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dc.contributor.advisorPérez Pérez, León Darío
dc.creatorDíaz Ariza, Ivonne Lorena
dc.date.accessioned2020-09-03T03:57:31Z
dc.date.available2020-09-03T03:57:31Z
dc.date.created2020-02-14
dc.identifier.urihttps://repositorio.unal.edu.co/handle/unal/78367
dc.descriptionEl primer capítulo revisa los aspectos más importantes de los vectores poliméricos usados en terapia génica. Este tipo de terapia envuelve la introducción de material genético en células específicas para el tratamiento de ciertas enfermedades, usando vectores que median la internalización celular de los ácidos nucleicos y los protege de la degradación. Dentro de los vectores que han mostrado resultados prometedores se encuentran los polímeros catiónicos sintéticos, los cuales presentan ventajas como mayor seguridad, procesos sencillos de fabricación y posibilidad de modificar su estructura. La poli(etilenimina) (PEI) y el poli(metacrilato de 2-(dimetilamino) etilo) (PDMAEMA) se han destacado en este contexto debido a su alta eficiencia de transfección; sin embargo, su carácter no biodegradable y citotoxicidad asociada han limitado sus aplicaciones. Para superar estas desventajas, varios investigadores han estudiado la implementación de estrategias como la PEGilación, la copolimerización con segmentos hidrófobos y el uso de arquitecturas ramificadas, las cuales han mostrado reducir significativamente la toxicidad y aumentar la eficiencia de transfección. Además, estas modificaciones estructurales pueden realizarse empleando métodos sintéticos, como polimerizaciones controladas y acoplamientos selectivos, que permiten diseñar moléculas con propiedades bien definidas. El segundo capítulo describe la síntesis y caracterización de una serie de copolímeros compuestos por metoxi-polietilenglicol y un bloque hidrófobo de poli(caprolactona-co-carbonato de propargilo) injertado con segmentos de PDMAEMA o PEI lineal (lPEI) de bajo peso molecular, empleando una combinación de polimerización por apertura de anillo, química click y polimerización radicalaria por transferencia de átomo. De esta manera se obtuvieron materiales con diferentes densidades de injerto y longitudes del segmento hidrófobo. Siguiendo la ruta sintética planteada, se logró sintetizar copolímeros de injerto basados en PDMAEMA con una estructura y composición target, mientras que para los copolímeros compuestos por lPEI, se obtuvieron densidades de injerto menores a las esperadas. Estos materiales se auto-ensamblaron en medio acuoso para formar partículas cargadas positivamente con tamaños promedio entre 150 y 380 nm. El tercer capítulo cubre la formación y caracterización de los complejos copolímero/ADN, variando parámetros como la relación nitrógeno/fósforo (N/P) y la matriz de complejación. Los estudios de habilidad de condensación mostraron que todos los copolímeros de PDMAEMA pudieron complejar las moléculas de ADN a relaciones N/P ≥ 1, sin importar las condiciones empleadas, mientras que los copolímeros de lPEI, necesitaron relaciones N/P de 7 a 20, dependiendo de su composición y la solución de complejación. En el caso de los materiales compuestos por PDMAEMA, las mediciones de tamaño de partícula y potencial zeta indicaron la formación de complejos cargados positivamente con tamaños inferiores a 300 nm, los cuales son adecuados para la internalización celular. Por su parte, los complejos basados en lPEI exhibieron cargas superficiales negativas o neutras con diámetros hidrodinámicos por debajo de 500 nm. Estas propiedades podrían generan restricciones para la entrega efectiva del ADN. El cuarto capítulo describe la citotoxicidad in vitro de los copolímeros de injerto y la eficiencia de transfección de los poliplejos resultantes, empleando como controles lPEI 25 kDa y PDMAEMA 20 kDa. Los copolímeros fueron menos citotóxicos que los homopolímeros control en células L929, exhibiendo una mayor viabilidad celular al reducir la longitud del segmento hidrófobo y la cantidad de injertos catiónicos. Los poliplejos formados a partir de los copolímeros de PDMAEMA fueron más eficientes en la entrega de ADN que los policationes estándar en células HEK-293, sin afectar su viabilidad. Por su parte, los complejos basados en los copolímeros de lPEI exhibieron una baja eficiencia de transfección, incluso usando altas concentraciones de polímero y relaciones N/P. En general, los copolímeros compuestos por segmentos hidrófobos largos y mayor densidad de injerto presentaron un mejor desempeño. En especial, el material denominado como PP6D5 mostró las características más prometedoras para su aplicación como agente de transfección de ADN, tanto en ensayos in vitro como in vivo. Esta tesis representa el punto de partida para futuras investigaciones relacionadas con el diseño racional de estructuras injertadas basadas en polímeros catiónicos que puedan presentar mejores propiedades y desempeño para su aplicación como sistemas de entrega de genes.
dc.description.abstractThe first chapter reviews the most important aspects of the polymeric vectors used in gene therapy. This type of therapy involves the introduction of genetic material into specific cells to treat certain diseases, using vectors that mediate the cellular internalization of nucleic acids and protect them from degradation. Synthetic cationic polymers have shown promising results as gene vectors, due to their advantages such as higher safety, simple fabrication process and modifiable structure. Poly(ethyleneimine) (PEI) and poly(2- (dimethylamino)ethyl methacrylate) (PDMAEMA) have been highlighted in this context due to their high transfection efficiency; however, the non-biodegradable character and associated cytotoxicity have limited their applications. To overcome these disadvantages, several researchers have studied the implementation of strategies such as PEGylation, copolymerization with hydrophobic segments and the use of branched architectures, which have demonstrated to significantly reduce the toxicity and increase the transfection efficiency. In addition, these structural modifications can be perform using synthetic methods, such as controlled polymerizations and selective coupling reactions, which allow the design of molecules with well-defined properties. The second chapter describes the synthesis and characterization of copolymers composed of methoxy poly(ethylene glycol) and a hydrophobic block of poly(ɛ-caprolactone-co-propargyl carbonate) grafted with low molecular weight PDMAEMA or linear PEI (lPEI), using a combination of ring opening polymerization, click chemistry and atom transfer radical polymerization. In this way, materials with different grafting densities and lengths of the hydrophobic segment were obtained. Following the proposed synthetic route, the synthesis of graft copolymers based on PDMAEMA with target structure and composition was achieved, while for copolymers composed of lPEI, lower grafting densities than the target ones were obtained. These materials could self-assembled in aqueous medium to form positively charged particles with average sizes between 150 and 380 nm. The third chapter covers the formation and characterization of the copolymer/DNA complexes with different nitrogen/phosphorus (N/P) ratios and complexation matrices. The study of the condensation ability showed that all PDMAEMA copolymers were able to complex the DNA molecules at N/P ratios ≥ 1, regardless of the used conditions, while lPEI copolymers required N/P ratios from 7 to 20, depending on their composition and the complexation solution. In the case of the materials composed of PDMAEMA, the measurements of particle size and zeta potential indicated the formation of positively charged complexes with sizes below 300 nm, which are suitable for cellular uptake. On the other hand, the lPEI based complexes exhibited negative or neutral surface charges with hydrodynamic diameters below 500 nm. These physicochemical properties could generate restrictions for the effective delivery of DNA. The fourth chapter describes the in vitro cytotoxicity of graft copolymers and the transfection efficiency of the resulting polyplexes, using 25 kDa lPEI and 20 kDa PDMAEMA as controls. The copolymers were less cytotoxic to L929 cells than the control homopolymers, exhibiting higher cell viability by reducing the length of the hydrophobic segment and the amount of cationic grafts. Polyplexes formed from PDMAEMA copolymers were more efficient in the delivery of DNA in HEK-293 cells than the standard polycations, without affecting the cell viability. On the other hand, the complexes based on lPEI copolymers exhibited a low transfection efficiency, even using high polymer concentrations and N/P ratios. In general, copolymers composed of long hydrophobic segments and higher grafting density showed a better performance. In particular, the material PP6D5 showed the most promising features for its application as DNA transfection agent, for both in vitro and in vivo assays. This thesis represents the starting point for future research related to the rational design of grafted structures based on cationic polymers that may have improved properties and performance for their application as gene delivery systems.
dc.format.extent155
dc.format.mimetypeapplication/pdf
dc.language.isoeng
dc.rights.urihttp://creativecommons.org/licenses/by-nd/2.5/co/
dc.subjectPDMAEMA
dc.subjectPEI
dc.subjectterapia génica
dc.subjectvectores poliméricos
dc.subjectcopolímeros de injerto catiónicos
dc.subjecttransfección de ADN
dc.subject.ddc660 - Ingeniería química
dc.subject.ddc547 - Química orgánica
dc.titleGraft copolymers based on Poly(2-(dimethylamino)ethyl methacrylate) and Poly(ethyleneimine): synthesis and evaluation as vectors for DNA transfection
dc.typeOther
dc.rights.spaAcceso abierto
dc.contributor.institutionUniversidad Nacional de Colombia - Sede Bogotá
dc.subject.keywordPDMAEMA
dc.subject.keywordPEI
dc.subject.keywordDNA transfection
dc.subject.keywordgene therapy
dc.subject.keywordpolymeric vectors
dc.subject.keywordcationic graft copolymers
dc.type.spaOtro
dc.type.hasversionAccepted Version
dc.coverage.programDepartamento de Química
dc.contributor.gruplacMacromoléculas
dc.coverage.modalityDoctorado
dc.rights.accessRightsOpen Access
dc.rights.ccAtribución-SinDerivadas 2.5 Colombia
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