Chronic constriction injury-induced nociception is relieved by nanomedicine-mediated decrease of rat hippocampal tumor necrosis factor.

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.

Increasing TNF levels solely in the rat hippocampus produces persistent pain-like symptoms.

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.

Gold nanorod--siRNA induces efficient in vivo gene silencing in the rat hippocampus.

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.

Gold nanorod delivery of an ssRNA immune activator inhibits pandemic H1N1 influenza viral replication.

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.

Hyperbranched polysiloxysilane nanoparticles: surface charge control of nonviral gene delivery vectors and nanoprobes.

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.

Nanotechnology approach for drug addiction therapy: gene silencing using delivery of gold nanorod-siRNA nanoplex in dopaminergic neurons.

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.

Covalently dye-linked, surface-controlled, and bioconjugated organically modified silica nanoparticles as targeted probes for optical imaging.

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.