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CN111135307B - 一种基于car-t细胞的药物传递系统的制备方法及应用 - Google Patents

一种基于car-t细胞的药物传递系统的制备方法及应用 Download PDF

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CN111135307B
CN111135307B CN202010080284.5A CN202010080284A CN111135307B CN 111135307 B CN111135307 B CN 111135307B CN 202010080284 A CN202010080284 A CN 202010080284A CN 111135307 B CN111135307 B CN 111135307B
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CN111135307A (zh
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袁苗苗
徐洋
付雪梅
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Southern Medical University
Eighth Affiliated Hospital of Sun Yat Sen University
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Abstract

本发明提供了一种基于CAR‑T细胞的药物传递系统，包括CAR‑T细胞、载药纳米材料以及抗肿瘤药物，所述抗肿瘤药物包封于载药纳米材料中形成纳米药物，所述CAR‑T细胞吞噬所述纳米药物，利用CAR‑T细胞上的嵌合抗体靶向定位于与该抗体结合的特定抗原。本发明制备的基于CAR‑T细胞的药物传递系统，能显著提高药物的肿瘤靶向性，增加药物在作用部位浓度，从而增强药物疗效，同时降低了药物在外周器官的分布，减少了药物的毒副作用。本发明开发了CAR‑T细胞的新用途，提高了CAR‑T细胞的经济价值，也为CAR‑T细胞在药物传递系统的应用提供了理论和实践基础。

Description

一种基于CAR-T细胞的药物传递系统的制备方法及应用
技术领域
本发明涉及肿瘤治疗技术领域，具体涉及一种基于CAR-T细胞的药物传递系统的制备方法及应用。
背景技术
药物治疗疾病的首要条件是药物能够在靶组织内达到有效药物浓度，但是由于受限于身体内的各种生理屏障和潜在的毒副作用，目标药物到达靶组织是一个充满挑战的过程。目前即使是一些已经在临床使用的传统药物，也存在药代动力学行为不良而导致治疗效率有限、毒副作用严重的问题。因此，为了获得更好的治疗效果，合适剂量的药物必须在适当的时间转运到需要治疗的部位并且维持一定浓度来保证治疗效果。此外，为了降低药物剂量和潜在的毒副作用，就需要尽量降低药物在作用位置和代谢器官之外的组织分布。基于此，研究和应用新的药物传递系统来改善药物传递变得尤为重要。
基于细胞的药物传递系统是一种有效治疗疾病的药物输送平台。细胞本身是一种高度智能的药物传递装置，它们可以感受各种信号，并迁移到需要的部位，整合微环境中的各种输入信号，然后做出相应的复杂行为。此外，基于细胞的药物传递系统，细胞本身具有肿瘤导引能力，自身带有靶向性，不需要再连接靶向识别分子。近年来，免疫细胞作为药物运输工具引起了人们的广泛关注，因为它们不仅可以通过血液流动迁移到损伤、炎症或肿瘤的部位，且免疫清除率降低，还可以延长生物半衰期。
CAR-T细胞除了具有免疫细胞药物传递系统具有的固有的靶向性、药物传递性及较低的免疫清除率之外，更增加了其本身对肿瘤细胞等的杀伤作用的功能，有望成为一种更有效的药物传递系统。基于此，我们旨在开发一种基于CAR-T细胞的能够主动靶向疾病的药物传递系统，研究其在体内循环时间、肿瘤组织的靶向作用、肿瘤细胞的摄取以及体内抗肿瘤活性，为药物的靶向治疗肿瘤奠定基础。
发明内容
为实现上述目的，本发明采取的技术方案为：一种基于CAR-T细胞的药物传递系统，所述系统包括CAR-T细胞、载药纳米材料以及抗肿瘤药物，所述抗肿瘤药物包封于载药纳米材料中形成纳米药物，所述CAR-T细胞吞噬所述纳米药物，利用CAR-T细胞上的嵌合抗体靶向定位于与该抗体结合的特定抗原。
优选地，所述特定抗原包括CD19、CD140a、EGFR、HER2。
优选地，所述嵌合抗体包括CD19抗体、CD140a抗体、EGFR抗体、Her2抗体。
优选地，所述CAR-T细胞为CD19-CAR-T细胞、CD140a-CAR-T细胞、EGFR-CAR-T细胞、HER2-CAR-T细胞。
优选地，所述载药纳米材料为介孔硅、脂质体、聚乳酸-羟基乙酸共聚物(PLGA)、白蛋白、外泌体。
优选地，所述抗肿瘤药物为阿霉素类、紫杉烷类、喜树碱类、葱醒类及其衍生物中的一种或几种。
本发明还提供了如上所述基于CAR-T细胞的药物传递系统在制备抗肿瘤药物中的应用。
优选地，所述肿瘤为脑胶质瘤、肝癌、乳腺癌、卵巢癌、肺癌、皮肤癌或恶性黑色素瘤中的任意一种。
本发明还提供了如上所述基于CAR-T细胞的药物传递系统的制备方法，包括以下步骤：
(1)制备载药纳米材料；
(2)将步骤(1)制备的载药纳米材料将抗肿瘤药物溶液混合，将混合物冷冻干燥，获得纳米药物；
(3)将引入了编码嵌合抗体基因的CAR质粒转染T细胞，将转染后的T细胞置于含有白介素2的培养基中培养，激活T细胞，获得靶向特定抗原的CAR-T细胞；
(4)将CAR-T细胞与纳米药物共培养，形成基于CAR-T细胞的药物传递系统。
优选地，所述特定抗原包括CD19、CD140a、EGFR、HER2。
优选地，所述嵌合抗体为CD19抗体、CD140a抗体、EGFR抗体、Her2抗体。
优选地，所述CAR-T细胞为CD19-CAR-T细胞、CD140a-CAR-T细胞、EGFR-CAR-T细胞、Her2-CAR-T细胞。
优选地，所述载药纳米材料为介孔硅、脂质体、聚乳酸-羟基乙酸共聚物(PLGA)、白蛋白、外泌体。
优选地，所述抗肿瘤药物为阿霉素类、紫杉烷类、喜树碱类、葱醒类等及其衍生物中的一种或几种。
本发明的有益效果：
1、经实验证明，本发明制备的基于CAR-T细胞的药物传递系统，能显著提高药物的肿瘤靶向性，增加药物在作用部位浓度，从而增强药物疗效，同时降低了药物在外周器官的分布，减少了药物的毒副作用。
2、本发明开发了CAR-T细胞的新用途，提高了CAR-T细胞的经济价值，也为CAR-T细胞在药物传递系统的应用提供了理论和实践基础。
附图说明
图1为实施例1中制备的四氧化三铁介孔硅核壳结构材料的电镜图(其中A为制备的材料的扫描电镜图；B为制备的材料的透射电镜元素分布图)；
图2为流式细胞仪检测CAR-T细胞内DOX荧光强度(其中A为未吞噬D-FMSNs的CAR-T细胞，B为与D-FMSNs共孵育以后的CAR-T细胞)；
图3基于CAR-T细胞的药物传递系统对肿瘤细胞杀伤效果的测定；
图4基于CAR-T细胞的药物传递系统在动物体内的靶向性测定；
图5基于CAR-T细胞的药物传递系统对动物生存期的影响。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例及其附图对本发明做进一步的详细描述。
实施例1制备本发明的基于CAR-T细胞的药物传递系统
1、制备载药纳米材料——四氧化三铁介孔硅纳米材料
(1)将10mL 0.25mol/L FeCl3·6H2O与10mL 0.25mol/L FeSO4·7H2O混合溶液加入到烧瓶中，在氮气保护下将加入适量1mol/L的NH3·H2O溶液到反应体系中，使体系的p H≥10，剧烈搅拌30min后结束反应，用蒸馏水反复洗涤直至中性，倾去上层清液，在60℃下真空干燥后，即得Fe3 O4纳米颗粒。
(2)将2.5mg的Fe3 O4纳米颗粒加入到0.2mL氯仿中，将2mL十六烷基三甲基溴化铵(CTAB)水溶液(40mg CTAB，54mmol/L)加入到Fe3O4胶体溶液中，并用盖子密封后将混合物超声处理10min，然后蒸发氯仿，得到透明的良好分散的Fe3O4胶体水溶液，并将其命名为Fe3O4@CTAB。
(3)在100mL的烧瓶中将40mg CTAB溶于18mL水，再加入120uL NaOH溶液(2M)；然后，将步骤(2)得到的2ml Fe3O4@CTAB胶体溶液加入到该反应溶液中，剧烈搅拌并将溶液的温度升至70℃；将200uL正硅酸乙酯(TEOS)和1.2mL乙酸乙酯逐滴添加到溶液中，搅拌2小时后，将40μL 3-氨丙基三乙氧基硅烷(APTES)逐滴加入溶液中并再搅拌2小时，然后将溶液冷却至室温，离心后并用乙醇洗涤3次，得到介孔二氧化硅纳米粒子颗粒。
(4)将步骤(3)中得到的纳米颗粒加入含有120mg NH4NO3的20mL乙醇中，并在60℃下搅拌1小时，此步骤重复两次后，用去离子水和乙醇多次洗涤纳米颗粒。
将步骤(4)制备的纳米颗粒经透射电子显微镜表征其形貌及成分，结果如图1所示，由图1A可知：制备的材料的粒径约为50nm左右；由图1B元素分布分析可知：本发明成功制备出四氧化三铁介孔硅核壳结构材料，标记为FMSNs。
2、制备纳米药物
(1)将20mg四氧化三铁介孔硅核壳结构加入到20ml阿霉素(DOX)PBS溶液中，避光，搅拌过夜，得到阿霉素纳米药物，标记为D-FMSNs。
(2)离心收集D-FMSNs，并且在真空下冷冻干燥。通过λ＝480nm的UV-Vis光谱测量FMSNs加载DOX的效率。
3、制备CAR-T细胞
首先构建CD140a CAR慢病毒载体，其包含来自临床药物Olaratumab的ScFv序列(Olaratumab的VH和VL区通过3X GGGGS接头连接)，CD8铰链和跨膜结构域，4-1BB和CD3ζ细胞内结构域。CD140a CAR的质粒载体DNA由IGE基因公司(广州)进行优化和合成，然后使用Gibsson Assembly方法将其克隆到NheI和AgeI限制性酶切位点之间的慢病毒转移载体pLenti-EF1a。然后，梯度离心法获得外周血单个核细胞(PBMC)，用Dynabeads磁珠分选出T细胞，将培养在含有白介素2的1640培养基中，以便刺激T细胞生长。将T细胞暴露于含有抗CD140a-CAR慢病毒载体的上清液，开始传导。扩增这些传导的细胞，通过针对CAR蛋白的流式细胞荧光分选技术(FACS)分析，确定成功的CAR基因转移，获得CD140a CAR-T细胞。
4、制备基于CAR-T细胞的药物传递系统
将CAR-T细胞与D-FMSNs纳米药物在37℃下于新鲜无血清培养基中孵育一小时，离心用冰冷的PBS洗涤2次后，获得CAR-T+D-FMSNs悬浮液。取部分CAR-T+D-FMSNs悬浮液，通过流式细胞仪PE-A通道检测CAR-T细胞内的DOX荧光。
由图2A可知：单独CAR-T细胞的荧光强度为0.73％；由图2B可知与D-FMSNs共孵育以后的CAR-T细胞的荧光强度为99.7％。结果表明D-FMSNs能进入CAR-T细胞，本发明基于CAR-T的药物传递系统已成功制备。
实施例2基于CAR-T细胞的药物传递系统体外对肿瘤细胞的杀伤效果
本实施例采用Luciferase法与共聚焦实验检测以CAR-T细胞为载体的药物传递系统对U87细胞的毒性作用，检测CAR-T细胞药物传递系统体外抗肿瘤活性，具体操作如下：
将U87胶质瘤细胞(由上海细胞库提供用)培养于含有10％胎牛血清，1％双抗的DMEM培养基中，置于37℃，5％CO2的细胞培养箱内孵育，隔天换液，2-3天后用0.25％胰蛋白酶(含0.02％EDTA)消化传代一次。取对数生长期细胞，常规消化为单细胞悬液后计数，调整待测细胞密度至5×103个/孔(边缘孔用无菌PBS填充)，铺U型96孔板，每孔加入200μL培养基，然后置于37℃含有5％CO2的细胞孵育箱中培养。待24h细胞全部贴壁后，弃去原培养基。
将细胞分为实验组、对照组和空白组。实验组中分别加入与肿瘤细胞体积比为1:8，1:4，1:2，1:1，2:1，4:1，8:1的CAR-T+D-FMSNs悬浮液；对照组、中按照同样比例加入D-FMSNs复合物的悬浮液，前述每种比例设置5个复孔；空白组加入培养基。将三组细胞继续置于培养箱培养24h后终止培养。将U型96孔板取出，平角离心机离心1000rpm/min，用排枪轻轻去除上清液，再加入报告基因细胞裂解液每孔100uL吹打混匀细胞，将细胞转移至黑色不透明的96孔板中，每孔加入100μL的萤光素酶检测试剂，立即在荧光照度计中检测荧光值；杀伤比例计算：以不加T细胞的靶细胞孔为参照，荧光值降低的比例即为杀伤百分比，公式为：杀伤百分比＝(空白孔荧光值-目的孔荧光值)/空白孔×100％。
由图3可知：D-FMSNs，CAR-T+D-FMSNs分别与U87人胶质瘤细胞共培养后，实验组(CAR-T+D-FMSNs)对胶质瘤细胞的杀伤效果显著高于没有使用CAR-T细胞的对照组(D-FMSNs)。结果表明基于CAR-T细胞的药物传递系统具有显著的肿瘤细胞杀伤效果。
实施例3基于CAR-T细胞的药物传递系统在动物体内的靶向效果
首先将FMSNs连接CY7荧光标记物，30mg FMSNs，345mg NHS,576mg EDC和1.5mgCY7混合，加入至30mL PBS中，在室温下，避光搅拌24小时。搅拌完毕后，离心使用PBS冲洗混合物数次，获得FMSNs-CY7。并按照上述CAR-T细胞药物传递系统的制备方法构建D-FMSNs-CY7，Normal T+D-FMSNs-CY7(该组使用的T细胞为正常普通的T细胞，不包含抗-CD140a CAR慢病毒载体)和CAR-T+D-FMSNs-CY7。然后建立胶质瘤原位模型，使用立体定向固定装置向NSG小鼠脑右半球颅内注5×105个U87-Luc细胞。通过腹腔内注射D-肝素钾盐并使用体内成像系统(Caliper IVIS Spectrum)观察脑中U87-Luc细胞的发光强度来监测脑肿瘤生长。在将神经胶质瘤细胞移植到小鼠脑中后8天，随机分3组，并分别通过尾静脉给予一定量的D-FMSNs-CY7，Normal T+D-FMSNs-CY7和CAR-T+D-FMSNs-CY7。随后，用含20％氧气的1-2％异氟烷麻醉小鼠，并在给药后1、2、4、8和24小时使用IVIS光谱成像系统观察及拍照。
由图4可知：将胶质瘤小鼠随机分组，并分别给予治疗注射D-FMSNs-CY7，Normal T+D-FMSNs-CY7和CAR-T+D-FMSNs-CY7，其中CAR-T+D-FMSNs-CY7组能明显聚集于小鼠脑部胶质瘤位置。结果表明基于CAR-T细胞的药物传递系统具有显著的靶向胶质瘤的效果，提高药物在作用部位浓度。
实施例4基于CAR-T细胞药物传递系统在动物体内的治疗效果
建立胶质瘤原位模型，使用立体定向固定装置向NSG小鼠脑右半球颅内注5×105个U87-Luc细胞。通过腹腔内注射D-肝素钾盐并使用体内成像系统(Caliper IVISSpectrum)观察脑中U87-Luc细胞的发光强度来监测脑肿瘤生长。在将神经胶质瘤细胞移植到小鼠脑中后8天，随机分2组，分别接受静脉注射，如下：(1)D-FMSNs；(2)CAR-T+D-FMSNs(每只小鼠1×107个细胞)；记录每组的存活时间。
由图5可知：结果表明本发明基于CAR-T细胞药物传递系统能显著提高原位胶质瘤小鼠模型的存活时间，与对照组(D-FMSNs)相比有显著的抗肿瘤效果。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (7)

1.一种基于CAR-T细胞的药物传递系统，其特征在于，所述系统包括CAR-T细胞、载药纳米材料以及抗肿瘤药物，所述抗肿瘤药物包封于载药纳米材料中形成纳米药物，所述CAR-T细胞吞噬所述纳米药物，利用CAR-T细胞上的嵌合抗体靶向定位于与该抗体结合的特定抗原；
所述CAR-T细胞为CD140a-CAR-T细胞；
所述载药纳米材料为四氧化三铁介孔硅；所述抗肿瘤药物为阿霉素；
所述纳米药物的制备方法，包括以下步骤：
1）将四氧化三铁介孔硅核壳结构加入到阿霉素 PBS溶液中，避光，搅拌过夜，得到纳米药物D-FMSNs；
2）离心收集纳米药物D-FMSNs，并且在真空下冷冻干燥；
将CAR-T细胞与纳米药物D-FMSNs在37℃下于新鲜无血清培养基中孵育一小时，离心用PBS洗涤，获得CAR-T+D-FMSNs悬浮液，形成基于CAR-T细胞的药物传递系统。
2.如权利要求1所述的基于CAR-T细胞的药物传递系统，其特征在于，所述特定抗原为CD140a。
3.如权利要求1所述的基于CAR-T细胞的药物传递系统，其特征在于，所述嵌合抗体为CD140a抗体。
4.如权利要求1所述的基于CAR-T细胞的药物传递系统在制备抗肿瘤药物中的应用。
5.如权利要求4所述的应用，其特征在于，所述肿瘤为脑胶质瘤、肝癌、乳腺癌、卵巢癌、肺癌、皮肤癌或恶性黑色素瘤中的任意一种。
6.一种如权利要求1所述的基于CAR-T细胞的药物传递系统的制备方法，其特征在于，包括以下步骤：
（1）制备载药纳米材料；
（2）将步骤（1）制备的载药纳米材料将抗肿瘤药物溶液混合，将混合物冷冻干燥，获得纳米药物；
（3）将引入了编码嵌合抗体基因的CAR质粒转染T细胞，将转染后的T细胞置于含有白介素2的培养基中培养，激活T细胞，获得靶向特定抗原的CAR-T细胞；
（4）将CAR-T细胞与纳米药物共培养，形成基于CAR-T细胞的药物传递系统。
7.如权利要求6所述的制备方法，其特征在于，所述特定抗原为CD140a；所述嵌合抗体为CD140a抗体；所述CAR-T细胞为CD140a-CAR-T细胞；所述载药纳米材料为四氧化三铁介孔硅；所述抗肿瘤药物为阿霉素。
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