Transformative metal–ligand nanocoating for dual antibacterial and antithrombotic functionality in hemodialysis catheters

Abstract

Hemodialysis catheters are life-saving for end-stage renal disease patients but suffer from catheter-related bloodstream infections and thrombosis, which impair device function and threaten patient survival. Although functional coatings have been employed to mitigate some of these complications, the development of coatings that simultaneously exhibit both antibacterial and antithrombotic properties remains a significant challenge. To address these issues, we developed a PDA/Hep/Cu nanocomposite coating through sequential metal–ligand coordination, polymerization, and covalent immobilization. By employing copper–ammonia complexation, we achieved stable and high-density Cu²⁺ incorporation into the polydopamine (PDA) framework under alkaline conditions, avoiding precipitation problems of conventional methods. Heparin was then covalently conjugated to PDA via carbodiimide chemistry, preserving its antithrombotic bioactivity. Characterization confirmed a hierarchical structure with atomically dispersed Cu²⁺ in square-planar CuN₂O₂/CuN₄ coordination and effective heparin immobilization. In vitro assays showed initial antibacterial efficacy exceeding 99% against S. aureus and E. coli, with sustained activity (99.2% for S. aureus and 98.6% for E. coli) after 10-day PBS immersion. The coating reduced platelet adhesion by 32.4%, prolonged partial thromboplastin time by 24 seconds, and exhibited excellent biocompatibility (hemolysis <5%, cell viability 99.66%). In vivo porcine model validated 87.2–91.1% reduced bacterial colonization and 89% lower thrombus weight compared to uncoated catheters. This dual-functional coating synergizes Cu²⁺-mediated antibacterial activity and heparin-derived antithrombotic properties, offering a promising strategy to enhance hemodialysis catheter safety and longevity, with potential applications in other blood-contacting medical devices.

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Introduction

Hemodialysis catheters are life-saving for patients with end-stage renal disease but are plagued by two major complications: catheter-related bloodstream infections and thrombosis.^1,2 Upon implantation, they trigger a cascade of biointerfacial events, including protein adsorption, microbial colonization, platelet adhesion/activation, and intrinsic coagulation pathway activation. Together, these processes drive the dual risks of infection and thrombosis.^3,4 Such complications not only impair device function but also pose severe threats to patient survival.⁵ Thus, developing multifunctional coatings that concurrently prevent bacterial colonization and thrombus formation has become a critical research priority to enhance device safety and longevity.

Current strategies to address these dual risks are fragmented. Antimicrobial coatings typically use antimicrobial peptides, metal nanoparticles, or other biocidal agents to inhibit pathogens.^6,7 However, these materials inherently lack antithrombotic properties, leaving thrombotic risks unaddressed. Moreover, widespread use of antibiotic-loaded coatings raises concerns about antimicrobial resistance, and rapid leaching of metal ions in physiological environments can cause cytotoxicity at high concentrations—limiting clinical applicability.^6,8,9 In contrast, antithrombotic coatings incorporating heparin or other bioactive compounds effectively inhibit coagulation cascades by targeting antithrombin, thrombin, and platelet adhesion/activation.^10–12 Yet they offer no protection against microbial biofilm formation. Integrating these two functionalities into a single coating remains challenging, as achieving synergistic efficacy without compromising biocompatibility and stability is difficult.

Recent progress in metal–ligand polydopamine (PDA) framework coatings provides a promising direction.⁷ PDA, a bioinspired polymer that forms adherent coatings on diverse substrates, has a catechol-rich structure enabling robust chelation of Cu²⁺via stable metal–catechol coordination bonds—ensuring controlled ion release and reduced cytotoxicity.¹³ Additionally, PDA's abundant amine groups allow covalent conjugation of heparin via carbodiimide chemistry, incorporating antithrombotic functionality.¹⁴ This integrated design—combining PDA's interfacial stability, Cu²⁺'s antimicrobial efficacy, and heparin's antithrombotic properties—holds significant potential for developing synergistic antimicrobial–antithrombotic interfaces. However, practical implementation faces key challenges. Conventional PDA synthesis under alkaline conditions (e.g., pH 8.5) limits copper incorporation due to copper hydroxide precipitation. While trace copper ions can accelerate PDA polymerization in Tris buffer, only subtherapeutic Cu²⁺ concentrations are incorporated while maintaining coating stability.¹⁵ These constraints demand innovative strategies to optimize copper loading, control ion release, and preserve heparin bioactivity within a cohesive coating architecture.

Herein, we present a rationally designed PDA/Hep/Cu nanocomposite coating that addresses these challenges through sequential coordination, polymerization, and immobilization processes (Fig. 1). Our approach uses copper–ammonia complexation to stabilize Cu²⁺ during alkaline PDA deposition, enabling high-density metal incorporation without precipitation. Subsequent heparin conjugation preserves the glycosaminoglycan's antithrombotic properties while maintaining surface hydrophilicity and negative charge—critical for inhibiting platelet adhesion. Comprehensive in vitro and in vivo evaluations show that the coating simultaneously exhibits antimicrobial activity against clinically relevant pathogens, inhibits platelet activation, and prevents thrombus formation, demonstrating robust biological functionality in combating infection and clotting. Our results establish a versatile platform for enhancing hemodialysis catheter safety and durability through dual-pathway biointerface engineering.

Fig. 1 Illustration of the antibacterial and antithrombotic properties of PDA/Hep/Cu coating.

Results and discussion

Coating characterization

The morphological and chemical modifications of the PDA/Hep/Cu coating provide insights into its dual functionality. SEM analysis revealed that the PDA/Cu coating had a surface with numerous nanoparticles, in contrast to bare PU and PDA coatings, while heparinization did not alter the structure further (Fig. 2a). AFM measurements indicated that the PDA/Hep/Cu coating had an average roughness of 72 nm, significantly greater than that of the PU (4.15 nm) and PDA coatings (5.08 nm) (Fig. 2b). This increased roughness is attributed to the aggregation of Cu²⁺-chelated PDA nanoparticles (Fig. S1). The thickness of the PDA/Hep/Cu coating ranged from 451 to 571 nm (Fig. 2c), depending on the solution concentration and reaction time (Fig. S2a and d).

Fig. 2 Morphological characterization of PU and modified surfaces. (a) SEM images of PU, PU-PDA, PU-PDA/Cu, and PU-PDA/Hep/Cu. (b) AFM images of the samples. (c) Thickness profile of the PDA/Hep/Cu coating.

Variations in water contact angle (WCA) reflect the hierarchical functionalization of the coating. Initially, PDA deposition reduced hydrophobicity, with WCA decreasing from 109.9° to 66.9° (Fig. 3a). Chelation of Cu²⁺ then restored hydrophobicity to 110°, attributed to metal–catechol nanoparticles aggregation (Fig. 3a and Fig. S2c). Subsequent heparinization reintroduced hydrophilic sulfate and carboxyl groups, further reducing WCA to 66.1°. Additionally, zeta potential decreased after heparinization (Fig. 3b). The decreased WCA and negative zeta potential confirm effective heparin immobilization, introducing negative charges and hydrophilic groups (e.g., carboxyl and sulfate) that form a hemocompatible hydration layer to repel platelet adhesion.^16,17

Fig. 3 Physicochemical characterization of PU and modified surfaces. (a) Water contact angle, (b) zeta potential, (c) FTIR spectra, and (d) XPS wide-scan spectra of the samples. High-resolution XPS spectra of Cu 2p (e) and S 2p (f) for PU-PDA/Hep/Cu. Data in (a) and (b) are presented as mean ± standard deviation (n = 3).

FTIR spectroscopy confirmed stepwise chemical functionalization of the PU substrate. Pristine PU exhibited characteristic vibrations at 1726 cm⁻¹ (urethane C=O), 1529 cm⁻¹ (amide II), and 3329 cm⁻¹ (N–H), consistent with its polymer backbone (Fig. 3c).¹⁸ Following deposition of the PDA/Cu coating, significant spectral alterations were observed. The N–H stretching band redshifted to 3233 cm⁻¹ with broadening, indicating enhanced hydrogen bonding involving PDA's catechol/amine functionalities (Fig. 3c).¹⁹ A new intense peak at 610 cm⁻¹ in PU-PDA/Cu, assigned to metal–ligand bond stretching (M–O or M–N), provides direct evidence for coordination between copper ions and PDA (Cu–O or Cu–N bonds).²⁰ Heparin grafting introduced diagnostic sulfate ester vibrations at 890 cm⁻¹ and 943 cm⁻¹ (C–O–S stretching) and a prominent glycosidic linkage peak at 1035 cm⁻¹ (C–O–C)—features absent in PDA/Cu—confirming covalent immobilization via EDC/NHS catalysis (Fig. 3c).²¹

XPS analysis elucidated elemental composition and chemical bonding (Fig. 3d). For copper speciation, core-level peaks of Cu 2p_3/2 and Cu 2p_1/2 were observed at 934.1 eV and 954.3 eV, respectively, with satellite features characteristic of octahedrally coordinated Cu(II) (Fig. 3e).^22,23 Deconvolution revealed secondary components at 932.4 eV (Cu 2p_3/2) and 952.4 eV (Cu 2p_1/2), consistent with Cu⁺ (∼932.5 eV, 952.2 eV) or metallic Cu⁰ (∼932.7 eV, ∼952.7 eV) but lower than typical Cu(II) values (∼934.5 eV, ∼953.5 eV) (Fig. 3e).^22,24–26 Within the Cu(LMM) Auger spectra, a dominant peak at ∼570 eV (Fig. S2h) matches the characteristic binding energy of Cu⁺.^27,28 Given dopamine's reducibility, partial reduction of Cu²⁺ to Cu⁺ is plausible, resulting in a mixed valence state (+1 and +2).^29,30 For sulfur speciation, a S 2p_3/2 peak at 168.9 eV (Fig. 3f) confirmed heparin incorporation, assignable to N-sulfated moieties in heparin's highly sulfated domains.³¹ This peak is distinctly separated by >5 eV from Cu–S bond energies (162–164 eV for thiolate/disulfide species), ruling out significant Cu–S coordination.³² Notably, while no Cu–sulfate interactions were detected, PDA–Cu binding impacts heparin immobilization by depleting free amino groups. During dopamine's oxidative polymerization, free –NH₂ groups drive intramolecular cyclization (e.g., forming 5,6-dihydroxyindole) or covalent cross-linking.³³ Copper chelation accelerates this process via a Cu²⁺-dopamine quinone intermediate, enhancing cyclization and depleting amino groups.³⁴ This depletion reduces heparin grafting, supported by the inverse correlation between heparin grafting (2 mg mL⁻¹) and Cu²⁺ loading—with a peak at 0.1 M copper–ammonia (Fig. S2f).

High-resolution XPS elucidated critical bonding transformations for coating stability (Fig. 4). Pristine PU exhibited C 1s peaks at 284.8 eV (C–C), 285.8 eV (C–O/C–N), and 288.3 eV (O=C–N from urethane groups), consistent with its structure (Fig. 4a).^35,36 After forming the PDA/Hep/Cu layer, C 1s revealed new peaks at 287.2 eV (C=O quinone from oxidized PDA) and 286.0 eV (C–O/C–N), confirming PDA polymerization (Fig. 4b).^37–39 C 1s peaks at 289.1 eV were attributed to COO⁻ groups from heparin's uronic acid residues (Fig. 4b).⁴⁰ Peaks at 282.7 eV and 279.2 eV are rare and may relate to copper-interaction-induced carbon species (e.g., carbides) or fitting artifacts due to charging effects (Fig. 4b).^41,42 The N 1s spectrum of PU exhibited a characteristic peak at 399.4 eV, attributed to nitrogen in urethane linkages (–NH–COO⁻) – a core structural feature. After PDA/Hep/Cu construction, new N 1s peaks at 397.1 eV and 398.5 eV (Fig. 4c and d) – characteristic of metal nitrides – indicated N–Cu bond formation between Cu²⁺ and PDA's amine groups.^43–45 Regarding O 1s, PU displayed a peak at 533.2 eV corresponding to single-bond oxygen (C–O–C/C–O–H), primarily from polyether/polyester soft segments and urethane C–O–R moieties (Fig. 4e).⁴⁶ A less typical peak at 530.8 eV (uncommon in pure polymers) may arise from either metal oxide contamination or carbonyl oxygen with reduced binding energy under specific conditions (Fig. 4e). Following PDA/Hep/Cu formation, the 530.9 eV peak – typical of metal–oxygen bonds – indicated possible coordination between Cu²⁺ and PDA's phenolic hydroxyl oxygen (Fig. 4f).⁴⁷

Fig. 4 High-resolution spectra of C 1s, N 1s, and O 1s for PU (a), (c) and (e) and PU-PDA/Hep/Cu (b), (d) and (f).

XAFS analysis at the Cu K-edge elucidated the local structure of Cu centers in PDA/Hep/Cu particles (Fig. 5). X-ray absorption near-edge structure (XANES) spectra showed the absorption edge of PDA/Hep/Cu lies beyond those of CuO (Cu(II)) and CuPc (planar CuN₄), consistent with a predominant Cu(II) state—this indicates the antibacterial activity is primarily mediated by Cu(II) rather than Cu(I) (Fig. 5a). Notably, XAFS is a bulk-averaging technique with limited sensitivity to minority species, whereas XPS probes the outermost 3–4 nm (surface-sensitive). This distinction implies that subtle contributions from trace Cu⁺ (<5% of total Cu) may be masked by signal noise and the dominant bulk Cu²⁺ signal.^48,49

Fig. 5 XAFS analysis of PDA/Hep/Cu. (a) XANES spectra at Cu K-edge and (b) Fourier transform (FT) EXAFS spectra of PDA/Hep/Cu and reference samples (Cu₂O, CuO, Cu foil, CuPc). (c) Fitting results of PDA/Hep/Cu at the Cu K-edge. Contour plots of WT spectra for (d) PDA/Hep/Cu, (e) Cu₂O, and (f) CuPc.

Extended X-ray Absorption Fine Structure (EXAFS) fitting further defined the local coordination of Cu(II), revealing a prominent Cu–N/O shell peak at R = 1.5 Å (R-space) and confirming atomically dispersed Cu²⁺ with no metallic or oxide clustering (Fig. 5b and c). First-shell fitting yielded a coordination number of 4.3 ± 0.2 and bond length of 1.98 ± 0.01 Å (σ² = 0.0056 Ų), consistent with square-planar geometry (e.g., CuN₂O₂ or CuN₄; Table S1).^50–53 The four-coordinate environment of Cu²⁺ suggests that carboxyl and sulfate groups are unlikely to participate in coordination. Additionally, the bulky heparin molecules (12–15 kDa) experience steric hindrance, limiting their access to Cu sites tightly encapsulated by PDA ligands.⁵⁴ Wavelet transform (WT) analysis further validated homogeneous Cu–N/O bonding, with no detectable Cu–Cu or Cu–S interactions (Fig. 5d–f), thus ruling out aggregated Cu species.

We performed Linear Combination Fitting (LCF) of XANES spectra against standards (Cu₂O, CuO, CuPc, Cu foil), but it failed to produce reliable results. This failure aligns with the structural insights from EXAFS: if surface Cu⁺ is present, it is likely an atomically dispersed species coordinated to PDA (analogous to the Cu²⁺ environment), rather than a standard bulk oxide (e.g., Cu₂O). Such a unique coordination would yield a XANES fingerprint not represented in conventional standard libraries, meaning the LCF failure does not confirm the absence of Cu⁺ but reflects the lack of matching reference spectra for this distinct species.

Collectively, these findings outline a hierarchical coordination framework: PDA serves as a platform where catechol and amine groups chelate copper ions via Cu–N/O bonds (Fig. 6). XAFS confirms predominantly atomically dispersed Cu(II) in square-planar coordination (e.g., CuN₂O₂ or CuN₄), with no Cu⁰ clusters or significant Cu(I). Heparin is covalently immobilized via amide bonds between its carboxyl groups and PDA's amine groups. This integration of copper-mediated antibacterial activity and heparin-derived antithrombotic properties provides a strategic approach for advanced biomedical coating design.

Fig. 6 Possible Cu–N₂O₂ (a) or Cu–N₄ (b) coordination geometry in PDA/Hep/Cu.

In vitro biocompatibility

The PDA/Hep/Cu coating's biocompatibility was evaluated via hemolysis and cytotoxicity assays. Both direct contact and 72-hour extract methods showed hemolysis rates below 5% (Fig. 7a), consistent with ISO 10993 standards, confirming satisfactory blood compatibility. The CCK-8 cytotoxicity assay indicated 99.66% cell viability after 72 hours of co-culture (Fig. 7b), and live/dead staining showed minimal cellular damage (Fig. 7c). These results reflect negligible toxicity from free Cu²⁺, attributed to stable metal–ligand bonds—consistent with literature highlighting controlled Cu²⁺ release as critical for balancing antibacterial efficacy, hemocompatibility, and platelet activation inhibition.^8,9

Fig. 7 Hemolytic rate of PDA/Hep/Cu coatings via direct contact and extraction methods (a) (n = 3). Cell viability assessed by CCK-8 assay (b) (n = 6) and CLSM with live/dead staining (c). Data are presented as mean ± standard deviation. Scale bars: 200 μm.

In vitro antibacterial property

The PDA/Hep/Cu coating demonstrated excellent antibacterial activity against S. aureus and E. coli, with this efficacy primarily attributed to Cu²⁺ (Fig. 8a). Cu²⁺ exerts broad-spectrum antibacterial effects through membrane disruption and reactive oxygen species generation.⁷ Controlled Cu²⁺ release—ranging from 0.09 ± 0.01 to 1.97 ± 0.01 μg per cm² per day over 21 days in PDA/Hep/Cu (Fig. S2i)—is critical to maintaining this activity. Given the elevated infection risk associated with temporary catheters used beyond 1 week, we assessed the antibacterial performance of the coating before and after immersion.⁵⁵ Unimmersed PDA/Hep/Cu achieved 99.7% and 99.6% bactericidal rates against S. aureus and E. coli, respectively. Importantly, these rates remained high (99.2% and 98.6%) following 10 days of immersion in PBS (Fig. 8a and Fig. S2j). This validates the sustained antibacterial activity of the PDA/Hep/Cu coating, which arises from its slow and consistent release of Cu²⁺. SEM confirmed minimal bacterial adhesion on treated surfaces (Fig. 8b). The few bacteria that did adhere exhibited deformed and collapsed morphologies. To elucidate the mechanism, bacteria on glass slides were treated with either pristine or 10-day-immersed PDA/Hep/Cu. SEM showed intact bacterial membranes on untreated surfaces (including PU), whereas treated specimens exhibited severe membrane disintegration (porosity, fissures, and fragmentation) (Fig. 8c), providing direct evidence of contact-mediated bactericidal activity via membrane disruption. These results highlight the coating's potential to prevent infections in blood-contacting devices.

Fig. 8 Antibacterial properties of PDA/Hep/Cu on day 0 and day 10 after immersion in PBS. (a) Results of the spread plate method. (b) Bacterial attachment on the PDA/Hep/Cu coating. (c) Observation of bacterial membranes after treatment with different materials.

In vitro antithrombotic performance

The PDA/Hep/Cu coating significantly reduced platelet adhesion compared to uncoated catheters (Fig. 9). LSCM showed abundant rhodamine 123-labeled platelets on uncoated surfaces, but minimal adhesion on coated surfaces (Fig. 9a). SEM confirmed extensive platelet accumulation on uncoated surfaces (omelet-like morphology with pseudopods), versus no significant adhesion on coated surfaces (Fig. 9a). Blood cell counter and LDH assay results substantiated superior anti-platelet properties: residual platelets were significantly higher in the coated group (Fig. 9b and Fig. S2k), with the coating reducing platelet loss by ∼32.4%. Red and white blood cell counts showed no significant differences between groups (Fig. 9b). LDH levels (from lysed adherent cells) were ∼71.1% lower on coated surfaces (Fig. S2k), confirming reduced blood cell adhesion.

Fig. 9 (a) Antiplatelet performance observed via CLSM and SEM. (b) Blood cell adhesion behavior of samples assessed by whole blood cytometry. (c) Protein adsorption of samples incubated in a single solution; (d) Protein adsorption in a mixed solution. (e) Anti-FXa activity of PDA/Hep/Cu coatings before and after 10-day immersion in PBS. (f) PTT assessment for all samples. PC, positive control; NC, negative control. Data in (b–f) are presented as mean ± standard deviation (n = 3); ***p < 0.001.

Multiple factors contribute to reduced platelet adhesion: the hydrophilic coating's hydration layer and negative charge repel negatively charged platelets; heparin directly inhibits thrombin-induced platelet adhesion/activation; and increased albumin adsorption forms a protein corona that impedes platelet interactions.^56–58 Albumin, a passivating protein lacking platelet receptors, contrasts with fibrinogen (which has platelet-binding sites).⁵⁹ Protein adsorption assays showed a higher albumin-to-fibrinogen ratio on the PDA/Hep/Cu coating, consistent in both single (Fig. 9c) and mixed (Fig. 9d) solutions. In mixed physiological solutions, fibrinogen adsorption did not differ significantly, but albumin adsorption was ∼79.2% higher on coated surfaces (Fig. 9d), resulting in a higher BSA/Fbg ratio (3.62 vs. 0.74). Enhanced albumin adsorption thus reduces platelet adhesion/activation, contributing to antithrombotic properties.

Heparin bioactivity on PDA/Hep/Cu was confirmed via a chromogenic anti-FXa assay. Heparin induces a conformational change in antithrombin III (ATIII), accelerating protease inhibition by up to 1000-fold to neutralize FXa efficiently. Heparin-free PDA/Cu films exhibited no FXa inhibition, with chromogenic substrate turnover comparable to that of the control group (Fig. 9e). In contrast, PDA/Hep/Cu exerted significant inhibition via the formation of heparin–ATIII complexes, resulting in the lowest chromogenic substrate turnover (Fig. 9e). Notably, heparin bioactivity and immobilization were preserved after 10 days in PBS (Fig. 9e and Fig. S2l), indicating durable antithrombotic activity.

To avoid obscuring material intrinsic properties, PTT—rather than activated partial thromboplastin time (APTT)—was used to evaluate antithrombotic performance.⁶⁰ Compared to uncoated catheters, PDA/Hep/Cu extended PTT by ∼24 seconds, within the 75–100% negative control range (Fig. 9f), indicating minimal activation of the intrinsic coagulation pathway (American Society for Testing and Materials, ASTM F2382-04). This extension is attributed to heparin, which binds ATIII to expose its reactive site, enhancing inhibitory capacity to irreversibly inhibit factor Xa and IIa, and prevent prothrombin-to-thrombin conversion.¹¹ These results confirm robust antithrombotic efficacy.

In vivo antibacterial and antithrombotic properties

Building on in vitro findings, a porcine hemodialysis model was developed. Uncoated and PDA/Hep/Cu-coated catheters were inserted into swine external jugular veins (Fig. 10). To simulate clinical infections, catheters were sealed with bacteria-saline suspensions. No complications occurred during catheterization; one swine from the control group developed local redness, swelling, and fever 24 hours post-surgery and received penicillin (800 000 units) for 3 days.

Fig. 10 In vivo assessment of PDA/Hep/Cu coating performance via hemodialysis catheter insertion into swine external jugular veins. (a) Gross view of swine and hemodialysis catheter placement. (b) Representative images of catheter insertion sites at 0 h and 24 h post-insertion.

After 24 hours, plate cultures of sealing fluid from uncoated catheters showed significant S. aureus and E. coli growth, while the coated group had minimal proliferation (Fig. 11a). PDA/Hep/Cu enhanced antibacterial efficacy by 87.2% (S. aureus) and 91.1% (E. coli) versus uncoated catheters (Fig. 11b). Thrombus quantification showed a significantly lower mean weight in the coated group (11 ± 4 mg) versus the uncoated group (102 ± 58 mg) (Fig. 11c). SEM revealed a fibrin network with red blood cells and activated platelets on uncoated surfaces, versus no significant thrombus on coated surfaces (Fig. 11d).

Fig. 11 Antibacterial (a) and (b) and antithrombotic (c) and (d) property of PDA/Hep/Cu coating in vivo after 24 h catheterization. Data in (b) and (c) are presented as mean ± standard deviation (n = 3).

Copper ions inhibited bacterial colonization even under blood flow and shear stress, while reduced thrombus formation reflected the coating's ability to prevent platelet adhesion/aggregation and heparin's in vivo fibrin inhibition. SEM confirmed coating durability under physiological conditions. These findings demonstrate that the dual-functional design mitigates both infectious and thrombotic complications, addressing key limitations of current hemodialysis catheters.

Conclusions

This study presents a multifunctional metal–ligand PDA/Hep/Cu nanocoating designed to address catheter-related infections and thrombosis in hemodialysis catheters. Copper–ammonia complexation under alkaline conditions enables optimal Cu²⁺ incorporation into the PDA framework, synergistically integrating Cu²⁺-mediated antibacterial activity with heparin's antithrombotic properties. The coating exhibits enhanced antibacterial and antithrombotic performance in vitro and in vivo, with excellent hemocompatibility. Synergies between nanostructured roughness, hydrophilicity, and chemical functionality establish a versatile platform for mitigating infections and thrombosis in medical devices. These findings advance safer hemodialysis catheters and propose a scalable approach to improve other blood-contacting devices.

Experimental section

Materials

Polyurethane (PU) films were purchased from Dongguan Huacheng Plastic Products Co., Ltd (China). Hemodialysis catheters (Gambro, GDHK1325/GDK1115) were sourced from Baxter International Inc. (USA). Anhydrous copper sulfate (CuSO₄) was obtained from Tianjin Zhiyuan Chemical Reagent Co., Ltd (China), and ammonium hydroxide from Tianjin Kemiou Chemical Reagent Co., Ltd (China). Dopamine hydrochloride (DA·HCl), sodium heparin, and toluidine blue O (TBO) were acquired from Aladdin (China). 2-N-Morpholino ethanesulfonic acid (MES) and rhodamine 123 were purchased from Sigma-Aldrich (USA). 1-Ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Shanghai Adamas Reagent Co., Ltd. Bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), bicinchoninic acid (BCA) protein assay kit, cell counting kit-8 (CCK8), lactate dehydrogenase (LDH) cytotoxicity assay kit, and calcein/PI live/dead viability/cytotoxicity assay kit were purchased from Beyotime Biotechnology (China). Bovine fibrinogen (Fbg) and Fbg ELISA kit were sourced from Shanghai Enzyme-linked Biotechnology Co., Ltd (China). Reagents for the anti-FXa activity assay were purchased from Seebio Biotechnology (Shanghai) Co., Ltd. New Zealand white rabbits were provided by the West China Experimental Animal Service Station, and domestic swines (male) were obtained from Chengdu Xiangba Li Fragrant Pig Specialized Cooperative (China).

Preparation of PDA/Hep/Cu coating

PU films (1 × 1 cm) were sequentially rinsed with deionized water and ethanol. A 0.1 M copper–ammonia complex solution was prepared by mixing 0.8 mL aqueous ammonia with 19.2 mL of 0.1 M CuSO₄. Subsequently, 40 mg of dopamine was dissolved in 20 mL of this copper–ammonia solution. For the control group, 20 mL of Tris–HCl buffer (pH 8.5, 2 mg mL⁻¹ dopamine) was used. PU films were immersed in the dopamine solutions and agitated in a water bath shaker (25 °C, 135 rpm) for 24 h. After reaction, PU-PDA/Cu samples were rinsed with deionized water and air-dried.

Heparin was covalently immobilized onto PU-PDA/Cu using EDC/NHS chemistry.⁶¹ Briefly, 2 mg mL⁻¹ heparin sodium was dissolved in 20 mL of 50 mM MES buffer, followed by adding 4.8 mg NHS and 20 mg EDC. PU-PDA/Cu films were immersed in this solution and shaken (25 °C, 60 rpm) for 12 h. Resulting PU-PDA/Hep/Cu samples were thoroughly rinsed and dried. For hemodialysis catheters (GDHK1325 and GDK1115), the coating procedure was adapted: GDHK1325 catheters were cut into 3.5-mm segments (surface area: ∼1 cm²), while GDK1115 catheters were coated under dynamic flow using a peristaltic pump to simulate luminal and external fluid environments.

Coating characterization

Surface morphology was analyzed via scanning electron microscopy (SEM). Wettability was assessed by water contact angle (WCA) measurements using a contact angle meter. Chemical composition and elemental analysis were performed via X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy, and surface roughness was quantified using atomic force microscopy (AFM). X-ray absorption fine structure (XAFS) measurements were conducted at beamline 07A1 of the Taiwan Light Source (TLS) at the National Synchrotron Radiation Research Center (NSRRC, China).

Copper ion release assay

Prepared PU-PDA/Hep/Cu membranes (25 pieces, total surface area 50 cm²) were air-dried and immersed in 50 mL phosphate-buffered saline (PBS). The solution was oscillated daily; 5 mL was aspirated for copper quantification via inductively coupled plasma optical emission spectrometry (ICP-OES), and 5 mL fresh PBS was replenished. This was repeated for 21 days to monitor cumulative Cu²⁺ release.

Biocompatibility evaluation

Hemolysis test. Citrated rabbit blood (2 mL) was diluted with 2.5 mL saline. For the extract method, samples were incubated in saline (37 °C, 72 h); the direct contact method omitted extraction. After incubating with diluted blood (37 °C, 1 h), supernatants were centrifuged (800×g, 5 min), and absorbance at 545 nm was measured. Hemolysis (%) was calculated as:
where A represents optical density (OD).

Cytotoxicity assay. GDHK1325 and GDHK1325-PDA/Hep/Cu samples were incubated with 6 mL complete culture medium (37 °C, 24 h). Extract medium and fresh medium were used to culture L929 cells (3000 cells per well) in 96-well plates for 24 h and 72 h, respectively. Medium was replaced with 1 : 9 CCK-8 reagent in fresh medium, incubated 1 h, and OD at 450 nm measured. Cell viability (%) was calculated as:
where A represents the OD value of different groups.

For live/dead staining, L929 cells (20 000 cells per well) were cultured with extract or normal medium (24 h), rinsed with PBS, and stained with propidium iodide (PI, 2 μmol L⁻¹) and calcein-AM (4.5 μmol L⁻¹) (37 °C, 30 min). Fluorescence microscopy visualized live (green) and dead (red) cells.

In vitro antibacterial property

Staphylococcus aureus (ATCC6538) and Escherichia coli (ATCC25922) were used. GDHK1325 and GDHK1325-PDA/Hep/Cu samples were sterilized under UV light (30 min) and co-cultured in 96-well plates with 200 μL bacterial solution (1 × 10⁵ CFU mL⁻¹ in RPMI1640). A diluted bacterial solution without intervention served as control (3 replicates/group). After 12 h, bacterial solutions were diluted, inoculated on agar plates, and incubated (37 °C, 12 h). Antibacterial rate (%) was calculated as:

Samples were rinsed with PBS, fixed in 2.5% glutaraldehyde (overnight), dehydrated with 50–100 vol% ethanol, and observed via SEM.

Antibacterial rates of 10-day PBS-immersed PU-PDA/Hep/Cu membranes were assessed similarly. To confirm membrane damage, glass slides and smooth PU were co-cultured with 500 μL bacterial solution (1 × 10⁴ CFU mL⁻¹) for 8 h. Glass slides were then treated with pristine or 10-day-immersed PU-PDA/Hep/Cu for 16 h. Samples were fixed, dehydrated, and observed via SEM.

Protein adsorption

GDHK1325 and GDHK1325-PDA/Hep/Cu were immersed in 200 μL BSA solutions (100 μg mL⁻¹ and 40 mg mL⁻¹) and incubated in a gas-bath oscillator (37 °C, 60 rpm, 12 h). Samples were rinsed with PBS, sonicated in 2 wt% SDS (200 μL, 30 min), and 20 μL of this solution added to 200 μL BCA working solution (30 min incubation). OD at 540 nm was measured, and BSA concentration calculated from a standard curve.

Similarly, samples were immersed in 200 μL Fbg solutions (50 μg mL⁻¹ and 4 mg mL⁻¹). After incubation, rinsing, and sonication, 50 μL solution was added to a zymography plate; Fbg content was calculated from OD at 450 nm using a standard curve.

A mixed solution of BSA (40 mg mL⁻¹) and Fbg (4 mg mL⁻¹) was tested using the same protocol.

Blood cell adhesion assessment

Citrate-anticoagulated rabbit whole blood (heparinized to 2 U mL⁻¹, recalcified to 10 mmol L⁻¹) was incubated with GDHK1325 and GDHK1325-PDA/Hep/Cu within 2 h of collection. Glass beads served as positive control, blank PP tubes as blank control. After 1 h (37 °C, 60 rpm), EDTA was added to 5 mmol L⁻¹, and blood was transferred to pre-cooled PP tubes for cytometry. Blood cell loss (%) was calculated as:
where B and A are blood cell counts from blank control and material groups, respectively.

Samples were rinsed with PBS, and adhered blood cells quantified using LDH working solution; absorbance was measured at 490 nm and 600 nm.

Platelet-rich plasma (PRP) from rabbit blood (centrifuged at 200g, 10 min) was incubated with samples (37 °C, 60 min). Samples were rinsed, fixed in 2.5% glutaraldehyde (overnight), dehydrated, and observed via SEM. Platelets were stained with rhodamine 123 (15 min) and observed via LSCM.

Anti-FXa activity assay

The bioactivity of heparin was assessed by a chromogenic anti-Fxa assay. Materials were incubated with antithrombin III (37 °C, 30 min), followed by FXa and chromogenic substrate S2765 (2-min incubation). EDTA quenched the reaction, and absorbance at 405 nm was measured.

Partial thromboplastin time (PTT) assessment

Platelet-poor plasma (PPP) from rabbit blood (centrifuged at 2000 g, 10 min) was incubated with GDHK1325, GDHK1325-PDA/Hep/Cu, and glass beads (positive control); bare PP tubes with PPP served as negative control. After incubation, 100 μL PPP was equilibrated (37 °C, 60 s), activated with 100 μL rabbit brain phospholipid (1 mg mL⁻¹, 2 min), and 100 μL CaCl₂ (25 mmol L⁻¹) added. PTT was measured using a semi-automatic thrombin analyzer.

In vivo antibacterial and antithrombotic properties

Animal experiments were conducted in strict accordance with the animal ethical guidelines and protocols of the China Council on Animal Care and Sichuan University, and were approved by the Experimental Animal Ethics Committee of West China Hospital of Sichuan University (ethical number: 20230207005).

Six male domestic swine were randomized into control (uncoated) and experimental (PDA/Hep/Cu-coated) groups (n = 3/group). PDA/Hep/Cu coatings were applied to GDK1115 catheters (inner/outer surfaces) using peristaltic pumps. Catheters were implanted into external jugular veins; arterial ends were sealed with 2 mL S. aureus solution, and venous ends with E. coli solution, each prepared by diluting 10 μL of a 1 × 10⁵ CFU mL⁻¹ stock suspension. After 24 h, sealing fluid was cultured, and catheters were removed, rinsed, sectioned, and fixed in 4% paraformaldehyde for 24 h. After fixation, they were rinsed with PBS to remove residual fixative, dehydrated through a graded ethanol series, and then vacuum-dried. Thrombus weight was calculated by subtracting the weight of unused catheters from that of the thrombus-bearing catheters. Surface morphology was observed via SEM.

Statistical analysis

Data are presented as mean ± standard deviation. Differences between two groups were evaluated by independent samples t-test (two-tailed), with subtype (Student's or Welch's) based on variance homogeneity. Multiple comparisons used one-way ANOVA with Tukey's HSD post-hoc test. P < 0.05 was considered significant.

Author contributions

C. L., Z. F. Z., M. X. R., and Y. Y. performed experiments, validated results, analyzed data, and drafted the manuscript. Y. C., L. Z., and P. F. contributed to conceptualization, critically reviewed, and edited the manuscript. All authors approved the final version.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

All data supporting the results are included in the article and its SI. See DOI: https://doi.org/10.1039/d5tb01459j

Acknowledgements

This work was supported by the Strategic Cooperation Special Funds Project of Sichuan University-Dazhou Municipal People's Government (2022CDDZ-20), Science and Technology Program of Sichuan Province (2023YFG0211), and the 1.3.5 project for disciplines of excellence from West China Hospital of Sichuan University (ZYGD23015).

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