Novel silicene-mesoporous silica nanoparticles conjugated gemcitabine induced cellular apoptosis via upregulating NF-κB p65 nuclear translocation suppresses pancreatic cancer growth in vitro and in vivo

Pancreatic cancer’s high fatality rates stem from its resistance to systemic drug delivery and aggressive metastasis, limiting the efficacy of conventional treatments. In this study, two-dimensional ultrathin silicene nanosheets were initially synthesized and near-infrared-responsive two-dimensional silicene-mesoporous silica nanoparticles (SMSNs) were successfully constructed to load the clinically-approved conventional pancreatic cancer chemotherapeutic drug gemcitabine. Experiments on nanoparticle characterization show that they have excellent photothermal conversion ability and stability. Then silicene-mesoporous silica nanoparticles loaded with gemcitabine nanoparticles (SMSN@G NPs) were employed in localized photothermal therapy to control pancreatic tumor growth and achieve therapeutic effects. Our research confirmed the functionality of SMSN@G NPs through immunoblotting and apoptotic assays, demonstrating its capacity to enhance the nuclear translocation of the NF-κB p65, further affect the protein levels of apoptosis-related genes, induce the apoptosis of tumor cells, and ultimately inhibit the growth of the tumor. Additionally, the study assessed the inhibitory role of SMSN@G NPs on pancreatic neoplasm growth in vivo, revealing its excellent biocompatibility. SMSN@G NPs have a nice application prospect for anti-pancreatic tumors.


Introduction
Pancreatic cancer presents an alarming mortality rate, boasting only a 12% 5 year survival rate [1].This dire prognosis is primarily rooted in the disease's remarkable resistance to treatment, which can be attributed to two key factors.Firstly, within the distinctive tumor microenvironment of pancreatic cancer, a dense fibrous interstitium is formed by extracellular matrices such as collagen fibers and hyaluronic acid [2].This unique environment renders pancreatic tumors highly resistant to conventional therapeutic approaches, namely the gemcitabine-based therapies [3,4].As an antimetabolite and analog of deoxycytidine nucleoside, Gemcitabine exerts its anti-cancer properties by impeding the synthesis of DNA [5].However, the therapeutic response rate remains 22%-35% due to the high resistance of the pancreatic tumor [5,6].Factors contributing to this resistance include a hypoxic and poorly vascularized mesenchymal barrier [7], impeding the effective delivery of radiotherapeutic or chemotherapeutic agents into the tumor tissue during systemic administration and inevitably causing severe side effects on normal tissues.Furthermore, surgical intervention remains a viable treatment option for fewer than 20% of pancreatic tumors [8].Furthermore, the propensity of pancreatic tumor cells to metastasize via the bloodstream and lymph nodes not only leads to a grim prognosis but also undermines the function of critical organs.Consequently, developing an effective treatment approach that can inhibit pancreatic tumor growth, eliminate tumor dissemination, and minimize systemic side effects poses a significant challenge.Therefore, ongoing research efforts are concentrated on overcoming the obstacles imposed by the pancreatic cancer tumor microenvironment and attaining optimal drug delivery to pancreatic cancer cells.
Photothermal therapy is a biomedical method wherein a photothermal agent is intravenously introduced, leading to its accumulation within tumor tissue.Subsequently, upon exposure to near-infrared light (NIR) with a wavelength range of 650-950 nanometers [9].These photothermal agents are capable of taking in light energy and transforming it into heat efficiently.The heat produced increases the temperature of the tumor cells to around 45-50 degrees Celsius for a period, causing irreversible damage to cellular membranes and inducing protein denaturation.It is this fundamental principle that underlies the efficacy of photothermal therapy in eliminating tumor cells.This method has garnered significant attention among researchers due to its non-invasive, precise, and direct localized treatment capabilities [10], facilitated by the ease of focusing and adjusting the applied NIR light.In this therapeutic process, photothermal agents based on nanomaterials, typically ranging in size from 20 to 200 nanometers, play a pivotal role [11].Intravenous administration of these substances can lead to their concentration in tumor tissues, as solid tumors possess a heightened permeability and retention effect [12].Importantly, in the absence of light exposure, these photothermal agents pose minimal risk to surrounding cells.Consequently, photothermal agents relying on various nanomaterials for photothermal conversion exhibit significant potential and promising prospects in the realm of tumor photothermal therapy.
In the context of pancreatic cancer treatment, an innovative approach emerges by combining local near-infrared photothermal therapy and chemotherapy, offering unique advantages [13].This strategy harnesses the potential of NDDS, offering a multitude of advantages, such as increased drug absorption, better tumor infiltration, and decreased systemic toxicity.NDDS provides a precise way to target stromal components, which could potentially improve the effectiveness of therapy through a variety of drug delivery methods [14].As a result, our research efforts are focused on coassembling gemcitabine with nanoparticles, aiming to achieve localized combined NIR photothermal chemotherapy for pancreatic tumors.
Recent research emphasis has been placed on selecting near-infrared light for photothermal agents, particularly those with a high degree of absorption and effective photothermal transformation within the NIR-II window.The NIR-II window offers advantages such as reduced tissue heating, larger maximum permissible exposure (MPE), greater depth of penetration, and decreased light scattering compared to the NIR-I window [15].Although the NIR-II window is widely used in biomedical optical imaging, its application in PTT has been limited due to the scarcity of high photothermal conversion efficiency NIR-II photothermal agents [16], especially those with nanoscale dimensions and excellent biocompatibility.In this study, we introduce the two-dimensional silicon-mesoporous silica nanoparticle loaded with Gemcitabine (SMSN@G NPs), which exhibits broad absorption and remarkable photothermal conversion efficiency (η = 37.63%) within the NIR-II region.This efficient NIR-II photothermal agent effectively ablates tumors through PTT with negligible side effects, as demonstrated comprehensively through in vitro and in vivo experiments.
The combination of photothermal therapy and chemotherapy presents unique advantages for the treatment of pancreatic tumors.By activating drugs within the 700-900 nm wavelength range for near-infrared photothermal conversion, these therapies enhance tissue penetration while minimizing adverse reactions.Ideally, thermotherapy should induce irreversible damage to tumor tissue that has been enriched with photothermal agents [17], offering particular benefits in targeting tumor metastasis.The local injection method surpasses the mesenchymal obstacles linked to pancreatic tumors, guaranteeing accurate and consistent drug delivery to the tumor area with a consistent therapeutic dosage [18].Therefore, we have developed a co-assembled formulation comprising gemcitabine and two-dimensional silicene-mesoporous silica nanoparticles (SMSNs) to create a silicene-mesoporous silica @ gemcitabine system (SMSN@G NPs) for localized combinatorial near-infrared photothermal chemotherapy of pancreatic tumors.
In this present study, we synthesized two-dimensional ultrathin silicon nanosheets (SN) using a wet chemical method.These nanosheets served as the basis for creating two-dimensional SMSNs endowed with near-infrared responsiveness.Subsequently, we loaded these SMSNs with the conventional pancreatic cancer therapeutic drug, Gemcitabine, resulting in the formation of SMSN@G NPs.Our investigations revealed that SMSN@G NPs exhibited exceptional photothermal conversion efficiency and stability.We observed that when exposed to near-infrared II window irradiation, SMSN@G NPs significantly enhanced tumor cell apoptosis by enhancing the nuclear translocation of the NF-κB p65.This led to effective inhibition of tumor growth while maintaining excellent biocompatibility.Together, these characteristics underscore the promising potential of SMSN@G NPs as an anti-tumor agent for the future of cancer treatment.Specifically, it holds great promise as a candidate for synergistic chemo-photothermal therapy in pancreatic cancer treatment.Hence, SMSN@G NPs, as we have developed it, represent a prospective nano-drug candidate for advancing the field of combined chemo-photothermal therapy for pancreatic cancer.

Synthesis and characterization of silicene-mesoporous silica @ gemcitabine (SMSNS@G NPs)
In this study, two-dimensional ultrathin SN were synthesized initially, following previously reported protocols.Subsequently, two-dimensional SMSNs were engineered to exhibit near-infrared responsiveness, allowing for the controlled and efficient delivery of Gemcitabine in tumor treatment (figure 1(a)).Transmission electron microscopy (TEM) imaging showed the ultrathin nature of the silicene nanosheets, displaying the characteristic two-dimensional planar structure (figure 1(b)).
To establish a two-dimensional silicene nanosheet system capable of drug loading, the surfactant cetyltrimethylammonium chloride (CTAC) was surface-modified onto the nanosheets through electrostatic interactions.Tetraethyl orthosilicate (TEOS) was subsequently introduced as a silicon source to facilitate the in situ growth of SMSNs.TEM images and corresponding elemental mapping provided clear visual evidence of the individual SMSNs structures and their composition, confirming the uniform distribution of silicon (Si) and oxygen (O) elements within the SMSNs, their ideal mesoporous structure, and the two-dimensional planar topology (figures 1(c)-(e)).After seven days of storage in PBS solution that simulates the physiological environment, the basic morphology of the nanomaterials did not change significantly (figure S1), which can further explain the stability of the nanoparticles under normal conditions.
To further validate the successful encapsulation of Gemcitabine, UV-visible near infrared spectrophotometer (UV-vis-NIR spectra) were obtained before and after Gemcitabine loading.The results demonstrated that SMSN@G NPs exhibited stronger absorption compared to SMSNs, providing additional confirmation of successful Gemcitabine encapsulation (figure 1(f)).Furthermore, DLS analysis revealed a gradual increase in the average hydrodynamic size of silicene, SMSNs, and SMSN@G NPs dispersions.This size increase was attributed to the surface encapsulation of mesoporous SiO 2 shells and the subsequent loading of Gemcitabine molecules (figure 1(g)).Additionally, the ζ-potential analysis indicated a reduction in the negative surface charge of drug-bound SMSN@G NPs in comparison to empty nanoparticles (figure 1(h)).This observation further supported the result of gemcitabine being successfully encapsulated within mesoporous SMSN nanoparticles.

NIR-II-mediated photothermal activation of SMSN@G NPs
To assess the photothermal performance of SMSN@G NPs, we conducted a series of experiments.SMSN@G NPs, containing silicene nanosheets, retained the distinctive photothermal conversion capacity inherent to pristine twodimensional silicene when subjected to NIR-II laser irradiation.We quantified the normalized adsorption intensity (A/L) of SMSN@G NPs at λ = 1064 nm across various concentrations (80, 40, 20, 10, and 5 ppm).A linear correlation between A/L and concentration was established adhering to the Lambert-Beer law (A/L = α c, where α represents the extinction coefficient), with an extinction coefficient measured at 1064 nm as 16.12 Lg −1 cm −1 (figures 2(a)-(b)).
To further assess the photothermal capabilities of SMSN@G NPs, we exposed it to 1064 nm laser irradiation at different power densities (0.25, 0.5, 0.75, and 1.0 Wcm −2 ) at relatively low SMSN@G NPs concentrations (100 ppm) A comprehensive set of experiments was conducted to evaluate the photothermal performance of SMSN@G NPs at different concentrations (100, 50, 25, 12.5 ppm, and ddH 2 O) using a laser power density of 1.0 W cm −2 .
Notably, the temperature of ddH 2 O showed minimal changes, underscoring the rapid and efficient conversion of near-infrared light into thermal energy by SMSN@G NPs (figures 2(d) and (h)).Additionally, the photothermal conversion efficiency (η), which serves as a crucial metric for assessing photothermal performance, was calculated by considering the entire heating/cooling process following laser treatment at 1064 nm.The η value for SMSN@G NPs was determined to be 37.63% (figure 2(e)), slightly surpassing that of conventional inorganic photothermal agents [19][20][21].
To evaluate the photothermal stability of SMSN@G NPs, the temperature changes during five consecutive laser on/off cycles were recorded.Each cycle involved 5 min of NIR-II laser irradiation (laser-on) followed by natural cooling to room temperature (laser-off).The photothermal performance of SMSN@G NPs remained relatively stable throughout the recovery process, indicating its potential as a durable photothermal agent for photothermal therapy in cancer treatment (figure 2(f)).

Safety evaluation of SMSN@G NPs on pancreatic cancer cells in vitro
Having established the biological properties of SMSN@G NPs, we proceeded to investigate cytotoxicity using a CCK-8 assay to confirm the safety of SMSN@G NPs.It has been reported in the literature [22] that nanoparticles are relatively safe in the absence of light stimulation, and only under the stimulation of light can they have toxic effects.Therefore, we evaluate the safety of SMSN@G with and without photothermal stimulation respectively.Figure 3(a) illustrates CCK-8 uptake following incubation with varying concentrations of SMSN@G NPs in the SW1990 and PANC1 cell lines.Remarkably, we observed that after 1, 3, 5, and 7 days of incubation in SW1990 and PANC1 cells, the different concentrations of SMSN@G resulted in a similar CCK-8 absorbance with no obvious and consistent trend compared with the CTL group, suggesting that in the absence of photothermal effects, the different concentrations of SMSN@G had no obvious toxic effects on the tumor cells.However, in the presence of photothermal stimulation, there was a significant reduction in cell proliferation, implying the presence of cytotoxicity.Thus, our results indicate that without the photothermal effect, the different concentrations of SMSN@G had no significant toxic effects, whereas, in the presence of the photothermal effect, SMSN@G showed significant toxic effects.This means that SMSN@G NPs have good biocompatibility.
To evaluate the impact of different concentrations of SMSN@G on pancreatic cancer cells in the absence of light, we performed an apoptotic assay using flow cytometry to assess their safety.The results depicted in figures 3(b)-(c) indicate that, within the SW1990 cell line, there were no (e) The photothermal-conversion efficiency at 1064 nm was calculated.In the experiments, the aqueous solution of SMSN@G NPs exhibited a red line in the heating curve, which represented the diffusion of the photothermal effect over time when the laser was switched off.The time constant (τs) of heat transfer in the system was determined using linear time data from the cooling period and represented by the blue line.(f) The heating curve of SMSN@G NPs suspension was measured over five laser switching cycles at a power density of 1.0 W cm −2 .The corresponding infrared thermal images for the heating curve were also captured (g) and (h).The infrared thermal images correspond to the heating curve (c) and (d).
substantial differences in the apoptosis rate among the control group (CTL), 2 ppm, and 100 ppm concentrations.Furthermore, although there was a statistical increase in the apoptosis rate at the 200 ppm concentration, the change observed was minimal (approximately 1%).In the case of PANC1 cells, there was no statistically significant difference in the apoptosis rate between the CTL group and the 2 ppm concentration.Additionally, at concentrations of 100 ppm and 200 ppm, although there were statistical differences, the observed changes were still negligible (around 2%).These findings suggest that increasing the drug concentration does not significantly impact the survival of pancreatic cancer cells, thus reinforcing the notion of the safety of gemcitabineloaded nanomaterials.

SMSN@G NPs induce apoptosis and inhibit proliferation in pancreatic cancer cells under NIR-II irradiation
Based on the results from the CCK8 experiment, figure 4(a) reveals that the combined application of nanoparticles and gemcitabine exhibits superior efficacy in inhibiting tumor cell proliferation compared to the individual use of nanoparticles or gemcitabine alone.The impact of NIR-II further enhances the inhibition of tumor cell proliferation by SMSN@G, as evidenced by the increasing magnitude of the effect with longer exposure to light.Moreover, as the treatment duration extends, the disparity between the various treatments becomes more pronounced, indicating that the photochemical combination treatment of SMSN@G under the influence of NIR-II demonstrates a remarkable anti-tumor effect.Flow cytometry analysis demonstrated that upon laser irradiation, SMSN@G NPs effectively induced significant apoptosis in SW1990 and PANC1 cells.As figure 4(b) depicted, in SW1990 cells, a noteworthy 17.59% of cells exhibited apoptosis after 30 min of NIR-II treatment, with a slightly higher rate of 18.02% observed after 15 min of NIR-II exposure.In contrast, the SMSN group and the SMSN@G NPs group showed merely 4% of apoptotic cells.Similarly, PANC1 cells exhibited a substantial increase in apoptosis rates under NIR-II treatment, with 16.6% of apoptotic cells after 30 min of NIR-II exposure and 12.25% after 15 min.These rates were significantly higher compared to those observed in the SMSN group and the SMSN@G NPs group.Notably, as the duration of NIR-II exposure prolonged, an increased apoptotic ratio of PANC1 tumor cells was evident, while this trend was not observed in SW1990 cells.These findings collectively reinforce the notion that SMSN@G NPs, when subjected to NIR-II irradiation, enhance the heat sensitivity of tumor cells.The comprehensive results from these systematic investigations provide evidence of the remarkable synergistic efficiency of SMSN@G NPs in combined photothermal and chemotherapeutical applications.p < 0.001, #### p < 0.0001, analyzed using student's t-test.

Evaluation of apoptosis-related protein expression in SW1990 and PANC1 cells
The NF-κB signaling pathway plays a pivotal role in regulating both the promotion and inhibition of apoptosis.This pathway is intricately linked to various cellular processes, including tumorigenesis, growth, and metastasis [23].To gain deeper insights into the specific mechanisms by which SMSN@G NPs affect apoptosis in pancreatic cancer SW1990 and PANC1 cells, we conducted immunoblotting experiments comparing the SMSN@G NPs group under 15 or 30 min NIR-II irradiation, with the SMSN group and the SMSN@G NPs group without irradiation.
Figures 5(b)-(c) revealed notable differences in the expression levels of key genes when subjected to photothermal treatment for 15 min (SMSN@G NPs, NIR-II (15 min)) and 30 min (SMSN@G NPs, NIR-II(30 min)).While the protein level of NF-κB p65 in whole cell lysate remains unchanged, the nuclear phosphorylated NF-κB p65 level, as well as pro-apoptotic genes such as p21 [24] and BID [25], along with anti-apoptotic gene BCL2 [24], exhibited distinct alterations.These findings suggest that SMSN@G NPs may enhance the nuclear translocation of the NF-κB p65, subsequently altering apoptosis-related genes, namely p21, BID, and BCL2.Ultimately, this cascade of events induced apoptosis.However, it is important to note that further investigations are warranted to elucidate the specific molecular mechanisms underlying this effect.

In vivo evaluation of the thermotherapeutic effects of SMSN@G NPs
To assess the thermal chemotherapeutic effects of SMSN@G NPs in an in vivo setting, murine SW1990 xenografts were subjected to an 808 nm laser at an intensity of 1.5 W cm −2 , administered 4 h following intravenous injection of SMSN@G NPs.Infrared thermography was employed for continuous monitoring of temperature changes within the tumor region.In the SMSN@G NPs group, the temperature within the tumor region notably surged by 14 °C within a mere 5 min, demonstrating a robust photothermal therapeutic effect.Conversely, the control group exhibited minimal temperature elevation (figure 6(b)).
To substantiate the enhanced anti-tumor activity and synergistic thermochemotherapy achievable with SMSN and SMSN@G NPs in vivo, SW1990 tumor-bearing nude mice were randomly allocated into three groups: (1) control group, (2) SMSN group, and (3) SMSN@G NPs group.When exposed to near-infrared laser light, as depicted in figure 6(c), relative to the control group (CTL group), the growth rate of the tumor in the SMSN group was significantly slowed down, but the tumor volume tended to be stable and did not achieve a significant reduction in tumor volume.In contrast, under NIR-II laser irradiation, the inhibitory effect of SMSN@G NPs on tumor growth was much more pronounced, and the relative size of the tumors was continuously reduced.
Furthermore, during the treatment period, which spanned two weeks post-injection, The body weight of SW1990 nude mice in the SMSN and SMSN@G NPs groups did not show significant fluctuations (figure 6(d)).Importantly, no fatalities were recorded among the tumor-bearing nude mice during the ongoing follow-up, indicating the absence of adverse effects on their health.
Following the completion of treatments for two weeks, mice were sacrificed and tumors were collected.The results demonstrated that both the SMSN and the SMSN@G NPs group exhibited significantly reduced tumor sizes compared to that of the control group.Notably, in the SMSN@G NPs group, tumors ultimately disappeared with time (figure 6(e)), underscoring the effectiveness of chemotherapeutic photothermal treatment using SMSN@G NPs.Collectively, these results affirm SMSN@G NPs as a promising biocompatible thermotherapeutic agent, showcasing both therapeutic efficacy and biosafety in preclinical models.

Characterizations
Imaging and analysis of the sample's characterizations were conducted using various instruments.Transmission electron microscopy (JEM-2100F, JEOL, Japan, TEM) and highresolution TEM (JEM-2100F, JEOL, Japan, HR-TEM) images were obtained using a JEM-2100F microscope operating at 200 kV.Energy-dispersive spectroscopy (EDS) analyses were also performed using the same microscope.Quantitative elemental analysis was carried out using an Agilent 700 Series instrument (Agilent 700 Series, Agilent Technologies, USA) with inductively coupled plasma-optical emission spectrometry (Agilent 5800 ICP-OES, Agilent Technologies, USA, ICP-OES).UV-visible-near-infrared (UV-vis-NIR) absorption spectra were recorded using a UV-3600 Shimadzu UV-vis-NIR spectrometer (UV-3600i Plus, Shimadzu, Japan, UV-vis-NIR).The Zetasizer Nano series instrument (Nano AS90, Malvern Panalytical, UK) was used to measure the hydrodynamic particle size and zeta potential.To induce hyperthermia through NIR-II laser irradiation, a 1064 nm laser source from Shanghai Connect Fiber Optics Co. was utilized (China).Temperature changes and thermal images were recorded using an infrared thermal imaging instrument (FLIR A325sc, FLIR, USA).

In vitro cytotoxicity assessment
SW1990 and PANC1 cells were plated in 96-well plates (1 × 10 4 cells per well) and routinely cultured overnight at 37 °C (5% CO 2 , humified incubator).Subsequently, 10 μl of SMSN@G NPs at different concentrations (2, 20, 50, 100, and 200 ppm) were added to each well, while the control group received PBS.Followed by 24 h incubation, the cells were subsequently irradiated with a laser (808 nm, 0.5 W cm −2 ) for 10 min.Afterward, the cells were rinsed with PBS thrice, added with fresh complete medium, and cultured overnight.Following removal of the medium, 10 μl per well of Cell Counting Kit-8 solution (Cat.160903202 C, Invigentech, USA, CCK-8) was added, and measured for absorbance at 450 nm using a microplate reader (ELx800, BioTek, USA) [32].

Apoptosis assessment
To determine the apoptotic rates of SW1990 and PANC1 cells, we analyzed the following groups: (1) SMSN (2) SMSN@G NPs (3) SMSN@G NPs with NIR irradiation (200 pm, 808 nm, 1 W cm −2 , for 15 min) (4) SMSN@G NPs with NIR irradiation (200 pm, 808 nm, 1 W cm −2 , for 30 min).SW1990 and PANC1 cells were inoculated at a density of 3 × 10 5 cells per well in 6-well plates and incubated for 24 h.Cells were subsequently treated the following treatments for an additional 24 h: SMSN, or SMSN@G NPs, each at a drug concentration of 200 pmol.Subsequently, the treated cell cultures were subjected to laser irradiation (808 nm, 1.5 W cm −2 , for 15 min or 30 min), followed by additional incubation for 24 h.To evaluate the cytotoxicity induced by NIR-II laser irradiation in both the control group and groups with varying concentrations of SMSN@G NPs, SW1990, and PANC1 cells were inoculated at a density of 3 × 10 5 cells per well in a 6-well plate and incubated for 24 h.PBS was added to the control group, while SMSN@G NPs were respectively added to the other three groups, each containing 2 ppm, 100 ppm, and 200 ppm of SMSN@G NPs.The cells were then subjected to laser irradiation (808 nm, 1.5 W cm −2 , for 15 min).For apoptosis assessment, cells were routinely collected, stained with an Apoptosis Detection Kit [32] (Cat.556547, BD Biosciences, USA) per manufacturers' protocol, and analyzed using a flow cytometer (CytoFLEX S, Beckman, USA).

Immunoblotting
The immunoblotting assay was conducted following established procedures as described in previous reports.Briefly, cells were lysed with Radio Immunoprecipitation Assay Lysis buffer (Cat.87787, Thermo Scientific, USA, RIPA) containing protease (Cat.04693124001, Roche, Switzerland) and phosphatase inhibitors (Cat.B15001-A, Bimake, USA) on ice for 30 min.Following centrifugation (13 000 rpm for 15 min at 4 °C), the supernatant was collected, and protein concentrations were quantified using a Bicinchoninic Acid Assay kit (Cat.YH372198, Thermo Scientific, USA, BCA) [30].The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat.78833, Thermo Scientific, USA) were used to isolate proteins from the cytoplasm and nucleus according to manufacturers' protocol.
To assess the safety measures of respective treatments, the body weight of each experimental animal was documented once every three days over two weeks after treatments.The tumor volume (V ) was calculated based on the tumor length (L) and width (W) using the following calculation formula: V = (L * W 2 )/2 [33].The relative tumor volume was defined as V at each time point (once every three days) divided by V at baseline (on the first day of treatment).

Statistical analysis
The data were presented as the mean ± standard deviation (SD) based on a minimum of three independent experiments.Statistical analysis was performed using GraphPad Prism 9.5.0 software.Group comparisons were assessed using student's t-test, and a p-value of less than 0.05 was considered statistically significant.

Discussion
Nanomaterial-based therapeutics exhibit distinctive biological properties that enable precise targeting, localized drug release, and improved therapeutic efficacy.The exploration of combining nanomaterials with other cancer treatments has garnered significant attention due to its wide-ranging potential and remarkable anti-cancer outcomes.Liu et al [34] demonstrated that nanomedicines can serve as delivery platforms for immune agents, enabling nano-immunotherapy through controlled release within biomaterial delivery systems.Zhu et al [35] highlighted the potential of nanomedicines to overcome chemotherapeutic drug resistance when used in combination with conventional chemotherapeutic drugs.In recent years, the advent of light-responsive nanomaterials has provided an additional avenue for enhancing the anti-cancer effects of nanomedicines.The progress in light-responsive nanomaterials for cancer therapy encompasses photothermal therapy, photodynamic therapy, light-responsive molecular delivery, and photocontrolled combination therapy.Photothermal therapy utilizing nanomedicines has been extensively investigated to validate its anti-cancer efficacy.Li et al [36] conducted a study wherein they designed Bi2Se3@AIPH nanoparticles, which exhibited excellent photothermal conversion efficiency (η = 31.2%)alongside favorable biocompatibility and anti-cancer effects.
Two-dimensional silicene, as the third topology of silicon materials, exhibits low cytotoxicity [37], excellent biocompatibility, high photothermal therapeutic ability, enhanced biodegradability, and superior photothermal conversion efficiency compared to inorganic nanoparticles.It outperforms most inorganic photothermal agents [36], including graphene oxide, C 3 N 4 , MnO 2 , and MoS 2 [38].Additionally, 2D Xene nanosheets possess reasonable maximum permissible exposure (MPE) and an ideal laser penetration depth [26].They have a broad absorption range spanning from the near-infrared (NIR-I) to the NIR-II biological window (750-1350 nm).The silicene nanosheets synthesized in this study exhibit superior photothermal conversion capabilities (η = 37.63%) compared to other 2D Xene materials.In conclusion, silicene nanosheets hold great potential as efficient photosensitizers in photothermal therapy, presenting promising applications.The developed SMSN@G, a two-dimensional silicene-based material, demonstrates favorable biological properties, including structural and photothermal functional stability, high photothermal conversion efficiency, low toxicity, and biocompatibility.These characteristics render it suitable for controlled and efficient tumor treatment protocols.
The cell phenotype experiments clearly illustrated the pro-apoptotic effect of SMSN@G NPs under photothermal therapy.Analyzing the pro-apoptotic mechanism of SMSN@G NPs under photothermal therapy, we observed elevated BID, p21, NF-κB p65 nuclear fraction levels.It is plausible that SMSN@G NPs promoted NF-κB p65 nuclear translocation, altering the transcription of apoptosis-related genes as illustrated by their promoted protein levels, and ultimately promoting tumor cell apoptosis.However, it is worth noting that this regulation includes both pro-apoptotic (BID, p21) and anti-apoptotic (BCL2) [24] genes.Further research is needed to elucidate the intricate interactions among these genes and whether other pathways are involved.
Additionally, in SW1990 cells, a slight decrease in apoptosis rate, NF-κB p65 nuclear fraction to total, and p21 protein levels were observed after 30 min of light exposure compared to 15 min.It is plausible that there are alternative mechanisms contributing to this observation in SW1990, such as feedback mechanisms, alternate programmed cell death [39], and cyclin or caspase-dependent or independent cell cycle arrests [40], these pathways may have influenced the outcome by exerting an anti-apoptotic effect.However, additional investigations are necessary to elucidate the precise mechanism of action underlying these findings, such as further refinement of experimental time and extension of intervention duration.
Overall, our findings highlight the promising potential of SMSN@G NPs as multifunctional anti-tumor agents in suppressing pancreatic cancer growth in vivo and in vitro.Further research will be essential to unravel the intricate molecular mechanisms involved and optimize its therapeutic applications.At the same time, nanomaterials are usually complex in design and composition, which undoubtedly creates difficulties in clinical translation, and the clinical translation of nanomaterials still needs further research.

Conclusion
We successfully designed and synthesized two-dimensional ultrathin silicon nanosheets (SN), further constructed twodimensional SMSNs with near-infrared responsiveness and successfully loaded gemcitabine to form SMSN@G NPs.SMSN@G demonstrated an excellent inhibitory effect on pancreatic cancer cell proliferation.Furthermore, the mechanism study revealed that SMSN@G potently induced tumor cell apoptosis by promoting NF-κB nuclear translocation and upregulating apoptosis-related protein levels under photothermal therapy.Through biosafety evaluation of the nanoparticles, we observed their excellent biocompatibility in pancreatic cancer models both in vitro and in vivo.The current study provides novel strategies for gemcitabine-loaded nanoparticles, thereby expanding the application of photothermal therapy with potential clinical translational value.

Figure 2 .
Figure2.The photothermal-conversion performance of SMSN@G NPs nanoparticles was evaluated under NIR-II laser irradiation.(a) UVvis spectra of SMSN@G NPs at different concentrations were measured, and (b) the corresponding mass extinction coefficient at 1064 nm was determined.(c) The photothermal heating curves of dispersed SMSN@G NPs were then recorded when exposed to a 1064 nm (NIR-II) laser at varying power densities (0.25, 0.5, 0.75, 1.0 W cm −2 ) and (d) different concentrations (0, 12.5, 25, 50, 100 ppm).(e) The photothermal-conversion efficiency at 1064 nm was calculated.In the experiments, the aqueous solution of SMSN@G NPs exhibited a red line in the heating curve, which represented the diffusion of the photothermal effect over time when the laser was switched off.The time constant (τs) of heat transfer in the system was determined using linear time data from the cooling period and represented by the blue line.(f) The heating curve of SMSN@G NPs suspension was measured over five laser switching cycles at a power density of 1.0 W cm −2 .The corresponding infrared thermal images for the heating curve were also captured (g) and (h).The infrared thermal images correspond to the heating curve (c) and (d).

Figure 3 .
Figure 3. Safety evaluation of SMSN@G NPs in vitro.(a) CCK8 absorbance at different concentrations of SMSN@G NPs with or without light; (b) apoptotic assay at different concentrations of SMSN@G NPs; (c)Histogram of apoptosis rates of SW1990 and PANC1 cells treated with different concentrations of SMSN@G NPs; Annexin V+/PI+ were defined as late apoptosis or necrosis, Annexin V+/PI− were defined as early apoptosis, * compared with SMSN without irradiation group.NS: No statistical difference, * p < 0.05, ** p < 0.01, *** p < 0.001, analyzed using student's t-test.

Figure 6 .
Figure 6.(a) A schematic depiction of murine xenograft establishment and intervention.(b) Thermal images captured during 808 nm laser irradiation (1.5 W m −2 ) of SW1990 xenograft in various groups.(c) Relative tumor volume (V/V0) was observed in the various treatment groups, V: the tumor volume at each time point, V0: the tumor volume on the first day of treatment.(d) Body weight measurements of SW1990 xenograft in each group.(e) Representative photographs showcasing SW1990 xenograft subjected to different treatments.