Publicação
Development of a multifunctional phage-based nanoparticle for targeted drug delivery
| Resumo: | Breast cancer is the most frequently diagnosed cancer in women and killed more than half a million people in 2012. Classic treatments, namely chemotherapy, continue to be the backbone of cancer therapies. However, their non-specificity and non-selectivity is an open window to develop new targeted therapies that will eventually eliminate only cancer cells. The goal of this work was to develop a phage-based nanoparticle that can be used as a carrier for multiple anticarcinogenic drugs, targeting and eliminating breast cancer cells, without damaging healthy cells. Specifically, it was planned to genetically manipulate M13KE phage with encoding cell-penetrating peptides (CPPs) and specific peptides for breast cancer cells, which would allow phage internalization towards human cells. HIV-Tat and Penetratin CPPs, as wells as a specific peptide for human breast cancer cell receptors (so-called 231 peptide) were used. Moreover, coupling anticarcinogenic drugs to the phage major coat protein would result in a lower in situ drug concentration required to eliminate those cells. Doxorubicin (DOX) and PD98059 were used for that purpose owing to their synergistic effect on cancer cells death and proliferation. Several approaches herein implemented provided clear evidences that chemical conjugation of DOX to the phage was achieved. For instance, Fourier transform near-infrared spectroscopy (FT-NIRS) enabled the discrimination between free DOX or phage solutions from conjugated phages. PD98059 conjugation was not successfully accomplished mainly due to solubility issues. In vitro cytotoxicity of the phages (control and conjugated with DOX) and free DOX was assessed against tumorigenic MDA-MB-231 and non-tumorigenic MCF-10-2A cell lines. Conjugated DOX induced cytotoxicity in an independent phage-manner but, as expected, the treatment was not specific to the cancer cells. The phage genome manipulation with HIV-Tat, Penetratin and 231 peptides could not be accomplished, thus limiting the phage internalization by human cells. Parallel to this cytotoxic analysis, an image segmentation algorithm was developed, able to provide information on cell confluence, regardless of the cell morphology. As a result, the operator variability during cell culturing was reduced. In summary, the results highlight the clear need of targeted therapies for cancer treatment, as well as the potential of phages to be used as therapy platforms. |
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| Autores principais: | Xavier, Mariana |
| Ano: | 2014 |
| 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: | Breast cancer is the most frequently diagnosed cancer in women and killed more than half a million people in 2012. Classic treatments, namely chemotherapy, continue to be the backbone of cancer therapies. However, their non-specificity and non-selectivity is an open window to develop new targeted therapies that will eventually eliminate only cancer cells. The goal of this work was to develop a phage-based nanoparticle that can be used as a carrier for multiple anticarcinogenic drugs, targeting and eliminating breast cancer cells, without damaging healthy cells. Specifically, it was planned to genetically manipulate M13KE phage with encoding cell-penetrating peptides (CPPs) and specific peptides for breast cancer cells, which would allow phage internalization towards human cells. HIV-Tat and Penetratin CPPs, as wells as a specific peptide for human breast cancer cell receptors (so-called 231 peptide) were used. Moreover, coupling anticarcinogenic drugs to the phage major coat protein would result in a lower in situ drug concentration required to eliminate those cells. Doxorubicin (DOX) and PD98059 were used for that purpose owing to their synergistic effect on cancer cells death and proliferation. Several approaches herein implemented provided clear evidences that chemical conjugation of DOX to the phage was achieved. For instance, Fourier transform near-infrared spectroscopy (FT-NIRS) enabled the discrimination between free DOX or phage solutions from conjugated phages. PD98059 conjugation was not successfully accomplished mainly due to solubility issues. In vitro cytotoxicity of the phages (control and conjugated with DOX) and free DOX was assessed against tumorigenic MDA-MB-231 and non-tumorigenic MCF-10-2A cell lines. Conjugated DOX induced cytotoxicity in an independent phage-manner but, as expected, the treatment was not specific to the cancer cells. The phage genome manipulation with HIV-Tat, Penetratin and 231 peptides could not be accomplished, thus limiting the phage internalization by human cells. Parallel to this cytotoxic analysis, an image segmentation algorithm was developed, able to provide information on cell confluence, regardless of the cell morphology. As a result, the operator variability during cell culturing was reduced. In summary, the results highlight the clear need of targeted therapies for cancer treatment, as well as the potential of phages to be used as therapy platforms. |
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