CN117442749A - 一种rvg15肽修饰的脑靶向性金属有机框架药物载体及其制备方法和应用 - Google Patents
一种rvg15肽修饰的脑靶向性金属有机框架药物载体及其制备方法和应用 Download PDFInfo
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- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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Abstract
本发明属于医药技术领域,公开一种基于狂犬病毒糖蛋白衍生肽15(RVG‑15)修饰的脑靶向性金属有机框架药物载体的制备方法和应用。本发明以脑靶向肽RVG为靶向基团,与PEG共价结合得到RVG‑PEG‑COOH靶向偶联物。然后将RVG‑PEG‑COOH通过正负电荷相互作用修饰在金属有机框架材料ZIF‑8表面,制得脑靶向性金属有机框架药物载体。本发明所制备的脑靶向性金属有机框架药物载体可以通过简单一步包载法实现对化疗药物的包载,具有载药率高、稳定性好、脑靶向效果强等优点。
Description
技术领域
本发明属于医药技术领域,具体涉及一种RVG肽修饰的脑靶向性金属有机框架药物载体的制备方法和应用。
背景技术
脑胶质瘤是中枢神经系统最常见的恶性肿瘤,预后差且易复发,严重威胁人类生命安全。目前临床上治疗脑胶质瘤主要采用手术的方式切除癌变组织,同时辅以化疗、放疗进行术后维持治疗,杀死残存的肿瘤细胞,以延长患者的生存时间。然而由于血脑屏障的存在,化疗药物普遍面临因透过血脑屏障效率低而发挥药效不理想的状况,并伴有全身毒副作用大等问题。因此,通过巧妙的设计脑靶向性药物载体以提高化疗药物透过血脑屏障效率,改变化疗药物在体内的分布,减少系统性毒性,对脑胶质瘤的治疗具有重要意义。
金属有机框架材料(MOFs)是一类新型多孔性纳米材料,近年来在药物装载和输送方面领域的运用备受关注。与传统的纳米材料相比,MOFs材料具有多孔性,较大的比表面积,对化疗药物可实现较高的载药量。同时MOFs材料由金属离子与有机配体通过配位键构成,具有优秀的化学稳定性和物理稳定性,可避免药物在血液循环过程中早泄或突释。沸石咪唑骨架-8(Zeolitic imidazolate frameworks-8,ZIF-8)是最具有代表性的一种MOFs材料,其骨架结构是由二甲基咪唑中的N原子与金属Zn离子通过配位键相连构成的四面体结构。除了具备MOFs的固有优势外,ZIF-8具有灵敏的pH敏感性,其结构在中性生理条件下可以高度稳定,而进入肿瘤细胞的内涵体/溶酶体环境中可以发生分解,可以实现负载药物在肿瘤部位的可控、精准释放,满足新型纳米载药体系在载药量、稳定性及药物释放等多方面的要求。因此,选择金属有机框架材料ZIF-8作为化疗药物的载体材料构建纳米载药体系,实现其减毒增效治疗。
一种高效的脑靶向性药物载体一般应该具备几个典型特征:脑部转运的血脑屏障(Blood brain barrier,BBB)通透性高、脑靶向性好、生物安全性高等特点。狂犬病毒衣壳糖蛋白(Rabies virus glycoprotein,RVG)是唯一暴露在狂犬病毒膜外的一种结构蛋白,在狂犬病毒的透过血脑屏障和侵袭脑组织过程中发挥重要作用。RVG可以作为一种关键配体,其介导狂犬病毒与内源性受体结合,继而通过受体介导的转胞吞(Receptor-mediatedtranscytosis,RMT)机制透过血脑屏障侵入脑组织。研究表明,RVG不仅可以使狂犬病毒侵入神经细胞,还能够携带其他病毒经突触逆行转移进入中枢神经系统(Central nervoussystem,CNS)。基于病毒能够进入神经系统的仿生原理,可将RVG用于协助治疗性药物分子透过血脑屏障快速进入脑内。RVG-29是狂犬病毒衣壳糖蛋白衍生的一种含有29个氨基酸序列的多肽,具有狂犬病毒衣壳糖蛋白相似的血脑屏障透过特征。RVG-29通过与血脑屏障上的乙酰胆碱受体进行特异性结合,协助治疗性化疗药物跨过血脑屏障进入脑部疾病病灶,因而被认为是一种高效的脑靶向材料。然而RVG-29具有结构过长、分子量高等特点,对所修饰的纳米粒的粒径具有不利影响,因而使其运用场景受限。本课题组前期对狂犬病毒衣壳糖蛋白衍生肽的氨基酸序列及对应的靶向功能进行考察,筛选出一种由15个氨基酸构成的短肽RVG-15。与RVG-29相比,RVG-15(序列YTIWCDIFTNSRGKRC)的氨基酸序列较短,分子量更小,而且同时保持高效的脑靶向效率,并有利于缩小所制备的纳米粒粒径。本课题组前期构建了以RVG-15为靶头修饰的脑靶向载体递送核酸入脑,用以治疗脑胶质瘤,体外实验与体内实验均得到较好效果(黄伟.一种狂犬病毒糖蛋白衍生肽修饰的脑靶向核酸递送载体及其应用:中国,201710277357.8[P].2017-4-25.)。本发明将该RVG-15修饰至羧基化的聚乙二醇中,构建RVG15-PEG-COOH脑靶向偶联物,然后将RVG-PEG通过正负电荷相互作用修饰在金属有机框架材料ZIF-8表面,制得脑靶向性金属有机框架药物载体。可包载递送传统化药入脑,对那些体外有效但体内难以跨越BBB的药物入脑治疗脑部疾病提供了一个种新方法。
发明内容
本发明解决的技术问题是克服现有化疗药物难以透过血脑屏障的缺陷,提供一种狂犬病毒糖蛋白衍生肽15(RVG15)修饰的脑靶向性金属有机框架药物载体的制备方法和应用。
具体地,本发明是通过如下所述的技术方案实现的:
本发明的第一方面,首次提供了一种具有脑靶向功能的狂犬病毒衍生肽-羧基化聚乙二醇偶联物(RVG15-PEG-COOH),其中狂犬病毒衍生肽(RVG15)可通过n型乙酰胆碱受体介导跨血脑屏障,脑靶向效率与已报道的RVG-29相当,而分子量更小,有利于缩小所制备的纳米粒粒径。本课题组前期已经对RVG-15的氨基酸序列进行公开报道(黄伟.一种狂犬病毒糖蛋白衍生肽修饰的脑靶向核酸递送载体及其应用:中国,201710277357.8[P].2017-4-25.)。狂犬病毒衍生肽-羧基化聚乙二醇偶联物(RVG15-PEG-COOH)的结构如图(Ⅱ)所示。其中RVG15的氨基酸序列可为D型或L型,优选L型。聚乙二醇的聚合度n为5~450,优选8~230,进一步优选18~115,更优选25~55,进一步更优选30~50,最优选40~45,进一步最优选45。R为羧酸基。
本发明还提供了上述狂犬病毒衍生肽-羧基化聚乙二醇偶联物(RVG15-PEG-COOH)的制备方法,其制备方法为:在PEG-COOH末端引入马来酰亚胺基团(MAL),RVG15末端引入半胱氨酸(Cys),二者可通过迈克尔加成反应得到RVG15-PEG-COOH,反应如图(III)示。
优选的制备方法如下:
(1)取摩尔比略大于1:1的RVG15-Cys和MAL-PEG2000-COOH溶于pH为8的HEPEs,氮气保护下搅拌下常温反应18h。
(2)选用截留分子量为200-25000的透析袋进行透析,优选截留分子量为3000的透析袋。去离子水中透析,透析时间为12h。
(3)液体冷冻干燥后得到冻干粉备用,即得具有脑靶向功能的狂犬病毒衍生肽-羧基化聚乙二醇偶联物(RVG15-PEG-COOH)。
本发明第二方面,首次提供了一种RVG15肽修饰的脑靶向性金属有机框架药物载体。其中脑靶向功能材料RVG15-PEG-COOH。金属有机框架材料可为ZIF-8、UiO-66-COOH或UiO-66-NH2,优选ZIF-8。活性药物可为雷公藤甲素、阿霉素、丝裂霉素C、丝裂霉素C前药、丝裂霉素C衍生物、多西紫杉醇或羟基喜树碱,优选多西紫杉醇。
本发明还提供一种上述RVG15肽修饰的脑靶向性金属有机框架药物载体的制备方法,其制备方法为:将含金属盐的二甲基亚砜溶液与含配体的水溶液,混合反应,通过离心纯化即得金属有机框架材料;将金属有机框架材料分散至RVG15-PEG-COOH脑靶向偶联物溶液中混合均匀,搅拌反应过夜后,离心纯化获得的固体即为RVG15肽修饰的脑靶向性金属有机框架药物载体。
当金属有机框架材料为ZIF-8时,优选的制备方法如下:
将80mg六水合硝酸锌溶于3ml二甲基亚砜中,加入含有800mg的二甲基咪唑水溶液7ml,室温反应30min,在15000rpm/min的转速下离心15min。然后用甲醇洗涤三次,即得金属有机框架材料ZIF-8。将10mg ZIF-8分散至浓度为0.4mg/mL的RVG15-PEG-COOH偶联物5ml水溶液中,搅拌反应12h后,离心纯化获得的固体即为RVG15-PEG@ZIF-8。
本发明还提供了RVG15肽修饰的脑靶向性金属有机框架药物载体包载活性药物的制备方法,载体可以通过简单一步包载法实现对化疗药物的包载,在形成金属有机框架材料过程中活性药物被荷载进入金属有机框架材料的孔隙中。其制备方法为:将抗肿瘤药物溶液加入金属盐的二甲基亚砜溶液中获得混合液,反应一段时间后,在搅拌条件下滴加含有配体的水溶液中形成混悬液,离心分离,洗涤,干燥得到固体。将固体分散至RVG15-PEG-COOH偶联物水溶液中混合均匀,反应结束后离心分离,洗涤,干燥。RVG15肽修饰的脑靶向性金属有机框架药物载体对活性药物的载药量为5%-30%。
当金属有机框架材料为ZIF-8,抗肿瘤药物为多烯紫杉醇(DTX)时,其优选的制备方法为:
将5mg多烯紫杉醇、80mg六水合硝酸锌溶于3ml二甲基亚砜中,搅拌30min后。在室温条件下,向其中加入含有800mg的二甲基咪唑水溶液,反应30min后。通过15000rpm/min的转速离心15min。用甲醇洗涤三次,即得DTX@ZIF-8。将10mg DTX@ZIF-8分散至浓度为0.4mg/mL RVG15-PEG-COOH偶联物5ml水溶液中,室温搅拌反应12h后,离心纯化获得的固体即为RVG15-PEG@DTX@ZIF-8。
本发明还提供了RVG15肽修饰的脑靶向性金属有机框架药物载体在体外药物递送中的应用。其方法和步骤如下:
将载药脑靶向性有机金属骨架用培养液稀释成预定浓度的溶液。每个浓度设6个复孔,并设对照组和调零组。将适量对数生长期的大鼠神经胶质瘤C6细胞接种于96孔板中,继续培养24h后,介质以含有有机金属骨架溶液的新鲜培养液替换,分别继续培养24h或48h。每孔加入浓度为10%的CCK-8试剂200μl。继续孵育2h,于450nm处测定吸收值,并以650nm为参考波长。计算细胞活力,公式如下:
细胞活力(%)=[(OD实验-OD调零)/(OD对照-OD调零)]×100
本发明还提供了RVG15肽修饰的脑靶向性金属有机框架药物载体在体内治疗脑胶质瘤的运用。其方法和步骤如下:
取荷C6脑胶质瘤小鼠随机分为4组,每组5只,静脉注射载药脑靶向性有机金属骨架0.2mL,同时设置生理盐水、游离活性药物及无RVG15肽修饰的载药有机金属骨架作为对照组。每三天给药一次,给药5次后将小鼠用异氟烷麻醉,用小动物核磁成像仪观察脑部瘤的大小。
本发明的有益效果如下:
1、本发明制备的狂犬病毒衍生肽-羧基化聚乙二醇偶联物(RVG15-PEG-COOH),具有狂犬病毒衣壳糖蛋白相似的血脑屏障透过特征,而且分子量小,更有利于形成小粒径的纳米粒,便于实现脑靶向递送,同时具有合成工艺简单、生产成本低的优势。
2、本发明制备的RVG15肽修饰的脑靶向性金属有机框架药物载体,具有良好的血脑屏障穿透能力,生物相容性和血液长循环的功能,可解决荷载的活性药物难以入脑的瓶颈问题,为脑胶质瘤治疗提供了一种全新的方案。
3、本发明制备的RVG15肽修饰的脑靶向性金属有机框架药物载体,可用于包载多种无法跨血脑屏障的活性药物,对开发那些体外药理活性好,但体内无法跨BBB入脑发挥作用的传统药物提供了一个新途径。
4、本发明制备的RVG15肽修饰的脑靶向性金属有机框架药物载体,制备工艺简单、快速,稳定性和可重复性高。
附图说明
图1:Mal-PEG-COOH的1H-NMR图谱
图2:RVG15-PEG-COOH的1H-NMR图谱
图3:RVG15的质谱图
图4:Mal-PEG-COOH的质谱图
图5:RVG15-PEG-COOH的质谱图
图6:DTX@ZIF-8的粒径图
图7:DTX@ZIF-8的电位图
图8:空白金属有机骨架ZIF-8的扫描电镜图
图9:载药金属有机骨架DTX@ZIF-8的扫描电镜图
图10:RVG15-PEG@DTX@ZIF-8的粒径图
图11:RVG15-PEG@DTX@ZIF-8的电位图
图12:RVG15-PEG@DTX@ZIF-8的扫描电镜图
图13:空白金属有机框架ZIF-8对C6细胞的毒性
图14:空白金属有机框架ZIF-8对HBMEC细胞的毒性
图15:24h载药金属有机框架对C6细胞增殖抑制情况
图16:48h载药金属有机框架对C6细胞增殖抑制情况
图17:香豆素6标记的金属有机骨架在大鼠神经胶质瘤C6细胞的摄取情况
图18:核磁共振成像MRI中小鼠脑部肿瘤成像
具体实施方式
以下实施例旨在说明本发明而不是对本发明的进一步限定。下面参照实施例进一步详细阐述本发明,但本发明并不限于这些实施例及使用的制备方法。而且,本领域技术人员根据本发明的描述可以对本发明进行等同替换、组合、改良或修饰,但这些都将包括在本发明的范围内。
实施例1:RVG15-PEG-COOH的合成
称取19mg RVG15-Cys以及26mg Mal-PEG-COOH溶于10ml的pH=8.0HEPEs缓冲液中,氮气保护下,室温搅拌反应18h。反应结束后,加入透析袋(MWCO=3000Da)中,使用去离子水透析12h,样品进行冷冻干燥,得到纯化的RVG15-PEG-COOH。
取冻干粉末RVG15-PEG-COOH溶解于CDCl3,采用400MHz的核磁共振氢谱(1H-NMR)进行结构确认,结果见图。从图1可知,化学位移6.7ppm归属于Mal-PEG-COOH结构中的马来酰亚胺基团(Mal)。从图2可知,Mal-PEG-COOH与RVG15-Cys的反应后生成RVG15-PEG-COOH,化学位移6.7ppm处的马来酰亚胺基团(Mal)消失,证明RVG15-Cys中的半胱氨酸残基中的巯基通过迈克尔加成反应制备,说明RVG15-PEG-COOH成功合成。
采用基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF-MS)分析,结果如图3、4及图5所示。RVG15-Cys分子量约为1963,Mal-PEG-COOH的分子量约为1900-2100,目标产物RVG15-PEG-COOH约为4000,接近两反应物的分子量和,且分子量分布与反应物Mal-PEG-COOH较为一致,说明成功合成目标产物且纯度较高。
实施例2:包载多烯紫杉醇的金属有机骨架ZIF-8的制备
在10ml离心管中,将5mg多烯紫杉醇、80mg六水合硝酸锌溶于3ml二甲基亚砜中,超声将其溶解,搅拌30min后。在室温条件下,向其中加入含有800mg的二甲基咪唑水溶液7ml,反应30min后。取反应液于2ml离心管中,15000rpm/min的转速下离心15min。然后用甲醇洗涤三次,即得DTX@ZIF-8。
DTX@ZIF-8NPs的物理性质:如图6和7所示,以动态光散射法测得粒径为94.75nm,PDI为0.043,Zeta电位为26.7V;经高效液相色谱法测定纳米粒的包封率为82%,载药量为16.5%。如图8和9所示,通过扫描电镜可见,空白ZIF-8材料为单面为菱形的十二面体,载药过后仍为单面为菱形的十二面体,说明载药并不影响材料本身的物理性质。
实施例3:脑靶向性金属有机骨架的RVG15-PEG@DTX@ZIF-8的制备
在10ml离心管中,将2mg RVG15-PEG-COOH溶于5ml水中,冰浴超声10min使其均匀分散于水溶液中,然后加入10mgDTX@ZIF-8。搅拌12h后结束反应。取反应液于2ml离心管中,10000rpm/min的转速下离心10min。然后用水洗涤三次,即得RVG15-PEG@DTX@ZIF-8。
RVG15-PEG@DTX@ZIF-8NPs的物理性质:如图10和11所示,以动态光散射法测得粒径为127.4nm,PDI为0.081,Zeta电位为4.84V。如图12所示,通过扫描电镜得到,通过靶头RVG15-PEG-COOH的链接单面为菱形的十二面体金属有机骨架ZIF-8转变为圆球形,证明靶头的成功连接。
实施例4:材料细胞毒性研究
将空白纳米粒溶液用培养液稀释成预定浓度的溶液。每个浓度设6个复孔,并设对照组和调零组。将适量对数生长期的大鼠神经胶质瘤C6细胞、人脑微血管内皮细胞HBMEC细胞接种于96孔板中,继续培养24h后,介质以含有不同空白金属有机骨架ZIF-8溶液的新鲜培养液替换,分别继续培养24h或48h。每孔加入浓度为10%的CCK-8试剂200μl。继续孵育2h,于450nm处测定吸收值计算细胞活力,公式如下:
细胞活力(%)=[(OD实验-OD调零)/(OD对照-OD调零)]×100
由图13和14的结果表明,空白金属有机骨架ZIF-8在所测定的浓度范围内,C6、HBMEC细胞的活力均在85%以上,表明材料本身对细胞毒性较小。说明本专利提供的金属有机骨架ZIF-8在安全性方面存在优势。
实施例5:细胞增殖抑制研究
将载药有机金属骨架RVG15-PEG@DTX@ZIF-8、DTX@ZIF-8和多烯紫杉醇注射液用培养液稀释成预定浓度的溶液。每个浓度设6个复孔,并设对照组和调零组。将适量对数生长期的大鼠神经胶质瘤C6细胞接种于96孔板中,继续培养24h后,介质以含有有机金属骨架溶液的新鲜培养液替换,分别继续培养24h或48h。每孔加入浓度为10%的CCK-8试剂200μl。继续孵育2h,于450nm处测定吸收值,并以650nm为参考波长。计算细胞活力,公式如下:
细胞活力(%)=[(OD实验-OD调零)/(OD对照-OD调零)]×100
图15和16结果表明,在药物浓度为0.01-10μg/mL的范围中,RVG15-PEG@DTX@ZIF-8对细胞的增殖抑制强于市售多烯紫杉醇注射液。
实施例6:激光共聚焦显微镜观察金属有机框架入胞情况
利用激光共聚焦定性观察大鼠神经胶质瘤C6细胞对金属有机框架的摄取情况。将C6细胞以1.5×105个细胞/孔接种于底部预先放置细胞爬片的12孔板中,在37℃、5%CO2培养箱中培养24小时,待细胞贴壁后,分别于不同的时间点,向孔板内加入由无血清培养基稀释的游离Cou-6、Cou-6@ZIF-8、RVG15-PEG@Cou-6@ZIF-8使最终Cou-6的浓度为5μg/ml,再置于37℃、5%CO2培养箱中培养2小时。培养结束后,弃去培养液并用PBS清洗两次,4%多聚甲醛避光固定20分钟,PBS清洗后用1μg/ml DAPI液染核15min,最终用PBS清洗后取出12孔板底部细胞爬片,将其置于用抗荧光淬灭剂处理后的载玻片上,最后,用激光共聚焦显微镜观察金属有机框架入胞情况。
结果如图17所示,RVG15-PEG@Cou-6@ZIF-8比其他组的具有较高的摄取效率,且具有明显的时间依赖性,随着时间延长,摄取效率逐渐升高。
实施例7:脑靶向金属有机骨架体内药效学评价
取4周龄的ICR小鼠,荷C6脑胶质瘤小鼠模型建立7天后,将小鼠随机分为4组,每组5只,按多烯紫杉醇5mg/kg的剂量分别尾静脉0.2mL的RVG15-PEG@DTX@ZIF-8、DTX@ZIF-8和多西紫杉醇注射液以及生理盐水。每三天给药一次,给药5次后将小鼠用异氟烷麻醉,用小动物核磁成像仪观察脑部瘤的大小,结果如图18所示,与无靶头的纳米粒相比,RVG15-PEG@DTX@ZIF-8给药组的瘤面积更小,表明RVG15-PEG@DTX@ZIF-8可显著提高穿透血脑屏障的能力,对脑胶质瘤具有良好的治疗效果。
Claims (13)
1.一种RVG15肽修饰的脑靶向性金属有机框架药物载体,其特征在于,所述脑靶向性金属有机框架药物载体是以脑靶向肽RVG15为靶向基团,与PEG共价结合得到RVG15-PEG-COOH脑靶向偶联物,然后通过正负电荷相互作用,制得RVG15肽修饰的脑靶向性金属有机框架药物载体。
2.根据权利要求1所述的RVG15肽修饰的脑靶向性金属有机框架药物载体,其特征在于,所述金属有机框架材料是ZIF-8、UiO-66-COOH或UiO-66-NH2,优选地,所述金属有机框架材料是ZIF-8。
3.根据权利要求1所述的RVG15肽修饰的脑靶向性金属有机框架药物载体,其特征在于,所述RVG15-PEG-COOH脑靶向偶联物,其结构组成为:聚乙二醇分子链的一端通过马来酰亚胺基和半胱氨酸连接狂犬病毒衍生肽RVG15,分子链另一端连接羧基,结构式如图(Ι)所示。
4.根据权利要求3所述的RVG15肽修饰的脑靶向性金属有机框架药物载体,其特征在于,聚乙二醇的聚合度n优选8~230,进一步优选18~115,更优选25~55,进一步更优选30~50,最优选40~45,进一步最优选45。
5.权利要求1所述的RVG15肽修饰的脑靶向性金属有机框架药物载体的制备方法,步骤为:将含金属盐的二甲基亚砜溶液与含配体的水溶液,混合反应,通过离心纯化即得金属有机框架材料,将金属有机框架材料分散至RVG15-PEG-COOH脑靶向偶联物溶液中混合均匀,搅拌反应过夜后,离心纯化获得的固体即为RVG15肽修饰的脑靶向性金属有机框架药物载体。
6.根据权利要求5所述的RVG15肽修饰的脑靶向性金属有机框架药物载体的制备方法,其特征在于:含金属盐的二甲基亚砜溶液中金属离子浓度为1-100mg/mL,优选为8mg/mL;含配体的水溶液浓度为0.5-1000mg/mL,优选为80mg/mL。金属盐与配体的摩尔比为1:1至1:72,优选为1:36;将含金属盐的二甲基亚砜溶液与含配体的水溶液,室温反应10-120min,优选为30min;脑靶向偶联物材料溶液浓度为0.1-20mg/mL,优选为0.4mg/mL;将金属有机框架材料分散至RVG15-PEG-COOH溶液中混合均匀,室温搅拌6-48h,优选为12h;将金属有机框架材料分散至RVG15-PEG-COOH水溶液中混合均匀,金属有机框架材料浓度为0.1-40mg/mL,优选为2mg/mL;金属有机框架材料与RVG15-PEG-COOH偶联物的质量比为20:1-1:1,优选为5:1。
7.根据权利要求6所述的RVG15肽修饰的脑靶向性金属有机框架药物载体制备方法,其特征在于,当金属有机框架材料为ZIF-8时,其制备步骤为:将六水合硝酸锌溶于3ml二甲基亚砜中,加入含有二甲基咪唑水溶液7ml,六水合硝酸锌与二甲基咪唑的摩尔比为1:36。室温反应30min,在15000rpm/min的转速下离心15min。然后用甲醇洗涤三次,即得金属有机框架材料ZIF-8;将ZIF-8分散至含有RVG15-PEG-COOH偶联物的5ml水溶液中,二者的质量比为5:1。搅拌反应12h后,离心纯化获得的固体即为RVG15-PEG@ZIF-8。
8.权利要求1所述的RVG15肽修饰的脑靶向性金属有机框架药物载体在包载活性药物中的应用,所述活性药物为脂溶性药物和亲水性药物。
9.根据权利要求8所述的RVG15肽修饰的脑靶向性金属有机框架药物载体的应用,其特征在于,包载的活性药物包括抗肿瘤药物雷公藤甲素、阿霉素、丝裂霉素C、丝裂霉素C前药、丝裂霉素C衍生物、多西紫杉醇或羟基喜树碱;优选地,所述肿瘤药物为多烯紫杉醇。
10.根据权利要求8所述的RVG15肽修饰的脑靶向性金属有机框架药物载体的应用,其特征在于,载体可以通过简单一步包载法实现对化疗药物的包载,在形成金属有机框架材料过程中活性药物被荷载进入金属有机框架材料的孔隙中;其步骤包括:将抗肿瘤药物溶液加入金属盐的二甲基亚砜溶液中获得混合液,反应一段时间后,在搅拌条件下滴加含配体的水溶液中形成混悬液,离心分离,洗涤,干燥得到固体。将固体分散至RVG15-PEG-COOH偶联物水溶液中混合均匀,反应结束后离心分离,洗涤,干燥。
11.根据权利要求10所述的RVG15肽修饰的脑靶向性金属有机框架药物载体的应用,其特征在于,活性药物总浓度为0.1-50mg/mL,优选为0.2mg/mL;将活性药物加入至金属盐溶液中,室温反应3-120min,优选为30min;抗肿瘤药物溶液,溶剂优选二甲基亚砜、水、甲醇;所述RVG15肽修饰的脑靶向性金属有机框架药物载体对活性药物的载药量为5%-30%。
12.根据权利要求10所述的RVG15肽修饰的脑靶向性金属有机框架药物载体的应用,其特征在于,当金属有机框架材料为ZIF-8,抗肿瘤药物为多烯紫杉醇时,其步骤包括:将1份多烯紫杉醇、40份六水合硝酸锌溶于3ml二甲基亚砜中,搅拌30min后。在室温条件下,向其中加入含有400份的二甲基咪唑水溶液,反应30min后。通过15000rpm/min的转速离心15min;用甲醇洗涤三次,即得DTX@ZIF-8;将5份DTX@ZIF-8分散至含1份的RVG15-PEG-COOH偶联物水溶液中,搅拌反应12h后,离心纯化获得的固体即为RVG15-PEG@ZIF-8。
13.根据权利要求1所述的RVG肽修饰的脑靶向性金属有机框架药物载体的的应用,优选地,所述肿瘤为脑胶质瘤。
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