Papain functionalized Prussian blue nanozyme colloids of triple enzymatic function.

Prussian blue nanozymes were surface engineered with papain enzyme to develop processable nanoparticle dispersions with antioxidant and hydrolytic activities for biocatalytic applications. Enzyme coating improved the colloidal stability of the nanozymes and the obtained papain-Prussian blue hybrid showed remarkable peroxidase (vmax = 8.82 × 10-9 M s-1, KM = 12.3 mM), superoxide dismutase (IC50 = 14.6 ppm) and protease-like (41.2 U L-1) activities.

Coamplified Nanozyme Cocktails for Cascade Reaction-Driven Antioxidant Treatments.

Antioxidant nanozymes are powerful tools to combat oxidative stress, which can be further improved by applying nanozyme mixtures of multiple enzymatic function. Here, cocktails of Prussian blue (PB) nanocubes and copper(II) exchanged ZSM-5 zeolites (CuZ) with enhanced reactive oxygen species (ROS) scavenging activity were developed. Surface functionalization of the particles was performed using polymers to obtain stable colloids, i.e., resistant to aggregation, under a wide range of experimental conditions. The nanozyme cocktails possessed advanced antioxidant properties with multiple enzyme-like functions, catalyzing the decomposition of ROS in cascade reactions. The activity of the mixture far exceeded that of the individual particles, particularly in the peroxidase assay, where an improvement of more than an order of magnitude was observed, pointing to coamplification of the enzymatic activity. In addition, it was revealed that the copper(II) site in the CuZ plays an important role in the decomposition of both superoxide radicals and hydrogen peroxide, as it directly catalyzes the former reaction and acts as cocatalyst in the latter process by boosting the peroxidase activity of the PB nanozyme. The results give important insights into the design of synergistic particle mixtures for the broad-spectrum scavenging of ROS to develop efficient tools for antioxidant treatments in both medical therapies and industrial manufacturing processes.

QbD Approach-Based Preparation and Optimization of Hydrophobic Ion-Pairing Complex of Lysozyme with Sodium Dodecyl Sulphate to Enhance Stability in Lipid-Based Carriers.

Hydrophobic ion pairing (HIP) complexation was found to be an efficient approach in modulating the release and enhancing the stability and encapsulation of hydrophilic macromolecules such as proteins in hydrophobic nano/microcarriers. The present work strives to develop and optimize the preparation of the HIP complex of the antimicrobial enzyme lysozyme (LYZ) with the ion-pairing agent (IPA) sodium dodecyl sulphate (SDS) relying on the quality-by-design (QbD) approach. The quality target product profile (QTPP) includes the achievement of maximal lipophilicity in a reversible manner to enable the maintenance of biological activity. The related critical quality attributes (CQAs) were defined as complexation efficacy, complex stability, enzyme recovery and activity. Three risk assessment (RA) tools were used to identify and rank the critical process parameters (CPPs) and critical material attributes (CMAs). From this assessment, the pH of the medium, LYZ:SDS molar ratio and drying conditions were determined as high-risk factors that need to be investigated. To the best of our knowledge, for the first time, electrostatic titration was used as a smart approach to determine the optimum molar ratio at different pH values. Based on the predefined CQAs, pH 8 with an LYZ/SDS molar ratio of 1:8 was found to be the optimal condition for complexation efficiency and recovery (%) of a biologically active enzyme. A cost-effective drying process based on a ventilated oven was developed, which resulted in complex qualities comparable to those obtained by the commonly used freeze-drying method. In a nutshell, the optimum conditions for the preparation of the LYZ/SDS HIP complex were efficiently facilitated by the rational application of QbD principles and the utilization of efficient electrostatic titration and ventilated oven-drying methods.

Comparative study of photocatalysis with bulk and nanosheet graphitic carbon nitrides enhanced with silver.

The main goal of this research is to investigate the effectiveness of graphitic carbon nitride (g-C3N4, g-CN) in both bulk and nanosheet forms, which have been surface-modified with silver nanoparticles (Ag NPs), as photocatalysts for the degradation of acid orange 7 (AO7), a model dye. The photodegradation of AO7 dye molecules in water was used to test the potential photocatalytic properties of these powder materials under two different lamps with wavelengths of 368 nm (UV light) and 420 nm (VIS light). To produce Ag NPs (Ag content 0.5, 1.5, and 3 wt%) on the g-CN materials, a new synthesis route based on a wet and low-temperature method was proposed, eliminating the need for reducing agents. The photodegradation activity of the samples increased with increasing silver content, with the best photocatalytic performances achieved for bulk g-CN samples and nanosheet silver-modified samples (with the highest content of 3 wt% Ag) under UV light, i.e., more than 75% and 78%, respectively. The VIS-induced photocatalytic activity of both examined series was higher than that of UV. The highest activities of 92% and 98% were achieved for the 1.5% Ag-modified g-CN bulk and nanosheet materials. This research presents an innovative, affordable, and environmentally friendly chemical approach to synthesizing photocatalysts that can be used for degrading organic pollutants in wastewater treatment.

Increased blood-brain barrier permeability of neuroprotective drug by colloidal serum albumin carriers.

Encapsulation possibilities of two neuroprotective drugs of slightly different structures, kynurenic acid (KYNA) and its more hydrophilic analogue (SzR72), are studied in bovine serum albumin (BSA) nanoparticles (NPs) to increase their permeability through the blood-brain barrier (BBB). The effect of various preparation conditions such as protein concentration, protein-to-drug ratio, pH, ionic strength, type, and amount of desolvation agent and cross-linker concentration are discussed. It was found that the encapsulation proved to be successful only if the drugs are added to the pre-prepared BSA NPs. If the pH of the medium is adjusted to 4.0 instead of 7.4 the drug loading increased (from 4.5 % to 20.7 % for KYNA) due to the electrostatic interaction between the oppositely charged functional groups accompanied by significant secondary structural changes verified by circular dichroism spectroscopy (CD) suggesting the drug insertion in the hydrophobic pockets of BSA. The in vitro polar brain lipid extract (porcine) based permeability test proved the aimed three-, or fourfold higher BBB specific penetration for KYNA in the carrier relative to the unformatted drug.

Antioxidant colloids via heteroaggregation of cerium oxide nanoparticles and latex beads.

Antioxidant colloids were developed via controlled heteroaggregation of cerium oxide nanoparticles (CeO2 NPs) and sulfate-functionalized polystyrene latex (SL) beads. Positively charged CeO2 NPs were directly immobilized onto SL particles of opposite surface charge via electrostatic attraction (SL/Ce composite), while negatively charged CeO2 NPs were initially functionalized with poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte and then, aggregated with the SL particles (SPCe composite). The PDADMAC served to induce a charge reversal on CeO2 NPs, while the SL support prevented nanoparticle aggregation under conditions, where the dispersions of bare CeO2 NPs were unstable. Both SL/Ce and SPCe showed enhanced radical scavenging activity compared to bare CeO2 NPs and were found to mimic peroxidase enzymes. The results demonstrate that SL beads are suitable supports to formulate CeO2 particles and to achieve remarkable dispersion storage stability. The PDADMAC functionalization and immobilization of CeO2 NPs neither compromised the peroxidase-like activity nor the radical scavenging potential. The obtained SL/Ce and SPCe artificial enzymes are foreseen to be excellent antioxidant agents in various applications in the biomedical, food, and cosmetic industries.

Rational Mitomycin Nanocarriers Based on Hydrophobically Functionalized Polyelectrolytes and Poly(lactide-co-glycolide).

Encapsulation of hydrophilic and amphiphilic drugs in appropriate colloidal carrier systems for sustained release is an emerging problem. In general, hydrophobic bioactive substances tend to accumulate in water-immiscible polymeric domains, and the release process is controlled by their low aqueous solubility and limited diffusion from the nanocarrier matrix. Conversely, hydrophilic/amphiphilic drugs are typically water-soluble and insoluble in numerous polymers. Therefore, a core-shell approach─nanocarriers comprising an internal core and external shell microenvironments of different properties─can be exploited for hydrophilic/amphiphilic drugs. To produce colloidally stable poly(lactic-co-glycolic) (PLGA) nanoparticles for mitomycin C (MMC) delivery and controlled release, a unique class of amphiphilic polymers─hydrophobically functionalized polyelectrolytes─were utilized as shell-forming materials, comprising both stabilization via electrostatic repulsive forces and anchoring to the core via hydrophobic interactions. Undoubtedly, the use of these polymeric building blocks for the core-shell approach contributes to the enhancement of the payload chemical stability and sustained release profiles. The studied nanoparticles were prepared via nanoprecipitation of the PLGA polymer and were dissolved in acetone as a good solvent and in an aqueous solution containing hydrophobically functionalized poly(4-styrenesulfonic-co-maleic acid) and poly(acrylic acid) of differing hydrophilic-lipophilic balance values. The type of the hydrophobically functionalized polyelectrolyte (HF-PE) was crucial for the chemical stability of the payload─derivatives of poly(acrylic acid) were found to cause very rapid degradation (hydrolysis) of MMC, in contrast to poly(4-styrenesulfonic-co-maleic acid). The present contribution allowed us to gain crucial information about novel colloidal nanocarrier systems for MMC delivery, especially in the fields of optimal HF-PE concentrations, appropriate core and shell building materials, and the colloidal and chemical stability of the system.

Investigation of Surface Properties and Free Volumes of Chitosan-Based Buccal Mucoadhesive Drug Delivery Films Containing Ascorbic Acid.

Nowadays, the buccal administration of mucoadhesive films is very promising. Our aim was to prepare ascorbic acid-containing chitosan films to study the properties and structures important for applicability and optimize the composition. During the formulation of mucoadhesive films, chitosan as the polymer basis of the film was used. Ascorbic acid, which provided the acidic pH, was used in different concentrations (2-5%). The films were formulated by the solvent casting method. The properties of films important for applicability were investigated, such as physical parameters, mucoadhesive force, surface free energy, and breaking strength. The fine structure of the films was analyzed by atomic force microscopy, and the free volume was analyzed by PALS, which can be important for drug release kinetics and the location of the drug in the film. The applicability of the optimized composition was also tested with two different types of active ingredients. The structure of the films was also analyzed by XRPD and FTIR. Ascorbic acid can be used well in chitosan films, where it can function as a permeation enhancer when reacting to chitosan, it is biodegradable, and can be applied in 2% of our studies.

Serum Albumin Nanoparticles: Problems and Prospects.

The present paper aims to summarize the results regarding serum albumin-based nanoparticles (NPs) for drug delivery purposes. In particular, it focuses on the relationship between their preparation techniques and synthesis parameters, as well as their successful clinical application. In spite of the huge amount of consumed material and immaterial sources and promising possibilities, products made from different types of albumin NPs, with the exception of a few, still have not been invented. In the present paper, promising applications of serum albumin nanoparticles (SANPs) for different biomedical purposes, such as carriers, delivery systems and contrast agents, are also discussed. The most frequent utilization of the NPs for certain diseases, i.e., cancer therapy, and future prospects are also detailed in this study.

Design of hybrid biocatalysts by controlled heteroaggregation of manganese oxide and sulfate latex particles to combat reactive oxygen species.

The preparation of an antioxidant hybrid material by controlled heteroaggregation of manganese oxide nanoparticles (MnO2 NPs) and sulfate-functionalized polystyrene latex (SL) beads was accomplished. Negatively charged MnO2 NPs were prepared by precipitation and initially functionalized with poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte to induce charge reversal allowing decoration of oppositely charged SL surfaces via simple mixing. The PDADMAC-functionalized MnO2 NPs (PMn) aggregated with the SL particles leading to the formation of negatively charged, neutral and positively charged (SPMn) composites. The charge neutralization resulted in rapidly aggregating dispersions, while stable samples were observed once the composites possessed sufficiently high negative and positive charge, below and above the charge neutralization point, respectively. The antioxidant assays revealed that SL served as a suitable substrate and that the PDADMAC functionalization and immobilization of MnO2 NPs did not compromise their catalase (CAT) and superoxide dismutase (SOD)-like activities, which were also maintained within a wide temperature range. The obtained SPMn composite is expected to be an excellent candidate as an antioxidant material for the efficient scavenging of reactive oxygen species at both laboratory and larger scales, even under harsh conditions, where natural antioxidants do not function.

Vitamin E-Loaded PLA- and PLGA-Based Core-Shell Nanoparticles: Synthesis, Structure Optimization and Controlled Drug Release.

The (±)-α-Tocopherol (TP) with vitamin E activity has been encapsulated into biocompatible poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) carriers, which results in the formation of well-defined nanosized (d ~200-220 nm) core-shell structured particles (NPs) with 15-19% of drug loading (DL%). The optimal ratios of the polymer carriers, the TP active drug as well as the applied Pluronic F127 (PLUR) non-ionic stabilizing surfactant, have been determined to obtain NPs with a TP core and a polymer shell with high encapsulation efficiency (EE%) (69%). The size and the structure of the prepared core-shell NPs as well as the interaction of the carriers and the PLUR with the TP molecules have been determined by transmission electron microscopy (TEM), dynamic light scattering (DLS), infrared spectroscopy (FT-IR) and turbidity studies, respectively. Moreover, the dissolution of the TP from the polymer NPs has been investigated by spectrophotometric measurements. It was clearly confirmed that increase in the EE% from ca. 70% (PLA/TP) to ca. 88% (PLGA65/TP) results in the controlled release of the hydrophobic TP molecules (7 h, PLA/TP: 34%; PLGA75/TP: 25%; PLGA65/TP: 18%). By replacing the PLA carrier to PLGA, ca. 15% more active substance can be encapsulated in the core (PLA/TP: 65%; PLGA65/TP: 80%).

The effect of synthesis conditions and tunable hydrophilicity on the drug encapsulation capability of PLA and PLGA nanoparticles.

Three drugs with different hydrophilicity are encapsulated in poly-lactide (PLA) and Poly(lactide-co-glycolide) (PLGA) drug delivery systems prepared by ring-opening polymerization (ROP). Formation of well-defined core-shell type nanoparticles (NPs) is observed for α-tocopherol (TP) and by systematically altering the hydrophilicity of the drug carrier NPs the entrapment efficiency (EE (%)) can be remarkably controlled. The highest (90%) of EE (%) is obtained for the most lipophilic TP from the applied three drugs in the 75% lactide-containing PLGA75 NPs, which is ca. 69% for PLA NPs. Subsequent to drug loading the detailed characterization of the polymers and the formed NPs was carried out. Precipitation titrations reveal that our PLGAs have narrower weight distribution than the commercially available polymer enabling favorable properties to obtain NPs with better size distribution. It is pointed out that during the synthesis the applied solvent and stabilizing agent play a decisive role in the size distribution and stability of the drug carrier NPs. The Pluronic F127-stabilized NPs have the smallest diameter (ca. 190 nm) with less polydispersity among the applied stabilizing agent in nanoprecipitation.

Effect of pH on stability and plasmonic properties of cysteine-functionalized silver nanoparticle dispersion.

Citrate-stabilized spherical silver nanoparticles (Ag NPs) with d=8.25±1.25 nm diameter were prepared and functionalized with L-cysteine (Cys) in aqueous dispersion. The nanosilver-cysteine interactions have been investigated by Raman and (1)H NMR spectroscopy. The effect of pH on stability of biofunctionalized Ag NPs was investigated. The cysteine-capped nanosilver dispersions remain stable at higher pH (pH>7), while the degree of aggregation increased as the pH decreased. Below pH ~7, the characteristic surface plasmon band of bare silver nanoparticles was back-shifted from λ(measured)(bareAgNP)=391 nm to λ(measured)(1)=387-391 nm, while the presence of a new band at λ(measured)(2)=550-600 nm was also observed depending on pH. Finite element method (FEM) was applied to numerically compute the absorption spectra of aqueous dispersions containing bare and cysteine-functionalized Ag NPs at different pH. Both the dynamic light scattering (DLS) measurements, Zeta potential values and the transmission electron microscopic (TEM) images confirmed our supposition. Namely, electrostatic interaction arose between the deprotonated carboxylate (COO(-)) and protonated amino groups (NH(3)(+)) of the amino acid resulting in cross-linking network of the Ag NPs between pH ~3 and 7. If the pH is measurable lower than ~3, parallel with the protonation of citrate and L-cysteine molecules the connection of the particles via l-cysteine is partly decomposed resulting in decrease of second plasmon band intensity.

Hydrothermal synthesis and humidity sensing property of ZnO nanostructures and ZnO-In(OH)3 nanocomposites.

Prism- and raspberry-like ZnO nanoparticles and ZnO-In(OH)(3) nanocomposites were prepared by template free hydrothermal method. XRD investigations and microscopic studies showed that pill-like In(OH)(3) particles with body-centered cubic crystal structure formed on the surface of ZnO nanoparticles resulting in increased specific surface area. TEM-EDX mapping images demonstrated that not only nanocomposite formation took place in the course of the synthesis, but zinc ions were also built into the crystal lattice of the In(OH)(3). However, only undoped In(OH)(3) was found on the surface of the pill-like particle aggregates by XPS analyses. The raspberry- and prism-like ZnO particles exhibit strong visible emission with a maximum at 585 and 595 nm, respectively, whose intensity significantly increase due to nanocomposite formation. Photoelectric investigations revealed that photocurrent intensity decreased with increasing indium ion concentration during UV light excitation, which was explained by increase in visible fluorescence emission. QCM measurements showed that morphology of ZnO and concentration of In(OH)(3) had an influence on the water vapor sensing properties.

Optical and structural properties of protein/gold hybrid bio-nanofilms prepared by layer-by-layer method.

Lysozyme/gold thin layers were prepared by layer-by-layer (LbL) self-assembly method. The build-up of the films was followed by UV-vis-absorbance spectra, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) techniques. The structural property of films was examined by X-ray diffraction (XRD) measurements, while their morphology was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). It was found that gold nanoparticles (NPs) had cubic crystalline structure, the primary particles form aggregates in the thin layer due to the presence of lysozyme molecules. The UV-vis measurements prove change in particle size while the colour of the film changes from wine-red to blue. The layer thickness of films was determined using the above methods and the loose, porous structure of the films explains the difference in the results. The vapour adsorption property of hybrid layers was also studied by QCM using different saturated vapours and ammonia gas. The lysozyme/Au films were most sensitive for ammonia gas among the tested gases/vapours due to the strongest interaction between the functional groups of the protein.

Structural, optical, and adsorption properties of ZnO(2)/poly(acrylic acid) hybrid thin porous films prepared by ionic strength controlled layer-by-layer method.

ZnO(2)/poly(acrylic acid) sandwich structures were prepared by layer-by-layer (LbL) self-assembly. The structure and optical behavior of the hybrid films were controlled by changing the surface charge and conformation of the poly(acrylic acid). The buildup of the films was followed by UV-vis absorption and reflection spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), and quartz crystal microbalance (QCM) measurements. It was found that the ionic strength of the polymer solution had a great influence on the film thickness which, in turn, affected the optical properties. The water vapor adsorption isotherms of the films determined by QCM showed an adsorption hysteresis characteristic of porous thin layer structures. The adsorption of water molecules inside the films changed the effective refractive index resulting in a change of the reflection properties. This phenomenon is shown to be exploited for the application of the films as optical sensors. The polarizability of water molecules in the adsorption layer was also determined. It was found that polarization of water molecules in the adsorption layer is much lower than in the liquid water when the surface coverage (Theta) is low.

Synthesis and stabilization of Prussian blue nanoparticles and application for sensors.

Prussian blue (PB) nanoparticles were synthesized by two methods from FeCl2 and K3Fe(CN)6 and from FeCl3 and K3Fe(CN)6 based on the method published by Fiorito et al., and stabilized by different polymers like polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyallylamine hydrochloride (PAH), polydiallyl-dimethyldiammonium chloride (PDDA) and polystyrene sulfonate (PSS). The effect of the monomer/Fe3+ ratio was studied regarding the average particle size and zeta-potential. The forming PB structure was checked by X-ray diffraction. The stabilization was successful for every applied polymer, but the average particle size significantly differs. Particle size distributions were determined by Malvern type nanosizer equipment and by transmission electron microscope (TEM) and zeta potential values were determined for the obtained stabile samples. The results revealed that by using FeCl2 and K3Fe(CN)6 for PB preparation particles with narrow size distribution and average diameter of 1.7 nm occurred but stabilization was necessary. By the other method the dispersion was stabile with 182 nm particles but the particle size exponentially decreased to 18 nm with increasing PVP concentration. Ultrathin nanofilms were prepared on glass support by the alternating layer-by-layer (LbL) method from PB particles and PAH. The morphology of the prepared films was investigated also by AFM. The films were immobilized on interdigitated microsensor electrodes (IME) and tested in sensing hydrogen peroxide and different acids like acetic acid, hydrochloric acid vapors.