Polyethylenimine-conjugated gold nanoparticles: Gene transfer potential and low toxicity in the cornea

Abstract

This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNPs) in the human cornea in vitro and rabbit cornea in vivo. PEI2-GNPs with nitrogen-to-phosphorus ratios of up to 180 exhibited significant transgene delivery in the human cornea without altering the viability or phenotype of these cells. Similarly, PEI2-GNPs applied to corneal tissues collected after 12 hours, 72 hours, or 7 days exhibited appreciable gold uptake throughout the rabbit stroma with gradual clearance of GNPs over time. Transmission electron microscopy detected GNPs in the keratocytes and the extracellular matrix of the rabbit corneas. Additionally, slit-lamp biomicroscopy in live animals even 7 days after topical PEI2-GNP application to the cornea detected no inflammation, redness, or edema in rabbit eyes in vivo, with only moderate cell death and immune reactions. These results suggest that PEI2-GNPs are safe for the cornea and can potentially be useful for corneal gene therapy in vivo.

From the clinical editor: This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles in the human cornea in vitro and rabbit cornea in vivo. The results suggest that PEI2-GNPs are safe for the cornea and can potentially be useful for corneal gene therapy in vivo.

Figure 1

Dose-dependent effect of PEI2-GNP on human corneal fibroblast viability. Cultures were exposed to five different concentrations of PEI2-GNP (N/P ratios of 60, 90, 120, 180, and 210) for 1 h, and cellular viability was quantified with a trypan blue assay.

Figure 2

Representative images showing cytotoxicity of PEI2-GNP to the rabbit cornea in vivo evaluated with TUNEL assay (A-F) and CD11b immunocytochemistry. The GNP-untreated (A, E) and GNP-treated (B, F) corneas did not show any statistically significant difference in TUNEL positive cells (red) at 12 h or 7days. However, a significant difference in the TUNEL-positive cells in GNP-untreated (C) and GNP-treated (D) corneas was detected at 72 h. The detection of apoptosis at 12 hours in untreated control and treated corneas was due to the vector-delivery technique, which involves removal of epithelium and is known to cause transient keratocyte apoptosis. The corneal epithelium in rabbit typically regenerates completely in 48-72h. The removal of epithelium is frequently used in eye clinic to treat epithelial defects, and was found useful for defining tissue-selective gene therapy modalities for the cornea. Images G-H show immunological response of cornea to PEI2-GNP application in vivo. The GNP-untreated (G) and PEI2-GNP–treated (H) rabbit corneas subjected to CD11b immunostaining showed moderate CD11b-positive cells (red) in the anterior stroma at 12 h. Less than 3 CD11b-positive cells were detected at 72 h or 7 days (data not shown). Nuclei are stained blue with DAPI. Scale bar denotes 100 μm.

Figure 3

Representative clinical eye examination images performed with slitlamp microscope in PEI2-GNP-treated and untreated (control) rabbit corneas at 12 h and 7 days post application. No opacity, redness or inflammation are seen. PEI2-GNP-treated corneas show mild purple coloration at 12 h confirming GNP uptake and clearing of coloration detected on day 7 suggest no GNP accumulation in the cornea.

Figure 4

Representative images of rabbit corneal sections showing localization and distribution of GNP detected with silver staining. Stained gold nanoparticles (black) were detected throughout the stroma of PEI2-GNP-treated rabbit corneas. Scale bar denotes 20 μm.

Figure 5

Representative transmission electron microscopy images of PEI2-GNP-treated rabbit corneas demonstrating the presence and intracellular trafficking of GNP in keratoctes (A) and extracellular matrix (B). GNP can be seen in the endosomes near the cell surface depicting their uptake by endocytosis. Ruptured endosome represents the release of GNP into the cytoplasm.

Figure 6

Florescence microscopy images of PEI2-GNP: plasmid treated human corneal fibroblast cultures showing delivered GFP expression. The cultures were exposed to mixtures made of PEI2-GNP and plasmid expressing GFP (N/P ratio of 180). Nuclei are stained blue with DAPI; scale bar denotes 100 μm.