Low-intensity low-frequency ultrasound mediates riboflavin delivery during corneal crosslinking

We employed the mechanical effect from 40 kHz ultrasound (US) to improve the delivery of riboflavin into corneal stroma for collagen crosslinking, which can benefit the treatment of keratoconus and other corneal ectasias. Experiments were conducted, first with porcine corneas ex vivo and then with New Zealand white rabbits in vivo, at varying mechanical index (MI) and sonication time. Results showed that 15 min of US applied on the cornea at MI = 0.8 in the presence of 0.5% of riboflavin solution enabled its delivery to deeper corneal stroma. Excessive heat was removed by a cooling setup to negate the thermal effect. The corneal absorption amount and penetration of riboflavin through cornea as detected by fluorotron, as well as the enhancement of corneal stiffness as measured by Young's modulus, were comparable to the conventional approach that requires complete corneal epithelium debridement. Histological analysis revealed minor exfoliation of superficial cell layers of corneal epithelium and loss of ZO-1 tight junctions immediately after US. Full recovery of the corneal epithelium and restoration of tight junctions occurred in 3-4 days. The study shows that low-intensity low-frequency ultrasound (LILF US) is a less invasive alternative to the conventional epithelium-off method for delivering riboflavin into the corneal stroma.