超声引导卡铂/乳酸羟基乙酸共聚物微球瘤内注射治疗胰腺癌实验研究
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摘要
目的：
1、制备广谱抗癌药卡铂的新剂型——注射型卡铂/乳酸羟基乙酸共聚物缓释微球（CBP-PLGA Ms），用于超声引导经皮穿刺肿瘤内注射治疗。
2、将介入超声与缓释技术结合，探讨卡铂/乳酸羟基乙酸共聚物微球瘤内注射治疗荷瘤裸小鼠胰腺癌的价值，旨在为胰腺癌治疗提供一条新途径。
3、超声动态观察荷瘤裸小鼠胰腺癌生长特性及声像图特征，建立评价超声引导瘤内注射化疗药物治疗胰腺癌效果新方法。
方法：
1、用均匀设计优化W/O/W型乳化溶剂挥发法制备卡铂/乳酸羟基乙酸共聚物微球的制备工艺，观察优化工艺制备微球形态特征、粒径分布、载药率和体外缓释性能。
2、SW1990人胰腺癌细胞株体外培养传代，BALB/c-nu裸小鼠（SPF级）背部皮下接种，建立胰腺癌动物模型，每5天用Acuson Sequoia512彩超（探头频率13MHz）观察肿瘤体积和生长曲线、二维声像图和彩色多谱勒特征。
3、将20只荷瘤裸小鼠分为实验组（即CBP-PLGA Ms注射组）、卡铂+空白微球注射组（CBP+Ms注射组）、游离卡铂注射组（CBP注射组）和5%葡萄糖溶液注射组（5%GS注射组），超声引导胰腺癌瘤内注射治疗。每5天超声观察肿瘤体积和生长曲线变化、二维声像图和彩色多谱勒变化，共30天，计算肿瘤倍增时间，观察各组小鼠血象变化和病理变化。
结果：
1、卡铂/乳酸羟基乙酸共聚物微球的优化制备工艺为：10%卡铂溶液1.5ml、PLGA浓度2.5%、搅拌速度1000转/分钟、油水相体积比1:5；用优化工艺制备的微球粒径范围为5～200μm，＜100μm的微球数为96.7%，符合注射要求；该微球载药率10.16%，包封率77.8%，体外缓释时间超过10天，释放稳定。
2、成功建立了裸小鼠皮下种植性胰腺癌动物模型，该肿瘤声像图表现为无包膜、边界清晰的低回声结节，彩色血流信号丰富；接种后第15～20天，肿瘤处于进入生长旺盛期，可作为治疗观察开始时期。
3、实验组肿瘤体积进行性缩小，瘤体倍增时间为-33.9天，与CBP+Ms注射组、
CBP注射组和5%GS注射组（分别为4.65天、4.32天、2.39天）比较，有显著差异，P＜0.0001；肿瘤结节边界较模糊，形态尚规则；超声观察到微球注入后肿瘤声像图变化规律为注射时和注射后初期呈不均质高回声，随观察时间延长，回声强度逐渐降低，注射后30天呈不均质低回声，血流信号少于对照组。实验组小鼠血象未受抑制，CBP+Ms注射组、CBP注射组小鼠在治疗后10天内其WBC、RBC、PLT均有降低，P＜0.05。病理学观察显示实验组小鼠肿瘤呈大片状凝固性坏死，部分伴有液化坏死、残留细胞空泡变性和小血管微血栓形成；CBP+Ms注射组、CBP注射组肿瘤组织以空泡变性为主，液化坏死少见，5%GS注射组无坏死和变性；实验组和CBP+Ms注射组残留肿瘤组织内均见少量微球存留。
结论：
1、采用W/O/W型乳化溶剂挥发法，经均匀设计优化微球制备工艺，成功制备了广谱抗癌药卡铂的新剂型——卡铂/乳酸羟基乙酸共聚物微球，其粒径大小符合注射要求，体外缓释效果较好，卡铂释放时间超过10天，可用于瘤内注射动物实验。
2、体内治疗实验研究结果表明该微球可使卡铂持久、有效地作用于荷瘤裸小鼠种植性胰腺癌细胞，造成肿瘤细胞变性、坏死，对小鼠骨髓无明显抑制作用，达到了延长卡铂释放时间、有效降低其毒副作用、提高化疗效果的目的。
3、超声引导下经皮穿刺将卡铂/乳酸羟基乙酸共聚物微球用于瘤内注射治疗胰腺癌，扩大了超声引导肿瘤局部化疗的研究范围，本研究所用方法简便易行、效果肯定，方法可望作为胰腺癌治疗的一条新途径。
4、超声动态观察裸小鼠皮下种植性胰腺癌具有灵敏、方便、准确、实用的优点，可作为主要观察指标。
Objective: First, to prepare carboplatin-loaded PLGA microspheres (CBP-PLGA Ms) for intra-tumoral injection (i.t.). Then the CBP-PLGA Ms would be injected directly in human pancreatic cancer in nude mice guided by ultrasound, and the anti-tumor effects would be evaluated. Our purpose is finding a novel therapy method for pancreatic cancer .
Method:W/O/W emulsion solvent evaporation method and uniform test design were applied to optimize the formulation and procedure in the preparation of CBP-PLGA Ms, then physical characteristics and drug loading (DL) of the microspheres and release time of carboplatin from the Ms in vitro were tested. Also the subcutaneous implanting nude mice model of human pancreatic cancer was established successfully with sw1990 cell strain. Its ultrasound imaging and biological characteristics including the curve of tumor growth were studied by high frequence ultrasound (HFU). Then 20 mice was divided into 4 groups of CBP-PLGA Ms, carboplatin and blank microspheres mixture(CBP+Ms), free CBP and 5% glucose injection(5%GS). Intra-tumoral injections were performed respectively. The changes of tumor growth and ultrasound imaging were observed every 5 days, and blood changes were checked from 3 to 15 days after i.t. And pathology was examinated at the end of 30days.
Result : The optimum factors of preparation of CBP-PLGA Ms were 10% CBP solution 1.5ml, PLGA concentration 2.5% in dichloromethane, stirring speed 1000 r/min, and the ratio of oil in water 1:5. The CBP-PLGA Ms were suitable for i.t. due to the rate of 96.7% of microspheres smaller than 100μm. in particle size.The DL was 10.16%, and release time of CBP from the microspheres was over 10 days. The human pancreatic cancer imaging in nude mice was low-echo and an-theca and clear border node. The CDFI and pathologic examination showed the vasculature was very rich in tumor. The suitable time for observation or injection was from 15 days to 20 days after implanting. The tumor growth double time (DT) of group of CBP-PLGA Ms was -33.9days, the difference from the other groups was significant (p<0.0001). The DT of those groups of CBP+Ms, free CBP, 5%GS were 4.65days, 4.32 days and 2.39 days respectively. The HFU also observed
injection process of CBP-PLGA Ms and its imaging characteristics in tumor, which was hyper-echo at the early stage and weakened bit by bit to low-echo at the end of 30 days. The tumor imaging of experimental group was unequal low-echo or weak-echo node with unclear border. The sign of color flow becomes insufficiency. The amount of WBC, RBC and PLT in the groups of CBP+Ms, free CBP became less after i.t., and lower than the group of CBP-PLGA Ms, p<0.05. The pathologic examinations showed that big coagulation necrosis masses with small colliquative necrosis and cell vacuolation were observed in the tumor in experimental mice, these signs were significantly different from the others. A few microspheres were found in some tumor tissues in the groups of CBP-PLGA Ms and CBP+Ms, which showed that the microspheres could be degradable slowly over 30 days in vivo.
Conclusion: CBP-PLGA Ms can be used to injection in animal experiment，due to its good physical characteristics and degradation. Because of sustaining degradation of microspheres, the CBP can cause tumor to necrosis and vacuolation effectively and continously in vivo. HFU is sensitive, convenience and accurate using for observation on human pancreatic cancer in nude mice. The method, intra-tumoral injection with CBP-PLGA Ms guided by ultrasound, should be a new pathway using for therapy pancreatic cancer.
1、制备广谱抗癌药卡铂的新剂型——注射型卡铂/乳酸羟基乙酸共聚物缓释微球（CBP-PLGA Ms），用于超声引导经皮穿刺肿瘤内注射治疗。
2、将介入超声与缓释技术结合，探讨卡铂/乳酸羟基乙酸共聚物微球瘤内注射治疗荷瘤裸小鼠胰腺癌的价值，旨在为胰腺癌治疗提供一条新途径。
3、超声动态观察荷瘤裸小鼠胰腺癌生长特性及声像图特征，建立评价超声引导瘤内注射化疗药物治疗胰腺癌效果新方法。
方法：
1、用均匀设计优化W/O/W型乳化溶剂挥发法制备卡铂/乳酸羟基乙酸共聚物微球的制备工艺，观察优化工艺制备微球形态特征、粒径分布、载药率和体外缓释性能。
2、SW1990人胰腺癌细胞株体外培养传代，BALB/c-nu裸小鼠（SPF级）背部皮下接种，建立胰腺癌动物模型，每5天用Acuson Sequoia512彩超（探头频率13MHz）观察肿瘤体积和生长曲线、二维声像图和彩色多谱勒特征。
3、将20只荷瘤裸小鼠分为实验组（即CBP-PLGA Ms注射组）、卡铂+空白微球注射组（CBP+Ms注射组）、游离卡铂注射组（CBP注射组）和5%葡萄糖溶液注射组（5%GS注射组），超声引导胰腺癌瘤内注射治疗。每5天超声观察肿瘤体积和生长曲线变化、二维声像图和彩色多谱勒变化，共30天，计算肿瘤倍增时间，观察各组小鼠血象变化和病理变化。
结果：
1、卡铂/乳酸羟基乙酸共聚物微球的优化制备工艺为：10%卡铂溶液1.5ml、PLGA浓度2.5%、搅拌速度1000转/分钟、油水相体积比1:5；用优化工艺制备的微球粒径范围为5～200μm，＜100μm的微球数为96.7%，符合注射要求；该微球载药率10.16%，包封率77.8%，体外缓释时间超过10天，释放稳定。
2、成功建立了裸小鼠皮下种植性胰腺癌动物模型，该肿瘤声像图表现为无包膜、边界清晰的低回声结节，彩色血流信号丰富；接种后第15～20天，肿瘤处于进入生长旺盛期，可作为治疗观察开始时期。
3、实验组肿瘤体积进行性缩小，瘤体倍增时间为-33.9天，与CBP+Ms注射组、
CBP注射组和5%GS注射组（分别为4.65天、4.32天、2.39天）比较，有显著差异，P＜0.0001；肿瘤结节边界较模糊，形态尚规则；超声观察到微球注入后肿瘤声像图变化规律为注射时和注射后初期呈不均质高回声，随观察时间延长，回声强度逐渐降低，注射后30天呈不均质低回声，血流信号少于对照组。实验组小鼠血象未受抑制，CBP+Ms注射组、CBP注射组小鼠在治疗后10天内其WBC、RBC、PLT均有降低，P＜0.05。病理学观察显示实验组小鼠肿瘤呈大片状凝固性坏死，部分伴有液化坏死、残留细胞空泡变性和小血管微血栓形成；CBP+Ms注射组、CBP注射组肿瘤组织以空泡变性为主，液化坏死少见，5%GS注射组无坏死和变性；实验组和CBP+Ms注射组残留肿瘤组织内均见少量微球存留。
结论：
1、采用W/O/W型乳化溶剂挥发法，经均匀设计优化微球制备工艺，成功制备了广谱抗癌药卡铂的新剂型——卡铂/乳酸羟基乙酸共聚物微球，其粒径大小符合注射要求，体外缓释效果较好，卡铂释放时间超过10天，可用于瘤内注射动物实验。
2、体内治疗实验研究结果表明该微球可使卡铂持久、有效地作用于荷瘤裸小鼠种植性胰腺癌细胞，造成肿瘤细胞变性、坏死，对小鼠骨髓无明显抑制作用，达到了延长卡铂释放时间、有效降低其毒副作用、提高化疗效果的目的。
3、超声引导下经皮穿刺将卡铂/乳酸羟基乙酸共聚物微球用于瘤内注射治疗胰腺癌，扩大了超声引导肿瘤局部化疗的研究范围，本研究所用方法简便易行、效果肯定，方法可望作为胰腺癌治疗的一条新途径。
4、超声动态观察裸小鼠皮下种植性胰腺癌具有灵敏、方便、准确、实用的优点，可作为主要观察指标。
Objective: First, to prepare carboplatin-loaded PLGA microspheres (CBP-PLGA Ms) for intra-tumoral injection (i.t.). Then the CBP-PLGA Ms would be injected directly in human pancreatic cancer in nude mice guided by ultrasound, and the anti-tumor effects would be evaluated. Our purpose is finding a novel therapy method for pancreatic cancer .
Method:W/O/W emulsion solvent evaporation method and uniform test design were applied to optimize the formulation and procedure in the preparation of CBP-PLGA Ms, then physical characteristics and drug loading (DL) of the microspheres and release time of carboplatin from the Ms in vitro were tested. Also the subcutaneous implanting nude mice model of human pancreatic cancer was established successfully with sw1990 cell strain. Its ultrasound imaging and biological characteristics including the curve of tumor growth were studied by high frequence ultrasound (HFU). Then 20 mice was divided into 4 groups of CBP-PLGA Ms, carboplatin and blank microspheres mixture(CBP+Ms), free CBP and 5% glucose injection(5%GS). Intra-tumoral injections were performed respectively. The changes of tumor growth and ultrasound imaging were observed every 5 days, and blood changes were checked from 3 to 15 days after i.t. And pathology was examinated at the end of 30days.
Result : The optimum factors of preparation of CBP-PLGA Ms were 10% CBP solution 1.5ml, PLGA concentration 2.5% in dichloromethane, stirring speed 1000 r/min, and the ratio of oil in water 1:5. The CBP-PLGA Ms were suitable for i.t. due to the rate of 96.7% of microspheres smaller than 100μm. in particle size.The DL was 10.16%, and release time of CBP from the microspheres was over 10 days. The human pancreatic cancer imaging in nude mice was low-echo and an-theca and clear border node. The CDFI and pathologic examination showed the vasculature was very rich in tumor. The suitable time for observation or injection was from 15 days to 20 days after implanting. The tumor growth double time (DT) of group of CBP-PLGA Ms was -33.9days, the difference from the other groups was significant (p<0.0001). The DT of those groups of CBP+Ms, free CBP, 5%GS were 4.65days, 4.32 days and 2.39 days respectively. The HFU also observed
injection process of CBP-PLGA Ms and its imaging characteristics in tumor, which was hyper-echo at the early stage and weakened bit by bit to low-echo at the end of 30 days. The tumor imaging of experimental group was unequal low-echo or weak-echo node with unclear border. The sign of color flow becomes insufficiency. The amount of WBC, RBC and PLT in the groups of CBP+Ms, free CBP became less after i.t., and lower than the group of CBP-PLGA Ms, p<0.05. The pathologic examinations showed that big coagulation necrosis masses with small colliquative necrosis and cell vacuolation were observed in the tumor in experimental mice, these signs were significantly different from the others. A few microspheres were found in some tumor tissues in the groups of CBP-PLGA Ms and CBP+Ms, which showed that the microspheres could be degradable slowly over 30 days in vivo.
Conclusion: CBP-PLGA Ms can be used to injection in animal experiment，due to its good physical characteristics and degradation. Because of sustaining degradation of microspheres, the CBP can cause tumor to necrosis and vacuolation effectively and continously in vivo. HFU is sensitive, convenience and accurate using for observation on human pancreatic cancer in nude mice. The method, intra-tumoral injection with CBP-PLGA Ms guided by ultrasound, should be a new pathway using for therapy pancreatic cancer.
引文
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