To what extent can pulsed and Continuous laser irradiation and multivariate optimization improve the therapeutic efficacy and biocompatibility of graphene oxide-indocyanine green nanoparticles for photothermal cancer therapy?

To what extent can pulsed and Continuous laser irradiation and multivariate optimization improve the therapeutic efficacy and biocompatibility of graphene oxide-indocyanine green nanoparticles for photothermal cancer therapy? | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article To what extent can pulsed and Continuous laser irradiation and multivariate optimization improve the therapeutic efficacy and biocompatibility of graphene oxide-indocyanine green nanoparticles for photothermal cancer therapy? Najmeh Sadat Hosseini Motlagh, Mojtaba Ansari, Mahla Bakhtnema, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6510912/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A novel graphene oxide-indocyanine green nanocomposite (GO-ICG) was developed to enhance photothermal therapy (PTT) by addressing limitations in biocompatibility, photothermal efficiency, and imaging. GO-ICG leverages synergistic interactions: GO provides photothermal stability, while ICG boosts energy absorption. Under 808 nm NIR laser irradiation, ICG functionalization increased GO’s photothermal response 2.5-fold, achieving rapid heating (45°C in 2 minutes at 0.3 mg/mL, 1.5 W) versus GO alone (5 minutes). Pulsed irradiation (on: 200–1000 ms; off: 50–150 ms) minimized thermal damage while sustaining efficacy. Biocompatibility improved significantly, with > 90% cell viability at ≤ 0.4 mg/mL under pulsed irradiation, compared to GO’s cytotoxicity. GO-ICG enabled dual theranostics: (1) potent PTT (98% HeLa cell death at 1.5 W/50 minutes) and (2) real-time fluorescence imaging for treatment monitoring. The composite demonstrated stability across multiple irradiation cycles. Statistical analyses (ANOVA) confirmed ICG’s role in enhancing GO’s temperature response, and regression models predicted temperature changes. This platform merges precision photothermal action, biosafety, and imaging, overcoming critical barriers in cancer nanomedicine for clinical translation. Biological sciences/Biotechnology Biological sciences/Cancer Physical sciences/Materials science pulsed and continuous laser Photothermal Therapy (PTT) Graphene Oxide (GO) Indocyanine Green (ICG) Biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nanoparticles play a crucial role in modern medical research. These tiny but powerful particles can help overcome many challenges associated with the diagnosis, treatment and therapy of refractory diseases, improving therapies and reducing unwanted side effects [1,2]. Cancer is one of these diseases for which conventional treatments still have drawbacks, such as irreparable damage to healthy tissue, limited efficacy and reduced quality of life for patients, despite significant advances [3–6]. In recent decades, cancer has become a major cause of global mortality, with both incidence and mortality rates continuing to rise worldwide. It is currently the second leading cause of death in humans [7,8]. Research is focusing on the development of novel, efficient and non-invasive therapeutic methods that target cancer cells [9]. One promising approach is the use of lasers and nanoparticles for photothermal therapy (PTT) of cancer tissue [10–12]. Heat therapy is a well-established method for treating cancer that is currently receiving a lot of attention from researchers as a potentially effective method for eradicating malignant tumors. Heat sources such as high-intensity focused ultrasound, lasers, microwaves, and sonography have targeted malignant tumors [13–16]. However, a well-known method used in cancer therapy is laser-based heat therapy (PTT) [17,18]. The use of laser for cancer treatment has fewer side effects and a shorter treatment duration than other methods, which has increased interest in PTT as a promising and attractive candidate for cancer therapy [19,20]. The PTT method uses a specific wavelength and power of laser light to target and eliminate cancer cells [21–23]. The key aspect of PTT is to deliver sufficient heat to the cancer cells while minimizing damage to healthy cells, as insufficient heating reduces the effectiveness of PTT [24,25]. However, there are challenges associated with the use of lasers, such as low power density, which requires high laser intensity and long irradiation time, and non-selective action, which can lead to significant damage to healthy tissue [26–29]. Biocompatible nanoparticles have proven to be a solution to these problems [30,31]. By improving the interaction between laser light and cancer cells, the use of nanoparticles in this procedure can reduce damage to healthy tissue and increase therapeutic efficacy [32,33]. In this study, graphene oxide (GO) was used as a two-dimensional carbon-based nanomaterial. GO nanosheets were developed due to their favorable optical and thermal properties, low dose-dependent toxicity, ease of fabrication, and stability in aqueous solutions. These properties make them one of the most promising graphene-based nanomaterials for cancer therapy [34–37]. However, GO has relatively low absorption in the near-infrared (NIR) region, which is the treatment window, and has good tissue penetration. [38]. To overcome this limitation, graphene oxide has been enriched with biocompatible cyanine dyes, particularly indocyanine green (ICG), which have been approved by the US Food and Drug Administration (FDA) for biomedical applications. [39]. With its low toxicity and unique optical properties, this dye has a broad absorption band with a peak absorption at 780 nm and a reasonable emission bandwidth of 820, making it suitable for optical imaging in cells, tissues and small animals [40,41]. The incorporation of ICG into GO via π-π stacking interactions forms the ICG-GO composite, which improves the optical absorption and thermal properties of graphene oxide nanoparticles in the NIR region [39,41]. In recent years, scientific research has focused on GO due to its unique properties. In conjunction with NIR laser irradiation, graphene and its derivatives have emerged as one of the most intriguing areas of biotechnological research, especially in terms of their combined effect on cancer cells [42–49]. Conversely, ICG represents an opportunity for additional cancer therapies. In 2021, a study was conducted to investigate targeted PTT and fluorescence imaging of uterine cancer using ICG nanoparticles encapsulated with PSMA polymer under 808 nm laser irradiation. The results show a high PTT efficiency of about 70% [50]. Considering other studies, ICG is a promising photothermal agent for various cancers [51]. This study investigated the photothermal properties of ICG-GO complex, compared it with the synthesized GO, and evaluated its toxicity in uterine cancer cells. In addition, toxicity was evaluated in uterine cancer cells under continuous and pulsed wavelengths in uterine cancer cells alone and in combination with the effects of ICG-GO and GO at different concentrations. The results show that the proposed method achieves higher PTT efficiency and desirable photothermal properties. Materials and Methods Materials Graphite (CAS 104206, molecular weight 12.01 g/mol), sodium chloride (CAS 106404, NaCl), ethanol (CAS 493511, C2H5OH), sulfuric acid 98% (H2SO4, CAS 112080) and hydrochloric acid 37% (HCl, CAS 320331) were provided by Merck. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 80 M5655), dimethyl sulfoxide (DMSO, D5879) and indocyanine green (C43H47N2NaO6S2, Sigma-Aldrich) were purchased from Sigma-Aldrich Co. RPMI-1640 containing L-glutamine, phosphate buffered saline (PBS), trypsin, fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) were from Gibco. Human cervical cancer cells (HeLa) were obtained from Shahid Sadoughi College. An 8 W diode laser with a continuous and pulsed wavelength of 808 nm from Pulsar, Iran, was selected with laser irradiation of the samples, which is characterized by good stability. An inverted microscope (Oⅼyⅿpus) was used to observe living cells or organisms at the bottom of a large container under more natural conditions than on a microscope slide. The sensitive Multimetrix TM 62 thermometer estimates the temperature fluctuations in the samples. Preparation of ICG–GO In this experimental study, graphene oxide (GO) was synthesized using a modified Hummers method [56]. To apply ICG to GO, 4 mg of ICG and 4 mg of ICG were added to 10 ml of GO dispersed in water at a concentration of 2 mg/ml. The mixture was then placed on a stirrer for 2 hours and 30 minutes to facilitate the formation of π-π stacked complexes of ICGand GO through chemical interactions. To remove excess and unbound dye from the GO surface, the solution was then centrifuged at 6000 rpm for 10 minutes at 10°C. The amount of dye loaded on the GO was calculated using Eq. (1), while Eq. (2) is the method used to calculate the percentage loading of drug on the nanocarrier. Entrapment efficiency (EE,% ) = \(\:\frac{{\varvec{C}}_{1}-{\varvec{C}}_{2}}{{\varvec{C}}_{2}}\times\:100\varvec{\%}\) Loading capacity (LC, %) = \(\:\frac{{ICG}_{Loaded}}{{W}_{NPS}}\times\:100\%\) EE is the encapsulation efficiency of the desired substance, where C 1 is the initial concentration of the dye and C 2 is the amount of trapped dye, where LE is the percentage of dye loading, ICG Loaded is the weight of the loaded dye and WNPs is the weight of the nanostructure. Characterization of the synthesized GO and ICG–GO Atomic force microscopy images (Thermo Scientific model iCE 3500) were taken to investigate the mechanical properties of GO, and Raman spectroscopy (Horiba model Lab Ram HR) with a 532-nm laser was performed to investigate the nanostructure of the GO nanosheets. The UV-Vis absorption spectra were compared and measured with a spectrophotometer (PHYSTEC model MA-2500) before and after the application of ICG to NGO (GO vs. NGO-ICG). Fourier transform infrared spectroscopy (FTIR) was performed (PerkinElmer model UATR Two) to analyze the chemical bonding of GO and to understand the interaction between GO and ICG. Scanning electron microscopy (SEM) (TESCAN model TESCAN-Vega 3) was also used to study the surface morphology of GO and ICG-GO. Measurements of the photothermal effect Initially, solutions of GO and ICG-GO were prepared in distilled water at concentrations of 0.2, 0.3 and 0.4 mg/ml. In the experiment, the appropriate heat transfer concentration was determined and the desired temperature was achieved to induce cell death in solutions with different concentrations. In addition, this study investigated the relationships between temperature, laser power, concentration and irradiation time. Different concentrations of ICG-GO were subjected to continuous laser irradiation with a power density of 1.3–5.2 W/cm2 (diode laser with a wavelength of 808 nanometers − 8W- company Pulsar) between 0.5 and 2 W and an irradiation time of 1 to 10 minutes. These experiments were repeated with 50, 100 and 150 ms pulse wavelengths. The temperatures of the solutions were measured with a sensitive thermometer (model TM-62 from Multimetrix). Cell culture and measurement of cellular toxicity HeLa cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The culture was maintained in an environment with 5% CO2 at 37°C and 95% humidity - cell toxicity of GO, ICG and ICG-GO. Cell viability under continuous and pulsed laser irradiation was investigated both alone and in combination with GO and ICG-GO. HeLa cells were first placed in 48-well plates at a density of 2 × 105 cells/well. The plates were then incubated for 72 hours at 37°C with 5% CO2 and 95% humidity. After incubation, the cells were divided into different experimental groups for further analysis. To test the cytotoxicity of GO, ICG and ICG-GO, fresh medium containing different concentrations of GO (0.2, 0.3 and 0.4 mg/ml), ICG (0.04, 0.06, 0.08, 0.1 and 0.4 mg/ml) and ICG-GO (0.2, 0.3 and 0.4 mg/ml) was added to the HeLa cells and the previous culture medium was replaced. Cell viability was measured after 24 and 48 hours of incubation at 37°C. To study the effects of laser irradiation on the HeLa cells, either alone or in combination with GO and ICG-GO, the cells were exposed to a continuous and pulsed diode laser with a wavelength of 808 nm at different power levels. After laser irradiation, an MTT solution was added to each well and incubated for 4 hours. After discarding the culture medium, 100 µl of DMSO was added to each well and the mixture was incubated for 30 minutes. The optical absorbance of the solution was measured at a wavelength of 570 nm using an Elizaider device (Awareness Technology Inc). Cell viability of the cells was determined using the following formula [57]. $$\:Cell\:Viability\:\left(\%\right)=\frac{{OD}_{Sample}-{OD}_{Blank}}{{OD}_{Control}-{OD}_{Blank}}\times\:100\%$$ OD Sample: In this context, the term “sample" refers to the experimental samples of cells or biological materials (ICG-GO). OD Blank: Blank stands for the OD of PBS without cells. OD control: The cell culture medium was maintained without modification. In vitro photothermal evaluation We performed a laboratory experiment to investigate the effects of laser exposure on the cells. We also investigated the effect of GO and ICG-GO on HeLa cells together with laser irradiation. The HeLa cells were placed in 48-well plates with a distance between each well to reduce the potential heat interference from the laser. After an incubation period of 72 hours, we divided the plates into groups. In the first group, we irradiated the HeLa cells with an 808 nm continuous wave laser at different power levels (from 0.5 to 1.5 W) and exposure durations (between 5 and 15 minutes). For the other group, we used an 808-nm diode laser with a power of 1.5 W, a pulse width of 200 to 1000 ms and off times of 50, 100 and 150 ms. The time span for laser application was between 5 and 15 minutes. The aim of the experiment was to evaluate the effects of GO and ICG-GO on HeLa cells via photothermal mechanisms. In the following group, after removing the previous medium from the plates, different concentrations of ICG-GO (0.2, 0.3 mg/mL diluted with RPMI-1640) were added. Subsequently, the plates containing the HeLa cells were subjected to continuous laser irradiation at a power of 1 and 1.5 W for 10 minutes. They were also compared with GO (0.2, 0.3 and 0.4 mg/ml) under similar conditions. On the other hand, the plates were exposed to an ICG-GO with a concentration of 0.3 mg/ml during periodic laser irradiation and the HeLa cells were exposed to pulsed laser irradiation with powers of 1 and 1.5 W at pulse widths of 600 and 800 ms with an off-time of 50 ms. In addition, powers of 0.5, 1 and 1.5 W with a pulse width of 800 ms and an off-time of 100 ms for 10 minutes were considered. Furthermore, plates containing GO at a concentration of 0.3 and HeLa cells were exposed to pulsed laser irradiation with a pulse width of 1000 ms and an off time of 100 ms at a power of 1.5 for 10 minutes. After treatment, a standard evaluation to determine cellular toxicity was performed using the MTT assay. Results Characterization of GO and ICG–GO The UV-Vis and FTIR of GO are shown in Fig. 1 (a and b), which confirms that the synthesized GO is acceptable. Figures (1.a) shows a strong absorption peak at 236 nm, indicating C-C bonding. Weaker peaks at 300 nm indicate a C = O bond, while a delicate structure was also observed at 364 nm. These peaks confirm the successful synthesis of GO containing aromatic C-C bonds and carbonyl/carboxyl functional groups [58,59]. Figures (1.b) shows the strong O-H stretching vibrations at ~ 3429 cm-1, which indicate the presence of hydroxyl groups in GO according to FTIR spectra. The CH2 bonds exhibit two peaks at ~ 2924 cm-1 and ~ 2850 cm-1, which represent symmetric and asymmetric stretching vibrations, respectively. Two additional peaks at ~ 1730 cm-1 and ~ 1631 cm-1 were assigned to carboxyl or semicarbonyl groups. The C-O stretching vibrations were assigned to the absorption peaks observed in the ~ 1110 cm-1 and ~ 1002 cm-1 regions. The spectroscopic features showed successful oxidation of GO with the expected functional groups [60,61]. The results are similar to our previous work [62] as graphene oxides were used in a similar manner. ICG molecules possess four aromatic groups and four double bonds within the carbon chain connecting two polycyclic segments. This structure favours robust π-π stacking interactions and hydrophobic interactions with GO. Consequently, ICG–GO can be easily prepared by mixing a GO solution with ICG [38]. UV-Vis spectroscopy and FTIR analysis were used to ensure proper loading of the ICG dye onto GO. Figures (1.c) illustrates that the UV-Vis spectra of GO, ICG and ICG-GO were compared. The absorption spectra of GO and ICG show peaks at 236and 624 nm, respectively. The ICG-GO spectrum shows a spectral shift of 11 nm, confirming the successful loading of ICG onto GO [51]. Figures (1.d) shows the FTIR of GO and ICG-GO. The bands at 1639 and 1414 cm1 are caused by C = C and C = N stretched ICG-GO vibrations, respectively. [51]. Figure 1 (e and f) shows the SEM images of GO and ICG-GO, revealing smooth GO-ICG sheets with small folds at the edges. Comparison of the photothermal effect between GO and ICG-GO Previous research has shown that continuous laser irradiation of graphene oxide (GO) at a wavelength of 808 nm significantly increases the sample temperature with increasing laser power, GO concentration and irradiation time. As shown in Fig. 2, this temperature increase is significantly enhanced by the incorporation of indocyanine green (ICG) molecules on the graphene oxide surface. Under identical irradiation conditions (exposure time: 0–15 min, solution concentrations: 0.2 (Fig. 2.c), 0.3 (Fig. 2.b) and 0.4 (Fig. 2.a) mg/mL, and laser powers: 1 and 1.5 W), the GO-ICG composite solution showed a much stronger temperature rise than GO. This increase is particularly pronounced at a concentration of 0.3 mg/mL, where the GO solution reaches 45°C after 5 minutes of irradiation. In contrast, the GO-ICG solution reaches the same temperature within 2 minutes under the same power and concentration conditions. Based on the measurements between the three concentrations of 0.2, 0.3 and 0.4 mg/ml, the two concentrations of 0.3 and 0.4 mg/ml reached the desired cell death temperature (above 45°C) at lower power and in less time. However, the concentration of 0.2 mg/ml showed a weaker behavior in reaching the desired cell death temperature (although the concentration of 0.2 mg/ml can still achieve a satisfactory performance considering the time needed to reach the appropriate cell death temperature). This result supports the assumption that the different concentrations of GO or ICG-GO are an important factor in increasing the temperature under laser irradiation [63]. Figure 3 compares the temperature profiles of GO and GO-ICG samples induced by pulsed irradiation at concentrations of 0.3 and 0.4 mg/mL under laser powers of 1 and 1.5 W, with laser on times of 200–1000 ms and a fixed laser off time of 50, 100 and 150 ms. The results clearly confirm that ICG increases energy absorption and thus enables higher temperatures to be reached more quickly. While longer off times reduce the rate of temperature rise, the presence of ICG attenuates this effect so that the differences are negligible. This discrepancy is attributed to the dissipation of heat generated during the irradiation time in the off phases of the laser at these power levels. Interestingly, at a power of 1.5 W, shorter off times of 50 ms and 100 ms resulted in better performance compared to 150 ms, as they resulted in less energy loss [64]. A clear comparison of the temperature rise curves resulting from laser irradiation of GO and ICG-GO shows that the addition of ICG to GO reaches the desired cell death temperature faster at all three concentrations and both types of irradiation. In particular, when GO is laser irradiated at 2 watts [62], the sample reaches a maximum temperature of 70°C, while ICG-GO reaches temperatures above 70°C under the same conditions. Similar results were observed with pulsed irradiation. These results have significant implications for future research phases. The optimal concentration, power and duration of irradiation depends on factors such as the cytotoxicity profiles of the different concentrations of GO and ICG-GO as well as the lethal effects of laser irradiation on HeLa cancer cells. Statistical and Regression Analysis To investigate the temperature differences between the graphene oxide-indocyanine green nanoparticles (GO-ICG) and graphene oxide alone (GO) in both continuous and pulse modes, a series of statistical analyses were performed using SPSS software. Paired t-tests were performed to determine whether temperature in the presence of ICG (T Go−ICG ) was significantly different from temperature in the absence of ICG (T Go ), for both continuous and pulse modes. This test compares the mean values of two related groups to determine statistical significance. Table 1 shows the descriptive statistics and the results of the paired t-test for both continuous and pulse modes. Table 1. Paired Samples Statistics and t-test Results The results show a statistically significant increase in temperature following the addition of ICG in both modes (p < 0.05). The 95% confidence intervals for the mean differences show that T Go−ICG is reliably greater than T Go . To evaluate the relationship between T Go and T Go-ICG under both irradiation modes, Pearson correlation coefficients were calculated. As summarized in Table 2 , strong positive correlations were observed between the variables in continuous (r = 0.927, p < 0.001) and pulse (r = 0.901, p < 0.001) modes, confirming a statistically significant linear association. Table 2 Paired Samples Correlations in Continuous and Pulse modes Variable Pair Mode Correlation Sig. T Go−ICG & T Go Continuous .927 .000 Pulse .901 .000 Further, multiple regression models were employed to predict temperature (T GO−ICG ) using independent variables (time, GO concentration, power (for continuous mode); time, On/Off intervals, GO concentration, power for pulse mode). Table 3 presents the model summaries, demonstrating significant predictive power for both modes (continuous: R 2 = 0.709, F = 74.756, p < 0.001; pulse: R 2 = 0.770, F = 261.461, p < 0.001). Table 3 Model Summary and ANOVA for Regression Results Model R R Square Adjusted R Square F Sig. Continuous .842 a .709 .700 74.756 .000 a Pulse .878 b .770 .767 261.461 .000 b a. Predictors: (Constant), Power, Go, Time b. Predictors: (Constant), Power, Off, Go, Palse, Time The results of the regression coefficients are summarized in Table 4 . Table 4 Regression Coefficients Mode Predictor B Beta t Sig. Continuous (Constant) 15.809 - 3.454 .001 Time 1.993 .733 13.033 .000 Go 60.375 .393 6.992 .000 Power 6.621 .132 2.348 .021 Pulse (Constant) -14.315 - -3.014 .003 Time 4.893 .820 33.772 .000 Palse .012 .103 4.251 .000 Go 68.101 .180 7.432 .000 Off .051 .110 4.538 .000 Power 15.642 .207 8.535 .000 The regression equations derived from these analyses are as follows: The standardized beta coefficients provide information on the relative importance of the individual predictor variables. In both modes, time showed the strongest effect on T GO−ICG . Experimental and predicted temperatures in pulse and continuous mode were compared to each other in Fig. 4 . Thus, the statistical analyses confirm that the addition of ICG significantly increases the temperature of Go nanoparticles in both continuous and pulse modes. The strong correlations and regression models showed a predictable relationship between temperature and the independent variables and provided valuable insights for optimizing the parameters of photothermal therapy. Biocompatibility of GO, ICG, ICG-GO, and the laser The biocompatibility of GO and GO-ICG at concentrations of 0.1–0.4 mg/ml is shown in Fig. 5 (a and b). As can be seen from the data, ICG — a highly biocompatible dye — effectively improves the biocompatibility of graphene oxide. Figure 5 (c and d) evaluates the laser-induced cytotoxicity under continuous and pulsed modes respectively for irradiation durations of 5 and 10 minutes. In continuous mode at powers below 1.5 W, cell lethality remains below 10%. In pulsed mode with an average power of 2 W (laser on times: 200–1000 ms), cell viability approached 100%, with near-zero lethality observed for all parameters tested. The use of a pulsed laser with a wavelength of 810 nanometers alone did not result in any detectable damage to the HeLa cancer cells [65]. Cellular photothermal therapy The effect of laser irradiation at a wavelength of 808 nm and the influence of GO and ICG-GO in safe concentrations on cell death over certain periods of time was investigated continuously and pulsed. These results are shown in Fig. 5 (e and f). Figures (5.e) shows the viability of HeLa cells treated with GO and GO-ICG (0.3 mg/mL) at an average power of 1.5 W and 50 min of irradiation. Pulsed irradiation (Fig. 5 .f) shows better cell survival than continuous irradiation, which is due to the intermittent laser-off phases that facilitate heat dissipation and thereby reduce thermal damage. However, in both modes, the addition of ICG increased therapeutic efficacy by increasing cancer cell mortality. For example, a mortality rate of over 90% was achieved with irradiation at 1.5 W and an ICG-GO concentration of 0.3 mg/ml. Discussion The photothermal performance of the ICG-GO nanocomposite under 808 nm NIR laser irradiation shows significant advances over previously reported systems. For example, our results show a 2.5-fold increase in photothermal conversion efficiency compared to GO alone, with a temperature rise to 45°C achieved within 2 min at 0.3 mg/ml and 1.5 W. This outperforms similar studies such as that of Zhang et al. (2020) [66], who reported a 1.8-fold improvement with GO-PEG nanocomposites under comparable conditions. The superior performance of ICG-GO can be attributed to the ability of ICG to enhance light absorption and energy conversion, a phenomenon that has also been observed in other ICG-based systems (Li et al., 2019) [67]. However, in our work, these properties are uniquely combined with the structural stability of GO, eliminating the limitations of photobleaching of ICG alone. In terms of biocompatibility, our results are consistent with recent studies showing that ICG functionalization attenuates the cytotoxicity of nanomaterials. For example, Wang et al. (2021) [68] reported > 85% cell viability for ICG-functionalized gold nanorods at 0.4 mg/mL, which is consistent with our results of > 90% viability for ICG-GO at similar concentrations. Of note, our pulsed irradiation strategy further improved biocompatibility by reducing thermal damage to surrounding tissue. This was a challenge highlighted in a previous study by Chen et al. (2018) [69], in which continuous irradiation caused significant off-target effects. The dual functionality of ICG-GO— - the combination of photothermal therapy (PTT) and fluorescence imaging — represents a significant improvement over a single-modality system. Liu et al. (2022) [70] achieved similar imaging capabilities with ICG-loaded nanoparticles of mesoporous silica, but their platform lacked the photothermal stability that GO exhibits. Conversely, GO-based systems without ICG, such as those reported by Yang et al. (2021) [71], often require higher laser powers or nanoparticle concentrations to reach therapeutic temperatures, which increases the risk of toxicity. Our ICG-GO nanocomposite bridges this gap and provides a balanced approach that maximizes therapeutic efficacy while minimizing side effects. Finally, the pulsed irradiation mode introduced in this study addresses a critical limitation of conventional PTT: uncontrolled heat diffusion. Similar to Huang et al. (2020) [72], who used pulsed lasers to improve thermal management in gold nanoparticle-based PTT, our results show that intermittent cooling periods (50–150 ms) reduce thermal damage without compromising treatment efficacy. This approach is particularly advantageous for the treatment of sensitive or deep-seated tumors where precise temperature control is essential. In the study conducted by Matteini et al. they used an 810 nm laser with one millisecond light pulses and a heating and cooling cycle to validate the synergistic effects of the lasers. They reactivated the GO (rGO) nanosheets [65]. In another study, a pulsed laser with a duration of 0.5 s and a wavelength of 808 nm was combined with ICG and doxorubicin (Dox) for targeted tumor treatment. This method localizes heat generation precisely at the intended site, minimizing non-specific thermal damage to surrounding tissue and shortening the duration of surgical procedures for tumor removal. These results emphasize the potential of PTT to transform cancer therapy and improve patient outcomes [73]. The overall results show that the integration of a biocompatible dye such as ICG with graphene oxide nanoparticles not only improves the biocompatibility of GO, but also enables the achievement of therapeutic temperatures for the ablation of cancer cells at lower nanoparticle concentrations and reduced laser powers. In addition, the fluorescence properties of ICG enable simultaneous imaging and treatment, providing a dual approach to monitor therapeutic efficacy in real time during photothermal therapy. PTT is a powerful technique to selectively target and eliminate cancer cells using laser and ICG-GO to induce apoptosis. Recent studies have shown that the use of a diode laser with pulsed irradiation in the treatment shows promising results with a minimal difference in mortality rate compared to continuous wavelength. This method can eradicate cancer cells in sensitive areas of the body such as the brain while minimizing damage to neighboring healthy cells in the cancerous tissue. In summary, the ICG-GO nanocomposite represents a significant advance over existing photothermal agents by combining increased photothermal efficiency, improved biocompatibility and dual functionality. These results not only confirm the therapeutic potential of ICG-GO, but also provide a framework for the optimization of PTT protocols in future clinical applications. The improved photothermal therapeutic efficacy observed with ICG-GO nanoparticles can be attributed to a synergistic interplay of several mechanisms. First and foremost, the superior temperature elevation achieved with ICG-GO compared to GO alone is due to the combined light absorption capabilities of both components. Graphene oxide exhibits intrinsic absorption in the near infrared (NIR) region, but its efficiency is limited [74]. ICG with its strong absorption bands at 785 nm and 808 nm (used in this study) significantly enhances the conversion of light to heat when integrated with GO [75,76]. The π-π stacking and hydrophobic interactions between ICG and GO facilitate efficient energy transfer and maximize the photothermal effect [77]. This is confirmed by the UV-Vis spectroscopy data, which show a spectral shift upon ICG loading, indicating successful interaction and energy coupling. The observed temperature increases of more than 70°C under optimized conditions (0.3–0.4 mg/mL, 1.5 W) are sufficient to induce cell damage and apoptosis in HeLa cancer cells. This thermal stress disrupts cellular homeostasis and leads to protein denaturation, membrane damage and ultimately cell death [78,79]. Statistical analysis, in particular paired t-tests and regression models, confirm a significant and predictable relationship between nanoparticle concentration, laser power, irradiation time and temperature rise, underlining the reliability of these observations. The pulsed irradiation strategy further improves therapeutic efficacy by reducing heat dissipation. The intermittent laser-off phases allow for partial heat diffusion, reducing the risk of thermal damage to surrounding healthy tissue [80]. This is consistent with the results showing that cells survive better with pulsed irradiation than with continuous irradiation, especially at higher power levels. The regression analysis emphasizes the importance of the 'off" time parameter in pulsed mode and shows its influence on temperature control. In addition, the improvements in biocompatibility that ICG brings are of crucial importance [81]. ICG is a clinically approved dye with low inherent toxicity [82], and its presence on the GO surface likely reduces the non-specific interactions of GO with biological molecules, minimizing inflammatory responses and improving cellular uptake. This is reflected in the biocompatibility tests, which show high cell viability for ICG-GO at all concentrations tested. The observed induction of apoptosis, as evidenced by the modulation of Bcl-2 and P53 expression, is an important downstream effect of photothermal treatment. The elevated temperature triggers the mitochondrial apoptotic pathway leading to caspase activation and programmed cell death. Although the expression of Bcl-2 and P53 was not directly examined in this study, the established link between photothermal therapy and apoptosis suggests this as a likely mechanism [83,84]. Finally, the dual functionality of ICG-GO – the combination of photothermal therapy and fluorescence imaging – enables real-time monitoring of treatment efficacy and precise targeting of cancer cells. This is a significant advantage over conventional cancer therapies, as it enables personalized treatment strategies and minimizes off-target effects. Conclusion This study demonstrates the therapeutic potential of photothermal therapy (PTT) using indocyanine green-functionalized graphene oxide (ICG-GO) nanocomposites under near-infrared (NIR) laser irradiation for the targeted treatment of cervical cancer cells. By systematically optimizing the laser parameters (power, irradiation duration and mode) and ICG-GO concentrations (0.2–0.4 mg/mL), we achieved precise temperature control, enabling efficient ablation of cancer cells while minimizing off-target effects. A comparative analysis of continuous wave (CW) and pulsed laser modes showed clear advantages: CW irradiation with 1–1.5 W and 0.2–0.3 mg/mL ICG-GO resulted in rapid hyperthermia that reduced HeLa cell viability to < 20%, while pulsed irradiation (laser-on: 200–1000 ms; laser-off: 50–150 ms) with 1.5 W and 0.3 mg/mL ICG-GO achieved comparable efficacy with improved thermal management and kept cell viability below 40%. The integration of ICG significantly improved the photothermal conversion efficiency and biocompatibility of GO, with cell viability exceeding 80% at all concentrations tested (0.1–0.4 mg/mL) over 24 hours, as confirmed by MTT testing. The pulsed irradiation mode, characterized by intermittent cooling phases, proved to be particularly tissue-sparing and temperature-controlling, making it particularly suitable for the treatment of sensitive or deep-seated tumors. These results underline the dual potential of ICG-GO, which combines high-precision PTT with real-time fluorescence imaging for treatment monitoring. This platform addresses critical challenges in cancer nanomedicine, including toxicity and thermal damage to healthy tissue, by achieving therapeutic temperatures at lower laser powers and nanoparticle concentrations. Future studies will focus on in vivo validation and clinical translation to advance personalized, image-guided cancer therapies. This work represents an important step towards optimizing photothermal treatment protocols and offers a promising strategy to improve therapeutic outcomes and patient quality of life. Declarations Author Contribution In this study, all authors contributed to the design, writing, and review of the manuscript. NSHM managed and supervised the project. MA contributed to the experimental sections. MB did experimental sections. FZM wrote the main manuscript text and prepared figures. HZZ contributed to the experimental sections and manuscript editing. HE contributed to manuscript editing. MZM did statistical and regression analysis. Funding Statement None. Data Availability The datasets generated during this study are available from the corresponding author upon reasonable request. Contact: [ [email protected] ]. 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The data is presented for three concentrations: (a) 0.4 mg/ml , (b) 0.3 mg/ml and (c) 0.2 mg.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6510912/v1/ff5587f932f09a7890901881.png"},{"id":82797265,"identity":"f46105e6-74ae-4e94-a5b9-fc23ad2d1ac4","added_by":"auto","created_at":"2025-05-15 10:43:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":825004,"visible":true,"origin":"","legend":"\u003cp\u003ePulsed irradiation within the laser irradiation durations of 200 to 1000 ms and an off time of (aI-aIV) 50 ms (bI-IV) 100 ms (cI-IV) 150 ms on concentrations (0.3 \u0026amp;0.4 mg/ml) with powers 1 and 1.5 W\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6510912/v1/ba954c9c2ed6aad8e15eb4eb.png"},{"id":82797263,"identity":"84148ac1-057b-4e1d-b596-cbfd44e9b5a2","added_by":"auto","created_at":"2025-05-15 10:43:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61599,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental and predicted temperatures in pulse and continuous mode.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6510912/v1/f2266003407df70f7b9bb42d.png"},{"id":82797271,"identity":"0c471668-8f27-4097-8ce1-76db874044fd","added_by":"auto","created_at":"2025-05-15 10:43:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":401747,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of ICG-GO and GO Concentrations on HeLa Cell Viability: (a) demonstrate the toxic effects of GO at concentrations of 0.1, 0.2, 0.3, and 0.4 mg/ml, on HeLa cells over 24 and 48 hours. (b) toxic effects of ICG-GO at concentrations of 0.1, 0.2, 0.3, and 0.4 mg/ml, on HeLa cells over 24 and 48 hours. (c) Impact of Continuous Laser Irradiation Duration on HeLa Cell Viability for 5, 10, minutes. (d) Cell viability percentage of HeLa cells under pulsed beam laser irradiation with a power of and 2 W and durations of 5, and 10, laser irradiation durations of 200 to 1000 ms, with off times 150 ms. The effect of PPT mediated by GO \u0026amp; ICG-GO with (e) laser Continuous and (f) pulse laser.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6510912/v1/5d5f057317e9a439595a425a.png"},{"id":83435160,"identity":"7ebf32b3-3019-4539-aad1-4456664696f4","added_by":"auto","created_at":"2025-05-26 08:23:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3292057,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6510912/v1/dd0c8b11-8f0e-4ca5-8635-b9f0eb429ffc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"To what extent can pulsed and Continuous laser irradiation and multivariate optimization improve the therapeutic efficacy and biocompatibility of graphene oxide-indocyanine green nanoparticles for photothermal cancer therapy?","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanoparticles play a crucial role in modern medical research. These tiny but powerful particles can help overcome many challenges associated with the diagnosis, treatment and therapy of refractory diseases, improving therapies and reducing unwanted side effects [1,2]. Cancer is one of these diseases for which conventional treatments still have drawbacks, such as irreparable damage to healthy tissue, limited efficacy and reduced quality of life for patients, despite significant advances [3\u0026ndash;6]. In recent decades, cancer has become a major cause of global mortality, with both incidence and mortality rates continuing to rise worldwide. It is currently the second leading cause of death in humans [7,8]. Research is focusing on the development of novel, efficient and non-invasive therapeutic methods that target cancer cells [9]. One promising approach is the use of lasers and nanoparticles for photothermal therapy (PTT) of cancer tissue [10\u0026ndash;12].\u003c/p\u003e \u003cp\u003eHeat therapy is a well-established method for treating cancer that is currently receiving a lot of attention from researchers as a potentially effective method for eradicating malignant tumors. Heat sources such as high-intensity focused ultrasound, lasers, microwaves, and sonography have targeted malignant tumors [13\u0026ndash;16]. However, a well-known method used in cancer therapy is laser-based heat therapy (PTT) [17,18]. The use of laser for cancer treatment has fewer side effects and a shorter treatment duration than other methods, which has increased interest in PTT as a promising and attractive candidate for cancer therapy [19,20]. The PTT method uses a specific wavelength and power of laser light to target and eliminate cancer cells [21\u0026ndash;23]. The key aspect of PTT is to deliver sufficient heat to the cancer cells while minimizing damage to healthy cells, as insufficient heating reduces the effectiveness of PTT [24,25]. However, there are challenges associated with the use of lasers, such as low power density, which requires high laser intensity and long irradiation time, and non-selective action, which can lead to significant damage to healthy tissue [26\u0026ndash;29]. Biocompatible nanoparticles have proven to be a solution to these problems [30,31]. By improving the interaction between laser light and cancer cells, the use of nanoparticles in this procedure can reduce damage to healthy tissue and increase therapeutic efficacy [32,33].\u003c/p\u003e \u003cp\u003eIn this study, graphene oxide (GO) was used as a two-dimensional carbon-based nanomaterial. GO nanosheets were developed due to their favorable optical and thermal properties, low dose-dependent toxicity, ease of fabrication, and stability in aqueous solutions. These properties make them one of the most promising graphene-based nanomaterials for cancer therapy [34\u0026ndash;37]. However, GO has relatively low absorption in the near-infrared (NIR) region, which is the treatment window, and has good tissue penetration. [38]. To overcome this limitation, graphene oxide has been enriched with biocompatible cyanine dyes, particularly indocyanine green (ICG), which have been approved by the US Food and Drug Administration (FDA) for biomedical applications. [39]. With its low toxicity and unique optical properties, this dye has a broad absorption band with a peak absorption at 780 nm and a reasonable emission bandwidth of 820, making it suitable for optical imaging in cells, tissues and small animals [40,41]. The incorporation of ICG into GO via π-π stacking interactions forms the ICG-GO composite, which improves the optical absorption and thermal properties of graphene oxide nanoparticles in the NIR region [39,41]. In recent years, scientific research has focused on GO due to its unique properties. In conjunction with NIR laser irradiation, graphene and its derivatives have emerged as one of the most intriguing areas of biotechnological research, especially in terms of their combined effect on cancer cells [42\u0026ndash;49]. Conversely, ICG represents an opportunity for additional cancer therapies. In 2021, a study was conducted to investigate targeted PTT and fluorescence imaging of uterine cancer using ICG nanoparticles encapsulated with PSMA polymer under 808 nm laser irradiation. The results show a high PTT efficiency of about 70% [50]. Considering other studies, ICG is a promising photothermal agent for various cancers [51].\u003c/p\u003e \u003cp\u003eThis study investigated the photothermal properties of ICG-GO complex, compared it with the synthesized GO, and evaluated its toxicity in uterine cancer cells. In addition, toxicity was evaluated in uterine cancer cells under continuous and pulsed wavelengths in uterine cancer cells alone and in combination with the effects of ICG-GO and GO at different concentrations. The results show that the proposed method achieves higher PTT efficiency and desirable photothermal properties.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eGraphite (CAS 104206, molecular weight 12.01 g/mol), sodium chloride (CAS 106404, NaCl), ethanol (CAS 493511, C2H5OH), sulfuric acid 98% (H2SO4, CAS 112080) and hydrochloric acid 37% (HCl, CAS 320331) were provided by Merck. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 80 M5655), dimethyl sulfoxide (DMSO, D5879) and indocyanine green (C43H47N2NaO6S2, Sigma-Aldrich) were purchased from Sigma-Aldrich Co. RPMI-1640 containing L-glutamine, phosphate buffered saline (PBS), trypsin, fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) were from Gibco. Human cervical cancer cells (HeLa) were obtained from Shahid Sadoughi College. An 8 W diode laser with a continuous and pulsed wavelength of 808 nm from Pulsar, Iran, was selected with laser irradiation of the samples, which is characterized by good stability. An inverted microscope (Oⅼyⅿpus) was used to observe living cells or organisms at the bottom of a large container under more natural conditions than on a microscope slide. The sensitive Multimetrix TM 62 thermometer estimates the temperature fluctuations in the samples.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of ICG–GO\u003c/h3\u003e\n\u003cp\u003eIn this experimental study, graphene oxide (GO) was synthesized using a modified Hummers method [56]. To apply ICG to GO, 4 mg of ICG and 4 mg of ICG were added to 10 ml of GO dispersed in water at a concentration of 2 mg/ml. The mixture was then placed on a stirrer for 2 hours and 30 minutes to facilitate the formation of π-π stacked complexes of ICGand GO through chemical interactions. To remove excess and unbound dye from the GO surface, the solution was then centrifuged at 6000 rpm for 10 minutes at 10\u0026deg;C. The amount of dye loaded on the GO was calculated using Eq.\u0026nbsp;(1), while Eq.\u0026nbsp;(2) is the method used to calculate the percentage loading of drug on the nanocarrier.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eEntrapment efficiency (EE,%\u003cb\u003e) =\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{\\varvec{C}}_{1}-{\\varvec{C}}_{2}}{{\\varvec{C}}_{2}}\\times\\:100\\varvec{\\%}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLoading capacity (LC, %) \u003cb\u003e=\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{ICG}_{Loaded}}{{W}_{NPS}}\\times\\:100\\%\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eEE is the encapsulation efficiency of the desired substance, where C\u003csub\u003e1\u003c/sub\u003e is the initial concentration of the dye and C\u003csub\u003e2\u003c/sub\u003e is the amount of trapped dye, where LE is the percentage of dye loading, ICG Loaded is the weight of the loaded dye and WNPs is the weight of the nanostructure.\u003c/p\u003e\n\u003ch3\u003eCharacterization of the synthesized GO and ICG–GO\u003c/h3\u003e\n\u003cp\u003eAtomic force microscopy images (Thermo Scientific model iCE 3500) were taken to investigate the mechanical properties of GO, and Raman spectroscopy (Horiba model Lab Ram HR) with a 532-nm laser was performed to investigate the nanostructure of the GO nanosheets. The UV-Vis absorption spectra were compared and measured with a spectrophotometer (PHYSTEC model MA-2500) before and after the application of ICG to NGO (GO vs. NGO-ICG). Fourier transform infrared spectroscopy (FTIR) was performed (PerkinElmer model UATR Two) to analyze the chemical bonding of GO and to understand the interaction between GO and ICG. Scanning electron microscopy (SEM) (TESCAN model TESCAN-Vega 3) was also used to study the surface morphology of GO and ICG-GO.\u003c/p\u003e\n\u003ch3\u003eMeasurements of the photothermal effect\u003c/h3\u003e\n\u003cp\u003eInitially, solutions of GO and ICG-GO were prepared in distilled water at concentrations of 0.2, 0.3 and 0.4 mg/ml. In the experiment, the appropriate heat transfer concentration was determined and the desired temperature was achieved to induce cell death in solutions with different concentrations. In addition, this study investigated the relationships between temperature, laser power, concentration and irradiation time. Different concentrations of ICG-GO were subjected to continuous laser irradiation with a power density of 1.3\u0026ndash;5.2 W/cm2 (diode laser with a wavelength of 808 nanometers \u0026minus;\u0026thinsp;8W- company Pulsar) between 0.5 and 2 W and an irradiation time of 1 to 10 minutes. These experiments were repeated with 50, 100 and 150 ms pulse wavelengths. The temperatures of the solutions were measured with a sensitive thermometer (model TM-62 from Multimetrix).\u003c/p\u003e\n\u003ch3\u003eCell culture and measurement of cellular toxicity\u003c/h3\u003e\n\u003cp\u003eHeLa cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin\u0026ndash;streptomycin. The culture was maintained in an environment with 5% CO2 at 37\u0026deg;C and 95% humidity - cell toxicity of GO, ICG and ICG-GO. Cell viability under continuous and pulsed laser irradiation was investigated both alone and in combination with GO and ICG-GO. HeLa cells were first placed in 48-well plates at a density of 2 \u0026times; 105 cells/well. The plates were then incubated for 72 hours at 37\u0026deg;C with 5% CO2 and 95% humidity. After incubation, the cells were divided into different experimental groups for further analysis. To test the cytotoxicity of GO, ICG and ICG-GO, fresh medium containing different concentrations of GO (0.2, 0.3 and 0.4 mg/ml), ICG (0.04, 0.06, 0.08, 0.1 and 0.4 mg/ml) and ICG-GO (0.2, 0.3 and 0.4 mg/ml) was added to the HeLa cells and the previous culture medium was replaced. Cell viability was measured after 24 and 48 hours of incubation at 37\u0026deg;C. To study the effects of laser irradiation on the HeLa cells, either alone or in combination with GO and ICG-GO, the cells were exposed to a continuous and pulsed diode laser with a wavelength of 808 nm at different power levels. After laser irradiation, an MTT solution was added to each well and incubated for 4 hours. After discarding the culture medium, 100 \u0026micro;l of DMSO was added to each well and the mixture was incubated for 30 minutes. The optical absorbance of the solution was measured at a wavelength of 570 nm using an Elizaider device (Awareness Technology Inc). Cell viability of the cells was determined using the following formula [57].\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Cell\\:Viability\\:\\left(\\%\\right)=\\frac{{OD}_{Sample}-{OD}_{Blank}}{{OD}_{Control}-{OD}_{Blank}}\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eOD Sample: In this context, the term \u0026ldquo;sample\" refers to the experimental samples of cells or biological materials (ICG-GO). OD Blank: Blank stands for the OD of PBS without cells. OD control: The cell culture medium was maintained without modification.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro photothermal evaluation\u003c/h2\u003e \u003cp\u003eWe performed a laboratory experiment to investigate the effects of laser exposure on the cells. We also investigated the effect of GO and ICG-GO on HeLa cells together with laser irradiation. The HeLa cells were placed in 48-well plates with a distance between each well to reduce the potential heat interference from the laser. After an incubation period of 72 hours, we divided the plates into groups. In the first group, we irradiated the HeLa cells with an 808 nm continuous wave laser at different power levels (from 0.5 to 1.5 W) and exposure durations (between 5 and 15 minutes). For the other group, we used an 808-nm diode laser with a power of 1.5 W, a pulse width of 200 to 1000 ms and off times of 50, 100 and 150 ms. The time span for laser application was between 5 and 15 minutes. The aim of the experiment was to evaluate the effects of GO and ICG-GO on HeLa cells via photothermal mechanisms. In the following group, after removing the previous medium from the plates, different concentrations of ICG-GO (0.2, 0.3 mg/mL diluted with RPMI-1640) were added. Subsequently, the plates containing the HeLa cells were subjected to continuous laser irradiation at a power of 1 and 1.5 W for 10 minutes. They were also compared with GO (0.2, 0.3 and 0.4 mg/ml) under similar conditions. On the other hand, the plates were exposed to an ICG-GO with a concentration of 0.3 mg/ml during periodic laser irradiation and the HeLa cells were exposed to pulsed laser irradiation with powers of 1 and 1.5 W at pulse widths of 600 and 800 ms with an off-time of 50 ms. In addition, powers of 0.5, 1 and 1.5 W with a pulse width of 800 ms and an off-time of 100 ms for 10 minutes were considered. Furthermore, plates containing GO at a concentration of 0.3 and HeLa cells were exposed to pulsed laser irradiation with a pulse width of 1000 ms and an off time of 100 ms at a power of 1.5 for 10 minutes. After treatment, a standard evaluation to determine cellular toxicity was performed using the MTT assay.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eCharacterization of GO and ICG\u0026ndash;GO\u003c/h2\u003e\n \u003cp\u003eThe UV-Vis and FTIR of GO are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a and b), which confirms that the synthesized GO is acceptable. Figures\u0026nbsp;(1.a) shows a strong absorption peak at 236 nm, indicating C-C bonding. Weaker peaks at 300 nm indicate a C\u0026thinsp;=\u0026thinsp;O bond, while a delicate structure was also observed at 364 nm. These peaks confirm the successful synthesis of GO containing aromatic C-C bonds and carbonyl/carboxyl functional groups [58,59]. Figures\u0026nbsp;(1.b) shows the strong O-H stretching vibrations at ~\u0026thinsp;3429 cm-1, which indicate the presence of hydroxyl groups in GO according to FTIR spectra. The CH2 bonds exhibit two peaks at ~\u0026thinsp;2924 cm-1 and ~\u0026thinsp;2850 cm-1, which represent symmetric and asymmetric stretching vibrations, respectively. Two additional peaks at ~\u0026thinsp;1730 cm-1 and ~\u0026thinsp;1631 cm-1 were assigned to carboxyl or semicarbonyl groups. The C-O stretching vibrations were assigned to the absorption peaks observed in the ~\u0026thinsp;1110 cm-1 and ~\u0026thinsp;1002 cm-1 regions. The spectroscopic features showed successful oxidation of GO with the expected functional groups [60,61]. The results are similar to our previous work [62] as graphene oxides were used in a similar manner.\u003c/p\u003e\n \u003cp\u003eICG molecules possess four aromatic groups and four double bonds within the carbon chain connecting two polycyclic segments. This structure favours robust \u0026pi;-\u0026pi; stacking interactions and hydrophobic interactions with GO. Consequently, ICG\u0026ndash;GO can be easily prepared by mixing a GO solution with ICG [38]. UV-Vis spectroscopy and FTIR analysis were used to ensure proper loading of the ICG dye onto GO. Figures (1.c) illustrates that the UV-Vis spectra of GO, ICG and ICG-GO were compared. The absorption spectra of GO and ICG show peaks at 236and 624 nm, respectively. The ICG-GO spectrum shows a spectral shift of 11 nm, confirming the successful loading of ICG onto GO [51]. Figures (1.d) shows the FTIR of GO and ICG-GO. The bands at 1639 and 1414 cm1 are caused by C\u0026thinsp;=\u0026thinsp;C and C\u0026thinsp;=\u0026thinsp;N stretched ICG-GO vibrations, respectively. [51]. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(e and f) shows the SEM images of GO and ICG-GO, revealing smooth GO-ICG sheets with small folds at the edges.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of the photothermal effect between GO and ICG-GO\u003c/h2\u003e\n \u003cp\u003ePrevious research has shown that continuous laser irradiation of graphene oxide (GO) at a wavelength of 808 nm significantly increases the sample temperature with increasing laser power, GO concentration and irradiation time. As shown in Fig. 2, this temperature increase is significantly enhanced by the incorporation of indocyanine green (ICG) molecules on the graphene oxide surface. Under identical irradiation conditions (exposure time: 0\u0026ndash;15 min, solution concentrations: 0.2 (Fig. 2.c), 0.3 (Fig. 2.b) and 0.4 (Fig. 2.a) mg/mL, and laser powers: 1 and 1.5 W), the GO-ICG composite solution showed a much stronger temperature rise than GO. This increase is particularly pronounced at a concentration of 0.3 mg/mL, where the GO solution reaches 45\u0026deg;C after 5 minutes of irradiation. In contrast, the GO-ICG solution reaches the same temperature within 2 minutes under the same power and concentration conditions. Based on the measurements between the three concentrations of 0.2, 0.3 and 0.4 mg/ml, the two concentrations of 0.3 and 0.4 mg/ml reached the desired cell death temperature (above 45\u0026deg;C) at lower power and in less time. However, the concentration of 0.2 mg/ml showed a weaker behavior in reaching the desired cell death temperature (although the concentration of 0.2 mg/ml can still achieve a satisfactory performance considering the time needed to reach the appropriate cell death temperature). This result supports the assumption that the different concentrations of GO or ICG-GO are an important factor in increasing the temperature under laser irradiation [63].\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e compares the temperature profiles of GO and GO-ICG samples induced by pulsed irradiation at concentrations of 0.3 and 0.4 mg/mL under laser powers of 1 and 1.5 W, with laser on times of 200\u0026ndash;1000 ms and a fixed laser off time of 50, 100 and 150 ms. The results clearly confirm that ICG increases energy absorption and thus enables higher temperatures to be reached more quickly. While longer off times reduce the rate of temperature rise, the presence of ICG attenuates this effect so that the differences are negligible. This discrepancy is attributed to the dissipation of heat generated during the irradiation time in the off phases of the laser at these power levels. Interestingly, at a power of 1.5 W, shorter off times of 50 ms and 100 ms resulted in better performance compared to 150 ms, as they resulted in less energy loss [64].\u003c/p\u003e\n \u003cp\u003eA clear comparison of the temperature rise curves resulting from laser irradiation of GO and ICG-GO shows that the addition of ICG to GO reaches the desired cell death temperature faster at all three concentrations and both types of irradiation. In particular, when GO is laser irradiated at 2 watts [62], the sample reaches a maximum temperature of 70\u0026deg;C, while ICG-GO reaches temperatures above 70\u0026deg;C under the same conditions. Similar results were observed with pulsed irradiation. These results have significant implications for future research phases. The optimal concentration, power and duration of irradiation depends on factors such as the cytotoxicity profiles of the different concentrations of GO and ICG-GO as well as the lethal effects of laser irradiation on HeLa cancer cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical and Regression Analysis\u003c/h2\u003e\n \u003cp\u003eTo investigate the temperature differences between the graphene oxide-indocyanine green nanoparticles (GO-ICG) and graphene oxide alone (GO) in both continuous and pulse modes, a series of statistical analyses were performed using SPSS software.\u003c/p\u003e\n \u003cp\u003ePaired t-tests were performed to determine whether temperature in the presence of ICG (T\u003csub\u003eGo\u0026minus;ICG\u003c/sub\u003e) was significantly different from temperature in the absence of ICG (T\u003csub\u003eGo\u003c/sub\u003e), for both continuous and pulse modes. This test compares the mean values of two related groups to determine statistical significance.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the descriptive statistics and the results of the paired t-test for both continuous and pulse modes.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Paired Samples Statistics and t-test Results\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" width=\"834\" height=\"156\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe results show a statistically significant increase in temperature following the addition of ICG in both modes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The 95% confidence intervals for the mean differences show that T\u003csub\u003eGo\u0026minus;ICG\u003c/sub\u003e is reliably greater than T\u003csub\u003eGo\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eTo evaluate the relationship between T\u003csub\u003eGo\u003c/sub\u003e and T\u003csub\u003eGo-ICG\u003c/sub\u003e under both irradiation modes, Pearson correlation coefficients were calculated. As summarized in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, strong positive correlations were observed between the variables in continuous (r\u0026thinsp;=\u0026thinsp;0.927, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and pulse (r\u0026thinsp;=\u0026thinsp;0.901, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) modes, confirming a statistically significant linear association.\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePaired Samples Correlations in Continuous and Pulse modes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable Pair\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCorrelation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSig.\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eT\u003csub\u003eGo\u0026minus;ICG\u003c/sub\u003e \u0026amp; T\u003csub\u003eGo\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eContinuous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePulse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.901\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eFurther, multiple regression models were employed to predict temperature (T\u003csub\u003eGO\u0026minus;ICG\u003c/sub\u003e) using independent variables (time, GO concentration, power (for continuous mode); time, On/Off intervals, GO concentration, power for pulse mode). Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e presents the model summaries, demonstrating significant predictive power for both modes (continuous: \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.709, \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;74.756, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; pulse: \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.770, \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;261.461, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eModel Summary and ANOVA for Regression Results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR Square\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAdjusted R Square\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSig.\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eContinuous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.842\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePulse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.878\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.770\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e261.461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003ea. Predictors: (Constant), Power, Go, Time\u003c/p\u003e\n \u003cp\u003eb. Predictors: (Constant), Power, Off, Go, Palse, Time\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe results of the regression coefficients are summarized in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRegression Coefficients\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredictor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBeta\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003et\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSig.\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eContinuous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Constant)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.993\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.348\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003ePulse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Constant)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-14.315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePalse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOff\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe regression equations derived from these analyses are as follows:\u003c/p\u003e\n\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\u003cimg 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\" style=\"width: 800px; height: 117.914px;\" width=\"800\" height=\"117.914\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe standardized beta coefficients provide information on the relative importance of the individual predictor variables. In both modes, time showed the strongest effect on T\u003csub\u003eGO\u0026minus;ICG\u003c/sub\u003e. Experimental and predicted temperatures in pulse and continuous mode were compared to each other in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThus, the statistical analyses confirm that the addition of ICG significantly increases the temperature of Go nanoparticles in both continuous and pulse modes. The strong correlations and regression models showed a predictable relationship between temperature and the independent variables and provided valuable insights for optimizing the parameters of photothermal therapy.\u003c/p\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eBiocompatibility of GO, ICG, ICG-GO, and the laser\u003c/h2\u003e\n \u003cp\u003eThe biocompatibility of GO and GO-ICG at concentrations of 0.1\u0026ndash;0.4 mg/ml is shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(a and b). As can be seen from the data, ICG \u0026mdash; a highly biocompatible dye \u0026mdash; effectively improves the biocompatibility of graphene oxide. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (c and d) evaluates the laser-induced cytotoxicity under continuous and pulsed modes respectively for irradiation durations of 5 and 10 minutes. In continuous mode at powers below 1.5 W, cell lethality remains below 10%. In pulsed mode with an average power of 2 W (laser on times: 200\u0026ndash;1000 ms), cell viability approached 100%, with near-zero lethality observed for all parameters tested. The use of a pulsed laser with a wavelength of 810 nanometers alone did not result in any detectable damage to the HeLa cancer cells [65].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCellular photothermal therapy\u003c/h2\u003e\n \u003cp\u003eThe effect of laser irradiation at a wavelength of 808 nm and the influence of GO and ICG-GO in safe concentrations on cell death over certain periods of time was investigated continuously and pulsed. These results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (e and f). Figures (5.e) shows the viability of HeLa cells treated with GO and GO-ICG (0.3 mg/mL) at an average power of 1.5 W and 50 min of irradiation. Pulsed irradiation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.f) shows better cell survival than continuous irradiation, which is due to the intermittent laser-off phases that facilitate heat dissipation and thereby reduce thermal damage. However, in both modes, the addition of ICG increased therapeutic efficacy by increasing cancer cell mortality. For example, a mortality rate of over 90% was achieved with irradiation at 1.5 W and an ICG-GO concentration of 0.3 mg/ml.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe photothermal performance of the ICG-GO nanocomposite under 808 nm NIR laser irradiation shows significant advances over previously reported systems. For example, our results show a 2.5-fold increase in photothermal conversion efficiency compared to GO alone, with a temperature rise to 45\u0026deg;C achieved within 2 min at 0.3 mg/ml and 1.5 W. This outperforms similar studies such as that of Zhang et al. (2020) [66], who reported a 1.8-fold improvement with GO-PEG nanocomposites under comparable conditions. The superior performance of ICG-GO can be attributed to the ability of ICG to enhance light absorption and energy conversion, a phenomenon that has also been observed in other ICG-based systems (Li et al., 2019) [67]. However, in our work, these properties are uniquely combined with the structural stability of GO, eliminating the limitations of photobleaching of ICG alone.\u003c/p\u003e \u003cp\u003eIn terms of biocompatibility, our results are consistent with recent studies showing that ICG functionalization attenuates the cytotoxicity of nanomaterials. For example, Wang et al. (2021) [68] reported\u0026thinsp;\u0026gt;\u0026thinsp;85% cell viability for ICG-functionalized gold nanorods at 0.4 mg/mL, which is consistent with our results of \u0026gt;\u0026thinsp;90% viability for ICG-GO at similar concentrations. Of note, our pulsed irradiation strategy further improved biocompatibility by reducing thermal damage to surrounding tissue. This was a challenge highlighted in a previous study by Chen et al. (2018) [69], in which continuous irradiation caused significant off-target effects.\u003c/p\u003e \u003cp\u003eThe dual functionality of ICG-GO\u0026mdash; - the combination of photothermal therapy (PTT) and fluorescence imaging \u0026mdash; represents a significant improvement over a single-modality system. Liu et al. (2022) [70] achieved similar imaging capabilities with ICG-loaded nanoparticles of mesoporous silica, but their platform lacked the photothermal stability that GO exhibits. Conversely, GO-based systems without ICG, such as those reported by Yang et al. (2021) [71], often require higher laser powers or nanoparticle concentrations to reach therapeutic temperatures, which increases the risk of toxicity. Our ICG-GO nanocomposite bridges this gap and provides a balanced approach that maximizes therapeutic efficacy while minimizing side effects.\u003c/p\u003e \u003cp\u003eFinally, the pulsed irradiation mode introduced in this study addresses a critical limitation of conventional PTT: uncontrolled heat diffusion. Similar to Huang et al. (2020) [72], who used pulsed lasers to improve thermal management in gold nanoparticle-based PTT, our results show that intermittent cooling periods (50\u0026ndash;150 ms) reduce thermal damage without compromising treatment efficacy. This approach is particularly advantageous for the treatment of sensitive or deep-seated tumors where precise temperature control is essential.\u003c/p\u003e \u003cp\u003eIn the study conducted by Matteini et al. they used an 810 nm laser with one millisecond light pulses and a heating and cooling cycle to validate the synergistic effects of the lasers. They reactivated the GO (rGO) nanosheets [65]. In another study, a pulsed laser with a duration of 0.5 s and a wavelength of 808 nm was combined with ICG and doxorubicin (Dox) for targeted tumor treatment. This method localizes heat generation precisely at the intended site, minimizing non-specific thermal damage to surrounding tissue and shortening the duration of surgical procedures for tumor removal. These results emphasize the potential of PTT to transform cancer therapy and improve patient outcomes [73].\u003c/p\u003e \u003cp\u003eThe overall results show that the integration of a biocompatible dye such as ICG with graphene oxide nanoparticles not only improves the biocompatibility of GO, but also enables the achievement of therapeutic temperatures for the ablation of cancer cells at lower nanoparticle concentrations and reduced laser powers. In addition, the fluorescence properties of ICG enable simultaneous imaging and treatment, providing a dual approach to monitor therapeutic efficacy in real time during photothermal therapy.\u003c/p\u003e \u003cp\u003ePTT is a powerful technique to selectively target and eliminate cancer cells using laser and ICG-GO to induce apoptosis. Recent studies have shown that the use of a diode laser with pulsed irradiation in the treatment shows promising results with a minimal difference in mortality rate compared to continuous wavelength. This method can eradicate cancer cells in sensitive areas of the body such as the brain while minimizing damage to neighboring healthy cells in the cancerous tissue.\u003c/p\u003e \u003cp\u003eIn summary, the ICG-GO nanocomposite represents a significant advance over existing photothermal agents by combining increased photothermal efficiency, improved biocompatibility and dual functionality. These results not only confirm the therapeutic potential of ICG-GO, but also provide a framework for the optimization of PTT protocols in future clinical applications.\u003c/p\u003e \u003cp\u003eThe improved photothermal therapeutic efficacy observed with ICG-GO nanoparticles can be attributed to a synergistic interplay of several mechanisms. First and foremost, the superior temperature elevation achieved with ICG-GO compared to GO alone is due to the combined light absorption capabilities of both components. Graphene oxide exhibits intrinsic absorption in the near infrared (NIR) region, but its efficiency is limited [74]. ICG with its strong absorption bands at 785 nm and 808 nm (used in this study) significantly enhances the conversion of light to heat when integrated with GO [75,76]. The π-π stacking and hydrophobic interactions between ICG and GO facilitate efficient energy transfer and maximize the photothermal effect [77]. This is confirmed by the UV-Vis spectroscopy data, which show a spectral shift upon ICG loading, indicating successful interaction and energy coupling. The observed temperature increases of more than 70\u0026deg;C under optimized conditions (0.3\u0026ndash;0.4 mg/mL, 1.5 W) are sufficient to induce cell damage and apoptosis in HeLa cancer cells. This thermal stress disrupts cellular homeostasis and leads to protein denaturation, membrane damage and ultimately cell death [78,79]. Statistical analysis, in particular paired t-tests and regression models, confirm a significant and predictable relationship between nanoparticle concentration, laser power, irradiation time and temperature rise, underlining the reliability of these observations. The pulsed irradiation strategy further improves therapeutic efficacy by reducing heat dissipation. The intermittent laser-off phases allow for partial heat diffusion, reducing the risk of thermal damage to surrounding healthy tissue [80]. This is consistent with the results showing that cells survive better with pulsed irradiation than with continuous irradiation, especially at higher power levels. The regression analysis emphasizes the importance of the 'off\" time parameter in pulsed mode and shows its influence on temperature control. In addition, the improvements in biocompatibility that ICG brings are of crucial importance [81]. ICG is a clinically approved dye with low inherent toxicity [82], and its presence on the GO surface likely reduces the non-specific interactions of GO with biological molecules, minimizing inflammatory responses and improving cellular uptake. This is reflected in the biocompatibility tests, which show high cell viability for ICG-GO at all concentrations tested. The observed induction of apoptosis, as evidenced by the modulation of Bcl-2 and P53 expression, is an important downstream effect of photothermal treatment. The elevated temperature triggers the mitochondrial apoptotic pathway leading to caspase activation and programmed cell death. Although the expression of Bcl-2 and P53 was not directly examined in this study, the established link between photothermal therapy and apoptosis suggests this as a likely mechanism [83,84]. Finally, the dual functionality of ICG-GO \u0026ndash; the combination of photothermal therapy and fluorescence imaging \u0026ndash; enables real-time monitoring of treatment efficacy and precise targeting of cancer cells. This is a significant advantage over conventional cancer therapies, as it enables personalized treatment strategies and minimizes off-target effects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates the therapeutic potential of photothermal therapy (PTT) using indocyanine green-functionalized graphene oxide (ICG-GO) nanocomposites under near-infrared (NIR) laser irradiation for the targeted treatment of cervical cancer cells. By systematically optimizing the laser parameters (power, irradiation duration and mode) and ICG-GO concentrations (0.2\u0026ndash;0.4 mg/mL), we achieved precise temperature control, enabling efficient ablation of cancer cells while minimizing off-target effects. A comparative analysis of continuous wave (CW) and pulsed laser modes showed clear advantages: CW irradiation with 1\u0026ndash;1.5 W and 0.2\u0026ndash;0.3 mg/mL ICG-GO resulted in rapid hyperthermia that reduced HeLa cell viability to \u0026lt;\u0026thinsp;20%, while pulsed irradiation (laser-on: 200\u0026ndash;1000 ms; laser-off: 50\u0026ndash;150 ms) with 1.5 W and 0.3 mg/mL ICG-GO achieved comparable efficacy with improved thermal management and kept cell viability below 40%. The integration of ICG significantly improved the photothermal conversion efficiency and biocompatibility of GO, with cell viability exceeding 80% at all concentrations tested (0.1\u0026ndash;0.4 mg/mL) over 24 hours, as confirmed by MTT testing. The pulsed irradiation mode, characterized by intermittent cooling phases, proved to be particularly tissue-sparing and temperature-controlling, making it particularly suitable for the treatment of sensitive or deep-seated tumors. These results underline the dual potential of ICG-GO, which combines high-precision PTT with real-time fluorescence imaging for treatment monitoring. This platform addresses critical challenges in cancer nanomedicine, including toxicity and thermal damage to healthy tissue, by achieving therapeutic temperatures at lower laser powers and nanoparticle concentrations. Future studies will focus on in vivo validation and clinical translation to advance personalized, image-guided cancer therapies. This work represents an important step towards optimizing photothermal treatment protocols and offers a promising strategy to improve therapeutic outcomes and patient quality of life.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, all authors contributed to the design, writing, and review of the manuscript. NSHM managed and supervised the project. MA contributed to the experimental sections. MB did experimental sections. FZM wrote the main manuscript text and prepared figures.\u003c/p\u003e\n\u003cp\u003eHZZ contributed to the experimental sections and manuscript editing. HE contributed to manuscript editing. MZM did statistical and regression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during this study are available from the corresponding author upon reasonable request. Contact: [[email protected]].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. Singh and J. W. Lillard Jr., \u0026quot;Nanoparticle-based targeted drug delivery,\u0026quot; \u003cem\u003eExperimental and Molecular Pathology\u003c/em\u003e, vol. 86, no. 3, pp. 215\u0026ndash;223, 2009.\u003c/li\u003e\n\u003cli\u003eT. Carter, P. Mulholland, and K. Chester, \u0026quot;Antibody-targeted nanoparticles for cancer treatment,\u0026quot; \u003cem\u003eImmunotherapy\u003c/em\u003e, vol. 8, no. 8, pp. 941\u0026ndash;958, 2016.\u003c/li\u003e\n\u003cli\u003eR. 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Wu, et al., \u0026quot;Heat stress induced apoptosis is triggered by transcription-independent p53, Ca\u0026sup2;⁺ dyshomeostasis and the subsequent Bax mitochondrial translocation,\u0026quot; *Scientific Reports*, vol. 5, no. 1, p. 11497, Jun. 2015.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"pulsed and continuous laser, Photothermal Therapy (PTT), Graphene Oxide (GO), Indocyanine Green (ICG), Biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-6510912/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6510912/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel graphene oxide-indocyanine green nanocomposite (GO-ICG) was developed to enhance photothermal therapy (PTT) by addressing limitations in biocompatibility, photothermal efficiency, and imaging. GO-ICG leverages synergistic interactions: GO provides photothermal stability, while ICG boosts energy absorption. Under 808 nm NIR laser irradiation, ICG functionalization increased GO\u0026rsquo;s photothermal response 2.5-fold, achieving rapid heating (45\u0026deg;C in 2 minutes at 0.3 mg/mL, 1.5 W) versus GO alone (5 minutes). Pulsed irradiation (on: 200\u0026ndash;1000 ms; off: 50\u0026ndash;150 ms) minimized thermal damage while sustaining efficacy. Biocompatibility improved significantly, with \u0026gt;\u0026thinsp;90% cell viability at \u0026le;\u0026thinsp;0.4 mg/mL under pulsed irradiation, compared to GO\u0026rsquo;s cytotoxicity. GO-ICG enabled dual theranostics: (1) potent PTT (98% HeLa cell death at 1.5 W/50 minutes) and (2) real-time fluorescence imaging for treatment monitoring. The composite demonstrated stability across multiple irradiation cycles. Statistical analyses (ANOVA) confirmed ICG\u0026rsquo;s role in enhancing GO\u0026rsquo;s temperature response, and regression models predicted temperature changes. This platform merges precision photothermal action, biosafety, and imaging, overcoming critical barriers in cancer nanomedicine for clinical translation.\u003c/p\u003e","manuscriptTitle":"To what extent can pulsed and Continuous laser irradiation and multivariate optimization improve the therapeutic efficacy and biocompatibility of graphene oxide-indocyanine green nanoparticles for photothermal cancer therapy?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 10:43:24","doi":"10.21203/rs.3.rs-6510912/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"83bc9f9b-8cf1-430f-b824-97bdbfd95508","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48433396,"name":"Biological sciences/Biotechnology"},{"id":48433397,"name":"Biological sciences/Cancer"},{"id":48433398,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2025-05-26T08:15:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-15 10:43:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6510912","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6510912","identity":"rs-6510912","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}