Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids
[eng] Cationic Solid Lipid Nanoparticles (cSLNs) are one of the most promising nonviral vectors for gene therapy and DNA / RNA delivery. The aim of this thesis is the development and research of new formulations of SLNs that will improve the formulation already existing in the research group. Thus,...
| Author: | |
|---|---|
| Format: | doctoral thesis |
| Status: | Published version |
| Publication Date: | 2019 |
| Country: | España |
| Institution: | Universidad de Barcelona |
| Repository: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/139340 |
| Online Access: | https://hdl.handle.net/2445/139340 http://hdl.handle.net/10803/667442 |
| Access Level: | Open access |
| Keyword: | Desenvolupament de medicaments Lípids Nanopartícules Teràpia genètica Sistemes d'alliberament de medicaments Drug development Lipides Nanoparticles Gene therapy Drug delivery systems |
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Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| title |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| spellingShingle |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids Suñé Pou, Marc Desenvolupament de medicaments Lípids Nanopartícules Teràpia genètica Sistemes d'alliberament de medicaments Drug development Lipides Nanoparticles Gene therapy Drug delivery systems |
| title_short |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| title_full |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| title_fullStr |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| title_full_unstemmed |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| title_sort |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acids |
| dc.creator.none.fl_str_mv |
Suñé Pou, Marc |
| author |
Suñé Pou, Marc |
| author_facet |
Suñé Pou, Marc |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Suñé i Negre, Carles Maria Suñé i Negre, Josep M. (Josep Maria) Universitat de Barcelona. Facultat de Farmàcia i Ciències de l'Alimentació |
| dc.subject.none.fl_str_mv |
Desenvolupament de medicaments Lípids Nanopartícules Teràpia genètica Sistemes d'alliberament de medicaments Drug development Lipides Nanoparticles Gene therapy Drug delivery systems |
| topic |
Desenvolupament de medicaments Lípids Nanopartícules Teràpia genètica Sistemes d'alliberament de medicaments Drug development Lipides Nanoparticles Gene therapy Drug delivery systems |
| description |
[eng] Cationic Solid Lipid Nanoparticles (cSLNs) are one of the most promising nonviral vectors for gene therapy and DNA / RNA delivery. The aim of this thesis is the development and research of new formulations of SLNs that will improve the formulation already existing in the research group. Thus, after different experiments, SLNs with cholesteryl oleate as matrix lipid have been developed, improving the transfection efficiency of the nanoparticles without affecting cell viability. For this development, 5 different lyophilized formulations with different proportions of cholesteryl oleate were manufactured, to study and understand its influence depending on the amount to be incorporated in morphology, stability, physicochemical characteristics and biological characteristics (such as efficiency of transfection and cytotoxicity). Thus, this study would also allow identifying the most appropriate formulation to continue the investigation. The Reference 14 was identified as the formulation with the best physicochemical characteristics and efficiency of transfection in assays in vitro. Once a definitive formulation was identified, the next step was to study more thorougly its physical and chemical characteristics once the lyophilized was reconstituted, applying different techniques such as DSC, DRX or TEM microscopy. The objective of this complete characterization was to know the formulation and its key process to be able to reproduce exactly the formulation. In addition, stability studies were conducted, and it was observed that lyophilized SLNs were stable for at least 1 year. Subsequently, different tests were performed in the Biology laboratory to test the efficiency and safety of the SLNs in different cell lines, such as HeLa, HEK293T, Jurkat or A549 cells. Thus, it was found that Reference 14 was capable of transfecting about 45% of HEK293T cells in vitro, and was capable of transfecting siRNA giving about 35% silencing in HeLa cells. These results were also confirmed by confocal microscopy. Finally, in vitro experiments were performed that indicated a possible therapeutic application of the formulation: the treatment of flavivirus infections such as Dengue virus, giving a decrease of about 85% of the infection in A549 cells treated with siRNA by silencing RPLP1/2 transfected by reference 14. This promising result opens avenues to deepen in such a line, as well as to find other therapeutic applications of this formulation and make the transition to the design of future in vivo experiments. |
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2019 |
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2019 |
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info:eu-repo/semantics/doctoralThesis info:eu-repo/semantics/publishedVersion |
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doctoralThesis |
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publishedVersion |
| dc.identifier.none.fl_str_mv |
https://hdl.handle.net/2445/139340 http://hdl.handle.net/10803/667442 |
| url |
https://hdl.handle.net/2445/139340 http://hdl.handle.net/10803/667442 |
| dc.language.none.fl_str_mv |
Inglés |
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Inglés |
| dc.rights.none.fl_str_mv |
(c) Suñé, 2019 info:eu-repo/semantics/openAccess |
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(c) Suñé, 2019 |
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openAccess |
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application/pdf |
| dc.publisher.none.fl_str_mv |
Universitat de Barcelona |
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Universitat de Barcelona |
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Tesis Doctorals - Facultat - Farmàcia i Ciències de l'Alimentació reponame:Dipòsit Digital de la UB instname:Universidad de Barcelona |
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Universidad de Barcelona |
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Dipòsit Digital de la UB |
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Dipòsit Digital de la UB |
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| _version_ |
1869415745853587456 |
| spelling |
Cholesteryl oleate-loaded solid lipid nanoparticles for the vectorization of nucleic acidsSuñé Pou, MarcDesenvolupament de medicamentsLípidsNanopartículesTeràpia genèticaSistemes d'alliberament de medicamentsDrug developmentLipidesNanoparticlesGene therapyDrug delivery systems[eng] Cationic Solid Lipid Nanoparticles (cSLNs) are one of the most promising nonviral vectors for gene therapy and DNA / RNA delivery. The aim of this thesis is the development and research of new formulations of SLNs that will improve the formulation already existing in the research group. Thus, after different experiments, SLNs with cholesteryl oleate as matrix lipid have been developed, improving the transfection efficiency of the nanoparticles without affecting cell viability. For this development, 5 different lyophilized formulations with different proportions of cholesteryl oleate were manufactured, to study and understand its influence depending on the amount to be incorporated in morphology, stability, physicochemical characteristics and biological characteristics (such as efficiency of transfection and cytotoxicity). Thus, this study would also allow identifying the most appropriate formulation to continue the investigation. The Reference 14 was identified as the formulation with the best physicochemical characteristics and efficiency of transfection in assays in vitro. Once a definitive formulation was identified, the next step was to study more thorougly its physical and chemical characteristics once the lyophilized was reconstituted, applying different techniques such as DSC, DRX or TEM microscopy. The objective of this complete characterization was to know the formulation and its key process to be able to reproduce exactly the formulation. In addition, stability studies were conducted, and it was observed that lyophilized SLNs were stable for at least 1 year. Subsequently, different tests were performed in the Biology laboratory to test the efficiency and safety of the SLNs in different cell lines, such as HeLa, HEK293T, Jurkat or A549 cells. Thus, it was found that Reference 14 was capable of transfecting about 45% of HEK293T cells in vitro, and was capable of transfecting siRNA giving about 35% silencing in HeLa cells. These results were also confirmed by confocal microscopy. Finally, in vitro experiments were performed that indicated a possible therapeutic application of the formulation: the treatment of flavivirus infections such as Dengue virus, giving a decrease of about 85% of the infection in A549 cells treated with siRNA by silencing RPLP1/2 transfected by reference 14. This promising result opens avenues to deepen in such a line, as well as to find other therapeutic applications of this formulation and make the transition to the design of future in vivo experiments.[spa] Las nanopartículas lipídicas sólidas catiónicas (cSLN) son uno de los vectores no virales más prometedores para la terapia génica y la vectorización de ADN / ARN. El objetivo de esta tesis es el desarrollo e investigación de nuevas formulaciones de SLN que mejorarán la formulación ya existente en el grupo de investigación. Así, después de diferentes experimentos, se han desarrollado SLN con colesteril oleato como lípido matricial, mejorando la eficiencia de transfección de las nanopartículas sin afectar la viabilidad celular. Para este desarrollo, se fabricaron 5 formulaciones liofilizadas diferentes con diferentes proporciones de colesteril oleato, para estudiar y comprender su influencia según la cantidad a incorporar en la morfología, estabilidad, características físicoquímicas y características biológicas (como la eficiencia de transfección y la citotoxicidad). Por lo tanto, este estudio también permitiría identificar la formulación más adecuada para continuar la investigación. La Referencia 14 se identificó como la formulación con las mejores características fisicoquímicas y la eficiencia de la transfección en ensayos in vitro. La composición de la referencia 14 es 600 mg de octadecilamina, 300 mg de oleato de colesterilo, 200 mg de ácido esteárico y 100 mg de poloxámero 188. Una vez que se identificó una formulación definitiva, el siguiente paso fue estudiar mejor sus características físicas y químicas una vez que se reconstituyó el liofilizado, aplicando diferentes técnicas como la microscopía DSC, DRX o TEM. El objetivo de esta caracterización completa fue conocer la formulación y su proceso clave para poder reproducir exactamente la formulación. Además, se realizaron estudios de estabilidad y se observó que las SLN liofilizadas se mantuvieron estables durante al menos 1 año. Esto mejoró la estabilidad de las SLN en suspensión, que tienen una estabilidad máxima de 15 días a 4 ° C. Posteriormente, se realizaron diferentes pruebas en el laboratorio de biología para probar la eficacia y seguridad de las SLN en diferentes líneas celulares, como HeLa, HEK293T, Jurkat o A549. Por lo tanto, se encontró que la Referencia 14 era capaz de transfectar aproximadamente el 45% de las células HEK293T in vitro, y era capaz de transfectar el siRNA dando un silenciamiento de aproximadamente el 35% en las células HeLa. Estos resultados también fueron confirmados por microscopía confocal. Finalmente, se llevaron a cabo experimentos in vitro que indicaron una posible aplicación terapéutica de la formulación: el tratamiento de infecciones por flavivirus como el virus del dengue, que produce una disminución de aproximadamente el 85% de la infección en células A549 tratadas con siRNA silenciando la RPLP1/2, transfectado con la referencia 14. Este resultado prometedor abre vías para profundizar en dicha línea, así como para encontrar otras aplicaciones terapéuticas de esta formulación y hacer la transición al diseño de futuros experimentos in vivo.Universitat de BarcelonaSuñé i Negre, Carles MariaSuñé i Negre, Josep M. (Josep Maria)Universitat de Barcelona. Facultat de Farmàcia i Ciències de l'Alimentació2019info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/139340http://hdl.handle.net/10803/667442Tesis Doctorals - Facultat - Farmàcia i Ciències de l'Alimentacióreponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglés(c) Suñé, 2019info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/1393402026-05-27T06:46:51Z |
| score |
15.300719 |