CN109091678A - 一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法及其应用 - Google Patents
一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法及其应用 Download PDFInfo
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Abstract
一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法及其应用,构筑单元以β‑环糊精修饰的透明质酸为主体,以八肽修饰的磁纳米粒子为客体,通过超分子主客体相互作用构筑的纳米超分子纤维聚集体。本发明的优点是:该超分子组装体在地磁场或弱磁场的诱导下定向聚集,并且可以光控诱导超分子组装体聚集;另一方面,该超分子组装体可以在纳米纤维网状结构中特定地吸引癌细胞,并且该组装体可以对线粒体造成损伤;该具有磁场和光照双重调控的超分子组装体制备方法简单、易于实施且原料成本低,使其在肿瘤治疗领域,尤其是在主动抑制肿瘤细胞侵袭和扩散方面具有广阔的应用前景。
Description
技术领域
本发明涉及抑制肿瘤侵袭和扩散技术,特别是一种抑制肿瘤侵袭和扩散的具有磁场和光照双重调控的超分子组装体的制备方法及其应用。
背景技术
尽管在早期原发性肿瘤的药物治疗和实用技术方面取得了很大进展,然而侵袭性和转移性仍然是癌症治疗中最神秘的方面和最棘手的问题。据统计,由于外科手术无法操作的性质和临床上缺乏阻止癌细胞扩散的治疗药物,癌细胞向其他部位转移导致的相关癌症死亡率高达90%。此外,我们对肿瘤细胞的侵袭和转移的发病机制认识不足也阻碍了发展有效的抗转移治疗。因此,建立新的、有希望的治疗策略势在必行,在这种基础上,即使在晚期癌症阶段,也能有效地减少肿瘤的生长和转移。
随着具有生物功能的纳米组装体在治疗癌症方面展现巨大的潜力,上述想法迅速改变与发展。非共价键相互作用的动态可逆和多重响应特性可以赋予这些超分子组装以应对各种内源性和外源性刺激的响应能力。因此,大量科研人员一直致力于探索可控和具有靶向性的药物以及基因(共)传递体系的抗癌作用,如纳米载体,纳米粒子,水凝胶,(聚)轮烷等。然而,相比于传统的被动识别和跟踪转移性恶性肿瘤细胞的肿瘤疗法,对肿瘤的生长和迁移的主动防御无疑会加快生物功能纳米组装体从基础研究到临床应用的发展。
发明内容
本发明的目的是针对上述技术分析和存在问题,提供了一种可以抑制肿瘤细胞侵袭和转移,并且具有磁场和光照双重调控的超分子组装体,同时提供了该组装体的制备方法。
本发明中的超分子组装体是一种能够通过光照和磁场诱导的形貌转化的纳米纤维聚集体。这些独特的能力是通过将生物相容性的靶向肽连接在氧化铁磁性纳米颗粒下与β-环糊精修饰的透明质酸非共价交联来完成的。更重要的是,由于癌细胞的表面的透明质酸受体过度表达,得到地磁定向聚合的多糖为基础的组装体,其可以在纳米纤维网状结构中特定地吸引癌细胞,从而抑制肿瘤细胞的迁移和挽救肿瘤细胞迁移的小鼠。本发明是实现生物超分子组装体对较弱的地磁场精确响应的第一个实例,为减少肿瘤细胞转移造成的死亡提供了一种新型的刺激响应性纳米超分子生物材料。
本发明的技术方案:
一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体,其构筑单元以β-环糊精修饰的透明质酸为主体,以八肽修饰的磁纳米粒子为客体,通过超分子主客体相互作用构筑的纳米超分子纤维聚集体;其中β-环糊精修饰的透明质酸为单 -6-脱氧-6-乙二胺基-β-环糊精与透明质酸钠通过酰胺缩合得到,八肽修饰的磁纳米粒子为硅烷化的Fe3O4磁纳米粒子与异硫氰酸荧光素标记的线粒体靶向八肽通过共价连接得到。
一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,包括以下步骤:
1)β-环糊精修饰的透明质酸(HACD)的合成
将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺磺酸钠盐(NHSS)添加到透明质酸钠的磷酸缓冲溶液中,混合物在25℃下搅拌30分钟,然后将单-6-脱氧-6-乙二胺基-β-环糊精的磷酸缓冲溶液加入到反应体系中,并在室温下搅拌24小时,反应结束后,在超纯水中透析5天后冻干,得到白色粉末状产物;
2)磁纳米粒子(MNPs)的合成
采用共沉淀法制备了本发明中所用的磁纳米粒子,将FeCl2和FeCl3的水溶液混合,然后将氢氧化钠溶液慢慢加入到混合物中,得到黑色溶液,加热溶液,得到沉淀并抽滤,用蒸馏水洗涤至中性后,在室温下干燥得到磁纳米粒子;
3)八肽修饰的磁纳米粒子(MitP-MNPs)的合成
将所得磁纳米粒子悬浮在乙醇中,向其中加入氨基丙基三乙氧基甲硅烷(APTES),混合物在80℃下搅拌2小时,离心分离得到产物,并用乙醇洗涤三次,超纯水洗涤两次,得到有游离氨基的磁纳米粒子(MNP-NH2);
将制备的有游离氨基的磁纳米粒子(MNP-NH2)悬浮在8%的戊二醛的磷酸缓冲液中,混合物在室温下摇动6小时,离心分离得到小球状的带有戊二醛的磁纳米粒子,将这些小球用磷酸缓冲液洗涤3次后,悬浮在磷酸缓冲液中,然后加入异硫氰酸荧光素标记的线粒体靶向八肽的磷酸缓冲溶液,混合物在4℃下,以120rpm的速度摇动24小时,离心分离得到小球,在用超纯水洗涤两次后,真空冻干得到八肽修饰的磁纳米粒子(MitP-MNPs);
4)抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备
将八肽修饰的磁纳米粒子(MitP-MNPs)的水溶液与β-环糊精修饰的透明质酸(HACD)的水溶液混合在一起,并且超声5分钟,即制备得到超分子组装体
以上所述具有磁场和光照双重调控的超分子组装体,其分子组装过程受磁场和光照调控,特别是地磁场和弱磁场。该超分子组装体可以诱导线粒体功能障碍和在细胞间的聚集,最终在体内和体外条件下导致对肿瘤侵袭和转移的特异性抑制。
所述透明质酸钠的溶液用量为3.33g/L;单-6-脱氧-6-乙二胺基-β-环糊精的溶液用量为0.1mol/L,反应液中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐 (EDC),N-羟基琥珀酰亚胺磺酸钠盐(NHSS),单-6-脱氧-6-乙二胺基-β-环糊精和透明质酸钠的摩尔比为1:1:1.143:0.00248;
所述β-环糊精修饰的透明质酸(HACD)的合成中的磷酸缓冲液为PBS, 0.1M,pH=7.2;
所述FeCl2和NaOH的溶液浓度均为0.3mol/L,FeCl3的溶液浓度为0.6 mol/L,FeCl2,FeCl3和NaOH溶液所用体积均为100ml;
所述八肽为异硫氰酸荧光素标记的线粒体靶向八肽(MitP, FITC-ACP-Fx-r-Fx-K-Fx-r-Fx-K,Mw=1701);
所述磁纳米粒子质量为40mg,乙醇的体积为40ml,氨基丙基三乙氧基甲硅烷(APTES)的体积为2ml,磷酸缓冲溶液为PBS,pH 7.4,体积为20ml,八肽溶液浓度为1mM,体积为400uL;
所述具有磁场和光照双重调控的超分子组装体的应用,在体内和体外用于有效抑制肿瘤细胞侵袭和转移,具体实施方法为:将八肽修饰的磁纳米粒子 (MitP-MNPs)的水溶液与β-环糊精修饰的透明质酸(HACD)的水溶液混合在一起,并且超声5分钟,即制备得到超分子组装体
所述八肽修饰的磁纳米粒子(MitP-MNPs)的水溶液与β-环糊精修饰的透明质酸(HACD)的水溶液的浓度均为0.2mg/mL。
本发明的优点是:1)Fe3O4纳米粒子是一种对磁响应性纳米粒子,其可以使得超分子组装体在地磁场或弱磁场的诱导下定向聚集;2)β-环糊精修饰的透明质酸一方面有着环糊精的大环空腔,可以选择性键合特定分子,利用偶氮苯类分子顺反结构与环糊精键合能力的区别可以光控诱导超分子组装体聚集,另一方面,癌细胞的表面的透明质酸受体过度表达,超分子组装体可以在纳米纤维网状结构中特定地吸引癌细胞;3)异硫氰酸荧光素标记的线粒体靶向八肽含有环己烷单元,可以通过主客体作用与环糊精形成组装体,从而使得八肽修修饰的磁纳米粒子与环糊精修饰的透明质酸共组装形成超分子组装体,并且该组装体具有吸引线粒体并对线粒体造成损伤的特性;4)该具有磁场和光照双重调控的超分子组装体制备方法简单、易于实施且原料成本低,使其在肿瘤治疗领域,尤其是在主动抑制肿瘤细胞侵袭和扩散方面具有广阔的应用前景。
附图说明
图1为β-环糊精修饰的透明质酸(HACD)的合成路线图。
图2磁纳米粒子(MNPs)的表征图。
图3为肽修饰的磁纳米粒子(MitP-MNPs)的合成路线。
图4为超分子组装体的制备和表征。
图5为超分子组装体在不同磁场不同时间下的光学显微镜图。
图6为超分子组装体在不同光照下的共聚焦显微镜图。
图7为超分子组装体在体内和体外有效抑制肿瘤细胞侵袭和迁移。
图8为本发明超分子组装体在抑制肿瘤侵袭和转移应用示意图。
图9为本发明纳米纤维聚集体构筑单元的结构。
具体实施方式
下面通过实例对本发明做进一步的说明:
实施例:
一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体,其构筑单元以β-环糊精修饰的透明质酸为主体,以八肽修饰的磁纳米粒子为客体,通过超分子主客体相互作用构筑的纳米超分子纤维聚集体。其中β-环糊精修饰的透明质酸为单 -6-脱氧-6-乙二胺基-β-环糊精与透明质酸钠通过酰胺缩合得到,八肽修饰的磁纳米粒子为硅烷化的Fe3O4磁纳米粒子与异硫氰酸荧光素标记的线粒体靶向八肽通过共价连接得到。该纳米纤维聚集体构筑单元的结构参见附图9。
一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,步骤如下:
1)β-环糊精修饰的透明质酸(HACD)的合成
将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)(167.7mg,0.875 mmol)和N-羟基琥珀酰亚胺磺酸钠盐(NHSS)(190mg,0.875mmol)添加到透明质酸钠(100mg)的磷酸缓冲溶液(PBS,0.1M,pH 7.2)中,混合物在25℃下搅拌30分钟。然后将单-6-脱氧-6-乙二胺基-β-环糊精(1177mg,1.0mmol) 的磷酸缓冲溶液(10ml)加入到反应体系中,并在室温下搅拌24小时。反应结束后,在超纯水中透析5天后冻干,得到白色粉末状产物;
图1为β-环糊精修饰的透明质酸(HACD)的合成路线图。
2)磁纳米粒子(MNPs)的合成
采用共沉淀法制备了本发明中所用的磁纳米粒子。将FeCl2(0.3M,100ml) 和FeCl3(0.6M,100ml)的水溶液混合,然后将氢氧化钠溶液(0.3M,100ml) 慢慢加入到混合物中,得到黑色溶液。加热溶液,得到沉淀并抽滤,用蒸馏水洗涤至中性后,在室温下干燥得到磁纳米粒子;
图2为磁纳米粒子(MNPs)的透射电子显微镜图。图中表明所述磁纳米粒子(MNPs)的直径大约为10-20nm。
3)八肽修饰的磁纳米粒子(MitP-MNPs)的合成
将所得磁纳米粒子(40mg)悬浮在乙醇(40ml)中,向其中加入氨基丙基三乙氧基甲硅烷(APTES)(2ml),混合物在80℃下搅拌2小时。离心分离得到产物,并用乙醇洗涤三次,超纯水洗涤两次,得到有游离氨基的磁纳米粒子 (MNP-NH2)。
将制备的有游离氨基的磁纳米粒子悬浮在8%的戊二醛的磷酸缓冲液(PBS, pH7.4,30ml)中,混合物在室温下摇动6小时,离心分离得到小球状的带有戊二醛的磁纳米粒子。将这些小球用磷酸缓冲液洗涤3次后,悬浮在磷酸缓冲液(20ml)中,然后加入异硫氰酸荧光素标记的线粒体靶向八肽的磷酸缓冲(1 mM,400uL)溶液,混合物在4℃下,以120rpm的速度摇动24小时。离心分离得到小球,在用超纯水洗涤两次后,真空冻干得到八肽修饰的磁纳米粒子 (MitP-MNPs)。
本发明中所述八肽为异硫氰酸荧光素标记的线粒体靶向八肽(MitP, FITC-ACP-Fx-r-Fx-K-Fx-r-Fx-K,Mw=1701)。
图3为肽修饰的磁纳米粒子(MitP-MNPs)的合成路线。
4)抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备
将八肽修饰的磁纳米粒子(MitP-MNPs)的水溶液(0.2mg/ml)与β-环糊精修饰的透明质酸(HACD)的水溶液(0.2mg/ml)混合在一起,并且超声5分钟,即制备得到超分子组装体
图4为超分子组装体的制备和表征。(A)该超分子纳米组装体的制备示意图,(B)八肽修饰的磁纳米粒子(MitP-MNPs)的透射电子显微镜图,(C)超分子纳米组装体的透射电子显微镜图,(D)八肽修饰的磁纳米粒子(MitP-MNPs)和超分子纳米组装体的动态光散射图。图中表明所述纳米纤维的直径为几百纳米,长度大约为几微米,能够有效地吸引和聚集肿瘤细胞,抑制肿瘤细胞的侵袭和扩散。
以上所得超分子纳米组装体的磁场和光照响应性的实验验证:
将该超分子纳米组装体加入培养皿中并立即在普通室内用共聚焦显微镜或光学显微镜观察。为了检查地磁效应,在配备有场消除系统的房间中观察纳米聚集体的形成,该室可以屏蔽任何人造磁场。并在传统指南针不能准确地工作的金属包裹的TEM室中观察纳米聚集体的形成,该房间在很大程度上屏蔽地磁场。为了探索磁场强度与组装生长速率之间的关系,记录超分子纳米纤维的延伸长度与生长时间的关系,并且根据不同的磁场强度计算这些组装体的生长速率。图5表明,该超分子组装体有着对地磁场或弱磁场响应性,并且磁场强度越大,超分子纳米组装体的生长速率越快。
自组装纳米纤维的光响应调控使用竞争客体分子一种偶氮类分子(arylazopyrazoles carboxylate,AAP)进一步测试。这种新型的光敏分子在反式和顺式光致异构化过程中与β-CD具有可逆的包合作用。将反式AAP添加到该超分子组装体中可以严重破坏纤维组织,并且仅形成无序纳米聚集体。当在 365nm(UV)下照射时,由于顺式AAP不能再被封装在β-CD,腔中规则纳米纤维结构重现并显示出磁性定向响应能力。在交替的UV和Vis光照射下对这种光驱动的形态转化的可逆性进行研究。图6表明,含有HACD,MitP-MNPs和 trans-AAP的三元体系显示随机分布的纳米团簇和纳米粒子。然而,在365nm 照射10分钟后,无序纳米团簇逐渐转变为规则的纳米纤维,并随着地球磁场的方向而生长。纳米纤维通过在520nm进一步照射而被分解,并且在365nm的 UV照射下重新组装。
具体应用效果:
将RFP标记的A549细胞株A549-Luc2-tdT-2加入到已经胶凝的Matrigel基质胶中,然后加入MitP-MNP或超分子组装体,在培养12 小时之后,在共聚焦显微镜下观察,位于基质胶中不同高度的细胞荧光强度通过Image J软件检测。并用胰蛋白酶(2.5%)处理基质胶来确定细胞内是否形成超分子纳米纤维。体外A549-Luc2-tdT-2细胞株的迁移是通过刮取六孔板中粘附细胞来检测的,随后加入MitP-MNP或超分子组装体培养 12小时,之后在间隙观察细胞迁移。图7表明,超分子组装体对肿瘤细胞侵袭和转移有着强烈的抑制作用,并且在细胞间形成了纳米纤维,其可能作为细胞迁移的屏障。
四周的BALB/c雌性小鼠预先用环磷酰胺(200mg/kg)处理两天,之后皮下注射200uLA549-Luc2-tdT-2细胞悬浮液(1×108细胞/ml)。小鼠分成3组,每组5只。三天之后,MitP-MNP(80mg/kg)或(MitP-MNP 80mg/kg,HACD 80mg/kg)超分子组装体静脉注射到小鼠体内,对照组仅注射盐水。在2天之后观察A549-Luc2-tdT-2的分布,记录小鼠存活率。图7表明,在体内肿瘤转移模型中,强烈抑制RFP标记的肿瘤细胞的迁移,肿瘤细胞在肿瘤细胞注射部位附近受到限制。与之对照的是,MitP-MNP不能抑制肿瘤转移,并且肿瘤细胞远离注射部位迁移到颈部。并且,所有肿瘤负荷小鼠在治疗后存活,而对照组和MitP-MNP处理的小鼠在肿瘤细胞感染6天后死亡。此外,组织病理学观察显示超分子聚集体对网状内皮系统(RES)器官包括肝脏,脾脏和肾脏没有影响,表明聚集体具有良好的生物相容性。同时,流式细胞仪显示组装体显示出与RFP标记的肿瘤细胞比未标记的293T正常细胞具有更强的结合亲和力,表明含有HA 的组装体可以专一地和主动地靶向结合肿瘤细胞。生物相容的 超分子组装体可以有效抑制肿瘤侵袭和转移,图8示出了本发明超分子组装体在抑制肿瘤侵袭和转移应用示意图。
Claims (8)
1.一种抑制肿瘤侵袭和扩散的双重调控的超分子组装体,其构筑单元以β-环糊精修饰的透明质酸为主体,以八肽修饰的磁纳米粒子为客体,通过超分子主客体相互作用构筑的纳米超分子纤维聚集体;其中β-环糊精修饰的透明质酸为单-6-脱氧-6-乙二胺基-β-环糊精与透明质酸钠通过酰胺缩合得到,八肽修饰的磁纳米粒子为硅烷化的Fe3O4磁纳米粒子与异硫氰酸荧光素标记的线粒体靶向八肽通过共价连接得到。
2.根据权利要求1所述抑制肿瘤侵袭和扩散的双重调控的超分子组装体,其特征在于:纳米纤维的直径为几百纳米,长度大约为几微米,能够有效地吸引和聚集肿瘤细胞,抑制肿瘤细胞的侵袭和扩散。
3.一种如权利要求1所述的抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,其特征在于包括以下步骤:
1)β-环糊精修饰的透明质酸HACD的合成
将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和N-羟基琥珀酰亚胺磺酸钠盐NHSS添加到透明质酸钠的磷酸缓冲溶液中,混合物在25℃下搅拌30分钟,然后将单-6-脱氧-6-乙二胺基-β-环糊精的磷酸缓冲溶液加入到反应体系中,并在室温下搅拌24小时,反应结束后,在超纯水中透析5天后冻干,得到白色粉末状产物;
2)磁纳米粒子MNPs的合成
采用共沉淀法制备了本发明中所用的磁纳米粒子,将FeCl2和FeCl3的水溶液混合,然后将氢氧化钠溶液慢慢加入到混合物中,得到黑色溶液,加热溶液,得到沉淀并抽滤,用蒸馏水洗涤至中性后,在室温下干燥得到磁纳米粒子;
3)八肽修饰的磁纳米粒子MitP-MNPs的合成
将所得磁纳米粒子悬浮在乙醇中,向其中加入氨基丙基三乙氧基甲硅烷APTES,混合物在80℃下搅拌2小时,离心分离得到产物,并用乙醇洗涤三次,超纯水洗涤两次,得到有游离氨基的磁纳米粒子MNP-NH2;
将制备的有游离氨基的磁纳米粒子悬浮在8%的戊二醛的磷酸缓冲液中,混合物在室温下摇动6小时,离心分离得到小球状的带有戊二醛的磁纳米粒子,将这些小球用磷酸缓冲液洗涤3次后,悬浮在磷酸缓冲液中,然后加入异硫氰酸荧光素标记的线粒体靶向八肽的磷酸缓冲溶液,混合物在4℃下,以120rpm的速度摇动24小时,离心分离得到小球,在用超纯水洗涤两次后,真空冻干得到八肽修饰的磁纳米粒子MitP-MNPs;
4)抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备
将八肽修饰的磁纳米粒子MitP-MNPs的水溶液与β-环糊精修饰的透明质酸HACD的水溶液混合在一起,并且超声5分钟,即制备得到超分子组装体
4.根据权利要求3所述抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,其特征在于:步骤1)中透明质酸钠的溶液用量为3.33g/L;单-6-脱氧-6-乙二胺基-β-环糊精的溶液用量为0.1mol/L,反应液中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC,N-羟基琥珀酰亚胺磺酸钠盐NHSS,单-6-脱氧-6-乙二胺基-β-环糊精和透明质酸钠的摩尔比为1:1:1.143:0.00248;所述β-环糊精修饰的透明质酸HACD的合成中的磷酸缓冲液为PBS,0.1M,pH 7.2。
5.根据权利要求3所述抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,其特征在于:步骤2)中所述FeCl2和NaOH的溶液浓度均为0.3mol/L,FeCl3的溶液浓度为0.6mol/L,FeCl2,FeCl3和NaOH溶液所用体积均为100ml;所述磁纳米粒子MNPs的直径为10-20nm。
6.根据权利要求3所述抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,其特征在于:步骤3)中所述八肽为异硫氰酸荧光素标记的线粒体靶向八肽MitP,FITC-ACP-Fx-r-Fx-K-Fx-r-Fx-K,Mw=1701;所述磁纳米粒子质量为40mg,乙醇的体积为40ml,氨基丙基三乙氧基甲硅烷APTES的体积为2ml,磷酸缓冲溶液为PBS,pH 7.4,体积为20ml,八肽溶液浓度为1mM,体积为400uL。
7.根据权利要求3所述抑制肿瘤侵袭和扩散的双重调控的超分子组装体的制备方法,其特征在于:步骤4)中所述八肽修饰的磁纳米粒子MitP-MNPs的水溶液与β-环糊精修饰的透明质酸HACD的水溶液的浓度均为0.2mg/mL。
8.一种如权利要求1所述抑制肿瘤侵袭和扩散的双重调控的超分子组装体的应用,其特征在于:其分子组装过程受磁场和光照调控,特别是地磁场和弱磁场,该超分子组装体可以诱导线粒体功能障碍和在细胞间的聚集,最终在体内和体外条件下导致对肿瘤侵袭和转移的特异性抑制,具体实施方法为:将八肽修饰的磁纳米粒子MitP-MNPs的水溶液与β-环糊精修饰的透明质酸HACD的水溶液混合在一起,并且超声5分钟,即制备得到超分子组装体 所述八肽修饰的磁纳米粒子MitP-MNPs的水溶液与β-环糊精修饰的透明质酸HACD的水溶液的浓度均为0.2mg/mL。
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