雷公藤甲素脂质体的制备及质量的初步研究
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摘要
雷公藤甲素(又称雷公藤内酯、雷公藤内酯醇;或Triptolide, TP),是中药雷公藤(Tripterygium wilfordii Hook. F.)中分离出的活性最高的环氧化二萜内酯化合物,雷公藤甲素(TP)在治疗各种免疫性疾病、炎性疾病及癌症方面起了很大作用。但其临床不良反应的发生频率高,主要发生在消化系统、泌尿系统、生殖系统、心血管系统、骨髓和血液系统等。因此,改良雷公藤甲素单体剂型、研究新型给药系统以降低其毒性具有十分重要的意义。本文是将雷公藤甲素(简称TP)制备成为雷公藤甲素脂质体(简称TP-LP),以达到药物缓释的目的,为后续体内增效减毒研究奠定基础。
本文分为三部分:
第一部分:雷公藤甲素脂质体的处方前研究
目的:建立雷公藤甲素脂质体的含量测定方法、测定雷公藤甲素在PBS中的溶解度及筛选包封率的测定方法,为雷公藤甲素脂质体的制备处方筛选、工艺研究、质量研究及体外分析奠定基础。
方法:采用紫外-可见全波长扫描测定雷公藤甲素和空白脂质体的最大吸收峰,确定检测波长。采用紫外分光光度法测定雷公藤甲素的含量,建立标准曲线;用紫外分光光度法测定雷公藤甲素在PBS中的溶解度;采用凝胶过滤法、高速离心法、透析法及超滤离心法这四种方法测定TP-LP的包封率,考察方法回收率,并以包封率为指标,进行方法的可行性分析。
结果:全波长扫描结果:空白脂质体在202nm有最大吸收,雷公藤甲素在220nm有最大吸收,雷公藤甲素发生显色反应后在540nm有最大吸收;绘制雷公藤甲素在0.1539μg/ml~ 12.467μg/ml浓度范围和1.3852μg/ml~ 112μg/ml浓度范围的标准曲线,浓度与吸光度的线性关系良好,回归方程分别为:A = 0. 0128 C + 0. 0022( r = 0. 9984 ,n = 5)和A = 0. 0097 C + 0. 02( r = 0. 998,n = 5);测定雷公藤甲素在pH 7.0 PBS中溶解度约为21.55μg/ml;四种包封率测定方法对游离药物回收率分别为:107.1%、92.2%、107.8%、95.3%,对脂质体回收率分别为较好、不好、较好、好;
结论:对结果分析,雷公藤甲素在220nm处最大吸收与空白脂质体于202nm处最大吸收峰相近,在含量测定中易导致干扰,最终确定540nm作为雷公藤甲素的检测波长;PBS溶解度测定结果表明,雷公藤甲素在PBS中有微量溶解,对包封率测定方法的筛选提供了一定参考;经可行性分析,相对于其他包封率测定方法,超滤离心法对游离药物和脂质体回收率好,因此确定采用超滤离心法测定雷公藤甲素脂质体的包封率。
第二部分雷公藤甲素脂质体制备方法筛选及制备工艺的研究
目的:以包封率为指标,筛选TP-LP的最佳制备方法;以脂质体粒径、稳定性及包封率为指标优化TP-LP的制备工艺。
方法:分别采用薄膜分散法、机械匀浆法、乙醇注入法制备TP-LP,以包封率为衡量指标,采用超滤离心法分离脂质体和游离药物,测定包封率,筛选最佳制备方法;在空白脂质体的制备工艺研究中,以空白脂质体粒径和稳定性为指标,对制备条件:磷脂与胆固醇比例、氯仿用量及超声情况进行优化;在TP-LP制备工艺研究中,以包封率为衡量指标,采用正交优化方法优化影响脂质体包封率的三个制备条件:载药温度、药物与磷脂质量比(药脂比)和载药时间,采用超滤离心法分离脂质体和游离药物,紫外分光光度法测定游离药物含量,计算TP-LP的包封率,筛选最佳制备工艺,并验证最佳条件的准确性。
结果:制备方法的筛选中,薄膜分散法、机械匀浆法、乙醇注入法制备TP-LP的包封率分别为:12.7%、14%、13.6%;空白脂质体制备工艺研究中,磷脂-胆固醇比为4:1制备的脂质体稳定性好;超声100w/5min处理的脂质体粒径分布好;用20ml氯仿制备的空白脂质体粒径分布较好。TP-LP制备工艺研究中,经正交优化实验,制备条件:载药温度37℃、药脂比1:50、载药时间60min所制备的雷公藤甲素脂质体包封率最高;最后按最佳优化条件同时制备三批TP-LP,测定其平均包封率为46.4%±0.0295。
结论:以包封率为指标,比较三种制备方法,机械匀浆法制备的TP-LP包封率较高,经可行性分析,该方法简单、可重复性好,因此确定机械匀浆法制备TP-LP;空白脂质体单因素优化结果显示,制备最佳条件为:磷脂-胆固醇比例为4:1,超声100w/5min,氯仿用量20ml;TP-LP制备工艺正交优化结果显示,最佳制备条件为载药温度37℃、药脂比1:50、载药时间60min。最佳条件重复验证实验结果验证了正交优化方法的准确性。
第三部分雷公藤甲素脂质体的质量研究及体外释药的初步研究
目的:对TP-LP进行质量研究,为提高TP-LP稳定性研究奠定基础;进行TP-LP体外药效初步研究,为其体内药效研究奠定基础。
方法:按最佳工艺制备TP-LP,用透射电镜显微观察TP-LP形态,用动态光散射仪测定粒径分布,用zeta电位分析仪测定TP-LP的zeta电位,从以上几个指标对脂质体进行质量评价;分别加入不同冻干保护剂制备TP-LP冻干粉,以脂质体冻干粉的外观、复水情况及复水后包封率为衡量指标,筛选最佳冻干保护剂;在体外释药实验,采用透析法分别观察TP和TP-LP的体外释药情况,采用紫外分光光度法测定不同时间点的累计释药量;体外药效实验,用TP、TP-LP处理经PHA活化的人外周血单个核细胞,以MTT法为检测方法,分别于24h、48h、72h测定TP、TP-LP抑制T淋巴细胞增殖的效果。
结果:最佳优化工艺制备的TP-LP,粒径范围在40nm~250nm之间,平均粒径为89.2nm。粒径对称分布,分散均匀,zeta电位平均值为-67.55mV ,绝对值大于60mv;冻干粉的制备结果,5%蔗糖-甘露醇混合物(w:w=1:1)作为冻干保护剂所制备的冻干粉外观良好,复水过程良好,无肉眼结晶。相对于其他保护剂制备的冻干粉,复水后的包封率下降程度小;TP-LP的体外释药实验结果,44小时后,脂质体中TP全部释放,而游离TP在3h已完全释放;体外药效实验结果显示,24h TP-LP抑制T淋巴细胞增殖效果低于游离的TP;48hTP-LP的抑制效果与游离TP相当;72hTP-LP的抑制效果稍高于游离TP。
结论:TP-LP形态、粒径分布基本符合后续研究的要求;zeta电位结果显示,TP-LP稳定性相当好,有利于后续研究;体外释药结果显示,与游离的TP相比,TP-LP有一定的缓释作用;体外药效结果表明,TP经脂质体包裹后具有缓释功能。
Triptolide (TP), is the highest activity of isolated diterpene lactone epoxide compounds from traditional Chinese medicine Tripterygium wilfordii. Thunder triptolide played a significant role in the treatment of various autoimmune diseases, inflammatory diseases and cancer, meanwhile it cause adverse reaction in iateria and the clinical adverse events mainly were in the digestive system, urinary system, reproducti- vesystem, cardiovascular system, bone marrow and blood system,especially has high frequency in digestive system.Therefore, it is very significant to improve triptolide single dosage form, investigate new drug delivery system in order to reduce its toxicity. We have prepared triptolide liposome in order to synergia and Attenuation.
This study was divided into three parts as follows.
Part I: the prophas research of triptolide liposome’s preparation
Objective: We set up the method of measuring Triptolide (TP)and Triptolide liposome (TP-LP) content , and the triptolide solubility in PBS, in order to anaphase study of TP-LP.
Methods: We determined the maximum absorption of Triptolide and blank liposomes with UV-VIS wavelength scanning. Established standard curve of triptolide and measured the triptolide solubility by ultraviolet spectrophotometry ; Determined the encapsulation efficiency of TP-LP by gel filtration, high-speed centrifugation, dialysis and ultrafiltration centrifugal respectively, and analysis feasibility of the four methods.
Results: The wavelength scan results: the maximum absorption wavelength of blank liposomes is 202nm, triptolide is 220nm and the triptolide after color reaction is 540nm; Drawed the triptolide standard curve in 0.1539μg/ml~12.467μg/ml concentrat- ion range and 1.3852μg/ml ~ 112μg/ml respectively. The linear relationship between concentration and absorbance is good, regression equations were: A = 0. 0128 C + 0. 0022 ( r = 0. 9984, n = 5) and A = 0. 0097 C + 0. 02 (r = 0. 998, n = 5); The triptolide solubility in pH 7.0 PBS is about 21.55μg/ml ; The recovery rate of entrapment efficie- ncy with four method for free TP were: 107.1%, 92.2%, 95.3% and 107.8%, and the ultrafiltration centrifugal has good recovery rate for liposome.
Conclusions: Because of the wavelength between TP and blank liposome are adjoin, it is easy to cause interference within later period measure of entrapment efficiency, so determined the detect wavelength of TP is 540nm; Solubility test of TP showed that triptolide has trace dissolve in PBS, which provide a reference for the determination of entrapment efficiency. Through feasibility analysis, compared to other encapsulation methods, ultrafiltration has good recovery rate for free drug and blank liposome, so adopt ultrafiltration centrifugal for the determination of triptolide liposome encapsulation efficiency.
Part II : the study of liposome’s preparation method and technology
Objective: Determine the optimal preparation method of liposome according to entrapment efficiency, and optimized preparation technology of liposome .
Methods: Prepare TP-LP respectively with film dispersion method, mechanical homogenization and ethanol injection method. Separated free drug from liposome by ultrafiltration centrifugation inoder to calculate the encapsulation efficiency of TP-LP;
In the technology study of blank liposome, we optimized the preparation conditions ( proportion of phospholipids and cholesterol, volume of chloroform and ultrasound conditions) with single factor investigation on the basis of liposome size and stability. In the technology study of TP-LP, we optimized the preparation conditions(the ratio between drug and liposome, loading temperature and drug loading time) with orthogonality optimization on the basis of liposome entrapment efficiency as target.
Results: The entrapment efficiency of TP-LP which was prepared by film dispersion method, mechanical homogenization and ethanol injection method is respective 12.7%, 14% and 13.6%; The blank liposome via the ratio between phospholipid and cholesterol of 4:1 (w/w) is better stability than others, blank liposome size with 20ml of CHCl3 has better uniformity than 10ml liposomes, and the liposome size with ultrasonic 100w/5min is better uniformity ; The optimized preparation conditions is drug loading temperature 37℃, drug lipid ratio of 1:50 and the drug preparation time of 60min; Final the average encapsulation efficiency of TP-LP with optimal conditions was measured 46.4%±0.0295.
Conclusion: The entrapment efficiency of TP-LP which was prepared by mechanical homogenization is highest, so determine the preparation method is mechanical homogenization; The best preparation conditions of blank liposome are: the ratio between phospholipid and cholesterol of 4:1 (w/w), ultrasound 100w/5min, 20ml of chloroform; The best preparation conditions for TP-LP is drug loading temperature of 37℃, drug-lipid ratio of 1:50, drug loading time of 60min. Moreover, the replicated experimental results verify the accuracy of the orthogonal optimization method.
We also compare four measure method of entrapment efficiency, and carrying out feasibility analysis, determine that separate liposomes and free drug by ultrafiltration centrifugation, to survey free drug and calculate the encapsulation efficiency indirectly.
Part III: the study of liposome’s quality and release in vitro
Objective: Do quality research of TP-LP in order to improve the stability of TP-LP; Do study of TP-LP’s release in vitro in order to establish groundwork for their efficacy in vivo.
Methods: Prepared TP-LP with optimal conditions ,and measured liposome size via electron microscope, measured particle size distribution by dynamic light scattering, and determined zeta potential analysis. We also prepared freeze-dried powder of liposome with adding different cryoprotectant. Do release experiment of TP-LP by dialysis in vitro conditions, and measure cumulative release amount of TP by UV spectrophotometry at different time points; Finally, we designed the experiment to check the inhibition effect of TP-LP to T lymphocyte proliferation. Firstly, activate human peripheral blood mononuclear cells with the PHA, then detected the inhibition effect of TP-LP to T lymphocyte proliferation by MTT method respectively at 24h, 48h and 72h .
Results: The particle size of TP-LP is range of 40nm ~ 250nm, with an average diameter of 89.2nm; Symmetric particle size distribution, dispersed, zeta potential of mean-67.55mV; Compared with other cryoprotectant, the freeze-dried powders of TP-LP with adding 5% sucrose-mannitol (w: w = 1:1) has better appearance, and the process of rehydration is well without naked eye crystal, in addition to have higher entrapment efficiency. In the release experiments of TP-LP in vitro, TP had been released from TP-LP completely after 44 hours, but had been released completely from free TP after 3h; In the experiment of inhibition effect to T lymphocyte proliferation, the inhibition effect of TP-LP are lower than free TP after 24h, the inhibition effect of TP-LP are equality to free TP after 48h and the inhibition effect of TP-LP are higher than free TP after 72h.
Conclusion: In the morphology study of TP-LP, the particle size distribution of TP-LP were satisfied to the follow study; The results of zeta potential show that TP-LP has good stability; Prepare freeze-dried powder of liposome with adding 5% sucrose-mannitol (w: w = 1:1) as cryoprotectant; The release result of TP-LP show that TP-LP has delayed release compared with free TP,and the experiment of inhibition effect to T lymphocyte proliferation also authenticate delayed release of TP- LP.
本文分为三部分:
第一部分:雷公藤甲素脂质体的处方前研究
目的:建立雷公藤甲素脂质体的含量测定方法、测定雷公藤甲素在PBS中的溶解度及筛选包封率的测定方法,为雷公藤甲素脂质体的制备处方筛选、工艺研究、质量研究及体外分析奠定基础。
方法:采用紫外-可见全波长扫描测定雷公藤甲素和空白脂质体的最大吸收峰,确定检测波长。采用紫外分光光度法测定雷公藤甲素的含量,建立标准曲线;用紫外分光光度法测定雷公藤甲素在PBS中的溶解度;采用凝胶过滤法、高速离心法、透析法及超滤离心法这四种方法测定TP-LP的包封率,考察方法回收率,并以包封率为指标,进行方法的可行性分析。
结果:全波长扫描结果:空白脂质体在202nm有最大吸收,雷公藤甲素在220nm有最大吸收,雷公藤甲素发生显色反应后在540nm有最大吸收;绘制雷公藤甲素在0.1539μg/ml~ 12.467μg/ml浓度范围和1.3852μg/ml~ 112μg/ml浓度范围的标准曲线,浓度与吸光度的线性关系良好,回归方程分别为:A = 0. 0128 C + 0. 0022( r = 0. 9984 ,n = 5)和A = 0. 0097 C + 0. 02( r = 0. 998,n = 5);测定雷公藤甲素在pH 7.0 PBS中溶解度约为21.55μg/ml;四种包封率测定方法对游离药物回收率分别为:107.1%、92.2%、107.8%、95.3%,对脂质体回收率分别为较好、不好、较好、好;
结论:对结果分析,雷公藤甲素在220nm处最大吸收与空白脂质体于202nm处最大吸收峰相近,在含量测定中易导致干扰,最终确定540nm作为雷公藤甲素的检测波长;PBS溶解度测定结果表明,雷公藤甲素在PBS中有微量溶解,对包封率测定方法的筛选提供了一定参考;经可行性分析,相对于其他包封率测定方法,超滤离心法对游离药物和脂质体回收率好,因此确定采用超滤离心法测定雷公藤甲素脂质体的包封率。
第二部分雷公藤甲素脂质体制备方法筛选及制备工艺的研究
目的:以包封率为指标,筛选TP-LP的最佳制备方法;以脂质体粒径、稳定性及包封率为指标优化TP-LP的制备工艺。
方法:分别采用薄膜分散法、机械匀浆法、乙醇注入法制备TP-LP,以包封率为衡量指标,采用超滤离心法分离脂质体和游离药物,测定包封率,筛选最佳制备方法;在空白脂质体的制备工艺研究中,以空白脂质体粒径和稳定性为指标,对制备条件:磷脂与胆固醇比例、氯仿用量及超声情况进行优化;在TP-LP制备工艺研究中,以包封率为衡量指标,采用正交优化方法优化影响脂质体包封率的三个制备条件:载药温度、药物与磷脂质量比(药脂比)和载药时间,采用超滤离心法分离脂质体和游离药物,紫外分光光度法测定游离药物含量,计算TP-LP的包封率,筛选最佳制备工艺,并验证最佳条件的准确性。
结果:制备方法的筛选中,薄膜分散法、机械匀浆法、乙醇注入法制备TP-LP的包封率分别为:12.7%、14%、13.6%;空白脂质体制备工艺研究中,磷脂-胆固醇比为4:1制备的脂质体稳定性好;超声100w/5min处理的脂质体粒径分布好;用20ml氯仿制备的空白脂质体粒径分布较好。TP-LP制备工艺研究中,经正交优化实验,制备条件:载药温度37℃、药脂比1:50、载药时间60min所制备的雷公藤甲素脂质体包封率最高;最后按最佳优化条件同时制备三批TP-LP,测定其平均包封率为46.4%±0.0295。
结论:以包封率为指标,比较三种制备方法,机械匀浆法制备的TP-LP包封率较高,经可行性分析,该方法简单、可重复性好,因此确定机械匀浆法制备TP-LP;空白脂质体单因素优化结果显示,制备最佳条件为:磷脂-胆固醇比例为4:1,超声100w/5min,氯仿用量20ml;TP-LP制备工艺正交优化结果显示,最佳制备条件为载药温度37℃、药脂比1:50、载药时间60min。最佳条件重复验证实验结果验证了正交优化方法的准确性。
第三部分雷公藤甲素脂质体的质量研究及体外释药的初步研究
目的:对TP-LP进行质量研究,为提高TP-LP稳定性研究奠定基础;进行TP-LP体外药效初步研究,为其体内药效研究奠定基础。
方法:按最佳工艺制备TP-LP,用透射电镜显微观察TP-LP形态,用动态光散射仪测定粒径分布,用zeta电位分析仪测定TP-LP的zeta电位,从以上几个指标对脂质体进行质量评价;分别加入不同冻干保护剂制备TP-LP冻干粉,以脂质体冻干粉的外观、复水情况及复水后包封率为衡量指标,筛选最佳冻干保护剂;在体外释药实验,采用透析法分别观察TP和TP-LP的体外释药情况,采用紫外分光光度法测定不同时间点的累计释药量;体外药效实验,用TP、TP-LP处理经PHA活化的人外周血单个核细胞,以MTT法为检测方法,分别于24h、48h、72h测定TP、TP-LP抑制T淋巴细胞增殖的效果。
结果:最佳优化工艺制备的TP-LP,粒径范围在40nm~250nm之间,平均粒径为89.2nm。粒径对称分布,分散均匀,zeta电位平均值为-67.55mV ,绝对值大于60mv;冻干粉的制备结果,5%蔗糖-甘露醇混合物(w:w=1:1)作为冻干保护剂所制备的冻干粉外观良好,复水过程良好,无肉眼结晶。相对于其他保护剂制备的冻干粉,复水后的包封率下降程度小;TP-LP的体外释药实验结果,44小时后,脂质体中TP全部释放,而游离TP在3h已完全释放;体外药效实验结果显示,24h TP-LP抑制T淋巴细胞增殖效果低于游离的TP;48hTP-LP的抑制效果与游离TP相当;72hTP-LP的抑制效果稍高于游离TP。
结论:TP-LP形态、粒径分布基本符合后续研究的要求;zeta电位结果显示,TP-LP稳定性相当好,有利于后续研究;体外释药结果显示,与游离的TP相比,TP-LP有一定的缓释作用;体外药效结果表明,TP经脂质体包裹后具有缓释功能。
Triptolide (TP), is the highest activity of isolated diterpene lactone epoxide compounds from traditional Chinese medicine Tripterygium wilfordii. Thunder triptolide played a significant role in the treatment of various autoimmune diseases, inflammatory diseases and cancer, meanwhile it cause adverse reaction in iateria and the clinical adverse events mainly were in the digestive system, urinary system, reproducti- vesystem, cardiovascular system, bone marrow and blood system,especially has high frequency in digestive system.Therefore, it is very significant to improve triptolide single dosage form, investigate new drug delivery system in order to reduce its toxicity. We have prepared triptolide liposome in order to synergia and Attenuation.
This study was divided into three parts as follows.
Part I: the prophas research of triptolide liposome’s preparation
Objective: We set up the method of measuring Triptolide (TP)and Triptolide liposome (TP-LP) content , and the triptolide solubility in PBS, in order to anaphase study of TP-LP.
Methods: We determined the maximum absorption of Triptolide and blank liposomes with UV-VIS wavelength scanning. Established standard curve of triptolide and measured the triptolide solubility by ultraviolet spectrophotometry ; Determined the encapsulation efficiency of TP-LP by gel filtration, high-speed centrifugation, dialysis and ultrafiltration centrifugal respectively, and analysis feasibility of the four methods.
Results: The wavelength scan results: the maximum absorption wavelength of blank liposomes is 202nm, triptolide is 220nm and the triptolide after color reaction is 540nm; Drawed the triptolide standard curve in 0.1539μg/ml~12.467μg/ml concentrat- ion range and 1.3852μg/ml ~ 112μg/ml respectively. The linear relationship between concentration and absorbance is good, regression equations were: A = 0. 0128 C + 0. 0022 ( r = 0. 9984, n = 5) and A = 0. 0097 C + 0. 02 (r = 0. 998, n = 5); The triptolide solubility in pH 7.0 PBS is about 21.55μg/ml ; The recovery rate of entrapment efficie- ncy with four method for free TP were: 107.1%, 92.2%, 95.3% and 107.8%, and the ultrafiltration centrifugal has good recovery rate for liposome.
Conclusions: Because of the wavelength between TP and blank liposome are adjoin, it is easy to cause interference within later period measure of entrapment efficiency, so determined the detect wavelength of TP is 540nm; Solubility test of TP showed that triptolide has trace dissolve in PBS, which provide a reference for the determination of entrapment efficiency. Through feasibility analysis, compared to other encapsulation methods, ultrafiltration has good recovery rate for free drug and blank liposome, so adopt ultrafiltration centrifugal for the determination of triptolide liposome encapsulation efficiency.
Part II : the study of liposome’s preparation method and technology
Objective: Determine the optimal preparation method of liposome according to entrapment efficiency, and optimized preparation technology of liposome .
Methods: Prepare TP-LP respectively with film dispersion method, mechanical homogenization and ethanol injection method. Separated free drug from liposome by ultrafiltration centrifugation inoder to calculate the encapsulation efficiency of TP-LP;
In the technology study of blank liposome, we optimized the preparation conditions ( proportion of phospholipids and cholesterol, volume of chloroform and ultrasound conditions) with single factor investigation on the basis of liposome size and stability. In the technology study of TP-LP, we optimized the preparation conditions(the ratio between drug and liposome, loading temperature and drug loading time) with orthogonality optimization on the basis of liposome entrapment efficiency as target.
Results: The entrapment efficiency of TP-LP which was prepared by film dispersion method, mechanical homogenization and ethanol injection method is respective 12.7%, 14% and 13.6%; The blank liposome via the ratio between phospholipid and cholesterol of 4:1 (w/w) is better stability than others, blank liposome size with 20ml of CHCl3 has better uniformity than 10ml liposomes, and the liposome size with ultrasonic 100w/5min is better uniformity ; The optimized preparation conditions is drug loading temperature 37℃, drug lipid ratio of 1:50 and the drug preparation time of 60min; Final the average encapsulation efficiency of TP-LP with optimal conditions was measured 46.4%±0.0295.
Conclusion: The entrapment efficiency of TP-LP which was prepared by mechanical homogenization is highest, so determine the preparation method is mechanical homogenization; The best preparation conditions of blank liposome are: the ratio between phospholipid and cholesterol of 4:1 (w/w), ultrasound 100w/5min, 20ml of chloroform; The best preparation conditions for TP-LP is drug loading temperature of 37℃, drug-lipid ratio of 1:50, drug loading time of 60min. Moreover, the replicated experimental results verify the accuracy of the orthogonal optimization method.
We also compare four measure method of entrapment efficiency, and carrying out feasibility analysis, determine that separate liposomes and free drug by ultrafiltration centrifugation, to survey free drug and calculate the encapsulation efficiency indirectly.
Part III: the study of liposome’s quality and release in vitro
Objective: Do quality research of TP-LP in order to improve the stability of TP-LP; Do study of TP-LP’s release in vitro in order to establish groundwork for their efficacy in vivo.
Methods: Prepared TP-LP with optimal conditions ,and measured liposome size via electron microscope, measured particle size distribution by dynamic light scattering, and determined zeta potential analysis. We also prepared freeze-dried powder of liposome with adding different cryoprotectant. Do release experiment of TP-LP by dialysis in vitro conditions, and measure cumulative release amount of TP by UV spectrophotometry at different time points; Finally, we designed the experiment to check the inhibition effect of TP-LP to T lymphocyte proliferation. Firstly, activate human peripheral blood mononuclear cells with the PHA, then detected the inhibition effect of TP-LP to T lymphocyte proliferation by MTT method respectively at 24h, 48h and 72h .
Results: The particle size of TP-LP is range of 40nm ~ 250nm, with an average diameter of 89.2nm; Symmetric particle size distribution, dispersed, zeta potential of mean-67.55mV; Compared with other cryoprotectant, the freeze-dried powders of TP-LP with adding 5% sucrose-mannitol (w: w = 1:1) has better appearance, and the process of rehydration is well without naked eye crystal, in addition to have higher entrapment efficiency. In the release experiments of TP-LP in vitro, TP had been released from TP-LP completely after 44 hours, but had been released completely from free TP after 3h; In the experiment of inhibition effect to T lymphocyte proliferation, the inhibition effect of TP-LP are lower than free TP after 24h, the inhibition effect of TP-LP are equality to free TP after 48h and the inhibition effect of TP-LP are higher than free TP after 72h.
Conclusion: In the morphology study of TP-LP, the particle size distribution of TP-LP were satisfied to the follow study; The results of zeta potential show that TP-LP has good stability; Prepare freeze-dried powder of liposome with adding 5% sucrose-mannitol (w: w = 1:1) as cryoprotectant; The release result of TP-LP show that TP-LP has delayed release compared with free TP,and the experiment of inhibition effect to T lymphocyte proliferation also authenticate delayed release of TP- LP.
引文
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