5-氟尿嘧啶磁性固体脂质纳米粒的制备及其药效学研究
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摘要
磁靶向药物能有效减小化疗药物的毒副作用,提高药效,在治疗恶性肿瘤方面前景广阔。本文综述了磁靶向给药系统的研究进展,采用化学共沉淀法制备磁性纳米Fe3O4,复乳-溶剂挥发法制备5-氟尿嘧啶磁性固体脂质纳米粒(5-FU-MSLN),研究了5-FU-MSLN对H22肝癌实体瘤的抑瘤作用,期望5-FU浓集于肿瘤靶组织,定位释放,减少不良反应,提高药物的治疗效果。
1、磁性纳米Fe3O4的制备研究
以FeCl3·6H2O、FeSO4·7H2O、氨水为原料,用化学共沉淀法制备磁性纳米Fe3O4。采用单因素实验,研究了制备条件(pH值、反应温度、铁盐浓度、Fe3+/Fe2+、陈化时间)对磁性纳米Fe3O4粒径、磁响应性、颜色的影响；在此基础上采用正交设计对制备工艺进行了优化。得到较为理想的工艺条件为：反应过程中保持pH值在9～10之间,Fe3+浓度为0.2mol/L, Fe3+/Fe2+为1.25：1,表面活性剂为SDS,机械搅拌速率为1000r/min,反应温度为65℃,机械搅拌后超声分散30min,并在此温度下保温陈化3h。在此条件下制备的磁性纳米Fe304呈黑色,粒径为15.72nm,在强度为1000Gauss的磁场下磁响应时间为48.67s。采用化学分析方法对产物的组成进行了检测,结果表明其中铁和亚铁的比例为1.96：1,略小于2。通过SEM、XRD对制备的磁性纳米Fe304形貌和结构进行表征,磁性纳米Fe3O4呈球形,XRD图谱与Fe3O4标准衍射图谱一致。
2、5-氟尿嘧啶磁性固体脂质纳米粒的制备研究
为提高5-氟尿嘧啶(5-FU)制剂的疗效,降低毒副作用,以单硬脂酸甘油酯为载体,氢化大豆卵磷脂、泊洛沙姆为乳化剂,磁性纳米Fe3O4为磁体,采用复乳-溶剂挥发法制备5-FU-MSLN.通过单因素实验,考察了各因素对粒径、Zeta电位、包封率的影响,初步确定了制备工艺。在单因素实验基础上,以包封率为考察指标,通过正交设计对制备工艺进行优化,得到较佳的制备条件为：单硬脂酸甘油酯含量为4.0%,药脂比为1/10,类脂比为1/10,磁性纳米Fe3O4用量为0.1%,乳化温度为55℃,乳化时间为1h,初乳400W超声分散30s,终乳200W超声分散60s。所制备的5-FU-MSLN粒径为81.49nm,PDI为0.151,Zeta电位为-25.28mV,包封率为58.35%。用透射电镜观察,5-FU-MSLN外观形态圆整,粒径分布均匀,在倒置的显微镜下观察,其体外磁响应性良好。5-FU-MSLN具有一定的稳定性,是有希望的静脉给药靶向制剂。
3、5-FU-MSLN对H22肝癌实体瘤小鼠的抑瘤作用研究
观察5-FU-MSLN对小鼠H22肝癌实体瘤的抑瘤作用。昆明种小鼠H22肝癌实体瘤模型建立后,随机分为5组,每组10只,分别为生理盐水对照组,空白磁性固体脂质纳米粒组,5-FU溶液组,5-FU-MSLN组(不加磁场),5-FU-MSLN组(加磁场)。尾静脉给药,连续7天。给药期间,每天观察各组小鼠生活状态,隔天测量各组小鼠的体重和瘤体积。给药结束后,颈椎脱臼处死各组小鼠,剥取瘤块,称重,计算抑瘤率。瘤块做常规切片进行病理检查,SP免疫组织化学染色法检测肿瘤的VEGF、Ki-67、MVD。5-FU-MSLN组(加磁场)与其他各组相比,肿瘤生长缓慢,瘤重、VEGF、Ki-67、MVD检测结果与各组相比都有显著性差异。结果表明,5-FU-MSLN组(加磁场)对肿瘤生长有显著抑制作用。
用生物相容性好的磁性纳米Fe3O4为磁体制备的5-FU-MSLN工艺稳定,满足用药需求,荷瘤鼠体内的药物在外磁场的作用下能在肿瘤区定位释放,提高了疗效,有希望成为5-FU的新剂型。
Magnetic targeted drugs can effectively reduce the toxic effect of chemotherapy drugs to improve efficacy, which have demonstrated a very bright prospect in the treatment of malignant tumors. In this paper, the research and development of magnetic targeted drugs delivery system was reviewed; the preparation of Fe3O4 magnetic nanoparticles and 5-fluorouracil magnetic solid lipid nanoparticles was introduced and the inhibitory effect of 5-fluorouracil magnetic solid lipid nanoparticles on H22 liver cancer solid tumor was studied.5-fluorouracil was expected to concentrate in tumor target tissue, release positioning and reduce side effect.
1、The preparation of Fe3O4 magnetic nanoparticles
Chemical co-precipitation method was adopted to prepare Fe3O4 magnetic nanoparticles with FeCl3·6H2O, FeSO4·7H2O, ammonia as the raw materials. Single factor tests were designed to study the influence of preparation conditions (pH, reaction temperature, concentration of iron salt, Fe3+/Fe2+, aging time) on the size, magnetic response and colour of Fe3O4 magnetic nanoparticles. On this basis, an orthogonal test was used to optimize the preparation. The results indicated that the more ideal technical process was as follows:the mechanical stirring rate was 1000r/min for 30 min at 65℃maintaining pH value in the range of 9～10, the concentration of Fe3+ was 0.2mol/L, Fe3+/Fe2+ was 1.25:1, with SDS as surfactant. After 30min mechanical stirring and ultrasonic dispersion, the aging time was 3h at 65℃. Under these conditions, the magnetic Fe3O4 nanoparticle was black, particle size was 15.72nm and response time in the strength of the magnetic field for the 1000Gauss was 48.67s. The composition of the products was tested by chemical analysis to find that the proportion of Fe3+/Fe2+ was 1.96:1, which was slightly less than 2. The structure and particle size of Fe3O4 was characterized by means of XRD and SEM. The results showed that the Fe3O4 magnetic nanoparticles were spherical, whose XRD pattern was consistent with Fe3O4 standard diffraction pattern.
2、The preparation of 5-fluorouracil magnetic solid lipid nanoparticles
To improve the therapeutic efficacy and reduce the toxicity of 5-fluorouracil (5-FU),5-fluorouracil magnetic solid lipid nanoparticles (5-FU-MSLN) were prepared by a W/O/W double emulsion solvent evaporation technique, using monostearin as the carrier, hydrogenated soybean lecithin and poloxamer as emulsifier, magnetic Fe3O4 nanoparticles as magnets. Single factor tests were designed to study the influence of preparation on the size, Zeta potential, encapsulation efficiency of 5-FU-MSLN to set the initial preparation. Base on the single factor tests, with the index of encapsulation efficiency, the preparation process was optimized by orthogonal test. The best preparation condition was:the monostearin content was 4.0%, the ratio of drug to lipid ratio was 1/10, the hydrogenated soybean lecithin to monostearin was 1/10, the Fe3O4 magnetic nanoparticles dosage was 0.1%, emulsifying temperature was 55℃, emulsifying time was 1h, the ultrasound power of foremilk was 400W last 30s, the ultrasound power of final milk was 200W last 60s. The result showed that the average diameter in size, PDI, Zeta potential, drug encapsulation efficiency of 5-FU-MSLN were 81.49nm,0.151,-25.28mV,58.35%. TEM presented 5-FU-MSLN as spherical particles, evenly distributed; 5-FU-MSLN were observed under the inverted microscope, whose vitro magnetic responsiveness was good. Such magnetic solid lipid nanoparticles seem appropriate for vascular administration followed by drug targeting.
3、The inhibitory effect of 5-FU-MSLN on H22 liver cancer tumor-bearing mice
The aim was to evaluate the inhibitory effect of 5-FU-MSLN on the growth of H22 liver cancer solid tumor in mice. Fifty Kunming mice with H22 liver cancer solid tumor were divided into 5 groups evenly at random, namely, saline control group、blank magnetic solid lipid nanoparticles group、5-FU solution group、5-FU-MSLN group(without magnetic field) and 5-FU-MSLN group(plus magnetic field), which were received tail vein injection for seven days. The living condition of mice was observed everyday and the body weight and tumor volume were measured every other day. After the administration, the mice were killed in each group through cervical dislocation and the tour was stripped, weighed to calculate inhibition rate. The pathology of tumor was examined through conventional biopsy and the VEGF, Ki-67, MVD of tour were detected by SP immunohistochemical staining. 5-FU-MSLN group (plus magnetic field) was compared with other groups:the tumors grew slowly, tumor weight compared with the significant difference, VEGF, Ki-67, MVD detection and each group has a significantly compared to differences. The results show that 5-FU-MSLN (plus magnetic field) can significantly inhibit tumor growth.
5-FU-MSLN was prepared with Fe3O4 magnetic nanoparticles which had good biocompatibility for the magnets, whose process was stable, meeting the needs for drugs. The drug in tumor-bearing mice under the influence of external magnetic field can locate and release, whose efficacy was improved. The results showed that 5-FU-MSLN was prospective for a new formulation.
1、磁性纳米Fe3O4的制备研究
以FeCl3·6H2O、FeSO4·7H2O、氨水为原料,用化学共沉淀法制备磁性纳米Fe3O4。采用单因素实验,研究了制备条件(pH值、反应温度、铁盐浓度、Fe3+/Fe2+、陈化时间)对磁性纳米Fe3O4粒径、磁响应性、颜色的影响；在此基础上采用正交设计对制备工艺进行了优化。得到较为理想的工艺条件为：反应过程中保持pH值在9～10之间,Fe3+浓度为0.2mol/L, Fe3+/Fe2+为1.25：1,表面活性剂为SDS,机械搅拌速率为1000r/min,反应温度为65℃,机械搅拌后超声分散30min,并在此温度下保温陈化3h。在此条件下制备的磁性纳米Fe304呈黑色,粒径为15.72nm,在强度为1000Gauss的磁场下磁响应时间为48.67s。采用化学分析方法对产物的组成进行了检测,结果表明其中铁和亚铁的比例为1.96：1,略小于2。通过SEM、XRD对制备的磁性纳米Fe304形貌和结构进行表征,磁性纳米Fe3O4呈球形,XRD图谱与Fe3O4标准衍射图谱一致。
2、5-氟尿嘧啶磁性固体脂质纳米粒的制备研究
为提高5-氟尿嘧啶(5-FU)制剂的疗效,降低毒副作用,以单硬脂酸甘油酯为载体,氢化大豆卵磷脂、泊洛沙姆为乳化剂,磁性纳米Fe3O4为磁体,采用复乳-溶剂挥发法制备5-FU-MSLN.通过单因素实验,考察了各因素对粒径、Zeta电位、包封率的影响,初步确定了制备工艺。在单因素实验基础上,以包封率为考察指标,通过正交设计对制备工艺进行优化,得到较佳的制备条件为：单硬脂酸甘油酯含量为4.0%,药脂比为1/10,类脂比为1/10,磁性纳米Fe3O4用量为0.1%,乳化温度为55℃,乳化时间为1h,初乳400W超声分散30s,终乳200W超声分散60s。所制备的5-FU-MSLN粒径为81.49nm,PDI为0.151,Zeta电位为-25.28mV,包封率为58.35%。用透射电镜观察,5-FU-MSLN外观形态圆整,粒径分布均匀,在倒置的显微镜下观察,其体外磁响应性良好。5-FU-MSLN具有一定的稳定性,是有希望的静脉给药靶向制剂。
3、5-FU-MSLN对H22肝癌实体瘤小鼠的抑瘤作用研究
观察5-FU-MSLN对小鼠H22肝癌实体瘤的抑瘤作用。昆明种小鼠H22肝癌实体瘤模型建立后,随机分为5组,每组10只,分别为生理盐水对照组,空白磁性固体脂质纳米粒组,5-FU溶液组,5-FU-MSLN组(不加磁场),5-FU-MSLN组(加磁场)。尾静脉给药,连续7天。给药期间,每天观察各组小鼠生活状态,隔天测量各组小鼠的体重和瘤体积。给药结束后,颈椎脱臼处死各组小鼠,剥取瘤块,称重,计算抑瘤率。瘤块做常规切片进行病理检查,SP免疫组织化学染色法检测肿瘤的VEGF、Ki-67、MVD。5-FU-MSLN组(加磁场)与其他各组相比,肿瘤生长缓慢,瘤重、VEGF、Ki-67、MVD检测结果与各组相比都有显著性差异。结果表明,5-FU-MSLN组(加磁场)对肿瘤生长有显著抑制作用。
用生物相容性好的磁性纳米Fe3O4为磁体制备的5-FU-MSLN工艺稳定,满足用药需求,荷瘤鼠体内的药物在外磁场的作用下能在肿瘤区定位释放,提高了疗效,有希望成为5-FU的新剂型。
Magnetic targeted drugs can effectively reduce the toxic effect of chemotherapy drugs to improve efficacy, which have demonstrated a very bright prospect in the treatment of malignant tumors. In this paper, the research and development of magnetic targeted drugs delivery system was reviewed; the preparation of Fe3O4 magnetic nanoparticles and 5-fluorouracil magnetic solid lipid nanoparticles was introduced and the inhibitory effect of 5-fluorouracil magnetic solid lipid nanoparticles on H22 liver cancer solid tumor was studied.5-fluorouracil was expected to concentrate in tumor target tissue, release positioning and reduce side effect.
1、The preparation of Fe3O4 magnetic nanoparticles
Chemical co-precipitation method was adopted to prepare Fe3O4 magnetic nanoparticles with FeCl3·6H2O, FeSO4·7H2O, ammonia as the raw materials. Single factor tests were designed to study the influence of preparation conditions (pH, reaction temperature, concentration of iron salt, Fe3+/Fe2+, aging time) on the size, magnetic response and colour of Fe3O4 magnetic nanoparticles. On this basis, an orthogonal test was used to optimize the preparation. The results indicated that the more ideal technical process was as follows:the mechanical stirring rate was 1000r/min for 30 min at 65℃maintaining pH value in the range of 9～10, the concentration of Fe3+ was 0.2mol/L, Fe3+/Fe2+ was 1.25:1, with SDS as surfactant. After 30min mechanical stirring and ultrasonic dispersion, the aging time was 3h at 65℃. Under these conditions, the magnetic Fe3O4 nanoparticle was black, particle size was 15.72nm and response time in the strength of the magnetic field for the 1000Gauss was 48.67s. The composition of the products was tested by chemical analysis to find that the proportion of Fe3+/Fe2+ was 1.96:1, which was slightly less than 2. The structure and particle size of Fe3O4 was characterized by means of XRD and SEM. The results showed that the Fe3O4 magnetic nanoparticles were spherical, whose XRD pattern was consistent with Fe3O4 standard diffraction pattern.
2、The preparation of 5-fluorouracil magnetic solid lipid nanoparticles
To improve the therapeutic efficacy and reduce the toxicity of 5-fluorouracil (5-FU),5-fluorouracil magnetic solid lipid nanoparticles (5-FU-MSLN) were prepared by a W/O/W double emulsion solvent evaporation technique, using monostearin as the carrier, hydrogenated soybean lecithin and poloxamer as emulsifier, magnetic Fe3O4 nanoparticles as magnets. Single factor tests were designed to study the influence of preparation on the size, Zeta potential, encapsulation efficiency of 5-FU-MSLN to set the initial preparation. Base on the single factor tests, with the index of encapsulation efficiency, the preparation process was optimized by orthogonal test. The best preparation condition was:the monostearin content was 4.0%, the ratio of drug to lipid ratio was 1/10, the hydrogenated soybean lecithin to monostearin was 1/10, the Fe3O4 magnetic nanoparticles dosage was 0.1%, emulsifying temperature was 55℃, emulsifying time was 1h, the ultrasound power of foremilk was 400W last 30s, the ultrasound power of final milk was 200W last 60s. The result showed that the average diameter in size, PDI, Zeta potential, drug encapsulation efficiency of 5-FU-MSLN were 81.49nm,0.151,-25.28mV,58.35%. TEM presented 5-FU-MSLN as spherical particles, evenly distributed; 5-FU-MSLN were observed under the inverted microscope, whose vitro magnetic responsiveness was good. Such magnetic solid lipid nanoparticles seem appropriate for vascular administration followed by drug targeting.
3、The inhibitory effect of 5-FU-MSLN on H22 liver cancer tumor-bearing mice
The aim was to evaluate the inhibitory effect of 5-FU-MSLN on the growth of H22 liver cancer solid tumor in mice. Fifty Kunming mice with H22 liver cancer solid tumor were divided into 5 groups evenly at random, namely, saline control group、blank magnetic solid lipid nanoparticles group、5-FU solution group、5-FU-MSLN group(without magnetic field) and 5-FU-MSLN group(plus magnetic field), which were received tail vein injection for seven days. The living condition of mice was observed everyday and the body weight and tumor volume were measured every other day. After the administration, the mice were killed in each group through cervical dislocation and the tour was stripped, weighed to calculate inhibition rate. The pathology of tumor was examined through conventional biopsy and the VEGF, Ki-67, MVD of tour were detected by SP immunohistochemical staining. 5-FU-MSLN group (plus magnetic field) was compared with other groups:the tumors grew slowly, tumor weight compared with the significant difference, VEGF, Ki-67, MVD detection and each group has a significantly compared to differences. The results show that 5-FU-MSLN (plus magnetic field) can significantly inhibit tumor growth.
5-FU-MSLN was prepared with Fe3O4 magnetic nanoparticles which had good biocompatibility for the magnets, whose process was stable, meeting the needs for drugs. The drug in tumor-bearing mice under the influence of external magnetic field can locate and release, whose efficacy was improved. The results showed that 5-FU-MSLN was prospective for a new formulation.
引文
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