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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.
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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.
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Parálisis Cerebral , Dendrímeros , Animales , Conejos , Glutamato Carboxipeptidasa II , Parálisis Cerebral/tratamiento farmacológico , Microglía , Endotoxinas , GlutamatosRESUMEN
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.
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Dendrímeros , Glaucoma , Ratas , Animales , Microglía , Neuroprotección , Modelos Animales de EnfermedadRESUMEN
Oxidative stress, inflammation and neovascularization are the key pathological events that are implicated in human age-related macular degeneration (AMD). There are a limited number of animal models available for evaluating and developing new therapies. Most models represent late exudative or neovascular AMD (nAMD) but there is a relative paucity of models that mimic early events in AMD. The purpose of this study is to characterize the evolution of oxidative stress, inflammation, retinal degeneration and neovascularization in a rat model of AMD, created by subretinal injection of human lipid hydroperoxide (HpODE) that found in the sub-macular region in aged and AMD patients. Subretinal HpODE induced retinal pigment epithelium (RPE) and retinal degeneration resulting in loss of RPE cells, photoreceptors and retinal thinning. RPE degeneration and atrophy were detected by day 5, followed by neural tissue degeneration at day 12 with robust TUNEL positive cells. Western blot analysis confirmed an increase in pro-apoptotic Bak protein at day 12 in retinal tissues. Oxidative damage biomarkers (4-hydroxynonenal, malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and nitrotyrosine) increased in retinal tissue from days 5-12. Müller glial activation was observed in the HpODE injected area at day 5 followed by its remodeling and migration in the outer retina by day 20. RT-qPCR analysis further indicated upregulation of pro-inflammatory genes (TNF-α, IL-1ß and IL-6) both in retinal and RPE/choroidal tissue as early as day 2 and persisted until day 12. Upregulation of oxidative stress markers such as NADPH oxidase (NOX and DOUX family) was detected early in retinal tissue by day 2 followed by its upregulation in choroidal tissue at day 5. Neovascularization was demonstrated from day 12 to day 20 post HpODE injection in choroidal tissue. The results from this study indicate that subretinal HpODE induces advanced AMD phenotypes comprising many aspects of both dry/early and late) and neovascular/late AMD as observed in humans. Within 3 weeks via oxidative damage, upregulation of reactive oxygen species and pro-inflammatory genes, pro-apoptotic Bak and pro-angiogenic VEGF upregulation occurs leading to CNV formation. This experimental model of subretinal HpODE is an appropriate model for the study of AMD and provides an important platform for translational and basic research in developing new therapies particularly for early/dry AMD where currently no viable therapies are available.
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Neovascularización Coroidal/etiología , Atrofia Geográfica/inducido químicamente , Inflamación/etiología , Peróxidos Lipídicos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Neovascularización Retiniana/etiología , Degeneración Macular Húmeda/inducido químicamente , Animales , Biomarcadores/metabolismo , Western Blotting , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Atrofia Geográfica/patología , Etiquetado Corte-Fin in Situ , Inflamación/metabolismo , Inflamación/patología , Microscopía Confocal , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Degeneración Macular Húmeda/patologíaRESUMEN
Over two million people die of liver disorders every year globally. Hepatocytes are the key cells affected in several acute and chronic liver diseases. The current clinical outcomes of liver-targeted nanoparticles are limited, necessitating the need to develop smart hepatocyte-targeted drug delivery systems. Here, we present the rational design and development of a hepatocyte-targeting glycodendrimer (GAL-24) built from biocompatible building blocks, using expedite and facile chemical methodology. GAL-24 is designed to inherently target asialoglycoprotein receptor 1 (ASGP-R) on hepatocytes and shows significant accumulation in the liver (20% of injected dose), just 1 h after systemic administration. This is highly specific to hepatocytes, with over 80% of hepatocytes showing GAL-24-Cy5 signal at 24 h. GAL-24-Cy5 maintains hepatocyte-targeting capabilities in both a mouse model of severe acetaminophen poisoning-induced hepatic necrosis and a rat model of nonalcoholic steatohepatitis (NASH). This GAL-24 nanoplatform holds great promise for improved drug delivery to hepatocytes to combat many liver disorders.
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Dendrímeros , Hepatopatías , Preparaciones Farmacéuticas , Animales , Sistemas de Liberación de Medicamentos , Galactosa , Hepatocitos , Hígado , Hepatopatías/tratamiento farmacológico , Ratones , RatasRESUMEN
BACKGROUND: Hypothermic circulatory arrest (HCA) is associated with neurologic morbidity, in part mediated by activation of the N-methyl-D-aspartate glutamate receptor causing excitotoxicity and neuronal apoptosis. Using a canine model, we hypothesized that the N-methyl-D-aspartate receptor antagonist MK801 would provide neuroprotection and that MK801 conjugation to dendrimer nanoparticles would improve efficacy. MATERIALS AND METHODS: Male hound dogs were placed on cardiopulmonary bypass, cooled to 18°C, and underwent 90 min of HCA. Dendrimer conjugates (d-MK801) were prepared by covalently linking dendrimer surface OH groups to MK801. Six experimental groups received either saline (control), medium- (0.15 mg/kg) or high-dose (1.56 mg/kg) MK801, or low- (0.05 mg/kg), medium-, or high-dose d-MK801. At 24, 48, and 72 h after HCA, animals were scored by a standardized neurobehavioral paradigm (higher scores indicate increasing deficits). Cerebrospinal fluid was obtained at baseline, eight, 24, 48, and 72 h after HCA. At 72 h, brains were examined for histopathologic injury in a blinded manner (higher scores indicate more injury). RESULTS: Neurobehavioral deficit scores were reduced by low-dose d-MK801 on postoperative day two (P < 0.05) and by medium-dose d-MK801 on postoperative day 3 (P = 0.05) compared with saline controls, but free drug had no effect. In contrast, high-dose free MK801 significantly improved histopathology scores compared with saline (P < 0.05) and altered biomarkers of injury in cerebrospinal fluid, with a significant reduction in phosphorylated neurofilament-H for high-dose MK801 versus saline (P < 0.05). CONCLUSIONS: Treatment with MK-801 demonstrated significant improvement in neurobehavioral and histopathology scores after HCA, although not consistently across doses and conjugates.
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Paro Circulatorio Inducido por Hipotermia Profunda/efectos adversos , Maleato de Dizocilpina/farmacología , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Encéfalo/patología , Cognición , Perros , MasculinoRESUMEN
BACKGROUND: Neuroinflammation mediated by microglia plays a central role in the pathogenesis of perinatal/neonatal brain injury, including cerebral palsy (CP). Therapeutics mitigating neuroinflammation potentially provide an effective strategy to slow the disease progression and rescue normal brain development. Building on our prior results which showed that a generation-4 hydroxyl poly(amidoamine) (PAMAM) dendrimer could deliver drugs specifically to activated glia from systemic circulation, we evaluated the sustained efficacy of a generation-6 (G6) hydroxyl-terminated PAMAM dendrimer that showed a longer blood circulation time and increased brain accumulation. N-acetyl-L-cysteine (NAC), an antioxidant and anti-inflammatory agent that has high plasma protein binding properties and poor brain penetration, was conjugated to G6-PAMAM dendrimer-NAC (G6D-NAC). The efficacy of microglia-targeted G6D-NAC conjugate was evaluated in a clinically relevant rabbit model of CP, with a mild/moderate CP phenotype to provide a longer survival of untreated CP kits, enabling the assessment of sustained efficacy over 15 days of life. METHODS: G6D-NAC was conjugated and characterized. Cytotoxicity and anti-inflammatory assays were performed in BV-2 microglial cells. The efficacy of G6D-NAC was evaluated in a rabbit model of CP. CP kits were randomly divided into 5 groups on postnatal day 1 (PND1) and received an intravenous injection of a single dose of PBS, or G6D-NAC (2 or 5 mg/kg), or NAC (2 or 5 mg/kg). Neurobehavioral tests, microglia morphology, and neuroinflammation were evaluated at postnatal day 5 (PND5) and day 15 (PND15). RESULTS: A single dose of systemic 'long circulating' G6D-NAC showed a significant penetration across the impaired blood-brain-barrier (BBB), delivered NAC specifically to activated microglia, and significantly reduced microglia-mediated neuroinflammation in both the cortex and cerebellum white matter areas. Moreover, G6D-NAC treatment significantly improved neonatal rabbit survival rate and rescued motor function to nearly healthy control levels at least up to 15 days after birth (PND15), while CP kits treated with free NAC died before PND9. CONCLUSIONS: Targeted delivery of therapeutics to activated microglia in neonatal brain injury can ameliorate pro-inflammatory microglial responses to injury, promote survival rate, and improve neurological outcomes that can be sustained for a long period. Appropriate manipulation of activated microglia enabled by G6D-NAC can impact the injury significantly beyond inflammation.
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Materiales Biocompatibles/administración & dosificación , Parálisis Cerebral/tratamiento farmacológico , Dendrímeros/administración & dosificación , Modelos Animales de Enfermedad , Nanomedicina/métodos , Animales , Animales Recién Nacidos , Línea Celular , Parálisis Cerebral/inducido químicamente , Parálisis Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Lipopolisacáridos/toxicidad , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Embarazo , Conejos , Factores de Tiempo , Resultado del TratamientoRESUMEN
Glioblastoma exhibits high mortality rates due to challenges with drug delivery to the brain and into solid tumors. This two-pronged barrier necessitates high doses of systemic therapies, resulting in significant off-target toxicities. Recently, dendrimer-nanomedicines (without ligands) have shown promise for targeting specific cells in brain tumors from systemic circulation, for improved efficacy and amelioration of systemic toxicities. A dendrimer-rapamycin conjugate (D-Rapa) is presented here that specifically targets tumor-associated macrophages (TAMs) in glioblastoma from systemic administration. D-Rapa improves suppression of pro-tumor expression in activated TAMs and antiproliferative properties of rapamycin in glioma cells in vitro. In vivo, D-Rapa localizes specifically within TAMs, acting as depots to release rapamycin into the tumor microenvironment. This targeted delivery strategy yields improved reduction in tumor burden and systemic toxicities in a challenging, clinically relevant orthotopic syngeneic model of glioblastoma, demonstrating the significant potential of dendrimers as targeted immunotherapies for improving glioblastoma treatment, still an unmet need.
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Neoplasias Encefálicas , Dendrímeros , Glioblastoma , Neoplasias Encefálicas/tratamiento farmacológico , Dendrímeros/uso terapéutico , Sistemas de Liberación de Medicamentos , Glioblastoma/tratamiento farmacológico , Humanos , Sirolimus/farmacología , Microambiente Tumoral , Macrófagos Asociados a TumoresRESUMEN
Mitochondria mediate critical cellular processes, including proliferation, apoptosis, and immune responses; as such, their dysfunction is pathogenic in many neurodegenerative disorders and cancers. In glioblastoma, targeted delivery of mitochondria-focused anticancer therapies has failed to translate into clinical success due to the nonspecific cellular localization, heterogeneity of receptor expression across patients, poor transport across biological barriers to reach the brain, tumor, and mitochondria, and systemic side effects. Strategies that can overcome brain and solid tumor barriers and selectively target mitochondria within specific cell types may lead to improvements in glioblastoma treatment. Developments in dendrimer-mediated nanomedicines have shown promise targeting tumor-associated macrophages (TAMs) in glioblastoma, following systemic administration. Here, we present a novel dendrimer conjugated to the translocator protein (18 kDa) (TSPO) ligand 5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-ylacetamide (DPA). We developed a clickable DPA for conjugation on the dendrimer surface and demonstrated in vitro that the dendrimer-DPA conjugate (D-DPA) significantly increases dendrimer colocalization with mitochondria. Compared to free TSPO ligand PK11195, D-DPA stimulates greater antitumor immune signaling. In vivo, we show that D-DPA targets mitochondria specifically within TAMs following systemic administration. Our results demonstrate that dendrimers can achieve TAM-specific targeting in glioblastoma and can be further modified to target specific intracellular compartments for organelle-specific drug delivery.
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Dendrímeros , Glioblastoma , Línea Celular Tumoral , Dendrímeros/farmacología , Dendrímeros/uso terapéutico , Glioblastoma/tratamiento farmacológico , Humanos , Ligandos , Mitocondrias , Receptores de GABA/uso terapéutico , Macrófagos Asociados a TumoresRESUMEN
OBJECTIVE: X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder due to mutations in the peroxisomal very long-chain fatty acyl-CoA transporter, ABCD1, with limited therapeutic options. ALD may manifest in a slowly progressive adrenomyeloneuropathy (AMN) phenotype, or switch to rapid inflammatory demyelinating cerebral disease (cALD), in which microglia have been shown to play a pathophysiological role. The aim of this study was to determine the role of patient phenotype in the immune response of ex vivo monophagocytic cells to stimulation, and to evaluate the efficacy of polyamidoamine dendrimer conjugated to the antioxidant precursor N-acetyl-cysteine (NAC) in modulating this immune response. METHODS: Human monophagocytic cells were derived from fresh whole blood, from healthy (n = 4), heterozygote carrier (n = 4), AMN (n = 7), and cALD (n = 4) patients. Cells were exposed to very long-chain fatty acids (VLCFAs; C24:0 and C26:0) and treated with dendrimer-NAC (D-NAC). RESULTS: Ex vivo exposure to VLCFAs significantly increased tumor necrosis factor α (TNFα) and glutamate secretion from cALD patient macrophages. Additionally, a significant reduction in total intracellular glutathione was observed in cALD patient cells. D-NAC treatment dose-dependently reduced TNFα and glutamate secretion and replenished total intracellular glutathione levels in cALD patient macrophages, more efficiently than NAC. Similarly, D-NAC treatment decreased glutamate secretion in AMN patient cells. INTERPRETATION: ALD phenotypes display unique inflammatory profiles in response to VLCFA stimulation, and therefore ex vivo monophagocytic cells may provide a novel test bed for therapeutic agents. Based on our findings, D-NAC may be a viable therapeutic strategy for the treatment of cALD. Ann Neurol 2018;84:452-462.
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Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/metabolismo , Dendrímeros/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Acetilcisteína/metabolismo , Adulto , Anciano , Antioxidantes/metabolismo , Encéfalo/metabolismo , Niño , Femenino , Humanos , Masculino , Microglía/metabolismo , Persona de Mediana Edad , Fenotipo , Adulto JovenRESUMEN
After publication of the article [1], it has been brought to our attention that an author's name has been formatted incorrectly.
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Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system (CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.
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Materiales Biocompatibles/farmacocinética , Parálisis Cerebral/patología , Dendrímeros/farmacocinética , Microglía/citología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Lipopolisacáridos/efectos adversos , Microglía/química , Microglía/inmunología , Microscopía Confocal , Neuroglía/química , Neuroglía/citología , Neuroglía/inmunología , ConejosRESUMEN
BACKGROUND: Rett 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. METHODS: To 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. RESULTS: We 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. CONCLUSIONS: These 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.
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Acetilcisteína/uso terapéutico , Encéfalo/patología , Dendrímeros/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Microglía/efectos de los fármacos , Síndrome de Rett/tratamiento farmacológico , Acetilcisteína/farmacología , Animales , Encéfalo/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Dendrímeros/farmacología , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Lipopolisacáridos/farmacología , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Mutación/genética , Síndrome de Rett/genética , Síndrome de Rett/patología , Distribución Tisular/efectos de los fármacos , Distribución Tisular/genéticaRESUMEN
Brain-related disorders have outmatched cancer and cardiovascular diseases worldwide as the leading cause of morbidity and mortality. The lack of effective therapies and the relatively dry central nervous system (CNS) drug pipeline pose formidable challenge. Superior, targeted delivery of current clinically approved drugs may offer significant potential. Minocycline has shown promise for the treatment of neurological diseases owing to its ability to penetrate the blood-brain barrier (BBB) and potency. Despite its potential in the clinic and in preclinical models, the high doses needed to affect a positive therapeutic response have led to side effects. Targeted delivery of minocycline to the injured site and injured cells in the brain can be highly beneficial. Systemically administered hydroxyl poly(amidoamine) (PAMAM) generation-6 (G6) dendrimers have a longer blood circulation time and have been shown to cross the impaired BBB. We have successfully prepared and characterized the in vitro efficacy and in vivo targeting ability of hydroxyl-G6 PAMAM dendrimer-9-amino-minocycline conjugate (D-mino). Minocycline is a challenging drug to carry out chemical transformations due to its inherent instability. We used a combination of a highly efficient and mild copper catalyzed azide-alkyne click reaction (CuAAC) along with microwave energy to conjugate 9-amino-minocycline (mino) to the dendrimer surface via enzyme responsive linkages. D-mino was further evaluated for anti-inflammatory and antioxidant activity in lipopolysaccharides-activated murine microglial cells. D-mino conjugates enhanced the intracellular availability of the drug due to their rapid uptake, suppressed inflammatory cytokine tumor necrosis factor α (TNF-α) production, and reduced oxidative stress by suppressing nitric oxide production, all significantly better than the free drug. Fluorescently labeled dendrimer conjugate (Cy5-D-mino) was systematically administered (intravenous, 55 mg/kg) on postnatal day 1 to rabbit kits with a clinically relevant phenotype of cerebral palsy. The in vivo imaging study indicates that Cy5-D-mino crossed the impaired blood-brain barrier and co-localized with activated microglia at the periventricular white matter areas, including the corpus callosum and the angle of the lateral ventricle, with significant implications for positive therapeutic outcomes. The enhanced efficacy of D-mino, when combined with the inherent neuroinflammation-targeting capability of the PAMAM dendrimers, may provide new opportunities for targeted drug delivery to treat neurological disorders.
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Antiinflamatorios/administración & dosificación , Parálisis Cerebral/tratamiento farmacológico , Dendrímeros/química , Sistemas de Liberación de Medicamentos , Inflamación/tratamiento farmacológico , Microglía/efectos de los fármacos , Minociclina/administración & dosificación , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Parálisis Cerebral/complicaciones , Parálisis Cerebral/inmunología , Portadores de Fármacos/química , Inflamación/complicaciones , Inflamación/inmunología , Microglía/inmunología , Minociclina/química , Minociclina/uso terapéutico , ConejosRESUMEN
Perinatal hypoxic-ischemic encephalopathy (HIE) can result in neurodevelopmental disability, including cerebral palsy. The only treatment, hypothermia, provides incomplete neuroprotection. Hydroxyl polyamidoamine (PAMAM) dendrimers are being explored for targeted delivery of therapy for HIE. Understanding the biodistribution of dendrimer-conjugated drugs into microglia, neurons and astrocytes after brain injury is essential for optimizing drug delivery. We conjugated N-acetyl-L-cysteine to Cy5-labeled PAMAM dendrimer (Cy5-D-NAC) and used a mouse model of perinatal HIE to study effects of timing of administration, hypothermia, brain injury, and microglial activation on uptake. Dendrimer conjugation delivered therapy most effectively to activated microglia but also targeted some astrocytes and injured neurons. Cy5-D-NAC uptake was correlated with brain injury in all cell types and with activated morphology in microglia. Uptake was not inhibited by hypothermia, except in CD68+ microglia. Thus, dendrimer-conjugated drug delivery can target microglia, astrocytes and neurons and can be used in combination with hypothermia for treatment of HIE.
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Acetilcisteína/administración & dosificación , Antioxidantes/administración & dosificación , Dendrímeros/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Acetilcisteína/farmacocinética , Acetilcisteína/uso terapéutico , Animales , Animales Recién Nacidos , Antioxidantes/farmacocinética , Antioxidantes/uso terapéutico , Parálisis Cerebral/tratamiento farmacológico , Parálisis Cerebral/patología , Modelos Animales de Enfermedad , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipotermia Inducida , Hipoxia-Isquemia Encefálica/patología , Hipoxia-Isquemia Encefálica/terapia , Ratones , Microglía/efectos de los fármacos , Microglía/patología , Distribución TisularRESUMEN
BACKGROUND: Microglial cells have been implicated in neuroinflammation-mediated injury in the brain, including neurodevelopmental disorders such as cerebral palsy (CP) and autism. Pro-inflammatory activation of microglial cells results in the impairment of their neuroprotective functions, leading to an exaggerated, ongoing immune dysregulation that can persist long after the initial insult. We have previously shown that dendrimer-mediated delivery of an anti-inflammatory agent can attenuate inflammation in a rabbit model of maternal inflammation-induced CP and significantly improve the motor phenotype, due to the ability of the dendrimer to selectively localize in activated microglia. METHODS: To elucidate the interactions between dendrimers and microglia, we created an organotypic whole-hemisphere brain slice culture model from newborn rabbits with and without exposure to inflammation in utero. We then used this model to analyze the dynamics of microglial migration and their interactions with dendrimers in the presence of neuroinflammation. RESULTS: Microglial cells in animals with CP had an amoeboid morphology and impaired cell migration, demonstrated by decreased migration distance and velocity when compared to cells in healthy, age-matched controls. However, this decreased migration was associated with a greater, more rapid dendrimer uptake compared to microglial cells from healthy controls. CONCLUSIONS: This study demonstrates that maternal intrauterine inflammation is associated with impaired microglial function and movement in the newborn brain. This microglial impairment may play a role in the development of ongoing brain injury and CP in the offspring. Increased uptake of dendrimers by the "impaired" microglia can be exploited to deliver drugs specifically to these cells and modulate their functions. Host tissue and target cell characteristics are important aspects to be considered in the design and evaluation of targeted dendrimer-based nanotherapeutics for improved and sustained efficacy. This ex vivo model also provides a rapid screening tool for evaluation of the effects of various therapies on microglial function.
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Movimiento Celular/efectos de los fármacos , Dendrímeros/toxicidad , Inflamación/patología , Microglía/efectos de los fármacos , Microglía/patología , Nanopartículas/toxicidad , Animales , Animales Recién Nacidos , Encéfalo/citología , Encéfalo/efectos de los fármacos , Femenino , Inflamación/inducido químicamente , Microglía/ultraestructura , Técnicas de Cultivo de Órganos , Embarazo , ConejosRESUMEN
The development of synthetic bone grafts with requisite mechanical and morphological properties remains a key challenge in orthopaedics. Supercritical carbon dioxide (scCO2)-processed nanocomposites consisting of organically-modified montmorillonite clay dispersed in poly-d-lactide (PDLA) have shown structural and mechanical properties similar to corticocancellous bone. Using quantitative undecalcified histology and micro-computed tomography (µCT), time and material-dependent influences on in vivo bone formation, and inflammatory response were characterized. This represents the first in vivo evidence of the ability of scCO2-processed PDLA-nanoclay constructs to support osteogenesis, while eliciting an inflammatory response comparable to PDLA-hydroxyapatite materials. Histologic analyses demonstrated that the in vivo performance of nanoclay-containing PDLA constructs was similar to pure PDLA constructs, though nanocomposites demonstrated more radiodense bone at all time points (µCT analysis), and higher bone volume at 6 weeks. Taken with previous structural and mechanical studies, these in vivo analyses suggest that scCO2-processed, polymer-clay nanocomposites may be suitable structural bone graft materials. FROM THE CLINICAL EDITOR: With advances in science, orthopedic researchers have devoted significant amount of time in developing synthetic bone graft materials. Many of which are indeed currently in clinical use. In their previous studies, the authors described and studied supercritical carbon dioxide (scCO2)-processed nanocomposites consisting of organically modified montmorillonite clay dispersed in poly-D-lactide (PDLA) in in-vitro experiments. Here, in-vivo experiments were performed to investigate if this new material had improved mechanical properties, as well as the induction of inflammatory response. The overall positive findings may mean that this material could be used for future bone graft substitute applications.
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Bentonita/química , Sustitutos de Huesos/química , Nanocompuestos/química , Osteogénesis , Poliésteres/química , Animales , Bentonita/efectos adversos , Sustitutos de Huesos/efectos adversos , Femenino , Inflamación/etiología , Ratones Endogámicos BALB C , Nanocompuestos/efectos adversos , Nanocompuestos/ultraestructura , Poliésteres/efectos adversos , Microtomografía por Rayos XRESUMEN
Intrauterine inflammation is associated with preterm birth and can lead to fetal neuroinflammation and neurobehavioral disorders in newborns. Dendrimers can intrinsically target and deliver drugs for the treatment of neuroinflammation. We explore whether hydroxyl polyamidoamine (PAMAM) dendrimer (G4-OH)-based nanomedicines can be delivered to the fetus by intra-amniotic administration, in a mouse model of intrauterine inflammation. The time-dependent accumulation of G4-OH-fluorophore conjugate was quantified by fluorescence. These studies suggest that, after intra-amniotic administration, there is significant accumulation of dendrimer in the fetus gut and brain. In addition, there is some fetal-maternal transport of the dendrimer. Confocal microscopy confirmed the presence of G4-OH in the fetal brain, with a large accumulation in the brain blood vessels and the brain parenchyma, and some microglial uptake. We believe that intra-amniotic administration of G4-OH-drug nanomedicines may enable the treatment of diseases related to intrauterine inflammation and fetal neuroinflammation. FROM THE CLINICAL EDITOR: Using a mouse model of intrauterin inflammation leading to neuroinflammation in the fetus, these investigators demonstrate that intra-amniotic delivery of hydroxyl polyamidoamine (PAMAM) dendrimer (G4-OH)-based nanomedicines may provide an effective method in preventing this complication.
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Amnios/metabolismo , Dendrímeros/administración & dosificación , Dendrímeros/farmacocinética , Sistemas de Liberación de Medicamentos , Feto/metabolismo , Animales , Modelos Animales de Enfermedad , Vías de Administración de Medicamentos , Femenino , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/patología , Ratones , Nacimiento Prematuro/tratamiento farmacológico , Nacimiento Prematuro/etiología , Nacimiento Prematuro/patología , Útero/patologíaRESUMEN
Drug discovery is challenging task with numerous obstacles in translating drug candidates into clinical products. Dendrimers are highly adaptable nanostructured polymers with significant potential to improve the chances of clinical success for drugs. Yet, dendrimer-based drug products are still in their infancy. However, Hydroxyl polyamidoamine (PAMAM) dendrimers showed significant promise in drug discovery efforts, owning their remarkable potential to selectively target and deliver drugs specifically to activated microglia and astrocytes at the site of brain injury in several preclinical models. After a decade's worth of academic research and pre-clinical efforts, the hydroxyl PAMAM dendrimer-N-acetyl cysteine conjugate (OP-101) nanomedicine has made a significant advancement in the field of nanomedicine and targeted delivery. The OP-101 conjugate, primarily developed and validated in academic labs, has now entered clinical trials as a potential treatment for hyperinflammation in hospitalized adults with severe COVID-19 through Ashvattha Therapeutics. This chapter, we delve into the journey of the hydroxyl PAMAM dendrimer-N-acetylcysteine (NAC) OP-101 formulation from the laboratory to the clinic. It will specifically focus on the design, synthesis, preclinical, and clinical development of OP-101, highlighting the potential it holds for the future of medicine and the positive Phase 2a results for treating severe COVID-19.