Mesoporous silica nanoparticles functionalized with folic acid and zinc for enhanced targeted delivery of daunorubicin to breast cancer cells

Figures

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  Fig. 1. Physicochemical characterization of mesoporous silica nanoparticles. (A) Transmission electron microscopy (TEM) image of Zn/MSN nanoparticles showing spherical morphology and uniform size distribution (scale bar = 50 nm). Particle size distribution analysis using ImageJ software indicated that the average particle size is 99 nm. (B,C) Dynamic light scattering (DLS) size distribution results of Zn/MSN, DAN-Zn/MSN, and FA-DAN-Zn/MSN formulations showing hydrodynamic diameter and polydispersity index (PDI). (D) Zeta potential values of MSN, DAN-Zn/MSN, and FA-DAN-Zn/MSN indicate successful stepwise surface functionalization.

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  Fig. 2. pH-responsive drug release profile of daunorubicin (DAN). Cumulative release percentage of DAN from free drug solution and FA-DAN-Zn/MSN at pH 5.4, 6.5, and 7.4 over time. Release studies were performed using a dialysis membrane (MWCO = 12 kDa). Drug concentration was 300 µg/mL and nanoparticle concentration was 1000 µg/mL. Data are expressed as mean ± SD (n = 3).

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  Fig. 3. Stability evaluation of DAN-Zn/MSN and FA-DAN-Zn/MSN formulations during storage. (A) Size stability evaluation of DAN-Zn/MSN, (B) Size stability evaluation of FA-DAN-Zn/MSN, (C) PDI stability evaluation of DAN-Zn/MSN, (D) PDI stability evaluation of FA-DAN-Zn/MSN, (E) EE (%) stability evaluation of DAN-Zn/MSN, (F) EE (%) stability evaluation of FA-DAN-Zn/MSN after 1 months of storage at 4 ± 2 and 25 ± 2 °C; EE for FA-DAN-Zn/MSN is = 86.72 ± 2.12% (Loading capacity (LC) = 30.24 ± 0.52), and for DAN-Zn/MSN is = 69.24 ± 1.85% (Loading capacity (LC) = 25.71 ± 0.50); Data are represented as mean ± SD and n = 3; ***p < 0.001, **p < 0.01, *p < 0.05.

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  Fig. 4. Cytotoxicity evaluation of DAN-based formulations in breast cell lines. Viability of (A) MCF-7 (B) MCF-10A cell line treated with various concentrations of DAN, Zn/MSN, DAN-Zn/MSN, and FA-DAN-Zn/MSN in comparison with control (0, 0.25, 0.5, 1, 2, and 4 µM, Drug concentration in free form or loaded in prepared formulations). A control sample is MCF-7 cells without incubation with any drug or nanomaterials. The data were examined utilizing one-way ANOVA, followed by Tukey’s post hoc test to determine statistical significance among groups. Standard deviations were obtained from triplicate wells in each biological replicate, and mean values were calculated over three replicates. (C) Viability of MCF-7 cell line treated with various concentrations of MSN, Zn/MSN, DAN/MSN, and DAN-Zn/MSN in comparison with control (0, 0.25, 0.5, 1, 2, and 4 µM, Drug concentration in free form or loaded in prepared formulations). (D) Viability of MCF-7 cell line treated with various concentrations of FA-DAN/MSN and FA-DAN-Zn/MSN in comparison with control (0, 0.25, 0.5, 1, 2, and 4 µM, Drug concentration in free form or loaded in prepared formulations). Data represent mean ± SD (n = 3). The statistical…

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  Fig. 5. Relative gene expression analysis in MCF-7 cells following treatment at IC50 concentrations. Variations in the expression levels of (A) Caspase-9, (B) BAX, (C) BCL-2, (D) MMP-2, and (E) MMP-9 in the MCF-7 cancer cell line following treatment with Zn/MSN, DAN, DAN-Zn/MSN, and FA-DAN-Zn/MSN compared to control. Data are mean ± SD (n = 3). *: p < 0.05, **: p < 0.01 and ***: p < 0.001.

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  Fig. 6. Anti-metastatic and oxidative stress effects of DAN-based formulations in MCF-7 cells. (A) Wound healing (scratch) assay showing percentage migration rate after 48 h of treatment. (B) Intracellular reactive oxygen species (ROS) levels were measured using the DCF fluorescence assay. Data are presented as mean ± SD (n = 3). Statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 7. Caspase-9 activity in MCF-7 cells following treatment. Relative Caspase activity (fold change compared to control) in cells treated with Zn/MSN, DAN, DAN-Zn/MSN, and FA-DAN-Zn/MSN. Data are expressed as mean ± SD (n = 3). Statistical significance: *p < 0.05, ***p < 0.001.