Study Of Oral Liposome Loaded With Ginsenoside Compound K For Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a systemic autoimmune disease which can cause chronic inflammation and joint deformity.The main disease features include joint swelling and synovial hyperplasia.At present,the pathogenesis of RA is still not fully understood,and it is currently believed that the pathogenesis of RA involves different immune system mechanisms,resulting in a variety of immune responses.A large number of activated macrophages will accumulate in the diseased site of RA and secrete pro-inflammatory cytokines to aggravate the pathogenesis of RA.Therefore,activated macrophages are a target for RA therapy.Ginseng has a variety of medicinal values,and its main component,ginsenosides,exerts most of the pharmacological effects.Ginsenoside CK（Ginsenoside compound K,GCK）is a diol-type ginsenoside,which is converted from natural diol-type ginsenosides such as Rb1,Rb2,and Rc by deglycosylation of gastrointestinal flora.Current studies have shown that GCK can inhibit the secretion of pro-inflammatory cytokines and have immune and anti-inflammatory functions,thereby exerting a therapeutic effect on RA.However,its low water solubility,poor permeability to the intestinal cell membrane,and serious intestinal efflux have limited its application.Liposomes are commonly used nano-drug carriers with low toxicity,which can be further improved for drug delivery by various modifications on their surfaces.Vitamin E polyethylene glycol succinate（D-alphatocopheryl polyethylene glycol succinate,TPGS）,which is esterified from vitamin E succinic acid and polyethylene glycol,can enhance cellular uptake of drugs and inhibit Pglycoprotein mediators.guided drug transport.Targeted modification of liposomes can specifically deliver drugs to diseased sites,thereby improving efficacy.Folate receptors are highly expressed in activated macrophages at the site of RA disease and are a target for RA therapy.Therefore,folic acid（FA）-modified liposomes can accumulate at activated macrophages,thereby improving the therapeutic effect.Therefore,in this paper,a folic acid-targeting liposome was designed,its physicochemical properties were investigated,and the treatment of RA in vitro and in vivo was studied.1.Establishment of in vitro methodology of ginsenoside CKIn this study,GCK was quantitatively analyzed by high performance liquid chromatography,and the methodology was validated.The experimental results show that in the range of 5.00-200.00 μg/m L,the standard curve R2 of GCK is greater than 0.999,and the linear relationship is good.The method validation results showed that GCK had good specificity,and the relative standard deviations of the precision,blank recovery and stability test results within 24 hours were all less than 2.00%.The experimental results show that the analytical method has good precision and stability,and can be used to detect the concentration of GCK in liposomes.2.Preparation and characterization of ginsenoside CK liposomesIn this study,ginsenoside CK liposomes（LP-GCK）were prepared by ethanol injection,and the conditions of particle size,PDI,potential,and encapsulation efficiency were investigated from three aspects: phosphorus-cholesterol ratio,TPGS concentration,and lipid-drug ratio.indicators to filter the best prescriptions.The results show that when the ratio of phosphorus to bile is 2:1,the concentration of TPGS is 14%,and the ratio of lipid to drug is 8:1,the liposome encapsulation efficiency is the largest,the particle size is suitable,and the dispersibility is good,which is the best liposome.prescription.At this time,the particle size of the liposome was 221.1±2.8 nm,the encapsulation efficiency was94.46±0.22%,and the PDI was less than 0.2.Using DSPE-m PEG-FA to replace the amount equivalent to 10 percent TPGS molar ratio for folic acid-targeted preparation of folic acid-targeted liposomes（FA-LP-GCK）,the measured particle size of FA-LP-GCK liposomes was 249.13±1.40 nm,the encapsulation efficiency was 92.86±0.28%,and the PDI was less than 0.2.The morphology of LP-GCK and FA-LP-GCK liposomes was smooth and spherical.The addition of FA made the phospholipid layer of the liposomes thicker and the particle size increased slightly.The in vitro release results showed that FALP-GCK liposomes had a certain sustained release effect,had certain stability in gastrointestinal fluid,and could be more absorbed by the intestinal tract in the form of liposomes to enter the blood circulation.3.In vitro evaluation of ginsenoside CK liposomesMouse mononuclear macrophage leukemia cells（RAW 264.7）are a commonly used model to study inflammation.First,the safety of the carrier was evaluated by cytotoxic experiments.The results showed that the empty liposome had almost no growth inhibitory effect on cells within 24 h,indicating that the liposome was a safe drug delivery carrier.Next,the cytotoxicity of different concentrations of GCK,LP-GCK,and FA-LP-GCK was investigated,and the results showed that FA-LP-GCK liposomes exhibited stronger growth inhibitory effects on activated macrophages within 24 h.In the cell uptake experiment,laser confocal microscopy intuitively showed that with the increase of time,the green fluorescence of both LP-GCK and FA-LP-GCK groups continued to increase,indicating that the uptake of liposomes by cells continued to increase,and at the same time The green fluorescence of FA-LP-GCK group was stronger than that of LP-GCK group.The results of flow cytometry showed that after the cells were incubated with the drug for 4 h,the fluorescence intensity of the FA-LP-GCK group was about 2 times that of the LP-GCK group,which proved that the activated RAW 264.Sexual enhancement.4.In vivo evaluation of ginsenoside CK liposomesThe effect of liposomes in the treatment of RA was evaluated by establishing a complete adjuvant-induced rat arthritis model.The results showed that the FA-LP-GCK group had significantly less joint swelling and lower arthritis scores compared with the saline,GCK,and LP-GCK groups.The determination of inflammatory factors in serum of rats and the observation of joint histopathology further showed that the FA-LP-GCK group could significantly inhibit the expression of pro-inflammatory factors and significantly improve synovial joint hyperplasia.In addition,the FA-LP-GCK group could significantly reduce the spleen index and improve the pathological changes of the spleen in rats.It can be seen from rat liver slices that liposomes have no toxic and side effects on rats,and are a safe and reliable way of administration.In conclusion,the FA-LP-GCK liposomes prepared in this study have suitable particle size and high encapsulation efficiency.In vitro experiments show that it has a strong growth inhibitory effect and cell uptake ability on activated macrophages.In vivo experiments showed that FA-LP-GCK liposomes can effectively inhibit the incidence of RA without obvious toxic and side effects.Therefore,FA-LP-GCK liposome is a potential new way to treat RA.