A wound repair material for multiple bacterial infections and a preparation method and application thereof

Abstract

The application discloses a wound repair material for multiple bacterial infections and a preparation method and application thereof, and belongs to the technical field of biomaterials. The gallium-polyphenol nanoparticle (GaED NP) without a carrier is prepared by one-step self-assembly of trivalent gallium, diethyl dithiocarbamate (DEDTC) and natural polyphenol epigallocatechin gallate (EGCG), can improve the oxidation instability problem of the natural polyphenol, and significantly improves the bioavailability. The gallium-polyphenol nanoparticle has strong bacteriostatic effect and good biological safety, can effectively inhibit the formation of a biofilm, can destroy a mature mixed bacterial biofilm, and can accelerate the healing of a skin wound infected by multiple bacteria.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a wound repair material for multiple bacterial infections, its preparation method, and its application. Background Technology

[0002] Biofilm-associated bacterial infections pose a significant threat to global public health. Skin, as the body's primary physical barrier against exogenous pathogens, is susceptible to damage from surgery and trauma, providing entry points for bacteria. Common pathogens include Gram-negative bacteria (Pseudomonas aeruginosa), Gram-positive bacteria (Staphylococcus aureus), and drug-resistant bacteria (methicillin-resistant Staphylococcus aureus (MRSA)), which can colonize wounds individually or in combination, forming difficult-to-remove biofilms. Biofilms are structured bacterial communities encapsulated within a matrix of secreted extracellular polymers, inducing wound infection and delaying healing. Compared to planktonic bacteria, bacteria encapsulated in biofilms exhibit significantly increased resistance to antibiotics, directly leading to prolonged treatment cycles, increased medical costs, and higher infection rates and mortality. Adding to the woes, the development of new antibiotics lags far behind the rapid evolution of "superbugs," necessitating the development of novel formulations to meet the treatment needs of wounds with multiple bacterial infections. Meanwhile, antibiotic resistance has become an increasingly serious threat to the treatment of wounds with multiple bacterial infections.

[0003] Pathogenic bacteria are highly dependent on iron to maintain core physiological processes such as metabolism, respiration, and proliferation. Trivalent gallium (Ga(III)) and trivalent iron (Fe(III)) have highly similar physicochemical properties, including ionization energy, electron affinity, and octahedral / tetrahedral ionic radius, allowing gallium to competitively dominate bacterial iron uptake and utilization pathways. Iron can cycle between trivalent Fe(III) and divalent Fe(II) oxidation-reduction, while gallium exists only in a stable trivalent oxidation state. When bacteria mistakenly ingest Ga(III) into iron-dependent pathways, it disrupts their own iron metabolism, damages iron-containing enzymes, and thus inhibits bacterial growth and survival. More importantly, antibacterial therapies targeting iron metabolism are less likely to induce drug resistance because iron-dependent physiological processes are essential for the survival and proliferation of pathogenic bacteria. However, existing gallium compounds and gallium complexes readily hydrolyze at physiological pH (approximately 7.4) to form gallium hydroxide (Ga(OH)3) and tetrahydroxygallate (Ga(OH)4). - This significantly reduces the bioavailability and pharmacological activity of gallium; furthermore, free trivalent gallium can interfere with normal cellular iron metabolism, causing off-target tissue toxicity. Gallium ions (trivalent gallium, Ga(III)), as a competitive inhibitor of oxidizing inert trivalent iron, can exert antibacterial effects by disrupting bacterial iron metabolism, with a low risk of inducing drug resistance. However, traditional gallium preparations (such as gallium compounds and gallium complexes) still face challenges such as low bioavailability, limited efficacy, and off-target safety risks. Summary of the Invention

[0004] To address the aforementioned deficiencies, this invention proposes a wound repair material for multiple bacterial infections and its preparation method. Through one-step self-assembly of trivalent gallium, diethyl dithiocarbamate (DEDTC), and the natural polyphenol epigallocatechin gallate (EGCG), carrier-free gallium-polyphenol nanoparticles (GaED NPs) are prepared. This improves the oxidative instability of natural polyphenols, significantly enhancing bioavailability. It exhibits strong antibacterial activity and good biosafety, effectively inhibiting biofilm formation, disrupting mature mixed bacterial biofilms, and accelerating the healing of skin wounds infected with multiple bacteria.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] The first objective of this invention is to provide a wound repair material for multiple bacterial infections, comprising composite nanoparticles GaED NPs formed from epigallocatechin gallate, diethyl dithiocarbamate, and trivalent gallium.

[0007] The second objective of this invention is to provide a method for preparing a wound repair material for multiple bacterial infections, comprising the following steps: mixing an aqueous solution of epigallocatechin gallate with an aqueous solution of diethyldithiocarbamate at room temperature, then adding an aqueous solution of trivalent gallium, allowing the system to spontaneously assemble for 1-2 minutes, washing the mixture with deionized water, and freeze-drying to obtain composite nanoparticles.

[0008] Furthermore, the concentration of the aqueous solution of epigallocatechin gallate is 2 mmol / L.

[0009] Furthermore, the concentration of the aqueous solution of diethyldithiocarbamate is 1 mmol / L.

[0010] Furthermore, the trivalent gallium is gallium nitrate.

[0011] Furthermore, the concentration of the trivalent gallium aqueous solution is 10 mmol / L.

[0012] Furthermore, the volume ratio of the epigallocatechin gallate aqueous solution, the diethyldithiocarbamate aqueous solution, and the trivalent gallium aqueous solution is 3:1:0.5-1. Preferably, the volume ratio of the epigallocatechin gallate aqueous solution, the diethyldithiocarbamate aqueous solution, and the trivalent gallium aqueous solution is 3:1:0.8.

[0013] The third objective of this invention is to provide an application of a wound repair material for multiple bacterial infections in the preparation of antibacterial biofilms, or antimicrobial drugs or materials.

[0014] This invention discloses a wound repair material for multiple bacterial infections, its preparation method, and its application. The beneficial effects are as follows:

[0015] (1) EGCG is the catechin with the highest content in green tea. It has antibacterial, antioxidant and anti-inflammatory activities. However, the phenolic hydroxyl group is easily oxidized and degraded, and the physicochemical stability is poor. The composite nanoparticles GaED NPs obtained in this invention can effectively inhibit the hydrolysis of trivalent gallium under physiological conditions, alleviate the oxidative degradation of EGCG, and improve the bioavailability of the drug. At the same time, after acting on the infected wound, the three active substances Ga(III), DEDTC and EGCG released by decomposition can synergistically fight bacteria.

[0016] (2) GaED NPs have broad-spectrum antibacterial activity against common pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA), and can effectively inhibit biofilm formation and destroy mature mixed bacterial biofilms; the composite nanoparticles of this invention can accelerate the healing of skin wounds infected by multiple bacteria.

[0017] (3) This invention prepares carrier-free composite nanoparticles (GaED NPs) through one-step self-assembly of trivalent gallium, diethyl dithiocarbamate (DEDTC) and natural polyphenol epigallocatechin gallate (EGCG). The preparation method is simple and efficient. The three antibacterial components released by the system, namely trivalent gallium, DEDTC and EGCG, can synergistically exert antibacterial effects, and have good biosafety and biocompatibility. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This section presents the physical characterization of GaED NPs composite nanoparticles. A shows a schematic diagram of the GaED NPs synthesis process; B shows photographs of EGCG, DEDTC, gallium nitrate, and GaED NPs dispersed in deionized water; C shows the UV-Vis spectrum of GaED NPs; D and E show the Fourier transform infrared spectra of GaED NPs; F shows the energy spectrum of GaED NPs; G shows the dynamic light scattering (DLS) particle size distribution of GaED NPs; H shows the Zeta potential of GaED NPs; I shows the SEM image of GaED NPs; J shows the typical TEM morphology of GaED NPs; K shows the TEM-EDS elemental mapping of GaED NPs; and L, M, N, and O show the X-ray photoelectron energy (XPS) spectra of GaED NPs.

[0020] Figure 2The in vitro antibacterial activity of GaED NP composite nanoparticles is shown in Figure A, where A represents colony images of Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa after treatment with different samples at a concentration of 100.0 μg / mL; B represents the colony counts for each group; C represents the relative survival rate of the corresponding bacteria in each group; D represents the turbidity of bacterial solutions treated with different concentrations of GaED NPs; E represents the colony results on agar plates; F represents the bacterial growth curves of Staphylococcus aureus (S. aureus) after treatment with gradient concentrations of GaED NPs; G represents the bacterial growth curves of MRSA after treatment with gradient concentrations of GaED NPs; H represents the bacterial growth curves of Pseudomonas aeruginosa (P. aeruginosa) after treatment with gradient concentrations of GaED NPs; and I represents the relative survival rate of Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa after treatment with gradient concentrations of GaED NPs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0021] Figure 3 The images depict the inhibition and destruction of biofilms by GaED NPs composite nanoparticles. A shows a schematic diagram of the mechanism by which GaED NPs inhibit biofilm formation and degrade mature biofilms; B shows crystal violet-stained images of newly formed biofilms from Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa treated with different materials; C shows quantitative statistics of biofilm biomass; D shows images of mature biofilms degraded by different materials; E shows quantitative analysis of mature biofilm biomass for each bacterium; F shows confocal imaging of live bacteria stained with SYTO9 green fluorescent nucleic acid dye to evaluate the removal effect of each group on newly formed and mature biofilms of mixed bacteria. *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0022] Figure 4 This study evaluated the in vivo wound repair efficacy of GaED NPs composite nanoparticles against multiple bacterial infections. A shows a schematic diagram of the experimental procedure involving treatment with GaED NPs on wounds infected with Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa; B shows representative photographs of wounds from each group of mice, with embedded contour maps illustrating dynamic changes in wound healing (scale bar: 10 mm); C shows dynamic statistics of wound healing area in each group; D shows a comparison of wound area at the treatment endpoint among the groups; E shows hematoxylin-eosin (H&E) stained sections and Masson trichrome stained sections of wound edge tissue from each group, with blue arrows indicating wound length; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0023] Figure 5Histological quantitative analysis of markers related to wound inflammation, tissue repair, and angiogenesis in each group was performed. A represents the number of infiltrating inflammatory cells in each group's H&E sections; B represents the density of new blood vessels in each group; C represents the collagen deposition rate in each group's Masson-stained sections; D represents CD31 immunofluorescence imaging (vascular endothelial labeling) of infected wounds in each group; E represents the statistical analysis of the mean CD31 fluorescence intensity (MFI) in each group; F represents α-SMA immunofluorescence imaging (myofibroblast labeling) in each group; G represents the statistical analysis of the mean α-SMA fluorescence intensity in each group. Scale bar: 50 μm. *p < 0.05, ****p < 0.0001;

[0024] Figure 6 To evaluate the biocompatibility of GaED NPs composite nanoparticles, A represents the cell viability of NIH-3T3 cells after co-culturing with different concentrations of GaED NPs for 24 h, with the x-axis representing the GaED NPs concentration gradient (0, 25, 50, 75, 100, 200, 400 μg / mL). B represents the cell viability of NIH-3T3 cells after co-culturing with different concentrations of GaED NPs for 48 h, with the x-axis representing the GaED NPs concentration gradient (0, 25, 50, 75, 100, 200, 400 μg / mL). C represents the quantitative statistics of in vitro hemolysis experiments with different concentrations of GaED NPs, with an embedded hemolysis diagram. Ctrl (for the saline negative control group) and the 25-400 μg / mL gradient represent GaED NPs. NPs group and DI are deionized water positive control groups; D is an in vitro coagulation qualitative test diagram, where Ctrl is the CaCl2+ rabbit whole blood blank control group and the 25-400μg / mL gradient is the GaED NPs experimental group; E is the H&E pathological sections of the main organs of mice in each group (heart, liver, spleen, lung, kidney), scale bar: 100μm; F is the dynamic detection of blood routine indicators of mice in each group during the experimental period (days 0, 2, 4, 6, 8, and 10).

[0025] Figure 7 Optical images of EGCG, DEDTC, Ga(NO3)3 and GaED NPs dispersed in deionized water, where the obvious Tyndall effect indicates that GaED nanoparticles have been formed.

[0026] Figure 8The water solubility and stability of the composite nanoparticles GaED NPs were investigated. In aqueous solution (pH 7.4), the dissociated EGCG was rapidly oxidized, and Ga(III) ions underwent significant hydrolysis, while the GaED nanoparticles maintained good water solubility and stability: (A) Optical image of the composite nanoparticles GaED NPs in aqueous solution (pH 7.4); (B) Optical image of the composite nanoparticles GaED NPs in acidic environment (pH 5.5); (C) DLS analysis of the composite nanoparticles GaED NPs in aqueous solution (pH 7.4); and (D) zeta potential analysis of the composite nanoparticles GaED NPs in aqueous solution (pH 7.4).

[0027] Figure 9 The colony morphology of Staphylococcus aureus after different sample treatments (100.0 μg / mL).

[0028] Figure 10 Colony morphology of MRSA after different sample treatments (100.0 μg / mL).

[0029] Figure 11 Colony morphology of Pseudomonas aeruginosa after different sample treatments (100.0 μg / mL).

[0030] Figure 12 The minimum inhibitory concentration (MIC) of GaED NPs against Staphylococcus aureus is given.

[0031] Figure 13 The minimum inhibitory concentration (MIC) of GaED NPs against MRSA.

[0032] Figure 14 The minimum inhibitory concentration (MIC) of GaED NPs against Pseudomonas aeruginosa.

[0033] Figure 15 The antibacterial activity of GaED NPs against Staphylococcus aureus at different concentrations was investigated.

[0034] Figure 16 Antibacterial activity of GaED NPs against MRSA at different concentrations;

[0035] Figure 17 The antibacterial activity of GaED NPs against Pseudomonas aeruginosa at different concentrations;

[0036] Figure 18 The relative colony counts of Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa after treatment with different concentrations of GaED NPs;

[0037] Figure 19 To investigate the inhibitory effects of different samples on Staphylococcus aureus (S. aureus) biofilm formation;

[0038] Figure 20 The inhibitory effects of different samples on MRSA biofilm formation;

[0039] Figure 21 To investigate the inhibitory effects of different samples on biofilm formation by Pseudomonas aeruginosa;

[0040] Figure 22 To investigate the effects of different samples on the destruction of existing Staphylococcus aureus biofilms and to quantitatively analyze the OD570 of the corresponding biofilms;

[0041] Figure 23 To analyze the effects of different samples on the destruction of existing biofilms in MRSA biofilms and to quantitatively analyze the OD570 of the corresponding biofilms;

[0042] Figure 24 To investigate the effects of different samples on the destruction of biofilms formed by Pseudomonas aeruginosa and to quantitatively analyze the OD570 of the corresponding biofilms;

[0043] Figure 25 Visualize the penetrability of different samples in a gelatin tissue model using colorimetric methods;

[0044] Figure 26 Changes in body weight of mice in different sample treatment groups;

[0045] Figure 27 The image shows the results of a 96-well plate assay (3 replicates per group) for the coagulation of blood by GaED NPs composite nanoparticles. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Example 1

[0048] Wound repair materials for multiple bacterial infections include composite nanoparticles GaED NPs formed from epigallocatechin gallate, diethyl dithiocarbamate, and gallium nitrate.

[0049] In this embodiment, the composite nanoparticles GaED NPs are prepared as follows: 600 μL of 2 mmol / L EGCG aqueous solution (pH 8–9) is mixed with 200 μL of 1 mmol / L DEDTC solution, and 160 μL of 10 mmol / L gallium nitrate aqueous solution is added dropwise. The system spontaneously assembles to generate GaED NPs. The mixture is washed and freeze-dried at -60 to -80°C to obtain composite nanoparticles GaED NPs.

[0050] Physical property testing of GaED composite nanoparticles NPs

[0051] EGCG, DEDTC, and gallium nitrate are all colorless and transparent solutions when dissolved in water individually. Figure 1 B); After mixing, EGCG, acting as a coordinating ligand, and DEDTC, acting as a coordination modifier, rapidly form a metal-polyphenol network with trivalent gallium. The system exhibits a unique light purple color, and the Tyndall effect can be observed in aqueous solution. Figure 7 GaED NPs can be uniformly dispersed in neutral physiological solution (pH 7.4) without significant precipitation within several days, demonstrating excellent solubility and stability. Figure 8 In contrast, free EGCG undergoes rapid oxidation due to a large number of phenolic hydroxyl groups, while free trivalent gallium undergoes violent hydrolysis within 24 hours in neutral solution. Figure 8 Incubating GaED NPs in a pH 5.5 aqueous solution (simulating the acidic microenvironment of an infected wound) for 24 h resulted in significant degradation of the nanoparticles, facilitating the subsequent release of trivalent gallium, EGCG, and DEDTC. Figure 8 ).

[0052] The coordination of nanoparticles was analyzed using ultraviolet-visible spectroscopy (UV-vis) and Fourier transform infrared spectroscopy (FTIR). For example... Figure 1 As shown in Figure C, the characteristic ultraviolet absorption peak of GaED NPs is red-shifted, proving that trivalent gallium undergoes coordination bonding with EGCG and DEDTC; the infrared spectrum at 1250 cm⁻¹... -1 CN characteristic peak appears at 1210 cm⁻¹ -1 A CO peak appeared at 3360 cm⁻¹. -1 The peak at 1630 cm⁻¹ is a broad peak representing the phenolic hydroxyl C-OH group. -1 The presence of a carbonyl C=O peak confirms the successful integration of EGCG and DEDTC into GaED nanoparticles. Figure 1 D, 1E (Table 1).

[0053] Dynamic light scattering (DLS) was used to further verify the morphology and elemental composition of the nanoparticles. The results showed that the hydrodynamic particle size of GaEDNPs was approximately 110.0 nm. Figure 1G); the Zeta potentials of EGCG, DEDTC, free Ga(III), and GaED NPs were -8.57 ± 1.51 mV, -20.76 ± 1.11 mV, 13.14 ± 1.52 mV, and -38.70 ± 3.58 mV, respectively. These potential changes not only confirm successful coordination assembly but also reflect the high stability of the nanoparticles in aqueous solution. Figure 1 H).

[0054] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images confirmed that GaED NPs had uniform particle size and good dispersion. Figure 1 I, Figure 1 J); Energy dispersive spectroscopy (EDS) and transmission electron microscopy elemental mapping detected gallium, carbon, oxygen, nitrogen, and sulfur, all originating from the raw material components (J). Figure 1 F, Figure 1 K).

[0055] X-ray photoelectron spectroscopy (XPS) further verified the elemental composition and coordination bonds of the nanoparticles: characteristic peaks of C, O, S, and Ga were detected, along with corresponding signals for Ga-O and Ga-S coordination bonds. Figure 1 L, Figure 1 M, Figure 1 N、 Figure 1 O).

[0056] Table 1. FTIR characteristic bands of DEDTC, EGCG and GaED NPs

[0057]

[0058] Example 2

[0059] In vitro antibacterial activity of GaED NP composite nanoparticles

[0060] Three standard bacterial strains were selected: *Staphylococcus aureus* (ATCC 25923), *MRSA* (ATCC 43300), and *Pseudomonas aeruginosa* (ATCC 27853). After bacterial resuscitation, the bacteria were inoculated onto LB (Luria-Bertani) liquid medium and cultured at 37°C and 180 r / min in a shaker until the logarithmic growth phase. The bacterial suspension was then diluted with sterile LB medium to adjust the final bacterial concentration to 1 × 10⁻⁶. 6 CFU / mL (CFU, colony-forming unit) is determined by plate colony counting, bacterial growth curve determination, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC).

[0061] Plate colony counting method: The bacterial suspension was diluted to a bacterial density of approximately 5 × 10³ CFU / mL and co-incubated with different samples (EGCG, DEDTC, gallium nitrate, vancomycin, GaED NPs, final concentration 100.0 μg / mL) for 4 h. 100 μL of the treated bacterial suspension was spread onto LB agar plates and incubated at 37 °C for 24 h. The colony count was then recorded and photographed. Parallel experiments were also conducted using gradient concentrations of GaED NPs (0.0, 25.0, 50.0, 75.0, 100.0 μg / mL).

[0062] Growth curve method: Dilute the bacterial culture to OD 600 =0.1 (absorbance at 600 nm), add gradient concentrations of GaED NPs to 96-well plates, measure the absorbance at 600 nm every 1 hour using a microplate reader, and record the bacterial growth curve.

[0063] Minimum inhibitory concentration (MIC): GaED NPs were initially diluted 1000.0 μg / mL, then serially diluted twice, and co-incubated with bacteria at a density of 5 × 10³ CFU / mL to determine the minimum inhibitory concentration for each strain.

[0064] Experimental group setup:

[0065] Blank control group (Control group): (LB bacterial culture only);

[0066] Free EGCG group: An equal volume of 100.0 μg / mL EGCG aqueous solution was added to LB bacterial culture;

[0067] Free DEDTC group: An equal volume of 100.0 μg / mL DEDTC aqueous solution was added to LB bacterial culture;

[0068] Trivalent gallium ion (Ga(NO3)3) group: An equal volume of gallium nitrate aqueous solution of 100.0 μg / mL was added to the LB bacterial culture;

[0069] Positive control group (Van group): An equal volume of vancomycin (Van) solution of 100.0 μg / mL was added to LB bacterial culture;

[0070] GaED NPs experimental group: Add an equal volume of 100.0 μg / mL GaED NPs aqueous solution to LB bacterial culture.

[0071] Each group was incubated on a shaker at 37℃ and 180 r / min. After incubation, colony counting was performed using the serial dilution plating method, and colony photographs visually confirmed the results. Figure 2 A, Figure 9-11 );from Figure 2As can be seen from A, compared with the positive control group (commercial antibacterial drug vancomycin), EGCG, DEDTC, and Ga(III) form GaED NPs with stronger synergistic antibacterial and bacteriostatic effects.

[0072] The concentration-dependent antibacterial effect of GaED NPs was investigated by setting final concentration gradients of GaED NPs: 0, 25.0, 50.0, 75.0, and 100.0 μg / mL, and a final concentration of 1×10⁻⁶ μg / mL. 6 The LB bacterial culture was mixed with CFU / mL and incubated at 37°C. The absorbance at 600 nm was measured using a microplate reader, and the turbidity of the culture medium was recorded every 1 hour to plot the bacterial growth curve (Figure 2D, Figures 2F-2H). The results showed that the proliferation of Gram-positive bacteria (Staphylococcus aureus, MRSA) was significantly inhibited with increasing nanoparticle concentration. However, the concentration-dependent antibacterial effect was weak against the Gram-negative model bacterium Pseudomonas aeruginosa, mainly due to its outer membrane barrier.

[0073] The MICs of GaED NPs against Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa were 62.5, 62.5, and 125.0 μg / mL, respectively. Figure 12-13 Plate count results showed that 75.0 μg / mL GaED NPs completely inhibited the growth of Staphylococcus aureus and MRSA, and this concentration is the MBC of the two bacteria (…). Figure 2 E, 2I, Figure 15 , Figure 16 , Figure 18 Complete eradication of Pseudomonas aeruginosa requires even higher concentrations (>100.0 μg / mL). Figure 2 E, 2I, Figure 17 , Figure 18 ).

[0074] In summary, GaED NPs exhibit selective antibacterial activity, with significantly better inhibitory effects against Gram-positive bacteria than against Gram-negative bacteria.

[0075] Example 3

[0076] The ability of GaED composite nanoparticles to inhibit biofilm formation and remove mature biofilms

[0077] Grouping:

[0078] Blank control group (Control group): (bacterial solution only);

[0079] Free EGCG group: An equal volume of 100.0 μg / mL EGCG aqueous solution was added to the bacterial culture;

[0080] Free DEDTC group: An equal volume of 100.0 μg / mL DEDTC aqueous solution was added to the bacterial culture;

[0081] Trivalent gallium ion (Ga(NO3)3) group: Add an equal volume of 100.0 μg / mL gallium nitrate aqueous solution to the bacterial culture;

[0082] Positive control group (Van group): An equal volume of 100.0 μg / mL vancomycin solution was added to the bacterial culture;

[0083] GaED NPs experimental group: Add an equal volume of 100.0 μg / mL GaED NPs aqueous solution to the bacterial culture.

[0084] This embodiment uses two types of biofilm models, single-species and mixed-species, to evaluate the effects of GaED NPs on inhibiting biofilm formation and disrupting mature biofilms. Figure 3 A).

[0085] The inhibitory and scavenging effects of EGCG, DEDTC, trivalent gallium ions (Ga(NO3)3), vancomycin (Van), and GaED NPs on biofilms of single bacterial species *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and methicillin-resistant *Staphylococcus aureus* (MRSA) were detected using crystal violet staining. After treatment with 100.0 μg / mL of materials in each experimental group, crystal violet staining was used for imaging. Figure 3 B Figure 19-21 ) and quantitative statistics of biofilms ( Figure 3 C) Clearly demonstrates the differences in biofilm inhibition effects among the groups.

[0086] GaED NPs significantly inhibited biofilm formation in three types of bacteria, with the core mechanism being the continuous release of multiple active components, including EGCG, DEDTC, and trivalent gallium ions, from the nanoparticles. Simultaneously, GaED NPs also exhibited a strong ability to disrupt mature biofilms. Figure 3 D, 3E, Figure 22-24 This is attributed to the stronger biomembrane penetration performance of nanoparticles. Figure 25 ).

[0087] The SYTO9 live bacteria fluorescence staining method was used to verify the inhibitory and scavenging effects of EGCG, DEDTC, trivalent gallium ions (Ga(NO3)3), vancomycin (Van), and GaED NPs on mixed biofilms of these three bacteria. Laser confocal microscopy imaging was employed. Figure 3 F) shows that the blank control group (untreated) has a dense and intact biofilm structure with extremely strong green fluorescence signal; the DEDTC group, Ga(NO3)3 group, and vancomycin (Van) group have a weak effect on inhibiting biofilm formation and destroying mature biofilms; free EGCG, with its own antibacterial activity, can inhibit biofilm formation and disintegrate mature biofilms to a certain extent.

[0088] The three-bacterial mixed biofilm treated with GaED NPs showed almost complete dissolution of its membrane structure, whether it was a newly formed biofilm or a mature biofilm, leaving only a weak green fluorescence. Its anti-biofilm performance was far superior to the other groups. This superior performance stemmed from the fact that the nanoparticles enhanced the bioavailability of the active components and produced a synergistic antibacterial effect: destroying bacterial cell membranes, interfering with bacterial energy metabolism, and efficiently clearing biofilm infections.

[0089] In summary, GaED NPs can effectively inhibit biofilm formation and remove mature biofilms, with particularly outstanding advantages in complex wound environments with mixed bacterial infections.

[0090] Example 4

[0091] Effects of GaED NPs composite nanoparticles on wound repair in vivo with multiple bacterial infections

[0092] A mouse model of multi-species full-thickness skin infection was constructed to evaluate the in vivo wound-healing ability of GaED NPs. Figure 4 A).

[0093] Model Construction: 6–8 week old SPF (specific pathogen-free) grade female ICR mice were used. After hair removal from the back of the mice, a 10 mm diameter full-thickness skin wound was prepared using a circular punch. The wound was inoculated with a mixed bacterial suspension of Staphylococcus aureus, Pseudomonas aeruginosa, and MRSA. 24 h after infection (when obvious redness and swelling appeared), the mice were randomly divided into 6 groups (n=5 per group). Each group was then given a different formulation for local administration, and the treatment was monitored throughout. The local administration concentration for each group was uniformly 100.0 μg / mL, administered for 3 consecutive days (days 1, 2, and 3), 25 μL each time. No significant fluctuations in mouse body weight were observed throughout the experiment, demonstrating that none of the formulations had significant in vivo toxic side effects. Figure 26 ).

[0094] The groups are as follows:

[0095] Uninfected control group (Uninf): Full-thickness skin defects were created on the backs of mice, without bacterial infection or drug application;

[0096] PBS-infected control group: Phosphate-buffered saline (PBS) was administered to the infected wound.

[0097] Free EGCG group: EGCG was administered to the infected wound;

[0098] Gallium nitrate (Ga(NO3)3) group: Ga(NO3)3 was administered to infected wounds;

[0099] Positive control group (Van): Vancomycin (Van) was administered to the infected wound.

[0100] GaED NPs group: GaED NPs were administered to infected wounds.

[0101] Throughout the experimental period, mouse body weight, blood count, and wound area changes were monitored regularly. Wound area (e.g.) Figure 4 As shown in B, the embedded wound area outline map and quantitative statistics ( Figure 4 C, 4D) Visually demonstrate the dynamic healing process of each group of wounds:

[0102] On day 0, all wounds showed normal skin color with no obvious redness, swelling, or exudation; inoculation was performed with a mixed bacterial suspension (Staphylococcus aureus, Pseudomonas aeruginosa, and MRSA mixed in a 1:1:1 ratio, with a single bacterial concentration of 10). 6 After 24 hours (day 1) with CFU / mL and total bacterial solution of 50μL, obvious infection and biofilm accumulation appeared on the wound, proving that the multi-bacterial infection wound model was successfully constructed.

[0103] GaED NPs exhibit excellent wound repair effects thanks to the continuous release of active components from nanoparticles, which can penetrate deep into the biomembrane to exert their effects.

[0104] To further evaluate the treatment effect, the core indicators of wound repair were detected by tissue sections (heart, liver, spleen, lung and kidney) of ICR mice (mice after modeling treatment, i.e. mice on day 12) and immunofluorescence staining: tissue remodeling, collagen deposition, myofibroblast generation and angiogenesis.

[0105] On day 12 of treatment, one mouse from each group was randomly sacrificed, and the wound tissue and organs of heart / liver / spleen / lung / kidney were separated; fixed with 4% paraformaldehyde, routinely embedded in paraffin, and stained by sectioning.

[0106] H&E staining results showed that the wound in the blank control group was not completely closed, and obvious tissue defects were visible at the wound edge. Figure 4 (E magnified area); The uninfected group, PBS group, free EGCG group, and gallium nitrate group showed severe inflammatory cell infiltration in the wound (deeply stained blue-purple areas on the sections; quantitative statistics can be found in...). Figure 5 A); Vancomycin group showed improved wound healing, but the epidermis was abnormally thickened. GaEDNPs treatment group showed the best wound repair effect: complete epidermal regeneration, stratum corneum formation, and the lowest amount of inflammatory cell infiltration. Figure 4 E, Figure 5 A); at the same time, the number of newly formed blood vessels in the wound of this group was significantly greater than that of the other groups ( Figure 5 B).

[0107] Masson-stained sections ( Figure 4 E) and quantitative collagen deposition ( Figure 5C) Confirmed that: collagen distribution in all control group wounds was sparse, disordered, and loose; collagen fibers in infected wounds treated with GaED NPs were dense and orderly, resulting in better tissue remodeling and wound repair.

[0108] Immunofluorescence staining results showed that weak fluorescence signals of platelet-endothelial cell adhesion molecules (CD31, a vascular endothelial marker) and α-smooth muscle actin (α-SMA, a myofibroblast marker) were observed in the wounds of each infection control group, suggesting insufficient angiogenesis and impaired myofibroblast-mediated tissue remodeling. Figure 5 (D, 5E, 5F, 5G). GaED NPs leverage the advantages of nano-formulation and the synergistic effect of multiple components to effectively activate fibroblasts and promote angiogenesis.

[0109] Histological and immunofluorescence results demonstrate that GaED NPs accelerate wound repair by reducing inflammatory infiltration, increasing collagen deposition, promoting angiogenesis and myofibroblast-mediated tissue remodeling.

[0110] In this embodiment, H&E staining, Masson staining, immunofluorescence staining, and CCK-8 (Cell Counting Kit-8) working solution are all conventional techniques in the art, and their methods and steps will not be described in detail here.

[0111] Example 5

[0112] Biocompatibility evaluation of GaED composite nanoparticles NPs

[0113] (a) Cytotoxicity detection

[0114] Different concentrations of GaED NPs (0, 25.0, 50.0, 75.0, 100.0, 200.0, 400.0 μg / mL) were co-cultured with mouse embryonic fibroblasts NIH-3T3 for 24 h and 48 h, and the cell viability was then assessed. Specifically, NIH-3T3 cells were digested with trypsin, centrifuged and resuspended, and then cultured at 1×10⁻⁶ μg / mL. 4 Cells were seeded per well in a 96-well plate, with PBS added to the edge wells to prevent evaporation. The plates were then incubated at 37°C in a 5% CO2 incubator until adherence was achieved. The old culture medium was discarded, and different concentrations of GaED NPs (0, 25.0, 50.0, 75.0, 100.0, 200.0, 400.0 μg / mL) were added for treatment. A normal cell control group and a blank culture medium group were set up, and incubation was continued. After incubation, the supernatant was discarded, and the cells were washed with PBS to remove drug residues. Pre-prepared CCK-8 working solution was added to each well, and the plates were incubated in the dark for 50 min. Finally, the absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated according to the formula.

[0115] Relative cell survival rate (%) = [(OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0116] The results are as follows Figure 6 A and Figure 6 As shown in Figure B, the cell survival rate was close to 100%, demonstrating that GaED NPs have extremely low cytotoxicity.

[0117] (ii) In vitro blood compatibility testing

[0118] Set up groups:

[0119] Grouping for hemolysis experiments:

[0120] ① Positive control group: Rabbit blood samples diluted with deionized water;

[0121] ② Negative control group: Rabbit blood samples diluted with physiological saline;

[0122] ③ Experimental group: Gradient concentrations of GaED NPs (0, 25.0, 50.0, 75.0, 100.0, 200.0, 400.0 μg / mL) were mixed with an equal volume of diluted rabbit blood.

[0123] Grouping for coagulation qualitative test:

[0124] ① Blank control group: 0.1 mol / L CaCl2+ rabbit whole blood;

[0125] ② Experimental group: gradient concentrations of GaED NPs (25, 50, 75, 100, 200, 400 μg / mL), combined with 0.1 mol / L CaCl2 and rabbit whole blood.

[0126] Hemolysis assay: Rabbit whole blood was diluted 50-fold with 0.9 w / v % physiological saline. Then, equal volumes of diluted blood and equal masses of GaED NPs at gradient concentrations (0, 25.0, 50.0, 75.0, 100.0, 200.0, 400.0 μg / mL) were mixed in 5 mL test tubes and incubated at 37°C for 2 hours. After incubation, the supernatant was recovered by centrifugation (1000 rpm, 10 min), and 100 μL aliquots were transferred to 96-well plates. The absorbance at 545 nm was measured using a multi-mode microplate reader. Three parallel experiments were performed for each group, and the hemolysis volume was calculated using the formula.

[0127] Hemolysis rate (%) = (Experimental group absorbance - negative control absorbance) ÷ positive control absorbance × 100%.

[0128] The results are as follows Figure 6As shown in C, compared with the pure water positive control, the hemolysis rate of blood samples treated with GaED NPs at all concentrations was <5.0%, with no obvious hemolysis phenomenon;

[0129] Coagulation qualitative test: 5 μL of sample solutions prepared with 0.1 mol CaCl2 at different concentrations (25, 50, 75, 100, 200, 400 μg / mL) were pre-placed at the bottom of a 96-well plate. Then, 45 μL of rabbit whole blood (50 μL total rabbit blood plus drug) was added to each well. At selected time points, the wells were immediately washed with 0.9 w / v% physiological saline to stop coagulation and remove all soluble blood components. Coagulation was recorded using an optical microscope. Blank wells containing 0.1 mol CaCl2 and rabbit whole blood served as controls. Three parallel test wells were set up for each group.

[0130] The results are as follows Figure 6 As shown in D, the blood clotting time in the GaED NPs treatment group was not different from that in the blank control group; it neither accelerated nor inhibited coagulation, and there was no risk of coagulation abnormalities. Figure 27 ).

[0131] (III) In vivo blood safety testing

[0132] During the treatment period for infected wounds, the complete blood count of mice in each group in Example 4 was periodically monitored: white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT). The results are as follows: Figure 6 As shown in F, all indicators are within the normal physiological range, and the fluctuations in a few indicators are only normal physiological reactions caused by wound inflammation.

[0133] (iv) Organ and tissue toxicity

[0134] At the end of the treatment cycle (i.e., after 3 consecutive days of administration followed by 9 days of observation, up to day 12), the heart, liver, spleen, lungs, and kidneys of mice from each group in Example 4 were subjected to H&E pathological staining. The results are as follows: Figure 6 As shown in Figure E, no obvious pathological damage or lesions were observed in any of the organs, demonstrating that topical application of GaED NPs to the skin does not produce significant tissue toxicity to the organs of mice.

[0135] The composite nanoparticles GaED NPs constructed in this invention exhibit excellent biocompatibility.

[0136] In summary, this invention utilizes a one-step self-assembly method to synthesize trivalent gallium ions, diethyl dithiocarbamate (DEDTC), and the natural polyphenol EGCG to prepare carrier-free gallium-phenol nanoparticles (GaED NPs). This nanoformulation improves the aqueous stability of trivalent gallium under physiological conditions, while mitigating the oxidative degradation of EGCG, thus significantly enhancing the physicochemical stability of the active components.

[0137] Upon local application to infected wounds, GaED NPs dissociate, continuously releasing bioactive trivalent gallium, DEDTC, and EGCG, with the multiple components synergistically exerting a broad-spectrum antibacterial effect. In vitro and in vivo experiments have jointly confirmed that GaED NPs possess potent antibacterial activity against common wound pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and MRSA; they can significantly inhibit biofilm formation and clear mature mixed bacterial biofilms; and, while maintaining good biosafety, efficiently accelerate the healing of multi-bacterial infected wounds. This provides a new approach for the rational design of carrier-free gallium-phenol nanomedicines, achieving highly efficient intervention in multi-bacterial wound infections by improving the bioavailability of active components and amplifying synergistic therapeutic effects.

[0138] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A wound repair material for multiple bacterial infections, characterized in that: It includes composite nanoparticles formed from epigallocatechin gallate, diethyl dithiocarbamate, and trivalent gallium.

2. A method for preparing a wound repair material for multiple bacterial infections according to claim 1, characterized in that: The process includes the following steps: mixing an aqueous solution of epigallocatechin gallate with an aqueous solution of diethyldithiocarbamate at room temperature, then adding an aqueous solution of trivalent gallium. After the system spontaneously assembles for 1-2 minutes, the mixture is washed and freeze-dried to obtain composite nanoparticles.

3. The method for preparing wound repair material for multiple bacterial infections according to claim 2, characterized in that: The concentration of the aqueous solution of epigallocatechin gallate was 2 mmol / L.

4. The method for preparing the wound repair material for multiple bacterial infections according to claim 2, characterized in that: The concentration of the aqueous solution of diethyl dithiocarbamate is 1 mmol / L.

5. The method for preparing wound repair material for multiple bacterial infections according to claim 2, characterized in that: The trivalent gallium is gallium nitrate.

6. The method for preparing the wound repair material for multiple bacterial infections according to claim 2, characterized in that: The concentration of the trivalent gallium aqueous solution was 10 mmol / L.

7. The method for preparing wound repair material for multiple bacterial infections according to claim 2, characterized in that: The volume ratio of the epigallocatechin gallate aqueous solution, the diethyldithiocarbamate aqueous solution, and the trivalent gallium aqueous solution is 3:1:0.5-1.

8. Application of wound repair materials for multiple bacterial infections in the preparation of antibacterial biofilms, or antimicrobial drugs or materials.

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