Preparation Of A Novel Liposome Loaded With Cantharidin Based On Glycyrrhizic Acid And Its Anti Hepatoma Effect

Cantharidin(CTD)is an essential bioactive ingredient isolated from poisonous traditional Chinese medicine(PTCM)Mylabris with distinctive therapeutic efficacy against hepatocellular carcinoma(HCC),lung cancer and breast cancer.Nevertheless,the strongly irritation and corrosive actions of CTD on the gastrointestinal and urinary system limits its clinical application.Currently,there are mainly two approaches to address these problems.Firstly,several CTD derivatives have been synthesized by chemical structural modifications,which contribute to the reduction of toxicity,but with less biological activity than CTD.Secondly,CTD is encapsulated in drug carriers by modern formulation techniques,which can reduce toxicity while retaining the original drug activity,thus receiving widespread attention.Liposome,a special carrier similar to biological membranes,has non-immunogenic characteristics and high loading capacity.The application of cholesterol,which are usually used to stabilize the structure of liposomes,has been questioned due to various disadvantages.The oxidation products of cholesterol are prone to mutagenicity and potential carcinogenicity,and the enrichment of cholesterol is prone to trigger "complement mediated pseudoallergic reactions".The novel drug delivery system designed by replacing cholesterol with natural active substances such as sterols and saponins can avoid the problems caused by cholesterol and has unique superiorities.For example,a novel liposome prepared using ginsenoside Rg3 instead of cholesterol not only improves the stability and targeting ability of liposomes,but also enhances the efficacy of chemotherapy drugs by regulating the tumor microenvironment and synergistically killing cancer cells.Glycyrrhizic acid(GA)has various biological functions,such as anti-inflammatory,anti-tumor and hepatoprotective effect.Also,GA has the ability to enhance the stability of phospholipid bilayer.Moreover,the delivery carrier modified by specific ligand GA can realize the active targeting of drugs on hepatoma cells to reduce the side effects on normal tissues.Based on the excellent physicochemical properties and pharmacological activities of GA,an innovative GA-LP system was developed using GA instead of cholesterol for delivering CTD(CTD-GA-LP)to effectively overcome the obstacles faced by cholesterol in common liposomes,and to achieve the purpose of enhancing the anti-tumor effect of CTD and reducing its systemic toxicity.Preparation and process optimization of CTD-GA-LP.With mean size,PDI,potential,encapsulation efficiency(EE)and loading efficiency(LE)as evaluation indices,the liposomal prescription was determined by method selection,single factor test and Box-Behnken experiment.The preparation method was ethanol injection method,the mass ratio of phospholipid(EYPC)to GA was 20:7,the mass ratio of EYPC to CTD was 10:1,the concentration of ethanol was 90%,the volume of organic phase was10 m L,and the volume of aqueous phase was 10 m L.The time and power of ultrasound were 10 min and240 W,respectively.Quality evaluation of CTD-GA-LP.The mean size,PDI and potential of liposomes were measured using a laser particle size analyzer.The EE and LE were determined by ultrafiltration centrifugation-HPLC and freeze drying methods.Transmission electron microscopy(TEM)was used to observe the morphology of liposomes,and the safety and drug release behavior were investigated by hemolysis and dialysis methods.The mean size of CTD-GA-LP was(76.12±4.01)nm,the PDI was0.18±0.02,the potential was(-27.47±1.45)m V,the EE and LE were(90.81±3.32)% and(6.24±0.21)%.It was found that all liposomes were spherical structure with smooth surface.CTD-C-LP has flocculation and precipitation,increased size and PDI,and serious drug leakage within 15 days of storage.CTD-GA-LP has excellent stability,and the size,PDI change and drug leakage were negligible.After co-incubation with BSA,0.9% Na Cl and 200 U/m L heparin sodium,the size distribution of CTD-GA-LP remained unchanged.CTD-GA-LP has good biocompatibility,with a hemolysis rate of less than 5% in the range of 0.2-2 mg/m L.CTD-GA-LP presented a sustained and slow release characteristics in p H 7.4 medium,and accelerated drug release rate in p H 5.4 medium,while the drug release of CTD-C-LP in different p H media was not different.In vivo and in vitro anti-hepatoma studies of CTD-GA-LP.MTT assay was used to evaluate the effect of CTD-GA-LP on the activity of L-02 and Hep G2 cells,and the apoptosis of Hep G2 cells was observed by DAPI and AO/EB staining.Hepatocarcinoma tumor(H22)model was established to assess the therapeutic efficiency of CTD-loaded liposomes in vivo.After treatment of L-02 cells for 48 h,CTD significantly inhibited cell growth,and the cytotoxicity of CTD-C-LP and CTD-GA-LP was significantly reduced.The cell activity of CTD-GA-LP was significantly increased,and the IC50 was(5.94±0.57)μg/m L,3.71 and 1.73 times that of CTD and CTD-C-LP.After treatment with Hep G2 cells for 48 h,CTD-GA-LP showed the strongest inhibitory effect with IC50 of(2.37±0.27)μg/m L,which was 2.40 and 1.69 times lower than CTD and CTD-C-LP.CTD-GA-LP exhibited a stronger effect on promoting tumor cell apoptosis compared with the same dose of CTD and CTD-C-LP.CTD-GA-LP exhibited strong killing ability on cancer cells,and the tumor inhibition rate reached 74.49%,which was 2.12 and 1.33 times of that of equal-dose CTD and CTD-C-LP.The necrotic area of tumor tissues was significantly larger than that of CTD and CTD-C-LP.Also,CTD-GA-LP significantly improved the prognosis and survival status of mice,promoted the organ coefficient of mice to normal levels and prolonged the survival time.In vivo and in vitro targeting studies of GA-LP.Coumarin 6(C6)or DIR were used as simulated drugs to replace non-fluorescent CTD for preparation of liposomes loaded with fluorescent dyes.The uptake of C6,C6-loaded C-LP and GA-LP by L-02,Hep G2 and H22 cells was estimated by fluorescence microscopy and multifunctional fluorescence enzyme marker.The fluorescence distribution of DIR,DIR-loaded C-LP and GA-LP in vivo was real-time observed via small animal imaging.After incubation for 2 h,the fluorescence intensity of GA-LP in Hep G2 and H22 cells was about 1.74 and 2.24 times that of C-LP,which significantly increased the intracellular drug accumulation.After pretreatment with abundant free GA,the uptake of GA-LP was inhibited,while the uptake of C-LP was not affected,suggesting that GA-LP was actively transported into the cells through the endocytosis pathway mediated by GA receptors.The inhibitory effect of free GA on uptake of GA-LP by Hep G2 and H22 cells was strong,while the inhibitory effect on L-02 cells was relatively weak,indicating that high expression of GA receptors on the surface of cancer cells.In vivo results showed that the fluorescence of GA-LP in liver and tumor tissue was 1.71 times and 3.53 times of C-LP.GA-LP can promote the accumulation of DIR in tumor and prolong the circulation time in the vivo.In this study,a novel GA-LP system was developed utilizing GA as a cholesterol replacement.GA-LP can not only increase the stability of liposomes,but also enhance the tumor targeting efficiency of drugs via active targeting mediated by GA receptor.CTD-GA-LP obviously increased the accumulation of drug by effectively deliver CTD to the tumor site in comparison with injection CTD-C-LP,maximize in vitro cytotoxicity and in vivo tumor inhibition efficacy of CTD,and minimize the side effect of CTD on normal cells and tissues.