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CHEMISTRY

Biological silver

Nanoparticles from fungi are successfully tested on antibacterial fabrics and on sores

Fungi isolated from mangroves in the state of São Paulo, grown in a laboratory at the Butantan Institute

Léo RamosFungi isolated from mangroves in the state of São Paulo, grown in a laboratory at the Butantan InstituteLéo Ramos

They decompose organic substances in nature and they contaminate our food and industrialized products. They are fungi, and they come in all shapes, colors and sizes, from mushroom to microscopic form. Present in a wide range of industrial fermentation processes, these microorganisms are also able to produce biological nanoparticles of silver through a chemical process mediated by their enzymes. These particles can then be used to make antibacterial fabrics for things like bedsheets, pillow cases, and hospital scrubs; coatings for ceramic tiles; and treatments for toenail fungal infections and sores caused by cutaneous leishmaniasis.

Since 2003, two professors at the University of Campinas (Unicamp) — Nélson Duran, coordinator of the Biological Chemistry Laboratory, and Oswaldo Luiz Alves, head of the Solid State Chemistry Laboratory — have been dedicating some of their research efforts to studying processes and uses involving these unicellular beings. “The effect that biological silver nanoparticles have on the treatment of cutaneous leishmaniasis sores is much superior to that of commercially available fungicides, as shown by our research in collaboration with Professor Bartira Rossi-Bergmann from the Biophysics Institute at the Federal University of Rio de Janeiro (UFRJ),” says Duran, who is also a visiting professor at the Natural and Human Science Center of the Federal University of the ABC in the city of Santo André.

The tests examined the effects of treatment with silver nanoparticles obtained via chemical or biological methods and compared them to the commercially available antifungal drug Amphotericin B. “Our biological system is about 300 times more efficient than the antifungal medicine used in conventional treatments and three times better than chemically obtained nanoparticles,” says Duran. The results of the experiments on the fungus that affects nails,  especially toenail fungus, were also very promising. The next stage of the research on toenail mycoses (fungal infections) will be the clinical trials, which will be assigned to researchers at Unicamp’s School of Medicine.

The fungi used in the studies are Fusarium oxysporum, the species that causes fusariosis, a disease that afflicts agricultural crops and makes them wilt. The first strains of the fungus were obtained from the Fusarium bank maintained by microbiologist João Lúcio de Azevedo, a retired professor from the Luiz de Queiroz School of Agriculture (Esalq) at the University of São Paulo (USP), Piracicaba campus. “We ran tests to select strains that have the chemical potential to transform silver ions [by adding electrons] to metallic silver, a process that results in the formation of the nanoparticles,” says Alves. Enzymes found in the phytopathogen, such as nitrate reductase, are responsible for this chemical transformation. “In some cases, the conditions are present in the fungus, but instead of monodispersed nanoparticles, they produce large aggregates of particles,” he says.

After the fungi are selected, they are cultivated until they reach the ideal size, then the fungal biomass is incubated in distilled water to extract enzymes, proteins and extracellular chemicals. The mixture is then filtered to separate the biomass from the liquid. Next comes the addition of silver nitrate, which triggers the chemical reduction process that produces the nanoparticles. Using the technique known as ultraviolet-visible absorption spectroscopy, it is possible to watch the metallic silver’s formation.

“The advantage to using the biological method as opposed to the chemical one is that the fungus leaves a part of its proteins on the surface of the nanoparticles, giving them special characteristics such as greater adhesion to fabrics, making them antibacterial.” The researchers tested the particles by infusing cotton and polyester fabrics with the biological silver and exposing them to colonies of Staphylococcus aureus, a type of bacteria commonly found in hospital environments, where they are a major cause of infection. “After 30 washes, the fabrics’ antibacterial properties remained virtually unchanged,” says Alves.

Transmission electron microscope in Unicamp laboratory

Léo RamosTransmission electron microscope in Unicamp laboratoryLéo Ramos

Isolated from mangroves
A separate study conducted at the Butantan Institute, coordinated by Professor Ana Olívia de Souza from the Biochemistry and Biophysics Laboratory, is also testing the antimicrobial properties of silver nanoparticles incorporated into fabrics. The fungi used in this case are Aspergillus tubingensis and Bionectria ochroleuca, isolated from mangroves in the state of São Paulo during a project funded by FAPESP and coordinated by Professor Itamar Soares de Melo from the Brazilian Agricultural Research Corporation (Embrapa), Environmental Division, based in the city of Jaguariúna. Professor Souza also took part in that project.

“Among the strains used in the study, the 15 most interesting fungi from a biotechnological viewpoint were selected and evaluated in the Butantan laboratory,” says Souza, who was advised by Duran during her PhD. Five strains made the cut after the initial screening. The subsequent stage, an evaluation of nanoparticle-forming capacity and of whether the resulting particles had antimicrobial properties, was developed with support from FAPESP through its BIOTA program, which studies Brazilian biodiversity.

A new screening resulted in the selection of two fungal species. “We did a physical-chemical characterization of the nanoparticles obtained from these fungi in order to determine whether they were uniform in size, an important requirement for incorporating silver into cotton or polyester fabrics or plastic materials,” says Souza. According to the researcher, in addition to antimicrobial fabrics for use in hospitals, a major market for biological silver nanoparticles will be plastic materials for household use.

The researchers at Unicamp went beyond merely producing the nanoparticles by studying the enzymes of the selected fungi. “We separated the enzymes and were able to build nanoparticles that were identical to those manufactured by the microorganism,” says Duran. The paper Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains, published on July 13, 2005 in the open-access Journal of Nanobiotechnology, has been downloaded more than 30,000 times. The researchers also studied the antibacterial properties of silver — in other words, how the metal destroys bacteria. The chosen target was Escherichia coli. Using a transmission electron microscope, the researchers watched as the nanoparticles started acting on the bacteria’s surface, then clustered together to cover the cells entirely, and finally penetrated and killed them.

Fungus in liquid culture medium in the Butantan Institute collection

Léo RamosFungus in liquid culture medium in the Butantan Institute collectionLéo Ramos

A different study assessed the biological nanoparticles’ effect on isolates of antibiotic-resistant enterobacteria — those that cause blood and urinary tract infections as well as pneumonias. “In this study, we proved that silver is very effective, even when used as an isolated treatment without antibiotics,” says Duran. A diverse team of professors participated in the two research projects: Marcelo Brocchi from the Biosciences Institute, and Ljubica Tasica from the Chemistry Institute at Unicamp; Simone Picoli from Feevale University in the city of Novo Hamburgo (state of Rio Grande do Sul); Gerson Nankazato from Londrina State University (state of Paraná); Priscyla Marcato from the USP School of Pharmaceutical Sciences in Ribeirão Preto; and Mahendra Rai, an Indian professor from Amravati University who is currently at Unicamp.

Duran and Alves, the trailblazers of biological silver nanoparticle study in Brazil, initiated their line of research at the same time as the first related work began being published in India. Instead of fungi, the Indian researchers use plants to obtain the silver, which is used mainly to fight agricultural pests. The Brazilian researchers are now preparing to ramp up production of silver nanoparticles by building a pilot plant. Another recent development at Unicamp’s Chemistry Institute is the merger of the two laboratories involved in the project — the Biological Chemistry and Solid State labs — into a single laboratory called NanoBioss. The “merger” was approved in February by Brazil’s Ministry of Science, Technology and Innovation as part of SisNano — Brazil’s national nanotechnology system. “As a member lab, our knowledge and techniques will be made available to Brazilian industry and academia,” says Alves.

Project
Use of mangrove fungi in biosynthesis of silver nanoparticles, and application in production of antimicrobial textiles (nº 10/50186-5); Grant mechanism Regular Line of Research Project Award — BIOTA Program; Coordinator Ana Olívia de Souza – Butantan Institute; Investment R$ 248,424.03 (FAPESP).

Scientific articles
MARCATO, P.D. et al. Biogenic silver nanoparticles: antibacterial and cytotoxicity applied to textile fabrics. Journal of Nano Research. v. 20, p. 69-76. 2012.
RODRIGUES, A.G. et al. Biogenic antimicrobial nanoparticles produced by fungi. Applied Microbiology and Biotechnology.  v. 95, p. 1-8. 2012.

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