Mussel-inspired citric acid crosslinked antifouling and bactericidal coatings constructed using sulfobetaine zwitterionic/quaternary ammonium cationic copolymers

Abstract

Inhibiting and reducing bacterial infections associated with biomedical implants and devices remains a significant challenge. In this study, we successfully grafted crosslinked antifouling and bactericidal coatings onto a polyurethane (PU) surface using sulfobetaine (SB) zwitterionic and quaternary ammonium cationic (QAC) copolymers through a combination of PDA-assisted co-deposition and amidation reactions. The successful formation and surface properties of the crosslinked coatings were characterized using Fourier transform infrared spectroscopy (FT-IR), water contact angle (WCA) measurements, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), electrochemical corrosion tribometry (MFT-EC), and atomic force microscopy (AFM). The antifouling performance was evaluated via protein adsorption, platelet adhesion, whole blood adhesion, and cytotoxicity assays. Additionally, the antibacterial and bactericidal efficacy was evaluated using E. coli, P. aeruginosa and S. aureus as models. Our results indicate that the molar ratio of SB and QAC critically influences the antifouling and bactericidal properties, and a relatively high SB content (60 mol%) combined with a low QAC content (20 mol%) achieves an optimal balance between antifouling and bactericidal properties. This combination of zwitterionic and quaternary ammonium cationic copolymer modifications not only effectively kills bacteria upon contact but also prevents the adhesion of dead bacteria, demonstrating promising potential for applications in biomedical implants and devices.

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Introduction

Polyurethane (PU) is a class of synthetic polymers composed of phase-separated hard and soft segments.¹ PU has numerous advantages as a biomaterial, including structural diversity, superior mechanical properties, and exceptional biocompatibility with surrounding tissues.^2–4 Presently, the most prominent applications of PU in the biomedical field include a variety of medical devices, such as pacemakers, heart valves, vascular prostheses, breast implants and catheters.⁵ However, the poor hydrophilicity of PU results in a higher propensity for irreversible adhesion of microbial adhesins and extracellular polysaccharides.^6,7 This adhesion facilitates the formation of organized bacterial aggregates, ultimately leading to biofilm development and subsequent bacterial adhesion and infections. Biofilm-associated bacteria exhibit 10–1000 times greater resistance to conventional antibiotics compared to planktonic bacteria.⁸ Therefore, developing surfaces with antifouling and bactericidal properties that can inhibit bacterial aggregation and biofilm adhesion is currently an urgent challenge.

The ideal antifouling surface, in addition to possessing antibacterial properties, should also confer the substrate with superior hemocompatibility and minimal cell and tissue adhesion. During the past two decades, considerable attention has been given to developing antifouling coatings using hydrophilic polymers such as polyethylene glycol (PEG)^9–11 and zwitterionic including sulfobetaine (SB), carboxybetaine (CB), or phosphorylcholine (PC) polymers^12–22 on diverse substrate surfaces. However, the in vivo oxidative degradation of PEG elevates the risk of cell and tissue adhesion, thereby limiting its practical application.^7,23 Therefore, zwitterionic polymers have emerged as promising next-generation materials for antifouling applications, receiving extensive research focus.^12–20 The antifouling mechanism of zwitterionic polymer coatings stems from the robust interactions between the surface charges of the coating and water molecules, leading to the formation of a well-ordered, highly hydrogen-bonded hydration layer on the polymer surface. This hydration layer serves as an effective physical and energetic barrier between the substrate and biomolecules, thereby significantly inhibiting the adhesion of contaminants.^19,24 However, existing research indicates that antifouling coatings are incapable of killing bacteria or fully preventing their adhesion. Over time, a few adhered bacteria can eventually form a biofilm.^25,26 Therefore, incorporating bactericides during the construction of antifouling coatings can effectively address this issue. To date, various bactericides have been utilized in the construction of bactericidal coatings, including silver-based compounds,²⁷ antibiotics,^28,29 antimicrobial peptides,^30,31 and quaternary ammonium cations (QACs).^32–34 Unfortunately, surfaces that possess only bactericidal properties cannot prevent the adhesion of foulants and dead bacteria. The accumulation of these contaminants can result in a decline or loss of antibacterial efficacy. Consequently, polymers with antifouling properties are frequently combined with bactericides to develop dual-functional coatings that exhibit both antifouling and bactericidal capabilities, thereby enhancing the overall fouling resistance of the materials. Recently, by leveraging the universal adhesion properties of mussel-inspired PDA chemistry^{15–18,35–38} and the distinctive chemical structures of the synthesized copolymers, we reported a strategy to synergistically enhance both the antifouling and bactericidal properties of PU catheters by integrating the antifouling effect of zwitterions with the bactericidal effect of quaternary ammonium cations.¹⁵ The results showed that by optimizing the ratio of zwitterionic and cationic copolymer units, a surface with good biocompatibility, antifouling properties and bactericidal effect could be successfully constructed. However, the limited stability of the coating restricts its broader application. To enhance the stability of the coatings while maintaining their excellent biocompatibility, antifouling properties, and bactericidal efficacy, in this study, citric acid (CA) was introduced as a small-molecule crosslinker during the construction of the copolymer coatings. It is worth mentioning that CA is an inexpensive, non-toxic tri-carboxylic acid that is often used as a cross-linker or an additive to improve the performance properties of cellulose, chitosan,^39,40 proteins⁴¹ and hydrogels⁴² due to the three –COOH groups of CA participating in esterification or amidation reactions to obtain mono-, di-, tri-esters or amides.⁴³ However, to the best of our knowledge, the method of simultaneously constructing a zwitterionic polymer coating by crosslinking a dopamine-mediated layer with CA has not been reported previously. The formation process of the crosslinked copolymer coatings and the possible mechanism are illustrated in Scheme 1.

Scheme 1 The chemical structure and compositions of the synthesized copolymers as well as the chemical structure of the small molecule crosslinker, CA (A). Schematic illustration of the construction of the crosslinked copolymer coatings on the surface of PU catheters (B), and the anticipated chemical structures of the PDA crosslinked coating, and the subsequent cross-linked copolymer coatings using CA as the crosslinker (C).

Materials and methods

Materials

PU catheters were supplied by Xi'an Xijing Medical Supplies Co., Ltd (China). Dopamine hydrochloride (DA·HCl) was purchased from Beijing Bailingwei Technology Co., Ltd (China). Polyethyleneimine (PEI) with a molecular weight of approximately 600, 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) and N-hydroxy succinimide (NHS) were obtained from Adamas (China). Citric acid (CA) was purchased from Shanghai Shaoyuan Reagent Co., Ltd (China). Bovine serum albumin (BSA) and bovine blood fibrinogen (Fg) proteins, along with 4,6-diamino-2-phenyl indole (DAPI) and propidium iodide (PI) cell stains, were purchased from Youbo Biotechnology Co., Ltd (China). Three types of bacteria, namely Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), were acquired from Xi’jing Hospital (China). The L929 cell line, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and NCTC clone L929 cell medium with MEM (including NEAA), 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution (P/S solution) were purchased from Procell Life Science &Technology Co., Ltd (China). The copolymers, PCSB, PCAS60, PCAS40 and PCAC bearing a molar ratio of carboxylic side chain units of about 20%, were synthesized through free radical random copolymerization according to our previous report.¹⁵ The detailed synthetic procedures and relevant characterization data, including proton nuclear magnetic resonance spectroscopy (¹H NMR) and gel permeation chromatography (GPC), are provided in that report.¹⁵ The molecular structures and compositions of the four copolymers are shown in Fig. S1 and Table S1. Unless otherwise specified, all other chemicals used in this study were of analytical grade and used without further purification. The pH value of PBS without specific labeling was 7.4.

Surface modification and characterization

PU catheters were surface modified using a previously reported method involving PDA-assisted co-deposition and amidation reaction,^15–18 employing our synthesized copolymers PCSB, PCAS60, PCAS40, and PCAC using CA as a crosslinker to enhance the stability of the coatings. Firstly, cleaned PU catheters (1 cm length) were placed in a vial containing PBS (pH 8.5) solution of DA·HCl (2 mg mL⁻¹) and PEI (0.5 mg mL⁻¹). After stirring at 30 °C for 6 h, the catheters in the vial were washed three times with distilled water to form the PU catheters with NH₂ groups on the surface, PU/PDA. Subsequently, NH₂ groups on the PU/PDA coatings reacted with the copolymers and CA activated by EDC (16 mg mL⁻¹) and NHS (12 mg mL⁻¹) in PBS buffer (pH 5.6) at 60 °C for 12 h. After successive washing with ethanol and deionized (DI) water successively, followed by drying with cold air, we obtained the CA crosslinked copolymer coated PU coatings. In the experiment, we optimized the addition order of CA and copolymers as well as the concentration of CA to investigate their influence on the hydrophilic and hydrophobic properties of the resulting crosslinked coatings. For convenience, we denoted the modified coating obtained by adding CA first followed by PCSB as PU/PDA/CA/PCSB; similarly, we denoted the modified coating obtained by adding PCSB first followed by CA as PU/PDA/PCSB/CA; finally, we denoted the modified coating obtained by simultaneously adding both CA and PCSB as PU/PDA/CA&PCSB.

The surface wettability, morphology, elemental composition, and roughness of the crosslinked coatings were measured using a water contact angle (WCA) analyzer, a scanning electron microscope (SEM), an X-ray photoelectron spectrometer (XPS), a Fourier transform infrared (FTIR) spectrometer, and an atomic force microscope (AFM), respectively. And, the coefficient of friction (COF) of the crosslinked coatings was characterized using an electrochemical corrosion tribometer (MFT-EC). The detailed information on surface characterization is given in the SI.

Coating stability

The modified PU catheters (1 cm length) were placed in a PBS solution (pH 7.4) and artificial urine (pH 5.6) at 37 °C and shaken at 180 rpm for 30 d. WCA values and element contents were determined through WCA measurements and XPS to evaluate the stability of the crosslinked coatings, respectively.

Protein adsorption

Bovine serum albumin (BSA) and fibrinogen (Fg) were selected as the standard proteins, and the protein adsorption properties of the crosslinked coatings were tested by the Bradford method.⁴⁴ The detailed experimental procedures are provided in the SI.

Platelet adhesion

The platelet adhesion experiment of the crosslinked coatings was conducted according to the previously reported methods.^15,45 And, the adhered platelets on the surfaces were observed after sputtering with gold for SEM observation. The detailed experimental procedures are provided in the SI.

Whole blood adhesion

The whole blood adhesion experiment of the crosslinked coatings was conducted according to the previously reported methods.^15,17 The thrombus adhered on the different surfaces at different time points was observed using an upright microscope (Nikon Eclipse Ti-U, Japan). The detailed experimental procedures are provided in the SI.

In vitro cell attachment and cytotoxicity assay

The cell attachment and cytotoxicity of the crosslinked coatings were performed using L929 cells as a model. The detailed experimental procedures are provided in the SI. The cell attachment was observed by fluorescence microscopy (Eclipse Ni-U, Nikon) and quantitatively analyzed using Image J. And, the cytotoxicity was evaluated by the MTT method and calculated using the following formula, with 0.64% phenol as the positive control.

Cell viability (%) = OD_sample/OD_control × 100%

In vitro bacterial adhesion and bactericidal performance

The bacterial adhesion and bacterial performance of the crosslinked coatings were evaluated by fluorescent staining labeling (DAPI/PI) using E. coli, P. aeruginosa and S. aureus as models. The detailed experimental procedures are provided in the SI. Fluorescence microscopy (Eclipse Ni-U, Nikon) was used to observe the bacterial cells attached to the substrate surface, and threshold analysis of the microscopy images was performed using the ImageJ software package.

Statistical analysis

Every test was repeated at least 3 times and the results were expressed as the mean ± standard deviation. Statistical significance was evaluated using one-way analysis of variance (ANOVA), with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 suggesting significant and highly significant differences, respectively.

Results and discussion

Coating fabrication and characterization

Considering the absence of chemical reactive groups on the PU surface for covalent immobilization of modification molecules, we employed a PDA universal adhesive coating strategy in this study to provide chemically reactive –NH₂ groups on the PU surface. This approach has been extensively utilized for surface functionalization of diverse materials.^{15–18,35–37} Subsequently, an amide bond was formed between the –COOH groups in the copolymers' chains and the –NH₂ groups on the PU/PDA surface, as previously reported by us.¹⁵ Additionally, we selected the crosslinker CA with –COOH groups to enhance the stability of these coatings.

Taking the construction of PCSB copolymer coatings as a model, the WCA and the WCA changes of the coatings in PBS and artificial urine at 37 °C for 15 d were selected as parameters in the experiment. We optimized the addition orders of the CA and PCSB, as well as the concentration of CA. As depicted in Fig. S2 and S3, the hydrophilicity and stability of PCSB copolymer crosslinked coatings formed by the addition of CA and copolymers simultaneously (PU/PDA/CA&PCSB), specifically at a concentration of 1.5 mg mL⁻¹ CA, were significantly improved with smaller changes in the WCA.

In addition, to confirm that a cross-linking reaction actually occurred during the formation of the polymer coating after the introduced of CA, FT-IR spectroscopy was performed on the PCSB copolymer, the uncrosslinked PU/PDA/PCSB coating, and the cross-linked PU/PDA/CA&PCSB coating, respectively. The results are presented in Fig. 1A. The peak at 3324 cm⁻¹ (black curve) represents the characteristic absorption of the carboxylic hydroxyl in PCSB. And, all the FT-IR spectra of PCSB, PU/PDA/PCSB and PU/PDA/CA&PCSB display prominent peaks at 1732 cm⁻¹, 1184 cm⁻¹, 1043 cm⁻¹, 932 cm⁻¹ and 605 cm⁻¹, which are attributed to the C=O stretching vibration, S=O asymmetric/symmetric stretching vibration, S–O and C–S absorptions.^46,47 These results confirm the successful formation of the PCSB copolymer coatings. More importantly, new strong peaks appeared at 1690 cm⁻¹ in the IR spectra of both PU/PDA/PCSB and PU/PDA/CA&PCSB, indicating the occurrence of the amidation reaction. Especially, for PU/PDA/CA&PCSB, the absorption intensity of the amide bond at 1690 cm⁻¹ was stronger than that of the ester bond at 1732 cm⁻¹, suggesting a higher density of amide bonds in this coating. This result can be attributed to the fact that during the formation of the PU/PDA/CA&PCSB, in addition to the reaction between the PCSB and the PDA-mediated layer, the amidation reaction also occurred between CA and the PDA-mediated layer.

Fig. 1 Chemical compositions of the copolymer PCSB modifier coatings. FT-IR spectra of PCSB (KBr) and coatings (ATR) (A) and XPS high-resolution spectra of C 1s (B).

Finally, the optimized conditions were employed to prepare three other copolymer crosslinked coatings, namely PU/PDA/CA&PCAS60, PU/PDA/CA&PCAS40, and PU/PDA/CA& PCAC, respectively.

Elemental analysis of crosslinked coatings. During the crosslinking coating construction process, the reaction between the –COOH groups in the CA structure and the NH₂ groups on the PU/PDA surface competes with the reaction between the –COOH groups in the copolymer chains and the –NH₂ groups on the PU/PDA surface. Because CA will consume –NH₂ groups on the PU/PDA surface by modifying with EDC and NHS, and it will possibly decrease the linkage between PEI and the copolymer. The surface elemental composition of the PU surfaces before and after modification was characterized using XPS. As depicted in Fig. S4 (black line) and Table S2, the sulfur (S) content on the surface of the crosslinked coatings remained comparable to that of the non-crosslinked counterpart (in parentheses, Table S2). This indicates that the introduction of CA has minimal impact on altering the content of the introduced zwitterions. The possible reason may be attributed to the relatively low concentration of CA, the optimized concentration being 1.5 mg mL⁻¹, which ensures the stability and hydrophilicity of the coating, as shown in Fig. S3. More importantly, the occurrence of the cross-linking reaction was further confirmed by the comparison of the XPS high-resolution spectra of C 1s of both PU/PDA/PCSB and PU/PDA/CA&PCSB. As shown in Fig. 1B, the C 1s high-resolution spectrum of the PU/PDA/CA&PCSB is similar to that of the PU/PDA/PCSB. The C signals at 284.6 eV, 286.3 eV, 287.2 eV, and 288.7 eV correspond to the C atoms in C–H/C–C, C–O, HN–C=O, and O–C=O groups, respectively.⁴⁸ Notably, the C content in HN–C=O in PU/PDA/CA&PCSB (11.29%) is significantly higher than that in PU/PDA/PCSB (4.69%), further indicating that the amidation reaction has occurred between CA and the PDA-mediated layer.

Wettability of crosslinked coatings. The wettability of the PU surfaces before and after modification was evaluated by measuring the WCA of the surfaces. As shown in Fig. 2A, both PU/PDA and crosslinked copolymer coatings exhibited a significant reduction in WCA compared to the original PU (81.1°), indicating an increase in surface hydrophilicity due to the covalent bonding of the hydrophilic copolymers. In comparison with our previous report on non-crosslinked coatings,¹⁵ there was no substantial change observed in the WCA of the crosslinked coatings, suggesting that crosslinking had negligible impact on their hydrophilicity. The pronounced hydrophilicity of these coatings is essential for their excellent antifouling performance.

Fig. 2 WCA (A) and SEM images (B) of the PU surfaces before and after modification.

Morphology of the crosslinked coatings. The morphology of the PU surfaces before and after modification were characterized by SEM and AFM. Fig. 2B presents the SEM images, revealing a smooth and flat surface for PU. After PDA deposition, the PU/PDA surface remained relatively flat due to the introduction of PEI, with no abundant aggregation of PDA nanoparticles observed. This observation is consistent with previous studies.^38,49,50 The crosslinked PCSB copolymer coating also exhibited a smooth and flat surface, However, when copolymers containing the MAC unit (PCAS60, PCAS40, and PCAC) were used as coatings, sub-micrometer structured domains were evident in the SEM images. In addition, AFM was employed to assess the surface roughness of the PU surfaces before and after modification. The root-mean-square roughness (R_q) results shown in Fig. 3A and Fig. S5 mirrored those obtained from the SEM analysis.

Fig. 3 The average root-mean-square roughness (R_q) (A) and the average coefficient of friction (B) of the PU surfaces before and after modification: PU (a), PU/PDA (b), PU/PDA/CA&PCSB (c), PU/PDA/CA &PCAS60 (d), PU/PDA/CA&PCAS40 (e), and PU/PDA/CA&PCAC (f).

Lubrication of crosslinked coatings. The surface lubrication of the coatings can effectively mitigate potential skin damage during catheterization. Therefore, the friction tests were conducted to evaluate the lubrication properties of PU surfaces before and after modification in PBS and artificial urine. The results (Fig. 3B and Fig. S6) show that the coefficient of friction (COF) of the crosslinked coatings (0.041–0.062) is lower than that of the non-crosslinked coating (0.067–0.079)¹⁵ possibly attributed to a slightly increased roughness enabling the penetration of PBS and artificial urine between the upper friction PTFE ball and lower friction coatings, thereby forming an effective hydrophilic crosslinked coating with reduced COF.⁵¹ The improvement of lubrication performance of the coatings holds promise for its application in medical catheters.

Stability of the crosslinked coatings

The stability of the PU surfaces before and after modification was characterized through WCA measurements and XPS. As can be seen from Fig. 4A and B, compared with the non-crosslinked coatings in our previous report,¹⁵ the WCA values of the crosslinked coatings in PBS and artificial urine at different time periods showed a similar or smaller trend of change. Taking the WCA changes of the PCSB copolymer coating as an example, the WCA was increased by 12.6° (from 24.4° to 37.0°) for the non-crosslinked PCSB coating, that is, PU/PDA/PCSB immersed in PBS for 10 days.¹⁵ But, the WCA was increased by only 7.9° (from 27.5° to 35.4°) for the crosslinked PCSB coating, i.e. PU/PDA/CA&PCSB after immersion in PBS for 10 days. From the change of WCA, it can be inferred that the stability of the coating after crosslinking is improved. Additionally, based on the previously reported method,⁵² the stability of the coatings was further characterized by assessing the retention rate of the S element on their surface after immersion in PBS and artificial urine for a specific duration (XPS, Table S3 and Fig. S4). The retention rate was calculated as (S_t/S₀ × 100%), where S_t and S₀ represent the S element content on the surface compared to the non-crosslinked coatings' S element retention rate after 10 days of shaking in PBS and artificial urine; even after 30 days under identical conditions, the three crosslinked coatings exhibited significantly higher levels of S element retention. The XPS results further confirmed that crosslinking substantially enhanced the stability of these coatings.

Fig. 4 WCA values of the crosslinked copolymer coatings after shaking in PBS (A) and in artificial urine (B) at 37 °C for 30 d; S content retention rates of non-crosslinked (10 d) and crosslinked copolymer coatings (30 d) calculated by XPS results (C): PU/PDA/PCSB (a), PU/PDA/PCAS60 (b), PU/PDA/PCAS40 (c), PU/PDA/CA& PCSB (d), PU/PDA/CA&PCAS60 (e), and PU/PDA/CA&PCAS40 (f).

Hemocompatibility

Hemocompatibility is a crucial requirement for blood-contacting materials and devices. According to the existing literature,^17,53–55 these materials and devices must not adversely interact with or activate blood components, nor should they cause damage or destruction to these components. In this study, we evaluate the hemocompatibility of crosslinked copolymer coatings by evaluating protein adsorption, platelet adhesion, whole blood adhesion, and cell adhesion.

Similar to our previous research on non-crosslinked polymer coatings,¹⁵ we selected BSA and Fg as model proteins to investigate the adsorption performance of crosslinked copolymer coatings towards these two proteins, and compare it with the protein adsorption behavior of non-crosslinked copolymer coatings. From Fig. 5A, it is evident that the protein adsorption trends for both the crosslinked and non-crosslinked copolymer coatings¹⁵ are largely consistent. The three coatings constructed from copolymers containing SBMA units exhibit a significant resistance to protein adsorption. Furthermore, compared with the unmodified PU surface used as a control (with a protein adsorption capacity set at 100%), the protein adsorption (48 h) on the non-crosslinked copolymer coatings was reduced by 87–79% (Fig. 5B(a–c), calculated by the average value). The decreased rate of protein adsorption of the crosslinked copolymer coatings (Fig. 5B(d–f), calculated by the average value) is marginally lower, ranging from 82 to 75%. Regarding the phenomenon of reduced protein adsorption in the crosslinked copolymer coating, a plausible explanation involves both the inhibitory effect of the zwitterionic polymer hydration layer on protein adsorption and the potential contribution of unreacted –COOH groups, which reduce protein adsorption via electrostatic repulsion.

Fig. 5 Adsorption capacity of the two proteins BSA and Fg on different coating surfaces after 2 h and 48 h adsorption, (A). The adsorption change rate of the two proteins BSA and Fg on different non-crosslinked surfaces [PU/PDA/PCSB (a), PU/PDA/PCAS60 (b), and PU/PDA/PCAS40 (c)] and crosslinked surfaces [PU/PDA/CA&PCSB (d), PU/PDA/CA&PCAS60 (e), and PU/PDA/CA&PCAS40 (f)] after 48 h adsorption (B). SEM images of adhered platelets (C). Asterisks represent statistically significant differences (**p < 0.01 and ***p < 0.001).

In addition, the three crosslinked coatings constructed from copolymers containing SBMA units also exhibit a significant improvement in suppressing platelet adhesion and activation (Fig. 5C), whole blood adhesion (Fig. 6) and cell adhesion using L929 as a model (Fig. 7A and B), which matches the results of the protein adsorption (Fig. 5A). More importantly, to ensure that the copolymer-modified PU catheters described in this study do not pose secondary risks to human health during use, we evaluated the cytotoxicity of the leaching liquors obtained from both modified and unmodified PU catheters after a 12-hour immersion using the MTT assay, with phenol as a positive control and L929 cells as the model. As shown in Fig. 7C, the cell viability of the 0.64% phenol solution was about 27%, suggesting its significant cytotoxicity to L929 cells. In contrast, after 48 h of coincubation with the series of leaching liquors, the cell viability remained consistently above 90%, which is higher than the 82% observed for the non-crosslinked copolymer-modified PU catheters in our previous study.¹⁵ This minimal cytotoxicity confirms the stability of the coatings, which is consistent with the findings from the stability study presented in Fig. 4.

Fig. 6 Optical photographs (0 h, 2 h, 12 h and 24 h adhesion) of whole blood contacted at 37 °C: non-crosslinked surfaces [PU/PDA/PCSB (a), PU/PDA/PCAS60 (b), and PU/PDA/PCAS40 (c)] and crosslinked surfaces [PU/PDA/CA&PCSB (d), PU/PDA/CA&PCAS60 (e), and PU/PDA/CA&PCAS40 (f)].

Fig. 7 Fluorescence images (A) and quantitative analysis (B) of resistance to L929 cell adhesion on the surfaces of PU catheters before and after modification, evaluated at 24 h and 48 h co-culture; and the cell viability of L929 cells exposed to leaching liquors from the corresponding modified and unmodified PU catheters after a 12 h immersion (C). Asterisks represent statistically significant differences (*p < 0.01).

The aforementioned experiments on protein adsorption, platelet adhesion, whole blood adhesion, and cell-related experimental results have confirmed that the hemocompatibility of the crosslinked copolymer coatings ranks from low to high as follows: PU/PDA/CA&PCAC < PU/PDA/CA&PCAS40 < PU/PDA/CA&PCAS60 < PU/PDA/CA&PCSB, and this order is consistent with our reported non-crosslinked coatings.

Antibacterial and bactericidal properties

The results of hemocompatibility related experiments indicate that the incorporation of MAC cationic units in the copolymers used for modifying the PU surface adversely affects the hemocompatibility of the coatings. Furthermore, as the proportion of MAC units in the copolymer increases, the hemocompatibility deteriorates progressively. This indicates that while the introduction of MAC cationic units is detrimental to hemocompatibility, the surface cationic charge has been established as an effective bactericidal component.^54–56 Therefore, we attempt to find a good balance between the antifouling performance of the zwitterionic component (SBMA) and the bactericidal activity of the cationic unit (MAC).

The antibacterial and bactericidal performance of the crosslinked coatings was evaluated by co-culturing them with E. coli, P. aeruginosa and S. aureus, respectively. As shown in Fig. S7, after 1 day of co-culture, large aggregates of live bacteria (blue fluorescence) were observed on the surfaces of PU and PU/PDA. After 3 days (Fig. S8) and 7 days (Fig. 8), the number of live bacteria increased significantly, and almost no dead bacteria (red fluorescence) were observed. The above results indicate that both PU and PU/PDA surfaces have no antibacterial activity against the three types of bacteria, and their killing efficacy is close to zero. In contrast, on the surface of PU/PDA/CA&PCAC, a substantial amount of red fluorescence was detected. This indicates that the cationic structure of PCAC could cause membrane permeation and destabilization upon contact, leading to the death of bacteria.⁵⁷ Notably, very few live or dead bacteria were detected on the surfaces of PU/PDA/CA&PCSB, PU/PDA/CA&PCAS60, and PU/PDA/CA&PCAS40 even after 7 days of co-culture. The excellent antibacterial and bactericidal properties of these coatings are mainly attributed to the fact that zwitterionic SBMA in the copolymer coating effectively inhibits bacterial adhesion on the surface, while the QAC has the ability to efficiently kill the bacteria from adhering the surfaces via selective bacterial membrane lysis. More importantly, the partial coverage of the QAC by the zwitterionic copolymer layer does not compromise the bactericidal efficacy of the QAC. Similar findings have been reported in the relevant literature.⁵⁸ Furthermore, the bacterial adhesion on the PU surface, which served as the control group after 7 days of co-culture with various bacteria, was defined as 100%. Quantitative analysis revealed that the bactericidal rates of PU/PDA/CA&PCAS60, PU/PDA/CA&PCAS40 and PU/PDA/CA&PCAC exceed 99%, surpassing those of their respective non-crosslinked copolymer coatings (∼95%) as reported in our previous study.¹⁵ The bactericidal performance (7 d) of the crosslinked versus non-crosslinked coatings constructed by the three copolymers containing the SB zwitterionic segment against three bacteria, E. coli, P. aeruginosa and S. aureus using PU as a control (Fig. S9). This indicates that crosslinking modification not only compensated for the deficiencies of the original coatings but also enhanced their stability, thereby significantly improving both antibacterial and bactericidal efficacy. Finally, it should be emphasized that the long-term antibacterial and bactericidal effects of the substrate are closely related to the chemical composition, structural integrity, and stability of the substrate surface. As long as the functional polymer exhibiting antibacterial and bactericidal properties remains stably attached to the surface, its sustained antibacterial and bactericidal efficacy can be ensured. From the stability evaluation results of the polymer coatings constructed in this study (Fig. 4), it can be inferred that the constructed cross-linked coating has the potential to achieve long-term anti-fouling and bactericidal effects. Nevertheless, there is still potential for further improvement in terms of modification methods and stability.

Fig. 8 Fluorescence microscopy images (A) and quantitative analysis for the number (B) of adherent bacteria, E. coli, P. aeruginosa and S. aureus on the crosslinked copolymer-modified coatings after 7 d incubation, respectively (n = 3): PU (a), PU/PDA (b), PU/PDA/CA&PCSB (c), PU/PDA/CA&PCAS60 (d), PU/PDA/CA&PCAS40 (e), and PU/PDA/CA&PCAC (f).

However, it should be noted that this study still has several limitations that require careful consideration. On the one hand, the PDA-mediated layer not only promotes cell adhesion and spreading⁵⁹ but may also interact specifically with cellular receptors, particularly dopamine receptor D1 (DRD1).⁶⁰ Given that the zwitterionic polymer coatings constructed in this study may not completely cover the PDA-mediated layer, and considering the potential for gradual coating delamination during prolonged use, the underlying PDA layer may become increasingly exposed over time. Therefore, the potential biological effects and long-term biocompatibility of the PDA-mediated layer need to be fully considered. On the other hand, for biomedical catheter applications, a long-term assessment of the expected degradation behavior and in vivo performance of the constructed coatings would be essential to ensure clinical reliability and safety.

Conclusions

On the basis of the previously reported methods that combine PDA-assisted co-deposition and amidation reactions to prepare functional antifouling and bactericidal coatings on PU surfaces, which have relatively poor stability,¹⁵ this work has developed effective and robust dual functional antifouling and bactericidal coatings for PU by introducing CA as a crosslinker during the amidation reaction. The FT-IR and XPS results proved that a crosslinked structure is formed due to the reactions between small molecule CA's –COOH groups and copolymer side chain's –COOH groups with –NH₂ on the PU/PDA surface. And the crosslinked copolymer containing PSBMA segment coatings showed better lubrication, stability, and antifouling and bactericidal efficiency compared to the corresponding non-crosslinked copolymer coatings. The optimal molar ratio of MSA, SBMA and MCA is determined to be 2 : 6 : 2. Specifically, the ratio of SBMA and MCA components plays a critical role in balancing the degree of hydration and the positive charge on the coatings, which directly impacts the antifouling and bactericidal performance of the coatings. This study provides a foundation for the development of stable dual-functional antifouling and antibacterial surfaces. Further studies are currently underway to evaluate potential biological effects and long-term biocompatibility of the PDA-mediated layer as well as their in vivo performance.

Author contributions

Shengnan Cui: writing – original draft, formal analysis, investigation, methodology, and visualization. Henan Wei: writing – original draft, conceptualization, methodology, formal analysis, investigation, and data curation. Xiaolin Zhang: conceptualization, methodology, and data curation. Haimei Cao: conceptualization and methodology. Shiping Zhang: conceptualization, writing – review and editing, supervision, project administration, and funding acquisition.

Conflicts of interest

There are no conflicts to declare.

Data availability

Data will be made available upon request.

Supplementary information: the detailed information of surface characterization, the experimental procedures of protein adsorption, platelet adhesion, whole blood adhesion, in vitro antibacterial and cell attachment, and cytotoxicity. The chemical structures of the synthesized copolymers (Fig. 1), the variation of WCA during the optimization process (Fig. 2 and 3), XPS spectra (Fig. 4), roughness (Fig. 5), COF (Fig. 6), fluorescent microscopy images and quantitative analysis for the number of adherent bacteria after 1 d (Fig. 7) and 3 d (Fig. 8) incubation, and the bactericidal performance after 7 d incubation (Fig. 9). Syntheses and characterization of the prepared copolymers (Table 1), elemental composition of the newly constructed coatings (Table 2), and after immersion in PBS and artificial urine for 30 d (Table 3) as determined from XPS spectra. See DOI: https://doi.org/10.1039/d5tb01829c.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (NSFC 21004048) and the Natural Science Foundation of Shaanxi Province (2019JM-222). The authors express their gratitude to Prof. Kewu Yang for help in the antibacterial performance test and Prof. Jinjun Lv for help in the friction performance test.

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Footnote

† These two authors contributed equally to this work.

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