Influence of Formulation Factors on the Preparation of Zein Nanoparticles

The main objective of the present study was to investigate the influence of various formulation parameters on the preparation of zein nanoparticles. 6,7-dihydroxycoumarin (DHC) was used as a model hydrophobic compound. The influence of pH of the aqueous phase, buffer type, ionic strength, surfactant, and zein concentration on particle size, polydispersity index, and zeta potential of DHC-loaded zein nanoparticles were studied. Smaller nanoparticles were formed when the pH was close to the isoelectric point of zein. DHC-loaded zein nanoparticles prepared using citrate buffer (pH 7.4) was better than phosphate buffer in preventing particle aggregation during lyophilization. The ionic strength did not have a significant influence on the particle size of DHC-loaded zein nanoparticles. A combination of Pluronic F68 and lecithin in 2:1 ratio stabilized the zein nanoparticles. An increase in zein concentration led to increase in particle size of DHC-loaded zein nanoparticles. The use of optimal conditions produced DHC-loaded nanoparticles of 256 ± 30 nm and an encapsulation efficiency of 78 ± 7%. Overall, the study demonstrated the optimal conditions to prepare zein nanoparticles for drug encapsulation.KEY WORDS: drug delivery, particle size distribution, pH nanoprecipitation, protein polymers, zein, zeta potential     

Thermoresponsive self-assemblies of cyclic and branched oligosaccharide-block-poly(N-isopropylacrylamide) diblock copolymers into nanoparticles

This paper discusses the thermoresponsive nanoparticles obtained by self-assemblies of nonlinear oligosaccharide-based diblock copolymer systems. These diblock copolymers were synthesized by Cu(I)-catalyzed 1,3-dipolar azide/alkyne cycloaddition ("click" reaction) of propargyl-functionalized β-cyclodextrin (βCyD) and xyloglucooligosaccharide (XGO) with poly(N-isopropylacrylamide) (PNIPAM) having a terminal azido group prepared by atom transfer radical polymerization (ATRP). Elastic and quasi-elastic light scattering analysis of the dibock copolymers in H(2)O indicated that thermodynamic phase transitions of the PNIPAM blocks at their cloud points (T(cp)s ≈ 34 °C), around lower critical solution temperatures (LCSTs), triggered their self-assemblies into the nanoparticles. These nanoparticles had narrow size distributions and small interphases (i.e., sharp boundaries). The mean hydrodynamic radii (R(h)s) of the βCyD and XGO-based nanoparticles were determined to be around 150 and 250 nm upon slow heating (i.e., step-by-step heating), and 364 and 91.5 nm upon fast heating, respectively, depending on a predominance of the interchain association or the intrachain contraction. Transmission electron microscope (TEM) and field emission gun-scanning electron microscopy (FEG-SEM) images of the nanoparticles clearly showed compact spherical nanoparticles whose cores are mainly made with the PNIPAM blocks, whereas the rough shells consist in the oligosaccharidic blocks.     

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.     

Sweet block copolymer nanoparticles: preparation and self-assembly of fully oligosaccharide-based amphiphile

The preparation of biocompatible nanocarriers that have potential applications in the cosmetic and health industries is highly desired. The self-assembly of amphiphilic block copolymers displaying biosourced polysaccharides at the surface is one of the most promising approaches. In the continuity of our works related to the preparation of "hybrid" amphiphilic oligosaccharide-based block copolymers, we present here the design of a new generation of self-assembled nanoparticles composed entirely of oligosaccharide-based amphiphilic block co-oligomers (BCO). These systems are defined by a covalent linkage of the two saccharidic blocks through their reducing end units, resulting in a sweet "head-to-head" connection. As an example, we have prepared and studied a BCO in which the hydrophilic part is composed of a free maltoheptaosyl derivative clicked to a hydrophobic part composed of a peracetylated maltoheptaosyl derivative. This amphiphilic BCO self-assembles to form spherical micelles in water with an average diameter of 30 nm. The efficient enzymatic hydrolysis of the maltoheptaose that constitutes the shell of the micelles was followed by light scattering and colorimetric methods.     

Fabrication of curcumin-loaded zein nanoparticles stabilized by sodium caseinate/sodium alginate: Curcumin solubility,thermal properties,rheology, and stability

Curcumin is a polyphenol with multiple biological activities, but its extremely poor water solubility severely limits its application in the food industry. The purposes of this work were to study the effect of nano-encapsulation on the water solubility of curcumin (C), the interaction of curcumin with zein (Z), the thermal properties, rheological properties, and the stability under different environmental pressures of the nanoparticles. The results of particle size, zeta potential, and surface hydrophobicity (H₀) indicated that the combination of coating materials including sodium caseinate (SC) and sodium alginate (SA) with zein nanoparticles by electrostatic interaction led to a gradual increase in the particle size of composite nanoparticles and a decrease in surface hydrophobicity. The nano-encapsulation significantly improved the water solubility of curcumin and causing its crystal structure to change to an amorphous state. Fourier transform infrared spectroscopy confirmed that curcumin bound to zein through hydrogen bonding. Rheological test results showed that the coating materials combined with zein led to an increase in the apparent viscosity of the nanoparticles. The stability analysis results indicated that the composite nanoparticles with a sodium alginate coating have excellent stability of pH, salt solution and storage, and excellent anti-gastrointestinal fluids digestion characteristics when compared to pure protein nanoparticles.     

Selective separation of human serum albumin with copper(II) chelated poly(hydroxyethyl methacrylate) based nanoparticles

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 300 nm in diameter and with a polydispersity index of 1.156 were produced by surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 996 m²/g. Metal-chelating ligand 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was covalently attached to the PHEMA nanoparticles. IMEO content was 0.97 mmol IEMO/g. The morphology and properties of these nanoparticles were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The Cu²⁺-chelated PHEMA–IMEO nanoparticles were used in the adsorption-elution studies of human serum albumin (HSA) in a batch system. Maximum HSA adsorption amount of the Cu²⁺ chelated nanoparticles was 680 mg HSA/g. The PHEMA–IMEO–Cu²⁺ nanoparticles exhibited a quite high adsorption capacity and fast adsorption rate due to their high specific surface area and the absence of internal diffusion resistance.     

Selective Interactions of Zein Microspheres with Different Class of Drugs: An In Vitro and In Silico Analysis

In this study, we have evaluated the interactions of zein microspheres with different class of drugs (hydrophobic, hydrophilic, and amphiphilic) using in vitro and in silico analysis. Zein microspheres loaded with aceclofenac, metformin, and promethazine has been developed by solvent evaporation technique and analyzed for its compatibility. The physical characterization depicted the proper encapsulation of hydrophobic drug in the microspheres. The in vitro release study revealed the sustaining ability of the microspheres in the following order: hydrophobic > hydrophilic > amphiphilic. In silico analysis also confirmed the better binding affinity and greater interactions of hydrophobic drug with zein. The above results revealed that zein is more suitable for hydrophobic drugs in the development of sustained drug delivery systems using solvent evaporation technique. The study therefore envisages a scope for identifying the most suitable polymer for a sustained drug delivery system in accordance with the nature of the drug.KEY WORDS: hydrophilic drugs, hydrophobic drugs, in silico analysis, protein-drug interactions, solvent evaporation, zein microspheres     

Structural effects on the biodistribution and positron emission tomography (PET) imaging of well-defined (64)Cu-labeled nanoparticles comprised of amphiphilic block graft copolymers

The synthesis of poly(methyl methacrylate-co-methacryloxysuccinimide-graft-poly(ethylene glycol)) (PMMA-co-PMASI-g-PEG) via living free radical polymerization provides a convenient route to well-defined amphiphilic graft copolymers having a controllable number of reactive functional groups, variable length PEG grafts, and low polydispersity. These copolymers were shown to form PMMA-core/PEG-shell nanoparticles upon hydrophobic collapse in water, with the hydrodynamic size being defined by the molecular weight of the backbone and the PEG grafts. Functionalization of these polymeric nanoparticles with a 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) ligand capable of chelating radioactive 64Cu nuclei enabled the biodistribution and in vivo positron emission tomography of these materials to be studied and directly correlated to the initial structure. Results indicate that nanoparticles with increasing PEG chain lengths show increased blood circulation and low accumulation in excretory organs, suggesting the possible use of these materials as stealth carriers for medical imaging and systemic administration.     

Effect of Hydrophilic and Lipophilic Compounds on Zein Microstructures

Zein, the prolamine of corn, contains nearly an equal amount of hydrophilic and lipophilic amino acid residues. Its tertiary structure has a regular geometry measuring 17 × 4.5 × 1.2 nm. The structure of zein allows it to function as a polymeric amphiphile. Zein had been observed to self-assemble into periodic bilayer structures and nanotubes. This work investigated the structural development of zein self-assemblies as affected by the hydrophilic–lipophilic balance (HLB) of the system. The formation of several structures, including spheres, sponge, and lamellae, were identified. Images were obtained by SEM, AFM, and FIB/SEM. The radius of curvature of the observed assemblies was affected by the HLB of the components in the system. Thus, lipophilic flavor oils increased the curvature of zein spheres producing smaller spheres. Curvature decreased in the presence of amphiphilic fatty acids forming sponges with interconnected channels. Hydrophilic compounds decreased the curvature to the point of forming smooth films. The results of this study see a future in microencapsulation and controlled release systems for flavor and bioactive compounds in the food industry.     

Application of polyhydroxyalkanoate binding protein PhaP as a bio-surfactant

PhaP or phasin is an amphiphilic protein located on surfaces of microbial storage polyhydroxyalkanoates granules. This study aimed to explore amphiphilic properties of PhaP for possible application as a protein surfactant. Following agents were used to conduct this study as controls including bovine serum albumin, sodium dodecyl sulfate (SDS), Tween 20, sodium oleate, a commercial liquefied detergent together with the same amount of PhaP. Among all these tested control surfactants, PhaP showed the strongest effect to form emulsions with lubricating oil, diesel, and soybean oil, respectively. PhaP emulsion stability study compared with SDS revealed that PhaP had a stronger capability to maintain a very stable emulsion layer after 30 days while SDS lost half and two-thirds of its capacity after 2 and 30 days, respectively. When PhaP was more than 200 μg/ml in the water, all liquids started to exhibit stable emulsion layers. Similar to SDS, PhaP significantly reduced the water contact angles of water on a hydrophobic film of biaxially oriented polypropylene. PhaP was thermally very stable, it showed ability to form emulsion and to bind to the surface of polyhydroxybutyrate nanoparticles after a 60- min heating process at 95 °C. It is therefore concluded that PhaP is a protein with thermally stable property for application as natural and environmentally friendly surfactant for food, cosmetic, and pharmaceutical usages.     

Characteristics of an Acid protease from maize endosperm

An assay has been developed to measure protease activity in endosperm extracts of maize seeds. With hemoglobin as substrate, the enzyme(s) has a pH optimum of 3.8 and a temperature optimum of 46 C. It also degrades gliadin, edestin, bovine serum albumin, and partially hydrolyzed zein and glutelin under standard assay conditions. The enzyme(s) has endopeptidase activity with all substrates tested. When undenatured zein and glutelin are suspended in an agar gel, both are efficiently degraded. Using this assay, the protease activity increases from day 3 to day 8 after inhibition and then declines.     

Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15′-oxygenase (BCO1) and the β-carotene-9′,10′-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15′-apocarotenoids, C₂₀), whereas eccentric cleavage by BCO2 produces long-chain (>C₂₀) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1^−/−, Bco2^−/−, and Bco1^−/−Bco2^−/− double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10′-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.     

Hyaluronate-covered nanoparticles for the therapeutic targeting of cartilage

Hyaluronic acid (HA) has a high affinity for the CD44 receptor present at the surface of articular cells, particularly of chondrocytes. HA-covered polylactide nanoparticles containing bioactive compounds such as HA and chondroitin sulfate (CS) were thus prepared in order to achieve a controlled delivery targeted to cartilage cells after injection near articular alterations/erosions. Such nanoparticles (diameter = 700 nm) were prepared by double emulsion/solvent evaporation, using amphiphilic derivatives of HA, as stabilizer of the secondary emulsion. These nanoparticles were incubated with articular cells, and several tests were carried out. First, they proved that the nanospheres provoked no decrease in cell viability, even after 72 h of contact. Second, a confocal microscopy analysis on fluorescent HA-covered particles showed that they were captured by articular cells, while with those covered with poly(vinyl alcohol), the uptake was far lower. Third, a scattering electron microscopy analysis proved that the HA-coated nanoparticles were localized in the cell intracytoplasmic area.     

Synthesis of lipophosphoramidyl-cyclodextrins and their supramolecular properties

The synthesis of lipophosphoramidyl-β-CD was obtained by an Atherton-Todd (AT) reaction that involved dioleylphosphite and either functionalized permethylated or native β-cyclodextrin. This AT reaction that produced dioleylphosphoramide by making use of the amino group grafted on cyclodextrin, was optimized for these cyclic oligosaccharides. These new amphiphilic compounds were fully characterized, and their self-assembling properties were investigated: the mean size diameter and polydispersity measured by Dynamic Light Scattering (DLS) were affected by the nature of the aqueous media and the temperature of storage. The encapsulation properties of these nanoparticles have been evaluated using carboxyfluorescein and scopolamine derivatives as model of guests.     

Emulsifying properties of biodegradable polylactide-grafted dextran copolymers

Amphiphilic glycopolymers, polylactide-grafted dextran copolymers (Dex-g-PLA), were synthesized with a well-controlled architecture obtained through a three-step procedure: partial silylation of the dextran hydroxyl groups, ring-opening polymerization of D,L-lactide initiated from remaining hydroxyl groups, silylether deprotection under very mild conditions. Depending on their proportion in polylactide (PLA), these copolymers exhibited solubility either in water or in organic solvents. The emulsifying properties of these glycopolymers were studied: depending on their PLA-to-dextran ratio, they were able to stabilize either direct or inverse emulsions. Droplet size was related to the amount of amphiphilic copolymer in the continuous phase. The aging mechanism of both direct and inverse emulsions was shown to be Ostwald ripening in the first weeks following preparation. Finally inverse miniemulsion copolymerization of acrylamide and N, N'-methylenebisacrylamide was performed in the presence of an amphiphilic Dex-g-PLA stabilizer. Polyacrylamide hydrogel nanoparticles were prepared in that way.     

钛- 载药纳米复合材料的组装及其性质的研究

目的:构建一种能结合钛表面的载药纳米粒及钛-载药纳米复合材料的组装和性质研究。方法:(1)多巴胺修饰的非离子表面活性剂多巴胺-泊洛沙姆188(Dop-Poloxamer188)的合成和检测;(2)Dop-Poloxamer188作为表面活性剂、PLGA作为油相基质,制备纳米粒及纳米粒载药和表征;(3)钛片的预处理及钛片与修饰后的纳米粒的结合;(4)纳米粒修饰后的钛表面的表征。结果:新合成的Dop-Poloxamer188在285 nm左右有紫外吸收峰,说明多巴胺成功的修饰在Poloxamer188的两端;Dop-Poloxamer188能和PLGA制备出很好的纳米粒,平均粒径在110 nm左右,PDI小于0.1;多巴胺修饰的纳米粒与钛片通过简单的浸渍过程结合后,通过水接触角、场发射扫面电镜(Fe-SEM)、荧光显微镜、X射线光电子能谱(XPS)等仪器检测都显示多巴胺修饰的纳米粒成功且牢固的修饰在钛片表面。结论:成功达到钛表面的载药纳米粒修饰的目的,为钛种植体的载药系统提供了新的思路和方法。     

Silver Nanoparticles Modified by Gelatin with Extraordinary pH Stability and Long-Term Antibacterial Activity

The potential for application of any nanoparticles, including silver nanoparticles (AgNPs), is strongly dependent on their stability against aggregation. Therefore, improvement of this parameter is a key task, especially in the case of AgNPs, because a correlation between size and biological activity has been demonstrated. In the present work, a natural stabilizer, gelatin, was investigated for the stabilization of AgNPs in an aqueous dispersion. The particles were prepared via a modified Tollens process, and the gelatin modifier was added prior to the reducing agent. The stability against aggregation of the AgNPs prepared by this method was more than one order of magnitude higher (on the basis of the critical coagulation concentration (CCC)) than that of AgNPs prepared via a similar method but without the assistance of gelatin. Their high stability against aggregation was confirmed over wide pH range (from 2 to 13) in which the particles did not exhibit rapid aggregation; such stability has not been previously reported for AgNPs. Additionally, gelatin not only fulfills the role of a unique stabilizer but also positively influences the modified Tollens process used to prepare the AgNPs. The diameter of the gelatin-modified AgNPs was substantially smaller in comparison to those prepared without gelatin. The polydispersity of the dispersion significantly narrowed. Moreover, the gelatin-stabilized AgNPs exhibited long-term stability against aggregation and maintained high antibacterial activity when stored for several months under ambient conditions.     

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Spray-Dried Gliadin Powders Inclusive of Linoleic Acid (Microcapsules): Their Preservability,Digestibility and Application to Bread Making

An aqueous-alcohol solution containing gliadin (or zein) and linoleic acid was pulverized by spray-drying and the powders were examined for their stability, digestibility and application. Linoleic acid in the powders was kept stable during three months’ storage in each case of low, moderate and high water activity. When the spray-dried powders were treated with such digestive enzymes as pepsin and pancreatin, linoleic acid was effectively changed into the hexane-extractable form concomitantly with the digestion of gliadin. The powders were administered to rats, instead of gluten and essential fatty acid in a semi-purified diet, and there was no abnormal excretion of fatty acid into the faeces. These facts indicate that two components of the powders could be digested and absorbed in the gut as usual. On the other hand, supplementation of bread with gliadin and linoleic acid in the form of spray-dried powders led to a considerable increase in bulk after baking, without affecting the taste and flavor. Linoleic acid in a loaf of bread seemed to be fairly resistlant to peroxidation during baking in an oven and during storage under aerobic conditions for a week.