CN116196291A - 一种rgd修饰的雷公藤红素白蛋白纳米粒及其制备方法和应用 - Google Patents
一种rgd修饰的雷公藤红素白蛋白纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
一种RGD修饰的雷公藤红素白蛋白纳米粒及其制备方法和应用，属于生物医药技术领域。一种雷公藤红素白蛋白纳米粒，是白蛋白包载雷公藤红素形成纳米粒。RGD修饰的雷公藤红素白蛋白纳米粒是精氨酸‑甘氨酸‑天冬氨酸环状三肽(RGD环肽)修饰于雷公藤红素白蛋白纳米粒表面而形成。RGD修饰的雷公藤红素白蛋白纳米粒可以特异性靶向并穿透中性粒细胞，诱导中性粒细胞凋亡，在治疗类风湿性关节炎和炎症疾病方面具有良好的效果。
Description
技术领域
本发明属于生物医药技术领域，公开了一种RGD修饰的雷公藤红素白蛋白纳米粒及其制备方法和应用。
背景技术
类风湿关节炎(Rheumatoid arthritis,RA)是一种病因不明的不可治愈的慢性炎症性自身免疫性疾病，RA影响全球约1％的人口(Ariyo O,Victor H.Estimating MaternalMortality:Surveillance methods and agenda for improvement[J].The RoutledgeHandbook of African Demography,2022:787-799.)。疾病进展逐渐导致残疾，发病率很高，死亡率增加(H.J.Byeon,S.Y.Min,I.Kim,E.S.Lee,K.T.Oh,B.S.Shin,et al.,Humanserum albumin-TRAIL conjugate for the treatment of rheumatoid arthritis,Bioconjugate Chem.25(2014)2212–2221.)。在炎症的发展过程中，中性粒细胞(Neutrophils)被激活并且率先迁移到关节腔内。中性粒细胞占人类外周血白细胞的50-70％，是提供系统保护的第一道防线(Zhang,C.Y.,Dong,X.,Gao,J.,Lin,W.,Liu,Z.,Wang,Z.J.,2019a.Nanoparticle-induced neutrophil apoptosis increases survival insepsis and alleviates neurological damage in stroke.Sci.Adv.5,7964–7977.)。在炎症过程中中性粒细胞不断迁移产生各种炎症因子，从而使炎症的炎症状态持续存在。因此，通过抑制中性粒细胞的迁移，减少中性粒细胞在疾病部位的积累，将为炎症治疗提供新策略。
雷公藤红素(Celastrol，CEL)是一个具有多种生物活性的天然产物，来源于中药雷公藤(拉丁学名：Tripterygium wilfordii Hook.f.)的根皮，它是治疗类风湿病雷公藤片、雷公藤多苷片等制剂的有效成分之一。现代研究表明：CEL具有广泛的抗炎、抗氧化和抗癌活性。雷公藤红素在慢性炎症和自身免疫性疾病(如类风湿性关节炎、系统性红斑狼疮和银屑病等)等一系列慢性疾病的实验模型中都显示出良好的预防/治疗效果。尽管雷公藤红素具有显著的药理活性，但其临床应用仍受到严重副作用的严格限制，主要原因水稳定性差、生物利用度低、治疗窗口窄和不良副作用等。因此亟需找到改善其水稳定性差、生物利用度低、靶向药物递送等问题的方法！
发明内容
为了解决现有技术存在的问题，本发明提供了一种RGD修饰的雷公藤红素白蛋白纳米粒及其制备方法和应用。本发明所述的RGD修饰的雷公藤红素白蛋白纳米粒，以雷公藤红素白蛋白纳米粒作为中间体，并在其表面修饰精氨酸-甘氨酸-天冬氨酸环状三肽(RGD环肽)组成。
将雷公藤红素包载与白蛋白纳米粒中，得到的雷公藤红素白蛋白纳米粒，可以提高雷公藤红素的生物利用度，延长其半衰期，提高疗效。此外，白蛋白纳米粒可以与炎症中性粒细胞高度表达的Fcγ受体结合，精氨酸-甘氨酸-天冬氨酸环状三肽(RGD环肽)被修饰于雷公藤红素白蛋白纳米粒表面，能够提高对中性粒细胞的靶向性和穿透性。从而实现对雷公藤红素的高效递送。
本发明提供的包载雷公藤红素的RGD修饰白蛋白纳米粒可以特异性靶向炎症中性粒细胞，并且释放雷公藤红素诱导中性粒细胞凋亡，从而阻断中性粒细胞向炎症部位的迁移，大大增强了抗炎疗效。对雷公藤红素的靶向递送和类风湿性关节炎和炎症疾病治疗具有重要意义。
为实现上述目的，本发明采用以下技术方案：
本发明的雷公藤红素白蛋白纳米粒，是白蛋白包载雷公藤红素形成纳米粒。
本发明的RGD修饰的雷公藤红素白蛋白纳米粒，是RGD环肽修饰于雷公藤红素白蛋白纳米粒表面，从而形成的RGD修饰的雷公藤红素白蛋白纳米粒。
所述白蛋白选自哺乳动物白蛋白。更优选地，所述哺乳动物白蛋白选自人血清白蛋白或牛血清白蛋白。
进一步的，所述雷公藤红素和白蛋白的质量比为1：(20～35)。
所述RGD环肽和白蛋白的摩尔比为1:(5～40)。
所述的RGD修饰的雷公藤红素白蛋白纳米粒的粒径为(112.5±2.9)nm。
本发明的雷公藤红素白蛋白纳米粒的制备方法为去溶剂法，具体为：
将白蛋白溶于PBS缓冲液，搅拌至溶解，根据比例，加入雷公藤红素的无水乙醇溶液，沉淀白蛋白，30min后，滴加戊二醛交联白蛋白，制得雷公藤红素白蛋白纳米粒。
本发明的RGD修饰的雷公藤红素白蛋白纳米粒的制备方法，具体为：以水为溶剂，以2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)和N,N-二异丙基乙胺DIPEA为催化剂，将RGD环肽在0℃搅拌20-30min，得到羧基被活化的RGD环肽；
将羧基被活化的RGD环肽和雷公藤红素白蛋白纳米粒混合，搅拌16～20h，得到RGD修饰的雷公藤红素白蛋白纳米粒。
按质量比，催化剂：RGD环肽＝1:2；
搅拌速率为200～400rpm/min。
本发明的雷公藤红素白蛋白纳米粒和RGD修饰的雷公藤红素白蛋白纳米粒在用于治疗类风湿性关节炎药物或治疗炎症疾病药物中的应用。
本发明的一种RGD修饰的雷公藤红素白蛋白纳米粒及其制备方法和应用，其有益效果在于：
本发明的一种RGD修饰的雷公藤红素白蛋白纳米粒可以极大提高雷公藤红素的稳定性，延长循环时间，提高生物利用度。RGD修饰的雷公藤红素白蛋白纳米粒可以特异性靶向炎症中性粒细胞，并且释放雷公藤红素诱导中性粒细胞凋亡，从而阻断中性粒细胞向炎症部位的迁移，大大增强了抗炎疗效。对雷公藤红素的靶向递送和关节炎治疗具有重要意义。
本发明在制备过程中通过调节白蛋白和雷公藤红素的质量比，提高包载率。
在RGD修饰过程中，通过选择合适的催化剂，从而实现了RGD对白蛋白纳米粒的稳定修饰。
附图说明
图1为本发明RGD修饰的雷公藤红素白蛋白纳米粒的组装示意图。
图2为CEL@BSA纳米粒的粒径分布图。
图3为CEL@BSA-RGD纳米粒的粒径分布图。
图4为CEL@BSA纳米粒的透射电镜图。
图5为CEL@BSA-RGD纳米粒的透射电镜图。
图6为关节炎小鼠足肿胀体积变化图，Control代表正常小鼠，CIA组代表关节炎小鼠模型组，CEL代表给予雷公藤红素溶液组的关节炎小鼠，CEL@BSA代表给予CEL@BSA纳米粒治疗的关节炎小鼠，CEL@BSA-RGD代表给予CEL@BSA-RGD纳米粒治疗的关节炎小鼠。
图7为不同CEL和BSA比例下，CEL的包封率。
图8为使用EDC和NHS作为催化剂时，CEL@BSA-RGD纳米粒的粒径图。
图9为RGD与BSA不同比例时，CEL@BSA-RGD纳米粒的粒径变化图。
图10为对比例形成的纳米粒的图片。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例提供一种RGD修饰的雷公藤红素白蛋白纳米粒，其组装示意图见图1，所述RGD修饰的雷公藤红素白蛋白纳米粒由雷公藤红素，RGD环肽和白蛋白组成，所述RGD修饰的雷公藤红素白蛋白纳米粒的制备方法包括以下步骤：
本实施例提供一种RGD修饰的雷公藤红素白蛋白纳米粒，所述雷公藤红素与白蛋白的比例通过雷公藤红素的包封率(Encapsulateefficiency,EE％)筛选：
步骤1：在25℃条件下，将20mgBSA溶于1mL PBS缓冲液，搅拌至溶解，搅拌条件为300rpm/min；加入3.5mL雷公藤红素(CEL)无水乙醇溶液沉淀蛋白，CEL：BSA质量比为1:(5～60)；以0.3mL/min缓慢滴加100μL戊二醛(浓度为2％)，充分搅拌交联纳米粒，高转速(12000rpm/min)离心除去无水乙醇以及多余的戊二醛溶液，用PBS重悬即得CEL@BSA纳米粒。由表1可知，在CEL：BSA质量比为1:25时，CEL的包封率最高，可达(87.2±3.5)％。
表1不同CEL：BSA质量比的CEL的包封率
根据表1中的数据构建图7，通过图7可以确定，当CEL：BSA质量比比例为1:25～1:30，CEL的包封率才能够达到50％。
实施例2
本实施例提供一种RGD修饰的雷公藤红素白蛋白纳米粒的制备方法，具体为在CEL@BSA纳米粒表面修饰RGD环肽，具体过程如下：
取2mg RGD环肽加入1mL水溶解。加入催化剂2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯，HATU(1.5mg)和N,N-二异丙基乙胺DIPEA(1mg)，冰水浴中搅拌30min，转速为300rpm/min。接下来，在300rpm/min转速下，加入1mL包载雷公藤红素的BSA纳米粒(CEL@BSA纳米粒)溶液，搅拌16～20h。即得RGD修饰的雷公藤红素白蛋白(CEL@BSA)纳米粒。
以粒径为指标，考察了RGD和BSA的比例，通过图9可以确定，当比例为1:5～1:40，其粒径稳定在200nm以下。
对比例
同实施例2，不同之处在于，催化剂为1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC)3.2mg和N-羟基琥珀酰亚胺(NHS)2.5mg，冰水浴中搅拌30min，转速为300rpm/min。接下来，在300rpm/min转速下，加入1mL按实施例1方法制备包载雷公藤红素的BSA纳米粒(CEL@BSA纳米粒)溶液，搅拌16～20h。即得RGD修饰的雷公藤红素白蛋白(CEL@BSA)纳米粒。采用该催化剂会使RGD和BSA发生严重的自交联，难以形成稳定的纳米粒，极易发生聚沉(图10)，其粒径为(1913.5±66.3)nm，如图8所示。说明催化体系的选择影响纳米粒的稳定性。
实施例3
本实施例提供一种RGD修饰的雷公藤红素白蛋白纳米粒，所述RGD修饰的雷公藤红素白蛋白纳米粒表面修饰RGD环肽，对RGD修饰成功与否进行验证。使用电泳光散射法测量RGD修饰前后纳米粒的电位，CEL@BSA纳米粒电位为(-2.96±0.5)mV，CEL@BSA-RGD纳米粒电位为(-39.2±1.7)mV，证明RGD成功修饰于CEL@BSA纳米粒表面。
实施例4
本实施例提供一种RGD修饰的雷公藤红素白蛋白纳米粒，使用动态光散射法和透射电镜检测纳米粒的粒径。由图2、图3可知，CEL@BSA纳米粒和CEL@BSA-RGD纳米粒的粒径分别为(115.8±3.7)，(112.5±2.9)nm。透射电镜结果如图4、图5所示。
实施例5
本实施例对CEL@BSA纳米粒和CEL@BSA-RGD纳米粒对类风湿性关节炎小鼠的治疗效果进行研究。
使用C57BL/6RA类风湿性关节炎小鼠评估CEL@BSA纳米粒和CEL@BSA-RGD纳米粒在体内的关节炎疗效。CIA小鼠被随机分为4组(n＝6)。Control代表正常小鼠，CIA组代表关节炎小鼠模型组，CEL代表给予雷公藤红素溶液组的关节炎小鼠，CEL@BSA代表给予CEL@BSA纳米粒治疗的关节炎小鼠，CEL@BSA-RGD代表给予CEL@BSA-RGD纳米粒治疗的关节炎小鼠。治疗组在第28、31、34、37和40天注射DP/BTST NPs。在试验期间，观察小鼠足肿胀程度并拍照记录，见图6通过图6可以看出给予CEL@BSA纳米粒和CEL@BSA-RGD纳米粒治疗后的RA小鼠，关节肿胀程度显著降低，表明CEL@BSA纳米粒和CEL@BSA-RGD纳米粒对RA具有良好的治疗效果。
Claims (10)
1.一种雷公藤红素白蛋白纳米粒，其特征在于，雷公藤红素白蛋白纳米粒是白蛋白包载雷公藤红素形成的纳米粒。
2.根据权利要求1所述的雷公藤红素白蛋白纳米粒，其特征在于，所述白蛋白选自哺乳动物白蛋白。
3.根据权利要求2所述的雷公藤红素白蛋白纳米粒，其特征在于，所述哺乳动物白蛋白选自人血清白蛋白或牛血清白蛋白。
4.根据权利要求1所述的雷公藤红素白蛋白纳米粒，其特征在于，所述雷公藤红素和白蛋白的质量比为1：(20～35)。
5.权利要求1-4任意一项所述的雷公藤红素白蛋白纳米粒的制备方法，为去溶剂法，其特征在于，具体为：
将白蛋白溶于PBS缓冲液，搅拌至溶解，根据比例，加入雷公藤红素的无水乙醇溶液，沉淀白蛋白，30min后，滴加戊二醛交联白蛋白，制得雷公藤红素白蛋白纳米粒。
6.一种RGD修饰的雷公藤红素白蛋白纳米粒，其特征在于，是RGD环肽修饰于权利要求1-4任意一项所述的雷公藤红素白蛋白纳米粒表面，从而形成的RGD修饰的雷公藤红素白蛋白纳米粒。
7.根据权利要求6所述的RGD修饰的雷公藤红素白蛋白纳米粒，其特征在于，按摩尔比，RGD环肽：白蛋白为1:(5～40)。
8.根据权利要求6所述的RGD修饰的雷公藤红素白蛋白纳米粒，其特征在于，所述的RGD修饰的雷公藤红素白蛋白纳米粒的粒径为(112.5±2.9)nm。
9.权利要求6所述的RGD修饰的雷公藤红素白蛋白纳米粒的制备方法，其特征在于，具体为：以水为溶剂，以2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐和N,N-二异丙基乙胺为催化剂，将RGD环肽在0℃搅拌20-30min，得到羧基被活化的RGD环肽；按质量比，催化剂：RGD环肽＝1:2；
将羧基被活化的RGD环肽和雷公藤红素白蛋白纳米粒混合，搅拌16～20h，得到RGD修饰的雷公藤红素白蛋白纳米粒。
10.权利要求1-4任意一项所述的雷公藤红素白蛋白纳米粒或权利要求6-8任意一项所述的RGD修饰的雷公藤红素白蛋白纳米粒在用于治疗类风湿性关节炎药物或治疗炎症疾病药物中的应用。
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