Elizabeth Nance

Drug delivery to the brain involves challenging transportation across multiple biological barriers. Despite the great tunability of polymeric nanoparticles, the synthetic challenge of varying the physical property of a nanoparticle without varying its chemical formulation only adds to our limited understanding of this transport. Here, we introduce a method for tuning the rigidity of poly(lactic-co-glycolic acid) (PLGA) nanoparticles without altering the chemical formulation by using UV irradiation to control the crosslinking chemistry. Using atomic force microscopy (AFM), we were able to show that Young’s modulus of nanoparticles increased with UV exposure time. Crosslinked nanoparticles with greater rigidity were found to have greater diffusive ability through the brain parenchyma than their non-crosslinked counterparts. However, even with identical chemical formulations, minor differences in the chemical structure of crosslinked and non-crosslinked particles were found to influence transport, highlighting the importance of decoupling the physical properties of nanoparticles from their chemistry.

Nanoparticles represent a major advancement in drug delivery, enhancing stability, solubility, and targeted delivery while reducing non‐specific toxicity. Despite promising preclinical results, few nanoparticle platforms have successfully translated to clinical use due to limited pharmacokinetic (PK) data, off‐target effects, manufacturing complexity, and a lack of long‐term studies. Emphasizing the pivotal role of PK in this development process is essential for harnessing the full therapeutic potential of nanoparticle applications in clinical practice. In this review, we introduce the concept of model‐informed nanoparticle development (MIND), an extension of model‐informed drug development (MIDD) principles to nanomedicine that integrates knowledge of nanoparticle interactions with biological systems and state‐of‐the‐art pharmacokinetic modeling to support evidence‐based decision‐making. Physiologically‐based PK (PBPK) models simulate the absorption, distribution, metabolism, and elimination (ADME) of nanoparticles within the body, incorporating physiological parameters to predict their PK behavior. Population‐based PK (PopPK) models utilize population variability to characterize nanoparticle PK across diverse patient groups, optimizing dosing strategies and personalized medicine approaches. Mechanistic models elucidate the intricate interactions between nanoparticles and biological systems, integrating cellular pathways and disease mechanisms to predict therapeutic outcomes and guide nanoparticle design. Through the MIND framework, these modeling approaches can enhance our understanding of nanoparticle PK, foster innovation in active agent delivery systems, and accelerate nanoparticle translation from research to clinical applications.

Brain injury after hypoxia–ischemia (HI) is the leading cause of morbidity and mortality in term and near-term neonates worldwide. The ferret is a promising translational model to study HI due to its gyrified brain and white-to-gray matter ratio that more closely resembles humans compared to rodents. Caffeine, an adenosine A2A receptor (A2AR) antagonist, shows neuroprotective potential after HI, but its effects have not been fully characterized. We sought to evaluate caffeine’s effect on neuronal cell death, cytotoxicity, and inflammatory and oxidative stress markers in a term-equivalent ferret organotypic brain slice model of HI. Slices were cultured for 72 h, exposed to two hours of oxygen–glucose deprivation (OGD), and randomized to OGD alone, OGD with caffeine (20 or 50 mg/L), or OGD with caffeine and an A2AR agonist. Healthy slices served as controls. Outcomes included global cell death, regional cell death, microglial morphology, and expression of inflammatory and oxidative stress genes (46–48 slices/group for cell death assays and 18 slices/group for imaging, balanced by sex). Caffeine 50 mg/L significantly reduced global cell death compared to OGD (p = 0.02), and this effect persisted despite co-administration of an A2AR agonist (p = 0.01), suggesting that protection was not primarily mediated through A2AR signaling. Caffeine also did not change regional pyknotic nuclei counts (p > 0.05). Caffeine altered microglial morphology, increasing the proportion of microglia with features characteristic of control conditions. OGD significantly increased expression of inflammatory and oxidative stress-related genes (p < 0.05) compared with control slices, whereas caffeine did not significantly alter gene expression. In summary, caffeine partially reversed global cell death after OGD and altered microglial morphology. Larger, higher-powered studies are needed to further investigate caffeine’s effects on neonatal HI.

Acute brain injuries such as perinatal asphyxia, stroke, and traumatic brain injury result in ischemia, oxidative stress, excitotoxicity, and inflammation, leading to a depletion of ATP. Nicotinamide adenine dinucleotide (NAD+) is crucial for ATP regeneration and DNA repair during postinjury recovery. However, the therapeutic benefits of NAD+ and its precursors, such as nicotinamide (NAM), are limited by challenges in achieving effective cell-specific intracellular delivery. In this study, we use a nanopeptoid delivery strategy to replenish the cellular redox state and increase energy production in the acutely injured brain. By self-assembling peptoids into tubular structures, we created biocompatible NAM-conjugated peptoid nanotubes (NAM-PNTs) that vary in tubular length. NAM-PNTs demonstrated significant therapeutic benefits by enhancing cell viability and replenishing intracellular ATP levels within 24 h of treatment in oxygen-glucose-deprived (OGD) BV-2 cells. In organotypic brain slices, NAM-PNT treatment promoted glial proliferation, reduced proinflammatory cytokines, and increased anti-inflammatory cytokines after OGD, an ex vivo model of hypoxia-ischemia. The effect of NAM-PNTs is associated with their uptake into microglia via fluid-phase phagocytosis and caveolae-mediated endocytosis. A single systemic dose of NAM-PNTs localized in microglia in the injured hemisphere and reduced brain tissue loss and improved neuropathology after hypoxia-ischemia in term-equivalent rats. These findings highlight the therapeutic potential of NAM-PNTs for cell-specific targeted delivery and energy restoration in the acutely injured neonatal brain. In the neonatal brain injury field, this work demonstrates the development of an innovative nanoparticle platform from first-principles design and synthesis to in vitro screening and then demonstration of efficacy in vivo.

Preterm brain injury affects both white and gray matter, including altered cortical development and gyrification, with associated neurodevelopmental sequelae such as cerebral palsy and learning deficits. The preterm brain also displays regionally heterogeneous responses to both injury and treatment, suggesting that drug combinations may be needed to provide global neuroprotection. We developed an extremely preterm‐equivalent organotypic whole hemisphere (OWH) slice culture mild injury model using the gyrencephalic ferret brain to probe treatment mechanisms of promising therapeutic agents and their combination. Regional and global responses to injury and treatment were assessed by cell death quantification, machine learning‐augmented morphological microglia assessments, and digital transcriptomics. Using two promising therapeutic agents, azithromycin (Az) and erythropoietin (Epo), we show minimal neuroprotection by either therapy alone, but evidence of synergistic neuroprotection by Az*Epo both globally and regionally. This effect of Az*Epo involved augmentation of transcriptomic responses to injury related to neurogenesis and neuroplasticity and downregulation of transcripts involved in cytokine production, inflammation, and cell death. With the increasing need to develop therapies for extremely preterm brain injury, the ferret OWH slice culture model provides a high‐throughput platform to examine combinations of therapeutics as part of a preclinical therapeutic pipeline.

Summary: Preterm brain injury affects both white and grey matter, including altered cortical development and gyrification, with associated neurodevelopmental sequelae such as cerebral palsy and learning deficits. The preterm brain also displays regionally heterogeneous responses to both injury and treatment, supporting the need for drug combinations to provide global neuroprotection. We developed an extremely preterm-equivalent organotypic whole hemisphere (OWH) slice culture injury model using the gyrencephalic ferret brain to probe treatment mechanisms of promising therapeutic agents and their combination. Regional and global responses to injury and treatment were assessed by cell death quantification, machine learning-augmented morphological microglia assessments, and digital transcriptomics. Using two promising therapeutic agents, azithromycin (Az) and erythropoietin (Epo), we show minimal neuroprotection by either therapy alone, but evidence of synergistic neuroprotection by Az*Epo both globally and regionally. This effect of Az*Epo involved emergent augmentation of transcriptomic responses to injury related to neurogenesis and neuroplasticity and downregulation of transcripts involved in cytokine production, inflammation, and cell death. This study supports the use of the ferret OWH slice culture model to provide a powerful high-throughput platform to examine combinations of therapeutics for extremely preterm brain injury.

Monitoring H₂O₂ dynamics in conjunction with key biological interactants is critical for elucidating the physiological outcome of cellular redox regulation. Optogenetic hydrogen peroxide sensor with HaloTag with JF635 (oROS-HT₆₃₅) allows fast and sensitive chemigenetic far-red H₂O₂ imaging while overcoming drawbacks of existing red fluorescent H₂O₂ indicators, including oxygen dependency, high pH sensitivity, photoartifacts and intracellular aggregation. The compatibility of oROS-HT₆₃₅ with blue-green-shifted optical tools allows versatile optogenetic dissection of redox biology. In addition, targeted expression of oROS-HT₆₃₅ and multiplexed H₂O₂ imaging enables spatially resolved imaging of H₂O₂ targeting the plasma membrane and neighboring cells. Here we present multiplexed use cases of oROS-HT₆₃₅ with other green fluorescence reporters by capturing acute and real-time changes in H₂O₂ with intracellular redox potential and Ca²⁺ levels in response to auranofin, an inhibitor of antioxidative enzymes, via dual-color imaging. oROS-HT₆₃₅ enables detailed insights into intricate intracellular and intercellular H₂O₂ dynamics, along with their interactants, through spatially resolved, far-red H₂O₂ imaging in real time.

Acute brain injuries such as perinatal asphyxia, stroke, and traumatic brain injury result in ischemia, oxidative stress, excitotoxicity, and inflammation, leading to a depletion of ATP, the brain’s cellular energy store. Nicotinamide adenine dinucleotide (NAD+), a key regulator of cellular homeostasis, is crucial for energy regeneration and DNA repair in post-injury recovery. However, the therapeutic benefits of NAD+ and its precursors, such as nicotinamide (NAM), are limited by the complexity of their metabolic pathways and challenges in effective cell-specific intracellular delivery. Therefore, cellular delivery strategies are needed to capture the potential of an NAD+-regenerating therapeutic approach. In this study, we introduce a nanopeptoid delivery strategy to replenish cellular redox state and energy production in the acutely injured brain. By self-assembling peptoids into tubular structures, we created biocompatible NAM-conjugated peptoid nanotubes (NAM-PNTs) that vary in tubular length. NAM-PNTs demonstrated significant therapeutic benefits by enhancing cell viability and replenishing intracellular ATP levels within 24 hours of treatment in oxygen-glucose deprived (OGD) BV-2 cells. In organotypic brain slices, NAM-PNT treatment promoted glial proliferation, reduced pro-inflammatory cytokine levels, and increased anti-inflammatory cytokines after OGD, an ex vivo model of hypoxia-ischemia. A single systemic dose of NAM-PNTs also reduced brain tissue loss and improved neuropathology after hypoxia-ischemia in term-equivalent rats. These findings highlight the strong therapeutic potential of NAM-PNTs for cell-specific targeted delivery and energy restoration in the acutely injured neonatal brain. In the neonatal brain injury field, this is the first demonstration of a novel nanoparticle platform development from first principles design and synthesis to in vitro screening and then demonstration of efficacy in vivo.

Fetal growth restriction (FGR) affects 5% to 10% of all pregnancies in developed countries and is the second most leading cause of perinatal mortality and morbidity. Life-long consequences of FGR range from learning and behavioral issues to cerebral palsy. To support the newborn brain following FGR, timely and accessible neuroprotection strategies are needed. Curcumin-loaded polymeric nanoparticles, which have been widely explored for the treatment of cancer, neurological disorders, and bacterial infections, have the potential to prevent and mitigate pathogenic inflammatory processes in the FGR brain. Curcumin is a hydrophobic molecule with poor aqueous solubility and therefore has been incorporated into nanoparticles to improve solubility and delivery. However, curcumin loading in many nanoparticles can be limited to 10% by weight or lower. Here, we first optimize the formulation process of curcumin-loaded polymeric nanoparticles to find a tunable, reproducible, and stable formulation with high curcumin loading and encapsulation efficiency. We establish a curcumin formulation with 39% curcumin loading and > 95% curcumin encapsulation efficiency. Using this formulation, we assessed the biodistribution of polymeric nanoparticles in FGR piglets and normally grown (NG) piglets following different administration routes and evaluated brain cellular uptake. We show a significant amount of nanoparticle accumulation in the brain parenchyma of neonatal piglets as early as 4 h after intranasal administration. Nanoparticles colocalized in microglia, a therapeutic target of interest in FGR brain injury. This study demonstrates the potential of curcumin-loaded nanoparticles to treat neuroinflammation associated with FGR in the newborn. Graphical abstract