Downregulation of VEGF mRNA expression by triamcinolone acetonide acetate-loaded chitosan derivative nanoparticles in human retinal pigment epithelial cells

Abstract

Background: The purpose of this study was to investigate the downregulation of mRNA expression of vascular endothelial growth factor (VEGF) by triamcinolone acetonide acetate (TAA)-loaded chitosan nanoparticles in human retinal pigment epithelial cells.

Methods: TAA-loaded deoxycholic acid-modified chitosan (TAA/DA-Chit) nanoparticles were prepared via a self-assembly mechanism, and their morphology and zeta potential were examined by transmission electron microscopy and zeta potential analysis, respectively. DA-Chit and TAA/DA-Chit nanoparticle toxicity was evaluated using a Cell Counting Kit-8 assay. The efficiency of cellular uptake was determined using fluorescein isothiocyanate-labeled DA-Chit nanoparticles, in place of TAA/DA-Chit nanoparticles, assessed by both inverted fluorescence microscopy and flow cytometry. Downregulation of VEGF mRNA expression by TAA/DA-Chit nanoparticles was further investigated by real-time reverse transcription polymerase chain reaction (RT-PCR) assay of the treated human retinal pigment epithelial cells.

Results: TAA/DA-Chit nanoparticles were prepared with a TAA-loading capacity in the range of 12%-82%, which increased the water solubility of TAA from 0.3 mg/mL to 2.1 mg/mL. These nanoparticles showed oblate shapes 100-550 nm in size in transmission electron microscopic images and had positive zeta potentials. The Cell Counting Kit-8 assay indicated that the DA-Chit and TAA/DA-Chit nanoparticles had no toxicity and low toxicity, respectively, to human retinal pigment epithelial cells. Fluorescein isothiocyanate-labeled DA-Chit nanoparticle uptake by human retinal pigment epithelial cells was confirmed by inverted fluorescence microscopy and flow cytometry. Real-time RT-PCR assay showed that the VEGF mRNA level decreased after incubation of human retinal pigment epithelial cells with TAA/DA-Chit nanoparticles.

Conclusion: TAA/DA-Chit nanoparticles had a downregulating effect on VEGF mRNA expression in human retinal pigment epithelial cells and low cytotoxicity, which might be beneficial characteristics for the development of future treatment for diabetic retinopathy.

Keywords: chitosan; human retinal pigment epithelial cells; mRNA; nanoparticle; triamcinolone acetonide acetate; vascular endothelial growth factor.

Figure 1

Structural characterization of DA-Chit. (A) Fourier transform infrared spectrum and (B) ¹H nuclear magnetic resonance spectrum. Abbreviation: DA-Chit, deoxycholic acid-modified chitosan.

Figure 2

Water solubility of TAA and zeta potential of TAA/DA-Chit nanoparticles at various TAA-loading capacities (n = 3, mean ± standard deviation). Abbreviations: TAA, triamcinolone acetonide acetate; DA-Chit, deoxycholic acid-modified chitosan.

Figure 3

Transmission electron microscopy images of (A) deoxycholic acid-modified chitosan nanoparticles and triamcinolone acetonide acetate/deoxycholic acid-modified chitosan nanoparticles with different triamcinolone acetonide acetate-loading capacities, ie, (B) 12%, (C) 29%, and (D) 82%.

Figure 4

Morphology of cultured hRPE cells. (A) primary cultured hRPE cells, (B) Giemsa staining hRPE cells, (C) immunocytochemical staining hRPE cells, and (D) hRPE cells lacking immunohistochemical staining in control preparation (×100). Abbreviation: hRPE, human retinal pigment epithelial.

Figure 5

Cytotoxicity assay for DA-Chit at various concentrations compared with control hRPE cells (after a 24-hour incubation period, n = 5, mean ± standard deviation, *P < 0.05, statistically significant difference versus controls). Abbreviations: DA-Chit, deoxycholic acid-modified chitosan; hRPE, human retinal pigment epithelial.

Figure 6

Cytotoxicity assay for DA-Chit, TAA, and TAA/DA-Chit nanoparticles compared with controls in human retinal pigment epithelial cells (at different incubation times and TAA concentrations of 0.1 mg/mL for TAA and TAA/DA-Chit nanoparticles, ^▲P < 0.05, statistically significant difference versus controls, *P < 0.05, statistically significant difference versus the TAA group, n = 5, mean ± standard deviation). Abbreviations: TAA, triamcinolone acetonide acetate; DA-Chit, deoxycholic acid-modified chitosan.

Figure 7

Typical fluorescence images of (I) hRPE cells incubated with FITC/DA-Chit nanoparticles for 24 hours at concentrations of (A) 0.3 mg/mL, (B) 0.5 mg/mL, and (C) 1.0 mg/mL; (II) hRPE cells incubated with FITC/DA-Chit nanoparticles at 1.0 mg/mL for (A) 3 hours, (B) 6 hours, and (C) 24 hours. (III) hRPE cells further incubated for different times following removal of FITC/DA-Chit nanoparticles from the culture medium, ie, (A) one day, (B) 3 days, and (C) 5 days, after incubation with 1.0 mg/mL of FITC/DA-Chit nanoparticles for 24 hours (scale bar 50 μm, 1 fluorescence field images, 2 bright field images). Abbreviations: hRPE, human retinal pigment epithelial; FITC/DA-Chit, fluorescein isothiocyanate-labeled deoxycholic acid-modified chitosan.

Figure 8

Cellular uptake efficiency in response to FITC/DA-Chit nanoparticles by flow cytometry analysis. (A) hRPE cells incubated with FITC/DA-Chit nanoparticles for 24 hours at concentrations of (a) 0.3 mg/mL, (b) 0.5 mg/mL, and (c) 1.0 mg/mL. (B) hRPE cells incubated with FITC/DA-Chit nanoparticles at 1.0 mg/mL for (a) 3 hours, (b) 6 hours, and (c) 12 hours. (C) Proportions of hRPE cells taking up FITC/DA-Chit versus nanoparticle concentrations. (D) Proportions of hRPE cells taking up FITC/DA-Chit nanoparticles versus incubation time. Abbreviations: hRPE, human retinal pigment epithelial; FITC/DA-Chit, fluorescein isothiocyanate-labeled deoxycholic acid-modified chitosan; SSC, side scatter.

Figure 9

VEGF₁₆₅ expression in hRPE cells assessed by real-time reverse transcription polymerase chain reaction (*P < 0.01, statistically significant difference versus controls; ^▲P < 0.05, statistically significant difference versus the TAA-treated group). Abbreviations: hRPE, human retinal pigment epithelial; TAA, triamcinolone acetonide acetate; DA-Chit, deoxycholic acid-modified chitosan; VEGF, vascular endothelial growth factor.