CN114470201B - 一种装载kcna5抗体的金铂纳米颗粒及其制备方法和应用 - Google Patents
一种装载kcna5抗体的金铂纳米颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种装载KCNA5抗体的金铂纳米颗粒及其制备方法和应用，具体为该纳米颗粒在制备治疗肺动脉高压药物中的应用，所述装载KCNA5抗体的金铂纳米颗粒，包括以下重量百分含量的各组分：Au@Pt纳米颗粒大于90％；KCNA5抗体0.4‑0.6％；活性酯PEG巯基小于10％。本发明结合了KCNA5抗体和纳米技术，构建了负载KCNA5蛋白的纳米药物。核壳结构的纳米载体为靶向药物的负载提供了物理三维空间，在药物传递和纳米动力学方面有优势，同时KCNA5具有肺血管靶向性，该纳米药物可以高效的将KCNA5蛋白递送至肺血管部位，发挥一定肺血管钾通道的功能，缓解肺动脉高压症状。
Description
技术领域
本发明涉及纳米材料技术领域，具体地，涉及一种装载KCNA5抗体的金铂纳米颗粒及其制备方法和应用。
背景技术
纳米级结构材料简称为纳米材料，是指其结构单元的尺寸介于1～100nm范围之间。由于它的尺寸已经接近电子的相干长度，它的性质因为强相干所带来的自组织使得性质发生很大变化。并且，其尺度与可见光处于同一量级，加上其具有大的表面积体积比，因此其所表现的物理特性，例如熔点、磁性、光学、导热、导电特性等等，往往不同于该物质在整体状态时所表现的性质。
Au@Pt纳米颗粒具有核壳结构，外层覆盖较小尺寸的铂纳米颗粒增加纳米材料与底物之间的接触面积，并将铂作为催化中心，可以催化体内过氧自由基(ROS)并转换为氧，从而打开钾离子通道，抑制细胞增生。内核的金纳米颗粒由于等离激元效应可以在红外光照射下发热起到光热转换的作用。而这种热能通过脉冲激光，在金纳米颗粒表面周期性的产生，并引起纳米颗粒尺寸周期性的变化，从而产生光声效应(Photoacoustic Effect)，用来追踪纳米材料在生物体内位置。
KCNA5，钾通道亚家族成员3，已有越来越多的实验证据证明肺动脉平滑肌细胞钾通道功障碍是肺动脉高压的标志，其中KCNA5(Kv1.5)的功能障碍是肺动脉高压发病机制中的关键事件。Stephen L.Archer课题组2003年在Circulation杂志报道：尽管肺动脉高压患者机体仍处于缺氧状态，Kv1.5基因转移将使肺循环正常化，恢复肺血管收缩力和改善肺动脉高压。
发明内容
针对现有技术不足，本发明的目的在于提供一种装载KCNA5抗体的金铂纳米颗粒及其制备方法和应用。
本发明的目的是通过以下技术方案实现的：
第一方面，本发明提供了一种装载KCNA5抗体的金铂纳米颗粒在制备治疗肺动脉高压药物中的应用，所述装载KCNA5抗体的金铂纳米颗粒包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 大于90％；
KCNA5抗体 0.4-0.6％；
活性酯PEG巯基(HS-PEG-NHS) 小于10％。
优选地，所述活性酯PEG巯基的分子量为3500。
优选地，所述装载KCNA5抗体的金铂纳米颗粒包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 95-98％；
KCNA5抗体 0.4-0.6％；
活性酯PEG巯基 1-5％。
第二方面，本发明提供了一种装载KCNA5抗体的金铂纳米颗粒，包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 大于90％；
KCNA5抗体 0.4-0.6％；
活性酯PEG巯基(HS-PEG-NHS) 小于10％。
优选地，所述活性酯PEG巯基的分子量为3500，本发明中采用活性酯PEG巯基的主要作用是通过聚乙二醇一端的巯基连接金、铂纳米颗粒，而聚乙二醇(PEG)另一端是NHS羧基活化基团，用来连接抗体或者药物分子。
优选地，所述装载KCNA5抗体的金铂纳米颗粒包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 95-98％；
KCNA5抗体 0.4-0.6％；
活性酯PEG巯基 1-5％。
第三方面，本发明提供了一种根据权利要求4所述的装载KCNA5抗体的金铂纳米颗粒的制备方法，包括以下步骤：
A、Au@Pt纳米颗粒制备：将氯铂酸和氯金酸配置成溶液后与泊洛沙姆混合，再加入抗坏血酸后进行超声处理，即得Au@Pt纳米颗粒；
B、将KCNA5抗体与活性酯PEG巯基混合后孵育，再加入步骤A制备的Au@Pt纳米颗粒混合后再孵育，即得所述装载KCNA5抗体的金铂纳米颗粒。
优选地，步骤B中，所述KCNA5抗体与活性酯PEG巯基混合后孵育的条件为：先室温下孵育0.5-1.5h，再在0-5℃下孵育12-20h。
优选地，步骤B中，所述加入步骤A制备的Au@Pt纳米颗粒混合后再孵育的条件为：室温下孵育15-25min。
优选地，步骤A中，所述氯铂酸和氯金酸的摩尔比为1:1；
所述超声处理的时间为12-18mins。
本发明通过将KCNA5抗体与金铂纳米颗粒结合，使抗体与病人基因表达的受体定向结合，有利于形成靶向药物治疗，减少对正常细胞的损害和药物剂量的节约。同时利用铂纳米颗粒的催化作用，消耗体内自由基产生活性氧打开钾离子通道抑制细胞增生。目前传统只含抗体的靶向药物无法实现跟踪效果，因此我们设计的金属载体纳米颗粒中含有的金纳米颗粒能够光热光声成像作用，除此之外整个核壳结构提供更大抗体结合表面积优势，由此实现一加一大于二的效果。因此本发明通过KCNA5抗体与金纳米颗粒、铂纳米颗粒制备的结合药物是一种将靶向驱动、药物治疗、成像检测三位一体的治疗药物。
与现有技术相比，本发明具有如下有益效果：
本发明结合了KCNA5抗体和纳米技术，构建了负载KCNA5蛋白的纳米药物。核壳结构的纳米载体为靶向药物的负载提供了物理三维空间，在药物传递和纳米动力学方面有优势，同时KCNA5具有肺血管靶向性，该纳米药物可以高效的将KCNA5蛋白递送至肺血管部位，发挥一定肺血管钾通道的功能，缓解肺动脉高压症状。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为本发明实施例1制备得到的Au@Pt-Kv1.5纳米颗粒表征；其中图1A为Au@Pt-Kv1.5纳米颗粒尺寸和透射电镜图(scale bar＝50nm)；图1B为透射电镜图(scale bar＝100nm)；图1C为紫外可见吸收光谱图；
图2为Au@Pt-Kv1.5纳米颗粒的制备流程；
图3为实施例2中各实验组处理PAMSC细胞后的荧光强度对比结果；
图4为实施例3中模型小鼠的右心室超声结果；其中，上图为右心室长轴图，下图为肺动脉血流过肺动脉瓣图；
图5为实施例3中模型小鼠的PAT/PET比值。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下实施例中提供了一种装载KCNA5抗体的金铂纳米颗粒，包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 大于90％；
KCNA5抗体 0.4-0.6％；
活性酯PEG巯基(HS-PEG-NHS) 小于10％。
以下实施例中还提供了一种装载KCNA5抗体的金铂纳米颗粒的制备方法，包括以下步骤：
A、Au@Pt纳米颗粒制备：将氯铂酸和氯金酸配置成溶液后与泊洛沙姆混合，再加入抗坏血酸后进行超声处理，即得Au@Pt纳米颗粒；
B、将KCNA5抗体与活性酯PEG巯基混合后孵育，再加入步骤A制备的Au@Pt纳米颗粒混合后再孵育，即得所述装载KCNA5抗体的金铂纳米颗粒。
步骤B中，所述KCNA5抗体与活性酯PEG巯基混合后孵育的条件为：先室温下孵育0.5-1.5h，再在0-5℃下孵育12-20h。
步骤B中，所述加入步骤A制备的Au@Pt纳米颗粒混合后再孵育的条件为：室温下孵育15-25min。
步骤A中，所述氯铂酸和氯金酸的摩尔比为1:1；
所述超声处理的时间为12-18mins。
在上述条件下，均能制备得到本发明所述的装载KCNA5抗体的金铂纳米颗粒，下述实施例中不再进行一一列举。
下面结合附图和具体实施例对本发明作进一步说明，但不作为对本发明的限定。
实施例1、Au@Pt-Kv1.5纳米颗粒的制备
本实施例提供一种装载KCNA5抗体的金铂纳米颗粒，按以下重量百分比含量配置各组分：
Au@Pt纳米颗粒 96.15％；
KCNA5抗体 0.5％；
活性酯PEG巯基 3.35％。
所述装载KCNA5抗体的金铂纳米颗粒的制备工艺如图2所示，具体步骤如下：
第一步：将氯铂酸和氯金酸粉末分别加入去离子水配制成25mM的氯铂酸溶液和氯金酸溶液，将制备的氯铂酸溶液和氯金酸溶液各1毫升与0.06g泊洛沙姆混合。随后向混合液中加入6ml浓度为0.1M的抗坏血酸溶液。超声处理15mins。室温放置24h。
第二步：将155μg活性酯PEG巯基(分子量为3500)粉末加入到44.5μl浓度为0.1M的HEPES缓冲液中，立即用漩涡混合器混合。室温放置30mins后加入74.5μl浓度为310μg/ml的KCNA5抗体，室温孵育1h，再在4℃冰箱中孵育过夜。
第三步：取100μl第一步制得的混合液在12500rpm下离心10mins，倒掉上层清液再加入乙醇重新分散，重复离心洗涤3次。得到的金铂纳米颗粒沉淀(即Au@Pt纳米颗粒)重新分散在500μl浓度为0.1M的HEPES缓冲液中，用漩涡混合器混合均匀并超声分散处理15mins。在得到的分散液中加入52μl第二步制成的混合液，用漩涡混合器混合均匀，并在室温下孵育20mins。
本实施例成功合成了装载KCNA5抗体的金铂纳米颗粒(Au@Pt-Kv1.5)，在形貌上使用电子透射显微镜进行了表征，纳米颗粒(Au@Pt-Kv1.5)大小在50nm左右(图1A)，并呈现明显的核壳结构(图1B)。紫外可见吸收光谱的测试显示纳米颗粒(Au@Pt-Kv1.5)在红外区域有一定的吸收(图1C)。
实施例2、体外PAMSC实验验证
2.1实验材料
本实施例中采用的大鼠肺动脉平滑肌细胞为PAMSC,由ATCC公司,ScienCell提供；Kv1.5抗体购自proteintech公司。
2.2实验方法
提前将PAMSC细胞放在由3％O2，2％CO2，95％N2组成的低氧培养箱内培养24小时作为低氧组PAMSC，常氧条件下培养的PAMSC作为常氧组PAMSC。将常氧组PAMSC和低氧组PAMSC中分别加入溶媒作为对照组1(图3中的Control+Vehicle组)和对照组2(图3中的Hypoxia+Vehicle组)，将低氧组PAMSC中分别加入Au@Pt-Kv1.5(图3中的Au@Pt-Kv1.5+Hypoxia组)，Au@Pt(图3中的Au@Pt+Hypoxia组)作用2小时后，各处理组分别用PBS洗涤，免疫荧光固定液固定10min，Kv1.5抗体4℃敷育过夜；第二天，再用PBST洗涤，用相应的二抗常温敷育1小时，洗涤，共聚焦显微镜(蔡司Zeiss LSM880)20x拍照，Image J软件分析数据。
2.3实验结果
如图3所示，与常氧组PAMSC相比，低氧组PAMSC的Kv1.5荧光强度明显下降。给予Au@Pt-Kv1.5干预2小时后，低氧组PAMSC中Kv1.5荧光强度明显增加(图3)，而给予Au@Pt干预的低氧组PAMSC中Kv1.5荧光强度基本未增加。提示Au@Pt-Kv1.5纳米颗粒在体外PAMSC中的有效性。
实施例3、体内实验验证
构建小鼠肺高压模型，具体步骤为：C57BL6近交系小鼠,8-10周龄，雄性，20±2.5g。置于常压低氧舱中，舱内氧浓度维持在10％，持续3周，且每周第一天皮下注射SU541620mg/kg。SU5416：血管生成抑制剂，血管内皮生长因子受体2抑制剂；购于Sigma-Aldrich。
处理方法为：对照组(Control)置于常压常氧条件下；低氧加纳米载体组(Hypoxia+Au@Pt)先间隔三天腹腔注射含金铂纳米颗粒溶液(即Au@Pt纳米颗粒)(4.0mg/kg)两次后置于常压低氧舱中；纳米抗体组(hypoxia+Au@Pt-Kv1.5)先间隔三天腹腔注射含装载KCNA5抗体的金铂纳米颗粒溶液两次(Au@Pt-Kv1.5)(4.0mg/kg)后置于常压低氧舱中。三组饲料、光照等其他条件一致。
如图4所示，与对照组(Control)相比，低氧加纳米载体组(Hypoxia+Au@Pt)的右心室功能下降，而给予纳米抗体(hypoxia+Au@Pt-Kv1.5)干预后，可以部分逆转低氧诱导的小鼠右心室功能下降。
同时，如图5所示，与对照组(Control)相比，低氧加纳米载体组(Hypoxia+Au@Pt)的PAT/PET比值明显下降，而给予纳米抗体组(hypoxia+Au@Pt-Kv1.5)干预后，可以部分逆转低氧诱导的小鼠PAT/PET的下降。提示纳米抗体(Au@Pt-Kv1.5)对低氧诱导的肺高压有一定的保护作用。
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本发明说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。
Claims (5)
1.一种装载KCNA5抗体的金铂纳米颗粒在制备治疗肺动脉高压药物中的应用，其特征在于，所述装载KCNA5抗体的金铂纳米颗粒包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 大于90%；
KCNA5抗体 0.4-0.6%；
活性酯PEG巯基 小于10%；
所述金铂纳米颗粒具有核壳结构；
所述活性酯PEG巯基的分子量为3500；
所述装载KCNA5抗体的金铂纳米颗粒的制备方法，包括以下步骤：
A、Au@Pt纳米颗粒制备：将氯铂酸和氯金酸配置成溶液后与泊洛沙姆混合，再加入抗坏血酸后进行超声处理，即得Au@Pt纳米颗粒；
B、将KCNA5抗体与活性酯PEG巯基混合后孵育，再加入步骤A制备的Au@Pt纳米颗粒混合后再孵育，即得所述装载KCNA5抗体的金铂纳米颗粒。
2.根据权利要求1所述的应用，其特征在于，所述装载KCNA5抗体的金铂纳米颗粒包括以下重量百分含量的各组分：
Au@Pt纳米颗粒 95-98%；
KCNA5抗体 0.4-0.6%；
活性酯PEG巯基 1-5%。
3.根据权利要求1所述的应用，其特征在于，步骤B中，所述KCNA5抗体与活性酯PEG巯基混合后孵育的条件为：先室温下孵育0.5-1.5h，再在0-5℃下孵育12-20h。
4.根据权利要求1所述的应用，其特征在于，步骤B中，所述加入步骤A制备的Au@Pt纳米颗粒混合后再孵育的条件为：室温下孵育15-25min。
5.根据权利要求1所述的应用，其特征在于，步骤A中，所述氯铂酸和氯金酸的摩尔比为1:1；
所述超声处理的时间为12-18mins。
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