RESUMEN
Neuropathic pain is a chronic pain syndrome that arises from nerve injury. Current treatments only offer limited relief, clearly indicating the need for more effective therapeutic strategies. Previously, we demonstrated that proinflammatory tumor necrosis factor-alpha (TNF) is a key mediator of neuropathic pain pathogenesis; TNF is elevated at sites of neuronal injury, in the spinal cord, and supraspinally during the initial development of pain. The inhibition of TNF action along pain pathways outside higher brain centers results in transient decreases in pain perception. The objective of this study was to determine whether specific blockade of TNF in the hippocampus, a site of pain integration, could prove efficacious in reducing sciatic nerve chronic constriction injury (CCI)-induced pain behavior. Small inhibitory RNA directed against TNF mRNA was complexed to gold nanorods (GNR-TNF siRNA; TNF nanoplexes) and injected into the contralateral hippocampus of rats 4 days after unilateral CCI. Withdrawal latencies to a noxious thermal stimulus (hyperalgesia) and withdrawal to innocuous forces (allodynia) were recorded up to 10 days and compared with baseline values and sham-operated rats. Thermal hyperalgesia was dramatically decreased in CCI rats receiving hippocampal TNF nanoplexes; and mechanical allodynia was transiently relieved. TNF levels (bioactive protein, TNF immunoreactivity) in hippocampal tissue were decreased. The observation that TNF nanoplex injection into the hippocampus alleviated neuropathic pain-like behavior advances our previous findings that hippocampal TNF levels modulate pain perception. These data provide evidence that targeting TNF in the brain using nanoparticle-protected siRNA may be an effective strategy for treatment of neuropathic pain.
Asunto(s)
Hipocampo/metabolismo , Nanomedicina/métodos , Nanotubos , Dolor Nociceptivo/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Constricción , Hipocampo/efectos de los fármacos , Masculino , Dolor Nociceptivo/tratamiento farmacológico , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
The manifestation of chronic, neuropathic pain includes elevated levels of the cytokine tumor necrosis factor-alpha (TNF). Previously, we have shown that the hippocampus, an area of the brain most notable for its role in learning and memory formation, plays a fundamental role in pain sensation. Using an animal model of peripheral neuropathic pain, we have demonstrated that intracerebroventricular infusion of a TNF antibody adjacent to the hippocampus completely alleviated pain. Furthermore, intracerebroventricular infusion of rTNF adjacent to the hippocampus induced pain behavior in naïve animals similar to that expressed during a model of neuropathic pain. These data support our premise that enhanced production of hippocampal-TNF is integral in pain sensation. In the present study, TNF gene expression was induced exclusively in the hippocampus, eliciting increased local bioactive TNF levels, and animals were assessed for pain behaviors. Male Sprague-Dawley rats received stereotaxic injection of gold nanorod (GNR)-complexed cDNA (control or TNF) plasmids (nanoplasmidexes), and pain responses (i.e., thermal hyperalgesia and mechanical allodynia) were measured. Animals receiving hippocampal microinjection of TNF nanoplasmidexes developed thermal hyperalgesia bilaterally. Sensitivity to mechanical stimulation also developed bilaterally in the rat hind paws. In support of these behavioral findings, immunoreactive staining for TNF, bioactive levels of TNF, and levels of TNF mRNA per polymerase chain reaction analysis were assessed in several brain regions and found to be increased only in the hippocampus. These findings indicate that the specific elevation of TNF in the hippocampus is not a consequence of pain, but in fact induces these behaviors/symptoms.
Asunto(s)
Hipocampo/metabolismo , Hiperalgesia/metabolismo , Dolor/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , ADN Complementario , Expresión Génica , Calor , Masculino , Nanotubos , Plásmidos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Tacto , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Inhibition of Matrix metalloproteinase-9 (MMP-9) activity using delivery of short interfering RNA (siRNA) molecules to brain microvascular endothelial cells (BMVECs) that constitute the BBB may have a significant impact on reducing the BBB permeability. Gold nano rods (GNRs) can electrostatically bind with MMP-9 siRNA to form a nanoplex and the uptake of this nanoplex by BMVEC cells can result in suppression of MMP-9 expression. The current study explores if this GNR-MMP-9 siRNA nanoplex gene silencing modulates the expression of tight junction (TJ) proteins in the BMVEC. The endothelial TJ's of the BBB play a critical role in controlling cellular traffic into the central nervous system. We hypothesize that silencing of the MMP-9 gene expression in BMVEC will increase the expression of TJ proteins thereby decrease endothelial permeability. Our results showed a significant increase in the gene and protein expression of TJ proteins: ZO-1, Occludin and Claudin-5 in BMVEC cells that were transfected with the GNRs-siRNA-MMP-9 nanoplex suggesting that BBB disruption, which results from loss of TJ function due to MMP-9 activation during neuroinflammation can be prevented by silencing MMP-9 expression.
Asunto(s)
Encéfalo/metabolismo , Endotelio Vascular/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Nanotubos , ARN Interferente Pequeño/metabolismo , Supervivencia Celular , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Activación Enzimática , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Microvasos/citología , Nanotubos/química , Nanotubos/ultraestructura , Tamaño de la Partícula , ARN Interferente Pequeño/química , Electricidad Estática , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , TransfecciónRESUMEN
AIM: Gold nanorods (GNRs), cellular imaging nanoprobes, have been used for drug delivery therapy to immunologically privileged regions in the brain. We demonstrate that nanoplexes formed by electrostatic binding between negatively charged RNA and positively charged GNRs, silence the expression of the target housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) within the CA1 hippocampal region of the rat brain, without showing cytotoxicity. MATERIALS & METHODS: Fluorescence imaging with siRNA(Cy3)GAPDH and dark-field imaging using plasmonic enhanced scattering from GNRs were used to monitor the distribution of the nanoplexes within different neuronal cell types present in the targeted hippocampal region. RESULTS & CONCLUSION: Our results show robust nanoplex uptake and slow release of the fluorescent gene silencer with significant impact on the suppression of GAPDH gene expression (70% gene silencing, >10 days postinjection). The observed gene knockdown using nanoplexes in targeted regions of the brain opens a new era of drug treatment for neurological disorders.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Oro/química , Hipocampo/metabolismo , Nanotubos/química , Animales , Ganglios Espinales/citología , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/metabolismo , Masculino , Nanotecnología , ARN Interferente Pequeño/química , Ratas , Ratas Sprague-DawleyRESUMEN
The emergence of the pandemic 2009 H1N1 influenza virus has become a world-wide health concern. As drug resistance appears, a new generation of therapeutic strategies will be required. Here, we introduce a nanotechnology approach for the therapy of pan-demic and seasonal influenza virus infections. This approach uses gold nanorods (GNRs) to deliver an innate immune activator, pro-ducing a localized therapeutic response. We demonstrated the utility of a biocompatible gold nanorod, GNR-5'PPP-ssRNA nanoplex, as an antiviral strategy against type A influenza virus. In human respiratory bronchial epithelial cells, this nanoplex activated the retinoic acid-inducible gene I (RIG-I) pathogen recognition pathway, resulting in increased expression of IFN-beta and other IFN-stimulated genes (ISGs) (e.g., PKR, MDA5, IRF1, IRF7, and MX1). This increase in type I IFN and ISGs resulted in a decrease in the replication of H1N1 influenza viruses. These findings suggest that further evaluation of biocompatible nanoplexes as unique antivirals for treatment of seasonal and pandemic influenza viruses is warranted.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Nanopartículas del Metal/administración & dosificación , ARN/administración & dosificación , Replicación Viral/efectos de los fármacos , Antivirales/administración & dosificación , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Sistemas de Liberación de Medicamentos , Oro , Humanos , Inmunidad Innata/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Interferón beta/metabolismo , Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de Transmisión , Nanotubos/ultraestructura , ARN/inmunología , Receptores Inmunológicos , Transducción de Señal/efectos de los fármacos , Resonancia por Plasmón de SuperficieRESUMEN
The 32-kDa dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) is recognized to be critical to the pathogenesis of drug addiction. Opiates via the mu-receptor act on the dopaminergic system in the brain and modulates the expression of DARPP-32 phosphoprotein which is an important mediator of the activity of the extracellular signal-regulated kinase (ERK) signaling cascades, the activation of which represents an exciting nexus for drug-induced changes in neural long-term synaptic plasticity. Silencing of DARPP-32 using an siRNA against DARPP-32 may provide a novel gene therapy strategy to overcome drug addiction. In this study, we investigated the effect of the opiate (heroin) on D1 receptor (D1R) and DARPP-32 expression and additionally, evaluated the effects of DARPP-32-siRNA gene silencing on protein phosphatase-1 (PP-1), ERK, and cAMP response element-binding (CREB) gene expression in primary normal human astrocytes (NHA) cells in vitro. Our results indicate that heroin significantly upregulated both D1R and DARPP-32 gene expression, and that DARPP-32 silencing in the NHA cells resulted in the significant modulation of the activity of downstream effector molecules such as PP-1, ERK, and CREB which are known to play an important role in opiate abuse-induced changes in long-term neural plasticity. These findings have the potential to facilitate the development of DARPP32 siRNA-based therapeutics against drug addiction.
Asunto(s)
Fosfoproteína 32 Regulada por Dopamina y AMPc/efectos de los fármacos , Heroína/farmacología , Narcóticos/farmacología , Trastornos Relacionados con Opioides/metabolismo , Células Cultivadas , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador , Análisis de Secuencia por Matrices de Oligonucleótidos , Trastornos Relacionados con Opioides/genética , ARN Interferente Pequeño , Receptores de Dopamina D1/biosíntesis , Receptores de Dopamina D1/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
New hyperbranched polysiloxysilane (HBPS) materials containing terminal carboxylic acid and quaternary ammonium groups were designed and synthesized to obtain fluorescent-dye-encapsulated nanoparticles. These polymers exhibited desirable characteristics, including amphiphilicity for nanoparticle formation, and contained various terminal groups for surface-charge control on the nanoparticles or for further bioconjugation for targeted imaging. Nanoprobes composed of polysiloxysilane nanoparticles encapsulating two-photon dyes were also prepared for optical bioimaging with controlled surface charge density (zeta potential) for modulation of cellular uptake. Intracellular delivery of these structurally similar polysiloxysilane nanoparticles, with substantially different surface charges, was investigated using confocal and two-photon fluorescence microscopy as well as flow cytometry. Finally, the use of these nanoparticles as efficient gene delivery vectors was demonstrated by means of in vitro transfection study using beta-galactosidase plasmid and pEGFP-N1 plasmid and the most efficient combination was obtained using HBPS-CN30:70.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Colorantes Fluorescentes/farmacocinética , Técnicas de Transferencia de Gen , Vectores Genéticos , Nanopartículas/química , Siloxanos/síntesis química , Transfección/métodos , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Portadores de Fármacos/síntesis química , Portadores de Fármacos/farmacocinética , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Microscopía Fluorescente , Modelos Químicos , Estructura Molecular , Nanopartículas/administración & dosificación , Plásmidos/genética , Siloxanos/administración & dosificación , Siloxanos/química , beta-Galactosidasa/genéticaRESUMEN
The matrix-degrading metalloproteinases (MMPs), particularly MMP-9, are involved in the neuroinflammation processes leading to disrupting of the blood brain barrier (BBB), thereby exacerbating neurological diseases such as HIV-1 AIDS dementia and cerebral ischemia. Nanoparticles have been proposed to act as non-viral gene delivery vectors and have great potential for therapeutic applications in several disease states. In this study, we evaluated the specificity and efficiency of quantum dot (QD) complexed with MMP-9-siRNA (nanoplex) in downregulating the expression of MMP-9 gene in brain microvascular endothelial cells (BMVEC) that constitute the BBB. We hypothesize that silencing MMP-9 gene expression in BMVECs and other cells such as leukocytes may help prevent breakdown of the BBB and inhibit subsequent invasion of the central nervous system (CNS) by infected and inflammatory cells. Our results show that silencing of MMP-9 gene expression resulted in the up-regulation of extracellular matrix (ECM) proteins like collagen I, IV, V and a decrease in endothelial permeability, as reflected by reduction of transendothelial resistance across the BBB in a well validated in-vitro BBB model. MMP-9 gene silencing also resulted in an increase in expression of the gene tissue inhibitor of metalloproteinase-1 (TIMP-1). This indicates the importance of a balance between the levels of MMP-9 and its natural inhibitor TIMP-1 in maintaining the basement membrane integrity. These studies promise the application of a novel nanoparticle based siRNA delivery system in modulating the MMP-9 activity in BMVECs and other MMP-9 producing cells. This will prevent neuroinflammation and maintain the integrity of the BBB.
Asunto(s)
Barrera Hematoencefálica , Inhibidores de la Metaloproteinasa de la Matriz , Puntos Cuánticos , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Membrana Basal/efectos de los fármacos , Membrana Basal/enzimología , Barrera Hematoencefálica/enzimología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Encefalitis/enzimología , Encefalitis/genética , Encefalitis/prevención & control , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Proteínas de la Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Técnicas de Transferencia de Gen , Humanos , Metaloproteinasa 9 de la Matriz/genética , Nanopartículas/uso terapéutico , ARN Interferente Pequeño/genética , Inhibidor Tisular de Metaloproteinasa-1/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
Drug abuse is a worldwide health concern in which addiction involves activation of the dopaminergic signaling pathway in the brain. Here, we introduce a nanotechnology approach that utilizes gold nanorod-DARPP-32 siRNA complexes (nanoplexes) that target this dopaminergic signaling pathway in the brain. The shift in the localized longitudinal plasmon resonance peak of gold nanorods (GNRs) was used to show their interaction with siRNA. Plasmonic enhanced dark field imaging was used to visualize the uptake of these nanoplexes in dopaminergic neurons in vitro. Gene silencing of the nanoplexes in these cells was evidenced by the reduction in the expression of key proteins (DARPP-32, ERK, and PP-1) belonging to this pathway, with no observed cytotoxicity. Moreover, these nanoplexes were shown to transmigrate across an in vitro model of the blood-brain barrier (BBB). Therefore, these nanoplexes appear to be suited for brain-specific delivery of appropriate siRNA for therapy of drug addiction and other brain diseases.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Oro , Nanotubos , Neuronas/metabolismo , ARN Interferente Pequeño/administración & dosificación , Trastornos Relacionados con Sustancias/terapia , Barrera Hematoencefálica , Dopamina , Fosfoproteína 32 Regulada por Dopamina y AMPc/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Silenciador del Gen , Humanos , Nanotecnología/métodos , ARN Interferente Pequeño/farmacologíaRESUMEN
A water-soluble nitrosyl complex with large two-photon absorption was synthesized by incorporating a two-photon absorbing chromophore with tetra(ethylene glycol) units, into the Roussin's red salt. The nitrosyl complex exhibits quenched emission due to energy transfer from the two-photon chromophore to the Roussin's red salt. The nitric oxide (NO) release induced by one- or two-photon irradiation was detected by EPR spectroscopy with a chemical probe, the Fe(II)- N-(dithiocarbamoyl)- N-methyl- d-glucamine (Fe-MGD) complex. Increased one- or two-photon excited fluorescence, with a concomitant photochemical release of NO, was observed upon one- or two-photon light irradiation. With the observed light-dependent cytotoxicity against cancer cells of the water-soluble nitrosyl complex, it was demonstrated that two-photon-functionalized nitrosyl complexes can be effective NO donors for light-activated treatment.
Asunto(s)
Compuestos de Hierro/química , Donantes de Óxido Nítrico/química , Óxido Nítrico/química , Fotones , Fototerapia , Espectroscopía de Resonancia por Spin del Electrón , Estructura Molecular , Solubilidad , Agua/químicaRESUMEN
We report pH sensing for biological applications based on surface enhanced Raman scattering (SERS) from silver nanoparticles functionalized with 2-aminothiophenol (2-aminobenzenethiol, 2-ABT). pH-dependent SERS spectra of the attached 2-ABT molecules enable one to sense the pH over the range of 3.0-8.0. We have performed the first demonstration of SERS detection in living cells kept in different pH buffer solutions and show that the pH sensitivity is retained in that case. Thus, the nanoparticles can be used as probes delivering spatially localized chemical information from biological environments and provide a new way to monitor chemical changes at cellular level.
Asunto(s)
Compuestos de Anilina/química , Técnicas Biosensibles/métodos , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/química , Plata/química , Espectrometría Raman/métodos , Propiedades de SuperficieRESUMEN
In this paper we report the synthesis and characterization of organically modified silica (ORMOSIL) nanoparticles, covalently incorporating the fluorophore rhodamine-B, and surface-functionalized with a variety of active groups. The synthesized nanoparticles are of ultralow size (diameter approximately 20 nm), highly monodispersed, stable in aqueous suspension, and retain the optical properties of the incorporated fluorophore. The surface of the nanoparticles can be functionalized with a variety of active groups such as hydroxyl, thiol, amine, and carboxyl. The carboxyl groups on the surface were used to conjugate with various bioactive molecules such as transferrin, as well as monoclonal antibodies such as anti-claudin 4 and anti-mesothelin, for targeted delivery to pancreatic cancer cell lines. In vitro experiments have revealed that the cellular uptake of these bioconjugated (targeted) nanoparticles is significantly higher than that of the nonconjugated ones. The ease of surface functionalization and incorporation of a variety of biotargeting molecules, combined with their observed noncytotoxicity, makes these fluorescent ORMOSIL nanoparticles potential candidates as efficient probes for optical bioimaging, both in vitro and in vivo.