CN107417769A - 一种介导药物递送的新型穿膜肽及其应用 - Google Patents
一种介导药物递送的新型穿膜肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种新型穿膜肽，可以高效介导药物递送至细胞内，同时亦提供了该发明在肿瘤治疗中的应用。
Description
技术领域
本发明涉及生物技术领域，更具体地，涉及一种穿膜肽以及该穿膜肽在药物递送和肿瘤治疗中的应用。
背景技术
恶性肿瘤，即通常所指的癌症是人体细胞不正常增生，且这些增生的细胞可能侵犯身体毗邻部位和扩散到其它器官并最终致死。肿瘤已成为威胁人类生命的最严重的疾病之一。在现行临床治疗方法中，利用化学药物杀伤肿瘤细胞并抑制肿瘤细胞增殖的化学治疗是主要手段。然而，许多肿瘤治疗药物都不能自由穿透细胞膜，故而治疗药物的递送过程成为化学治疗的关键环节。
在目前药物递送过程主要有两大方式，一是纳米给药体系，包括脂质体、聚合物纳米颗粒、胶束等等，其药物递呈主要通过胞吞来实现。但目前此类药物递送依然有包裹效率较低；生物相容性较差；靶向性分子和纳米药物的连接效率较低以及连接后活性的降低等问题。更重要该方法通过胞吞作用实现递呈，药物分子由于内吞而形成胞内体，继而须从胞内体中逃逸出来才能发挥药效，导致药物释放困难；同时，药物从胞内体释放需要溶酶体，影响药效；此外，形成的胞内体亦有可能被细胞外排，削弱递送。而另一种则是通过理化机械递送将被转运物质直接导入细胞质或细胞核的方法，例如电穿孔和显微注射等方法。但普遍存在导入效率低、难于推广和易造成细胞损伤甚至死亡等问题。总言之，目前仍缺乏一种高效可行的治疗药物直接导入的方法。
细胞穿膜肽又称为蛋白转导域或膜转导肽，因为其无毒且高效的穿膜就被认为是最具前景的胞内有效转导的工具。多数穿膜肽由30个以下残基组成，具有净正电荷性，其穿膜浓度在mM级别以下。通过CPPs介导，可以使一些本身膜不可透过性的分子通过细胞膜进入胞质,甚至进入细胞核。目前，穿膜肽主要的穿膜机制假说有直接穿膜和胞吞穿膜。前者指穿膜肽不经胞吞作用，不形成胞内体直接穿膜；或直接与细胞膜相互作用成孔穿膜。而后者的过程与脂质体类似，通过与细胞膜相互作用内吞穿膜。尽管穿模机制依然尚不完全明确，但显然确有部分穿膜肽的递送过程是不依赖胞吞作用的直接穿膜，所以药物递送过程中通过CPPs的直接转运是最理想的。
发明内容
本发明的目的在于提供一种可以高效介导药物递送的新型穿膜肽。
本发明所述穿膜肽其特征在于，所述亲和肽的氨基酸序列如SEQ ID No.1和2中的任一项：
Arg-Arg-Arg-Arg-Arg-Trp-Trp(SEQ ID No:1),
Arg-Arg-Arg-Arg-Arg-Trp-Trp-Pro-Pro(SEQ ID No:2)。
本发明所述穿膜肽可以与药物小分子非共价作用相互结合，介导药物递送至细胞内。
本发明所述穿膜肽可介导的药物选自下组：丝裂霉素、柔红霉素、顺铂、卡铂、喜树碱、博莱霉素、5-氟尿啶、环磷肽酰、吉西他滨、甲氨蝶呤、卡培他滨、洛莫司汀、紫杉醇、依托泊苷和阿霉素。
本发明与现有技术相比，该穿膜肽分子量小，免疫原性低；其残基序列取自20种必需氨基酸，安全无毒。穿膜肽由固相合成而得，便于质量管控。
本发明与现有技术相比，该穿膜肽突出的优点是能够高效介导药物递送至细胞内。在肿瘤治疗中，该穿膜肽可以将抗肿瘤药物高效导入肿瘤细胞，其抑制效果较单纯给药组大幅提高。同时在诊断和检测领域的药物递送过程应用潜力广泛。
附图说明
附图是对本发明提供进一步说明解释，但并不构成对本发明的限制。
图1为本发明穿膜肽对小鼠神经成纤维细胞毒性试验结果显示图。
图2为本发明中的穿膜肽FITC荧光标记后，孵育宫颈癌(hela)细胞，荧光检测成像图。
图3为本发明中的穿膜肽介导抗肿瘤药物紫杉醇进入宫颈癌、肺癌和肝癌细胞的抑制结果。
图4为本发明中的穿膜肽介导抗肿瘤药物紫杉醇递送治疗肿瘤的结果。
具体实施方式
以下是对本发明的具体实施方式进行详细说明，仅用于对本发明提供进一步说明解释，但并不构成对本发明的限制。
实施例1：穿膜肽合成
1)活化树脂：称取1000mg Fmoc-Trp wang Resin或Fmoc-Pro wang Resin，加入10-15ml(浸没全部树脂)DMF浸泡30min，使其充分溶胀。
2)脱保护：浸泡树脂的DMF压滤除去，加入10ml含有20％哌啶的DMF溶液，氮气吹沸反应15min，然后压滤除去，脱除氨基的FMOC基团，用10ml异丙醇洗涤树脂三次，再用10ml DMF洗涤三次，而后用茚三酮法检测树脂应成黑色或紫色。
3)缩合反应：连接下一个氨基酸，称取Fmoc-氨基酸的用量是1.4mmol/g树脂，910mg TBTU，加入10ml DMF和0.45g HOBt混和均匀后，加入0.52mlDIEA配成反应液，室温下氮气吹沸反应2h。反应完毕后，用异丙醇洗涤树脂三次，然后用DMF洗涤树脂三次。检测氨基。
4)重复步骤2)-3)过程：按多肽的顺序自C端向N端延伸多肽。重复脱保护.洗涤.缩合的过程至剩余的氨基酸连接完毕，完成多肽的连接。
5)多肽切割：用氮气将多肽-树脂复合物吹干，按TFA/phenol/H20/thioanisole/EDT/TIS(80/5/5/5/3/2)的比例配成混和切割试剂。将肽树脂置于圆底烧瓶中，加入切割液磁力搅拌3小时，用200目砂芯过滤器除去树脂，滤液直接抽入冷冻乙醚中，3000r/min离心使粗肽沉淀，冻干至恒重即得粗肽，干燥称重。
6)粗肽用HPLC纯化。
所合成的肽的序列为Arg-Arg-Arg-Arg-Arg-Trp-Trp。
实施实例2：细胞毒性实验
1)取96孔板，每孔加入含有7×103个细胞培养液，37℃，5％二氧化碳培养箱24小时，使细胞贴壁。
2)配置含不同浓度的穿膜肽的培养基，以无穿膜肽的培养基为阴性对照孔(Control)，37℃，5％二氧化碳培养1-5h。
3)贴壁细胞每孔加入20μl MTT，继续孵育24h之后弃掉培养液，每孔加入150μl DMSO(二甲基亚砜)，震荡10min。
4)选择490nm或570nm波长，在酶标仪免疫检测仪上通过光吸收值计算细胞存活率。结果见图1。
结果显示该穿膜肽无明显细胞毒性。
实施实例3：穿膜肽穿膜暨荧光检测实验
1)在贴壁细胞中加入含有一定浓度FITC荧光标记的穿膜肽的培养基，孵育30min-4h后小心吸去培养基，并PBS洗涤贴壁细胞三次。
2)加入固定液，10min-20min，固定细胞后PBS洗涤细胞2-3次。
3)加入DAPI染色液染色5min-10min后PBS洗涤2-3次。
4)分别在488nm和543nm的激发光下用荧光显微镜观察，结果见图2。
结果显示该穿膜肽成功穿膜并将荧光标志物递送于细胞内。
实施实例4：介导抗肿瘤药物紫杉醇递送实验
1)加入含有5微克/毫升紫杉醇和一定浓度穿膜肽的培养基孵育宫颈癌(Hela)、肝癌和肺癌细胞，孵育30min后小心吸去培养基，并PBS洗涤贴壁细胞三次。以无穿膜肽和紫杉醇的培养基为基准，同时设置仅含有5微克/毫升紫杉醇的培养基作为阴性对照(Control)。
2)加入Hela细胞培养基培养24h。
3)Hela细胞每孔加入20μl MTT，继续孵育3h之后弃掉培养液，每孔加入150μl DMSO(二甲基亚砜)，震荡10min。
4)选择490nm或570nm波长，在酶标仪免疫检测仪上通过光吸收值计算细胞存活率。结果见图3。
结果显示该穿膜肽成功将紫杉醇递送入细胞内，加入穿膜肽后肿瘤细胞存活率明显下降，肿瘤细胞抑制率明显上升。
实施实例5：介导抗肿瘤药物紫杉醇递送治疗肿瘤实验
1)接种Hela细胞于无胸腺裸鼠体内至肿瘤体积50mm3。
2)每日原位注射一定量含有5微克/毫升紫杉醇和一定浓度穿膜肽的无菌注射液，同时注射仅含紫杉醇(Control)和无任何药物(Blank)的无菌注射液作为阴性对照。
3)一月后杀鼠取瘤，测量瘤体积。结果见图4。
结果显示单纯的紫杉醇组治疗效果不明显，而穿膜肽则成功将紫杉醇递送入肿瘤细胞内，其瘤体积较对照组明显下降，治疗效果显著。
申请人声明，本发明通过上述实施例来说明本发明的详细特征以及方法，但本发明并不局限于上述详细特征以及方法，即不意味着本发明必须依赖上述详细特征以及方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用材料和步骤的等效替换以及辅助材料和步骤的增加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
Claims (6)
1.其特征在于，所述亲和肽的氨基酸序列如SEQ ID No.1和2中的任一项所示。
2.根据权利要求1所述的穿膜肽，其特征在于所述穿膜肽与药物小分子非共价作用相互结合，介导药物递送至细胞内。
3.根据权利要求1所述的穿膜肽，其特征在于所述穿膜肽可介导的药物选自下组：丝裂霉素、柔红霉素、顺铂、卡铂、喜树碱、博莱霉素、5-氟尿啶、环磷肽酰、吉西他滨、甲氨蝶呤、卡培他滨、洛莫司汀、紫杉醇、依托泊苷和阿霉素。
4.根据权利要求1所述的穿膜肽在药物递送中的应用。
5.根据权利要求1所述的穿膜肽在诊断和检测中的应用。
6.根据权利要求1所述的穿膜肽在肿瘤治疗中的应用。
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| CN109608519A (zh) * | 2018-11-06 | 2019-04-12 | 南阳师范学院 | 一种穿膜肽及其应用 |
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| CN109999202B (zh) * | 2019-01-18 | 2022-03-22 | 南阳师范学院 | 一种介导紫杉醇递送的多功能肽及其应用 |
| CN114835775A (zh) * | 2022-02-24 | 2022-08-02 | 南阳师范学院 | 一种新型细胞穿膜肽及其应用 |
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