Publicação
Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors
| Resumo: | Throughout the last decades, several liposomal formulations have been developed with the goal of condensing, transporting and releasing complementary DNA (cDNA) into cells, thus allowing the treatment of several genetic diseases via DNA replacement therapy. Although this technology has been partially substituted with the more contemporary use of small interference RNA (siRNA) therapy, the intracellular delivery of cDNA still remains the only viable gene therapy approach for stable host genome modification instead of transient transfection. The success of lipofection technique greatly depends on the structural and physicochemical properties of the liposomal carrier and ultimately, on the molecular attributes of the components of the lipid mixture. The empirical testing of diverse families of lipids such as cationic surfactants, pH-sensitive tensioactives or other lipid conjugates has allowed the development of highly efficient nanoparticles that stably condense the genetic material and withstand the harsh destabilizing conditions of the biological environment. However, it is this very same increased carrier stability that is responsible for limiting the gene release from the nucleic acid (NA)/cationic liposome complex (lipoplex) and reducing its final cell transfection efficiency. One recent approach to overcome this problem has come with the inclusion of non-lamellar forming lipids (also called helper lipids) in the liposomal formulation, which potentiate the formation of membrane fusion intermediates that disrupt the lamellar organization of the lipoplexes and favour the release of the genetic content. Molecules such as dioleoylphosphatidyl ethanolamine (DOPE) and cholesterol (Chol) have been employed with success as helper lipids in several liposomal formulations, enhancing the lipofection efficiency through the formation of inverted hexagonal structures (HII). Adopting a similar strategy, the work plan for this thesis was focused on the development of a new nucleic acid (NA)/cationic liposome formulation for gene delivery purposes comprising the synthetic cationic lipid Dioctadecyl Dimethylammonium Bromide (DODAB) and the non-ionic and non-lamellar forming lipid Monooleoyl-rac-glycerol (monoolein, MO). The structural and physicochemical characterization of DODAB:MO liposomes and DNA/DODAB:MO lipoplexes has been done through light scattering, microscopy, spectroscopy (with emphasis in UV/Visible fluorescence emission) and calorimetry techniques. The lipoplex formation process has been studied through the same techniques referred above at varying molar fractions of cationic lipid/DNA and cationic lipid/neutral lipid, while lipoplex destabilization was assessed by incubation of the pre-formed lipoplexes in several physiological simulation environments (salt, serum, temperature, pH, anionic lipids). Finally, the cell transfection efficiency and cytotoxicity profile of these non-viral gene delivery vectors was assessed by β-galactosidase reporter gene expression and Lactate Dehydrogenase (LDH) cell viability assays, respectively, in the 293T human embryonic kidney cell line. The results attained substantiate the high potential for DNA/DODAB:MO lipoplexes to be used as nonviral vectors in gene delivery, and validate the application of MO as helper lipid in cationic liposome formulations, being a viable alternative to classic DOPE and Chol molecules. |
|---|---|
| Autores principais: | Silva, João Paulo Neves da |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| _version_ | 1867438629360500736 |
|---|---|
| author | Silva, João Paulo Neves da |
| author_facet | Silva, João Paulo Neves da |
| author_role | author |
| contributor_name_str_mv | Real Oliveira, M. Elisabete C.D. Coutinho, Paulo J. G. Coutinho, O. P. RepositóriUM - Universidade do Minho |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Silva, João Paulo Neves da\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Real Oliveira, M. Elisabete C.D. Coutinho, Paulo J. G. Coutinho, O. P. RepositóriUM - Universidade do Minho |
| datacite.creators.creator.creatorName.fl_str_mv | Silva, João Paulo Neves da |
| datacite.date.Accepted.fl_str_mv | 2013-12-12T00:00:00Z |
| datacite.date.available.fl_str_mv | 2014-07-08T08:45:38Z |
| datacite.date.embargoed.fl_str_mv | 2014-07-08T08:45:38Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_16ec |
| datacite.titles.title.fl_str_mv | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| dc.contributor.none.fl_str_mv | Real Oliveira, M. Elisabete C.D. Coutinho, Paulo J. G. Coutinho, O. P. RepositóriUM - Universidade do Minho |
| dc.creator.none.fl_str_mv | Silva, João Paulo Neves da |
| dc.date.Accepted.fl_str_mv | 2013-12-12T00:00:00Z |
| dc.date.available.fl_str_mv | 2014-07-08T08:45:38Z |
| dc.date.embargoed.fl_str_mv | 2014-07-08T08:45:38Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | https://hdl.handle.net/1822/29565 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_16ec |
| dc.title.fl_str_mv | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_db06 |
| description | Throughout the last decades, several liposomal formulations have been developed with the goal of condensing, transporting and releasing complementary DNA (cDNA) into cells, thus allowing the treatment of several genetic diseases via DNA replacement therapy. Although this technology has been partially substituted with the more contemporary use of small interference RNA (siRNA) therapy, the intracellular delivery of cDNA still remains the only viable gene therapy approach for stable host genome modification instead of transient transfection. The success of lipofection technique greatly depends on the structural and physicochemical properties of the liposomal carrier and ultimately, on the molecular attributes of the components of the lipid mixture. The empirical testing of diverse families of lipids such as cationic surfactants, pH-sensitive tensioactives or other lipid conjugates has allowed the development of highly efficient nanoparticles that stably condense the genetic material and withstand the harsh destabilizing conditions of the biological environment. However, it is this very same increased carrier stability that is responsible for limiting the gene release from the nucleic acid (NA)/cationic liposome complex (lipoplex) and reducing its final cell transfection efficiency. One recent approach to overcome this problem has come with the inclusion of non-lamellar forming lipids (also called helper lipids) in the liposomal formulation, which potentiate the formation of membrane fusion intermediates that disrupt the lamellar organization of the lipoplexes and favour the release of the genetic content. Molecules such as dioleoylphosphatidyl ethanolamine (DOPE) and cholesterol (Chol) have been employed with success as helper lipids in several liposomal formulations, enhancing the lipofection efficiency through the formation of inverted hexagonal structures (HII). Adopting a similar strategy, the work plan for this thesis was focused on the development of a new nucleic acid (NA)/cationic liposome formulation for gene delivery purposes comprising the synthetic cationic lipid Dioctadecyl Dimethylammonium Bromide (DODAB) and the non-ionic and non-lamellar forming lipid Monooleoyl-rac-glycerol (monoolein, MO). The structural and physicochemical characterization of DODAB:MO liposomes and DNA/DODAB:MO lipoplexes has been done through light scattering, microscopy, spectroscopy (with emphasis in UV/Visible fluorescence emission) and calorimetry techniques. The lipoplex formation process has been studied through the same techniques referred above at varying molar fractions of cationic lipid/DNA and cationic lipid/neutral lipid, while lipoplex destabilization was assessed by incubation of the pre-formed lipoplexes in several physiological simulation environments (salt, serum, temperature, pH, anionic lipids). Finally, the cell transfection efficiency and cytotoxicity profile of these non-viral gene delivery vectors was assessed by β-galactosidase reporter gene expression and Lactate Dehydrogenase (LDH) cell viability assays, respectively, in the 293T human embryonic kidney cell line. The results attained substantiate the high potential for DNA/DODAB:MO lipoplexes to be used as nonviral vectors in gene delivery, and validate the application of MO as helper lipid in cationic liposome formulations, being a viable alternative to classic DOPE and Chol molecules. |
| dirty | 0 |
| eu_rights_str_mv | restrictedAccess |
| format | doctoralThesis |
| fulltext.url.fl_str_mv | https://repositorium.uminho.pt/bitstreams/5a3ea7d4-42ab-4b9c-a9b0-cac34f67b5fd/download |
| id | rum_dc09a48482ec8527fc2c025e7c4fed49 |
| identifier.url.fl_str_mv | https://hdl.handle.net/1822/29565 |
| instacron_str | repositorium |
| institution | Universidade do Minho |
| instname_str | Universidade do Minho |
| language | eng |
| network_acronym_str | rum |
| network_name_str | RepositóriUM - Universidade do Minho |
| oai_identifier_str | oai:repositorium.uminho.pt:1822/29565 |
| organization_str_mv | urn:organizationAcronym:repositorium |
| person_str_mv | Silva, João Paulo Neves da |
| publishDate | 2013 |
| reponame_str | RepositóriUM - Universidade do Minho |
| repository_id_str | urn:repositoryAcronym:rum |
| service_str_mv | urn:repositoryAcronym:rum |
| spelling | engporThroughout the last decades, several liposomal formulations have been developed with the goal of condensing, transporting and releasing complementary DNA (cDNA) into cells, thus allowing the treatment of several genetic diseases via DNA replacement therapy. Although this technology has been partially substituted with the more contemporary use of small interference RNA (siRNA) therapy, the intracellular delivery of cDNA still remains the only viable gene therapy approach for stable host genome modification instead of transient transfection. The success of lipofection technique greatly depends on the structural and physicochemical properties of the liposomal carrier and ultimately, on the molecular attributes of the components of the lipid mixture. The empirical testing of diverse families of lipids such as cationic surfactants, pH-sensitive tensioactives or other lipid conjugates has allowed the development of highly efficient nanoparticles that stably condense the genetic material and withstand the harsh destabilizing conditions of the biological environment. However, it is this very same increased carrier stability that is responsible for limiting the gene release from the nucleic acid (NA)/cationic liposome complex (lipoplex) and reducing its final cell transfection efficiency. One recent approach to overcome this problem has come with the inclusion of non-lamellar forming lipids (also called helper lipids) in the liposomal formulation, which potentiate the formation of membrane fusion intermediates that disrupt the lamellar organization of the lipoplexes and favour the release of the genetic content. Molecules such as dioleoylphosphatidyl ethanolamine (DOPE) and cholesterol (Chol) have been employed with success as helper lipids in several liposomal formulations, enhancing the lipofection efficiency through the formation of inverted hexagonal structures (HII). Adopting a similar strategy, the work plan for this thesis was focused on the development of a new nucleic acid (NA)/cationic liposome formulation for gene delivery purposes comprising the synthetic cationic lipid Dioctadecyl Dimethylammonium Bromide (DODAB) and the non-ionic and non-lamellar forming lipid Monooleoyl-rac-glycerol (monoolein, MO). The structural and physicochemical characterization of DODAB:MO liposomes and DNA/DODAB:MO lipoplexes has been done through light scattering, microscopy, spectroscopy (with emphasis in UV/Visible fluorescence emission) and calorimetry techniques. The lipoplex formation process has been studied through the same techniques referred above at varying molar fractions of cationic lipid/DNA and cationic lipid/neutral lipid, while lipoplex destabilization was assessed by incubation of the pre-formed lipoplexes in several physiological simulation environments (salt, serum, temperature, pH, anionic lipids). Finally, the cell transfection efficiency and cytotoxicity profile of these non-viral gene delivery vectors was assessed by β-galactosidase reporter gene expression and Lactate Dehydrogenase (LDH) cell viability assays, respectively, in the 293T human embryonic kidney cell line. The results attained substantiate the high potential for DNA/DODAB:MO lipoplexes to be used as nonviral vectors in gene delivery, and validate the application of MO as helper lipid in cationic liposome formulations, being a viable alternative to classic DOPE and Chol molecules.application/pdfporPhysicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectorsSilva, João Paulo Neves daReal Oliveira, M. Elisabete C.D.Coutinho, Paulo J. G.Coutinho, O. P.HostingInstitutionOrganizationalRepositóriUM - Universidade do Minhoe-mailmailto:repositorium@usdb.uminho.ptrepositorium@usdb.uminho.ptTID1014245902014-07-08T08:45:38Z2013-12-122013-112013-12-12T00:00:00ZHandlehttps://hdl.handle.net/1822/29565http://purl.org/coar/access_right/c_16ecrestricted access14691392 bytesliteraturehttp://purl.org/coar/resource_type/c_db06doctoral thesishttp://purl.org/coar/access_right/c_16ecapplication/pdffulltexthttps://repositorium.uminho.pt/bitstreams/5a3ea7d4-42ab-4b9c-a9b0-cac34f67b5fd/download |
| spellingShingle | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors Silva, João Paulo Neves da |
| status | SINGLETON |
| title | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| title_full | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| title_fullStr | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| title_full_unstemmed | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| title_short | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| title_sort | Physicochemical characterization of DNA/DODAB:MO Cationic Liposome complexes and study of its potential as nonviral vectors |
| url | https://hdl.handle.net/1822/29565 |
| visible | 1 |