CN113616804B - 靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物 - Google Patents
靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物 Download PDFInfo
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- CN113616804B CN113616804B CN202111029111.1A CN202111029111A CN113616804B CN 113616804 B CN113616804 B CN 113616804B CN 202111029111 A CN202111029111 A CN 202111029111A CN 113616804 B CN113616804 B CN 113616804B
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Abstract
本发明涉及靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物。所述多功能纳米药物载体由下式所示结构的多聚硒代氨基酸两亲性嵌段共聚物自组装,继而通过乳铁蛋白修饰而成;其中,22≤n≤454、2≤x≤50、2≤y≤50,n、x、y均为整数。本发明药物载体能够负载药物分子,制成缓释控释的靶向药物输送***,能够将药物分子定向的送到病变部位,降低给药频率、提高治疗效果等。该药物载体兼具一般氨基酸类药物载体优势的同时,既具备了硒的多种生物学功能,又具备了靶向乳铁蛋白受体的特异性,是一类新型、靶向、多功能药物载体,适用于乳铁蛋白受体异常相关多种疾病的各类药物的药物载体研究、开发及临床应用。
Description
技术领域
本发明涉及生物医用药物载体与缓释材料技术领域,尤其涉及靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物。
背景技术
人体内拥有十分复杂的生理环境,药物从摄取到发挥作用需要经过多重障碍,往往最终只有一小部分药物能发挥药效,严重影响治疗效果,同时还会带来毒副作用。如何增强药物的利用率、安全性等对于提升疾病的治疗效果和人类健康具有极为重要的意义。近些年来,不同类型的药物载体相关研究受到人们的高度重视。
药物载体主要是天然或合成的高分子材料,以不同的形式与药物分子通过化学键合、物理吸附或包裹,构成药物控制***,可在不降低原药物分子药效并抑制其副作用的情况下,通过一系列物理、化学及生物控制,实现药物定时、定位、定量释放,协助增强其疗效。药物载体已经被运用于多种给药途径,包括注射、口服、透皮吸收等。药物载体种类较多,纳米药物载体是指粒径在10~1000nm的一类新型载体,由于其粒径比毛细血管通路小,且具有降低药物毒副作用、提高药物稳定性、缓释控释药物和药物靶向释放等优点,近年来,纳米药物载体逐渐引起人们的高度重视。纳米药物载体有聚合物胶束、纳米囊和纳米球、纳米脂质体、固体脂质纳米粒以及磁性纳米颗粒等。多种高分子材料可用于纳米药物载体的研究开发,但生物相容性、生物降解性和安全性等是必须要考虑的重要问题。
氨基酸是生物功能大分子蛋白质的基本组成单位,是构成机体营养所需蛋白质的基本物质。采用天冬氨酸、谷氨酸、赖氨酸、丙氨酸、苯丙氨酸等制备的聚氨基酸是一类低毒、生物相容性好、容易被机体吸收和代谢的生物全降解高分子材料,在药物载体领域发展潜力极大。但由于氨基酸之间的强氢键作用等,该类药物载体材料存在水溶性较差、在体内降解速率和周期难以掌控,且难以实现靶向传输等不足。
因此,现有技术仍有待于改进和发展。
发明内容
功能化、智能化已是当前纳米药物载体发展的战略性趋势。发明人研究发现,聚乙二醇(PEG)具有柔韧的亲水性长链,无毒无免疫原性,FDA已经批准用于临床使用,是目前已知亲水载体中最具有发展前途的材料之一。PEG与聚氨基酸结合形成嵌段共聚物,能够改善聚氨基酸的亲水性,减少体内蛋白质在材料表面的吸附和细胞的粘附等。由于PEG具有在体内循环时不易被免疫***识别的优点,可以保护聚氨基酸不受免疫***的破坏,延长材料在体内的循环时间等。此外,PEG两端可引入多种官能团,显著增强聚氨基酸类纳米药物载体的综合性能。
进一步研究发现,人体必需微量元素硒具有抗氧化、调节免疫、抗有害重金属、抗衰老等重要生物功能。硒缺乏与许多人类疾病的发病有关,包括糖尿病、癌症和神经退行性疾病等。人体摄入硒主要有两种方式:无机硒,利用率低,毒性大;有机硒,如硒代氨基酸,生物相容性更好,利用率高,更容易被人体吸收,具有更低的毒性和更高的安全性。故,硒代氨基酸的引入有望积极推动功能化纳米聚氨基酸类药物载体的研究开发。
乳铁蛋白(Lactoferrin,简称Lf)是一种多功能铁离子转运糖蛋白,属于转铁蛋白家族,主要在腺上皮细胞表达和分泌,主要调节内环境离子稳定、抗微生物感染、调节细胞生长、抑制血小板聚集、增强免疫等,而其生物学功能主要是由乳铁蛋白受体介导实现的。最近研究发现,阿尔兹海默病、帕金森病等中枢神经细胞退行性疾病等患者脑部神经元和微血管上皮细胞乳铁蛋白受体表达量特异性显著增加,其异常直接诱发、促进了上述多种复杂疾病。故,乳铁蛋白受体可作为功能纳米药物载体靶向传输药物的重要靶点。
为此,鉴于当前聚氨基酸类纳米载体主要功能单一,只能作为药物载体,缺乏靶向性等不足,本发明提供了一种靶向乳铁蛋白受体的多功能纳米药物载体,兼具一般氨基酸类纳米药物载体优势的同时,既具备了硒的多种生物学功能,又特异性靶向乳铁蛋白受体,是一类新型、靶向、多功能纳米药物载体。
具体地,本发明的技术方案如下:
一种靶向乳铁蛋白受体的多功能纳米药物载体,其中,所述多功能纳米药物载体包括纳米胶束和结合于所述纳米胶束表面的铁乳蛋白,所述纳米胶束由式(Ⅰ)所示结构的多聚硒代氨基酸两亲性嵌段共聚物自组装而成,所述纳米胶束通过所述R1与所述铁乳蛋白结合;
其中,22≤n≤454、2≤x≤50、2≤y≤50,n、x、y均为整数;
-R1选自-OCH3、-COOH、-NH2或-MAL;
-R2选自-CH(CH3)CH3、-H、-CH3、-CH2CH(CH3)CH3、-CH(CH3)CH2CH3、/>-CH2OH、/>-CH2SH、-CH2CH2SCH3、/>-CH(OH)CH3、/>
-R3选自-CH2CH2SeCH3或-CH2SeH。
可选地,所述纳米胶束以纳米球形、纳米棒状或纳米囊泡的形式存在。
一种本发明所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其中,包括以下步骤:
提供纳米胶束;
取乳铁蛋白分散到缓冲液中,加入EDCI和NHS,依次进行搅拌和避光室温活化处理;
将活化处理后的反应液中未反应的EDCI和NHS去除,然后分散于缓冲液中,得到活化的乳铁蛋白分散液;
向所述活化的乳铁蛋白分散液中加入所述纳米胶束,搅拌下反应过夜;
将反应后体系进行纯化处理,得到靶向乳铁蛋白受体的多功能纳米药物载体;
其中,所述纳米胶束为氨基修饰的纳米胶束,所述乳铁蛋白为羧基修饰的乳铁蛋白;
或者,所述纳米胶束为羧基修饰的纳米胶束,所述乳铁蛋白为氨基修饰的乳铁蛋白。
可选地,所述取乳铁蛋白分散到pH=6的0.01mol/L KH2PO4缓冲液中;
所述乳铁蛋白与EDCI、NHS用量比为1mg:2-6mg:4-8mg;
所述避光室温活化处理的时间为15min-12h。
一种本发明所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其中,包括以下步骤:
提供纳米胶束;
取乳铁蛋白溶解于硼酸钠溶液中,加入Traut’s试剂,进行搅拌;
将搅拌后反应液过Zeba脱盐柱,除去杂质,得到巯基化乳铁蛋白溶液;
将所述纳米胶束用NaH2PO4分散后,加入所述巯基化乳铁蛋白溶液,进行室温搅拌反应;
将所述室温搅拌反应后体系进行纯化处理,得到靶向乳铁蛋白受体的多功能纳米药物载体。
可选地,所述乳铁蛋白与Traut’s试剂的摩尔比为1:30-50;
所述巯基化乳铁蛋白与纳米胶束的用量比为1g:2-100mg;
所述室温搅拌反应的时间为6~12h。
一种载药组合物,其中,包括本发明所述的靶向乳铁蛋白受体的多功能纳米药物载体和包裹于所述多功能纳米药物载体内的药物。
可选地,所述药物选自抗AD药物、抗肿瘤药物、甾体类抗炎药物、非甾体抗炎药物、代谢调节类药物、抗生素、心血管药物、抗病毒药物、抗真菌药物、免疫调节剂中的一种或多种。
可选地,所述多功能纳米药物载体与药物的质量比为1mg:(0.1~10)mg。
有益效果:本发明提供的靶向乳铁蛋白受体的多功能纳米药物载体由式(Ⅰ)所示结构的多聚硒代氨基酸两亲性嵌段共聚物自组装,然后通过PEG外端官能团进行乳铁蛋白修饰,得到靶向乳铁蛋白受体的多功能纳米药物载体。该多功能纳米药物载体能够将药物分子定向的送到病变部位,提高药效,同时制成缓释控释的药物输送***,所包载的药物可以根据需要从输送***中缓释控释的释放,从而降低给药频率、提高治疗效果,并减少药物的毒副作用。与现有技术相比,本发明提供的多功能纳米药物载体兼具一般氨基酸类纳米药物载体优势的同时,既具备了硒的多种生物学功能,又具备了靶向乳铁蛋白受体的特异性,是一类新型、靶向、多功能药物载体,适用于乳铁蛋白受体异常相关多种疾病的各类药物的药物载体研究、开发及临床应用。
附图说明
图1为本发明实施例中所述多聚硒代氨基酸纳米球形胶束的TEM(透射电镜)图。
图2为本发明实施例中所述乳铁蛋白修饰多聚硒代氨基酸纳米球形胶束的TEM(透射电镜)图。
图3为本发明实施例中所述多聚硒代氨基酸纳米球形胶束的纳米激光粒度图。
图4为本发明实施例中所述乳铁蛋白修饰多聚硒代氨基酸纳米球形胶束的纳米激光粒度图。
图5为本发明实施例中所述乳铁蛋白修饰多聚硒代氨基酸纳米球形胶束的细胞靶向摄取激光共聚焦图。
具体实施方式
本发明提供靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明实施例提供一种靶向乳铁蛋白受体的多功能纳米药物载体,其中,所述多功能纳米药物载体包括纳米胶束和结合于所述纳米胶束表面的铁乳蛋白,所述纳米胶束由式(Ⅰ)所示结构的多聚硒代氨基酸两亲性嵌段共聚物自组装而成,所述纳米胶束通过所述R1与所述铁乳蛋白结合;
其中,22≤n≤454、2≤x≤50、2≤y≤50,n、x、y均为整数;
-R1选自-OCH3、-COOH、-NH2或-MAL(马来酰亚胺基团)等;
-R2选自-CH(CH3)CH3、-H、-CH3、-CH2CH(CH3)CH3、-CH(CH3)CH2CH3、/>-CH2OH、/>-CH2SH、-CH2CH2SCH3、/>-CH(OH)CH3、/>
-R3选自-CH2CH2SeCH3或-CH2SeH。
本发明实施例所述靶向乳铁蛋白受体的多功能纳米药物载体由所述纳米胶束中聚乙二醇外端的-OCH3、-COOH、-NH2或-MAL等官能团与乳铁蛋白的羧基或者氨基通过酯化或者酰胺缩合等多种方式进行化学连接,在纳米胶束表面覆盖一层蛋白“外衣”,得到乳铁蛋白修饰的靶向乳铁蛋白受体的多功能纳米药物载体。利用所述多功能纳米药物载体将药物分子定向的送到病变部位,提高药效,同时制成缓释控释的药物输送***,所包载的药物可以根据需要从输送***中缓释控释的释放,从而降低给药频率、提高治疗效果,并减少药物的毒副作用。与现有技术相比,本发明实施例提供的多功能纳米药物载体兼具一般氨基酸类纳米药物载体优势的同时,既具备了硒的多种生物学功能,又具备了靶向乳铁蛋白受体的特异性,是一类新型、靶向、多功能药物载体,适用于乳铁蛋白受体异常相关多种疾病的各类药物的药物载体研究、开发及临床应用。
具体的,本发明实施例式(Ⅰ)所示结构中的聚乙二醇胺一方面用于提供亲水端,形成纳米胶束的外壳,这样可以延长纳米胶束在体内循环时间;另一方面用于提供靶向修饰基团,不仅提高药物的治疗效果,还能降低药物的毒副性。聚硒代氨基酸提供疏水端,用于载药,以提供一种稳定、既具有药物缓释控释功能,同时又具备硒的多种生物学功能的纳米药物载体。离子型聚氨基酸中的活泼离子基团可用于嵌段共聚物之间的交联,以提供一种更稳定,药物缓释控释功能更优的纳米药物载体。
本发明实施例提供一种如上所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其中,包括以下步骤:
S10、提供纳米胶束;
S11、取乳铁蛋白分散到缓冲液中,加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)和N-羟基丁二酰亚胺(NHS),依次进行搅拌和避光室温活化处理;
S12、将活化处理后的反应液中未反应的EDCI和NHS去除,然后分散于缓冲液中,得到活化的乳铁蛋白分散液;
S13、向所述活化的乳铁蛋白分散液中加入所述纳米胶束,搅拌下反应;
S14、将反应后体系进行纯化处理,得到靶向乳铁蛋白受体的多功能纳米药物载体;
其中,所述纳米胶束为氨基修饰的纳米胶束(即PEG外端的官能团为氨基),所述乳铁蛋白为羧基修饰的乳铁蛋白;
或者,所述纳米胶束为羧基修饰的纳米胶束(即PEG外端的官能团为羧基),所述乳铁蛋白为氨基修饰的乳铁蛋白。
在一种实施方式中,所述乳铁蛋白与EDCI、NHS用量比为1mg:2-6mg:4-8mg。
在一种实施方式中,所述KH2PO4缓冲液pH可以适当上下浮动1-2个单位。所述KH2PO4缓冲液为0.01mol/L~0.1mol/L。
在一种实施方式中,所述取乳铁蛋白分散到pH=6的0.01mol/L KH2PO4缓冲液中。
在一种实施方式中,所述乳铁蛋白与KH2PO4缓冲液用量比为1mg:1-5mL。
在一种实施方式中,所述避光室温活化处理的时间为15min-12h,优选时间为20min。
本实施例中,所述纳米胶束由式(Ⅰ)所示结构的多聚硒代氨基酸两亲性嵌段共聚物(式(Ⅰ)中,-R1为-COOH或-NH2)自组装而成,所述纳米胶束和所述共聚物的制备方法详见申请号为CN202011344949.5,专利名称为一种多聚硒代氨基酸两亲性嵌段共聚物及制备方法与应用的专利文件中,在此不再赘述。
本发明实施例提供一种如上所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其中,包括以下步骤:
S20、提供纳米胶束;
S21、取乳铁蛋白溶解于硼酸钠溶液中,加入Traut’s试剂,进行搅拌;
S22、将搅拌后反应液过Zeba脱盐柱,除去杂质,得到巯基化乳铁蛋白溶液;
S23、将所述纳米胶束用NaH2PO4分散后,加入所述巯基化乳铁蛋白溶液(巯基能够与纳米胶束的-MAL反应),进行室温搅拌反应;
S24、将所述室温搅拌反应后体系进行纯化处理,得到靶向乳铁蛋白受体的多功能纳米药物载体。
需说明的是,本文中的室温均指的是16-25℃。
本实施例中,所述纳米胶束由式(Ⅰ)所示结构的多聚硒代氨基酸两亲性嵌段共聚物(式(Ⅰ)中,-R1为-OCH3或-MAL)自组装而成,所述纳米胶束和所述共聚物的制备方法详见申请号为CN202011344949.5,专利名称为一种多聚硒代氨基酸两亲性嵌段共聚物及制备方法与应用的专利文件中,在此不再赘述。
在一种实施方式中,所述乳铁蛋白与Traut’s试剂的摩尔比为1:30-50,最佳摩尔比为1:40。
在一种实施方式中,所述巯基化乳铁蛋白与纳米胶束的用量比为1g:2-100mg,最佳用量比为1g:2mg。
在一种实施方式中,所述室温搅拌反应的时间为6~12h,优选时间为9h。
本发明实施例提供一种载药组合物,其中,包括本发明实施例所述的靶向乳铁蛋白受体的多功能纳米药物载体和包裹于所述多功能纳米药物载体内的药物。
在一种实施方式中,所述药物选自抗AD药物、抗肿瘤药物、甾体类或非甾体抗炎药物、代谢调节类药物、抗生素、心血管药物、抗病毒药物、抗真菌药物、免疫调节剂等各类药物中的一种或多种,但不限于此。
在一种实施方式中,所述多功能纳米药物载体与药物的质量比为1mg:(0.1~10)mg。
下面通过具体的实施例对本发明作进一步地说明。
实施例1:
纳米球形胶束的制备
取10mg mPEG45-PV2-PMet(Se)2(P=0.28)和10mg NH2-PEG45-PV2-PMet(Se)2(P=0.28)完全溶解于2mL DMSO中,振荡使聚合物混合均匀,得到聚合物溶液;然后向其中逐滴滴加去离子水5mL,搅拌2小时,得到溶液;将得到的溶液直接加入截留分子量为2000DA的透析袋中,在2L去离子中室温透析7天,每天换一次水,然后使用水相滤头过滤(孔径450nm)透析袋内液,过滤掉大分子沉淀和聚集的胶束颗粒,得到目的产物纳米球形胶束。
实施例2:
纳米棒状胶束的制备
取10mg mPEG45-PV2-PMet(Se)4(P=0.46)和10mg NH2-PEG45-PV2-PMet(Se)4(P=0.46)完全溶解于2mL二氯甲烷中,在茄形瓶中旋转蒸发制成均匀薄膜,抽真空过夜除去残留的有机溶剂。在茄形瓶中加入5mL双蒸水于60℃水化1h,震荡混匀,水浴下超声即得纳米棒状胶束分散液,3000r/min离心30min,除去大的聚集粒子,清液经冷冻干燥,得到目的产物纳米棒状胶束。
实施例3:
纳米囊泡的制备
量取16mL的0.25%聚乙烯醇的水溶液于50mL烧杯中,搅拌。称量10mg mPEG45-PV2-PMet(Se)6(P=0.64)和10mg NH2-PEG45-PV2-PMet(Se)6(P=0.64)溶于4mL的CH2Cl2溶液,然后用超声波细胞粉碎机进行两次超声,一次超声工作时间为4s,间歇时间4s,总工作时间30s,两次超声间隔时间为30s。在开始第一次超声时用注射器吸取0.1mL0.1%聚乙烯醇的水溶液注入该溶液中,观察乳化情况。超声完毕,用注射器将溶液全部吸出,缓慢均匀注入质量分数为0.25%聚乙烯醇的水溶液中,室温下搅拌2h。最后经反复洗涤,冷冻、干燥,得到目的产物纳米囊泡。
实施例4:
阴阳离子复合纳米球形胶束的制备
取5mg NH2-PEG45-PAsp2-PMet(Se)2(P=0.30)、5mg mPEG45-PAsp2-PMet(Se)2(P=0.30)和10mg mPEG45-PLL2-PMet(Se)2(P=0.30)完全溶解于2mL DMSO中,振荡使聚合物混合均匀,得到聚合物溶液;然后向其中逐滴滴加去离子水5mL,搅拌2小时,得到溶液;将得到的溶液直接加入截留分子量为2000DA的透析袋中,在2L去离子中室温透析7天,每天换一次水,然后使用水相滤头过滤(孔径450nm)透析袋内液,过滤掉大分子沉淀和聚集的胶束颗粒,得到目的产物纳米球形胶束。用透射电子显微镜观察形成的纳米球形胶束,胶束呈规则圆球状形状,结果见图1,测得粒径约为100nm,结果见图2。
实施例5:
阴阳离子复合纳米球形胶束的制备
取5mg MAL-PEG45-PAsp2-PMet(Se)2(P=0.30)、5mg mPEG45-PAsp2-PMet(Se)2(P=0.30)和10mg mPEG45-PLL2-PMet(Se)2(P=0.30)完全溶解于2mL DMSO中,振荡使聚合物混合均匀,得到聚合物溶液;然后向其中逐滴滴加去离子水5mL,搅拌2小时,得到溶液;将得到的溶液直接加入截留分子量为2000DA的透析袋中,在2L去离子中室温透析7天,每天换一次水,然后使用水相滤头过滤(孔径450nm)透析袋内液,过滤掉大分子沉淀和聚集的胶束颗粒,得到目的产物纳米球形胶束。用透射电子显微镜观察形成的纳米球形胶束,胶束呈规则圆球状形状。
实施例6:
阴阳离子复合纳米棒状胶束的制备
取5mg mPEG45-PAsp2-PMet(Se)4(P=0.47)、5mg NH2-PEG45-PAsp2-PMet(Se)4(P=0.47)和10mg mPEG45-PLL2-PMet(Se)4(P=0.49)完全溶解于2mL二氯甲烷中,在茄形瓶中旋转蒸发制成均匀薄膜,抽真空过夜除去残留的有机溶剂。在茄形瓶中加入5mL双蒸水于60℃水化1h,震荡混匀,水浴下超声即得纳米棒状胶束分散液,3000r/min离心30min,除去大的聚集粒子,清液经冷冻干燥,得到目的产物纳米棒状胶束。用透射电子显微镜观察形成的纳米棒状胶束,纳米囊呈棒球状。
实施例7:
阴阳离子复合纳米囊泡的制备
量取16mL的0.25%聚乙烯醇的水溶液于50mL烧杯中,搅拌。称量5mg mPEG45-PAsp2-PMet(Se)6(P=0.65)、5mg NH2-PEG45-PAsp2-PMet(Se)6(P=0.65)和10mg mPEG45-PLL2-PMet(Se)6(P=0.67)溶于4mL的CH2Cl2溶液,然后用超声波细胞粉碎机进行两次超声,一次超声工作时间为4s,间歇时间4s,总工作时间30s,两次超声间隔时间为30s。在开始第一次超声时用注射器吸取0.1mL0.1%聚乙烯醇的水溶液注入该溶液中,观察乳化情况。超声完毕,用注射器将溶液全部吸出,缓慢均匀注入质量分数为0.25%聚乙烯醇的水溶液中,室温下搅拌2h。最后经反复洗涤,冷冻、干燥,得到目的产物纳米囊泡。用透射电子显微镜观察形成的纳米囊泡,纳米球呈圆球形。
实施例8:
阴阳离子复合型载阿霉素纳米球形胶束的制备
取5mg mPEG45-PAsp2-PMet(Se)2(P=0.30)、5mg NH2PEG45-PAsp2-PMet(Se)2(P=0.30)和10mg mPEG45-PLL2-PMet(Se)2(P=0.30)与2.7mg阿霉素分别完全溶解于适量DMSO中。振荡前者使聚合物混合均匀,得到聚合物溶液,然后在快速搅拌下缓慢逐滴加入阿霉素溶液,在上述聚合物溶液中加入2.7mg阿霉素,轻微搅拌使两者混合均匀;然后向其中逐滴滴加去离子水5mL,搅拌2小时,得到溶液;将得到的溶液直接加入截留分子量为2000DA的透析袋中,在1L去离子中室温透析7天,每天换一次水,然后使用水相滤头过滤(孔径450nm)透析袋内液,过滤掉大分子沉淀和聚集的胶束颗粒,得到目的产物载阿霉素纳米球形胶束。
实施例9:
阴阳离子复合型载多奈哌齐纳米囊泡的制备
量取16mL的质量分数为0.25%的聚乙烯醇的水溶液于50mL烧杯中,搅拌。称量5mgmPEG45-PAsp2-PMet(Se)6(P=0.65)、5mg OH-PEG45-PAsp2-PMet(Se)6(P=0.65)和10mgmPEG45-PLL2-PMet(Se)6(P=0.67)与2.7mg多奈哌齐完全溶于4mL的CH2Cl2溶液,然后用超声波细胞粉碎机进行两次超声,一次超声工作时间为4s,间歇时间4s,总工作时间30s,两次超声间隔时间为30s。在开始第一次超声时用注射器吸取0.1mL0.1%聚乙烯醇的水溶液注入该溶液中,观察乳化情况。超声完毕,用注射器将溶液全部吸出,缓慢均匀注入0.25%聚乙烯醇的水溶液中,室温下搅拌2h。最后经反复洗涤,冷冻、干燥,得到目的产物mPEG45-PAsp2-PMet(Se)6-多奈哌齐纳米囊泡。
实施例10:
阴阳离子复合型载CYP纳米球形胶束的制备
取5mg mPEG45-PAsp2-PMet(Se)2(P=0.30)、5mg NH2-PEG45-PAsp2-PMet(Se)2(P=0.30)和10mg mPEG45-PLL2-PMet(Se)2(P=0.30)与1.0mgCYP分别完全溶解于适量DMSO中。振荡前者使聚合物混合均匀,得到聚合物溶液,然后在快速搅拌下缓慢逐滴加入CYP溶液,轻微搅拌使两者混合均匀;然后向其中逐滴滴加去离子水5mL,搅拌2小时,得到溶液;将得到的溶液直接加入截留分子量为2000DA的透析袋中,在1L去离子中室温透析7天,每天换一次水,然后使用水相滤头过滤(孔径450nm)透析袋内液,过滤掉大分子沉淀和聚集的胶束颗粒,得到目的产物载CYP纳米球形胶束。
实施例11:
阴阳离子复合型载CYP纳米球形胶束的制备
取5mg mPEG45-PAsp2-PMet(Se)2(P=0.30)、5mg MAL-PEG45-PAsp2-PMet(Se)2(P=0.30)和10mg mPEG45-PLL2-PMet(Se)2(P=0.30)与1.0mgCYP分别完全溶解于适量DMSO中。振荡前者使聚合物混合均匀,得到聚合物溶液,然后在快速搅拌下缓慢逐滴加入CYP溶液,轻微搅拌使两者混合均匀;然后向其中逐滴滴加去离子水5mL,搅拌2小时,得到溶液;将得到的溶液直接加入截留分子量为2000DA的透析袋中,在1L去离子中室温透析7天,每天换一次水,然后使用水相滤头过滤(孔径450nm)透析袋内液,过滤掉大分子沉淀和聚集的胶束颗粒,得到目的产物载CYP纳米球形胶束。
实施例12:
阴阳离子复合型载药纳米球形胶束的体外释放试验
精确称取4mg实施例8所制备的阴阳离子复合载阿霉素纳米球形胶束放入透析袋(截留分子量为2000DA)内,透析袋内加入5mL磷酸盐缓冲溶液(lmol/L,pH 7.4)分散。将透析袋放入三角瓶中,加入20mL PBS(lmol/L,pH7.4)作为释放介质。将三角瓶置于37℃、100r/min的恒温摇床中进行释放实验,每隔1h取样2mL,取样后再补充相同体积的新鲜PBS保证释药外液体积恒定。通过紫外-可见分光光度计测量样品在λ=483nm的紫外吸光度,绘制阿霉素纳米胶束的体外药物释放曲线。按照上述操作绘制阿霉素纯品的体外药物释放标准曲线。由阿霉素纳米胶束体外药物释放曲线可知,阴阳离子复合型纳米胶束的载药量约为10.0%,包封率50.5%。且与阿霉素体外药物释放标准曲线对比,发现本发明提供的载药纳米胶束在体外释放过程中未出现突释现象,说明胶束表面吸附的药物比较少,绝大多数的药物被包裹在其内部,证明载药纳米胶束具有较好的控释效果,有利于减少药物的毒副作用。
实施例13:
纳米药物载体中硒抗氧化功能的检测
将乳腺癌MDA-MB-231细胞株(以下简称231细胞)用含10%胎牛血清的DMEM培养基,置于5%CO2、37℃培养箱中培养,1~2d传代一次,取对数生长期的细胞进行实验。取对数生长期的231细胞,以8×103/孔细胞接种于96孔板,培养24h后药物处理。实验分为四组:对照组、纳米胶束组、阿霉素组和纳米胶束+阿霉素组,给药剂量为纳米胶束(100μmol/L),阿霉素(2.0μmol/L),每孔加100μl,设6个复孔,对照孔加等量不含药物的DMEM培养基。24h后收集各组细胞,加入细胞裂解液冰上裂解,离心收取细胞全蛋白。BCA法测定蛋白浓度,再由WST-1法测定细胞总SOD活性、TBA法测定细胞MDA含量,操作步骤严格按照试剂盒要求。通过测定231细胞内SOD、MDA水平可知,处理后各组与对照组相比细胞SOD活力均降低,MDA水平均升高(P<0.05);其中纳米胶束联合阿霉素组细胞总SOD活力下降、MDA水平升高最为显著。由此表明,纳米药物载体具备了硒的抗氧化功能,且纳米药物载体与阿霉素的联合具有相加作用。
实施例14:
靶向阴阳离子复合型载CYP纳米球形胶束的制备
取适量羧基修饰的乳铁蛋白超声分散到10mL PH=6的0.01mol/L KH2PO4缓冲液中,加入80mg EDCI,120mg NHS,磁力搅拌混合均匀,避光室温活化20min。反应液转移至超滤管(截留分子量10K)中,超滤除去未反应的EDCI、NHS。然后以PH=7.4的0.01mol/L KH2PO4缓冲液分散,加入适量实施例10所制备的阴阳离子复合型载CYP纳米球形胶束,磁力搅拌下反应过夜。转移至超滤管(截留分子量100K)中,超滤除去未结合的乳铁蛋白,得到乳铁蛋白修饰的靶向、多功能纳米药物载体,用透射电子显微镜观察形成的纳米球形胶束,胶束呈规则圆球状形状,结果见图3,测得粒径约为200nm,结果见图4。
实施例15:
靶向阴阳离子复合型载CYP纳米球形胶束的制备
取适量乳铁蛋白溶解于0.15mol/L硼酸钠溶液(PH=8.5)中,加入1mg/mL Traut’s试剂,室温低速搅拌1h,过Zeba(7KMW,10mL)脱盐柱,除去小分子杂质,收集巯基化乳铁蛋白组分。取适量实施例10所制备的阴阳离子复合型载CYP纳米球形胶束用0.2mol/L NaH2PO4分散后,加入计算量的巯基化乳铁蛋白溶液,室温搅拌反应9h,将反应液转移至超滤管中,超滤除去未结合的乳铁蛋白,得到乳铁蛋白修饰的靶向、多功能纳米药物载体。
实施例16:
细胞摄取乳铁蛋白修饰阴阳离子复合型载CYP纳米球形胶束的定性观察
231细胞以2×104个/孔密度接种于48孔培养板中培养,24h后用乳铁蛋白修饰载CYP纳米球形胶束的DMEM溶液在37℃条件下孵育1h,纳米球形胶束的浓度分别50,100,200,400,600μg/mL。弃去纳米球形胶束溶液,细胞用PBS冲洗3次,洗去吸附在细胞表面的纳米球形胶束,加入3.7%的甲醛溶液固定10min,然后用100ng/mL的DAPI溶液染核10min,再用PBS漂洗3次,荧光显微镜下观察不同时间条件下细胞对乳铁蛋白修饰载CYP纳米球形胶束的摄取情况,结果见图5。从图5可知,细胞核被DAPI染成蓝色以定位细胞位置,载红色荧光染料的纳米胶束分布细胞核周围,充满细胞质中,证明乳铁蛋白修饰载CYP纳米球形胶束顺利进入细胞,被细胞摄取。
综上所述,本发明提供的靶向乳铁蛋白受体的多功能纳米药物载体由本发明所述纳米胶束中含聚乙二醇外端官能团化(-COOH、-NH2或-MAL等)的两亲性三嵌段共聚物与乳铁蛋白进行化学连接,在纳米胶束表面覆盖一层蛋白“外衣”,得到乳铁蛋白修饰靶向乳铁蛋白受体的多功能纳米药物载体。将药物分子定向的送到病变部位,提高药效,同时制成缓释控释的药物输送***,所包载的药物可以根据需要从输送***中缓释控释的释放,从而降低给药频率、提高治疗效果,并减少药物的毒副作用。与现有技术相比,本发明提供的纳米药物载体兼具一般氨基酸类纳米药物载体优势的同时,既具备了硒的多种生物学功能,又具备了靶向乳铁蛋白受体的特异性,是一类新型、靶向、多功能药物载体,适用于乳铁蛋白受体异常相关多种疾病的各类药物的药物载体研究、开发及临床应用。
应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (8)
1.一种靶向乳铁蛋白受体的多功能纳米药物载体,其特征在于,所述多功能纳米药物载体包括纳米胶束和结合于所述纳米胶束表面的乳铁蛋白,所述纳米胶束由式(Ⅰ)所示结构的多聚硒代氨基酸两亲性嵌段共聚物自组装而成,所述纳米胶束通过所述R1与所述乳铁蛋白结合;
式(Ⅰ);
其中,n=45、x=2、y=2;
-R1选自-NH2;
-R2选自、/>;
-R3选自-CH2CH2SeCH3。
2.根据权利要求1所述的靶向乳铁蛋白受体的多功能纳米药物载体,其特征在于,所述纳米胶束以纳米球形、纳米棒状或纳米囊泡的形式存在。
3.一种权利要求1~2任一项所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其特征在于,包括以下步骤:
提供纳米胶束;
取乳铁蛋白分散到缓冲液中,加入EDCI和NHS,依次进行搅拌和避光室温活化处理;
将活化处理后的反应液中未反应的EDCI和NHS去除,然后分散于缓冲液中,得到活化的乳铁蛋白分散液;
向所述活化的乳铁蛋白分散液中加入所述纳米胶束,搅拌下反应;
将反应后体系进行纯化处理,得到靶向乳铁蛋白受体的多功能纳米药物载体;
其中,所述纳米胶束为氨基修饰的纳米胶束,所述乳铁蛋白为羧基修饰的乳铁蛋白。
4.根据权利要求3所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其特征在于,取乳铁蛋白分散到pH=6的0.01mol/L KH2PO4缓冲液中;
所述乳铁蛋白与EDCI、NHS用量比为1 mg:2-6 mg:4-8mg;
所述避光室温活化处理的时间为15min-12h。
5.一种权利要求1~2任一项所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其特征在于,包括以下步骤:
提供纳米胶束;
取乳铁蛋白溶解于硼酸钠溶液中,加入Traut’s试剂,进行搅拌;
将搅拌后反应液过Zeba脱盐柱,除去杂质,得到巯基化乳铁蛋白溶液;
将所述纳米胶束用NaH2PO4分散后,加入所述巯基化乳铁蛋白溶液,进行室温搅拌反应;
将所述室温搅拌反应后体系进行纯化处理,得到靶向乳铁蛋白受体的多功能纳米药物载体。
6.根据权利要求5所述的靶向乳铁蛋白受体的多功能纳米药物载体的制备方法,其特征在于,所述乳铁蛋白与Traut’s试剂的摩尔比为1:30-50;
所述巯基化乳铁蛋白与纳米胶束的用量比为1g:2-100mg;
所述室温搅拌反应的时间为6~12h。
7.一种载药组合物,其特征在于,包括权利要求1-2任一项所述的靶向乳铁蛋白受体的多功能纳米药物载体和包裹于所述多功能纳米药物载体内的药物,所述药物为阿霉素。
8.根据权利要求7所述的载药组合物,其特征在于,所述多功能纳米药物载体与药物的质量比为1mg:(0.1~10)mg。
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