Isolating and analyzing tumor-derived exosomes (TEX) can provide important information about the state of a tumor, facilitating early diagnosis and prognosis. Since current isolation methods are mostly laborious and expensive, we propose herein a fast and cost-effective method based on a magnetic nanoplatform to isolate TEX. In this work, we have tested our method using three magnetic nanostructures: (i) Ni magnetic nanowires (MNWs) (1500 × 40 nm), (ii) Fe3O4 nanorods (NRs) (41 × 7 nm), and (iii) Fe3O4 cube-octahedral magnetosomes (MGs) (45 nm) obtained from magnetotactic bacteria. The magnetic response of these nanostructures has been characterized, and we have followed their internalization inside canine osteosarcoma OSCA-8 cells. An overall depiction has been obtained using a combination of Fluorescence and Scanning Electron Microscopies. In addition, Transmission Electron Microscopy images have shown that the nanostructures, with different signs of degradation, ended up being incorporated in endosomal compartments inside the cells. Small intra-endosomal vesicles that could be precursors for TEX have also been identified. Finally, TEX have been isolated using our magnetic isolation method and analyzed with a Nanoparticle tracking analyzer (NanoSight). We observed that the amount and purity of TEX isolated magnetically with MNWs was higher than with NRs and MGs, and they were close to the results obtained using conventional non-magnetic isolation methods.

Isolation of Cancer-Derived Exosomes Using a Variety of Magnetic Nanostructures: From Fe3O4 Nanoparticles to Ni Nanowires.

Nanomaterials are increasingly being used in new products and devices with a great impact on different fields from sensoristics to biomedicine. Biosynthesis of nanomaterials by microorganisms is recently attracting interest as a new, exciting approach towards the development of 'greener' nanomanufacturing compared to traditional chemical and physical approaches. This review provides an insight about microbial biosynthesis of nanomaterials by bacteria, yeast, molds, and microalgae for the manufacturing of sensoristic devices and therapeutic/diagnostic applications. The last ten-year literature was selected, focusing on scientific works where aspects like biosynthesis features, characterization, and applications have been described. The knowledge, challenges, and potentiality of microbial-mediated biosynthesis was also described. Bacteria and microalgae are the main microorganism used for nanobiosynthesis, principally for biomedical applications. Some bacteria and microalgae have showed the ability to synthetize unique nanostructures: bacterial nanocellulose, exopolysaccharides, bacterial nanowires, and biomineralized nanoscale materials (magnetosomes, frustules, and coccoliths). Yeasts and molds are characterized by extracellular synthesis, advantageous for possible reuse of cell cultures and reduced purification processes of nanomaterials. The intrinsic variability of the microbiological systems requires a greater protocols standardization to obtain nanomaterials with increasingly uniform and reproducible chemical-physical characteristics. A deeper knowledge about biosynthetic pathways and the opportunities from genetic engineering are stimulating the research towards a breakthrough development of microbial-based nanosynthesis for the future scaling-up and possible industrial exploitation of these promising 'nanofactories'.

Microbial Nanotechnology: Challenges and Prospects for Green Biocatalytic Synthesis of Nanoscale Materials for Sensoristic and Biomedical Applications.

Next to plants, bacteria account for most of the biomass on Earth. They are found everywhere, although certain species thrive only in specific ecological niches. These microorganisms biosynthesize a plethora of both primary and secondary metabolites, defined, respectively, as those required for the growth and maintenance of cellular functions and those not required for survival but offering a selective advantage for the producer under certain conditions. As a result, bacterial fermentation has long been used to manufacture valuable natural products of nutritional, agrochemical and pharmaceutical interest. The interactions of secondary metabolites with their biological targets have been optimized by millions of years of evolution and they are, thus, considered to be privileged chemical structures, not only for drug discovery. During the last two decades, functional genomics has allowed for an in-depth understanding of the underlying biosynthetic logic of secondary metabolites. This has, in turn, paved the way for the unprecedented use of bacteria as programmable biochemical workhorses. In this Review, we discuss the multifaceted use of bacteria as biological factories in diverse applications and highlight recent advances in targeted genetic engineering of bacteria for the production of valuable bioactive compounds. Emphasis is on current advances to access nature’s abundance of natural products. This Review highlights bacteria as biological factories in diverse applications, with an emphasis on targeted genetic engineering for the production of bioactive natural products.

Bacteria as genetically programmable producers of bioactive natural products

The emergence of bacterial resistance to antibiotics has led to the search for alternate antimicrobial treatment strategies. Engineered nanoparticles (NPs) for efficient penetration into a living system have become more common in the world of health and hygiene. The use of microbial enzymes/proteins as a potential reducing agent for synthesizing NPs has increased rapidly in comparison to physical and chemical methods. It is a fast, environmentally safe, and cost-effective approach. Among the biogenic sources, fungi and bacteria are preferred not only for their ability to produce a higher titer of reductase enzyme to convert the ionic forms into their nano forms, but also for their convenience in cultivating and regulating the size and morphology of the synthesized NPs, which can effectively reduce the cost for large-scale manufacturing. Effective penetration through exopolysaccharides of a biofilm matrix enables the NPs to inhibit the bacterial growth. Biofilm is the consortia of sessile groups of microbial cells that are able to adhere to biotic and abiotic surfaces with the help extracellular polymeric substances and glycocalyx. These biofilms cause various chronic diseases and lead to biofouling on medical devices and implants. The NPs penetrate the biofilm and affect the quorum-sensing gene cascades and thereby hamper the cell-to-cell communication mechanism, which inhibits biofilm synthesis. This review focuses on the microbial nano-techniques that were used to produce various metallic and non-metallic nanoparticles and their "signal jamming effects" to inhibit biofilm formation. Detailed analysis and discussion is given to their interactions with various types of signal molecules and the genes responsible for the development of biofilm.

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Microbiologically-Synthesized Nanoparticles and Their Role in Silencing the Biofilm Signaling Cascade.

Inorganic nanomaterials are widely used in chemical, electronics, photonics, energy and medical industries. Preparing a nanomaterial (NM) typically requires physical and/or chemical methods that involve harsh and environmentally hazardous conditions. Recently, wild-type and genetically engineered microorganisms have been harnessed for the biosynthesis of inorganic NMs under mild and environmentally friendly conditions. Microorganisms such as microalgae, fungi and bacteria, as well as bacteriophages, can be used as biofactories to produce single-element and multi-element inorganic NMs. This Review describes the emerging area of inorganic NM biosynthesis, emphasizing the mechanisms of inorganic-ion reduction and detoxification, while also highlighting the proteins and peptides involved. We show how analysing a Pourbaix diagram can help us devise strategies for the predictive biosynthesis of NMs with high producibility and crystallinity and also describe how to control the size and morphology of the product. Here, we survey biosynthetic inorganic NMs of 55 elements and their applications in catalysis, energy harvesting and storage, electronics, antimicrobials and biomedical therapy. Furthermore, a step-by-step flow chart is presented to aid the design and biosynthesis of inorganic NMs employing microbial cells. Future research in this area will add to the diversity of available inorganic NMs but should also address scalability and purity. The biosynthesis of inorganic nanomaterials in microorganisms is an environmentally friendly alternative to chemical synthesis. This Review describes the engineering of microorganisms to rationally prepare nanomaterials for diverse applications.

Biosynthesis of inorganic nanomaterials using microbial cells and bacteriophages

Magnetotactic bacteria (MTB) biomineralize magnetosomes, which are defined as intracellular nanocrystals of the magnetic minerals magnetite (Fe3O4) or greigite (Fe3S4) enveloped by a phospholipid bilayer membrane The synthesis of magnetosomes is controlled by a specific set of genes that encode proteins, some of which are exclusively found in the magnetosome membrane in the cell Over the past several decades, interest in nanoscale technology (nanotechnology) and biotechnology has increased significantly due to the development and establishment of new commercial, medical and scientific processes and applications that utilize nanomaterials, some of which are biologically derived One excellent example of a biological nanomaterial that is showing great promise for use in a large number of commercial and medical applications are bacterial magnetite magnetosomes Unlike chemically-synthesized magnetite nanoparticles, magnetosome magnetite crystals are stable single-magnetic domains and are thus permanently magnetic at ambient temperature, are of high chemical purity, and display a narrow size range and consistent crystal morphology These physical/chemical features are important in their use in biotechnological and other applications Applications utilizing magnetite-producing MTB, magnetite magnetosomes and/or magnetosome magnetite crystals include and/or involve bioremediation, cell separation, DNA/antigen recovery or detection, drug delivery, enzyme immobilization, magnetic hyperthermia and contrast enhancement of magnetic resonance imaging Metric analysis using Scopus and Web of Science databases from 2003 to 2018 showed that applied research involving magnetite from MTB in some form has been focused mainly in biomedical applications, particularly in magnetic hyperthermia and drug delivery

Applications of Magnetotactic Bacteria, Magnetosomes and Magnetosome Crystals in Biotechnology and Nanotechnology: Mini-Review.

The unique extreme abiotic conditions of Antarctic environments like cold, high salinity, and ultraviolet light incidence shaped the diversity and metabolic capability of marine and continental microbial communities in this heterogeneous environment. Besides the vast diversity of psychrophilic/psychrotolerant microorganism inhabiting continental as well as the marine environment in Antarctica, the challenging access to this continent contributes for the slow advances in the characterization of the inhabiting populations/communities of living beings. Therefore the bioprospection of microorganisms in this extreme environment might reveal novel biological processes, which are applicable in several biotechnological/industrial approaches. In this chapter, we focus on Antarctic microorganisms–derived enzymes and give some insights about microbial-related products, such as drugs, antifreezing biomolecules, and nanoparticles.

Antarctic microorganisms as sources of biotechnological products

Nanotechnological developments, including fabrication and use of magnetic nanomaterials, are growing at a fast pace. Magnetic nanoparticles are exciting tools for use in healthcare, biological sensors, and environmental remediation. Due to better control over final-product characteristics and cleaner production, biogenic nanomagnets are preferable over synthetic ones for technological use. In this sense, the technical requirements and economic factors for setting up industrial production of magnetotactic bacteria (MTB)-derived nanomagnets were studied in the present work. Magnetite fabrication costs in a single-stage fed-batch and a semicontinuous process were US$ 10,372 and US$ 11,169 per kilogram, respectively. Depending on the variations of the production process, the minimum selling price for biogenic nanomagnets ranged between US$ 21 and US$ 120 per gram. Because these prices are consistently below commercial values for synthetic nanoparticles, we suggest that microbial production is competitive and constitutes an attractive alternative for a greener manufacturing of magnetic nanoparticles nanotools with versatile applicability.

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Why Does Not Nanotechnology Go Green? Bioprocess Simulation and Economics for Bacterial-Origin Magnetite Nanoparticles.

Magnetotactic bacteria, which synthesize biological magnetite nanoparticles (BMs), are the main microbial source of magnetic nanomaterials. Although the use of BMs has been explored in vitro and in vivo for new anticancer formulations, targeted treatments of fungal and parasitic diseases would also benefit from biogenic magnetic nanoformulations. Due to the necessity of new formulations of amphotericin B, we developed a magnetic-nanoparticle based conjugate of this drug using bacterial magnetosomes. Different amphotericin B preparations were obtained using BMs extracted from Magnetovibrio blakemorei strain MV-1T as well as glutaraldehyde and poly-L-lysine as linking reagents. The highest capture efficiencies and drug loadings were achieved using 0.1‰ poly-L-lysine as the only linking agent (52.7 ± 2.1%, and 25.3 ± 1.9 μg per 100 μg, respectively) and 0.1‰ poly-L-lysine and glutaraldehyde 12.5% (45.0 ± 5.4%, and 21.6 ± 4.9 μg per 100 μg, respectively). Transmission electron microscopy and infrared spectroscopy analyses confirmed the association of amphotericin B to the BM surface. Moreover, controlled drug release from these nanoparticles was achieved by applying an alternating magnetic field. In this condition the release of amphotericin B in PBS increased approximately four-fold as compared to the release under standard conditions with no applied magnetic fields. Hence, the functionalization of BMs with amphotericin B produces stable nanoformulations with a controllable drug release profile, thus, enabling its potential in the treatment of fungal and parasitic diseases.

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A rapid and simple preparation of amphotericin B-loaded bacterial magnetite nanoparticles

Biomineralization in the microbial realm usually gives origin to finely structured inorganic nanomaterials. Perhaps, one of the most elegant bioinorganic processes found in nature is the iron biomineralization into magnetosomes, which is performed by magnetotactic bacteria. A magnetosome gene cluster within the bacterial genome precisely regulates the mineral synthesis. The spread and evolution of this ability among bacteria are thought to be a 2,7-billion-year process mediated by horizontal gene transfers. The produced magnetite or greigite nanocrystals coated by a biological membrane have a narrow diameter dispersibility, a highly precise morphology, and a permanent magnetic dipole due to the molecular level control. Approaches inspired by this bacterial biomineralization mechanism can imitate some of the biogenic nanomagnets characteristics in the chemical synthesis of iron oxide nanoparticles. Thus, this chapter will give a concise overview of magnetosome synthesis’s main steps, some hypotheses about the evolution of magnetosomes’ biomineralization, and approaches used to mimic this biological phenomenon in vitro.

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Tarcisio Correa

Papers

Applications of Magnetotactic Bacteria, Magnetosomes and Magnetosome Crystals in Biotechnology and Nanotechnology: Mini-Review.

Antarctic microorganisms as sources of biotechnological products

Why Does Not Nanotechnology Go Green? Bioprocess Simulation and Economics for Bacterial-Origin Magnetite Nanoparticles.

A rapid and simple preparation of amphotericin B-loaded bacterial magnetite nanoparticles

Biomineralization of Magnetosomes: Billion-Year Evolution Shaping Modern Nanotools