Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers

Abstract

Background and methods: Chondroitin sulfate-chitosan (ChS-CS) nanoparticles and positively and negatively charged fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA)-loaded ChS-CS nanoparticles were prepared and characterized. The properties of ChS-CS nanoparticles, including cellular uptake, cytotoxicity, and transepithelial transport, as well as findings on field emission-scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were evaluated in human epithelial colorectal adenocarcinoma (Caco-2) fibroblasts. ChS-CS nanoparticles with a mean particle size of 250 nm and zeta potentials ranging from -30 to +18 mV were prepared using an ionic gelation method.

Results: Standard cell viability assays demonstrated that cells incubated with ChS-CS and FITC-BSA-loaded ChS-CS nanoparticles remained more than 95% viable at particle concentrations up to 0.1 mg/mL. Endocytosis of nanoparticles was confirmed by confocal laser scanning microscopy and measured by flow cytometry. Ex vivo transepithelial transport studies using Caco-2 cells indicated that the nanoparticles were effectively transported into Caco-2 cells via endocytosis. The uptake of positively charged FITC-BSA-loaded ChS-CS nanoparticles across the epithelial membrane was more efficient than that of the negatively charged nanoparticles.

Conclusion: The ChS-CS nanoparticles fabricated in this study were effectively endocytosed by Caco-2 fibroblasts without significant cytotoxicity at high nanoparticle concentrations. ChS-CS nanoparticles represent a potential novel delivery system for the transport of hydrophilic macromolecules.

Keywords: cell uptake; chitosan; chondroitin sulfate; cytotoxicity; nanoparticles.

Figure 1

Influence of log(M_ChS/M_CS) on the particle size and the zeta potential of FITC-BSA-loaded ChS-CS nanoparticles. (◆) Particle size, (■) zeta potential (A), particle size distribution of the formulation with a ChS/CS volume ratio of 2.8/4 (B) and 4/1 (C). Initial concentrations were ChS (4 mg/mL) and CS (4 mg/mL). Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.

Figure 2

Images of FE-SEM (I) and TEM (II) micrographs of ChS-CS nanoparticles and FITC-BSA-loaded ChS-CS nanoparticles. (A) Blank ChS-CS nanoparticles (+). (B) FITC-BSA-loaded ChS-CS nanoparticles (+). (C) Blank ChS-CS nanoparticles (−). (D) FITC-BSA-loaded ChS-CS nanoparticles (−). Note: Bar 100 nm. Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate; FE-SEM, field emission scanning electron microscopy; TEM, transmission electron microscopy.

Figure 3

Cytotoxicity profile of positively (blue) and negatively (red) charged ChSCS nanoparticles, and of positively (green) and negatively (purple) charged FITC-BSA loaded ChS-CS nanoparticles after 72 hours of incubation with Caco-2 cells as determined by WST-1 assay. Notes: Percent viability of fibroblasts is expressed relative to control cells (results are represented as the mean ± standard deviation, n = 6). Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.

Figure 4

Uptake of positively charged nanoformulated FITC-BSA-loaded ChS-CS nanoparticles by Caco-2 cells following phagocyte cocultivation as described in the Methods section; confocal laser scanning imaging of Caco-2 incubated with FITC-BSA-loaded ChS-CS nanoparticles for 0, 1, 2, 4, and 6 hours (from top to bottom). (A) Cell nucleus stained with TO-PRO-3 iodide (blue part), (B) FITC-BSA-loaded ChS-CS nanoparticles (green part), (C) cell membranes stained with BoDiPY-phalloidin (red part), and (D) merged A, B, and C pictures. Note: Bar 20 μm. Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.

Figure 5

Time course for uptake of FITC-BSA-loaded ChS-CS nanoparticles by Caco-2 cells quantified by fluorescence-activated cell sorting analysis using flow cytometry at hours 0, 0.5, 1, 1.5, 2, 4, and 6. Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.

Figure 6

Fluorescent intensity according to flow cytometry data, elucidating the effect of charge modifications on the internalization efficiency of positively charged FITC-BSA-loaded ChS-CS nanoparticles ( ), negatively charged FITC-BSA ChS-CS nanoparticles ( ), and FITC-BSA solution (0.1 mL; 0.1 mg/mL) ( ) on Caco-2 cells at varied time incubations. Note: Results are represented as the mean ± standard deviation, n = 6. Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.

Figure 7

Confocal laser scanning imaging of Transwell membrane of Caco-2 incubated with positively charged FITC-BSA-loaded ChS-CS nanoparticles at 37°C for hours 0, 1, 2, 4, and 6 (from top to bottom) as described in the Methods section. (A) Cell nucleus stained with TO-PRO-3 iodide (blue part), (B) FITC-BSA-loaded ChS-CS nanoparticles (green part), (C) cell membrane stained with BoDiPY-phalloidin (red part), and (D) merged A, B, and C pictures. Note: Scale bar 20 μm. Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.

Figure 8

Time courses for the Caco-2 cell monolayer transepithelial transport efficiency study. The fluorescent intensity of FITC-BSA data; positive charge of FITC-BSA-loaded ChS-CS nanoparticles ( ); negative charge of FITC-BSA-loaded ChS-CS nanoparticles ( ), and FITC-BSA solution (0.1 mL; 0.1 mg/mL) ( ). Notes: All values are shown as the mean ± standard deviation; n = 6. Abbreviations: BSA, bovine serum albumin; ChS, chondroitin 4-sulfate sodium salt; CS, chitosan; FITC, fluorescein isothiocyanate.