Introduction

Zirconia, Also known as zirconium dioxide (ZrO2), is a white crystalline material that exists as an oxide of zirconium1,2. The Zirconia systems consist of about 2.5–3.5% of Yttrium oxide (Y2O3) is called Yttria-tetragonal zirconia polycrystal (Y-TZP) and is widely used in dental and biomedical applications due to its excellent mechanical properties3,4.The standard density of zirconia is 6 g/cm3, while its theoretical density is calculated as 6.51 g/cm3. The surface strength of the material increases with the reduction in particle area, which is to approximate the theoretical and practical values of the zirconia densities5. The ZrO2-nanoparticles (ZrO2NPs) have resulted in outstanding developments in industrial, biomedical, and dental applications in recent years. Industrial uses of Zirconia NPs (ZrO2-NPs) include applications in foundry sands, refractories, and ceramics. ZrO2NPs are also applied in biomedicine and dentistry, e.g., in the coatings of hip joints end-prostheses, dental prostheses, bone defect materials, biosensors, and drug-loaded carriers6,7,8,9. Moreover, the NPs are difficult to degrade and can easily accumulate in organs. Because of their huge surface areas, NPs impact the enzymes and other proteins, thereby disrupting a series of biological processes in the body10,11. Thus, the extensive use of ZrO2-NPs may lead to uncalled for environmental and human exposure, and raise concerns about their potential impact and toxicity, especially, also through occupational exposure during manufacturing. ZrO2 NPs have also been utilized in various applications as antimicrobial agents, nanopowder filling, sintering raw material, nanocoating, and anticancer and antioxidant agents. The functionalization of ZrO2 NPs as hybrid substances has received increased attention in tissue-engineering scaffolds, microfluidic devices, bone prostheses, drug delivery carriers, as well as their uses in other medical devices owing to their bionics, biocompatible and physico-mechanical properties12,13. Bio-medically, the ZrO2-based materials are efficiently used in meat packaging14, dentistry15, artificial scaffolds for body tissues16. At the tissue levels, ZrO2 was observed to be biocompatible as equivalent to the titanium. Cultured osteoblasts proliferate and differentiate on zirconia without causing any adverse effects. The ZrO2 is also a bioinert ceramic material, because, after implantations, it showed only morphological fixation with its surrounding tissues without creating any biological/chemical bonding. Notwithstanding, towards antimicrobial activity of the Zirconia NPs, the bacterial adhesion, towards bacterial action of infecting organisms, in some cases, led to bacterial colonization and biofilm formations. Circumstantially, the general source of bacteria includes the environment of the hospitals’ operation rooms, in certain cases the hospital premises and patients, as well as surgical equipment for facilities lacking fool-proof sterility. The bacterial infection during, or after implantations can lead to early complications or permanent contamination, wherein remedy for both these situations accounts for an enormous medical cost, that includes the surgical removal of the infected implant becoming the sole solution in some cases17. Searching for new, inherent, and modified antimicrobial agents as a robust antimicrobial implant support, and as a day to day use antimicrobial material is a priority. Y2O3 does not have antibacterial capacity18, but it helps improve the fracture toughness of zirconia by mixing with it19,20. The antibacterial activity of ZrO2 NPs can be attributed to the interaction of surface-ionized ions with negatively charged cell membranes. The resulting change in the permeability of the bacterial cell membrane causes subsequent leakage of the cytosol, leading to the death of the bacteria21,22. Biofilm acts as a chemical barrier by adsorbing the harmful reactive oxygen species from all treatment of ZrO2 NPs from reaching the cell surface, thereby decreasing the effect of ROS23. Zirconia (ZrO2) nanoparticles offer both improved mechanical stability and antibacterial function, particularly when integrated into other materials like dental composites. Zirconia’s inherent strength and biocompatibility contribute to mechanical enhancements, while its positive surface charge and potential for oxidative stress generation enable it to disrupt bacterial cell walls and inhibit growth. The antibacterial efficacy often increases with higher concentrations of zirconia nanoparticles, making them a promising material for applications such as dental fillings and restorative materials. Dental composite has been widely used in dentistry because of its desirable optical properties. However, several problems remain unsolved such as polymerization shrinkage, unsatisfactory mechanical properties and bactericidal function of dental composites24,25. The aim of this study is provides information on the effect of the ZrO2, binary mixture (ZrO2+ Y2O3),and triple mixture (ZrO2 + Y2O3 + Al2O3) as antibacterial agents against some pathogenic bacteria, inhibitory effect on biofilm formation and anticancer effect.

Materials and methods

Preparation of the nanocomposite powder Zirconia – Yttria system

Zirconia-Yttria binary system was prepared by adding the matrix powder (ZrO2) stabilized (3% mol. Y2O3) with four different concentrations (2%, 5%, 7%, and 10% weight by weight), and dispersed by ultra-sonication with ethyl alcohol (1 h), followed by stirring and drying at 1500 °C for 2 h. The purpose of this process was to blend the matrix and the homogenize the Zirconia reinforcement material. A high dispersibility of the Zirconia-Yttria system was achieved. Following oxidized materials were employed for dental mixture preparation (Table 1).

Table 1 Materials used in Preparation of the dental mixture.
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Forming process

Specimens were formed by a semi-dry pressing method, binder was added to all specimens, and formed by a mold and pressed by a hydraulic press under a pressure of 2.5 Tons. Zirconia- yttria system binary and tertiary (Yttria and alumina) composite nanopowders were prepared according to the percentage listed (Table 2). The binder solution was prepared according to the dilution law as shown in the following Eq. 126:

$${\text{M1 }} \times {\text{ V1}}\,=\,{\text{M2 }} \times {\text{ V2}}$$
(1)

Where;

M1: initial solution concentration (before dilution).

M2: final solution concentration (after dilution - more solvent is added).

V1: volume of the initial solution (before dilution - before adding the solvent).

V2: volume of the final solution (after dilution - after adding the solvent).

Table 2 Symbols and code for partially stabilizer zirconia systems.
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Physical properties

Following equations (Eqs. 1–4) provided the apparent porosity, water absorption ratio, and bulk density of the prepared materials in accordance with the ASTM protocol27.

$$\:(\text{A}.\text{D})\:=\:\frac{\text{W}\mathsf{d}}{\text{W}\mathsf{s}-\text{W}\text{i}}\times \text{D}$$
(2)
$$\:(\:\text{A}.\text{P})\text{\%}\:=\:\frac{Ws-Wd}{\text{W}\mathsf{s}-\text{W}\text{i}}\times\:100$$
(3)
$$\:(\text{W}.\text{A})\:\text{\%}\:=\frac{Ws-Wd}{Wd}\times\:100$$
(4)

\(\:\text{W}\text{s}\::\:\text{t}\text{h}\text{e}\:\text{m}\text{a}\text{s}\text{s}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{w}\text{a}\text{t}\text{e}\text{r}-\text{i}\text{n}\text{f}\text{i}\text{l}\text{t}\text{r}\text{a}\text{t}\text{e}\text{d}\:\text{s}\text{p}\text{e}\text{c}\text{i}\text{m}\text{e}\text{n}\text{s}\:\)

\(\:\text{W}\text{d}\::\:\text{t}\text{h}\text{e}\:\text{d}\text{r}\text{y}\:\text{s}\text{p}\text{e}\text{c}\text{i}\text{m}\text{e}\text{n}\text{s}\:\text{m}\text{a}\text{s}\text{s}.\)

\(\:\text{W}\text{i}:\:\text{m}\text{a}\text{s}\text{s}\:\text{o}\text{f}\:\text{s}\text{p}\text{e}\text{c}\text{i}\text{m}\text{e}\text{n}\text{s}\:\text{b}\text{e}\text{i}\text{n}\text{g}\:\text{i}\text{m}\text{m}\text{e}\text{r}\text{s}\text{e}\text{d}\:\text{i}\text{n}\:\text{w}\text{a}\text{t}\text{e}\text{r}\)

\(\:\text{D}:\:\text{w}\text{a}\text{t}\text{e}\text{r}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}\:(1\:\text{g}/\text{c}\text{m}^{3}.\)

Structural properties

  1. 1.

    Field Emission-Scanning Electron Microscope (FE-SEM).

    Scanning electron microscope was observed through the FE-SEM (Nova 200 Nano Lab) machine.

  2. 2.

    X-Ray Diffraction:

    Shimadzu-6000 X-ray Diffractometer, Jaban located at the Nano Technology Center, University of Technology was employed with graphite monochromatic and copper anode for K radiation. At a wavelength of 0.154 nm, the X-ray tube was operated at 40 kV and the 30mAhe scanning range was (20.0000–70.0000) degrees with a step size of 0.02° and 0.10 s for each step.

Bacterial isolates

Pathogenic bacterial isolates i.e. E. coli, S. aureus, and Strep. mutants were obtained from the microbiology laboratory of the Medical City Hospital, Baghdad, Iraq. Every patient was informed of the goal of these studies and consented to the protocol. Isolates were identified using the VITEK system (VITEK, Biomérieux, Marcy-l’Etoile, France), kindly supplied by the Department of Biotechnology/ College of Applied Science, University of Technology, Baghdad, Iraq. Informed Consent was obtained from all subjects and/or their legal guardian. S. mutans is frequently used in bacterial adhesion tests, particularly in studies related to dental materials and biofilm formation. It’s a primary bacterium associated with dental caries, making it a relevant subject for investigating how bacteria adhere to and colonize different surfaces, and we used two types of bacteria E. coli and S. aureus because they were available for the purpose of making a comparison between Gram-positive and Gram-negative bacteria.

Antibacterial activity

The effect of Nano-zirconia (Zirconia –Yttria(Systems on the growth of bacterial isolates was evaluated by the agar well diffusion method. The McFarland standard was used to obtain suspensions at 1.5 × 108 colony-forming units (CFU) mL− 1. Mueller–Hinton agar medium plates were swabbed with each bacterial suspension, and then wells at 8 mm diameter were punched out of each agar plate using a sterilized well cutter. The wells were loaded with 80 µL from different concentration (25, 50, 75, and 100)µgmL− 1 of all treatments for both tested bacteria E. coli and S. aureus. Deionized water was used as the control negative. After incubation at 37 °C for 24 h. Results were obtained by measuring the inhibition zone28,29,30. Three replicates were made for each experment.

Bacterial adhesion tests

To perform an adhesion test between epithelial cells isolated from the oral cavity and bacteria cells S. mutans, equal volumes (100) µl of both solutions (bacterial suspension + epithelial cell suspension) were mixed in sterile Eppendorf tubes containing different concentrations of zirconia to study its effect on the adhesion rate. They were well and left at a temperature of 37 ° C for a whole hour, then out the tubes and mixed them with a micro pipe and took a drop of this solution and spread it on a clean glass slide. The slides were left to dry at room temperature, then they were stained with Kamza dye (2%) for 3 min, and the slides were washed with water It was then left to dry again, at room temperature, and then examined using a light microscope at a magnification of (40X) and the adhesion percentage was calculated from a thousand examined epithelial cells according to the Eq. 5 below.

$$\:\text{I}\text{R}\:=\:\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{a}\text{d}\text{h}\text{e}\text{r}\text{e}\text{n}\text{t}\:\text{e}\text{p}\text{i}\text{t}\text{h}\text{e}\text{l}\text{i}\text{a}\text{l}\:\text{c}\text{e}\text{l}\text{l}\text{s}}{\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{e}\text{p}\text{i}\text{t}\text{h}\text{e}\text{l}\text{i}\text{a}\text{l}\:\text{c}\text{e}\text{l}\text{l}\text{s}\:\left(1000\right)}\text{x}\:100\text{\%}\:$$
(5)

Where: 1000: examined cells; IR: inhibition Rate %.

Flow cytometry assay

Flow cytometry was employed to evaluate the generation of reactive oxygen species (ROS) in bacterial cells following treatment with ZrO2, ZrO2 + Y2O3, and ZrO2 + Y2O3 + Al2O3 at concentration 50 µg/mL− 1. Briefly, bacterial strains E. coli and S. aureus were exposed to the ZrO2, ZrO2 + Y2O3, and ZrO2 + Y2O3 + Al2O3 for 24 h at 37 °C. After incubation, the bacterial cells were collected by centrifugation and resuspended in phosphate-buffered saline (PBS) to obtain a homogeneous suspension with a final volume of 1 mL. Subsequently, the cells were incubated with 1.5 mL of 50 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) at 37 °C for 45 min to enable ROS detection. The ROS levels were then quantified using a flow cytometer (FACSVerse, BD Biosciences).

Bacterial biofilm formation assay

Bacterial strains were inoculated into 24-well plates at a concentration of 1 × 10⁶ CFU/mL and incubated in the presence of Nano-zirconia (Zirconia –Yttria(Systems, for 24 h. Following treatment, the wells were gently washed with PBS to remove non-adherent bacterial cells. The remaining attached biofilm was stained with 0.1% crystal violet (C.V.) and subsequently rinsed with PBS to eliminate excess dye. To quantify biofilm formation, 0.2 mL of 95% ethanol was added to each well to solubilize the bound CV, followed by incubation with shaking for 2 h. The absorbance was then measured at 595 nm to assess biofilm density.

Metabolic activity for bacterial biofilm formation

To assess the bactericidal effect of Nano-zirconia System on biofilms, the MTT assay was employed. After completing the biofilm formation procedure described above, 150 µL of MTT solution at concentration (4 mg/mL) was added to each well, followed by incubation with shaking for three hours. Subsequently, DMSO was added to dissolve the resulting formazan crystals. The absorbance was measured at 570 nm for each well, and the percentage of bactericidal activity was calculated accordingly.

Cytotoxicity test

MTT assay was performed using 96-well plates to determine the cytotoxic effect of different treatments against Rat embryonic fibroblast (REF) cells (Purchased from Sigma). REF cells were seeded at 1 × 104 cells/well31,32. After 24 h the attached monolayer was obtained. Furthermore, the cells were treated with the tested compounds at different concentrations. Cell viability was measured after 24, 48, 72 h of treatment by removing the medium, adding 28 µL of 2 mg/mL solution of MTT, and incubating the cells for 2.5 h at 37 °C. After removing the MTT solution, the crystals remaining in the wells were solubilized by adding 130 µL of DMSO (Dimethyl Sulphoxide) and then incubating at 37 °C for 15 min by shaking. The absorbency was determined on a microplate reader at 492 nm; the assay was performed in triplicate. The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as the following Eq. 633.

$${\text{Inhibition rate}}\,=\,{\text{A}} - {\text{ B}}/{\text{A}}*{\text{1}}00$$
(6)

Where A is the optical density of control, and B is that of the samples.

Ethics approval and consent to participate

All the protocols used in these experiments were approved by the Human Care and Ethics Committee, department of Biotechnology, college of Applied Sciences, University of Technology—Iraq (Ref. No. 022/CAS/2022).“all methods were carried out in accordance with the Guidelines of the U.S. National Institutes of Health (NIH Publication No. 86–23, Revised 1996) “.

Statistical analysis

The data were analyzed with one-way analysis of variance (ANOVA) using SPSS v.24 software at a 0.05 level of statistical significance. The data were presented as mean ± SD. All experiments were carried out in triplicate.

Results and discussion

Scanning electron microscopic (SEM) analysis

Figure 1 shows images of a scanning electron microscope, From (Fig. 1A) it was clear that the zirconia particles were sintered with yttria and alumina nanoparticles in the electrical furnace at 1570 °C as a result of the clear cohesion of the particles. (Fig. 1B) shows the homogeneous distribution of nanoparticles without the appearance of aggregates due to the homogeneous mixing of the Nano powdered materials with good sintering factors high sintering temperature of 1570 °C and 2 h as a socking time .Homogeneous distribution of pores, Pores were small, isolated, and not connected, with each other. this is a very important point for artificial dental since dental is at all-time exposed to saliva and liquids. Closed, non-apparent pores it is suitable for artificial dental works, as shown in (Fig. 1C). The percentage of porosity was low and this is consistent with the results of the apparent porosity. Small pores isolated from each other are considered as a good factor for artificial dental. Teeth in the mouth cavity are immersed in saliva continuously and are exposed to different acidic and alkaline liquids. So, the presence of open pores negatively affected the teeth and the penetration of saliva and liquids through the apparent pores inside the artificial dental body which is suitable invariant to the growth of bacteria inside artificial dental. As a result, contamination and damage would occur to that tooth. (Fig. 1D), showed the surfaces of the granules of the nanoparticles take a scattered and irregular spherical shape, which some together and forms large particles.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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SEM for zirconia with yttria alumina in a thermal well at 1570 °C (A). (B) Homogeneous mixing of the powdery materials. (C) Pores were small and not connect. (D) Showed surfaces of the granules of the alumina nanoparticles take a scattered and irregular spherical shape.

Figure 2 shows appearance of tetragonal zirconia phase in high proportions, as it is considered the main phase. Hexa-alumina phase (corundum) appeared in a good proportion at the peaks of (33:61). So, (zirconia - yttria - alumina) system was prepared with oxide of the Nano powder system. Monoclinic phase was appeared in a very low percentage, and this indicated improved properties due to the effect of nanoparticles.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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XRD patterns of ZrO2 + Y2O3 + Al2O3.

Antibacterial activity against pathogenic bacteria

The results in the (Table 3; Fig. 3) showed that the inhibitory effect of nanoparticles increased with the increase in concentration. The diameter of inhibitory zone shows the degree of susceptibility of microorganisms. All the treatments had a good antibacterial properties, Gram-positive bacteria were the most affected compared to Gram-negative bacteria. This is evident from the diameters of the inhibition zones, where the highest inhibition zone diameter was recorded at (27.33 mm) for the ternary mixture, followed by (24.66 mm) diameter for the inhibition zone for the binary mixture, and finally (24 mm) for the inhibition zone diameter for zirconium alone, for Staph.aureus bacteria. While the highest inhibition zone diameter was (22.66 mm) for the ternary mixture in intestinal bacteria E.coli, followed by (21 mm) for the inhibition zone diameter for the binary mixture, and finally, the lowest inhibition zone diameter was recorded for zirconium alone, which was (20 mm) .The biological activity of nano zirconia can be attributed to several mechanisms. The increased surface area of nano zirconia allows for more interactions with surrounding molecules such as proteins, enzymes, and ions. This high surface reactivity enables nano zirconia to participate in various biological processes34. Nano zirconia can also release Zr4+ ions into its surrounding environment. These ions can interact with biological molecules and affect cellular processes. For example, Zr4+ ions can influence enzyme activity, gene expression, and cell signaling pathways35. Nano zirconia particles can induce oxidative stress in biological systems. Owing to their high reactivity, they can generate reactive oxygen species (ROS) when in contact with biological fluids or cellular components. ROS can lead to cellular damage and trigger inflammatory responses. Nano zirconia particles possess a surface charge that can interact with charged molecules, such as proteins and DNA. This interaction can affect the conformation, activity, and stability of these biomolecules, thereby influencing biological processes36. The results show that the triple mixture was the best in terms of its effect on the growth of the pathogenic bacterial species used in this study, as it recorded the highest diameters of the inhibition zones. As previously explained, it is known that zirconium has an antibacterial effect, and that adding yttria to it increases its stability. Therefore, we find that the effect of the binary mixture increased slightly compared to what zirconium showed alone, but when mixing the triple mixture, we find that this showed a difference in the results, due to the appearance of the synergistic effect of the materials together.

Various investigations showed that the cell wall of bacteria has a great role against different materials. Generally, in Gram (+ ve) and (− ve) bacteria, its behavior is different. NPs via the assistance of electrostatic interaction, can connect to the outer membrane of bacteria and cause disturbing the membrane, suppressing periplasmic enzymes, rupture of bacteria cells in the culture media, and ultimately restraining DNA, RNA, and protein synthesis. The NPs preferably attack the respiratory chain, the cell division finally leading to cell death37. Furthermore, functionalized NPs release their metallic ions in the vicinity of the cell wall which would diffuse into the cell and then based on the toxicity mechanism cause injury to bacteria38. Because of their complex cell wall structure, Gram –bacteria are more resistant to NPs. Due to the inability to obtain references similar to this work, we did not discuss the results as required. ZrO2 nanoparticles lead to an increase in the formation of reactive oxygen species (R.O.S.s), leading to the destruction of bacterial cells. These elevated R.O.S. have many effects on the bacteria, such as lipid peroxidation. As a result, the collapse of bacteria’s cell wall leads to the death of bacteria with increasing concentration of ZrO2 nanoparticles. It is important to note that the ability of nanoparticles to bind to bacteria depends on the available surface area. Nanoparticles’ ability to penetrate bacteria is not well understood. However, according to specific research, nanoparticle treatment of bacteria has been linked to alterations in cell membrane shape. Due to their distinctive shape, nanoparticles have been proven in general to be able to penetrate bacteria. The size of nanoparticles has a direct relationship with their diffusion over the membrane of bacteria. Bacterial membrane damage is more likely to occur when nanoparticles are smaller. Ion channels and transporter proteins have facilitated nanoparticle transportation across the plasma membrane. Cell death is caused by zirconium ions having a positive charge. A similar result was obtained with silver nanoparticles39. In contrast, microorganisms have a negative charge, while metal oxides do. Metal oxide nanoparticles induce oxidation in microorganisms, which results in their death40. Antibacterial properties decrease over time as active agents are depleted and bacteria develop resistance or tolerance, leading to reduced effectiveness under repeated challenges in physiologically relevant conditions. While some antibacterial agents can show long-term stability in storage, their efficacy in a dynamic biological environment is limited by factors like bacterial growth rate, environmental stress, and inherent bacterial mechanisms like persistence and biofilm formation that reduce the impact of the antibacterial effect. The factors affecting antibacterial properties over time are the concentration of the active antibacterial agent can decrease due to degradation, consumption by the bacteria, or interactions with the physiological environment, bacteria evolve to resist antibiotics through genetic changes, a process known as antimicrobial resistance (AMR), which is a natural and continuous phenomenon, even when fully susceptible, bacteria can survive antibiotic exposure by entering a dormant, slow-growing state called persistence. These persisters can survive under stress conditions, including the presence of antibiotics, and can later resume growth and cause infection relapses, the effectiveness of an antibacterial agent is influenced by the physiological state of the bacteria, with some agents being less effective against non-growing or stress-induced bacteria, the formation of biofilms creates a self-protective, gel-like matrix that limits the diffusion of antibacterial agents and provides a protected environment for bacteria to survive and develop resistance, repeated exposure to antibacterial agents allows for the selection and enrichment of more resistant strains, while the active agents themselves are gradually depleted, leading to a progressive loss of effectiveness, and the overall antibacterial effect is not sustained; rather, the challenge shifts to dealing with bacteria that have developed resistance or persistence. This can result in infections that are difficult or impossible to treat effectively, increasing risks of severe illness and death41.

The relationship between a substance’s antibacterial effects and its cytotoxicity toward host cells is a critical trade-off, where desired antibacterial activity can inadvertently cause harm to host cells, especially at higher concentrations. A substance with good antibacterial activity and low host cell cytotoxicity is highly desirable for biomedical applications, but the ideal balance is difficult to achieve. The key is to develop agents that are selective for bacteria over host cells, a characteristic that is often dependent on the agents’ mechanisms of action and interaction with cell. Factors influencing the relationship: (*) Concentration: A substance might be harmless at low concentrations but toxic at higher concentrations. (*) Mode of Action: Substances with selective membrane-disrupting abilities are more likely to be host-cell-compatible, as they target specific bacterial structures rather than host cells.(* ) Bacterial vs. Host Cell Characteristics: The differences in bacterial cell walls and eukaryotic cell membranes allow for selective targeting.(*) Bacterial Factors: Some bacteria, like S. aureus can cause cytotoxicity through the production of toxins or by becoming intracellular.(*) Bacterial-Cell Interactions: How bacteria interact with host cells during an infection can significantly alter both antibacterial and cytotoxic outcomes, highlighting the importance of complex, realistic models for testing novel agents. Strategies for achieving selectivity(*) Targeted Design: New compounds can be designed to target specific bacterial features, minimizing interaction with host cells.(7) Host-Cell-Compatible Peptides: Antimicrobial peptides (AMPs), such as d-CONGA, have been developed with a focus on being host cell-compatible, meaning they have antibacterial activity without being toxic to host cells.(*) Nanomaterials: Some silver nanoparticles can exhibit antibacterial properties without causing damage to host cells at their effective bactericidal concentrations42. Zirconia based on its biocompatible properties & improved Osseo integration, lesser bacterial adhesion takes importance in new generation of dental implants. From the various in vivo and in vitro studies zirconia seems to be a bio inert material which supports the implementation of this material in dental implant ology43.

Table 3 Effects of all treatments on the growth of pathogenic bacteria.
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Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Antibacterial effect of ZrO2, ZrO2 + Y2O3, and ZrO2 + Y2O3 + Al2O3) against S. aureus and E. coli. (A) Control untreated bacteria, (B) 25 µg/mL− 1 treated bacteria, (C) 50 µg/mL− 1 treated bacteria, (D) 75 µg/mL− 1 treated bacteria, and (E) 100 µg/mL− 1 treated bacteria.

Several previous studies support the findings of our research regarding the antibacterial efficacy of nanoparticles. For instance, research by Khan et al.44 demonstrated that Ag-Zr nanoparticles with graphene oxide exhibited significant antibacterial activity against Gram-positive bacteria, similar to our observations with zirconia nanoparticles. Additionally, [Aktug et al. (2021] found that the combination of nanoparticles with other materials enhanced their antibacterial properties, corroborating our results with the ternary mixture. These studies highlight the importance of nanoparticle size, surface area, and charge in influencing antibacterial effectiveness, aligning well with our findings that smaller and more reactive nanoparticles lead to greater inhibition zones. Furthermore, the mechanisms by which nanoparticles induce oxidative stress in bacteria, as noted in prior research, align with our observations on the role of reactive oxygen species in bacterial cell death. Collectively, these studies reinforce the potential of utilizing nanoparticles, particularly zirconia-based materials, in developing effective antibacterial treatments. This is consistent with our results, which demonstrated that the combination of zirconium nanoparticles (ZrO2) with other agents exhibited a more potent antibacterial effect than either component alone. As shown in Table 4, the synergistic interactions of zirconium nanoparticles with various materials enhanced their antibacterial properties, particularly against Gram-positive bacteria. This reinforces the notion that the inclusion of zirconium in formulations can significantly improve their efficacy in combating pathogenic bacteria.

The antibacterial effects of ZrO2, ZrO2 + Y2O3, and ZrO2 + Y2O3 + Al2O3 at concentration 50 µg/mL− 1were assessed against bacterial strains. Intracellular reactive oxygen species (ROS) levels were quantified using the DCFDA assay following exposure to the ZrO2, ZrO2 + Y2O3, and ZrO2 + Y2O3 + Al2O3, as presented in Fig. 4. Flow cytometry analysis revealed that the untreated bacterial cells (control group) exhibited minimal ROS production. In contrast, treatment with ZrO2, ZrO2 + Y2O3, and ZrO2 + Y2O3 + Al2O3 at concentration 50 µg/mL− 1 resulted in a marked increase in ROS levels. Notably, treatment with the ZrO2 + Y2O3 + Al2O3 induced the most pronounced rightward shift in fluorescence intensity, indicating the highest ROS production in both bacterial strains. These findings suggest that the ZrO2 + Y2O3 + Al2O3 significantly enhances intracellular oxidative stress, surpassing the effects observed with ZrO2 nanoparticles a lone.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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ROS generation in bacterial strains after been treated as indicated. A, Control untreated bacteria. B, bacterial strains were treated with ZrO2 at concentration 50 µg/mL− 1. C, bacterial strain were treated with ZrO2 + Y2O3 at concentration 50 µg/mL− 1. D, bacterial strains were treated with ZrO2 + Y2O3 + Al2O3 at concentration 50 µg/mL− 1.

Table 4 Overview of antibacterial activities involving zirconium nanoparticles in pathogen control.
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Bacterial adhesion tests

The adherence results represented in (Table 5), indicated that all clinical strains showed low adherence to modified surface materials. After the incubation period 24 h. at 37 °C, it was observed that bacterial adhesion was decreased with an increase in the concentrations of the material (zirconia + yttria + alumina). Bacterial adhesion to a material surface is the first step in biofilm development60,61. It is determined by the combination of interactions between bacterial surface, the substrate surface and the environment around them. It depends largely on the structure and atomic composition of the surface of implanted biomaterials, surface charge, hydrophobicity, and its roughness and surface morphology62. YSZ is among the most used ceramic materials in dental implants, due to its mechanical properties and biocompatibility, because YSZ implants are bio-inert and have excellent resistance to wear and corrosion. Due to the high flexural strength and fracture resistance63,64. The mechanism of the antimicrobial effect shown by YSZ coating is not clear. During bacterial adhesion, depending on the specific resistivity of the substratum, bacteria either donate to or accept electrons from the substratum. It could be hypothesized that YSZ donates electrons to the bacteria which produces a repulsive force between the bacteria and the substratum and leads to a lowered bacterial adhesion65. The purpose of this test was to examine the adhesion of S.mutans bacteria to zirconia in laboratory- prepared samples. The results in the laboratory, after 24 h of incubation at 37 ° C of S. mutants bacteria, showed that the adhesion decreased with the increase in the concentration of the material which was a mixture of (zirconia + yttria + alumina), and since the material dimension were in nano, its effect was better because the nanoparticles that were characterized by their small size worked to close the sites of adhesion which prevented the bacteria from sticking and growing. Bacteria adhesion was generally affected by the physical and chemical properties of the material surface including surface roughness. The water absorbtion property played an important role in the formation and maintenance of bacterial biofilms. Reduction in bacterial colonization was studied by Groessner-Schreiber et al.66 who concluded that zirconium nitride-coated titanium discs harbor had less amount of bacteria when compared to other coatings and controls. In addition, it was concluded that zirconia surfaces accumulated significantly fewer bacteria when compared with titanium ones. Tables (4 − 2) shows the number of adherent epithelial cells. Surface wettability as the point of contact between bacteria and the bulk of a material, the surface plays a pivotal role in promoting or preventing bacterial adhesion. Surface wettability is a central property that governs the interactions between solid and liquid phases in biological systems. Briefly, the liquid phase “wets” the surface of a solid surface by maximizing its area in contact with the surface. This in turn increases the interaction between the liquid and the solid surface. In general, surfaces with low surface energy and liquids with high surface tension tend to reduce surface wettability. On the other hand, surfaces with high surface energy and liquids with low surface tension tend to increase surface wettability67,68 .

Table 5 Number of adherent epithelial cells.
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Quantitative detection of biofilm formation

The biofilm detection method using the tissue culture plate made of polystyrene showed gradient in the formation of the biofilm according to the concentration of the materials used. The amount of pigment that stained the negative control wells was very small, while the positive control wells were strongly pigmented, indicating the presence of dense biofilm. Figures 5 and 6 shows that pathogens show significant removal of biofilm formation when treated with a different concentration of all treatments under this study But in different proportions. S. aureus was the most affected than E.coli. At higher concentrations, the viability of the cell may lead to a toxic effect69. The exopolysaccharides were responsible for induced biofilm formation with the receptor70 Glucan-binding domains of glucosyltransferase play a vital role in forming biofilms by glucan-binding proteins in virulent strains of S. aureus71. In some adverse conditions, the exuberance of pathogenic resistance to antibiotics reoccurrence occurs and may lead to rapid cell growth in the oral tissues72. This can be addressed using medication with natural products and the combination of the dual role by direct contact killing of pathogens as well as the release of ionic by the capped agents in the nanoparticles with monomers8. Biofilms are recognized to have greater resistance compared to bacterial isolates. This is attributed to their capability to produce exopolysaccharides that enhance their adhesion. This characteristic enhances their durability. To eliminate biofilms, it is believed that NaNPs impede the release of exopolysaccharides through targeted interactions, thereby hindering their adhesion to biofilms73. Zirconia has been utilized in pediatric dentistry since 2008 in the form of crowns. Apart from its esthetically pleasing color, it exhibits a highly polished surface and exceptional durability. It also accumulates less biofilm, which contributes to better gingival health and ultimately leads to higher satisfaction among parents74,75,76,77,78. Dental implants are one of the most frequently used treatment options for the replacement of missing teeth. The oral microflora and its dynamic interactions with the implant substrata seem to crucially influence the long-term success or failure of dental implants79,80,81,82,83,84,85. As soon as implant surfaces are exposed to the human oral cavity, they are immediately colonized by microorganisms86,87. The initial bacterial adhesion on implants is the first and essential step in the geneses of complex peri-implant biofilms, which, in turn, may result in peri-implantitis and loss of the supporting bone. E. coli can form implant-associated biofilms by initially adhering to the surfaces of medical implants, often following the deposition of a host-derived “conditioning film”. The bacteria then multiply, embedding themselves in a protective matrix of extracellular polymeric substances (EPS). This biofilm structure provides protection from the host immune system and antibiotics, leading to persistent infections, recurrent issues, or implant failure, particularly with indwelling devices like urinary catheters88. Antibiofilm performance can be due to preventing initial adhesion, inhibiting biofilm maturation, or disrupting established biofilms, or a combination of these mechanisms. Strategies can target any or all stages of biofilm development, from the initial attachment of bacteria to the surface, to the formation of the biofilm matrix and signaling between bacteria, to the eventual degradation of already mature biofilms. Response : Inhibiting Biofilm Maturation by Strategies include: Inhibiting extracellular polymeric substance (EPS) production: Reducing the amount of the protective matrix material, Interfering with quorum sensing (QS): Blocking the chemical communication between bacteria that coordinates the formation of the biofilm structure, and Directly killing attached bacteria: Using substances that eliminate bacteria while they are still in the early stages of colonization89,90. The MTT assay was utilized to evaluate the metabolic activity of bacterial cells within the biofilm, as shown in Fig. 7. Consistent with the observed effects on biofilm inhibition and disruption, the reduction in metabolic activity was found to be concentration dependent manner.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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(A) Quantitative detection of biofilm formation by E. coli, (B) Biofilm formation reduced after treatment with ZrO2, (C) ZrO2 + Y2O3, (D) ZrO2 + Y2O3 + Al2O3.

Fig. 6
Fig. 6The alternative text for this image may have been generated using AI.
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(A) Quantitative detection of biofilm formation by S. aureus, (B) Biofilm formation reduced after treatment with ZrO2, (C) ZrO2 + Y2O3, (D) ZrO2 + Y2O3 + Al2O3.

Fig. 7
Fig. 7The alternative text for this image may have been generated using AI.
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Metabolic activity assay for bacterial strains as indicated. A, bacterial strains were treated with ZrO2. B, bacterial strain were treated with ZrO2 + Y2O3. C, bacterial strains were treated with ZrO2 + Y2O3 + Al2O3 at concentration 50 µg/mL− 1.

Cells viability

The MTT test revealed that for the samples after 24, 48, and 72 h of incubation (Fig. 8). It was found that a concentration of 200 µg/mL showed cytotoxicity almost 13.33% than other lower concentrations. This activity was interpreted as the ability of reactive oxygen (ROS) to cause damage to cellular fibers such as DNA, proteins, and fats, thus leading to perturbation of the cell cycle and causing cell death34,35,91. Previous work found that ZrO2 NPs exhibited low cytotoxicity, as manifested in the in vitro studies with human umbilical vein endothelial cell lines (HUVEC)92. Therefore, it can be concluded that the ZrO2 NPs materials can be a promising bio-ceramic material for prosthetic dentistry, and dental implants92,93.

Fig. 8
Fig. 8The alternative text for this image may have been generated using AI.
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Biocompatibility of (zirconia + yttria + alumina) against REF cell. Cells were treated with (zirconia + yttria + alumina) for 24 h (Upper panel). Cells were treated with (zirconia + yttria + alumina) for 48 h (Middle panel). Cells were treated with (zirconia + yttria + alumina) for 72 h (Lowe panel). Data were represented as mean ± SD of three independent experiments.

Several previous studies support the findings of our research regarding the anticancer efficacy of nanoparticles. For instance, research by44 demonstrated that Ag-Zr nanoparticles with graphene oxide exhibited significant cytotoxic effects against cancer cell lines, similar to our observations with zirconia nanoparticles. Additionally48, found that the combination of nanoparticles with other materials enhanced their anticancer properties, corroborating our results with the ternary mixture.

These studies highlight the importance of nanoparticle size, surface area, and charge in influencing anticancer effectiveness, aligning well with our findings that smaller and more reactive nanoparticles lead to greater inhibition of cancer cell proliferation. Furthermore, the mechanisms by which nanoparticles induce oxidative stress in cancer cells, as noted in prior research, align with our observations on the role of reactive oxygen species in promoting apoptosis.

Collectively, these studies reinforce the potential of utilizing nanoparticles, particularly zirconia-based materials, in developing effective anticancer treatments. This is consistent with our results, which demonstrated that the combination of zirconium nanoparticles (ZrO2) with other agents exhibited a more potent anticancer effect than either component alone. As shown in Table 6, the synergistic interactions of zirconium nanoparticles with various materials enhanced their anticancer properties, particularly against specific cancer cell lines. This reinforces the notion that the inclusion of zirconium in formulations can significantly improve their efficacy in combating cancer.

Table 6 Overview of anticancer efficacy of nanoparticles in previous studies.
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Conclusion

Due to its biocompatibility, non-hazardous nature, excellent properties, the ZrO2NP-based nano-mixtures with remarkable characteristics have been used in recent times in the areas of biomedicine. This study is provides information on the effect of the ZrO2, binary mixture (ZrO2+ Y2O3),and triple mixture (ZrO2 + Y2O3 + Al2O3) as antibacterial agents against pathogenic bacteria, Anti- biofilm, and anticancer effect. The purpose of this study was to identify some biological properties of ZrO2 , binary mixture of (ZrO2+ Y2O3),and triple mixture of (ZrO2 + Y2O3 + Al2O3). They all showed varying effects in this respect, and from this we find that the best results were achieved for the ternary mixture, with the emergence of a synergistic effect of the mixed materials.