Study On Preparation And Properties Of W/O/W Multiple Emulsions Encapsulating Insulin

W/O/W multiple emulsions present many possibilities for the controlled release of active drug component initially entrapped in the internal aqueous compartment.In the present work, the preparation of a W/O/W emulsion,together with the factors influencing emulsion stability, and the encapsulation,release and bioavailability of insulin,a drug for diabetes treatment, were studied.First,primary W/O emulsions with insulin disolved in water phase,liquid paraffin as oil phase and Span 80/Tween 80 mixtures as emulsifier were prepared using different methods such as ultra-sonication,homogenization and high speed stirring.Then the primary W/O emulsions were dispersed in water phase at low speed stirring or using membrane emulsifying to obtain W/O/W multiple emulsions.By examining the emulsion stability and the encapsulation efficiency of the insulin,the optimized preparation technique parameters was determined.Second,the formulations of the hydrophiic emulifiers for stabilizing the primary W/O emulsions and that of the lipophilic emulsifiers for stabilizing the W/O/W emulsions were further optimized by means of an artificial neural network method.Centrifugal creaming, microscopy observation,rheological properties and droplet size distribution measurement,and HPLC analysis of the insulin concentration in the inner aqueous phase were employed for the assessment of the emulsions stability and encapsulation efficiency of insulin.The optimized preparation procedure and formulation is as follows:first,10 wt.%lipophilic emulsifier (Span80/Tween80 mixture at weight ratio 9:1)was dissolved in oil phase at 30℃,then the inner aqueous phase was added into the oil phase at a volume ratio of 1:1 under stirring rate of 9500 r.min^-1 for 6 minutes to form W/O primary emulsion,which was then added into another water phase containing 10%hydrophilic emulsifier(Span80/Tween80 mixture at weight ratio 1:1) under low speed stirring of 2000 r·min^-1 for 3 minutes to form W/O/W emulsions,the volume ratio of the primary emulsion to the outer water phase was 1:1.The influences of the phase ratio between inner aqueous phase and oil phase and that between primary emulsion and outer water phase,as well as the osmosis pressure of the inner compartment and viscosity of the outer water phase on W/O/W emulsin stability and encapsulation efficiency of insulin were studied too.To prevent the shrinkage of inner water droplets during storage induced by Oswald ripening,NaCl was added into the inner aqueous phase and it was found that 0.1%(w/v) NaCI(osmosis pressure value 4.78kPa) could counterbalance the influence of the Laplace pressure and enable a stable droplet size distribution and insulin encapsulation efficiency for at least 6 months at 4℃.Higher concentration of NaCI in the inner phase,however,will induce water flowing from outer aqueous phase to the inner aqueous phase,and result in swell-breakdown of some oily droplets,which was found to be proportional to the concentration of NaCI added.Increasing viscosity of the outer water phase by adding xanthan can improve stability of multiple emulsions,but too much xanthan(＞0.15%wt/v) will gel the aqueous phase and accelerate the creaming of the oily droplets,which is difficult to be redispersed by simple stirring.For measuring the release rate of insulin entrapped in the inner aqueous phase of the multiple emulsions,the multiple emulsions were diluted into deioned water,phosphate buffer solution of pH 6.8,and artificial intestinal liquid with gentle stirring at 37℃respectively.At different time intervals,the amount of the insulin in both the inner and outer aqueous phases were measured respectively and the release rate was calculated.The released amount in deioned water and phosphate buffer solution after 8 hours incubation were both less than 20%.It seems that the high molecular mass of the lipophilic emulsifiers inhibit the formation of reverse-micelles and the molecular diffusion the insulin is slow due to its high molecular mass.On contrast,in artificial intestinal liquid,the insulin entrapped can be released with an approximately constant rate during 8 hours incubation and the total amount released reaches 65.9%at the end of incubation.It seemes that the hydrolysis of the emulsifiers by the lipase added to the artificial intestinal liquid trigger the release.Experimental results indicated that insulin encapsulated in W/O/W multiple emulsions could be conserved until being released into artificial intestinal liquid.However,the insulin released was found to discompose quickly.Adding soybean protease inhibitor can effectively improve the stability of the released insulin by inhibiting the activity of the protease.The protective effect of the soybean protease inhibitor increases with increasing the amount of the inhibitor but declines with time.At a concentration of inhibitor more than 4.3 mg/ml,the insulin released could be totally protected in the initial 2 hours of incubation.At last the hypoglycemic effect of the prepared multiple emulsions entrapping insulin were evaluated using animal experiments.Results indicated that the multiple emulsions can release insulin in a controlled and sustained manner and show a slightly but important decrease of the blood glucose compared to using directly the insulin solution.Addition of poly-unsaturated fatty acid DHA as absorption enhancer of insulin shows no enhancement for hypoglycemic effect,it seems that DHA may also enhance the absorption of glucose.On the other hand both addition of soybean protease inhibitor in the inner aqueous phase(0.5 wt.%) and addition of bile salt in the outer aqueous phase(20mmol/L) can notably improve the hypoglycemic effect,as indicated by the cumulate decrease of serum glucose of 95.2%and 92.9%respectively.