Development of Reconstituted High-Density Lipoprotein Nanoparticles Utilizing Fluorescence Resonance Energy Transfer for Ocular Applications

Purpose:

Macular degeneration and glaucoma are considered age-related degenerative eye diseases. Both conditions can lead to vision change and loss. While glaucoma and macular degeneration have similarities, they affect different eye regions and require targeted drug-delivery systems. Significant limitations of current ocular therapies are poor bioavailability and delivery barriers in the eye. Developing an efficient ocular delivery system is thus critical to improving the efficacy of therapeutic agents. Specifically, reconstituted high-density lipoprotein (rHDL) mimics the structure and function of endogenous human plasma HDL and thus presents a non-toxic therapeutic strategy for delivering various drugs and imaging agents to ocular tissue. Moreover, rHDL nanoparticles (rHDL NPs) are ideal for transporting lipophilic therapeutic agents and imaging dyes since they are small in size, non-immunogenic, can circulate in the body fluids for an extended time, and have specific receptor-protein interactions to release their lipophilic payloads. Our study aims to employ a reconstituted rHDL drug delivery vehicle that mimics the structure and function of endogenous human plasma HDL and offers a novel strategy for the delivery of drugs and imaging agents to the eye.

Methods:

A stable rHDL-payload complex (rHDL NPs) was prepared by combining lipophilic fluorescent dyes using phosphatidylcholine and apolipoprotein A-I (Apo A-I) via a novel preparation method. Dual fluorescent rHDL NPs have been used as Förster resonance energy transfer (FRET) probes and were assessed by dynamic light scattering (DLS), spectrophotometry, and fluorescence spectroscopy.

Results:

Dual fluorescence rHDL NPs were generated with 64.4% and 79.2% encapsulation efficiency for the donor and acceptor fluorophores, respectively. rHDL NPs were found to have a polydispersity index (PDI) of 0.302 ± 0.023, an average size of 10.96 ± 1.47 nm, and a zeta potential of -7.65 ± 0.63 mV. The fluorescent signals were characterized by anisotropy measurements while the FRET signal was detected by the change in fluorescence lifetime between the donor and acceptor fluorophores.

Conclusions:

A stable rHDL NP formulation that includes a FRET pair was successfully prepared through an optimized protocol. The rHDL NPs can be utilized for biodistribution studies and dynamic kinetic characterization in vivo to assess the efficacy of drug-loaded rHDL NPs for the treatment of ocular degenerative diseases such as glaucoma and macular degeneration.