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| Author(s): |
Fernanda Santos de Oliveira
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Escola Politécnica (EP/BC) |
| Defense date: | 2023-03-10 |
| Examining board members: |
Adriano Rodrigues Azzoni;
Marianna Teixeira de Pinho Favaro;
Soraia Attie Calil Jorge
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| Advisor: | Adriano Rodrigues Azzoni |
| Abstract | |
Advanced therapies such as gene therapy have been widely studied for the treatment of cancer. Many of these studies are based on the use of viral vectors for the delivery of nucleic acids to target cells. However, there are still recurring problems in these studies, such as; 1) the difficulty of gene delivery to tumor cells due to the presence of physical, enzymatic and diffusional barriers; 2) the stimulation of inflammatory response by the immune system due to the use of viral vectors and; 3) the high costs of producing these vectors. An alternative for overcoming these obstacles is the use of recombinant proteins specifically designed for gene delivery and combating tumor cells. These proteins are, in general, modular, multifunctional proteins capable of mimicking the ability of viruses to exploit extra- and intracellular signals and responses to infect cells. Thus, the main objective of this master\'s degree was the development and characterization of multifunctional recombinant proteins capable of efficiently carrying out the transport of genetic material (interfering RNA) into tumor cells. For this, the GFP reporter protein was fused to a disintegrin (Echistatin) serving as a targeting domain for tumor cells. The protein also contains a polyarginine (R6) that interacts and condenses the genetic material to be delivered. Four different gene constructs were synthesized and expressed in Escherichia coli: construct 1 complete (H6-R6-GFP-ECH); construct 2 - without Echistatin (H6-R6-GFP); construct 3 - without the R6 sequence (H6-GFP-ECH); and construction 4 - (H6-R9-GFP-ECH), containing the R9 sequence with the potential to induce the formation of nanoparticles. The proteins were produced in E. coli and purified by affinity chromatography, with no obstacles regarding toxicity to the bacteria. The fluorescence of the different constructs was analyzed and compared. The hydrodynamic behavior of the proteins in solution was also evaluated by DLS, with and without the presence of pDNA, thus verifying interaction with nucleic acid and formation of nanoparticles. Biological activity studies carried out in vitro indicate that construct 1 was able to direct and facilitate the entry of siRNAs into cancer cells that overexpress integrin receptors (U87MG cells). In the cytotoxicity tests, the potential of the protein itself was also demonstrated, which was capable of reducing the viability of cells that overexpress integrins, being itself a potential antitumor agent. (AU) | |
| FAPESP's process: | 20/00616-5 - Nanoparticles formed by modular recombinant proteins: new platforms against tumor cells |
| Grantee: | Fernanda Santos de Oliveira |
| Support Opportunities: | Scholarships in Brazil - Master |