New Nanostructured Materials Based on Mesoporous Silica Loaded with Ru(II)/Ru(III) Complexes with Anticancer and Antimicrobial Properties

A new series of nanostructured materials was obtained by functionalization of SBA-15 mesoporous silica with Ru(II) and Ru(III) complexes bearing Schiff base ligands derived from salicylaldehyde and various amines (1,2-diaminocyclohexane, 1,2-phenylenediamine, ethylenediamine, 1,3-diamino-2-propanol, N,N-dimethylethylenediamine, 2-aminomethyl-pyridine, and 2-(2-aminoethyl)-pyridine). The incorporation of ruthenium complexes into the porous structure of SBA-15 and the structural, morphological, and textural features of the resulting nanostructured materials were investigated by FTIR, XPS, TG/DTA, zeta potential, SEM, and N2 physisorption. The ruthenium complex-loaded SBA-15 silica samples were tested against A549 lung tumor cells and MRC-5 normal lung fibroblasts. A dose-dependent effect was observed, with the highest antitumoral efficiency being recorded for the material containing [Ru(Salen)(PPh3)Cl] (50%/90% decrease in the A549 cells’ viability at a concentration of 70 μg/mL/200 μg/mL after 24 h incubation). The other hybrid materials have also shown good cytotoxicity against cancer cells, depending on the ligand included in the ruthenium complex. The antibacterial assay revealed an inhibitory effect for all samples, the most active being those containing [Ru(Salen)(PPh3)Cl], [Ru(Saldiam)(PPh3)Cl], and [Ru(Salaepy)(PPh3)Cl], especially against Staphylococcus aureus and Enterococcus faecalis Gram-positive strains. In conclusion, these nanostructured hybrid materials could represent valuable tools for the development of multi-pharmacologically active compounds with antiproliferative, antibacterial, and antibiofilm activity.


Introduction
One of the greatest challenges standing in front of modern biomedical science is the resistance to both antibacterial and antitumoral agents, raising an acute necessity to develop new, safe, and highly effective therapeutic strategies. Nowadays, the treatment of any malignancy is based on surgery, radiotherapy, and chemotherapy. Despite significant progress in understanding the molecular biology of cancer development, the design of novel cytotoxic anticancer drugs continues to be the cornerstone of modern antitumor therapy. After the discovery of cisplatin in 1960, the use of metallodrugs to treat cancer has been a great development. Since then, many metal-based drugs have been investigated for their activity against various types of cancer. Despite the discovery of antibiotics and vaccines, infectious diseases are still one of the top causes of mortality and morbidity, challenging ruthenium complex in cancer cells and dramatically enhances the anticancer efficacy of the hydrophobic ruthenium complex [30]. Sun et al. fabricated a ruthenium-loaded palmitoyl ascorbate (PA)-modified mesoporous silica that showed promising activity against human cancer cells in vitro and in vivo [31]. Martinez-Carmona et al. reported that the material obtained by encapsulation of [Ru(ppy-CHO)(phen) 2 ][PF 6 ] in mesoporous silica nanoparticles functionalized with amino groups shows very high anticancer activity against U87 glioblastoma cells [13]. Harun et al. demonstrated that encapsulation of novel ruthenium polypyridyl complexes (Ru-PIP) in mesoporous silica enhances significantly the cytotoxicity against Hela, A549, and T24 cancer cell lines, compared to unloaded Ru-PIP [32].
In this context and in continuation of our research work in the field of materials with biological activity, the aim of our study was to develop a new series of hybrid nanosystems based on Ru(II)/Ru(III) complexes with Schiff base ligands loaded in mesoporous silica and to evaluate their antimicrobial and anticancer properties. We were encouraged by the results obtained in one of our previous studies, in which the hybrid materials constructed through the immobilization of three Ru(III) complexes bearing Schiff base ligands derived from o-vanillin inside the mesoporous channels of SBA-15 exhibited very good cytotoxic activity against HeLa tumor cells [33].

Characterization Methods
FT-IR spectra on KBr pellets were acquired using a Jasco FT/IR-4700 spectrophotometer (Tokyo, Japan). UV-Vis spectra were recorded using a JASCO V-750 spectrophotometer (Tokyo, Japan). Thermogravimetric analyses (TGA) coupled with differential thermal analyses (DTA) were performed using a Mettler Toledo TGA/SDTA851e thermogravimeter (Greifensee, Switzerland), under 80 mL min −1 synthetic air atmosphere, at a heating rate of 10 • C min −1 . Sample composition was computed from the mass loss curves, with respect to the dry sample mass at 110 • C. A Micromeritics ASAP 2020 analyzer (Norcross, GA, USA) was used to measure the N 2 adsorption-desorption isotherms at −196 • C. Before analysis, the samples were heated at 80 • C for 6 h under vacuum to remove all of the adsorbed species. Specific surface areas (S BET ) were calculated using the Brunauer-Emmett-Teller (BET) method, while the amount adsorbed at a relative pressure of 0.99 was used to compute the total pore volume (V total ). The Barrett-Joyner-Halenda (BJH) method was applied to obtain the average pore diameter using the desorption data. Elemental analysis (C, H, N) was performed using an EuroEA elemental analyzer (HEKAtech GmbH, Wegberg, Germany). The magnetic properties were assessed at room temperature on a fully integrated Vibrating Sample Magnetometer system 7404 from Lake Shore (Westerville, OH, USA). XPS analysis was performed on a Kratos Ultra DLD Setup (Kratos Analytical Ltd., Manchester, UK) using a monochromatic Al-Kα source (hν = 1486.74 eV, X-ray source). A charge neutralizer was used for all samples and the conditions for recording XP spectra were as follows: power 240 W (20 kV × 12 mA), pressure 1 × 10 −7 Pa. The samples were calibrated to 284.6 eV (C 1s). Zeta potential measurements were performed on a Backman Coulter Delsa Nano C analyzer (Brea, CA, USA), at 25 • C. All samples for zeta potential measurements were suspended in water at a concentration of 250 µg mL −1 . The Pharmaceutics 2023, 15, 1458 4 of 25 morphology of the samples was analyzed by scanning electron microscopy (SEM) using a FEI Quanta 3D FEG microscope (FEI, Brno, Czech Republic).

Antibacterial Activity Assay
The antibacterial activity of the functionalized mesoporous silica was evaluated against four standard strains: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853.
The qualitative evaluation of the antimicrobial activity was performed following the CLSI (Clinical and Laboratory Standards Institute, Berwyn, PA, USA) guidelines using the agar diffusion method. Briefly, inoculums with a turbidity adjusted to 0.5 McFarland were prepared from fresh cultures and inoculated on Mueller-Hinton agar plates. A volume of 10 µL of each compound was placed on the agar surface, and after overnight incubation at 37 • C the growth inhibition zones diameters were measured with a ruler.
The quantitative analysis of the antimicrobial activity was carried out using the broth microdilutions assay. Two-fold dilutions of the Ru(II)-and Ru(III)-based compounds were prepared in culture liquid medium distributed in a 96-well plate, with the tested concentrations ranging from 5 to 0.002 mg/mL. Ciprofloxacin was used as a positive control. Each well was inoculated with a bacterial inoculum of 10 6 CFU/mL (colony forming units). Sterility controls and growth controls were used in order to determine the inhibitory effect. After overnight incubation at 37 • C, the bacterial growth was evaluated by reading the optical density at 620 nm (Multiskan FC Thermo Scientific, Waltham, MA, USA). The minimum inhibitory concentration (MIC) was determined as the lowest concentration that inhibits bacterial growth. The assays were performed in duplicate and the results were presented as mean ± standard deviation (SD). In order to determine the compounds' interference with the bacterial adherence to inert substrata and the subsequent biofilm development, the crystal violet assay was used. After MIC determination, the 96-well plates were discarded, washed with phosphate buffered saline, and fixed with cold methanol for 5 min in order to fix the adhered bacterial cells, which were further stained with 1% crystal violet solution for 20 min. Following the removal of the dye, a 33% acetic acid solution was added in each well, and after 10 min, the absorbance at 492 nm was read using a plate-reading spectrophotometer (Multiskan FC Thermo Scientific, Waltham, MA, USA). The assays were performed in duplicate and the results were presented as mean ± standard deviation (SD). The viability was quantified after incubating the cells with 1 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, Burlington, MA, USA) solution for 2 h at 37 • C. The purple formazan crystals formed in the live cells were dissolved with 2-propanol (Sigma-Aldrich, Burlington, MA, USA) and the absorbance was measured at 595 nm using a plate multireader (FlexStation 3, Molecular Device, San Jose, CA, USA). Compound concentrations that produce 50% cell growth inhibition (IC 50 ) were calculated from curves constructed by plotting cell survival (%) versus drug concentration (µg/mL) using the Quest Graph™ IC50 calculator (AAT Bioquest, Pleasanton, CA, USA).

Cytotoxicity Assay
The level of nitric oxide (NO) released in the culture medium was quantified with the Griess reagent, a stoichiometric solution (v/v) of 0.1% naphthylethylenediamine dihydrochloride and 1% sulphanilamide. Equal volumes of culture supernatants and Griess reagent were mixed, and the absorbance was read at 550 nm using the FlexStation 3 multireader.

Statistical Analysis
The in vitro assays were performed in triplicates, and the results were presented as the mean ± standard deviation (SD) of three independent experiments. The statistical significance was analyzed by Student's t-test, and values of P less than 0.05 were considered significant.

Characterization of the Ruthenium Complexes
Only the RuSalen complex was obtained as single crystals suitable for X-ray diffraction, and its structure was confirmed by X-ray crystallography. Because the SCXRD investigation of the RuSalen complex was previously reported by Tang et al. [40] in 2018, herein we will briefly describe its crystal structure. [Ru(Salen)(PPh 3 )Cl] is a mononuclear Ru(III) complex that crystallizes in the P 2 1 /c monoclinic space group. Its structure consists of discrete neutral [Ru(Salen)(PPh 3 )Cl] units, as shown in Figure 3. In this structure, the ruthenium atom is six-coordinated by two phenoxido oxygen atoms [Ru1 − O1 = 2.023(4), Ru1 − O2 = 2.011(4) Å] and two imino nitrogen atoms [Ru1 − N1 = 1.987(5), Ru1 − N2 = 2.000(5) Å] from the tetradentate Schiff base ligand (H 2 Salen), in the equatorial plane, and by one PPh 3 we will briefly describe its crystal structure. [Ru(Salen)(PPh3)Cl] is a mononuclear Ru(III) complex that crystallizes in the P 21/c monoclinic space group. Its structure consists of discrete neutral [Ru(Salen)(PPh3)Cl] units, as shown in Figure 3. In this structure, the ruthenium atom is six-coordinated by two phenoxido oxygen atoms [Ru1 − O1 = 2.023(4), Ru1 − O2 = 2.011(4) Å] and two imino nitrogen atoms [Ru1 − N1 = 1.987(5), Ru1 − N2 = 2.000(5) Å] from the tetradentate Schiff base ligand (H2Salen), in the equatorial plane, and by one PPh3 group [Ru1 − P1 = 2.349(2) Å] and one chloride atom [Ru1 − Cl1 = 2.4350 (19) Å] into the axial positions, building a distorted octahedral environment around the Ru(III) center. Electronic spectra of the Ru(III) and Ru(II) complexes have been recorded in the solid state in the 1000-200 nm range (spectra not shown). The UV-Vis spectra of all Ru(III) compounds show similar features and contain an intense broad band in the 200-1000 nm region, which is a multi-band coverage (three-structured absorption band in the ultraviolet and visible region, ~300, 400, 510, and 730 nm). The strong visible band in the range 500-1000 nm is due to the [Ru III N2O2PCl] chromophore (mainly charge-transfer transitions). In most of the Ru(III) complexes containing Schiff base ligands, charge-transfer transitions are prominent in the low-energy region, which obscures the weaker bands due to the d-d transition of the metal. It is therefore difficult to assign conclusively the bands of the ruthenium(III) complexes that appear in the visible region. The spectral profiles below 400 nm correspond to intra-ligand transitions (π-π* and n-π*) [33,41,42].
The magnetic moments, at room temperature, of all of the complexes, RuSalen, RuSalpnol, RuSalfen, and RuSaldiam, show that they are one-electron paramagnetic, confirming a low-spin d 5 , 5 t2g configuration for the ruthenium(III) ion (1.6 for RuSalen, 1.75 for RuSalpnol, 1.87 for RuSalfen, and 1.91 BM for RuSaldiam) [33,43,44]. The values of magnetic moments, close to expected for the spin-only value of a single unpaired electron species (1.73 BM), confirmed the (+3) state of ruthenium in these coordination compounds.
The absorption spectra of the RuSaldmen, RuSalampy, and RuSalaepy complexes are dominated in the visible region by absorption between 433 and 630 nm and in the UV region between 293 and 332 nm. The bands in the visible region are assigned to charge- Electronic spectra of the Ru(III) and Ru(II) complexes have been recorded in the solid state in the 1000-200 nm range (spectra not shown). The UV-Vis spectra of all Ru(III) compounds show similar features and contain an intense broad band in the 200-1000 nm region, which is a multi-band coverage (three-structured absorption band in the ultraviolet and visible region,~300, 400, 510, and 730 nm). The strong visible band in the range 500-1000 nm is due to the [Ru III N 2 O 2 PCl] chromophore (mainly charge-transfer transitions). In most of the Ru(III) complexes containing Schiff base ligands, charge-transfer transitions are prominent in the low-energy region, which obscures the weaker bands due to the d-d transition of the metal. It is therefore difficult to assign conclusively the bands of the ruthenium(III) complexes that appear in the visible region. The spectral profiles below 400 nm correspond to intra-ligand transitions (π-π* and n-π*) [33,41,42].
The magnetic moments, at room temperature, of all of the complexes, RuSalen, RuSalpnol, RuSalfen, and RuSaldiam, show that they are one-electron paramagnetic, confirming a low-spin d 5 , 5 t 2g configuration for the ruthenium(III) ion (1.6 for RuSalen, 1.75 for RuSalpnol, 1.87 for RuSalfen, and 1.91 BM for RuSaldiam) [33,43,44]. The values of magnetic moments, close to expected for the spin-only value of a single unpaired electron species (1.73 BM), confirmed the (+3) state of ruthenium in these coordination compounds.
The absorption spectra of the RuSaldmen, RuSalampy, and RuSalaepy complexes are dominated in the visible region by absorption between 433 and 630 nm and in the UV region between 293 and 332 nm. The bands in the visible region are assigned to charge-transfer transitions (MLCT) and in the UV region to ligand (π-π* and n-π*) transitions [45]. The experimental magnetic susceptibilities at room temperature of the RuSaldmen, RuSalampy, and RuSalaepy complexes were negative, indicating that these compounds are diamagnetic, with the ruthenium ion being in the (+2) oxidation state.  [46]. The pair of bands in the interval 2850-2940 cm −1 , characteristic of symmetric and asymmetric stretching aliphatic C-H bonds [47], can be observed in the spectra of SBA15-NH 2 and all the samples functionalized with ruthenium complexes. The bands at 3420 and 1630 cm −1 are assigned to O-H bond stretching and bending vibrations of the silanol groups of the materials and the adsorbed H 2 O molecules. The new band at 1553 cm −1 in the spectrum of SBA15-NH 2 , attributable to the bending vibration mode of N−H, confirms the grafting of aminopropyl groups on the surface of mesoporous silica. New bands of low intensity can be distinguished after functionalization of SBA-15 with ruthenium complexes, these bands being associated with the functional groups of the complexes. The most intense one, located at~1603 cm −1 , is attributed to the imine (C=N) stretching vibration of the Schiff bases in the structure of ruthenium complexes. This characteristic band of the ruthenium complexes confirms their presence in the mesoporous silica channels. The other bands of lower intensity, at around 1530 and 1436 cm −1 are assigned to C-N and C-C stretching vibrations and arise also from the attached ruthenium complex [33].

Characterization of SBA-15 Functionalized with Ruthenium Complexes
shown in Figure 4. The peaks located at 460 cm −1 (Si-O bending vibration), 798 cm −1 (symmetric Si-O-Si stretching vibration), 960 cm −1 (Si-OH stretching vibration), and 1077 cm −1 (asymmetric Si-O-Si stretching vibration) represent the fingerprint of silica framework in all materials [46]. The pair of bands in the interval 2850-2940 cm −1 , characteristic of symmetric and asymmetric stretching aliphatic C-H bonds [47], can be observed in the spectra of SBA15-NH2 and all the samples functionalized with ruthenium complexes. The bands at 3420 and 1630 cm −1 are assigned to O-H bond stretching and bending vibrations of the silanol groups of the materials and the adsorbed H2O molecules. The new band at ~1553 cm −1 in the spectrum of SBA15-NH2, attributable to the bending vibration mode of N−H, confirms the grafting of aminopropyl groups on the surface of mesoporous silica. New bands of low intensity can be distinguished after functionalization of SBA-15 with ruthenium complexes, these bands being associated with the functional groups of the complexes. The most intense one, located at ~1603 cm −1 , is attributed to the imine (C=N) stretching vibration of the Schiff bases in the structure of ruthenium complexes. This characteristic band of the ruthenium complexes confirms their presence in the mesoporous silica channels. The other bands of lower intensity, at around 1530 and 1436 cm −1 are assigned to C-N and C-C stretching vibrations and arise also from the attached ruthenium complex [33]. XPS analysis was conducted to obtain a more particular knowledge of the valence states of elements and the chemical composition of the samples. XPS spectra shown in Figure 5 confirm that, in all samples loaded with ruthenium complexes, ruthenium was successfully deposited and is present in the range between 0. XPS analysis was conducted to obtain a more particular knowledge of the valence states of elements and the chemical composition of the samples. XPS spectra shown in Figure 5 confirm that, in all samples loaded with ruthenium complexes, ruthenium was successfully deposited and is present in the range between 0.2-0.4% on the surface of SBA-15. The C1s core level was fitted with five components: the first component at lower binding energies (~279.9 eV), corresponding to the Ru-C bond; the second component at 283.2 eV, associated with C-Si-O bonds; the third one at 284.6 eV, corresponding to the C-C/C=C bond; the fourth at 285.7 eV, corresponding to the C-N/C-O bonds; and the fifth at 287.0 eV, associated with C=O bonds ( Figure S1). During modification with ruthenium complexes, an increase in the C-N component can be observed, which was expected since in the Ru complexes the carbon-nitrogen bond is present. The Si 2p core level presents three components: a component at low binding energies of 101.7 eV associated with the Si-C bond, the Si-O bond at 103.3 eV, and a component at higher binding energies of 104.5 eV, most probably due to some hydroxylated Si on the surface ( Figure S1). The O1s core level presents in all samples three components: the first one at 531.2 eV assigned to the C-O bond, the second one at 532.6 eV associated with the Si-O component, and the third component corresponding to -OH groups at higher binding energies (533.7 eV) ( Figure S1). The nitrogen is present in all samples in a relatively small amount (between 1.5 and 2.1%) and in all cases there are three components associated with imine N (398.2 eV), primary N (399.8 eV), and Ru-N at 401.3 eV ( Figure S1). The XPS spectra highlight the presence of chlorine in all samples containing ruthenium ( Figure S1), which suggests that the adsorption of the ruthenium complexes into the mesoporous silica channels is probably achieved through molecular interactions between the polar groups of the ruthenium complexes and the amino groups grafted onto the internal walls of SBA-15. component corresponding to -OH groups at higher binding energies (533.7 eV) ( Figure  S1). The nitrogen is present in all samples in a relatively small amount (between 1.5 and 2.1%) and in all cases there are three components associated with imine N (398.2 eV), pri mary N (399.8 eV), and Ru-N at 401.3 eV ( Figure S1). The XPS spectra highlight the pres ence of chlorine in all samples containing ruthenium ( Figure S1), which suggests that the adsorption of the ruthenium complexes into the mesoporous silica channels is probably achieved through molecular interactions between the polar groups of the ruthenium com plexes and the amino groups grafted onto the internal walls of SBA-15. The textural analysis of the samples was performed by recording the N2 adsorptiondesorption isotherms ( Figure 6). Specific surface area, pore diameter, and total pore vol ume were determined from the sorption isotherms, and the results are listed in Table 1 According to the IUPAC classification [48], the nitrogen adsorption-desorption analysis indicated type IV isotherms for all samples, accompanied by type H1 hysteresis loops characteristic for mesoporous materials with uniform cylindrical pores ( Figure 6). After functionalization of SBA-15 with aminopropyl groups, a significant decrease in surface area (about 46%) and in total pore volume (about 40%) was observed, as well as a corre sponding decrease in pore diameter (Table 1). A higher decrease in the values of surface area (58-63%) and total pore volume (52-58%) compared to the corresponding ones for SBA-15 was observed after the immobilization of ruthenium complexes. A decrease o 10-17% was also observed in the average pore size. These findings suggest a uniform im mobilization of the ruthenium complexes onto the internal pore walls of SBA-15, resulting in reduced accessible space for adsorbed nitrogen. The textural analysis of the samples was performed by recording the N 2 adsorptiondesorption isotherms ( Figure 6). Specific surface area, pore diameter, and total pore volume were determined from the sorption isotherms, and the results are listed in Table 1. According to the IUPAC classification [48], the nitrogen adsorption-desorption analysis indicated type IV isotherms for all samples, accompanied by type H1 hysteresis loops, characteristic for mesoporous materials with uniform cylindrical pores ( Figure 6). After functionalization of SBA-15 with aminopropyl groups, a significant decrease in surface area (about 46%) and in total pore volume (about 40%) was observed, as well as a corresponding decrease in pore diameter (Table 1). A higher decrease in the values of surface area (58-63%) and total pore volume (52-58%) compared to the corresponding ones for SBA-15 was observed after the immobilization of ruthenium complexes. A decrease of 10-17% was also observed in the average pore size. These findings suggest a uniform immobilization of the ruthenium complexes onto the internal pore walls of SBA-15, resulting in reduced accessible space for adsorbed nitrogen.   Zeta (ζ)-potential measurements were carried out to analyze the net surface of the samples, a very important parameter on which the internalization of nanop by cancer cells depends [49]. The obtained values are shown in Table 1. The neg potential of pristine SBA-15 (−24.7 mV) is due to the presence of silanol groups. Fu alization of SBA-15 with aminopropyl groups led to a positive ζ-potential (+24 while loading with ruthenium complexes further increased the ζ-potential of the o hybrid materials. Since all the ruthenium-loaded materials have positive and re high ζ-potential values, they can be expected to target cancer cells efficiently due trostatic attraction to their negatively charged membrane [50]. These ζ-potentia also reveal a relatively good colloidal stability of mesoporous silica loade Ru(II)/Ru(III) complexes in aqueous medium.
Bearing in mind that these compounds were synthesized in order to study th logical activity (antibacterial and cytotoxic activity), it is very important to kno stability in solution. For this purpose, a spectroscopic study was carried out using Vis technique, on the compounds suspended in deionized water (250 μg/mL), ( Figure S2). No significant spectral changes in the studied materials were observ Zeta (ζ)-potential measurements were carried out to analyze the net surface charge of the samples, a very important parameter on which the internalization of nanoparticles by cancer cells depends [49]. The obtained values are shown in Table 1. The negative ζ-potential of pristine SBA-15 (−24.7 mV) is due to the presence of silanol groups. Functionalization of SBA-15 with aminopropyl groups led to a positive ζ-potential (+24.4 mV), while loading with ruthenium complexes further increased the ζ-potential of the obtained hybrid materials. Since all the ruthenium-loaded materials have positive and relatively high ζ-potential values, they can be expected to target cancer cells efficiently due to electrostatic attraction to their negatively charged membrane [50]. These ζ-potential values also reveal a relatively good colloidal stability of mesoporous silica loaded with Ru(II)/Ru(III) complexes in aqueous medium.
Bearing in mind that these compounds were synthesized in order to study their biological activity (antibacterial and cytotoxic activity), it is very important to know their stability in solution. For this purpose, a spectroscopic study was carried out using the UV-Vis technique, on the compounds suspended in deionized water (250 µg/mL), at 37 • C ( Figure S2). No significant spectral changes in the studied materials were observed after 24 and 72 h, respectively, after the preparation of the suspensions, which shows their very good stability in the aqueous environment. Therefore, it can be said that the studied hybrid systems based on mesoporous silica functionalized with Ru(II) and Ru(III) complexes can be used in aqueous suspensions to determine their biological activity.
The morphologic characterization of the samples was performed by SEM, and the acquired images are shown in Figure 7. Pure SBA-15 consists of short-rod-like particles with typical wheat-like morphologies and relatively uniform sizes ranging between 0.5 and 1.0 µm. For all the ruthenium-containing mesoporous materials, no significant changes were observed in particle sizes and shapes. This suggests that loading with ruthenium complexes does not affect the macroscopic morphology of the materials. with typical wheat-like morphologies and relatively uniform sizes ranging between 0.5 and 1.0 μm. For all the ruthenium-containing mesoporous materials, no significant changes were observed in particle sizes and shapes. This suggests that loading with ruthenium complexes does not affect the macroscopic morphology of the materials. Thermogravimetric analyses coupled with differential thermal analyses were carried out in order to evidence the combustion of the functionalized organic groups. All samples exhibit variable mass loss from 25 to 110 °C, (Figure 8a) accompanied by an endothermic thermal effect (Figure 8b), which is likely caused by physisorbed water evaporation. SBA-15 shows a gradual mass loss above 200 °C, explained by the condensation of surface si-  Thermogravimetric analyses coupled with differential thermal analyses were carried out in order to evidence the combustion of the functionalized organic groups. All samples exhibit variable mass loss from 25 to 110 • C, (Figure 8a) accompanied by an endothermic thermal effect (Figure 8b), which is likely caused by physisorbed water evaporation. SBA-15 shows a gradual mass loss above 200 • C, explained by the condensation of surface silanol groups. The combustion of aminopropyl groups can be noticed for SBA15-NH 2 as a mass loss event between 250 and 650 • C, accompanied by an exothermic thermal effect. The samples containing the Ru complexes all exhibit similar mass loss effects between 200 and 500 • C, caused by the superposition of the combustion of organic ligand and silica functional groups. The composition of the samples was computed assuming that the silanol content of SBA-15 and the aminopropyl content of SBA15-NH 2 are persevered for all other materials ( Table 2). The SBA15-NH 2 matrix contains 11.7% wt. aminopropyl groups with respect to the dry sample mass. The materials containing the Ru complexes exhibit 8.4-11.6% weight loss associated with the ligand combustion. Thus, TGA analyses show the successful functionalization of SBA-15 and the incorporation of the complexes. Thermogravimetric analyses coupled with differential thermal analyses were carried out in order to evidence the combustion of the functionalized organic groups. All samples exhibit variable mass loss from 25 to 110 °C, (Figure 8a) accompanied by an endothermic thermal effect (Figure 8b), which is likely caused by physisorbed water evaporation. SBA-15 shows a gradual mass loss above 200 °C, explained by the condensation of surface silanol groups. The combustion of aminopropyl groups can be noticed for SBA15-NH2 as a mass loss event between 250 and 650 °C, accompanied by an exothermic thermal effect. The samples containing the Ru complexes all exhibit similar mass loss effects between 200 and 500 °C, caused by the superposition of the combustion of organic ligand and silica functional groups. The composition of the samples was computed assuming that the silanol content of SBA-15 and the aminopropyl content of SBA15-NH2 are persevered for all other materials ( Table 2). The SBA15-NH2 matrix contains 11.7% wt. aminopropyl groups with respect to the dry sample mass. The materials containing the Ru complexes exhibit 8.4-11.6% weight loss associated with the ligand combustion. Thus, TGA analyses show the successful functionalization of SBA-15 and the incorporation of the complexes.

Antimicrobial Activity
The qualitative evaluation of the functionalized mesoporous silica samples revealed that, except for SBA-15, all of the samples exhibited an inhibitory effect on the growth of Gram-positive strains (S. aureus and E. faecalis), the largest growth inhibition diameters being recorded for SBA15-RuSalaepy, SBA15-RuSalen, and SBA15-RuSaldiam (Table 3).
SBA-15 had no effect on any of the strains tested. The Ru(II)-and Ru(III)-based compounds inhibited to a lesser extent the development of the Gram-negative E. coli strain when tested on solid media, while the P. aeruginosa growth was not impaired by any of the tested compounds (Table 3). The quantitative evaluation of the antimicrobial activity confirmed the inhibitory effects of the mesoporous silica functionalized with Ru(II) and Ru(III) complexes, especially against the Gram-positive tested strains. Except for SBA-15, all of the other compounds had MIC values of maximum 156 µg/mL for the Gram-positive strains (Figure 9, Table 4), with SBA15-RuSaldiam and SBA15-RuSalen being the most active. Regarding the Gramnegative strains, most of the MIC values were higher (625 µg/mL for all samples, except for SBA15-RuSalfen with an MIC of 1250 µg/mL against E. coli) (Figure 9, Table 4).    The inhibition of bacterial adherence to the inert substrata was observed for most of the mesoporous silica compounds at a concentration of 625 µg/mL. SBA15-RuSaldiam, SBA15-RuSalen were the most active against S. aureus and E. faecalis strains, and SBA15-RuSalfen against the S. aureus strain ( Figure 10, Table 5).  Our results confirm the fact that the ruthenium-based compounds have good an crobial activity towards Gram-positive bacteria and, to a lesser extent, to Gram-neg species, with SBA15-RuSaldiam and SBA15-RuSalen being the most efficient agains tested bacterial strains.

Cytotoxicity Evaluation
The in vitro studies ( Figure 11) were carried out to assess the viability of A549  Our results confirm the fact that the ruthenium-based compounds have good antimicrobial activity towards Gram-positive bacteria and, to a lesser extent, to Gram-negative species, with SBA15-RuSaldiam and SBA15-RuSalen being the most efficient against the tested bacterial strains.

Cytotoxicity Evaluation
The in vitro studies ( Figure 11) were carried out to assess the viability of A549 lung tumor cells after 24 and 72 h of exposure to various concentrations (0-200 µg/mL) of Ru-based hybrid materials, as well as their potential to induce inflammation. The cellular viability assay revealed that all ruthenium complex-containing samples inhibited cell growth in a time-and dose-dependent manner compared to the control ( Figure 11). In contrast, SBA-15 did not show the same pattern of viability decrease at the highest concentration tested (200 µg/mL) reducing the number of live cells only by 10% of the control after 24 h. Among the investigated materials, those containing Ru(III) complexes with compartmental ligands (SBA15-RuSalpnol, SBA15-RuSalen, SBA15-RuSaldiam, and SBA15-RuSalfen) showed a higher cytotoxic activity on A549 lung tumor cells than those with open ligands (SBA15-RuSaldmen, SBA15-RuSalampy, and SBA15-RuSalaepy), regardless of the incubation time. It is worth mentioning that SBA15-RuSalen showed the highest cytotoxic potential, diminishing the viable cell population by half of the control at a concentration of 70 µg/mL after 24 h of incubation, and at 35 µg/mL after 72 h of incubation. Furthermore, higher concentrations of this compound reduced the viability of A549 cells by more than 90% of the control.
Ruthenium-based systems have gained great attention recently for their activity against cancer [50]. Previously, it was shown that ruthenium-loaded palmitoyl ascorbate (PA)-modified mesoporous silica was able to inhibit cancer cell growth and induce their apoptosis through superoxide generation and DNA damage [31]. Our results confirmed the good biological activity of Ru-based hybrid materials against cancer cell growth, inducing their death, most probably by oxidative stress activation. In addition, we noticed a higher potency of Ru(III) complexes than Ru(II) ones. Previous reports showed that Ru(II) complexes are more reactive than Ru(III) [51], but less cytotoxic [52]. These could interact with the thiol groups in the cell, modulating the activity of intracellular enzymes and signaling pathways.
In order to check if these Ru-containing hybrid materials could affect the viability of non-tumoral cells, the MTT assay was performed also after 24 and 72 h of incubation with normal lung fibroblasts MRC-5 ( Figure 12). A decrease in viable cell number was observed compared to the control after both periods of exposure for all types of materials tested, and no great difference was noticed between values obtained after 24 h and those after 72 h. The highest reduction in cell viability was determined after incubation with concentrations higher than 70 µg/mL of SBA-15-RuSaldiam, SBA-15-RuSalpnol, SBA-15-RuSalfen, and SBA-15-RuSalen. However, it is important to highlight that the viability percentages for non-tumor cells were higher compared to the values obtained in the case of A549 cancer cells after 72 h. These findings could suggest that MRC-5 cells were more sensitive to the compounds tested after the first day of exposure, the values being lower than those recorded for A549 epithelial cells, but after another 2 days, the tumor cells were much more affected, especially at high concentrations. This could confirm that despite the cytotoxicity exerted on normal cells, the ruthenium-containing hybrid materials possess a good anti-cancer potential.
the good biological activity of Ru-based hybrid materials against cancer cell growth, inducing their death, most probably by oxidative stress activation. In addition, we noticed a higher potency of Ru(III) complexes than Ru(II) ones. Previous reports showed that Ru(II) complexes are more reactive than Ru(III) [51], but less cytotoxic [52]. These could interact with the thiol groups in the cell, modulating the activity of intracellular enzymes and signaling pathways. In order to check if these Ru-containing hybrid materials could affect the viability of non-tumoral cells, the MTT assay was performed also after 24 and 72 h of incubation with normal lung fibroblasts MRC-5 ( Figure 12). A decrease in viable cell number was observed compared to the control after both periods of exposure for all types of materials tested, and no great difference was noticed between values obtained after 24 h and those after 72 of A549 cancer cells after 72 h. These findings could suggest that MRC-5 cells were more sensitive to the compounds tested after the first day of exposure, the values being lower than those recorded for A549 epithelial cells, but after another 2 days, the tumor cells were much more affected, especially at high concentrations. This could confirm that despite the cytotoxicity exerted on normal cells, the ruthenium-containing hybrid materials possess a good anti-cancer potential.  The IC50 values (Table 6) obtained from cell survival plots (Figures S3-S6) showed that the SBA-15-RuSalen compound has the most potent antitumor efficiency, with a concentration of 23.9 μg/mL being able to inhibit half of A549 cells' growth compared to control after 72 h. The selectivity index (the ratio between the IC50 for normal cell line and IC50 for the cancer cell line) for this compound was 2.4, proving the higher toxicity against The IC50 values (Table 6) obtained from cell survival plots (Figures S3-S6) showed that the SBA-15-RuSalen compound has the most potent antitumor efficiency, with a concentration of 23.9 µg/mL being able to inhibit half of A549 cells' growth compared to control after 72 h. The selectivity index (the ratio between the IC 50 for normal cell line and IC 50 for the cancer cell line) for this compound was 2.4, proving the higher toxicity against tumor cells than against normal ones. By comparing the result of SBA-15-RuSalen with a positive control, such as cisplatin, the standard therapy for patients with lung cancer [53], we observed an almost-similar IC 50 value (26 ± 3.0 µg/mL), as it was previously reported [54] after 72 h of incubation of this drug with A549 cells. Furthermore, it is important to highlight that all Ru-containing hybrid materials exhibited lower IC 50 values compared to the SBA-15 compound. NO is toxic to cells in high concentrations, and measuring its release in cell culture media can provide a valuable way to assess the toxic effects of nanoparticles, materials, drugs, or other compounds on cells [55]. This molecule is also involved in the inflammatory response, and quantifying its release can indicate the level of inflammation in the cells. The results of the Griess assay showed an increase in the NO release compared to the control only after 72 h of incubation with the highest concentration used (200 µg/mL) ( Figure 13). This could indicate that inflammation and high toxicity were induced only by the high quantity of compounds tested.
Regarding the effect on MRC-5 cells, Ru-containing hybrid materials induced an increase in NO release compared to the control only after the incubation with 200 µg/mL, but the values did not exceed those registered in the case of A549 cancer cells. The most elevated values were noticed in the case of SBA-15 ( Figure 14). drugs, or other compounds on cells [55]. This molecule is also involved in the inflammatory response, and quantifying its release can indicate the level of inflammation in the cells. The results of the Griess assay showed an increase in the NO release compared to the control only after 72 h of incubation with the highest concentration used (200 μg/mL) ( Figure 13). This could indicate that inflammation and high toxicity were induced only by the high quantity of compounds tested.  Regarding the effect on MRC-5 cells, Ru-containing hybrid materials induced an increase in NO release compared to the control only after the incubation with 200 μg/mL, but the values did not exceed those registered in the case of A549 cancer cells. The most elevated values were noticed in the case of SBA-15 ( Figure 14).

Conclusions
In this work, we have obtained and characterized by various methods a new series of nanostructured materials based on SBA-15 mesoporous silica loaded with Ru(II) and Ru(III) complexes bearing Schiff base ligands derived from salicylaldehyde and various amines. Their antimicrobial activity was evaluated against S. aureus, E. faecalis, E. coli, and P. aeruginosa, while the anticancer activity was investigated in vitro against A549 lung tumor cells and MRC-5 normal lung fibroblasts. The results of the antibacterial activity suggest the promising potential of SBA15-RuSaldiam, SBA15-RuSalen, and SBA15-RuSalaepy for the development of novel antibacterial drugs, efficient against S. aureus and E. faecalis Gram-positive strains, two of the most fearful resistant opportunistic nosocomial pathogens, both in planktonic and adherent growth states. The compounds SBA15-RuSalpnol, SBA15-RuSalen, SBA15-RuSaldiam, and SBA15-RuSalfen proved to have the highest cytotoxic potential demonstrated on the A549 tumor cells. Of these, SBA15-RuSalen stands out as the most potent, with an IC 50 index of 23.9 µg/mL and a selectivity index of 2.4. Thus, these activities open the avenue for the development of multi-pharmacologically active compounds with antiproliferative activity against prokaryotic and eukaryotic cells.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics15051458/s1, Figure S1: C1s, O1s, Si2p, N1s, Ru3p, and Cl2p deconvoluted photoelectron spectra for the investigated samples; Figure S2: UV-Vis spectra of the investigated mesoporous silica functionalized with Ru(II) and Ru(III) complexes in aqueous solution (250 µg/mL): (a) immediately after preparation; (b) after 24 h; (c) after 72 h; Figure S3: Cell survival graphs (%) obtained by MTT assay for Ru-containing hybrid materials after 24 h of incubation with A549 lung tumor cells; Figure S4: Cell survival graphs (%) obtained by MTT assay for Ru-containing hybrid materials after 72 h of incubation with A549 lung tumor cells; Figure S5: Cell survival graphs (%) obtained by MTT assay for Ru-containing hybrid materials after 24 h of incubation with MRC-5 lung non-tumoral cells; Figure S6: Cell survival graphs (%) obtained by MTT assay for Ru-containing hybrid materials after 72 h of incubation with MRC-5 lung non-tumoral cells.