Two-Sided Antibacterial Cellulose Combining Probiotics and Silver Nanoparticles.

The constant increase of antibiotic-resistant bacteria demands the design of novel antibiotic-free materials. The combination of antibacterials in a biocompatible biomaterial is a very promising strategy to treat infections caused by a broader spectrum of resistant pathogens. Here, we combined two antibacterials, silver nanoparticles (AgNPs) and living probiotics (Lactobacillus fermentum, Lf), using bacterial cellulose (BC) as scaffold. By controlling the loading of each antibacterial at opposite BC sides, we obtained a two-sided biomaterial (AgNP-BC-Lf) with a high density of alive and metabolically active probiotics on one surface and AgNPs on the opposite one, being probiotics well preserved from the killer effect of AgNPs. The resulting two-sided biomaterial was characterized by Field-Emission Scanning Electron Microscopy (FESEM) and Confocal Laser Scanning Microscopy (CLSM). The antibacterial capacity against Pseudomonas aeruginosa (PA), an opportunistic pathogen responsible for a broad range of skin infections, was also assessed by agar diffusion tests in pathogen-favorable media. Results showed an enhanced activity against PA when both antibacterials were combined into BC (AgNP-BC-Lf) with respect to BC containing only one of the antibacterials, BC-Lf or AgNP-BC. Therefore, AgNP-BC-Lf is an antibiotic-free biomaterial that can be useful for the therapy of topical bacterial infections.

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.

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.

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.

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.

Chemically and biologically harmless versus harmful ferritin/copper-metallothionein couples.

The simultaneous measurement of the decrease of available Fe(II) ions and the increase of available Fe(III) ions allowed the analysis of the ferroxidase activity of two distinct apoferritins. Although recombinant human apoferritin (HuFtH) rapidly oxidizes Fe(II) to Fe(III) , this iron is not properly stored in the ferritin cavity, as otherwise occurs in horse-spleen H/L-apoferritin (HsFt; H=heavy subunit, L=light subunit). Iron storage in these apoferritins was also studied in the presence of two copper-loaded mammalian metallothioneins (MT2 and MT3), a scenario that occurs in different brain-cell types. For HuFtH, unstored Fe(III) ions trigger the oxidation of Cu-MT2 with concomitant Cu(I) release. In contrast, there is no reaction with Cu-MT2 in the case of HsFt. Similarly, Cu-MT3 does not react during either HuFtH or HsFt iron reconstitution. Significantly, the combination of ferritin and metallothionein isoforms reported in glia and neuronal cells are precisely those combinations that avoid a harmful release of Fe(II) and Cu(I) ions.

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.

Monitoring lactoferrin iron levels by fluorescence resonance energy transfer: a combined chemical and computational study.

Three forms of lactoferrin (Lf) that differed in their levels of iron loading (Lf, LfFe, and LfFe2) were simultaneously labeled with the fluorophores AF350 and AF430. All three resulting fluorescent lactoferrins exhibited fluorescence resonance energy transfer (FRET), but they all presented different FRET patterns. Whereas only partial FRET was observed for Lf and LfFe, practically complete FRET was seen for the holo form (LfFe2). For each form of metal-loaded lactoferrin, the AF350-AF430 distance varied depending on the protein conformation, which in turn depended on the level of iron loading. Thus, the FRET patterns of these lactoferrins were found to correlate with their iron loading levels. In order to gain greater insight into the number of fluorophores and the different FRET patterns observed (i.e., their iron levels), a computational analysis was performed. The results highlighted a number of lysines that have the greatest influence on the FRET profile. Moreover, despite the lack of an X-ray structure for any LfFe species, our study also showed that this species presents modified subdomain organization of the N-lobe, which narrows its iron-binding site. Complete domain rearrangement occurs during the LfFe to LfFe2 transition. Finally, as an example of the possible applications of the results of this study, we made use of the FRET fingerprints of these fluorescent lactoferrins to monitor the interaction of lactoferrin with a healthy bacterium, namely Bifidobacterium breve. This latter study demonstrated that lactoferrin supplies iron to this bacterium, and suggested that this process occurs with no protein internalization.

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.

Living Cellulose Materials with Tunable Viscoelasticity through Probiotic Proliferation.

Probiotic cellulose (PC), a living material (LM) consisting of probiotics integrated into bacterial cellulose, is the first example where life (probiotic proliferation) is the input to tune the viscoelasticity of the biomaterial. The gradual proliferation of probiotics within the matrix acts as a key modulator of the cellulose viscoelasticity, providing from celluloses with lower-than-matrix viscoelasticity to celluloses with viscoelastic moduli closer to those of elastic solids. This concept is a promising approach to producing living bio-ink with tunable viscoelastic response of special interest for specific applications such as 3D printing. In contrast to the most common hydrogels with stimuli-tunable mechanical properties, which require external stimuli such as mechanical stress, UV radiation, or heat, this living bio-ink only requires time to tune from a fluid-like into a solid-like biomaterial.

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.

Repairing and Preventing Photooxidation of Few-Layer Black Phosphorus with ß-Carotene.

Few-layer black phosphorus (FLBP), a technologically important 2D material, faces a major hurdle to consumer applications: spontaneous degradation under ambient conditions. Blocking the direct exposure of FLBP to the environment has remained the key strategy to enhance its stability, but this can also limit its utility. In this paper, a more ambitious approach to handling FLBP is reported where not only is FLBP oxidation blocked, but it is also repaired postoxidation. Our approach, inspired by nature, employs the antioxidant molecule ß-carotene that protects plants against photooxidative damages to act as a protecting and repairing agent for FLBP. The mechanistic role of ß-carotene is established by a suite of spectro-microscopy techniques, in combination with computational studies and biochemical assays. Transconductance studies on FLBP-based field effect transistor (FET) devices further affirm the protective and reparative effects of ß-carotene. The outcomes indicate the potential for deploying a plethora of natural antioxidant molecules to enhance the stability of other environmentally sensitive inorganic nanomaterials and expedite their translation for technological and consumer applications.

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.

Metal-dependent glycosylation in recombinant metallothioneins.

We show for the first time glycosylation of recombinant metallothioneins (MTs) produced in E. coli. Interestingly, our results show that the glycosylation level of the recombinant MTs is inversely proportional to the degree of protein structuration, and reflects their different metal preferences.

Colon cancer therapy with calcium phosphate nanoparticles loading bioactive compounds from Euphorbia lathyris: In vitro and in vivo assay.

Amorphous calcium phosphate nanoparticles (ACP NPs) exhibit excellent biocompatibility and biodegradability properties. ACP NPs were functionalized with two coumarin compounds (esculetin and euphorbetin) extracted from Euphorbia lathyris seeds (BC-ACP NPs) showing high loading capacity (0.03% and 0.34% (w/w) for esculetin and euphorbetin, respectively) and adsorption efficiency (2.6% and 33.5%, respectively). BC-ACP NPs, no toxic to human blood cells, showed a more selective cytotoxicity against colorectal cancer (CRC) cells (T-84 cells) (IC50, 71.42 µg/ml) compared to non-tumor (CCD18) cells (IC50, 420.77 µg/ml). Both, the inhibition of carbonic anhydrase and autophagic cell death appeared to be involved in their action mechanism. Interestingly, in vivo treatment with BC-ACPs NPs using two different models of CRC induction showed a significant reduction in tumor volume (62%) and a significant decrease in the number and size of polyps. A poor development of tumor vasculature and invasion of normal tissue were also observed. Moreover, treatment increased the bacterial population of Akkermansia by restoring antioxidant systems in the colonic mucosa of mice. These results show a promising pathway to design innovative and more efficient therapies against CRC based on biomimetic calcium phosphate NPs loaded with natural products.

Magnetic nanoparticles--templated assembly of protein subunits: a new platform for carbohydrate-based MRI nanoprobes.

A new approach for the preparation of carbohydrate-coated magnetic nanoparticles is reported. In a first step, we show that the pH-driven assembly-disassembly natural process that occurs in apoferritin protein is effective for the encapsulation of maghemite nanoparticles of different sizes: 4 and 6 nm. In a second step, we demonstrate that the presence of functional amine groups in the outer shell of apoferritin allows functionalization with two carbohydrates, N-acetyl-D-glucosamine and d-mannose. High-resolution electron microscopy (HREM), high angle annular dark field scanning electron microscopy (HAADF-STEM), electron energy loss spectroscopy (EELS), X-ray diffraction (XRD), and SQUID technique have been used to characterize the magnetic samples, termed herein Apomaghemites. The in vivo magnetic resonance imaging (MRI) studies showed the efficiency in contrasting images for these samples; that is, the r(2) NMR relaxivities are comparable with Endorem (a commercial superparamagnetic MRI contrast agent). The r(2) relaxivity values as well as the pre-contrast and post-contrast T(2)*-weighted images suggested that our systems could be used as perspective superparamagnetic contrast agents for magnetic resonance imaging (MRI). The carbohydrate-functionalized Apomaghemite nanoparticles retained their recognition abilities, as demonstrated by the strong affinity with their corresponding carbohydrate-binding lectins.

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.

A bioinspired approach to the synthesis of bimetallic CoNi nanoparticles.

Bimetallic CoNi nanoparticles have been prepared within the apoferritin cavity. The protein shell controls size, prevents aggregation, and makes nanoparticles water-soluble. The CoNi series prepared in this way were structurally and magnetically characterized, the resulting magnetic properties varying accordingly with composition (Co(75)/Ni(25), Co(50)/Ni(50), Co(25)/Ni(75)). Co and Ni metals were associated in each nanoparticle, as demonstrated by high-angle annular dark field scanning electron microscopy and electron energy loss spectroscopy (EELS). After intentional oxidation, the CoNi nanoparticles were characterized by EELS, X-ray absorption near edge structure (XANES), and SQUID measurements to evaluate the importance of the oxidation on magnetic properties.

Structural and magnetic characterization of Pd nanoparticles encapsulated in apoferritin.

Pd nanoparticles exhibiting permanent magnetism at room temperature have been prepared within the apoferritin cavity. Pd nanoparticles in air and under an inert atmosphere were synthesized to study the influence of the aerobic and anaerobic conditions in the final magnetic properties. The surface of nanoparticles as well as the type of crystalline phase could determine the magnetic properties. X-ray powder diffraction, including Debye-function analysis, transmission electronic microscopy, and magnetization measurements have been used for characterizing the nanoparticles.