MPS-PLGA微球的制备及其治疗大鼠脊髓压迫损伤的实验研究
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摘要
目的:脊髓损伤有两个阶段,原发性损伤和继发性损伤,其中继发性损伤的危害更大。甲基强的松龙(MP)能够通过抑制脂质过氧化、抗炎、抑制脂质水解和花生四烯酸释放、改善损伤后脊髓血流、防止细胞内外Na、K及Ca失衡、防止脊髓细胞凋亡、抑制兴奋性氨基酸、增强神经兴奋性和突触传递等作用减轻脊髓继发性损伤、促进神经功能恢复,是临床是上唯一确定有效的药物之一,也是唯一被美国联邦食品药品管理局(Food Drug Administration,FDA)批准的治疗SIC药物。目前MP治疗脊髓损伤的用药方案主要是大剂量冲击疗法(30mg/kg,iv,之后5.4mg/kg/h,iv,23h),通过外周静脉给药,该方案的治疗效果已获临床证实,但是其全身副作用也引起医学专家的重视。本实验尝试通过蛛网膜下腔给药来减少用药量,减轻全身副作用,为避免反复多次注射引发感染,先将MP微球化,力求通过一次性蛛网膜下腔注射MPS-PLGA微球达到较好的治疗效果。
方法:(1)MPS-PLGA微球的制备及优化:以PLGA为囊材采用W/O/W型复乳法制备MPS-PLGA微球,通过单因素分析和正交设计优化微球制备工艺,检测优化条件下制得微球的形态、粒径、载药量、包封率、体外释药速度和稳定性。(2)动物实验:将240只成年将康SD大鼠(体重:230-250g,雌雄各半)随机分为A、B、C、D四组,A、B、C三组按Nystrom法(50g,5min)制备大鼠压迫损伤模型,A组蛛网膜下腔注射MPS-PLGA微球(3mg/kg),B组尾静脉注射MPS(30mg/kg),C组不予治疗,D组为假手术组(仅暴露脊髓而不压迫)。于术后各时相点进行BBB运动功能评分、MEP检测、损伤段脊髓组织NO含量和NOS活性检测、HE染色、Bcl-2和NF200免疫组织化学染色。
结果:(1)内水相和油相体积比1:10、初乳功率150W、MPS浓度60mg/ml、复乳时间3min、复乳转速2000转/分钟是制备MPS-PLGA微球的最佳工艺组合。(2)在最佳工艺条件下制备的微球形态规则(近圆形)、表面光滑、平均粒径12±9.41um、粒径分布集中(<18um的微球数占80.58±1.12%)、载药量26.98%、包封率67.46%、体外释药符合Higuchi方程(y=3.203t1/2+40.526)。(3)A、B、C三组动物在损伤后其运动功能评分(BBB评分)均降低,且随时间延长逐渐升高,14d时仍低于正常水平,各时相点组内存在显著差异(p<0.01),A、B、C三组组与假手术组比较也有显著差异(p<0.01),A、B组于术后3d、7d、14d明显高于C组(p<0.01),A组7d、14d时优于B组,但差异不显著(P<0.05)。(4)大鼠脊髓损伤后MEP N1波潜伏期明显延长,随时间增加逐渐恢复。A组于7天基本恢复正常,B组也于14天时恢复至正常水平,C组14天时与D组仍有显著统计学差异。(5)NO含量和NOS活性于脊髓损伤后2h已有升高,24h打高峰并持续到72h。A、B两组明显低于C组(p<0.01),但仍高于D组(P<0.01),且A组低于B组。(6)NF200免疫组织化学染色相对阳性染色面积和平均光密度于伤后3D达最低点,各时间点A、B组与C组间存在显著差异(p<0.01),A、B组间无统计学差异(p>0.05)。(7)Bcl-2免疫组织化学染色阳性细胞百分比和平均光密度在损伤后1d达高峰,并于7d时恢复正常水平。在8h、1d、3d三个时间点,A、B组与C组间存在显著统计学差异(p<0.01),A组和B组间也有明显差异(P<0.05)。
结论:(1)W/O/W型溶剂挥发法是MPS-PLGA微球很好的制备方法。(2)按正交设计优化工艺制备的MPS-PLGA微球符合药学要求。(3)MP早期应用能够减轻脊髓继发性损伤,促进神经功能恢复。(4)MPS-PLGA微球蛛网膜下腔注射给药治疗脊髓急性损伤的效果不亚于于MPS外周给药。
In traumatic spinal cord injury (SCI), the impact on the spinal cord induces the primary injury and the secondary damage. The secondary pathological changes following mechanical injury to the spinal cord contribute to the worsening of neurological functions. Methylprednisolone can cut down the disservice of the secondary damage and enhance the recovery of the neurofunction after SCI. The mechanisms probably included inhibiting lipid peroxidation, inflammatory reaction, lipid hydrolization and arachidonic acid releasing, improving microcirculation of the damaged spinal cord, preventing the disequilibrium of the Na, K and Ca between the internal and external, preventing apoptosis, suppressing excitatory amino acids, enhancing nervous excitation and synaptic transmission. MP is the one of those which has been certificated having effect with the SCI, and it is the only drug which having been approved to therapy the SCI by the FDA. At present, MP has been used to therapy the SCI with the high dose chemotherapy through intravenous injection. This drug delivery system usually has many adverse reactions. In this study, we try to inject the drug in the subarachnoid space in order to decrease the drug consumption and lessen the adverse reactions. First, we prepare the MPS-PLGA microspheres so as to therapy the SCI by one time injection without infection.
Materials and Methods:
Preparation of microspheres experiment: PLGA was choice as the capsule wall material and W/O/W double emulsion solvent evaporation-extraction method is used to prepare MPS-PLGA microspheres. The different variables of influencing factors on the preparation technology of PLGA microspheres were optimized by single factor analysis experiments and orthogonal design experiments. Prepare MPS-PLGA microspheres under the optimized conditions, and determine the shape, particle size, drug loading amount, encapsulation efficiency and release profile in vitro.
Animal experiment: 240 healthy adult SD rats (weight: 230-250g) of either sex half and half were randomly divided into four groups. Group A, B and C were spinal cord compression model (50g/5min) according to the letter reported by Nystrom. Group A was injected MPS-PLGA microspheres (110mg/kg) though subarachnoid route, Group B was injected MPS (30mg/kg) by vena caudalis, and nothing has to do with Group C. Group was exposed the spinal cord without compression. The evaluating indicators in two weeks include: BBB score, motor evoked potentials, amount of nitric oxide in the damaged spinal cord, activity of nitric oxide synthetase in the damaged spinal cord, expression of Bcl-2 and NF200 in the spinal cord.
Results:
1. The optimized conditions of Preparation of MPS-PLGA microspheres: the phase volume ratio of the inner aqueous phase and oil phase is 1: 10, ultrasonic power of emulsicication is 150w, the density of MPS is 60mg/ml, frequency of stir is 2000rpm, stir time is 3min.
2. The mirospheres prepared under the optimized conditions have nearly round integrated shape and slick appearance. Its average particle size is 12±9.41um, drug loading is 26.98%, drug trapping efficiency is 67.46%. The in vitro release profile was figured by Higuchi equation: y=3.203t1/2+40.526.
3. BBB score of Group A, Group B and Group C is lower than Group D at every time point after surgery(p<0.01), and that of Group A and Group B is higher than Group C(p<0.01) at every time point after SCI except 8h. There are obviously differences between Group A and Group B 7d and 14d after SCI (P<0.05).
4. The latent time of MEP N1 lengthen after SCI and then become short and short with the time increasing. The latent time of MEP N1 of Group A and Group B recovery nearly to the normal level at 7d and 14d after SCI.
5. The amount of NO and the activity of NOS in the damaged spinal cord of Group A and Group B are higher than Group C at every time point after SCI(p<0.01). So do the Group A and Group B.
6. The Area and AOD of the NF200 of the Group A and Group B are higher than the Group C 7d and 14d after SCI(p<0.01). There is no significant difference between Group A and Group B.
7. The expression of Bcl-2 increase and reach to the peak 1d after SCI, then it return to the normal level at the 7d time point. At the 8h,1d and 3d time point, the expression of Bcl-2 of the Group A and Group B is lower than Group C(p<0.01). There is also an obviously difference between Group A and Group B.
Conclusions:
1. W/O/W double emulsion solvent evaporation-extraction method is a excellent method to prepare MPS-PLGA microspheres.
2. The MPS-PLGA microspheres prepared under the optimized conditions according to the orthogonal design experiments consistent with the requisition of the pharmaceutical sciences.
3. Methylprednisolone can decrease the disservice of the secondary damage and enhance the recovery of the neurofunction after SCI.
4. The therapeutic effect of MPS-PLGA microspheres injected through subarachnoid route to the SCI is as good as MPS injected though intravenous.
方法:(1)MPS-PLGA微球的制备及优化:以PLGA为囊材采用W/O/W型复乳法制备MPS-PLGA微球,通过单因素分析和正交设计优化微球制备工艺,检测优化条件下制得微球的形态、粒径、载药量、包封率、体外释药速度和稳定性。(2)动物实验:将240只成年将康SD大鼠(体重:230-250g,雌雄各半)随机分为A、B、C、D四组,A、B、C三组按Nystrom法(50g,5min)制备大鼠压迫损伤模型,A组蛛网膜下腔注射MPS-PLGA微球(3mg/kg),B组尾静脉注射MPS(30mg/kg),C组不予治疗,D组为假手术组(仅暴露脊髓而不压迫)。于术后各时相点进行BBB运动功能评分、MEP检测、损伤段脊髓组织NO含量和NOS活性检测、HE染色、Bcl-2和NF200免疫组织化学染色。
结果:(1)内水相和油相体积比1:10、初乳功率150W、MPS浓度60mg/ml、复乳时间3min、复乳转速2000转/分钟是制备MPS-PLGA微球的最佳工艺组合。(2)在最佳工艺条件下制备的微球形态规则(近圆形)、表面光滑、平均粒径12±9.41um、粒径分布集中(<18um的微球数占80.58±1.12%)、载药量26.98%、包封率67.46%、体外释药符合Higuchi方程(y=3.203t1/2+40.526)。(3)A、B、C三组动物在损伤后其运动功能评分(BBB评分)均降低,且随时间延长逐渐升高,14d时仍低于正常水平,各时相点组内存在显著差异(p<0.01),A、B、C三组组与假手术组比较也有显著差异(p<0.01),A、B组于术后3d、7d、14d明显高于C组(p<0.01),A组7d、14d时优于B组,但差异不显著(P<0.05)。(4)大鼠脊髓损伤后MEP N1波潜伏期明显延长,随时间增加逐渐恢复。A组于7天基本恢复正常,B组也于14天时恢复至正常水平,C组14天时与D组仍有显著统计学差异。(5)NO含量和NOS活性于脊髓损伤后2h已有升高,24h打高峰并持续到72h。A、B两组明显低于C组(p<0.01),但仍高于D组(P<0.01),且A组低于B组。(6)NF200免疫组织化学染色相对阳性染色面积和平均光密度于伤后3D达最低点,各时间点A、B组与C组间存在显著差异(p<0.01),A、B组间无统计学差异(p>0.05)。(7)Bcl-2免疫组织化学染色阳性细胞百分比和平均光密度在损伤后1d达高峰,并于7d时恢复正常水平。在8h、1d、3d三个时间点,A、B组与C组间存在显著统计学差异(p<0.01),A组和B组间也有明显差异(P<0.05)。
结论:(1)W/O/W型溶剂挥发法是MPS-PLGA微球很好的制备方法。(2)按正交设计优化工艺制备的MPS-PLGA微球符合药学要求。(3)MP早期应用能够减轻脊髓继发性损伤,促进神经功能恢复。(4)MPS-PLGA微球蛛网膜下腔注射给药治疗脊髓急性损伤的效果不亚于于MPS外周给药。
In traumatic spinal cord injury (SCI), the impact on the spinal cord induces the primary injury and the secondary damage. The secondary pathological changes following mechanical injury to the spinal cord contribute to the worsening of neurological functions. Methylprednisolone can cut down the disservice of the secondary damage and enhance the recovery of the neurofunction after SCI. The mechanisms probably included inhibiting lipid peroxidation, inflammatory reaction, lipid hydrolization and arachidonic acid releasing, improving microcirculation of the damaged spinal cord, preventing the disequilibrium of the Na, K and Ca between the internal and external, preventing apoptosis, suppressing excitatory amino acids, enhancing nervous excitation and synaptic transmission. MP is the one of those which has been certificated having effect with the SCI, and it is the only drug which having been approved to therapy the SCI by the FDA. At present, MP has been used to therapy the SCI with the high dose chemotherapy through intravenous injection. This drug delivery system usually has many adverse reactions. In this study, we try to inject the drug in the subarachnoid space in order to decrease the drug consumption and lessen the adverse reactions. First, we prepare the MPS-PLGA microspheres so as to therapy the SCI by one time injection without infection.
Materials and Methods:
Preparation of microspheres experiment: PLGA was choice as the capsule wall material and W/O/W double emulsion solvent evaporation-extraction method is used to prepare MPS-PLGA microspheres. The different variables of influencing factors on the preparation technology of PLGA microspheres were optimized by single factor analysis experiments and orthogonal design experiments. Prepare MPS-PLGA microspheres under the optimized conditions, and determine the shape, particle size, drug loading amount, encapsulation efficiency and release profile in vitro.
Animal experiment: 240 healthy adult SD rats (weight: 230-250g) of either sex half and half were randomly divided into four groups. Group A, B and C were spinal cord compression model (50g/5min) according to the letter reported by Nystrom. Group A was injected MPS-PLGA microspheres (110mg/kg) though subarachnoid route, Group B was injected MPS (30mg/kg) by vena caudalis, and nothing has to do with Group C. Group was exposed the spinal cord without compression. The evaluating indicators in two weeks include: BBB score, motor evoked potentials, amount of nitric oxide in the damaged spinal cord, activity of nitric oxide synthetase in the damaged spinal cord, expression of Bcl-2 and NF200 in the spinal cord.
Results:
1. The optimized conditions of Preparation of MPS-PLGA microspheres: the phase volume ratio of the inner aqueous phase and oil phase is 1: 10, ultrasonic power of emulsicication is 150w, the density of MPS is 60mg/ml, frequency of stir is 2000rpm, stir time is 3min.
2. The mirospheres prepared under the optimized conditions have nearly round integrated shape and slick appearance. Its average particle size is 12±9.41um, drug loading is 26.98%, drug trapping efficiency is 67.46%. The in vitro release profile was figured by Higuchi equation: y=3.203t1/2+40.526.
3. BBB score of Group A, Group B and Group C is lower than Group D at every time point after surgery(p<0.01), and that of Group A and Group B is higher than Group C(p<0.01) at every time point after SCI except 8h. There are obviously differences between Group A and Group B 7d and 14d after SCI (P<0.05).
4. The latent time of MEP N1 lengthen after SCI and then become short and short with the time increasing. The latent time of MEP N1 of Group A and Group B recovery nearly to the normal level at 7d and 14d after SCI.
5. The amount of NO and the activity of NOS in the damaged spinal cord of Group A and Group B are higher than Group C at every time point after SCI(p<0.01). So do the Group A and Group B.
6. The Area and AOD of the NF200 of the Group A and Group B are higher than the Group C 7d and 14d after SCI(p<0.01). There is no significant difference between Group A and Group B.
7. The expression of Bcl-2 increase and reach to the peak 1d after SCI, then it return to the normal level at the 7d time point. At the 8h,1d and 3d time point, the expression of Bcl-2 of the Group A and Group B is lower than Group C(p<0.01). There is also an obviously difference between Group A and Group B.
Conclusions:
1. W/O/W double emulsion solvent evaporation-extraction method is a excellent method to prepare MPS-PLGA microspheres.
2. The MPS-PLGA microspheres prepared under the optimized conditions according to the orthogonal design experiments consistent with the requisition of the pharmaceutical sciences.
3. Methylprednisolone can decrease the disservice of the secondary damage and enhance the recovery of the neurofunction after SCI.
4. The therapeutic effect of MPS-PLGA microspheres injected through subarachnoid route to the SCI is as good as MPS injected though intravenous.
引文
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