Publicação
Terapia Génica com Ondas Fotoacústicas
| Resumo: | Gene therapy involves a process that requires the genetic manipulation of specific cells using nucleic acids, through the replacement or correction of altered genes for a therapeutic purpose. The main difficulty with existing gene therapy methods is in permeating the cell membrane for safe DNA delivery. The aim of this work was to use a non-viral transfection method mediated by laser-induced pressure waves. Light-pressure transducers, known as piezophotonic materials, are composed of a chromophore with a high light-absorption capacity confined in a material with high thermoelastic expansion. Laser pulses in the nanosecond range (8 ns) and picosecond range (30 ps), with a wavelength at 1064 nm, were absorbed by the piezophotonic material fuligem-PDMS, which exhibits a high efficiency in converting light into pressure, giving rise to photoacoustic waves capable of enhancing cell membrane permeability, facilitating the entry of the plasmid encoding the green fluorescent protein (gWizGFP).In order to achieve different pressure gradients, low laser fluences (60 mJ cm-2 and 100 mJ cm-2) were used, resulting in the generation of high absolute pressures (60 bar and 100 bar), and relatively low-frequency bandwidths (10 MHz), compared to other materials studied by our research group (MnTPP). This technique has proven to be safe, with cell viability exceeding 95% even after several minutes of exposure to photoacoustic waves. This is a highly advantageous feature, and it is also possible to expose the materials to successive laser pulses without damaging them.Transfection results were evaluated using fluorescence microscopy and flow cytometry techniques. These results showed that the nanosecond laser with an 8 ns pulse had lower efficiency than the picosecond laser with a 30 ps pulse, achieving a transfection efficiency of approximately 5% in COS-7 cell line. This result indicates that the presence of higher pressures, inducing higher pressure gradients, is highly relevant for cell membrane permeabilization. In the future, it is expected that after some optimizations, this technique can be introduced into clinical practice. |
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| Autores principais: | Seletchi, Evgheni |
| Assunto: | Gene Therapy Photoacoustic Waves Light-pressure transducers Fuligem-PDMS GFP Terapia Genética Ondas Fotoacústicas transdutores luz-pressão Fuligem-PDMS GFP |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | português |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Gene therapy involves a process that requires the genetic manipulation of specific cells using nucleic acids, through the replacement or correction of altered genes for a therapeutic purpose. The main difficulty with existing gene therapy methods is in permeating the cell membrane for safe DNA delivery. The aim of this work was to use a non-viral transfection method mediated by laser-induced pressure waves. Light-pressure transducers, known as piezophotonic materials, are composed of a chromophore with a high light-absorption capacity confined in a material with high thermoelastic expansion. Laser pulses in the nanosecond range (8 ns) and picosecond range (30 ps), with a wavelength at 1064 nm, were absorbed by the piezophotonic material fuligem-PDMS, which exhibits a high efficiency in converting light into pressure, giving rise to photoacoustic waves capable of enhancing cell membrane permeability, facilitating the entry of the plasmid encoding the green fluorescent protein (gWizGFP).In order to achieve different pressure gradients, low laser fluences (60 mJ cm-2 and 100 mJ cm-2) were used, resulting in the generation of high absolute pressures (60 bar and 100 bar), and relatively low-frequency bandwidths (10 MHz), compared to other materials studied by our research group (MnTPP). This technique has proven to be safe, with cell viability exceeding 95% even after several minutes of exposure to photoacoustic waves. This is a highly advantageous feature, and it is also possible to expose the materials to successive laser pulses without damaging them.Transfection results were evaluated using fluorescence microscopy and flow cytometry techniques. These results showed that the nanosecond laser with an 8 ns pulse had lower efficiency than the picosecond laser with a 30 ps pulse, achieving a transfection efficiency of approximately 5% in COS-7 cell line. This result indicates that the presence of higher pressures, inducing higher pressure gradients, is highly relevant for cell membrane permeabilization. In the future, it is expected that after some optimizations, this technique can be introduced into clinical practice. |
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