In vitro response of retinal pigment epithelial cells exposed to chitosan materials prepared with different cross-linkers

Abstract

The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S(N)2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

Keywords: chitosan; cross-linking; cytocompatibility; genipin; glutaraldehyde; retinal pigment epithelial cells.

Figure 1.

Cross-linking reaction scheme of chitosan with (A) genipin (GP), and (B) glutaraldehyde (GTA).

Figure 2.

Cross-linking index of GP and GTA treated chitosan membranes as a function of cross-linking time. An asterisk indicates statistically significant differences (*p < 0.05; n = 5) for the mean value of cross-linking index compared with the value at the previous time point.

Figure 3.

ATR-FTIR spectra of various chitosan membranes.

Figure 4.

Cell viability of ARPE-19 cultures was determined by staining with Live/Dead Viability/Cytotoxicity Kit in which the live cells fluoresce green and the dead cells fluoresce red. Green (A, C, E) and red (B, D, F) fluorescence images of cells after exposure to 5 mg of different types of chitosan membranes (A, B) Chi, (C, D) GP-chi, and (E, F) GTA-chi for 3 days at 37 °C. Scale bars indicate 100 μm.

Figure 5.

Mean percentage of live cells in the ARPE-19 cultures exposed to various chitosan membranes (5 mg) as measured by the Live/Dead assay. An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls (without materials).

Figure 6.

Gene expression of IL-6 in ARPE-19 cells incubated with various chitosan membranes (5 mg) for 3 days, measured by real-time RT-PCR. Normalization was done using GAPDH. Data in the experimental groups are percentages relative to that of control groups (without materials). An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls.

Figure 7.

Level of IL-6 released from ARPE-19 cell cultures after incubation with various chitosan membranes (5 mg) for 3 days. An asterisk indicates statistically significant differences (*p < 0.05; n = 4) as compared to controls (without materials).

Figure 8.

Apoptotic cells labeled with TUNEL assay in the ARPE-19 cultures. Fluorescence micrographs of control cells (without materials) (A), and cells after exposure to 5 mg of different types of chitosan membranes (B) Chi, (C) GP-chi, and (D) GTA-chi for 24 h at 37 °C. Blue fluorescence is DAPI nuclei staining. Green fluorescence is TUNEL-positive nuclei staining. Scale bars indicate 30 μm.

Figure 9.

Apoptotic index of ARPE-19 cells exposed to various indicated chitosan membranes (5 mg) as determined by the TUNEL assay. An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls (without materials).