Construction Of Compound Immunoliposomes Loaded With Melittin And Bufalin And Its Synergistic Mechanism In Inhibiting Sorafenib Resistance In Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma?HCC?is one of the major cancers that attract worldwide attention.As one of the health concern in China,the incidence of HCC ranks fourth among all kinds of cancers?466,100 new cases per year?,and the mortality ranks third?422,100 deaths per year?.The number of new cases and deaths accounts for more than half of the total number of cases in the whole world.HCC not only brings scourges of high mortality rate to the patients,but also heavy financial burden to families and individuals.Sorafenib is the first-line drug for patients with advanced HCC,but it is prone to developing drug resistance.Acquired drug resistance of Sorafenib usually occurs within 6 months of treatment.In our previous research,we found that both bufalin and melittin exhibit very strong anti-HCC effects,and their mechanisms are different.Therefore,the combined application of bufalin and melittin is expected to play a synergistic role in anti-HCC,especially in inhibiting Sorafenib resistance of HCC.However,the combined use of bufalin and melittin must overcome two key problems:?1?melittin has strong hemolytic side effects;?2?bufalin has low water solubility.In view of this,we chose the compound immunoliposome technology to achieve the co-loading of melittin and bufalin,and to further achieve the targeting of HCC.This compound immunoliposome is expected to play a synergistic role in inhibiting Sorafenib resistance of HCC.Methods1.Referring to Dr.Liu Dong's method of constructing Sorafenib acquired drug-resistant cell line,we constructed Sorafenib-resistant SMMC-7721 cells?SMMC-7721-R cells?.In addition,we verified the drug resistance of SMMC-7721-R cells in vitro and in vivo.In vitro,CCK-8 and flow cytometry were used to detect the proliferation inhibition and apoptosis resistance of both sensitive and drug-resistant cell lines.In vivo,Balb/c nude mice were used to construct subcutaneous transplanted tumor models of both sensitive and drug-resistant cell lines.And the inhibition of Sorafenib on these two transplanted tumor models was also studied and observed.2.CCK8 and flow cytometry were used to investigate the synergistic killing effect of melittin and bufalin on SMMC-7721-R cells.Compu Syn was used to screen the optimum proportion of melittin and bufalin in inhibiting SMMC-7721-R cells.Flow cytometry was used to detect the apoptotic rate of the selected drugs.These laid a foundation for the construction of the subsequent compound immunoliposomes.3.SMMC-7721-R cells were treated with single drug group of concentration(close to melittin and bufalin IC₅₀)treatment,PBS treatment was used in blank control group,1.0?mol/L melittin+0.2?mol/L bufalin treatment group was used in combined drug treatment group.After 48 hours of treatment,RNA was extracted and its quality and concentration were detected and frozen.The samples were sequenced by RNA-Seq transcription group.The mechanism of synergistic inhibition of melittin and bufalin on Sorafenib-resistant hepatocellular carcinoma was preliminarily screened by analyzing the results of detection.4.We established an HPLC method for simultaneous determination of melittin and bufalin.In addition,we investigated the properties of melittin and bufalin in equilibrium solubility,oil-water partition coefficient and Zeta potential.At the same time,we also use bioinformatics methods to obtain relevant data from Oncomine and TCGA databases.And we used R language to analyze the results and got the expression of HER1 in human clinical HCC samples and the impact of HER1 on prognosis of HCC.These provided a basis for the determination of antibodies linked by compound immunoliposomes.5.On the basis of the known physical and chemical properties of melittin and bufalin,the encapsulation process of melittin and bufalin was systematically optimized by single factor investigation,orthogonal experiment and response surface optimization methods by taking encapsulation efficiency and particle size as the main quality control indexes.On this basis,the physicochemical properties and stability of the optimized compound immunoliposomes were further evaluated.The physicochemical properties and stability of the compound immunoliposomes were investigated from the aspects of appearance,Zeta potential,leakage rate,drug release behavior,oxidation stability,hydrolysis stability,toxicity of blank liposomes,hemolysis and serum stability.6.The effect and mechanism of compound immunoliposomes on Sorafenib-resistant HCC were studied in vitro and in vivo.In vitro,we first evaluated the complement-dependent cytotoxicity?CDC?and antibody-dependent cell-mediated cytotoxicity?ADCC?of compound immunoliposomes in vitro,and investigated their effects on the proliferation inhibition and apoptosis of Sorafenib-resistant HCC cell lines.On this basis,combined with RNA-Seq transcriptome results,Western Blot was used to detect the related signaling pathways.In vivo,we constructed the SMMC-7721-R hepatoma nude mice model to evaluate the anti-tumor effect of the compound immunoliposomes.The toxicity of the compound immunoliposomes to the main organs of the tumor-bearing nude mice was investigated by HE staining of the main organs.Finally,the expression level of the protein in the tumor tissues was further verified by Western Blot.Results1.The acquired Sorafenib-resistant HCC cell line?SMMC-7721-R?constructed by us not only showed significant resistance to Sorafenib in vitro proliferation inhibition and flow cytometry apoptosis,but also showed resistance to Sorafenib in nude mice subcutaneously transplanted tumors.This indicates that the acquired Sorafenib-resistant human hepatocellular carcinoma cell line has been successfully constructed.2.IC₅₀ of bufalin at 24 h,48 h and 72 h was 1.52±0.23?mol/L,0.38±0.13?mol/L,and0.20±0.02?mol/L,respectively.IC₅₀ of melittin at 24 h,48 h and 72 h was 5.32±0.23?mol/L,1.64±0.54?mol/L,and 1.13±0.20?mol/L,respectively.There were significant differences between different time groups?P<0.05?.Compu Syn analysis showed that melittin and bufalin had synergistic anti-HCC effect.The optimum synergistic ratio of bufalin and melittin is 1.0?mol/L and 0.2?mol/L,which was consistent with the results of flow cytometry apoptosis detection.3.The results of RNA-Seq transcription group indicated that the transcription group of melittin combined with bufalin had obvious changes and the number of differentially expressed genes increased significantly.Further KEGG analysis showed that melittin and bufalin may play a synergistic role in inhibiting Sorafenib resistance by regulating endoplasmic reticulum stress.4.The solubility of bufalin in PBS increased with the increase of temperature,but the dissolution rate of bufalin was slower and the solubility was lower.It was saturated in 12hours.The equilibrium solubility of bufalin was less affected by pH under acidic conditions?pH<7.4?.However,under alkaline conditions?pH<7.4?,the solubility of bufalin was more affected by pH.And with the increase of pH,the solubility of bufalin also increased.The solubility of melittin in PBS was not different at 25,37 and 45?,but decreased significantly at 65?.The equilibrium solubility of melittin was lower under acidic conditions?pH<7.4?.However,under alkaline conditions?pH<7.4?,the solubility of melittin was significantly higher than that under acidic conditions.The oil-water partition coefficient of bufalin is larger?>3?,and it decreases slightly with the prolongation of time.The oil-water partition coefficient of melittin was negative,and it increased slightly with time.This shows that melittin has good water solubility and strong hydrophilicity.Bufalin has poor water solubility and belongs to hydrophobic drugs.Zeta potential showed that melittin itself was positively charged,whereas bufalin was poorly soluble in water.The Ztea potential of bufalin was 0.5.By response surface methodology and orthogonal test,we comprehensively investigated the factors related to the formulation and preparation process of compound immunoliposomes.We finally obtained the optimal formulation and preparation process of compound immunoliposomes.The optimal prescription of compound immunoliposomes is:Cholesterol/DOPC/DSPC/Mal-PEG₂₀₀₀-DSPE/Bufalin/Melittin/Vitamin E=40:10:30:8:10:1.4?molar ratio?.The optimum preparation process is as follows:thin film dispersion method,chloroform as organic solvent,oil-water ratio 1:2,speed 40 rpm,ultrasonic time 20 min,hydration temperature 45 C,hydration time 40 min.Compound immunoliposomes were positively charged with a potential of 9.03±0.25 mv.The hydrolysis stability and oxidation stability of compound immunoliposomes decreased with the increase of temperature,which was consistent with the results of external stability.The drug release behavior of compound immunoliposomes showed that bufalin was released faster than melittin.The cumulative drug release rate of bufalin at 12 hours was 72.21%±1.81%and that of melittin at 12 hours was62.73%±2.43%.Compared with free melittin,the hemolysis rate of compound immunoliposomes was significantly lower.6.Compound immunoliposomes can inhibit the proliferation and promote apoptosis of Sorafenib-resistant hepatocellular carcinoma cell lines in vitro,and can normally exert the effects of CDC and ADCC.In vivo,compound immunoliposomes have stronger anti-tumor effect than melittin/bufalin group.Compound immunoliposomes can more effectively prolong the survival time of nude mice bearing tumors.And compound immunoliposomes have better safety.Compound immunoliposomes can activate IRE1 and PERK pathways,up-regulate the level of apoptotic Chop protein and activate Caspase-3.ConclusionCompound immunoliposomes loaded with melittin and bufalin can kill Sorafenib-resistant cells?SMMC-7721-R?synergistically.It may play this role through endoplasmic reticulum activation pathway.