Assessment of persistent antimicrobial and anti-biofilm activity of p-HEMA hydrogel loaded with rifampicin and cefixime

Catheter-associated urinary tract infections (CAUTIs) are nosocomial infections causing more than one million hospital cases annually. The progress of CAUTIs leads to severe health complications. Infections result in blockage of the medical device due to biofilm formation, which necessitates the replacement of the device. The objective of this study is to improve urological biomaterials to minimize microbial growth and reduce the incidence of CAUTIs. Challenges from mixed biofilm are crucial and need to be addressed in the development of new coating materials. Herein, an investigation highlighted the reduction of mixed biofilm overgrowth and attachment tendency on poly-2-hydroxyethyl methacrylate (p-HEMA) surface by loading the hydrogel with rifampicin (RIF), cefixime trihydrate (CFX), and combined ratios of RIF and CFX. Mixed biofilm-formation ability in (3:1) RIF: CFX-loading p-HEMA (F6) surface showed best tendency to resist form biofilm. Persistent antimicrobial activity increased in p-HEMA loaded with combined ratios of RIF and CFX surface compared to p-HEMA alone, antimicrobial activity lasted for 8 days. All fabricated films exhibited %cell viability higher than 75% on HEK 293 cells. The addition of RIF and CFX may improve the duration of urological device employment before replacement.

Microbicides-coated catheters were a strategy adopted to inhibit or delay the onset of CAUTIs 29 . Rifampicin (RIF), and cefixime trihydrate (CFX) are effective against Gram-positive and Gram-negative pathogens with antibiofilm properties [30][31][32] . CFX is effective in the treatment of a broad variety of bacterial infections, such as urinary tract infection 32 .
The use of polymeric coating comprises approaches dictated by the physicochemical properties of the polymer. For example, pH-sensitive polymers are polyelectrolytes that have ionizable function groups in their structure that either lose or accept protons and dissociate in response to differences in environmental pH 33 . poly-2-hydroxyethyl methacrylate) (p-HEMA) is a pH-sensitive polymer that contains primary alcohol 34 , with hydrophilic and lipophilic balance, non-toxic, transparent, insoluble 35 , can't be absorbed by the body 36 , resistant to degradation 37 , has stable mechanical properties 38 . p-HEMA was used in contact lenses, wound dressings, controlled drug release delivery systems, and implants 39 . The aim of this study was to develop and characterize urological biomaterials fabricated from 2-hydroxyethyl methacrylate (2-HEMA) and loaded with RIF, CFX, and combined ratios. Moreover, we report for the first time the assessment of anti-fouling ability of RIF/CFX hydrogel against mixed biofilms formation.

Results
In this study, p-HEMA was loaded with RIF, CFX, and combined ratios of RIF and CFX. Characterization of the fabricated films takes place to determine T g , U.T.S, elongation%, and Young's modulus. Study the EWC% and evaluate HEK 293 cells cytotoxicity. Various types of bacteria were used to determine the persistence antimicrobial activity for 8 days and biofilm-formation ability.

Discussion
The recent advancement in biomaterial sciences and their versatile employment in all medical fields changed the typical medical guidelines and traditional approaches in providing health care 40,41 . Developing medical devices is undeniably a direct application of those advancements in the aforementioned fields. However, the welldocumented problem of developing sensitivity and microbial infections limited the use of those medical devices. For instance, the urinary medical devices (that include and are not limited to urinary catheters and stents) were reported to develop a microbial infection in 100% of all patients after catheterization within 1 month 42 . The only solution up to date is to replace the urinary device which will put the burden on the patient, medical staff, and cause cost-related sub-sequences 6 . The main challenge in developing a microbial infection is the formation of biofilm, which is a complex structure that requires a higher concentration of microbicide up to 500 times to eradicate the microorganism considering the drug difficulty in penetrating the complex structure of polysaccharide and glycocalyx 42 . Moreover, it was found that microorganisms involved in biofilm-formation are not single species, but multispecies which makes the management even harder 8 . To address the mixed biofilm problem, a documented method to employ p-HEMA hydrogel in urinary catheters was adapted here and the hydrogel was exposed to multispecies microorganisms to detect their ability to delay biofilm formation. RIF was utilized attributed to its amphoteric nature that may prevent bacterial colonization 43,44 . Moreover, CFX is prescribed routinely in UTI 32 . p-HEMA was formed as control, void of a drug. Also, it was loaded with either RIF or CFX or a combination of both in different ratios. The formation of mixed biofilm was detected. Biofilm-formation results showed that the addition of microbicides significantly decreased biofilm-formation compared to p-HEMA, indicating that the properties of p-HEMA surface alone are not adequate and the loading of the drugs is quite crucial in resisting biofilm-formation. Furthermore, the physicochemical properties of the drug may contribute to the suppression of biofilm formation. For instance, the observed reduction in fabricated films containing a high amount of RIF may be attributed to the zwitterionic nature of the microbicide. Zwitterionic compound contains cation and anion groups which makes it highly hydrophilic. The hydrophilicity enables the formation of a hydration layer on the surface and repel nonspecific protein deposition on the surface through steric repulsion which subsequently reduces the pre-dispositioning factor of biofilm formation 43,44 . www.nature.com/scientificreports/ The results from the persistence of antimicrobial activity were in compliance with the results obtained from the biofilm adhesion properties where the zone of inhibition over sequential transfers was affected by the type and amount of drug used. Essential to microbicide assessment was the evaluation of toxicity. HEK 293 cells cytotoxicity was evaluated by using an MTT assay that is based on the conversion of water-soluble yellow dye to an insoluble purple formazan by the action of mitochondrial reductase 45 . All fabricated films showed viable HEK 293 cells count higher than 80% and were deemed safe 46 , except RIF-loaded p-HEMA (F2) which showed the viability of 79.19 ± 14.92% which could be relatively safe. The viability lowering in (F2) may be attributed to the zwitterionic nature of the drug that allows its adsorption on the tested cell. Thus, the interaction between the p-HEMA and the tested drugs did not show toxicity on HEK 293 cells. Moreover, the fabricated films showed good swelling ability, which would allow oxygen permeability. Oxygen molecules can penetrate easily through free water network in the hydrogel to the cells allowing cellular activity 47 . The employment of polymeric coating layer in urinary stents and catheters should consider their capacity to swell. That is water uptake will expand the size and mass of the layer which-if not controlled-may lead to blockage. Therefore, swelling was studied at 2 different pH(s) considering normal and infected urine (if the infection was caused by urease forming bacteria). The observed lowering in EWC% of RIF-loaded p-HEMA (F2) at pH 5 could be attributed to the chemical nature of RIF which acts as zwitterion at pH 5 hindering its interaction with water 48 . Moreover, because RIF is zwitterionic, it would act as anion at pH 9 leading to higher ionization and subsequent enhancement in water uptake. The observed elevation in EWC% at pH 9 in all tested films is due to the ionization of the hydroxyl group in p-HEMA that leads to enhance water uptake and entrapment 49,50 . The protonation of the primary alcohol groups in p-HEMA would result in ionic repulsion leading to expansion and increasing the probabilities of interaction with water. Furthermore, the significant variations in ECW% in the tested films were correlated with T g . T g is the temperature at which change of materials from an amorphous glassy solid to a rubber-like liquid occurs 51 . T g reflects the polymer ability to move freely that at lower T g value, as seen in Fig. 2. Loading p-HEMA with ratios of RIF and CFX decreased the EWC% possibly due to the increase of the interaction between the drugs and the polymer. Where the increase of the interaction between the polymer chain and the drugs would decrease the void volume and therefore reduce polymer chain mobility 52 , this result is also supported by T g Fig. 2. Another important parameter affected by T g is the mechanical properties 53 . For example, U.T.S increases with increasing T g , that is higher T g means less void volume, and the required force to cause the brake, therefore, should be higher. On the contrary, elongation% increases when the void volume increases (lower T g ), that is the stretching ability and polymer chain movement elevated 54,55 . Young's modulus of the highest elongation% among the fabricated films has the lowest U.T.S. therefore, loaded p-HEMA with combined ratios of RIF and CFX (F4, F5, F6) elevate U.T.S, Young's modulus, and decrease elongation%. Preparation of p-HEMA films and loading them with RIF, CFX, and combined ratios. p-HEMA was prepared by the addition of a mixture of 1% (w/w) ethylene glycol-dimethacrylate (EGDMA) and 1% (w/w) of 2, 2′-azobisisobutyronitrile (AIBN) to a 98% (w/w) of 2-hydroxyethyl methacrylate (2-HEMA). After that, the mixture was continuously stirred for 10 min at 20 °C. Subsequently, the resulting mixture was injected slowly into the mold. The mold was in-house setup, containing two glass plates that were separated via a medical-grade silicone tube with a lumen diameter of 0.3 mm and wall thickness of 0.18 mm and clamped utilizing clips 56 . The polymerization reaction was carried out at 60 °C for 18 h. The fabricated film was peeled and soaked in deionized water to remove unreacted monomers, and this was confirmed by measuring the absorbance of acrylate till it reached zero. Acrylate absorbance was determined using a UV spectrophotometer (Shimadzu UV-1800 spectrometer (Kyoto, Japan)) at a maximum wavelength (λ max ) of 220 nm. The soaked film was cut into the preferred shape, then dried to reach constant weight in an oven at 30 °C to produce xerogels. For drugs loading, the same procedure was carried out taking into account the amount of the drugs that will be included, 0.5% (w/w) of drugs was added to a mixture of 97.5% (w/w) 2-HEMA, 1% (w/w) EGDMA, and 1% (w/w) AIBN, as presented in Table 1. Film preparation is presented in Fig. 8.

Materials and methods
Thermal analysis using dynamic mechanical thermal analyser (DMTA). The glass transition temperature (T g ) of the dried films of p-HEMA and drugs-loaded p-HEMA was determined by utilizing Q800 DMTA (TA, New Castle, USA). The heating rate was 3 °C/min over a temperature range of 35 °C to 160 °C using tensile mode at an oscillatory frequency was 1 Hz. Dried films (n = 3) were cut into rectangular shapes with a dimension (30 mm length, 10 mm width, and 0.83 mm thickness using a digital caliper). The T g values determined from the maximum peak of the tan δ, the dried films were fixed in the loading position inside the furnace 57,58 . Tan δ is represented as a dimensionless number, viewed as the mechanical damping factor represented as the ratio of loss and storage modulus, Eq. (1) 57 59 . Dried films (30 mm length, 10 mm width, and 0.83 mm, n = 6) were clamped between the static lower grip and moveable upper grip, assuring that the length of the films below stress was equal to 20 mm. The upper clamp was raised at a constant rate (0.5 mm/s) until the fracture of the films happened. According to the stress-strain plot, the U.T.S, elongation% at the breakpoint, and Young's modulus were calculated 54,55 .
Equilibrium water content (swelling). Films were cut into (30 mm length and 10 mm width, n = 6), then dried to reach constant weight in an oven at 30 °C. The dried films were carried into McCarthy bottles, each containing 15 mL of universal buffer pH 5 and pH 9 representing normal and infected urine with urease forming bacteria respectively at 20 °C. After 24 h, the soaked films were removed, blotted with filter paper to remove excess surface water, and reweighed. The equilibrium water content percent (EWC%) which is the percentage weight of water in the swollen polymer was calculated according to Eq. (2) 60 .
M i and M f represent the initial dried film mass and final film mass after buffer absorption, respectively 23 .
Cytotoxic evaluation of the fabricated hydrogel using (MTT assay). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium assay was applied to determine the cell proliferation, viability, and the potential cytotoxicity of fabricated films by MTT Cell Proliferation Assay (ATCC ® 30-1010 K) 61 . HEK 293 cells were seeded in a 96-well flat-bottom microtiter plate containing 100 μL medium at a density of 1 × 10 4 cells/well and allowed to adhere at 37 °C in a humidified 5% CO 2 incubator for 24 h. The dried films (discs, n = 6 in each case, with an average diameter of 3.735 ± 0.46 mm and average thickness of 0.83 ± 0.04 mm) sterilized using UV (FLUFRANCE, France) for 15 min and then soaked in RPMI 1640 media for softening before transferring to the wells and they were incubated for 24 h. Subsequently, 10 μL of MTT working solution was added to each well, and the plate placed in an incubator for 4 h. Then 100 μL Detergent Reagent (Stop Solution) was added to each well and the plate was placed for 2 h. Finally, the intensity of the formazan crystals (1) tanδ = Loss modulus/storage modulus. Statistical analysis. Data analysis was conducted using GraphPad Prism software version 7. The difference between groups was determined by one-way analysis of variance and two-way analysis (ANOVA) followed by the Tukey test. Data were represented as mean ± standard deviation (SD) and (p-value < 0.05) was deemed statistically significant.

Conclusion
This study describes the development of urological biomaterials to minimize microbial growth and therefore reduce the incidence of CAUTIs. The fabricated films were synthesized from p-HEMA successfully and loaded with RIF, CFX, and combined ratios of both. Drug loaded hydrogel showed better persistence to microbial growth and biofilm formation. The physicochemical nature of the drugs had impact on the films. Where loading p-HEMA with RIF alone or RIF in highest ratio led to lower biofilm formation. Results show the ability of employing the p-HEMA with the loaded drugs in urinary biomaterials to increase their lifespan and subsequently patient acceptance. www.nature.com/scientificreports/