(562g) Nicotinamide-Loaded Nanopeptoids for Energy Regeneration to Drive DNA Repair in Neonatal Brain Injury

Background: Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells that plays a crucial role in various cellular processes, including cell metabolism, energy production, and DNA repair. Neonatal brain injury results in DNA damage, which activates poly(ADP-ribose) polymerase-1 (PARP-1) and decreases cytosolic nicotinamide adenine dinucleotide (NAD+), decreasing adenosine triphosphate (ATP) production, impairing mitochondrial function, and inactivating cellular metabolism, leading to cell death. Increasing NAD+ levels could therefore restore cellular NAD+ to aid in DNA repair and increase cell survival. In the brain, NAD+ is more likely to be made by nicotinamide (NAM)-derived nicotinamide mononucleotide (NMN) available locally to brain cells. However, mammalian cells cannot import NAD+, and the cell-specific targeting of NAD+ and NAM is limited. Therefore, cellular delivery strategies are needed to capture the potential of an NAD+-restorative therapeutic approach.

Methods: In this study, we developed a nanopeptoid delivery strategy to replenish cellular redox state and energy production following acutely injured, energy-depleted brain cells (Fig. 1A). Peptoids are sequence-specific heteropolymers that were developed as protein mimetics possessing advantages of both synthetic polymers and biopolymers. We self-assembled peptoids into tubular form to create peptoid nanotubes (PNTs) with different functional groups. Varying lengths of PNT were achieved by sonication (Fig. 1B). NAD+ or NAM were electrostatically associated or conjugated to the PNT before sonication, and the drug conjugation stability post sonication was confirmed by measuring released drug concentration via liquid chromatography. Free NAD+ or NAM, blank PNT, and NAM-PNT, or NAD+-PNT at a dose of 20 µg/mL were applied topically to organotypic whole hemisphere (OWH) slices isolated from the postnatal day 10 (P10) rat brain. After 24 h of treatment, healthy and oxygen-glucose deprived (OGD) treated slices, an ex vivo model of neonatal hypoxic-ischemic (HI) injury, were evaluated for cell death and PNT-cellular localization using confocal microscopy. Intracellular ATP, cell death, and cell proliferation were also quantitively analyzed. In a P10 rat model of neonatal HI, NAM-PNT was systemically administered immediately after HI injury at a single dose of 50 µg/mL of NAM (500 mg/kg). Controls included HI pups treated with saline, free NAM, and blank PNTs. Animals were sacrificed 72 h after treatment to evaluate gross injury, area loss, and neuropathology.

Results: Peptoids are usually around 2 µm in length without sonication and around 10 nm in diameter and show tubular structure on atomic force microscopy (AFM). The length can be further reduced to hundreds of nanometers after 2 h of sonication without changing other characteristics. PNTs conjugated with NAD+ or NAM achieved high drug loading efficiency and no cytotoxicity in brain cells (Fig. 1C). NAM-PNTs localize in microglia and associate with neurons (Fig. 1D). NAM-PNTs improve cell viability in response to OGD in OWH brain slices (Fig. 1E). NAM-PNTs replenish intracellular ATP levels by 24h after treatment (Fig 1F) and drive glial proliferation (Fig 1G). We see an associated shift in reduced pro-inflammatory cytokine production and increased anti-inflammatory cytokines. In the P10 HI rat, a single dose of NAM-PNTs administered systemically decreased brain tissue area loss and improved neuropathology.

Conclusions: Our study demonstrates that NAM delivery via PNTs has strong therapeutic potential for cell-specific delivery and energy regeneration in the acutely injured neonatal brain.