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.

Tryptophanhydroxamic Acid-Stabilized Ultrasmall Gold Nanoclusters: Tuning the Selectivity for Metal Ion Sensing.

Sub-nanometer-sized gold nanoclusters (Au NCs) were prepared via the spontaneous reduction of [AuCl4]-- ions with a hydroxamate derivative of L-tryptophan (Trp) natural amino acid (TrpHA). The prepared TrpHA-Au NCs possess intense blue emission (λem = 470 nm; λex = 380 nm) with a 2.13% absolute quantum yield and 1.47 ns average lifetime. The Trp-stabilized noble metal NCs are excellent metal ion sensors for Fe3+, but in this work, we highlighted that the incorporation of the hydroxamate functional group with an excellent metal ion binding capability can tune the selectivity and sensitivity of these NCs, which is a promising way to design novel strategies for the detection of other metal ions as well. Moreover, their simultaneous identification can also be realized. By decreasing the sensitivity of our nano-sensor for Fe3+ (limit of detection (LOD) ~11 µM), it was clearly demonstrated that the selectivity for Cu2+-ions can be significantly increased (LOD = 3.16 µM) in an acidic (pH = 3-4) condition. The surface-bounded TrpHA molecules can coordinate the Cu2+ confirmed by thermodynamic data, which strongly generates the linking of the NCs via the Cu2+ ions in acidic pH, and a parallel fluorescence quenching occurs. In the case of Fe3+, the degree of quenching strongly depends on the metal ion concentration, and it only occurs when the NCs are not able to bind more Fe3+ (~10 µM) on the surface, causing the NCs' aggregation.

Thermodynamic Characterization of the Interaction of Biofunctionalized Gold Nanoclusters with Serum Albumin Using Two- and Three-Dimensional Methods.

Fluorescent gold nanoclusters have been successfully used as fluorescent markers for imaging of cells and tissues, and their potential role in drug delivery monitoring is coming to the fore. In addition, the development of biosensors using structure-tunable fluorescent nanoclusters is also a prominent research field. In the case of these sensor applications, the typical goal is the selective identification of, e.g., metal ions, small molecules having neuroactive or antioxidant effects, or proteins. During these application-oriented developments, in general, there is not enough time to systematically examine the interaction between nanoclusters and relevant biomolecules/proteins from a thermodynamic viewpoint. In this way, the primary motivation of this article is to carry out a series of tests to partially fill this scientific gap. Besides the well-known fluorescent probes, the mentioned interactions were investigated using such unique measurement methods as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). These two-dimensional (at the solid/liquid interface) and three-dimensional (in the bulk phase) measuring techniques provide a unique opportunity for the thermodynamic characterization of the interaction between different gold nanoclusters containing various surface functionalizing ligands and bovine serum albumin (BSA).

Lipid-Based Nanocarriers for Delivery of Neuroprotective Kynurenic Acid: Preparation, Characterization, and BBB Transport.

Encapsulation possibilities of an extensively investigated neuroprotective drug (kynurenic acid, KYNA) are studied via lipid-based nanocarriers to increase the blood-brain barrier (BBB) specific permeability. The outcomes of various preparation conditions such as stirring and sonication time, concentration of the lipid carriers and the drug, and the drug-to-lipid ratio are examined. Considering the experimentally determined encapsulation efficiency, hydrodynamic diameter, and ζ-potential values, the initial lipid and drug concentration as well as the stirring and sonication time of the preparation were optimized. The average hydrodynamic diameter of the prepared asolectin-(LIP) and water-soluble lipopolymer (WSLP)-based liposomes was found to be ca. 25 and 60 nm under physiological conditions. The physicochemical characterization of the colloidal carriers proves that the preparation of the drug-loaded liposomes was a successful process, and secondary interactions were indicated between the drug molecule and the polymer residues around the WSLP membrane. Dissolution profiles of the active molecule under physiological conditions were registered, and the release of the unformulated and encapsulated drug is very similar. In addition to this outcome, the in vitro polar brain lipid extract (porcine)-based permeability test proved the achievement of two- or fourfold higher BBB specific penetration and lipid membrane retention for KYNA in the liposomal carriers relative to the unformatted drug.

Plasmonic Effect of Gold-Patchy Silica Nanoparticles on Green Light-Photopolymerizable Dental Resin.

A low ratio of polymerization is a major problem in resin-based composites. In this paper, the plasmonic effect of gold-covered silica nanoparticles on the physicochemical and mechanical properties of bisphenol A diglycidyl dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) green light-photopolymerizable dental resin was investigated at an intensity of 1.4 mW/cm2 for 40 s. Transmission electron microscopy (TEM) showed silica of about 350 nm covered with 12-15 nm gold nanoparticles (Au NPs) at 100% nominal coverage. Five different concentrations of bare and patchy silica particles were used; in the latter composite, the calculated Au wt% were 0.0052 wt%, 0.0104 wt%, 0.0208 wt%, 0.04160 wt%, and 0.0823 wt%. The plasmon peak of patchy silica-filled nanocomposite overlapped with the absorption of Irgacure 784 photoinitiator and green LED light emission peak. The effect of plasmon-enhanced polymerization achieved with green light illumination was analyzed using diametral tensile strength (DTS), differential scanning calorimetry (DSC), surface plasmon resonance imaging (SPRi), and degree of conversion (DC) based on Raman spectroscopy. The values of the Au NP with 0.0208 wt% was found to be maximum in all the measured data. Based on our result, it can be concluded that the application of patchy silica particles in dental resin can improve the polymerization ratio and the mechanical parameters of the composite.

Deposition of Thin Electroconductive Layers of Tin (II) Sulfide on the Copper Surface Using the Hydrometallurgical Method: Electrical and Optical Studies.

Thin films of tin (II) sulfide (SnS) were deposited onto a 500 µm thick copper substrate by a chemical bath method. The effect of sodium (Na) doping in these films was studied. The synthesis of the films was performed at temperatures of 60, 70, and 80 °C for 5 min. The microstructure of the SnS films analyzed by scanning electron microscopy (SEM) showed a compact morphology of the films deposited at 80 °C. The edges of the SnS grains were rounded off with the addition of a commercial surfactant. The thickness of different SnS layers deposited on the copper substrate was found to be 230 nm from spectroscopic ellipsometry and cross-section analysis using SEM. The deposition parameters such as temperature, surfactant addition, and sodium doping time did not affect the thickness of the layers. From the X-ray diffraction (XRD) analysis, the size of the SnS crystallites was found to be around 44 nm. Depending on the process conditions, Na doping affects the size of the crystallites in different ways. A study of the conductivity of SnS films provides a specific conductivity value of 0.3 S. The energy dispersive analysis of X-rays (EDAX) equipped with the SEM revealed the Sn:S stoichiometry of the film to be 1:1, which was confirmed by the X-ray photoelectron spectroscopy (XPS) analysis. The determined band-gap of SnS is equal to 1.27 eV and is in good agreement with the literature data.

The Effect of Concentration, Temperature, and pH on the Formation of Hyaluronic Acid-Surfactant Nanohydrogels.

The assembly of colloidal hyaluronic acid (HyA, as a polysaccharide) based hydrogel particles in an aqueous medium is characterized in the present paper, with an emphasis on the particular case of nanohydrogels formed by surfactant-neutralized polysaccharide networks. The structural changes and particle formation process of polysaccharide- and cationic-surfactant-containing systems were induced by the charge neutralization ability and the hydrophobic interactions of cetyltrimethylammonium bromide (CTAB) under different conditions. Based on the rheological, light scattering, ζ-potential, turbidity, and charge titration measurements, it can be concluded that the preparation of the HyA-CTAB particles can be greatly controlled. The results indicate that more available negative charges can be detected on the polymer chain at smaller initial amounts of HyA (cHyA < 0.10 mg/mL), where a molecular solution can be formed. The change in the pH has a negligible effect on the formation process (particle aggregation appears at nCTAB/nHyA,monomer~1.0 in every case), while the temperature dependence of the critical micelle concentration (c.m.c.) of CTAB determines the complete neutralization of the forming nanohydrogels. The results of our measurements confirm that after the appearance of stable colloidal particles, a structural change and aggregation of the polymer particles take place, and finally the complete charge neutralization of the system occurs.

The Role of the Amino Acid Molecular Characteristics on the Formation of Fluorescent Gold- and Silver-Based Nanoclusters.

Role of amino acids like L-phenylalanine (Phe), L-glutamine (Gln) and L-arginine (Arg) is described and interpreted in terms of their potential for preparation of fluorescent molecular-like gold and silver nanostructures. We are among the first to demonstrate the effect of syntheses conditions as well as the molecular characteristics of Phe, Gln and Arg amino acids on the structure of the formed products. Comprehensive optical characterizations (lifetime, quantum yield (QY%)) of the blue-emitting products were also carried out. It was confirmed that for all Au-containing samples and for Gln-Ag system the characteristic fluorescence originates from few-atomic metallic nanoclusters (NCs) where the reduction of metal ions was promoted by citrate in some cases. Relatively high QY% (∼18 %) was obtained for Arg-stabilized Au NCs due to the existence of an electrostatic interaction between the electron rich, positively charged guanidium side chain of Arg and the negatively charged carboxylate group of citrate on the metallic surface. Size and structural analysis of the products were evaluated by infrared measurements and dynamic light scattering techniques.

Core-Shell Structured PLGA Particles Having Highly Controllable Ketoprofen Drug Release.

The non-steroid anti-inflammatory drug ketoprofen (KP) as a model molecule is encapsulated in different poly(lactide-co-glycolide) (PLGA) nanostructured particles, using Tween20 (TWEEN) and Pluronic F127 (PLUR) as stabilizers to demonstrate the design of a biocompatible colloidal carrier particles with highly controllable drug release feature. Based on TEM images the formation of well-defined core-shell structure is highly favorable using nanoprecipitation method. Stabile polymer-based colloids with ~200-210 nm hydrodynamic diameter can be formed by successful optimization of the KP concentration with the right choice of stabilizer. Encapsulation efficiency (EE%) of 14-18% can be achieved. We clearly confirmed that the molecular weight of the stabilizer thus its structure greatly controls the drug release from the PLGA carrier particles. It can be determined that ~20% and ~70% retention is available with the use of PLUR and TWEEN, respectively. This measurable difference can be explained by the fact that the non-ionic PLUR polymer provides a steric stabilization of the carrier particles in the form of a loose shell, while the adsorption of the non-ionic biocompatible TWEEN surfactant results in a more compact and well-ordered shell around the PLGA particles. In addition, the release property can be further tuned by decreasing the hydrophilicity of PLGA by changing the monomer ratio in the range of ~20-60% (PLUR) and 70-90% (TWEEN).

Promising Bioactivity of Vitamin B1-Au Nanocluster: Structure, Enhanced Antioxidant Behavior, and Serum Protein Interaction.

In the current work, we first present a simple synthesis method for the preparation of novel Vitamin-B1-stabilized few-atomic gold nanoclusters with few atomic layers. The formed nanostructure contains ca. eight Au atoms and shows intensive blue emissions at 450 nm. The absolute quantum yield is 3%. The average lifetime is in the nanosecond range and three main components are separated and assigned to the metal-metal and ligand-metal charge transfers. Based on the structural characterization, the formed clusters contain Au in zero oxidation state, and Vitamin B1 stabilizes the metal cores via the coordination of pyrimidine-N. The antioxidant property of the Au nanoclusters is more prominent than that of the pure Vitamin B1, which is confirmed by two different colorimetric assays. For the investigation into their potential bioactivity, interactions with bovine serum albumin were carried out and quantified. The determined stoichiometry indicates a self-catalyzed binding, which is almost the same value based on the fluorometric and calorimetric measurements. The calculated thermodynamic parameters verify the spontaneous bond of the clusters along the protein chain by hydrogen bonds and electrostatic interactions.

Synthesis and Catalytic Study of NiAg Bimetallic Core-Shell Nanoparticles.

This publication presents the synthesis of core-shell nanoparticles, where the core was Ni, and the shell was a Ag-Ni nano alloy. The synthesis was based on the reduction of Ni and Ag ions with sodium borohydride in the presence of trisodium citrate as a stabilizer. In order to determine the phase composition of the obtained nanoparticles, an XRD study was performed, and in order to identify the oxidation states of the nanoparticle components, an XPS spectroscopic study was performed. The composition and shape of the particles were determined using the HR-TEM EDS test. The obtained nanoparticles had a size of 11 nm. The research on catalytic properties was carried out in the model methylene blue reduction system. The investigation of the catalytic activity of colloids was carried out with the use of UV-Vis spectrophotometry. The Ag-Ni alloy was about ten times more active than were pure silver nanoparticles of a similar size.

Milk-Derived Carbon Quantum Dots: Study of Biological and Chemical Properties Provides Evidence of Toxicity.

Carbon dots (CDs) are carbon-based zero-dimensional nanomaterials that can be prepared from a number of organic precursors. In this research, they are prepared using fat-free UHT cow milk through the hydrothermal method. FTIR analysis shows C=O and C-H bond presence, as well as nitrogen-based bond like C-N, C=N and -NH2 presence in CDs, while the absorption spectra show the absorption band at 280 ± 3 nm. Next, the Biuret test was performed, with the results showing no presence of unreacted proteins in CDs. It can be said that all proteins are converted in CDs. Photo luminance spectra shows the emission of CDs is 420 nm and a toxicity study of CDs was performed. The Presto Blue method was used to test the toxicity of CDs for murine hippocampal cells. CDs at a concentration of 4 mg/mL were hazardous independent of synthesis time, while the toxicity was higher for lower synthesis times of 1 and 2 h. When the concentration is reduced in 1 and 2 h synthesized CDs, the cytotoxic effect also decreases significantly, ensuring a survival rate of 60-80%. However, when the synthesis time of CDs is increased, the cytotoxic effect decreases to a lesser extent. The CDs with the highest synthesis time of 8 h do not show a cytotoxic effect above 60%. The cytotoxicity study shows that CDs may have a concentration and time-dependent cytotoxic effect, reducing the number of viable cells by 40%.

Delamination of Layered Double Hydroxide in Ionic Liquids under Ambient Conditions.

Liquid phase delamination of layered materials into single- or few-layer nanosheets leads to stable nanoscale dispersions of 2D materials. The delamination of layered double hydroxide (LDH) to double hydroxide nanosheets was studied in two ionic liquids (ILs): ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium thiocyanate (BMIMSCN). The as-prepared lamellar structure of LDH disappeared upon dispersing it in ILs due to delamination into 2D nanosheets confirmed by X-ray scattering and diffraction techniques and further evaluated by height profile assessment of the nanoparticles by atomic force microscopy. The results showed that both the thickness and lateral size of the dispersed particles decreased in the IL-based samples, indicating that cleavage of the LDH materials can be observed in addition to delamination. The findings prove the concept of delamination of layered materials by ILs under ambient conditions─an excellent way to prepare 2D double hydroxide nanosheet dispersions in one step using nonvolatile green solvents.

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.

Fluorescence Quenching of Tyrosine-Ag Nanoclusters by Metal Ions: Analytical and Physicochemical Assessment.

A new synthesis method is described for the first time to produce silver nanoclusters (AgNCs) by using the tyrosine (Tyr) amino acid. Several important parameters (e.g., molar ratios, initial pH, reaction time etc.) were optimized to reach the highest yield. The formed Tyr-AgNCs show characteristic blue emission at λem = 410 nm, and two dominant fluorescence lifetime components were deconvoluted (τ1 ~ 3.7 and τ2 ~ 4.9 ns). The NCs contained metallic cores stabilized by dityrosine. For possible application, the interactions with several metal ions from the tap water and wastewater were investigated. Among the studied cations, four different ions (Cu2+, Ni2+, Fe3+, and Rh3+) had a dominant effect on the fluorescence of NCs. Based on the detected quenching processes, the limit of detection of the metal ions was determined. Static quenching (formation of a non-luminescent complex) was observed in all cases by temperature-dependent measurements. The calculated thermodynamic parameters showed that the interactions are spontaneous ranked in the following order of strength: Cu2+ > Fe3+ > Rh3+ > Ni2+. Based on the sign and relations of the standard enthalpy (ΔH°) and entropy changes (ΔS°), the dominant forces were also identified.

Tropaeolin OO as a Chemical Sensor for a Trace Amount of Pd(II) Ions Determination.

The selective determination of metals in waste solutions is a very important aspect of the industry and environmental protection. Knowledge of the contents and composition of the waste can contribute to design an efficient process separation and recovery of valuable metals. The problematic issue is primarily the correct determination of metals with similar properties such as palladium and platinum. Thus this paper focuses on the development of a selective method that enables Pd(II) determination in the presence of Pt(IV) ions using the azo-dye tropaeolin OO (TR). For this purpose, the process of the metalorganic complex formation and Pd(II) ions determination were studied by using UV-Vis spectrophotometry under different conditions: solvents (water and B-R buffer), pH (2.09-6.09), temperature (20-60 °C), anions and cations concentrations. The formed metalorganic complex between Pd and tropaeolin OO allows for distinguishing Pd(II) ions from both platinum complexes, i.e. Pt(II), Pt(IV). Moreover, the proposed method can be applied to solutions containing both chloride and chlorate ions. The obtained characteristic spectrum with two maxima allows the determination of palladium even in the presence of other cations (Na, K, Mg, Zn, Co, Ni, Al) and changed concentrations of Pt(IV) ions. Furthermore, the developed spectrophotometric method for the Pd(II) ions determination using tropaeolin OO is characterized by high selectivity towards palladium ions.

Evaluation of noble metal nanostructure-serum albumin interactions in 2D and 3D systems: Thermodynamics and possible mechanisms.

In this review, we clearly highlight the importance of the detailed study of the interactions between noble metal colloids (nanoparticles (NPs) and nanoclusters (NCs)) with serum albumins (SAs) due to their rapidly growing presence in biomedical research. Besides the changes in the structure and optical property of SA, we demonstrate that the characteristic localized surface plasmon resonance (LSPR) feature of the colloidal noble metal NPs and the size- and structure-dependent photoluminescence (PL) property of the sub-nanometer sized NCs are also altered differently because of the interactions between them. Namely, for plasmonic NPs - SA interactions the PL quenching of SA (mainly static) is identified, while the SA cause PL enhancement of the ultra-small NCs after complexation. This review summarizes that the thermodynamic nature and the possible mechanisms of the binding processes are dependent partly on the size, morphology, and type of the noble metals, while the chemical structure as well as the charge of the stabilizing ligands have the most dominant effect on the change in optical features. In addition to the thermodynamic data and proposed binding mechanisms provided by three-dimensional spectroscopic techniques, the quantitative and real-time data of "quasi" two-dimensional sensor apparatus should also be considered to provide a comprehensive evaluation on many aspects of the particle/cluster - SA interactions.

Detailed Calorimetric Analysis of Mixed Micelle Formation from Aqueous Binary Surfactants for Design of Nanoscale Drug Carriers.

While numerous papers have been published according to the binary surfactant mixtures, only a few articles provide deeper information on the composition dependence of the micellization, and even less work attempts to apply the enhanced feature of the mixed micelles. The most important parameter of the self-assembled surfactants is the critical micelle concentration (cmc), which quantifies the tendency to associate, and provides the Gibbs energy of micellization. Several techniques are known for determining the cmc, but the isothermal titration calorimetry (ITC) can be used to measure both cmc and enthalpy change (ΔmicH) accompanying micelle formation. Outcomes of our calorimetric investigations were evaluated using a self-developed routine for handling ITC data and the thermodynamic parameters of mixed micelle formation were obtained from the nonlinear modelling of temperature- and composition- dependent enthalpograms. In the investigated temperature and micelle mole fractions interval, we observed some intervals where the cmc is lower than the ideal mixing model predicted value. These equimolar binary surfactant mixtures showed higher solubilization ability for poorly water-soluble model drugs than their individual compounds. Thus, the rapid and fairly accurate calorimetric analysis of mixed micelles can lead to the successful design of a nanoscale drug carrier.

Optimization of layering technique and secondary structure analysis during the formulation of nanoparticles containing lysozyme by quality by design approach.

In our study, core-shell nanoparticles containing lysozyme were formulated with precipitation and layering self-assembly. Factorial design (DoE) was applied by setting the process parameters during the preparation with Quality by Design (QbD) approach. The factors were the concentration of lysozyme and sodium alginate, and pH. Our aim was to understand the effect of process parameters through the determination of mathematical equations, based on which the optimization parameters can be predicted under different process parameters. The optimization parameters were encapsulation efficiency, particle size, enzyme activity and the amount of α-helix structure. The nanoparticles were analysed with transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. Based on our results, we found that pH was the most important factor and pH 10 was recommended during the formulation. Enzyme activity and α-helix content correlated with each other very well, and particle size and encapsulation efficiency also showed very good correlation with each other. The results of the α-helix content of FTIR and CD measurements were very similar for the precipitated lysozyme due to the solid state of lysozyme. The mixing time had the best influence on the encapsulation efficiency and the particle size, which leads to the conclusion that a mixing time of 1 h is recommended. The novelty in our study is the presentation of a mathematical model with which the secondary structure of the protein and other optimization parameters can be controlled in the future during development of nanoparticle based on the process parameters.