Nanovectorización de ácidos nucleicos mediada por lipoplejos en terapia génica

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 26-11-2021Gene therapy is a very promising branch of biomedicine that aims to treat inherited or acquired diseases (such as cancer, cardiovascular, neurological and inflammatory diseases) at molecular level. The purpose is to restore correct cell function using nucleic acids (NAs) as therapeutic agents. Depending on the NAs inserted, there are different mechanisms of action. For instance, plasmids DNA (pDNAs) use the patient's biological machinery to replace the defective gene and express the healthy exogenous gene of interest. Other NAs, such as small interfering RNAs (siRNAs), prevent the synthesis of the pathogenic protein by knockdown the corresponding genes. In most cases, NAs require a carrier agent, known as vector, for their internalization into the target cells. Viruses were the source of inspiration during the early stages of gene therapy for the insertion of NAs into cells. Nowadays, viral vectors are still in use due to their high efficiency; however, the undesirable adverse effects associated with their use are forcing the search for alternatives. Among the different synthetic options for non-viral vectors, polymeric and colloidal systems stand out. Within colloidal systems, cationic lipids (CLs) are perhaps the most studied group due to their ability to interact electrostatically with anionic NAs, and spontaneously self-organize in aqueous solution forming structures similar to that of the cell membrane. The resulting complex between the CLs and NAs, well-known as lipoplex, must transport the NAs into cells and release them into the cytoplam without causing damage. The efficiency of this process will depend on the vector’s capacity to overcome the known biological barriers during its physiological journey...La terapia génica es una rama muy prometedora de la biomedicina que tiene como objetivo tratar enfermedades heredadas o adquiridas (como el cáncer o las enfermedades cardiovasculares, neurológicas e inflamatorias) a nivel molecular. En ella se utilizan ácidos nucleicos (NAs) como agentes terapéuticos para tratar de restablecer el correcto funcionamiento celular. Según los NAs insertados, existen distintos mecanismos de acción. Por ejemplo, los plásmidos de DNA (pDNAs) utilizan la maquinaria biológica del paciente para reemplazar el gen defectuoso y expresar el gen exógeno sano de interés. Otros NAs, como los pequeños RNAs de interferencia (siRNAs), bloquean la síntesis de la proteína defectuosa silenciando los genes específicos correspondientes. En la mayoría de los casos, los NAs necesitan un agente transportador, conocido como vector, para su internalización en las células. Los virus fueron la fuente de inspiración para insertar los NAs en las células durante las primeras etapas de la terapia génica. Aunque actualmente aún son utilizados como vectores dada su alta eficacia, los indeseados efectos adversos que provocan obligan a buscar otras alternativas. Entre las distintas opciones sintéticas de vectores no virales destacan los sistemas poliméricos y los coloidales. Dentro de este último grupo, los lípidos catiónicos (CLs) son quizás los más estudiados por su capacidad para interaccionar electrostáticamente con los NAs aniónicos y auto-organizarse espontáneamente en medio acuoso formando estructuras similares a las de la membrana celular. El complejo resultante, conocido como lipoplejo, debe transportar el material genético hasta el interior celular y liberarlo en el citoplasma sin causar daños. La eficacia de este proceso dependerá de la capacidad que muestre el vector durante su recorrido fisiológico para superar ciertas barreras biológicas...Fac. de Ciencias QuímicasTRUEunpu

Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presentedThis work has been funded by the Spanish Ministry of Science, Innovation and Universities (MICIU) (Grant RTI2018-095844-B-I00 and CTQ2017-88948-P), the University Complutense of Madrid (Spain) (project number UCMA05-33-010), and the Regional Government of Madrid (Grant P2018/NMT-4389). P.T. thanks Agencia Estatal de Investigación (AEI) through the Project MAT2016-80266-R and Xunta de Galicia (Grupo de Referencia Competitiva ED431C 2018/26; Agrupación Estratégica en Materiales-AEMAT ED431E 2018/08). ERDF funds are all greatly acknowledged. The proteomic analysis was performed in the Proteomics Unit of Complutense University of Madrid, a member of ProteoRed and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDFS

Intercellular Trafficking of Gold Nanostars in Uveal Melanoma Cells for Plasmonic Photothermal Therapy

Efficient plasmonic photothermal therapies (PPTTs) using non-harmful pulse laser irradiation at the near-infrared (NIR) are a highly sought goal in nanomedicine. These therapies rely on the use of plasmonic nanostructures to kill cancer cells while minimizing the applied laser power density. Cancer cells have an unsettled capacity to uptake, retain, release, and re-uptake gold nanoparticles, thus offering enormous versatility for research. In this work, we have studied such cell capabilities for nanoparticle trafficking and its impact on the effect of photothermal treatments. As our model system, we chose uveal (eye) melanoma cells, since laser-assisted eye surgery is routinely used to treat glaucoma and cataracts, or vision correction in refractive surgery. As nanostructure, we selected gold nanostars (Au NSs) due to their high photothermal efficiency at the near-infrared (NIR) region of the electromagnetic spectrum. We first investigated the photothermal effect on the basis of the dilution of Au NSs induced by cell division. Using this approach, we obtained high PPTT efficiency after several cell division cycles at an initial low Au NS concentration (pM regime). Subsequently, we evaluated the photothermal effect on account of cell division upon mixing Au NS-loaded and non-loaded cells. Upon such mixing, we observed trafficking of Au NSs between loaded and non-loaded cells, thus achieving effective PPTT after several division cycles under low irradiation conditions (below the maximum permissible exposure threshold of skin). Our study reveals the ability of uveal melanoma cells to release and re-uptake Au NSs that maintain their plasmonic photothermal properties throughout several cell division cycles and re-uptake. This approach may be readily extrapolated to real tissue and even to treat in situ the eye tumor itself. We believe that our method can potentially be used as co-therapy to disperse plasmonic gold nanostructures across affected tissues, thus increasing the effectiveness of classic PPTT

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*

Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12

Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications

Artificial Intelligence to evaluate the impact of COVID-19 disease on the elderly

Resumen del trabajo presentado a las II Jornadas Científicas PTI+ Salud Global, celebradas en Valencia del 5 al 6 de octubre de 2022.Peer reviewe

The gut microbiota, a hallmark of human aging, could implicate risk and effect of COVID19 in the elderly

Resumen del trabajo presentado a las II Jornadas Científicas PTI+ Salud Global, celebradas en Valencia del 5 al 6 de octubre de 2022.Peer reviewe

Longitudinal study of the immune response after

Resumen del trabajo presentado a las II Jornadas Científicas PTI+ Salud Global, celebradas en Valencia del 5 al 6 de octubre de 2022.Peer reviewe