Study On Nanoparticle In Oil In Water Submicron Emulsion

The way to increase the solubility of poorly soluble drugs is concerned in recent years.Both poorly water-soluble and poorly oil-soluble drugs are faced with a major challenge in dosage form development.In this study,nanoparticle and emulsion were designed and put together to prepare a novel emulsion which have both the advantages of nanoparticle and emulsion.An nanoparticle-in-oil-in-water(N/O/W) submicron emulsion was prepared with physiological excipients and achieved intravenous administration of poorly soluble drugs.Nanoparticles were incorporated in the oil phase of O/W emulsion to enhance the drug loading capacity of N/O/W emulsion.Furthermore,this novel emulsion could be converted into dry emulsion to increase the stablity of unstable drugs.Dihydroartemisinin(DHA)is the lactol reduction product of artemisinin and found to be effective against acute malaria and chloroquine resistant strains of falciparum malaria.In recent years,DHA has been proven to have potent antitumor activity, well-tolerated and relative non-toxicity in vitro and in vivo.DHA kills tumor cells under the therapeutic dose while common cells partly increased.These findings show that DHA offer great potential and a promising approach in the treatment of cancer, particularly in view of the relative non-toxicity.DHA is a poorly soluble drug in both water and oil and unstable under various conditions.DHA would be serious degraded in gastrointestinal tract.These properties restricts the application of its administration. In this study,DHA was used as a model drug and DHA N/O/W emulsion was prepared for intraveous administration of DHA.Furthermore,DHA N/O/W dry emulsion was prepared to increase the stablity of DHA.The method of high performance liquid chromatography(HPLC)was set up to determine the concentration of DHA.DHA exsist as a mixture of a andβanomers in solution and the ratio of a versusβremained constant after the equilibrium of the andβanomers had been reached.In our study,the ratio of a versusβremained constant around 2.3(α/β)and did not vary over a period of 10 h.The peak area of DHA could be calculated as the sum of a andβanomer to assure the correct results. The solubility of DHA in different solvents were also determined.The apparent solubility of DHA in distilled water and soybean oil was 0.119±0.021 and 1.24±0.01 mg/ml,respectively.The solubility could be enhanced by adding different emulsifiers into systems.The most effective one is Soybean Oil/SPL/Chol.DHA N/O/W emulsion containing DHA,soybean oil,SPL,stabilizer C and cholesterol as oily phase and Poloxamer 188 and glycerol as aqueous phase were prepared by microfluid technology.The DHA N/O/W emulsion were characterized and its droplet size,zeta potential and pH were 152±18 nm(PDI 0.078±0.018), -33.28±2.07 mV,and 7.18±0.03,respectively.The drug loading capacity of DHA N/O/W emulsion was 2.98±0.03 mg/ml and was nearly 3 times more than the highest concentration of DHA O/W emulsion.Light microscopy,Transmission electron microscopy(TEM)and Freeze-fracture transmission electron microscopy(FF-TEM) were used for the observation of DHA N/O/W emulsion structure and compared with DHA O/W emulsion.There were some particles existed in the oil droplets of N/O/W emulsion while not in O/W emulsion.We also used FF-TEM method to observe the internal structure of DHA N/O/W emulsion and compared with DHA O/W emulsion. Thermal analysis was performed to study the characterization of N/O/W emulsion. Compared with DHA powder,the endothermic peak of DHA N/O/W emulsion and DHA oil nanosuspension were decreased nearly 10 centi-degree,while the endothermic peak of DHA aqueous nanosuspension and the mixture of DHA aqueous nanosuspension and blank N/O/W emulsion were similar to DHA powder's.These results indicated that DHA nanoparticles were exsited in the oil phase of DHA N/O/W emulsion,which was conformed to the microscopy micrographs of DHA N/O/W emulsion.About 91.94%of DHA located in oil phase of DHA N/O/W emulsion. About 13.83%of DHA dissolved in oil phase and 8.06%dissolved in aqueous phase. Most of DHA distributed in the interface and nanoparticles(～78.11%).The lyophilized emulsion was developed to improve the stability of DHA N/O/W emulsion.Screening from various cryopotectants,Sucrose-mannitol(4:1)was optimized as a mixture of cryopotectants for DHA N/O/W emulsion.The ratio of cryopotectant and oil phase was 2:1(w/w).The emulsions were freezed at -40℃for 6 h,primary drying at -40℃for 4 hours,from-40℃to 0℃for 8 hours,and secondary drying at 20℃for 4 hours to allow for a complete solidification.The chamber pressure was maintained at 30-50 mTorr while the condenser temperature remained at -80℃during the drying process.A HPLC method was established to determine DHA and its related substances by optimizing the mobile phase.The samples in destroy tests of acid,alkali,strong light, heat and oxygen were prepared.We found and detected 7 decomposable compounds under different destroy conditions.This method was simple,sensitive and suitable for the determation of the related substances of DHA for its stability study.The stability of DHA,DHA N/O/W emulsion and DHA N/O/W dry emulsion were investigated.The chemical raw medicine of DHA was extremly unstable under strong light and heat condition.It was also not stable in high humidity environment and should be stored in a dry and light-proof enviroment with low temperature.The stability of DHA N/O/W emulsion was better than DHA solution,while it was still sensitive with strong light and heat.It should be stored at 4℃and fullfilled with N₂. The accelerated test of DHA N/O/W dry emulsion at 25℃,60%RH and long stability test at 4℃indicated that DHA N/O/W dry emulsion should be stored in a dry and light-proof enviroment at 4℃.The safety of DHA N/O/W emulsion was evaluted by abnormally toxic test,blood vessel irritation test and hemolysis test.The results indicated that DHA N/O/W emulsion had neither serious intravenous toxicity nor undesirable irritation to veins and no stimulation on blood vessel of rabbits,no hemolysis and agglomeration on vitro body red cells of rabbit.DHA N/O/W emulsion has no hemolysis and stimulative reaction.The pharmaceutical safety of DHA N/O/W emulsion was confirmed.To further understand and reveal the case of DHA N/O/W emulsion in vivo,a HPLC-MS method for determining DHA was developed.This method was simple, sensitive and suitable for the determation of DHA in biological samples.The plasma protein binding rate of DHA was determined with ultraflitration.The plasma protein binding rate of DHA with rabbit,rat and human plasma were 76.36%,66.34%and 63.94%,respectively.The binding rate of DHA with plasma is midding strength.After intraveous administration of DHA N/O/W emulsion,DHA O/W emulsion and DHA solution to rabbit,pharmacokinetics parameters were calculated.It turned out that DHA N/O/W emulsion and O/W emulsion significantly prolonged MRT of DHA.DHA N/O/W emulsion and O/W emulsion could extend the MRT of DHA solution by 10.39 and 3.42 times,when AUC was enhanced by 3.01 and 1.80 times, respectively.The biodistribution test was carried out on ICR mice.DHA N/O/W emulsion enhanced DHA content in plasma,stomach and brain,while decreased the ditribution in heart.Experimental tumor H₂₂mice was used to observe the anti-tumor effect of DHA on tumor growth.The tumor inhibitory rate of DHA N/O/W emulsion(45 mg/kg)group is 51.8%,which was better than other DHA different doses groups(P＜0.01).DHA N/O/W emulsion(45 mg/kg)can significantly inhibit H₂₂growth.The expression of VEGF and MVD were down regulated.Histopathological examination showed the tumor inhibition of DHA might related to the inhibition of angiogenesis in tumor.In this study,the physicochemical characteristics,stablility,safety test,and antitumor effect of DHA N/O/W emulsion were studied.The results show that this novel formulation has the following advantages:(1)Increasing drug-loaded capability of emulsion for the drugs which solubility are both poorly in oils and in water;(2) N/O/W emulsion was prepared with physiological excipients and achieved intravenous administration;(3)Submicron emulsion as carriers of nanoparticles can restrain nanoparticles aggregate with each other,and this can improve formulation's physical stability;(4)N/O/W emulsion can be further converted into dry emulsion for unstable drugs;(5)The pharmacokinetics and biodistribution could be changed and possible to be developed as a targeted agent;(6)ease of manufacture.We provide some academic and experimental data for DHA intraveous administration and N/O/W emulsion as carrier of poor solubility drugs.The study of DHA N/O/W emulsion could provided an emergency medicine for patients with severe malaria and a promising approach in the study of cancer treatment.