Assessment of stromal riboflavin concentration-depth profile in nanotechnology-based transepithelial corneal crosslinking.

PURPOSE: To determine the intrastromal concentration of riboflavin in nanotechnology-based transepithelial corneal crosslinking. SETTING: Consiglio Nazionale delle Ricerche, Messina, Italy. DESIGN: Experimental study. METHODS: Six human donor sclerocorneal tissues were used to evaluate penetration of nanotechnology-based riboflavin 0.1% solution in the stroma through the intact epithelium. Three additional tissues were deepithelialized and soaked with dextran 20.0%-enriched riboflavin 0.1% solution for 30 minutes. After corneal soaking with riboflavin, all tissues were irradiated using a 10 mW/cm2 device for 9 minutes. Two-photon emission fluorescence (TPEF) axial scanning measurements were collected in all specimens before treatment and immediately after corneal soaking with riboflavin and ultraviolet-A (UVA) irradiation of the cornea. The absorbance spectra of each tissue were collected at the same time intervals. The TPEF signals and absorbance spectra were used to calculate the concentration-depth profile of riboflavin in the corneal stroma during treatments. RESULTS: The mean stromal riboflavin concentration was 0.008% ± 0.003% (SD) and 0.017% ± 0.001% after transepithelial soaking with the nanotechnology-based solution and standard soaking, respectively (P = .001). After UVA irradiation of the cornea, the mean consumption of riboflavin was 52% ± 13% and 67% ± 2% in the study group and control group, respectively (P < .01). CONCLUSIONS: The nanotechnology-based platform was effective in enriching the anterior stroma with riboflavin through the intact epithelium, although the riboflavin concentration-depth profile rapidly decreased across the mid and posterior stroma. The treatment-induced stiffening effect on the corneal stroma was not assessed in this study.

All-Optical Method to Assess Stromal Concentration of Riboflavin in Conventional and Accelerated UV-A Irradiation of the Human Cornea.

PURPOSE: We investigated the concentration of riboflavin in human donor corneas during corneal cross-linking using two-photon optical microscopy and spectrophotometry. METHODS: Eight corneal tissues were de-epithelialized and soaked with 20% dextran-enriched 0.1% riboflavin solution for 30 minutes. After stromal soaking, three tissues were irradiated using a 3 mW/cm2 UV-A device for 30 minutes and three tissues irradiated using a 10 mW/cm2 device for 9 minutes. Two additional tissues were used as positive controls. A Ti:sapphire laser at 810 nm was used to perform two-photon emission fluorescence (TPEF) and second harmonic generation axial scanning measurements in all specimens before and after stromal soaking and after UV-A irradiation. In addition, spectrophotometry was used to collect the absorbance spectra of each tissue at the same time intervals. Analysis of the absorbance spectra and TPEF signals provided measures of the concentration depth profile of riboflavin in corneal stroma. RESULTS: After stromal soaking, the average peak concentration of riboflavin (0.020% ± 0.001%) was found between a stromal depth of 100 and 250 µm; the concentration of riboflavin was almost constant up to 320 ± 53 µm depth, then decreased toward the endothelium, though riboflavin was still enriched in the posterior stroma (0.016%% ± 0.001%). After conventional and accelerated UV-A irradiation, the concentration of riboflavin decreased uniformly 87% ± 2% and 67% ± 3% (P < 0.001), respectively. CONCLUSIONS: The combined use of two-photon optical microscopy and spectrophotometry provides relevant information for investigating the concentration of riboflavin in corneal stroma. The method can assist with the assessment of novel riboflavin formulations and different UV-A irradiation protocols.