Copper sulfate pentahydrate (CuSO₄·5H₂O), zinc acetate [Zn(OAc)₂], ferric chloride hexahydrate (FeCl₃·6H₂O), magnesium Sulfate (MgSO₄), manganous acetate [Mn(OAc)₂], chloroform, 3-morpholino-2-propanesulfonic acid, benzylsulfanilimide F, potassium bromide (KBr), disodium ethylenediaminetetraacetate, MeOH, dimethyl formamide, dimethyl sulfoxide (DMSO), 2,7-dichlorofluorescein diacetate (DCFH-DA), and 50% glutaraldehyde were purchased from Bide Pharmaceutical Technology Co., Ltd. (Shanghai, China). Lysozyme (egg white) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Biosharp Biotechnology Co., Ltd. (Beijing China). Middlebrook 7H9 broth medium and modified Lowenstein - Jensen medium base were obtained from Signature Biotechnology Co., Ltd. (Guangzhou, China). Sabouraud’s medium was obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). LD₄ was synthesized as previously described (Meng et al., 2015). The standard strain of Mtb H37Rv, the drug-sensitive strain 63, 64 and the drug-resistant strain 22,26 (resistant to rifampicin and isoniazid) were provided by the Tianjin Haihe Hospital (Tianjin, China). Ethical approval for the utilization of clinical strains was obtained from the Tianjin Haihe Hospital Medical Ethics Committee (Approval No. 2024HHQX-002). Mouse fibroblasts (3T3), human immortalized keratinocytes (HaCaT), human normal liver cells (LO2), and human bronchial epithelial cells (BEAS-2B) were obtained from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). Semiconductor laser (model 7404, Intense, North Brunswick, NJ, United States) and optical power meter (LM1; Carl Zeiss, Oberkochen, Germany) were used.

For C. albicans and Mtb, the MIC was determined using the broth microdilution method. Serial two-fold dilutions of metal ions solution were prepared in Sabouraud’s or Middlebrook 7H9 broth medium, and LD₄ was prepared in phosphate-buffered saline, ranging from 1,000–1.95 μg/mL. C. albicans strains were grown to the logarithmic phase and then adjusted to a standard inoculum density. The Mtb was inoculated onto the modified Löwenstein - Jensen medium and incubated at 37°C in a constant temperature incubator. After approximately 4 weeks of incubation, the culture reached the logarithmic growth phase. Subsequently, the Mtb colonies were harvested from the medium by scraping and resuspended in 2 mL of normal saline. Next, the bacterial suspension was sonicated for 120 s to ensure uniform dispersion. A bacterial concentration equivalent to 1 McFarland standard (approximately 3 × 10⁸ CFU/mL) was achieved and subsequently diluted to 10⁵ CFU for further experiments. Aliquots of the bacterial suspension (100 μL) were added to each well of the 96-well microdilution plate containing the metal ions or LD₄ concentrations (100 μL). For C. albicans, the fungal suspension and drug solution were co-incubated for 30 min; in contrast, for Mtb, the bacterial suspension and drug solution were co-incubated for 24 h. The plates were exposed to a 650 nm laser with an energy density of 6 J/cm² for 10 min and subsequently incubated at 37°C for either 24 h or 2 weeks, depending on the organism. The MIC was defined as the lowest concentration of the test compound that completely inhibited visible bacterial growth after incubation. The minimum bactericidal concentration was determined following the MIC assay. For C. albicans and Mtb, suspensions from wells showing no visible growth (i.e., at or above the MIC) were subcultured onto sterile Sabouraud’s agar or Lowenstein - Jensen medium plates, respectively. These plates were then incubated under the same conditions used for the respective MIC assays. The minimum bactericidal concentration was defined as the lowest concentration of the test compound that resulted in no visible growth on the agar plates, indicating a reduction in viable bacteria or fungi by at least 99.9% compared to the untreated control.

The antimicrobial synergistic effects of metal ions and LD₄ were evaluated against the bacterial strains C. albicans and Mtb using the Checkerboard dilution method. Serial concentrations of metal ions and LD₄ were prepared to achieve final concentrations of 4MIC, 2MIC, MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC, and 0 in the respective wells of a 96-well microdilution plate.

The fractional inhibitory concentration index (FICI) for each drug in combination was calculated using the following formulas (Barbara et al., 2006): FICI = FIC metal   ions + FIC LD 4 = MIC metal   ions   in   combination MIC metal   ion   independently + MIC LD 4   in   combination MIC LD 4   independently

Synergistic effects were defined as FICI ≤ 0.5, additive effects as 0.5 < FICI ≤ 1, indifferent effects as 1 < FICI ≤ 4, and antagonistic effects as FICI > 4.

To investigate the synergistic mechanism of Cu²⁺/Zn²⁺ and LD₄ against C. albicans and H37Rv, the intracellular uptake of LD₄ was quantified using flow cytometry. H37Rv was cultured on Löwenstein - Jensen medium at 37°C until the logarithmic growth phase. Bacterial colonies were harvested using sterile loops, suspended in normal saline, and homogenized by ultrasonication (5 s pulse/5 s pause, 12 cycles, 80% amplitude) to obtain a uniform suspension (5 McFarland standard). C. albicans was cultured on Sabouraud’s medium at 28°C until the logarithmic growth phase, and the suspension was concentrated to 5 McFarland standard. Three experimental groups were established: LD₄ alone (31.25 μg/mL); LD₄ (31.25 μg/mL) + Cu²⁺; and LD₄ (31.25 μg/mL) + Zn²⁺. Metal ion concentrations ranged from 3.13 to 50 μg/mL (two-fold serial dilution). For each condition, 1 mL of bacterial suspension was mixed with 500 μL of LD₄ (125 μg/mL stock solution) and 500 μL of metal ion solution in sterile tubes. After incubation for 24 h in the dark at 37°C, unbound LD₄ was removed by centrifugation (4,000 × g, 10 min, 4°C), and the bacteria were subsequently washed thrice with saline (0.9% NaCl, Signature Biotechnology Co., Ltd. ,Guangzhou, China). Washed bacteria were resuspended in 1 mL saline, filtered through a 40 μm cell strainer, and analyzed using a NovoCyte Quanteon flow cytometer (Agilent Technologies Inc. ,City of Santa Clara, CA, United States). PE-Cy7 channel (excitation/emission: 488/780 nm) was selected based on LD₄ fluorescence. Data from 100,000 events per sample were acquired at high flow rate (66 μL/min), with unstained bacteria serving as negative controls. Fluorescence compensation was applied using single-stained LD₄ samples. Three independent replicate experiments were conducted for each group.

A stock solution of LD₄ (10 mg/mL) was prepared in DMSO. Metal ion stock solutions (Zn²⁺ and Cu²⁺) were prepared at concentration of 2,000 ppm (weight/volume) in ultrapure water. Three experimental groups were designed to evaluate quenching effects: Control group with LD₄ alone (31.25 μg/mL); LD₄ + metal ions (31.25 μg/mL LD₄ + 50 μg/mL combined Zn²⁺ or Cu²⁺). Aliquots of diluted metal ions (500 μL) and LD₄ working solution (500 μL) were mixed in 2 mL microcentrifuge tubes, followed by incubation at 25°C for 10 min. Temperature-dependent experiments were conducted at 20°C, 10°C, and 0°C using a thermostatically controlled ethanol bath. Fluorescence spectra were acquired using a fluorescence spectrophotometer (Hitachi F4600, Japan) with excitation at 390 nm and emission scanning from 400 to 800 nm. Slit widths were set at 2 nm for both excitation and emission. The Stern - Volmer equation was applied to quantify quenching efficiency: I 0 I = 1 + K q τ 0 Q where I ₀ and I represent fluorescence intensities without and with quencher, respectively; Kq is the Stern - Volmer quenching constant; τ ₀ denotes the intrinsic fluorescence lifetime; and [Q] indicates quencher concentration. The quenching efficiency (η) was calculated using the following formula: η = F I L D 4 − F I L D 4 + i o n F I L D 4 × 100 % where FI _LD4 and FI _LD4+ion correspond to maximum fluorescence intensities of LD₄ in the absence and presence of metal ions, respectively.

Infrared spectra were acquired using a Thermo Fisher Scientific Nicolet iS20 Fourier-transform infrared (FTIR) spectrometer (Waltham, MA, United States). LD₄ samples were analyzed in three states: LD₄ alone; LD₄-Cu²⁺ complex; and LD₄-Zn²⁺ complex. The concentrations of LD₄, Cu²⁺/Zn²⁺ were 31.25 μg/mL and 50 μg/mL, respectively. The complex was prepared via freeze-drying. Each sample (5 mg) was homogeneously mixed with anhydrous KBr (spectroscopic grade, 500 mg) using a mortar and pestle for 15 min under ambient conditions. The mixture was pressed into transparent pellets (10 mm diameter) under 10 MPa pressure for 35 s using a hydraulic pellet press. Pellets were mounted in the sample compartment, and background spectra were collected from clean KBr crystals. FTIR measurements were performed over the wavenumber range of 400–4,000 cm⁻¹ with a spectral resolution of 4 cm⁻¹. Each spectrum represented the average of three independent scans to ensure reproducibility.

DCFH-DA was employed as the fluorescent probe for quantifying ROS production. The DCFH-DA powder was dissolved in DMSO to prepare a 10 mM stock solution, which was aliquoted and stored at −20°C in the dark. During the experiment, the stock solution was diluted 1:1,000 prior to use. LD₄ was used at a concentration of 31.25 μg/mL, while Cu²⁺ and Zn²⁺ metal ions were combined with LD₄ at concentrations of 50, 25, 12.5, 6.25, and 3.13 μg/mL. For each experimental group, 1 mL of solution was prepared, and 1 μL of the DCFH-DA stock solution was added. After thorough mixing, the samples were exposed to a 650 nm laser with a light density of 6 J/cm² for 10 min. Following irradiation, the samples were diluted three-fold, and their fluorescence intensities were measured using a fluorescence spectrometer. Change in the rate of ROS production was calculated using the following formula: R Φ Δ = F s × η s 2 F r × η r 2 × 100 %

F _s and F _r represent areas under the fluorescence curve of the sample and LD₄ control groups, respectively, at 525 nm; η _s is the refractive index of the sample solution, and η _r is the refractive index of the solvent in the LD₄ control group.

The dispersion of Mtb was performed as described in Section 2.2, and a bacterial suspension with a concentration of 10 MCF was prepared. Subsequently, the suspension was transferred to a 5 mL centrifuge tube and centrifuged at 4,000 rpm for 10 min. The supernatant was discarded. The stock solutions of Cu²⁺ and Zn²⁺ were diluted to a final concentration of 31.25 μg/mL and added to the treated bacterial suspension. The mixture was incubated at 37°C for 24 h. After incubation, the samples were centrifuged again at 4,000 rpm for 10 min, the supernatant was discarded, and the pellets were washed thrice with 1 mL of physiological saline under identical centrifugation conditions. Following washing, 5% glutaraldehyde solution (2 mL) was added to each sample for fixation. The samples were thoroughly mixed and fixed at 4°C for 72 h. Next, the fixed samples were observed under a TEM (FEI Tecnai F20, Hillsboro, OR, United States) for morphological analysis and image acquisition. Elemental mapping signals were subsequently collected to determine the distribution characteristics of Cu²⁺ and Zn²⁺ within the bacteria.

Cytotoxicity was assessed using the CCK-8 assay in various cell lines, including HaCaT, 3T3, BEAS-2B, and LO2. Concentrations for Cu²⁺ and Zn²⁺ monotherapy were set at 6.25, 12.5, 25, 50, and 100 μg/mL, while those for Cu²⁺/Zn²⁺-LD₄ were set at 7.81 + 3.13, 15.63 + 6.25, 31.25 + 12.5, 62.5 + 25, and 125 + 50 μg/mL based on the FIC results. After the cells reached the logarithmic growth phase, they were co-incubated with the respective treatment at 37°C for 30 min and subsequently irradiated with a 650 nm laser (energy density: 6 J/cm²) for 10 min. Thereafter, cells were transferred to a cell incubator and incubated for an additional 24 h. Following incubation, enhanced CCK-8 dilution solution (10 μL) was added to each well of both the experimental and control groups. The plates were then incubated at 37°C and 5% CO₂ for 30 min. Following incubation, the plates were removed, and the absorbance at 450 nm was measured for each well using an enzyme-linked spectrophotometer (Thermo Fisher Scientific, VARIOSKAN FLASH, Waltham, MA, United States).

The changes in particle size upon mixing LD₄ solution with Cu²⁺ and Zn²⁺ at specific concentrations were determined using a laser particle size analyzer (Malvern Panalytical ZETASIZER Ultra ZSU3305, Malvern, United Kingdom). Three mixed solutions were prepared with concentrations of 125 + 50, 62.5 + 25, and 31.25 + 12.5 μg/mL (LD₄ + Cu²⁺/Zn²⁺), respectively. The solutions were treated with ultrasound for 15 min to ensure homogeneity. Subsequently, the particle size changes of the mixed solutions were measured at 25°C. Prior to each measurement, the instrument was stabilized for 2 min to ensure measurement accuracy.

Data processing was performed using GraphPad Prism software (version 8.0.1). All experiments in this chapter were independently repeated three times. Statistical analyses were carried out using GraphPad Prism 8.0.2 software, and image processing as well as data export were conducted using ImageJ software. Results are presented as mean ± standard deviation (mean ± SD). Each variable was analyzed using one-way ANOVA, and the significance of differences between groups was assessed via Tukey’s multiple comparison test (p < 0.05).

The MIC values of each metal ion and LD₄ against C. albicans alone were determined using the broth microdilution method, providing a basis for investigating the synergistic effects between metal ions and LD₄. Significant antifungal activity of Cu²⁺, Zn²⁺, Fe³⁺, and Mn²⁺ against C. albicans was demonstrated, with MIC values of 40 μg/mL. In contrast, Mg²⁺ exhibited weaker antifungal efficacy, with an MIC value of 80 μg/mL. Additionally, LD₄ showed limited antifungal activity against C. albicans, with an MIC value of 125 μg/mL. Based on the above results, we observed that the antifungal activity of certain metal ions (Cu²⁺, Zn²⁺, Fe³⁺, and Mn²⁺) was significantly higher than that of LD₄. As a lysine-conjugated amino phenyl porphyrin photosensitizer, LD₄ had been suggested by previous studies to potentially exert synergistic effects through its ability to complex metal ions (Cu²⁺) and accumulate in pathogenic microorganisms (manuscript in preparation). Based on these findings, we propose that the antifungal activity of LD₄ against C. albicans can be enhanced through the addition of supplementary metal ions. To verify this hypothesis, the checkerboard dilution method was used to quantitatively assessed the synergistic effect of metal ions combined with LD₄ against C. albicans.

As shown in Table 1, the metal ions with the most effective synergistic effects were Cu²⁺, Zn²⁺, Mn²⁺, and Mg²⁺, all of which had FICI values < 0.5. Compared to their individual utilization, the MIC of these four metal ions decreased by 16- to 32-fold, while the MIC of LD₄ decreased by 64- to 16-fold. These results demonstrated that when combined with LD₄, Cu²⁺, Zn²⁺, Mn²⁺, and Mg²⁺ exhibited a significant synergistic antibacterial effect against C. albicans. The FICI value of Fe³⁺ was 0.75, indicating an additive effect, which was higher than those of other tested metal ions. Consequently, Cu²⁺, Zn²⁺, Mn²⁺, and Mg²⁺, which exhibited synergistic effects, were selected for further investigation of the synergistic antibacterial activity against Mtb with LD₄. We selected one standard Mtb strain, two sensitive strains, and two drug-resistant strains (resistant to rifampicin and isoniazid) for this study. The results are illustrated as a heatmap (Figure 1), while the corresponding FIC and FICI values are summarized in Table 2. The results demonstrated that Mg²⁺ exhibited no significant synergistic effect against Mtb, with an MIC value exceeding 50 μg/mL. Furthermore, no synergistic effect was observed when Mg²⁺ was combined with LD₄. In contrast, the combination of LD₄ with Cu²⁺, Zn²⁺, or Mn²⁺ resulted in pronounced growth inhibition of Mtb. Quantitative analysis demonstrated that the synergistic antibacterial effects of metal ions combined with LD₄ were more pronounced against sensitive strains and the standard strain H37Rv than resistant strains. The FIC values for these strains were lower than those for drug-resistant Mtb, suggesting that bacterial growth could be effectively inhibited at reduced drug combination concentrations (Eumkeb and Chukrathok, 2013). These three metal ions exhibited synergistic effects with LD₄, with FICI values consistently <0.5. Notably, Cu²⁺ and Zn²⁺ demonstrated the most potent synergistic efficacy against Mtb with LD₄. The MIC values of these metal ions were reduced by 8- to 128-fold, while the MIC of LD₄ decreased by 8- to 64-fold. Furthermore, their FICI values were all < 0.2, indicating a strong synergistic interaction. However, Mn²⁺ exhibited weaker synergistic effect compared to Cu²⁺ and Zn²⁺, although it still demonstrated synergistic efficacy. FIC value of Mn²⁺ (25 μg/mL) was higher than that of Cu²⁺ (0.78–1.56 μg/mL) and Zn²⁺ (0.78–12.5 μg/mL), and there may be a potential risk of manganese ion accumulation toxicity (Rodichkin and Guilarte, 2022). The concentration-dependent antibacterial activity of the LD₄-Cu²⁺ complex highlights a critical mechanistic subtlety. Specifically, complete inhibition of Mtb. Growth was observed exclusively at the synergistic ratio of 3.91 μg/mL LD₄ and 1.56 μg/mL Cu²⁺ (Figure 1, Row 1-Panel 4 and Row 2-Panel 2). Conversely, deviations from this optimal Cu²⁺ concentration paradoxically restored bacterial viability. This phenomenon is likely attributable to the dual interaction between the stoichiometric chelation of LD₄ and Cu²⁺ and the pathogen’s intrinsic metal stress adaptation mechanisms. At the defined ratio, LD₄ and Cu²⁺ may form a stable antimicrobial complex that disrupts membrane integrity or metabolic pathways via targeted interactions. However, concentrations of Cu²⁺ could activate bacterial copper detoxification systems, such as the CtpV efflux pump (a P-type ATPase essential for copper homeostasis in Mtb.), thereby diminishing the efficacy of LD₄. These adaptive responses are consistent with prior studies demonstrating mycobacterial resilience to metal stress through regulated efflux and redox balancing mechanisms (Ward et al., 2008). Based on these findings, Cu²⁺ and Zn²⁺ were selected for subsequent studies on mechanisms and cytotoxicity in combination with LD₄.

The uptake of LD₄ by C. albicans and Mtb was quantitatively analyzed using flow cytometry, with fluorescence intensity serving as the indicator (Setiawati et al., 2017). The results are presented in Figures 2A,B. Compared to the LD₄ alone group, the fluorescence intensity in the LD₄+Cu²⁺ group was significantly reduced (p < 0.05). Although the fluorescence intensity in the LD₄+Zn²⁺ group was higher than that in the LD₄+Cu²⁺ group, no significant difference was observed compared to the LD₄ alone group (p > 0.05). Notably, although Cu²⁺ and Zn²⁺ significantly enhanced the antibacterial efficacy of LD₄ against C. albicans and Mtb, this improvement did not correlate with drug uptake. The possible reason for this observation is the presence of a complexation-dissociation equilibrium between metal ions and LD₄ (Tabata et al., 1996). Consequently, the system comprises at least three components, namely, metal ions, LD₄, and their complexes. These constituents exhibit distinct modes of bacterial interaction, resulting in different biological outcomes. LD₄ initially binds to the cell wall and partial cytoplasmic membrane of Mtb. It disrupts copper transport proteins by photodynamic action, thereby facilitating massive influx of copper ions into the bacterial cells. This process culminates in intracellular copper overload, exerting toxicity and achieving bactericidal effects. Meanwhile, the excessive accumulation of copper ions disrupts bacterial homeostasis significantly, potentially leading to bacterial death. LD₄ enters bacterial cells via passive diffusion at a relatively low concentration. LD₄ may bind to the DNA of Mtb, disrupting protein synthesis through photodynamic action, which in turn suppresses replication and inhibits bacterial growth. Copper ions may complex with the LD₄ porphyrin ring, leading to fluorescence quenching of LD₄ and promoting its aggregation. Consequently, lower fluorescence intensity of LD₄ is observed in flow cytometry analysis.

To further investigate the underlying causes of this phenomenon, we lysed Mtb that had been treated with drugs and analyzed the resulting data using flow cytometry. The scatter plots, histograms, and the fluorescence intensities before and after lysis, are presented in Figures 2C–E. Significant differences were observed in the bacterial scatter plots before and after lysis. The scatter points predominantly shifted toward smaller volume and increased surface roughness, confirming the success of the lysis process (Gienger et al., 2019). Notably, bacterial morphology changes in the LD₄ combined with Cu²⁺ and Zn²⁺ group were more pronounced compared to the LD₄ group, indicating a greater lysis effect. These findings suggest that the synergy between metal ions and LD₄ exerts a substantial impact on bacterial homeostasis, reducing their ability to resist external damage and rendering them more susceptible to lysis. After bacterial lysis, the supernatants were colorless, suggesting that most of the LD₄ was bound to the cell wall, cell membrane, and intracellular components of Mtb; only a small proportion of LD₄ remained in a free and unbound state. Analysis of lysed Mtb following treatment with LD₄ alone showed a greater release of cellular components but reduced average fluorescence intensity. This evidence indicates that, compared to the other two groups, less LD₄ was bound to the bacterial contents. In contrast, in the LD₄ + Cu²⁺ group, lysis resulted in both an increased proportion of cellular contents and a significantly elevated average fluorescence intensity. These findings suggest that under metal ion co-catalysis conditions, Mtb exhibits enhanced LD₄ uptake capability and effectively transports LD₄ from the cell wall into the bacterial interior, thereby achieving efficient photodynamic antibacterial effects. When the concentration of metal ions reached 50 μg/mL, the fluorescence intensity peaked, which may be closely associated with the transport mechanisms of Mtb. In the Zn²⁺ group, a significant difference in LD₄ uptake was observed between pre- and post-lysis conditions. Under high-concentration conditions (25 μg/mL and 50 μg/mL), the average fluorescence intensity of LD₄ was significantly increased, indicating that leaked bacterial contents effectively bound to LD₄, enabling the exertion of the aPDT effect within bacteria. Conversely, under low-concentration conditions (3.13, 6.25, and 12.5 μg/mL), the LD₄ content decreased after lysis, suggesting that the binding capacity of bacterial contents to LD₄ was lower compared to that recorded under high-concentration conditions. Additionally, in the LD₄ group, the fluorescence intensity was significantly decreased, further supporting the aforementioned findings. The experimental results demonstrated that the synergistic interaction of Cu²⁺/Zn²⁺ with LD₄ enhances the uptake of LD₄ by Mtb, thereby significantly improving its antibacterial efficacy.

Based on the above findings, we systematically investigated the spectral properties of the complexes formed between metal ions (Cu²⁺ and Zn²⁺) and LD₄ by analyzing their fluorescence and infrared spectra at various temperatures following combined administration. The corresponding results are presented in Figures 3A–D. Both Zn²⁺ and Cu²⁺ induced fluorescence quenching of LD₄ under varying temperature conditions. Notably, Cu²⁺ exhibited the most pronounced quenching effect on LD₄ (Wu et al., 2020). At 20°C, the fluorescence quenching efficiency (η) was >95%. With decreasing temperature, the fluorescence quenching effect progressively intensified, reaching >98% at 0°C. At 0°C, Zn²⁺ induced an enhancement in the fluorescence intensity of LD₄. With increasing temperature, the η progressively increased from 18.148% to 71.628%. The fluorescence intensity of the LD₄ + Cu²⁺ group decreased as temperature increased, from 98.823% to 95.437%. In the LD₄ + Zn²⁺ group, the fluorescence intensity at 650 nm increased with rising temperature, whereas the fluorescence intensity at 612 nm decreased with increasing temperature. This observation is primarily attributed to Cu²⁺ being a tetrahedral metal ion with four coordination sites, which readily achieves coordination saturation. The resulting compounds or polymer structures exhibit stability, and the binding constant increases. Consequently, its overall structure, irrespective of its form, resembles that of a well-defined compound. Thus, its fluorescence behavior as a function of temperature can be explained by traditional molecular principles: as the temperature decreases, the fluorescence intensity increases, leading to highly efficient fluorescence quenching. Zn²⁺ can adopt a pentacoordinate state. The LD₄ molecule contains four lysine side chains, each providing two amino groups as potential ligands. Consequently, during the binding process, LD₄ tends to form a tetrahedral coordination structure, which cannot fully satisfy the pentacoordination requirement of Zn²⁺ (He et al., 2011). In the pentacoordinate state, Zn²⁺ exhibits an unsaturated coordination environment, and its structure achieves partial stability through dynamic adjustments. However, this relatively unstable configuration is prone to physical energy-dissipating processes, such as rotation, vibration, and oscillatory motions, which adversely affect fluorescence emission efficiency. When the temperature increases, the complex of LD₄ with zinc ions facilitates increased molecular collisions and structural reorganization, thereby transitioning the system from an unstable state to a more stable configuration. This reduction in instability leads to decreased internal energy dissipation within the molecules, which enhances fluorescence emission. Conversely, when the temperature decreases, the coordination-unsaturated state induces continuous internal mechanical adjustments within the molecules. These adjustments result in greater vibrational and oscillatory energy losses, which diminish luminescence efficiency. In conclusion, the response of the unstable structural state caused by coordination desaturation to temperature changes is opposite to that of stable structure molecules: when the temperature rises, the fluorescence signal intensifies; while when the temperature drops, the fluorescence intensity weakens.

Furthermore, the infrared spectra of LD₄, LD₄ + Cu²⁺, and LD₄ + Zn²⁺ were recorded to investigate the changes in their functional groups in response to metal ion coordination. The results are presented in Figure 3E. The absorption peaks observed at 964 cm⁻¹ correspond to the stretching vibration of -NH- in the inner ring of porphyrin; the peaks at 1,440 cm⁻¹ and 1,516 cm⁻¹ belong to the deformation vibration of -CH₂ on the porphyrin ring and the stretching vibration of -C=C-, respectively; the absorption peaks at 1,674 cm⁻¹ and 1,598 cm⁻¹ correspond to the deformation vibration of -C=O and -NH- in the amide bond. Moreover, the absorption peak at 797 cm⁻¹ indicated the presence of a benzene ring. In the LD₄ + Cu²⁺ group, the disappearance of the absorption peak at 964 cm⁻¹ indicated the suppression of -NH- stretching vibrations. The peak at 1,107 cm⁻¹ was attributed to the stretching vibration of -S=O, while the peak at 619 cm⁻¹ corresponds to the bending vibration of -NH- induced by the coordination of Cu²⁺ with the amino group (Zhao et al., 2019). These results confirmed successful coordination between Cu²⁺ and the free amino groups in the porphyrin ring. Additionally, a broad peak observed in the 3,000–3,500 cm⁻¹ region suggests the presence of hydrogen bonding and possible polymer formation. The peak at 3,192 cm⁻¹ is associated with the -NH- vibration of secondary amine groups, whereas the peak at 1,070 cm⁻¹ may represent the characteristic oxidation state band of metal porphyrins formed upon coordination of Cu²⁺ with the -NH- group in the inner porphyrin ring. The infrared spectroscopic analysis of the LD₄ + Zn²⁺ group revealed that the absorption peak at 697 cm⁻¹ was attributed to the deformation vibration of -NH- induced by the coordination of Zn²⁺ with -NH₂. The sharp peak at 1,549 cm⁻¹ showed a significantly increased intensity and reduced transmittance compared to the peak at 1,598 cm⁻¹ in the LD₄ spectrum. This finding confirmed the formation of N-Zn coordination bonds due to chemical bond changes on the amino group. The peak at 1,454 cm⁻¹ corresponded to the symmetric stretching vibration of COO⁻. Moreover, the higher transmittance of the peak at 3,427 cm⁻¹ compared to the peak at 3,419 cm⁻¹ in the LD₄ spectrum indicated a weakened stretching vibration of the amino group (Kurtikyan et al., 2014). This evidence suggested partial participation of amino groups in Zn²⁺ coordination, while some amino groups remained uncoordinated.

The determination of ROS yield serves as a critical indicator for evaluating the oxidative damage exerted by antibacterial agents on pathogen cells (Khorsandi et al., 2022). Measuring ROS yield aids in elucidating the antibacterial mechanism of aPDT, and directly reflects its therapeutic efficacy. The fluorescence spectra of DCFH-DA and the corresponding ROS yield results are presented in Figure 4. As the concentration of Zn²⁺ increased, the production of ROS after LD₄ irradiation gradually increased. In contrast, with increasing concentrations of Cu²⁺, the ability to generate ROS progressively diminished. Cu²⁺ induced a fluorescence quenching effect on LD₄, leading to a significantly reduced ROS yield rate compared to LD₄ alone. At a Cu²⁺ concentration of 50 μg/mL, the ROS production rate decreased to only 6.58% ± 0.13% of that observed with LD₄ alone. Conversely, the addition of Zn²⁺ significantly enhanced the ability of LD₄ to generate ROS, achieving an ROS production rate of 322.35% ± 7.30% at a Zn²⁺ concentration of 50 μg/mL. These experimental results demonstrated that the synergistic interaction between Zn²⁺ and LD₄ significantly increased the ROS yield, enabling efficient bacterial eradication. Although the combination of Cu²⁺ and LD₄ generated less ROS, it still exhibited notable antibacterial efficacy. The relatively low ROS yield can be attributed to the formation of stable complexes between Cu²⁺ and the amino groups on the side chains of LD₄. This interaction reduces non-radiative transitions while enhancing radiative transitions, thereby minimizing vibrational and rotational energy losses to the greatest extent (Kejík et al., 2021). The significant antibacterial activity observed may result from the ability of the Cu²⁺-LD₄ complex to promote the oxidation of bacterial cell wall lipids, which severely compromises the structural integrity of the bacterial cell wall.

TEM analysis was further utilized to explore the mechanism underlying the synergistic antibacterial effect of metal ions and LD₄. Bacterial morphological changes were examined (Figure 5). The cell wall of Mtb contains a high concentration of lipids (Chen et al., 2017). The results demonstrated that H37Rv exhibited intact structural integrity, with a tightly packed cell wall, an undamaged cell membrane, and observable ribosomes in the control group. In contrast, in the Cu²⁺ treatment group, partial indentations were observed in the bacterial cell wall compared to the control group. Additionally, no significant differences were noted between the light and dark condition groups, suggesting that light exposure did not significantly influence the Cu²⁺-induced damage to the Mtb cell wall. Bacterial deformation and cell wall indentation were observed in the Zn²⁺ treatment group. However, light exposure did not significantly compromise the structural integrity of the bacteria. In the LD₄ group, bacterial structures remained largely intact under dark condition, with no clear cell wall indentation or damage. Similarly, under light condition, the bacterial cell wall maintained its integrity. These findings suggest that under light reaction conditions, LD₄ does not significantly impair the viability of Mtb. In the LD₄ + Cu²⁺ group, detachment of the bacterial cell wall from the cell membrane was observed in the dark reaction group, suggesting that the dark reaction significantly affected the structural integrity of Mtb cells. The coordination of Cu²⁺ with the amino groups of LD₄ formed a copper-ammonia complex with strong oxidizing properties, which induced the separation of the cell wall and cell membrane. Under light condition, the cell wall was severely compromised, leading to leakage of bacterial contents, which severely impaired the physiological activity of Mtb and consequently resulted in a strong antibacterial effect. In the LD₄ + Zn²⁺ group, bacterial structures were relatively intact, and antibacterial activity was weak under dark condition. In contrast, under light condition, the bacterial cell wall exhibited damage, intracellular contents leaked out, and separation of the cell wall from the cell membrane as well as a blank region around the ribosomes were observed. Typically, hydrogen peroxide, peracetic acid, and chlorine dioxide are used as disinfectants for inactivating Mtb. However, their effective concentrations are relatively high; for example, hydrogen peroxide requires 0.8%, while peracetic acid requires 0.06%, and these agents must be maintained for 60–90 min to effectively kill Mtb. Chlorine dioxide requires an effective concentration of 1–10 g/L (Rutala et al., 1991). Additionally, these three disinfectants exhibit significant cytotoxicity and can cause strong irritation to the respiratory tract.

We further quantified the metal ion content within the bacteria using TEM mapping, and the results are presented in Figure 6. Under light condition, the combination of LD₄ with Cu²⁺/Zn²⁺ induced significant damage to the cell wall of H37Rv, resulting in the leakage of bacterial contents. Mapping analysis of Mtb in the combined treatment group revealed that Zn was almost undetectable in the control group, whereas Cu was only present in trace amounts within the bacteria. We observed that in the LD₄ combined with Cu²⁺/Zn²⁺ group, the Mapping images of Cu and Zn elements showed a high degree of overlap with bacteria cells. Additionally, Cu and Zn were detected in the bacterial efflux contents. These findings suggest that the complexes formed by Cu²⁺/Zn²⁺ and LD₄ can localize to the bacterial cell wall, cell membrane, and intracellular contents, exerting antibacterial effects via photodynamic action under 650 nm illumination. In the LD₄ group, a small amount of Cu was observed on the bacterial surface, whereas Zn was scarcely detectable within the bacteria. The results of the Cu²⁺ group showed that there was no significant difference between the bacteria in this concentration group and those in the Control group or the LD₄ group. This finding may be attributed to the cooperative action of the nutrient copper sensor Mac1p (Graden and Winge, 1997) and the toxic copper sensor Ace1p (Peña et al., 1998), which jointly regulate copper homeostasis in bacteria, ensuring that intracellular copper ions remain within a range that satisfies physiological activity requirements while avoiding toxic accumulation. Mapping results of the Zn²⁺ group demonstrated that intracellular Zn²⁺ levels were extremely low. Zinc homeostasis is primarily regulated by the zinc importer MtNramp1 (Juttukonda and Skaar, 2015) and the zinc efflux protein MtZnT1 in Mtb (Dow et al., 2021). MtNramp1 facilitates zinc uptake to fulfill physiological demands, whereas MtZnT1 plays a critical role in preventing cytotoxic levels of intracellular Zn²⁺ accumulation (Tejada-Jiménez et al., 2015). Mapping results from the Cu²⁺ and Zn²⁺ groups demonstrated that Mtb exhibited a higher uptake for copper compared to zinc. When LD₄ was applied alone, the corresponding TEM images revealed a modest increase in copper levels within bacterial cells. In contrast, in the LD₄ + Cu²⁺ group, where additional Cu²⁺ was supplemented, TEM images indicated a significant increase in intracellular copper ion levels. In the LD₄ + Zn²⁺ group, the intracellular Zn²⁺ concentration was significantly higher than that in the LD₄ group, the control group, and the Zn²⁺ group. Previous findings revealed that LD₄ can form complexes and nanoparticles with Zn²⁺, resulting in a higher singlet oxygen yield compared to LD₄ alone. Under 650 nm laser irradiation, this enhanced photodynamic antibacterial activity surpassed that of LD₄ alone. The increased intracellular Zn²⁺ levels in bacteria were found to inhibit the activity of key enzymes, induce oxidative stress, high ROS yield, and interfere with the synthesis of the bacterial cell wall, thereby contributing to its overall antibacterial efficacy. Under high concentration condition, the activities of proteins involved in maintaining Cu²⁺ and Zn²⁺ homeostasis may be inhibited, leading to excessive intracellular accumulation of Cu²⁺ and Zn²⁺, inducing toxicity and, thereby, achieving a greater antibacterial effect.

Mouse embryonic fibroblast cells (3T3), human immortalized keratinocytes (HaCaT), human normal liver cells (LO2), and human bronchial epithelial cells (BEAS-2B) were selected to evaluate the cytotoxicity under the synergistic cytotoxicity of metal ions and LD₄ using CCK-8 assays. The results are presented in Figures 7A–D. At low concentrations, the cell survival rate remained relatively high. Under therapeutic dose conditions, the cell viability was maintained >98% across all tested cell lines. However, when the concentration of LD₄ in combination with Cu²⁺ was gradually increased to 31.25 + 12.5 μg/mL, the survival rate of HaCaT cells decreased significantly to 52.43% ± 8.63%. Notably, at this concentration, no cytotoxic effects were observed in 3T3, BEAS-2B, or LO2 cells, with their survival rates remaining >90%. The survival rate of HaCaT cells gradually increased with the increasing concentration. When the concentration reached 125 + 50 μg/mL, the survival rate increased to 75.36% ± 6.47%. The experimental results for the LD₄ combined with Zn²⁺ group revealed that HaCaT, 3T3, and BEAS-2B cells exhibited a trend consistent with that observed in the LD₄ + Cu²⁺ group. Under therapeutic doses, the cell survival rate remained >90%, indicating low cytotoxicity. However, at high concentrations, the cytotoxicity of metal ions combined with LD₄ was observed. At 62.5 + 25 μg/mL, the viability of HaCaT, 3T3, and BEAS-2B cells was lowest, at 75.13% ± 0.94%, 63.34% ± 1.34%, and 63.48% ± 0.57%, respectively. At 125 + 50 μg/mL, the survival rate of LO2 cells was lowest, at 68.89% ± 7.08%. The possible reason for the initial increase followed by a decrease in cytotoxicity with increasing concentration is that LD₄ forms dimers or trimers at higher concentrations, thereby reducing metal ion-induced damage to cells and resulting in lower cytotoxicity at high concentrations. To verify the conjecture, the changes in particle size under various concentration combinations were determined.

The results of particle size determination are presented in Figures 7E–G. Upon mixing the LD₄ aqueous solution with the metal ion aqueous solution, significant aggregation was observed, with a significant increase in the proportion of particles measuring >1 μm and an overall rise in average particle size. In contrast to the observations, the LD₄ aqueous solution exhibited less particle aggregation. Notably, most of the particulate matter was retained in a dissolved colloidal state throughout the experimental duration, as evidenced by dynamic light scattering analysis. The cytotoxicity trend observed in Figures 7C,D may be attributed to the formation of polymers through the combination of some LD₄ molecules with metal ions. This process complexes the toxic metal ions, thereby reducing their cytotoxic effects and ultimately enhancing cell survival rates.