Electrochemical monitoring of TMZ derivative release from a novel chitosan-modified polymeric carrier for targeted glioma therapy

2.1. Chemicals and measurements

All the solvents and reagents were commercially sourced and don’t need to be further purified. Scanning electron microscopy (SEM) (Hitachi S-4700) with energy dispersive X-ray (EDX) spectroscopy (HORIBA EMAX XACT) was exploited to characterize the elemental composition and surface morphology of the composite materials. With a Rigaku D/max-B diffractometer (DMX-2200), X-ray diffraction (XRD) analysis was conducted to obtain crystallographic information. On a RENISHAW in Via Raman microscope (UK) with a He/Ne laser source of 514.4 nm, the Raman spectra were recorded.

2.2. Synthesis of compound 1

To prepare the target compound, 3-nitrobenzaldehyde (151 mg and 1.0 mmol), methyl 3-aminocrotonate (115 mg or about 0.97 mL, 1.0 mmol), and 1,3-cyclohexanedione (112 mg and 1.0 mmol) were added to anhydrous ethanol (10 mL), and then 3 drops of glacial acetic acid were added as a catalyst. For 8 h, the reaction mixture was refluxed at 100°C. The desired heterocyclic compound was acquired by filtering, cleaning with cold ethanol, and vacuum-drying the precipitate after the reaction was finished and allowed to cool to RT.

2.3. Preparation and characterization for [La₂(pbbp)(pbsd)₃] (CP1)

A mixture synthesized from 42.0 mg and 0.1 mmol H₂pbbp·2Cl, 41.2 mg and 0.1 mmol H₂pbsd and 0.1 mmol and 43.6 mg La(NO₃)₃·6H₂O was mixed in DMF-H₂O (6 mL, 3:3 in v/v) with a drop of HBF₄. The acquired mixture was stirred in air for 30 min, subsequently placed in a stainless-steel vessel lined with Teflon (23 mL) and heated at 120°C for 2 d. The mixture was cooled at 10°C-h^-1 to RT to produce light-yellow bulk crystals that were gathered by filtration, cleaned in water, and dried in air (about 53% yield on the basis of La). Anal. Calcd for C₄₀H₂₉LaN₄O₈S₃: N, 6.03; H, 3.15; C, 51.73%. Found: N 5.96, H 3.11, C 51.87%. IR (KBr pellets, cm⁻¹): 735 (m), 827 (m), 1055 (m), 1182 (m), 1395 (s), 1448 (s), 1562 (m), 1615 (s), 1641 (s), 1698 (m), 2933 (w), 3051 (w).

The Oxford Xcalibur E diffractometer was applied to obtain the X-ray data. The intensity data were investigated through CrysAlisPro software, which was later converted to HKL files. The SHELXS software formed the initial structural models with the direct method, and the Olex2 1.2 program, based on the least-squares method, was employed to modify them. Anisotropic parameters were employed to mix all the non-H atoms. AFIX commands were exploited to fix each H atom geometrically on its matching C atom. The crystallographic parameters, together with refinement details of CP1 and compound 1, have been displayed in Table S1.

2.4. Synthesis of Fru-APTMS@CP1@1

The reaction of APTMS (170 μL, 1.0 mmol) with fructose (180 mg, 1.0 mmol) in 10 mL of anhydrous methanol was followed by the incorporation of 12 mg, 0.1 mmol DMAP and 20.6 mg, 0.1 mmol DCC, which acted as a catalyst and coupling agent, separately. The mixture was stirred at RT for 24 h. The by-product dicyclohexylurea (DCU) was removed by filtration, and the solvent was evaporated to give Fru-APTMS. Subsequently, Fru-APTMS (1 mmol) and CP1 (123 mg, 1.0 mmol) were reacted in a mixed ethanol/water solvent (1:1, 10 mL) at 80°C for 8 h to form the CP Fru-APTMS@CP1. The reaction mixture was subsequently cooled, centrifuged, cleaned, and dried to give a solid product. Finally, Fru-APTMS@CP1 (50 mg) was dispersed in PBS buffer or an ethanol/water mixture (10 mL) containing compound 1 (10 mg) and shaken at 37°C for 12–24 hrs. After centrifugation and washing to remove unloaded drug, the final drug delivery system Fru-APTMS@CP1@1 was obtained.

2.5. Drug loading and encapsulation efficiency

In this study, the nanoparticles were separated from the dispersion via centrifugation at 22,000 rpm for 25 min. The collected supernatant was appropriately diluted and analyzed using a UV-Visible spectrophotometer (Shimadzu UV-2201, double-beam, Japan) at the characteristic absorption wavelength of Rg3 (497 nm) to determine the concentration of free (unencapsulated) drug. The entrapment efficiency (EE%) and loading capacity (LC%) of Rg3 in the Fru-APTMS@CP1@1 nanocarrier were calculated using the following formulas Eqs. (1,2):

where m_loaded is the amount of encapsulated Rg3, m_total is the total amount of compound 1 used, is the total mass of the nanocarrier system. These calculations enabled precise evaluation of the drug incorporation performance of the nanoplatform.

2.6. Electrochemical measurements

In this study, the electrochemical performance of Fru-APTMS@CP1@1 was systematically assessed via DPV along with CV. On a CHI 6171D electrochemical workstation with a traditional three-electrode system, all the measurements were implemented with a modified nickel foam electrode (5 × 5 mm), a saturated silver/silver chloride electrode (saturated with KCl), as well as a platinum wire as the working electrode, reference electrode, and counter electrode, separately. All the electrochemical experiments were carried out in 0.1 M PB solution.

2.7. CCK-8 assay

U251, the human glioma cell line, was acquired from the American Type Culture Collection (ATCC). The cells were inoculated in DMEM (HyClone) with 100 μg/mL streptomycin, 100 U/mL penicillin, as well as 10% foetal bovine serum (FBS) (Gibco). The cells were then subjected to routine culture in an incubator with 5% CO₂ at 37°C. Cells were inoculated into 96-well plates at 5×10³ cells/well and cultivated for 24 hrs. Fru-APTMS@CP1@1 was incorporated, and 10 μL of CCK-8 reagent (BeoTemi) was added to each well following cells adhered to the surface at 24, 48, and 72 h of treatment, separately. Afterwards, the cells were incubated for another 2 h, and OD values were then determined using a microplate reader (Bio-Rad) at 450 nm. The percentage of cell viability Eq. (3) was calculated as below :

2.8. Real-time quantitative PCR (RT-qPCR)

Based on the instructions of the kit, TRIzol reagent (Invitrogen) was exploited to extract the total cellular RNA following drug treatment for 48 h. Then, 1 μg of RNA was reverse transcribed into cDNA with M-MuLV Enzyme Mix (NEB) after identifying the purity and concentration of RNA through NanoDrop 2000 (Invitrogen). Amplification was conducted via SYBR Green Universal Master Mix (Thermo Fisher Scientific). Utilizing GAPDH as an internal reference, 2^-ΔΔCt approach was applied to detect the relative expression of target genes. GAPDH (F): GGAGCGAGATCCCTCCAAAAT. GAPDH (R): GGCTGTTGTCATACTTCTCATGG. LRP1B (F): GGAGCGAGATCCCTCCAAAAT. LRP1B (R): GGCTGTTGTCATACTTCTCATGG. ERBB4 (F): ACCTGGGCTACCCTGAAGAA. ERBB4 (R): AGGTAGCCACGGTCGTAGGT. BAX (F): CCCAGAGGCGGGGGACGAT. BAX (R): CGGAGGAAGTCCAATGTCCAG.

3.1. Characterization of Fru-APTMS@CP1@1

SCXRD was employed for the characterization of molecular structures of CP1 and compound 1, and the detailed crystallographic data have been given in Table S1 and Figures S1 and S2 of the Supporting Information. Based on this, a composite material, Fru-APTMS@CP1@1, was successfully constructed by first functionalizing the Fru backbone through an amidation reaction with APTMS, followed by the sequential incorporation of the CP1 and its active component, compound 1.

Figure 1(a) displays the FT-IR spectra of the materials. The FRU-APTMS spectrum exhibits characteristic stretching vibrations of –OH and –NH₂ groups at 3428 cm⁻¹, a C=O stretching peak near 1654 cm⁻¹, and a Si–O–Si symmetric stretching band at 1156 cm⁻¹, indicating the successful grafting of APTMS onto the Fru framework. Upon the introduction of CP1 and compound 1, notable enhancement and shifts were observed in the absorption bands within the 1500-500 cm⁻¹ region, corresponding to C=N stretching, aromatic ring vibrations, and metal–ligand coordination signals, confirming the effective loading of the target species.

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Figure 1.

(a) FTIR, (b) Raman spectra x and (c) SEM of Fru-APTMS@CP1@1.

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Figure 1(b) presents the Raman spectra of Fru-APTMS@CP1 and Fru-APTMS@CP1@1. Both samples display prominent characteristic peaks in the 500-1000 cm⁻¹ region, with significantly intensified signals in Fru-APTMS@CP1@1. These enhancements suggest that the incorporation of compound 1 altered the vibrational modes of the material, likely due to its intramolecular π–π conjugation and coordination interactions with metal centers. As shown in Figure 1(c), the SEM image confirms the successful synthesis of Fru-APTMS@CP1@1, which exhibits a well-defined layered structure with nanoscale features. The stacked sheet-like morphology, characterized by clear contours and surface roughness, provides high porosity and surface area, conducive to drug loading, potential functionalization as well as diffusion, highlighting its applicability in nanocarrier-based drug delivery and electrochemical sensing.

3.2. Electrochemical characterization

CV tests were implemented in an electrolyte solution with 0.1 M KCl and 5 mM [Fe(CN)₆] ^3−/4− to assess the composite materials’ electrochemical activity in more detail. Figure 2(a) indicates that three types of working electrodes were tested: bare nickel foam (NF), Fru-APTMS@CP1-modified NF, and Fru-APTMS@CP1@1-modified NF, with the scan rate set at 50 mV/s. The peak currents obtained were 65 mA, 86 mA, and 133 mA, respectively, with the Fru-APTMS@CP1@1 electrode exhibiting the highest current response. This indicates a larger electrochemically active surface area (EASA) and superior electron transfer capability. The improved performance can be ascribed to the enhanced electrical conductivity of the composites, the ion diffusion channels provided by its porous structure, and the contribution of surface functional groups that facilitate electron/ion transport, thereby significantly promoting redox reaction kinetics. Furthermore, Figure 2(b) displays that the Fru-APTMS@CP1@1 electrode’s redox peak currents gradually rose with the scan rate between 0.02 and 0.20 V/s, suggesting a typical diffusion-controlled electrochemical process. This mechanism is further supported by Figure 2(c), which displays a linear correlation between the square root of the scan rate and the anodic and cathodic peak currents, with fitting equations of I_pa = 462.11(ν ^1/2) + 2.2553 (R² = 0.9984) and I_pc = −410.63(ν ^1/2) − 2.1367 (R² = 0.9980).

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Figure 2.

(a) Electrochemical responses of various modified electrodes in [Fe(CN)₆]^3−/4− solution; (b) CV curves of Fru-APTMS@CP1@1 at different scan rates; (c) Linear fitting plots corresponding to various scan rates.

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Following the Randles–Sevcik equation Eq. (4):

Here, Ip represents the peak current (A), n indicates the number of electrons transferred, A stands for the EASA (cm²), D denotes the diffusion coefficient (cm²/s), C is the electroactive species concentration (mol/cm³), and ν represents the scan rate (V/s). By substituting the experimental data into the equation, the EASA of the Fru-APTMS@CP1@1 electrode was calculated to be 0.1866 cm², which is significantly higher than that of bare NF (0.117 cm²) and Fru-APTMS@CP1 (0.1705 cm²), further validating its superior electrochemical performance.

Figure 3(a) displays that the CV responses of the Fru-APTMS@CP1@1 electrode were recorded in phosphate buffer (PB, pH 5) at 0.05 V/s under various concentrations of compound 1 (50–200 μM). With the gradual increase in CP1 concentration, a marked enhancement of the peak anodic current was noted, indicating that the electrode maintains excellent electrochemical responsiveness and anti-fouling capability even at higher analyte concentrations. This improved performance can be attributed to the superior electron transfer ability and structural stability of the Fru-APTMS@CP1@1 composite, which facilitates efficient electron and ion transport. Figure 3(b) illustrates the linear correlation between the Concentration of compound 1 and peak anodic current, with the fitted regression equation I = 0.0588 × [Compound 1] + 0.2353 and a correlation coefficient of R² = 0.9975. This finding demonstrates that the system exhibits a wide linear response range and excellent electrocatalytic activity, making it highly suitable for sensitive electrochemical detection applications.

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Figure 3.

(a) Various additions (0 to 400 µM) of Fru-APTMS@CP1@1 (pH 5) were assayed, (b) calibration plot of Fru-APTMS@CP1@1 concentration versus peak current.

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3.3. Effect of scan rates and buffer pH

A series of CV determinations were implemented in phosphate buffer (PB, pH 5) with 200 μM Fru-APTMS@CP1@1 at different scan rates of 0.04-0.24 V/s to further examine the electrochemical behavior of the Fru-APTMS@CP1@1, as illustrated in Figure 4(a). The anodic peak current steadily elevated as the scan rate elevated, suggesting that the scan rate had a major impact on the oxidation process. Scan rate and peak current were found to be linearly related. There is a linear correlation between scan rate and peak current (Figure 4b), and the corresponding fitted equation was Ip = 33.06 × v + 5.6591, with R² = 0.9983, suggesting that the electrochemical process is mainly surface adsorption-controlled. Importantly, this electrochemical signal does not directly represent the drug release process but rather quantifies the concentration of compound 1 released into the medium. To further elucidate the charge transfer kinetics, a Tafel plot was created from plotting the logarithm of the current against the potential (Figure 4c), yielding a fitted equation of y = 2.2268x − 1.3268 with R² = 0.9907. Based on the Tafel equation Eq. (5):

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Figure 4.

(a) CV curves of 100 μM Fru-APTMS@CP1@1 at various scan rates; (b) Calibration plot of peak current vs. square root of scan rate; (c) Tafel plot of potential versus log(current); (d) Electrochemical responses of Fru-APTMS@CP1@1 (200 μM) under different pH conditions ranging from pH 3 to 11; (e) Calibration plot of peak current as a function of pH.

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In which, Δ represents the slope of Tafel plot, n denotes the number of electrons transferred (assumed to be 1), α stands for the charge transfer coefficient, F and R represents the Faraday constant (96,485 C/mol) and gas constant (8.314 J·mol⁻¹·K⁻¹), separately, and T indicates the temperature (300 K). Based on this equation, the calculated α value is approximately 0.57, suggesting a double electron transfer process in Fru-APTMS@CP1@1 oxidation. In addition, to optimize the electrochemical sensing conditions, the effect of pH (from 3 to 11) on the electrochemical response of Fru-APTMS@CP1@1 electrodes was examined, and the corresponding CV curves have been presented in Figure 4(d). The oxidation peak currents remarkably rose as the pH value was elevated from 3 to 5, while they decreased markedly at pH values above 5. In Figure 4(e), the current response was maximum at pH 5, which may be ascribed to the stronger surface activity and optimal electron transfer rate of the electrode under this condition. Consequently, pH 5 was chosen as the optimal operating condition for follow-up electrochemical measurements.

3.4. Kinetic release measurement

The in vitro release behavior of Fru-APTMS@CP1@1 nanoparticles was evaluated using the dialysis bag diffusion method. As shown in Figure 5, the cumulative drug release data were fitted to various kinetic models, including the zero-order (Figure 5a), first-order (Figure 5b), Higuchi (Figure 5c), and Korsmeyer-Peppas (Figure 5d) models, to investigate the release mechanism. Among them, the Korsmeyer-Peppas model exhibited the best fitting with the highest correlation coefficient (R² = 0.9675), indicating that the release followed a non-Fickian diffusion mechanism involving both diffusion and matrix relaxation. The Higuchi model also demonstrated strong linearity (R² = 0.9503), suggesting that diffusion played a predominant role. In contrast, the zero-order (R² = 0.8537) and first-order (R² = 0.7078) models showed relatively poor fitting.

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Figure 5.

Drug release kinetics plots: (a) Zero order plot, (b) First order plot, (c) Higuchi plot, and (d) Korsmeyer Peppas plot.

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Furthermore, the drug LC and EE of compound 1 in the nanocarrier were determined to be 4.82% and 73.67%, respectively. The cumulative drug release of compound 1 from the nanocomposite reached approximately 49.85% over the tested period. These results collectively demonstrate that the nanoplatform exhibits favorable loading capacity and sustained release characteristics, offering potential advantages for targeted and controlled therapeutic delivery in Crohn’s disease treatment.

3.5. Selectivity studies

Fru-APTMS@CP1@1 exhibits excellent electrochemical response, concentration-dependent behavior, and high selectivity, highlighting its potential as a smart drug delivery material. Measurements of DPV in phosphate buffer (PB, pH 5.0) exhibited an excellent linear relationship in the concentration of compound 1 between 12 and 60 μM (Figures 6a, b), with I = 0.0128x + 1.4180 and R² = 0.9894. According to the signal-to-noise ratio (S/N = 3), the detected limit of detection (LOD) were calculated to be 0.02229 μM and 0.0944 μM, and the sensitivities were 9.4 and 38.74 μA·μmol⁻¹·L⁻¹, respectively, indicating the material’s high responsiveness toward trace levels of drug molecules. In addition, Fru-APTMS@CP1@1 displayed excellent recognition selectivity for compound 1 (Figures 6c, d). Even in the presence of common biological small molecule interferents, for instance, ascorbic acid, catechol, hydroquinone, dopamine, glucose, uric acid, lactose, and fructose, the modified electrode maintained a strong and specific response toward compound 1. As shown in panels a-f, the modified electrode retained a strong and specific response toward compound 1 even in the presence of common biological small molecule interferents, including ascorbic acid (a), catechol (b), hydroquinone (c), dopamine (d), glucose (e), and uric acid (f), as well as lactose and fructose. The signal intensity for compound 1 was more than 10 times higher than that of the interferents, indicating remarkable specificity and anti-interference capability. These superior properties can be attributed to the biocompatibility and functionalization capacity provided by Fru, the active surface groups introduced by APTMS, and the specific coordination interactions of CP1, which together enhance the targeted binding efficiency. Furthermore, ionic adsorption, electrostatic interactions, and potential matching at the material surface provide a tunable platform for the loading and release of drugs.

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Figure 6.

(a) DPV response of the Fru-APTMS@CP1@1-modified electrode; (b) Calibration plot of CP1 concentration vs. peak current; (c) DPV curves of frequent interfering analytes in the presence of Fru-APTMS@CP1@1; (d) Bar diagram showing the current responses corresponding to different interfering species.

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3.6. Fru-APTMS@CP1@1 inhibited the proliferation of glioma cells

A CCK-8 assay was carried out to determine the viability of U251 glioma cells with Fru-APTMS@CP1@1 treatment for 24, 48, and 72 h, and the findings indicated a time-dependent decrease in cell viability with the increase in treatment time. After 72 h of treatment, cell survival was noted to be 68.3% lower than that of the control group (Figure 7a), suggesting a remarkable inhibitory effect of the drug on the proliferation of glioma cells. RT-qPCR results demonstrated that the levels of LRP1B mRNA were significantly increased in drug-treated glioma cells (Figure 7b). Concurrently, the pharmaceutical intervention demonstrated the capacity to potently decrease the ERBB4 expression in glioma cells (Figure 7c). In addition, Fru-APTMS@CP1@1 treatment induced apoptosis in glioma cells (Figure 7d). Our results suggested that Fru-APTMS@CP1@1 suppresses glioma cell proliferation via upregulation of LRP1B expression and suppression of ERBB4 expression to block its abnormally activated downstream signaling pathway.

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Figure 7.

(a) Fru-APTMS@CP1@1 inhibited the proliferation of U251 cells. (b) Fru-APTMS@CP1@1 regulated LRP1B expression. (c) Fru-APTMS@CP1@1 regulated ERBB4 expression. (d) Fru-APTMS@CP1@1 regulated Bax expression. * P < 0.05 and ** P < 0.01.

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