Generation and characterization of a zebrafish gain-of-function ACOX1 Mitchell disease model.

Background: Mitchell syndrome is a rare, neurodegenerative disease caused by an ACOX1 gain-of-function mutation (c.710A>G; p.N237S), with fewer than 20 reported cases. Affected patients present with leukodystrophy, seizures, and hearing loss. ACOX1 serves as the rate-limiting enzyme in peroxisomal beta-oxidation of very long-chain fatty acids. The N237S substitution has been shown to stabilize the active ACOX1 dimer, resulting in dysregulated enzymatic activity, increased oxidative stress, and glial damage. Mitchell syndrome lacks a vertebrate model, limiting insights into the pathophysiology of ACOX1-driven white matter damage and neuroinflammatory insults. Methods: We report a patient presenting with rapidly progressive white matter damage and neurological decline, who was eventually diagnosed with an ACOX1 N237S mutation through whole genome sequencing. We developed a zebrafish model of Mitchell syndrome using transient ubiquitous overexpression of the human ACOX1 N237S variant tagged with GFP. We assayed zebrafish behavior, oligodendrocyte numbers, expression of white matter and inflammatory transcripts, and analysis of peroxisome counts. Results: The patient experienced progressive leukodystrophy and died 2 years after presentation. The transgenic zebrafish showed a decreased swimming ability, which was restored with the reactive microglia-targeted antioxidant dendrimer-N-acetyl-cysteine conjugate. The mutants showed no effect on oligodendrocyte counts but did display activation of the integrated stress response (ISR). Using a novel SKL-targeted mCherry reporter, we found that mutants had reduced density of peroxisomes. Conclusions: We developed a vertebrate (zebrafish) model of Mitchell syndrome using transient ubiquitous overexpression of the human ACOX1 N237S variant. The transgenic mutants exhibited motor impairment and showed signs of activated ISR, but interestingly, there were no changes in oligodendrocyte counts. However, the mutants exhibited a deficiency in the number of peroxisomes, suggesting a possible shared mechanism with the Zellweger spectrum disorders.

miRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4.

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.

Systemic Dendrimer-Peptide Therapies for Wet Age-Related Macular Degeneration.

Wet age-related macular degeneration (AMD) is an end-stage event in a complex pathogenesis of macular degeneration, involving the abnormal growth of blood vessels at the retinal pigment epithelium driven by vascular endothelial growth factor (VEGF). Current therapies seek to interrupt VEGF signaling to halt the progress of neovascularization, but a significant patient population is not responsive. New treatment modalities such as integrin-binding peptides (risuteganib/Luminate/ALG-1001) are being explored to address this clinical need but these treatments necessitate the use of intravitreal injections (IVT), which carries risks of complications and restricts its availability in less-developed countries. Successful systemic delivery of peptide-based therapeutics must overcome obstacles such as degradation by proteinases in circulation and off-target binding. In this work, we present a novel dendrimer-integrin-binding peptide (D-ALG) synthesized with a noncleavable, "clickable" linker. In vitro, D-ALG protected the peptide payload from enzymatic degradation for up to 1.5 h (~90% of the compound remained intact) in a high concentration of proteinase (2 mg/mL) whereas ~90% of free ALG-1001 was degraded in the same period. Further, dendrimer conjugation preserved the antiangiogenic activity of ALG-1001 in vitro with significant reductions in endothelial vessel network formation compared to untreated controls. In vivo, direct intravitreal injections of ALG-1001 and D-ALG produced reductions in the CNV lesion area but in systemically dosed animals, only D-ALG produced significant reductions of CNV lesion area at 14 days. Imaging data suggested that the difference in efficacy may be due to more D-ALG remaining in the target area than ALG-1001 after administration. The results presented here offer a clinically relevant route for peptide therapeutics by addressing the major obstacles that these therapies face in delivery.

Dendrimer nanotherapy targeting of glial dysfunction improves inflammation and neurobehavioral phenotype in adult female Mecp2-heterozygous mouse model of Rett syndrome.

Rett syndrome is an X-linked neurodevelopmental disorder caused by mutation of Mecp2 gene and primarily affects females. Glial cell dysfunction has been implicated in in Rett syndrome (RTT) both in patients and in mouse models of this disorder and can affect synaptogenesis, glial metabolism and inflammation. Here we assessed whether treatment of adult (5-6 months old) symptomatic Mecp2-heterozygous female mice with N-acetyl cysteine conjugated to dendrimer (D-NAC), which is known to target glia and modulate inflammation and oxidative injury, results in improved behavioral phenotype, sleep and glial inflammatory profile. We show that unbiased global metabolomic analysis of the hippocampus and striatum in adult Mecp2-heterozygous mice demonstrates significant differences in lipid metabolism associated with neuroinflammation, providing the rationale for targeting glial inflammation in this model. Our results demonstrate that treatment with D-NAC (10 mg/kg NAC) once weekly is more efficacious than equivalently dosed free NAC in improving the gross neurobehavioral phenotype in symptomatic Mecp2-heterozygous female mice. We also show that D-NAC therapy is significantly better than saline in ameliorating several aspects of the abnormal phenotype including paw clench, mobility, fear memory, REM sleep and epileptiform activity burden. Systemic D-NAC significantly improves microglial proinflammatory cytokine production and is associated with improvements in several aspects of the phenotype including paw clench, mobility, fear memory, and REM sleep, and epileptiform activity burden in comparison to saline-treated Mecp2-hetereozygous mice. Systemic glial-targeted delivery of D-NAC after symptom onset in an older clinically relevant Rett syndrome model shows promise in improving neurobehavioral impairments along with sleep pattern and epileptiform activity burden. These findings argue for the translational value of this approach for treatment of patients with Rett Syndrome.

Microglial-Targeted nSMase2 Inhibitor Fails to Reduce Tau Propagation in PS19 Mice.

The progression of Alzheimer's disease (AD) correlates with the propagation of hyperphosphorylated tau (pTau) from the entorhinal cortex to the hippocampus and neocortex. Neutral sphingomyelinase2 (nSMase2) is critical in the biosynthesis of extracellular vesicles (EVs), which play a role in pTau propagation. We recently conjugated DPTIP, a potent nSMase2 inhibitor, to hydroxyl-PAMAM-dendrimer nanoparticles that can improve brain delivery. We showed that dendrimer-conjugated DPTIP (D-DPTIP) robustly inhibited the spread of pTau in an AAV-pTau propagation model. To further evaluate its efficacy, we tested D-DPTIP in the PS19 transgenic mouse model. Unexpectantly, D-DPTIP showed no beneficial effect. To understand this discrepancy, we assessed D-DPTIP's brain localization. Using immunofluorescence and fluorescence-activated cell-sorting, D-DPTIP was found to be primarily internalized by microglia, where it selectively inhibited microglial nSMase2 activity with no effect on other cell types. Furthermore, D-DPTIP inhibited microglia-derived EV release into plasma without affecting other brain-derived EVs. We hypothesize that microglial targeting allowed D-DPTIP to inhibit tau propagation in the AAV-hTau model, where microglial EVs play a central role in propagation. However, in PS19 mice, where tau propagation is independent of microglial EVs, it had a limited effect. Our findings confirm microglial targeting with hydroxyl-PAMAM dendrimers and highlight the importance of understanding cell-specific mechanisms when designing targeted AD therapies.

A new era in posterior segment ocular drug delivery: Translation of systemic, cell-targeted, dendrimer-based therapies.

Vision impairment and loss due to posterior segment ocular disorders, including age-related macular degeneration and diabetic retinopathy, are a rapidly growing cause of disability globally. Current treatments consist primarily of intravitreal injections aimed at preventing disease progression and characterized by high cost and repeated clinic visits. Nanotechnology provides a promising platform for drug delivery to the eye, with potential to overcome anatomical and physiological barriers to provide safe, effective, and sustained treatment modalities. However, there are few nanomedicines approved for posterior segment disorders, and fewer that target specific cells or that are compatible with systemic administration. Targeting cell types that mediate these disorders via systemic administration may unlock transformative opportunities for nanomedicine and significantly improve patient access, acceptability, and outcomes. We highlight the development of hydroxyl polyamidoamine dendrimer-based therapeutics that demonstrate ligand-free cell targeting via systemic administration and are under clinical investigation for treatment of wet age-related macular degeneration.

Galactosylated hydroxyl-polyamidoamine dendrimer targets hepatocytes and improves therapeutic outcomes in a severe model of acetaminophen poisoning-induced liver failure.

Toxicity to hepatocytes caused by various insults including drugs is a common cause of chronic liver failure requiring transplantation. Targeting therapeutics specifically to hepatocytes is often a challenge since they are relatively nonendocytosing unlike the highly phagocytic Kupffer cells in the liver. Approaches that enable targeted intracellular delivery of therapeutics to hepatocytes have significant promise in addressing liver disorders. We synthesized a galactose-conjugated hydroxyl polyamidoamine dendrimer (D4-Gal) that targets hepatocytes efficiently through the asialoglycoprotein receptors in healthy mice and in a mouse model of acetaminophen (APAP)-induced liver failure. D4-Gal localized specifically in hepatocytes and showed significantly better targeting when compared with the non-Gal functionalized hydroxyl dendrimer. The therapeutic potential of D4-Gal conjugated to N-acetyl cysteine (NAC) was tested in a mouse model of APAP-induced liver failure. A single intravenous dose of a conjugate of D4-Gal and NAC (Gal-d-NAC) improved survival in APAP mice, decreased cellular oxidative injury and areas of necrosis in the liver, even when administered at the delayed time point of 8 h after APAP exposure. Overdose of APAP is the most common cause of acute hepatic injury and liver transplant need in the United States, and is treated with large doses of NAC administered rapidly within 8 h of overdose leading to systemic side effects and poor tolerance. NAC is not effective when treatment is delayed. Our results suggest that D4-Gal is effective in targeting and delivering therapies to hepatocytes and Gal-D-NAC has the potential to salvage and treat liver injury with a broader therapeutic window.

Nanoparticle-based targeting of microglia improves the neural regeneration enhancing effects of immunosuppression in the zebrafish retina.

Retinal Müller glia function as injury-induced stem-like cells in zebrafish but not mammals. However, insights gleaned from zebrafish have been applied to stimulate nascent regenerative responses in the mammalian retina. For instance, microglia/macrophages regulate Müller glia stem cell activity in the chick, zebrafish, and mouse. We previously showed that post-injury immunosuppression by the glucocorticoid dexamethasone accelerated retinal regeneration kinetics in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation of microglia reactivity may therefore enhance the regenerative potential of Müller glia for therapeutic purposes. Here, we investigated potential mechanisms by which post-injury dexamethasone accelerates retinal regeneration kinetics, and the effects of dendrimer-based targeting of dexamethasone to reactive microglia. Intravital time-lapse imaging revealed that post-injury dexamethasone inhibited microglia reactivity. The dendrimer-conjugated formulation: (1) decreased dexamethasone-associated systemic toxicity, (2) targeted dexamethasone to reactive microglia, and (3) improved the regeneration enhancing effects of immunosuppression by increasing stem/progenitor proliferation rates. Lastly, we show that the gene rnf2 is required for the enhanced regeneration effect of D-Dex. These data support the use of dendrimer-based targeting of reactive immune cells to reduce toxicity and enhance the regeneration promoting effects of immunosuppressants in the retina.

Dendrimer-enabled targeted delivery attenuates glutamate excitotoxicity and improves motor function in a rabbit model of cerebral palsy.

Glutamate carboxypeptidase II (GCPII), localized on the surface of astrocytes and activated microglia, regulates extracellular glutamate concentration in the central nervous system (CNS). We have previously shown that GCPII is upregulated in activated microglia in the presence of inflammation. Inhibition of GCPII activity could reduce glutamate excitotoxicity, which may decrease inflammation and promote a 'normal' microglial phenotype. 2-(3-Mercaptopropyl) pentanedioic acid (2-MPPA) is the first GCPII inhibitor that underwent clinical trials. Unfortunately, immunological toxicities have hindered 2-MPPA clinical translation. Targeted delivery of 2-MPPA specifically to activated microglia and astrocytes that over-express GCPII has the potential to mitigate glutamate excitotoxicity and attenuate neuroinflammation. In this study, we demonstrate that 2-MPPA when conjugated to generation-4, hydroxyl-terminated polyamidoamine (PAMAM) dendrimers (D-2MPPA) localize specifically in activated microglia and astrocytes only in newborn rabbits with cerebral palsy (CP), not in controls. D-2MPPA treatment led to higher 2-MPPA levels in the injured brain regions compared to 2-MPPA treatment, and the extent of D-2MPPA uptake correlated with the injury severity. D-2MPPA was more efficacious than 2-MPPA in decreasing extracellular glutamate level in ex vivo brain slices of CP kits, and in increasing transforming growth factor beta 1 (TGF-ß1) level in primary mixed glial cell cultures. A single systemic intravenous dose of D-2MPPA on postnatal day 1 (PND1) decreased microglial activation and resulted in a change in microglial morphology to a more ramified form along with amelioration of motor deficits by PND5. These results indicate that targeted dendrimer-based delivery specifically to activated microglia and astrocytes can improve the efficacy of 2-MPPA by attenuating glutamate excitotoxicity and microglial activation.

Dendrimer-Conjugated Glutamate Carboxypeptidase II Inhibitor Restores Microglial Changes in a Rabbit Model of Cerebral Palsy.

We have previously shown that maternal endotoxin exposure leads to a phenotype of cerebral palsy and pro-inflammatory microglia in the brain in neonatal rabbits. "Activated" microglia overexpress the enzyme glutamate carboxypeptidase II (GCPII) that hydrolyzes N-acetylaspartylglutamate to N-acetylaspartate and glutamate, and we have shown previously that inhibiting microglial GCPII is neuroprotective. Glutamate-induced injury and associated immune signaling can alter microglial responses including microglial process movements for surveillance and phagocytosis. We hypothesize that inhibition of GCPII activity could alter microglial phenotype and normalize microglial process movement/dynamics. Newborn rabbit kits exposed to endotoxin in utero, when treated with dendrimer-conjugated 2-(phosphonomethyl)-pentanedioic acid (D-2PMPA), a potent and selective inhibitor of microglial GCPII, showed profound changes in microglial phenotype within 48 h of treatment. Live imaging of hippocampal microglia in ex vivo brain slice preparations revealed larger cell body and phagocytic cup sizes with less stable microglia processes in CP kits compared to healthy controls. D-2PMPA treatment led to significant reversal of microglial process stability to healthy control levels. Our results emphasize the importance of microglial process dynamics in determining the state of microglial function in the developing brain and demonstrate how GCPII inhibition specifically in microglia can effectively change the microglial process motility to healthy control levels, potentially impacting migration, phagocytosis, and inflammatory functions.

Targeted Microglial Attenuation through Dendrimer-Drug Conjugates Improves Glaucoma Neuroprotection.

Retinal microglial/macrophage activation and optic nerve (ON) microglial/macrophage activation are glaucoma biomarkers and potential therapeutic targets for this blinding disease. We report targeting of activated microglia by PAMAM dendrimers in a rat glaucoma model and neuroprotection by N-acetylcysteine-conjugated dendrimer (D-NAC) conjugates in a post-injury rescue experiment. Intravitreally delivered fluorescently labeled dendrimer (D-Cy5) conjugates targeted and were retained in Iba-1-positive cells (90% at 7 days and 55% after 28 days) in the retina following intraocular pressure (IOP) elevation, while systemically delivered D-Cy5 targeted ON cells. A single intravitreal D-NAC dose given 1 week after IOP elevation significantly reduced transcription of pro-inflammatory (IL-6, MCP-1, IL-1ß) and A1 astrocyte (Serping1, Fkbp5, Amigo2) markers and increased survival of retinal ganglion cells (39 ± 12%) versus BSS- (20 ± 15%, p = 0.02) and free NAC-treated (26 ± 14%, p = 0.15) eyes. These results highlight the potential of dendrimer-targeted microglia and macrophages for early glaucoma detection and as a neuroprotective therapeutic target.

Targeted Brain Delivery of Dendrimer-4-Phenylbutyrate Ameliorates Neurological Deficits in a Long-Term ABCD1-Deficient Mouse Model of X-Linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a genetic disorder that presents neurologically as either a rapid and fatal cerebral demyelinating disease in childhood (childhood cerebral adrenoleukodystrophy; ccALD) or slow degeneration of the spinal cord in adulthood (adrenomyeloneuropathy; AMN). All forms of ALD result from mutations in the ATP Binding Cassette Subfamily D Member (ABCD) 1 gene, encoding a peroxisomal transporter responsible for the import of very long chain fatty acids (VLCFA) and results mechanistically in a complex array of dysfunction, including endoplasmic reticulum stress, oxidative stress, mitochondrial dysfunction, and inflammation. Few therapeutic options exist for these patients; however, an additional peroxisomal transport protein (ABCD2) has been successfully targeted previously for compensation of dysfunctional ABCD1. 4-Phenylbutyrate (4PBA), a potent activator of the ABCD1 homolog ABCD2, is FDA approved, but use for ALD has been stymied by a short half-life and thus a need for unfeasibly high doses. We conjugated 4PBA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-4PBA) to a create a long-lasting and intracellularly targeted approach which crosses the blood-brain barrier to upregulate Abcd2 and its downstream pathways. Across two studies, Abcd1 knockout mice administered D-4PBA long term showed neurobehavioral improvement and increased Abcd2 expression. Furthermore, when the conjugate was administered early, significant reduction of VLCFA and improved survival of spinal cord neurons was observed. Taken together, these data show improved efficacy of D-4PBA compared to previous studies of free 4PBA alone, and promise for D-4PBA in the treatment of complex and chronic neurodegenerative diseases using a dendrimer delivery platform that has shown successes in recent clinical trials. While recovery in our studies was partial, combined therapies on the dendrimer platform may offer a safe and complete strategy for treatment of ALD.

Dendrimer-Conjugated nSMase2 Inhibitor Reduces Tau Propagation in Mice.

Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid-ß and hyperphosphorylated tau (pTau), which can spread throughout the brain via extracellular vesicles (EVs). Membrane ceramide enrichment regulated by the enzyme neutral sphingomyelinase 2 (nSMase2) is a critical component of at least one EV biogenesis pathway. Our group recently identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), the most potent (30 nM) and selective inhibitor of nSMase2 reported to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), modest brain penetration, and rapid clearance, limiting its clinical translation. To enhance its PK properties, we conjugated DPTIP to a hydroxyl-PAMAM dendrimer delivery system, creating dendrimer-DPTIP (D-DPTIP). In an acute brain injury model, orally administered D-DPTIP significantly reduced the intra-striatal IL-1ß-induced increase in plasma EVs up to 72 h post-dose, while oral DPTIP had a limited effect. In a mouse tau propagation model, where a mutant hTau (P301L/S320F) containing adeno-associated virus was unilaterally seeded into the hippocampus, oral D-DPTIP (dosed 3× weekly) significantly inhibited brain nSMase2 activity and blocked the spread of pTau to the contralateral hippocampus. These data demonstrate that dendrimer conjugation of DPTIP improves its PK properties, resulting in significant inhibition of EV propagation of pTau in mice. Dendrimer-based delivery of DPTIP has the potential to be an exciting new therapeutic for AD.

Dendrimer-siRNA Conjugates for Targeted Intracellular Delivery in Glioblastoma Animal Models.

Small interfering RNAs (siRNAs) are potent weapons for gene silencing, with an opportunity to correct defective genes and stop the production of undesirable proteins, with many applications in central nervous system (CNS) disorders. However, successful delivery of siRNAs to the brain parenchyma faces obstacles such as the blood-brain barrier (BBB), brain tissue penetration, and targeting of specific cells. In addition, siRNAs are unstable under physiological conditions and are susceptible to protein binding and enzymatic degradation, necessitating a higher dosage to remain effective. To address these issues and advance siRNA delivery, we report the development of covalently conjugated hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer-siRNA conjugates, demonstrated with a siRNA against GFP (siGFP) conjugate (D-siGFP) utilizing glutathione-sensitive linkers. This allows for precise nucleic acid loading, protects the payload from premature degradation, delivers the siRNA cargo into cells, and achieves significant GFP knockdown in vitro (∼40%) and in vivo (∼30%). Compared to commercially available delivery systems such as RNAi Max and Lipofectamine, D-siGFP retains the potency of the siRNA in vitro. In addition, the dendrimer-siGFP conjugate significantly enhances the half-life of siRNA in the presence of plasma and endonucleases and maintains the passive targeting ability of PAMAM dendrimers to reactive microglia. When administered intratumorally to orthotopic glioblastoma multiform tumors (GBM) in CX3CR-1GFP mice, D-siGFP localizes in tumor-associated macrophages (TAMs) within the tumor parenchyma, minimizing off-target effects in other cell populations. The facile conjugation strategy for dendrimer-siRNA conjugates presented here offers a promising approach for targeted, systemic intracellular delivery of siRNA, serving as a potential bridge for the clinical translation of RNAi therapies.

Dendrimer nanotherapy for severe COVID-19 attenuates inflammation and neurological injury markers and improves outcomes in a phase2a clinical trial.

Hyperinflammation triggered by SARS-CoV-2 is a major cause of disease severity, with activated macrophages implicated in this response. OP-101, a hydroxyl-polyamidoamine dendrimer-N-acetylcysteine conjugate that specifically targets activated macrophages, improves outcomes in preclinical models of systemic inflammation and neuroinflammation. In this multicenter, randomized, double-blind, placebo-controlled, adaptive phase 2a trial, we evaluated safety and preliminary efficacy of OP-101 in patients with severe COVID-19. Twenty-four patients classified as having severe COVID-19 with a baseline World Health Organization seven-point ordinal scale of ≥5 were randomized to receive a single intravenous dose of placebo (n = 7 patients) or OP-101 at 2 (n = 6), 4 (n = 6), or 8 mg/kg (n = 5 patients). All study participants received standard of care, including corticosteroids. OP-101 at 4 mg/kg was better than placebo at decreasing inflammatory markers; OP-101 at 4 and 8 mg/kg was better than placebo at reducing neurological injury markers, (neurofilament light chain and glial fibrillary acidic protein). Risk for the composite outcome of mechanical ventilation or death at 30 and 60 days after treatment was 71% (95% CI: 29%, 96%) for placebo and 18% (95% CI: 4%, 43%; P = 0.021) for the pooled OP-101 treatment arms. At 60 days, 3 of 7 patients given placebo and 14 of 17 OP-101-treated patients were surviving. No drug-related adverse events were reported. These data show that OP-101 was well tolerated and may have potential to treat systemic inflammation and neuronal injury, reducing morbidity and mortality in hospitalized patients with severe COVID-19.

Dendrimer-Based N-Acetyl Cysteine Maternal Therapy Ameliorates Placental Inflammation via Maintenance of M1/M2 Macrophage Recruitment.

Intrauterine inflammation (IUI) is the primary cause of spontaneous preterm birth and predisposes neonates to long-term sequelae, including adverse neurological outcomes. N-acetyl-L-cysteine (NAC) is the amino acid L-cysteine derivative and a precursor to the antioxidant glutathione (GSH). NAC is commonly used clinically as an antioxidant with anti-inflammatory properties. Poor bioavailability and high protein binding of NAC necessitates the use of high doses resulting in side effects including nausea, vomiting, and gastric disruptions. Therefore, dendrimer-based therapy can specifically target the drug to the cells involved in inflammation, reducing side effects with efficacy at much lower doses than the free drug. Towards development of the new therapies for the treatment of maternal inflammation, we successfully administered dendrimer-based N-Acetyl Cysteine (DNAC) in an animal model of IUI to reduce preterm birth and perinatal inflammatory response. This study explored the associated immune mechanisms of DNAC treatment on placental macrophages following IUI, especially on M1/M2 type macrophage polarization. Our results demonstrated that intraperitoneal maternal DNAC administration significantly reduced the pro-inflammatory cytokine mRNA of Il1ß and Nos2, and decreased CD45+ leukocyte infiltration in the placenta following IUI. Furthermore, we found that DNAC altered placental immune profile by stimulating macrophages to change to the M2 phenotype while decreasing the M1 phenotype, thus suppressing the inflammatory responses in the placenta. Our study provides evidence for DNAC therapy to alleviate IUI via the maintenance of macrophage M1/M2 imbalance in the placenta.

Dendrimer-2PMPA Delays Muscle Function Loss and Denervation in a Murine Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease where muscle weakness and neuromuscular junction (NMJ) denervation precede motor neuron cell death. Although acetylcholine is the canonical neurotransmitter at the mammalian NMJ synapse, glutamate has recently been identified as a critical neurotransmitter for NMJ development and maintenance. One source of glutamate is through the catabolism of N-acetyl-aspartyl-glutamate (NAAG), which is found in mM concentrations in mammalian motoneurons, where it is released upon stimulation and hydrolyzed to glutamate by the glial enzyme glutamate carboxypeptidase II (GCPII). Using the SOD1G93A model of ALS, we found an almost fourfold elevation of GCPII enzymatic activity in SOD1G93A versus WT muscle and a robust increase in GCPII expression which was specifically associated with activated macrophages infiltrating the muscle. 2-(Phosphonomethyl)pentanedioic acid (2PMPA) is a potent GCPII inhibitor which robustly blocks glutamate release from NAAG but is highly polar with limited tissue penetration. To improve this, we covalently attached 2PMPA to a hydroxyl polyamidoamine (PAMAM-G4-OH) dendrimer delivery system (D-2PMPA) which is known to target activated macrophages in affected tissues. Systemic D-2PMPA therapy (20 mg/kg 2PMPA equivalent; IP 2 × /week) was found to localize in muscle macrophages in SOD1G93A mice and completely normalize the enhanced GCPII activity. Although no changes in body weight or survival were observed, D-2PMPA significantly improved grip strength and inhibited the loss of NMJ innervation in the gastrocnemius muscles. Our finding that inhibiting elevated GCPII activity in SOD1G93A muscle can prolong muscle function and delay NMJ denervation may have early therapeutic implications for ALS patients.

Dendrimer-2PMPA selectively blocks upregulated microglial GCPII activity and improves cognition in a mouse model of multiple sclerosis.

Cognitive impairment is a common aspect of multiple sclerosis (MS) for which there are no treatments. Reduced brain N-acetylaspartylglutamate (NAAG) levels are linked to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate and glutamate. GCPII activity is upregulated multifold in microglia following neuroinflammation. Although several GCPII inhibitors, such as 2-PMPA, elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, none are clinically available due to poor bioavailability and limited brain penetration. Hydroxyl-dendrimers have been successfully used to selectively deliver drugs to activated glia. Methods: We attached 2-PMPA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-2PMPA) using a click chemistry approach. Cy5-labelled-D-2PMPA was used to visualize selective glial uptake in vitro and in vivo. D-2PMPA was evaluated for anti-inflammatory effects in LPS-treated glial cultures. In experimental autoimmune encephalomyelitis (EAE)-immunized mice, D-2PMPA was dosed biweekly starting at disease onset and cognition was assessed using the Barnes maze, and GCPII activity was measured in CD11b+ hippocampal cells. Results: D-2PMPA showed preferential uptake into microglia and robust anti-inflammatory activity, including elevations in NAAG, TGFß, and mGluR3 in glial cultures. D-2PMPA significantly improved cognition in EAE mice, even though physical severity was unaffected. GCPII activity increased >20-fold in CD11b+ cells from EAE mice, which was significantly mitigated by D-2PMPA treatment. Conclusions: Hydroxyl dendrimers facilitate targeted drug delivery to activated microglia. These data support further development of D-2PMPA to attenuate elevated microglial GCPII activity and treat cognitive impairment in MS.

Systemic administration of dendrimer N-acetyl cysteine improves outcomes and survival following cardiac arrest.

Cardiac arrest (CA), the sudden cessation of effective cardiac pumping function, is still a major clinical problem with a high rate of early and long-term mortality. Post-cardiac arrest syndrome (PCAS) may be related to an early systemic inflammatory response leading to exaggerated and sustained neuroinflammation. Therefore, early intervention with targeted drug delivery to attenuate neuroinflammation may greatly improve therapeutic outcomes. Using a clinically relevant asphyxia CA model, we demonstrate that a single (i.p.) dose of dendrimer-N-acetylcysteine conjugate (D-NAC), can target "activated" microglial cells following CA, leading to an improvement in post-CA survival rate compared to saline (86% vs. 45%). D-NAC treatment also significantly improved gross neurological score within 4 h of treatment (p < 0.05) and continued to show improvement at 48 h (p < 0.05). Specifically, there was a substantial impairment in motor responses after CA, which was subsequently improved with D-NAC treatment (p < 0.05). D-NAC also mitigated hippocampal cell density loss seen post-CA in the CA1 and CA3 subregions (p < 0.001). These results demonstrate that early therapeutic intervention even with a single D-NAC bolus results in a robust sustainable improvement in long-term survival, short-term motor deficits, and neurological recovery. Our current work lays the groundwork for a clinically relevant therapeutic approach to treating post-CA syndrome.

ssDNA nanotubes for selective targeting of glioblastoma and delivery of doxorubicin for enhanced survival.

Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.