Jon Zarate

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain

G‐protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G‐protein‐coupled receptors such as the serotonin 5‐HT1A receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5‐HT1A receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co‐expression of GIRK subunits with the 5‐HT1A receptor and cell markers of glutamatergic, γ‐aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double‐label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double‐labeling experiments show that GIRK subunits are present in most of the 5‐HT1A receptor‐expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases. J. Comp. Neurol. 510:581–606, 2008.

Biomaterials in Cell Microencapsulation

The field of cell encapsulation is advancing rapidly. This cell-based technology permits the local and long-term delivery ofa desired therapeutic product reducing or even avoiding the need ofimmunosuppressant drugs. The choice of a suitable material preserving the viability and functionality of enclosed cells becomes fundamental if a therapeutic aim is intended. Alginate, which is by far the most frequently used biomaterial in the field of cell microencapsulation, has been demonstrated to be probably the best polymer for this purpose due to its biocompatibility, easy manipulation, gel forming capacity and in vivo performance.

A novel cationic niosome formulation for gene delivery to the retina

Niosomes represent a recent promising approach for gene delivery purposes. We elaborated on a novel niosome formulation based on the 2,3-di(tetradecyloxy)propan-1-amine cationic lipid, combined with squalene and polysorbate 80 to evaluate the transfection efficiency in rat retinas. Niosomes prepared by the solvent emulsification-evaporation technique were mixed with the pCMSEGFP plasmid to form lipoplexes which were characterized in terms of morphology, size, surface charge, and DNA condensation, protection and release. In vitro studies were conducted to evaluate transfection efficiency, viability and internalization mechanism in HEK-293 and ARPE-19 cells. The efficacy of the most promising formulation was evaluated in rat eyes by monitoring the expression of the EGFP after intravitreal and subretinal injections. Lipoplexes at 15/1 ratio were 200nm in size, 25mV in zeta potential and exhibited spherical morphology. At this ratio, niosomes condensed and protected the DNA from enzymatic digestion. Lipoplexes successfully transfected HEK-293 and specially ARPE-19 cells, without affecting the viability. Whereas lipoplexes entered mainly retinal cells by clathrin-mediated endocytosis, HEK-293 cells showed a higher caveolae-dependent entry. After ocular administration, the expression of EGFP was detected in different cells of the retina depending on the administration route. This novel niosome formulation represents a promising approach to deliver genetic material into the retina to treat inherited retinal diseases.

Protamine/DNA/Niosome Ternary Nonviral Vectors for Gene Delivery to the Retina: The Role of Protamine

The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.

Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications

UNLABELLED This work demonstrates the successful long-term transfection in vivo of a DNA plasmid vector in rat visual cortex neurons using the magnetofection technique. The transfection rates reached values of up to 97% of the neurons after 30days, comparable to those achieved by viral vectors. Immunohistochemical treatment with anti-EGFP antibodies enhanced the detection of the EYFP-channelrhodopsin expression throughout the dendritic trees and cell bodies. These results show that magnetic nanoparticles offer highly efficient and enduring in vivo high-rate transfection in identified neurons of an adult mammalian brain and suggest that the magnetotechnique facilitates the introduction of large functional genetic material like channelrhodopsin with safe non-viral vectors using minimally invasive approaches. FROM THE CLINICAL EDITOR Gene therapy may be one of the treatment modalities for neurological diseases in the future. The use of viral transfection remains a concern due to restrictions to the size limit of the genetic material able to be packed, as well as safety issues. In this work, the authors evaluated magnetoplexes as an alternative vehicle. The results showed very promising data in that these nanoparticles could offer high transfection efficiency.

New Insights into Gene Delivery to Human Neuronal Precursor NT2 Cells: A Comparative Study between Lipoplexes, Nioplexes, and Polyplexes.

The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.13 mV), nioplexes (142.5 nm; +35.4 mV), and polyplexes (294.8 nm; +15.1 mV). We compared the transfection efficiency, cellular uptake, and intracellular trafficking of the three vectors in NT2 cell line. Lipoplexes exhibited the highest percentages of EGFP positive cells. The values obtained with polyplexes were lower compared to lipoplexes but higher than the percentages obtained with nioplexes. Cellular uptake results had a clear correlation with respect to the corresponding transfection efficiencies. Regarding the endocytosis mechanism, lipoplexes enter in the cell, mainly, via clathrin-mediated endocytosis (CME) while polyplexes via caveolae-mediated endocytosis (CvME). Nioplexes were discarded for this experiment due to their low cellular uptake. By simulating an artificial endosome, we demonstrated that the vectors were able to release the DNA cargo once inside the late endosome. The data collected from this assay showed that at 6 h the genetic material carried by polyplexes was still located in the late endosome, while DNA carried by lipoplexes was already in the nucleus. This result indicates a faster intracellular traffic of the lipid-based vectors. Overall, our work gives new insights into the transfection process of NT2 cells by different nonviral vectors as a first step in the development of ex vivo gene therapy platform.

The role of helper lipids in the intracellular disposition and transfection efficiency of niosome formulations for gene delivery to retinal pigment epithelial cells.

In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.

Cationic vesicles based on non-ionic surfactant and synthetic aminolipids mediate delivery of antisense oligonucleotides into mammalian cells

A formulation based on a synthetic aminolipid containing a double-tailed with two saturated alkyl chains along with a non-ionic surfactant polysorbate-80 has been used to form lipoplexes with an antisense oligonucleotide capable of inhibiting the expression of Renilla luciferase mRNA. The resultant lipoplexes were characterized in terms of morphology, Zeta potential, average size, stability and electrophoretic shift assay. The lipoplexes did not show any cytotoxicity in cell culture up to 150 mM concentration. The gene inhibition studies demonstrated that synthetic cationic vesicles based on non-ionic surfactant and the appropriate aminolipid play an important role in enhancing cellular uptake of antisense oligonucleotides obtaining promising results and efficiencies comparable to commercially available cationic lipids in cultured mammalian cells. Based on these results, this amino lipid moiety could be considered as starting point for the synthesis of novel cationic lipids to obtain potential non-viral carriers for antisense and RNA interference therapies.

A study of the toxic effect of oxidized sunflower oil containing 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal on cortical TrkA receptor expression in rats

Abstract Sunflower oil at a specific oxidation stage (when several oxygenated α,β-unsaturated aldehydes are generated, mainly 4-hydroperoxy-trans-2-alkenals and 4-hydroxy-trans-2-alkenals), caused at 70°C with aeration for 7 days, was administered intraperitoneally to rats. This oil was studied by means of solid phase micro-extraction followed by gas chromatography/mass spectrometry (SPME-GC/MS) and by proton nuclear magnetic resonance (1H-NMR). Oxidized sunflower oil (3 ml/kg/day) was administered to male Sprague–Dawley rats for 21 days. The control group was administered non-oxidized sunflower oil in the same volume and for the same duration as the experimental group. A significant decrease in the number of neural cells positively immunostained for TrkA receptor was detected in the frontal cortex of the experimental group, with respect to controls, suggesting both neuronal damage as well as a deficit in neuronal survival signalling at this level. This could lead to apoptosis of cholinergic neurons, which play a key role in memory and attention function. These results indicate that toxic substances present in the oxidized sunflower oil, among them 4-hydroxy-trans-2-nonenal (HNE) and 4-hydroperoxy-trans-2-nonenal (HPNE), could disrupt survival signalling of frontal cortex cholinergic neurons, which could lead to apoptosis and neurodegenerative diseases. In the case of humans, this fact reinforces the necessity of avoiding the re-utilization of oxidized sunflower oil, in order to contribute to long-term neurodegenerative diseases prevention.

Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification

Abstract Context: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications. Objective: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells. Materials and methods: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay. Results and discussion: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000™ showed decreased cell viability. Conclusion: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.