Studies On Nanostructured Lipid Carriers For Improved Oral Bioavailability Of Silymarin

Silymarin (SM), a mixed extract of polyphenolic flavonoids isolated from milk thistle (Silybum marianum), has been used in humans for the treatment of a variety of acute and chronic liver diseases. However, its clinical efficacy is compromised by its poor bioavailability due to poor water solubility and low permeability. Although some reported delivery systems are able to improve the bioavailability of silymarin to some extent, their efficacy is weakened by several problems, like low stability, low drug loading and poor biocompatibility. Therefore, it is vey important to develop new drug delievery systems or to optimaze current drug delivery systems to improve the bioavailability of silymarin, which is of high significance to further development of highly efficient SM preparations. In this study, binary lipids-based nanostructured lipid carriers (NLC) were prepared to improve the oral bioavailability of SM. Meantime, its oral absorption mechanism was studied preliminarily in both Caco-2 cell model and animal model.SM-loaded NLCs (SM-NLC) were prepared by the method of high pressure homogenization with glycerol distearates (PRECIROL ATO-5) and oleic acid as the solid and liquid lipids, respectively, and lecithin (Lipoid E 100) and Tween 80 as the emulsifiers. Lipids and water phase were heated to 80℃ respectively, and silymarin were firsted dissolved in ammonium hydroxide and ethanol solutions before being dispersed into the melted lipids. Lipids and water phase were mixed under high shearing to produce preliminary emulsion, and afterwards the preliminary emulsion was homogenized to form NLCs. The preparative variables that affected the responsive variables such as entrapment efficiency, loading and particle size were optimized. Among the preparative variables, SM/lipids ratio (w/w) and emulsifier/lipids ratio (w/w) strongly affect the resultant physicochemical properties of the NLCs; and the homogenization pressure substantially affects the size of resultant NLCs. To ensure high entrapment efficiency, loading capacity and an appropriate particle size, the optimum preparative conditions were selected as follows: SM/lipids ratio (w/w),4/29; emulsifier/lipids ratio (w/w),20/29; OA/ATO-5 ratio (w/w),9/20; lipids/water phase ratio (w/v),2.9/30; homogenization pressure,20,000 psi; homogenization cycles,5. The SM-NLCs prepared under optimum conditions were spherical in shape with mean particle size of about 80 nm, entrapment efficiency of 87.55%, loading capacity of 8.32% and zeta potential of about -65 mV, respectively.In vitro release of SM-NLCs in water was limited with 25% even after 12 h, much lower than 67% of marketed Legalon(?) after 12 h and 90% of SM solid dispersion pellets after 1 h. In vitro lipolysis, which mimicks the behavior of NLC entering into the small intestine, showed fast digestion of NLCs within 1 h. After centrifugation, lipolysis liquid was divided into three layers including the lipid phase, the aqueous phase and the precipitate phase. The proportions of SM in three phases were 4.63%,46.15% and 49.22%, respectively. The drug dissolved in the aqueous phase was probably in the form of micelles or mixed micelles and resulted in enhanced small intestinal drug absorption.The pharmacokinetic study of SM-NLCs was evaluated in beagle dogs using a crossover experimental design in comparison with marketed SM capsules (Legalon(?)) and fast-release SM solid dispersion pellets. Blood samples were withdrawn at time intervals in 8 h, and liquid-liquid extraction method was used to extract SM from the plasma. Drug content in the plasma was detected by an HPLC method. Pharmacokinetic results showed faster absorption of SM-NLC than marketed Legalon(?) and SM solid dispersion pellets. Relative oral bioavailability of SM-NLCs was 2.54- and 3.10- folds that of marketed Legalon(?) and SM solid dispersion pellets, respectively. It was concluded that NLCs were potential drug delivery systems to improve the bioavailability of silymarin.Six different lipid carriers were prepared in the absorption mechanism study. The NLC group included silymarin nanostructured lipid carriers (SM-NLC), the mixture of silymarin solid dispension and blank nanostructured lipid carriers (SMSD-NLC mixture) and the lipolysis liquid of silymarin nanostructured lipid carriers (SM-NLC-lipolysis liquid); the SLN group included silymarin solid lipid nanoparticles (SM-SLN), the mixture of silymarin solid dispension and blank solid lipid nanoparticles (SMSD-SLN mixture) and the lipolysis liquid of silymarin solid lipid nanoparticles (SM-SLN-lipolysis liquid). The bioavailability of these lipid carriers were compared in Beagle dogs. The bioavailabilities of lipid nanoparticles were substantially higher than the other lipid carriers in the same group. Relative oral bioavailability of SMSD-NLC mixture and SM-NLC-lipolysis liquid were 65.71% and 74.86% that of SM-NLC respectively; relative oral bioavailability of SMSD-SLN mixture and SM-SLN-lipolysis liquid were 40.09% and 58.56% that of SM-SLN, respectively. Moreover, the bioavailabilities of lipid carriers were much higher than fast-release preparations with significant difference. The bioavailability of SMSD-NLC mixture was about 2- fold that of SM-pellets. As a result, the predominant factors of the enhanced drug absorption are the lipid components and the integrity of lipid nanoparticles. It could be indicated that besides the lipolysis mechanism, there is probably other mechanisms that contribute to enhance oral absorption.Transport study was performed using Caco-2 cell monolayers on silymarin lipid nanoparticles (NLC/SLN), silymarin-blank lipid nanoparticles mixture (NLC/SLN) and silymarin solid dispension solution. The samples were added to the apical side, and the cumulative transport amounts at time intervals in 2 h were detected by HPLC and Papp values were calculated. The results showed that there were no significant differences among the cumulative transport amounts in 2 h and Papp values of 5 preparations. However, the bioavailability study in beagle dogs showed greater absorption of silymarin lipid nanoparicles than silymarin-blank lipid nanoparticles mixture and silymarin solid dispension pellets; this disparity between cell model and in vivo model indicated the probability of existence of other absorption mechanism. Morover, cellular uptake study was preformed using Caco-2 cell culture, and the culmulative cellular uptake amount after 2 h was detected by HPLC. The results demonstrated that lipid nanoparticles improved cell internalization and leaded to the enhancement of cumulative cellular uptake. The mechanisms of enhanced intestinal absorption need to be further investigated.In conclusion, SM-NLCs were prepared and characterized in vitro. The pharmacokinetic study in Beagle dogs indicated that the bioavailability of silymarin was strongly enhanced by NLC, which was higher than that of marketed Legalon(?) capsules and SM-pelltes. More studies on lipid nanoparticles (NLC/SLN) demonstrated that the lipid compositions and the integrated structure of drug loaded lipid nanoparticles were the predominant factors for the improvement of oral absorption; besides small intestinal lipolysis, other mechanisms may contribute to the absorption enhancement.