Abstract
Purpose :
miRNAs complexed to NPs offer great potential as therapeutic interventions to alter diseased tissues. A major hurdle to achieving this goal is to effectively formulate and deliver miRNAs to the cytoplasm of target cells in a stable form. With the demonstration that HDLs, isolated from human serum contained miRNAs and that these HDL-bound miRNAs had improved stability compared with naked miRNAs, we investigated whether HDL NPs could deliver functional miRNAs to the cornea.
Methods :
Spherical, HDL-like NPs containing apolipoprotein A-1, bind with high affinity to SCARB1 and selectively target cells that express SCARB1. Such HDL NPs were complexed with either miR-205 or -146a mimics to generate miR-205- and miR-146a-HDL NPs. miR-205-HDL NPs were delivered to primary human corneal epithelial cells (HCECs) and miR-146a-HDL-NPs were delivered to a J774.1 murine macrophage cells. Immunoblotting confirmed downstream targets regulated by miR-205-HDL-NPs. Colorimetic analyses confirmed downstream targets regulated by miR-146a. Cy-3-tagged HDLNP (1µM in PBS) were applied topically for 4 hrs to intact corneas. Twenty-four hrs post-treatment, eyes were processed for fluorescent microscopy.
Results :
HCECs exposed to miR-205-HDL NPs (48 hrs) decreased SHIP2 and increased p-Akt at 50 nM. This confirms our findings that miR-205 directly targets SHIP2 and positively regulates p-Akt signaling. Murine macrophages exposed to HDL NPs carrying miR146a significantly reduced the signal of LPS-induced secreted alkaline phosphatase, which is under the control of NF-kB, when compared with empty control and negative control particles. This is consistent with the observation that miR-146a can inhibit NF-kB transcriptional activity. Topically applied Cy3-taged HDL NPs to mouse corneas were detected in basal and superficial cells of the limbal/corneal epithelia, as well as in stromal keratocytes.
Conclusions :
Collectively, this is compelling evidence that our novel HDL NPs can deliver miRNAs topically to cells and tissues of the ocular anterior segment, with functional consequences. This has vast translational opportunities, and provides a basis for developing innovative treatment regimens based on miRNA delivery to enhance normal physiological processes (e.g., cell migration), or in patients with inflammatory diseases that affect the ocular anterior epithelia.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.