CN113679829B - 一种预防癌症术后复发的肿瘤疫苗及其制备方法和应用 - Google Patents
一种预防癌症术后复发的肿瘤疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种预防癌症术后复发的肿瘤疫苗及其制备方法和应用,所述肿瘤疫苗包括杂合细胞膜外壳和内核材料,所述杂合细胞膜外壳包括革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜。本发明所涉及的肿瘤疫苗以手术切除来源的实体肿瘤组织中的细胞膜作为肿瘤抗原,并创造性地以杂合膜中的革兰氏阴性菌内膜作为免疫佐剂,二者可共同递送到同一树突状细胞中,有利用肿瘤抗原的摄取和呈递,能够增强机体的先天性免疫和特异性免疫,且具有一定的***富集能力,具有显著的预防肿瘤切除手术后复发的功效,延长患者的生存期,并提供长效保护机制。且该肿瘤疫苗生物安全性良好,制备原料易得,制备成本低。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种肿瘤疫苗及其制备方法和应用,尤其涉及一种预防癌症术后复发的肿瘤疫苗及其制备方法和应用。
背景技术
乳腺癌是女性第一高发恶性肿瘤,严重威胁患者生命健康,且发病年龄日趋年轻化。早期乳腺癌往往不具备典型的症状和体征,不易引起重视,常通过体检或乳腺癌筛查发现。随着对乳腺癌生物学行为认识的不断深入,以及治疗理念的转变与更新,乳腺癌的治疗进入了综合治疗时代,形成了乳腺癌局部治疗与全身治疗并重的治疗模式。医生会根据肿瘤的分期和患者的身体状况,酌情采用手术、放疗、化疗、内分泌治疗、生物靶向治疗及中医药辅助治疗等多种手段。外科手术在乳腺癌的诊断、分期和综合治疗中发挥着重要作用。乳腺癌的外科手术包括乳腺和腋窝***两部分。
结肠癌是最为常见的癌症之一,近年来发病率呈不断上升的趋势,早期癌内镜下可以根治的病变可以采取内镜微创治疗,中晚期癌治疗方法是以手术为主、辅以化疗、免疫治疗、中药以及其他支持治疗的综合方案,以提高手术切除率,降低复发率,提高生存率。
目前对于癌症的治疗,外科手术仍然是首选的治疗方案,其在癌症的诊断、分期、重建和缓解方面具有很重要的作用。传统放化疗和新型辅助放化疗领域的进步在很大程度上也提高了癌症患者的预后。然而,尽管治疗技术在不断发展,局部肿瘤的复发和转移仍然成为了癌症患者发病率和死亡率的主要来源。很多研究发现,手术操作以及对肿瘤切缘的破坏都可能会使个别肿瘤细胞脱落到外周循环中。同时,手术应激反应还会影响神经-内分泌调节、新陈代谢、炎性反应以及肿瘤细胞周围的免疫微环境,导致免疫抑制。在大手术过后,细胞免疫抑制可以持续数天,而这段时间就可能成为肿瘤入侵免疫***的空窗期。这时期的免疫抑制常表现为对肿瘤细胞监督的缺失、外周血中NK细胞、细胞毒性T淋巴细胞、树突状细胞以及辅助T淋巴细胞的减少。
在人体免疫***中,机体自身的细胞免疫被认为是抗肿瘤免疫的主力,它们构成了抵御肿瘤入侵最主要的防线。细胞免疫主要包括T细胞亚群、Th1型细胞因子(IL-2、IFN-γ等)、Th2型细胞因子(IL-4、IL-6、IL-10等)、自然杀伤细胞(NK细胞)、自然杀伤T细胞(NKT细胞)、细胞因子诱导杀伤细胞(CIK细胞)等。其中,NK细胞、NKT细胞、CD4+Th1型细胞和CD8+细胞毒性淋巴细胞(CTL)是抗肿瘤主要的效应细胞,而Th2型细胞则会通过抑制机体抗肿瘤免疫,成为促进肿瘤形成、生长和发展的主要细胞。
目前,用于预防术后肿瘤复发的疫苗类型还比较有限,CN104619833A公开了一种针对癌症的肽疫苗,具体为一种引发CTL的衍生自UBE2T的表位肽,进一步提供了含有衍生自UBE2T的此类表位肽或编码该多肽的多核苷酸作为活性成分的药物组合物。此外,该发明提供了用于治疗和/或防范,和/或预防其术后复发的方法,及用于诱导CTL的方法,用于诱导抗肿瘤免疫的方法,其使用该发明的衍生自UBE2T的表位肽,编码肽的多核苷酸,或呈递肽的抗原呈递细胞,或药物组合物进行。CN109481418A公开了一种抗肿瘤纳米颗粒及其制备方法和应用,提供的抗肿瘤纳米颗粒,包括核心、包覆核心的包裹层及包覆包裹层的免疫层。由免疫佐剂包裹纳米颗粒得到的抗肿瘤纳米颗粒能够达到同时具有光动力治疗和免疫治疗的联合治疗效果,对靶组织及损伤程度具有可选择性,可减少对正常组织的损伤,同时通过增强患者自身免疫力来***,同时具有反应快、无副作用、疗效持久且对预防术后复发具有重要作用等特点。
因此,开发一种新型的效果显著的预防癌症术后复发的肿瘤疫苗及其制备方法是非常有意义的。
发明内容
针对现有技术的不足,本发明的目的在于提供一种肿瘤疫苗及其制备方法和应用,尤其提供一种预防癌症术后复发的肿瘤疫苗及其制备方法和应用。
为达到此发明目的,本发明采用以下技术方案:
一方面,本发明提供一种预防癌症术后复发的肿瘤疫苗,所述肿瘤疫苗包括杂合细胞膜外壳和内核材料,所述杂合细胞膜外壳包括革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜。
本发明所涉及的肿瘤疫苗是一种杂合膜包裹内核材料的纳米颗粒,本发明以杂合膜中的手术切除来源的实体肿瘤组织中的细胞膜作为肿瘤抗原,并创造性地以杂合膜中的革兰氏阴性菌内膜作为免疫佐剂,二者可共同递送到同一树突状细胞中,有利用肿瘤抗原的摄取和呈递,能够协同增强机体的先天性免疫和特异性免疫,且具有一定的***富集能力,具有显著的预防肿瘤切除手术后复发的功效,延长患者的生存期,并提供长效保护机制。
本发明所涉及的肿瘤疫苗杂合膜中包裹的内核材料能够帮助维持杂合膜的刚性结构和稳定性,进而维持杂合膜的免疫增强、淋巴富集、预防肿瘤切除手术后复发的功效;同时可以进一步包载其他生物活性成分例如siRNA或化疗药等,实现更多的功能。
另外,本发明所涉及的肿瘤疫苗生物安全性良好,制备原料易得,制备成本低;且适用范围广,可根据实际需要在疫苗表面修饰各种化学基团,具有广阔的应用前景。
优选地,所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为1:3-3:1,例如1:3、1:2、2:3、1:1、2:1或3:1等,优选(2-3):1,上述数值范围内的具体点值均可选择,在此便不再一一赘述。
所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例特定选择为1:3-3:1的数值范围,其中(2-3):1是效果最佳的数值范围。
优选地,所述杂合细胞膜外壳与内核材料的质量比为1:(1-10),例如1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9或1:10等,优选1:(4-6),上述数值范围内的具体点值均可选择,在此便不再一一赘述。
所述杂合细胞膜外壳与内核材料的质量比特定选择为1:(1-10)的数值范围,因为在此范围内,最终产物的表面电荷较为接近杂合细胞膜外壳的表面电荷。
优选地,所述革兰氏阴性菌包括大肠杆菌、肺炎杆菌、布氏杆菌、变形杆菌或不动杆菌中的任意一种或至少两种的组合。
本发明所涉及的革兰氏阴性菌包括但不限于上述类型,其他类型的革兰氏阴性菌的内膜均可获得本发明所涉及的技术效果。革兰氏阴性菌的细胞壁为多层结构,从外到内依次是:外膜层(OM)为磷脂层和脂多糖层;中间为薄薄的肽聚糖层(PGN);内膜IM(细胞质膜)为脂蛋白层和磷脂层。革兰氏阴性菌内膜基本上不含内毒素(脂多糖LPS),有较好的生物安全性。
优选地,所述肿瘤包括乳腺癌、结肠癌、肝癌、胃癌、肾细胞癌、胰腺癌、卵巢癌或食道癌中的任意一种或至少两种的组合。
本发明所涉及的肿瘤包括但不限于上述类型,其他类型的肿瘤均可应用于本发明所涉及的技术方案。
优选地,所述内核材料包括PLGA、MOF、PCL、PAMAM、碳纳米管、石墨烯、金纳米颗粒、介孔二氧化硅纳米颗粒或氧化铁纳米颗粒中的任意一种或至少两种的组合。
本发明所涉及的聚合物包括但不限于上述类型,其他具有生物相容性的聚合物均可获得本发明所涉及的技术效果。杂合细胞膜外壳结构为磷脂双分子层,易于包裹在各种内核材料表面,且方法简单,可根据实际需要进行不同组合。
优选地,所述预防癌症术后复发的肿瘤疫苗的粒径为100-300nm,例如100nm、150nm、200nm、250nm或300nm等,上述数值范围内的具体点值均可选择,在此便不再一一赘述。
优选地,所述肿瘤疫苗的内核材料中还包载免疫检查点抑制剂、IDO抑制剂、siRNA或化疗药中的任意一种或至少两种的组合;所述至少两种的组合例如免疫检查点抑制剂和IDO抑制剂的组合、siRNA和化疗药的组合等,其他任意的组合方式均可选择,在此便不再一一赘述。
另一方面,本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗的制备方法,其特征在于,所述制备方法包括如下步骤:
(1)分别提取革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜;
(2)将步骤(1)提取到的革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜混合,并用脂质体挤出器挤出,得到杂合膜纳米颗粒;
(3)将步骤(2)得到的杂合膜微粒与纳米颗粒内核混合,并用脂质体挤出器挤出,得到所述预防癌症术后复发的肿瘤疫苗。
优选地,步骤(2)所述混合的温度为20-45℃,例如20℃、25℃、30℃、35℃、40℃或45℃等,时间为10-20min,例如10min、12min、15min、18min或20min等,上述数值范围内的具体点值均可选择,在此便不再一一赘述。
优选地,步骤(2)所述脂质体挤出器的滤膜孔径为300-500nm,例如300nm、350nm、400nm、450nm或500nm等,上述数值范围内的具体点值均可选择,在此便不再一一赘述。
优选地,步骤(2)所述挤出是指累计来回挤压10-15次(一来一回算1次)。
优选地,步骤(3)所述混合的温度为20-45℃,例如20℃、25℃、30℃、35℃、40℃或45℃等,时间为10-20min,例如10min、12min、15min、18min或20min等,上述数值范围内的具体点值均可选择,在此便不再一一赘述。
优选地,步骤(3)所述脂质体挤出器的滤膜孔径为100-300nm,例如100nm、150nm、200nm、250nm或300nm等,上述数值范围内的具体点值均可选择,在此便不再一一赘述。
优选地,步骤(3)所述挤出是指累计来回挤压10-15次(一来一回算1次)。
优选地,步骤(3)所述纳米颗粒内核通过双乳法制备得到。具体地,示例性地可以为:
(1)将内核材料溶于有机溶剂,再加入无菌蒸馏水;
(2)混合物超声乳化;
(3)随后与胆酸钠溶液混合,超声乳化;
(4)将乳液逐滴加入胆酸钠溶液,搅拌;
(5)旋蒸;离心;无菌蒸馏水洗涤后重悬得到纳米颗粒内核。
优选地,所述有机溶剂包括二氯甲烷、三氯甲烷、丙酮或乙醇中的任意一种或至少两种的组合。
优选地,所述有机溶剂与无菌蒸馏水的体积比为(3-7):1,例如3:1、4:1、5:1、6:1或7:1等,上述数值范围内的任意具体点值均可选择,在此便不再一一赘述。
优选地,步骤(2)所述超声的温度为0-4℃,例如0℃、1℃、2℃、3℃或4℃等,时间为2-5min,例如2min、3min、4min或5min等,功率为20-30W,例如20W、22W、25W、28W或30W等,上述数值范围内的任意具体点值均可选择,在此便不再一一赘述。
优选地,步骤(3)所述胆酸钠溶液的质量分数为1-3%,例如1%、2%或3%等。
优选地,步骤(3)所述胆酸钠溶液与有机溶剂的体积比为(1-3):1,例如1:1、2:1或3:1等。
优选地,步骤(3)所述超声的温度为0-4℃,例如0℃、1℃、2℃、3℃或4℃等,时间为4-6min,例如4min、5min或6min等,功率为25-35W,例如25W、28W、30W、32W或35W等,上述数值范围内的任意具体点值均可选择,在此便不再一一赘述。
优选地,步骤(4)所述胆酸钠溶液的质量分数为0.3-0.7%,例如0.3%、0.5%或0.7%等。
优选地,步骤(4)所述胆酸钠溶液与有机溶剂的体积比为(8-12):1,例如8:1、10:1或12:1等。
优选地,步骤(4)所述搅拌的温度为20-30℃,例如20℃、25℃或30℃等,时间大于10min。
再一方面,本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备增强机体先天性免疫和特异性免疫应答的药物中的应用。
再一方面,本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备促进树突状细胞分泌促炎细胞因子的药物中的应用。
再一方面,本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备促进树突状细胞成熟的药物中的应用。
再一方面,本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备促进脾脏淋巴细胞分泌IFN-γ的药物中的应用。
再一方面,本发明提供一种如上所述的预防癌症术后复发的肿瘤疫苗在制备刺激全身免疫***分泌炎症因子和趋化因子的药物中的应用。
相对于现有技术,本发明具有以下有益效果:
(1)本发明所涉及的肿瘤疫苗是一种杂合膜包裹聚合物的纳米颗粒,本发明以杂合膜中的手术切除来源的实体肿瘤组织中的细胞膜作为肿瘤抗原,并创造性地以杂合膜中的革兰氏阴性菌内膜作为免疫佐剂,二者可共同递送到同一树突状细胞中,有利用肿瘤抗原的摄取和呈递,能够增强机体的先天性免疫和特异性免疫,且具有一定的***富集能力,具有显著的预防肿瘤切除手术后复发的功效,延长患者的生存期,并提供长效保护机制。在动物试验中,其很好地抑制了因患乳腺癌行肿瘤切除术的荷瘤小鼠的术后肿瘤复发,抑制率高达83.3%;完全抑制了因患结肠癌行肿瘤切除术的荷瘤小鼠的术后肿瘤复发,抑制率高达100%。
(2)本发明所涉及的肿瘤疫苗杂合膜中包裹的内核材料能够帮助维持杂合膜的刚性结构和稳定性,进而维持杂合膜的免疫增强、淋巴富集、预防肿瘤切除手术后复发的功效;同时可以进一步包载其他生物活性成分例如siRNA或化疗药等,实现更多的功能。
(3)另外,本发明所涉及的肿瘤疫苗生物安全性良好,制备原料易得,制备成本低;且适用范围广,可根据实际需要在疫苗表面修饰各种化学基团,具有广阔的应用前景。
附图说明
图1是EM-NPs,TM-NPs以及HM-NPs杂合膜纳米颗粒的透射电镜图,标尺为100nm;
图2是HM-NPs杂合膜纳米颗粒的动态光散射结果图;
图3是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组上清中IL-6、TNF-α以及IL-1β的水平对比图(a为IL-6水平、b为TNF-α水平、c为IL-1β水平);
图4是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组上清中CD80和CD86水平对比图(a为CD80水平、b为CD86水平);
图5是ELISPOT试剂盒检测对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组上清中IFN-γ分泌量的结果图;
图6是利用CTL-ImmunoSpot Plate Reader软件对对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组斑点数目进行统计的结果图;
图7是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组小鼠血清中炎症因子和趋化因子分泌水平的结果图(A、B、C、D、E、F、G、H、I、J、K、L、M分别对应于IL-6、IFN-γ、TNF-α、MCP-1、IL-12p70、IL-1β、IL-10、IL-23、IL-27、IL-17A、IFN-β、GM-CSF、IL-1α);
图8是实施例1制得的产品在体内***富集效果的活体成像图;
图9是实施例1制得的产品在体内***富集效果的统计结果图;
图10是HM-NPs杂合膜纳米颗粒的溶血试验结果图;
图11是实施例6的试验操作流程图;
图12是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤荧光强度的活体成像图;
图13是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤体积随时间变化图;
图14是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤体积随时间变化图;
图15是对照组、EM-NPs组,TM-NPs组,Mix NPs组以及HM-NPs组荷瘤小鼠的生存曲线图;
图16是实施例7的试验操作流程图;
图17是对照组、Mix NPs组以及HM-NPs组荷瘤小鼠肿瘤随时间变化的照片;
图18是对照组、Mix NPs组以及HM-NPs组荷瘤小鼠的生存曲线图;
图19是术后再激发试验中各组小鼠肿瘤随时间的变化结果图;
图20是术后再激发试验中各组小鼠血清中IL-6、TNF-α、IL-1β以及IFN-γ表达水平对比图(A、B、C、D分别对应于IL-6、TNF-α、IL-1β以及IFN-γ);
图21是杂合膜纳米颗粒中不同膜蛋白浓度比例产品对上清中IL-6、TNF-α以及IL-1β的水平对比图(A、B、C分别对应于IL-6、TNF-α以及IL-1β)。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
以下实施例所涉及的Elisa试剂盒购自美国Abcam公司;CD80和CD86抗体购自美国BioLegend公司;ELISPOT试剂盒购自北京达科为生物技术有限公司;ProcartaPlexmultiplex免疫分析试剂盒购自美国Thermo Fisher Scientific公司。
以下实施例所涉及的试验动物小鼠型号为BALB/c,鼠龄6-8周,体重16-18g,雌性,购自北京维通利华实验动物技术有限公司,饲养在国家纳米科学中心无病原体动物房内,所有动物实验都严格按照国家机构动物护理和使用委员会(IACUC)批准的指导方针进行。
以下实施例1-8使用大肠杆菌(E.coli DH5α)内膜作为革兰氏阴性菌内膜的研究模型,使用手术切除来源的4T1-luciferase三阴性乳腺癌组织中细胞的细胞膜或CT26结肠癌组织中细胞的细胞膜作为手术切除来源的实体肿瘤细胞膜的研究模型,使用PLGA作为内核材料的研究模型。
实施例1
本实施例制备四种膜纳米颗粒体系,分别为①大肠杆菌内膜包裹PLGA形成的纳米颗粒(后面以代号EM-NPs表示)体系;②4T1-luciferase三阴性乳腺癌组织中细胞的细胞膜包裹PLGA形成的纳米颗粒(后面以代号TM-NPs表示)体系;③将①和②的产品混合得到的纳米颗粒(后面以代号Mix NPs表示)体系;④大肠杆菌内膜和肿瘤细胞膜组成的杂合膜包裹PLGA形成的纳米颗粒(后面以代号HM-NPs表示)体系。四种产品中总膜蛋白浓度当量均保持一致,Mix NPs和HM-NPs中的两种膜蛋白的浓度当量各自保持一致。
其中HM-NPs的制备方法为如下步骤:
(1)提取大肠杆菌内膜EM,操作为:扩增大肠杆菌,过夜培养至OD600值为1.2;加溶菌酶(裂解细胞壁)至终浓度为2mg/mL,与细菌在37℃下共孵育1h;离心去上清得到原生质体;加入extraction buffer(提取buffer),利用密度梯度离心得到大肠杆菌内膜;重悬后保存在-80℃备用;
(2)提取4T1-luciferase三阴性乳腺癌组织中细胞的细胞膜TM,操作为:每只BALB/c小鼠皮下接种20万个肿瘤细胞,等平均肿瘤体积达到300mm3,无菌条件下手术切除肿瘤组织;将得到的肿瘤组织用剪刀初步剪碎,然后加入含有胶原酶IV(1.0mg·mL-1)、脱氧核糖核酸酶DNase(0.1mg·mL-1)以及透明质酸酶(0.1mg·mL-1)的GBSS溶液,37℃放置15min用来消化肿瘤组织;将消化的组织进行研磨,然后通过孔径为0.22μm的滤网进行过滤,滤液进行离心;将离心细胞重悬,加入含有甘露醇、蔗糖和EGTA的提取buffer,然后在冰浴条件下利用细胞破碎仪破碎细胞,最终通过离心得到肿瘤组织来源的肿瘤细胞膜;将细胞膜重悬并冻存于-80℃备用;
(3)将3mg提取的大肠杆菌内膜EM与1mg提取的手术切除肿瘤细胞膜TM(具有相同的膜蛋白浓度)混合于PBS中,37℃恒温摇床摇15min,之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器,累计来回挤压13次(一来一回算1次),得到HM杂合膜微粒;
(4)将10mg PLGA溶于1mL二氯甲烷,再加入0.2mL无菌蒸馏水;混合物冰上25W超声乳化3min;随后与2mL 1%胆酸钠溶液(表面活性剂)混合,冰上30W超声乳化5min;将乳液逐滴加入10mL 0.5%胆酸钠溶液中,25℃搅拌15min;37℃旋蒸10min;将乳液10000g离心15min;用无菌蒸馏水洗两次,重悬得到PLGA聚合物溶液;
(5)将50μL步骤(3)制备的2mg/mL HM杂合膜微粒与500μL步骤(4)制备的1mg/mLPLGA混合后通过滤膜孔径为200nm的脂质体挤出器,累计来回挤压13次(一来一回算1次),得到HM-NPs杂合膜纳米颗粒。
其中EM-NPs的制备方法为如下步骤:
(1)将4mg制备好的细菌内膜EM溶液置于37℃摇床震荡10min;然后通过滤膜孔径为400nm的脂质体挤出器,累计来回挤压13次(一来一回算1次);
(2)参照HM-NPs的制备方法中步骤(4)利用双乳法制备PLGA纳米颗粒,将50μL制备的2mg/mL细菌内膜EM与500μL制备的1mg/mL PLGA混合,然后通过滤膜孔径为200nm的脂质体挤出器,累计来回挤压13次(一来一回算1次),最终得到EM-NPs。
其中TM-NPs的制备方法为如下步骤:
(1)将4mg制备好的手术切除肿瘤细胞膜TM溶液置于37℃摇床震荡10min;然后通过滤膜孔径为400nm的脂质体挤出器,累计来回挤压13次(一来一回算1次);
(2)参照HM-NPs的制备方法中步骤(4)利用双乳法制备PLGA纳米颗粒,将50μL制备的2mg/mL手术切除肿瘤细胞膜TM与500μL制备的1mg/mL PLGA混合,然后通过滤膜孔径为200nm的脂质体挤出器,累计来回挤压13次(一来一回算1次),最终得到TM-NPs。
其中Mix NPs的制备方法为如下步骤:
(1)将制备得到的EM-NPs与TM-NPs在37℃下进行混合,其中两种膜蛋白的浓度当量与HM-NPs中的各自保持一致。
对制备得到的EM-NPs,TM-NPs以及HM-NPs杂合膜纳米颗粒用透射电镜(美国FEI,Tecnai G2 20S-TWIN,200KV)进行观察,结果如图1所示,结果显示上述三种膜纳米颗粒形状均呈规则圆形,标尺为100nm。
对制备得到的HM-NPs杂合膜纳米颗粒用动态光散射(Zetasizer NanoZS)进行表征,结果如图2所示,有图可知:制备而成的HM-NPs杂合膜纳米颗粒粒径主要分布在100-300nm之间,并且在164.2nm处达到峰值(占15.8%),说明制备的杂合膜纳米颗粒粒径分布较好,有利于进入细胞发挥生物学作用。
实施例2
本实施例对实施例1制得的四种产品增强先天性免疫和特异性免疫的效果进行探究,具体内容包括:
1、促进BMDC分泌促炎细胞因子实验:
(1)提取BALB/c小鼠骨髓来源的树突状细胞(BMDC);
(2)将BMDC培养至第5天,用1640完全培养基重悬,均分为5份以每孔5万个细胞接种在96孔板中,分别加入终浓度为1mg/mL的EM-NPs、TM-NPs、Mix NPs以及HM-NPs溶液进行共孵育,每组3个平行,以等体积的PBS溶液作为对照组(Control,Ctrl);
(3)37℃共孵育24h后,分别取出各组培养基上清;
(4)利用Elisa试剂盒检测上清中IL-6、TNF-α以及IL-1β的水平。
结果如图3所示,可知:HM-NPs杂合膜纳米颗粒与其他组相比具有最佳的增强机体先天性免疫的潜力。
2、促进BMDC成熟实验:
(1)提取BALB/c小鼠骨髓来源的树突状细胞(BMDC);
(2)将BMDC培养至第5天,用1640完全培养基重悬,均分为5份以每孔8万个细胞接种在24孔板中,分别加入终浓度为1mg/mLEM-NPs、TM-NPs、Mix NPs以及HM-NPs溶液进行共孵育,每组3个平行,以等体积的PBS溶液作为对照组(Control,Ctrl);
(3)37℃共孵育24h后,收集细胞,用PBS洗3次;
(4)用表面分子CD11c,CD80和CD86标志物对细胞进行染色分析;
(5)利用BD-C6流式细胞仪检测各组细胞表面表达的CD80和CD86水平,每组采集10000个细胞。
结果如图4所示,可知:HM-NPs杂合膜纳米颗粒与其他组相比具有最强刺激DC细胞成熟的潜力。
3、促进小鼠脾脏淋巴细胞分泌IFN-γ实验:
(1)将BALB/c小鼠随机分为5组,每组3只;
(2)分别在第1、3和第7天对各组小鼠右后背皮下注射EM-NPs,TM-NPs,Mix NPs以及HM-NPs疫苗(100μg/只);以等体积的PBS溶液作为对照组(Control,Ctrl);
(3)第8天分别取其脾脏细胞进行研磨,过滤网后得到混合脾脏淋巴细胞;
(4)将各组小鼠脾脏淋巴细胞与HM-NPs疫苗中相同的肿瘤细胞膜在37℃5%CO2培养箱内共孵育24h;
(5)取各组培养细胞上清液,用ELISPOT(酶联免疫斑点)试剂盒检测其中IFN-γ分泌量;
(6)利用CTL-ImmunoSpot Plate Reader软件对各组斑点数目进行统计,分析数据。
结果如图5和图6所示,可知:HM-NPs杂合膜纳米颗粒与其他组相比具有最强刺激脾脏淋巴细胞分泌IFN-γ的潜力。
综合上述三个试验结果,表明HM-NPs杂合膜纳米颗粒具有最好的增强先天性和特异性免疫的潜力。
实施例3
本实施例对实施例1制得的四种产品刺激小鼠全身免疫***分泌炎症因子和趋化因子的效果进行探究,具体步骤如下:
(1)将BALB/c小鼠随机分为5组,每组5只;
(2)分别在第1、3和第7天对各组小鼠右后背皮下注射EM-NPs,TM-NPs,Mix NPs以及HM-NPs疫苗(100μg/只);以等体积的生理盐水液作为对照组(Control,Ctrl);
(3)第7天皮下注射疫苗6小时后,分别取各组小鼠血清,用ProcartaPlexmultiplex免疫分析试剂盒检测其中炎症因子和趋化因子分泌水平;
(4)利用BD-C6流式细胞仪进行数据分析,利用ProcartaPlex Analysis软件进行数据处理。
结果如图7所示,可知:HM-NPs杂合膜纳米颗粒对全身免疫***分泌炎症因子和趋化因子具有一定影响,与空白对照组相比,只有促炎细胞因子IL-6和IL-1β上升较明显,其他11种炎症因子和趋化因子基本保持不变。表明HM-NPs杂合膜纳米疫苗具有较好的生物安全性。
实施例4
本实施例对实施例1制得的四种产品在体内***富集的效果进行探究,具体步骤如下:
(1)包载荧光染料IR780的各组膜纳米颗粒构建:利用双乳法将亲水性荧光染料IR780包载在PLGA中,具体为:取200μL浓度为1mg/mL亲水性荧光染料IR780,加入1mL浓度为10mg/mL的PLGA二氯甲烷溶液中,初乳3min;加入1%表面活性剂胆酸钠,复乳5min;避光旋蒸10min去除有机溶剂二氯甲烷;11000g离心15min,并用三次水洗两次,得到包载荧光染料IR780的PLGA纳米颗粒。然后与提前制备好的各组膜微粒混合。具体步骤及投料比例同实施例1;
(2)将BALB/c小鼠随机分为4组,每组4只;
(3)各组小鼠右后背皮下注射各组荧光标记的膜纳米颗粒,12h后取各组小鼠***,利用小动物光学3D活体成像***进行荧光成像,并用LivingImage软件进行分析。
结果如图8和图9所示,可知:HM-NPs杂合膜纳米颗粒与其他组相比在***荧光信号最强,表明HM-NPs杂合膜纳米颗粒有较强***富集潜力。
实施例5
本实施例对实施例1制得的HM-NPs杂合膜纳米颗粒的生物安全性进行探究,采用溶血试验评价,具体步骤如下:
(1)BALB/c小鼠取血液1mL,溶于2mL PBS溶液中,3000rpm离心10min;
(2)用PBS洗三次,轻轻吹打至血液上清无颜色;
(3)将离心出的红细胞加2mL PBS稀释,分别取0.1mL血液稀释液加入到0.9mL待测样品(不同浓度HM-NPs杂合膜纳米颗粒、PBS作为阴性对照、三次水作为阳性对照)中;
(4)所有样品置于摇床,150rpm,37℃摇晃3h;
(5)将摇晃后的混合物12000rpm离心10min,摆好拍照;用酶标仪检测上清液在541nm处的吸收值,计算溶血率。
结果如图10所示,可知:各浓度的HM-NPs杂合膜纳米颗粒与红细胞混合后,均未出现溶血现象,说明本发明所涉及的肿瘤疫苗具有好的生物安全性。
实施例6
本实施例对实施例1制得的四种产品抑制行肿瘤切除术的4T1-luciferase乳腺癌荷瘤小鼠术后肿瘤复发的效果进行探究,本实施例的试验操作流程图如图11所示。具体步骤如下:
(1)将雌性BALB/c小鼠随机分为5组,每组6只,每只小鼠右后背皮下接种20万个4T1-luciferase乳腺癌细胞,构建小鼠乳腺癌模型(第0天);
(2)每隔一天观察并记录小鼠肿瘤生长情况,并且每隔一周采用小动物荧光成像***检测荷瘤小鼠肿瘤荧光强度如图12所示;
(3)当平均肿瘤体积达到300mm3左右时,手术切除所有老鼠肿瘤,然后对伤口进行缝合(第7天);
(4)将切下来的瘤块研磨破碎,用胶原酶、DNA酶以及透明质酸酶混合溶液在37℃条件下对肿瘤组织进行消化,消化完的细胞悬液过滤网然后离心,离心弃上清后加入提取buffer,重悬得到细胞悬液;用细胞破碎仪对细胞悬液进行冰浴超声破碎(35W,5min);将破碎后的细胞悬液进行离心(3000g,5min,4℃);取上一步的上清再次进行离心(10000g,10min,4℃);取离心后的细胞上清加入到超离管中进行超速离心(100000g,2h,4℃);超速离心后弃上清,用1mL PBS重悬,得到手术切除来源的肿瘤细胞膜碎片TM,置于-80℃保存;
(5)参照实施例1中方法制备各种膜纳米颗粒;
(6)对术后小鼠腹腔注射荧光素钾,进行小动物荧光成像,观察并统计术后小鼠残余肿瘤体积(第9天);
(7)根据小动物荧光成像结果对小鼠重新随机分组,共分5组,每组6只;
(8)分别在第10、12以及16天给各组小鼠皮下注射EM-NPs,TM-NPs,Mix NPs以及HM-NPs(100μg/只),生理盐水组作为对照组(Control,Ctrl);
(9)每隔一天观察并记录小鼠肿瘤生长情况,当小鼠肿瘤体积生长达到1000mm3左右,即视为小鼠死亡。记录每组小鼠死亡的具体时间。
结果如图13-15所示,可知:本发明所涉及的HM-NPs杂合膜纳米颗粒与其他组相比具有更明显抑制乳腺癌术后小鼠肿瘤复发的潜力,抑制率达到83.3%。
实施例7
本实施例对实施例1制得的四种产品抑制行肿瘤切除术的CT26结肠癌荷瘤小鼠术后肿瘤复发以及肿瘤细胞再激发实验的效果进行探究,本实施例的试验操作流程图如图16所示。
具体步骤如下:
1、预防结肠癌术后复发实验:
(1)将雄性BALB/c小鼠随机分为5组,每组6只,每只小鼠右后背皮下接种20万个CT26结肠癌细胞,构建小鼠结肠癌模型(第0天);
(2)每隔一天观察并记录小鼠肿瘤生长情况,并且每隔一周采用小动物荧光成像***检测荷瘤小鼠肿瘤荧光强度;
(3)当平均肿瘤体积达到300mm3左右时,手术切除所有小鼠肿瘤,然后对伤口进行缝合(第7天);
(4)将切下来的瘤块研磨破碎,用胶原酶、DNA酶以及透明质酸酶混合溶液在37℃条件下对肿瘤组织进行消化,消化完的细胞悬液过滤网然后离心,离心弃上清后加入提取buffer,重悬得到细胞悬液;用细胞破碎仪对细胞悬液进行冰浴超声破碎(35W,5min);将破碎后的细胞悬液进行离心(3000g,5min,4℃);取上一步的上清再次进行离心(10000g,10min,4℃);取离心后的细胞上清加入到超离管中进行超速离心(100000g,2h,4℃);超速离心后弃上清,用1mL PBS重悬,得到手术切除来源的肿瘤细胞膜碎片TM,置于-80℃保存;
(5)参照实施例1中方法制备各种膜纳米颗粒;
(6)对术后小鼠腹腔注射荧光素钾,进行小动物荧光成像,观察并统计术后小鼠残余肿瘤体积(第9天);
(7)根据小动物荧光成像结果对小鼠重新随机分组,共分5组,每组15只;
(8)分别在第10、12以及16天给各组小鼠皮下注射EM-NPs,TM-NPs,Mix NPs以及HM-NPs(100μg/只),生理盐水组作为对照组(Control,Ctrl);
(9)每隔一天观察并记录小鼠肿瘤生长情况,当小鼠肿瘤体积生长达到1000mm3左右,即视为小鼠死亡。记录每组小鼠死亡的具体时间。
结果如图17-18所示,可知:本发明所涉及的HM-NPs杂合膜纳米颗粒与其他组相比具有更明显抑制结肠癌术后小鼠肿瘤复发的潜力,抑制率达到100%。
2、术后再激发试验:
(1)将1中存活的HM-NPs组小鼠在60天后重新分为3组,每组5只;
(2)一组小鼠右后背皮下接种20万个4T1乳腺癌细胞,另一组小鼠右后背皮下接种20万个CT26结肠癌细胞,剩下一组小鼠不接种肿瘤细胞(接种生理盐水作为对照Control,Ctrl);
(3)每隔一天观察并记录各组小鼠肿瘤生长情况,当小鼠肿瘤体积生长达到1000mm3左右,即视为小鼠死亡,记录每组小鼠死亡的具体时间。
结果如图19,可知:当CT26结肠癌细胞第二次“入侵”小鼠体内后,此前接种过结肠癌组织来源的肿瘤细胞膜制备的杂合膜疫苗组小鼠具有完全抵御肿瘤的能力;而当4T1乳腺癌细胞“入侵”小鼠体内后,由于小鼠此前未接触4T1乳腺癌细胞,未获得4T1乳腺癌细胞相关肿瘤抗原,因此不具有对4T1乳腺癌细胞的豁免能力。并在第90天对各组小鼠血清中的IL-6、TNF-α、IL-1β和IFN-γ进行测定,结果如图20所示,可知:二次接种CT26结肠癌细胞的小鼠分泌的促炎因子(IL-6,TNF-α以及IL-1β)明显高于另外两组,其分泌的IFN-γ与接种4T1乳腺癌细胞的小鼠相比也有一定程度升高。
实施例8
本实施例对本发明所涉及的HM-NPs杂合膜纳米颗粒中革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜的用量配比进行探究,具体包括如下内容:
(1)根据实施例1的方法制备不同膜蛋白浓度比例的杂合膜纳米颗粒,具体设置为:单独的革兰氏阴性菌内膜EM、单独的手术切除来源的实体肿瘤细胞膜TM、以及革兰氏阴性菌内膜EM与手术切除来源的实体肿瘤细胞膜TM的质量比为3:1、1:1、1:3的杂合膜,以等体积的PBS溶液作为对照组(Control);
(2)根据实施例2的方法提取并培养BALB/c小鼠骨髓来源的树突状细胞(BMDC);将BMDC培养至第5天,用1640完全培养基重悬,均分为6份每孔5万个细胞接种在96孔板中,分别加入不同膜蛋白比例的杂合膜纳米颗粒进行共孵育,每组3个平行;
(3)37℃共孵育24h后,分别取出各组培养基上清;利用Elisa试剂盒检测上清中IL-6、TNF-α以及IL-1β的水平。
结果如图21所示,可知:与其他组相比,杂合膜比例中EM:TM为3:1时有最强刺激促炎细胞因子(IL-1β,TNF-α、IL-6)的能力,说明其有最强刺激机体先天性免疫的潜力。
实施例9
本实施例制备一种膜纳米颗粒体系,为克雷白氏肺炎杆菌(CG43菌株)内膜和HepG2肝癌组织中细胞的细胞膜组成的杂合膜包裹的基于锆(Zr)的MOF(金属有机框架)形成的纳米颗粒(后面以代号HM-NPs-2表示)体系。其制备方法为如下步骤:
(1)提取肺炎杆菌内膜EM,具体操作同实施例1,包括:细菌增殖;加溶菌酶破裂细胞壁得到原生质体;加入提取buffer并利用密度梯度离心法最终得到肺炎杆菌内膜EM;
(2)提取HepG2肝癌组织中细胞的细胞膜TM,具体操作同实施例1,包括:BALB/c小鼠皮下接瘤;等肿瘤长到一定体积时手术切除;剪刀剪碎、加三种酶进行消化裂解肿瘤组织、研磨过滤网;加入提取buffer并利用超声细胞破碎仪进行破碎;最终离心得到肝癌组织来源的肝癌细胞膜;
(3)将3mg提取的肺炎杆菌内膜EM与1mg提取的手术切除肿瘤细胞膜TM(具有相同的膜蛋白浓度)混合于PBS中,37℃恒温摇床摇15min,之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器,累计来回挤压13次(一来一回算1次),得到HM杂合膜微粒。
(4)合成MOF:将300mg ZrOCl2·8H2O,100mg H2TCPP以及2.8mg苯甲酸(BA)溶于100mL二甲基甲酰胺(DMF)溶剂中,氮气保护条件下90℃搅拌5h;冷却至25℃后,10000g离心30min收集MOF产物并用DMF洗三次,合成好的MOF溶于DMF并储存在4℃条件下,实验使用前需用去离子水洗三次。
(5)将50μL步骤(3)制备的2mg/mL杂合膜微粒与500μL步骤(4)制备的1mg/mL MOF混合,将混合物在浴用超声仪(超声水浴锅)中超声30min,产物离心收集,得到HM-NPs-2杂合膜纳米颗粒。
实施例10
本实施例制备一种膜纳米颗粒体系,为布氏杆菌PBP39内膜和SN12-PM6SN12-PM6肾癌组织中细胞的细胞膜组成的杂合膜包裹MSN(介孔二氧化硅)形成的纳米颗粒(后面以代号HM-NPs-3表示)体系。其制备方法为如下步骤:
(1)提取布氏杆菌内膜EM,具体操作同实施例1,包括:细菌增殖;加溶菌酶破裂细胞壁得到原生质体;加入提取buffer并利用密度梯度离心法最终得到布氏杆菌内膜EM;
(2)提取SN12-PM6肾癌组织中细胞的细胞膜TM,具体操作同实施例1,包括:BALB/c小鼠皮下接瘤;等肿瘤长到一定体积时手术切除;剪刀剪碎、加三种酶进行消化裂解肿瘤组织、研磨过滤网;加入提取buffer并利用超声细胞破碎仪进行破碎;最终离心得到肝癌组织来源的肝癌细胞膜;
(3)将3mg提取的布氏杆菌内膜EM与1mg提取的手术切除肿瘤细胞膜TM(具有相同的膜蛋白浓度)混合于PBS中,37℃恒温摇床摇15min,之后将混合膜溶液通过滤膜孔径为400nm的脂质体挤出器,累计来回挤压13次(一来一回算1次),得到HM杂合膜微粒;
(4)合成MSN:1.5mL正硅酸乙酯(TEOS)和5mL丙酮作为油相,随后加入40mL溶有0.15g十六烷基三甲基溴化铵(CTAB)的水溶液;将混合物以10000rpm的速度搅拌剪切5min,形成乳液;在乳液中加入50μL氨水,随后以400rpm的速度磁力搅拌2h以蒸发有机溶剂;将悬液离心,沉淀物用去离子水洗三次然后冷冻干燥;将正硅酸乙酯(TEOS)被水解并在油/水界面浓缩形成二氧化硅壳;最后在反应或干燥过程中通过蒸发(挥发)除去液滴,获得介孔二氧化硅MSN。
(5)将50μL步骤(3)制备的2mg/mL HM杂合膜微粒与500μL步骤(4)制备的1mg/mLMSN混合,将混合物在浴用超声仪(超声水浴锅)中超声30min,产物离心收集,得到HM-NPs-3杂合膜纳米颗粒。
实施例11
本实施例对实施例9和实施例10制得的两种产品增强先天性免疫和特异性免疫的效果进行探究,具体内容包括:
1、促进BMDC分泌促炎细胞因子实验:
具体操作方法参见实施例2。
结果显示HM-NPs-2和HM-NPs-3杂合膜纳米颗粒促进BMDC分泌IL-6、TNF-α、IL-1β的水平与HM-NPs相似,具有显著的增强机体先天性免疫的潜力。
2、促进BMDC成熟实验:
具体操作方法参见实施例2。
结果显示HM-NPs-2和HM-NPs-3杂合膜纳米颗粒促进BMDC细胞表面表达的CD80和CD86水平与HM-NPs相似,具有显著的刺激DC细胞成熟的潜力。
3、促进小鼠脾脏淋巴细胞分泌IFN-γ实验:
具体操作方法参见实施例2。
结果显示HM-NPs-2和HM-NPs-3杂合膜纳米颗粒组的斑点数与HM-NPs相似,具有显著的刺激脾脏淋巴细胞分泌IFN-γ的潜力。
综合上述三个试验结果,表明HM-NPs-2和HM-NPs-3杂合膜纳米颗粒具有显著的增强先天性和特异性免疫的潜力。
申请人声明,本发明通过上述实施例来说明本发明的一种预防癌症术后复发的肿瘤疫苗及其制备方法和应用,但本发明并不局限于上述实施例,即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (9)
1.一种预防癌症术后复发的肿瘤疫苗,其特征在于,所述肿瘤疫苗包括杂合细胞膜外壳和内核材料,所述杂合细胞膜外壳由革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜组成;
所述革兰氏阴性菌为大肠杆菌、肺炎杆菌或布氏杆菌;
所述肿瘤为乳腺癌、结肠癌、肝癌或肾细胞癌;
所述内核材料为PLGA、MOF或介孔二氧化硅;
所述预防癌症术后复发的肿瘤疫苗的粒径为100-300nm。
2.如权利要求1所述的预防癌症术后复发的肿瘤疫苗,其特征在于,所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为1:3-3:1。
3.如权利要求2所述的预防癌症术后复发的肿瘤疫苗,其特征在于,所述革兰氏阴性菌内膜与手术切除来源的实体肿瘤细胞膜中蛋白质摩尔量的比例为(2-3):1。
4.如权利要求1所述的预防癌症术后复发的肿瘤疫苗,其特征在于,所述杂合细胞膜外壳与内核材料的质量比为1:(1-10)。
5.如权利要求4所述的预防癌症术后复发的肿瘤疫苗,其特征在于,所述杂合细胞膜外壳与内核材料的质量比为1:(4-6)。
6.如权利要求1-5中任一项所述的预防癌症术后复发的肿瘤疫苗的制备方法,其特征在于,所述制备方法包括如下步骤:
(1)分别提取革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜;
(2)将步骤(1)提取到的革兰氏阴性菌内膜和手术切除来源的实体肿瘤细胞膜混合,并用脂质体挤出器挤出,得到杂合膜纳米颗粒;
(3)将步骤(2)得到的杂合膜纳米颗粒与内核材料混合,并用滤膜孔径为100-300nm的脂质体挤出器挤出,得到所述预防癌症术后复发的肿瘤疫苗。
7.如权利要求6所述的预防癌症术后复发的肿瘤疫苗的制备方法,其特征在于,步骤(2)所述混合的温度为20-45℃,时间为10-20min。
8.如权利要求6所述的预防癌症术后复发的肿瘤疫苗的制备方法,其特征在于,步骤(3)所述混合的温度为20-45℃,时间为10-20min。
9.如权利要求1-5中任一项所述的预防癌症术后复发的肿瘤疫苗在制备增强机体先天性免疫和特异性免疫应答的药物中的应用。
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