Development of a three-dimensional polymeric structure to evaluate the effects of antifungals in the interaction with Aspergillus fumigatus

Author(s)

Bruna Nakanishi Fortes

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2023-06-02

Examining board members

Kelly Ishida; Patricia Cristina Baleeiro Beltrao Braga; Juliana Campos Junqueira; Sarah Gonçalves Tavares

• Advisor: Kelly Ishida

Abstract

Aspergillus fumigatus is primarily responsible for causing aspergillosis, and the invasive form is associated with greater severity in immunocompromised patients and high mortality rates. The study of the pathogenesis of this disease and the discovery of new treatments becomes essential, especially in methodologies that mimic the main target organ, the lung. The scaffold is a structure that mimics the extracellular matrix and improves cell adhesion and proliferation. Thus, this work aims to standardize the three-dimensional hydroxyethyl-alginate-gelatin (HAG) scaffold and evaluate the adhesion of pulmonary epithelial cells and A. fumigatus biofilm, as well as the treatment of this biofilm. The production of the scaffold is based on alginate and gelatin and the polymerization reaction occurs at low temperatures, forming a HAG cryogel. The HAG scaffold showed good stability when immersed in liquid and at body temperature, degrading only 20-30% of the weight in the first week and remaining stable for up to 5 weeks. The HAG scaffold showed high water swelling kinetics and elastic characteristics based on the rheology study. Light, fluorescence and scanning electron microscopy images showed pores on average 190 &#956m and homogeneously distributed, revealing a structure similar to the lung parenchyma. The HAG scaffold was not toxic to A. fumigatus or lung cells after immersion in glycine. Initially, the cells found it difficult to adhere and proliferate on the scaffold, but with the addition of laminin and the increase in cell culture time, cell proliferation and adhesion improved. However, this was not observed when laminin and collagen were added to the scaffold. The biofilm of A. fumigatus strains 1304 and ATCC 16913 established very well on the HAG scaffold, constituting a three-dimensional model for biofilm studies. The treatment with amphotericin B or voriconazole on the three-dimensional biofilms revealed to have the same action when the treatment was added in the two-dimensional model, showing an equivalence between the two models. The three-dimensional culture of lung cells and A. fumigatus 1304 on the HAG scaffold showed few cells present, but by scanning electron microscopy it was possible to observe interaction between them. Therefore, the HAG scaffold proved to be a good model for replicating the lung parenchyma with the establishment of a three-dimensional cell culture when laminin was added and was still effective in A. fumigatus biofilm formation and antifungal treatment. (AU)

• FAPESP's process: 20/11486-5 - Development of a three-dimensional polymeric structure to evaluate the effect of antimicrobials on the interaction of Aspergillus fumigatus
• Grantee: Bruna Nakanishi Fortes
• Support Opportunities: Scholarships in Brazil - Master