Synthesis of 5-Fluorouracil (5-FU) coated platinum nanoparticles and apoptotic effects on U87 human glioblastoma cells

Abstract

Background: 5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent; however, its clinical application is often limited by systemic toxicity and the development of drug resistance. To enhance its therapeutic efficacy, novel drug delivery strategies are under investigation. This study evaluated the use of platinum nanoparticles (PtNPs) as a nanocarrier system for 5-FU delivery to glioblastoma cells, focusing on their effects on apoptosis-related proteins.

Methods: The binding affinity and interactions of 5-FU with key apoptotic proteins (BAX, Bcl2, and Caspase-3) were assessed using molecular docking and validated through molecular dynamics (MD) simulations. PtNPs were synthesized and characterized via scanning electron microscopy (SEM), X-ray diffraction (XRD), and dynamic light scattering (DLS). Drug loading and encapsulation efficiency were determined, and cytotoxicity assays were conducted in U87 glioblastoma cells. The expression levels of apoptosis-related genes and proteins were evaluated to determine the biological impact of the formulations.

Results: Docking results confirmed effective binding of 5-FU to Bcl2, Caspase-3, and BAX, with MD simulations supporting stable complex formation, particularly with Bcl2 and Caspase-3. The synthesized PtNPs exhibited favorable physicochemical properties, including uniform morphology and high drug loading efficiency. In vitro release studies revealed a sustained release profile for the PtNPs/5-FU formulation. Furthermore, PtNPs/5-FU significantly downregulated the expression of EMT- and proliferation-related genes (cyclin D1, ZEB1, and Twist) and suppressed Bcl2 protein levels, resulting in enhanced apoptosis in U87 cells.

Conclusion: PtNPs effectively functioned as a delivery platform for 5-FU, improving its release kinetics and promoting apoptotic responses while potentially minimizing systemic toxicity. These findings support further exploration of PtNP-based drug delivery systems as a promising strategy for glioblastoma treatment.

Keywords: 5-Fluorouracil (5-FU); Apoptosis; Drug delivery; Glioblastoma; Molecular docking; Molecular dynamics simulation; Platinum nanoparticles (PtNPs).

Fig. 1

XRD pattern of Pt nanoparticles showing characteristic peaks at (111), (200), and (220) planes, confirming their face-centered cubic (fcc) structure

Fig. 2

FTIR spectrum of Pt nanoparticles showing key absorption peaks at 2922 and 2853 cm⁻¹ (C–H stretching), 1640 cm⁻¹ (C = O stretching), 1024 cm⁻¹ (C–O stretching), and additional fingerprint region peaks indicative of surface functional groups involved in stabilization

Fig. 3

SEM image of synthesized platinum nanoparticles (PtNPs), showing predominantly spherical morphology with size distribution primarily in the 100–120 nm range, consistent with nanoscale features suitable for biomedical applications

Fig. 4

Dynamic light scattering (DLS) analysis of PtNPs showing a narrow size distribution centered around 234.6 nm with a polydispersity index (PDI) of 1.0. The measured zeta potential was – 1.319 mV, indicating low surface charge and moderate colloidal stability

Fig. 5

Comparison of actual and predicted drug release profiles fitted to the biexponential kinetic model for free 5-FU and PtNPs-loaded 5-FU at pH 5.5 and 7.4. Each subplot displays the experimental cumulative release data (black diamonds) and the fitted biexponential model curve for: a free 5-FU at pH 5.5, b PtNPs-5FU at pH 5.5, c free 5-FU at pH 7.4, and d PtNPs-5FU at pH 7.4. The model captures both the initial burst and sustained release phases across all conditions

Fig. 6

Cytotoxic effects of A free 5-fluorouracil (5-FU), B platinum nanoparticles (PtNPs), and C PtNPs loaded with 5-FU (PtNPs/5-FU) on U87 glioblastoma cells after 72 h, as measured by MTT assay. Dose-dependent cell viability is plotted against different concentrations of each treatment. Trendline equations and R² values indicate the goodness of fit for each dose–response curve

Fig. 7

Flow cytometric analysis of apoptosis in U87 glioblastoma cells after 24-h treatment. Cells were stained with Annexin V-FITC and propidium iodide (PI) to distinguish between live, early apoptotic, late apoptotic, and necrotic populations. A Untreated control cells, B free 5-FU-treated cells, C PtNP-treated cells, and D PtNPs loaded with 5-FU (PtNPs/5-FU). The percentages in each quadrant indicate: Q1 (necrotic), Q2 (late apoptotic), Q3 (viable), and Q4 (early apoptotic) cells. PtNPs/5-FU notably increased apoptotic cell populations compared to control and individual treatments

Fig. 8

Quantitative real-time PCR analysis of gene expression levels in U87 glioblastoma cells following treatment. mRNA expression levels of A β-Catenin, B ZEB1, C Twist1, and D Cyclin D1 were evaluated in untreated cells, cells treated with free 5-FU, PtNPs, and the combination of PtNPs loaded with 5-FU (PtNPs/5-FU). Data are shown as log2 fold changes relative to the untreated control group, normalized to GAPDH. Combination treatment resulted in a significantly greater downregulation of all target genes compared to monotherapies. Statistical significance was determined using ANOVA with p-values indicated

Fig. 9

Western blot analysis of apoptosis-related proteins in U87 glioblastoma cells treated with free 5-FU, platinum nanoparticles (PtNPs), and 5-FU-loaded PtNPs (Complex). Expression levels of Bcl2 (anti-apoptotic), Bax and Caspase 3 (pro-apoptotic) were evaluated. β-actin served as the internal loading control. Combination treatment (Complex) notably reduced Bcl2 expression while enhancing Bax and Caspase 3 levels, indicating a stronger apoptotic response compared to single-agent treatments

Fig. 10

Molecular docking analysis of 5-fluorouracil (5-FU) with Bcl2 (PDB ID: 1YSW). The left panel shows the 3D structure of Bcl2 with 5-FU bound in the active site, and the inset highlights key interactions with residues Lys15 and His91. The right panel presents a 2D interaction map generated by LigPlot + , showing hydrogen bonds and hydrophobic contacts stabilizing the 5-FU–Bcl2 complex

Fig. 11

Molecular docking visualization of 5-fluorouracil (5-FU) bound to the pro-apoptotic protein BAX (PDB ID: 6E6B). The left panel illustrates the 3D binding pocket, highlighting interactions between 5-FU and key residues Lys57 and Gln32. The inset provides a magnified view of the interaction interface. The right panel shows a 2D interaction diagram generated by LigPlot + , indicating hydrogen bonds and hydrophobic contacts stabilizing the 5-FU–BAX complex

Fig. 12

Molecular docking visualization of 5-fluorouracil (5-FU) bound to caspase-3 (PDB ID: 5IBP). The left panel shows the 3D binding conformation of 5-FU within the active site of caspase-3, highlighting interactions with key amino acid residues Arg64, Arg207, and Gln161. The inset provides an enlarged view of hydrogen bonding between 5-FU and the active site residues. The right panel presents a 2D LigPlot + interaction map, showing hydrogen bonds and hydrophobic interactions stabilizing the 5-FU–caspase-3 complex

Fig. 13

Molecular dynamics simulation results of 5-FU complexes with BAX, Bcl2, and Caspase-3. A RMSD (Root Mean Square Deviation) plot representing backbone stability of each complex over 100 ns simulation, indicating higher structural stability for Bcl2–5-FU and Caspase-3–5-FU compared to BAX–5-FU, B RMSF (Root Mean Square Fluctuation) plot showing atomic flexibility of protein residues upon 5-FU binding