Publicação
Internalization of resveratrol nanoparticles in yeast and bioactivity
| Resumo: | Resveratrol (RSV) is a phenolic compound, belonging to a class of chemical compounds derived from phenylalanine with a reactive hydroxyl group (-OH) attached to an aromatic hydrocarbon ring. Several pharmacological effects were attributed to RSV, including antioxidant properties. Different studies have also shown that RSV has a very low bioavailability, which has led to the suspicion that many of the observed health benefits may not be attainable in humans due to its rapid metabolism. To improve bioavailability and pharmacokinetic profile some attempts have been made to encapsulate RSV into a lipid based nanocarrier system, such as liposomes, which are clinically established for drug delivery. The therapeutic efficiency of liposomes systems is highly dependent on the characteristics of lipid molecules, which influence the structure and stability of this type of nanocarriers under physiological conditions. Previous work by our group has shown that liposomes prepared with DODAB:MO (1:2) are promising nanocarriers for delivery of nucleic acids and proteins. Also, these nanocarriers were exploited to load with RSV and important parameters critical for their efficient biological application were assessed. In the scope of the previous research work developed by Inês Ferreira (Master thesis in Biophysics and Bionanosystems, University of Minho, 2015) the DODAB:MO (1:2) liposomal system loaded with RSV revealed adequate characteristics for drug release purposes, high stability and homogeneity, high positive surface charge, and a good as well as a good encapsulation efficiency i.e. the percentage of RSV which is incorporated by liposomal system, (EE%), and the percentage of RSV which is incorporated by total lipid concentration (RL%). In the context of the present study the yeast Saccharomyces cerevisiae W303-1A was then used as a cell model to evaluate the biological activity of RSV loaded DODAB:MO (1:2) liposomes. Internalization studies of RSV-loaded liposomes were then performed using fluorescent probes and fluorescence microscopy, and their biological activity was compared with free RSV using ROS-sensitive probes coupled to flow cytometry analysis. After 5 h incubation yeast cells readily internalized liposomes, that appeared as bright blue spots in the periphery of the central vacuole. Moreover, when yeast cells were incubated with DPH alone no fluorescence was detected in the cytosol. When yeast cells were incubated with the lipophilic fluorescent probe FM1-43, results showed that its green fluorescence co-localized with the blue fluorescence of DPH, suggesting that liposomes are efficiently internalized by cells and follow the endocytic pathway. To study the effect of RSV in yeast mitochondrial morphology, the strain S. cerevisiae W303-1A Pyx232-mtGFP, which expresses a mitochondrial matrix-targeted GFP was used. Results showed that cells displaying non-fragmented/tubular mitochondrial networks predominate up to 5 h mostly in control conditions, while a the vacuolar/diffuse cytosolic fluorescence predominate after 40 h of incubation either with free or encapsulated RSV. Moreover, the incubation with liposomes containing 200 μM RSV during 45 h was the experimental condition where cells exhibiting the latter phenotype were more abundant. |
|---|---|
| Autores principais: | Barbosa, Célia de Jesus Sousa |
| Assunto: | Ciências Naturais::Outras Ciências Naturais |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| _version_ | 1867437959359234048 |
|---|---|
| author | Barbosa, Célia de Jesus Sousa |
| author_facet | Barbosa, Célia de Jesus Sousa |
| author_role | author |
| contributor_name_str_mv | Gerós, H. Real Oliveira, M. Elisabete C.D. RepositóriUM - Universidade do Minho |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Barbosa, Célia de Jesus Sousa\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Gerós, H. Real Oliveira, M. Elisabete C.D. RepositóriUM - Universidade do Minho |
| datacite.creators.creator.creatorName.fl_str_mv | Barbosa, Célia de Jesus Sousa |
| datacite.date.Accepted.fl_str_mv | 2017-01-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2021-01-01T07:00:33Z |
| datacite.date.embargoed.fl_str_mv | 2021-01-01T07:00:33Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | Ciências Naturais::Outras Ciências Naturais |
| datacite.titles.title.fl_str_mv | Internalization of resveratrol nanoparticles in yeast and bioactivity Internalização e bioatividade do resveratrol em nanopartículas em células de levedura |
| dc.contributor.none.fl_str_mv | Gerós, H. Real Oliveira, M. Elisabete C.D. RepositóriUM - Universidade do Minho |
| dc.creator.none.fl_str_mv | Barbosa, Célia de Jesus Sousa |
| dc.date.Accepted.fl_str_mv | 2017-01-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2021-01-01T07:00:33Z |
| dc.date.embargoed.fl_str_mv | 2021-01-01T07:00:33Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | https://hdl.handle.net/1822/50060 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | Ciências Naturais::Outras Ciências Naturais |
| dc.title.fl_str_mv | Internalization of resveratrol nanoparticles in yeast and bioactivity Internalização e bioatividade do resveratrol em nanopartículas em células de levedura |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_bdcc |
| description | Resveratrol (RSV) is a phenolic compound, belonging to a class of chemical compounds derived from phenylalanine with a reactive hydroxyl group (-OH) attached to an aromatic hydrocarbon ring. Several pharmacological effects were attributed to RSV, including antioxidant properties. Different studies have also shown that RSV has a very low bioavailability, which has led to the suspicion that many of the observed health benefits may not be attainable in humans due to its rapid metabolism. To improve bioavailability and pharmacokinetic profile some attempts have been made to encapsulate RSV into a lipid based nanocarrier system, such as liposomes, which are clinically established for drug delivery. The therapeutic efficiency of liposomes systems is highly dependent on the characteristics of lipid molecules, which influence the structure and stability of this type of nanocarriers under physiological conditions. Previous work by our group has shown that liposomes prepared with DODAB:MO (1:2) are promising nanocarriers for delivery of nucleic acids and proteins. Also, these nanocarriers were exploited to load with RSV and important parameters critical for their efficient biological application were assessed. In the scope of the previous research work developed by Inês Ferreira (Master thesis in Biophysics and Bionanosystems, University of Minho, 2015) the DODAB:MO (1:2) liposomal system loaded with RSV revealed adequate characteristics for drug release purposes, high stability and homogeneity, high positive surface charge, and a good as well as a good encapsulation efficiency i.e. the percentage of RSV which is incorporated by liposomal system, (EE%), and the percentage of RSV which is incorporated by total lipid concentration (RL%). In the context of the present study the yeast Saccharomyces cerevisiae W303-1A was then used as a cell model to evaluate the biological activity of RSV loaded DODAB:MO (1:2) liposomes. Internalization studies of RSV-loaded liposomes were then performed using fluorescent probes and fluorescence microscopy, and their biological activity was compared with free RSV using ROS-sensitive probes coupled to flow cytometry analysis. After 5 h incubation yeast cells readily internalized liposomes, that appeared as bright blue spots in the periphery of the central vacuole. Moreover, when yeast cells were incubated with DPH alone no fluorescence was detected in the cytosol. When yeast cells were incubated with the lipophilic fluorescent probe FM1-43, results showed that its green fluorescence co-localized with the blue fluorescence of DPH, suggesting that liposomes are efficiently internalized by cells and follow the endocytic pathway. To study the effect of RSV in yeast mitochondrial morphology, the strain S. cerevisiae W303-1A Pyx232-mtGFP, which expresses a mitochondrial matrix-targeted GFP was used. Results showed that cells displaying non-fragmented/tubular mitochondrial networks predominate up to 5 h mostly in control conditions, while a the vacuolar/diffuse cytosolic fluorescence predominate after 40 h of incubation either with free or encapsulated RSV. Moreover, the incubation with liposomes containing 200 μM RSV during 45 h was the experimental condition where cells exhibiting the latter phenotype were more abundant. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | masterThesis |
| fulltext.url.fl_str_mv | https://repositorium.uminho.pt/bitstreams/c99d28c8-c737-4f5a-bfc4-fa93164ac95f/download |
| id | rum_0ed3fa0a42e24092da4e21c13be6aadb |
| identifier.url.fl_str_mv | https://hdl.handle.net/1822/50060 |
| 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:1822/50060 |
| organization_str_mv | urn:organizationAcronym:repositorium |
| person_str_mv | Barbosa, Célia de Jesus Sousa |
| publishDate | 2017 |
| reponame_str | RepositóriUM - Universidade do Minho |
| repository_id_str | urn:repositoryAcronym:rum |
| service_str_mv | urn:repositoryAcronym:rum |
| spelling | engporResveratrol (RSV) is a phenolic compound, belonging to a class of chemical compounds derived from phenylalanine with a reactive hydroxyl group (-OH) attached to an aromatic hydrocarbon ring. Several pharmacological effects were attributed to RSV, including antioxidant properties. Different studies have also shown that RSV has a very low bioavailability, which has led to the suspicion that many of the observed health benefits may not be attainable in humans due to its rapid metabolism. To improve bioavailability and pharmacokinetic profile some attempts have been made to encapsulate RSV into a lipid based nanocarrier system, such as liposomes, which are clinically established for drug delivery. The therapeutic efficiency of liposomes systems is highly dependent on the characteristics of lipid molecules, which influence the structure and stability of this type of nanocarriers under physiological conditions. Previous work by our group has shown that liposomes prepared with DODAB:MO (1:2) are promising nanocarriers for delivery of nucleic acids and proteins. Also, these nanocarriers were exploited to load with RSV and important parameters critical for their efficient biological application were assessed. In the scope of the previous research work developed by Inês Ferreira (Master thesis in Biophysics and Bionanosystems, University of Minho, 2015) the DODAB:MO (1:2) liposomal system loaded with RSV revealed adequate characteristics for drug release purposes, high stability and homogeneity, high positive surface charge, and a good as well as a good encapsulation efficiency i.e. the percentage of RSV which is incorporated by liposomal system, (EE%), and the percentage of RSV which is incorporated by total lipid concentration (RL%). In the context of the present study the yeast Saccharomyces cerevisiae W303-1A was then used as a cell model to evaluate the biological activity of RSV loaded DODAB:MO (1:2) liposomes. Internalization studies of RSV-loaded liposomes were then performed using fluorescent probes and fluorescence microscopy, and their biological activity was compared with free RSV using ROS-sensitive probes coupled to flow cytometry analysis. After 5 h incubation yeast cells readily internalized liposomes, that appeared as bright blue spots in the periphery of the central vacuole. Moreover, when yeast cells were incubated with DPH alone no fluorescence was detected in the cytosol. When yeast cells were incubated with the lipophilic fluorescent probe FM1-43, results showed that its green fluorescence co-localized with the blue fluorescence of DPH, suggesting that liposomes are efficiently internalized by cells and follow the endocytic pathway. To study the effect of RSV in yeast mitochondrial morphology, the strain S. cerevisiae W303-1A Pyx232-mtGFP, which expresses a mitochondrial matrix-targeted GFP was used. Results showed that cells displaying non-fragmented/tubular mitochondrial networks predominate up to 5 h mostly in control conditions, while a the vacuolar/diffuse cytosolic fluorescence predominate after 40 h of incubation either with free or encapsulated RSV. Moreover, the incubation with liposomes containing 200 μM RSV during 45 h was the experimental condition where cells exhibiting the latter phenotype were more abundant.application/pdfporInternalization of resveratrol nanoparticles in yeast and bioactivityAlternativeTitleporInternalização e bioatividade do resveratrol em nanopartículas em células de leveduraBarbosa, Célia de Jesus SousaGerós, H.Real Oliveira, M. Elisabete C.D.HostingInstitutionOrganizationalRepositóriUM - Universidade do Minhoe-mailmailto:repositorium@usdb.uminho.ptrepositorium@usdb.uminho.ptTID2017398952021-01-01T07:00:33Z201720172017-01-01T00:00:00ZHandlehttps://hdl.handle.net/1822/50060http://purl.org/coar/access_right/c_abf2open accesshttp://www.oecd.org/science/inno/38235147.pdfFields of Science and Technology (FOS)Ciências Naturais::Outras Ciências Naturais3401272 bytesliteraturehttp://purl.org/coar/resource_type/c_bdccmaster thesishttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorium.uminho.pt/bitstreams/c99d28c8-c737-4f5a-bfc4-fa93164ac95f/download |
| spellingShingle | Internalization of resveratrol nanoparticles in yeast and bioactivity Barbosa, Célia de Jesus Sousa Ciências Naturais::Outras Ciências Naturais |
| status | SINGLETON |
| subject.other.fl_str_mv | Ciências Naturais::Outras Ciências Naturais |
| title | Internalization of resveratrol nanoparticles in yeast and bioactivity |
| title_full | Internalization of resveratrol nanoparticles in yeast and bioactivity |
| title_fullStr | Internalization of resveratrol nanoparticles in yeast and bioactivity |
| title_full_unstemmed | Internalization of resveratrol nanoparticles in yeast and bioactivity |
| title_short | Internalization of resveratrol nanoparticles in yeast and bioactivity |
| title_sort | Internalization of resveratrol nanoparticles in yeast and bioactivity |
| topic | Ciências Naturais::Outras Ciências Naturais |
| topic_facet | Ciências Naturais::Outras Ciências Naturais |
| url | https://hdl.handle.net/1822/50060 |
| visible | 1 |