Carboxylated dendritic mesoporous silica nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses carboxylated dendritic mesoporous silica nanoparticles as well as a preparation method and application thereof. The preparation method of the carboxylated dendritic mesoporous silica nanoparticles comprises the following step: carrying out nucleophilic substitution reaction on a mixture of aminated dendritic mesoporous silica nanoparticles and a solvent and suberic anhydride. The carboxylated dendritic mesoporous silica nanoparticle prepared by the preparation method is used as a carrier to load the semeglutide to obtain the semeglutide-loaded nanoparticle, namely MSN-coated C8OOH-semeglutide, the release of the semeglutide-loaded nanoparticle is less when the pH value is 2.0, and the release of the semeglutide-loaded nanoparticle is basically equivalent to that of a free drug when the pH value is 6.8, which indicates that the semeglutide-loaded nanoparticle can effectively protect the semeglutide, and the semeglutide-loaded nanoparticle can be used as an anti-tumor drug. The medicine is prevented from being released too early in the stomach and finally reaches the intestinal tract to release the medicine. Pharmacokinetic experiments show that when the MSN (at) C8OOH-semeglutide is adopted, the higher bioavailability is achieved.

Description

Technical Field

[0001] This invention relates to a carboxylated dendritic mesoporous silica nanoparticle, its preparation method, and its application. Background Technology

[0002] Semaglutide is a new-generation GLP-1 (glucagon-like peptide-1) analog with hypoglycemic and anti-obesity effects. However, semaglutide is usually administered by injection, and the bioavailability of marketed oral tablets is poor, which limits its application. Summary of the Invention

[0003] To address the issue of poor bioavailability of oral semaglutide tablets in existing technologies, this invention provides carboxylated dendritic mesoporous silica nanoparticles, their preparation method, and their applications.

[0004] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0005] This invention provides a method for preparing carboxylated dendritic mesoporous silica nanoparticles, comprising the following steps:

[0006] A mixture of aminated dendritic mesoporous silica nanoparticles and solvent undergoes a nucleophilic substitution reaction with succinic anhydride.

[0007] Preferably, the concentration of the aminated dendritic mesoporous silica nanoparticles in the mixture is 2-20 mg / mL, for example, 3.33 mg / mL, 7.5 mg / mL or 12 mg / mL.

[0008] Preferably, the mass ratio of the aminated dendritic mesoporous silica to octanoic anhydride is 1:(1.67-20).

[0009] Preferably, the nucleophilic substitution reaction is carried out at a temperature of 20-40°C, for example, 30°C.

[0010] Preferably, the nucleophilic substitution reaction takes 12-24 hours, for example, 18 hours.

[0011] Preferably, the solvent is DMF or DMSO.

[0012] Preferably, the nucleophilic substitution reaction is followed by a post-treatment process. This post-treatment preferably includes centrifugation, precipitate collection, alcohol washing, drying, and grinding.

[0013] Preferably, the method for preparing the aminated dendritic mesoporous silica includes the following steps:

[0014] A first mixture of dendritic mesoporous silica nanoparticles and a first solvent undergoes an amination reaction with an amination reagent.

[0015] In the first mixture, the concentration of the dendritic mesoporous silica is preferably 1.25-15 mg / mL, for example, 2.5 mg / mL, 5.0 mg / mL or 7.5 mg / mL.

[0016] The preferred mass ratio of the dendritic mesoporous silica to the amination reagent is 1:(0.79-14.2), for example, 1:2.37, 1:3.15 or 1:4.73.

[0017] The amination reaction is preferably carried out at a temperature of 100-115°C, for example, 110°C.

[0018] The amination reaction is preferably carried out over a period of 3-5 hours.

[0019] The amination reagent can be a conventionally selected amination reagent in the art, such as 3-aminopropyltriethoxysilane or diethylenetriaminopropyltrimethoxysilane.

[0020] The first solvent may be a solvent commonly used in the art, such as toluene or ethanol.

[0021] Preferably, the amination reaction is followed by a post-treatment step. More preferably, the post-treatment includes centrifugation, precipitate collection, alcohol washing, drying, and grinding.

[0022] The method for preparing the dendritic mesoporous silica can be a conventional method used in the art, and preferably includes the following steps:

[0023] The oil phase and the aqueous phase are mixed and reacted; the aqueous phase includes a second mixture of surfactant, water and catalyst; the oil phase includes an organic solution of tetraethyl orthosilicate.

[0024] The surfactant is preferably one or both of hexadecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride.

[0025] In the process of preparing the aqueous phase, the surfactant is preferably added in the form of an aqueous surfactant solution; more preferably, the surfactant in the aqueous surfactant solution has a mass percentage of 10%-30%, for example 25%, where the percentage is the mass percentage of the surfactant relative to the mass of the aqueous surfactant solution.

[0026] The surfactant is preferably one or both of hexadecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride.

[0027] The catalyst is preferably one or more of sodium hydroxide, triethanolamine, and triethylamine.

[0028] The organic solvent in the organic solution of the tetraethyl orthosilicate is preferably chlorobenzene, cyclohexane, or octane.

[0029] Preferably, the concentration of tetraethyl orthosilicate in the organic solution of tetraethyl orthosilicate is 0.1-0.2 g / mL, for example, 0.15 g / mL.

[0030] The preferred mass ratio of the tetraethyl orthosilicate to the surfactant is (4-10):(3-8), for example, 4.5:3 or 9:6.

[0031] The preferred mass ratio of tetraethyl orthosilicate to catalyst is (4-10):(0.1-0.5), for example, 4.5:0.1, 8:0.5 or 9:0.2.

[0032] The preferred mass ratio of the surfactant to the catalyst is (3-8):(0.1-0.5), for example, 6:0.5 or 6:0.2.

[0033] The mixing method is preferably stirring. The stirring temperature is more preferably 40-60℃. The stirring time is more preferably 12-24 hours. The stirring speed is more preferably 300-800 rpm.

[0034] The preparation method of the second mixture can be a conventional preparation method in the art, preferably including the following steps: mixing surfactant, water and catalyst. The mixing method is preferably stirring. The stirring temperature is more preferably 40-60°C. The stirring time is more preferably 0.5-2.5 h. The stirring speed is more preferably 50-150 rpm.

[0035] The method for preparing dendritic mesoporous silica preferably includes post-treatment after the reaction. The post-treatment preferably includes collecting the precipitate, washing with water, washing with alcohol, washing with acid, centrifuging, collecting and washing the precipitate again, drying, and grinding it into powder. The acid washing more preferably includes dispersing the precipitate in methanol containing HCl and refluxing it; the molar concentration of HCl in the methanol containing HCl is preferably 0.2-0.3 mol / L.

[0036] This invention provides carboxylated dendritic mesoporous silica nanoparticles, which are prepared by the method described above for preparing carboxylated dendritic mesoporous silica nanoparticles.

[0037] The present invention provides nanoparticles loaded with smegglutinin, comprising a carrier and smegglutinin; wherein the carrier is carboxylated dendritic mesoporous silica nanoparticles as described above.

[0038] This invention provides a method for preparing nanoparticles loaded with smegglutinin, which uses carboxylated dendritic mesoporous silica nanoparticles as described above, and includes the following steps:

[0039] The mixture of carboxylated dendritic mesoporous silica nanoparticles, buffer solution, and smegglutinin is obtained by post-treatment; wherein the pH value of the buffer solution is 4.0-5.0.

[0040] The mass ratio of the smegglutinin and the carboxylated dendritic mesoporous silica nanoparticles is preferably 1:(1-5), for example, 1:2.

[0041] The buffer solution is preferably a citrate-disodium hydrogen phosphate buffer solution or a phosphate buffer solution.

[0042] In the mixture, the concentration of the carboxylated dendritic mesoporous silica nanoparticles is preferably 1-2 mg / mL, for example, 1.5 mg / mL.

[0043] In the mixture, the concentration of smegglutinin is preferably 1-2 mg / mL.

[0044] In the method for preparing the nanoparticles loaded with smegglutinin, the post-treatment preferably includes centrifugation, collection of precipitate, and washing of precipitate.

[0045] The preparation method of the mixture can be a conventional preparation method used in the art, and preferably includes the following steps:

[0046] The first mixture of carboxylated dendritic mesoporous silica nanoparticles and buffer solution is then mixed with smegglutinin to obtain the second mixture.

[0047] Preferably, the first mixture can be prepared using conventional methods in the art, such as mixing carboxylated dendritic mesoporous silica nanoparticles with a buffer solution. The mixing method is preferably ultrasonication. The ultrasonication time is preferably 10-20 minutes.

[0048] Preferably, the second mixing method is stirring. The stirring time is preferably 10-20 hours. The stirring speed is preferably 50-100 rpm.

[0049] This invention provides a method for preparing smegglutinin-loaded nanoparticles as described above, resulting in smegglutinin-loaded nanoparticles.

[0050] This invention provides the application of the smegglutinin-loaded nanoparticles described above in the preparation of hypoglycemic drugs.

[0051] This invention provides the application of the nanoparticles loaded with smegglutinin as described above in the preparation of anti-obesity drugs.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows:

[0055] This invention provides carboxylated dendritic mesoporous silica nanoparticles, their preparation method, and applications. The obtained carboxylated dendritic mesoporous silica nanoparticles are used as a carrier to load semagraviride, resulting in semagraviride-loaded nanoparticles, namely MSN@C8OOH-semagraviride. These nanoparticles release less semagraviride at pH 2.0, but release is essentially equivalent to that of the free drug at pH 6.8, indicating that they effectively protect semagraviride, preventing premature release in the stomach and ensuring the drug reaches the intestines for final release. Pharmacokinetic experiments show that MSN@C8OOH-semagraviride exhibits significantly higher bioavailability, exceeding 10 times that of the free drug. Attached Figure Description

[0056] Figure 1 This is a transmission electron microscope (TEM) image of the dendritic mesoporous silica obtained in Example 1.

[0057] Figure 2 This is a scanning electron microscope image of the dendritic mesoporous silica obtained in Example 1.

[0058] Figure 3 This is a particle size distribution diagram of the different modified dendritic mesoporous silica in Example 5.

[0059] Figure 4 The image shows the zeta potential of different modified dendritic mesoporous silica in Example 5.

[0060] Figure 5 The image shows adsorption / desorption isotherms of different modified dendritic mesoporous silica in Example 5.

[0061] Figure 6 This is a pore size distribution diagram of the different modified dendritic mesoporous silica in Example 5.

[0062] Figure 7 The infrared spectra of different modified dendritic mesoporous silica in Example 5 are shown.

[0063] Figure 8 Thermogravimetric curves of different modified dendritic mesoporous silica in Example 5 are shown.

[0064] Figure 9 This is a diagram showing the mucin binding capacity of different modified dendritic mesoporous silica in Example 5.

[0065] Figure 10 This is a comparison chart of the apparent permeability coefficients obtained from the mucus layer permeation experiment of different modified dendritic mesoporous silica in Example 5.

[0066] Figure 11 The graph shows the effect of different modified dendritic mesoporous silica on the survival of Caco-2 cells in Example 5, and the change in cell viability with concentration.

[0067] Figure 12 The in vitro release curves of MSN-semaglutide, MSN@NH2-semaglutide obtained in Comparative Examples 1-2 and MSN@C8OOH-semaglutide obtained in Example 8 are shown at a pH of 2.0 in the release medium.

[0068] Figure 13 The in vitro release curves of MSN-semaglutide, MSN@NH2-semaglutide obtained in Comparative Examples 1-2 and MSN@C8OOH-semaglutide obtained in Example 8 are shown at a pH of 6.8 in the release medium.

[0069] Figure 14 The graph shows the percentage change in blood glucose over time for MSN-semaglutide, MSN@NH2-semaglutide obtained in Comparative Examples 1-2, and MSN@C8OOH-semaglutide obtained in Example 8. Detailed Implementation

[0070] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0071] Octanoic anhydride (CAS: 10521-06-9)

[0072] 3-Aminopropyltriethoxysilane (CAS: 919-30-2)

[0073] Example 1

[0074] The preparation method of dendritic mesoporous silica (MSN) is as follows:

[0075] 12 mL of hexadecyltrimethylammonium chloride aqueous solution (25% by mass) was added to 18 mL of deionized water, followed by 0.1 g of triethanolamine. The mixture was stirred at 40 °C for 0.5 h, then 30 mL of tetraethyl orthosilicate chlorobenzene solution (0.15 g / mL) was added. The mixture was stirred slowly for another 12 h. The precipitate was collected by centrifugation and washed repeatedly with deionized water and ethanol. After washing, the precipitate was dispersed in 150 mL of methanol containing HCl (0.2 mol / L) and refluxed (70 °C for 6 h). The precipitate was collected by centrifugation and washed repeatedly with ethanol and deionized water. The precipitate was dried in an oven (60 °C for 18 h), and ground to obtain a white, dry powder (electron microscopy showed a particle size of approximately 110 nm, and DLS particle size was approximately 230 nm), which was then stored at room temperature.

[0076] The dendritic mesoporous silica nanoparticles prepared in Example 1 were characterized and analyzed, and the results are as follows:

[0077] Characterization of dendritic mesoporous silica

[0078] The specific procedure is as follows: 1 mg of dendritic mesoporous silica was diluted with 10 mL of ethanol and its morphology was observed under a transmission electron microscope. 5 mg of dendritic mesoporous silica was sputter-coated with gold (10 nm thick) and its morphology was observed under a scanning electron microscope (ZEISS Gemini SEM 300, Germany).

[0079] The results are as follows Figure 1 , 2 As shown, from Figure 1 The nanoparticles are observed to be uniformly dispersed, with a particle size of approximately 100 nm, and exhibit a dendritic structure. Figure 2 As can be seen, the nanoparticles have a uniform morphology and a relatively large pore size.

[0080] Example 2

[0081] The preparation method of dendritic mesoporous silica is as follows:

[0082] Take 24 mL of hexadecyltrimethylammonium chloride aqueous solution (hexadecyltrimethylammonium chloride mass percentage 25%) and add it to 36 mL of deionized water. Then add 0.5 g of triethanolamine. Stir at 60 °C for 2.5 h, then add 80 mL of tetraethyl orthosilicate chlorobenzene solution (tetraethyl orthosilicate concentration 0.1 g / mL). Continue stirring slowly for 24 h. Centrifuge to collect the precipitate and wash it repeatedly with deionized water and ethanol. After washing, disperse the precipitate in 150 mL of methanol containing HCl (HCl molar concentration 0.3 mol / L) and reflux (temperature 75 °C, time 4 h). Centrifuge to collect the precipitate and wash it repeatedly with ethanol and deionized water. Place the obtained precipitate in an oven to dry (drying temperature 80 °C, time 18 h), grind it to obtain a white dry powder, and store it at room temperature.

[0083] The other implementation steps are the same as in Example 1.

[0084] Example 3

[0085] The preparation method of dendritic mesoporous silica is as follows:

[0086] Take 24 mL of hexadecyltrimethylammonium chloride aqueous solution (hexadecyltrimethylammonium chloride mass percentage 25%) and add it to 36 mL of deionized water. Then add 0.2 g of triethanolamine and stir at 50 °C for 1 h. Then add 60 mL of tetraethyl orthosilicate chlorobenzene solution (tetraethyl orthosilicate concentration 0.15 g / mL) and continue stirring slowly for 18 h. Centrifuge to collect the precipitate and wash it repeatedly with deionized water and ethanol. After washing, disperse the precipitate in 120 mL of methanol containing HCl (HCl molar concentration 0.2 mol / L) and reflux (temperature 77 °C, time 3 h). Centrifuge to collect the precipitate and wash it repeatedly with ethanol and deionized water. Place the obtained precipitate in an oven to dry (drying temperature 70 °C, time 18 h), grind it to obtain a white dry powder, and store it at room temperature.

[0087] The other implementation steps are the same as in Example 1.

[0088] Example 4

[0089] The preparation method of carboxylated dendritic mesoporous silica (MSN@C8OOH) is as follows:

[0090] (1) The preparation method of aminated dendritic mesoporous silica (MSN@NH2) is as follows: Take 100 mg of dendritic mesoporous silica (obtained from Example 1), disperse it in 40 mL of toluene (the concentration of dendritic mesoporous silica is 2.5 mg / mL), add 0.5 mL of 3-aminopropyltriethoxysilane (mass is 473 mg), stir, heat at 100 °C for 3 h, centrifuge, take the precipitate, collect the solid, wash with alcohol, centrifuge, dry, grind, and obtain aminated dendritic mesoporous silica.

[0091] (2) Take 100 mg of aminated dendritic mesoporous silica, add 30 mL of DMF and stir evenly (the concentration of aminated dendritic mesoporous silica is 3.33 mg / mL), then add 1 g of octanoic anhydride and react at 20 °C for 12 h. Centrifuge, wash with alcohol, dry, and grind to obtain MSN@C8OOH.

[0092] Example 5

[0093] The preparation method of carboxylated dendritic mesoporous silica is as follows:

[0094] (1) The preparation of aminated dendritic mesoporous silica is as follows: 600 mg of dendritic mesoporous silica (obtained from Example 3) was dispersed in 80 mL of toluene (the concentration of dendritic mesoporous silica was 7.5 mg / mL), 1.5 mL of 3-aminopropyltriethoxysilane (mass 1419 mg) was added and stirred, heated at 115 °C for 5 h, centrifuged, the precipitate was collected, the solid was washed with alcohol, centrifuged, dried, and ground to obtain aminated dendritic mesoporous silica.

[0095] (2) Take 600 mg of aminated dendritic mesoporous silica, add 50 mL of DMF and stir evenly (the concentration of aminated dendritic mesoporous silica is 12 mg / mL), then add 2 g of octanoic anhydride and react at 40 °C for 24 h. Centrifuge, wash with alcohol, dry and grind to obtain MSN@C8OOH.

[0096] Example 6

[0097] The preparation method of carboxylated dendritic mesoporous silica is as follows:

[0098] (1) The preparation method of aminated dendritic mesoporous silica is as follows: Take 300 mg of dendritic mesoporous silica (obtained from Example 2), disperse it in 60 mL of toluene (the concentration of dendritic mesoporous silica is 5.0 mg / mL), add 1 mL of 3-aminopropyltriethoxysilane (mass is 946 mg), stir, heat at 110 °C for 3-5 h, centrifuge, take the precipitate, collect the solid, wash with alcohol, centrifuge, dry, grind, and obtain aminated dendritic mesoporous silica.

[0099] (2) Take 300 mg of aminated dendritic mesoporous silica, add 40 mL of DMF and stir until the concentration of aminated dendritic mesoporous silica is 7.5 mg / mL, then add 1.5 g of octanoic anhydride and react at 30 °C for 18 h. Centrifuge, wash with alcohol, dry and grind to obtain MSN@C8OOH.

[0100] The MSN obtained in Example 3, MSN@NH2 and MSN@C8OOH obtained in Example 5 are characterized as follows:

[0101] The specific steps are as follows:

[0102] (1) Take 1 mg of different modified dendritic mesoporous silica, dilute it with 10 mL of deionized water, and then use NICOMP nano laser particle size analyzer to detect the particle size and distribution.

[0103] (2) Take 1 mg of different modified dendritic mesoporous silica, dilute it with 10 mL of deionized water, and then use NICOMP nano laser particle size analyzer to detect the ζ potential.

[0104] (3) The nitrogen adsorption / desorption isotherms were measured using a fully automated specific surface area and porosity analyzer to analyze the specific surface area, pore volume, and pore size of the prepared modified dendritic mesoporous silica. The test temperature was 77.3 K, and the samples were degassed at 150 °C for 8 h before testing.

[0105] (4) Infrared spectroscopy was performed using a Fourier transform infrared spectrometer, and samples of different modified dendritic mesoporous silica were prepared and analyzed using the KBr pellet method.

[0106] (5) Thermogravimetric analysis was performed on different modified dendritic mesoporous silica using a thermogravimetric analyzer. The silica was first equilibrated at 100℃ for 20 min, and then heated to 850℃ at a rate of 10℃ / min.

[0107] The above test results are as follows Figure 3 , 4 As shown in figures 5, 6, 7, and 8. From Figure 3 It can be seen that the particle size of MSN and MSN@NH2 is around 230 nm, while the particle size of MSN@C8OOH decreases to 113 nm, which is close to the measurement by electron microscopy. This is mainly because the modification of the hydrophilic chain makes the particles more dispersed. Figure 4 As can be seen, the zeta potentials of different modified dendritic mesoporous silicas are different, confirming the successful modification. From Figure 5 It can be seen that the isotherms of dendritic mesoporous silica with different modifications are typical type IV isotherms, and the specific surface area, pore volume, and pore size are shown in Table 1. From Figure 7 It can be determined that 1550cm -1 NH band, 1718cm -1 The C=O band and 3089-2864 cm⁻¹-1 The CH bands on the spectrum confirm the successful grafting of the octanoic acid group. Figure 8 It can be seen that the organic content of dendritic mesoporous silica increases with the degree of modification.

[0108] Table 1. Specific surface area, pore volume, and pore size of MSN, MSN@NH2, and MSN@C8OOH

[0109] type <![CDATA[Specific surface area / (m 2 / g)]]> <![CDATA[Pore volume / (cm 3 / g)]]> Aperture / nm MSN 613.548 4.48 21.248 <![CDATA[MSN@NH2]]> 374.121 2.43 21.149 <![CDATA[MSN@C8OOH]]> 314.149 1.78 13.375

[0110] (6) Mucin binding experiment

[0111] The specific procedure was as follows: A mucin solution (concentration 2 mg / mL) was prepared using porcine gastric mucin (Type II, manufacturer: Sigma-Aldrich, model: M2378). 10 mL of different modified mesoporous silica solutions were added to 2 mL of the mucin solution, and the mixture was incubated in a shaker (37℃, 150 rpm). Samples were taken at different time points (0, 1, 2, 3 h) to detect particle size.

[0112] The results are as follows Figure 9 As shown in the figure. In the mucin binding experiment, MSN@C8OOH decreased the degree of mucin binding while the particle size remained essentially unchanged, while MSN@NH2 increased the degree of mucin binding.

[0113] (7) Mucus layer penetration experiment

[0114] The specific procedure was as follows: 15 mg of differently modified mesoporous silica and 2 mg of fluorescein isothiocyanate (FITC, model: Yuanye #S19127) were co-incubated in 10 mL of anhydrous ethanol to prepare fluorescent particles. 50 mg / mL porcine gastric mucin solution was added to a Transwell (6.5 mm) solution. An 8.0μm pore size polycarbonate membrane chamber (manufacturer: Corning, model: 3422) with a thickness of 2mm was used. A suspension of fluorescent particles was added to the top of the mucus layer.

[0115] Liquid samples were collected from the receiving cell at 20, 60, 120, and 240 minutes, and the concentration was measured to calculate the apparent permeability coefficient. The specific calculation formula is: P app = (V / (C0×A))×(ΔQ / Δt)cm / s. Where V represents the volume of the solution in the lower chamber, in mL; A represents the membrane area, which is 0.3 cm². 2 C0 represents the initial concentration of fluorescent particles, in μg / mL; ΔQ / Δt represents the concentration of fluorescent particles permeating the lower chamber per unit time, which is calculated by dividing the final concentration in the lower chamber by the transport time, in μg / mL·s.

[0116] The results are as follows Figure 10 As shown in the figure, in the mucus layer permeation experiment, MSN@C8OOH had the highest apparent permeability coefficient, followed by MSN, and then MSN@NH2.

[0117] (8) Cytotoxicity test

[0118] The specific procedure was as follows: Caco-2 cells (manufacturer: Pronosei, model: CL-0050) were cultured in 96-well plates for 48 hours. The cells were grouped into blank wells, control wells, and experimental wells (experimental wells were divided into 3 groups based on different mesoporous silica, with 5 concentrations in each group), with 5 samples in each group. After incubation for 24 hours, the absorbance was measured at 450 nm using the CCK8 method. Cell viability was calculated using the following method: Cell viability = (Experimental group absorbance - Blank control absorbance) / (Control group absorbance - Blank control absorbance) × 100%).

[0119] The results are as follows Figure 11 As shown, within the given concentration range, the viability of Caco-2 cells was essentially unaffected.

[0120] Example 7

[0121] The preparation method of MSN@C8OOH supported semaglutide nanoparticles is as follows:

[0122] Take 10 mg of MSN@C8OOH (prepared in Example 5) and add it to 10 mL of citrate-disodium hydrogen phosphate buffer solution (pH 4.0). Sonicate the solution for 10 min with a probe, and then add 10 mg of smegglutinin to obtain a mixture. The concentration of smegglutinin is 1 mg / mL and the concentration of MSN@C8OOH is 1 mg / mL. Stir the mixture at room temperature in the dark for 10 h, centrifuge to collect the precipitate, wash it with ultrapure water, and store the washed precipitate at -20℃ to obtain MSN@C8OOH-smegglutinin.

[0123] Example 8

[0124] The preparation method of MSN@C8OOH supported semaglutide nanoparticles is as follows:

[0125] Take 20 mg of MSN@C8OOH (prepared in Example 5) and add it to 10 mL of citrate-disodium hydrogen phosphate buffer solution (pH 5.0). Sonicate the solution for 20 min with a probe, and then add 10 mg of smegglutinin to obtain a mixture. The concentration of smegglutinin is 1 mg / mL and the concentration of MSN@C8OOH is 2 mg / mL. Stir the mixture at room temperature in the dark for 20 h, centrifuge to collect the precipitate, wash it with ultrapure water, and store the washed precipitate at -20℃ to obtain MSN@C8OOH-smegglutinin.

[0126] Example 9

[0127] Preparation of MSN@C8OOH nanoparticles loaded with semaglutide:

[0128] Take 15 mg of MSN@C8OOH (prepared in Example 5) and add it to 10 mL of citrate-disodium hydrogen phosphate buffer solution (pH 4.5). Sonicate the solution for 15 min with a probe, and then add 15 mg of smegglutinin to obtain a mixture. The concentration of smegglutinin is 1.5 mg / mL and the concentration of MSN@C8OOH is 1.5 mg / mL. Stir the mixture at room temperature in the dark for 15 h, centrifuge to collect the precipitate, wash it with ultrapure water, and store the washed precipitate at -20 °C to obtain MSN@C8OOH-smegglutinin.

[0129] Comparative Example 1

[0130] Based on Example 8, only MSN@C8OOH was changed to MSN to obtain MSN-semaglutide nanoparticles loaded with MSN.

[0131] Comparative Example 2

[0132] Based on Example 8, only MSN@C8OOH was changed to MSN@NH2 to obtain MSN@NH2-semaglutide nanoparticles loaded with MSN@NH2.

[0133] The MSN-semaglutide, MSN@NH2-semaglutide obtained in Comparative Examples 1-2 and the MSN@C8OOH-semaglutide obtained in Example 8 were characterized as follows:

[0134] (1) The supernatant obtained by centrifugation and the washing solution obtained by washing during the above preparation process were collected for HPLC detection. Specifically, the collected supernatant and washing solution were diluted 1:1 with acetonitrile and then subjected to HPLC detection to calculate the encapsulation efficiency and drug loading of the nanoparticles loaded with semaglutide. The specific calculation method is as follows: Encapsulation efficiency = (mass of added semaglutide - mass of semaglutide in supernatant and washing solution) / mass of added semaglutide × 100%. Drug loading = (mass of added semaglutide - mass of semaglutide in supernatant and washing solution) / (mass of mesoporous silica + mass of added semaglutide - mass of semaglutide in supernatant and washing solution) × 100%.

[0135] Table 2 shows the encapsulation efficiency and drug loading of smegglutinin by different modified dendritic mesoporous silica.

[0136] Table 2. Coating efficiency and drug loading of nanoparticles loaded with smegglutinin

[0137] type Encapsulation efficiency (%) Drug loading (%) MSN-Smeglucopyranoside 99.68±0.19 33.28±0.03 <![CDATA[MSN@NH2-Semaglutide]]> 99.38±0.23 33.23±0.04 <![CDATA[MSN@C8OOH-Semaglutide]]> 99.46±0.31 33.24±0.09

[0138] (2) In vitro release experiment

[0139] The specific procedures were as follows: Prepare enzyme-free simulated intestinal fluid with a pH of 6.8 or enzyme-free simulated gastric fluid with a pH of 2.0. Resuspend 1.5 mg of differently modified semaglutide-loaded nanoparticles in 2 mL of ultrapure water and add them to a 300 kDa dialysis bag. Add 28 mL of enzyme-free simulated intestinal fluid to a 50 mL centrifuge tube, then add the aforementioned dialysis bag. Perform in vitro release on a shaker (temperature 37°C, motor speed 150 rpm) to obtain MSN-semaglutide group 1, MSN@NH2-semaglutide group 1, and MSN@C8OOH-semaglutide group 1, respectively. Additionally, perform an in vitro release experiment with an equal dose of semaglutide using the same method to obtain blank group 1. At 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h, 1mL of the enzyme-free simulated intestinal fluid obtained after release was collected, and an equal volume of enzyme-free simulated intestinal fluid was added to obtain samples. Each group was divided into triplicates. The concentration of semagraviride was obtained by high performance liquid chromatography. In vitro release was performed using enzyme-free simulated gastric fluid as the release medium in the same way to obtain MSN-semagraviride group 2, MSN@NH2-semagraviride group 2, and MSN@C8OOH-semagraviride group 2. In addition, an in vitro release experiment was performed with an equal dose of semagraviride using the same method to obtain blank group 2.

[0140] The results are as follows Figure 12-13 As shown, the different release media at different pH values ​​indicate that different modified mesoporous silicas have different pH sensitivities. Specifically, MSN@C8OOH-semaglutide releases less semaglutide at pH 2.0, while at pH 6.8, the release is almost equivalent to that of free drug. This suggests that it effectively protects semaglutide, preventing premature release in the stomach and ensuring that the drug ultimately reaches the intestines for release.

[0141] (3) Pharmacodynamic experiments

[0142] Establishment of a type 2 diabetes model in C57BL / L6 mice: After one week of acclimatization, mice were fed a high-fat diet for four weeks. Starting from the fifth week, mice were intraperitoneally injected daily with 40 mg / kg streptozotocin (STZ) citrate solution for three consecutive days. The streptozotocin citrate solution was prepared fresh each time. Preparation method: 2.10 g of citric acid was added to 100 mL of double-distilled water to prepare citric acid stock solution (solution A); 2.94 g of trisodium citrate was added to 100 mL of double-distilled water to prepare sodium citrate stock solution (solution B); solutions A and B were mixed in a 1:1 ratio, and the pH was measured and adjusted to 4.2-4.5 to obtain the required 0.1 mol / L sodium citrate buffer solution for streptozotocin. Before intraperitoneal injection, 100 mg of STZ was weighed and dissolved in 10 mL of citrate buffer solution to obtain a 10 mg / mL streptozotocin citrate solution.

[0143] The specific procedure was as follows: Seven days later, all experimental mice were fasted, their tails were clipped, and their fasting blood glucose levels were measured using a blood glucose meter. Normal blood glucose levels in mice should be between 3-7 mmol / L; if a mouse's blood glucose level was greater than 11.1 mmol / L, the type 2 diabetes model was considered successfully established. The successfully modeled mice were randomly divided into six groups: a blank control group, a subcutaneous injection group, a free drug gavage group, three MSN-semaglutide groups, three MSN@NH2-semaglutide groups, and three MSN@C8OOH-semaglutide groups, with n=5 mice in each group.

[0144] The blank control group received 0.2 mL of D-PBS solution via gavage. The subcutaneous injection group received 0.2 mL of semaglutide-D-PBS solution (semaglutide concentration: 4.55 mg / kg) subcutaneously. The free drug gavage group received 0.2 mL of semaglutide-D-PBS solution (semaglutide concentration: 16 mg / kg) via gavage. The MSN-semaglutide group (3) received 0.2 mL of MSN@NH2-semaglutide group (3) received 0.2 mL of MSN@C8OOH-semaglutide group (3) received 0.2 mL of MSN@C8OOH-D-PBS solution (semaglutide concentration: 16 mg / kg) via gavage. The nanoparticles loaded with smegglutinin were derived from Example 8.

[0145] Mice were subjected to tail clipping at 0h before drug administration and at 2h, 4h, 6h, 8h, 12h, and 36h after drug administration. Blood glucose levels were measured using a blood glucose meter (manufacturer: Johnson & Johnson OneTouch Verio Flex) to obtain a blood glucose percentage change-time graph. The formula for calculating the blood glucose percentage was: blood glucose value / initial blood glucose value (0h) × 100%.

[0146] The results are as follows Figure 14 As shown, MSN@C8OOH-semaglutide group 3 exhibits efficacy second only to subcutaneous injection, and at the same dosage, its efficacy is greater than that of the free drug gavage group, MSN-semaglutide group 3, and MSN@NH2-semaglutide group 3.

[0147] (4) Pharmacokinetic experiments

[0148] After one week of acclimatization feeding, all experimental rats were fasted and randomly divided into 5 groups: subcutaneous injection group, free drug gavage group, MSN-semaglutide group 4, MSN@NH2-semaglutide group 4 and MSN@C8OOH-semaglutide group 4, with n=3 mice in each group.

[0149] The subcutaneous injection group received 0.3 mL of semaglutide-Dubor's phosphate-buffered saline (D-PBS) solution (semaglutide concentration: 0.18 mg / kg) subcutaneously. The free drug gavage group received 2 mL of semaglutide-D-PBS solution (semaglutide concentration: 16.7 mg / kg) by gavage. The MSN-semaglutide group received 2 mL of MSN@NH2-D-PBS solution (semaglutide concentration: 16.7 mg / kg) by gavage. The MSN@C8OOH-semaglutide group received 2 mL of MSN@C8OOH-D-PBS solution (semaglutide concentration: 16.7 mg / kg) by gavage. The nanoparticles loaded with smegglutinin were derived from Example 8. The results are shown in Table 3.

[0150] Table 3 Pharmacokinetic parameters

[0151]

[0152] Note: t 1 / 2 To eliminate the half-life, T max To reach peak time, C max To achieve peak concentration, AUC is the area under the curve at drug concentration, and CL is the concentration of the drug under the curve. z / F, where MRT is plasma clearance rate, BA is mean retention time, and BA is bioavailability.

[0153] As shown in Table 3, MSN@C8OOH-smegglutide group 4 has a higher bioavailability than MSN-smegglutide group 4, MSN@NH2-smegglutide group 4 and the free drug gavage group, reaching more than 10 times that of the free drug.

[0154] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for preparing carboxylated dendritic mesoporous silica nanoparticles, characterized in that, It includes the following steps: A mixture of aminated dendritic mesoporous silica nanoparticles and solvent undergoes a nucleophilic substitution reaction with succinic anhydride.

2. The method for preparing carboxylated dendritic mesoporous silica nanoparticles as described in claim 1, characterized in that, It satisfies one or more of the following conditions (a)-(f): (a) In the mixture, the concentration of the aminated dendritic mesoporous silica nanoparticles is 2-20 mg / mL; (b) The mass ratio of the aminated dendritic mesoporous silica to octanoic anhydride is 1:(1.67-20); (c) The nucleophilic substitution reaction is carried out at a temperature of 20-40°C; (d) The nucleophilic substitution reaction takes 12-24 hours; (e) The solvent is DMF or DMSO; (f) The nucleophilic substitution reaction is followed by a post-treatment process; wherein the post-treatment process preferably includes centrifugation, collection of precipitate, alcohol washing, drying and grinding.

3. The method for preparing carboxylated dendritic mesoporous silica nanoparticles as described in claim 1, characterized in that, The preparation method of the aminated dendritic mesoporous silica includes the following steps: A first mixture of dendritic mesoporous silica nanoparticles and a first solvent undergoes an amination reaction with an amination reagent. In the first mixture, the concentration of the dendritic mesoporous silica is preferably 1.25-15 mg / mL; And / or, the mass ratio of the dendritic mesoporous silica to the amination reagent is preferably 1:(0.79-14.2); And / or, the temperature of the amination reaction is preferably 100-115°C; And / or, the amination reaction is preferably carried out for 3-5 hours; And / or, the amination agent is preferably 3-aminopropyltriethoxysilane or diethylenetriaminopropyltrimethoxysilane; And / or, the first solvent is preferably toluene or ethanol; And / or, the amination reaction preferably includes a post-treatment step; wherein the post-treatment preferably includes centrifugation, collection of precipitate, alcohol washing, drying and grinding; And / or, the method for preparing the dendritic mesoporous silica preferably includes the following steps: mixing an oil phase and an aqueous phase for reaction; the aqueous phase includes a surfactant, water, and a catalyst; the oil phase includes an organic solution of tetraethyl orthosilicate; In the process of preparing the aqueous phase, the surfactant is preferably added in the form of an aqueous surfactant solution; more preferably, in the aqueous surfactant solution, the mass percentage of the surfactant is 10%-30%, where the percentage is the mass percentage of the surfactant relative to the mass of the aqueous surfactant solution. Preferably, the surfactant is one or both of hexadecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride; And / or, the catalyst is preferably one or more of sodium hydroxide, triethanolamine, and triethylamine; And / or, the organic solvent in the organic solution of the tetraethyl orthosilicate is preferably chlorobenzene, cyclohexane or octane; And / or, the concentration of tetraethyl orthosilicate in the organic solution of the tetraethyl orthosilicate is preferably 0.1-0.2 g / mL; And / or, the mass ratio of the tetraethyl orthosilicate to the surfactant is preferably (4-10):(3-8), for example, 4.5:3 or 9:6; And / or, the mass ratio of the tetraethyl orthosilicate to the catalyst is preferably (4-10):(0.1-0.5), for example, 4.5:0.1, 8:0.5 or 9:0.2; And / or, the mass ratio of the surfactant to the catalyst is preferably (3-8):(0.1-0.5), for example, 6:0.5 or 6:0.

2.

4. A type of carboxylated dendritic mesoporous silica nanoparticle, characterized in that, It is prepared by the method for preparing carboxylated dendritic mesoporous silica nanoparticles according to any one of claims 1-3.

5. A nanoparticle loaded with smegglutinin, characterized in that, It includes a carrier and smegglutinin; the carrier is carboxylated dendritic mesoporous silica nanoparticles as described in claim 4 and smegglutinin.

6. A method for preparing nanoparticles loaded with smegglutinin, characterized in that, It employs carboxylated dendritic mesoporous silica nanoparticles as described in claim 4, and includes the following steps: The mixture of carboxylated dendritic mesoporous silica nanoparticles, buffer solution, and smegglutinin is obtained through post-treatment; wherein the pH value of the buffer solution is 4.0-5.

0.

7. The method for preparing nanoparticles loaded with smegglutinin as described in claim 6, characterized in that, It satisfies one or more of the following conditions (a)-(d): (a) The mass ratio of the smegglutinin and the carboxylated dendritic mesoporous silica nanoparticles is 1:(1-5); (b) The buffer solution is a citrate-disodium hydrogen phosphate buffer solution or a phosphate buffer solution; (c) In the mixture, the concentration of the carboxylated dendritic mesoporous silica nanoparticles is 1-2 mg / mL; (d) In the mixture, the concentration of smegglutinin is 1-2 mg / mL.

8. Nanoparticles loaded with smegglutinin prepared by a method according to any one of claims 6-7.

9. The use of nanoparticles loaded with smegglutinin as described in claim 8 in the preparation of hypoglycemic drugs.

10. The use of nanoparticles loaded with smegglutinin as described in claim 8 in the preparation of anti-obesity drugs.

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