Preparation And In Vitro And In Vivo Evaluation Of Specific Targeting Hepatic Stellate Cells With Saikosaponins A/d Liposomes

The Chinese herb Chai Hu is the dried root of Bupleurum chinense DC or Bupleurum scorzonerifolium Willd^[1],which has a long history of application in the prevention and treatment of liver diseases and is supported by profound TCM theories,with the efficacy of reconciling the exterior and the interior,draining the liver and raising the yang,and the main active ingredients The main active constituents are saponins,of which Saponin a（SSa）and Saponin d（SSd）account for about 80%（1:1）^[2,3].Modern pharmacological studies have shown that chaihu saponins have significant antihepatic fibrosis effects^[4],and studies have confirmed that SSa and SSd are antagonistic to reverse the occurrence of hepatic fibrosis through multiple pathways such as anti-inflammatory,anti-hepatic stellate cell（HSCs）activation and antioxidant,SSa and SSd are isomers of each other,and combined application can increase the efficacy and produce synergistic effects^[5-7].Although SSa and SSd have significant efficacy in antihepatic Although SSa and SSd have significant efficacy in anti-fibrosis,they have many problems such as poor solubility,low bioavailability,and high hemolytic toxicity,which seriously limit their clinical application research and development^[8].Liposomes are small vesicles with bilayers,similar to biological membrane structures,and have a high encapsulation rate for both water-and lipid-soluble drugs,which are mainly taken up by cells through membrane fusion and endocytosis,and have become one of the excellent drug carriers for improving drug therapeutic effects.However,cholesterol（CHOL）in liposomes complexes with SSd,destroying its overall stability and limiting drug release into the body^[9,10].Meanwhile,common liposome delivery of SSa and SSd is susceptible to phagocytosis by the reticuloendothelial system（RES）^[11],resulting in reduced drug accumulation at the site of liver fibrosis lesions and a significant decrease in their targeting.In addition,it was confirmed^[12-14]that the key to blocking liver fibrosis lies in the inhibition of the activation of HSCs;therefore,the targeted delivery of SSa and SSd with significant anti-fibrotic effects to HSCs would be expected to achieve more desirable effects.In this paper,we construct VA-SSa/SSd-Lips with HSCs targeting based on the presence of a large number of retinol-binding protein（RBP）receptors expressed on HSCs^[15,16]and the specific binding of RBP to vitamin A（VA）.This delivery system can significantly reduce drug toxicity,enhance the accumulation concentration of drugs at the focal site,and improve drug bioavailability.Laying the foundation for the realization of targeted therapy for liver fibrosis.In the first part of this paper,DSPE-PEG-NH2 and retinoic acid（RA）were used as raw materials,EDC·HCl and NHS were used as catalysts to activate the carboxyl group of retinoic acid,and the ratio of each API dosage was adjusted to synthesize the targeting ligand DSPE by amide reaction to piggyback the VA structure on the phospholipid derivative DSPE-PEG-NH₂ which has good biocompatibility.-The structure of the product was identified and analyzed by hydrogen nuclear magnetic resonance（¹H-NMR）,and the molecular weight of DSPE-PEG-VA was verified by MALDI-TOF-MS,and the results showed that the targeting materials were successfully connected.The retinoid protein receptor（RBPR）-based VA-targeted hepatic stellate cell liposome delivery system VA-SSa/SSd-Lips（CHEM）was successfully prepared using a thin film dispersion ultrasound method.And the effect of VA-SSa/SSd-Lips（CHEM）on the inhibition rate of HSC-T6 cells at different DSPE-PEG-VA percentage densities was investigated by CCK₈ method,and the optimal targeting ligand concentration of 1%was screened.The second part of this paper examines the physicochemical properties（particle size potential,encapsulation rate（EE%）and stability）of the formulation VA-SSa/SSd-Lips.The preparation method was based on a thin film dispersion sonication method,where the drug was passively accumulated between the phospholipid bilayers of lipids by the action of lipophilic groups of phospholipids.The drug-lipid ratio was set at 0.1,and three methods for EE%determination were firstly screened and selected based on the results of physicochemical properties of the drug,and the EE%was determined by fisetin precipitation method.The DLE was measured as 97.56%（SSa）and 98.92%（SSd）;the drug loading capacity（DLC）was 4.57%（SSa）and 4.64%（SSd）,respectively.The in vitro release behaviors of SSa/SSd-Sol,SSa/SSd-Lips and VA-SSa/SSd-Lips were investigated by dynamic dialysis,and the results showed that common SSa/SSd-Sol released 89.53%（SSa）and 89.89%（SSd）within 24 h,and the drug release was basically complete;SSa/SSd-Lips and VA-SSa/SSd-Lips released relatively little within 24 h,and VA-SSa/SSd-Lips released relatively slowly during this period.Therefore,the release rate of SSa/SSd-Lips and VA-SSa/SSd-Lips was relatively slow compared to SSa/SSd-Sol with the encapsulation of drug delivery system.The appearance,particle size,zeta potential and morphology of the VA-SSa/SSd-Lips formulation were investigated by dynamic light scattering（DLS）and TEM,and VA-SSa/SSd-Lips exhibited a clear and transparent liquid substance with white opalescence,a particle size of about 140 nm,a narrow particle size distribution and a negative charge,and the morphology was observed by TEM to be sphere-like.In the third part of this paper,a high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）analytical method was developed to determine SSa/SSd concentrations in rat plasma and to investigate the pharmacokinetic behavior of SSa/SSd solution,SSa/SSd-Lips and VA-SSa/SSd-Lips in rats,as well as to investigate the in vivo pharmacokinetic behavior of SSa/SSd by in vivo animal imaging and in vitro cellular uptake.The targeting of the three formulations of SSa/SSd was investigated by in vivo animal imaging and in vitro cellular uptake.The results of pharmacokinetic experiments showed that SSa/SSd and the internal standard（Panax ginseng saponin R1）were well separated and did not interfere with the determination of SSa/SSd,and had the advantages of short determination time,high sensitivity and good reproducibility,which can be used to determine the plasma drug concentrations in rats after the administration of SSa/SSd solution,SSa/SSd-Lips and VA-SSa/SSd-Lips.It can be used to determine the plasma drug concentrations in rats after SSa/SSd solution,SSa/SSd-Lips and VA-SSa/SSd-Lips administration,and to evaluate the distribution and metabolism of the drugs.The results after tail vein injection of SSa/SSd solution,SSa/SSd-Lips and VA-SSa/SSd-Lips in rats showed that SSa and SSd in the free group SSa/SSd-Sol were eliminated faster in mice,and the t1/2 of SSa and SSd in the SSa/SSd-Sol group were 0.958 and 0.759 h,respectively,and the t1/2 of SSa/SSd-Lips were 1.17（SSa）and 1.25（SSd）times higher than those of the SSa/SSd-Sol group,while the t1/2 of VA-SSa/SSd-Lips were 2.46（SSa）and 3.19（SSd）times higher than those of the SSa/SSd-Sol group,respectively;and after administration of VA-SSa/SSd-Lips,t1/2 was significantly higher than that of SSa/SSd solution,indicating that VA-SSa/SSd-Lips had some slow-release effect.In vivo live imaging examined that the fluorescence of VA-C6-Lips preparation was mainly concentrated in the liver region and less distributed in other organs,and compared with C6-Lips,VA-C6-Lips had significant liver tropism.Rat hepatic stellate cells were selected and the uptake of SSa/SSd-Sol,SSa/SSd-Lips and VA-SSa/SSd-Lips by activated hepatic stellate cells was observed by fluorescence microscopy,and the results showed that the fluorescence intensity of the VA-SSa/SSd-Lips group was significantly stronger than that of the SSa/SSd-Lips group,and the free SSa/SSd group fluorescence intensity was the weakest.Fluorescence co-localization experiments were performed using laser confocalization,and the results showed that the targeting group preparation VA-SSa/SSd-Lips group was more efficient in targeting activated hepatic stellate cells compared to the non-targeting group SSa/SSd-Lips,and the results also showed that the new membrane-stabilized CHEM had little effect on the targeting of HSCs compared to the common membrane stabilizer CHOL,and finally,quantitative analysis by flow cytometry The targeting efficiency of VA-SSa/SSd-Lips（CHEM）,SSa/SSd-Lips,and VA-SSa/SSd-Lips（CHOL）on HSCs was finally quantified by flow cytometry.The combined results indicate that VA-SSa/SSd-Lips（CHEM）has a good targeting effect on HSCs.In the fourth part of this study,a mouse model of liver fibrosis was established with 20%carbon tetrachloride solution,and the therapeutic effects of high and low concentrations of VA-SSa/SSd-Lips preparation groups on liver fibrosis were evaluated from serum biochemical indexes（AST/ALT）,liver pathological examination（H&E staining/Masson staining）and liver immunohistochemistry（α-SMA/COIⅠ）to investigate the effect of VA-SSa/SSd-Lips anti-liver fibrosis mechanism.From the results of ALT and AST experiments,it was found that compared with the saline group,the AST and ALT in the serum of mice in the modeling group（without drug intervention treatment）were significantly elevated（P<0.05）,indicating a higher level of inflammatory factors in the modeling group;while after treatment with high and low concentrations of VA-SSa/SSd-Lips preparation and positive drug group（silymarin）,the serum of rats in each group showed a significant decrease in AST and After treatment with high,medium and low concentrations of VA-SSa/SSd-Lips and silymarin,the serum levels of AST and ALT in rats in each group decreased to different degrees,and compared with the modeling group,ALT in the positive drug group decreased significantly;compared with the modeling group,AST and ALT levels in the high,medium and low concentrations of VA-SSa/SSd-Lips preparations decreased,and the difference was significant（P<0.01）.Secondly,the liver tissue staining of the mice in the saline group（unmodified group）showed that the liver lobules in the H&E stained tissue sections were intact,the liver structures were regularly arranged,no fibrous intervals were formed,and there was no tissue cell damage.In contrast,H&E staining of liver tissue in the modeling group showed significant neutrophil infiltration and large areas of tissue cell damage and death;Masson staining microscopically showed a large number of collagen fibers proliferation collagen fiber band formation and pseudobullet formation.After treatment by the high and low VA-SSa/SSd-Lips preparation group,the collagen proliferation was significantly improved.By semi-quantitative analysis using Image J,it was found that the area of collagen discoloration was significantly higher in the modeling group than in all other groups（P<0.05）.The collagen chromogenic area of each group was reduced to different degrees after treatment.In this experiment,the collagen chromogenic area of the positive drug group was reduced to a lesser extent than that of the VA-SSa/SSd-Lips preparations at high,medium and low concentrations,and the smallest collagen chromogenic area could be found in the low dose group,but the difference between the collagen chromogenic areas of the three dose groups was not significant.In the immunohistochemical study,the mouse liver tissue was positive forα-SMA as well as COIⅠimmunohistochemical staining brownish yellow.A slight positive expression was observed in the normal group（saline group）;whereas the positive expression ofα-SMA and COIⅠwas significantly increased in the modeling group.According to the semi-quantitative analysis of brown expression on immunohistochemically stained slices,the expression was significantly increased in the modeling group compared with the normal group（P<0.05）;the expression was reduced to varying degrees in each of the treated preparation groups.There was a relative decrease in the brownish yellow region in the positive drug group（P<0.05）and the low-intermediate and high-concentration groups（P<0.05）compared with the modeling group.The cytostatic assay（CCK₈）examined the cytotoxicity of blank liposome material as well as novel membrane stabilizers and the in vitro anti-liver fibrosis activity of drug-loaded liposomes,and the blank liposomes modified by DSPE-PEG-VA were less cytotoxic;VA-SSa/SSd-Lips（CHEM）（IC50=31）was less toxic than SSa/SSd-Lips（IC50=53）as well as free SSa/SSd solution（IC50=43）,while the cytotoxicity of the targeted liposome formulation VA-SSa/SSd-Lips（CHOL）（IC50=41）,a common membrane-stabilized construct,was not significantly different from that of free SSa/SSd solution.The combined results suggest that VA-SSa/SSd-Lips（CHEM）has a good inhibitory effect on HSCs.The in vitro hemolysis study and in vivo safety evaluation experiments showed that VA-SSa/SSd-Lips has a good safety profile.