Curcumin-Loaded Polysaccharide Nanoparticles Enhance Aqueous Dispersibility and In Vitro Cytotoxicity in Breast Cancer Cell Lines

Abstract

Curcumin (CUR) is a natural compound with anticancer potential; however, its poor water solubility, instability, and rapid degradation limit its therapeutic use. To address these issues, we developed CUR-loaded nanoparticles (CUR-NPs) based on chitosan, hyaluronic acid, and alginate, the TEM-measured diameter of 29.3 ± 9.0 nm. Dynamic light scattering (DLS) analysis further confirmed good aqueous dispersibility, revealing hydrodynamic diameters of 39.8 ± 7.1 nm for UL-NPs and 46.1 ± 18.1 nm for CUR-NPs. Cytotoxicity assays revealed significant anticancer activity in both MCF-7 and MDA-MB-231 cells, with IC₅₀ values of 17.5 ± 1.9 μg/mL and 39.9 ± 5.4 μg/mL after 72 h, respectively, indicating cell line-dependent sensitivity with MCF-7 cells being more susceptible to CUR-NP treatment. Time-dependent uptake was confirmed using fluorescence imaging and flow cytometry, which demonstrated faster and higher NP uptake by MCF-7 cells than by MDA-MB-231 cells. Collectively, these data support a cell line-dependent cell death response: MCF-7 cells displayed earlier and more pronounced changes consistent with apoptosis, whereas MDA-MB-231 cells showed slower uptake with delayed apoptosis and partial necrosis. Subcellular localization dynamics, particularly perinuclear aggregation, have emerged as determinants of NP-induced cytotoxicity, highlighting the potential for tailoring NP design to specific cellular contexts to improve therapeutic efficacy.

Keywords: alginate; anticancer; breast cancer; chitosan; curcumin; cytotoxicity; hyaluronic acid; nanocarrier; nanoparticle.

Figure 1

Morphological characterization of NPs immediately after preparation using SEM, TEM, and particle size distribution analysis. (a–c) UL-NPs; (d–f) CUR-NPs. All scale bars = 100 nm.

Figure 2

Morphological stability of CUR-NPs during storage. (a) Representative SEM images of NPs after 110 and 195 days of storage. (b) Particle size measurements at 0, 21, 35, 110, and 195 days. No significant difference was observed compared with day 0. Data are presented as mean ± SD (n = 3). Scale bars = 100 nm. Red dots represent the mean particle size measured at each time point (0, 21, 35, 110, and 195 days), and black error bars indicate the standard deviation.

Figure 3

(a) FTIR spectra of free CUR, UL-NPs, and CUR-NPs. (b) The enlarged region of the characteristic peaks (1400–1800 cm⁻¹) is shown for better visualization. Vertical dotted lines indicate the characteristic peaks of curcumin, and the black frame highlights the magnified region of these characteristic peaks.

Figure 4

EE% and DL% of CUR-NPs at different CUR concentrations. The results indicate that the prepared NPs exhibit consistently high encapsulation efficiency (approximately 90–100%) across all tested conditions, while drug loading gradually increases (from ~5% to ~12%) with higher CUR concentrations, demonstrating excellent encapsulation ability and concentration-dependent drug-loading capacity. Data are presented as mean ± SD (n = 3). Red dots represent the mean encapsulation efficiency (EE%) at each curcumin concentration. Blue dots represent the mean drug loading (DL%) at each curcumin concentration. Black lines indicate the corresponding error bars.

Figure 5

Cytotoxicity of free CUR and CUR1.4-NPs in breast cell lines. Cell viability of (a) MCF-10A, (c) MDA-MB-231, and (e) MCF-7 after 24–72 h of treatment with CUR + DI water or CUR1.4-NPs. Comparisons at 208 µg/mL are shown in (b,d,f). Data are presented as mean ± SD (n = 6). Statistical analysis was performed using Tukey’s post hoc test. * p < 0.05 and ** p < 0.001 vs. CUR + DI water group at the same time point. Black lines indicate the corresponding error bars.

Figure 6

Fluorescence microscopy images of MDA-MB-231 incubated with Fl-CUR-NPs at 37 °C for 0.5, 1, 3, 6, and 24 h. Green fluorescence represents captured Fl-CUR-NPs and blue fluorescence indicates nuclei stained with Hoechst 33342. Red arrows indicate regions showing notable cellular morphological changes. Scale bars = 20 μm.

Figure 7

Fluorescence microscopy images of MCF-7 incubated with Fl-CUR-NPs at 37 °C for 0.5, 1, 3, 6, and 24 h. Green fluorescence indicates the captured Fl-CUR-NPs and blue fluorescence indicates nuclei stained with Hoechst 33342. Scale bars = 20 μm. Red arrows indicate pronounced organelle extrusion, while yellow arrows highlight partial Fl-CUR-NP release.

Figure 8

Cellular uptake of Fl-CUR-NPs by MDA-MB-231 analyzed using flow cytometry. (a) Untreated control cells, (b) cells treated with non-fluorescent CUR-NPs, and (c–e) cells treated with Fl-CUR-NPs for 0.5, 3, and 6 h, respectively. (f) Quantitative analysis of mean fluorescence intensity at each time point, reflecting the relative amount of NPs incorporated by each cell. The black line in panels (a–e) represents the percentage of fluorescence intensity, whereas the black line in panel (f) indicates the error bars.

Figure 9

Cellular uptake of Fl-CUR-NPs by MCF-7 analyzed using flow cytometry. (a) Untreated control cells, (b) cells treated with non-fluorescent CUR-NPs, and (c–g) cells treated with Fl-CUR-NPs for 0.5, 1, 3, 6, and 24 h, respectively. (h) Quantitative analysis of mean fluorescence intensity at each time point, reflecting the relative amount of NPs incorporated by each cell. The black line in panels (a–g) represents the percentage of fluorescence intensity, whereas the black line in panel (h) indicates the error bars.