接着单细胞与带有荧光偶联的抗体共孵育,用流式细胞仪进行检测。
体内免疫反应分析实验检测结果显示(图13),在mRDZ处理下引流淋巴结(TDLN)内M1型巨噬细胞数量增多(图13中A),M2型巨噬细胞数量减少(图13中B),相对其它给药处理,mRDZ诱导TDLN内M1/M2的细胞数量比升高程度最大(图13中C)。此外,在TDLN中,经mRDZ处理后,成熟DC数量明显增加(图13中D)。表明肿瘤抗原可诱导到达TDLN并激活其中髓系的免疫细胞。在脾脏中,CD4+T(图13中E)及CD8+T(图13中F)细胞数量减少,免疫抑制性细胞如髓系抑制性细胞(MDSCs)(图13中G)及调节性T细胞(Tregs)(图13中H)数量减少,记忆性CD4+T细胞数量增多(图13中I),而记忆性CD8+T细胞数量减少(图13中J)。表明在脾脏中T细胞可进一步被激活,激活的T细胞迁移至肿瘤组织,导致脾脏中的CD8+T及其记忆性T细胞数量的减少,此外,免疫抑制性细胞数量的有效降低表明,mRDZ不仅增强了抗肿瘤免疫细胞数量,同时亦削弱了抑制性免疫细胞,该双重效应,使得mRDZ产生强大的抗肿瘤免疫响应,且该响应具有免疫记忆性。
以上结果表明,mRDZ具有激活局部及全身性抗肿瘤免疫反应的能力,且该免疫反应具有强大的记忆性。
实施例7:体内抑制肿瘤复发
肿瘤再攻击。先在6周大的Balb/c小鼠左边背部皮下注射CT26细胞悬液(5×105个细胞)。当肿瘤体积增大近50mm3时,将小鼠随机分为5组(n=6/group)。小鼠每隔两天尾静脉分别注射生理盐水,DOX,DZ,RDZ和mRDZ,其中DOX剂量为2.5mg/kg,一共治疗3次。在第3次治疗后的第4天在小鼠原始肿瘤对侧(右边)背部皮下注射CT26细胞悬液(5×105个细胞)。在预定的时间点监测小鼠两边肿瘤体积和小鼠体重,计算肿瘤体积公式为:V=长度×宽度2/2。
肿瘤再攻击研究实验监测结果显示(图14),在mRDZ治疗后,小鼠对侧肿瘤的发生率显著降低,六只小鼠中仅有一只小鼠的对侧出现肿瘤,肿瘤生长极其缓慢,并且肿瘤大小在再攻击后的第八天开始变小。
以上所述仅为本发明的优选实施例,对本发明而言仅是说明性的,而非限制性的;本领域普通技术人员理解,在本发明权利要求所限定的精神和范围内可对其进行许多改变,修改,甚至等效变更,但都将落入本发明的保护范围。
Claims (10)
1.一种多功能纳米免疫调节制剂,其特征在于,所述的多功能纳米免疫调节制剂由金属有机框架材料包裹化疗药物并负载免疫抑制阻断剂之后,再经巨噬细胞膜包膜而得。
2.如权利要求1所述的多功能纳米免疫调节制剂,其特征在于,金属有机框架材料为ZIF-8。
3.如权利要求1所述的多功能纳米免疫调节制剂,其特征在于,化疗药物为DOX。
4.如权利要求1所述的多功能纳米免疫调节制剂,其特征在于,免疫抑制阻断剂为siRNA。
5.如权利要求1所述的多功能纳米免疫调节制剂,其特征在于,巨噬细胞膜为RAW264.7细胞中提取的细胞膜。
6.一种如权利要求1所述的多功能纳米免疫调节制剂的制备方法,其特征在于,所述的制备方法包括如下步骤:
(1)将六水合硝酸锌溶液和DOX溶液混合,搅拌下加入2-甲基咪唑溶液,继续搅拌0.5~2h,离心,收集沉淀,得到DZ载药纳米颗粒;
(2)将步骤(1)所得DZ载药纳米颗粒与siRNA混合,室温静置,通过静电吸附作用siRNA被吸附到DZ载药纳米颗粒表面,形成RDZ纳米颗粒;
(3)从RAW264.7细胞中提取细胞膜,将步骤(2)所得RDZ纳米颗粒与提取所得细胞膜混合进行冰浴超声包膜,之后离心,收集得到mRDZ纳米颗粒,即所述的多功能纳米免疫调节制剂。
7.如权利要求6所述的制备方法,其特征在于,步骤(1)中,六水合硝酸锌溶液的浓度为2mg/mL,溶剂为纯水;DOX溶液的浓度为1mg/mL,溶剂为纯水;2-甲基咪唑溶液的浓度为40mg/mL,溶剂为纯水;六水合硝酸锌溶液、DOX溶液、2-甲基咪唑溶液的体积比为4:1:4。
8.如权利要求6所述的制备方法,其特征在于,步骤(2)中,DZ载药纳米颗粒包封的DOX与siRNA的质量比为25:1。
9.如权利要求6所述的制备方法,其特征在于,步骤(3)中,从RAW264.7细胞中提取细胞膜后,用BCA试剂盒定量,RDZ纳米颗粒与巨噬细胞膜膜蛋白量的质量比为10:1。
10.如权利要求1所述的多功能纳米免疫调节制剂在制备免疫系统重塑的抗肿瘤药物中的应用。
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