Electrically assisted delivery of macromolecules into the corneal epithelium

Abstract

Electrically assisted delivery is noninvasive and has been investigated in a number of ocular drug delivery studies. The objectives of this study were to examine the feasibility of electrically assisted delivery of macromolecules such as small interfering RNA (siRNA) into the corneal epithelium, to optimize the iontophoresis and electroporation methods, and to study the mechanisms of corneal iontophoresis for macromolecules. Anodal and cathodal iontophoresis, electroporation and their combinations were the methods examined with mice in vivo. Cyanine 3 (Cy3)-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA and fluorescein isothiocyanate (FITC)-labeled dextran of different molecular weights (4-70 kDa) were the macromolecules studied. Microscopy and histology after cryostat sectioning were used to analyze and compare the delivery of the macromolecules to the cornea. Iontophoresis was effective in delivering siRNA and dextran up to 70 kDa into the cornea. The electroporation method studied was less effective than that of iontophoresis. Although both iontophoresis and electroporation alone can deliver the macromolecules into the cornea, these methods alone were not as effective as the combination of iontophoresis and electroporation (iontophoresis followed by electroporation). The significant enhancement of dextran delivery in anodal iontophoresis suggests that electroosmosis can be a significant flux-enhancing mechanism during corneal iontophoresis. These results illustrate the feasibility of electrically assisted delivery of macromolecules such as siRNA into the cornea.

Figures

Fig. 1

Schematic diagram of the dosing device.

Fig. 2

Representative images of the cornea after iontophoretic and passive delivery of FITC-labeled dextrans (n ≥ 3). The fluorescence images were acquired immediately after the treatments. A: in vivo image, 2 mA anodal iontophoresis of dextran 70 kDa; B: cryostat section image, 2 mA anodal iontophoresis of dextran 70 kDa; C: in vivo image, 2 mA anodal iontophoresis of dextran 20 kDa; D: in vivo image, 2 mA cathodal iontophoresis of dextran 20 kDa; E: in vivo image, 0.5 mA anodal iontophoresis of dextran 20 kDa; F: in vivo image, passive delivery of dextran 20 kDa; and G: cryostat section image, passive delivery of dextran 20 kDa. All in vivo images: magnification 20×; all cryostat section images: magnification 400×.

Fig. 3

Representative images of the cornea after iontophoresis, electroporation, and passive delivery of Cy3-labeled GAPDH siRNA (n ≥ 3). A: 6-hr in vivo image, combination of 0.5 mA cathodal iontophoresis and 20 V anodal electroporation; B: 6-hr in vivo image, 0.5 mA cathodal iontophoresis: C: 6-hr in vivo image, 20 V anodal electroporation; D: 24-hr in vivo image, combination of 0.5 mA cathodal iontophoresis and 20 V anodal electroporation; E: 24-hr in vivo image, 0.5 mA cathodal iontophoresis; F: 24-hr in vivo image, 20 V anodal electroporation; G: 0-hr in vivo image, 20 V anodal electroporation; H: 0-hr in vivo image, 20 V cathodal electroporation; I: 0-hr in vivo image, 0.5 mA anodal iontophoresis; J: 0-hr cryostat section image, combination of 0.5 mA cathodal iontophoresis and 20 V anodal electroporation; K: 6-hr cryostat section image, combination of 0.5 mA cathodal iontophoresis and 20 V anodal electroporation; L: 24-hr cryostat section image, combination of 0.5 mA cathodal iontophoresis and 20 V anodal electroporation; M: 0-hr in vivo image, passive delivery; N: 0-hr cryostat section image, passive delivery. All in vivo images: magnification 20×; all cryostat section images: magnification 400×.

Fig. 4

Illustration of the mechanisms of anodal (left panel) and cathodal (right panel) iontophoretic transport of (a) neutral dextrans and (b) negatively charged siRNA across the negatively charged cornea under the normal physiological condition. From the results of dextrans, the rank order of the effects is: electroosmosis > electro-permeabilization. From the results of siRNA, the rank order of the effects is: electrophoresis > electroosmosis > electro-permeabilization.