AN EXPERIMENTAL APPROACH USING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY ON TEARS ANALYSES FOR OPTIMAL TREATMENT OF DRY EYE SYNDROME ASSOCIATED WITH KERATOCONUS DISEASE Funding Source:The Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI) PN-III-P2-2.1-PED-2016-0187 Contract No. 153PED/2017 Project leader: Dr. Elena Gabriela Ionita Institute of Physical Chemistry “Ilie Murgulescu ” Partner : Oftaclinic Grup SRL Prof. Dr. Catalina Corbu Funding: 550.000,00 lei Co-funding: 28.005,00 2017 -2018

Summary This project is intended to generate a new protocol in optimisation of the eye drops treatment for dry eye syndrome (DES) associated with keratoconus (KC) disease, based on data provided by electron paramagnetic resonance (EPR) spectroscopy. DES seems to become more prevalent disorder, it is considered that almost 30% of the world -wide population it is affected. KC is an eclectic/dystrophy disease of the eye which causes an irregularly shaped cornea leading to severe impairment of vision, with a prevalence of approximately 1:2000. Dry eye is also a symptom in KC, some studies indicating that dry eye is one of multifactorial etiology of KC. Corneal collagen cross-linking (CXL) based on the combined use of the photosensitizer riboflavin and UVA light of 370 nm is a relatively new method in KC treatment. Summary

Taking the advantage of EPR spectroscopy methods, the objectives of our project are: 1 - analysis of the EPR spectra of various spin probes introduced in  tears samples obtained from normal patients and those affected by DES; 2 - identification of reactive radical species in tear film formed after instillation of keratoconus eye with riboflavin followed by UVA irradiation; 3- analysis of the hyaluronic acid influence on physico-chemical properties of tears secretion. This will provide information on the optimal concentration of the hyaluronic acid in eye drops. The project starts from a general technological concept TLR2 reflecting the potential of the EPR spectroscopy to describe changes in dynamic and structure of the proteins which can be apply to correlate with the symptoms of a specific disease in particular, dry eye disease. In this way, we will generate an analytical proof of the disease based on collection of data which will be an early proof of concept in laboratory models.  Thus, our proposal can be considered that start from a TRL2 product and is materialized in a technology validated in laboratory (TRL3).

TEAMS Institute of Physical Chemistry Ionita Gabriela Matei Iulia Mocanu Sorin Aricov Ludmila Carpen Rodica OFTACLINIC Corbu Catalina Constantin Mihaela Cristea Miruna Potop Vasile

Activity report 2017 Institute of Physical Chemistry SPIN probe method study on model systems for lacrimal secretion - EPR study on model systems for lacrimal secretion - EPR measurements using spin probes for lacrimal secretions. - Biochemical analysis of tear secretion (total protein, albumin, lactoferrin, lyzozime, interleukine) Spin labelling of some components from lacrimal secretion - Albumin, - Hyaluronic acid Analysis of the EPR data and ophthalmologic parameters for patients introduced in this study Evidence of reactive radical species in model systems and in tear secretion speciilor radicalice reactive in sisteme model pentru filmul lacrimal UVA irradiation of solutions containing prevalent proteins found in tear secretion in the absence and in the presence of hyaluronic acid. evidence of reactive radical species in tear secretion during the crosslink procedue at patients with keratoconus. Oftaclinic Selection of patients with dry eye syndrome and keratoconus Obtaining the consent from patients to provide tear secretions Ophthalmologic examination Collection of tear secretion for biochemical analysis and EPR measurements Correlation of ophthalmologic, EPR data and biochemical analysis (common task) Dissemination of the results to the patients.

Dissemination – Papers 2017 M.M. Constantin, C. Corbu, V. Potop, M. Burcel, C. Ionescu, S. Mocanu, G. Ionita “EVALUATION of DRY EYE SYMPTOMATOLOGY at PATIENTS with KERATOCONUS” Revista de Chimie , under review Conference presentations

Dissemination - Conference presentations 2017 I. Matei, S. Mocanu, M. M. Constantin, C.G. Corbu, G. Ionita “Spin probes behaviour in model systems of tear films” The 50th Annual International Meeting of the ESR Spectroscopy Group of the Royal Society of Chemistry, Oxford, Uk, 2-7 April 2017, poster   Gabriela Ionita, Iulia Matei, Sorin Mocanu, Catalina Corbu, Mihaela Constantin, Catalina Ionescu, Miruna Burcel „Spin probes behaviour in tear secretions of patients with drye eye syndrome” SPIN-2017: THE 8th CONFERENCE ON NITROXIDES, Padova, 10-14 septembrie 2017, oral communication Sorin Mocanu, Iulia Matei, Gabriela Ionita „Dual molecular probes with fluorescent and paramagnetic moieties linked by short alkyl chains: reporters for host-guest interactions” 20th Romanian International Conference on Chemistry and Chemical Engineering Poiana Brasov, ROMANIA - September 6-9, 2017, poster M.M.Constantin, C. Corbu, M. Cristea, V. Potop, D. Dascalescu, C. Ionescu, G.Ionita, I. Matei „Evaluation of dry eye symptomatology at patients with keratoconus” The Xth Regional Conference of Ophthalmology, Timisoara, 20-22 April 2017, oral communication M.M. Constantin, C. Corbu, M. Cristea, V. Potop, C. Ionescu, G.Ionita, I. Matei „Studiu prospectiv privind corelatia parametrilor oftalmologice ai filmului lacrimal cu parametrii spectrelor de rezonanta electronica de spin” A 53-a Reunine anuala a oftalmologilor cu participare internationala, Iasi, 18-20 May, 2017, oral communication