Antibacterial properties of enzymatically treated PET fibers functionalized by nitric oxide

.


Introduction
Serious infections caused by pathogens are the most common in community-acquired and hospital-acquired.Cholecystitis, bacteremia, enterocolitis, urinary tract infections (UTI), and other clinical illnesses such as meningitis and pneumonia are frequently caused by Escherichia coli [1,2].In addition, Staphylococcus aureus causes a wide range of infections, including life-threatening conditions such as pneumonia, meningitis, endocarditis, and bacteremia [3,4].Currently, E. coli accounts for the majority of clinical infections requiring hospitalization (17.3%) after S. aureus infection (18.8%) [5].
Approximately one in every 31 hospital patients has a healthcare-associated infection (HAI) in the United States and the most common HAIs are related to medical devices [6].
Infections of medical devices are often caused by contaminated tools that are implanted or bacteria attaching to the device's surface and then colonizing the surface, leading to a serious infection in human health [7].To overcome these issues, antifouling and antibacterial strategies have been mainly investigated using various materials [8,9].Antifouling is the ability of specific materials to impede the initial attachment of unwanted microorganisms by coating or surface treatment [10].However, the film formed by these coatings usually has a short lifetime and then it becomes ineffective [11].Meanwhile, antibacterial refers to any substances that can kill, inhibit, or slow down the growth of bacteria [12].Antibacterial agents such as disinfectants (hydrogen peroxide, hypochlorite), antibiotics, and antiseptics have several shortfalls including environmental toxicity problems and low activity due to bacteria resistance [13,14].Therefore, an effective and long-term antibacterial agent is needed to achieve biomedical demands.
In this work, recombinant PETase was used to enzymatically treat waste PET fibers, increasing the number of hydroxyl and carboxyl groups on the surface.Due to its environmentally friendly and moderate reaction conditions, enzymatic hydrolysis has attracted significant interest in industries [25].Here, the porous structure of carboxylated PET fibers has the potential to be utilized as a matrix for the production of NO-conjugated PET fibers.In addition, polyethylenimine (PEI) grafted PET fiber was employed as a secondary amine to conjugate with NO gas releasing NONOate product for an antibacterial agent.The antibacterial activity was performed using the colony forming units (CFU) method against E. coli and S. aureus.

Preparation of PEI-grafted PET fibers
First, PET fibers were hydrolyzed by PETase (20 µg/mL) at 30°C for 3 days to obtain carboxylated PET fibers.After that, EDC/NHS was utilized to activate the carboxylated surface before adding PEI.The carboxylated fibers (10 mg) were incubated with 0.1 wt% EDC for 10 min and 0.1 wt% NHS for 60 min.Next, PEI solutions (3 wt% and 5 wt%) were added to PET fibers solution with a volume of 1 mL.The solutions were incubated at room temperature for 12 h avoiding light with shaking (200 rpm) to obtain PEI-grafted fibers.Subsequently, the products were washed with distilled water and dried at 30⁰C overnight for further analysis.

Preparation of NO-conjugated PET fibers
For NO loading, aminated PET fibers (20 mg) were dispersed in 2 mL of solvents (water, methanol, or acetonitrile).The glass vials were inserted in a stainless-steel reactor with vigorous stirring.The pressure of the nitrogen-purged reactor was kept at 10 atm for 3 days.NO-conjugated PET fibers were obtained after washing twice with its solvent and drying the products in the oven at 30⁰C overnight.

Characterization
The number of amino groups in PEI-grafted PET fibers was evaluated by ninhydrin test.While, the chemical bonds of the functionalized PET fibers were identified by fourier transform infrared spectroscopy (FTIR) (FTS 3500, Bio-Rad Laboratories Sadtler Division, USA).The fiber samples were placed on the glass surface by double tape that formed a flat surface before analysis.
Furthermore, the amount of NO released was measured by Cayman's nitrate/nitrite colorimetric assay kit in a two-step process.NO-functionalized PET fibers (2 mg) were added to 1 mL phosphate-buffered saline (PBS) pH 7.4 and incubated at different time points (37°C).Then, NO release in supernatant was centrifugated at 6000 rpm for 5 min.80 µL of supernatant was mixed with 10 µL of each cofactor and nitrate reductase followed by incubation at room temperature for 1 h.After the required incubation time, 50 µL of each Griess reagent (R1 and R2) was added to the solution and incubated for 10 min to develop a purple color.The absorbance of samples was measured at 540 nm using a plate reader and total NO content was determined using nitrate standard curve.

Antibacterial test
The antibacterial activity of NO-conjugated PET fibers against E. coli and S. aureus was determined based on the number of colony forming units (CFU).Bacteria cultures were grown in media at 37°C overnight.Subsequently, the bacteria pellets were centrifugated at 6000 rpm for 10 min, resuspended in sterile PBS to reach OD600 of 1, and then diluted (1/10 6 ) in sterile PBS.The functionalized fibers (2 mg) were added to 0.5 ml diluted bacteria solution.Each bacteria mixture was incubated at 37℃ for 7 h to initiate NO release.Then, the 0.1 ml bacteria solution was spread on an agar medium followed by incubation at 37℃ overnight.The number of bacteria colonies was counted and the results were compared with the control samples (pure bacteria and aminated PET fibers).The antibacterial activity was calculated based on the colony numbers (CFU/mL).

Materials preparation and characterization of PEI-grafted carboxylated PET fibers
Briefly, PET fibers were enzymatically hydrolyzed with PETase to obtain carboxyl groups on the surface followed by EDC/NHS activation.First, the carboxyl groups were activated using EDC, forming an amine-reactive O-acylisourea intermediate that spontaneously reacted with amine groups to establish an amide bond.The addition of Sulfo-NHS led to NHS ester formation which stabilized amine reactive intermediate for efficient conjugation to the amine groups.Subsequently, the secondary amine of PEI was grafted to the active carboxylated PET fibers.
As shown in Fig. 1, the surface morphologies of PET fibers before and after PEI grafting were observed using SEM analysis.The surface of untreated PET fibers was smooth, while PETase enzyme caused obvious cracks and porous on the surface as reported in previous studies [27].PEI reacted with carboxyl groups on PET fibers successfully forming a layer that coated the porous structure of enzyme-treated PET fibers.
Ninhydrin assay has been widely used for qualitative and quantitative determination of primary and secondary amines in peptides, proteins, or nanomaterials [28].Based on the purple color intensity developed in the reaction solution, the presence of amine groups in the PEI-grafted PET fibers was confirmed.However, the absence of EDC/NHS activation in the preparation of PEI-grafted PET fibers did not produce Ruhemann's purple due to the lack of stable amine-reactive intermediate as previously described.The amine contents were determined according to the ninhydrin standard curve to be 2.78 and 4.14 μmol/g for carboxylated PET fibers treated with EDC/NHS-PEI 3% and EDC/NHS-PEI 5%, respectively (Table 1).Therefore, PEI-grafted PET fibers prepared from 5 wt% PEI solution were further used for analysis and preparation of NO-conjugated PET fibers because of their higher amine content.The chemical composition of the functionalized PET fibers was analyzed by FTIR as shown in Fig. 2. The FTIR spectra of hydrolyzed PET fibers show adsorption at 2900 cm -1 for C-H stretching, 1716 cm -1 for carbonyl group (CO), 1100-1250 cm -1 for asymmetrical C-O-C stretching, and 518-1100 cm -1 for aromatic rings.Meanwhile, untreated PET fibers exhibit much smaller amount of C-H stretching and lower intensity of characteristic spectra compared to hydrolyzed PET.In addition, N-H groups appeared after PEI treatment at 3325 cm -1 and 1600 cm -1 indicating the secondary amine of PEI had been successfully grafted onto PET fibers.

NO conjugation to PEI-grafted PET fibers
Since PET fibers have poor solubility in organic solvents [29], various solvents have been used to study NO release profile of NO-conjugated fibers in PBS buffer at different time points.Based on the calibration curve, the total NO concentration was determined by the Griess method.As shown in Fig. 3, untreated PET fibers as a control showed the absence of NO release.Both NO-fiber/without solvent and NO-fiber/water showed rapid release of NO in the first 7 h followed by a steady NO release over 12 h to reach maximum NO concentrations of 6.72 μM and 7.24 μM, respectively.Besides, NO-fiber/methanol and NOfiber/acetonitrile exhibited relatively high NO flux in the first 9 h and thereafter a steady release of NO for 12 h with maximum NO concentrations of 15.09 μM and 12.16 μM, respectively.Interestingly, NO-fiber/methanol released NO about twice than NO-fiber/no solvent, indicating that methanol is a suitable carrier for PEI-grafted PET fibers to obtain a higher NO payload.The continuous release of NO-fiber/methanol over 12 h has great potential for biomedical applications such as antibacterial agent which requires sustained NO release.PET fiber NO-fiber/no solvent NO-fiber/water NO-fiber/methanol NO-fiber/acetonitrile Fig. 3. NO release profile of NO-conjugated PET fibers (2 mg/mL) prepared in different solvents over a period of 12 h at 37°C.

Antibacterial activity of NO-conjugated PET fibers
The antibacterial activities of the NO-conjugated PET fibers that grafted in various solvents against E. coli and S. aureus were carried out by counting the number of CFUs (Fig. 4).The activity of PEI-grafted PET fibers was also evaluated since PEI is known as antibacterial agent due to its polycationic nature [30].PEI-fibers resulted in a 61.7% reduction of E. coli and a 55.9% reduction of S. aureus.Furthermore, NO gas was reacted with a secondary amine of PEI to improve the killing effect because of its excellent antibacterial properties [31].Evidently, NO-fiber/methanol and NO-fiber/acetonitrile showed an increase in the percentage reduction of both bacteria as compared to PEI-fibers.The highest bacterial reduction by ~1 log (90.2% reduction) against E. coli and ~0.5 log (71.1% reduction) against S. aureus was observed with 2 mg of NO-fiber/methanol.This result was supported by the highest NO release profile of NO-fiber/methanol in solution indicating that the improving killing activity could be attributed to the NO content.NO is able to kill bacterial cells by direct or indirect oxidation, especially through the formation of peroxynitrite (-OONO) [32,33].Meanwhile, NO prepared in other solvents did not exhibit significant antibacterial activity due to lower NO release.NO-conjugated PET fibers effectively killed E. coli as compared to S. aureus.It is speculated that the thick peptidoglycan in gram-positive S. aureus serves as a protective barrier that inhibits the effect of NO release on the bacteria.

Conclusions
In this study, the first strategy for antibacterial applications using NO-releasing PETasehydrolyzed PET fibers was demonstrated.NO-conjugated PET fibers were successfully prepared via conjugation of NO gas with PEI-grafted PET fibers which exhibit a continuous NO release profile over 12 h.Antibacterial efficacy experiments resulted in up to a 90.2% reduction in Gram-negative E. coli and a 71.1% reduction in Gram-positive S. aureus after exposure to the diazeniumdiolate-PET fibers material.Overall, this novel antibacterial agent may offer great potential applications as a medical device coating to prevent deviceassociated infections.

Table 1 .
Amine content on PEI-grafted PET fibers by ninhydrin assay.