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.This work is supported by the Spanish Ministry of Education (Grant CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208) and the Instituto de Salud Carlos III (CB06_01_0019). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The authors wish to thank CIBER-BBN Research Infrastructures, in particular S. Vilchez and C. Fornaguera from the Nanostructured liquid characterization unit for the characterization and stability measurements of the lipoplexes by Dynamic Light Scattering. Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged.N

Kudeaketa, Plangintza, Legeria eta Deontologia

Duración (en horas): Más de 50 horas Destinatario: EstudianteEn esta asignatura, el alumnado deberá aplicar diferentes conceptos necesarios para gestionar eficientemente la oficina de farmacia. Para ello, trabajará diferentes conceptos relaccionados con el marketing, la contabilidad, el control de stock, la gestión de compras y de ventas, etc. Todo ello, con la finalidad de conseguir la máxima rentabilidad posible en un establecimiento de interés sanitario público y gestión privada como es la oficina de farmacia, respetando siempre las normas y leyes que regulan su funcionamiento. Para el desarrollo de la mayor parte del programa se utilizarán metodologías activas, principalmente Aprendizaje Basado en Problemas (ABP), aunque también se llevarán a cabo otras actividades colaborativas como por ejemplo el aprendizaje a través del Puzzle de Aronson o GPS (Gided Problem Solving) cuyas carcaterísticas diferenciales se irán explicando en el momento de aplicarlas en el aula. Con el ABP lo que se pretende es que el alumnado sea capaz, a través de una situación inicial cercana a lo que le ocurrirá en su futuro profesional, de pensar qué información necesita para poder hacer frente con éxito a ese probelma o situación. Es decir, el alumnado debe identificar las necesidades u objetivos de aprendizaje. Una vez identificados, será necesario un trabajo de búsqueda e interiorización de información, así como de su aplicación a ciertas situaciones de una manera autónoma, aunque siempre guiada por el profesorado, que facilitará el aprendizaje continuado del alumnado. Al contrario que en el desarrollo de la actividad de una manera más tradicional, en esta metodología, el profesorado no explica una teoría que después se ha de aplicar, sino que es el alumnado el que ha de llegar a buscar y comprender la teoría que antes se explicaba en las clases magistrales para poder resolver una situación hasta ese momento desconocida

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

Design of double functionalized carbon nanotube for amphotericin B and genetic material delivery.

In the present work, single wall carbon nanotubes (SWCNT) were successively functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine (EDA), to obtain double functionalized single wall carbon nanotube (DFSWCNT). Then, DFSWCNT was applied as a carrier for delivering amphotericin B (Amb) and EGFP plasmid. FSWCNT’s concentration obtained via UV–visible analysis was 0.99 mg/mL. The TGA analysis results provided the lost weights of DSPE-PEG-COOH, EDA, Amb and SWCNT impurities. XPS results showed that carbon atoms’ percentage decreased during the functionalization processes from 97.2% (SWCNT) to 76.4% (FSWCNT) and 69.9% (DFSWNCT). Additionally, the oxygen atoms’ percentage increased from 2.3% (SWCNT) to 21% and 22.5% for FSWCNT and DFSWCNT, respectively. New bonds such as C–N and N–C=O appeared in the synthesized nanocarrier. The IG/ID ratio in Raman analysis decreased from 7.15 (SWCNT) to 4.08 (FSWCNT). The amount of Amb released to phosphate buffer saline medium was about 33% at pH = 5.5 and 75% at pH = 7.4 after 48 h. CCK8 results confirmed that the toxicity of functionalized SWCNT had decreased. In a 2:1 ratio of DFSWCNT/EGFP plasmid, the cell viability (87%) and live transfected cells (56%) were at their maximum values. The results indicate that carbon nanotubes have the potential to be applied as drug/gene delivery systems with outstanding properties such as high loading capacity and easy penetration to cell membrane.This work was supported by the Basque Country Government (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)

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

Mesenchymal Stem Cells as a Gene Delivery Tool: Promise, Problems, and Prospects

The cell-based approach in gene therapy arises as a promising strategy to provide safe, targeted, and efficient gene delivery. Owing to their unique features, as homing and tumor-tropism, mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in gene therapy. Nevertheless, non-viral transfer of nucleic acids into MSCs remains limited due to various factors related to the main stakeholders of the process (e.g., nucleic acids, carriers, or cells). In this review, we have summarized the main types of nucleic acids used to transfect MSCs, the pros and cons, and applications of each. Then, we have emphasized on the most efficient lipid-based carriers for nucleic acids to MSCs, their main features, and some of their applications. While a myriad of studies have demonstrated the therapeutic potential for engineered MSCs therapy in various illnesses, optimization for clinical use is an ongoing challenge. On the way of improvement, genetically modified MSCs have been combined with various novel techniques and tools (e.g., exosomes, spheroids, 3D-Bioprinting, etc.,) aiming for more efficient and safe applications in biomedicine.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. The Department of Education, University and Research of the Basque Country Government (Consolidated Groups, IT907-16); the Spanish Ministry of Science and Innovation (GrantsPID2019-106199RB-C21)

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

Therapeutic Opportunities and Delivery Strategies for Brain Revascularization in Stroke, Neurodegeneration, and Aging

[EN] Central nervous system (CNS) diseases, especially acute ischemic events and neurodegenerative disorders, constitute a public health problem with no effective treatments to allow a persistent solution. Failed therapies targeting neuronal recovery have revealed the multifactorial and intricate pathophysiology underlying such CNS disorders as ischemic stroke, Alzheimers disease, amyotrophic lateral sclerosis, vascular Parkisonism, vascular dementia, and aging, in which cerebral microvasculature impairment seems to play a key role. In fact, a reduction in vessel density and cerebral blood flow occurs in these scenarios, contributing to neuronal dysfunction and leading to loss of cognitive function. In this review, we provide an overview of healthy brain microvasculature structure and function in health and the effect of the aforementioned cerebral CNS diseases. We discuss the emerging new therapeutic opportunities, and their delivery approaches, aimed at recovering brain vascularization in this context. SIGNIFICANCE STATEMENT: The lack of effective treatments, mainly focused on neuron recovery, has prompted the search of other therapies to treat cerebral central nervous system diseases. The disruption and degeneration of cerebral microvasculature has been evidenced in neurodegenerative diseases, stroke, and aging, constituting a potential target for restoring vascularization, neuronal functioning, and cognitive capacities by the development of therapeutic pro-angiogenic strategies.This work was supported by the University of the Basque Country (UPV/EHU) [Grant ESPDOC19/47] (postdoctoral fellowship to I.V.B.); and the Basque Country Government (Consolidated Groups) [Grant IT907-16]

Medial septal beta-amyloid 1-40 injections alter septo-hippocampal anatomy and function

Degeneration of septal neurons in Alzheimer’s disease (AD) results in abnormal information processing at cortical circuits and consequent brain dysfunction. The septum modulates the activity of hippocampal and cortical circuits and is crucial to the initiation and occurrence of oscillatory activities such as the hippocampal theta rhythm. Previous studies suggest that amyloid β peptide (Aβ) accumulation may trigger degeneration in AD. This study evaluates the effects of single injections of Aβ 1–40 into the medial septum. Immunohistochemistry revealed a decrease in septal cholinergic (57%) and glutamatergic (53%) neurons in Aβ 1–40 treated tissue. Additionally, glutamatergic terminals were significantly less in Aβ treated tissue. In contrast, septal GABAergic neurons were spared. Unitary recordings from septal neurons and hippocampal field potentials revealed an approximately 50% increase in firing rates of slow firing septal neurons during theta rhythm and large irregular amplitude (LIA) hippocampal activities and a significantly reduced hippocampal theta rhythm power (49%) in Aβ 1–40 treated tissue. Aβ also markedly reduced the proportion of slow firing septal neurons correlated to the hippocampal theta rhythm by 96%. These results confirm that Aβ alters the anatomy and physiology of the medial septum contributing to septohippocampal dysfunction. The Aβ induced injury of septal cholinergic and glutamatergic networks may contribute to an altered hippocampal theta rhythm which may underlie the memory loss typically observed in AD patients

How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.This work was supported by the Basque Country Government (Department of Education, University and Research, Consolidated Groups IT907-16) and by the Spanish Ministry of Science and Innovation (Grant PID2019-106199RB-C21). 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. Additional funding was provided by the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), an initiative of the Carlos III Health Institute (ISCIII)