Optimized Synthesis of Small and Stable Silver Nanoparticles Using Intracellular and Extracellular Components of Fungi: An Alternative for Bacterial Inhibition.

Silver nanoparticles (AgNPs) represent an excellent option to solve microbial resistance problems to traditionally used antibiotics. In this work, we report optimized protocols for the production of AgNPs using extracts and supernatants of Trichoderma harzianum and Ganoderma sessile. AgNPs were characterized using UV-Vis spectroscopy and transmission electron microscopy, and the hydrodynamic diameter and Z potential were also determined. The obtained AgNPs were slightly larger using the fungal extract, and in all cases, a quasi-spherical shape was obtained. The mean sizes of AgNPs were 9.6 and 19.1 nm for T. harzianum and 5.4 and 8.9 nm for G. sessile using supernatant and extract, respectively. The AgNPs were evaluated to determine their in vitro antibacterial effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was determined, and in all cases the AgNPs showed an antimicrobial effect, with a MIC varying from 1.26-5.0 µg/mL, depending on the bacterial strain and type of nanoparticle used. Cytotoxicity analyses of AgNPs were carried out using macrophages and fibroblast cell lines. It was determined that the cell viability of fibroblasts exposed for 24 h to different concentrations of AgNPs was more than 50%, even at concentrations of up to 20 µg/mL of silver. However, macrophages were more susceptible to exposure at higher concentrations of AgNPs as their viability decreased at concentrations of 10 µg/mL. The results presented here demonstrate that small AgNPs are obtained using either supernatants or extracts of both fungal strains. A remarkable result is that very low concentrations of AgNPs were necessary for bacterial inhibition. Furthermore, AgNPs were stable for more than a year, preserving their antibacterial properties. Therefore, the reported optimized protocol using fungal supernatants or extracts may be used as a fast method for synthesizing small AgNPs with high potential to use in the clinic.

Chitosan Nanoparticles Containing Lipoic Acid with Antioxidant Properties as a Potential Nutritional Supplement.

The addition of the antioxidant α-lipoic acid (ALA) to a balanced diet might be crucial for the prevention of comorbidities such as cardiovascular diseases, diabetes, and obesity. Due to its low half-life and instability under stomach-like conditions, α-lipoic acid was encapsulated into chitosan nanoparticles (Ch-NPs). The resulting chitosan nanoparticles containing 20% w/w ALA (Ch-ALA-NPs) with an average diameter of 44 nm demonstrated antioxidant activity and stability under stomach-like conditions for up to 3 h. Furthermore, fluorescent Ch-ALA-NPs were effectively internalized into 3T3-L1 fibroblasts and were able to cross the intestinal barrier, as evidenced by everted intestine in vitro experiments. Thus, chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.

Asparaginase-Phage P22 Nanoreactors: Toward a Biobetter Development for Acute Lymphoblastic Leukemia Treatment.

Asparaginase (ASNase) is a biopharmaceutical for Acute Lymphoblastic Leukemia (ALL) treatment. However, it shows undesirable side effects such as short lifetimes, susceptibility to proteases, and immunogenicity. Here, ASNase encapsidation was genetically directed in bacteriophage P22-based virus-like particles (VLPs) (ASNase-P22 nanoreactors) as a strategy to overcome these challenges. ASNase-P22 was composed of 58.4 ± 7.9% of coat protein and 41.6 ± 8.1% of tetrameric ASNase. Km and Kcat values of ASNase-P22 were 15- and 2-fold higher than those obtained for the free enzyme, respectively. Resulting Kcat/Km value was 2.19 × 105 M-1 s-1. ASNase-P22 showed an aggregation of 60% of the volume sample when incubated at 37 °C for 12 days. In comparison, commercial asparaginase was completely aggregated under the same conditions. ASNase-P22 was stable for up to 24 h at 37 °C, independent of the presence of human blood serum (HBS) or whether ASNase-P22 nanoreactors were uncoated or PEGylated. Finally, we found that ASNase-P22 caused cytotoxicity in the leukemic cell line MOLT-4 in a concentration dependent manner. To our knowledge, this is the first work where ASNase is encapsulated inside of VLPs, as a promising alternative to fight ALL.

Determination of conjugated protein on nanoparticles by an adaptation of the Coomassie blue dye method.

A new accurate spectrophotometric method for protein determination on nanoparticles is described. The method is based on the Coomassie blue dye that binds to the basic and aromatic amino acid residues of proteins, especially arginine and lysine. A known amount of reagent dye was mixed with a variety of protein-loaded nanoparticles. Thereafter the unconjugated reagent was mixed with excess protein (bovine serum albumin) and titrated. In this method, the reacted dye on the protein coating of nanoparticle is directly determined, in opposite to the conventional method, in which the conjugated protein is determined as the difference between the non-conjugated protein found in the supernatant after centrifugation, and the total amount of protein originally used. This method is able to measure amounts of coated protein lower than 1 ppm. •Simple and accurate method especially adapted for protein-coated nanoparticles.•The amino acid residues of protein in the nanoparticle surface react with Coomassie brilliant blue dye.•The unreacted dye is titrated with an excess of a standard protein.

PEGylation of cytochrome P450 enhances its biocatalytic performance for pesticide transformation.

Pesticide intoxication is a major public health concern, and unfortunately there is not an effective treatment for severe organophosphorus pesticide intoxication. In this work, a non-immunogenic enzymatic bioconjugate based on cytochrome P450 was assayed for organophosphorus pesticide transformation. Enzyme therapy is an alternative approach to inactivate pesticides in the bloodstream, transforming them into less toxic metabolites. A variant of cytochrome P450 (CYPBM3 F87A) from Bacillus megaterium was chemically modified with polyethylene glycol. The PEGylated enzyme showed enhanced pesticide transformation activity when compared with the unmodified protein. The transformation rates were higher than those obtained with the unmodified enzyme for all six pesticides transformed. The specific activity of PEGylated preparation for parathion and dichlorophen was up to 9-times higher than these obtained with the unmodified enzyme. In addition, the modified CYP (CYP-PEG) remained active at extremely high pHs, maintaining 90% of its maximal activity at pH 11, as opposed to the unmodified CYP that retained less than 20% of its maximal activity at that pH. In addition, the bioconjugate showed good catalytic activity in blood serum and innocuousness on immune cells. The potential use of PEGylated CYP as a detoxification strategy for pesticide poisoning is demonstrated and discussed.

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed.

SERS properties of different sized and shaped gold nanoparticles biosynthesized under different environmental conditions by Neurospora crassa extract.

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these 'green' synthesized gold nanoparticles might have potential applicability in the field of biological sensing.

Biosynthesis and microscopic study of metallic nanoparticles.

Nanobiotechnology, bionanotechnology, and nanobiology are terms that have emerged in reference to the combination of nanotechnology and biology. Through the convergence of these disciplines, the production of metallic nanoparticles (NPs) using biological material as reducing agents is rapidly progressing. In the near future, the application of clean, non-toxic, and eco-friendly nanostructured material will be possible in industry and/or biomedicine. Currently, there is a wide range of organisms that have been reported to be useful in producing NPs. However, the development of finer protocols and the applicability of biosynthesized nanostructures are presently under study. Silver and gold are among the most studied metals due to their potential use in medical treatment. In fact, silver NPs have been evaluated as antimicrobial agents, having been successfully used against several types of fungi and bacteria. However, the use of such material in our daily life must be carefully evaluated. This article summarizes some of the most significant results using organisms to produce metallic NPs as well as the microscopic analyses used to characterize the nanostructured material obtained, providing a valuable database for future research.