A method for preparing enteric slow-release defensin mesoporous silica nanoparticles

Abstract

The application discloses a preparation method of enteric sustained-release defensin mesoporous silica nanoparticles. The enteric sustained-release defensin mesoporous silica nanoparticles prepared by the method have good defensin loading capacity, good responsiveness to trypsin and good enteric targeting and sustained-release defensin performance.

Description

Technical Field

[0001] This invention belongs to the field of drug controlled release technology, and specifically relates to a method for preparing mesoporous silica nanoparticles containing intestinal sustained-release defensins. Background Technology

[0002] Defensins are natural polypeptides expressed by the genes of humans and animals, possessing antibacterial and immunomodulatory functions. They typically consist of 20-50 amino acids and exhibit secondary spatial structures such as α, β, and θ sheets. Human intestinal defensins mainly include two types: human α-defensin 5 (HD5) and human α-defensin 6 (HD6). Both HD5 and HD6 are expressed and secreted by Paneth cells in the small intestine. Their propeptides, after the signal peptide is cleaved by trypsin and other enzymes, become mature peptides that exert their biological functions in the intestinal lumen. Numerous studies have shown that HD5 and 6 are key regulators of human intestinal microecology and mucosal immune homeostasis. HD5 and 6 not only regulate the composition and structure of the intestinal flora but also inhibit intestinal mucosal inflammatory responses and provide a beneficial microenvironment for the proliferation, differentiation, and physiological cycle turnover of intestinal stem cells. Recent studies suggest that abnormal expression of intestinal defensins is associated with inflammatory bowel disease and intestinal tumors. Defensins have emerged as a promising new type of antibiotic and immunomodulatory molecule. However, defensin amino acid peptides are easily degraded by various proteases in gastric juice, and the traditional oral administration route limits the research and application of defensins.

[0003] Mesoporous silica nanoparticles (MSNs), widely used in the field of controlled drug release in recent years, possess characteristics such as three-dimensional network channels, large specific surface area, easy surface modification, and good biocompatibility. The particle size of MSNs can be adjusted within the range of 50–1000 nm, and their pore size can also be adjusted within the range of 2–20 nm through optimized synthesis conditions, thus meeting the requirements for efficiently loading drug molecules of different sizes. The surface of mesoporous silica nanoparticles is rich in silanol groups, making it easy to modify with functional groups possessing specific biological functions. After being combined with fluorescent substances, peptides, etc., they can achieve targeted cell and tissue drug delivery and imaging functions for specific organs, tissues, or cells. Modified MSNs can respond to specific stimuli, such as pH and proteases, enabling sustained release of loaded drugs to target tissues or cells. This avoids drug degradation or waste during in vivo transport and delivery, reduces drug side effects, improves drug utilization, and lowers drug dosage and cost. Therefore, MSNs have become a hot research topic in biomedicine, chemistry, materials science, and other disciplines.

[0004] The design principle of enzyme-responsive drug delivery systems is based on the fact that different tissues and cells in the body express and secrete different enzyme profiles. In the small intestinal fluid of humans and mammals, a characteristic feature is the presence of enterokinase, which activates trypsinogen. The high abundance of activated trypsin in the small intestine provides favorable enzyme response conditions for drug delivery systems. However, existing enteric-coated sustained-release drug delivery systems still suffer from problems such as poor targeting of peptide drugs, poor enzyme or pH responsiveness, and unsatisfactory drug loading capacity and release performance. Summary of the Invention

[0005] The main objective of this invention is to provide a method for preparing intestinal sustained-release defensin mesoporous silica nanoparticles, in order to solve the problem of poor intestinal-targeted drug delivery performance of defensin peptides in the prior art.

[0006] The technical solution of this invention is:

[0007] The preparation method of intestinal sustained-release defensin mesoporous silica nanoparticles includes the following steps:

[0008] 1) Add 3-aminopropyltrimethoxysilane to a toluene dispersion of mesoporous silica nanoparticles, reflux under nitrogen protection, wash with acetone and ultrapure water after the reaction is complete, and dry under vacuum to obtain aminated mesoporous silica nanoparticles.

[0009] 2) The aminated mesoporous silica nanoparticles obtained in step 1) were dispersed in hexamethylenetetramine-HCl (HMTA-HCl) buffer, and a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and trypsin-responsive peptides were added. After the reaction was completed, the mixture was centrifuged, washed with ethanol and ultrapure water, and freeze-dried to obtain trypsin-responsive mesoporous silica nanoparticles.

[0010] 3) Take the trypsin-responsive mesoporous silica nanoparticles obtained in step 2), add defensin peptide PBS solution for loading, add gelatin for encapsulation after loading, centrifuge after encapsulation, wash the precipitate with ultrapure water, freeze dry, and obtain intestinal sustained-release defensin mesoporous silica nanoparticles loaded with defensin.

[0011] Step 1) The mesoporous silicon nanoparticles are obtained by the following method:

[0012] Triethanolamine solution was added to hexadecyltrimethylammonium chloride solution, the temperature was adjusted to 55-60℃, and the reaction was stirred for 1 h. Then, cyclohexane solution of ethyl silicate was slowly added, and the temperature was maintained at 55-60℃ for 12 h with stirring. The mixture was washed with ultrapure water and ethanol, and vacuum dried at 60-65℃ for 12-18 h to obtain mesoporous silica nanoparticles with a pore size of 5-8 nm.

[0013] The mass-to-volume ratio of triethanolamine to ultrapure water is 1:200.

[0014] The mass-to-volume ratio of the hexadecyltrimethylammonium chloride to ultrapure water is 1:4.

[0015] The volume ratio of the ethyl silicate to cyclohexane is 1:9;

[0016] The volume ratio of the triethanolamine solution, the hexadecyltrimethylammonium chloride solution, and the cyclohexane solution of ethyl silicate is 9:6:5.

[0017] After the vacuum drying described in step 1), the following steps are also included:

[0018] Mesoporous silica nanoparticles were dispersed in anhydrous toluene, sonicated, and stirred for 15 min. 3-Aminopropyltrimethoxysilane was added, and the mixture was kept at 55-60 °C under nitrogen protection and refluxed for 24-28 h. After cooling and centrifugation, the precipitate was washed with acetone. The precipitate was then dispersed in N,N-dimethylformamide, kept at 60 °C, and stirred for 5 h. After centrifugation, the precipitate was washed with ultrapure water and ethanol, and dried under vacuum at 60-65 °C for 12-18 h to obtain aminated mesoporous silica nanoparticles.

[0019] Preferably, the mass-to-volume ratio of mesoporous silica nanoparticles to anhydrous toluene is 1:2;

[0020] Preferably, the volume ratio of the toluene dispersion of mesoporous silica nanoparticles to 3-aminopropyltrimethoxysilane is 125:1.

[0021] In step 2), the mass-to-volume ratio of the reaction solution system is 100:1:4:1:8000 for MSN-NH2 (mass):TrpResPep (mass):1-ethyl-(3-dimethylaminopropyl)carbodiimide (mass):N-hydroxysuccinimide (mass):hexamethylenetetramine-HCl buffer solution (volume).

[0022] Preferably, the pH of the hexamethylenetetramine-HCl buffer solution is 5.0-5.5.

[0023] Step 3) The loading method is as follows: trypsin-responsive mesoporous silica nanoparticles and defensin peptide PBS solution are dialyzed in a dialysis bag with a molecular weight cutoff of 2 kDa at a temperature of 4°C. The loading is stirred at a stirring speed of 250 rpm, and the dialysate is changed every 12 hours.

[0024] Preferably, the loading time is 32 to 36 hours;

[0025] Preferably, the dialysate is ultrapure water.

[0026] Preferably, the volume ratio of the reaction solution of trypsin-responsive mesoporous silica nanoparticles and defensin polypeptide PBS solution to the dialysis solution is 1:10.

[0027] Step 3) The gelatin encapsulation is performed by slowly adding a catalyst dropwise to the gelatin solution under ice-water bath conditions, stirring, and reacting for 3 hours.

[0028] Preferably, the catalyst is an ultrapure aqueous solution containing 1 mM vitamin C and 1 mM CuSO4;

[0029] Preferably, the gelatin is an ultrapure aqueous solution of propyneated gelatin with a mass percentage concentration of 1.0 to 1.5%.

[0030] Step 3) The mass-to-volume ratio of the trypsin-responsive mesoporous silica nanoparticles to the defensin peptide solution is 1:2; the concentration of the defensin peptide PBS solution is 0.5 mg / mL.

[0031] Step 2) The compositional sequence of the trypsin-responsive peptide is SEQ ID NO:1 (azidoacetyl-AAKRAAKRAA).

[0032] Step 3) The defensin polypeptide is human intestinal α-defensin 5, and its amino acid sequence is SEQ ID NO:2 (ATCYCRTGRCATRESLSGVCEISGRLYRLCCR).

[0033] The method of this invention uses mesoporous silica nanoparticles with a specific pore size (5-8 nm) as the carrier material for defensin peptides. Amino groups are synthesized on the surface of the mesoporous silica nanoparticles around the pores using 3-aminopropyltrimethoxysilane to obtain aminated mesoporous silica nanoparticles. Then, the carboxyl groups in the trypsin-responsive peptide (TrpResPep) and the amino groups on the surface of the mesoporous silica nanoparticles are activated to generate amide bonds. After loading defensins (such as HD5 peptides), gelatin is used to seal the residue, thus obtaining trypsin-responsive, intestinal-release, sustained-release defensin mesoporous silica nanoparticles.

[0034] This invention employs a protease-responsive sustained-release system. In the small intestinal environment where trypsinogen is activated, the trypsin-responsive peptide is cleaved, and simultaneously, the gelatin, acting as a gating group, detaches, allowing defensin peptides to be slowly released from the pores on the surface of mesoporous silica nanoparticles. After oral administration (swallowing), this sustained-release system releases almost no defensin peptides during the several hours it remains in the stomach. However, once it enters the small intestinal lumen, it remains for a longer period, resulting in a higher abundance of trypsin in the small intestinal mucus. This allows for the slow release of more defensins after the system (intestinal sustained-release defensin mesoporous silica nanoparticles) enters the small intestine. The released defensin peptides then continuously exert biological functions such as microecological regulation and inhibition of inflammatory responses within the intestinal lumen.

[0035] The beneficial effects of this invention are:

[0036] The method of this invention uses mesoporous silica nanoparticles with a specific pore size (5-8 nm) as carrier materials for defensin peptides. Amino groups are synthesized on the surface of the mesoporous silica nanoparticles around the pores using 3-aminopropyltrimethoxysilane to obtain aminated mesoporous silica nanoparticles. Then, activated amino and carboxyl groups are used to generate amide bonds, enabling trypsin-responsive peptides to be attached to the pores of the mesoporous silica nanoparticles. After loading defensins (such as human intestinal α-defensin 5), gelatin is used to seal the pores, thereby forming mesoporous silica nanoparticles with trypsin-responsive, slow-release intestinal defensin.

[0037] The intestinal sustained-release defensin mesoporous silica nanoparticles obtained by the method described in this invention exhibit good responsiveness to pancreatic enzymes. While their release rate in the stomach is extremely low, in the small intestinal mucus microenvironment, the trypsin-responsive peptides are cleaved by pancreatic enzymes, causing gelatin to detach from the surface of the mesoporous silica, resulting in the release of a large amount of defensin from the pores of the mesoporous silica nanoparticles. Therefore, the degradation and inactivation of defensin peptides by pepsin and other enzymes in the stomach is avoided, allowing more defensin with preserved biological structure and activity to reach the intestine and exert its biological effects.

[0038] The applicant's experiments show that the intestinal sustained-release defensin mesoporous silica nanoparticles of the present invention have good defensin loading capacity, good responsiveness to trypsin, and good performance of intestinal targeted sustained-release defensin peptides. Attached Figure Description

[0039] Figure 1 This diagram illustrates the preparation of mesoporous silica nanoparticles for intestinal sustained-release defensin and the intestinal-targeted sustained-release defensin.

[0040] Figure 2 The images show electron micrographs of mesoporous silica nanoparticles containing intestinal sustained-release defensins, where A is a scanning electron microscope image and B is a transmission electron microscope image.

[0041] Figure 3 Infrared spectra of mesoporous silica nanoparticles in response to trypsin.

[0042] Figure 4 Standard curve of concentration-fluorescence intensity for FITC-labeled human intestinal α-defensin 5 peptide (FITC-HD5).

[0043] Figure 5 The sustained-release curves of FITC-HD5 from mesoporous silica nanoparticles for intestinal defensin in simulated gastric and intestinal fluids are shown. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] Unless otherwise stated, the raw materials, reagents, and equipment used in the embodiments of the present invention can all be purchased from the market or prepared by existing methods.

[0046] The compositional sequence of the trypsin-responsive peptide described in this invention is shown in SEQ ID NO.1: azidoacetyl-AAKRAAKRAA (amino acid sequence).

[0047] The defensin polypeptide is human intestinal α-defensin 5, and its amino acid sequence is SEQ ID NO.2: amino-terminal-ATCYCRTGRCATRESLSGVCEISGRLYRLCCR-carboxyl-terminal. Example

[0048] A method for preparing mesoporous silica nanoparticles containing intestinal sustained-release defensins includes the following steps:

[0049] (1) Synthesis of mesoporous silica nanoparticles with specific pore sizes

[0050] Weigh 6g of hexadecyltrimethylammonium chloride (CTAC) and 0.18g of triethanolamine (TEA) and dissolve them in 24mL and 36mL of deionized water, respectively. Mix the solutions and keep them at 60℃ for 1h. Then slowly add 20mL of cyclohexane (CYH) (10%, v / v) to dissolve tetraethyl orthosilicate (TEOS). Keep the solution at 60℃ and stir for 12h until a white precipitate is formed. After centrifugation, wash the precipitate three times with ethanol and ultrapure water and vacuum dry it at 60-65℃ for 12-18h to obtain mesoporous silica nanoparticles.

[0051] (2) Amination modification of mesoporous silica nanoparticles

[0052] Weigh 50 mg of the prepared mesoporous silica nanoparticles and disperse them in 100 mL of anhydrous toluene. Then, add 0.8 mL of 3-aminopropyltrimethoxysilane (APTES) directly. Keep the mixture at 60 °C with stirring and reflux under nitrogen protection for 24 h. After cooling, remove the toluene by centrifugation and wash the precipitate three times with acetone. Then, disperse the washed precipitate in 100 mL of N,N-dimethylformamide (DMF), keep the mixture at 60 °C with stirring for 5 h, centrifuge, wash the precipitate three times with ethanol and ultrapure water, and dry it under vacuum at 60-65 °C for 12-18 h to obtain aminated mesoporous silica nanoparticles (MSN-NH2).

[0053] (3) The carboxyl group of the trypsin-responsive peptide reacts with the amino group to form an amide bond.

[0054] 40 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in 40 ml of hexamethylenetetramine-HCl buffer (HMTA-HCl) at pH 5.4. Then, 10 mg of trypsin-responsive peptide (TrpResPep) was added, and the mixture was placed in a water bath at 4 °C and stirred at approximately 250 rpm for 4 h. Then, 40 ml of hexamethylenetetramine-HCl buffer containing 1.0 g of aminated mesoporous silica was added, and the mixture was stirred for another 24 h. After centrifugation, the mixture was washed three times with ethanol and ultrapure water to obtain (intestinal) trypsin-responsive mesoporous silica nanoparticles (MSN-NH-TrpResPep). The nanoparticles were freeze-dried and stored at low temperature for later use.

[0055] (4) Loading of defensin peptides

[0056] Weigh 5 mg of the prepared MSN-NH-TrpResPep and add 10 mL of 0.5 mg / mL defensin (Def) [using human intestinal α-defensin 5 (HD5) peptide as an example] PBS solution. Place the solution in a dialysis bag with a molecular weight cutoff of 2 kDa and load with stirring at 4°C for 24 h. Then add 10 mL of 1.0% propyneated gelatin (guar gum) ultrapure aqueous solution, followed by slowly adding 20 μl of catalyst [1.67 mg vitamin C added to 10 mL of CuSO4 ultrapure aqueous solution (CuSO4 concentration 1 mM), magnetically stirred for 10 min. Prepare fresh for use]. Continue stirring and reacting at 4°C for 3 h. Then, centrifuge, collect the precipitate, and freeze-dry to obtain defensin-loaded intestinal sustained-release defensin mesoporous silica nanoparticles (Def@MSN).

[0057] Figure 1 This is a schematic diagram illustrating the preparation of mesoporous silica nanoparticles for intestinal sustained-release defensin and the intestinal targeted sustained-release defensin in this embodiment. Figure 2 This is an electron microscope image of the mesoporous silicon nanoparticles prepared in this embodiment; Figure 3 Infrared characteristic spectra of MSN, MSN-NH2, and MSN-NH-TrpResPep prepared in this embodiment.

[0058] Example 2: Defensive loading of mesoporous silica nanoparticles for intestinal sustained-release defensin

[0059] Loading mimicry defensin---FITC-labeled human intestinal alpha defensin 5 (FITC-HD5):

[0060] Different concentrations of FITC-HD5 PBS solutions were prepared, and the average fluorescence intensity was measured (excitation wavelength 488 nm, emission wavelength 525 nm). A standard curve of FITC-HD5 concentration-fluorescence intensity was plotted. The fluorescence intensity of the reaction solution (washing solution) was measured to calculate its concentration, and then the loading and release rate of the defensin could be calculated. Figure 4 This is the standard curve of FITC-HD5 fluorescence intensity.

[0061] 2.5 mg of the prepared MSN-NH-TrpResPep was weighed and dispersed in 5 mL of ultrapure water. 5 mL of 1 mg / mL FITC-HD5 PBS solution was added, and the mixture was loaded into a dialysis bag with a molecular weight cutoff of 2 kDa and stirred at 4°C for 24 h. Then, 5 mL of 1.0% propyneated gelatin (guar gum) ultrapure water was added, followed by the slow addition of 10 μl of catalyst [1.67 mg of vitamin C was added to 10 mL of CuSO4 ultrapure water (1 mM concentration), and the mixture was magnetically stirred for 10 min. The reaction was then continued at 4°C for 3 h. Finally, the mixture was centrifuged, the precipitate was collected, and freeze-dried to obtain intestinal sustained-release defensin mesoporous silica nanoparticles (FITC-HD5@MSN) loaded with defensins.

[0062] Example 3: Detection of defensin release rate of FITC-HD5@MSN in simulated gastric and intestinal fluids:

[0063] Preparation of simulated gastric juice: 16.4 ml of dilute hydrochloric acid (containing 9.5% to 10.5% HCl), add about 800 ml of water and 10 g of pepsin, shake well, dilute with water to 1000 ml, filter with a sterile filter membrane and set aside for use.

[0064] Preparation of simulated intestinal fluid: Weigh 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve, and adjust the pH to 6.8 with 0.4% sodium hydroxide solution; separately take 10g of pancreatic enzyme, add an appropriate amount of water to dissolve; mix the two solutions, add water to dilute to 1000ml, filter with a sterile filter membrane and set aside for use.

[0065] The prepared simulated gastric and intestinal fluids were used as FITC-HD5@MSN release solutions. Equal volumes (1.5 mg) of FITC-HD5@MSN were weighed and dispersed in 3 mL of simulated gastric and intestinal fluids, respectively. The mixtures were reacted slowly at 37°C, with samples taken at intervals to determine the FITC-HD5 concentration in the release solutions. The release experiment was repeated three times in parallel, and the FITC-HD5 release at each time point was calculated based on the standard curve. Figure 5 The in vitro release curves of defensins from FITC-HD5@MSN in simulated gastric and intestinal fluids are shown. Figure 5It can be seen that the release rate of defensins in intestinal fluid containing trypsin is close to 50% after 5 hours and close to 70% after 8 hours, but the release rate of defensins in simulated gastric fluid is very low.

[0066] Conclusion: The mesoporous silica nanoparticles for sustained-release defensin of the present invention exhibit good stability in gastric juice and good trypsin responsiveness and sustained-release performance in intestinal juice.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any modifications or equivalent substitutions made to the present invention without departing from the spirit and scope thereof should be covered within the protection scope of the claims of the present invention.

Claims

1. A method for preparing mesoporous silica nanoparticles containing intestinal sustained-release defensin, characterized in that, The steps are as follows: 1) Add 3-aminopropyltrimethoxysilane to a toluene dispersion of mesoporous silica nanoparticles, reflux under nitrogen protection, wash with acetone and ultrapure water after the reaction is complete, and dry under vacuum to obtain aminated mesoporous silica nanoparticles. 2) The aminated mesoporous silica nanoparticles obtained in step 1) were dispersed in hexamethylenetetramine-HCl buffer, and a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, hexamethylenetetramine-HCl buffer and trypsin-responsive peptide was added. After the reaction was completed, the mixture was centrifuged, the precipitate was washed with ethanol and ultrapure water, and then freeze-dried to obtain trypsin-responsive mesoporous silica nanoparticles. The trypsin-responsive mesoporous silica nanoparticles have a pore size of 5-8 nm. The mass ratio of the amino-mesoporous silica nanoparticles, trypsin-responsive peptide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and hexamethylenetetramine-HCl buffer in the reaction solution system is 100:1:4:1:8000. The compositional sequence of the trypsin-responsive peptide is SEQ ID NO:1; 3) Take the trypsin-responsive mesoporous silica nanoparticles obtained in step 2), add defensin peptide PBS solution for loading, add gelatin for encapsulation after loading, centrifuge after encapsulation, wash the precipitate with ultrapure water, freeze dry, and obtain intestinal sustained-release defensin mesoporous silica nanoparticles loaded with defensin. The mass-to-volume ratio of the trypsin-responsive mesoporous silica nanoparticles to the defensin peptide PBS solution was 1:2; the concentration of the defensin peptide PBS solution was 0.5 mg / mL. The defensin polypeptide is human intestinal α-defensin 5, and its amino acid sequence is SEQ ID NO:

2.

2. The method according to claim 1, characterized in that, Step 1) The mesoporous silicon nanoparticles are obtained by the following method: Triethanolamine solution was added to hexadecyltrimethylammonium chloride solution, the temperature was adjusted to 55-60℃, and the reaction was stirred for 1 h. Then, cyclohexane solution of ethyl silicate was slowly added, and the temperature was maintained at 55-60℃ for 12 h. The precipitate was washed with ultrapure water and ethanol, and dried under vacuum at 60-65℃ for 12-18 h to obtain mesoporous silica nanoparticles with a pore size of 5-8 nm.

3. The method according to claim 2, characterized in that, The mass-to-volume ratio of triethanolamine to ultrapure water is 1:

200. The mass-to-volume ratio of the hexadecyltrimethylammonium chloride to ultrapure water is 1:

4. The volume ratio of the ethyl silicate to cyclohexane is 1:9; The volume ratio of the triethanolamine solution, the hexadecyltrimethylammonium chloride solution, and the cyclohexane solution of ethyl silicate is 9:6:

5.

4. The method according to claim 1, characterized in that, Step 1) also includes the following steps: Mesoporous silica nanoparticles were dispersed in anhydrous toluene to obtain a toluene dispersion of mesoporous silica nanoparticles. The dispersion was ultrasonically treated and stirred for 15 min. 3-aminopropyltrimethoxysilane was added, and the mixture was kept at 55-60 °C under nitrogen protection and refluxed for 24-28 h. After cooling and centrifugation, the precipitate was washed with acetone. The precipitate was then dispersed in N,N-dimethylformamide, and the mixture was kept at 60 °C and stirred for 5 h. After centrifugation, the precipitate was washed with ultrapure water and ethanol, and dried under vacuum at 60-65 °C for 12-18 h to obtain aminated mesoporous silica nanoparticles.

5. The method according to claim 4, characterized in that, The volume ratio of the toluene dispersion of the mesoporous silica nanoparticles to 3-aminopropyltrimethoxysilane is 125:

1.

6. The method according to claim 4, characterized in that, The mass-to-volume ratio of the mesoporous silica nanoparticles to anhydrous toluene is 1:

2.

7. The method according to claim 1, characterized in that, Step 2) The pH of the hexamethylenetetramine-HCl buffer solution is 5.0-5.

5.

8. The method according to claim 1, characterized in that, Step 3) The loading method is as follows: trypsin-responsive mesoporous silica nanoparticles and defensin peptide PBS solution are dialyzed in a dialysis bag at a temperature of 4°C, with stirring during loading at a stirring speed of 250 rpm, and the dialysis solution is changed every 12 hours.

9. The method according to claim 8, characterized in that, The loading time is 32 to 36 hours.

10. The method according to claim 8, characterized in that, The dialysis bag is made of a dialysis material with a molecular weight cutoff of 2 kDa.

11. The method according to claim 8, characterized in that, The dialysis fluid is ultrapure water.

12. The method according to claim 8, characterized in that, The reaction solution of the trypsin-responsive mesoporous silica nanoparticles and the defensin polypeptide PBS solution has a volume ratio of 1:10 to the dialysate.

13. The method according to claim 1, characterized in that, Step 3) The gelatin encapsulation is performed by slowly adding a catalyst dropwise to the gelatin solution under ice-water bath conditions, stirring, and reacting for 3 hours.

14. The method according to claim 13, characterized in that, The catalyst is an ultrapure aqueous solution containing 1 mM vitamin C and 1 mM CuSO4.

15. The method according to claim 13, characterized in that, The gelatin is an ultrapure aqueous solution of propyneated gelatin with a mass percentage concentration of 1.0 to 1.5%.

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