Anticancer Activity of Au/CNT Nanocomposite Fabricated by Nanosecond Pulsed Laser Ablation Method on Colon and Cervical Cancer

Gold nanoparticles (AuNPs) and carbon nanotubes (CNTs) are increasingly being investigated for cancer management due to their physicochemical properties, low toxicity, and biocompatibility. This study used an eco-friendly technique (laser synthesis) to fabricate AuNP and Au/CNT nanocomposites. AuNPs, Au/CNTs, and CNTs were tested as potential cancer nanotherapeutics on colorectal carcinoma cells (HCT-116) and cervical cancer cells (HeLa) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, the non-cancer embryonic kidney cells HEK-293 were taken as a control in the study. The cell viability assay demonstrated a significant reduction in cancer cell population post 48 h treatments of AuNPs, and Au/CNTs. The average cell viabilities of AuNPs, Au/CNTs, and CNTs for HCT-116 cells were 50.62%, 65.88%, 93.55%, and for HeLa cells, the cell viabilities were 50.88%, 66.51%, 91.73%. The cell viabilities for HEK-293 were 50.44%, 65.80%, 93.20%. Both AuNPs and Au/CNTs showed higher cell toxicity and cell death compared with CNT nanomaterials. The treatment of AuNPs and Au/CNTs showed strong inhibitory action on HCT-116 and HeLa cells. However, the treatment of CNTs did not significantly decrease HCT-116 and HeLa cells, and there was only a minor decrease. The treatment of AuNPs, and Au/CNTs, on normal HEK-293 cells also showed a significant decrease in cell viability, but the treatment of CNTs did not produce a significant decrease in the HEK-293 cells. This study shows that a simplified synthesis technique like laser synthesis for the preparation of high-purity nanomaterials has good efficacy for possible future cancer therapy with minimal toxicity.


Introduction
Nanomaterials are increasingly explored for use in biomedical applications owing to their unique and outstanding properties, which render them suitable for developing new and enhanced products for diagnosis and therapy. Along this line, nanotechnology has been employed to remedy various metabolic and pathological disorders, including requirements of medical deployment, in this work, we report the laser synthesis of AuNP and Au/CNT nanocomposites in water. The fabricated materials demonstrate the merits of being prepared by a green technique and containing AuNPs as drug delivery probes, due to their small size, hydrophilic character, and other unique properties as well as incorporation of the promising nanocarrier CNTs due to both their ability to cross the plasma membrane and their high surface area. The impact of the prepared nanostructures was tested on two cancer cell lines, human colorectal carcinoma (HCT-116) and human cervical cancer cells (HeLa), on their viability and proliferation. Also, the non-cancer and healthy cell line (embryonic kidney cells, HEK-293) was taken as the control in the study.

Materials
This work used a gold metal target (Au) with high purity to fabricate AuNPs. Multiwall carbon nanotubes (MWCNTs) were sourced from Cheap Tubes (>95% purity and electrical conductivity >100 S/cm).

Synthesis of Au/CNT Nanocomposite
A pulsed laser ablation (PLA) technique was used to synthesize AuNP and Au/CNT nanocomposites. This experiment used two types of Q-switched Nd: YAG pulsed lasers model (PS-2225, LOTIS TII Ltd., Minsk, Belarus) operation. The first type was set up with a wavelength of 1064 nm, 50 mJ pulse energy, and a 10 Hz repetition rate, while a pulse width of 10 ns, was used to prepare gold nanoparticles (AuNPs). The Au target was set at the bo om of a 10 mL glass vial filled with 4 mL of deionized water. and ablated for 15 min as investigated earlier in Ref. [30]. The height of the water above the Au target was 12 mm. The obtained Au NPs as a product were kept in a clean vial to use later. The second type of laser was set up with a wavelength of 355 nm, 140 mJ pulse energy, and 10 Hz repetition rate, while a pulse width of 10 ns was used as another laser source to fabricate Au/CNTs after mixing the composite. An amount of 10 mL of deionized water containing 0.7 mg of AuNPs, with 20 mg of CNTs, was ablated by the second type of laser for 30 min in an 18 mL cylindrical glass vial to create the new product, Au/CNT nanocomposite. The focus of the UV laser beam was adjusted below the surface of the liquid to prevent high fluence that might have ablated the surface air interface and in turn avoided splashing of the liquid. Vigorous magnetic stirring was undertaken with the samples which were irradiated for 30 min. Figure 1 shows the work schematic of the pulsed laser ablation process. The prepared nanoparticles and the nanocomposite details were reported in our recent work [23,[31][32][33][34].

Morphological Analysis
UV-vis spectrophotometry was used to study the absorbance spectra of the Au/CNT nanocomposites with the SolidSpec-3700 apparatus at scanning wavelengths from 200 to 800 nm.
Surface morphological analysis of nanostructures was investigated by a high-resolution transmission electron microscope (HRTEM) (Morgani 268) at 200 kV. Elemental distribution analysis of the samples was conducted using energy dispersive X-ray (EDS) spectroscopy (EDAX, Octane Elect EDS System).
The powder XRD patterns were obtained to study the crystal structures of CNT, Au, and Au/CNT samples by using a Rigaku Ultima IV powder diffractometer with CuK α radiation (λ = 1.5406 Å) and a scan speed of 0.02 • /min, in the 2θ range from 10 • to 100 • at room temperature. The crystalline phases were identified by comparing the diffraction patterns with those of the standard powder XRD files (JCPDS: Joint Committee on Powder Diffraction Standards and COD: Crystallography Open Database).

Anti-Cancer Activity
Human colorectal carcinoma (HCT-116) and human cervical cancer cells (HeLa) were purchased from ATCC, USA, and were used to examine the impact of AuNP, Au/CNT, and CNT cancer cell viability [35,36]. In addition, healthy cell embryonic kidney cells, HEK-293, were also included in the study as the control. First, cells were seeded in the 96 well plates containing special media of Dulbecco's Modified Eagle Medium (DMEM) and kept in a CO 2 incubator. AuNPs, Au/CNTs, and CNTs (2.0 µg to 40 µg/mL) were added in each, containing HCT-116, HeLa, and HEK-293 for 48 h and processed for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay [37,38]. The AuNPs, Au/CNTs, and CNTs were not added in the 2 control wells. Thereafter cells were exposed to MTT (5.0 mg/mL) for 4 h, and the media were replaced with dimethyl sulfoxide (DMSO). The plates were examined under a plate reader supplied by Bio-Tek Instruments, USA, at 570 nm wavelength, and the optical density (OD) was obtained to calculate the percentage of cell viability. The data, presented in the graph as mean ± standard deviation obtained from triplicates, were statistically evaluated by GraphPad Version 6.0 Prism Software USA.

Apoptotic DAPI Staining
To examine the impact of the treatment of AuNPs, Au/CNTs, and CNTs on the nucleus of cancer cells, we stained cells with 4 ,6-diamidino-2-phenylindole (DAPI) staining post-48 h with 40 µg/mL. The blue, fluorescent staining was examined under a confocal scanning microscope (Zeiss, Munich, Germany). In brief, cells were treated with paraformaldehyde and labeled with DAPI dye.

Results and Discussion
In this study, the absorption spectra of three samples are shown in Figure 2. These are the AuNP, CNT, and Au/CNT nanocomposites in the 200 to 800 nm range.
AuNPs demonstrate one major peak at 525 nm due to surface plasmon resonance (SPR), as reported by Chinh et al. [39]. The second sample, CNTs, shows one prominent peak at 250 nm, as investigated in the literature [40,41]. However, when AuNPs decorated the CNTs, we observed covalent bonds attributed to a sensible broadening and red shift of the Plasmon resonance band in the nanocomposite Au/CNTs as reported by Chinh et al. [39]. However, Au/CNTs showed a slight shift to 625 nm, which occurs due to aggregated bonds between two materials [39]. Figure 3a,b shows HRTEM images of CNT samples obtained at different magnifications. Tubular structures with an average diameter of 10 nm are visible in a uniform distribution in Figure 3a. The TEM image obtained at high magnification in Figure 3b confirms the tubular structure. TEM analysis of Au nanoparticles was achieved in different magnifications, as shown in Figure 3c,d. The images obtained show that the average particle size is between 3-20 nm and has a uniform distribution.   Detailed HRTEM analysis of AuNP-loaded CNT structures is shown in Figure 4a,b. As seen in Figure 4a,b, Au nanoparticles of different sizes are a ached along the tubular structure. The a achment between CNT and Au nanostructures reveals the compatibility    Detailed HRTEM analysis of AuNP-loaded CNT structures is shown in Figure 4a,b. As seen in Figure 4a,b, Au nanoparticles of different sizes are a ached along the tubular structure. The a achment between CNT and Au nanostructures reveals the compatibility Detailed HRTEM analysis of AuNP-loaded CNT structures is shown in Figure 4a,b. As seen in Figure 4a,b, Au nanoparticles of different sizes are attached along the tubular structure. The attachment between CNT and Au nanostructures reveals the compatibility of both nanostructures. These features were also confirmed by HRTEM images obtained at high magnification, as shown in Figure 4b.  Elemental distribution of the Au/CNT sample was performed using energydispersive X-ray spectroscopy to examine the percentages of all respective elements in the sample. The SEM image was used to scan the EDX analysis to obtain the elemental distribution. Figure 5b-d shows the elemental mapping of C, O, and Au elements. The mapping confirms a uniform elemental distribution, and the elemental composition shows that 22% of Au doping was successfully achieved.  Figure 6 displays the Raman spectra of Au nanoparticles, CNTs, and Au nanoparticle decorated CNTs obtained using a LabRam HR evolution Raman Spectrometer Horiba Scientific at room temperature with a 455 nm laser light. As can be observed, at this excitation wavelength (455 nm), Raman sca ering from pure Au nanoparticles was not observed. The Raman spectrum of CNTs demonstrates a typical disorder-induced D band (defect) at 1372 cm −1 and a G band (graphite band) at 1581 cm −1 indicating the formation of multi-walled CNTs [42]. Three other band peaks were observed at 2475 cm −1 , 2728 cm −1 , and 2959 cm −1 and a ributed to highly oriented pyrolytic graphite (HOPG), 2D-band, and G + D band, respectively. The Raman spectrum of the Au nanoparticle decorated CNT Elemental distribution of the Au/CNT sample was performed using energy-dispersive X-ray spectroscopy to examine the percentages of all respective elements in the sample. The SEM image was used to scan the EDX analysis to obtain the elemental distribution. of both nanostructures. These features were also confirmed by HRTEM images obtain at high magnification, as shown in Figure 4b. Elemental distribution of the Au/CNT sample was performed using energ dispersive X-ray spectroscopy to examine the percentages of all respective elements in sample. The SEM image was used to scan the EDX analysis to obtain the elemen distribution. Figure 5b-d shows the elemental mapping of C, O, and Au elements. T mapping confirms a uniform elemental distribution, and the elemental compositi shows that 22% of Au doping was successfully achieved.  Figure 6 displays the Raman spectra of Au nanoparticles, CNTs, and Au nanoparti decorated CNTs obtained using a LabRam HR evolution Raman Spectrometer Hor Scientific at room temperature with a 455 nm laser light. As can be observed, at t excitation wavelength (455 nm), Raman sca ering from pure Au nanoparticles was n observed. The Raman spectrum of CNTs demonstrates a typical disorder-induced D ba (defect) at 1372 cm −1 and a G band (graphite band) at 1581 cm −1 indicating the formati of multi-walled CNTs [42]. Three other band peaks were observed at 2475 cm −1 , 2728 cm and 2959 cm −1 and a ributed to highly oriented pyrolytic graphite (HOPG), 2D-band, a G + D band, respectively. The Raman spectrum of the Au nanoparticle decorated CN  Figure 6 displays the Raman spectra of Au nanoparticles, CNTs, and Au nanoparticle decorated CNTs obtained using a LabRam HR evolution Raman Spectrometer Horiba Scientific at room temperature with a 455 nm laser light. As can be observed, at this excitation wavelength (455 nm), Raman scattering from pure Au nanoparticles was not observed. The Raman spectrum of CNTs demonstrates a typical disorder-induced D band (defect) at 1372 cm −1 and a G band (graphite band) at 1581 cm −1 indicating the formation of multi-walled CNTs [42]. Three other band peaks were observed at 2475 cm −1 , 2728 cm −1 , and 2959 cm −1 and attributed to highly oriented pyrolytic graphite (HOPG), 2D-band, and G + D band, respectively. The Raman spectrum of the Au nanoparticle decorated CNT sample shows that the D-band and G-band shifted to higher wavenumbers (1382, and 1591 cm −1 , respectively). The shift in D-band and G-band is often ascribed to structural defects after functionalization, and a substantial charge transfer interaction between the AuNPs and CNTs, respectively [43,44]. In addition, one can observe that the intensity of the Raman signal of the Au nanoparticle decorated CNTs is higher than that of pure CNT, which is most likely due to the localized surface plasmon resonance effect caused by Au nanoparticles [45].
Micromachines 2023, 14, x FOR PEER REVIEW 7 of 14 sample shows that the D-band and G-band shifted to higher wavenumbers (1382, and 1591 cm −1 , respectively). The shift in D-band and G-band is often ascribed to structural defects after functionalization, and a substantial charge transfer interaction between the AuNPs and CNTs, respectively [43,44]. In addition, one can observe that the intensity of the Raman signal of the Au nanoparticle decorated CNTs is higher than that of pure CNT, which is most likely due to the localized surface plasmon resonance effect caused by Au nanoparticles [45].  The XRD diffraction pattern of Au/CNT nanocomposite displays a broad diffraction peak from 2θ = 18.0 • -28.0 • of the nanocomposite corresponding to the (002) plane of CNT [50].
The characteristic diffraction reflections of crystalline AuNPs are evident in the Au/CNT combination [51]. In addition, in the Au/CNT composite, two reflections are observed between 28.0 • -32.0 • degrees. It can be seen from Figure 7 that the peak around 32.08 • degrees initially accompanies the CNT diffraction peaks. Based on the XRD qualitative analysis results and literature review, it is thought that these two peaks may be of graphite-graphene oxide origin (COD: 96-100-0066) [52]. This behavior is supported by the literature on CNT-doped nanocomposites [37,53]. The XRD diffraction pattern of Au/CNT nanocomposite displays a broad diffraction peak from 2θ = 18.0°-28.0° of the nanocomposite corresponding to the (002) plane of CNT [50].
The characteristic diffraction reflections of crystalline AuNPs are evident in the Au/CNT combination [51]. In addition, in the Au/CNT composite, two reflections are observed between 28.0°-32.0° degrees. It can be seen from Figure 7 that the peak around 32.08° degrees initially accompanies the CNT diffraction peaks. Based on the XRD qualitative analysis results and literature review, it is thought that these two peaks may be of graphite-graphene oxide origin (COD: 96-100-0066) [52]. This behavior is supported by the literature on CNT-doped nanocomposites [37,53].

Impact of AuNPs, CNTs, and Au/CNTs on Cell Viability
The impact of AuNPs, CNTs, and Au/CNTs on HCT-116 and HeLa cells was examined. Post 48 h of treatment; we found a significant decrease in cancer cell posttreatments of AuNP and Au/CNT (Figures 8 and 9). The treatment of AuNPs, and Au/CNTs showed strong inhibitory action on HCT-116 and HeLa cells (Figures 8 and 9). However, the treatment of CNTs did not produce a significant decrease in the HCT-116 and HeLa cells; there was only a minor reduction (Figures 8 and 9).

Impact of AuNPs, CNTs, and Au/CNTs on Cell Viability
The impact of AuNPs, CNTs, and Au/CNTs on HCT-116 and HeLa cells was examined. Post 48 h of treatment; we found a significant decrease in cancer cell post-treatments of AuNP and Au/CNT (Figures 8 and 9). The treatment of AuNPs, and Au/CNTs showed strong inhibitory action on HCT-116 and HeLa cells (Figures 8 and 9). However, the treatment of CNTs did not produce a significant decrease in the HCT-116 and HeLa cells; there was only a minor reduction (Figures 8 and 9).     The treatment of AuNPs and Au/CNTs on HEK-293 cells showed a decline in the cancer cell population, but the treatment of CNTs did not show a significant decline in HEK-293 as in Figure 10. Our results demonstrate that the prepared AuNPs and Au/CNTs showed cytotoxicity against HCT-116 and HeLa cells. Many studies have shown that different nanomaterials and biomaterials produce anti-cancer activities [37,[54][55][56]. The cell viability assay based on the in vitro assay showed that the treatment of nanoparticles on normal cells produced significant cytotoxicity, which may be controlled or reduced by using appropriate biomaterials as a combination therapy approach, to reduce the cell toxicity. In future studies, this cell toxicity can be reduced with appropriately functionalized nanomaterials. The impact of different concentrations (2 ug/mL, 10 ug/mL, 20 ug/mL, and 40 ug/mL) of AuNPs, Au/CNTs, and CNTs on cancer cells were examined, it was found that AuNPs, Au/CNTs produced significant cytotoxicity on both HCT-116 and HeLa cells. Whereas different concentrations (2 ug/mL, 10 ug/mL, 20 ug/mL, and 40 ug/mL) of CNTs produced non-significant cytotoxicity on both HCT-116 and HeLa cells. Lower contraction (2 ug/mL) produced less cytotoxicity than higher concentrations (10 ug, 20 ug, and 40 ug) on the cancer cells. HEK-293 as in Figure 10. Our results demonstrate that the prepared AuNPs and Au/CNTs showed cytotoxicity against HCT-116 and HeLa cells. Many studies have shown that different nanomaterials and biomaterials produce anti-cancer activities [37,[54][55][56]. The cell viability assay based on the in vitro assay showed that the treatment of nanoparticles on normal cells produced significant cytotoxicity, which may be controlled or reduced by using appropriate biomaterials as a combination therapy approach, to reduce the cell toxicity. In future studies, this cell toxicity can be reduced with appropriately functionalized nanomaterials. The impact of different concentrations (2 ug/mL, 10 ug/mL, 20 ug/mL, and 40 ug/mL) of AuNPs, Au/CNTs, and CNTs on cancer cells were examined, it was found that AuNPs, Au/CNTs produced significant cytotoxicity on both HCT-116 and HeLa cells. Whereas different concentrations (2 ug/mL, 10 ug/mL, 20 ug/mL, and 40 ug/mL) of CNTs produced non-significant cytotoxicity on both HCT-116 and HeLa cells. Lower contraction (2 ug/mL) produced less cytotoxicity than higher concentrations (10 ug, 20 ug, and 40 ug) on the cancer cells.

Anti-Apoptotic Impact of Nanocomposites
Many studies suggest that the treatment of nanoparticles causes significant loss of cancer cells due to programmed cell death [36,37,54,57]. To be er understand the reason for cancer cell death, we took HCT-116 cells to examine the cancer cell nuclei by DAPI (4',6-diamidino-2-phenylindole), which is a blue-fluorescent DNA stain used in identifying apoptosis. The treatment of AuNPs, and Au/CNTs produced a considerably

Anti-Apoptotic Impact of Nanocomposites
Many studies suggest that the treatment of nanoparticles causes significant loss of cancer cells due to programmed cell death [36,37,54,57]. To better understand the reason for cancer cell death, we took HCT-116 cells to examine the cancer cell nuclei by DAPI (4 ,6-diamidino-2-phenylindole), which is a blue-fluorescent DNA stain used in identifying apoptosis. The treatment of AuNPs, and Au/CNTs produced a considerably high cancer cell death Figure 11A. In addition, we observed chromatin condensation and formation of apoptotic bodies post-AuNP and Au/CNT treated HCT-166 cells as in Figure 11B,C. The treatment of CNT did not produce any significant change in the morphology of the cancer cell nuclei as in Figure 11D.

Conclusions
In this study, nanostructured Au and Au/CNT nanocomposites were fabricated u the PLA technique. The XRD technique confirmed the crystalline nature of the mate TEM, EDX, and SEM confirmed the morphology of the materials. The AuNPs, Au/C and CNTs were tested as potential cancer nanotherapeutics on HCT-116 and HeLa u an MTT assay. The result showed a significant reduction in cancer cells posttreatments of AuNPs and Au/CNTs. The treatment of AuNPs and Au/CNTs showed st inhibitory action on HCT-116 and HeLa cells. However, the treatment of CNTs did produce a significant decrease in both types of cells, as there was a minor decreas addition, the treatment of AuNPs and Au/CNTs also affected the cancer cell nucl cancer death occurred because of programmed cell death or apoptosis. The choice o

Conclusions
In this study, nanostructured Au and Au/CNT nanocomposites were fabricated using the PLA technique. The XRD technique confirmed the crystalline nature of the materials. TEM, EDX, and SEM confirmed the morphology of the materials. The AuNPs, Au/CNTs, and CNTs were tested as potential cancer nanotherapeutics on HCT-116 and HeLa using an MTT assay. The result showed a significant reduction in cancer cells post-48 h treatments of AuNPs and Au/CNTs. The treatment of AuNPs and Au/CNTs showed strong inhibitory action on HCT-116 and HeLa cells. However, the treatment of CNTs did not produce a significant decrease in both types of cells, as there was a minor decrease. In addition, the treatment of AuNPs and Au/CNTs also affected the cancer cell nuclei as cancer death occurred because of programmed cell death or apoptosis. The choice of an eco-friendly laser technique and the incorporation of both AuNPs and CNTs as efficient drug-delivery vehicles afforded nanocomposites that possess promising anti-colon and anti-cervical cancer capabilities.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.