CN116236473A - 一种抗肝纤维化的药物组合物及其制备方法 - Google Patents
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Abstract
本发明公开了一种抗肝纤维化的药物,它包括外泌体和连翘脂素;所述外泌体是经透明质酸修饰的外泌体。本发明利用透明质酸修饰牛奶外泌体mEXO,使被修饰后的mEXO装载的PHI具有肝脏靶向性,制成的靶向纳米药物在口服给药后治疗肝纤维化的疗效得到显著的提高。经试验证明:本发明制成的靶向口服制剂PHI‑HA‑mEXO不仅稳定性好,安全性高,还具有高效靶向能力,以及优越的肝纤维化作用,具备实际推广应用的价值。
Description
技术领域
本发明具体涉及一种抗肝纤维化的药物组合物及其制备方法。
背景技术
肝纤维化是影响全球数百万人健康的严重问题。它是各种急性或慢性刺激反复损伤肝脏的无效可逆伤口愈合反应。在各种原因导致的肝损伤后,肝星状细胞被凋亡的肝细胞和枯否细胞分泌的促炎细胞因子和活性氧激活,转化为肌成纤维细胞样细胞。活化的肝星状细胞(aHSC)会产生大量以胶原蛋白为主的细胞外基质,如果此时不干预肝纤维化的发展,肝纤维化将发展成更为严重的肝硬化甚至肝癌。
目前肝纤维化尚无标准的治疗方法。现阶段主要是通过抗炎、抗氧化、抗病毒及免疫调节等消除病因的治疗方法缓解肝纤维化。但以上疗法都有各种副作用,不能长期使用。因此,寻找安全有效的抗肝纤维化药物具有十分重要的意义。据报道,传统医药在抗肝纤维化方面有着独特的优势,比如中药,可通过抗炎、抗氧化等多种途径发挥保肝作用。其中,连翘脂素(PHI)具有显著的抗肝纤维化能力,其能通过降低NF-κB的表达发挥抗炎作用进而逆转肝纤维化。除此之外,它还能通过调节肠道微生物群及胆汁酸代谢发挥抗肝纤维化作用。尽管PHI表现出显著的抗肝纤维化特性,但由于其水溶性差和口服吸收不完全限制了其口服生物利用度,阻碍了PHI作为潜在肝纤维化治疗剂的临床开发。
目前有必要将PHI开发成一种具有肝脏靶向能力的药物,以期在口服给药后最大限度地提高药物疗效。
发明内容
为解决上述问题,本发明提供了一种抗肝纤维化的药物,它包括外泌体和连翘脂素;所述外泌体是经透明质酸修饰的外泌体。
进一步地,所述外泌体是经透明质酸修饰的牛奶外泌体。
进一步地,所述牛奶外泌体与透明质酸混合孵育,得经透明质酸修饰的牛奶外泌体;所述牛奶外泌体与透明质酸的质量比为20~40:1,优选20:1。
更进一步地,所述牛奶外泌体是牛奶经差速超速离心得到的沉淀,用PBS溶液洗涤并重悬的混悬液;所述混悬液中蛋白质含量为4~8mg/ml。
更进一步地,所述差速超速离心的方法为:
牛奶先10,000×g离心30min,取溶液100,000×g离心90min,收集上清135,000×g离心70min,收集沉淀,即得。
进一步地,所述药物在制备时外泌体和连翘脂素的制备比为4~8:1。
进一步地,所述药物在制备时外泌体和连翘脂素的制备比为6:1。
本发明还提供了一种前述的药物组合物的制备方法,其特征在于:它包括如下步骤:
1)按配比称取原料;取外泌体和透明质酸混合,孵育,离心,取上清得透明质酸修饰的牛奶外泌体;
2)取步骤1)所得透明质酸修饰的牛奶外泌体,与连翘脂素混合,孵育,超滤,即得。
进一步地,步骤1)所述孵育的温度为37℃,时间48h;离心速度3500g,时间25min;
步骤2)所述孵育是室温避光孵育,时间3h;所述超滤的超滤管10KDa,速度3500g,时间10min
本发明还提供了一种前述药物在制备治疗肝纤维化的药物中的用途。
本发明利用透明质酸修饰牛奶外泌体mEXO,使被修饰后的mEXO装载的PHI具有肝脏靶向性,制成的靶向纳米药物在口服给药后治疗肝纤维化的疗效得到显著的提高。经试验证明:本发明制成的靶向口服制剂PHI-HA-mEXO不仅稳定性好,安全性高,还具有高效靶向能力,以及优越的肝纤维化作用,具备实际推广应用的价值。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1mEXO和PHI-HA-mEXO的理化特性(A.mEXO和PHI-HA-mEXO的TEM图像;B.用PBS稀释后,mEXO和PHI-HA-mEXO通过NTA测量粒径和通过ζ电位测量表面电荷;C.mEXO和PHI-HA-mEXO标志蛋白CD9、CD81和Calnexin的鉴定)
图2PHI-HA-mEXO的稳定性及溶出度(A.PHI和PHI-HA-mEXO的HPLC图;B.低温储存1个月后,PHI-HA-mEXO的NTA、ζ电位图和电镜图;C.在PH7.4的PBS中,PHI-HA-mEXO的释放曲线)
图3PHI-HA-mEXO的摄取及CD44表达测定(A.qHSC和aHSC中DIR标记的PHI-HAmEOX和PHI-mEXO的摄取;B.正常和TAA诱导的斑马鱼幼虫肝纤维化中DIR标记的PHI-HAmEOX和PHI-mEXO的摄取;C.正常和TGF-β1激活的LX2细胞中CD44信号的代表性共焦图像;D.流式细胞仪检测正常和TGF-β1激活的LX2细胞中CD44信号的代表性图像)
图4PHI-HA-mEXO体外抗肝纤维化作用(A.TGF-β1和PHI-HA-mEXO(3.75、7.5、15、30、60和120μg/mL)处理后LX2细胞的细胞活力;B.PHI-HA-mEXO显著下调α-SMA和Col1α1mRNA水平;C.LX2细胞凋亡率的流式细胞术分析;D.TGF-β1和PHI-HA-mEXO、mEXO、HA-mEXO、PHI、PHI-mEXO处理后LX2细胞的细胞活力;E.PHI-HA-mEXO(3.75、7.5、15、30、60和120μg/mL)处理后LX2细胞和LO2细胞的细胞活力。每个条形代表平均值±SD(n=3)。#p<0.05,##p<0.01和###p<0.001vs.control group,*p<0.05,**p<0.01和***p<0.001vs.modelgroup)
图5PHI-HA-mEXO在斑马鱼模型中的抗肝纤维化作用(A.暴露于TAA、TAA+PHI-HA-mEXO(25、50、100μM)、TAA+HAmEXO、TAA+PHI-mEXO、TAA+PHI和TAA+mEXO的斑马鱼幼鱼的荧光图像;B.PHI-HA-mEXO对斑马鱼幼鱼ALT、AST和ALP水平的影响;C.斑马鱼肝脏的HE染色;D.斑马鱼肝脏的天狼星红染色;E.斑马鱼Col1α1和α-SMA表达的定量结果。每个条形代表平均值±SD(n=6)。#p<0.05,##p<0.01和###p<0.001vs.control group,*p<0.05,**p<0.01和***p<0.001vs.model group)
图6PHI-HA-mEXO对TAA诱导的斑马鱼肝纤维化中自噬相关基因的影响(A.Beclin1和LC3的基因表达水平;B.P62的基因表达水平;C.Atg5和Atg7的基因表达水平。每个条形代表平均值±SD(n=3)。#p<0.05,##p<0.01和###p<0.001vs.control group,*p<0.05,**p<0.01和***p<0.001vs.model group)
具体实施方式
实施例1、本发明牛奶外泌体的制备
将鲜牛乳装入若干50mL离心管中,于普通离心机中10,000×g离心30min,收集脱脂牛奶;将脱脂牛奶转移到若干38.4mL开口超速离心管中并配平,于超速离心机中100,000×g离心90min,收集上层乳清并将其过0.22μm的滤膜,减少可能夹杂的杂蛋白;得到的乳清于超速离心机中135,000×g离心70min,取外泌体沉淀,使用预冷PBS溶液洗涤外泌体沉淀三次,并用PBS溶液将该外泌体沉淀重悬成溶液,通过0.22μm微孔滤膜过滤获得蛋白质浓度为6mg/mL的外泌体溶液,即得。
实施例2本发明靶向药物制剂的制备
1)取20mg:1mg的外泌体(mEXO)和透明质酸(DSPE-PEG2000-HA)混合,37℃孵育48h,离心25分钟(3500g),收集上清液,得到透明质酸修饰的牛奶外泌体HA-mEXO;
2)取步骤1)得到的HA-mEXO,与连翘脂素按6mg:1mg混合,室温避光孵育3h,用10KDa的超滤管3500g超滤10min除去未结合的成分,得到PHI-HA-mEXO。
以下通过试验例来说明本发明的有益效果。
试验例1连翘脂素靶向性复合外泌体的制备及其抗肝纤维化研究
一、材料和方法
1、材料
牛奶购于本地超市。连翘脂素(Cat.No.21080708)从成都MUST生物技术有限公司(中国成都)获得。DSPE-PEG2000-HA(Cat.No.R-A66001-2k)由瑞禧生物科技有限公司(陕西)合成。转化生长因子-β1(Cat.No.CA59)从近岸蛋白质科技股份有限公司(上海)获得。LX-2细胞由中南大学(长沙,中国)赠予。人正常肝细胞LO2(iCell-h054)购自中国上海iCell生物科技有限公司。三卡因购自西格玛奥德里奇贸易有限公司(中国上海)。BCA蛋白检测试剂盒(Cat.No.G2026-1000T)购于联科生物技术有限公司(中国杭州)。HA(Cat.No.CPA182Ge21;Dilution 1:1000)、CD63(Cat.No.YT5525;Dilution 1:1000)、CD81(Cat.No.YT5394;Dilution 1:1000)和Calnexin(Cat.No.YT0613;Dilution 1:1000)抗体购于云克隆公司(武汉)和美国Immunoway生物科技公司(美国)。山羊抗兔IgG-HRP(Cat.No.05-4030-05;Dilution1:5000)购于联科生物技术有限公司(中国杭州)。高效液相色谱法甲醇(Cat.No.216565)级购自默克化工(上海,中国)。RPMI Medium 1640basic(1x)(Cat.No.8122651)和DMEM basic(1x)(Cat.No.8122340)从gibco(澳大利亚)获得。CD44(Cat.No.AF6186)来自Affinity biosciences(江苏)。Annexin-V-FITC凋亡检测试剂盒(Cat.No.E-CK-A211)来源于伊莱瑞特生物科技股份有限公司(武汉)。DIR(Cat.No.D4006)购自苏州优逸兰迪生物科技有限公司(中国苏州)。4%多聚甲醛(Cat.No.G1101)购自塞维尔科技有限公司(中国武汉)。总RNA分离试剂盒(Cat.No.RE-03014)从成都福际生物技术有限公司获得。ABScript III RT Master Mix for qPCR(Cat.No.20428)和Genious 2X SYBRGreen Fast qPCR Mix(No ROX)(Cat.No.21205)从爱博泰克生物技术有限公司(武汉,中国)获得。PCR引物序列由北京擎科生物科技有限公司(成都分部)合成。
2、方法
2.1外泌体的提取分离
本实验提取外泌体的方法为差速超速离心法,所有离心的过程温度均设置为4℃。简单来讲,将鲜牛乳装入若干50mL离心管中,于普通离心机中10,000×g离心30min去除脂肪球、酪蛋白聚集体及细胞碎片等物质并收集脱脂牛奶。将脱脂牛奶转移到若干38.4mL开口超速离心管中并配平,于超速离心机中100,000×g离心90min,弃除底部蛋白质沉淀,收集上层乳清并将其过0.22μm的滤膜,减少可能夹杂的杂蛋白;得到的乳清于超速离心机中135,000×g离心70min,弃去上清液收获外泌体沉淀。使用预冷PBS溶液洗涤外泌体沉淀三次,并用PBS溶液将该外泌体沉淀重悬成溶液,通过0.22μm微孔滤膜过滤获得外泌体溶液,通过BCA蛋白试剂盒测定牛奶外泌体的蛋白质浓度并将其标准化为6mg/mL。将外泌体重悬液储存于-80℃冰箱中备用。
2.2PHI药物装载及载药量测定
取外泌体(mEXO)和透明质酸(DSPE-PEG2000-HA)(20mg:1mg)混合,37℃孵育48h,离心25分钟(3500g)得到HA-mEXO。将连翘脂素与HA-mEXO分别按照1:2,1:4,1:6,1:8比例混合,室温避光孵育3h,用10KDa的超滤管3500g超滤10min除去未结合的药物,得到PHI-HA-mEXO。
为了评估mEXO对PHI的装载能力,通过高效液相色谱法测定了PHI的载药率。对照品溶液的制备:精密称取一定量的PHI对照品于5mL量瓶中,加入甲醇定容,即得母液。供试品溶液的制备:取PHI-HA-mEXO悬液10μL,加入790μL体积的甲醇破乳,即得。色谱柱:Agilent ZORBAX Eclipse C18(4.6×250mm,5um);检测波长:280nm;流动相:甲醇-水;柱温:25℃;按照表1梯度洗脱顺序进行洗脱,流速:1mL/min;进样量:10μL。得到的样品用高效液相仪器检测峰面积,依据PHI标准曲线计算浓度和包封率,包封率计算公式如下。包封率=[(纯化样品中连翘脂素浓度*样品体积)/连翘脂素总质量]×100%。
2.3mEXO及PHI-HA-mEXO表征
取从牛奶中提取的外泌体或PHI-HA-mEXO 10μL,滴加于200目碳膜铜网上沉淀1min,滤纸吸去浮液。吸取样品10μL滴加于铜网上沉淀1min,用滤纸小心吸取剩余的液体,加1%醋酸双氧铀10μL负染5min,滤纸吸去多余负染液,室温静置30min晾干,100KV进行电镜检测成像,观察mEXO以及PHI-HA-mEXO的形貌。将提取得到的外泌体或PHI-HA-mEXO用超纯水稀释1000倍,充分混匀后,上机(纳米颗粒跟踪分析仪)检测,记录外泌体粒径分布范围、外泌体膜zeta电位及外泌体浓度(囊泡数/mL)。将mEXO或PHI-HA-mEXO与蛋白上样缓冲液按照4:1的比例混合均匀,并于100℃处理10min;取10μL处理后的样品上样,进行电泳、转膜、洗涤封闭,分别将CD9、CD81和Calnexin一抗按照说明书比例稀释并将膜放入一抗溶液过夜孵育。孵育完成后,用TBST溶液清洗3次PVDF膜,每次10min。清洗完成后,用二抗孵育2h,孵育完成后,用TBST溶液清洗3次PVDF膜,每次10min。按照超敏ECL化学发光试剂盒中的说明书对PVDF膜进行成像。
2.4储存稳定性研究
由于所构建的载药体系主要基于mEXO,该生物膜材料不适合长期置于4℃保存。为探究该载药体系的储存条件以满足后续实验,将PHI-HA-mEXO体系以溶液状态于-80℃冰箱中储存,在储存1个月解冻后通过纳米颗粒跟踪分析仪检测外泌体粒径及电位变化,以评价PHI-HA-mEXO的稳定性。
2.5体外释放
2.5.1用DIR染料标记的PHI-HA-mEXO的细胞摄取研究
将qHSC和aHSC细胞以5×104个的细胞密度接种在共聚焦培养皿中孵育过夜。过夜孵育后,用DIR染料标记的PHI-mEXO和PHI-HA-mEXO以30μg/ml的浓度添加到每个孔中。孵育4小时后,用PBS洗涤细胞并用4%多聚甲醛溶液固定。然后使用激光共聚焦显微镜观察细胞。
2.5.2用DIR染料标记的PHI-HA-mEXO的体内生物分布研究
用DIR染料标记的PHI-HA-mEXO分别处理肝纤维化斑马鱼幼鱼和正常斑马鱼幼鱼。然后再将斑马鱼幼鱼麻醉侧放于共聚焦小皿上,并使用共聚焦显微镜在750和779nm的激发和发射波长下进行荧光观察。
2.6CD44的表达研究
将LX2细胞按1×106/孔接种在六孔板上,分为空白对照组、模型组、PHI-mEXO和PHI-HA-mEXO组。对照组为LX2细胞不加药物培养24h,模型组在LX2细胞中加入15nM TGF-β处理24h,PHI-mEXO和PHI-HA-mEXO组在加入15nM TGF-β的LX2细胞中分别加入PHI浓度为30μg/ml的PHI-mEXO和PHI-HA-mEXO培养24h。药物处理完毕后将细胞用PBS洗涤两次,加入CD44抗体在暗处孵育30min后用流式细胞仪进行分析,计算各组CD44表达的水平。
LX2细胞种植在共聚焦小皿上24h,干预后用4%多聚甲醛室温固定,正常非免疫血清(羊)室温封闭,一抗CD44-PE(1∶375)4℃孵育过夜,羊抗兔红色荧光二抗(1∶2000)室温孵育30min,DAPI试剂染核后封片,使用共聚焦显微镜观察、拍摄。
2.7细胞培养、细胞活力和细胞毒性
LO2细胞和LX-2细胞在添加了10%FBS的1640培养基中培养。将LO2细胞和LX-2细胞以每孔5×103个细胞的密度接种到96孔板中并孵育过夜。然后用不同药物浓度(3.75、7.5、15、30、60和120μg/mL)的PHI-HA-mEXO处理24小时。之后,丢弃含药培养基并加入20μLMTT溶液孵育4小时。最后用酶标仪测量490nm处的吸光度。
2.8动物试验
2.8.1实验动物
使用TAA溶液诱导2dpf斑马鱼幼鱼肝纤维化3天。第一组是对照组其中未给与斑马鱼幼鱼TAA及PHI-HA-mEXO。除第一组外,其他组斑马鱼幼鱼均接受TAA处理。第二组是TAA对照组,其中斑马鱼幼鱼未接受PHI-HA-mEXO处理。第三、四、五组分别给与PHI-HA-mEXO(25、50、100μM)处理。在实验结束时,将所有样本固定在4%多聚甲醛中用于石蜡包埋进行组织病理学检查。所有程序均遵循成都中医药大学动物实验伦理委员会。
2.8.2生化分析和肝脏组织组织病理学检查
肝组织样品用4%多聚甲醛固定24h,乙醇脱水、透明处理后,浸蜡、包埋、5μm切片,将石蜡切片依次经过脱蜡、复水、苏木精染色、分化、伊红染色等步骤进行常规苏木精-伊红染色(Hematoxylin-eosin staining,HE染色)及Siriusred染色,在显微镜下进行组织病理学检查。
首先准确选取30条斑马鱼幼鱼,加入9倍体积的匀浆液在冰水浴下匀浆。然后,将匀浆以2500r/min离心10分钟。收集上清液,根据制造商的说明,用ALT、AST和ALP商业试剂盒测量丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和碱性磷酸酶(ALP)的水平。
2.8.3实时定量PCR(RT-qPCR)分析
根据制造商的说明,使用溶解在50μL无RNase水中的总RNA分离试剂盒从大约30条的斑马鱼幼鱼中提取总RNA。RNA的纯度通过核酸/蛋白质分析仪测得的OD260/280值来表征。ⅢAll-in-one RT SuperMix Perfect for qPCR进行逆转录以合成cDNA。反应条件如下:25℃5min、42℃15min和85℃5s。RT-qPCR在StepOnePlus Real-Time PCRSystem上进行,方法是根据制造商的方案添加2×Taq Pro Universal SYBR qPCR MasterMix。反应条件设置如下:95℃1min,然后在95℃5s和60℃30s 40个循环。2-△△Ct法用于计算目的基因的相对表达量。
2.8.4统计分析
采用SPSS 26.0软件进行统计学分析。实验结果均以平均值±标准差表示,采用单因素方差分析(One-Way ANOVA)进行统计学分析,P<0.05为差异有统计学意义。
3、结果
3.1 mEXO及PHI-HA-mEXO的理化表征
透射电镜结果显示,差速离心法提取的外泌体分散性较好,mEXO以及PHI-HA-mEXO多呈圆形或类圆形的茶托样/杯状结构,具有明显的膜状结构,边界较清楚,分布较集中,提示所得外泌体大小均一性较好(图1A)。
纳米颗粒跟踪分析结果显示外泌体总体粒径范围在80~140nm之间,平均粒径为114.2nm(图1B),膜表面带负电荷,zeta电位为:-36.41±1.63mV(图1C),浓度为6.0E+10Particles/mL。PHI-HA-mEXO总体粒径范围为80~130nm,平均粒径为120.7nm(图1B),膜表面带负电荷,zeta电位为:-36.77±1.21mV(图1D),浓度为2.7E+11Particles/mL。
Western blot实验结果显示所得mEXO以及PHI-HA-mEXO均有特征性膜蛋白CD9、CD81表达,无阴性蛋白Calnexin的表达(图1C)。
3.2PHI-HA-mEXO载药量、稳定性和体外释放研究
根据PHI标准品的进样浓度和峰面积,计算得出标准曲线的线性回归方程为Y=42.457X-70.798(R2=0.9997),PHI在3.125~700mg/L的质量浓度范围内线性关系良好。如图2A所示为连翘脂素标准品和PHI-HA-mEXO的色谱图,通过PHI标准曲线回归方程,计算PHI的载药率。实验测得mEXO对PHI的载药率在连翘脂素与HA-mEXO比例为1:2,1:4,1:6,1:8比例下分别为34.10%,40.97%,57.06%,48.93%,该结果表明当连翘脂素与HA-mEXO比例为1:6时载药率最高,为57.06%,表明mEXO对PHI有良好的装载效率,有利于后续药理学实验的开展。
通过纳米颗粒跟踪分析仪测定在-80℃冰箱中储存1个月后的PHI-HA-mEXO的粒径及电位变化。如图2B结果显示,低温储存1个月后,PHI-HA-mEXO体系的粒径集中于120.5nm,zeta电位为-33.21±0.00mV。与新鲜制备的样品相比,纳米颗粒性质未发生显著变化,这些发现表明,PHI-HA-mEXO可以长时间储存,而不会导致其理化性质发生显著变化。
在PBS缓冲溶液中,使用透析袋测定PHI-HA-mEXO的药物释放动力学。结果表明,PHI-HA-mEXO表现出时间依赖性释放。在1、2、4、8h后,PHI的累积释放量分别为2%、46%、70%、83%,几乎所有药物(92%)在12小时后释放(图2C)。
3.3.细胞摄取、靶细胞上CD44的表达评估和体内生物分布
使用激光共聚焦显微镜观察qHSC和aHSC对PHI-HA-mEXO的摄取,结果如图3A所示,aHSC比qHSC对PHI-HA-mEXO的摄取更高。aHSC会表达过量的CD44,而qHSC则不会上调该蛋白的表达。此外,我们分析了用DIR染料标记的PHI-HA-mEXO在斑马鱼幼鱼的肝纤维化和正常肝脏中的生物分布(图3B)。共聚焦图像显示纤维化肝脏中的荧光强度明显强于正常肝脏。以上发现指出PHI-HA-mEXO对aHSC的高效靶向能力,以及改善肝纤维化的潜力。为了评估PHI-HA-mEXO的靶向特异性,我们通过流式细胞术检测aHSC和qHSC上CD44的表达,结果如图3C所示,在aHSC中观察到高水平的CD44表达,而在qHSC中发现低水平的CD44表达,PHI-HA-mEXO组CD44因和透明质酸结合其表达水平明显低于模型组。此外,我们使用共聚焦显微镜对肝星状细胞上CD44荧光进行了测定,结果如图3D所示,CD44在模型组PHI-mEXO组表达最多,在模型组PHI-HA-mEXO中有所下调,其原因是靶向药结合CD44从而导致荧光强度减弱。而空白组中靶向和未靶向组因肝星状细胞并没有活化所以显示出较弱的荧光强度,且二者无显著性差异。这些结果表明,PHI-HA-mEXO可以进入aHSC中,并且对于qHSC相互作用的趋势较低。
3.4.PHI-HA-mEXO对aHSC的抗肝纤维化作用
我们使用TGF-β1诱导的LX-2细胞肝纤维化模型评估了PHI-HA-mEXO的抗肝纤维化作用,并将其与游离的PHI等进行了比较。我们首先检测了PHI-HA-mEXO(3.75、7.5、15、30、60和120μg/mL)对aHSC的细胞毒性。结果如图4A所示,细胞活力随着PHI-HA-mEXO的浓度增加而降低,并在30μg/mL时恢复至正常水平。此外,qRT-PCR结果也表现出相似的结果,如图4B所示,PHI-HA-mEXO可以显著抑制LX-2细胞中Col1α1和α-SMA的表达水平,而mEXO、HA-mEXO、PHI以及PHI-mEXO组不能显著抑制LX-2细胞中Col1α1和α-SMA的表达水平。此外,流式细胞仪结果也表明,PHI-HA-mEXO能够促进LX-2细胞凋亡(图4C)。为了排除mEXO及透明质酸对结果的影响,我们在30μg/mL的给药浓度下进行了平行对照实验,结果如图4D所示,外泌体组、靶向外泌体组、游离的PHI组和PHI-mEXO组与模型组相比均未显示出差异,而PHI-HA-mEXO组则显著的表现出细胞毒作用。
为了评估PHI-HA-mEXO的生物安全性,我们研究了PHI-HA-mEXO对正常肝细胞和肝星状细胞的细胞毒性。将PHI-HA-mEXO(3.75、7.5、15、30、60和120μg/mL)与正常的肝细胞和肝星状细胞共孵育24小时后,我们没有观察到显著的细胞毒性(图4E)。这表明PHI-HA-mEXO是专门作用于aHSC的,对其他细胞是安全的、生物惰性的,并具有良好的生物相容性。
3.5.PHI-HA-mEXO对硫代乙酰胺诱导的肝纤维化的改善作用
2天大斑马鱼幼鱼浸泡在硫代乙酰胺(TAA)3天以诱导肝纤维化,各组于此同时分别给与PHI-HA-mEXO(25、50、100μM)处理3天。如图5A所示,荧光区域代表斑马鱼幼虫的肝组织。正常对照组幼虫显示出清晰而强烈的荧光信号。然而,用TAA处理的幼虫呈现出相对微弱且暗的荧光信号,肝脏面积明显减少,这表明TAA暴露肯定会导致幼虫的肝脏形态学改变。相反,PHI-HA-mEXO治疗显著改善了TAA激发引起的肝脏变化,并剂量依赖性地增强了肝脏荧光和面积。此外,用PHI-HA-mEXO(25、50、100μM)处理的斑马鱼显示出肝脏的显著改善,表明PHI-HA-mEXO比HA-mEXO、PHI-mEXO、PHI和mEXO具有更好的抗肝纤维化作用。然后收集样本对其进行生化指标(ALT、AST、ALP)评估,结果如图5B所示,与空白组相比,模型组斑马鱼幼鱼ALT、AST、ALP均显著升高,反应了相当大的肝损伤。随着PHI-HA-mEXO剂量(25、50、100μM)的增加,ALT、AST、ALP都显著的降低至空白组水平,表现出优异的保肝作用。
为了进一步探索PHI-HA-mEXO在肝纤维化中的作用,对模型斑马鱼肝脏进行了H&E和天狼星红染色。H&E染色结果如图5C所示,空白组肝脏肝细胞表现出可见的核仁、圆形核和分散在外周的染色质,而没有出现细胞的损失和结构的改变。与空白组相比,模型组则显示出明显的细胞缺失和结构改变。PHI-HA-mEXO则以剂量依赖性的方式恢复了肝损伤。与H&E染色变化一致,在模型组中观察到肝切片Sirius red染色中肝细胞之间的胶原沉积明显多于空白组,而PHI-HA-mEXO组(25、50、100uM)则以剂量依赖性的方式减轻了肝细胞之间的胶原沉积(图5D)。Col1α1和α-SMA作为细胞外基质的重要组成成分,其表达水平可反应肝纤维化的严重程度。我们使用了PCR检测了不同组肝组织中Col1α1和α-SMA的基因表达变化(图5E)。结果显示模型组Col1α1和α-SMA的表达水平显著升高,而PHI-HA-mEXO则以剂量依赖性的方式抑制了这种改变。以上数据清楚地表明,PHI-HA-mEXO处理显著地抑制了TAA诱导的肝纤维化。
3.6.PHI-HA-mEXO通过下调Beclin-1和LC3抑制自噬
自噬可以通过降解脂滴和细胞蛋白为aHSC提供能量来加剧肝纤维化。Beclin-1、LC3和P62是自噬过程中最为关键的自噬体形成的重要标志物。为了评估PHI-HA-mEXO处理后肝脏中的自噬水平,使用qRT-PCR测量这些指标的基因水平。结果表明,Beclin-1和LC3等蛋白在模型组中表达水平显著增加,而翘脂素靶向性复合外泌体组(25、50、100uM)中的Beclin-1和LC3水平则以剂量依赖性的方式逐渐降低(图6A)。P62作为一种自噬相关转运蛋白在模型组中显著减少,在PHI-HA-mEXO处理后以剂量依赖性的方式增加(图6B)。为了进一步证实我们的发现,我们还分析了参与将LC3-Ⅰ转化为LC3-Ⅱ的Atg5和Atg7的水平。结果如图6C所示,PHI-HA-mEXO显著降低了Atg5和Atg7的mRNA水平。以上结果表明,PHI-HA-mEXO至少部分通过抑制aHSC中的自噬减轻肝纤维化。
4、讨论
PHI已被证明是一种具有较好抗炎及抗纤维化作用的药物。然而由于其水溶性差、口服吸收不完全,因此其口服生物利用度低。为了克服与口服给药相关的限制,多种递药系统已被开发出来用于药物递送。比如,王等人开发出了一种新型的脂质体递药系统用于PHI的递送;然而,由于其成本高昂、缺乏广泛应用性以及包括安全性在内的固有限制,它们没有进入临床应用。我们的研究旨在通过使用牛奶衍生的外泌体来研究PHI的口服靶向递送,以克服PHI生物利用度低及靶向治疗的问题。药物装载是通过将外泌体(mEXO)和透明质酸(DSPE-PEG2000-HA)混合,37℃孵育48h,离心25分钟(3500g)得到HA-mEXO。取HA-mEXO,与连翘脂素混合,室温避光孵育3h,用10KDa的超滤管3500g超滤10min除去未结合的药物,得到PHI-HA-mEXO。与游离外泌体(115.9nm)相比,PHI-HA-mEXO(116nm)的形态大小并未出现显著变化。纳米颗粒和细胞膜的相互作用明显受到纳米复合物的表面电荷影响。并且,带正电荷的纳米颗粒更能诱导细胞氧化应激且扰乱肝细胞的抗氧化系统。而外泌体表现出明显的负电性,保证了外泌体载药系统的安全性。
当我们尝试使用外泌体递送PHI时,至关重要的一点在于外泌体制剂的稳定性。我们的稳定性实验结果显示,将PHI-HA-mEXO在-80℃储存一个月后其粒径和ζ电位值与新鲜制备的样品无明显变化,表明PHI-HA-mEXO体系具有一定的储存稳定性。体外释放是另一项重要指标,可用于预测给药后药物的体内动力学。我们的体外释放实验结果显示,PHI-HA-mEXO大约在2小时内会释放46%的负载药物,8小时内释放出83%的负载药物,表明PHI-HA-mEXO药物溶出较为完全。
CD44作为一种常见的透明质酸受体,常在癌细胞中高表达,多用于癌细胞的靶向治疗点。本发明将透明质酸用于靶向肝纤维化细胞产生了预料不到的效果。细胞摄取实验表明,与qHSC相比,aHSC表现出更高的摄取,这表明大量的CD44有助于PHI-HA-mEXO在肝脏中肝星状细胞所处的Disse腔中富集。此外在生物分布研究中,DIR标记的PHI-HA-mEXO明显聚集在肝脏中,显示出优异的靶向性。
尽管有透明质酸保证PHI-HA-mEXO的靶向性,但也有可能会出现脱靶现象导致PHI作用于正常细胞。PHI-HA-mEXO的无细胞毒性对于在抗肝纤维化治疗期间防止正常肝星状细胞和肝细胞的凋亡很重要。我们的细胞毒性实验显示,在给药后,正常肝星状细胞和肝细胞的细胞存活率分别为73.54%和83.01%,表现出较好的安全性。因此,基于透明质酸的PHI-HA-mEXO可用于临床试验以治疗肝纤维化。
PHI-HA-mEXO的体外抗增殖功效在aHSC上进行了测试,结果显示与游离的PHI和PHI-mEXO相比,PHI-HA-mEXO具有更高的细胞毒性。基于细胞毒性数据,我们使用硫代乙酰胺诱导斑马鱼幼鱼肝纤维化模型进一步探索了PHI-HA-mEXO的功效。PHI-HA-mEXO以三种剂量进行了研究:25μmol/L、50μmol/L、100μmol/L。给与PHI-HA-mEXO的肝纤维化斑马鱼幼鱼,其生化指标ALT、AST和ALP的改变表明肝功能随着肝纤维化的程度而改变。与空白组相比,模型组斑马鱼幼鱼的ALT、AST和ALP显著升高,反应出相当大的肝功能损伤。给与PHI-HA-mEXO治疗后,所有的测试指标均显著的降低,表现出良好的抗纤维化作用。此外,与模型组相比,PHI-HA-mEXO治疗组的肝脏组织病理结果显示胶原纤维形成和局灶性坏死减少,且呈剂量依赖性。总之,PHI-HA-mEXO可抑制肝星状细胞活化,减少col1α1和α-SMA形成,以防止肝纤维化。
鉴于肝星状细胞活化是肝纤维化的典型特征,而自噬作为一种高度保守的细胞内降解途径,其主要是通过自噬相关基因调控溶酶体降解自身受损的细胞器和大分子物质。现目前已经发现,自噬能通过降解脂滴为HSC提供能量促进活化,而抑制自噬能显著抑制HSC活化。我们的研究发现,PHI-HA-mEXO能靶向抑制HSC中的自噬,如自噬相关蛋白LC3、Beclin1、P62、Atg5和Atg7水平在PHI-HA-mEXO处理之后恢复了正常水平。我们的研究结果表明PHI-HA-mEXO可能通过下调自噬部分抑制HSC的活化。
这些研究得出的数据表明,口服给药后,负载PHI的外泌体显著提高了PHI的生物利用度并增强了药物疗效。虽然外泌体负载并没有完全解决PHI溶解度差的问题,但也使得PHI更有希望走向临床应用中。连翘脂素作为疏水性药物,通过常规的孵育、超声等方法得到的载药外泌体效率不高,为了提高外泌体对药物的装载效率,我们预先使用DMSO对药物进行溶解后再与外泌体一起孵育,从而极大提高了外泌体对连翘脂素的负载效率。此外,本实验所制备的基于牛奶外泌体的抗肝纤维化靶向给药体系在细胞和动物实验中均呈现出良好的靶向性,证明HA功能化是一种高效的牛奶外泌体靶向修饰策略。为了进一步验证牛奶外泌体载药体系口服递药的潜力,我们还构建了硫代乙酰胺诱导的斑马鱼肝纤维化模型,以探索该策略构建的牛奶外泌体靶向给药体系在体内的抗肝纤维化活性。
5、结论
通过本研究开发了PHI-HA-mEXO,PHI是连翘中的一种天然保肝剂,可诱导活化的肝星状细胞凋亡并减轻硫代乙酰胺诱导的斑马鱼幼鱼肝纤维化。我们的结果表明,PHI-HA-mEXO的粒径约为116.3nm。该药物系统可显著诱导活化的肝星状细胞凋亡而不影响正常的肝星状细胞和肝细胞。此外,PHI-HA-mEXO的治疗效果与相同剂量的游离PHI相比,显示出更为有效的治疗效果。良好的生物安全性和显著的抗肝纤维化作用表明PHI-HA-mEXO在治疗肝纤维化方面具有很大的应用潜力。
Claims (10)
1.一种抗肝纤维化的药物,其特征在于:它包括外泌体和连翘脂素;所述外泌体是经透明质酸修饰的外泌体。
2.根据权利要求1所述的药物,其特征在于:所述外泌体是经透明质酸修饰的牛奶外泌体。
3.根据权利要求2所述的药物,其特征在于:所述牛奶外泌体与透明质酸混合孵育,得经透明质酸修饰的牛奶外泌体;所述牛奶外泌体与透明质酸的质量比为20~40:1,优选20:1。
4.根据权利要求3所述的药物,其特征在于:所述牛奶外泌体是牛奶经差速超速离心得到的沉淀,用PBS溶液洗涤并重悬的混悬液;所述混悬液中蛋白质含量为4~8mg/ml。
5.根据权利要求4所述的药物,其特征在于:所述差速超速离心的方法为:
牛奶先10,000×g离心30min,取溶液100,000×g离心90min,收集上清135,000×g离心70min,收集沉淀,即得。
6.根据权利要求1所述的药物,其特征在于:所述药物在制备时外泌体和连翘脂素的质量比为4~8:1。
7.根据权利要求1所述的药物,其特征在于:所述药物在制备时外泌体和连翘脂素的质量比为6:1。
8.一种权利要求1~7任一项所述的药物的制备方法,其特征在于:它包括如下步骤:
1)按配比称取原料;取外泌体和透明质酸混合,孵育,离心,取上清得透明质酸修饰的牛奶外泌体;
2)取步骤1)所得透明质酸修饰的牛奶外泌体,与连翘脂素混合,孵育,超滤,即得。
9.根据权利要求8所述的制备方法,其特征在于:步骤1)所述孵育的温度为37℃,时间48h;离心速度3500g,时间25min;步骤2)所述孵育是室温避光孵育,时间3h;所述超滤的超滤管10KDa,速度3500g,时间10min。
10.权利要求1~7任一项所述的药物在制备治疗肝纤维化的药物中的用途。
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