Supplementary information:
SI1. Thermographic analysis of the corneal surface
Three rabbits were treated with WST-D for 20 minutes, followed by NIR
illumination (755nm, 10mW/cm2) for 30 minutes as described in the In-vivo studies
section of the manuscript. Temperature measurements on the corneal surface were
performed during WST-D/NIR treatment using an IR thermocamera (Thermal
imager InfRec R300, NEC Avio Infrared Technologies Co., Ltd., Tokyo, Japan) with
thermal resolution 0.05℃, temperature accuracy of ±1℃ and a spatial resolution of
120µm. Images were recorded before irradiation, every 7 minutes during irradiation
and at the conclusion (last seconds) of irradiation. Selected thermographic images
were processed with InfReC Analyzer NS9500 Lite (NEC Avio Infrared
Technologies Co., Ltd., Tokyo, Japan).
A constant temperature gradient from T=32℃ at the corneal center to T=37.5℃ at
the limbal periphery was measured before, during and after treatment. Deviations of
less than 1℃ were observed throughout the whole procedure (data not shown).
SI2. Electron Spin Resonance (ESR) spectroscopy
All ESR measurements were carried out using a ESR Miniscope MS 100
Spectrometer (Magnettech Germany), with a Microwave X-band Bridge. The ESR
spectrometer operates at 9.3-9.55GHz, 20mW microwave power. ESR measurements
were carried out at room temperature in glass capillaries or flat cells. Sample
solutions of WST11 or RF with/without dextran were illuminated at 755nm or UVA
respectively as previously described by our group15. To each solution the spin-trap α(4-Pyridyl N-oxide)-N-tert-butylnitrone (4-POBN, 65mM) and ethanol (8%) were
added. Controls contained illuminated 4-POBN in saline with/without dextran, and
non-illuminated solutions of RF or WST11 with/without dextran, also with/ without
4-POBN.
Ex-vivo ESR measurements: Eight eyes of 4 rabbits were enucleated post mortem.
The corneas were de-epithelialized mechanically and corneal strips, 5-mm in width,
were cut with a self-constructed double-blade cutter. The strips were immediately
immersed for 30 minutes in solutions containing 4-POBN (65mM), ethanol (8%),
and either WST11 in saline or RF in dextran. Next, the stripes were washed with
saline, and put in flat cells (0.5×5mm2) for NIR/UVA illumination followed by ESR
measurements with the aforementioned Miniscope.
SI3. Palladium based measurement of systemic absorption of topically applied
WST-D
Six rabbits were anesthetized as described in the In-vivo studies section. After deepithelialization one cornea of each rabbit was treated with WST-D 2.5mg/mL (N=3)
and 10mg/mL (N=3) for 20 minutes using an eye cap. Blood samples (~0.5mL) were
taken from the ear vein before application (time 0), and at 10, 20, 40 and 60 minutes
after application began, placed in pre-weighed polyethylene 1.5mL test tubes,
weighted and lyophilized. The dry samples were digested with nitric acid, and Pd
concentrations were determined by inductively-coupled plasma mass spectrometry
(ICP-MS) using a set of Pd standards (High-purity Standards, USA) as previously
described.16
ICP-MS measurement of blood samples drawn from rabbits during and after topical
application of WST-D could not detect any significant levels of Pd+2, as an evidence
for no leakage of WST11 to the circulation of the treated animals at all measured time
points.