Example 1:
 1 Instruments and materials
 1.1 Instruments
 Waters high performance liquid chromatograph (Waters e2695 quaternary pump, 2998 PDA detector, American Waters company);
 Nano-ZS 90 laser particle size analyzer (Malvern Instrument Co., Ltd., UK);
 Optima MAX ultra-low temperature centrifuge (Beckman Coulter Co., Ltd., USA);
 Mill-Q ultrapure water meter (Millpore, USA);
 PH acidity meter (Mettler-Toledo, Switzerland);
 JEM-1200EX transmission electron microscope (JEOL, Japan);
 Vector 22 Fourier transform infrared spectrometer (BRUKER, Germany);
 TRISTAR II3020 automatic specific surface and porosity analyzer (Micromeritics Instrument Company, USA);
 Rigaku D/max 2550PC automatic polycrystalline X-ray diffractometer (Japan Rigaku Electric Co., Ltd.);
 Thermo Forma ultra-low temperature refrigerator (Thermo Fisher Scientific, USA);
 TGL-16B high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory);
 KQ5200DE CNC Ultrasound Instrument (Kunshan Ultrasound Instrument Co., Ltd.);
 CP225D electronic balance (Beijing Sartorius Instrument System Co., Ltd.);
 SCIENTZ-ⅡD ultrasonic cell crusher (Ningbo Xinzhi Biotechnology Co., Ltd.);
 DF-101S collector type constant temperature heating magnetic stirrer (Zhengzhou Kechuang Instrument Co., Ltd.);
 HZ-9212S constant temperature oscillator (Taicang Science and Education Equipment Factory);
 VORTEX-5 vortex mixer (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.); etc.
 1.2 Drugs and Reagents
 Resveratrol (Nanjing Zelang Pharmaceutical Technology Co., Ltd., purity>98%);
 Resveratrol reference substance (China Institute for Food and Drug Control, 11535-200502);
 Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES) (Aladdin Reagent Shanghai Jingchun Biochemical Technology Co., Ltd.) ;
 0.25% trypsin, D-Hanks buffer (Gibco, USA);
 DMEM (containing 4.5g·L -1 Glucose, 3.7g L -1 Sodium bicarbonate, 10% fetal bovine serum, 1% non-essential amino acids, 1% glutamine, 100U·mL -1 Penicillin and 100 μg·mL -1 Streptomycin) (Hangzhou Jinuo Biomedical Technology Co., Ltd.);
 Methanol (Honeywell Burdick & Jackson Company, USA), and other reagents were of analytical grade.
 1.3 Experimental animals and cells
 18 clean-grade SD rats, both male and female, weighing (280±20) g (provided by the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine, SCXK Shanghai 2012-0002); Caco-2 cells (frozen in the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine, Originally purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences);
 2 methods
 2.1H 2 Preparation of N-MSN
 MSN was prepared according to the classical Stober method. On this basis, this experiment improved the classical Stober method and synthesized H in one step. 2 N-MSN: 1.0g CTAB was dissolved in 480mL ultrapure water, 3.5mL 2M NaOH solution was added, stirred at a constant temperature of 80°C for 2h, then 3mL TEOS was added rapidly, 2mL APTES was slowly dripped at a constant rate after 30min, and the dripping was controlled for 5min. Continue to react for 2h, after the reaction is finished, let stand for aging for 24h, 15000r·min -1 The white solid obtained by centrifugation for 30 min. Disperse the white solid obtained by centrifugation in 200 mL of NH 4 NO 3 (10mg·mL -1 ) in an ethanol solution of 80 °C for 4 h, the template agent CTAB was removed after 6 repeated operations, and the final centrifugation was followed by vacuum drying to obtain a white powder H 2 N-MSN. Transmission electron microscopy (TEM) was used to analyze the MSN and H 2 N-MSN was observed, particle size and Zeta potential were measured by particle size analyzer, and H was characterized by Fourier transform infrared spectroscopy (FTIR). 2 Modified amino group on N-MSN.
 2.2H 2 Preparation of N-MSN-RES
 The present invention adopts the repeated saturated solution adsorption method to prepare H 2 N-MSN-RES: Weigh 100mgH 2 N-MSN (denoted as W 10 ) was dispersed into 20 mL of RES saturated ethanol solution, stirred at room temperature 25°C in the dark, sonicated for 3 minutes every 30 minutes, and stirred for 2 hours to make H 2 N-MSN fully adsorbed RES saturated ethanol solution, and then 15000r·min -1 Centrifuge for 30min, and weigh the white solid obtained by centrifugation after drying under reduced pressure at 35°C (denoted as W 11 ), and then repeat dispersion, adsorption, centrifugation, drying, and weighing, and each repeated weighing is recorded as W 12 , W 13 , W 14 … Similarly, weigh 100 mg of H 2 Disperse N-MSN into 20 mL of ethanol solution, and operate in the same way except that RES is not added. Each weighing is recorded as W. 01 , W 02 , W 03 , W 04..., the control group MSN-RES was prepared according to the same method.
 2.3H 2 Determination of in vitro release of N-MSN-RES
 Weigh RES, MSN-RES, H 2 An appropriate amount of N-MSN-RES (equivalent to 10 mg of RES) was dispersed in 10 mL of PBS (pH 7.4) buffer, placed in a dialysis bag (molecular weight cut-off 3500KDa), and the dialysis clip was sealed and placed in 1000 mL of release medium PBS. ℃ at 50r·min -1 Shake in a constant temperature water bath, and operate 3 copies in parallel. At 15, 30, 45, 60 min, 2, 4, 6, 8, 12, 24, and 48 h, 1 mL of dialysis medium was taken and supplemented with corresponding PBS. The dialysis medium taken out was filtered with a 0.45 μm microporous membrane, and the RES concentration of the subsequent filtrate was determined by HPLC, and the cumulative release rate was calculated.
 2.4 HPLC chromatographic conditions
 Chromatographic column: SunFire C18 (4.6×250mm, 5μm); mobile phase: methanol-water (50:50); column temperature: 35°C; flow rate: 1.0mL·min -1; Detection wavelength: 306 nm; Injection volume: 20 μL. Taking the peak area of resveratrol as the ordinate (Y) and its concentration as the abscissa (X) for linear regression, the regression equation is: Y=132917X+776.27, r=0.9999, indicating that RES is between 0.25 and 10 μg·mL -1 The internal linear relationship is good. The intra-day precision RSD of the three concentration solutions, high, medium and low, was less than 2%, and the inter-day precision RSD was less than 3%.
 2.5H 2 N-MSN cytotoxicity
 Caco-2 cells were placed in a culture flask, DMEM medium was added, and the cells were placed in a 37°C incubator (5% CO 2 , relative humidity 90%) continuous culture. The cells were digested with trypsin containing 0.02% EDTA every 2-3 days and subcultured at a ratio of 1:3.
 Caco-2 cells in logarithmic growth phase were seeded in 96-well flat-bottom cell culture plates at a density of 1 × 10 5 pc.mL -1 , 190 μL of culture medium was added to each well and incubated for 12 h. Then the experimental groups were added with different concentrations of MSN and H 2 The suspension of N-MSN was 10 μL, and the final concentrations were 0.01, 0.1, 0.5, 1, 5, 20, 50, and 100 μg·mL, respectively. -1 , sterile saline was added to the blank control group, and 6 parallel wells were set for each concentration in each group. After culturing for 24 h, add 5 mg·mL to each well -1 10 μL of MTT solution of 10 μL, shake on a micro-shaker for 3-5 min, continue to culture for 4 h, discard the supernatant, add 150 μL of DMSO, shake on a micro-shaker for 10 min, and measure the optical density (OD) value at a wavelength of 570 nm with a microplate reader . Take the average OD value of 6 wells to calculate the cell survival rate (IC), IC=(OD value of experimental group/OD value of blank control group)×100%.
 2.6H 2 Transmembrane transport of N-MSN-RES
 References for the establishment of the Caco-2 cell monolayer model (Hilgers A R, Conradi R A, Burton P S. Caco-2 Cell Monolayers as a Model for Drug Transport Across the Intestinal Mucosa [J]. Pharm Res. 1990; 7(9): 902-910; Rieux A, Ragnarsson EG, Gullberg E, Préat V, Schneider YJ, Artursson P.Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium[J].Eur J Pharm Sci.2005;25( 4): 455-65.). The cells in logarithmic growth phase were prepared with complete culture medium at a concentration of 1 × 10 6 pc.mL -1 The cell suspension was seeded in Transwell 12-well plate (3402 type of Corning Company, USA, membrane area 1.12cm) per 1mL. 2 , the membrane pore size is 3 μm), after three weeks of culture, the transmembrane resistance measured by the transmembrane resistance meter is greater than 500Ω·cm 2 , the established Caco-2 cell monolayer model was verified with propranolol, and the model was confirmed to be successful after comparing with the literature.
 The successfully modeled Transwell chambers were placed in 12-well nested plates, and each plate was divided into 3 groups (RES, MSN-RES, H 2 N-MSN-RES), parallel 6 wells, each group contains 2 μg·mL RES -1 Transport of the 3 formulations. Rinse 3 times with D-Hanks solution pre-incubated at 37°C. When transmembrane transfer from AP side to BL side, add 1.5mL blank D-Hanks solution to the BL chamber to completely saturate the outer surface of the AP; then add 1.5mL to the AP chamber Drug-containing medium; when transmembrane transport from BL side to AP side, add 1.5mL of drug-containing medium to the BL chamber, and add 1.5mL of blank D-Hanks solution to the AP chamber. After adding the liquid medicine, carefully sample 0.15 mL from the BL or AP room at 0.5, 1, 2, 4, 8, and 12 h, and supplement with 0.15 mL of blank D-Hanks. The drug concentration of the sample was determined by HPLC, the drug absorption curve was drawn, and the apparent permeability coefficient P was calculated. app , P app =ΔQ/(Δt·A·C 0 ), ΔQ is the cumulative drug transport amount (μg); ΔQ/Δt is the drug transport rate (μg·min -1 ); C 0 is the initial concentration of the drug (μg·mL -1 ); A is the surface area of the cell monolayer (cm 2 ).
 2.7 Pharmacokinetic studies
 2.7.1 Treatment of plasma samples Precisely aspirate 100 μL of plasma supernatant, add 400 μL methanol, vortex for 30 s, 8000 r·min -1 After centrifugation for 10 min, 300 μL of the supernatant was taken and evaporated to dryness under nitrogen at 35°C, the residue was redissolved in 150 μL methanol, vortexed for 30 s, sonicated for 2 min, passed through a 0.22 μm microporous membrane, and 20 μL was injected for analysis.
 2.7.2 Dosing schedule and blood sample collection 18 SD rats were randomly divided into 3 groups, fasting for 12 h before administration, and drinking water freely. RES solution, MSN-RES and H were administered by gavage, respectively 2N-MSN-RES (equivalent to RES dose of 200 mg·kg -1 ). At 15, 30, 45 min, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, and 12 h after administration, 0.5 mL of blood was collected through femoral artery cannula, and placed in a centrifuge tube treated with heparin sodium. Rapidly 3500r·min -1 After centrifugation for 5 min, 100 μL of the supernatant was taken and processed according to the method under “2.7.1”, and 100 μL of normal saline was added to the remaining plasma, shaken and shaken, and then slowly injected back into the rat through the jugular vein. The samples were determined by HPLC and the blood drug concentration was calculated, the drug-time curve was drawn and the pharmacokinetic parameters were calculated.
 3 results
 3.1H 2 Preparation and characterization of N-MSN
 In this experiment, a modified classical Stober method was used to synthesize H in one step. 2 N-MSN. By TEM observation, H 2 Before removing the template agent CTAB, N-MSN had a large number of agglomerates and adhesion, poor dispersibility, and no mesoporous structure ( figure 1.A);H 2 After removing the template agent, N-MSN presents a round spherical shape, regular shape, good dispersibility, no aggregation phenomenon, and the mesoporous structure can be clearly observed when magnified to 40k times ( figure 1.B). H 2 The particle size of N-MSN is 98.4±2.8nm and the Zeta potential is 13.2±1.8mv after being measured by the particle size analyzer. The particle size of MSN synthesized by the classical Stober method is 77.8±3.4nm and the Zeta potential is -24.6±0.9mv ( figure 2.and image 3 ). H 2 The particle size of N-MSN is significantly larger than that of MSN, mainly because the 3-aminopropyl triethoxy group in APTES increases H 2 The particle size of N-MSN, and 3-aminopropyltriethoxy makes MSN change from negatively charged to positively charged H 2 N-MSN. H 2 N-MSN was characterized by infrared spectroscopy and compared with the peak shape of MSN at 3000cm -1 double peaks ( Figure 4 ), which can be inferred to be the stretching vibration peak of N-H, indicating the successful synthesis of H 2 N-MSN.
 3.2H 2 Preparation of N-MSN-RES
 Preparation of H by Repeated Saturated Solution Adsorption 2 N-MSN-RES, by Figure 5 Shown: As the number of adsorption increases, H 2 The weight of N-MSN was continuously lost, with an average loss rate of 3.21 ± 0.50 mg each time; although H 2 The total weight of N-MSN-RES also showed a downward trend, but H 2 N-MSN-RES minus H 2 However, the weight of N-MSN keeps increasing [Fig. 2 N-MSN-RES) i -(H 2 N-MSN) i curve], indicating that as the number of adsorption increases, H 2 The amount of N-MSN loaded into RES keeps increasing, and the average increase in drug loading for each adsorption is 1.69±0.22 mg; it can also be seen from the figure that after 8 adsorptions, the loaded drug is basically unchanged, and it can be inferred that H 2 The mesopores of N-MSN are close to the fully loaded state. Therefore, this experiment determined the repeated saturated solution adsorption method to prepare H 2 The adsorption times of N-MSN-RES were 8 times, and the final drug loading was 19.26±2.51%.
 3.3 Evaluation of drug carrier cytotoxicity
 MSN and H were determined by MTT method 2 Cytotoxicity of N-MSN on Caco-2. like Image 6 As shown, after 24h incubation, the nanoparticle concentration reached 20ug·mL -1 The cell viability remained above 90%. In addition, H 2 The toxicity of N-MSN is slightly higher than that of MSN, which may be due to the toxicity of 3-aminopropyltriethoxy to cells, and its toxicity mechanism needs further study. The experimental results show that at 0~20ug·mL -1 range, MSN and H 2 N-MSN nanocarriers had no obvious toxic effects on Caco-2 cells.
 3.4 In vitro drug release evaluation
 Examined MSN-RES and H 2 Drug release behavior of N-MSN-RES in simulated physiological environment (PBS, pH 7.4). It can be seen from the in vitro drug release curve that the RES solution released the drug rapidly, the drug release amount within 3h reached 90%, and the drug was basically completely released in 4h; MSN-RES and H 2 The cumulative drug release of N-MSN-RES within 12h was 70.1% and 62.5%, and the drug release was stable after 12h, and the drug release within 48h was 81.4% and 73.3%, respectively. MSN-RES and H 2 N-MSN-RES showed obvious sustained-release properties.
 3.5 Evaluation of transmembrane transport
 In transmembrane transport experiments, RES solution, MSN-RES and H 2 The amount of N-MSN-RES transported across the membrane increased with time ( Figure 8 ). RES solution, MSN-RES and H 2 The transport of N-MSN-RES in the AP→BL and BL→AP directions was very similar, with ER values of 0.99, 1.07, and 1.06, respectively, indicating that the three preparations were all passively transported across the membrane without obvious efflux ( Table 1). The RES solution basically reached a steady state after 2h, while the MSN-RES reached a steady state after 4h, but the apparent transmission absorption rate of MSN-RES was very low, and the P in both directions app The values of only 10.58±0.76 and 9.89±0.40 are significantly smaller than those of the RES solution. H 2 N-MSN-RES reached a plateau after 4 h, but its P app values are much higher than RES solution and MSN-RES, P in both directions app They were 17.86±0.59 and 16.85±0.38, respectively, indicating that the amino-modified MSN had strong transmembrane ability.
 Table 1: Carrier P in Caco-2 monolayer cell transport app determination of
 3.5 Pharmacokinetic studies
 Rats were administered RES solution, MSN-RES and H by one-time gavage 2 Post-N-MSN-RES plasma concentration-timeline as Figure 9 As shown, data analysis was performed by PKSolver software, and the AIC minimum method was used to select the most suitable compartment model. RES solution is a one-compartment model, while MSN-RES and H 2 N-MSN-RES is a two-compartment model, and the main pharmacokinetic parameters are shown in Table 2.
 Depend on Figure 9 and Table 2 it can be seen that MSN-RES and H 2 N-MSN-RES half-life (T 1/2 ) and time to peak (T max ) was significantly larger than that of the RES solution, indicating that the RES was transformed by MSN and H 2 After N-MSN encapsulation, not only the drug metabolism rate is reduced, but also the sustained release characteristics of the carrier make the T of RES max Significant delay, mean residence time (MRT) extended several-fold, and fluctuations in drug concentration leveled off. H 2 The peak concentration of N-MSN-RES (C max ) was significantly larger than that of RES solution and MSN-RES, indicating that amino modification could promote the gastrointestinal absorption of MSN, which was consistent with the results of transmembrane transport. H 2 The area under the drug-time curve (AUC) of N-MSN-RES 0-t ) is 2.37 times that of the RES solution, while H 2 The clearance (CL) of N-MSN-RES was only 29.2% of that of the RES solution, indicating that H 2 N-MSN-RES can effectively increase the effect time of RES in the body and improve the bioavailability of RES.
 Table 2: Main pharmacokinetic parameters (n=6)
 4 Discussion
 Most poorly soluble drugs have low oral bioavailability and poor clinical effects due to poor water solubility. contact and therefore cannot be fully absorbed. The advantage of the oral nano-drug delivery system is to increase the biomembrane permeability of the drug: the nano-carrier has a very large surface area due to its small particle size. The contact time and contact area of the drug and the intestinal wall, thereby improving the bioavailability of oral absorption of the drug, etc. The charged properties of the surface of the nanocarriers also affect the biofilm permeability of the drug. The positively charged nanocarriers can interact electrostatically with the negatively charged mucins on the surface of the gastrointestinal mucosa, resulting in strong mucoadhesion. Extend the residence time of the carrier, promote drug absorption and the transmembrane transport of the carrier.
 MSN can effectively improve the oral bioavailability of drugs by virtue of its huge specific surface area, and the amino-modified MSN can more effectively promote the absorption of drugs through mutual adhesion with mucins on the surface of the gastrointestinal tract. Likewise, the experimental results show that MSN and NH 2 -MSN has certain cytotoxicity at higher concentrations. On the one hand, its huge specific surface area destroys the stability of the cell membrane when it is in contact with cells. On the other hand, it may cause cytotoxicity due to the failure to remove the template agent CTAB during preparation. The former is unavoidable in oral administration, because reducing the specific surface area will also reduce the contact area with the gastrointestinal tract and reduce absorption, and the latter can be solved by improving the synthesis process in subsequent studies.
 The repeated saturated solution adsorption method adopted in the present invention is a very effective drug loading method. Compared with other drug loading methods such as dipping method, conjugate combination method, acid-base adsorption method, etc., the repeated saturated solution adsorption method is simple in operation and heavy in operation. It has good performance and does not require expensive equipment and instruments. The most important thing is that with the increase of adsorption times, the drug loading capacity of the drug can be significantly improved, and the problem of low drug loading capacity of nanocarriers can be effectively solved. However, the repeated saturated solution adsorption method can only be limited to nanocarriers with specific rigid structures such as MSN, carbon nanotubes, and nanometals.
 Nano-drug delivery systems can change the pharmacokinetic characteristics of drugs, and pharmacokinetic research is also one of the criteria for evaluating whether nano-drug delivery systems are successfully constructed, providing a reference for the final determination of drug dosage. After the RES was loaded into the nanocarrier, it was changed from a single-compartment model to a two-compartment model, while T 1/2 , T max and MRT significantly prolonged, Cmax and AUC 0-t markedly increased, while CL decreased exponentially, mainly due to NH 2 - Caused by the controlled release properties of MSN and its adhesion to the gastrointestinal tract. Therefore, amino-modified mesoporous silica is an excellent oral nanocarrier material, which can effectively improve the bioavailability of poorly soluble drugs, and has broad research and application prospects.