Publicação
HIV-based virus-like particles: design and evaluation as a targeted therapy tool
| Resumo: | Nanomedicine has emerged in recent years as a field aimed at surpassing the shortcomings of standard medicine. Among the different types of nanoparticles (NPs), virus-like particles (VLPs) arise as promising nanostructures comprised of assembled viral proteins that take structural cues from viruses whilst lack- ing viral genetic material. Production of these platforms can be carried out in countless expression sys- tems, namely insect cells and mammalian cells. VLPs are characterized by being modifiable, biocompat- ible, capable of self-assembly, immunogenic, and able to cross cell membranes, and can therefore be harnessed as vaccines, delivery platforms and carriers of imaging molecules. Antibody fragments, such as single-chain variable fragments (scFv), have been widely studied as targeting moieties. This thesis aimed to characterize and evaluate human immunodeficiency virus type 1 (HIV-1)- based VLPs that expresses on its surface a scFv derived from trastuzumab, an antibody against the human epidermal growth factor receptor 2 (HER2). The design of these nanoplatforms will be used as a proof- of-concept for their application as specific drug and/or imaging agents carriers. VLPs were produced by transient transfection with a mammalian cell line, HEK-293T. After VLPs production, they were charac- terized by Western Blot, which confirmed the presence of scFv from trastuzumab. The radiolabelling tests showed that fac-[99mTc(CO)3(H2O)3]+ could be used for radiolabelling His-tagged VLPs and its sta- bility and uptake in cellular lines were performed. Evaluation of VLP-HER2 binding was carried out by ELISA. According to the results, HIV1-based VLPs have an extremely high affinity towards HER2 proteins. Finally, cytotoxicity assays to ascertain the antiproliferative activity of VLPs were conducted in two breast cancer cell lines, a HER2-overexpressing cell line (SK-BR-3) and a cell line that lacks HER2 overexpression (MDA-MB-231). The VLPs do not appear to be cytotoxic, which is an important feature for targeted delivery tools. Overall, this work set up a novel protocol to produce a modified HIV-1-based VLP that can potentially be used as a targeted delivery platform for various targets in multiple diseases. |
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| Autores principais: | Travassos, Rafael José Cartaxo |
| Assunto: | Human Immunodeficiency Virus Type 1 (HIV-1)-Based Virus-Like-Particles Human Ep- idermal Growth Factor Receptor 2 (HER2) Single-Chain Variable Fragment (scFv) Targeted Therapy |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Nanomedicine has emerged in recent years as a field aimed at surpassing the shortcomings of standard medicine. Among the different types of nanoparticles (NPs), virus-like particles (VLPs) arise as promising nanostructures comprised of assembled viral proteins that take structural cues from viruses whilst lack- ing viral genetic material. Production of these platforms can be carried out in countless expression sys- tems, namely insect cells and mammalian cells. VLPs are characterized by being modifiable, biocompat- ible, capable of self-assembly, immunogenic, and able to cross cell membranes, and can therefore be harnessed as vaccines, delivery platforms and carriers of imaging molecules. Antibody fragments, such as single-chain variable fragments (scFv), have been widely studied as targeting moieties. This thesis aimed to characterize and evaluate human immunodeficiency virus type 1 (HIV-1)- based VLPs that expresses on its surface a scFv derived from trastuzumab, an antibody against the human epidermal growth factor receptor 2 (HER2). The design of these nanoplatforms will be used as a proof- of-concept for their application as specific drug and/or imaging agents carriers. VLPs were produced by transient transfection with a mammalian cell line, HEK-293T. After VLPs production, they were charac- terized by Western Blot, which confirmed the presence of scFv from trastuzumab. The radiolabelling tests showed that fac-[99mTc(CO)3(H2O)3]+ could be used for radiolabelling His-tagged VLPs and its sta- bility and uptake in cellular lines were performed. Evaluation of VLP-HER2 binding was carried out by ELISA. According to the results, HIV1-based VLPs have an extremely high affinity towards HER2 proteins. Finally, cytotoxicity assays to ascertain the antiproliferative activity of VLPs were conducted in two breast cancer cell lines, a HER2-overexpressing cell line (SK-BR-3) and a cell line that lacks HER2 overexpression (MDA-MB-231). The VLPs do not appear to be cytotoxic, which is an important feature for targeted delivery tools. Overall, this work set up a novel protocol to produce a modified HIV-1-based VLP that can potentially be used as a targeted delivery platform for various targets in multiple diseases. |
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