Desenvolvimento de filmes finos à base de polímeros e polipeptídios recombinantes do tipo elastina a partir de métodos de triagem de alta produtividade

Author(s)

Rogério Aparecido Bataglioli

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Química
• Defense date: 2021-04-08

Examining board members

Marisa Masumi Beppu; Everson Alves Miranda; Jorge Vicente Lopes da Silva; Osvaldo Novais de Oliveira Júnior

• Advisor: Marisa Masumi Beppu; Bradley D Olsen

Abstract

Surface functionalization has been explored to overcome biotechnologically relevant challenges, from the targeted drug delivery to the selective capture of bioactive agents for diagnosis. Recombinant protein plays an important role in designing bioactive surfaces by designing macromolecules with the desired functionalities. The layer-by-layer (LbL) technique became a popular strategy for surface functionalization with minimal restrictions regarding the coated surface and the materials deposited. Nevertheless, both strategies demand substantial efforts to determine the appropriate conditions to prepare the newly designed materials. This thesis aimed to streamline the production of biopolymer- and recombinant protein-based materials through high-throughput methods. At first, the LbL method was adapted to deposit multilayer films on 96-well plates, enabling one to investigate different assembly conditions into the same batch. This method effectively produced polysaccharide-based films, indicating the dependence of free amino and carboxylate groups on multilayers to the assembly pH conditions. Hybrid polysaccharide/recombinant elastin-like polypeptides (ELPs) were also assembled based on electrostatic interactions. ELP variants containing a higher number of hydrophobic residues and assembly pH conditions that promoted lower ionization degrees of both polyelectrolytes led to higher film growth. This thesis also explored protein engineering's versatility for designing a library ELPs fused with complementary isopeptide binding domains for self-assembly LbL films. Spy-Tag/Spy-Catcher and leucine zipper ZE/ZR interactions, among others, were effective to drive the self-assembly deposition of multilayer films. Spy-Tag/Spy-Catcher also led to resistant multilayers over a wide range of pH (3.0 – 12.0) and ionic strength (5.0 mol/L NaCl), which may disassemble traditional polyelectrolyte-based multilayers. The high-throughput-LbL method was also used to investigate the role of film assembly conditions on the loading and release of a model drug molecule (calcein) on poly(acrylic acid)/poly(allylamine hydrochloride) multilayer films. Higher values of pH (> 8.5) and the presence of NaCl or MgCl2 salts (both at 0.05 mol/L) in the polyelectrolyte solutions favored the model drug loading in multilayer films. The drug loading method also affected the film morphology and drug transport mechanisms, leading to slower drug release rates for films loaded during polyelectrolyte multilayer deposition. Finally, this thesis explored the use of the high-throughput approach for recombinant protein test expression. Molecular biology protocols, from cell transformation to protein biosynthesis, were automated to screen tens of different cell strain-vector combinations per batch. The study also focused on automating analytical total (Bradford) and histidine-tagged (immunoassay) protein quantification to determine the most favorable condition for mCherry biosynthesis (model protein). Overall, this thesis sheds light on using high-throughput methods to facilitate the development of biopolymer- or protein-based materials that demand intense testing and optimization, contributing to accelerating the innovation cycles in the biotechnology arena (AU)

• FAPESP's process: 16/10193-9 - New mechanisms for controlling triggered release of anticancer drugs in cellular backpacks
• Grantee: Rogério Aparecido Bataglioli
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)