CN113144171A - 一种氧化响应形貌转变多肽纳米药物 - Google Patents
一种氧化响应形貌转变多肽纳米药物 Download PDFInfo
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- CN113144171A CN113144171A CN202110316337.3A CN202110316337A CN113144171A CN 113144171 A CN113144171 A CN 113144171A CN 202110316337 A CN202110316337 A CN 202110316337A CN 113144171 A CN113144171 A CN 113144171A
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Abstract
本发明涉及一种氧化响应形貌转变多肽纳米药物。基于含有甲硫氨酸并且能够发生活性氧响应形貌转变的多肽序列EIMIME,分别合成了含有光敏剂或化疗药物的多肽衍生物,通过共组装的方式制备多肽纳米药物。形貌表征及氧化响应性能测试表明该多肽纳米药物能够在激光照射下产生活性氧,原位产生的活性氧一方面起到光动力治疗的作用,另一方面能够氧化多肽中的甲硫氨酸,进而促使纳米药物的组装形貌发生转变。本发明的优点是:该多肽纳米药物具有较高的肿瘤细胞杀伤性,具有良好的肿瘤部位富集能力以及向肿瘤深处渗透能力,具有优异的肿瘤治疗效果。制备方法简单,易于工业化生产,应用领域广阔。
Description
技术领域
本发明涉及多肽纳米药物技术领域,具体涉及一种氧化响应形貌转变多肽纳米药物及性能表征方法。
技术背景
利用生物相容性刺激调控多肽组装体的纳米结构已被证明是一种制备先进功能生物材料的有效策略,在疾病诊断和治疗方面具有巨大的潜力。在众多的生物相容性刺激中,活性氧作为一种活性化学物质或自由基,通常在病理性损伤组织中过表达,这赋予了活性氧能够作为一种重要的生物标志物来识别病理损伤的功能,同时也促进了活性氧响应的自组装多肽在生物材料中的应用和发展。迄今为止,一些活性氧响应的基团已经被引入到多肽序列中用以制备活性氧响应性的多肽缀合物。尽管如此,如何利用活性氧响应性来调控天然短肽高效且精准的自组装结构仍然具有挑战性,因此很大程度上限制了短肽在生物材料应用中的易于合成以及良好生物安全性等方面的优势。
在活性氧响应基团中,甲硫氨酸能够在氧化刺激下由疏水性硫醚转化为亲水性亚砜或砜,这种可靠而稳定的转化使甲硫氨酸成为创建活性氧响应性多肽体系的理想候选之一。例如,甲硫氨酸已被嵌入多肽和多肽缀合物中用以制备活性氧响应性胶束、囊泡和水凝胶,并在癌症治疗中表现出巨大的潜力。然而,由于甲硫氨酸在形成β-折叠结构中的温和趋势,使甲硫氨酸残基的氧化在介导β-折叠结构组装体中的应用仍然具有挑战性。此外,单个甲硫氨酸的氧化反应及其相互作用在活性氧响应构象转变中的作用依然难以捉摸,从而限制了基于甲硫氨酸的活性氧响应性多肽体系的进一步开发。因此,通过合理设计含有甲硫氨酸的活性氧响应多肽纳米药物递送体系具有重要意义和广阔的应用前景。
发明内容
本发明的目的是解决现有技术存在的上述缺陷,提供一种通过合理设计含有甲硫氨酸的氧化响应形貌转变的多肽纳米药物。这种氧化响应性形貌转变多肽纳米药物在激光照射下,多肽中的甲硫氨酸发生氧化,由疏水性硫醚转变为亲水性的砜,从而使多肽的组装结构由β-折叠转变为无规卷曲,多肽纳米药物的组装形貌由纳米纤维转变为纳米颗粒,进而提高肿瘤细胞的摄取量,促进纳米药物向肿瘤深处的渗透,提高肿瘤治疗效果。该多肽纳米药物制备方法简单,反应条件温和,操作简便。
为实现上述目的,本发明提供的技术方案为:
一种氧化响应形貌转变多肽纳米药物,该药物包括:多肽序列EIMIME基元,含有光敏剂具有光动力治疗功能的多肽组装基元一和含有化疗药物具有药物治疗功能的多肽组装基元二组成;所述多肽纳米药物通过如下方法制备而成:
S1:设计合成含有甲硫氨酸的能够产生氧化响应发生形貌转变的多肽序列EIMIME,该序列能够自组装形成β-折叠结构的组装体,当序列中的甲硫氨酸被氧化,由疏水性硫醚转化为亲水性砜,进而使氧化后的组装体二级结构发生转变,由β-折叠转变为无规卷曲,从而使多肽组装体的形貌发生转变,由氧化前的纤维转变为纳米颗粒。多肽序列的合成是通过标准Fmoc固相多肽合成(SPPS)方法合成,以哌啶作为脱保护剂,苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)为缩合剂;
S2:在步骤S1设计合成的氧化响应形貌转变多肽序列EIMIME的基础上,将光敏剂通过酰胺缩合的方法共价连接到氧化响应性多肽上,赋予其光动力治疗功能,得到含有光敏剂具有光动力治疗功能性的多肽组装基元一。
S3:在步骤S1设计合成的氧化响应形貌转变多肽序列EIMIME的基础上接半胱氨酸,得到七肽CEIMIME,将传统化疗药物通过有谷胱甘肽响应性的二硫键共价连接到七肽CEIMIME上,药物释放后起到化疗的效果,得到含有化疗药物具有药物治疗功能的多肽组装基元二。
S4:将步骤S1,S2和S3所述的三种多肽及其衍生物多肽组装基元在水溶液中按照一定的组分比例共组装,得到多肽共组装体系溶液,再经过退后得到一系列具有特定组装结构的多肽纳米药物。
多肽纳米药物的性能表征
S5:将步骤S4得到的含有光敏剂组分的共组装多肽纳米药物置于氧化条件下,氧化性能测试以及形貌表征证明,多肽纳米药物氧化后,纳米药物的形貌发生转变,由纳米纤维转变为纳米颗粒。
S6:将步骤S4得到的纳米药物进行进一步的细胞实验和动物实验,证明该多肽纳米药物的细胞摄取量得到改善,具有优异的肿瘤深部渗透能力和肿瘤治疗性能。
在本发明的进一步实施方案中,所述的氧化响应形貌转变的多肽序列EIMIME为含有甲硫氨酸的能够产生氧化响应发生形貌转变的多肽序列,例如谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸、谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-异亮氨酸-谷氨酸和谷氨酸-异亮氨酸-异亮氨酸-异亮氨酸-甲硫氨酸-谷氨酸等。
在本发明的进一步实施方案中,所述的含有光敏剂具有光动力治疗功能的多肽组装基元一是包括二氢卟吩、5-氨基酮戊酸、卟啉类、酞菁类等中的一种光敏剂的功能化的多肽。
在本发明的进一步实施方案中,所述的含有化疗药物具有药物治疗功能的多肽组装基元二是包括喜树碱、紫杉醇、多西紫杉醇、阿霉素或柔红霉素等中的一种化疗药物的功能化的多肽。
在本发明的进一步实施方案中,步骤S4中:含有光敏剂具有光动力治疗功能的多肽组装基元一占多肽共组装体系的总摩尔数的比例在0.1%到50%之间;含有化疗药物具有药物治疗功能的多肽组装基元二占多肽共组装体系的总摩尔数的比例在0.1%到50%之间;所述多肽共组装体系溶液的物质的量浓度在0.1微摩尔每升到10毫摩尔每升;所述多肽共组装体系溶液的退火温度在10-100℃之间,所述的退火时间为0.1-100小时,优选1-48小时,溶剂为缓冲液或水,其中水为超纯水、去离子水或Milli-Q水。
在本发明的进一步实施方案中,步骤S5中:氧化响应形貌转变多肽纳米药物的氧化条件为双氧水氧化,或通过激光照射使光敏剂产生活性氧氧化;双氧水浓度范围在0.1微摩尔每升到1摩尔每升,温度范围在0-100℃之间,氧化时间为0.1-100小时;激光照射波长范围在300-800nm之间,强度范围在0.1-1.2W/cm2,照射时间为0.1-100小时,温度范围在0-100℃之间。
在本发明的进一步实施方案中,步骤S6中:细胞实验所用的正常细胞为小鼠胚胎成纤维细胞(3T3);细胞实验中所用的癌细胞为小鼠乳腺癌细胞(4T1)。
本发明提供的多肽纳米药物在激光照射后,原位产生的活性氧不仅能起到光动力治疗的效果,还能够氧化纳米药物中的甲硫氨酸,从而使纳米药物的形貌由纳米纤维转变为纳米颗粒。
本发明提供的多肽纳米药物具有良好的肿瘤富集能力,以及由于形貌转变赋予纳米药物优异的肿瘤细胞摄取能力和肿瘤深处渗透能力。所述的多肽纳米药物能够联合光动力治疗和化疗,实现光动力治疗和化疗的级联肿瘤治疗。所述多肽纳米药物在肿瘤治疗领域具有应用价值。
本发明的优点和有益效果:
(1)本发明采用的多肽序列具有良好的生物相容性、生物活性以及生物降解性,多肽作为生物体内重要的生物活性物质,具有良好的生物兼容性,可以通过合理设计和调控组装序列中的分子结构,实现对组装过程中的热力学和动力学控制。(2)本发明通过合理设计含有甲硫氨酸的β-折叠组装结构的多肽序列,得到具有氧化响应形貌转变性能的多肽组装体。(3)本发明通过将具有光动力治疗功能的光敏剂和具有药物治疗功能的化疗药物共价连接到氧化响应形貌转变多肽中,通过共组装的方式得到一种多肽纳米药物。该多肽纳米药物在激光照射下产生的活性氧不仅能够氧化多肽中的甲硫氨酸,使纳米药物的形貌发生转变,由纳米纤维转变为纳米颗粒,还起到了光动力治疗的功能。(4)本发明提供的氧化响应形貌转变多肽纳米药物的形貌能够从氧化前的纳米纤维转变为氧化后的纳米颗粒,从而有利于肿瘤细胞对多肽纳米药物的摄取,使多肽纳米药物向肿瘤深处渗透,提高治疗效果。(5)本发明提供的氧化响应形貌转变多肽纳米药物能够在肿瘤部位有效积累和保留,并且能够联合光动力治疗和化疗,实现光动力治疗和化疗的级联治疗。(6)本发明提供的氧化响应形貌转变多肽纳米药物制备方法简单,反应条件温和,操作简便,易于工业化,在抗炎、抗肿瘤、抗菌等方面具有巨大的应用潜力。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
图1为本发明实施例中的部分多肽序列以及功能化多肽的分子结构式图(1:谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸,2:谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-异亮氨酸-谷氨酸,3:谷氨酸-异亮氨酸-异亮氨酸-异亮氨酸-甲硫氨酸-谷氨酸,4:二氢卟吩-谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸,5:喜树碱-谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸);
图2为本发明实施例中的氧化响应形貌转变多肽纳米药物组装得到的原子力显微镜图和透射电子显微镜图;
图3为本发明实施例中的氧化响应形貌转变多肽纳米药物氧化后组装得到的原子力显微镜图和透射电子显微镜图;
图4为本发明实施例中的氧化响应形貌转变多肽纳米药物在氧化前后的圆二色表征图。
图5为本发明实施例中的氧化响应形貌转变多肽纳米药物在激光照射后,在活性氧探针1,3-二苯基异苯并呋喃(DPBF)存在下的紫外可见吸收光谱以及单独DPBF的紫外可见吸收光谱;
图6为本发明实施例中的氧化响应形貌转变多肽纳米药物的细胞摄取流式细胞仪分析;
图7为本发明实施例中的氧化响应形貌转变多肽纳米药物的溶酶体共定位激光共聚焦图;
图8为本发明实施例中的氧化响应形貌转变多肽纳米药物在细胞摄取4小时和12小时后,通过激光照射,在活性氧探针2,7-二氯荧光黄双乙酸盐(DCFH-DA)存在下的激光共聚焦图;
图9为本发明实施例中的氧化响应形貌转变多肽纳米药物的细胞毒性分析;
图10为本发明实施例中的氧化响应形貌转变多肽纳米药物的细胞凋亡检测;
图11为本发明实施例中的氧化响应形貌转变多肽纳米药物的体内生物分布成像;
图12为本发明实施例中的氧化响应形貌转变多肽纳米药物的各器官分布研究;
图13为本发明实施例中的氧化响应形貌转变多肽纳米药物的肿瘤渗透冷冻切片激光共聚焦图片;
图14为本发明实施例中的氧化响应形貌转变多肽纳米药物氧化前后细胞摄取的激光共聚焦图片。
图15为本发明实施例中的氧化响应形貌转变多肽纳米药物的活体治疗小鼠体重记录。
图16为本发明实施例中的氧化响应形貌转变多肽纳米药物的活体治疗抗肿瘤功效的研究。
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,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的试验材料,如无特殊说明,均为常规生化试剂商店购买得到的。
以下实施例中的定量试验,均设置三次重复实验,数据为三次重复实验的平均值或平均值±标准差。
下面结合具体实施例对本发明提供的一种含有甲硫氨酸的氧化响应形貌转变多肽纳米药物的制备方法作进一步说明。
实施例1
本实施例提供的是一种含有甲硫氨酸的氧化响应形貌转变多肽纳米药物,制备过程包括:
S1:通过固相多肽合成的方法设计合成多肽序列:
多肽自组装基元:谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸;
含有光敏剂具有光动力治疗功能的多肽组装基元一:二氢卟吩-谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸;
含有化疗药物具有药物治疗功能的多肽组装基元二:喜树碱-谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸三种多肽序列;
固相树脂投料均为0.25毫摩尔,多肽序列的合成是通过标准Fmoc固相多肽合成(SPPS)方法合成,以哌啶作为脱保护剂,苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)为缩合剂,产率90%,通过高效液相色谱分离提纯,产物纯度≥99%。
S2:将步骤S1中设计合成的三种多肽如:多肽自组装基元谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸,含有光敏剂具有光动力治疗功能的多肽组装基元一二氢卟吩-谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸以及含有化疗药物具有药物治疗功能的多肽组装基元二喜树碱-谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸三种多肽基元以摩尔比90:10:10的配方,在水溶液中进行退火组装,退火温度为80℃,组装时间为48小时,得到多肽纳米药物,将得到的组装结构在原子力显微镜以及场发射透射电子显微镜下观察,如图2所示。
实验结果:从原子力显微镜图片以及场发射透射电子显微镜图片中能够观察到明显的纤维结构,说明多肽纳米药物能够成功组装成有序的纤维结构。
实施例2
将本发明实施例1制备得到的多肽纳米药物,在双氧水的氧化环境下氧化,双氧水浓度为5毫摩尔,温度为37℃,氧化时间为12小时。氧化完成后,在水溶液中进行退火组装,退火温度为80℃,组装时间为48小时,将得到的组装结构在原子力显微镜以及场发射透射电子显微镜下观察,如图3所示。
实验结果:从原子力显微镜图片以及场发射透射电子显微镜图片中能够观察到,氧化后,多肽纳米药物的纤维结构消失,转变为纳米颗粒,说明多肽纳米药物能够实现氧化响应,并发生形貌转变,由纳米纤维转变为纳米颗粒。
实施例3
将本发明实施例1制备得到的多肽纳米药物和实施例2得到的氧化后的多肽纳米药物进行圆二色表征,如图4所示。
实验结果:从圆二色光谱中可以看出,多肽纳米药物在203nm处有一个正峰,在223nm处有一个负峰,是典型的β-折叠二级结构;氧化后的多肽纳米药物在205nm处有一个负峰,是典型的无规卷曲二级结构。说明该多肽纳米药物能够由氧化前的β-折叠二级结构转变为氧化后的无规卷曲二级结构,实现氧化前后二级结构的变化。
实施例4
将本发明实施例1制备得到的多肽纳米药物中加入活性氧探针DPBF,用激光照射多肽纳米药物,照射波长为660nm,强度为0.1W/cm2,照射时间为15min,温度为37℃。在激光照射后的不同时间点,用紫外光谱仪进行测试,得到DPBF的吸收光谱如图5所示。
实验结果:经过激光照射后多肽纳米药物中的DPBF探针在408nm处的紫外吸收强度逐渐降低,而单独DPBF探针在408nm处的紫外吸收强度几乎没有变化,说明了多肽纳米药物在激光照射后能成功产生活性氧。
实施例5
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,通过流式细胞仪检测细胞摄取情况。
将4T1细胞以1×105/孔的细胞密度铺在12孔板中,在培养箱内培养24小时。加入含有多肽纳米药物的新鲜培养基,继续培养1、2、4、8或12小时。培养至预定时间后,弃培养基,用PBS洗涤细胞三次后,使用胰蛋白酶消化细胞,收集细胞并用PBS洗涤两次,将细胞重悬于PBS中进行流式细胞仪分析,如图6所示。
实验结果:流式细胞术的结果显示,随着孵育时间的延长,4T1细胞中多肽纳米药物的荧光强度逐渐提高,说明多肽纳米药物成功被4T1细胞摄取,并且随着摄取时间的延长,摄取量不断增加。另外我们还观察到与单独光敏剂相比,多肽纳米药物处理的4T1细胞具有更高的荧光强度,说明细胞摄取效率有所提高。
实施例6
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,通过共聚焦激光扫描显微镜监测纳米药物的细胞摄取途径。
将4T1细胞以1×105的细胞密度铺在共聚焦玻璃皿中,在CO2培养箱内培养24小时。加入含有多肽纳米药物的新鲜培养基,继续培养1、2、4、8或12小时。培养至预定时间后,弃培养基,用PBS洗涤细胞三次后,加入含有溶酶体绿色荧光探针的新鲜培养基孵育30分钟,然后,将细胞用4%多聚甲醛固定20分钟,再用细胞核染料DAPI染20分钟。通过共聚焦激光扫描显微镜进行观察,如图7所示。
实验结果:从共聚焦图片中可以看出,随着孵育时间的延长,细胞内的荧光强度逐渐提高,说明多肽纳米药物成功被细胞摄取。当细胞孵育至4小时,多肽纳米药物与内体/溶酶体之间的皮尔森相关系数达到了0.9,说明这时多肽纳米药物与内体/溶酶体具有较好的共定位;当孵育时间延长至10小时,皮尔森相关系数降低至0.45,这说明多肽纳米药物是通过内体/溶酶体-介导的内吞途径被细胞摄取,并且能够实现内体/溶酶体逃逸进入细胞质。
实施例7
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,利用活性氧探针DCFH-DA,通过共聚焦激光扫描显微镜监测多肽纳米药物在细胞内产生的活性氧。
将4T1细胞以1×105的细胞密度接种与共聚焦玻璃皿中,在CO2培养箱内培养24小时。加入含有多肽纳米药物的新鲜培养基,继续培养4或12小时。培养至预定时间后,弃培养基,用PBS洗涤细胞三次。随后,用含有DCFH-DA的无血清培养基孵育30分钟。最后,弃培养基并用PBS洗涤细胞三次,用波长为660nm,激光强度为0.6W/cm2的激光照射3分钟,立即在共聚焦扫描显微镜下进行观察,如图8所示。
实验结果:从共聚焦图片中可以看出,经激光照射后,4T1细胞中可以观察到明显的绿色荧光信号,而没有激光照射处理的细胞中却很难观察到绿色荧光信号,这说明纳米药物被细胞摄取后,在激光照射下能够成功产生活性氧。
实施例8
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,通过细胞毒性和细胞凋亡的检测来评价该纳米药物的肿瘤细胞杀伤能力。
通过MTT的方法检测多肽纳米药物的细胞毒性:将4T1细胞以6×103/孔的细胞密度接种在96孔板中,在CO2培养箱内培养24小时。向96孔板中加入含有不同浓度多肽纳米药物的新鲜培养基,继续培养24小时。随后,向每孔中加入10微升MTT溶液,在培养箱内继续孵育4小时。4小时后,弃培养液,向每孔中加入100微升二甲基亚砜,用酶标仪测定在490nm处的吸光度。
通过流式细胞仪检测多肽纳米药物诱导细胞凋亡:将4T1细胞以1×105/孔的细胞密度接种在12孔板中,在CO2培养箱内培养24小时。向孔板中加入含有多肽纳米药物的新鲜培养基,孵育24小时后,弃培养基并用PBS洗涤细胞3次,用不含有EDTA的胰酶消化细胞,收集细胞并在结合缓冲液中用膜联蛋白V-FITC和PI溶液在黑暗中染色15分钟,在流式细胞仪下进行检测。
实验结果:如图9和图10所示,该氧化响应性多肽对细胞存活率几乎没有影响,表明其具有良好的生物兼容性。此外,与游离CPT和游离Ce6相比,多肽纳米药物具有更高的肿瘤细胞毒性,这可能是由于多肽纳米药物具有更高的细胞摄取量,从而提高了药物利用率。同样,流式细胞术结果也表明由多肽纳米药物处理的细胞中超过70%的细胞凋亡率,表明该多肽纳米药物具有强大的诱导细胞凋亡的能力。
实施例9
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,进行体内生物分布和各组织分布的研究。
将4T1细胞(3×106)皮下注射到雌性BALB/c小鼠右侧腋下,建立荷瘤小鼠模型。当肿瘤生长到200mm3时,将小鼠随机分组,对小鼠尾静脉注射多肽纳米药物,随后,分别在注射后2、4、8、12、24和36小时后,使用活体成像系统对小鼠进行成像,监测纳米药物的活体分布情况。36小时以后,处死小鼠,收集主要器官(心脏,肝脏,脾脏,肺,肾)和肿瘤组织,进行离体组织成像以观察纳米药物在各组织中的分布情况,如图11和图12所示。
实验结果:活体成像结果显示,在肿瘤组织附近能够长时间观察到明显的多肽纳米药物荧光信号,说明多肽纳米药物能够成功在肿瘤部位富集。另外,在小鼠给药36小时后,在肿瘤部位依然能够观察到明显的多肽纳米药物荧光信号,这意味着该纳米药物在肿瘤部位具有较长的保留时间。各离体主要器官(心脏,肝脏,脾脏,肺,肾)和肿瘤组织荧光成像结果表明,多肽纳米药物在肿瘤组织中的荧光强度明显高于各正常器官,这些结果说明多肽纳米药物能够在肿瘤部位富集并且具有较长的滞留时间。
实施例10
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,进行实体肿瘤渗透性能研究。
将4T1细胞(3×106)皮下注射到雌性BALB/c小鼠右侧腋下,建立荷瘤小鼠模型。当肿瘤体积接近200mm3时,将小鼠随机分组,对小鼠尾静脉注射多肽纳米药物,给药2小时后,其中一组小鼠接受激光照射处理(660nm,0.3W/cm2,3min),另一组不处理。给药8小时后,解剖获得肿瘤组织,进行冷冻切片和DAPI染色,在激光共聚焦扫描显微镜下观察肿瘤切片中多肽纳米药物的肿瘤渗透情况,如图13所示。
实验结果:共聚焦图片中可以看出,在肿瘤切片中能够观察较强的多肽纳米药物荧光信号,说明多肽纳米药物能够在肿瘤组织处富集。另外,能够观察到经过激光照射处理后的肿瘤组织切片中,在肿瘤的深部区域有较强的多肽纳米药物荧光信号,而未经激光照射处理的肿瘤组织切片中纳米药物的荧光信号主要集中在肿瘤边缘处。这说明经过激光照射后,多肽纳米药物向肿瘤组织深部的渗透性增强。这可能是由于激光照射后,多肽纳米药物原位产生的活性氧使多肽中的甲硫氨酸被氧化,从而使多肽组装体形貌发生了转变,从纳米纤维转变为纳米颗粒,从而提高了多肽纳米药物的肿瘤细胞摄取量以及向肿瘤组织的渗透能力。
实施例11
将本发明实施例1制备得到的多肽纳米药物以及本发明实施例2制备得到的氧化后的多肽纳米药物,进行多肽纳米药物氧化前后对细胞摄取影响的研究。
将4T1细胞以1×105的细胞密度接种于共聚焦玻璃皿中,在CO2培养箱内培养24小时。分别加入含有氧化前后多肽纳米药物的新鲜培养基,继续培养1、2、4、8或12小时。培养至预定时间后,弃培养基,用PBS洗涤细胞三次后,将细胞用4%多聚甲醛固定20分钟,再用细胞核染料DAPI染20分钟。通过共聚焦激光扫描显微镜进行观察,如图14所示。
实验结果:从共聚焦图片中可以观察到,用氧化前后多肽纳米药物处理的4T1细胞中均观察到多肽纳米药物的荧光信号,并且随着时间的延长多肽纳米药物荧光信号强度逐渐提高,说明氧化前后的多肽纳米药物均能够成功被细胞摄取。另外,可以观察到在相同的孵育时间条件下,氧化后的多肽纳米药物在细胞中的荧光信号强度高于氧化前的多肽纳米药物,说明多肽纳米药物氧化后的细胞摄取量有所提高,细胞内化得到改善。这是因为氧化后的多肽纳米药物的组装形貌由纳米纤维转变为纳米颗粒,从而更有利于细胞摄取。
实施例12
将本发明实施例1制备得到的氧化响应形貌转变多肽纳米药物,进行体内抗肿瘤治疗效果的研究。
将4T1细胞(3×106)皮下注射到雌性BALB/c小鼠右侧腋下,建立荷瘤小鼠模型。当肿瘤体积达到100mm3时,将小鼠随机分组,通过尾静脉注射对小鼠进行不同的处理,氧化响应形貌转变多肽纳米药物作为实验组,PBS作为对照组。每两天给药一次,给药4小时后,对光照组进行激光照射处理(660nm,0.3W/cm2,3min),在实验开始的第1天、第3天和第5天分别进行尾静脉给药,共给药三次。另外,每隔一天监测和记录每只小鼠的体重和肿瘤体积,如图15和图16所示。
实验结果:如图15所示,多肽纳米药物实验组和对照组小鼠的体重相差不大且体重稳定,这说明多肽纳米药物具有良好的生物相容性,其全身毒性可忽略不计。图16中的结果显示,与游离药物CPT和游离光敏剂Ce6组相比,多肽纳米药物治疗组的小鼠肿瘤体积明显减小,说明多肽纳米药物能够联合光动力治疗和化疗,实现光动力治疗和化疗的级联治疗,具有较强的治疗效果,能够显著抑制肿瘤生长。
需要说明的是,除了上述实施例1至实施例12列举的情况,选用其他的原料配比和制备方法参数也是可行的。
Claims (7)
1.一种氧化响应形貌转变多肽纳米药物,其特征在于,该药物包括:多肽序列EIMIME基元,含有光敏剂具有光动力治疗功能的多肽组装基元一和含有化疗药物具有药物治疗功能的多肽组装基元二组成;所述多肽纳米药物通过如下方法制备而成:
S1:设计合成含有甲硫氨酸的能够产生氧化响应发生形貌转变的多肽序列EIMIME,该序列能够自组装形成β-折叠结构的组装体,当序列中的甲硫氨酸被氧化,由疏水性硫醚转化为亲水性砜,进而使氧化后组装体的二级结构发生转变,由β-折叠转变为无规卷曲,从而使多肽组装体的形貌发生转变,由氧化前的纤维转变为纳米颗粒;多肽序列的合成是通过标准Fmoc固相多肽合成(SPPS)方法合成,以哌啶作为脱保护剂,苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)为缩合剂;
S2:在多肽序列EIMIME的基础上,通过酰胺缩合的方法将光敏剂共价连接到多肽序列EIMIME上,得到含有光敏剂具有光动力治疗功能的多肽组装基元一;另外,在多肽序列EIMIME上接半胱氨酸,得到七肽CEIMIME,将化疗药物通过二硫键连接到七肽CEIMIME上,得到含有化疗药物具有药物治疗功能的多肽组装基元二;以多肽序列EIMIME作为自组装基元,将多肽序列EIMIME与含有光敏剂具有光动力治疗功能的多肽组装基元一和含有化疗药物具有药物治疗功能的多肽组装基元二共组装,得到多肽共组装体系溶液,再经过退火后得到具有氧化响应形貌转变的多肽纳米药物。
2.根据权利要求1所述的氧化响应形貌转变多肽纳米药物,其特征在于:
所述多肽序列EIMIME包含但不限于谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-甲硫氨酸-谷氨酸、谷氨酸-异亮氨酸-甲硫氨酸-异亮氨酸-异亮氨酸-谷氨酸和谷氨酸-异亮氨酸-异亮氨酸-异亮氨酸-甲硫氨酸-谷氨酸。
3.根据权利要求1所述的氧化响应形貌转变多肽纳米药物,其特征在于:
所述含有光敏剂具有光动力治疗功能的多肽组装基元一是包含但不限于二氢卟吩、5-氨基酮戊酸、卟啉类或酞菁类中的一种光敏剂的功能化多肽。
4.根据权利要求1所述的氧化响应形貌转变多肽纳米药物,其特征在于:
所述含有化疗药物具有药物治疗功能的多肽组装基元二是包含但不限于喜树碱、紫杉醇、多西紫杉醇、阿霉素或柔红霉素中的一种化疗药物的功能化多肽。
5.根据权利要求1所述的氧化响应形貌转变多肽纳米药物,其特征在于:
含有光敏剂具有光动力治疗功能的多肽组装基元一占多肽共组装体系的总摩尔数的比例在0.1%到50%之间;含有化疗药具有药物治疗功能的多肽组装基元二占多肽共组装体系的总摩尔数的比例在0.1%到50%之间;所述多肽共组装体系溶液的物质的量浓度在0.1微摩尔每升到10毫摩尔每升;所述多肽共组装体系溶液的退火温度在10-100℃之间,所述的退火时间为0.1-100小时,溶剂为缓冲液或水,其中水为超纯水、去离子水或Milli-Q水。
6.根据权利要求1所述的氧化响应形貌转变多肽纳米药物,其特征在于:
所述氧化响应形貌转变多肽纳米药物的氧化条件包含但不限于双氧水氧化,或通过激光照射使光敏剂产生活性氧氧化;双氧水浓度范围在0.1微摩尔每升到1摩尔每升,温度范围在0-100℃之间,氧化时间为0.1-100小时;激光照射波长范围在300-800nm之间,强度范围在0.1-1.2W/cm2,照射时间为0.1-100小时,温度范围在0-100℃之间。
7.根据权利要求1所述的氧化响应形貌转变多肽纳米药物的制备方法,其特征在于:
所述氧化响应形貌转变多肽纳米药物的细胞实验中,所用的正常细胞为小鼠胚胎成纤维细胞(3T3);细胞实验中所用的癌细胞为小鼠乳腺癌细胞(4T1)。
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