Carbon monoxide-releasing molecule-3 eradicates mature Enterococcus faecalis biofilms and inhibits recolonization

CORM-3 solution preparation and CO release kinetics

CORM-3 was dissolved in PBS to prepare a 10 mM stock solution, freshly prepared before use. Inactivated control (iCORM-3) was obtained by incubating CORM-3 solution at 37°C for 24 h to completely exhaust CO release. ¹⁴

CO release kinetics were measured using the deoxymyoglobin assay. ¹⁵ Deoxymyoglobin (deoxy-Mb, final concentration 60 μM) was incubated with CORM-3 (final concentration 40 μM) in PBS buffer (pH 7.4) at 37°C. Absorbance changes at 540 nm were continuously monitored by UV spectrophotometry. CO release was calculated using the molar extinction coefficient, and time-CO release curves were plotted to determine the half-release time (t_1/2).

Experimental groups

The following experimental groups were established: (1) Control group: PBS treatment; (2) iCORM-3 group: inactivated CORM-3 treatment (400 μM) as vehicle control; (3) CORM-3 groups: low-dose (200 μM) and high-dose (400 μM) for dose-response evaluation. All experiments were independently repeated at least three times.

Mature biofilm eradication assay

E. faecalis ATCC 29212 suspension (1 × 10⁷ CFU/mL) was inoculated into 96-well plates and cultured anaerobically at 37°C for 72 h to establish mature biofilm models. ⁴ After removing the culture medium, wells were gently washed three times with PBS. Fresh BHI medium containing various concentrations of CORM-3 (0, 200, 400 μM) or iCORM-3 (400 μM) was added, followed by continued incubation for 24 h.

Biofilm colonization inhibition assay and SEM observation

Sterile round coverslips (14 mm diameter) were placed in 24-well plates. E. faecalis suspension (1 × 10⁷ CFU/mL) was co-cultured with various concentrations of CORM-3 (0, 200, 400 μM) under anaerobic conditions at 37°C for 24 h to assess the inhibitory effect of CORM-3 on initial bacterial colonization and biofilm formation.

For SEM sample preparation, coverslips were washed with PBS after culture, fixed overnight with 2.5% glutaraldehyde at 4°C, dehydrated through a graded ethanol series, substituted with tert-butanol, and freeze-dried. After gold sputter-coating, samples were observed using scanning electron microscopy (Hitachi SU8010) at 5 kV accelerating voltage. Images were acquired at 2000× and 10,000× magnifications.

RT-qPCR analysis of biofilm-related gene expression

Biofilm bacteria from control and CORM-3-treated groups (400 μM, 24 h) were collected. Total RNA was extracted using TRIzol method, and concentration and purity were assessed by NanoDrop (A₂₆₀/A₂₈₀ ratio 1.8-2.0). After reverse transcription to cDNA, real-time quantitative PCR was performed. Target genes included: esp (enterococcal surface protein, mediating initial adhesion), ¹⁷ gelE (gelatinase, involved in tissue invasion), ¹⁸ fsrB (core component of fsr quorum sensing system), ⁵ and cylL (cytolysin precursor). ¹⁹ 23S rRNA served as the internal reference gene, and relative expression was calculated using the 2^−ΔΔCt method.

ROS generation and membrane integrity assays

Intracellular reactive oxygen species (ROS) generation and membrane integrity were evaluated in mature E. faecalis biofilms treated for 24 h with PBS (control), iCORM-3 (400 μM), or CORM-3 (200 or 400 μM). For ROS detection, treated bacteria were collected, washed twice with PBS, and incubated with 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C for 30 min in the dark. After washing to remove the unloaded probe, fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 488 nm and 525 nm, respectively, and expressed as relative fluorescence units (RFU). To assess membrane damage, culture supernatants were collected after treatment and centrifuged at 12,000 × g for 10 min to remove residual cells. The concentration of DNA released into the supernatant was determined from the absorbance at 260 nm, and the amount of leaked protein was quantified using a bicinchoninic acid (BCA) protein assay kit with bovine serum albumin as the standard. All assays were performed with three independent biological replicates.

Cytocompatibility assessment

HOK and HGE cells were seeded into 96-well plates at 5 × 10³ cells/well. After cell attachment, medium was replaced with culture medium containing various concentrations of CORM-3 (0, 100, 200, 400, 800 μM). Cell viability was assessed using CCK-8 assay at 24 h and 48 h. Ten microliters of CCK-8 solution was added to each well, and after 2 h incubation at 37°C, absorbance was measured at 450 nm. Cells treated with 0.1% Triton X-100 served as positive control (complete killing), and untreated cells served as negative control. Relative viability (%) = (OD of treatment group−OD of positive control)/(OD of negative control−OD of positive control) × 100%. According to ISO 10993-5 standard, cell viability >70% indicates no significant cytotoxicity.

Animal experiments

SPF-grade male SD rats (180–220 g, 6–8 weeks old) were randomly assigned to two groups (n = 6/group): control group (intraperitoneal injection of equal volume saline) and CORM-3 group (intraperitoneal injection of CORM-3, 8 mg/kg/day, equivalent to approximately 400 μM plasma concentration). Administration was performed once daily for seven consecutive days. Animal experiments were approved by the institutional ethics committee.

Statistical analysis

All experiments were independently repeated at least three times. Data are presented as mean ± standard deviation (x̄ ± s). Statistical analysis was performed using GraphPad Prism 9.0. Comparisons between two groups were analyzed by independent samples t-test, and comparisons among multiple groups were analyzed by one-way ANOVA. p < 0.05 was considered statistically significant.

CO release characteristics of CORM-3

The chemical structure of CORM-3 is shown in Figure 1(A). Its molecular formula is [Ru(CO)₃Cl(glycinate)], a water-soluble ruthenium carbonyl compound. The ruthenium atom (Ru) coordinates with three carbonyl groups (CO), which gradually dissociate under physiological conditions to release CO. ¹² The activation mechanism of CORM-3 primarily depends on hydrolysis. In aqueous environments, the chloride and glycinate ligands are progressively displaced, destabilizing the Ru-CO bonds and triggering CO release. CO release kinetics were measured using the deoxymyoglobin assay (Figure 1(B)). In PBS buffer at 37°C and pH 7.4, CORM-3 (40 μM) incubated with deoxymyoglobin (60 μM) showed continuous CO release and binding to form carboxymyoglobin (MbCO). CO release exhibited rapid initial kinetics followed by gradual deceleration. Release reached 12.8 ± 1.6 μM at 1 min, 30.4 ± 2.5 μM at 5 min, and plateaued at 32.8 ± 2.7 μM by 10 min, with a half-release time (t_1/2) of approximately 1.5 min. This rapid onset with sustained release pattern is advantageous for rapidly establishing effective CO concentrations at the treatment site. ¹⁴ In contrast, iCORM-3 released only 1.36 ± 0.16 μM under identical conditions at 10 min, confirming complete loss of CO-releasing activity and validating its use as a negative control.OPEN IN VIEWER

CORM-3 eradicates mature E. faecalis biofilms

To address the more clinically challenging mature biofilms, we constructed 72-h E. faecalis biofilm models and evaluated CORM-3 eradication efficacy. ⁴ Crystal violet staining visually demonstrated dense dark-purple staining in control and iCORM-3 groups, while CORM-3-treated groups showed markedly reduced staining in a dose-dependent manner (Figure 2(A)). Quantitative analysis revealed no significant difference between iCORM-3 (400 μM) and control groups, confirming that the antibacterial activity of CORM-3 originates from released CO rather than the ruthenium carrier (Figure 2(B)). After 24 h treatment with 200 μM CORM-3, mature biofilm eradication rate reached 42.8 ± 5.8%, increasing significantly to 65.3 ± 4.7% at 400 μM. XTT metabolic activity results were consistent with crystal violet quantification (Figure 2(C)). The iCORM-3 group showed no significant difference in metabolic activity compared to control. Treatment with 200 μM CORM-3 reduced residual biofilm metabolic activity by 38.5 ± 6.2%, while 400 μM treatment achieved 61.7 ± 5.3% metabolic inhibition. These results indicate that CORM-3 not only physically disrupts biofilm structure but also effectively kills or inhibits the metabolic activity of surviving bacteria within the biofilm.OPEN IN VIEWER

CORM-3 inhibits E. faecalis biofilm colonization

SEM observation directly revealed the inhibitory effect of CORM-3 on E. faecalis initial colonization and biofilm formation (Figure 3(A)). In the control group, abundant E. faecalis adhered to coverslip surfaces. Bacteria were tightly connected by extensive extracellular matrix, forming dense three-dimensional biofilm network structures. Following CORM-3 treatment, bacterial adhesion was substantially reduced. Only scattered individual bacteria or small clusters were observed, with virtually no organized biofilm structure formation. Additionally, some bacteria exhibited morphological alterations including surface wrinkling and irregular depressions, suggesting compromised cell membrane integrity. These results demonstrate that CORM-3 effectively inhibits initial E. faecalis adhesion to substrate surfaces and subsequent biofilm colonization.OPEN IN VIEWER

CORM-3 downregulates biofilm-related gene expression

To elucidate the molecular mechanisms underlying CORM-3 anti-biofilm activity, we analyzed biofilm-related gene expression changes by RT-qPCR (Figure 3(B)). Results showed significant downregulation of multiple key virulence genes in E. faecalis after 24 h CORM-3 treatment. Expression of esp was downregulated by 64.2 ± 7.5%, gelE by 68.7 ± 5.8%, fsrB by 55.3 ± 8.1%, and cylL by 42.1 ± 9.3%. These findings indicate that CORM-3 simultaneously inhibits E. faecalis adhesion capacity (esp), extracellular matrix metabolism (gelE), and the quorum sensing system (fsrB) at the transcriptional level, thereby blocking biofilm colonization and maturation through multiple pathways.^5,17,20

CORM-3 induces ROS generation and compromises membrane integrity

To further elucidate the antibacterial mechanism of CORM-3, we examined intracellular ROS accumulation and bacterial membrane integrity in mature biofilms following treatment (Figure 4). Intracellular ROS levels detected by the DCFH-DA fluorescence assay increased in a dose-dependent manner after CORM-3 treatment (Figure 4(A)). Relative to the control group (1398 ± 172 RFU), ROS fluorescence rose to 3085 ± 298 RFU at 200 μM and 4592 ± 412 RFU at 400 μM, corresponding to approximately 2.2- and 3.3-fold increases, respectively, whereas the iCORM-3 group (1487 ± 195 RFU) showed no significant difference from the control. Membrane damage was assessed by quantifying the leakage of intracellular contents into the culture supernatant. Extracellular DNA leakage increased from 7.1 ± 1.0 μg/mL in the control group to 23.8 ± 3.1 μg/mL at 200 μM and 37.2 ± 4.2 μg/mL at 400 μM CORM-3 (Figure 4(B)), and protein leakage rose correspondingly from 19.2 ± 2.5 μg/mL to 64.8 ± 6.7 μg/mL and 101.5 ± 8.9 μg/mL (Figure 4(C)). For both indicators, the iCORM-3 group remained comparable to the control, confirming that these effects originate from released CO rather than the ruthenium carrier. Collectively, these findings demonstrate that CORM-3 induces intracellular oxidative stress and compromises membrane integrity, resulting in leakage of cellular contents and bacterial death. Together with the transcriptional downregulation of biofilm-related genes, this mechanism accounts for the antibacterial and anti-biofilm activity of CORM-3 against E. faecalis (Figure 4(D)).OPEN IN VIEWER

Cytocompatibility of CORM-3

To evaluate CORM-3 safety for oral tissue cells, we assessed its cytotoxicity to HOK and HGE cells using CCK-8 assay (Figure 5). For HOK cells (Figure 5(A) and (B)), cell viability after 24 h treatment with 100, 200, 400, and 800 μM CORM-3 was 96.8 ± 2.6%, 93.4 ± 3.4%, 87.3 ± 3.6%, and 68.5 ± 5.2%, respectively. At 48 h, viability was 94.5 ± 3.0%, 90.7 ± 3.5%, 83.6 ± 4.1%, and 61.2 ± 6.4%, respectively. For HGE cells (Figure 5(C) and (D)), cell viability after 24 h treatment at corresponding concentrations was 97.2 ± 2.1%, 94.6 ± 2.9%, 89.1 ± 4.3%, and 70.3 ± 6.7%, respectively. At 48 h, viability was 95.3 ± 2.6%, 91.8 ± 3.3%, 85.2 ± 4.7%, and 63.4 ± 6.9%, respectively. According to ISO 10993-5 standard, CORM-3 demonstrated favorable biocompatibility toward both oral cell types at concentrations ≤400 μM. Notably, 400 μM represents the optimal anti-biofilm concentration in this study, indicating acceptable cellular safety at effective antibacterial concentrations.OPEN IN VIEWER

In vivo safety evaluation of CORM-3

To further evaluate CORM-3 systemic toxicity, acute toxicity experiments were performed in SD rats (Figure 6). Histopathological examination revealed no obvious pathological changes in H&E-stained sections of heart, liver, spleen, lung, and kidney from both control and CORM-3 groups (Figure 6(A)). Specifically, cardiac muscle fibers were regularly arranged, hepatocyte cords showed normal organization, splenic red and white pulp structures were preserved, alveolar structures remained intact, and glomerular morphology appeared normal. Complete blood count analysis showed no significant differences in WBC, RBC, HGB, and PLT between CORM-3-treated rats (8 mg/kg/day for 7 days) and control group (Figure 6(B)), indicating that CORM-3 at this dose does not cause significant hematological toxicity. These results demonstrate that CORM-3 exhibits no apparent toxic damage to major organs in rats at the experimental dose and possesses favorable in vivo safety.OPEN IN VIEWER