Preparation, Characteristic And Tumor Inhibition Ratio Of Long-circulating Liposomes Containing Etoposide

To date,cancer is still threatening the quality of human life seriously.Because of the difficulties in early-diagnosis and discovery,a considerable number of cancer patients have to rely on chemical treatment,so reseach on anti-cancer drug is very important.Since 1946,nitrogen mustard is used in the treatment of malignant lymphoma,and then research on anti-cancer drugs has been rapidly developed and improved.As an efficient method of tumor treatment,anti-cancer drugs can extend the lives of cancer patients and improve the quality of life.However,lacking of specificity, several anti-cancer agents are utilized limitedly in clinic because of their severe side effects,and they not only act on the cancer cells but also damage normal cells.Side effects should not be ignored,such as bone marrow suppression,gastrointestinal reactions.Liposomes is a promising and effective vector in tumor treatment.Liposomal antineoplastic agents are characterized by targeting tumor and controlled-release,and thus they can increase therapeutic index(TI),improve clinical effect and reduce toxicity. However,the general ease of liposome leakage,and plasma-derived material,the existence of poor stability,short half-life in the blood and so on,restricting the development and applications of liposomes.Recent reseach showed that the surface-modified liposomes can improve the stability of liposomes and change the fate of liposomes in vivo.PEG-modified liposome surface is often named as long-circulating liposomes(LCL),which can reduce macro-phagocyte system(MPS) and prevent destruction of plasma membrane lipid composition on the the drug.LCL can significantly prolong the cycle time in the body and increase the release ratio of drug in tumor site,play EPR(enhanced permeability and retention) effect.For the above reasons,research and application of LCL have been paid on attention. In this paper,we selected etoposide,one of semisynthetic derivatives of the podophyllotoxins from Podophyllum peltatum and Podophyllus emodii Wall.etoposide is clinical drug of small cell lung and ovarian cancer in the first line,by inhibiting DNA topoisomeraseâ…¡to play the anti-tumor effect of activity,with broad-spectrum anti-tumor activity,which is mainly acted as the treatment of small cell lung cancer, malignant lymphoma,malignant germ cell tumors,leukemia,neuroblastoma, rhabdomyosarcoma,ovarian cancer,non-small cell lung cancer,gastric cancer,esophageal cancer and so on.The injection preparation and oral soft capsule of etoposide had been manufactured,but they have some shortcomings,such as low bioavailability and poor stability,which restrict the application of its use.To overcome these problems,we design the long-circulating liposomes preparations containing etoposide.PEG modifying in the surface of liposomes,forming a compact conformation of the three-dimensional clouds and thick steric layer,not only enhance the role of the solvent,but prevent the surface of liposome opsonization.In conclusion, we desire that adoption of long-circulating liposome vectors as etoposide,to increase drug stability,extend the drug half-life in the blood,reduce side effects and low toxicity and improve tumor-targeted drug properties.Around these objectives,this paper carried out the reseach as follows.Firstly,we research on the pre-prescription of etoposide,such as establishment of etoposide by HPLC analysis method,oil-water distribution coefficient and determination method of encapsulation efficiency.Base on these reseaches,screen on etoposide long-circulating liposome prescription and research process.In this study,we research on the phospholipid material,the buffer medium,PEG-modified material usage,as well as the lipid concentration of antioxidants,and then ensure these parameters.After choosing several common methods of preparation of liposomes,such as film dispersion method,freezing dryness method and ethanol injection method, encapsulation efficiency and trait characteristics as indicators,we finalize the adoption of ethanol injection method as preparation method.Through a single factor and orthogonal design optimization,we achieve the best preparation process.Ethanol injection method is a good process to avoid using of toxic organic solvents,and the products have shown characteristic and good stability,fit encapsulation efficiency, appropriate particle size and be in accordance with the normal distribution.To observe on etoposide long-circulating liposome's physical and chemical properties systematically,including particle size,morphology,Zeta potential,in vitro release rate,determination of encapsulation efficiency,stability reseach and so on.The appearance of etoposide long-circulating liposomes was slightly translucent opalescence,color uniformity,good mobility.Through the TEM observation,liposomes have uniform particle size,good dispersion,internal space of the water-based nanoparticles.Liposome samples have narrow particle size distribution,with an average diameter of 112nm,showed single-peak distribution.Entrapment efficiency is over 80%.Resarch on chemical stability show that oxidation index is low.Physical stability test results show that percent ratio of leakage for 24h were(38.27±3.15) and(46.72±2.20)%in pH7.4 phosphate buffer and rat plasma,show that etoposide long-circulating liposomes have good physical stability.Reseach on lofting stability results show that etoposide long-circulating liposome situated at 0～4℃place for three months to maintain stability.Dilution tests show that by forms of clinical use dilution,ETP-LCL remain stable,and there is no any drugs leakage and precipitation.Furthermore,we observe on the pharmacokinetic behavior and distribution of etoposide long-circulating liposomes.Both the two formulations and free etoposide were administered via tail vein at a dose of 10mg/kg.Etoposide was separated from the plasma component by solvent extract.The t_1/2,α,t_1/2,β and the AUC values of etoposide long-circulating liposomes are 10,17 and 3.5 times as ordinary etoposide injection.The t_1/2,α,t_1/2,β and AUC values of conventional liposomes are 4,2.5 and 1.4 folds as etoposide injection in thesame dose.The idistribution and elimination rate of the of long-circulating liposome is slower than the etoposide injection and conventional liposomes.Long-circulating liposome can be maintained in the body and keep longer cycle time,to have more opportunities to reach the target site,target to achieve the role played by EPR effect.Cl value of etoposide injection is 3.5 times that the long-circulating liposome,which show that in vivo elimination of etoposide is very slow.Determination of tissue distribution results showed that compared with etoposide and ordinary injection liposome,etoposide long-circulating liposomes in the liver, spleen drug distribution in a significant decrease in the distribution in the plasma increased.To carry out acute toxicity tests of etoposide long-circulating liposome,and acquire LD₅₀ values by Karber method.Measurement results show that the LD₅₀ value of etoposide injection is 86.6mg/kg,LD₅₀ value of etoposide long-circulating liposome is 179.1 mg/kg,is 2 times more than the injection group,which shows that etoposide liposomes have the low toxicity and more drug safety. For predicting tumor inhibition ratio of ETP-LCL,use the C₅₇BL/6J mice inoculated by Lewis lung cancer cell(LCC),after the administration of the establishment of the tumor model,and study the inhibition ratio.The results show that inhibition ratio of etoposide injection group was 47.92%,conventional liposomes group was 58.49%,ETP-LCL group was 64.15%.Biopsy of tumor tissue showed that etoposide long-circulating liposome group shows a visible tumor necrosis and rupture of nuclear solidification phenomenon dissolved.In summary,using ethanol injection method,we have successfully prepared long-circulating liposome containing etoposide and the method is repeatable with satisfactory results.Physical and chemical stability,particle size,encapsulation efficiency,drug loading and the leakage rate of the products meet the requirements and so on.It was demonstrated that ETP-LCL not only prolonged the resident time of the drug in blood circulation,but also showed a higher anti-neoplastic activity.