CN111617264A - 一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法 - Google Patents
一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法 Download PDFInfo
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Abstract
本发明公开了一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法，该方法具体包括如下步骤：第一步：采用EDC‑NHS酰胺反应法建立达拉菲尼与DSPE‑PEG2000‑COOH的共价连接，合成DSPE‑PEG2000‑Dabrafenib；第二步：采用薄膜水化法配制脂质悬混液；第三步：采用机械振荡法制备高浓度脂质纳米微泡超声造影剂。惰性气体C3F8构成了内核，使纳米微泡具有良好的稳定性；DSPE‑PEG2000‑Dabrafenib作为脂质纳米微泡壳膜的主要组成成分，实现了Dabrafenib与纳米微泡有效、可靠的连接，本发明能够实现BRAFV600E基因突变型肿瘤的一站式超声造影成像与精准治疗。
Description
技术领域
本发明属于超声分子影像学技术领域，涉及一种纳米微泡超声造影剂，具体的说是涉及一种用于治疗BRAFV600E基因突变型疾病的携载达拉菲尼脂质纳米微泡超声造影剂制备方法。
背景技术
BRAFV600E基因突变与甲状腺癌、黑色素瘤等恶性肿瘤的临床生物学行为密切相关，如侵袭和转移。美国国家食品药品监督管理局（FDA）已于2013年5月批准BRAFV600E抑制剂达拉菲尼（Dabrafenib）进入临床治疗。2018年，达拉菲尼在我国获批上市。达拉菲尼虽然具备一定的治疗效果，然而在患者治疗过程中，口服达拉菲尼存在靶标专一性低、副作用大的根本性问题。而且，长期、高浓度地用药会导致癌细胞对药物脱敏，最终迫使患者不断加大用药剂量而产生严重的耐药情况。临床资料表明，在肿瘤发生的早期，其内部的新生血管壁一般是不完整的，内皮细胞之间存在许多380nm～780nm大小的裂隙；另外，新生血管缺乏基底膜和平滑肌组织，因此，相比于正常组织血管，肿瘤新生血管具有更高的通透性。
不同于大粒径尺度的微米级微泡超声造影剂，纳米微泡超声造影剂的纳米级微气泡平均粒径小于肿瘤血管的管壁裂隙，能够穿过肿瘤组织的毛细血管壁而进入组织内部聚集，产生超声增强显影。如果将与肿瘤治疗药物连接的纳米微泡超声造影剂注入体内，不仅可获得肿瘤组织的高质量超声造影成像，还可利用超声靶向微泡破坏技术（UTMD）促使微泡破裂融合于肿瘤组织，实现直达肿瘤病灶的针对性药物递送与精准治疗。目前现有技术中尚缺乏一种锚定BRAFV600E基因突变型肿瘤超声成像与治疗的携载达拉菲尼纳米微泡超声造影剂。
发明内容
本发明为了克服现有技术中存在的不足，提供一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法，用以实现BRAFV600E基因突变型肿瘤的“一站式”超声造影成像与精准治疗。
本发明是通过以下技术方案实现的：本发明公开了一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法，该方法具体包括如下步骤：
第一步：采用EDC-NHS酰胺反应法建立达拉菲尼与DSPE-PEG2000-COOH（二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-羧基交连物）的共价连接，合成DSPE-PEG2000-Dabrafenib；第二步：采用薄膜水化法配制脂质悬混液；第三步：采用机械振荡法制备高浓度脂质纳米微泡超声造影剂。
第一步的具体操作方法为：首先将DSPE-PEG2000-COOH溶于医用级二甲基亚砜（DMSO）中，按照一定摩尔比加入EDC（1-乙基-3-二甲氨基丙基碳化二亚胺盐酸盐）和NHS（N-羟基琥珀酰胺），在室温下进行振荡活化；然后将Dabrafenib与活化后生成的DSPE-PEG2000-COO-琥珀酰亚胺按照一定摩尔比混合使之发生酰胺反应，合成DSPE-PEG2000-Dabrafenib；待充分反应后，使用透析袋内除去未发生反应的EDC、NHS和Dabrafenib。
第二步的具体操作方法为：旋蒸瓶中，按照一定摩尔比将的二棕榈磷脂酰胆碱（DPPC）、DSPE-PEG2000-Dabrafenib和DSPE-PEG2000（二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000）充分溶解于三氯甲烷（氯仿）中，均匀晃动旋蒸瓶至物质充分溶解、混合；将盛有混合物的旋蒸瓶放置于旋转蒸发仪上，待三氯甲烷充分挥发后，在瓶壁上形成一层厚度均匀的干性脂质薄膜；往旋蒸瓶中加入已被预热至37℃的甘油和PBS水合液水化薄膜，在恒温摇床上，待瓶壁薄膜脱落，形成均匀的脂质悬混液。
第三步的具体操作方法为：将脂质悬混液置入盖有橡胶瓶塞的西林瓶中，然后使用注射器抽真空后注入惰性气体C3F8；将西林瓶置于银汞胶囊调和器上振荡90s，完成高浓度脂质纳米微泡超声造影剂的制备；最后，对制备好的造影剂进行Co60照射消毒，置于冰箱冷藏保存备用。
第二步和第三步可以转变为第一步和第二步，转变的前提是第二步脂质悬混液配制时采用DPPC、DSPE-PEG2000-COOH和DSPE-PEG2000，两步中涉及的摩尔配比和制备方法不变。
第一步EDC-NHS酰胺反应可以后移至第三步进行，即向经前两步制备的脂质纳米微泡悬混液中加入EDC和NHS，保持DSPE-PEG2000-COOH、EDC、NHS的摩尔配比不变，室温下振荡活化1h，然后以摩尔比1:1.5将Dabrafenib与活化后生成的DSPE-PEG2000-COO-琥珀酰亚胺混合发生酰胺反应，使纳米微泡壳膜成分中的DSPE-PEG2000-COOH转化为DSPE-PEG2000-Dabrafenib，最后将样品置于4℃下的透析袋内除去未发生反应的EDC、NHS和Dabrafenib。
第一步中，DSPE-PEG2000-COOH、EDC和NHS的摩尔比为1:2:2。
第二步中，DPPC、DSPE-PEG2000-Dabrafenib和DSPE-PEG2000的摩尔比为5:1:1。
本发明的有益效果为：本发明涉及一种携载达拉菲尼脂质纳米微泡超声造影剂的制备方法，用以实现BRAFV600E基因突变型肿瘤的“一站式”超声造影成像与精准治疗。本发明中的制备方法还具有如下优点：第一，惰性气体C3F8构成了内核，使纳米微泡具有良好的稳定性；第二，DSPE-PEG2000-Dabrafenib作为脂质纳米微泡壳膜的主要组成成分，实现了Dabrafenib与纳米微泡有效、可靠的连接；第三，本发明制备过程中，可将DSPE-PEG2000替换为DSPE-PEG2000-Biotin（生物素化二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000），使DSPE-PEG2000-Biotin成为脂质纳米微泡的另一组成成分，其目的在于可引入生物素-亲和素法，通过链霉亲和素法将抗体结合在肿瘤细胞表面特异性高表达抗原上，实现针对相应肿瘤的特异性寻靶功能；第四，携载Dabrafenib纳米微泡穿过肿瘤血管管壁裂隙进入肿瘤组织后，在超声辐照下可获得显著的组织超声造影增强显影，且经UTMD后，DSPE-PEG2000-Dabrafenib可在37℃条件下发生水解，驱使Dabrafenib与脂质分子脱离，与肿瘤组织融合达到精准抑癌效果，从而克服Dabrafenib口服治疗的效果不足、全身不良反应和耐药性。
附图说明
图1是本发明Dabrafenib的分子式。
图2是通过EDC-NHS酰胺反应法构建DSPE-PEG2000-Dabrafenib示意图。
图3是通过机械振荡法合成携载Dabrafenib纳米微泡示意图。
具体实施方式
以下结合附图和具体实施方式对本发明作详细描述。
本发明公开了一种携载达拉菲尼脂质纳米微泡超声造影剂的制备方法，具体实施步骤为：
第一步：建立Dabrafenib与DSPE-PEG2000-COOH（二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-羧基交连物）的共价连接，合成DSPE-PEG2000-Dabrafenib。参照图1所示的分子式，Dabrafenib含有氨基（-NH2）官能团，因此，引入EDC与NHS，通过酰胺反应，建立Dabrafenib与DSPE-PEG2000-COOH的共价结合，从而合成DSPE-PEG2000-Dabrafenib，参照图2。具体实施过程为：首先，医用级DMSO中加入DSPE-PEG2000-COOH、EDC和NHS，摩尔比为1:2:2，室温下振荡活化1h；然后，将活化后生成的 DSPE-PEG2000-COO-琥珀酰亚胺与Dabrafenib发生酰胺反应，DSPE-PEG2000-COO-琥珀酰亚胺与Dabrafenib摩尔比为 1:1.5，合成DSPE-PEG2000-Dabrafenib；最后，反应结束后将其置于 4℃下的透析袋内(MWCO，2000)除去未参与反应的 EDC、NHS和Dabrafenib。
第二步：采用薄膜水化法配制脂质悬混液，具体实施过程为：首先，将DPPC、DSPE-PEG2000-Dabrafenib和DSPE-PEG2000按照摩尔比5:1:1加入旋蒸瓶中；然后，向旋蒸瓶中加入氯仿，使用铝箔纸包裹旋蒸瓶并均匀晃动旋蒸瓶至瓶中物质充分溶解、混合；接着，将旋蒸瓶置于旋转蒸发仪上，温度设定为55℃，转速设定为130 rpm，时间设定为12 min，目的是使瓶中的氯仿充分挥发，在瓶壁上形成一层厚度均匀的干性脂质薄膜；最后，向旋蒸瓶中加入已被预热至37℃的水合液（甘油：PBS的体积比=1:9）使薄膜水化，将其置于恒温摇床上，温度设定为37℃，转速设定为120 rpm，时间设定为60 min，待瓶壁薄膜脱落，形成均匀的脂质悬混液。
第三步：采用机械振荡法合成高浓度脂质纳米微泡超声造影剂，如图3所示，具体实施过程为：首先，将脂质悬混液置入盖有橡胶瓶塞西林瓶中；然后，使用50ml注射器抽真空后注入惰性气体C3F8；进一步，将西林瓶置于银汞胶囊调和器上振荡90s，即完成高浓度脂质纳米微泡超声造影剂制备；最后，对制备的造影剂进行Co60照射30 min消毒后置入冰箱中4℃冷藏保存备用。
尽管以上对本发明的实施方案进行了详细说明，但是本发明并不局限于此方案。此实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下，在不脱离本发明权利要求所保护的范围情况下，还可以做出多种形式的技术方案修改，或对其中部分技术特征进行等同替换，这些修改或替换均属于本发明保护范围之内。
Claims (8)
1.一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法，其特征在于：所述携载达拉菲尼的脂质纳米微泡超声造影剂制备方法具体包括如下步骤：
第一步：采用EDC-NHS酰胺反应法建立达拉菲尼与DSPE-PEG2000-COOH的共价连接，合成DSPE-PEG2000-Dabrafenib；第二步：采用薄膜水化法配制脂质悬混液；第三步：采用机械振荡法制备高浓度脂质纳米微泡超声造影剂。
2.根据权利要求1所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法，其特征在于：所述第一步的具体操作方法为：首先将DSPE-PEG2000-COOH溶于医用级二甲基亚砜中，按照一定摩尔比加入EDC和NHS，在室温下进行振荡活化；然后将Dabrafenib与活化后生成的DSPE-PEG2000-COO-琥珀酰亚胺按照一定摩尔比混合使之发生酰胺反应，合成DSPE-PEG2000-Dabrafenib；待充分反应后，使用透析袋内除去未发生反应的EDC、NHS和Dabrafenib。
3.根据权利要求1所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法, 其特征在于：所述第二步的具体操作方法为：旋蒸瓶中，按照一定摩尔比将的DPPC、DSPE-PEG2000-Dabrafenib和DSPE-PEG2000充分溶解于三氯甲烷中，均匀晃动旋蒸瓶至物质充分溶解、混合；将盛有混合物的旋蒸瓶放置于旋转蒸发仪上，待三氯甲烷充分挥发后，在瓶壁上形成一层厚度均匀的干性脂质薄膜；往旋蒸瓶中加入已被预热至37℃的甘油和PBS水合液水化薄膜，在恒温摇床上，待瓶壁薄膜脱落，形成均匀的脂质悬混液。
4.根据权利要求1所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法, 其特征在于：所述第三步的具体操作方法为：将脂质悬混液置入盖有橡胶瓶塞的西林瓶中，然后使用注射器抽真空后注入惰性气体C3F8；将西林瓶置于银汞胶囊调和器上振荡90s，完成高浓度脂质纳米微泡超声造影剂的制备；最后，对制备好的造影剂进行Co60照射消毒，置于冰箱冷藏保存备用。
5.根据权利要求1所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法, 其特征在于：所述第二步和第三步能够转变为第一步和第二步，转变的前提是第二步脂质悬混液配制时采用DPPC、DSPE-PEG2000-COOH和DSPE-PEG2000，两步中涉及的摩尔配比和制备方法不变。
6.根据权利要求1所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法, 其特征在于：所述第一步EDC-NHS酰胺反应能够后移至第三步进行，即向经前两步制备的脂质纳米微泡悬混液中加入EDC和NHS，保持DSPE-PEG2000-COOH、EDC、NHS的摩尔配比不变，室温下振荡活化1h，然后以摩尔比1:1.5将Dabrafenib与活化后生成的DSPE-PEG2000-COO-琥珀酰亚胺混合发生酰胺反应，使纳米微泡壳膜成分中的DSPE-PEG2000-COOH转化为DSPE-PEG2000-Dabrafenib，最后将样品置于4℃下的透析袋内除去未发生反应的EDC、NHS和Dabrafenib。
7.根据权利要求2所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法, 其特征在于：所述第一步中，DSPE-PEG2000-COOH、EDC和NHS的摩尔比为1:2:2。
8.根据权利要求3所述的一种携载达拉菲尼的脂质纳米微泡超声造影剂制备方法, 其特征在于：所述第二步中，DPPC、DSPE-PEG2000-Dabrafenib和DSPE-PEG2000的摩尔比为5:1:1。
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