CN113952468B - 一种治疗心肌缺血再灌注损伤的环孢菌素a纳米药物 - Google Patents
一种治疗心肌缺血再灌注损伤的环孢菌素a纳米药物 Download PDFInfo
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Abstract
本发明涉及医药领域,具体公开了一种治疗心肌缺血再灌注损伤的环孢菌素A纳米药物,环孢菌素A甘氨酸酯‑酮缩硫醇‑聚L‑精氨酸通过化学键连接的双亲分子,在水中自组装形成荷正电的纳米胶束,表面静电吸附荷负电的P‑选择素单抗得到的纳米药物。此药物可主动靶向作用于缺血部位活化的内皮细胞,阻断中性粒细胞募集,P‑选择素解吸附后,纳米粒表面荷正电增加入胞效率,在ROS的作用下,酮缩硫醇断裂并消耗ROS,通过电子传递效应,R8‑TK‑CsA酯键与酰胺键断裂,释放出L‑Arginine和环孢菌素A,进一步作用于缺血部位的内皮细胞和心肌细胞,序贯发挥“抗炎抗凋亡”作用,提高MI/RI治疗的效果。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种缺血心肌组织靶向和活性氧响应的自组装纳米药物的合成及应用方案,此药物为环孢菌素A甘氨酸酯-酮缩硫醇-聚精氨酸通过化学键连接的双亲分子,在水中可自组装形成荷正电的纳米胶束,纳米胶束表面静电吸附荷负电的P-选择素单抗。
背景技术
缺血性心脏病是严重威胁人类生命健康的“头号杀手”,在我国其死亡率呈持续增长趋势,急性心肌梗死(Acute Myocardial Infarction,AMI)是缺血性心脏病最主要的致病因素。再灌注是目前AMI的标准治疗策略,也是挽救患者生命的首要手段。然而,临床研究发现,再灌注在恢复血供的同时会随之产生严重的心肌缺血再灌注损伤(MyocardialIschemia-Reperfusion Injury,MI/RI),导致持久性心肌收缩功能低下,心律失常,原有梗死面积增大等,加剧心室重塑及心力衰竭发展。目前临床上针对MI/RI的药物尚属空白,随着经皮冠状动脉介入术(PCI)的广泛应用,MI/RI已成为制约AMI临床疗效的瓶颈问题,探索治疗MI/RI新策略具有重要的临床意义和社会效益。
环孢菌素A(CyclosporinA,CsA)是最经典的线粒体通透性转换孔(mPTP)抑制剂之一,它能与CyP-D结合并形成复合物,阻碍CyP-D与ANT结合,从而抑制mPTP开放,降低caspase-3活性,减少细胞凋亡,起到心肌保护作用,并在多种动物体内得到证实。然而,CsA的治疗窗非常窄,在不同群体体内的代谢差异大,本身作为免疫抑制剂在其他组织分布会产生较大的免疫抑制副作用。因此,为了最大限度发挥CsA的作用,需要设计药物,将其靶向递送至缺血心肌细胞,使其能够在作用部位释放,发挥疗效。
一氧化氮(NO)是重要的血管扩张剂,通过一氧化氮合酶(NOS)、结合四氢生物蝶呤(BH4)和L-精氨酸产生。研究表明,生理和治疗水平的NO供体在心脏缺血再灌注后有明显的细胞保护作用,最主要的途径之一就是存在于内皮细胞的eNOS可通过eNOS/NO途径催化生成NO,减轻缺血再灌注损伤。
MI/RI时,缺血心肌细胞线粒体是ROS的主要来源,纳米药物引入ROS响应基团能够增加药物在缺血心肌细胞线粒体中的释放速度。酮缩硫醇(TK)对线粒体活性氧(以O2-为主)具有特异性响应,能够在1mM O2-水溶液中快速降解。
P-选择素是心肌梗死后活化血管内皮细胞表面产生的特异性黏附因子,中性粒细胞必须首先通过其表面的配体与P-选择素结合,才能浸润进入缺血心肌组织。研究发现P-选择素的单克隆抗体(mAb)能够有效减少再灌注后中性粒细胞在缺血心肌组织的浸润,降低血清中肌钙蛋白I和CK-MB的含量。
因此,根据心肌缺血再灌注发生后,心肌组织mPTP开放,ROS增加,内皮细胞功能受损,中性粒细胞募集的特点,我们采用TK将亲水性的聚L-精氨酸(R8)和亲脂性的CsA连接,R8-TK-CsA能在水中自组装形成活性氧响应并荷正电的纳米胶束,通过静电吸附作用与mAb结合,主动靶向作用于缺血部位活化的内皮细胞,阻断中性粒细胞募集,mAb解吸附后,纳米胶束表面荷正电增加入胞效率,在ROS的作用下,酮缩硫醇断裂并消耗ROS,通过电子传递效应,R8-TK-CsA酯键与酰胺键断裂,释放出聚L-Arginine和环孢菌素A(CsA),进一步作用于缺血部位的内皮细胞和心肌细胞,从而发挥MI/RI防治的作用。
发明内容
本发明的目的是提供一种治疗心肌缺血再灌注损伤的环孢菌素A纳米药物,是一种缺血心肌组织靶向和活性氧响应的无载体自组装纳米药物的合成及应用方案。通过纳米药物的设计,可同时发挥CsA和L-精氨酸的心肌保护作用和P-选择素的抗炎作用,抑制心肌细胞凋亡和中性粒细胞募集,并阻断二者之间的恶性循环,为心肌缺血再灌注损伤的治疗提供新的思路。
本发明的技术方案是:一种治疗心肌缺血再灌注损伤的环孢菌素A(CsA)纳米药物,其特征是将水溶性R8(八聚L-精氨酸)与脂溶性CsA通过酮缩硫醇(Thioketal,TK)连接,在水中自组装形成荷正电的纳米胶束,再利用静电作用吸附P-选择素单抗,得到自递送纳米药物;其中
(1)R8-TK-CsA的制备:
称取环孢菌素A(CsA)(3651.7mg,3.04mmol)溶于无水吡啶(15.0mL),通入氮气保护,冰水浴搅拌10min,加入氯乙酸酐(1560.2mg,9.13mmol),继续冰水浴搅拌10min,除去冰浴,室温下搅拌24h。加入50mL乙醚,并用20mL水洗涤3次,无水硫酸钠干燥后,经硅胶柱色谱纯化,洗脱剂为二氯甲烷:乙酸乙酯(V/V=1:1),收集目标组分,减压旋蒸至干燥,乙醚重结晶,得到环孢菌素A氯乙酸酯(1573.6mg,1.23mmol),产率40.5%;
将所得环孢菌素A氯乙酸酯(1573.6mg,1.23mmol)溶于二甲基亚砜10.0mL DMSO中,加入浓度为0.5mol/L的叠氮钠的DMSO溶液9.8mL,充入氮气保护,室温下避光搅拌6h,反应结束后,用40mL乙醚萃取产物,10mL水洗涤3次,无水硫酸钠干燥1h后,减压旋蒸至干燥,得到环孢菌素A叠氮乙酸酯(1073.1mg,0.83mmol),将产物继续溶解于10mL甲醇,缓慢加入0.5mL去离子水,配制浓度为1.0mol/L的无水氯化亚锡的甲醇溶液,取4.2mL加入反应烧瓶,室温下搅拌6h,而后缓慢加入氨水(5%)至白色沉淀不再增加,40mL乙醚萃取后,用10mL饱和盐水洗涤3次,无水硫酸钠干燥1h,减压旋蒸至干燥,得到环孢菌素A甘氨酸酯(800.1mg,0.64mmol),产率52.0%;
环孢菌素A甘氨酸酯(800.1mg,0.64mmol)溶于5.0mL二氯甲烷,依次加入240.0mg5,5-二甲基-4,6-二硫代-壬二酸(TK)、23.5mg 4-二甲氨基吡啶、198.1mg二环己基碳二亚胺,室温下搅拌反应4h,硅胶柱色谱纯化,洗脱剂为二氯甲烷:甲醇(V/V=30:1),收集目标组分,减压旋蒸至干燥,得到CsA-TK(650.2mg,0.43mmol),产率68.1%;
称取CsA-TK 137mg,溶于5.0mL DMSO,加入36.0mg NHS(N-羟基琥珀酰亚胺)和24.0mg EDCI(1-(3-二甲氨基丙基)-3-乙基碳二亚胺),氮气保护下室温避光活化12h,然后加入80.0mg R8(八聚L-精氨酸),继续反应24h,然后将反应液转移至透析袋(MWCO=100D)中,对去离子水透析24h,0.8μm孔径滤器过滤,3kD超滤离心浓缩,冻干后得19.8mg R8-TK-CsA;
(2)mAb@R8-TK-CsA的制备:
称取5.0mg R8-TK-CsA,在超声水浴中逐渐分散于5mL去离子水中,由于R8-TK-CsA分子具有双亲特性,在临界胶束浓度以上时,自组装形成纳米胶束,取P-selectin单克隆抗体(mAb)13μL分散于50μL去离子水中,然后在超声水浴条件下缓慢分散于1mL R8-TK-CsA溶液中,涡旋振荡30s,4℃静置1h,即得mAb@R8-TK-CsA纳米药物溶液。
本发明构建了一种新型缺血心肌组织靶向和“抗炎抗凋亡”的自组装纳米胶束药物,该药物是主动靶向且活性氧响应的无载体自组装纳米药物,此药物由环孢菌素A甘氨酸酯-酮缩硫醇-聚精氨酸通过化学键连接的双亲分子,在水中自组装形成荷正电的纳米胶束,并在表面静电吸附荷负电P-选择素单抗的纳米药物。该纳米药物可通过微环境靶向和活性氧双响应,协同发挥“抗炎抗凋亡”作用,从而提高心急缺血再灌注损伤的防治效果。
附图说明
图1是R8-TK-CsA的质谱图。
图2是R8-TK-CsA及mAb@R8-TK-CsA的粒径及电位分布;A:R8-TK-CsA粒径分布图;B:R8-TK-CsA电位图;C:P-选择素与R8-TK-CsA不同质量比吸附后的粒径变化;D:P-选择素与R8-TK-CsA不同质量比吸附后的电位变化;n=3,
具体实施方式
mAb@R8-TK-CsA的表征及其对心肌缺血再灌注损伤的防治作用
1目的:评价环孢菌素A纳米药物对MIRI的治疗作用。
2研究方法:
2.1 mAb@R8-TK-CsA的表征
称取适量R8-TK-CsA,溶于色谱级甲醇,得到100ng/mL溶液,使用MALDI-TOF对其分子量进行鉴定。
称取5.0mg R8-TK-CsA,在超声水浴中分散于5mL去离子水中,充分震荡后,测定其粒径与电位。然后将上述溶液稀释至150μg/mL,各取1mL分为4组,在超声水浴振荡下,分别加入P-选择素0、1、2、3μg,充分混合均匀后,4℃静置1h,分别测定粒径与电位。
2.2细胞保护作用评价
取对数生长期的大鼠心肌H9C2细胞接种于96孔板中,密度为1.2×105,培养36h。正常对照组将培养基更换为无血清DMEM,继续培养;模型组换为缺血液,在95%N2,5%CO2的条件下37℃孵育3h,然后取出后吸除缺血液,分别加入不含血清的培养液、环孢菌素A及不同浓度的R8-TK-CsA;4h后各组同时加入20μL噻唑兰溶液继续孵育4h,弃去培养液,加200μLDMSO溶解紫色结晶,酶标仪于492nm下读取OD值,绘制细胞存活率曲线图,按公式计算细胞存活率。
细胞存活率(%)=(试验组OD值/正常对照组OD值)×100%
2.3药物对小鼠心肌缺血再灌注损伤的防治作用评价
(1)手术操作
小鼠仰卧位固定,左胸前手术区域剔除鼠毛并消毒。距胸骨左缘约1~2mm皮肤处做长约1cm纵向切口,切口处行垂直外翻褥式缝合预留缝线。逐层钝性分离胸壁肌肉,从第3或第4肋间隙快速进入胸腔,用止血钳撑开肋间隙,配合心脏跳动左手轻轻挤压使心脏从孔隙中弹出。在左心耳下缘1~2mm、肺动脉圆锥旁0.5mm处以6-0带线缝合针穿过冠状动脉前降支将其结扎,松紧适宜,控制进针深度(以隐约可见细针为宜)和行针宽度(2mm左右)。由于不借助通气装置,要求开胸时间不要超过30s,结扎操作尽量控制在10s左右完成。结扎完成后轻柔地将心脏送回胸腔,挤压胸腔排出空气同时收紧结扎切口处预留缝线,完成手术,sham组只穿线不结扎。结扎左冠状动脉前降支使心肌缺血30min后,松开结扎线,使缺血心肌再灌注。
(2)实验分组
36只小鼠随机分成6组,每组6只。
sham组:只将缝合针穿过冠状动脉前降支将而不结扎,其余过程相同;
model组:结扎左冠状动脉前降支30min后,松开结扎线,使缺血心肌再灌注;
CsA组:再灌注前5min,按照1.25mg/Kg尾静脉注射CsA溶液,术后第3天和第5天分别给予相同剂量药物;
R8-TK-CsA组:再灌注前5min,按照2.85mg/Kg尾静脉注射R8-TK-CsA溶液,术后第3天和第5天分别给予相同剂量药物;
IgG@R8-TK-CsA组:再灌注前5min,按照2.85mg/Kg尾静脉注射IgG@R8-TK-CsA溶液,术后第3天和第5天分别给予相同剂量药物;
mAb@R8-TK-CsA组:再灌注前5min,按照2.85mg/Kg尾静脉注射mAb@R8-TK-CsA溶液,术后第3天和第5天分别给予相同剂量药物;
将各组小鼠于28天行心脏超声检查,采集超声心动图,统计左心室射血分数和缩短分数评价心功能。
3实验结果:
3.1 mAb@R8-TK-CsA的表征
质谱测得结果显示化合物分子量为目标分子量,证实所合成化合物为R8-TK-CsA
3.2细胞保护作用评价
将大鼠心肌R8-TK-CsA细胞进行3h缺氧4h复氧损伤后,游离环孢菌素A和R8-TK-CsA浓度为12.5μM、25μM时均具有较强的细胞保护作用,其中游离药物保护效果更好,这是由于R8-TK-CsA在发挥作用时需要释放游离药物,这一过程可能使其发挥药效比CsA更慢。总之,R8-TK-CsA显示了对H/R H9c2细胞的保护作用,提示可通过这一方式提高CsA溶解度,从而更好发挥保护作用。
3.3药物对小鼠心肌缺血再灌注损伤的防治作用评价
相比于同剂量游离CsA,R8-TK-CsA能够显著地提高小鼠心脏缺血再灌注28天后的左室射血分数(LVEF)和缩短分数(FS);相比于IgG@R8-TK-CsA,mAb@R8-TK-CsA更好地显示了心脏保护作用,说明mAb@R8-TK-CsA能够提高环孢菌素A在缺血心肌组织和细胞的分布,从而提高治疗效果。
4结论:
体内外的实验结果表明,与游离环孢菌素A相比,mAb@R8-TK-CsA可显著提高心肌缺血再灌注损伤的保护作用,具有一定的开发前景。
Claims (1)
1.一种治疗心肌缺血再灌注损伤的环孢菌素A CsA纳米药物的制备方法,其特征是将水溶性R8八聚L-精氨酸与脂溶性CsA通过酮缩硫醇TK连接,在水中自组装形成荷正电的纳米胶束,再利用静电作用吸附P-选择素单抗,得到自递送纳米药物;其中
(1)R8-TK-CsA的制备:
称取3651.7 mg,3.04 mmol的环孢菌素A溶于15.0 mL无水吡啶,通入氮气保护,冰水浴搅拌10 min,加入1560.2 mg,9.13 mmol氯乙酸酐,继续冰水浴搅拌10 min,除去冰浴,室温下搅拌24 h,加入50 mL乙醚,并用20 mL水洗涤三次,无水硫酸钠干燥后,经硅胶柱色谱纯化,洗脱剂为二氯甲烷:乙酸乙酯以体积比计算等于1:1,收集目标组分,减压旋蒸至干燥,乙醚重结晶,得到1573.6 mg,1.23 mmol环孢菌素A氯乙酸酯,产率40.5%;
将所得1573.6 mg,1.23 mmol环孢菌素A氯乙酸酯溶于二甲基亚砜10.0 mL DMSO中,加入浓度为0.5 mol/L的叠氮钠的DMSO溶液9.8 mL,充入氮气保护,室温下避光搅拌6 h,反应结束后,用40 mL乙醚萃取产物,10 mL水洗涤三次,无水硫酸钠干燥1 h后,减压旋蒸至干燥,得到1073.1 mg,0.83 mmol环孢菌素A叠氮乙酸酯,将产物继续溶解于10 mL甲醇,缓慢加入0.5 mL去离子水,配制浓度为1.0 mol/L的无水氯化亚锡的甲醇溶液,取4.2 mL加入反应烧瓶,室温下搅拌6 h,而后缓慢加入5%氨水至白色沉淀不再增加,40 mL乙醚萃取后,用10 mL饱和盐水洗涤三次,无水硫酸钠干燥1 h,减压旋蒸至干燥,得到800.1 mg,0.64 mmol环孢菌素A甘氨酸酯,产率52.0%;
将800.1 mg,0.64 mmol环孢菌素A甘氨酸酯溶于5.0 mL二氯甲烷,依次加入240.0 mg5,5-二甲基-4,6-二硫代-壬二酸、23.5 mg 4-二甲氨基吡啶、198.1 mg二环己基碳二亚胺,室温下搅拌反应4 h,硅胶柱色谱纯化,洗脱剂为二氯甲烷:甲醇以体积比计算等于30:1,收集目标组分,减压旋蒸至干燥,得到650.2 mg,0.43 mmol CsA-TK,产率68.1%;
称取CsA-TK 137 mg,溶于5.0 mL DMSO,加入36.0 mg NHS N-羟基琥珀酰亚胺和24.0mg EDC 1-(3-二甲氨基丙基)-3-乙基碳二亚胺,氮气保护下室温避光活化12 h,然后加入80.0 mg R8,继续反应24h,然后将反应液转移至MWCO=100 D透析袋中,对去离子水透析24h,0.8 μm孔径滤器过滤,3 kD超滤离心浓缩,冻干后得19.8 mg R8-TK-CsA;
(2)mAb@R8-TK-CsA的制备:
称取5.0 mg R8-TK-CsA,在超声水浴中逐渐分散于5 mL去离子水中,由于R8-TK-CsA分子具有双亲特性,在临界胶束浓度以上时,自组装形成纳米胶束,取P-selectin单克隆抗体mAb 13 μL分散于50 μL去离子水中,然后在超声水浴条件下缓慢分散于1 mL R8-TK-CsA溶液中,涡旋振荡30 s,4℃静置1 h,即得mAb@R8-TK-CsA纳米药物溶液。
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