| Lymphoma is a serious disease threatening human health.For relapsed or refractory patients,high-dose chemotherapy followed by an autologous stem cell transplant was the clinical standard therapeutic method.Etoposide(VP 16 or EPEG)is recommended as the first-line chemotherapeutic drug for high-dose chemotherapy of lymphoma.The mechanism of VP 16 is to interfere with DNA topoisomerase ? and indirectly induce DNA breakage,thus blocking tumor cells at late S and G2 phases.At present,the market preparations of VP16 are soft capsules and injections,in which soft capsules administered orally 50 mg daily,once a day.The specifications of VP 16 injections are 100 mg(5 mL),500 mg(25 mL)and 1 g(50 mL).It is difficult to satisfy high-dose treatment of lymphoma due to poor drug solubility,severe gastrointestinal reactions and dose-limiting side effects of VP 16 in clinical application.Therefore,it is urgent to develop a new dosage form of VP 16 to solve the above problems.In recent years,the rapid development of nanomedicine showed great potential in the treatment of tumors.Lipid-based nanosuspensions(LNS),as a promising nanoparticulate based drug delivery system,has the following unique advantages:(1)Lipid materials are served as stabilizers,which could ensure the low systemic toxicity,good biocompatibility and safety of nanoformulation.(2)LNS have high drug loading efficiency and no drug leakage problem,thus the volume of administrated nanoformulation could be dramatically reduced,which is more suitable for clinical high-dose administrated drugs such as VP 16.(3)The prescription contains only appropriate amount of stabilizers,avoiding a large number of stimulating adjuvants,which can reduce side effects greatly and improve patient compliance.(4)The enhanced permeation and retention effect(EPR effect)of LNS might increase the accumulation of drugs in the tumor sites to enhance the antitumor effect while reducing the toxicity of drugs on normal tissues.(5)The preparation method of LNS is relatively simple,feasible and suitable for industrialization.In this study,soybean lecithin and polyethylene glycol 1000 vitamin E succinate(TPGS)were used as stabilizers to prepare etoposide loaded lipid-based nanosuspensions(VP16-LNS)for intravenous administration.It was hoped that VP16-LNS could solve the problems of poor drug solubility of the commercial formulations,large administrated volume caused by high-dose cilinical application of VP 16 toward lymphoma therapy and poor compliance of patients caused by the use of irritant excipients.VP16-LNS was expected to enhance the therapeutic effect while reduce the side effects in the treatment of lymphoma.The main research contents and results were as follows:1.Determination of VP 16 contentHPLC method was established for the determination of VP 16 in vitro.The specificity,limit of detection and limit of quantitation,linearity,intra-day and inter-day precision,method recovery and sample recovery were investigated.The results showed that HPLC method was highly specific,the peak area A of VP 16 in the range of 25?500 ?g/mL had a good linear relationship with the concentration C,and the precision and recovery also met the requirements of content determination methodology.Therefore,HPLC could be used to determine the content of VP16.2.Formulation optimization and in vitro evaluation of VP16-LNSIn this study,VP16-LNS was prepared by nano-precipitation method.The optimum prescription and technological conditions were screened by single factor investigation and orthogonal design.Three batches of VP16-LNS were prepared follow the optimum formulation and physicochemical properties of VP16-LNS were evaluated,including appearance,micro-morphology,particle size,zeta potential and drug loading.Mannitol with the content of 6%(w/v)was served as lyoprotectant to prepare lyophilized VP16-LNS in order to further improve the stability of VP16-LNS and facilitate storage and transportation.The crystalline nature of lyophilized VP16-LNS was investigated by differential scanning calorimetry and X-ray diffraction.The release behavior of VP16-LNS in pH 7.4 PBS(containing 1%Tween-80,w/v)was evaluated by dynamic membrane dialysis method.The stability of VP16-LNS was investigated by measuring the particle size of VP16-LNS in different media.In vitro hemolysis assay was conducted to assess preliminary safety of VP16-LNS and whether it was suitable for intravenous administration.Finally,Human Raji lymphoma cells and murine A20 lymphoma cells were used to study the in vitro cytotoxicity of VP16-LNS by MTT assay.The morphology of VP16-LNS prepared by the optimal prescription was spherical or quasi-spherical particles with relative uniform size distribution.The average particle size was(87.50± 7.44)nm,PDI was 0.224 ± 0.039,zeta potential was(-7.38 ± 0.26)mV and drug loading was(6.81 ± 0.30)%.The appearance of lyophilized VP16-LNS was uniform,exquisite and loose white powder with good dispersion.The results of differential scanning calorimetry and X-ray diffraction exhibited that the lyophilized VP16-LNS existed in amorphous or no crystalline state.The release of VP16-LNS in vitro showed comparative sustained release property and the cumulative release of VP 16 was over 80%in the first 5 hours.In vitro stability assay verified that VP16-LNS was stable in pH 7.4 PBS,cell culture medium PRMI 1640 and plasma.In vitro hemolysis test proved that VP16-LNS did not cause hemolysis and was suitable for intravenous administration.MTT results showed that both VP 16-injection and VP16-LNS had a clear concentration-dependent cytotoxicity on lymphoma cells.Moreover,IC50 values of VP16-LNS were 0.508 ?g/mL and 0.219 p,g/mL on Raji and A20 lymphoma cells,respectively and they were 0.735?g/mL and 0.531 ?g/mL for VP 16-injection group.IC50 values of VP16-LNS were significantly lower than that of VP 16-injection on Raji and A20 lymphoma cells(p<0.05).3.In vivo evaluation of VP16-LNSThe pharmacokinetic properties of VP16-LNS and VP 16-injection were studied by HPLC method using female Kunming mice through tail vein injection,and the pharmacokinetic parameters were processed by DAS 2.0 software.DiR-LNS was prepared by using lipophilic near-infrared fluorescent dye DiR iodide instead of VP16.The distribution of DiR-LNS in mice after tail vein injection was observed by near-infrared fluorescence imaging technology in vivo.The antitumor activity of VP16-LNS in vivo was evaluated in murine A20 lymphoma bearing female BALB/c mice.In addition,the safety of VP16-LNS was evaluated by investigating body weight change,organ coefficients,peripheral hemogram,liver and kidney indexes,histopathological observation of various organs of mice and ear vein irritation of New Zealand rabbits.Pharmacokinetic study showed that VP16-LNS could significantly increase drug concentration of plasma in mice compared with VP 16-injection.The pharmacokinetic parameters of t1/2?,AUC0-t,AUMC0-t and MRT0-t in VP16-LNS group increased to 6.37,1.87,6.14 and 3.00 times of VP16-injection group respectively,and CL decreased to 48%of VP 16-injection group.The results indicated that VP16-LNS might slow down the clearance of VP 16 and prolong the circulation time in vivo compared with VP 16-injection,which contributed to enhancing the antitumor effect.Near-infrared fluorescence imaging in vivo showed that DiR-LNS treated mice had stronger fluorescence signal than free DiR treated mice at tumor site,which was 5.21 times higher than that of free DiR treated mice,suggesting that LNS could remarkably increase drug accumulation at tumor sites.The results of pharmacodynamic study in vivo exhibited that the tumor volume of VP16-LNS group was significantly smaller than that of VP 16-injection group(p<0.05).The average tumor weight of VP16-LNS group was significantly lower than that of other treatment groups and the tumor inhibition rate of VP16-LNS group(25 mg/kg)was as high as 93.68%(p<0.01).The results of H&E,Ki-67 and TUNEL staining of tumor tissues in different groups also verified that VP16-LNS could prominently enhance the therapeutic effect of lymphoma(p<0.05).The results of body weight change,organ coefficients,peripheral hemogram,liver and kidney indexes,histopathological observation of various organs in mice and ear vein irritation in New Zealand rabbits showed that VP16-LNS was safer than VP 16-injection.In conclusion,VP16-LNS exhibited spherical shape,small particle size and high drug loading.Moreover,the preparation process of VP16-LNS was simple,feasible and reproducible.VP16-LNS could remarkedly improve the solubility of VP16,prolong the blood circulation time,enhance antitumor effect and reduce the systemic toxicity of the nanoformulation,which proved that VP16-LNS was a potential nanomedicine for lymphoma therapy and had broad clinical application prospects. |
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