CN115350145A - 细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用 - Google Patents
细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用 Download PDFInfo
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Abstract
本发明涉及细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用，并提出了一种制备细菌介导负载二甲双胍的多肽水凝胶的方法，实验证明该多肽水凝胶不仅可以杀伤肿瘤细胞，还具备调节肿瘤免疫微环境，激活机体免疫系统的功能，可抑制肿瘤复发和转移，为肿瘤免疫治疗提供了新的策略。
Description
技术领域：
本发明涉及肿瘤免疫治疗用品领域，具体涉及细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用。
背景技术：
肿瘤免疫微环境是一个免疫抑制微生态系统，决定肿瘤生物学特性的良恶性，由肿瘤相关成纤维细胞、肿瘤相关巨噬细胞、肿瘤相关脂肪细胞、某些类型的淋巴细胞组成。抗炎基质细胞和M2型巨噬细胞包围肿瘤细胞，阻止促炎性基质细胞和M1型巨噬细胞浸润至肿瘤内部，并导致肿瘤免疫逃逸。TIME的这种特征似乎也具有肿瘤内的异质性，即更具侵袭性和抵抗力的肿瘤细胞和肿瘤干细胞通常位于肿瘤块的边缘，这可能导致肿瘤在手术切缘复发。肿瘤中免疫微环境的另一个障碍是抵抗免疫细胞的浸润，这使得PD-1和PD-L1抑制剂对肿瘤患者无效。除了其自身复杂的细胞和非细胞成分，肿瘤免疫微环境的棘手之处还在于它可以根据不同的外部治疗情况，动态地、持续地改变内部生物信号网络，使肿瘤细胞逐渐对原始敏感药物产生耐药性。因此，单一药物和一成不变的治疗方式往往难以达到令人满意的临床效果。
细菌介导的治疗方式是一种足智多谋、动态变化的抗肿瘤策略。梭状芽孢杆菌是一种只在缺氧条件下生长和繁殖的细菌。因此，实体肿瘤内部是梭状芽孢杆菌的完美生态位。无限的繁殖能力和不断产生的细菌毒素使细菌有望成为灵活的药物来控制肿瘤生长，但是治疗同时产生的严重炎症限制了其临床应用。多肽自组装材料是由自组装多肽分子之间通过电荷互补自发形成稳定的β折叠结构，继而自组装形成的纳米级纤维凝胶材料，已广泛应用于药物缓释载体、三维细胞培养和骨、血管以及中枢神经系统等组织的缺损修复。二甲双胍在改善机体免疫功能状态的方面具有很大的潜力。具体而言，二甲双胍在促进固有免疫系统，例如促进NKs功能实现和浸润水平，促进M1巨噬细胞极化，以及唤醒适应性免疫细胞，包括增加CD8+T细胞的数量和质量，抑制促瘤免疫细胞的功能。此外，二甲双胍调节肿瘤免疫微环境内的非细胞成分(如HIF-1α和PDL1)对抗肿瘤反应也有积极作用。然而，免疫系统的重塑离不开二甲双胍的长期调节。因此，创建缓慢释放二甲双胍的相关纳米药物已成为目前肿瘤免疫治疗领域研究中最热门的项目之一。
发明内容：
(一)解决的技术问题
针对上述背景，本发明提出细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用，并提出了一种制备细菌介导负载二甲双胍的多肽水凝胶的方法，实验证明该多肽水凝胶不仅可以杀伤肿瘤细胞，还具备调节肿瘤免疫微环境，激活机体免疫系统的功能，可抑制肿瘤复发和转移，为肿瘤免疫治疗提供了新的策略。
(二)技术方案
为解决上述技术问题，本发明采用如下技术方案：
细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用。
所述多肽水凝胶的制备方法如下：
S1，将二甲双胍溶解在0.9％的盐水中，制备浓度为40mM的溶液；
S2，向每毫升二甲双胍溶液中添加107CFU C-novyi-NT Spores和10mg RADA32-蜂毒素融合肽(RADA32-GG-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2)，并反复吹打直至形成均一溶液；
S3，将上述S2获得的混合物放在4℃冰箱中过夜保存，以形成MRM-Coated Spores，即为所述的多肽水凝胶，其中MRM表示RADA32-蜂毒素融合肽+MET，Spores表示C-novyi-NTSpores。
其中，C-novyi-NT Spores表示梭状芽孢杆菌的减毒菌株，不仅增强化疗药物的抗肿瘤作用，同时激活机体的免疫反应；MET表示二甲双胍。
C-ovyi-NT Spores为C-novyi-NT的孢子，为便于表示直接使用C-novyi-NTSpores表示。C-novyi-NT Spores不仅可以增强化疗药物的抗肿瘤作用，也激活了机体的免疫反应，这表现在C-novyi-NT Spores治愈的小鼠能够抵抗相同肿瘤细胞的再次定植。
所述多肽水凝胶以RADA-32为基础肽，连接活性成分蜂毒肽，形成具有抗肿瘤作用的蜂毒肽水凝胶，同时负载二甲双胍，形成β折叠结构隐藏肽链之间的酶切位点，所述多肽水凝胶缓慢释放活性成分蜂毒肽和二甲双胍，持续杀伤肿瘤细胞的同时，调节肿瘤免疫微环境内的非细胞成分(如HIF-1α和PDL1)，实现对免疫系统的长期调节，延长抗肿瘤成分在体内的作用时间。
(三)有益效果
本发明产生的有益效果是：通过提出细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用，并提出了一种制备细菌介导负载二甲双胍的多肽水凝胶的方法，实验证明该多肽水凝胶不仅可以杀伤肿瘤细胞，还具备调节肿瘤免疫微环境，激活机体免疫系统的功能，可抑制肿瘤复发和转移，为肿瘤免疫治疗提供了新的策略。
附图说明：
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1：MRM-Coated Spores的合成和表征。其中A为各材料合成后的图示，从左向右依次为罗丹明染液，MR(蜂毒肽水凝胶)，MRM(蜂毒肽水凝胶+二甲双胍)，MRM-CoatedSpores(蜂毒肽水凝胶+二甲双胍+C-novyi-NT的孢子)，Rho-MRM-Coated Spores(连接有罗丹明燃料的蜂毒肽水凝胶+二甲双胍+C-novyi-NT的孢子)；B为MRM的扫描电镜结构图；C为C-novyi-NT的孢子的扫描电镜的结构图；D为MRM-Coated Spores的扫描电镜结构图；E为MRM，C-novyi-NT spores,MRM-Coated Spores的表面电位情况；F为MRM-Coated Spores在应力为0.1％下的扫描流变学分析，表明MRM-Coated Spores有良好的储存模量和损耗模量；G为MRM-Coated Spores在角频率为1rad/s下的跃迁时间相关的流变学分析，MRM-Coated Spores在低应变力下保持良好的胶体结构，应力提高破坏其胶体结构后在恢复较低的应力，其凝胶结构逐渐恢复；H为MR，MRM，MRM-Coated Spores在存在或不存在蛋白酶K(PK)的条件下的降解情况，表明MRM-Coated Spores在蛋白酶K消化下18天左右降解完全；I为在pH值为7.4时，在存在或不存在蛋白酶K(PK)的情况下，MRM和MRM-Coated Spores的二甲双胍的释放曲线。
图2:MRM-Coated Spores的体外抗肿瘤作用图。其中A为克隆形成实验，表明MRM-Coated Spores具有最好的抗肿瘤效果；B为各组材料和细胞共培养后第一天和第三天的细胞活性；C为各组材料和细胞共培养后细胞的凋亡状况；D为为各组材料和细胞共培养后进行死活细胞染色的结果。其中MRM+Bac模仿MRM-Coated Spores的体外作用。
图3:MRM-Coated Spores的体内抗肿瘤作用展示。其中A为各组小鼠进行治疗后利用活体成像技术检测各组肿瘤的生长状况；B为各组治疗小鼠的肿瘤体积监测图；C为各组小鼠的肿瘤组织的展示；D为各组肿瘤质量的测量结果；E为各组小鼠体重的监测结果；F为各组小鼠的生存监测。
图4：MRM-Coated Spores激活了机体的免疫系统结果展示。其中A为流式结果展示MRM-Coated Spores组DC细胞是对照组的3.19倍；B为MRM-Coated Spores组NK细胞是对照组的6.63倍；C为MRM-Coated Spores组CD8+T细胞是对照组的1.49倍；D为MRM-CoatedSpores组CD8+T/CD4+T的比率是对照组的2.94倍；E为MRM-Coated Spores组CD8+IFNγ+T细胞是对照组的9.79倍；F为免疫荧光染色展示MRM-Coated Spores组具有最高的CD8+IFNγ+T细胞的浸润；G为MRM-Coated Spores组M1型巨噬细胞的浸润水平远高于其它各组；H为MRM-Coated Spores组M2型巨噬细胞的浸润水平低于其它各组；I为MRM-Coated Spores组M1/M2的比率高于其它各组。
图5：小鼠的血液学检查的结果。A为白细胞，B为血红蛋白，C为红细胞，D为乳酸脱氢酶，E为谷丙转氨酶，F为谷草转氨酶，G肌酸激酶，H为C反应蛋白。
图6：小鼠心、肝、脾、肺的HE染色结果。
图7：小鼠心、肝、脾、肺的的革兰氏染色结果。
图8：MRM-coated spores激活免疫记忆的结果展示。A为实验的基本流程示意图；B为肿瘤的照片展示；C为肿瘤生长的监测情况；D为各组肿瘤的重量；E为小鼠的体重监测；F为小鼠的生存状况监测；G、H为小鼠淋巴结记忆细胞的水平；I、J为小鼠脾脏记忆细胞的水平。
图9：MRM-coated spores的颅内运用。A为实验的基本流程；B为活体成像显示对照组和MRM-coated spores组小鼠的颅内肿瘤生长情况；C为两组小鼠肿瘤的HE染色；D为小鼠的生存监测；E为小鼠的体重监测；F为MRM-coated spores组小鼠颅内肿瘤的HE染色；G为免疫荧光展示肿瘤浸润的CD8+IFNγ+T细胞的浸润水平；H为免疫荧光展示肿瘤浸润的M1型巨噬细胞的浸润水平；I为免疫荧光展示肿瘤浸润的M2型巨噬细胞的浸润水平。
具体实施方式：
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
一、纳米材料可持续在体内作用：通过蛋白激酶K在体外模仿纳米材料在体内的消化状况，观察发现，蛋白激酶作用18天后纳米材料才完全被消化。而将纳米材料注入至小鼠的皮下后，观察发现，纳米材料可以在体内存在10天以上。
二、纳米材料在体外具有良好的抗肿瘤作用：
进行克隆形成实验，实验步骤如下：
胶质瘤细胞GL261按照每孔600个铺于6孔板上，第二天加入各组药物，于细胞生长至明显的克隆团块后甲醛固定后结晶紫染色，计数分析。
实验结果见图2A，结果显示，细胞的生存率从PBS、MR、MRM到MRM+Bac(MRM+Bac模仿MRM-Coated Spores的体外作用)依次降低，分别为1、0.78、0.42、0.11。CCK-8实验见图2B展示了与药物培养1天和3天时的细胞活性，MRM+Bac组分别为0.28(第1天)和0.10(第3天)。流式细胞术和活/死细胞染色显示死亡细胞的情况，结果见图2C和2D同样说明MRM+Bac组的细胞凋亡率最高(54.8％)，GL261细胞的活细胞比例最低(43.6％)。这说明MRM+Bac可通过提高肿瘤细胞的凋亡水平，发挥抗肿瘤作用。
三、纳米材料在体内具有良好的抗肿瘤作用：
在GL261肿瘤体积增长至约50mm3时，使用不同的治疗药物进行治疗。治疗方案为50μL PBS、MET、C-novyi-NT Spores、MR、MRE、MR-Coated Spores或MRM-Coated Spores。PBS组小鼠的肿瘤荧光信号在注射后15天内迅速增强，表明该组小鼠的肿瘤生长迅速(见图3A)。PBS组在第15天内肿瘤体积约为1500mm3(见图3B-D)。MRM-Coated Spores具有最强的抗肿瘤作用，抑制率达到95.5％。注射5天后，MRM-Coated Spores组肿瘤荧光强度开始下降，第15天观察到微弱荧光，表明MRM-Coated Spores在体内对肿瘤有持续的抑制作用。各组小鼠的体重没有显著差异(见图3E)。MRM-Coated Spores组小鼠的存活状态明显优于其他组，只有一只小鼠在观察截止日期前死亡(见图3F)。
四、MRM-Coated Spores激活了机体的免疫反应：
在治疗截止时间点，收获小鼠肿瘤组织，消化后进行流式细胞学和免疫荧光染色。
实验结果见图4，流式显示MRM-Coated Spores处理组小鼠DCs(CD11c+MHCII+)的细胞显著提高，是对照组的3.19倍(图4A)；CD3-CD11c+NK1.1+细胞的比例是PBS组的6.64倍(图4B)；CD8+T细胞的比例增加(图4C)，CD8+/CD4+T细胞的比率显著升高(图4D)，MRM-Coated Spores组CD8+IFN-γ+T细胞的比例明显高于其余各组(图4E)。免疫荧光染色也证实MRM-Coated Spores可增加GBM中CD8+IFN-γ+T细胞的浸润水平(图4F)。MRM-CoatedSpores处理组的M1型巨噬细胞比例显著增加(图4G)，比PBS组高1.95倍，M2型巨噬细胞的比例显著降低(图4H，I)。
五、MRM-coated spores拥有良好的生物安全性：
在治疗的第八天，取小鼠的血液进行血液学指标的检测；在治疗截止时间点，分别获取小鼠的心、肝、脾、肺进行HE染色和革兰氏染色。
实验结果见图5-7，MRM-coated spores治疗组白细胞、血红蛋白、红细胞水平均正常，小鼠LDH、ALT、AST和CK水平正常(图5)。小鼠的心脏、肝脏、脾脏和肺进行HE(图6)和革兰氏染色(图7)显示小鼠重要器官未受侵犯。
六、MRM-coated spores激活免疫记忆：
选取手术和MRM-Coated Spores治疗后肿瘤完全消除的小鼠，再次接种相同的肿瘤细胞(GL261细胞)，并观察肿瘤形成(图8A)。结果见图8显示，手术组中的GL261细胞再次定植并快速形成肿瘤组织，而MRM-Coated Spores组中的肿瘤细胞仅形成小的组织块(图8B，C)。手术组的肿瘤重量明显高于MRM-Coated Spores组(图8D)。两组之间的体重没有显著差异(图8E)。两组小鼠的生存率有显著差异(图8F)。大约23天，手术组小鼠开始死亡，而MRM-Coated Spores组小鼠全部存活至观察终点。从小鼠淋巴结和脾脏中提取免疫细胞，分析记忆细胞是否被激活，结果显示，淋巴结和脾脏中的CD8+CD44+T分别增加了55.13％和292.53％(图8G-J)。上述结果表明，MRM-Coated Spores具有激活免疫记忆的潜力。
七、MRM-coated spores在颅内运用也安全有效：
在小鼠颅内接种GL261细胞，第7天注射MRM-coated spores进行治疗，同时对照组注射PBS。在第14天处死小鼠，获取小鼠的颅脑组织进行后续实验。期间检测小鼠的体重和生存情况。
实验结果见图9颅内治疗组小鼠肿瘤生长明显较慢(图9B，C)。在治疗一周后，对照组小鼠因肿瘤生长开始死亡，所有小鼠在三周内死亡。治疗组的第一只小鼠在治疗18天后死亡，50％的小鼠存活超过21天(图9D)。对照组小鼠的体重在实验期间急剧下降，而治疗组小鼠的体重在治疗后以较慢的速度下降(图9E)。通过免疫荧光技术证实与PBS对照组相比，在用MRM-Coated Spores治疗的小鼠的脑瘤中发现了更高比例的CD8+IFN-γ+T细胞(图9G)，并且M1表型在巨噬细胞中占据了主要地位(图9H，I)。说明MRM-Coated Spores凝胶具有激活颅内免疫反应的潜能。革兰氏染色检测细菌在颅内的定位情况(图9F)。结果发现，细菌只在厌氧部位的肿瘤中生长，在正常脑组织中未发现定植。
综上所述，本发明通过提出细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用，并提出了一种制备细菌介导负载二甲双胍的多肽水凝胶的方法，实验证明该多肽水凝胶不仅可以杀伤肿瘤细胞，还具备调节肿瘤免疫微环境，激活机体免疫系统的功能，可抑制肿瘤复发和转移，为肿瘤免疫治疗提供了新的策略。
最后需要说明的是，以上实施例仅用于说明本发明而非限制本发明的保护范围。另外，在阅读了本发明的技术内容之后，本领域技术人员可以对本发明作各种改动、修改或变型，所有的这些等价形式同样属于本申请所要求限定的保护范围之内。
Claims (3)
1.细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用。
2.根据权利要求1所述的细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用，其特征在于，所述多肽水凝胶的制备方法如下：
S1，将二甲双胍溶解在0.9％的盐水中，制备浓度为40mM的溶液；
S2，向每毫升二甲双胍溶液中添加107CFU C-novyi-NT Spores和10mg RADA32-蜂毒素融合肽，并反复吹打直至形成均一溶液；
S3，将上述S2获得的混合物放在4℃冰箱中过夜保存，以形成MRM-Coated Spores，即为所述的多肽水凝胶，其中MRM表示RADA32-蜂毒素融合肽+MET，Spores表示C-novyi-NTSpores；
其中，C-novyi-NT Spores表示梭状芽孢杆菌的减毒菌株，不仅增强化疗药物的抗肿瘤作用，同时激活机体的免疫反应；MET表示二甲双胍。
3.根据权利要求2所述的细菌介导负载二甲双胍的多肽水凝胶在肿瘤免疫治疗中的应用，其特征在于，所述多肽水凝胶以RADA-32为基础肽，连接活性成分蜂毒肽，形成具有抗肿瘤作用的蜂毒肽水凝胶，同时负载二甲双胍，形成β折叠结构隐藏肽链之间的酶切位点，所述多肽水凝胶缓慢释放活性成分蜂毒肽和二甲双胍，持续杀伤肿瘤细胞的同时，调节肿瘤免疫微环境内的非细胞成分，实现对免疫系统的长期调节，延长抗肿瘤成分在体内的作用时间。
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