Cellular Uptake and Cytotoxicity of β-Lactoglobulin Nanoparticles: The Effects of Particle Size and Surface Charge

Abstract

It is necessary to understand the cellular uptake and cytotoxicity of food-grade delivery systems, such as β-lactoglobulin (β-lg) nanoparticles, for the application of bioactive compounds to functional foods. The objectives of this study were to investigate the relationships between the physicochemical properties of β-lg nanoparticles, such as particle size and zeta-potential value, and their cellular uptakes and cytotoxicity in Caco-2 cells. Physicochemical properties of β-lg nanoparticles were evaluated using particle size analyzer. Flow cytometry and confocal laser scanning microscopy were used to investigate cellular uptake and cytotoxicity of β-lg nanoparticles. The β-lg nanoparticles with various particle sizes (98 to 192 nm) and zeta-potential values (-14.8 to -17.6 mV) were successfully formed. A decrease in heating temperature from 70°C to 60°C resulted in a decrease in the particle size and an increase in the zeta-potential value of β-lg nanoparticles. Non-cytotoxicity was observed in Caco-2 cells treated with β-lg nanoparticles. There was an increase in cellular uptake of β-lg nanoparticles with a decrease in particle size and an increase in zeta-potential value. Cellular uptake β-lg nanoparticles was negatively correlated with particle size and positively correlated with zeta-potential value. Therefore, these results suggest that the particle size and zeta-potential value of β-lg nanoparticles play an important role in the cellular uptake. The β-lg nanoparticles can be used as a delivery system in foods due to its high cellular uptake and non-cytotoxicity.

Keywords: Cellular Uptake; Cytotoxicity; Nanoparticle; Particle Size; Zeta-potential; β-Lactoglobulin.

Figure 1

Particle size of β-lactoglobulin (β-lg) and fluorescein isothiocyanate-conjugated β-lg nanoparticles prepared at various heating temperature (60°C, 65°C, and 70°C). NPs, nanoparticles; HT, heating temperature; ns, not significant (p>0.05).

Figure 2

Zeta-potential of fluorescein isothiocyanate-conjugated β-lactoglobulin nanoparticles prepared at various heating temperature (60°C, 65°C, and 70°C). Different letters on a column indicate significant differences (p<0.05).

Figure 3

Viability of Caco-2 cells treated with β-lactoglobulin (β-lg) nanoparticles prepared at various heating temperature (60°C, 65°C, and 70°C). The β-lg nanoparticles (250 μg/mL) were added to Caco-2 cells and incubated at 37°C for 2 h. Different letters on a column indicate significant differences (p<0.05).

Figure 4

Effects of nanoparticle concentration and incubation time on the cellular uptake of β-lactoglobulin (β-lg) nanoparticles in Caco-2 cells. (A) Various concentration of fluorescein isothiocyanate (FITC)-conjugated β-lg nanoparticles (100, 250, and 500 μg/mL) were added to Caco-2 cells and incubated at 37°C for 2 h. (B) FITC-conjugated β-lg nanoparticles (250 μg/mL) were added to Caco-2 cells and incubated at 37°C for 0, 0.5, 1, 2, 3, 4 h. Different letters on a column indicate significant differences (p<0.05).

Figure 5

Effect of heating temperature on the cellular uptake of fluorescein isothiocyanate (FITC)-conjugated β-lactoglobulin (β-lg) nanoparticles in Caco-2 cells. FITC-conjugated β-lg nanoparticles (250 μg/mL) were added to Caco-2 cells and incubated at 37°C for 2 h. Different letters on a column indicate significant differences (p<0.05).

Figure 6

Confocal laser scanning microscopy images of Caco-2 cells after 2 h incubation at 37°C with fluorescein isothiocyanate (FITC)-conjugated β-lactoglobulin (β-lg) nanoparticles (250 μg/mL) prepared at (A) 60°C, (B) 65°C, and (C) 70°C heating temperatures. To observe the cellular uptake of β-lg nanoparticles, cells were stained with propidium iodide (PI) and FITC-conjugated β-lg nanoparticles were used. Cellular uptake of FITC-conjugated β-lg nanoparticles was imaged by PI channel (left panel), FITC channel (middle panel), and combined PI channel and FITC channel (right panel), respectively.