CN115006528B - 一种辐射敏感性水凝胶及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种辐射敏感性水凝胶及其制备方法和应用，所述水凝胶的制备包括如下步骤：在常温条件下，向质量浓度3~9%PVA溶液中加入药物，混匀，然后将质量浓度6~9%TSPBA快速加入到该混合液中混合，静止，即可。在药物为AAV‑PD1和ADU‑S100时，将含有AAV‑PD1和ADU‑S100的水凝胶和RT联合治疗胶质母细胞瘤（GBM）时，对比传统的免疫治疗联合放射治疗能显著抑制胶质母细胞瘤（GBM）复发，经联合治疗的小鼠组未发生肿瘤复发；本发明的联合治疗方案对比传统的免疫治疗联合放射治疗能延长术后小鼠生存期，60%的小鼠实现了长期生存。
Description
技术领域
本发明属于肿瘤治疗领域，具体涉及一种辐射敏感性水凝胶及其制备方法和应用。
背景技术
尽管免疫治疗改变了许多肿瘤的治疗格局，但仍不能提高多形性胶质母细胞瘤(GBM)患者的生存率。手术和术后辅助放化疗仍是GBM的主要治疗方法。放疗(RT)是预防GBM术后复发的第一种治疗方法，但临床疗效仍然很差。大多数GBM患者在放疗过程中由于抗辐射而发生肿瘤复发。因此，如何有效地抑制GBM术后复发仍是一个令人困惑的临床问题。
近年来，依赖于RT对肿瘤微环境(TME)的调节作用的放射免疫治疗已成为治疗各种恶性肿瘤的一种有前途的方法。RT诱导免疫原性细胞死亡(ICD) 并促进肿瘤CXCL10的转录，从而将更多T细胞招募到TME中。然而，大量证据表明，GBM的免疫原性明显较差，仅RT只能诱导低水平的ICD，这不足以诱导免疫应答。此外，GBM独特的免疫抑制TME进一步限制了RT治疗后T细胞的浸润。
重组腺相关病毒(rAAV)是一种新兴的高效基因治疗载体，能够将治疗基因有效地携带到宿主细胞中，并持续表达治疗蛋白。克服血脑屏障(BBB)的阻断和抗PD-1药物的靶向效应，提高脑肿瘤部位的药物效率。我们将治疗性PD-1 基因引入rAAV，并希望治疗性rAAV(AAV-PD1)能够有效地转运肿瘤细胞并稳定表达治疗性PD-1蛋白，其可以结合到肿瘤细胞的PD-L1受体，从而阻断肿瘤细胞与T细胞之间的PD-1/PD-L1相互作用。这只是提高GBM放射免疫治疗效果的第一步。另一个问题是如何招募更多的T细胞进入GBM的TME。
ADU-S100，一种STING激动剂，可激活STING通路并诱导趋化因子的产生，以促进T细胞渗入TME。STING通路的激活还可以激活固有免疫系统，从而产生强大的抗肿瘤免疫反应。
为了更好地发挥药物的治疗效果。我们开发了一种辐射敏感性水凝胶药物组合，凝胶作为AAV-PD1和ADU-S100的药物库，可以持续释放药物，提高药物效率。肿瘤微环境中的ROS浓度相对较低，可能难以触发药物从水凝胶中释放。但是RT可以诱导活性氧的持续产生，活性氧被用来触发药物释放。
发明内容
针对现有问题的不足，本发明的目的是提供一种辐射敏感性水凝胶及其制备方法和应用。
本发明解决其技术问题采用的技术方案是：
本发明首先保护一种辐射敏感性水凝胶的制备方法，包括如下步骤：在常温条件下，向质量浓度3～9％PVA溶液中加入药物，混匀，然后将质量浓度6～9％ TSPBA快速加入到该混合液中混合，静止，即可。
作为本申请的优选技术方案，所述药物为AAV-PD1和/或ADU-S100。
作为本申请的优选技术方案，所述AAV-PD1质量浓度为3×107IU/mL。
作为本申请的优选技术方案，所述ADU-S100的浓度为质量浓度0.04％。
一种辐射敏感性水凝胶的制备方法，在常温条件下，向质量浓度3～9％PVA 溶液中加入AAV-PD1和ADU-S100，充分混匀，然后将质量浓度6～9％TSPBA 快速加入到该混合液中混合，静止，构建成治疗药物ADU-AAV-PD1@Gel药物。
作为本申请的优选技术方案，在常温条件下，向质量浓度3～9％PVA溶液中加入AAV-PD1和ADU-S100，涡旋使其充分混匀，然后将质量浓度6％TSPBA 快速加入到该混合液中混合，静止5～30秒钟，构建成治疗药物 ADU-AAV-PD1@Gel药物。
作为本申请的优选技术条件，常温条件下，向质量浓度6％PVA溶液中加入 6×107IU/mL AAV-PD1和质量浓度0.08％的ADU-S100，涡旋使其充分混匀，然后将质量浓度6％TSPBA快速加入到该混合液中混合，静止10秒钟，构建成治疗药物ADU-AAV-PD1@Gel药物。
作为本申请的优选技术方案，所述TSPBA可商业购买或者自行制备。
具体的，TSPBA的制备如下：将N1、N1、N3、N3-四甲基丙烷-1,3-二胺(PBA) 加入过量4-(溴甲基)苯硼酸进行季铵化反应，即可。
N1、N1、N3、N3-四甲基丙烷-1,3-二胺(PBA)一种ROS敏感的连接剂，通过N1、N1、N3、N3-四甲基丙烷-1,3-二胺在过量的4-(溴甲基)苯硼酸存在下的季铵化反应即可获得TSPBA。
本发明还保护前述制备方法制备得到的辐射敏感性水凝胶。
本发明还保护前述水凝胶在制备治疗多形性胶质母细胞瘤的药物中的应用。
有益效果
本发明提供的一种辐射敏感性水凝胶及其制备方法和应用，与现有技术相比，具有以下有益效果：
(1)ADU-AAV-PD1@Gel+RT的治疗方案对比传统的免疫治疗联合放射治疗能显著抑制肿瘤复发，经ADU-AAV-PD1@Gel联合RT治疗的小鼠组未发生肿瘤复发。
(2)ADU-AAV-PD1@Gel+RT的治疗方案对比传统的免疫治疗联合放射治疗能延长术后小鼠生存期，60％的小鼠实现了长期生存。
附图说明
图1为ADU-AAV-PD1@Gel的电镜图片；
图2为鼠肿瘤生长曲线；
图3为鼠治疗后生存期曲线；
图4为ADU-AAV-PD1@Gel药物安全性评价；
图5为ADU-AAV-PD1@Gel的血清生化分析结果。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过市场购买的常规产品。
实施例1TSPBA的制备
将PBA加入过量4-(溴甲基)苯硼酸进行季铵化反应，即可。
实施例2凝胶形成剂量摸索
将PVA溶于水，形成PVA溶液，向其中加入溶于水中的TSPBA溶液，混匀后静置10秒，以评估水凝胶的柔韧性；其中PVA浓度分别为3％、6％和9％， TSPBA浓度也分别为3％、6％和9％。
结果如图1所示，TSPBA浓度为9％，无论PVA浓度如何，都会形成固体凝胶。相比之下，当TSPBA的浓度为3％时，9％的PVA会形成液态水凝胶，而降低浓度的PVA不会形成水凝胶。
因此，TSPBA与PVA的最佳比例为6％：6％。
实施例3构建ADU-AAV-PD1@Gel
试剂：TSPBA：C21H34O2N2Br2，PVA：[C2H4O]n，AAV-PD1：1×108IU/mL(汉恒生物)，ADU-S100(MedChemExpress，HY-12885)。
在常温条件下，向质量浓度6％PVA溶液中加入6×107IU/mL AAV-PD1和质量浓度0.08％的ADU-S100，涡旋使其充分混匀，然后将质量浓度6％TSPVA快速加入到该混合液中混合，静止10秒钟。TSPBA上的硼酸基团和PVA上的羟基迅速发生络合反应形成交联键。图2为扫描电子显微镜观察形成凝胶后的微观结构。
实施例4：治疗实验
GL261-Luciferase：小鼠胶质母细胞瘤细胞系并持续表达荧光素酶；
PierceTM D-荧光素单钠盐(赛默飞；货号：88294)；
PD1治疗抗体：(BioXCell；货号：BE0146)；
首先在小鼠体内原位注射GL261-Luciferase细胞构建胶质瘤原位模型，在第 10天用活体成像检测模型是否构建成功，构建成功后在第11天在显微镜下部分切除胶质瘤，同时用活体成像检测残余肿瘤大小并随机分组：不做治疗的对照组、 RT组(仅进行放疗)、ADU+RT组(给药4μg ADU-S100联合放射治疗)、 anti-PD1+RT组(给药200μg的anti-PD1联合放射治疗)、ADU+anti-PD1+RT 组(给药4μg ADU-S100和200μg的anti-PD1联合放射治疗)和ADU-AAV-PD1@Gel+RT组(给药10μL的ADU-AAV-PD1@Gel联合RT)，每组小鼠在第11天给予药物治疗，在第12、13、14天给予2Gy的放射治疗，在第15、20、25、30天进行活体成像观测肿瘤大小，并记录实验小鼠的生存时间。
如图3所示，ADU-AAV-PD1@Gel+RT组对比传统的免疫治疗联合放射治疗 (anti-PD1+RT组)能显著抑制肿瘤复发，经ADU-AAV-PD1@Gel联合RT治疗的小鼠组未发生肿瘤复发，而其他组均出现不同程度的肿瘤复发，表明ADU-AAV-PD1@Gel联合RT治疗组的肿瘤复发率较低对肿瘤生长产生了强烈的抑制作用。
如图4所示，ADU-AAV-PD1@Gel+RT组对比传统的免疫治疗联合放射治疗 (anti-PD1+RT组)能延长术后小鼠生存期，60％的小鼠实现了长期生存。
实施例5：安全性评价
取接受不同方案治疗的小鼠外周血血清，分别使用天门冬氨酸氨基转移酶 (谷草转氨酶/AST/GOT)测试盒、丙氨酸氨基转移酶(谷丙转氨酶/ALT/GPT)测试盒、碱性磷酸酶(ALP/AKP)测定试剂盒和γ－谷氨酰转移酶(γ－GT/GGT)测试盒，血尿素氮(BUN)和肌酐(CREA)，根据制造商说明书进行检测。图5 所示，所有组之间的血清生化分析结果没有显示出明显差异。综上所述，所有结果表明ADU-AAV-PD1@Gel在肿瘤治疗中表现出良好的生物相容性。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。
Claims (4)
1.一种辐射敏感性水凝胶，其特征在于，其通过如下步骤制备：在常温条件下，向质量浓度6% PVA溶液中加入药物，混匀，然后将质量浓度6% TSPBA快速加入到该混合液中混合，静止；其中，所述药物为AAV-PD1和ADU-S100；
其中，所述AAV-PD1的浓度为3×107 IU/mL；
所述ADU-S100的质量浓度为0.04%。
2.根据权利要求1所述的一种辐射敏感性水凝胶，其特征在于，其制备包括如下步骤：常温条件下，向质量浓度6% PVA溶液中加入3×107 IU/mL AAV-PD1和质量浓度0.04%的ADU-S100，涡旋使其充分混匀，然后将质量浓度6%TSPBA快速加入到该混合液中混合，静止10秒钟，构建成治疗药物ADU-AAV-PD1@Gel药物。
3.根据权利要求1-2任一所述的一种辐射敏感性水凝胶，其特征在于，所述TSPBA的制备如下：将PBA加入过量4-（溴甲基）苯硼酸进行季铵化反应，即可。
4.权利要求1所述的辐射敏感性水凝胶在制备治疗多形性胶质母细胞瘤的药物中的应用。
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