Long Range Forster Resonance Energy Transfer for rHDL Nanoparticles Drug Payload Estimation




Raut, Sangram
Fudala, Rafal
Shah, Sunil
Sabnis, Nirupama
Gryczynski, Zygmunt
Borejdo, Julian
Lacko, Andras G.
Gryczynski, Ignacy


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Purpose: Over the last 30 years, there has been a dramatic increase in the application of fluorescence technology in the biomedical sciences. These advances include the application of fluorescence anisotropy for the assessment of the location and rigidity of the drug payload within the lipoprotein nanocomplexes. Forster Resonance Energy Transfer (FRET) is the long range through space interaction between the two chromophores (one energy donor and one energy acceptor) that very strongly depends on the chromophores separation. In the studies presented here, we selected the optimal combination and synthesized a FRET pair using doxorubicin within the rHDL nanoparticles as acceptor, and carboxyfluorescein linked to the rHDL via an 18-carbon lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) [18:1 PE-CF]) as the donor. The emission spectra of donor (18:1 PE-CF) has a good overlap with the absorption spectra of an acceptor (doxorubicin), making this combination suitable for FRET studies. In this communication, we used our previous findings and FRET methodology for the assessment of the localization of the payload (doxorubicin) within the respective nanoparticles and their corresponding encapsulation/entrapment efficiencies. These findings validate the diameter and the three-dimensional array of the drug transporting (rHDL) nanoparticles, while additional measurements also allowed the estimation of the number of drug molecules carried by the individual nano-assemblies using non-destructive sampling procedure. The approach described here could likely to be extended to the investigation of the loading capacity of a variety of drug carrying nanostructures, including micellar nanoparticles and exosomes.