CN109999204A - 用于靶向投递疏水性抗肿瘤药物的小肽及其应用 - Google Patents

用于靶向投递疏水性抗肿瘤药物的小肽及其应用 Download PDF

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CN109999204A
CN109999204A CN201910293389.6A CN201910293389A CN109999204A CN 109999204 A CN109999204 A CN 109999204A CN 201910293389 A CN201910293389 A CN 201910293389A CN 109999204 A CN109999204 A CN 109999204A
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吴国球
蒋兴禄
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Southeast University
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Abstract

用于靶向投递疏水性抗肿瘤药物的小肽及其应用，氨基酸序列如SEQ ID NO.1所示。本发明通过在疏水性抗肿瘤药物的激发下自组装形成均一性，分散性，稳定性等较好的球形纳米颗粒，并能够将该疏水性抗肿瘤药物靶向的投递至肿瘤细胞组织，并在特定的肿瘤细胞内环境中释放，高密度的富集在肿瘤细胞核，最终高效的杀死肿瘤细胞，降低抗肿瘤药物副作用，实现靶向治疗。

Description

用于靶向投递疏水性抗肿瘤药物的小肽及其应用
技术领域
本发明属于生物医药技术领域，具体涉及一种用于靶向投递疏水性抗肿瘤药物的小肽及其应用。
背景技术
据世界卫生组织报道，恶性肿瘤目前是全球第二大死因，全球约六分之一的死亡由其造成；而且近几年它的发病率还在不断增高。随着对恶性肿瘤的生物特性认识不断深入，各种新治疗手段也达到有效的推广，如靶向治疗，免疫治疗等；但是化学治疗仍是目前最主要的治疗。化学治疗药物的原理通常是借由干扰细胞分裂的机制来抑制癌细胞的生长，譬如抑制DNA复制或是阻止染色体分离。虽然恶性肿瘤与正常细胞最大的差异在于快速的细胞分裂及生长，但是目前大多数药物都缺乏专一性，同样会杀死处于细胞分裂的正常组织细胞造成不同程度的毒副作用，特别是人体中生长发育旺盛的血液、淋巴组织细胞等。
药物递送系统（Drug Delivery System，DDS）是指在空间、时间及剂量上全面调控药物在生物体内分布的技术体系，从而增加药物的利用效率，提高疗效，降低成本，减少毒副作用。这为恶性肿瘤的化学治疗提供了一个新的途径。基于纳米颗粒的药物递送系统由于其独特的药代动力学和生物学特征，更是激起了广大科研工作的兴趣。然而，常见的纳米材料不仅价格昂贵、非治疗成分众多，而且在生物可降解性、长期毒性等方面的作用尚待观察；同时，在载药纳米制备（如成膜、水化、载药等）过程中存在诸多影响因素，操作极其繁琐复杂，造成纳米粒径、Zeta电位及载药率批间差异极大，给该制剂的规模化制备及相应质量标准的制定带来极大困难，限制了其应用和发展，因此，寻找成分相对单一，生物安全性高，制备简便快速，易于纳米药物规模化制备及质量控制的新型纳米材料成为研究热点之一。
天然肽材料（尤其人源肽）由于具有高生物相容性、无毒、无免疫源性等优点成为纳米药物递送系统研究的热点。而且改变氨基酸序列和修饰特殊的基团可使多肽具有新的功能，如实现对肿瘤的主动靶向，穿越生物屏障，更好的转载特殊药物，具有某种生物活性等。因此多功能化的多肽研究吸引很多研究者的兴趣。
发明内容
解决的技术问题：本发明设计合成一条用于靶向投递疏水性抗肿瘤药物的小肽及其应用，通过在疏水性抗肿瘤药物的激发下自组装形成均一性，分散性，稳定性等较好的球形纳米颗粒，并能够将该疏水性抗肿瘤药物靶向的投递至肿瘤细胞组织，并在特定的肿瘤细胞内环境中释放，高密度的富集在肿瘤细胞核，最终高效的杀死肿瘤细胞，降低抗肿瘤药物副作用，实现靶向治疗。
技术方案：用于靶向投递疏水性抗肿瘤药物的小肽，氨基酸序列如SEQ ID NO.1所示。
上述小肽在制备用于靶向投递疏水性抗肿瘤药物中的应用。
具体制备方法为：首先将小肽溶液和抗肿瘤药物混合后超声处理，室温静置至少2小时，然后离心收集上清液即得。
上述抗肿瘤药物为阿霉素、吡柔比星或紫杉醇。
有益效果：1. 人源肽天然有高生物相容性、无毒、无免疫源性；2. 在人源肽上设计靶向序列，释放序列，组装序列等多个功能，实现高效负载药物并靶向递送至恶性肿瘤细胞组织，在恶性肿瘤细胞内的特定环境下释放，如酸性环境、GSH、MMP2等作用下释放，提高药物靶向作用，更好的降低药物副作用。3.人源肽通过自组装作用装载疏水性抗肿瘤药物,如吡柔比星，阿霉素、紫杉醇等，提高疏水性药物溶解性，为疏水性药物运用提供理论支撑。
附图说明
图1为药物投递系统的电镜图，可以清晰见中间吡柔比星被组装在球形纳米颗粒，大小为50nm左右。
图2为动态光散射粒径分析仪（DLS）分析药物投递系统的粒径大小图，同电镜结果基本相似。
图3为药物投递系统在不同pH环境下，GSH以及MMP作用下后的电泳分析结果图，和对照组比较，可以明显发现位置和大小有明显变化，在酸性情况下位置更下；在GSH以及MMP作用下出现两条明显条带，说明小肽被GSH以及MMP切断。
图4为通过检测吡柔比星荧光强度分析其释放率图，在不同时间点测定荧光强度分析和计算药物投递系统释放率。
图5为CCK-8检测药物投递系统中的吡柔比星对乳腺癌细胞MDA-231的杀伤作用图，吡柔比星浓度分别为0.625，1.25，2.5，5.0，10.0 μg/mL，对照细胞为正常细胞人肾上皮细胞系293T。
图6为本发明作用机制示意图。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1：小肽自组装同时装载吡柔比星，并进行表征
设计和合成人源肽的序列如下：
Arg-Gly-Asp-Ser-Glu-Glu-Asp-Pro-Cys-s-s-Cys-Glu-Ser-Pro-Val-Gly-Leu-Ile-Gly-Gly-Leu-Val-Lys-Phe-Gln-Ala-Lys-Val-Glu-Gly-Leu-Leu-Gln-Ala-Leu-Thr-Arg-Lys-Leu-Glu-Ala-Val-Ser-Lys-Arg-Leu-Ala-Ile-Leu-Glu-Asn-Thr
合成方法：首先将配制1mg/mL的小肽溶液和称取1mg/mL的抗肿瘤药物吡柔比星（Pirarubicin，C32H37NO12）。两者混合涡旋1min后在超声（100w）作用下继续反应30min，再继续室温静置2h让其充分自组装并让未结合的吡柔比星沉淀下来，最后10000转离心5min收集上清液即可。
表征：运用透射电镜，动态光散射粒径分析仪（DLS）研究药物投递系统形态外貌及粒径大小，发现其为球形颗粒，直径约为50nm左右。
实施例2：药物投递系统中吡柔比星释放
释放环境验证：运用不同酸性环境，GSH以及MMP-2作用于药物投递系统，通过检测吡柔比星荧光强度分析其释放，通过电泳分析小肽的大小来判断其小肽断裂情况。荧光测试结果显示在该药物投递系统对酸性环境最为敏感，释放率约为40%；在GSH以及MMP-2存在的情况下也能释放，效率约为10%。在三者同时存在的情况下，释放效率可以达到60%。
实施例3：药物投递系统对肿瘤细胞的高效杀伤
运用CCK-8检测药物投递系统中的吡柔比星对乳腺癌细胞MDA-231的杀伤作用，设计运用正常细胞人肾上皮细胞系293T作为对照。药物投递系统中的吡柔比星的浓度分别是0.625，1.25，2.5，5.0，10.0 μg/mL。处于低浓度水平的时候，相同浓度的作用情况下，吡柔比星对乳腺癌细胞MDA-231的杀伤作用更为明显。
实施例4：人源肽通过自组装作用装载其他疏水性抗肿瘤药物，阿霉素和紫杉醇
运用实施例1同样的方法，人源肽仍然可以通过自组装作用装载阿霉素、紫杉醇等疏水性抗肿瘤药物。验证该人源肽是疏水性抗肿瘤药物的很好载体，为该类药物体内输送提供一个可行的载体，为肿瘤靶向治疗提供理论和技术支持。
序列表
<110> 东南大学
<120> 用于靶向投递疏水性抗肿瘤药物的小肽及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 51
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Gly Asp Ser Glu Glu Asp Pro Cys Cys Glu Ser Pro Val Gly Leu
1 5 10 15
Ile Gly Gly Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu Leu Gln
20 25 30
Ala Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Arg Leu Ala Ile Leu
35 40 45
Glu Asn Thr
50

Claims (4)

1.用于靶向投递疏水性抗肿瘤药物的小肽，其特征在于氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述小肽在制备用于靶向投递疏水性抗肿瘤药物中的应用。
3.权利要求2所述的应用，其特征在于制备方法为：首先将小肽溶液和抗肿瘤药物混合后超声处理，室温静置至少2小时，然后离心收集上清液即得。
4.根据权利要求2所述的应用，其特征在于抗肿瘤药物为阿霉素、吡柔比星或紫杉醇。
CN201910293389.6A 2019-04-12 2019-04-12 用于靶向投递疏水性抗肿瘤药物的小肽及其应用 Pending CN109999204A (zh)

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Publication number Priority date Publication date Assignee Title
CN110655559A (zh) * 2019-10-23 2020-01-07 东南大学 用于构建肿瘤pH响应微型机器人的人源性多肽及其应用
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