An accurate spectrophotometric method for chitosan quantification.

Chitosan is a biopolymer obtained from chitin, one of the most abundant biopolymers in nature. Numerous applications of chitosan are well known in the biomedical, environmental, and industrial fields, and the potential applications are considerable. This work reports a new spectrophotometric method to determine chitosan concentration accurately. The method is based on the deamination of chitosan by nitrite in acidic conditions, followed by a carbohydrate determination by the anthrone reagent.

Glucose oxidase virus-based nanoreactors for smart breast cancer therapy.

BACKGROUND: Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects. METHODS AND MAJOR RESULTS: Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome mosaic virus (BMV) and containing the catalytic activity of glucose oxidase (GOx) enzyme. The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactor treatment on triple-negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity. CONCLUSIONS AND IMPLICATIONS: The nanoreactors containing GOx activity are entirely suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors, a strategy designed for selective cancer targeting, showed no improvement in the cytotoxic effect. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are ongoing to reinforce the effectiveness of this treatment strategy.

Antibody-Functionalized Copper Oxide Nanoparticles with Targeted Antibacterial Activity.

Copper oxide nanoparticles (CuO-NPs) were functionalized with specific antibodies to target their antibacterial activity against Gram-positive or Gram-negative bacteria. The CuO-NPs were covalently functionalized to cover their surface with specific antibodies. The differently prepared CuO-NPs were characterized by X-ray diffraction, transmission electron microscopy and dynamic light scattering. The antibacterial activities of the unmodified CuO-NPs and the antibody-functionalized nanoparticles (CuO-NP-AbGram- and CuO-NP-AbGram+ ) were determined for both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria. The antibody-functionalized NPs showed a differential increase of their antibacterial activity according to the specific antibody. The CuO-NP-AbGram- in E. coli showed reduced half maximal inhibitory concentration (IC50 ) and minimum inhibitory concentration (MIC) values when compared with unfunctionalized CuO-NPs. On the other hand, the CuO-NP-AbGram+ also showed reduced IC50 and MIC values in B. subtilis, when compared with non-functionalized CuO-NPs. Thus, the functionalized CuO nanoparticles with specific antibodies showed enhanced specificity of their antibacterial activity. The advantages of "smart" antibiotic nanoparticles are discussed.

Virus-like nanoparticles as enzyme carriers for Enzyme Replacement Therapy (ERT).

Enzyme replacement therapy (ERT) has been used to treat a few of the many existing diseases which are originated from the lack of, or low enzymatic activity. Exogenous enzymes are administered to contend with the enzymatic activity deficiency. Enzymatic nanoreactors based on the enzyme encapsulation inside of virus-like particles (VLPs) appear as an interesting alternative for ERT. VLPs are excellent delivery vehicles for therapeutic enzymes as they are biodegradable, uniformly organized, and porous nanostructures that transport and could protect the biocatalyst from the external environment without much affecting the bioactivity. Consequently, significant efforts have been made in the production processes of virus-based enzymatic nanoreactors and their functionalization, which are critically reviewed. The use of virus-based enzymatic nanoreactors for the treatment of lysosomal storage diseases such as Gaucher, Fabry, and Pompe diseases, as well as potential therapies for galactosemia, and Hurler and Hunter syndromes are discussed.

Monolayer (2D) or spheroids (3D) cell cultures for nanotoxicological studies? Comparison of cytotoxicity and cell internalization of nanoparticles.

Two-dimensional (2D) cell culture monolayers are commonly used for toxicological assessments of nanomaterials. Despite their facile handling, they exhibit several constraints due to their structural and complexity differences with three-dimensional (3D) in vitro cell models, such as spheroids. Here, we conducted a comparative nanotoxicological study of fibroblasts (L929) and melanoma (B16-F10) cells, grown in 2D and 3D arrangements. The cytotoxicity, reactive oxygen species (ROS) production, genotoxicity, cell morphology complexity, and uptake of silver nanoparticles (AgNPs) and folic acid-functionalized upconversion nanoparticles (FA-UCNPs) were compared in the two culture arrangements. AgNPs cytotoxicity was higher in spheroids than in monolayer cultures. Furthermore, apoptotic cell percentages and ROS production were higher in 3D than in 2D cell cultures. More importantly, 2D cultures required twice the concentration of AgNPs than the 3D cell models to reach a considerable DNA damage index (c.a. 200). Therefore, spheroids are more sensitive to the genotoxic effects of AgNPs. FA-UCNPs exerted negligible cell toxicity in 2D and 3D cell models. Moreover, AgNPs induced disaggregation and downsizing of spheroids in a facile and concentration-dependent manner. Internalization of FA-UCNPs in spheroids was 20% higher than in the 2D cell arrangements. Collectively, our findings, demonstrated that spheroids are a more sensitive model than monolayers for the assessment of nanoparticle biocompatibility and internalization.

Targeted Enzymatic VLP-Nanoreactors with ß-Glucocerebrosidase Activity as Potential Enzyme Replacement Therapy for Gaucher's Disease.

Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29 mM) and the functionalized nanoreactors (KM =0.32 mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pH 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.

Virus-like Particles: Fundamentals and Biomedical Applications.

Nanotechnology is a fast-evolving field focused on fabricating nanoscale objects for industrial, cosmetic, and therapeutic applications. Virus-like particles (VLPs) are self-assembled nanoparticles whose intrinsic properties, such as heterogeneity, and highly ordered structural organization are exploited to prepare vaccines; imaging agents; construct nanobioreactors; cancer treatment approaches; or deliver drugs, genes, and enzymes. However, depending upon the intrinsic features of the native virus from which they are produced, the therapeutic performance of VLPs can vary. This review compiles the recent scientific literature about the fundamentals of VLPs with biomedical applications. We consulted different databases to present a general scenario about viruses and how VLPs are produced in eukaryotic and prokaryotic cell lines to entrap therapeutic cargo. Moreover, the structural classification, morphology, and methods to functionalize the surface of VLPs are discussed. Finally, different characterization techniques required to examine the size, charge, aggregation, and composition of VLPs are described.

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.

Nonlinear charge regulation for the deposition of silica nanoparticles on polystyrene spherical surfaces.

HYPOTHESIS: We describe the deposition behavior of monodispersed silica nanoparticles on polystyrene spherical particles by using modified pairwise DLVO (Derjaguin-Landau-Verwey-Overbeek) interaction force profiles at pH values between two and twelve. Our modified model contains a new nonlinear charge regulation parameter that considers redistribution of ions, which allows us to realistically express the electrical double layer (EDL) interaction forces. EXPERIMENTS: Silanol-terminated silica nanoparticles (7.6 ± 0.4 nm), l-lysine-covered silica nanoparticles (7.8 ± 0.4 nm), and polyallylamine hydrochloride-covered polystyrene (PAH/PS) particles (348 ± 1 nm) were synthesized. Then, each type of silica nanoparticle was deposited on the PAH/PS particles at a range of pH values. FINDINGS: Our new regulation parameter describes the realistic redistribution of charges governed by pH, total salt concentration, ionic strength of solution, and separation distance of particles. We find that this regulation parameter can be roughly approximated from the absolute values of theoretically calculated surface charge density and potential distributions, as well as experimentally measured ζ-potentials. Morphological analysis using electron microscopy of the experimental systems shows that the modified pairwise DLVO interaction forces exceptionally describe the deposition behavior of the silica nanoparticles physically adsorbed on the PAH/PS particle substrates.

Enzymatic characterization of agmatinase (AGM-1) from the filamentous fungus Neurospora crassa.

Agmatinase is a metallohydrolase involved in the hydrolysis of agmatine to produce urea and putrescine. Although its role in organisms is still under study, there are no reports of this family of enzymes in filamentous fungi. Recently, a protein showing agmatinase activity was reported in Neurospora crassa. Therefore, the aim of this work is to determine if the protein (AGM-1) found in the filamentous fungus N. crassa is a true agmatinase. The protein AGM-1was purified directly from N. crassa cultures, and its enzymatic characterization was carried out. The catalytic parameters such as optimum pH, thermostability, transformation kinetics, and activity in the presence of a cofactor were determined. The results show that AGM-1 can use manganese as a cofactor for its enzymatic activity, showing a transformation rate constant (kcat) of 77 s-1 and an affinity constant (KM) of 50.5 mM. The protein loses 50% of its activity when incubated 15 min at 30 °C and reaches maximal enzymatic activity at a pH range of 8-8.5. Our results indicate that the AGM-1 from N. crassa shows similar characteristics to true agmatinases already reported in other organisms. Thus, our findings strongly support that the protein annotated as hypothetical agmatinase in N. crassa is a true agmatinase.

Discretizing Three-Dimensional Oxygen Gradients to Modulate and Investigate Cellular Processes.

With the increased realization of the effect of oxygen (O2 ) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O2 concentrations and establish biomimetic O2 gradients to study and manipulate cellular behavior. Nonetheless, O2 gradients present in 3D engineered platforms result in diverse cell behavior across the O2 gradient, making it difficult to identify and study O2 sensitive signaling pathways. Using a layer-by-layer assembled O2 -controllable hydrogel, the authors precisely control O2 concentrations and study uniform cell behavior in discretized O2 gradients, then recapitulate the dynamics of cluster-based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O2 concentrations. Using RNA sequencing, it is found that time-dependent regulation of cyclic adenosine monophosphate signaling enables cell survival and clustering in the high stress microenvironments. Various extracellular matrix modulators orchestrate hypoxia-driven endothelial cell clustering. Finally, clustering is facilitated by regulators of cell-cell interactions, mainly vascular cell adhesion molecule 1. Taken together, novel regulators of hypoxic cluster-based vasculogenesis are identified, and evidence for the utility of a unique platform is provided to study dynamic cellular responses to 3D hypoxic environments, with broad applicability in development, regeneration, and disease.

Virus-Based Nanoreactors with GALT Activity for Classic Galactosemia Therapy.

Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.

Simple in situ functionalization of carbon nanospheres.

Functionalized carbon nanospheres have been synthesized in situ via a facile chemical vapor deposition strategy, fabricated by the pyrolysis of toluene/ethanol mixtures at different percentages (0, 1, 2, 3, 4, and 5 wt% of ethanol). The as-grown nanospheres have been characterized using transmission electron microscopy, scanning electron microscopy, Raman and Fourier transform infrared spectroscopy, x-ray diffraction, nitrogen adsorption, zeta potential measurements and x-ray photoelectron spectroscopy. Results indicate that the incorporation of ethanol in the precursor solution reflected in the presence of oxygen and hydrogen functional groups, the highest functionalized nanospheres without compromising the morphology of the sample were yielded at 3 wt% concentration. These in situ added functional groups rendered the carbon nanostructures enhancedly dispersible and stable in water, avoiding post-synthesis and harsh chemicals processing; envisaging thus applications of the nanospheres in the biomedical field where hydrophilicity of the nanomaterials is mandatory.

3D printer waste, a new source of nanoplastic pollutants.

Plastics pollution has been recognized as a serious environmental problem. Nevertheless, new plastic uses, and applications are still increasing. Among these new applications, three-dimensional resin printers have increased their use and popularity around the world showing a vertiginous annual-sales growth. However, this technology is also the origin of residues generation from the alcohol cleaning procedure at the end of each printing. This alcohol/resin mixture can originate unintentionally very small plastic particles that usually are not correctly disposed, and as consequence, could be easily released to the environment. In this work, the nanoparticle generation from 3D printer's cleaning procedure and their physicochemical characterization is reported. Nano-sized plastic particles are easily formed when the resin residues are dissolved in alcohol and placed under UV radiation from sunlight. These nanoparticles can agglomerate in seawater showing an average hydrodynamic diameter around 1 µm, whereas the same nanoparticles remain dispersed in ultrapure water, showing a hydrodynamic diameter of ≈300 nm. The formed nanoparticles showed an isoelectric point close to pH 2, which can facilitate their interaction with other positively charged pollutants. Thus, these unexpected plastic nanoparticles can become an environmental issue and public health risk.

Chemical Profiling Provides Insights into the Metabolic Machinery of Hydrocarbon-Degrading Deep-Sea Microbes.

Marine microbes are known to degrade hydrocarbons; however, microbes inhabiting deep-sea sediments remain largely unexplored. Previous studies into the classical pathways of marine microbial metabolism reveal diverse chemistries; however, metabolic profiling of marine microbes cultured with hydrocarbons is limited. In this study, taxonomic (amplicon sequencing) profiles of two environmental deep-sea sediments (>1,200 m deep) were obtained, along with taxonomic and metabolomic (mass spectrometry-based metabolomics) profiles of microbes harbored in deep-sea sediments cultured with hydrocarbons as the sole energy source. Samples were collected from the Gulf of México (GM) and cultured for 28 days using simple (toluene, benzene, hexadecane, and naphthalene) and complex (petroleum API 40) hydrocarbon mixtures as the sole energy sources. The sediment samples harbored diverse microbial communities predominantly classified into Woeseiaceae and Kiloniellaceae families, whereas Pseudomonadaceae and Enterobacteriaceae families prevailed after sediments were cultured with hydrocarbons. Chemical profiling of microbial metabolomes revealed diverse chemical groups belonging primarily to the lipids and lipid-like molecules superclass, as well as the organoheterocyclic compound superclass (ClassyFire annotation). Metabolomic data and prediction of functional profiles indicated an increase in aromatic and alkane degradation in samples cultured with hydrocarbons. Previously unreported metabolites, identified as intermediates in the degradation of hydrocarbons, were annotated as hydroxylated polyunsaturated fatty acids and carboxylated benzene derivatives. In summary, this study used mass spectrometry-based metabolomics coupled to chemoinformatics to demonstrate how microbes from deep-sea sediments could be cultured in the presence of hydrocarbons. This study also highlights how this experimental approach can be used to increase the understanding of hydrocarbon degradation by deep-sea sediment microbes.IMPORTANCE High-throughput technologies and emerging informatics tools have significantly advanced knowledge of hydrocarbon metabolism by marine microbes. However, research into microbes inhabiting deep-sea sediments (>1,000 m) is limited compared to those found in shallow waters. In this study, a nontargeted and nonclassical approach was used to examine the diversity of bacterial taxa and the metabolic profiles of hydrocarbon-degrading deep-sea microbes. In conclusion, this study used metabolomics and chemoinformatics to demonstrate that microbes from deep-sea sediment origin thrive in the presence of toxic and difficult-to-metabolize hydrocarbons. Notably, this study provides evidence of previously unreported metabolites and the global chemical repertoire associated with the metabolism of hydrocarbons by deep-sea microbes.

Camouflaged, activatable and therapeutic tandem bionanoreactors for breast cancer theranosis.

The one-pot cascade reaction of naturally occurring enzymes is exciting for highly selective complex reaction and biodegradable approaches. Tamoxifen is the main drug against breast cancer for decades and induces an anticancerous effect upon metabolic activation by cytochrome P450 (CYP450). Herein, bi-enzymatic nanoreactors (NRs) are developed as a multimodality platform for smart action against breast tumors. CYPBM3 of Bacillus magaterium (CYP) is co-confined with glucose oxidase (GOx) where GOx produces H2O2 in the presence of glucose that elicits the CYP-mediated transformation of tamoxifen. The scintillating and mesoporous LaF3:Tb as nanocarrier showed advantages like a wide range of pore size and positive surface charge for efficient loading of enzyme couple, while the smallest pores were available for substrate/product diffusion. The obtained NRs were camouflaged with human serum albumin (HSA) to overcome premature enzyme leaching and provide active stealth properties. The nanocomposite was characterized for physicochemical properties and glucose-mediated sequential catalysis. The in vitro studies demonstrated the cell internalization of NRs in both ER+ and triple-negative breast cancer cell lines and showed significant cytotoxicity. The developed NRs not only improve the outcomes of endocrine therapy in ER+ cells but also synergistically act with oxidation therapy for enhanced therapeutic effect. Importantly, inhibition of triple-negative cells was also achieved. Thus, the development of the new multimodal nanomedicine of the present work should afford new tools towards the theranosis of breast cancer with minimized adverse effects.

Brome mosaic virus-like particles as siRNA nanocarriers for biomedical purposes.

There is an increasing interest in the use of plant viruses as vehicles for anti-cancer therapy. In particular, the plant virus brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are novel potential nanocarriers for different therapies in nanomedicine. In this work, BMV and CCMV were loaded with a fluorophore and assayed on breast tumor cells. The viruses BMV and CCMV were internalized into breast tumor cells. Both viruses, BMV and CCMV, did not show cytotoxic effects on tumor cells in vitro. However, only BMV did not activate macrophages in vitro. This suggests that BMV is less immunogenic and may be a potential carrier for therapy delivery in tumor cells. Furthermore, BMV virus-like particles (VLPs) were efficiently loaded with small interfering RNA (siRNA) without packaging signal. The gene silencing was demonstrated by VLPs loaded with siGFP and tested on breast tumor cells that constitutively express the green fluorescent protein (GPF). After VLP-siGFP treatment, GFP expression was efficiently inhibited corroborating the cargo release inside tumor cells and the gene silencing. In addition, BMV VLP carring siAkt1 inhibited the tumor growth in mice. These results show the attractive potential of plant virus VLPs to deliver molecular therapy to tumor cells with low immunogenic response.

Bi-enzymatic virus-like bionanoreactors for the transformation of endocrine disruptor compounds.

Endocrine disruptor compounds (EDCs) are pollutants able to alter both hormone synthesis and their regulation in animals and humans, thus, EDCs represent a risk for public health and for the environment. Cytochrome P450 enzymes (CYPs) are involved in the detoxification of a wide range of compounds, and it has been established that these enzymes produce the initial biotransformation of many EDCs. In this work, a bionanoreactor based on the encapsulation of an enhanced peroxygenase CYPBM321B3 inside the capsid of bacteriophage P22 virus-like particles (VLPs) was designed and characterized. VLPs were functionalized with glucose oxidase to generate in situ hydrogen peroxide necessary to activate the transformation of bisphenol A, nonylphenol, 17ß-estradiol, triclosan, and resorcinol. Catalytic parameters, as well as the chemical nature of reaction products are presented. The enzymatic nanoreactors showed specific activities varying from 0.175 to 0.456 min-1 in the transformation of these EDCs, which are equivalent to 22-77% of the activity obtained with free CYP. The capacity to transform structurally diverse compounds, easy production and glucose fueled catalytic activity make these enzymatic nanoreactors an interesting platform for enzyme delivery in the biomedical field.

Nanoparticle-plasma Membrane Interactions: Thermodynamics, Toxicity and Cellular Response.

Nanomaterials have become part of our daily lives, particularly nanoparticles contained in food, water, cosmetics, additives and textiles. Nanoparticles interact with organisms at the cellular level. The cell membrane is the first protective barrier against the potential toxic effect of nanoparticles. This first contact, including the interaction between the cell membranes -and associated proteins- and the nanoparticles is critically reviewed here. Nanoparticles, depending on their toxicity, can cause cellular physiology alterations, such as a disruption in cell signaling or changes in gene expression and they can trigger immune responses and even apoptosis. Additionally, the fundamental thermodynamics behind the nanoparticle-membrane and nanoparticle-proteins-membrane interactions are discussed. The analysis is intended to increase our insight into the mechanisms involved in these interactions. Finally, consequences are reviewed and discussed.

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.