Abstract
Purpose :
Recently, it has emerged that mineralisation in the retina with hydroxyapatite (HAP) and other species is correlated with the development of AMD and ALzheimer disease. While we have used legacy stains including classic tetracycline antibiotics as selective fluorescent stains for HAP in the retina, these compounds have some limitations and novel HAP-specific fluorescent stains may offer useful new properties including fluorescence imaging contrast using new mechanisms. These properties may further elucidate the HAP deposition process mechanism(s) and suggest novel treatments.
Methods :
Methods: Syntheses Details of the syntheses and purification of ((4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)phenyl)phosphonic acid) (BODIPY-PPA), (2,7-dichlorofluorescein-bis(methyliminodiphosphonic acid) (DCF-tetraphosphonate), and 5,10,15,20-tetrakis[p-phenylphosphonic acid] porphyrin (p-H8TPPA) will appear elsewhere.
Characterization of Probes: The structure and purity of the probes were characterized by liquid chromatography, infrared, NMR, and/or mass spectrometry. Their optical properties were characterized by absorption and fluorescence spectrophotometry in the presence and absence of authentic hydroxyapatite, whitlockite, and other doped apatites. The dyes were also tested on mouse ribs as an example of hydroxyapatite in tissue.
Results :
We found that several of the synthesized probes bound HAP tightly, in some cases with alterations of their fluorescence properties, which we attribute to synergistic interactions arising from conjugation of the sp2 carbons of the aryl fluorophore moiety with the phosphonate moiety. Staining a cross section of mouse rib with meso-tetra(4-phosphorylphenyl)porphyrin (red,) and DAPI (blue), together with autofluorescence (green) is depicted in the figure (A), with a DAPI-stained control section from the same mouse (B). Based on these preliminary results, staining appears specific for hydroxyapatite.
Conclusions :
We conclude that these new fluorescent labels offer novel features that will be of use in elucidating the biology of retinal mineralisation and its relationship to macular and peripheral deposit formation seen in AMD and Alzheimer disease, respectively.
This is a 2020 ARVO Annual Meeting abstract.