Broad-spectrum targeted virulence factor response type phototherapy nano-particles, preparation method and application of broad-spectrum targeted virulence factor response type phototherapy nano-particles in preparation of antibacterial drugs

Abstract

The invention discloses a broad-spectrum targeted virulence factor response type phototherapy nano-particle, a preparation method and application of the broad-spectrum targeted virulence factor response type phototherapy nano-particle in preparation of antibacterial drugs. The nano-particle comprises a shell PSP formed by heterozygosis of lipid PS and a platelet cell membrane P, and the lipid PS is synthesized with phosphatidylcholine through sphingomyelin; a photosensitizer entrapped in the lipid PS bilayer; the photosensitizer and the perfluorocarbon are entrapped in the shell PSP, and the perfluorocarbon carries oxygen. According to the present invention, with the application of the compound, the broad-spectrum targeting of gram-negative bacteria and gram-positive bacteria can be achieved, the treatment accuracy can be improved, the dual functions of virulence factor neutralization and antibacterial effect enhancement can be provided, and the phototherapy antibacterial treatment effect can be significantly improved.

Description

Technical Field

[0001] This invention relates to biomedicine, specifically to broad-spectrum targeted virulence factor-responsive phototherapy nanoparticles, their preparation method, and their application in the preparation of antibacterial drugs. Background Technology

[0002] With the continued high incidence of bacterial infections globally, especially the rapid spread of multidrug-resistant strains, global public health faces unprecedented challenges. To date, antibiotics remain the primary means of clinical treatment for bacterial infections. However, the long-standing inappropriate overuse of antibiotics has significantly accelerated the emergence of drug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (PA), severely limiting the therapeutic efficacy of traditional drugs.

[0003] In recent years, phototherapy has shown great promise in the field of antibacterial therapy as an emerging non-drug antibacterial strategy. Phototherapy is mainly divided into photothermal therapy (PTT) and photodynamic therapy (PDT). PTT uses near-infrared (NIR) light to activate photosensitizers, converting light energy into heat energy to kill bacteria through high temperature; while PDT involves photosensitizers being excited by light and transferring energy to oxygen molecules to produce reactive oxygen species (ROS), which can also cause oxidative damage to bacteria.

[0004] However, commonly used photosensitizers, such as phthalocyanine IR780, generally lack targeting specificity to bacterial infection sites and are prone to causing non-specific damage to surrounding normal tissues during light irradiation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a broad-spectrum targeted virulence factor-responsive phototherapy nanoparticle, its preparation method, and its application in the preparation of antibacterial drugs. This nanoparticle can broadly target both Gram-negative and Gram-positive bacteria, improving the precision of treatment. It also possesses the dual functions of neutralizing virulence factors and enhancing antibacterial effects, thereby significantly improving the efficacy of phototherapy for antibacterial purposes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A broad-spectrum targeted virulence factor-responsive phototherapy nanoparticle, comprising... The outer shell PSP is formed by the hybridization of lipid PS with platelet cell membrane P. Lipid PS is synthesized through sphingomyelin and phosphatidylcholine. Photosensitizers encapsulated in lipid PS bilayers; The photosensitizer and perfluorocarbon compound, which carries oxygen, are encapsulated within the outer casing of the PSP.

[0007] As a further improvement of the present invention, the perfluorocarbon compound is perfluorooctane (PFC).

[0008] As a further improvement of the present invention, the photosensitizer is IR780.

[0009] As a further improvement of the present invention, the photosensitizer, in combination with near-infrared (NIR) light, forms a photothermal effect; The outer shell PSP is triggered by bacterial virulence factors to rupture, releasing photosensitizers and oxygen carried in perfluorooctane PFC.

[0010] As a further improvement of the present invention, the lipid PS contains sphingomyelin and phosphatidylcholine in a mass ratio of 1:2.

[0011] A method for preparing virulence factor-responsive phototherapy nanoparticles is also provided, comprising the following steps: Platelet membrane P was collected; IR780-encapsulated lipid PS was prepared by phosphatidylcholine, sphingomyelin, and IR780 thin film hydration method. The lipid PS was fused with platelet membrane P at a ratio of 1:1 w / w by extrusion to obtain the outer shell PSP; The PSP shell and the perfluorooctane PFC were ultrasonically bathed together to encapsulate the perfluorooctane PFC inside the PSP shell, thus obtaining PSP@IR780-PFC. PSP@IR780-PFC(O2) is obtained by filling with oxygen.

[0012] As a further improvement of the present invention, the sphingomyelin, phosphatidylcholine and IR780 are in a mass ratio of 66:33:1.

[0013] The invention also provides the application of virulence factor-responsive phototherapy nanoparticles in the preparation of antibacterial drugs, wherein the virulence factor-responsive phototherapy nanoparticles are those described in any of the above-mentioned embodiments.

[0014] The invention also provides the application of virulence factor-responsive phototherapy nanoparticles in the preparation of antibacterial drugs, wherein the virulence factor-responsive phototherapy nanoparticles are prepared by any one of the above-described methods for preparing virulence factor-responsive phototherapy nanoparticles.

[0015] The beneficial effects of this invention are that platelet membranes naturally have broad-spectrum targeting of bacteria, and the biomimetic nanoparticles that are virulence factor responsive phototherapy nanoparticles can be specifically enhanced by bacterial virulence factors to increase the intensity of phototherapy. These nanoparticles can not only broadly target Gram-negative and Gram-positive bacteria, improving the accuracy of treatment, but also have the dual functions of neutralizing virulence factors and enhancing antibacterial effects, thereby significantly improving the efficacy of phototherapy for antibacterial purposes. Attached Figure Description

[0016] Figure 1 This is a basic characterization diagram of the PSP@IR780-PFC(O2) of the present invention; Figure 2 This is a diagram showing the broad-spectrum targeting capability of the PSP@IR780-PFC(O2) of this invention for MRSA and PA. Figure 3 This is a diagram showing the responsive release of photosensitizer and oxygen from the virulence factor PSP@IR780-PFC(O2) of the present invention. Figure 4 This is a targeting diagram of the PSP@IR780-PFC(O2) of the present invention for subcutaneous MRSA infection; Figure 5 This is a targeting diagram of PSP@IR780-PFC(O2) of the present invention for subcutaneous PA infection; Figure 6 This image shows the therapeutic effect of the PSP@IR780-PFC(O2) of the present invention on subcutaneous bacterial infection in mice. Figure 7 This image shows the therapeutic effect of PSP@IR780-PFC(O2) of the present invention on subcutaneous MRSA infection in mice. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to the embodiments shown in the accompanying drawings.

[0018] Because Gram-positive and Gram-negative bacteria differ significantly in cell wall structure, lipid composition, and virulence factors, existing targeting strategies are often limited to specific species and lack broad-spectrum targeting. For example, amphotericin B can specifically bind to LPS on the surface of Gram-negative bacteria, but has no targeting effect on Gram-positive bacteria. Similarly, some peptide molecules, such as vancomycin, can bind to the peptidoglycan portion of Gram-positive bacteria to enhance their targeting, but have no recognition ability for Gram-negative bacteria. Nucleic acid aptamers can achieve highly specific species recognition, but their ligand specificity is high, development cycles are long, and their applicability is limited. Therefore, constructing a broad-spectrum targeting strategy that can simultaneously recognize both Gram-positive and Gram-negative bacteria has become an urgent need for current technological development.

[0019] This study found that platelet membranes, as naturally derived biomembrane materials, inherit the excellent targeted binding ability of platelets to inflammation and infection. Proteins on the platelet membrane surface, such as integrin αIIbβ3, LFA-1, and CD62P, can specifically bind to P-selectin and E-selectin on the surface of inflammatory endothelial cells, achieving targeting of the inflammatory site. Furthermore, platelet membranes can bind to various bacteria through receptors such as FcγRIIa, forming stable binding with Staphylococcus aureus's agglutination factor A (ClfA) and fibronectin-binding protein A (FnBPA) via fibrin bridges. In addition, platelet membranes can also interact with Gram-negative bacteria such as Pseudomonas aeruginosa (PA) and Helicobacter pylori. These characteristics provide an ideal biological interface for constructing a broad-spectrum phototherapy platform targeting bacteria using platelet membranes.

[0020] Preparation and characterization of PSP@IR780-PFC(O2) phototherapy nanoplatform Platelet membrane (P) isolation: Whole blood was collected from the cheek of mice and mixed with 10 mM EDTA-PBS buffer at a 1:1 volume ratio. The mixture was centrifuged at 300 × g for 20 minutes at room temperature. The lower layer of red blood cells was discarded, and the supernatant was collected. The collected supernatant was centrifuged at 2000 × g for 10 minutes at 4°C, and the precipitate was retained. The precipitate was resuspended in 5 mM EDTA-ultrapure water containing 1% protease inhibitor and frozen at -80°C for 30 minutes. The cryovials were then removed, thawed at room temperature, and 20 × PBS was added to adjust the osmotic pressure to 1 ×. Finally, the mouse platelet membrane was obtained by centrifugation at 14000 rpm for 10 minutes at 4°C. The membrane protein content was quantitatively analyzed using the BCA method.

[0021] Construction and Characterization of PSP@IR780-PFC(O2): Phosphatidylcholine and sphingomyelin (PS) liposomes were prepared using a thin-film hydration method. First, sphingomyelin (Sm), phosphatidylcholine (PC-98T), and photosensitizer IR780 were dissolved in 10 mL of chloroform at a mass ratio of 66:33:1 and thoroughly mixed. Then, the organic solvent was evaporated to dryness using a rotary evaporator at 65°C for 1 hour, forming a lipid film in the flask. The resulting lipid film was resuspended in a preheated 10% (w / v) sucrose solution to form a preliminary liposome solution. PS liposomes were then fused with platelet membranes (P) at a 1:1 (w / w) ratio using an extrusion method to obtain PSP. Different volumes of perfluorooctane (PFC, 0, 1, 2, 4, and 8 μL per mg of liposomes) were added to the above PSP nanoparticles, and the mixtures were ultrasonically emulsified for 10 minutes in an ice bath using a probe-type sonicator (60 W power, 2 seconds of sonication followed by a 3-second pause). The particle size and surface potential of PSP@IR780-PFC(O2) were determined by dynamic light scattering (DLS), and the morphology of PSP@IR780-PFC(O2) was determined by TEM. All PSP@IR780-PFC(O2) used in subsequent experiments were pre-filled with 30 mL of oxygen before use.

[0022] PSP@IR780-PFC(O2) targeting adhesion ability to MRSA and PA MRSA and PA were labeled with BacLight™ Bacterial Stains (B-35000) green fluorescent markers. The resulting product contained 2 × 10⁻⁶ ppm. 7 CFU of MRSA or PA bacterial suspension was mixed with 1 mg / mL PSP@IR780-PFC(O2) to a total volume of 1 mL and incubated at 37°C for 30 minutes. After incubation, the bacterial pellet was collected by centrifugation at 5000 × g for 10 minutes. The pellet was then washed three times with PBS and resuspended in PBS. 5 μL of the pellet was placed on a glass slide, and the fluorescence colocalization of PSP@IR780-PFC(O2) with MRSA or PA was observed under a fluorescence confocal microscope. Untreated MRSA or PA, and bacterial samples treated with PSR@IR780-PFC(O2) constructed by encapsulating IR780 and PFC with PSR formed by erythrocyte membrane (R) and PS lipids served as control groups.

[0023] PSP@IR780-PFC(O2) responsively releases photosensitizer and oxygen as virulence factors. Temperature change of PSP@IR780-PFC(O2) under NIR irradiation after addition of toxins: 100 μg / mL PSP@IR780-PFC(O2) solution was mixed with bacterial secreted toxins sMT (toxin secreted by MRSA) or sPT (toxin secreted by PA), and then irradiated with an 808 nm near-infrared laser (power density 1.0 W / cm²). 2 The samples were irradiated for 300 seconds. During irradiation, the temperature change of the samples was recorded every 30 seconds using a thermal imager (Fotric 322pro). The PSP@IR780-PFC(O2) sample without added toxins served as a control group.

[0024] Determination of oxygen release curves in PSP@IR780-PFC(O2) induced by toxins: 1 mL of PSP@IR780-PFC(O2) solution with a concentration of 10 mg / mL was pre-oxygenated and added to 20 mL of PBS solution that had been deoxygenated (N2 purging O2) at 25°C. The dissolved oxygen content in the solution was measured every 30 seconds using a portable dissolved oxygen meter (Leici JPB-607A) for a total continuous measurement time of 360 seconds. Bacterial toxins sMT or sPT were added at time point 120 seconds to observe their effect on oxygen release behavior.

[0025] Evaluation of the in vitro antibacterial effect of PSP@IR780-PFC(O2) under the action of toxins: To evaluate the in vitro antibacterial effect of biomimetic phototherapy nanoparticles, a total of 2 × 10⁻⁶ PSP@IR780-PFC(O2) was subjected to toxins. 7 CFU MRSA or 5 × 10 7 CFU PAs were dispersed in PBS and mixed with PSP@IR780-PFC(O2) nanoparticles containing or without bacterial toxins. The mixture was then subjected to a reaction at 1.0 W / cm². 2 Irradiate the bacteria under near-infrared laser at a high power density for 5 minutes. After irradiation, transfer the treated bacterial suspension to 100 mL of TSB liquid medium and incubate for 8 hours at 37°C with shaking at 120 rpm. Samples are taken at 0, 2, 4, 6, and 8 hours, and the OD values ​​are measured using a microplate reader. 600 The bacterial growth was assessed using the PSR@IR780-PFC(O2) value. PBS treatment, toxin-only treatment, and PSR@IR780-PFC(O2) treatment were used as controls.

[0026] Targeted antibacterial phototherapy for bacterial infections in vivo Construction of MRSA or PA skin infection model: 100 μL of 2 × 10 7 A CFU / mL MRSA bacterial suspension was injected subcutaneously into the right back of mice. The PA infection model was established by injecting 100 μL of a 5 × 10⁻⁶ CFU / mL suspension. 7A PA suspension of CFU / mL was prepared. It was ready for experimental use 24 hours after injection and infection.

[0027] Targeted distribution experiment of PSP@IR780-PFC(O2) in mice with MRSA or PA infection models: PSP@IR780-PFC(O2) was injected intravenously at a dose of 50 mg / kg into mice with established MRSA or PA infection models. Fluorescence intensity at the infection site was monitored at preset time points (0, 1, 2, 4, 8, 12, 24, 48, and 72 hours) using an IVIS Lumina XRMS Series III, PerkinElmer small animal in vivo imaging system to assess the accumulation of the nanoplatform in the infected area. 72 hours after administration, mice were sacrificed, and major organs (heart, liver, spleen, lung, kidney, brain, and bone tissue) and infected lesion tissue were collected, weighed, and analyzed in vitro. Imaging parameters were set to excitation wavelength 785 nm and emission wavelength 810 nm. A PBS-treated group and a PSR@IR780-PFC(O2) treatment group containing an equal amount of IR780 were used as controls to compare tissue distribution and targeting ability.

[0028] Evaluation of the in vivo phototherapy effect of PSP@IR780-PFC(O2): To evaluate the in vivo phototherapy effect of intravenously injected PSP@IR780-PFC(O2), an MRSA infection model was first constructed by injecting 100 μL of PSP@IR780-PFC(O2) at a concentration of 2 × 10⁻⁶. 7 CFU / mL MRSA bacterial suspension was injected into the backs of mice. Twenty-four hours after infection, mice were randomly divided into three groups: a PBS control group, a PSR@IR780-PFC(O2) group, and a PSP@IR780-PFC(O2) treatment group. Another 24 hours later, all infected areas of the mice were irradiated with near-infrared laser (808 nm, 1.0 W / cm²). 2 Irradiation was performed for 5 minutes. On days 1, 5, 9, and 13 post-treatment, the size of the infected lesions in each group of mice was recorded. At the end of the experiment, the lesion tissue was homogenized and inoculated onto TSB agar plates for 24 hours to count MRSA colony-forming units (CFU).

[0029] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A broad-spectrum targeted toxicity factor-responsive phototherapy nanoparticle, characterized in that, include The outer shell PSP is formed by the hybridization of lipid PS with platelet cell membrane P. Lipid PS is synthesized through sphingomyelin and phosphatidylcholine. Photosensitizers encapsulated in lipid PS bilayers; The photosensitizer and perfluorocarbon compound, which carries oxygen, are encapsulated within the outer casing of the PSP.

2. The virulence factor-responsive phototherapy nanoparticles according to claim 1, characterized in that, The perfluorocarbon compound is perfluorooctane (PFC).

3. The virulence factor-responsive phototherapy nanoparticles according to claim 1, characterized in that, The photosensitizer is IR780.

4. The virulence factor-responsive phototherapy nanoparticles according to claim 2, characterized in that, The photosensitizer, in combination with near-infrared (NIR) light, forms a photothermal effect; The outer shell PSP is triggered by bacterial virulence factors to rupture, releasing photosensitizers and oxygen carried in perfluorooctane PFC.

5. The virulence factor-responsive phototherapy nanoparticles according to claim 1, characterized in that, The lipid PS contains sphingomyelin and phosphatidylcholine in a mass ratio of 1:

2.

6. A method for preparing virulence factor-responsive phototherapy nanoparticles, characterized in that, Includes the following steps Platelet membrane P was collected; IR780-encapsulated lipid PS was prepared by phosphatidylcholine, sphingomyelin, and IR780 thin film hydration method; The lipid PS was fused with platelet membrane P at a ratio of 1:1 w / w by extrusion to obtain the outer shell PSP; The PSP shell and the perfluorooctane PFC were ultrasonically bathed together to encapsulate the perfluorooctane PFC inside the PSP shell, thus obtaining PSP@IR780-PFC. PSP@IR780-PFC(O2) is obtained by filling with oxygen.

7. The preparation method according to claim 6, characterized in that, The sphingomyelin, phosphatidylcholine, and IR780 are in a mass ratio of 66:33:

1.

8. The application of a virulence factor-responsive phototherapy nanoparticle in the preparation of antibacterial drugs, characterized in that, The virulence factor-responsive phototherapy nanoparticles described herein are those as described in any one of claims 1 to 5.

9. The application of a virulence factor-responsive phototherapy nanoparticle in the preparation of antibacterial drugs, characterized in that, The virulence factor-responsive phototherapy nanoparticles are prepared using the method described in any one of claims 6 to 7.

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