新型栓塞剂温敏纳米凝胶的应用基础研究
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摘要
第一部分PIB纳米凝胶温敏相变特性及其生物相容性的实验研究
目的：研究聚(N-异丙基丙烯酰胺-co-甲基丙烯酸丁酯)(PIB)纳米凝胶的温敏相转变特性及其生物相容性。
材料和方法：采用目视法结合瓶倒转法和体外推注试验来观察PIB纳米凝胶的温敏溶胶-凝胶相转变特性。体外培养L929细胞,以不同浓度的PIB纳米凝胶浸提液培养细胞3天后,采用CCK-8比色试验法检测PIB纳米凝胶浸提液对L929细胞活性的影响。体外培养L929细胞,以不同浓度的PIB纳米凝胶培养细胞2天,采用钙黄绿素-AM/碘化丙啶荧光双染活/死细胞后,激光共聚焦显微镜下观察、细胞计数的方法评价PIB纳米凝胶对L929细胞存活率的影响。日本大耳白兔15只,在其背部一侧肌肉注射PIB纳米凝胶,另一侧注射生理盐水,观察术后1、2、4、12、26周材料周围的组织反应情况。
结果：PIB纳米凝胶在36.5℃时发生溶胶-凝胶相转变。它对L929细胞具有良好的细胞相容性且无明显细胞毒性存在。兔背部植入部位肌肉组织无异常病变,没有严重的炎症反应,组织病理学检查结果符合材料植入后局部反应的国家标准。
结论：PIB纳米凝胶具有良好的生物相容性和温度敏感的相转变特性。
第二部分PIB纳米凝胶栓塞兔肾动脉的实验研究
目的：我们研发了一种新型液体栓塞剂,聚(N-异丙基丙烯酰胺-co-甲基丙烯酸丁酯)(PIB)温敏纳米凝胶,它可以在体温下通过溶胶-凝胶相变转变而固化。我们进一步用兔肾动脉栓塞模型,研究它在血管内的分布模式、炎症反应及栓塞血管的持久性。
材料和方法：实验1中,PIB纳米凝胶栓塞18只实验兔的右肾动脉,按不同的注射速度均分为3组。栓塞后1小时处死动物,取出肾脏,接触X线和组织学观察评价PIB纳米凝胶在肾动脉内的分布模式。实验2中,PIB纳米凝胶以合适的注射速度注射,栓塞20只实验兔的右肾动脉,按栓塞后不同的处死时间点(1周、1月、2月、3月)均分成4组,每组5只。处死前复查血管造影评价血管再通,处死后病理学评价栓塞的有效性和对PIB纳米凝胶的炎症反应。
结果：所有实验兔均能成功栓塞。在实验1中,注射速度越高,PIB纳米凝胶越能到达更小的动脉分支。以0.10ml/s的速度注射,可以使PIB纳米凝胶均匀地分布在段动脉到肾小球毛细血管前水平。在实验2中,后续的血管造影复查未发现血管再通。没有发现血管壁的破裂和血管内膜下出血,没有发现栓塞剂的外溢和新生血管形成。组织学上只有轻微的炎症反应,表现为少数淋巴细胞和单核细胞浸润,无异物肉芽肿形成。
结论：PIB纳米凝胶栓塞肾动脉容易而可控,可以产生均匀和持久的栓塞效果,不伴有严重的炎症反应。因此,PIB纳米凝胶是一种较理想的栓塞材料。
第三部分PIB纳米凝胶栓塞免VX2移植性肝癌模型的实验研究
目的：观察聚(M异丙基丙烯酰胺-co-甲基丙烯酸丁酯)(PIB)温敏纳米凝胶经肝动脉栓塞对兔肝功能及VX2肝癌生长的影响。
材料和方法：MR已证实的30只兔VX2肝癌模型随机分为3组,每组10只,经肝动脉分别给予生理盐水(A组)、超液态碘油+明胶海绵粉(B组)、PIB纳米凝胶(C组)。治疗前1天及治疗后3、7天检测血清ALT和AST水平。治疗后7天处死动物,取肝肿瘤组织进行病理学观察,计算肿瘤坏死率。处死前MR扫描,计算肿瘤体积、生长率。
结果：治疗前1天的肿瘤体积,治疗前1天及治疗后7天的ALT、AST水平,3组之间互相对比无统计学差异(P>0.05)。治疗后3天血清ALT和AST水平,C组低于B组、大于A组,组间对比均有统计学差异(P<0.05)。治疗后7天A组肿瘤生长率为(392.79±40.39)%,B组为(157.47±26.25)%,C组为(111.85±20.16)%,组间对比均有统计学差异(P<0.05)。
结论：PIB纳米凝胶肝动脉栓塞对兔VX2肝癌生长有较明显的抑制作用,而对其肝功能影响较小。
PART I:An experimental study on the thermosensitive phase transition characteristics and biocompatibility of PIB nanogel
Purpose:To study the thermosensitive phase transition characteristics and biocompatibility of poly(N-isopropylacrylamide-co-butyl methylacrylate)(PIB) nanogel.
Materials and methods:The thermosensitive sol-gel phase transitions characteristics of PIB nanogel was measured by the inverting-vial method and in vitro injection test. L929cells were in vitro cultured. The Cells were treated with different concentrations of aqueous extract of PIB nanogel for3days. Cell viability was determined by Cell Counting-8assay. The Cells were also treated with different concentrations of PIB nanogel aqueous for2days. Calcein-AM and propidium iodide Double Staining Kit was utilized for simultaneous fluorescence staining of viable and dead cells. Cell survival rate was determined by cell count method, which was observed under laser confocal microscope. PIB nanogel was injected in one side of the back muscle of15rabbits, and normal saline was injected in another side. Pathological examination of the operative muscle was performed at1,2,4,12and26weeks after operation to evaluate the histocompatibility of PIB nanogel.
Results:PIB nanogel underwent a sol-gel transition at its lower critical solution temperature of36.5℃. PIB nanogels have good cytocompatibility to L929cells, demonstrating no obvious cytotoxicity of the material. There was no severe inflammatory response and no evidence of abnormal pathological changes was found in muscular tissue. The postoperative histopathologic changes were within the authoritative ranges according to the national standards for local effects after implanation.
Conclusions:PIB nanogel has good biocompatibility and showed satisfactory thermosensitive sol-gel phase transitions character.
PART II:An experimental study on the embolization of rabbit renal arteries with PIB nanogel
Purpose:We have developed a new thermosensitive liquid embolic agent, poly(N-isopropylacrylamide-co-butyl methylacrylate)(PIB) nanogel that can be solidfied by a sol-gel phase transition at body temperature. We thus further investigated the distribution, durability of vascular occlusion, and inflammatory reactions of PIB in embolization of the renal artery of rabbits.
Materials and methods:In experiment1,18rabbits were evenly divided into3groups according to the injection rate. The right renal arteries were embolized by PIB with various injection rates. The animals were sacrificed at1hour after embolization. To evaluate the distribution pattern of PIB, the kidneys were examined by contact radiography and tissue section microscopy. In experiment2. we embolized the right renal artery of20rabbits with PIB with the injection rate of0.10ml/s. Angiography was performed before sacrifice1week (n=5),1month (n=5),2months and3months (n=5) after embolization. Pathologic and histologic examination of the kidneys was performed to assess the embolization efficacy and the specific inflammatory response of PIB.
Results:The procedure was performed successfully in all rabbits. In experiment1. the higher injection rate, the smaller arteries PIB could reach, and the rate of0.10ml/s produced a homogeneous distribution of PIB from segmental arteries to the precapillary level. In experiment2, no angiographic recanalization appeared during follow-up. We found no disruption of the vessel wall and subintimal bleeding, no extravasation of PIB and no evidence of neocapillarization. Histologically, there was only mild inflammatory response manifested by few lymphocytic and monocellular infiltration, without foreign body granuloma formation.
Conclusions:Embolization renal artery with PIB was easy and controllable, which could lead to a homogeneous and persistent occlusion without severe inflammatory changes. PIB might be a suitable material for intravascular embolization.
PART Ⅲ:An experimental study on the embolization of rabbit VX2liver tumors with PIB nanogel
Purpose:To observe the effect of transcatheter arterial embolization using thermosensitive poly(N-isopropylacrylamide-co-butyl methacrylate)(PIB) nanogel on hepatic function and growth of hepatic tumor of rabbits bearing VX2tumor.
Materials and methods:Thirty rabbits implanted liver VX2tumors diagnosed by MR were randomly divided into3groups of10each. A microcatheter was inserted into the hepatic artery and injection was performed via the hepatic artery using physiological saline (group A), lipiodol and gelfoam (group B), and PIB nanogel (group C), respectively. The level of AST and ALT in serum was respectively tested at1day before treatment,3and7days after treatment. All experimental rabbits were sacrificed7days after treatment. Hepatic tumor tissue was obtained for pathological examination to calculate necrotic rate of tumors. The volume and growth rate of tumors were calculated by the examination of MR before sacrifice.
Results:There was no significant difference in the volume of tumors at1day before treatment, the level of AST and ALT at1day before treatment and7days after treatment, among3groups (P>0.05). There was significant difference in the level of AST and ALT between each group3days after treatment (P<0.05). The level of AST and ALT in group C was lower than that in group B and higher than that in group A. There was significant difference in the growth rate of tumor between each group7days after treatment (P<0.05). The growth rate of tumor was (392.79±40.39)%in group A,(157.47±26.25)%in group B, and (111.85±20.16)%in group C.
Conclusions:Transcatheter arterial embolization using PIB nanogel can inhibit the growth of the implanted VX2tumor, and there is no obvious liver toxicity related it.
目的：研究聚(N-异丙基丙烯酰胺-co-甲基丙烯酸丁酯)(PIB)纳米凝胶的温敏相转变特性及其生物相容性。
材料和方法：采用目视法结合瓶倒转法和体外推注试验来观察PIB纳米凝胶的温敏溶胶-凝胶相转变特性。体外培养L929细胞,以不同浓度的PIB纳米凝胶浸提液培养细胞3天后,采用CCK-8比色试验法检测PIB纳米凝胶浸提液对L929细胞活性的影响。体外培养L929细胞,以不同浓度的PIB纳米凝胶培养细胞2天,采用钙黄绿素-AM/碘化丙啶荧光双染活/死细胞后,激光共聚焦显微镜下观察、细胞计数的方法评价PIB纳米凝胶对L929细胞存活率的影响。日本大耳白兔15只,在其背部一侧肌肉注射PIB纳米凝胶,另一侧注射生理盐水,观察术后1、2、4、12、26周材料周围的组织反应情况。
结果：PIB纳米凝胶在36.5℃时发生溶胶-凝胶相转变。它对L929细胞具有良好的细胞相容性且无明显细胞毒性存在。兔背部植入部位肌肉组织无异常病变,没有严重的炎症反应,组织病理学检查结果符合材料植入后局部反应的国家标准。
结论：PIB纳米凝胶具有良好的生物相容性和温度敏感的相转变特性。
第二部分PIB纳米凝胶栓塞兔肾动脉的实验研究
目的：我们研发了一种新型液体栓塞剂,聚(N-异丙基丙烯酰胺-co-甲基丙烯酸丁酯)(PIB)温敏纳米凝胶,它可以在体温下通过溶胶-凝胶相变转变而固化。我们进一步用兔肾动脉栓塞模型,研究它在血管内的分布模式、炎症反应及栓塞血管的持久性。
材料和方法：实验1中,PIB纳米凝胶栓塞18只实验兔的右肾动脉,按不同的注射速度均分为3组。栓塞后1小时处死动物,取出肾脏,接触X线和组织学观察评价PIB纳米凝胶在肾动脉内的分布模式。实验2中,PIB纳米凝胶以合适的注射速度注射,栓塞20只实验兔的右肾动脉,按栓塞后不同的处死时间点(1周、1月、2月、3月)均分成4组,每组5只。处死前复查血管造影评价血管再通,处死后病理学评价栓塞的有效性和对PIB纳米凝胶的炎症反应。
结果：所有实验兔均能成功栓塞。在实验1中,注射速度越高,PIB纳米凝胶越能到达更小的动脉分支。以0.10ml/s的速度注射,可以使PIB纳米凝胶均匀地分布在段动脉到肾小球毛细血管前水平。在实验2中,后续的血管造影复查未发现血管再通。没有发现血管壁的破裂和血管内膜下出血,没有发现栓塞剂的外溢和新生血管形成。组织学上只有轻微的炎症反应,表现为少数淋巴细胞和单核细胞浸润,无异物肉芽肿形成。
结论：PIB纳米凝胶栓塞肾动脉容易而可控,可以产生均匀和持久的栓塞效果,不伴有严重的炎症反应。因此,PIB纳米凝胶是一种较理想的栓塞材料。
第三部分PIB纳米凝胶栓塞免VX2移植性肝癌模型的实验研究
目的：观察聚(M异丙基丙烯酰胺-co-甲基丙烯酸丁酯)(PIB)温敏纳米凝胶经肝动脉栓塞对兔肝功能及VX2肝癌生长的影响。
材料和方法：MR已证实的30只兔VX2肝癌模型随机分为3组,每组10只,经肝动脉分别给予生理盐水(A组)、超液态碘油+明胶海绵粉(B组)、PIB纳米凝胶(C组)。治疗前1天及治疗后3、7天检测血清ALT和AST水平。治疗后7天处死动物,取肝肿瘤组织进行病理学观察,计算肿瘤坏死率。处死前MR扫描,计算肿瘤体积、生长率。
结果：治疗前1天的肿瘤体积,治疗前1天及治疗后7天的ALT、AST水平,3组之间互相对比无统计学差异(P>0.05)。治疗后3天血清ALT和AST水平,C组低于B组、大于A组,组间对比均有统计学差异(P<0.05)。治疗后7天A组肿瘤生长率为(392.79±40.39)%,B组为(157.47±26.25)%,C组为(111.85±20.16)%,组间对比均有统计学差异(P<0.05)。
结论：PIB纳米凝胶肝动脉栓塞对兔VX2肝癌生长有较明显的抑制作用,而对其肝功能影响较小。
PART I:An experimental study on the thermosensitive phase transition characteristics and biocompatibility of PIB nanogel
Purpose:To study the thermosensitive phase transition characteristics and biocompatibility of poly(N-isopropylacrylamide-co-butyl methylacrylate)(PIB) nanogel.
Materials and methods:The thermosensitive sol-gel phase transitions characteristics of PIB nanogel was measured by the inverting-vial method and in vitro injection test. L929cells were in vitro cultured. The Cells were treated with different concentrations of aqueous extract of PIB nanogel for3days. Cell viability was determined by Cell Counting-8assay. The Cells were also treated with different concentrations of PIB nanogel aqueous for2days. Calcein-AM and propidium iodide Double Staining Kit was utilized for simultaneous fluorescence staining of viable and dead cells. Cell survival rate was determined by cell count method, which was observed under laser confocal microscope. PIB nanogel was injected in one side of the back muscle of15rabbits, and normal saline was injected in another side. Pathological examination of the operative muscle was performed at1,2,4,12and26weeks after operation to evaluate the histocompatibility of PIB nanogel.
Results:PIB nanogel underwent a sol-gel transition at its lower critical solution temperature of36.5℃. PIB nanogels have good cytocompatibility to L929cells, demonstrating no obvious cytotoxicity of the material. There was no severe inflammatory response and no evidence of abnormal pathological changes was found in muscular tissue. The postoperative histopathologic changes were within the authoritative ranges according to the national standards for local effects after implanation.
Conclusions:PIB nanogel has good biocompatibility and showed satisfactory thermosensitive sol-gel phase transitions character.
PART II:An experimental study on the embolization of rabbit renal arteries with PIB nanogel
Purpose:We have developed a new thermosensitive liquid embolic agent, poly(N-isopropylacrylamide-co-butyl methylacrylate)(PIB) nanogel that can be solidfied by a sol-gel phase transition at body temperature. We thus further investigated the distribution, durability of vascular occlusion, and inflammatory reactions of PIB in embolization of the renal artery of rabbits.
Materials and methods:In experiment1,18rabbits were evenly divided into3groups according to the injection rate. The right renal arteries were embolized by PIB with various injection rates. The animals were sacrificed at1hour after embolization. To evaluate the distribution pattern of PIB, the kidneys were examined by contact radiography and tissue section microscopy. In experiment2. we embolized the right renal artery of20rabbits with PIB with the injection rate of0.10ml/s. Angiography was performed before sacrifice1week (n=5),1month (n=5),2months and3months (n=5) after embolization. Pathologic and histologic examination of the kidneys was performed to assess the embolization efficacy and the specific inflammatory response of PIB.
Results:The procedure was performed successfully in all rabbits. In experiment1. the higher injection rate, the smaller arteries PIB could reach, and the rate of0.10ml/s produced a homogeneous distribution of PIB from segmental arteries to the precapillary level. In experiment2, no angiographic recanalization appeared during follow-up. We found no disruption of the vessel wall and subintimal bleeding, no extravasation of PIB and no evidence of neocapillarization. Histologically, there was only mild inflammatory response manifested by few lymphocytic and monocellular infiltration, without foreign body granuloma formation.
Conclusions:Embolization renal artery with PIB was easy and controllable, which could lead to a homogeneous and persistent occlusion without severe inflammatory changes. PIB might be a suitable material for intravascular embolization.
PART Ⅲ:An experimental study on the embolization of rabbit VX2liver tumors with PIB nanogel
Purpose:To observe the effect of transcatheter arterial embolization using thermosensitive poly(N-isopropylacrylamide-co-butyl methacrylate)(PIB) nanogel on hepatic function and growth of hepatic tumor of rabbits bearing VX2tumor.
Materials and methods:Thirty rabbits implanted liver VX2tumors diagnosed by MR were randomly divided into3groups of10each. A microcatheter was inserted into the hepatic artery and injection was performed via the hepatic artery using physiological saline (group A), lipiodol and gelfoam (group B), and PIB nanogel (group C), respectively. The level of AST and ALT in serum was respectively tested at1day before treatment,3and7days after treatment. All experimental rabbits were sacrificed7days after treatment. Hepatic tumor tissue was obtained for pathological examination to calculate necrotic rate of tumors. The volume and growth rate of tumors were calculated by the examination of MR before sacrifice.
Results:There was no significant difference in the volume of tumors at1day before treatment, the level of AST and ALT at1day before treatment and7days after treatment, among3groups (P>0.05). There was significant difference in the level of AST and ALT between each group3days after treatment (P<0.05). The level of AST and ALT in group C was lower than that in group B and higher than that in group A. There was significant difference in the growth rate of tumor between each group7days after treatment (P<0.05). The growth rate of tumor was (392.79±40.39)%in group A,(157.47±26.25)%in group B, and (111.85±20.16)%in group C.
Conclusions:Transcatheter arterial embolization using PIB nanogel can inhibit the growth of the implanted VX2tumor, and there is no obvious liver toxicity related it.
引文
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