The chimeric lysin, ClyC, was expressed as described previously (Li et al., 2021). Concentration of ClyC was determined by BCA assay and visualized by SDS-PAGE. The ClyC protein solution was freeze-dried and then stored at −80°C until use. An optimized ClyC-AH formulation was prepared using a one-step procedure at room temperature as proposed by Deng and Feng (Deng et al., 2015; Feng et al., 2020), with slight modification. Briefly, sodium alginate was dissolved in distilled water and magnetically stirred for 3 h until it completely dissolved. Freeze-dried ClyC was mixed with 0.68% calcium chloride solution (Hushi, Shanghai, China). Then, the mixture was added into 1.5% alginate solution at a ratio of 1:1 (v/v) and homogenized. The gelation time was measured by the inversion tube method at room temperature. In brief, 400 μL ClyC-AH was taken in a 1.5-ml Eppendorf tubes and incubated at room temperature. The gelation time was confirmed by inverting the tube every 5 s until the gel stopped flowing. Each solution was filtered separately through a sterile 0.22-μm membrane into a sterile container.

Hydrogel alone and ClyC-AH were separately immersed in Tris buffer at room temperature until reached swelling equilibrium, cut into sections (10 mm × 7 mm × 5 mm). The samples were lyophilized using the LyoBeta 6 PL (Telstar, Spain) instrument, coated with gold (Hummer VI; Technic Inc., Anaheim, CA, United States). The microstructure of the sample was evaluated using the SEM (SU8010, Hitachi, Japan) at an accelerating voltage of 3 kv.

The toxicity of ClyC released from hydrogel was evaluated using the CCK-8 assay. Alginate hydrogels with different ClyC concentrations (0, 31.25, 62.5, 125, 250, 500, and 1,000 μg/ml) were incubated in equal-volume Dulbecco’s modified Eagle’s medium (DMEM; Sigma, United States) at 37°C. After 24 and 48 h, the whole medium was collected. To investigate the cytotoxicity of the ClyC in the harvested medium, Baby hamster Syrian kidney (BHK)-21 cells were used. The cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C under 5% CO₂. BHK-21 cells were seeded into 96-well at a density of 1 × 10⁴ cells/well. After 24 h, the cell culture medium was exchanged with the harvested medium (100 µL) and the same concentration of soluble ClyC as a control. After incubation for 24 h, the cell viability was measured by CCK-8 assay. The normal cell culture medium without ClyC was used as a positive control (PC), while medium without ClyC and BHK-21 cells served as a negative control (NC). The relative cell viability was determined by using the formula: %cytotoxicity = [1- (OD_sample–OD_NC)/(OD_PC–OD_NC)] × 100%.

To investigate the release amount of ClyC from alginate hydrogels, the ClyC-AH (250 µg/ml, 400 µL) was incubated in Tris buffer (100 µL in 1.5-ml Eppendorf tubes) at 37°C. At predesignated time intervals (1, 3, 6, 10, 14, 24, 36, 48, and 72 h). At each timepoint, the supernatant was sampled, and the same volume of fresh Tris buffer was added. The amount of released ClyC from alginate hydrogels at each time point was determined using the BCA method and calculated based on the standard curve. The structural integrity of ClyC after release was examined using SDS-PAGE analysis. The ClyC released from hydrogel was suspended in SDS-PAGE loading buffer, destabilized by boiling for 10 min, processed and analyzed by SDS-PAGE, following standard protocol. Free native ClyC was also processed similarly and analyzed along with the test sample as a reference.

The bacteria strain used in the study was S. aureus T23. The inoculum was prepared by culturing S.aureus T23 overnight in lysogeny broth (LB) medium with shaking at 37°C, washing three times in Tris buffer, by centrifugation, and adjusting the optical density at OD₆₀₀ (Synergy H1). A preliminary assessment of the anti-bacterial effect of ClyC released from hydrogel was conducted via turbidity reduction experiment and inhibition zone method. For turbidity reduction experiment, the decrease of samples in OD₆₀₀ was monitored by a Synergy H1 microplate reader every 1 min for 10 min at 37°C. For the inhibition zone method, 10 μL ClyC and ClyC-AH were spotted onto agar plates overlaid with T23 and incubated overnight at 37°C. Bactericidal activity was assessed by clear zones on the agar plates. Comparison of bactericidal activity of different timepoint sample was done by measuring colony count. Samples from each timepoint (50 µL) incubated with 50 µL T23 inoculum at 37°C for 1 h, serial dilution, and plating on lysogeny broth agar. Free ClyC solutions (125 μg/ml) incubated at 37°C were also prepared at each timepoint.

This study was approved by the Ethics Committee for Animal Research, Wuhan University, China (No. 2019A79). C57BL/6 mice were housed in 12-h light/dark cycles and freely provided food and water. The mice were assigned to four groups (bacteria infected, bacteria infected with hydrogel alone, bacteria infected with soluble ClyC alone, and bacteria infected with ClyC-AH groups; n = 5 or 6 per group). Briefly, after anesthetized, the skin on the right knee of mice was shaved and sterilized with 75% alcohol. A lateral incision was made, and the muscle was bluntly dissected to expose the femur. The proximal end of the femur was perforated using a high-speed drill with a 0.5-mma sterile round bur (Cassat and Skaar, 2013; Klopfenstein et al., 2021). T23 (3 × 10⁶ cell CFU/mL in 2 μL of PBS) was inoculated into the medullary cavity of the femur using 10 μL-pipet tips and then injected 5 μL of different treatments. For animals receiving hydrogel alone, the hydrogel was pipetted over the hole to polymerize. For animals receiving soluble ClyC, the concentration of ClyC was equal to that of ClyC-AH. Then the wound was double-stitched by surgical silk suture.

After 1 week, all mice were sacrificed. The skin on the right hind leg was disinfected with 75% alcohol. After making a lateral incision, the femur and surrounding tissue were separated, weighed, and placed in 1.5-ml microcentrifuge tubes (with saline). Following dissection and removal, all samples were maintained on ice. The tissue and femur samples were subsequently homogenized using the MD100 by bead beating (5,000 rpm, 20 s) (NewZongKe, China) (Johnson et al., 2018). Water bath sonication was used to create single-cell bacterial suspensions after homogenization (sonicate 10 min, vortex 30 s). Homogenates were serially diluted, plated on Baird-Parker agar, and incubated overnight at 37°C. Colonies were acounted, normalized to sample weight, and transformed using the formula CFU = log₁₀ (1 + X) to avoid negative values.

Experimental data were expressed as mean ± standard deviation. Statistical analyses were conducted using Prism v 8.0.2 (GraphPad Software, United States). Statistical comparisons between two groups were made with Student’s t test. ANOVA for multivariate parametric data with a Tukey’s post hoc test or a Kruskal–Wallis test with Dunn’s post hoc test for nonparametric data. p-value of <0.05 was considered as statistically significant.

ClyC-AH were prepared simply by mixing 1.5% alginate solution and 0.68% calcium chloride solution-containing ClyC, which was the most common method to prepare the hydrogel from the sodium alginate solution (Lee and Mooney, 2012). From a macroscopic view, there was no significant difference between ClyC-AH and alginate hydrogel without ClyC, which were both transparent and colorless (Figure 2A). The morphology of the samples was observed using SEM. The surface morphology of hydrogel alone and ClyC-AH was similar, which showed porous structure with good continuity. There were tiny pores observed in the structure which was thought to be beneficial for the sustain ClyC release (Figure 2C). The gelation time of ClyC-hydrogel was about 20 s, short but sufficient for injection into the lesion (Figure 2B). Meanwhile, it was conducive for the rapid fixation of ClyC-AH in the lesion.

Considering that high concentration of ClyC could cause cytotoxicity in the local infection, we loaded ClyC into alginate hydrogels. The results showed that the cytotoxicity of ClyC-AH was reduced compared with soluble ClyC (Figure 3). At 24 h after incubation, with the concentration of ClyC up to 250 μg/ml, the cytotoxicity of ClyC-AH showed no significant difference among different concentrations. Soluble ClyC alone had toxic effect on BHK-21 cells. Compared with soluble ClyC, ClyC-AH significantly improved cell viability. It could be explained by the slow release of ClyC from alginate hydrogels. The results suggested that the blank and loaded 250 μg/ml ClyC-AH were not cytotoxic to BHK-21 cells.

The amount of ClyC released from the alginate hydrogels are showed in Figure 4A. After 72 h, the cumulative released amount of ClyC from alginate hydrogels was approximately 23 ± 0.45%. To test the structural integrity of ClyC released from the alginate hydrogels, SDS-PAGE analysis was used and showed a single band corresponding to the molecular weight of free ClyC, as could be seen in Figure 4B. No additional band below the ClyC from the alginate hydrogels band, proving that ClyC was not hydrolyzed or degraded during the process of hydrogel gelation and release.

As reported before, ClyC had highly anti-bacterial against S.aureus and showed enhanced bactericidal activity in the presence of calcium. In the turbidity reduction experiment, the OD₆₀₀ values of T23 bacterial suspensions reduced rapidly (Figure 5A). In support, clear zones could also be seen in the plates after dipping ClyC and ClyC-AH on the lawn of S. aureus (Figure 5B), which showed that the released ClyC retained bactericidal activity. The bactericidal activity against S.aureus T23, by ClyC released from the hydrogel after different time intervals, was compared. The results showed that the ClyC released from hydrogel had high bactericidal activity within 24 h. After incubation in 37°C incubator for 24 h, ClyC could lead to a reduction of 2.89 log₁₀ in viable cell numbers. Moreover, after incubation for 72 h, there was still a 1.14 log₁₀ reduction in viable cells, indicating a therapeutic effect for acute infection (Figure 5C).

The osteomyelitis mouse model was established to evaluate the ability of ClyC-AH to inhibit S. aureus infection in vivo. ClyC-AH was injected into the medullary cavity using 10 μL-pipet tips and subsequently transformed into hydrogels which allow stable injection into the cavity without leakage. The number of bacteria CFU in the femur and surrounding tissue was measured 7 days after surgery to evaluate the severity of osteomyelitis. Both the infection-only and hydrogel-only groups had high number of bacteria in the femur and surrounding tissue (Figure 6), indicating alginate hydrogels alone had no effect against S. aureus. The number of bacteria of the soluble ClyC group was significantly reduced in the surrounding tissue (Figure 6B). For the ClyC-AH group, the amount of bacteria was significantly reduced compared with the infection-only group or the hydrogel-only group both in the femur and surrounding tissue (Figure 6). In summary, these results demonstrated that ClyC-AH decreased S. aureus infections, the effect of which was comparable with that of soluble ClyC.