Acid response type hollow mesoporous organic silicon nanoparticles and preparation method thereof

Abstract

The invention discloses an acid response type hollow mesoporous organic silicon nanoparticle and a preparation method thereof. The method comprises the following steps: 1) reacting hexadecyl trimethyl ammonium chloride, triethanolamine and tetraethyl silicate in ultrapure water; (2) dropwise adding a mixed solution of 1, 2-bis (triethoxysilyl) ethane and tetraethyl silicate to form silicon nanoparticles with a shell-core structure; 3) dispersing in ethanol and hydrochloric acid solution, refluxing, centrifuging and collecting solid; 4) repeating the step 3) to form meso-porous silicon balls; (5) reacting in ammonia water to obtain mesoporous hollow silicon spheres HMONs-OH; (6) preparing the HMONs-NH2 nano particles; and 7) modifying the surface of the HMONs-NH2 with pyridine betaine to obtain the acid response type hollow mesoporous organic silicon nanoparticles. The nano-particle has pH responsiveness and surface charge switchability, can effectively permeate into a bacterial biofilm, and is combined with an anti-infection drug for use to efficiently kill bacteria in the film.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to acid-responsive hollow mesoporous organosilicon nanoparticles and a preparation method thereof. Background Art

[0002] Biofilm is the main form in which bacteria exist and function. Bacteria are tightly attached to the surface of various matrices and surrounded by a protective capsule composed of extracellular polymers. Extracellular polymers contain polysaccharides, proteins, lipids and nucleic acids, which together provide a stable microenvironment for the microbial community, promote intercellular communication, and enhance resistance to external environmental pressures. The formation of bacterial biofilms can enhance the survival and resistance of pathogens in the environment, thereby increasing the risk of disease. The presence of bacterial biofilms not only increases the complexity of infection, but may also cause persistent, recurrent, aggravated or acute attacks of infection. This is because bacterial biofilms can intermittently release planktonic bacteria, which spread to surrounding tissues or spread to other parts of the body through the blood, causing new infections.

[0003] There are many treatments for bacterial biofilm infections, including antibiotic therapy, anti-biofilm agents (such as quorum sensing inhibitors and extracellular polymer degrading enzymes), debridement therapy, photodynamic therapy, etc. However, extracellular polymers significantly limit the penetration of antibiotics, making bacteria associated with bacterial biofilms up to 1000 times more resistant to antibiotics than their planktonic forms. Anti-biofilm agents may have problems with sensitivity to serum and proteases and biosafety, and have limited effects on the removal of mature biofilms. Debridement therapy may have shortcomings such as more bleeding and higher surgical costs, and may pollute the surgical environment. Using photosensitizers to produce reactive oxygen species under light of a specific wavelength to kill bacteria has a non-specific killing effect on bacteria and is not easy to induce bacterial resistance. However, if it is impossible to penetrate into the bacterial biofilm, it is still limited to kill the superficial surface of the bacterial biofilm. Therefore, if the penetration ability of the antibacterial system can be enhanced, it is of great significance to improve the treatment effect.

[0004] Bacterial biofilms create a microenvironment that is distinct from normal tissues and exhibits unique physicochemical properties. One notable feature is the formation of a slightly acidic environment, which is caused by the byproducts of bacterial carbohydrate metabolism and the presence of extracellular DNA. Studies have shown that the pH of bacterial biofilms is usually below 7.0, and there is a pH gradient from the surface to the inside, which may be due to the accumulation of acidic substances along the flow path. Although traditional cationic nanoparticles interact with the negative charges on the surface of bacterial biofilms and are adsorbed and accumulated on the surface, they often find it difficult to further penetrate the bacterial biofilms; anionic and neutral nanoparticles can neither penetrate nor adsorb bacterial biofilms and are easily washed away. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an acid-responsive hollow mesoporous organosilicon nanoparticle.

[0006] The second object of the present invention is to provide a method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles.

[0007] The third object of the present invention is to provide an application of acid-responsive hollow mesoporous organosilicon nanoparticles as a drug carrier.

[0008] The technical solution of the present invention is summarized as follows:

[0009] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0010] 1) Dissolve hexadecyltrimethylammonium chloride and triethanolamine in ultrapure water, heat to 70-90°C, stir at 800-1000rpm for 20-40 minutes, add tetraethyl silicate dropwise, and continue to react for 30-60 minutes after the addition is complete;

[0011] 2) adding a mixed solution 1 consisting of 1,2-bis(triethoxysilyl)ethane and tetraethyl silicate to the material obtained in step 1), and after the addition is complete, continuing the reaction for 1-2 hours, cooling to room temperature, centrifuging at 10000-12000 rpm for 30 minutes, washing, and harvesting core-shell structured silicon nanoparticles;

[0012] 3) Dispersing 500 mg of the core-shell silicon nanoparticles in 90-110 mL of a mixed solution 2 consisting of ethanol and hydrochloric acid according to the ratio, reflux for 8-12 hours, cool to room temperature, centrifuge at 10000-12000 rpm for 20-30 minutes, collect the solid, and wash; the volume ratio of the ethanol to the hydrochloric acid is 80-100:1;

[0013] 4) Repeat step 3) twice, and the obtained solid is a mesoporous silicon sphere;

[0014] 5) dispersing the mesoporous silicon spheres in an aqueous ammonia solution with a volume concentration of 8%-10%, reacting at 80-95° C. for 3-5 hours to obtain mesoporous hollow silicon spheres, cooling to room temperature, centrifuging at 10000-12000 rpm for 20-30 minutes, washing the obtained solid, and obtaining mesoporous hollow HMONs-OH silicon spheres;

[0015] 6) dispersing the mesoporous hollow HMONs-OH silica spheres and 3-aminopropyltriethoxysilane in anhydrous ethanol, reflux at 80-90° C. for 3-6 hours, cooling to room temperature, centrifuging at 10000-12000 rpm for 20-30 minutes, washing, and obtaining amino-modified mesoporous hollow silica nanoparticles HMONs-NH2;

[0016] 7) Proceed in one of the following two ways:

[0017] Method 1:

[0018] The HMONs-NH2 obtained in step 6) and 2,3-dimethylmaleic anhydride and triethylamine are added to N,N-dimethylformamide, stirred at 0-10°C for 3-6 hours, centrifuged at 10000-12000 rpm for 10 minutes, and washed to obtain an acid-responsive hollow mesoporous organosilicon nanoparticle, referred to as HMONs-DA;

[0019] Method 2:

[0020] The HMONs-NH2, isonicotinic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine obtained in step 6) are added to anhydrous N,N-dimethylformamide, protected by argon, stirred at room temperature for 3-6 hours, centrifuged at 10000-12000 rpm for 20-30 minutes, and washed to obtain a solid product; bromoacetic acid and the solid product are added to acetonitrile, heated to reflux for 8-12 hours; after cooling to room temperature, the solid precipitate is collected by centrifugation, washed, and an acid-responsive hollow mesoporous organosilicon nanoparticle, referred to as HMONs-PyB, is obtained.

[0021] Preferably, in step 1), the mass ratio of hexadecyltrimethylammonium chloride, triethanolamine and tetraethyl silicate is 2:(0.1-0.4):(0.8-2).

[0022] Preferably, the mass ratio of 1,2-bis(triethoxysilyl)ethane to tetraethyl silicate in step 2) is 1:(0.25-2), and the mass ratio of the material obtained in step 1) to the mixed solution 1 is (10-40):1.

[0023] Preferably, in step 5), the mass ratio of the silicon spheres with the mesoporous structure to the aqueous ammonia solution with a volume concentration of 8%-10% is 0.2:(40-400).

[0024] Preferably, in step 6), the mass ratio of the mesoporous hollow HMONs-OH silicon spheres, 3-aminopropyltriethoxysilane and anhydrous ethanol is: 100:(10-40):(5000-50000).

[0025] Preferably, in step 7) method 1, the mass ratio of HMONs-NH2, 2,3-dimethylmaleic anhydride, triethylamine and N,N-dimethylformamide is 100:(10-100):(10-100):(2000-10000).

[0026] Preferably, in step 7) of method 2, the mass ratio of HMONs-NH2, isonicotinic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and anhydrous N,N-dimethylformamide is 11:(11-55):(11-110):(1.1-5.5):(3300-6600); the mass ratio of the bromoacetic acid, the solid product and acetonitrile is (5.5-22):11:(1100-5500).

[0027] The preparation method mentioned above prepares an acid-responsive hollow mesoporous organosilicon nanoparticle.

[0028] The use of the above-mentioned acid-responsive hollow mesoporous organosilicon nanoparticles as a drug carrier comprises the following steps:

[0029] The acid-responsive hollow mesoporous organosilicon nanoparticles, the drug and the solvent for dissolving the drug are uniformly mixed in a mass ratio of 10:(0.1-10):(500-5000), and the mixture is dropped into water 5-20 times the mass of the solvent, stirred for 10-60 minutes, dialyzed in deionized water for 10-12 hours using a dialysis bag, filtered through a 3 μm membrane, and centrifuged at 10000-12000 rpm for 10 minutes to obtain drug-loaded nanoparticles.

[0030] The above-mentioned drug is preferably at least one of an antibacterial drug, a protease, a quorum infection signal molecule inhibitor, an active peptide, a photosensitizer, a photothermal agent, a saturated fatty acid and an anti-biofilm agent.

[0031] Preferred antibacterial drugs are: ciprofloxacin, levofloxacin, penicillin G, cefepime, ceftazidime, imipenem, aztreonam, amikacin, gentamicin, streptomycin, roxithromycin, doxycycline, polymyxin B, vancomycin or rifampicin.

[0032] Preferred proteases are: trypsin or chymotrypsin.

[0033] Preferred inhibitors of quorum infection signal molecules are: acylhomoserine lactone degrading enzyme, AHL-acyltransferase, isopentenyl caffeic acid, curcumin, furanocoumarin or dodecanoyl sulfonyl ethyl ester.

[0034] The preferred active peptides are: cecropin, gramicidin or antimicrobial peptide trifluoroacetate.

[0035] Preferred photosensitizers: Bengal rose, Chlorin e6 (MedChemExpress), aluminum phthalocyanine, TBTDC

[0036] (MedChemExpress), HPPH (MedChemExpress), or temoporfin.

[0037] The photothermal agent is preferably: heptamethyl cyanine, porphyrin ring or indocyanine green.

[0038] Preferred saturated fatty acids are: lauric acid, stearic acid or myristic acid.

[0039] Preferred anti-biofilm agents include: RNA-III inhibitory peptide, allicin, salicylic acid or cinnamaldehyde.

[0040] The solvent is: methanol, ethanol, acetone, tetrahydrofuran, N,NN,N-dimethylformamide, dimethyl sulfoxide, dimethylacetamide, 1,4-dioxane, hydroxypropionic acid, ethylenediamine, glycerol, ether, butyl sulfone, diethylene glycol dimethyl ether or hexamethylphosphoramide. (The solvent is selected by conventional technical means to select a solvent that can dissolve the selected drug).

[0041] Advantages of the present invention:

[0042] The acid-responsive hollow mesoporous organosilicon nanoparticles of the present invention have pH-responsive and surface charge switchable capabilities. The nanoparticles of the present invention have enhanced bacterial biofilm penetration effects, overcome the barrier of bacterial biofilm penetration, and are combined with anti-infective drugs to achieve efficient killing of bacteria in bacterial biofilms. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a characterization of the physical and chemical properties of each nanoparticle.

[0044] (A) Transmission electron microscopy images of nanoparticles HMONs-OH, HMONs-NH2, HMONs-DA and HMONs-PyB. Scale bar: 100 nm.

[0045] (B) Transmission electron microscopy images of four drug-loaded nanoparticles AIE / LA@HMONs. Scale bar: 100 nm.

[0046] (C) Hydrated particle size of drug-loaded nanoparticles AIE / LA@HMONs-OH (AIE / LA@HMONs-OH is referred to as OH in each figure).

[0047] (D) Hydrated particle size of drug-loaded nanoparticles AIE / LA@HMONs-NH2 (AIE / LA@HMONs-OH is referred to as NH2 in each figure).

[0048] (E) Hydrated particle size of drug-loaded nanoparticles AIE / LA@HMONs-DA (AIE / LA@HMONs-OH is referred to as DA in each figure).

[0049] (F) Hydrated particle size of drug-loaded nanoparticles AIE / LA@HMONs-PyB (AIE / LA@HMONs-OH is referred to as PyB in each figure).

[0050] (G) Stability of four drug-loaded nanoparticles AIE / LA@HMONs in water for seven days.

[0051] (H) Relationship between the relative fluorescence intensity of dichlorodihydrofluorescein solution at 525 nm and irradiation time under different conditions (AIE@HMONs-PyB is referred to as PyB (w / o LA) in each figure).

[0052] (I) Relationship between relative fluorescence intensity of dihydrorhodamine 123 solution at 525 nm and irradiation time under different conditions.

[0053] (J) Relationship between the relative fluorescence intensity of 9,10-anthracenediyl-bis(methylene)dicarboxylic acid at 378 nm and irradiation time under different conditions.

[0054] Figure 2 Figure 3 shows the potential changes of four drug-loaded nanoparticles AIE / LA@HMONs in different pH buffers and their penetration ability in bacterial biofilms.

[0055] (A) Potential changes of drug-loaded nanoparticles AIE / LA@HMONs in different pH buffers.

[0056] (B) Changes in potential of drug-loaded nanoparticles AIE / LA@HMONs-DA in pH = 5.5 buffer over time.

[0057] (C) Changes in potential over time of drug-loaded nanoparticles AIE / LA@HMONs-PyB in pH = 6.5 buffer.

[0058] (D) Penetration of four drug-loaded nanoparticles AIE / LA@HMONs into the 3D structure of bacterial biofilms after incubation with bacterial biofilms for 2 h.

[0059] (E) Relative fluorescence intensity of four drug-loaded nanoparticles AIE / LA@HMONs in bacterial biofilms after incubation with bacterial biofilms for 0.5 h.

[0060] (F) Relative fluorescence intensity of four drug-loaded nanoparticles AIE / LA@HMONs in bacterial biofilms after incubation with bacterial biofilms for 1 h.

[0061] (G) Relative fluorescence intensity of four drug-loaded nanoparticles AIE / LA@HMONs in bacterial biofilms after incubation with bacterial biofilms for 2 h.

[0062] Figure 3 The killing and removal effects of four drug-loaded nanoparticles AIE / LA@HMONs on bacterial biofilms.

[0063] (A) Photographs of bacterial colonies formed on agar plates after different treatments.

[0064] (B) Bacterial survival rate in different treatment groups. Compared with the blank light-free group, ***p<0.001.

[0065] (C) SEM images of bacterial biofilms after different treatments. Scale bar: 2 μm.

[0066] (D) Laser scanning confocal microscopy images of bacterial biofilms after different treatments. Scale bar: 100 μm.

[0067] (E) Relative fluorescence intensity of bacterial biofilm in different treatment groups. Compared with the blank no-light group, ***p<0.001.

[0068] (F) Crystal violet stained photographs of bacterial biofilms after different treatments.

[0069] (G) Relative biomass of bacterial biofilm in different treatment groups. ***p<0.001 compared with the blank light-free group.

[0070] Figure 4 The ability of drug-loaded nanoparticles AIE / LA@HMONs-PyB (abbreviated as AIE in this figure) and antimicrobial drug combination therapy to eliminate bacterial biofilm in vitro.

[0071] (A) Checkerboard experiment of drug-loaded nanoparticles AIE / LA@HMONs-PyB and ciprofloxacin.

[0072] (B) Photographs of bacterial colonies formed on agar plates by bacterial biofilms treated with different conditions.

[0073] (C) Logarithmic statistical analysis of the number of bacterial colonies surviving in bacterial biofilms treated with different conditions. ***p<0.001 compared with the blank light-free group.

[0074] (D) SEM images of bacterial biofilms treated with different conditions. Scale bar: 2 μm.

[0075] (E) Laser scanning confocal microscopy images of bacterial biofilms treated with different conditions after SYTO 9 staining.

[0076] Scale bar: 100 μm.

[0077] (F) Relative fluorescence intensity statistics of laser scanning confocal microscopy images of bacterial biofilms treated with different conditions after staining with SYTO 9. Compared with the blank no-light control group, ***p<0.001.

[0078] (G) Photographs of bacterial biofilms treated with different conditions stained with crystal violet.

[0079] (H) Relative biomass of bacterial biofilm in the crystal violet staining experiment. ***p<0.001 compared with the blank control group.

[0081] Figure 5 It is the application of drug-loaded nanoparticles AIE / LA@HMONs-PyB (abbreviated as AIE in this figure) and antibacterial drug combination therapy in in vivo antibacterial experiments.

[0082] (A) Schematic timeline of the treatment process.

[0083] (B) Wound photos of MRSA-infected diabetic mice in different treatment groups. Scale bar: 0.5 cm.

[0084] (C) Relative infection area of ​​wounds in different treatment groups. Compared with the blank light-free group, ***p<0.001. Compared with AIE / L, ***p<0.001.

[0085] (D) Body weight changes of mice during the treatment period.

[0086] (E) Survival rate of mice during the treatment period.

[0087] (F) Logarithmic statistics of bacterial colonies in the wound on day 11.

[0088] (G) Photograph of bacterial colonies formed on the agar plate at the wound site on day 11.

[0089] Figure 6 Tissue analysis of the infection site after animal infection treated with drug-loaded nanoparticles AIE / LA@HMONs-PyB and antimicrobial combination therapy.

[0090] (A) Hematoxylin and eosin staining and Masson staining of wound tissue sections. Scale bar: 100 μm.

[0091] (B) Immunofluorescence staining images of tumor necrosis factor-α and interleukin-6 in wound tissue. Scale bar: 100 μm.

[0092] (C) Quantitative analysis of the relative fluorescence intensity of TNF-α.

[0093] (D) Quantitative analysis of the relative fluorescence intensity of interleukin-6.

[0094] (E) Quantitative analysis of the relative intensities of platelet-endothelial cell adhesion molecules.

[0095] (F) Quantitative analysis of relative intensity of type I collagen.

[0096] (G) Quantitative analysis of the relative intensity of type III collagen.

[0097] (H) Immunohistochemical staining images of platelet-endothelial cell adhesion molecule, type I collagen, and type III collagen. Scale bar:

[0098] 100μm. DETAILED DESCRIPTION

[0099] The present invention will be further described below in conjunction with specific embodiments.

[0100] Example 1

[0101] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0102] 1) Dissolve hexadecyltrimethylammonium chloride (2 g) and triethanolamine (0.2 g) in ultrapure water (20 mL), heat to 80°C, stir at 900 rpm for 30 minutes, add tetraethyl silicate (1 g) dropwise, and continue to react for 45 minutes after the addition is complete (to achieve the purpose of generating nuclei);

[0103] 2) adding a mixed solution 1 consisting of 1,2-bis(triethoxysilyl)ethane (1 g) and tetraethyl silicate (1 g) dropwise to the material obtained in step 1), and after the addition was complete, continuing the reaction for 1.5 hours, cooling to room temperature, centrifuging at 10000 rpm for 30 minutes, washing twice with water and twice with ethanol, and harvesting core-shell silicon nanoparticles;

[0104] The mass ratio of the material obtained in step 1) to the mixed solution 1 is 20:1;

[0105] 3) Dispersing 500 mg of the core-shell silicon nanoparticles in 100 mL of a mixed solution 2 consisting of ethanol and hydrochloric acid, wherein the volume ratio of the ethanol to the hydrochloric acid is 100:1; reflux for 10 hours, cool to room temperature, centrifuge at 11000 rpm for 25 minutes, collect the solid, and wash twice with ethanol.

[0106] 4) Repeat step 3) twice, and the obtained solid is a mesoporous silicon sphere;

[0107] 5) The mesoporous silica spheres (0.2 g) were dispersed in an aqueous ammonia solution (200 g) with a volume concentration of 9%, reacted at 90° C. for 4 hours to obtain mesoporous hollow silica spheres, cooled to room temperature, centrifuged at 11000 rpm for 25 minutes, and the obtained solid was washed twice with water and twice with ethanol to obtain mesoporous hollow HMONs-OH silica spheres (referred to as nanoparticles HMONs-OH). Figure 1 A);

[0108] 6) The mesoporous hollow HMONs-OH silica spheres (100 mg) and 3-aminopropyltriethoxysilane (20 mg) were dispersed in anhydrous ethanol (20 g), refluxed at 80° C. for 6 hours, cooled to room temperature, centrifuged at 10,000 rpm for 25 minutes, and washed twice with ethanol to obtain amino-modified mesoporous hollow silica nanoparticles HMONs-NH2 (referred to as nanoparticles HMONs-NH2). Figure 1 A);

[0109] 7) The HMONs-NH2 (100 mg) obtained in step 6) and 2,3-dimethylmaleic anhydride (50 mg) and triethylamine (50 mg) were added to N,N-dimethylformamide (5 g), stirred at 5°C for 4 hours, centrifuged at 10000 rpm for 10 minutes, washed with dimethylformamide 3 times, and washed with ethanol 3 times to obtain an acid-responsive hollow mesoporous organosilicon nanoparticle (referred to as nanoparticle HMONs-DA); the structure of the nanoparticle was confirmed by transmission electron microscopy, see Figure 1 A.

[0110] Example 2

[0111] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0112] 1) Dissolve hexadecyltrimethylammonium chloride (2 g) and triethanolamine (0.1 g) in ultrapure water (20 mL), heat to 70°C, stir at 800 rpm for 40 minutes, add tetraethyl silicate (0.8 g) dropwise, and continue to react for 30 minutes after the addition is complete;

[0113] 2) adding a mixed solution 1 consisting of 1,2-bis(triethoxysilyl)ethane (1 g) and tetraethyl silicate (0.25 g) dropwise to the material obtained in step 1), and after the addition was completed, continuing the reaction for 1 hour, cooling to room temperature, centrifuging at 11000 rpm for 30 minutes, washing twice with water and twice with ethanol, and harvesting core-shell structured silicon nanoparticles;

[0114] The mass ratio of the material obtained in step 1) to the mixed solution 1 is 10:1;

[0115] 3) dispersing 500 mg of the core-shell silicon nanoparticles in 90 mL of a mixed solution 2 consisting of ethanol and hydrochloric acid, wherein the volume ratio of the ethanol to the hydrochloric acid is 80:1; reflux for 8 hours, cool to room temperature, centrifuge at 10,000 rpm for 30 minutes, collect the solid, and wash twice with ethanol;

[0116] 4) Repeat step 3) twice, and the obtained solid is a mesoporous silicon sphere;

[0117] 5) dispersing the mesoporous silica spheres (0.2 g) in an aqueous ammonia solution (40 g) with a volume concentration of 8%, reacting at 80° C. for 5 hours to obtain mesoporous hollow silica spheres, cooling to room temperature, centrifuging at 10,000 rpm for 30 minutes, washing the obtained solid twice with water and twice with ethanol to obtain mesoporous hollow HMONs-OH silica spheres;

[0118] 6) dispersing the mesoporous hollow HMONs-OH silica spheres (100 mg) and 3-aminopropyltriethoxysilane (10 mg) in anhydrous ethanol (5 g), refluxed at 85° C. for 4 hours, cooled to room temperature, centrifuged at 11000 rpm for 30 minutes, and washed twice with ethanol to obtain amino-modified mesoporous hollow silica nanoparticles HMONs-NH2;

[0119] 7) The HMONs-NH2 (100 mg) obtained in step 6) and 2,3-dimethylmaleic anhydride (10 mg) and triethylamine (10 mg) were added to N,N-dimethylformamide (2 g), stirred at 0°C for 6 hours, centrifuged at 11000 rpm for 10 minutes, washed with dimethylformamide 3 times and ethanol 3 times to obtain acid-responsive hollow mesoporous organosilicon nanoparticles, referred to as HMONs-DA.

[0120] Example 3

[0121] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0122] 1) Dissolve hexadecyltrimethylammonium chloride (2 g) and triethanolamine (0.4 g) in ultrapure water (20 mL), heat to 90°C, stir at 1000 rpm for 20 minutes, add tetraethyl silicate (2 g) dropwise, and continue to react for 60 minutes after the addition is complete;

[0123] 2) Adding a mixed solution 1 consisting of 1,2-bis(triethoxysilyl)ethane (1 g) and tetraethyl silicate (2 g) dropwise to the material obtained in step 1), continuing the reaction for 2 hours after the addition is complete, cooling to room temperature, centrifuging at 12000 rpm for 30 minutes, washing twice with water, washing twice with ethanol, and harvesting core-shell structured silicon nanoparticles;

[0124] The mass ratio of the material obtained in step 1) to the mixed solution 1 is 40:1;

[0125] 3) dispersing 500 mg of the core-shell silicon nanoparticles in 110 mL of a mixed solution 2 consisting of ethanol and hydrochloric acid, wherein the volume ratio of ethanol to hydrochloric acid is 90:1; reflux for 12 hours, cool to room temperature, centrifuge at 12000 rpm for 20 minutes, collect the solid, and wash twice with ethanol;

[0126] 4) Repeat step 3) twice, and the obtained solid is a mesoporous silicon sphere;

[0127] 5) dispersing the mesoporous silica spheres (0.2 g) in a 10% volume concentration ammonia aqueous solution (400 g), reacting at 95° C. for 3 hours to obtain mesoporous hollow silica spheres, cooling to room temperature, centrifuging at 12,000 rpm for 20 minutes, washing the obtained solid twice with water and twice with ethanol to obtain mesoporous hollow HMONs-OH silica spheres;

[0128] 6) dispersing the mesoporous hollow HMONs-OH silica spheres (100 mg) and 3-aminopropyltriethoxysilane (40 mg) in anhydrous ethanol (50 g), reflux at 90° C. for 3 hours, cooling to room temperature, centrifuging at 12,000 rpm for 20 minutes, and washing twice with ethanol to obtain amino-modified mesoporous hollow silica nanoparticles HMONs-NH2;

[0129] 7) The HMONs-NH2 (100 mg) obtained in step 6) and 2,3-dimethylmaleic anhydride (100 mg) and triethylamine (100 mg) were added to N,N-dimethylformamide (10 g), stirred at 10° C. for 3 hours, centrifuged at 12000 rpm for 10 minutes, washed with dimethylformamide 3 times and ethanol 3 times to obtain acid-responsive hollow mesoporous organosilicon nanoparticles, referred to as HMONs-DA.

[0130] Example 4

[0131] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0132] Steps 1) to 6) are the same as steps 1) to 6) of Example 1;

[0133] 7) The HMONs-NH2 (11 mg), isonicotinic acid (30 mg), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (52 mg) and 4-dimethylaminopyridine (2 mg) obtained in step 6) were added to anhydrous N,N-dimethylformamide (5 g), protected by argon, stirred at room temperature for 4 hours, centrifuged at 10000 rpm for 30 minutes, and washed to obtain a solid product; bromoacetic acid (11 mg) and the solid product (11 mg) were added to acetonitrile (5 g), heated to reflux for 8 hours; after cooling to room temperature, the solid precipitate was collected by centrifugation and washed three times with ethanol to obtain an acid-responsive hollow mesoporous organosilicon nanoparticle (referred to as nanoparticle HMONs-PyB), and the structure of the nanoparticle was confirmed by transmission electron microscopy, see Figure 1 A.

[0134] Example 5

[0135] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0136] Steps 1) to 6) are the same as steps 1) to 6) of Example 2;

[0137] 7) The HMONs-NH2 (11 mg), isonicotinic acid (11 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (11 mg) and 4-dimethylaminopyridine (1.1 mg) obtained in step 6) were added to anhydrous N,N-dimethylformamide (3.3 g), protected by argon, stirred at room temperature for 3 hours, centrifuged at 11000 rpm for 20 minutes, and washed to obtain a solid product; bromoacetic acid (5.5 mg) and the solid product (11 mg) were added to acetonitrile (1.1 g), heated to reflux for 10 hours; after cooling to room temperature, the solid precipitate was collected by centrifugation and washed to obtain an acid-responsive hollow mesoporous organosilicon nanoparticle, referred to as HMONs-PyB.

[0138] Example 6

[0139] A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles comprises the following steps:

[0140] Steps 1) to 6) are the same as steps 1) to 6) of Example 3;

[0141] 7) The HMONs-NH2 (11 mg), isonicotinic acid (55 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (110 mg) and 4-dimethylaminopyridine (5.5 mg) obtained in step 6) were added to anhydrous N,N-dimethylformamide (6.6 g), protected by argon, stirred at room temperature for 6 hours, centrifuged at 12000 rpm for 25 minutes, and washed to obtain a solid product; bromoacetic acid (22 mg) and the solid product (11 mg) were added to acetonitrile (5.5 g), heated to reflux for 12 hours; after cooling to room temperature, the solid precipitate was collected by centrifugation and washed to obtain an acid-responsive hollow mesoporous organosilicon nanoparticle, referred to as HMONs-PyB.

[0142] Example 7

[0143] Application of acid-responsive hollow mesoporous organosilicon nanoparticles as drug carriers comprises the following steps:

[0144] An acid-responsive hollow mesoporous organosilicon nanoparticle HMONs-DA (10 mg) prepared in Example 1, two drugs, the first one (lauric acid, abbreviated as LA (2 mg)), the second one ((Z)-2-{2-[(5-{4-[bis(4-methoxyphenyl)amino]phenyl}thiophen-2-yl)methylene]-3-oxo-2,3-dihydro-1H-indene-1-ylidene}malononitrile) (abbreviated as TPAI, 160 μg) which belongs to an aggregation-induced emission photosensitizer, referred to as AIE, and a solvent (500 mg of dimethyl sulfoxide) for dissolving the above drugs were mixed uniformly, added dropwise to water equivalent to 10 times the mass of the solvent, stirred for 10 minutes, dialyzed in deionized water for 12 hours using a dialysis bag with a molecular weight cutoff value of 3000 Da, filtered through a 3 μm membrane, and centrifuged at 10000 rpm for 10 minutes to obtain drug-loaded nanoparticles AIE / LA@HMONs-DA, see Figure 1 B.

[0145] The second drug preparation method is described in: V.Jeux, O.Segut, D.Demeter, O.Alévêque, P.Leriche, J.Roncali, ChemPlusChem 2015, 80, 697.

[0146] Example 8

[0147] Application of acid-responsive hollow mesoporous organosilicon nanoparticles as drug carriers comprises the following steps:

[0148] The acid-responsive hollow mesoporous organosilicon nanoparticles HMONs-PyB (10 mg) prepared in Example 4, two drugs, the first LA (2 mg), the second TPAI (160 μg), and the solvent (500 mg of dimethyl sulfoxide) for dissolving the above drugs were mixed evenly, added dropwise to water equivalent to 10 times the mass of the solvent, stirred for 10 minutes, dialyzed in deionized water for 12 hours using a dialysis bag with a molecular weight cutoff value of 3000 Da, filtered through a 3 μm membrane, and centrifuged at 10000 rpm for 10 minutes to obtain drug-loaded nanoparticles AIE / LA@HMONs-PyB. Figure 1 B.

[0149] Example 9

[0150] The nanoparticles HMONs-OH prepared in step 5) of Example 1 were used to replace the nanoparticles HMONs-DA in Example 7, and the rest was the same as in Example 7 to obtain the drug-loaded nanoparticles AIE / LA@HMONs-OH. Figure 1 B.

[0151] The nanoparticles HMONs-NH2 prepared in step 5) of Example 1 were used to replace the nanoparticles HMONs-DA in Example 7, and the rest was the same as in Example 7 to obtain the drug-loaded nanoparticles AIE / LA@HMONs-NH2. Figure 1 B.

[0152] Referring to Example 8, only TPAI was selected as the drug, and the rest was the same as Example 8 to obtain drug-loaded nanoparticles AIE@HMONs-PyB.

[0153] Example 10

[0154] The hydrated particle size and seven-day stability of four drug-loaded nanoparticles AIE / LA@HMONs (AIE / LA@HMONs-OH, AIE / LA@HMONs-NH2, AIE / LA@HMONs-DA, AIE / LA@HMONs-PyB) in water were measured using a Malvern instrument (model: Zetasizer Nano ZS90). Figure 1 BG.

[0155] The following examples relate to drug-loaded nanoparticles, wherein AIE / LA@HMONs-DA is prepared by Example 7, and AIE / LA@HMONs-PyB is prepared by Example 8.

[0156] Embodiment 11

[0157] Photosensitization ability of four drug-loaded nanoparticles AIE / LA@HMONs and drug-loaded nanoparticles AIE@HMONs-PyB.

[0158] The total reactive oxygen species generation capacity, type II reactive oxygen species generation capacity and type I reactive oxygen species generation capacity of the drug-loaded nanoparticles were verified using three commercial probes, 2', 7'-dichlorofluorescein (DCFH), 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA) and dihydrorhodamine 123 (DHR-123), respectively. In DCFH solution, the relative emission intensities (I / I0) of the four drug-loaded nanoparticles AIE / LA@HMONs and the drug-loaded nanoparticles AIE@HMONs-PyB were approximately 100 and 24 times, respectively. This indicates that the AIE self-aggregates formed in the highly crystalline LA matrix enhance the photosensitization ability. In DHR-123 solution, the relative emission intensities (I / I0) of the four drug-loaded nanoparticles AIE / LA@HMONs and the drug-loaded nanoparticles AIE@HMONs-PyB were approximately 9.6 and 2.5 times, respectively. In ABDA solution, the absorption ratio of all drug-loaded nanoparticles at 378 nm did not change significantly compared with the blank group. These results indicate that the four drug-loaded nanoparticles AIE / LA@HMONs mainly produce type I reactive oxygen species under light irradiation. Figure 1 HJ.

[0159] Example 12

[0160] The response ability of four drug-loaded nanoparticles (AIE / LA@HMONs-OH, AIE / LA@HMONs-NH2, AIE / LA@HMONs-DA and AIE / LA@HMONs-PyB) to different pH environments was evaluated. The specific experimental steps are as follows:

[0161] The zeta potential of the four drug-loaded nanoparticles was measured at 25 °C by dynamic light scattering technique in phosphate buffer (5 mM sodium dihydrogen phosphate and 5 mM sodium hydrogen phosphate) with a pH range of 5.0 to 7.4.

[0162] Between pH 7.4 and 5.0, the zeta potential of AIE / LA@HMONs-DA was −19.4 mV at pH 7.4 and flipped to +15.7 mV at pH 5.0.

[0163] AIE / LA@HMONs-PyB showed a similar trend, with the zeta potential changing from −3.4 mV at pH 7.4 to +17.5 mV at pH 5.0.

[0164] AIE / LA@HMONs-OH and AIE / LA@HMONs-NH2 have no charge reversal phenomenon.

[0165] For charge reversal, AIE / LA@HMONs-DA requires a more acidic pH (5.5) and more than 1 hour to complete the process. In contrast, AIE / LA@HMONs-PyB complexes with water, undergoes protonation, and rapidly achieves charge reversal, achieving charge reversal within 10 minutes at a less acidic pH (6.5). Figure 2 AC.

[0166] Embodiment 13

[0167] Four drug-loaded nanoparticles for bacterial biofilm penetration:

[0168] 1) Methicillin-resistant Staphylococcus aureus (MRSA, commercial product) biofilm culture, the specific steps are:

[0169] Transfer a single colony on the solid agar plate to 5 mL of tryptic soy broth (TSB) containing tetracycline (10 μg / mL) (abbreviated as: TSB broth containing TeT (10 μg / mL)) and culture at 37°C in a shaker at 170 rpm. After overnight culture, collect the bacterial cells by centrifugation at 3500 g for 3 min and then wash with PBS. Discard the supernatant and resuspend the bacterial cells in PBS. Adjust the optical density of the bacterial suspension to 1.0 (OD 600) at 600 nm with PBS. 600 =1.0) for subsequent use. 100 μL of bacterial suspension (OD 600 =0.1) were placed in a 96-well plate and incubated at 37°C for 2 h to allow bacteria to adhere, then gently washed three times with PBS to remove non-adherent bacteria, and then added with TeT (10 μg / mL) in TSB liquid medium, and then incubated at 37°C for 24 h. The supernatant was aspirated, and then added with TeT (10 μg / mL) in TSB liquid medium, and then incubated at 37°C for 24 h to form bacterial biofilm;

[0170] 2) Four drug-loaded nanoparticles were used in bacterial biofilm penetration experiments. The specific steps were as follows:

[0171] The MRSA bacterial biofilm was carefully washed with PBS and then incubated with AIE / LA@HMONs at a concentration of 40 μM for different times (0, 0.5, 1, and 2 h) at 37 °C. After the incubation, the excess drug-loaded nanoparticles were washed with PBS, and the bacterial biofilm was stained with a live bacterial fluorescent indicator (SYTO 9, 3.34 μM) for 30 min. After washing away the unbound dye molecules with PBS, the bacterial biofilm was imaged in 3D using the z-axis scanning mode of a laser scanning confocal microscope (SYTO9, Ex = 488 nm, Em = 490-540 nm; AIE / LA@HMONs, Ex = 638 nm, Em = 640-730 nm). The fluorescence intensity of the bacterial biofilm at each time point was quantified.

[0172] As the incubation time was extended from 0.5h to 2h, the red fluorescence intensity gradually increased, indicating that the accumulation of drug-loaded nanoparticles in bacterial biofilms was time-dependent. Specifically, AIE / LA@HMONs-OH exhibited the weakest red fluorescence signal, which may be due to the limited electrostatic interaction between the negatively charged AIE / LA@HMONs-OH and bacterial cells, resulting in the easy washing away of AIE / LA@HMONs-OH. In contrast, AIE / LA@HMONs-NH2 showed relatively strong fluorescence intensity, which was attributed to the electrostatic interaction with the outer layer of bacterial biofilm. Nevertheless, the persistent positive charge partially hindered their deeper penetration into the bacterial biofilm. Compared with AIE / LA@HMONs-OH and AIE / LA@HMONs-NH2, AIE / LA@HMONs-DA showed stronger penetration ability and bacterial adhesion, which can be explained by the formation of positively charged guanidine groups under acidic conditions. In addition, AIE / LA@HMONs-PyB exhibited significantly higher fluorescence intensity, indicating that AIE / LA@HMONs-PyB could effectively penetrate and accumulate in bacterial biofilms. When exposed to the gradient acidic microenvironment of bacterial biofilms, AIE / LA@HMONs-PyB was rapidly protonated and became positively charged. The protonated AIE / LA@HMONs-PyB interacted electrostatically with the negatively charged bacterial membrane, promoting the adhesion of AIE / LA@HMONs-PyB to bacteria. These findings suggest that the ability of nanoparticles to penetrate MRSA bacterial biofilms can be improved by redesigning surface properties, providing a promising approach for the development of more effective anti-bacterial biofilm treatment strategies. Figure 2 DG.

[0173] Embodiment 14

[0174] Relationship between the penetration capacity of four drug-loaded nanoparticles and the efficiency of bacterial biofilm removal:

[0175] Four drug-loaded nanoparticles for bacterial biofilm removal:

[0176] 1) The bactericidal effects of drug-loaded nanoparticles at different concentrations (0, 10, 20, 30, and 40 μM, with PBS as solvent) were tested in the presence and absence of light.

[0177] The group treated with PBS only was denoted as blank.

[0178] Specifically, the MRSA biofilm cultured according to step 1) in Example 13 was mixed with the four drug-loaded nanoparticles, respectively, and then incubated in the dark for 2 hours (37° C.). Next, a 660 nm laser was used at 400 mW / cm 2 The bacterial biofilm was irradiated with a power density of 100 μg / cm2 for 30 min or kept away from light for 30 min. The supernatant was then carefully aspirated and the bacterial biofilm was gently washed three times with PBS.

[0179] 2) For the bacterial biofilm obtained in step 1), in order to study the number of surviving bacteria in the bacterial biofilm after different treatments, the bacterial biofilm was peeled off from the bottom of the 96-well plate by repeated blowing and suspended in PBS (200 μL). The suspension was diluted with PBS to 1×10 4 100 μL of the diluted bacterial suspension was spread on TSB solid medium containing TeT (10 μg / mL) and then incubated at 37°C for 12-16 hours. The number of CFU in each group was determined by counting the bacterial colonies formed, which was used to quantitatively evaluate the bacterial biofilm removal effect (n=3 per group);

[0180] 3) For the bacterial biofilm obtained in step 1), SYTO 9 (3.34 μM) was then stained for 30 minutes to mark live bacterial cells for 3D imaging. After removing unbound dye molecules with PBS, the bacterial biofilm was imaged in 3D using the z-axis scanning mode of a laser scanning confocal microscope (SYTO9, Ex=488 nm, Em=490-540 nm). The fluorescence intensity in the bacterial biofilm image at each time point was quantified by ImageJ software (n=3);

[0181] 4) For the bacterial biofilm obtained in step 1), fix it with formalin for 10 minutes. Subsequently, stain the bacterial biofilm with an ethanol solution of crystal violet (content 0.25%) for 30 minutes and gently rinse three times with PBS to remove unbound dye. Finally, add ethanol (150 μL) to each well and record the absorbance at 590 nm with a microplate reader (Tecan, Infinite M Nano, n=3). At the same time, take pictures using a digital camera.

[0182] 5) Prepare a 24-well plate and place a glass slide in each well for culturing bacterial biofilm. Then, add MRSA suspension (OD 600 =0.1, 500μL), let stand for 2h to wait for bacteria to adhere, and then gently wash three times with PBS to remove non-adherent bacterial cells. Add TSB liquid culture medium with TeT (10μg / mL) and incubate at 37°C for 24h. This operation is repeated once to form a 48-h bacterial biofilm. Treat the bacterial biofilm according to the method in step 1) of this embodiment. Subsequently, immerse the slide in 2.5% glutaraldehyde and fix it at room temperature overnight. The sample is then washed with water twice, and then dehydrated with gradient ethanol (including 30%, 50%, 70%, 80%, 90%, 100%, each concentration 6min×2 times), spray gold after the sample is fully dried, and observe under a scanning electron microscope.

[0183] We deeply analyzed the role of different penetration abilities in photodynamic therapy (PDT) to remove bacterial biofilms. The experimental results showed that without light irradiation, all drug-loaded nanoparticle groups did not show obvious bactericidal activity. Under 660nm laser irradiation, the AIE / LA@HMONs-PyB group showed a bactericidal rate of approximately 99%, and the AIE / LA@HMONs-DA group showed a bactericidal rate of approximately 75%, which was significantly higher than the AIE / LA@HMONs-OH and AIE / LA@HMONs-NH2 groups. This result indicates that enhanced penetration ability plays a key role in the photodynamic removal of bacterial biofilms by the PyB group. See Figure 3 .

[0184] Embodiment 15

[0185] Application of drug-loaded nanoparticles AIE / LA@HMONs-PyB combined with antimicrobial drugs in bacterial biofilm removal:

[0186] The drug-loaded nanoparticles AIE / LA@HMONs-PyB prepared in Example 8 are used as a photodynamic nanosystem in combination with antibacterial drugs:

[0187] 1) Cultivating bacterial biofilm according to step 1) in Example 13, setting up experimental groups under conditions of light irradiation and no light irradiation, including:

[0188] The control group treated with PBS (denoted as Blank),

[0189] The group treated with ciprofloxacin (CIP) only (denoted as CIP; [CIP] = 10 μg / mL; ciprofloxacin was added to the wells with or without light),

[0190] The group treated with AIE / LA@HMONs-PyB prepared in Example 8 (abbreviated as AIE; [TPAI] = 40 μM);

[0191] The group treated with AIE / LA@HMONs-PyB prepared in Example 8 and ciprofloxacin (denoted as AIE+CIP; [TPAI]=40 μM, [CIP]=10 μg / mL).

[0192] First, MRSA biofilms were incubated with AIE / LA@HMONs-PyB (for AIE and AIE+CIP groups) or PBS (for Blank and CIP groups) at 37 °C for 2 h. Next, a 660 nm laser was used at a power density of 400 mW / cm 2 Irradiate the bacterial biofilm for 30 min under the conditions of 40 °C or place it in the dark for 30 min. Then carefully aspirate the supernatant, add 100 μL of ciprofloxacin or PBS to the bacterial biofilm, and incubate at 37 °C for 8 h. Finally, carefully aspirate the supernatant and gently wash the bacterial biofilm three times with PBS.

[0193] 2) Collect CFU count, crystal violet staining, 3D imaging and scanning electron microscopy image data according to steps 2), 3), 4) and 5) in Example 14.

[0194] Since the three-dimensional structure of bacterial biofilms is partially damaged after photodynamic therapy, it is expected that the subsequent application of antibiotics can reduce the required dose of antibiotics. The damage to the bacterial biofilm structure caused by photodynamic therapy can enhance the permeability of drugs in the bacterial biofilm, achieving the purpose of efficient sterilization. Ciprofloxacin is a broad-spectrum antibiotic that works by accumulating in the bacterial nucleus, inhibiting DNA replication, and inducing the production of reactive oxygen species, thereby aggravating bacterial oxidative damage. Based on the property analysis of four drug-loaded nanoparticles, AIE / LA@HMONs-PyB, which exhibits superior bacterial biofilm penetration ability, was selected as the evaluation object for combined use with ciprofloxacin.

[0195] The bactericidal effect of PDT+CIP on MRSA biofilm was evaluated by plate counting. By comparative analysis, the combined therapy showed a bactericidal rate of 99.999%. Scanning electron microscopy images provided further support for this result, confirming the destruction of the bacterial biofilm structure and effective bacterial killing after combined treatment. In addition, laser scanning confocal microscopy results showed that the bacterial biofilm was significantly thinned and dispersed, reflecting the serious structural damage of the bacterial biofilm under combined treatment. Fluorescence intensity quantification confirmed that the PDT+CIP group had the lowest signal among all groups, indicating an enhanced ability to clear bacterial biofilms. See Figure 4 .

[0196] Example 16

[0197] Application of drug-loaded nanoparticles AIE / LA@HMONs-PyB combined with antimicrobial drugs in the healing of chronic infected wounds:

[0198] Encouraged by the significant in vitro anti-bacterial biofilm activity, we applied this combined treatment strategy to a diabetic mouse model infected with MRSA biofilm to evaluate its in vivo potential. Difficult diabetic wound healing is one of the common complications of diabetic patients, and bacterial biofilm infection plays a key role in it. Therefore, establishing a diabetic wound model infected with MRSA biofilm is of great significance for evaluating the therapeutic effect of photodynamic therapy combined with ciprofloxacin. First, mice were induced to develop diabetes by injection of streptozotocin (STZ), ensuring that their blood glucose was stable above 16.7mM, indicating the successful establishment of the diabetic model. Then, a circular skin wound was made on the right back of the mouse, and MRSA bacterial solution was inoculated to establish a bacterial biofilm-infected wound, followed by treatment and observation of the healing effect. The infected diabetic mice were randomly divided into eight groups and treated with or without light:

[0199] The groups treated with PBS only (denoted as Blank and Blank / L),

[0200] The group treated with ciprofloxacin (denoted as CIP and CIP / L, [CIP] = 20 μg / mL),

[0201] The groups treated with AIE / LA@HMONs-PyB (denoted as AIE and AIE / L, [TPAI] = 40 μM), and the groups treated with AIE / LA@HMONs-PyB and ciprofloxacin (denoted as AIE+CIP and AIE+CIP / L, [CIP] = 20 μg / mL, [TPAI] = 40 μM).

[0202] During the experimental period, the wound size and the weight of the mice were recorded every day. It can be seen intuitively from the photos that although the wound size of all groups gradually decreased with the increase of days, it is obvious that the AIE+CIP / L group showed a significantly faster healing speed, and the superimposed wound size images clearly showed the rapid shrinkage of the wound. The relative infection area of ​​mice in the four unilluminated groups, Blank / L group and CIP / L group was reduced to about 40% on the 8th day. The AIE / L group showed a moderate therapeutic effect, with the relative infection area reduced to about 20%, which was mainly attributed to the effect of photodynamic therapy on bacterial biofilms. In contrast, the wounds of mice in the AIE+CIP / L group were almost completely healed, and the relative infection area was reduced to only about 2%, highlighting the superior effect of combining photodynamic therapy and ciprofloxacin in the treatment of bacterial biofilms.

[0203] The weight loss of mice in all groups on day 1 was mainly due to the stress response during the establishment of the wound model. Figure 5 As shown in D, as the wounds recovered, the weight of the mice gradually increased, and the weight gain of the mice in the AIE+CIP / L group was the most significant, which was consistent with the good trend of their wound healing. All groups of mice survived throughout the treatment period, indicating the biosafety of the treatment strategy. On day 11, we collected half of the infected skin of the surviving mice in each group for homogenization in order to count the colonies on the infected site. Figure 5 As shown in F and G, the number of bacterial colonies in the AIE+CIP / L group decreased by three orders of magnitude, while the number of bacterial colonies in the AIE / L group decreased by two orders of magnitude. These results clearly show that compared with single therapy, the combination of photodynamic therapy and ciprofloxacin significantly improved the treatment effect and effectively reduced the number of bacteria in the infected area. These results emphasize the potential value of photodynamic therapy combined with ciprofloxacin in clinical applications, which can significantly improve the treatment efficiency and maintain good biosafety. Figure 5 .

[0204] To elucidate the effects of combined photodynamic therapy and ciprofloxacin treatment, we performed histological analysis on the collected skin tissues. First, we used hematoxylin-eosin staining to analyze the degree of dermal regeneration and inflammatory cell infiltration. The AIE+CIP / L group had the least inflammatory cells, but all groups observed varying degrees of inflammatory cell infiltration. It can also be seen that the AIE+CIP / L group showed new hair follicles and a thinner dermis, which means that the degree of skin healing and reconstruction was the highest. Masson staining uses the different affinities of different tissue components for dyes to observe the content of collagen fibers in the tissue. Collagen deposition is a key marker for tissue regeneration, granulation tissue, and re-epithelialization. The blue color, which symbolizes collagen fibers, was more prominent in the AIE+CIP / L group, which means that collagen fiber expression was significantly higher than in all other groups, indicating that AIE+CIP / L treatment significantly accelerated skin regeneration.

[0205] We characterized and quantified various cytokines in the wound area using immunofluorescence and immunohistochemical staining. First, we used immunofluorescence to study the changes in inflammatory factors near the wound. Interleukin-6 is a proinflammatory cytokine mainly secreted by activated T cells and fibroblasts, while tumor necrosis factor α is mainly produced by activated macrophages and T lymphocytes, both of which are key mediators of inflammation. Compared with other groups, the yellow fluorescence (tumor necrosis factor α) and green fluorescence (interleukin-6) of the AIE / L group were slightly reduced, and the reduction was most obvious in the AIE+CIP / L group, indicating that photodynamic therapy and ciprofloxacin synergistic treatment significantly downregulated proinflammatory cytokines. This downregulation helps to reduce the inflammatory response and create a more favorable environment for wound healing. Further, we evaluated the wound healing process by immunohistochemistry. Platelet-endothelial cell adhesion molecule is a cell surface glycoprotein that is mainly present on vascular endothelial cells. Platelet-endothelial cell adhesion molecule positivity indicates that the cells have endothelial differentiation characteristics and are closely related to the angiogenesis process. Compared with other groups, the AIE+CIP / L group showed a significant increase in new blood vessels, indicating an enhanced state of skin regeneration. Type I collagen and type III collagen are two common types of collagen, which are characterized by strong toughness and high elasticity, respectively, and are involved in the synthesis and repair of the extracellular matrix. The degree and quality of wound healing can be evaluated by detecting the expression of type I collagen and type III collagen. Compared with the other groups, the expression of type I collagen and type III collagen in the AIE+CIP / L group was significantly increased, which once again proved that AIE+CIP / L treatment can promote the synthesis and deposition of collagen, thereby accelerating the wound healing process. In summary, the combination of photodynamic therapy and ciprofloxacin effectively downregulated proinflammatory factors and upregulated the expression of CD31 and collagen, significantly reduced wound tissue inflammation, and accelerated wound healing. These findings highlight the application potential of PDT+CIP combination therapy, which has the characteristics of improving treatment efficiency and maintaining good biosafety. See Figure 6 .

[0206] Embodiment 17

[0207] Application of acid-responsive hollow mesoporous organosilicon nanoparticles as drug carriers comprises the following steps:

[0208] An acid-responsive hollow mesoporous organosilicon nanoparticle HMONs-DA (10 mg) prepared in Example 2, two drugs, the first LA (0.05 mg), the second TPAI (0.05 mg), and a solvent for dissolving the above drugs (1000 mg of dimethyl sulfoxide) were mixed evenly, added dropwise to water equivalent to 5 times the mass of the solvent, stirred for 30 minutes, dialyzed in deionized water for 10 hours using a dialysis bag with a molecular weight cutoff value of 3000 Da, filtered through a 3 μm membrane, and centrifuged at 11000 rpm for 10 minutes to obtain drug-loaded nanoparticles AIE / LA@HMONs-DA.

[0209] Embodiment 18

[0210] Application of acid-responsive hollow mesoporous organosilicon nanoparticles as drug carriers comprises the following steps:

[0211] An acid-responsive hollow mesoporous organosilicon nanoparticle HMONs-DA (10 mg) prepared in Example 3, two drugs, the first LA (5 mg) and the second TPAI (5 mg), and a solvent for dissolving the above drugs (5000 mg of dimethyl sulfoxide) were mixed evenly, added dropwise into water equivalent to 20 times the mass of the solvent, stirred for 60 minutes, dialyzed in deionized water for 11 hours using a dialysis bag with a molecular weight cutoff value of 3000 Da, filtered through a 3 μm membrane, and centrifuged at 12000 rpm for 10 minutes to obtain drug-loaded nanoparticles AIE / LA@HMONs-DA.

[0212] The drug-loaded nanoparticles AIE / LA@HMONs-DA obtained in Examples 17 and 18 have similar hydrated particle size in water, stability for seven days, photosensitization ability, response ability to different pH environments, and ability to penetrate bacterial biofilms as those in Example 7.

[0213] Embodiment 19

[0214] Application of acid-responsive hollow mesoporous organosilicon nanoparticles as drug carriers comprises the following steps:

[0215] An acid-responsive hollow mesoporous organosilicon nanoparticle HMONs-PyB (10 mg) prepared in Example 5, two drugs, the first LA (0.06 mg), the second TPAI (0.04 mg), and a solvent for dissolving the above drugs (1000 mg of dimethyl sulfoxide) were mixed evenly, added dropwise to water equivalent to 5 times the mass of the solvent, stirred for 30 minutes, dialyzed in deionized water for 10 hours using a dialysis bag with a molecular weight cutoff value of 3000 Da, filtered through a 3 μm membrane, and centrifuged at 11000 rpm for 10 minutes to obtain drug-loaded nanoparticles AIE / LA@HMONs-PyB.

[0216] Embodiment 20

[0217] Application of acid-responsive hollow mesoporous organosilicon nanoparticles as drug carriers comprises the following steps:

[0218] An acid-responsive hollow mesoporous organosilicon nanoparticle HMONs-PyB (10 mg) prepared in Example 6, two drugs, the first LA (7 mg) and the second TPAI (3 mg), and a solvent for dissolving the above drugs (5000 mg of dimethyl sulfoxide) were mixed evenly, added dropwise into water equivalent to 20 times the mass of the solvent, stirred for 60 minutes, dialyzed in deionized water for 11 hours using a dialysis bag with a molecular weight cutoff value of 3000 Da, filtered through a 3 μm membrane, and centrifuged at 12000 rpm for 10 minutes to obtain drug-loaded nanoparticles AIE / LA@HMONs-PyB.

[0219] The drug-loaded nanoparticles AIE / LA@HMONs-PyB obtained in Examples 19 and 20 have similar hydrated particle size in water, stability for seven days, photosensitization ability, response ability to different pH environments, and ability to penetrate bacterial biofilms as those in Example 8.

Claims

1. A method for preparing acid-responsive hollow mesoporous organosilicon nanoparticles, characterized in that The steps include: 1) Dissolve hexadecyltrimethylammonium chloride and triethanolamine in ultrapure water, heat to 70-90°C, stir at 800-1000rpm for 20-40 minutes, add tetraethyl silicate dropwise, and continue to react for 30-60 minutes after the addition is complete; 2) adding a mixed solution 1 consisting of 1,2-bis(triethoxysilyl)ethane and tetraethyl silicate to the material obtained in step 1), and after the addition is complete, continuing the reaction for 1-2 hours, cooling to room temperature, centrifuging at 10000-12000 rpm for 30 minutes, washing, and harvesting core-shell structured silicon nanoparticles; 3) Dispersing 500 mg of the core-shell silicon nanoparticles in 90-110 mL of a mixed solution 2 consisting of ethanol and hydrochloric acid according to the ratio, reflux for 8-12 hours, cool to room temperature, centrifuge at 1000-12000 rpm for 20-30 minutes, collect the solid, and wash; the volume ratio of ethanol to hydrochloric acid is 80-100:1; 4) Repeat step 3) twice, and the obtained solid is a mesoporous silicon sphere; 5) dispersing the mesoporous silicon spheres in an aqueous ammonia solution with a volume concentration of 8%-10%, reacting at 80-95° C. for 3-5 hours to obtain mesoporous hollow silicon spheres, cooling to room temperature, centrifuging at 10000-12000 rpm for 20-30 minutes, washing the obtained solid, and obtaining mesoporous hollow HMONs-OH silicon spheres; 6) dispersing the mesoporous hollow HMONs-OH silica spheres and 3-aminopropyltriethoxysilane in anhydrous ethanol, reflux at 80-90° C. for 3-6 hours, cooling to room temperature, centrifuging at 10000-2000 rpm for 20-30 minutes, washing, and obtaining amino-modified mesoporous hollow silica nanoparticles HMONs-NH2; 7) Proceed in one of the following two ways: Method 1: The HMONs-NH2 obtained in step 6) and 2,3-dimethylmaleic anhydride and triethylamine are added to N,N-dimethylformamide, stirred at 0-10°C for 3-6 hours, centrifuged at 10000-12000 rpm for 10 minutes, and washed to obtain an acid-responsive hollow mesoporous organosilicon nanoparticle, referred to as HMONs-DA; Method 2: The HMONs-NH2, isonicotinic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine obtained in step 6) are added to anhydrous N,N-dimethylformamide, protected by argon, stirred at room temperature for 3-6 hours, centrifuged at 10000-12000 rpm for 20-30 minutes, and washed to obtain a solid product; bromoacetic acid and the solid product are added to acetonitrile, heated to reflux for 8-12 hours; after cooling to room temperature, the solid precipitate is collected by centrifugation, washed, and an acid-responsive hollow mesoporous organosilicon nanoparticle, referred to as HMONs-PyB, is obtained.

2. The preparation method according to claim 1, characterized in that In the step 1), the mass ratio of hexadecyltrimethylammonium chloride, triethanolamine and tetraethyl silicate is 2:(0.1-0.4):(0.8-2).

3. The preparation method according to claim 1, characterized in that The mass ratio of 1,2-bis(triethoxysilyl)ethane to tetraethyl silicate in step 2) is 1:(0.25-2), and the mass ratio of the material obtained in step 1) to the mixed solution 1 is (10-40):

1.

4. The preparation method according to claim 1, characterized in that In the step 5), the mass ratio of the silicon spheres with the mesoporous structure to the aqueous ammonia solution with a volume concentration of 8%-10% is 0.2:(40-400).

5. The preparation method according to claim 1, characterized in that In the step 6), the mass ratio of the hollow mesoporous HMONs-OH silicon spheres, 3-aminopropyltriethoxysilane and anhydrous ethanol is: 100:(10-40):(5000-50000).

6. The preparation method according to claim 1, characterized in that Step 7) In method 1, the mass ratio of HMONs-NH2, 2,3-dimethylmaleic anhydride, triethylamine and N,N-dimethylformamide is 100:(10-100):(10-100):(2000-10000).

7. The preparation method according to claim 1, characterized in that Step 7) In method 2, the mass ratio of HMONs-NH2, isonicotinic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and anhydrous N,N-dimethylformamide is 11:(11-55):(11-110):(1.1-5.5):(3300-6600); the mass ratio of the bromoacetic acid, the solid product and acetonitrile is (5.5-22):11:(1100-5500).

8. Acid-responsive hollow mesoporous organosilicon nanoparticles prepared by the preparation method of any one of claims 1 to 7.

9. Use of an acid-responsive hollow mesoporous organosilicon nanoparticle as a drug carrier according to claim 8, characterized in that The steps include: The acid-responsive hollow mesoporous organosilicon nanoparticles, the drug and the solvent for dissolving the drug are uniformly mixed in a mass ratio of 10:(0.1-10):(500-5000), and the mixture is dropped into water 5-20 times the mass of the solvent, stirred for 10-60 minutes, dialyzed in deionized water for 10-12 hours using a dialysis bag, filtered through a 3 μm membrane, and centrifuged at 10000-12000 rpm for 10 minutes to obtain drug-loaded nanoparticles.

10. The use according to claim 9, characterized in that The drug is at least one of an antibacterial drug, a protease, a quorum infection signal molecule inhibitor, an active peptide, a photosensitizer, a photothermal agent, a saturated fatty acid and an anti-biofilm agent.

Images