Journal of Materials Chemistry B

In this study, we examined the modification of polypropylene non-woven fabrics (PP NWFs) via a one-step oxidation treatment using photo-activated chlorine dioxide radicals (ClO 2 (cid:2) ). The oxidised PP NWFs exhibited excellent antibacterial activity against both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). The mound structure and antibacterial activity in the modified PP NWFs disappeared upon washing with a polar organic solvent. After washing, nanoparticles of around 80 nm in diameter were observed in the solution. The results of several mechanistic studies suggest that nanoparticles can contribute to the antimicrobial activity of oxidised PP NWFs.


Introduction
Polypropylene non-woven fabrics (PP NWFs) have been prevalent in industries such as medicine, sanitation and packaging, owing to their high mechanical strength and biocompatibility.However, in medical or sanitary applications, the presence of bacteria on the surface of PP NWFs can lead to serious problems, such as bacterial colonization and biofilm formation. 1][4][5] The antibacterial modification of device surfaces is a prevalent technique for preventing bacterial infections. 6Various antibacterial agents, including antibiotics and antibacterial drugs, have been utilised as coatings on medical devices. 6lthough many antibiotics and antibacterial drugs have been developed, 7,8 the emerging antimicrobial resistance (AMR) of bacteria has made the control and treatment of bacterial infections difficult. 9,10For example, methicillin-resistant Staphylococcus aureus (MRSA) shows resistance against most b-lactam antibiotics, and the spread of MRSA in hospitals is an urgent problem. 11,12Hence, an antibacterial strategy that is independent of antibiotics is a desirable solution.
Recently, antimicrobial peptides, 13,14 cationic compounds, 15-17 metal nanoparticles, 18,19 and polymer nanoparticles [20][21][22] have received considerable attention because of their broad-spectrum activities.They absorb on the membranes of bacterial cell and induce leakage of the cell contents.Membrane disruption is also effective against drug-resistant bacteria.For example, a surface modified with Ag nanoparticles has shown antibacterial activity against MRSA. 23y contrast, the direct modification of PP materials with antibacterial agents is difficult because they are hydrophobic and exhibit poor interactions with other molecules.5][26][27][28] Hence, there is a need for an efficient and cost-effective method for the antibacterial modification of PP NWFs.
In recent years, our group has developed a surface-modification method for polymer materials that uses photo-activated chlorine dioxide radicals (ClO 2 ). [29][30][31][32] The photo-activated ClO 2 act as a strong oxidizing agent that is capable of cleaving C-H bonds, thus introducing oxygen-containing functional groups onto the polymer surface.This technique improves the hydrophilicity of PP films for better reactivity.In addition, owing to the gaseous oxidants used, our method is applicable to the oxidative modification of threedimensional network structures such as PP NWFs. 32n this study, we found that oxidised PP NWFs exhibited excellent antibacterial properties against Escherichia coli (Gramnegative) and Staphylococcus aureus (Gram-positive).The modified PP NWFs were characterised using Fourier-transform infrared (FT-IR) spectroscopy, field-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and toluidine blue O (TBO) assays.The antibacterial mechanism of the modified PP NWFs was also investigated by analysing the wash solvent used for the washing of PP NWF products.Unlike other methods, the as-described antibacterial modification process does not require antibiotics or antibacterial agents.In addition, the treatment described herein is convenient, is completed in one step, and is expected to be used practically.

Oxidation of PP NWFs using photo-activated ClO 2
The PP NWFs (50-60 mg; dimensions of 4 cm Â 4 cm) were oxidised using ClO 2 generated from an aqueous solution (20 mL) of NaClO 2 (200 mg) and 35% HCl aq.(100 mL), under irradiation with a 60 W LED lamp (l = 365 nm, 5 mW cm À2 ).0][31][32] An aqueous solution of ClO 2 was added to the outer moat of the glassware, while the sample was placed on the inner side.During the reaction, the samples were protected from direct UV irradiation using an aluminium sheet.
The reaction was conducted for 20 min at 30 1C, 60 1C or 80 1C, and the oxidised samples were washed with distilled water and dried in vacuo -these are designated as Ox-30, Ox-60, and Ox-80, respectively (Scheme 1a).

Solvent washing of oxidised PP NWFs
Two solvents (ethanol (70%) and acetone) were used for washing.Solvent washing was performed by placing the oxidised PP NWF samples in each solvent and shaking for 1 min.The washed samples were then rinsed with distilled water three times, followed by drying in vacuo.The samples washed in ethanol and acetone, e.g., for Ox-80, are denoted as Ox-80-EtOH and Ox-80-Ac, respectively (Scheme 1b).

Antibacterial testing of PP NWFs
Antibacterial testing of the PP NWF samples was performed according to the ''absorption method'' described in JIS L 1902 (corresponding to ISO 20743), a quantitative test to determine the antibacterial activity of textiles. 33In this study, the sample weight was modified from 400 AE 50 mg to 14 AE 1 mg, the nontreated (control) and oxidised (test) PP NWF samples were cut into pieces (14 AE 1 mg; dimensions of 2 cm Â 2 cm), and E. coli (Gram-negative) and S. aureus (Gram-positive) were selected as the test bacteria.The bacterial suspensions used to inoculate the samples were prepared as follows.One colony of bacteria was added to 20 mL of nutrient broth (NB) and incubated at 37 1C for 24 h (110 rpm).Next, 400 mL of the previous suspension was added to 20 mL of NB and incubated for 3 h at 37 1C (110 rpm).The bacterial concentration was measured and diluted with 1/20 NB (in distilled water) to approximately (1-3) Â 10 5 colony-forming units mL À1 (CFU mL À1 ).Then, each sample (six pieces each of the test sample and control sample) was inoculated with 200 mL of the prepared bacterial suspension.Three test samples and three control samples were mixed with 20 mL of physiological saline solution (8.5 g NaCl, 2.0 g polysorbate 80, in 1 L distilled water) immediately after inoculation.The remaining three test samples and three control samples were incubated at 37 1C for 20 h.Then, 20 mL of physiological saline solution was added to each sample and shaken.The pour-plate method was used to measure the number of live bacteria before and after incubation in each physiological saline solution used for washing.A series of 10fold dilutions of the supernatant in physiological saline solution was performed, and 1 mL of each supernatant was plated on standard agar medium.The colonies in each plate were counted after 24 h of incubation at 37 1C.The antibacterial activity (A) was calculated using the following formula, and the values are defined in Table 1: where F is the growth value of the control sample {i.e., (log 10 CFU post-incubation ) À (log 10 CFU pre-incubation )}, and G is the growth value of the test sample {i.e., (log 10 CFU post-incubation ) À (log 10 CFU pre-incubation )}.The percentage reduction in bacteria (%) after incubation for 20 h was calculated using the following formula: The S. aureus culture medium was prepared using 10 g of Hipolypepton, 2 g of yeast extract, 1 g of MgSO 4 Á7H 2 O, and 1 L of distilled water instead of NB.Incubation was performed at 30 1C instead of 37 1C according to recommended culture conditions of the NBRC.

Surface characterization of PP NWFs
The surface functional groups of the PP NWF samples were determined via room-temperature FT-IR spectroscopy using a Spectrum TWO spectrophotometer (PerkinElmer, USA) with a diamond window.The surface elemental compositions and bonding states of the PP NWF samples were determined via XPS using an ESCA 3057 instrument (Ulvac-PHI, Japan) with a dual-anode Mg Ka source (1253.6 eV) operating at 400 W. A pass energy (PE) of 117.40 eV was used to acquire wide-range survey spectra, while a PE of 29.35 eV was used to acquire narrow C 1s spectra.The XPS data were analysed using Multi-Pak software (version 9).All spectra were calibrated with the hydrocarbon C 1s photoemission set at the 285.0 eV binding energy.The surface morphologies of the PP NWF samples were observed via FE-SEM using a JSM-F100 microscope (JEOL, Japan) set at 3.0 kV.

Quantification of surface carboxy groups on the PP NWFs
The surface carboxy (-COOH) groups of the PP NWF samples were quantified using the toluidine blue O (TBO) assay, based on the 1 : 1 formation of TBO with -COOH via electrostatic interactions. 34First, PP NWF samples (2 cm Â 2 cm) were incubated in 4 mL of the TBO solution (0.1% TBO in 1 mM NaOH) at 40 1C for 15 min (120 rpm).The samples were washed with 4 mL of 1 mM NaOH at 40 1C for 5 min with stirring (120 rpm).The washing process was repeated at least five times until the supernatant was clear.Finally, TBO was desorbed via incubation in 4 mL 20% SDS solution at 40 1C for 30 min (120 rpm).The TBO concentration in the SDS supernatant was calculated from the absorption at 630 nm using an ultravioletvisible (UV-Vis) spectrophotometer V-750 (Jasco, Japan).The results were calculated as the average of three replicates.

Acetone washing of oxidised PP NWFs
First, 10 pieces of the PP NWFs samples (4 cm Â 4 cm), including the non-treated, Ox-30, Ox-60, and Ox-80 samples, were prepared.The prepared samples were then placed in acetone (20 mL) and shaken for 1 min.

Optical microscopy observation of acetone washing solution
Optical microscopy of the acetone washing solution of the PP NWF samples was performed using a Spotlight 200i FT-IR microscope with a Spectrum Two spectrometer (PerkinElmer, USA).

Characterization of nanoparticles in the acetone washing solution of the PP NWFs
Optical microscopy measurements were performed using a BX53M optical microscope (OLYMPUS, Tokyo, Japan).The diameter of the nanoparticles was evaluated via dynamic light scattering (DLS) using an ELSZ-2 particle size analyser (Otsuka Electronics Co., Ltd, Japan).The samples for DLS measurements were prepared as follows.The PP NWF samples oxidised at 80 1C were placed in acetone and washed with distilled water.The acetone solution was shaken for 1 min, and the PP NWF samples were removed.The acetone solution was centrifuged at 12 000 rpm for 1 min for separation of the fibrous dispersion from the solution, and the supernatant including the nanoparticles was used for the DLS measurements.The zeta potential values of the nanoparticles in H 2 O were measured using the ELSZ-2 instrument (Otsuka Electronics Co., Ltd, Japan).For the zeta potential measurements, the nanoparticle solution was prepared as follows.The Ox-80 samples were placed in acetone and then washed with distilled water.The acetone solution was shaken for 1 min and the PP NWFs were removed.The acetone solution was centrifuged at 12 000 rpm for 1 min for separation of the fibrous dispersion from the solution, and the supernatant including the nanoparticles was heated at 70 1C until the acetone had evaporated.The residue was dispersed in 60 mL of acetone followed by adding 3 mL of distilled water.Aqueous solutions were used for the zeta potential measurements.The results were calculated as the average of 10 replicates.

Results and discussion
Antibacterial testing of PP NWFs oxidised using photoactivated ClO 2 We evaluated the antibacterial properties of the PP NWFs oxidised using photo-activated ClO 2 . The antibacterial properties of the as-prepared PP NWF samples, Ox-30 and Ox-80, were evaluated according to JIS L 1902. 33The results are summarised in Table 2. Ox-80 showed a high antibacterial activity of over 3 in both E. coli and S. aureus (5.57against E. coli and 5.12 against S. aureus) reduced by more than 99.9% compared with the nontreated PP NWFs, as per Table 1.Unlike Ox-80, Ox-30 showed an antibacterial activity of less than 2 (0.27 against E. coli and 1.45 against S. aureus), indicating that Ox-30 has no antibacterial activity against E. coli and S. aureus.We also evaluated the antibacterial activity of Ox-60 to examine the relationship between the antibacterial activity and the reaction temperature.As a result, Ox-60 showed antibacterial activity against E. coli, and the bacterial growth was inhibited by 99.9% (Table S1, ESI †).Next, we performed an antibacterial test for Ox-80 against E. coli after solvent washing with 70% ethanol and acetone.Table 3 presents the antibacterial test results.The antibacterial activity of Ox-80-EtOH and Ox-80-Ac against E. coli was 0.78 and À0.08, respectively, indicating no antibacterial activity against E. coli, in contrast to Ox-80.

Characterization of oxidised PP NWFs
Chemical analyses and surface observations of the oxidised PP NWF samples were performed.First, the functional groups were characterised using FT-IR spectroscopy (Fig. 1, and Fig. S1, ESI †).In the oxidised samples, new peaks were observed at 1720 cm À1 , corresponding to the stretching of the carbonyl (CQO) groups. 32Next, the oxidised PP NWF samples were washed with 70% ethanol and acetone.After washing, the characteristic carbonyl peaks at 1720 cm À1 were diminished, especially in Ox-80, indicating that the newly incorporated carbonyl groups were unstable in polar organic solvents.The FE-SEM images obtained (Fig. 2) revealed mound structures on the surface of Ox-80, as previously reported. 32After washing with ethanol and acetone, the mounds on Ox-80 disappeared, indicating that the surface deposits were washed away by polar organic solvents.Considering the diminished 1720 cm À1 peaks, the surface deposits are presumed to be rich in carbonyl groups.
To further characterize the surface of the PP NWF samples, the surface elemental and chemical compositions were determined via XPS (Fig. S2 in the ESI †: the wide-scan XPS spectra).Only carbon was detected in the control samples, whereas Ox-30 and Ox-80 contained 15.1% and 12.0% oxygen, respectively (Table 4).By contrast, Ox-80-EtOH contained 5.0% oxygen, indicating a decreased oxygen ratio after washing with ethanol.To further our understanding, the C 1s XPS spectra of the PP NWF samples were analysed.The C 1s XPS spectra exhibit five distinct peaks at 285.0, 286.3, 287.0, 288.3, and 289.1 eV, which are attributed to C-C, C-O, C-Cl, C-Cl 2 /CQO, and -COO groups, respectively (Fig. S3 in ESI †).Table 5 lists the ratios of the functional groups.In Ox-30 and Ox-80, the ratio of -COO groups was 2.8% and 4.2%, respectively, suggesting that more carboxy groups were introduced to the PP NWFs during   View Article Online oxidation at 80 1C than at 30 1C.In Ox-80-EtOH, the ratio of -COO groups was only 0.1%, which is consistent with the decreased intensity of the carbonyl peak at 1720 cm À1 after ethanol washing.The C 1s XPS spectra showed a decrease in carboxy groups upon washing with polar organic solvents.Here, we quantified the surface carboxy groups per unit mass of PP NWFs using the toluidine blue O (TBO) assay.Table 6 present the results of the TBO assay (the data are shown in Fig. S4 (ESI †)).The non-treated samples exhibited limited TBO adsorption, whereas the oxidised samples exhibited good TBO absorption.The calculated surface carboxy groups on the non-treated, Ox-30, and Ox-80 samples were 0.5 AE 0.2, 2.7 AE 0.1, and 10.0 AE 1.0 mmol g À1 , respectively.By contrast, the calculated surface carboxy groups on Ox-80-EtOH and Ox-80-Ac were 0.8 AE 0.1 and 12.0 AE 0.1 mmol g À1 , respectively.These results suggest a decrease in the surface carboxy groups after ethanol washing.

Antibacterial mechanism of oxidised PP NWFs
In recent years, antibacterial nanoparticles with various surface potential values have been reported. 20,21,35In particular, Sarian et al. reported negatively charged cyanoacrylate nanoparticles (20-250 nm) that show a growth inhibitory effect against Grampositive and Gram-negative bacteria. 20The authors considered that the antibacterial activity was derived from disruption of the bacterial membrane and the subsequent generation of reactive oxygen species (ROS) via physical interactions. 20The cyanoacrylate nanoparticles also induced disorder of the cell walls and inhibited growth in green microalgae, such as Chlamydomonas reinhardtii, 36,37 and several non-green algae. 38Here, we considered the formation of nanoparticles during oxidation with photoactivated ClO 2 as a factor that governs the antibacterial activity.
The experimental results showed that Ox-80 exhibited excellent antibacterial activity, whereas Ox-80-EtOH and Ox-80-Ac did not, indicating that washing with polar organic solvents affected the antibacterial activity.The physical and chemical characteristics of the samples washed with polar organic solvents were examined to identify their antibacterial mechanism.Acetone was selected as the washing solvent because it has a lower boiling point than ethanol.The soluble and insoluble components of the acetone washing solution of Ox-80 were analysed.The results from the examination of the acetone-soluble components are shown in the ESI.† Ten pieces of the Ox-80 sample (571.2 mg) were prepared and washed with acetone (20 mL).After washing and drying, the weight decreased to 563.5 mg, which is a 1.35% loss (7.7 mg).Ten pieces of the non-treated and Ox-30 samples were also washed with acetone.By contrast, the weight loss from acetone washing in the non-treated and Ox-30 samples was only 0.04% and 0.34%, respectively.The difference in weight change is consistent with the disappearance of the surface mounds after washing with a polar solvent, as shown in Fig. 2. Therefore, we predict that this weight change is due to cleavage of the PP main chains during oxidation to form smaller products that are acetone-soluble.
The acetone-insoluble components of the washing solution were also examined.Fibrous dispersions were observed in the acetone solutions of both the non-treated and Ox-80 samples (Fig. S5 (ESI †)).An optical microscope (Spotlight 200i) was used to for further examination, and revealed that PP fibers and middle-sized particles (B50 mm) are present in both samples (Fig. S6 and S7, ESI †).By contrast, small particles (o1 mm) were observed only in the Ox-80 sample (Fig. S6, ESI †).Dynamic light scattering (DLS) measurements were carried out, and it was determined that 94.4% of the small Ox-80 particles were distributed in the range of 65-140 nm (Fig. 3).The average diameter of the nanoparticles in this area was 82.6 AE 16.1 nm.
The zeta potential of nanoparticles in H 2 O was À25.0 AE 2.7 mV. Here, we predict that the B80 nm nanoparticles found on the PP fibers are related to its antibacterial activity, and that the oxidation reaction temperature affects the number of  Based on these results, we suggest that the excellent antibacterial activity of the Ox-60 and Ox-80 PP NWFs can be attributed to the B80 nm nanoparticles formed during oxidation.There was a correlation between the antibacterial activity of the PP NWFs and the number of nanoparticles formed, and the acetone extracts of Ox-80 that included the nanoparticles showed the growth inhibition of E. coli (ESI); however, other factors could not be ruled out in this experiment.Whether the nanoparticles themselves impart antibacterial properties to the PP NWFs remains to be examined.Further investigations focusing on the antibacterial mechanism of nanoparticles are ongoing in our laboratory.
In medical situations, PP NWFs are used for disposable equipment, such as masks or gowns, because of their low cost.Hence, the antibacterial modification method for PP NWFs is expected to be inexpensive for practical and industrial use.By contrast, our modification method has an advantage regarding the cost.The ClO 2 utilized for our modification can be prepared simply using inexpensive reagents (NaClO 2 and HCl).In addition, our method requires only a single step and the modification effects are stable in air and H 2 O (Fig. S9, ESI †).
Considering the above, our findings are of scientific and practical interest.

Conclusions
We demonstrated that PP NWFs oxidised at 80 1C using photoactivated ClO 2 exhibit excellent antibacterial activity against E. coli (Gram-negative) and S. aureus (Gram-positive).To investigate the underlying mechanism of antibacterial activity, we analysed the polar organic solvents used to wash the oxidised PP NWFs.We found nanoparticles of B80 nm in diameter in the solvent, particularly when the oxidation step was carried out at high temperature (60 1C and 80 1C).Our findings suggest that the nanoparticles are formed during oxidation, and are involved in the antibacterial properties of the oxidised PP NWFs.The detailed structure of the nanoparticles, their mechanism of antimicrobial activity, and the long-term stability of the oxidized non-woven fabrics are still under investigation and will be reported in the near future.

Fig. 3
Fig.3Diameter of nanoparticles as measured using DLS.The nanoparticles were extracted from the Ox-80 PP NWF samples using acetone.The blue solid line and the orange dotted line show the number (%) and the cumulative distribution (%), respectively.

Table 1
Antibacterial activity rating associated with the percentage reduction in bacterial growth This journal is © The Royal Society of Chemistry 2023

Table 2
Results of antibacterial testing in Ox-30 and Ox-80

Table 3
Results of antibacterial testing in Ox-80-EtOH and Ox-80-Ac

Table 6
Amount of adsorbed TBO for quantification of the surface -COOH groups in the PP NWF samples (n = 3)

Table 5
Ratios of carbon-containing functional groups (%) on the PP NWF samples at specified peaks (eV), as determined via C 1s XPS