31 research outputs found

    Agile delivery of protein therapeutics to CNS

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    A variety of therapeutic proteins have shown potential to treat central nervous system (CNS) disorders. Challenge to deliver these protein molecules to the brain is well known. Proteins administered through parenteral routes are often excluded from the brain because of their poor bioavailability and the existence of the blood-brain barrier (BBB). Barriers also exist to proteins administered through non-parenteral routes that bypass the BBB. Several strategies have shown promise in delivering proteins to the brain. This review, first, describes the physiology and pathology of the BBB that underscore the rationale and needs of each strategy to be applied. Second, major classes of protein therapeutics along with some key factors that affect their delivery outcomes are presented. Third, different routes of protein administration (parenteral, central intracerebroventricular and intraparenchymal, intranasal and intrathecal) are discussed along with key barriers to CNS delivery associated with each route. Finally, current delivery strategies involving chemical modification of proteins and use of particle-based carriers are overviewed using examples from literature and our own work. Whereas most of these studies are in the early stage, some provide proof of mechanism of increased protein delivery to the brain in relevant models of CNS diseases, while in few cases proof of concept had been attained in clinical studies. This review will be useful to broad audience of students, academicians and industry professionals who consider critical issues of protein delivery to the brain and aim developing and studying effective brain delivery systems for protein therapeutics

    SOD1 nanozyme salvages ischemic brain by locally protecting cerebral vasculature

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    Copper/zinc superoxide dismutase (CuZnSOD; SOD1) is widely considered as a potential therapeutic candidate for pathologies involving oxidative stress, but its application has been greatly hindered by delivery issues. In our previous study, nano-formulated SOD1 (cl-nanozyme) was shown to decrease infarct volume and improve sensorimotor functions after single intravenous (IV) injection in a rat middle cerebral artery occlusion (MCAO) model of ischemia/reperfusion (I/R) injury. However, it remained unclear how cl-nanozyme was able to deliver SOD1 to the brain and exert therapeutic efficacy. Present study aims to answer this question by exploring micro-distribution pattern of cl-nanozyme in the rat brain after stroke. Immunohistochemistry studies demonstrated cl-nanozyme co-localization with fibrin along damaged arteries and capillaries in the ischemic hemisphere. We further found that cl-nanozyme can be cross-linked into thrombi formed after I/R injury in the brain, and this effect is independent of animal species (rat/mouse) used for modeling I/R injury. This work is also the first report reinforcing therapeutic potential of cl-nanozyme in a well-characterized mouse MCAO model of I/R injury

    A Low Protein Binding Cationic Poly(2-oxazoline) as Non-Viral Vector: A Low Protein Binding Cationic Poly(2-oxazoline)…

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    Developing safe and efficient non-viral gene delivery systems remains a major challenge. We present a new cationic poly(2-oxazoline) (CPOx) block copolymer for gene therapy that was synthesized by sequential polymerization of non-ionic 2-methyl-2-oxazoline and a new 2-oxazoline monomer, 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline, followed by deprotection of the pendant secondary amine groups. Upon mixing with plasmid DNA (pDNA), CPOx forms small (diameter ≈ 80 nm) and narrowly dispersed polyplexes (PDI < 0.2), which are stable upon dilution in saline and against thermal challenge. These polyplexes exhibited low plasma protein binding and very low cytotoxicity in vitro compared to the polyplexes of pDNA and poly(ethylene glycol)-b-poly(l-lysine) (PEG-b-PLL). CPOx/pDNA polyplexes at N/P = 5 bound considerably less plasma protein compared to polyplexes of PEG-b-PLL at the same N/P ratio. This is a unique aspect of the developed polyplexes emphasizing their potential for systemic delivery in vivo. The transfection efficiency of the polyplexes in B16 murine melanoma cells was low after 4 h but increased significantly for 10 h exposure time, indicative of slow internalization of polyplexes. Addition of Pluronic P85 boosted the transfection using CPOx/pDNA polyplexes considerably. The low protein binding of CPOx/pDNA polyplexes is particularly interesting for the future development of targeted gene delivery

    Nano-particle delivery of brain derived neurotrophic factor after focal cerebral ischemia reduces tissue injury and enhances behavioral recovery

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    Low levels of brain-derived neurotrophic factor (BDNF) are linked to delayed neurological recovery, depression, and cognitive impairment following stroke. Supplementation with BDNF reverses these effects. Unfortunately, systemically administered BDNF in its native form has minimal therapeutic value due to its poor blood brain barrier permeability and short serum half-life. In this study, a novel nano-particle polyion complex formulation of BDNF (nano-BDNF) was administered to mice after experimental ischemic stroke

    Neuronal uptake of nanoformulated superoxide dismutase and attenuation of angiotensin II-dependent hypertension after central administration

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    Excessive production of superoxide (O2•−) in the central nervous system has been widely implicated in the pathogenesis of cardiovascular diseases, including chronic heart failure and hypertension. In an attempt to overcome the failed therapeutic impact of currently available antioxidants in cardiovascular disease, we developed a nanomedicine-based delivery system for the O2•− scavenging enzyme, copper/zinc superoxide dismutase (CuZnSOD), in which CuZnSOD protein is electrostatically bound to poly-L-lysine (PLL50)-polyethylene glycol (PEG) block co-polymer to form CuZnSOD nanozyme. Different formulations of CuZnSOD nanozyme are covalently stabilized by either reducible or non-reducible crosslinked bonds between the PLL50-PEG polymers. Herein, we tested the hypothesis that PLL50-PEG CuZnSOD nanozyme delivers active CuZnSOD protein to neurons and decreases blood pressure in a mouse model of AngII-dependent hypertension. As determined by electron paramagnetic resonance (EPR) spectroscopy, nanozymes retain full SOD enzymatic activity as compared to native CuZnSOD protein. Non-reducible CuZnSOD nanozyme delivers active CuZnSOD protein to central neurons in culture (CATH.a neurons) without inducing significant neuronal toxicity. In vivo studies conducted in adult male C57BL/6 mice demonstrate that hypertension established by chronic subcutaneous infusion of AngII is significantly attenuated for up to 7 days following a single intracerebroventricular (ICV) injection of non-reducible nanozyme. These data indicate the efficacy of non-reducible PLL50-PEG CuZnSOD nanozyme in counteracting excessive O2•− and decreasing blood pressure in AngII-dependent hypertensive mice following central administration. Additionally, this study supports the further development of PLL50-PEG CuZnSOD nanozyme as an antioxidant-based therapeutic option for hypertension

    Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins

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    Active targeted transport of the nanoformulated redox enzyme, catalase, in macrophages attenuates oxidative stress and as such increases survival of dopaminergic neurons in animal models of Parkinson’s disease. Optimization of the drug formulation is crucial for the successful delivery in living cells. We demonstrated earlier that packaging of catalase into a polyion complex micelle (‘nanozyme’) with a synthetic polyelectrolyte block copolymer protected the enzyme against degradation in macrophages and improved therapeutic outcomes. We now report the manufacture of nanozymes with superior structure and therapeutic indices

    Superoxide Dismutase 1 Nanozyme for Treatment of Eye Inflammation

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    Use of antioxidants to mitigate oxidative stress during ocular inflammatory diseases has shown therapeutic potential. This work examines a nanoscale therapeutic modality for the eye on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), termed “nanozyme.” The nanozyme is produced by electrostatic coupling of the SOD1 with a cationic block copolymer, poly(L-lysine)-poly(ethyleneglycol), followed by covalent cross-linking of the complexes with 3,3′-dithiobis(sulfosuccinimidylpropionate) sodium salt. The ability of SOD1 nanozyme as well as the native SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with immunogenic uveitis. Results suggested that topical instillations of both enzyme forms demonstrated anti-inflammatory activity; however, the nanozyme was much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as the intensities of corneal and iris edema, hyperemia of conjunctiva, lens opacity, fibrin clots, and the protein content in aqueous humor. Clinical findings were confirmed by histological data. Thus, SOD1-containing nanozyme is potentially useful therapeutic agent for the treatment of ocular inflammatory disorders

    Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

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    Lipidoid nanoparticles (LNPs) are clinically successful carriers for nucleic acid delivery to liver and muscle targets. The ability of LNPs to load and deliver small molecule drugs has not been reported yet. We propose that the delivery of adenosine triphosphate (ATP) to brain endothelial cells (BECs) lining the blood–brain barrier may increase cellular energetics of the injured BECs. We formulated and studied the physicochemical characteristics of ATP-loaded LNPs using the C12-200 ionizable cationic lipid and other helper lipids. Polyethylene glycol-dimyristoyl glycerol (PEG-DMG), one of the helper lipids, played a crucial role in maintaining colloidal stability of LNPs over time whereas the inclusion of both ATP and PEG-DMG maintained the colloidal stability of LNPs in the presence of serum proteins. ATP-LNPs formulated with PEG-DMG resulted in a 7.7- and 6.6- fold increased uptake of ATP into normoxic and hypoxic BECs, respectively. Altogether, our results demonstrate the potential of LNPs as a novel carrier for the delivery of small molecular mass actives to BECs—a CNS target

    Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain.

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    Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown
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