Introduction

The prevalence of various infections especially in the context of global crisis caused by detrimental microorganisms contributes to the destruction of public health and it is the source of serious problems, particularly since the transmission of COVID-19. The impact on public well-being is also connected with the spread of multidrug-resistant bacteria (Brown and Wright 2016; Pascuta and Vodnar 2022). Every day, materials employed in healthcare systems can accumulate infectious agents such as the coronavirus, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus (MRSA). It is noteworthy, that certain microbes exhibit remarkable resilience with the capability to persist for over 90 days (Neely and Maley 2000). Thus, the shortcomings of antimicrobial medicines can be overcome by the integration of bactericidal materials, photodynamic inactivation and personal protective equipment (PPE) against infections to control the spread of diseases (Duan et al.

Fig. 3
figure 3

1H, 13C and 29Si NMR (500 Hz) spectra of 3-[(Triethoxysilylpropyl)thio]propylsuccinic anhydride (TESSA) in CDCl3

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Fig. 4
figure 4

The comparison of the 1H-13C HSQC NMR (500 Hz) spectra: a 3-[(Triethoxysilylpropyl)thio]propylsuccinic anhydride (TESSA) in CDCl3, and b Triethoxysilyl-substituted phenosafranin (TESPSF) in CD3OD in the range of (0–4.2 ppm)

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Finally, the signals proving the presence of alkoxysilyl groups needed for further reactions are found as a triplet and quartet at 1.21 and 3.81 ppm in the 1H spectrum, and at 18.3 and 58.4 ppm respectively in the 13C spectrum. The Fig. 4 illustrates the 1H-13C HSQC NMR spectra in the range up to 4.2 ppm before and after binding with PSF. A significant difference is between CH2 protons No. 6 and 8 and CH proton No. 7, shifted from 3.11 to 2.78 ppm at the same carbon position assigned to 40.5 ppm. The ring-opening process removes the non-equivalence of protons number 6 from the five-membered anhydride structure. In the TESSA spectrum, we observed them at 1.75 and 2.01 ppm, and after the reaction was completed, they approached to 1.73 ppm. The resonance signals from 5.80 to 8.3 ppm correspond to the aromatic protons and the NH amide proton (8.25 ppm) in TESPSF.

The fabrication of light-induced antibacterial cellulose handsheets

The cellulosic handsheets were prepared from commercial bleached soft-wood pulp (BSK) using a laboratory paper machine according to the industrial standard ISO 5269-1 (2005). No additional pulp milling, no other mechanical or chemical fiber modification methods were performed, therefore resulting sheets were very porous and relatively mechanically resistant. Silica-starch emulsions based on MTES were prepared from the PSF hydrochloride solution E1 and the TESPSF solution E2. The photosensitive dye was dissolved in the sol and formed a homogeneous emulsion that uniformly coated the surface of the cellulose sheets according to the procedure described earlier (Ganicz and Rozga-Wijas 2021). The process of manufacturing antibacterial cellulose materials is illustrated in Scheme 2.

Scheme 2
scheme 2

The diagram shows the manufacturing process of the new photosensitive cellulosic materials

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The surface concentration of PSF on modified papers was calculated based on the concentration of PSF in the starting siloxane sol and the weight of the paper cover listed in Table 1 and Table 5s in data repository. Besides, the basic building block of the silsesquioxane network is composed of a repeating unit of MeSi(O–)OH with a mass of 76.13.

The hydrolysis of a trialkoxysilane leads to silanetriol (Pietschnig and Spirk 2016), which can be stabilized by bifunctional secondary and tertiary amines (e.g., bipyridine or piperazine) to maximize the possible interaction sites (Prabusankar et al. 2004). Therefore, we expected that PSF endowed with two primary amine groups will form stable adducts with the silanol (SiOH), siloxide (SiO–), and siloxane (Si–O–Si) surface groups (Scheme 3).

Scheme 3
scheme 3

Plausible structure highlighting the role of PSF in a sol–gel process to produce a photosensitive coating for cellulose sheets

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The solid-state 29Si and 13C NMR spectra were taken to illustrate the chemical structure of the covering on the cellulose fibers. For this purpose, the siloxane-starch emulsion E2.4 loaded with TESPSF was maintained at room temperature for 30 days and then centrifuged. The precipitate was washed several times with methanol, dried, and heated to 110 °C under reduced pressure. The 29Si NMR spectrum of the light violet powder shows a change in the silicon shift from − 45.47 ppm characteristic of TESPSF to − 56.17 and − 66.01 ppm for the coating (Fig. 5a and b and Fig. 2s in data repository). Both broad peaks were assigned to the units T2 = (HO)(SiO)2SiCH2−, and T3 = (SiO)3SiCH2−, which confirmed the formation of a T-type silsesquioxane network (Sato et al. 2022). The central silicon atom is connected to three siloxane bonds (T3) or two siloxane bonds and one –OH group (T2). There are no signals for the M or Q structures. The sol–gel process is not selective, therefore the polycondensation of trifunctional silanes results in a variety of silsesquioxane structures, including e.g., linear, cyclic, ladder, cage, and incomplete cage.

Fig. 5
figure 5

a The 29Si NMR spectrum of the TESPSF in CD3OD; b 29Si MAS NMR spectrum of the silica-starch E2.4 gel; c The FTIR spectrum of the handsheets of E1.5 coated and uncoated papers

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The reflection FTIR analysis was performed to compare the handsheets before and after coating the cellulose fibers with the siloxane sol (Fig. 5c). The ATR-FTIR spectrum of native paper displays the classical bands with maxima wavenumbers at 3340 cm−1 (OH stretching), and signals at following frequencies 1325, 1015, 1094 and 1163 cm−1 are assigned to sv(C–O), v(C–H) bending, and sv(C–O and C–C) mode, respectively (Kizil et al. 2002; Meng et al. 2015). The methyl silsesquioxane-modified paper, both with and without PSF, indicated two new bands at 1270 and 770 cm−1 assigned to sv(Si–CH3) and v(Si–CH3) bending, confirming the formation of the silsesquioxane polymer by the polycondensation reaction (Siuda et al. 2019). Moreover, signals in the range of (1127–1004 cm−1) were attributed to svs(Si–O–Si) and svas(Si–O–C) modes in polymers, but they overlapped with the bands of cellulose vibrations. Absorption bands characteristic of the PSF dye are obscured in the FTIR spectrum of modified handsheets due to the low concentration of PSF. The molar ratio of PSF to the alkoxysilyl group (PSF/CH3SiO) ranges from 8.0 × 10–3 to 8.0 × 10–6. Thus, the concentration of the methylsilyl group was three orders of magnitude higher than PSF, and the surface concentration of dye molecules increased from 9.0 × 10–7 to 1.0 × 10–3 mol per m2 for E1 and from 6.0 × 10–7 to 3.0 × 10–4 mol per m2 for E2 (Table 1).

X-ray energy dispersive spectrometer (EDS) analyses provided images of the elemental dispersion of carbon, oxygen, and silicon on cellulose coated with PSF embedded in a siloxane material. The measurement of selected samples (Fig. 6) shows that silicon was well dispersed on the modified surface E1 and E2 of the cellulose sheets. The EDS elemental analysis allowed us to estimate the amount of silicon and carbon in the silsesquioxane layer. However, it is not possible to determine the amount of photosensitizer anchored into the inorganic structure because nitrogen is not detectable. A decrease in the amount of carbon was observed for the E1.4 and E2.5 materials compared to the pure cellulose sheet due to covering of the cellulose surface with methylsilsesquioxane groups CH3Si(O–)3 (see Table 1).

Fig. 6
figure 6

The X-ray energy dispersive spectrometer (EDS) analyses of PSF-silica cellulose materials images display the elemental dispersion of C, O, and Si (yellow zone) at the micrometric level: a SEM images of cellulose natural handsheets and covered b E1.4 with MTES sol–gel with PSF and c E2.5 with MTES sol–gel with TESPSF

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Surface properties

The surface of the cellulose fibers was coated with the silsesquioxane gel and starch, then dried using a rotary press machine and applying elevated temperature in the range of 95–100 °C to obtain the uniform coating. The coating was not a continuous layer but formed a cluster of spherical nanoparticles on the surface of the fibers after using aqueous emulsions of MTES (Ganicz and Rozga-Wijas 2021). The surfaces of E1/S and E1.1–E1.5 papers modified with PSF were examined by SEM (Fig. 6b and Fig. 3s in data repository). The photomicrographs did not show visible differences between the aforementioned samples. However, the change in morphology was revealed in the case of the E2 sol–gel sample. The presence of the new sol–gel precursor TESPSF initiates the formation of a layer instead of clusters of microspheres on the E2/S and E2.1–E2.5 cellulose fibers (Fig. 6c and Fig. 3s in data repository).

The presence of PSF and its grafting in the silsesquioxane network was confirmed (apart from the visible color) by the measurement of relative intensities of reflected light, ISO brightness, and microbiological tests under light irradiation. The first experiment shows the dependence of the intensity of the reflected light on the type of covering of the paper sheet. Dye-loaded cellulose sheets coated with E1 and E2 emulsions show the maximum absorption at wavelengths of 527 and 537 nm, respectively (Fig. 7a and b) which correspond to the absorption of native PSF in methanol (529 nm) (Blauz et.al 2021). Studies of polymer-coated sheets with a surface concentration of PSF at 0.1 and 1.17 mmol/m2 for E1 and 0.01 and 0.32 mmol/m2 for E2 samples indicate a strong convergence of pre- and post-Cobb measurements.

Fig. 7
figure 7

Relative reflection light intensity for the modified cellulose surface a E1.4 and E1.5; b E2.3 and E2.5; ISO brightness and light reflection intensity as a function of calculated PSF surface concentration c for E1.4 and E1.5; d for E2.3 and E2.5

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Comparisons of the reflected light curves allowed us to assess the high stability of the immobilized PSF dye in the silsesquioxane coating. The polycondensation of alkoxysilanes can occur and causes internal voids due to the formation of branched and cyclic structures and incomplete condensation, leaving free SiOH groups to interact with the dye (Sato et al. 2022; Catauro and Ciprioti 2021). We indicated in the 29Si NMR spectrum (Fig. 5b), that approximately 20% of the Si atoms remain substituted on a free hydroxyl group corresponding to T2 units at − 56.17 ppm. It is likely that PSF is readily located in these spaces because of electrostatic interactions or hydrogen bonding, and then becomes blocked there during the drying process. It should be noted that the sol into which the photosensitizer is introduced is a polymer (polysiloxane-polyols) which has caused multi-point electrostatic interactions due to the repeating units of (–O)Si(OH) (Nakai et al. 2017). The second parameter, ISO brightness, decreased with increasing PSF concentration in the emulsion because starch and siloxane moieties did not change this parameter significantly. The comparison of relative reflection VIS spectra of samples (Fig. 7) clearly proved that the main factor changing the ISO brightness of the samples had a strong absorption of PSF/TESPSF at a maximum wavelength of 520–540 nm that increased with the surface concentration of both compounds.

In our previous studies we proved that the ethoxysilanes solution was a very effective hydrophobization agent leading to the materials with strong surface hydrophobicity that improved barrier properties as measured by Cobb test (up to 250% better than uncoated paper) (Ganicz and Rozga-Wijas 2021). Unfortunately, the addition of PSF and TESPSF to the mixtures strongly decreases the effect of hydrophobicity on the surface. For example, samples with a small amount of PSF indicated the water contact angles over 90° due to the low concentration of a dye on the surface. For higher surface concentrations of PSF than 0.009 mmol/m2 the measured contact angles were below 90° and decreased with an increasing its concentration. The addition of a very low concentration of TESPSF caused a slight decrease in contact angles below 90°, but they were still in the range of 90–82° at higher concentrations of TESPSF, and water drops were swallowed after 2–3 min (Table 1).

Photoinactivation of gram-positive and gram-negative bacteria and erythrocyte lysis assay of the modified cellulose surfaces

The antibacterial properties of the modified paper were examined against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria with exposure samples to green light for 15 and 30 min. Firstly, it appears necessary to show the differences between paper sheets left in a bacteria environment in the darkness and after irradiation to demonstrate no inhibition of bacterial growth in the dark conditions, as shown in (Fig. 8a and b). As an example, the number of bacteria was compared for E1.4 sheets with a PSF density of 0.1 mmol/m2 and E2.4 sheets with a density of 0.04 mmol/m2. In the darkness, living bacteria remain on the surface, unchanged. After 15 min of green light irradiation, all S. aureus on E1.4 and E2.4 were killed, while the number of living E. coli remained unchanged on both sheets. Increasing the exposure time to 30 min led to E. coli killing.

Fig. 8
figure 8

Number of viable S. aureus ATCC 6538 and E. coli ATCC 8739 cells, calculated from the colony forming unit (CFU) count in dark conditions and after irradiation for 15 in (a) and 30 min in (b) on the surface of the E1.4 (0.1 mmol/m2) and E2.4 (0.04 mmol/m2) handsheets. c.1—control 1—cellulosic discs covered with bacteria of appropriate optical density, c. 2—control 2—cellulosic discs with an appropriate emulsion and then covered with bacteria of appropriate optical density

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Inhibition of the growth of S. aureus and E. coli bacteria was enhanced by increasing the photosensitizer concentration on the surfaces of E1 and E2 sheets. The handsheets E2 despite a lower concentration of PSF on the surface showed better antibacterial effectiveness compared to E1 (Fig. 9a and b). Thus, the complete killing of S. aureus was observed after 15 min irradiation of E2.4 at a concentration of 0.04 mmol/m2, and the number of bacteria decreased by two orders at a concentration of 0.01 mmol/m2. However, the E1.4 sample reveals the complete killing of S. aureus after 15 min irradiation of the surface at a concentration of 0.1 mmol/m2. The E. coli bacteria were less sensitive to the ROS effect generated by PSF from the silsesquioxane network included in the cellulose surface, and test results for the E2 sample were illustrated in (Fig. 9c). For both E1 and E2 surfaces, 100% inhibition was achieved after extending the irradiation time to 30 min.

Fig. 9
figure 9

The number of S. aureus ATCC 6538 bacteria for the E1 (a) and E2 (b) handsheets as a function of the irradiation time (0, 15, 30 min) for a different surface concentration of PSF; c The number of E. coli ATCC 8739 strain for the E2 handsheets. The PSF concentration is expressed in (mmol/m2)

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The photodynamic deactivation of bacteria was observed in E2 samples with covalently bound photosensitizer molecules, as well as in E1 including the silsesquioxane polymer with a dye-complex. In both cases, PSF molecules were well dispersed and strongly fixed with the inorganic silica matrix, generating singlet oxygen under irradiation using green light. The effectiveness of bacteria-killing was proportional to the concentration of PSF, so the singlet oxygen was the cell-deactivating factor (Rozga-Wijas et al. 2021). The greater efficiency of the E2 surface with shorter irradiation time and a lower PSF concentration resulted from the greater homogeneity of the cellulose coverage.

Moreover, the erythrocyte lysis assay was performed to evaluate the toxicity of modified sheets of paper on red blood cells and is described in Section “Erythrocyte lysis assay”. Hemolysis was observed only in the case of disc E1.5 (the highest PSF concentration of 1.17 mmol/m2 in the E1 sheet series) before and after irradiation using green light for 15 and 30 min, and it amounted to 8.78%, 9.05% and 13.25%, respectively. Studies with the rest of the tested modified sheets of paper showed no hemolysis in the dark conditions, after 15 and 30 min of irradiation (Fig. 10).

Fig. 10
figure 10

The effect of modified sheets of papers on erythrocytes. The results are presented as a percentage of hemolysis of red blood cells. Lack of bars in tested papers means no hemolysis. Triton X-100 was taken at 100%, and saline at 0% hemolysis. Data are presented as mean ± SD

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