(G-267) Peptoid nanotube delivery for cellular energy regeneration in brain injury

Introduction:: NAD is a coenzyme found in all living cells that play a crucial role in various cellular processes, including cell metabolism, energy production, and DNA repair.¹ DNA damage commonly occurs in acute brain injuries which consumes an increasing amount of NAD⁺.² This quick consumption of NAD⁺ would disrupt the regular cell energy metabolism, and reduce ATP production, which would result in mitochondrial dysfunction and further lead to permanent cell death.³ Currently, there is no direct way to mediate cell metabolism and replenish cell energy when DNA damage occurs. Thus, strategies to deliver NAD⁺ in injured cells and the ability to promote DNA recovery have significant clinical potential. Peptoid, a synthetic analog of peptide, has emerged as an important class of molecule for developing new therapeutics, materials, and biotechnology applications due to their unique properties and ease of synthesis.^4,5 With tunable sequence and functional group, PNTs can load various drugs such as NAM and NAD⁺ either by conjugation or molecular interaction. Here, we utilized peptoid nanotubes loaded with NAD⁺ or NAM to replenish NAD⁺ levels in acutely injured, energy-depleted brain cells with a primary target outcome to see a reduction in DNA damage and cell death.


Materials and Methods:: Nbrpm₆Nce₃Nae₃ (Nbrpm = N-[(4-bromophenyl) methyl] glycine, Nce = N-(2-carboxyethyl) glycine, and Nae = N-(2-aminoethyl) glycine) peptoids was synthesized using solid-phase synthesis (Jin, 2018). NAM was directly conjugated onto the peptoid and thymine functionalized Nbrpm₆Nce₃Nae₃ peptoid was used to load NAD⁺ by electrostatic interaction. The peptoid nanotubes (PNTs) were obtained by performing self-assembly of peptoids in aqueous environment using an evaporation-induced crystallization approach followed the “rolling-up and closure of nanosheet” process.⁶ BV-2 cells were chosen as an in vitro glial model and exposed to rotenone (1, 10, 100 nM) to induce dysfunction of mitochondrial complex I, and oxygen-glucose deprivation (OGD) times (10 and 30 min). Cells were seeded in a 96 well plate at 10,000 or 20,000 cells per well and allowed to proliferate for 2 days before the experiment. The cell viability was identified by MTT assay, and the cellular metabolic state was assessed by alamarBlue assay. The luminescent ATP detection kit was applied to discover the ATP level before and after the treatment. For cell experiments, cells exposed to blank PNTs (2, 20, 50 µg/mL), equivalent dosages of free NAD⁺ and free NAM (0.11, 1.1, 2.7 µg/mL), and no treatment will serve as a control. The drug exposure time was selected as 24 h.


Results, Conclusions, and Discussions:: On healthy cells, PNTs indicate little cytotoxicity. NAM-PNTs gave overall improved cell viability under all three rotenone dosages compared to nontreated rotenone exposed groups. At high rotenone concentrations, it was also noted that the therapeutic threshold was 2 µg/mL (Fig. 1a). Specifically for the addition of NAM and NAM-PNTs, the number exceeding 100% can be explained by more cells generated and implies that NAM promotes cell growth and proliferation (Fig. 1b). In an OGD model, 30 min of OGD killed >75% of cells, and the addition of treatments did not improve cell viability after 24 h of treatment (Fig. 2a). When the OGD time was reduced to 10 min, free NAM resulted in a 2-fold increase in cell viability in OGD-exposed cells. Free NAD⁺ could also promote cell recovery but at lower drug concentrations (Fig. 2b). We observed strong seeding density dependence: at lower seeding density, the OGD damage was more prominent. It was also discovered that the cell viability increased with increasing NAM dosages after the 24 h treatment (Fig. 2b, c).

By giving comparable results of free NAM and NAM-PNT in rotenone-treated cells, it proves that PNT is a reliable drug delivery method. The increased cell viability after adding free NAM, NAD^+, or NAM-PNTs on rotenone or OGD-treated cells further identified the importance of NAM and NAD+ in cellular health. However, previous literature illustrated that NAD⁺ cannot pass through cellular membranes and the result of adding free NAM should be the same as control. The therapeutic effect of NAD⁺ can be possibly explained by the conversion of NAD⁺ in cell culture media to its precursors.

In this project, we demonstrated the ability to recover cells after injury by using a nano-based delivery strategy of NAD or NAD precursors. This PNT-mediated delivery of NAM or NAD⁺ can promote cellular function and increase cytosolic NAD⁺ levels following acute injury, which can result in improved cell viability. The success in DNA recovery will lead to a new design of therapeutic method in brain diseases, where energy depletion and DNA damage also plays a critical role.


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References (Optional): : 1 Rajman L, Chwalek K, Sinclair DA. Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence. Cell Metabolism 2018;27:529–47. https://doi.org/10.1016/j.cmet.2018.02.011.

2 Alano CC, Garnier P, Ying W, Higashi Y, Kauppinen TM, Swanson RA. NAD+ Depletion Is Necessary and Sufficient forPoly(ADP-Ribose) Polymerase-1-Mediated Neuronal Death. J Neurosci 2010;30:2967–78. https://doi.org/10.1523/JNEUROSCI.5552-09.2010.

3 Brennan AM, Suh SW, Won SJ, Narasimhan P, Kauppinen TM, Lee H, et al. NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation. Nat Neurosci 2009;12:857–63. https://doi.org/10.1038/nn.2334.

4 Li Z, Cai B, Yang W, Chen C-L. Hierarchical Nanomaterials Assembled from Peptoids and Other Sequence-Defined Synthetic Polymers. Chem Rev 2021;121:14031–87. https://doi.org/10.1021/acs.chemrev.1c00024.

5 Jin H, Ding Y-H, Wang M, Song Y, Liao Z, Newcomb CJ, et al. Designable and dynamic single-walled stiff nanotubes assembled from sequence-defined peptoids. Nat Commun 2018;9:270. https://doi.org/10.1038/s41467-017-02059-1.

6 Jin H, Ding Y-H, Wang M, Song Y, Liao Z, Newcomb CJ, et al. Designable and dynamic single-walled stiff nanotubes assembled from sequence-defined peptoids. Nat Commun 2018;9:270. https://doi.org/10.1038/s41467-017-02059-1.