A targeted antibacterial nanoliposome preparation and its preparation method

Abstract

A nanoliposome preparation, which is a liposome prepared with distearoylphosphatidylglycerol (DSPG), cholesterol and Biotin‑PEG3000‑cholesterol as a carrier, is surface-modified with ICAM‑1 monoclonal antibody, and is loaded with an AIE photosensitizer and Nicorandil. The nanoliposomes prepared by the present invention have targeted and responsive drug delivery to infected lesions, improve the enrichment of drugs at bacterial infection sites, and the components of the nanosystem can be cleaved by phospholipases in the infected microenvironment, which can achieve responsive release of drugs at bacterial infection sites and enhance the therapeutic effect. The present invention utilizes the hydrophobic shell and hydrophilic core of the liposome, and simultaneously loads hydrophobic NO donor molecules and hydrophilic AIE photosensitizers to achieve the synergistic treatment of bacterial infections with "photodynamic therapy" and "gas therapy", and has a better killing effect on drug-resistant bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine preparation, and in particular to a targeted antibacterial nanoliposome preparation and a preparation method thereof. Background Art

[0002] In the current medical field, diseases and deaths caused by bacterial infections are one of the most challenging natural threats facing humanity, and the use of antibiotics to inhibit or kill bacteria is a conventional treatment strategy. However, the misuse of antibiotics leads to bacterial resistance, which not only increases the risk of complications and mortality in patients, but also increases public health care costs. In addition, bacteria adhere to surfaces to form biofilms, further increasing the difficulty of treatment. Faced with the increasingly serious threat of drug-resistant bacteria and biofilm infections, there is an urgent need to develop new drugs and new therapies that can replace antibiotics to treat infections caused by drug-resistant pathogens.

[0003] Photodynamic therapy (PDT) is a novel antibacterial approach that has recently developed. Photosensitizers generate reactive oxygen species (ROS) through photodynamic reactions, effectively killing pathogens. This therapy has become one of the most promising new antibacterial therapies due to its high antibacterial efficacy, non-invasive nature, resistance resistance, and strong spatiotemporal selectivity. However, traditional organic photosensitizers experience fluorescence quenching and reduced ROS production in aqueous environments and during nanoformation. The use of photosensitizers with aggregation-induced emission (AIE) properties can effectively address this issue and enhance the efficacy of photodynamic therapy. However, the uncontrolled consumption of ROS by high levels of glutathione (GSH) in the bacterial infection microenvironment results in unsatisfactory antibacterial efficacy of PDT alone. Therefore, the combined use of GSH-depleting agents and AIE photosensitizers for synergistic antibacterial activity is a promising new therapeutic approach. Nitric oxide (NO), a unique bioactive molecule, can promote GSH depletion. Furthermore, NO reacts with ROS generated by PDT to generate reactive nitrogen species (RNS), which have even stronger bactericidal potential. Therefore, GSH-responsive NO donors can be selected for synergistic antibacterial effects in combination with AIE photosensitizers. However, as small molecule formulations, NO donors and AIE photosensitizers suffer from shortcomings such as poor targeted enrichment at the site of infection, rapid clearance from the body, low bioavailability, and high toxicity and side effects. Therefore, developing a delivery system that can effectively and responsively deliver both drugs to the site of infection simultaneously is a major challenge. Summary of the Invention

[0004] The purpose of the present invention is to provide a dual-therapy synergistic antibacterial liposome nanoformulation that targets and responds to the infection microenvironment. The preparation is a liposome that is simultaneously loaded with a water-soluble AIE photosensitizer and a fat-soluble NO donor, and introduces an ICAM monoclonal antibody with a targeted effect. It can achieve a dual-therapy synergistic antibacterial effect through targeted delivery to the infection site and responsive release of drugs using photodynamic / gas therapy.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned antibacterial liposome nanoformulation. This method effectively solves the problem that ICAM monoclonal antibodies cannot attach to the liposome surface for modification, or the monoclonal antibodies are inactivated after attachment, making it difficult to achieve targeting.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] A nanoliposome preparation, characterized in that: the nanoliposome preparation is a nano preparation using liposomes prepared with distearoylphosphatidylglycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol as a carrier, with ICAM-1 monoclonal antibody modified on the surface, and loaded with an AIE photosensitizer and nicorandil.

[0008] Furthermore, the nanoparticle preparation is a spherical structure with a particle size range of 100-150 nm.

[0009] Furthermore, the nanoformulation is prepared by first preparing a lipid membrane loaded with nicorandil using distearoylphosphatidylglycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol, then loading the AIE photosensitizer to form liposomes, adding avidin and then mixing with biotinylated ICAM-1 monoclonal antibody to obtain the nanoformulation.

[0010] Furthermore, the ICAM-1 was biotinylated by diluting the ICAM-1 antibody with sodium bicarbonate buffer (pH 8.0) and dialyzing to obtain an antibody solution, dissolving N-hydroxysuccinimide biotin (NHSB) with DMSO to obtain an NHSB solution, adding the NHSB solution to the antibody solution, stirring continuously at room temperature, and incubating for 2 to 4 hours. An NH4Cl solution was added, and stirring was continued at room temperature for 10 minutes. After removing free biotin, the solution was passed through a molecular sieve column and slowly eluted with PBS to collect the biotinylated ICAM-1.

[0011] Furthermore, the concentration of the sodium bicarbonate buffer is 0.08-0.12 mol / L, and the concentration of the antibody solution after dilution and dialysis is 0.8-1.2 mg / mL.

[0012] Furthermore, the concentration of the NHSB solution is 0.85-1.2 mg / mL, and the volume ratio of the antibody solution to the NHSB solution is 1:0.1-0.14.

[0013] Furthermore, the concentration of the NH4Cl solution is 1 mol / L, and the volume ratio of the NH4Cl solution to the NHSB solution is 12-13:1.

[0014] A method for preparing a targeted antibacterial nanoliposome preparation is characterized by preparing a lipid membrane loaded with nicorandil using distearoylphosphatidylglycerol (DSPG), cholesterol, and biotin-PEG3000-cholesterol, adding an AIE photosensitizer solution for loading to obtain a liposome solution, then adding avidin for reaction, and then adding biotinylated ICAM-1 monoclonal antibody for further reaction to obtain targeted dual-drug loaded liposomes, denoted as AN@ALips.

[0015] Furthermore, the lipid membrane loaded with Nicorandil is prepared by distearoylphosphatidylglycerol (DSPG), cholesterol, Biotin-PEG3000-cholesterol and Nicorandil in chloroform to form a mixed solution, which is then evaporated under reduced pressure to form a lipid membrane.

[0016] Furthermore, the molar ratio of DSPG, cholesterol, Biotin-PEG3000-cholesterol and Nicorandil is 2:0.8:0.15-0.25:1.2-1.4.

[0017] Furthermore, the AIE solution is loaded into the lipid membrane by water bath stirring and ultrasonic hydration. The water bath stirring temperature is 20~30°C, the stirring speed is 180~220rpm, the ultrasonic hydration temperature is 40~45°C, the ultrasonic power is 100~150W, and the ultrasonic time is 12~15min.

[0018] Furthermore, the AIE solution is obtained by mixing the AIE photosensitizer TTVP with normal saline in a ratio of 2 mg: 1-1.2 mL.

[0019] Furthermore, avidin is added according to a molar ratio of avidin to Biotin-PEG3000-cholesterol in the liposome of 2.5-3.5:2. After adding avidin, the mixture is stirred at 800-1000 rpm for 4-8 minutes and then centrifuged.

[0020] Furthermore, the liposomes were resuspended in physiological saline to a resuspension with a mass-to-volume ratio of 2 mg:1 mL, and a biotinylated ICAM-1 monoclonal antibody solution with a concentration of 0.1 mg / L was added. The mixture was stirred at 800-1000 rpm for 4-8 minutes and then centrifuged. The volume ratio of the biotinylated ICAM-1 monoclonal antibody solution to the resuspension was 1:1.

[0021] Furthermore, the biotinylated ICAM-1 monoclonal antibody is prepared by diluting the ICAM-1 antibody with sodium bicarbonate buffer (pH 8.0) and dialyzing to obtain an antibody solution, dissolving N-hydroxysuccinimide biotin (NHSB) with DMSO to obtain an NHSB solution, adding the NHSB solution to the antibody solution, continuously stirring at room temperature, and incubating for 2 to 4 hours. An NH4Cl solution is added, and stirring is continued at room temperature for 10 minutes. After removing free biotin, the solution is passed through a molecular sieve column and slowly eluted with PBS to collect the biotinylated ICAM-1.

[0022] Furthermore, the concentration of the sodium bicarbonate buffer is 0.08-0.12 mol / L, and the concentration of the antibody solution after dilution and dialysis is 0.8-1.2 mg / mL.

[0023] Furthermore, the concentration of the NHSB solution is 0.85-1.2 mg / mL, and the volume ratio of the antibody solution to the NHSB solution is 1:0.1-0.14.

[0024] Furthermore, the concentration of the NH4Cl solution is 1 mol / L, and the volume ratio of the NH4Cl solution to the NHSB solution is 12-13:1.

[0025] Among numerous nanocarrier materials, liposomes are highly attractive due to their excellent biocompatibility, low immunogenicity, ability to simultaneously load both hydrophilic and lipophilic drugs, and ease of surface modification. The flexible and adjustable formulation of nanoliposomes makes them ideal for the preparation of targeted and responsive delivery vehicles. The bacterial infection microenvironment is characterized by localized acidification, elevated levels of reduced GSH, overexpressed phospholipases, and upregulated expression of the intercellular adhesion molecule (ICAM-1) on endothelial cells. Therefore, using ICAM-1 as a target for the phospholipase response at the infection site, a nanodelivery system modified with an ICAM-1 monoclonal antibody can achieve targeted and responsive delivery of two drugs. However, during the preparation process, effective modification of the liposomes with the ICAM-1 monoclonal antibody is difficult, hindering targeted and responsive drug delivery.

[0026] In the present invention, Biotin-PEG3000-cholesterol of a specific molecular weight is selected to participate in the preparation of liposomes during the liposome preparation process. Through the combination of avidin and biochemically treated ICAM-1 monoclonal antibody and the steric hindrance synergistic effect of Biotin-PEG3000-cholesterol, the introduction efficiency of the ICAM-1 monoclonal antibody is improved, so that the ICAM-1 monoclonal antibody is effectively attached to the liposome surface to form a modification, thereby achieving targeted delivery of the liposome nanoformulation, improving the enrichment of the drug at the bacterial infection site, and achieving responsive release of the drug at the bacterial infection site through the shearing effect of phospholipases in the bacterial infection microenvironment on the liposome, thereby improving the therapeutic effect.

[0027] The NO donor Nicorandil is released from the liposomes and reacts with GSH to generate NO. The reduction of GSH can inhibit the consumption of POS. The generated NO reacts with the ROS produced by PDT to generate reactive nitrogen RNS with strong bactericidal ability.

[0028] A method for preparing a targeted antibacterial nanoliposome preparation, characterized by comprising the following steps:

[0029] S1. Distearoylphosphatidylglycerol (DSPG), cholesterol, Biotin-PEG3000-cholesterol, and nicorandil were dissolved and mixed in chloroform at a molar ratio of 2:0.8:0.15-0.25:1.2-1.4 to obtain a mixture, and the mixture was subjected to vacuum rotary evaporation to form a film;

[0030] S2. Water-soluble TTVP was mixed with normal saline at a ratio of 2 mg:1-1.2 mL to obtain a photosensitizer solution. The photosensitizer solution was added to the membrane prepared in step S1, and the membrane was stirred in a water bath and ultrasonically hydrated, followed by dialysis to obtain a liposome solution. The water bath stirring temperature was 20-30°C, the stirring speed was 180-220 rpm, the ultrasonic hydration temperature was 40-45°C, the ultrasonic power was 100-150 W, and the ultrasonic time was 12-15 min.

[0031] S3. The liposome solution in step S2 was extruded using a liposome extruder, and avidin was added to the extruded solution at a molar ratio of avidin: Biotin-PEG3000-cholesterol = 3:2, stirred at 800~1200 rpm for 4~8min, and then centrifuged to collect the solid;

[0032] S4. The solid collected in S3 was resuspended in physiological saline to form a resuspension (mass-to-volume ratio of 20:1 (mg / ml)), and a biotinylated ICAM-1 monoclonal antibody solution with a mass concentration of 0.1 mg / kg was added. The volume ratio of the biotinylated ICAM-1 monoclonal antibody solution to the resuspension was 1:1. The mixture was stirred at 800-1200 rpm for 4-8 minutes, and then the solid was further collected by centrifugation to obtain a targeted antibacterial liposome nanoformulation. The biotinylated ICAM-1 was prepared by diluting the ICAM-1 antibody to 1 mg / mL with 0.08-0.12 mol / L sodium bicarbonate buffer (pH 8.0). The protein was fully dialyzed with 0.08-0.12 mol / L sodium bicarbonate buffer (pH 8.0), and then lysed with DMSO. Dissolve N-hydroxysuccinimide biotin (NHSB) to obtain a 0.85-1.2 mg / mL NHSB solution. Mix the antibody solution and NHSB solution at a volume ratio of 1:0.1-0.14. Stir continuously at room temperature for 2-4 hours. Add 1 mol / L NH4Cl at a volume ratio of 12-13:1 between NH4Cl and NHSB solution. Stir at room temperature for 10 minutes. Dilute thoroughly against PBS at 4°C to remove free biotin. Load the sample onto a 1 ml molecular sieve column and slowly elute with PBS, collecting 1 ml per tube.

[0033] Water-soluble TTVP is a compound containing tetraphenylethylene, triphenylamine, ethylene and benzene rings, and these structural units together constitute AIE materials.

[0034] The present invention has the following technical effects:

[0035] The present invention uses Biotin-PEG-cholesterol, cholesterol and DSPG that can be cleaved by phospholipase as nanoliposome components, as well as avidin-biotin interaction, introduces monoclonal antibodies on the surface of the nanosystem, and gives the nanosystem targeted and responsive drug delivery to the infected lesions, thereby improving the enrichment of drugs at the bacterial infection site. The components of the nanosystem can be cleaved by phospholipases in the infected microenvironment, which can achieve responsive release of drugs at the bacterial infection site and enhance the therapeutic effect. The present invention utilizes the hydrophobic shell and hydrophilic core of the liposome, and simultaneously loads hydrophobic NO donor molecules and hydrophilic AIE photosensitizers to achieve the synergistic treatment of bacterial infections with "photodynamic therapy" and "gas therapy", which has a better killing effect on drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Transmission electron microscope image of AN@ALips in Example 2.

[0037] Figure 2: Transmission electron microscopy image of AN@ALips (prepared with high molecular weight Biotin-PEG-cholesterol) in Example 2.

[0038] Figure 3 : Particle size, zeta potential and storage stability of AN@ALips in Example 2.

[0039] Figure 4 : The PAGE image of AN@ALips in Example 2 verifies the coupling of the antibody.

[0040] Figure 5 : The expression of ICAM-1 in HUVECs after TNF-α treatment for different time periods in Example 3.

[0041] Figure 6 : Study on the cellular targeting of AN@ALips in Example 3.

[0042] Figure 7 : Fluorescence microscopy results of HUVECs pretreated with TNF-α treated with targeted liposomes containing different antibody contents (biotin-PEG-cholesterol: cholesterol) in Example 3.

[0043] Figure 8 : Evaluation of enzyme-responsive release of AN@ALips, GSH-responsive NO release, and GSH consumption in biofilms in Example 4.

[0044] Figure 9 : Evaluation of cytotoxicity and hemolytic activity of AN@ALips in Example 4.

[0045] Figure 10 : Colony growth of MRSA bacteria in Example 5 after being treated with PBS, N@ALips, A@ALips+Light and AN@ALips+Light.

[0046] Figure 11 : Scanning electron microscopy results of MRSA after treatment with different groups of AN@ALips in Example 5.

[0047] Figure 12 : Crystal violet staining results of MRSA biofilms treated with PBS, N@ALips, A@ALips+Light, and AN@ALips+Light in Example 5, respectively.

[0048] Figure 13 : The production of ROS, NO and RNS in the infected wound site after AN@ALips treatment in Example 6.

[0049] Figure 14: Fluorescence of various tissues in mice after tail vein injection of PBS, AN@Lips and AN@ALips in Example 6.

[0050] Figure 15 : Wound healing of infected tissues of mice in Example 6, and the infected tissues within one week after treatment with N@ALips, A@ALips+Light, and AN@ALips+Light, respectively.

[0051] Figure 16 : Results of bacterial counts in infected tissues of mice in Example 6, and in tissues after the infected tissues were treated with A@ALips+Light, N@ALips, and AN@ALips+Light, respectively.

[0052] Figure 17 : HE staining results of healthy skin tissue, infected tissue, and infected tissue of mice in Example 6 after being treated with A@ALips, A@ALips+Light, N@ALips, and AN@ALips+Light, respectively.

[0053] Figure 18 : Analysis results of blood biochemical indicators after tail vein injection of PBS, A@ALips, N@ALips and AN@ALips in mice in Example 7.

[0054] Figure 19 : HE staining results of various tissues after tail vein injection of PBS, A@ALips, N@ALips and AN@ALips in mice in Example 7. DETAILED DESCRIPTION

[0055] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0056] Example 1

[0057] A method for preparing a targeted antibacterial nanoliposome preparation comprises the following steps:

[0058] 1.1 Preparation of dual-drug loaded liposomes AN@Lips

[0059] Distearoylphosphatidylglycerol (DSPG), cholesterol, Biotin-PEG3000-cholesterol, and Nicorandil were dissolved in chloroform at a molar ratio of 2:0.8:0.2:1.3 to obtain a mixture, which was then subjected to vacuum rotary evaporation at a water bath temperature of 40°C and a rotation speed of 100 rpm to form a film. 30 mg of water-soluble TTVP was dissolved in 15 mL of normal saline and added to the prepared film. The film was stirred in a water bath at 200 rpm at 25°C for 5 min, then ultrasonically hydrated at 45°C at a power of 120 W for 15 min, and then dialyzed to obtain a liposome solution, i.e., the AN@Lips solution.

[0060] 1.2 Preparation of targeted dual-drug loaded liposomes AN@ALips

[0061] The liposome AN@Lips solution prepared in step 1.1 was extruded using a liposome extruder. Avidin was added to the extruded solution at a molar ratio of avidin: biotin-PEG3000-cholesterol = 3:2. The mixture was stirred at 1000 rpm for 5 minutes, then centrifuged to collect the solid, and resuspended with normal saline to form a resuspension with a mass volume ratio of 20 mg:1 mL. A biotinylated ICAM-1 monoclonal antibody solution with a mass concentration of 0.1 mg / kg was added, and the volume of the monoclonal antibody solution and the resuspension was 1:1. The mixture was stirred at 1000 rpm for 5 minutes, then further centrifuged to collect the solid to obtain the targeted antibacterial liposome nanoformulation AN@ALips. The biotinylated ICAM-1 was prepared by diluting the ICAM-1 antibody to be biotinylated to 1 mg / mL with 0.1 mol / L sodium bicarbonate buffer (pH 8.0), and the protein was fully dialyzed with 0.1 mol / L sodium bicarbonate buffer (pH 8.0). The protein was then diluted with 1 mL DMSO. Dissolve 1 mg of N-hydroxysuccinimide biotin (NHSB) and add 120 μL of NHSB solution (containing 120 μg of NHSB) to 1 mL of antibody solution (containing 1 mg of antibody). Stir continuously at room temperature and incubate for 2-4 hours. Add 9.6 μL of 1 mol / L NH4Cl solution and stir at room temperature for 10 minutes. Dilute thoroughly against PBS at 4°C to remove free biotin. Apply the solution to a 1 mL molecular sieve column and slowly elute with PBS. Collect 1 mL / tube to obtain biotinylated ICAM-1 antibody.

[0062] A@ALips loaded only with AIE photosensitizer and N@ALips loaded only with NO donor were prepared using the same method.

[0063] Example 2: Characterization of AN@ALips

[0064] 2.1 Observation of the morphology of AN@ALips using transmission electron microscopy (TEM)

[0065] Weigh an appropriate amount of AN@ALips and disperse it in physiological saline, drop 10 μL onto the carbon support film copper grid, wait for it to dry at room temperature and then observe. Figure 1 As shown in Figure 2, the prepared AN@ALips have good monodispersity and the particle size is about 100 nm. In addition, we used Biotin-PEG5000-cholesterol with a larger molecular weight to prepare dual-drug loaded liposomes according to the above method, and the electron microscopy results are shown in Figure 2. Figure 2 As shown, its uniformity and dispersibility deteriorated, and the average particle size increased to approximately 110 nm. We calculated the drug loading efficiency of liposomes prepared using Biotin-PEG3000-cholesterol and Biotin-PEG5000-cholesterol. The drug loading efficiency of liposomes prepared using Biotin-PEG3000-cholesterol for the AIE photosensitizer TTVP and NO donor was 18% and 9.83%, respectively, while the drug loading efficiency of liposomes prepared using Biotin-PEG5000-cholesterol was 11.16% and 5.91%, respectively. Overall, liposomes prepared using Biotin-PEG3000-cholesterol have a greater advantage.

[0066] 2.2 Dynamic Light Scattering (DLS) Detection of Particle Size, Zeta Potential, and Storage Stability of AN@ALips

[0067] Take an appropriate amount of AN@ALips solution and disperse it thoroughly for DLS analysis. Figure 3 As shown in Figure A, the particle size of the prepared AN@ALips is about 100 nm, which is consistent with the TEM results. Figure 3 As shown in B, the Zeta potential of AN@ALips is -25.1±0.3 mV. Figure 3 As shown in Figure C, AN@ALips can maintain its particle size well within one week. The above results show that the prepared AN@ALips are relatively uniform in size, with a moderate particle size, and contain PEG and negative surface charge, which is conducive to the long-term circulation of nanomedicines in vivo.

[0068] 2.3 Verification of Antibody Coupling by PAGE

[0069] Take an appropriate amount of AN@ALips solution, centrifuge to collect the solid, add RIPA lysis buffer containing PMSF, and lyse and extract the protein in an ice bath. The extracted protein solution is treated with 5x loading buffer, treated in a metal bath at 100℃ for 5 minutes, and loaded on PAGE electrophoresis. The results are as follows Figure 4As shown in the figure, the results of protein electrophoresis in liposomes were consistent with those of free antibodies, indicating that the antibody was successfully coupled to the liposome surface.

[0070] Example 3: Verification of AN@ALips targeting at the cell level

[0071] 3.1 Verification of upregulation of target protein ICAM-1 expression

[0072] Using vascular endothelial cells (HUVECs) as a cell model, the HUVEC cell concentration was 2 x 10 5 The cells were seeded in 6-well plates. When the cell confluence exceeded 80%, the inflammatory factor TNF-α (50 ng / ml) was added and incubated for 4 h or 8 h. The cells without TNF-α incubation served as the control group. The treated cells were collected to extract proteins, and the expression of ICAM-1 was studied by Western blotting. Figure 5 As shown, cells not treated with TNF-α expressed only a small amount of ICAM-1. However, cells treated with TNF-α showed a significant upregulation of ICAM-1 expression, indicating that ICAM-1 expression is upregulated on the surface of endothelial cells in the infected microenvironment, which facilitates the accumulation, binding, and penetration of targeted liposomes.

[0073] 3.2 Analysis of cellular targeting of AN@ALips using fluorescence microscopy

[0074] The HUVEC cells were plated at a concentration of 1 x 10 5 The cells were seeded in 12-well plates and cultured until the confluence reached more than 80%. They were divided into four experimental groups, namely, group one [-TNF-α (untreated), +AN@ALips (containing 5μM AIE photosensitizer) incubated for 2h]; group two [+TNF-α (50 ng / ml incubated for 8h), +ICAM-1 antibody pre-incubated for 1h, +AN@ALips (containing 5μM AIE photosensitizer) incubated for 2h]; group three [+TNF-α (50 ng / ml incubated for 8h), +AN@Lips (containing 5μM AIE photosensitizer) incubated for 2h]; group four [+TNF-α (50 ng / ml incubated for 8h), +AN@ALips (containing 5μM AIE photosensitizer) incubated for 2h]. Cell nuclei were stained with DAPI dye. After treatment, the cells were observed under a fluorescence microscope. The blue color was DAPI fluorescence and the red color was AIE photosensitizer fluorescence. Figure 6As shown, the red fluorescence in the fourth group was the strongest, indicating that the liposomes were able to be targeted and enriched on the cell surface. As a control, the cells in the first group were not treated with TNF-α, with low target expression and very weak red fluorescence. The antibody pre-incubation in the second group occupied the cell surface target, making the targeted liposomes unrecognizable and the red fluorescence very weak. The liposomes in the third group were non-targeted liposomes, with very weak red fluorescence. Taken together, these results indicate that AN@ALips can effectively target the cell surface where ICAM-1 expression is upregulated, verifying the cellular targeting of AN@ALips.

[0075] 3.3 Analysis of the effect of targeting ligand content on liposome targeting using fluorescence microscopy

[0076] Under the steric hindrance of PEG, the biotin in Biotin-PEG-cholesterol binds to the biotinylated antibody through the avidin cross-linking effect. Using different molecular weights of Biotin-PEG-cholesterol results in varying steric hindrance, significantly affecting the binding of biotin to the antibody. The content of Biotin-PEG-cholesterol in the liposomes also influences the antibody content. Both of these factors influence the antibody content. Experimental studies have shown that when the molecular weight of PEG in Biotin-PEG-cholesterol is too small, the steric hindrance is minimal, resulting in excessive electron density between the bonding atoms, strong repulsion, and weakened covalent bond stability. This results in poor antibody stability after binding and facilitates dissociation of the formed covalent bond, resulting in low antibody content in the final liposome. On the other hand, when the molecular weight of PEG is too large, greater intramolecular tension is generated, increasing bond lengths and hindering covalent bonding, which is detrimental to increasing antibody surface content on the liposome. In the present invention, we have repeatedly tried to find that when the molecular weight of PEG in Biotin-PEG-cholesterol is about 3000, it is the best for increasing the content of biotinylated ICAM-1 antibody in liposomes. If the molecular weight of PEG is too large or too small, the content of biotinylated ICAM-1 antibody will decrease. On this basis, in order to study the effect of the antibody content on the liposome surface on the binding ability of liposomes to cell membranes, we prepared AN@ALips with different Biotin-PEG3000-cholesterol:cholesterol ratios (molar ratio 0:10, 1:9, 1:7, 1:4, 1:3), and incubated HUVEC cells incubated with 50 ng / ml TNF-α for 8 hours with them for 2 hours, and observed the results under a fluorescence microscope. Figure 7 As shown in the figure, as the antibody content increases, the red fluorescence on the cell membrane surface gradually increases, with the best effect at 1:4. However, when the antibody content is further increased to 1:3, the fluorescence decreases. Therefore, we chose 1:4 as the dosage of Biotin-PEG3000-Cholesterol in subsequent experiments.

[0077] Example 4: In vitro performance test of AN@ALips

[0078] 4.1 Phospholipase-responsive drug release of AN@ALips

[0079] The drug release behavior of liposomes was studied using the fluorescence aggregation-induced quenching effect of doxorubicin. When preparing AN@ALips, TTVP dye was replaced with an equimolar amount of doxorubicin (Dox) to prepare Dox@ALips. The prepared Dox@ALips was resuspended in saline and divided into three parts. Equal volumes of PBS, PBS containing phospholipase (PLA2), and saline were added respectively, mixed well, and incubated for 2 hours. After incubation, the fluorescence intensity of each solution was tested, and the same concentration of free Dox (5 μM) was used as a control. Figure 8 As shown in Figure A, compared to free Dox, after forming Dox@ALips, Dox fluorescence is quenched due to aggregation, resulting in very weak fluorescence. In the PBS-treated group, a small amount of Dox is released, and fluorescence recovers somewhat. Fluorescence recovery is greatest in the phospholipase PLA2-treated group. This demonstrates that liposomes exhibit phospholipase-responsive drug release. Specifically, under the action of phospholipase, the liposomes in the AN@ALips prepared according to the present invention exhibit responsive drug release for TTVP and nicorandil.

[0080] 4.2 Study on the responsive cumulative release of NO from AN@ALips

[0081] Prepare 2 mL of AN@ALips solution, in which the NO release amount is 100 μg / mL. Add phospholipase and incubate it to release the NO donor. Prepare 4 μM and 16 mM GSH solutions respectively. Take three portions of 0.5 mL of AN@ALips solution incubated with phospholipase, add 0.5 mL of PBS, 0.5 mL of 4 μM GSH solution and 16 mM GSH solution respectively, so that the final concentrations of GSH in the solution are 0 μM, 2 μM and 8 mM respectively. The time point after mixing is recorded as 0 h, and then samples are taken at 1, 2, 3 and 6 h, respectively, and the NO content in the solution is determined using a NO content kit. Figure 8 As shown in Figure B, low concentrations of GSH hardly induce the release of NO, while 8 mM GSH can induce significant release of NO.

[0082] 4.3 Study on GSH consumption in AN@ALips biofilms

[0083] Plate the cultured bacteria and calculate the concentration of the bacterial solution. Dilute the bacterial solution to 10 8CFU / mL, take 100 μL and add it to a 96-well plate, and add 100 μL of culture medium. Culture in a 37°C incubator. Replace fresh culture medium every 24 hours. After 96 hours, a biofilm is obtained. The cultured biofilm is divided into 4 groups, with 3 replicates in each group. Treat with PBS, A@ALips, N@ALips and AN@ALips for 6 hours respectively, and then use a GSH content kit to determine the GSH content in the biofilm. Figure 8 As shown in Figure C, compared with the PBS group, A@ALips did not consume GSH because it did not contain an NO donor. However, both N@ALips and AN@ALips were able to significantly reduce the GSH level in the biofilm.

[0084] 4.4 Cytotoxicity of AN@ALips

[0085] HUVEC cells were plated at 1.5х10 per well. 4 Inoculate into 96-well plates. When the cell confluence exceeds 80%, different concentrations of AN@ALips (0, 1, 2, 3, 4, and 5 μM in terms of AIE photosensitizer concentration) are co-cultured with the cells for 24 h. After removing the supernatant, add MTS reagent and incubate for another 1 h. Use a microplate reader to record the absorbance at 490 nm. Figure 9 As shown in Figure A, when the concentration of TTVP in AN@ALips reached 5 μM, the cell viability was still above 80%, indicating low cytotoxicity.

[0086] 4.5 Hemolytic activity of AN@ALips

[0087] Blood was collected from mice, centrifuged, and washed to prepare a 2% erythrocyte suspension. Various concentrations of AN@ALips (0, 1, 2, 3, 4, and 5 μM, based on the AIE photosensitizer concentration) were added. PBS was used as a negative control, and pure water was used as a positive control. After incubation at 37°C for 2 hours, the cells were centrifuged, photographed, and the supernatant collected. The absorbance of the supernatant was measured using a UV-vis spectrophotometer.

[0088] like Figure 9 As shown in Figures B and 9C, when AN@ALips (measured as AIE photosensitizer TTVP concentration of 0, 1, 2, 3, 4, and 5 μM) was used, no hemolysis was observed in either the photographic results or the UV-vis absorption spectrophotometer results.

[0089] Example 5: In vitro antibacterial performance test of AN@ALips

[0090] 5.1 Plate count study of the antibacterial ability of AN@ALips

[0091] Prepare MRSA bacterial suspension (~10 6CFU) and were divided into four groups. Group 1: treated with PBS and cultured overnight; Group 2: treated with phospholipase-treated N@ALips and cultured overnight; Group 3: treated with phospholipase-treated A@ALips and incubated in a 37°C incubator for 10 minutes, then exposed to green LED light at 80 J cm -2 The cells were irradiated for 10 minutes and then cultured overnight. The fourth group was incubated with AN@ALips treated with phospholipase at 37°C for 10 minutes and then irradiated with green LED at 80 J cm -2 Irradiate for 10 minutes and then culture overnight. After overnight culture, dilute the bacterial solution of each group at different times and count the cells on the plate. Figure 10 As shown in the figure, compared with the PBS group, the colony counts in other groups were significantly reduced, among which the colony counts in the AN@ALips+Light group were less than those in the other groups, indicating that the photosensitizer and NO donor have synergistic antibacterial ability.

[0092] 5.2 Scanning electron microscopy study of the antibacterial ability of AN@ALips

[0093] We used SEM to study the morphological changes of MRSA bacteria before and after different treatments of AN@ALips. 6 CFU / ml), and divided them into four groups. The first group: AN@ALips after +phospholipase treatment, without GSH and without light; the second group: AN@ALips after +phospholipase treatment, with GSH and without light; the third group: AN@ALips after +phospholipase treatment, without GSH and with light; the fourth group: AN@ALips after +phospholipase treatment, with GSH and with light. The treated bacterial solution was collected by centrifugation and washed three times with saline. The bacterial solution precipitate was resuspended in 2.5% fixative and fixed at 4°C overnight. The fixed bacteria were collected by centrifugation and washed three times with saline. The bacterial solution was gradient dehydrated in an ethanol solution, and finally stored in 100% ethanol. The samples were prepared and sent for SEM testing. As Figure 11 As shown, in the Light(-)GSH(-) group, MRSA exhibited a uniform spherical structure with a relatively smooth and rounded bacterial surface, indicating that the bacterial membrane was not damaged under this treatment mode. In the other three groups, however, the bacterial surface became rough and damaged and adhered, indicating that the bacterial membrane was damaged, leading to bacterial death. The Light(+)GSH(+) group showed the most significant damage to the bacterial membrane.

[0094] 5.3 Biofilm Elimination Ability of AN@ALips

[0095] The biofilms were cultured according to the method in 4.3 and divided into four groups. The first group was treated with PBS and cultured overnight; the second group was treated with N@ALips treated with phospholipase and cultured overnight; the third group was treated with A@ALips treated with phospholipase and incubated in a 37°C incubator for 10 minutes, and then illuminated with green LED light at 80 J cm -2 The cells were irradiated for 10 minutes and then cultured overnight. The fourth group was incubated with AN@ALips treated with phospholipase at 37°C for 10 minutes and then irradiated with green LED at 80 J cm -2 Irradiate for 10 minutes and then incubate overnight. After incubation overnight, pour out the incubation solution, wash the wells 3 times with PBS, add 200 μL of methanol to each well, incubate at room temperature for 15 minutes, remove the methanol and dry thoroughly at room temperature, add 150 μL of 0.1% crystal violet to each well for staining for 15 minutes. Remove the staining solution, rinse the well plate with water 5 times and air-dry at room temperature. Dissolve completely with 33% (v / v) glacial acetic acid, aspirate 150 μL of solution from each well to a new 96-well plate, take pictures and record, and use a microplate reader to measure the absorbance at OD590 nm, with three replicates per group. Figure 12 As shown in the figure, compared with the PBS group, the biofilms in the other groups were reduced to a certain extent, and the reduction of biofilms was most obvious in the dual-drug-loaded liposome light-irradiated group, indicating that photodynamic therapy and gas therapy can achieve synergistic antibacterial effects at the bacterial level and biofilm level.

[0096] Example 6: Animal Level Performance Test of AN@ALips

[0097] 6.1 Production of ROS, NO, and RNS at the Wound Infection Site after AN@ALips Administration

[0098] First, a skin wound infection model was constructed. We divided acclimated mice (female, 6-week-old, Kunming white mice) into three groups. A wound with a diameter of approximately 1 cm was made on the back of the mice and 50 μl of MRSA liquid (10 7 CFU / ml) and cultured for 24 hours. After infection was established, AN@ALips were injected into mice through the tail vein. After 8 hours, the infected area was illuminated by a green LED at 80 J cm -2 Irradiation was performed for 10 min. DCFH-DA (λex / λem: 488 / 525 nm) was then added to the wounds of the mice to detect ROS generation, DAF-FM DA (λex / λem: 488 / 515 nm) was used to detect NO generation, and R21 (λex / λem: 488 / 516 nm) was used to detect RNS generation. Figure 13As shown in the figure, luminescence was detected at the wound site, indicating that ROS, NO, and RNS were generated at the site. This shows that AN@ALips can be targeted and enriched at the site of bacterial infection and can release the AIE photosensitizer TTVP and NO donor Nicorandil to produce effector ROS, NO, and RNS.

[0099] 6.2 Studying the Targeting of AN@ALips in Animals Using Small Animal Imaging

[0100] The same method as 6.1 was used to construct infected mice. After infection, the mice were randomly divided into three groups. PBS, AN@Lips and AN@ALips were injected into the tail vein of the mice respectively. 4 hours after injection, the mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney and infected tissues were collected, rinsed with physiological saline, and imaged in an imager. Figure 14 As shown, no fluorescence was detected in the PBS group. In the AN@Lips and AN@ALips groups, fluorescence was produced in the liver, kidney, and infected tissues. The fluorescence in the infected tissues of the AN@ALips group was significantly stronger than that of the AN@Lips group, while the fluorescence in the liver and kidney tissues was weaker than that of the AN@Lips group. This suggests that the nanoliposomes are primarily metabolized in the liver and kidneys and that the presence of the targeting ligand increases the accumulation of the nanoparticles at the site of infection.

[0101] 6.3 In vivo antibacterial efficacy test of AN@ALips

[0102] Infected mice were constructed using the same method as in 6.1. After infection, the mice were randomly divided into four groups. The mice were treated with tail vein injection of PBS, N@ALips, A@ALips+Light, and AN@ALips+Light. After treatment, wound location images were collected on days 0, 1, 3, 5, and 7. Figure 15 As shown, wound size gradually decreased over time in all mice. In the untreated group, wounds did not heal significantly and formed large scabs. All three treatment groups demonstrated good wound healing, demonstrating that the nanosystem can kill bacteria at the site of infection and promote wound healing and tissue regeneration to a certain extent. The AN@ALips+Light treatment was the most effective.

[0103] After the wounds of the four groups of mice were treated, the residual fluid was extracted from the wound tissue, cultured with TSB medium for 24 hours, and then measured by plate count method. Figure 16 The results showed that a large number of bacteria were present in the untreated group, while the number of colonies in the treated group was significantly reduced. In the targeted dual-drug-loaded light-irradiated group, there were almost no bacterial colonies in the wound tissue. The results show that AN@ALips has the strongest antibacterial ability.

[0104] Histological analysis was used to evaluate the healing effect of regenerated wound tissue. After 7 days of treatment, skin tissue from the infected area was collected and stained with hematoxylin and eosin (H&E). Figure 17 As shown, MRSA-infected tissue in the control group displayed severe inflammatory cell infiltration, with severe damage to blood vessels and hair follicles. Treatment with A@ALips alone did not significantly improve these findings. In the A@ALips+Light and N@ALips groups, some new blood vessels and hair follicles were observed. The AN@ALips+Light group, on the other hand, exhibited well-organized stratified epithelium and orderly granulation tissue, with some new blood vessels and hair follicles. These results suggest that AN@ALips can be used as an effective nanoformulation for treating bacterial infections and promoting the skin wound healing process.

[0105] Example 7: In vivo biocompatibility of AN@ALips

[0106] Based on tissue distribution, drug-loaded liposomes are primarily metabolized in the liver and kidneys. Therefore, we analyzed liver and kidney-related biochemical markers to investigate the in vivo biosafety of the nanoliposomes. Healthy mice were randomly divided into four groups. PBS, A@ALips, N@ALips, and AN@ALips were injected intravenously into the tail vein. Seven days after administration, blood, heart, liver, spleen, lung, and kidney samples were collected. The blood was centrifuged to separate serum, and serum levels of ALT, BUN, CRE, TG, TCHO, and TP were measured using kits. Organ tissues of the mice were fixed with 4% paraformaldehyde for 48 hours, dehydrated, embedded, sectioned, and stained with hematoxylin and eosin for analysis of histopathological changes.

[0107] from Figure 18 It can be seen that no obvious abnormalities in biochemical indicators were found in different groups compared with the PBS control group, indicating that these three nanoliposomes did not cause obvious liver and kidney damage.

[0108] like Figure 19 As shown, no obvious histopathological changes were observed in the main organs of mice in different groups compared with the PBS control group.

Claims

1. A nanoliposome preparation, characterized in that: The nanoliposome preparation is a nanoformulation prepared by using distearoylphosphatidylglycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol as a carrier, with an ICAM-1 monoclonal antibody modified on the surface and loaded with an AIE photosensitizer TTVP and nicorandil. The molar ratio of DSPG, cholesterol, Biotin-PEG3000-cholesterol and nicorandil is 2:0.8:0.15-0.25:1.2-1.

4.

2. The nanoliposome preparation according to claim 1, wherein: The nanoformulation is prepared by first preparing a lipid membrane loaded with nicorandil using distearoylphosphatidylglycerol DSPG, cholesterol and Biotin-PEG3000-cholesterol, then loading the AIE photosensitizer TTVP to form liposomes, adding avidin and reacting with biotinylated ICAM-1 monoclonal antibody.

3. The nanoliposome preparation according to claim 1 or 2, wherein: The ICAM-1 was biotinylated by diluting the ICAM-1 antibody with sodium bicarbonate buffer (pH 8.0) and dialyzing to obtain an antibody solution. N-hydroxysuccinimide-biotin (NHSB) was dissolved in DMSO to obtain an NHSB solution. The NHSB solution was added to the antibody solution, and the mixture was stirred continuously at room temperature for 2 to 4 hours. An NH4Cl solution was added, and the mixture was stirred at room temperature for 10 minutes to remove free biotin. The mixture was then passed through a molecular sieve column and slowly eluted with PBS to collect the biotinylated ICAM-1.

4. A method for preparing a targeted antibacterial nanoliposome preparation, characterized in that: A lipid membrane loaded with nicorandil is prepared using distearoylphosphatidylglycerol (DSPG), cholesterol, and biotin-PEG3000-cholesterol. A TTVP solution is then added for loading to obtain a liposome solution. Avidin is then added for reaction, and then a biotinylated ICAM-1 monoclonal antibody is added for further reaction to obtain targeted dual-drug-loaded liposomes. The molar ratio of DSPG, cholesterol, biotin-PEG3000-cholesterol, and nicorandil is 2:0.8:0.15-0.25:1.2-1.

4.

5. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 4, wherein: The lipid membrane loaded with Nicorandil is prepared by distearoylphosphatidylglycerol DSPG, cholesterol, Biotin-PEG3000-cholesterol and Nicorandil being dissolved in chloroform to form a mixed solution, which is then evaporated under reduced pressure to form a lipid membrane.

6. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 5, wherein: The TTVP solution is added to the lipid membrane by water bath stirring and ultrasonic hydration for loading. The water bath stirring temperature is 20-30° C., the stirring speed is 180-220 rpm, the ultrasonic hydration temperature is 40-45° C., the ultrasonic power is 100-150 W, and the ultrasonic time is 12-15 minutes.

7. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 6, wherein: Avidin was added at a molar ratio of 2.5-3.5:2 between avidin and Biotin-PEG3000-cholesterol in the liposomes. After adding avidin, the mixture was stirred at 800-1000 rpm for 4-8 minutes and then centrifuged.

8. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 7, wherein: The liposomes were resuspended in physiological saline to a resuspension with a mass-to-volume ratio of 2 mg:1 mL. A 0.1 mg / L biotinylated ICAM-1 monoclonal antibody solution was added, stirred at 800-1000 rpm for 4-8 minutes, and then centrifuged. The volume ratio of the biotinylated ICAM-1 monoclonal antibody solution to the resuspension was 1:

1.

9. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 8, wherein: The biotinylated ICAM-1 monoclonal antibody is prepared by diluting the ICAM-1 antibody with sodium bicarbonate buffer (pH 8.0) and dialyzing to obtain an antibody solution. N-hydroxysuccinimide biotin (NHSB) is dissolved in DMSO to obtain an NHSB solution. The NHSB solution is added to the antibody solution, and the mixture is continuously stirred at room temperature for 2 to 4 hours. An NH4Cl solution is then added, and the mixture is stirred at room temperature for 10 minutes to remove free biotin. The mixture is then passed through a molecular sieve column and slowly eluted with PBS to collect the biotinylated ICAM-1.

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