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      Dendrimers Target the Ischemic Lesion in Rodent and Primate Models of Nonarteritic Anterior Ischemic Optic Neuropathy

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          Abstract

          Introduction

          Polyamidoamine dendrimer nanoparticles (~ 4 nanometers) are inert polymers that can be linked to biologically active compounds. These dendrimers selectively target and accumulate in inflammatory cells upon systemic administration. Dendrimer-linked compounds enable sustained release of therapeutic compounds directly at the site of damage. The purpose of this study was to determine if dendrimers can be used to target the optic nerve (ON) ischemic lesion in our rodent and nonhuman primate models of nonarteritic anterior ischemic optic neuropathy (NAION), a disease affecting >10,000 individuals in the US annually, and for which there currently is no effective treatment.

          Methods

          NAION was induced in male Long-Evans rats (rNAION) and in one adult male rhesus monkey (pNAION) using previously described procedures. Dendrimers were covalently linked to near-infrared cyanine-5 fluorescent dye (D-Cy5) and injected both intravitreally and systemically (in the rats) or just systemically (in the monkey) to evaluate D-Cy5 tissue accumulation in the eye and optic nerve following induction of NAION.

          Results

          Following NAION induction, Cy-5 dendrimers selectively accumulated in astrocytes and circulating macrophages. Systemic dendrimer administration provided the best penetration of the ON lesion site when injected shortly after induction. Systemic administration 1 day post-induction in the pNAION model gave localization similar to that seen in the rats.

          Conclusions

          Dendrimers selectively target the ischemic ON lesion after induction of both rNAION and pNAION. Systemic nanoparticle-linked therapeutics thus may provide a powerful, targeted and safe approach to NAION treatment by providing sustained and focused treatment of the cells directly affected by ischemia.

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          Most cited references22

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          Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications.

          Dendrimers are discrete nanostructures/nanoparticles with 'onion skin-like' branched layers. Beginning with a core, these nanostructures grow in concentric layers to produce stepwise increases in size that are similar to the dimensions of many in vivo globular proteins. These branched tree-like concentric layers are referred to as 'generations'. The outer generation of each dendrimer presents a precise number of functional groups that may act as a monodispersed platform for engineering favourable nanoparticle-drug and nanoparticle-tissue interactions. These features have attracted significant attention in medicine as nanocarriers for traditional small drugs, proteins, DNA/RNA and in some instances as intrinsically active nanoscale drugs. Dendrimer-based drugs, as well as diagnostic and imaging agents, are emerging as promising candidates for many nanomedicine applications. First, we will provide a brief survey of recent nanomedicines that are either approved or in the clinical approval process. This will be followed by an introduction to a new 'nanoperiodic' concept which proposes nanoparticle structure control and the engineering of 'critical nanoscale design parameters' (CNDPs) as a strategy for optimizing pharmocokinetics, pharmocodynamics and site-specific targeting of disease. This paradigm has led to the emergence of CNDP-directed nanoperiodic property patterns relating nanoparticle behaviour to critical in vivo clinical translation issues such as cellular uptake, transport, elimination, biodistribution, accumulation and nanotoxicology. With a focus on dendrimers, these CNDP-directed nanoperiodic patterns are used as a strategy for designing and optimizing nanoparticles for a variety of drug delivery and imaging applications, including a recent dendrimer-based theranostic nanodevice for imaging and treating cancer. Several emerging preclinical dendrimer-based nanotherapy concepts related to inflammation, neuro-inflammatory disorders, oncology and infectious and ocular diseases are reviewed. Finally we will consider challenges and opportunities anticipated for future clinical translation, nanotoxicology and the commercialization of nanomedicine.
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            Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model.

            Cerebral palsy (CP) is a chronic childhood disorder with no effective cure. Neuroinflammation, caused by activated microglia and astrocytes, plays a key role in the pathogenesis of CP and disorders such as Alzheimer's disease and multiple sclerosis. Targeting neuroinflammation can be a potent therapeutic strategy. However, delivering drugs across the blood-brain barrier to the target cells for treating diffuse brain injury is a major challenge. We show that systemically administered polyamidoamine dendrimers localize in activated microglia and astrocytes in the brain of newborn rabbits with CP, but not healthy controls. We further demonstrate that dendrimer-based N-acetyl-l-cysteine (NAC) therapy for brain injury suppresses neuroinflammation and leads to a marked improvement in motor function in the CP kits. The well-known and safe clinical profile for NAC, when combined with dendrimer-based targeting, provides opportunities for clinical translation in the treatment of neuroinflammatory disorders in humans. The effectiveness of the dendrimer-NAC treatment, administered in the postnatal period for a prenatal insult, suggests a window of opportunity for treatment of CP in humans after birth.
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              Systemic inflammatory challenges compromise survival after experimental stroke via augmenting brain inflammation, blood- brain barrier damage and brain oedema independently of infarct size

              Background Systemic inflammation impairs outcome in stroke patients and experimental animals via mechanisms which are poorly understood. Circulating inflammatory mediators can activate cerebrovascular endothelium or glial cells in the brain and impact on ischaemic brain injury. One of the most serious early clinical complications of cerebral ischaemia is brain oedema, which compromises survival in the first 24-48 h. It is not understood whether systemic inflammatory challenges impair outcome after stroke by increasing brain injury only or whether they have direct effects on brain oedema, cerebrovascular inflammation and blood-brain barrier damage. Methods We used two different systemic inflammatory stimuli, acute endotoxin treatment and anaphylaxis to study mechanisms of brain injury after middle cerebral artery occlusion (MCAo). Ischaemic brain injury, blood-brain barrier damage and oedema were analysed by histological techniques. Systemic cytokine responses and inflammatory changes in the brain were analysed by cytometric bead array, immunofluorescence, in situ hibridization and quantitative real-time PCR. Results Systemic inflammatory challenges profoundly impaired survival in the first 24 h after experimental stroke in mice, independently of an increase in infarct size. Systemic lipopolysaccharide (LPS) dose-dependently increased mortality (50-100%) minutes to hours after cerebral ischaemia. Acute anaphylactic challenge in ovalbumin-sensitised mice affected stroke more seriously when induced via intraperitoneal administration compared to intravenous. Both LPS and anaphylaxis induced inflammatory changes in the blood and in the brain prior to experimental stroke. Plasma cytokine levels were significantly higher after LPS, while increased IL-10 levels were seen after anaphylaxis. After MCAo, both LPS and anaphylaxis increased microglial interleukin-1α (IL-1α) expression and blood-brain barrier breakdown. LPS caused marked granulocyte recruitment throughout the ipsilateral hemisphere. To investigate whether reduction of ischaemic damage can improve outcome in systemic inflammation, controlled hypothermia was performed. Hypothermia reduced infarct size in all treatment groups and moderately improved survival, but failed to reduce excess oedema formation after anaphylaxis and LPS-induced neuroinflammation. Conclusions Our results suggest that systemic inflammatory conditions induce cerebrovascular inflammation via diverse mechanisms. Increased brain inflammation, blood-brain barrier injury and brain oedema formation can be major contributors to impaired outcome in mice after experimental stroke with systemic inflammatory stimuli, independently of infarct size.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 April 2016
                2016
                : 11
                : 4
                : e0154437
                Affiliations
                [1 ]Department of Ophthalmology and Visual Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, United States of America
                [2 ]Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD, 21287, United States of America
                [3 ]Division of Neuro-Ophthalmology, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD, 21287, United States of America
                [4 ]Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, 21201, United States of America
                Hanson Institute, AUSTRALIA
                Author notes

                Competing Interests: The dendrimer technology utilized in this manuscript is protected by awarded and pending patents, listed below. Dr. Kannan Rangaramanujam is co-inventor on these patents, and is a co-founder and chief technology officer of two companies, Ashvattha Therapeutics, LLC and Orpheris Inc., that are commercializing this technology. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. (1) Dendrimer-containing particles for sustained release of compounds, Kannan RM, Iezzi R, Kannan S, US provisional patent filed 10/5/07, Regular patents filed in US (#12/681,516, pending), Canada (#2,701,291), European Union (#08835693.6), Japan (#2010-528216, awarded) and India (1247/ELNP/2010) (Apr. 2010). Japanese Patent Awarded. (2) Dendrimer-based therapeutic nanodevices for therapeutic and imaging applications, Kannan RM, Kannan S, Romero R, Navath R, Dai H, Kurtoglu Y, Wang B, Menjoge A, Provisional patent filed # 61/187263, 5/09, US patent (#12/797,657, awarded 2014). (3) Novel Drug Delivery Systems for Systemic Therapies for Back of the Eye Diseases, Kannan RM, Lutty G, Kambhampati S, Mishra M, Bhutto I, US Provisional Patent, #61/986,495, Provisional PCT Application No. US2015/028386.

                Conceived and designed the experiments: SLB NRM MAJ RK. Performed the experiments: YG ZM. Analyzed the data: SLB NRM MAJ YG ZM RK. Contributed reagents/materials/analysis tools: MKM RK. Wrote the paper: SLB NRM MAJ RK ZM YG.

                Article
                PONE-D-16-03142
                10.1371/journal.pone.0154437
                4851377
                27128315
                265e3b4f-b846-4d37-aa43-d236496b54f4
                © 2016 Guo et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 2 February 2016
                : 13 April 2016
                Page count
                Figures: 5, Tables: 2, Pages: 14
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000053, National Eye Institute;
                Award ID: EY105304
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000053, National Eye Institute;
                Award ID: EY019529
                Award Recipient :
                Funded by: Johns Hopkins University Fund
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000070, National Institute of Biomedical Imaging and Bioengineering;
                Award ID: EB018306
                Award Recipient :
                This work was funded by NEI RO1EY01530 and RO1EY019529 to SLB, RO1EY025304 to RK ( https://nei.nih.gov), NIBIB RO1 EB018306 to RK ( http://www.nibib.nih.gov), and Johns Hopkins Optic Nerve Research Fund to NRM.
                Categories
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