CN115590986A - 携载丹参酮ⅱa的脂质纳米微泡超声造影剂制备方法与应用 - Google Patents
携载丹参酮ⅱa的脂质纳米微泡超声造影剂制备方法与应用 Download PDFInfo
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Abstract
本发明公开了携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法与应用,涉及超声分子影像学技术领域,所述携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法包括以下步骤:步骤一:制备脂质溶液;步骤二:对制备得到的脂质溶液进行除杂处理获得纯净的脂质体溶液;步骤三:向脂质体溶液中加入丹参酮ⅡA脂类纳米粒及冻干保护剂进行均匀混合,然后再分装至盛装容器内进行真空冷冻干燥处理;步骤四:向盛装容器内注入全氟丙烷气体。本发明通过采用脂类物质对丹参酮ⅡA的脂质纳米粒的包封与本身为脂类物质的靶向超声造影剂的微泡壳之间的良好的互溶性和生物安全性,能够有效地提高丹参酮ⅡA的生物利用度,降低它的不良反应,使其达到更好的药效。
Description
技术领域
本发明涉及超声分子影像学技术领域,尤其涉及携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法与应用。
背景技术
丹参是唇形科植物丹参的干燥根和根茎,是我国传统中药之一,具有抗缺血缺氧、改善微循环、抑制血小板黏附聚集等作用;临床上主要用于冠心病、急性脑梗死等心脑血管疾病的治疗。丹参中的有效成分主要包括两大类:水溶性的芬酸类及脂溶性的二萜醌类;其中丹参酮是从丹参中提取的脂溶性菲醌类化合物,包括丹参酮Ⅰ、ⅡA、ⅡB,隐丹参酮,异丹参酮Ⅰ、ⅡA。丹参酮ⅡA是丹参主要脂溶性成分,可用于治疗心绞痛、心肌梗死、动脉粥样硬化、心律失常及糖尿病等多种疾病。
丹参酮ⅡA作为脂溶性化合物,其原药在水中溶解度差,理化性质不稳定,生物利用度低,这样大大限制了其临床应用,故优化成分提取工艺及选择合适的药物传递系统显得尤为重要。
人们陆续研制了丹参酮ⅡA的多种新工艺及新剂型,以提高丹参酮ⅡA的生物利用度,降低它的不良反应,使其达到更好的药效。其中固体脂质纳米粒是一种新兴的亚微粒给药系统,具有较好的靶向性、毒性小、可控制药物的释放、药物的稳定性高并可大规模生产等优点。
近年来,靶向治疗技术在众多领域都显示出其应用优势。要达到理想的治疗效果,将基因片段或者药物递送进入靶组织或器官是非常重要的,早期的研究中基因片段或者药物是直接注入到靶组织和器官,然而此方法存在很大的局限性,为了改善这些问题,研究人员开发了很多传递技术。如腺病毒成为基因片段或者药物治疗作用的有效载体,但因存在肝毒性、致癌、致畸等作用而难以推广应用。裸质粒虽避免了上述不利影响,但转染率低。
因此需要提出携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法解决上述问题。
发明内容
本发明的目的是解决现有技术中丹参酮ⅡA的生物利用度低的缺点,而提出的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法。
为了实现上述目的,本发明采用了如下技术方案:
携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,所述携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法具体包括以下步骤:
步骤一:制备脂质溶液;
步骤二:对制备得到的脂质溶液进行除杂处理获得纯净的脂质体溶液;
步骤三:向脂质体溶液中加入丹参酮ⅡA脂类纳米粒及冻干保护剂进行均匀混合,然后再分装至盛装容器内进行真空冷冻干燥处理;
步骤四:向盛装容器内注入全氟丙烷气体,获得携载丹参酮ⅡA脂类纳米粒的靶向超声造影剂。
进一步地,所述步骤一的具体操作方法为:秤取二棕榈酰磷脂酰胆碱10mg、二棕榈酰磷脂酰乙醇胺4mg及胆固醇2mg,将上述二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺以及胆固醇溶于20ml无水乙醇中制成脂质溶液。
进一步地,所述步骤二的具体操作方法为:采用注射器吸取脂质溶液缓慢地加入到54℃的去离子水溶液中,搅拌2h去除残留的乙醇,最后通过离心处理去除杂质,获取纯净的脂质体溶液。
进一步地,所述步骤三的具体操作方法为:向脂质体溶液加入丹参酮ⅡA脂类纳米粒及冻干保护剂均匀混合,分装至容量为5ml的西林瓶中,并且保证每瓶的盛装量为1ml,然后进行真空冷冻干燥处理。
进一步地,所述冷冻干燥剂为浓度为5%的葡萄糖。
进一步地,所述步骤三中的丹参酮ⅡA脂类纳米粒的制备方法包括以下步骤:
S1、通过聚乙二醇的单硬脂酸酯和二硬脂酸酯混合物制备水相;
S2、通过丹参酮ⅡA、卵磷脂、单硬脂酸甘油酯及山嵛酸甘油酯制备有机相;
S3、将有机相注入水相中,通过搅拌至有机溶剂挥发,经过微孔滤膜过滤后即可得到丹参酮ⅡA脂类纳米粒。
进一步地,所述丹参酮ⅡA脂类纳米粒的制备方法的S1具体操作方法为:秤取聚乙二醇的单硬脂酸酯和二硬脂酸酯混合物600mg,加入去离子水10ml,水浴75℃使溶解作为水相。
进一步地,所述丹参酮ⅡA脂类纳米粒的制备方法的S2具体操作方法为:秤取丹参酮ⅡA1mg、卵磷脂200mg、单硬脂酸甘油酯70mg及山嵛酸甘油酯30mg溶于5ml无水乙醇,加热使溶解构成有机相。
进一步地,所述丹参酮ⅡA脂类纳米粒的制备方法的S3具体操作方法为:将有机相缓慢注入600r/min搅拌的水相中,继续搅拌至有机溶剂挥发完全至5ml,经微孔滤膜过滤后,即可制得丹参酮ⅡA脂类纳米粒。
本发明还包括携载丹参酮ⅡA的脂质纳米微泡超声造影剂的应用,用心肌超声造影技术观察大鼠心肌血流灌注,验证定量分析靶向介导丹参酮ⅡA脂类纳米粒大鼠心肌的血流灌注,具体包括:应用1%戊巴比妥钠注射于大鼠腹腔麻醉后,经尾静脉注射携载丹参酮ⅡA脂类纳米粒的超声造影剂,使用彩色多普勒超声诊断仪,转换成超声造影模式,取乳头肌水平左心室短轴观,当图像显示心肌内心肌大量造影剂充盈时即用造影模式以高机械指数脉冲“闪烁”充分破坏心肌内造影剂微泡,再观察上述短轴切面并采集动态图像并保存,获得心肌灌注参数,验证携载丹参酮ⅡA脂类纳米粒的靶向超声造影剂在评价心肌血流灌注的应用。
本发明的有益效果为:
通过采用二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺及胆固醇等原材料制成纯净的脂质体溶液与丹参酮ⅡA脂类纳米粒均匀混合,对丹参酮ⅡA脂类纳米粒进行包封,从而使得包封后的丹参酮ⅡA脂类纳米粒与靶向超声造影剂的微泡壳均为脂类物质,既丹参酮ⅡA脂类纳米粒与本身为脂类物质的靶向超声造影剂的微泡壳具有较好的互溶性和生物安全性,能够有效地提高丹参酮ⅡA的生物利用度,降低丹参酮ⅡA的不良反应,使其达到更好的药效,改善了基因片段或者药物注入到靶组织和器官中的局限性的问题。
附图说明
图1为本发明的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法的流程框图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
实施例一
携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法具体包括以下步骤:
步骤一:首先秤取二棕榈酰磷脂酰胆碱10mg、二棕榈酰磷脂酰乙醇胺4mg及胆固醇2mg,将上述二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺以及胆固醇溶于20ml无水乙醇中制成脂质溶液。
步骤二:采用注射器吸取上述脂质溶液缓慢地加入到54℃的去离子水溶液中,搅拌2h去除残留的乙醇,最后通过离心处理去除杂质,获取纯净的脂质体溶液。
步骤三:秤取聚乙二醇的单硬脂酸酯和二硬脂酸酯混合物600mg,加入去离子水10ml,水浴75℃使溶解作为水相,再秤取丹参酮ⅡA1mg、卵磷脂200mg、单硬脂酸甘油酯70mg及山嵛酸甘油酯30mg溶于5ml无水乙醇,加热使溶解构成有机相,再将有机相缓慢注入600r/min搅拌的水相中,继续搅拌至有机溶剂挥发完全至5ml,经微孔滤膜过滤后,制得丹参酮ⅡA脂类纳米粒;
然后向脂质体溶液加入上述制得的丹参酮ⅡA脂类纳米粒及5%葡萄糖的冻干保护剂均匀混合,分装至容量为5ml的西林瓶中,并且保证每瓶的盛装量为1ml,然后进行真空冷冻干燥处理
步骤四:向盛装容器内注入全氟丙烷气体,获得携载丹参酮ⅡA脂类纳米粒的靶向超声造影剂。
实施例二
用心肌超声造影技术观察大鼠心肌血流灌注,验证定量分析靶向介导丹参酮ⅡA脂类纳米粒大鼠心肌的血流灌注。
首先设置对比组,使用1%戊巴比妥钠注射于其中一组大鼠腹腔麻醉后,经尾静脉注射超声微泡对比造影剂,使用彩色多普勒超声诊断仪,转换成超声造影模式,取乳头肌水平左心室短轴观,当图像显示心肌内心肌大量造影剂充盈时即用造影模式以高机械指数脉冲“闪烁”充分破坏心肌内造影剂微泡,再观察上述短轴切面并采集动态图像并保存。
然后再使用1%戊巴比妥钠注射于另一组大鼠腹腔麻醉后,经尾静脉注射携载丹参酮ⅡA脂类纳米粒的超声造影剂,使用彩色多普勒超声诊断仪,转换成超声造影模式,取乳头肌水平左心室短轴观,当图像显示心肌内心肌大量造影剂充盈时即用造影模式以高机械指数脉冲“闪烁”充分破坏心肌内造影剂微泡,再观察上述短轴切面并采集动态图像并保存。
然后对两组图像进行分析对比,获得心肌灌注参数,验证携载丹参酮ⅡA脂类纳米粒的靶向超声造影剂在评价心肌血流灌注的应用。
图像分析包括:软件生成ROI置于左心室每个节段心肌中央,取样框大小尽量包含整个心肌厚度,并避开心内膜和心外膜的干扰;软件自动生成时间灌注强度曲线并通过拟合函数:Y=A(1-e-βt)+B,曲线峰值强度(A值)反映局部心肌血容量;曲线斜率(β值)反映心肌血流速度;B为背景信号强度;灌注量(A值×β值)反映心肌血流量。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
1.携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法具体包括以下步骤:
步骤一:制备脂质溶液;
步骤二:对制备得到的脂质溶液进行除杂处理获得纯净的脂质体溶液;
步骤三:向脂质体溶液中加入丹参酮ⅡA脂类纳米粒及冻干保护剂进行均匀混合,然后再分装至盛装容器内进行真空冷冻干燥处理;
步骤四:向盛装容器内注入全氟丙烷气体,获得携载丹参酮ⅡA脂类纳米粒的靶向超声造影剂。
2.根据权利要求1所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述步骤一的具体操作方法为:秤取二棕榈酰磷脂酰胆碱10mg、二棕榈酰磷脂酰乙醇胺4mg及胆固醇2mg,将上述二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺以及胆固醇溶于20ml无水乙醇中制成脂质溶液。
3.根据权利要求1所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述步骤二的具体操作方法为:采用注射器吸取脂质溶液缓慢地加入到54℃的去离子水溶液中,搅拌2h去除残留的乙醇,最后通过离心处理去除杂质,获取纯净的脂质体溶液。
4.根据权利要求1所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述步骤三的具体操作方法为:向脂质体溶液加入丹参酮ⅡA脂类纳米粒及冻干保护剂均匀混合,分装至容量为5ml的西林瓶中,并且保证每瓶的盛装量为1ml,然后进行真空冷冻干燥处理。
5.根据权利要求4所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述冷冻干燥剂为浓度为5%的葡萄糖。
6.根据权利要求4所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述步骤三中的丹参酮ⅡA脂类纳米粒的制备方法包括以下步骤:
S1、通过聚乙二醇的单硬脂酸酯和二硬脂酸酯混合物制备水相;
S2、通过丹参酮ⅡA、卵磷脂、单硬脂酸甘油酯及山嵛酸甘油酯制备有机相;
S3、将有机相注入水相中,通过搅拌至有机溶剂挥发,经过微孔滤膜过滤后即可得到丹参酮ⅡA脂类纳米粒。
7.根据权利要求6所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述丹参酮ⅡA脂类纳米粒的制备方法的S1具体操作方法为:秤取聚乙二醇的单硬脂酸酯和二硬脂酸酯混合物600mg,加入去离子水10ml,水浴75℃使溶解作为水相。
8.根据权利要求6所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述丹参酮ⅡA脂类纳米粒的制备方法的S2具体操作方法为:秤取丹参酮ⅡA1mg、卵磷脂200mg、单硬脂酸甘油酯70mg及山嵛酸甘油酯30mg溶于5ml无水乙醇,加热使溶解构成有机相。
9.根据权利要求6所述的携载丹参酮ⅡA的脂质纳米微泡超声造影剂制备方法,其特征在于,所述丹参酮ⅡA脂类纳米粒的制备方法的S3具体操作方法为:将有机相缓慢注入600r/min搅拌的水相中,继续搅拌至有机溶剂挥发完全至5ml,经微孔滤膜过滤后,即可制得丹参酮ⅡA脂类纳米粒。
10.携载丹参酮ⅡA的脂质纳米微泡超声造影剂的应用,用心肌超声造影技术观察大鼠心肌血流灌注,验证定量分析靶向介导丹参酮ⅡA脂类纳米粒大鼠心肌的血流灌注,具体包括:应用1%戊巴比妥钠注射于大鼠腹腔麻醉后,经尾静脉注射携载丹参酮ⅡA脂类纳米粒的超声造影剂,使用彩色多普勒超声诊断仪,转换成超声造影模式,取乳头肌水平左心室短轴观,当图像显示心肌内心肌大量造影剂充盈时即用造影模式以高机械指数脉冲“闪烁”充分破坏心肌内造影剂微泡,再观察上述短轴切面并采集动态图像并保存,获得心肌灌注参数,验证携载丹参酮ⅡA脂类纳米粒的靶向超声造影剂在评价心肌血流灌注的应用。
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