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

Scientific Reports volume 15, Article number: 21498 (2025) Cite this article

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.

Silibinin (2,3-dihydro-3-[4-hydroxy-3-methoxyphenyl]−2-[hydroxymethyl]−6-[3,5,7-trihydroxy-4-oxobenzopyran-2-yl]benzodioxin, C25H22O10) is a naturally occurring flavonoid derived from Silybum marianum. It is primarily used for treat liver-related disorders and has demonstrated significant potential as an anticancer agent by targeting processes such as cancer cell growth, angiogenesis, inflammation, and cell cycle regulation1,2,3. In addition to its anticancer properties, silibinin exhibits a wide range of biological and medicinal effects, including antibacterial4, hepatoprotective antioxidant5,6, and cardioprotective activities7. However, its hydrophobic nature results in poor water solubility, which significantly limits its therapeutic effects8,9,10. To overcome this challenge, nanotechnology-based drug delivery strategies have been developed to enhance the aqueous solubility of hydrophobic components, improve their stability, and modulate their pharmacological activity11,12,13.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic nosocomial pathogen14,15. Its inherent and acquired resistance to multiple drugs, coupled with its ability to form biofilms, presents significant challenges to effective treatment16. Biofilm formation driven by extracellular polysaccharides such as alginate, psl, and Pel, plays a crucial role in the development of high-level drug resistance17,18. Therefore, strategies that inhibit or disrupt biofilms are essential for combating P. aeruginosa infections17. Bacteria that form biofilms secrete various extracellular polymeric substances that embed the bacterial population within a protective matrix. Polysaccharides are key structural components of biofilms and contribute to the enhanced antibiotic resistance observed in these bacterial communities19,20. Specifically, in P. aeruginosa, the exopolysaccharides psl, pel, and alginate are critical for biofilm development21,22. psl, a mannose-rich exopolysaccharide, is particularly important during the early stages of biofilm formation18. It is a neutral, branched pentasaccharide composed of repeating units of D-mannose, glucose, and L-rhamnose, synthesized by proteins encoded by 15 co-transcribed genes within the psl operon (pslA-pslO)23,24. Upon surface contact, psl is organized helically around bacterial cells, facilitating attachment and intercellular connection. As the bacterium moves, it leaves behind psl trails that enhance surface motility and promote microcolony development25,26. Psl sensing also contributes to biofilm maturation by increasing the the intracellular levels of cyclic di-guanosine monophosphate (c-di-GMP), a keysecondary messenger21. In addition to biofilm formation, multidrug resistance in P. aeruginosa is mediated by several mechanisms, including the upregulation of efflux pumps, reduced outer membrane permeability (via porin downregulation), and mutations in target enzymes16. Upregulation of efflux pump genes decreases intracellular drug accumulation by activity exporting antibiotics from the cell27. Among these, the mexAB-oprM and mexXY-oprM efflux systems, belonging to the resistance-nodulation-division (RND) family, play a critical role in the multidrug resistance observed in P. aeruginosa isolates16.

Liver cancer is among the most prevalent malignancies worldwide and is influenced by a range of pathogenic factors. Hepatocellular carcinoma (HCC) is the most common form of liver cancer28. Apoptosis, a form of programmed cell death, plays a crucial role in maintaining tissue homeostasis in long-lived mammals. DNA damage or uncontrolled cell proliferation can trigger apoptosis through intrinsic and/or extrinsic pathways, making the stimulation of apoptosis a key target in anticancer therapy29,30.

The integration of plant extracts into nanoparticle synthesis represents a promising green synthesis approach. This trend is driven by the ability of plant-derived biomolecules to act as efficientreducing agents during nanoparticle formation, thereby minimizing environmental impact31,32.

Magnetic nanoparticles (MNPs), defined as solid colloidal particles ranging in size from 1 to 100 nanometers, have attracted considerable attention in biomedicine due to their versatility33,34. Advances in nanotechnology have enabled the synthesis of nanomaterials with diverse sizes, shapes, and surface charges, facilitating targeted interactions with bacterial and cancer cells and the development of potent antimicrobial and anticancer agents35. Several studies reported that particle size of nanoparticles up to 100 nm can playing a critical role in cellular uptake36. Docetaxel-loaded solid lipid nanoparticless with size of 120 nm increased cellular uptake, stability and cytotoxity on alzimer’s diseaes with less than side effects on adjecent normal cells37. Polyphenols are powerfull anti-oxidant38 that is considered to using therapeiutic strategies. This study aimed to design recyclable, silibinin-functionalized, silica-coated Fe3O4 (Fe3O4@SiPr@Silibinin) nanocomposites with multifunctional therapeutic properties. Additionally, the antimicrobial and antitumor activities of Fe3O4@SiPr@Silibinin were evaluated againest P. aeruginosa isolates and HepG2 hepatocellular carcinoma cells.

Silibinin (C25H22O10, MW: 482.44 g/mol) was purchased from SigmaAldrich (purity ≥ 98%, München, Germany). Triethylamine (Et3N, 99.5%), xylene, and (3-Chloropropyl) triethoxysilane (CPTES) were obtained from Merck (Darmstadt, Germany) and Fluka Chemie GmbH (Buchs, Switzerland), respectively, and were used without further purification.

Fe3O4 magnetic nanoparticles (MNPs) were synthesized according to previously reported method39,40,41. To synthesis Fe3O4@SiPrCl MNCs, 1.0 g of Fe3O4 MNPs was dispersed in 5.0 mL of xylene and ultrasonicated for 30 min. Subsequently, 2.0 mL of CPTES was added dropwise to the dispersion, followed by another 30 min of ultrasonication. The mixture was then centrifuge (10 min), and the product (Fe3O4@SiPrCl MNCs) was separated using a super magnet (1.4 T) and dried in 60 oC for 6 h. Next, Fe3O4@SiPrCl MNPs (0.2 g), silibinin (0.2 g), 5 mL of 10% (w/v) NaOH solution, and 20 mL of distilled water were mixed and stirred continuously for 24 h. After 5 h of stirring, 5 mL of Et3N was added to the mixture. The reaction mixture was further stirred, magnetically separated, and incubated in an oven at 50 °C for 24 h.

The structure of the synthesized Fe3O4@SiPr@Silibinin MNCs was confirmed using various charactrization techniques, including transmission electron microscopy (TEM), field-emission scanning electron microscopy (FE-SEM), thermogravimetric analysis (TGA), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), and Fourier-transform infrared (FT-IR) spectroscopy.

Fourier transform infrared (FTIR) spectroscopy is an technique for the identification of molecular structures and the finding of chemical bonding in nanoparticles42. Fourier-transform infrared (FT-IR) spectra of silibinin, nano-Fe3O4, Fe3O4@SiPr, and Fe3O4@SiPr@Silibinin magnetic nanoparticles were recorded using a Shimadzu FT-IR-8400 S spectrometer (Shimadzu Europe). Spectra were collected over a range of 400 to 4000 cm−1.

The TEM imaging can used to assess particle size, size distribution and morphology of the nanoparticles43. The particle size of the Fe3O4@SiPr@Silibinin MNCs was evaluated using a Zeiss-EM10C-100 KV transmission electron microscope (Germany). Sample were diluted in distilled water to a concentration of 1 mg/mL, and a 20 µL drop was placed onto a carbon-coated copper grid. After two minutes, excess liquid was carefully removed with filter paper. The sample was stained with 2% uranyl acetate, and any remaining solution was absorbed, and the grid was then air-dried at ambient temperature prior to e microscopic examination and imagingy.

Field emission scanning electron microscopy (FE-SEM) is an advanced type of SEM used for high-resolution analysis of nanoparticle morphology, surface topography, and crystallographic structure, offering significantly greaterresolution than conventional SEM44. The surface morphology and crystal structure of Fe3O4@SiPr@Silibinin magneic nanoparticles were examinedd using a MIRA3 TESCAN field-emission scanning electron microscope (FE-SEM) (Czech Republic), equipped with energy-dispersive X-ray (EDX) analysis capabilities. FE-SEM imaging was also used to determine the particle size distribution.

Thermogravimetric analysis (TGA) is used to the amount of surface coating and decomposition temperatures45. The thermal stability ofFe3O4@SiPr@Silibinin magnetic nanoparticles was evaluated using a TA Q600 thermogravimetric analyzer (TA Instruments, USA). Magnetic properties were characterized with a vibrating sample magnetometer (VSM, Lakeshore 7403, USA).

X-Ray diffraction analysis (XRD) is applicated to determine chemical composition and crystallographic structure of a material46. The crystallinity of the synthesized MNCs was confirmed by X-ray diffraction (XRD) analysis, conducted with a Philips XPert X-ray powder diffractometer (PANalytical, Netherlands). The diffractometer operated at a scanning rate of 2°/min over a 2θ range of 10° to 80°, using a Cu Kα radiation source (λ = 0.154 nm).

Energy dispersive X-ray Spectroscopy (EDX) analysis was used to determine elemental composition of materials46. The elemental composition of Fe3O4@SiPr@Silibinin magnetic nanoparticles was determined by via energy-dispersive X-ray (EDX) spectrometry. Elemental point analysis was performed using a VEGA\TESCAN-LMU instrument (Czech Republic).

A total of 250 clinical samples were collected from various laboratories and hospitals in Tehran (Tehran province, Iran), resulting in the identification of 40 nosocomial isolates of Pseudomonas aeruginosa. Informed consent was obtained from all participants and/or their legal guardiancs prior to sample collection. The isolates were confirmed through Gram staining, citrate utilization, catalase, and oxidase tests, as well as cultivation on cetrimide agar, followed by incubation at 42 °C. P.aeruginosa ATCC 9027, obtained from the Pasteur Institute (Tehran, Iran), was used as the control (reference) strain. Antibiotic susceptibility profilng was performed using the disk diffusion (Kirby-Bauer) method according to CLSI guidelines (2023). The antibiotics tested included imipenem (10 µg), amikacin (30 µg), piperacillin (100 µg), ceftazidime (30 µg), meropenem (10 µg), levofloxacin (5 µg), and ciprofloxacin (5 µg). Additionally, the minimum inhibitory concentration (MIC) of ciprofloxacin was determined using the broth-dilution method (CLSI 2023) for all P. aeruginosa isolates.

The checkerboard dilution assay was employed to assess the synergistic effects of Fe3O4@SiPr@Silibinin and ciprofloxacin against P. aeruginosa. In Mueller-Hinton Broth (MHB), ciprofloxacin and Fe3O4@SiPr@Silibinin were serially diluted to obtain eight different concentrations each.

Equal volumes of Fe3O4@SiPr@Silibinin (Drug A) were dispensed into each column of the microtiter plate wells, while equal volumes of ciprofloxacin (Drug B) were dispensed into each row. Approximately 5–7.5 × 105 CFU/mL of bacterial suspension was added to each well. The fractional inhibitory concentration index (FICI) was calculated to determine the interaction between the two agents, using the following formula:

FICI = (MICdrugA combined with/MICdrug A used alone) + (MIC drug B combined with/MICdrug B alone).

The interaction between the drugs was classified based on FICI values as follows: FICI ≤ 0.5 indicated synergy, 0.5 < FICI ≤ 0.75 indicated partial synergy, 0.76 < FICI ≤ 1 indicated additivity, 1 < FICI ≤ 4 denoted indifference, and FICI > 4 indicated antagonism47.

The P. aeruginosa ATCC 9027 strain and ciprofloxacin-resistant clinical isolates were cultured for 24 h. A 0.5 McFarland Standard solution (1–1.5 × 108 CFU/mL) was prepared and serially diluted to achieve a bacterial concentration of 106 CFU/mL. Subsequently, 1000 µL of the bacterial suspension (106 CFU/ml) was added to 1000 µL of Mueller–Hinton broth, resulting in a final bacterial suspension of approximately 5–7.5 × 105 CFU/mL. The cultures were subjected to the following conditions: (1) 1/2 MICFe3O4@SiPr@Silibinin + 1/2 MICCiprofloxacin, (2) 1/4 MICFe3O4@SiPr@Silibinin + 1/2 MICCiprofloxacin, (3) 1/2 MICCiprofloxacin and (4) Untreated bacteria. All samples were incubated at 37 °C for 24 h.

Biofilm formation was evaluated by crystal violet staining48. Forty clinical isolates and the P. aeruginosa ATCC 9027 strain were diluted to a 0.5 McFarland standardn and cultured in a 96-well microplate at 37 °C for 24 h. Following incubation, biofilm formation was assessed in all isolates. Additionally, six ciprofloxacin–resistant isolates and the ATCC 9027 strain were diluted to approximately 5–7.5 × 105 CFU/mL and treated with ciprofloxacin and Fe3O4@SiPr@Silibinin under thr previously described conditions at 37 °C for 24 h.

To evaluate biofilm formation, treated and untreated bacterial cells were washed with phosphate-buffered saline (PBS, pH 7.0) to remove planktonic cells. Subsequently, 96% methanol was added to the wells for 5 min to fix the adherent bacteria. After rinsing the wells five times with sterile distilled water, the bacteria were stained with 1% crystal violet for 10 min. The adherent bacteria were then solubilized with 33% glacial acetic acid. The optical density (OD) was measured at 575 nm using an ELISA plate reader (Biotek ELx800, USA)49.

An in vitro model to evaluate bactericidal or bacteriostatic property of antibacterial agent is time–kill kinetics50. The time-kill assay was performed as described by Eleftheriadou et al.51. The P. aeruginosa ATCC 9027 strain and six clinical isolates were exposed to four different treatments. Bacterial cells (5 × 105 CFU/mL) were diluted in Mueller-Hinton broth (MHB)and incubated for 0–8 h. Colony counts was measured at different time intervals, and the final counts were recorded after 8 h.

This evaluation was conducted to investigate the function of Fe3O4@SiPr@Silibinin as efflux pump inhibitors by measuring the accumulation of ciprofloxacin inside antibiotic-resistant bacterial cells. Seven antibiotic-resistant isolates were treated with Fe3O4@SiPr@Silibinin and ciprofloxacin for 24 h. After treatment, the cells were lysed using RNX-Plus™ kit (Cinaclon, Tehran, Iran) following the protocol up to step 8, in order to isolate the cytoplasmic fraction from the aqueous phase. Finally, the amount of ciprofloxacin inside the cells was measured by measuring the optical absorption at a wavelength of 327 nm using a Agilent Cary 60 UV-Vis spectrophotometer(USA).

Q-RT-PCR is the gold standard test for the quantitative measurement of genes in RNA level52. The transcription levels of the mexX, mexA, and pslA genes were evaluated by quantitative real-time PCR (Q-RT-PCR) in bacterial isolates treated with: Sub-MIC concentrations of ciprofloxacin combined with Fe3O4@SiPr@Silibinin, and Sub-MIC concentrations of ciprofloxacin alone. Total RNA was isolated using the RNX-Plus™ kit (Sinagen, Iran). cDNA synthesis was performed using the cDNA synthesis kit (YektaTajhiz, Tehran). Q-RT-PCR was conducted using SYBR Green qPCR Master Mix (YektaTajhiz, Tehran) on a Rotor-Gene® Q instrument (QIAGEN, Germany).

The Q-RT-PCR protocol included: Initial denaturation at 95 °C for 3 min, Followed by 40 cycles of 95 °C for 5 s, 60 °C for 5 s, and 75 °C for 10 s. The rpsL gene was used as the internal control for normalization. Primer sequences used in the Q-RT-PCR assays are listed in Table 1. Gene expression levels were calculated using the 2−ΔΔCT methode.

HepG2 cells (a human liver cancer cell line) and MCF10 cells (non-cancerous mammary epithelial cells) were obtained from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran). Both cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (pen/strep), and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The cytotoxic effects of Fe3O4@SiPr@Silibinin on HepG2 cells were evaluated using the MTT assay (Sigma-Aldrich, USA). HepG2 cells (6 × 104 cells/well) were seeded into 96-well flat-bottom plates and incubated for 24 h. Subsequently, the cells were treatedwith varying concentrations of Fe3O4@SiPr@Silibinin (0–100 µg/mL) for 24, 48, and 72 hoursys. After treatment, the cells were washed with PBS, and 100 µL of RPMI 1640 medium containing 10 µL of MTT solution (0.5 mg/mL) was added to each well. The plates were incubated at 37 °C for 3 h to allow for formazan crystal formation via mitochondrial succinate dehydrogenase activity. After incubation, the medium was removal, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the purple formazan crystals. The absorbance was measured at 570 nm using a microplate reader (ELx800, BioTek, USA), with a reference wavelength of 630 nm, after 15 min of incubation at room temperature. All experiments were performed in triplicate. The IC50 value (half-maximal inhibitory concentration) was calculated using the GraphPad Prism®9.4.1 software(GraphPad Software, USA).

The ability of Fe3O4@SiPr@Silibinin to induce apoptosis in HepG2 was evaluated using flow cytometry with a FITC Annexin V Apoptosis Detection kit (BioLegend, USA). HepG2 cells (1 × 106 cells/flask) were treated with 35.79 µg/mL of Fe3O4@SiPr@Silibinin for 24 h, while untreated cells served as controls. After incubation the cells were washed and stained with Annexin V-FITC and propidium iodide (PI), following the manufacturer’s protocol. The stained cells were immediately analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, USA).

Quantitative real-time PCR (Q-RT-PCR) was conducted to assess the expression of p53 and Bcl2 genes in Fe₃O₄@SiPr@Silibinin-treated and untreated HepG2 cells. Total RNA was extracted using the RNX-Plus™ kit (Cinaclon, Tehran, Iran), and first-strand cDNA was synthesized using a cDNA synthesis kit (Yekta Tajhiz Azma, Iran). Gene expression was quantified using SYBR® Green qPCR Master Mix (Yekta Tajhiz Azma, Iran) on a Rotor-Gene® Q instrument (QIAGEN, Germany). The thermal cycling program consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 5 s,, and extension at 75 °C for 10 s. The GAPDH gene was used as the internal reference (housekeeping gene).Primer sequences are listed in Table 2. Gene expression was analyzed using the 2−ΔΔCT method.

To evaluate significant differences between groups, t-tests, as well as one-way and two-way ANOVA, were performed. A p-value of less than 0.05 (p ˂0.05) was considered statistically significant. All statistical analyses were conducted using GraphPad Prism® version 9.4.1 (LaJolla, CA, USA).

As shown in Fig.ure 1 A, Fe3O4@SiPr@Silibinin MNCs were successfully synthesized using inexpensive and readily available materials. Figure 1B displays the FT-IR spectra of both Silibinin and Fe3O4@SiPr@Silibinin MNCs, confirming the conjunction of Silibinin to the Fe3O4@SiPr surface. A broad absorption band in the 3100–3500 cm−1 region corresponds to the O-H stretching vibrations,, indicating the presence of phenolic or alcoholic groups in both Silibinin and the modified nanocomposites. The peak at 1715 cm−1 is associated with the C = O stretching of the ketone group, attributed to Silibinin. Distinct peaks at 1450 and 1490 cm−1 are characteristic of C = C stretching vibrations from aromatic and vinyl groups. A strong band at 1120 cm−1 corresponds to Si-O-Si vibrations, confirming the silica shell structure, while peaks near 625 cm−1 indicate Fe-O bending, validating the presence of Fe3O4 in the MNCs (Fig. 1B).

FE-SEM and TEM analyses were performed to evaluate the size and morphology of the synthesized MNCs. The FE-SEM image (Fig. 1C) shows spherical and uniform particles with diameters ranging from 40 to 80 nm. The TEM images (Fig. 1D) reveals slight aggregation, likely due to intermolecular interactions between Silibinin and the Fe3O4@SiPr nanoparticles.

(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.

The magnetic properties of uncoated iron oxides (Fe3O4), Fe3O4@SiPr, and Fe3O4@SiPr@Silibinin magnetic nanocomposites (MNCs) were investigated at room temperature using a vibrating sample magnetometer (VSM) over a field range of ± 15 kOs. As shown in the hysteresis curves (Fig. 2B), the saturation magnetization values of Fe3O4@SiPr and Fe3O4@SiPr@Silibinin MNCs are comparable to that of bare Fe3O4. This similarity indicates that the magnetic functionality of the core is preserved after surface modification, enabling efficient magnetic separation of Fe3O4@SiPr@Silibinin MNCs from aqueous solutions under an external magnetic field.The elemental composition of the synthesized Fe3O4@SiPr@Silibinin MNCs was confirmed by energy-dispersive X-ray spectroscopy (EDX), as shown in Fig. 2C. The EDX spectrum revealed the presence of Fe (55.41 w/w %), O (36.03 w/w %), Si (0.16 w/w %), N (2.67 w/w %), and C (4.90 w/w %), confirming the successful functionalizationof Fe3O4 with Silibinin.Based on the elemental analysis, the nanocomposite is composed of approximately 76% Fe₃O₄ and approximately 8% Silibinin by weight.

The thermal stability of Fe3O4@SiPr@Silibinin MNCs was assessed using thermalgravimetric analysis (TGA) and derivative thermogravimetry (DTG). The TGA-DTG curve (Fig. 2D) display an initial weight loss of about 3% at 49.0 °C, followed by additional weight loss between 46 and 100 °C, likely due to the evaporation of adsorbed moisture and surface hydroxyl groups. No significant weight loss was observed between 100 and 600 °C, indicating the absence of degradation of organic functional groups and confirming the high thermal stability of the nanocomposite.

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

In this study, 40 clinical isolates of Pseudomonas aeruginosa were collected from blood, burn wounds, shunt infections, and urine samples (Fig. 3B). Antibiotic susceptibility testing revealed that 90% of the isolates exhibited multidrug resistance (MDR). The highest resistance rates were observed against imipenem (90%), levofloxacin (75%), and ciprofloxacin (72%) (Fig. 3A). Furthermore, minimum inhibitory concentration (MIC) analysis indicated that all isolates were resistant to ciprofloxacin, with MIC values ranging from 128 to1024 µg/mL (Fig. 3C).

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.

The synergistic activity of Fe3O4@SiPr@Silibinin in combination with ciprofloxacin against P. aeruginosa isolates was evaluated using the fractional inhibitory concentration (FIC) index. Results from the checkerboard dilution assay (Table 3), demonstrate a synergistic interaction between ciprofloxacin and Fe3O4@SiPr@Silibinin. This finding suggests that the co-administration of these agents may enhance antibacterial efficacy and reduce the required ciprofloxacin dosage to inhibit bacterial growth.

Biofilm formation in the ATCC 9027 reference strain and 40 clinical P. aeruginosa isolates was assessed using the crystal violet staining method47 (Fig. 3D). In the reference strain and six selected clinical isolates, treatment with combinations of 1/2 MICFe3O4@SiPr@Silibinin + 1/2 MICCIP and 1/4 MICFe3O4@SiPr@Silibinin + 1/2 MICCIP significantly reduced biofilm formation compared with ciprofloxacin alone (1/2 MIC) and untreated controls (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.

A time-kill kinetics assay was conducted to evaluate the synergistic effects of Fe3O4@SiPr@Silibinin and ciprofloxacin (CIP) on P. aeruginosa isolates. The antibacterial activity of Fe3O4@SiPr@Silibinin was time-dependent, with the optimal bactericidal time varying across isolates: 2 h for isolate No.122 isolate, 4 h for isolates No.49 and No.61, and 8 h for the ATCC 9027 strain and isolates No.6, No.35, and No.37. The combination of 1/2 MICFe3O4@SiPr@Silibinin +1/2 MICCIP showed the greatest inhibitory effect, significantly reducing P. aeruginosa growth after 8 h of treatment (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 MICFe3O4@SiPr@Silibinin + 1/2 MICCIP, ¼ MICFe3O4@SiPr@Silibinin+ 1/2 MICCIP, and 1/2 MICCIP alone. Results are presented as mean ± SD.

Silibinin nanoparticles were found to enhance ciprofloxacin retention in cells by inhibiting efflux pump activity. Spectrophotometric analysis showed that higher doses of silibinin nanoparticles led to increased intracellular accumulation of ciprofloxacin (according to Table 4).

Quantitative RT-PCR analysis revealed the downregulation of mexA (Fig. 6A), mexX (Fig. 6B), and pslA (Fig. 6C) genes in the ATCC9027 reference strain and six clinical isolates treated with sub-MIC concentrations of Fe3O4@SiPr@Silibinin + CIP, compared to those treated with sub-MIC CIP alone.

The combination of 1/2 MICFe3O4@SiPr@Silibinin +1/2 MICCIP resulted in more pronounced gene suppression than the 1/4 MICFe3O4@SiPr@Silibinin +1/2 MICCIP treatment.

A schematic model illustrating the effects of Fe3O4@SiPr@Silibinin-CIP combinationon the transcription of genes associated with efflux pump function and biofilm formation in both treated and untreated P. aeruginosa isolates is presented in Fig. 7.

The nanocomposite appears to modulate gene expressionby reducing mRNA levels for key efflux and adhesion factors. This suppression likely enhance ciprofloxacin uptake and promote bacterial cell death through diminished efflux activity and biofilm integrity.

Quantitative expressions levels of (A) mexA, (B) mexX, and (C) pslA genes in P. aeruginosa isolates treated with 1/2 MICFe3O4@SiPr@Silibinin +1/2 MICCIP and 1/4 MIC Fe3O4@SiPr@Silibinin+1/2 MICCIP, compared to cells treated with 1/2 MICCIP 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).

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

The viability of HepG2 cancer cells and MCF10 normal cells was assessed after treatment various concentrations of Fe3O4@SiPr@Silibinin (0–100 µg/ml) over 1, 2, and 3 days. The results indicated that Fe3O4@SiPr@Silibinin inhibited cell viability and proliferation in a dose- and time-dependent manner. The half-maximal inhibitory concentrations (IC₅₀) of Fe3O4@SiPr@Silibinin in HepG2 cells were 35.79 µg/mL, 19.3 µg/mL, and 9.03 µg/mL at 24, 48, and 72 h, respectively (Fig. 8A). Furthermore,, Fe3O4@SiPr@Silibinin exhibited negligible toxicity toward Hff2 normal cells (Fig. 8B).

The apoptosis effect of Fe3O4@SiPr@Silibinin (0 and 35.79 µg/mL) was assessed using flow cytometry in HepG2 cells. The proportion of apoptotic cells increased to 28.23% in in the treated group, compared to 6.68% in the untreated controls (Fig. 8C). Additionally, the percentage of necrotic cells rose from 0.25% in the untreated group to 1.97% following treatment.

Quantitative real-time PCR revealed a significant upregulation of p53 expression (1.93 ± 0.89) and a notable downregulation of the Bcl-2 (0.12 ± 0.08) in HepG2 cells treated with 35.79 µg/mL Fe₃O₄@SiPr@Silibinin compared tountreated controls (Fig. 8E).

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.

Silibinin, a principal bioactive compound derived from Silybum marianum, has traditionally been used to treating various liver disorders. More recently, it has exhibited significant inhibitory effects including anti-tomor53, anti-inflamatory, anti-bacterial and anti-oxidant propertis. Due low solubilty of silybin in water and body fliud54, in the present study, we synthetized silibinin-functionalized silica-coated iron oxide nanoparticles (Fe3O4@SiPr@Silibinin) and investigated its antibacterial and anticancer activities against clinical isolates of Pseudomonas aeruginosa and HepG2 hepatocellular carcinoma cells.

Silybin insulublity in water is an important problem in designing new therapeutic strategis. Therefore, it is necessary to design new drugs that increased solublity and bioavailebilty of low soluble agent such as silibinin. It seems taht magnetic nanoparticles can confer these properties to silibinin and other polyphenol compounds55. Magnetic nanoparticles, guided by an external magnetic field, deliver drugs to specific tissues in a targeted manner, increasing the effectiveness of treatment and reducing side effects. Also, their small size and high active surface area allow for surface modification and attachment of targeting agents or stimuli-responsive coatings56,57. On the other hand, magnetite (Fe₃O₄) particles have shown good biocompatibility, low toxicity and stability in biological fluids, making them a suitable option for clinical applications58,59. Pourasgar et al. showed that curcumin-functionalized silica-coated Fe3O4 nanoparticles with particle sizes between 40 and 80 nm with high thermal stability (> 600 °C) and paramagnetic properties, increased drug efficiency against bacterial cells60.Similarly, Ramya et al. revealed that silibinin-Fe2O3 nanoparticles with aspherical morphology and particle size 70 nm had effeicient delivery in colon cancer inhibition61. Xia et al. revealed that nanoparticles in the 40–80 nm size range strike a good balance between cellular uptake and biocompatibility. These dimensions allow for efficient cell entry via clathrin- and receptor-mediated endocytosis. In particular, the 40–50 nm subset has shown the highest uptake efficiency in many cells62. Particles smaller than about 30 nm may have lower uptake due to insufficient cell membrane tortuosity, while larger particles (about 60–80 nm) may show reduced uptake due to receptor saturation. Also, although smaller nanoparticles (about 40 nm) may have higher uptake, they are also more likely to be toxic due to greater intracellular accumulation. In contrast, larger nanoparticles (up to about 80 nm) generally show lower toxicity. Accordingly, the selected range (40–80 nm) in this study, based on reliable scientific sources, is considered appropriate in terms of the balance between therapeutic efficacy and biological safety63,64. In our study, ferromagnetic Fe3O4@SiPr@Silibinin nanocomposite was synthetized with particle size 40–80 nm and thermal stability more than 300 °C.

Silibinin as hepatoprotective and important component of silymarin exhibits antibacterial properties. Checker board analysis by Lee et al. demonstrated that the MIC of ampicillin nn combination with silibinin was reduced to ≤ 4- to 8-fold in all standard strains confirming synergistic effect of silibinin and ampicillin65. Kang et al. showed the MIC of oxacillin or ampicillin decreased by ≤ 4-fold in the combination with silibinin against clinical methicillin-resistant isolates of Staphylococcus aureus66. Our checkerboard analysis demonstrated that Fe3O4@SiPr@Silibinin increased the susceptibility to ciprofloxacin by 4- to 16-fold in resistant isolates P. aeruginosa.

Time-kill kinetics was used to identify antimicrobial activity of different agants67. Petersen et al. showed that time-kill kinetics with tigecycline demonstrated 1 to 2 log10 CFU/mL decrease in bacterial counts in the most clinical pathogens. But, tigecycline reveals bactericidal activity against select isolates68. Rezk et al. revealed that gold-silver core-shell nanoparticles (Au@AgNPs) synthesized with propolis extract demonstrated strong bactericidal effects against P. aeruginosa and S. enterica in a time-kill assay. At 50 µg/mL, these effects lasted up to 5 h69. Our time-killing experiments also showed that the combination of Fe₃O₄@SiPr@Silibinin and ciprofloxacin exerted a bactericial effect on P. aeruginosa isolates. The optimal time for bacterial inhibition varied among the isolates, with the combination treatment (1/2 MIC of each agents) achieving the greatest inhibitory effect at 8 h. These findings underscore the potential of this combined therapy for effectively combating drug-resistant bacteria.

Biofilm formation plays a critical role in the development of bacterial drug resistance. For example, planctonic forms of Staphylococcus epidermidis and Klebsiella pneumoniae strains were found to be sensitive to antibiotic; whereas cells isolated from biofilms exhibited drugs resistance70. Fydrych et al. reported that phytochemicals such as apigenin, quercetin, gallic acid, and rutin exert antibaterial effects by inhibiting biofilm formation71. Similarly, Makled et al. showed that silver nanoparticles (AgNPs), administered at concentrations ranging from 6.25 to 50 µg/ml, demonstrated potent antibacterial and anti-biofilm activity against P.aeruginosa isolates72. Pourasgar et al. reported that curcumin-functionalized magnetic nanoparticles (MNPs) combined with ciprofloxacin significantly reduced biofilm formatiom and antibiotic resistance in Acinetobacter baumannii partly through downregaulating bap (biofilm-associated protein) gene47. Montazeri et al. demonstrated that Ag@Glu/Tsc nanoparticles inhibited biofilm formation in MRSA isolates by suppressing the expression of two intercellular adhesion molecules (icaA and icaD genes)73. Omer et al. reported that in resistant isoaltes of Klebsiella oxytoca sylibin and curcumin decreased the expression fimA gene mediated adhesion and biofilm formation74. Shang et al. showed that Trp-Containing Antibacterial Peptides can inhibited biofilm formation with downregulating pslA, pelA and algD extracellular polysaccharide genes75. The Psl polysaccharide in P. aeruginosa is encoded by a 15-geneoperon (pslA-pslO) and is essential for promoting cell-surface and cell-to-cell adhesion, playing a critical role in the initiation and maintenance of biofilm structure76. In our study, the combination of Fe3O4@SiPr@Silibinin with ciprofloxacin significantly reduced biofilm formation and bacterial adherence in drug-resistant -isolated, at least in part due to the downregulation of the pslA gene.

The overexpression of efflux pumps, as a key mechanism of multidrug resistance77, enhances the active extrusion of antibiotics from the intracellular to the extracellular environment, thereby reducing their intracellular concentration and therapeutic efficacy. Thus, suppressing these pumps (efflux pump inhibitors)78 can be an therapieutic strategy over to drug resistance. Efflux pump inhibitors (EPI) increase susceptibility to the antibiotics in antibiotic resistant isolates79. Adabi et al. demonstrated that carbonyl cyanide-m-chlorophenylhydrazone (CCCP) as an EPI inhanced ciprofloxacin susceptibility80. Shang et al. reported that tryptophan-containing peptides acted synergistically with piperacillin and ceftazidime against multidrug-resistant P. aeruginosa, by downregulating efflux pump related genes mexA, oprM and mexX, thereby inhibiting the bacterial efflux pump system81. According to Rahbar Takrami et al.., curcumin encapsulated in nanoparticles in combination with ciprofloxacin, markedly decreased the expression of efflux pump genes (mexX and oprM) in comparison with ciprofloxacin alone82. Sharma et al. demonstrated that silibinin act as an EPI with targeting efflux pump norA gene78. In our investigation, Fe3O4@SiPr@Silibinin was found to enhance the susceptibility of ciprofloxacin-resistant P. aeruginosa isolates by downregulating the mexA and mexX efflux pump genes. This downregulation likely increases intracellular drug accumulation, thereby enhancing ciprofloxacin efficacy and promoting bacterial cell death. Our findings also suggest that silibinin may function as an efflux pump inhibitor (EPI), potentially through modulation of efflux pump-related gene expression.

Silibinin is known as hepatoprotective, anti-oxidant, anti-cancer and anti-inflamatory agent83. Me et al. showed that silymarin-functionalized selenium nanoparticles had cytotoxic effect against AGS cancer cells without demonstrating toxicity on normal cells84. While, Namvar et al. showed that magnetic iron oxide nanoparticles can demonstrated low cytotoxicity effects on normal cells85. Habibi et al. reported that Fe3O4 can have cytotoxic effects at high concentration (1802 µg/mL) on normal cells86. Thus, our results confirmed that silibinin can non-toxic on normal cells, while, magnetic nanoparticles can have low cytotoxic effects on normal cells.

Silibinin as an polyphenolic flavonolignan have anticancer activities on different caner cells87. Varghese et al. demonstrated that silibinin (IC50 = 50 µM) inhibited cell growth, induced apoptosis, and arrested the cell cycle at the G1 and G2/M phases in HepG2 and Hep3B liver cancer cells88. Ray, et al. emphasize that silibinin can inhibit cancer cell growth, induce apoptosis, and be considered as a promising treatment for cancers by inhibiting various signaling pathways such as PI3K/Akt, Wnt/β-catenin, NF-κB, and MAPK2. Khakinezhad Tehrani et al. evaluated the effects of silybin encapsulated in polymersomes on pancreatic cancer cells, reporting significant apoptotic and anti-proliferative activity.,which was characterized by increased expression of the Bax, Caspase−9, and P53 genes, along with decreased expression of the anti-apoptotic Bcl-2 gene89. Similarly, Sameri et al. reorted that silibinin with IC50 = 50 µmol/L upregulated the expression of apoptotic genes (Bax and Caspase−3) and autophagy related genes (ATG5, ATG7, and BECN1) in CT26 colorectal cancer cells90. Flow cytometry and Q-RT-PCR analysis in this study suggested Fe3O4@SiPr@Silibinin could inbited HepG2 cancer cells with induction of apoptosis partly with upregulation of P53 gene and down regulation of anti-apoptotic gene Bcl-2.

Future studies should focus on evaluating the pharmacokinetic and biodistribution profiles of Fe₃O₄@SiPr@Silibinin nanocomposites in in vivo models to better understand their circulation time, tissue accumulation, and metabolic fate. Additionally, incorporating other therapeutic agents such as antibiotics, chemotherapeutics, or efflux pump inhibitors into the nanocomposite platform may further enhance its multifunctionality and therapeutic index. Such multi-drug loading strategies could allow for synergistic actions against resistant infections and tumors while enabling personalized medicine approaches tailored to patient-specific resistance patterns and tumor profiles.

Our study revealed that Fe3O4@SiPr magnetic nanocomposites (MNCs), functionalized with silibinin, with sized between 40 and 80 nm, exhibited high thermal stability (up to 600 °C). The synergistic antibacterial effects of Fe3O4@SiPr@Silibinin combined with ciprofloxacin were demonstrated via checkerboard dilution assays, showing a significant reduction in biofilm formation compared to ciprofloxacin alone. Inhibition of P. aeruginosa growth was observed within 2 to 8 h of treatment. Furthermore, the downregulation of efflux pump genes (mexX, mexA) and the biofilm- associated pslA gene indicated enhanced intracellular retention of ciprofloxacin, thereby improving antibacterial efficacy. In addition to its antimicrobial activity, Fe3O4@SiPr@Silibinin MNCs exhibited cytotoxic effects against HepG2 liver cancer cells, with an IC50 value of 35.79 µg/mL. Apoptotic induction was confirmed by flow cytometry, as well as by the upregulation of the tumor suppressor gene p53 and downregulation of the anti-apoptotic Bcl-2 gene.These findings suggest that Fe3O4@SiPr MNCs can serve as stable, biodegradable nanocarriers for the targeted delivery of silibinin and other poorly soluble therapeutic agents to both pathogenic bacteria and cancer cells.

The data supporting this study are available upon request from Mahdi Shahriarinour (Email: [email protected]).

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Department of Biology, Ra.C., Islamic Azad University, Rasht, P.O. Box: 3516-41335, Iran

Sanaz Borji, Mahdi Shahriarinour & Najmeh Ranji

Department of Chemistry, Ra.C., Islamic Azad University, Rasht, Iran

Shahab Shariati & Mohammad Nikpassand

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M. S, S. S., N. R., and M. N. contributed to the conception and design of this article. S. B,, M. S., S. S., N. R., and M. N., contributed analyzed and interpreted the data. M. S., S. S., and N. R., were involved in statistical analysis. S. B., M. S., N. R., and M. N. contributed to writing and critically revised this article. M. S., S. S., N. R., and M. N. gave final approval for this article. All authors agree to be accountable for all aspects of the work.

Correspondence to Mahdi Shahriarinour.

The authors declare no competing interests.

This study was conducted in full accordance with the guidelines and recommendations of the ethics committee. The study protocol was reviewed and approved by the Ethics Committee of Human Experiments at the Rasht Branch of Islamic Azad University, Rasht, Iran (Approval Code: IR.IAU.RASHT.REC.1401.040).

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Borji, S., Shahriarinour, M., Shariati, S. et al. Enhanced therapeutic efficacy of silibinin loaded silica coated magnetic nanocomposites against Pseudomonas aeruginosa in Combination with Ciprofloxacin and HepG2 cancer cells. Sci Rep 15, 21498 (2025). https://doi.org/10.1038/s41598-025-07529-x

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Received: 03 November 2024

Accepted: 16 June 2025

Published: 01 July 2025

DOI: https://doi.org/10.1038/s41598-025-07529-x

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