Laser Ablated Citrate-Stabilized Silver Nanoparticles Display Size and Concentration Dependant Biological Effects

. Silver nanoparticles (AgNPs) have been recognized for widespread biological applications due to their antimicrobial and anti-infammatory efect, especially in dentistry and for wound healing. Many features determine their benefcial or toxic potential, such as their synthesis type, size, morphology, coating, and concentration. Most synthesis types rely on the use of synthetic chemicals, which contributes to their toxicity. We present an environmentally friendly method for “green” synthesis of AgNPs from the silver target by pulsed laser ablation in liquid (PLAL) using citrate as the stabilizing agent. Since AgNPs already have many dental applications, we examined their antibacterial efect against supragingival bioflm-forming bacteria and bacterial strains known to cause resistant dental infections. Teir impact on human fbroblast cells’ cytotoxicity, proliferation (measured by XTT and Ki-67 immunofuorescence), pro/antioxidant balance, and lipid peroxidation (measured by PAB and LPP) was evaluated. AgNPs1 (21nm) and AgNPs2 (15 nm) spherical nanoparticles with good overall stability were obtained. Te highest tested dose of smaller nanoparticles (AgNPs2) displays not only an efective antibacterial efect against the tested oral bacteria strains but also a pro-oxidant and cytotoxic efect on fbroblast cells. Lower doses do not afect bacterial survival but increase the cell proliferation and metabolic activity and show an antioxidative efect, suggesting that diferent concentrations display a substantially opposite efect. Compared to larger AgNPs1, smaller AgNPs2 possess more potent biological efects, indicating that size plays a pivotal role in their activity. Such opposite outcomes could be important for their medical application, and high concentrations could be used for the inhibition of dental bioflm formation and resistant dental infections as well as proliferative conditions, while low doses could be benefcial in the treatment of atrophic and infammatory disorders. Finally, we showed that silver-targeted PLAL, using citrate as a stabilizing agent, produces biologically potent nanoparticles that could have many applications depending on their size and concentration.


Introduction
Silver nanoparticles (AgNPs) have been extensively studied over the past decades as a promising agent in various felds, from the food and paint industry to the wide spectrum of medical applications such as wound healing, material coating, dentistry, or even as potential anticancer agents [1][2][3][4][5]. Most of these applications rely on their well-known anti bacterial [6] and bioflm inhibitory activities, antifungal, anti-viral, anti-infammatory, antiangiogenic, and anticancer agents [7], as well as antiparasitic, antioxidant, and anticoagulant activities [8]. On the other hand, the apparent adverse efects of AgNPs have also been reported, varying from cyto-and genotoxicity, neurotoxicity, cardiotoxicity, and nanoparticle-induced oxidative stress [9]. Several mechanisms have been described to contribute to AgNPsinduced toxicity. Te induction of oxidative stress is one of the major mechanisms responsible for the antibacterial effects of AgNPs and also its geno-and cytotoxicity [10][11][12][13]. It has been reported that AgNPs increase the oxidative stress by ROS generation and antioxidant enzymes inhibition, which leads to lipid, DNA, and protein oxidative damage [14]. Tese efects combined with proliferation suppression are considered to be the main causes of AgNPs-induced cytotoxicity. Also, the literature data indicate that AgNPs could also lead to enhanced cell proliferation and have overall benefcial efects on human cells [2,15]. It appears that many features determine their toxic or benefcial potential, such as their size, morphology, dose, crystallinity, agglomeration, surface coating, and synthesis type [9,[16][17][18][19][20]. Te synthesis type and the nanoparticle size were proven to be the major factors afecting nanoparticleinduced toxicity [9]. Among the many types of AgNPs synthesis the most common are chemical-using silver nitrate as a precursor and various reducing agents as a stabilizer; green-using natural precursors such as plant extracts [21,22], bacteria, enzymes, electrochemical, and photochemical [23]. Chemically synthetized AgNPs are demonstrated to have the most toxic efects since synthetic chemicals are used as reducing agents, such as sodium borohydride and dimethylformamide [16]. On the other hand, laser synthesis from a metal target by pulsed ablation in liquid (PLAL) is the method that avoids the use of hazardous chemicals while simultaneously allowing the formation of stable nanoparticles [24]. Te antibacterial efect of nanoparticles synthetized in this manner is relatively rarely investigated although it is believed to possess bactericidal potential without uncontrollable parameters [25,26]. Te liquid used for the synthesis could be supplemented with capping or stabilizing molecules. Among those, citric acid or sodium citrate is considered to be effective in size and shape control throughout the synthesis process, generation of monodispersed nanoparticles, and act as both reducing and stabilizing agents [27]. Its main advantage is the possibility of further AgNPs functionalization due to the highly negatively charged surface and weak interaction of citrate molecules with metal surfaces and because of this, they can be easily exchanged by many other molecules or ligands [28]. However, capping or stabilizing molecules can afect the biological response to nanoparticles directly or indirectly through the modulation of their physicochemical characteristics, with regards to their toxicity [29]. Terefore, our study aimed to evaluate the biological efects of laser ablated AgNPs synthetized with the addition of citric acid and using diferent laser energies. Since AgNPs have already found signifcant topical prophylactic or therapeutic use in dentistry in recent years, [5,30] the investigation of laser ablated citrate-stabilized AgNPs antibacterial efects on oral contaminants could provide important fndings for their further application. For this purpose, we have chosen bacteria commonly involved in supragingival dental bioflm formation, [31,32] as well as bacterial strains that may be responsible for the failure of the diferent dental infection treatments [33].
Additionally, considering reported adverse efects of AgNPs that are not negligible, [9][10][11][12][13][14] their efect on the human fbroblasts regarding cytotoxicity as well as proliferation suppression and their relation to oxidative stress parameters were also determined.

AgNPs Synthesis.
AgNPs were produced by the laser ablation method. A high purity (>99.99% metal basis, 1.0 mm thick, Alfa Aesar, USA) silver foil was placed in a glass and covered with 6 mL of deionized water and 200 μL of a 0.6 mg/L water solution of citric acid (citric acid monohydrate p.a., Alkaloid Skopje). Before ablation, the silver plate was cleaned by using an ultrasonic bath for 30 minutes to remove contamination. Te laser applied was the picosecond Nd:YAG system (Nd:YAG EKSPLA SL 212/ SH/FH, LT), operating at the fundamental wavelength of 1064 nm with a 150-ps pulse length, pulse energies were 6 mJ (further referred to as AgNPs1) and 12 mJ (further referred to as AgNPs2), giving fuencies of 0.12 and 0.24 J/cm 2 , respectively. During the irradiation, the laser was focused on the Ag target using a 16 cm lens. Each sample was irradiated for 20 minutes at room temperature, leading to the formation of a pale-yellow colloidal suspension.

AgNPs Characterization.
To determine the nanoparticles' concentration, size, shape, composition and structure, several measurements were performed. First of all, the total concentration of silver in these colloidal solutions was determined using the ICP-OES method (ICP-OES, iCap7400 DUO, Termo Fisher, USA). Te ultravioletvisible spectrum of AgNPs colloidal solutions was recorded in the wavelength range from 190 to 1100 nm using a UV-Vis spectrophotometer (LLG-UNISPEC 2, LLG Labware, USA).
Te size, shape, and dispersity of Ag nanoparticles were analysed by transmission electron microscopy (TEM), using a FEI Talos F200X microscope at 200 keV with an X-FEG source and point-to-point resolution below 0.24 nm. Te micrographs were captured in the conventional mode and recorded on a CCD camera with a resolution of 4096 × 4096 pixels using the User Interface software package. Te samples for TEM examination were prepared by a standard procedure in which a drop of the very dilute suspension was placed on a carbon-coated copper grid, which was allowed to dry in the air.
Te size distributions were determined by manually measuring 60-120 particles using the public domain software ImageJ [34]. For obtaining the mean particles' size (d TEM ) and an index of polydispersity (PdI), the collected data were fnally ftted to a log-normal function as follows: Te averaged hydrodynamic diameters (d H ) were estimated by dynamic light scattering (DLS), measured on a NanoZS90 apparatus (Malvern, UK) with a 4 mW He-Ne laser source (λ � 633 nm). Te measurements were performed in disposable polystyrene cuvettes (DTS0012) at ambient temperature (25 ± 0.1°C). (MIC). Te antimicrobial activity of AgNPs in comparison to standard was investigated on bacterial strains responsible for the formation of of supragingival dental bioflm such as Streptococcus mutans (ATCC 25175), Aggregatibacter actinomycetemcomitans (ATCC 29552), Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC 25586), as well as on bacteria responsible for the failure of the treatment of diferent dental infections: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Streptococcus β haemoliticus (ATCC 27956) using the microdilution method according to ISO20776-1. Te aerobic bacterial stock was cultured on Mueller-Hinton Agar (HiMedia Laboratories, Dindhori, India) at 37°C for 24 hours, while the anaerobic stocks were cultured on the Shaedler broth (Termo Fisher Scientifc, Waltham, MA, USA) with vitamin K and hemin at 37°C for 72 hours in an anaerobic chamber.

Determination of Minimal Inhibitory Concentration
After being cultured, the bacterial strains were suspended in sterile saline to obtain the fnal cell concentration of 5 × 10 5 CFU/mL. Te minimum inhibitory concentrations (MICs) were determined after 24 hours of incubation of twofold serial dilutions of the investigated samples (100 μL) prepared in the liquid growth medium inoculated with bacterial strains using 96-well plates under aseptic conditions. Te plates that did not contain the investigated samples were used as controls.

Cell Culture and Treatment.
Te human primary dermal fbroblast cell line (PCS-201-012 ™ ) was commercially available (https://www.atcc.com). Te cells were grown in Dulbecco`s modifed eagle`s medium (DMEM, Gibco, Termo Fisher Scientifc, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Capricorn Scientifc, Germany) at 37°C and 5% CO 2 . Te cell cultures were treated in the log phase and at the subconfuence level, with four concentrations of AgNPs determined MIC concentration (results section) and three lower concentrations of 0.05, 0.1, and 1 ppm (parts per million) for 24 hours.

XTT Assay.
Te viability assay (XTT) was performed according to the standard procedure [35]. Shortly, after treatment and medium discharge, the cultured cells were supplemented with XTT reagent activated with phenazine methosulfate (PMS) and placed at 37°C until the colour developed. Absorbance was measured at 470 nm on a microplate reader (Sunrise, Tecan Group Ltd., Switzerland).

Ki-67
Immunofuorescence. Ki-67 immunofuorescent staining was performed to elucidate the efects of AgNPs not only on cellular survival but also on the proliferation rate. To perform immunofuorescent staining, the cells were grown on polylysine-coated slides (Merk, Germany) and after adhering to the polylysine surface, treated with AgNPs for 24 hours. After the treatment, the cells were fxed with 4% formaldehyde solution in 1 x PBS, permeabilized with 0.25% Triton-X (Merk, Germany), and blocked with 1% BSA solution. Directly labelled Ki-67 (SolA15, FITC, eBioscience ™ , Termo Fisher Scientifc, USA) antibody was hybridized overnight at 4°C. Te slides were washed in 1 x PBS solution, dehydrated in series of ethanol, and stained using DAPI-Vectashield solution (Vector laboratories, United Kingdom). Te results are presented as the proliferative index (PI), the ratio between immunoreactive (Ki-67+) cells, and the total number of cells. At least 500 cells per treatment were analysed using a Zeiss-Axioimager A1 microscope and the ISIS imaging software package (MetaSystems, Altlussheim, Germany).

Pro-Oxidant/Antioxidant Balance (PAB) Assay.
Following 24 hours of treatment, the medium was discarded, and cells were detached from the fasks using the trypsin solution (0.5% Trypsin-EDTA, Capricorn Scientifc, Germany). After washing with PBS, the cells were lysed by repeated freezing and thawing. Results for both the PAB assay and lipid peroxidation products were presented per mg of proteins in the samples, according to Lowry [36].
PAB assay was performed as previously described in [37]. Two oxidation-reduction processes in the same sample are in the basis of the following assay: oxidation of chromogen 3,3′,5,5′-tetramethylbenzidine (TMB) to a coloured cation by peroxides, catalysed by the horseradish peroxidase (HRP), and a reduction of coloured cation by antioxidants to a colourless compound. Te optical density (OD) of the coloured product was measured at 450 nm, with a reference wavelength of 570 nm. Te values are presented as arbitrary Hamidi-Koliakos (HK) units and calculated from the standard curve of H 2 O 2 in the standard solution.

Statistical Analysis.
Experiments were set up in triplicate and repeated twice. Te results were presented as means ± standard error of the mean (SEM). Te one-way ANOVA statistical test was used for statistical analysis using SPSS 10 for Windows (IBM, Armonk, NY, USA). Te level of signifcance was set to < 0.05.

AgNPs Synthesis and Characterization.
Two diferent laser energies applied during the laser ablation of silver foil led to two types of nanoparticles, whose size and concentration were diferent; AgNPs1 (laser energy: 6 mJ) and AgNPs2 (laser energy: 12 mJ). Te concentrations of AgNPs were 17.4 ± 0.1 and 28.7 ± 0.2 µg/mL for AgNPs1 and AgNPs2, respectively. UV-Vis spectroscopy is the simplest way to confrm the formation of nanoparticles. Te appearance of a peak near 430 nm (Figure 1) demonstrates the surface plasmon resonance properties of AgNPs [39]. Also, there is a clear enhancement in the intensity of the surface plasmon resonance (SPR) band with the increasing nanoparticle concentration.
Te morphology and particle size were obtained by statistical analysis of prepared nanoparticles. Typical TEM micrographs of AgNPs1 and AgNPs2 are shown in Figures 2(a)  and 2(b). Te nanoparticles are mainly spherical and pseudospherical, with larger particles tending to form hexagonal shapes. Te particles are partially agglomerated on each other, and their sizes are subjected to the log-normal size distribution (Figures 2(c) and 2(d)). After the statistical analysis, the mean particle size of AgNPs1 was d TEM � 21.2 ± 6.8 nm (the polydispersity index was 32.1%), while the size of AgNPs2 was d TEM � 15.3 ± 4.6 nm (PdI≈30%).
As is generally accepted, the very negative zeta potential of nanoparticles indicates their good colloidal stability [16]. Furthermore, zeta potential values for colloidal suspensions stabilized by electrostatic repulsion must be at least ± 30 mV [40]. Te zeta potentials of the produced silver colloidal solutions were − 32.1 ± 5.22 mV and − 33.2 ± 5.61 mV for AgNPs1 and AgNPs2, respectively.
Te hydrodynamic diameters of nanoparticles' aqueous suspension at pH 7 were determined by the DLS method ( Figure 2). Te obtained d H values for AgNPs1 and AgNPs2 were 81 ± 19 nm and 78 ± 18 nm, respectively. Te diference between the measured d TEM and d H is because the d H value presents the size of the nanoparticles in their hydrated state, together with the surrounding layers of water molecules that extend the hydrodynamic diameter and make the hydrated shell around the particle signifcantly larger than the particles themselves. Unlike DLS, this cannot be observed by microscopic techniques because of the low background originating from low electron density. Te measured d H values of synthesized nanoparticles were notably higher than the d TEM , which may be a hint of a certain degree of particles' agglomeration in suspension. Tat can lead to an unwanted destabilization of the colloid. Nevertheless, the obtained d H values were still inside the acceptable biomedical application range (less than 100 nm).

Determination of Minimal Inhibitory Concentration (MIC).
MICs represent the lowest concentration of samples that completely inhibit visible bacterial growth. AgNPs2 with a higher concentration and smaller particle size (15.3 ± 4.6 nm) showed antibacterial potential at 14.35 μg/ mL against all tested oral pathogens (Streptococcus mutants, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum) as well as one gram-positive (Staphylococcus aureus) and one gramnegative bacteria Pseudomonas aeruginosa, but had no effect against Escherichia coli and Streptococcus β haemoliticus. Unlike AgNPs2, AgNPs1 did not display an antibacterial efect against any of the tested bacteria, not even when concentrated.
Antibacterial efects of AgNPs against all tested oral pathogens, as well as Pseudomonas aeruginosa and Staphylococcus aureus have previously been reported in the literature, whereas reported minimal inhibitory concentrations varied to a great extent from approximately 5 to 50 µg/mL, depending on the tested strain, clinical or commercial, as well as the nanoparticle size [41]. Also, all of these nanoparticles were chemically synthetized, where toxic waste is produced during the synthesis process and could have a synergistic efect with the silver. Barabadi et al. (2021) compared the antibacterial efects of photosynthetized AgNPs using the Zataria multifora plant extract (green synthesis) with commercially available and chemically synthetized citrate-reduced AgNPs against Staphylococcus aureus and Pseudomonas aeruginosa. Tey stated that green synthetized AgNPs had the most potent antibacterial efects against tested bacterial strains in planktonic, as well as in the bioflm form [42]. Hamida et al. reported similar fndings when compared biogenic AgNPs and silver nitrate. Tey found out that the efects are a result of intracellular ROS generation leading to biomolecules oxidation [43]. Reduction in ATP levels in AgNPs-treated bacteria support these fndings [44]. However, reports on the antimicrobial activity of laser synthetized AgNPs are rather limited, [24,41,45,46] and to the best of our knowledge, they are not available for the oral pathogens.
Since undiluted AgNPs1 have greater concentration than MIC for AgNPs2, the probable reason for AgNPs2 efcacy is the smaller particle size. Other research groups have also reported that the antimicrobial efect of AgNPs correlates with smaller particle sizes and nanoparticle shape [41,47,48]. Since both synthetized nanoparticles are similar in shape (spherical and pseudospherical), the size seems to be the determining factor for the antibacterial activity in this case. Comparing AgNPs with diferent sizes, shapes, and surface coatings revealed that shape had a greater infuence on the antibacterial activity than surface coatings [49]. Besides size, shape, and concentration, other factors could contribute to their antimicrobial efects, such as direct bacterial cell membrane damage, protein and/or enzyme inhibition and denaturation, and the generation of reactive oxygen species (ROS) with subsequent oxidation of the cellular macromolecules [6,9,50]. Apparently, ROS production plays a major role in the antimicrobial activity since it was demonstrated that it interacts with thioredoxin system of Staphylococcus aureus, one of the most important systems in the processes of oxidative stress. Tis interaction leads to depletion of intracellular thiol which in turn activates the oxidative stress by the disruption of the protective components [51]. Te proposed antibacterial mechanisms of AgNPs are presented in Figure 3. Comparing the efects of two types of synthesized nanoparticles, the increase was signifcantly higher after AgNPs2 treatment for PAB values (p < 0.001, Figure 4). Tese results are consistent with the literature data, where the antibacterial efects of AgNPs are mostly attributed to the generation of ROS [52]. Te higher pro-oxidant state and increased lipid peroxidation products after AgNPs2 treatment suggest that the particle size plays a pivotal role in ROS production, which is mostly accredited to the larger surface areas that enable interaction with the cellular macromolecules [17]. Consistent with these results are the efects on cellular survival and proliferation. Te same concentration of AgNPs led to a dramatic loss of viable cells compared to the control (p < 0.001); also, the reduction in cellular survival rate was more pronounced for AgNPs2 (p < 0.001). Tis viability reduction might be a consequence of either cell death, proliferation suppression, or their combination [16]. AgNPs inhibition of cell proliferation and migration by activation of caspase 3 and 7 was previously reported [53]. To assess whether viability reduction was a result of cell death or suppression of proliferation, we performed Ki-67 immunofuorescent staining, which showed that at these concentrations, no proliferative active cells were detected (no Ki-67+ cells), while also cells with altered morphology (small, condensed nuclei) were observed, suggesting that viability reduction was a combination of both cytotoxic efect and proliferation suppression.
Te efects of low concentrations were quite opposite. Both AgNPs1 and AgNPs2 decreased PAB levels (p < 0.001) at concentrations of 1, 0.1, and 0.05 µg/mL. Interestingly, the same concentrations had diferent efect on lipid peroxidation; AgNPs1 elevated lipid peroxidation products compared to control (p < 0.01 for 1 and 0.1 μg/mL), while only the lowest AgNPs2 concentration of 0.05 μg/mL increased LPP (p < 0.001). Te concentration of 0.1 μg/mL had no efect and the concentration of 1 μg/mL slightly, albeit insignifcantly, reduced lipid peroxidation.
Te efects on PAB levels could be explained by mild-free radicals' production which promptly activates enzymes involved in antioxidant defence. Te enzymes can lower overall pro-oxidant/antioxidant balance to a certain extent, but at high concentrations, such as our determined MIC (14.35 μg/mL), their capacity is overcoming and pro-oxidant state occurs. In vivo study reported by Docea et al. [54] demonstrated that the subacute administration of AgNPs also lower pro-oxidant/antioxidant balance due to the activation of nonenzymatic (reduced GSH) and enzymatic (increased catalase activity) defence systems.
On the other hand, lipid peroxidation products are the result of interactions between ROS and polyunsaturated fatty acids (PUFA) [55]. Regarding the efects of lower AgNPs concentrations on LPP levels, two types of nanoparticles showed diferent trends. Larger AgNPs1 nanoparticles led to a small but consistent linear increase in lipid peroxidation ( Figure 4). Furthermore, only the lowest (0.05 μg/mL) concentration of AgNPs2 increased LPP levels, while with an increase in concentration up to 1 μg/mL, LPP levels decreased. Tis discrepancy could be explained by the nanoparticle size, i.e., a similar trend was observed in the previously mentioned in vivo study [54]-PVP-coated nanoparticles that were larger increased TBARS dosedependently, while EG-coated nanoparticles (smaller in size) increased TBARS at the lowest concentration, subsequently decreasing it at all higher concentrations, similar to AgNPs2. Te authors explained that this diference was due to diferent coating materials; however, since in our experiment, the surface-stabilizing agent was the same for both types of AgNPs, the particle size should not be neglected. Another possible explanation is the interaction between nanosilver and enzymes involved in antioxidant defence, where it was shown that AgNPs sized 20 nm interact with catalase, thus suppressing its activity and disabling antioxidative defence to a certain extent [56]. Additionally, smaller nanoparticles could display their efects beyond the cell membrane, i.e., the cellular uptake and interplay with mitochondria could be the cause of antioxidative enzymes activation, ROS quenching, and reduction of LPP levels at the lower applied concentrations [20,57]. Bressan et al. demonstrated that AgNPs smaller than 20 nm enter human dermal fbroblasts by endocytosis, upon which they form aggregates in the close proximity of mitochondria and impair their function without altering other subcellular compartments. If ROS overaccumulate, the mitochondrial membrane could breakdown, leading to the release of mitochondrial antioxidative enzymes into the cytoplasm and ROS quenching or activation of regulatory genes involved in cellular defence, which results in overall cellular protection [58]. Consistent with this, it was shown that LPP production, depending on its extent, could not only lead to cellular damage but also display a protective efect [55]. Our results demonstrated that all three concentrations of both nanoparticles elevated XTT levels and the proliferation index (Ki-67 + cells) compared to the control (Figure 4(c), Figure 5).
Interestingly, for AgNPs2, this elevation was in inverse correlation with LPP, suggesting the previously mentioned activation of alternative regulatory genes with the prosurvival function. A mild increase in XTT levels was observed for AgNPs2 (statistically signifcant for 0.1 and 1 μg/ mL, p < 0.01, and p < 0.001, respectively) and great concentration-dependent induction of cellular proliferation was observed for all three concentrations (p < 0.001). On the contrary, AgNPs1 led to slightly higher, albeit insignifcant, increase in XTT levels compared to AgNPs2 (Figure 4(c)), and a statistically signifcant lower increase in the proliferation index compared to AgNPs2 (p < 0.001). Since XTT assay essentially represents a measure of the mitochondrial activity in living cells, i.e., not only the number of viable cells but also their metabolic activity, [59] higher proliferation rates after AgNPs2 could be a sign of activation of additional prosurvival pathways, rather than only mitochondrial that favour cellular proliferation. Our previous research also demonstrated that the proliferation ability of AgNPs is size dependent and probably mediated by ILGF-1 production in peripheral blood mononuclear cells [19]. In addition, other research studies also showed concentration-and size-  dependant efects on the proliferation rates of human keratinocytes and dermal fbroblasts [2,15,60]. Similar to our results, high concentrations displayed a cytotoxic efect, while lower concentrations led to an increase in keratinocyte proliferation, without altering fbroblast proliferation rates. A positive efect on proliferation is mostly attributed to the suppression of proinfammatory pathways and the activation of anti-infammatory pathways. Unlike these studies, we showed that lower concentrations also increase the proliferation rate of human dermal fbroblasts and that this increase is more pronounced after treatment with smaller nanoparticles ( Figure 5). We suppose that this efect is related to the activation of alternative pathways, but the exact mechanism remains to be elucidated.

Conclusion
Te biological efects of diferent concentrations and sizes of two types of AgNPs synthesized by picosecond laser ablation of silver foil in a water solution of citric acid were examined in this study. Nanoparticles with good colloidal stability were obtained, whereas higher laser energy (12 mJ) produced overall smaller AgNPs (15.3 ± 4.6 nm) with higher concentration (28.7 ± 0.2 μg/mL). High concentrations of these nanoparticles, AgNPs2, displayed a potent antibacterial effect against bioflm forming bacteria, as well as against Staphylococcus aureus and Pseudomonas aeruginosa. Te same concentration also led to a strong pro-oxidant and cytotoxic efect with the complete inhibition of cell proliferation. However, when applied at lower doses, the antimicrobial efect was absent, but a positive efect on the cell proliferation and metabolic activity was observed, suggesting that diferent concentrations display a substantially opposite efect. Additionally, the low concentration showed an antioxidative efect probably due to the antioxidative enzymes activation. Tese fndings could be of importance for the treatment of various conditions with essentially diferent pathophysiological mechanisms, ranging from dental bioflm inhibition and resistant dental infections, as well as proliferative conditions when applied at high concentrations, to atrophic and infammatory conditions, when applied at low concentrations.

Data Availability
Te data that support the fndings of this study are available on request from the corresponding author.

Conflicts of Interest
Te authors declare that there are no conficts of interest.