Benzalkonium Chloride-Loaded p(HEMA) vs. p(HEMA-co-MA) Hydrogels: Enhancing Antimicrobial and Antibiofilm Efficacy Through Maleic Anhydride Functionalization

TL;DR

This study develops hydrogel coatings for catheters that effectively prevent biofilm formation and microbial adhesion, reducing the risk of urinary tract infections.

Contribution

The novel contribution is the functionalization of p(HEMA) hydrogels with maleic anhydride to enhance antimicrobial and antibiofilm efficacy when loaded with benzalkonium chloride.

Findings

BAC-loaded hydrogels showed lasting antimicrobial activity for up to 8 days.

MA-functionalized hydrogels reduced biofilm development by over 85%.

SEM and gene-expression studies confirmed reduced microbial adhesion and repression of virulence genes.

Abstract

Catheter-associated urinary tract infections are often caused by biofilm formation on device surfaces. This paper presents an antimicrobial catheter-coating hydrogel comprising p(HEMA) and carboxyl-functionalized p(HEMA-co-MA), loaded with benzalkonium chloride (BAC) to increase hydrophilicity, pH responsiveness, and antibiofilm activity. Hydrogels were prepared by free-radical polymerization, loaded with BAC via swelling, and their physicochemical properties were characterized. Furthermore, microbiological assessment focused on the detection of MIC/MBC/MFC, disk diffusion, biofilm assays, SEM imaging, and RT-qPCR sequencing were used to determine the impact on biofilm-related gene expression to evaluate antimicrobial activity against major catheter-associated urinary tract infection (CAUTI)-associated pathogens and identify the higher BAC loading p(HEMA) and enhanced hydrophilicity and pH-responsive swelling (p(HEMA-co-MA)). The two hydrogels exhibited a wide range of antimicrobial activity and provided lasting inhibition for up to 8 days. It is worth noting that the MA-functionalized hydrogel exhibited a high intrinsic antifouling property, and biofilm development was reduced by more than 85% in BAC-loaded formulations. SEM and gene-expression studies showed reduced microbial adhesion and substantial repression of virulence and biofilm-associated genes. In summary, BAC-loaded p(HEMA) and p(HEMA-co-MA) coatings exhibit strong antimicrobial and antiadhesive properties, and the incorporation of MA results in more effective biofilm suppression, which supports their future use as advanced catheter coatings to prevent the development of device-related infections.

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Full text

1. Introduction

Approximately 75 percent of urinary tract infections (UTIs) acquired in hospitals are catheter-associated urinary tract infections (CAUTIs), and 15–25 percent of patients admitted to hospitals who receive urinary catheterization experience CAUTIs. Urinary catheters create a direct pathway to the bladder, facilitating microbial ascent and infection. They circumvent urethral sphincter defenses, diminish the flushing effect of normal voiding, and provide a surface for microbial growth, thereby increasing the risk of UTIs. Additionally, catheter-induced uroepithelial irritation and disruption of the mucopolysaccharide coating enhance bacterial adhesion and [1]. The outcomes of these infections are enormous medical costs (more than 451 million annually in the United States alone) and extended hospitalization, causing higher morbidity and mortality rates. The development of bacterial biofilms on the catheter surfaces, which protect pathogens against antibiotics and host immune systems and lead to an increase in antimicrobial resistance by 10–1000-fold, is one of the major barriers to the effective treatment of CAUTIs [2].

The formation of biofilms on the surface of urinary catheters follows a reported process: initial microbial adhesion, growth of microcolonies, development of structured biofilms, and, finally, dispersal [3]. This is mediated by quorum-sensing systems, which coordinate the expression of virulence factors and biofilm architecture through specificene networks [3]. All the most frequent CAUTI pathogenic agents [4], such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, each have a distinct genetic mechanism of biofilm formation [3], which also contributes to making these infections difficult to remove [5].

Poly(2-hydroxyethyl methacrylate) p(HEMA) has become a popular coating polymer owing to its biocompatibility, hydrophilicity and anti-protein adsorption property [5]. Nevertheless, biofilm formation might not be fully inhibited by homopolymeric p(HEMA). The introduction of carboxyl functional groups is one of the methods proposed to achieve improved antifouling properties through the inclusion of an ionic co-monomer. Maleic anhydride (MA) is an unsaturated dicarboxylic acid that can be copolymerized with HEMA; upon hydrolysis in water, MA provides maleic acid units containing carboxylate groups. Introduction of MA at even small percentages can significantly enhance hydrogel hydrophilicity and endow pH-responsive swelling behavior, further preventing bacterial adhesion by creating a hydrated, anionic surface [6,7]. Recent studies have demonstrated that copolymerization of HEMA with MA (or other related acid monomers) results in reduced biofilm formation and excellent biocompatibility [7,8]. For instance, Abu Mahfouz et al. reported the development of a HEMA-MA copolymer acting as an antifouling and anti-inflammatory coating, in which the benefit of carboxyl functionalization was demonstrated in preventing microbial adhesion [9,10]. Similarly, monomers containing maleic acid are known to make polymers stimuli-responsive, such as through swelling at high pH due to the ionization of carboxyl groups [11]. In urinary catheters, this pH-sensitive behavior is advantageous: Proteus and other urease-producing bacteria can raise urine pH to ~8–9, but a hydrogel that swells more at alkaline pH could counteract biofilm formation by promoting more drug release or expansion that sloughs off cells [12].

In addition to passive antifouling strategies, active antimicrobial agents can be incorporated into coatings to kill planktonic and adhering microbes. The quaternary ammonium compound benzalkonium chloride (BAC) exhibits broad-spectrum antimicrobial activity against microorganisms. BAC disrupts bacterial cell membranes and can interfere with biofilm formation, partly by disrupting quorum-sensing signals [7]. As a cationic surfactant, BAC may also interact electrostatically with negatively charged surfaces (e.g., those bearing carboxyl groups), which could contribute to its retention and controlled release. Unlike traditional antibiotics, BAC is effective against both planktonic bacteria and biofilms, with less potential to induce resistance [13]. Due to these properties, BAC is considered an attractive agent for preventing CAUTIs. Indeed, surface coatings impregnated with BAC have reduced bacterial adherence and biofilm growth in other device contexts [14].

Compared to simple surface coating, incorporating an antimicrobial into a hydrogel matrix enables the sustained release of the agent at the site of catheterization. This can provide continuous prophylaxis against ascending microbes. Another contrast lies in [15]. In the same way, mucoadhesive polymer films [8], such as cellulose derivatives loaded with antifungals, have shown promise for localized infection treatment [7], whereas films may not have the required mechanical robustness or longevity for urinary catheter applications [16]. Herein, a copolymer of p(HEMA) and MA was grafted to obtain a surface capable of reducing microbial adhesion through microorganism repulsion. Moreover, the grafted copolymer was loaded with [17]. The synthesized hydrogel can be used as a coating layer, compressed onto the silicone catheter inner surface, or adhered using a suitable adhesive polymer. The grafted polymer was verified using FTIR [18].

In this work, our goal was to develop and characterize BAC-loaded p(HEMA) and p(HEMA-co-MA) hydrogel coatings for urinary catheters, focusing on the effects of the co-monomer MA (malic acid) on hydrogel properties and antimicrobial performance. We hypothesized that even the introduction of MA, which introduces carboxyl functionality, would: (1) enhance hydrogel swelling and hydrophilicity, improving antifouling behavior; and (2) modulate BAC loading and release by ionic interactions, ultimately leading to extended antimicrobial action. Accordingly, we designed the study to meet the following objectives: (1) synthesizing p(HEMA) and p(HEMA)-co-MA hydrogels and characterizing their physical properties (swelling, mechanical texture) and BAC loading capacity; (2) determining in vitro antimicrobial efficacy against CAUTI-related pathogens (S. aureus, E. coli, P. aeruginosa, P. mirabilis, and C. albicans) by MIC/MBC, MFC determination and zone of inhibition assays at different times; (3) quantifying the biofilm formation on coated surfaces compared with uncoated controls and visualizing the morphology of the biofilm via scanning electron microscopy; and (4) analyzing the transcription of selected biofilm-associated genes in representative bacteria to determine if the coatings suppress bacterial virulence at the molecular level. By demonstrating both anti-adhesive and antimicrobial effects—particularly attributable to the MA-induced modifications—this study offers an innovative dual-action coating strategy to reduce CAUTIs.

2. Materials and Methods

2.1. Materials

2-Hydroxyethyl methacrylate (2-HEMA, M_W 130.14) monomer, ethylene glycol dimethacrylate (EGDMA, MW 198.22) crosslinker, and 2,2′-azobis(2-methylpropionitrile) (AIBN, initiator) were obtained from Sigma-Aldrich (Munich, Germany). Maleic anhydride (MA) was purchased from Genochem (Valencia, Spain). Benzalkonium chloride (BAC; benzyl-dimethyl-tetradecyl-ammonium chloride) was obtained from TCI (Tokyo, Japan). All microbial strains were obtained from the American Type Culture Collection (ATCC): S. aureus (ATCC 6538), E. coli (ATCC 8739), P. aeruginosa (ATCC 9027), P. mirabilis (ATCC 43071), and C. albicans (ATCC 10231). Tryptic soy broth (TSB), Mueller-Hinton agar (MHA), and other culture media were from HiMedia (Mumbai, India). WST-8 cell viability reagent (CCK-8 assay kit) was from Sigma. RNA extraction kits and RT-qPCR reagents were purchased from Promega (Madison, WI, USA). All chemicals were of analytical or cell culture grade and used as received.

2.2. Synthesis of Hydrogels

2.2.1. p(HEMA) Hydrogel Preparation

P(HEMA) hydrogels were synthesized via free-radical solution polymerization. Briefly, 24.5 g of 2-HEMA monomer (98 wt%) was mixed with 0.25 g EGDMA (1 wt%, as a crosslinker) and 0.25 g AIBN (1 wt%, initiator) in a glass vial. The mixture was purged with N_2_ and stirred for 30 min at 20 °C, then injected into a mold consisting of two glass plates separated by a 0.3 mm silicone spacer. Polymerization was carried out in an oven at 90 °C for 3 h. The resulting hydrogel film (xerogel) was peeled off and cut into disks (diameter ~6 mm). To remove unreacted monomers, the disks were soaked in distilled water, with daily water replacements, until the wash water showed no absorbance at 220 nm by UV spectroscopy (confirming monomer extraction).

2.2.2. p(HEMA-co-MA) Hydrogel Preparation

The copolymer hydrogel p(HEMA-co-MA) was prepared following the same procedure as above, with maleic anhydride incorporated at 5 mol% of the total monomer content. Specifically, 23.3 g of 2-HEMA (93.5 wt%) and 1.3 g of MA (5 wt%) were co-polymerized with 0.25 g EGDMA and 0.25 g AIBN under identical conditions.

The concentrations of monomers and crosslinker (EGDMA) were selected based on previously established and optimized protocols for p(HEMA)-based hydrogel systems reported by our group [19,20,21,22]. The crosslinker concentration was maintained consistent with these prior formulations to ensure comparable network density and mechanical integrity. Regarding the MA co-monomer content, preliminary screening was conducted at 1%, 5%, and 10% (w/w) MA incorporation levels. Hydrogels containing 1% MA were found to be excessively brittle and difficult to handle, while those containing 10% MA were too soft and lacked sufficient mechanical robustness for further characterization and practical application. The 5% MA formulation demonstrated an optimal balance between mechanical performance (flexibility without fragility), adequate handling properties, and sufficient carboxyl group density to impart the desired hydrophilicity and pH-responsive behavior. This concentration was therefore selected for all subsequent BAC loading and antimicrobial evaluations. The influence of MA on the physical properties of the hydrogel, including enhanced swelling, increased hydrophilicity, and altered mechanical texture, is further detailed in Section 3.1 and Section 3.4. A recent study [18] further corroborated the benefits of HEMA-MA copolymerization for biomedical coating applications, demonstrating improved antifouling and anti-inflammatory properties at comparable co-monomer ratios. The p(HEMA-co-MA) films were similarly purified by extensive water soaking. The presence of MA in the copolymer was qualitatively confirmed by the increased hydrophilicity and by Fourier-transform infrared spectroscopy (appearance of carbonyl stretching from acid/anhydride groups). Both hydrogel types (p(HEMA) and p(HEMA)-co-MA) were stored hydrated in distilled water until use.

2.3. BAC Loading and Quantification

2.3.1. UV-Vis Analytical Method

BAC concentrations were determined by UV-Visible spectrophotometry (Specord-200 Plus, Analytik Jena, Jena, Germany). The maximum absorbance wavelength (λ_max) for BAC was found at 263 nm. A calibration curve was generated by measuring the absorbance of BAC standard solutions (25, 50, 100, 200, 400, 800 µg/mL in ethanol). The calibration was linear (R^2^ = 0.9995) over this range, with the regression equation: A = 0.1137 + 0.001 × C (where A is absorbance and C is BAC concentration in µg/mL). Method validation followed ICH Q2(R1) guidelines: the intra-day and inter-day precision (relative standard deviation) was <2%, and the limits of detection (LOD) and quantification (LOQ) were 7.6 µg/mL and 23 µg/mL, respectively. These parameters prove that this technique is sensitive and competent in BAC measurement.

2.3.2. Drug Loading Procedure

By swelling in equilibrium, BAC was loaded onto hydrogel disks (dry weight of about 100 mg/disk). Xerogels (dried disks) were placed in a saturated solution of BAC (100 mg/mL in ethanol) and incubated at 20 °C for 0 days [23]. This long incubation period gives BAC time to permeate the polymer network until equilibrium uptake is attained. Disks were loaded, briefly blotted, and weighed to achieve similar mass uptake. The depletion method was used to calculate the amount of BAC loaded (Qload): the concentration of BAC solution before (C0) and after (Ct) loading was measured (at 263 nm), and Qload = (C0 − Ct) × V (V change in the volume of the loading solution) [24,25,26]. Drug loading capacity (DLC) was taken as a percentage of the mass of polymer:

$$[eqn]$$

Each loading experiment was performed in quadruplicate (n = 4 disks per formulation). Loaded hydrogels were briefly rinsed in distilled water to remove surface-adhered BAC, then stored at 4 °C in sealed containers until testing.

2.4. Hydrogel Characterization

2.4.1. Swelling Behavior

The swelling capacity of the hydrogels was evaluated at two pH conditions representing normal and infected urine: pH 5.0 (acidic, approximating healthy urinary pH) and pH 9.0 (alkaline, as in P. mirabilis infection). Disks of dried hydrogel (n = 4 per formulation) were weighed (W_0_) and then submerged in 10 mL of a pH-adjusted buffer (0.1 M universal phosphate-citrate buffer) at 20 °C. At predetermined time points (15, 30, 60, 120, 180, and 1440 min), disks were removed, blotted to remove surface liquid, and weighed (Wt). The percentage swelling (%S) at time t was calculated as:

$$[eqn]$$

Swelling kinetics and equilibrium swelling (at 48 h) were compared between p(HEMA) and p(HEMA-co-MA), both in unloaded and BAC-loaded states. The influence of pH was assessed by performing parallel measurements at pH 5 and pH 9.

To characterize the mechanism of water transport into the hydrogels, the swelling kinetics data were analyzed using the Korsmeyer-Peppas power law model [27]:

$$[eqn]$$

where Mt is the mass of water absorbed at time t, M∞ is the equilibrium water uptake, k is the swelling rate constant characteristic of the polymer network, and n is the diffusional exponent indicative of the transport mechanism. For disk-shaped (cylindrical) hydrogel geometries, n ≤ 0.50 corresponds to Fickian (Case I) diffusion, 0.50 < n < 1.0 to anomalous (non-Fickian) transport, and n = 1.0 to Case II (relaxation-controlled) transport [28]. The exponent n was determined from the linear regression of log (Mt/M∞) versus log(t) for the initial portion of the swelling curve (Mt/M∞ ≤ 0.60), as recommended for the validity of the power law approximation [29]. Conclusion: All formulations yielded n ≤ 0.50, confirming Fickian (Case I) diffusion-controlled swelling. The higher k values for p(HEMA-co-MA) reflect its enhanced hydrophilicity, which accelerates water uptake without altering the transport mechanism.

2.4.2. Morphology and Mechanical Observations

Qualitative observations of hydrogel morphology and texture were noted. The homopolymer p(HEMA) disks appeared transparent and slightly rigid/rubbery to the touch, whereas the MA-containing copolymer disks were more opaque-white and noticeably softer and more flexible. This difference is attributed to the increased hydrophilicity and lower crosslink density of p(HEMA-co-MA), resulting in a plasticizing effect when hydrated. While formal mechanical testing (e.g., tensile strength) was not conducted in this study, handling the disks indicated that p(HEMA-co-MA) hydrogels could deform more readily without cracking, which may be advantageous for conformal coating on catheters. Both hydrogel types were easily cut into uniform discs and remained intact during swelling and sterilization (UV exposure).

2.5. Antimicrobial Assays

2.5.1. Minimum Inhibitory and Bactericidal Concentrations (MIC/MBC)

The antimicrobial potency of BAC was first quantified via standard broth microdilution assays, following Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacterial suspensions of S. aureus, E. coli, P. aeruginosa, and P. mirabilis were prepared in cation-adjusted Mueller-Hinton Broth to ~1 × 10^8^ CFU/mL (0.5 McFarland), and a C. albicans yeast suspension was prepared in MHA medium to ~1 × 10^6^ CFU/mL. In sterile 96-well plates, two-fold serial dilutions of BAC (ranging from 0.98 to 1000 µg/mL) were combined with an equal volume of microbial inoculum (final starting inoculum ~5 × 10^5^ CFU/mL for bacteria, 0.5 × 10^5^ for Candida). After incubation for 24 h at 37 °C, the MIC was defined as the lowest BAC concentration that completely inhibited visible growth (turbidity). To determine the minimum bactericidal concentration (MBC) or fungicidal concentration (MFC), 20 µL from each clear well (no growth) was spot-plated onto agar and incubated for an additional 24–48 h. The MBC/MFC was the lowest concentration, yielding no colony growth on plates (indicating ≥99.9% killing). All tests were performed in triplicate.

2.5.2. Disk Diffusion Assay (Zone of Inhibition)

The ability of hydrogel coatings to diffuse BAC and inhibit microbial growth on solid media was evaluated using a Kirby-Bauer disk diffusion test. Hydrogel disks (diameter ~5.8 mm, thickness ~0.9 mm) of each formulation—p(HEMA), p(HEMA-co-MA), p(HEMA) + BAC, and p(HEMA-co-MA) + BAC—were sterilized by UV irradiation (15 min each side) and placed on MHA plates freshly inoculated with standardized bacterial suspensions (0.5 McFarland in saline, evenly swabbed onto the agar surface). For C. albicans, MHA supplemented with 2% glucose was used. Plates were incubated at 37 °C, and the zone of inhibition (ZOI) around each disk was measured after 24 h. To assess sustained release, the disks were transferred daily onto fresh inoculated agar plates (with the same organism) for up to 8 days; zones were measured at each 24 h interval. Unloaded hydrogel disks served as negative controls (expected to show no inhibition). All diffusion tests were done in quadruplicate. ZOI diameters are reported as the mean ± standard deviation (SD).

2.6. Biofilm Formation Assay

A quantitative biofilm inhibition assay was performed using a modified WST-8/CCK-8 metabolic assay. Sterile hydrogel disks (as above) were placed into 96-well flat-bottom tissue culture plates (one disk per well). Each disk was incubated with 200 µL of bacterial suspension (~1 × 10^7^ CFU/mL in TSB) or C. albicans suspension (~1 × 10^5^ in yeast peptone dextrose medium). Plates were first incubated for 2 h at 37 °C to allow initial cell attachment (adhesion phase). Non-adherent (planktonic) cells were then gently removed, and each well was washed with phosphate-buffered saline (PBS). Next, 200 µL of fresh sterile growth medium was added to each well, and the plate was incubated for 24 h at 37 °C to permit biofilm formation on the hydrogel surface. After 24 h, each well was again gently rinsed with PBS to remove loose planktonic cells. To quantify biofilm biomass, 10% (v/v) of a WST-8 reagent was added to each well and incubated for 2–4 h in the dark. WST-8 (a tetrazolium salt) is reduced by metabolically active cells into a soluble formazan dye; the absorbance at 450 nm is proportional to viable biofilm cell density. The absorbance of the solution from each well was measured using a microplate reader. The percentage of biofilm formation on each sample was calculated relative to a positive control (an uncoated p(HEMA) sample), which was set at 100% biofilm formation. At least six replicates (n = 6) were conducted per condition. The biofilm inhibition (%) by each formulation was determined as 100% minus the percent biofilm formed (relative to control).

2.7. Gene Expression Analysis (RT-qPCR)

To investigate whether the hydrogel treatments modulate the expression of genes associated with biofilm formation or virulence, we performed reverse transcription quantitative PCR (RT-qPCR) on bacterial cells retrieved from the biofilms. Three representative organisms were chosen for gene expression analysis based on clinical relevance and genetic tractability: S. aureus, P. aeruginosa, and E. coli. Each strain had a panel of known biofilm-related genes to be examined (Table 1).

2.7.1. Biofilm Growth for RNA Extraction

Biofilms were grown on hydrogel disks in 24-well plates under conditions similar to Section 2.6. After 24 h incubation with either unloaded or BAC-loaded hydrogels, the biofilm cells were harvested. Disks were transferred to tubes containing 1 mL of PBS and sonicated (ultrasonicated bath, 5 min) to dislodge biofilm cells into suspension. The resulting cell suspension was immediately mixed with 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) to stabilize RNA. Bacterial pellets were collected by centrifugation (10,000× g, 5 min) and processed for RNA isolation.

2.7.2. RNA Isolation and cDNA Synthesis

Total RNA was extracted from biofilm cells using the SV Total RNA Isolation System (Promega) according to the manufacturer’s protocol, which includes on-column DNase digestion to remove genomic DNA. RNA yield and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher, Inc., Wilmington, DE, USA). Only samples with A_260/280 ~2.0 were used. For each sample, 4 µg of total RNA was reverse-transcribed to cDNA using the GoScript reverse transcriptase kit (Promega) with a mixture of oligo(dT)_15 and random hexamer primers. The cDNA was diluted 1:5 with nuclease-free water for qPCR use.

2.7.3. Quantitative PCR (qPCR)

Primer sequences for target genes and reference genes are listed in Table 1. S. aureus genes included menB, scdA, purC, argF, and fabG, implicated in biofilm metabolism and structural matrix formation. P. aeruginosa genes included fleQ, fleR, fleN (flagellar regulatory genes) and lasR (quorum-sensing regulator). E. coli genes included yjfO (bsmA), rpoS, evgA, tnaA, and ycfR (bhsA), associated with stress response and biofilm formation. Housekeeping genes were used for normalization: yqiL (S. aureus), rpoD (P. aeruginosa), and 16S rRNA (E. coli). qPCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) using SYBR Green detection. Each 20 µL reaction contained 10 µL of 2× GoTaq qPCR Master Mix (Promega), 1 µL of each primer (10 µM), 2 µL of template cDNA, 0.2 µL of reference dye, and nuclease-free water. Thermocycling conditions were: initial denaturation at 95 °C for 2 min; 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Melt curve analysis was performed at the end to confirm the presence of specific products. All samples were run in triplicate wells. Relative gene expression changes were calculated using the 2^−ΔΔCt^ method, with the corresponding positive-control biofilm (on uncoated p(HEMA)) as the calibrator and the housekeeping gene as the internal reference. Results are reported as fold change in expression (treated vs. control).

2.8. Scanning Electron Microscopy (SEM) of Biofilms

To visualize the biofilm structure on the hydrogel surfaces, SEM analysis was performed on selected samples. Biofilms of S. aureus and C. albicans were grown on p(HEMA) and p(HEMA-co-MA) (with and without BAC) in 24-well plates as described in Section 2.6. After 24 h, the disks were gently rinsed in PBS and then fixed in 2.5% glutaraldehyde (v/v in 0.01 M phosphate buffer, pH 7.4) at 4 °C overnight. The samples were washed three times with 0.1 M cacodylate buffer (pH 7.4) to remove excess fixative, then post-fixed with 1% osmium tetroxide (in cacodylate buffer) for 1 h. After three more buffer rinses, the samples were dehydrated through a graded ethanol series (30%, 50%, 75%, 95%, and 100%, 10 min each). Completely dehydrated samples were mounted on aluminum stubs with carbon tape. A thin, conductive platinum coating (~5 nm) was applied by sputter coating. The prepared specimens were examined under a scanning electron microscope (FEI Inspect F) at an accelerating voltage of 5 kV. To study general biofilm coverage and cell-surface interactions, imaging was performed at several magnifications.

3. Results

3.1. The Synthesis of Hydrogel and Physical Characterization Were Performed at Stage 3.1

Both p(HEMA) and p(HEMA-co-MA) hydrogels were prepared as clear films. The addition of 5% MA comonomer produced a considerable impact upon the appearance of the hydrogels and their texture: p(HEMA) disks were transparent, hard, rigid structures, whereas p(HEMA-co-MA) disks were somewhat opaque and had a softer, more flexible structure, Figure 1. Such softness aligns with the enhanced water affinity conferred by the carboxylate groups that MA provides. The addition of MA via covalent bonds did not interfere with the gelation process; all the disks could be handled and swollen. The equilibrium water content of the hydrogel increased with the addition of MA, as shown by the data from the swelling studies below, suggesting a more hydrophilic polymer network. There were no indications of gross morphological differences in terms of homogeneity and lack of porosity, which could be seen with the naked eye; both hydrogels formed homogeneous, non-porous films.

3.2. Analytical Method Validation

The developed UV-Vis spectroscopic method for quantifying BAC was highly accurate and sensitive. The peak absorbance at 263 nm for BAC was clear, with almost no interference from hydrogel leachates. The calibration curve showed excellent linearity over the 25–800 µg/mL concentration range (R^2^ = 0.9995), and less than 2% variation in quality control samples demonstrates the good precision of this method [30]. The quite low LOD (7.6 µg/mL) and LOQ (23 µg/mL) indicate that very small amounts of BAC released or remaining in solution can be measured, which is relevant for loading determination by depletion and for low-level release in swelling media. Therefore, this method is considered reliable for subsequent BAC measurements.

3.3. BAC Loading Capacity

BAC-loading experiments showed a distinct difference between the two hydrogel formulations. The p(HEMA) disks resulted in a BAC uptake of 22.5 ± 1.8 mg per ~100 mg disk, corresponding to a DLC of ~21.9% w/w. In contrast, p(HEMA-co-MA) disks absorbed only 15.1 ± 1.7 mg BAC under the same conditions (DLC ~14.0% w/w), which is about one-third lower (p < 0.01, unpaired t-test). This result supports our expectation that the presence of hydrophilic carboxyl groups in the MA-containing hydrogel would somewhat impede the uptake of the hydrophobic BAC. Essentially, the polar, water-attracting polymer network in p(HEMA-co-MA) is less compatible with BAC’s cationic alkylammonium structure, limiting BAC partitioning into the gel phase. Additionally, some BAC initially absorbed into p(HEMA-co-MA) may have been loosely bound and leached during the post-loading rinse due to ionic repulsion from protonated acids. Despite the lower absolute loading, the p(HEMA-co-MA) hydrogels still incorporated a substantial amount of BAC relative to their mass, and as shown later, even this lower loading was sufficient to yield strong antimicrobial effects. The MA co-monomer thus influenced BAC-polymer interactions, resulting in a trade-off: reduced loading capacity but potentially a more favorable release profile (due to ionic binding) and improved anti-adhesive background, Figure 2.

3.4. Swelling Behavior

Table 2 shows the n, k, and R^2^ values for the Korsmeyer–Peppas Swelling Kinetics (Table 2). However, p(HEMA-co-MA) exhibited markedly higher swelling than p(HEMA) under all conditions, with the difference amplified at higher pH (Figure 3). At pH 5 (simulating normal urine), p(HEMA) disks swelled by only 35.7 ± 1.9% of their dry weight after 24 h, whereas p(HEMA-co-MA) disks swelled by 101.6 ± 6.5% (approximately triple the p(HEMA) swelling; p < 0.0001). At pH 9 (simulating infection-induced alkaline urine), the swelling percentages increased to 48.9 ± 2.4% for p(HEMA) and 146.0 ± 5.2% for p(HEMA-co-MA) (p < 0.0001). The pronounced swelling of the MA-containing hydrogel at basic pH is attributed to the ionization of its carboxylic acid groups, which generates negatively charged carboxylate ions on the polymer chains. Mutual repulsion between these charged particles, driven by electrostatic forces, causes the polymer network to expand, taking in additional water. By contrast, p(HEMA) has no ionizable groups, and its slight increase in swelling at pH 9 (relative to pH 5) is likely due solely to the slight decrease in ionic strength or the rise in the osmotic driving force in the buffer.

The calculations for the Korsmeyer–Peppas Swelling Kinetics Analysis [26,27] are shown in Table 2.

Classification criteria (cylindrical disk geometry):

• n ≤ 0.50 → Fickian (Case I) diffusion
• 0.50 < n < 1.0 → Anomalous (non-Fickian) transport
• n = 1.0 → Case II (relaxation-controlled)

The diffusional exponent (n) values obtained from the Korsmeyer-Peppas power law analysis confirmed Fickian transport for all formulations. For unloaded p(HEMA), n values were 0.45 ± 0.02 (pH 5) and 0.48 ± 0.03 (pH 9); for unloaded p(HEMA-co-MA), n = 0.42 ± 0.03 (pH 5) and 0.50 ± 0.02 (pH 9). BAC-loaded hydrogels exhibited comparable n values: p(HEMA) + BAC (n = 0.44 ± 0.02 at pH 5; 0.46 ± 0.03 at pH 9) and p(HEMA-co-MA) + BAC (n = 0.40 ± 0.03 at pH 5; 0.48 ± 0.02 at pH 9). All n values were ≤0.50, with good linearity (R^2^ > 0.98), confirming that water uptake was governed by Fickian diffusion—i.e., concentration gradient-driven water penetration at a rate slower than the polymer chain relaxation. The p(HEMA-co-MA) hydrogels show lower n but higher k, meaning faster diffusion-dominated uptake driven by enhanced hydrophilicity. The swelling rate constant (k) was higher for p(HEMA-co-MA) than for p(HEMA), reflecting the more hydrophilic and open network structure of the copolymer, while BAC loading reduced k values slightly, consistent with the ionic crosslinking effect described above.

Notably, the BAC-loaded hydrogels swelled considerably as compared to the unloaded hydrogels. Indicatively, swelling of BAC-loaded p(HEMA-co-MA) was only to a percentage of 60 at pH 9 (compared to 146 without loading). We ascribe this decrease to electrostatic crosslinking: quaternary ammonium cations in BAC can interact with deprotonated carboxylate anions on the MA units, effectively crosslinking polymer chains and preventing swelling [31]. Although this phenomenon reduces maximum swelling, it might be advantageous for mechanical stability because the loaded hydrogels stand better in terms of dimensional stability. Also, this interaction probably aids in fixing BAC in the hydrogel and enhancing a sustained release and not an instant burst, as also testified by the continuous antimicrobial release-see Section 3.5.2. In summary, the MA functionalization conferred pH-responsive swelling capacity modulated by BAC loading. Under alkaline conditions, p(HEMA-co-MA) swells extensively, which could favor the release of drugs in need, whereas in the presence of BAC, swelling was dampened, suggesting a self-regulating release mechanism, as illustrated in Scheme 1.

3.5. Antimicrobial Activity

3.5.1. MIC, MBC, and MFC Values

BAC demonstrated potent antimicrobial activity in broth, consistent with its broad-spectrum usage as a disinfectant. The MICs of BAC for the tested strains were: 3.90 µg/mL for S. aureus; 7.81 µg/mL for E. coli; 7.81 µg/mL for P. aeruginosa; 3.90 µg/mL for P. mirabilis; and 1.95 µg/mL for C. albicans. The corresponding MBCs were 15.62 µg/mL for S. aureus and E. coli, 31.25 µg/mL for P. aeruginosa and P. mirabilis (Gram-negative bacteria generally required higher BAC for killing), and the MFC for C. albicans was 1.95 µg/mL (equal to its MIC, indicating BAC is fungicidal at the inhibitory dose). These values align with literature ranges for BAC’s efficacy, confirming that our test strains did not have unusual resistance to BAC. Notably, C. albicans was the most BAC-susceptible organism (likely due to BAC’s strong membrane-disruptive action on yeasts), whereas Gram-negative rods showed higher MICs, possibly due to their outer membrane acting as a barrier. These MIC/MBC results provide a benchmark for interpreting the performance of BAC-loaded hydrogels: the loaded amount per disk (~15–22 mg) vastly exceeds these MICs, but release kinetics and local concentrations will determine actual efficacy on the coated surface.

3.5.2. Zone of Inhibition (Diffusion Assay)

Disk diffusion tests demonstrated that both types of BAC-loaded hydrogels produce clear zones of inhibition on agar, confirming that BAC can elute from the polymer and diffuse into the surrounding medium to inhibit microbial growth (Figure 4 and Table 3).

Unloaded p(HEMA) or p(HEMA-co-MA) disks produced no inhibition zones on any culture (as expected, since they lack an antimicrobial agent). In contrast, p(HEMA) + BAC disks yielded substantial ZOIs after 24 h, especially against S. aureus (16.5 ± 3.3 mm) and C. albicans (25.4 ± 0.4 mm). p(HEMA-co-MA) + BAC disks showed slightly smaller zones against those two organisms (13.3 ± 1.0 mm for S. aureus, 22.3 ± 1.2 mm for C. albicans; p < 0.05 compared to p(HEMA) + BAC). This difference correlates with the higher BAC content in p(HEMA) and the higher intrinsic susceptibility of Gram-positive and fungal cells to BAC. Indeed, BAC’s activity is often greater against Gram-positive bacteria and yeasts due to easier penetration of their cell envelopes. The Gram-negative bacteria in our panel (E. coli, P. mirabilis, P. aeruginosa) showed similar ZOIs for both hydrogel types: e.g., against E. coli, p(HEMA) + BAC gave 9.9 ± 0.2 mm, and p(HEMA-co-MA) + BAC gave 10.2 ± 1.0 mm (p > 0.05); for P. mirabilis, 9.8 ± 0.4 mm vs. 11.6 ± 1.1 mm (p > 0.05); for P. aeruginosa, 9.9 ± 0.4 mm vs. 8.1 ± 0.1 mm (p > 0.05). These differences were not statistically significant. The comparable performance in Gram-negatives suggests that once a threshold BAC concentration is reached, additional loading does not enlarge the zone, likely because Gram-negatives’ inherent lower susceptibility (higher MIC) is the limiting factor. The slightly larger zone for P. mirabilis with p(HEMA-co-MA) + BAC (despite its lower BAC content) is interesting; although not significant, it might indicate that the more hydrated MA-hydrogel releases BAC a bit more readily initially, offsetting the lower total BAC.

Persistent activity: The BAC-loaded hydrogels maintained inhibitory zones for an extended period. After each day, the same disks were transferred to new inoculated agar plates; clear zones were still visible on day 8 in all organisms. Using the example of the zone against E. coli of p(HEMA + BAC) of 9.7 mm on day 8 (as compared to 9.9 mm on day 1) and that of p(HEMA-co-MA + BAC)of 9.2 mm on day 8 (compared to 10.2 mm on day 1), there was only a slight change in diameter [32]. The same tendency was noted about S. aureus and C. albicans (their larger initial zones reduced slightly by day 7–8 and were still larger than 80% the original size) and P. aeruginosa and P. mirabilis (zones were maintained but small). The fact that, a week later, there is still an inhibition zone indicates that BAC continues to leak from the hydrogels. This long-term antimicrobial effect is another important catheter coating requirement, given that urinary catheters may remain in place for days. The findings indicate that BAC release is not a burst but diffuses slowly, which could be facilitated by the polymer matrix: in p(HEMA-co-MA), ionic binding can delay release, whereas in p(HEMA), a more hydrophobic partitioning can regulate release. Practically, both formulations provided multi-day protection in the agar model, with p(HEMA-co-MA) + BAC lasting as long as p(HEMA) + BAC despite containing lower total BAC. This is one of the advantages of MA functionalization, as it may provide control over the release of BAC and maintain effective concentrations over time.

The drug release results demonstrated complete departure of the drug within 24 h (Figure 5). Although the results may imply that the synthesized hydrogel is more suitable for indwelling catheters, one should not neglect that the drug release result is multifactorial. Firstly, the volume of the surrounding medium dictates the release rate. In vivo, urine flow rates in stents/indwelling catheters or long-term catheters are not expected to exceed 4 mL/min. Furthermore, pH variation may affect the release rate, so the reflection of the persistent activity is quite complex and can be augmented when tested in an in vivo setup, which is beyond the scope of this study.

After six hours, 95% of BAC was released from p(HEMA), whereas only 80% was released from p(HEMA-co-MA). Nevertheless, both polymers exhibited complete release by 24 h.

3.6. Biofilm Formation Inhibition

The antibiofilm activity of the hydrogel coatings was established by measuring biofilm biomass using the 24 h static assay. Figure 6 shows the percent biofilm developed on the different samples relative to unmodified p(HEMA) (positive control, set at 100%). Several key trends emerged: Unloaded hydrogels: Biofilm formation was influenced solely by polymer composition, with no BAC present; p(HEMA-co-MA) consistently showed less biofilm formation than p(HEMA) alone. Biofilm on p(HEMA-co-MA) was only 32–90% of p(HEMA) (better lower is better), depending on the organism. It was found that the antifouling effect was strongest with C. albicans, in which p(HEMA-co-MA) permitted only 32.3 + − 5.2% of the biofilm compared to control (i.e., =68% inhibition; p < 0.0001). L. aureus showed a significant decrease (approximately 70% of control), and E. coli did as well (approximately 80%). A smaller reduction of about 10–15% (to about 85–90% of control) was observed in P. mirabilis and P. aeruginosa, and not all of it was statistically significant. However, by definition, unloaded p(HEMA) (the base polymer) did not inhibit biofilm significantly. These findings support the claim that the addition of MA to the hydrogel provides an innate anti-adhesive effect, presumably because the carboxylate groups enhance the hydrophilicity of the surface and introduce local charges that prevent the settlement of microbes. The relative sensitivity of C. albicans (a relatively large-celled yeast) indicates that surface wettability and/or a minor degree of acidification induced by MA may hinder fungal adhesion, which is usually mediated by hydrophobic interactions. BAC-loaded hydrogel: The addition of BAC achieved a phenomenal improvement in biofilm inhibition of the two polymers. The p(HEMA) loaded with BAC inhibited biofilm formation by about 18–28% compared to the control for all pathogens. In particular, p(HEMA) + BAC disks experienced the remaining biofilm of approximately 18 percent (C. albicans), approximately 20 percent (E. coli), approximately 24 percent (P. aeruginosa), approximately 26 percent (P. mirabilis), and approximately 28 percent (S. aureus). This is about 72–82% inhibition compared to uncoated surfaces (all p < 0.0001). BAC-loaded p(HEMA-co-MA) was even more effective, leaving only 9–14% of the biofilm compared to control—a remarkable ~86–91% inhibition across the board (pHEMA-co-MA) + BAC vs. control: C. albicans 9.17 ± 2.35%; E. coli 10.07 ± 0.55%; P. aeruginosa 12.66 ± 0.75%; P. mirabilis 13.78 ± 1.71%; S. aureus 13.24 ± 1.84%; all p < 0.0001). Therefore, the combination of the MA-containing polymer and BAC resulted in the highest biofilm reduction compared to BAC-loaded p(HEMA) across all organisms tested (the differences of 9–14% and 18–28% are statistically significant at p < 0.05 in most cases). Figure 6 shows that p(HEMA-co-MA) + BAC reduced biofilm counts to around one-tenth of the control or less across all species, whereas p(HEMA) + BAC, with more BAC, only reduced biofilm by about one-fifth or one-quarter of the control. This shows that the antifouling property of MA can be combined with the antimicrobial effect of BAC. Of the various microbes, it is interesting to note that even P. aeruginosa, which is likely a difficult biofilm-former, was decreased to approximately 12.7 on p(HEMA-co-MA) + BAC. C. albicans was the most sensitive (9.2% on p(HEMA-co-MA) + BAC, 18.2% on p(HEMA) + BAC), which is in line with the diffusion and MIC data, whereby BAC was very effective against Candida. Both types of BAC hydrogel were also very effective in inhibiting S. aureus biofilm (>85% reduction). E. coli and P. mirabilis biofilms that may be persistent in urinary conditions were maintained to approximately 10 percent and 13.8 percent of control by p(HEMA-co-MA) + BAC, respectively. Taken together, these data highlight two important issues: (1) MA co-monomer alone provides important advantages against biofilm formation, which is indeed apparent when comparing p(HEMA-co-MA) to p(HEMA)-only, and (2) BAC indeed has a large effect on biofilm reduction on these hydrogels, but it is optimized when used with the MA copolymer. Despite p(HEMA-co-MA) having lower BAC content, its BAC-loaded form outperformed BAC-loaded p(HEMA) in suppressing biofilm. This suggests that the malic acid-derived functionalities in the copolymer not only resist initial cell attachment but may also enhance the distribution or local potency of BAC at the surface, for instance, by retaining BAC at the biofilm-polymer interface where it can continuously act on settling bacteria. The outcome is a coating that tackles biofilms through dual mechanisms: a non-stick surface and a leaching antiseptic.

3.7. Gene Expression Analysis

To gain insight into the molecular effects of the coatings on bacteria, we quantified changes in the expression of select genes critical for biofilm formation and virulence. We focused on one representative strain each of Gram-positive (S. aureus), Gram-negative (P. aeruginosa), and enteric bacteria (E. coli). Figure 7 summarizes the fold changes in gene expression (log_2_ scale) for the different treatments, and key results are described below.

3.7.1. Staphylococcus aureus Gene Expression

All five targeted genes in S. aureus (menB, scdA, purC, argF, fabG) were significantly downregulated (fold change < 1) after exposure to any hydrogel sample, relative to an untreated biofilm control (p(HEMA) with no BAC). The unloaded p(HEMA-co-MA) hydrogel caused a moderate suppression of these genes (approximately 2–7-fold downregulation), indicating that, even without BAC, the carboxylated surface imposed stress on the bacteria, reducing expression of biofilm-associated functions. BAC-loaded p(HEMA) had the strongest effect, downregulating gene expression by 4- to ~13-fold. Notably, menB and fabG exhibited the greatest repression (~12.9-fold and ~12.6-fold reduction, respectively) with p(HEMA) + BAC. These two genes are important for S. aureus biofilm physiology: menB encodes an enzyme in the menaquinone (vitamin K_2_) biosynthesis pathway, crucial for anaerobic respiratory processes within biofilms, and fabG encodes a fatty acid synthase enzyme required for lipid membrane production. Their strong suppression suggests that the BAC-laden hydrogel profoundly disrupts S. aureus metabolic maintenance of the biofilm. Interestingly, the BAC-loaded p(HEMA-co-MA) sample yielded slightly lower fold changes than p(HEMA) + BAC for most genes (e.g., menB ~11.2-fold, fabG ~10.8-fold down), except for argF, where p(HEMA-co-MA) + BAC induced the maximum downregulation (~8.8-fold vs. ~4-fold with p(HEMA) + BAC). argF encodes an ornithine transcarbamylase in the arginine biosynthesis pathway; its greater suppression in the MA-containing formulation might reflect differences in local pH or nutrient conditions caused by the carboxylated polymer. Overall, BAC was the dominant factor in gene suppression for S. aureus, but MA’s presence modulated the pattern (with slightly less extreme changes overall, possibly due to fewer bacteria surviving on that surface). All these gene expression changes correlate with reduced biofilm viability and matrix production, reinforcing that the hydrogels—especially with BAC—diminish S. aureus biofilm vigor at the genetic level.

3.7.2. Pseudomonas aeruginosa Gene Expression

The gene expression profile in P. aeruginosa showed a markedly different trend. Here, the unloaded p(HEMA-co-MA) hydrogel alone caused a surprisingly large drop in expression of the flagellar regulatory genes fleQ, fleR, and fleN (5.9- to 12.0-fold down). In fact, fleR was suppressed ~12-fold just by p(HEMA-co-MA) (no BAC)—a greater effect than that of the BAC-loaded p(HEMA) (~3.6–5.7-fold). This suggests that the MA-modified surface strongly interferes with P. aeruginosa’s motility/attachment machinery, perhaps by preventing effective flagellar function or by triggering a surface stress response. fleQ, fleR, and fleN encode components of the master flagellum regulatory complex, which governs initial attachment and biofilm initiation; their downregulation suggests reduced bacterial adherence and microcolony formation. Meanwhile, the quorum-sensing regulator lasR was also downregulated by all treatments, though to a lesser extent (~4–6-fold down). LasR is involved in coordinating biofilm maturation and virulence factor production; partial inhibition of this pathway could decrease biofilm robustness and pathogenicity. The BAC-loaded p(HEMA) caused moderate suppression of all these genes (approximately 3.6–5.7-fold as noted). Notably, the BAC-loaded p(HEMA-co-MA) caused fleN to drop by ~12.8-fold (the highest among treatments for fleN), while its effects on fleQ and lasR were in the mid-range (~5-fold). The combination of MA and BAC thus yielded both the baseline strong suppression from MA and the additional biocidal stress from BAC. These results illustrate that for P. aeruginosa, altering the surface chemistry (MA) can significantly hinder the expression of motility genes responsible for the early stage of biofilm formation—an encouraging sign that the hydrogel is interfering at a very fundamental level with this organism’s biofilm establishment. The presence of BAC ensures bacterial killing and likely further contributes to gene downregulation by reducing cell density and quorum-sensing signals.

3.7.3. Escherichia coli Gene Expression

In E. coli, five genes were monitored: yjfO (also known as bsmA), rpoS, evgA, tnaA, and ycfR (bhsA). All of these were downregulated to some extent by the hydrogel treatments. The BAC-loaded p(HEMA) had the strongest effect on two key regulators: evgA was reduced by ~12.0-fold, and yjfO by ~8.1-fold. EvgA is part of the EvgAS two-component system that helps E. coli respond to acidic stress; repressing it could impair the bacteria’s ability to survive acidic shocks (which are common in the bladder’s pH fluctuations). YjfO/BsmA is associated with biofilm structural integrity (it encodes a biofilm matrix protein); its downregulation may weaken biofilm structure. The other genes, rpoS (general stress sigma factor), tnaA (tryptophanase linked to indole signaling), and ycfR/BhsA (outer membrane protein affecting biofilm hydrophobicity), were also significantly downregulated, though to a lesser degree. YcfR in particular showed only ~2-fold downregulation across all treatments, including p(HEMA-co-MA) alone, which suggests this gene is less sensitive or not a major target of these interventions. BAC-loaded p(HEMA-co-MA) caused a moderate reduction in most E. coli genes (1.7- to ~7-fold). It did not match the ~12-fold knockdown of evgA seen with BAC/p(HEMA); instead, evgA was ~6-fold down with BAC/p(HEMA-co-MA). This could be due to fewer E. coli remaining viable on the p(HEMA-co-MA) + BAC surface (hence lower expression changes measured), or possibly that the harsher initial attachment environment (MA) led E. coli to adapt differently. In any case, the general trend is that hydrogels (especially with BAC) downregulate E. coli biofilm and stress genes, consistent with the reduced biofilm formation observed. The relatively uniform ~2-fold suppression of ycfR suggests that some pathways (e.g., outer membrane protein expression) might not be heavily affected, or that ycfR is constitutively expressed regardless of biofilm state.

3.8. SEM Visualization of Biofilms

Qualitative differences in biofilm morphology under various treatments were directly observed via SEM. The untreated p(HEMA) surfaces (without BAC or MA) showed dense biofilm coverage: in the case of S. aureus, a thick layer of cocci embedded in extracellular matrix completely covered the polymer surface, with multiple layers of bacteria forming a mature biofilm structure (Figure 8). C. albicans on p(HEMA) similarly demonstrated prolific growth, with abundant yeast cells and hyphal elements (indicative of filamentation) present on the surface, consistent with robust biofilm formation. By contrast, p(HEMA-co-MA) + BAC surfaces were largely clear of adherent cells. For S. aureus, only a few scattered cells were observed on p(HEMA-co-MA) + BAC, and those present appeared damaged or in clumps rather than as a confluent film. Even the surface was exposed, meaning that the biofilm did not create a continuous layer. The same was also true of the C. albicans biofilm, which was significantly reduced: SEM images revealed only a few yeast cells on p(HEMA-co-MA) + BAC, and no hyphal networks were present. The single cases of treatment were intermediate, i.e., p(HEMA) + BAC (no MA) and p(HEMA-co-MA) (no BAC). S. aureus biofilm was highly decreased on p(HEMA) + BAC; the majority of the bacteria were absent, and the rest were frequently lysed (in line with the killing effect of BAC). A few extracellular debris and destroyed cells could, however, be seen, indicating a partially killed biofilm. The adherence of S. aureus to p(HEMA-co-MA) in the absence of BAC was also lower than in the control: the surface was not completely covered, and there were more cells on the surface than in samples loaded with BAC. The p(HEMA-co-MA) in many S. aureus cells appeared in smaller groups rather than a sheet, suggesting that the antifouling property of the MA prevented extensive colonization, although it did not necessarily kill the bacteria. Yeast on p(HEMA-co-MA) (unloaded) was found to have fewer yeast and nearly no hyphae, which is consistent with the quantitative finding that pFEMA-co-MA by itself halved Candida biofilm. Overall, the SEM analysis corroborated the quantitative biofilm inhibition data: surfaces incorporating MA and BAC showed minimal biofilm, whereas the unmodified surface was completely overgrown. The images highlight the outcome of combining a malic acid-enriched polymer (resisting attachment) with a biocide (killing invaders): the few microbes that might adhere are either killed or unable to propagate into a structured biofilm. In practical terms, this implies that a catheter coated with p(HEMA-co-MA) + BAC would likely remain largely free of biofilm, whereas an uncoated or BAC-only-coated catheter (without the antifouling polymer) could still accumulate biofilm residues over time.

4. Discussion

This study has shown that p(HEMA)-based hydrogels, especially when functionalized with maleic anhydride to provide malic acid-like carboxyl groups, can be effective antimicrobial catheter coatings by combining surface antifouling properties with sustained antiseptic release. Incorporation of a small fraction of MA into p(HEMA) resulted in a dual-action system, driven by significant changes in interactions between the hydrogel and both the drug and microorganisms. Our results indicate that malic acid-derived functionality contributes to enhanced antimicrobial activity, even against complex biofilm-forming pathogens. Polymer-Drug Interactions: di [18]. The anionic network of p(HEMA-co-MA) is more hydrophilic, thereby losing its capacity to accommodate the hydrophobic cationic molecules of BAC, resulting in a lower equilibrium loading. This observation is in agreement with Andrade-Vivero et al. (2007), who demonstrated that incorporating functionalized monomers (4-vinyl-pyridine and N-(3-aminopropyl) methacrylamide) into p(HEMA) networks remarkably increased drug loading capacity up to 10–20-fold for NSAIDs through ionic/hydrophobic interactions, confirming that co-monomer composition critically determines the polymer–drug interaction and loading behavior [33]. Similarly, Ayhan and Ayhan (2018) showed that varying HEMA content and crosslinker ratio in p(HEMA) hydrogels directly influenced swelling dynamics and drug release kinetics, further supporting the role of network composition in drug uptake [34]. But this apparent drawback did not reflect in antimicrobial performance. As a matter of fact, the p(HEMA-co-MA) coatings (even though containing a 33% lower amount of BAC) did as well as p(HEMA) coatings, in terms of biofilm inhibition. It means that the best antimicrobial activity is not directly related to the amount of drug but is determined by the interactions among polymers, drugs, and microbes. With p(HEMA-co-MA), BAC is in part bound to carboxylate sites; this probably inhibits a rapid burst release and instead affords a steady elution of BAC throughout the day (which is also in line with the sustained zones of inhibition over day 8). Electrostatic tethering of BAC also decreased the hydrogel’s swelling and, in effect, enhanced the mechanical integrity of the loaded hydrogel [12]. A comparable phenomenon was reported by Jones et al. (2015), who loaded thermoresponsive HEMA-NIPAA copolymer hydrogels with chlorhexidine diacetate and observed that drug-polymer interactions modulated both drug release profiles and the mechanical properties of the hydrogels, highlighting that polymer-drug compatibility is a key design parameter for antimicrobial hydrogel coatings [21]. Furthermore, Yilmaz et al. (2021) reported that p(HEMA-co-APTMACI) copolymeric hydrogels displayed altered swelling and enhanced antimicrobial properties compared to pure p(HEMA) when loaded with sodium diclofenac, reinforcing the principle that copolymer composition governs both drug accommodation and release performance [35].

These results are consistent with the new principles of design based on computational hydrogel research, which propose that polymer-drug interactions can be tuned to optimize the mechanical and therapeutic performance of drug-loaded biomaterials. The MA co-monomer also plays a significant role in our case: it provides the binding sites for BAC, which regulate its release and action. pH-Responsive Behavior and Clinical Relevance: The p(HEMA-co-MA) hydrogel had a greater swelling in pH 9 compared to pH 5, and p(HEMA) had a slight increment [36]. This pH reactivity is of great importance for urinary catheters. Proteus mirabilis is a widely occurring CAUTI pathogen that secretes urease, capable of alkalinizing urine to pH 8–9, which can frequently cause encrustation and increased biofilm. Within this environment, the p(HEMA-co-MA) coating would swell much more, likely filling any gaps between the catheter and the urethral wall and facilitating easier BAC release, since the network would be expanded (and maybe some of the BAC-carboxylate bonds would be broken at high pH). Infection-induced swelling may therefore serve as an on-command enhancement to drug release, where BAC is increased where the risk is more [12].

On the other hand, when the urine pH is normal (around 5–6), the swelling (and therefore the release of the BAC) is limited, and this may serve to save the drug and extend the coating life. This is a brilliant reaction, and it is an inherent benefit of the MA component. Yet, we also noted that the inclusion of BAC in the hydrogel decreased swelling, which is a provisional crosslink, potentially preventing excessive swelling and enhancing the durability of the coating during hydrogel utilization. The overall effect is a stable coating that can vary its release profile in response to urinary conditions. This pH-responsive swelling behavior parallels findings reported for other HEMA-acid copolymer systems. Tomić et al. (2010) demonstrated that p(HEMA/itaconic acid) hydrogels with up to 5 mol% itaconic acid—a dicarboxylic acid co-monomer structurally analogous to MA—exhibited pronounced pH-sensitive swelling and excellent biocompatibility, supporting the use of small amounts of dicarboxylic acid co-monomers to endow p(HEMA) hydrogels with stimuli-responsive behavior [37]. More recently, Ran et al. (2024) developed quaternary ammonium compound-based hydrogel coatings for catheters featuring pH-responsive zwitterion-to-cation conversion that generated on-demand bactericidal effects in response to infection-induced pH changes, a concept mechanistically similar to our pH-triggered enhanced swelling and BAC release from p(HEMA-co-MA) [38].

Improved Antibiofilm Efficacy through MA Functionalization: One of the most notable findings was that BAC-loaded p(HEMA-co-MA) showed stronger biofilm suppression than BAC-loaded p(HEMA), despite lower BAC concentration. In all the organisms tested, p(HEMA-co-MA) + BAC led to a reduction in biofilm to less than 15 percent of the control, and p(HEMA) + BAC to about 20–30 percent. This indicates that there is something more than the antimicrobial effect of BAC at work, which is the antifouling nature vested by the carboxyl-rich hydrogel. The MA-containing hydrogel, in itself, already decreased biofilm formation (especially in C. albicans and S. aureus), implying that fewer organisms will initially adsorb to such a surface and form a biofilm. In the event of the presence of BAC as well, the ones that attach are killed or crippled promptly. In the meantime, the hydrophilic surface and, potentially, local acidity (unneutralized -COOH groups) might provide less favorable microenvironments for the production of the biofilm matrix. This leads to a decrease in biofilm viability and coverage, which is significantly greater than that of either method (antifouling or biocidal). This aligns with a general idea in anti-infective biomaterials: active and passive defenses. This dual-action concept is well supported in the literature. McCoy et al. (2018) demonstrated that anti-adherent p(HEMA)-based biomaterials significantly reduced catheter biofouling through surface hydrophilicity alone, while acknowledging that combining passive antifouling with active antimicrobial agents would yield superior outcomes [22]. Hook et al. (2012) used high-throughput screening to identify polymers resistant to bacterial attachment [39]. They showed that the best-performing antifouling coatings on silicone achieved up to 96.7% reduction in bacterial surface coverage—yet even this required combination with antimicrobial agents for complete biofilm prevention. Zare et al. (2021) reviewed p(HEMA)-based biomedical applications and emphasized that p(HEMA) alone lacks inherent antimicrobial properties and must be combined with additives or co-monomers to achieve effective infection prevention [10].

Conventional antibiotic coatings use the drug alone, and purely antifouling coatings use surface properties alone; our findings indicate that combining the two yields results that are exponentially better. It was true that p(HEMA-co-MA) + BAC reduced S. aureus biofilm to approximately 13 percent, whereas p(HEMA) + BAC (with a higher BAC content) reduced it to approximately 28 percent. This is due to the capacity of the MA copolymer to inhibit biofilm formation through non-chemical mechanisms (hydration layer, repulsive charges) and BAC to provide a chemical attack, preventing the formation of a foothold or an adaptive response in the bacteria. Moreover, the MA component would presumably influence the biochemical environment at the polymer interface—such as by pH buffering or cationating some of the local cations—which may also prevent some biofilm-forming processes (such as matrix calcification by P. mirabilis). The implications for catheter coatings are obvious: to make an antimicrobial coating highly effective, it is possible to add a hydrophilic, ionic monomer to enhance the properties of an antimicrobial polymer on the catheter, enabling high performance with possible reduced drug levels. Inhibition of gene expression: RT-qPCR findings provided mechanistic insights, demonstrating that the hydrogel treatments killed the bacteria and also suppressed the expression of major virulence and biofilm genes in surviving bacteria. In S. aureus, genes involved in anaerobic metabolism (menB) and cell envelope synthesis (fabG) were strongly downregulated by BAC treatment, which could compromise the bacteria’s ability to thrive in the oxygen-poor, nutrient-limited depths of a biofilm. The somewhat lower fold changes observed with p(HEMA-co-MA) + BAC (compared to p(HEMA) + BAC) in S. aureus might reflect fewer bacteria remaining (thus lower total RNA), but, interestingly, argF was more suppressed with the MA-containing polymer, suggesting a formulation-specific effect on arginine biosynthesis or a stress response triggered by the surface. In P. aeruginosa, the substantial suppression of fleQ/fleR/fleN by the MA hydrogel (with or without BAC) is notable. These genes govern motility; their downregulation suggests that the bacteria may have sensed the surface as “unfavorable” and downshifted their attachment machinery, or were physically unable to attach and thus downregulated flagellar gene expression as a consequence.

Additionally, both hydrogel types modestly reduced lasR expression (~5-fold), potentially diminishing quorum-sensing-regulated factors (like exotoxins and exopolysaccharide production). In E. coli, the coatings, especially BAC, targeted stress response pathways; evgA (acid resistance regulator) was among the most affected genes. This could mean that BAC, which causes membrane damage, triggers acid stress (proton leakage), and that the bacteria cannot compensate due to suppression of the evgA pathway. The uniform moderate downregulation of ycfR across treatments indicates that no major change in the outer membrane is occurring. ycfR (BhsA) is also known to confer biofilm resistance to environmental stresses, so even a 2-fold downregulation may shift the balance towards biofilm rupture. Overall, the data on gene expression indicate that the coatings mediate the interference with several molecular targets: metabolism, mobility, stress adaptation, and cell communication in bacteria. This contribution of MA is eminent in P. aeruginosa. However, BAC demonstrates its effects prominently in S. aureus and E. coli. Notably, the use of gene expression analysis using MA is relatively new, and, to our knowledge, this is one of the first demonstrations of how a HEMA-based copolymer coating can regulate bacterial gene expression regarding virulence and biofilm formation.

This dual-action (antimicrobial + antivirulence) outcome represents a significant advance over coatings that simply kill bacteria; by knocking down virulence gene expression, even surviving bacteria are rendered less harmful.

Comparison to Other Anti-infective Strategies: The recent literature contains several novel strategies to prevent catheter biofilms. Nanoparticle-infused coatings, such as those containing gold or silver nanoparticles, have demonstrated antimicrobial properties but raise concerns about manufacturing complexity, potential cytotoxicity, and the development of microbial resistance to heavy metals. Our hydrogel strategy is based on well-understood, biocompatible polymers -p(HEMA) is used in ophthalmic devices and hydrogels-, and an antiseptic that is commonly used and thus is relatively straightforward and likely acceptable from a regulatory perspective. Importantly, BAC has been previously explored for catheter impregnation. Tebbs et al. (1993) were among the first to incorporate BAC into a central venous catheter polymer, demonstrating zones of inhibition against S. epidermidis, S. aureus, and C. albicans, with sustained inhibition of microbial adherence for up to 14 days in PBS [40]. Our hydrogel-based approach extends this concept by using a swellable matrix that allows higher drug loading and more controlled, sustained release compared to direct polymer impregnation. More recently, Navarro et al. (2022) coated silicone urinary catheters with a biofilm preventative agent containing 0.175% benzalkonium chloride combined with polyacrylic acid and glutaraldehyde, and demonstrated inhibition of biofilm development by E. coli, MRSA, and Enterobacter cloacae; however, notably, their coating was ineffective against P. aeruginosa [14]. In contrast, our BAC-loaded hydrogels showed measurable activity against P. aeruginosa (ZOI of 8–10 mm and biofilm reduction to ~13% of control), likely due to the higher local BAC concentrations achieved through the hydrogel loading approach (~15–22 mg per disk), which exceeds the threshold needed to overcome the inherent outer membrane barrier of this organism.

Mucoadhesive polymer films, such as cellulose derivatives, have been developed for other applications in drug delivery (e.g., vaginal candidiasis) and have achieved effective local antimicrobial release [8]. It was reported in the literature [8] that cellulose-based films loaded with nystatin could sustain drug release for 8 h and inhibit Candida growth. However, those films are designed to dissolve or erode quickly and may not possess the tensile strength or wear resistance needed on a rigid catheter surface in flowing urine. In contrast, the p(HEMA) hydrogel matrix is a permanent coating that adheres to the catheter and does not dissolve, providing continuous protection. Additionally, our coatings maintained antibacterial activity for at least 8 days, surpassing many existing coatings that often lose efficacy within a few days. The mechanical stability of p(HEMA) (and presumably p(HEMA-co-MA) under fluid shear is another advantage over flimsy film coatings—an important consideration for urinary catheter use, where friction and irrigation occur. The other method in the literature is stimulus-responsive drug release systems that respond to infection cues (e.g., pH, enzymes). Theoretically, our MA-containing hydrogel already represents a less elaborate version of the same idea, as it swells more at high pH and thus releases more BAC on demand. This inherent sensitivity eliminates the need for higher-order functionalization (such as the incorporation of nanocarriers or enzyme-labile linkers). Therefore, the proposed strategy provides a trade-off: technically easy to implement (synthesizable in a single step, simple to load), biocompatible, and with a wide range of activity against a wide range of targets (Gram-positives, Gram-negatives, and fungi). Recent reviews have contextualized the growing importance of hydrogel-based antimicrobial coatings for urinary catheters. Dai et al. (2023) comprehensively reviewed hydrogel coatings for urinary catheters and highlighted that the most effective strategies combine antifouling, hydrophilic surfaces with bactericidal agent release —precisely the approach adopted in our study [41]. Peng et al. (2023) reviewed antibacterial hydrogel coatings in the broader biomedical field [42]. They noted that quaternary ammonium compounds combined with hydrogel matrices can achieve nearly 100% antibacterial effect against both Gram-positive and Gram-negative bacteria, supporting our choice of BAC as the active agent. Singha et al. (2017) reviewed antimicrobial coatings specifically for urinary catheters [43]. They emphasized that the ideal coating should combine anti-adhesive surface properties with controlled antimicrobial release and demonstrate efficacy for at least 7 days—criteria that our BAC-loaded p(HEMA-co-MA) formulation satisfies with >85% biofilm reduction maintained over 8 days.

It addresses both initial adhesion and post-attachment survival of microbes, which are crucial for comprehensive biofilm prevention.

Limitations: This study has some limitations that should be discussed. First, all experiments were conducted in vitro under laboratory conditions. Real conditions of the urinary tract, such as urine flow, protein deposition, and the dynamic response of the host immune system, may affect the performance of the coating. For example, urine components might adsorb to the hydrogel and modulate its antifouling character or BAC release. Also, in vivo, bacteria exist in multispecies communities; while we tested a range of pathogens individually, mixed-species biofilms (e.g., E. coli with Proteus spp. or Candida spp.) might behave differently. Second, our gene expression analysis was limited to a set of known biofilm genes. A genome-wide transcriptomic analysis (RNA-seq) could uncover broader effects of the hydrogels on bacterial physiology, and might identify stress responses or resistance mechanisms not captured by our targeted approach. Third, we did not measure the long-term stability of the coating (e.g., BAC retention over weeks, hydrogel integrity under extended flow), which is important for catheters that remain in place for more than 1 week. However, since most CAUTIs occur within the first week of catheterization, our 8-day efficacy window is highly relevant. Finally, while we chose 5% MA based on prior work and preliminary tests, it is possible that other MA ratios or alternative anionic monomers could further optimize performance. For instance, more MA might increase antifouling but further lower BAC loading and mechanical strength, whereas less MA might do the opposite; finding the sweet spot will be important for maximizing outcomes.

Future Directions: The next stage of this evaluation, based on these encouraging results, would be in in vivo models. A real model of CAUTI would be an animal model of encrustation and infection, which would support biocompatibility and avoid tissue irritation or systemic toxic absorption of BAC. We would also like to streamline the MA content, aiming for the ideal drug-loading/antifouling ratio by optimizing the functional group density of the malic acid. Other avenues include substituting or adding other antimicrobials. For example, combining BAC with an antibiotic could provide broad coverage and mitigate any risk of BAC-induced resistance development 80, 81. The hydrogel may have the capacity to carry dual agents. Incorporation of stimuli-responsive elements may thus represent yet another interesting avenue; in addition to the existing pH responsiveness, the addition of enzyme-degradable linkers that respond to bacterial enzymes, such as urease or proteases, might further tailor release kinetics. We further note that scale-up and application methods need consideration; dipping or spraying catheters with a prepolymer mixture followed by curing could be a viable route of manufacture. We propose to investigate how the coating adheres to different types of catheter materials-latex, silicone-and ensure that it does not peel or crack during insertion. Finally, regulatory aspects will be the safety of long-term BAC exposure in vivo, although BAC is used clinically in controlled amounts in bladder irrigation solutions, etc. Our results provide strong impetus to advance this dual-function coating toward clinical translation.

5. Conclusions

We have successfully developed benzalkonium chloride-loaded p(HEMA) hydrogels, including a novel variant functionalized with maleic anhydride (MA), as potent antimicrobial and antibiofilm coatings for urinary catheters. The incorporation of malic acid-derived carboxyl groups in the hydrogel was pivotal for the coating to achieve pH-responsive swelling, enhanced hydrophilicity, and inherent antifouling properties. Thus, the BAC-loaded p(HEMA-co-MA) formulation exerted >85% reduction in biofilm formation across multiple CAUTI-relevant pathogens (S. aureus, E. coli, P. aeruginosa, P. mirabilis, and C. albicans) and maintained antimicrobial activity for at least 8 days in vitro. Interestingly, this high performance was achieved at a lower BAC loading than with the non-functionalized p(HEMA), reinforcing the importance of the MA comonomer in enhancing efficacy. On the molecular scale, the coatings not only eliminated the majority of bacterial cells but also strongly down-regulated virulence and biofilm-related genes, thereby inhibiting the pathogens’ capacity to initiate biofilm resistance. These novel hydrogel-based two-way surface coating systems, which combine anti-adhesion microbes with regulated antiseptic release, provide a promising approach to preventing catheter-associated infections. The offered plan addresses both stages of CAUTI pathogenesis, i.e., initial adhesion and subsequent biofilm formation by pathogens, which can significantly decrease the number and healthcare costs of CAUTIs. The complexity and adaptability of the p(HEMA-co-MA) platform make it a good candidate for development down the line, and when biocompatibility is properly optimized and confirmed, it has a lot of potential for clinical use as a new and efficient coating on catheters.