Engineering Mesoporous Silica Hosts for Ultrasmall ZnO Nanoparticles: A Dendritic Polymer-Assisted Strategy Towards Sustainable, Safe, and Effective Antibacterial Systems

Academic Editor: Anne Kahru Received: 9 October 2025 Revised: 1 November 2025 Accepted: 5 November 2025 Published: 9 November 2025 Citation: Papavasiliou, A.; Lyra, K.M.; Sakellis, E.; Lozano Násner, A.M.; Gallego, J.; Katsaros, F.K.; Sideratou, Z. Engineering Mesoporous Silica Hosts for Ultrasmall ZnO Nanoparticles: A Dendritic Polymer-Assisted Strategy Towards Sustainable, Safe, and Effective Antibacterial Systems. Nanomaterials 2025,15, 1697. https://doi.org/ 10.3390/nano15221697 Copyright: © 2025 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/). Article Engineering Mesoporous Silica Hosts for Ultrasmall ZnO Nanoparticles: A Dendritic Polymer-Assisted Strategy Towards Sustainable, Safe, and Effective Antibacterial Systems Aggeliki Papavasiliou 1,*, Kyriaki Marina Lyra 1, Elias Sakellis 1,2 , Albany Milena Lozano Násner 3, Jose Gallego 3, Fotios K. Katsaros 1and Zili Sideratou 1,* 1Institute of Nanoscience and Nanotechnology, National Center for Scientific Research “Demokritos”, 15310 Aghia Paraskevi, Greece; [email protected] (K.M.L.); [email protected] (E.S.); [email protected] (F.K.K.) 2Condensed Matter Physics Section, Physics Department, National and Kapodistrian University of Athens, Panepistimiopolis, Zografos, 15784 Athens, Greece 3Association for Research and Development of Innovations and Technologies for the Protection of Environmental, Social, and Cultural Heritage, 48 Avenue Mont-Rabeau, 06200 Nice, France; [email protected] (A.M.L.N.); [email protected] (J.G.) *Correspondence: [email protected] (A.P.); [email protected] (Z.S.); Tel.: +30-2106503913 (A.P.); +30-2106503616 (Z.S.) Abstract In response to the urgent need for sustainable antibacterial solutions against antibioticresistant pathogens, this study presents a facile dendritic polymer-assisted approach for synthesizing highly active ZnO/mesoporous silica nanocomposites (SBA-15, SBA-16, KIT-6, MSU-X). Two hyperbranched polymers—polyethyleneimine (PEI) and carboxy-methylated polyethyleneimine (Trilon-P, TrP)—were employed as templating and metal-trapping agents. The influence of pore geometry, polymer functionality, and polymer-loading method (wet or dry impregnation) on ZnO nanoparticle (NP) formation was systematically examined. All nanocomposites exhibited high structural homogeneity, incorporating ultrasmall or amorphous ZnO NPs (1–10 nm) even at 8 wt.% Zn loading. Zn uptake was strongly dependent on polymer end groups, while the spatial distribution of ZnO NPs was dictated by the silica host structure. Antibacterial assays against Staphylococcus aureus revealed remarkable activity, particularly for ZnO/SBA-15_PEI, ZnO/SBA-16_PEI, and ZnO/MSUX_TrP nanocomposites, with minimum inhibitory concentrations of 1–2.5 µ g mL −1 Zn and over 90% mammalian cell viability. Life Cycle Assessment identified energy use as the main environmental factor, with ZnO/SBA-15_PEI_WI displaying the lowest impact. Overall, the interplay between silica pore architecture, polymer type, and impregnation method governs ZnO accessibility and bioactivity, establishing a versatile strategy for designing next-generation ZnO/SiO 2 nanocomposites with tunable antibacterial efficacy and minimal cytotoxic and environmental footprint. Keywords: ZnO nanoparticles; nanocomposites; mesoporous silica; hyperbranched polyethyleneimine; carboxy-methylated hyperbranched polyethyleneimine; hyperbranched polymers; dendritic polymer-assisted impregnation technique; antibacterial properties 1. Introduction Infectious diseases remain a major global threat, impacting both public health and economic stability. They account for over 20% of worldwide mortality, with bacterial infecNanomaterials 2025,15, 1697 https://doi.org/10.3390/nano15221697 Nanomaterials 2025,15, 1697 2 of 35 tions alone being responsible for approximately 7.7 million deaths annually, underscoring the urgent need for sustainable and effective antibacterial strategies [ 1 ]. Compounding this challenge is the growing crisis of antibacterial resistance [ 2 , 3 ]. Antibiotics, which constitute the most common practice to treat these infections, present nowadays a limited efficacy due to the growth of antibiotic-resistant bacteria [ 4 – 6 ]. The improper and excessive prescription and use of antibiotics, coupled with the genetic flexibility of the bacteria, are identified as the primary cause for the appearance and prevalence of antibiotic resistance [ 4 , 5 ]. To tackle the hazards related to the escalation of infections and of antibiotic-resistant pathogens, inorganic nanoparticles have emerged as an efficient alternative solution to conventional antibiotics [ 4 , 6 – 8 ]. Au, Ag, CuO, TiO 2 , and ZnO NPs are amongst the most widely studied inorganic antimicrobial agents [ 7 , 8 ], with ZnO singled out as a highly potent material in view of its low cost, abundance, and long-term effectiveness [ 4 , 9 – 11 ]. Although ZnO nanoparticles (NPs) may still pose health risks depending on their concentration and route of exposure, such as skin contact, ingestion, or inhalation, ZnO in its bulk form is classified as ‘generally recognized as safe’ and is approved by the U.S. Food and Drug Administration (FDA) [ 12 ]. Despite their broad potential in diverse sectors, such as pharmaceuticals, cosmetics, textiles, and agriculture, the widespread use of ZnO NPs has been restricted due to concerns about their toxicological impact on both human health [ 13 , 14 ] and the environment [15,16]. The antibacterial effect of ZnO, bactericidal or bacteriostatic, relies on multiple mechanisms mediated by chemical and physical interactions with the bacterial cell [ 10 ]. Chemical interactions include the generation of reactive oxygen species (ROS) and the release of Zn 2+ ions, while physical interactions mostly include electrostatic, hydrophobic interactions and van der Waals forces, leading to the attachment of ZnO nanoparticles onto the bacterial cell wall [ 10 ]. The antimicrobial mechanisms can act simultaneously and induce cell envelope rupturing, cellular internalization, oxidative stress, and interaction and impairment of intracellular components, such as DNA, lysosomes, ribosomes, enzymes, etc. [7–11,17]. Properties of ZnO nanoparticles, such as shape/morphology, size, and surface modification, are considered key factors determining, to a great extent, their antimicrobial effect [ 10 , 17 – 20 ]. Downsizing ZnO particles to the nanoscale region has proved a highly beneficial approach in view of the size-dependency of several ZnO properties [ 8 , 10 , 17 ]. Particularly, oxygen defect-induced ROS production, dissolution of ZnO NPs into Zn 2+ , accumulation on the bacterial cell membrane, and ZnO internalization, all contributing towards antibacterial activity, are notably enhanced by decreasing particle size [ 10 , 17 , 21 ]. A clear correlation between ZnO particle size, cellular internalization and ROS generation was reported by G. Applerot et al. [ 21 ]. According to their findings, nanoscale ZnO particles displayed pronounced antibacterial activity, attributable to their facile penetration into bacterial cells and the subsequent formation of ROS, predominantly OH radicals, resulting in bacterial cell damage. Notably, this antibacterial mechanism was not observed with microscale ZnO particles. Yet, despite their promising features, ZnO NPs present some significant drawbacks associated with cytotoxicity and ecotoxicity effects in a dose-dependent manner, as previously mentioned, and with the great aggregation tendency, induced either by their intrinsic nature or by the defense bacteria mechanism [ 11 , 19 , 22 – 24 ]. Lately, the encapsulation or immobilization of inorganic nanoparticles on mesoporous silica hosts is adopted as an effective strategy to address these issues [ 25 – 27 ]. Ordered mesoporous silicas, owing to their appealing textural features, such as high surface area, large pore volume, well-defined porous network, and adjustable pore size in the nanometer range—coupled with their biocompatibility, high loading capacity, and facile functionalization due to the silanol-enriched surface—have been established as ideal nanocarriers for drug delivery and currently for Nanomaterials 2025,15, 1697 3 of 35 antibacterial agents [ 28 – 32 ]. Still, in order to ensure a high NP dispersion state, apart from the utilization of porous media, special attention must be given to the deposition procedure employed. Up to now, significant advances have been made towards engineering mesoporous silicas with metal or metal oxide NPs for a wide variety of applications. To this end, numerous methods have been applied, including wet impregnation, solid-state mixing, precipitation, co-condensation, template ion-exchange, and post-grafting [ 33 ]. Conventional impregnation or precipitation techniques, although quite simple to conduct, usually lack control over the final NP size and dispersion [ 33 ]. Direct incorporation of metal ions via co-condensation routes affords uniform metal distribution; however, it also results in insufficient material use, since a large amount of metal is buried within the siliceous skeleton. On the other hand, silica functionalization with organic molecules able to entrap metal ions can yield easily accessible and finely dispersed metal species [ 33 ]. However, only limited work has been reported referring to the antibacterial properties of such systems. Specifically, Vallet-Regí and co-workers in their work describe a synthetic strategy for the design of a multicomponent antimicrobial nanosystem, combining the effect of an antibiotic molecule and of antimicrobial Zn 2+ or Ag + ions [ 34 ]. In this case, a third-generation polypropyleneimine dendrimer was employed for the external silica functionalization, enabling not only an efficient internalization in Gram-negative bacteria but also an effective decoration of the silica surface with Zn 2+ and Ag + ions through complexation with the tertiary amine groups. In another study, Kankala et al. developed an antibacterial nanocomposite by externally depositing a hyperbranched polyethyleneimine/Ag NPs complex layer on copper-doped mesoporous silica already loaded with tetracycline guest molecules [ 35 ]. Moreover, the group of Zhou has focused on the production of antibacterial nanomaterials through the modification of mesoporous silicas with specific organic moieties forming coordinate bonds with several metal ions, including Zn 2+ and Cu 2+ , affording at the end ZnO and/or CuO NPs either deposited or embedded into the siliceous matrix [36,37]. In this context, a novel facile dendritic polymer-templating strategy is proposed for the fabrication of ZnO NPs/mesoporous silica antibacterial nanocomposites. Hyperbranched polymers possess a key role in this strategy, serving as metal-trapping and -templating agents and hence enabling the geometric restriction of ZnO NPs within the siliceous mesoporous frameworks in an easy and precise manner. Specifically, by introducing these polymers into mesoporous silicas, abundant fixed-metal-anchoring sites, through metal– ligand complex formation, are furnished, controlling in this way the size, dispersion, and location of the generated ZnO NPs. In the past, our group has produced a number of novel nanoporous catalytic materials by harnessing hyperbranched polymers’ intriguing chemical and chelating properties [ 33 , 38 – 40 ]. Herein, this knowledge is transferred to the formulation of antibacterial nanocomposites and further enriched by investigating the effect of the siliceous hosts’ pore geometries in combination with the method of polymer’s loading and the type of its terminal functional groups, on the final ZnO NPs properties. For this purpose, four mesoporous silicas with different pore structural characteristics were surveyed as hosts, i.e., SBA-15, SBA-16, KIT-6, and MSU-X. SBA-15 possesses large, uniform mesopores arranged in a two-dimensional hexagonal symmetry with intra-wall micropores [ 41 ]. SBA-16 exhibits a three-dimensional body-centered cubic structure (Im3m) with interconnected cage-like mesopores, enhancing molecular accessibility and mass transport compared to the unidirectional channels of SBA-15 [ 42 ]. MSU-X displays a less ordered wormhole-like framework with uniform channels and thick pore walls, enhancing reactants diffusion rate and enabling synthesis under near-neutral pH conditions [ 43 ]. KIT6 exhibits a cubic Ia3d symmetry with interpenetrating bicontinuous channels, providing a highly open structure that facilitates transport of guest species without pore blockage [ 44 ]. In addition, two hyperbranched polymers were employed, specifically hyperbranched Nanomaterials 2025,15, 1697 4 of 35 polyethyleneimine (PEI) and a carboxy-methylated hyperbranched polyethyleneimine (Trilon-P, TrP), which possess different functional end groups, i.e., primary amines and carboxylate groups, respectively. Polymers were introduced into the siliceous framework either through the wetor dry-impregnation method. After thorough physicochemical characterization, the antibacterial activity of the resulting ZnO/SiO 2 nanocomposites was evaluated against the Gram-positive bacterium Staphylococcus aureus (S. aureus), while their cytotoxicity was assessed in mammalian cell lines. In addition, Life Cycle Assessment (LCA) methodology was employed as a robust tool to examine the environmental impacts associated with the synthesis of the most effective ZnO/SiO 2 nanocomposites, which demonstrated superior antibacterial performance. 2. Materials and Methods 2.1. Chemicals and Reagents Hyperbranched polyethyleneimine (PEI) of 5000 Da molecular weight (Lupasol ® WF, water-free, 99%) and carboxy-methylated polyethyleneimine (Trilon-P, TrP) were kindly donated by BASF (Ludwigshafen, Germany). Sodium trisilicate solution (Water glass), tetraethyl orthosilicate (TEOS) 98%, pluronic ® F-127, pluronic ® P-123, TWEEN ® 60, zinc nitrate hexahydrate ( ≥ 99%), HCl (37%), glutaraldehyde (solution, 25%), sodium cacodylate sodium chloride (NaCl), sodium hydroxide (NaOH), sodium fluoride (NaF), tryptic soy broth (TSB), and agar were purchased from Sigma-Aldrich (St. Louis, MA, USA). RPMI 1640 medium was purchased from Biowest (Nuaillé, France). Dulbecco’s phosphate-buffered saline (PBS), penicillin/streptomycin, trypsin/EDTA, and fetal bovine serum (FBS) were obtained from Biochrom GmbH (Berlin, Germany). Thiazolyl blue tetrazolium bromide (MTT) and isopropanol were purchased from Merck KGaA (Calbiochem ® , Darmstadt, Germany). Peptone from Casein was purchased from AppliChem GmbH (Darmstadt, Germany). 2.2. Synthesis of Mesoporous Silica Hosts The synthetic protocols employed for the mesoporous silica hosts’ production are based on hydrothermal routes with the aid of commercially available non-ionic triblock poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) copolymers as structure-directing agents (Pluronic P123 and F127), in a strong acidic or mild acidic environment. Specifically, the synthetic protocol selected for SBA-15, described in the work of Kosuge et al. [ 41 ], utilizes low-cost water-soluble sodium silicate as the silica source without hydrothermal treatment of the obtained solid. Therefore, this route offers many advantages, such as lower costs, shorter time, and simplicity in preparation, especially when intended for commercial applications. In brief, P123 (1.739 g) was dissolved in 68 mL of 2 M HCl (mixture A), and a dilute sodium silicate solution was prepared, consisting of 4 g sodium silicate solution ( ≈ 26.5 wt.%) and 12 g of deionized water (DW, mixture B). Mixture A was added to mixture B, with the temperature maintained at 30 ◦ C and stirring at 600 rpm applied only for 30 s. The reaction solution was subsequently kept under static conditions for 6 h. The obtained solid was filtrated and washed repeatedly with warm DW (~300 mL warmed at 30–35 ◦ C). After drying at room temperature for 2 days and then at 50 ◦ C for another 2 days, the product was calcined at 550 ◦ C for 6 h at a heating rate of 1 ◦C min−1. For the production of SBA-16, the synthetic procedure proposed by Grudzien et al. [ 42 ] was followed, involving the dissolution of Pluronic F127 (2 g) and sodium chloride (7.05 g) into a solution containing 20 mL of HCl (2 M) and 60 mL DW at 40 ◦ C. Then, TEOS (8.4 g) was added dropwise and left stirring for 20 h at 40 ◦ C. The produced sol was hydrothermally treated for 24 h at 100 ◦ C under static conditions. The precipitate was Nanomaterials 2025,15, 1697 5 of 35 filtered, washed with DW, dried in an oven at 80 ◦ C, and then calcined under the same conditions as previously mentioned in the case of SBA-15. KIT-6 was produced through the synthetic route reported by Kleitz et al. [ 44 ]. Typically, 1.94 g of P123 were dissolved in 70 mL of distilled water and 3.2 mL of HCl (37 wt.%). After complete dissolution, 2.4 mL of butanol was added. The mixture was left stirring at 35 ◦ C for 1 h, followed by the addition of TEOS (4.16 g). After another 24 h of stirring at 35 ◦ C, the mixture was hydrothermally treated at 100 ◦ C for 24 h under static conditions. The solid product was filtered without washing, dried at 100 ◦C, and calcined under the same conditions as in the case of SBA-15. A modified synthetic protocol based on the one described by Boissiere et al. [ 43 ] was adopted for the synthesis of MSU-X. First, an acidified aqueous solution of Tween 60 (0.02 M) was prepared with a proper amount of dilute HCl to adjust the pH to 2.3. Then, the solution was magnetically stirred at mild heating (45 ◦ C) until homogenization, followed by the addition of TEOS (molar Si/surfactant = 8). After about 2 h to allow for hydrolysis, sodium fluoride (molar NaF/Si = 0.04) was introduced to this solution to initiate silica condensation and left stirring for 30 min. Subsequently, the obtained mixture was placed in a thermostated water bath for 24 h at 45 ◦ C without any disturbance of the solution. Finally, the derived material was filtrated, dried, and calcined at 550 ◦ C for 6 h at a heating rate of 1◦C min−1. 2.3. Synthesis of ZnO/SiO2Nanocomposites For the production of the ZnO NPs/mesoporous silica nanocomposites, a dendritic polymer-templating route was employed, which comprises the following synthetic steps, as illustrated in Scheme 1. Scheme 1. Schematic illustration of ZnO/SiO 2 nanocomposites synthesis via a dendritic polymertemplating route. At first, organic modification of the different siliceous supports took place with the introduction of the hyperbranched polymers using wet or dry impregnation procedures. This modification is based on the polymers’ physical attachment onto the silica surface via electrostatic interactions and/or hydrogen bonding and thus requires only a 24 h outgassing of the powder as a pretreatment step so as to facilitate the polymer’s penetration Nanomaterials 2025,15, 1697 6 of 35 into the porous network. In the case of wet impregnation, 500 mg of silica powder was suspended in a polymeric aqueous solution (0.2 wt.%) and left stirring at room temperature (RT) for 24 h. In contrast, in the case of dry impregnation, a polymer solution of appropriate volume (depending on the pore volume of each silica) was added dropwise to the silica powder with simultaneous stirring until a homogeneous paste was formed. In both cases, given the high aqueous solubility of the hyperbranched polymers employed herein, the obtained polymer/silica materials were washed with DW to take off the free hyperbranched macromolecules accumulated mainly at the external SBA-15 surface. For metal sorption, 350 mg of the dried organically modified mesoporous silicas were dispersed in 100 mL of zinc nitrate hexahydrate aqueous solution (Zn(NO 3 ) 2· 6H 2 O) with an initial concentration (C 0 ) of 10 g/L and then stirred for 24 h at RT. Solution’s pH was adjusted around 5 to 5.5 in view of reaching the highest Zn adsorption capacity avoiding at the same time the precipitation of Zn in the form of hydroxides, Zn(OH)2. More precisely, at low pH values hyperbranched polymer’s protonation impedes metal binding, whereas at pH values approaching the precipitation limit, the formation of zinc oxo-species becomes unavoidable. Therefore, maintaining this specific pH range ensures a controlled ZnO growth mechanism mediated solely by the hyperbranched polymer. Following the 24 h sorption, solids were filtered, washed, dried, and calcined at 550 ◦ C for 5 h with a heating rate of 1 ◦ C min −1 under air flow for the decomposition of the hyperbranched polymers and the acquisition of the final ZnO-loaded silicas. 2.4. Materials Characterization Thermogravimetric analysis was carried out on a Setaram SETSYS Evolution 18 thermal analyzer (Setaram Instrumentation, Caluire, France), with a heating rate of 10 ◦ C min −1 , in an alumina crucible and by employing dried air as the carrier gas. FTIR spectroscopy was performed over the range of 4000–500 cm −1 using a Thermo Scientific Nicolet 6700 FTIR (Thermo Scientific, Waltham, MA, USA), equipped with a Specac Quest ATR (Attenuated Total Reflection) having a diamond crystal (Specac Ltd., Orpington, Kent, UK). For each sample, 128 scans were averaged at a resolution of 4 cm −1 , with all spectra being normalized against a single-beam spectrum of the clean ATR crystal and converted into absorbance units. Lowand wide-angle XRD diffraction patterns were acquired using a Rigaku rotating anode X-ray generator (operating at 50 kV, 100 mA, using Ni-filtered Cu Ka1 radiation) equipped with an R-AXIS IV image plate (Rigaku Co., Tokyo, Japan). All samples were mounted in Lindemann capillaries (Hilgenberg-Mark tubes of 0.7 mm inner diameter). N 2 adsorption measurements were performed at 77 K on an automated volumetric system (AUTOSORB-1-Krypton version—Quantachrome Instruments, Boynton Beach, FL, USA). Prior to measurements, samples were degassed at 250 ◦ C for 12 h. The specific surface area was determined using the Brunauer–Emmet–Teller method. The pore size distribution was calculated using the Nonlocal Density Functional Theory (NLDFT) model (Quantachrome Instruments, AS1Win software, Version 2.01, 2016). Data reduction parameters: Calc. Model: N 2 at 77 K on silica (cylindrical pore, NLDFT equilibrium model or adsorption branch). SEM analysis was conducted on a JEOL JSM 7401F Field Emission Scanning Electron Microscope (JEOL, Tokyo, Japan) equipped with a Gentle Beam mode operating at 2 kV acceleration voltage. The samples were placed on metallic (brass) substrates with double-coated carbon conductive tape. For the Si, Zn, and O elemental mapping analysis and determination of the final Zn loading, an EDS microanalysis was employed at a 20 kV acceleration voltage, utilizing an Xplore-15 SDD detector (Oxford Instruments, Abingdon, UK) with a 15 mm 2 surface area. TEM, HRTEM, STEM imaging, along with Si, Zn, and O elemental mapping distributions at the nanoscale, were obtained via a FEI Talos F200i field-emission (scanning) transmission electron microscope (Thermo Fisher Nanomaterials 2025,15, 1697 7 of 35 Scientific Inc., Waltham, MA, USA), operating at 200 kV. The microscope was equipped with a windowless energy-dispersive spectroscopy microanalyzer (6T/100 Bruker, Hamburg, Germany). TEM samples were prepared by dispersing the sample in ethanol and then drop-casting it onto a carbon-coated Cu grid. 2.5. Evaluation of Antibacterial Activity The antibacterial activity of Zn-loaded nanoporous silica materials was studied against Gram (+) Staphylococcus aureus (S. aureus strain ATCC 25923) following the CLSI guidelines (documents M07-A9 and M26-A) [ 45 , 46 ]. Bacteria were grown at 37 ◦ C in tryptic soy broth (TSB) for 16 h, in aerobic conditions, using a Stuart SI500 orbital shaker (~200 rpm shaking speed, Bibby Scientific Ltd., Staffordshire, UK). Then, the bacterial suspension was diluted with TSB to a concentration equal to 0.5 McFarland Standard (~10 8 CFU/mL) as confirmed by measuring their absorbance at 600 nm on a Cary 100 Conc UV–vis spectrophotometer (Varian Inc., Mulgrave, Australia). The resulting dispersion was one-fold-diluted and used for the subsequent tests. Minimum Inhibitory Concentration (MIC) determination: The broth macro-dilution method was employed to calculate the MIC values for all ZnO-loaded mesoporous silicas, following the standard CLSI M07-A9 protocol [ 46 ]. Specifically, dispersions of ZnO-loaded mesoporous silicas in TSB were prepared at various concentrations ranging from 25 to 1700 µ g/mL. Subsequently, 20 µ L of bacterial suspension containing ~10 −7 CFU/mL of S. aureus was inoculated into 2 mL of each dispersion of mesoporous silicas and incubated in a Stuart SI500 orbital shaker (~200 rpm rotation speed) at 37 ◦ C for 16 h. Untreated bacteria and bacteria-free growth media served as positive and negative controls, respectively. Following the incubation period, the MIC was defined as the lowest concentration at which no visible bacterial growth was detected. Minimum Bactericidal Concentration (MBC) determination: The colony-counting method was used to determine the MBC values for all ZnO-loaded mesoporous silicas, following the standard CLSI M26-A protocol [ 45 ]. In brief, 100 µ L of aliquots were taken from the tube at the MIC value and from tubes at three higher concentrations than MIC, one-fold-diluted, and plated on agar plates. After incubation at 37 ◦ C for 24 h, bacterial colonies (CFU/mL) were counted, and the reduction in bacterial growth was calculated for each sample. The MBC was defined as the lowest concentration that resulted in a 99.9% reduction in the initial bacterial inoculum. All experiments for MIC and MBC determination were conducted at least in triplicate to ensure reproducibility. Morphological analysis of bacteria: To determine the morphology of S. aureus bacteria after treatment with ZnO-loaded mesoporous silicas, Scanning Electron Microscopy (Jeol JSM 7401F Field Emission SEM, Jeol, Tokyo, Japan) was used. Briefly, bacteria were incubated with mesoporous silica materials at a concentration equal to MIC. After overnight incubation, bacteria were fixed with 3% glutaraldehyde in sodium cacodylate buffer (100 mM, pH = 7.1) for 12 h. Subsequently, the bacteria were collected by centrifugation as a pellet, washed, and resuspended in sodium cacodylate buffer. Subsequently, a 50 µ L aliquot of each suspension was placed on poly(L-lysine)-coated glass coverslips. Samples were dehydrated in graded ethanol (50%, 70%, 95%, 100%, 10 min each), air-dried, and coated with gold using a sputter coater prior to SEM imaging [47,48]. 2.6. Evaluation of In Vitro Cytotoxicity The cytotoxicity of ZnO-loaded mesoporous silicas was evaluated using the standard MTT assay. Human embryonic kidney cells (HEK293) and human prostate cancer cell lines (PC3) were cultured in an RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 µ g/mL). Cells were maintained at Nanomaterials 2025,15, 1697 8 of 35 37 ◦ C in a humidified atmosphere with 5% CO 2 and sub-cultured twice weekly using a trypsin (0.05% w/v)/EDTA (0.02% w/v) solution. For the MTT assay, 10 4 cells/well were seeded in 96-well plates and incubated in complete medium for 24 h. Following this pre-incubation period, cells were treated with ZnOloaded mesoporous silicas at MIC-related concentrations ranging from 50 to 500 µ g/mL (total material) for 24 h (the same exposure time used in the antibacterial tests). Subsequently, the medium was replaced with 100 µ L of MTT solution (10 µ g/mL in complete RPMI) and incubated for an additional 4 h under the same conditions. Formazan crystals formed during this period were dissolved in 100 µ L of 2-propanol per well, and absorbance was measured at 540 nm using an Infinite M200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Each concentration was tested in six replicates, and each experiment was repeated three times independently. Cell viability (%) was calculated relative to untreated controls (cells incubated in complete medium). Blank values from wells containing only 2-propanol were subtracted from all measurements. To evaluate the statistical significance of differences in cell viability, a two-tailed paired Student’s t-test was performed comparing treated samples to controls. Statistical significance was categorized as follows: p< 0.05 (*), p< 0.01 (**), p< 0.001 (***), p< 0.0001 (****) , and not significant (ns) for p> 0.05. 2.7. LCA of the ZnO/SiO2Nanocomposites Environmental impacts of ZnO/SiO 2 nanocomposites with superior antibacterial performance were evaluated using standardized Life Cycle Assessment (LCA). The methodology encompassed defining the goal and scope, compiling the life cycle inventory (LCI), performing the life cycle impact assessment, and interpreting the results, quantified through SimaPro 9.3 software. The functional unit refers to the production of 1 g of nanocomposite (total mass without considering the antibacterial activity), and the inventory data belongs to the laboratory-scale formulation (Table 1). The impact assessment method used was the Environmental Footprint 3.1 Method (EF3.1 adapted), as recommended by the European Commission (Commission Recommendations 2013/179/EU) [49]. Table 1. Functional unit and the systems introduced in SimaPro software. Functional Unit 1 g of Synthesized ZnO/SiO2 Method EF3.1 (adapted) Geography Europe Dataset Ecoinvent v3.11 Data type Primary data (experimental cases at laboratory scale under the scope of this work) 3. Results and Discussion 3.1. Characterization of the Pristine Mesoporous Silicas The pure siliceous materials employed as hosts for ZnO NPs were characterized using a combination of techniques, including lowand wide-angle XRD, nitrogen adsorption– desorption isotherms, and scanning electron microscopy (SEM). As derived by the lowangle XRD analysis (Figure S1A), SBA-15, SBA-16, and KIT-6 samples demonstrate order mesoporous structures of very high quality. Specifically, the low-angle XRD pattern of SBA15 material exhibits three diffractions corresponding to the (100), (110), and (200) planes of 2D hexagonal p6mm structure; in the case of SBA-16, the one major and the two less intensive peaks are indexed as (110), (200) and (211) reflections in the cubic space group (Im 3 m), Nanomaterials 2025,15, 1697 9 of 35 while in KIT-6 pattern all the characteristic reflections ascribed to the cubic Ia 3 dsymmetry could be identified [ 41 – 43 ]. In contrast, in the case of MSU-X, only a broad diffraction peak could be detected, indicative of the disordered worm-hole-like structure of MSU-X-type materials [ 44 ]. Wide-angle XRD diffractograms of all silica samples (Figure S1B) present only a single very broad low-intensity peak around 22 ◦ , denoting the amorphous nature of the pore walls. Additional information about the pore architecture was obtained by N 2 adsorption analysis. The derived N 2 adsorption–desorption isotherms together with the corresponding pore size distribution curves are shown in Figure S2, whereas N 2 sorption properties, such as specific surface area, total pore volume, and average pore size, are summarized in Table 2. All materials’ isotherm plots are of type IV, as defined by IUPAC classification, typical of mesoporous solids [ 50 ]. However, differences, particularly in the hysteresis loops, can be distinguished. N 2 sorption isotherms of SBA-15 and KIT-6 samples present a steep capillary condensation step and a H1 type hysteresis loop, revealing uniform mesochannels of cylindrical geometry (Figure S2A) [ 41 , 43 ]. This pore uniformity is further substantiated by the pore size distribution (PSD) analysis, calculated by applying the nonlocal density functional theory (NLDFT) equilibrium method on the desorption branch. As seen in Figure S2B , monomodal, narrow, and symmetrical PSD curves are obtained, located though in different pore diameters, larger for KIT-6 material at about 8.5 nm. In the case of the SBA16 sample, a broad H2 type hysteresis loop is observed with a delayed and steep desorption at ≈ 0.42 P/P 0 , related to ink-bottle or cage-like pore geometry ( Figure S2A ). This type of mesoporous material consists of cage-like mesopores interconnected by rather narrow pore entrances [ 42 ]. Pore size distribution (PSD) is estimated by applyingthe NLDFT method, using the model isotherm of N 2 adsorbed on silica with cylindrical pores acquired from the adsorption branch (Figure S2B). According to the obtained PSD, the SBA-16 material consists of uniform mesopore bodies/cages with a mean size around 6.5 nm and narrow pore apertures smaller than 2.5 nm. The MSU-X sample demonstrates a smaller, ill-defined hysteresis loop (Figure S2A), located at lower P/P 0 values, indicative of less uniform and smaller mesopores, also confirmed via the PSD analysis calculated by applying the NLDFT equilibrium method on the desorption branch (Figure S2B) [ 50 ]. As a result of the smaller pores, this sample possesses the largest specific surface area (SSA) value (1001 m 2 /g), while SBA-15, demonstrating the lower microporosity content, possesses the lowest specific surface area (SSA) value. Regarding the total pore volume (TPV) values, the KIT-6 sample with the largest pores presents the highest TPV value (1.16 cc/g). Table 2. Pore structural properties of pristine mesoporous silicas. Samples SSA (m2g−1) TPV (cc g−1) Average Pore Diameter (nm) SBA-15 546 0.49 3.6 KIT-6 876 1.16 5.3 SBA-16 871 0.53 2.4 MSU-X 1001 0.82 3.3 Morphological properties were investigated by means of scanning electron microscopy. SEM micrographs of the SBA-15 sample, depicted in Figure 1A,B, reveal a highly homogeneous morphology of loosely aggregated plate-like particles. At higher magnification, (Figure 1B), well-ordered channels of very short length, not exceeding 300 nm, and aligned along the thickness of the platelets could be distinguished. KIT-6 and SBA-16 samples consist of dense monolithic pieces with orderly arrayed mesopores, as detected Nanomaterials 2025,15, 1697 16 of 35 Figure 3. Low- (A,C,E) and wide-angle (B,D,F,G) XRD patterns of ZnO/SiO2nanocomposites. Nanomaterials 2025,15, 1697 17 of 35 Figure 4. N 2 -physisorption isotherms (A,C,E,G) and pore size distribution curves (B,D,F,H) of ZnO/SiO2nanocomposites. Nanomaterials 2025,15, 1697 18 of 35 Figure 5. STEM micrographs of (A) ZnO/SBA-16_PEI_WI, (B) ZnO/SBA-15_PEI_WI, (C) ZnO/SBA15_PEI_DI, and (D) ZnO/KIT-6_PEI_DI samples. In-depth insight into the ZnO particle size, spatial distribution, and dispersion state within the siliceous hosts was obtained using TEM, HAADF-STEM micrographs, selected area diffraction (SAED) patterns, and EDS mapping analysis. As shown in Figure 6, very small ZnO nanoparticles were uniformly dispersed within all silica matrices, confirming the stabilization effect imparted by the proposed methodology. This observation is further corroborated by the EDS mapping analysis, illustrated in Figure 7, revealing a very high compositional uniformity and homogeneous spatial distribution of Zn species at the nanoscale without the detection of Zn-rich nanodomains. Despite the excellent dispersion attained, ZnO NPs size and location differ amongst the samples, evidencing the significant role of the porous host. More precisely, in the ZnO/SBA-16_PEI_WI sample, ultra-small ZnO NPs with a very narrow distribution of 1 to 2 nm were predominantly deposited on the external amorphous silica particles. In contrast, the other samples exhibited a broader size distribution (1–10 nm). Specifically, for ZnO/SBA-15_PEI_WI and ZnO/KIT-6_PEI_DI, a significant fraction of the ZnO NPs was located within the mesoporous channels (Figure 6B,D) , whereas in ZnO/SBA-15_PEI_DI, the ZnO NPs were mainly situated at the pore entrances (Figure S37). Finally, the diffuse rings recorded in all the acquired SAED patterns (Figure 6, inserts) revealed the amorphous state of ZnO NPs, in accordance with XRD results. Nevertheless, lattice fringes could be detected in certain nanoparticles by TEM or HRTEM (Figure S37), indicating partial crystallization, which may have been induced by electron beam irradiation. Nanomaterials 2025,15, 1697 19 of 35 Figure 6. TEM and HAADF-STEM micrographs of (A) ZnO/SBA-16_PEI_WI, (B) ZnO/SBA15_PEI_WI, (C) ZnO/SBA-15_PEI_DI, and (D) ZnO/KIT-6_PEI_DI samples. Figure 7. HAADF-STEM micrographs and elemental mappings of (A) ZnO/SBA-16_PEI_WI, (B) ZnO/SBA-15_PEI_WI, (C) ZnO/SBA-15_PEI_DI, and (D) ZnO/KIT-6_PEI_DI samples. Nanomaterials 2025,15, 1697 20 of 35 3.4. Antibacterial Activity of ZnO/SiO2Nanocomposites The antibacterial activity of the as-prepared ZnO-loaded mesoporous silica nanocomposites was assessed against Staphylococcus aureus by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), with a critical focus on the effective Zn concentration ( µ g/mL Zn), not total composite mass. This Zn-based approach provides a more accurate assessment of antimicrobial efficiency, as it directly reflects the bioavailable antibacterial agent (ZnO) rather than inert support material. The MIC and MBC values were determined followed the Clinical and Laboratory Standards Institute (CLSI) guidelines M07-A9 and M26-A, utilizing the broth macro-dilution and colonycounting methods, respectively [45,46]. The results are summarized in Tables 5and 6. Table 5. MIC and MBC values of ZnO-loaded siliceous nanocomposites via PEI, employing either wet impregnation (WI) or dry impregnation (DI) against S. aureus bacteria. Both total concentrations and the corresponding Zn actual concentrations are shown for all nanocomposites. Samples MIC (µg/mL) MBC (µg/mL) ZnO/SBA-16_PEI_WI Total 50 1200 Zn content 0.9 21.6 ZnO/SBA-15_PEI_WI Total 100 1000 Zn content 2.6 26.0 ZnO/MSU-X_PEI_WI Total 100 600 Zn content 4.3 25.8 ZnO/KIT-6_PEI_WI Total 100 500 Zn content 5.9 29.5 ZnO/SBA-16_PEI_DI Total 50 1200 Zn content 0.65 15.6 ZnO/SBA-15_PEI_DI Total 50 900 Zn content 1.35 24.3 ZnO/MSU-X_PEI_DI Total 100 600 Zn content 4.2 25.3 ZnO/KIT-6_PEI_DI Total 200 500 Zn content 16.2 40.5 Table 6. MIC and MBC values of ZnO-loaded siliceous nanocomposites via Trilon-P (TrP), employing either wet impregnation (WI) or dry impregnation (DI) against S. aureus bacteria. Both total concentrations and the corresponding Zn actual concentrations are shown for all nanocomposites. Samples MIC (µg/mL) MBC (µg/mL) ZnO/SBA-16_TrP_WI Total 400 1500 Zn content 3.0 11.25 ZnO/SBA-15_TrP_WI Total 400 1200 Zn content 3.6 10.8 ZnO/MSU-X_TrP_WI Total 200 500 Zn content 2.0 5.0 ZnO/KIT-6_TrP_WI Total 200 600 Zn content 3.6 10.8 Nanomaterials 2025,15, 1697 21 of 35 Table 6. Cont. Samples MIC (µg/mL) MBC (µg/mL) ZnO/SBA-15_TrP_DI Total 300 500 Zn content 3.0 5.0 ZnO/KIT-6_TrP_DI Total 400 900 Zn content 3.6 8.1 Among all ZnO-loaded mesoporous silica nanocomposites, those employing PEI generally exhibited the lowest MIC and MBC values based on Zn concentrations ( Table 5 ). This superior performance is directly related to the effective ZnO loading and the formation of homogeneously distributed ultrasmall ZnO nanoparticles. Specifically, ZnO/SBA16_PEI_WI and ZnO/SBA-16_PEI_DI achieved the lowest MIC (0.9 and 0.65 µ g/mL Zn, respectively) and MBC (21.6 and 15.6 µ g/mL Zn, respectively) values. Despite their modest Zn loading (1.3–1.8 wt.%), their antibacterial activity is remarkable, likely due to the formation of 1–2 nm ZnO NPs deposited on the amorphous silica phase, as well as the presence of highly accessible external ZnO sites and uniform nanoparticle distribution without aggregation, which enhance surface interactions and possibly Zn 2+ ion release. In case of ZnO/SBA-15_PEI nanocomposites, antibacterial activity was almost comparable to that of ZnO/SBA-16_PEI nanocomposites. Specifically, the sample prepared via the DI method demonstrated slightly stronger antibacterial efficiency (MIC: 1.35 µ g/mL Zn, MBC: 24.3 µ g/mL Zn) than its WI analog (MIC: 2.6 µ g/mL Zn, MBC: 26 µ g/mL Zn), despite their similar Zn content (2.7 wt.% DI vs. 2.6 wt.% WI). This performance is likely due to the DI method concentrating ZnO nanoparticles near pore entrances, thereby increasing Zn availability and therefore enhancing surface interactions and possibly Zn 2+ ion release. For ZnO/MSU-X_PEI nanocomposites, both samples exhibited similar antibacterial performance (MIC: ~4 µ g/mL Zn, MBC: ~25 µ g/mL Zn), which was slightly lower than that of ZnO/SBA-15_PEI and ZnO/SBA-16_PEI nanocomposites. Interestingly, ZnO/KIT-6_PEI nanocomposites, despite having the highest Zn content coupled with an excellent dispersion (ZnO/KIT-6_PEI_WI 5.9 wt.%, ZnO/KIT-6_PEI_DI 8.1 wt.%), showed the lowest antibacterial efficacy (MIC values 5.9 and 16.2 µ g/mL Zn for ZnO/KIT-6_PEI_WI and ZnO/KIT-6_PEI_DI, respectively, and MBC values of 29.5 and 40.5 µ g/mL Zn content for ZnO/KIT-6_PEI_WI and ZnO/KIT-6_PEI_DI, respectively). As previously reported (Section 3.3), ZnO NPs in these samples are predominantly confined within the siliceous hosts, thereby limiting their accessibility to bacterial cells as well as potentially retarding the release of Zn 2+ ions from the siliceous network. Although this uniform dispersion of ZnO NPs within the porous network seems to adversely affect their antibacterial activity, it could be proven highly beneficial, especially at such high metal loading, enhancing materials’ potential for other applications, such as catalysis. On the other hand, ZnO/MSU-X_TrP_WI (MIC: 2 µ g/mL Zn, MBC: 5 µ g/mL Zn) and ZnO/SBA-15_TrP_DI (MIC: 3 µ g/mL Zn, MBC: 5 µ g/mL Zn) exhibited the strongest antibacterial activity among the ZnO-loaded siliceous nanocomposites via Trilon-P, as indicated by their lower MIC and MBC values (Table 6). They were followed in effectiveness by ZnO/KIT-6_TrP_DI, ZnO/KIT-6_TrP_WI, ZnO/SBA-15_TrP_WI, and ZnO/SBA-16_TrP_WI. It should be noted that pure mesoporous silicas, such as SBA-15, SBA-16, MSU-X, and KIT-6, are well recognized as biocompatible materials lacking intrinsic antibacterial activity [ 52 – 54 ]. Antimicrobial properties arise only upon suitable surface functionalization [ 55 ] or incorporation of active nanoparticles, such as ZnO NPs [ 36 , 37 , 54 ] or active compounds [ 56 – 58 ]. On the other hand, it is well established that ZnO NPs exhibited strong antibacterial properties. The MIC values reported for bare ZnO NPs vary considerably, Nanomaterials 2025,15, 1697 22 of 35 typically ranging from 10 to 1000 µ g/mL, depending on particle size, morphology, surface area-to-volume ratio, synthesis route, and the bacterial strain tested [13,17,21,48,59–62] . For example, in our previous publication, ZnO NPs with a polyhedral plate-like morphology and an average particle size of 28 nm exhibited pronounced antibacterial activity against the same S. aureus strain, with MIC and MBC values of 20 and 50 µ g/mL, respectively [ 48 ]. In another study, spherical ZnO nanoparticles with an average diameter of ~7 nm exhibited inhibitory activity against S. aureus at a concentration of 250 µ g/mL, whereas larger particles (~150 nm) required concentrations up to 1000 µ g/mL to achieve comparable antibacterial effects [ 19 ]. Immobilization of ZnO NPs on silica supports has been reported to enhance antibacterial performance [ 27 , 36 , 37 ]. Shehata et al. reported that silver/zinc-incorporated mesoporous silica nanoparticles (MCM-48) exhibited strong antibacterial activity against S. aureus,E. coli, and methicillin-resistant S. aureus (MRSA), with MICs ranging from 7.8 to 62.5 µ g/mL [ 63 ]. Likewise, ZnO nanoparticles supported on mesoporous silica SBA-3 inhibited the growth of E. coli and S. aureus, displaying MIC values of 1.24 and 0.31 mg/mL, respectively [ 36 ]. More recently, Trinh et al. demonstrated that Zn-loaded mesoporous silicas (SBA-1 and SBA-15) showed pronounced antibacterial activity against E. coli and Bacillus subtilis at a concentration of 20 µ g/mL [ 54 ]. Notably, in the present study, superior antibacterial activity was achieved, with comparable inhibition reached at substantially lower concentrations (MICs 1–2.5 µ g/mL Zn content). Specifically, the optimum antibacterial performance was achieved by ZnO NPs loaded on SBA-15 and SBA-16 via PEI regardless of the loading method as well as by ZnO NPs loaded on MSU-X via TrP (MICs 1–2.5 µ g/mL Zn). These findings underscore the key role of the siliceous host properties, particularly morphology and pore architecture. In SBA-16 and MSU-X samples, ZnO NPs are finely dispersed either on or near the external siliceous surface as a result of the narrow pore entrances in the former and the small-sized mesopores in the latter, which impede the penetration of the larger Trilon-P molecule. These features probably enhance the interactions of ZnO NPs with the bacterial cell envelope and promote the release of Zn 2+ ions. In SBA-15, the plate-like morphology generates short mesochannels with abundant pore openings, yielding a high population of exposed ZnO NPs. In contrast, the open three-dimensional pore network of KIT-6 facilitates the confinement of ZnO NPs, but in combination with its dense monolithic morphology, this restricts NPs accessibility and thereby limits their interaction with the bacteria and probably retards the release of Zn 2+ ions. Thus, bioactivity appears to depend more on nanoparticle accessibility, dispersion, and location than on total Zn loading, highlighting the importance of balancing ZnO content with suitable pore structure and diffusion efficiency. The morphology of S. aureus after treatment was examined by scanning electron microscopy (SEM). Bacterial cells were exposed to the most active ZnO/mesoporous silica nanocomposites at MIC for 12 h, and changes in the surface morphology and membrane structure were evaluated. Figure 8shows SEM images of untreated cells (control) and those treated with the nanocomposites. The untreated bacteria (Figure 8A) display a typical spherical morphology with intact cell walls, whereas cells treated with all ZnO-loaded nanocomposites (Figure 8B–F) exhibit pronounced morphological alterations. Treated cells appear wrinkled and structurally damaged, with evidence of intracellular content leakage, deformation, and extensive cell death. These observations are consistent with previous reports, which describe ZnO NPs as initially disrupting bacterial membranes and cell walls, followed by leakage of intracellular components and eventual cell death. The antibacterial mechanism of ZnO nanoparticles (ZnO NPs) is not yet fully understood [64–66] , although several complementary pathways have been proposed. These include (a) direct interaction of ZnO NPs with bacteria cell walls, leading to membrane disruption and subsequent internalization either through the cell envelope or via endocytic-like processes; (b) gen- Nanomaterials 2025,15, 1697 23 of 35 eration of reactive oxygen species (ROS), which induce oxidative stress and inhibit DNA replication and protein synthesis [ 64 ]; and (c) release of Zn 2+ ions upon ZnO dissolution, leading to membrane integrity loss, collapse of the proton motive force, and blockage of ion channels [ 67 ]. In a related study, Trinh et al. demonstrated that the antibacterial activity of Zn-loaded mesoporous silicas (SBA-1 and SBA-15) against E. coli and Bacillus subtilis was primarily attributed to their strong interaction with bacterial cell walls and the sustained release of Zn 2+ ions [ 54 ]. In line with these findings, the ZnO/mesoporous silica nanocomposites investigated in this study likely exert their antibacterial action through a synergistic interplay of physical and chemical mechanisms, involving interaction with the bacterial cell envelope, release of Zn 2+ ions, and ROS generation, ultimately leading to membrane destabilization, impairment of metabolic and enzymatic functions, and bacterial cell death. 3.5. In Vitro Cytotoxicity Evaluation of ZnO/SiO2Nanocomposites The widespread application of ZnO NPs in the pharmaceutical, cosmetic, textile, and agricultural sectors has raised increasing concerns regarding their potential toxicological impacts on both the environment and human health. ZnO NPs are known to induce cytotoxicity primarily through the release of Zn 2+ ions and the generation of reactive oxygen species (ROS), which together trigger oxidative stress, mitochondrial dysfunction, DNA damage, and ultimately cell death [ 68 , 69 ]. Among these mechanisms, Zn 2+ dissolution is generally regarded as the predominant contributor to cytotoxicity, while intracellular ROS formation amplifies the oxidative imbalance and accelerates apoptotic pathways [ 70 , 71 ]. The magnitude of these effects is strongly influenced by the physicochemical characteristics of the nanoparticles—particularly their size, morphology, and surface chemistry [ 19 , 68 ]. Smaller (<50 nm) and anisotropic ZnO nanostructures, such as nanorods, display enhanced reactivity and faster Zn 2+ release due to their high surface-to-volume ratio, resulting in stronger cytotoxic responses compared with spherical or microscale particles [19]. In view of these findings, systematic evaluation of the cytotoxicity of ZnO-based materials is essential prior to their practical use. In the present study, the cytotoxic effects of the as-prepared ZnO-loaded mesoporous silica nanocomposites were investigated at MICrelated concentrations. Specifically, the in vitro cytotoxicity was assessed using HEK293 (normal human embryonic kidney) and PC3 (prostate cancer) cell lines via the MTT assay following 24 h of exposure. In the case of ZnO NPs loaded on mesoporous silicas via PEI, cell viability decreased in a dose-dependent manner (Figures 9and S38). Specifically, both HEK293 and PC3 cells treated with ZnO NPs loaded on SBA-15, SBA-16, or MSU-X via PEI using WI-loading method, maintained relatively high viability (>80%) at concentration up to 300 µ g/mL (corresponding to 7.8, 5.4, and 12.9 µ g/mL Zn content, respectively), while at 500 µ g/mL (corresponding to 13, 9, and 21.5 µ g/mL Zn content, respectively), cell viability was slightly reduced to 60–70%. On the other hand, the DI-prepared counterparts exhibited similar cytotoxicity towards HEK293 cells, whereas in PC3 cells, these nanocomposites were found to be less toxic (cell viability: >75% at 500 µ g/mL, corresponding to 6.5–21 µg/mL Zn content). It should be noted that all these nanocomposites, regardless of the loading method, were non-toxic to both tested cell lines at MIC-related concentrations (cell viability ≥ 90% at their MIC; Figures 9and S38). Among them, ZnO/SBA-16_PEI_WI and ZnO/SBA-15_PEI_DI exhibited negligible toxicity, showing nearly 100% cell viability at their MIC (50 µ g/mL, corresponding to 0.9 and 1.35 µ g/mL Zn content, respectively; Table 5and Figures 9and S38 ). ZnO NPs loaded on KIT-6 via PEI, regardless of the loading method, were non-toxic only at low concentrations, close to their MIC (survival: >80% at 100–200 µg/mL , corresponding to 5.9–16.2 µ g/mL Zn content). However, at higher concentrations, HEK293 cell viability was further reduced, dropping below 60%, while PC3 cells retained slightly higher viability at Nanomaterials 2025,15, 1697 24 of 35 equivalent doses. This higher toxicity of both ZnO/KIT-6_PEI nanocomposites compared to the others may be attributed to their higher Zn content (ZnO/KIT-6_PEI_WI 5.9 wt.%, ZnO/KIT-6_PEI_DI 8.1 wt.%). Figure 8. SEM images of S. aureus bacteria: (A) untreated cells (control) and cells after treatment with (B) ZnO/SBA-16_PEI_WI, (C) ZnO/SBA-15_PEI_WI, (D) ZnO/SBA-16_PEI_DI, (E) ZnO/SBA15_PEI_DI, and (F) ZnO/MSU-X_TrP_WI for 12 h at their MIC. The scale bar in each micrograph corresponds to 1 µm. Nanomaterials 2025,15, 1697 25 of 35 0 20 40 60 80 100 120 140 ZnO/SBA-16_PEI_WI ZnO/SBA-15_PEI_WI ZnO/MSU-X_PEI_WI ZnO/KIT-6_PEI_WI A * * * * * Concentration (μg/mL) % Cell viabillity control 50 100 200 300 500 * * * * * * * *** ** * * * * * * * * * * * * * * 0 20 40 60 80 100 120 ZnO/SBA-16_PEI_WI ZnO/SBA-15_PEI_WI ZnO/MSU-X_PEI_WI ZnO/KIT-6_PEI_WI * * Concentration (μg/mL) % Cell viabillity control 50 100 200 300 500 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * B 0 20 40 60 80 100 120 140 * Concentration (μg/mL) % Cell viabillity ZnO/SBA-16_PEI_DI ZnO/SBA-15_PEI_DI ZnO/MSU-X_PEI_DI ZnO/KIT-6_PEI_DI control 50 100 200 300 500 * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * C 0 20 40 60 80 100 120 ZnO/SBA-16_PEI_DI ZnO/SBA-15_PEI_DI ZnO/MSU-X_PEI_DI ZnO/KIT-6_PEI_DI Concentration (μg/mL) % Cell viabillity control 50 100 200 300 500 * * * * * * * * * * * * * ** ** * * * * * * * * * * * * * * * * * * * * * * * * * * * D Figure 9. Comparative cytotoxicity of ZnO-loaded mesoporous silica nanocomposites via PEI, using either wet impregnation (WI) or dry impregnation (DI), on HEK293 (A,C) and PC3 (B,D) cells, assessed by MTT assay after 24 h incubation. Results are presented as mean ± SD from six values obtained in at least three independent experiments. Statistical significance (Student’s paired twotailed t-test) is denoted as * p< 0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001; absence of annotation indicates no significance (p> 0.05). For ZnO NPs loaded on mesoporous silicas via TrP (Figures 10 and S39), cytotoxic effects were generally milder in both cell lines compared to PEI analogs. Particularly, the WIprepared nanocomposites induced only moderate reductions in PC3 cell viability (75–80% at 500 µ g/mL). In the case of HEK293, ZnO/SBA-16_PEI_WI and ZnO/MSU-X_PEI_WI retained higher viability (>70% at 500 µ g/mL) compared to the other two nanocomposites. On the other hand, the DI-prepared nanocomposites were essentially non-toxic to HEK293 cells even at the higher tested concentration, whereas in PC3 cells, they demonstrated slightly stronger cytotoxic effects, with viability dropping to ~60% at 500 µ g/mL. However, these nanocomposites exhibited a slightly toxicity in both tested cell lines at their MIC (cell survival: 70–90%), except ZnO/MSU-X_TrP_WI, which did not exhibit any toxicity at its MIC (cell viability 85–90% at 200 µ g/mL, corresponding to 2 µ g/mL Zn content; Table 6 and Figures 10 and S39). Taken together, the results demonstrate that the cytotoxicity of ZnO/mesoporous silica nanocomposites is mainly influenced by the type of mesoporous silica host, while the loading method and polymer type exert comparatively minor effects. Correlation of the antibacterial and cytotoxicity data further reveals that the nanocomposites exhibiting the most effective antibacterial performance—namely, ZnO NPs loaded on SBA-15 and SBA-16 via PEI (both WI and DI) as well as ZnO NPs loaded on MSU-X via TrP employing WI—did not display significant cytotoxicity (cell survival > 90%) at MIC-related concentrations (50–100 µg/mL corresponding to 0.65–2 µg/mL Zn content). Nanomaterials 2025,15, 1697 32 of 35 PSD Pore Size Distribution ROS Reactive Oxygen Species RT Room Temperature S. aureus Staphylococcus aureus SBA-15 Santa Barbara Amorphous-15 SBA-16 Santa Barbara Amorphous-16 SEM Scanning Electron Microscopy SiO2Silicon Dioxide SSA Specific Surface Area STEM Scanning Transmission Electron Microscopy TGA Thermogravimetric Analysis TEM Transmission Electron Microscopy TEOS Tetraethyl Orthosilicate TPV Total Pore Volume TSB Tryptic soy broth TrP Carboxy-Methylated Hyperbranched Polyethyleneimine (Trilon-P) WI Wet Impregnation UV–vis Ultraviolet-visible spectroscopy XRD X-ray Diffraction ZnO Zinc Oxide References 1. Okeke, I.N.; De Kraker, M.E.A.; Van Boeckel, T.P.; Kumar, C.K.; Schmitt, H.; Gales, A.C.; Bertagnolio, S.; Sharland, M.; Laxminarayan, R. The scope of the antimicrobial resistance challenge. Lancet 2024,403, 2426–2438. [CrossRef] [PubMed] 2. de Kraker, M.E.A.; Stewardson, A.J.; Harbarth, S. Will 10 million people die a year due to antimicrobial resistance by 2050? PLoS Med. 2016,13, e1002184. [CrossRef] [PubMed] 3. Prestinaci, F.; Pezzotti, P.; Pantosti, A. Antimicrobial resistance: A global multifaceted phenomenon. Pathog. Glob. Health 2015, 109, 309–318. [CrossRef] 4. Tsikourkitoudi, V.; Henriques-Normark, B.; Sotiriou, G.A. Inorganic nanoparticle engineering against bacterial infections. Curr. Opin. Chem. Eng. 2022,38, 100872. [CrossRef] 5. Lushniak, B.D. Antibiotic resistance: A public health crisis. Public Health Rep. 2014,129, 314–316. [CrossRef] 6. Paladini, F.; D’Urso, F.; Broccolo, F.; Pollini, M. Combating Healthcare-Associated Infections in Modern Hospitals: Nanotechnology-Based Approaches in the Era of Antimicrobial Resistance. Nanomaterials 2025,15, 1405. [CrossRef] 7. Mubeen, B.; Ansar, A.N.; Rasool, R.; Ullah, I.; Imam, S.S.; Alshehri, S.; Ghoneim, M.M.; Alzarea, S.I.; Nadeem, M.S.; Kazmi, I. Nanotechnology as a Novel Approach in Combating Microbes Providing an Alternative to Antibiotics. Antibiotics 2021,10, 1473. [CrossRef] [PubMed] 8. Wang, L.; Hu, C.; Shao, L. The antimicrobial activity of nanoparticles: Present situation and prospects for the future. Int. J. Nanomed. 2017,12, 1227–1249. [CrossRef] 9. Jiang, S.; Lin, K.; Cai, M. ZnO nanomaterials: Current advancements in antibacterial mechanisms and applications. Front. Chem. 2020,8, 580. [CrossRef] 10. Kumar, R.; Umar, A.; Kumar, G.; Nalwa, H.S. Antimicrobial properties of ZnO nanomaterials: A review. Ceram. Int. 2016,43, 3940–3961. [CrossRef] 11. Laurenti, M.; Grochowicz, M.; Dragoni, E.; Carofiglio, M.; Limongi, T.; Cauda, V. Biodegradable and drug-eluting inorganic composites based on mesoporous zinc oxide for urinary stent applications. Materials 2020,13, 3821. [CrossRef] [PubMed] 12. Sánchez-López, E.; Gomes, D.; Esteruelas, G.; Bonilla, L.; Lopez-Machado, A.L.; Galindo, R.; Cano, A.; Espina, M.; Ettcheto, M.; Camins, A.; et al. Metal-based nanoparticles as antimicrobial agents: An overview. Nanomaterials 2020,10, 292. [CrossRef] [PubMed] 13. Raha, S.; Ahmaruzzaman, M. ZnO nanostructured materials and their potential applications: Progress, challenges and perspectives. Nanoscale Adv. 2022,4, 1868–1925. [CrossRef] [PubMed] 14. Pal, P.; Pareek, A. Zinc oxide nanoparticles: A comprehensive review on its synthesis, characterization, and role in biomedical applications as well as health risks. Inorg. Chem. Commun. 2025,181, 115314. [CrossRef] 15. Franklin, N.M.; Rogers, N.J.; Apte, S.C.; Batley, G.E.; Gadd, G.E.; Casey, P.S. Comparative Toxicity of Nanoparticulate ZnO, Bulk ZnO, and ZnCl 2 to a freshwater Microalga (Pseudokirchneriella subcapitata): The importance of particle solubility. Environ. Sci. Technol. 2007,41, 8484–8490. [CrossRef] Nanomaterials 2025,15, 1697 33 of 35 16. Bathi, J.R.; Wright, L.; Khan, E. Critical Review of Engineered Nanoparticles: Environmental Concentrations and Toxicity. Curr. Pollut. Rep. 2022,8, 498–518. [CrossRef] 17. Sirelkhatim, A.; Mahmud, S.; Seeni, A.; Kaus, N.H.M.; Ann, L.C.; Bakhori, S.K.M.; Hasan, H.; Mohamad, D. Review on Zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Lett. 2015,7, 219–242. [CrossRef] 18. Raghupathi, K.R.; Koodali, R.T.; Manna, A.C. Size-Dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir 2011,27, 4020–4028. [CrossRef] 19. Babayevska, N.; Przysiecka, Ł.; Iatsunskyi, I.; Nowaczyk, G.; Jarek, M.; Janiszewska, E.; Jurga, S. ZnO size and shape effect on antibacterial activity and cytotoxicity profile. Sci. Rep. 2022,12, 8148. [CrossRef] 20. Zhu, X.; Wang, J.; Cai, L.; Wu, Y.; Ji, M.; Jiang, H.; Chen, J. Dissection of the antibacterial mechanism of zinc oxide nanoparticles with manipulable nanoscale morphologies. J. Hazard. Mater. 2022,430, 128436. [CrossRef] 21. Applerot, G.; Lipovsky, A.; Dror, R.; Perkas, N.; Nitzan, Y.; Lubart, R.; Gedanken, A. Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS--mediated cell injury. Adv. Funct. Mater. 2009,19, 842–852. [CrossRef] 22. Donnadio, A.; Cardinali, G.; Latterini, L.; Roscini, L.; Ambrogi, V. Nanostructured zinc oxide on silica surface: Preparation, physicochemical characterization and antimicrobial activity. Mater. Sci. Eng. C 2019,104, 109977. [CrossRef] 23. Niño-Martínez, N.; Orozco, M.F.S.; Martínez-Castañón, G.-A.; Méndez, F.T.; Ruiz, F. Molecular mechanisms of bacterial resistance to metal and metal oxide nanoparticles. Int. J. Mol. Sci. 2019,20, 2808. [CrossRef] 24. Tang, S.; Wang, J.; Zhu, X.; Shen, D. Ecological risks of zinc oxide nanoparticles for early life stages of obscure puffer (Takifugu obscurus). Toxics 2024,12, 48. [CrossRef] [PubMed] 25. Bai, X.; Lin, C.; Wang, Y.; Ma, J.; Wang, X.; Yao, X.; Tang, B. Preparation of Zn doped mesoporous silica nanoparticles (Zn-MSNs) for the improvement of mechanical and antibacterial properties of dental resin composites. Dent. Mater. 2020,36, 794–807. [CrossRef] 26. Hou, Y.-X.; Abdullah, H.; Kuo, D.-H.; Leu, S.-J.; Gultom, N.S.; Su, C.-H. A comparison study of SiO 2 /nano metal oxide composite sphere for antibacterial application. Compos. B Eng. 2018,133, 166–176. [CrossRef] 27. Krakor, E.; Saniternik, S.; Gessner, I.; Frohnhoven, R.; Wilhelm, M.; Drexelius, M.; Tosun, N.; Neundorf, I.; Mathur, S. Hollow mesoporous silica capsules loaded with copper, silver, and zinc oxide nanoclusters for sustained antibacterial efficacy. J. Am. Ceram. Soc. 2022,105, 1685–1696. [CrossRef] 28. Colilla, M.; Vallet-Regí, M. Organically modified mesoporous silica nanoparticles against bacterial resistance. Chem. Mater. 2023, 35, 8788–8805. [CrossRef] 29. González, B.; Colilla, M.; Díez, J.; Pedraza, D.; Guembe, M.; Izquierdo-Barba, I.; Vallet-Regí, M. Mesoporous silica nanoparticles decorated with polycationic dendrimers for infection treatment. Acta Biomater. 2018,68, 261–271. [CrossRef] 30. Zhuang, J.; Yu, Y.; Lu, R. Mesoporous silica nanoparticles as carrier to overcome bacterial drug resistant barriers. Int. J. Pharm. 2023,631, 122529. [CrossRef] 31. Colilla, M.; Vallet-Regí, M. Targeted stimuli-responsive mesoporous silica nanoparticles for bacterial infection treatment. Int. J. Mol. Sci. 2020,21, 8605. [CrossRef] 32. Kankala, R.K.; Han, Y.-H.; Na, J.; Lee, C.-H.; Sun, Z.; Wang, S.; Kimura, T.; Ok, Y.S.; Yamauchi, Y.; Chen, A.-Z.; et al. Nanoarchitectured structure and surface biofunctionality of mesoporous silica nanoparticles. Adv. Mater. 2020,32, 1907035. [CrossRef] 33. Deze, E.G.; Papavasiliou, A.; Papageorgiou, S.K.; Katsaros, F.K.; Kouvelos, E.P.; Romanos, G.E.; Boukos, N.; Xin, Q.; Nyalosaso, J.L.; Cool, P. Metal loaded nanoporous silicas with tailor-made properties through hyperbranched polymer assisted templating approaches. Micropor. Mesopor. Mat. 2016,235, 107–119. [CrossRef] 34. Álvarez, E.; Estévez, M.; Jiménez-Jiménez, C.; Colilla, M.; Izquierdo-Barba, I.; González, B.; Vallet-Regí, M. A versatile multicomponent mesoporous silica nanosystem with dual antimicrobial and osteogenic effects. Acta Biomater. 2021,136, 570–581. [CrossRef] 35. Kankala, R.K.; Lin, W.-Z.; Lee, C.-H. Combating antibiotic resistance through the synergistic effects of mesoporous silica-based hierarchical nanocomposites. Nanomaterials 2020,10, 597. [CrossRef] 36. Qiu, S.; Zhou, H.; Shen, Z.; Hao, L.; Chen, H.; Zhou, X. Synthesis, characterization, and comparison of antibacterial effects and elucidating the mechanism of ZnO, CuO and CuZnO nanoparticles supported on mesoporous silica SBA-3. RSC Adv. 2020,10, 2767–2785. [CrossRef] [PubMed] 37. Wen, H.; Zhou, X.; Shen, Z.; Peng, Z.; Chen, H.; Hao, L.; Zhou, H. Synthesis of ZnO nanoparticles supported on mesoporous SBA-15 with coordination effect -assist for anti-bacterial assessment. Colloids Surf. B Biointerfaces 2019,181, 285–294. [CrossRef] [PubMed] 38. Papavasiliou, A.; Tsiourvas, D.; Deze, E.G.; Papageorgiou, S.K.; Katsaros, F.K.; Poulakis, E.; Philippopoulos, C.J.; Boukos, N.; Xin, Q.; Cool, P. Hyperbranched polyethyleneimine towards the development of homogeneous and highly porous CuO–CeO 2 –SiO 2 catalytic materials. Chem. Eng. J. 2016,300, 343–357. [CrossRef] Nanomaterials 2025,15, 1697 34 of 35 39. Tsiourvas, D.; Papavasiliou, A.; Deze, E.; Papageorgiou, S.; Katsaros, F.; Romanos, G.; Poulakis, E.; Philippopoulos, C.; Xin, Q.; Cool, P. A green route to copper loaded silica nanoparticles using hyperbranched Poly(Ethylene imine) as a biomimetic template: Application in heterogeneous catalysis. Catalysts 2017,7, 390. [CrossRef] 40. Papavasiliou, A.; Deze, E.G.; Papageorgiou, S.K.; Sideratou, Z.; Boukos, N.; Poulakis, E.; Philippopoulos, C.J.; Glisenti, A.; Van Everbroeck, T.; Cool, P.; et al. A hyperbranched polymer synthetic strategy for the efficient fixation of metal species within nanoporous structures: Application in automotive catalysis. Chem. Eng. J. 2021,421, 129496. [CrossRef] 41. Kosuge, K.; Sato, T.; Kikukawa, N.; Takemori, M. Morphological control of rodand fiberlike SBA-15 type mesoporous silica using water-soluble sodium silicate. Chem. Mater. 2004,16, 899–905. [CrossRef] 42. Grudzien, R.M.; Grabicka, B.E.; Jaroniec, M. Adsorption studies of thermal stability of SBA-16 mesoporous silicas. Appl. Surf. Sci. 2007,253, 5660–5665. [CrossRef] 43. Boissière, C.; Larbot, A.; Van Der Lee, A.; Kooyman, P.J.; Prouzet, E. A new synthesis of mesoporous MSU-X silica controlled by a two-step pathway. Chem. Mater. 2000,12, 2902–2913. [CrossRef] 44. Kleitz, F.; Choi, S.H.; Ryoo, R. Cubic Ia3d large mesoporous silica: Synthesis and replication to platinum nanowires, carbon nanorods and carbon nanotubes. Chem. Commun. 2003,17, 2136–2137. [CrossRef] 45. CLSI M26; Methods for Determining Bactericidal Activity of Antimicrobial Agents. Clinical and Laboratory Standards Institute (CLSI): Malvern, PA, USA, 1999. 46. CLSI M07; Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Clinical and Laboratory Standards Institute (CLSI): Malvern, PA, USA, 2012. 47. Panagiotaki, K.N.; Lyra, K.-M.; Papavasiliou, A.; Stamatakis, K.; Sideratou, Z. Synthesis of N--sulfopropylated hyperbranched polyethyleneimine with enhanced biocompatibility and antimicrobial activity. ChemPlusChem 2025,90, e202400454. [CrossRef] 48. Piffet, C.; Thomassin, J.-M.; Stierlin, E.; Tchoumtchoua, J.; Fernández, C.; Mateo, M.; Hernández, L.; Lyra, K.M.; Papavasiliou, A.; Sakellis, E.; et al. Sustainable antibacterial chitin nanofiber/ZnO nanohybrid materials: Ex situ and in situ synthesis, characterization and evaluation. Nanomaterials 2025,15, 809. [CrossRef] 49. EC-European Commission. Commission Recommendation 2013/179/EU of 9 April 2013 on the use of common methods to measure and communicate the life cycle environmental performance of products and organisations. ANNEX II: Product Environmental Footprint (PEF) Guide. Off. J. Eur. Union 2013,L124, 1–210. 50. Thommes, M.; Kaneko, K.; Neimark, A.V.; Olivier, J.P.; Rodriguez-Reinoso, F.; Rouquerol, J.; Sing, K.S.W. Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report). Pure Appl. Chem. 2015,87, 1051–1069. [CrossRef] 51. Kaplun, M.; Sandström, M.; Boström, D.; Shchukarev, A.; Persson, P. Crystal structures and spectroscopic properties of palladium complexes isolated from Pd–EDTA solutions. Inorg. Chim. Acta 2005,358, 527–534. [CrossRef] 52. Croissant, J.G.; Fatieiev, Y.; Almalik, A.; Khashab, N.M. Mesoporous silica and organosilica nanoparticles: Physical chemistry, biosafety, delivery strategies, and biomedical applications. Adv. Healthcare Mater. 2018,7, 1700831. [CrossRef] [PubMed] 53. He, Q.; Shi, J. Mesoporous silica nanoparticle based nano drug delivery systems: Synthesis, controlled drug release and delivery, pharmacokinetics and biocompatibility. J. Mater. Chem. 2011,21, 5845–5855. [CrossRef] 54. Trinh, H.T.; Tran, T.K.A.; Arora, S.; George, S.M.; Sheri, J.; Li, Z.; Yang, J.H.; Naruphontjirakul, P.; Balani, K.; Karakoti, A.; et al. Zn-Loaded SBA-1 and SBA-15 Molecular Sieves for Combined Antimicrobial and Osteogenic Activity. Adv. Mater. Technol. 2023, 8, 2201169. [CrossRef] 55. Kodeh, F.S.; El-Nahhal, I.M. Exploring of Potential Antibacterial Activity of Hypochlorite and Chloroamine Ad-sorbed Ammonium Functionalized Mesoporous SBA-15 Silica. Chem. Afr. 2021,4, 599–605. [CrossRef] 56. Shahriarinour, M.; Divsar, F.; Eskandari, Z. Synthesis, characterization, and antibacterial activity of thymol loaded SBA-15 mesoporous silica nanoparticles. Inorg. Nano-Metal Chem. 2019,49, 182–189. [CrossRef] 57. Xu, Z.; Xu, D.; Li, S.; Liu, Z.; Gao, X.; Cheng, L.; Liu, Y.; Sun, J. Preparation, sustained-release and antibacterial activity of SBA-15/CG antibacterial agent. Mater. Lett. 2023,344, 134432. [CrossRef] 58. Banafti, S.; Jahanshahi, M.; Peyravi, M.; Khalili, S. Controllable release activity of antibacterial Ag/SBA-16 cage-like synthesized by one-pot method. Micropor. Mesopor. Mat. 2020,299, 110107. [CrossRef] 59. Gudkov, S.V.; Burmistrov, D.E.; Serov, D.A.; Rebezov, M.B.; Semenova, A.A.; Lisitsyn, A.B. A mini review of antibacterial properties of ZnO nanoparticles. Front. Phys. 2021,9, 641481. [CrossRef] 60. Abebe, B.; Zereffa, E.A.; Tadesse, A.; Murthy, H.C.A. A review on enhancing the antibacterial activity of ZnO: Mechanisms and microscopic investigation. Nanoscale Res. Lett. 2020,15, 190. [CrossRef] 61. Siddiqi, K.S.; Rahman, A.U.; Tajuddin, N.; Husen, A. Properties of zinc oxide nanoparticles and their activity against microbes. Nanoscale Res. Lett. 2018,13, 141. [CrossRef] 62. Jiang, Y.; Zhang, L.; Wen, D.; Ding, Y. Role of physical and chemical interactions in the antibacterial behavior of ZnO nanoparticles against E. coli.Mater. Sci. Eng. C 2016,69, 1361–1366. [CrossRef] Nanomaterials 2025,15, 1697 35 of 35 63. Shehata, S.; Elkholy, Y.N.; Hussien, M.S.; Yahia, I.S.; Aboshanab, K.M. Antibacterial, antibiofilm and cyto-toxic activity of synthesized metal-incorporated mesoporous silica nanoparticles. AMB Express 2025,15, 130. [CrossRef] 64. Agarwal, N.H.; Menon, N.S.; Kumar, S.V.; Rajeshkumar, S. Mechanistic study on antibacterial action of zinc oxide nanoparticles synthesized using green route. Chem. Biol. Interact. 2018,286, 60–70. [CrossRef] 65. Mendes, C.R.; Dilarri, G.; Forsan, C.F.; De Moraes Ruy Sapata, V.; Lopes, P.R.M.; De Moraes, P.B.; Montagnolli, R.N.; Ferreira, H.; Bidoia, E.D. Antibacterial action and target mechanisms of zinc oxide nanoparticles against bacterial pathogens. Sci. Rep. 2022, 12, 2658. [CrossRef] 66. da Silva, B.L.; Abuçafy, M.P.; Manaia, E.B.; Oshiro-Junior, J.A.; Chiari-Andréo, B.G.; Pietro, R.C.R.; Chiavacci, L.A. Relationship between structure and antimicrobial activity of zinc oxide nanoparticles: An overview. Int. J. Nanomed. 2019,14, 9395–9410. [CrossRef] 67. Patra, J.K.; Baek, K.-H. Antibacterial Activity and Synergistic Antibacterial Potential of Biosynthesized Silver Na-noparticles against Foodborne Pathogenic Bacteria along with Its Anticandidal and Antioxidant Effects. Front. Microbiol. 2017,8, 167. [CrossRef] [PubMed] 68. Canta, M.; Cauda, V. The investigation of the parameters affecting the ZnO nanoparticle cytotoxicity behaviour: A tutorial review. Biomater. Sci. 2020,8, 6157–6174. [CrossRef] [PubMed] 69. Vagena, I.-A.; Gatou, M.-A.; Theocharous, G.; Pantelis, P.; Gazouli, M.; Pippa, N.; Gorgoulis, V.G.; Pavlatou, E.A.; Lagopati, N. Functionalized ZnO-Based Nanocomposites for Diverse Biological Applications: Current Trends and Future Perspectives. Nanomaterials 2024,14, 397. [CrossRef] 70. Liao, C.; Jin, Y.; Li, Y.; Tjong, S.C. Interactions of Zinc Oxide Nanostructures with Mammalian Cells: Cytotoxicity and Photocatalytic Toxicity. Int. J. Mol. Sci. 2020,21, 6305. [CrossRef] 71. Song, W.; Zhang, J.; Guo, J.; Zhang, J.; Ding, F.; Li, L.; Sun, Z. Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles. Toxicol. Lett. 2010,199, 389–397. [CrossRef] 72. Camaioni, A.; Massimiani, M.; Lacconi, V.; Magrini, A.; Salustri, A.; Sotiriou, G.A.; Singh, D.; Bitounis, D.; Bocca, B.; Pino, A.; et al. Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion. Part. Fibre Toxicol. 2021,18, 33. [CrossRef] [PubMed] 73. Vieira, C.O.; Grice, J.E.; Roberts, M.S.; Haridass, I.N.; Duque, M.D.; Lopes, P.S.; Leite-Silva, V.R.; Martins, T.S. ZnO: SBA-15 nanocomposites for potential use in sunscreen: Preparation, properties, human skin penetration and toxicity. Skin Pharmacol. Physiol. 2018,32, 32–42. [CrossRef] [PubMed] 74. Chia, S.L.; Leong, D.T. Reducing ZnO nanoparticles toxicity through silica coating. Heliyon 2016,2, e00177. [CrossRef] [PubMed] 75. ISO 14040:2006; Environmental Management—Life-Cycle Assessment—Principles and Framework. International Organization for Standardization: Geneva, Switzerland, 2006. 76. ISO 14044:2006; Environmental Management—Life Cycle Assessment—Requirements and Guidelines. International Organization for Standardization: Geneva, Switzerland, 2006. 77. Ros-Lis, J.V.; Vetter, S.; Smith, P. A comparative life cycle assessment of the synthesis of mesoporous silica materials on a small and a large scale. Green Chem. 2024,26, 10107–10114. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.