Construction Of A Multifunctional Mesoporous Silica Drug Delivery System Loaded With Puerarin And Study On Its Oral Delivery

Puerariae Lobatae Radix is a widely used traditional Chinese medicine in clinical practice.It has the effects of producing fluid and stopping thirst,relieving muscle fever,raising yang and stopping diarrhea,relieving alcohol and protecting liver.Puerarin,the main active ingredient in Pueraria,is commonly used in the treatment of fever,diabetes and cardiovascular and cerebrovascular diseases.Modern pharmacological studies have shown that puerarin has pharmacological activities such as anti-inflammatory,anti-oxidation,hypoglycemic,vascular protection and anti-cancer.It can also be used to treat ulcerative colitis,improve intestinal mucus barrier,and play an anti-inflammatory and antioxidant role.However,the water solubility and fat solubility of puerarin are poor,and the oral absorption is affected by the efflux of P-glycoprotein（P-gp）,and the bioavailability is only about 7%,which seriously limits its clinical efficacy.In this paper,puerarin was used as a model drug.In view of the multiple barriers that affect its oral absorption,vitamin E polyethylene glycol succinate（TPGS）,dodecyl sulfobetaine（SB12）and Pluronic P-123（P123）were used to modify mesoporous silica nanoparticles（MSN）to construct a puerarin multifunctional MSN drug delivery system to improve the mucus penetration performance and epithelial cell uptake of MSN.In order to simultaneously solve the poor solubility,mucus barrier and P-gp efflux barrier faced by oral absorption of drugs,and improve the oral delivery efficiency of puerarin.The main research of this experiment is as follows:（1）Preparation and characterization of multifunctional mesoporous silica nanoparticlesMSN was prepared by template method.The results of Na Sal dosage showed that the particle size of MSN was smaller when the dosage of Na Sal was 84 and 105 mg,which were（257.9±6.5）nm and（308.9±10.0）nm,respectively.After amination,TPGS modification,P123 and SB12 coating,the particle size and PDI of MSN（84 mg）increased,and the particle sizes of MSN-TPGS@P123（105 mg）and MSN-TPGS@SB12（105 mg）decreased to（321.0±17.4）nm and（254.8±1.4）nm,Zeta potential decreased from（34.36±1.17）m V to（12.43±1.10）m V and（4.19±1.66）m V,respectively.Neutral charge was beneficial to penetrate mucus.The results of Scanning Electron Microscope（SEM）and Transmission Electron Microscope（TEM）showed that the mesoporous channels of MSN（105mg）were clear,the number and pore size were moderate.Based on the results of particle size and potential,105mg Na Sal was selected to prepare MSN.SEM,TEM and Infrared spectrum（IR）were used to characterize the different modified nanoparticles.The results showed that the morphology of the nanoparticles before and after modification did not change significantly,-NH₂ and TPGS were successfully connected to MSN.P123 and SB12only had a simple coating effect with nanoparticles.The results of Thermal Gravimetric（TG）showed that the grafting rates（W/W）of-NH₂ and TPGS on MSN were 10.27%and 20.14%,and the coating rates of P123 and SB12 were 3.06%and 2.65%,indicating that MSN was successfully modified.The results of Powder X Ray Diffraction（PXRD）and Differential scanning calorimetry（DSC）further indicate that multifunctional modification such as amination does not affect the structure of MSN.（2）Mucus penetration performance evaluation of multifunctional mesoporous silica nanoparticlesThe interaction between different modified MSN and mucus was investigated.The apparent permeability coefficient of different modified MSN mucus diffusion was quantitatively studied by Transwell plate.The retention ability of different modified MSN in rat intestine was evaluated by laser confocal microscopy.The results of mucin interaction showed that P123 and SB12 could effectively reduce the binding of nanoparticles to mucin.The Papp values（4.05±0.22）and（3.03±0.10）(×10^-6 cm/s)of MSN-TPGS@P123 and MSN-TPGS@SB12 were higher than that of MSN-TPGS.The results of intestinal retention fluorescence imaging showed that MSN-TPGS@P123 and MSN-TPGS@SB12 were evenly dispersed on the surface of intestinal mucosa and epithelial cells.P123 and SB12 effectively improved the mucus penetration performance of MSN.（3）Preparation and physicochemical properties evaluation of PUE-loaded multifunctional mesoporous silica nanoparticlesThe PUE-loaded multifunctional mesoporous silica was prepared by solvent precipitation method.The drug loading rates of MSN-TPGS-PUE,MSN-TPGS@P123-PUE and MSN-TPGS@SB12-PEU were（13.63±4.02）%,（11.81±0.61）%and（11.57±0.56）%,respectively.SEM and TEM results showed that the structure of nanoparticles did not change significantly after drug loading,and the mesoporous channels inside MSN decreased significantly,indicating that PUE was successfully loaded in mesoporous channels.IR results showed that the drug loading process had no effect on the structure of PUE.DSC results showed that PUE existed in amorphous state in mesoporous channels,which was beneficial to the dissolution and release of drugs.The results of in vitro release showed that different modified nanoparticles showed rapid release in the early stage and slow release in the later stage,which was beneficial to the absorption of drugs.（4）In vitro cell evaluation of multifunctional mesoporous silica nanoparticlesThe biological safety of different modified MSN nanoparticles was investigated by Cell Counting Kit-8（CCK-8）method.The inhibition rate of nanoparticles on Caco-2 cells below 250μg/m L was less than 10%,and the biological safety was good.The results of effect of mucus layer on the cellular uptake of different modified MSN nanoparticles showed that the mucus layer had a significant effect on the cellular uptake of MSN-NH₂ and MSN-TPGS,while MSN-TPGS@P123 and MSN-TPGS@SB12were less affected by the mucus layer,indicating that P123 and SB12 coatings can effectively improve the mucus penetration performance of nanoparticles.Quantitative and qualitative evaluation studies of cell uptake showed that MSN-TPGS@SB12 had the highest cell uptake rate.The results of cell transport showed that TPGS could improve the transport efficiency of nanoparticles,and MSN-TPGS@SB12 had the highest cell transport efficiency.The cellular uptake mechanism of MSN-TPGS@SB12 is mainly based on caveolin-mediated endocytosis,which can carry drugs to avoid the efflux of P-gp.（5）Pharmacokinetic evaluation of drug-loaded multifunctional MSN oral administration in vivoHealthy SD rats were used as a model.The drug concentration in rats was detected by high-sensitivity LC-MS/MS technology,the PUE drug-time curve was drawn,and the pharmacokinetic parameters were calculated and analyzed.The results showed that the oral bioavailability of MSN-TPGS@P123-PUE and MSN-TPGS@SB12-PUE was 3.30 times and 3.88 times higher than that of PUE,and 2.29 times and 2.70 times higher than that of MSN-TPGS-PUE,respectively.The coating of P123 and SB12increased the hydrophilicity of MSN-TPGS,while the zwitterionic surfactant SB12made the surface charge of nanoparticles close to neutral,which was more conducive for nanoparticles to penetrate the mucus layer.The modification of TPGS and SB12simultaneously improved the mucus penetration and cell uptake of the nanoparticles,providing a new strategy for improving the oral bioavailability of puerarin.