Gold nanoparticle-coated apoferritin conductive nanowires.

Gold-metallic nanofibrils were prepared from three different iso-apoferritin (APO) proteins with different Light/Heavy (L/H) subunit ratios (from 0% up to 100% L-subunits). We show that APO protein fibrils have the ability to in situ nucleate and grow gold nanoparticles (AuNPs) simultaneously assembled on opposite strands of the fibrils, forming hybrid inorganic-organic metallic nanowires. The AuNPs are arranged following the pitch of the helical APO protein fiber. The mean size of the AuNPs was similar in the three different APO protein fibrils studied in this work. The AuNPs retained their optical properties in these hybrid systems. Conductivity measurements showed ohmic behavior like that of a continuous metallic structure.

Magneto-optical hyperthermia agents based on probiotic bacteria loaded with magnetic and gold nanoparticles.

Probiotic bacteria were used as carriers of metallic nanoparticles to develop innovative oral agents for hyperthermia cancer therapy. Two synthetic strategies were used to produce the different therapeutic agents. First, the probiotic bacterium Lactobacillus fermentum was simultaneously loaded with magnetic (MNPs) and gold nanoparticles (AuNPs) of different morphologies to produce AuNP + MNP-bacteria systems with both types of nanoparticles arranged in the same layer of bacterial exopolysaccharides (EPS). In the second approach, the probiotic was first loaded with AuNP to form AuNP-bacteria and subsequently loaded with MNP-EPS to yield AuNP-bacteria-EPS-MNP with the MNP and AuNP arranged in two different EPS layers. This second strategy has never been reported and exploits the presence of EPS-EPS recognition which allows the layer-by-layer formation of structures on the bacteria external wall. The AuNP + MNP-bacteria and AuNP-bacteria-EPS-MNP samples were characterized by scanning (SEM) and transmission electron microscopy (TEM), and UV-vis spectroscopy. The potential of these two heterobimetallic systems as magnetic hyperthermia or photothermal therapy agents was assessed, validating their capacity to produce heat either during exposure to an alternating magnetic field or near-infrared laser light. The probiotic Lactobacillus fermentum has already been proposed as an oral drug carrier, able to overcome the stomach medium and deliver drugs to the intestines, and it is actually marketed as an oral supplement to reinforce the gut microbiota, thus, our results open the way for the development of novel therapeutic strategies using these new heterobimetallic AuNP/MNP-bacteria systems in the frame of gastric diseases, using them, for example, as oral agents for cancer treatment with magnetic hyperthermia and photothermal therapy.

Understanding the Formation of Apoferritin Amyloid Fibrils.

We present the optimization of experimental conditions to yield long, rigid apoferritin protein amyloid fibrils, as well as the corresponding fibrillation pathway. Fibril growth kinetics was followed using atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), circular dichroism (CD), fourier-transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among the morphologies identified, we show that the conditions result in small aggregates, as well as medium and long fibrils. Extended incubation times led to progressive unfolding and hydrolysis of the proteins into very short peptide fragments. AFM, SDS-PAGE, and CD support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role. These collective results provide convincing evidence that protein unfolding and complete hydrolysis of the proteins into very short peptide sequences are essential for the formation of the final apoferritin amyloid-like fibrils.

Probiotic cellulose: Antibiotic-free biomaterials with enhanced antibacterial activity.

The alarming increase of antibiotic-resistant bacteria, causing conventional treatments of bacterial infections to become increasingly inefficient, is one of the biggest threats to global health. Here, we have developed probiotic cellulose, an antibiotic-free biomaterial for the treatment of severe skin infections and chronic wounds. This composite biomaterial was in-depth characterized by Gram stain, scanning electron microscopy (SEM) and confocal fluorescence microscopy. Results demonstrated that probiotic cellulose consists of dense films of cellulose nanofibers, free of cellulose-producing bacteria, completely invaded by live probiotics (Lactobacillus fermentum or Lactobacillus gasseri). Viability assays, including time evolution of pH and reducing capacity against electrochromic polyoxometalate, confirmed that probiotics within the cellulose matrix are not only alive but also metabolically active, a key point for the use of probiotic cellulose as an antibiotic-free antibacterial biomaterial. Antibacterial assays in pathogen-favorable media, a real-life infection scenario, demonstrated that probiotic cellulose strongly reduces the viability of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA), the most active pathogens in severe skin infections and chronic wounds. Likewise, probiotic cellulose was also found to be effective to inhibit the proliferation of methicillin-resistant SA (MRSA). The combination of the properties of bacterial cellulose as wound dressing biomaterial and the antibacterial activity of probiotics makes probiotic cellulose an alternative to antibiotics for the treatment of topical infections, including severe and hard-to-heal chronic wounds. In addition, probiotic cellulose was obtained by a one-pot synthetic approach under mild conditions, not requiring the long and expensive chemical treatments to purify the genuine bacterial cellulose.

Apoferritin Amyloid-Fibril Directed the In Situ Assembly and/or Synthesis of Optical and Magnetic Nanoparticles.

The coupling of proteins that can assemble, recognise or mineralise specific inorganic species is a promising strategy for the synthesis of nanoscale materials with a controllable morphology and functionality. Herein, we report that apoferritin protein amyloid fibrils (APO) have the ability to assemble and/or synthesise various metal and metal compound nanoparticles (NPs). As such, we prepared metal NP-protein hybrid bioconjugates with improved optical and magnetic properties by coupling diverse gold (AuNPs) and magnetic iron oxide nanoparticles (MNPs) to apoferritin amyloid fibrils and compared them to the well-known ß-lactoglobulin (BLG) protein. In a second approach, we used of solvent-exposed metal-binding residues in APO amyloid fibrils as nanoreactors for the in situ synthesis of gold, silver (AgNPs) and palladium nanoparticles (PdNPs). Our results demonstrate, the versatile nature of the APO biotemplate and its high potential for preparing functional hybrid bionanomaterials. Specifically, the use of apoferritin fibrils as vectors to integrate magnetic MNPs or AuNPs is a promising synthetic strategy for the preparation of specific contrast agents for early in vivo detection using various bioimaging techniques.

Magnetic and Golden Yogurts. Food as a Potential Nanomedicine Carrier.

Yogurt is one of the most emblematic and popular fermented foods. It is produced by the fermentation of milk lactose by bacteria such as Streptococcus thermophilus and Lactobacillus acidophilus. Magnetic (MNPs) and gold nanoparticles (AuNPs) were incorporated into the exopolysaccharides (EPSs) of these bacteria. The functionalized bacteria were characterized by UV-vis spectroscopy and transmission electron microscopy. A large number of MNPs and AuNPs were bound to the bacterial EPS. Interestingly, the nanoparticles' (NPs) presence did not affect the bacteria's capacity to ferment milk and to produce magnetic and golden yogurts. Magnetic and golden yogurts represent the perfect combination of emblematic food and nanoparticles and have a range of potential biomedical applications: use in iron-deficiency anemia, diagnosis and hyperthermia treatment of appropriate digestive diseases, and interest in glamour cuisine.

Apoferritin Protein Amyloid Fibrils with Tunable Chirality and Polymorphism.

Ferritin, a soluble and highly robust protein with subunits packed into well-defined helices, is a key component of the iron regulatory system in the brain and thus is widely recognized as a crucial protein for iron metabolism, but may also bear possible implications in some neurodegenerative disorders. Here, we present evidence of how human recombinant apoferritin can convert into an unusual structure from its folded native state; that is, amyloid fibrils analogue to those found in pathological disorders such as Alzheimer's and Parkinson's diseases. An extensive combination of advanced microscopy, spectroscopy and scattering techniques concur to reveal that apoferritin fibrils possess a common double stranded twisted ribbon structure which can result in a mesoscopic right-handed chirality. We highlight a direct connection between the chirality and morphology of the resulting amyloid fibrils, and the initial protein subunits composition, advancing our understanding on the possible role of misfolding in some ferritin-related pathologies and posing new bases for the design of chiral 1D functional nanostructures.

Bacteria-Carried Iron Oxide Nanoparticles for Treatment of Anemia.

The efficiency of maghemite nanoparticles for the treatment of anemia was sensibly higher when nanoparticles were incorporated onto the probiotic bacterium Lactobacillus fermentum (MNP-bacteria) than when administrated as uncoated nanoparticles (MNP). Plasma iron and hemoglobin, intestine expression of divalent metal transporter 1 (DMT1) and duodenal Cytochrome b (DcytB), as well as hepatic expression of the hormone hepcidin were fully restored to healthy levels after administration of MNP-bacteria but not of MNP. A magnetic study on biodistribution and biodegradation showed accumulation of maghemite nanoparticles in intestine lumen when MNP-bacteria were administrated. In contrast, MNP barely reached intestine. In vivo MRI studies suggested the internalization of MNP-bacteria into enterocytes, which did not occur with MNP. Transmission electronic microscopy confirmed this internalization. The collective analysis of results point out that L. fermentum is an excellent carrier to overcome the stomach medium and drive maghemite nanoparticles to intestine, where iron absorption occurs. Due the probiotic ability to adhere to the gut wall, MNP-bacteria internalize into the enterocyte, where maghemite nanoparticles are delivered, providing an adequate iron level into enterocyte. This paper advances a new route for effective iron absorption in the treatment of anemia.

Varying iron release from transferrin and lactoferrin proteins. A laboratory experiment.

Iron metabolism is an important subject of study for undergraduate students of chemistry and biochemistry. Relevant laboratory exercises are scarce in the literature but would be very helpful in assisting students grasp key concepts. The experiment described here deals with different iron release mechanisms of two protagonists in iron metabolism: serum transferrin (Tf) and lactoferrin (Lf). Despite having very similar structures and iron-binding sites, Tf releases practically all its iron at pH 5.5 while Lf requires a significantly lower pH of 3. This difference in behavior is directly related to their respective biological functions as Tf blood-borne iron into the cell, while Lf competes with pathogens to sequester iron in biological fluids at more acidic pHs. During this experiment, the students will carry out iron loading and unloading on both human Lf and Tf and monitor the iron release at different pHs using UV-Vis spectroscopy. With this simple approach, the students will discover the different patterns of iron release of Tf and Lf and how this variance in behavior relates to their biological functions. Furthermore, this laboratory practice can be expanded to allow students to investigate a variety of iron proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):521-527, 2017.

Hybrid Nanoscopy of Hybrid Nanomaterials.

The combination of complementary techniques to characterize materials at the nanoscale is crucial to gain a more complete picture of their structure, a key step to design and fabricate new materials with improved properties and diverse functions. Here it is shown that correlative atomic force microscopy (AFM) and localization-based super-resolution microscopy is a useful tool that provides insight into the structure and emissive properties of fluorescent ß-lactoglobulin (ßLG) amyloid-like fibrils. These hybrid materials are made by functionalization of ßLG with organic fluorophores and quantum dots, the latter being relevant for the production of 1D inorganic nanostructures templated by self-assembling peptides. Simultaneous functionalization of ßLG fibers by QD655 and QD525 allows for correlative AFM and two-color super-resolution fluorescence imaging of these hybrid materials. These experiments allow the combination of information about the topography and number of filaments that compose a fibril, as well as the emissive properties and nanoscale spatial distribution of the attached fluorophores. This study represents an important step forward in the characterization of multifunctionalized hybrid materials, a key challenge in nanoscience.

Identification of the key excreted molecule by Lactobacillus fermentum related to host iron absorption.

We have taken a vital step towards understanding why probiotic bacteria increase iron absorption in the gastrointestinal tract. We show here that Lactobacillus fermentum, one of the main probiotics of the microbiota, exhibits an extraordinary ferric-reducing activity. This activity is predominantly due to an excreted molecule: p-hydroxyphenyllactic acid (HPLA). Reduction of Fe(III) to Fe(II) is essential for iron absorption in the gastrointestinal tract. By reducing Fe(III), HPLA boosts Fe(II) absorption through the DMT1 channels of enterocytes. An in vitro experiment tested and confirmed this hypothesis. This discovery opens new avenues for the treatment of iron deficiency in humans, one of the most common and widespread nutritional disorders in the world.

Iron chemistry at the service of life.

Iron is an essential element for almost all organisms on Earth. It is necessary for a number of crucial processes such as hemoglobin and myoglobin transport and storage of oxygen in mammals; electron transfer support in a variety of iron-sulfur protein or cytochrome reactions; and activation and catalysis of reactions of a wide range of substrate like alkanes, olefins, and alcohols. Living organisms adopted iron as the main metal to carry out all of these functions due to the rich coordination chemistry of its two main redox states, Fe2+ and Fe3+ , and because of its abundance in the Earth's crust and oceans. This paper presents an overview of the coordination chemistry of iron that makes it suitable for a large variety of functions within biological systems. Despite iron's chemical advantages, organisms were forced to manage with some drawbacks: Fe3+ insolubility and the formation of toxic radicals, especially the hydroxyl radical. Iron chemistry within biology is an example of how organisms evolved by creating molecular machinery to overcome these difficulties and perform crucial processes with extraordinary elegance and efficiency. © 2017 IUBMB Life, 69(6):382-388, 2017.

Magnetic study on biodistribution and biodegradation of oral magnetic nanostructures in the rat gastrointestinal tract.

We have undertaken a magnetic study on the oral biodistribution and biodegradation of nude maghemite nanoparticles of 10 nm average size (MNP) and probiotic bacteria, Lactobacillus fermentum, containing thousands of these same nanoparticles (MNP-bacteria). Using AC magnetic susceptibility measurements of the stomach, small intestine, cecum and large intestine obtained after rat sacrifice, and iron content determination by ICP-OES, we have monitored the biodistribution and biodegradation of the maghemite nanoparticles along the gastrointestinal tract, after oral administration of both MNP and MNP-bacteria. The results revealed that the amount of magnetic nanoparticles accumulated in intestines is sensibly higher when MNP-bacteria were administered, in comparison with MNP. This confirms our initial hypothesis that the use of probiotic bacteria is a suitable strategy to assist the magnetic nanoparticles to overcome the stomach medium, and to achieve their accumulation in intestines. This finding opens doors to different applications. Since iron absorption in humans takes place precisely in the intestines, the use of MNP-bacteria as an iron supplement is a definite possibility. We have actually illustrated how the administration of MNP-bacteria to iron-deficient rats corrects the iron levels after two weeks of treatment.

Insights on the (Auto)Photocatalysis of Ferritin.

Traditionally, ferritin has been considered a photocatalyst capable of photo-oxidizing organic molecules and transferring electrons to external electron acceptors when irradiated by UV-visible light. We have designed new approaches to resolve the uncertainties regarding its photocatalytical mechanism. Experiments with an Fe(II) chelator, an electrochromic indicator, and recombinant ferritin proteins indicate that the excited electrons at the conduction band of the ferritin core do not cross the protein shell. Instead, irradiation causes the electrons to reduce the ferrihydrite core to produce Fe(II) ions. These Fe(II) ions exit the protein shell to reduce electron acceptors. In the absence of electron acceptors or chelators, Fe(II) re-enters ferritin.

Apoferritin fibers: a new template for 1D fluorescent hybrid nanostructures.

Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by ß-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials.

Apomaghemite as a doxorubicin carrier for anticancer drug delivery.

Protein cages have well-defined structures and can be chemically and biologically engineered in many ways, making them useful platforms for drug delivery applications. Taking advantage of the unique structure feature of apoferritin, a new theranostic nanocarrier is proposed herein. The apoferritin protein is effective for the encapsulation of maghemite nanoparticles and for loading a significant dose of doxorubicin (DOX) drug. This simultaneous loading of maghemite nanoparticles and DOX has been achieved using either co-encapsulation or surface-binding approaches. Maghemite nanoparticles coated with the protein apoferritin are an effective long-term MRI liver contrast agent and we report here that additionally they can serve as an anticancer drug-delivery system. In particular we show that maghemite-containing apoferritin can sustain the DOX delivery under period of 10 to 25 days depending on the environmental conditions.

Nanopatterning of Magnetic CrNi Prussian Blue Nanoparticles Using a Bacterial S-Layer as a Biotemplate.

We have developed a simple process to fabricate on a bioplatform patterns of nanoparticles of a molecule-based magnet. Nanoparticles of the ferromagnetic Prussian blue derivative CsxNi[Cr(CN)6] were orderly deposited onto S-layers of Lysinibacillus sphaericus, forming a dense carpet of nanoparticles following the square lattice (p4) pattern of the biotemplate. These results are encouraging to extend this approach by focusing on molecule-based magnets patterned into domains with controlled shapes and positions on a biosurface.

Electrochromic polyoxometalate material as a sensor of bacterial activity.

Lactobacillus fermentum, a bacterium of human microbiota, acts as an electron donor for the electrochromic [P2Mo(VI)18O62](6-). Since the reductive capacity of L. fermentum correlates with its metabolic activity, the reaction with [P2Mo(VI)18O62](6-) affords a means of evaluating its activity. Following this logic, we have concluded that vancomycin severely affects the activity of L. fermentum whereas omeprazole does not.

Bioinspired magneto-optical bacteria.

"Two-in-one" magneto-optical bacteria have been produced using the probiotic Lactobacillus fermentum for the first time. We took advantage of two features of bacteria to synthesize this novel and bifunctional nanostructure: their metal-reducing properties, to produce gold nanoparticles, and their capacity to incorporate iron oxide nanoparticles at their external surface. The magneto-optical bacteria survive the process and behave as a magnet at room temperature.

In vivo long-term magnetic resonance imaging activity of ferritin-based magnetic nanoparticles versus a standard contrast agent.

New long-circulating maghemite nanoparticles of 4 and 6 nm, coated with an apoferritin protein capsid, exhibit useful properties to act as magnetic resonance imaging (MRI) contrast agents. A full in vivo study of the so-called apomaghemites reveals that their long-term MRI properties are better than those of a standard superparamagnetic iron oxide (SPIO) widely used in biomedical applications. The biodistribution of apomaghemites and standard SPIO was investigated by MRI in mice at two different concentrations, 6 and 2.5 mg of Fe·kg(-1), over 60 days. Significant differences are found at low dose (2.5 mg of Fe·kg(-1)). Thus, whereas apomaghemites are active for MR bioimaging of liver for 45 days, standard SPIO is not effective beyond 7 days. On the basis of our data, we may concluded that apomaghemites can act as new long-term MRI liver contrast agents, allowing first the diagnosis of a liver pathology and then monitoring after treatment without the need for a second injection.