CN108210883B - 一种基于蜂毒肽的纳米试剂及其制备方法和应用 - Google Patents
一种基于蜂毒肽的纳米试剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于蜂毒肽的纳米试剂及其制备方法和应用，该纳米试剂由蜂毒肽、有机染料和透明质酸制备而成。本发明的纳米试剂制备简单，能够极大地降低蜂毒肽的溶血性，并且可以实现体内的肿瘤荧光成像和显著的抗肿瘤疗效。本发明的纳米试剂在静脉注射入体内后能够有效富集在肿瘤区域，并且能够通过荧光信号跟踪其在体内的分布情况，除了蜂毒肽的化疗抗癌活性之外，该纳米试剂中的有机染料成分可以对激光照射产生响应并进一步提高抗癌效果，从而实现化疗及光疗联合抗癌的应用。此外，该试剂的生物安全性良好，可以广泛应用于制备体内癌症诊疗一体化药物，实现蜂毒肽的体内治疗应用。
Description
技术领域
本发明属于生物材料领域，涉及一种基于蜂毒肽的纳米试剂，具体涉及一种基于蜂毒肽的癌症诊疗纳米试剂及其制备方法和应用。
背景技术
蜂毒肽(melittin)是一种来源于蜜蜂毒液中的细胞溶解性多肽。大量研究表明，蜂毒肽具有极强的药理作用和生物学活性，在抗炎、阵痛、抗微生物、抗病毒及抗肿瘤等诸多领域都具有潜在的临床应用价值。尤其在癌症治疗方面，蜂毒肽与传统的化疗药物相比更不易使癌细胞产生耐药性。然而，蜂毒肽在经静脉注射到体内后会引起严重的溶血反应，并且会对正常组织造成损伤，因此蜂毒肽的体内应用一直受到很大的限制。近年来，随着纳米技术的不断发展，包括高分子纳米颗粒、脂质体和纳米乳液在内的许多纳米材料被用来装载蜂毒肽来降低其在给药过程中产生的生物毒性。此外，一些研究组还将纳米载体表面进行靶向分子修饰，实现了蜂毒肽的体内肿瘤靶向运输。然而，目前的蜂毒肽载药体系还存在以下缺陷：首先，许多装载有蜂毒肽的纳米颗粒稳定性不足，可能会存在蜂毒肽泄露和潜在的溶血问题；其次，大部分蜂毒肽递送体系都没有荧光，因此不能示踪蜂毒肽在生物体内的分布和富集情况；更重要的是，这些装载在纳米载体内的蜂毒肽与游离蜂毒肽相比，抗癌疗效大大降低，所需的杀死癌细胞的剂量成倍增加。为了实现蜂毒肽的体内应用，开发一种稳定性高，抗癌疗效好、具有肿瘤靶向且能够荧光示踪的蜂毒肽载药体系至关重要。
发明内容
发明目的：为了解决上述技术问题，本发明提供了一种基于蜂毒肽的纳米试剂。该纳米试剂制备简单，血液相容性良好且抗癌活性高。在静脉注射入体内后能够有效富集在肿瘤区域，并且能够通过荧光信号跟踪其在体内的分布情况。另外，除了蜂毒肽的化疗抗癌活性之外，该纳米试剂中的有机染料成分可以对激光照射产生响应(比如产生单线态氧或热等)并进一步提高抗癌效果，从而实现化疗及光疗联合抗癌的应用。
技术方案：为了实现上述目的，本发明公开了一种基于蜂毒肽的纳米试剂，由蜂毒肽、有机染料和透明质酸自组装构成。
其中，所述的蜂毒肽氨基酸序列为SEQ ID NO.1：GIGAVLKVLTTGLPALISWIKRKRQQ。
作为优选，所述有机染料为二氢卟吩e6、碳菁染料Cypate、原卟啉或5,10,15,20-四(4-羧基苯基)卟啉。
作为优选，所述的透明质酸重均分子量为3500-1500000。
更进一步地，所述的透明质酸重均分子量为200000-1000000。最优选的，所述的透明质酸重均分子量为700000。
本发明所述的纳米试剂的制备方法，包括以下步骤：
(1)将蜂毒肽的水溶液与有机染料的水溶液等体积混合，水浴超声后，室温下搅拌过夜；反应结束后，经超纯水透析纯化，形成复合物水溶液；
(2)将步骤(1)制得的复合物水溶液逐滴滴加至透明质酸溶液中，并在室温下搅拌过夜得到基于蜂毒肽的纳米试剂。
作为优选，所述蜂毒肽、有机染料和透明质酸的质量比为1：(2-10)：(1-10)。
本发明所述的纳米试剂在制备癌症诊疗药物中的应用。
其中，所述癌症诊疗药物包括体内肿瘤荧光成像制剂。
进一步地，所述癌症诊疗药物包括化疗和光疗联合治疗药物。
本发明所述的纳米试剂由蜂毒肽、有机染料和透明质酸自组装构成。其中，蜂毒肽可以与有机染料通过疏水相互作用形成纳米复合物，这种纳米复合物能够有效降低蜂毒肽的溶血性。在对该纳米复合物表面进行透明质酸包覆后，其溶血性得到进一步降低。由于透明质酸能够特异性识别癌细胞表面过量表达的透明质酸受体，这种纳米试剂具有较强的肿瘤靶向性。在经静脉注射进入体内后，该纳米试剂能够有效富集在肿瘤区域，并实现肿瘤区域的荧光成像。在光照条件下，这种纳米试剂能够实现化疗和光疗的联合疗法并抑制肿瘤生长，从而实现了体内的癌症诊疗一体化。
有益效果：相对于现有技术，本发明具有以下优势：
(1)极低的溶血性：该纳米试剂即使在较高的浓度下对红细胞的溶血也很低，极大提高了蜂毒肽的血液相容性；
(2)肿瘤靶向性：本发明所述的纳米试剂能够特异性识别肿瘤细胞表面过量表达的透明质酸受体，具有主动靶向能力；
(3)体内荧光示踪：传统的蜂毒肽纳米试剂一般不具有荧光，不能示踪纳米颗粒在体内的分布。本发明所述的纳米试剂包含有机荧光染料，因此可以实现体内肿瘤区域的荧光成像；
(4)体内抗癌效果明显：本发明所述的纳米试剂整合了蜂毒肽的化疗效果以及有机染料的光疗效果，实现了体内联合抗癌疗法；
(5)毒副作用低：本发明所述的纳米试剂生物安全性良好，几乎无全身毒性。
(6)本发明的纳米试剂制备简单，原料来源广，可以广泛应用于制备体内的癌症诊疗一体化药物。
附图说明
图1是本发明的纳米试剂制备示意图；
图2是本发明试剂对红细胞的溶血评价结果；
图3是本发明试剂对人肺癌细胞的毒性评价结果；
图4是本发明试剂在荷瘤小鼠体内的荧光成像图；
图5是本发明试剂在荷瘤小鼠体内的抗肿瘤结果。
具体实施方式
下面结合附图和具体对本发明作进一步说明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
以下实施例中，原料蜂毒肽(melittin)、二氢卟吩e6(Ce6)、碳菁染料Cypate、原卟啉、5,10,15,20-四(4-羧基苯基)卟啉以及透明质酸(HA)均可以通过市售获得。
实施例1
基于蜂毒肽的癌症诊疗纳米试剂合成过程如下：
步骤1：将1mL的蜂毒肽水溶液(1mg/mL)与1mL的Ce6水溶液(2mg/mL)混合，水浴超声5分钟，室温下搅拌过夜；随后用截留分子量为10kDa的透析袋在超纯水中进行透析纯化；
步骤2：将上述得到的复合物水溶液逐滴滴加至6mL的HA水溶液中(0.5mg/mL)，HA的重均分子量为700000，并在室温下搅拌过夜，得到基于蜂毒肽的纳米试剂。
上述方法所得的试剂记为melittin/Ce6@HA，其制备过程示意图见图1。
实施例2
基于蜂毒肽的癌症诊疗纳米试剂合成过程如下：
步骤1：将1mL的蜂毒肽水溶液(1mg/mL)与1mL的碳菁染料Cypate水溶液(3mg/mL)混合，水浴超声5分钟，室温下搅拌过夜；随后用截留分子量为10kDa透析袋在超纯水中进行透析纯化；
步骤2：将上述得到的复合物水溶液逐滴滴加至6mL的HA水溶液中(0.5mg/mL)，HA的平均分子量为3500，并在室温下搅拌过夜，得到基于蜂毒肽的纳米试剂。
实施例3
基于蜂毒肽的癌症诊疗纳米试剂合成过程如下：
步骤1：将1mL的蜂毒肽水溶液(1mg/mL)与1mL的原卟啉水溶液(10mg/mL)混合，水浴超声5分钟，室温下搅拌过夜；随后用截留分子量为10kDa的透析袋在超纯水中进行透析纯化；
步骤2：将上述得到的复合物水溶液逐滴滴加至6mL的HA水溶液中(1mg/mL)，HA的平均分子量为1500000，并在室温下搅拌过夜，得到基于蜂毒肽的纳米试剂。
实施例4
基于蜂毒肽的癌症诊疗纳米试剂合成过程如下：
步骤1：将1mL的蜂毒肽水溶液(1mg/mL)与1mL的5,10,15,20-四(4-羧基苯基)卟啉(10mg/mL)混合，水浴超声5分钟，室温下搅拌过夜；随后用截留分子量为10kDa的透析袋在超纯水中进行透析纯化；
步骤2：将上述得到的复合物水溶液逐滴滴加至5mL的HA水溶液中(2mg/mL)，HA的平均分子量为200000，并在室温下搅拌过夜，得到基于蜂毒肽的纳米试剂。
实施例5
将实施例1中Ce6水溶液的浓度变为3mg/mL，HA水溶液的浓度变为1.5mg/mL，HA的平均分子量为1000000，其他反应参数与实施例1中的相同。
实施例6
将实施例1中Ce6水溶液的浓度变为2mg/mL，HA水溶液取5mL浓度变为0.2mg/mL，其他反应参数与实施例1中的相同。
实施例7
评价实施例1所制得的melittin/Ce6@HA对红细胞的溶血行为，其方法如下：
取1mL来源于小鼠的新鲜血液并离心获得红细胞，并用磷酸缓冲液清洗3次。在1.5mL离心管中配置不同浓度的melittin溶液和melittin/Ce6@HA溶液，每个离心管中溶液体积为300μL。随后，向上述各管溶液中加入10μL的红细胞悬液并混合均匀，于室温下放置2小时。孵育结束后，将红细胞离心并收集上清溶液，利用紫外分光光度计测取在540nm处的吸光度值。该实验的阴性和阳性对照分别是将红细胞与磷酸缓冲液和纯水孵育得到的。最后，根据以下公式计算溶血率：
溶血率(％)＝(实验组吸光度值－阴性对照吸光度值)/(阳性对照吸光度值-阴性对照吸光度值)*100％
结果见图2，由图可见，游离蜂毒肽具有极强的溶血能力，在5μM左右就能够使红细胞完全溶破。而实施例1所制得的纳米试剂即使在40μM时也几乎没有溶血产生，证明其具有很好的血液相容性。
实施例8
评价实施例1所制得的melittin/Ce6@HA的细胞暗毒性，步骤如下：将A549细胞按5×104个/mL的密度接种于96孔板中，培养24小时后，再与不同浓度的蜂毒肽和melittin/Ce6@HA避光孵育24小时，最后利用MTT检测法评价该探针的细胞毒性，结果见图3。实验结果表明，该纳米试剂具有跟蜂毒肽同样的抗癌活性。
实施例9
观察实施例1所制得的纳米试剂在小鼠体内的分布情况，步骤如下：取健康状况良好的4周龄BALA/c无胸腺的雌性裸鼠若干只，并在其皮下注射鼠源宫颈癌细胞(U14)。当肿瘤体积达到100mm3左右时，将实施例1所得的纳米试剂通过尾静脉注射的方式注射到裸鼠体内。然后将裸鼠经异氟烷气体麻醉后放入成像暗箱平台，在小动物活体成像系统中用600nm激发光激发并接收670nm波段的发射光，曝光时间为750ms。分别在不同时间点(0、2、6、12和24小时)采集成像数据，并进行数据分析。实验结果见附图4a至图4g。
由图4可见，实施例1所得纳米试剂能够显著地富集到肿瘤区域，并可实现优异的体内荧光成像。
实施例10
评价实施例1所制得的纳米试剂对荷瘤小鼠的肿瘤抑制能力，步骤如下：当肿瘤体积达到50mm3左右时，将实施例1所得光疗纳米制剂通过尾静脉注射的方式注射到裸鼠体内，注射完成4小时后，用670nm的红色激光照射肿瘤区域，激光功率为20mW/cm2，照射时间为20分钟。随后每隔一天测量肿瘤体积，肿瘤体积＝0.5×肿瘤长度×肿瘤宽度2，结果见图附图5。
实验结果表明，实施例1所制得的纳米试剂能够有效抑制肿瘤的生长。
序列表
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<120> 一种基于蜂毒肽的纳米试剂及其制备方法和应用
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Claims (9)
1.一种基于蜂毒肽的纳米试剂，其特征在于，由蜂毒肽、有机染料和透明质酸制备而成，所述有机染料为二氢卟吩e6、碳菁染料Cypate、原卟啉或5,10,15,20-四（4-羧基苯基）卟啉。
2.根据权利要求1所述的纳米试剂，其特征在于，所述的蜂毒肽氨基酸序列为SEQ IDNO.1。
3.根据权利要求1所述的纳米试剂，其特征在于，所述的透明质酸重均分子量为3500-1500000。
4.根据权利要求3所述的纳米试剂，其特征在于，所述的透明质酸重均分子量为200000-1000000。
5.一种权利要求1-4任一项所述的纳米试剂的制备方法，其特征在于，包括以下步骤：
（1）将蜂毒肽的水溶液与有机染料的水溶液等体积混合，水浴超声后，室温下搅拌过夜；反应结束后，经超纯水透析纯化，形成复合物水溶液；
（2）将步骤（1）制得的复合物水溶液逐滴滴加至透明质酸水溶液中，并在室温下搅拌过夜得到基于蜂毒肽的纳米试剂。
6.根据权利要求5所述的制备方法，其特征在于，所述蜂毒肽、有机染料和透明质酸的质量比为1：2-10：1-10。
7.一种权利要求1-4任一项所述的纳米试剂在制备癌症诊疗药物中的应用。
8.根据权利要求7所述的应用，其特征在于，所述癌症诊疗药物包括体内肿瘤荧光成像制剂。
9.根据权利要求7所述的应用，其特征在于，所述癌症诊疗药物包括化疗和光疗联合治疗药物。
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