Evaluation of the biophysical performance upon the integration of helper components into cationic niosomes for the treatment of both retinal and brain diseases by genetherapy approach

227 p.The integration of novel compounds into formulations may open up new avenues and opportunities for improving the treatment of various diseases using a genetherapytechnique. To this purpose, my thesis aimed to study the impact of incorporation of biomaterials such sphingolipids or nanodiamonds as a helper component into a cationic niosome formulation for gene delivery, the formulations and related complexes were studied in terms of particle size,surface charge, dispersity, and morphology. Furthermore, biological research were conducted inboth in vitro and in vivo experimental trials to evaluate cellular uptake, intracellular trafficking pathways, and transfection efficiency

Gene Therapy for Cystic Fibrosis: Hurdles to Overcome for Successful Clinical Translation

Cystic fibrosis (CF) is a genetic disease that hampers the lung function. Despite that the main defective gene has been deeply characterized, some relevant concerns still need to be resolved before considering gene therapy as a realistic medical choice. One of the major issues that need to be strongly considered in order to succeed in the search for an effective gene therapy approach for CF is the design of the appropriate genetic material to be delivered. Other relevant factors to take into consideration include the design of safe and effective gene delivery systems, the biological barriers that need to be overcome in order to reach the nucleus of the target cells, and the problems related to the design of a drug formulation suitable for lung delivery purposes. Furthermore, some problems related to the commercialization of gene therapy products also need to be resolved. In this chapter, we discuss the up-to-date strategies to overcome such hurdles in order for gene therapy to become a routine treatment modality for CF

Sphingolipid extracts enhance gene delivery of cationic lipid vesicles into retina and brain

[EN]The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.This work was supported by the Basque Country Government (Department of Education, University and Research, Consolidated Groups IT907-16) . Additional funding was provided by the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) , an initiative of the Carlos III Health Institute (ISCIII) . I.V.B. and M.S.R. thank the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (ESPDOC19/47) and the granted pre-doctoral fellowship (PIF17/79) , respectively. Authors wish to thank the intel-lectual 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) . Technical and human sup-port provided by SGIKER (UPV/EHU) is also gratefully acknowledged

Niosome-Based Approach for In Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.This work was supported by the Basque Country Government (Department of Education, University and Research, pre-doctoral grant PRE_2016_2_0302 and Consolidated Groups IT907-16). Additional funding was provided by the University of Basque Country UPV/EHU (predoctoral grant PIF17/19), the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), and initiative of the Carlos III Health Institute (ISCIII)

Niosome-Based Approach for In Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed

Construction of a recombinant mu opioid receptor tagged with FLAG epitope and yellow fluorescent protein to generate a FLIP-In HEK293 stable cell line

Máster en Biología Molecular y Biomedicin

Correlation between Biophysical Properties of Niosomes Elaborated with Chloroquine and Different Tensioactives and Their Transfection Efficiency

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation.Authors wish to thank: ICTS “NANBIOSIS”, specifically the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) for the intellectual and technical assistance; SGIker (UPV/EHU) for technical and human support; the Department of Education, University and Research of the Basque Country Government (Consolidated Groups, IT907-16); the Spanish Ministry of Science and Innovation (Grants PID2019-106199RB-C21 and RTI2018-099985-B-I00); also the support of CIBER of Respiratory Diseases (CIBERES-ISCIII) and the Cystic Fibrosis Association of the Basque Country. M.S.-R. thanks the University of the Basque Country (UPV/EHU) for the granted pre-doctoral fellowship (PIF17/79). I.V.-B. thanks the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (call for the Specialization of Doctor Researcher Personnel of the UPV/EHU, grant reference: ESPDOC19/47).Peer reviewe

Nanodiamond Integration into Niosomes as an Emerging and Efficient Gene Therapy Nanoplatform for Central Nervous System Diseases

[Image: see text] Nanodiamonds (NDs) are promising materials for gene delivery because of their unique physicochemical and biological features, along with their possibility of combination with other nonviral systems. Our aim was to evaluate the biophysical performance of NDs as helper components of niosomes, named nanodiasomes, to address a potential nonviral gene delivery nanoplatform for therapeutic applications in central nervous system (CNS) diseases. Nanodiasomes, niosomes, and their corresponding complexes, obtained after genetic material addition at different ratios (w/w), were evaluated in terms of physicochemical properties, cellular uptake, intracellular disposition, biocompatibility, and transfection efficiency in HEK-293 cells. Nanodiasomes, niosomes, and complexes fulfilled the physicochemical features for gene therapy applications. Biologically, the incorporation of NDs into niosomes enhanced 75% transfection efficiency (p < 0.001) and biocompatibility (p < 0.05) to values over 90%, accompanied by a higher cellular uptake (p < 0.05). Intracellular trafficking analysis showed higher endocytosis via clathrins (p < 0.05) in nanodiaplexes compared with nioplexes, followed by higher lysosomal colocalization (p < 0.05), that coexisted with endosomal escape properties, whereas endocytosis mediated by caveolae was the most efficient pathway in the case of nanodiaplexes. Moreover, studies in CNS primary cells revealed that nanodiaplexes successfully transfected neuronal and retinal cells. This proof-of-concept study points out that ND integration into niosomes represents an encouraging nonviral nanoplatform strategy for the treatment of CNS diseases by gene therapy

Niosome-Based Approach for In Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.Acknowledgments: The authors 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). Funding: This work was supported by the Basque Country Government (Department of Education, University and Research, pre-doctoral grant PRE_2016_2_0302 and Consolidated Groups IT907-16). Additional funding was provided by the University of Basque Country UPV/EHU (predoctoral grant PIF17/19), the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), and initiative of the Carlos III Health Institute (ISCIII).Peer reviewe