CN110575546A - 一种高度核靶向性抗肿瘤纳米药物的制备方法及其应用 - Google Patents
一种高度核靶向性抗肿瘤纳米药物的制备方法及其应用 Download PDFInfo
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Abstract
本发明属于纳米药物与生物医学领域，公开了一种高度核靶向性抗肿瘤纳米药物的制备方法及其应用。本发明将具有细胞核靶向能力的多肽与石墨粉共混球磨，在常温常压下，制备具有高度肿瘤靶向能力和核靶向能力的多肽功能化石墨烯（TG）。进而采用乙酸等离子技术在TG上引入羧基，将抗肿瘤药物丝裂霉素C（MMC）通过接载在TG上。所制备的的抗肿瘤纳米药物MMC‑TG具有良好的肿瘤细胞核靶向能力，可高度选择性的靶向到肿瘤细胞，肿瘤杀伤率达95%以上，但同时对正常细胞的损伤很小。实验结果证实了所研制的纳米药物最先在细胞核发挥作用，具有高效的肿瘤细胞核靶向能力、肿瘤杀伤能力和肿瘤转移抑制能力。
Description
技术领域
本发明属于纳米药物与生物医学领域，具体涉及一种高度核靶向性抗肿瘤纳米药物的制备方法及其应用。
背景技术
肿瘤细胞的快速增殖性和易转移性，造成了大多数癌症的不可治愈性，给人类生命健康带来了极大的威胁。譬如脉络膜黑色素瘤，是一种成人最常见的眼部恶性肿瘤，病死率高，五年存活率低于50％，主要就是由于这类肿瘤的全身高转移率造成的。尽管临床上已经开发了多种技术治疗脉络膜黑色素瘤，包括手术切除，激光治疗，放疗和全身化疗等，但是这些方法都只针对原位肿瘤，对转移瘤束手无策，除了给病人造成了巨大的痛苦，并未能提高病人的存活率。肿瘤细胞的易转移性，根源在细胞核，因为细胞核是基因遗传和转录的源头。因此，针对细胞核设计高靶向的抗肿瘤药物，将会为抑制肿瘤细胞的转移提供一条高效便捷的途径。纳米技术和纳米材料的快速发展，开发了一系列可以穿透某些生理屏障的纳米药物载体，不仅有望提高药物的生物利用率，也为研制高度核靶向的纳米药物提供了可行性。当前已开发的纳米载体材料，比如由金属，金属氧化物，半导体和聚合物等，虽然能够将药物靶向递送至肿瘤微环境，但效率低下，并且纳米药物无法穿透细胞核膜。
石墨烯及其衍生物具有独特的尺寸效应和理化性能。石墨烯内在结构上独特的大Π键使得基于石墨烯的纳米药物载体具有一定的负电性，对于具有弱酸正电性的肿瘤微环境具有较好的亲和力。同时直径小于200nm的石墨烯纳米片，能够穿过通透性较高的肿瘤区新生血管，聚集到肿瘤微环境，形成纳米富集效应。因此石墨烯纳米片被认为是高性能的肿瘤靶向药物载体。本发明进一步在石墨烯上修饰具有核靶向能力的TAT多肽，从而实现对于肿瘤细胞核的高度靶向识别和药物输送。TAT是一种阳离子穿膜肽，具有很强的细胞膜穿透性和核膜穿透性，能够到达细胞核，为纳米药物载体提供高效的细胞膜穿透能力和核靶向能力。我们进一步在所制备的TG纳米载体上共价接载常规抗肿瘤药物丝裂霉素C(MMC)，构建高度核靶向的抗肿瘤纳米药物MMC-TG。MMC能够作用于细胞核，通过解聚DNA，抑制DNA 的复制来发挥作用。
综上所述，本发明针对肿瘤细胞易转移的临床瓶颈问题，致力于构建高效核靶向性的抗肿瘤纳米药物MMC-TG，为抗肿瘤药物的下一步发展提供新的思维与方向。
发明内容：
本发明针对肿瘤细胞易转移的临床瓶颈问题，提供了一种简便高效的核靶向药物纳米载体TG与核靶向抗肿瘤药物MMC-TG的制备方法。
本发明的另一目的在于提供了一种核靶向抗肿瘤药物MMC-TG的制备方法的应用，该药物能够准确靶向到肿瘤细胞，并高效穿透细胞膜和核膜，进入细胞核，对肿瘤细胞(如脉络膜黑色素瘤)的抑制率高达95％以上，并同时不对正常细胞造成较大损伤。
为解决上述技术问题，本发明采用如下技术方案：
一种简便高效的核靶向药物纳米载体TG的制备方法，包括下述步骤：
多肽TAT(RKKRRQRRR)和石墨粉按质量比1：1～1：5的比例混合后置于球磨罐内，以300-500rpm的转速室温球磨3-5个小时。球磨结束后加入去离子水，洗出其中的产物，分别采用1000-3000转速离心处理5-15分钟，之后再采用5000-8000rpm的高速离心处理5-15 分钟，依次除去沉淀中的杂质。然最后将沉淀分散于去离子水中，即为TAT多肽功能化的石墨烯(TG)。
一种高度核靶向性抗肿瘤纳米药物的制备方法包括：
1)将上述制得的TG在-80℃通过真空冷冻干燥制成粉末，采用乙酸等离子技术对TG粉末表面进行羧基化修饰；
3)采用摩尔浓度比为1:1-1:3的N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化TG表面羧基后，加入丝裂霉素C(MMC)(TG与MMC的质量比为 1:1-1:3)，4℃搅拌1-3天，3000-5000rpm离心5-15分钟取沉淀，即得到纳米药物MMC-TG。
本发明的保护内容还包括，制备的高度核靶向性抗肿瘤纳米药物在肿瘤核靶向、抑制肿瘤转移、以及高效杀伤肿瘤细胞中的应用。
与现有技术相比，本发明具有以下优点：
TAT容易在生物环境溶解，普通方法合成的TAT加石墨烯加药物后，很难进细胞核。通过本发明的方法，减小了纳米复合物尺寸，又能保持TAT的穿膜活性进入细胞。MMC起作用是通过抑制DNA。但是传统用法不能进入细胞核，所以无法发挥作用，肿瘤抑制率很有限。我们的制备方法能够把药物送到细胞核起作用，从而使得肿瘤抑制率达95％以上。
本发明制备的高核靶向抗肿瘤纳米药物MMC-TG从肿瘤细胞转移的源头细胞核着手，解决了肿瘤细胞易转移的瓶颈问题。所制备的纳米药物能够高效靶向到肿瘤微环境，穿透细胞膜和核膜，并进入到细胞核发挥作用。抗肿瘤药物直接在肿瘤细胞核内发挥效能，有效抑制了细胞核的分裂增殖与迁移，大大提高了药物的杀伤作用，并有效抑制了肿瘤的侵袭性和转移性。所合成的MMC-TG纳米药物对于肿瘤细胞如脉络膜黑色素瘤细胞的杀伤率高达95％以上，但对正常细胞的损伤很小。
附图说明
图1为高核靶向抗肿瘤纳米药物载体TG的合成示意图。
图2为所制备的纳米载体TG的实物图。
图3为所合成的高度核靶向性抗肿瘤纳米药物MMC-TG的原子力表征图。
图4为所制备的纳米药物MMC-TG的靶向抗肿瘤效果评价；
其中，(a)为纳米药物MMC-TG与正常细胞视网膜色素上皮细胞(ARPE-19)和脉络膜黑色素细胞(OCM-1)共培养72h后的细胞存活率检测结果；(b)为未处理的MMC与两种细胞共培养72h后的细胞存活率检测结果。
图5为所制备的纳米药物载体TG经过蓝色荧光剂FITC标记后与肿瘤细胞OCM-1共培养后的共聚焦显微镜图；
其中(a)为未用纳米材料处理的对照组OCM-1细胞的荧光共聚焦显微镜图；(b)为TG与OCM-1共培养后的共聚焦荧光显微镜图。实验中，细胞核采用DAPI染料标记成蓝色荧光，细胞骨架用红色荧光的鬼比环肽标记。
图6为所制备的纳米药物MMC-TG与OCM-1共培养后的细胞切片透射电镜表征图；
其中，(a)为对照组未经处理的OCM-1细胞的透射电镜图；(b)为MMC-TG与OCM-1 细胞共培养后24h后的细胞透射电镜图；(c)为MMC-TG与OCM-1共培养后72h后的细胞透射电镜图。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
下面结合附图以及具体实施例，可以更好地说明本发明。
实施例1.
一种高度核靶向性抗肿瘤纳米药物的制备方法，包括下述步骤：
1)将TAT多肽与石墨粉按质量比1：1-1：5的比例混合后置于球磨罐内。以300-500rpm 的转速室温球磨3-5个小时。球磨结束后加入去离子水，洗出其中的产物。分别采用1000-3000转速离心处理5-15分钟，之后再采用5000-8000rpm的高速离心处理5-15分钟转速的高速离心处理，依次除去沉淀中的杂质。然最后将沉淀分散于去离子水中，即为TAT多肽功能化的石墨烯(TG)。从图1可以看出，通过边缘功能化球磨法，能够简便高效的将TAT多肽接载到石墨烯二维平面。从图2中可以看出，所制备的纳米药物载体TG具有很好的水向分散性。并且我们研究发现，将TG分散在去离子水中7天之后，依然能看到均匀的分散液，未发现明显的团聚和沉淀现象。
2)将TG在-80℃通过真空冷冻干燥制成粉末，采用乙酸等离子技术对TG粉末表面进行羧基化修饰。
3)采用摩尔浓度比为1:1-1:3的N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化TG表面羧基后，加入丝裂霉素C(MMC)(TG与MMC的质量比为 1:1-1:3)，4℃搅拌48h，离心取沉淀，即得到纳米药物MMC-TG。
实施例2：
实施例1制备的MMC-TG纳米药物的理化性质：
从图3中可以看出，所制备的石墨烯片的高度在0.8nm左右，说明是单层石墨烯。在共价接载MMC药物之后，纳米材料的厚度上升到了4.3nm。
实施例3.
实施例1制备的纳米药物MMC-TG的靶向抗肿瘤效果评价。
我们采用Transwell双细胞共培养体系考察MMC-TG的靶向抗肿瘤效果。Transwell板是一种特殊的细胞培养板，分为上层和下层，两层之间是通透性的膜，膜上培养液中的成分可以自由穿过通透膜而细胞却不能穿过。本实验在Transwell板的上室和下室同时分别培养 ARPE-19正常细胞和OCM-1肿瘤细胞，然后加入MMC-TG和MMC与细胞共同培养72h后，检测细胞活力。从图3中(a)和(b)中可以看出，MMC-TG与两种细胞共培养72h后， MMC浓度达到4μg/mL时，OCM-1肿瘤细胞的存活率下降到5％以下，而ARPE-19正常细胞的存活率依然在50％以上。单纯的药物MMC同时作用于两种细胞时，两种细胞的存活率基本相似，甚至部分实验组中正常ARPE-19细胞被杀灭得更多。说明实施例1制备的MMC-TG 纳米对肿瘤细胞具有良好的靶向杀伤性，而未经处理的MMC药物不具有肿瘤靶向抑制性。
实施例4.
实施例1制备的纳米药物载体TG的肿瘤细胞核靶向性检测
从图4中可以看出，绿色荧光标记的TG纳米药物载体在与OCM-1细胞作用24h之后，大部分纳米材料就出现在了细胞核中。在纳米材料与肿瘤细胞作用72h之后，大部分纳米材料依然只出现在细胞核中，证实了TG纳米材料优异的肿瘤细胞膜穿透能力和核靶向能力。
实施例5.
实施例1制备的纳米药物MMC-TG的肿瘤细胞核靶向性检测与抗肿瘤机制分析
从图5中可以看出，MMC-TG与OCM-1共同作用24h之后，细胞核内容物出现溶解现象。共同作用72h后，核膜完全溶解，细胞核消失，同时细胞质也开始溶解。结果再次证实了MMC-TG具有优异的肿瘤细胞膜穿透能力和核靶向能力，能够进入细胞核，并最先在细胞核发挥作用，从而从源头上抑制肿瘤细胞的快速增殖与全身转移。
序列表
<110> 温州医科大学
<120> 一种高度核靶向性抗肿瘤纳米药物的制备方法及其应用
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Claims (3)
1.一种高度核靶向性抗肿瘤纳米药物的制备方法，包括：
1）将TAT多肽与石墨粉按质量比1：1-1：5的比例混合后置于球磨罐内；以300-500 rpm的转速室温球磨3-5个小时;球磨结束后加入去离子水，洗出其中的产物；分别采用1000-8000 rpm转速的高速离心处理，依次除去沉淀中的杂质；最后将沉淀分散于去离子水中，得到TAT多肽功能化的石墨烯（TG）;
2) 将TG在-80℃通过真空冷冻干燥制成粉末，采用乙酸等离子技术对TG粉末表面进行羧基化修饰;
3）采用N-羟基琥珀酰亚胺（NHS）和1-（3-二甲氨基丙基）-3-乙基碳二亚胺（EDC）活化TG表面羧基后，加入丝裂霉素C（MMC），4℃搅拌24-48h，离心取沉淀，即得到纳米药物MMC-TG。
2.根据权利要求1所述的制备方法，其特征在于：球磨原料TAT和石墨粉的比例为1：1-1：5，球磨转速为300-500 rpm, 球磨时间为3-5个小时;球磨法制备得到的TG通过乙酸等离子技术引入羧基，然后通过酰胺键共价接载丝裂霉素（MMC），制备得到新型抗肿瘤纳米药物MMC-TG。
3.权利要求1的方法制备的抗肿瘤纳米药物MMC-TG在肿瘤细胞核靶向、高效杀伤肿瘤、以及抑制肿瘤转移中的应用。
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| CN113181120A (zh) * | 2021-02-07 | 2021-07-30 | 香港理工大学深圳研究院 | 一种制备用于靶向肿瘤干细胞的产品的方法、产品及应用 |
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|---|---|---|---|---|
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-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Non-Patent Citations (2)
| Title |
|---|
| 林蜜蜜 等: "核靶向性石墨烯药物载体在抑制脉络膜黑色素瘤上的应用", 《第八届中国眼科学和视觉科学研究大会论文集》 * |
| 贾书娟: "核靶向性石墨烯药物载体在抑制脉络膜黑色素瘤上的应用", 《万方数据知识服务平台》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111870702A (zh) * | 2020-06-22 | 2020-11-03 | 温州医科大学 | 一种纳米眼用药物的制备方法及其应用 |
| CN111870702B (zh) * | 2020-06-22 | 2023-08-18 | 温州医科大学 | 一种纳米眼用药物的制备方法及其应用 |
| CN113181120A (zh) * | 2021-02-07 | 2021-07-30 | 香港理工大学深圳研究院 | 一种制备用于靶向肿瘤干细胞的产品的方法、产品及应用 |
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