Photodynamic synergistic targeted drug-loaded nanocomposite, preparation method and application thereof

Abstract

The application provides a kind of photodynamic synergistic targeted drug-loaded nanocomposite, uses BPN as drug delivery carrier, PMb and photosensitizer as loaded drug, constructs nanometer drug delivery system by electrostatic adsorption method, the obtained nanocomposite can be highly targeted mixed bacterial biofilm, and under the premise of the destruction of BPN to mixed bacterial biofilm, gram-positive bacteria and gram-negative bacteria are effectively killed by combining PMb and photosensitizer, so as to achieve the purpose of ablation of mixed bacterial biofilm.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery systems, and more specifically to a photodynamic synergistic targeted drug-loaded nanocomposite and its preparation method, as well as its applications, particularly in the preparation of drugs for ablating mixed bacterial biofilms. Background Technology

[0002] More than ten million indwelling or implantable devices are placed in the human body globally each year. However, these implants are highly susceptible to bacterial infections, which can lead to the formation of bacterial biofilms. Due to the emergence of antibiotic-resistant bacteria and the physical protection of extracellular polymeric substances (EPS), antibiotic treatment is often ineffective in eradicating bacterial biofilms. If left untreated, the bacterial biofilm on the implant surface can cause chronic or recurrent diseases, necessitating replacement of the biomedical implant, which brings patients more suffering and financial burden.

[0003] Biofilms are three-dimensional bacterial communities with coordinated functions, composed of bacteria and their secreted extracellular polymeric matrix. Especially in clinical settings, biofilms are not typically composed of a single bacterium but rather a mixture of multiple bacteria. Different bacterial species in mixed bacterial biofilms can work synergistically and influence each other, leading to more diverse functional expressions, such as cooperative antibiotic secretion and sharing of nutrient resources. Therefore, mixed bacterial biofilms are more resistant to antibiotics and immune system tolerance, more pathogenic, and more difficult to remove than single-bacterial biofilms.

[0004] However, current technologies primarily focus on the ablation of biofilms formed by single bacteria, such as using photodynamic therapy (PDT). For mixed bacterial biofilms composed of both Gram-negative and Gram-positive bacteria, the removal efficiency remains poor. Summary of the Invention

[0005] The purpose of this invention is to address the problem of poor removal efficiency of mixed bacterial biofilms in existing technologies by providing a targeted and highly efficient photodynamic synergistic drug-loaded nanocomposite. Using BPN as the drug delivery carrier and PMb and a photosensitizer as the loaded drugs, a nano-drug delivery system is constructed via electrostatic adsorption. The resulting nanocomposite can highly target mixed bacterial biofilms and, while BPN disrupts the mixed bacterial biofilm, effectively kills both positive and negative bacteria in combination with PMb and the photosensitizer, thereby achieving the purpose of dissolving the mixed bacterial biofilm.

[0006] The first aspect of the present invention relates to a photodynamic synergistic targeted drug-loaded nanocomposite, the nanocomposite comprising a carrier, polymyxin B (PMb) and a photosensitizer, wherein the carrier is black phosphorus nanosheets (BPN).

[0007] The polymyxin B and photosensitizer are sequentially adsorbed onto the carrier via electrostatic interaction to obtain the nanocomposite.

[0008] As an optional implementation, the thickness of the black phosphorus nanosheets is 5 nm to 50 nm.

[0009] As an optional embodiment, the mass content of polymyxin B in the nanocomposite is 2.3% to 2.6%, and the mass content of the photosensitizer is 22.6% to 25.4%.

[0010] As an optional implementation, the nanosheets have a particle size of 150 nm to 170 nm and a potential of -26 mV to -18 mV.

[0011] As an optional implementation, the photosensitizer is Ce6.

[0012] The second aspect of this invention relates to a method for preparing the aforementioned photodynamic synergistic targeted drug-loaded nanocomposite, comprising the following steps:

[0013] S1. Disperse black phosphorus (BP) in deionized water and remove dissolved oxygen from the solution with nitrogen to obtain BP dispersion;

[0014] The BP dispersion was ultrasonically exfoliated under ice bath conditions, followed by centrifugation to remove unexfoliated BP and obtain BPN.

[0015] S2. Add polymyxin B (PMb) to the BPN dispersion, so that PMb is adsorbed onto the BPN through electrostatic interaction to form PMb@BPN, and obtain the first mixed solution, and PMb@BPN is uniformly dispersed in the first mixed solution.

[0016] S3. Add the photosensitizer to the first mixed solution obtained in step S2, so that the photosensitizer is adsorbed onto PMb@BPN through electrostatic interaction to obtain the second mixed solution.

[0017] After removing the free photosensitizer by ultrafiltration, the second mixed solution was washed and redispersed to obtain a dispersion of the desired nanocomposite.

[0018] As an optional implementation, the concentration of BPN in the BPN dispersion is 100 μg / mL to 120 μg / mL.

[0019] As an optional implementation, the mass ratio of PMb to BPN in the first mixed solution is (1:25) to (1:33).

[0020] As an optional implementation, the mass ratio of photosensitizer to BPN in the second mixed solution is (3:10) to (7:20).

[0021] The third aspect of this invention relates to the application of the aforementioned photodynamic synergistic targeted drug-loaded nanocomposite in the preparation of ablative biofilm drugs.

[0022] The fourth aspect of the present invention relates to a nanomedicine delivery system comprising the aforementioned photodynamic synergistic targeted drug-loaded nanocomposite.

[0023] Compared with the prior art, the significant advantages of the present invention are as follows:

[0024] 1. The photodynamic synergistic targeted drug-loaded nanocomposite of the present invention uses BPN as a drug delivery carrier and loads PMb and photosensitizer on its surface through electrostatic adsorption, thereby constructing a novel nanocomposite for ablation of mixed bacterial biofilms. After PMb highly targets mixed bacterial biofilms containing Gram-negative bacteria, the nanocomposite, under the premise of BPN destroying the mixed bacterial biofilm, effectively kills Gram-positive and Gram-negative bacteria in combination with PMb and photosensitizer. Finally, through the linkage of various parts, the purpose of ablation of mixed bacterial biofilms is achieved, which has broad application potential in the biomedical field.

[0025] 2. The nanocomposite of the present invention, by loading a small amount of PMb onto BPN, can reduce its toxicity in vivo, while improving its stability and reducing the probability of side effects.

[0026] 3. The nanocomposite of the present invention loads the photosensitizer onto BPN, which helps to improve the dispersibility and stability of the photosensitizer in vivo, thereby improving the effect of the photosensitizer in the photodynamic therapy process.

[0027] 4. The preparation method of the photodynamic synergistic targeted drug-loaded nanocomposite of the present invention is simple. It uses electrostatic adsorption to load PMb and Ce6 onto BPN. The method is easy to achieve mass production and is conducive to promotion and application. Attached Figure Description

[0028] Figure 1 This is a flowchart (a) of the preparation process of the photodynamic synergistic targeted drug-loaded nanocomposite of the present invention, and a diagram (b) of its working principle.

[0029] Figure 2 These are the particle size and thickness of the S-BPN as characterized by atomic force microscopy in the embodiments of the present invention.

[0030] Figure 3This is a quantitative measure of the residual A. baumannii biomass after treating A. baumannii biofilms with different thicknesses of BPN (100 μg / mL) in the embodiments of the present invention.

[0031] Figure 4 The hydration particle size is the S-BPN and three types of black phosphorus nanosheet systems in the embodiments of the present invention.

[0032] Figure 5 This refers to the potential of the S-BPN and the three nano-black phosphorus nanosheet systems in the embodiments of the present invention.

[0033] Figure 6 This is a TEM image of Ce6&PMb@S-BPN in an embodiment of the present invention.

[0034] Figure 7 These are the ultraviolet-visible absorption spectra of S-BPN, Ce6, Ce6@S-BPN, and Ce6&PMb@S-BPN in the embodiments of the present invention.

[0035] Figure 8 The relative cell viability of L929 and Raw 264.7 cells after 12 hours of treatment with Ce6&PMb@S-BPN (2-50 μg / mL, equivalent to BPN concentration) in the embodiments of the present invention.

[0036] Figure 9 This is a quantitative fluorescence intensity diagram of A. baumannii after treatment with PBS, Ce6, Ce6@S-BPN and Ce6&PMb@S-BPN for 30 min in the embodiments of the present invention.

[0037] Figure 10 These are CLSM images of A. baumannii biofilms after treatment with PBS, Ce6, Ce6@S-BPN, and Ce6&PMb@S-BPN in embodiments of the present invention.

[0038] Figure 11 This is the quantitative fluorescence intensity of A. baumannii biofilms treated with PBS, Ce6, Ce6@S-BPN, and Ce6&PMb@S-BPN in the embodiments of this invention.

[0039] Figure 12 This is the crystal violet quantitative data after treating A. baumannii biofilm with PBS, PMb@S-BPN+NIR, Ce6@S-BPN, Ce6@S-BPN+NIR, Ce6&PMb@S-BPN and Ce6&PMb@S-BPN+NIR (40 μg / mL, equivalent to BPN concentration) for 30 min in the embodiments of this invention.

[0040] Figure 13These are plate coating data of surviving bacteria in the *A. baumannii* biofilm after treatment with PBS, PMb@S-BPN+NIR, Ce6@S-BPN, Ce6@S-BPN+NIR, Ce6&PMb@S-BPN, and Ce6&PMb@S-BPN+NIR (40 μg / mL, equivalent to BPN concentration) in the embodiments of this invention.

[0041] Figure 14 These are the crystal violet quantitative data of A. baumannii-MRSA mixed biofilms treated with PBS, PMb@S-BPN+NIR, Ce6@S-BPN, Ce6@S-BPN+NIR, Ce6&PMb@S-BPN and Ce6&PMb@S-BPN+NIR (40 μg / mL, equivalent to BPN concentration) for 30 min in the embodiments of this invention.

[0042] Figure 15 These are plate coating data of surviving bacteria in the A. baumannii-MRSA mixed biofilm after treatment with PBS, PMb@S-BPN+NIR, Ce6@S-BPN, Ce6@S-BPN+NIR, Ce6&PMb@S-BPN and Ce6&PMb@S-BPN+NIR (40 μg / mL, equivalent to BPN concentration) in the embodiments of the present invention.

[0043] Figure 16 This refers to the survival rate of mice implanted with A. baumannii-MRSA coated catheters on day 5 after treatment in this embodiment of the invention.

[0044] Figure 17 These are quantitative data on live bacteria at the site of the A. baumannii-MRSA coated catheter implanted in this embodiment of the invention.

[0045] Figure 18 This is a quantitative ROS fluorescence image obtained by using DCFH-DA as the ROS probe and co-incubating PBS, Ce6, Ce6@S-BPN and Ce6&PMb@S-BPN with A. baumannii for 30 min in an embodiment of the present invention.

[0046] Figure 19 This refers to the survival rate of MRSA after treatment with PMb, PMb@S-BPN, Ce6&PMb and Ce6&PMb@S-BPN (2μg / mL, equivalent to BPN concentration) in the embodiments of the present invention. Detailed Implementation

[0047] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0048] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0049] Photodynamic therapy (PDT), as a non-invasive and low-toxicity treatment method, has received widespread attention in the field of antibacterial therapy in recent years. PDT generates highly reactive singlet oxygen (SO4) through the excitation of a specific wavelength light source by a photosensitizer. 1 O2 and other reactive oxygen species cause bacteria to die.

[0050] Based on this, the present invention provides a black phosphorus nanosheet simultaneously loaded with polymyxin B (PMb) and a photosensitizer to address the poor therapeutic effects of existing technologies on mixed bacterial biofilms, as well as the limitations in the application of PMb and Ce6.

[0051] Combination Figure 1 As shown, in a preferred embodiment of the present invention, a photodynamic synergistic targeted drug-loaded nanocomposite is provided, the nanocomposite comprising a carrier, polymyxin B (PMb) and a photosensitizer, wherein the carrier is black phosphorus nanosheets (BPN).

[0052] The polymyxin B and photosensitizer are sequentially adsorbed onto the carrier via electrostatic interaction to obtain the nanocomposite.

[0053] As an optional implementation, the thickness of the black phosphorus nanosheets is 5nm to 50nm, and more preferably 5nm to 10nm.

[0054] As an optional embodiment, the mass content of polymyxin B in the nanocomposite is 2.3% to 2.6%, and the mass content of the photosensitizer is 22.6% to 25.4%.

[0055] As an optional implementation, the nanosheets have a particle size of 150 nm to 170 nm and a potential of -26 mV to -18 mV.

[0056] As an optional implementation, the photosensitizer is Ce6.

[0057] like Figure 1 As shown in part a, in another preferred embodiment of the present invention, a method for preparing the aforementioned photodynamic synergistic targeted drug-loaded nanocomposite is provided, comprising the following steps:

[0058] S1. Disperse black phosphorus (BP) in deionized water and remove dissolved oxygen from the solution with nitrogen to obtain BP dispersion;

[0059] The BP dispersion was ultrasonically exfoliated under ice bath conditions, followed by centrifugation to remove unexfoliated BP and obtain BPN.

[0060] S2. Add polymyxin B (PMb) to the BPN dispersion, so that PMb is adsorbed onto the BPN through electrostatic interaction to form PMb@BPN, and obtain the first mixed solution, and PMb@BPN is uniformly dispersed in the first mixed solution.

[0061] S3. Add the photosensitizer to the first mixed solution obtained in step S2, so that the photosensitizer is adsorbed onto PMb@BPN through electrostatic interaction to obtain the second mixed solution.

[0062] After removing the free photosensitizer by ultrafiltration, the second mixed solution was washed and redispersed to obtain a dispersion of the desired nanocomposite.

[0063] As an optional implementation, the concentration of BPN in the BPN dispersion is 100–120 μg / mL.

[0064] As an optional implementation, the mass ratio of PMb to BPN in the first mixed solution is (1:25) to (1:33).

[0065] As an optional implementation, the mass ratio of photosensitizer to BPN in the second mixed solution is (3:10) to (7:20).

[0066] As an optional implementation, the photosensitizer is Ce6.

[0067] In another preferred embodiment of the present invention, the application of the aforementioned photodynamic synergistic targeted drug-loaded nanocomposite in the preparation of ablative biofilm drugs is provided.

[0068] In another preferred embodiment of the present invention, a nanodrug delivery system is provided, comprising the aforementioned photodynamic synergistic targeted drug-loaded nanocomposite.

[0069] The exemplary method for preparing the photodynamic-synergistic targeted drug-loaded nanocomposite of the present invention includes the following specific steps:

[0070] Flake-shaped BPN solution was prepared using liquid exfoliation technology:

[0071] Black phosphorus (BP) was dispersed in deionized water and dissolved oxygen in the dispersion was removed with nitrogen. Next, the BP dispersion was placed in an ultrasonic cell disruptor and ultrasonicated for 2 seconds every 4 seconds for 8 hours at 19-25 kHz and 15% power, during which time the process was carried out in an ice bath. After 8 hours of sharp ultrasonication, the BP dispersion was continuously ultrasonicated in an ultrasonic water bath at 10W power for 8 hours, during which time the process was carried out in an ice bath.

[0072] Then, centrifugation was performed to obtain black phosphorus nanosheets (BPN) of different sizes and thicknesses by adjusting the centrifugation speed. The precipitate was then resuspended in deoxygenated water and stored at 4°C in the dark to obtain BPN solution, which was ready for subsequent experiments.

[0073] Preparation of PMb solution:

[0074] Dissolve PMb in deionized water to prepare a 0.9–1.1 mg / mL PMb solution, and store it in a refrigerator at 4°C protected from light for later use.

[0075] Preparation of Ce6 solution:

[0076] Prepare a 4.9–5.1 mg / mL Ce6 solution by dissolving Ce6 in DMSO and store it in a refrigerator at 4°C protected from light.

[0077] Preparation of BPN dispersion:

[0078] Centrifuge the prepared flake BPN solution, discard the supernatant, and add deionized water to the precipitate to uniformly disperse the BPN, resulting in a BPN dispersion with a concentration of 100–120 μg / mL.

[0079] Preparation of PMb@BPN mixed solution:

[0080] The PMb solution was added to the BPN dispersion and ultrasonically vibrated to ensure that the BPN was uniformly dispersed in the mixed solution, thus obtaining the PMb@BPN mixed solution. At this time, PMb was adsorbed on the surface of the sheet-like BPN through electrostatic interaction.

[0081] Preparation of Ce6 & PMb@BPN:

[0082] Add Ce6 solution to PMb@BPN mixed solution, stir overnight at room temperature in the dark, remove excess free Ce6 by ultrafiltration using Amicon Ultra-15 centrifuge tube, wash 3 times with PBS buffer to obtain Ce6&PMb@BPN.

[0083] Combination Figure 1 As shown in part b, the nanocomposite of the present invention, after highly targeting a mixed bacterial biofilm containing Gram-negative bacteria with PMb, utilizes the two-dimensional sheet-like structure and sharp edges of BPN to directly insert into the bacterial biofilm matrix and disrupt the biofilm structure, causing the biofilm to rupture and break down, thus losing its integrity. On the other hand, combined with the high lipophilicity of black phosphorus nanosheets, it penetrates into the lipid layer of the bacterial biofilm, interfering with the structure and stability of the lipid layer, thereby disrupting the integrity of the biofilm. Furthermore, combined with the photothermal effect of BPN when exposed to light, it causes denaturation and destruction of lipids and proteins in the biofilm, leading to structural rupture and loss of function of the biofilm.

[0084] Based on the disruption of biofilm integrity by BPN, the nanocomposite enters the biofilm. On the one hand, the photosensitizer exerts photodynamic antibacterial effect under light conditions, penetrating and interfering with the thick cell wall of Gram-positive bacteria, causing intracellular substances to leak out, ultimately leading to bacterial colony inactivation.

[0085] In addition, the interaction between photosensitizers and the cell membranes and key enzyme targets of Gram-positive bacteria can disrupt bacterial integrity and interfere with their biological metabolic processes, thereby effectively inhibiting bacterial growth.

[0086] Furthermore, photosensitizers generate reactive oxides through photoactivation, which interfere with the cell structure of Gram-positive and Gram-negative bacteria and cause them to die, thereby achieving a bactericidal effect.

[0087] On the other hand, PMb in the system binds to lipid A in the cell membrane of Gram-negative bacteria, interfering with the integrity and function of the cell membrane and causing the death of Gram-negative bacteria, while increasing the sensitivity to Gram-positive bacteria and synergistically killing Gram-positive bacteria.

[0088] In this way, the ablation of mixed bacterial biofilms can be achieved through the coordinated action of the various parts of the nanocomposite.

[0089] The following will provide exemplary experiments and comparisons of the preparation and delivery performance of the aforementioned delivery system, using specific examples and tests.

[0090] Optionally, the PMb used in the following embodiments of the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and Ce6 was purchased from Frontier Scientific, USA. Of course, the embodiments of the present invention are not limited thereto.

[0091] Example 1

[0092] PMb solution: Weigh 10 mg of PMb and dissolve it in 10 mL of deionized water to prepare a 1 mg / mL PMb solution. Store the solution in a refrigerator at 4°C, protected from light, for later use.

[0093] Ce6 solution: Weigh 10 mg of Ce6 and dissolve it in 2 mL of DMSO to prepare a 5 mg / mL Ce6 solution. Store the solution in a refrigerator at 4°C protected from light.

[0094] Bulk BP crystals were prepared according to the literature. The specific procedures are as follows:

[0095] Tin, tin tetraiodide, and red phosphorus were sealed in quartz glass ampoules at a mass ratio of 2:1:50 and then placed in a muffle furnace. The furnace was first raised to 923 K and held for 1 hour, then cooled to 773 K and held for 1 hour, followed by a slower cooling to 550 K, and finally allowed to cool naturally to room temperature. This process yielded black crystalline BP, and residual mineralizing agents were removed by ultrasonic treatment with ethanol. Finally, the vacuum-dried BP was stored in argon gas for subsequent experiments.

[0096] Example 2

[0097] Flake-shaped BPN solution was prepared using liquid exfoliation technology:

[0098] First, 50 mg of BP (obtained by the method in Example 1) was added to 50 mL of deionized water, and then nitrogen was used to remove dissolved oxygen to prevent oxidation. Next, the BP dispersion was placed in an ultrasonic cell disruptor and sonicated for 8 hours at 25 kHz and 15% power, with 2 seconds every 4 seconds, during which time the process was carried out in an ice bath. After 8 hours of sharp sonication, the BP dispersion was continuously sonicated in an ultrasonic water bath at 10 W for 8 hours, during which time the process was carried out in an ice bath. Afterwards:

[0099] 1. Collect the supernatant and centrifuge at 12000 rpm for 30 min to collect the BPN (name it S-BPN);

[0100] 2. Collect the supernatant and centrifuge at 10,000 rpm for 15 min to collect the BPN (name it M-BPN);

[0101] 3. Collect the supernatant and centrifuge at 6000 rpm for 10 min to collect BPN (name it L-BPN).

[0102] The collected BPN was resuspended in deoxygenated water and stored at 4°C in the dark for use in subsequent experiments.

[0103] Example 3

[0104] Preparation of S-BPN dispersion:

[0105] The S-BPN prepared above was placed in a 50 mL centrifuge tube and centrifuged at 12000 rpm at 4 °C for 30 min, and then the supernatant was discarded.

[0106] DW was added to the precipitate to ensure uniform dispersion of S-BPN. The concentration of BPN was determined by ICP, and the concentration of S-BPN was 100 μg / mL.

[0107] Preparation of PMb@S-BPN:

[0108] Add 60 μL of PMb solution (prepared according to the method in Example 1) to 2 mL of S-BPN dispersion and sonicate for 15 min to ensure that S-BPN is uniformly dispersed in the mixed solution, thus obtaining PMb@S-BPN mixed solution.

[0109] Preparation of Ce@S-BPN:

[0110] Add 40 μL of Ce6 solution (prepared according to the method in Example 1) to 2 mL of S-BPN dispersion and stir overnight at room temperature in the dark. Remove excess free Ce6 by ultrafiltration using an Amicon Ultra-15 centrifuge tube, and wash three times with PBS buffer to obtain Ce6@S-BPN.

[0111] Preparation of Ce6 & PMb@S-BPN:

[0112] Add 40 μL of Ce6 solution (prepared by the method in Example 1) to the PMb@S-BPN mixed solution and stir overnight at room temperature in the dark.

[0113] Excess free Ce6 was removed by ultrafiltration using Amicon Ultra-15 centrifuge tubes, and the mixture was washed three times with PBS buffer to obtain Ce6&PMb@S-BPN.

[0114] Example 4

[0115] BPN thickness characterization

[0116] Atomic force microscopy (AFM) imaging of BPN samples on Si / SiO2 substrates was performed using an atomic force microscope (Asylum Research, USA) in AC mode in air. All BPN samples were characterized using AFM to evaluate their thickness. The thicknesses of S-BPN, M-BPN, and L-BPN ranged from 5-10 nm, 15-20 nm, and 20-50 nm, respectively; among them, the particle size and thickness of S-BPN are as follows: Figure 2 As shown.

[0117] Example 5

[0118] Investigation of the ablation capacity of BPN of different thicknesses for A. baumannii biofilm

[0119] To evaluate the ablation ability of different thicknesses on A. baumannii biofilms, this study employed crystal violet staining. First, mature biofilms were gently washed with PBS to remove free bacteria.

[0120] Next, 100 μL of BPN at different thicknesses (1 mg / mL) was added to 24-well plates and incubated at 37°C for 30 min to select the optimal BPN thickness. PBS served as the control group.

[0121] Subsequently, the perforated plate was exposed to an LED light (660nm, 1W / cm²). 2 After 10 min of light exposure, add crystal violet reagent for staining, and then treat with acetic acid (33%, w / v).

[0122] Finally, the absorbance of the supernatant was measured at 595 nm to quantitatively analyze the ablation effect.

[0123] Quantitative results of crystal violet staining are as follows Figure 3 As shown, when the BPN concentration is 100 μg / mL, BPN of different thicknesses all have varying degrees of ablation ability against *A. baumannii* biofilm. S-BPN exhibits superior ablation ability, with a significantly lower biofilm residue (55.7%) compared to the M-BPN (77.3%) and L-BPN groups (88.7%). This is because the thinner BPN has sharper edges that can more effectively break through the biofilm surface matrix, thus ablating the bacterial biofilm.

[0124] Example 6

[0125] Particle size, potential and absorption spectrum characterization

[0126] The black phosphorus nanosheets (S-BPN), PMb-modified black phosphorus nanosheets (PMb@S-BPN), Ce6-modified black phosphorus nanosheets (Ce6@S-BPN), and nanocomposite (Ce6&PMb@S-BPN) prepared according to the method in Example 3 were diluted with deionized water to prepare solutions with a concentration of 20 μg / mL, and then their particle size and potential were measured at 25 °C.

[0127] like Figure 4 As shown, the average particle size of S-BPN is 151.3 nm; the average particle size of PMb@S-BPN is 152.2 nm; the average particle size of Ce6@S-BPN is 157.9 nm; and the average particle size of Ce6&PMb@S-BPN is 160 nm.

[0128] The results showed that the hydrated particle size gradually increased with the modification of PMb and Ce6, that is, Ce6&PMb@S-BPN was successfully prepared.

[0129] like Figure 5As shown, the average potentials of BPN, PMb@S-BPN, Ce6@S-BPN, and Ce6&PMb@S-BPN are -25.8, -18.3, -28.2, and -21.7 mV, respectively. The zeta potential of Ce6&PMb@S-BPN is significantly higher than that of S-BPN and Ce6@S-BPN, which is due to the successful loading of positively charged PMb onto the BPN surface.

[0130] Example 7

[0131] Morphology test

[0132] Ce6&PMb@S-BPN prepared according to the method in Example 3 was dissolved in deionized water to form a solution with a concentration of 20 μg / mL (the concentration of S-BPN), and its morphology was then observed by transmission electron microscopy.

[0133] The morphological characterization results of transmission electron microscopy are as follows: Figure 6 As shown, Ce6&PMb@S-BPN exhibits a thin planar structure with black dot-like substances on its surface, which are the loaded drugs.

[0134] Example 8

[0135] Absorption spectroscopy characterization

[0136] BPNs, Ce6@S-BPN and Ce6&PMb@S-BPN, prepared according to the method in Example 3, were dispersed in 80% isopropanol and diluted to appropriate concentrations with PBS. Their UV-Vis absorption spectra in the wavelength range of 350 nm to 800 nm were then measured using a microplate reader.

[0137] like Figure 7 As shown, Ce6@S-BPN and Ce6&PMb@S-BPN exhibit two distinct absorption peaks, located at 400 nm and 663 nm, respectively. These two peaks correspond to the Soret band and Q band of Ce6, indicating that Ce6 was successfully loaded onto the surfaces of S-BPN and PMb@S-BPN.

[0138] As can be seen from Examples 5-7 above, the present invention successfully prepared black phosphorus nanosheets simultaneously loaded with PMb and photosensitizer.

[0139] Example 9

[0140] Cytotoxicity assay

[0141] Raw 264.7 and L929 cells were respectively loaded with 10 5The nanosheets were seeded at a density of 1000 cells / well in a 96-well plate and incubated for 12 h at 37 °C, 5% CO2, and 95% humidity. Next, different concentrations (2, 4, 6, 8, 10, 20, 30, 40, and 50 μg / mL) of Ce6&PMb@S-BPN nanosheets prepared according to the method in Example 3 were added to each well, and incubation continued for another 12 h. Afterward, the supernatant was removed, and 180 μL of culture medium and 20 μL of 5 mg / mL MTT solution were added, and incubation continued at 37 °C for 4 h. Then, the supernatant was removed, 150 μL of DMSO was added, and the plate was shaken for 10 min on a microplate reader until formazan was completely dissolved. Finally, the OD was measured. 570 The absorbance was measured. Six duplicate wells were set up, with the wells without Ce6&PMb@S-BPN nanosheets serving as a blank control group.

[0142] The results are as follows Figure 8 As shown, after 12 hours of co-incubation, when the concentration of Ce6&PMb@S-BPN was less than 30 μg / mL, the survival rates of L929 and Raw 264.7 cells were both greater than 90%. Even at higher concentrations (50 μg / mL, equivalent to BPN concentration), the relative cell viability of Ce6&PMb@S-BPN nanosheets after co-treatment with L929 and Raw 264.7 cells still exceeded 85%, indicating that Ce6&PMb@S-BPN of the present invention has good biocompatibility.

[0143] Example 10

[0144] Adhesion behavior on bacterial surfaces

[0145] To confirm the effect of PMb in enhancing the interaction between Ce6 & PMb@S-BPN and bacterial cells, flow cytometry was used to observe the adhesion behavior of Ce6 & PMb@S-BPN prepared according to the method in Example 3 on the bacterial surface.

[0146] The experimental procedure involved taking 900 μL of A. baumannii solution (10 8 The bacteria were incubated with 100 μL of Ce6 & PMb@S-BPN (Ce6 concentration of 10 μM) at 37 °C for 30 min. The bacteria were then centrifuged, washed twice with PBS, and the resuspended bacterial solution was analyzed by flow cytometry. PBS, Ce6, and Ce6 & S-BPN (Ce6 concentration of 10 μM) were used as control groups, with the same procedures as the Ce6 & PMb@S-BPN group.

[0147] The results are as follows Figure 9The results showed that free Ce6 and Ce6@S-BPN had limited absorption or adsorption capacity by *A. baumannii*, while Ce6 & PMb@S-BPN exhibited a significant enhanced interaction. Specifically, the fluorescence intensity of *A. baumannii* treated with Ce6 & PMb@S-BPN was significantly higher than that of the free Ce6 group and the Ce6@S-BPN group, being 3.87 times higher than that treated with Ce6@S-BPN.

[0148] This demonstrates that Ce6&PMb@S-BPN exhibits higher targeting specificity for A. baumannii compared to free Ce6 and Ce6@S-BPN.

[0149] Example 11

[0150] Permeability to biofilms

[0151] To investigate the permeability of Ce6&PMb@S-BPN to biofilms, the distribution of Ce6&PMb@S-BPN in A. baumannii biofilms was studied using CLSM.

[0152] After static incubation of the *A. baumannii* biofilm for 48 h, the supernatant culture medium was removed, and the remaining free bacteria were washed with PBS. Then, PBS, Ce6, Ce6@S-BPN prepared according to the method in Example 2, and Ce6&PMb@S-BPN (Ce6 concentration of 10 μM) were added to the biofilm, and incubated for 30 min. Unpermeated drugs were washed away with PBS, and then analyzed and quantified using CLSM.

[0153] Figure 10 The images show fluorescence images of *A. baumannii* after different treatments. In the biofilm treated with free Ce6, almost no red fluorescence is visible throughout, while in the biofilm treated with Ce6@S-BPN, weak red fluorescence is visible in localized areas. However, abundant and bright fluorescence was detected in the biofilm treated with Ce6&PMb@S-BPN.

[0154] Quantitative results ( Figure 11 The results showed that Ce6&PMb@S-BPN had a biomembrane penetration capacity three times that of Ce6@S-BPN. This may be because the PMb-mediated targeting effect caused the BPN nanosheets to rapidly disrupt the surface integrity of the biomembrane, creating a large number of pores in the extracellular polymeric material, thereby effectively accumulating Ce6.

[0155] This demonstrates that the Ce6&PMb@S-BPN of the present invention has strong biofilm permeability.

[0156] Example 12

[0157] In vitro ablation of A. baumannii biofilm experiment

[0158] To evaluate the ablation ability of Ce6&PMb@S-BPN prepared according to the method in Example 3 against A. baumannii biofilm, the dilution plate count method and crystal violet staining method were used for evaluation.

[0159] First, mature biofilms were gently washed with PBS to remove free bacteria. Next, 100 μL of Ce6 & PMb@S-BPN at a concentration of 400 μg / mL was added to 24-well plates and incubated at 37°C for 30 min to select the optimal antibacterial concentration. The control group consisted of PBS, PMb@S-BPN, and Ce6@S-BPN. Subsequently, the plates were exposed to an LED lamp (660 nm, 1 W / cm²). 2 After 10 min, the bacterial biofilm was dispersed in PBS and transferred to sterile EP tubes for vortex redispersion. After the above operations, 100 μL of the mixed bacterial suspension was diluted and plated on LB solid medium and incubated at 37°C for 24 h. Finally, the colony count was recorded.

[0160] The procedure for crystal violet staining assay is the same as that for plate counting assay, except that crystal violet reagent is added and a drying process is performed. The residual biofilm after crystal violet treatment is treated with acetic acid (33%, w / v), and the absorbance of the supernatant is measured at 595 nm for quantitative analysis of the ablation effect.

[0161] Quantitative results of crystal violet staining ( Figure 12 The results showed that the percentage of residual biofilm after treatment with 40 μg / mL Ce6 & PMb@S-BPN was 41.87%; after NIR irradiation, the ability to remove biofilm was further enhanced, leaving only 12.83% of the biofilm biomass. Furthermore, the percentage of residual biofilm in the PMb@S-BPN+NIR, Ce6@S-BPN, and Ce6@S-BPN+NIR groups all exceeded 60%.

[0162] Therefore, it can be seen that the Ce6&PMb@S-BPN of the present invention has a strong ability to ablate single bacterial biofilms under NIR light irradiation.

[0163] Coating data such as Figure 13 As shown, the number of bacteria was significantly reduced after Ce6&PMb@S-BPN+NIR treatment, with the bacterial count dropping to 4.52. Other groups of nanomedicine treatments also showed a certain degree of reduction in the number of bacteria. The bacterial count in the PMb@S-BPN+NIR group dropped to 7.74, while the bacterial count in the Ce6@S-BPN+NIR group only dropped to 8.09.

[0164] The above results fully demonstrate that the Ce6&PMb@S-BPN+NIR of the present invention can reduce bacterial activity within a single bacterial biofilm.

[0165] Example 13

[0166] In vitro ablation of A. baumannii-MRSA mixed biofilm experiment

[0167] To evaluate the ablation ability of Ce6&PMb@S-BPN prepared according to the method in Example 3 against A. baumannii-MRSA mixed biofilms, the dilution plate count method and crystal violet staining method were used for evaluation.

[0168] First, mature biofilms were gently washed with PBS to remove free bacteria. Next, 100 μL of Ce6 & PMb@S-BPN at a concentration of 400 μg / mL was added to 24-well plates and incubated at 37°C for 30 min to select the optimal antibacterial concentration. The control group consisted of PBS, PMb@S-BPN, and Ce6@S-BPN. Subsequently, the plates were exposed to an LED lamp (660 nm, 1 W / cm²). 2 After 10 min, the bacterial biofilm was dispersed in PBS and transferred to sterile EP tubes for vortex redispersion. After the above operations, 100 μL of the mixed bacterial suspension was diluted and plated on LB solid medium and incubated at 37°C for 24 h. Finally, the colony count was recorded.

[0169] The procedure for crystal violet staining assay is the same as that for plate counting assay, except that crystal violet reagent is added and a drying process is performed. The residual biofilm after crystal violet treatment is treated with acetic acid (33%, w / v), and the absorbance of the supernatant is measured at 595 nm for quantitative analysis of the ablation effect.

[0170] Similar to the results observed with ablation of single biofilms, the A. baumannii-MRSA mixed bacterial biofilm was effectively destroyed under the combined action of Ce6&PMb@S-BPN at a concentration of 40 μg / mL and NIR irradiation.

[0171] Figure 14 The figure represents the percentage of residual biofilm biomass after treatment. As can be seen from the figure, the percentage of biomass in the Ce6&PMb@S-BPN+NIR group decreased to 34.88%, while the residual biomass in the other four groups remained above 70%. Furthermore, the quantitative results of bacteria within the biofilm showed (…). Figure 15The mean logarithmic number of surviving colonies in the mixed bacterial biofilm of Ce6&PMb@S-BPN+NIR group decreased to 4.43, which was much lower than 7.44 in the PMb@S-BPN+NIR group and 8.18 in the Ce6@BPN+NIR group.

[0172] This indicates that the Ce6&PMb@S-BPN of the present invention can reduce bacterial activity in mixed bacterial biofilms under NIR light irradiation and has a strong ability to ablate mixed bacterial biofilms.

[0173] Example 14

[0174] In vivo ablation of A. baumannii-MRSA mixed biofilm experiment

[0175] To prepare the implant, 21G × 0.75” × 7” commercial catheters were first cut into 1 cm lengths, then immersed in 75% ethanol for 15 min for sterilization, and finally placed in a biosafety cabinet for UV sterilization and drying. The catheters were then placed in a mixed bacterial suspension of *A. baumannii*-MRSA and statically cultured in sugar-containing TSB medium at 37°C for 48 h. Afterwards, the catheter surfaces were washed with PBS to remove any loosely adhered bacteria.

[0176] To induce neutropenia in mice, cyclophosphamide (150 mg / kg) was injected intraperitoneally for 3 consecutive days before establishing a catheter infection model. A catheter coated with a biofilm of *A. baumannii* and MRSA was subcutaneously implanted into the back of the mouse, and the wound was sealed with physiological adhesive. Infection was allowed for 5 hours. Infected mice were divided into 7 groups: PBS+NIR group, PMb@S-BPN+NIR group, Ce6@S-BPN+NIR group, Ce6&PMb+NIR group, Ce6&PMb@S-BPN group, Ce6&PMb@S-BPN group (5 mg / kg, BPN at the same concentration), and PMb (3 mg / kg) group. In the Ce6&PMb+NIR group, the dosages of Ce6 and PMb were 1.63 mg / kg and 0.15 mg / kg, respectively. An equal volume of PBS was injected as a control group.

[0177] Then, the catheter implantation sites of the mice in the light-illuminated group were irradiated with an NIR laser (λ = 660 nm) for 15 min at a light intensity of 1 W / cm². 2 The mice were treated for 5 days, and their survival rate was recorded. After 5 days of treatment, the mice were euthanized, the implanted catheter was surgically removed, and tissue fluid from the catheter implantation site was collected using a sterile swab. The sterile swab was then immersed in 2 mL of LB liquid culture medium and placed in a constant temperature incubator with shaking for 4 h. 100 μL of the suspension was then used for dilution gradient plating and counting.

[0178] The survival rate of mice was recorded within 5 days of treatment. Results are as follows: Figure 16 As shown, the survival rate of mice in the Ce6&PMb@S-BPN+NIR group remained at 100% after 5 days of treatment. Furthermore, the bacterial count at the catheter implantation site was quantified using the dilution plate count method. Figure 17 As shown, compared with the PBS+NIR group, the bacterial counts after treatment with PMb@S-BPN+NIR, Ce6@S-BPN+NIR, PMb&Ce6+NIR, and Ce6&PMb@S-BPN decreased by only approximately 18-fold, 10-fold, 6-fold, and 1-fold, respectively. Meanwhile, the viable bacterial count after treatment with Ce6&PMb@S-BPN+NIR was 6.3 × 10⁻⁶. 5 The CFU / mL count was lower than that of viable bacteria after treatment with PMb (3 mg / kg) (6.8 × 10⁻⁶). 5 (CFU / mL)

[0179] The above results indicate that the Ce6&PMb@S-BPN+NIR group can reduce bacterial activity in mixed bacterial biofilms more effectively than high-dose PMb. The Ce6&PMb@S-BPN of the present invention can reduce bacterial activity in mixed bacterial biofilms in vivo under NIR light irradiation and has a strong ability to ablate mixed bacterial biofilms.

[0180] Example 15

[0181] Antibacterial mechanism

[0182] 1. To reveal the antibacterial mechanism of Ce6 & PMb@S-BPN, the reactive oxygen species probe DCFH-DA was used to assess the generation of intracellular ROS in bacterial cells, as ROS can directly affect the antibacterial effect.

[0183] First, bacteria were treated with 100 μL Ce6, Ce6@S-BPN and Ce6&PMb@S-BPN (Ce6 concentration of 10 μM) prepared according to the method in Example 2 at 200 rpm and 37 °C. 8 CFU / mL) for 30 min. After treatment, the bacterial pellet was collected by centrifugation, washed three times with PBS, and then treated with 10 μM DCFH-DA in the dark for 15 min. After staining, excess dye was washed away with PBS, and the ROS production capacity of bacteria after different sample treatments was evaluated by flow cytometry.

[0184] The results are as follows Figure 18The results showed that under 660nm light irradiation, the amount of ROS produced by bacteria treated with free Ce6 and Ce6@S-BPN was almost equal and limited. However, *A. baumannii* treated with Ce6&PMb@S-BPN+light irradiation exhibited a considerable amount of ROS production. Specifically, the ROS production of Ce6&PMb@S-BPN was 7.08 times and 8.26 times that of free Ce6 and Ce6@S-BPN treatments, respectively.

[0185] In summary, the Ce6&PMb@S-BPN of the present invention, through the synergistic effect of PMb-mediated chemotherapy and targeted PDT, when acting on mixed bacterial biofilms, the ROS generated by Ce6 kills positive bacteria. At the same time, through the targeting ability of PMb, the Ce6&PMb@S-BPN target site is driven to the negative bacteria site. The ROS generated by Ce6 can also accumulate around the negative bacteria as PMb targets the negative bacteria, thereby better killing both positive and negative bacteria simultaneously through PDT.

[0186] 2. The dilution plate count method was used to conduct antibacterial research on MRSA.

[0187] The specific experimental steps are as follows:

[0188] 900 μL MRSA (10 6 CFU / mL was mixed with 100 μL of PMb, PMb@S-BPN, Ce6&PMb, and Ce6&PMb@S-BPN (20 μg / mL, equivalent to BPN concentration) and incubated for 30 min. Then, 100 μL of the mixed bacterial suspension was obtained, diluted, and spread onto LB agar plates, and incubated at 37°C for 24 h to count colonies. The PBS-treated group served as a negative control.

[0189] like Figure 19 As shown, compared with the PBS group, the PMb group and the Ce6&PMb group exhibited negligible antibacterial effects, with MRSA survival rates all exceeding 90%. The PMb@S-BPN group showed a relatively lower MRSA survival rate, with an average survival rate of 84.6%. The Ce6&PMb@S-BPN group demonstrated the strongest antibacterial activity, with a MRSA survival rate of only 65.2% after treatment.

[0190] This indicates that the Ce6&PMb@S-BPN system of the present invention has the ability of PMb to fight both Gram-positive and Gram-negative bacteria simultaneously, and the antibacterial effect of PMb against Gram-positive bacteria is improved under the conditions of this system.

[0191] Therefore, it can be seen that the photodynamic synergistic targeted drug-loaded nanocomposite of the present invention achieves ablation of mixed bacterial biofilms through the linkage of various parts of the nanocomposite.

[0192] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A photodynamic-synergistic targeted drug-loaded nanocomposite, characterized in that, The nanocomposite includes a carrier, polymyxin B (PMb), and a photosensitizer; The carrier is black phosphorus nanosheets (BPN), which are prepared into sheets with a thickness of 5 nm to 50 nm using liquid exfoliation technology. The preparation process is as follows: Black phosphorus (BP) was dispersed in deionized water and dissolved oxygen in the dispersion was removed with nitrogen. Next, the BP dispersion was placed in an ultrasonic cell disruptor and ultrasonicated for 2 seconds every 4 seconds for 8 hours at 19-25 kHz and 15% power, during which time the process was carried out in an ice bath. After 8 hours of sharp ultrasonication, the BP dispersion was continuously ultrasonicated in an ultrasonic water bath at 10W power for 8 hours, during which time the process was carried out in an ice bath. The precipitate was then centrifuged to obtain black phosphorus nanosheets (BPN), and the precipitate was resuspended in deoxygenated water and stored at 4°C in the dark to obtain BPN solution. The polymyxin B and the photosensitizer were sequentially adsorbed onto the carrier via electrostatic interaction to obtain the nanocomposite; the photosensitizer was Ce6. Specifically, the two-dimensional sheet structure and sharp edges of BPN are used to directly insert into the mixed bacterial biofilm matrix to destroy the biofilm structure, causing the biofilm to rupture and break down, thus losing its integrity. Under the premise of BPN destroying the mixed bacterial biofilm, combined with PMb and photosensitizer, it causes the death of Gram-negative bacteria while increasing the sensitivity to Gram-positive bacteria, synergistically killing Gram-positive bacteria, effectively killing both Gram-positive and Gram-negative bacteria, and achieving the ablation of the mixed bacterial biofilm.

2. The photodynamic synergistic targeted drug-loaded nanocomposite according to claim 1, characterized in that, In the nanocomposite, the mass content of polymyxin B is 2.3%~2.6%, and the mass content of photosensitizer is 22.6%~25.4%.

3. The photodynamic synergistic targeted drug-loaded nanocomposite according to claim 1, characterized in that, The nanosheets have a particle size of 150 nm to 170 nm and a potential of -26 mV to -18 mV.

4. A method for preparing a photodynamic synergistic targeted drug-loaded nanocomposite according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Disperse black phosphorus (BP) in deionized water and remove dissolved oxygen from the solution with nitrogen to obtain BP dispersion; The BP dispersion was ultrasonically exfoliated under ice bath conditions, followed by centrifugation to remove unexfoliated BP and obtain BPN. S2. Add polymyxin B (PMb) to the dispersion of BPN, so that PMb is adsorbed onto BPN through electrostatic interaction to form PMb@BPN, and obtain the first mixed solution, and PMb@BPN is uniformly dispersed in the first mixed solution. S3. Add the photosensitizer to the first mixed solution obtained in step S2, so that the photosensitizer is adsorbed onto PMb@BPN through electrostatic interaction to obtain the second mixed solution. After removing the free photosensitizer by ultrafiltration, the second mixed solution was washed and redispersed to obtain a dispersion of the desired nanocomposite.

5. The method for preparing the photodynamic synergistic targeted drug-loaded nanocomposite according to claim 4, characterized in that, In the BPN dispersion, the concentration of BPN is 100 μg / mL to 120 μg / mL.

6. The method for preparing the photodynamic synergistic targeted drug-loaded nanocomposite according to claim 4, characterized in that, In the first mixed solution, the mass ratio of PMb to BPN is (1:25) to (1:33); in the second mixed solution, the mass ratio of photosensitizer to BPN is (3:10) to (7:20), and the photosensitizer is Ce6.

7. The use of the photodynamic synergistic targeted drug-loaded nanocomposite according to any one of claims 1-3 in the preparation of ablation biofilm drugs.

8. A nanomedicine delivery system, characterized in that, The photodynamic synergistic targeted drug-loaded nanocomposite as described in any one of claims 1-3.

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