Interactions between prokaryotic polysaccharides and colloidal magnetic nanoparticles for bacteria removal: A strategy for circumventing antibiotic resistance.

Highly stable, colloidal iron oxide nanoparticles with an oxyhydroxide-like surface were used as bacteria-capturing nano-baits. Peptidoglycan isolated from Listeria spp was used as bacteria polysaccharide model, and the nanoparticle binding was characterized showing a Langmuir isotherm constant, KL, equal to 50 ± 3 mL mg-1. The chemical affinity was further supported by dynamic light scattering, transmission electron microscopy, and infrared and UV-Vis data, pointing at the occurrence of extended, coordinative multiple point bindings. The interaction with Gram (+) (Listeria spp) and Gram (-) (Aeromonas veronii) bacteria was shown to be effective and devoid of any toxic effect. Moreover, a real sample, containing a population of several oligotrophic bacteria strains, was incubated with 1 g L-1 of nanoparticle suspension, in the absence of agitation, showing a 100 % capture efficiency, according to plate count. A nanoparticle regeneration method was developed, despite the known irreversibility of such bacterial-nanosurface binding, restoring the bacteria capture capability. This nanomaterial represents a competitive option to eliminate microbiological contamination in water as an alternative strategy to antibiotics, aimed at reducing microbial resistance dissemination. Finally, beyond their excellent features in terms of colloidal stability, binding performances, and biocompatibility this nanoparticle synthesis is cost effective, scalable, and environmentally sustainable.

Chiroplasmonic DNA Scaffolded "Fusilli" Structures.

DNA is an ideal template for the design of nanoarchitectures with molecular-like features. Here, we present an optimized assembly strategy for the concatenation of DNA quasi-rings into long scaffolds. Ionic strength, which played a major role during self-assembly, produced the expected high quality only at 15 mM MgCl2. Atomic force microscopy (AFM) characterization showed several micrometer long tubular structures that were used as templates for the positioning of plasmonic nanoparticles (NPs) along a three-dimensional helical path using DNA tethers. As imaged by high-resolution scanning transmission electron microscopy (HR-STEM) and modeled by theoretical calculations, the NPs distributed into a "fusilli" fashion (i.e., a helical pasta shape), displaying chiroptical activity as revealed by a bisignated CD absorption, centered at the plasmon resonance wavelength. The present structures contribute to enrich the ever-developing arena of chiroplasmonic DNA-based nanomaterials and demonstrate that large assemblies are attainable for their future application to develop metamaterials.

Special Issue "Bio-Nano Interactions 2.0".

In 1961, USA's blues legend Howlin' Wolf released the single entitled "Down in the Bottom" (Figure 1) [...].

Highly specific colloidal ɣ-Fe2O3-DNA hybrids: From bioinspired recognition to large-scale lactoferrin purification.

The industry transfer of laboratory-use magnetic separation is still hampered by the lack of suitable nanoparticles, both in terms of their features and large-scale availability. Surface Active Maghemite Nanoparticles (SAMNs) characterized by a unique surface chemistry, low environmental impact, scalable synthesis and functionalization were used to develop a bio-inspired lactoferrin (LF) recognition system. Based on the LF affinity for DNA, a self-assembly process was optimized for obtaining a SAMN@DNA hybrid displaying chemical and colloidal stability and LF specificity. SAMN@DNA was successfully tested for the affinity purification of LF from crude bovine whey. Advantages, such as high selectivity and loading capacity, nanoparticle re-usability, outstanding purity (96 ± 1%), preservation of protein conformation and short operational time, were highlighted. Finally, scalability was demonstrated by an automatic system performing continuous purification of LF from 100 liters day-1 of whey. This study responds to essential prerequisites, such as efficiency, re-usability and industrialization feasibility.

Spermine Oxidase-Substrate Electrostatic Interactions: The Modulation of Enzyme Function by Neighboring Colloidal ɣ-Fe2O3.

Protein-nanoparticle hybridization can ideally lead to novel biological entities characterized by emerging properties that can sensibly differ from those of the parent components. Herein, the effect of ionic strength on the biological functions of recombinant His-tagged spermine oxidase (i.e., SMOX) was studied for the first time. Moreover, SMOX was integrated into colloidal surface active maghemite nanoparticles (SAMNs) via direct self-assembly, leading to a biologically active nano-enzyme (i.e., SAMN@SMOX). The hybrid was subjected to an in-depth chemical-physical characterization, highlighting the fact that the protein structure was perfectly preserved. The catalytic activity of the nanostructured hybrid (SAMN@SMOX) was assessed by extracting the kinetics parameters using spermine as a substrate and compared to the soluble enzyme as a function of ionic strength. The results revealed that the catalytic function was dominated by electrostatic interactions and that they were drastically modified upon hybridization with colloidal ɣ-Fe2O3. The fact that the affinity of SMOX toward spermine was significantly higher for the nanohybrid at low salinity is noteworthy. The present study supports the vision of using protein-nanoparticle conjugation as a means to modulate biological functions.

Integration of DNA-RNA-triplex-based regulation of transcription into molecular logic gates.

In recent years, increasing numbers of noncoding RNA molecules were identified as possible components of endogenous DNA-RNA hybrid triplexes involved in gene regulation. Triplexes are potentially involved in complex molecular signaling networks that, if understood, would allow the engineering of biological computing components. Here, by making use of the enhancing and inhibiting effects of such triplexes, we demonstrate in vitro the construction of triplex-based molecular gates: 'exclusive OR' (XOR), 'exclusive NOT-OR' (XNOR), and a threshold gate, via transcription of a fluorogenic RNA aptamer. Precise modulation was displayed by the biomolecular-integrated systems over a wide interval of transcriptional outputs, ranging from drastic inhibition to significant enhancement. The present contribution represents a first example of molecular gates developed using DNA-RNA triplex nanostructures.

Rational design of hybrid DNA-RNA triplex structures as modulators of transcriptional activity in vitro.

Triplex nanostructures can be formed in vitro in the promoter region of DNA templates, and it is commonly accepted that these assemblies inhibit the transcription of the downstream genes. Herein, a proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA-RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.

Crossing the Borders of Nanomedicine.

Nanomedicine, being pressured by the increasing demands for fighting menacing diseases such as cancer, relies pragmatically on consolidated knowledge, namely on therapeutic strategies that are at an advanced stage of experimentation [...].

Acidic Shift of Optimum pH of Bovine Serum Amine Oxidase upon Immobilization onto Nanostructured Ferric Tannates.

Protein-nanoparticle hybrids represent entities characterized by emerging biological properties that can significantly differ from those of the parent components. Herein, bovine serum amine oxidase (i.e., BSAO) was immobilized onto a magnetic nanomaterial constituted of surface active maghemite nanoparticles (i.e., SAMNs, the core), surface-modified with tannic acid (i.e., TA, the shell), to produce a biologically active ternary hybrid (i.e., SAMN@TA@BSAO). In comparison with the native enzyme, the secondary structure of the immobilized BSAO responded to pH variations sensitively, resulting in a shift of its optimum activity from pH 7.2 to 5.0. Conversely, the native enzyme structure was not influenced by pH and its activity was affected at pH 5.0, i.e., in correspondence with the best performances of SAMN@TA@BSAO. Thus, an extensive NMR study was dedicated to the structure-function relationship of native BSAO, confirming that its low activity below pH 6.0 was ascribable to minimal structural modifications not detected by circular dichroism. The generation of cytotoxic products, such as aldehydes and H2O2, by the catalytic activity of SAMN@TA@BSAO on polyamine oxidation is envisaged as smart nanotherapy for tumor cells. The present study supports protein-nanoparticle conjugation as a key for the modulation of biological functions.

Colorimetric Kit for Rapid Porcine Circovirus 2 (PCV-2) Diagnosis.

The aim of the current study is to present a low-cost and easy-to-interpret colorimetric kit used to diagnose porcine circovirus 2 (PCV-2) to the naked eye, without any specific equipment. The aforementioned kit used as base hybrid nanoparticles resulting from the merge of surface active maghemite nanoparticles and gold nanoparticles, based on the deposition of specific PCV-2 antibodies on their surface through covalent bonds. In total, 10 negative and 40 positive samples (≥102 DNA copies/µL of serum) confirmed by qPCR technique were tested. PCV-1 virus, adenovirus, and parvovirus samples were tested as interferents to rule out likely false-positive results. Positive samples showed purple color when they were added to the complex, whereas negative samples showed red color; they were visible to the naked eye. The entire color-change process took place approximately 1 min after the analyzed samples were added to the complex. They were tested at different dilutions, namely pure, 1:10, 1:100, 1:1000, and 1:10,000. Localized surface plasmon resonance (LSPR) and transmission electron microscopy (TEM) images were generated to validate the experiment. This new real-time PCV-2 diagnostic methodology emerged as simple and economic alternative to traditional tests since the final price of the kit is USD 4.00.

An Iron Shield to Protect Epigallocatehin-3-Gallate from Degradation: Multifunctional Self-Assembled Iron Oxide Nanocarrier Enhances Protein Kinase CK2 Intracellular Targeting and Inhibition.

Protein kinase CK2 is largely involved in cell proliferation and apoptosis and is generally recognized as an Achilles' heel of cancer, being overexpressed in several malignancies. The beneficial effects of (-)-epigallocatechin-3-gallate (EGCG) in the prevention and treatment of several diseases, including cancer, have been widely reported. However, poor stability and limited bioavailability hinder the development of EGCG as an effective therapeutic agent. The combination of innovative nanomaterials and bioactive compounds into nanoparticle-based systems demonstrates the synergistic advantages of nanocomplexes as compared to the individual components. In the present study, we developed a self-assembled core-shell nanohybrid (SAMN@EGCG) combining EGCG and intrinsic dual-signal iron oxide nanoparticles (Surface Active Maghemite Nanoparticles). Interestingly, nano-immobilization on SAMNs protects EGCG from degradation, preventing its auto-oxidation. Most importantly, the nanohybrid was able to successfully deliver EGCG into cancer cells, displaying impressive protein kinase CK2 inhibition comparable to that obtained with the most specific CK2 inhibitor, CX-4945 (5.5 vs. 3 µM), thus promoting the phytochemical exploitation as a valuable alternative for cancer therapy. Finally, to assess the advantages offered by nano-immobilization, we tested SAMN@EGCG against Pseudomonas aeruginosa, a Gram-negative bacterium involved in severe lung infections. An improved antimicrobial effect with a drastic drop of MIC from 500 to 32.7 µM was shown.

Toward the Specificity of Bare Nanomaterial Surfaces for Protein Corona Formation.

Aiming at creating smart nanomaterials for biomedical applications, nanotechnology aspires to develop a new generation of nanomaterials with the ability to recognize different biological components in a complex environment. It is common opinion that nanomaterials must be coated with organic or inorganic layers as a mandatory prerequisite for applications in biological systems. Thus, it is the nanomaterial surface coating that predominantly controls the nanomaterial fate in the biological environment. In the last decades, interdisciplinary studies involving not only life sciences, but all branches of scientific research, provided hints for obtaining uncoated inorganic materials able to interact with biological systems with high complexity and selectivity. Herein, the fragmentary literature on the interactions between bare abiotic materials and biological components is reviewed. Moreover, the most relevant examples of selective binding and the conceptualization of the general principles behind recognition mechanisms were provided. Nanoparticle features, such as crystalline facets, density and distribution of surface chemical groups, and surface roughness and topography were encompassed for deepening the comprehension of the general concept of recognition patterns.

Environmental implications of one-century COPRs evolution in a single industrial site: From leaching impact to sustainable remediation of CrVI polluted groundwater.

The Stoppani factory manufactured chromium for more than one century, dumping millions of tons of Chromite Ore Processing Residues (COPRs) over decades. The massive presence of COPRs resulted in an intense CrVI leaching and consequent contamination of percolating groundwater. The site offers a unique opportunity to follow COPRs evolution from the primary roasting process to the aged Cr-bearing mineral phases. Herein, new insights on COPRs mineralogy evolution and their role in CrVI release are provided by a dry sample preparation protocol, coupled with in-depth multi-technique characterization. Besides typical COPRs mineral assemblages, highly soluble Na2CrO4 and the first evidence of crocoite (PbCrO4) in a COPR contaminated site are revealed. Selective extraction experiments confirmed a strong reactivity for Cr-bearing minerals as confirmed by concentrations as high as 375 mg L-1 of leached CrVI. The mineralogical approach was combined with a nanotechnological solution for CrVI wastewater remediation. The application of naked colloidal maghemite (γ-Fe2O3) nanoparticles (SAMNs) on the complex industrial wastewater, led to > 90% CrVI removal, either under acidic or in-situ conditions. The present case study of a highly polluted site, ranging from mineral characterization to wastewater remediation, highlights the use of multidisciplinary approaches to cope with complex environmental issues.

Colloidal Iron Oxide Formulation for Equine Hoof Disinfection.

The presence of bacteria of various origins on horse hoofs enables the onset of infections following trauma or even post-surgical wounds. Thus, the analysis of new antibacterial substances is of fundamental importance. In this study, the antibacterial efficacy of Iron Animals (IA), a stable colloidal suspension of iron oxide, organic acids, and detergents, was tested in vitro and in vivo. In vitro assays were performed to test the unspecific inhibitory effect of IA on both gram-positive and gram-negative bacteria monitoring the microorganism growth by spectrophotometry (optical density OD600) at 37 °C for 24 h. In vivo test consists on the quantification of the bacterial load in colony forming units per gram (CFU/g) of specimens collected from the frog region of the anterior hooves of 11 horses. Sampling followed the application of four disinfectant protocols consisting of two consecutive 3 min scrubs with 50 mL of 10% Povidone-iodine (PI) or 4% Chlorhexidine (CHx), with or without an additional application for 15 min of 10 mL of Iron Animals (PI+IA and CHx+IA). In vitro, IA completely suppressed the bacterial growth of all the tested microorganisms, resulting in effectiveness also against CHx-resistant bacteria, such as Staphylococcus aureus. In vivo, PI emerged as an ineffective protocol; CHx was effective in 18% of cases, but with the addition of IA (CHx + IA) its use emerged as the best disinfectant protocol for horse hoof, achieving the lowest bacterial load in 55% of cases. The addition of IA, after PI or CHx, improves the effectiveness of both disinfectants leading to the highest bactericidal activity in 82% of cases.

Intracellular protein kinase CK2 inhibition by ferulic acid-based trimodal nanodevice.

Protein kinase CK2, a pleiotropic and constitutively active kinase, is strictly involved in different diseases, especially in cancer. Many efforts have been carried out to develop specific CK2 inhibitors and recently, it has been evidenced that ferulic acid (FA) represents a promising, albeit cell impermeable, CK2 inhibitor. In the present study, the potential of a nanotechnological approach to cope with intracellular CK2 regulation was explored. Surface-Active Maghemite Nanoparticles (SAMNs), coupling magnetism with photoluminescence, a new feature of SAMNs here described for the first time, were chosen as dual imaging nanocarrier for FA. The self-assembled nanodevice (SAMN@FA) displayed a significant CK2 inhibitory activity in vitro. Moreover, effective cellular internalization of SAMN@FA in cancer cells was proved by direct visualization of the photoluminescent nanocarrier by confocal microscopy and was corroborated by phosphorylation levels of endogenous CK2 targets. The proposed trimodal nanodevice, representing the first example of cellular CK2 nano-inhibition, paves the way for novel active nanocarriers as appealing theranostic tool for future biomedical applications.

Role of carboxylic group pattern on protein surface in the recognition of iron oxide nanoparticles: A key for protein corona formation.

The knowledge of protein-nanoparticle interplay is of crucial importance to predict the fate of nanomaterials in biological environments. Indeed, protein corona on nanomaterials is responsible for the physiological response of the organism, influencing cell processes, from transport to accumulation and toxicity. Herein, a comparison using four different proteins reveals the existence of patterned regions of carboxylic groups acting as recognition sites for naked iron oxide nanoparticles. Readily interacting proteins display a distinctive surface distribution of carboxylic groups, recalling the geometric shape of an ellipse. This is morphologically complementary to nanoparticles curvature and compatible with the topography of exposed FeIII sites laying on the nanomaterial surface. The recognition site, absent in non-interacting proteins, promotes the nanoparticle harboring and allows the formation of functional protein coronas. The present work envisages the possibility of predicting the composition and the biological properties of protein corona on metal oxide nanoparticles.

Smart synthetic maghemite nanoparticles with unique surface properties encode binding specificity toward AsIII.

Pristine ɣ-Fe2O3 nanoparticles, called surface active maghemite nanoparticles (SAMNs) display unprecedented colloidal stability and specific binding properties. Herein, the interactions of SAMNs with AsV and AsIII as surface molecular probes were comparatively studied. Thermodynamic and kinetic characterizations, along with chemical and structural analysis of SAMN@As complexes, evidenced two distinct binding modalities. Arsenite, emerged as an elective and specific ligand for SAMNs, whereas arsenate adsorption was more labile, pH dependent and ruled by different binding possibilities. In particular, AsIII oxyacid exclusively interacts through inner-sphere coordination occupying available surface crystal positions resembling a key-lock fitting, while AsV leads to both outer-sphere and inner-sphere complexes. Noteworthy, discrimination between AsV and AsIII was never reported for nanostructured maghemite evidencing the importance of synthetic route on surface properties of the nanomaterial. The present report, besides enriching the chemistry of nanosized iron oxides, suggests SAMNs application for the remediation of water contaminated by AsIII, the most threatening As species in water.

Nanotechnology-Based Strategies to Develop New Anticancer Therapies.

The blooming of nanotechnology has made available a limitless landscape of solutions responding to crucial issues in many fields and, nowadays, a wide choice of nanotechnology-based strategies can be adopted to circumvent the limitations of conventional therapies for cancer. Herein, the current stage of nanotechnological applications for cancer management is summarized encompassing the core nanomaterials as well as the available chemical-physical approaches for their surface functionalization and drug ligands as possible therapeutic agents. The use of nanomaterials as vehicles to delivery various therapeutic substances is reported emphasizing advantages, such as the high drug loading, the enhancement of the pay-load half-life and bioavailability. Particular attention was dedicated to highlight the importance of nanomaterial intrinsic features. Indeed, the ability of combining the properties of the transported drug with the ones of the nano-sized carrier can lead to multifunctional theranostic tools. In this view, fluorescence of carbon quantum dots, optical properties of gold nanoparticle and superparamagnetism of iron oxide nanoparticles, are fundamental examples. Furthermore, smart anticancer devices can be developed by conjugating enzymes to nanoparticles, as in the case of bovine serum amine oxidase (BSAO) and gold nanoparticles. The present review is aimed at providing an overall vision on nanotechnological strategies to face the threat of human cancer, comprising opportunities and challenges.

Enzyme Immobilization on Maghemite Nanoparticles with Improved Catalytic Activity: An Electrochemical Study for Xanthine.

Generally, enzyme immobilization on nanoparticles leads to nano-conjugates presenting partially preserved, or even absent, biological properties. Notwithstanding, recent research demonstrated that the coupling to nanomaterials can improve the activity of immobilized enzymes. Herein, xanthine oxidase (XO) was immobilized by self-assembly on peculiar naked iron oxide nanoparticles (surface active maghemite nanoparticles, SAMNs). The catalytic activity of the nanostructured conjugate (SAMN@XO) was assessed by optical spectroscopy and compared to the parent enzyme. SAMN@XO revealed improved catalytic features with respect to the parent enzyme and was applied for the electrochemical studies of xanthine. The present example supports the nascent knowledge concerning protein conjugation to nanoparticle as a means for the modulation of biological activity.

Nano-immobilized flumequine with preserved antibacterial efficacy.

Flumequine was nano-immobilized by self-assembly on iron oxide nanoparticles, called surface active maghemite nanoparticles (SAMNs). The binding process was studied and the resulting core-shell nanocarrier (SAMN@FLU) was structurally characterized evidencing a firmly immobilized organic canopy on which the fluorine atom of the antibiotic was exposed to the solvent. The antibiotic efficacy of the SAMN@FLU nanocarrier was tested on a fish pathogenic bacterium (Aeromonas veronii), a flumequine sensitive strain, in comparison to soluble flumequine and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were assessed. Noteworthy, the MIC and MBC of soluble and nanoparticle bound drug were superimposable. Moreover, the interactions between SAMN@FLU nanocarrrier and microorganism were studied by transmission electron microscopy evidencing the ability of the complex to disrupt the bacterial wall. Finally, a preliminary in vivo test was provided using Daphnia magna as animal model. SAMN@FLU was able to protect the crustacean from the fatal consequences of a bacterial infection and showed no sign of toxicity. Thus, in contrast with the strength of the interaction, nano-immobilized FLU displayed a fully preserved antimicrobial activity suggesting the crucial role of fluorine in the drug mechanism of action. Besides the importance for potential applications in aquaculture, the present study contributes to the nascent field of nanoantibiotics.