| Bifendate is widely used in China for the treatment of chronic hepatitis by lowering alanine transaminase (ALT) in patients. Currently, there are only oral preparations on market because DDB is insoluble in water, which results in low bioavailability. However, for acute hepatitis patients and thosewith decreased liver functions after surgical operations parenteral dosages would provide the best benefit for them.In order to prepare the DDB solution for intravenous injection, several groups had tried to improve the solubility of DDB in water. In the present work, based on the solubility properties of DDB, which show high or sufficient solubility in liquid oils but poor solubility in solid lipids, DDB-loaded NLC was prepared by the emulsion-evaporation technique using glycerol monostearate (GMS) and stearic acid (SA) as solid lipids with medium chain triglycerides (MCT) as liquid lipid.As carrier material, lipids have been widely developed as the carrier of nanoparticles due to its desirable biocompatible and biodegradable properties as well as minimally toxicity. Therefore, in our investigation, bifendate was taken as the model drug, mixed lipids of glyceryl monostearate, stearic acid and medium chain triglycerides as the drug carrier, bifendate-loaded nanostructured lipid carriers (DDB-NIC) were prepared by the method of emulsion evaporation at a high temperature and solidification at a low temperature. The formula and technology for preparing DDB-NLC colloidal dispersion were optimized by the uniform design method, with the entrapment efficiency as the criterion, and its lyophilization injection was also systematically studied. The release kinetics in vitro, characteristics of distribution and pharmacokinetics in vivo were examined. DDB-NLC were expected to reach the aim of enhancing its therapeutic efficacy of hepatitis through hepatic targeting and decreasing its side effects.The optimized parameters were as follows:The ratio of drug to lipid materials was 3:20, the ratio of liquid lipid to solid lipid materials was 1:4, the amount of surfactants was 450 mg, the mount of Pluronic F68 was 3%, the stirring rate was 800rmp and emulsifying temperature was 75℃.The entrapment efficiency, and actual drug loading of the nanoparticles were (91.52±1.76)% and (12.07±0.17)%, respectively. Through the observation of transmission electron microscope, we found that the nanoparticles prepared were sphere-like and regular. The size distribution of the nanoparticles was narrow, with the average particle size of 277.0 nm.The Zeta potential was-21.91 mV and the pH was 5.95.To increase the shelf life of the DDB-NLC and to further study the dissolution behavior and physical state of the formulation, the DDB-NLC dispersions were freeze-dried. Based on the appearance, color and redispersibility of the nanoparticles, we studied the major factors of the freeze-dried procedure. The final established freeze-dried method was as follows:First the DDB-NLC dispersions were poured into glass flasks and pre-frozen using an ultra-cold freezer at -80℃for 24h; then the samples were freeze-dried using a lyophilizer at -40℃and at 0.10 mbar of pressure for 48h to yield dry powder. The comparison between the nanoparticles before and after freeze-dried procedure showed that lyopyilization had little effects on the particle size, entrapment efficiency, drug-loading, Zeta potential and pH of the nanoparticles. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that DDB existed in the form of amorphous in the nanoparticles. The in vitro release properties of the freeze-dried DDB-NLC were evaluated by ultrafiltration-centrifugation. The results showed that the drug release pattern was in accord with two phases kinetics equation,having sustained-release character:Ln(100-Q)=-0.1951 t+4.6128, r=0.9985. The initiatory stability research showed that the freeze-dried Sily-NLC could be stored at 25℃or 4℃for 3 months.We utilized the HPLC method to determine and compare the content of bifendate in different tissues of mice following the tail intravenous injection of DDB-Sol and DDB-NLC. The results showed that being packed in nanoparticles, the distribution of silybin in liver were all enhanced. Moreover, the mean retention time in the tissue was also prolonged. Therefore, nanostructured lipid carriers were helpful for bifendate to improve its therapertic efficiency and achieve a long-term effect.The results of pharmaceutics showed that, the encapsulation of bifendate in nanostructured lipid carriers was remarkably effective in prolonging its blood circulation time. Pharmacokinetic parameters were obtained using the DAS 2.0. The major calculated parameters of the DDB-Sol group were as follows:T1/2z=5.92h, MRT=1.5730h, AUC=4.678 h·μg/ml, CLz=5.344 mg/kg/h/(μg/ml). Meanwhile, the major calculated parameters of the DDB-NLC group were as follows:T1/2Z=6.973 h, MRT=4.114h, AUC=9.109 h·μg/ml, CLz=2.836 mg/kg/h/(μg/ml). The results indicated that nanostructured lipid carriers could be a potential carrier for bifendate to obtain prolonged elimination half life as well as retention time.MTT results showed that DDB-NLC could effectively protect the primary cultured human hepatocytes against CCl4 induced injury as compared with DDB-Sol.It is the first report on the preparation of bifendate-loaded nanostructured lipid carriers, the results of our studies contributes to the development of injection of poorly soluble drugs, and plays a very important role in clinical application of bifendate. |
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