Introduction

Infectious diseases caused by microorganisms, such as bacteria and viruses, are a major public health problem [1]. Antibiotics significantly reduce mortality caused by infectious diseases and are one of the most important discoveries in the history of medicine [2]. However, the frequent use of antibiotics has caused the emergence of antibiotic-resistant strains that cannot be easily treated, even in the hospital environment [1]. Antibiotic resistance has created a need to develop viable alternatives to antibiotics for the prevention of infectious diseases [3, 4].

Singlet oxygen (1O2) is highly reactive and able to attack virion membranes, cell membranes, essential enzymes, and nucleic acids, which leads to inactivation or death by oxidative stress [5,6,7,8]. The use of 1O2 is nonspecific to the target microorganism, has few side effects, prevents the regrowth of microbes and presents limited opportunities for resistance mechanisms to develop because of the mode of action and type of biochemical targets (lethal multitarget process) [9,10,11]. Moreover, mutants resistant to this approach have never been detected [12]. Therefore, the development of materials that generate 1O2 by photoactivation is a promising approach to infectious disease prevention that may replace or reduce the burden on antibiotics and the unintended consequences of their use [1, 4].

The spread of microbes and viruses, and resulting infections, is a serious global health concern with increasingly severe consequences as the world becomes more interconnected. The effects of outbreaks can be suppressed to some extent by reducing direct contact with infected individuals or indirect contact with contaminated objects and surfaces [13, 14]. Self-disinfecting surfaces such as fabrics, paper, films, and coatings may play a vital role in preventing the transmission of pathogens. Accordingly, films and structures containing a photosensitizer (PS) that generates 1O2 upon irradiation with visible light have been researched because they may be able to continuously self-disinfect hospitals or other indoor environments [11, 13,14,15,16].

Numerous studies have examined organic PS molecules to induce the lethal photosensitization of bacteria, including phenothiazines such as toluidine blue O [17] and halogenated xanthenes such as rose bengal (RB) [5], acridines [18], porphyrins [19], and phthalocyanine (Pc) derivatives [20,21,22]. RB is a well-known 1O2 sensitizer [23]. It is excited to its excited species upon irradiation with visible light, and the excited state species activate molecular oxygen to 1O2. Decraene et al. reported cellulose acetate (CA) films containing 25 μM toluidine blue O and the same amount of RB killed microbes when illuminated with white light [24] and reduced the microbial load in a clinical environment [25]. Ideally, self-disinfecting surfaces should be activated by room light, easy to prepare, nonspecific to pathogens, scalable, and durable. However, most PSs, such as phenothiazines [22, 26, 27], RB [28, 29], and porphyrins [30], undergo photobleaching under room light irradiation, which restricts their long-term utilization.

Pcs have been shown to be more stable than RB with respect to photooxidation [28] when homogenously distributed throughout the polymer matrix. However, Pc molecules tend to aggregate when dispersed in CA films [31], which affects their optical properties. Preventing aggregation relies on inducing steric isolation of the Pc core, and substitution with bulky groups in solution has led to reduced aggregation of Pc or prevented it entirely [32,33,34,35]. Furthermore, the packing structure of Pc might be inhibited by introducing a substituent with large steric hindrance, such as a phenoxy group, to improve solubility in and compatibility with the resin. The phenoxy group can also shift the absorption wavelength to improve the transmittance (transparency) of visible light.

Considering the balance between the transmission of visible light and the light sterilization effect of its absorption, we addressed these issues by synthesizing and using phenoxy-substituted zinc Pc with the name [2,3,9,10,16,17,23,24-octakis(2-chloro-4-methoxyethoxycarbonylphenoxy)-1,4,8,11,15,18,22,25-octafluoro-29H,31H-phthalocyaninato(2-)-N29, N30, N31, N32] zinc (PPcZn). The structure of PPcZn is expected to confer chemical robustness against the generated 1O2 [36]. The bulky phenoxy groups in PPcZn should effectively protect this molecule from aggregation and facilitate its homogeneous distribution in the polymer matrix. Site isolation without aggregation in the polymer matrix is an important feature, since aggregation diminishes the 1O2 yield, shortens the lifetime of the excited states, and produces inefficient intersystem crossing. PPcZn is also chemically and thermally stable while being photoreactive, features that make it an attractive choice for research.

The photometric monitoring of 1,3-diphenylisobenzofuran (DPBF) degradation is a widely used method for indirect 1O2 detection. The mechanism of 1O2 detection by DPBF is its reaction with 1O2 to form endoperoxide, which then irreversibly decomposes to 1,2-dibenzoylbenzene [37]. Most recently, we detected 1O2 generated by irradiating an RB-containing film using a solid film containing DPBF [38].

In this study, we synthesized PPcZn in which bulky phenoxy groups were arranged and dispersed it in a CA film to create a transparent film. We confirmed the aggregation state of the PS molecule in the films by changing the concentration of PPcZn [39] and comparing the rate of 1O2 formation under visible light irradiation using a film containing DPBF. The antiviral property of the film was evaluated, and the continuity of 1O2 generation was tested after 6 months. Film durability was also measured to evaluate its practicality.

Experimental procedure

Materials

CA with a molecular weight of 50,000 (39.53 wt% acetyl content) and zinc iodide (II) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). RB (2,4,5,7-tetraiodo-3′,4′,5′,6′-tetrachlorofluorescein disodium salt; Acid Red 94) and methyl cellosolve were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). EC (ethyl cellulose 100 with a viscosity of 100 mPa s) was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). Benzonitrile was obtained from Nippon Shokubai Co., Ltd. (Osaka, Japan). DPBF, acetone, methanol, tetrahydrofuran (THF), and dimethyl acetoamide (DMAc) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All the chemicals and reagents used in the study were of analytical grade and used as received.

Measurement

NMR spectra were measured with an ECZ600R spectrometer (JEOL Ltd., Tokyo, Japan) at 20 °C. Proton spectra were referenced to Si(CH3)4 as the internal standard. 19F NMR spectra were referenced to monofluorobenzene as an external standard at δ = ­−113.15 ppm. Mass measurements were performed with liquid chromatography-mass spectrometry (Synapt G2, Waters Corporation, Milford, MA, USA) in LC-QTOF/MS ESI mode (negative) and with MALDI-TOFMS (Autoflex III Bruker, Billerica, MA, USA). The thickness of the films was measured at least three positions using a dial thickness gauge (Mitutoyo Corporation, Kanagawa, Japan), and an average value for each film was obtained. For the detection of 1O2 production, films were irradiated with an F32W-T fluorescent light source (Nichido Ind. Co., Ltd., Osaka, Japan). The illuminance of the light was measured using a general light class A illuminance meter (YOKOGAWA 510 Luxmeter model 51011; Yokogawa Test & Measurement Corporation, Tokyo, Japan). A white fluorescent lamp (FL20SSW/18, Mitsubishi Electronic Corporation, Tokyo, Japan) with a sharp cut filter (Type B) was used as the light source for the antiviral experiment. The absorption spectra of the films were obtained using a UV–Vis spectrophotometer (UV-1650PC; Shimadzu Corp., Kyoto, Japan). The tensile tests were performed using a tensile testing machine (Autograph AGS-1kNX; Shimadzu Corp., Kyoto Japan) at room temperature (controlled at 25 °C).

Synthesis of PPcZn

PPcZn was synthesized in the two steps illustrated in Fig. 1 based on a reported procedure [40]. The yield of the first step to obtain intermediate (1) was 64.8%. 1H NMR (600 MHz, CDCl3, 20 °C, Fig. S1): δ 8.07 (s, 2H), 7.90 (d, 2H), 6.85 (d, 2H), 4.47 t, 4H), 3.72 (t, 4H), 3.42 (s, 6H); 19F{1H} NMR (CDCl3, 20 °C, Fig. S2): δ 118.13 (s, 2F); Exact Mass: 620.06, Calc. for C28H20Cl2F2N2O8 (621.37): C 54.12; H 3.24; Cl 11.41; F 6.11, N 4.51%; Found: C 54.11; H 3.21; Cl 11.39; F 6.13; N 4.52%. Exact mass refers the measured mass value and calc. indicates the calculated average molecular weight. In the second step, 20.0 g (0.032 mol) of intermediate (1) was added to a 200 mL four-necked flask; 2.57 g (0.0081 mol) of zinc iodide (II) and 30.0 g of benzonitrile were charged into the flask and reacted at 160 °C for 24 h with stirring. After the reaction was complete, 52.7 g of methyl cellosolve were added to the reaction solution. The mixture was added dropwise to a solution of methanol and water to induce crystallization, and the crystals were separated by vacuum filtration. The obtained cake was then washed with a solution of methanol and water, followed by another vacuum filtration step. The washed cake was dried at 90 °C for 24 h using a vacuum dryer to obtain 17.35 g (yield 86.7%) of the target phthalocyanine (PPcZn). 1H NMR (600 MHz, CDCl3, 20 °C, Fig. S3): δ 7.79 (m, 8H), 7.76 (m, 8H), 7.18 (m,8H), 7.17 (m,8H), 4.22 (m, 16H), 3.57 (m, 16H), 3.27 (m, 24H); 19F{1H} NMR (CDCl3, 20 °C, Fig. S4): δ 131.23 (m, 8 F); Exact Mass: 2544.16, UV–Vis (DMAc): λmax 695 nm (ε = 3.1 × 105), Calc. for C112H80Cl8F8N8O32Zn (2550.86): C 52.74; H 3.16; Cl 11.12; F 5.96, N 4.39%; Found: C 52.51; H 3.22; Cl 11.05; F 5.91; N 4.35%.

Fig. 1
figure 1

Synthesis of PPcZn

Full size image

Fabrication of PS-containing films

Films containing PSs (PPcZn and RB) and evaluated as self-disinfecting surfaces were prepared using the procedures described in the upper part of Fig. 2. In a typical preparation, 3 mg of PPcZn and 1.2 g of CA were dissolved in 12 mL of acetone. THF was available to use as a solvent, but acetone was used in this study based on a previous report [24]. The resulting solution was cast to form a film on a glass plate, which was dried at 25 °C and is referred to as a P1.3C film (based on the PS, its concentration, and the polymer matrix). The PS molar concentrations were calculated using [PS] = n/m [41], where n/m is the ratio between the number of moles of PS and the mass of the film and is calculated using the molar mass of PPcZn (2550.86 g mol−1), the molar mass of RB (973.67 g mol−1), and the density of CA (1.34 g cm−3). The films were defined as XiY, where X represents the PS (P for PPcZn and R for RB), the subscript i represents the concentration of the PS (mM), and Y represents the polymer matrix (C for CA and E for EC). For example, a film composed of 1.3 mM PPcZn in CA would be a P1.3C film. We fabricated four different films: P0.5C, P1.3C, P2.1C, and R1.3C. All fabrication procedures were carried out in the dark. The thickness of each film ranged from 30 ± 5 μm. In all cases, optically clear films were obtained.

Fig. 2
figure 2

Illustration showing the fabrication process for PS-containing films

Full size image

For the detection of 1O2, CA and EC films containing DPBF were prepared using the same method described above. The solution of DPBF was then cast on the preformed self-disinfecting film to obtain an integrated film, as shown in the lower part of Fig. 2. Using the molar mass of DPBF (270.33 g mol−1) and the densities of CA and EC, the concentration of DPBF in the films was calculated to be 19.8 mM (DC, where D represents DPBF) and 16.9 mM (DE, where E represents EC). The integrated films were named according to the rule that the first layer was indicated before the second layer, the individual layers were defined with the letter representing the PS placed before the letter(s) for the polymer, and the subscript represents the concentration of the PS in the film. For example, films prepared by casting a DE solution on a P1.3C film were referred to as P1.3CDE. Notably, a subscript is not used for the DPBF concentration in CA or EC because it does not vary as the PS concentration does. The DE film was prepared using THF as the solvent instead of acetone. The thickness of the double-layer films ranged from 60 ± 5 μm. The dried films were stored in foil laminate bags until required. The films tested in this study are listed in Table 1.

Table 1 Films fabricated for evaluation
Full size table

Detection of 1O2 using DPBF

The generation of 1O2 from the self-disinfecting surfaces was determined using integrated films, such as the P1.3CDE film. The test films were 20 × 40 mm in size. Figure 3 illustrates the integrated film sandwiched between plates of glass with no gap. The layered laminate was then irradiated from the upper side at 2000 lx using the F32W-T fluorescent light source. The absorbance level of the films was measured after 30, 60, 120, 300, and 600 s. Similarly, an experiment without a self-disinfecting surface film was conducted for comparison. All experiments were repeated more than three times.

Fig. 3
figure 3

Cross-sectional illustration of 1O2 detection during irradiation with visible light

Full size image

Durability tests of self-disinfecting surfaces

The durability of the films as self-disinfecting surfaces was evaluated by performing a water-resistance test and an indoor exposure test. The water-resistance test involved the immersion of the film sample (20 × 40 mm in size) in 50 mL of pure water in a 100 mL beaker. The absorbance was measured before and after stirring for 2 h in the dark, and the ratio was compared. The indoor exposure test involved the illumination of the film sample (50 × 50 mm in size) for 6 months in a laboratory with room lights at 610 lx (the lights were turned off at night). The absorptance of the film was measured before and after illumination, and the ratio was used to determine the photostability of PS in the film.

Antiviral activity of P1.3C film

Using Bacteriophage Qβ (NBRC 20012) as the test microorganism, antiviral activities of the P1.3C film and a CA film (the control) were assessed according to the Japanese Industrial Standard JIS R 1756 2020. A sterilized humidity control filter paper was placed on the bottom of a sterilized storage petri dish, an appropriate amount of sterilized water was added to the humidity control paper, and a U-shaped glass tube was placed on it. An ultraviolet-sterilized test piece (length: 50 ± 2 mm, width: 50 ± 2 mm, thickness: 30 ± 5 μm) was placed on a U-shaped glass tube, and 0.15 mL of the test bacteriophage solution was dropped onto the test piece. The bacteriophage solution was covered with a polyethylene film, and the moisturizing glass was placed on the petri dish. Light irradiation tests were performed immediately after inoculation with the test bacteriophage solution using the white fluorescent lamp (FL20SSW/18) as the light source, except for the control pieces. The experiment was performed for 4 h both in the dark and in visible light (1000 lx). After experiments, the test pieces were immediately washed with 10 mL of Soybean-Casein Digest Broth with Lecithin & Polysorbate 20 Medium (SCDLP), and the solution was diluted 100, 101, 102, and 103-fold. The diluted solution was added to a separately prepared plate on which Escherichia coli was grown, incubated at 35 °C for 16 h, and the number of plaques formed was visually counted. All tests were carried out three times to obtain an average value.

Results and discussion

Aggregation behavior of PPcZn in the CA film

PS should be present in a nonaggregated state for self-disinfecting applications because aggregation induces fast, radiationless deactivation and inhibits 1O2 generation [33, 39]. Generally, Pc exhibits a strong tendency to aggregate that derives oligomers in solution due to its extended π-conjugation [33, 39, 42, 43]. Additionally, tetra-tert-phthalocyanine has been reported to be present in an aggregated state in CA films [31]. The aggregation behavior was assessed using UV–Vis spectroscopy. As expected, the absorption spectra of PiC films (P0.5C, P1.3C, and P2.1C) showed a single sharp Q-band at λmax 664 nm, which is typical of nonaggregated species (Fig. 4a).

Fig. 4
figure 4

a UV–Vis absorption spectra of P2.1C, P1.3C, and P0.5C; and b plot of the PPcZn concentration in the CA film versus absorbance at 664 nm

Full size image

The aggregation behavior of PPcZn in the CA matrix was examined at different concentrations. Figure 4b shows that the appearance of the Q-band absorption maxima remained unchanged as the concentration increased. Furthermore, its apparent molar extinction coefficient remained almost constant, suggesting a purely monomeric form, which obeys the Beer–Lambert Law in the studied concentration range. PPcZn was confirmed to be uniformly dispersed in the CA film without aggregation, and the CA film containing PPcZn should be expected to generate 1O2 upon visible light irradiation.

1O2 generation from CA films containing PSs upon visible light irradiation

The rate of 1O2 formation is associated with a decrease in the absorbance of DPBF as a function of the irradiation time [44], and the reaction of DPBF with 1O2 in solution has been reported to follow a pseudofirst-order kinetic model [34]. The change in the absorption spectrum of the P1.3CDE film irradiated with visible light is shown in Fig. 5a, and a dramatic reduction in absorption at 416 nm corresponding to DPBF absorption was observed. The degradation kinetics of DPBF by 1O2 generated from P1.3CDC and P1.3CDE were determined by monitoring the photooxidation of DPBF. The degradation rate of DPBF, k, was determined using the following equation [45]:

$$k{\mathrm{t}} = \ln \frac{{\left[ {{\mathrm{DPBF}}} \right]0}}{{\left[ {{\mathrm{DPBF}}} \right]t}}$$
(1)

where [DPBF]0 and [DPBF]t are the concentrations of DPBF determined by measuring the absorbance at reaction times zero and t, respectively. As shown in Fig. 5b, the reaction kinetics follow a pseudofirst-order rate law. This result suggests that the concentrations of oxygen and PS did not change during the experiment [46].

Fig. 5
figure 5

a Changes in the UV–Vis spectra of the P1.3CDE film during irradiation and b time-dependent absorption intensity during irradiation reported as ln [DPBF]0/[DPBF]t

Full size image

According to our previous study [38], the DPBF photodegradation rates in DE and DC films that contained only DPBF as the PS were 0.0004 and 0.0002 s−1, respectively. In the present study, the degradation rates of DPBF in the P1.3CDE and P1.3CDC films were 0.0067 and 0.0028 s−1, respectively. Thus, the P1.3C film produced 1O2 upon visible light irradiation, and the 1O2 reacted with DPBF. Additionally, the rate of the reaction between 1O2 and DPBF in the DC film was slower than that in the DE film because of the difference in oxygen permeability coefficients between the CA and EC films. The reaction rate of 1O2 with DPBF has been reported to depend on the solvent [34], and a similar outcome is expected when a polymer film is used.

P2.1CDE, P0.5CDE, and R1.3CDE films were evaluated, and the results are shown in Fig. 6. Interestingly, the rate of 1O2 generation from the film depended on the concentration of PS. When the PS concentration in the film was increased 2.6 times (1.3 mM against 0.5 mM) and 4.2 times (2.1 mM against 0.5 mM), the rate of 1O2 production increased 2.9 times (0.0067 s−1 against 0.0023 s−1) and 4.5 times (0.0104 s−1 against 0.0023 s−1), respectively. In this concentration range, the higher the concentration of PPcZn, the higher the amount of 1O2 produced per unit time. The P2.1C, P1.3C, and P0.5C films reduced the initial concentration of DPBF almost completely (0.2% or less), to a large extent (2% or less), and by 24% at 600 s, respectively. 1O2 was likely first generated at the interface with the PS-containing film and diffused into the deeper region of the DE or DC film to reach the unreacted DPBE molecule. Additionally, at the same concentration, PPcZn produced more 1O2 per unit time than RB. PPcZn is therefore expected to possess antiviral properties upon visible light irradiation.

Fig. 6
figure 6

Comparison of the rate of 1O2 generation upon the visible light irradiation of CA films containing RB and PPcZn at different concentrations

Full size image

Antiviral activity of the P1.3C film

The antiviral activity of the P1.3C film, a representative CA film containing PPcZn, was analyzed, and the test satisfied the requirements for validity as specified in JIS R 1756. Log reduction [47] was used to quantify the live microbes. The number of visible plaques after irradiation and the number of visible plaques in the dark were counted, and the infection value (pfu/sample) was calculated based on the number of visible plaques. Equation (2) was then used to evaluate antiviral activity. The results are summarized in Table 2.

$${\mathrm{Log}}\;{\mathrm{reduction}} = \log _{10}\left( {\mathrm{A}} \right) - \log _{10}\left( {\mathrm{B}} \right)$$
(2)

where A and B are the number of active microbes that infect target cells, as described by the number of virus particles capable of forming plaques per unit volume in the dark and after irradiation with visible light, respectively. After incubation under visible light, the antiviral activity of the P1.3C film increased compared with incubation in the dark. This result indicates that the photodynamic effect on Bacteriophage Qβ was achieved with ordinary, daily light conditions (1000 lx). In an actual indoor environment, sunlight containing infrared light of 700 nm or more will be present, and a greater photoantiviral effect is expected based on the high durability of the P1.3C film, as described below.

Table 2 Antiviral activity of the P1.3C film compared to that of the blank film
Full size table

Durability of PC films compared to the RC film

The absorbance of the R1.3C film decreased to 84.3% compared to the film before immersion in water in the water-resistance test, which is likely due to the leaching of RB. Conversely, no leaching of PS was observed for any of the PPcZn-containing films in the UV–Vis spectra. This observation is likely because of the difference in the water solubility of PS.

The results of the indoor exposure test using the absorbance intensity ratio at 561 nm for the R1.3C film were 71.3% after only one day of exposure and 42.4% after seven days of exposure. The P0.5C, P1.3C, and P2.1C films had absorbance intensity ratios of 96.4%, 95.5%, and 98.5%, respectively, after 6 months of exposure at 664 nm. CA films containing PPcZn displayed a photobleaching degree of less than 5% even when incubated indoors under white light for 6 months. Additionally, the results of tensile tests on P2.1C films before and after 6 months of indoor exposure were performed 5 times for each sample and are summarized in Table 3. The data are presented as means ± standard deviations. These results suggest that the CA matrix containing PPcZn is a highly durable film both optically and physically.

Table 3 Tensile strength of P2.1C films before and after 6 months of indoor exposure
Full size table

The 1O2 generation rates of the P1.3C films before and after exposure were compared by measuring the oxidation rate of DPBF by casting a DE solution onto each P1.3C film to confirm the lack of change in the 1O2 production capacity of the PC film after 6 months of indoor exposure. As shown in Fig. 7, the P1.3C film displayed a similar 1O2 production rate before exposure and after 6 months of indoor exposure. The P1.3C film maintained the 1O2 photogeneration efficiency over 6 months and was expected to maintain its antiviral effect. Furthermore, when comparing the transmittance of the P1.3C film with air in the visible light region (470–530 nm) in which PPcZn and CA have no absorption, the effect of exposure of the film for 6 months was investigated. The transmittance values both before and after exposure were >91%, confirming that the P1.3C film maintained its transparency for more than 6 months. These results prove that the CA film containing PPcZn exhibits excellent durability under indoor exposure conditions in terms of its function (1O2 generation ability with light irradiation), appearance (transparency and nonfading properties), and mechanical strength.

Fig. 7
figure 7

Comparison of 1O2 production rates from the DE film before (orange) and after (blue) indoor exposure for 6 months

Full size image

A representative application of the CA film containing PPcZn

The CA film containing PPcZn was used in origami to determine whether it has the appropriate strength and stiffness for practical applications. Figure 8 shows an example of a windmill that had the same appearance after 1 month and after 6 months of indoor exposure and was expected to exhibit antiviral function when placed in air flow. The self-sterilizing film obtained in this study has the potential to be used in indoor interior design because it maintained its 1O2 photogeneration ability for a long period while maintaining its transparency without fading and it could be manipulated without losing strength or stiffness.

Fig. 8
figure 8

A picture of an “origami (windmill)” created with the P1.3C film. a Initial film (before exposure), b after 1 month of exposure, and c after 6 months of exposure

Full size image

Conclusions

We fabricated CA films containing PPcZn using a simple casting procedure. This phthalocyanine was successfully dispersed into CA films without aggregation, as confirmed by the fit to the Beer–Lambert law. Visible light illumination resulted in the photogeneration of 1O2 from CA films containing PPcZn by CA and EC films containing DPBF. PPcZn in CA showed good photostability with no significant degradation over 6 months of continuous exposure to room light while maintaining its ability to generate 1O2 with minimal degradation over time. We have shown that room light can serve as an economical and efficient light source for the photodynamic inactivation of viruses, and these data suggested that the photodynamic disinfection of the CA film containing PPcZn persists for over 6 months. These findings are promising for the implementation of this film as a self-disinfecting surface in real-world applications.