Publicado em 29/09/2016
Agência FAPESP em 07/09/2016
In order to reproduce in a host organism, a virus must undergo a process of adsorption to host cells, whereby the receptors in the viral envelope become attached to complementary receptors on the cell membrane. Researchers at Brazil’s National Energy & Materials Research Center (CNPEM) have developed a strategy to prevent viral infection by means of nanoparticles loaded with chemicals.
The nanoparticles are engineered to attract viruses, bind to them, and occupy the available sites that can adsorb to cell receptors.
When the surface of a virus is occupied by chemicals from the nanoparticles, it cannot infect host cells.
This innovative viral inactivation strategy was developed as part of the research project “Functionalization of silica nanoparticles: increasing biological interaction”, supported by FAPESP, with Mateus Borba Cardoso as principal investigator.
The study is the first to demonstrate viral inactivation based on surface chemistry of functionalized nanoparticles.
“Viral inhibition is performed by nanoparticles that have been modified in the laboratory. We coat them with chemicals capable of attracting and binding to viral particles,” Cardoso said.
“This steric effect, which relates to the fact that each atom in a molecule occupies a certain amount of space on the surface, prevents viruses from reaching their targets, the cells, and from binding to them, because the sites that can bind to the targets are already ‘occupied’ by nanoparticles.”
The researchers synthesized silica nanoparticles with different surface properties and evaluated their biocompatibility and antiviral efficacy.
They performed in vitro assays on cell cultures to evaluate the nanoparticles’ efficacy against human immunodeficiency virus (HIV) and vesicular stomatitis virus (VSV) in infected HEK293T cells (human embryonic kidney cells that express the large T antigen).
Virus particles were engineered to express the green fluorescent protein (GFP) reporter gene, labeling infected cells for detection by fluorescence microscopy and flow cytometry.
The innovation follows a strategy already implemented by the researchers to functionalize nanoparticles for the delivery of chemotherapy drugs at high concentrations to cancer cells, avoiding damage to healthy cells and minimizing the adverse effects of chemotherapy (read more in Portuguese at agencia.fapesp.br/23210).
Mesoporous silica nanoparticles were again chosen because of their high surface reactivity, which enables effective functionalization by the addition of chemicals through the pores. The chemical reactions induced in this manner are designed to attract specific viral proteins.
After synthesis and chemical loading, the researchers characterized the nanoparticles by measuring their size and checking that they were correctly functionalized using techniques ranging from microscopy to analysis of zeta-potential to determine the nanoparticles’ surface charge. They then correlated these data with the already known chemical properties of the viral envelope to increase the likelihood that the nanoparticles would become anchored in specific regions of the virus.
They also deployed small angle X-ray scattering (SAXS) using the particle accelerator at the National Synchrotron Light Laboratory (LNLS), which belongs to CNPEM. The SAXS procedure entailed emitting radiation from the LNLS beamline to analyze the shape and spatial organization of the functionalized silica nanoparticles.
“The functionalized nanoparticles were then incubated with the viral particles for a period,” Cardoso said. “During incubation, they interacted because of their respective surface properties. The strong attraction created by the chemicals on the surface of the nanoparticles led the viruses to bind to them instead of binding to host cells.”
After functionalizing the nanoparticles, analyzing load and other properties, and incubating them with the viruses, the researchers performed in vitro assays in which they infected HEK293T cells with HIV and VSV-G that had been prepared to express the fluorescent protein.
They used fluorescence microscopy to track the infection process and detect uninfected cells. Flow cytometry, a technique that uses light to analyze the properties of cells or particles in a heterogeneous fluid, enabled them to count the number of cells that respond positively or negatively to exposure to the viruses.
The findings showed that the nanoparticles reduced viral infection by up to 50%, demonstrating the efficacy of the strategy.
“This result could reach 100% if we increased the number of functionalized nanoparticles during the incubation period, but in vitro assays have to take place within an optimal range of viral inactivation so that the effects on infected cells can be observed, highlighting the differences for the sake of comparison,” Cardoso explained.
The assays also showed cell morphology was maintained and not influenced by the nanoparticles.
According to Cardoso, the strategy could be used to detect and eliminate viruses in blood bags before transfusion, for example. For this purpose, he said, the researchers are studying magnetic nanoparticles that can bind to any viruses in the blood bag, inactivate them, and then be separated from the blood by a magnet, taking the viral particles with them. The affinity between the chemicals loaded onto the nanoparticles and the viruses could also be leveraged to facilitate the development of novel techniques for detecting HIV and other viruses.
The results of the research have been published in the journal Applied Materials & Interfaces. The article “Viral inhibition mechanism mediated by surface-modified silica nanoparticles” was authored by Juliana Martins de Souza e Silva, Talita D. M. Hanchuk, Murilo Izidoro Santos, Jörg Kobarg, Marcio Chaim Bajgelman and Mateus Borba Cardoso, and it can be retrieved frompubs.acs.org/doi/abs/10.1021/acsami.6b03342.