A high-throughput biophotonics instrument to screen for novel ocular photosensitizing therapeutic agents.
OBJECTIVE
High-throughput techniques are needed to identify and optimize novel photodynamic therapy (PDT) agents with greater efficacy and to lower toxicity. Novel agents with the capacity to completely ablate pathologic angiogenesis could be of substantial utility in diseases such as wet age-related macular degeneration (AMD).


METHODS
An instrument and approach was developed based on light-emitting diode (LED) technology for high-throughput screening (HTS) of libraries of potential chemical and biological photosensitizing agents. Ninety-six-well LED arrays were generated at multiple wavelengths and under rigorous intensity control. Cell toxicity was measured in 96-well culture arrays with the nuclear dye SYTOX Green (Invitrogen-Molecular Probes, Eugene, OR).


RESULTS
Rapid screening of photoactivatable chemicals or biological molecules has been realized in 96-well arrays of cultured human cells. This instrument can be used to identify new PDT agents that exert cell toxicity on presentation of light of the appropriate energy. The system is further demonstrated through determination of the dose dependence of model compounds having or lacking cellular phototoxicity. Killer Red (KR), a genetically encoded red fluorescent protein expressed from transfected plasmids, is examined as a potential cellular photosensitizing agent and offers unique opportunities as a cell-type-specific phototoxic protein.


CONCLUSIONS
This instrument has the capacity to screen large chemical or biological libraries for rapid identification and optimization of potential novel phototoxic lead candidates. KR and its derivatives have unique potential in ocular gene therapy for pathologic angiogenesis or tumors.
Butler
Mark C
MC
Department of Ophthalmology, SUNY University at Buffalo, Buffalo, NY 14215, USA.
Itotia
Patrick N
PN
Sullivan
Jack M
JM
D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000633:toxicity; D006801:Humans; D055095:Optics and Photonics / Q000295:instrumentation*; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000032:analysis* / Q000633:toxicity
D017319:Photosensitizing Agents; C508771:killer red protein, Anthomedusae; D049452:Green Fluorescent Proteins