The Preparation Of CAT-loaded Solid Lipid Nanoparticles And Its Stability

Catalase has a good prospect in the pharmaceutical industry, because it could eliminate free radicals in vivo through transfer hydrogen peroxide into water. However, as a therapeutic protein, its poor stability and easily inactive in acid, alkali and enzyme conditions, the half-life in vivo is short, and it does not possess the required physicochemical properties to be absorbed at their target sites. To overcome these disadvantages and improve its performance and get a carrier system that have high entrapment efficiency, stable particle and sustained release behavior, this paper chose the solid lipid nanoparticles as the carrier to encapsulate catalase.Nanoparticles were prepared by the double emulsion method (w/o/w) and solvent evaporation techniques. To optimize the conditions of preparation, we conducted the factorial tests and response surface methodology. Enzyme activity as the evaluation index and considering emulsification results, we concluded that methylene chloride/acetone (1/1) led to lowest activity and the first step of emulsifying with sonication for 20 seconds and the second step of emulsifying with sonication for 30 seconds. Based on the results of factorial tests, response surface methodology was employed in order to further investigate the effects of the concentration of emulsifier in the inner phase, the concentration of emulsifier in the outer phase and the volume ratio of w/o within the w/o/w on the encapsulation efficiency. Under the optimum conditions, i.e. the concentration of emulsifier in the inner phase is 15.24%(w/w), the concentration of emulsifier in the outer phase is 1%(w/v), the volume ratio of w/o within the w/o/w is 1:4, the encapsulation efficiency reached its maximum of 74.51% and the loading capacity of 3.58%. Significance of the three factors to affect encapsulation efficiency was in the order: the concentration of emulsifier in the outer phase > the volume ratio of w/o within the w/o/w > the concentration of emulsifier in the inner phase. The interaction of the volume ratio of w/o within the w/o/w and the concentration of emulsifier in the outer phase is significant. We also found that under the optimum conditions the particles we get were the morphology is spherical, the distribution is homogeneous.Under the optimum conditions, we compared the effects of tripalmitolein with monoglyceride as the lipid matrix on encapsulation efficiency and particles morphologies, the results revealed that using tripalmitolein as lipid matrix produced homogeneous particles, while using monoglyceride as lipid matrix achieved high encapsulation efficiency and loading capacity.Under the optimum conditions, we made a comparison of the effects of tripalmitolein and mixed lipids(tripalmitolein/MCT) on encapsulation efficiency, particles distribution and stability of release, we found that adding a bit of MCT into tripalmitolein(5%) can improve encapsulation efficiency(81.40%) and loading capacity(3.89%), and showed no obvious result to particle size and potential distribution. Also in vitro release research showed that glycine can act as a stabilizer; the addition of MCT can accelerate the initial stage release behavior, which makes the aims of quick function and maintaining a long stable plasma drug concentration in vivo is possible. We carried out model fittings using release curves, the prolong the action time of the catalase.To compare the structure of catalase during the whole procedure, we use the far-UV CD spectra and SDS-PAGE to detect the structure of catalase and released catalase, Also we detect the catalase activity of the released catalase. The results revealed that during this process the catalase activity did not have the bigger lose; the content ofα-helixes had no change, but the content ofβ-sheets andβ-turns made some difference. We found that during the procedure someβ-sheets turned intoβ-turns,while the secondary structure of enzyme was not damaged during the whole process; and electrophoresis results also showed there was no change of the subunit.