Study On The Block Copolymer Nanoparticle Drug Delivery System For Chinese Medicine Multicomponents (Ⅱ)

I this study, Syringopicroside and Hydroxytyrosol were used as model drugs, which had exact curative effect on hepatitis B in Folium Syringac to prepare mPEG-PLGA nanoparticle which co-loaded two Chinese medicine ingredients to build a long-acting and targeting of nano drug delivery system.The regular of using mPEG-PLGA block copolymer as a carrier encapsulating and releasing traditional Chinese medicine were discussed which was expected to achieve intracellular deliver medication by the research in vivo and in vitro.1.Process and prescription optimization of SH dual drug loaded nanoparticlesThe nanoparticles particle size, entrapment efficiency and drug loading were studied as the evaluation indexes, the single factor method and the central composite design-response surface method were combined to optimize process and prescription.The optimization results showed that, with the mPEgG-PLGA block copolymer as carrier material, F68 as stabilizer, were prepared by precipitation of SH dual drug loaded nanoparticles solution appearance with light blue opalescence, total envelopment rate and total drug loadings were (32.38±1.21 )% and (12.01±0.32)%, respectively. by means of comparison of process research, results showed that, the concentration of 5%mannitol as a freeze-drying protective agent, freeze-dried SH-NPs appearance was loose and white powder, solubility was good. The three batches of verification of product display good reproducibility, meanwhile the process was feasible.2.Research on characteristics of SH dual drug loaded nanoparticle The morphology of freeze-dried SH dual drug loaded nanoparticles was observed by transmission electron microscope; Zetasizer Nano-ZS90 laser particle size analyzer determined the Zeta potential, particle size and PDI.The results showed that the freeze-dried SH dual drug loaded nanoparticles morphology was spherical, uniform distribution, particle size and PDI value of SH-NPs were 91.70±2.11nm and 0.22±0.01,and Zeta potential was-24.9±1.16 mV. Using dynamic dialysis method to study vitro drug release,PBS (pH7.4) was drug release medium, under the condition of 37℃ and constant stirring speed (100rpm).The results showed that it has certain sustained release effects that nanoparticles in vitro drugrelease conformed to Higuchi equation, the fitting coefficient R2 was 0.979 and 0.984 respectively. SH dual drug loaded nanoparticles had a slow-release effect. Data show that small molecular weight, small volume of HT had higher encapsulation efficiency and release rate than the SYR.3.Pharmacokinetic study of SH dual drug loaded nanoparticles drugSH saline solution was used as control group to study dynamic parameters of SH dual drug loaded nanoparticles. On designed time point to extract the blood of Rat eyes, the blood concentration of SYR and HT were measured by UPLC, used DAS2.0 software to process data, pharmacokinetic parameters fitting medicine.The results showed that the control group and the nanoparticles group concentration time course were fitted to the two-compartment model. In the same dose, the elimination half-life T1/2β of SYR and HT was 3.17 and 2.32 times respectively compared with control group, and AUC was 3.70 and 5.55 times respectively.T1/2β significantly prolonged, AUC increased significantly, indicating that the SYR and HT made nanoparticles, prolonged drug circulation time in vivo, improved the bioavailability, had certain long cycle effect.4.Targeted research of SH dual drug loaded nanoparticlesThe research on targeting was taken on Kunming mice. After tail vein of mice were injected the SH dual drug loaded nanoparticles solution and SH saline solution, using UPLC method to detect the SYR and HT concentration in various tissues. Relative targeting efficiency and relative uptake efficient drug was used to evaluate the distribution of SH dual drug loaded nanoparticles administered mice. Experiments showed that, compared with SH normal saline group, SH dual drug loaded nanoparticles group could significantly increase the SYR and HT content in mice liver tissue after administration, from the liver of two drugs relative uptake efficiency and relative targeting efficiency, SH dual drug loaded nanoparticles could significantly improve the distribution of SYR and HT in the liver.It indicated the SH dual drug loaded nanoparticles had a certain liver targeting property.Mice in vivo real time level of cell size was observed by fluorescence endoscopic laser confocal imaging system after tail vein injection of SH dual drug loading nanoparticles solution and FITC solutioin in mice.Then the level of cell size nanoparticles could deliver medication in the cell was discussed.The results showed that FITC solution had no fluorescence,eliminates the interference FITC. For FITC labeled SH-NPs group, with the extension of time, the nanoparticles by extracellular into the cell, the number increases gradually which had a positive correlation.Therefore, SH dual drug loaded nanoparticles could be targeted into the hepatocytes.5. Research on cytotoxicity of SH dual drug loaded nanoparticlesMTT method was used to examine SH dual drug loaded nanoparticles on the cytotoxicity of HepG2.2.15 cells, the cell cycle by flow cytometry. The results showed that, the free drug group and nanoparticles can inhibit the proliferation of HepG2.2.15 cells, and with the increase of drug concentration the inhibition rate increased; At the same concentration, inhibition rate of each drug group increased as time goes on; nanoparticles group with incubation time prolonged, the inhibition rate gradually increased and reached the level of inhibition of free drug, show nanoparticles group had obvious sustained release property. With the increase of concentration of drug, G1 and G2 had no obvious change, DNA content of S phase had significantly increased, suggesting that the drug-loaded nanoparticles can block the cell cycle at the S phase.6. Research on the cellular uptake SH dual drug loaded nanoparticles(1) Study on the cellular uptakeCellular uptake was analyzed by fluorescence microscope to qualitative uptake in HepG2.2.15 cells, and quantitative uptake by flow cytometry.Fluorescence microscopy study showed that no FITC labeled blank NPs and FITC soution incubated cells did not fluoresce, eliminates the interference carrier and free FITC. Fluorescence can be observed after NPs and cell function, spot density and fluorescence intensity gradually increased with the increase of NPs concentration. Flow cytometry analysis showed, the experimental group and HepG2.2.15 cells after incubation. blanknanoparticles group and FITC solution group showed no fluorescence,eliminating the influence of blank NPs and FITC molecules on experimental results. With the increase of drug concentration and incubation time prolonged, the fluorescence intensity increases, indicates that cellular uptake and drug concentration, incubation time was positively related to the amount;the cells uptake of FITC-SH-NPs was higher than 4℃ to 37℃. illustrates the process of cellular uptake of active transport.(2) The effect of inhibitory on the cellular uptakeFlow cytometry was performed with colchicine and chloroquine on cellular uptake inhibition study. The results showed that, with colchicine and chloroquine on cellular uptake of FITC-SH-NPs was inhibited, with prolonging of incubation time, diminish the inhibitory effect of the two inhibitor. Inhibition effect of cellular uptake of chloroquine is weaker than that of colchicines. With the increasing of FITC-SH-NPs concentration, the percentage of positive cells did not increase significantly. FITC-SH-NPs concentration had the smaller Influence for the inhibition effect of two kinds inhibitor.7. Study on Pharmacodynamics of SH dual drug loaded nanoparticlesTo establish the rat acute liver injury model induced by D-GalN, the analysis of serum ALT. AST activity in semi automatic biochemical analyzer,using ultraviolet visible spectrophotometer to deterermine SOD activity and MDA content in liver homogenate, using optical microscope to observe HE dyeing pathological to explore the liver protecting effect of nanoparticles.The results showed the medicine group can reduce the ALT, AST activity in serum of rats, improved the liver homogenate SOD activity, decrease the MDAcontent in liver homogenate, also could reduce the liver index; it could improve the liver tissue damage. Nanoparticles group and SH group could obviously reduce the liver tissue degeneration and necrosis, and inflammatory reaction, and nanoparticles had more significant effect, indicating that SH dual drug loaded nanoparticles had protective effect on rats with acute liver injury induced by D-GalN.ELISA method was used to detect the expression levels of HBsAg and-HBeAg of HepG2.2.15 in cell supernatant. The results showed that the positive control group and nanoparticles group could inhibit the secretion of IBsAg and HBeAg of HepG2.2.15,had a significant difference compared with the blank control group (P<0.05,P<0.01).Nanoparticles group at 144h,the concentration was 31.2μg/mL, the HBsAg inhibition rate was the highest,up to 44.6%, inhibition effect was stronger than the positive control drug.Inhibitory effect of nanoparticles on HBsAg and HBeAg showed dose effect and the time effect was obvious relation. SH dual drug loaded nanoparticles had anti-HBV effect of certain.To sum up, we successful prepared SH dual drug loaded nanoparticles and constructed a water-soluble multicomponent drug nano delivery system which was suitable for the high entrapment efficiency,high drug loading in this article. mPEG-PLGA nanocarrier preparation of water-soluble components of the nanoparticles drug encapsulation and release rules was discussed. The data demonstrated that, small molecular weight and small volume of drugs such as HT encapsulation rate, the release rate was greater than the high molecular weight and large volume SYR. SH dual drug loaded nanoparticles had long circulating and liver targeting, could realize the intracellular targeting,and had protective effect on acute liver injury induced by D-GalN,Which had inhibitory effect on the proliferation of HepG2.2.15 cells, HBsAg and HBeAg.This article Provided methods and techniques for the study of water soluble multicomponent of traditional Chinese medicine nano drug delivery system.