Dendrimer-mediated delivery of N-acetyl cysteine to microglia in a mouse model of Rett syndrome

Abstract

BackgroundRett syndrome (RTT) is a pervasive developmental disorder that is progressive and has no effective cure. Immune dysregulation, oxidative stress, and excess glutamate in the brain mediated by glial dysfunction have been implicated in the pathogenesis and worsening of symptoms of RTT. In this study, we investigated a new nanotherapeutic approach to target glia for attenuation of brain inflammation/injury both in vitro and in vivo using a Mecp2-null mouse model of Rett syndrome.MethodsTo determine whether inflammation and immune dysregulation were potential targets for dendrimer-based therapeutics in RTT, we assessed the immune response of primary glial cells from Mecp2-null and wild-type (WT) mice to LPS. Using dendrimers that intrinsically target activated microglia and astrocytes, we studied N-acetyl cysteine (NAC) and dendrimer-conjugated N-acetyl cysteine (D-NAC) effects on inflammatory cytokines by PCR and multiplex assay in WT vs Mecp2-null glia. Since the cysteine-glutamate antiporter (Xc−) is upregulated in Mecp2-null glia when compared to WT, the role of Xc− in the uptake of NAC and l-cysteine into the cell was compared to that of D-NAC using BV2 cells in vitro. We then assessed the ability of D-NAC given systemically twice weekly to Mecp2-null mice to improve behavioral phenotype and lifespan.ResultsWe demonstrated that the mixed glia derived from Mecp2-null mice have an exaggerated inflammatory and oxidative stress response to LPS stimulation when compared to WT glia. Expression of Xc− was significantly upregulated in the Mecp2-null glia when compared to WT and was further increased in the presence of LPS stimulation. Unlike NAC, D-NAC bypasses the Xc− for cell uptake, increasing intracellular GSH levels while preventing extracellular glutamate release and excitotoxicity. Systemically administered dendrimers were localized in microglia in Mecp2-null mice, but not in age-matched WT littermates. Treatment with D-NAC significantly improved behavioral outcomes in Mecp2-null mice, but not survival.ConclusionsThese results suggest that delivery of drugs using dendrimer nanodevices offers a potential strategy for targeting glia and modulating oxidative stress and immune responses in RTT.Electronic supplementary materialThe online version of this article (10.1186/s12974-017-1004-5) contains supplementary material, which is available to authorized users.

Scalable synthesis and validation of PAMAM dendrimer‐N‐acetyl cysteine conjugate for potential translation

“…We found that the Cy5‐D‐NAC conjugate co‐localized with activated microglia, indicated by ameboid soma with shortened processes, in the periventricular white matter region (PVR), including corpus callosum, and in the lateral ventricle in the cortex (Figure B). These results indicate that D‐NAC crossed the blood‐brain barrier, reached the injured white matter region, and targeted activated microglia in a cell‐specific manner consistent with our many previous findings . These properties make D‐NAC an ideal therapeutic reagent in the treatment of neonatal brain injuries.…”

“…Before proceeding to in vitro efficacy experiments, we evaluated the cell cytotoxicity of PAMAM‐G4‐OH, NAC and D‐NAC in BV‐2 cells to establish the therapeutic window. We have previously reported that D‐NAC does not induce any cytotoxicity to brain primary mixed glial cultures . In this study, we analyzed the results from three different batches of D‐NAC synthesized using new route and compared them for reproducibility (Figure A,B, Table ).…”

“…We have previously demonstrated that the dendrimer uptake is increased when the microglial cells are activated . D‐NAC treatment improves intracellular glutathione (GSH) levels while suppressing glutamate release significantly compared to NAC …”

Brain Delivery of Multifunctional Dendrimer Protein Bioconjugates

“…For example, Tyler et al demonstrated a decrease in gene expression of neurotensin receptor using peptide nucleic acids at a brain molecular concentration almost fivefold lower than G3‐DSA 54. Nance et al found a % ID of 0.003% (healthy animal) for G4‐OH PAMAM dendrimer loaded with N‐acetylcysteine to efficiently deliver the drug load in the diseased animal with 100‐fold greater brain accumulation compared to free drug 26. Additionally, from observed data it could be supposed that DSA might protect drug load from lysosomal entrapment holding the possibility of even higher efficiency.…”

“…Furthermore, a successful antineuroinflammatory effect could be obtained for the treatment of the Rett syndrome, a pervasive developmental disorder. Although in this case D‐NAC significantly improved behavioral outcomes, it could not prevent the lethal outcome 26. The need of very careful investigations presenting new CNS therapeutic strategies was revealed by the case of poly(propyleneimine) glycodendrimers, which offer antiamyloidogenic effects but at the same time cause cognitive decline in WT mice 18.…”