Antibacterial and Non-Toxic to Mammalian Cell Composite Material Based on Polymethyl-Methacrylate-like Resin Containing Grain-Shaped Copper Oxide Nanoparticles

Abstract

Granular copper oxide nanoparticles (CopOx NPs), synthesized via laser ablation (100 nm, ζ-potential +30 mV), were introduced into photolithographic polymethyl methacrylate (PMMA) resin at concentrations of 0.001–0.1%. The resulting composite material enables the fabrication of high-resolution (up to 50 μm) parts with a high degree of surface quality after polishing using the MSLA method. CopOx NPs increased the degree of resin polymerization (decrease by almost 4× in unpolymerized components at 0.1% CopOx NPs) and induced the in situ formation of self-organized periodic structures visible under a modulation interference microscope. The composite samples exhibit pronounced oxidative activity: they intensify the generation of hydrogen peroxide and hydroxyl radicals and cause the oxidative modification of biomolecules (formation of 8-oxoguanine in DNA and long-lived reactive forms of proteins). A key property of the materials is their selective biological activity. While lacking cytotoxicity for human fibroblasts, they exhibit a strong antibacterial effect against E. coli, leading to cell death within 24 h. Thus, the developed composite photolithographic resin combines improved technological characteristics (high printing resolution, degree of polymerization) with functional properties (selective antibacterial activity) and holds promise for application in biomedicine, as well as in the food and agricultural industries.

1. Introduction

Today, there are several main 3D printing methods: Fused Deposition Modeling (FDM), Stereolithography (SLA), Selective Laser Sintering (SLS), Selective Laser Melting (SLM), and Binder Jetting (BJ) [1]. Polymer parts are often manufactured using FDM and SLA, and less commonly using SLS [2]. FDM is the most common and accessible 3D printing method, used in both industry and everyday life [3]. An extrusion head melts and precisely forces a polymer filament through a nozzle, sequentially creating a part layer by layer [4]. The SLA method operates by polymerizing liquid photolithographic resin using ultraviolet radiation [5]. Despite the higher cost, the SLA method has a number of key advantages: high strength of parts (especially under loads directed parallel to the layers), high precision (the ability to produce thin-walled elements and micro-parts), excellent surface quality (minimizing subsequent processing) and low noise levels during operation [6]. In modern 3D printing technologies using the SLA method, materials based on modified methacrylates, thermoplastic polymers, derivatives of acrylic, methacrylic or cyanoacrylic acid esters are widely used [7]. 3D printing based on methacrylates is actively used in optics and electronics, engineering and architecture, in the jewelry and chemical industries [8]. It should be noted that methacrylates are a popular structural material for the creation of high-precision products not only using 3D printing [9]. Products made from methacrylates initially did not have significant biocompatibility, but, at the very beginning of this millennium, number of resins based on polymethyl methacrylates (PMMA) were approved by the Food and Drug Administration (FDA) for use in medical devices [10]. Currently, PMMA-based resins are widely used in dentistry (printing dental models, dental products, temporary crowns) [11], ophthalmology (intraocular lenses) [12], and surgery (bone cement) [13]. However, products printed from PMMA have two significant drawbacks, namely, susceptibility to bacterial contamination and incomplete polymerization of the resin, which leads to the release of resin monomers into the environment [14].

Quite often, the addition of nanoparticles to polymeric materials significantly modifies their biological and physical properties [15,16]. In terms of biological properties, metal and metal oxide nanoparticles have demonstrated significant antibacterial and fungicidal effects [17]. Polymerization of polymethyl methacrylate resin occurs via a radical mechanism, and therefore one can assume the potential effectiveness of nanoparticles of metals and metal oxides of variable valence capable of participating in redox reactions [18]. The most common variable valence metals are iron, copper, titanium, cobalt, nickel, chromium and manganese. Cobalt, nickel, chromium and manganese are quite toxic and are not suitable for use in living systems [19]. Titanium is quickly oxidized and chemically inactivated in living systems [20]. In addition, titanium oxide is photoactive, which can also be significant limitations [17]. Iron and copper are also rapidly oxidized to oxides in living systems, but they can actively participate in redox reactions with similar efficiency [21].

A comparative analysis of the literature data shows that iron and copper oxide nanoparticles differ significantly in terms of the mechanisms and efficiency of their antibacterial action [22,23]. The antimicrobial activity of FexOy nanoparticles strongly depends on their size, crystalline phase (magnetite, hematite, maghemite), and synthesis conditions, which makes it difficult to reproduce the effect [22,24]. In contrast, copper oxide nanoparticles demonstrate consistently high and reproducible bactericidal activity, which is less dependent on the oxidation state of the metal and production methods [25]. This effect is associated with a combination of several mechanisms, including “contact” membrane damage, release of Cu²⁺ ions, and induction of oxidative stress in the bacterial cell due to catalytic generation of ROS [23,26]. Moreover, there are a number of studies indicating the possible role of transition metal nanoparticles as co-initiators or catalysts of radical polymerization, which potentially allows for the problem of residual monomers to be solved [27,28]. Despite the growing number of studies on filling polymers with antimicrobial nanoparticles, most of them are focused on systems for FDM printing (composite filaments) [29,30] or classical casting [31,32]. There are virtually no studies in the literature devoted to the targeted creation and comprehensive study of photolithographic PMMA resins modified with copper oxide nanoparticles specifically for MSLA printing (as a high-performance variant of SLA). The key distinction of this study is not simply imparting antibacterial properties to the material, but rather a comprehensive approach aimed at simultaneously addressing two key challenges of PMMA resins for biomedical applications: the material’s essential antimicrobial properties and its high degree of photopolymerization.

As a consequence, the aim of this work is to obtain a series of new composite materials for lithographic printing based on polymethyl methacrylate-like photopolymer resin containing CopOx NPs, as well as to study the physicochemical and biological properties of products obtained from such resins.

2. Materials and Methods

2.1. Synthesis of Copper Oxide Nanoparticles (CopOx NPs)

The CopOx NPs used in the experiments were produced via laser ablation in deionized water. A ground copper plate with a purity of 99.99% was used as the ablated target. The copper target was attached to the bottom of a glass cuvette with a volume of just over 50 mL. The thickness of the deionized water layer above the target surface was approximately 3 mm. The cuvette is connected via an inlet and outlet hose to an additional reservoir of approximately 60 mL. The total volume of deionized water in the system is approximately 100 cm³. Water is pumped through a hose exiting the cuvette using a peristaltic pump, ensuring constant fluid circulation in the system. The laser source employed was a P-Mark TT 100 ytterbium fiber laser (Pokkels, Moscow, Russia), operating at a wavelength of 1064 nm. It delivered pulses with an energy of 1.5 mJ, a duration of 200 ns, and a repetition rate of 35 kHz. The beam was directed and focused onto the target using an LScanH galvano-optical scanning system (Ateko-TM, Moscow, Russia) equipped with an F-Theta lens (90 mm focal length). The scanning pattern consisted of parallel straight lines within a 2 cm square area, with a line spacing of approximately 20 μm and a scanning speed of 3000 mm/s.

2.2. Characterization of CopOx NPs

The hydrodynamic diameter, ζ-potential, and nanoparticle concentration were determined using dynamic light scattering on a Malvern Zetasizer Ultra analyzer (Malvern Panalytical Ltd., Worcestershire, UK). A 10 × 10 mm quartz cuvette and a polyetheretherketone cuvette with ‘Dip’ Cell ZEN1002 palladium electrodes were used for the measurements. UV-Vis spectra were obtained using a Cintra 4040 differential dual-beam spectrophotometer (GBC Scientific Equipment Pty Ltd., Melbourne, Australia). Nanoparticle morphology was studied using a Libra 200 FE HR transmission electron microscope (TEM) (Carl Zeiss, Oberkochen, Germany). The details of the experiments were described previously in [33].

2.3. Addition of CopOx NPs to Photolithographic Resin

Before adding to the photolithographic resin, the CopOx NPs were transferred from water to acetone. Using a 3-16KL rotary centrifuge (Sigma, Esslingen, Germany), the aqueous CopOx NP colloid was centrifuged at 22 °C for 40 min at 7000 rpm. After centrifugation, the supernatant was replaced with acetone and sonicated (40 kHz, 22 °C) for 3 min, followed by shaking on a shaker for 5 min. The centrifugation procedure, replacement of the supernatant with acetone, and mechanical shaking were repeated three times. The resulting CopOx NPs colloid in acetone was poured into a glass vial and sealed. The resulting CopOx NPs colloid in acetone was mixed with Dental Clear PRO lithographic resin (Harz Labs, Moscow, Russia) to final nanoparticle concentrations of 0.001%, 0.01%, and 0.1% by weight. After mixing, the resin with nanoparticles was subjected to mechanical action on a shaker for 5 min. and ultrasonic treatment (40 kHz, 22 °C) for 3 min. The finished lithographic resin samples with nanoparticles were stored protected from light in sealed 500 cm³ dark glass bottles. The resulting colloidal systems of CopOx NPs in lithographic resin demonstrated high stability over time. Observations over 14 days confirmed the absence of visible sediment or particle agglomeration.

2.4. Additive Manufacturing of Composite Material Samples

A Saturn 3 Ultra 12K MSLA printer (Elegoo, Shenzhen, China) was used to fabricate various samples. The samples were made from photolithographic resins containing 0.001%, 0.01%, and 0.1% CopOx NPs. Control samples were made from pure photolithographic resin without nanoparticles. Composite material samples printed from PMMA/CopOx resin were post-processed as follows. (1) Primary washing by immersion for 6 min in a cylinder filled with absolute isopropanol (99.9%) moving along a ring-shaped trajectory due to a magnetic stirrer; (2) Ultrasonic cleaning by treatment for 6 min in an ultrasonic bath with isopropanol; (3) Drying by exposure for 10 min at room temperature under a fume hood; (4) Finishing treatment by applying glycerin followed by UV curing for 30 min on a rotating platform (UW-02, Creality3D, China); (5) Repeated ultrasonic treatment; (6) Repeated drying; (7) Heat treatment by exposure for 30 min at 80 °C in a dry-heat oven. The finished samples were stored in closed containers at room temperature.

2.5. Methods for Characterizing Composite Materials

The surface topography of the printed specimens at the micro- and nanoscales was examined with an atomic force microscopy (AFM) system (NT-MDT, Zelenograd, Russia). To investigate the interaction between the nanoparticles and the polymer matrix, Fourier-transform infrared (FTIR) spectroscopy was conducted using an IR-8000 spectrometer ((ORTHODYNE S.A., Ans, Belgium) MIM-321-Moscow, Russia) fitted with a ZnSe Sealed Flat Plate accessory (Pike Technologies, Madison, WI, USA). Additionally, optical absorption was measured with a Cintra 4040 dual-beam UV/Vis spectrometer (GBC, Sydney, Australia). The dispersion of nanoparticles within the matrix was assessed via a modulation interference microscope MIM-321 (Amphora Laboratories, Moscow, Russia).

2.6. Measurement of Hydrogen Peroxide Concentration

The concentration of hydrogen peroxide was quantified via chemiluminescence with a high-sensitivity Biotox-7A-USE chemiluminometer (Engineering Center-Ecology, Moscow, Russia). The assay was based on a reaction mixture of luminol, 4-iodophenol, and horseradish peroxidase in Tris-HCl buffer (pH 8.5). Samples of the composite material, prepared as 10 × 10 × 0.5 mm plates, were immersed in 20 cm³ polypropylene vials containing deionized water. Following incubation at 40 °C for 180 min., chemiluminescent measurements were conducted. This method provides a detection limit for hydrogen peroxide of 0.1 nM. A detailed description of the experimental protocol has been reported previously [34].

2.7. Measurement of Hydroxyl Radicals

A fluorimetric method based on the formation of 7-hydroxycoumarin-3-carboxylic acid as a result of the reaction of hydroxyl radicals with coumarin-3-carboxylic acid was used to quantify hydroxyl radicals. The test samples, in the form of 10 × 10 × 0.5 mm plates, were placed in 20 cm³ polypropylene vials filled with an aqueous solution of coumarin-3-carboxylic acid. The vials were incubated at 80 °C for 120 min., after which the pH of the medium was raised to 8.5 and measurements were taken. The resulting fluorescent product, 7-hydroxycoumarin-3-carboxylic acid, was recorded using a 8300 spectrofluorimeter (JASCO, Tokyo, Japan) at excitation wavelengths of 400 nm and emission wavelengths of 450 nm. Experimental details have been published previously [35].

2.8. Quantitative Determination of 8-Oxoguanine in DNA

Quantitative analysis of DNA samples was performed via an enzyme-linked immunosorbent assay using specific monoclonal antibodies to 8-oxoguanine. Composite material samples were used in the form of 10 × 10 × 0.5 mm plates. The samples were incubated in 5 mL of DNA colloid at 40 °C for 180 min. After DNA immobilization at the bottom of the wells of the plates, primary antibodies to 8-oxoguanine (diluted 1:2000) were added to the wells. DNA incubation with antibodies was carried out at 40 °C for 180 min. After washing, secondary antibodies conjugated with horseradish peroxidase (diluted 1:1000) were added. Incubation with secondary antibodies was carried out at 37 °C for 90 min. Horseradish peroxidase enzymatic activity was measured on a Feyond-A400 plate reader at 405 nm using 18.2 mM ABTS in the presence of 2.6 mM H₂O₂ in 75 mM citrate buffer, pH 4.2. Experimental details have been published previously [36].

2.9. Quantitative Determination of Long-Lived Reactive Protein Species (LRPS)

Composite material samples took the form of 10 × 10 × 0.5 mm plates. The samples were incubated in 10 mL of a 0.1% aqueous colloid solution of bovine serum albumin (BSA) at 40 °C for 120 min. The LRPS concentration was determined by measuring the chemiluminescence of the protein solutions after their incubation for 2 h at 40 °C. Following incubation, the samples were kept in the dark at room temperature for 30 min. Measurements were performed in complete darkness at ambient temperature using 20-mL polypropylene vials (Beckman, Brea, CA, USA). A high-sensitivity “Biotox-7A” chemiluminometer (Engineering Center “Ecology”, Moscow, Russia) served as the measuring instrument. Unheated BSA solutions were used as the control group. All described procedures were carried out in accordance with a previously published protocol [37].

2.10. Dynamics of E. coli Broth Culture Reproduction

To study the effect of composite materials on bacterial proliferation dynamics, 10 mm diameter samples were prepared and sterilized and placed into the wells of a 24-well plate. A 1 mL sample of a bacterial culture (106 CFU/mL) was added to each well of the plate, after which the plate, without a lid, was placed in a plate reader equipped with a thermostat and shaking system. Cultivation took place at 37 °C, shaking once per hour. The optical density of the bacterial cultures was recorded at wavelength of 600 nm. Experimental details have been published previously [38].

2.11. Evaluation of Antibacterial Activity by Flow Cytofluorimetry

After completion of the culturing process, 1 mL of phosphate-buffered saline (PBS) containing 4 μM propidium iodide (PI) was added to each well of the 24-well plate. After adding the dye, the plate was incubated for 60 min in the absence of light. Before analysis, the samples were thoroughly resuspended and transferred to sterile 1.5 mL Expell Microcentrifuge Tubes (Capp, Wolnzach, Germany). For each sample, event counting, side (SS) and forward (FS) scattering measurements, and PI fluorescence intensity assessment were performed. Fluorescence was recorded using a LongCyte C3110 cytofluorimeter (ChBio, Qingdao, China) in the orange-red channel (excitation 535 nm, emission 617 nm). This mode allows for accurate determination of the number of damaged bacterial cells with compromised membrane integrity. Experimental details have been published previously [7].

2.12. Cytotoxicity Assessment on Eukaryotic Cells

The effect of printed composite materials on a culture of human spleen fibroblasts HSF (ATCC line #PCS-201-012) was studied. Cells were cultured under standard conditions. A cell suspension at a density of 105 cells per well was applied to prepared slides placed in the wells of a 6-well plate. Cell cultivation was performed in an S-Bt Smart Biotherm CO₂ incubator (Biosan, Riga, Latvia) at 37 °C and a CO₂ concentration of 5% for 72 h. After incubation, samples were removed, and the cell cultures were analyzed microscopically. To assess cell viability, a combination of fluorescent dyes was used: Hoechst 33,342 for nuclei staining, Rhodamine for mitochondria visualization, and propidium iodide for detecting dead cells. A DMI 4000B (Leica, Wetzlar, Germany) system was used for cell visualization and recording, and the resulting images were analyzed using ImageJ software v. 1.54p. At least 200 cells per field of view were analyzed during microscopic examinations. Each experimental variant was repeated five times to ensure statistical reliability of the results. All experimental details have been described previously [39].

2.13. Statistical Data Analysis and Visualization

Data processing and statistical analyses were conducted with GraphPad Prism software (version 8.3.0). The data are presented as means ± standard error of the mean (SEM), derived from a minimum of three independent experiments.

3. Results

CopOx NPs were obtained using laser ablation of copper target in deionized water with nanosecond laser pulses. Laser ablation produces monodisperse aqueous nanoparticle colloids (Figure 1a) with an average hydrodynamic size of approximately 100 nm. The colloid potentially contains nanoparticles with hydrodynamic sizes in the range of 40–200 nm. The size distribution width at half-maximum is approximately 70 nm (from 60 to 130 nm). These sizes account for the overwhelming majority of nanoparticles in the colloid.

CopOx NPs in an aqueous colloid have pronounced positive electrokinetic potential (Figure 1b). The average electrokinetic potential of nanoparticles is about +30 mV. Nanoparticles with electrokinetic potentials in the range of −20 to +70 mV are potentially detected in the colloid. The width of the distribution for the value of electrokinetic potential at half-maximum is about 35 mV (from +15 to +50 mV). This is the electrokinetic potential of the overwhelming majority of nanoparticles in the colloid. Using differential double-beam spectrometry, the optical properties of the colloid of nanoparticles were studied (Figure 1c). In the range from 300 to 550 nm, decrease in the optical density of the colloid is observed, with an absorption minimum around 550 nm. At 350 nm, the colloid of nanoparticles shows a slight increase in absorption. Pronounced absorption maximum in the spectrum is observed only at 660 nm. CopOx NPs produced by laser ablation are known to not always have a shape close to spherical. The morphology of the nanoparticles was studied using transmission electron microscopy (Figure 1d). It was shown that the nanoparticles have the shape of grains with an aspect ratio of 1/2–1/3 (length over 100 nm, width approximately 50 nm). The nanoparticles are pointed at the ends, and zones with varying degrees of contrast are visible in the pointed areas.

CopOx NPs were obtained in water. The presence of water in lithographic resins is unacceptable. To solve this problem, an aqueous colloid of nanoparticles was centrifuged at 27,000 rpm for 20 min. The supernatant (water) was discarded, and the nanoparticle sediment was diluted with pure acetone in an ultrasonic bath. After replacing the solvent, approximately 0.3–0.8% water remains in the colloid. It was shown that defects may occur in the final products when using this composite lithographic resin. When repeating the procedure and replacing acetone containing 0.3–0.8% water with pure acetone, defects in the final products were extremely rare. A third liquid replacement produces resin suitable for defect-free (visually) printing. Various massive products (round plates, complex parts, scaffold networks) have been manufactured using this resin. It has been demonstrated that optically transparent parts can be manufactured from a composite lithographic resin containing CopOx NPs (Figure 2a). Furthermore, using this resin, maximum MSLA printer resolution of approximately 50 µm can be achieved (Figure 2b).

Optically transparent circular plates were polished until a specular reflection appeared, meaning a clear, distortion-free image of the objects was achieved. The polished samples were examined using atomic force microscopy. Even at the highest CopOx NP concentration of 0.1%, no cracks, cavities, or craters were detected on the surface of the polished plates. A typical three-dimensional surface profile is shown in Figure 3. It is shown that, over areas of approximately 100 µm², the maximum height difference does not exceed 50–60 nm.

It was shown that samples made of composite lithographic resin containing CopOx NPs, even at maximum concentration of 0.1 wt. %, are polished perfectly and are free of surface defects. After polishing, the samples appear uniform even at 1000× magnification. To obtain information on the arrangement of the nanoparticles, the samples were examined using MIM (Figure 4). It was shown that the samples without CopOx NPs do not contain any distinct areas with uniform phase incursion. At CopOx NP concentration of 0.001%, areas with uniform phase incursion are observed in the samples. The size of the areas with uniform phase incursion often does not exceed 1 µm². With an increase in the CopOx NP concentration to 0.01%, areas with uniform phase incursion up to 4–6 µm² in size are observed in the samples. In some cases, it can even be assumed that regions with uniform phase shifts are ordered relative to one another. At the maximum CopOx NP concentration of 0.1%, the samples exhibit ordered regions with uniform phase shifts. A clear alternation in regions with minimum and maximum phase shifts is visible. Regions with uniform phase shifts are longer than 10 µm.

Using a Fourier transform infrared spectrometer, spectra of wafers made of composite lithographic resin with or without CopOx NPs were obtained (Figure 5a). As expected, even in samples with CopOx NP content of approximately 0.1%, weakly pronounced Cu-O lines are not detected. However, all pronounced lines characteristic of organic compounds are detected. The most interesting spectral changes are observed in the lines associated with C=C double bonds. In wafers made of composite lithographic resin without CopOx NPs, the absorption in this spectral region is approximately 25%. With the addition of nanoparticles to the resin, absorption in the region associated with C=C double bonds decreases. At the maximum concentration of CopOx NPs in the resin (0.1%), absorption is approximately 6%. Using differential dual-beam UV-Vis spectrometer, spectra were obtained for wafers made from composite lithographic resin with or without CopOx NPs (Figure 5b). All studied absorption spectra exhibited a local minimum near 335 nm and a local maximum near 375 nm. CopOx NPs, even at a concentration of 0.1%, had no significant effect on the spectra.

Using enhanced chemiluminescence, the effect of wafers made from composite lithographic resin containing or not containing CopOx NPs on hydrogen peroxide generation was studied (Figure 6a). Under control conditions, approximately 3 nM of hydrogen peroxide was formed. Adding composite lithographic resin sample containing no CopOx NPs to the medium resulted in approximately 5 nM of hydrogen peroxide. Adding composite lithographic resin sample containing CopOx NPs at concentration of 0.001% resulted in approximately 7 nM of hydrogen peroxide. At nanoparticle concentrations of 0.01% and 0.1%, approximately 11 and 16 nM of hydrogen peroxide were formed, respectively.

Using a CCA fluorescent probe, the effect of wafers made of composite lithographic resin containing or not containing CopOx NPs on the generation of hydroxyl radicals was studied (Figure 6b). It was shown that, under control conditions, approximately 20 nM of hydroxyl radicals are formed. When a sample of composite lithographic resin containing no CopOx NPs is added to the medium, approximately 27 nM of hydroxyl radicals are formed. When a sample of composite lithographic resin containing CopOx NPs at concentration of 0.001% is added to the medium, more than 30 nM of hydroxyl radicals are formed. However, no statistical differences were found between the PMMA group and the PMMA+CopOx NPs 0.001% group. Increasing the nanoparticle concentration in the polymer to 0.01% and 0.1% resulted in the formation of more than 40 and 55 nM hydroxyl radicals, respectively.

The impact of PMMA+CopOx NP composite plates on in vitro DNA oxidation was determined by quantifying 8-oxoguanine levels via a specific ELISA (Figure 6c). Under control conditions, approximately 1.5 molecules of 8-oxoguanine bases per 10⁵ guanines were formed in DNA. When a sample of composite lithographic resin containing no CopOx NPs was added to the medium, approximately two molecules of 8-oxoguanine bases per 10⁵ guanines of DNA were formed. Adding 0.001% CopOx NPs-containing composite lithographic resin sample to the medium produced approximately 2.5 molecules of 8-oxoguanine bases per 10⁵ DNA guanines, which is statistically higher than in the control group. At 0.01% and 0.1% nanoparticle concentrations, approximately 3.2 and 3.9 molecules of 8-oxoguanine bases per 10⁵ DNA guanines were formed, respectively.

The effect of wafers made of composite lithographic resin containing or not containing CopOx NPs on the formation of LRPS was studied using induced luminescence (Figure 6d). It was shown that under control conditions, long-lived reactive protein species are formed. When a sample of the composite lithographic resin containing no CopOx NPs was added to the medium, the amount of LRPS formed did not differ from the control. When sample of the composite lithographic resin containing CopOx NPs at concentration of 0.001% was added to the medium, 30% more long-lived reactive protein species were formed compared to the control. At concentration of nanoparticles in the polymer of 0.01% and 0.1%, 1.5 and 2 times more long-lived reactive protein species were formed relative to the control. Interestingly, the average half-life of long-lived reactive protein species in all groups was almost the same and equal to 4 h.

The influence of composite photolithographic resin materials with and without embedded CopOx NPs on the growth and development of E. coli suspension cultures was investigated (Figure 7).

In control experiments, the lag phase lasted approximately 4 h, with a logarithmic phase observed up to and including 16 h. In the presence of lithographic resin containing no nanoparticles, the lag phase also lasted approximately 4 h, with a logarithmic phase also observed up to and including 16 h. The stationary phase was achieved at a bacterial cell concentration 25% lower than in the control. In the presence of lithographic resin containing 0.001% nanoparticles, the lag phase lasted approximately 5 h. After this, a slight increase in bacterial numbers was observed, but a pronounced logarithmic phase never occurred. By 20–24 h, bacterial cell concentrations were 90% lower than in the control, but approximately twice as high as at the beginning of the experiment. In the presence of lithographic resin containing CopOx NPs at concentration of 0.01%, the lag phase lasted approximately 5 h, followed by slight increase in bacterial numbers without pronounced logarithmic phase. By 20–24 h, bacterial cell concentrations were 95% lower than in the control and approximately 30% higher than at the beginning of the experiment. In the presence of lithographic resin containing CopOx NPs at concentration of 0.1%, no statistically significant increase in bacterial numbers was observed by 24 h relative to the beginning of the experiment.

It was found that materials made from composite lithographic resin containing CopOx NPs significantly affect the growth rate of bacterial cells; however, this experimental setup does not allow us to determine the extent of the effect of the manufactured materials on the viability of bacterial cells. Flow cytofluorometry was used to evaluate the effect of materials made from composite lithographic resin containing CopOx NPs on the viability of bacterial cells (Figure 8). No significant decrease in bacterial cell viability was observed in the control and when grown with samples made from lithographic resin containing no nanoparticles. In the presence of lithographic resin containing CopOx NPs at all concentrations, both living and dead cells were observed. Moreover, the number of non-viable cells exceeded the number of living cells by orders of magnitude. Furthermore, in the presence of lithographic resins containing CopOx NPs, a broadening of the fluorescent signal in the range of 10³–10⁴ GMFI was observed.

An analysis of the histograms of bacterial cell distribution by the geometric mean value of PI intensity during incubation with samples of composite lithographic resin containing or not containing CopOx NPs allowed us to numerically determine the proportion of dead cells (Figure 9a) and the concentration of viable E. coli bacteria (Figure 9b). It was shown that, when grown in control medium and in the presence of samples not containing nanoparticles, no significant decrease in the viability of bacterial cells was observed. In the presence of lithographic resin containing CopOx NPs at a concentration of 0.001%, about three-quarters of the cells were nonviable. In the presence of lithographic resin containing CopOx NPs at concentrations of 0.01% and 0.1%, the proportion of nonviable cells averaged over 95%, and in some experiments the value was closer to 100%. It was shown that, when bacteria were grown without external stimulation (control), approximately 6 × 10⁷ cells/mL of viable cells were detected in the culture medium. When nanoparticle-free resin samples were added to the culture medium, approximately 3 × 10⁷ cells/mL of viable cells were detected. In the presence of lithographic resin containing CopOx NPs at all concentrations, the viable cell concentration did not exceed 10⁶ cells/mL.

It was shown that plates made of composite lithographic resin containing oxide nanoparticles significantly affect the growth and development of bacterial cells. We investigated whether the prepared samples would be toxic to mammals; to this end, we studied the effect of plates made of composite lithographic resin on a HSF cell culture (Figure 10). Transmitted light microscopy did not reveal any differences between all the studied cell groups (Figure 10a–c). It was shown that, on all the studied materials (culture plastic, lithographic resin samples containing and not containing nanoparticles), the cells attached to the surface and spread out on it. The viability of the cell cultures was studied using fluorescence microscopy (Figure 10d–f). In general, very few non-viable cells were detected upon visual inspection of the images. It is possible that non-viable cells were slightly more common when cultured with samples containing the highest concentration of CopOx NPs. To illustrate this potential pattern, we specifically selected photograph that contains two dead cells (Figure 10f).

Stream-based image processing revealed that the proportion of viable cells exceeding 95% was observed in all groups except for the samples with the highest concentration of CopOx NPs (Figure 10g). In the group of samples with the highest concentration of CopOx NPs, cell viability was greater than 90% but less than 95%. It should be noted that the groups did not have statistically significant differences from each other; therefore, we cannot conclude that our samples reduce cell viability. The degree of cell spreading on the surfaces was assessed (Figure 10h). It was shown that cells on the surface of wafers made of composite lithographic resin containing or not containing CopOx NPs occupied 15–20% more space than on the control surfaces. The largest cell area was occupied by cells on the surface of the composite lithographic resin containing CopOx NPs at concentration of 0.001%. The effect of our manufactured materials on cell nuclear size was assessed (Figure 10i). It was shown that cell nuclear size did not differ across all study groups.

4. Discussion

Laser ablation and laser fragmentation allow precise control over the size of nanoparticles and their main characteristics [40]. Nanoparticles consisting of copper oxide have previously been obtained many times using the laser ablation method; these were mainly spherical nanoparticles [41], less often ellipsoid-shaped nanoparticles [42] or filiform nanoparticles [43]. Typically, filiform nanoparticles consist only of copper oxide. Such particles are characterized by a broad absorption spectrum with maximum at 660 nm [44], which is also typical for the nanoparticles obtained in this research (Figure 1c). In this study, we obtained grain-shaped nanoparticles (Figure 1d), which are pointed at the ends; zones with varying degrees of contrast are visible in the pointed areas. It should be noted that such nanoparticles are formed only in the selected range of experimental setup settings. It can be assumed that the grains are formed by the condensation of thread-like nanoparticles; however, this should produce products of varying shapes and sizes due to the different conditions within the reactor [45]. The nanoparticles we obtained have a narrow, monomodal size distribution (Figure 1a) and fairly narrowly distributed electrokinetic potential (Figure 1b).

A method for transferring the obtained CopOx NPs into lithographic resin has been developed. Optically transparent products (Figure 2a) with minimum achievable resolution of 50 μm (Figure 2b) can be manufactured from composite lithographic resin with nanoparticles. It is known that the addition of nanoparticles to resins for MSLA printing can lead to the appearance of defects in the final products [46]. The addition of nanoparticles to a polymer can complicate its processing [47] and interfere with polishing [48]. Products manufactured from composite lithographic resin containing CopOx NPs polish remarkably well and have an ultra-low surface roughness after polishing (Figure 3). Mechanical problems, including those associated with processing and polishing, are often associated with the uneven distribution of nanoparticles in the polymer matrix [49]. It should be noted that the uneven distribution of nanoparticles in polymers is extremely common [50,51,52,53]. This is often associated with the aggregation of nanoparticles in the polymer [54], less often with the adhesive properties, charge and surface energy of the polymer chains and the rheological properties of the nanoparticle–polymer system [55]. At the same time, using modulation-interference microscopy, it was established that there are regions with uniform incursion in the samples. At microscope wavelength of 405 nm, the refractive index of copper oxide is 2.63 [56], while for polymethyl methacrylate it is 1.50 [57]. Such large difference in refractive indices makes it possible to distinguish between the nanoparticles and the lithographic resin. Interestingly, at a CopOx NP concentration of 0.1%, extended alternating regions with uniform phase incursion are observed in the samples (Figure 4). This arrangement of zones is partly similar to the arrangement of lines in diffraction gratings [58]. The presence of self-organized periodic structures similar to diffraction gratings opens up prospects for using these composites as active elements in integrated optics, for example, as diffraction gratings for spectral optical sensors [59], as well as for creating metasurfaces that control the phase [60,61] and polarization of light [62,63] in micro-optical devices. The ability of such structures, formed in situ during photopolymerization, to modulate light makes this material a promising platform for the one-step fabrication of functional polymer elements for photonics.

One of the key problems in lithographic photo printing is the incomplete polymerization of resins, which is often estimated by the number of double bonds in the final product [64]. With the incomplete polymerization of the resin in the final product, there is release of residual monomers, which leads to an increase in the toxicity of the material [65]. On the other hand, incomplete polymerization affects the mechanical, tribological and strength characteristics of the product [66]. A high degree of resin polymerization is achieved by changing the duration of exposure to UV radiation and increasing the concentration of the photoinitiator. In this case, it is necessary to find the optimal time of exposure to UV radiation and the optimal concentration of the photoinitiator. In terms of why this is so, with an infinite time of exposure to UV radiation, the concentration of unpolymerized components will obviously tend to zero, but, at the same time, the number of damaged polymer structures will increase [67]. In this regard, it is important to find the “golden mean” at which the degree of polymerization of the lithographic resin is already quite high, and the number of damaged polymer structures is still quite small [68]. A similar balance must be sought with the photoinitiator concentration. At low photoinitiator concentrations, achieving significant polymerization is quite difficult [69]. At high photoinitiator concentrations, the mechanical properties of the final product will suffer [70]. It should also be taken into account that the photoinitiator is usually the most difficult-to-manufacture and expensive component of the lithographic resin [71]. In this study, it was shown that the addition of CopOx NPs increases the degree of polymerization (Figure 5). The observed effect can be explained, according to the literature, by several possible mechanisms typical for nanodispersed fillers. First, CopOx NPs, like other transition metal nanoparticles, can act as catalysts for photoinitiator decomposition or generate active radicals due to their photocatalytic properties under the UV radiation used in printing [72,73]. Second, they can promote the cross-linking of polymer chains by acting as multifunctional centers through the interaction of their surface groups with the reactive ends of macroradicals [74]. Third, the alteration of the composite’s rheological properties and the restricted diffusion of growing chains in the presence of nanoparticles may reduce the probability of termination reactions, thereby increasing the average molecular weight of the polymer [75,76].

Moreover, this phenomenon may be based on the ability of CopOx NPs to generate reactive oxygen species (ROS) [77] and oxidize organic molecules under the influence of light [78]. The discovered phenomenon is important for the technological process of product manufacturing in itself, regardless of other beneficial properties that lithographic resins acquire with the addition of CopOx NPs. Beyond imparting targeted functional properties to the material, the nanoparticles perform an important technological function by serving as an efficient agent that modifies the kinetics and degree of photopolymerization. This paves the way for creating safer and more processable composite materials for 3D printing, offering the potential to optimize process parameters such as exposure time and the concentration of expensive photoinitiators.

In this study, it was shown (Figure 6a,b) that ROS generation in the presence of CopOx NPs occurs even in the dark. The longest-lived ROS, hydrogen peroxide, and the most reactive ROS, hydroxyl radicals, are formed [79]. The ability to influence ROS formation in aqueous systems is inherent in most transition metals [80]. First of all, this is associated with the ability of transition metals to both donate and accept an electron [81]. The most well-known reaction in which ROS are formed in the presence of transition metals is the Fenton reaction [82]. On the one hand, an increase in the concentration of ROS can have both direct [83] and indirect [84] positive effects on biological systems; on the other hand, an increase in the concentration of ROS above the level of antioxidant protection leads to significant problems [85]. In this study, it was shown that, in the presence of lithographic resin, more intense damage to DNA and proteins is observed (Figure 6c,d). Moreover, CopOx NPs somewhat sensitize the action of the resin.

It has been shown that lithographic resins containing CopOx NPs significantly reduce the rate of growth and development of E. coli bacteria (Figure 7), leading to a loss of viability in bacterial cells (Figure 8 and Figure 9). The antibacterial action of CopOx NPs is realized through several alternative mechanisms of action, often characteristic of other nanoparticles. The most frequently discussed mechanism of antibacterial activity is the contact interaction of CopOx NPs with the surface of bacterial cells (“contact killing”) [86]. Another frequently discussed mechanism of antibacterial activity of CopOx NPs is the release of copper cations into the medium, which subsequently interact with cell wall molecules [87], and can be absorbed and accumulated by the bacterial cell interacting with intracellular targets [88]. Some researchers associate the antibacterial action of CopOx NPs with the overproduction of ROS [89], including due to photocatalytic activity [90]. Despite the pronounced antibacterial properties, lithographic resins containing CopOx NPs did not have significant effects on the viability of eukaryotic cells, their surface spreading, or nuclear size (Figure 10). Although the observed increase in the concentration of reactive oxygen species near the material confirms its pronounced antibacterial activity, the localized and controlled nature of this process is key for biomedical applications. Our data indicate that the generated ROS levels are spatially limited and do not lead to oxidative stress sufficient to damage mammalian cells, which is clearly confirmed by high viability rates (over 95%) in cytotoxicity tests. The observed selective effect of inducing bactericidal concentrations of ROS upon contact with material surfaces, while remaining safe for eukaryotic cells, is consistent with current understanding of the mechanism of action of copper oxide-based nanomaterials described in a number of studies [91,92]. Thus, the identified imbalance between antibacterial efficacy and cytocompatibility can be explained by the different sensitivity of prokaryotic and eukaryotic cells to oxidative stress, as well as differences in the contact area with the active surface of the material.

CopOx NPs have previously been shown to exhibit cytotoxicity relative to mammalian cells. It is important to note that these were almost always transformed cell cultures. Thus, the cytotoxicity of CopOx NPs has been demonstrated against transformed cultures of MCF-7 [93], L929 [94], A549 [95], SNU-16 [96], HT-29 [97], AGS [98], LoVo, MKN-45, HDF [99] and others, while the effect of CopOx NPs against normal cells was less pronounced. It is possible that materials and pharmaceuticals based on CopOx NPs will find applications in cancer therapy.

5. Conclusions

• A series of photopolymerizable resins based on a methacrylate-like rubber and containing CopOx NPs grains at concentrations of 0.001%, 0.01%, and 0.1% was successfully synthesized.
• The resulting polymeric materials demonstrated a range of valuable properties, which we attribute to the unique shape and morphology of the incorporated nanoparticles.
• The materials exhibited excellent polishability, making them suitable for the fabrication of optical elements.
• It was found that the addition of CopOx NPs enhanced the polymerization conversion of the resin. Specifically, in the sample containing 0.1% nanoparticles, the number of unreacted monomers decreased by almost a factor of four, representing a significant technological advantage for the manufacturing process.
• Modulation interference microscopy revealed that samples with 0.1% CopOx NPs contained extended, alternating regions with a uniform phase shift, a structure reminiscent of the lines in a diffraction grating. This suggests the material’s potential for applications in modulation-interference optics.
• The developed composites demonstrated pronounced antibacterial properties while showing no acute cytotoxicity towards mammalian cells.
• Owing to their combination of optical, processing, and biological characteristics, these materials show promise for applications in biomedicine, the food industry, and agriculture.