Targeted antibacterial nano-liposome preparation and preparation method thereof

Abstract

The invention relates to a nano-liposome preparation, which is characterized in that a liposome prepared from distearoyl phosphatidyl glycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol is used as a carrier, the surface of the nano-liposome preparation is modified with an ICAM-1 monoclonal antibody, and the nano-liposome preparation is loaded with an AIE photosensitizer and Nicodil. The nano-liposome prepared by the invention has targeting and responsive drug delivery to infected lesions, the enrichment of drugs at bacterial infected parts is improved, nano-system components can be cut by phospholipase in an infected microenvironment, responsive release of the drugs at the bacterial infected parts can be realized, and the treatment effect is improved. According to the invention, a hydrophobic shell layer and a hydrophilic core of the liposome are utilized, and hydrophobic NO donor molecules and a hydrophilic AIE photosensitizer are loaded at the same time, so that synergistic treatment of bacterial infection by a photodynamic therapy and a gas therapy is realized, and a better killing effect on drug-resistant bacteria is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparation, and particularly relates to a targeted antibacterial nano-liposome preparation and a preparation method thereof. Background Art

[0002] In the current medical field, diseases and deaths induced by bacterial infections are one of the most challenging natural threats faced by humans. Using antibiotics to inhibit or kill bacteria is a conventional treatment strategy. However, the abuse of antibiotics has led to the emergence of bacterial drug resistance, which not only increases the risk of complications and mortality of patients, but also increases the cost of public healthcare. In addition, bacteria can adhere to the surface to form biofilms, further increasing the difficulty of treatment. In the face of the increasing threat of drug-resistant bacteria and biofilm infections, there is an urgent need to develop new drugs and new therapies for treating drug-resistant pathogen infections that can replace antibiotics.

[0003] Photodynamic antibacterial therapy (PDT) is a new antibacterial method developed in recent years. Photosensitizers can generate reactive oxygen species (ROS) through photodynamic reactions, which can effectively kill pathogenic bacteria. Because this therapy has advantages such as high antibacterial efficiency, non-invasiveness, low probability of generating drug resistance, and strong spatiotemporal selectivity, it has become one of the most promising new antibacterial therapies. However, traditional organic photosensitizers will produce fluorescence quenching and reduce the ROS yield in the aqueous physiological environment and the nanonization process. Using photosensitizers with aggregation-induced emission (AIE) properties can better solve this problem and improve the photodynamic therapy effect. However, the high level of glutathione (GSH) in the bacterial infection microenvironment causes the unprovoked consumption of ROS, resulting in the unsatisfactory antibacterial effect of the single PDT therapy. Therefore, using GSH depletors in combination with AIE photosensitizers for synergistic antibacterial is a new treatment method worthy of research. Nitric oxide (NO) is a special bioactive molecule that can promote the consumption of GSH. Moreover, NO can react with the ROS generated by PDT to generate reactive nitrogen species (RNS) with stronger bactericidal ability. Therefore, a NO donor molecule with GSH responsiveness can be selected to be used in combination with AIE photosensitizers for synergistic antibacterial. However, as small molecule preparations, NO donor molecules and AIE photosensitizers have disadvantages such as poor targeted enrichment at the infection site, fast in vivo clearance rate, low bioavailability, and high toxicity and side effects. Therefore, developing a delivery system that can effectively target and respond to deliver the two drugs to the infection site at the same time is a major challenge. Summary of the Invention

[0004] The purpose of the present invention is to provide a dual-therapy synergistic antibacterial liposome nano-preparation that targets and responds to the infection microenvironment. This preparation is a liposome that simultaneously loads a water-soluble AIE photosensitizer and a lipid-soluble NO donor, and introduces an ICAM monoclonal antibody with a targeting effect, which can deliver and release drugs in a targeted manner to the infection site and exert a dual-therapy synergistic antibacterial effect by using photodynamic / gas therapy.

[0005] Another object of the present invention is to provide a method for preparing the above antibacterial liposome nano - preparation. This method effectively solves the problem that the ICAM monoclonal antibody cannot be attached to the surface of the liposome for modification, or the monoclonal antibody is inactivated after attachment, making it difficult to achieve the targeting effect.

[0006] The object of the present invention is achieved by the following technical solutions: A nano - liposome preparation, characterized in that: the nano - liposome preparation is a nano - preparation with a liposome prepared from distearoyl phosphatidylglycerol (DSPG), cholesterol and Biotin - PEG3000 - cholesterol as a carrier, surface - modified with ICAM - 1 monoclonal antibody and loaded with AIE photosensitizer and nicorandil.

[0007] Furthermore, the nano - preparation is a spherical structure with a particle size range of 100 - 150 nm.

[0008] Furthermore, the nano - preparation is prepared by first preparing a lipid membrane loaded with nicorandil from distearoyl phosphatidylglycerol (DSPG), cholesterol and Biotin - PEG3000 - cholesterol, then loading the AIE photosensitizer to form liposomes, and after adding avidin, mixing and reacting with biotinylated ICAM - 1 monoclonal antibody.

[0009] Furthermore, the ICAM - 1 is biotinylated. Specifically, the ICAM - 1 antibody is diluted and dialyzed with sodium bicarbonate buffer (pH 8.0) 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 continuous stirring is carried out at room temperature for 2 - 4 hours for incubation. NH 4 Cl solution is added, and stirring is continued at room temperature for 10 minutes. After removing free biotin, it is passed through a molecular sieve column and slowly eluted with PBS to collect biotinylated ICAM - 1.

[0010] 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.

[0011] 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.

[0012] Furthermore, the concentration of the NH 4 Cl solution is 1 mol / L, and the volume ratio of the NH 4 Cl solution to the NHSB solution is 12 - 13:1.

[0013] A preparation method of a targeted antibacterial nanoliposome preparation, characterized in that: a lipid membrane loaded with nicorandil is prepared from distearoyl phosphatidylglycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol, and then an AIE photosensitizer solution is added for loading to obtain a liposome solution. Then, after adding avidin and reacting, biotinylated ICAM-1 monoclonal antibody is added and reacted continuously to obtain a targeted dual-loaded drug liposome, denoted as AN@ALips.

[0014] Furthermore, the lipid membrane loaded with nicorandil is formed by dissolving DSPG, cholesterol, Biotin-PEG3000-cholesterol and nicorandil in chloroform to form a mixed solution, and then rotary evaporating under reduced pressure to form a lipid membrane.

[0015] Furthermore, the molar ratio of DSPG, cholesterol, Biotin-PEG3000-cholesterol and nicorandil is 2:0.8:0.15 - 0.25:1.2 - 1.4.

[0016] Furthermore, adding the AIE solution to the lipid membrane is carried out by water bath stirring and ultrasonic hydration for loading. The temperature of the water bath stirring is 20 - 30 °C, the stirring speed is 180 - 220 rpm, the temperature of the ultrasonic hydration is 40 - 45 °C, the ultrasonic power is 100 - 150 W, and the ultrasonic time is 12 - 15 min.

[0017] Furthermore, the AIE solution is obtained by mixing the AIE photosensitizer TTVP and physiological saline in a ratio of 2 mg:1 - 1.2 mL.

[0018] Furthermore, avidin is added according to the molar ratio of avidin to Biotin-PEG3000-cholesterol in the liposome of 2.5 - 3.5:2. After adding avidin, it is stirred at 800 - 1000 rpm for 4 - 8 min, and then centrifuged.

[0019] Furthermore, the liposome is resuspended with physiological saline to form a resuspension with a mass-volume ratio of 2 mg:1 mL, and a biotinylated ICAM-1 monoclonal antibody solution with a concentration of 0.1 mg / L is added. It is stirred at 800 - 1000 rpm for 4 - 8 min, and then centrifuged. The volume ratio of the biotinylated ICAM-1 monoclonal antibody solution to the resuspension is 1:1.

[0020] Furthermore, the biotinylated ICAM-1 monoclonal antibody is obtained by diluting and dialyzing the ICAM-1 antibody with sodium bicarbonate buffer (pH 8.0) to obtain an antibody solution. Dissolve N-hydroxysuccinimide biotin (NHSB) with DMSO to obtain an NHSB solution. Add the NHSB solution to the antibody solution, continuously stir at room temperature, incubate for 2 - 4 hours, and add NH 4The Cl solution was continuously stirred at room temperature for 10 minutes. After removing free biotin, it was slowly eluted through a molecular sieve column with PBS, and biotinylated ICAM-1 was collected.

[0021] Furthermore, the concentration of the sodium bicarbonate buffer solution 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.

[0022] 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.

[0023] Furthermore, the NH 4 Cl solution concentration is 1 mol / L, and the volume ratio of the NH 4 Cl solution to the NHSB solution is 12 - 13:1.

[0024] Among many nano-carrier materials, liposomes have characteristics such as good biocompatibility, low immunogenicity, the ability to simultaneously carry hydrophilic and lipophilic drugs, and easy surface modification. The flexible and adjustable formulation of nano-liposomes is very suitable for the preparation of targeted and responsive delivery carriers. The bacterial infection microenvironment has characteristics such as local acidification, high levels of reducing GSH, overexpressed phospholipase, and up-regulated expression of intercellular adhesion molecule (ICAM-1) on vascular endothelial cells. Therefore, a phospholipase-responsive nanodelivery system modified with ICAM-1 monoclonal antibody targeting the infection site can achieve targeted and responsive release of two drugs. However, in the preparation process, it is difficult for ICAM-1 monoclonal antibody to effectively modify liposomes, and drug targeting and responsive release cannot be achieved.

[0025] In the present invention, during the preparation of liposomes, Biotin-PEG3000-cholesterol with a specific molecular weight is selected to participate in the preparation of liposomes. Through the binding of avidin and biotinylated ICAM-1 monoclonal antibody, and the synergistic effect of the steric hindrance of Biotin-PEG3000-cholesterol, the introduction efficiency of ICAM-1 monoclonal antibody is improved, enabling ICAM-1 monoclonal antibody to effectively adhere to the surface of liposomes to form a modification, thereby achieving targeted delivery of liposome nano-formulations, enhancing the enrichment of drugs at the bacterial infection site. Through the shearing effect of phospholipase in the bacterial infection microenvironment on liposomes, responsive release of drugs at the bacterial infection site is achieved, improving the therapeutic effect.

[0026] By releasing the NO donor nicorandil from liposomes, which reacts with GSH to generate NO, the reduction of GSH can inhibit the consumption of POS, and the generated NO reacts with ROS produced by PDT to generate reactive nitrogen species (RNS) with strong bactericidal ability.

[0027] A preparation method of a targeted antibacterial nano-liposome preparation, characterized by comprising the following steps: S1. Dissolve and mix distearoyl phosphatidylglycerol (DSPG), cholesterol, Biotin-PEG3000-cholesterol and nicorandil in chloroform according to a molar ratio of 2:0.8:0.15 - 0.25:1.2 - 1.4 to obtain a mixed solution, and perform rotary evaporation under reduced pressure on the mixed solution to form a film; S2. Mix water-soluble TTVP and physiological saline according to a ratio of 2 mg:1 - 1.2 mL to obtain a photosensitizer solution. Add the photosensitizer solution to the film prepared in step S1, perform water bath stirring and ultrasonic hydration in sequence, and then perform dialysis to obtain a liposome solution. The temperature of the water bath stirring is 20 - 30 °C, the stirring speed is 180 - 220 rpm, the temperature of the ultrasonic hydration is 40 - 45 °C, the ultrasonic power is 100 - 150 W, and the ultrasonic time is 12 - 15 min; S3. Use a liposome extruder to extrude the liposome solution in step S2, add avidin to the extruded solution according to a molar ratio of avidin:Biotin-PEG3000-cholesterol = 3:2, stir at 800 - 1200 rpm for 4 - 8 min, and then centrifuge to collect the solid; S4. Resuspend the solid collected in S3 with physiological saline to form a resuspension (mass-to-volume ratio 20:1 (mg / ml)), add a biotinylated ICAM-1 monoclonal antibody solution with a mass concentration of 0.1 mg / kg, and the volume ratio of the biotinylated ICAM-1 monoclonal antibody solution to the resuspension is 1:1. Stir at 800 - 1200 rpm for 4 - 8 min, and then further centrifuge to collect the solid to obtain a targeted antibacterial liposome nano-preparation. The biotinylated ICAM-1 is obtained by diluting the ICAM-1 antibody with 0.08 - 0.12 mol / L sodium bicarbonate buffer (pH 8.0) to 1 mg / mL, fully dialyzing the protein with 0.08 - 0.12 mol / L sodium bicarbonate buffer (pH 8.0) alternately, dissolving N-hydroxysuccinimide biotin (NHSB) with DMSO to obtain an NHSB solution with a concentration of 0.85 - 1.2 mg / mL, mixing the antibody solution and the NHSB solution according to a volume ratio of 1:0.1 - 0.14, continuously stirring at room temperature, incubating for 2 - 4 hours, adding a 1 mol / L NH 4 Cl, and the volume ratio of the NH 4 Cl solution to the NHSB solution is 12 - 13:1. Stir at room temperature for 10 minutes, dialyze against PBS at 4 °C to remove free biotin, load the sample onto a 1 ml molecular sieve column, elute slowly with PBS, and collect 1 ml / tube.

[0028] Water-soluble TTVP is a compound containing tetraphenylethylene, triphenylamine, ethylene, and benzene rings, and these structural units together constitute the AIE material.

[0029] The present invention has the following technical effects: In the present invention, Biotin-PEG-cholesterol, cholesterol, and DSPG that can be cleaved by phospholipase are used as nano-liposome components, and through the avidin-biotin interaction, monoclonal antibodies are introduced on the surface of the nano-system, endowing the nano-system with targeted and responsive drug delivery to the infected lesion, improving the enrichment of drugs at the bacterial infection site. The components of this nano-system can be cleaved by phospholipase in the infected microenvironment, enabling the responsive release of drugs at the bacterial infection site and enhancing the therapeutic effect. In the present invention, by utilizing the hydrophobic shell and hydrophilic core of the liposome, hydrophobic NO donor molecules and hydrophilic AIE photosensitizers are simultaneously loaded, realizing the synergistic treatment of bacterial infection by "photodynamic therapy" and "gas therapy", and having a better killing effect on drug-resistant bacteria. Description of the Drawings

[0030] Figure 1 : Transmission electron microscope image of AN@ALips in Example 2.

[0031] Figure 2 : Transmission electron microscope image of AN@ALips (prepared with large molecular weight Biotin-PEG-cholesterol) in Example 2.

[0032] Figure 3 : Particle size, Zeta potential, and storage stability of AN@ALips in Example 2.

[0033] Figure 4 : PAGE diagram of AN@ALips in Example 2 to verify antibody conjugation.

[0034] Figure 5 : Expression of ICAM-1 after TNF-α treatment of HUVECs for different times in Example 3.

[0035] Figure 6 : Cellular level targeting study of AN@ALips in Example 3.

[0036] Figure 7 : Fluorescence microscope results of targeted liposomes with different antibody contents (Biotin-PEG-cholesterol: cholesterol) treating TNF-α-pretreated HUVECs in Example 3.

[0037] Figure 8 : Evaluation of enzyme-responsive release, GSH-responsive NO release, and GSH consumption in biofilms of AN@ALips in Example 4.

[0038] Figure 9 : Evaluation of the cytotoxicity and hemolytic activity of AN@ALips in Example 4.

[0039] Figure 10 : Colony growth of MRSA after treatment with PBS, N@ALips, A@ALips+Light, and AN@ALips+Light in Example 5.

[0040] Figure 11 : Scanning electron microscopy results of MRSA after treatment with different groups of AN@ALips in Example 5.

[0041] Figure 12 : Crystal violet staining results of MRSA biofilms after treatment with PBS, N@ALips, A@ALips+Light, and AN@ALips+Light in Example 5.

[0042] Figure 13 : Production of ROS, NO, and RNS at the infected wound site after treatment with AN@ALips in Example 6.

[0043] Figure 14 : Fluorescence of various tissues in mice after tail vein injection of PBS, AN@Lips, and AN@ALips in Example 6.

[0044] Figure 15 : Wound healing of infected tissues in mice and infected tissues treated with N@ALips, A@ALips+Light, and AN@ALips+Light within one week in Example 6.

[0045] Figure 16 : Results of plate counting of bacterial loads in tissues of mice infected tissues and infected tissues treated with A@ALips+Light, N@ALips, and AN@ALips+Light in Example 6.

[0046] Figure 17 : HE staining results of healthy skin tissues, infected tissues, and infected tissues treated with A@ALips, A@ALips+Light, N@ALips, and AN@ALips+Light in mice in Example 6.

[0047] Figure 18 : Analysis results of blood biochemical indexes in mice after tail vein injection of PBS, A@ALips, N@ALips, and AN@ALips in Example 7.

[0048] Figure 19:HE staining results of various tissues in Example 7 after mouse tail vein injection of PBS, A@ALips, N@ALips, and AN@ALips. Detailed implementation manner

[0049] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and should not be construed as limiting the protection scope 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 content of the present invention.

[0050] Example 1 A preparation method of a targeted antibacterial nanoliposome preparation, comprising the following steps: 1.1 Preparation of dual-loaded liposome AN@Lips Distearoyl phosphatidylglycerol (DSPG), cholesterol, Biotin-PEG3000-cholesterol, and nicorandil were added to chloroform in a molar ratio of 2:0.8:0.2:1.3 to dissolve to obtain a mixed solution. The mixed solution was subjected to reduced-pressure rotary evaporation to form a film at a water bath temperature of 40 °C and a rotation speed of 100 rpm; 30 mg of water-soluble TTVP was dissolved in 15 mL of physiological saline, added to the film prepared above, and stirred in a water bath at 25 °C at 200 rpm for 5 min, and then sonicated for hydration at a power of 120 W for 15 min at 45 °C, and then dialyzed to obtain a liposome solution, that is, AN@Lips solution; 1.2 Preparation of targeted dual-loaded liposome AN@ALips 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, and stirred at 1000 rpm for 5 min. Then, it was centrifuged, and the solid was collected and resuspended in physiological saline to form a suspension with a mass-to-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. The volume ratio of the monoclonal antibody solution to the suspension was 1:1, and it was stirred at 1000 rpm for 5 min. Then, it was further centrifuged, and the solid was collected to obtain the targeted antibacterial liposome nanoplatform AN@ALips. The biotinylated ICAM-1 was prepared by diluting the ICAM-1 antibody to be biotinylated with 0.1 mol / L sodium bicarbonate buffer (pH 8.0) to 1 mg / mL, and dialyzing the protein thoroughly with 0.1 mol / L sodium bicarbonate buffer (pH 8.0) alternately. 1 mg of N-hydroxysuccinimide biotin (NHSB) was dissolved in 1 mL of DMSO. 120 μL of NHSB solution (i.e., containing 120 μg of NHSB) was added to 1 mL of antibody solution (i.e., containing 1 mg of antibody), and continuously stirred at room temperature for 2 - 4 hours. 9.6 μL of 1 mol / L NH 4 Cl solution was added, and stirred at room temperature for 10 minutes. At 4 °C, it was dialyzed thoroughly against PBS to remove free biotin. The solution was applied to a 1 mL molecular sieve column and slowly eluted with PBS, collecting 1 mL / tube to obtain the biotinylated ICAM-1 antibody.

[0051] A@ALips loaded only with AIE photosensitizer and N@ALips loaded only with NO donor were prepared by the same method.

[0052] Example 2: Characterization of AN@ALips 2.1 Observation of the morphology of AN@ALips using transmission electron microscopy (TEM) An appropriate amount of AN@ALips was weighed and dispersed in physiological saline, and 10 μL was dropped onto a carbon support film copper mesh and observed after drying at room temperature. As Figure 1 shown, the prepared AN@ALips had good monodispersity and a particle size of about 100 nm. In addition, we prepared a dual-loaded liposome using Biotin-PEG5000-cholesterol with a larger molecular weight according to the above method, and the electron microscopy results are as Figure 2As shown, its uniformity and dispersibility deteriorated, and the average particle size also increased to around 110 nm. We calculated the drug loading rates of liposomes prepared using Biotin-PEG3000-cholesterol and Biotin-PEG5000-cholesterol. The drug loading rates of liposomes prepared using Biotin-PEG3000-cholesterol for the AIE photosensitizer TTVP and NO donor were 18% and 9.83% respectively, while those of liposomes prepared using Biotin-PEG5000-cholesterol for the two were 11.16% and 5.91% respectively. Overall, liposomes prepared using Biotin-PEG3000-cholesterol had more advantages.

[0053] 2.2 Detection of the particle size, Zeta potential of AN@ALips using a dynamic light scattering nanosizer (DLS) and monitoring of storage stability An appropriate amount of AN@ALips solution was taken, fully dispersed and used for DLS analysis. As Figure 3 shown in A, the prepared AN@ALips had a particle size of around 100 nm, which was consistent with the TEM results. As Figure 3 shown in B, the Zeta potential of AN@ALips was -25.1 ± 0.3 mV. As Figure 3 shown in C, AN@ALips could maintain its particle size well within one week. From the above results, it can be seen that the prepared AN@ALips had relatively uniform size, moderate particle size, and contained PEG and surface negative charges, which was beneficial for the long circulation of nano-drugs in vivo.

[0054] 2.3 Verification of antibody conjugation using PAGE An appropriate amount of AN@ALips solution was taken, the solid was collected by centrifugation, and the protein was extracted by lysing on ice with RIPA lysis buffer containing PMSF. The extracted protein solution was treated with 5x loading buffer, treated in a metal bath at 100 °C for 5 min, and loaded onto a PAGE gel for electrophoresis. The results were as Figure 4 shown. The results of protein electrophoresis in liposomes were consistent with those of free antibodies, indicating that the antibody was successfully conjugated to the liposome surface.

[0055] Example 3: Verification of the cellular level targeting of AN@ALips 3.1 Verification of upregulated expression of the target protein ICAM-1 Using human umbilical vein endothelial cells (HUVEC) as a cell model, the HUVEC cell concentration was adjusted to 2x10 per well 5Inoculate into 6-well plates. When the cell confluence exceeds 80%, add the inflammatory factor TNF-α (50 ng / ml) and incubate for 4 h or 8 h. Cells without TNF-α incubation serve as the control group. Collect the treated cells to extract proteins, and study the expression of ICAM-1 through Western blot assay. As Figure 5 shown, only a small amount of ICAM-1 is expressed in cells without TNF-α treatment. In cells treated with TNF-α, the expression of ICAM-1 is significantly up-regulated, indicating that ICAM-1 is up-regulated on the surface of endothelial cells in the infected microenvironment, which is beneficial to the enrichment, binding, and penetration of targeted liposomes.

[0056] 3.2 Analyze the cellular targeting of AN@ALips using a fluorescence microscope Seed HUVEC cells at a concentration of 1×10 5 per well into 12-well plates and culture until the cell confluence is above 80%. Divide them into four experimental groups: Group 1 [-TNF-α (untreated), +AN@ALips (containing 5 μM AIE photosensitizer) incubated for 2 h]; Group 2 [+TNF-α (incubated with 50 ng / ml for 8 h), +ICAM-1 antibody pre-incubated for 1 h, +AN@ALips (containing 5 μM AIE photosensitizer) incubated for 2 h]; Group 3 [+TNF-α (incubated with 50 ng / ml for 8 h), +AN@Lips (containing 5 μM AIE photosensitizer) incubated for 2 h]; Group 4 [+TNF-α (incubated with 50 ng / ml for 8 h), +AN@ALips (containing 5 μM AIE photosensitizer) incubated for 2 h]. Stain cell nuclei with DAPI dye. After the treatment, observe under a fluorescence microscope. The blue color is the fluorescence of DAPI, and the red color is the fluorescence of the AIE photosensitizer. As Figure 6 shown, the red fluorescence in the fourth group is the strongest, indicating that the liposomes can target and enrich on the cell surface. As a control, in the first group, the cells are not treated with TNF-α, with less target expression and weak red fluorescence. In the second group, the pre-incubation with the antibody occupies the cell surface target, resulting in the inability of the targeted liposomes to recognize and weak red fluorescence. In the third group, the liposomes are non-targeted liposomes with weak red fluorescence. Considering comprehensively, the above results show that AN@ALips can well target the cell surface with up-regulated ICAM-1 expression, verifying the cellular targeting of AN@ALips.

[0057] 3.3 Analyze the effect of the content of the targeting ligand on the liposome targeting using a fluorescence microscope Under the steric hindrance of PEG, the Biotin in Biotin-PEG-cholesterol will bind to the biotinylated antibody through the cross-linking effect of avidin. The use of Biotin-PEG-cholesterol with different molecular weights has different steric hindrance effects, which has a more obvious effect on the binding effect of Biotin and antibodies. The content of Biotin-PEG-cholesterol in liposomes will also affect the change in antibody content. The above two aspects are factors that affect the change in antibody content. It was found in continuous experiments that when the molecular weight of PEG in Biotin-PEG-cholesterol is too small and the steric hindrance is small, the electron cloud density between the bonding atoms will be too high, resulting in strong repulsion, weakening the stability of the covalent bond, and the antibody has poor stability after binding. The covalent bond formed is easy to dissociate, resulting in a low antibody content in the final liposome. If the molecular weight of PEG is too large, a large tension will be formed in the molecule, the bond length will increase, and the covalent bond connection will be blocked, which is not conducive to increasing the antibody content on the liposome surface. In the present invention, continuous attempts have found that when the molecular weight of PEG in Biotin-PEG-cholesterol is about 3000, it is optimal for increasing the content of biotinylated ICAM-1 antibody in liposomes. Too large or too small a PEG molecular weight will lead to a decrease in the content of biotinylated ICAM-1 antibody. On this basis, in order to study the effect of the antibody content on the surface of liposomes on the binding ability of liposomes to cell membranes, we prepared AN@ALips with different Biotin-PEG3000-cholesterol: cholesterol ratios (molar ratios of 0:10, 1:9, 1:7, 1:4, and 1:3), and used them to incubate HUVEC cells that had been incubated with 50 ng / ml TNF-α for 8 hours for 2 hours, and observed the results under a fluorescence microscope. Figure 7 As shown in the figure, with the increase of antibody content, the red fluorescence on the cell membrane surface gradually increased, and the best effect was achieved at 1:4. When the antibody content was further increased to a ratio of 1:3, the fluorescence decreased. Therefore, we chose 1:4 as the dosage of Biotin-PEG3000-cholesterol in subsequent experiments.

[0058] Example 4: In vitro performance test of AN@ALips 4.1 Phospholipase-responsive drug release of AN@ALips The drug release behavior of liposomes was studied by using the fluorescence aggregation-induced quenching effect of doxorubicin. When preparing AN@ALips, TTVP dye was replaced with an equal molar amount of doxorubicin (Dox) to prepare Dox@ALips. The prepared Dox@ALips were resuspended in saline and divided into three portions. PBS, phospholipase (PLA) containing 1% phospholipase (1% phospholipase) and 1% phospholipase (1% phospholipase) were added in equal volumes. 2) of PBS and normal saline, and incubated for 2 h after mixing evenly. After incubation, the fluorescence intensity of each solution was measured, and free Dox (5 μM) at the same concentration was used as a control. As Figure 8 shown in A, compared with free Dox, after the formation of Dox@ALips, the fluorescence of Dox was quenched due to aggregation and was very weak. In the PBS treatment group, a small amount of Dox was released, and the fluorescence recovered to a certain extent. While in the phospholipase PLA 2 treatment group, the fluorescence recovered the most. It can be shown that the liposome has the ability of phospholipase-responsive drug release, that is, under the action of phospholipase, the liposome in AN@ALips prepared in the present invention has the ability of responsive drug release for TTVP and nicorandil.

[0059] 4.2 NO-responsive cumulative release study of AN@ALips Prepare 2 mL of AN@ALips solution, in which the NO release amount is 100 μg / mL. Incubate with phospholipase to release the NO donor therein. Prepare 4 μM and 16 mM GSH solutions respectively. Take three 0.5 mL aliquots of the AN@ALips solution incubated with phospholipase, and 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 solutions 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 measured using a NO content kit. As Figure 8 shown in B, low-concentration GSH hardly causes the release of NO, and 8 mM GSH can cause obvious release of NO.

[0060] 4.3 GSH consumption study of AN@ALips on biofilms The cultured bacteria were spread on plates to calculate the bacterial liquid concentration. The bacterial liquid was diluted to 10 8 CFU / mL with fresh medium, take 100 μL and add it to a 96-well plate, and supplement 100 μL of medium. Incubate in a 37 °C incubator. Replace the fresh medium every 24 h. After 96 h, a biofilm was obtained. The cultured biofilm was divided into 4 groups, with 3 replicates in each group. Treat with PBS, A@ALips, N@ALips and AN@ALips for 6 h respectively, and then use a GSH content kit to measure the GSH content in the biofilm. As Figure 8 shown in C, compared with the PBS group, A@ALips did not consume GSH because it does not contain a NO donor. While both N@ALips and AN@ALips can significantly reduce the GSH level in the biofilm.

[0061] 4.4 Cytotoxicity of AN@ALips Seed HUVEC cells at a density of 1.5×10 4 per well in a 96-well plate. When the cell confluence exceeds 80%, co-culture the cells with different concentrations of AN@ALips (0, 1, 2, 3, 4, and 5 μM in terms of the AIE photosensitizer concentration) for 24 h. After removing the supernatant, add MTS reagent and incubate for another 1 h. Record the absorbance at 490 nm using a microplate reader. As Figure 9 shown in A, when the concentration of TTVP in AN@ALips reaches 5 μM, the cell viability is still above 80%, indicating low cytotoxicity.

[0062] 4.5 Hemolytic activity of AN@ALips Collect mouse blood, centrifuge and wash it to prepare a 2% red blood cell suspension. Add different concentrations of AN@ALips (0, 1, 2, 3, 4, and 5 μM in terms of the AIE photosensitizer concentration). Use PBS as the negative control and pure water as the positive control. After incubating at 37 °C for 2 h, centrifuge, take pictures to record the results, collect the supernatant, and record the absorbance of the supernatant using a UV-vis absorption spectrophotometer.

[0063] As Figure 9 shown in B and 9C, no hemolysis was observed in the pictures and the results of the UV-vis absorption spectrophotometer when AN@ALips (0, 1, 2, 3, 4, and 5 μM in terms of the AIE photosensitizer TTVP concentration).

[0064] Example 5: In vitro antibacterial performance test of AN@ALips 5.1 Plate counting to study the antibacterial ability of AN@ALips Prepare an MRSA bacterial suspension (∼10 6 CFU) and divide it into four groups. The first group: treated with PBS and cultured overnight; the second group: treated with N@ALips after phospholipase treatment and cultured overnight; the third group: co-incubated with A@ALips after phospholipase treatment in an incubator at 37 °C for 10 minutes, then irradiated with green light LED at 80 J cm -2 for 10 minutes, and then cultured overnight; the fourth group: co-incubated with AN@ALips after phospholipase treatment in an incubator at 37 °C for 10 minutes, then irradiated with green light LED at 80 J cm -2 for 10 minutes, and then cultured overnight. After culturing overnight, dilute the bacterial suspensions of each group by different multiples and perform plate counting. As Figure 10 shown, compared with the PBS group, the number of colonies in other groups decreased significantly, and the AN@ALips + Light group had fewer colonies than the other groups, indicating that the photosensitizer and NO donor have synergistic antibacterial ability.

[0065] 5.2 Scanning Electron Microscope Study on the Antibacterial Ability of AN@ALips We used SEM to study the morphological changes of MRSA bacteria before and after different treatments of AN@ALips. Prepare an MRSA bacterial suspension (10 6 CFU / ml), and divide it into four groups. The first group: AN@ALips after phospholipase treatment, without GSH, without light; the second group: AN@ALips after phospholipase treatment, with GSH, without light; the third group: AN@ALips after phospholipase treatment, without GSH, with light; the fourth group: AN@ALips after phospholipase treatment, with GSH, with light. Centrifuge to collect the treated bacterial solution, and wash it three times with physiological saline. Resuspend the bacterial precipitate with 2.5% fixative and fix it overnight at 4°C. Centrifuge to collect the fixed bacteria and wash them three times with physiological saline. Gradient dehydrate the bacterial solution in an ethanol solution and finally store it in 100% ethanol, and prepare samples for SEM testing. As Figure 11 shown, in the Light(-)GSH(-) group, MRSA presented a uniform spherical structure, and the surface of the bacterial cells was relatively smooth and round, indicating that the bacterial membrane was not damaged under this treatment mode. In the other three groups, the surface of the bacterial cells became rough and showed breakage and adhesion, indicating that their bacterial membranes were damaged, which in turn led to the death of the bacteria. Among them, the Light(+)GSH(+) group had the most obvious damage to the bacterial membrane.

[0066] 5.3 Biofilm Elimination Ability of AN@ALips Culture the biofilm according to the method in 4.3, and divide it into four groups. The first group: treated with PBS and cultured overnight; the second group: treated with N@ALips after phospholipase treatment and cultured overnight; the third group: co-incubated with A@ALips after phospholipase treatment in an incubator at 37°C for 10 minutes, then irradiated with green light LED at 80 J cm -2 for 10 minutes, and then cultured overnight; the fourth group: co-incubated with AN@ALips after phospholipase treatment in an incubator at 37°C for 10 minutes, then irradiated with green light LED at 80 J cm -2 for 10 minutes, and then cultured overnight. After culturing overnight, pour out the incubation solution, then wash the wells 3 times with PBS, add 200 μL of methanol to each well, incubate at room temperature for 15 min, remove the methanol and air dry thoroughly at room temperature. Add 150 μL of 0.1% crystal violet to each well and stain for 15 min. Remove the staining solution, rinse the well plate 5 times with water and air dry at room temperature. Dissolve completely with 33% (v / v) glacial acetic acid, aspirate 150 μL of the solution from each well into a new 96-well plate, take pictures and record, and measure the absorbance at OD590 nm using a microplate reader, with three replicates for each group. As Figure 12As shown, compared with the PBS group, the biofilms in other groups were reduced to a certain extent, and the reduction of the biofilm was most obvious in the dual-drug-loaded liposome light irradiation group, indicating that photodynamic therapy and gas therapy can achieve synergistic antibacterial effects at the bacterial level and the biofilm level.

[0067] Example 6: Animal-level performance test of AN@ALips 6.1 Generation of ROS, NO, and RNS at the wound infection site after administration of AN@ALips First, a skin wound infection model was constructed. We divided the mice (female, 6-week-old Kunming white mice) after adaptive feeding into three groups. A wound with a diameter of about 1 cm was made on the back of the mice, and 50 μl of MRSA bacterial solution (10 7 CFU / ml) was inoculated and cultured for 24 h. After the infection was formed, AN@ALips was injected into the mice by tail vein injection. After 8 h, the infected site was irradiated with green light LED at 80 J cm -2 for 10 min. Then, DCFH-DA (λex / λem: 488 / 525 nm) was added to the wounds of the mice to detect the generation of ROS, DAF-FM DA (λex / λem: 488 / 515 nm) was used to detect the generation of NO, and R21 (λex / λem: 488 / 516 nm) was used to detect the generation of RNS. As Figure 13 shown, luminescence was detected at the wound site, indicating the generation of ROS, NO, and RNS at this site. This shows that AN@ALips can target and accumulate at the bacterial infection site, and can release the AIE photosensitizer TTVP and the NO donor nicorandil, generating reactive oxygen species (ROS), nitric oxide (NO), and reactive nitrogen species (RNS).

[0068] 6.2 Investigation of the animal-level targeting of AN@ALips using small animal imaging Infected mice were constructed using the same method as in 6.1. After the infection was established, the mice were randomly divided into three groups. The mice were injected with PBS, AN@Lips, and AN@ALips via the tail vein, respectively. At 4 h after injection, the mice were sacrificed by cervical dislocation, and the heart, liver, spleen, lungs, kidneys, and infected tissues were collected. After rinsing with normal saline, they were imaged in an imager. As Figure 14 shown, no fluorescence was detected in the PBS group. In the AN@Lips and AN@ALips groups, fluorescence was generated in the liver, kidneys, and infected tissues. Comparing the two, the fluorescence in the infected tissues of the AN@ALips group was significantly stronger than that in the AN@Lips group, while the fluorescence in the liver and kidney tissues was weaker than that in the AN@Lips group. This indicates that the nanoliposomes are mainly metabolized by the liver and kidneys, and also shows that the presence of the targeting ligand increases the enrichment of the nanoparticles at the infection site.

[0069] 6.3 In vivo antibacterial effect test of AN@ALips Mice with infections were constructed using the same method as in 6.1. After the infection was established, the mice were randomly divided into four groups. The mice were treated by tail vein injection with PBS, N@ALips, A@ALips+Light, and AN@ALips+Light, respectively. After the treatment, images of the wound sites were collected on days 0, 1, 3, 5, and 7. As Figure 15 shown, for all mice, the wound size gradually decreased over time. In the untreated infection group, the wound did not heal significantly and large crusts formed. All three drug-administered groups showed good wound healing effects, indicating that the nanosystem could kill bacteria at the infection site and promote wound healing and tissue regeneration to a certain extent. Among them, the effect of the AN@ALips+Light treatment was the most obvious.

[0070] After the infected wounds of the above four groups of mice were treated, the residual fluid was extracted from the wound tissue, cultured in TSB medium for 24 h, and then measured by the plate counting method. As Figure 16 shown, a large number of bacteria were present in the untreated infection group, while the number of colonies in the treatment groups was significantly reduced. In the targeted dual-drug light group, there were almost no bacterial colonies in the wound tissue. The results showed that AN@ALips had the strongest antibacterial ability.

[0071] Histological analysis was used to evaluate the healing effect of the regenerated wound tissue. Seven days after the treatment, the skin tissue at the infection site was collected and stained with hematoxylin and eosin (H&E). As Figure 17 shown, the tissue infected with MRSA in the control group showed severe inflammatory cell infiltration, and blood vessels and hair follicles were severely damaged. There was no obvious improvement with the treatment of A@ALips alone. For the A@ALips+Light and N@ALips groups, some new blood vessels and hair follicles were observed. The AN@ALips+Light group showed well-layered epithelium and orderly granulation tissue in the tissue, with some new blood vessels and hair follicles. The results showed that AN@ALips could be used as an effective nanoplatform for treating bacterial infections and promoting the skin wound healing process.

[0072] Example 7: In vivo biocompatibility of AN@ALips In terms of tissue distribution, the drug-loaded liposomes are mainly metabolized by the liver and kidney. Therefore, we analyzed the biochemical indicators related to the liver and kidney to study the in vivo biosafety of the nano-liposomes. Healthy mice were randomly divided into four groups. PBS, A@ALips, N@ALips and AN@ALips were injected into the tail veins of the mice respectively. After 7 days of administration, the blood, heart, liver, spleen, lung and kidney of the mice were collected. The blood was centrifuged to separate the serum, and ALT, BUN, CRE, TG, TCHO and TP in the serum were measured using kits. The organ tissues of the mice were fixed with 4% paraformaldehyde solution for 48 h, dehydrated, embedded, sectioned and stained with HE for analyzing the histopathological changes.

[0073] From Figure 18 It can be seen that in different groups, compared with the control group PBS, no obvious abnormalities in biochemical indicators were found, indicating that these three nano-liposomes do not cause obvious liver and kidney damage.

[0074] As Figure 19 shown, in different groups, compared with the control group PBS, no obvious histopathological changes were observed in the main organs of the mice.

Claims

1. 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 carriers, with ICAM-1 monoclonal antibody modified on the surface and loaded with AIE photosensitizer and Nicorandil.

2. The nanoliposome preparation according to claim 1, characterized in that: The nanoformulation is prepared by first preparing a lipid membrane loaded with nicorandil using distearoylphosphatidylglycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol, then loading an AIE photosensitizer to form a liposome, adding avidin and reacting with a biotinylated ICAM-1 monoclonal antibody.

3. The nanoliposome preparation according to claim 1 or 2, characterized in that: The ICAM-1 was biotinylated, specifically, the ICAM-1 antibody was diluted with sodium bicarbonate buffer (pH 8.0) and dialyzed to obtain an antibody solution, N-hydroxysuccinimide biotin (NHSB) was dissolved with DMSO to obtain an NHSB solution, the NHSB solution was added to the antibody solution, and the mixture was continuously stirred at room temperature for 2 to 4 hours, and an NH4Cl solution was added, and the mixture was continuously stirred at room temperature for 10 minutes. After removing free biotin, the mixture was 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 was prepared with distearoylphosphatidylglycerol (DSPG), cholesterol and Biotin-PEG3000-cholesterol, and then an AIE photosensitizer solution was added for loading to obtain a liposome solution. After avidin was added for reaction, biotinylated ICAM-1 monoclonal antibody was added for further reaction to obtain targeted dual-drug-loaded liposomes.

5. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 4, characterized in that: The lipid membrane loaded with nicorandil is prepared by dissolving 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.

6. A method for preparing a targeted antibacterial nanoliposome preparation as claimed in claim 4 or 5, characterized in that: The molar ratio of DSPG, cholesterol, Biotin-PEG3000-cholesterol and Nicorandil is 2:0.8:0.15-0.25:1.2-1.

4.

7. A method for preparing a targeted antibacterial nanoliposome preparation according to any one of claims 4 to 6, characterized in that: The AIE solution is added to the lipid film 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.

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

9. A method for preparing a targeted antibacterial nanoliposome preparation according to any one of claims 4 to 8, characterized in that: 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, stirred at 800-1000 rpm for 4-8 min, and then centrifuged. The volume ratio of the biotinylated ICAM-1 monoclonal antibody solution to the resuspension was 1:

1.

10. The method for preparing a targeted antibacterial nanoliposome preparation according to claim 9, characterized in that: 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, keeping the temperature for 2 to 4 hours, adding NH4Cl solution, continuing to stir at room temperature for 10 minutes, removing free biotin, passing through a molecular sieve column, and slowly eluting with PBS to collect the biotinylated ICAM-1.

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