A novel facile synthesis of metal nitride@metal oxide (BN/Gd2O3) nanocomposite and their antibacterial and anticancer activities

In this study, a novel core/shell nanocomposite structure (h-BN@Gd2O3 NCs) was created for the first time by combining hexagonal boron nitride (h-BN) with doped gadolinium oxide (Gd2O3) using different laser pulse numbers, i.e., 150, 338, and 772 pulses. We employed various analytical techniques, including mapping analysis, FE-SEM, EDS, HRTEM, SAED, XRD, zeta potential analysis, DLS, FTIR, Raman spectroscopy, and PL measurements, to characterize the synthesized h-BN, c-Gd2O3, and h-BN@Gd2O3 NCs (338 pulses). XRD results indicated hexagonal and cubic crystal structures for BN and Gd2O3, respectively, while EDS confirmed their chemical composition and elemental mapping. Chemical bonds between B–N–Gd, B–N–O, and Gd–O bands at 412, 455, 474, and 520 cm−1 were identified by FTIR analysis. The antimicrobial and anticancer activities of these NCs using agar well diffusion and MTT assays. They exhibited potent antibacterial properties against both Gram-positive and Gram-negative pathogens. Furthermore, NCs have reduced the proliferation of cancerous cells, i.e., human colon adenocarcinoma cells (HT-29) and human breast cancer cells (MCF-7), while not affecting the proliferation of the normal breast cell line (MCF-10). The anticancer efficacy of NCs was validated by the AO/EtBr assay, which confirmed apoptotic cell death. Blood compatibility on human erythrocytes was also confirmed by hemolytic and in vitro toxicity assessments. The compiled results of the study proposed these nanoparticles could be used as a promising drug delivery system and potentially in healthcare applications.

Nanotechnology has revolutionized therapeutic approaches by creating materials at the nanoscale.Nanoparticles possess unique properties that differ from larger particles, influencing their interactions within biological systems.Size is important in biological processes such as crossing barriers and immune recognition.Nanotechnology offers advantages over conventional methods by enhancing cellular retention and penetration.Particles larger than 100 nm but with exceptional properties are also considered nanomaterials 1,2 .Currently, there is a pressing need for an advanced treatment approach that can effectively overcome the barrier posed by the cell membrane and ensure targeted drug delivery and sustained retention at the desired site.In comparison to conventional agents, drugs engineered at the nanoscale can offer a multitude of pharmacological advantages.The creation of unique and superior "Nano-drugs" is possible by fusing the extensive understanding of nanoparticles (NPs) with the most recent knowledge of cellular and molecular activities 3 .These NPs possess the remarkable ability to encapsulate, incorporate, or conjugate various drug molecules, enabling precise delivery to specific targets [3][4][5] .Cancer, among other devastating diseases, remains a major cause of mortality and demands urgent intervention.Breast cancer, in particular, continues to claim a significant number of lives worldwide 6 .The utilization of existing chemotherapy drugs has its drawbacks, including high costs, toxicity, and severe side effects.Therefore,

Synthesis of h-BN@Gd 2 O 3 hybrid NCs
To create h-BN@Gd 2 O 3 HNCs, we followed a two-step process.First, we prepared an h-BN suspension using similar steps as described in "Synthesis of h-BN and Gd 2 O 3 NPs".In the second step, we ablated the Gd 2 O 3 pellet using the same laser with a fluence of 1.27 Jcm -2 in colloid h-BN Ns solution at different numbers of laser pulses (150, 338, and 772).A schematic diagram of the procedure for the formation of h-BN@Gd 2 O 3 NCs via the hybrid technique is shown in Fig. 1. Figure 2 shows a schematic diagram of the mechanisms of the formation of h-BN@Gd 2 O 3 NCs via laser ablation in liquid technique.Selecting the appropriate parameter, such as the number of laser pulses, is crucial in antibacterial and cancer treatment experiments.This choice enables us to achieve the necessary high concentration for optimal results in eliminating bacteria or cancer cells.In addition, it helps us determine the ideal parameter for synthesis, which is 10 mJ with 338 pulses, as it yields the most favorable outcome.Several methods were used to examine the materials' morphology, structure, chemical, and optical properties.Field Emission Scanning Electron Microscopy (FE-SEM) with Energy Dispersive Spectroscopy (EDS) and mapping analysis were conducted using the Inspect F50 system (a small amount, one drop, of the prepared sample was deposited on a glass substrate).Transmission electron microscopy with high resolution (HR-TEM) from JEOL in Tokyo, Japan, was employed with Selected Area Electron Diffraction (SAED).A small amount (one drop) of the prepared sample was deposited on a gold mish, and then the samples underwent a coating process with a thin layer of gold to enhance imaging quality and reduce charging effects.X-ray crystallography (XRD) data were collected using a Shimadzu X-ray diffractometer at 40 kV and 25 mA with Cu-Kα radiation at 1.5405 Å. Nano plus DLS Nano Particle Sizer was used to assess the particle size and zeta potential (the colloidal sample was placed in a glass cuvette).Instruments of various types were used to evaluate chemical and optical qualities.A Perkin-Elmer spectrometer was used to collect FT-IR spectra in the 400-4000 cm −1 range.A THERMO SCIENTIFIC DXR FT-Raman spectrometer was used to record the Raman spectra.The laser source had a 2-mW output at 532 nm.Using a Shimadzu UV-2600 spectrometer, the UV absorbance spectra of the colloidal suspensions were determined between 200 and 500 nm.Shimadzu Spectra fluorophotometer model RF-5301pc was used to measure Photoluminescence Spectroscopy) PL (, and the colloidal sample was placed in a quartz cuvette.

Antibacterial activity of h-BNNs, c-Gd 2 O 3 , and h-BN@Gd 2 O 3 NCs
Using an agar well diffusion experiment, the antimicrobial activity of the produced materials (h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs) was examined against both Gram-ve (Escherichia coli and Proteus mirabilis) and Gram + ve (Streptococcus mutans and Staphylococcus aureus) bacterial isolates, which were identified using the VITEK system (VITEK, Biomérieux, Marcy-l'Etoile, France), kindly provided by the Medical Microbiology Laboratory, Biotechnology Division, Department of Applied Science, University of Technology, Baghdad, Iraq.www.nature.com/scientificreports/ The McFarland standard was used to obtain suspensions at 1.5 × 10 8 colony-forming units (CFU) mL -1 .Muller-Hinton (MH) agar was aseptically put onto sterile Petri plates in an amount of 20 mL.Using a sterile wire loop, the bacteria were extracted from their stock cultures.Using a sterile point, 6 mm-diameter wells were drilled into the agar plates after the organisms had been cultured.In the dreary wells.Before measuring and recording the average zones of inhibition diameter, the cultivated plates containing the test organisms and samples (h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs at a concentration of 50 µg mL −1 of each) were incubated overnight at 37 °C for 24 h in an upright position, and inhibition zones were measured in millimeters by measuring the diameter of circular inhibition zones around the well using a physical ruler.Deionized distilled water (DDW) was used as a control.The assay for antibacterial activity was performed in triplicate 31 .

Culturing of cell lines
The human colon adenocarcinoma cells (HT-29), Michigan Cancer Foundation-7 (MCF-7; human breast cancer cells), and normal breast MCF-10 were provided by the American Type Culture Collection (ATCC, Manassas, USA).The cells were grown in DMEM supplemented with 10% FBS, 2 mM l-glutamine, and 20 mM HEPES and employed using tissue culture-treated flasks (T 25 cm 2 ; Falcon, USA) under the conditions of 5% CO 2 and 37 °C.

MTT assay
To assess the cell viability and cytotoxicity of h-BNNs, c-Gd where Abc and Abs were the optical density of the control and tested samples, respectively.

Dual staining assay
To assess the apoptosis effects of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs in HT-29 and MCF-7 cells, dual-labeling using AO/EtBr dye was carried out according to the method of Al-Jubori and his co-workers 33 .Briefly, cells were seeded (density 1 × 10 5 cells per mL) using DMEM in 96-well microtiter plates and incubated overnight.IC 50 concentrations of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs were then applied for 24 h, followed by washing with PBS, applying dual fluorescent dyes (100 μL) to the cells, and finally visualized under a fluorescent microscope.

Hemocompatibility test
Fresh samples of blood were taken from five healthy donors and distributed into heparin-coated tubes based on the method of the NIH (National Institute of Health) and FDA (Food and Drug Administration) and as per the declaration and regulation of Helsinki of 1975 as a statement of ethical principles.Permission was obtained from the hospitals of the medical city in Baghdad, Iraq, and approved by the institutional ethical committee of the Department of Applied Sciences, University of Technology, Baghdad, Iraq (Ref.No. 216AS18/1/2021).Study participants were informed about the value of the study before we collected any samples.Informed consent was obtained from the study participants.
A hemolysis test was performed for the h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs samples based on a previously described method 33 .Briefly, whole blood (100 μL) was mixed with PBS (700 μL) and 100 μL of h-BNNs, c-Gd 2 O 3 Ns, or h-BN@Gd 2 O 3 NCs at seven concentrations (1, 5, 10, 15,25,50, and 100 μg mL −1 ) were added; phosphate buffer saline served as a negative control (0% hemolysis), whereas deionized distilled water served as a positive control (100% hemolysis).After shaking for 1 h at 37 °C, the reaction mixture is centrifuged at 700 rpm for 5 min, and the absorbance of the supernatant at 541 nm is measured.The hemolysis value percentages were therefore expressed in the following equation (2).

Statistical analysis
We utilized Graph Pad Prism version 8, Image J, and Origin 2021 software to analyze the results obtained from the conducted experiments.A one-way ANOVA was used to analyze the data, and then significant differences were set at * p ≤ 0.05, ** p ≤ 0.01, or *** p ≤ 0.01.Data were presented as mean ± standard deviation. (1) Figures 3 and 4 illustrate the morphology, structure, and particle size of the BNNs, Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 obtained with varying numbers of laser pulses.The FESEM analysis revealed that the structures change as the number of laser pulses increases in DDW.In Fig. 3a, the h-BNNs exhibit small particles with irregular spherical shapes and nanosheets, these results validate those of Guerra et al., Thangasamy et al., and Kumar et al. [34][35][36] .On the other hand, Gd 2 O 3 Ns (Fig. 3b) display nanofiber-like, needle-like, and sheets (leaf-like) morphologies with significant heterogeneity, clearly demonstrating the flaky structure of the prepared Gd 2 O 3 .The Gd 2 O 3 nanocrystalline flakes are flat and have irregular shapes, as observed in the micrographs, these results validate those of Almeida et al., and Jeon et al. 37,38 .However, in the case of the nanocomposites during the synthesis of h-BN@ Gd 2 O 3 , the h-BN and c-Gd 2 O 3 particles tend to coalesce, as depicted in Fig. 3c-e.The results indicated that the h-BNNs consisted of spherical-shaped particles, while Gd 2 O 3 exhibited numerous flake-like and sheets (leaf-like) morphologies with a layered structure, making it easy to identify the Nano type.Most of the nanostructures are interconnected, forming networks that do not exhibit well-ordered patterns, and their sizes and thicknesses vary.
The HRTEM was used to capture images of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs, which were synthesized using different numbers of laser pulses, resulting in a variety of morphologies.The TEM analysis findings were consistent with those obtained from the FESEM images.Specifically, Fig. 4a shows well-dispersed and www.nature.com/scientificreports/uniform h-BNNs with both spherical and nanosheet shapes 36 .These NPs' average particle size was found to be 30 nm.On the other hand, Fig. 4b reveals the presence of diverse Gd 2 O 3 nanostructures, including needle-like and sheet-like structures.These NPs' average particle size indicated that these Gd 2 O 3 Ns had varying lengths ranging from 40 to 140 nm and diameters ranging from 5 to 30 nm.It is interesting that the number of laser pulses had a considerable impact on the size and morphology of the synthesized h-BN@Gd 2 O 3 NCs, as observed in Fig. 4c-e.As the number of laser pulses increased, the average crystallite size of the Ns decreased.The core-shell www.nature.com/scientificreports/structure of the prepared h-BN and c-Gd 2 O 3 NPs was confirmed by the TEM images.In this structure, the dark points represent the h-BNNs (core) surrounded by the light points, which represent the Gd 2 O 3 Ns (shell).The h-BN@Gd 2 O 3 NCs, synthesized with 338 pulses, underwent EDS analysis to determine their chemical composition.Figure 5 (left lane) presents the EDS spectra of the h-BN@Gd 2 O 3 NCs deposited on a glass substrate along with the corresponding weight percentages of elements.The analysis revealed the presence of boron (B), nitrogen (N), gadolinium (Gd), oxygen (O), and silicon (Si) elements in the h-BN@Gd 2 O 3 NCs.The O elements can be attributed to interactions with the atmosphere, Gd 2 O 3 target, and DDW.As a result, the prepared NCs are obviously devoid of additional contaminants.Due to the poor electron scattering by light components, this method is not suited for directly measuring the presence of hydrogen (H) in the Nanocomposite.Nevertheless, micro-Raman, FTIR, and XRD analyses can be used to infer the existence of the H element.The findings of the EDS mapping and spectrum show that the h-BN@Gd 2 O 3 NCs (core-shell) successfully formed and that the distribution of the elements in the sample was uniform.In the right lane of Fig. 5a, the SEM image of the sample is shown, while Fig. 5b-f correspond to the formation of silicon (from the glass substrate), boron, nitrogen, gadolinium, and oxygen nanoparticles in the sample, respectively.
where is Bragg's diffraction angle, is the angular peak width at half maximum (in rad), and is the X-ray wavelength (= 1.54060).The average crystallite sizes for h-BNNs, c-Gd 2 O 3 Ns, h-BN@Gd 2 O 3 (150 pulses), h-BN@ Gd 2 O 3 (338 pulses), and h-BN@Gd 2 O 3 (772 pulses) were determined to be 7.04 nm, 0.68 nm, 2.59 nm, 3.75 nm, and 4.88 nm, respectively.Table 1 provides the calculated crystallite size for each sample.These findings corroborate the FESEM results, indicating the successful formation of nanometer-sized particles.These results are in full agreement with previously reported data conducted by other researchers 29,34,37,39 .When compared to the XRD patterns of h-BNNs and c-Gd 2 O 3 , the diffraction patterns of h-BN@Gd 2 O 3 produced at different laser pulse levels correspond to both the h-BNNs peak and the c-Gd 2 O 3 Ns peak.This confirmation supports the creation of a hybridized material (core/shell) without any chemical interaction between them.A slight increase in the intensity of the diffraction peaks has been observed, but there has not been a significant broadening of the peak.This is because more laser pulses lead to a higher quantity of NCs in the colloidal solution [40][41][42] .This observation implies an enhancement in crystallinity due to doping.The data indicate that both BN and Gd 2 O 3 influenced the crystallite sizes of the NCs.
The insights garnered from HRTEM and SAED analyses provide a pivotal understanding of the structural attributes of various materials, including h-BNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs, as illustrated in Fig. 7.In Fig. 7A, the presence of a 3.2 Å d-spacing and the alignment with hkl (002) planes affirm crystallinity, validating the successful synthesis of the hexagonal phase with anticipated interplanar distances.SAED patterns further verify crystallographic orientation and atomic arrangement.The congruence among d-spacing, hkl planes, and SAED reinforces the high-quality synthesis of h-BN through laser exfoliation and fragmentation in deionized water (DDW).Both c-Gd 2 O 3 Ns and h-BN@Gd 2 O 3 NC samples exhibit robust crystallinity, evident from welldefined lattice fringes across a significant proportion of particles (Fig. 7B, C).HRTEM images vividly capture regular lattice fringes, reflecting consistent atom spacing, while SAED patterns present concentric rings, indicating a polycrystalline nature of the sample.The d-spacing values deduced from HRTEM and SAED analyses were cross-referenced with those derived from XRD data, resulting in alignment and confirming the cubic crystal structure for c-Gd 2 O 3 Ns (hkl (100), ( 211), ( 222), (123), and (411)) and for h-BN@Gd 2 O 3 NCs (hkl (100), ( 211), (002), ( 411), ( 123), ( 100), (004), ( 444), (110), and (411)).These findings collectively endorse the credibility of HRTEM and SAED methodologies in characterizing nanoscale crystal structures.Notably, these results harmonize seamlessly with the XRD findings discussed earlier, solidifying their reliability.These revelations hold promise for potential applications spanning electronics, optoelectronics, and nanocomposites.
Using DLS, it was possible to ascertain the hydrodynamic diameter of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@ Gd 2 O 3 NCs (338 pulses) in DDW medium, which provides information about their particle size (Fig. 8).The hydrodynamic size of h-BNNs was measured at 981 nm with a polydispersity of 0.332 (Fig. 8A).For c-Gd 2 O 3 Ns, the hydrodynamic size was 432 nm with a polydispersity of 0.240 (Fig. 8B).As for h-BN@Gd 2 O 3 NCs (338 pulses), the hydrodynamic size was recorded as 573.64 nm with a polydispersity of 0.241 (Fig. 8C).It's crucial to remember that the DLS particle size was discovered to be greater than the results from XRD and TEM, as it represents the hydrodynamic size.Furthermore, the results indicate that h-BNNs exhibited higher dispersibility in DDW compared to Gd 2 O 3 Ns, as evidenced by their respective hydrodynamic sizes.The value of the zeta potential (ζ) provides important details regarding the potential stability of the colloidal system.Charged particles are repelled by an increase in zeta potential, which prevents them from aggregating in the suspension.A ζ value of at least 30 mV is often displayed by stable systems 41 .When a ζ nanoparticle's value is between −10 and + 10 mV, it is said to be virtually neutral, however when it ζ exceeds + 30 mV or drops below −30 mV, it is classified as strongly cationic or strongly anionic.In our study, we measured ζ of three samples: h-BNNs  and the results show that the h-BNNs sample has a high modulus value of ζ = −15.20 mV.This is explained by the fact that the laser exfoliation and fragmentation process leaves hydroxyl groups (OH) on the surface of the BNNs, which improves the suspension's stability 43 .Understanding the zeta potential is important as it helps to comprehend the in vivo destination of materials as it relates to cellular processes like aggregation, adhesion, and activation.This evaluation of stability in the synthesized materials is essential when they are dispersed in DDW.However, it is important to note that other factors such as particle size, surface charge density, and the presence of stabilizing agents also play significant roles in determining the overall stability of nanoparticles 44 .
The FTIR measurements provide more evidence that the final product is a combination of h-BN and c-Gd 2 O 3 Ns.Figure 10 illustrates the results of FTIR studies to examine the chemical compositions and bonding properties of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs.The three samples' spectra have the same characteristics, including a strong absorption peak at 1631 cm −1 and a wide absorption band spanning 3000 cm −1 to 3700 cm −145 .Both of these absorptions were linked to the O-H vibration modes of bending and stretching, respectively.However, it was found that the three materials' IR absorption varied noticeably between 400 and 550 cm −1 (Fig. 10 inset).The Black spectrum reveals characteristic peaks at 416 cm −1 , 455 cm −1 , 490 cm −1 , and 520 cm −1 , attributed to the bending vibration of B-N and B-N-O, which are indicative of h-BNNs 34 .The red spectrum displays characteristic peaks at 404, 428, 447, 514, and 524 cm −1 , attributed to the bending vibration of Gd-O and Gd-OH,    Raman spectra, the professional technique for material analysis, were utilized in this study to investigate various characteristics such as crystallographic phase, impurities, and structural defects.The Raman analyses employed a laser wavelength of (632.8 nm), and the obtained spectra ranged from 0 to 2000 cm −1 (Fig. 11).A comparison was made between the Raman spectra of h-BN@Gd 2 O 3 prepared using different laser pulses, h-BNNs, and c-Gd 2 O 3 Ns prepared by the same technique.The Raman spectrum of the BNNs material exhibited two distinct bands at 1372, 1426, and a small peak in reign < 808 cm −1 , corresponding to h-BN.The peak at < 808 cm −1 was attributed to the B1g phonon mode (which corresponds to the breathing mode of boron and nitrogen atoms in the hexagonal BN lattice).The peak at 1372 cm −1 was attributed to the E 2g phonon mode (it corresponds to the in-plane vibration of boron and nitrogen atoms in a hexagonal lattice), similar to the G peak observed in graphene.The peak at 1426 cm −1 was attributed to the A 1g phonon mode (it is related to the outof-plane vibrations of boron and nitrogen atoms in a hexagonal BN structure) 35,47,48 .In contrast, the Raman spectra of Gd 2 O 3 revealed five prominent peaks at approximately 210, 304, 360, 483, 681, and 722 cm -1 , which corresponded to F 2g (This mode is associated with oxygen vibrations in the lattice), A g translatory (It is attributed to stretching vibrations of oxygen atoms), F g + A g , and E 1g liberation modes 49,50 , showing the existence of c-Gd 2 O 3 Ns for the doped sample's h-BN phase, c-Gd 2 O 3 at the location of nine significant Raman peaks is seen at 210 cm −1 , 242 cm −1 , 304 cm −1 , 360 cm −1 , 483 cm −1 , 681 cm −1 , 722 cm −1 , 1372 cm −1 , and 1426 cm −1 .These findings suggest that the nanostructured h-BN@Gd 2 O 3 samples consist of a mixture of h-BN and c-Gd 2 O 3 nanostructures.
The wave numbers of the corresponding Raman peaks for the hexagonal 47 and cubic 51 phases of doped and undoped h-BN and c-Gd 2 O 3 are listed in Table 2. Notably, the intensity of the Raman bands in the spectra increased with the number of pulses, indicating a correlation between the concentration of the studied molecules and the intensity of the Raman spectral bands.The observed Raman bands align with those reported by other researchers 48,[52][53][54][55] .Additionally, this study represents the first Raman analysis conducted on the h-BN@Gd 2 O 3 cubic phase.Furthermore, the Raman spectra of h-BN exhibited two broad bands around 1600 cm −1 , associated with O 2 and H-O-H bending modes, while Gd 2 O 3 's Raman spectra showed a wide band at 1000 cm −1 , corresponding to the H-O-H bending mode, both originating from the liquid environment.The shift of Raman modes can occur due to various factors, including the presence of oxygen vacancies, defects induced by disorder, or the effects of phonon confinement 56 .
Figure 12A illustrates the UV-vis intensity of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs, synthesized using a laser energy of 10 mJ with varying numbers of laser pulses (150, 338, and 772).The recorded spectra show www.nature.com/scientificreports/ a progressive enhancement in absorption intensity between 200 and 250 nm as the number of laser pulses increases. 57,58This trend is likely attributed to all samples' heightened concentration and reduced size of nanoparticles.The UV spectra of h-BNNs and NCs samples reveal an absorption peak below 380 nm, due to fine structural vibrations and band-to-band electron transitions (as depicted in Fig. 12A).Moreover, a smooth decline is observed at longer wavelengths.The higher energy of laser pulses causes target evaporation, melting, and phase transformation at elevated temperatures and pressures, accompanied by an extended irradiation time.Several parameters, including stoichiometry, morphologies, and size distribution, influence the absorption behavior of .Figure 12B illustrates the optical band gap of the samples, offering a window for the PL spectrum of the nanoparticles (NPs).The impact of nanocrystal size on the electronic structure of semiconducting NPs is highlighted by the rise in band gap energy coupled with the decrease in particle size.This phenomenon is widely recognized  www.nature.com/scientificreports/as the quantum confinement effect, and its assessment was conducted using the Tauc method.The Tauc method postulates that the energy-dependent absorption coefficient α can be expressed by Eq. ( 4) 63,64 : (4) (α.hv)1/γ = β hv − Eg .www.nature.com/scientificreports/Here, the symbol h represents the Planck constant, v indicates the frequency of the photon, Eg represents the band gap energy, and β is a constant.The γ factor varies based on the nature of the electron transition and takes values of 1/2 or 2 for direct and indirect transition band gaps, respectively 60 .Band gap energies of 5.6, 5.7, 5.6, 4.9, and 5.5 eV were recorded for different materials, including h-BN Ns, c-Gd 2 O 3 Ns, and three unique h-BN@Gd 2 O 3 NC samples produced using varying laser pulse numbers (150, 338, and 772).The decrease in band gap energy in the doped samples confirms the effective incorporation of c-Gd 2 O 3 Ns into the h-BN structure, highlighting their potential in materials science applications.
Figure 13A-C shows the spectrum using a 532 nm excitation wavelength and deconvolution peaks of PL emission for three different samples: h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs (338 pulses) in DDW.The deconvolution process utilized multi-Gaussian functions to obtain these contributions.In Fig. 13, the PL spectra underwent deconvolution, specifically focusing on the PL peaks, using Origin Lab software, while the other components were fitted with a Gaussian line shape.Following the deconvolution, we assigned labels to the peaks based on their wavelength and color-coded each peak according to its emission band for each sample.Our findings indicate that the PL spectra of h-BNNs exhibit four distinguishable spectral features in the blue range (~ 300 to 550 nm): Violet (370 nm, 3.35 eV), Indigo (422 nm, 2.9 eV), Blue (470 nm, 2.64 eV), and Cyan (500 nm, 2.48 eV) emission.For c-Gd 2 O 3 Ns, three distinguishable spectral features were observed in the UVblue-green range (~ 300 to 500 nm): Violet (369 nm, 3.36 eV), Blue (460 nm, 2.7 eV), and Green (532 nm, 2.3 eV) emission.However, for h-BN@Gd 2 O 3 NCs (338 pulses), three spectral features were detected in the UV-bluegreen range (~ 300 to 550 nm): Violet (368 nm, 3.36 eV), Blue (470 nm, 2.64 eV), and Green (535 nm, 2.3 eV) emission.Figure 13C represents the ultraviolet, blue, and green regions, the green emission peak corresponds to the single ionized oxygen vacancy 65 .It is evident from Fig. 13A  of the UV, Blue, and Green bands also increases.Therefore, the fitting results demonstrate that the PL spectra can be described by four PL components, which exhibit different trends with changes in NPs.The above results indicate the successful synthesis of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs, all of which exhibit stable and strong fluorescence in water.The introduction of Gd 2 O 3 to BN nanoparticles can elicit various effects on PL spectra.These effects depend on factors such as the concentration and specific properties of the Gd 2 O 3 and BN NPs.One notable effect is the PL intensity enhancement, where Gd 2 O 3 nanoparticles, under certain conditions, act as sensitizers, boosting the PL intensity of BN nanoparticles.This phenomenon is often observed in rareearth-doped materials, where Gd 2 O 3 transfers energy to BN, resulting in heightened luminescence (Fig. 13C).
Another effect is the emission wavelength shift, where the inclusion of Gd 2 O 3 induces a shift in the emission wavelength of BN nanoparticles.This shift can manifest as a movement toward longer or shorter wavelengths, contingent on the specific interactions between these materials.The doping-related bands have a significant effect, as Gd 2 O 3 introduction can result in new PL bands associated with gadolinium emissions.These bands may intersect or interact with the PL bands of BN, causing changes in the PL spectrum.Gd 2 O 3 's capacity for energy transfer is also noteworthy, influencing the excitation and relaxation dynamics of BN nanoparticles.This can lead to alterations in PL kinetics and spectral characteristics.Surface effects are yet another factor to consider, as the characteristics of the surface and interactions at the Gd 2 O 3 -BN interface can influence PL spectra.Surface states or introduced energy levels due to the presence of the Gd 2 O 3 layer can affect PL characteristics.The specific effects observed depend on variables like Gd 2 O 3 concentration, nanoparticle size, structure, and synthesis conditions.Understanding these effects is crucial when exploring the potential applications of Gd 2 O3-enhanced BN nanoparticles in various fields, from optoelectronics to biomedicine.The well diffusion method has been used to investigate the antibacterial activity of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs (338 pulses) against S. aureus and S. mutans and E. coli and P. mirabilis.The inhibition zones of the prepared colloidal Ns are listed in the graphical representation of all measured zones of inhibition after 24 h of incubation period at 37C ˚.The results show that the prepared nanostructures were more effective in the growth of Gram-ve bacteria than Gram + ve bacteria.For h-BNNs, the highest activity was recorded against P. mirabilis with the inhibition zone reaching 16.3 mm, followed by E. coli which exhibited the highest diameter of inhibition zone that reached 15.8 mm after treatment with h-BN@Gd 2 O 3 NCs, and the lowest diameter of the inhibition zone was 15.4 mm after treatment with c-Gd 2 O 3 Ns.For Gram + ve bacteria, the highest effect for h-BNNs and h-BN@Gd 2 O 3 NCs) was in S. mutans with an inhibition zone diameter reached 16.7 and 16.2 mm, and the lowest inhibition zone diameter was 13.6 mm for the c-Gd 2 O 3 Ns.The inhibition zone of S. aureus was 15.2 mm for h-BNNs, followed by (c-Gd 2 O 3 Ns) in diameter reached 15 mm, and the lowest inhibition zone diameter was 13 mm for h-BN@Gd 2 O 3 NCs (Fig. 14).
Table 3 presents a comparison between our synthesized nanocomposite and previously reported antibacterial nanomaterials.The results in Table 3 indicate that our prepared h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs nanocomposite were more potent than recorded previously.Therefore, it can be concluded that h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs have the potential to serve as next-generation antibacterial agents for a range of biomedical applications.
The antibacterial efficacy of the synthesized Ns can be attributed to their interaction with cell wall constituents, causing structural changes potentially affecting cell membranes.These findings stem from the close contact between the c-Gd 2 O 3 Ns (shell) and the h-BNNs (core), facilitating direct engagement with the membrane and ultimately leading to the rupture of contracted bacteria.The escalating integration of nanostructures in medicine has sparked numerous investigations into their potential antibacterial mechanisms.Metal nitride and oxides, for instance, can modulate bacterial metabolic activity, offering a promising strategy for disease treatment.The effective antibacterial action of nanostructures hinges on their direct interaction with bacterial cells.This interaction is achieved through mechanisms such as electrostatic attraction 71 , the forces of van der Waals 72 , the interactions between receptors and ligands 73 , and hydrophobic bonds 74 .Subsequently, nanoparticles (NPs) traverse bacterial membranes and accumulate along metabolic pathways, influencing cellular membrane structure and function.NPs also engage with fundamental components of bacterial cells, including DNA, where they induce disruptions through various means, including endogenous and exogenous damage, lysosomes, ribosomes, and enzymes, among others 75 .This interaction causes oxidative stress, as endogenous sources trigger the formation of ROS during normal cellular metabolism 76 .These highly unstable free radicals can instantaneously react with other substances.The interaction of free radicals with DNA initiates a cascade of events resulting in genotoxic lesions.This process initiates diverse changes, including heterogeneous alterations, shifts in cell membrane permeability, and disruptions in electrolyte balance, enzyme inhibition, protein deactivation, and modifications in gene expression [77][78][79][80] .Notably, contemporary research has proposed several prevalent mechanisms, which include oxidative stress 81 , the release of metal ions 82 , and non-oxidative pathways 83 .The various mechanisms through which nanoparticles combat bacteria are explained in Fig. 15.
The cellular toxicity of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs was performed to evaluate the viability of MCF-7, MCF-10, and HT-29 cell lines using the MTT assay.Different concentrations (1, 5, 10, 15, 25, 50, and 75 µg/mL) of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs were used and incubated for 24 h.As the concentration of nanomaterials increases, the violet color of the MTT assay becomes progressively lighter.This color change indicates a decrease in the number of viable cells.The efficacy of nanomaterials against biological systems and cells holds considerable importance for fulfilling biomaterial criteria, rendering them suitable for medical applications.The outcomes of the MTT assay are illustrated in Fig. 16A-C.
The findings illustrated that treatment with h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs inhibited the growth of cells significantly (P < 0.05) as compared to those of control cultures, and the reduction was concentrationdependent.The highest inhibition was found in the HT-29 cells, followed by MCF-7 cells, with less cytotoxicity against MCF-10.For HT-29, the inhibitory concentration value (IC 50 ) was 12 µg mL −1 , 10 µg mL −1 , and 15 µg mL −1 , respectively.While IC 50 for MCF-7 cells reveals 14 µg mL −1 , 15 µg mL −1 , and 28 µg mL −1 , respectively.The Ns used in this investigation had particles that were 30 nm in size.The outcomes from XRD and the results of PL analysis showed that there are multiple interstitial oxygen vacancies (O i ) at 532 nm linked to the sample's oxygen vacancies (O v ).These alterations are attributed to the substantial quantity of ROS generated across the three samples.These defects allow a greater number of electron-hole pairs to travel toward the nanomaterial matrix's surface within the specimens.Additionally, the samples' aqueous environment (DDW) contains singlet oxygen ( 1 O 2 ), hydroxyl radicals (OH), and superoxide anions (O 2 ), which the electrons and holes interact with and may cause to produce ROS.These free radicals can trigger oxidation and reduction reactions in macromolecules, including proteins, lipids, and nucleic acids, which leads to oxidative stress when they come into contact with the cellular environment.As a result, a state of disequilibrium develops inside the cells as a result of the buildup of ROS, outpacing the biological system's ability to quickly remove these reactive radicals or repair the harm done by tumor cells [84][85][86] .
In the context of h-BNNs and c-Gd 2 O 3 Ns, MTT testing has been conducted to assess their cytotoxicity in MCF-10 cells.The results indicate that both h-BNNs and c-Gd 2 O 3 Ns demonstrate minimal cytotoxicity and are deemed compatible with MCF-10 cells, aligning with previously published studies [87][88][89][90][91] .However, uncertainties persist regarding the toxicity of h-BNNs and c-Gd 2 O 3 Ns, and their full biocompatibility remains unproven 4,92 .These findings suggest the potential utility of these materials in biomedical applications requiring interaction with living tissues.Nevertheless, further research is imperative to comprehensively comprehend their behavior in various biological systems and ensure their safety for medical applications.www.nature.com/scientificreports/Previous studies revealed that the cell viability of cancer and normal cells after treatment with boron nitride decreased as a function of concentration and time.Cancer cell lines MCF-7 and Hela showed 45% and 60% cytotoxicity, respectively at a concentration of 2 mg/mL for 24 h treatment, which further increased up to 60% and 70%, respectively in 48 h treatment.Whereas in normal cell line (HEK-293) 30% cytotoxicity was observed at a higher concentration (2 mg mL −1 ) and 24 h of treatment, which increased up to 50% after 48 h.It is evident that the cytotoxicity of BN nanostructures is higher in cancer cells as compared with normal cell lines 93 .
Cytotoxicity associated with h-BNNs, c-Gd  One proposed mechanism of the anticancer activity of these nanoparticles can be attributed to the ability of NPs to produce ROS, which can cause changes in biomacromolecules, such as proteins, nucleic acids, and lipids in response to the effects of the generated oxidative stress in the cells and tissues.ROS produce free radicals that are short-lived and unstable, which influence the nuclear viability and healthiness conditions of the affected organisms, and finally lead to cell death.ROS also causes oxidation of proteins and peroxidation of lipids that leads to damaging the fluidity of the cell membrane, thereby changing the permeability of fluids and ion transports across it, and causing inhibition of metabolic processes 31,32 .
Finally, h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs emerge as highly promising materials for cancer therapy due to their notable attributes, including strong chemical stability, uniformity, and excellent dispersibility in solution.Unique structural features, such as the spherical nanosheets of BN and the needle-like surface of c-Gd 2 O 3 Ns, position h-BNNs and c-Gd 2 O 3 as ideal nanocarriers for targeted delivery of anticancer drugs, potentially enhancing their penetration into endosomes.Future research efforts can be directed towards further functionalizing h-BNNs to enhance their biological performance, similar to BNNTs.
Previous study has elucidated diverse responses of cancer cell lines to treatments involving boron nitride (BN) and gadolinium oxide (Gd 2 O 3 ).BN has demonstrated effectiveness against neoplastically transformed IAR-6-1 cells 87 , breast cancer cells (4T1) 89 , prostate cancer cells (DU145 and PC3) 94 , as well as BV2 mouse glioma cells, GMI-R1 rat microglial cells, and human cervical carcinoma cells (HeLa), although its efficacy in other cancer types remains relatively unexplored 95 .The cytotoxicity of h-BNNDs against HUVEC cells (human umbilical vein endothelial cells) was also investigated by Mao et al. 96 .They found that the assessment of cell viability combined with cell counting, viability/cytotoxicity assays, and cell apoptosis detection demonstrated that BNNDs could not elicit significant acute cytotoxicity.They proved that the cell viability remained higher than 80% even at high concentrations and long incubation (200 μg mL −1 , 48 h).
On the other hand, Gd 2 O 3 nanoparticles have exhibited promise in treating M109 (malignant tumors of the mouse pulmonary system), 4T1 (epithelial breast carcinoma) cell lines 88 , mouse colon adenocarcinoma CT26 cell line 97 , HeLa, and COC1/DDP cells in the context of ovarian cancers 98 .Previous results on the viability of the Ba/F3 cells (a murine interleukin-3 dependent pro-B cell line) and THP-1 cells (human monocytic cell line) showed that the cells were viable after incubation with Gd 2 O 3 nanoparticles and remained intact at the time of MRI measurement 99 .
The in vitro cytotoxicity test on normal (HaCaT) and cancerous (HeLa) cells reveals that the CNT/ Gd 2 O 3 hybrid nanostructure is bio-compatible in comparison to the pure CNTs 99 .
However, this study underscores the potential of BN, Gd 2 O 3 , and their combination in cancer treatment.Nevertheless, further research is imperative to elucidate the underlying mechanisms and optimize their therapeutic applications across wide spectrum of cancer cell lines.However, toxicology investigations using in vivo studies of prepared nanoparticles need more thorough viability studies, and long-term biological effects need to be evaluated.
Additionally, an assessment of the hemolytic effect using an ex vivo incubation with erythrocytes revealed that hexagonal boron nitride (h-BN) nanosheets, cubic gadolinium oxide (c-Gd 2 O 3 ) nanoparticles, and h-BN@Gd 2 O 3 nanocomposites demonstrated minimal hemolysis rates, registering at less than 5%, even when exposed to concentrations up to 100 μg mL −1 (Fig. 18).The data is consistent with a previous study that demonstrated Gd 2 O 3 @ PCD-FA showed no cytotoxic effect on MCF10A cells (normal human breast cells) and exhibited acceptable hemocompatibility against human RBCs, revealing their biocompatible properties 100 .Another study also revealed that BN has no significant hemolysis rate on human RBCs even when the concentration reached 100 μg mL −190 .The hemolysis assay serves as a preliminary analytical approach for ascertaining the compatibility of nanoparticles (NPs) with blood and for assessing their effects on erythrocyte well-being.These observations underscore the potential of h-BN@Gd 2 O 3 as a viable candidate for drug delivery and demonstrate its good cytocompatibility for biomedical applications.

Conclusion
In summary, a novel, cost-effective hybrid method was utilized to synthesize new nanomaterials, namely h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs.The LEFL process involved the use of Nd: YAG lasers with varying wavelengths (1064 and 532 nm) and pulse numbers (150, 338, and 772).The resulting h-BNNs displayed small particles with irregular spherical shapes and nanosheets, while Gd 2 O 3 Ns exhibited nanofiber-like, needle-like, and sheet-like structures (leaf-like).TEM analysis corroborated the findings from FESEM images.Phase analysis unveiled hexagonal and cubic structures, with average crystallite sizes of 7.04 nm for h-BNNs, 0.68 nm for c-Gd 2 O 3 Ns, and 3.75 nm for h-BN@Gd 2 O 3 NCs.These findings collectively endorse the credibility of HRTEM and SAED methodologies in characterizing nanoscale crystal structures.Notably, these results harmonize seamlessly with the XRD findings discussed earlier, solidifying their reliability.Raman spectroscopy and FTIR spectroscopy provide complementary insights into the structure and composition of materials.Raman spectroscopy detects homo-nuclear molecular bonds and reveals details about both intra-and intermolecular vibrations.On the other hand, FTIR spectroscopy is attuned to hetero-nuclear functional group vibrations and polar bonds, thereby enhancing the understanding of material properties.All samples showed absorption band edge values

Figure 1 .
Figure 1.(A) Illustration of the hybrid approach to preparing h-BN@c-Gd 2 O 3 colloids.(B) Photographs for the prepared colloids of h-BN@c-Gd 2 O 3 NCs.

Figure 2 .
Figure 2. Illustration of the mechanism for the formation of h-BN@c-Gd 2 O 3 NCs colloids.

Figure 6 .
Figure 6.XRD pattern of the prepared sample, h-BN Ns, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs with different laser pulses.
www.nature.com/scientificreports/characteristic of c-Gd 2 O 3 Ns38,46 .Lastly, the blue spectrum exhibits characteristic peaks at 412 cm −1 , 435 cm −1 , 455 cm −1 , 474 cm −1 , 513 cm −1 , and 520 cm −1 , ascribed to the bending vibration of the triangular B-N-Gd, B-N-O, and Gd-O, which suggests the existence of h-BN@Gd 2 O 3 NCs.The strength of other bands found in the FTIR spectrum is noticeably lower.The resulting product is a combination of h-BN and c-Gd 2 O3 Ns, as further evidenced by the FTIR data.The shifts in FTIR absorption bands in h-BNNs, c-Gd 2 O 3 Ns, and h-BN@ Gd 2 O 3 NCs result from various factors, including the effect of the size.Smaller nanoparticles exhibit blue shifts in their FTIR bands due to quantum size effects.Additionally, differences in surface chemistry, confinement

Figure 16 .
Figure 16.Cytotoxicity effects of (h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs) treated with (A) HT-29, (B) MCF-7, and (C) MCF-10 cell lines.The data were collected over a 24-h treatment, and the values reflect the mean standard deviation of three experiments.
https://doi.org/10.1038/s41598-023-49895-4www.nature.com/scientificreports/ between 200 and 250 nm.The PL spectrum highlighted the presence of numerous O v that brought on cell death and the creation of ROS.The samples exhibited higher antibacterial activity against both bacterial strains.Furthermore, all three samples demonstrated substantial anticancer effects against HT-29 and MCF-7 cells.Moving forward, we anticipate that h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs could potentially serve as promising anticancer agents and biocompatible, with further in vivo experiments worthy of exploring their antitumor effect.
2 O 3 Ns, and h-BN@Gd 2 O 3 NCs, cells were seeded (MCF-7, MCF-10, and HT-29: 4-6 × 10 4 cells per well in 100 μl culture media) in 96-well plates (SPL Life Sciences, Korea) and cultivated overnight.Afterward, cells with 70-80% of confluence were treated with diverse concentrations of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs (1, 5, 10, 15, 25, 50, and 75 µg/ml) and incubated for 24 h.Prior to conducting the experiment, the plates were incubated at 37 °C for 24 h with 20 μL of MTT (5 mg/ml in PBS), specifically thiazolyl blue tetrazolium bromide solution.Subsequently, the plates were kept in the dark for 4 h.The change from yellow MTT dye to purple formazan indicates the metabolic activity of the cells.Afterward, the MTT solution was removed, and each well was treated with 100 μL of dimethyl sulfoxide (DMSO) to dissolve the formazan.Using a Sat Fax 2100 microplate reader from Stat Fax, USA.Absorbance at a wavelength of 545 nm was measured to quantify the extent of dye conversion.The proportion of dye conversion by untreated cells was utilized to determine the viability and/or metabolic activity of the cells 32 .The formula (1) below was applied to determine the inhibition percentage: . The diffraction peaks of Gd 2 O 3 Ns were observed at 2θ = 11.525°,17.025°, 20.875°, 23.225°, 28.475°, 31.275°, and 35.225°, corresponding to (

Table 2 .
Data obtained via the Raman spectra of h-BN Ns, C-Gd 2 O 3 Ns, h-BN@Gd 2 O 3 NCs (150, 338, and 772 pulses) prepared by LEFL in DDW at room temperature.vw very weak, w weak, m medium, s strong, vs very strong, sh shoulder.
while the EB dye exclusively interacted with deceased cells, resulting in red fluorescence.Within viable cells (non-treated cells), distinct bright uniform normal green nuclei with an organized structure.While treated cells were significantly enhanced and distinguished according to the fluorescence emission the apoptotic cells have yellow nuclei.(depicted in Fig.17A-D.The anticancer activity ascribed to BN, Gd, and O v in h-BN, c-Gd 2 O 3 , and h-BN@Gd 2 O 3 NCs, corresponds with increased ROS production.MTT assays performed on HT-29 and MCF-7 cells revealed the potential anticancer effects of h-BNNs, c-Gd 2 O 3 Ns, and h-BN@Gd 2 O 3 NCs.Moreover, the demise of cancer cells induced by nanoparticles occurred through apoptosis, as visualized by AO/EtBr staining. Vol.:(0123456789) Scientific Reports | (2023) 13:22749 | https://doi.org/10.1038/s41598-023-49895-4www.nature.com/scientificreports/