CN114181286A - 一种纳米蛋白复合物、药物递送系统及应用 - Google Patents

一种纳米蛋白复合物、药物递送系统及应用 Download PDF

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CN114181286A
CN114181286A CN202111284287.1A CN202111284287A CN114181286A CN 114181286 A CN114181286 A CN 114181286A CN 202111284287 A CN202111284287 A CN 202111284287A CN 114181286 A CN114181286 A CN 114181286A
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protein
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刘凯
李博
徐沪济
张洪杰
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Tsinghua University
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Abstract

本发明涉及生物医学材料技术领域，尤其涉及一种纳米蛋白复合物、药物递送系统及应用。该纳米蛋白复合物包括高等电点蛋白和阴离子表面活性剂。分别溶解在水中，通过静电力作用形成蛋白‑表面活性剂自组装纳米药物递送系统，可以在665nm激光激发下发出红外荧光，并对一些脂溶性药物进行包裹。本发明能够制备具有高生物相容性的蛋白纳米药物递送系统，可用于肿瘤及类风湿关节炎的成像诊断及治疗。

Description

一种纳米蛋白复合物、药物递送系统及应用
技术领域
本发明涉及生物医学材料技术领域，尤其涉及一种纳米蛋白复合物、药物递送系统及应用。
背景技术
类风湿关节炎(Rheumatoid Arthritis,RA)是一种累及机体多个部位和系统的自身免疫性疾病，通常以关节的肿胀、疼痛为先发症状，随着病程的进展可能导致关节严重变形、僵硬，并会累及心血管系统、呼吸系统、消化系统、内分泌系统以及皮肤、眼、神经等多个系统和器官。流行病学统计类风湿关节炎患者的致残率和死亡率较正常人明显提高。世界范围内人群患病率约为1％，中国人口中患病率约为0.4％，总数超过500万。女性患病率约为男性的2-4倍，且病人多集中在20-55岁的青壮年。因为缺乏自愈性，所以类风湿关节炎的治疗是一个复杂而漫长的过程。有文献报道到2021年预计全球治疗类风湿关节炎的药物花费总额将达到360亿美元。因此，无论是对个人的工作生活和家庭而言，还是对于整个社会的人口和经济而言，类风湿关节炎的治疗一直是一个沉重的负担。
类风湿关节炎的发病机制目前还没有完全阐明，因此治疗措施和临床疗效也比较局限。临床常用的一线抗类风湿药物主要是非甾体类抗炎药，包括布洛芬、双氯芬酸钠、吲哚美辛、塞来昔布等常用解热镇痛药物。此类药物价格便宜，但是只能暂时缓解关节疼痛的症状，对于疾病进展本身并没有实质的治疗作用，且长期服用会导致胃肠道溃疡、肝肾脏器损害等副损伤。一线药物还有糖皮质激素，虽然抗炎作用明显，但是停药后的复发和长期服用的副作用一直困扰着患者和医生。临床目前应用较多的是疾病修饰的抗类风湿药(DMARDs)。主要包括甲氨蝶呤、柳氮磺胺吡啶、氯喹、来氟米特等。此类药物多可以减缓关节炎症和畸形的进展，但是起效慢。患者通常需要长期服用并定期监测肝肾功能以防止药物带来的脏器损伤。同时还有很多患者会出现对药物的不敏感导致疗效不佳。随着对类风湿关节炎发病机制的研究深入，生物制剂类DMARDs(bDMARDs)给类风湿关节炎的治疗带来了新的希望。主要代表药物包括肿瘤坏死因子α(TNF-α)抑制剂(依那西普、英夫利昔、阿达木单抗)，人工重组白介素1(IL-1β)受体抑制剂(阿那白滞素)，乌帕替尼等。此类药物针对炎症通路中的靶点发挥治疗作用，成为了近年来类风湿关节炎治疗领域的研究热点。bDMARDs虽然疗效突出且副作用少，但目前仍然存在一些问题，比如价格昂贵、长期应用会致机体产生抗体导致疗效不佳、半衰期短需要反复多次用药而导致患者依从性差等。这些问题是bDMARDs药物面临的比较普遍的问题。因此，如何提高现有bDMARDs的生物相容性(降低免疫源性)、提高半衰期并降低成本成为目前提高类风湿关节炎治疗效率亟待解决的问题。
癌症，是人类第一大疾病，目前临床常用的癌症药物有化学类药物，单抗类药物及多肽类药物。多肽类药物作为癌症治疗的有效用药，越来越受到业界关注，但由于其较短的半衰期及较差的生物利用度限制了其在临床中的使用次数。由此，如何有效的提高此类药物的半衰期及生物利用度是现阶段亟待解决的临床问题。
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用于体外成像及药物递送的高等电点蛋白，该蛋白具有生物相容性好，纯度高的优点。
为了实现上述发明目的，本发明提供以下技术方案：
一种高等电点蛋白，具有如SEQ ID NO.1所示的氨基酸序列；或具有在SEQ IDNO.1所示氨基酸序列中取代、缺失或添加氨基酸且蛋白活性不发生改变的氨基酸序列。
本发明中，所述高等电点蛋白由原核表达系统制备获得。所述原核表达系统采用的骨架载体为pET25b，转化的宿主为大肠杆菌，具体的的，所述高等电点蛋白的制备方法为：
将编码所述高等电点蛋白的核酸克隆至骨架载体pET25b，获得重组载体；
将所述重组载体转化大肠杆菌，经表达、筛选获得稳定表达菌株，发酵后收集菌体。
本发明还提供一种纳米蛋白复合物，由本发明所述的高等电点蛋白、胆绿素和阴离子表面活性剂组成。
一些实施方案中，所述高等电点蛋白和阴离子表面活性剂的摩尔比为1:(360～720)，具体可为1：360或1:720。
一些实施方案中，所述阴离子表面活性剂为含羧基的阴离子表面活性剂，包括羧基聚乙二醇、硫酸软骨素等多糖中的至少一种。
本发明还提供了所述的纳米蛋白复合物的制备方法，包括：
将高等电点蛋白与胆绿素混合，获得蛋白复合物；
将所述蛋白复合物和阴离子表面活性剂在水中进行混合，经自组装获得纳米蛋白复合物。
一些具体实施例中，所述纳米蛋白复合物的制备方法具体为：
(1)按照摩尔比1：3将氨基酸序列如SEQ ID NO.1所示的高等电点蛋白与胆绿素进行混合，用纯水透析除去多余胆绿素，得到蛋白复合物；
(2)将步骤(1)得到的蛋白复合物用超纯水配置成浓度为2mg/ml的蛋白复合物溶液，同时配置浓度为102.9mg/ml的阴离子表面活性剂水溶液。
(3)将步骤(2)中所述的蛋白复合物溶液和阴离子表面活性剂水溶液按照体积比为1：1的比例进行混合，确保高等电点蛋白与阴离子表面活性剂的摩尔比为1：720，然后透析除去多余的阴离子表面活性剂，得到纳米蛋白复合物。
本发明中，所述阴离子表面活性剂为羧基聚乙二醇。步骤(1)-(3)中，透析取分子量大于5000的产物。
本发明中，所述高等电点蛋白和阴离子表面活性剂在水中混合，通过静电力作用形成蛋白-表面活性剂自组装纳米蛋白复合物，可以在665nm激光激发下发出红外荧光，并对一些脂溶性药物进行包裹。研究表明，本发明纳米蛋白复合物具有高生物相容性，可用于肿瘤及类风湿关节炎的成像诊断及治疗。
本发明还提供一种纳米药物递送系统，包括本发明所述的纳米蛋白复合物和包裹在所述纳米蛋白复合物中的药物。
本发明提供的药物递送系统以本发明所述的高等电点蛋白为核心，与阴离子表面活性剂PEG-COOH上的羧基进行静电力结合，通过物理吸附作用包裹药物。
本发明中，所述药物为脂溶性药物，包括包括硫链丝菌素、抗癌肽(如PMI)、乌帕替尼中的至少一种。
一些实施方案中，所述纳米药物递送系统的制备方法及结构示意图如图1所示，具体包括如下步骤：
(1)按照摩尔比1：3将氨基酸序列如SEQ ID NO.1所示的高等电点蛋白与胆绿素进行混合，用纯水透析除去多余胆绿素，得到蛋白复合物；
(2)将步骤(1)得到的蛋白复合物用超纯水配置成浓度为2mg/ml的蛋白复合物溶液，同时配置浓度为102.9mg/ml的阴离子表面活性剂水溶液。
(3)将步骤(2)中所述的蛋白复合物溶液和阴离子表面活性剂水溶液按照体积比为1：1的比例进行混合，确保高等电点蛋白与阴离子表面活性剂的摩尔比为1：720，然后透析除去多余的阴离子表面活性剂，得到纳米蛋白复合物。
(4)取5mg药物加入到步骤(3)得到的纳米蛋白复合物中，之后进行搅拌，离心，收取上清，透析后得到纳米药物递送系统。
本发明中，所述阴离子表面活性剂为羧基聚乙二醇。步骤(1)-(4)中，透析取分子量大于5000的产物。
本发明中，纳米药物递送系统的粒径为180-200nm。
本发明还提供所述的纳米蛋白复合物或所述的纳米药物递送系统在制备治疗和/或预防肿瘤、类风湿关节炎的药物中的应用。
其中，所述肿瘤包括乳腺肿瘤、转移性肝癌、黑色素瘤、肺癌中的至少一种。
本发明还提供了所述的纳米蛋白复合物或所述的纳米药物递送系统在生物医学成像中的应用。
本发明提供的纳米药物递送系统以蛋白为核心，将阴离子表面活性剂(如PEG-COOH)与疏水药物结合在上面，用于肿瘤及类风湿关节炎的成像及治疗，实验表明，该纳米药物递送系统在生物医学成像及治疗实验中成像效果非常好，具有良好的医学前景。同时，本发明纳米蛋白复合物以及纳米药物递送系统的制备方法极其简单，反应效率高，收率高，具有较高的工业应用前景。
附图说明
图1为本发明的高等电点蛋白复合物的结构及合成示意图；
图2为本发明的高等电点蛋白纯化结果示意图；
图3为本发明的高等电点蛋白纳米药物递送系统的TEM图；
图4为本发明的高等电点蛋白纳米药物递送系统的粒径分布图；
图5为本发明的高等电点蛋白纳米药物递送系统的细胞毒性结果图；
图6为本发明的高等电点蛋白纳米药物递送系统体外荧光成像图；其中，6-a为蛋白复合物和血液混合后成像，6-b为蛋白复合物成像；
图7为本发明的高等电点蛋白纳米药物递送系统对小鼠皮下肺肿瘤的成像；
图8为本发明的高等电点蛋白纳米药物递送系统对小鼠皮下肺肿瘤的治疗作用；
图9为本发明的高等电点蛋白纳米药物递送系统对类风湿关节炎SD大鼠踝关节的成像作用；
图10为本发明的高等电点蛋白纳米药物递送系统对类风湿关节炎SD大鼠踝关节的治疗效果；
图11为本发明的高等电点蛋白纳米药物递送系统对药物体内半衰期的提升作用。
具体实施方式
本发明提供了一种纳米蛋白复合物、药物递送系统及应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
(1)取氨基酸序列如SEQ ID NO.1所示的高等电点蛋白纯化(见图2)，按照摩尔比1：3将高等电点蛋白与胆绿素进行混合，用纯水透析除去多余胆绿素，得到蛋白复合物；
(2)将步骤(1)得到的蛋白复合物用超纯水配置成浓度为2mg/ml的蛋白复合物溶液，同时配置浓度为102.9mg/ml的阴离子表面活性剂水溶液。
(3)将步骤(2)中所述的蛋白复合物溶液和表面活性剂水溶液按照体积比为1：1的比例进行混合，确保高等电点蛋白与聚乙二醇的摩尔比为1：720，然后透析除去多余的阴离子表面活性剂，得到纳米蛋白复合物。
(4)取5mg硫链丝菌素加入到步骤(3)得到的纳米蛋白复合物中，之后进行搅拌，离心，收取上清，透析后得到纳米药物递送系统。
步骤(1)-(4)中，透析取分子量大于5000的产物。
获得的纳米药物递送系统用于乳腺肿瘤及转移性肝癌的诊断及治疗。TEM图如图3所示，粒径分布情况见图4。
结果显示，本发明纳米药物递送系统具有均匀的纳米形态和良好的稳定性，在水中具有很好的分散性和均匀性。
实施例2
(1)取氨基酸序列如SEQ ID NO.1所示的高等电点蛋白纯化(见图2)，按照摩尔比1：3将高等电点蛋白与胆绿素进行混合，用纯水透析除去多余胆绿素，得到蛋白复合物；
(2)将步骤(1)得到的蛋白复合物用超纯水配置成浓度为2mg/ml的蛋白复合物溶液，同时配置浓度为102.9mg/ml的阴离子表面活性剂水溶液。
(3)将步骤(2)中所述的蛋白复合物溶液和羧基聚乙二醇水溶液按照体积比为1：1的比例进行混合，确保高等电点蛋白与羧基聚乙二醇的摩尔比为1：720，然后透析除去多余的阴离子表面活性剂，得到纳米蛋白复合物。
(4)取5mg乌帕替尼加入到步骤(3)得到的纳米蛋白复合物中，之后进行搅拌，离心，收取上清，透析后得到纳米药物递送系统。
步骤(1)-(4)中，透析取分子量大于5000的产物。
所述纳米药物递送系统用于风湿性关节炎的诊断及治疗。该纳米药物递送系统具有均匀的纳米形态和良好的稳定性，在水中具有很好的分散性和均匀性。
实施例3性能测试
1、蛋白纳米药物递送系统对肺癌细胞的细胞毒性。
采用标准MTT法测定体外细胞毒性。待细胞长满后，取活化好的细胞培养基悬浮液，用PBS洗三次，加入1mL胰酶消化，后加入1mL培养基终止消化，将Hela细胞接种在96孔板中，使细胞浓度至每孔约6000个，加入100μLDMEM+10％FBS培养基，放入含有5％CO2、37℃恒温培养箱中培养24h。待细胞约长满孔面积的80％后，将加入的培养基除去，向每个孔中添加100μL以培养基为溶剂的浓度为800,400,200,100,50,25,0nm/mL的实施例1包裹硫链丝菌素的蛋白纳米药物递送系统红外蛋白复合物溶液，左右轻轻摇晃，置于含有5％CO2，37℃恒温培养箱中培养24小时。吸去上清液，然后向每个孔中添加100μL10％MTT溶液(PBS缓冲液中为5mg/ml)，在37℃下培养4h。然后弃去MTT，每孔加入150μL DMSO来溶解细胞内的紫色甲臜晶体。置于摇床上平摇10min后，酶标仪检测测量490nm处的吸光度，计算细胞生长的活力。以空白浓度为0的孔为对照，按照下面的公式分别计算不同药物浓度作用于肺癌细胞时的细胞抑制率。
抑制率(％)＝(对照组吸光值－实验组吸光值)/对照组吸光值×100％
存活率(％)＝1－抑制率(％)，结果如图5所示，由图5可以看出，本发明的不带药物的蛋白复合物细胞存活率均在80％以上，进一步也证实了本发明的蛋白复合物是安全低毒甚至无毒的，可以被生物体所接受，而加入多肽的细胞存活率非常低，说明了其在体内肿瘤治疗方面的潜力。
2、高等电点蛋白纳米药物递送系统体外荧光成像效果。
取2mg/mL实施例1～2的蛋白纳米药物递送系统，将其与PBS或小鼠血液等比例混合，在665nm波长激光激发下对其进行荧光成像，结果见图6。
如图6b中可以看出，纳米蛋白复合物具有相当高的荧光强度，其和血液混合后的结果如图6a所示，其仍然具有相当好的信号强度，说明此种递送系统的荧光成像效果不会被血液等体内组织阻挡，具有较好的穿透能力。
3、高等电点蛋白纳米药物递送系统体内荧光成像及治疗效果。
将高等电点蛋白纳米药物递送系统取1mg，溶于PBS缓冲液之中，得步骤如下：
1.小鼠皮下肺部肿瘤模型建立
取4-6周的Balb/c-nu裸鼠，左后腿皮下注射1×107个A549人肺腺癌细胞，之后等待14天，进行下一步实验。
2.大鼠胶原诱导关节炎模型建立
取4-6周的SD大鼠，左后腿及颈部皮下分别注射100μl胶原-弗氏佐剂乳浊液，每14天注射一次，总计注射两次，第二次注射14天后造模完成。
3.纳米药物递送系统注射剂制备
将实施例1～2高等电点蛋白纳米药物递送系统溶解在PBS中，配置成终浓度1mg/ml的澄清溶液。
4.纳米药物递送系统成像及治疗研究
将上述配置好的实施例1的纳米药物递送系统注射剂尾静脉注射进小鼠皮下肺部肿瘤模型上述两种模型小鼠体内，将上述配置好的实施例2的纳米药物递送系统注射剂尾静脉注射进大鼠胶原诱导关节炎模型体内，在665nm波长激光激发下对其进行荧光成像，治疗周期结束后，评估其治疗效果
由图7中看到，对于小鼠皮下肺肿瘤的成像，在3小时后，纳米探针材料开始在肿瘤、肺部以及肝肾等部位富集，之后随着时间增加，荧光强度开始增强，一直到24h都没有衰减。同样在图9中，对于大鼠类风湿关节炎模型的成像，可以看到在12h后纳米探针材料开始在肿瘤部位与肝肾部位慢慢富集，在24h达到最大。对于其治疗效果，从图8中可以看出，其在注射后肿瘤大小得到明显抑制。而图10结果显示其较好的类风湿性关节炎治疗效果。综上，本发明的蛋白纳米药物递送系统材料在快速诊断治疗肿瘤及类风湿关节炎都具有很好的应用前景。
4、高等电点蛋白纳米药物递送系统体内半衰期测定。
打药后1,2，4,6,12,24,48,96,148H后取全血，37℃静置30min，之后离心，取上层血清检测其中药物含量，从图11中可以看到，相比于单独乌帕替尼药物，包裹后的乌帕替尼的半衰期及生物利用度显著增高。综上，本发明的蛋白纳米药物递送系统材料在快速诊断治疗肿瘤及类风湿关节炎都具有很好的应用前景。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

1.一种高等电点蛋白，其特征在于，具有如SEQ ID NO.1所示的氨基酸序列；或具有在SEQ ID NO.1所示氨基酸序列中取代、缺失或添加氨基酸且蛋白活性不发生改变的氨基酸序列。
2.根据权利要求1所述的高等电点蛋白，其特征在于，所述高等电点蛋白由原核表达系统制备获得。
3.一种纳米蛋白复合物，其特征在于，由权利要求1或2所述的高等电点蛋白、胆绿素和阴离子表面活性剂组成。
4.根据权利要求2所述的纳米蛋白复合物，其特征在于，所述高等电点蛋白和阴离子表面活性剂的摩尔比为1:360-1:720。
5.根据权利要求1～4任一项所述的纳米蛋白复合物，其特征在于，所述阴离子表面活性剂包括羧基聚乙二醇、硫酸软骨素中的一种或两种。
6.权利要求5所述的纳米蛋白复合物的制备方法，其特征在于，包括：
将高等电点蛋白与胆绿素混合，获得蛋白复合物；
将所述蛋白复合物和阴离子表面活性剂在水中进行混合，经自组装获得纳米蛋白复合物。
7.一种纳米药物递送系统，其特征在于，包括权利要求3～5任一项所述的纳米蛋白复合物和包裹在所述纳米蛋白复合物中的药物。
8.根据权利要求7所述的纳米药物递送系统，其特征在于，所述药物为脂溶性药物，包括硫链丝菌素、PMI抗癌肽、乌帕替尼中的至少一种。
9.权利要求7或8所述的纳米药物递送系统，其特征在于，其粒径为180-200nm。
10.权利要求3～5任一项所述的纳米蛋白复合物或权利要求7～9任一项所述的纳米药物递送系统在制备治疗和/或预防肿瘤、类风湿关节炎的药物中的应用。
11.根据权利要求10所述的应用，其特征在于，所述肿瘤包括乳腺肿瘤、转移性肝癌、黑色素瘤、肺癌中的至少一种。
12.权利要求3～5任一项所述的纳米蛋白复合物或权利要求7～9任一项所述的纳米药物递送系统在制备生物医学成像产品中的应用。
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