Characterization of Ultrasound-mediated Trans-corneal Delivery of Riboflavin by Modified Fluorotron™ Master

Abstract

Purpose : The corneal drug penetration is commonly detected by measuring fluorescence profile of labelled drug in corneal tissues by cryosection. However, such method requires sacrificing animals and the data is often debased by artefacts. Although ocular fluorophotometer may provide a better alternative, problems of Inner Filter Effect (IFE) and low resolution have been reported. This study reports a modified Fluorotron to circumvent this issue to allow quantitative measurement of both the absorption and penetration of riboflavin into the cornea. Using the new approach, the penetration depth of riboflavin by ultrasound-mediated delivery was characterized.

Methods : The objective lens of ocular fluorophotometer was stabilised with plexiglass and the angle was increased between excitation and emission paths from 28 degree to 53 degrees that resulted in decreasing depth of focus to about 85 microns. The modified fluorophotometer was used to detect porcine eyes (Epi-on) which were soaked in 0.5% riboflavin for 30 minutes followed by washing and 0.025% riboflavin solution for 15 minutes by dropping different concentration riboflavin separately. The cryosection method was employed after ultrasound-mediated riboflavin delivery with different operating parameters of sonification which will be used to validate the result in modified fluorophotometer.

Results : The riboflavin penetration profile of cornea which detected by the modified fluorophotometer can have a higher resolution and minimized IFE (Fig.1). We anticipate that the modified flurophotometer will expedite the optimization of new modalities, such as mediation by ultrasound, in corneal drug delivery.

Conclusions : This modified Fluorotron fluorophotometer can detect the riboflavin penetration depth and concentration across the corneal sections with higher resolution and minimized IFE. The absorption and penetration of riboflavin in cornea by ultrasound-mediated delivery is distinguishable in cryosection fluorescence microscope image.

This is a 2021 ARVO Annual Meeting abstract.

 

Fig.1 Scanning profile for porcine eye (Epi-on) in 0.5% riboflavin for 30 minutes A) Original Fluorotron. B) Modified Fluorotron.
Porcine eye (Epi-on) in 0.025% riboflavin for 15 minutes. C) Followed by 0.05% riboflavin drop. D) Followed by 0.25% riboflavin drop.

 

Fig.2 Riboflavin cryosection fluorescence microscope A) Epi-on control. B, C) After ultrasound treatment varying in sonication parameters (10x magnification).