神经生长因子柔性脂质体及其脑靶向性的初步研究
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摘要
神经生长因子(nerve growth factor，NGF)属于典型的大分子多肽类药物，本论文试图通过对NGF的研究来考察大分子多肽类药物的脑靶向传递系统。NGF是由神经元、神经支配的靶组织或胶质细胞等产生的，能促进中枢和外周神经元分化、生长、存活的活性蛋白因子，用于治疗神经系统疾病。NGF在水溶液中很快失活，易受温度、酸碱等多种因素的影响，在体内易被生物酶降解。此外NGF本身不能穿透血脑屏障(blood-brain-barrier，BBB)，上述原因限制了NGF的应用，本文试图通过制剂手段解决NGF稳定性的问题，初步尝试提高NGF的脑组织靶向性。
本文建立了氯化银胶体溶液散射光强度猝灭法测定痕量多肽浓度的分析方法；以白细胞介素-2(IL-2)为模型药物代替NGF采用改良的反向蒸发法制备了脂质体；建立了DEAE-sephadex A50微柱离心法测定脂质体包封率；以脂质体包封率为评价指标进行了单因素影响实验；在此基础上进行了L_9(3~4)正交设计实验优化了制剂的处方，采用最优处方和改良的反向蒸发法制备的脂质体包封率为33.11％(n=3)。根据多肽类药物自身特殊的性质采用0.22μm微孔滤膜过滤的方法除菌，比较灭菌前后脂质体包封率，基本没有发生变化；为了提高脂质体混悬液的稳定性将其进一步制成冻干脂质体，在冻干处方中加入不同种类和剂量的冻干支撑剂，以制剂的外观、复溶情况、包封率为考察指标筛选最优冻干处方。
考察了脂质体的物理、化学稳定性：进行了IL-2脂质体高温、强光稳定性影响因素实验，在此条件下冻干脂质体和脂质体混悬液均不稳定，但冻干脂质体的稳定性要优于脂质体混悬液；进行了IL-2脂质体稳定性加速实验，结果表明冻干脂质体在3个月基本保持稳定。在考察了制剂
沈阳药科人学硕士论文 中文稠要
的物理、化学稳定性基础上，针对NGF自身特殊的性质对第一部分的冻
干处方加以改进应用与IL－2相同的脂质体制备工艺制备了NGF冻干脂质
体，并且采用 7刁日龄鸡胚背根神经节Norsal root ganglia，DRG讨经纤
维生长状况为判断指标考察了NGF冻于脂质体和NGF对照品的生物学稳
定性，结果表明将NGF制成冻干脂质体之后NGF的生物活性没有降低，
证明了该剂型的可行性。NGF冻干脂质体在 4 C贮存 3个月生物活性不降
低。在 40OC、40OC＋饱和湿度条件下保存同样时间生物活性部分丧失，
但在此条件下冻干脂质体的稳定性优于对照品。
对注射用NGF脂质体的理化性质进行了考察：冻干脂质体复溶后的
透射电镜照片和激光散射法测得粒径分布结果可以看出制剂为纳米级微
粒，平均粒径为39．6urn，粒度分布比较均匀；冻干脂质体的g电位实验
表明制剂荷正电，即提高了它穿透BBB的能力又可以在一定程度上防止
粒子的聚集。
对注射用NGF脂质体的安全性进行了考察，家兔耳血管刺激性实验。
滴眼刺激性实验及溶血实验结果表明该制剂的刺檄性小；小鼠异常毒性
实验合格。
进行了小鼠体内的药动学初步实验，采用‘丫标记 NGF将 SDS干AGE
电泳和单光于发射型计算机断层显像（SPECT）技术联用测定小鼠血浆中
微量NGF浓度，药动学特征符合双隔室模型。采用‘丫标记NGF通过SPECT
技术初步考察了制剂的脑组织靶向性。
Nerve growth factor (NGF) belongs to the kind of classic molecular peptide which is produced by nerve and target cell managed by nerve . The essay tried to institute the delivery system of peptide according to NGF. The NGF can promote the cell to grow , divide and survive, and also can cure nerve system disease. NGF is ready to lose biological activity quickly in water solution and is sensitive to many conditions such as pH,high temperature, and so on. The use of NGF is limited ,as it can't cross the BBB freely .In my experiment I try to change dosage in order to solve the question of stability and attempt to elevate the penetration of the BBB.
First,establish the analytic method of peptide by quenching of scattering light of silver chloride gel .Then, select the IL-2 as model drug and prepare liposome suspension . Establish the analynatic method of encapsulation efficiency(EE) through DEAE-sephadex A50 minicolumn centrifuge;Select the EE as the parameter and make the single factor experiment . Based on the single factor experiment , the optimal formulation is obtained by orthogonal design test. With the optimal formulation ,the EE of the liposome is 33.11%. Use the 0.22
μm micropore sterilization on account of the specific nature of the NGF. In order to enhance the NGF's stability , prepare the lyophilized liposome . Select the optimal lyophilized formulation according to EE, appearance,rehydration .
Discuss the physical and chemical stability of the NGF liposome suspension and lyophilized liposome. Under high temperature or intense light, the liposome suspension and lyophilized liposome both have feeble stability , but the lyophilized liposome is stronger than the liposome suspension.The accelerated stabilitv experiment indicates the shelf life of lyonhilized linosome is about two
years. On the basis of physical and chemical character , discuss the biological stability by means of outgrowth of chicken embryo dorsal ganglia. The result of experiment indicate the lyophilized liposome can maintain the biological activity for three months under 4 ℃.
The physical and chemical properties of lyophilized liposome were
described.Transmission election microscopy and the Laser scattering mersurement
showed that NGF particle was nanoparticle with mean diameter of 39.6 nm. The
, experiment showed that NGF lyophilized liposome was positive which
enhance the NGF penetration of the BBB.
The safety experiments of rehydrated liposome such as vessel irritation and hemolysis ,were tested for intravenous administration. It was showed that lyophilized liposome was safe formulation and nonirritant.
The preliminary studies of pharmacokinetics were evaluated in mouses. The plasma NGF concenteation was determined by labeling with99Tc with the SDS-PAGE and SPECT .The pharmacokinetic characteristics of rehydrated lyophilized liposome conformed two compartments model.The preliminary studies of brain tissue distribution were determined by SPECT .
本文建立了氯化银胶体溶液散射光强度猝灭法测定痕量多肽浓度的分析方法；以白细胞介素-2(IL-2)为模型药物代替NGF采用改良的反向蒸发法制备了脂质体；建立了DEAE-sephadex A50微柱离心法测定脂质体包封率；以脂质体包封率为评价指标进行了单因素影响实验；在此基础上进行了L_9(3~4)正交设计实验优化了制剂的处方，采用最优处方和改良的反向蒸发法制备的脂质体包封率为33.11％(n=3)。根据多肽类药物自身特殊的性质采用0.22μm微孔滤膜过滤的方法除菌，比较灭菌前后脂质体包封率，基本没有发生变化；为了提高脂质体混悬液的稳定性将其进一步制成冻干脂质体，在冻干处方中加入不同种类和剂量的冻干支撑剂，以制剂的外观、复溶情况、包封率为考察指标筛选最优冻干处方。
考察了脂质体的物理、化学稳定性：进行了IL-2脂质体高温、强光稳定性影响因素实验，在此条件下冻干脂质体和脂质体混悬液均不稳定，但冻干脂质体的稳定性要优于脂质体混悬液；进行了IL-2脂质体稳定性加速实验，结果表明冻干脂质体在3个月基本保持稳定。在考察了制剂
沈阳药科人学硕士论文 中文稠要
的物理、化学稳定性基础上，针对NGF自身特殊的性质对第一部分的冻
干处方加以改进应用与IL－2相同的脂质体制备工艺制备了NGF冻干脂质
体，并且采用 7刁日龄鸡胚背根神经节Norsal root ganglia，DRG讨经纤
维生长状况为判断指标考察了NGF冻于脂质体和NGF对照品的生物学稳
定性，结果表明将NGF制成冻干脂质体之后NGF的生物活性没有降低，
证明了该剂型的可行性。NGF冻干脂质体在 4 C贮存 3个月生物活性不降
低。在 40OC、40OC＋饱和湿度条件下保存同样时间生物活性部分丧失，
但在此条件下冻干脂质体的稳定性优于对照品。
对注射用NGF脂质体的理化性质进行了考察：冻干脂质体复溶后的
透射电镜照片和激光散射法测得粒径分布结果可以看出制剂为纳米级微
粒，平均粒径为39．6urn，粒度分布比较均匀；冻干脂质体的g电位实验
表明制剂荷正电，即提高了它穿透BBB的能力又可以在一定程度上防止
粒子的聚集。
对注射用NGF脂质体的安全性进行了考察，家兔耳血管刺激性实验。
滴眼刺激性实验及溶血实验结果表明该制剂的刺檄性小；小鼠异常毒性
实验合格。
进行了小鼠体内的药动学初步实验，采用‘丫标记 NGF将 SDS干AGE
电泳和单光于发射型计算机断层显像（SPECT）技术联用测定小鼠血浆中
微量NGF浓度，药动学特征符合双隔室模型。采用‘丫标记NGF通过SPECT
技术初步考察了制剂的脑组织靶向性。
Nerve growth factor (NGF) belongs to the kind of classic molecular peptide which is produced by nerve and target cell managed by nerve . The essay tried to institute the delivery system of peptide according to NGF. The NGF can promote the cell to grow , divide and survive, and also can cure nerve system disease. NGF is ready to lose biological activity quickly in water solution and is sensitive to many conditions such as pH,high temperature, and so on. The use of NGF is limited ,as it can't cross the BBB freely .In my experiment I try to change dosage in order to solve the question of stability and attempt to elevate the penetration of the BBB.
First,establish the analytic method of peptide by quenching of scattering light of silver chloride gel .Then, select the IL-2 as model drug and prepare liposome suspension . Establish the analynatic method of encapsulation efficiency(EE) through DEAE-sephadex A50 minicolumn centrifuge;Select the EE as the parameter and make the single factor experiment . Based on the single factor experiment , the optimal formulation is obtained by orthogonal design test. With the optimal formulation ,the EE of the liposome is 33.11%. Use the 0.22
μm micropore sterilization on account of the specific nature of the NGF. In order to enhance the NGF's stability , prepare the lyophilized liposome . Select the optimal lyophilized formulation according to EE, appearance,rehydration .
Discuss the physical and chemical stability of the NGF liposome suspension and lyophilized liposome. Under high temperature or intense light, the liposome suspension and lyophilized liposome both have feeble stability , but the lyophilized liposome is stronger than the liposome suspension.The accelerated stabilitv experiment indicates the shelf life of lyonhilized linosome is about two
years. On the basis of physical and chemical character , discuss the biological stability by means of outgrowth of chicken embryo dorsal ganglia. The result of experiment indicate the lyophilized liposome can maintain the biological activity for three months under 4 ℃.
The physical and chemical properties of lyophilized liposome were
described.Transmission election microscopy and the Laser scattering mersurement
showed that NGF particle was nanoparticle with mean diameter of 39.6 nm. The
, experiment showed that NGF lyophilized liposome was positive which
enhance the NGF penetration of the BBB.
The safety experiments of rehydrated liposome such as vessel irritation and hemolysis ,were tested for intravenous administration. It was showed that lyophilized liposome was safe formulation and nonirritant.
The preliminary studies of pharmacokinetics were evaluated in mouses. The plasma NGF concenteation was determined by labeling with99Tc with the SDS-PAGE and SPECT .The pharmacokinetic characteristics of rehydrated lyophilized liposome conformed two compartments model.The preliminary studies of brain tissue distribution were determined by SPECT .
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