Silver-loaded ruthenium-based nano photosensitizer as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of photodynamic antibiosis, and discloses a silver-loaded ruthenium-based nano photosensitizer as well as a preparation method and application thereof. The chemical formula of the silver-loaded ruthenium-based nano photosensitizer (Ag-Ru) is Ag-[Ru (phen) 2Cur] X2, phen is 1, 10-phenanthroline, Cur is coumarin-6, and X is an anion. Ag-[Ru (phen) 2Cur] X2 molecules are prepared by reacting AgNO3 with [Ru (phen) 2Cur] 2 +, and Ag-Ru shows a remarkable photoresponsive release characteristic: under a dark condition, silver ion release kinetics is relatively slow, and under an illumination condition, silver ions are in a continuous release mode, and the 24-hour accumulative release amount is remarkably increased to 44.17%. And the in-vitro antibacterial activity on pseudomonas aeruginosa is improved by 4 times, the sterilization efficiency on all test strains is close to 100%, and the antibacterial peptide shows an excellent broad-spectrum antibacterial effect and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of photodynamic antibacterial technology, specifically relating to a silver-loaded ruthenium-based nanophotosensitizer, its preparation method, and its application. Background Technology

[0002] Bacterial biofilm infections pose a significant challenge to clinical treatment. Biofilms form a three-dimensional network structure composed of biomacromolecules such as polysaccharides, proteins, and nucleic acids by secreting extracellular polymeric matrix (EPS). This dense physical barrier significantly hinders the penetration and diffusion of antimicrobial drugs. More seriously, the unique microenvironment formed within biofilms (such as hypoxia and acidity) further reduces the bioactivity of antimicrobial drugs and inhibits the generation of reactive oxygen species (ROS) during photodynamic therapy (PDT), leading to a significant decrease in the efficacy of traditional antibiotics and conventional PDT. PDT, as an emerging non-antibiotic antimicrobial strategy, relies heavily on photosensitizers (PSs). The photophysicochemical properties of photosensitizers, particularly their light absorption and excited-state characteristics, directly determine the efficiency of ROS generation and the final therapeutic effect. Therefore, developing novel photosensitizers that maintain high photodynamic activity and good penetration capabilities within the harsh microenvironment of biofilms has become a critical technical challenge urgently needing to be addressed in this field.

[0003] In previous studies, we successfully developed a ruthenium-based photosensitizer (RuCur NPs) that maintains significant photodynamic activity even in hypoxic and acidic environments, demonstrating its adaptability to the harsh microenvironment of bacterial biofilms. However, photodynamic therapy alone still has inherent limitations in practical anti-biofilm applications: firstly, the reactive oxygen species (ROS) it generates have a limited range and short duration of action, making it difficult to completely cover and kill bacteria in the deep layers of biofilms; secondly, its killing effect on certain specific bacterial species, such as Gram-negative bacteria, is relatively poor. Therefore, the ability of photodynamic therapy alone to clear bacteria in the deep layers of biofilms remains relatively limited. Silver ions (Ag) + Silver ions, as a classic broad-spectrum antibacterial agent, are considered a highly promising antibacterial material due to their high antibacterial activity and low tendency to induce drug resistance. However, the application of silver ions in anti-biofilm therapy also faces severe challenges. The dense extracellular polymeric substance (EPS) matrix of biofilms severely hinders the penetration of silver ions, making it difficult for them to effectively reach the deep layers of the biofilm to exert their effects; their release kinetics are difficult to control, and excessively rapid or slow release can lead to decreased efficacy or toxicity risks.

[0004] In summary, given the respective shortcomings of PDT and silver ion anti-biofilm, there is an urgent need in this field for an innovative strategy that can overcome the aforementioned dual dilemmas. Summary of the Invention

[0005] Addressing the limitations of existing technologies such as "single PDT" and the "silver ion penetration-release dilemma," the primary objective of this invention is to provide a silver-loaded ruthenium-based nanophotosensitizer. This aims to utilize photodynamic processes to disrupt the initial structure of biofilms, paving the way for deep penetration of silver ions. Simultaneously, it achieves controllable loading and sustained release of silver ions, avoiding the toxic side effects of rapid release. Furthermore, it leverages the long-lasting antibacterial properties to compensate for the short-term effects of ROS. Ultimately, through the synergistic effect of photodynamic killing and the multiple antibacterial mechanisms of silver ions, it achieves comprehensive and efficient removal of both the surface and deep layers of biofilms.

[0006] Another object of the present invention is to provide a method for preparing silver-loaded ruthenium-based nanophotosensitizers.

[0007] Another object of the present invention is to provide the application of silver-loaded ruthenium-based nanophotosensitizers.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A silver-loaded ruthenium-based nanophotosensitizer with the chemical formula Ag-[Ru(phen)2Cur]X2, where phen is 1,10-phenanthroline, Cur is coumarin-6, and X is an anion.

[0010] Preferably, the cationic chemical structure of the silver-loaded ruthenium-based photosensitizer is as follows:

[0011] .

[0012] Preferably, the anion is selected from NO3. - .

[0013] Preferably, the silver-loaded ruthenium-based photosensitizer is a spherical nanoparticle with a particle size of 5-10 nm and a surface zeta potential of 10-40 mV.

[0014] A method for preparing a silver-loaded ruthenium-based nanophotosensitizer includes the following steps:

[0015] (1) First, [Ru(phen)2Cur]Cl2 was synthesized, and then subjected to ion exchange resin to obtain [Ru(phen)2Cur]. 2+ ;

[0016] (2) [Ru(phen)2Cur] 2+ AgNO3 solution was added to an organic solvent and reacted under light-protected and stirred conditions to obtain a crude solution containing the target product.

[0017] (3) Add the crude solution containing the target product to water and stir continuously to obtain a mixed solution. Dialyze the mixed solution to purify it. Dry the purified solution to obtain silver-loaded ruthenium-based nanophotosensitizer.

[0018] Preferably, [Ru(phen)2Cur] 2+ The molar ratio with AgNO3 is 1:2~2.5.

[0019] Preferably, the reaction time in step (2) is 2-4 hours, and the stirring speed is 800-1000 rpm;

[0020] The concentration of the AgNO3 solution in step (2) is 20~50 mg / mL, and the organic solvent is DMSO.

[0021] Preferably, the dialysis in step (3) uses a dialysis bag with a molecular weight cutoff of 800~1200 Da, the dialysis time is 72~96h, and the dialysis solution is changed every 8~10h.

[0022] Preferably, the drying in step (3) is freeze drying, and the drying time is 96~120h.

[0023] Preferably, in step (3), the volume ratio of the crude solution containing the target product to water is 1:50~150, and the water is double-distilled water or ultrapure water.

[0024] Preferably, the [Ru(phen)2Cur]Cl2 is prepared according to the method disclosed in the document DOI: 10.1021 / acsami.5c01822.

[0025] The above-mentioned silver-loaded ruthenium-based photosensitizers are used in the preparation of reagents to inhibit or eliminate bacteria on the surface and deep layers of biofilms.

[0026] Preferably, the biofilm is a biofilm formed by Gram-positive bacteria, including at least one of Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis; or the biofilm is a biofilm formed by Gram-negative bacteria, including at least one of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] (1) By constructing silver-loaded ruthenium-based nanophotosensitizers (Ag-Ru NPs) with photocontrolled release characteristics, this invention successfully achieved efficient synergy between photodynamic therapy and silver ion antibacterial activity, achieving complementary advantages: the photodynamic process may disturb the biomembrane structure, creating favorable conditions for silver ion penetration; while the continuous antibacterial properties of silver ions can compensate for the shortcomings of short duration and limited range of reactive oxygen species, solving the limitations of single photodynamic therapy and the dilemma of silver ion penetration-release.

[0029] (2) The material exhibits significant photoresponsive release characteristics. Under light conditions, the cumulative release of silver ions in 24 hours is as high as 44.17%, which is significantly higher than the cumulative release of 10.39% under dark conditions.

[0030] (3) The material can quickly penetrate the biofilm under light conditions. Through the synergistic mechanism of "photodynamic barrier breaking - deep cleaning of silver ions", it exhibits strong killing power against bacteria deep in the biofilm, with a bacterial mortality rate of 89.7%, which is significantly higher than the dark treatment group (21.4%), the single RuCur NPs group (33.6%) and the RuCur NPs+AgNO3 physical mixture group (44.5%). At the same time, it has been shown to significantly promote tissue repair in infected wounds and keratitis models.

[0031] (4) The photosensitive material loaded with silver ions of this invention has good cell compatibility and biosafety, and achieves a comprehensive anti-biofilm effect of photodynamic destruction of biofilm and silver ion killing of residual bacteria, providing a new material for efficient and safe intervention in biofilm infection. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the synthesis of Ag-[Ru(phen)2Cur](NO3)2.

[0033] Figure 2 These are the XPS spectra of Ag-[Ru(phen)2Cur](NO3)2; (A) fine spectrum of S 2p, (B) fine spectrum of Ag 3d.

[0034] Figure 3 This is the UV spectrum of Ag-[Ru(phen)2Cur](NO3)2.

[0035] Figure 4 These are TEM images (A) and Zeta potential maps (B) of Ag-Ru NPs, n = 3.

[0036] Figure 5 This is an evaluation of the photodynamic properties of Ag-Ru NPs, (A) superoxide anions (·O2) generated by Ag-Ru NPs under illumination. - (a) Electron paramagnetic resonance (EPR) signals of hydroxyl radicals (·OH), (b) UV-Vis spectrum of photocatalytic oxidation of TMB by Ag-Ru NPs, (c) UV-Vis spectrum of photocatalytic oxidation of ABTS by Ag-Ru NPs, "L" indicates light conditions, "Ag-Ru" indicates Ag-Ru NPs, "Ru" indicates RuCur NPs, and the same applies below.

[0037] Figure 6This is the silver ion release curve of Ag-Ru NPs in PBS (pH = 7.4), where "L" represents the light condition and n = 3.

[0038] Figure 7 This is a comparative analysis of the minimum inhibitory concentrations (MICs) of Ag-Ru NPs, RuCur NPs, and their physical mixtures with AgNO3 against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. In the figure, ">" indicates that the MIC value is higher than the test concentration, and "L" represents the light conditions.

[0039] Figure 8 This is the result of a surface contact antimicrobial experiment. Representative images of bacterial plating plates treated with different materials are shown (A) and the corresponding E. coli colony counts (B), P. aeruginosa colony counts (C), and S. aureus colony counts (D). n=3 *** P<0.001.

[0040] Figure 9 These are the results of a live / dead staining experiment for Pseudomonas aeruginosa. "L" indicates that the experiment was conducted under light conditions.

[0041] Figure 10 These are SEM images of Pseudomonas aeruginosa from different treatment groups. "L" indicates that the experiment was conducted under light conditions.

[0042] Figure 11 This is a preliminary investigation into the anti-biofilm properties of Ag-Ru NPs; (A) the penetration effect of Ag-Ru NPs on Pseudomonas aeruginosa biofilm, (B) the green / blue fluorescence intensity ratio in Figure (A); where Green represents the fluorescence intensity of Ag-Ru NPs or RuCur NPs, Blue represents the fluorescence intensity of DAPI, n = 3, *** P<0.001.

[0043] Figure 12 This is a preliminary investigation into the anti-biofilm properties of Ag-Ru NPs; (A) Images of live / dead Pseudomonas aeruginosa biofilms after different treatments, (B) The red / blue fluorescence intensity ratio in Figure (A), where Ag-Ru represents Ag-Ru NPs, Ru represents RuCur NPs, and "L" represents the illumination condition; where Red represents the fluorescence intensity of PI, Blue represents the fluorescence intensity of DAPI, n = 3, *** P<0.001.

[0044] Figure 13 This is a schematic diagram of the in vitro anti-biofilm activity assessment of Ag-Ru NPs.

[0045] Figure 14The crystal violet staining method was used to evaluate the inhibitory and clearance effects of different treatments on biofilms; (A) representative crystal violet-stained images of Pseudomonas aeruginosa biofilms in different treatment groups, (B) biofilm formation inhibition rate, (C) mature biofilm clearance rate; n = 3, ** P<0.01 and *** P<0.001.

[0046] Figure 15 It uses laser confocal microscopy imaging to evaluate the inhibitory and clearance effects of different treatments on biofilms.

[0047] Figure 16 This involves scanning electron microscopy characterization analysis of biofilms under different treatment conditions.

[0048] Figure 17 The cell viability after treatment with (A) Ag-Ru NPs, (B) RuCur NPs, (C) Ag NPs and (D) AgNO3 for 24 h is n = 3.

[0049] Figure 18 This is a schematic diagram illustrating the construction and treatment of a mouse model of Pseudomonas aeruginosa infection in skin wounds.

[0050] Figure 19 Ag-Ru NPs promote the healing of wounds infected with Pseudomonas aeruginosa; (A) at different time points, Control, Model, Ag + Representative images of wounds in the Dres, Ag-Ru, Ru + L, and Ag-Ru + L groups; (B) Schematic diagram of wound area at different time points; (C) Control, Model, and Ag groups at different time points. + Wound area analysis (%) of Dres, Ag-Ru, Ru + L, and Ag-Ru + L groups; n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0051] Figure 20 This is a histological analysis of wound tissue on day 15; (A) representative images of the wound tissue, including H&E staining, Masson staining, and CD31 immunohistochemical staining; (B) relative quantitative analysis of wound collagen content on day 15 based on (A); (C) quantitative assessment of angiogenesis on day 15 based on the results of (A); n = 3. ** p < 0.01, *** p < 0.001.

[0052] Figure 21This is the result of monitoring mouse weight changes over 15 days.

[0053] Figure 22 These are H&E staining images of the major organs of mice 15 days after treatment.

[0054] Figure 23 This is a schematic diagram of the experimental protocol for constructing and treating a mouse model of keratitis induced by Pseudomonas aeruginosa.

[0055] Figure 24 These are (A) representative slit-lamp photographs and (B) clinical scores of mice with bacterial keratitis during treatment; Ag-Ru represents Ag-Ru NPs, L represents illumination conditions; n = 3, * p < 0.05, ** p < 0.01 and *** p < 0.001.

[0056] Figure 25 The images show representative plating plots (A) and corneal colony counts (B) of mice after different treatments, n = 3. * p < 0.05, *** p < 0.001.

[0057] Figure 26 The results include HE staining of corneal tissue and immunohistochemical analysis of IL-1β and TNF-α after treatment. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the implementation of the present invention is not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0059] Ru(phen)2Cur]Cl2 was prepared according to the method disclosed in the literature (DOI: 10.1021 / acsami.5c01822).

[0060] Example 1: Preparation of the silver-ruthenium complex Ag-[Ru(phen)2Cur](NO3)2

[0061] The Ag-[Ru(phen)2Cur](NO3)2 molecule used in this invention is formed by reacting AgNO3 with [Ru(phen)2Cur](NO3)2. 2+ It is prepared by reaction, and the synthesis diagram is shown below. Figure 1 As shown, the specific steps are as follows:

[0062] 1) After synthesizing [Ru(phen)₂Cur]Cl₂ according to the published method, it was subjected to ion exchange using an ion exchange resin. Subsequently, the ion-exchanged [Ru(phen)₂Cur]Cl₂ was... 2+With 40 mg / ml AgNO3 solution (according to [Ru(phen)2Cur] 2+ The product is dissolved in DMSO along with AgNO3 (at a molar ratio of 1:2). The mixture is then magnetically stirred for 2 hours at 800-1000 rpm under ambient temperature and light-protected conditions to obtain a crude solution containing the target product.

[0063] (2) The solution was added dropwise to ddH2O with continuous stirring at a volume ratio of 1:100. The mixture was then transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in deionized water for 72 hours to retain nanoparticles. The dialysate was changed every 8 hours during the process. Finally, the purified solution was aliquoted into centrifuge tubes and freeze-dried for 96 hours to obtain Ag-[Ru(phen)2Cur](NO3)2.

[0064] Test Example 1

[0065] Characterization of the silver-ruthenium complex using Ag-[Ru(phen)2Cur](NO3)2

[0066] The Ag-[Ru(phen)2Cur](NO3)2 was characterized by X-ray photoelectron spectroscopy, and the results are as follows: Figure 2 As shown, this confirms Ag + The effective introduction of .

[0067] like Figure 3 The optical properties of [Ru(phen)2Cur]Cl2 (hereinafter referred to as Ru) and its silver complex Ag-[Ru(phen)2Cur](NO3)2 (hereinafter referred to as Ag-Ru) were compared using ultraviolet-visible absorption spectroscopy (UV-Vis). The results showed that the introduction of Ag... + The absorption characteristics of the formed Ag-[Ru(phen)2Cur](NO3)2 (Ag-Ru) in the visible light region (400-800 nm) did not change significantly.

[0068] like Figure 4 Transmission electron microscopy (TEM) characterization results showed that the Ag-[Ru(phen)2Cur](NO3)2 nanoparticles (Ag-Ru NPs) prepared by solvent displacement method exhibited a regular spherical morphology, uniform size, and a particle size of approximately 7.2 nm. Zeta potential testing indicated that the nanoparticles had a positively charged surface with a surface Zeta potential of 31.2 mV.

[0069] like Figure 5 EPR detected significant O2. -The presence of characteristic ·OH signal peaks confirmed the efficient generation of reactive oxygen species (ROS); the TMB and ABTS oxidation experiments showed that Ag-Ru NPs have good photodynamic activity.

[0070] like Figure 6 The release behavior of silver ions from Ag-Ru NPs was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). In a PBS buffer system at pH 7.4, Ag-Ru NPs exhibited significant photoresponsive release characteristics: under dark conditions, the release kinetics of silver ions were relatively slow, with a cumulative release of only 10.39% over 24 hours; while under light conditions, silver ions exhibited a continuous release pattern, with a significant increase in the cumulative release to 44.17% over 24 hours.

[0071] Test Example 2

[0072] In vitro antibacterial activity of silver-ruthenium complex nanoparticles (Ag-Ru NPs)

[0073] To systematically evaluate the in vitro antibacterial properties of Ag-Ru NPs, the minimum inhibitory concentration (MIC) against representative Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was determined using the microbroth dilution method.

[0074] After culturing the bacteria to the logarithmic growth phase, a concentration of 5 × 10⁻⁶ was prepared. 5 CFU / mL bacterial suspension; the drug was serially diluted twofold in a 96-well plate, with 100 μL of drug solution added to each well, followed by 100 μL of bacterial suspension, to make a final volume of 200 μL per well; the 96-well plate was then exposed to light (wavelength ≥ 420 nm, light intensity 0.11 W / cm², time 10 min) or protected from light; after treatment, the 96-well plate was incubated in a 37℃ incubator for 24 hours, and the lowest drug concentration at which no obvious turbidity was observed was determined as the minimum inhibitory concentration (MIC) of the drug.

[0075] The results are as follows Figure 7As shown, under illumination, Ag-Ru NPs exhibited significant dose-dependent antibacterial activity against all three tested strains, with MIC values ​​of 12.5 μM, 12.5 μM, and 3.13 μM, respectively. Of particular note, in the test against Pseudomonas aeruginosa, the MIC value of Ag-Ru NPs under illumination (12.5 μM) was significantly lower than that of un-silver-loaded RuCur NPs (MIC = 50 μM), representing a four-fold improvement in antibacterial performance. This fully demonstrates the enhancing effect of silver ions on the antibacterial properties of the material.

[0076] To comprehensively evaluate the antibacterial properties of Ag-Ru NPs, a contact antibacterial experiment was further conducted, with unmodified commercial silver nanoparticles (Ag NPs) with the same molar concentration of silver, AgNO3 solution, single RuCur NPs, and a physical mixture of RuCur NPs / AgNO3 as controls. The results are as follows: Figure 8 As shown, under light conditions, no bacterial colonies were observed on LB agar plates in the experimental group treated with Ag-Ru NPs. Quantitative analysis indicated that its bactericidal efficiency against all tested strains was close to 100%, demonstrating excellent broad-spectrum antibacterial effects. Systematic comparative analysis revealed that:

[0077] (1) Under light conditions, the antibacterial effect of Ag-Ru NPs was significantly better than that of the single RuCur NPs treatment group and the RuCurNPs / AgNO3 physical mixture group, indicating that the improvement of its antibacterial performance was due to the synergistic effect of ruthenium photosensitizer and silver ions, rather than a simple physical mixing effect.

[0078] (2) The antibacterial activity of Ag-Ru NPs was also significantly higher than that of commercial Ag NPs and AgNO3 solution treatment group with the same molar concentration of silver, proving that its unique nanostructure and composition are beneficial to enhancing the antibacterial efficacy of silver ions.

[0079] (3) Compared with the control group of Ag-Ru NPs under dark conditions, light can significantly activate its antibacterial properties, further confirming that photodynamic effect plays a key role in the synergistic sterilization process.

[0080] The above comparison results fully demonstrate that the introduction of silver ions and the synergistic effect of photodynamic therapy significantly enhance the photodynamic antibacterial properties of Ag-Ru NPs.

[0081] The killing effect of DAPI / PI dual-fluorescence staining on Pseudomonas aeruginosa was detected using this method. Figure 9 This fully verifies that Ag-Ru NPs can be activated by photodynamics and Ag... + They work synergistically to kill bacteria.

[0082] like Figure 10As shown, scanning electron microscopy observations revealed significant differences in the degree of damage to the morphology and structure of Pseudomonas aeruginosa among different treatment groups, and the degree of bacterial damage was positively correlated with the antibacterial activity of the material. This provides intuitive structural evidence that Ag-Ru NPs exert their bactericidal efficacy through photodynamic effects and synergistic effects with silver ions.

[0083] Test Example 3

[0084] Biofilm permeability of Ag-Ru NPs and their bactericidal effect on deep-seated bacteria

[0085] The concentration of Pseudomonas aeruginosa in LB liquid medium was adjusted to 1×10⁻⁶. 7 CFU / mL was used to obtain the bacterial suspension for the experiment. Under aseptic conditions, sterilized circular smears were placed in each well of a 24-well plate, and 400 μL of the bacterial suspension was added. The 24-well plate was incubated at 37°C for 48 hours. The formation of a pale yellow, viscous flocculent substance in the wells indicated successful bacterial biofilm formation. Subsequently, the bacterial suspension in the wells was carefully aspirated, and the plate was gently washed three times with PBS buffer (pH 7.4) to thoroughly remove any unattached free bacteria. Next, the test material was added and co-cultured with the biofilm. After treating different time points, the plate was gently washed three times again with PBS buffer (pH 7.4) to remove free bacteria and unbound material. Then, DAPI staining solution at a concentration of 10 μg / mL was added, and staining was performed for 30 minutes in the dark. Finally, the permeation of the material in the biofilm was observed using a laser confocal microscope (CLSM), and a three-dimensional structural image of the biofilm was obtained.

[0086] like Figure 11 As shown, the permeation behavior of RuCur NPs and Ag-Ru NPs in Pseudomonas aeruginosa biofilms was monitored using confocal laser scanning microscopy. Quantitative analysis based on fluorescence intensity ratios indicated that both materials exhibited continuously enhanced diffusion kinetics: at 15 min, the permeabilities were 12.2% (RuCur NPs) and 13.4% (Ag-RuNPs), respectively; at 30 min, these increased to 41.6% and 53.4%; and at 60 min, they reached 80.4% and 84.9%, respectively. Ag-Ru NPs exhibited slightly better diffusion capacity while retaining the permeation performance of the parent materials.

[0087] like Figure 12 The antibacterial effect was further evaluated using a live / dead bacteria staining method.

[0088] Pseudomonas aeruginosa biofilms were prepared according to the aforementioned method. After biofilm formation, the culture medium in the wells was removed, and the biofilm surface was gently washed with PBS buffer (pH 7.4). Subsequently, different test material solutions were added to each experimental group, and the mixtures were incubated at 37°C for 60 minutes. The biofilms were then subjected to either light irradiation (wavelength ≥ 420 nm, light intensity 0.11 W / cm², duration 10 minutes) or light-protected treatment. After treatment, the biofilms were gently washed again with PBS buffer to remove unbound material. Then, a mixed staining solution consisting of DAPI (10 μg / mL) and propidium iodide (PI, 10 μg / mL) was added, and the mixture was stained for 30 minutes in the dark. Finally, the three-dimensional distribution images of bacterial survival status in the biofilms were observed and acquired using a confocal laser scanning microscope with multi-channel scanning mode and Z-axis tomography.

[0089] The Ag-Ru NPs light-treated group exhibited the strongest bactericidal activity, with a bacterial mortality rate of 89.7%, significantly higher than the dark-treated group (21.4%), the single RuCur NPs group (33.6%), and the physical mixture group (44.5%). The experimental results confirm that after Ag-Ru NPs disrupt the biofilm structure through photodynamic action, they utilize their permeability to deliver silver ions to deeper layers, ultimately achieving synergistic clearance through the sustained antibacterial effect of silver ions.

[0090] Test Example 4

[0091] In vitro anti-biofilm activity of Ag-Ru NPs

[0092] This invention further evaluated the in vitro anti-biofilm activity of Ag-Ru NPs. A systematic evaluation framework was established targeting the two key aspects of biofilm infection treatment: inhibiting biofilm formation and clearing mature biofilms. Figure 13 As shown, using *Pseudomonas aeruginosa* as a model strain, the anti-biofilm effect of Ag-Ru NPs was comprehensively evaluated using various characterization methods, including confocal microscopy, crystal violet staining, and scanning electron microscopy. In the biofilm inhibition experiment, during the initial bacterial adhesion stage (10... 7 Different groups of test materials (CFU / mL) were added to evaluate the ability of each group to block biofilm formation; in the mature biofilm removal experiment, mature biofilms that had been pre-cultured for 48 hours were treated to systematically examine the effect of each group on destroying the formed biofilm.

[0093] like Figure 14 As shown, the inhibitory and scavenging effects of Ag-Ru NPs on Pseudomonas aeruginosa biofilms were quantitatively analyzed using crystal violet staining. The results showed that Ag-Ru NPs exhibited significant effects on both inhibiting biofilm formation and disrupting mature biofilms under light conditions.

[0094] Confocal laser scanning microscopy (CLSM) observations further validated the anti-biofilm activity of Ag-Ru NPs. Figure 15 As shown: In the biofilm formation inhibition experiment, the Ag-Ru NPs light-treated group showed the weakest blue fluorescence intensity, indicating that it effectively inhibited bacterial adhesion and biofilm formation; in the mature biofilm destruction experiment, this treatment group also showed the most significant structural destruction effect, and the integrity of the biofilm was significantly disrupted.

[0095] like Figure 16 As shown, scanning electron microscopy revealed that the untreated mature biofilm exhibited a complete three-dimensional network structure, with bacteria tightly encapsulated by abundant extracellular polymers (EPS), forming highly organized aggregates. After treatment with Ag-Ru NPs, the biofilm structure underwent significant changes: (1) the continuous EPS matrix network completely disintegrated; (2) the bacteria changed from an aggregated state to a clearly dispersed free state; and (3) significant damage characteristics appeared on the bacterial surface. In contrast, other control groups only showed partial dissociation of bacterial aggregates into scattered small clusters, and the degree of bacterial damage was relatively mild.

[0096] The above structures collectively confirm that Ag-Ru NPs exert their anti-biofilm activity through a synergistic mechanism with silver ions via photodynamic action: under light irradiation, the ROS generated by Ag-Ru NPs effectively disrupts the three-dimensional structure of biofilms, undermining their stability; simultaneously, Ag... + The antibacterial effect further enhances the killing effect on bacteria, together achieving efficient inhibition and removal of biofilm.

[0097] Test Example 5

[0098] Cytotoxicity of Ag-Ru NPs

[0099] To assess the biosafety of Ag-Ru NPs, human immortalized keratinocytes (HaCaT) were used as an in vitro model. Mitochondrial succinate dehydrogenase activity was detected using the MTT assay to quantitatively analyze cell viability. HaCaT cells were co-incubated for 24 hours with different concentrations (1.56 μM, 3.31 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM) of Ag-Ru NPs, RuCur NPs, AgNPs, and AgNO3. The results are as follows: Figure 17 As shown, within the concentration range of 0–50 μM, the cell viability of each experimental group remained above 80%, demonstrating good cell compatibility.

[0100] Test Example 6

[0101] In vivo antibacterial activity of Ag-Ru NPs

[0102] This invention uses a mouse model of skin wounds infected with Pseudomonas aeruginosa to evaluate in vivo antibacterial activity. For example... Figure 18 As shown, after successful model establishment, the experimental groups were treated with 100 µL of Ag-Ru NPs solution and subjected to light exposure and light-protection treatments, respectively. The positive control group used the commercially available silver ion dressing Mepilex. ® Treatment was performed with Ag (Mepicon Silver). To exclude the influence of non-specific healing factors, the model group (Model group) was treated with only 100 µL PBS buffer for infected wounds, while the control group (Control group) consisted of uninfected wounds. During the experiment, wounds in each group were photographed regularly, and the wound contraction rate was quantitatively calculated using image analysis software to systematically evaluate the impact of different treatments on the wound healing process.

[0103] like Figure 19 As shown, observations of infected wounds at different time points revealed that on day 3, the wound areas in the model group, silver ion dressing group, Ag-Ru NPs light-protected group, and RuCur NPs light-exposed group remained large, indicating delayed healing. By day 6, the Ag-Ru NPs light-exposed group began to show a significant shrinkage trend, while the wounds in the other groups remained large. By day 10, the wound area in the Ag-Ru NPs light-exposed group was significantly smaller than that in the other treatment groups. By day 15 of treatment, the infected wounds in this group were almost completely healed, while the other groups still had significant unhealed wounds. These results fully demonstrate that Ag-Ru NPs can significantly accelerate the healing process of infected wounds under light exposure.

[0104] like Figure 20 In the histopathological evaluation of Pseudomonas aeruginosa-infected wounds, Ag-Ru NPs exhibited a significant healing-promoting effect under light exposure. H&E staining showed complete wound closure, continuous epidermal regeneration, and minimal inflammatory infiltration in this group. Masson staining confirmed that collagen deposition was significantly higher than in the control group, with well-organized and clearly layered fibers. CD31 immunohistochemistry further revealed a significantly increased angiogenesis density. These results indicate that Ag-Ru NPs, activated by light, can simultaneously accelerate epithelial regeneration, promote orderly collagen deposition, and enhance angiogenesis, thereby systematically improving the repair quality and healing process of infected wounds.

[0105] Test Example 7

[0106] Biosafety of Ag-Ru NPs

[0107] like Figure 21As shown, during the treatment of bacterial wound infections, the body weight of mice was monitored at days 0, 3, 6, 10, and 15. The results showed no significant difference in body weight changes among the groups, and no obvious abnormal behaviors were observed throughout the observation period. Figure 22 Histopathological analysis of the major organs of the experimental mice showed that the organ structures in each experimental group were intact, with no pathological changes observed. These results confirm that Ag-Ru NPs did not cause significant organ structural damage at the experimental dose, demonstrating good biocompatibility.

[0108] Test Example 8

[0109] In vivo anti-biomembrane activity of Ag-Ru NPs

[0110] like Figure 23 The in vivo anti-biofilm activity was evaluated using a mouse keratitis model induced by *Pseudomonas aeruginosa*. Treatment began 24 hours post-infection (day 0), and corneal condition was observed and recorded using a slit lamp on days 1, 3, and 7. After treatment, corneal tissue was harvested for quantitative bacterial culture and histological analysis to systematically evaluate the antibacterial effect.

[0111] like Figure 24 As shown in (A) and (B), slit-lamp observation and clinical scoring results showed that the corneal lesions in the model group mice improved slowly, with a clinical score of 9.7 on day 3, indicating large-area corneal ulcers; the positive control group (Ag) showed slow improvement. + Eyedrops showed only mild efficacy; the Ag-Ru NPs light-avoidance group showed limited improvement; while the Ag-Ru NPs light-exposed group showed the most significant efficacy, with obvious relief of inflammation on the 3rd day, and the cornea basically returning to transparency by the 7th day, with the clinical score dropping to 0.3 points, confirming its outstanding therapeutic effect on Pseudomonas aeruginosa keratitis under light conditions.

[0112] After treatment, corneal tissue from each group of mice was collected to prepare homogenates, and the amount of residual bacteria was detected by plate count method. Figure 25 As shown, the corneal bacterial load in the Ag-Ru NPs irradiation group was significantly lower than that in the model group and other treatment groups, with a relative colony count of only 15.1%, confirming its significant in vivo antibacterial effect in the keratitis model.

[0113] Mouse corneal tissue was analyzed using H&E staining and immunohistochemistry. Figure 26As shown, the model group exhibited typical pathological features of bacterial keratitis: disordered corneal structure, extensive inflammatory cell infiltration in the stroma, high expression of pro-inflammatory factors IL-1β and TNF-α, and abnormally increased corneal thickness. Among the treatment groups, the Ag-Ru NPs light irradiation group showed the best recovery effect: the corneal structure was basically normal, the inflammatory infiltration was significantly reduced, the expression of IL-1β and TNF-α was significantly downregulated, and the corneal thickness was restored to near normal levels, confirming that it effectively improved bacterial keratitis under light conditions through anti-inflammatory and tissue repair effects.

[0114] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A silver-loaded ruthenium-based nanophotosensitizer, characterized in that, The chemical formula is Ag-[Ru(phen)2Cur]X2, where phen is 1,10-phenanthroline, Cur is coumarin-6, and X is an anion.

2. The silver-loaded ruthenium-based nanophotosensitizer according to claim 1, characterized in that, The cationic chemical structure of the silver-loaded ruthenium-based photosensitizer is as follows: 。 3. The silver-loaded ruthenium-based photosensitizer according to claim 1, characterized in that, The anion is selected from NO3. - .

4. The silver-loaded ruthenium-based nanophotosensitizer according to claim 1, characterized in that, The silver-loaded ruthenium-based photosensitizer is a spherical nanoparticle with a particle size of 5-10 nm and a surface zeta potential of 10-40 mV.

5. A method for preparing the silver-loaded ruthenium-based photosensitizer according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) First, [Ru(phen)2Cur]Cl2 was synthesized, and then subjected to ion exchange resin to obtain [Ru(phen)2Cur]. 2+ ; (2) [Ru(phen)2Cur] 2+ AgNO3 solution was added to an organic solvent and reacted under light-protected and stirred conditions to obtain a crude solution containing the target product. (3) Add the crude solution containing the target product to water and stir continuously to obtain a mixed solution. Dialyze the mixed solution to purify it. Dry the purified solution to obtain silver-loaded ruthenium-based nanophotosensitizer.

6. The method for preparing the silver-loaded ruthenium-based nanophotosensitizer according to claim 5, characterized in that, [Ru(phen)2Cur] 2+ The molar ratio with AgNO3 is 1:2~2.

5.

7. The method for preparing the silver-loaded ruthenium-based nanophotosensitizer according to claim 5, characterized in that, The reaction time in step (2) is 2-4 hours, and the stirring speed is 800-1000 rpm; The concentration of the AgNO3 solution in step (2) is 20~50 mg / mL, and the organic solvent is DMSO.

8. The method for preparing the silver-loaded ruthenium-based nanophotosensitizer according to claim 5, characterized in that, The dialysis in step (3) uses a dialysis bag with a molecular weight cutoff of 800~1200 Da, and the dialysis time is 72~96 hours. The dialysis solution is changed every 8~10 hours.

9. The method for preparing the silver-loaded ruthenium-based nanophotosensitizer according to claim 5, characterized in that, The drying process described in step (3) is freeze drying, and the drying time is 96~120h.

10. The use of the silver-loaded ruthenium-based photosensitizer according to any one of claims 1 to 4 in the preparation of reagents for inhibiting or eliminating bacteria on the surface and deep layers of biofilms.

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