Enhanced therapeutic efficacy of silibinin loaded silica coated magnetic nanocomposites against Pseudomonas aeruginosa in Combination with Ciprofloxacin and HepG2 cancer cells

Abstract

Silibinin, a major bioactive compound extracted from Silybum marianum, possesses notable antioxidant, antitumor, hepatoprotective, and antibacterial activities. However, its poor solubility limits its clinical applications. This study aimed to enhance the delivery of silibinin by synthesizing magnetic nanocomposites (MNCs) and evaluating their efficacy against clinical isolates of Pseudomonas aeruginosa and HepG2 cancer cells. The physicochemical properties of the Fe₃O₄@SiPr@Silibinin nanocomposites were characterized by FT-IR, TGA-DTG, TEM, FE-SEM, XRD, and VSM analysis. Clinical isolates and a standard strain of P. aeruginosa were treated with Fe₃O₄@SiPr@Silibinin (at sub-MIC level) in combination with ciprofloxacin (sub-MIC), and the results were compared to treatment with ciprofloxacin alone. Additionally, the anticancer effects of Fe₃O₄@SiPr@Silibinin were evaluated in HepG2 cells. The nanocomposites, with particle sizes ranging from 40 to 80 nm, significantly enhanced the antimicrobial activity of ciprofloxacin when used in combination. Treatment with Fe₃O₄@SiPr@Silibinin plus ciprofloxacin led to a downregulation of biofilm and efflux pump-related gene expression compared to ciprofloxacin treatment alone. Furthermore, Fe₃O₄@SiPr@Silibinin exhibited anti-cancer activity against HepG2 cells, with an IC₅₀ value of 35.79 µg/mL In Silibinin-treated HepG2 cells, upregulation of the P53 gene and downregulation of the Bcl2 gene were observed. Our findingssuggest that Fe₃O₄@SiPr@Silibinin MNCs, with high stability and water solublity, can efficiently deliver silibinin into pathogenic and tumorigenic cells, thereby enhancing its therapeutic effects against P. aeruginosa and HepG2 cells. Given the antimicrobial and antitumor properties of silibinin, these magnetic nanocarriers represent a promising strategy for its targeted delivery.

Keywords: Pseudomonas aeruginosa; Apoptosis; Fe3O4@SiPr; HepG2 cells; Silibinin.

Fig. 1

(A) Schematic illustration of the synthesis of Fe₃O₄@SiPr@Silibinin magnetic nanoparticles (MNPs). (B) FT-IR spectra of Silibinin and Fe₃O₄@SiPr@Silibinin magnetic nanocomposites (MNCs). (C) FE-SEM image and (D) TEM image o of Fe₃O₄@SiPr@Silibinin MNCs. Figure 2A shows the X-ray diffraction (XRD) patterns of Fe₃O₄, Fe₃O₄@SiPr and Fe₃O₄@SiPr@Silibinin MNCs. The XRD spectra display characteristic peaks at 2θ = 31.1° (220), 35.7° (311), 43.5° (400), 54.2° (422), 57.7° (511), and 63.4° (440), which correspond well with the standard diffraction pattern of magnetite (Fe₃O₄) with a face-centered cubic crystal structure (JCPDS card no. 19–0629) These diffraction peaksconfirm the presence of magnetite as the core istructure in both Fe₃O₄@SiPr and Fe₃O₄@SiPr@Silibinin MNCs. Furthermore, a broad peak observed in the 10–20° range in the XRD patterns of Fe₃O₄@SiPr and Fe₃O₄@SiPr@Silibinin MNCs is attributed to the amorphous structureof the SiO₂ shell.

Fig. 2

(A) The XRD, (B) the VSM, (C) the EDX image (D) the TGA-DTG of Fe₃O₄@SiPr@Silibinin MNCs.

Fig. 3

Antibiotic susceptibility profiling of P. aeruginosa clinical isolates. (A) Antibiotic susceptibility profile of P. aeruginosa isolates determined by the disc diffusion assay. (B) Distribution of P. aeruginosa isolates from various clinical sources. (C) Minimum inhibitory concentrations (MIC) of ciprofloxacin determined by the broth dilution method in all 40 nosocomial P. aeruginosa isolates. (D) Biofilm formation capacity of nosocomial P. aeruginosa isolates, assessed using crystal violet staining.

Fig. 4

Biofilm formation assay in P. aeruginosa isolates. The optical density (OD) of crystal violet staining in ATCC 9027 strain and six clinical P. aeruginosa isolates across four groups untreated, treated with Fe₃O₄@SiPr@Silibinin, treated with ciprofloxacin, and co-treated. Results are expressed as mean ± standard deviation (SD). Statistical significance compared to control groups was determined as *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5

Time kill kinetics of Fe₃O₄@SiPr@Silibinin and ciprofloxacin in A) P. aeruginosa ATCC 9027 (*P < 0.023), and six clinical isolates: No.06 (*P < 0.025), No.35 (*P < 0.021), No.37 (*P < 0.018), No.49 (*P < 0.029), No.61 (ns) and No.122 (*P < 0.019). Bacterial cells were treated or untreated with 1/2 MIC_{Fe3O4@SiPr@Silibinin} + 1/2 MIC_CIP, ¼ MIC_{Fe3O4@SiPr@Silibinin}+ 1/2 MIC_CIP, and 1/2 MIC_CIP alone. Results are presented as mean ± SD.

Fig. 6

Quantitative expressions levels of (A) mexA, (B) mexX, and (C) pslA genes in P. aeruginosa isolates treated with 1/2 MIC_{Fe3O4@SiPr@Silibinin} +1/2 MIC_CIP and 1/4 MIC _{Fe3O4@SiPr@Silibinin}+1/2 MIC_CIP, compared to cells treated with 1/2 MIC_CIP alone. Results are presented as mean ± SD. All experiments were performed in triplicate. Asterisks indicate statistically significant differences between treated and untreated cells (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 7

Schematic illustration of gene expression in Pseudomonas aeruginosa isolates treated with (A) Fe₃O₄@SiPr@Silibinin + CIP (sub-MIC) and (B) CIP (sub-MIC) alone.

Fig. 8

Evaluation of cell viability and apoptosis. (A) Viability of HepG2 cancer cells and (B) HFF2 normal cells treated with various concentrations (0–100 µg/ml) of Fe₃O₄@SiPr@Silibinin for 24, 48, and 72 h, assessed using the MTT assay. C–D) Flow cytometry analysis of apoptosis in HepG2 cells, comparing on C, D) untreated controls with cells treated with Fe₃O₄@SiPr@Silibinin at the IC₅₀ concentration (35.79 µg/mL). E) Relative gene expression of pro-apoptotic (TP53) and anti-apoptotic (Bcl-2) markers in treated and untreated HepG2 cells, measured by qRT-PCR. Data are presented at mean ± SD from at least three independent experiments. Statistical significancee between treated and control groups is denoted as *P < 0.05, **P < 0.01, and ***P < 0.001.