Study On Silymarin-loaded Solid Lipid Nanoparticles: Preparation, Oral Bioavailability And Targeting Effect On Liver

Silymarin(SM), a standardized extract from Silybum marianum L. (Asteraceae), is used as human medicine for its heptaprotective effects. Silibinin (SB) is the main flavonolignan of SM, and SM also include other flavonolignans: silidianin, silichristin, silibonol, etc. Clinical studies indicated that SM has a therapeutical effect on chronic hepatitis, various liver damages, early hepatocirrhosis and other liver diseases. However, SM's clinical application is limited because of lower oral bioavailability caused by its poor water-solubility.In the present study, silymarin-loaded solid lipid nanoparticles(SM-SLN) using Compritol 888 ATO as material, was prepared by high pressure homogenization. SM-SLN was stabilized by lecithin and poloxamer 188. SM-SLN was expected to enhance oral bioavailability of SM and have a good targeting effect on liver. All aspects of SM-SLN were investigated in detail and systematically, including the physical and chemical characteristic, properties of pharmacy, pharmacokinetics and distribution in vivo etc.The particle size, entrapment ratio(ER) and drug loading (DL) were taken as criterions to evaluate the suitability of preparing PLGA nanoparticles, nano-liposome and SLN. The experimental results indicated that SLN was a good drug delivery system for SM. In the following study of SLN, when both centrifugal ultrafiltration and sephadex gel-chromatography methods were used to evaluate the ER, the results of two methods were not equal completely, andthe extent of differences was closely related to the parameters of production. Therefore, based on the different principle of two methods, drug incorporation mechanism of SM-SLN was investigated. The results showed that drug in nanoparticles by cold -homogenization existed in two patterns: encapsulation and adsorption, while nanoparticles prepared by hot-homogenization mainly formed lipid core and drug was absorbed in surface. As a result, cold-homogenization was chosen to prepare SM-SLN.The particle size is a key factor to improve oral absorption of poorly soluble drugs and its fate in vivo. When SLN was administrated peroral route, appropriate size should be considered firstly, and then its effect on liver-targeting. 150nm, 500nm and lOOOnm SLN were prepared, and the effect of size on improving oral absorption in rat was also investigated. The results showed the AUC of 150nm, 500 and lOOOnm were 7.11, 3.41 and 2.81, respectively. Therefore, 150nm was chosen to carry out the following experiments.The formulation of SM-SLN was optimized using central composite design and response surface method. SM-SLN was prepared by cold-homogenization technique. The effects of influence factors such as the ratio of drug to Compritol 888 ATO, the concentration of emulsifier, the ratio of poloxamer, homogenization pressure on mean diameter, entrapment efficiency, drug loading, Zeta potential and the amount of drug absorbed on the surface ofSLN (M%=ERcentrifugal ultrafiltration-ERsephadex gel-chromatography) Were investigated. Theoverall desirability (OD) were produced and treated by a statistic analystical system to optimize the formulation. The data were initated using multi-linear equation and second-order polynimial equation. The latter was prior to the former considering from multiple correlation coefficient and P value. Under the optimal conditions, the mean diameter, entrapment ratio, drug loading , Zeta potential and M% of the SM-SLN were 151.5nm, 88.88%, 2.79%, -21.74 and 10.69%, respectively.The formulation of SM-SLN lyophilyzation powder was optimized based on its appearance, color and dispersing properties. SM-SLN was freeze-dried with the mixture of 2% glucose and 2% lactose as cryoprotectant, as a result, the problem that the colloidal solution was not stable and difficult to disperse in water was resolved. The stability of SM-SLN was not affected by freeze-drying process with nearly unchanged particle size, entrapment ratio, drug loading, etc.. Results of hydroscopicity test showed that CRH of SM-SLN lyophilyzation powder was 75.04%. The stability of SM-SLN lyophilyzation powder was studied based on its particle size, entrapment ratio, drug loading,etc, and results showed that SM-SLN lyophilyzation powder was stable within three months. The in vitro release kinetics in saline and PBS (pH=7.4) was investigated and the results showed that the release of SM-SLN lyophilization powder could be well characterized by Weibull equation. Compared to SM-SLN prepared by hot-homogenization, a burst release of SM-SLN prepared by cold-homogenization within 2 h was observed, and since then, drug was sustained -released from SM-SLN.Legalon capsules and Yiganling tablets were used as references to investigate oral bioavailability of SM-SLN in rat. Program 3p97 was applied to the calculation of pharmacokinetic parameters. The plasma drug concentration-time courses of SM-SLN lyophilization powder, Legalon capsules and Yiganling tablets were both conformed to two-compartment model. Statistic test showed that there was significant difference between SM-SLN lyophilization powder and references for Cmax, AUC, MRT and CLs (PO.01). The relative bioavailability of SM-SLN was 310% (Legalon capsules as reference), 459% (Yiganling tablets as reference). In addition, oral bioavailability of SM-SLN in Beagle dog was also studied, and Yiganling tablets were used as reference preparation. The relative bioavailability of SM-SLN in dog was 190%.The mice were employed as experimental animal to study bodydistribution of SM-SLN after oral and intravenous administration. The results of student test and one-way analysis of variance showed that SM-SLN lyophilization powder could distinctly change the distribution of SM in vivo and greatly increased the concentrations of silibinin in liver in contrast to SM after p.o. and i.v.. Overall targeting efficiency (TEC), targeting index (TIC) and relative overall targeting efficiency (RTEC) of SM-SLN was calculated and compared to SM to evaluate the liver targeting property of SM-SLN. Compared to SM suspension, the liver AUC of p.o. SM-SLN was 7.50-fold higher and MRT was 1.30-fold longer; similarly, the liver AUC of i.v. SM-SLN was 6.60-fold higher than that of SM solution and MRT was 7.41-fold longer. The results indicated that both p.o. and i.v. SM-SLN have good liver-targeting effect. The liver bioavailability (F=p.o. AUCtargeted tissue/i.v. AUCtargeted tissue) was 39.97%.The preparation method of SM-SLN was simple and reproducible. Lyophilization is a promising way to increase chemical and physical SLN stability over extended periods of time, and also offers principle possibilities for SLN incorption into pellets, tablets or capsules. Meanwhile, Accu Sizer 780 (Single particle optical seasing,, SPOS) was used to analyse the absolute number of the large particles in the micrometer ranging lum to 400 um after lyophilization. This offers a new thought for increasing the administration route ofSM.In our study, both centrifugal ultrafiltration and sephadex gel-chromatograph methods were used to investigate the drug incorporation mechanism and in vitro release was also carried out to testify the drug-loaded mechanism. The experimental design and method can enrich the content of SLN about drug loaded and in vitro release mechanism. The study of oral bioavailability of SM-SLN in rat and Beagle dog was firstly reported systematically, and the effect of p.o. and i.v. SM-SLN on liver targeting was also firstly investigated.The study opens up new perspectives for the formulation of poorly soluble drugs. It appears that SLNs offer a promising delivery system for the enhancement of the bioavailability of poorly soluble drugs, and are good liver-targeted drug delivery system. The accomplishment of this project is a great achievement for SLN and will definitely propel the clinic application of SLN.