Preparation And Quality Study Of Triptolide Liposome

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 preparationObjective: 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 technologyObjective: 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 vitroObjective: 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.