Localization of Novel Chlorin Photosensitizers and Commercial Stains in the AY-27 Cell Model Using Advanced Fluorescence Microscopy

Abstract

Gjennom et samarbeid mellom Linköping Universitet og Norges Tekniske og Naturvitenskapelige Universitet, har flere nye kloriner for bruk i fotodynamisk terapi blitt syntetisert. To av disse klorinene, KA20-11H og KA20-11H-proto har vist størst potensial, og har blitt videre undersøkt. Kvanteeffektiviteten og Fluorescens levetid til klorinene, og flere kommersielle fargestoffer, har blitt bestemt gjennom spektroskopieksperimenter. Kvanteeffektiviteten til KA20-11H og KA20-11H-proto ble bestemt til henholdsvis 0.04 og 0.12. Fluorescens levetid ble målt på to lignende kloriner, KA20-15H og KA20-15H-proto, og ble bestemt til henholdsvis 1.40 ns og 10.50 ns. Konfokal laser skanning mikroskopi bilder har blitt tatt av AY-27, blærekreftceller fra rotter, der cellene har inkubert med klorinene i 2 timer, 4 timer, 8 timer og 24 timer. Gjennom bildebehandling i python har ko-lokasjonen mellom klorinene og kommersielle fargestoff, som farger organellene: lysosomene, mitokondriene og cellemembranen, blitt kvantifisert. Manders Korrelasjons Koeffisienter (MCC), M1 og M2, ble sammen med prosentvis overlap, brukt som mål for ko-lokasjon. Klorinene ble funnet til å ko-lokalisere mest med lysosomer, og viste ikke signifikant ko-lokalisering med mitokondriene eller cellmembranen. I tillegg ble fluorescens levetid bildemikroskopi (FLIM) tatt av AY-27 celler med KA20-11H-proto, og levetiden sammenlignet med levetider målt gjennom spektroskopi i laboratoriet. For KA20-11H-proto var levetiden kortere når den ble målt under FLIM, alstå når den var inni cellen.

As a part of an ongoing collaboration between Linköping Unviersity and Norwegian University of Science and Technology, seven novel chlorin photosensitizers have been investigated for use in photodynamic therapy. After preliminary research, two chlorins, KA20-11H and KA20-11H-proto, showed the greatest potential, and are the focus in this project. The quantum yield and the fluorescent lifetimes of the chlorins, and of different commercial stains were determined through spectroscopy measurements. The quantum yield of KA20-11H and KA20-11H-proto were determined to be approximately 0.04 and 0.12, respectively. The fluorescent lifetimes were measured on two similar chlorins, KA20-15H and KA20-15H-proto, and were determined to be around 1.40 ns and 10.50 ns, respectively, depending on the solvent that was used. AY-27, rat bladder cancer cells, incubated with the photosensitizers (2h, 4h, 8h, 24h) and commercial stains were imaged through confocal laser scanning microscopy. An automated thresholding algorithm based on Costes' method of thresholding was developed in python 3, which together with other automatic segmentation tools were used to process the images digitally. The co-localization between the photosensitizers and the commercial stains were quantified and evaluated statistically. Mander's correlation coefficients, (M1 and M2), and overlap percentage were used as measures of co-localization. The photosensitizers were found to have the highest degree of co-localization with lysosomes, and did not show significant co-localization with either the cell membrane or mitochondria. Fluorescent lifetime imaging microscopy (FLIM) of KA20-11H-proto in the AY-27 cell line was also carried out. The fluorescent lifetime of KA20-11H-proto from FLIM measurements was shorter than the lifetimes obtained through measurements in the laboratory with different solvents (ethanol, buthanol, methanol and PBS).