CN111544594A - 基于免疫疗法和化学疗法相结合的多功能纳米材料及其制备方法和应用 - Google Patents
基于免疫疗法和化学疗法相结合的多功能纳米材料及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了基于免疫疗法和化学疗法相结合的多功能纳米材料及其制备方法和应用,所述多功能纳米材料是通过免疫激活子、化学药物与纳米载体材料组装形成。本发明的多功能纳米材料制备工艺简单,绿色经济、环境友好、成本低廉、工艺简单、可大批量生产高等优点。通过该技术路线制备得到的多功能纳米材料具有很好的抗肿瘤效果。
Description
技术领域
本发明属于多功能纳米材料技术领域,具体涉及基于免疫疗法和化学疗法相结合的多功能纳米材料及其制备方法和应用。
背景技术
免疫疗法是对抗疾病的重要方式,通过增强免疫反应或启动免疫***来对抗或预防疾病。免疫疗法作为一种高效率治疗和预防方式,因其对抗性强、疗效好,长期以来受医疗界的好评。尤其在高致死率肿瘤病例中,利用免疫疗法大大提高病患生存率,日趋成为患者的优选。目前已经FDA批准的免疫疗法包括免疫检查点阻断疗法在一些肿瘤病例中取得了较好的治疗效果,然而该系列免疫疗法只对表达特定抗原和活化淋巴细胞浸润的肿瘤起作用,因而整体而言该种免疫疗法存在一定的局限性。
以肿瘤为例,在肿瘤区域表达特定的抗原现阶段是一种不可控状态,因而启动或增强肿瘤区域的淋巴细胞成为另一重要研究点。目前基于已知的增强免疫反应的信号通路,如CpG寡核苷酸可激活Toll样受体蛋白9(TLR9)信号通路,进而启动或增强免疫反应用于肿瘤辅助治疗。由于分子量小,易降解等方面影响,目前可用于治疗的CpG寡核苷酸通常采用瘤内注射,对不能采取瘤内注射的肿瘤而言具有很大局限性。
对于高致死肿瘤治疗而言,联合用药明显有助于提高病患生存率。多次给药以及药物之间的相互作用,将会造成药物效果降低。同时就化学药物治疗而言,单纯静脉给药会给病患带来严重的副反应,如疼痛、呕吐以及造成其他器官损伤。因而选择合适的载体给药递送***将会具有很好的应对性。
近年来,纳米技术介导的药物传递***发展迅速,借助纳米材料载体作用,可实现靶向性和高效率药物传递。同时作为载体功能的纳米材料还具有多功能性,如铁的氧化物作为载体还可以用于T2核磁成像,锰的氧化物作为载体除核磁成像外还可用于氧气产生以有助于改善肿瘤的乏氧环境,因而借助纳米材料可实现高效药物递送,同时会体现多功能性。
发明内容
发明目的:为了应对以上挑战,我们设计了基于免疫疗法和化学疗法相结合的多功能纳米材料及其制备方法和应用,提出了选用纳米材料为载体实现免疫疗法和化学相结合的治疗新策略。实验效果表明,借助纳米材料递送免疫激活子和化学药物明显提高抗肿瘤效率,同时选用纳米载体***实现静脉注射,增强药物疗效,降低药物副反应。
技术方案:为了解决上述技术问题,本发明提供了基于免疫疗法和化学疗法相结合的多功能纳米材料,所述多功能纳米材料是通过免疫激活子、化学药物与纳米载体材料自组装形成。
本发明所述免疫疗法包括抗原、抗体介导的免疫激活,免疫增强等方式。
本发明所述的自组装是指免疫激活子和化学药物通过特定作用与纳米载体材料相结合,可以通过多种方式组装但不仅限于自主装(物理作用,结构特征等引发自主装),化学组装(通化学键方式组装、共价或非共价组装、静电作用等)、范德华力等。
其中,所述免疫激活子是指可以促发免疫反应的核酸分子,也可以是其他类型促发/增强免疫反应的分子,本发明中免疫激活子以双链DNA分子为例。
其中,所述化学药物包括但不仅限于指用于疾病治疗的化学类药物如抗肿瘤药物(紫杉醇类,喜树碱性类,抗叶酸类,铂类,长春碱类,抗嘧啶类,蒽环类,亚硝脲类等),也可以为抗其他疾病类化学药物,本发明中以抗肿瘤类药物阿霉素DOX为例。
其中,所述纳米载体材料是指可以用于药物递送功能的纳米材料,包括但不仅限于各种纳米颗粒,如硅的氧化物(中空二氧化硅),铁的氧化物,锰的氧化物等纳米颗粒,本发明中以四氧化三锰纳米颗粒为例。
作为优选地,所述免疫激活子包括但不仅限于Au-dsDNA。
作为优选地,所述纳米载体材料包括但不仅限于四氧化三锰纳米颗粒。
其中,所述免疫激活子Au-dsDNA制备方法包括以下步骤:
1)氯金酸溶液经油浴煮至沸腾;
2)向沸腾的氯金酸溶液加入抗坏血酸溶液还原,冷却备用;
3)先将单链巯基修饰的DNA分子与Au纳米颗粒混合,经搅拌逐步加盐获得Au-ssDNA;
4)Au-ssDNA与互补DNA分子通过杂交获得免疫激活子Au-dsDNA。
其中,所述四氧化三锰纳米颗粒的制备方法如下:
1)油酸还原高锰酸钾后获得前躯体,后经200℃高温空气炉烧制获得四氧化三锰前驱体;
2)四氧化三锰前驱体在200℃温度下进行烧制反应5小时;
3)自然冷却至室温,将产物取出即得多功能的纳米载体Mn3O4纳米颗粒。
本发明内容还包括所述的基于免疫疗法和化学疗法相结合的多功能纳米材料的制备方法,所述多功能纳米材料制备方法如下:
1)将Au-dsDNA纳米颗粒与Mn3O4纳米颗粒经自组装后,获得Mn3O4@Au-dsDNA;
2)将Mn3O4@Au-dsDNA与化学药物组装获得Mn3O4@Au-dsDNA/化学药物。
其中,所述化学药物为阿霉素DOX。
本发明内容还包括所述的基于免疫疗法和化学疗法相结合的多功能纳米材料在制备抗肿瘤药物或其他疾病类化学药物方面的应用。
有益效果:本发明通过简单合成方法获得免疫激活子和多功能纳米载体材料,并将免疫激活子和化学药物共组装到多功能纳米材料载体上,动物实验水平实验取得很好的抗肿瘤疗效。通过改变免疫激活子类型,化学药物以及纳米载体材料,可以推广至其他疾病的协同治疗。本发明的多功能纳米材料制备工艺简单,绿色经济、环境友好、成本低廉、工艺简单、可大批量生产高等优点。通过该技术路线制备得到的多功能纳米材料具有很好的疾病协同治疗效果。
附图说明
图1基于免疫疗法和化学疗法相结合的多功能纳米材料用于肿瘤协同治疗的流程图;
图2免疫激活子,自组装多功能纳米材料学表征;a)免疫激活子Au-dsDNA的扫描电子显微镜图片;b)多功能纳米载体四氧化三锰的透射电子显微镜图片;c)Au-dsDNA与多功能纳米载体四氧化三锰自组装获得Mn3O4@Au-dsDNA的透射电子显微镜图片;d)Mn3O4@Au-dsDNA与化学药物阿霉素DOX自组装后获得Mn3O4@Au-dsDNA/DOX的透射电子显微镜图片。
图3免疫疗法和化学疗法相结合的多功能纳米材料在细胞水平进行免疫激活验证(以免疫激活子Au-dsDNA激活STING信号通路为例);a)细胞实验验证STING信号通路激活的流程图;b)肿瘤细胞分泌的细胞因子干扰素β检测(干扰素β最为STING介导免疫激活的特异性指标);c)巨噬细胞分泌的细胞因子干扰素β检测;
图4免疫疗法和化学疗法相结合的多功能纳米材料在小动物水平的抗肿瘤验证(以免疫激活子Au-dsDNA和阿霉素DOX共组装到纳米材料Mn3O4为例);a)以黑色素瘤的小鼠模型,借助纳米材料联合免疫疗法和化学疗法后肿瘤体积随时间变化曲线;b)剥离肿瘤块的图片;c)借助纳米材料联合免疫疗法和化学疗法对抗肿瘤后对小鼠生存率的影响;
图5多功能纳米材料Mn3O4作为载体多功能性;a)纳米载体Mn3O4小鼠体内核磁共振成像;b)纳米载体Mn3O4分解双氧水产生氧气能力。
具体实施方式
下面对本发明技术方案进行详细说明,但是本发明的保护范围不局限于所述实施例。
实施例1基于免疫激活子Au-dsDNA的制备
1、100mL 0.4mg/mL氯金酸溶液经油浴煮至沸腾;
2、向沸腾的氯金酸溶液加入2mL 50mg/mL抗坏血酸溶液还原,冷却备用;
3、先将单链巯基修饰的1mL 50μM的DNA分子(59-nt poly(dT)在3’端有巯基修饰)与50mL 0.4mg/mL Au纳米颗粒(13nm)混合,经搅拌加NaCl溶液(5mL 3M缓慢加入,并搅拌过夜)获得Au-ssDNA;
4、Au-ssDNA与互补DNA分子(59-nt poly(dA))通过杂交获得免疫激活子Au-dsDNA。
实施例2基于多功能纳米载体材料的制备
1、向250mL 2mg/mL高锰酸钾溶液中加入5mL油酸,经搅拌充分还原后获得四氧化三锰前躯体;
2、四氧化三锰前躯体在200℃温度下进行烧制反应5小时;
3、自然冷却至室温,将产物取出即得多功能的纳米载体四氧化三锰纳米颗粒,命名为Mf。
实施例3基于免疫疗法和化学疗法相结合多功能纳米材料的制备
1、将实施例1制备的Au-dsDNA纳米颗粒作为免疫激活子与实施例2制备的Mn3O4颗粒经混匀室温搅拌半小时后,自组装获得Mn3O4@Au-dsDNA,命名为MfAN;
2、将Mn3O4@Au-dsDNA与化学药物阿霉素DOX混匀轻微超声搅拌半小时自组装获得Mn3O4@Au-dsDNA/DOX,命名为MfAND。
实施例4基于免疫疗法和化学疗法相结合多功能纳米材料抗肿瘤疗效
1、将实施例2制备的Mn3O4纳米载体材料,作为一组对照组,命名为Mf;将实施例1制备的Au-dsDNA免疫激活子,作为一组对照组,命名为AN;将阿霉素DOX与纳米材料Mn3O4自组装形成Mn3O4@DOX,作为一组对照组,命名为MfD;将实施例3制备的MfAN作为一组对照组;将实施例3制备的MfAND作为一组实验组;将给予PBS作为一组对照组,命名为Ctrl;
2、将黑色素瘤选做模式肿瘤模型,C57BL/6小鼠经皮下给予鼠源的B16F10肿瘤细胞(来源于美国ATCC细胞库)后形成黑色素瘤;
3、将已成瘤的小鼠随机分成6组;
4、对6组小鼠分别经尾静脉给予200μL药物,给药如下:Mf(5mg/kg)、MfD(Mf::5mg/kg,DOX:6mg/kg)、AN(AN:10mg/kg)、MfAN(Mf:5mg/kg,AN:10mg/kg)、MfAND(Mf:5mg/kg,AN:10mg/kg,DOX:6mg/kg)和Ctrl(0.01M PBS);
5、将测定各组小鼠肿瘤体积变化,考察抗肿瘤效果。
由图4a和图4b可以看出抗肿瘤效果,通过借助Mn3O4纳米载体联合运用免疫激活子AN和化学药物DOX显示出最好的治疗效果。经治疗后MfAND肿瘤体积仅为Ctrl组的1/6约为350mm2,且60天的生存率高达50%。
实施例5多功能纳米材料Mn3O4作为载体多功能性
a)纳米载体Mf和MfAN小鼠体内核磁共振成像
1、将实施例2制备的Mf作为一组实验组;实施例3制备的MfAN作为另外一组对照组;同时将PBS作为一组对照组,命名为Ctrl;
2、将黑色素瘤选做模式肿瘤模型,C57BL/6小鼠经皮下给予鼠源的B16F10肿瘤细胞后形成黑色素瘤;
3、将已成瘤的小鼠随机分成3组;
4、对3组小鼠分别经尾静脉给予200μL药物,给药如下:Mf(5mg/kg)、MfAN(Mf:5mg/kg,AN:10mg/kg)和Ctrl(0.01M PBS);
5、对上述4中的各组小鼠给药后12h,进行核磁共振成像。
由图5a可以观察到Mf和MfAN具有核磁共振成像功能。
b)纳米载体Mn3O4分解双氧水产生氧气能力
1、将实施例2制备的Mn3O4作为实验组,设立不同实验浓度如下:40mg/mL,20mg/mL,10mg/mL,5mg/mL,2.5mg/mL,1.25mg/mL和0mg/mL;
2、将不同浓度的双氧水加入1所述含有各种浓度Mn3O4的离心管中,双氧水浓度如下:0mM,5mM,10mM和20mM。
3、反应5分钟后进行拍照观察。
如图5b所示意,Mn3O4能够分解双氧水产生氧气泡,且随着Mn3O4浓度增加产生的氧气泡增多;在相同Mn3O4浓度下,双氧水浓度增加,氧气泡也增加。由此可见Mn3O4具有分解双氧水产生氧气的能力,这将为肿瘤乏氧的微环境提供氧气。
Claims (10)
1.基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述多功能纳米材料是通过免疫激活子、化学药物与纳米载体材料组装形成。
2.根据权利要求1所述的基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述免疫激活子为dsDNA、ssDNA、RNA、polyI:C、Au-dsDNA或其他能激活和增强机体免疫反应的免疫原物中的一种或几种。
3.根据权利要求1所述的基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述化学药物包括抗肿瘤药物紫杉醇类、喜树碱性类、抗叶酸类、铂类、长春碱类、抗嘧啶类、蒽环类、亚硝脲类或其他疾病类化学药物中的一种或几种。
4.根据权利要求1所述的基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述纳米载体材料为硅的氧化物、铁的氧化物或锰的氧化物的纳米颗粒中的一种或几种,所述锰的氧化物的纳米颗粒为四氧化三锰纳米颗粒。
5.根据权利要求1所述的基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述组装方式为物理作用自组装、结构特征引发的自主装、通化学键方式的化学组装、共价或非共价的化学组装、静电作用组装或范德华力组装。
6.根据权利要求2所述的基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述免疫激活子Au-dsDNA制备方法包括以下步骤:
1)氯金酸溶液经油浴煮至沸腾;
2)向沸腾的氯金酸溶液加入抗坏血酸溶液还原,冷却备用;
3)先将单链巯基修饰的DNA分子与Au纳米颗粒混合,经搅拌逐步加盐获得Au-ssDNA;
4)Au-ssDNA与互补DNA分子通过杂交获得免疫激活子Au-dsDNA。
7.根据权利要求4所述的基于免疫疗法和化学疗法相结合的多功能纳米材料,其特征在于,所述四氧化三锰纳米颗粒的制备方法如下:
1)油酸还原高锰酸钾后获得前躯体,后经200℃高温空气炉烧制获得四氧化三锰前驱体;
2)四氧化三锰前驱体在200℃温度下进行烧制反应5小时;
3)自然冷却至室温,将产物取出即得多功能的纳米载体Mn3O4纳米颗粒。
8.权利要求1~7任一项所述的基于免疫疗法和化学疗法相结合的多功能纳米材料的制备方法,其特征在于,所述多功能纳米材料具体制备方法如下:
1) 将Au-dsDNA纳米颗粒与Mn3O4纳米颗粒经组装后,获得Mn3O4@Au-dsDNA;
2)将Mn3O4@Au-dsDNA与化学药物组装获得Mn3O4@Au-dsDNA/化学药物。
9.权利要求8任一项所述的基于免疫疗法和化学疗法相结合的多功能纳米材料的制备方法,其特征在于,所述化学药物为阿霉素DOX。
10.根据权利要求1~7任一项所述的基于免疫疗法和化学疗法相结合的多功能纳米材料在制备抗肿瘤药物或其他疾病类化学药物方面的应用。
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Application publication date: 20200818 |
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