Preparation Of Sophorolipids-Etoposide Submicron Emulsion And Its Evaluation In Vitro And In Vivo

Cancer is now a major problem in the medical field.For the treatment of cancer,the current mainstream treatment methods include surgery,radiotherapy,chemotherapy,etc.Among them,chemotherapy is one of the most commonly used treatments.Etoposide（ETO）is a widely used topoisomerase inhibitor chemotherapeutic drug.The low bioavailability and the chemosensitivity of tumor cells are the biggest problems in its application.The development of new ETO dosage forms will help to improve its bioavailability.The new ETO formulations have been reported to include（suspensions,liposomes,microemulsions,submicron emulsions,nanoemulsions,solid dispersions,implants,microcapsules,microspheres,etc.）.Combined chemotherapy refers to a method of several chemotherapeutic drugs simultaneously or sequentially be used in a course of chemotherapy.Combined chemotherapy can synergistically kill tumor cells,thereby improving the efficacy;in addition,combined chemotherapy can remove drug resistance factors and reduce the possibility of cancer cell resistance.Due to the superiority of combined chemotherapy,the current single drug regimen has been eliminated and become a history.Submicron emulsion（SE）is a kind of stable drug-dispersing system which combines oil-water two-phase fusion under the action of surfactant and co-surfactant with particle size below 200nm,can effectively improve the bioavailability of the drug.In recent years,submicron emulsion preparations of diazepam,amphotericin B,and carbamazepine have been marketed,and the research on drug-loaded submicron emulsion has made important progress both in theory and in practice.Tween 80 is a kind of commonly used surfactant excipients,which play an important role in solubilization and emulsification in the preparation of submicron emulsion.However,the widespread use of Tween 80 has been reported to cause adverse reactions such as allergies and hemolytic,leading to serious drug safety problems.Sophorolipids（SLs）are a class of the most promising biosurfactants currently reported.They are mainly classified into acidic sophorolipid（ASL）and lactonic sophorolipid（LSL）.Two types,of which ASL has better foaming and emulsifying ability,LSL has better anti-tumor and other physiological activities.In summary,this study first explored and investigated the theoretical and practical significance of ASL as a surfactant instead of Tween 80 in the preparation of new ETO submicron emulsion;then examined the anti-tumor effect and safety of the combined drug of LSL and ETO.Through the above research,we explored the potential of sophorolipids as a surfactant excipient and antitumor active substance and expanded the application range of sophorolipids.The main research contents and research results of this paper are as follows:1.Optimization and determination of the preparation process of ETO submicron emulsion.Optimize the formulation and preparation process of submicron emulsion preparation,and prepare etoposide submicron emulsion by replacing tween 80 with sophorolipid,and obtain the optimal process:MCT:LCT=1:1 for oil phase,1.8%lecithin,stir to Dissolved,then mixed with water,800 bar,homogenized 8 times,acid sophorolipid added 0.8 g/L.A method for the determination of etoposide in etoposide submicroemulsion was established to avoid the influence of the excipients on the peak time and peak shape.At the same time,the characterization method of the submicron emulsion preparation was optimized,and the 2.5%glycerol solution was finally determined.Two submicron emulsions were successfully prepared and characterized by dilution 20 times as a characterization condition.2.Comparison of physical and chemical properties of ASL-ESE and Tween-ESE.The two etoposide submicron emulsions prepared by using acid sophorolipid and Tween80 as surfactants were used as research objects to detect and compare the particle size,zeta potential and PDI of two submicron emulsions and indicators such as pH,drug loading,and encapsulation efficiency.The experimental results show that the ASL-ESE prepared in this study has a particle size of 197.73 nm,a PDI of 0.051,a zeta potential of-41.40 mV,and an entrapment efficiency of 95.95%.Tween-ESE has a particle size of 195.8 nm,a PDI of 0.167,a zeta potential of-43.47 mV,and an entrapment efficiency of 93.01%,indicating ASL-ESE particle size distribution and drug loading.The amount of the drug was significantly better than Tween-ESE,and the drug loading was increased by 112.6%.The morphology of the submicron emulsion was observed by transmission electron microscopy and in vitro release experiments were performed.The in vitro release experiments showed that the in vitro release characteristics of the two ETO submicron emulsions were superior to the traditional ETO injection,and the in vitro release characteristics of ASL-ESE were better than Tween-ESE.3.Comparison of in vitro antitumor activity of ASL-ESE and Tween-ESE.The in vitro anti-tumor test results of the two submicron emulsion preparations showed that the excipients in the submicron emulsion preparations were less toxic to the cells,The submicroemulsion type has obvious cell proliferation inhibition effect compared with the traditional ETO injection.The inhibition rate of 40μg/mL ASL-ESE on ovarian cancer A2780 cells was 78%,while the inhibition rate of Tween-ESE was only 36.64%at the same concentration.The IC₅₀0 of ASL-ESE is only 12.77μg/mL,which is much lower than 158.35μg/mL of ETO injection and lower than 84.8μg/mL of Tween-ESE.The effect of different submicron emulsion on apoptosis of ovarian cancer A2780 cells was detected by flow cytometry.The results indicate that the submicron emulsion preparation can inhibit the proliferation of tumor cells by inducing apoptosis.Among them,the proportion of ovarian cancer A2780 viable cells in the control group was94.04%,and the apoptotic rate was 5.87%.Compared with the control group,the ovarian cancer A2780 viable cells in the ETO injection group were 59.33%,and the apoptotic rate was 37.8%;in the Tween-ESE group,the ovarian cancer A2780 viable cells were 46.47%,and the apoptotic rate was 52.1%;In the ASL-ESE group,the A2780viable cells of ovarian cancer were only 1.29%,and the apoptotic rate reached 95.58%.In addition,the safety evaluation of the obtained ETO submicron emulsion showed that the submicron emulsion preparation had good safety,no vascular irritation and allergy,and no hemolysis.4.In vitro anti-tumor activity and mechanism of LSL combined with ETO.Firstly,the anti-tumor activity of LSL against breast cancer MCF-7,murine breast cancer 4T1,ovarian cancer A2780,nasopharyngeal carcinoma CEN-28,melanoma B-16,gastric cancer SGC-7901 was investigated.The results showed that LSL had the best inhibitory effect on ovarian cancer A2870,IC₅₀0 was only 30.34μg/mL,and inhibited the proliferation of breast cancer 4T1 cells with IC₅₀0 of 36.17μg/mL.Therefore,the subsequent studies were based on ovarian cancer cell A2780 and breast cancer cell 4T1.The results of the MTT assay for the combined drug showed that for the ovarian cancer A2780 cells,the addition of LSL can significantly reduce the IC₅₀0 of the drug.When LSL:ETO is 1:1,the IC₅₀0 is only 95.36μg/mL,which is 30.21%lower than that of ETO alone.For breast cancer 4T1,when LSL:ETO is 0:4,1:3,2:2,3:1,4:0,the corresponding IC₅₀0 is 93.36μg/mL,74.49μg/mL,53.65μg/mL,38.56μg/mL,36.17μg/mL.After comprehensive consideration,choose LSL:ETO is 1:1 for the optimal dose ratio in subsequent trials.DAPI staining was used to observe the morphological changes of the nucleus of the two cancer cell lines after drug.It was found that the nuclear chromatin of the two cancer cells had obvious shrinkage and even nuclear fragmentation.Flow cytometry confirmed that the two cancer cell lines selected for this study had a synergistic effect in combination drug.In the case of ovarian cancer A2780,when etoposide was used alone,the apoptotic rate was only 8.06%;when the concentration of LSL 40μg/mL was drug alone,the apoptotic rate was 15.01%;The death rate reached 23.95%.For breast cancer4T1,cell survival rate was 83.5%when etoposide was used alone,and cell viability was84.0%when LSL 40μg/mL was administered alone;cell viability decreased to 80.8when administered in combination.The combineddrug group can increase the efficacy of etoposide relative to drug alone and inhibit cancer cells by inducing apoptosis.5.In vivo antitumor activity of LSL combined with ETO.The mouse breast cancer cell 4T1 cells were subcutaneously injected with 1*10⁶-1*10⁷ viable cells subcutaneously in the right side of each mouse,and the injection amount was 0.1 mL.After inoculation,the physiological state of the mice and the tumor formation under the arm were observed every day.Thirty-six experimental animals were divided into model group,etoposide group,lactionic type sophorolipid group,low-dose combined drug group,medium-dose combined drug-administered group,and high-dose combined drug-administered group 6 in each group.After the tumor formation was observed,the group drug was carried out by intragastric drug,and the drug amount was 0.2 mL,and the drug was continued for 7 days.The results showed that both the average body weight of the mice and the body weight growth rate of the mice were the lowest in the etoposide group,while the weights of the mice in the sophorolipid drug group and the combineddrug group did not change significantly.The inhibition rate of the etoposide group was at least 19.45%（p<0.001）compared with the model group.The tumor inhibition rate of the combination group was higher than that of the single drug group,and the high dose combination group had the best inhibition effect,reaching 75.38%（p<0.001）.