CN115590820A - 一种靶向肝脏的脂质体载药系统及其制备方法 - Google Patents
一种靶向肝脏的脂质体载药系统及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种靶向药物载体,特别涉及一种靶向肝脏的脂质体载药系统及其制备方法。靶向肝脏的脂质体载药系统以半乳糖修饰的红细胞膜与载药脂质体共挤出,得到红细胞膜仿生修饰的肝靶向纳米载药系统。制备方法如下:1、制备靶向材料;2、制备半乳糖修饰红细胞膜;3、制备脂质体;4、制备半乳糖修饰红细胞膜融合脂质体。本发明以非诺贝特为模型药物,将其制备脂质体的基础上,成功将红细胞膜与脂质体融合,再外接有肝脏靶向功能的半乳糖基团,提高载体的生物相容性,延长了药物在体内的代谢时间,提高载体的肝脏靶向性。
Description
技术领域
本发明涉及一种靶向药物载体,特别涉及一种靶向肝脏的脂质体载药系统及其制备方法。
背景技术
根据全球疾病负担项目统计,2020年约有超过200万人死于重大肝病,包括急性肝炎、肝硬化和肝癌,约占全球死亡人数的4%。中国超过五分之一的人口受到某种形式肝病的影响,如病毒性肝炎,包括乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)等;慢性肝脏疾病,包括非酒精性脂肪肝肝病(NAFLD)、酒精性脂肪肝(AFLD)和药物性肝损伤(DILI)等;恶性肝脏疾病,如肝癌。针对发展日益严重的肝脏疾病,近20年来,世界范围内展开无数针对此类疾病的临床研究。在越发强调精准化医疗的今天,人们不约而同地把药物的精准递送作为新的治疗手段。精准靶向递送给药平台改善了药物的药代动力学,减少了毒副作用,并提高患者的依从性,还可以使因毒性、快速清除和脱靶沉积而被认为不切实际的化学品和生物制剂的临床应用成为可能。
在靶向递送系统中,药物载体决定着药物的载药量、释放程度、生物相容性等生物学功能。脂质体是由磷脂双分子层组成的单层或多层封闭式囊泡,其组成与人体细胞的生物膜结构相似,故脂质体具有很强的生物亲和力和良好的生物相容性。由于磷脂类同时具有两亲性的分子结构,脂质体可包封亲水性或亲脂性药物,通过注射、口服和经皮给药等方式作用于有机体,通过膜融合、内吞等方式被细胞摄取,所以脂质体作为药物载体成功递送不同类别药物时,可提高药物稳定性,增加药物的溶解度。但是单纯脂质体在体内的主要根据细胞间隙大小不同被动靶向,容易被巨噬细胞清除或被非靶器官摄取,目标组织细胞摄取量有限。所以如何提高其在体内的长循环能力以及对目标组织的靶向能力是亟待解决的问题。
发明内容
针对现有技术的不足,本发明提供一种靶向肝脏的脂质体载药系统及其制备方法。
本发明采用的技术方案是:一种靶向肝脏的脂质体载药系统,以半乳糖修饰的红细胞膜与载药脂质体共挤出,得到红细胞膜仿生修饰的肝靶向纳米载药系统。
一种靶向肝脏的脂质体载药系统的制备方法,包含如下步骤:
步骤一,靶向材料的制备:
将二硬脂酰基磷脂酰乙醇胺-聚乙二醇3400-琥珀酰亚胺(DSPE-PEG3400-NHS)溶解于无水的N,N二甲基甲酰胺(DMF)中,调节溶液PH为8~9,超声12h,加入半乳糖铵盐酸盐室温下避光反应24h;取出有机溶剂及未反应完的杂质,然后冷冻干燥,得到靶向材料;
步骤二,半乳糖修饰红细胞膜(Gal-RBC)的制备:
取大鼠全血于预先用肝素钠溶液润洗过的离心管中,加入预冷的1×PBS溶液,离心,弃去上层血浆和白细胞,收集下层积压的红细胞层;用1×PBS溶液重复清洗3次;加入0.25×PBS溶液,低渗处理,离心,弃去上清,得到干净的近白色的红细胞膜(RBC);将制得的RBC重悬于1×PBS溶液中,加入由步骤一制备的靶向材料,将溶液置于摇床上,避光孵育,离心,弃去上清,收集下层沉淀;用1×PBS溶液重复清洗3次后,即得半乳糖修饰红细胞膜(Gal-RBC);
步骤三,脂质体的制备:
将磷脂、胆固醇、FNB置于甲醇中超声溶解,减压旋蒸2h,值得脂质薄膜,随后加入1×PBS溶液水化形成脂质体;将脂质体超声破碎得到脂质体混悬液;将脂质体混悬液挤出,即得到非诺贝特脂质体(FNB-Lip)混悬液;
步骤四,半乳糖修饰红细胞膜融合脂质体的制备:
取步骤二所制备的Gal-RBC,破碎后加入步骤三制备的FNB-Lip混悬液,混合均匀后,挤压得到半乳糖修饰的红细胞膜融合非诺贝特脂质体(Gal-RBC-FNB-Lip)。
所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤一为,精密称取40 mg的二硬脂酰基磷脂酰乙醇胺-聚乙二醇3400-琥珀酰亚胺(DSPE-PEG3400-NHS),将其溶解在5 mL无水的N,N二甲基甲酰胺(DMF)中,加入适量的三乙胺(TEA)调节溶液的pH值为8~9,待DSPE-PEG3400-NHS完全溶解后,超声12 h,最后加入半乳糖铵盐酸盐4 mg,室温下避光反应24h;随后以去离子水作为透析介质,使用截留分子量为3500Da的透析袋对反应产物溶液避光透析48 h,从而除去有机溶剂以及未反应完的包含半乳糖胺的杂质;将透析后的产物冷冻干燥,即得靶向材料DSPE-PEG3400-Gal。
所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤二为,经眼眶采血后取得SD大鼠全血;取5 mL新鲜大鼠全血于50 mL预先用肝素钠溶液润洗过的离心管中,加入10倍体积预冷的1×PBS溶液,4℃,6000 rpm,离心10 min,弃去上层血浆和白细胞,收集下层积压的红细胞层;用1×PBS溶液重复清洗3次;加入4倍体积的0.25×PBS溶液,置于4℃的摇床中,低渗处理30 min,4 ℃,8000 rpm,离心15 min,弃去上清,重复3次,得到干净的近白色的红细胞膜(RBC);将制得的RBC重悬于1mL1×PBS溶液中,加入50μg制备好的DSPE-PEG3400-Gal冻干粉末,将溶液置于37℃的摇床上,100 rpm,避光孵育2 h,4℃,8000 rpm,离心10 min,弃去上清,收集下层沉淀;用1×PBS溶液重复清洗3次后,即得半乳糖修饰的红细胞膜(Gal-RBC)。
所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤三为,精密称取磷脂225 mg、胆固醇25mg、FNB25mg于100 mL茄形瓶中,加入20 mL甲醇,超声溶解,减压旋蒸2 h,制得脂质薄膜;随后加入10 mL1×PBS溶液水化30 min形成脂质体;将脂质体转移至10 mL EP管内,240w,室温,超声破碎30min得到脂质体混悬液;使用脂质体挤出器通过200 nm碳酸脂膜将脂质体混悬液挤出15-20次,再经过100 nm 碳酸脂膜挤出15-20次,即得非诺贝特脂质体(FNB-Lip)混悬液。
所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤四为,取1mL上述制备的Gal-RBC置于10 mL EP管内,100 W, 破碎30 s,间隙10 s,循环10 min;破碎完成后加入1mL步骤三制备的FNB-Lip混悬液,混合均匀后,使用脂质体挤出器通过200 nm碳酸脂膜将混合液反复挤压20次,再经过100 nm 碳酸脂膜挤压10次,即得半乳糖修饰的红细胞膜融合非诺贝特脂质体(Gal-RBC-FNB-Lip)。
本发明以非诺贝特(FNB)为模型药物,将其制备脂质体的基础上,成功将红细胞膜与脂质体融合,再外接有肝脏靶向功能的半乳糖基团,提高载体的生物相容性,延长了药物在体内的代谢时间,提高载体的肝脏靶向性。
附图说明
图1为 DSPE-PEG3400-Gal,半乳糖胺,DSPE-PEG3400-NHS核磁共振氢谱图;
图2 为FNB-Lip、Gal-RBC-FNB-Lip透射电镜图;
图3为Gal-RBC-FNB-Lip的稳定性图;
图4 为Gal-RBC-FNB-Lip和FNB的体外累积释放图;
图5 为不同浓度FNB和Gal-RBC-FNB-Lip对细胞存活率的影响图;
图6 Gal-RBC-RhB-Lip电镜图;
图7 为各组HL-02细胞摄取罗丹明B类的荧光情况图;
图8为 ICR小鼠尾静脉注射ICG、ICG-Lip和Gal-RBC-ICG-Lip后的体内外荧光图;
图9 为ICR小鼠尾静脉注射24 h后的离体器官荧光图;
图10为预防给药对体外肝细胞中甘油三酯沉积的影响;
图11为预防给药对体外肝细胞中总胆固醇沉积的影响。
具体实施方式
一种靶向肝脏的脂质体载药系统,采用如下方法制备:
步骤一,靶向材料的制备:
精密称取40 mg的二硬脂酰基磷脂酰乙醇胺-聚乙二醇3400-琥珀酰亚胺(DSPE-PEG3400-NHS),将其溶解在5 mL无水的N,N二甲基甲酰胺(DMF)中,加入适量的三乙胺(TEA)调节溶液的pH值为8~9,待DSPE-PEG3400-NHS完全溶解后,超声12 h,最后加入半乳糖铵盐酸盐4 mg,室温下避光反应24h;随后以去离子水作为透析介质,使用截留分子量为3500Da的透析袋对反应产物溶液避光透析48 h,从而除去有机溶剂以及未反应完的半乳糖胺等杂质。将透析后的产物冷冻干燥,即得靶向材料DSPE-PEG3400-Gal。
步骤二,半乳糖修饰红细胞膜(Gal-RBC)的制备:
经眼眶采血后取得SD大鼠全血;取5 mL新鲜大鼠全血于50 mL预先用肝素钠溶液润洗过的离心管中,加入10倍体积预冷的1×PBS溶液,4℃,6000 rpm,离心10 min,弃去上层血浆和白细胞,收集下层积压的红细胞层;用1×PBS溶液重复清洗3次;加入4倍体积的0.25×PBS溶液,置于4℃的摇床中,低渗处理30 min,4 ℃,8000 rpm,离心15 min,弃去上清,重复3次,得到干净的近白色的红细胞膜(RBC);将制得的RBC重悬于1mL1×PBS溶液中,加入50μg制备好的DSPE-PEG3400-Gal冻干粉末,将溶液置于37℃的摇床上,100 rpm,避光孵育2 h,4℃,8000 rpm,离心10 min,弃去上清,收集下层沉淀;用1×PBS溶液重复清洗3次后,即得半乳糖修饰的红细胞膜(Gal-RBC)。
步骤三,脂质体的制备:
精密称取磷脂225 mg、胆固醇25mg、FNB25mg于100 mL茄形瓶中,加入20 mL甲醇,超声溶解,减压旋蒸2 h,制得脂质薄膜;随后加入10 mL1×PBS溶液水化30 min形成脂质体;将脂质体转移至10 mL EP管内,240w,室温,超声破碎30min得到脂质体混悬液;使用脂质体挤出器通过200 nm碳酸脂膜将脂质体混悬液挤出15-20次,再经过100 nm 碳酸脂膜挤出15-20次,即得非诺贝特脂质体(FNB-Lip)混悬液。
步骤四,半乳糖修饰红细胞膜融合脂质体的制备:
取1mL上述制备的Gal-RBC置于10 mL EP管内,100 W, 破碎30 s,间隙10 s,循环10 min。破碎完成后加入1mL上述制备的FNB-Lip混悬液,混合均匀后,使用脂质体挤出器通过200 nm碳酸脂膜将混合液反复挤压20次,再经过100 nm 碳酸脂膜挤压10次,即得半乳糖修饰的红细胞膜融合非诺贝特脂质体(Gal-RBC-FNB-Lip)。
验证实验:
1. DSPE-PEG3400-Gal的核磁共振氢谱
由图1可知,DSPE-PEG3400-Gal,1H NMR(400 MHz,DMSO)显示DSPE-PEG3400-Gal既有半乳糖的特殊质子化学位移峰(3.35 ppm),有显示出DSPE的特殊质子化学位移峰(0.92ppm)。同时NHS的特殊质子化学位移峰(2.75 ppm)在图中消失,说明化合物DSPE-PEG3400-Gal合成成功。
图1中,A:DSPE-PEG3400-Gal;B:半乳糖胺;C:DSPE-PEG3400-NHS。
2. Gal-RBC-FNB-Lip的形态学表征
图2中A视图,FNB-Lip透射电镜图表明,脂质体呈现出完整的球形且形状良好,符合脂质体形态学的一般要求。粒径大小为88.32 ± 2.38 nm,PDI为0.263 ± 0.018。图2中B视图,Gal-RBC-FNB-Lip的透射电镜图表明,相比于FNB-Lip,融合了RBC后,RBC将脂质体包裹起来,在单层膜结构的脂质体外面包裹了一层浅色的RBC,RBC与脂质体完全融合,脂质体本身的单层膜结构无法观察清楚。Gal-RBC-FNB-Lip的粒径约为94.28±1.77 nm,相较于FNB-Lip粒径增大。说明Gal-RBC成功修饰在FNB-Lip上。
图2 中,比例尺:100 μm,A:FNB-Lip,B:Gal-RBC-FNB-Lip。
3. 稳定性考察
取新配置的Gal-RBC-FNB-Lip溶液,置于4℃条件下保存,在0、7、14、21、30天时测定其粒径大小和PDI,评价载药系统的稳定性。结果如图3所示,附图3中,A为粒径稳定性图,B为PDI稳定性图,Gal-RBC-FNB-Lip的粒径维持在95 nm左右,PDI维持在0.26~0.27左右,表明Gal-RBC-FNB-Lip在30天内稳定性良好。
4. 体外释放研究
采用透析法测定Gal-RBC-FNB-Lip与FNB在含2% Cremophor EL 的PBS溶液中的释放度。在0.5、1、2、4、8、12、24和48h时的累积释放度如图4所示。在含2% Cremophor EL 的PBS溶液中,FNB在24h内释放迅速,在48h时,FNB的累积释放度为85%,说明48 h已基本释放完全。而Gal-RBC-FNB-Lip在48h是累积释放度为38%,且释放速度较为恒定,延缓了FNB的释放。
图4中,释放介质:2% Cremophor EL的PBS。
5. 细胞毒性试验
取半乳糖修饰的红细胞膜融合非诺贝特脂质体(Gal-RBC-FNB-Lip)用培养基稀释配置为0.05、0.1、0.5、1μmol/L(以FNB含量计)等系列浓度,取1×105个处于生长对数期的HL7702细胞,接种于96孔板中。放置于培养箱中使其贴壁后培养24 h进行后续实验。24 h后,分别设置空白组,给药组,对照组。空白组每孔分别加入培养液100μL,给药组中每孔中分别加入用培养液稀释成不同浓度的Gal-RBC-FNB-Lip100μL,对照组每孔中分别加入用培养液稀释成不同浓度的FNB100μL。培养24h后,弃去上清液,每孔加入100μL含MTT的DMEM培养液(0.5 μg/μL),在培养箱中孵育4h。然后弃去上清,每个孔再加入100 μL的DMSO。待甲瓒充分溶解后,用酶标仪在490 nm处测定其吸光度,计算细胞活力。细胞存活率=(OD给药组-OD空白组)/(OD对照组-OD空白组)×100%。
结果如图5所示,用不同浓度的FNB和Gal-RBC-FNB-Lip溶液培养细胞24 h后,FNB组细胞和Gal-RBC-FNB-Lip细胞存活率均在85%左右,说明在此浓度范围内FNB和Gal-RBC-FNB-Lip无明显细胞毒性。
6. 半乳糖红细胞膜融合脂质体的肝细胞靶向性研究
1)半乳糖修饰的红细胞膜融合罗丹明B脂质体(Gal-RBC-RhB-Lip)的制备
精密称取磷脂,胆固醇,罗丹明B(RhB)(225 mg : 25mg : 25mg)于100 mL的茄形瓶中,加入20 mL甲醇,超声溶解,然后减压旋蒸2 h,蒸去溶剂形成脂质薄膜。加入10 mL 1×PBS水溶液水化30 min形成脂质体。将脂质体转移至10 mL EP管内,240w,室温,超声破碎30min得到脂质体混悬液。使用脂质体挤出器通过200 nm碳酸脂膜将脂质体混悬液挤出15-20次,再经过100 nm 碳酸脂膜挤出15-20次,即得罗丹明B脂质体(RhB-Lip)混悬液。
取1mL制备好的Gal-RBC置于10 mL EP管内,100 W, 破碎30 s,间隙10 s,循环10min。破碎完成后加入1 mL上述制备的RhB-Lip混悬液,混合均匀后,使用脂质体挤出器通过200 nm碳酸脂膜将混合液反复挤压20次,再经过100 nm 碳酸脂膜挤压10次,即得半乳糖修饰的红细胞膜融合罗丹明B脂质体(Gal-RBC-RhB-Lip)。
2)Gal-RBC-RhB-Lip的形态学表征
结果如图6所示,Gal-RBC-RhB-Lip的透射电镜图表明,RBC将脂质体包裹起来,在单层膜结构的脂质体外面包裹了一层浅色的RBC,RBC与脂质体完全融合,脂质体本身的单层膜结构无法观察清楚。
图6 中,比例尺:100 μm。
3)体外的靶向性研究
取生长对数期的HL7702细胞1×105个接种于6孔板中,待细胞贴壁生长24h后,将细胞分为RhB组,RhB-Lip组,Gal-RBC-RhB-Lip组。弃去旧的培养基,RhB组加入10 μmol/L的罗丹明B荧光探针,RhB-Lip组加入10 μmol/L的RhB-Lip,Gal-RBC-RhB-Lip组加入10 μmol/L的Gal-RBC-RhB-Lip,继续培养2 h,弃去培养基,用PBS溶液清洗三次直至无多余的荧光染料、将孔板置于荧光倒置显微镜下观察荧光情况,并用Image J软件对荧光进行定量分析。
结果如图7所示,细胞荧光共孵育2h后,与RhB组和RhB-Lip组细胞相比,Gal-RBC-RhB-Lip组细胞中红色荧光明显增强,说明Gal-RBC-RhB-Lip相对于RhB和RhB-Lip对肝细胞有更好的靶向性。
图7中,A:RhB组;B:RhB-Lip组;C:Gal-RBC-RhB-Lip组;D:各组细胞平均荧光强度,*p<0.05,**p<0.01。
4)半乳糖修饰的红细胞膜融合吲哚菁绿脂质体(Gal-RBC-ICG-Lip)的制备
精密取磷脂,胆固醇,吲哚菁绿((90 mg : 10 mg : 10 mg)于100 mL的茄形瓶中,加入20 mL甲醇,超声溶解,然后减压旋蒸2 h,蒸去溶剂形成脂质薄膜。加入10 mL 1×PBS水溶液水化30 min形成脂质体。将脂质体转移至10 mL EP管内,240w,室温,超声破碎30min得到脂质体混悬液。使用脂质体挤出器通过200 nm碳酸脂膜将脂质体混悬液挤出15-20次,再经过100 nm 碳酸脂膜挤出15-20次,即得吲哚菁绿脂质体(ICG-Lip)混悬液。
取1mL制备好的Gal-RBC置于10 mL EP管内,100 W, 破碎30 s,间隙10 s,循环10min。破碎完成后加入1 mL上述制备的ICG-Lip混悬液,混合均匀后,使用脂质体挤出器通过200 nm碳酸脂膜将混合液反复挤压20次,再经过100 nm 碳酸脂膜挤压10次,即得半乳糖修饰的红细胞膜融合吲哚菁绿脂质体(Gal-RBC-ICG-Lip)。
5)体内靶向研究
ICR小鼠提前24 h剃毛,禁食不禁水。实验时将小鼠随机分为3组,分别尾静脉注射给药ICG、ICG-Lip和Gal-RBC-ICG-Lip(浓度为1 mg/mL,以ICG计)。给药后,10 min,1 h,2h,4 h,8 h,12 h,24 h使用异氟烷麻醉小鼠后,将小鼠放入小动物活体成像仪中,设置为ICG荧光通道(激发波长780 nm,发射波长845 nm)观察小鼠体内的荧光强度和荧光分布。尾静脉给药24 h,处死小鼠,取出小鼠的心、肝、脾、肺和肾,用PBS溶液清洗后放入活体成像仪测定离体器官的荧光强度和荧光分布。
结果如图8所示,ICR小鼠静脉注射ICG、ICG-Lip和Gal-RBC-ICG-Lip后,ICG先是随着血液弥漫全身,随着时间的推移,荧光逐渐集中于小鼠的腹腔部位。再随着时间的推移,代谢过程开始进行,与ICG、ICG-Lip相比,Gal-RBC-ICG-Lip组代谢速度明显减慢,Gal-RBC-ICG-Lip能显著性地延长ICG在小鼠体内的保留时间,起到长循环缓释作用。离体器官中荧光分布情况如图9所示。给药24 h后,ICG组小鼠器官中基本没有荧光物质残留,可以认为已基本代谢完毕。在ICG-Lip组中,除肝脏和肾脏外,脾脏部位和肺部也呈现明显的荧光分布,说明ICG-Lip对肝脏靶向性较差,而在Gal-RBC-ICG-Lip组中,荧光更加集中的分布在肝脏,而在脾脏和肺部未观察到明显的荧光,说明Gal-RBC-ICG-Lip可以将药物精准靶向地递送至肝脏,避免药物在小鼠心、脾、肺和肾等其他重要器官吸收,有效提高药物在肝脏的浓度和药效,同时降低药物在其他组织器官的毒副作用。
7. Gal-RBC-FNB-Lip对体外脂质沉积细胞模型的修复作用
1) 取对数生长期的HL7702细胞1×106个,接种于直径10cm培养皿中。待细胞生长24 h后,将细胞分为对照组(不作处理),模型组(5%棕榈酸),阳性对照组1(5%棕榈酸和FNB,1μmol/L)、阳性对照组2(5%棕榈酸和FNB,0.5μmol/L)、给药组1(5%棕榈酸和Gal-RBC-FNB-Lip,1μmol/L)、给药组2(5%棕榈酸和Gal-RBC-FNB-Lip,0.5μmol/L),分别给药后作用24 h。胰酶消化收集细胞,用冷的PBS清洗3次后置于冰上超声破碎。取细胞破碎液用PBS稀释后,使用BCA蛋白定量法进行蛋白定量。根据TC和TG试剂盒说明书操作,测定不同组细胞内TG和TC含量。
结果如图10,图11所示,与对照组相比,模型组细胞内TG和TC含量均显著上升。当给与1μmol/L FNB和1μmol/L Gal-RBC-FNB-Lip 24 h后,与模型组相比,细胞内TG和TC含量均发生显著下降,说明Gal-RBC-FNB-Lip和FNB都可以有效减轻肝细胞中脂质积累,并且制备的靶向载体并没有影响到FNB本身的降脂作用。
本发明中缩写词中英文对照表
Claims (6)
1.一种靶向肝脏的脂质体载药系统,其特征是:以半乳糖修饰的红细胞膜与载药脂质体共挤出,得到红细胞膜仿生修饰的肝靶向纳米载药系统。
2.一种靶向肝脏的脂质体载药系统的制备方法,其特征是:包含如下步骤:
步骤一,靶向材料的制备:
将二硬脂酰基磷脂酰乙醇胺-聚乙二醇3400-琥珀酰亚胺(DSPE-PEG3400-NHS)溶解于无水的N,N二甲基甲酰胺(DMF)中,调节溶液PH为8~9,超声12h,加入半乳糖铵盐酸盐室温下避光反应24h;取出有机溶剂及未反应完的杂质,然后冷冻干燥,得到靶向材料;
步骤二,半乳糖修饰红细胞膜(Gal-RBC)的制备:
取大鼠全血于预先用肝素钠溶液润洗过的离心管中,加入预冷的1×PBS溶液,离心,弃去上层血浆和白细胞,收集下层积压的红细胞层;用1×PBS溶液重复清洗3次;加入0.25×PBS溶液,低渗处理,离心,弃去上清,得到干净的近白色的红细胞膜(RBC);将制得的RBC重悬于1×PBS溶液中,加入由步骤一制备的靶向材料,将溶液置于摇床上,避光孵育,离心,弃去上清,收集下层沉淀;用1×PBS溶液重复清洗3次后,即得半乳糖修饰红细胞膜(Gal-RBC);
步骤三,脂质体的制备:
将磷脂、胆固醇、FNB置于甲醇中超声溶解,减压旋蒸2h,值得脂质薄膜,随后加入1×PBS溶液水化形成脂质体;将脂质体超声破碎得到脂质体混悬液;将脂质体混悬液挤出,即得到非诺贝特脂质体(FNB-Lip)混悬液;
步骤四,半乳糖修饰红细胞膜融合脂质体的制备:
取步骤二所制备的Gal-RBC,破碎后加入步骤三制备的FNB-Lip混悬液,混合均匀后,挤压得到半乳糖修饰的红细胞膜融合非诺贝特脂质体(Gal-RBC-FNB-Lip)。
3.根据权利要求2所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤一为,精密称取40 mg的二硬脂酰基磷脂酰乙醇胺-聚乙二醇3400-琥珀酰亚胺(DSPE-PEG3400-NHS),将其溶解在5 mL无水的N,N二甲基甲酰胺(DMF)中,加入适量的三乙胺(TEA)调节溶液的pH值为8~9,待DSPE-PEG3400-NHS完全溶解后,超声12 h,最后加入半乳糖铵盐酸盐4 mg,室温下避光反应24h;随后以去离子水作为透析介质,使用截留分子量为3500Da的透析袋对反应产物溶液避光透析48 h,从而除去有机溶剂以及未反应完的包含半乳糖胺的杂质;将透析后的产物冷冻干燥,即得靶向材料DSPE-PEG3400-Gal。
4.根据权利要求2所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤二为,经眼眶采血后取得SD大鼠全血;取5 mL新鲜大鼠全血于50 mL预先用肝素钠溶液润洗过的离心管中,加入10倍体积预冷的1×PBS溶液,4℃,6000 rpm,离心10 min,弃去上层血浆和白细胞,收集下层积压的红细胞层;用1×PBS溶液重复清洗3次;加入4倍体积的0.25×PBS溶液,置于4℃的摇床中,低渗处理30 min,4 ℃,8000 rpm,离心15 min,弃去上清,重复3次,得到干净的近白色的红细胞膜(RBC);将制得的RBC重悬于1mL1×PBS溶液中,加入50μg制备好的DSPE-PEG3400-Gal冻干粉末,将溶液置于37℃的摇床上,100 rpm,避光孵育2 h,4℃,8000 rpm,离心10 min,弃去上清,收集下层沉淀;用1×PBS溶液重复清洗3次后,即得半乳糖修饰的红细胞膜(Gal-RBC)。
5.根据权利要求2所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤三为,精密称取磷脂225 mg、胆固醇25mg、FNB25mg于100 mL茄形瓶中,加入20 mL甲醇,超声溶解,减压旋蒸2 h,制得脂质薄膜;随后加入10 mL1×PBS溶液水化30 min形成脂质体;将脂质体转移至10 mL EP管内,240w,室温,超声破碎30min得到脂质体混悬液;使用脂质体挤出器通过200 nm碳酸脂膜将脂质体混悬液挤出15-20次,再经过100 nm 碳酸脂膜挤出15-20次,即得非诺贝特脂质体(FNB-Lip)混悬液。
6.根据权利要求2所述的一种靶向肝脏的脂质体载药系统的制备方法,其特征是:所述步骤四为,取1mL上述制备的Gal-RBC置于10 mL EP管内,100 W, 破碎30 s,间隙10 s,循环10 min;破碎完成后加入1mL步骤三制备的FNB-Lip混悬液,混合均匀后,使用脂质体挤出器通过200 nm碳酸脂膜将混合液反复挤压20次,再经过100 nm 碳酸脂膜挤压10次,即得半乳糖修饰的红细胞膜融合非诺贝特脂质体(Gal-RBC-FNB-Lip)。
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