Hyperbranched Poly‐L‐Lysine‐Based Water‐Insoluble Complexes as Antibacterial Agents with Efficient Antibacterial Activity And Cytocompatibility

Abstract

Despite the advances in technology, bacterial infection associated with biomedical devices is still one of the most challenging issues in clinical practice. Incorporation of antimicrobial agents is regarded as an efficient way to combat medical device associated infectious. However, most of antimicrobial agents have high toxicity to host cells. Thus, fabrication of novel antimicrobial agents that simultaneously fulfill the requirements of antibacterial activity as well as biocompatibility is urgently needed. Herein, a series of water‐insoluble antibacterial complexes based on hyperbranched poly‐L‐lysine (HBPL) and four different surfactants through non‐covalent interactions are developed. Such kinds of surfactants have great effects on the antibacterial property of poly(ɛ‐caprolactone) (PCL) films that incorporate with the HBPL‐based complexes. The results reveal that the PCL films that doped with HBPL/phosphate ester surfactant complexes showed the highest bacterial killing efficiency. Moreover, the cytocompatibility of the composite films is also investigated. Hemolysis experiments indicate that all the films  had low hemolytic activities. Considering the excellent antimicrobial and cytocompatibility properties, this work believes that the optimized complexes have great potential to be used as antimicrobial agents in biomedical field.

In this study, a series of water‐insoluble antibacterial complexes based on hyperbranched poly‐L‐lysine (HBPL) and different surfactants through non‐covalent interactions are developed. The complexes that contained surfactant with phosphate ester group show excellent bactericidal capabilities even at low dopant content. In addition, they also display outstanding cytocompatibility. This work would provide a novel method for design of safe and high antibacterial efficiency materials to inhibit biomedical device associated infection.

1. Introduction

The incidence of the bacterial‐associated infections is increasing on the back of the increased use of biomedical device. Once a bacterial biofilm is firmly established on the surface of the biomedical device, replacement surgery would be the only choice.^{[ 1 ]} The clinical risks of biomedical device‐associated infections can be reduced by using antibacterial agents. During the last 20 years, great efforts have been made to synthesize difunctional bactericidal agents that have high antimicrobial activity and low toxicity to host cells to meet the clinical requirement.^{[ 2} , ³ , ^{4 ]}

Antibacterial peptides (AMPs) can be used as potential alternative for antibiotics to solve the antimicrobial resistance threaten.^{[ 5} , ⁶ , ^{7 ]} The AMPs can selectively target the microbial membrane over host's cell membrane.^{[ 8 ]} The inherent cationic feature promotes the interaction of AMPs with microbial cell membranes.^{[ 9 ]} Moreover, AMPs have many other biological functions, such as immune regulation, wound healing and antitumor activity.^{[ 10 ]} However, the AMPs are sensitive to hydrolysis by pH changes or proteases, and the high cost limits the application of AMPs. Fortunately, a chemical synthetic AMP analog – hyperbranched poly‐L‐lysine (HBPL) showed high bacterial killing efficiency, good biocompatibility, and enhanced stability.^{[ 11 ]} More importantly, large‐scale synthesis of HBPL through thermal polymerization method can cut its cost and expand the applications.^{[ 12 ]} Recently, Yang et al. covalently immobilized HBPL onto the surface of titanium, and the functionalized coating showed strong antibacterial property and good cytocompatibility.^{[ 11 ]} However, bacterial attachment on the hyperbranched polypeptides grafted surface may occur because the inevitable defects caused by mechanical damage or degradation of the polymer in vivo.

Embedding bactericidal agents, such as nanoparticles,^{[ 13} , ¹⁴ , ^{15 ]} antibiotics,^{[ 16 ]} or polycation,^{[ 17} , ^{18 ]} into a matrix material has been considered as an effective strategy to combat biomedical device associated infections. To our knowledge, physical blending HBPL into polymer matrix to impart antibacterial property is a well‐studied strategy.^{[ 19} , ²⁰ , ^{21 ]} However, the antibacterial property may loss with time after implantation due to the water solubility of the HBPL.^{[ 22 ]} Katano et al. reported a water‐insoluble complex formed by water soluble ε‐poly‐l‐lysine (ε‐PL) and anionic surfactant, and the complex can be dissolved in organic solvents.^{[ 23 ]} This kind of complexes formed by electrostatic and hydrophobic interaction have attracted great attention in antibacterial field. Compared to the linear ε‐poly‐l‐lysine, HBPL possesses both units of ε‐PL and α‐poly‐l‐lysine (α‐PL). The ε‐PL contains abundant amino groups and has broad antimicrobial spectrum.^{[ 24} , ^{25 ]} Previous studies showed that the α‐PL can promote cell attachment and proliferation in the α‐PL‐grafted poly(ethylene glycol) diacrylate hydrogel system.^{[ 26 ]} Thus, the HBPL demonstrated outstanding antibacterial property and good compatibility.^{[ 27 ]} However, similar to ε‐PL, the water solubility of HBPL limit its wide range of applications in the fields of biomedical devices. Fortunately, the idea of polyelectrolyte‐surfactant complexes inspires us to prevent the loss of antibacterial function.^{[ 28} , ²⁹ , ³⁰ , ^{31 ]}

The study presented here described a series of water‐insoluble antimicrobial complexes based on HBPL and different surfactants, which could be blended with poly(ε‐caprolactone) (PCL) through solution mixing method to investigate the composition‐activity relationship. In this study, the PCL was chosen due to its great biocompatible and biodegradability. The PCL as biocompatible polymer has been applied as mesh for hernia repair, plate for bone fixation and suture.^{[ 32} , ^{33 ]} Importantly, biodegradable polyesters have attracted wide attention in biomedical device field during recent years.^{[ 34} , ^{35 ]} The antimicrobial activity of the complexes‐blended PCL composite films was investigated against clinically relevant bacterial – Staphylococcus aureus (S. aureus). The cell viability and hemolytic property of the as‐prepared films were also carefully characterized. We anticipate that this work will provide a series of novel HBPL‐surfactant‐based antibacterial agents with high bactericidal efficiency and excellent biocompatibility.

2. Results and Discussion

2.1. Characterization of the HBPL and the Complexes

In the present sduty, the thermal polymerization method was used to synthesize the HBPL following reported procedure.^{[ 12 ]} The structure of the HBPL was then characteried by ¹H NMR at ambient temperature (Figure S1, Supporting Information). The ¹H NMR spectrum indicated that the synthesis of HBPL is successful with characteristic proton resonances agreement with literature reports.^{[ 12 ]} In 2022, Ji et al. demonstrated that HBPL had excllent antibaterial and antifungal property.^{[ 27 ]} However, the HBPL has highly water soluble, which limitted its application in biomedical filed. The utilizaion of oppositely charged surfactants to transform water‐soluble polymer into polyelectrolyte‐surfactant complexes (PSC) has attracted increasing attention.^{[ 36 ]} Until now, organic sulfonate componds were the commonly used agents. Recent advances in PSC are enabling unique solution to solve the water soluble challenge of the polyelectrolyte in the blending system.

In this investigation, the HBPL‐based water‐insoluble materials were prepared by using organic sulfonate or phosphate esters surfacatants with differernt alkyl chain, such as sodium dodecyl benzene sulfonate (SDBS), or dioctyl sulfosuccinate sodium salt (DSS), isooctyl phosphate (IP) and bis (2‐ethylhexyl) phosphate (BP) (Figure 1 ). We anticipate that the differtent surfactants may have significantly effect on the antimicrobial activity of the complexes against bateria. The molar ratio of ε‐NH₂ of HBPL and surfctant molecules mixed in water is 1:1.1. As shown in Figure 2 , the HBPL can be dissolved in water, acid solution (pH = 1) and PBS solution, while the complexes were insoluble. The chemical composition data of the complexes were obtained by XPS analysis. As shown in Figure 3 the appearance of a new N_1s peak at 401.4 eV (C‐N⁺) for the HBPL/SDBS, HBPL/DSS, HBPL/IP and HBPL/BP, was a solid indication of the formation of protonated amino group. It reveals that the electrostatic interaction promotes the complexes formation of HBPL/SDBS, HBPL/DSS, HBPL/IP and HBPL/BP. Moreover, as shown in Table 1 , S element was only detected for HBPL/SDBS (3.45%) and HBPL/DSS (2.73%), while P element was only found in HBPL/IP (4.31%) and HBPL/BP (3.96%). The presence of the S or P elements in the complexes is attributed to the incorporation of surfactants. We evaluated the antibacterial property of the four kinds of complexes against S. aureus. As shown in Figure S2, Supporting Information, compared to positive control, S. aureus grow slowly in the wells with HBPL/SDBS, HBPL/DSS, HBPL/IP and HBPL/BP. Next, the hemolysis of rabbit red blood cells was evaluated after incubation with the complexes. The absorbance of the released hemoglobin from the complexes treated red blood cells was detected. The hemolysis ratio of HBPL/SDBS, HBPL/DSS, HBPL/IP and HBPL/BP was 9%, 15%, 7% and 17%, respectively (Figure S3, Supporting Information). It revealed that the complexes can accelerate the hemolysis of red blood cells.

Figure 1.

Chemical structures of surfactants used for preparing HBPL‐based complexes.

Figure 2.

The solubility of the HBPL and the HBPL‐based complexes in deionized water, acid solution (pH = 1) and PBS solution after incubation for 24 h. a) HBPL, b) HBPL/SDBS, c) HBPL/DSS, d) HBPL/IP, and e) HBPL/BP.

Figure 3.

The high‐resolution N 1s XPS spectra and their peak‐fitting curves of the complexes. a) HBPL/SDBS, b) HBPL/DSS, c) HBPL/IP, and d) HBPL/BP.

Table 1.

The compositions of the various complexes.

Sample	C [%]	N [%]	O [%]	S [%]	P [%]
HBPL/SDBS	75.53	5.53	15.48	3.45	n/a
HBPL/DSS	70.55	4.98	21.74	2.73	n/a
HBPL/IP	67.69	6.07	21.93	n/a	4.31
HBPL/BP	73.40	3.69	18.95	n/a	3.96

2.2. Surface Characterization of the PCL‐Complexes Films

The water‐insoluble and organic solvent‐soluble property (Figure S4, Supporting Information) of the above complexes facilitate their mixing with polymer substrates via solution blending method. The surface wettability of the as‐prepared films is presented in Figure S5, Supporting Information. The contact angel of the PCL film was about 85.3° ± 0.8. After introduction of the complexes into the PCL matrix, the contact angel of all the composite films decreased, indicating the improvement of hydrophilicity of the PCL film. The chemical compositions of the films were then investigated by ATR‐FTIR. The results showed that new peaks appeared at about 1100–1200 cm⁻¹ (S─O or P─O) and 936–1046 cm⁻¹ (S═O or P═O) (Figure S6, Supporting Information). XPS is an analytical technique to precisely probe the elemental composition and bonding states of the surface. Herein, we used XPS to further characterize surface elemental category (Figures 4 and S7, Supporting Information) and composition (Table 2 ) of the films. As shown in Figure S7, Supporting Information, no N 1s curve was observed for the pure PCL film. As for P‐HBPL/SDBS (1.5 wt%), P‐HBPL/DSS (1.5 wt%), P‐HBPL/IP (1.5 wt%) and P‐HBPL/BP (1.5 wt%), the high‐resolution N 1s curves were generally decomposed into two peaks: C‐N (at 399.7 eV) and C‐N⁺ (at 401.4 eV). The atom composition of S was 0.48% (P‐HBPL/SDBS (1.5 wt%)) and 0.37% (P‐HBPL/DSS (1.5 wt%)), respectively. And the atom composition of P detected in P‐HBPL/IP (1.5 wt%) film was 3.31% and P‐HBPL/BP (1.5 wt%) film was 0.65%. Compared to the pure complexes, the atom composition of S or P decreased in the blending system. The above results imply the successful incorporation of complexes into PCL matrix.

Figure 4.

The high‐resolution N 1s XPS spectra and their peak‐fitting curves of the films. a) P‐HBPL/SDBS (1.5 wt%), b) P‐HBPL/DSS (1.5 wt%), c) P‐HBPL/IP (1.5 wt%), and d) P‐HBPL/BP (1.5 wt%).

Table 2.

The surface compositions of the films.

Sample	C [%]	N [%]	O [%]	S [%]	P [%]
PCL	74.27	n/a	25.73	n/a	n/a
P‐HBPL/SDBS (1.5 wt %)	73.20	1.18	25.14	0.48	n/a
P‐HBPL/DSS (1.5 wt %)	73.81	0.83	24.98	0.37	n/a
P‐HBPL/IP (1.5 wt %)	67.06	5.37	24.25	n/a	3.31
P‐HBPL/BP (1.5 wt %)	74.12	0.78	24.46	n/a	0.65

2.3. Antibacterial Performance

Biomedical care‐associated infections account for big part of hospital‐acquired infections.^{[ 37 ]} Various pathogens can cause biomedical infections. Among them, S. aureus, a kind of gram‐positive and sphere‐shaped bacteria, can cause high risk of morbidity and mortality.^{[ 38 ]} It's reported that nearly 30% of the human population is colonized with S. aureus. Cutting off the transmission channel of bacteria colonization on the surface of biomedical devices is consequently a priority. Endowing the biomedical devices bactericidal property is regarded as a useful method to solve the above problem. In this study, we chose S. aureus as a model organism to investigate the antibacterial performance of the composite films. The bactericidal property of the composite films was judged by the number and morphology of the adhered S. aureus. The results revealed that most S. aureus kept around shape on the surface of PCL (Figure S8, Supporting Information). As for P‐HBPL/SDBS and P‐HBPL/DSS, at low dopant content of complexes (0.25 or 0.5 wt %), there are still some round S. aureus can be found on their surfaces. When the dopant content of HBPL/SDBS or HBPL/DSS reached 0.75%, nearly no S. aureus can be seen on their surfaces (Figure 5 ). By contrast, no bacteria were observed on the surfaces of P‐HBPL/IP and P‐HBPL/BP, suggesting the strong bactericidal ability of the HBPL/IP and HBPL/BP.

Figure 5.

Antibacterial assessment in vitro: morphology of the adherent S. aureus on different samples characterized by FESEM after incubation for 24 h.

The bactericidal activity of the composite films was then quantitatively investigated by the spreading plate method (Figures 6 and  7 ). The spread plate technique is a viable counting method that can count the live bacteria on the surface of the films. As shown in Figure S9, Supporting Information, many live S. aureus can be seen on the plate for the PCL film. The CFU of S. aureus for the PCL was measured to be 4.00 ± 0.12 [log₁₀(CFU mL⁻¹)]. As for P‐HBPL/SDBS film, the CFU of S. aureus was 3.71 ± 0.29 (0.25 wt %), 1.81 ± 0.10 (0.5 wt %), 0 (0.75 wt %) and 0 (1.5 wt %), respectively. The number of CFU on the surface of P‐HBPL/DSS film was 3.77 ± 0.26 (0.25 wt %), 1.82 ± 0.03 (0.5 wt %), 0 (0.75 wt %), and 0 (1.5 wt %), respectively. The quantitative data of the S. aureus show that the P‐HBPL/SDBS or P‐HBPL/DSS surfaces had relatively weak bactericidal ability with low content of complexes (0.25 wt %). When increasing the dopant content of HBPL/SDBS or HBPL/DSS, the killing efficacy against Gram‐positive S. aureus was increased for the P‐HBPL/SDBS and P‐HBPL/DSS (Figure 7d,c). The antibacterial activity of P‐HBPL/SDBS or P‐HBPL/DSS showed strong content dependence of the complexes. The results of P‐HBPL/IP or P‐HBPL/BP surfaces that treated with the S. aureus, showed that these surfaces were able to inhibit this bacterial growth effectively even at low dopant content (Figure 6). It was found that when the P‐HBPL/IP (0.25 wt%) or P‐HBPL/BP (0.25 wt%) surface was treated with 10⁵ CFU mL⁻¹ S. aureus for 24 h, bacterial proliferation was completely inhibited (Figure 7d,e). The CFU of S. aureus of P‐HBPL/IP and P‐HBPL/BP was found to be zero. Compared to PCL, there was a ≈4 log reduction in colony counts for P‐HBPL/IP and P‐HBPL/BP.

Figure 6.

Bactericidal activity in vitro: agar plate images of the as‐prepared films incubated with S. aureus for 24 h.

Figure 7.

Quantitative number of S. aureus on different films: a) PCL, b) P‐HBPL/SDBS, c) P‐HBPL/DSS, d) P‐HBPL/IP, and e) P‐HBPL/BP.

In summary, both the qualitative and quantitative results suggested that all the P‐HBPL/IP or P‐HBPL/BP surfaces showed excellent bactericidal ability against S. aureus after incubation for 24 h. Moreover, inhibition prowess of the composite films against S. aureus revealed that the bactericidal activity of P‐HBPL/IP (P‐HBPL/BP) is higher than P‐HBPL/SDBS (P‐HBPL/DSS) with same complexes content. While the pure PCL has no bactericidal activity, previously study reported that the antibacterial activity of HBPL was attributed to the synergistic actions of reactive oxygen species, DNA damage and membranes destruction.^{[ 11 ]} In this work, the composite films may kill the bacteria via a membrane rupture/lysis mechanism owing to the bacterial properties of the C‐N⁺. The P‐HBPL/IP (or P‐HBPL/BP) exhibited higher antibacterial activity than that of P‐HBPL/SDBS (or P‐HBPL/DSS), which may be due the lager radius and lower electronegativity of the phosphorus atom than that of sulfur atom.^{[ 39} , ^{40 ]}

2.4. Cytocompatibility and Hemocompatibility

Biocompatibility of biomedical devices with antibacterial property is critical for the successful translation in clinical. Herein, L929 cells were used to investigate cytocompatibility of the composite films due to that they are expected to be used in biomedical filed. The cell viability was measured by a cell counting kit‐8 (CCK‐8) viability assay. As illustrated in Figure 8 , although the P‐HBPL/BP (1.5 wt%) suppressed cell viability at 1 day (Figure 8A), and the cell viability was increased at 3 (Figure 8B) and 5 days (Figure 8C). As for other composite films, the L929 cell viability was similar to that of the pure PCL film after incubation for certain time.

Figure 8.

Cytocompatibility and hemocompatibility of the as‐prepared films. The cytotoxicity assay of the films incubated with L929 cells for A) 1 d, B) 3 d, and C) 5 d. D) Hemolysis assay of the films against red cells. a) PCL, b) P‐HBPL/SDBS, c) P‐HBPL/DSS, d) P‐HBPL/IP, e) P‐HBPL/BP, and f) positive control.

Relevant composite film toxicity against erythrocytes was also evaluated via hemolytic assay using rabbit red blood cells (RBC). The hemolytic assay is based on the ability of material to induce RBC lysis. Herein, RBC solution was dropped onto surface of films for 2 h incubation at 37 °C. As shown in Figure 8D and Figure S10, Supporting Information, the results revealed that all the films have low hemolytic activity (less than 0.5%), indicating excellent anti‐hemolytic property. The good biocompatibility is in favor of the potential clinical application of these novel antibacterial complexes.

3. Conclusion

In conclusion, we have described the preparation of a series of water‐insoluble HBPL/surfactant complexes formed by non‐covalent interaction. These novel complexes blended with PCL matrix exhibited tunable bactericidal properties that depend on the kind of surfactants. The bactericidal efficiency of the composite films was varied by altering the content of complexes containing sulfonate group in the polymer matrix. Most notably, the P‐HBPL/IP (or P‐HBPL/BP) that contained surfactant with phosphate ester group showed excellent bactericidal capabilities even at low dopant content. In addition, all the composite films displayed outstanding biocompatibility. Therefore, these novel complexes based on HBPL and surfactants with phosphate ester group may be ideal antimicrobial agents.

4. Experimental Section

Materials

Poly(ɛ‐caprolactone) (PCL, Mw = 60 kDa) was provided by Shenzhen Esun Industrial Co., Ltd. Isooctyl phosphate (IP), bis (2‐ethylhexyl) phosphate (BP) and 2,2,2‐trifluoroethanol (TFEA) was purchased from Shanghai Macklin Biochemical Co., Ltd. Dioctyl sulfosuccinate sodium (DSS) salt was obtained from Beijing Innochem Science & Technology Co., Ltd. Sodium dodecyl benzene sulfonate (SDBS) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. L‐Lysine monohydrocholoride was provided by GL Biochem Shanghai Co., Ltd. Zirconium (IV) butoxide (80% in 1‐butanol) was purchased from Merck Chemical Technology Shanghai Co., Ltd. o‐Vanillin was purchased from Anhui Zesheng Technology Co., Ltd. Gram‐positive Staphylococcus aureus (S. aureus, ATCC 6538), Mueller‐Hinton (MH) broth, and Luria‐Bertani (LB) agar were obtained from Qingdao Hope Bio‐Technology Co., Ltd. Cell Counting Kit‐8 (CCK‐8) was purchased from Beijing Solarbio Science & Technology Co., Ltd. All other reagents were AR grade and used without further purification.

Synthesis of Hyperbranched Poly‐L‐lysine

The HBPL was synthesized according to previous published work.^{[ 12 ]} Briefly, a mixture of L‐Lysine HCl (18.27 g, 100 mmol) and KOH (5.61 g, 100 mmol) was mixed in a mortar, and grinded to a pulp like consistence. The pulp was transferred into an open 200 mL single standard mouth round bottomed flask equipped with a PTFE stir bar and subsequently heated up to 150 °C. When the mixture was molten, zirconium (IV) butoxide was then added. The reaction was stopped by air cooling the flask to room temperature after 12 h. The crude product was dissolved in methanol and then purified. Finally, the aqueous solution was lyophilized to afford the polymer as a yellow‐brown solid. ¹H NMR spectra of the HBPL was recorded at room temperature on a Bruker AVANCE NEO 400 MHz spectrometer. ¹H NMR (400 MHz, D₂O, 298K): 4.14 (br, 1 H, COCH(R)N^αH), 4.08 (br, 1 H, COCH(R)N^αH), 3.29 (br, 1 H, COCH(R)N^αH₂), 3.23 (br, 1 H, COCH(R)N^αH₂), 3.12 (br, 2H, CH ₂‐N^ɛH), 2.82 (br, 2H, CH ₂‐N^ɛH₂).

Synthesis of the HBPL‐Surfactant Complex

The complexes were prepared by two different ways. The SDBS or DSS formed complex (abbreviated as HBPL/SDBS and HBPL/DSS) was prepared according to a previous report.^{[ 23 ]} The anionic surfactant was dissolved in 60% (v/v) ethanol and then added dropwise to the HBPL solution (1.5% (w/v) HBPL in 0.2 mol L⁻¹ of HCl) at room temperature. The mixture was then centrifuged at 10 000 rpm for min, and the precipitate was washed three times with 30 mL of water, and lyophilized to obtain the orange precipitate.

The other two kinds complexes were prepared by dropwise addition of surfactants/ ethanol IP or BP into the HBPL solution at room temperature under stirring, which were namely as HBPL/IP and HBPL/BP. The centrifugalizing, washing and dried process was the same as the above.

Preparation of PCL/complex Film

The PCL/complex films were prepared by dissolving 0.25%, 0.5%, 0.75%, and 1.5 wt % of complex in TFEA with 10 wt% PCL. Then the PCL composite films were dried at room temperature. The as‐prepared composite films were named as P‐HBPL/surfactant (X wt%), which the X represented the percentage of complex in the polymer matrix.

Surface Characterization

The static water contact angle (WCA) of the films was measured by dynamic/static optical contact angle meter/interfacial tensiometer (SL200KS). The change of the chemical compositions of the membranes were examined by attenuated total reflectance‐Fourier transform infrared spectroscopy (ATR‐FTIR, Bruker Vertex 70) in the range from 400 to 4000 cm⁻¹. The composition of the complexes and the surface composition of the films was detected by X‐ray photoelectron spectroscopy at room temperature (XPS, VG Scientific ESCA MK II Thermo Avantage V3.20 analyzer equipped with an Al K αanode mono‐X‐ray source (hν = 1486.6 eV)). The atomic concentrations of the elements were calculated by the peak area ratios.

Antibacterial Test

Gram‐positive S. aureus was chosen as the representative bacteria to evaluate the antibacterial activities of all the films. The freshly prepared bacterial MH broth suspension (S. aureus) with a concentration of 10⁷ CFU mL⁻¹ was calculated by testing the OD₆₀₀ nm = 0.07 using a microplate reader (Tecan Sunrise, Swiss). Then, the bacterial cells were diluted with MH broth to obtain a final concentration of 10⁵ CFU mL⁻¹.

The bactericidal property of the complexes was evaluated. Different complexes (0.5 mg) were added into the bacteria suspension (0.6 mL), and then incubated at 37 °C for 24 h. The optical density (OD) value of bacteria cells in 48 well plate was measured by a microplate reader (Tecan Sunrise, Swiss) after incubation with the complexes.

To evaluate the antibacterial activity, the UV sterilized (irradiation time: 2 min) films were put into 24‐well plates. Bacterial suspension (50 µL) was then added onto the surfaces for co‐culture in a 37 °C incubator for 24 h. Subsequently, the films were replaced in centrifuge tubes containing 5 mL of MH broth and subjected to a vortex mixer for 2 min to remove the adhered bacteria. Afterward, 100 µL of the diluted bacterial solution was coated on a pre‐prepared agar plate, which was incubated in an incubator at 37 °C for 24 h.

The films after contacting with bacteria were also gently rinsed with ultrapure to remove loosely adherent cells, and then immersed in a 4% glutaraldehyde solution for 4 h. After freeze‐drying, the morphologies of attached bacteria on the films were observed by field emission scanning electron microscope (FESEM, Quanta 250 FEG).

Cytotoxicity Evaluation

The experiment as carried out according to our previously report.^{[ 41 ]} The cytotoxicity of the films was evaluated by utilizing L929 cells. Films (with diameter 1 cm) were placed in 24‐well plate and sterilized by ultraviolet irradiation for 3 min. L929 cells were seeded into the 24‐well plate at a density of 1.5 × 10⁴ cell cm⁻² and incubated for 4 h at 37 °C in 5% CO₂. Following that, the films were added into the well. Films were taken out after incubated for 1, 3, or 5 days. CCK‐8 reagent was added to the culture dishes, and incubated for 4 h, the absorbance of the supernatant was measured at 450 nm on a microplate reader (Tecan Sunrise, Swiss).

Hemolysis Assay

The hemolytic potentials of the films were evaluated according to the method reported.^{[ 41 ]} Briefly, the fresh whole blood containing anticoagulation was obtained legally from healthy rabbits. Then the fresh rabbit blood was centrifugated (1000 rpm, 15 min) at 4 °C. Erythrocytes were separated from plasma and lymphocytes and diluted with PBS. The diluted erythrocytes were then placed on the films (1 cm × 0.5 cm) or the complexes. After incubation on a thermostatic shaker for a certain time at 37 °C, the plate was centrifuged at 3000 rpm for 5 min. The supernatant from each well was transferred to 96‐well plates. The hemolysis percentage was calculated using the following formula:

$$\mathrm{Hemolysis}\mathrm{ratio}\left(\%\right)=\left(\frac{\mathrm{A}-\mathrm{C}1}{\mathrm{C}2-\mathrm{C}1}\right)\times 100\%$$ (1)

Where A is the absorbance of the sample (%), C1 is the absorbance of the negative control (%), and C2 is the absorbance of the positive control (%). The hemolysis data were expressed as the mean of three replicates.

Statistical Analysis

All quantitative data were expressed as mean ± standard deviation (SD) for n = 3. The statistical analysis was performed using one‐way analysis of variance (ANOVA). P < 0.05 is considered statistically significant.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

Qi Chen: investigation, data curation. Yuanjing Xu: investigation. Jingyi Feng: data curation. Xingshuang Lv: data curation. Xiaohui Fu: data curation, formal analysis. Shuaishuai Yuan: conceptualization, data curation, formal analysis, writing – original draft. Zhibo Li: conceptualization, formal analysis, writing – review & editing, supervision.

Supporting information

Supporting Information

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.