| Objective:1、We choose multicomponent Chinese medicine with different polarities as research objective, using evaluation parameters including encapsulation efficiency, morphology, Zeta potential, particle size and its distribution as indexes to explore the preparation method of three-component compound liposomes, and then evaluate the reasonability of the administration system.2、We research in vitro release behavior, in vivo pharmacokinetics and tissue distribution of GTS-lip to investigate the reasonability of compatibility of compound liposomes which contain ingredients with different polarities from couplet herbs Salvia miltiorrhiza Glycyrrhiza glabra, and provide experimental evidence to evaluate the feasibility of the compound liposomes.Method:This paper contains four parts.Firstly, preformulation studies were performed. HPLC method was established to determine Sal B, TSN and GA simultaneously. Oil-water partition coefficient of Sal B under different pH conditions was determined. Then, we use thermostat concussion method to explore the solution properties of TSN and GA in different media such as PBS buffer (pH=6.8),0.2% tween,0.5% tween,0.2% SDS and 0.5% SDS. Methods for determining the entrapment efficiency of hydrophobic components and hydrophilic component were developed separately. The liposomes and free drugs were separated by sephadex filtration and low-speed centrifugation methods respectively. Accuracy of the two methods was evaluated by recovery. Then, high-speed centrifugation and centrifugal-ulrrafiltration methods were investigated to determine the encapsulation efficiency of water-soluble component Sal B. The optimal method got optimized and validated.Secondly, the co-encapsulation process of Salvianolic acid B (Sal B), Tanshinone Ⅱ A (TSN) and Glycyrrhetinic acid (GA) into liposomes was systematically optimized and characterized. Encapsulation efficiency of three drugs and particle size of GTS-lip were selected as evaluation indexes of the whole screening process which was divided into two primary steps to obtain optimal conditions with less and reasonable experiments. During step one, encapsulation method of two hydrophobic components were chosen from film hydration combined with probe sonication method, ethanol injection method and reverse evaporation method. Three mainly effective factors (ratio of lipid to chloroform (w/w) (A), ratio of lip id to TSN (n/n) (B) and ratio of lipid to GA (n/n) (C)) among six key single ones (type of phospholipid, ratio of lipid to chloroform, ratio of lipid to TSN, ratio of lipid to GA, water bath temperature, power of ultrasonic probe) associated with this process got further optimized using orthogonal L9(34) test design. The three factors were tested at three different levels choosing encapsulation efficiency of TSN (EETSN) as evaluation index. During step two, ratio of GA and Sal B to lipid, pH of buffer got further optimized and were subjected to response surface methodology (RSM) with a three factor-three coded level as they were considered to be key ones based on single factor test, which was used to screen various new factors involved in this step (incubation time and temperature, ratio of GA and Sal B to lipid, pH of buffer). Lyophilization process got optimized to improve the stability of GTS-lip. At the end of each step, morphology, Zeta potential, particle size and its distribution were researched to evaluate the quality of liposomes. The release of three drugs from GTS-lip was investigated through two different dialysis methods with opposite dialysis directions, and was compared with drugs in physically mixed solution.Thirdly, GTS-lip’s in vitro effects on proliferation of hepatic stellate cells were investigated. To evaluate the GTS-lip’activity on proliferative inhibition of hepatic stellate cells (HSC), an in vitro cell culture system using human hepatic stellate cell (HHSC) line was developed. The proliferative inhibition rate of the HSCs affected by GTS-lip with various dosages at different time points was determined by MTT assay, and compared with TSN、GA、 Sal B mixed solution group. The conformation of HSCs was monitored by phase-contrast microscopy.Finally, in vivo pharmacokinetics and tissue distribution of GTS-lip were studied. A reliable UPLC-PDA method was developed for simultaneous determination of Sal B, TSN and GA in mouse plasma, heart, liver, spleen, lung, kidney and brain tissues. After intravenous injection administration, blood and organs were obtained at 0.083,0.17, 0.33,0.5,0.75,1,1.5,2,4,6 h time points. Drug concentration of blood and tissues was detected and processed by the WinNonlin 5.2 software to calculate pharmacokinetic parameters. Then, plasma drug concentration-time curves and tissue distribution bar graphs of three drugs were drawn and compared with corresponding drugs in mixed solution. Furthermore, liver targeting characteristic of three drugs encapsulated in GTS-lip was evaluated using relative liver uptake rate Re and peak concentration ratio Ce.Results:A reliable HPLC method was developed for simultaneous determination of Sal B, TSN and GA with a high concentration linear range of 0～600μg·mL-1 and low linear range of 8-120μg·mL-1 for Sal B; a high concentration linear range of 16～400μg-mL-1 and low linear range of 0.4～30μg·mL-1 for TSN; a high concentration linear range of 30～600μg·mL-1 and low linear range of 0.4～30μg·mL-1 for GA. Under acidic conditions the oil-water partition coefficient of Sal B tended to increase whenpH got lower, and it turned to be lipophilic at pH 3.32. Both TSN and GA were insoluble drugs which exhibited poor solubility in PBS.0.5% SDS could improve its solubility to a certain degree. Sephadex filtration method with low recovery of glycyrrhetinic acid and tanshinone ⅡA might absorb GT-Lip, while low-speed centrifugation method with centrifuge speed of 2000 r·min-1 for 5 min could separate liposomes and free drugs effectively. High-speed centrifugation combined with ultrafiltration method was chosen to separate Sal B. Two methods were both efficient and accurate.The liposomes (GTS-lip) were prepared using film hydration method combined with probe sonication to encapsulate two hydrophobic components (TSN and GA), and using pH gradient method to load hydrophilic component Sal B. Systematic optimization of encapsulation process was performed using single factor test, orthogonal test in combination with Box-Behnken Design.Optimum conditions were as follows:ratio of GA to lipid (w/w) =0.08, ratio of Sal B to lipid (w/w)=0.12 and pH of buffer=3.3. Based on the conditions mentioned above, encapsulation efficiency of TSN, GA and Sal B reached target levels: (96.03±0.28)%, (80.63±0.91)% and (88.56±0.17)% respectively. The GTS-lip had a unimodal size-distribution and a mean diameter of 191.3±6.31 nm. Morphology determination of the GTS-lip indicated that the liposomes were spherical, and there was no free drug crystal in the visual field of transmission electron microscopy. Also, the ξ, potential of GTS-lip was detected to be-11.6±0.35 mV.9% saccharum was chosen as protective agent during the freeze drying process of GTS-lip. In vitro release investigation of GTS-lip suggested that the release rate of GTS-lip significantly decreased compared to drug solution. The accumulative release percentage of TSN, GA and Sal B were 10% in 36h,4% in 36h and 77% in 24 h.GTS-lip exhibited definite activity on proliferative inhibition of hepatic stellate cells (HSC). GTS-lip decreased the viability of the HSC to higher than 75% at two high drug concentration groups in 24h. At the same time, GTS-lip of two low drug concentration groups increased the inhibition rates by 2.3 folds and 1.9 folds separately at 48h compared to 24h. By contrast, inhibition activity of G-T-S solution group showed less change between 48h and 24h. The prolonged and enhanced activity in 48h which GTS-lip group manifested might contribute to its sustained release effect.Plama and tissue samples were extracted with ethyl acetate two times and detected by UPLC-PDA. All the three drugs could be detected within 5 minutes,and the process were effective and accurate. Pharmacokinetic results indicated that AUC of Sal B in GTS-lip increased from 20.37±1.58μg-h/mL to 636.06±27.73μg·h/mL, and MRT prolonged from 0.28±0.01 h to 1.97±0.09 h compared to mixed solution group, while the AUC of TSN also increased. Tissue distribution results indicated that the distribution behavior of all the three drugs changed compared to mixed solution group, and AUC got significantly increased in liver especially. The overall tissue distribution amount of Sal B was higher than mixed solution group which indicated that GTS-lip could improve the stability and bioavailability of Sal B effectively. Meanwhile, tissue distribution of TSN in each organ all increased, especially significant in liver. Liver tissue distribution pharmacokinetic parameters showed that AUC of Sal B, TSN and GA were 2.11、9.07 and 2.59 fold compared to mixed solution group respectively, while Cmax was 1.76、2.08 and 1.73 fold correspondingly, which meant the compound liposome could improve liver targeting of all the three drugs simultaneously.Conclusion:After systematical screening and optimization including single factor test, orthogonal test and BBD, the particle size of liposomes could be controlled between 100 nm and 200 nm. EE of three drugs could all be kept over 80% with little variation. We successfully obtained liposomes containing TSN, GA and Sal B simultaneously.The present study suggested that GTS-lip could inhibit the proliferation of human HSCs effectively. Meanwhile, all three drugs displayed sustained release trend to some extent. Results of tissue distribution suggested that the distribution behavior of all the three drugs changed compared to mixed solution group, and AUC got significantly increased in liver especially. All the three drugs exhibited liver targeting characteristic in liposomes. The results provide basis for GTS-lip’s effect against liver fibrosis. |
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