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Background: Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB. Methodology/Principal Findings: In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes. Conclusions/Significance: With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.
Fluorescently labeled nanoparticles are widely used for evaluating their distribution in the biological environment. However, dye leakage can lead to misinterpretations of the nanoparticles’ biodistribution. To better understand the interactions of dyes and nanoparticles and their biological environment, we explored PLGA nanoparticles labeled with four widely used dyes encapsulated (coumarin 6, rhodamine 123, DiI) or bound covalently to the polymer (Cy5.5.). The DiI label was stable in both aqueous and lipophilic environments, whereas the quick release of coumarin 6 was observed in model media containing albumin (42%) or liposomes (62%), which could be explained by the different affinity of these dyes to the polymer and lipophilic structures and which we also confirmed by computational modeling (log PDPPC/PLGA: DiI—2.3, Cou6—0.7). The importance of these factors was demonstrated by in vivo neuroimaging (ICON) of the rat retina using double-labeled Cy5.5/Cou6-nanoparticles: encapsulated Cou6 quickly leaked into the tissue, whereas the stably bound Cy.5.5 label remained associated with the vessels. This observation is a good example of the possible misinterpretation of imaging results because the coumarin 6 distribution creates the impression that nanoparticles effectively crossed the blood–retina barrier, whereas in fact no signal from the core material was found beyond the blood vessels.
Little attention so-far has been paid to the influence of chronobiology on the processes of nanoparticle uptake and transport into the brain, even though this transport appears to be chronobiologically controlled to a significant degree. Nanoparticles with specific surface properties enable the transport across the blood–brain barrier of many drugs that normally cannot cross this barrier. A clear dependence of the central antinociceptive (analgesic) effects of a nanoparticle-bound model drug, i.e., the hexapeptide dalargin, on the time of day was observable after intravenous injection in mice. In addition to the strongly enhanced antinociceptive effect due to the binding to the nanoparticles, the minima and maxima of the pain reaction with the nanoparticle-bound drug were shifted by almost half a day compared to the normal circadian nociception: The maximum in the pain reaction after i.v. injection of the nanoparticle-bound dalargin occurred during the later rest phase of the animals whereas the normal pain reaction and that of a dalargin solution was highest during the active phase of the mice in the night. This important shift could be caused by an enhanced endo- and exocytotic particulates transport activity of the brain capillary endothelial cells or within the brain during the rest phase.
The genetic treatment of neurodegenerative diseases still remains a challenging task since many approaches fail to deliver the therapeutic material in relevant concentrations into the brain. As viral vectors comprise the risk of immune and inflammatory responses, human serum albumin (HSA) nanoparticles were found to represent a safer and more convenient alternative. Their ability to cross the blood-brain barrier (BBB) and deliver drugs into the brain in order to enhance gene-based therapy has been previously demonstrated. The present study deals with the development of pGL3-PEI-coated HSA nanoparticles and subsequent in vitro testing in cerebellar granular and HeLa cells. The luciferase control vector pGL3 was chosen as reporter plasmid encoding for the firefly luciferase protein, linear polyethylenimine (22 kDa) as endosomolytic agent for enhancing the cells’ transfection. Studies on particle characteristics, their cellular uptake into aforementioned cell lines and on subcellular localisation, and transfection efficiency in the cerebellar cells proved the feasibility of nanoparticle-based gene delivery.
Background: Chemotherapy of glioblastoma is largely ineffective as the blood-brain barrier (BBB) prevents entry of most anticancer agents into the brain. For an efficient treatment of glioblastomas it is necessary to deliver anti-cancer drugs across the intact BBB. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with poloxamer 188 hold great promise as drug carriers for brain delivery after their intravenous injection. In the present study the anti-tumour efficacy of the surfactant-coated doxorubicin-loaded PLGA nanoparticles against rat glioblastoma 101/8 was investigated using histological and immunohistochemical methods. Methodology: The particles were prepared by a high-pressure solvent evaporation technique using 1% polyvinylalcohol (PLGA/PVA) or human serum albumin (PLGA/HSA) as stabilizers. Additionally, lecithin-containing PLGA/HSA particles (Dox-Lecithin-PLGA/HSA) were prepared. For evaluation of the antitumour efficacy the glioblastoma-bearing rats were treated intravenously with the doxorubicin-loaded nanoparticles coated with poloxamer 188 using the following treatment regimen: 3×2.5 mg/kg on day 2, 5 and 8 after tumour implantation; doxorubicin and poloxamer 188 solutions were used as controls. On day 18, the rats were sacrificed and the antitumour effect was determined by measurement of tumour size, necrotic areas, proliferation index, and expression of GFAP and VEGF as well as Isolectin B4, a marker for the vessel density. Conclusion: The results reveal a considerable anti-tumour effect of the doxorubicin-loaded nanoparticles. The overall best results were observed for Dox-Lecithin-PLGA/HSA. These data demonstrate that the poloxamer 188-coated PLGA nanoparticles enable delivery of doxorubicin across the blood-brain barrier in the therapeutically effective concentrations.
Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood.
Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor.
Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.
The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that - after first-line in vitro assays - characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.
An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.
Objective: Establishment of an immunocompetent mouse model representing the typical progressive stages observed in malignant human gliomas for the in vivo evaluation of novel target-specific regimens.
Methods: Isolated clones from tumours that arose spontaneously in GFAP-v-src transgenic mice were used to develop a transplantable brain tumour model in syngeneic B6C3F1 mice. STAT3 protein was knocked down by infection of tumour cells with replication-defective lentivirus encoding STAT3-siRNA. Apoptosis is designed to be induced by soluble recombinant TRAIL + chemical Bcl-2/Bcl-xL inhibitors.
Results: Striatal implantation of 105 mouse tumour cells resulted in the robust development of microscopically (2 – 3 mm) infiltrating malignant gliomas. Immunohistochemically, the gliomas displayed the astroglial marker GFAP and the oncogenic form of STAT3 (Tyr-705-phosphorylated) which is found in many malignancies including gliomas. Phosphorylated STAT3 was particularly prominent in the nucleus but was also found at the plasma membrane of peripherally infiltrating glioma cells. To evaluate the role of STAT3 in tumour progression, we stably expressed siRNA against STAT3 in several murine glioma cell lines. The effect of STAT3 depletion on proliferation, invasion and survival will be first assessed in vitro and subsequently after transplantation in vivo. Upstream and downstream components of the STAT3 signalling pathway as well as possible non-specific side effects of STAT3-siRNA expression after lentiviral infection will be examined, too.
Conclusions: Its high rate of engraftment, its similarity to the malignant glioma of origin, and its rapid locally invasive growth should make this murine model useful in testing novel therapies for malignant gliomas.