Hierarchical silver-tannic acid hydrophobic coating via layer-by-layer assembly for antibiofilm applications on urinary catheters

Abstract

Catheter-associated urinary tract infections (CAUTIs) are a serious global concern due to the emergence of drug-resistant bacteria and the biofilm formation on urinary catheters (UCs). Surface modification strategies have been identified as a prominent and cost-effective method to address this issue owing to its tunable properties, which effectively combat the biofilm formation on UCs. This study reports the development of a hydrophobic-silver nanoparticles (Ag NPs) decorated tannic acid-based coating on silicone UC using a layer-by-layer (LBL) approach that can effectively eradicate Escherichia coli (E. coli) biofilms. The LBL coating (PFDT-Ag-Dex) consists of a tannic acid (TA)-(3-aminoprophyl) triethoxysilane (APTES) NPs deposition, followed by Ag NPs decoration and 1H,1H,2H,2H-perfluorodecane-thiol (PFDT) layers, which impart the hydrophobicity, biocompatibility, antibacterial activity, and coating adhesion, respectively. Formation of the LBL coating on the Si catheter was successfully confirmed through extensive characterizations. In vitro and in vivo investigations showed that the PFDT-Ag-Dex coated Si catheter significantly inhibited the E. coli biofilm formation with ∼95 % efficiency due to the combined effects of the hydrophobic properties, tannic acid and Ag⁺ ions causing the cell membrane disruption. Furthermore, in vivo studies using mouse and rabbit animal models confirmed the biosafety of the PFDT-Ag-Dex-coated Si catheter, which exhibited a negligible inflammatory response. The studies suggest that the Ag-TA-hydrophobic coated catheter is a promising solution for combating the urinary tract infections.

Graphical abstract

Highlights

• •Developed a hierarchical structured Ag-tannic acid hydrophobic coating for urinary catheters.
• •Demonstrated improved biofilm inhibition via sustained release of Ag ⁺ ions and a hydrophobic barrier.
• •Biosafety and bactericidal efficacy validated in subcutaneous mouse and rabbit models.

1. Introduction

Catheter-associated urinary tract infections (CAUTIs) remain a significant challenge for healthcare systems worldwide. These infections account for nearly 40 % of all healthcare-associated infections, largely because of pathogenic bacteria can adhere to and grow on the urinary catheter surfaces [1]. In the United States, it is reported that approximately one million CAUTI cases occur each year, generating an economic burden that surpasses $350 million [2,3]. Various preventive strategies have been explored to address this issue, including antibiotic administration, catheter material modifications, regular catheter replacement, and developing surface coatings [4]. Among these approaches, antimicrobial surface coatings have gained attention due to their ease of preparation, tunable multifunctional characteristics, cost-efficiency, and superior antibacterial performance [3,5,6]. Antibacterial coatings typically function through either contact-inhibition or release-inhibition, both of which are widely used to inhibit bacterial growth on catheter surfaces [7].

Mussel-inspired surface coatings are renowned for their remarkable adhesive capabilities, due to the existence of catechol groups in mussel foot silk proteins [8]. These catechol groups facilitate adhesion through hydrogen bonding, electrostatic interactions, hydrophobic effects, and other molecular forces. These multifaceted interactions allow for strong binding to various material surfaces [[9], [10], [11]]. Recent research has identified tannic acid-3-aminopropyltriethoxysilane (TA-APTES) coatings as a promising alternative to conventional polydopamine coatings. TA-APTES coatings offer advantages including mild synthesis conditions, compatibility with various shapes and materials, biocompatibility, tunable secondary functionalities, cost-effectiveness, and unique hierarchical architecture [[12], [13], [14]]. The abundance of hydroxyl groups in tannic acid, along with its distinct capacity for secondary functionalization, allows for the incorporation of various macromolecules and metal ions via hydrogen bonding, Michael addition, and Schiff-base reactions. This versatility supports the fabrication of films, coatings, hydrogels, and nanoparticles [15]. Furthermore, the hierarchical nano/microstructures inherent to these coatings impart hydrophobic or superhydrophobic properties that effectively minimize bacterial adherence [16,17]. Tannic acid is a natural polyphenolic compound originating from plant sources (like red wine, tea and grapes) and it is recognized for its biocompatibility and antimicrobial activity. It can damage bacterial cell membranes, making it suitable for biomedical device applications [14,18]. Recent studies have demonstrated that surfaces with hierarchical nano/microstructures can enhance urine flow, facilitate the removal of bacteria, and prevent biofilm formation and associated complications [16,17,19]. Despite existing research, limited studies have investigated TA-APTES coatings with hierarchical nano/microstructures that incorporate antibacterial agents for use in urinary catheters.

Silver nanoparticles (Ag NPs) have attracted considerable interest in the biomedical field due to their peculiar characteristics, such as, (1) facile preparation, (2) versatility in formulation with the hydrogels, films, and coatings, (3) broad-spectrum antimicrobial activity at low doses due to cell membrane damage and reactive active oxygen species (ROS), and (4) anti-inflammatory and antioxidant properties [20]. Many studies have investigated the development of Ag NP-based coatings for treating urinary tract infections (UTIs). Notably, experimental studies and commercial products, such as the silver-coated catheter (ConocoPhillips) and the Silverglide catheter (Teleflex), utilize Ag NPs as bactericidal agents within surface coatings [3]. Despite these benefits, Ag NPs have limitations, such as cytotoxicity, rapid ion release, and limited longevity during UTI treatment [21]. For example, mussel-inspired polydopamine (PDA) coatings embedded with Ag NPs have demonstrated cytotoxic effects on fibroblast cell lines due to the rapid release of Ag⁺ ions when inhibiting biofilm on urinary catheters. However, applying an additional superhydrophobic layer has been shown to enable sustained Ag⁺ ion release while minimizing toxicity [16]. Therefore, developing new coating solutions that effectively combat bacteria, are highly biocompatible, and have a long functional lifespan has become imperative.

The layer-by-layer (LBL) coating method has gained attention as an effective surface modification strategy, enabling the incorporation of various functional components-including drugs, enzymes, and antimicrobial agents-onto medical devices through various deposition processes such as dipping, electrodeposition, and spraying. This method facilitates the construction of multilayered structures via electrostatic interactions between charged polymers and nanoparticles, and has been widely applied in both biomedical and other technological fields [22]. The LBL approach offers several notable advantages, such as the ability to precisely tailor the structure and composition of the coatings, control over thickness and uniformity across substrates of varying shapes, and cost-effective fabrication [23]. Recent advancements have led to the development of a variety of LBL-based coatings, including multilayers of poly(sodium 4-styrenesulfonate) and poly(allylamine hydrochloride) [24], aminocellulose nanospheres loaded with hyaluronic acid [25], chlorhexidine-micelle/polyacrylic acid composites [26], polydopamine-Cu-F nanoparticle hybrids [17], polydopamine-silver nanoparticle hybrids [16,27], silver nanoparticle-embedded polyelectrolyte films [28], and aminoglycoside-integrated coatings [29]. Jiru Miao et al. has recently reported the formation of a superhydrophobic PDA-Cu-F coating on a PDMS sheet using a LBL approach for the UTI applications against Proteus mirabilis (P. mirabilis) and demonstrated excellent antibacterial performance and encrustation properties in vitro [17]. These coatings have been applied to a range of biomedical devices to enhance their antibacterial performance. Although, previous studies have reported superhydrophobic LBL coatings on model substrates for antibacterial applications, research on realistic medical UCs and their biofilm model studies in vitro and in vivo remains limited. In this report, we present a facile and robust hierarchical LBL coating composed of TA-APTES, Ag-Dex, and PFDT directly on the medical UCs. This multifunctional coating provides enhanced hydrophobicity, sustained Ag ion release, and superior antibiofilm performance suitable for long-term UTI prevention in UC applications.

Briefly, this study presents the development of a silver-hydrophobic coating (PFDT-Ag-Dex) on silicone urinary catheters using a LBL assembly via a dipping method. This coating integrates hierarchical nano/microstructures, hydrophobicity, and silver NPs-assisted antibacterial activity. It aims to address the limitations of current UTI prevention strategies. The hierarchical structure, derived from TA-APTES templating, provides anti-adhesion properties and controls the release of Ag⁺ ions. The incorporation of the top PFDT layer onto Ag-Dex was selected due to its strong bonding affinity toward Ag-Dex NPs through thiol groups, its hydrophobic surface characteristics arising from multiple fluoro groups, and its ability to control Ag ion release, prevent bacterial adhesion and maintain biocompatibility. The multifunctional catheter coating achieved through the LBL approach exhibits significant antibacterial performance and biosafety properties. We characterized the silver-tannic acid hydrophobic coating systematically and investigated its in vitro and in vivo antibacterial activities and biosafety. The results demonstrate that the PFDT-Ag-Dex coating is biocompatible and provides effective, sustained biofilm inhibition through the controlled release of Ag⁺ ions on urinary catheters. An overview of the work is depicted schematically in Fig. 1a.

Fig. 1.

(a) Schematic diagram showing the synthesis route of Ag-Dex NPs and the fabrication of their hydrophobic PFDT-Ag-Dex coating on urinary catheters by dipping method, along with the possible mechanism, and followed by an illustration of their antibiofilm application. Characterization of Ag-Dex NPs: (b) XRD pattern, (c) UV–visible spectra, and (d) TEM image (along with their particle distribution in inset image).

2. Results and discussion

2.1. Characterizations: Phase, optical absorption, surface morphology and thickness

The powder XRD patterns of the prepared Ag-Dex NPs exhibited peaks at 38.14°, 44.26°, 64.55°, and 77.51°, which belong to the (111), (200), (220), and (311) crystallographic planes of the FCC-Ag (JCPDS No. 04–0783), as illustrated in Fig. 1b [30]. The Ag-Dex NPs exhibited optical absorption behavior with a broad peak at ∼425 nm due to their characteristic surface plasmon resonance effect (Fig. 1c) [31]. Furthermore, the TEM analysis was performed to understand the shape and particle size of Ag-Dex NPs as shown in Fig. 1d. Analysis confirmed the Ag NPs in spherical form with a size of 7.6 ± 3.2 nm (ranging from ∼6 to 30 nm) integrated with dextran molecules.

FESEM analysis was carried out to explore the surface features of the LBL coatings and the Bare Si catheter surface, as depicted in Fig. 2a–d. Spherical TA-APTES NPs formed on the TA-APTES coated Si catheter (Fig. 2b), while the Bare Si appeared plain (Fig. 2a). The Ag-Dex coating (Fig. 2c) demonstrated the presence of Ag-Dex NPs embedded on the surface of the TA-APTES NPs and its surface via electrostatic interactions between the Ag NPs and -OH groups originating from the tannic acid. Similarly, the PFDT-Ag-Dex NPs retained the same surface morphology (Fig. 2d) after the curing process. However, the coating surface appeared denser and defect-free. The diameter of the TA-APTES-Ag-Dex NPs was estimated to be ∼341 ± 79 nm. Due to the nanoscale formation of Ag NPs, they could not be distinguished in Ag-deposited coatings. Furthermore, the EDS analysis was done to understand the uniformity of the coating formation (Fig. 2e). The results indicate the presence of the elements Si, C, O, N, Ag and F, as well as their uniform distribution. The results displayed in Fig. 2e, further substantiate the elements observed in the EDS analysis. However, the EDS analysis could not detect the S element, possibly due to its minimal quantity. Additionally, the thickness of the prepared LBL coatings on the Si catheter was examined at their cross sections, as illustrated in Fig. 2f–h. The estimated thicknesses are ∼0.98 ± 0.01, 1.37 ± 0.03, and 1.39 ± 0.02 μm (highlighted by dotted lines), corresponding to the TA-APTES, Ag-Dex and PFDT-Ag-Dex coatings, respectively. Digital photographs of these LBL-coated Si catheter samples are shown in Fig. 2i. The photographs demonstrate the feasibility of uniform coating formation on the surface of the Si catheter. The slight increase in the coating thickness of the latter coating is attributed to the additional layers of Ag and PFDT polymer.

Fig. 2.

(a–d) FESEM surface morphologies of (a) bare Si catheter, (b) TA-APTES, (c) Ag-Dex, and (d) PFDT-Ag-Dex coated SCs. (e) FESEM-EDS elemental mapping of PFDT-Ag-Dex along with EDX spectrum. SEM cross-sectioned images show the coating thickness of (f) TA-APTES-, (g) Ag-Dex-, and (h) PFDT-Ag-Dex-coated UCs, as well as their (i) appearance in digital images.

2.2. Surface chemical properties

The chemical structure of the TA-APTES NPs was investigated using FTIR, ¹H NMR and XPS analyses. The results are presented in the supporting information (Fig. S2.1), including a discussion. Furthermore, ATR-FTIR was employed to study the chemical properties of the prepared LBL coatings and the Bare Si catheter. The spectra are shown in Fig. 3a. The LBL coatings (TA-APTES, Ag-Dex and PFDT-Ag-Dex) exhibited a broad band between 3100 and 3600 cm⁻¹ as compared to the Bare Si catheter. This can be assigned to the stretching (stret) vibrations of the residual phenolic groups of TA molecules and the hydrolyzed product of APTES molecules (-OH/NH_2stret). It suggests the presence of intramolecular hydrogen bonds [32]. Additionally, the peaks observed at ∼1717 and ∼1619 cm⁻¹ in the LBL coatings spectra are due to the stretching vibrations of -C=O_stret and -C=C_stret, respectively, originating from the TA molecules. The peaks identified at ∼1556 and ∼1507 cm⁻¹ indicate the presence of -C=N bonds and N-H_bend vibrations, respectively. Together, these findings support the bond formations between the TA (via quinone groups) and APTES (via amino groups) molecules through the Michael addition or Schiff base reactions [33]. In the PFDT-Ag-Dex coating, the appearance of absorption peaks at ∼1198 and ∼1145 cm⁻¹ are attributed to the vibrations of -C-S-C_stret and -C-F_stret, respectively (insert Fig. 3a). This is due to the interaction between the -SH group of the PFDT polymer and the phenolic group of the TA molecules via a Michael addition reaction [34]. The ATR-FTIR results confirm the TA-APTES NPs formation and the presence of the PFDT polymer in the final LBL coating.

Fig. 3.

Characterization of coated urinary catheters: (a) FTIR curves reveal the functional groups, and (b–f) XPS analysis: (b) Survey scan spectra show the elements present on the coatings, (c) High-resolution spectra of C 1s peak comparing the coatings and bare Si catheter, (d) High-resolution spectra of Ag 3d peak compare the silver-coated SCs, and (e) High-resolution spectra of the S 2p and (f) F 1s peaks of the PFDT-Ag-Dex coated SC.

The surface chemical features of the developed with and without Si catheters were examined by the XPS method, and the results are shown in Fig. 3b–f. The Si 1s, C 1s, and O 1s XPS peaks indicates the existence of elements in all the coated and uncoated catheters. Additionally, the Ag-Dex coating revealed the presence of silver (Ag 3p and Ag 3d peaks), while the PFDT-Ag-Dex showed the presence of fluorine (F 1s), sulphur (S 2p), and Ag elements. This was confirmed by their survey scan spectra in Fig. 3b. The C 1s XPS high spectrum (Fig. 3c) of the TA-APTES coating, compared to Bare Si catheter (B.E. of C-H at ∼284.62 eV and C-Si at ∼285.57 eV) showed the formation of additional chemical bonds, such as, the B.E. of C=C at 284.6 eV, C-C/C-Si at ∼285.59 eV, C-OH at ∼286.56 eV and C=O at ∼288.51 eV. This suggests that the tannic acid was successfully integrated with the APTES via Schiff base and Michael addition reactions on the Si catheter [14]. Similarly, the base coating of TA-APTES in the Ag-Dex (B.E. of C=C at ∼284.6 eV, C-C/C-Si at ∼285.60 eV, C-OH at ∼286.72 eV and C=O at ∼288.31 eV) and PFDT-Ag-Dex (B.E. of C=C at ∼284.60 eV, C-C/C-Si at ∼285.56 eV, C-OH at ∼287.03 eV, and C=O/CH₂-CF₂ at ∼288.44 eV) coatings showed the corresponding chemical bonding as seen in their deconvoluted XPS high-scan spectra (Fig. 3c). Furthermore, in the PFDT-Ag-Dex coating showed additional XPS peaks at 285.97 eV, 291.7 eV and 294.06 eV, which were assigned to CH₂-CF₂, CF₂ and CF₃ linkage, respectively, originating from the PFDT polymer [35].

The Ag 3d XPS high-scan spectrum of the Ag-Dex and PFDT-Ag-Dex coatings, displayed in Fig. 3d, shows the Ag 3d peaks showed the doublet peaks (Ag 3d_3/2 and 3d_5/2 due to spin-orbit coupling) at the B.E. of 374.21 and 368.23 eV, and at B.E. values of 374.68 and 368.65 eV, respectively. The occurrence of a chemical shift in Ag 3d peaks of the PFDT-Ag-Dex coating was attributed to the formation of Ag_x-S and Ag-F bonding as well as the more electronegative nature of the F content [36]. Due to the curing process at 100 °C and the chemical affinity of Ag to S, the presence of thiol group (SH) in the PFDT-Ag-Dex coating transformed to Ag_x-S/C-S and SO_x bonding [37], as it is depicted in Fig. 3e. XPS high-scan spectrum observation of F 1s peak at B.E. of 688.96 eV substantiates the successful deposition of the PFDT coating, illustrated in Fig. 3f. Additionally, the variation in the element content is provided in Table S2.2, which describes the purity of the LBL deposition. Overall, the combined ATR-FTIR and XPS results suggest successful the LBL deposition of the PFDT polymer, Ag NPs and TA-APTES coatings on the Si catheter.

Furthermore, the coating formulation was systematically optimized based on the TA-APTES formation time and the amount of silver loading to achieve a uniform coating with an appropriate silver content (Figure S2.3 and Table S2.3.1).

2.3. Surface wettability

The water contact angle (WCA) of the LBL-coated and Bare Si catheters was measured to understand their hydrophilic/hydrophobic properties, as shown in Fig. 4a. The TA-APTES-coated Si catheter exhibited a WCA value of 104.90 ± 1.40°, whereas the values were increased to 117.60 ± 2.10° and 132.0 ± 4.30° for the Ag-Dex- and PFDT-Ag-Dex-coated Si catheters, respectively. These results indicate that the latter coating demonstrated the hydrophobic behavior by reducing the surface tension of the surface due to the presence of perfluoroalkyl chains in the PFDT oligomer [16,38]. Additionally, the formation of a nano/micro hierarchical structure of TA-APTES NPs is conducive to the hydrophobic properties. The hydrophobic surface of urinary catheter device surface could impact the bacterial adhesion and may enhance the biofilm reduction efficacy. Earlier works have reported that the generation of a nano/micro hierarchical structure of polydopamine NPs integrated with Ag NPs and a PFDT polymer is beneficial for the anti-biofilm (against E. coli and P. mirabilis) and anti-fouling (against Phaeodactylum tricornutum) properties [16,34,38]. Furthermore, the stability of the hydrophobic coating properties was verified over 10 days, illustrated in Fig. 4b. A slight reduction in the WCA value of about 4 ± 2.10° was observed after 10 days; however, the PFDT-Ag-Dex-coated catheter maintained its hydrophobic properties. This indicates stable performance, which could favour the non-adhesion of bacteria to its surface.

Fig. 4.

(a) Water contact angle measurements of the LBL-coated SCs and (b) PFDT-Ag-Dex SC demonstrate their hydrophobic behavior and stability of their hydrophobicity, respectively. Bacterial growth curves of (c) S. aureus and (d) E. coli treated with Ag-Dex NPs in suspension. (e) The dissolution behavior of Ag⁺ ions of Ag-Dex- and PFDT-Dex- coated UCs in a simulated urine with pH = 7.0 at 37 °C, as measured by ICP-OES method. (f) Cytotoxicity performance of the LBL-coated UCs using MTT assay in the presence of L929 cells (For the Independent t-test, the data are shown as mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.).

2.4. Antibacterial activity by growth behavior and inhibition zone

To examine the antimicrobial performance, the synthesized Ag-Dex NPs were tested using a Gram-positive (G + ve) and Gram-negative (G−ve) models. The effect on bacterial growth inhibition behavior was monitored for 24 h and the data are displayed in Fig. 4c and d. The Ag-Dex NPs exhibited a minimum inhibitory concentration (MIC) of 25 μg mL⁻¹ for S. aureus and 50 μg mL⁻¹ for E. coli. In case both cases, the OD values were slowly increased to over 0.8 for 5–10 μg mL⁻¹ in 18 h and under ∼0.12 for 25–50 μg mL⁻¹ in 24 h. Further, the corresponding suspensions at 15–50 μg mL⁻¹ were used for plate colony counting as illustrated in Fig. S2.4a. Results demonstrate that the prepared NPs were relatively more effective against S. aureus than E. coli. This may be due to differences in cell structure and chemical composition of the cell walls. However, the previous studies have reported that Ag NPs are more sensitive to G-ve bacteria than to G +ve bacteria. G +ve and G-ve bacterial cell walls are composed of a thick LPS (Lipopolysaccharide) and thin peptidoglycan layers, and thin LPS and thin peptidoglycan layers, respectively. Due to the presence of a thick peptidoglycan layer, the Ag NP penetration into the cell is reduced. Additionally, variations in the physical properties of Ag NPs, such as shape, size, surface charge and diffusion state, can alter their antibacterial performance [39]. In this study, the dextran coating on the Ag NPs may have affected their antibacterial activity. The zeta potential of the prepared Ag-Dex NPs was measured to be −15.2 ± 0.6 mV due to the presence of hydroxyl groups in the dextran molecules. A recent study showed that the capping agents (citric acid, polyvinyl pyrrolidone, and dextran) influence the stability and antibacterial performance of Ag NPs against methicillin-resistant S. aureus (MRSA) and E. coli [40].

The inhibition zone method was used to evaluate the bactericidal effect of the developed LBL coatings (Ag-Dex and PFDT-Ag-Dex) against S. aureus and E. coli, as shown in Fig. S2.4b,c. Transparent zones indicate the bactericidal effect of the coatings [41]. The inhibition zones increased for the Ag-Dex- and PFDT-Ag-Dex- coated Si catheters, meanwhile, Bare-Si catheter (control) did not exhibit the inhibition zones. In addition, the time profile of bacterial growth, both the coated and uncoated Si UCs against S. aureus and E. coli, showed a complete reduction in bacterial growth due to the bactericidal effect of Ag NPs (shown in Fig.S2.4d-e). The bactericidal effect observed against both G +ve and G-ve bacterial strains is attributed to the slow release of Ag⁺ ions, which inhibits the bacteria by damaging their cell membranes. The ICP-OES method was used to evaluate the controlled release of Ag⁺ ions for 10 days and the data are depicted in Fig. 4e. These demonstrates that the developed LBL coatings (Ag-Dex and PFDT-Ag-Dex) possess excellent antibacterial properties, verifying their applicability in combating the biofilm formation on urinary devices.

Additionally, the stability of the PFDT-Ag-Dex coating was studied using the surface morphological changes and variations in Ag content using FESEM analysis with elemental mapping and EDS spectrum, and the antibacterial performance over time using inhibition zone and plating assays. The results are presented in Fig. S2.5(a-c) followed by a discussion. Collectively, the results demonstrate that the PFDT-Ag-Dex coating maintains morphological and compositional, and antibacterial stability under simulated urine conditions, confirming its potential for long-term performance in urinary catheter applications.

2.5. Cytotoxicity assay

It is crucial to ascertain the biocompatibility of the coated Si catheters before in vivo application [14,42]. The MTT assay was performed to examine the cytotoxicity of the developed LBL coatings using a fibroblast cell line, L929 cells for three and five days, as illustrated in Fig. 4f. The L929 cell viability was determined to be above 93 % for Bare Si after three and five days, whereas viability was decreased to above 80 % for the TA-APTES-coated Si catheter. The biocompatibility of the TA-Si NPs (derived from TA-APTES) and TA-APTES coatings for blood catheter was also confirmed in previous studies [14,[42], [43], [44]]. A slight reduction in cell viability could be due to the APTES molecules. For Ag-Dex coated Si catheter over three and five days as well as for PFDT-Ag-Dex-coated Si catheters after 3 days, cell viability was observed to be ∼50–55 %. A decrease in cell viability occurred due to impaired cellular activity resulting from the interactions between intracellular components and dissolved Ag⁺ ions. The Ag-Dex coating exhibited some toxicity. However, the PFDT-Ag-Dex coating showed a marked recovery in cell viability over time, reaching above 70 % and 83 % after five and seven days of incubation, respectively. This improvement suggests enhanced surface stability due to the fluorinated (PFDT) layer. The live/dead assay results obtained by FDA-PI staining (Fig.S2.6) corroborate the MTT results. In this assay, the green fluorescence (FDA-stained) indicates the cells are alive, whereas the red fluorescence (PI-stained) denotes dead cells. Representative fluorescent images of the PFDT-Dex coating show that, after three days of incubation, the number of live (green) cells are increased, while, the dead (red) cells decreased, demonstrating an improvement in biocompatibility over time. Previous cytotoxicity assessments of silver NPs and their coated samples with mammalian cells (e.g., L929 fibroblasts, RAW 264.7 macrophages, and human peripheral blood mononuclear cells) have shown that cell viability more than 70–75 % is considered non-toxic or within negligible risk region (ISO 10993–1:2018 standard) [[45], [46], [47]]. These results suggest that the PFDT-Ag-hydrophobic coating exhibits a negligible toxicity, favouring its applicability in biomedical devices.

2.6. Biofilm studies

To validate the biofilm inhibition activity of the PFDT-Ag-Dex coated Si catheter, biofilm studies were performed against E. coli using a dynamic flow biofilm formation model. Most clinical and antibiofilm coating studies have identified E. coli (28.1 %) as most frequently occurring biofilm-producing bacterium in the urinary catheter infections, followed by other bacteria, including Candida sp. (17.8 %), klebsiella pneumoniae (15.9 %) and Enterococcus faecalis (13.1 %)) [43,44]. Therefore, E. coli was chosen for the biofilm evaluation in this study. Fig. 5a shows the digital photographs of the commercial and modified Si urinary catheters, including TA-APTES-, Ag-Dex-, and PFDT-Ag-Dex-coated samples. The distinct color change observed in the TA-APTES-modified catheter, compared to the commercial catheter, confirms the successful deposition of the TA-APTES layer. Similarly, the characteristic brown color of the Ag-Dex- and PFDT-Ag-Dex-coated catheters indicates the successful Ag-Dex NPs incorporation. Inset images in Fig. 5a further reveal uniform coating coverage, particularly in the inner lumen of the catheter, suggesting effective surface modification.

Fig. 5.

Photographs of (a) uncoated and LBL-coated SCs, as well as (b) an experimental setup used for the biofilm studies under dynamic flow conditions at 37 °C. In vitro antibacterial performance of the (c) TA-APTES-, (d) Ag-Dex-, and (e) PFDT-Ag-Dex-coated SCs compared with uncoated UCs, as determined by plate colony counts against E. coli under dynamic growth conditions for four days at 37 °C, (f and g) the corresponding (f) CFU values and (g) bacterial reduction efficiency. (h–k) FESEM images showing the biofilm adhesion activity on the uncoated and LBL-coated UCs. For the Independent t-test, the data are shown as mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

The antibiofilm properties of the LBL-coated catheters were evaluated using a dynamic biofilm formation model for four days (Fig. 5b). At designated time intervals (days 1, 3, and 4), bacterial adhesion on both uncoated and coated catheters was estimated using the plate colony counts method. Representative images of the bacterial colonies grown from extracted catheter samples are shown in Fig. 5c–e. After four days of incubation, substantial bacterial colonization was observed on the TA-APTES-coated catheter, though it was still markedly lower than that of the Bare Si catheter. In contrast, both the Ag-Dex- and PFDT-Ag-Dex-coated catheters demonstrated significantly reduced bacterial attachment. Quantitative analysis of colony-forming unit (CFU), as shown in Fig. 5f and g, further substantiates these observations. Specifically, the PFDT-Ag-Dex coating yielded the lowest bacterial counts, with an average CFU of ∼2.40 × 10⁴ CFU cm⁻². The values calculated for the Ag-Dex- and TA-APTES-coated, and uncoated Si catheters were substantially higher: ∼1.05 × 10⁵, ∼1.16 × 10⁶, and ∼3.28 × 10⁶ CFU cm⁻², respectively. In terms of antibacterial performance, the Ag-Dex- and PFDT-Ag-Dex- coatings demonstrated inhibition efficiencies exceeding 96 %, compared to the Bare Si catheter. The TA-APTES-coated catheter, while less effective, still achieved a reduction of approximately 43 % in bacterial adhesion. The moderate antibacterial effect observed for the TA-APTES surface can be attributed to its nano/micro-structured topography with hydrophobic characteristics, and tannic acid. These features likely contribute to partial resistance against bacterial colonization.

Moreover, the antibacterial mechanism of biofilm inhibition by the LBL-coated Si catheter was evaluated using FESEM and a live/dead assay. Fig. 5h–k shows the FESEM images of Bare and TA-APTES-, Ag-Dex- and PFDT-Ag-Dex-coated Si catheters after the biofilm studies were conducted for 4 days. A complete E. coli colonization was observed on the Bare Si catheter, indicating a lack of antibiofilm characteristics. The TA-APTES-coated Si catheter was also seen with biofilm formation; however, it was lower than that of counterparts. Interestingly, the Ag-Dex- and PFDT-Ag-Dex- coated Si catheters were exhibited no bacterial adhesion, demonstrating excellent anti biofilm properties. Additionally, the coated remained well-anchored even after the biofilm studies, supporting their stability. The quantitative analysis of the bacterial colony attachment, as counted from the FESEM images, is shown in Fig. S2.7e. The bacterial inhibition efficiency was estimated to be 35 % for the TA-APTES-coated Si catheter and over 96 % for the Ag-Dex- and PFDT-Ag-Dex-coated Si catheters. Representative live/dead results after the biofilm studies were carried out for 4 days are depicted in Fig. S2.7a-d. The images show more live bacteria and fewer dead bacteria in the Bare Si and TA-APTES coated Si catheters. In contrast, only a few bacterial cells were observed in the Ag-Dex- and PFDT-Ag-Dex-coated Si catheters, revealing a remarkably high number of dead bacteria. The corresponding quantitative analysis of live and dead bacteria is shown in Fig. S2.7f. The excellent antibiofilm performance observed in the Ag-Dex- and PFDT-Ag-Dex-coated Si catheters is mainly due to the release of silver ions from the coatings and their hydrophobic properties. Combinedly, the numerous hydroxyl groups in tannic acid and Ag ⁺ ions can interact the with cell structure of E. coli, resulting in membrane disruption and the release of cellular components. Additionally, the positively charged amino groups in the TA-APTES NPs can interact electrostatically with the negatively charged outer membrane of E. coli [42]. However, the Ag-Dex coated Si catheter lacks potential for the real-time applications due to their cytotoxic properties. A previous study also reported a reduction in the cytotoxicity of Ag NPs incorporated into a polydopamine coated Si catheter [16].

2.7. In vivo studies in subcutaneous mouse model

2.7.1. Antibacterial performance

The antibacterial efficacy of the LBL-coated urinary catheters (UCs) against E. coli infection was assessed using a subcutaneous mouse model. Quantitative bacterial counts obtained from agar plate cultures are presented in Fig. 6a. A comparative analysis was performed for both coated and uncoated catheters, with and without bacterial infection. All catheter samples implanted in mice without bacterial infection showed no signs of infection, confirming that the animal models were infection-free initially. Catheters coated only with TA-APTES, as well as commercial UC, demonstrated limited antibacterial activity, as evidenced by the high number of bacterial colonies present. In contrast, a significant reduction in bacterial colonies was observed for the Ag-Dex and PFDT-Ag-Dex coated catheters. The Ag-Dex coated UC achieved an inhibition efficiency of approximately 81 ± 6.3 % (3.2 × 10⁴ ± 6.45 × 10³ CFU/0.5 cm²), while the PFDT-Ag-Dex UC exhibited even higher efficacy, with around 90 ± 5.1 % inhibition (8.0 × 10³ ± 2.01 × 10³ CFU/0.5 cm²), as illustrated in Fig. 6b and c. The relatively modest antibacterial effect observed with the TA-APTES coating (∼25.7 ± 4.9 % inhibition, 2.08 × 10⁵ ± 7.59 × 10³ CFU/0.5 cm²) was attributed to the synergistic contributions of tannic acid and the hydrophobic surface [48]. The PFDT-Ag-Dex coating demonstrated superior antibacterial performance, which can be primarily due to the sustained release of Ag⁺ ions that damage the bacterial cell membranes. The ICP-OES results (Fig. 4e) confirmed the slow-release profile of Ag⁺ ions. These observations are similar with the earlier reports on Ag NP-functionalized silicone catheters that exhibited prolonged ion release [41,49]. Furthermore, the PFDT top layer, which has a hierarchical nano/microstructure of TA-APTES, imparted a slippery surface that significantly impeded bacterial adhesion [16,48]. A visual inspection of the explanted (+) groups (Fig. 6d) revealed clear differences in surface integrity and infection at the implantation sites after seven days. The sutured areas were visible and the PFDT-Ag-Dex coating maintained its appearance.

Fig. 6.

In vivo studies performed with a subcutaneous mouse model: antibacterial activity of (a) the uncoated and LBL-coated UCs with (+) and without (−) infections as assessed by plate colony counts against E. coli, the corresponding (b) bacterial inhibition efficiency and (c) CFU values. (d) Digital photographs of mice implanted with uncoated and LBL-coated UCs (inset images show the extracted UCs) captured after seven days and the respective (e) body weight changes. For the Independent t-test, the data are shown as mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Mice health was also monitored by tracking changes in body weight of (±) control and PFDT-Ag-Dex UC groups over the seven-day period (Fig. 6e). A slight weight loss was noted in all groups during the first three days, likely due to stress and reduced appetite after surgery. However, weight gain resumed subsequently, indicating a recovery trend. Notably, mice implanted with PFDT-Ag-Dex-coated UCs under infectious conditions exhibited no adverse health effects, underscoring the coating's therapeutic efficacy.

2.7.2. Biosafety evaluation

To evaluate the biosafety of the (±) control and PFDT-Ag-Dex UC groups, the inflammatory response was examined in skin tissue under the implant sites using the H&E staining. Representative results are displayed in Fig. 7a. The H&E-stained images of other (±) groups (TA-APTES and Ag-Dex-coated UCs) are presented in Fig. S2.8. Nearby bacterial infection or wound sites exhibits infiltration of immune cells, such as, neutrophils (a type of white blood cell) from the circulating blood infiltrates into soft tissues in response to the foreign species or particles entering the body, indicating inflammation [[49], [50], [51]]. Changes in the subcutaneous tissue length and neutrophil quantification in the (±) groups are shown in Fig. 7b and c. Relatively, a high subcutaneous tissue lengths with many neutrophil infiltrations were detected in the control groups (± uncoated groups) and the TA-APTES coated UC (Fig. S2.8). Conversely, the Ag-Dex (Fig. S2.8) and PFDT-Ag-Dex-coated UCs (±groups) exhibited minimal subcutaneous tissue length and neutrophil infiltration, validating their biocompatibility. However, the PFDT-Ag-Dex coated catheter (+ group) performed better than the Ag-Dex-coated catheter. Notably, there were no considerable pathological differences in the H&E-stained images of the extracted organs (heart, kidney, lung, liver and spleen) of the controls and coated UCs after 7 days, as illustrated in Fig. 7d. For comparison, H&E-stained images of the extracted organs from TA-APTES- and Ag-Dex- coated UCs are displayed in Fig. S2.8, no differences, which implies that no leaching occurred from the coatings.

Fig. 7.

Histological results from the mouse subcutaneous model include (a) representative H&E-stained skin tissue images surrounding the control and PFDT-Ag-Dex coated urinary catheter implantation sites (±), (b) tissue lengths (ST = subcutaneous tissue), and (c) neutrophil infiltration score seven days post-implantation (red circles indicate neutrophils). (d) H&E-stained tissues of vital organs, including heart, kidney, lung, liver, and spleen in each group. For the Independent t-test, the data are shown as mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.8. In vivo studies in rabbit urinary tract model-antibacterial and biosafety performance

To further validate the antibacterial activity and biosafety of the PFDT-Ag-Dex coated UC (+), a rabbit urinary tract infection model was used [6]. The rabbits were implanted with UCs monitored for five days and urine samples were collected daily. The antibacterial activity of the urine samples collected from the PFDT-Ag-Dex coated UC (+) and control (+) groups was compared using the plate colony counts and the results are displayed in Fig. 8a. An increasing trend in the bacterial colonies was observed in the control UC (+) over five days. In contrast, the bacterial colonies substantially decreased over time in the PFDT-Ag-Dex coated UC (+) group. The corresponding estimated CFU values are shown in Fig. 8b. Similarly, the antibacterial activity of the bladder and urethral regions of the extracted UCs was tested using plate colony counts, as shown in Fig. 8a. Many colonies were observed in all tested portions of the control group. Conversely, a significant reduction in the bacterial colonies was seen in all portions tested from the PFDT-Ag-Dex coated UC (+) group. However, a partial crystalline deposition was noticed in the few urethral regions after extraction in the PFDT-Ag-Dex coated UC. Earlier studies have reported that the urine with an alkaline pH condition favours the crystal formation in the wall of urethral regions [52,53]. On day five, the measured pH value of the urine sample was around 8.5–9.0. The crystal deposition occurs due to the formation of calcium- and magnesium-based phosphates and calcium oxalate [52,53]. Here, the crystal deposition could have occurred for several reasons such as, the urine flowing through a long pathway (15 cm from the bladder neck), an upward flow during urination (the urinary shaft was attached to the leg of the rabbit outward to avoid the catheter removal) and urease-generating bacteria (Proteus mirabilis, Pseudomonas aeruginosa and Corynebacterium). Crystal deposition may rub the urethral mucosa during UC explantation, leading to pain and hematuria [54]. Further research is needed to address these issues for clinical application. Although, the combined antibacterial evaluation of the urine samples infected with E. coli and the explanted UCs suggests that the hydrophobic PFDT-Ag-Dex coating has the potential to reduce the biofilm formation on urinary catheters in UTIs.

Fig. 8.

In vivo study results of the control and PFDT-Ag-Dex coated UCs (8 Fr) in a rabbit model for five days: (a) antibacterial activity of urine samples over time, and the extracted UCs in the bladder and urethral regions, evaluated by plate colony counts against E. coli and (b) their corresponding CFU values. (c) images of the extracted control and PFDT-Ag-Dex coated UCs after five days of catheterization, (d) gross images of the urinary tract catheterized by the control and PFDT-Ag-Dex coated UCs for five days (the black dashed line indicates the bladder neck) and (e) H&E-stained histological images of the bladder and urethral tissues catheterized by the control and PFDT-Ag-Dex coated UCs for five days. For the Independent t-test, the data are presented as mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 8c and d shows the extracted UCs and the urinary tract of the rabbits after 5 days. In Fig. 8d, the surface of urinary tract catheterized with uncoated catheter shows redness compared to the coated catheter. Representative H&E-stained images of the urinary bladder and urethral tissues from the respective groups are presented in Fig. 8e. In the uncoated group (control group, +), congestion was observed in the submucosa and tunica muscularis of the urinary tract, accompanied by lymphocytic infiltration in the mucosal layer. Both congestion and lymphocytic infiltration are indicative of an inflammatory response. Bacterial infection could have caused the infiltration of inflammatory cells [53]. Importantly, the PFDT-Ag-Dex-coated UC (+) group showed drastically reduced infiltration, revealing an insignificant sign of inflammation (Fig. 8e). Thus, the results of the rabbit urinary tract model demonstrate that the developed PFDT-Ag-Dex coating is non-toxic and confirms its biocompatibility performance while inhibiting biofilm formation.

3. Conclusion

A new coating formulation, PFDT-Ag-Dex layer, with antibacterial and hydrophobic properties to prevent the biofilm formation was successfully fabricated on a silicone urinary catheter using a layer-by-layer assembly through hierarchical nano-micro structures of TA-APTES. In antibacterial studies, the Ag-Dex NPs and their hydrophobic coating were found to be effectively inhibit S. aureus and E. coli. Notably, the LBL silver-hydrophobic coating exhibited ∼90 % efficacy in inhibiting E. coli in in vitro dynamic flow biofilm studies due to the sustained release of Ag ⁺ ions. The biosafety and antibacterial properties of the coated urinary catheter were confirmed using an in vivo mouse subcutaneous model. A silver-hydrophobic coated catheter implanted in a rabbit model also demonstrated notable antibacterial activity, and its biosafety was evaluated in the urethra. This study revealed that a silver-hydrophobic coating on urinary catheters can effectively prevent the urinary tract infections. However, further work is needed to assess its in vivo stability, its antimicrobial durability, and the effectiveness against other UTI pathogens, such as, proteus mirabilis and Citrobacter species, as well as fungi. Additionally, molecular biology studies are required to gain mechanistic insight, and the feasibility of large-scale production deserves further consideration.

4. Material and methods

4.1. In vitro studies: Coating preparation, characterizations, biosafety, and biofilm studies

The details of the material section and fabrication of the PFDT-Ag-Dex coated urinary catheter, as well as its antibacterial and cytotoxicity studies, are given in the supplementary information (S1). The biofilm study was performed using a dynamic flow method against E. coli (pure culture, strain KCTC-2571, Jeollabuk-do, Republic of Korea) and the setup is shown in Fig. 5b. Briefly, it consists of the peristatic pumps (P1 (pumped at 1.5 mL min⁻¹) and P2 (pumped at 50 mL min⁻¹)), a water bath containing 10 L of sterile water, an air supply (to prevent the backflow of the medium), a 500 mL Erlenmeyer flask to collect the used TSB medium, uncoated (sterilized, 7 mm and 10 mm-inner and outer diameter, respectively) and coated (3.5 mm and 6 mm-inner and outer diameter, respectively) Si tubes and 3-way T-tube connecters. The entire assembly was placed in a laminar flow cabinet to prevent the airborne pathogen contamination. Initially, the P1 was used to fill the bacterial culture medium. The continuous flow of medium into the Si tubes (3.5 mm and 6 mm-inner and outer diameter, respectively) was controlled by P2 and incubated at 37 °C in the water bath. Biofilm formation was carried out for 4 days and each day, the circulating TSB medium was replaced with the fresh medium. On each day, a ∼50 mL of fresh culture medium was used and inoculated with E. coli suspension (1 mL, ∼10⁶ CFU mL⁻¹, OD₆₀₀ = 0.57) on the initial day. On each day, a ∼3 cm length of coated and uncoated SC was cut to assess the bacterial adhesion using colony counting method. This setup was adopted to form the biofilm in vitro on the Si tube surface to mimic the clinically formed E. coli biofilm on the Si catheter surface, however, the experimental conditions may differ from the previous works [50,[55], [56], [57]]. The adherent bacteria on both the samples were ultrasonically detached in 1x PBS solution (2 mL per sample) in 5 min and quantified by the plate colony count method. The bacterial suspension of E. coli (5 μL) from the PBS solution was plated on the EMB agar and incubated for one day at 37 °C. The antibacterial efficiency was estimated using a previously reported method [31]. Furthermore, the bactericidal effects of the prepared coatings were assessed by FESEM analysis and live/dead assay (S1).

4.2. In vivo studies of the Ag-hydrophobic coated UCs in a mouse model

The in vivo studies were conducted to assess the antibacterial efficacy and biosafety of the prepared PFDT-Ag-Dex SC using a mouse subcutaneous infection model according to the ASTM standard protocol (F1408-97). The animal study was performed as per the national ethical guidelines at PKNU (PKNUIACUC-2024-08). Female BALB/C mice (Hana Biotech, Pyengtaek, Republic of Korea) with ∼8–10 weeks old and ∼20 g body weight, were used for the validation and acclimated for one week preceding the studies. During the study, the mice were provided with food and water ad libitum and were monitored for 7 days. The animal studies were divided into two main groups with (+) and without (−) E. coli infection. Each group was subdivided into four different groups (n = 3), such as (1) commercial UC (+/−, controls), (2) TA-APTES UC (±), (3) Ag-Dex UC (±) and PFDT-Ag-Dex UC (±). Mice were anesthetized with isoflurane (Terrell™, Piramal Critical Care, USA) along with the supply of oxygen at 0.8 L min⁻¹. Their flanks were then shaved and cleaned with 70 % isopropyl alcohol medical wipes. After the incision (∼0.7 cm) was made along the spine of the mice (close to the subcutaneous tissue), the uncoated and coated UC (∼0.5 cm length, 8Fr/Ch, 3–5 mL/cc, Boho, China) with (+) and without (−) E. coli infection were separately inserted and sutured. Infection was induced by spreading 10 μL of E. coli suspension on the surface of UCs (+groups) and kept for incubation at 37 °C for 2 h. The body weights of the animals were monitored at 0–7 days after implantation. The animals were then euthanized and the coated and uncoated UCs with (+) and without (−) infection were collected. They were ultrasonicated to detach the adherent bacteria in 1 mL of 1xPBS solution, colonized on EMB agar and incubated at 37 °C for 24 h to determine the antibacterial activity. The biosafety analysis was evaluated using the H & E staining method (S1).

CRediT authorship contribution statement

Sivakumar Bose: Writing – original draft, Validation, Methodology, Investigation, Data curation, Conceptualization. Myungji Kang: Validation, Methodology, Investigation, Formal analysis, Data curation. Seonho Jung: Validation, Methodology, Investigation, Formal analysis, Data curation. Mijeong Kim: Methodology, Investigation, Data curation. Chanwoo Yoon: Methodology, Investigation, Data curation. Priya Ranganathan: Methodology, Data curation. Seung Yun Nam: Visualization, Resources, Formal analysis. Hyun Wook Kang: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

Data will be made available on request.