Fabrication of Poly(vinyl alcohol)/Chitosan Composite Films Strengthened with Titanium Dioxide and Polyphosphonate Additives for Packaging Applications

Eco-innovation through the development of intelligent materials for food packaging is evolving, and it still has huge potential to improve food product safety, quality, and control. The design of such materials by the combination of biodegradable semi-synthetic polymers with natural ones and with some additives, which may improve certain functionalities in the targeted material, is continuing to attract attention of researchers. To fabricate composite films via casting from solution, followed by drying in atmospheric conditions, certain mass ratios of poly(vinyl alcohol) and chitosan were used as polymeric matrix, whereas TiO2 nanoparticles and a polyphosphonate were used as reinforcing additives. The structural confirmation, surface properties, swelling behavior, and morphology of the xerogel composite films have been studied. The results confirmed the presence of all ingredients in the prepared fabrics, the contact angle of the formulation containing poly(vinyl alcohol), chitosan, and titanium dioxide in its composition exhibited the smallest value (87.67°), whereas the profilometry and scanning electron microscopy enlightened the good dispersion of the ingredients and the quality of all the composite films. Antimicrobial assay established successful antimicrobial potential of the poly(vinyl alcoohol)/chitosan-reinforced composites films against Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Cytotoxicity tests have revealed that the studied films are non-toxic, presented good compatibility, and they are attractive candidates for packaging applications.


Introduction
Plastic packaging continues, in recent years, to be the most significant industrial use in the world (representing, on average, 30% of the total) [1][2][3]. Particularly, polymers, like polyethylene terephthalate, low-density polyethylene, polystyrene, polypropylene, and high-density polyethylene, were widely used as single-use packaging materials in the food and beverage industry because of their mechanical properties that could provide effective barriers to oxygen and carbon dioxide as well as their relative affordability and ease of availability [4][5][6][7][8]. Nevertheless, when plastic packaging based on the abovementioned polymers reaches the end life of its use, a significant amount frequently eludes formal first, it strengthens and functionalizes the matrix by improving its dispersibility, and second, it gives the mixture antibacterial capabilities [37].
In composite materials, PVA is frequently utilized as a polymer matrix, and CS is frequently employed as a reinforcing agent in an effort to broaden the application of PVA in the field of high-strength materials [26,38]. Thus, the co-existence of the hydroxyl groups in both PVA and CS, doubled by the presence of amino groups in CS, can produce intermolecular interactions, which may result in an improvement of performances of PVA composites. The use of polyphosphonate containing sulfur and phosphorus in the main chain and in the side chain in phosphaphenanthrene-type heterocycles is expected to induce thermal stability in the composite, then, to synergistically bring its own antimicrobial contribution to the composite, as was described in few studies in the recent past [39].
In the present study, optimized solutions of PVA/CS, PVA/CS/TiO 2 , or PVA/CS/ TiO 2 /polyphosphonate have been used to obtain xerogels composite films via casting from solution method, followed by drying in ambient conditions. Schematic representation of the composites is presented in Figure 1.
can aid to lessen the spontaneous agglomeration of TiO2, improving the functional asp of the composite [36]. The addition of nanoparticles to the polymer matrix has two b fits: first, it strengthens and functionalizes the matrix by improving its dispersibility, second, it gives the mixture antibacterial capabilities [37].
In composite materials, PVA is frequently utilized as a polymer matrix, and C frequently employed as a reinforcing agent in an effort to broaden the application of in the field of high-strength materials [26,38]. Thus, the co-existence of the hydr groups in both PVA and CS, doubled by the presence of amino groups in CS, can pro intermolecular interactions, which may result in an improvement of performances of composites. The use of polyphosphonate containing sulfur and phosphorus in the m chain and in the side chain in phosphaphenanthrene-type heterocycles is expected t duce thermal stability in the composite, then, to synergistically bring its own antimicr contribution to the composite, as was described in few studies in the recent past [39] In the present study, optimized solutions of PVA/CS, PVA/CS/TiO2 PVA/CS/TiO2/polyphosphonate have been used to obtain xerogels composite film casting from solution method, followed by drying in ambient conditions. Schematic resentation of the composites is presented in Figure 1. The effect of all the ingredients on the structure-properties relation of the develo xerogel composite films was determined and discussed. Physical, chemical, morpho cal properties, surface properties, antimicrobial activity, and cytocompatibility o  The effect of all the ingredients on the structure-properties relation of the developed xerogel composite films was determined and discussed. Physical, chemical, morphological properties, surface properties, antimicrobial activity, and cytocompatibility of the matrices were studied by means of FTIR, SEM, swelling behavior, profilometry, contact angle measurements, antimicrobial, and cytotoxicity assays.

Preparation and Structural Characterization
Binary PVA/CS films and the composite PVA/CS films containing TiO 2 or TiO 2 with polyphosphonate, PFR-3, have been obtained by the casting from solution procedure. The details of this procedure are presented in the Materials and Methods section. The composition of the composite polymer matrices expressed in mass ratio for all the ingredients utilized in the preparation and the codes of the as-prepared composites are listed in Table 1. The chemical structure of the products was introspected by FTIR spectroscopy. Figure 2 presents the FTIR spectra of the binary PVA/CS-0 sample and of the PVA/CS composite films. In the spectrum of the PVA/CS-0 sample, the characteristic strong and wide band appeared at approximately 3320 cm −1 due to hydroxyl stretching vibration. matrices were studied by means of FTIR, SEM, swelling behavior, profilometry, contact angle measurements, antimicrobial, and cytotoxicity assays.

Preparation and Structural Characterization
Binary PVA/CS films and the composite PVA/CS films containing TiO2 or TiO2 with polyphosphonate, PFR-3, have been obtained by the casting from solution procedure. The details of this procedure are presented in the Materials and Methods section. The composition of the composite polymer matrices expressed in mass ratio for all the ingredients utilized in the preparation and the codes of the as-prepared composites are listed in Table 1. The chemical structure of the products was introspected by FTIR spectroscopy. Figure 2 presents the FTIR spectra of the binary PVA/CS-0 sample and of the PVA/CS composite films. In the spectrum of the PVA/CS-0 sample, the characteristic strong and wide band appeared at approximately 3320 cm −1 due to hydroxyl stretching vibration. Common characteristic absorption bands were found also at about 2930 cm −1 with a shoulder at 2860 cm −1 due to asymmetric and symmetric stretching vibrations of C-H, at 1414 cm −1 (δC-H), 1378 cm −1 (ωC-H), 1243 cm −1 (ωC-H), 1074 cm −1 , 1023 cm −1 (νC-O), and 830 cm −1 (ρCH2) [40]. Due to C=O and C-O-C units, respectively, characteristic absorption bands for the non-hydrolyzed ester groups−O-CO-CH3 were observed at 1735 cm −1 and 1245 cm −1 . The characteristic bands for CS could be also assigned in the FTIR spectra of the samples, at 1556 cm −1 (band characteristic for amide II, νN-H), 2920 cm −1 (νC-H), and Common characteristic absorption bands were found also at about 2930 cm −1 with a shoulder at 2860 cm −1 due to asymmetric and symmetric stretching vibrations of C-H, at 1414 cm −1 (δ C -H ), 1378 cm −1 (ω C -H ), 1243 cm −1 (ω C -H ), 1074 cm −1 , 1023 cm −1 (ν C -O ), and 830 cm −1 (ρ CH2 ) [40]. Due to C=O and C-O-C units, respectively, characteristic absorption bands for the non-hydrolyzed ester groups−O-CO-CH 3 were observed at 1735 cm −1 and 1245 cm −1 . The characteristic bands for CS could be also assigned in the FTIR spectra of the samples, at 1556 cm −1 (band characteristic for amide II, ν N -H ), 2920 cm −1 (ν C -H ), and approximately 3300 cm −1 (wide band with νNH vibration overlapping with νO-H of polyvinyl alcohol). The characteristic band appearing at 1557 cm −1 assigned for δ NH (amide II) vibration of the NH 2 group, and the band observable at 1654 cm −1 assigned for amide I (ν C=O ) of O=C-NHR groups, were revealed to be diminished and slightly shifted in the FTIR spectra of the ternary and quaternary composite films (samples PVA/CS-2 and PVA/CS-3), indicating some interactions between CS and PVA that could be catalyzed by the presence of the additives in the samples. Additionally, the band appearing enlarged at 846 cm −1 in the multi-component composites (samples PVA/CS-1, PVA/CS-2, and PVA/CS-3) is the indicative of occurrence of hydrogen bonds formed between OH groups of the ingredients present in the composites.
From the FTIR spectra of the PVA/CS-2 and PVA/CS-3 samples, characteristic absorption bands at 1470 cm −1 , 1210 cm −1 (appearing as small shoulders), and 1140 cm −1 (appearing as a distinctive peak), were assigned to stretching vibration of the P-Ar, P-O and P-O-C, respectively. Another distinctive band confirming the presence of the PFR-3 additive into the composition of the PVA/CS-2 and PVA/CS-3 samples could be observed as a sharp peak near 755 cm −1 , which is attributed to the P-O-Ph group.

Surface Characteristics of the PVA/CS Composite Films
Due to their outstanding biocompatibility, resistance to bodily fluids, mechanical qualities, anticorrosive capability, and flexibility, titanium-based composites are preferred for use in biomedical applications [41,42]. However, their properties depend on the surface, which is related with the mixing capacity of nanomaterials with titanium oxide [43]. Accordingly, it has been suggested that the interaction between titanium oxide nanoparticles (TiO 2 ) and biopolymers (starch, gums, and chitosan) can aid in reducing the spontaneous agglomeration of nanoparticles, thereby enhancing the functional qualities of the composite [36].
PVA/CS composite films were analyzed using a profilometer. Microscopic images of the composite films were taken (histograms generated by the profilometer) and are presented in Figure 3, and the average roughness values were calculated ( Table 2). According to the histograms and the microscopic images, it has been revealed that the addition of titanium dioxide to the polymeric matrix based on chitosan and PVA resulted in the production of composite films with a homogeneous composition and a surface devoid of cracks. The average roughness, Ra, representing the arithmetic mean of the highs and lows profiles, was in the interval of 65.5-570.3 nm. The surface roughness parameters decreased when introducing the PFR-3 in the PVA/CS matrix, according to the Ra value ( Table 2), suggesting its contribution to the homogeneity and quality of the multi-component composites.  The wetting characteristics, such as work of adhesion (Wa), the total solid surface free energy (γSV), solid-liquid interfacial tension (γSL), etc., of the PVA/CS samples with respect to the W and EG were investigated. A summary of all contact angle study parameters is provided. Table 2 provides a summary of all the contact angle investigations' parameters. W and EG were used as the test liquids, whereas PVA samples were examined using contact angle measurements at room temperature. Figure 4 displays the results of measuring the contact angle for the PVA sample in W. The wetting characteristics, such as work of adhesion (Wa), the total solid surface free energy (γ SV ), solid-liquid interfacial tension (γ SL ), etc., of the PVA/CS samples with respect to the W and EG were investigated. A summary of all contact angle study parameters is provided. Table 2 provides a summary of all the contact angle investigations' parameters. W and EG were used as the test liquids, whereas PVA samples were examined using contact angle measurements at room temperature. Figure 4 displays the results of measuring the contact angle for the PVA sample in W.  The wetting characteristics, such as work of adhesion (Wa), the total solid surface free energy (γSV), solid-liquid interfacial tension (γSL), etc., of the PVA/CS samples with respect to the W and EG were investigated. A summary of all contact angle study parameters is provided. Table 2 provides a summary of all the contact angle investigations' parameters. W and EG were used as the test liquids, whereas PVA samples were examined using contact angle measurements at room temperature. Figure Table 2). The same behavior, in terms of contact angle and roughness decreases, has been noticed when adding 0.2172 g of polyphosphonate PFR-3, which resulted in a further decrease of the contact angle as a result of the presence of phenyl-phosphonate groups within the macromolecule of the polymeric additive that could influence the molecular hydrogen bonding; these results are consistent with the FTIR findings. Thus, in the case of PVA/CS-2, the contact angle value decreased to 87.67 • due to the orientation of macromolecules and the reorganization of the polar groups from the surface of the composite sample. After continuing to add the PFR-3 additive (0.4344), the hydrophobic character was preserved as a result of the reduced concentration of OH groups, whereas the phenyl-phosphonate group's impact was disrupted by the presence of bulky, rigid phosphaphenanthrene groups.
When polyphosphonate PFR-3 was added into the PVA matrix, the work of adhesion  Table 2 revealed that samples PVA/CS-1 and PVA/CS-2 show lower polar surface parameters γ P SV due to the presence of nano powders in the polymeric matrices. After continuing to add PFR-3 additive, a decrease of the Wa value and an increase of the polar surface parameters γ P SV were achieved, probably due to the increase in the hydrophobicity of the surface of the sample PVA/CS-3. Additionally, from Table 2 it can be observed that the dispersive component values for the PVA/CS samples are greater than the polar component values, which leads to the conclusion that there are more hydrophobic groups than hydrophilic ones on the surface of the PVA/CS films.

Swelling Behavior and Morphology of PVA/CS Composite Films
The swelling behavior of the samples PVA/CS revealed that the PVA/CS-0 matrix without TiO 2 and PFR-3 additive exhibited the highest maximum swelling degree achieved at equilibrium, 834% ( Figure 5). For the sample containing TiO 2 , the maximum swelling degree achieved at equilibrium was 688%, whereas introducing the polyphosphonate additive into the systems further decreased the swelling behavior of the composite in the series, with the swelling degree achieved at equilibrium being 639% and 593% in the case of the PVA/CS-2 and PVA/CS-3 sample, respectively. Another observation relates to a higher initial water uptake in the first 10 min when the samples PVA/CS-0 and PVA/CS-1 reached the maximum swelling degree, whereas in the case of the samples PVA/CS-2 and PVA/CS-3, the maximum swelling degree was achieved after approximately 30 min.  The morphology of PVA/CS composite films was investigated by scanning electron microscopy. Microphotographs for PVA/CS-0 in comparison with the samples containing additive are presented in Figure 6. The morphology of the PVA/CS-0 sample appeared uniform and smooth, presenting a porous behavior. The aspect of the main polymeric matrix was also preserved in the samples containing either titanium dioxide nanoparticles  The morphology of PVA/CS composite films was investigated by scanning electron microscopy. Microphotographs for PVA/CS-0 in comparison with the samples containing additive are presented in Figure 6. The morphology of the PVA/CS-0 sample appeared uniform and smooth, presenting a porous behavior. The aspect of the main polymeric matrix was also preserved in the samples containing either titanium dioxide nanoparticles or both TiO 2 and PFR-3 additive in their structure. A slight increase in the porosity could be observed in the sample PVA/CS-3 as the content of polyphosphonate PFR-3 increased.

In Vitro Evaluation of Antimicrobial Potential of the PVA/CS Composite Films
Due to the chitosan and the specific conditions of the culture medium and incubatio parameters (37 °C/24 h), the tested matrices changed their disc-like shape and/or lost co tact with the microbial culture, leading to a limitation in the interpretation of the antim crobial activity.

In Vitro Evaluation of Antimicrobial Potential of the PVA/CS Composite Films
Due to the chitosan and the specific conditions of the culture medium and incubation parameters (37 • C/24 h), the tested matrices changed their disc-like shape and/or lost contact with the microbial culture, leading to a limitation in the interpretation of the antimicrobial activity.
However, qualitative evaluation of the antimicrobial potential of the PVA/CS composites films against Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 90028 (Table 3)   Comparing the results obtained for the PVA/CS-0 film (the sample containing only PVA and chitosan) and the functionalized composite films (PVA/CS-1, PVA/CS-2, PVA/CS-3), it can be seen that the PVA/CS-1 sample (containing PVA, CS, and TiO 2 nanopowder) has the best antimicrobial activity against both Gram-positive bacteria (Staphylococcus aureus, MRSA) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa). The yeast Candida albicans also proved to be sensitive to the action of the compounds in the PVA/CS-3 sample.
In the case of the PVA/CS-2 and PVA/CS-3 composite films containing both TiO 2 nanoparticles and PFR-3 additive in their structure, the presence of TiO 2 at a similar concentration as in PVA/CS-1 highlights the antimicrobial effect of the sample on all strains tested. The inclusion of PRF in their composition did not significantly alter the antimicrobial potential of the tested materials.
The tests performed in our study confirmed the well-recognized antimicrobial capability of TiO 2 . The antimicrobial potential and mechanisms of action of TiO 2 on bacterial cells are complex and diverse, exhibiting a broad spectrum of antimicrobial activity against bacteria (Gram-positive and Gram-negative), yeasts, and implicitly antibiotic-resistant microorganisms [44]. The mechanism thought to be responsible for the antimicrobial effect of TiO 2 is commonly associated with reactive oxygen species (ROS), which, when irradiated with bandgaps, produce photoinduced charges in the presence of O 2 [45]. Thus, the oxidative effect alters several cellular structures, but the first to be affected are the cell wall and cell membranes, leading to cell lysis and loss of cell integrity [46]. Specific studies on antimicrobial mechanisms have shown that microorganisms exposed to photocatalytic TiO 2 NPs exhibited cell inactivation at the level of the regulatory network and signal transduction, a significant reduction in respiratory chain activity, and inhibition of the ability to assimilate and transport iron and phosphorus. These processes, with the extensive cell wall and membrane changes, were the main factors explaining the biocidal activity of TiO 2 NPs [44].

In Vitro Citotoxicity and Proliferation Assay
The cytotoxicity tests undertaken in this step of the study (MTT and cell morphology analysis) have a screening value and are performed and analyzed in accordance with ISO-10993-5 standards recommendations for cytotoxicity as a first-intended and eliminatory requirement for biocompatibility.
PVA/CS composite films have been subjected to cytocompatibility tests that were performed by culturing MCF 7 in the physical presence of material samples (Figure 7). The MCF 7 cells are a standard line for cytocompatibility testing, as recommended by ASTM.

Analysis of Cell Morphology
The effect of direct exposure of cultured cells to the sample material was asse microscopically by phase contrast, overlapped to fluorescence images of DAPI-sta nuclear DNA. The images of the fixed and stained cells at 20× objective magnificatio shown in Figure 8.   Figure 7 shows the results of the MTT test, in which cell viability was expressed as an average and standard variation, obtained from the processing of absorbance values. Cell viability in control cultures was considered 100% ± SD and experimental valuespercentage of control value. Figure 7 shows the cell viability after 48 and 72 h of direct cell interaction with the material samples.
The test revealed that all analyzed material samples decrease the cell viability by about 20% compared to control cultures. On the other hand, cell viability in the experimental cultures is approximatively the same, regardless of the TiO 2 ratio, which means that TiO 2 does not influence the biocompatibility of the materials. The decreasing of the cell viability in experimental cultures with the same value could be explained through the mechanical action of the direct contact of the membranes on the cells. The in vitro experimental condition for cell viability testing leads to the conclusion that polyvinyl alcohol/chitosan matrices with TiO 2 nanoparticles and/or polyphosphonate additive PFR-3 express a cytocompatibility, according to ISO 10993-5 recommendations.

Analysis of Cell Morphology
The effect of direct exposure of cultured cells to the sample material was assessed microscopically by phase contrast, overlapped to fluorescence images of DAPI-stained nuclear DNA. The images of the fixed and stained cells at 20× objective magnification are shown in Figure 8.
From the images showing the experimental cultures, no differences of cell morphology were observed between the different compositions of the samples. All analyzed cultures contain epithelial cells with a shape typical of the MCF 7 cell line. It is observed that at 72 h of culture, the cells grow in confluent monolayers, with a cell density without significant differences between the binary PVA/CS sample and multi-component PVA/CS films containing TiO 2 and/or PFR-3 additives.

Analysis of Cell Morphology
The effect of direct exposure of cultured cells to the sample material was assesse microscopically by phase contrast, overlapped to fluorescence images of DAPI-staine nuclear DNA. The images of the fixed and stained cells at 20× objective magnification ar shown in Figure 8.

Conclusions
PVA/CS composite films were obtained by the casting from solution technique, starting from appropriate amounts of PVA and CS, as polymer matrices, and TiO 2 and PFR-3 polyphosphonate were utilized as reinforcing additives. Infrared spectroscopy enabled structural validation and the identification of distinctive bands for certain functionalities in the prepared composite films. According to the Ra value, the surface roughness characteristics decreased when the PFR-3 was introduced into the PVA/CH matrix, indicating its contribution to the homogeneity and quality of the four-component composites. Scanning electron microscopy demonstrated a small increase in porosity in the PVA/CS-3 sample as the polyphosphonate PFR-3 level increased. In vitro evaluation of antimicrobial potential in the series confirmed the antimicrobial role of TiO 2 in the PVA/CS-TiO 2 matrices. Cell viability testing revealed that polyvinyl alcohol/chitosan matrices containing TiO 2 nanoparticles or TiO 2 /polyphosphonate additive PFR-3 exhibit cytocompatibility in accordance with ISO 10993-5 guidelines.

Preparation of PVA/CS Films following the Casting Procedure
The first step in preparing these materials as composite films involved the treating of polyvinyl alcohol with oligophosphonate, a reaction which takes place in polar organic solvent, in this case DMF, working with concentrations of 5% PVA, by heating the mixtures at 95 • C for about 4-5 h. After the dissolution of the components is complete, the mixture is allowed to cool down to about 40 • C; then, the solution is poured into crystallizers, and the solvent is allowed to evaporate. The second stage consists in transferring the film obtained after drying in the crystallizer over a previously obtained chitosan solution (1% solution in water treated with glacial acetic acid 2%). After complete dissolution of the reaction mixture, the mixture was treated with an aqueous suspension of a predetermined amount of titanium dioxide nanoparticles, homogenized, and previously dispersed by ultrasound for 15 min. The resulting homogeneous composite mixture was poured into 10 × 10 cm 2 Teflon plates to slowly evaporate the solvent in ambient conditions. Afterwards, they were placed in the oven for an advanced drying treatment by heating to approximately 40-50 • C, under a vacuum, for a period of 8 h. The obtained films are stored and used for the characterization and testing of new materials. The complete data on the synthesis of composites in this series are presented in Table 1, whereas the schematic diagram of the preparation is given in Figure 9. mixture is allowed to cool down to about 40 °C; then, the solution is poured into crystallizers, and the solvent is allowed to evaporate. The second stage consists in transferring the film obtained after drying in the crystallizer over a previously obtained chitosan solution (1% solution in water treated with glacial acetic acid 2%). After complete dissolution of the reaction mixture, the mixture was treated with an aqueous suspension of a predetermined amount of titanium dioxide nanoparticles, homogenized, and previously dispersed by ultrasound for 15 min. The resulting homogeneous composite mixture was poured into 10 × 10 cm 2 Teflon plates to slowly evaporate the solvent in ambient conditions. Afterwards, they were placed in the oven for an advanced drying treatment by heating to approximately 40-50 °C, under a vacuum, for a period of 8 h. The obtained films are stored and used for the characterization and testing of new materials. The complete data on the synthesis of composites in this series are presented in Table 1, whereas the schematic diagram of the preparation is given in Figure 9. The chemical structure of the PVA/CS/TiO2 films was analyzed using Bio-Rad 'FTS 135′ FTIR spectrometer equipped with a Specac "Golden Gate" ATR accessory. To record scans between 4000 and 500 cm −1 at a resolution of 4 cm −1 , a LUMOS Microscope Fourier Transform Infrared (FTIR) spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany), equipped with an attenuated total reflection (ATR) device was used. The chemical structure of the PVA/CS/TiO 2 films was analyzed using Bio-Rad 'FTS 135 FTIR spectrometer equipped with a Specac "Golden Gate" ATR accessory. To record scans between 4000 and 500 cm −1 at a resolution of 4 cm −1 , a LUMOS Microscope Fourier Transform Infrared (FTIR) spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany), equipped with an attenuated total reflection (ATR) device was used.

Roughness and Contact Angle Measurements
The roughness of the composite films was analyzed at a recording speed of 0.10 mm/s using the Tencor Alpha-Step D-500 High Sensitivity Stylus Profiler (KLA-Tencor Corporation, Milpitas, CA, USA). By applying a stylus force of 15 mg and a long-range cutoff filter of 60 µm, arithmetic mean roughness, Ra, was obtained, which is the average of the absolute value along the sampling length.
Two test liquids-double-distilled water (W) and ethylene glycol (EG)-were used to test their static contact angles on the composite film surfaces. The measurements were performed in triplicate, at room temperature, on a CAM 101 system (KSV Instruments, Helsinki, Finland) equipped with a liquid dispenser, video camera, and drop shape analysis software. The geometric mean approach was employed to calculate the surface tension parameters. This measurement allows for the calculation of parameters that describe the composite film's surface and its capacity for absorption, such as free surface energy (SV), solid-liquid interfacial tension (SL), and work of adhesion (Wa). Detailed experimental setup and calculations were described in a previous publication [34].

Degree of Swelling
Swelling behavior of the PVA/CS composite films prepared via the solution casting method was studied. Thus, dried samples of 0.5 × 0.5 cm 2 were weighted and submerged in 10 mL Millipore water in a closed bottle that was set in a thermostatic bath at 37 • C. The composite film samples were taken at predetermined intervals, and, after the extra water was removed with filter paper, the films weights were measured. The following equation was used to compute the swelling ratio, which represents the water absorption of each sample: where SD (%) represents the amount of absorbed Millipore water; W d -weight of the dry composite film; W t -weight of the hydrated sample.

Microscopic Morphology
Microscopic examinations have been performed on Environmental Scanning Electron Microscope Type Quanta 200, operating at 10 kV with secondary electrons in low vacuum mode (LFD detector). The composite samples were fractured, and their cross-section surfaces were analyzed using scanning electron microscopy (SEM). An Energy Dispersive X-ray (EDX) system is a feature of the Quanta 200 microscope that allows for qualitative, quantitative analysis, and elemental mapping.

Evaluation of Antimicrobial Activity
Diffusimetric determinations were performed to highlight the antimicrobial activity of the PVA/CS samples containing PFR-3 and/or TiO 2 additives. This technique is common for such tests and is based on the principle of contact of the test matrices with the surface of a culture medium inoculated with different microbial species. The antimicrobial tests were performed with standardized strains: Staphylococcus aureus ATCC 25923, MRSA ATCC 43300, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 90028. Standard microbial suspensions with a density of 0.5 McFarland, prepared with a spectrophotometer, were used. An aliquot (500 µL) of the bacterial suspension was applied to the surface of the Mueller-Hinton aggregate culture medium (BioRad, Taufkirchen, Germany) using an exudate swab. After drying for 10 min in a thermostat with the lid open, the test matrices were spread on the surface of the medium. The results were evaluated after incubation at 37 • C for 24 h. The antimicrobial activity was highlighted by identifying the areas of microbial inhibition formed upon contact with the tested matrices.

Evaluation of Cytotoxicity and Cell Morphology
The PVA/CS composite films were cut into 4 mm Ø discs, decontaminated with 70% aqueous ethyl alcohol solution for 20 min and then repeatedly washed in sterile ultrapure water and HBSS (Hank's Balanced Salt Solution) to remove any remaining contaminants. Following washing, the samples were equilibrated for 24 h in culture media (DMEM Ham/F12, supplemented with 10% fetal bovine serum and 1% antibiotic mixture). For equilibration, 0.5 mL of culture media were utilized for each piece of 4 mm Ø material. Using the MTT assay, the cytotoxicity of the growth medium and the material sample were both evaluated. Additionally, phase-contrast microscopy and fluorescence microscopy were used to examine the direct effects of the material samples on cell morphology. For biocompatibility study, the human epithelial cell line MCF 7, at the density of 20 × 10 4 /well cells, was plated in 48-well culture plates and DMEM Ham/F12 culture medium supplemented with 10% bovine fetal serum and 1% mixture of Penicillin-Streptomycin-Neomycin antibiotics (all for in vitro use). Thus, material samples or the media of their extraction were put over the cell monolayers in the 48-well culture plates in order to assess the viability, density, and morphology of the cells.
The MTT technique uses tetrazolium salts as an oxidized substrate for mitochondrial dehydrogenases to measure the activity of cellular metabolism. The basic idea behind the procedure is that the yellow MTT compound is reduced into an insoluble purple (formazan) product. The formazan salts are solubilized with isopropyl alcohol to obtain a blue-violet solution, the intensity of which is directly proportional to the number of living cells in the culture. Briefly, for MTT experiments, the culture medium of the cell cultures was replaced with work MTT solution of 0.25 mg/mL and incubated with cells for 3 h. The MTT solution was replaced with an equal volume of isopropyl alcohol to solubilize the formazan crystals. The absorbance of the formazan solution was measured spectrophotometrically at 570 nm, using a TECAN UV/VIS plate reader. An equivalent amount of isopropanol was used as the blank reference sample. The cell viability was calculate applying the equation: Cell viability (%) = A Sample A control × 100 Each membrane sample was evaluated in triplicate, at 48 and 72 h of cell incubation with materials. The cultures developed in the absence of the material were used as growth control.
The cells were fixed in 4% paraformaldehyde solution and permeabilized for 30 min at +40 • C, in 0.05% Triton X 100 in PBS. After 72 h of interaction between the cells and material, the DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidines) staining protocol was carried out for the cell morphology examination. PBS was used to properly clean the permeabilized cells before they were incubated with DAPI 10 µg/mL work solution for 30 min at room temperature. After being washed in PBS, stained cells were examined using fluorescence microscopy at λex 340 nm and λem 488 nm. To emphasize the cell nucleus and form, phase contrast was overlapped with the fluorescence.

Data Availability Statement:
The data that support the findings of the current study are listed within the article.