Browsing by Author "Petty, R. Max"
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Item Assessment of WIN 55,212-2 Loaded Reconstituted High-Density Lipoprotein Nanoparticles for Ocular Delivery(2024-03-21) Petty, R. Max; Ranjan, Rajiv; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras; Gryczynski, Zygmunt; Krishnamoorthy, Raghu; Stankowska, DorotaPurpose: Overcoming challenges in glaucoma therapy, such as biological barriers and retina delivery, led us to develop innovative reconstituted high-density lipoprotein nanoparticles (rHDL NPs) for effective drug delivery. Optic nerve head astrocytes (ONHAs) are vital in maintaining retinal ganglion cell (RGC) axon integrity. This study describes the encapsulation of WIN 55,212-2 (WIN) in rHDL NPs and investigates the delivery mechanism of these nanoparticles in ONHAs. Methods: Using a novel preparation method, a stable rHDL-payload complex was created by combining lipophilic fluorescent dye IR780 or therapeutic agent WIN with phosphatidylcholine and apolipoprotein A-I (Apo A-I). Fluorescent rHDL (rHDL-IR780) was used to assess cellular uptake in human primary ONHAs in vitro. Scavenger receptor class B1 (SR-B1) expression was confirmed in retinal cell lysates by SDS-PAGE followed by western blot analysis. Receptor-mediated payload release through SR-B1 was confirmed by receptor blocking using BLT-1 as a specific SR-B1 receptor-blocking agent. Results: Fluorescent rHDL NPs exhibited an IR780 encapsulation efficiency of 68.7% (103 M), a polydispersity index (PDI) of 0.287 ± 0.013, a size of 14.01 ± 4.36 nm, and a zeta potential of -7.44 ± 0.90 mV. Additionally, drug-loaded rHDL-WIN NPs displayed a WIN encapsulation efficiency of 44.6% (341.4 M), a PDI of 0.280 ± 0.011, a size of 62.04 ± 25.06 nm, and a zeta potential of -20.13 ± 0.86 mV. Western blot analysis on human retinal lysates, ONHA lysates, and RGC lysates indicated the expression of SR-B1 (57/82 kDa (unmodified/glycosylated)). Cellular uptake studies confirmed the ability of rHDL to deliver payloads to ONHAs and RGCs. Receptor blocking with 10 nM BLT-1 highlighted the role of SR-B1 in specific cellular uptake from rHDL to ONHAs (p < 0.01). Conclusions: Our study highlights the role of SR-B1 in facilitating the delivery of rHDL payloads to ONHAs, offering the potential for targeted drug delivery in glaucoma. We anticipate that the cellular uptake by RGCs will follow the same SR-B1-mediated pathway. Successful WIN encapsulation in rHDL NPs suggests a potential avenue for targeting therapies to treat and prevent glaucomatous damage. Further studies are needed to determine the neuroprotective effects of rHDL-WIN and develop the potential of rHDL NPs to be used as an agent to target therapies in glaucoma.Item Development of Reconstituted High-Density Lipoprotein Nanoparticles Utilizing Fluorescence Resonance Energy Transfer for Ocular Applications(2023) Petty, R. MaxPurpose: 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.