CN113425700B - 一种基于蛋白自组装的纳米药物载送系统的构建及应用 - Google Patents
一种基于蛋白自组装的纳米药物载送系统的构建及应用 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
本发明属于生物制药技术领域,具体涉及一种基于蛋白自组装的纳米药物载送系统的构建及应用,本发明通过非共价键方式,将载药前体小分子与可对小分子结构域进行响应的血清白蛋白体系,以及功能药物分子,纳米晶体(无机晶体)相结合;首先通过将待包载药物溶于有机溶剂中,再将该有机溶剂滴入血清白蛋白溶液中完成自组装,而后除去有机溶剂并纯化溶液,得到纳米药物载送系统。本发明利用小分子和血清白蛋白的自组装构建得到的纳米药物载送系统,可以包载多种药物、粒径均一、稳定性强、包裹效率高、生物相容性好、药物体内滞留时间长,同时本发明的制备成本低,可用于磁学成像、药物递送、肿瘤杀伤等领域,具有良好的医用前景。
Description
技术领域
本发明属于生物制药技术领域,具体涉及一种基于蛋白自组装的纳米药物载送系统的构建及应用。
背景技术
纳米药物的研究在生物医学等领域有着广泛的应用与潜在的价值。当药物颗粒的尺寸接近纳米尺度时,会发生一些有利的变化,如颗粒表面和溶剂的相互作用力增强,使药物能够克服因密度差异而导致的颗粒聚沉或沉降问题。然而,许多纳米药物具有疏水性,如超顺磁性四氧化三铁纳米晶体(SPIO)、硫化锰纳米晶体(MnS)、亚硫化铁纳米晶体(FeS2)、药物分子阿霉素、紫杉醇、喜树碱、IR780、吲哚菁绿(ICG)以及金属离子Mn2+等,导致其出现难以被细胞摄取从而出现不易被生物体吸收、药代动力学不稳定、药物体内循环时间短等问题;同时,不少纳米药物还具有无靶向性以及全身毒性的特点,从而不利于治疗。因此,如何增强疏水纳米材料以及疏水性药物的水溶性是当前的研究重点。
目前,应用于疏水药物的增溶与稳定改性的生物材料主要为脂质体、类脂质体两亲性分子,如二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、聚乙二醇-己内酯(PEG-PCL)等。在水溶液中,这些两亲性分子可以利用其分子结构中的疏水基团,在疏水相互作用下自发聚集成疏水核心,同时亲水性基团也会相互聚集形成外围亲水冠层,从而达到更稳定的状态,而在组装过程中形成的疏水核腔可以有效包载疏水性分子、纳米粒子等疏水性材料。然而,有研究表明,PEG分子类生物材料,在体内环境下会刺激机体产生PEG特异性免疫球蛋白,缩短PEG药物递送系统在体内循环的半衰期,从而影响药物疗效,并造成非特异性组织沉积从而产生毒性。
近年来,基于蛋白的递送系统逐渐受到人们的重视。其中以白蛋白纳米粒为主的药物递送系统由于具有提高药物稳定性、增加体内循环时间、提高药物的肿瘤靶向性以及降低毒副作用等优点而成为药学领域的研究热点,具有巨大的发展潜力及应用前景。当前,白蛋白药物载体的构建主要有两种方式,第一种是采用化学共价键连接的方式使载体与药物分子相结合,但这种方式需要利用蛋白结构上的氨基、羧基与药物分子进行化学偶联,而蛋白分子的结构较为精细,化学基团的改变极易造成结构的破坏,从而影响其生物功能。另一种方式是通过蛋白上的巯基将蛋白分子极性交联形成纳米粒,在交联过程中将药物分子进行组装,这种方法同样会造成蛋白的化学结构发生改变,从而影响蛋白的生物功能,同时这种方式装载的药物种类有限,难以实现纳米晶体的包裹。
综上可见,很有必要开发一种新的方式来构建纳米药物载送系统,并且使构建得到的药物载送系统具有可以实现多种药物包载需求、粒径适中且分布均一、热力学稳定性高、机体免疫原性低、对正常细胞具有良好生物相容性、体内滞留时间长、靶向性好等特点。
发明内容
为了克服上述现有技术的不足,本发明提出了一种基于蛋白自组装的纳米药物载送系统的构建方法,本发明利用小分子和血清白蛋白的自组装构建纳米药物载送系统,该系统可以包载多种药物、粒径均一、稳定性强、包裹效率高、生物相容性好、药物体内滞留时间长,可用于磁学成像等医学领域。
为了实现上述目的,本发明所采用的技术方案是:
本发明提供了一种基于蛋白自组装的纳米药物载送系统的构建方法,包括以下步骤:
S1、将药物、烟酸、2-甲基咪唑溶解于有机溶剂,制得溶液A,所述药物包括药物分子或/和无机晶体,所述药物为一种或多种;
S2、将血清白蛋白溶解,制得溶液B;
S3、将溶液A逐滴超声滴入溶液B中,混匀后制得溶液C;
S4、溶液C蒸发去除有机溶剂后经超速离心制备得到基于蛋白自组装的纳米药物载送系统。
在本发明的另一个优选实施例中,步骤S4中,在去除有机溶剂后还可以加入金属基化合物水溶液,经超速离心后制备得到金属离子负载的纳米药物载送系统,实现纳米体系的多功能化。进一步地,所述金属基化合物为含有Mn2+、Fe2+、Fe3+、Zn2+其中之一的金属基氯化物。
在本发明的另一个优选实施例中,当所述药物为无机晶体时,还可以通过单独添加金属阳离子化合物与功能化阴离子化合物的形式在纳米药物载送系统中原位形成无机盐晶体结构。进一步地,所述金属阳离子化合物为Mn2+或Fe2+金属氯化物,所述功能化阴离子化合物为硫化物。最常见的硫化物如Na2S。具体的,金属阳离子化合物添加于步骤S2中,功能化阴离子化合物则在步骤S4的超速离心前加入。可按需求,适当添加金属阳离子化合物和功能化阴离子化合物,当逐渐加大离子加入量,体系出现沉淀时,为离子的最大加入量。
烟酸、2-甲基咪唑在本发明中作为载药前体小分子,优选地,所述载药前体小分子也可替换为其他可以达到相同或相似效果的两亲性小分子或能形成两亲性小分子的混合物。
本发明构建的纳米药物载送系统先将载药前体烟酸和2-甲基咪唑的混合物与待包载药物溶于有机溶剂中,通过芳香杂环间π-π共轭和疏水相互作用形成功能域,同时利用烟酸上羧基与2-甲基咪唑上亚氨基的化学极性使二者形成氢键,随后将该有机溶剂滴入含有多个疏水子结构域和氢键位点的血清白蛋白溶液中,使上述有机溶剂与血清白蛋白溶液在非共价相互作用下完成自组装,随后通过蒸发除去有机溶剂并通过超速离心法纯化溶液,制备得到基于蛋白自组装的纳米药物载送系统。在纳米递送系统制备过程中,还可以通过滴加金属基化合物水溶液的方式使纳米药物载送系统负载金属离子,从而实现纳米系统的多功能化,满足生物医用需求。其机理如图1所示,待包载药物(如IR780等)及无机金属离子(如Mn2+、Fe2+、Fe3+、Zn2+等)在烟酸与2-甲基咪唑通过疏水相互作用下形成功能域,该功能域与含多个疏水子结构域的血清白蛋白进行自组装,从而构建出一种基于蛋白自组装的纳米药物载送系统。
本发明的纳米药物载送系统可以负载光热试剂如IR780;多种抗肿瘤药物如两亲性的盐酸阿霉素,疏水性的紫杉醇、喜树碱、SPIO、FeS2;多种金属离子如Mn2+、Fe2+、Fe3+、Zn2+等;本发明通过小分子和血清白蛋白的自组装提供了一种可以包载多种药物、粒径均一、稳定性强、包裹效率高、生物相容性好、药物体内滞留时间长的纳米药物载送系统,同时本发明的制备成本低,可用于磁学成像、药物递送、肿瘤杀伤等领域,具有良好的医用前景。
优选地,所述有机溶剂为药物与载药前体混合后的优良溶剂。进一步地,所述有机溶剂为四氢呋喃。
优选地,所述药物分子包括但不限于盐酸阿霉素(Doxorubicin,DOX)、IR780、紫杉醇、喜树碱、吲哚菁绿(ICG),所述无机晶体包括但不限于SPIO、MnS、FeS、FeS2、MnFe2O4。进一步地,所述药物可以是单独的药物分子,或者多种药物分子,也可以是单独的无机晶体,或者多种无机晶体,还可以是无机晶体与药物的混合物体系。
优选地,所述血清白蛋白包括但不限于牛血清白蛋白、人血清白蛋白。
优选地,所述烟酸和2-甲基咪唑的总量与所述药物的质量比为(16-32):1,所述烟酸与2-甲基咪唑的质量比为(1-4):(2-4),所述药物与血清白蛋白的质量比为(1-4):(10-20)。进一步地,所述烟酸和2-甲基咪唑的总量与所述药物的质量比为20:1,所述烟酸与2-甲基咪唑的质量比为1:1,所述药物与血清白蛋白的质量比为2:10。
优选地,所述烟酸在有机溶剂中的浓度为(1-3)mg/500uL,血清白蛋白在溶液B中的浓度为(10-20)mg/(5-8)mL。进一步地,所述烟酸在有机溶剂中的浓度为2mg/500uL,血清白蛋白在溶液B中的浓度为10mg/5mL。具体的,所述溶液B为血清白蛋白的水溶液。
优选地,溶液A与溶液B的体积比为1:(10-16)。进一步地,溶液A与溶液B的体积比为1:10。
优选地,所述超速离心的离心速度为2000-8000rpm,分子截留量(MWCO)为3-10kDa,时间为5-30min。进一步地,所述超速离心的离心速度为5000rpm,分子截留量(MWCO)为10kDa,时间为20min。
优选地,去除有机溶剂的方法为旋转蒸发法,转速为10-60rpm,温度为20-40℃,时间为5-30min。进一步地,转速为30rpm,温度为30℃,时间为20min。
优选地,步骤S3的混匀采用超声处理的方式,超声的时间控制在30s-2min,例如:30s-1min,1min-1min30s,1min30s-2min。
本发明还提供了采用上述方法构建得到的基于蛋白自组装的纳米药物载送系统。
优选地,所述应用领域包括但不限于药物递送,造影成像,制备治疗肿瘤药物。
与现有技术相比,本发明的有益效果是:
本发明通过非共价键方式,将载药前体小分子与可对小分子结构域进行响应的血清白蛋白体系,以及功能药物分子,纳米晶体(无机晶体)相结合;首先通过将待包载药物溶于有机溶剂中,再将该有机溶剂滴入血清白蛋白溶液中完成自组装,而后除去有机溶剂并纯化溶液,得到纳米药物载送系统。总体而言,本发明具有以下优点:
(1)本发明采用血清白蛋白与小分子体系自组装,构建纳米药物载送系统,实现药物分子、纳米晶体的单一组装与协同组装,装载成分可以更换且具备多样性;
(2)构建得到的纳米药物载送系统以非共价键结合方式为主,未破坏蛋白原有的化学键;
(3)血清白蛋白作为血液组成成分,具有较低的免疫原性,生物相容性良好;
(4)血清白蛋白作为生物大分子,为药物提供多种类型的氢键、疏水空间结构,使得该系统可以包载多种药物分子,可得到良好的药物/无机晶体包载率,本发明的多种药物、无机晶体包载率达到65%-85%;
(5)体系中烟酸上的羧基与2-甲基咪唑上的亚氨基的化学极性强,二者形成氢键,可以与含有多个疏水子结构域和氢键位点的蛋白自组装,蛋白的生物相容性与载药前体烟酸、2-甲基咪唑的增溶效果共同作用于系统,使制得的纳米药物载送系统热力学稳定性高,粒径均一且适中;
(6)制备过程中所需的外界能量为通过超声法将有机溶液逐滴滴入蛋白溶液完成自组装;同时,本发明的超声时长短,功率低,不会使系统由于过热而解组装。
可见,本发明利用小分子和血清白蛋白的自组装构建得到的纳米药物载送系统,可以包载多种药物、粒径均一、稳定性强、包裹效率高、生物相容性好,同时本发明的制备成本低,可用于磁学成像、药物递送、肿瘤杀伤等领域,具有良好的医用前景。
附图说明
图1为纳米药物载送系统自组装的原理图;
图2为BSA@DOX的紫外可见光谱谱图;
图3为(烟酸+2-甲基咪唑)与IR780的质量比对药物载送系统包封率的影响;
图4为SPIO@BSA的透射电镜图;
图5为IR780&SPIO@BSA纳米药物载送系统的透射电镜图;
图6为DOX@BSA@Mn2+的透射电镜(a)、粒径分布(b)以及元素组成分析(c)图像。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
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,均为可通过常规的商业途径购买得到的。
第一类:基于蛋白的自组装可以实现药物分子(单种或多种药物)的装载
实施例1紫杉醇纳米药物载送系统的构建
(1)取2.0mg紫杉醇、20.0mg烟酸、20.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;
(2)取10.0mg牛血清白蛋白溶解于5.0mL超纯水中,制得溶液B;
(3)将溶液A逐滴超声(功率为150W,频率为20kHZ)滴入溶液B中(即将溶液B置于超声波处理机上,然后在超声状态下逐滴滴入溶液A),滴完后继续处理1.5min,制得溶液C;
(4)溶液C在30℃下以30rpm的转速旋转蒸发20min,去除有机溶剂四氢呋喃,制得溶液D;
(5)在10kDa的分子截留量(MWCO)以及5000rpm的转速条件下对溶液D进行超速离心(20min),得到蛋白@紫杉醇纳米粒子复合物,即紫杉醇纳米药物载送系统,并将其命名为BSA@紫杉醇。
实施例2喜树碱药物纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,将实施例1中的紫杉醇替换为喜树碱,得到蛋白@喜树碱纳米粒子复合物,即喜树碱药物纳米药物载送系统,并将其命名为样品BSA@喜树碱。
实施例3阿霉素纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,将实施例1中的紫杉醇替换为阿霉素(DOX),得到蛋白@DOX纳米粒子复合物,即阿霉素纳米药物载送系统,并将其命名为样品BSA@DOX。
采用多角度粒度分析仪(BrookhavenOmni)测得BSA@DOX的平均粒径为91.3nm;同时,通过超滤法分离游离药物,并根据《中国药典》中公布的包封率计算方法【包封率=(系统中包封的药量/系统中包封及未包封的药量)×100%】测定得到BSA@DOX的包载率为70%。其中,药量的测定方法为:(1)针对药物分子,先对纯药物进行吸光度-浓度的线性拟合得到标准曲线,再将待测药物的吸光度代入该标准曲线得出待测药物浓度,最后根据待测药物的体积计算得到药量;(2)针对无机晶体(后续实施例涉及),采用原子吸收光谱检测离子含量。
采用紫外可见分光光度计(仪器型号为UV—2600;购买自津岛仪器(苏州)有限公司)对制备得到的BSA@DOX进行紫外可见光谱分析,如图2所示,纳米系统与FreeDOX的吸收峰一致,说明纳米系统不影响DOX的紫外吸收。
实施例4不同小分子与被包载药物的质量比对IR780纳米药物载送系统的影响
制备方法同实施例1,不同之处在于,将实施例1中的紫杉醇替换为IR780,并且将(烟酸+2-甲基咪唑)与IR780的质量比分别设置为:10,16,20,24,28,32,烟酸与2-甲基咪唑的质量比为1:1,IR780的用量为2.0mg。得到蛋白@IR780纳米粒子复合物,即IR780纳米药物载送系统,并将其命名为样品BSA@IR780。
以小分子与被包载药物的质量比【(烟酸+2-甲基咪唑)与IR780的质量比】为横纵标,以包封率为纵坐标构建曲线图。如图3所示,小分子与被包载药物的质量比为20:1时包封率最好,包封率高达85%;在20:1至32:1之间略有下降,10:1为包封的最小质量比,包封率在35%左右。可见,当小分子与被包载药物的质量比在16-32:1时,具有较好的包封率,包载率达到65%-85%。
实施例5ICG纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,实施例5的步骤(1)为:取2.0mgICG、16.0mg烟酸、16.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;步骤(2)为:取20.0mg牛血清白蛋白溶解于5.0mL超纯水中,制得溶液B;
最后制备得到蛋白@ICG纳米粒子复合物,即ICG纳米药物载送系统,并将其命名为BSA@ICG。
实施例6IR780&紫杉醇纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,实施例6的步骤(1)为:取1.0mgIR780、1.0mg紫杉醇、20.0mg烟酸、20.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;
最后制备得到蛋白@IR780&紫杉醇纳米粒子复合物,即IR780&紫杉醇纳米药物载送系统,并将其命名为BSA@IR780&紫杉醇。
实施例7IR780&喜树碱药物纳米药物载送系统的构建
制备方法同实施例6,不同之处在于,将实施例6中的IR780和紫杉醇替换为IR780和喜树碱,得到蛋白@IR780&喜树碱纳米粒子复合物,即IR780&喜树碱药物纳米药物载送系统,并将其命名为样品BSA@IR780&喜树碱。
实施例8IR780&DOX药物纳米药物载送系统的构建
制备方法同实施例6,不同之处在于,实施例8的步骤(1)为:取1.0mgIR780、1.0mgDOX、16.0mg烟酸、16.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;
步骤(2)为:取20.0mg牛血清白蛋白溶解于5.0mL超纯水中,制得溶液B;
最后制备得到蛋白@IR780&DOX纳米粒子复合物,即IR780&DOX纳米药物载送系统,并将其命名为BSA@IR780&DOX。
采用多角度粒度分析仪(BrookhavenOmni)测得BSA@IR780&DOX的平均粒径为95.5nm;同时,按照实施例1的包封率测定方法测得BSA@IR780&DOX的包载率为70%。
第二类:基于蛋白的自组装可以实现无机晶体的装载
无机晶体既具有对肿瘤细胞的杀伤作用(MnS与FeS中S元素的作用),又因含金属元素而具有成像功能,可用于造影及肿瘤杀伤。
实施例9超顺磁氧化铁(SPIO)药物纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,将实施例1中的紫杉醇替换为SPIO,得到蛋白@SPIO纳米粒子复合物,即SPIO药物纳米药物载送系统,并将其命名为样品BSA@SPIO。
采用多角度粒度分析仪(BrookhavenOmni)测得BSA@SPIO的平均粒径为90.2nm。同时,按照实施例1的包封率测定方法测得BSA@SPIO的包封率为80%。
对制备得到的BSA@SPIO进行透射电镜(JEM1400;厂商:JEOL)分析,如图4所示,表明SPIO@BSA纳米系统成球状,可从表观上证明无机晶体SPIO成功被包封。
实施例10硫化锰(MnS)药物纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,将实施例1中的紫杉醇替换为MnS,得到蛋白@MnS纳米粒子复合物,即MnS药物纳米药物载送系统,并将其命名为样品BSA@MnS。
实施例11硫化铁(FeS)药物纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,将实施例1中的紫杉醇替换为FeS,得到蛋白@FeS纳米粒子复合物,即FeS药物纳米药物载送系统,并将其命名为样品BSA@FeS。
第三类:基于蛋白的自组装可以实现无机晶体与药物分子的协同装载
无机晶体与药物共同负载,在肿瘤治疗中可以增强对肿瘤细胞的杀伤作用,并且具有成像功能。
实施例12IR780&SPIO药物纳米药物载送系统的构建
制备方法同实施例1,不同之处在于,实施例12的步骤(1)为:取2.0mgIR780、2.0mgSPIO、32.0mg烟酸、32.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;
最后制备得到蛋白@IR780&SPIO纳米粒子复合物,即IR780&SPIO纳米药物载送系统,并将其命名为BSA@IR780&SPIO。
采用多角度粒度分析仪(BrookhavenOmni)测得BSA@IR780&SPIO的平均粒径为34.9nm;包载率为60%。
对制备得到的BSA@IR780&SPIO进行透射电镜分析,如图5所示,表明BSA@IR780&SPIO纳米系统成球状,可从表观上证明IR780&SPIO成功被包封【药物和无机晶体被共同包封在同一微囊中,与图4(只含无机晶体)对比来看,深颜色颗粒为无机晶体,浅颜色颗粒为药物】。
实施例13IR780&FeS药物纳米药物载送系统的构建
制备方法同实施例12,不同之处在于,将实施例12中的IR780和SPIO替换为IR780和FeS,得到蛋白@IR780&FeS纳米粒子复合物,即IR780&FeS药物纳米药物载送系统,并将其命名为样品BSA@IR780&FeS。
实施例14IR780&MnS药物纳米药物载送系统的构建
制备方法同实施例12,不同之处在于,将实施例12中的IR780和SPIO替换为IR780和MnS,得到蛋白@IR780&MnS纳米粒子复合物,即IR780&MnS药物纳米药物载送系统,并将其命名为样品BSA@IR780&MnS。
采用多角度粒度分析仪(BrookhavenOmni)测得BSA@IR780&MnS的平均粒径为98.5nm,包载率为65%。
实施例15DOX&SPIO纳米药物载送系统的构建
制备方法同实施例12,不同之处在于,将实施例12中的IR780和SPIO替换为DOX和SPIO,得到蛋白@DOX&SPIO纳米粒子复合物,即DOX&SPIO纳米药物载送系统,并将其命名为样品BSA@DOX&SPIO。
第四类:基于蛋白的自组装可以实现药物的协同装载与无机金属离子的螯合富集
在药物对肿瘤细胞的杀伤作用基础上,装载无机金属离子可使药物具有造影成像的功能。
实施例16DOX@Mn2+纳米药物载送系统的构建
(1)取2.0mgDOX、20.0mg烟酸、20.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;
(2)取10.0mg牛血清白蛋白溶解于5.0mL超纯水中,制得溶液B;
(3)将溶液A逐滴超声(功率为150W,频率为20kHZ)滴入溶液B中,滴完后继续超声处理30s,制得溶液C;
(4)溶液C在30℃下以40rpm的转速旋转蒸发20min,去除有机溶剂四氢呋喃,制得溶液D;
(5)向溶液D中超声滴入(采用手持式超声波处理器UltrasonicProcessor)500uL浓度为500ug/mL的MnCl2水溶液,然后在10kDa的分子截留量(MWCO)以及8000rpm的转速条件下对溶液D进行超速离心(10min),得到蛋白@DOX@Mn2+纳米粒子复合物,即DOX@Mn2+纳米药物载送系统,并将其命名为DOX@BSA@Mn2+。
采用多角度粒度分析仪(BrookhavenOmni)测得DOX@BSA@Mn2+的平均粒径为91.6nm;同时,按照实施例1的包封率测定方法测得DOX@BSA@Mn2+的包载率为70%。
对DOX@BSA@Mn2+进行透射电镜、粒径分布以及元素组成分析,如图6所示,说明DOX@BSA@Mn2+纳米系统呈球形,成功被包封(图6a);同时说明DOX@BSA@Mn2+纳米系统的粒径集中分布在100nm附近(图6b);还说明DOX@BSA@Mn2+纳米系统含有Mn2+(图6c)。
第五类:基于蛋白的自组装可以实现药物的协同装载与无机晶体的原位合成
与第二类体系相比,第二类体系采用的无机晶体原料需预先制备得到,而制备无机晶体较复杂,第五类则直接采用离子原位矿化的方式实现无机晶体的装载,最终效果相同,但耗时更短。同时,离子原位矿化不会影响白蛋白的自组装。
实施例17IR780@MnS纳米药物载送系统的构建
(1)取1.0mgIR780、10.0mg烟酸、10.0mg2-甲基咪唑溶解于500uL四氢呋喃中,制得溶液A;
(2)取10.0mg牛血清白蛋白、3.1mg无水MnCl2溶解于8.0mL超纯水中,制得溶液B;
(3)将溶液A加入溶液B中,超声处理2min,制得溶液C;
(4)溶液C在30℃下以30rpm的转速旋转蒸发20min,去除有机溶剂四氢呋喃,制得溶液D;
(5)将1mL浓度为8.87mg/mL的Na2S·9H2O溶液搅拌加入溶液D中,在4℃下搅拌孵育3h,制得溶液E;
(6)在10kDa的分子截留量(MWCO)以及3500rpm的转速条件下对溶液E进行超速离心(30min),并将pH调节至7.35,得到蛋白@IR780@MnS纳米粒子复合物,即IR780@MnS纳米药物载送系统,并将其命名为DOX@IR780@MnS。
采用多角度粒度分析仪(BrookhavenOmni)测得DOX@IR780@MnS的平均粒径为97.5nm;同时,按照实施例1的包封率测定方法测得DOX@IR780@MnS的包载率为65%。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (7)
1.一种基于蛋白自组装的纳米药物载送系统的构建方法,其特征在于,包括以下步骤:
S1、将药物、烟酸、2-甲基咪唑溶解于有机溶剂,制得溶液A,所述药物包括药物分子或/和无机晶体,所述药物为一种或多种,所述烟酸和2-甲基咪唑的总量与所述药物的质量比为(16-30):1,所述烟酸与2-甲基咪唑的质量比为(1-4):(2-4);
S2、将血清白蛋白溶解,制得溶液B,所述血清白蛋白与药物的质量比为(10-20): (1-4);
S3、将溶液A逐滴超声滴入溶液B中,混匀后制得溶液C;
S4、溶液C蒸发去除有机溶剂后经超速离心制备得到基于蛋白自组装的纳米药物载送系统;
步骤S4中,在去除有机溶剂后还可以加入金属基化合物水溶液,经超速离心后制备得到金属离子负载的纳米药物载送系统,实现纳米体系的多功能化;
当所述药物为无机晶体时,还可以通过单独添加金属阳离子化合物与功能化阴离子化合物的形式在纳米药物载送系统中原位形成无机盐晶体结构。
2.根据权利要求1所述的一种基于蛋白自组装的纳米药物载送系统的构建方法,其特征在于,所述药物分子包括盐酸阿霉素、IR780、紫杉醇、喜树碱、吲哚菁绿,所述无机晶体包括SPIO、MnS、FeS、FeS2、MnFe2O4。
3.根据权利要求1所述的一种基于蛋白自组装的纳米药物载送系统的构建方法,其特征在于,所述烟酸在有机溶剂中的浓度为(1-3)mg/500uL,血清白蛋白在溶液B中的浓度为(10-20)mg/(5-8)mL。
4.根据权利要求1所述的一种基于蛋白自组装的纳米药物载送系统的构建方法,其特征在于,溶液A与溶液B的体积比为1:(10-16)。
5.根据权利要求1所述的一种基于蛋白自组装的纳米药物载送系统的构建方法,其特征在于,所述超速离心的离心速度为2000-8000rpm,分子截留量(MWCO)为3-10kDa,时间为5-30min。
6.采用权利要求1-5任一项所述方法构建得到的基于蛋白自组装的纳米药物载送系统。
7.权利要求6所述的基于蛋白自组装的纳米药物载送系统的应用,其特征在于,所述应用包括制备药物递送载体,制备造影成像剂,制备治疗肿瘤药物。
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