A macrophage-nanozyme delivery system for Parkinson's disease

Selective delivery of antioxidants to the substantia nigra pars compacta (SNpc) during Parkinson's disease (PD) can potentially attenuate oxidative stress and as such increase survival of dopaminergic neurons. To this end, we developed a bone-marrow-derived macrophage (BMM) system to deliver catalase to PD-affected brain regions in an animal model of human disease. To preclude BMM-mediated enzyme degradation, catalase was packaged into a block ionomer complex with a cationic block copolymer, polyethyleneimine-poly(ethylene glycol) (PEI-PEG). The self-assembled catalase/PEI-PEG complexes, "nanozymes", were ca. 60 to 100 nm in size, stable in pH and ionic strength, and retained antioxidant activities. Cytotoxicity was negligible over a range of physiologic nanozyme concentrations. Nanozyme particles were rapidly, 40-60 min, taken up by BMM, retained catalytic activity, and released in active form for greater than 24 h. In contrast, "naked" catalase was rapidly degraded. The released enzyme decomposed microglial hydrogen peroxide following nitrated alpha-synuclein or tumor necrosis factor alpha activation. Following adoptive transfer of nanozyme-loaded BMM to 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine-intoxicated mice, ca. 0.6% of the injected dose were found in brain. We conclude that cell-mediated delivery of nanozymes can reduce oxidative stress in laboratory and animal models of PD.     

Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP.

Pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) was used for in vitro delivery of [gamma-32P]ATP into intact Jurkat cells. Negatively charged ATP molecules are not able to penetrate cell plasma membrane. Hence, exogenous [gamma-32P]ATP added to a cell culture does not participate in phosphorylation of intracellular proteins. The addition to cells of [gamma-32P]ATP solubilized in positively charged (containing dodecylamine) pluronic micelles results in considerable increase of protein phosphorylation. In this case the treatment of intact cells with alkaline phosphatase (resulting in dephosphorylation of external proteins) causes no essential decrease of [32P]-incorporation in cell proteins. That gives an evidence of delivery of solubilized ATP into a cell. Under the experimental conditions used, pluronic micelles neither influence the viability of cells nor permeabilize cell plasma membrane.     

Pluronic micelles as a tool for low-molecular compound vector delivery into a cell: effect of Staphylococcus aureus enterotoxin B on cell loading with micelle incorporated fluorescent dye.

Micelles of pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) are used as microcontainers for in vitro delivery of fluorescein into Jurkat and MDCK cells. In order to target the fluorescein containing micelles into the cell, Staphylococcus aureus enterotoxin B (SEB) is covalently conjugated with a pluronic molecule and the conjugate is incorporated into the micelle content. The incorporation of SEB capable of receptor-mediated endocytosis results in a drastic enhancement of the efficiency of cell loading with the fluorescent dye. This effect is not observed under the conditions (4 degrees C) when endocytosis is abolished.     

Polyion complex micelles with protein-modified corona for receptor-mediated delivery of oligonucleotides into cells.

Graft-copolymers, containing poly(ethylene glycol) (PEG) and polyethyleneimine (PEI) chains have been proposed as carriers for delivery of phosphorothioate oligonucleotides (SODNs). Complexes of such copolymers with SODN self-assemble into particles having a core of neutralized PEI and SODN and a corona of PEG. Transferrin molecules are attached to the PEG corona using avidin/biotin construct. For this purpose, biotin moieties are covalently linked to the free ends of the PEG chains in the PEG-g-PEI copolymer. SODNs are reacted with mixtures of biotinylated and biotin-free PEG-g-PEI copolymers of various compositions to adjust the number of the biotin moieties in the complex. Resulting complexes have small size (ca. 40 nm) and do not aggregate in aqueous solutions for at least several days. To attach transferrin, they are supplemented first with avidin and then with biotin-transferrin conjugate. This increases the effective diameter of the particles to ca. 75-103 nm, depending on the composition of the complex. Cellular accumulation and fluorescence microscopy studies characterize the effects of these modifications on interaction of fluorescently labeled SODNs with KBv cell monolayers. The data suggest significant enhancement of SODN association with cells resulting from modification of the complex with transferrin. SODN complimentary to the site 546-565 of human mdr 1-mRNA was used to inhibit expression of the drug efflux transporter, P-glycoprotein (P-gp), in multiple drug resistant (MDR) cancer cells (KBv, MCF-7 ADR). Accumulation of a P-gp specific probe, rhodamine 123, in the cell monolayers is used to characterize the effects on P-gp efflux system following the treatment of the cells with antisense SODN or its complexes. This study suggests that antisense SODN incorporated in the complexes retain the ability to inhibit P-gp efflux system, while complexes of the randomized control SODN are inactive. Therefore, the antisense SODN is released from the complex and interacts with its intracellular target upon interaction of the complexes with the cells. Furthermore, modification of the complexes with transferrin leads to a significant increase of the effects of the antisense SODN on the P-gp efflux system in the cells. Overall, this study suggests that polyion complex micelles with protein-modified corona are promising tools for the delivery of antisense SODN.     

GDNF-Transfected Macrophages Produce Potent Neuroprotective Effects in Parkinson's Disease Mouse Model

The pathobiology of Parkinson''s disease (PD) is associated with the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) projecting to the striatum. Currently, there are no treatments that can halt or reverse the course of PD; only palliative therapies, such as replacement strategies for missing neurotransmitters, exist. Thus, the successful brain delivery of neurotrophic factors that promote neuronal survival and reverse the disease progression is crucial. We demonstrated earlier systemically administered autologous macrophages can deliver nanoformulated antioxidant, catalase, to the SNpc providing potent anti-inflammatory effects in PD mouse models. Here we evaluated genetically-modified macrophages for active targeted brain delivery of glial cell-line derived neurotropic factor (GDNF). To capitalize on the beneficial properties afforded by alternatively activated macrophages, transfected with GDNF-encoded pDNA cells were further differentiated toward regenerative M2 phenotype. A systemic administration of GDNF-expressing macrophages significantly ameliorated neurodegeneration and neuroinflammation in PD mice. Behavioral studies confirmed neuroprotective effects of the macrophage-based drug delivery system. One of the suggested mechanisms of therapeutic effects is the release of exosomes containing the expressed neurotropic factor followed by the efficient GDNF transfer to target neurons. Such formulations can serve as a new technology based on cell-mediated active delivery of therapeutic proteins that attenuate and reverse progression of PD, and ultimately provide hope for those patients who are already significantly disabled by the disease.     

Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.     

Nanogels for oligonucleotide delivery to the brain

Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood-brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine ("nanogel"). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain.     

Polypeptide point modifications with fatty acid and amphiphilic block copolymers for enhanced brain delivery

There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood-brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain.     

Influence of magnetic field on the spatial orientation in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) (Cyprinidae) and Roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis>) (Cyprinidae)

Preferred direction of motion under influence of geomagnetic field and its modifications was registered in zebrafish (Danio rerio) raised in laboratory culture and in roach (Rutilus rutilus) from the Rybinsk Reservoir. In the geomagnetic field, specimens of zebrafish prefer two opposite directions oriented towards the north and south, while they prefer towards east and west at 90° turning of the horizontal component of geomagnetic field. The specimens of roach in the geomagnetic field prefer only the direction oriented towards east–northeast. This direction coincides with the direction along the canal where roach was sampled to the main river channel part of the Rybinsk Reservoir. At 90° rotation of the horizontal component of geomagnetic field, the direction turns to the south–southeast. The reasons for selection of certain directions in the geomagnetic field are discussed.