Oligokitosano ultrapuruetan oinarritutako poliplexoak : disenua,, karakterizazioa eta terapia genikorako aplikazioak

253 p.Esta tesis doctoral se basa en el uso de oligoquitosanos ultrapuros (UOC) como vectores no-virales para el transporte de genes. El quitosano (Ch) es un polímero natural compuesto de cadenas ß-(1-4) D-glucosamina y N-acetil-D-glucosamina unidas por enlaces (1-4) glucosídicos. Gracias a los grupos amino del quitosano cargados positivamente es posible la unión electrostática con moléculas de carga negativa, como el ADN, dando lugar a lo que llamamos poliplexos (poliplexos Ch/ADN). En esta tesis hemos trabajado con quitosanos de bajo peso molecular y alto grado de deacetilación, Novafect O15 y O25 concretamente, ya que presentan unas características adecuadas para el transporte de genes. A pesar de que numerosos trabajos describen las prometedoras características de este polímero para su uso en terapia génica, el problema relacionado con su baja eficiencia de transfección aún persiste. Por ello, en este trabajo hemos evaluado in vitro la influencia de factores fisicoquímicos, especialmente el pH, en el proceso de transfección de poliplexos basados en quitosanos de bajo peso molecular. También hemos analizado la capacidad de estos poliplexos para transportar un plásmido de gran tamaño y transfectar células pulmonares. Y por último, hemos tratado de conocer el propio proceso de transfección de lo poliplexos en células precursoras neuronales, mediante su comparación con otros dos tipos de vectores no-virales

First Insights into Non-invasive Administration Routes for Non-viral Gene Therapy

Gene delivery has attracted increasing interest as a highly promising therapeutic method to treat various diseases, including both genetic and acquired disorders. However, its clinical application is still hampered by the lack of safe and effective gene delivery techniques, as well as by the need of non-invasive routes of administration in gene delivery platforms. Among the different approaches used to transport nucleic acids into target cells, non-viral vectors represent promising and safer alternatives to viruses. Non-invasive administration routes are currently being studied, such as intranasal administration to target the brain, topical retinal administration for ocular diseases and aerosolized formulations for inhalation for the treatment of pulmonary diseases. Reasonable evidence suggests that future gene delivery systems might be based on effective non-viral vectors administered through non-invasive routes, which would constitute a safe, easy to produce, cheap and customizable alternative to the current viral gene delivery platforms. In this review, after briefly introducing the basis of gene therapy, we discuss the up-to-date and possible future strategies to improve DNA transfection efficiency using non-viral vectors and focusing on the non-invasive routes of administration

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.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), the National Council of Science and Technology (CONACYT), Mexico, Reg. # 217101, the Spanish Ministry of Education (Grants CTQ2010-20541, CTQ2010- 14897), the Basque Government (Department of Education, University and Research, predoctoral BFI- 2011-2226 grant) and by Spanish grants MAT2012-39290-C02-01 and IPT-2012-0574- 300000. Technical and human support provided by SGIker (UPV/ EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU). GC acknowledges support by the Italian Minister for University and Research (MIUR) (Futuro in Ricerca, Grant No. RBFR08TLPO).Peer reviewe

Niosomes based on synthetic cationic lipids for gene delivery: The influence of polar head-groups on the transfection efficiency in HEK-293, ARPE-19 and MSC-D1 cells

We designed niosomes based on three lipids that differed only in the polar-head group to analyze their influence on the transfection efficiency. These lipids were characterized by small-angle X-ray scattering before being incorporated into the niosomes which were characterized in terms of pKa, size, zeta potential, morphology and physical stability. Nioplexes were obtained upon the addition of a plasmid. Different ratios (w/w) were selected to analyze the influence of this parameter on size, charge and the ability to condense, release and protect the DNA. In vitro transfection experiments were performed in HEK-293, ARPE-19 and MSC-D1 cells. Our results show that the chemical composition of the cationic head-group clearly affects the physicochemical parameters of the niosomes and especially the transfection efficiency. Only niosomes based on cationic lipids with a dimethyl amino head group (lipid 3) showed a transfection capacity when compared with their counterparts amino (lipid 1) and tripeptide head-groups (lipid 2). Regarding cell viability, we clearly observed that nioplexes based on the cationic lipid 3 had a more deleterious effect than their counterparts, especially in ARPE-19 cells at 20/1 and 30/1 ratios. Similar studies could be extended to other series of cationic lipids in order to progress in the research on safe and efficient non-viral vectors for gene delivery purposes.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), the National Council of Science and Technology (CONAYT), Mexico, Reg. # 217101, the Spanish Ministry of Education (Grant CTQ2010-20541, CTQ2010-14897), the Basque Government (Department of Education, University and Research, predoctoral BFI-2011-2226 grant), the Generalitat de Catalunya (2009SGR208, 2009SGR1331) and the Instituto de Salud Carlos III. Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the platform for Drug Formulation (NANBIOSIS) CIBER-BBN.Peer reviewe

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.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), by the Research Chair in Retinosis Pigmentosas “Bidons Egara”, the National Council of Science and Technology (CONACYT), Mexico, Reg. No. 217101, the Spanish Ministry of Education (Grant Nos. CTQ2010-20541, CTQ2010-14897), the Basque Government (Department of Education, University and Research, predoctoral BFI-2011-2226 grant), and by Spanish Grant Nos. MAT2012-39290-C02-01 and IPT-2012-0574-300000. Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged. The authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU).Peer reviewe

The influence of the polar head-group of synthetic cationic lipids on the transfection efficiency mediated by niosomes in rat retina and brain

The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain. © 2015 Elsevier Ltd.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), the National Council of Science and Technology (CONACYT), Mexico, Reg. # 217101, the Spanish Ministry of Education (Grant CTQ2010-20541, CTQ2010- 14897), the Basque Government (Department of Education, University and Research, predoctoral BFI-2011-2226 grant) and by Spanish grants MAT2012-39290-C02-01 and IPT-2012-0574- 300000. Technical and human support provided by SGIker (UPV/ EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU).Peer reviewe

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.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32 and UFI11/35), the Basque Government (Department of Education, GIC-12/150 to J.S, University and Research, for the predoctoral BFI-2011-2226 fellowship, and Department of Industry SAIOTEK S-PE13UN193) and the National Council of Science and Technology (CONACYT, Mexico, Reg. 217101). Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically, by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU).Peer reviewe

Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

Non-viral gene delivery vectors are emerging as a safer alternative to viral vectors. Among natural polymers, chitosan (Ch) is the most studied one, and low molecular weight Ch, specifically, presents a wide range of advantages for non-viral pDNA delivery. It is crucial to determine the best process for the formation of Low Molecular Weight Chitosan (LMWC)-pDNA complexes and to characterize their physicochemical properties to better understand their behavior once the polyplexes are administered. The transfection efficiency of Ch based polyplexes is relatively low. Therefore, it is essential to understand all the transfection process, including the cellular uptake, endosomal escape and nuclear import, together with the parameters involved in the process to improve the design and development of the non-viral vectors. The aim of this review is to describe the formation and characterization of LMWC based polyplexes, the in vitro transfection process and finally, the in vivo applications of LMWC based polyplexes for gene therapy purposes

Oligokitosano ultrapuruetan oinarritutako poliplexoak : disenua,, karakterizazioa eta terapia genikorako aplikazioak

253 p.Esta tesis doctoral se basa en el uso de oligoquitosanos ultrapuros (UOC) como vectores no-virales para el transporte de genes. El quitosano (Ch) es un polímero natural compuesto de cadenas ß-(1-4) D-glucosamina y N-acetil-D-glucosamina unidas por enlaces (1-4) glucosídicos. Gracias a los grupos amino del quitosano cargados positivamente es posible la unión electrostática con moléculas de carga negativa, como el ADN, dando lugar a lo que llamamos poliplexos (poliplexos Ch/ADN). En esta tesis hemos trabajado con quitosanos de bajo peso molecular y alto grado de deacetilación, Novafect O15 y O25 concretamente, ya que presentan unas características adecuadas para el transporte de genes. A pesar de que numerosos trabajos describen las prometedoras características de este polímero para su uso en terapia génica, el problema relacionado con su baja eficiencia de transfección aún persiste. Por ello, en este trabajo hemos evaluado in vitro la influencia de factores fisicoquímicos, especialmente el pH, en el proceso de transfección de poliplexos basados en quitosanos de bajo peso molecular. También hemos analizado la capacidad de estos poliplexos para transportar un plásmido de gran tamaño y transfectar células pulmonares. Y por último, hemos tratado de conocer el propio proceso de transfección de lo poliplexos en células precursoras neuronales, mediante su comparación con otros dos tipos de vectores no-virales

Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

Non-viral gene delivery vectors are emerging as a safer alternative to viral vectors. Among natural polymers, chitosan (Ch) is the most studied one, and low molecular weight Ch, specifically, presents a wide range of advantages for non-viral pDNA delivery. It is crucial to determine the best process for the formation of Low Molecular Weight Chitosan (LMWC)-pDNA complexes and to characterize their physicochemical properties to better understand their behavior once the polyplexes are administered. The transfection efficiency of Ch based polyplexes is relatively low. Therefore, it is essential to understand all the transfection process, including the cellular uptake, endosomal escape and nuclear import, together with the parameters involved in the process to improve the design and development of the non-viral vectors. The aim of this review is to describe the formation and characterization of LMWC based polyplexes, the in vitro transfection process and finally, the in vivo applications of LMWC based polyplexes for gene therapy purposes