羟基喜树碱半固体脂质纳米粒的制备和体外药剂学性质研究
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摘要
研究背景和目的
随着上世纪70年代，Birrenbach等首次提出纳米粒的概念和制备方法，药物制剂的研究进入了一个全新的微观领域——纳米制剂。与常规药物相比，纳米药物具有比表面积大、表面反应活性高、活性中心多、吸附能力强等特性，还可以改变药物在体内的分布，增加药物在靶器官的分布量，从而提高疗效。固体脂质纳米粒(Solid lipid nanoparticles，SLN)是以毒性低、生物相容性好的脂质材料作为载体，将药物吸附或包裹于脂质膜中制成的新一代纳米给药体系。除了减轻毒副作用，固体脂质又使它具有聚合物纳米粒的优点，如可以控制药物的释放，避免药物的降解或泄漏以及具有良好的靶向性等。
羟基喜树碱(10-hydroxycamptothecin，HCPT)是从珙桐科植物喜树中提取的一种微量生物碱，是拓扑异构酶Ⅰ特异性抑制剂，广泛应用于肝癌、胃癌、白血病等多种恶性肿瘤的治疗。但其特殊的理化性质：水不溶脂难溶、内酯环结构不稳定等因素，限制了临床应用，普遍存在疗效降低、半衰期短的缺点。本研究以HCPT为模型药物制备半固体脂质纳米粒(Semisolid lipid nanoparticles，SSLN)并对其体外药剂学性质进行了研究，以期达到提高HCPT的稳定性，延长其生物半衰期，缓释，提高药效，降低毒性的目的。
主要内容
以固态和液态脂质为载体，大豆卵磷脂为表面活性剂，采用乳化蒸发—低温固化法制备了HCPT半固体脂质纳米粒。以纳米粒外观、粒径、包封率为指标，对处方和工艺进行了单因素考察，内容包括有机相种类、固态脂质／液态脂质摩尔比、药脂比、表面活性剂浓度以及乳化蒸发搅拌时间、搅拌速度等等，继而通过正交设计，确定了最优处方和制备工艺。为了提高制剂的稳定性，采用冷冻干燥法制成HCPT-SSLN冻干粉剂。
采用Sephadex G-50凝胶过滤法，先用pH5.0PBS洗脱下HCPT-SSLN，再用pH12.0的NaOH溶解并洗脱下吸附在柱顶端的游离药物，从而分离HCPT-SSLN和游离药物。利用NaOH破坏脂质膜结构并溶解包封在其中的HCPT，氯仿消除磷脂等脂质膜材的干扰后，依据建立的羧酸盐形式存在的HCPT紫外分光光度法测定HCPT的含量。
考察了HCPT-SSLN混悬液的理化性质，电镜下纳米粒为圆整的球状体，大小比较均匀，边缘光滑，平均粒径130.5nm，Zeta电位-33.1mV，pH6.29，包封率79.19；冻干品的粒径稍有增大，电位和pH几乎不变，包封率下降了约5％，载药量为2.51％。差示扫描量热分析(DSC)和X射线衍射分析(XRD)皆表明HCPT被脂质包裹后已经构成了一种新的物相。
以外观、粒径、pH、包封率、水中再分散性及显微镜检等为指标，考察了HCPT-SSLN混悬液和冻干品在影响因素实验、留样观察中的稳定性。结果表明：HCPT-SSLN最宜制成冻干粉剂，充氮密封避光置于4℃条件下保存。
选择含有30％甲醇的pH7.4磷酸盐缓冲液做释药介质，在(37±0.5)℃避光的条件下对HCPT原料药和自制的HCPT-SSLN进行了0～72h体外释药的研究。以内酯形式存在的HCPT的HPLC含量测定结果表明：HCPT-SSLN0～3h内有一定程度的突释，72h累积释药量不超过70％，对照药物72h只有24.7％，可见HCPT-SSLN增强了HCPT在体内的吸收，且显示出一定的缓释效应。
以HCPT钠盐注射液作对比，对HCPT-SSLN进行了小鼠的急性毒性实验。HCPT钠盐注射液组的半数致死量是93.47mg／kg，HCPT-SSLN的最大耐受剂量(MTD)大于214.6mg／kg，HCPT-SSLN毒性降低。
结论
本实验为HCPT的剂型开发作了有益的探索，为它的临床应用提供了一种可能的新剂型，有较好的研究开发价值。
Background & objectiveSince 1970s, Birrenbach introduced the concept and preparative method of nanoparticle firstly, the study of the pharmaceutical preparation has entered into anew micro realm——nanometric praeparatum. Compared to routine drugs,nanometric praeparatums have many good characters such as large specific surface area,high surface reaction activity, plenty of active centers, strong adsorbability and so on. They can also change the intracorporal distribution of the medicine to increase the dose distribution in target organs and raise therapeutic effects. Solid lipid nanoparticles(SLN) is a latest generation of nanometric drug delivery system. It uses lipid materials with low toxicity and fine biocompatibility as carrier, then absorbs the drug and wraps it up into the lipid membrane. Besides descreasing poison and side effects, solid lipid make it control drug release, just like PVP, so it avoids the degradation or leakage of the drug and has favourable target characteristics.
10-hydroxycamptothecin(HCPT) is a kind of microamount alkaloids extracted from Camptotheca acuminata Decaisne, and is one of the specific DNA topoisomeraseⅠinhibitors. It is widely used to cure fiver cancer, stomach cancer, leukemia and many malignant tumors. However, its clinical applications are confined by its special physico-chemical properties(for example, it is insolubilize in water and difficultly dissolves in fipids, the lactone band is also instable), so its curative effects decrease and the intracorporal half life is too short. This study chose it as model drug to prepare semisolid lipid nanoparticles(HCPT-SSLN) and investigated its pharmaceutical characteristics in vitro, intending to enhance its stability and prolong biological half-fife, make it release slowly in vivo to reinforce drug action and lower the toxicity.
Mean content
HCPT-SSLN was prepared by the method of "emulsion evaporation at a high temperature and solidification at a low temperature", using solid & liquid lipids as carrier materials, Soya lecithin as surfactant. According to the appearance, particle size and entrapment efficiency, the single factor experiment of prescription and technology were carried out. There were many factors such as "organic phase category", "mole ratio of solid lipids/liquid lipids", "ratio of HCPT/Lipids", "concentration of surfactant", "stir time", "stir velocity" and so on. Then the optimized prescription was obtained by orthogonal design. In order to keep the stability, HCPT-SSLN was lyophilized.
Sephadex G-50 filtration chromatography was adopted to separate free drug from HCPT-SSLN. pH5.0PBS was used to elute HCPT-SSLN and then pH12.0 sodium hydroxide solution was applied to dissolve and elute free HCPT. pH12.0 sodium hydroxide solution was added to the HCPT-SSLN solution to destroy the structure of lipid membrane and dissolve HCPT, and then chloroform was added to eliminate the lecithoid. The concentration of HCPT in sodium hydroxide solution was in carboxylate form and determined at wavelength of 414nm by ultraviolet spectrophotometry.
The physicochemical properties of HCPT-SSLN were studied. The morphology was examined by transmission electron microscope and the TEM graph showed that the morphological characteristic of most nanoparticles were spheroplasts, well distributed with smooth surface. The mean particle size of the prepared HCPT-SSLN was 130.5nm, zeta potential was-33.1mV, pH was 6.29, entrapment efficiency was 79.19%; After lyophilization, the mean particle size of the prepared HCPT-SSLN turned a little larger, zeta potential and pH were almost the same, the entrapment efficiency decreased about 5% and the drug loading was 2.51%. The results of DSC and XRD both showed that HCPT was wrapped up into lipid materials and constituted a new physical phase.
According to the appearance, particle size, pH, entrapment efficiency, the redispersibility after hydrating and microscopic examination, the stabilities of HCPT-SSLN samples before, and after lyophilization were tested in influencing factor experiment and remained sample observation experiment. The results indicated that the HCPT-SSLN should be lyophilized, filled with nitrogen, tightly preserved from light and stored at low temperature (4℃).
pH7.4 phosphate buffered saline containing 30%(volume ratio) methanol was chosen as release medium in vitro. At (37±0.5)℃, away from light, the release rules in vitro of HCPT and the HCPT-SSLN during 72h were studied. The result by the method of high performance liquid chromatography(HPLC) which was set up to determine the concentration of 10-hydroxycamptothecin(HCPT) in lactone form indicated that in the first 3 hours, there was a break through of HCPT. At 72h,The cumulate percentage of releasing HCPT from HCPT-SSLN was about 70%,while that from HCPT crude drug was only 24.7%.So HCPT-SSLN could increase the absorption of HCPT in vivo and showed some slow-release effect.
Compared with HCPT sodium injections, the acute toxicity testing of the prepared HCPT-SSLN to mice was carried out. The medial lethal dose(LD_(50)) of HCPT sodium injections was 93.47mg/kg,and the maximum tolerated dose of HCPT-SSLN exceeded 214.6mg/kg. So HCPT-SSLN could lower the toxicity.
Conclusion
To sum up, this study took a helpful exploration for the development of HCPT dosage form, and could be expected to apply to clinical application as a new form of medication, which has important meanings to the following research and development.
随着上世纪70年代，Birrenbach等首次提出纳米粒的概念和制备方法，药物制剂的研究进入了一个全新的微观领域——纳米制剂。与常规药物相比，纳米药物具有比表面积大、表面反应活性高、活性中心多、吸附能力强等特性，还可以改变药物在体内的分布，增加药物在靶器官的分布量，从而提高疗效。固体脂质纳米粒(Solid lipid nanoparticles，SLN)是以毒性低、生物相容性好的脂质材料作为载体，将药物吸附或包裹于脂质膜中制成的新一代纳米给药体系。除了减轻毒副作用，固体脂质又使它具有聚合物纳米粒的优点，如可以控制药物的释放，避免药物的降解或泄漏以及具有良好的靶向性等。
羟基喜树碱(10-hydroxycamptothecin，HCPT)是从珙桐科植物喜树中提取的一种微量生物碱，是拓扑异构酶Ⅰ特异性抑制剂，广泛应用于肝癌、胃癌、白血病等多种恶性肿瘤的治疗。但其特殊的理化性质：水不溶脂难溶、内酯环结构不稳定等因素，限制了临床应用，普遍存在疗效降低、半衰期短的缺点。本研究以HCPT为模型药物制备半固体脂质纳米粒(Semisolid lipid nanoparticles，SSLN)并对其体外药剂学性质进行了研究，以期达到提高HCPT的稳定性，延长其生物半衰期，缓释，提高药效，降低毒性的目的。
主要内容
以固态和液态脂质为载体，大豆卵磷脂为表面活性剂，采用乳化蒸发—低温固化法制备了HCPT半固体脂质纳米粒。以纳米粒外观、粒径、包封率为指标，对处方和工艺进行了单因素考察，内容包括有机相种类、固态脂质／液态脂质摩尔比、药脂比、表面活性剂浓度以及乳化蒸发搅拌时间、搅拌速度等等，继而通过正交设计，确定了最优处方和制备工艺。为了提高制剂的稳定性，采用冷冻干燥法制成HCPT-SSLN冻干粉剂。
采用Sephadex G-50凝胶过滤法，先用pH5.0PBS洗脱下HCPT-SSLN，再用pH12.0的NaOH溶解并洗脱下吸附在柱顶端的游离药物，从而分离HCPT-SSLN和游离药物。利用NaOH破坏脂质膜结构并溶解包封在其中的HCPT，氯仿消除磷脂等脂质膜材的干扰后，依据建立的羧酸盐形式存在的HCPT紫外分光光度法测定HCPT的含量。
考察了HCPT-SSLN混悬液的理化性质，电镜下纳米粒为圆整的球状体，大小比较均匀，边缘光滑，平均粒径130.5nm，Zeta电位-33.1mV，pH6.29，包封率79.19；冻干品的粒径稍有增大，电位和pH几乎不变，包封率下降了约5％，载药量为2.51％。差示扫描量热分析(DSC)和X射线衍射分析(XRD)皆表明HCPT被脂质包裹后已经构成了一种新的物相。
以外观、粒径、pH、包封率、水中再分散性及显微镜检等为指标，考察了HCPT-SSLN混悬液和冻干品在影响因素实验、留样观察中的稳定性。结果表明：HCPT-SSLN最宜制成冻干粉剂，充氮密封避光置于4℃条件下保存。
选择含有30％甲醇的pH7.4磷酸盐缓冲液做释药介质，在(37±0.5)℃避光的条件下对HCPT原料药和自制的HCPT-SSLN进行了0～72h体外释药的研究。以内酯形式存在的HCPT的HPLC含量测定结果表明：HCPT-SSLN0～3h内有一定程度的突释，72h累积释药量不超过70％，对照药物72h只有24.7％，可见HCPT-SSLN增强了HCPT在体内的吸收，且显示出一定的缓释效应。
以HCPT钠盐注射液作对比，对HCPT-SSLN进行了小鼠的急性毒性实验。HCPT钠盐注射液组的半数致死量是93.47mg／kg，HCPT-SSLN的最大耐受剂量(MTD)大于214.6mg／kg，HCPT-SSLN毒性降低。
结论
本实验为HCPT的剂型开发作了有益的探索，为它的临床应用提供了一种可能的新剂型，有较好的研究开发价值。
Background & objectiveSince 1970s, Birrenbach introduced the concept and preparative method of nanoparticle firstly, the study of the pharmaceutical preparation has entered into anew micro realm——nanometric praeparatum. Compared to routine drugs,nanometric praeparatums have many good characters such as large specific surface area,high surface reaction activity, plenty of active centers, strong adsorbability and so on. They can also change the intracorporal distribution of the medicine to increase the dose distribution in target organs and raise therapeutic effects. Solid lipid nanoparticles(SLN) is a latest generation of nanometric drug delivery system. It uses lipid materials with low toxicity and fine biocompatibility as carrier, then absorbs the drug and wraps it up into the lipid membrane. Besides descreasing poison and side effects, solid lipid make it control drug release, just like PVP, so it avoids the degradation or leakage of the drug and has favourable target characteristics.
10-hydroxycamptothecin(HCPT) is a kind of microamount alkaloids extracted from Camptotheca acuminata Decaisne, and is one of the specific DNA topoisomeraseⅠinhibitors. It is widely used to cure fiver cancer, stomach cancer, leukemia and many malignant tumors. However, its clinical applications are confined by its special physico-chemical properties(for example, it is insolubilize in water and difficultly dissolves in fipids, the lactone band is also instable), so its curative effects decrease and the intracorporal half life is too short. This study chose it as model drug to prepare semisolid lipid nanoparticles(HCPT-SSLN) and investigated its pharmaceutical characteristics in vitro, intending to enhance its stability and prolong biological half-fife, make it release slowly in vivo to reinforce drug action and lower the toxicity.
Mean content
HCPT-SSLN was prepared by the method of "emulsion evaporation at a high temperature and solidification at a low temperature", using solid & liquid lipids as carrier materials, Soya lecithin as surfactant. According to the appearance, particle size and entrapment efficiency, the single factor experiment of prescription and technology were carried out. There were many factors such as "organic phase category", "mole ratio of solid lipids/liquid lipids", "ratio of HCPT/Lipids", "concentration of surfactant", "stir time", "stir velocity" and so on. Then the optimized prescription was obtained by orthogonal design. In order to keep the stability, HCPT-SSLN was lyophilized.
Sephadex G-50 filtration chromatography was adopted to separate free drug from HCPT-SSLN. pH5.0PBS was used to elute HCPT-SSLN and then pH12.0 sodium hydroxide solution was applied to dissolve and elute free HCPT. pH12.0 sodium hydroxide solution was added to the HCPT-SSLN solution to destroy the structure of lipid membrane and dissolve HCPT, and then chloroform was added to eliminate the lecithoid. The concentration of HCPT in sodium hydroxide solution was in carboxylate form and determined at wavelength of 414nm by ultraviolet spectrophotometry.
The physicochemical properties of HCPT-SSLN were studied. The morphology was examined by transmission electron microscope and the TEM graph showed that the morphological characteristic of most nanoparticles were spheroplasts, well distributed with smooth surface. The mean particle size of the prepared HCPT-SSLN was 130.5nm, zeta potential was-33.1mV, pH was 6.29, entrapment efficiency was 79.19%; After lyophilization, the mean particle size of the prepared HCPT-SSLN turned a little larger, zeta potential and pH were almost the same, the entrapment efficiency decreased about 5% and the drug loading was 2.51%. The results of DSC and XRD both showed that HCPT was wrapped up into lipid materials and constituted a new physical phase.
According to the appearance, particle size, pH, entrapment efficiency, the redispersibility after hydrating and microscopic examination, the stabilities of HCPT-SSLN samples before, and after lyophilization were tested in influencing factor experiment and remained sample observation experiment. The results indicated that the HCPT-SSLN should be lyophilized, filled with nitrogen, tightly preserved from light and stored at low temperature (4℃).
pH7.4 phosphate buffered saline containing 30%(volume ratio) methanol was chosen as release medium in vitro. At (37±0.5)℃, away from light, the release rules in vitro of HCPT and the HCPT-SSLN during 72h were studied. The result by the method of high performance liquid chromatography(HPLC) which was set up to determine the concentration of 10-hydroxycamptothecin(HCPT) in lactone form indicated that in the first 3 hours, there was a break through of HCPT. At 72h,The cumulate percentage of releasing HCPT from HCPT-SSLN was about 70%,while that from HCPT crude drug was only 24.7%.So HCPT-SSLN could increase the absorption of HCPT in vivo and showed some slow-release effect.
Compared with HCPT sodium injections, the acute toxicity testing of the prepared HCPT-SSLN to mice was carried out. The medial lethal dose(LD_(50)) of HCPT sodium injections was 93.47mg/kg,and the maximum tolerated dose of HCPT-SSLN exceeded 214.6mg/kg. So HCPT-SSLN could lower the toxicity.
Conclusion
To sum up, this study took a helpful exploration for the development of HCPT dosage form, and could be expected to apply to clinical application as a new form of medication, which has important meanings to the following research and development.
引文
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