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Dive into the research topics where Gustavo Puras is active.

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Featured researches published by Gustavo Puras.


Journal of Controlled Release | 2014

A novel cationic niosome formulation for gene delivery to the retina

Gustavo Puras; Mohamed Mashal; Jon Zarate; Mireia Agirre; Edilberto Ojeda; Santiago Grijalvo; Ramon Eritja; A. Diaz-Tahoces; G. Martínez Navarrete; M. Avilés-Trigueros; Eduardo Fernández; José Luis Pedraz

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.


European Journal of Pharmaceutical Sciences | 2013

Oligochitosan polyplexes as carriers for retinal gene delivery.

Gustavo Puras; J. Zarate; A. Díaz-Tahoces; Marcelino Avilés-Trigueros; Eduardo Fernández; José Luis Pedraz

Non-viral gene therapy represents a promising approach for the treatment of retinal diseases. However, the lack of an efficient carrier hampers the implementation of this therapy. In this study, we evaluated low molecular weight ultrapure oligochitosans for the delivery of the pCMS-EGFP plasmid into the rat retina cells after subretinal and intravitreal administrations. Polyplexes were technologically characterized. Resulting polyplexes based on ultrapure oligochitosans were slightly spherical, protected the plasmid against enzymatic digestion, and their charge and size values ranged from 8 to 14 millivolts and from 150 to 69 nm respectively depending on the N/P ratio. In HEK-293 cultured cells, transfection efficiency significantly increased from 12% to 30% when pH decreased from 7.4 to 7.1 (data normalized to Lipofectamine™ 2000). However, no significant transfection was detected in ARPE-19 cultured cells. Subretinal administrations transfected mainly the pigmented cells of the retinal pigment epithelium and the light sensitive photoreceptor cells, whereas intravitreal injections transfected cells in the ganglion cell layer, blood vessels in the inner layers of the retina and photoreceptors. These results support the potential use of oligochitosans for delivering genetic material into retinal cells in vivo.


Molecular Pharmaceutics | 2015

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

Gustavo Puras; Gema Martínez-Navarrete; Mohamed Mashal; Jon Zarate; Mireia Agirre; Edilberto Ojeda; Santiago Grijalvo; Ramon Eritja; Ariadna Díaz-Tahoces; Marcelino Avilés-Trigueros; Eduardo Fernández; José Luis Pedraz

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.


Nanomedicine: Nanotechnology, Biology and Medicine | 2015

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

Cristina Soto-Sánchez; Gema Martínez-Navarrete; Lawrence Humphreys; Gustavo Puras; Jon Zarate; José Luis Pedraz; Eduardo Fernández

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.


Molecular Pharmaceutics | 2015

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

Mireia Agirre; Edilberto Ojeda; Jon Zarate; Gustavo Puras; Santiago Grijalvo; Ramon Eritja; García del Caño G; Barrondo S; González-Burguera I; López de Jesús M; Sallés J; José Luis Pedraz

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.


International Journal of Pharmaceutics | 2016

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

Edilberto Ojeda; Gustavo Puras; Mireia Agirre; Jon Zarate; Santiago Grijalvo; Ramon Eritja; Luca Digiacomo; Giulio Caracciolo; José Luis Pedraz

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.


Colloids and Surfaces B: Biointerfaces | 2014

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

Santiago Grijalvo; Adele Alagia; Gustavo Puras; Jon Zarate; José Luis Pedraz; Ramon Eritja

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.


Journal of Controlled Release | 2017

Retinal gene delivery enhancement by lycopene incorporation into cationic niosomes based on DOTMA and polysorbate 60

Mohamed Mashal; Noha Attia; Gustavo Puras; Gema Martínez-Navarrete; Eduardo Fernández; José Luis Pedraz

&NA; The present study aimed to evaluate the incorporation of the natural lipid lycopene into niosome formulations based on cationic lipid DOTMA and polysorbate 60 non‐ionic surfactant to analyze the potential application of this novel formulation to deliver genetic material into the rat retina. Both niosomes with and without lycopene were prepared by the reverse phase evaporation method and physicochemically characterized in terms of size, zeta potential, polydispersity index and capacity to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed in ARPE‐19 cells after complexion of niosomes with pCMS‐EGFP plasmid at appropriate cationic lipid/DNA ratios. At 18/1 mass ratio, nioplexes containing lycopene had nanometric size, positive zeta potential, low polydispersity and were able to condense, release and protect DNA. Percentage of transfected cell was around 35% without compromising cell viability. The internalization pathways studies revealed a preference to caveolae mediated endocytosis and macropinocytosis, which could circumvent lysosomal degradation. Both subretinal and intravitreal administrations to the rat retina showed that nioplexes were able to transfect efficiently the outer segments of the retina, which offer reasonable hope for the treatment of many inherited retinal diseases by a safe non‐viral vector formulation after the less invasive intravitreal administration.


Drug Delivery | 2015

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

Mireia Agirre; Jon Zarate; Gustavo Puras; Edilberto Ojeda; José Luis Pedraz

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.


International Journal of Pharmaceutics | 2017

Non-viral vectors based on magnetoplexes, lipoplexes and polyplexes for VEGF gene delivery into central nervous system cells

Ilia Villate-Beitia; Gustavo Puras; Cristina Soto-Sánchez; Mireia Agirre; Edilberto Ojeda; Jon Zarate; Eduardo Fernández; José Luis Pedraz

Nanotechnology based non-viral vectors hold great promise to deliver therapeutic genes into the central nervous system (CNS) in a safe and controlled way. Vascular endothelial growth factor (VEGF) is a potential therapeutic gene candidate for CNS disorders due to its specific roles in brain angiogenesis and neuroprotection. In this work, we elaborated three different non-viral vectors based on magnetic, cationic lipid and polymeric nanoparticles complexed to the phVEGF165aIRESGFP plasmid, which codifies the VEGF protein -extracellular- and the green fluorescent protein (GFP) -intracellular-. Nanoparticles and corresponding nanoplexes -magnetoplexes, lipoplexes and polyplexes- were characterized in terms of size, zeta potential, polydispersity index, morphology and ability to bind, release and protect DNA. Transfection efficiencies of nanoplexes were measured in terms of percentage of GFP expressing cells, mean fluorescent intensity (MFI) and VEGF (ng/ml) production in HEK293, C6 and primary neuronal culture cells. Magnetoplexes showed the highest transfection efficiencies in C6, followed by lipoplexes, and in primary neuronal culture cells, followed by polyplexes. Lipoplexes were the most efficient in HEK293 cells, followed by magnetoplexes. The biological activity of VEGF was confirmed by its proliferative effect in HUVEC cells. Overall, these results provide new insights for VEGF gene delivery into CNS cells using non-viral vectors.

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José Luis Pedraz

University of the Basque Country

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Santiago Grijalvo

Spanish National Research Council

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Laura Saenz del Burgo

University of the Basque Country

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Jesús Ciriza

University of the Basque Country

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