Construction And In Vivo And In Vitro Evaluation Of Functional Mesoporous Silica Nanoparticles Loading Andrographolide Nanocrystals

Andrographolide（AG）has the effect of clearing heat,detoxifying,anti-inflammatory and antibacterial,and is mainly used for the treatment of upper respiratory tract infection and dysentery caused by bacteria and viruses in clinical application.The pharmacology study shows that AG has anti-inflammatory,antibacterial,hepatoprotective,anti-tumour,anti-viral and other pharmacological activities,and is known as a natural antibiotic.In this study,andrographolide was loaded into mesoporous silica nanoparticles in the form of nanocrystals by combining nanocrystals technology with mesoporous silica nanoparticles nanotechnology,in order to improve the dissolution and oral bioavailability of andrographolide.However,the gastrointestinal mucus clearance is a natural barrier to oral absorption of nanoparticles,which can reduce the effective absorption of drugs in vivo.The many studies have found that PEG-coated nanoparticles with particle size less than 500nm were more likely to escape gastrointestinal mucus clearance.Owing to the easily functionalization of mesoporous silica nanoparticles surface,the methoxy polyethylene glycol（mPEG）modification can increase the hydrophilicity and steric resistance of the nanoparticles,reduce the interaction with the hydrophobic region of mucus,so that the nanoparticles could penetrate mucus as much as possible based on their own nano size,and enhance drug absorption.In summary,the proposed mPEG-modified intestinal mucus-penetrating andrographolide mesoporous silica nanoparticles were prepared by integrating nanocrystal technology and mesoporous silica nanotechnology to achieve the loading of andrographolide nanocrystal into the mesopores of mesoporous silica to improve drug dissolution,reduce mucus clearance and enhance uptake by intestinal mucosal cells.The main research of this experiment is as follows:（1）Preparation and characterization of functionalized mesoporous silica nanoparticlesThe mesoporous silica nanoparticles（MSNs）with narrow particle size distribution and stable dispersion were successfully prepared by the soft template method and modified with-NH₂ and-mPEG groups by the post-grafting method.The effect of different ratios of mPEG2000-NHS on the size,distribution and zeta potential of the prepared Methoxy Polyethylene Glycol Mesoporous Silica（mPEG-MSNs）was investigated.The mPEG-MSNs prepared in the ratio of 1:1 had the smallest particle size of about 233 nm and the smallest PDI,indicating a more uniform particle size distribution.Zeta potential decreased with the increase of mPEG2000-NHS ratio.The mPEG-MSNs made in the 1:1 and 1:2 ratios were weakly positively charged,and the mPEG-MSNs made in the 1:4 ratio were neutrally charged.Therefore,the mPEG-MSNs were initially prepared with a ratio of 1:1 and 1:4 of MSN-NH₂ to mPEG2000-NHS.The nanoparticles were characterized using a high resolution Scanning Electron Microscope（SEM）,Transmission Electron Microscope（TEM）and Infrared spectrum（IR）spectrometer.The results of SEM and TEM showed that the MSNs had a homogeneous spherical morphology and mesoporous structure,and the modification of NH₂-and mPEG did not change their spherical morphology and mesoporous structure.The grafting rate of mPEG was about 6.59%and that of mPEG in mPEG-MSNs（1:4）was about 6.90%,further indicating the successful modification of NH₂-and mPEG on MSNs.The wide-angle XRD and DSC results showed that the MSNs,MSN-NH₂ and mPEG-MSNs were all present in amorphous form.The small-angle XRD results showed that the MSNs had an ordered mesoporous structure,further indicating that the modification of NH₂-and mPEG did not affect their mesoporous structure.（2）Evaluation of the mucus-penetrating ability of mesoporous silica nanoparticlesThe mucin interaction with mPEG-MSNs in vitro,mucus diffusion assay,in vitro mucus penetration evaluation and in vivo fluorescence imaging study of each intestinal tract were used to evaluate their mucus-penetration properties.The results of in vitro interaction between mucin and mPEG-MSNs showed that the particle size and PDI of mPEG-MSNs（1:1）were small in the range of 0～4h,indicating that little interaction of mPEG-MSNs（1:1）with the mucin,and the ratio 1:1 of MSN-NH₂ to mPEG2000-NHS was used to prepare mPEG-MSNs.The Papp value of MSN-NH₂ was 15911.5×10^-6 and that of mPEG-MSNs was 40672.7×10^-6 in mucus diffusion assay,indicating that mPEG-MSNs had greater penetration ability in mucus compared to MSN-NH₂.In vitro mucus penetration evaluation also showed that mPEG-MSNs could penetrate the mucus layer.In vivo fluorescence imaging studies of each intestinal tract showed that mPEG-MSNs were uniformly distributed on the mucosal surfaces of the three intestinal segments and could penetrate the mucus layer into cells.（3）Preparation and physicochemical characterization of mesoporous silica nanoparticles loading andrographolide nanocrystalsAndrographolide loaded mesoporous silica（AG-MSNs）was prepared by solvent evaporation method.The results of drug loading efficiencies showed that the best preparation process for AG-MSNs was:water bath condition at 60°C,AG concentration of 20mg/m L,the ratio of andrographolide to MSNs was 1:0.5,the stirring time was 2hours,and the drying method of nanoparticles was vacuum drying.SEM and TEM results showed that mPEG-MSNs and andrographolide loaded with polyethylene glycol mesoporous silica（AG-mPEG-MSNs）were homogeneous spheres with a rough surface,and the drug loading did not change the morphology of the mesoporous silica.The IR results showed no chemical reaction between the AG and the carrier.The DSC and PXRD results indicated that the drug may exist in a nanocrystalline state,but the crystalline characteristics were not altered.The BET analysis showed that andrographolide was successfully loaded into the mPEG-MSNs nanopore structure.In vitro release results showed that the p H of the gastrointestinal tract had no effect on the release of AG-mPEG-MSNs.（4）Evaluation of in vitro cytotoxicity and anti-inflammatory activity of AG-mPEG-MSNsThe cytotoxicity of mPEG-MSNs and MSN-NH₂ was evaluated using the Cell Counting Kit-8 method.With the increase of mPEG-MSNs or MSN-NH₂ concentration,the activity of Caco-2 and Raw264.7 cells did not change significantly and basically remained above 95%,indicating low cytotoxicity and good safety.The visualized evaluation results showed that MSN-NH₂ and mPEG-MSNs could be taken up by Caco-2 cells,and mPEG-MSNs were taken up by the cells more than MSN-NH₂.In vitro anti-inflammatory effect assays showed that AG-mPEG-MSNs were able to effectively inhibit the inflammatory response induced by LPS in RAW246.7 cells and effectively reduce the levels of IL-1β,IL-6 and TNF-αinflammatory factors.（5）In vivo pharmacokinetics and in vivo anti-inflammatory pharmacodynamic evaluation of AG-mPEG-MSNs administered orally to ratsUsing glipizide as the internal standard,a sensitive,specific and efficient LC-MS/MS analytical method was developed to investigate the pharmacokinetic parameters in rats after the administration of equal doses of AG and AG-mPEG-MSNs by gavage.The results showed that the oral bioavailability of AG-mPEG-MSNs was approximately 2.078 times higher than AG,indicating that the bioavailability of AG-mPEG-MSNs prepared by combining nanocrystal technology with mesoporous silica technology was significantly improved.In vivo anti-inflammatory experiments showed that the AG-mPEG-MSNs could effectively inhibit toe swelling caused by carrageenan,increase SOD levels and reduce NO,IL-1βand TNF-αlevels in mice serum,with significant anti-inflammatory effects.