Publicação
Development of liposomal vectors for effective delivery of pleiotropic recombinant leukemia inhibitory factor (LIF) cytokine
| Resumo: | Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that can trigger therapeutic effects in leukemia disease, muscle tissue regeneration, neurological diseases and infertility problems. This cytokine has high rates of clearance in vivo and the capability to trigger beneficial and detrimental functions in different cell types. The main goal of this work was to encapsulate LIF in dimethyldioctadecylammonium bromide (DODAB): monoolein (MO) (1:2) liposomes and validate the system in two cell models, C2C12 and M1 cell lines. The DODAB:MO liposomal system was previously well studied by our research group for protein delivery but not cytokines in particular. The DODAB:MO (1:2) system is characterized by the ability to form inverted non-lamellar phases inside the liposome core, solubilizing high amounts of protein. In order to validate these nanocarriers in cell models, lower lipid concentrations were used to minimize cytotoxicity. Using 0.4mM of DODAB:MO (1:2) and 10 pg/mL LIF, it was possible to produce a stable nanoparticle at two pH conditions (5 and 7.4). This nanocarrier was positively charged (60-70mV), small (-170 nm mean size) with high encapsulation efficiency (>80%). Based on the physical characteristics of this nanocarrier, it may have potential to be applied as a therapeutic option. The cell models used were murine myoblasts (C2C12 cell line) and myeloid leukemia cells (M1 cells). In C2C12 cells, lower LIF concentrations are responsible for cell proliferation and differentiation delay. In cultures with an initial seeding cell density of 1x104 cells/mL, 0.001 and 0.1 ng/mL LIF led to higher proliferation rates, assessed with SRB and Hoechst assays. C2C12 cells are capable to differentiate forming myotubes, and 2% of horse serum (HS) instead of 5% HS in culture medium demonstrated to boost differentiation in presence of LIF, without masking proliferation. Low LIF concentrations can stimulate M1 proliferation but not their differentiation into macrophages. Proliferation was studied with the Hoechst assay and viability with the trypan blue assay. The results show that at 3x104 cells /mL, lower proliferation rates were observed with 0.01 and 1 ng/mL LIF concentrations, but also higher viability. In future experiments, it is necessary to (i) test other in vitro assays to validate LIF concentrations, (ii) validate the DODAB:MO carrier encapsulating LIF in the cell models and (iii) produce another carrier with LIF adsorbed at the liposome surface. |
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| Autores principais: | Pinho, Vanessa Tatiana |
| Assunto: | DODAB:MO Liposomes LIF cytokine Myoblasts Myeloblasts DODAB:MO Lipossomas Citocina LIF Mieloblastos Mioblastos |
| Ano: | 2020 |
| 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 |
| Resumo: | Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that can trigger therapeutic effects in leukemia disease, muscle tissue regeneration, neurological diseases and infertility problems. This cytokine has high rates of clearance in vivo and the capability to trigger beneficial and detrimental functions in different cell types. The main goal of this work was to encapsulate LIF in dimethyldioctadecylammonium bromide (DODAB): monoolein (MO) (1:2) liposomes and validate the system in two cell models, C2C12 and M1 cell lines. The DODAB:MO liposomal system was previously well studied by our research group for protein delivery but not cytokines in particular. The DODAB:MO (1:2) system is characterized by the ability to form inverted non-lamellar phases inside the liposome core, solubilizing high amounts of protein. In order to validate these nanocarriers in cell models, lower lipid concentrations were used to minimize cytotoxicity. Using 0.4mM of DODAB:MO (1:2) and 10 pg/mL LIF, it was possible to produce a stable nanoparticle at two pH conditions (5 and 7.4). This nanocarrier was positively charged (60-70mV), small (-170 nm mean size) with high encapsulation efficiency (>80%). Based on the physical characteristics of this nanocarrier, it may have potential to be applied as a therapeutic option. The cell models used were murine myoblasts (C2C12 cell line) and myeloid leukemia cells (M1 cells). In C2C12 cells, lower LIF concentrations are responsible for cell proliferation and differentiation delay. In cultures with an initial seeding cell density of 1x104 cells/mL, 0.001 and 0.1 ng/mL LIF led to higher proliferation rates, assessed with SRB and Hoechst assays. C2C12 cells are capable to differentiate forming myotubes, and 2% of horse serum (HS) instead of 5% HS in culture medium demonstrated to boost differentiation in presence of LIF, without masking proliferation. Low LIF concentrations can stimulate M1 proliferation but not their differentiation into macrophages. Proliferation was studied with the Hoechst assay and viability with the trypan blue assay. The results show that at 3x104 cells /mL, lower proliferation rates were observed with 0.01 and 1 ng/mL LIF concentrations, but also higher viability. In future experiments, it is necessary to (i) test other in vitro assays to validate LIF concentrations, (ii) validate the DODAB:MO carrier encapsulating LIF in the cell models and (iii) produce another carrier with LIF adsorbed at the liposome surface. |
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