Synergistic antibacterial nanoplatform based on CO and AIEgens, its preparation method and application

Abstract

The invention belongs to the technical field of biomedical polymers, and particularly relates to a synergistic antibacterial nano-platform based on CO and AIEgens, comprising: a dendritic polypeptide nano-gel carrier, which is a third-generation lysine dendritic macromolecule G3-Lys with polyhedral oligomeric silsesquioxane POSS as a core and lysine as a branching unit, and is cross-linked with a cross-linking agent disulfide bis (succinimidyl propionate) DSP to obtain a blank gel G3DSP; wherein the inner cavity of G3DSP is loaded with photosensitizer AIEgens and a CO release donor. The beneficial effects of the invention are as follows: the construction of a synergistic antibacterial nano-platform based on CO and AIEgens has created a new strategy for overcoming the outer membrane (OM) barrier of Gram-negative bacteria, has excellent biofilm ablation performance and good biosafety and blood compatibility, can have a specific targeted killing effect on Gram-negative bacteria, and provides a new mode for combating Gram-negative bacterial infection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical polymers, and in particular to a synergistic antibacterial nano-platform based on CO and AIEgens, and a preparation method and application thereof. Background Art

[0002] The emergence of multidrug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Acinetobacter baumannii (A. baumannii), poses a significant and growing challenge to global public health. These bacteria are increasingly responsible for mortality associated with antibiotic-resistant infections. The robust resistance of these bacteria to antibiotics, including resistance of P. aeruginosa to polymyxins, a last-resort drug used to treat infections caused by resistant Gram-negative bacteria, has been primarily attributed to their unique outer membrane (OM) architecture. This membrane consists of an inner monolayer composed of asymmetric glycerophospholipids (GPLs) that play a crucial role in maintaining the structural stability of the OM. The outer monolayer is densely populated with lipopolysaccharide (LPS) molecules, forming an effective permeability barrier. This composition creates a strong barrier that effectively blocks the entry of small hydrophobic molecules, including a wide range of clinically valuable antibiotics. Due to the lack of effective drugs against these Gram-negative pathogens, these bacteria are increasingly associated with severe and even fatal hospital-acquired infections. These infections cover a wide range of diseases, including pneumonia, peritonitis, bloodstream infections, wound or surgical site infections, urinary tract infections, and bacterial keratitis. Therefore, the global biomedical research community is working to develop innovative strategies to effectively combat multidrug-resistant Gram-negative bacterial infections.

[0003] Antimicrobial photodynamic therapy (aPDT) represents an innovative approach that is distinct from traditional antibiotics. It exploits reactive oxygen species (ROS) generated by photosensitizers (PSs) upon irradiation with visible or infrared light to achieve therapeutic effects. Due to its non-invasive, precisely controlled, broad-spectrum antimicrobial activity, and independence from specific bacterial targets, aPDT has attracted significant attention in antibacterial and anti-biofilm applications. Photosensitizers with aggregation-induced emission (AIE) properties, called AIE luminogens (AIEgens), especially those with the ability to induce ROS generation, have been widely used in the diagnosis and fight against Gram-positive bacterial infections. The porous monolayer cell membrane of Gram-positive bacteria facilitates the efficient penetration of AIEgens, resulting in a powerful bactericidal effect due to the high level of ROS generated intracellularly.

[0004] However, the effectiveness of AIEgens-based aPDT (AIEgens-aPDT) against Gram-negative bacteria is often limited. This limitation stems from the hydrophobic nature of many AIEgens, which have difficulty penetrating the outer membrane (OM) of Gram-negative bacteria, hindering the generation of sufficient ROS inside the cell, thereby affecting the bactericidal effect. Therefore, innovative therapeutic strategies are urgently needed that can not only promote the penetration of AIEgens by disrupting the structural stability and permeability of the OM, but also synergize with AIEgens-aPDT to enhance its antibacterial effect. These advances provide great possibilities for expanding the application potential of AIEgens-aPDT in combating Gram-negative bacterial infections. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a synergistic antibacterial nanoplatform based on CO and AIEgens and a preparation method and application thereof to solve the above problems.

[0006] The technical solution of the present invention is achieved as follows: a synergistic antibacterial nano-platform based on CO and AIEgens, comprising:

[0007] Dendritic polypeptide nanogel carrier, which uses polyhedral oligomeric silsesquioxane POSS as the core and lysine as the branching unit of the third-generation lysine dendrimer G3-Lys, and is cross-linked with a cross-linking agent disulfide bis (succinimidyl propionate) DSP to obtain a blank gel G3DSP;

[0008] Among them, the inner cavity of G3DSP is loaded with photosensitizer AIEgens and CO release donor.

[0009] Furthermore, the CO releasing donor is CORM-401.

[0010] Furthermore, the preparation method of the synergistic antibacterial nanoplatform based on CO and AIEgens comprises the following steps:

[0011] Step 1: The third-generation lysine dendrimer G3-Lys with polyhedral oligomeric silsesquioxane POSS as the core and lysine as the branching unit is cross-linked with a cross-linking agent disulfide bis (succinimidyl propionate) DSP to form a blank gel G3DSP;

[0012] Step 2: Dissolve 100 mg of G3DSP in 10 mL of water to form a G3DSP solution;

[0013] Step 3: Dissolve 10 mg of AIEgens in 1 mL of DMSO to prepare an AIE solution, and dissolve 50 mg of CORM-401 in 1 mL of anhydrous methanol to prepare a CORM-401 solution;

[0014] Step 4: Add CORM-401 solution to the G3DSP solution under the protection of light and nitrogen, stir for 2 h, then add AIE solution and continue the reaction for 22 h;

[0015] Step 5: After the solution reaction in step 4 is completed, place it in a dialysis bag and dialyze it in a deionized water solution for 24 hours;

[0016] Step 6: The solution obtained in step 5 is freeze-dried to obtain a rose-red flocculent solid product AIE&CO@G3DSP.

[0017] Furthermore, the specific implementation steps of step one include:

[0018] a. Take 1.00 g, 0.12 mmol of G3-Lys and 0.10 g, 0.25 mmol of DSP and dissolve them in 10 mL and 5 mL of anhydrous DMF respectively to obtain a G3-Lys solution and a DSP solution;

[0019] b. Then, slowly add the DSP solution dropwise into the G3-Lys solution under stirring;

[0020] The solution obtained in step b was stirred at room temperature. After 24 hours, it was dialyzed in DMF for 4 hours and then in pure water for 24 hours. Then, the product G3DSP was obtained after freeze-drying.

[0021] Furthermore, the molecular weight cut-off of the dialysis bag is 1000Da.

[0022] Furthermore, a synergistic antibacterial nanoplatform based on CO and AIEgens is provided for application in the field of high-efficiency antibacterial properties.

[0023] The beneficial effects of the present invention are:

[0024] 1. The AIE&CO@G3DSP prepared in this application can efficiently penetrate into the interior of bacteria and has a specific targeted killing effect on Gram-negative bacteria;

[0025] 2. AIE&CO@G3DSP has excellent biofilm ablation performance;

[0026] 3. AIE&CO@G3DSP can effectively and safely treat bacterial infections in vivo and promote wound healing;

[0027] 4. AIE&CO@G3DSP has good biosafety and blood compatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 (a) particle size and (b) transmission electron microscopy image of AIE&CO@G3DSP according to a specific embodiment of the present invention;

[0030] Figure 2 The particle size and ζ-potential of four nanogels according to the specific embodiment of the present invention are shown;

[0031] Figure 3 AIE standard curve of the specific embodiment of the present invention;

[0032] Figure 4 The loading amount and drug loading efficiency of CORM-401 and AIE in four nanogels according to the specific embodiments of the present invention;

[0033] Figure 5 This is a specific embodiment of the present invention showing the generation of CO by different materials after being exposed to light for different periods of time;

[0034] Figure 6 This is a specific embodiment of the present invention showing the generation of H2O2 by different materials after being exposed to light for different periods of time;

[0035] Figure 7 The CO release behavior of AIE&CO@G3DSP under different H2O2 concentrations in the specific embodiment of the present invention;

[0036] Figure 8 This is the ROS release behavior of different materials under 808nm laser irradiation in a specific embodiment of the present invention;

[0037] Fig. 9 Specific embodiments of the present invention a) AIE molecular structure; b) CO release mechanism;

[0038] Fig.10 In vitro antimicrobial activities of different materials at different concentrations according to the specific embodiments of the present invention a) MRSA and b) PA;

[0039] Fig.11 a) MRSA and b) PA survival rates after treatment with different materials according to the specific embodiments of the present invention;

[0040] Fig.12 In vitro antimicrobial activities of different materials at different light exposure times for the specific embodiments of the present invention MRSA and PA;

[0041] Fig.13 a) MRSA and b) PA survival rates after treatment with different materials according to the specific embodiments of the present invention;

[0042] Fig.14 The survival of S. aureus, MRSA, E. coli, AB and PA after treatment with different materials in the specific embodiment of the present invention;

[0043] Fig.15 The survival rates of S. aureus, MRSA, E. coli, AB and PA after treatment with different materials in the specific embodiments of the present invention;

[0044] Fig.16 SEM images of a) MRSA and b) PA treated with different materials according to the specific embodiments of the present invention;

[0045] Fig.17 TEM images of a) MRSA and b) PA treated with different materials according to the specific embodiments of the present invention;

[0046] Fig.18 a) hemolysis, b) hemolysis rate, and toxicity to c) L929 and d) L926 cells of different materials according to the specific embodiments of the present invention;

[0047] Fig.19 Specific embodiments of the present invention include (a) treatment plan for the urinary catheter biofilm model, (b) wound photos, and (c) photos of the catheter removal;

[0048] Fig. 20 (a) qualitative and (b) quantitative analysis of residual bacteria on a urinary catheter in accordance with a specific embodiment of the present invention;

[0049] Fig.21 The surface morphology of the residual biofilm on the catheter of the specific embodiment of the present invention;

[0050] Fig. 22 (a) H&E staining, (b) Masson staining, and (c) inflammatory factor detection of skin tissue in a specific embodiment of the present invention;

[0051] Fig.23 The treatment scheme of the mouse abscess model according to the specific embodiment of the present invention is as follows;

[0052] Fig.24 The healing condition of the wound at the abscess site of mice in the specific embodiment of the present invention;

[0053] Fig.25 It is a quantitative analysis of the number of residual bacteria in the abscess site according to a specific embodiment of the present invention;

[0054] Fig.26 H&E, (c) Masson staining, and (d) immunohistochemical analysis of wound tissues on (a) day 2 and (b) day 10 for a specific embodiment of the present invention;

[0055] Fig. 27 The present invention is a specific embodiment of the present invention showing (a) the concentration of HbCO in the blood of mice after the treatment and (b) the changes in the weight of mice during the treatment period;

[0056] Fig.28 HE staining of heart, liver, spleen, lung and kidney according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] In the description of the present application, it should be noted that the terms used herein are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. For ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The technology, methods and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be regarded as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0059] This embodiment discloses a synergistic antibacterial nano-platform based on CO and AIEgens, and a preparation method and application thereof. The synergistic antibacterial nano-platform includes:

[0060] Dendritic polypeptide nanogel carrier, which uses polyhedral oligomeric silsesquioxane POSS as the core and lysine as the branching unit of the third-generation lysine dendrimer G3-Lys, and is cross-linked with a cross-linking agent disulfide bis (succinimidyl propionate) DSP to obtain a blank gel G3DSP;

[0061] Among them, the inner cavity of G3DSP is loaded with photosensitizer AIEgens and CO release donor CORM-401;

[0062] The preparation steps of the above-mentioned synergistic antibacterial nano-platform are as follows:

[0063] G3DSP Synthesis:

[0064] POSS is synthesized by polymerization of (3-aminopropyl)triethoxysilane (APTES) under the catalysis of concentrated hydrochloric acid.

[0065] The reaction steps are as follows: at normal room temperature, add 400 mL of methanol to a three-necked reaction bottle, heat and stir to 50 ° C, add APTES (15 mL, 60 mmol) dropwise with a dropping funnel, slowly add 30 mL of 37% hydrochloric acid, and finally raise the temperature to 90 ° C, cool and reflux, and react for 24 hours. After the temperature drops to room temperature, add the reaction solution to 200 mL of tetrahydrofuran (THF) and stir at room temperature for 6 hours. After centrifugation (4000 rpm, 5 min) and washing three times, put the product into a vacuum drying oven to finally obtain dry white powder POSS;

[0066] Subsequently, POSS (1.00 g, 0.85 mmol), tert-butyloxycarbonyl (Boc)-protected lysine Boc-Lys(Boc)-OH (2.80 g, 8.1 mmol), HOBt (1.36 g, 10.12 mmol), and HBTU (3.80 g, 10.02 mmol) were weighed respectively, dissolved in anhydrous DMSO (50 mL) under nitrogen protection, stirred for 30 min in an ice bath until completely dissolved, and 7 mL of DIPEA was added to the reaction liquid, and the reaction was stirred at room temperature for 48 h.

[0067] Then, 100 mL of chloroform was added to the solution, and then the solution was washed three times with 200 mL of saturated NaHCO3, 1 mmol of HCl, and saturated NaCl to remove the residual DMSO and impurities in the solution, and then anhydrous calcium sulfate was added to the solution to dry overnight. The next day, the solution was filtered and the liquid was evaporated at low pressure on a rotary evaporator until the solution became viscous.

[0068] Then, 2 mL of acetonitrile was added to the product and placed in a -20°C refrigerator for recrystallization for 4 h. Then, acetonitrile was added to precipitate and centrifuged (3200 rpm, 5 min) for washing until the supernatant was clear. The precipitate was collected and placed in a vacuum drying oven to finally obtain a white solid product G1-Lys (Boc).

[0069] The obtained product was then dissolved in chloroform, and trifluoroacetic acid ten times the equivalent of the Boc group was added to react for 8 hours. The trifluoroacetic acid was removed by rotary evaporation, and after the solution became viscous, it was slowly added dropwise to the ether solution. After stirring for 0.5 hours, it was centrifuged (3500 rpm, 3 min), and then ether was added for centrifugal washing 3-5 times, and it was placed in a vacuum drying oven for vacuum drying to finally obtain a white solid product G1-Lys.

[0070] Similarly, the synthesis method of G2-Lys and G3-Lys is the same as that of G1-Lys, that is, G1-Lys (Boc) is synthesized by reacting the eight amino groups connected to the top of POSS with activated Boc-protected lysine, and TFA is added to remove Boc. Then, it reacts with Boc-protected lysine to synthesize Boc-protected G2-Lys, and then TFA is added to remove Boc to expose the amino group to obtain G2-Lys. The synthesis method of G3-Lys is the same as that of G2-Lys. Boc-protected lysine is connected to the 32 peripheral amino groups outside G2-Lys, and then TFA is used to remove Boc to form G3-Ly. The structures of G2-Lys and G3-Lys were characterized by hydrogen nuclear magnetic resonance spectroscopy;

[0071] G3-Lys (1.00 g, 0.12 mmol) and dithiobis(succinimidyl propionate) (DSP, 0.10 g, 0.25 mmol) were dissolved in anhydrous DMF (10 mL, 5 mL) respectively. The DSP solution was slowly added dropwise to the G3-Lys solution under stirring. After stirring at room temperature for 24 hours, the solution was dialyzed in DMF for 4 hours and then in pure water for 24 hours. The product G3DSP was obtained after freeze drying.

[0072] The spherical macromolecule G3DSP has a hydrophobic inner cavity and a hydrophilic amino group on the outside. It loads the hydrophobic AIE molecules and CO donor CORM-401 in the water phase through hydrophobic association. The specific steps are as follows:

[0073] 100 mg G3DSP was dissolved in 10 mL water, 10 mg AIE was dissolved in 1 mL DMSO, and 50 mg CORM-401 was dissolved in 1 mL anhydrous methanol. CORM-401 solution was slowly added under light protection and nitrogen protection. After stirring for 2 hours, AIE solution was added and the reaction was continued for 22 hours. After the reaction was completed, the mixed solution was placed in a dialysis bag (1000 Da) and dialyzed in a deionized water solution for 24 hours. The obtained solution was freeze-dried to obtain a rose-red flocculent solid product AIE&CO@G3DSP.

[0074] This application has pioneered a new strategy to overcome the OM barrier of Gram-negative bacteria and provided a new model for combating Gram-negative bacterial infections. This application has cleverly combined carbon monoxide gas therapy with AIEgens-aPDT based on the antibacterial nanoplatform constructed based on CO and AIEgens. For Gram-negative bacterial infections, CORM-401 and AIEgens are encapsulated in the poly(L-lysine) dendrimer-based nanogel G3DSP. Subsequent LED light irradiation activates the production of ROS, which can oxidize CORM-401 and release CO. The released CO then penetrates the bacterial cell mold, inhibits energy metabolism, and destroys the stability and permeability of OM, thereby promoting the entry of AIE&CO@G3DSP. This process allows more ROS and CO to be produced inside the cell; thereby exerting significant synergistic antibacterial and anti-biofilm effects.

[0075] CO can efficiently penetrate the outer membrane of Gram-negative bacteria, thereby inhibiting the energy metabolism inside the bacteria, and on this basis, by destroying the synthesis of glycerophospholipids, the entire process of LPS synthesis, transport and anchoring on the outer membrane; these effects make the outer membrane of Gram-negative bacteria unstable and increase the permeability, so the nanogel of the present application can efficiently penetrate into the bacteria and produce a large amount of CO and ROS in the bacteria to exert a significant synergistic antibacterial and anti-biofilm effect.

[0076] Test Example 1:

[0077] A small amount of G3DSP, AIE&CO@G3DSP, CO@G3DSP and AIE@G3DSP were weighed and dissolved in water respectively, and the particle size and potential of the nanogel were detected by dynamic light scattering (DLS); the morphology of the nanogel was analyzed by transmission electron microscopy (TEM). CO@G3DSP and AIE@G3DSP were prepared according to the method of Example 1.

[0078] Determination of drug loading of AIE&CO@G3DSP: In order to determine the drug loading efficiency of CORM-401 in nanogel AIE&CO@G3DSP, the concentration of Mn ions in the solution was detected by inductively coupled plasma optical emission spectroscopy (ICP-OES), and the amount of CORM-401 was further determined. The specific operation is as follows: AIE&CO@G3DSP material is 16 mg, 1 mg CO&AIE@G3DSP is dissolved in 1 mL of nitric acid to obtain 1 mL 1 mg / mL AIE&CO@G3DSP solution, 50 μL is diluted 100 times with water to obtain 5 mL 10 mg / L AIE&CO@G3DSP solution, and the content of Mn ions in the solution is detected by ICP-OES:

[0079] The drug loading capacity and drug loading efficiency of CORM-401 were calculated by the following formulas:

[0080]

[0081] In order to determine the drug loading efficiency of AIE in nanogel AIE&CO@G3DSP, the drug loading amount and drug loading efficiency of AIE were calculated by the standard curve of AIE. The specific method is as follows: 1 mg / mL of AIEDMSO solution was diluted to the following concentrations: 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.0025 mg / mL, and the UV-visible absorption light of different concentrations of AIE in the range of 200-800nm ​​was scanned by UV-visible spectrophotometer, and the absorbance value at 515nm was fitted to the AIE standard curve.

[0082] The drug loading capacity and drug loading efficiency of AIE were calculated by the following formulas:

[0083]

[0084] Stability test of AIE&CO@G3DSP: 1 mg CO@G3DSP, AIE@G3DSP and AIE&CO@G3DSP were dissolved in 1 mL PBS, and the dispersion state of the solution was photographed by a digital camera. The particle size and PDI of AIE&CO@G3DSP on days 0, 1, 2, 3, 4, 5, 6 and 7 were monitored by dynamic light scattering (DLS).

[0085] CO release behavior of AIE&CO@G3DSP: The CO release behavior of AIE&CO@G3DSP was measured according to the method reported by UraraHasegawa et al. First, 2AIE&CO@G3DSP solution (1 mg / mL) and CO detector were placed in a sealable cylindrical glass container, and then the system was sealed and illuminated with white light of 3 W cm -2 The AIE&CO@G3DSP solution was irradiated with an output power of . The irradiation time was set to 0, 10, 20, 30, 40, 50, and 6 min, and the CO concentration was measured at each time point. In addition, 1 mL of H2O2 solutions of different concentrations (0, 100 μM, 500 μM, 1 mM, 2 mM) were injected into AIE&CO@G3DSP using a syringe, and the CO gas concentration at each time point was recorded. 1 mL of H2O2 solutions of different concentrations (0, 50 μM, 100 μM, 200 μM, 500 μM) were injected into CO@G3DSP using a syringe, and the CO gas concentration at each time point was recorded. The amount of CO released was calculated according to the following formula:

[0086]

[0087] Where p is the partial pressure of CO, Vg is the gas volume, Vl is the liquid volume, and R is the gas constant (0.008205Latmmol-1·K -1 ), T is the temperature, c is the concentration of CO in the liquid phase, and k is the Henry's law constant in the aqueous solution (1052.63 L·atm·mol at 25°C). -1 PBS, G3DSP (1 mg / mL), CO&G3DSP (1 mg / mL), AIE&G3DSP (1 mg / mL), and non-illuminated AIE&CO@G3DSP (1 mg / mL) were used as controls.

[0088] ROS release behavior of AIE&CO@G3DSP: The ROS generation curve of AIE&CO@G3DSP under white light irradiation was detected using an active oxygen fluorescence probe. First, 100 μL AIE&CO@G3DSP (1 mg / mL) was mixed with 100 μL DCFH-TA working solution in a 96-well plate, incubated at 37°C in the dark for 30 min, and irradiated with white light. The illumination time was set to 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6 min. At each time point, a microplate reader (VICTOR Nivo TM )Measure the fluorescence intensity of AIE&CO@G3DSP at an excitation wavelength of 488nm and an emission wavelength of 525nm. PBS, G3DSP (1mg / mL), CO&G3DSP (1mg / mL), AIE&G3DSP (1mg / mL), and non-illuminated AIE&CO@G3DSP (1mg / mL) were used as controls.

[0089] H2O2 release behavior of AIE&CO@G3DSP: The H2O2 generation of AIE&CO@G3DSP under white light irradiation was measured using a hydrogen peroxide fluorescent probe. First, 100 μL AIE&CO@G3DSP (1 mg / mL) was mixed with 100 μL H2O2 working solution in a 96-well plate and incubated at 37°C in the dark for 30 min. Then, the mixture was irradiated with white light at a laser intensity of 3 W cm -2 The irradiation time was set to 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6 min. At each time point, the microplate reader (VICTOR Nivo TM)Measure the absorbance of the mixture at 560nm, and calculate the H2O2 content through the standard curve. PBS, G3DSP (1mg / mL), CO&G3DSP (1mg / mL), AIE&G3DSP (1mg / mL), and non-illuminated AIE&CO@G3DSP (1mg / mL) were used as controls.

[0090] The results of the above test example are as follows:

[0091] Characterization of physical and chemical properties of AIE&CO@G3DSP: AIE&CO@G3DSP is a three-dimensional network structure formed by G3DSP encapsulating AIE and CORM-401 at the same time. The method of encapsulating CORM-401 first and then AIE can improve the drug loading rate of nanogels. This is because encapsulating CORM-401 increases the volume of nanogels and increases the encapsulation capacity of AIE. Figure 1 As shown in the figure, the particle size of the blank nanogel is 207.4nm, the PDI is 0.08, and the potential is 44.9mV. This shows that AIE&CO@G3DSP has good stability and dispersibility in aqueous solution. The TEM image shows that AIE&CO@G3DSP is a spherical structure with uniform size and good dispersibility. The particle size and potential of the four nanogels are shown in the figure. Figure 2 shown.

[0092] Determination of drug loading of AIE&CO@G3DSP: The blank nanogel (G3DSP) prepared by chemical cross-linking has a rich three-dimensional network structure inside and is a drug carrier with excellent performance. In order to calculate the loading amount of AIE in AIE&CO@G3DSP, Figure 3 As shown, I first determined the relationship between AIE absorbance and concentration, and calculated the standard curve of AIE. The AIE&CO@G3DSP material is 10 mg, and 0.1 mg is taken to prepare a solution with a concentration of 0.1 mg / mL. The average absorption value at 515 nm is 0.734, and the concentration calculated by substituting it into the formula is 5.6 μg / mL, so the AIE loading amount is 5.1%, and the AIE loading rate is 56.3%. The AIE@G3DSP material is 10 mg, and 0.1 mg is taken to prepare a solution with a concentration of 0.1 mg / mL. The average absorption value at 700 nm is 0.83, and the concentration calculated by substituting it into the formula is 6.3 μg / mL, so the AIE loading amount is 5.7%, and the AIE loading rate is 63.1%. Similarly, the AIE loading amount in AIE@G3DSP is 5.7%, and the AIE loading rate is 63.1%.

[0093] In order to calculate the loading amount and loading rate of CORM-401 in AIE&CO@G3DSP. ICP-OES was used to detect the concentration of Mn ions in the solution. According to elemental analysis, the Mn ion concentration in 1mL of 1mg / mL AIE&CO@G3DSP solution is 0.25352mg / L×100=25.352mg / L. According to concentration×volume=solute mass, the mass of Mn ions in the solution is calculated to be 0.025352mg, that is, 1mgAIE&CO@G3DSP contains 0.025352mg of Mn ions, so 16mg of AIE&CO@G3DSP material contains 0.4056mg of Mn ions. The relative atomic mass of Mn ion is 54.93, the relative molecular mass of CORM-401 is 274.89, the molar amount of Mn ion and CORM-401 is equal, the mass of CORM-401 in 16mgAIE&CO@G3DSP = (mass of Mn ion / relative atomic mass of Mn ion) × relative molecular mass of CORM-401 = (331.2mg / 54.93) × 0.4056 = 2.446mg. Encapsulation amount = mass of CORM-401 in AIE&CO@G3DSP / mass of AIE&CO@G3DSP × 100% = 2.446mg / 16 × 100% = 15.3%. Encapsulation rate = mass of CORM-401 in AIE&CO@G3DSP / mass of CORM-401 × 100% = 2.446mg / 5 × 100% = 48.9%. Similarly, the loading amount of CORM-401 in CO@G3DSP is 14.8%, and the loading rate is 44.4%. The loading amount and loading rate of CORM-401 and AIE in the four nanogels are shown in Figure 2. Figure 4 shown.

[0094] CO release behavior of AIE&CO@G3DSP: The CO release situation within 60 minutes can be obtained through the records of the carbon monoxide gas detector, such as Figure 5 As shown in the figure, under white light irradiation, AIE&CO@G3DSP generated 2μmol of CO, and the CO@G3DSP group released 0μmol of CO within 60min. From the above, it can be inferred that AIE can promote the release of CO. In addition, AIE&CO@G3DSP does not release CO in the absence of light and CO@G3DSP does not release CO in the presence of light, which proves that AIE&CO@G3DSP is stable enough under in vitro conditions. CO will only be released when light and AIE are present at the same time, which proves the controllability of CO release. In summary, AIE&CO@G3DSP has a high response to white light that triggers CO release.

[0095] CO release mechanism of AIE&CO@G3DSP: In order to explore the release mechanism of CO under the action of light and AIE, we conducted the following experiment. The generation of H2O2 within 6 minutes was measured by hydrogen peroxide fluorescent probe. Figure 6 As shown in the figure, under white light irradiation, the AIE&CO@G3DSP group released 50μmol / L of H2O2 within 6 minutes; the AIE@G3DSP group released 25μmol / L of H2O2 within 6 minutes; and the CO@G3DSP group did not contain AIE, so the amount of H2O2 released was 0. This shows that the difference in the generation of H2O2 between AIE&CO@G3DSP and AIE@G3DSP is due to the generation of CO, that is, H2O2 promotes the generation of CO;

[0096] In order to further verify the dependence of CO release from AIE&CO@G3DSP on H2O2, we mixed AIE&CO@G3DSP with H2O2 of different concentrations and measured the CO release. Figure 7 As shown in the figure, in a H2O2 mixed solution with a concentration of 1mM, the CO release amount reached 1.5μmol in 60min; in a H2O2 mixed solution with a concentration of 100μM, the CO release amount reached 0.75mol; in a H2O2 mixed solution with a concentration of 50μM, the CO release amount reached 0.25μmol; and there was no CO release in H2O. This shows that the release of CO depends on the concentration of H2O2. It is known that there is no H2O2 in the normal environment of the human body, which shows that AIE&CO@G3DSP is very stable in the human body and will not produce side effects.

[0097] The generation of ROS within 60 minutes was detected by reactive oxygen species fluorescence probe. Figure 8 As shown in the figure, under the irradiation of white light, the release of AIE&CO@G3DSP was almost completed within 3 minutes, and the relative fluorescence intensity reached 70; the relative fluorescence intensity of ROS released by the AIE@G3DSP group was 80; the CO@G3DSP group did not contain AIE, so 1 The release of O2 was 0. 1 The release of O2 is almost the same, so the difference in ROS release is mainly caused by H2O2, which also indirectly proves that H2O2 is an important factor in inducing CO release.

[0098] In summary, the generation of H2O2 by AIE under white light irradiation is the key to stimulate CO release, such as Fig. 9 .

[0099] In summary, the particle size of AIE&CO@G3DSP is about 259nm, and the potential is 23.4mV. Transmission electron microscopy observation shows that AIE&CO@G3DSP is a spherical structure similar to nanogel, with good dispersibility and stability in aqueous solution; AIE&CO@G3DSP has efficient photodynamic ability and CO release behavior; AIE&CO@G3DSP can quickly release CO under the drive of photodynamics, and the release of CO will not affect the photodynamic behavior, which provides an important prerequisite for the two to achieve synergistic antibacterial effects.

[0100] In order to explore the optimal anti-MRSA and PA concentration of AIE&CO@G3DSP, Fig.10 and 11 As shown in the figure, four concentration gradients were set. Under the irradiation of white light, it was found that the antibacterial effect increased exponentially with the increase of concentration. For MRSA bacteria, the antibacterial rate of 10μg / mLAIE&CO@G3DSP reached 95.1%, the antibacterial rate of 25μg / mLAIE&CO@G3DSP was 99.71%, the antibacterial rate of 50μg / mLAIE&CO@G3DSP was 99.998%, and the bactericidal rate of 100μg / mLAIE&CO@G3DSP was almost 100%. There was almost no bacterial colonization on the agar plate and all of them were killed. Compared with PA bacteria, the antibacterial rate of 10μg / mLAIE&CO@G3DSP reached 99.59%, the antibacterial rate of 25μg / mLAIE&CO@G3DSP reached 99.90%, the antibacterial rate of 50μg / mLAIE&CO@G3DSP reached 99.995%, and the bactericidal rate of 100μg / mLAIE&CO@G3DSP almost reached 100%. Therefore, 100μg / mLAIE&CO@G3DSP was finally selected as the best antibacterial concentration. G3DSP, CO@G3DSP and un-illuminated AIE&CO@G3DSP groups also had certain and similar antibacterial activity compared with the PBS group, because the dendritic lysine-rich nanogel enhanced the ability to associate with bacteria. It is worth noting that the antibacterial rate of 100μg / mL AIE@G3DSP against MRSA also reached 99.98%, while the antibacterial rate against PA was only 84.94%. This is attributed to the unique cell wall structure of Gram-negative bacteria. AIE@G3DSP cannot enter the bacteria and the antibacterial effect is greatly weakened, which indirectly highlights the excellent synergistic antibacterial properties of AIE&CO@G3DSP.

[0101] For the optimal illumination time of AIE&CO@G3DSP synergistic antibacterial, such as Fig.12 and 13As shown in the figure, the fixed concentration is 100 μg / mL, and 4 illumination time gradients are set. Under the irradiation of white light, the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 0.5 min for MRSA bacteria is 99.58%, the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 1 min is 99.97%, the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 2 min is 99.998%, and the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 3 min is almost 100%. The sterilization rate of the AIE&CO@G3DSP group with an illumination time of 0.5 min for PA bacteria is 92.09%, the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 1 min is 99.82%, the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 2 min is 99.95%, and the sterilization rate of the AIE&CO@G3DSP group with an illumination time of 3 min is almost 100%. The antibacterial rate of AIE@G3DSP against Gram-positive bacteria MRSA reached 99.997% after irradiation for 3 minutes, while for Gram-negative bacteria PA, a large number of bacteria still survived and the antibacterial rate was only 69.5%. These results further indicate that PDT and CO have excellent synergistic antibacterial effects.

[0102] like Fig.14 and 15 As shown in the figure, the antibacterial activity of AIE&CO@G3DSP against S.aureus, MRSA, E.coli, AB and PA was investigated by plate counting method. The material concentration was set to 100μg / mL, and the illumination time was 3min. The antibacterial effects of PBS, G3DSP, CO@G3DSP, AIE@G3DSP, AIE&CO@G3DSP and non-illumination group AIE&CO@G3DSP on S.aureus, MRSA, E.coli, AB and PA were investigated respectively. Under illumination conditions, AIE&CO@G3DSP had almost no bacterial residues on the agar plate, whether for Gram-positive bacteria S.aureus and MRSA, or for Gram-negative bacteria E.coli, AB and PA, and the antibacterial efficiency reached 100%. However, the AIE@G3DSP group can clearly see that there are still a large number of bacterial tribes, especially for Gram-negative bacteria E.coli, AB and PA, the antibacterial rates are only 88%, 99.4% and 84.94%. These results further illustrate the excellent antibacterial properties of AIE&CO@G3DSP and the outstanding synergistic antibacterial ability of PDT and CO against bacteria.

[0103] We mainly observe the morphology of bacteria by SEM and TEM, such as Fig.16 and 17After PBS treatment, the surfaces of the two bacteria were smooth and round, and the structural morphology remained intact without any damage. After G3DSP, CO@G3DSP and non-illumination AIE&CO@G3DSP treatment, the material can be seen adhering to the bacterial surface, wrinkles appeared on the bacterial surface, and a small number of bacteria were ruptured, indicating that the lysine-rich nanogel system enhanced the association effect between the material and bacteria and had certain antibacterial properties. After AIE@G3DSP treatment, a large number of MRSA bacteria were ruptured, serious wrinkles appeared on the surface, the cell wall was destroyed, and the cytoplasm leaked. However, for PA, although a large amount of material can be seen adhering to the surface, the integrity of the bacteria is still maintained, indicating that the unique cell wall structure of Gram-negative bacteria seriously affects AIE@G3DSP. However, after AIE&CO@G3DSP treatment, the cell morphology of both MRSA and PA was completely deformed and ruptured, accompanied by a large amount of cytoplasm leakage, which proves that AIE&CO@G3DSP not only retains the excellent antibacterial activity of PDT against Gram-positive bacteria, but also has strong antibacterial activity in the face of the unique cell wall barrier of Gram-negative bacteria under the synergistic effect of CO.

[0104] The hemolytic and cytotoxic properties of nanomaterials can reflect whether the materials can be used in vivo. Fig.18 As shown in (a, b), when the concentration of the nanomaterial gradually increased from 10μg / mL to 100μg / mL, the hemolysis rate also gradually increased. However, even at the highest concentration of 100μg / mL, the hemolysis rate of AIE&CO@G3DSP was less than 5%, proving that AIE&CO@G3DSP has low hemolysis and good biocompatibility. The cytotoxicity of AIE&CO@G3DSP was explored by CCK-8 experiment. Fig.18 As shown in (c, d), when the material concentration increases from 100μg / mL to 300μg / mL, the cell survival rate can be maintained above 80%. When the material concentration is 100μg / mL, the cell survival rate is basically around 90%. This proves that the material has low cytotoxicity, does not cause much harm to normal cells, and has good biocompatibility.

[0105] Animal Experimentation:

[0106] In this experiment, all animal experiments were performed in accordance with the requirements of the Animal Care Committee of Nanjing Tech University. The experimental groups were: PBS group, G3DSP (1 mg / mL), CO@G3DSP (1 mg / mL), AIE@G3DSP (1 mg / mL), AIE&CO@G3DSP (1 mg / mL) and non-light group AIE&CO@G3DSP (1 mg / mL), with 5 parallel samples in each group.

[0107] Since biofilms protect bacteria from harsh environments, bacterial infections caused by bacterial biofilms are more difficult to eradicate. While AIE&CO@G3DSP showed excellent biofilm ablation performance in vitro, we further evaluated its ability to remove biofilms in vivo by using a subcutaneously embedded biofilm catheter model. Fig.19 (a) The experimental scheme of the in vivo biofilm catheter model from establishment to treatment and finally to the evaluation of biofilm ablation effect is shown. PA biofilm was first cultured on the catheter and implanted subcutaneously on the left side of the mouse spine. AIE&CO@G3DSP was injected in situ 30 minutes later and then irradiated with white light for 3 minutes. Three days later, the implanted catheter and tissue were collected for digital camera photography, bacterial counts, SEM observations, and H&E and Masson staining of wound skin tissue.

[0108] Fig.19 (b) shows the skin tissue at the subcutaneous catheter implantation site after treatment. Inflammation and skin decay are visible to the naked eye in the PBS, G3DSP, and AIE@G3DSP groups, and the inflammation site is large, indicating that the wound has not healed on its own. The CO@G3DSP and AIE&CO@G3DSP groups without light have scarred, and the relative wound area is smaller, indicating that CO has a certain anti-inflammatory effect. The AIE&CO@G3DSP group not only has efficient sterilization, but also has excellent anti-inflammatory effects, as shown by the skin remaining smooth, without large-scale decay, and only healing scars at the modeled wound.

[0109] At the same time, take a photo after the catheter is removed after treatment. Fig.19 As shown in (c), the results show that after treatment with AIE&CO@G3DSP plus light, there is almost no biofilm residue and mucus adhesion secreted by the wound on the catheter surface, and the treatment effect is outstanding.

[0110] Fig. 20 (a) It can be clearly seen that after 3 days of treatment, there are almost no residual bacteria on the agar plate of the AIE&CO@G3DSP group, which further confirms the excellent in vivo biofilm ablation effect of AIE&CO@G3DSP. Fig. 20 (b) Quantitative analysis of bacterial residues on the catheter showed that the number of bacteria in the G3DSP group was 87.29% of that in the PBS group, the number of bacteria in the CO@G3DSP group was 82.2% of that in the PBS group, and the number of bacteria in the AIE@G3DSP group was 72.03% of that in the PBS group. The number of bacteria after AIE&CO@G3DSP plus light treatment was only 0.00015% of that in the PBS group. The results showed that the synergistic treatment effect was much greater than that of the single treatment effect, proving the excellent anti-biofilm effect of AIE&CO@G3DSP.

[0111] In order to more intuitively observe the effect of AIE&CO@G3DSP on ablating biofilm, the surface morphology of the catheter removed from the mouse was observed by SEM. Fig.21 As shown in the figure, after treatment with PBS, G3DSP, AIE@G3DSP and un-illuminated AIE&CO@G3DSP, the catheter surface was still covered with a large number of bacteria and biofilms, as well as a large number of inflammatory cells. However, after treatment with AIE&CO@G3DSP, the biofilm was almost completely eliminated and the catheter surface was smooth and flat, which confirmed the excellent anti-biofilm effect of AIE&CO@G3DSP.

[0112] H&E staining was used to detect the generation of inflammatory cells in the wound site. Fig. 22 As shown in (a), after treatment with AIE&CO@G3DSP, almost no inflammatory cells were observed, and there was no sign of tissue damage. Fig. 22 (b) Through Masson staining of collagen, it can be intuitively seen that a large amount of collagen is deposited after treatment with AIE&CO@G3DSP, proving that AIE&CO@G3DSP has the effect of promoting wound healing. Through the detection of inflammatory factors IL-6, IL-1β and TNF-α in the skin tissue of the infected site after treatment with antibacterial nanogel materials, it can be observed that after three days of treatment, the inflammatory factors in the AIE&CO@G3DSP+Light group were significantly reduced. This indirectly shows that the antibacterial nanogel AIE&CO@G3DSP in vivo has a good immune response and anti-inflammatory effect.

[0113] In view of the excellent in vitro antibacterial effect of AIE&CO@G3DSP, we then used the MRSA-infected subcutaneous abscess model to investigate the in vivo antibacterial effect of AIE&CO@G3DSP. Fig.23 The treatment scheme of the mouse abscess model is shown. One day before administration, 100 μL of MRSA bacterial solution was injected subcutaneously into the mouse to construct the abscess model. 100 μL of AIE&CO@G3DSP was injected in situ, followed by irradiation with white light for 3 minutes. Finally, the therapeutic effect was observed by colony counts and inflammatory cells on days 2 and 10.

[0114] like Fig.24The figure shows the healing of mouse wounds after treatment with different materials. It can be clearly seen that after treatment with PBS, the pustules of mice began to scab on the 4th day, but there was still a protruding abscess under the skin that did not disappear until the tenth day; after treatment with G3DSP, CO@G3DSP and AIE&CO@G3DSP without light, although scabs began to form on the second day, there were still bacterial abscesses under the skin on the 10th day, which shows that the treatment effect of G3DSP, CO@G3DSP and AIE&CO@G3DSP without light was not ideal. After treatment with AIE@G3DSP, the wound healing speed was faster than that of CO@G3DSP, but there was still a wound and a protruding abscess under the skin on the 10th day, indicating that there was still a small amount of bacteria remaining in the wound site; after treatment with AIE&CO@G3DSP, the wound healing speed was very fast, and the existence of the wound was basically invisible on the 10th day, and the wound site was flat and smooth, which further proved that AIE&CO@G3DSP also has strong antibacterial ability and promotes wound healing in vivo.

[0115] like Fig.25 After treatment with different materials, the antibacterial activity of AIE&CO@G3DSP was explored by qualitative and quantitative analysis of the number of colonies in the abscess wound. The results showed that after treatment with AIE&CO@G3DSP, the number of residual bacteria was the least on the 10th day, and the antibacterial rate was as high as 99.97%. These results clearly show that the combination of PDT and CO can effectively eliminate bacteria in infected wounds.

[0116] H&E staining was used to observe the infiltration of inflammatory cells in the wound. Fig.26 (a) and Fig.26 As can be seen in (b). After 2 days of administration, inflammatory cells were basically not seen in the AIE&CO@G3DSP group, proving that the material has an immediate and efficient bactericidal effect. After 10 days, the wound was basically completely recovered, proving that AIE&CO@G3DSP has the ability to effectively fight bacteria and accelerate wound healing. Fig.26 In (c), we can see that the collagen deposition in the AIE&CO@G3DSP group is the highest, which also proves that AIE&CO@G3DSP has the effect of promoting wound healing. As we all know, the wound repair process is accompanied by the repair and regeneration of microvessels. CD31 antibody is used to mark the microvessels in the wound site. Fig.26 (d) It can be seen that the number of microvessels in the wound of mice treated with AIE&CO@G3DSP increased significantly. In summary, the MRSA subcutaneous abscess infection model verified that AIE&CO@G3DSP has high antibacterial, anti-inflammatory and wound healing effects.

[0117] In order to investigate whether AIE&CO@G3DSP has the potential to cause CO poisoning, we detected the concentration of carboxyhemoglobin in the blood of mice after treatment. Fig. 27 As shown in (a), after the treatment, the CO@G3DSP group, AIE&CO@G3DSP group, and the AIE&CO@G3DSP group without light treatment had a relatively high concentration of carboxyhemoglobin in the blood of mice, but there was no statistical significance between the data of the several groups, which shows that the trace amount of CO produced by the material will not cause toxic side effects to mice. In order to investigate the effect of the AIE&CO@G3DSP treatment process on mice, we weighed the mice every day and drew the weight growth curve as follows Fig. 27 (b) It was found that the weight of mice grew normally within 10 days, indicating that AIE&CO@G3DSP did not affect the normal growth of mice.

[0118] In addition, we also investigated the effects of AIE&CO@G3DSP on important organs, such as Fig.28 As shown, no signs of inflammation and tissue necrosis were observed in the heart, liver, spleen, lung, and kidney of mice after treatment, which is consistent with the situation of normal tissues. This shows that AIE&CO@G3DSP will not cause side effects on important organs and proves that it is also biosafe in vivo.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a synergistic antibacterial nanoplatform based on CO and AIEgens, characterized in that: The steps include: Step 1: The third-generation lysine dendrimer G3-Lys with polyhedral oligomeric silsesquioxane POSS as the core and lysine as the branching unit is cross-linked with a cross-linking agent disulfide bis (succinimidyl propionate) DSP to form a blank gel G3DSP; Step 2: Dissolve 100 mg of G3DSP in 10 mL of water to form a G3DSP solution; Step 3: Dissolve 10 mg of AIEgens in 1 mL of DMSO to prepare an AIE solution, and dissolve 50 mg of CORM-401 in 1 mL of anhydrous methanol to prepare a CORM-401 solution; Step 4: Add CORM-401 solution to the G3DSP solution under the protection of light and nitrogen, stir for 2 h, then add AIE solution and continue the reaction for 22 h; Step 5: After the solution reaction in step 4 is completed, place it in a dialysis bag and dialyze it in a deionized water solution for 24 hours; Step 6: freeze-dry the solution obtained in step 5 to obtain a rose red flocculent solid product AIE&CO@G3DSP; The specific implementation steps of step one include: a. Dissolve 1.00 g, 0.12 mmol of G3-Lys and 0.10 g, 0.25 mmol of DSP in 10 mL and 5 mL of anhydrous DMF, respectively, to obtain a G3-Lys solution and a DSP solution; b. Then slowly add the DSP solution dropwise into the G3-Lys solution under stirring; The solution obtained in step b was stirred at room temperature, and after 24 hours, dialyzed in DMF for 4 hours, and then dialyzed in pure water for 24 hours, and then freeze-dried to obtain the product G3DSP; In step 1, the interior of the blank gel G3DSP is a three-dimensional network structure.

2. The method for preparing a synergistic antibacterial nanoplatform based on CO and AIEgens according to claim 1, characterized in that: In step 5, the molecular weight cut-off of the dialysis bag is 1000 Da.

3. A synergistic antibacterial nanoplatform based on CO and AIEgens, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 2.

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