BASIC INVESTIGATIONS Ultrasound-Enhanced Transcorneal Drug Delivery Vesna Zderic, PhD,*† John I. Clark, PhD,‡ Roy W. Martin, PhD,*† and Shahram Vaezy, PhD*† Purpose: Ultrasound has been shown to enhance, by up to 10 times, the corneal permeability to different compounds such as -blockers and fluorescein. Here, we report on our investigation of the mecha- nisms of ultrasound-enhanced drug delivery through the cornea using light and electron microscopy. Methods: Enhancement of permeability for a hydrophilic com- pound, sodium fluorescein, in rabbit cornea in vitro was achieved us- ing ultrasound at a frequency of 880 kHz and intensities of 0.19–0.56 W/cm 2 with an exposure duration of 5 minutes. Light and electron microscopy (transmission and scanning) were used to observe ultra- sound-induced structural changes in the cornea. Results: The permeability increased by 2.1, 2.5, and 4.2 times when ultrasound was applied at 0.19, 0.34, and 0.56 W/cm 2 , respectively (P < 0.05). The surface cells of corneal epithelium exposed to ultrasound appeared swollen and lighter in color (indications of membrane rup- ture) as compared with the control cells. Some of the surface epithelial cells were absent. The cells in the inner layers of the epithelium were occasionally lighter in color. Also, holes 3–10 μm in diameter were observed on the epithelial surface. No structural changes were ob- served in the stroma. Conclusion: Ultrasound enhancement of drug delivery through the cornea appears to result from minor structural alterations in the epi- thelium. Careful investigation of the recovery of cornea structure and barrier function after the ultrasound application, in vivo, is needed. Key Words: ultrasound, drug delivery, cornea, sonophoresis, phono- phoresis (Cornea 2004;23:804–811) T he cornea is the main route for penetration of ophthalmic drugs, even though it is an inefficient pathway because of its low permeability. 1,2 Short residence time of the drugs on the corneal surface and small surface area contribute to poor pen- etration through the cornea. 2,3 Fewer than 10% of all topically applied drugs can penetrate through the cornea into the anterior chamber, with virtually no penetration into the back of the eye. 1,4 Most topically applied drugs that penetrate the conjunc- tiva and sclera are carried away by the systemic circulation before diffusing into the inner structures of the eye. 5 Drug de- livery by systemic administration is also ineffective because of blood–aqueous and blood–retina barriers. 6 In some cases, in- traocular injection of drugs is used clinically, but this method is invasive and not very efficient. 7 We are investigating the use of ultrasound to increase the permeability of the corneas. ULTRASOUND ENHANCEMENT OF DRUG DELIVERY It has been shown that the barrier properties of the skin can be modified using low-frequency ultrasound (20 kHz) to enhance the efficiency of transdermal drug delivery. 8 Im- provement of as much as 1000-fold was achieved in the deliv- ery of hydrophilic and/or large compounds without long-term damage to the barrier properties of the skin. 9 These results mo- tivated us to investigate the use of ultrasound to enhance drug delivery through the cornea. However, the skin barrier layer, stratum corneum, is a dead structure, and ultrasound effects on the barrier function of live corneal epithelium may be signifi- cantly different. In preliminary studies, we found that 20-kHz ultrasound applied at intensity of 14 W/cm 2 resulted in a 4-fold increase in the corneal permeability for -blocker drugs (aten- olol, carteolol, timolol, and betaxolol) in a rabbit model in vitro, with significant disorganization of the epithelium. 10 The cells from the outer layers of the epithelium were absent, and the cells from the inner layers appeared swollen and ruptured. Thus, the epithelial healing was expected to be slow (within 5 days) with the possibility of complications such as corneal haze and infection. 11 Ultrasound application (phonophoresis) at medium fre- quencies (470–880 kHz) and intensities of 0.2–0.3 W/cm 2 has been used in the USSR and Russia for transcorneal drug deliv- ery, to improve treatment of corneal inflammation, wounds, and retinal dystrophy. 12–17 However, the Russian research on ocular phonophoresis is widely unknown in the United States, Europe, and Japan. Ultrasound application was reported to produce up to a 10-fold increase in the corneal permeability to Received for publication May 30, 2003; revision received April 5, 2004; ac- cepted April 7, 2004. From the *Department of Bioengineering, University of Washington, Seattle, WA; †Center for Industrial and Medical Ultrasound, Applied Physics Laboratory, University of Washington, Seattle, WA; and ‡Departments of Biological Structure and Ophthalmology, University of Washington, Seattle, WA. Reprints: Vesna Zderic, Center for Industrial and Medical Ultrasound, Ap- plied Physics Laboratory, 1013 NE 40th Street, Seattle, WA 98105 (e-mail: vesna@u.washington.edu). Copyright © 2004 by Lippincott Williams & Wilkins 804 Cornea • Volume 23, Number 8, November 2004