CN111053923B - 一种靶向肿瘤的超声造影剂 - Google Patents
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Abstract
本发明公开了一种靶向肿瘤的超声造影剂。包括包裹多西他赛的聚赖氨酸高分子造影剂以及与之相连的凝溶胶蛋白单抗。本发明制备的靶向肿瘤的超声造影剂，包封率及载药量均较高，且缓释性能较好，造影剂靶向肝癌组织的特异性强，体外显影效果亦较好。为早期肝癌的诊断和辅助治疗开辟了新的思路。
Description
技术领域
本发明属于超声造影剂技术领域，具体涉及一种定向靶向肝癌肿瘤的超声造影剂。
背景技术
肝癌是我国常见的恶性肿瘤之一，在恶性肿瘤死亡顺位中占第2位，在城市中仅次于肺癌；农村中仅次于胃癌。由于血清甲胎蛋白的临床应用和各种影像学技术的进步，特别是AFP和超声显像用于肝癌高危人群的监测，使肝癌能够在无症状和体征的亚临床期作出诊断，加之外科手术技术的成熟，以及各种局部治疗等非手术治疗方法的发展，使肝癌的预后较过去有了明显提高。
然而，目前常规影像学检查在肿瘤转移的预警及早期识别能力上具有明显的局限性。目前临床上广泛使用的超声造影剂属于血池显像，直径多在 2.5μm 以上而无法透过血管内皮间隙，对于低灌注区或无灌注区隐匿性微小转移病灶的检测敏感度及准确率仍然较低。因此，设计一种针对肝癌肿瘤抗原特异性显像的分子靶向造影剂，将为肝癌肿瘤转移的早期发现、精准诊断和精准治疗提供重要帮助。
发明内容
本发明的目的在于提供一种靶向肿瘤的超声造影剂，为肝癌肿瘤转移的早期发现、精准诊断和精准治疗提供重要帮助。
一种靶向肿瘤的超声造影剂，包括包裹多西他赛的聚赖氨酸高分子造影剂以及与之相连的凝溶胶蛋白单抗。
所述包裹多西他赛的聚赖氨酸高分子造影剂采用双乳化溶剂挥发法制成。
所述包裹多西他赛的聚赖氨酸高分子造影剂的制备方法为：
（1）称取 100mg 聚赖氨酸和10mg多西他赛，充分溶解于2ml二氯甲烷中，作为连续相，然后加入0.2ml双蒸水作为分散相，在-2-2℃条件下，声振仪声振，得乳白色的一次乳化液；
（2）加入 5ml 5%聚乙烯醇溶液，-2-2℃条件下，进行二次声振，得二次乳化液；
（3）向所得乳化液内加入15ml 3%异丙醇溶液，室温下磁力搅拌2-4h，使二氯甲烷充分挥发，微球表面固化，高速离心机7000-9000 转/分离心，收集造影剂，再以双蒸水重悬，反复离心洗涤收集微球，冷冻干燥24-48h 后充入氟碳气体，即得包裹多西他赛的聚赖氨酸高分子造影剂冻干粉，于4℃冰箱保存。
步骤（1）所述声振仪声振的参数为：95W，开5s关2s，共100s。
步骤（2）所述声振仪声振的参数为：80W，开3s关1s，共80s。
所述凝溶胶蛋白单抗与包裹多西他赛的聚赖氨酸高分子造影剂相连的方法为：
（1）将包裹多西他赛的聚赖氨酸高分子造影剂分散溶解于MES缓冲液中，加入耦联活化剂，室温下轨道式振荡器上振荡孵育35-50min，再以双蒸水6000-9000转/分反复洗涤离心，收集表面活化的造影剂；
（2）MES缓冲液重悬表面活化的造影剂，然后加入凝溶胶蛋白单抗，凝溶胶蛋白单抗终浓度为1.0-2.0m M，冰浴条件下振荡孵育1-2h，再以双蒸水 7000-8000转/分反复离心洗涤，收集微球，冷冻干燥 24-48h后充入氟碳气体得 冻干粉，于4℃冰箱保存。
所述耦联活化剂为EDC/NHS，EDC/NHS最终浓度比为3mM/4mM。
本发明的有益效果：本发明制备的靶向肿瘤的超声造影剂，包封率及载药量均较高，且缓释性能较好，造影剂靶向肝癌组织的特异性强，体外显影效果亦较好。为早期肝癌的诊断和辅助治疗开辟了新的思路。
附图说明
图1为肝癌常规超声造影。
图2为实施例2制备的造影剂超声造影。
具体实施方式
下面结合具体实施例对本发明做进一步说明。
实施例1
包裹多西他赛的聚赖氨酸高分子造影剂的制备方法：
（1）称取 100mg 聚赖氨酸和10mg多西他赛，充分溶解于2ml二氯甲烷中，作为连续相，然后加入0.2ml双蒸水作为分散相，在0℃条件下，声振仪声振（声振仪声振的参数为：95W，开5s关2s，共100s。），得乳白色的一次乳化液；
（2）加入 5ml 5%聚乙烯醇溶液，0℃条件下，进行二次声振（声振仪声振的参数为：80W，开3s关1s，共80s），得二次乳化液；
（3）向所得乳化液内加入15ml 3%异丙醇溶液，室温下磁力搅拌3h，使二氯甲烷充分挥发，微球表面固化，高速离心机7500 转/分离心，收集造影剂，再以双蒸水重悬，反复离心洗涤收集微球，冷冻干燥30h 后充入氟碳气体，即得包裹多西他赛的聚赖氨酸高分子造影剂冻干粉，于4℃冰箱保存。
实施例2
凝溶胶蛋白单抗与包裹多西他赛的聚赖氨酸高分子造影剂相连的方法：
（1）将包裹多西他赛的聚赖氨酸高分子造影剂分散溶解于MES缓冲液中，加入EDC/NHS（EDC/NHS最终浓度比为3mM/4mM），室温下轨道式振荡器上振荡孵育40min，再以双蒸水6800转/分反复洗涤离心，收集表面活化的造影剂；
（2）MES缓冲液重悬表面活化的造影剂，然后加入凝溶胶蛋白单抗，凝溶胶蛋白单抗终浓度为1.5m M，冰浴条件下振荡孵育1.5h，再以双蒸水 7500转/分反复离心洗涤，收集微球，冷冻干燥 36h后充入氟碳气体得靶向肿瘤的超声造影剂冻干粉，于4℃冰箱保存。
实施例3 靶向高分子造影剂一般性质检测
称取适量实施例2制备的靶向载药造影剂充分溶解于双蒸水中，取少量滴于载玻片，光镜下观察制备出的靶向载造影剂的形态、分布；另取 1ml 造影剂溶液于激光粒径仪检测专用皿内，检测其粒径大小、分布以及 zeta 电位。
靶向载药高分子造影剂外观为白色粉末状，复溶后大小较均一，呈球形，形态较规则且分散均匀，Malvern 激光粒径仪检测其粒径分布均匀，平均粒径大小为335.23±4.18）nm，造影剂表面电位为（-11.29±1.23）m V。
实施例4 靶向高分子造影剂体外缓释实验
准备截留分子量为9000 的透析袋，取用10cm 长一小段并置于100℃水中煮沸消毒15 min，取出并用双蒸水将透析袋内外进行彻底漂洗备用。精确称取 10mg的造影剂冻干粉充分溶解于2ml PBS 缓冲液中，带无菌手套将造影剂溶液转移至透析袋中，透析袋两端密闭并完全浸没于装有150ml 缓释介质的蓝口瓶内，于 37℃，120 转/分恒温振荡器内持续震荡，分别于 3h、6h、9h、12h、ld、2d、3d、8d、10d、20d、30d不同时间点从缓冲液中取样 lml，每次取样后及时补加 l ml PBS，经 0.22μm PVDF 膜过滤，取 20μl 进样，计算造影剂的药物累积释放百分率，并绘制释放时间曲线。
使用高效液相色谱分析仪测得到多西他赛各浓度标准品溶液的色谱图，并根据所得数据绘制得直线回归方程Y=19923X+17890，R2=0.9978，Y 值为色谱图中被检测样品的峰面积值，X 为被检测样品溶液的浓度，R2为决定系数，其数值的大小可反映所得趋势线与实际所得数据的拟合度，根据此方程计算得所制备的造影剂的平均包封率为（82.29±2.31）%，载药量平均为（8.48±0.24）%，药物包载率较高。体外缓释实验显示本发明所制备的靶向载药超声造影剂缓释性能较好，第20d 时多西他赛缓释率约为 91.22%。
实施例5 超声造影检测实验
实验样本选取，选取肝癌患者，早期肝癌，无明显阳性体征，症状常无特异性，仅类似肝硬化体征。
超声检查设备使用GE LOG E9彩色多普勒超声诊断仪，凸阵探头，探头频率为3.5~5 MHz。超声造影检查的检查者：均由同一名超声科副主任医师，在同一台机器进行操作。超声造影检查前，患者空腹8小时，取平卧位，对肝脏病灶行多切面检查，探查肝脏病灶大小、部位、边界及回声情况，选择最佳切面，固定探头位置，调节超声设备，进入造影程序，调节机械指数。经肘部浅静脉建立超声造影静脉通道。研究对象静脉注射本发明制备的超声造影剂，步骤为将造影剂冻干粉使用5 ml生理盐水溶解，震荡摇匀，抽取1.5 ml经静脉通道团注，随后立即用5 ml生理盐水冲管，实时存储图像，连续观测10分钟；对照采用采用意大利Bracco公司生产的SonoVue (声诺维)超声造影剂，步骤为将造影剂冻干粉使用5 ml生理盐水溶解，震荡摇匀，抽取2.4 ml经静脉通道团注，随后立即用5 ml生理盐水冲管，实时存储图像，连续观测10分钟。
图1为肝癌常规超声造影，SonoVue (声诺维)超声造影剂被注入后，肿块表现出不均匀增强，而且灌注时流速快，消退也快，呈快进快退灌注模式。动态观察肝癌的内部灌注情况，肝癌在动脉期为强化，病灶呈均匀或不均匀回声增强，延迟相呈回声减低，增强开始时间、到达最大强度时间和消退时间均较短，峰值强度较高，消退时间较短。SonoVue (声诺维)超声造影剂属于血池造影，富含微气泡的超声造影剂经外周静脉注入，通过目标肿物的毛细血管循环灌注情况判断肿物性质，对于乏血供类型的肿瘤难以诊断。血流显像无明显特异性，肿瘤边缘与周围正常组织分界欠清晰。
图2实施例2制备的造影剂超声造影，具有肿瘤靶向特异性，造影剂微泡能主动靶向聚集于肿瘤组织，动态观察肝癌的内部灌注情况，肿瘤组织表现出快速、均匀增强，到达最大强度时间短，能耐受血流的切应力，具有高稳定性，动脉期至延迟期均明显增强，肿瘤强化持续时间明显延长，有利于病灶的定性及动态观察。可实现肿瘤的靶向诊断，其携带的肿瘤治疗药物释放后可实现肿瘤的靶向诊断和治疗为一体化。本发明制备的靶向超声造影剂注射所需剂量小于常规超声造影剂，包封率及载药量均较高。
Claims (6)
1.一种靶向肿瘤的超声造影剂，其特征在于，包括包裹多西他赛的聚赖氨酸高分子造影剂以及与之相连的凝溶胶蛋白单抗；所述包裹多西他赛的聚赖氨酸高分子造影剂采用双乳化溶剂挥发法制成。
2.根据权利要求1所述靶向肿瘤的超声造影剂，其特征在于，所述包裹多西他赛的聚赖氨酸高分子造影剂的制备方法为：
（1）称取 100mg 聚赖氨酸和10mg多西他赛，充分溶解于2ml二氯甲烷中，作为连续相，然后加入0.2ml双蒸水作为分散相，在-2-2℃条件下，声振仪声振，得乳白色的一次乳化液；
（2）加入 5ml 5%聚乙烯醇溶液，-2-2℃条件下，进行二次声振，得二次乳化液；
（3）向所得乳化液内加入15ml 3%异丙醇溶液，室温下磁力搅拌2-4h，使二氯甲烷充分挥发，微球表面固化，高速离心机7000-9000 转/分离心，收集造影剂，再以双蒸水重悬，反复离心洗涤收集微球，冷冻干燥 24-48h 后充入氟碳气体，即得包裹多西他赛的聚赖氨酸高分子造影剂冻干粉，于 4℃冰箱保存。
3.根据权利要求2所述靶向肿瘤的超声造影剂，其特征在于，步骤（1）所述声振仪声振的参数为：95W，开 5s 关 2s，共 100s。
4.根据权利要求2所述靶向肿瘤的超声造影剂，其特征在于，步骤（2）所述声振仪声振的参数为：80W，开3s 关 1s，共 80s。
5.根据权利要求1所述靶向肿瘤的超声造影剂，其特征在于，所述凝溶胶蛋白单抗与包裹多西他赛的聚赖氨酸高分子造影剂相连的方法为：
（1）将包裹多西他赛的聚赖氨酸高分子造影剂分散溶解于MES缓冲液中，加入耦联活化剂，室温下轨道式振荡器上振荡孵育35-50min，再以双蒸水6000-9000转/分反复洗涤离心，收集表面活化的造影剂；
（2）MES缓冲液重悬表面活化的造影剂，然后加入凝溶胶蛋白单抗，凝溶胶蛋白单抗终浓度为1.0-2.0m M，冰浴条件下振荡孵育1-2h，再以双蒸水 7000-8000转/分反复离心洗涤，收集微球，冷冻干燥 24-48h后充入氟碳气体得 冻干粉，于4℃冰箱保存。
6.根据权利要求5所述靶向肿瘤的超声造影剂，其特征在于，所述耦联活化剂为EDC/NHS，EDC/NHS最终浓度比为3m M/4m M。
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