CN115177739A - 一种组织-细胞-细胞器三级靶向的仿生制剂及其制备方法和应用 - Google Patents
一种组织-细胞-细胞器三级靶向的仿生制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种组织‑细胞‑细胞器三级靶向的仿生制剂,包括载生物活性物质的纳米颗粒和覆盖在纳米颗粒表面的经高尔基体靶向肽修饰的活化血小板膜,纳米颗粒为聚合物纳米颗粒或脂质体,高尔基体靶向肽包含氨基酸序列SXYQRL,其中X代表任意氨基酸。该制剂可实现对关节炎‑滑膜成纤维细胞‑高尔基体的三级靶向药物递送,可通过扰乱高尔基体的功能,从而阻断滑膜成纤维细胞分泌造成滑膜炎和骨侵蚀的细胞因子和酶,最终实现RA的有效治疗。还公开了该仿生制剂的制备方法,操作简单,成本低廉,制备高效。
Description
技术领域
本发明属于生物医药领域,特别是一种组织-细胞-细胞器三级靶向的仿生制剂及其制备方法和应用。
背景技术
类风湿性关节炎(RA)是一种以慢性侵蚀性关节炎为特征的自身免疫性疾病,随着疾病进展,RA将导致患者关节变性甚至造成残疾。通常认为,滑膜巨噬细胞是RA关节滑膜中重要的免疫细胞,在RA的发生和发展过程中具有重要作用,常常作为RA的治疗靶点,药物递送***的研究也主要围绕着巨噬细胞展开。抑制滑膜巨噬细胞的活化或阻断其介导的细胞因子信号,通常可一定程度降低RA滑膜炎症,但这些方法对滑膜炎的持续性进展和骨破坏的保护作用有限。另外,这些治疗方式对30%的患者无效,仍需要寻找更有效的治疗方式。
近年来,研究者逐渐认识到RA关节滑膜成纤维细胞可通过分泌多种细胞因子和酶,从而造成滑膜炎和骨侵蚀。因此,将药物有效递送至滑膜成纤维细胞并有效阻断其分泌的病理性细胞因子和酶对于RA治疗具有重要意义。
发明内容
本发明所要解决的技术问题是,克服以上背景技术中提到的不足和缺陷,提供一种可实现组织-细胞-细胞器三级靶向的仿生制剂,最终实现RA的有效治疗。
为解决上述技术问题,本发明提出的技术方案为:
一种组织-细胞-细胞器三级靶向的仿生制剂,包括载生物活性物质的纳米颗粒和覆盖在所述纳米颗粒表面的经高尔基体靶向肽修饰的活化血小板膜,所述纳米颗粒为聚合物纳米颗粒或脂质体,所述高尔基体靶向肽包含氨基酸序列SXYQRL,其中X代表任意氨基酸,如序列表SEQ ID NO:1所示。
本发明的仿生制剂可通过活化血小板膜对炎症的趋向性实现对RA的炎性关节靶向,然后通过整合素α2β1介导的内吞,靶向滑膜成纤维细胞,最后在高尔基体靶向肽(含氨基酸序列SXYQRL的多肽可实现从内涵体到高尔基体的逆转运,从而定位在高尔基体上)的介导下,富集在滑膜成纤维细胞的高尔基体上。利用该仿生制剂递送高尔基体干扰剂(如全反式维甲酸),可以特异性地高效破坏RA滑膜成纤维细胞的高尔基体结构,以有效地阻断基于滑膜成纤维细胞的多重致病因子(如致炎性细胞因子、趋化因子以及基质降解酶等),从而实现对RA滑膜炎以及骨侵蚀的有效治疗。
上述的组织-细胞-细胞器三级靶向的仿生制剂,优选的,所述活化血小板膜为经血小板活化剂激活后所得的血小板膜或血小板微粒膜;所述聚合物纳米颗粒为PLGA纳米粒;所述生物活性物质为高尔基体干扰剂。
优选的,所述高尔基体干扰剂为全反式维甲酸及其衍生物。
优选的,所述经高尔基体靶向肽修饰的活化血小板膜通过化学合成或基因工程的方式制备得到。
基于一个总的发明构思,本发明还提供一种组织-细胞-细胞器三级靶向的仿生制剂的制备方法,包括如下步骤:
(1)将经过N-羟基琥珀酰亚胺活化的高尔基体靶向肽与活化血小板膜共同孵育,得到经高尔基体靶向肽修饰的活化血小板膜;
(2)制备聚合物纳米颗粒混悬液或脂质体液;
(3)将所述经高尔基体靶向肽修饰的活化血小板膜和所述聚合物纳米颗粒混悬液或脂质体液共同孵育,然后通过超声或挤压过微孔滤膜的方式制成仿生制剂,即得。
上述的制备方法,优选的在步骤(1)中,所述共同孵育时间为2-6h,未反应的高尔基体靶向肽通过超滤除去;所述活化血小板膜由以下方法制备得到:通过梯度离心制得血小板,然后加入血小板激活剂,孵育4-8h后,离心,取富含血小板微粒的上层液,通过反复冻融、梯度离心,除去血小板微粒内容物,即得;另外,所述活化血小板膜还可以通过以下方法制得:通过梯度离心制得血小板,然后加入血小板激活剂,孵育4-8h后,通过反复冻融、梯度离心,除去血小板内容物,即得;所述血小板激活剂为脂多糖、凝血酶、胶原、含钙离子试剂中的一种或多种。
优选的,在步骤(2)中,所述聚合物纳米颗粒混悬液由以下方法制备得到:将聚合物材料和生物活性物质溶于二氯甲烷中,得有机相;另外以质量-体积浓度为1%的PVA水溶液为水相,将所述有机相加入到水相中,有机相与水相的体积比例为1:5-20,进行200-400W的探头超声5-15min,即得聚合物纳米颗粒混悬液。
优选的,在步骤(2)中,所述脂质体液由以下方法制备得到:将磷脂、胆固醇以及生物活性物质溶解于乙醇中,通过旋转蒸发法除去有机试剂(可采用旋转蒸发仪操作),然后加入水脱膜,进行200-400W的探头超声5-15min,即得脂质体液。
优选的,在步骤(3)中,所述共同孵育的具体操作包括如下步骤:将所述经高尔基体靶向肽修饰的活化血小板膜和所述聚合物纳米颗粒混悬液或脂质体液混合得到混合液,将所述混合液在冰水浴中进行200-400W探头超声处理5-15min;或将所述混合液依次挤压通过直径为400nm、200nm、100nm的微孔滤膜。
基于一个总的发明构思,本发明还提供一种上述仿生制剂在制备防治类风湿性关节炎药物中的应用。
与现有技术相比,本发明的有益效果为:
1、本发明的仿生制剂具有关节炎内滑膜成纤维细胞高尔基体靶向的作用,该制剂可实现对关节炎-滑膜成纤维细胞-高尔基体的三级靶向药物递送,可通过扰乱高尔基体的功能,从而阻断滑膜成纤维细胞分泌造成滑膜炎和骨侵蚀的细胞因子和酶,最终实现RA的有效治疗。
2、本发明的制备方法,操作简单,成本低廉,制备高效。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是实施例1制备得到的仿生制剂的表征;其中:a)NPs、PMNPs和Gol-PMNPs的粒径;b)NPs、PMNPs和Gol-PMNPs的zeta电位;c)NPs、PMNPs和Gol-PMNPs粒径的多分散系数;d)NPs、PMNPs和Gol-PMNPs的透射电镜图。
图2是实施例1制备得到的仿生制剂的细胞摄取实验;其中:a)激光共聚焦显微镜观察NPs、PMNPs和Gol-PMNPs在人源滑膜成纤维细胞上的摄取;b)NPs、PMNPs和Gol-PMNPs被人源滑膜成纤维细胞摄取的流式直方图;c)NPs、PMNPs和Gol-PMNPs被人源滑膜成纤维细胞摄取的流式定量分析柱状图(n=3),**P<0.01;d)激光共聚焦显微镜观察NPs、PMNPs和Gol-PMNPs与人源滑膜成纤维细胞高尔基体共定位;e)NPs、PMNPs和Gol-PMNPs与人源滑膜成纤维细胞高尔基体共定位的皮尔森相关系数(n=3),**P<0.01;f)Gol-PMNPs在人源滑膜成纤维细胞上的摄取途径(n=3),**P<0.01,***P<0.001。
图3是实施例1制备得到的仿生制剂的体外生物活性考察实验;其中:a)人源滑膜成纤维细胞高尔基体的透射电镜图;b)人源滑膜成纤维细胞高尔基体的激光共聚焦图;c)人源滑膜成纤维细胞中绿色荧光的流式定量柱状图;d-j)药物处理24h后,细胞培养上清液的蛋白浓度。
图4是实施例1制备得到的仿生制剂的体内分布实验;其中:a)NPs、PMNPs和Gol-PMNPs在RA模型大鼠体内分布的小动物活体荧光成像图(A、心;B、肝;C、脾;D、肺;E、肾;F、血;G、炎性关节);b)NPs、PMNPs和Gol-PMNPs在RA模型大鼠炎性关节分布的半定量柱状图(n=6,3只动物的6个炎性关节),***P<0.001;c)NPs、PMNPs和Gol-PMNPs在RA模型大鼠主要脏器分布的半定量柱状图(n=3),*P<0.05,**P<0.01;d)NPs、PMNPs和Gol-PMNPs与RA模型大鼠炎性关节滑膜成纤维细胞共定位。
图5是实施例1制备得到的仿生制剂的药效学实验;其中:a)给药方案示意图;b)RA模型大鼠后肢脚踝直径变化图(n=7),*P<0.05;c)RA模型大鼠后肢脚掌直径变化图(n=7),**P<0.01;d)结束治疗48h后,各组大鼠后肢的照片及其切片的H&E(标尺为100μm)、safranin-O(标尺为200μm)和toluidineblue(标尺为200μm)染色分析;e)结束治疗48h后,各组大鼠后肢踝关节的Micro-CT分析图;f-g)各组大鼠后肢踝关节Micro-CT定量分析柱状图(n=3),*P<0.05,**P<0.01,***P<0.001。
图6是实施例2制备得到的仿生制剂的细胞摄取实验;其中:a)Lip、PM-Lip和Gol-PM-Lip被人源滑膜成纤维细胞摄取的流式定量分析柱状图(n=3),***P<0.001;b)Lip、PM-Lip和Gol-PM-Lip与人源滑膜成纤维细胞高尔基体共定位的皮尔森相关系数(n=3),**P<0.01,***P<0.001。
具体实施方式
为了便于理解本发明,下文将结合说明书附图和较佳的实施例对本发明做更全面、细致地描述,但本发明的保护范围并不限于以下具体实施例。
除非另有定义,下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的,并不是旨在限制本发明的保护范围。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
实施例1:
一种组织-细胞-细胞器三级靶向的仿生制剂,由聚合物纳米颗粒(PLGA纳米粒)和覆盖在聚合物纳米颗粒表面的经高尔基体靶向肽修饰的活化血小板膜组成,高尔基体靶向肽的氨基酸序列如SEQ ID NO:2所示,序列缩写为ASDYQRLN。
该仿生制剂的制备方法如下:
1)活化血小板膜(PM)的提取
将收集的全血样品在4℃下200g离心10min,取上层富血小板血浆;同样条件下,重复离心一次进行纯化,得纯化的富血小板血浆;在纯化的富血小板血浆中加入***素E1,在4℃下2000g离心10min,得血小板沉淀;将血小板沉淀重新分散在含有1mMCaCl2和5μg/ml胶原的生理盐水中,孵育4h,在4℃下2000g离心20min,取富含血小板微粒的上清液,反复冻融,通过梯度离心,除去血小板微粒内容物,即得PM。
2)高尔基体靶向肽修饰血小板膜(Gol-PM)的制备
将NHS(N-羟基琥珀酰亚胺)活化的高尔基体靶向肽(氨基酸序列为ASDYQRLN)加入到含血小板膜的PBS中,室温搅拌4h,通过超滤除去未反应的高尔基体靶向肽,即得。
3)载全反式维甲酸(ATRA)的PLGA纳米粒(NPs)的制备
将5mg全反式维甲酸、500mgPLGA50/50(平均分子量4万)溶于二氯甲烷中,制成有机相;将PVA溶于水中配置成1%质量-体积浓度的PVA水溶液,作为水相。按照有机相/水相体积比1:10将两相混合,冰水浴中探头超声(300W,8min),再通过旋转蒸发仪除掉有机试剂,即得。
4)仿生制剂的制备
将上述所得的PM溶液和NPs混合(PM与PLGA的质量比为1:2),冰水浴中探头超声(300W,8min),即得血小板膜包裹的纳米粒(PMNPs);将PM溶液换成将Gol-PM溶液(Gol-PM与PLGA的质量比为1:2),按照上述相同方法操作,即得具有高尔基体靶向的仿生制剂(Gol-PMNPs)。
5)制剂的表征
用纯水将上述所得仿生制剂PMNPs和Gol-PMNPs进行稀释至有淡蓝色乳光,通过激光粒度仪测定其粒径;所得制剂不经稀释直接测定其zeta电位;通过透射电镜观察其形貌。
从图1可知,NPs是粒径为150nm左右,zeta电位为-30eV左右的类球形颗粒,制备成仿生制剂PMNPs和Gol-PMNPs后,其粒径略有增长,而电位升高到-20eV之上。从透射电镜图(图1d)可以看出,仿生制剂PMNPs和Gol-PMNPs表面有一层明显的膜结构。以上结果均证实仿生制剂的成功制备。
6)体外摄取实验
取RA患者进行关节置换手术后所得的关节滑膜,剪碎,并用II型胶原消化,将消化后的组织通过70μm细胞滤网,收集滤过的细胞悬液;将细胞悬液放入细胞培养皿中培养,去除不贴壁的细胞后,将贴壁的成纤维细胞接种在12孔细胞培养板上,培养24h,待其贴壁后,将培养基替换成含DiD标记的NPs、PMNPs和Gol-PMNPs的无血清培养基(DiD浓度为1μ/ml),孵育4h后,用流式细胞仪检测细胞的荧光强度,并用激光共聚焦显微镜对细胞的荧光分布进行观察和拍照。为验证纳米粒的内吞途径,将成纤维细胞用不同抑制剂(25μM的TCI-15,30μM的氯丙嗪,50μM阿米洛利或5mM的M-β-CD)预处理1h,然后加入DiD标记的Gol-PMNPs,再孵育4h,不加抑制剂处理的成纤维细胞作为对照组,用流式细胞仪检测细胞的荧光强度。
从图2可知,Gol-PMNPs在人源滑膜成纤维细胞上的摄取明显高于NPs(图2a-c);进一步地,Gol-PMNPs与成纤维细胞高尔基体共定位能力明显高于NPs和PMNPs(图2d-e)。另外,经TCI-15(整合素α2β1抑制剂)预处理后,Gol-PMNPs在滑膜成纤维细胞上摄取显著降低(图2f)。以上结果说明Gol-PMNPs可以通过整合素α2β1介导的内吞高效被滑膜成纤维细胞摄取,而且能靶向分布于高尔基体上。
7)体外生物学活性考察
取RA患者进行关节置换手术后所得的关节滑膜,剪碎,并用II型胶原消化,将消化后的组织通过70μm细胞滤网,收集滤过的细胞悬液;将细胞悬液放入细胞培养皿中培养,去除不贴壁的细胞后,将贴壁的成纤维细胞接种在12孔细胞培养板上,培养24h,待其贴壁后,在培养基中分别加入载ATRA的NPs、PMNPs和Gol-PMNPs(ATRA的浓度为200ng/mL,不加药的培养基处理作为对照),孵育24h后,取上清液,使用ELLISA法测定其中collagense、MMP-1、CX3CL、IL-8、IL-6、MCP-1以及MMP-13的浓度;通过透射电镜观察每组细胞高尔基体的形态;使用带绿色荧光的GM130抗体标记细胞高尔基体,通过激光共聚焦显微镜观察并使用流式细胞仪测定细胞的荧光强度。
由图3可知,相对于其他处理方法,ATRA-Gol-PMNPs处理可以更有效地破坏成纤维细胞高尔基体的形态并显著降低成纤维细胞蛋白分泌能力。这些结果进一步证明,Gol-PMNPs可有效靶向滑膜成纤维细胞高尔基体并实现有效的药物递送。
8)RA大鼠模型
将完全弗氏佐剂通过皮下注射到大鼠尾根部处,待大鼠后肢出现关节“强直”现象,即得RA大鼠模型。
9)体内分布实验
将DiD溶液以及DiD标记的NPs、PMNPs和Gol-PMNPs分别通过尾静脉注射到大鼠体内,24h后,通过小动物活体成像仪观察大鼠炎性关节处的荧光强度并拍照。小动物活体成像仪观察结束后,将大鼠处死,分离各组大鼠炎性关节的滑膜,对其进行冰冻切片,然后分别用DAPI和绿色荧光标记的波形蛋白抗体对切片中细胞的细胞核和滑膜成纤维细胞进行染色,通过激光共聚焦显微镜进行观察并拍照。
由图4可知,相对于NPs,Gol-PMNPs可以有效地靶向分布于RA大鼠的炎性关节并在富集于滑膜成纤维细胞的位置。这些结果表明,Gol-PMNPs可以实现对关节炎和滑膜成纤维细胞的靶向。
10)药效学研究
通过尾静脉注射方式,对RA大鼠分别使用生理盐水、ATRA溶液、ATRA-NPs、ATRA-PMNPs以及ATRA-Gol-PMNPs进行治疗。ATRA的给药剂量为1mg/kg,隔两天给药一次,共给药5次。从给药第一天起,隔天测量一次各组大鼠踝关节和脚掌厚度的直径。
由图5可知,相对于其他方式来说,ATRA-Gol-PMNPs治疗可以更有效的缓解RA大鼠的关节炎症和实现更好的骨保护作用。这些结果充分说明实现了关节炎-成纤维细胞-高尔基体三级靶向递送药物对RA治疗的有效性,也证明本发明的仿生制剂在制备防治类风湿性关节炎药物中具有很好的应用效果。
实施例2:仿生脂质体
一种组织-细胞-细胞器三级靶向的仿生制剂,由仿生脂质体和覆盖在仿生脂质体表面的经高尔基体靶向肽修饰的活化血小板膜组成,高尔基体靶向肽的氨基酸序列如SEQID NO:2所示,序列缩写为ASDYQRLN。
该仿生制剂的制备方法如下:
1)活化血小板膜(PM)的提取
将收集的全血样品在4℃下200g离心10min,取上层富血小板血浆;同样条件下,重复离心一次进行纯化,得纯化的富血小板血浆;在纯化的富血小板血浆中加入***素E1,在4℃下2000g离心10min,得血小板沉淀;将血小板沉淀重新分散在含有100ng/ml胶原的生理盐水中,孵育8h,反复冻融,通过梯度离心,除去血小板内容物,即得PM。
2)高尔基体靶向肽修饰血小板膜(Gol-PM)的制备
将NHS(N-羟基琥珀酰亚胺)活化的高尔基体靶向肽(氨基酸序列为ASDYQRLN)加入到含血小板膜的PBS中,室温搅拌24h,通过超滤除去未反应的高尔基体靶向肽,即得。
3)DiD标记脂质体(Lip)的制备
将DiD、磷脂、胆固醇、全反式维甲酸溶于乙醇中,通过旋转蒸发仪除掉有机试剂,然后加水脱膜,冰水浴中探头超声(350W,6min),即得。激光粒度仪测定其粒径和电位分别为118.5±6.5nm和-6.1±1.4mV。
4)仿生制剂的制备
将上述所得的PM溶液和Lip混合(PM与脂质的质量比为1:5),将混合液依次挤压通过直径为400nm、200nm、100nm的微孔滤膜,即得血小板膜包裹的脂质体(PM-Lip),激光粒度仪测定其粒径和电位分别为126.3±4.2nm和-14.4±3.1mV;将PM溶液换成将Gol-PM溶液(Gol-PM与脂质的质量比为1:5),按照上述相同方法操作,即得具有高尔基体靶向的仿生制剂(Gol-PM-Lip),激光粒度仪测定其粒径和电位分别为127.1±3.5nm和-15.2±2.8mV。
5)体外摄取实验
取RA患者进行关节置换手术后所得的关节滑膜,切碎,并用II型胶原消化,将消化后的组织通过70μm细胞滤网,收集滤过的细胞悬液;将细胞悬液放入细胞培养皿中培养,去除不贴壁的细胞后,将贴壁的成纤维细胞接种在12孔细胞培养板上,培养24h,待其贴壁后,将培养基替换成含DiD标记的Lip、PM-Lip和Gol-PM-Lip的无血清培养基(DiD浓度为1μ/ml),孵育4h后,用流式细胞仪检测细胞的荧光强度,并通过激光共聚焦显微镜观察细胞的荧光分布和计算脂质体的高尔基体共定位系数。
从图6可知,Gol-PM-Lip在人源滑膜成纤维细胞上的摄取明显高于Lip(图6a);进一步地,Gol-PM-Lip与成纤维细胞高尔基体共定位能力明显高于Lip和PM-Lip(图6b)。以上结果说明Gol-PM-Lip不仅可以更高效被滑膜成纤维细胞摄取,而且能靶向分布于高尔基体上。
综上所述,本发明的仿生制剂,具有关节炎内滑膜成纤维细胞高尔基体靶向的工程化血小板膜仿生制剂,该制剂可实现对关节炎-滑膜成纤维细胞-高尔基体的三级靶向药物递送,可通过扰乱高尔基体的功能,从而阻断滑膜成纤维细胞分泌造成滑膜炎和骨侵蚀的细胞因子和酶,最终实现RA的有效治疗。且本发明的制备方法,操作简单,成本低廉,制备高效。
序列表
<110> 中南大学湘雅医院
<120> 一种组织-细胞-细胞器三级靶向的仿生制剂及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ser Xaa Tyr Gln Arg Leu
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ser Asp Tyr Gln Arg Leu Asn
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Claims (10)
1.一种组织-细胞-细胞器三级靶向的仿生制剂,其特征在于,包括载生物活性物质的纳米颗粒和覆盖在所述纳米颗粒表面的经高尔基体靶向肽修饰的活化血小板膜,所述纳米颗粒为聚合物纳米颗粒或脂质体,所述高尔基体靶向肽包含氨基酸序列SXYQRL,其中X代表任意氨基酸。
2.根据权利要求1所述的组织-细胞-细胞器三级靶向的仿生制剂,其特征在于,所述活化血小板膜为经血小板活化剂激活后所得的血小板膜或血小板微粒膜;所述聚合物纳米颗粒为PLGA纳米粒;所述生物活性物质为高尔基体干扰剂。
3.根据权利要求2所述的组织-细胞-细胞器三级靶向的仿生制剂,其特征在于,所述高尔基体干扰剂为全反式维甲酸及其衍生物。
4.一种如权利要求1-3中任一项所述组织-细胞-细胞器三级靶向的仿生制剂的制备方法,其特征在于,包括如下步骤:
(1)将经过N-羟基琥珀酰亚胺活化的高尔基体靶向肽与活化血小板膜共同孵育,得到经高尔基体靶向肽修饰的活化血小板膜;
(2)制备载生物活性物质的聚合物纳米颗粒混悬液或脂质体液;
(3)将所述经高尔基体靶向肽修饰的活化血小板膜和所述聚合物纳米颗粒混悬液或脂质体液共同孵育,然后通过超声或挤压过微孔滤膜的方式制成仿生制剂,即得。
5.根据权利要求4所述的制备方法,其特征在于,在步骤(1)中,所述活化血小板膜由以下方法制备得到:通过梯度离心制得血小板,然后加入血小板激活剂,孵育4-8h后,离心,取富含血小板微粒的上层液,通过反复冻融、梯度离心,除去血小板微粒内容物,即得;所述血小板激活剂为脂多糖、凝血酶、胶原、含钙离子试剂中的一种或多种;所述共同孵育时间为2-6h,未反应的高尔基体靶向肽通过超滤除去。
6.根据权利要求4所述的制备方法,其特征在于,在步骤(1)中,所述活化血小板膜由以下方法制备得到:通过梯度离心制得血小板,然后加入血小板激活剂,孵育4-8h后,通过反复冻融、梯度离心,除去血小板内容物,即得;所述血小板激活剂为脂多糖、凝血酶、胶原、含钙离子试剂中的一种或多种;所述共同孵育时间为2-6h,未反应的高尔基体靶向肽通过超滤除去。
7.根据权利要求4所述的制备方法,其特征在于,在步骤(2)中,所述聚合物纳米颗粒混悬液由以下方法制备得到:将聚合物材料和生物活性物质溶于二氯甲烷中,得有机相;另外以质量-体积浓度为1%的PVA水溶液为水相,将所述有机相加入到水相中,有机相与水相的体积比例为1:5-20,进行200-400W的探头超声5-15min,即得聚合物纳米颗粒混悬液。
8.根据权利要求4所述的制备方法,其特征在于,在步骤(2)中,所述脂质体液由以下方法制备得到:将磷脂、胆固醇以及生物活性物质溶解于乙醇中,通过旋转蒸发法除去有机试剂,然后加入水脱膜,进行200-400W的探头超声5-15min,即得脂质体液。
9.根据权利要求4所述的制备方法,其特征在于,在步骤(3)中,所述共同孵育的具体操作包括如下步骤:将所述经高尔基体靶向肽修饰的活化血小板膜和所述聚合物纳米颗粒混悬液或脂质体液混合得到混合液,将所述混合液在冰水浴中进行200-400W探头超声处理5-15min;或将所述混合液依次挤压通过直径为400nm、200nm、100nm的微孔滤膜。
10.一种如权利要求1-3中任一项所述的仿生制剂或由权利要求4-9所述制备方法制备得到的仿生制剂在制备防治类风湿性关节炎药物中的应用。
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