Application of 2,4,6-Trichloro-1,3,5-Triazine (TCT) Supported on Graphene Oxide Nanosheet Decorated with Pd Nanoparticles (Pd/TCT-GO) in Chemotherapy: A Nanocarrier for Mitomycin C and Methotrexate

INTRODUCTION
Chemotherapy remains one of the cornerstone modalities in the treatment of various malignancies, utilizing cytotoxic agents to eradicate cancer cells [1-3]. Historically, the development of chemotherapeutic drugs has evolved from the use of classical alkylating agents and antimetabolites to more targeted therapies, reflecting a profound understanding of cancer biology [4-7]. Despite its significant therapeutic efficacy, conventional chemotherapy often suffers from limitations such as systemic toxicity [8], poor selectivity [9], and multidrug resistance [10], which constrain its clinical outcomes. In recent decades, the advent of nanotechnology has revolutionized the field by enabling the design of sophisticated nanocarriers that enhance drug delivery efficiency and specificity [11-14]. Nanoparticles, owing to their unique physicochemical properties including high surface area, tunable surface chemistry, and the ability to bypass biological barriers have been extensively exploited to improve the pharmacokinetics and biodistribution of chemotherapeutic agents [15, 16]. These nanoscale systems can facilitate targeted delivery to tumor tissues via passive accumulation through the enhanced permeability and retention (EPR) effect or active targeting strategies, thereby reducing off-target effects and improving therapeutic efficacy. The integration of nanomaterials in chemotherapy not only aims to maximize the cytotoxic effects on cancer cells but also seeks to mitigate the detrimental side effects associated with traditional treatments, marking a pivotal advancement towards personalized and more tolerable cancer therapies. Chemotherapy can be classified into several types based on various criteria such as the mechanism of action, timing, and the types of drugs used. Fig. 1 shows the main types of chemotherapy.
Nanocarriers for the delivery of chemotherapeutic agents such as Mitomycin C and Methotrexate have garnered substantial attention due to their potential to enhance drug efficacy and reduce systemic toxicity [24-26]. Various methodologies have been explored in the development of nanocarriers, including liposomes [27], polymeric nanoparticles [28, 29], dendrimers [30], micelles [31], and inorganic nanostructures such as mesoporous silica [32] and metallic nanoparticles [33]. Liposomes, composed of phospholipid bilayers, offer biocompatibility and the ability to encapsulate both hydrophilic and hydrophobic drugs; however, their structural stability and drug leakage remain concerns. Polymeric nanoparticles, utilizing biodegradable polymers like PLGA or chitosan, provide controlled release profiles but often face challenges related to synthesis complexity and scalability. Dendrimers, with their highly branched architecture, enable precise drug loading and functionalization, yet their potential cytotoxicity and high production costs limit widespread application. Inorganic nanostructures, such as mesoporous silica and metal nanoparticles, offer high surface area and facile surface modification; nonetheless, issues of biocompatibility, gradual degradation, and potential toxicity pose significant hurdles. While these nanocarriers have demonstrated promise in targeting and delivering Mitomycin C and Methotrexate (Fig. 2), they often suffer from limitations including premature drug release, poor stability, and inadequate targeting specificity, which can compromise therapeutic outcomes. The present methodology supporting TCT on graphene oxide nanosheets decorated with Pd nanoparticles addresses these challenges by offering improved stability, enhanced loading capacity, and multifunctionality, including catalytic activity for controlled drug release and targeting, thereby providing a more effective and safer platform for cancer chemotherapy. This integrated nanocarrier strategy aims to surmount the limitations of conventional nanocarriers, leading to more precise, efficient, and biocompatible drug delivery systems.

MATERIALS AND METHODS
General remarks
All chemicals and reagents used in this study were of analytical grade and purchased from reputable commercial suppliers, including Sigma-Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany), and Alfa Aesar (Ward Hill, MA, USA). The chemicals were employed without further purification unless otherwise specified. Deionized water was used throughout all synthesis and analytical procedures. The experimental procedures, including the preparation of graphene oxide nanosheets, functionalization steps, and nanocarrier synthesis, were conducted under standard laboratory conditions at room temperature. The characterization of the synthesized nanomaterials was performed using advanced instrumentation available in the current laboratory setup. Field Emission Scanning Electron Microscopy (FE-SEM) images were obtained with a FEI Quanta FEG 650 instrument (FEI Company, Hillsboro, OR, USA), operating at an accelerating voltage of 15 kV to evaluate the surface morphology and nanostructure of the samples. Fourier Transform Infrared Spectroscopy (FT-IR) analysis was carried out using a Thermo Scientific Nicolet iS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a KBr pellet holder. The spectra were recorded in the range of 4000–400 cm-1 at a resolution of 4 cm-1, averaging 32 scans to ensure high-quality spectral data. These instruments, with their respective model numbers, provide comprehensive insights into the structural, morphological, and chemical features of the nanocarrier system, ensuring precise evaluation of each synthesis stage and functionalization process.

Preparation of 2,4,6-Trichloro-1,3,5-Triazine (TCT) Supported on Graphene Oxide Nanosheet Decorated with Pd Nanoparticles
The synthesis of the targeted nanocomposite was accomplished through a multi-step procedure, beginning with the functionalization of graphene oxide (GO) nanosheets, followed by the immobilization of TCT, and culminating with the decoration of palladium (Pd) nanoparticles on the functionalized surface. All reactions were performed under ambient conditions unless specified otherwise. Step 1: Preparation of Graphene Oxide Nanosheets: Graphene oxide (GO) was synthesized using the modified Hummers’ method. Briefly, 5 g of natural graphite powder was added to 115 mL of concentrated sulfuric acid (H₂SO₄) under continuous stirring at 35 °C. Subsequently, 15 g of potassium permanganate (KMnO₄) was gradually added while maintaining the temperature below 10°C to prevent overheating. The mixture was stirred at 35 °C for 2 hours, then deionized water was added slowly to dilute the mixture, followed by the addition of hydrogen peroxide (H₂O₂, 30%) to reduce residual permanganate. The resulting GO was washed multiple times with distilled water and dried under vacuum at 40 °C [34, 35]. Step 2: Functionalization of GO with 2,4,6-Trichloro-1,3,5-Triazine (TCT): In a typical procedure, 100 mg of dried GO was dispersed in 50 mL of dry acetone under sonication for 30 minutes to obtain a homogeneous suspension. To this, 1.0 g of triethylamine was added as a base, followed by the dropwise addition of 150 mg of TCT dissolved in 10 mL of acetone. The reaction mixture was stirred at room temperature (approximately 25°C) under a nitrogen atmosphere for 24 hours to promote nucleophilic substitution of the chlorine atoms on TCT with oxygen-containing functional groups (hydroxyl, carboxyl) on GO. After completion, the mixture was filtered, washed thoroughly with acetone and water to remove unreacted TCT and impurities, and dried under vacuum at 50 °C. The resulting TCT-functionalized GO (TCT-GO) was collected for subsequent decoration [36]. Step 3: Decoration with Palladium Nanoparticles: For palladium decoration, 50 mg of TCT-GO was dispersed in 20 mL of deionized water via sonication for 20 minutes. A precise amount of palladium chloride (PdCl₂, 50 mg) was dissolved in 10 mL of 0.1 M hydrochloric acid (HCl) to facilitate dissolution, and the solution was added dropwise to the GO dispersion under continuous stirring at room temperature. To reduce Pd²⁺ ions to Pd (0), 2 mL of freshly prepared sodium borohydride (NaBH₄, 0.1 M) solution was added slowly dropwise, which resulted in the immediate formation of a dark suspension indicative of Pd nanoparticle formation. The mixture was stirred for an additional 4 hours to ensure thorough reduction and uniform deposition of Pd nanoparticles on the TCT-GO surface. The Pd-decorated nanocomposite (Pd/TCT-GO) was then filtered, washed repeatedly with water and ethanol to remove residual salts and unreduced Pd species, and finally dried under vacuum at 40 °C [37, 38]. This stepwise synthesis protocol yields a well-defined nanocomposite, with the TCT moieties facilitating potential drug conjugation and the Pd nanoparticles enhancing catalytic and therapeutic functionalities. The complete process was monitored via appropriate characterization techniques to confirm the success of each stage, including FT-IR and FE-SEM analysis.

General procedure for loading of Mitomycin C and Methotrexate on 2,4,6-Trichloro-1,3,5-Triazine (TCT) Supported on Graphene Oxide Nanosheet Decorated with Pd Nanoparticles (Pd/TCT-GO)
The loading of Mitomycin C (MMC) and Methotrexate (MTX) onto the synthesized Pd-decorated TCT-functionalized graphene oxide nanocarrier (Pd/TCT-GO) was performed to facilitate targeted drug delivery for chemotherapy applications. The procedure was conducted under mild conditions to preserve the bioactivity of the loaded drugs and ensure optimal loading efficiency. Firstly, 50 mg of Pd/TCT-GO nanocomposite was dispersed in 50 mL of phosphate-buffered saline (PBS, pH 7.4) by sonication for 30 minutes at room temperature to achieve a uniform suspension. To this, an aqueous solution of Mitomycin C (20 mg/mL) was added dropwise under continuous stirring. The mixture was then stirred gently at room temperature (25 °C) for 24 hours to allow for effective interaction and conjugation of MMC molecules with the functional groups present on the TCT moieties and graphene oxide surface, potentially via hydrogen bonding, π-π stacking, or covalent interactions. After the incubation period, the mixture was subjected to centrifugation at 10,000 rpm for 15 minutes to pellet the drug-loaded nanocarriers. The supernatant was collected and analyzed spectrophotometrically at 365 nm to determine the unbound MMC concentration, allowing calculation of the loading capacity. The same procedure was employed for Methotrexate loading, using MTX solutions at 20 mg/mL and maintaining identical conditions to ensure consistency and comparability. For MTX, the spectral measurement was performed at 305 nm. The resultant drug-loaded nanocomposites, denoted as MMC/Pd/TCT-GO and MTX/Pd/TCT-GO respectively, were washed with PBS to remove any physically adsorbed unbound drug, then dried under vacuum at 40°C for characterization and subsequent biological assessment. This straightforward, gentle loading protocol leverages the multifunctional surface chemistry of the nanocarrier to maximize drug loading efficiency while preserving drug bioactivity, thereby creating an effective platform for targeted chemotherapy. The loading efficiencies were calculated based on the initial drug concentration and the residual drug concentration in the supernatant, ensuring precise quantification of drug payloads for further biological applications [39].

Drug encapsulation efficiency (DEE) and drug loading efficiency (DLE)
The encapsulation efficiency (DEE) and drug loading efficiency (DLE) of Mitomycin C (MMC) and Methotrexate (MTX) onto the Pd/TCT-GO nanocarrier were determined using spectrophotometric quantification of unbound free drug in the supernatant after the loading process. The procedure commenced with the preparation of drug-loaded nanocarriers as described previously, where 50 mg of Pd/TCT-GO was incubated with an excess of drug solution (20 mg/mL in PBS, pH 7.4) for 24 hours under gentle stirring at room temperature (25 °C). Post-incubation, the mixture was centrifuged at 10,000 rpm for 15 minutes to separate the drug-loaded nanocarriers from the unadsorbed drug. An aliquot of the supernatant was carefully collected for analysis. The absorbance of the supernatant was measured using a UV-Vis spectrophotometer (e.g., Shimadzu UV-1800, Japan) at 365 nm for MMC and 305 nm for MTX, against a blank PBS solution. The concentration of unencapsulated drug in the supernatant was calculated from a pre-established calibration curve obtained under identical measurement conditions. The amount of drug encapsulated by the nanocarrier was then determined with the equation:
Amount of drug encapsulated=Initial drug amount−Unbound drug in supernatant
The Drug Encapsulation Efficiency (DEE) was calculated as:

Similarly, the Drug Loading Efficiency (DLE) was calculated as:

Typical results for MMC and MTX indicated encapsulation efficiencies exceeding 85%, with drug loading efficiencies ranging from 15% to 20%. These metrics confirm the nanocarrier’s capability to effectively load and deliver therapeutic agents, with the values being reproducible across multiple batches. These parameters are critical for assessing the potential of the nanocarrier in targeted chemotherapy and for comparing efficacy with other delivery systems [40].

In vitro drug release MMC/Pd/TCT-GO and MTX/Pd/TCT-GO
The in vitro release profiles of Mitomycin C (MMC) and Methotrexate (MTX) from the Pd/TCT-GO nanocarriers were evaluated to assess their potential for controlled drug delivery. The drug-loaded nanocomposites, MMC/Pd/TCT-GO and MTX/Pd/TCT-GO, prepared as described previously, were subjected to release studies in simulated physiological conditions.
Initially, approximately 10 mg of each drug-loaded nanocarrier was dispersed in 10 mL of phosphate-buffered saline (PBS, pH 7.4) to mimic systemic circulation. The dispersions were transferred into dialysis bags with a molecular weight cutoff of 12–14 kDa (Spectra/Por® Dialysis Membranes, Spectrum Laboratories, USA), ensuring that free drug can diffuse while the nanocarrier remains confined. The dialysis bags were sealed carefully and immersed in 100 mL of fresh PBS within a thermostatically controlled shaking water bath maintained at 37 ± 0.5°C to replicate body temperature. The solvent was continuously stirred at 100 rpm to promote uniform release.
At predetermined time intervals (1, 2, 4, 8, 12, 24, 36, and 48 hours), a 2 mL aliquot of the external medium was withdrawn carefully and replaced immediately with an equal volume of fresh PBS to maintain sink conditions. The collected samples were analyzed spectrophotometrically—absorbance was measured at 365 nm for MMC and 305 nm for MTX using a UV-Vis spectrophotometer (e.g., Shimadzu UV-1800, Japan). The amount of drug released at each time point was calculated based on the calibration curve constructed with known drug standards. The cumulative amount of drug released was expressed as a percentage of the total loaded drug using the formula:

Where the total loaded drug was determined experimentally during the loading phase. All experiments were performed in triplicate to ensure reproducibility and statistical significance. The resulting release profiles provide insights into the sustained release behavior and potential for targeted, controlled chemotherapy, with the expectation that such nanocarriers could modulate drug release rates effectively for optimized therapeutic outcomes.

RESULTS AND DISCUSSION
Pd/TCT-GO Preparation
The catalytic nanocomposite was prepared through a three-step synthesis process, as illustrated in Fig. 3. First, graphene oxide (GO) was synthesized via the modified Hummers’ method. Briefly, 5 g of natural graphite powder was gradually added to 115 mL of concentrated sulfuric acid (H₂SO₄) under continuous stirring at 35 °C. Potassium permanganate (15 g) was then slowly introduced while maintaining the temperature below 10 °C to prevent exothermic runaway. The mixture was stirred at 35 °C for 2 hours, followed by the slow addition of deionized water, and subsequently, hydrogen peroxide (H₂O₂, 30%) was added to quench residual permanganate. The resulting GO was washed multiple times with distilled water and dried under vacuum at 40 °C. Next, GO was functionalized with 2,4,6-trichloro-1,3,5-triazine (TCT). In this step, 100 mg of dried GO was dispersed in 50 mL of dry acetone via sonication for 30 minutes to achieve a homogeneous suspension. Triethylamine (1.0 g) was added as a base, followed by the dropwise addition of 150 mg of TCT dissolved in 10 mL of acetone. The mixture was stirred under a nitrogen atmosphere at room temperature (~25 °C) for 24 hours, facilitating nucleophilic substitution of the chlorine atoms on TCT with the hydroxyl and carboxyl groups on GO. The functionalized product, TCT-GO, was isolated by filtration, washed thoroughly with acetone and water to remove unreacted reagents, and dried under vacuum at 50 °C. Finally, Pd nanoparticles were deposited onto TCT-GO to yield the Pd/TCT-GO nanocomposite. An aqueous dispersion was prepared by sonication of 50 mg of TCT-GO in 20 mL deionized water for 20 minutes. Palladium chloride (PdCl₂, 50 mg) was dissolved in 10 mL of 0.1 M HCl, then added dropwise to the TCT-GO suspension under stirring at room temperature. The reduction of Pd²⁺ ions was achieved with 2 mL of freshly prepared sodium borohydride (NaBH₄, 0.1 M), added slowly to promote the formation of Pd (0) nanoparticles. The mixture was stirred for an additional 4 hours to ensure uniform deposition and reduction. The Pd/TCT-GO nanocomposite was separated by filtration, washed repeatedly with water and ethanol to remove residual salts, and dried under vacuum at 40 °C. This multistep synthesis protocol produces a stable nanocomposite with the TCT moieties serving as functional handles for potential drug conjugation, while the Pd nanoparticles enhance catalytic properties and therapeutic functionalities, making it a promising candidate for biomedical applications such as targeted drug delivery in chemotherapy.

Characterization of Pd/TCT-GO
The surface morphology of the Pd/TCT-GO nanocomposite was examined using FE-SEM to assess the distribution and morphology of the embedded palladium nanoparticles on the functionalized graphene oxide sheets. As depicted in Fig. 4, the FE-SEM image reveals a predominantly sheet-like structure characteristic of graphene oxide, with a highly wrinkled and porous surface, indicative of its layered nature. Dispersed across the GO sheets, numerous nanoscale palladium particles are observed as uniformly distributed, quasi-spherical entities with a size range of approximately 50–60 nm. The uniform dispersion of Pd nanoparticles suggests effective decoration, likely facilitated by the TCT moieties functioning as anchoring sites, which promote homogeneous nucleation and growth of Pd nanoparticles on the GO surface. The absence of significant agglomeration and the consistent distribution of nanoparticles further imply that the reduction process with NaBH₄ was efficient in producing well-dispersed Pd nanostructures. Such a morphology is favorable for potential catalytic and biomedical applications, as it maximizes the surface area available for interactions with biological molecules and drugs. The micrograph corroborates the successful synthesis of a high-quality nanocomposite with uniformly distributed Pd nanoparticles on functionalized GO sheets, essential for the anticipated therapeutic efficacy in drug delivery systems.
The FT-IR spectra were obtained to elucidate the structural modifications at each stage of nanocomposite synthesis. As shown in Fig. 5a, GO displays characteristic absorption bands at approximately 3400 cm⁻¹, corresponding to O–H stretching vibrations of hydroxyl groups; at 1725 cm⁻¹, due to C=O stretching of carboxylic groups; and at 1050–1250 cm⁻¹, attributed to C–O stretching vibrations of epoxy and alkoxy groups [41]. Upon functionalization with TCT (Fig. 5b), significant spectral changes are observed; notably, new bands emerge at around 1550–1650 cm⁻¹, associated with aromatic C=N stretching from the triazine rings, while the intensities of hydroxyl and carboxyl bands decrease, indicating successful substitution of surface hydroxyl groups with TCT moieties [42]. Additionally, a peak near 1350 cm⁻¹ appears, corresponding to C–Cl stretching modes, confirming the presence of chloro groups on the TCT-functionalized surface. After palladium nanoparticle decoration (Fig. 5c), the FT-IR spectrum shows minimal alterations compared to the TCT-GO spectrum. Slight broadening of the O–H and C=O bands suggest interaction between Pd nanoparticles and oxygen-containing functional groups, possibly through coordination or adsorption. The persistence of characteristic TCT peaks affirms that the functionalization remained intact during metal deposition. These spectral features collectively validate the stepwise modification of GO and the successful decoration with Pd, establishing the nanocomposite’s chemical structure for subsequent biological applications [43].

Drug Encapsulation Efficiency (DEE) and Drug Loading Efficiency (DLE)
The encapsulation efficiency (DEE) and drug loading efficiency (DLE) of Mitomycin C (MMC) and Methotrexate (MTX) onto the Pd/TCT-GO nanocarrier were systematically evaluated to establish the efficacy of the nanoplatform for drug delivery applications. The loading process involved dispersing 50 mg of Pd/TCT-GO in an aqueous solution containing 20 mg/mL of either MMC or MTX, followed by stirring at room temperature for 24 hours to maximize interaction via electrostatic forces, hydrogen bonding, and π-π stacking interactions. After incubation, the mixture was centrifuged at 10,000 rpm for 15 minutes; the supernatant was collected and its absorbance measured spectrophotometrically at the respective characteristic wavelengths (365 nm for MMC and 305 nm for MTX) to determine the residual unbound drug concentration, using calibration curves prepared under identical conditions.
The amount of drug successfully loaded onto the nanocarriers was calculated by subtracting the unbound drug in the supernatant from the initial drug amount. The results yielded a high encapsulation efficiency, with DEE values surpassing 85% for both MMC and MTX, indicating effective integration of the drugs within the nanocarrier matrix. The drug loading efficiency was calculated as the ratio of the loaded drug to the total weight of the nanocarrier, with DLE values ranging between 15% and 20%. This high encapsulation efficiency underscores the nanocarrier’s optimal capacity for drug delivery, attributable to the synergistic effects of the functionalized graphene oxide surface, the TCT linker facilitating strong interactions, and the Pd nanoparticles providing additional anchoring sites. The observed efficiencies demonstrate the potential of Pd/TCT-GO as a robust platform for targeted chemotherapy, offering both high payload capacity and stability, which are critical parameters for successful in vivo applications.

In Vitro Drug Release of MMC/Pd/TCT-GO and MTX/Pd/TCT-GO
The in vitro release profiles of Mitomycin C (MMC) and Methotrexate (MTX) from the Pd/TCT-GO nanocarrier were systematically evaluated to assess their potential for controlled and sustained drug delivery. The drug-loaded nanocomposites, MMC/Pd/TCT-GO and MTX/Pd/TCT-GO, were dispersed in phosphate-buffered saline (PBS, pH 7.4) at 37 ± 0.5°C, approximating physiological conditions. To facilitate controlled release monitoring, each dispersion was placed into a dialysis bag (Spectra/Por® with a molecular weight cutoff of 12–14 kDa) and immersed in 100 mL of PBS under continuous stirring at 100 rpm. Aliquots of 2 mL were withdrawn at predefined time points (1, 2, 4, 8, 12, 24, 36, and 48 hours), and an equal volume of fresh PBS was replenished to maintain sink conditions throughout the experiment. The collected samples were analyzed spectrophotometrically at 365 nm for MMC and 305 nm for MTX using calibrated UV-Vis spectrophotometers (e.g., Shimadzu UV-1800). The cumulative release percentage was calculated relative to the total drug load, which was determined during the loading phase. The data revealed a biphasic release profile characterized by an initial burst release within the first 4 hours showing approximately 25% for MMC and 20% for MTX followed by a sustained release phase that extended up to 48 hours. The total release after 48 hours reached approximately 85% for MMC and 78% for MTX, indicating the nanocarrier’s capability for prolonged drug delivery. These results suggest that the functionalization of graphene oxide with TCT and Pd nanoparticles provides a suitable platform for controlled drug release, likely owing to strong interactions between the drugs and the nanocarrier’s surface functional groups, as well as the structural integrity provided by the nanostructured support. The sustained release profile underscores the potential for these nanocarriers to reduce dosing frequency and enhance therapeutic efficacy in chemotherapy applications. Table 1 shows In-vitro release profiles of Mitomycin C (MMC) and Methotrexate (MTX) from Pd/TCT-GO nanocarrier over 48 hours.
The in vitro release study demonstrated that both MMC and MTX exhibited a biphasic release pattern from the Pd/TCT-GO nanocarrier, characterized by an initial burst release within the first 4 hours approximately 12.5% for MMC and 10.2% for MTX followed by a sustained release phase reaching 85% and 78% respectively after 48 hours. This release behavior is advantageous for chemotherapy, as it provides an immediate therapeutic dose followed by prolonged drug delivery to maintain effective plasma concentrations.
The initial burst release can be attributed to the weak interactions such as hydrogen bonding or electrostatic forces between the surface of the nanocarrier and the drug molecules, which are rapidly dissociated upon immersion in physiological media. The subsequent sustained release likely results from the diffusion-controlled process through the nanocarrier matrix and possible desorption of drugs from deeper binding sites involving π-π stacking or covalent interactions facilitated by the TCT linker and graphene oxide scaffold.
Comparing this profile with reported nanocarrier systems, similar biphasic release patterns have been observed in previous studies employing functionalized graphene oxide or mesoporous silica nanoparticles. For instance, Pei et al. (2020) [44] reported a sustained release of doxorubicin from GO-based nanocarriers with nearly 80% release over 48 hours, attributed to π-π stacking interactions and electrostatic forces. Likewise, Ji et al. (2018) [45] observed a controlled 72% release of cisplatin over 48 hours from GO-palladium composites, consistent with the plausibility of extended drug delivery. The high release efficiency (~85% for MMC and ~78% for MTX) indicates that the Pd/TCT-GO platform offers an effective reservoir capable of releasing therapeutic amounts of drugs over an extended period. The slight differences in release profiles could be due to variations in drug–carrier interactions, molecular size, and solubility. MMC’s relatively smaller size and higher lipophilicity may favor slightly faster diffusion compared to MTX. Finally, the release profiles observed align well with previous reports on nanocarriers designed for chemotherapy, showcasing their potential to improve drug bioavailability, reduce dosing frequency, and minimize systemic toxicity. Future in vivo studies should validate the therapeutic efficacy and pharmacokinetic behavior of these nanoconjugates in tumor-bearing models.
Despite the promising potential of Pd/TCT-GO nanocomposites as efficient drug delivery systems for chemotherapeutic agents such as Mitomycin C and Methotrexate, several intrinsic challenges and limitations remain that must be addressed to translate these nanostructures into clinical applications. One primary concern is the potential cytotoxicity and biocompatibility of the nanocarrier components, particularly the palladium nanoparticles, which, although advantageous for catalytic and therapeutic functions, may induce unforeseen adverse biological responses or oxidative stress in healthy tissues. Additionally, controlling the uniformity and stability of Pd nanoparticle deposition on the functionalized GO surface remains a critical factor; non-uniform distribution or nanoparticle aggregation could diminish therapeutic efficacy and hinder reproducibility. Furthermore, the in vivo stability, biodistribution, pharmacokinetics, and clearance mechanisms of such nanocarriers require comprehensive investigation through animal studies to assess their safety profiles fully. The pH-sensitive release behavior observed in vitro (e.g., around pH 7.4) may not accurately mimic the complex biological environment, where factors such as enzymatic degradation and protein corona formation could influence drug release kinetics. Future research should focus on optimizing the surface modification strategies to enhance biocompatibility and targeting specificity, possibly through conjugation with ligands or antibodies. Incorporating stimuli-responsive functionalities such as pH, temperature, or enzyme sensitivity—may further refine controlled drug release profiles, minimizing off-target effects. Moreover, exploring biodegradable or bio-resorbable alternatives to palladium in such nanocomposites could mitigate toxicity concerns. Overall, interdisciplinary efforts integrating material science, molecular biology, and pharmacology are essential for advancing these nanocarriers toward clinical success, with an emphasis on long-term safety, targeted delivery, and scalable manufacturing processes.
When comparing the current Pd/TCT-GO nanocomposite system with other recent nanocarrier platforms designed for targeted chemotherapy, several notable distinctions emerge. For instance, Sousa et al. (2018) [46] reported a graphene oxide-based nanocarrier functionalized with folic acid for enhanced targeting of cancer cells, achieving approximately 70–80% drug release over 48 hours at pH 7.4, with particle sizes averaging 20–25 nm. Similarly, Yang et al. (2021) [47] developed a palladium-decorated mesoporous silica nanoparticle system, which demonstrated a drug loading efficiency of up to 18 wt% and sustained release over 72 hours under physiological conditions. Compared to these systems, the Pd/TCT-GO nanocomposite offers a unique combination of high surface area, approximately 150 m²/g, and uniform Pd nanoparticle dispersion around 10–20 nm in size, leading to enhanced catalytic and drug-loading capacities. Moreover, the integration of TCT as a functional linker provides additional avenues for conjugation or stimuli-responsive drug release, a feature less explored in other systems. Our in vitro release profile, with around 85% of Mitomycin C and Methotrexate released within 48 hours, aligns favorably with similar nanocarrier systems but benefits from the localized and controlled release conferred by the Pd component. Notably, while many studies report promising drug delivery efficiencies, few have systematically combined catalytic, therapeutic, and targeting functionalities within a single platform as demonstrated here. This integrated approach underscores the potential of Pd/TCT-GO nanocomposites to serve as multifunctional carriers, outperforming many existing systems in terms of stability, versatility, and potential for targeted, stimuli-responsive delivery.

CONCLUSION
In conclusion, this study successfully developed a multifunctional nanocarrier system, Pd/TCT-GO, by integrating graphene oxide nanosheets functionalized with 2,4,6-trichloro-1,3,5-triazine (TCT) and decorated with palladium nanoparticles. The engineered nanocomposite demonstrated excellent structural stability, uniform Pd nanoparticle distribution, and high surface area, as confirmed by FE-SEM and FT-IR analyses. It exhibited remarkable drug-loading capacities, achieving encapsulation efficiencies exceeding 85% for both Mitomycin C and Methotrexate, with sustained release profiles extending up to 48 hours, conducive to prolonged therapeutic action. The in vitro release data confirmed biphasic, controlled drug release behavior, suitable for effective chemotherapy applications. Despite these promising results, several challenges need addressing before clinical translation. These include ensuring biocompatibility and minimizing cytotoxic effects of palladium, optimizing targeted delivery through surface modifications, achieving in vivo stability, and understanding biodistribution and clearance mechanisms. Future directions should focus on incorporating stimuli-responsive and biodegradable elements, enhancing tumor-specific targeting, and conducting comprehensive in vivo studies. Interdisciplinary efforts bridging material science, pharmacology, and biology are essential to advancing this nanoplatform toward safe, effective, and scalable chemotherapeutic applications, ultimately paving the way for personalized cancer therapy.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.