CN112546245A - 一种用于缺血性心脏病基因靶向治疗的载基因显影微泡及其制备方法 - Google Patents
一种用于缺血性心脏病基因靶向治疗的载基因显影微泡及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于缺血性心脏病基因靶向治疗的载基因显影微泡及其制备方法。所述的载基因显影微泡包括阳离子显影微泡以及分散于显影微泡外壳表面的治疗基因质粒,其中,所述的显影微泡具有正电性的脂质双分子层外壳以及包裹在外壳内部的生物惰性气体,所述的治疗基因质粒包括含有基质金属蛋白酶抑制因子‑3(TIMP‑3)和沉默信息调节因子3(SIRT3)基因的质粒。该基因微泡制剂在超声作用下集中空化并释放目的基因,快速对靶器官进行基因转染,转染效率高、靶向性强、安全无创、可反复操作。另外,该基因显影微泡可以同时在超声下显影,对缺血心肌的灌注情况进行检测和诊断,在治疗的同时能够准确的评估,使治疗更精确。
Description
技术领域
本发明涉及一种用于缺血性心脏病基因靶向治疗的载基因显影微泡及其制备方法。本发明属于生物医药技术领域。
背景技术
世界范围内每年大概有近2000万人死于心血管疾病,而这个数字至2030年将会迅速增长至2630万人[1-2]。随着生活水平的提高以及生活方式的改变,近40年来,我国缺血性心脏病的发病率及死亡率亦呈逐年上升态势,在所有心脏疾病中居首位。缺血性心脏病可引发心脏功能细胞的不可逆性坏死、炎症细胞浸润、心肌间质重构等一系列的内在变化,最终导致难以控制的心力衰竭甚至死亡[3-4]。目前,国际上针对缺血性心脏疾病的标准临床治疗手段包括药物溶栓治疗、介入支架及冠脉搭桥手术等,这些治疗方式均以重建缺血区域的血运为主要目的称为急诊冠脉再血管化(CR),虽然能够通过恢复残存心肌供血、改善心脏功能达到治疗的效果,然而并不能彻底阻止或者逆转缺血对心脏组织造成的病理损害,所以仍有相当一部分患者在治疗的近期,尤其是远期出现心力衰竭影响患者的生存质量,甚至造成死亡。近20年来缺血性心脏病患者的死亡率始终没有明显下降[5-8]。因此,如何在恢复血供的同时,针对缺血损伤的病理进程及病理损害进行精准的调控,以有效降低急诊CR治疗后心衰的发生率,改善患者的生存质量并降低死亡率,是目前心血管病医生迫切希望解决的一个难题。
缺血性心脏病会造成心脏功能细胞的调亡、炎症反应、心肌间质重构等一系列病理生理改变,造成心肌纤维化、瘢痕形成等病理损伤,影响心脏的舒缩功能,最终造成心力衰竭甚至死亡[3-4]。而对缺血心肌给予急诊CR治疗后,上述的病理改变并不会完全逆转。有文献显示,缺血心肌在急诊CR后会在一定程度上继续存在心脏细胞凋亡、炎性细胞浸润、炎症因子释放、基质金属蛋白酶激活和胶原组织增生等现象[9-12],其中心脏功能细胞的继续丧失会直接导致心脏功能单位的减少,同时会进一步诱导炎性因子释放,加重周围心肌细胞的进一步损伤及间质的纤维化;另一方面,炎症反应、基质金属蛋白酶激活和胶原组织增生等会造成不良心室间质重构,最终可导致纤维瘢痕形成和心功能下降。而在目前的临床治疗过程中,在急诊CR后并没有针对上述病理改变进行精准、有效的后续治疗手段,从而导致急诊CR治疗后近期、尤其是远期心衰发生率高、治疗效果差。
应用基因治疗以达到对缺血性心脏病的精准治疗一直是心血管研究领域力求实现突破性进展的努力方向,其在实验室阶段的治疗效果已经在大量的体内、体外实验中得到强有力的印证。本发明人既往通过多项研究证实以骨髓间充质干细胞为载体,应用基质金属蛋白酶抑制因子-3(TIMP-3)、血管内皮生长因子(VEGF)和沉默信息调节因子3(SIRT3)等基因对缺血性心脏疾病进行基因治疗能够在一定程度上逆转缺血组织的相应病理改变,改善心脏功能,最终达到对缺血性心脏病的有效治疗[13-17]。相较于体外细胞基因转染的可控性,体内基因转染因其在转染效率以及在安全性、有效性、靶向性等多方面存在较多制约因素而无法有效开展,限制了其临床的应用。
目前,常规的体内基因转染方式主要包括将病毒、质粒或者携带基因的细胞直接注射到靶器官或直接输注到血液循环系统中[18-20]。这些转染方式主要存在以下缺陷:①转染途径受限:将携带目的基因的载体直接注射于靶器官内,虽可实现目的基因向靶器官的直接定向传输,但往往会造成组织侵入性的创伤,存在一定的风险性,且可重复操作性差;而将携带目的基因的载体输注于血液循环系统中,虽然是一种微创的转染手段,但是由于载体随血液系统流经全身,所以不能准确定位,且转染效率低下、转染效果不稳定;②基因载体受限:质粒载体相对安全,但是会在短时间内被血浆中的DNA酶快速清除而造成转染效率低下,病毒类载体转染率相对较高但因存在潜在的毒性、致癌、致畸作用等而在安全性方面存在较大问题,而利用转染了目的基因的细胞移植尤其是干细胞移植虽然能够参与受损组织的修复与再生,但目前干细胞移植仍存在种子细胞类型的选择、细胞不稳定分化、移植细胞存活率低和临床应用安全性等问题尚未解决。而对于缺血性心脏病CR后的进一步治疗,需要针对缺血损伤的不同病理阶段的不同病理损伤有针对性的应用不同的基因进行重复的精准治疗方能达到最佳的治疗效果。这就更需要一种损伤小、操作简单以便于实现反复操作,能够高效精准的将目的基因转染靶器官,可以针对不同的病理阶段存在多种剂型的体内基因转染方式以实现对缺血性心脏病最有效的精准治疗。
近年来,随着超声波靶向微泡击碎技术(UTMD)的高速发展以及其介导基因转染时无需长时间准备即能够达到无创、靶向、高效转染效果的特性[21-25]为实现上述治疗需求带来了可能。本发明人前期研究已经证实了应用UTMD技术可将携带特定基因的脂质微泡击碎从而实现动物体内基因的靶向转染,即实现对大鼠及小鼠缺血性心脏疾病的无创、靶向基因治疗,从而显著改善心脏功能[18-20,26]。因此,在本申请中我们将SIRT3的抗氧化应激损伤的特性、TIMP-3的抗间质重构能力和UTMD的体内基因转染优势结合起来,针对缺血性心脏损伤的不同病理阶段及不同病理损伤,分别制造两种剂型的基因显影微泡,应用UTMD无创靶向技术分别在缺血后的不同时间点,应用特定的频次,在急诊CR后的心肌组织中改变损伤局部SIRT3及TIMP-3的表达含量,提高心肌组织自身的抗氧化应激损伤和抗间质重构的能力,进而改善急诊CR后的近期及远期的治疗效果,为进一步改善临床对缺血性心脏病的治疗效果,并提高患者的生存质量及生存率探索出一种易于临床转化、无创、高效、精准的治疗方法。
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发明内容
本发明是以当今社会主要健康问题——缺血性心脏病为基点,克服了目前临床上药物溶栓、经皮冠状动脉导管介入(PCI)、冠脉搭桥等急诊CR治疗手段无法完全阻断缺血的病理损伤,造成近期、尤其是远期心力衰竭的发生,降低患者生存质量及生存率的缺点,提供了一种针对缺血性心脏损伤的病理进程及病理损伤,应用无创、靶向基因转染技术,实现心脏组织的精准、个体化基因调控的多剂型制剂。从而有效完成急诊CR后的进一步治疗,已达到对缺血性心脏病的最佳治疗效果。
为了达到上述目的,本发明采用了以下技术手段:
本发明公开了一种用于缺血性心脏病靶向治疗的载基因显影微泡,所述的载基因显影微泡包括阳离子显影微泡以及分散于显影微泡外壳表面的治疗基因质粒,其中,所述的显影微泡具有正电性的脂质双分子层外壳以及包裹在外壳内部的生物惰性气体,所述的治疗基因质粒包括含有基质金属蛋白酶抑制因子-3(TIMP-3)和沉默信息调节因子3(SIRT3)基因的质粒。
其中,优选的,所述的阳离子显影微泡通过以下方法制备得到:
将二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、DC胆固醇(DC-Chol)按照质量比4-6:1-3:0.1-1的比例进行混合,再加入三氯甲烷使其充分溶解;旋转蒸发仪减压蒸发,去除有机溶剂形成脂膜;在脂膜中加入甘油与PBS混合液,42℃水浴30min-1h,取脂膜和甘油、PBS混悬液加入管形瓶中;冷却后用C3F8置换管形瓶内空气2次,机械震荡,即得高浓度阳离子显影微泡,经60Co伽马射线辐射或紫外线消毒灭菌,正常放置4℃冰箱保存,-20℃冰箱可稳定保存1月。
其中,优选的,所述的生物惰性气体为C3F8。
其中,优选的,二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、DC胆固醇(DC-Chol)按照质量比5:2:0.5的比例进行混合。
其中,优选的,旋转蒸发仪50℃减压蒸发。
其中,优选的,甘油与PBS混合液中甘油与PBS的体积比为1:9。
其中,优选的,所述的载基因显影微泡通过以下方法制备得到:
分别制备含有人SIRT3或TIMP-3基因的质粒,以真核表达载体pCDNA3.1做为骨架载体;先将制备好的阳离子显影微泡高频震荡60S,按0.06ml/kg体重计算并获取相应体积的阳离子显影微泡,再按28.5μg/kg体重计算并获取相应体积的含有人SIRT3或TIMP-3基因质粒,如超过微泡最大结合率,则按微泡最大结合率计算即100ul阳离子微泡与40μg质粒结合,与准备好的微泡溶液混合,加0.9%生理盐水稀释至50ml,室温下孵育20min,制得含有人SIRT3或TIMP-3基因的载基因显影微泡制剂。
进一步的,本发明还提出了所述的载基因显影微泡在制备缺血性心脏病靶向治疗的药物制剂中的应用。
其中,优选的,所述的治疗所采用的手段为超声波靶向微泡击碎技术。
再进一步,本发明还提出了所述的药物制剂的应用方法及应用时间。
在常规急诊冠状动脉再血管化治疗(CR)后24小时,建立静脉通路,应用微量输液泵以150ml/h的速度将含有SIRT3基因质粒的微泡制剂持续泵入,同时应用超声诊断仪在二次谐波模式(传送:1.6MHz;接收:3.2MHz)对患者心脏进行超声波冲击,频率由心电记录仪自动触发装置自动控制,每两次心脏收缩触发一次,共持续20分钟,并分别于此后的24小时和48小时各重复一次相同治疗过程。
在常规急诊冠状动脉再血管化治疗(CR)后的第5天,应用微量输液泵以150ml/h的速度将TIMP-3基因微泡制剂持续泵入,同时应用超声诊断仪在二次谐波模式(传送:1.6MHz;接收:3.2MHz)对患者心脏进行超声波冲击,频率由心电记录仪自动触发装置自动控制,每两次心脏收缩触发一次,共持续20分钟,并分别于CR治疗后的第7天和第9天各重复一次相同的治疗过程。
上述过程中SIRT3基因微泡制剂和TIMP-3基因微泡制剂治疗的次数可根据患者的具体病情做适当的增减,但治疗的时间点应尽量遵守,以达到最佳的治疗效果。
为了验证本制剂的应用效果,我们建立了猪的心肌缺血再灌注损伤模型。具体方法如下:首先使用丙泊酚注射液臀部注射法麻醉实验香猪(雄性,2年龄),以8F插管进行气管插管,呼吸机以氧气和空气混合气体及七氟烷进行正压通气及麻醉维持,呼吸频率20次/min,并根据体重调整潮气量(10-12ml/kg)。猪采取右侧卧位,消毒,铺巾,左侧第Ⅳ肋间开胸,切开心包暴露心脏,使用自制无菌套管以4-0prolene缝线阻断冠脉前降支中下1/3处,40min后开放进行血管再通,关胸。送回实验动物中心专用饲养室饲养。模型建立后依照上述方法在不同时间点应用不同的制剂剂型对猪的心肌缺血再灌注损伤进行治疗,并应用超声检测治疗后2周和4周实验动物的心脏功能,结果显示,治疗后实验动物的心脏功能得到了显著的提高。
相较于现有技术,本发明的有益效果是:
缺血性心脏病发病率与死亡率激增已经成为目前国际医疗领域内的重大难题。目前临床上标准的治疗方式包括药物溶栓、介入治疗以及冠脉搭桥手术等急诊冠脉再血管化(CR)治疗。这些治疗方式,虽然能够实现犯罪血管供应区域的再灌注,但并不能完全阻止或逆转缺血对心肌组织损伤的病理改变。所以,仍有大部分患者近期、尤其是远期治疗效果不理想出现心力衰竭甚至死亡,这已经成为目前心血管病治疗的瓶颈。基因治疗可针对CR治疗后仍存在的缺血病理损伤进行精准治疗,但是目前应用于体内基因转染的方式包括将病毒、质粒或者携带基因的细胞直接注射到靶器官或输入血液循环系统中,均由于转染效率低、靶向性差、安全性不确定、无法反复操作等原因限制了其临床应用。本发明将临床已有的超声显影微球和体内基因转染相结合即UTMD技术,与本人既往针对缺血性心脏病病理损伤的研究以及应用SIRT3、TIMP-3等基因对缺血性心脏病进行基因治疗的经验相结合,发明出了此种多剂型载基因量更高的基因显影微泡制剂,操作简单、容易应用、效果确切、治疗精准,对患者不产生生理上的负担。同时能够在治疗的同时,对缺血区域的供血进行深入的评估,做到真正的有的放矢。本发明的载基因显影微泡在对猪的心肌缺血再灌注损伤后的治疗中,已经发现显著改善了实验动物的心脏功能(如图5)。本发明的产生能够进一步改善急诊CR的后续治疗效果,提高患者的生存率、改善患者的生存质量,成为一种安全、有效、精准的临床治疗方式。
附图说明
图1为基因显影微泡制剂的结构和外观;
图2为基因显影微泡制剂的显微镜下形态;
图3为基因显影微泡注射后在心脏超声下逐渐显影的情况;
图4为急诊CR后利用不同基因显影微泡制剂对缺血性心脏病进行治疗的疗程图;
图5为急诊CR经过不同基因显影微泡制剂治疗后实验动物的心脏功能得到了显著改善。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将随着描述而清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。
实施例1载基因显影微泡的制备
1、阳离子显影微泡的制备
将二棕榈酰磷脂酰胆碱(DPPC)5mg、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)2mg、DC胆固醇(DC-Chol)0.5mg进行混合,再加入5ml三氯甲烷使其充分溶解;旋转蒸发仪50℃减压蒸发1h,去除有机溶剂形成脂膜;在脂膜中加入甘油与0.1mol/L,pH 7.0的PBS混合液0.5ml(甘油:PBS=1:9,体积比),42℃水浴30min,取脂膜和甘油、PBS混悬液加入1.5ml管形瓶中;冷却后用C3F8置换管形瓶内空气2次,机械震荡50s,即得高浓度阳离子微泡。制备好的阳离子微泡外观呈乳白色混悬液(如图1),经60Co伽马射线辐射或紫外线消毒灭菌。应用马尔文粒径电位检测仪检测自制阳离子微泡的电位和粒径。应用库尔特计数仪检测微泡浓度,按标准调成统一浓度制剂。制备好的阳离子显影微泡放置4℃冰箱保存,-20℃冰箱可稳定保存1月。
阳离子显影微泡的理化性质:
所述显影微泡为阳离子微泡,电位为24.97±2.07mv,最高质粒结合率为37.9±1.14%。显微镜下观察微泡呈圆形,大小均一一致,粒径在1.55±0.2um(如图2)。制备的微泡浓度在7.18±0.33×109/ml左右,超声下显影良好(如图3)。
2、载基因显影微泡的制备
分别制备含有人SIRT3(NM_012239.6)或TIMP-3(NM_000362)基因质粒,以真核表达载体pCDNA3.1做为骨架载体。将两种质粒按照指定浓度分别制成制剂并分装。使用前先将制备好的阳离子显影微泡高频震荡60S,按0.06ml/kg体重计算并获取相应体积的阳离子微泡,再按28.5μg/kg体重计算并获取相应体积的含有人SIRT3(NM_012239.6)或TIMP-3(NM_000362)基因质粒(如超过微泡最大结合率,则按微泡最大结合率计算即100ul阳离子微泡与40μg质粒结合)与准备好的微泡溶液混合,加0.9%生理盐水稀释至50ml,室温下孵育20min,制得含有人SIRT3(NM_012239.6)或TIMP-3(NM_000362)基因质粒的显影微泡制剂。
实施例2载基因显影微泡在缺血性心脏病靶向治疗中的应用
为了验证本制剂的应用效果,我们建立了猪的心肌缺血再灌注损伤模型。具体方法如下:首先使用丙泊酚注射液臀部注射法麻醉实验香猪(雄性,2年龄),以8F插管进行气管插管,呼吸机以氧气和空气混合气体及七氟烷进行正压通气及麻醉维持,呼吸频率20次/min,并根据体重调整潮气量(10-12ml/kg)。猪采取右侧卧位,消毒,铺巾,左侧第Ⅳ肋间开胸,切开心包暴露心脏,使用自制无菌套管以4-0prolene缝线阻断冠脉前降支中下1/3处,40min后进行常规急诊冠状动脉再血管化治疗(CR),关胸。送回实验动物中心专用饲养室饲养。
在常规急诊冠状动脉再血管化治疗(CR)后24小时,建立静脉通路,应用微量输液泵以150ml/h的速度将含有SIRT3基因质粒的微泡制剂持续泵入,同时应用超声诊断仪在二次谐波模式(传送:1.6MHz;接收:3.2MHz)对模型心脏进行超声波冲击,频率由心电记录仪自动触发装置自动控制,每两次心脏收缩触发一次,共持续20分钟,并分别于此后的24小时和48小时各重复一次相同治疗过程。
在常规急诊冠状动脉再血管化治疗(CR)后的第5天,应用微量输液泵以150ml/h的速度将含有TIMP-3基因质粒的微泡制剂持续泵入,同时应用超声诊断仪在二次谐波模式(传送:1.6MHz;接收:3.2MHz)对模型心脏进行超声波冲击,频率由心电记录仪自动触发装置自动控制,每两次心脏收缩触发一次,共持续20分钟,并分别CR治疗后的第7天和第9天各重复一次相同的治疗过程。急诊CR后利用不同基因显影微泡制剂对缺血性心脏病进行治疗的疗程图如图4所示。
上述过程中SIRT3基因微泡制剂和TIMP-3基因微泡制剂治疗的次数可根据具体病情做适当的增减,但治疗的时间点应尽量遵守,以达到最佳的治疗效果。
应用超声检测治疗后2周和4周实验动物的心脏功能,结果显示,治疗后实验动物的心脏功能得到了显著的提高(如图5)。
Claims (9)
1.一种用于缺血性心脏病靶向治疗的载基因显影微泡,其特征在于,所述的载基因显影微泡包括阳离子显影微泡以及分散于显影微泡外壳表面的治疗基因质粒,其中,所述的显影微泡具有正电性的脂质双分子层外壳以及包裹在外壳内部的生物惰性气体,所述的治疗基因质粒包括含有基质金属蛋白酶抑制因子-3(TIMP-3)和沉默信息调节因子3(SIRT3)基因的质粒。
2.如权利要求1所述的载基因显影微泡,其特征在于,所述的生物惰性气体为C3F8。
3.如权利要求1或2所述的载基因显影微泡,其特征在于,所述的阳离子显影微泡通过以下方法制备得到:
将二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、DC胆固醇(DC-Chol)按照质量比4-6:1-3:0.1-1的比例进行混合,再加入三氯甲烷使其充分溶解;旋转蒸发仪减压蒸发,去除有机溶剂形成脂膜;在脂膜中加入甘油与PBS混合液,42℃水浴30min-1h,取脂膜和甘油、PBS混悬液加入管形瓶中;冷却后用C3F8置换管形瓶内空气2次,机械震荡,即得高浓度阳离子显影微泡,经60Co伽马射线辐射或紫外线消毒灭菌,正常放置4℃冰箱保存,-20℃冰箱可稳定保存1月。
4.如权利要求3所述的载基因显影微泡,其特征在于,二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、DC胆固醇(DC-Chol)按照质量比5:2:0.5的比例进行混合。
5.如权利要求3所述的载基因显影微泡,其特征在于,旋转蒸发仪50℃减压蒸发。
6.如权利要求3所述的载基因显影微泡,其特征在于,甘油与PBS混合液中甘油与PBS的体积比为1:9。
7.如权利要求1所述的载基因显影微泡,其特征在于,所述的载基因显影微泡通过以下方法制备得到:
分别制备含有人SIRT3或TIMP-3基因的质粒,以真核表达载体pCDNA3.1做为骨架载体;先将制备好的阳离子显影微泡高频震荡60S,按0.06ml/kg体重计算并获取相应体积的阳离子显影微泡,再按28.5μg/kg体重计算并获取相应体积的含有人SIRT3或TIMP-3基因质粒,如超过微泡最大结合率,则按微泡最大结合率计算即100ul阳离子微泡与40μg质粒结合,与准备好的微泡溶液混合,加0.9%生理盐水稀释至50ml,室温下孵育20min,制得含有人SIRT3或TIMP-3基因的载基因显影微泡制剂。
8.权利要求1-7任一项所述的载基因显影微泡在制备缺血性心脏病靶向治疗的药物制剂中的应用。
9.如权利要求8所述的应用,其特征在于,所述的治疗所采用的手段为超声波靶向微泡击碎技术。
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