Preparation And In Vitro And In Vivo Evaluation Of Saikosaponin A, Saikosaponin D Compound Liposomes Targeting Star Cells

Objective:1.The previous research results showed that both of Saikosaponin a (SSa) and Saikosaponin d (SSd) were effective material basics of the total saikoside of Radix Bupleuri in anti liver fibrosis. It was speculated that SSd in combination with SSa according to mass ratio of 1:1 would have obvious synergies effects to treat fibrosis of the liver, and the assumption would be verified the rationality by cell experiments and animal experiments in our studies.2.Based on the efficacy results and basic characteristics of SSa and SSd, the SSa and SSd compound liposome delivery system for targeting hepatic stellate cell (HSC) were designed and prepared to reduce the hemolysis and improve security.3.The rationalities of the pharmaceutical dosage form were evaluated by the drug-release effects in vivo and liver targeting characteristics through the pharmacokinetics and nude mice in vivo imaging technology.4.The HSC targeting characteristics of the modified liposome were studied to further evaluate on the rationalities of the pharmaceutical dosage form. The studies could also preliminarily clarify the SSa and SSd composite synergy mechanism of anti liver fibrosis.Method:The strength of the inhibited effects of SSa and SSd mixture on HSC-T6 by MTT, the synergy anti liver fibrosis effects of SSa and SSd mixture by using liver fibrosis models induced by CCl4 and DMN, were studied to determine the mass ratio of SSa and SSd. The hemolysis in vitro, the oil-water partition coefficient and stabilities of SSa and SSd were studied. The Plackett-Burman design was used as an efficient tool to screen for significant factors among a large number of variables while minimising the number of experiments. After finding the critical factors, a Box-Behnken design was chosen to optimise the actual values of these process factors combined with a desirability function. The optimal formulation and process of VA modified SSa-SSd-Lip were studied and the VA binding ratio was evaluated. The pharmacokinetics of VA-SSa-SSd-Lip in rats was determined by LC/MS/MS and the liver targeting characteristics were evaluated by nude mice in vivo imaging technology. The HSC targeting characteristics was determined by the inhibited effects of VA-SSa-SSd-Lip on HSC-T6 by MTT. The apoptosis of HSC-T6 induced by VA-SSa-SSd-Lip was studied by using flow cytometry. In order to preliminarily clarify the SSa and SSd composite synergy mechanism of anti liver fibrosis, Western blot was used to detect the important proteins expression associated with liver fibrosis.Results:1.Both SSa and SSd have obvious inhibiting effects on the cell proliferation of HSC-T6. The levels of inhibition effects were concerned with the concentrations of drugs. In other words, inhibition effect increased with increasing concentration of drugs. The results showed that SSd has stronger inhibiton effect on HSC-T6 than SSa. Different proportions of SSa and SSd mixture have different levels of inhibition effect, the weakest when the ratio of 1:2, the ratio of 2:1 was second, the strongest when the ratio of 1:1 and even more than same dosage of SSd. When the concentration of SSa and SSd were both 120μg·L-1, the inhibition rate of HSC-T6 was 96.35%. The liver fibrosis model induced by CCl4 experiments showed that parts of the index of SSa.SSd group were better than that of SSa group or SSd group, or even better than positive medicine control group. Parts of the index of SSd group, the same as positive medicine control group, were better than that of SSa group. It was indicated that the joint application of SSa and SSd has certain synergy. The liver fibrosis model induced by intraperitoneal injection of porcine serum showed that the rat liver coefficient of model rats in all treated groups could be significantly reduced, and the concentrations of HyP、HA、 LA、TNF-α、 TGF-β and PDGF in the serum and tissue homogenate were changed with different degrees. Compared with other dosage group, rat liver tissue lesions in SSa.SSd group was improved the most, liver hyperplasia of fibrous tissue was alleviated, liver fibrosis area was reduced, and no false flocculus formed. The researches were further confirmed that SSd in combination with SSa according to mass ratio of 1:1 would have obvious synergies effects to treat fibrosis of the liver.2.Based on the basic characteristics of SSa and SSd, we designed and prepared VA modified SSa-SSd-Lip. The researches of basic characteristics of SSa and SSd demonstrated that:(1) Hemolytic properties of SSa and SSd have showrn strong, and SSd were stronger than SSa; (2) the Oil-water partition coefficients showed that SSa and SSd were hydrophilic and lipophilic drugs; (3) SSa and SSd in neutral alkaline environment showed good stability. The researches above have laid a good foundation for the subsequent design of drug delivery system. The results of liposome preparation technology as follows:(1) the film dispersion method was used for the preparation of SSa and SSd compound liposomes; (2) Encapsulation efficiency of SSa and SSd, particle size and hemolysis were selected as evaluation indexes, the Plackett-Burman design was used as an efficient tool to screen for significant factors and Box-Behnken design was chosen to optimise the actual values of three process factors; (3) the ideal value predicted by Box-Behnken design was verified by experiments; (4) By comparing the pre-inserting and post-inserting methods, the results showed that the VA binging rate of VA-SSa-SSd-Lip prepared by post-inserting method was higher, and the entrapment efficiencies of SSa and SSd were unaffected; (6) the type and quantity of cryoprotectants were optimized and high entrapment efficiencies and stability of VA-SSa-SSd-Lip freeze-dried powder were prepared.3.The pharmacokinetic properties of different dosage forms by using LC/MS/MS showed that SSa and SSd have similar pharmacokinetic properties in vivo, after the rats was injected SSa-SSd-Lip, SSa-SSd-Sol and VA-SSa-SSd-Lip respectively, because of the similar structure, similar physical and chemical properties. The elimination rate of SSa and SSd in SSa-SSd-Lip group and VA-SSa-SSd-Lip group were lower and elimination half life were greater than that in SSa-SSd-Sol group. It indicated that both SSa-SSd-Lip and VA-SSa-SSd-Lip have certain sustained-release effects. MRT, CL. AUC0-t and AUC0-∞ of SSa and SSd in SSa-SSd-Lip group and VA-SSa-SSd-Lip group were similar, but Vc in VA-SSa-SSd-Lip group was smaller which indicated that the VA-SSa-SSd-Lip may have more targeted. Nude mice imaging studies showed that fluorescence determination of nude mice injected with saline blank group without interference; nude mice injected DiR-Sol did not reflect specific organs of targeting; the intensity of fluorescence of nude mouse liver area was stronger than other areas when nude mouse was injected DiR-Lip and VA-DiR-Lip respectively, and remaining time of the fluorescence were longer than DiR-Sol, within 72 hours fluorescence can be still detected in liver area. The intensity of fluorescence of nude mouse liver area after 72 hours could be detected stronger in VA-DiR-Lip group than that in DiR-Lip, and the liver distribution time was shorter in VA-DiR-Lip group, which indicated that VA-DiR-Lip was more easily absorbed by liver. In addition, after the injection of VA-DiR-Lip 12 hours, fluorescence was mainly distributed in nude mice liver area and difficult to be detected in other areas, but fluorescence; on the contrary, fluorescence distribution area in vivo was significantly greater after the injection of DiR-Lip. The researches above indicated that VA-DiR-Lip had showed better liver targeting property than DiR-Lip.4.The proliferation rates of HSC-T6 induced with different concentrations of SSa-SSd-Lip, SSa-SSd-Sol and VA-SSa-SSd-Lip were significantly reduced with the control group, and the inhibition of strength were dose-dependent. On the condition of the same dose, the inhibitory effect of VA-SSa-SSd-Lip was the strongest, followed by SSa-SSd-Lip, the inhibitory effect of SSa-SSd-Sol was weakest, and the inhibitory time of VA-SSa-SSd-Lip was longest. Flow cytometry results indicated that the dosage groups compared with blank group have significant difference; apoptosis effects of HSC-T6 induced by VA-SSa-SSd-Lip were related to the added amount of RBP, in other words, the stronger the addition amount, the greater the apoptosis effect. After adding RBPAb, because of its antagonism of RBP activation RBPR, apoptosis effects of HSC-T6 induced by VA-SSa-SSd-Lip were weak. Whether or not to join RBP and RBPAb has no effect on the apoptosis of HSC-T6 induced by SSa-SSd-Lip. Western Blotting results showed that all of SSa-SSd-Sol, SSa-SSd-Lip and VA-SSa-SSd-Lip had the inhibitory effects on the expressions of CTGFn TGF-β1、α-SMA and PDGF in HSC-T6 after 48 hours, the inhibitory effect of VA-SSa-SSd-Lip was the strongest, followed by SSa-SSd-Lip, the inhibitory effect of SSa-SSd-Sol was weakest. All of CTGF, TGF-β1, a-SMA and PDGF secreted by activated HSC-T6 are key proteins associated with liver fibrosis. By inhibiting the expression of these proteins to inhibit the activity of HCS-T6, VA-SSa-SSd-Lip gave play to the role of good anti liver fibrosis.Conclusion:Researches showed that SSd in combination with SSa according to mass ratio of 1:1 have had obvious synergies effects to treat fibrosis of the liver. High entrapment efficiencies, good stability of SSa-SSd-Lip and VA-SSa-SSd-Lip were prepared and have delayed the drugs release, reduced hemolysis, and showed a good liver-targeting efficiency in vivo. The HSC-T6 targeting characteristics of were verified by cytological experiments in vitro. VA-SSa-SSd-Lip could inhibit HSC-T6 activity and induce its apoptosis, and has significant intervention effects on the important protein of MAPK associated with liver fibrosis, and therefore the anti-liver fibrosis mechanism of SSa and SSd was preliminary clarified. The results of the study have reached the expected purpose.