Electrospun Mucoadhesive Zein/PVP Fibroporous Membrane for Transepithelial Delivery of Propranolol Hydrochloride.

Mucoadhesive drug delivery systems have been extensively studied to effectively reduce the limitations of conventional drug delivery systems. Zein and polyvinyl pyrrolidone (PVP) are appraised for mucoadhesive properties. This study focuses on developing a mechanically stable zein/PVP electrospun membrane for propranolol hydrochloride (PL) transport. Fourier transform infrared, Raman spectra, and swelling studies gave evidence for PVP crosslinking, whereas circular dichroism spectroscopy revealed crosslinking of zein owing to the conformational change from α-helix to ß-sheet. A 10 h thermal treatment of zein/PVP imparted 3.92 ± 0.13 MPa tensile strength to the matrix. Thermally crosslinked electrospun zein/PVP matrix showed 22.1 ± 0.1 g mm work of adhesion in porcine buccal mucosa tissue. Qualitative and quantitative evaluation of cytotoxicity in RPMI 2650 has been carried out. The in vitro drug release profile of PL from thermally crosslinked zein/PVP best fitted with the Korsmeyer-Peppas model. Immunostaining of ß-catenin adherens junctional protein confirmed the absence of paracellular transport through the junctional opening. Still, drug permeation was observed through the porcine buccal mucosa, attributed to the transcellular transport of PL owing to its lipophilicity. The ex vivo permeation of PL through porcine buccal mucosa was also evaluated.

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Carbonized zein nanosheets with intrinsic enzyme-mimicking activities and high photothermal conversion efficiency for synergistic cancer therapy.

With the rapid development of biology and nanotechnology, designing nanomaterials with intrinsic enzyme-like activities has attracted huge attention in recent years. Herein, for the first time, we use zein as a new protein precursor to prepare N-rich carbonized zein nanosheets (C-Zein) via facile pyrolysis. Zein is an inert, biodegradable and sustainable natural biopolymer. After high-temperature carbonization, zein can be converted into highly catalytically active C-Zein, which can possess excellent peroxidase- and oxidase-like catalytic activities. Such intrinsic enzyme-like activities of C-Zein are closely related to its graphitization degree, the ratio of graphitic nitrogen and the formation of disordered graphene. Intriguingly, C-Zein also exhibits high photothermal conversion efficiency in the near-infrared (NIR) region. Coupling their unique photothermal and catalytic properties, the as-prepared C-Zein can act as a robust agent for synergistic photothermal-catalytic cancer treatment under the irradiation of NIR light. We expect that this work paves the way to use zein for designing efficient artificial enzymes and accelerate further growth in exploring its new biomedical and pharmaceutical applications.

Tuning of Molecular Interactions between Zein and Tannic Acid to Modify Sunflower Sporopollenin Exine Capsules: Enhanced Stability and Targeted Delivery of Bioactive Macromolecules.

There are multiple obstacles for the storage and digestion of orally administered bioactive macromolecules. This study developed a low-cost and sustained-release delivery system (sporopollenin exine capsules with zein/tannic acid modification) of proteins with excellent storage stability, and at the same time provided insights into the sustained-release mechanism through exploring the interaction between zein and tannic acid (TA). ß-Galactosidase (ß-Gal) was utilized as a model protein and loaded into sporopollenin exine capsules (SECs), which were then coated with the zein/TA system. Under the optimized zein/TA conditions, the zein/TA system showed better performance than the zein alone system in the sustained release of ß-Gal, with the residual activity of about 70.26% after 24 h of simulated digestion. Evaluation of the storage stability demonstrated a ß-Gal residual activity of nearly 90% for 28 days at 25 °C. Additionally, FTIR analysis demonstrated that the stability of the zein/TA system depends on both hydrogen bonding and certain covalent bonding through the Schiff-base reaction, and the sustained release is regulated by the bonding strength.

Enzyme- and Relative Humidity-Responsive Antimicrobial Fibers for Active Food Packaging.

Active food packaging materials that are sustainable, biodegradable, and capable of precise delivery of antimicrobial active ingredients (AIs) are in high demand. Here, we report the development of novel enzyme- and relative humidity (RH)-responsive antimicrobial fibers with an average diameter of 225 ± 50 nm, which can be deposited as a functional layer for packaging materials. Cellulose nanocrystals (CNCs), zein (protein), and starch were electrospun to form multistimuli-responsive fibers that incorporated a cocktail of both free nature-derived antimicrobials such as thyme oil, citric acid, and nisin and cyclodextrin-inclusion complexes (CD-ICs) of thyme oil, sorbic acid, and nisin. The multistimuli-responsive fibers were designed to release the free AIs and CD-ICs of AIs in response to enzyme and RH triggers, respectively. Enzyme-responsive release of free AIs is achieved due to the degradation of selected polymers, forming the backbone of the fibers. For instance, protease enzyme can degrade zein polymer, further accelerating the release of AIs from the fibers. Similarly, RH-responsive release is obtained due to the unique chemical nature of CD-ICs, enabling the release of AIs from the cavity at high RH. The successful synthesis of CD-ICs of AIs and incorporation of antimicrobials in the structure of the multistimuli-responsive fibers were confirmed by X-ray diffraction and Fourier transform infrared spectrometry. Fibers were capable of releasing free AIs when triggered by microorganism-exudated enzymes in a dose-dependent manner and releasing CD-IC form of AIs in response to high relative humidity (95% RH). With 24 h of exposure, stimuli-responsive fibers significantly reduced the populations of foodborne pathogenic bacterial surrogates Escherichia coli (by ∼5 log unit) and Listeria innocua (by ∼5 log unit), as well as fungi Aspergillus fumigatus (by >1 log unit). More importantly, the fibers released more AIs at 95% RH than at 50% RH, which resulted in a higher population reduction of E. coli at 95% RH. Such biodegradable, nontoxic, and multistimuli-responsive antimicrobial fibers have great potential for broad applications as active and smart packaging systems.

Topography and biocompatibility of patterned hydrophobic/hydrophilic zein layers.

The topography and biocompatibility of zein layers adsorbed on patterned templates containing hydrophilic and hydrophobic regions were investigated. Nanopatterned templates consisting of hydrophilic lines on a hydrophobic background were drawn by dip-pen nanolithography (DPN) on gold-coated surfaces. 16-Mercaptohexadecanoic acid (COOH(CH(2))(15)SH, MHA) was used as primary ink to generate hydrophilic lines. Unpatterned surfaces were backfilled with 18-octadecanethiol (CH(3)(CH(2))(17)SH, ODT), which generated hydrophobic regions. Zein was allowed to adsorb on patterned surfaces from alcohol-water solutions. The topography of zein deposits was observed by atomic force microscopy (AFM). Height profiles from AFM measurements revealed that zein deposits followed closely the nanopatterned templates. The biocompatibility of zein layers assembled over hydrophilic/hydrophobic micropatterned templates was investigated. Templates containing MHA lines and ODT regions were generated by micro-contact printing (microCP). Mouse fibroblasts seeded on patterned zein layers proliferated on zein deposited over MHA lines, but not on zein over ODT. The experiment indicated that fibroblast cells were able to respond to variations in the underlying surface chemistry, transmitted by the different orientation adopted by zein on the different substrates. This property may be useful in controlling the spatial distribution of cells on patterned protein layers.

Mechanical properties and in vitro biocompatibility of porous zein scaffolds.

A porous scaffold utilizing hydrophobic protein zein was prepared by the salt-leaching method for tissue engineering. The scaffolds possessed a total porosity of 75.3-79.0%, compressive Young's modulus of (28.2+/-6.7)MPa-(86.6+/-19.9)MPa and compressive strength of (2.5+/-1.2)MPa-(11.8+/-1.7)MPa, the percentage degradation of 36% using collagenase and 89% using pepsin during 14 days incubation in vitro. The morphology of pores located on the surface and within the porous scaffolds showed good pore interconnectivity by scanning electron microscopy (SEM). Rat mesebchymal stem cells (MSCs) could adhere, grow, proliferate and differentiate toward osteoblasts on porous zein scaffold. With the action of dexamethasone, the cells showed a relative higher activity of alkaline phosphatase (ALP) and a higher proliferating activity (p<0.05) than those of MSCs without dexamethasone.

Antioxidant activity of zein hydrolysates in a liposome system and the possible mode of action.

Maize zein was hydrolyzed for 0.5-5 h by alcalase or papain. Protein solubility increased (P < 0.05) with the degree of hydrolysis (DH) and was higher for alcalase-hydrolyzed zein than for papain-hydrolyzed zein. The zein hydrolysates with both enzymes consisted mostly of small peptides or amino acids nondetectable by 15% acrylamide gel electrophoresis. Alcalase-hydrolyzed zein exhibited a stronger (P < 0.05) antioxidant activity than papain-hydrolyzed zein, as indicated by peroxide and thiobarbituric acid-reactive substance values in a liposome-oxidizing system. Zein hydrolysates possessed strong Cu(2+) chelation ability and marked reducing power, both of which were accentuated with hydrolysis time. The protein hydrolysates also showed strong radical-scavenging ability, which was not influenced by hydrolysis time. The antioxidant activity of alcalase-hydrolyzed zein at some specific low concentrations was close or comparable to those of butylated hydroxyanisole, alpha-tocopherol, and ascorbate. Although intact zein displayed an antioxidative effect, it was far less potent than hydrolyzed zein. The results demonstrated that enzyme-hydrolyzed zein can act as a metal ion chelator or a hydrogen donor, as well as a radical stabilizer to inhibit lipid oxidation. The effectiveness of the protein hydrolysates appeared to depend on both the concentration and the peptide/amino acid composition of the soluble protein fraction.

Compositional and moisture content effects on the biodegradability of zein/ethylcellulose films.

The effect of moisture content and film composition on biodegradability is the focus of this study. Flexible films were first characterized for the effect on water sorption isotherms of relative humidity, temperature, zein content, and the addition of the plasticizers stearic acid, poly(ethylene glycol), or etoxylated ricine oil. Zein/ethylcellulose (EC) mixture films had a behavior between that for pure zein and EC films, which had the lowest water sorption. For films with plasticizer, the lowest water sorption at 25 degrees C was observed for those with stearic acid. Biodegradability of zein/EC films, evaluated using bacterial cultures selected for their zein proteolytic activity and isolated from a local solid waste landfill and a lagoon, showed no plasticizer effect even though its effect on moisture content was significant. Large differences were observed at different film zein concentration with the highest biodegradability for 100% zein. However, biodegradability did not mimic the water sorption behavior of zein/EC mixture films.

NMR techniques in drug delivery: application to zein protein complexes.

Zein is a protein containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids. NMR techniques were used to detect binding interactions and measure affinity between zein and three different drugs: tetracycline, amoxicillin and indomethacin. The release study of zein microparticle formulations containing any of these drugs was confronted with the affinity results, showing a remarkable correlation. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising for the rational design of appropriate drug vehicles for drug delivery.

Cytocompatible cross-linking of electrospun zein fibers for the development of water-stable tissue engineering scaffolds.

This paper reports a new method of cross-linking electrospun zein fibers using citric acid as a non-toxic cross-linker to enhance the water stability and cytocompatibility of zein fibers for tissue engineering and other medical applications. The electrospun structure has many advantages over other types of structures and protein-based biomaterials possess unique properties preferred for tissue engineering and other medical applications. However, ultrafine fiber matrices developed from proteins have poor mechanical properties and morphological stability in the aqueous environments required for medical applications. Efforts have been made to improve the water stability of electrospun protein scaffolds using cross-linking and other approaches, but the current methods have major limitations, such as cytotoxicity and low efficiency. In this research electrospun zein fibers were cross-linked with citric acid without using any toxic catalysts. The stability of the cross-linked fibers in phosphate-buffered saline and their ability to support the attachment, spreading and proliferation of mouse fibroblast cells were studied. The cross-linked electrospun fibers retained their ultrafine fibrous structure even after immersion in PBS at 37 degrees C for up to 15 days. Citric acid cross-linked electrospun zein scaffolds showed better attachment, spreading and proliferation of fibroblast cells than uncross-linked electrospun zein fibers, cross-linked zein films and electrospun polylactide fibers.

Membrane-bound protein in giant vesicles: induced contraction and growth.

Cell-sized giant vesicles, produced by electroformation, were composed of phospholipids and zein (a hydrophobic protein that occupied a substantial percentage of the vesicle surface). Addition of sodium dodecyl sulfate removed the protein into the bulk phase, which led to a shrinkage of the vesicles. The vesicle bilayers were able to heal themselves from the damage caused by the departure of the zein, allowing the bilayers to maintain their spherical morphology. Giant vesicle growth was also observed when the following components were mixed (all four being necessary): (a) negatively charged giant vesicles, (b) membrane-incorporated zein, (c) positively charged submicroscopic vesicles (almost 103 times smaller than the giant vesicles), and (d) sodium dodecyl sulfate. The simplest mechanism consistent with literature data involves electrostatically promoted binding of the small vesicles (weakened by the surfactant) onto the giant vesicle surface, followed by the merging of membranes at protein-induced "fusion hot spots". The "feeding" of small vesicles by giant vesicles then leads to growth.

Exploring the interaction of the surfactant N-terminal domain of gamma-Zein with soybean phosphatidylcholine liposomes.

Zeins are maize storage proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner surface of the protein body membrane, and its N-terminal, proline-rich, repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation and forms cylindrical micelles in aqueous solution. Here we explore the interaction of (VHLPPP)(8) with soybean phosphatidylcholine unilamellar lipid vesicles and examine its effect on the stability and permeability of the liposome membrane. The amphipathic N-terminal domain of gamma-zein interacts with the membrane and assembles to form extended domains over the phospholipid membrane. The interaction between the peptide and the membrane increases the stability and permeability of the liposome membrane. The spontaneous amphipathic aggregation of (VHLPPP)(8) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies.

The effect of varying the quality of dietary protein and energy on food intake and growth in the Zucker rat.

1. Food intake and rates of protein, lipid and energy deposition were measured for lean and obese (fatty) Zucker rats offered to appetite from 34 d of age to slaughter at 66 d of age, one of sixteen semi-synthetic diets. Measurements were also made of the digestibility of dietary protein and the metabolizability of dietary energy. Total carcasses were analysed for protein and lipid, and body energy was calculated thereby. Changes in body constituents were calculated by the comparative-slaughter technique. 2. In Expt 1, four rats of each phenotype and sex were offered one of four diets, each of which contained either 150 or 300 g casein (150 C and 300 C respectively)/kg and either 150 or 300 g cellulose (150 cell and either 150 or 300 g casein (150 C and 300 C respectively)/kg and either 150 or 300 g cellulose (150 CELL and 300 CELL respectively)/kg (diets 150 C/150 CELL, 150 C/300 CELL, 300 C/150 CELL and 300 C/300 CELL. As expected, males ate more and had higher rates of protein deposition than female animals of the same phenotype on all diets. These sex differences were greater for the lean phenotype. The results for animals in this experiment are presented with, and discussed in relation to, those obtained previously for animals of both sexes fed on cellulose-free diets having these two levels of casein. 3. In Expt 2, four female animals of each phenotype were fed one of twelve semi-synthetic diets, each of which contained casein, gluten or zein at one of the following levels (g crude protein (nitrogen x 6.25)/kg diet): 93, 132, 267 or 627. On all diets containing zein both fatty and lean rats had similar, low food intakes and failed to grow. Fatty rats fed on diets containing casein or gluten had higher rates of food intake, weight gain, lipid and energy deposition than lean rats, but similar rates of protein deposition. Rats fed on diets having the two lower levels of casein ate more and grew better than animals of the same phenotype fed on the two corresponding diets containing gluten but at higher protein levels differences in food intake and growth attributable to differences in protein quality disappeared and furthermore, the rate of protein deposition became similar and maximal for both phenotypes. 4. The results from both experiments are discussed in relation to previous work on appetite control in the Zucker rat. It appears that fatty and lean rats eat during growth to attain the maximal rate of protein deposition of which they are capable. The rate of lipid deposition would appear to be of no importance in the food intake regulation of animals depositing protein maximally. 5. Rats given diets that fail to support maximal rates of protein deposition appear to regulate their intake of digestible energy rather than that of digestible protein. They do not overeat protein-deficient diets in order to acquire sufficient protein for maximal growth although the factors that induce satiety in these animals are unknown.