Surface energetics of antibiofilm property of dental material added with green synthesized copper nanoparticles

Abstract

Dental caries and lesions are difficult to treat during cement repairs. A remarkable antimicrobial therapeutic biomaterial is needed to fight dental caries and recurrent necrotic lesions. This study used Mentha longifolia extract to synthesize Copper nanoparticles (CuNPs) with distinctive properties at room temperature (22–25 °C). These CuNPs were supplemented with cephalosporin antibiotics that act as a capping agent to explore their synergistic antibacterial potency. These nanoparticles were subjected to FTIR, XRD, UV-Vis spectrophotometry, and SEM for characterisation. These CuNPs capped with antibiotics were added to glass ionomer (GIC) cement. These GIC samples were divided into pure GIC and modified GIC samples. Antibiotic-supplemented CuNPs, conjugated with GIC, showed good effect against Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae and Pseudomonas aeruginosa as compared to conventional GIC, tested through a modified direct contact test. Among them, GIC enriched with cefotaxime-supplemented CuNPs exhibited excellent antibacterial effects, followed by Cefepime and Ceftriaxone-supplemented CuNPs, respectively. Pure GIC has the most negligible antibacterial effect. Further, the interaction of these modified GICs with the selected bacterial strains was studied using the extended Derjaguin–Landau–Verwey–Overbeek (XDLVO) approach. The results show that the modified GIC effectively inhibited biofilm formation on dental implants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13568-024-01820-2.

Introduction

Numerous methods to synthesize metal nanoparticles follow the principles of green chemistry. Nanoparticles have been synthesized using environmentally safe and nontoxic chemicals. Animals, plants, and microorganisms have all been used as sources for the green synthesis of nanoparticles (Molnár et al. 2018; Podstawczyk et al. 2019). There are several uses for plant extracts for copper-based nanoparticles, including catalysis, photocatalysis, antibacterial activity, DNA binding and sensing, cytotoxicity, antioxidants, etc. (Ghosh et al. 2020; Nazar et al. 2018; Vasantharaj et al. 2019; Zhao et al. 2017). Researchers have explored nanoparticle production utilizing natural extracts of plants and microorganisms (fungi and bacteria) (Narayanan and Sakthivel 2010; Yu et al. 2019). By secreting redox-active substances, microorganisms convert metal into metal-protein conjugate nanocrystals. It is difficult to purify and separate metal particles following production (Dhillon et al. 2012). Reducing metal salt to metal nanoparticles using plant extracts is more efficient than other processes and microbial synthesis as it is environmentally safe, non-toxic, and simple to process. Secondary metabolites abundant in plants, such as flavonoids, alkaloids, steroids, terpenoids, proteins, and sugars, enable bio-reduction (nucleation and growth), stability, and capping in a single process (El-Seedi et al. 2019). Various industries, including agriculture, medicine, pharmaceuticals, and cosmetics, can benefit from the multifunctional properties imparted by coating metal nanoparticles with adaptable bioactive chemicals from plant extracts (Powar et al. 2019; Rana et al. 2020).

Several Mentha species are frequently grown worldwide for their essential oils and are widely used for flavouring, creating perfumes, and medical purposes (İşcan et al. 2002; Monfared et al. 2002). Stem and leaves of Mentha spp. are commonly used in herbal teas or commercial spice blends for various dishes to add flavour and aroma (Kothari and Singh 1995; Moreno et al. 2002). It is used as a folk remedy for the treatment of flatulence, anorexia, nausea, bronchitis, liver complaints, and ulcerative colitis, anti-inflammatory, carminative, antiemetic, diaphoretic, antispasmodic, analgesic, emmenagogue, stimulant and anticatarrhal activities (Cowan 1999; İşcan et al. 2002; Moreno et al. 2002). Furthermore, it is well known that some Mentha species have essential oils or extracts that have antimicrobial and antioxidant properties (Daferera et al. 2003; Economou et al. 1991). Nevertheless, an attempt has yet to be made to investigate the biological effects of the extracts and essential oils from the Mentha longifolia ssp. in copper nanoparticle synthesis.

Dental caries, a prevalent chronic oral disease affecting people worldwide, is associated with various types of microorganisms, both Gram-positive and Gram-negative (Naik et al. 2016; Wang et al. 2016). Currently, the primary approach for caries treatment involves repairing impacted teeth through fillings.

Consequently, developing novel materials with anti-caries properties has become a significant focus in this field. Metals, such as silver, gold, copper, zinc, titanium, and others, have been utilized for centuries as antimicrobial agents to combat this issue (Liu et al. 2018; Mahal et al. 2014). Each metal possesses distinct properties and a unique range of activity. Silver and CuNPs have garnered substantial attention in recent years due to their delivery capabilities, biocidal attributes, and ability to hinder microbial adhesion (Allaker and Yuan 2019). While silver nanoparticles have been extensively studied for their antimicrobial properties against oral microbes, there are fewer investigations on the antimicrobial potential of CuNPs. CuNPs, however, have shown the ability to bind to sulfhydryl (SH) groups, disrupting bacterial nucleic acids and vital enzymes. Moreover, these nanoparticles exhibit high thermal and electrical conductivity while being more cost-effective than their silver counterparts (Gutiérrez et al. 2019).

Controlling the formation of biofilms on solid surfaces is difficult for all biomedical and industrial field researchers. Many researchers focus on several anti-biofilm strategies that involve coating devices’ surfaces to enhance their characteristics (Park et al. 2004; Weng et al. 1999). There is a growing need for knowledge to explain and regulate microbial adhesion. Calculating the interaction energies between microbial cells and adhesion surfaces allows the investigation of microbial adhesion onto solid substrates (Busscher et al. 2010). Many approaches like thermodynamics, classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, and extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory are used to determine the interaction between cells and substrate (Sharma and Rao 2002). The adhesion of microbial cells to solid surfaces can be described by neither the thermodynamic nor the DLVO approach. Therefore, a different strategy was developed, combining the traditional DLVO approach with thermodynamics, including hydrophobic and hydrophilic interactions. This expanded version of DLVO theory is also known as the XDLVO or the extended DLVO approach (Van Oss 1989).

Since inorganic nanoparticles and antibiotics can act together and often have enhanced effectiveness, they are frequently used as advanced antimicrobial agents. Combining antibiotics and inorganic nanoparticles allows bacteria to be killed by a different agent if they resist one of the components. The antimicrobial activity of antibiotics conjugated with various nanoparticles has been extensively documented in the literature (Grace and Pandian 2007; Gu et al. 2003; Li et al. 2005; Saha et al. 2007). Still, before they can be used, they need to bind antibiotics to the surface of nanoparticles and undergo a series of steps, including surface functionalization, synthesis, and binding of a significant amount of the antibiotic. Until now, no study has been published on cephalosporin-capped CuNPs in the presence of Mentha extract as a reducing agent in one pot experiment. The current study was designed to investigate the green synthesis of cephalosporin-capped CuNPs from Mentha longifolia extract and evaluate their antibiofilm properties through the XDLVO approach while considering the significance of CuNPs and their therapeutic advantages.

Methods and materials

Materials

Copper sulfate pentahydrate and Nutrient agar were purchased from Sigma Aldrich (Burlington, MA, United States). Milli Q water was used for contact angle measurement, while distilled water was used throughout the experiment. Formamide and I-bromonapthalene were purchased from Fluka (Buch, Switzerland). GIC^® was supplied by Shanghai New Century Dental Materials Co., Ltd. Cefepime was purchased from Barrett Hodgson. In contrast, Cefotaxime and Ceftriaxone were purchased from Bosch.

Methods

Collection and sample preparation

Mentha longifolia was collected locally (identified and confirmed at the Department of Botany, University of Malakand). The plants were washed thoroughly with distilled water, shade-dried, and blended into powder form. The plant extract was prepared by boiling 2.5 g of plant powder in 80 ml of deionised water. The extract was then filtered with Whitman No.1 filter paper and stored at 20 ⁰C for further use.

Preparation of nanoparticles

CuNPs were prepared by mixing 2 g of copper sulfate pentahydrate (CuSO₄. 5H₂O) with 50 mL of menthe extract leaf. The mixture was combined under a magnetic stirrer for 4 h. CuNPs were then separated by centrifugation at 4000 rpm for 15 min (Model 16 K, Biored). The supernatant was discarded, and the pellet was washed several times with deionized water. The nanoparticles were oven-dried at 40 ⁰C. The same procedure was followed for preparing antibiotic-supplemented nanoparticles (Cefepime, Cefotaxime, and Ceftriaxone), but an antibiotic solution of 20 ml (1 mg/ml) was added at the start of the reaction. The lowest antibiotic concentration that capped the nanoparticles was chosen from concentrations (0.5-3 mg/ml).

Characterization of nanoparticles

Chemical characterization

UV-Vis spectroscopy

Moreover, the reduction of copper ions into CuNPs with the help of plant extract was monitored using a UV-Vis spectrophotometer. The spectra for each sample were recorded at a resolution of 1 nm from 200 to 800 nm (Shimadzu UV-1700, Japan).

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) was performed using the KBr pellet method within a predetermined spectral range of 4000–400 cm^− 1 (Model Tensor 27, Brucker, Germany) to find out various functional groups involved in the reduction of copper ions to CuNPs (Groiss et al. 2017).

X-ray diffraction studies of nanoparticles

The XRD pattern of CuNPs was investigated using an X-ray diffractometer (Model Kristalloflex D500, Siemens, Germany) (Ismail 2020). Cu Ka radiation with a wavelength of 1.540 Angstroms was used to study XRD patterns of CuNPs at room temperature. The diffraction pattern was obtained in the range of diffraction angle (2 h) from 0 to 80.

Scanning electron microscopy

Copper nanoparticle morphological features were studied using scanning electron microscopy (Model MIRA3 FEG-SEM, Tescan, Czech Republic) (Groiss et al. 2017). A voltage of 20 kV was used for SEM.

Biological activities of copper nanoparticles

Antimicrobial activity

The Agar disc diffusion method (Adil et al. 2019) was used to test the antibacterial activity of green synthesized CuNPs against four pathogenic bacterial species, i.e., Methicillin-resistant S. aureus (Gram-positive), E. faecalis (Gram-positive, ATCC 29212), K. pneumoniae (Gram-negative, ATCC 43816) and P. aeruginosa (Gram-negative ATCC 25619). Bacteria were overnight cultured in freshly prepared LB media. Cotton swabs were used to wipe the new bacterial culture onto the solidified nutrient agar plates. The 6 mm disc was prepared, placed onto the solidified nutrient agar plates, and loaded with CuNPs at concentrations (6.75, 13.5, 25, and 50 mg/ml). The exact concentration of pure antibiotics equal to the final volume of the initial nanoparticle suspension was taken as control.

Minimum inhibitory concentration of copper nanoparticles

The antibacterial efficacy of the green synthesized CuNPs was evaluated utilizing various clinically significant microorganisms. These strains included Methicillin-resistant S. aureus, K. pneumoniae, P. aeruginosa, and E. faecalis. Micro broth dilution experiments were conducted to determine the MIC of the nanoparticles produced for each bacterium (Rajapriya et al. 2020). Bacteria were cultured in nutrient broth overnight. The cultured bacteria were added to new LB media and compared with McFarland Standards. 96 well plates were used for the test. These wells were loaded with 50 µl each of the nutritional broth and nanoparticles (1 mg/ml), which were then serially diluted. Inoculum was taken as a negative control, and sterile media was taken as a positive control. The plates were incubated overnight at 37 ⁰C. The results were recorded at 490 nm on a microplate reader.

Hemolytic assay for cytotoxicity of copper nanoparticles

Fresh human blood samples (2 ml) were collected from a healthy person in EDTA vail and mixed gently to stop coagulation. The samples were centrifuged at 1000 rpm for 10 min. The platelet-deficient plasma-containing supernatant was discarded after red blood cells were collected as a pellet (Rajapriya et al. 2020). Pellets were rinsed three times with phosphate-buffered saline (pH 7.4). To make an equivalent suspension, the pellet was resuspended in 20 ml of PBS (7.4 pH). The experiment was performed in a microplate. Carefully, 800 µL of suspended RBC were treated with 200 µL of CuNPs at different concentrations (0.75, 1.25, 2.5, 5, 10 mg/mL). Triton X-100 (1%) was used as a positive control, while PBS was a negative control. The microplate was incubated at 37 C for two hours, and optical density was recorded at 405, 490, 540, 570, and 630 nm. The percentage of hemolysis is calculated according to the formula:

1

Where SA represents Sample Absorbance, NA represents Negative control absorbance and PA. Represents Positive control absorbance.

Bacterial DNA damage analysis

According to the literature, nanoparticles were subjected to the study of bacterial DNA damage (Ali et al. 2023). Bacteria strains were treated with 40 micrograms per milliliter (µg/mL) of bare and antibiotic-supplemented CuNPs. DNA was extracted by subjecting the broth cultures to centrifugation at 13,000 revolutions per minute (rpm) for 10 min. Subsequently, the resulting pellets were re-suspended in 1 milliliter (mL) of lysis buffer and subjected to another round of centrifugation at 13,000 rpm for 10 min. The pellets were once again re-suspended in lysis buffer and subjected to a 30-minute boiling process in a water bath. After cooling the samples to room temperature, centrifugation was performed at 13,000 rpm for 10 min. The resulting supernatants containing the DNA were collected. Gel electrophoresis was carried out to confirm the successful isolation of DNA. 500 µg/mL suspensions of DNA samples were prepared in deionised water (dH2O) and subsequently scanned within 200 to 300 nanometers (nm) using a UV spectrophotometer to assess DNA damage.

Zeta potential measurement

All measurements were made in triplicate using a Zetasizer Nano ZS from Malvern Instruments (Worcestershire, UK) to measure zeta potential values. Measurements were performed for all green synthesised nanoparticles and GIC using 2 mg/mL solutions in a 20 mM phosphate buffer (pH 7). Henry’s equation was applied to determine zeta potentials from the electrophoretic mobility data in the instrument software (Aasim et al. 2013; Van Oss 2006).

Stabilization of nanoparticles

The method described in the literature was adopted to study the stabilization of nanoparticles by PEG (Radziuk et al. 2007). After the preparation of green synthesized nanoparticles, 2 mg of nanoparticles were suspended in 5% of PEG 4000, 6000, 8000, and PEG 10,000 solution. Nanoparticles were sonicated to obtain a colloidal solution. The solution was then centrifuged at 7000 rpm. The pellets were collected and washed thrice and suspended in Milli Q water. The samples were then checked under UV spectroscopy at 200–800 nm.

Incorporation of CuNPs to GIC

Standard GIC^® Shanghai New Century Dental Materials Co., Ltd., powder and liquid were employed. GIC powder and liquid recommended 1:2 (one scoop powder and two drops of liquid) was mixed with green synthesized CuNPs at a concentration of 3% based on previous literature (Gutiérrez et al. 2019) (safe in hemolytic assay) to create the experimental group.

Contact angle measurement

The agar glycerol surface was prepared to determine the contact angle. Briefly, 2% agar and 10% glycerol were mixed, boiled, and poured into Petri plates. After mixing the GIC and CuNPs, it was spread evenly on the glycerol agar plates and waited till it dried. To calculate surface parameters, contact angles were measured using a goniometer (OCA20, Data Physics Instruments, Germany) by the sessile drop approach (Vennapusa et al. 2008). Water, formamide, and 1-bromonaphthalene were used as the probe liquids. In the instrumental software, Van Oss AB theory (Van Oss 2006) was used to calculate surface energy utilizing contact angle values (SCA 20). The contact angle of K. pneumoniae and MRSA was measured using the same procedure described earlier. Bacterial colonies were transferred to LB broth and incubated overnight for an active culture. The cells were harvested by centrifugation at 37º C for 5 min and washed three times with phosphate buffer saline (PBS) to measure the contact angle. The surface tension parameters γ^LW and γ^AB, as well as the electron-donating (γ–) and the electron-accepting (γ +) parameters of the probe liquids, are available (Aasim et al. 2013).

Surface energy calculations and Interaction free energy as a function of distance

The total interaction energy between a Bacteria and GIC can be expressed in terms of the extended DLVO theory as:

2

U^XDLVO represents the total interaction energy in aqueous media. ULW is the Lifshitz Van der Waals interaction energy, UEL is the electrostatic interaction energy, and UAB is the acid-base interaction energy between the GIC and the bacteria. Contact angle data was used to calculate the free energies of interaction between GIC and bacteria using the Lifshitz van der Waals–acid-base equation. ∆G donates interaction energy per unit area between the GIC and bacteria. The interaction energy between the two surfaces was evaluated at the closest distance of approximation, i.e. h ≈ 0.167 nm. The corresponding energy distance profile was calculated utilizing ∆the G value.

Antibacterial assay of modified GIC

The modified direct contact test was used in 96-well microtiter plates measuring bacterial growth’s colony forming unit (CFU) (Lewinstein et al. 2005) The wells and floor of the microtiter plate were coated with a thin layer of modified GIC. The plates were allowed to dry in a controlled environment. 50 µL of experimental bacteria were placed on the GIC with100 µL LB media. A positive control, as prepared by placing 50 µL of bacterial suspension along with 100 µL of LB broth in a separate well without the GIC, was considered. After 24 h, the bacterial suspension was withdrawn from the wells and spread on the nutrient agar plates. After incubation for approximately 24 h, the CFUs of the suspension were recorded and compared.

Results

UV-Vis spectrum characterization

The UV-Vis absorption spectra of green synthesized CuNPs were analyzed between 200 and 800 nm (Fig. 1). Green synthesized CuNPs display an absorption peak at 280.82 nm, while Cefepime (Cefe), Cefotaxime (Cefo), and Ceftriaxone (Cfx) show absorption peaks at 284.00, 298.09, and 292.06 nm, respectively. A clear difference was observed in the absorption peaks of all nanoparticles (Fig. 1).

Fig. 1.

UV-Vis absorption spectrum of bare CuNPs and antibiotics-supplemented CuNPs

Fourier transform infrared analysis of functional groups associated with nanoparticles

FTIR analysis was conducted to discover the possible biomolecules forming green synthesized CuNPs from Mentha longifolia extract. FTIR spectra showed the functional groups in mentha extract, antibiotics, and nanoparticles and the functional groups in antibiotics-supplemented green synthesized CuNPs (Fig. 2). Peaks observed around 3,584 cm^− 1 and 3,698 cm^− 1 are characteristics of alcohol (O-H stretching). Peaks at 2,842 cm^− 1 to 2980 correspond to C-H stretching of alkane, while the peak around 2322 arises due to N = C = O stretching of isocyanate. Peaks observed at 2,160 cm^− 1 to 2260 cm^− 1 correspond to the S-C ≡ N stretching of thiocyanate and the ≡ C stretching of alkyne, respectively. Peaks at 1,981 to 2,054 correspond to N = C = S stretching due to isothiocyanate. The peaks observed around 1650–1580 cm^− 1 correspond to the N-H bending of amine, and 1,450-1,465 cm^− 1 is a characteristic of the C-H bending of alkane. The peaks at 1,040−1,050 correspond to the CO-O-CO stretching of anhydride, and the band at 1,033 cm^− 1 is a characteristic of the S=O stretching of sulfoxide. The FTIR spectra showed that the alcohol, alkaloids, isocyanate thiocyanate, isothiocyanate, amine molecules, and other metabolites in the Mentha extract reduce copper ions to Cu nanoparticles. It is relatively straightforward from the FTIR spectrum that the peaks of the functional groups of the spectra of the Mentha extract and those of their corresponding synthesized CuNPs are almost similar, but the absorption was different. Also, it was observed from the FTIR spectrum that antibiotic conjugation also took place by contributing to the functional groups.

Fig. 2.

FT-IR spectrum (A) Cefepime (B) Cefotaxime (C) Ceftriaxone (D) Extract (E) bare CuNPs (F) Cefepime supplemented CuNPs (G) Cefotaxime supplemented CuNPs (H) Ceftriaxone supplemented CuNPs

X-ray diffraction analysis

The XRD pattern of the synthetic CuNPs synthesized by the green reduction of copper ions using Mentha extracts were analyzed. CuNPs synthesized from Mentha extract have experimental diffraction peaks of 18.72, 21.30, and 25.34 in patterns at 2θ. The three different diffraction peaks for the Mentha-derived CuNPs correspond to the (111), (200), and (220) lattice planes of the CuNPs’ face-centred cubic structure (FCC). When these nanoparticles were supplemented with antibiotics, additional peaks were observed at different 2θ, as depicted in Fig. 3.

Fig. 3.

X-ray (XRD) pattern of (A) bare CuNPs (B) Cefotaxime supplemented CuNPs (C) Ceftriaxone supplemented CuNPs (D) Cefepime supplemented CuNPs

Scanning electron microscopy

The CuNPs synthesized from Mentha extract are shown in Fig. 4, obtained from scanning electron microscopy (SEM). SEM images of CuNPs synthesized from Mentha extract depict the distributed CuNPs with irregular edges.

Fig. 4.

Scanning electron microscopy (SEM) images of (A) Bare CuNPs (B) Cefepime supplemented CuNPs (C) Cefotaxime supplemented CuNPs (D) Ceftriaxone supplemented CuNPs

Assessment of zeta potential for copper nanoparticles and bacterial strains

The zeta potential of E. faecalis, K. pneumoniae and P. aeruginosa were adopted from the literature (Abdallah et al. 2014; Gallardo-Moreno et al. 2002, 2004). In contrast, the zeta potential of dental material and nanoparticles was calculated at pH 7 (Fig. 5). The zeta potential of GIC and Methicillin-resistant S. aureus was − 20.01 mV and − 9.33 mV, respectively. Zeta potential determines the stability of particles in a colloidal solution. CuNPs have zeta potentials of -18.5.2 mV (Fig. 5A) while CuNPs-Cefo, CuNPs-Cfx, and CuNPs-Cefe (Fig. 5 (C) (E) (G) were − 12.3 mV, -19.2 mV, and − 20.9 mV, respectively. A colloidal solution of nanoparticles with a zeta potential value greater than + 25 mV or less than − 25 mV indicates a high stability level (Hanaor et al. 2012), as the colloidal solution will resist aggregation. Our findings show that CuNPs have a negative surface charge and are unstable. CuNPs-Cfx showed less stability when compared to other antibiotics-supplemented CuNPs, so further study was extended to stabilize it in the polymer solution.

Fig. 5.

Zeta potential of bare CuNPs and antibiotics conjugated nanoparticles. Figures A, C, E, and G represent the zeta potential of bare CuNPs, cefotaxime-supplemented CuNPs, ceftriaxone-supplemented CuNPs, and cefepime-supplemented CuNPs. Figures B, D, F, and H represent the PdI and hydrodynamic radius of bare CuNPs, cefotaxime-supplemented CuNPs, ceftriaxone-supplemented CuNPs, and cefepime-supplemented CuNPs, respectively

Figure 5 represents the hydrodynamic radius and polydispersity index of CuNP nanoparticles. CuNPs were larger than size because biomolecules and the water layer that covered their surface were included in the measurements (Bhakya et al. 2015). The PDI (polydispersity index) is a measure of particle homogeneity. According to the results, the PDI of green synthesized copper particles was between 0.194 and 0.480.

Antimicrobial activity and minimum inhibitory concentration

The antibacterial activity of green synthesis CuNPs was tested against Methicillin-resistant S. aureus (MRSA), E. faecalis, K. pneumoniae and P. aeruginosa using disk diffusion assay shown in Table 1. The zone of inhibition in millimeters (mm) (Table 1) depicts the antibacterial of antibiotics supplemented with green synthesized CuNPs. The supplementation of nanoparticles with antibiotics boosted the antibacterial activity.

Table 1.

Antibacterial activity and minimum inhibitory concentration of bare CuNPs and antibiotics-supplemented nanoparticles

	Bare and antibiotics supplemented CuNPs				Pure Antibiotics
	CuNPs	CuNPs-Cefe	CuNPs-Cefo	CuNPs-Cfx	Cefe	Cefo	Cfx
Bacteria	Activity (mm)
MRSA	13 ± 0.81	28 ± 1.19	31 ± 1.21	22 ± 1.47	26 ± 1.21	31 ± 1.32	23 ± 1.11
K. pneumonia	16 ± 1.10	34 ± 1.23	23 ± 0.43	22 ± 1.14	28 ± 1.04	21 ± 1.57	24 ± 1.43
P. aeruginosa	14 ± 1.37	24 ± 1.57	23 ± 1.27	15 ± 1.52	24 ± 1.29	23 ± 1.09	15 ± 1.37
E. faecalis	14 ± 0.39	25 ± 1.16	31 ± 1.73	25 ± 1.67	24 ± 1.84	30 ± 1.34	25 ± 1.27
Bacteria	MIC (µg/ml)
MRSA	400 ± 2.54	250 ± 1.98	300 ± 1.76	300 ± 1.93	360 ± 1.09	370 ± 0.63	350 ± 1.64
K. pneumonia	400 ± 1.67	200 ± 2.79	200 ± 1.48	250 ± 1.67	330 ± 1.44	330 ± 1.43	370 ± 1.27
P. aeruginosa	500 ± 2.43	350 ± 1.36	300 ± 2.00	350 ± 1.94	440 ± 1.73	380 ± 1.17	430 ± 2.06
E. faecalis	400 ± 1.87	200 ± 1.16	200 ± 1.58	250 ± 1.79	350 ± 1.15	340 ± 2.10	370 ± 1.17

The antibacterial activity of antibiotic-supplemented nanoparticles was better than pure antibiotics despite antibiotics being used in their pure form. In all cases, P. aeruginosa seemed slightly resistant to conjugated nanoparticles, as shown in Table 1 (images in supplementary files).

Microdilution assay was carried out to find the minimum concentration that inhibits the growth of bacteria. Different concentrations of green synthesized CuNPs and antibiotics-supplemented CuNPs were tested against MRSA, E. faecalis, K. pneumoniae, and P. aeruginosa. The findings (Table 1) show that the copper oxide nanoparticles’ minimum inhibitory concentration falls between 200 and 500 µg/ml. The MIC values of CuNPs against Methicillin-resistant S. aureus, E. faecalis, and K. pneumoniae recorded in this work were lower than those published by Das et al. (2019), where they synthesized CuNPs utilizing Moringa oleifera extract. The MIC of green synthesized produced nanoparticles for P. aeruginosa and were 500 µg/mL. As depicted in Table 1, CuNPs supplemented with antibiotics have reduced bacterial growth compared to bare CuNPs.

Hemolytic assay

As the CuNP nanoparticles encounter the RBC cells’ surfaces, they exhibit the highest percentage of hemolytic activity, which suggests that they release haemoglobin into the blood’s plasma. To compare the hemolytic activity, 1% Triton X 100 and PBS were used as positive and negative controls, respectively. Positive control shows 100% hemolysis, while no hemolysis was observed in negative control. In this study, no hemolysis was found for CuNPs at 20 mg/ml compared to antibiotics-supplemented nanoparticles, as shown in Table 2, as hemolysis less than 5% was considered safe. The hemolysis of washed RBC was safe at 10 mg/ml or less (Table 2), demonstrating that larger concentrations of CuNP nanoparticles will harm health. Hence, hemolytic activity was concentration dependent.

Table 2.

Hemolysis percentage of bare CuNPs and antibiotics-supplemented nanoparticles

mg/ml	Hemolysis %
	CuNPs	CuNPs-Cefe	CuNPs-Cefo	CuNPs-Cfx	Triton X	PBS
10	1.77 ± 1.31	2.81 ± 1.84	2.36 ± 1.23	2.76 ± 1.61	100	0
20	2.80 ± 1.05	4.30 ± 1.69	4.70 ± 1.74	4.68 ± 1.48	–	–
30	3.26 ± 1.78	5.16 ± 1.13	5.16 ± 1.17	4.91 ± 1.35	–	–
40	4.56 ± 0.75.	12.3 ± 1.47	12.1 ± 1.04	5.16 ± 1.68	–	–
50	5.70 ± 1.39	17.4 ± 0.48	24.2 ± 1.37	16.38 ± 1.19	–	–

Bacterial DNA damage analysis

Our findings demonstrate the potential for plant-based CuNPs and antibiotics-conjugated plant-based CuNPs such as Cefepime (CuNPs-Cefe), Cefotaxime (CuNPs-Cefo), and Ceftriaxone (CuNPs-Cfx) to cause DNA damage in Methicillin-resistant S. aureus, K. pneumoniae and P. aeruginosa. The findings demonstrate that when compared to the control (untreated DNA suspensions), all types of CuNPs significantly damaged the DNA of all bacterial strains. , except CuNPs against E. faecalis (abs = 1.97 at 280 nm) and control (abs = 1.65) while against K. pneumoniae CuNPs (abs = 2.10) and CuNPs-Cefe (abs = 1.69), which were ineffective in causing DNA damage (Figures in Supplementary file). According to the results, all plant-based CuNPs, especially antibiotics-supplemented CuNPs, were more effective against the tested bacterial strain.

Stabilization of copper nanoparticles in polyethylene glycol

CuNPs were stabilized by PEG of different molecular weights (MW). The results were confirmed by UV-Vis absorption spectra. The absorption peak of CuNPs stabilized by PEG 4000 shows a much broader bandwidth than stabilized by PEG 6000, 8000, and 10,000 in all cases ( graphs in Supplementary file). The absorption peak was also different; as the molecular weight of PEG increases, the absorption line tends to be straight. PEG with lower molecular weight showed a broader peak than higher molecular weight.

Contact angle and surface free energies

Contact angle measurements were carried out using three probe liquids, i.e., water, formamide, and I-Bromonepthalene. The surface tension parameters of the three probe liquids are available in the literature (Gallardo-Moreno et al. 2004). The Contact angle values and surface tension (γ^LW, γ^Acid, γ^Base, and γ^Total) parameters of E. faecalis and P. aeruginosa were adopted from the literature (Abdallah et al. 2014; Das et al. 2013; Gallardo-Moreno et al. 2002). In contrast, the protocol described in the literature was used to experimentally find the contact angles of Methicillin-resistant S. aureus, K. pneumoniae, and all other samples, including GIC and nanoparticles incorporated GIC (Gallardo-Moreno et al. 2004) (Table 3).

Table 3.

Contact angle values and surface tension (γ^LW, γ^Acid, γ^Base, and γ^Total) parameters of MRSA, E. faecalis, K. pneumoniae, P. Aeruginosa, GIC and nanoparticles incorporated GIC

	θW	θF	θB	γLW	γAcid	γBase	γTotal
Bacteria
MRSA	54.89 ± 1.09	76.47 ± 1.57	59.13 ± 1.85	28.70	0.00	62.35	28.70
K. pneumonia	21.3 ± 1.34	24.08 ± 1.00	39.88 ± 1.23	34.67	1.52	52.51	52.55
P. aeruginosaa	49.9 ± 0.79	39.4 ± 1.76	49.1 ± 0.79	34.77	1.06	28.84	45.82
E. faecalisb	49 ± 1.10	37 ± 1.37	47 ± 1.95	35.93	1.12	28.46	47.24
GIC
Pure GIC	60.09 ± 1.93	75.10 ± 1.07	34.30 ± 1.56	36.82	0.00	50.51	36.82
GIC-CuNPs	51.00 ± 0.23	57.10 ± 1.37	37.25 ± 1.28	34.21	0.00	39.23	34.22
GIC-CuNPs Cfx	61.70 ± 1.41	58.00 ± 1.61	36.66 ± 1.42	35.51	0.00	25.85	35.51
GIC-CuNPs Cefo	63.30 ± 1.02	56.20 ± 1.24	39.10 ± 1.19	35.01	0.02	23.39	36.36
GIC-CuNPs Cefe	65.00 ± 0.21	59.10 ± 1.63	36.50 ± 1.27	35.53	0.00	22.25	35.53

^aAbdallah et al. 2014, Das et al. (2013), ^bGallardo-Moreno et al. (2002)

The contact angle values were slightly higher for the GIC than the nanoparticles associated with GIC. The reason might be the incorporation of CuNPs into GIC, suggesting its hydrophilic nature.

Using Young’s equation, the surface free energy components, i.e., the Lifshitz-van der Waals (LW) and AB (+ and -) components of the bacteria and modified GIC with nanoparticles, were directly computed from their contact angles.

The LW component of bacteria and GIC was derived from the utilization (equ: 3) of I- Bromonapthalene while electron-denoting and accepting parameters were calculated from water and formamide (equ: 4).

3

4

The contact angle values of the bacteria and GIC were further utilized to calculate surface-free energies using the acid-base approach (Bos et al. 1999). Calculated parameters show that different energy components, such as Lifhsitz-van der Waals, electron acceptors, and electron donors, contribute to a material’s overall surface energy.

Methicillin-resistant S. aureus showed a lower surface energy of 28.72 mJ m^− 1 compared to other bacteria used in the study, which showed surface energy between 45 and 53 mJ m^− 1. It is a well-known fact that high contact angle values produce lower surface energy values (Vennapusa et al. 2009). On the other hand, the surface energy of pure GIC (34.22 mJ m^− 1) was lower than that of pure GIC and modified GIC with nanoparticles (35.51–36.82 mJ m^− 1). A comparison of surface energies of all bacteria and dental surfaces, i.e., GIC, is presented in Table 4.

Table 4.

Surface free energies and Interaction Energy (U) vs. Distance profile (H) for bacteria, GIC and nanoparticles incorporated GIC according to extended DLVO theory

Bacteria	Support	∆GLW mJ m− 1	∆GAB mJ m− 1	∆G TOTAL mJ m− 1	U (kT)
MRSA	Pure GIC GIC-CuNPs GIC- CuNPs Cfx GIC- CuNPs Cefo GIC- CuNPs Cefe	-1.93 -1.63 -1.78 -1.72 -1.78	49.69 41.10 28.38 25.88 25.48	47.75 39.49 26.60 24.16 23.70	-14.91 -12.13 -13.50 -13.16 -13.73
K. pneumonia	Pure GIC GIC-CuNPs GIC- CuNPs Cfx GIC- CuNPs Cefo GIC- CuNPs Cefe	-3.41 -2.88 -3.15 -3.04 -3.15	37.87 31.42 22.42 19.94 19.65	34.46 28.54 19.30 16.89 16.50	-29.31 -23.86 -27.13 -25.92 -26.88
P. aeruginosa	Pure GIC GIC-CuNPs GIC- CuNPs Cfx GIC- CuNPs Cefo GIC- CuNPs Cefe	-3.34 -2.90 -3.17 -3.43 -3.17	1.43 13.00 3.51 19.55 0.56	-1.91 10.10 0.35 16.11 -2.61	-32.51 -26.44 -30.63 -30.64 -32.15
E. faecalis	Pure GIC GIC-CuNPs GIC- CuNPs Cfx GIC- CuNPs Cefo GIC- CuNPs Cefe	-3.71 -3.13 -3.42 -3.31 -3.42	19.30 12.59 3.15 1.09 0.22	15.59 9.44 -0.26 -2.21 -3.20	-30.06 -24.15 -27.27 -25.97 -27.17

Calculation of surface free energies of Bacteria to GIC

The values of surface tension of bacteria and dental material were used to calculate the free energy of interaction, i.e. ∆G^AB and ∆G^LW were calculated using the equation described in the literature (Aasim et al. 2013) at the shortest distance of approximation (1.67 Å).

Table 4 represents the interaction free energies for bacteria and modified GIC. It was noted that ∆G^LW, i.e., the apolar component, is lower than ∆G^AB, the polar component, in all the cases. The apolar interaction component for Methicillin-resistant S. aureus and K. pneumoniae to GIC was higher than for P. aeruginosa and E. faecalis. The same trend was also observed for ∆G^TOTAL.

Interaction energy calculation as a function of distance

Refined energy to distance profiles can be used to understand how bacteria interact with dental material (U vs. H). Both the fundamental data from the experimentally determined contact angle and data from the zeta potential determinations were needed for these calculations.

The G^LW, G^AB, and zeta potential values calculated earlier were used to calculate the interaction energy (U) vs. distance (H) profiles using the XDLVO approach. All parameters were taken into account for these calculations. The interaction energy (U) vs. Distance profile for pathogenic bacteria on GIC, nanoparticles incorporated in GIC, and antibiotics-supplemented nanoparticles incorporated into GIC is shown in Table 4. Negative values of secondary energy − 14.91 kT, -12.13 kT, -13.73 kT, -13.16 kT and − 13.50 kT were observed for Methicillin-resistant S. aureus onto surfaces like pure GIC, GIC with bare CuNPs, GIC with cefepime incorporated CuNPs, GIC with cefotaxime incorporated CuNPs and GIC with ceftriaxone incorporated CuNPs respectively, indicating strong interaction between GIC and bacteria. This interaction of MRSA was weaker among all the bacteria tested. P. aeruginosa (Table 4) demonstrates the negative value of secondary energy minimum of -32.51 kT, -26.44 kT, -30.64 kT, − 32.15 kT and − 30.63 kT onto dental surfaces like pure GIC, GIC with bare CuNPs, GIC with cefepime incorporated CuNPs, GIC with cefotaxime incorporated CuNPs and GIC with ceftriaxone incorporated CuNPs respectively, indicating high interaction between GIC and bacteria.

E. faecalis (Table 4) depicts the negative value of secondary energy − 30.06 kT, -24.15 kT, -27.17 kT, -25.97 kT and − 27.27 kT for onto surfaces like pure GIC, GIC with bare CuNPs, GIC with cefepime incorporated CuNPs, GIC with cefotaxime incorporated CuNPs and GIC with ceftriaxone incorporated CuNPs respectively (indicating strong interaction between GIC and bacteria). For K. pneumonia, the negative value of secondary energy − 29.31 kT, -23.86 kT, -26.88 kT, − 25.92 kT, and − 27.13 kT was observed (Table 4) onto dental surfaces like pure GIC, GIC with CuNPs, GIC with cefepime incorporated CuNPs, GIC with cefotaxime incorporated CuNPs and GIC with ceftriaxone incorporated CuNPs respectively, indicating strong interaction between GIC and bacteria.

It became evident from Table 4 that the interaction of bacteria with pure GIC has more negative kT values, thus indicating a strong interaction of all selected bacteria onto GIC. It was also observed that the (U) kT values were lower among all the bacteria onto nanoparticles incorporated GIC, indicating weak interactive behaviour, and repulsion was observed as a result. Furthermore, when antibiotics-supplemented nanoparticles were incorporated into GIC, lower (U) kT values were observed compared to bare nanoparticles for all the bacteria, thus showing strong interactions with bacteria. It became evident from that bacterial interaction with GICS had less negative kT values when CuNPs were incorporated into GIC and eventually became more negative with antibiotics conjugated GIC and pure GIC.

According to the XDLVO approach, the interfacial free energy (U) vs. distance (H) profile can be assessed using the ∆G^LW and G^AB interfacial energies (Bos et al. 1999). To determine the Hamaker constant (A) for the interactions between bacteria and dental material, one can use the interfacial free energy component (∆G^LW). The zeta for selected bacteria and the modified dental surfaces needed for these calculations were determined earlier in the study. The information from ∆G^LW, ∆G^AB, and zeta potential values can be used to assess how selected bacteria interact with modified and unmodified dental surfaces.

Antibacterial activity of dental material

The modified direct contact test assay of pure GIC and modified GIC with green synthesized CuNPs was performed against K. pneumoniae, E. faecalis, P. aeruginosa and Methicillin-resistant S. aureus. The modified GIC showed a significant antimicrobial effect against all the tested bacteria compared to the antimicrobial effects of pure GIC. These were modified after one hour. The modified GIC groups with CuNPs with antibiotics were more effective than other groups. It was noted that cefotaxime had the highest antimicrobial efficacy against all the bacteria compared to the other groups (Fig. 6). The antimicrobial efficacy of ceftriaxone-conjugated CuNPs was slightly lower than that of cefepime-conjugated CuNPs and cefotaxime-conjugated CuNPs for all the tested bacteria on dental material, as shown in Fig. 6. It was noted that, compared to the other groups, GIC combined with antimicrobial agents (CuNPs + cefotaxime) demonstrated superior antibacterial activity against all bacteria.

Fig. 6.

Antibacterial activity of pure GIC and GIC modified with CuNPs against Methicillin-resistant S. aureus, E. faecalis, P. aeruginosa, and K. pneumoniae

Discussion

Plants are recognized as an invaluable source for future novel medicines due to their bioactive components. Several significant phytochemicals have been identified in the Mentha family as having antioxidant and antibacterial effects (Daferera et al. 2003; Economou et al. 1991). Due to their hydroxyl groups, Phenolic compounds are effective hydrogen donors, explaining their various biological activities. Plants can bioaccumulate metal ions, and their active phytochemicals can act as reductants and stabilizers, facilitating the green synthesis of CuNPs. The reduction of copper sulphate to CuNPs, indicated by a color change, is attributed to the excitation of surface plasmon vibrations, leading to surface plasmon resonance. CuNPs exhibit a broad absorption peak between 270 and 330 nm, typically peaking at 280.82 nm, without other peaks, confirming the synthesis of CuNPs aided by plant biomolecules in reduction and stabilization processes. This peak corresponds to the intrinsic band-gap absorption of CuNPs due to electron transitions from the valence band to the conduction band, as detailed by Podstawczyk et al. (2019). The addition of antibiotics, slightly red-shifted from the bulk value of 280.82 nm, is due to quantum confinement effects in the nanoparticles. The strong UV absorption of CuNPs suggests their potential use in medical applications, such as sunscreens or antiseptic ointments. The band gap and catalytic activity of metal oxide nanoparticles significantly influence their cytotoxic effects on biological systems. FTIR analysis revealed additional peaks indicating the presence of enzymes, proteins, and metabolites such as alkaloids, flavonoids, polyphenols, and carboxylic acids, which remain bound to CuNPs despite washing. These compounds, particularly flavonoids and phenolics, reduce zinc ions to CuNPs. The addition of antibiotics facilitated the production of CuNPs as it cleared from the shift in the absorption peak. Various functional groups from the antibiotics were observed to play a key role in the synthesis of nanoparticles. XRD analysis showed diffraction peaks corresponding to Cu nanocrystals and the inorganic Structural Data Base (ICSD), which these diffraction data values met (Gültekin et al. 2016). SEM characterization has provided valuable insights into the properties of these CuNPs, highlighting their unique features. The morphologies of the CuNPs synthesized from Mentha extract seemed dispersed. The Morphology and size of nanoparticles are influenced by factors such as plant species, the presence of different reducing agents in the extracts, and the conditions during the production process (Mittal et al. 2013). The findings agreed with those in the literature (Gültekin et al. 2016; Wu et al. 2020). These factors also play a role in determining the properties and bioactivity of the nanoparticles (Merin et al. 2010). The morphology suggests the nanoparticles have irregular patterns with varying size distribution. According to Mittal et al., (Mittal et al. 2013) the size difference was due to the reducing agents in the extract and antibiotics that contributed to the formation of nanoparticles.

The disc diffusion assay depicts the enhanced antibacterial activity of cephalosporin-capped CuNPs. This may be due to the incorporation of the β-lactam ring into these nanoparticles. As discussed in the literature (Rai et al. 2010), the MIC value of antibiotic-capped nanoparticles was far better than CuNPs and antibiotics alone. A concentration of almost 30 mg/ml was safer in the hemolytic assay and indicated less than 5% of red blood cell lysis except for cefepime (5.16 ± 1.13) and cefotaxime (5.16 ± 1.17), which caused the lysis of red blood cells at the concentration mentioned above. All the synthesized nanoparticles showed DNA damage in all four bacteria, i.e. K. pneumoniae, E. faecalis, P. aeruginosa and Methicillin-resistant S. aureus, showed the ability of CuNPs to bind to sulfhydryl (SH) groups, disrupting bacterial nucleic acids as stated in the literature (Gutiérrez et al. 2019).

Surface electrostatic effects, vital in determining the level of interaction between surfaces, cannot be calculated by contact angle measurements. In comparison, it is only practicle to evaluate a material’s surface charge by calculating a surface’s electrostatic potential at the hydrodynamic plane of shear. The zeta potential results showed unstable nanoparticles indicating the formation of stable aggregation solutions as the PDI was closer to 0.3 (Ahmed et al. 2016), which were stabilized by polyethylene glycol, forming a coating that prevents nanoparticle agglomeration. As the molecular weight increases, the length of the PEG molecular increases and the nanoparticles are best surrounded by the polymer, thus inhibiting the undesirable aggregation. The peak width of bare CuNPs was broader when stabilized by PEG and eventually narrower when antibiotics were conjugated to these nanoparticles. They may be the contribution of varying refractive index or because the initial aggregates present in the solution could be redispersed after PEG stabilization.

Bacterial biofilm, a major cause of dental caries worldwide, drives the search for new dental material with antibiofilm and antibacterial effects and higher efficacy. The supplementation of CuNPs with cephalosporin antibiotics and their subsequent combination with GIC have shown significant efficacy against various pathogenic bacteria commonly implicated in dental infections. The contact angle values of pure GIC were slightly higher than the nanoparticles associated with GIC, suggesting the hydrophilic nature of CuNPs associated with GIC.

Notably, the modified GIC samples exhibited superior antibacterial effects compared to conventional GIC, as evidenced by the modified direct contact test. The enhanced potency against Methicillin-resistant S. aureus, E. faecalis, K. pneumoniae, and P. aeruginosa underscores the potential of this innovative approach in combating antibiotic-resistant strains, a pressing concern in modern dentistry.

Among the antibiotic-supplemented CuNPs, those enriched with cefotaxime demonstrated the most pronounced antibacterial activity, followed by Cefepime and Ceftriaxone-supplemented CuNPs, respectively. This hierarchy suggests the importance of antibiotic selection and highlights the versatility of this composite material in targeting specific bacterial strains.

The size and shape of nanoparticles are critical factors influencing their antibacterial activity, particularly against pathogens (Tripathi and Goshisht 2022). In our study, smaller nanoparticles, typically below 50 nm, exhibited enhanced antibacterial effects due to their larger surface area-to-volume ratio, allowing better interaction with bacterial membranes, penetration, and disruption of essential cellular processes such as protein synthesis and DNA replication (Azam et al. 2012). Spherical nanoparticles are particularly effective because of their uniform charge distribution, which facilitates even adhesion to bacterial surfaces and efficient biofilm penetration, critical for Gram-negative bacteria like P. aeruginosa (Alshareef et al. 2017). Conversely, rod-shaped nanoparticles demonstrate greater efficacy against robust biofilms, highlighting the shape-dependent variability in antibacterial action (Li et al. 2005). Incorporating CuNPs into glass ionomer cement (GIC) retains these size- and shape-specific properties, enhancing their bioactivity against pathogens. Additionally, the conjugation of CuNPs with cephalosporin antibiotics such as cefotaxime synergistically increases antibacterial potency, as the small nanoparticles disrupt bacterial membranes and provide a larger surface for antibiotic binding, intensifying their combined effects. With their thick peptidoglycan layers, gram-positive bacteria like MRSA and E. faecalis are effectively targeted by smaller, spherical CuNPs that generate reactive oxygen species (ROS) to damage cell walls (Hajipour et al. 2012). Similarly, Gram-negative bacteria, such as K. pneumoniae, benefit from the enhanced penetration capabilities of these nanoparticles, particularly in biofilm inhibition. Overall, the size and shape of CuNPs and antibiotic capping play a pivotal role in determining their antibacterial efficacy, as demonstrated by the superior activity of cefotaxime-capped CuNPs against biofilm-associated infections in the current study.

Previous studies by Gulluce et al. (2007) reported the antimicrobial and antioxidant activities of the essential oil and methanol extract from Mentha longifolia. GC–MS analysis of the oil identified 45 constituents, cis-piperine epoxide, pulegone and piperitenone oxide being the main components (Gulluce et al. 2007). CuNPs nanostructures have shown promise in targeting bacterial cells, supporting observations that CuNPs are less toxic to haemolytic cells. Moshaverina, A. et al. (2008) reviewed the effect of the incorporation of hydroxyapatite and fluorapatite nano bioceramics into conventional GICs to study the mechanical strength (Moshaverinia et al. 2008). The exact mechanisms by which metal nanoparticles may control the growth of biofilms are still debated. Still, it is believed that metal ion causes polymeric disruption (Allaker 2010).

Furthermore, the extended Derjaguin–Landau–Verwey Overbeek (XDLVO) approach elucidated the interaction between modified GICs and bacterial strains, providing valuable insights into their mechanism of action. The addition of bare CuNPs to dental material inhibits the interaction of bacteria to dental material, while synthesised in the presence of antibiotics showed excellent antibacterial properties. The effective inhibition of biofilm formation on dental implants by modified GIC further underscores their potential in preventing recurrent infections and promoting oral health.

In conclusion, the findings of this study unveil the remarkable potential of Mentha longifolia extract as a natural and eco-friendly resource for the synthesis of CuNPs. These nanoparticles are synthesized by reducing copper salts facilitated by phenolic compounds and other enzymes within the extract. These CuNPs were supplemented with antibiotics to enhance their antibacterial properties while reducing the load of antibiotics. Further, these supplemented CuNPs were added to GIC, creating novel nanobiomaterials. The GIC containing bare CuNPs and antibiotic-supplemented CuNPs demonstrated a higher antibiofilm effect than pure GIC.

Overall, the findings of this study hold significant implications for the development of advanced therapeutic strategies in dentistry. Integrating natural extracts, nanotechnology, and antibiotic therapy offers a multifaceted approach to combating dental caries and lesions, addressing the challenges posed by microbial resistance and enhancing patient outcomes. Further research in this direction holds promise for the continued advancement of dental biomaterials and the improvement of clinical practices in oral healthcare.

Electronic supplementary material

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Authors contribution

HS conducted research work, Draft preparation, MA Designed Research, TK: Critical revision of Manuscript, AK, HMK, UA assisted in the software portion of the Research.

Funding

The Authors received no funding for this Research.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

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Not applicable.

Competing interests

The authors have no competing interests to declare.