Enhanced Cellular Uptake in an Electrostatically Interacting Fucoidan-L-Arginine Fiber Complex

Abstract

Fucoidan is an abundant marine sulfated polysaccharide extracted from the cell wall of brown macroalgae (seaweed). Recently, fucoidan has been highly involved in various industrial applications, such as pharmaceuticals, biomedicals, cosmetics, and food. However, the presence of a sulfate group (negative surface charge) in the fucoidan structure limits its potential and biological activity for use in biomedical applications during cellular uptake. Thus, we aimed to improve the uptake of fucoidan by using an L-arginine uptake enhancer within an in vitro study. A Fucoidan-L-Arginine (Fuc-L-Arg) fiber complex was prepared via α-helical electrostatic interactions using a freeze-drying technique and confirmed using field-emission scanning electron microscopy, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. In addition, fucoidan was conjugated with cyanine 3 (Cy3) dye to track its cellular uptake. Furthermore, the results of Fuc-L-Arg (1:1, 1:2.5) complexes revealed biocompatibility >80% at various concentrations (5, 10, 25, 50, 100 µg/mL). Owing to the higher internalization of the Fuc-L-Arg (1:5) complex, it exhibited <80% biocompatibility at higher concentrations (25, 50, 100 µg/mL) of the complex. In addition, improved cellular internalization of Fuc-L-Arg complexes (1:5) in HeLa cells have been proved via flow cytometry quantitative analysis. Hence, we highlight that the Fuc-L-Arg (1:5) fiber complex can act as an excellent biocomplex to exhibit potential bioactivities, such as targeting cancers, as fucoidan shows higher permeability in HeLa cells.

Keywords: L-arginine; cyanine 3 dye; fucoidan.

Figure 1

(a) UV absorbance of fucoidan, L-arginine, and their complexes; (b) FT-IR characterization of fucoidan, L-arginine, and their complexes.

Figure 2

Schematic representation of the electrostatic interaction between fucoidan and L-arginine.

Figure 3

FE-SEM morphology: (a) pristine fucoidan, (b) pristine L-arginine, (c) Fuc-L-Arg (1:1) complex, (d) Fuc-L-Arg (1:2.5) complex, and (e) Fuc-L-Arg (1:5) complex.

Figure 4

(a) UV absorbances of the Cy3 NHS ester and Fuc-Cy3 dyes, (b) two-dimensional fluorescence intensities of the Cy3 NHS ester and Fuc-Cy3 dyes, (c) three-dimensional fluorescence intensity of the Cy3 NHS ester dye, (d) three-dimensional fluorescence intensity of the Fuc-Cy3 dye, (e) FT-IR studies of the Fuc-Cy3 fiber complex.

Figure 5

Schematic representation of the conjugation of Cy3 dye with Fucoidan (Fuc-Cy 3).

Figure 6

Fluorescence intensities of the Fuc-Cy3 dye and Fuc-Cy3-L-Arg (1:1, 1:2.5, and 1:5) complexes.

Figure 7

Cell viabilities of pristine fucoidan, pristine L-arginine, and the Fuc-L-Arg complexes (1:1, 1:2.5, and 1:5) in HeLa cells. Where * represents p < 0.05, ** p < 0.01.

Figure 8

Cellular uptake of (a) control HeLa cells, (b) Cy3-treated HeLa cells, (c) Fuc-Cy3-treated HeLa cells, (d) Fuc-Cy3-L-Arg (1:1)-treated HeLa cells, (e) Fuc-Cy3-L-Arg (1:2.5)-treated HeLa cells, and (f) Fuc-Cy3-L-Arg (1:5)-treated HeLa cells.

Figure 9

Quantitative flow cytometry analysis of (a,b) fluorescence intensity of control HeLa cells (without treatment), Cy3 dye-treated HeLa cells, Fuc-Cy3 dye-treated HeLa cells, Fuc-L-Arg (1:1)-treated HeLa cells, Fuc-L-Arg (1:2.5)-treated HeLa cells, and Fuc-L-Arg (1:5)-treated HeLa cells. Where * represents p < 0.05 and *** represents p < 0.001.