CV was not able to suppress biofilm development of PaeS and PaeG (Figure 5c). Biofilm biomass of PaeS was 5.1 × 106 ± 1.6 × 106 µm3 on CBVF vs. 5.3 × 106 ± 4.2 × 105 µm3 on CV. However, CV seemingly induced a deeper stress when compared to CBVF, since the sessile biomass of both *P. aeruginosa* strains displayed an increased percentage of dead cells (Table 1, Figure 5c). As expected, CG displayed greater activity in preventing *P. aeruginosa* biofilm formation by significantly reducing the biomass of both isolates, being more effective against PaeS (5.1 × 106 ± 1.6 × 106 µm3 on CBVF vs. 2.1 × 106 ± 2.9 × 105 µm3 on CG) than against PaeG (5.2 × 106 ± 4.5 × 105 µm3 on CBVF vs. 2.7 × 106 ± 1.7 × 105 µm3 on CG). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 17

**Figure 2.** Biofilm biomass secreted by (**a**) *Staphylococcus aureus*, (**b**) *Staphylococcus epidermidis* and (**c**) **Figure 2.** Biofilm biomass secreted by (**a**) *Staphylococcus aureus*, (**b**) *Staphylococcus epidermidis* and (**c**) *Pseudomonas aeruginosa* isolates on ST and CBVF. Statistical significance: *p* < 0.001 (\*\*\*).

*Pseudomonas aeruginosa* isolates on ST and CBVF. Statistical significance: *p* < 0.001 (\*\*\*).

Magnification: 20x.

2.2.1. *Staphylococcus aureus*

Magnification: 20x.

2.2.1. *Staphylococcus aureus*

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 6 of 17

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 6 of 17

**Figure 3.** Representative confocal laser scan microscopy (CLSM) 3D images of sessile methicillinresistant *Staphylococcus aureus* on ST, CBVF, CG and CV stained with Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit. Magnification: 20x. **Figure 3.** Representative confocal laser scan microscopy (CLSM) 3D images of sessile methicillin-resistant *Staphylococcus aureus* on ST, CBVF, CG and CV stained with Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit. Magnification: 20×. **Figure 3.** Representative confocal laser scan microscopy (CLSM) 3D images of sessile methicillinresistant *Staphylococcus aureus* on ST, CBVF, CG and CV stained with Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit. Magnification: 20x.

**Figure 4.** Representative CLSM 3D images of the surface characteristics of ST and CBVF. **Figure 4.** Representative CLSM 3D images of the surface characteristics of ST and CBVF. **Figure 4.** Representative CLSM 3D images of the surface characteristics of ST and CBVF.Magnification: 20×.

CSLM analysis allowed quantification of the biofilm biomass at ~6.2 × 106 ± 1.2 × 106 µm3 on

CSLM analysis allowed quantification of the biofilm biomass at ~6.2 × 106 ± 1.2 × 106 µm3 on CBVF for SauS (Figure 5a). The biofilm biomass was significantly reduced to ~1.2 × 106 ± 7.4 × 104 µm3

on CG and to ~1.4 × 106 ± 3.9 × 105 µm3 on CV. In MRSA and GISA, similar patterns were found with a significant reduction of the biofilm biomass on CG or CV. For SauG, only CV was able to significantly decrease the biofilm production, whereas CG did not show a significant reduction. However, as reported in Table 1 and depicted in Figure 5a, CG displayed a markedly increased mortality rate of MRSA and GISA, as compared to CV (55.3% and 48.9% vs. 38.6% and 20.6%,

**Figure 5.** Ratio of live:dead cells (green:red) of (**a**) *Staphylococcus aureus*, (**b**) *Staphylococcus epidermidis* and (**c**) *Pseudomonas aeruginosa* isolates in biofilm formed on CBVF, CG and CV. Statistical **Figure 5.** Ratio of live:dead cells (green:red) of (**a**) *Staphylococcus aureus*, (**b**) *Staphylococcus epidermidis* and (**c**) *Pseudomonas aeruginosa* isolates in biofilm formed on CBVF, CG and CV. Statistical significance: *p* < 0.05 (\*), *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*).

significance: *p* < 0.05 (\*), *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*). **Table 1.** Mortality rate by CLSM analysis. Data are expressed as percentage (%) of dead cells against the **Table 1.** Mortality rate by CLSM analysis. Data are expressed as percentage (%) of dead cells against the whole biofilm biomass. CBVF: CeramentTM Bone Void Filler; CG: CeramentTM G; CV: CeramentTM V.

#### *2.3. Resistance Selection to Eluted Vancomycin and Gentamicin*

In the tested experimental conditions, no adaptation in the presence of CG or CV was observed by means of the zone of inhibition (ZOI) measurements on a solid agar medium. When repeatedly exposed to a freshly prepared antibiotic-loaded disc, all the tested strains displayed uniform ZOIs with no colonies of viable bacteria inside the inhibition halo.

When in the presence of a new disc, all the strains of *S. aureus* and *P. aeruginosa* maintained a comparable susceptibility profile (ZOI diameter) at all the time points, as depicted in Figure 6a,c, respectively (∆ ZOI = ± 2 mm between T1 and T7), with the exception of the MRSA strain, which showed a slight decrease in susceptibility to gentamicin after seven consecutive cycles (ZOI 10.3 ± 0.6 mm and 4.3 ± 0.6 mm at T1 and T7, respectively). In contrast, *S. epidermidis* strains, except for SepG, displayed an increased ZOI diameter after the second CG cycle (14.7 ± 0.6 mm vs. 19.3 ± 0.6 mm for SepS, 14.3 ± 0.6 mm vs. 17.3 ± 1.2 mm for MRSE and 14.3 ± 0.6 mm vs. 17 ± 1 mm for GISE at T1 and T2, respectively) (Figure 6b). Nevertheless, the increased susceptibility profile proved to be transitory as the growth–inhibition diameters reduced during the following cycles, reaching values like those observed at T1 (∆ ZOI = ± 2 mm between T1 and T7). All other differences observed were not significant.

When the same disc was used from T1 to T7, all the tested staphylococcal strains showed an absence of inhibition halo at the later time points in the presence of CV, comprising both the glycopeptide-intermediate strains (GISA and GISE). Otherwise, in the presence of CG, the gentamicin-resistant strains (SauG and SepG) were not inhibited from T4 and T3, respectively. The absence of any stable or transient adaptation was then confirmed by measuring MIC values for all the tested clinical isolates exposed to both gentamicin and vancomycin released from antibiotic-loaded discs. No significant changes in the susceptibility profile were observed. Indeed, MIC fluctuations recorded never exceeded 1-fold dilution (Tables 2 and 3).

**Table 2.** Resistance profile of the tested strains to gentamicin before (T0) and after (T7) exposure to CG and after cycle on antibiotic-free medium (T14). Numbers indicate gentamicin concentrations (µg/mL). (=), exposure to the same CG disc; (,), exposure to a fresh CG disc.

CG/CV disc.

**Figure 6.** Zone of inhibition (ZOI) of (**a**) *Staphylococcus aureus*, (**b**) *Staphylococcus epidermidis* and (**c**) *Pseudomonas aeruginosa* growth on agar plates before (T0) and after (T7) exposure to CG/CV. ZOI (mm ± SD) are plotted against time (days). (=), exposure to the same CG/CV disc; (≠), exposure to a fresh **Figure 6.** Zone of inhibition (ZOI) of (**a**) *Staphylococcus aureus*, (**b**) *Staphylococcus epidermidis* and (**c**) *Pseudomonas aeruginosa* growth on agar plates before (T0) and after (T7) exposure to CG/CV. ZOI (mm ± SD) are plotted against time (days). (=), exposure to the same CG/CV disc; (,), exposure to a fresh CG/CV disc.

#### **3. Discussion**

One of the most feared complications in orthopedic surgery is the bacterial colonization of abiotic surfaces causing PJI and FRI, occurring in 1% to 2% of the patients receiving orthopedic implants [23]. Antibiotic-eluting BGS represent an attractive clinical option in the management of PJI and FRI. Gentamicin-loaded ceramic fillers have been extensively employed in clinics and their successful use reported in the literature [20–22,24–28]. The increasing emergence of aminoglycoside resistance has led to the development of a new BGS formulation enriched with vancomycin, an additional option alongside gentamicin in the orthopedic field [29]. Although these are promising strategies, there is a lack of in vitro data supporting the safe use of both CG and CV in the treatment of biofilm-related infections. Indeed, their impact on bacterial adhesion, biofilm formation and selection of resistance has not been systematically evaluated in vitro.

#### *3.1. Bacterial Adhesion*

Clinically relevant strains of *S. aureus*, *S. epidermidis* and *P. aeruginosa* were tested for their ability to adhere in the first two hours of contact to solidified calcium sulfate/hydroxyapatite discs, to evaluate a possible contamination upon surgical implantation. This scenario can be expected in a contaminated open fracture (e.g., Gustilo–Anderson IIIb), where, even after the initial extensive debridement and irrigation, bacteria may persist and attach to bone fragments or internal fixation devices. When adhesion to unloaded CBVF was compared to ST, staphylococcal isolates displayed a heterogeneous trend, which was mainly similar or lower to that of ST, with the exception of the two susceptible strains. On the contrary, all the *P. aeruginosa* isolates showed a greater affinity for CBVF discs than for ST under the tested experimental conditions. It is well known that this reversible event not only varies according to the physical characteristics of the implant, but also relies on differences between bacterial species and even strains. Indeed, the surface hydrophilicity of the material, the presence of functional groups and electric charge, as well as the physicochemical characteristics of the microbial membrane modulate initial physicochemical interactions influencing early bacterial adhesion [30]. In this context, the use of antibiotic-loaded CG or CV disks drastically affected the ability of the selected strains to adhere to the tested compounds. CG inhibited the adhesion of the gentamicin-susceptible bacterial strains (SauS, GISA, SepS and GISE) and reduced the bacterial load of the gentamicin-resistant staphylococci (SauG, SepG, MRSA and MRSE), but could not completely impede their adhesion. Since gentamicin is active against Gram-negative bacteria, CG inhibited the attachment of PaeS and PaeG, whereas CV completely avoided the adhesion of all staphylococci strains. As expected, CV was not able to impair the adhesion of the Gram-negative *P. aeruginosa* strains.

In the clinical setting, this finding could be used in the prophylaxis of infection in contaminated open fractures. If a bone void or fracture gap is present during the initial debridement, it could be filled with CG. Since open fractures might be contaminated by Gram-positive and Gram-negative bacteria [31], the shown effect of CG on *P. aeruginosa* might be beneficial in this setting. Aljawadi and co-workers recently presented their clinical results on the use of CG in addition to systemic antibiotics in Gustilo–Anderson IIIb open fractures in a single-stage "Fix and Flap" procedure in 80 patients [24]. The limb salvage rate in this challenging patient group was 96.25% and the infection rate was 1.25%. A randomized controlled trial for CG as part of the surgical repair for open tibial diaphyseal fractures is on its way and recruitment of 200 patients has been finalized (www.clincaltrials.gov, Identifier: NCT02820363).

#### *3.2. Evaluation of Biofilm Formation*

The ability to form biofilm on the tested substrates was evaluated by CLSM. Despite the porous structure of CBVF, all the tested strains displayed a significantly greater biomass volume on sandblasted titanium than on CBVF.

Interestingly, biofilm formation by all the tested staphylococcal strains was negatively affected by the presence of antibiotic-eluting BGS, highlighting the efficacy of the initial burst release of an antimicrobial even in such an in vitro setting conducted under ideal conditions, which does not accurately recapitulate in vivo conditions. This experimental design does not consider important aspects such as the lack of surrounding tissues, the physiological supply of nutrients, cellular resorption and immune system action, which might concur in impairing bacterial contamination and help to reduce the number of bacteria on the site of contamination. The high bacterial inoculum (much greater than that of a possible contamination event, which is likely to happen from few cells) and a nutrient-rich growth medium represent a "worst case scenario", which understandably supports our results.

CG was able to significantly suppress biofilm formation of all staphylococci strains, except for SauG and both *P. aeruginosa* strains. CV reduced biofilm formation of all staphylococci, including MRSA and MRSE, but had no impact on Gram-negative *P. aeruginosa* biofilm biomass. The observed increase of mortality rate of cells encased within the biofilm matrix could be ascribed to the effects of the high vancomycin concentration, which might cause the perturbation of cell shape, as observed on Gram-negative strains by others [32].

To date, clinical outcomes of CG and CV have been published in PJI [22], FRI [25,27], chronic Osteomyelitis (cOM) [20,21], and Diabetic Foot Osteomyelitis (DFO) [26,28]. McNally and co-workers found a reduction of the recurrence rate of cOM of 4% in a one-stage procedure compared to common reports in the literature of about 10% [21]. Briefly, they evaluated the use of CG in the management of deep bone infection in a one-stage procedure. Considering the unknown bacterial resistance pattern, the broader Gram-negative and Gram-positive spectrum and the unavailability of CV at the time of the study, CG proved effective in the treatment of chronic Osteomyelitis, even in the presence of gentamicin-resistant clinical isolates.

The results of our in vitro investigation showed that CV might be beneficial in the management of staphylococcal infections. Selecting between CG and CV is only possible if the causative bacteria have been identified in the first stage of a two-stage procedure. Logoluso et al. used a two-stage approach in the management of PJI, where CG was used in 13 patients and CV in 7 (only staphylococcal and enterococcal infections), finding only one recurrence of PJI in their series of 20 consecutive cases [22]. In FRI, the recurrence rate was reported in 7.7% [25] and 8.5% of cases [27], and in DFO in 10% [26].

#### *3.3. Resistance Selection by Eluted Vancomycin and Gentamicin*

There is an ongoing discussion if local antibiotics can induce resistance or lead to a selection of resistant bacteria. The slow penetration rate of antibiotics through the biofilm matrix may give time to the cells to adapt phenotypically and genotypically. Sessile bacteria encased within the biomass are able to incorporate eDNA from the biofilm matrix, and in vitro tests suggested that horizontal gene transfer may even be 10,000 times more likely in biofilms compared to their planktonic counterparts [33–36]. Furthermore, cells in a biofilm are inherently prone to spontaneous mutations, possibly due to an increased response to stress and consequent DNA damage [37,38]. In the orthopedic field, this topic is gaining particular interest with regard to reduced staphylococcal susceptibility to glycopeptide antibiotics, which are commonly employed in the treatment of orthopedic infections because of their noteworthy bone penetration and the diffusion of methicillin-resistant strains [39]. While all ZOIs of gentamicin-resistant strains (SauG, SepG and PaeG) were notably inferior to those of non-resistant strains, such marked difference was not observed in the presence of vancomycin for the glycopeptide-intermediate strains. This result confirms that aminoglycoside resistance in staphylococci is derived from the acquisition of a gene (i.e., aph(3')-IIIa), whereas resistance to glycopeptide mainly consists of a reduced susceptibility by accumulation of point mutations [40]. However, it is worth noting that glycopeptide-intermediate staphylococci behavior in the presence of CV was like that of the other strains.

For all the aforementioned reasons, it is extremely important to test whether the exposure to antibiotic-loaded biomaterials might lead to the acquisition of a resistance profile. The results obtained from the present study suggest that prolonged exposure either to the same or new antibiotic-eluting hydroxyapatite/calcium sulfate discs did not lead to stable or transient adaptations in either of the tested bacterial strains.

Despite the aforementioned limitations of the study, the emergence of antibiotic-resistant strains in the presence of an antibiotic-enriched compound in a clinical scenario seems extremely unlikely considering the initial burst of high concentrations of active drugs and the subsequent retention of a sustained amount of antibiotics, as also reported from previous studies [19]. Furthermore, the combined use of both gentamicin and vancomycin-eluting compounds might be considered for clinical cases presenting high-risk of infection recurrence due to polymicrobial contamination. This low-risk profile, together with the clinically confirmed regenerative properties of the tested resorbable materials [20,41], confirms further the pertinence of antibiotic-containing BGS as prophylactic and treatment options and proves them potentially safe for clinical application in orthopedic infection surgery [42].

#### **4. Materials and Methods**

#### *4.1. Tested Bacterial Strains and Biomaterials*

Clinically relevant bacterial strains from patients with IRI were isolated at the Laboratory of Clinical Chemistry and Microbiology of the IRCCS Galeazzi Orthopedic Institute and were used in this study. Biofilm-producing strains of *S. aureus* (one susceptible strain, SauS; one gentamicin-resistant strain, SauG; one methicillin-resistant strain, MRSA; one glycopeptide-intermediate strain, GISA), *S. epidermidis* (one susceptible strain, SepS; one gentamicin-resistant strain, SepG; one methicillin-resistant strain, MRSE; one glycopeptide-intermediate strain, GISE) and *P. aeruginosa* (one gentamicin-susceptible strain, PaeS and one gentamicin-resistant strain, PaeG) were selected. The bacterial ability to adhere and to produce biofilm, as well as the emergence of antibiotic resistance in vitro were tested on the following substrates: sandblasted titanium (ST; used as growth control) (Geass, Pozzuolo del Friuli, Italy), CERAMENTTM|BONE VOID FILLER, a biphasic degradable bone graft substitute composed of 40% hydroxyapatite and 60% calcium sulfate (without antibiotic, used as growth control), CERAMENTTM|G, containing 17.5 mg gentamicin/mL paste and CERAMENTTM|V, containing 66 mg vancomycin/mL paste. Sterile kits of the products were supplied by BONESUPPORT AB (Lund, Sweden) and mixed according to the instructions for use provided by the manufacturer.

#### *4.2. Evaluation of Bacterial Adhesion on Material Surface*

The effects of different formulations of the calcium sulfate/hydroxyapatite bone graft substitute on bacterial adhesion were tested. To standardize the experimental conditions, 6 × 2.5 mm discs of unloaded CBVF and CG and CV were produced by pouring freshly prepared injectable pastes into a custom-made mold of polydimethylsiloxane (PDMS, Farnell, Lainate, Italy). The theoretical concentration of gentamicin in each disc was approximately 1.23 mg, whereas that of vancomycin was approximately 4.66 mg per disc. Discs were placed in a 96-well microplate containing 180 µL of Brain Heart Infusion (BHI) broth (BioMérieux, Craponne, France). Each well was inoculated with 20 µL of a bacterial overnight culture to obtain a final density of 1.0 × 106 CFU/mL. To evaluate the first phases of bacterial attachment upon tested substrates, after 30, 60 and 120 min of incubation at 37 ◦C, the discs were rinsed three times with sterile saline to remove non-adherent bacteria [43,44]. Then, discs were immersed in 200 µL of a 0.1% *w*/*v* dithiothreitol (DTT; Sigma-Aldrich, Milan, Italy) solution and mechanically stirred for 15 min at room temperature to detach bacteria adhering to the discs [45]. Finally, 10 µL from each well was serially diluted and drop-plated on Tryptic Soy Agar (TSA; Sigma-Aldrich, Milan, Italy) plates and incubated at 37 ◦C for 24 h for colony forming units (CFU) count [46]. The adhesion tests were performed in triplicates for each strain.

#### *4.3. Analysis of Biofilm Formation by CLSM*

The ability of the tested clinical strains to form biofilm on the antibiotic-containing and unloaded biphasic bone graft substitutes was evaluated using CLSM [47]. Discs were placed in a 24-well microplate with 950 µL of BHI broth (Biomérieux, Craponne, France) and 50 µL of 1.0 × 107 CFU/mL bacterial suspension. After 48 h of incubation at 37 ◦C, discs were rinsed three times with sterile saline to remove non-adherent bacteria and stained with Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit (Thermo Fisher Diagnostics, Monza, Italy), according to the manufacturer's instructions. Briefly, the staining solution was prepared by adding 3 µL of SYTO9 and 3 µL of propidium iodide to 1 mL of filter-sterilized water. Samples were incubated with 20 µL of staining solution at room temperature in the dark for 15 min. Afterwards, samples were washed with sterile saline and examined with an upright TCS SP8 (Leica Microsystems CMS GmbH, Mannheim, Germany) using a 20× dry objective (HC PL FLUOTAR 20×/0.50 DRY). Images from at least three randomly selected areas were acquired for each sample. The obtained images were processed with Las X (Leica Microsystems CMS GmbH, Mannheim, Germany) and analyzed with Fiji software (Fiji, ImageJ, Wayne Rasband National Institutes of Health). The test was performed in triplicates for each strain.

#### *4.4. Determination of Minimum Inhibitory Concentration*

The MIC for each strain was determined by the broth microdilution method, following the guidelines of the European Committee on Antimicrobial Susceptibility Testing [48]. A microbial suspension was prepared for each bacterial strain in Mueller–Hinton (MHB, Millipore, Milan, Italy) broth to an optical density equal to a standard turbidity of 0.5 McFarland (~1.5 × 108 CFU/mL). After obtaining a microbial load of 5 × 106 CFU/mL, 10 µL of each suspension was inoculated in a 96-well microplate containing 90 µL of a serial 2-fold dilution of either gentamicin (Sigma-Aldrich, Milan, Italy) or vancomycin (VWR, Milan, Italy). The MIC was defined as the lowest antibiotic concentration that inhibited the bacterial growth after 24 h of incubation at 37 ◦C, as assessed by the unaided eye. The experiment was performed in triplicates.

#### *4.5. Evaluation of Selection for Bacterial Resistance after Exposure to Eluted Antibiotics*

The emergence of bacterial resistance to the antibiotics released from the drug-loaded formulations was investigated by the modified Kirby–Bauer method as reported by others [49]. Briefly, either gentamicin or vancomycin-eluting discs were placed on TSA plates previously seeded with a bacterial suspension corresponding to a standard turbidity of 0.5 McFarland (~5 × 108 CFU/mL) (T0). After incubation at 37 ◦C for 24 h, the zone of inhibition (ZOI) was measured and bacterial cells growing on the edge thereof were subcultured on a fresh TSA plate for 7 days (from T1 to T7). Specifically, two different procedures were applied: (i) the same exhausted antibiotic-containing disc was used throughout the whole experiment (up to T7); (ii) the antibiotic-containing substrate was replaced with a fresh disc every 24 h for 7 days. The ZOI was measured and recorded daily. After overnight incubation in antibiotic-free BHI broth, bacteria were further subcultured on antibiotic-free TSA plates for an additional 7 days (T14) to assess the stability of the potentially acquired resistance. MIC values were determined before (T0) and after exposure to the eluted antibiotics (T7) and after the last cycle on antibiotic-free TSA plates (T14). The acquisition of bacterial resistance to an antimicrobial compound was defined as ≥4-fold increase in MIC values [50].

#### **5. Conclusions**

From the results obtained in this in vitro study, it can be concluded that CV efficacy is comparable to CG in the treatment of Gram-positive biofilm and has been proven to be effective against gentamicin-resistant strains. The use of antibiotic-containing hydroxyapatite/calcium sulfate bone graft substitutes could be a feasible strategy in the prevention of bacterial colonization and biofilm development by microorganisms and may prove to be a promising approach for dead-space management in the treatment of cOM, FRI, DFO and PJI, always combined with systemic antibiotic therapy.

**Author Contributions:** Conceptualization, N.L. and E.D.V.; methodology, A.B. and M.B.; formal analysis, A.B. and M.B.; data curation, A.B. and M.B.; writing—original draft preparation, A.B. and M.B.; writing—review and editing, N.L. and E.D.V.; visualization, M.B.; supervision, E.D.V.; funding acquisition, E.D.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was partly sponsored by BONESUPPORT AB and partially funded by the Italian Ministry of Health (RC Project).

**Acknowledgments:** The authors would like to thank Michael Diefenbeck and Maria Eugenia Butini for technical and scientific support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **Abbreviations**

#### **References**

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## *Article* **Oxo-Titanium(IV) Complex**/**Polymer Composites—Synthesis, Spectroscopic Characterization and Antimicrobial Activity Test**

**Piotr Piszczek 1,\* , Barbara Kubiak 1 , Patrycja Goli ´nska 2 and Aleksandra Radtke 1,\***

Received: 2 November 2020; Accepted: 15 December 2020; Published: 18 December 2020

**Abstract:** The emergence of a large number of bacterial strains resistant to many drugs or disinfectants currently used contributed to the search of new, more effective antimicrobial agents. In the presented paper, we assessed the microbiocidal activity of tri- and tetranuclear oxo-titanium(IV) complexes (TOCs), which were dispersed in the poly(methyl methacrylate) (PMMA) matrix. The TOCs were synthesized in reaction to Ti(OR)4 (R = iPr, iBu) and HO2CR' (R' = 4-PhNH2 and 4-PhOH) in a 4:1 molar ratio at room temperature and in Ar atmosphere. The structure of isolated oxo-complexes was confirmed by IR and Raman spectroscopy and mass spectrometry. The antimicrobial activity of the produced composites (PMMA + TOCs) was estimated against Gram-positive (*Staphylococcus aureus* ATCC 6538 and *S. aureus* ATCC 25923) and Gram-negative (*Escherichia coli* ATCC 8739 and *E. coli* ATCC 25922) bacteria and yeasts of *Candida albicans* ATCC 10231. All produced composites showed biocidal activity against the bacteria. Composites containing {Ti4O2} cores and the {Ti3O} core stabilized by the 4-hydroxybenzoic ligand showed also high activity against yeasts. The results of investigations carried out suggest that produced (PMMA + TOCs) composites, due to their microbiocidal activity, could find an application in the elimination of microbial contaminations in various fields of our lives.

**Keywords:** antimicrobial activity; oxo-titanium(IV) complexes; polymer-inorganic composites; physicochemical properties; thermal properties

#### **1. Introduction**

The unique physical and chemical properties of materials based on titanium dioxide, i.e., photocatalytic activity, hydrophilicity, or strong absorption of UV radiation, contribute to their wide-application inter alia in various fields of chemical, cosmetics, biomedical, pharmaceutical, and disinfecting applications [1–5]. The photocatalytic activity of TiO2 nanoparticles or nanocoatings, associated with the electron transfer between the valence band and the conduction one (the O2*p*-Ti3*d* charge transfer transition), which can take place in the presence of photons of wavelengths smaller than 350 nm (this corresponds to ca. 5% of the daylight), is of particular importance [3,4,6]. The above-mentioned phenomenon initiates the oxidation and reduction reactions, which proceed on the surface of TiO2 nanoparticles/nanocoatings and lead to the formation of the reactive oxygen species (ROS) responsible for the antimicrobial activity of these materials, among others [3]. Aiming to extend the absorption range of TiO2 in daylight, attention was paid to the oxo-titanium(IV) complexes (TOCs), which contain {TiaOb} cores of different architectures in their structures and their potential

use as antimicrobial and photocatalytic active substrates [7–11]. These compounds are studied for the possibility of their use for the photoinduced degradation of organic dyes or water splitting [8,12]. In the case of TOCs, the electron transfer takes place also between the 2*p* electron shell of the oxygen atom and the 3*d* shell of the titanium atom; however, the presence of ligands, which stabilize the oxo-titanium(IV) complex cluster, act as photosensitizers, and in this way, the radiation can be absorbed in a wider range [13,14]. By placing the ligands in the coordination sphere (e.g., carboxylate ligands (-O2CR')), the orbitals HOMO-LUMO of ligands and the {TiaOb} core can be mixed. As a result, the distance for electrons to move is smaller. The charge transfer from the ligands to the cluster core is possible due to its low-energy absorption band [15,16]. The photocatalytic activity of the oxo-titanium(IV) complexes, of the different {TiaOb} core architectures and possessing various carboxylic ligands, was most often assessed based on the photodegradation processes of organic dye aqueous solutions, such as methylene orange (MO), methylene blue (MB), and rhodamine B (RB) [16–19]. The samples, during the photocatalytic experiments were mostly irradiated by UV light in the range 315–400 nm; however, also, visible light was used. Kim et al. studied the oxo-Ti(IV) complexes with the {Ti6O6} core, and stabilized by such carboxylate ligands as 4-aminobenzoic, 4-amino-2-fluoro-benzoic, 4-amino-2-chloro-benzoic, 4-amino-3-chloro-benzoic, and 5-dichlorobenzoic acids, the anions were especially interesting. They showed a direct dependence of oxo-complex photocatalytic activity versus the way of -O2CR' group functionalization. This effect is closely related to the change in the distance between HOMO and LUMO orbitals in the tested compounds [19]. The results of our earlier studies on the photocatalytic activity of oxo-clusters with {Ti3O} and {Ti4O2} cores also exhibited a significant influence in the way carboxylate ligands functionalize [20–22]. According to these works, the oxo-Ti(IV) complexes stabilized by 9-fluorenecarboxylate and 4-aminobenzonate ligands revealed the best photocatalytic properties in the photodegradation of the methylene blue (MB) solution [20,21].

An interesting issue that may be related to the photocatalytic activity of oxo-Ti(IV) complexes are their antimicrobial properties. Nowadays, searching for new antimicrobial materials results from the necessity of their wide use in everyday life and from their importance in the public health system. Analyzing the literature data, we turned our attention on the antibacterial activity of compounds that contain metal-oxo cores, e.g., oxo-iron(III) complexes [23] or polyoxometalates [24], in their structures. The antibacterial assay of the (Fe3O(PhCO2)6(MeOH)3)(NO3)(MeOH)2 cluster showed a significant growth inhibition of *Bacillus cereus* MTCC 1272, *Staphylococcus epidermidis* MTCC 3086, and *Salmonella typhimurium* MTCC 98 but not of *Escherichia coli* MTCC 723 [23]. The results of studies on the antibacterial properties of polyoxometalates against *Moraxella catarrhalis* seem to be particularly interesting. It has been revealed that microbiocidal activity of these types of compounds mainly depends on their composition; metal-oxide anion core shape and size; and the type of the central metal in MO6 unit (M = Mo, V, and W) [24]. Antimicrobial properties have been found in the mixed ligand titanium dioxide complex, which was produced from TiO2 nanoparticles and 8-hydroxyquinoline and glycine as the ligands [25]. This complex showed good antifungal activity against *Alternaria alternata*, *Rhizoctania solani*, *Helminthosporium oryzae*, *Fusarium oxysporum*, *Curvularia lunata*, and *Aspergillus niger*, as well as antibacterial activity against *Salmonella*, *E. coli*, *S. epidermis*, and *Enterococcus faecalis*. However, this compound did not show biocidal activity against *Aspergillus fumigatus*, *Aspergillus terreus*, *Trichoderma viride*, and *Cladosporium herbarium* [25]. The investigations of Kaushal et al. revealed the promising biological activities of titanium complexes synthesized by reacting to TiCl4 with Schiff bases (SBs) [26,27]. The synthesized complexes were tested for their antimicrobial activity against pathogenic bacterial strains i.e., *B. cereus* MTCC 6728, *Micrococcus luteus* MTCC 1809, *S. aureus* MTCC 3160, *S. epidermidis* MTCC 3086, *Aeromonas hydrophila* MTCC 1739, *Aclaligenes faecalis* MTCC 126, *Shigella sonnei* MTCC 2957, *Klebsiella pneumoniae* MTCC 3384, *Pseudomonas aeruginosa* MTCC 1035, and *S. typhimurium* MTCC 1253. The (TiCl2(SB)2) complexes showed higher antimicrobial activity than their parent Schiff bases [27]. The analysis of the literature reports have shown that the antimicrobial activity of multinuclear oxo-titanium(IV) complexes have not been fully explored so far. Attention should be drawn to the results of the study by Svensson et al., which revealed the antibacterial activity of the oxo-complexes that

consisted of {Ti4O2} cores stabilized by two triclosan ligands against *S. aureus* [28]. However, in this case, the antibacterial activity of the hydrolyzed form of synthesized complex was studied by the authors.

It should be noted that all the above-mentioned antimicrobial agents can be used in liquid form, which distinguishes them from TOCs systems, which are discussed in the presented paper. The studied compounds (TOCs) containing {Ti3O} and {Ti4O2} cores due to their hydrophobic natures and their resistance to hydrolysis processes [20–22] were dispersed in a poly(methyl methacrylate) (PMMA) matrix (i.e., films (PMMA + TOCs) were produced). The aim of our investigations was the synthesis of a (PMMA + TOCs) composite characterized by very high surface antimicrobial activity. PMMA is an important material, which, due to sufficient mechanical properties and minimal inflammatory response, is used in different fields of our lives—inter alia, in dentistry [29]. An important direction of the research related to the use of PMMA-based materials is their modification aimed to improving their antimicrobial activity. This effect can be achieved by the addition of inorganic agents such as silver nanoparticles, titanium dioxide, a mixture of titanium dioxide and silicon dioxide nanoparticles, and anion powder (Na2SiO3) [29–34]. In this paper, the results of the modification of the PMMA films by the introduction of TOC micro-grains are discussed. The influence of TOC structures on the physicochemical properties and photocatalytic and microbicidal activity of the PMMA + TOC systems was assessed. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 19 {Ti4O2} cores stabilized by two triclosan ligands against *S. aureus* [28]. However, in this case, the antibacterial activity of the hydrolyzed form of synthesized complex was studied by the authors. It should be noted that all the above-mentioned antimicrobial agents can be used in liquid form, which distinguishes them from TOCs systems, which are discussed in the presented paper. The studied compounds (TOCs) containing {Ti3O} and {Ti4O2} cores due to their hydrophobic natures and their resistance to hydrolysis processes [20–22] were dispersed in a poly(methyl methacrylate) (PMMA) matrix (i.e., films (PMMA + TOCs) were produced). The aim of our investigations was the synthesis of a (PMMA + TOCs) composite characterized by very high surface antimicrobial activity. PMMA is an important material, which, due to sufficient mechanical properties and minimal inflammatory response, is used in different fields of our lives—inter alia, in dentistry [29]. An important direction of the research related to the use of PMMA-based materials is their modification aimed to improving their antimicrobial activity. This effect can be achieved by the addition of inorganic agents such as silver nanoparticles, titanium dioxide, a mixture of titanium dioxide and silicon dioxide nanoparticles, and anion powder (Na2SiO3) [29–34]. In this paper, the results of the modification of the PMMA films by the introduction of TOC micro-grains are discussed. The influence of TOC structures on the physicochemical properties and photocatalytic and microbicidal

#### **2. Results** activity of the PMMA + TOC systems was assessed.

Studied oxo-complexes (TOCs) were isolated from a the reaction mixture of appropriate titanium alkoxides and organic acids (4:1 alkoxide: acid molar ratio) at room temperature (RT) and at an inert gas atmosphere (Ar, Schlenk line), according to earlier described procedures (Table 1) [20–22]. 4-aminobenzoic acid (HOOC-4-PhNH2) and 4-hydroxobenzoic acid (HOOC-4-PhOH) were used in these reactions. The type of the titanium alkoxide (OR; R = iPr and iBu) is the factor that directly influences the {TiaOb} core architecture. **2. Results** Studied oxo-complexes (TOCs) were isolated from a the reaction mixture of appropriate titanium alkoxides and organic acids (4:1 alkoxide: acid molar ratio) at room temperature (RT) and at an inert gas atmosphere (Ar, Schlenk line), according to earlier described procedures (Table 1) [20–22]. 4-aminobenzoic acid (HOOC-4-PhNH2) and 4-hydroxobenzoic acid (HOOC-4-PhOH) were used in these reactions. The type of the titanium alkoxide (OR; R = iPr and iBu) is the factor that

**Table 1.** The compositions of the reaction mixtures. THF: tetrahydrofuran.

directly influences the {TiaOb} core architecture.

Unfortunately, the weak quality of the formed crystals that caused the structure of isolated solid reaction products were determined based on the analysis of IR, Raman spectra (Figures 1 and 2 and Table 2), and mass spectrometry ones (Table 3). Unfortunately, the weak quality of the formed crystals that caused the structure of isolated solid reaction products were determined based on the analysis of IR, Raman spectra (Figures 1 and 2 and Table 2), and mass spectrometry ones (Table 3).

**Figure 1.** IR and Raman spectra of the (**1**) and (**2**) complexes.

**Figure 1.** IR and Raman spectra of the (**1**) and (**2**) complexes.

The vibrational spectra analysis of the (**1**)–(**4**) compounds (Table 2) confirmed the presence of the coordinated carboxylate ligands 1514–1536 cm−1 νas(COO)) containing 1,4-substituted Ph groups 780–784 cm−1 (ν(CH), IR spectra) functionalized by NH2 or OH groups (3100–3500 cm−1) and, also, alkoxide groups 1014–1036 cm−1 and 948–988 cm−1 (ν(Ti-OR)) in their structures. The identification of normal vibrations bands, which descended from titanium-oxide bridge modes, was the spectral proof of the formed {TiaOb} core type. For this purpose, the results of our previous DFT calculations were used [20–22]. The medium bands at 483 and 483 cm−1 in the IR spectra of (**1**) and (**2**) and weak and very weak bands, which were found at 719–728 cm−1, 539–564 cm−1, and 342–419 cm−1 in the IR

**Figure 2.** IR and Raman spectra of the (**3**) and (**4**) complexes. **Figure 2.** IR and Raman spectra of the (**3**) and (**4**) complexes.

**Table 2.** Results of the vibrational spectra studies of the (**1**)–(**3**) complexes (the band intensity: strong (s), middle (m), weak (w), very weak (vw). **(1) (2) (3) (4) Modes IR R IR R IR R IR R** ν(OH) ν(NH2) 3200–3450 3162 (w) 3300–3500 3200–3450 3175 (w) 3300–3500 ν(CC) (Ph) 1622 (m) 1601 (m) 1603 (s) 1622 (m) 1603 (m) 1595 (s) 1621 (m) 1603 (m) 1602 (s) 1622 (m) 1603 (m) 1594 (s) δ(NH2) 1587 (m) 1589 (m) νas(COO) 1533 (m) 1516 (m) 1524 (m) 1536 (m) 1516 (m) 1519 (w) 1523 (m) 1524 (m) 1536 (m) 1510 (w) 1518 (w) νs(COO) 1462 (w) 1436 (m) 1464 (w) 1450 (w) 1497 (m) 1459 (w) 1498 (m) 1445 The vibrational spectra analysis of the (**1**)–(**4**) compounds (Table 2) confirmed the presence of the coordinated carboxylate ligands 1514–1536 cm−1 νas(COO)) containing 1,4-substituted Ph groups 780–784 cm−1 (ν(CH), IR spectra) functionalized by NH2 or OH groups (3100–3500 cm−1 ) and, also, alkoxide groups 1014–1036 cm−1 and 948–988 cm−1 (ν(Ti-OR)) in their structures. The identification of normal vibrations bands, which descended from titanium-oxide bridge modes, was the spectral proof of the formed {TiaOb} core type. For this purpose, the results of our previous DFT calculations were used [20–22]. The medium bands at 483 and 483 cm−1 in the IR spectra of (**1**) and (**2**) and weak and very weak bands, which were found at 719–728 cm−1 , 539–564 cm−1 , and 342–419 cm−1 in the IR and Raman spectra of these compounds, were attributed to vibrations of {Ti3-(µ3-O)} cores (Figure 1 and Table 2).

δ(NH2) 1362 (m) 1306 (m) 1299 (m) 1300 (m) ν(C-O) + δ(CC) 1014 (s) 1016 (m) 1036 (m) 1016 (m) **Table 2.** Results of the vibrational spectra studies of the (**1**)–(**3**) complexes (the band intensity: strong (s), middle (m), weak (w), very weak (vw).

(w)

Analysis of the electrospray ionization-mass spectrometry (ESI-MS) spectra of (**1**) and (**2**) confirm the presence of peaks, which can be assigned to the fragmentation ions containing {Ti3-(µ3-O)} cores (Table 3). Considering these data, we can state that the trinuclear oxo-Ti(IV) complexes, i.e., [Ti3O(OiPr)8(O2C-4-PhNH2)2] (**1**) and [Ti3O(OiPr)8(O2C-4-PhOH)2] (**2**) were isolated from the 4:1 reaction mixture of Ti(OiPr)4 and HO2CR'; R' = 4-PhNH2 and 4-PhOH using the tetrahydrofuran (THF)/PriOH (1:1 mixture) as a solvent. The bands, which were detected at 702 and 703 cm−1 and 687 and 690 cm−1 in the IR and Raman spectra of (**3**) and (**4**), were attributed to stretching modes of Ti4(µ4-O) bridges, whereas bands at 634–639 cm−1 and 532–556 cm−1 (IR) and 687–690 cm−1 and 536–548 cm−1 (Raman) were assigned to vibrations of Ti4-(µ-O) bridges of {Ti4O2} cores (Figure 2 and Table 2). Additionally, in these cases, the presence of peaks attributed to fragmentation ions containing {Ti4O2} cores in MS spectra of (**3**) and (**4**) confirms the tetranuclear structures of these complexes (Table 2). The results obtained indicate the isolation of the tetranuclear Ti(IV) oxo-complexes [Ti4O2(OiBu)10(O2C-4-PhNH2)2] (**3**) and [Ti4O2(OiBu)10(O2C-4-PhOH)2] (**4**) from the mother liquor composed of a 4:1 mixture of Ti(OiBu)4 and the above-mentioned organic acids and a 1:1 mixture of THF and BuiOH as a solvent.

**Table 3.** The results of electrospray ionization-mass spectrometry (ESI-MS) spectra studies of the (**1**)–(**4**) complexes. TOCs: tetranuclear oxo-titanium(IV) complexes.

#### *2.1. UV–Vis Di*ff*use Reflectance Spectra (UV-Vis-DRS) of the (1)–(4) Oxo-Complexes and HOMO-LUMO Gap Determination*

UV-Vis-DRS spectra of the (**1**)–(**4**) oxo-complexes were registered at room temperature using magnesium oxide as a standard reference (Figure 3). The HOMO-LUMO gap values were determined basing on the Kubelka-Munk (K-M) function versus light energy, i.e., K = f(*h*ν), where K = (1−R)2/2R and R is the reflectance, which was used for the optical band gap determination (Figure 3). According to these data, trinuclear oxo-complexes (**1**) and (**2**) exhibit absorption on the border of the UV and Vis range, λmax = 400 nm for (**1**) and λmax = 360 nm for (**2**), and with a sharp absorption edge at ca. 520 nm and 480 nm for complexes (**1**) and (**2**), respectively (Figure 3a). The wide absorption bands with maxima at 405 nm and 390 nm and with the same sharp absorption edge at 500 nm were found in the spectra of (**3**) and (**4**), respectively (Figure 3b). The determined HOMO-LUMO energy gap values (∆*E*) revealed significant differences between the oxo-complexes with a different type of stabilized carboxylate groups. The lowest ∆*E* values were found for (**1**), ∆*E* = 2.0 eV, and (**3**), ∆*E* = 2.15 eV, i.e., oxo-complexes containing -O2C-4PhNH2 ligands. The HOMO-LUMO energy gap values of complexes that contain the -O2C-4-PhOH groups in their structures were the highest and were equal to ∆*E* = 2.38 eV for (**2**) and ∆*E* = 2.25 eV for (**4**) (Figure 3).

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 6 of 19

**Figure 3.** Solid-state UV-Vis-diffuse reflectance spectra (DRS) of the (**1**)–(**4**) micro-grains (the left side) and Kubelka-Munk function versus light energy plot for the HOMO-LUMO gap determination (the right side): (**a**) [Ti3O(OiPr)8(O2CC6H4NH2)2] (**1**) and [Ti3O(OiPr)8(O2CC6H4OH)2] (**2**) and (**b**) [Ti4O2(OiBu)10(O2CC6H4NH2)2] (**3**) and [Ti4O2(OiBu)10(O2CC6H4OH)2] (**4**). **Figure 3.** Solid-state UV-Vis-diffuse reflectance spectra (DRS) of the (**1**)–(**4**) micro-grains (the left side) and Kubelka-Munk function versus light energy plot for the HOMO-LUMO gap determination (the right side): (**a**) [Ti3O(OiPr)8 (O2CC6H4NH2 )2 ] (**1**) and [Ti3O(OiPr)8 (O2CC6H4OH)2 ] (**2**) and (**b**) [Ti4O2 (OiBu)10(O2CC6H4NH2 )2 ] (**3**) and [Ti4O2 (OiBu)10(O2CC6H4OH)2 ] (**4**). side) and Kubelka-Munk function versus light energy plot for the HOMO-LUMO gap determination (the right side): (**a**) [Ti3O(OiPr)8(O2CC6H4NH2)2] (**1**) and [Ti3O(OiPr)8(O2CC6H4OH)2] (**2**) and (**b**) [Ti4O2(OiBu)10(O2CC6H4NH2)2] (**3**) and [Ti4O2(OiBu)10(O2CC6H4OH)2] (**4**). *2.2. (PMMA + TOC) Composites*

#### *2.2. (PMMA + TOC) Composites 2.2. (PMMA* + *TOC) Composites* The photocatalytic properties of the (**1**)–(**4**) titanium(IV) oxo-complexes (TOCs) and their

dispersed in the PMMA matrix.

The photocatalytic properties of the (**1**)–(**4**) titanium(IV) oxo-complexes (TOCs) and their antimicrobial activity were estimated using (PMMA + TOC) composite foils produced by the dispersion of 20 wt.% TOC micro-grains in the PMMA matrix [21,22]. SEM images, presented in Figure 4, show that composite foils (PMMA + (**1**), (**2**), and (**4**)) contain the dispersed TOC grains of diameters 100–300 μm. In the case of (PMMA + (**3)**), fine grains (of diameters 3–5 μm) of the oxo-complex that may form larger aggregates are dispersed in the polymer matrix. The registration of Raman microscopy maps allowed to investigate the TOC grain presence and distribution in the The photocatalytic properties of the (**1**)–(**4**) titanium(IV) oxo-complexes (TOCs) and their antimicrobial activity were estimated using (PMMA + TOC) composite foils produced by the dispersion of 20 wt.% TOC micro-grains in the PMMA matrix [21,22]. SEM images, presented in Figure 4, show that composite foils (PMMA + (**1**), (**2**), and (**4**)) contain the dispersed TOC grains of diameters 100–300 µm. In the case of (PMMA + (**3)**), fine grains (of diameters 3–5 µm) of the oxo-complex that may form larger aggregates are dispersed in the polymer matrix. The registration of Raman microscopy maps allowed to investigate the TOC grain presence and distribution in the PMMA matrix (Figure 5). antimicrobial activity were estimated using (PMMA + TOC) composite foils produced by the dispersion of 20 wt.% TOC micro-grains in the PMMA matrix [21,22]. SEM images, presented in Figure 4, show that composite foils (PMMA + (**1**), (**2**), and (**4**)) contain the dispersed TOC grains of diameters 100–300 μm. In the case of (PMMA + (**3)**), fine grains (of diameters 3–5 μm) of the oxo-complex that may form larger aggregates are dispersed in the polymer matrix. The registration of Raman microscopy maps allowed to investigate the TOC grain presence and distribution in the PMMA matrix (Figure 5).

500 μm 100 μm **Figure 4.** SEM images of poly(methyl methacrylate) (PMMA) + TOCs and TOCs= (**1**)–(**4**) composites **Figure 4.** SEM images of poly(methyl methacrylate) (PMMA) + TOCs and TOCs= (**1**)–(**4**) composites dispersed in the PMMA matrix. **Figure 4.** SEM images of poly(methyl methacrylate) (PMMA) + TOCs and TOCs= (**1**)–(**4**) composites dispersed in the PMMA matrix.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 7 of 19

**Figure 5.** Raman microscopy maps, registered for PMMA + TOC composites (TOCs (**2**) and (**4**)). The Raman spectra, in selected points (a)–(c) on the surface of PMMA + TOCs, are presented alongside. **Figure 5.** Raman microscopy maps, registered for PMMA + TOC composites (TOCs (**2**) and (**4**)). The Raman spectra, in selected points (a)–(c) on the surface of PMMA + TOCs, are presented alongside.

An analysis of the Raman spectra of composite foils proved the presence of tri- and tetranuclear titanium(IV) oxo-complexes of the earlier determined structure. The use of an ultrasonic bath for the dispersion of TOC grains in the polymer solution allowed the appropriate uniform distribution of the fine grains of TOCs throughout the entire polymer volume (Figure 5). However, probably, the solvent evaporation led to the formation of larger TOC grains and places dominated by the polymer. An analysis of the Raman spectra of composite foils proved the presence of tri- and tetranuclear titanium(IV) oxo-complexes of the earlier determined structure. The use of an ultrasonic bath for the dispersion of TOC grains in the polymer solution allowed the appropriate uniform distribution of the fine grains of TOCs throughout the entire polymer volume (Figure 5). However, probably, the solvent evaporation led to the formation of larger TOC grains and places dominated by the polymer.

#### *2.3. Thermal Analysis of PMMA*/*TOC Composites*

*2.3. Thermal Analysis of PMMA/TOC Composites* The use of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) allowed the thermal characterization of (PMMA + TOCs) composites and the PMMA matrix in the temperature range 30–500 °C at inert atmosphere. The results of these investigations are presented in Table 4 and Figure 6. The analysis of the TGA data of all studied samples revealed a slight weight loss (10–15%), which occurred below 280 °C (I Stage), and the major one (70–85%) between 280 and 450 °C (II Stage). Considering the previous investigations, Stage I can be attributed to the loss of the residual solvent and/or monomer, while Stage II was due the samples' thermal decomposition The use of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) allowed the thermal characterization of (PMMA + TOCs) composites and the PMMA matrix in the temperature range 30–500 ◦C at inert atmosphere. The results of these investigations are presented in Table 4 and Figure 6. The analysis of the TGA data of all studied samples revealed a slight weight loss (10–15%), which occurred below 280 ◦C (I Stage), and the major one (70–85%) between 280 and 450 ◦C (II Stage). Considering the previous investigations, Stage I can be attributed to the loss of the residual solvent and/or monomer, while Stage II was due the samples' thermal decomposition [35,36].

[35,36]. **Table 4.** Thermal parameters received from thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of the poly(methyl methacrylate) (PMMA) and (PMMA + TOCs) **Table 4.** Thermal parameters received from thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of the poly(methyl methacrylate) (PMMA) and (PMMA + TOCs) composites (Tg = glass transition temperature, T = evaporation temperature of an unreacted monomer, Tm = melting temperature. Tmax = temperature in the thermal transition maximum, ∆m = thermal transition weight loss).

(PMMA+(**2**)) 101.2 - 375.7 183.9/11 365.4/74 15 (PMMA+(**3**)) 99.0 - 377.8 159.9/15 372.7/76 9 (PMMA+(**4**)) 115.2 - 364.5 171.0/10 374.0/79 11

An analysis of the DSC thermograms exhibits that the PMMA matrix has a glass transition temperature (Tg) at 99.6 °C, while the exothermic region at 150.8 °C (T), according to previous reports, can be assigned to the evaporation of an unreacted monomer [37] (Figure 6d). The latter effect in the DSC curves of (PMMA + (**1**)–(**4**)) samples was not observed. The endothermic peak at 368.6 °C, which was found on the PMMA DSC thermogram, can be assigned to the PMMA melting temperature (Tm). In the case of composite films enriched with (**1**), (**2**), and (**3**) oxo-complexes, the

**Figure 6.** Thermogravimetric curves (TGA) (**a**,**c**) and the differential scanning calorimetry curves of (DSC) (**b**,**d**) of the produced composite materials (PMMA + (**1**)–(**4**)) and PMMA. **Figure 6.** Thermogravimetric curves (TGA) (**a**,**c**) and the differential scanning calorimetry curves of(DSC) (**b**,**d**) of the produced composite materials (PMMA + (**1**)–(**4**)) and PMMA.

*2.4. Estimation of Photocatalytic Activity of the (1)–(4) Oxo-Complexes* The photocatalytic activity of the (PA + TOCs) systems, with TOCs (**1**)–(**4**), was estimated on the basis of the photodecolorization process of the methylene blue (MB) solution irradiated in the UVA range, according to the ISO 10678:2010 procedure [38,39]. The changes of MB concentrations versus irradiation times for the studied (PMMA + TOCs) composites and the reaction observed rate constants are presented in Figure 7 and Table 5. According to these data, the (PMMA + (**3**)) and (PMMA + (**4**)) composites exhibit the best photocatalytic activity. Simultaneously, in the case of the (PMMA + (**1**)) and (PMMA (**2**)) samples, the very weak photocatalytic activity that was noticed An analysis of the DSC thermograms exhibits that the PMMA matrix has a glass transition temperature (Tg) at 99.6 ◦C, while the exothermic region at 150.8 ◦C (T), according to previous reports, can be assigned to the evaporation of an unreacted monomer [37] (Figure 6d). The latter effect in the DSC curves of (PMMA + (**1**)–(**4**)) samples was not observed. The endothermic peak at 368.6 ◦C, which was found on the PMMA DSC thermogram, can be assigned to the PMMA melting temperature (Tm). In the case of composite films enriched with (**1**), (**2**), and (**3**) oxo-complexes, the strong endothermic peaks appear at 370.4 ◦C, 375.7 ◦C, and 377.8 ◦C; this is above the Tm of the pure PMMA matrix. The exception is (PMMA + (**4**)), for which the Tm was registered at about 364.5 ◦C.

#### ((PMMA + (**1**)) has activity similar to the pure PMMA (difference in absorbance during 180-min *2.4. Estimation of Photocatalytic Activity of the (1)–(4) Oxo-Complexes*

measurements, Δ*A*180 = 0.011). The calculated values of Δ*A*180 are presented in Table 5. Moreover, the photodegradation of Rhodamine B of the above-mentioned composite films was carried out under VIS light, and the obtained results are presented in Figure 7 and in Table 5 [40]. The photocatalytic activity of the (PA + TOCs) systems, with TOCs (**1**)–(**4**), was estimated on the basis of the photodecolorization process of the methylene blue (MB) solution irradiated in the UVA range, according to the ISO 10678:2010 procedure [38,39]. The changes of MB concentrations versus irradiation times for the studied (PMMA + TOCs) composites and the reaction observed rate constants are presented in Figure 7 and Table 5. According to these data, the (PMMA + (**3**)) and (PMMA + (**4**)) composites exhibit the best photocatalytic activity. Simultaneously, in the case of the (PMMA + (**1**)) and (PMMA (**2**)) samples, the very weak photocatalytic activity that was noticed ((PMMA + (**1**)) has activity similar to the pure PMMA (difference in absorbance during 180-min measurements, ∆*A*180 = 0.011). The calculated values of ∆*A*180 are presented in Table 5. Moreover, the photodegradation of Rhodamine B of the above-mentioned composite films was carried out under VIS light, and the obtained results are presented in Figure 7 and in Table 5 [40].

**Figure 7.** Changes in the concentrations of the methylene blue (MB) and Rhodamine B (RhB) solutions as a function of time for the respective composite materials irradiated with UV and Vis **Figure 7.** Changes in the concentrations of the methylene blue (MB) and Rhodamine B (RhB) solutions as a function of time for the respective composite materials irradiated with UV and Vis light.

light. **Table 5.** Organic dye decolorization percentages and ∆*A*180 parameters for the studied reactions in relation to the composites.

PMMA+(**3**) 1.14 0.011 0 PMMA+(**4**) 1.75 0.018 0.007 a Methylene blue (MB) decolorization at the end of the measurements (t = 180 min). b Rhodamine B (RhB) decolorization at the end of the measurements (t = 180 min).

#### PMMA(irradiated) 1.14 0.011 - *2.5. Antimicrobial Activity of (PMMA* + *TOCs) Composites*

{Ti3O} core and stabilized by the -O2C-4-PhNH2 ligand.

aMethylene blue (MB) decolorization at the end of the measurements (t = 180 min). bRhodamine B (RhB) decolorization at the end of the measurements (t = 180 min). *2.5. Antimicrobial Activity of (PMMA + TOCs) Composites* The results of the (PMMA + (**1**)–(**4**)) antimicrobial activity tests against Gram-positive (*S. aureus* ATCC 6538 and *S. aureus* ATCC 25923) and Gram-negative (*E. coli* ATCC 8739 and *E. coli* ATCC 25922) bacteria and yeasts of *Candida albicans* ATCC 10231 of (PMMA + TOCs) composites, where TOCs (**1**)–(**4**) are presented in Table 6. According to these data, all of the (PMMA + TOCs) composites showed high antimicrobial activity against all tested bacteria (*R* between 2.5–6.0). In case The results of the (PMMA + (**1**)–(**4**)) antimicrobial activity tests against Gram-positive (*S. aureus* ATCC 6538 and *S. aureus* ATCC 25923) and Gram-negative (*E. coli* ATCC 8739 and *E. coli* ATCC 25922) bacteria and yeasts of *Candida albicans* ATCC 10231 of (PMMA + TOCs) composites, where TOCs (**1**)–(**4**) are presented in Table 6. According to these data, all of the (PMMA + TOCs) composites showed high antimicrobial activity against all tested bacteria (*R* between 2.5–6.0). In case of tests performed on the yeasts of *C. albicans* biocidal activity*, R* ≥ 2 or 99% was found for PMMA (**2**)–(**4**)—namely, the complex containing the {Ti3O} core and stabilized by the 4-hydroxybenzoic ligand, as well as for complexes with {Ti4O2} cores but not for PMMA (**1**), which is a complex with a {Ti3O} core and stabilized by the -O2C-4-PhNH2 ligand.

of tests performed on the yeasts of *C. albicans* biocidal activity*, R* ≥ 2 or 99% was found for PMMA (**2**)–(**4**)—namely, the complex containing the {Ti3O} core and stabilized by the 4-hydroxybenzoic ligand, as well as for complexes with {Ti4O2} cores but not for PMMA (**1**), which is a complex with a

**Table 6.** Antimicrobial activity of the (PMMA +TOCs) systems (*R* = biocidal rate (%), *R*% = reduction percentage).

*R* ≥ 2 is a biocidal effect when the microbial growth is reduced at least 100 times (99.0%).

#### **3. Discussion**

The multinuclear oxo-titanium(IV) complex applicability as a potentially new group of agents with antimicrobial properties was assessed based on carried out investigations. Considering the results of previous works, the oxo-clusters forming the small {TiaOb} core structures (i.e., {Ti4O2} and {Ti3O}), and stabilized by -O2C-4-PhR' (R' = -NH2, -OH) ligands have been chosen to further microbiological experiments [20–23]. The complexes were isolated as micro-grain powders from the reaction mixtures of appropriate titanium alkoxides and organic acids (4:1 molar ratio) at room temperature and in inert atmosphere. The structure of the isolated compounds such as (Ti3O(OiPr)8(O2C-4-PhR')2) (R' = -NH2 (**1**), -OH (**2**)), (Ti4O2(OiBu)10(O2C-4-PhR')2) (R' = -NH2 (**3**), and -OH (**4**)) were determined as a result of IR, Raman, and ESI-MS spectra analyses. Studies of UV-Vis-DRS spectra revealed that the absorption maximum of the (**1**)–(**4**) compounds is shifted towards the visible range, i.e., 360–405 nm, with a sharp absorption edge between 480–520 nm (Figure 3). The results of the previous investigations proved that the {TiaOb} cores were absorbed in the UV range, which is due to the O2*p*-Ti3*d* charge transfer transition [41]. The shifting of the absorption to the visible range, which was registered in the spectra of the (**1**)–(**4**) compounds, can be explained by the ligand-to-core charge transfer (LCCT) from the -O2CR' (R' = 4-PhNH2 or 4-PhOH) ligands to the tri- or tetranuclear titanium-oxo cores. A similar effect was discussed by Cui et al. for a group of multinuclear Ti(IV)-oxo complexes stabilized by 4-chlorosalicylate ligands [42]. The designated values of the HOMO-LUMO gap energy for the studied oxo-complexes changes in the row (**1**) < (**3**) < (**4**) < (**2**) (Figure 3). The analysis of these data confirms that the type of carboxylate ligand, which stabilizes the {TiaOb} skeleton, is the main factor allowed to effectively control the HOMO-LUMO energy gap of multinuclear oxo-complexes. This effect was highlighted during earlier studies of Ti(IV)-oxo clusters of similar core structures and significant different carboxylate ligands [13,14,21,22,41,42]. The influence of the {TiaOb} cluster core size on the HOMO-LUMO energy gap (*E*g) is definitely more negligible, e.g., the results of Cui et al.'s investigations revealed that, for the {Ti4O}, {Ti11O9}, and {Ti14O12} clusters stabilized by OiPr and 4-chlorosilicylate ligands, the *E*g values were 3.0, 2.8, and 2.9 eV, i.e., they differed from each other by 0.1 and 0.2 eV [42]. In the case of the studied {Ti3O} ((**1**) and (**2**)) and {Ti4O2} ((**3**) and (**4**)) clusters, these differences amounted to 0.13 and 0.15 eV, respectively (Figure 3).

The research carried out led to the isolation of micro-grained powders of TOCs containing {Ti3O} ((**1**) and (**2**)) and {Ti4O2} ((**3**) and (**4**)} cores, in which the absorption maximum was shifted towards the UVA-Vis range in comparison to TiO2 (UV, λmax = 300–350 nm [43]). The type of the carboxylate ligand, which stabilizes the {TiaOb} core, influences the HOMO-LUMO gap values (∆*E*), i.e., ∆*E* (**1**) and (**2**) < ∆*E* (**3**) and (**4**).

Due to the hydrophobic characters of studied powders, their photocatalytic and microbiocidal activity were estimated using (PMMA + TOCs) composite films of 0.25–0.50-µm thickness. The analysis of the SEM images proved that the uniform dispersion of TOC fine grains (3–5 µm) in the PMMA matrix was found only in the (PMMA + (**3**)) composite, while, in the cases of (PMMA + TOCs and TOC = (**1**), (**2**), and (**4**)) composites, the studied materials contained the dispersed, larger (100–300 µm) TOC grains (Figure 4). The nonuniform distribution of TOCs in composite films revealed the analysis of Raman microscopy maps of the (PMMA + (**2**) and (**4**)) films (Figure 5). The large grains of TOCs

are separated from each other by the pure polymer sites and by the areas that are formed by fine grains dispersed in PMMA matrices. The thermal characterization of (PMMA + (**1**)–(**4**)) showed a lack of significant differences between their thermal properties and the PMMA matrix. However, higher thermal decomposition temperatures of composite films versus the pure polymer indicate that the Ti(IV)-oxo complex addition to the PMMA matrix improves its thermal stability. A similar effect was observed for the PMMA matrix containing 20% BaTiO3 [37]. While analyzing the data presented in Table 3, attention should be paid to the differences in the thermal stability of (PMMA + TOCs) composites resulting from the type of added oxo-cluster. The dispersion of trinuclear oxo-complexes in the PMMA matrix slightly improves the thermal stability of the composite in comparison to the pure polymer, i.e., 0.3–2.7 ◦C, while this difference is clearer and was found to be 7.6–8.9 ◦C for the tetranuclear ones (Table 4).

For the reason that studied oxo-complexes can be a potential source of the reactive oxygen species (ROS), before the antimicrobial tests, their photocatalytic activity was determined. Luo et al. [18] and Lv et al. [44] demonstrated the promising photocatalytic activity of the oxo-Ti(IV) clusters dispersed in solutions of MO (methyl orange) and MB. Considering the potential application of TOCs (i.e., (**1**)–(**4**)) clusters, as an antimicrobial agent introduced to the polymer matrix (e.g., PMMA), the photocatalytic activity assessment was made for their (PMMA + (**1**)–(**4**)) composites. Our earlier studies drew our attention to the good photocatalytic activity of (Ti4O2(OiPr)10(O2C-4-PhNH2)2), which the 20 wt.% addition to the polystyrene (PS) matrix or PMMA was one, clearly improved the photodecolorization effect of methylene blue (MB) solution [21,22]. Additionally, trinuclear systems exhibited good photocatalytic activity, which, in the case of (Ti3O(OiBu)8(O2CR')2), (R' = -C13H9, -3-PhNO2) was proven to be better than for the appropriate tetranuclear oxo-Ti(IV) systems [22]. In the case of the (PMMA + (**1**)–(**4**)) films, the best photocatalytic activity revealed the (PMMA + (**4**)) system irradiated with UVA and Visible light for the MB solution and RhB one, respectively. The PMMA film containing uniformly dispersed fine grains of (**3**) showed slightly weaker activity also in the UVA-Vis range. Cui et al. drew attention to the importance of the size of the oxo-cluster and the related specific surface area [42]. In our case, the cluster size was similar, while the sizes of the grains dispersed in the PMMA matrix were different (Figure 4), which can be associated with the influence of the grain surfaces on their photocatalytic activity. It is interesting that a decrease the of Ti3O core size of the oxo-clusters influences the significant decrease of the (PMMA + TOCs) composites (where the TOC (**1**) and (**2**)) have photocatalytic activity, especially in Vis range, and practically no significant changes in UV activity.

The (PMMA + TOCs) systems were sterilized with the use of UVC light, which, according to our UV–Vis-DRS measurements, did not activate the studied samples. In the next stage, the samples were irradiated with natural indoor light directly before inoculation with microorganisms. Considering the results of our earlier works regarding the photocatalytic activity of the (polymer + TOCs) systems [21,22], we assumed that the antimicrobial mechanism of the action of the TOCs could be similar to that of the TiO2-assisted ultraviolet treatment (TUV). TUV is a well-known technique used in water disinfection [45,46] or the food industry [47] for the inactivation of microorganisms. The degree of photoinduced deactivation of the microorganisms depends on various treatment parameters, the nature of the microorganisms, and environmental conditions [45,48]. The mechanisms for the bactericidal action of TiO2 photocatalysis are associated with the generation of reactive oxygen species (ROSs), such as superoxide radical anion (O2 −·), hydrogen peroxide (H2O2), and hydroxyl radical (OH·), which cause oxidative damage to living organisms [47,49]. Many authors suggested that the cell membrane is the primary site of ROS attack [50–52]. This attack of the cell membrane by ROS induce lipid peroxidation. The cell membrane damage directly leads to the leakage of minerals, proteins, and genetic materials, which is the root cause of cell death [51]. Apart from the cytoplasmic membrane, the supercoiled plasmid DNA, genomic DNA, and internal organelles are destructed when the bacteria are exposed to TUV [47]. Similar mechanisms of PMMA coated with TiO2 action against *E. coli* and *S. aureus* were suggested by Su et al. [49]. Moreover, they claimed that a further oxidative attack of internal cellular components accelerates the cell death and, ultimately, results in the decrease of

the survival ratio of both tested bacterial strains, namely *S. aureus* and *E. coli* BL21. Furthermore, Pan et al. [53] evaluated the antibacterial properties of nano-trititanate (H2Ti3O7 nanomaterial) against *E. coli* MC1061 and observed that the bactericidal capabilities of these nano-trititanates were more significant compared to nano-TiO2, both with and without exposure to UV light. Various nano-titanates activated with UV light inhibited the bacterial growth in the range of 11.9–57.1%, while these nonirradiated in the range of 25.7–94.7%. The dependence between the photocatalytic properties and antimicrobial activity was also noticed for studied foils of (PMMA + TOCs) composites, containing the 20 wt.% of (**1**)–(**4**) TOCs. The weakest biocidal activity was observed for the (PMMA + (**1**)), which simultaneously showed the worst photocatalytic properties, even irradiated by the light in the UVA-Vis range. The photocatalytic activity of the (PMMA + (**2**)–(**4**)) films was higher, which can explain the strong inhibition of microorganism growth (>99.99%) on their surfaces. It should be noted that the biocidal activity of the (PMMA + TOC) composite films was assessed for 20 wt.% of TOC micro-grain contents due to our earlier studies of (polymer + (**3**)) composite photocatalytic activity (polymer PS and PMMA) [22]. Although ROS generation is the main proposed mechanism of antimicrobial activity of titanium nanomaterials or titanium nanomaterial-containing complexes, the other mechanism of action should be also considered. It is well-known that some nanomaterials, especially metal-based ones, exhibit strong inhibitory effects towards a broad spectrum of bacterial strains. It is claimed that the biocidal effect of these materials, including titanium oxide nanoparticles, results from the "electromagnetic" attraction between the microbe and metal oxide nanoparticles, as microorganisms carry a negative charge while metal oxides carry a positive charge [54]. Overall, metal-based nanoparticles may interact with the sulfur-containing proteins present in the cell envelopes causing irreversible changes in the cell wall structure, resulting in its disruption and affecting the permeability of the cell membrane by altering transport activity through the plasma [55] or disrupting the ATP production [56]. Nanoparticles can further penetrate inside the microbial cells and interact with ribosomes and biomolecules such as proteins, lipids, and DNA, which may alter translation, replication, and other processes in microbial cells. Moreover, the ion release, as a secondary oxidation process, contributes to the biocidal properties of nanomaterials. Metal ions also affect the losses in the ability to replicate DNA, translation process in ribosomes, and protein activities [57–59]. They can affect membrane transport and the release of potassium (K+) ions from the microbial cells. The increase of membrane permeability caused by both nanomaterials and released ions may lead to the leakage of cellular contents, including ions, proteins, reducing sugars, and, sometimes, cellular energy reservoirs (ATP) [58,60–62]. It should, however, be noted that the above-mentioned mechanism can be the result of the direct contact between the nanoparticles and bacterial cell. In the discussed case, the TOC micro-grains were surrounded by the polymer matrix, thus limiting their mutual contact. Therefore, the received results suggested that a mechanism based on the ROS formation seems to be the most probable.

In further investigations, the lowest but still highly active content of TOCs as an antimicrobial agent in the polymer matrix should be determined. Moreover, the possible cytotoxicity of studied (PMMA + TOCs) composites should be assessed, which is extremely important in the case of the potential biomedical applications of these systems. The antimicrobial activity of nanomaterials prevents bacterial adhesion and biofilm formation. This activity is especially important for medical applications of nanomaterials or nanomaterial-containing composites. Biofilms protect the underlying microbes from antibiotics and host defense mechanisms and, thus, may lead to serious infections. Moreover, the antimicrobial properties of nanomaterials may be used to prevent microbial contaminations that cause food spoilage, the spread of foodborne diseases, and bio-fouling of materials [29,63].

#### **4. Materials and Methods**

#### *4.1. Materials*

Titanium(IV) isopropoxide (Aldrich, St. Louis, MO, USA), titanium(IV) isobutoxide (Aldrich, St. Louis, MO, USA), 4-aminobenzoic acid (Aldrich, St. Louis, MO, USA), and 4-hydroxybenzoic

acid (Aldrich, St. Louis, MO, USA) were purchased commercially and were used without further purification. All solvents used in the synthesis, i.e., tetrahydrofuran (THF), isobutanol (HOiBu), and isopropanol (HOiPr) were distilled before their use and stored in argon atmosphere. The processes of Ti(IV) oxo-complexes synthesis were carried out using the standard Schlenk technique in the inert gas atmosphere (Ar) and at room temperature (RT).

#### *4.2. Synthesis of Ti(IV) Oxo-Complexes (TOCs) and (PMMA* +*TOCs) Composites*

The synthesis of [Ti3O(OiPr)8(O2CC6H4NH2)2] (**1**): 0.12 g of 4-aminobenzoic acid (0.875 mmol) was added to the solution of 1-mL titanium(IV) isopropoxide (3.5 mmol) in 2 mL of THF/PriOH (1:1), leading to a clear yellow solution. The solution was left for crystallization. The yield based on acid: 68% (0.31 g). Anal. Calc. for C38H68O13Ti3N2: C, 50.44; H, 7.52; N, 3.10; Ti, 15.93. Found: C, 50.38; H, 7.48; N, 2.99; Ti, 15.96.

The synthesis of [Ti3O(OiPr)8(O2CC6H4OH)2] (**2**): 0.12 g of 4-hydroxybenzoic acid (0.875 mmol) was added to the solution of 1-mL titanium(IV) isopropoxide (3.5 mmol) in 2 mL of THF/PriOH (1:1), leading to a clear yellow solution. The solution was left for crystallization. The yield based on acid: 74% (0.33 g). Anal. Calc. for C38H66O15Ti3: C, 50.33; H, 7.28; Ti, 15.89. Found: C, 50.81; H, 7.22; Ti, 15.80.

The synthesis of [Ti4O2(OiBu)10(O2CC6H4NH2)2] (**3**): complex was synthesized, as reported [21]. 0.12 g of 4-aminobenzoic acid (0.875 mmol) was added to the solution of 1.19-mL titanium(IV) isobutoxide (3.5 mmol) in 2 mL of toluene, leading to a clear yellow solution. The solution was left for crystallization. The yield based on acid: 41% (0.22 g). Anal. Calc. for C54H102O14Ti4N2: C, 52.86; H, 8.38; N, 2.28; Ti, 15.61. Found: C, 53.14; H, 7.83; N, 2.05; Ti, 15.56.

The synthesis of [Ti4O2(OiBu)10(O2CC6H4OH)2] (**4**): 0.12 g of 4-hydroxybenzoic acid (0.875 mmol) was added to the solution of 1.19-mL titanium(IV) isobutoxide (3.5 mmol) in 2 mL of THF/BuiOH (1:1), leading to a clear yellow solution. The solution was left for crystallization. The yield based on acid: 63% (0.30 g). Anal. Calc. for C54H100O18Ti4: C, 52.77; H, 8.14; Ti, 15.64. Found: C, 51.36; H, 7.92; Ti, 16.01.

The polymer foils containing 20 wt.% of synthesized Ti(IV)oxo-clusters (TOCs) were prepared by an addition of the solution of the TOC solution (ca. 0.25 g of (**1**)–(**4**) TOCs were dispersed in 1 cm3 of THF) to the poly(methyl methacrylate) (PMMA) solution (1.0 g of PMMA dissolved in 5 cm3 of THF). The resulting mixtures were stirred in an ultrasonic bath for 120 min; in the next step, they were poured into a glass Petri dish and left for the evaporation of the solvent at RT. The composite foil thickness ca. 50 µm were characterized by Raman and IR spectroscopy and scanning electron microscopy.

#### *4.3. Analytical Procedures*

The structures of the isolated solid reaction products (crystals and powders) were confirmed using vibrational spectroscopy methods, i.e., IR spectrophotometry (Perkin Elmer Spectrum 2000 FTIR spectrophotometer (400–4000 cm−1 range, KBr pellets)) and Raman spectroscopy (RamanMicro 200 spectrometer (PerkinElmer, Waltham, MA, USA)). Raman spectra were registered using a laser with the wavelength 785 nm, with a maximum power of 350 mW, in the range 200–3200 cm−1 , using a 20 × 0.40/FN22 objective lens and an exposure time of 15 s each time. Elemental analyses were performed on an Elemental Analyser vario Macro CHN Elemental Analyser vario Macro CHN Elementar Analysen Systeme GmbH (Elementar, Hanau, Germany). The mass spectra were recorded using the ESI-MS method using a QToFSynapt G2 Si (Waters Corporation, Mundelein, IL, USA) spectrometer. The main measurement parameters: capillary voltage: 2.5–3 kV, source temperature: 110 ◦C, sampling cone voltage 20–50 V, source offset 40–60 V, desolvation temperature 250–350 ◦C, cone gas flow: 50.0 dm3 /h, and desolation gas flow: 798.0–900 dm3 /h. The produced PMMA + TOCs foil surfaces were studied using a scanning electron microscope with field emission (SEM, Quanta 3D FEG, Houston, TX, USA). Composite materials underwent thermal treatment (Bruker Optik, Ettingen, Germany) in the range 20–500 ◦C, with the heating speed of 5 ◦C/min in the atmosphere of nitrogen.

#### *4.4. HOMO-LUMO Gap Determination*

The HOMO-LUMO gap energy values of isolated (**1**)–(**4**) complexes were determined by using diffuse reflectance UV-Vis spectra (UV-VIS-DRS), which were registered between 200 and 800 nm. Jasco V-750 spectrophotometer was used (JASCO Deutschland GmbH, Pfungstadt, Germany). The recorded spectra were evaluated in terms of energy band gap values via Spectra Manager TM CFR software.

#### *4.5. The Photocatalytic Activity Evaluation of (PMMA* + *TOCs) Composites*

The photocatalytic activity of PMMA + TOCs foils (TOCs = (**1**)–(**4**)) was studied by monitoring the degradation processes of MB aqueous solution according to ISO 10678:2010 procedure and, also, Rhodamine B (RhB) solution [38–40]. Foil samples of sizes 10 × 10 mm were preconditioned by exposure to UVA or Vis light for 30 h. In the next step, foils were placed in quartz cuvettes with both dye solutions (V = 3.5 cm3 and C = 2.0 × 10−5 M). After 12 h in the dark, the solutions were replaced by the appropriate test of MB and RhB solutions (for both: V = 3.5 cm3 and C = 1.0 × 10−5 M). The prepared samples were exposed to UVA irradiation (18-W lamp, 340–410-nm range, with a maximum at 365 nm) and Vis light (77-W tungsten halogen lamp, range of 350–2200 nm). All cuvettes were covered with quartz glass panes during irradiation. MB absorbance at 660 nm and RhB absorbance at 554 nm were registered (Metertech SP-830 PLUS, Metertech, Inc., Taipei, Taiwan) after 0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 min of irradiation. Percentage of MB decolorization was calculated using the equation

$$\% \text{ dye decoderization} = ((\text{C}\_0 - \text{C}\_l)(\text{C}\_0) \times 100 = ((A\_0 - A\_l)/A\_0) \times 100 \tag{1}$$

where *C*0 is an initial concentration of dye, *Ct* is a dye concentration at a given time *t*, and *A*0 and *At* are absorbances at 0 and *t* times [38,64].

The rate of both photodegradation processes was estimated by a simple calculation of a difference in absorbance during 180-min measurements (∆*A*180). Such kind of calculation is a result of a low degradation degree of dyes.

#### *4.6. The Evaluation of Antimicrobial Properties of (PMMA* + *TOCs) Systems*

Antimicrobial activity of PMMA + TOCs (TOCs = (**1**)–(**4**)) composite foils (30 × 30 × 0.05 mm) was studied against Gram-positive (*S. aureus* ATCC 6538 and *S. aureus* ATCC 25923) and Gram-negative (*E*. *coli* ATCC 8739 and *E. coli* ATCC 25922) bacteria and yeasts of *Candida albicans* ATCC 10231 using method according to the ISO 22196:2011 standard [65]. All strains were purchased from the American Type Culture Collection (Manassas, VA, USA). PMMA +TOC specimens were sterilized using a UVC lamp for 30 min in the laminar hood (Bioquell, Hampshire, UK) and placed in sterile Petri plates. The earlier investigations revealed that the absorption maximum of the (PMMA + TOCs) samples was found at the UVA-Vis border, which enabled the use of UVC radiation in order for their sterilization. Microbial suspension (1.0–1.8 × 106 colony-forming units (cfu) cm3 / −1 ) prepared in sterile deionized water was placed on the surfaces of the appropriate PMMA + TOCs samples and covered with sterile foil films (polypropylene; PP) and incubated for 24 h at 37 ◦C in a humid atmosphere. The PP film was then removed and microbial suspension collected into a 2-cm3 centrifuge tube. Subsequently, serial ten-fold dilutions of each sample were prepared. Aliquots (100 µL) of each dilution were aseptically spread over the surface of Tryptic Soy Agar (TSA, Becton Dickinson, Sparks Glencoe, MD, USA) or Sabouraud Dextrose Agar (SDA, Becton Dickinson) in Petri plates for the bacteria and fungi, respectively. Inoculated samples were incubated at 37 ◦C for 24 h. Assays were performed in triplicate. After incubation, colony-forming units (cfu) were counted on the agar plates. Control was PMMA inoculated with the test microorganism.

The antimicrobial activity was calculated using Formula (2), according to the ISO 22196:2011 standard [65]:

$$R = \mathcal{U}I\_t - A\_t \tag{2}$$

where *Ut* is the average of the common logarithm of the number of viable bacteria in cells/cm2 recovered from the untreated test specimens (PMMA) after 24 h, and *At* is the average of the common logarithm of the number of viable bacteria in cells/cm2 recovered from the treated test specimens (PMMA + TOCs) after 24 h. *R* ≥ 2 determines the biocidal activity

The percentage reduction (*R*%) of the bacterial or fungal growth was calculated using Formula (3):

$$R\% = ((B - A)\% \text{B}) \times 100\tag{3}$$

where *R* is the biocidal rate (%), *B*—the average number of microorganisms on unmodified PMMA in T0, and *A*—the average number of microorganisms on the surfaces of the studied (PMMA + TOCs) composites after 24 h. The 100-times reduction of the number of colonies determines at least 99% of the reduction of microbial growth (biocidal activity).

#### **5. Conclusions**

The work carried out led to the production of oxo-titanium(IV) complexes (TOCs), i.e., (Ti3O(OiPr)8(O2C-4-PhR')2) (**1**) and (**2**) and (Ti4O2(OiBu)10(O2C-4-PhR') (**3**) and (**4**) (R' = -NH2 and -OH), which structures were confirmed by IR, Raman spectroscopy, and ESI-MS spectrometry. The dispersion of 20 wt.% of TOCs (**1**)–(**4**) in the PMMA matrix allowed the formation of (PMMA + TOCs) composite films for which the thermal and photocatalytic properties and biocidal activity were estimated. The obtained results suggest that (Ti4O2(OiBu)10(O2C-4-PhOH)2) (**4**) can be used for the production of composites (polymer + TOC), which, in the form of films, coatings, or resin additives, can be used as a bactericidal agent in various areas of our lives. It should be noted that the potential use of materials based on PMMA with the addition of TOCs in dentistry would require medical experiments carried out in accordance with the procedures specified by Isola et al. [66,67].

**Author Contributions:** Conceptualization, P.P.; methodology, P.P., B.K., P.G., and A.R.; formal analysis, P.P., B.K., P.G., and A.R.; investigation, B.K. and P.G.; data curation, P.P. and B.K.; writing—original draft preparation, P.P.; writing—review and editing, P.P. and A.R.; and supervision, P.P. and A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Valorization of** *Gleditsia triacanthos* **Invasive Plant Cellulose Microfibers and Phenolic Compounds for Obtaining Multi-Functional Wound Dressings with Antimicrobial and Antioxidant Properties**

**Ioana Cristina Marinas 1,2 , Eliza Oprea 3,\* , Elisabeta-Irina Geana 4 , Oana Tutunaru 5 , Gratiela Gradisteanu Pircalabioru 1 , Irina Zgura 6 and Mariana Carmen Chifiriuc 1**

**Abstract:** *Gleditsia triacanthos* is an aggressive invasive species in Eastern Europe, producing a significant number of pods that could represent an inexhaustible resource of raw material for various applications. The aim of this study was to extract cellulose from the *Gleditsia triacanthos* pods, characterize it by spectrophotometric and UHPLC–DAD-ESI/MS analysis, and use it to fabricate a wound dressing that is multi-functionalized with phenolic compounds extracted from the leaves of the same species. The obtained cellulose microfibers (CM) were functionalized, lyophilized, and characterized by ATR-FTIR and SEM. The water absorption and retention capacity as well as the controlled release of phenolic compounds with antioxidant properties evaluated in temporal dynamics were also determined. The antimicrobial activity against reference and clinical multidrug-resistant *Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Candida albicans,* and *Candida parapsilosis* strains occurred immediately after the contact with the tested materials and was maintained for 24 h for all tested microbial strains. In conclusion, the multi-functionalized cellulose microfibers (MFCM) obtained from the reproductive organs of an invasive species can represent a promising alternative for the development of functional wound dressings with antioxidant and antimicrobial activity, as well as being a scalable example for designing cost-effective, circular bio-economy approaches to combat the accelerated spread of invasive species.

**Keywords:** multi-functionalized cellulose microfibers; controlled release; wound dressing; antimicrobial activity; antioxidant activity

#### **1. Introduction**

Micro- and nanomaterials derived from biological macromolecules are used in a wide range of industrial, technological, and biomedical applications, e.g., for adsorption, ultrafiltration, packaging, preservation of historical artefacts, thermal insulators, and fire

**Citation:** Marinas, I.C.; Oprea, E.; Geana, E.; Tutunaru, O.; Pircalabioru, G.G.; Zgura, I.; Chifiriuc, M.C. Valorization of *Gleditsia triacanthos* Invasive Plant Cellulose Microfibers and Phenolic Compounds for Obtaining Multi-Functional Wound Dressings with Antimicrobial and Antioxidant Properties. *Int. J. Mol. Sci.* **2021**, *22*, 33. https://dx.doi.org/10.3390/ ijms22010033

Received: 16 November 2020 Accepted: 17 December 2020 Published: 22 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

retardants, energy extraction and storage, acoustics, sensory, controlled delivery of drugs, and especially tissue engineering. Due to their mechanical robustness, hydrophilicity, biocompatibility, and good biodegradability, micro- and nanocellulose have specific functions, such as tissue repair, regeneration, and healing, and they are also antimicrobial nanomaterials with shape memory and intelligent membranes [1,2].

Different types of cellulose with a well-defined and functionalized morphology with different active principles can be used to obtain advanced wound dressings with high performance for preventing wound infections and accelerated wound healing [1]. Previous studies of wood-based nanofibrillated cellulose (NFC) have shown a high capacity to absorb liquids and form translucent films, suggesting the benefits of using cellulose fibers as a dressing. The property of maintaining a moist environment is necessary for the proper healing of chronic wounds, while translucency makes it possible to evaluate the wound progress without requiring the dressing removal [3]. In addition, these nanomaterials can provide various drug delivery properties and other effects, depending on the raw materials they come from [4] and can be used for the design of functional wound dressing or for topical drug delivery, offering several advantages over other administration pathways, including eliminating internal metabolism, minimizing pain, prolonging the drug release, and controlling drug absorption by removing skin bandage [5]. On the other side, Nie (2019) showed that the nanocellulose fibers (NF) have lower thermal stability compared with raw microcellulose fibers (MF) due to the reduced particle size, increased specific surface area, and increased exposed carbon and reactive group activity. The thermal degradation of NF generates low molecular weight segments and breakpoint defects. The same effect was produced after introducing the carboxylate group on the cellulose surface of the nano-sized fiber (prepared by oxidation of TEMPO ((2,2,6,6-tetramethyl-1-piperidinyl)oxidanyl) [2]. Microcellulose fibers can be obtained from different plant species (including invasive ones), such as bamboo [6], *Artemisia vulgaris* [2], and banana plant wastes [7].

*Gleditsia triacanthos* L. (*Fabaceae*) is a native tree to North America, with an extraordinary tolerance to high temperatures and drought, conditions that paradoxically favor its spread. This species is also very tolerant of industrial and transport emissions with relatively high resistance to soils with extreme pH. The combined clonal and sexual reproduction, the short juvenile period, the high seed production and the high germination rate of the seeds are considered defining traits for the degree of invasiveness of these species [8]. Initially introduced as an ornamental and aromatic species, it spread rapidly, invading different ecoregions, being presently considered one of the most aggressive legume invaders in the world [9]. This species invasiveness poses serious risks for ecosystems biodiversity, structure, and functions, as well as conservation of the protected areas, and requires high costs for its eradication [10]. The selective harvesting of reproductive organs of such invasive species for practical use could stop their uncontrolled spread. Taking into account that plant extracts offer unlimited therapeutic options, one of the sectors that could benefit from the potential use of invasive plants is the biomedical field.

Thus, the therapeutic valorization of these species could be both an ecological solution to stop their uncontrolled spreading and an economic solution, providing low costs raw materials. The purpose of the study was to obtain cellulose microfibers (CM) from the reproductive organs of *Gleditsia triachantos* and functionalize them with active principles (phenolic compounds from *G. triachantos* leaves) with antioxidant and antimicrobial activity in order to obtain functional wound dressings with improved performance in preventing wound infections, therefore, accelerating the wound healing process.

#### **2. Results**

#### *2.1. Chemical Characterization of Phenolic Compounds of G. triachantos Leaves*

2.1.1. Total Phenolic Compounds (TPC)

The quantification of total phenolic compounds (TPC) was performed using the calibration curve, resulting in a content of 113.62 ± 1.09 mg TPC (GAE, gallic acid equivalents)/g

dry plant, and the flavonoid content was 35.24 ± 0.55 mg (QE, quercetin equivalents)/g dry plant for the *G. triachantos* leaves extract.

#### 2.1.2. Ultra-High-Performance Liquid Chromatography Diode Array Detector Electrospray Ionization Tandem Mass Spectrometry (UHPLC–DAD-ESI/MS)

Phenolic compound profile by UHPLC–DAD-ESI/MS (ultra-high-performance liquid chromatography diode array detector electrospray ionization tandem mass spectrometry). The concentrations of phenolic acids (PAs), flavonoids, and flavonoid heterosides are shown in Table S1 of Supplementary Materials.

The main phenolic compounds identified in the alcoholic extract of G. triachantos were vanillic acid (773.88 µg/L), 4-hydroxy benzoic acid (570.37 µg/L), ferulic acid (319.81 µg/L), catechin (34,271.15 µg/L), quercetin (3,007.89 µg/L), and epicatechin (1,431.47 µg/L). The extract was rich in chlorogenic acid (11,984.43 µg/L), cinnamic acid (2,736.42 µg/L, an acid that lacks phenolic groups but is still usually included in the phenolic compounds class), and rutin (1,302.31 µg/L). The following compounds were also evidenced by UHPLC– DAD-ESI/MS: abscisic acid (16.10 µg/L), a sesquiterpenoid plant hormone, and ellagic acid (165.13 µg/L), a dimer of gallic acid.

#### *2.2. Multi-Functionalized Cellulose Microfiber Fabrication*

*G. triachantos* pods were ground into powder (to pass through a 100 mesh sieve) with a structure characterized by ATR-FTIR (Attenuated Total Reflectance—Fourier Transform Infrared) to highlight the specific bands of the three major components (see Section 2.3.1), which are hemi-cellulose (39.07% ± 1.49%), cellulose (29.78% ± 1.28%), and lignin (27.71% ± 0.75%) [11]. Then, the wood powder was subjected to purification by successive extractions to remove the extractable substances, hemicellulose (toluene and ethanol), and lignin. The lignocellulosic material required six cycles of delignification. The sample was taken from each cycle to quantify the total lignin content by a similar protocol to that used to determine the hydroxycinnamic acid content. The delignification process was continued for two more cycles after the lignin content was below the detection limit. After the last delignification cycle, the aqueous suspension of the obtained cellulose fibers was washed until no chloride ions were found, quantified by the Mohr titrimetric method. After extraction, the freeze-dried cellulosic material was characterized to highlight the lack of lignin content and compared to CM functionalized with phenolic compounds extracted from the leaves of the same species.

#### *2.3. Characterization of Cellulose Microfibers (CM) and Multi-Functionalized Cellulose Microfibers (MFCM)*

#### 2.3.1. ATR-FTIR Analysis

The ATR-FTIR spectra of the studied materials, CM and multi-functionalized cellulose microfibers (MFCM), are presented in Figure 1. According to Danial (2015) [12], the lack of the C=O band in both spectra that should have been observed at the wave number ≈1733 cm−1 highlights the successful removal of hemicelluloses. The complete removal of lignin was given by the lack of bands at ≈1509 cm−1 and ≈1592 cm−1 , bands attributed to the aromatic C=C vibrations in the plane as well as the lack of the band located at 1264 cm−1 , which would have been assigned C–O–C specific to ether bonds from lignin. The C–C band breathing rings (≈1158 cm−1 ) and the C–O–C glycosidic ether band (≈1100 cm−1 ) were attributed to the presence of cellulose. The band at ≈2900 cm−1 , observed in all samples, corresponded to the C–H stretching vibration that reflects the organic content in general [13].

eral [13].

were attributed to the presence of cellulose. The band at ≈2900 cm−1, observed in all samples, corresponded to the C–H stretching vibration that reflects the organic content in gen-

have come from phenolic compounds specific to the extract [14,15].

and 1558 cm−1 (C=C–C aromatic ring stretch) [19].

From the literature data, it is known that the bands in the region 1680–900 cm−1 may

Among others, several specific aromatic C–H vibrations were present between the wavenumbers 670–900 cm−1 and 950–1225 cm−1 [16]. The band at the level of 1247 cm−1 corresponded to the extension of pyran rings, typical for flavonoid compounds [17].

Theoretically, the cellulose–PAs and cellulose–flavonoids bonds are the ester and ether ones, respectively, which were indeed highlighted at the wavenumbers 1338 cm−1 and 1260–1230 cm−1, respectively. The bands specific to the ester groups were similar to those in the dry extract because PAs are found in the form of glycosidic esters according to Qian et al. [18]. By comparing the FTIR spectra of CM, MFCM, and the dry extract, we identified four specific bands for MFCM that were not found in the other spectra, at the

**Figure 1.** Attenuated Total Reflectance—Fourier Transform Infrared (ATR-FTIR) spectra of multifunctionalized cellulose microfibers (MFCM) (black line), CM (red line), lignin (blue line), initial **Figure 1.** Attenuated Total Reflectance—Fourier Transform Infrared (ATR-FTIR) spectra of multi-functionalized cellulose microfibers (MFCM) (black line), CM (red line), lignin (blue line), initial dry pods (pink line), and dry extract (green line).

2.3.2. Morphology of CM and MFCM From the literature data, it is known that the bands in the region 1680–900 cm−1 may have come from phenolic compounds specific to the extract [14,15].

SEM (scanning electron microscopy) analysis revealed that the control sample (CM) used for multi-functionalization with phenolic compounds showed the presence of microfibers (Figure 2) with diameters between 5 and 10 μm. The surface morphology of the Among others, several specific aromatic C–H vibrations were present between the wavenumbers 670–900 cm−1 and 950–1225 cm−1 [16]. The band at the level of 1247 cm−1 corresponded to the extension of pyran rings, typical for flavonoid compounds [17].

CM sample indicated a relatively smooth surface, while that of the MFCM highlighted a rougher surface due to the adherence of the phenolic compounds. In the case of MFCM (impregnated with the extract from *G. triachantos* leaves), it can be observed from Figure 3 that the chemical components specific to the extract adhered to the cellulose microfiber Theoretically, the cellulose–PAs and cellulose–flavonoids bonds are the ester and ether ones, respectively, which were indeed highlighted at the wavenumbers 1338 cm−1 and 1260–1230 cm−1 , respectively. The bands specific to the ester groups were similar to those in the dry extract because PAs are found in the form of glycosidic esters according to Qian et al. [18]. By comparing the FTIR spectra of CM, MFCM, and the dry extract, we identified four specific bands for MFCM that were not found in the other spectra, at the wavenumbers 779 cm−1 (aromatic C–H out-of-plane bending), 1338 cm−1 (C–O stretching (phenyl), C–H (bending and CH2), 1442 cm−1 (C–C stretching vibration in aromatic ring), and 1558 cm−1 (C=C–C aromatic ring stretch) [19].

#### 2.3.2. Morphology of CM and MFCM

dry pods (pink line), and dry extract (green line).

SEM (scanning electron microscopy) analysis revealed that the control sample (CM) used for multi-functionalization with phenolic compounds showed the presence of microfibers (Figure 2) with diameters between 5 and 10 µm. The surface morphology of the CM sample indicated a relatively smooth surface, while that of the MFCM highlighted a rougher surface due to the adherence of the phenolic compounds. In the case of MFCM (impregnated with the extract from *G. triachantos* leaves), it can be observed from Figure 3 that the chemical components specific to the extract adhered to the cellulose microfiber surface without requiring the presence of a fixing or crosslinking agent. These components (of MFCM) had a thickness between 40 and 120 nm and a length 0.2–1 µm.

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 5 of 17

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 5 of 17

**Figure 2.** SEM morphology of the reference CM surface obtained from *Gleditsia triacanthos* pods, used for functionalization with active principles from *G. triacanthos* leaves: (**A**) 1000×, (**B**) 5000×, (**C**) 10,000×, and (**D**) 20,000×. **Figure 2.** SEM morphology of the reference CM surface obtained from *Gleditsia triacanthos* pods, used for functionalization with active principles from *G. triacanthos* leaves: (**A**) 1000×, (**B**) 5000×, (**C**) 10,000×, and (**D**) 20,000×. (**C**) (**D**) **Figure 2.** SEM morphology of the reference CM surface obtained from *Gleditsia triacanthos* pods, used for functionalization with active principles from *G. triacanthos* leaves: (**A**) 1000×, (**B**) 5000×, (**C**) 10,000×, and (**D**) 20,000×.

**Figure 3.** SEM morphology of the MFCM (multi-functionalized cellulose microfiber): (**A**) 1000×, (**B**) 5000×, (**C**) 10,000×, and (**D**) 20,000×.

#### 2.3.3. Surface Wettability (**B**) 5000×, (**C**) 10,000×, and (**D**) 20,000×.

The CA (contact angle) measurements revealed that CM and MFCM samples showed a superhydrophilic behavior (CA ≈ 0 ◦ ), with the water drops being absorbed in the sample volume (Video Supplementary Materials). 2.3.3. Surface Wettability The CA (contact angle) measurements revealed that CM and MFCM samples showed a superhydrophilic behavior (CA ≈ 0°), with the water drops being absorbed in the sample

#### 2.3.4. Water Absorptivity and Retention Properties volume (Video Supplementary Materials).

**Figure 3.** SEM morphology of the MFCM (multi-functionalized cellulose microfiber): (**A**) 1000×,

The percentage of PBS (phosphate-buffered saline) uptake was slightly improved from 674.4% for the CM (control sample) to 721.51% for the MFCM. The freeze-dried phenolic compounds adhered to microfibers could be responsible for the increased absorption capacity, most probably due to the presence of the hydroxyl groups. 2.3.4. Water Absorptivity and Retention Properties The percentage of PBS (phosphate-buffered saline) uptake was slightly improved from 674.4% for the CM (control sample) to 721.51% for the MFCM. The freeze-dried phenolic compounds adhered to microfibers could be responsible for the increased absorption

The difference in terms of water retention capacity (PBS) between the CM (control) and MFCM remained constant after 4 h, however, MFCM had better water retention properties (Figure 4). capacity, most probably due to the presence of the hydroxyl groups. The difference in terms of water retention capacity (PBS) between the CM (control) and MFCM remained constant after 4 h, however, MFCM had better water retention properties (Figure 4).

**Figure 4.** The water retention capacity of CM and MFCM matrices over time (*p* < 0.05). **Figure 4.** The water retention capacity of CM and MFCM matrices over time (*p* < 0.05).

2.3.5. In Vitro Phenolic Compound Release Studies and Antioxidant Activity

#### The release kinetics of the active principles were evaluated by determining the TPC 2.3.5. In Vitro Phenolic Compound Release Studies and Antioxidant Activity

content by the Folin–Ciocalteu method and through monitoring the antioxidant activity over time. The process of releasing the phenolic compounds from the MFCM varied considerably in time (Figure 5A)—in the first 6 h, the release was linear (*R*2 = 0.9935), with a velocity of 3.1083 μg GAE/mL \* h. The release of the phenolic compounds from the cellulose-based dressings was relatively short, reaching an almost complete release of the extract from MFCM within 120 h. A sudden release of a high concentration of TPC in PBS was observed after 2 h, with the rest being released in inverse proportion to the contact time. All experiments were performed in triplicate and the results have statistical significance (*p* < 0.05). The TEAC (Trolox equivalent antioxidant capacity) results have shown that the highest concentration of compounds with antioxidant potential was released in the first 6 h The release kinetics of the active principles were evaluated by determining the TPC content by the Folin–Ciocalteu method and through monitoring the antioxidant activity over time. The process of releasing the phenolic compounds from the MFCM varied considerably in time (Figure 5A)—in the first 6 h, the release was linear (*R* 2 = 0.9935), with a velocity of 3.1083 µg GAE/mL \* h. The release of the phenolic compounds from the cellulose-based dressings was relatively short, reaching an almost complete release of the extract from MFCM within 120 h. A sudden release of a high concentration of TPC in PBS was observed after 2 h, with the rest being released in inverse proportion to the contact time. All experiments were performed in triplicate and the results have statistical significance (*p* < 0.05).

[20], while in the DPPH (1,1-diphenyl-2-picrylhydrazyl radical) assay, the antioxidant activity was inversely proportional to the contact time, observing a constant release until the complete reduction of the phenolic compound content in the material (Figure 5B). The TEAC (Trolox equivalent antioxidant capacity) results have shown that the highest concentration of compounds with antioxidant potential was released in the first 6 h [20], while in the DPPH (1,1-diphenyl-2-picrylhydrazyl radical) assay, the antioxidant activity was inversely proportional to the contact time, observing a constant release until the complete reduction of the phenolic compound content in the material (Figure 5B).

**Figure 5.** Kinetics of controlled release of phenolic compounds (**A**) and antioxidant effects (**B**) in temporal dynamics for multi-functionalized cellulose microfibers (MFCM), *p* < 0.05. **Figure 5.** Kinetics of controlled release of phenolic compounds (**A**) and antioxidant effects (**B**) in temporal dynamics for multi-functionalized cellulose microfibers (MFCM), *p* < 0.05.

#### *2.4. Antimicrobial Activity 2.4. Antimicrobial Activity*

The antimicrobial activity was performed by evaluating the viable cell counts (colony forming unit (CFU)/mL) and calculating the recovery factor initially and after 24 h. The data from Table 1 show that the active principles released from the cellulose matrix positively influenced the antimicrobial effect both compared to the strain and CM control. The MFCM showed a better antimicrobial activity than CM for all strains except *Enterococcus faecium* ATCC DMS 13590 after 24 h of incubation at 37 °C. The antimicrobial activity was performed by evaluating the viable cell counts (colony forming unit (CFU)/mL) and calculating the recovery factor initially and after 24 h. The data from Table 1 show that the active principles released from the cellulose matrix positively influenced the antimicrobial effect both compared to the strain and CM control. The MFCM showed a better antimicrobial activity than CM for all strains except *Enterococcus faecium* ATCC DMS 13590 after 24 h of incubation at 37 ◦C.

**Table 1.** Recovery rate of logarithmical colony forming unit/mL (lg CFU/mL) compared to micro-

The TEAC (Trolox equivalent antioxidant capacity) results have shown that the highest concentration of compounds with antioxidant potential was released in the first 6 h [20], while in the DPPH (1,1-diphenyl-2-picrylhydrazyl radical) assay, the antioxidant activity was inversely proportional to the contact time, observing a constant release until the complete reduction of the phenolic compound content in the material (Figure 5B).

bial strain control (the closer it is to 1, the lower the antimicrobial effect). **Strains Time CM MFCM Table 1.** Recovery rate of logarithmical colony forming unit/mL (lg CFU/mL) compared to microbial strain control (the closer it is to 1, the lower the antimicrobial effect).

MRSA: Methicillin-Resistant *Staphylococcus aureus*, ATCC: American Type Culture Collection.

and 7).

and 7).

terials.

terials.

200, and 500 µg/µL.

#### *2.5. Assessment of Cells Viability and Cytotoxicity* Among the tested compounds, CM (100 µg/uL) harbored the highest level of biocompatibility with high cell proliferation rates and low LDH. patibility with high cell proliferation rates and low LDH.

Cell viability and cell cytotoxicity were investigated by MTT ([3-(4,5-dimethylthia-

Cell viability and cell cytotoxicity were investigated by MTT ([3-(4,5-dimethylthia-

Among the tested compounds, CM (100 µg/uL) harbored the highest level of biocom-

drogenase) assay—L929 cells, respectively, co-cultured with CM and MFCM (Figures 6

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*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 8 of 16

*2.5. Assessment of Cells Viability and Cytotoxicity*

*2.5. Assessment of Cells Viability and Cytotoxicity*

Cell viability and cell cytotoxicity were investigated by MTT ([3-(4,5-dimethylthiazol-2yl)]- 2,5-diphenyltetrazolium bromide) assay—L929 cells, and by LDH (lactate dehydrogenase) assay—L929 cells, respectively, co-cultured with CM and MFCM (Figures 6 and 7). The high rates of cell viability in contact with the CM and MFCM demonstrated that these new cellulose microfibers obtained from *Gleditsia triachantos* are biocompatible ma-The high rates of cell viability in contact with the CM and MFCM demonstrated that these new cellulose microfibers obtained from *Gleditsia triachantos* are biocompatible ma-

**Figure 6.** Cell viability (%) investigated by MTT ([3-(4,5-dimethylthiazol-2yl)]-2,5-diphenyltetrazolium bromide) assay—L929 cells co-cultured with CM and MFCM at various concentrations: 100, **Figure 6.** Cell viability (%) investigated by MTT ([3-(4,5-dimethylthiazol-2yl)]-2,5-diphenyltetrazolium bromide) assay— L929 cells co-cultured with CM and MFCM at various concentrations: 100, 200, and 500 µg/µL. **Figure 6.** Cell viability (%) investigated by MTT ([3-(4,5-dimethylthiazol-2yl)]-2,5-diphenyltetrazolium bromide) assay—L929 cells co-cultured with CM and MFCM at various concentrations: 100, 200, and 500 µg/µL.

**3. Discussion 3. Discussion** Phenolic compounds are plant secondary metabolites with a strong antioxidant ef-Among the tested compounds, CM (100 µg/uL) harbored the highest level of biocompatibility with high cell proliferation rates and low LDH.

Phenolic compounds are plant secondary metabolites with a strong antioxidant effect, being known to initiate numerous processes involved in the wound healing process [21,22]. In this study, we aimed to extract the cellulosic material from the *G. triacanthos* pods, which was further multi-functionalized with a leaf extract of the same species, fect, being known to initiate numerous processes involved in the wound healing process [21,22]. In this study, we aimed to extract the cellulosic material from the *G. triacanthos* pods, which was further multi-functionalized with a leaf extract of the same species, The high rates of cell viability in contact with the CM and MFCM demonstrated that these new cellulose microfibers obtained from *Gleditsia triachantos* are biocompatible materials.

#### showing a significantly highphenolic compound content that in a first stage could exhibit showing a significantly highphenolic compound content that in a first stage could exhibit **3. Discussion**

Phenolic compounds are plant secondary metabolites with a strong antioxidant effect, being known to initiate numerous processes involved in the wound healing process [21,22]. In this study, we aimed to extract the cellulosic material from the *G. triacanthos* pods, which was further multi-functionalized with a leaf extract of the same species, showing a significantly highphenolic compound content that in a first stage could exhibit both antimicrobial and antioxidant activities, contributing to skin regeneration. Data regarding

UHPLC-DAD-ESI/MS analysis of PA and flavonoids from *G. triachantos* leaf extract have not been previously reported. However, Mohammed (2014) isolated and identified eight flavone glycosides and two flavone aglycones such as luteolin-7-O-β-glucopyranosid, luteolin-7-O-β-galactopyranoside, apigenin-7-O-β-glucopyranoside, luteolin, and apigenin from the aqueous ethanol extract of *G. triacanthos* L. leaves. Vitexin, luteolin, isovitexin, and quercetin have also been previously identified in this plant extract [23]. In addition, some identified compounds from the alcoholic extract of *G. triachantos*, such as syringic acid [24], gallic acid [25], p-coumaric acid [26], apigenin [27], quercetin [28], and myricetin [29], were reported to exhibit anti-inflammatory effects. Thus, PAs promise to be multivalent, bioinspired molecules that either alone or in the form of biomaterials additives can be very useful to prevent and treat medical device-associated and drug-resistant infections [30].

On the other hand, Saleh (2016) [31] reported the analgesic activity of the fruit methanolic extract of *G. triacanthos* and the saponins fraction derived, but the presence of these compounds could not be considered in our paper because the foaming test (for saponins) was negative.

To the best of our knowledge, the microcellulose obtained from *G. triacanthos* pods is herein reported for the first time, although some studies have been carried out on the characterization of other polysaccharides such as galactomannan [32], which is a heterogenous, water-soluble, non-toxic, and biodegradable polysaccharide used as a stabilizing, thickening, and emulsifying agent in the biomedical industry. The cellulose was extracted by treatment of grounded pods with toluene/ethanol (2:1) solution and six cycles of delignification method adapted after Zhuo (2017) [33]. The cellulose obtained was divided quantitatively into two parts—one was treated with 50% ethanol (control) in accordance with the second part, which contained the extract from the leaves of the same species. After lyophilization, the two materials were evaluated by ATR-FTIR analysis and compared to the commercial lignin. SEM analysis revealed that the control sample (CM) used for multi-functionalization with phenolic compounds showed the presence of microfibers and that the extract adhered to the cellulose microfibers surface, without requiring the presence of a fixing or crosslinking agent. The data were in accordance with Khalil (2020) [34], which concluded that by acid hydrolysis, the fiber size was in the range of 9–16 µm. In the wound dressing case, it was shown that cellulose microfibers improved the hemostasis of the scaffolds without affecting its cytotoxicity and the strength and flexibility of the films, which could be used in drug delivery or active wound dressing.

SEM (scanning electron microscopy) analysis revealed that the presence of microfibers with diameters between 5 and 10 µm and CM sample indicated a relatively smooth surface while the MFCM surface morphology highlighted a rougher surface compared to the CM due to the adherence of the phenolic compounds.

Microscopic characteristics, such as surface roughness, surface energy of materials, and thin surface coatings, play an important role in determining the wettability of material as well as hydrophobicity/hydrophilicity characteristics. The CM and MFCM highlighted a high wettability surface by CA measurements. The high wettability surface could be attributed to hydroxyl groups located at the equatorial positions of glucopyranose rings corresponding to higher planar orientation. [35]. On the other hand, it is well known that hydrophilicity is one of the most important surface characteristics of wound dressings. The water absorption study is a gravimetric test that has the main purpose of determining the maximum amount of water absorption and retention expressed in percentages. This assay was designed to determine how a dressing manifests its properties in conditions as close as possible to what can happen on the surface of a wound. In essence, the weight gain of the dressing after the absorption of the liquid and its swelling over a certain period of time was measured and used as an indication of water absorption and retention. MFCM had better water retention properties than CM, possibly due to the presence of phenolic compounds and glycosides. In general, these compounds increased the rate of hydration, which represents an advantage for wound dressing materials.

It was observed that MFCM had a statistically significant better retention capacity than CM, which means that it will be more helpful for wounds with moderate exudate and for dry wounds. Water loss could allow exudate and edema fluid to be picked up from wounds in the dressing through an active process directed upwards, as has been reported for some commercially available dressings [36,37].

Another important aspect for MFCM is the modulation of antioxidant activity during the release of active principles from the material. Thus, in wound healing, in addition to the antimicrobial potential of dressings, an important aspect is the modulation of reactive oxygen species (ROS) release. It is well known that low levels of ROS can exhibit positive effects, including an antimicrobial effect, but their excessive production can lead to oxidative stress, which will further inhibit the wound healing process, thus favoring wound chronicity [38]. In addition, the plant polyphenols inhibit the release of pro-inflammatory mediators and have no toxicity effects on human tissues [39].

There are many studies that have tried to establish some correlations between the antioxidant and antimicrobial activity, but only a few involved clinical strains isolated from hospital-acquired wound infections, such as *Escherichia coli* and *Staphylococcus aureus*, although the results were, however, contradictory [30]. MRSA and *E. coli* seem to be sensitive to cinnamic acid [40], p-coumaric acid [41,42], caffeic acid, vanillic acid, protocatechuic acid [41,43], while ferulic acid was effective only against *E. coli* [43]. On the contrary, in other studies, *E. coli* was not sensitive to p-coumaric acid, vanillic acid, or proocatechuic acid [44]. The MFCM proved to be more active on Gram-negative strains. Previous studies have shown only a minimal link between the antioxidant activity and antimicrobial efficacy [30]. In addition, the CM and MFCM are biocompatible materials with high cell proliferation rates and low cytotoxicity on mouse murine fibroblast cells.

#### **4. Materials and Methods**

#### *4.1. Plant Material*

*G. triachantos* leaves and shells were collected from Ramnicu Valcea, Romania, in September 2019 from spontaneous flora. Their taxonomic affiliation was confirmed, and voucher specimens were deposited in the herbarium of the Botanical Garden "Dimitrie Brândză" from the University of Bucharest (no. 40065). The plant materials were manually sorted and dried at room temperature.

#### *4.2. Phenolic Compounds Extraction from G. triachantos Leaves and Chemical Characterization* 4.2.1. Hydro-Alcoholic Extraction from *G. triachantos* Leaves

An amount of 2.5 g of previously ground dry leaves was weighed and brought into 50 mL of ethanol solution 50%. The extract obtained by the ultrasound-assisted extraction method was maintained at room temperature for 20 days, and after that, it was sonicated again for 30 min. Then, it was filtered and stored at −20 ◦C until it was incorporated in cellulose microfibers.

#### 4.2.2. Total Phenolic Compounds and Flavonoids Contents

TPC content was determined by the Folin–Ciocalteu method [45]. An aliquot was mixed with 0.1 mL Folin–Ciocalteu reagent, 1.8 mL distilled water, and 0.1 mL of saturated sodium carbonate. The tubes were vortexed for 15 s and allowed to stand at dark for 60 min for color development. Absorbance was then measured at 765 nm using a FlexStation 3 UV–VIS (Molecular Devices Company, Sunnyvale, CA, USA) spectrophotometer. A standard curve was prepared by using different concentrations of gallic acid in the same condition with samples (*R* 2 = 0.9985). TPC content was expressed as milligram gallic acid equivalent/g dry leaves (mg GAE/g DL). Analyses were performed in triplicate.

Flavonoid content was assessed through the AlCl3 method [46]. Briefly, 0.1 mL sample/standard solution was mixed with 0.1 mL sodium acetate 10% and 0.12 mL AlCl3 2.5%, with the final volume being adjusted to 1 mL with ethanol 50%. The samples were then vortexed and incubated in the dark for 45 min. The absorbances were measured at

λ = 430 nm. A standard curve was prepared by using different concentrations of quercetin (*R* 2 = 0.9958). Total flavonoid content was expressed as milligram quercetin equivalent/g dry leaves (mg QE/g DL). Analyses were performed in triplicate.

4.2.3. Ultra-High-Performance Liquid Chromatography Diode Array Detector Electrospray Ionization Tandem Mass Spectrometry

All experiments were performed using an UltiMate 3000 UHPLC system (ThermoFisher Scientific, Bremen, Germany), consisting of a quaternary pump, diode array detector (DAD) set at 280 nm, column oven, and autosampler coupled to a Q Exactive Focus Hybrid Quadrupole-OrbiTrap mass spectrometer equipped with heated electrospray ionisation (HESI) probe (ThermoFisher Scientific, Bremen, Germany).

Separations were performed on Kinetex (C18, 100 × 2.1 mm, 1.7 µm, Phenomenex, California, USA) column (reverse-phase UHPLC column) and a binary solvent system consisting of solvent A (water with 0.1% formic acid) and solvent B (methanol with 0.1% formic acid). The UHPLC gradient for mass screening, the flow rate, and the operation conditions of the mass spectrometer were previously optimized [47]. PAs and flavonoids from the extract were also identified and quantified according to Ciucure [47] (Table S1), as well as data acquisition and data analysis.

#### *4.3. CM and MFCM Obtaining and Characterization*

#### 4.3.1. CM and MFCM Obtaining

Cellulose extraction was performed according to Zhuo (2017) [33]. Briefly, an amount of 30 g ground shells of *G. triacanthos* was added over toluene/ethanol solution (2:1, *v*/*v*) and the mixture was further refluxed for 8 h at 90 ◦C. Then, the biomass was treated with sodium hypochlorite under the acidic condition (glacial acetic acid) at 70 ◦C for 6 h to remove the lignin, followed by treatment with 2% NaOH aqueous solution at 90 ◦C for 2 h to remove most of the hemicellulose. After that, the samples were again treated by sodium hypochlorite under acidic condition for 2 h, followed by final purification of 2% NaOH solution at 90 ◦C for 2 h. After each cycle, the presence of lignin was evaluated by hydroxycinnamic acid content according to British Pharmacopoeia [48] using a calibration curve with commercial lignin solubilized in DMSO (dimethylsulfoxide) (λ = 524 nm, *R* 2 = 0.9996). When lignin content was out of the detection limit, the cellulose was washed until it reached pH = 7.4, and each supernatant was evaluated by titration with AgNO3, indicating the removal of chloride ions. The cellulose fibers were divided into 2 samples, one being dispersed in 50% ethanol (control), and the second in the extract obtained previously. Both products were dispersed 3 times using IKA ULTRA-TURRAX (IKA®-Werke GmbH & Co. KG, Staufen, Germany) for 10 min, followed by an ultrasound treatment for 60 min. Finally, the samples were lyophilized at −55 ◦C and 1 Pa to obtain the cellulose microfibers (CM) and the multi-functionalized cellulose microfibers (MFCM), respectively.

#### 4.3.2. ATR-FTIR

The FTIR spectra for CM, MFCM, and lignin was recorded at room temperature using the Cary 630 FTIR Spectrometer in ATR mode (Agilent Technologies Inc., Santa Clara, CA, USA) and Agilent MicroLab Software FTIR System (Agilent Technologies, Inc., USA). The chosen measurement range was 4000–650 cm−1 , number of scans was 400, and resolution was 4 cm−1 . The FTIR spectra of dry extract were produced on the alcoholic extract of *G. tricanthos* leaves dried previously in an oven at 35 ◦C (for 24 h).

#### 4.3.3. Morphology of CM and MFCM

The morphology of CM and MFCM were performed by Nova NanoSEM 630 Scanning Electron Microscope (FEI Company, Hillsboro, OR, USA) using UHR detector (Through-Lens-Detector-TLD) at an acceleration voltage of 15 kV and a working distance of about 5 mm. The samples were fixed with a double tape on a conductive support of aluminum, which, afterward, was covered with a few nanometers of gold layer using sputtering for 30 s.

#### 4.3.4. Surface Wettability

Static (equilibrium) CAs were measured using a drop shape analysis instrument, model DSA100 (Krüss GmbH, Hamburg, Germany). The samples were positioned on a plane stage, under the tip of a stainless-steel needle, with a blunt end, with an outer diameter of 0.5 mm. The needle was attached to a syringe controlled by DSA3 software supplied with the instrument and was used to drop water with a controlled volume on the test surface as well as to evaluate the contact angle. The volume of water droplets was ≈1 µL with a pumping rate of 5 µL/min. The tests were carried out at room temperature [49].

#### 4.3.5. Water Absorptivity and Retention Properties

An amount of 0.1 g CM was treated with methanol and dried (60 ◦C for 24 h), then immersed in PBS (pH 7.4). The excess buffer was removed with filter paper and the material's wet mass was determined using the method adapted after Ngadaonye (2013) [36]. The same procedure was applied to MFCM, while the percentage of absorbed water was calculated for CM and MFCM as follows:

$$\text{Swelling ratio (\%)} = (\text{M}\_2 - \text{M}\_1) \times 100 / \text{M}\_1$$

where M1 and M2 represent the weights of dry and wet samples, respectively.