Comparative Analysis of the Physicochemical and Biological Characteristics of Freeze-Dried PEGylated Cationic Solid Lipid Nanoparticles

Cationic solid-lipid nanoparticles (cSLNs) have become a promising tool for gene and RNA therapies. PEGylation (PEG) is crucial in enhancing particle stability and protection. We evaluated the impact of PEG on the physicochemical and biological characteristics of cholesteryl-oleate cSLNs (CO-cSLNs). Several parameters were analyzed, including the particle size, polydispersity index, zeta potential, shape, stability, cytotoxicity, and loading efficiency. Five different formulations with specific PEGs were developed and compared in both suspended and freeze-dried states. Small, homogeneous, and cationic suspended nanoparticles were obtained, with the Gelucire 50/13 (PEG-32 hydrogenated palm glycerides; Gelucire) and DSPE-mPEG2000 (1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjungate-2000; DSPE) formulations exhibiting the smallest particle size (similar to 170 nm). Monodisperse populations of freeze-dried nanoparticles were also achieved, with particle sizes ranging from 200 to 300 nm and Z potential values of 30-35 mV. Notably, Gelucire again produced the smallest particle size (211.1 +/- 22.4), while the DSPE and Myrj S100 (polyoxyethylene (100) stearate; PEG-100 Stearate) formulations had similar particle sizes to CO-cSLNs (similar to 235 nm). The obtained PEGylated nanoparticles showed suitable properties: they were nontoxic, had acceptable morphology, were capable of forming SLNplexes, and were stable in both suspended and lyophilized states. These PEG-cSLNs are a potential resource for in vivo assays and have the advantage of employing cost-effective PEGs. Optimizing the lyophilization process and standardizing parameters are also recommended to maintain nanoparticle integrity

Dialysis is a key factor modulating interactions between critical process parameters during the microfluidic preparation of lipid nanoparticles

Manufacturing lipid nanoparticles through microfluidic mixing can be approached from a Quality by Design perspective. Research involving critical process parameters seems to focus on the total flow and flow rate ratio, thus other process variables, such as dialysis, are underestimated. This study used a Design of Experiments to identify the influence of critical process parameters on particle size, polydispersity index, and zeta potential. A response surface Design of Experiments modeled the influence of: total flow (400 to 4000 μL min-1); flow rate ratio (3 to 9) and dialysis (yes/no). Results suggest that dialysis is a crucial parameter that strongly influences particle size and zeta potential and moderately affects polydispersity index. The flow rate ratio's relevance decreases when dialysis is performed. As the purification method can change the influence of other process parameters, it should be an integrated part of the microfluidic manufacturing of lipid nanoparticles instead of an extra step.This work was supported by the University of Costa Rica [OAICE-64-2019]; Spanish Ministry of Science and Innovation grant PID2020-118859GB-100; and the Andalusian Government grants P20_01269 and P20_01271

Estrategia de aprendizaje interactivo para la mejora de la aplicación de la competencia matemática en el entorno de la Farmacia Galénica: Impacto de las primeras acciones de mejora sobre el rendimiento de los alumnos

Projecte: 2015PID-UB/039Se presentan los avances del Grupo de Innovación Docente de Tecnología Farmacéutica (GIDTF) en su línea de actuación relativa a la implementación de una estrategia de aprendizaje interactivo en los seminarios de problemas de Farmacia Galénica para el desarrollo de la competencia matemática en este ámbito por los estudiantes del grado de Farmacia. Las acciones realizadas incluyen actividades individuales de autoevaluación para detectar el nivel de capacidad resolutiva a modo de diagnóstico y otras grupales para fomentar un aprendizaje entre iguales. Se confirma que las acciones de mejora en la estrategia de enseñanza-aprendizaje implantada en curso 2015-16, respecto al desarrollo de la competencia matemática en Farmacia Galénica, han dado los resultados benéficos esperados.Universidad de Barcelon

Interplay between PRPF40B and the epigenetic modifier PRC2 (Polycomb Repressive Complex-2)".

Congresos y conferencias: Conferencia

Targeting Splicing in the Treatment of Human Disease

The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (grant number BFU2014-54660-R) and the Andalusian Government (Excellence Project BIO-2515/2012) to Carlos Suñé and from the Spanish Ministry of Economy and Competitiveness (grant number BFU2016-79699-P) and the Andalusian Government (Excellence Project CTS-6587) to Cristina Hernández-Munain Support from the European Region Development Fund (ERDF (FEDER)) is also acknowledged. We apologize to all researchers whose papers pertinent to this article we failed to citeWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Improved formulation of cationic solid lipid nanoparticles displays cellular uptake and biological activity of nucleic acids

Non-viral delivery using cationic solid lipid nanoparticles (SLNs) represents a useful strategy to introduce large DNA and RNA molecules to target cells. A careful selection of components and their amounts is critical to improve transfection efficiency. In this work, a selected and optimized formulation of SLNs was used to efficiently transfect circular DNA and linear RNA molecules into cells. We characterized the main physicochemical characteristics and binding capabilities of these SLNs and show that they deliver DNA and RNA molecules into cells where they display full bioactivity at nontoxic concentrations using fluorescence-and luminescence-based methodologies. Hence, we established a novel and simple SLN formulation as a powerful tool for future therapeutic use. (C) 2016 Elsevier B.V. All rights reserved.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (grant number BFU2014-54660-R) and the Andalusian Government (Excellence Project BIO-2515/2012) to C.S. and from the Spanish Ministry of Economy and Competitiveness (grant number BFU2013-44660-R) and the Andalusian Government (Excellence Project CTS-6587) to C.H.M. Support from the European Region Development Fund (ERDF [FEDER]), is also acknowledged.Peer reviewe

Improved synthesis and characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles with high transfection efficiency for gene therapy applications

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.We are grateful to many colleagues for their helpful suggestions, critical discussions, and comments. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (grant number BFU2017-89179-R) and the Andalusian Government (Excellence Project BIO-2515/2012) to C.S. and from the Spanish Ministry of Economy and Competitiveness (grant number BFU2016-79699-P) to C.H.M. Support from the European Region Development Fund (ERDF [FEDER]) is also acknowledged. M.S.-P. was supported by a fellowship from the Spanish Ministry of Education (FPU Program).Peer reviewe

Cholesteryl oleate-loaded cationic solid lipid nanoparticles as carriers for efficient gene-silencing therapy

Background: Cationic solid lipid nanoparticles (SLNs) have been given considerable attention for therapeutic nucleic acid delivery owing to their advantages over viral and other nanoparticle delivery systems. However, poor delivery efficiency and complex formulations hinder the clinical translation of SLNs. Aim: The aim of this study was to formulate and characterize SLNs incorporating the cholesterol derivative cholesteryl oleate to produce SLN-nucleic acid complexes with reduced cytotoxicity and more efficient cellular uptake. Methods: Five cholesteryl oleate-containing formulations were prepared. Laser diffraction and laser Doppler microelectrophoresis were used to evaluate particle size and zeta potential, respectively. Nanoparticle morphology was analyzed using electron microscopy. Cytotoxicity and cellular uptake of lipoplexes were evaluated using flow cytometry and fluorescence microscopy. The gene inhibition capacity of the lipoplexes was assessed using siRNAs to block constitutive luciferase expression. Results: We obtained nanoparticles with a mean diameter of approximately 150-200 nm in size and zeta potential values of 25-40 mV. SLN formulations with intermediate concentrations of cholesteryl oleate exhibited good stability and spherical structures with no aggregation. No cell toxicity of any reference SLN was observed. Finally, cellular uptake experiments with DNA- and RNA-SLNs were performed to select one reference with superior transient transfection efficiency that significantly decreased gene activity upon siRNA complexation. Conclusion: The results indicate that cholesteryl oleate-loaded SLNs are a safe and effective platform for nonviral nucleic acid delivery.We are grateful to many colleagues for their helpful suggestions, critical discussions, and comments. We thank Jose Alcami and Mayte Coiras (National Microbiology Center, Majadahonda, Madrid, Spain) for the TZM-bl cells. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (grant number BFU2014-54660-R) and the Andalusian Government (Excellence Project BIO-2515/2012) to CS and from the Spanish Ministry of Economy and Competitiveness (grant number BFU2016-79699-P) to CHM. Support from the European Region Development Fund (ERDF [FEDER]) is also acknowledged. MSP was supported by a fellowship from the Spanish Ministry of Education (FPU Program [Training Program for Academic Staff]).Peer reviewe

Characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles for the targeted delivery of nucleic acids

Nanoparticles have received considerable attention as vectors for gene delivery. However, the clinical translation of nanoparticles is limited due to poor delivery, immunogenicity, toxicity, high processing costs, and production scaling problems. In recent years, cationic solid lipid nanoparticles (SLNs) have gained considerable attention owing to their advantages over viral and other nanoparticle delivery systems. The use of the cell membrane component cholesterol in lipoplex formulations has attracted interest for successful transfection, thus opening new avenues for the synthesis of novel carriers using cholesterol or its derivatives, which could greatly enhance the delivery and activity of nucleic acids. Here, we describe the development and characterization of a stable and scalable formulation of SLNs containing cholesteryl oleate. In addition to studying the physicochemical properties of the SLNs, the effect of cholesteryl oleate on the cytotoxicity, plasmid DNA and RNA transfection efficiency, and gene inhibition capacity of the lipoplexes was also studied. We identified a formula optimized in terms of structure, morphology, and nucleic acid binding efficiency without cytotoxicity, as well as superior transfection efficiency for inducing potent biological activity

Estrategia de aprendizaje interactivo para la mejora de la aplicación de la competencia matemática en el entorno de la Farmacia Galénica: Impacto de las primeras acciones de mejora sobre el rendimiento de los alumnos

Projecte: 2015PID-UB/039Se presentan los avances del Grupo de Innovación Docente de Tecnología Farmacéutica (GIDTF) en su línea de actuación relativa a la implementación de una estrategia de aprendizaje interactivo en los seminarios de problemas de Farmacia Galénica para el desarrollo de la competencia matemática en este ámbito por los estudiantes del grado de Farmacia. Las acciones realizadas incluyen actividades individuales de autoevaluación para detectar el nivel de capacidad resolutiva a modo de diagnóstico y otras grupales para fomentar un aprendizaje entre iguales. Se confirma que las acciones de mejora en la estrategia de enseñanza-aprendizaje implantada en curso 2015-16, respecto al desarrollo de la competencia matemática en Farmacia Galénica, han dado los resultados benéficos esperados.Universidad de Barcelon