Localization of fluorescent gold nanoparticles throughout the eye after topical administration

Abstract

The human eye is a highly intricate sensory organ. When a condition requiring treatment occurs, eyedrops, which represent 90% of all ophthalmic treatments, are most frequently used. However, eyedrops are associated with low bioavailability, with less than 0.02% of therapeutic molecules reaching the anterior chamber. Thus, new delivery systems are required to ensure sufficient drug concentration over time at the target site. Gold nanoparticles are a promising avenue for drug delivery; however, they can be difficult to track in biological systems. Fluorescent gold nanoparticles, which have the same ultrastability and biocompatibility as their nonfluorescent counterpart, could act as an effective imaging tool to study their localization throughout the eye after administration. Thus, this study (1) synthesized and characterized fluorescent gold nanoparticles, (2) validated similar properties between nonfluorescent and fluorescent gold nanoparticles, and (3) determined their localization in the eye after topical application on ex vivo rabbit eyes. The fluorescent gold nanoparticles were synthesized, characterized, and identified in the cornea, iris, lens, and posterior segment of rabbit eyeballs, demonstrating tremendous potential for future drug delivery research.

Keywords: biodistribution; click chemistry; fluorescence imaging; gold nanoparticles; ophthalmology.

SCHEME 1

AuNPs-Nap synthesis. The proportion between the HS-PEG₂₀₀₀-Nap and HS-PEG₂₀₀₀ varies from 1 to 100%. For the experimental details, refer to the description in the Materials and Methods.

Figure 1

Characterization of the fluorescence emission of AuNPs-PEG2000 and AuNPs-PEG2000-Nap using ex vivo human corneas and fluorescence spectroscopy. (A) AuNPs-PEG2000 were deposited on a research-grade human cornea to be used as a control. (B) AuNPs-PEG2000-Nap 1% were placed on a human cornea, where i is the Bowman’s layer, ii is the stroma, and iii is the Descemet’s membrane. (C) AuNPs-PEG2000-Nap 100% were placed on a human cornea. (D) The fluorescence emission spectra of AuNPs-PEG2000-Nap 1 and 100%. Scale bar: 5 μm.

Figure 2

Quantitative analysis of mucins adsorbed on AuNPs. The calibration curve of the mucins is represented by the blue circles. The yellow triangle represents the absorbance coming from the concentration of mucins still free after the mix between the AuNPs (1 mg/mL) with an initial mucin concentration of 150 μg/mL (large blue diamond), and the pink square represents mucins still free after contact with AuNPs-PEG₂₀₀₀-Nap 1% (1 mg/mL). Insert: Histogram of the percentage of adsorbed mucins on AuNPs-PEG₂₀₀₀ (AuNPs 1) and AuNPs-PEG₂₀₀₀-Nap 1% (AuNPs 2). The data are reported as the mean ± standard deviation (n = 3).

Figure 3

Effect AuNPs-PEG₂₀₀₀ and AuNPs-PEG₂₀₀₀-Nap 1% on cell viability. The hCECs were cultured as monolayers (10,000 cells per well, three wells per condition, repeated using three different populations) and exposed to increasing doses of AuNPs-PEG₂₀₀₀ and AuNPs-PEG₂₀₀₀-Nap 1% for 18 h. Cell viability was assessed using an MTS assay. Results are presented as mean ± standard deviation.

Figure 4

Fluorescence assay of internalized of (A) control AuNPs-PEG₂₀₀₀ and (B) AuNPs-PEG₂₀₀₀-Nap 1% (green) by cultured hCECs with FITC filters. The arrows point to hCECs slightly showing autofluorescence. Scale bars: 10 μm.

Figure 5

Localization of AuNPs-PEG₂₀₀₀-Nap 1% (green) in rabbit cornea cross-sections observed under a fluorescence microscope using the FITC filter (first column), brightfield (middle column), and merge of the two (third column). Images of the cornea (A), (B), and (C) after 2-h PBS application; (D), (E), and (F) 3-min application of AuNPs-PEG₂₀₀₀-Nap 1% followed by a 2-h wait; and (G), (H), and (I) 2-h application of AuNPs-PEG₂₀₀₀-Nap 1%, where i is the multistratified epithelium, and ii is the stroma. Scale bars: 50 μm.

Figure 6

Localization of AuNPs-PEG₂₀₀₀-Nap 1% (green) in rabbit iris cross-sections observed under a fluorescence microscope using the FITC filter (first column), brightfield (middle column), and the merge of the two (third column). Images of the iris (A), (B), and (C) after 2-h PBS application; (D), (E), and (F) 3-min application of AuNPs-PEG₂₀₀₀-Nap 1% followed by a 2-h wait; and (G), (H), and (I) 2-h application of AuNPs-PEG₂₀₀₀-Nap 1%, where i represents the lumen of a blood vessel, ii represents a peripheral region where AuNPs-PEG₂₀₀₀-Nap 1% can be seen, and iii shows AuNPs-PEG₂₀₀₀-Nap 1% accumulation in the outermost structure of the iris. Scale bars: 50 μm.

Figure 7

Localization of AuNPs-PEG₂₀₀₀-Nap 1% (green) in the rabbit lens cross-sections observed under a fluorescence microscope using the FITC filter (first column) and the merge of the brightfield and FITC filter (second column) divided in two parts, where the anterior segment is closest to the cornea, and the posterior segment is closest to the back of the eye. Images of the anterior part of the lens (A) and (B) after 2-h PBS application; (C) and (D) 3-min application of AuNPs-PEG₂₀₀₀-Nap 1% followed by a 2-h wait; and (E) and (F) 2-h application of AuNPs-PEG₂₀₀₀-Nap 1%; (G) and (H) images of the posterior part of the lens after 2-h PBS application; (I) and (J) 3-min application of AuNPs-PEG₂₀₀₀-Nap 1% followed by a 2-h wait, and (K) and (L) 2-h application of AuNPs-PEG₂₀₀₀-Nap 1%. Scale bar: 50 μm.

Figure 8

Localization of AuNPs-PEG₂₀₀₀-Nap 1% (green) in rabbit posterior segment cross-sections observed under a fluorescence microscope using the FITC filter (first column), brightfield (middle column), and merge of the two (third column). Images of the posterior segment after (A), (B), and (C) 2-h PBS application; (D), (E), and (F) 3-min application of AuNPs-PEG₂₀₀₀-Nap 1% followed by a 2-h wait; and (G), (H), and (I) 2-h application of AuNPs-PEG₂₀₀₀-Nap 1%, where i AuNPs-PEG₂₀₀₀-Nap 1% form a thin line. Scale bars: 50 μm.