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BIOTECHNOLOGY

Green nanoparticles

Plants from the Cerrado are being used in the production of nanosystems designed to control agricultural pathogens and pests, among other applications

Pequi leaves...

Fábio Colombini Pequi leaves…Fábio Colombini

Nutshells, seeds, fruit and leaves from trees and plants of the Brazilian Cerrado – such as pequi, mangaba, sucupira, bureré, and a type of cashew, to name a few – as well as several species of mushrooms, are the raw materials of choice for researchers at Embrapa Genetic Resources and Biotechnology (Cenargen), based in the city of Brasília, for obtaining nanoparticles that can potentially be used in biosensors to detect viruses in plants and to control insect larvae, microorganisms, and cancer cells, among other applications. “We investigated several species and biological resources in order to find those most appropriate for use in the bioreduction process by which metallic nanoparticles are produced,” says Luciano Paulino da Silva, coordinator of the nanobiotechnology group at Embrapa Cenargen. Bioreduction is a biological process mediated by molecules such as enzymes, proteins, amino acids, polysaccharides, and metabolites found in extracts from leaves, nutshells, or seeds, for example. Through this process, silver ions gain electrons and are transformed into metallic silver, producing nanoparticles as a result. A major advantage of biological synthesis, as opposed to the traditional chemical method, is that some of the active molecules adhere to the surface of the nanoparticles, giving them antibacterial, antiviral, antiallergic, or other unique properties, depending on the plant or mushroom used. “This method for nanoparticle synthesis is called green nanotechnology.”

An aqueous extract from nutshells of Anacardium othonianum – a shrubby plant whose fruit closely resemble the common cashew – was the medium chosen by researcher and PhD student Cínthia Caetano Bonatto, at the University of Brasília (UnB), to study an alternative synthesis pathway that avoids using environmentally harmful solvents such as the traditionally employed sodium hydroxide. During her doctoral research, advised by Luciano Silva from Embrapa Cenargen, Bonatto verified that higher temperatures during the synthesis process will accelerate growth, modulate size, and increase the electrical conductivity of the nanoparticles, which were tested and shown to possess antifungal properties. “Some optical, structural, biological, and electrical properties of the particles are controlled by the temperature applied to the reaction medium during synthesis,” says Luciano Silva. The results were published in the journal Industrial Crops and Products, in July 2014.

The same strategy of applying higher temperatures during synthesis was used in a different research project by Bonatto, using leaves from the pequi tree (Caryocar brasiliense). “Compared to other plants tested in the laboratory, the aqueous extract from pequi has a high potential for bioreduction,” says the researcher. After synthesis and characterization, including an assessment of structural properties of the resulting nanomaterial based on size and shape, the researchers can determine the best applications for each type of nanoparticle. “For agriculture, for example, we can develop nanosystems to control pathogens and pests, including bacteria, fungi, and insect larvae,” says Luciano Silva. “Usage strategies will be assessed according to what applications are needed.” To give another example, pests like beetles or leafhoppers could be captured by putting nanosystems inside traps in the field. To fight leaf- or root-infesting fungi, nanoparticles could be sprayed directly onto affected leaves, plant tissues, or the soil.

cashew nutshells

Cínthia Bonattocashew nutshellsCínthia Bonatto

Laboratory tests
As part of the research involving pequi leaves, laboratory tests are underway to assess their biotechnological potential. “We tested the potential cytotoxicity of nanoparticles on bacteria, fungi, in vitro normal and malignant tumor cells from mammals, and nematodes used in pest control, and now we have started testing them on lab-grown plants to assess potential toxic effects when used in the field,” says Bonatto. She emphasizes that no visual changes were observed in healthy plants treated with pequi nanoparticles, when compared to untreated control plants. Because the plants are grown in containers in the laboratory, the nanoparticles are applied directly into the liquid culture medium in which the plants are grown. The next step in the research will consist in analyzing the plants’ morphology and biochemistry, assessing their cells and metabolism in order to determine whether any toxic effect has taken place.

Some particles exhibit particular properties that make them special, according to Luciano Silva, because of a difference in their surface plasmon resonance signals. “These particles are nanostructured, which gives them new optical properties, such as metallic silver absorbing light at a wavelength that the metal in its ionic form was not previously absorbing.” Due to this difference, these particles amplify the optical signal in the systems in which they are used, making them more sensitive. “Based on this unique characteristic, we are working to develop nanobiosensors to detect growth hormones and hormones secreted by tumor cells, as well as viruses in plants.”

The use of nanoparticles on surfaces like plastic or glass is an additional line of research being conducted at the laboratory. In this case, the study developed by Luciane Dias da Silva, a master’s student advised by Luciano Silva, focuses on silver nanoparticles synthesized from extracts of fruits and leaves of the mangaba tree (Hancornia speciosa), to be applied as a coating for plastics like polyethylene terephthalate (PET). The study aims to find an alternative method to control larvae of Aedes aegypti, the mosquito that transmits dengue fever. This new control mechanism would not involve capturing the insect. Instead, it would be based on the nanoparticles’ toxicity to mosquito larvae deposited on the PET. The possible ways of applying this method include using particles suspended in a liquid medium, or immobilizing and bonding some of the particles onto the plastic surface during the synthesis process. Separate extracts were obtained from the skins, flesh, and seeds of mangaba fruit. “The nanoparticles obtained from the silver salts with their respective extracts have reached the final stage of characterization, and some biological tests have already been completed,” says Dias da Silva. In the next stage of the project, the plastic tubes from which PET bottles are produced, known as PET pre-molds, will be used to immobilize and adhere the nanoparticles.

Mangaba fruit are raw materials used in biological synthesis

Fábio Colombini Mangaba fruit are raw materials used in biological synthesisFábio Colombini

Bioactive compounds
In addition to plants from the Cerrado, the group is also studying several species of macrofungi (mushrooms). “Mushrooms are sources of bioactive compounds with a wide range of biological effects, such as antitumoral, antiviral, antimicrobial, anti-inflammatory, or antioxidant properties, among others,” says researcher Vera Lúcia Perussi Polez, who is working with species obtained from the Embrapa Cenargen Mushroom Bank. “That is why we decided to use this material as a source, both for metal reduction and to stabilize the nanoparticles.” The biological material for silver nanoparticle synthesis can come from either the mushroom’s fruiting body (cap) or its mycelium, the vegetative part of the fungus.

Polez chose to use the mycelium because she believes it offers greater advantages than the cap – such as growing faster. “In addition, the chemical components in the mycelium are produced more homogeneously because we apply controlled conditions to the liquid culture medium, such as nutrients, pH, temperature, and oxygenation, among others. This enables us to maintain the reproducibility of this biological material,” she says. The quantity of chemicals present in these organisms depends on factors including species, chosen strain, development stage – fruiting body or mycelium –, and type of substrate used.

070-073_Nanoverde_223Infographic: Ana Paula Campos / Illustration: Alexandre AffonsoThe research into obtaining nanoparticles from mushrooms began two years ago. “We designed our experiments to make the biological material as uniformly reproducible as possible,” says Polez. After improving the conditions for growing the mycelia, synthesizing nanoparticles, and characterizing their physical, chemical, and structural properties, the research will now enter a new stage, at which it will investigate and describe the biological effects of the nanoparticles. The level of care exercised here is justified by the strong potential that compounds in mushrooms have shown for use in pharmaceuticals, medicine, agriculture, and industry. These bioactive compounds include beta-glucans and lecithins, which are respectively complex carbohydrates and proteins with immunoregulating and antitumor properties; triterpenes, which have antihypertensive, antiviral, antitumor, and antiallergic effects; phenolic compounds, with antiplatelet, antioxidant, and anti-inflammatory properties; and other, antimicrobial compounds.

In addition to the researchers from Embrapa and undergraduate researchers, and master’s and PhD students, the group’s collaborators also include researchers from other institutions, such as Professor Elmo Salomão Alves from the Physics Department at the Federal University of Minas Gerais (UFMG). “The group headed by Professor Alves develops surfaces containing graphenes [crystalline form of carbon], onto which recognition molecules like antibodies and receptor ligands are incorporated. This material is deposited onto the silver nanoparticles in order to produce nanobiosensors,” says Luciano Silva. Professor Eduardo Fernandes Barbosa, enzymology expert at the Federal University of Bahia (UFBA), and Professor Alexsandro Galdino, microorganism genetics expert at the Federal University of São João Del-Rei (UFSJ), are also partners in the research.

Mycelia (colored by computer), the vegetative part of mushrooms, grown in a controlled culture medium

Personal archives of Vera Lúcia Perussi Polez Mycelia (colored by computer), the vegetative part of mushrooms, grown in a controlled culture mediumPersonal archives of Vera Lúcia Perussi Polez

“We are working to develop nanostructured surfaces to immobilize enzymes that can be applied in industry to obtain food-grade hydrolyzed products from macromolecules like proteins and carbohydrates, in a process called enzymatic catalysis,” says Luciano Silva. According to the researcher, these enzymes are not yet applied on a large scale due to the high operating costs of using them. “With the immobilization process, this cost would drop significantly,” he explains.

Another biological synthesis model is also being tested and has already yielded good results: polymeric particles structured from chitosan, a substance found in the carapace of crustaceans. The particles could be used to transport both macromolecules and secondary metabolites, for applications in biomedicine and agriculture. “One of the molecules we transported using the polymer system is mellitin, an amino acid extracted from bee venom, which has antibacterial, antifungal, and anticancer properties,” says Luciano Silva. The model tested by PhD student Kelliane Almeida de Medeiros investigated the effects of mellitin-containing polymeric particles on breast cancer cells in vitro. Initial testing on mice is now underway, with promising results.

Scientific article
BONATTO, C. C.; SILVA, L. P. Higher temperatures speed up the growth and control the size and optoelectrical properties of silver nanoparticles greenly synthesized by cashew nutshells. Industrial Crops and Products. v. 58, p. 46-54. Jul. 2014.

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