5-methyl-pyrrolidinone chitosan films as carriers for buccal administration of proteins

The purpose of this research was to investigate 5-methyl-pyrrolidinone chitosan (MPC) films as carriers for buccal delivery of protein drugs. Placebo and protein-loaded MPC films were prepared by casting and were then cross-linked with tripolyphosphate at different pH conditions. Myoglobin (MHb) was chosen as the model protein because its molecular weight is under the permeability limit of the buccal mucosa. The observed characteristics like bioadhesiveness, swelling behavior, and in vitro release of MHb from loaded films furnish information on the functional behavior of these films. The results obtained show that the modulation of Mhb release was achieved only through chitosan cross-linking; the best results in release rate control were obtained by cross-linking performed at pH 6.5. Good bioadhesion properties were maintained even with high cross-linking degrees; the swelling index of MHb-loaded films at different cross-linking degrees evaluated at pH 7.4 and pH 6.4 were comparable to those of placebo films. By setting suitable tripolyphosphate cross-linking conditions for MPC films, one can control protein release without affecting bioadhesion. Published: September 1, 2006     

Swelling lecithin: cholesterol implants for the controlled release of proteins

This work demonstrated the effect of two salts as potential simple formulation excipients in modifying hydration properties, phase behavior, and protein release from lecithin-based implants. In vitro release of a model protein, bovine serum albumin (BSA), from cylindrical-shaped lecithin and lecithin:cholesterol (1:1 w/w) implants containing 0, 10, or 30% w/w NaCl or CaCl₂ was studied. In the absence of salts, BSA was released from lecithin and lecithin:cholesterol implants with a high monomer content and the release profiles were similar to those previously reported. Cholesterol increased the swelling, induced the formation of myelin structures, and reduced BSA release from the matrices. Addition of the salts to lecithin:cholesterol implants further enhanced the swelling, altered the hydrated morphology, and inhibited protein release. Analyses showed that BSA associated into multimers within these swollen lipid matrices but retained a high degree of protein native structure. Factors that may have contributed to the inhibition of the in vitro release included 1) the swollen multilamellar layers assembled as diffusional barriers, 2) adsorption of BSA onto the hydrated lipid vesicles, and 3) formation of protein aggregates.     

Curdlan gels as protein drug delivery vehicles

The use of curdlan, a natural -1,3-glucan, in protein drug delivery vehicles was studied by carrying out in vitro release studies with curdlan gels containing bovine serum albumin (BSA) as a model protein. Addition of urea (8 M) decreased the gel formation temperature to 37°C. Curdlan was hydroxyethylated in order to form gels under mild conditions such as physiological temperature and pH. In gels formed in 8 M urea solution, urea was almost released after 2 h while BSA was completely released after 45–100 h. The total time for complete release of BSA increased with curdlan concentration within gels. The strength of hydroxyethylated curdlan gels (385.7 dyne cm^–2) was weaker than that of curdlan gels formed in 8 M urea solution (6277 dyne cm^–2).     

Microemulsion for topical delivery of fenoprofen calcium: in vitro and in vivo evaluation

The aim of this study was to investigate microemulsion (ME) based topical delivery system for fenoprofen calcium (FPCa) to eliminate its oral gastrointestinal adverse effects. ME was prepared by the water titration method using oleic acid as oil phase, tween 80 as a surfactant and propylene glycol as a cosurfactant. Oleic acid was selected as oil phase due to its good solubilizing capacity. ME existence region was determined using pseudo-ternary phase diagrams for preparing different formulations. Six different formulations were selected with various values of oil (25–68%), water (2–3%), and the mixture of surfactant and cosurfactant (1:1) (24–67%). The selected ME formulae were characterized for optical birefringence, transmission electron microscopy (TEM), pH, % transmittance, electronic conductivity, drug content, droplet size, rheological properties and stability evaluation. In vitro release study of FPCa from ME s through the synthetic membrane and hairless rat skin were evaluated. The optimized formula ME5 consisting of 5% w/w FPCa, 60% w/w oleic acid as oil phase, 3% w/w aqueous phase, and 32% w/w of surfactant phase containing Tween 80 and propylene glycol (1:?1) showed the highest transdermal flux and highest skin permeation rate. Finally, the % inhibition of carrageenan-induced rat paw edema of the optimized formula ME5 was highly significant (p?0.001) as compared to plain gel of FPCa. In conclusion, ME is a promising technique for topical delivery of FPCa.     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Preparation, In Vitro Characterization and Preliminary In Vivo Evaluation of Buccal Polymeric Films Containing Chlorhexidine

The aim of this work was to investigate the suitability of some polymeric films as buccal systems for the delivery of the antiseptic drug chlorhexidine diacetate, considered as a valid adjunct in the treatment of oral candidiasis. Six different film formulations, mono- or double-layered, containing 5 or 10 mg of chlorhexidine diacetate, respectively, and alginate and/or hydroxypropylmethylcellulose and/or chitosan as excipients, were prepared by a casting-solvent evaporation technique and characterized in terms of drug content, morphology (scanning electron microscopy), drug release behavior, and swelling properties. Moreover, the in vivo concentrations of chlorhexidine diacetate in saliva were evaluated after application of a selected formulation on the oral mucosa of healthy volunteers. The casting-solvent evaporation proved to be a suitable technique for preparing soft, flexible, and easily handy mono- or double-layered chlorhexidine-loaded films. Some prepared formulations showed favorable in vitro drug release rates and swelling properties. The behavior of a selected formulation, chosen on the basis of its in vitro release results, was preliminarily investigated in vivo after application in the oral cavity of healthy volunteers. The films were well tolerated and the salivary chlorhexidine concentrations were maintained above the minimum inhibitory concentration for Candida albicans for almost 3 h. These preliminary results indicate that polymeric films can represent a valid vehicle for buccal delivery of antifungal/antimicrobial drugs.     

Encapsulation of proteins in biodegradable polymeric microparticles using electrospray in the Taylor cone-jet mode

Solvent extraction (or evaporation from a W(1)/O/W(2)-dispersion), coacervation, and spray drying methods are commonly employed to encapsulate protein drugs in polymeric microparticles for sustained delivery applications. To overcome the limitations of these methods, a novel electrospray method was developed to encapsulate a model protein drug-bovine serum albumin (BSA) in biodegradable polymeric microparticles and examine the feasibility of the process in not denaturing the protein. Microparticles of approximately 20 microm diameter with corrugated surfaces and smooth surfaces were observed by scanning electron microscope. Confocal laser scanning microscope images showed that BSA was distributed evenly in microparticles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to investigate the protein integrity of BSA released from the polymer matrix after 38 days. No protein degradation was observed during the 38 days release. The secondary structure of released BSA was characterized by Fourier transform infrared (FTIR) and circular dichroism (CD), which suggested that the released BSA was almost identical to native BSA. The encapsulation efficiency could reach 76% by adjusting the amount of the additive Pluronic F127 and processing parameters. The release profile could be tailored by the fabrication process and the sustained release of BSA could endure for more than 1 month. More than 80% of the bioactivity of BSA (evaluated by BSA ELISA kit) could be maintained after releasing from polymer matrix. Findings of the present study demonstrate that this novel electrospray method is a promising approach to encapsulate bioactive materials such as proteins, enzymes, antibiotics, and DNA fragments in biodegradable polymeric particles.     

Preparation and Evaluation of Diltiazem Hydrochloride Diffusion-Controlled Transdermal Delivery System

The objective was to investigate the suitable polymeric films for the development of diltiazem hydrochloride (diltiazem HCl) transdermal drug delivery systems. Hydroxypropyl methylcellulose (HPMC) and ethylcellulose (EC) were used as hydrophilic and hydrophobic film formers, respectively. Effects of HPMC/EC ratios and plasticizers on mechanical properties of free films were studied. Effects of HPMC/EC ratios on moisture uptake, in vitro release and permeation through pig ear skin of diltiazem HCl films were evaluated. Influence of enhancers including isopropyl myristate (IPM), isopropyl palmitate (IPP), N-methyl-2-pyrrolidone, oleic acid, polyethylene glycol 400, propylene glycol, and Tween80 on permeation was evaluated. It was found that addition of EC into HPMC film produced lower ultimate tensile strength, percent elongation at break and Young’s modulus, however, addition of EC up to 60% resulted in too hard film. Plasticization with dibutyl phthalate (DBP) produced higher strength but lower elongation as compared to triethyl citrate. The moisture uptake and initial release rates (0–1 h) of diltiazem HCl films decreased with increasing the EC ratio. Diltiazem HCl films (10:0, 8:2 and 6:4 HPMC/EC) were studied for permeation because of the higher release rate. The 10:0 and 8:2 HPMC/EC films showed the comparable permeation-time profiles, and had higher flux values and shorter lag time as compared to 6:4 HPMC/EC film. Addition of IPM, IPP or Tween80 could enhance the fluxes for approx. three times while Tween80 also shorten the lag time. In conclusion, the film composed of 8:2 HPMC/EC, 30% DBP and 10% IPM, IPP or Tween80 loaded with 25% diltiazem HCl should be selected for manufacturing transdermal patch by using a suitable adhesive layer and backing membrane. Further in vitro permeation and in vivo performance studies are required.     

From the Chef’s Mind to the Dish: How Scientific Approaches Facilitate the Creative Process

This work describes a practical way to optimize the high level of the chef creativity to produce rational approaches to food design. It is particularly focused on the preparation of two dishes: bubbly juice and false skin. For the first dish, three samples were prepared with egg white protein (EWP) and xanthan gum at pH 4.6 and pH 7.0. At pH 4.6 (isoelectric point), there were substantial differences of the interfacial dilational modulus of EWP when xanthan gum was added. At 1 mg/ml xanthan, the system showed a very strong interface (high viscoelasticity) compared to the other samples. Measuring half drainage time revealed which samples were the most stable. The properties discussed were related to stability. For the false skin dish, edible films were made by gelatin extracted from cod skins (A solution) and a mixture of cod skin gelatin and commercial gelatin (AG solution). The results showed that tensile strength (TS) of gelatin films increases almost by 25%, elongation at break (EAB) by 14%, and the Young modulus (E) by almost 100% when increasing protein concentration. To confirm water plasticizer effect, the results were compared to a gelatin film made with 30% glycerol (plasticizer). Water content affects to a great extent the mechanical properties of the films. Finally, images of the dishes are presented in order to have a full view of the purpose and the results obtained. This research has been supported by the Department of Agriculture and Fisheries from the Basque Government. This work was presented at the conference Delivery of Functionality in Complex Food Systems, Amherst, USA, October, 2007.     

Effect of temperature on the release of carvacrol and cinnamaldehyde incorporated into polymeric systems to control growth and biofilms of Escherichia coli and Staphylococcus aureus

This study assessed the effect of temperature on the release of essential oil components incorporated by melt compounding into polymeric films. Specifically, polyethylene-co-vinylacetate (EVA) films containing carvacrol (CAR) and cinnamaldehyde (ALD), alone and in combination, were prepared and their surface and mechanical properties and antibacterial and anti-biofilm activity against Escherichia coli and Staphylococcus aureus were evaluated. The addition of ALD and CAR did not provoke variation in the surface morphology of EVA and allowed their delivery. At 37°C, films containing CAR, ALD or their combination (25+75%) were found to have the strongest bactericidal effect, whereas at lower temperatures a lower killing rate was observed. There was no clear evidence of the influence of temperature on the anti-biofilm activity of the essential oil component-based polymeric films. The biomass formed on EVA containing ALD, CAR or their combination (25+75) was significantly lower (60–80% reduction) than that formed on the EVA control at both 37° and 22°C.     

Developmental toxicity assessment of thermoresponsive poly(N-isopropylacrylamide-co-acrylamide) oligomers in CD-1 mice

BACKGROUND: Although polymers and hydrogels are used successfully in biomedical applications, including implants and drug delivery devices, smaller molecular weight oligomers, such as those investigated here, have not been extensively studied in vivo. Poly(N‐isopropylacrylamide‐co‐acrylamide), or P(NIPAAm‐co‐AAm), has a unique thermoresponsive behavior and is under investigation as a novel drug delivery system for metastatic cancer treatment. To date, no studies have been published regarding the safety of P(NIPAAm‐co‐AAm) to the conceptus. METHODS: From gestation days (GD) 6–16, pregnant CD‐1 mice were dosed via i.p. injection with aqueous solutions containing 500, 750, or 1,000 mg/kg/d P(NIPAAm‐co‐AAm). Dams were sacrificed on GD 17 and their litters were examined for abnormalities. RESULTS: P(NIPAAm‐co‐AAm) caused no statistically significant difference in maternal weight gain or percent resorbed or dead fetuses compared to control values, but fetal weight was significantly decreased in the two highest dosage groups. CONCLUSIONS: At the highest dosages employed, maternal exposure to P(NIPAAm‐co‐AAm) was associated with decreased fetal weight. However, as the estimated human exposure levels for persons using this system would be some 1,500‐fold lower than the lowest dosage administered in this study, the authors feel that this oligomer was not shown to pose a biologically significant risk at relevant human dosages. Birth Defects Res (Part B), 2008. © 2008 Wiley‐Liss, Inc.     

Whey Protein/Polysaccharide-Stabilized Oil Powders for Topical Application—Release and Transdermal Delivery of Salicylic Acid from Oil Powders Compared to Redispersed Powders

Oil-in-water (o/w) emulsions are commonly converted into solid-like powders in order to improve their physical and chemical stabilities. The aim of this study was to investigate whether whey protein/polysaccharide-stabilized o/w emulsions could be converted into stable oil powders by means of freeze-drying. Moreover, during this study, the effects of pH and polymer type on release and trans(dermal) delivery of salicylic acid, a model drug, from these oil powders were investigated and compared to those of the respective template emulsions and redispersed oil powders. Physical characterization of the various formulations was performed, such as droplet size analysis and oil leakage, and relationships drawn with regards to release and trans(dermal) delivery. The experimental outcomes revealed that the oil powders could be redispersed in water without changing the release characteristics of salicylic acid. pH and polymer type affected the release of salicylic acid from the oil powders, template emulsions, and redispersed powders similarly. Contrary, the transdermal delivery from the oil powders and from their respective redispersed oil powders was differently affected by pH and polymer type. It was hypothesized that the release had been influenced by the electrostatic interactions between salicylic acid and emulsifiers, whereas the transdermal performance could have been determined by the particle or aggregate sizes of the formulations.KEY WORDS: carrageenan, chitosan, oil powders, release, salicylic acid, topical delivery, whey proteins     

Glucose-regulated protein 75 suppresses apoptosis induced by glucose deprivation in PC12 cells through inhibition of Bax conformational change

Glucose-regulated protein 75 (Grp75) is an important molecular chaperone that belongs to the heat shock protein 70 family and resides predominantly in mitochondria. Grp75 can protect cells from glucose deprivation (GD) injury. However, the molecular mechanisms by which it carries out this function are unknown. Here we report that Grp75 could delay the release of cytochrome c and reduce apoptosis induced by GD, and we also found that Grp75 could decrease Bax/Bcl-2 gene expression ratio and inhibit the conformational change of Bax during this process. In conclusion, these findings suggested that Grp75 overexpression was able to inhibit apoptosis induced by GD. Grp75 inhibited Bax conformational change to delay the release of cytochrome c, thus providing protection to PC12 cell which was used primarily as a neuron model against GD toxicity.     

Temperature-Sensitive Microemulsion Gel: An Effective Topical Delivery System for Simultaneous Delivery of Vitamins C and E

Microemulsions (ME)—nanostructured systems composed of water, oil, and surfactants—have frequently been used in attempts to increase cutaneous drug delivery. The primary objective addressed in this work has been the development of temperature-sensitive microemulsion gel (called gel-like ME), as an effective and safe delivery system suitable for simultaneous topical application of a hydrophilic vitamin C and a lipophilic vitamin E. By changing water content of liquid o/w ME (o/w ME), a gel-like ME with temperature-sensitive rheological properties was formed. The temperature-driven changes in its microstructure were confirmed by rotational rheometry, viscosity measurements, and droplet size determination. The release studies have shown that the vitamins’ release at skin temperature from gel-like ME were comparable to those from o/w ME and were much faster and more complete than from o/w ME conventionally thickened with polymer (o/w ME carbomer). According to effectiveness in skin delivery of both vitamins, o/w ME was found the most appropriate, followed by gel-like ME and by o/w ME carbomer, indicating that no simple correlation between vitamins release and skin absorption could be found. The cytotoxicity studies revealed good cell viability after exposure to ME and confirmed all tested microemulsions as nonirritant. This work was supported by a grant of Slovenian Research Agency.     

Preparation and characterization of a novel pachyman-based pharmaceutical aid. II: A pH-sensitive,biodegradable and biocompatible hydrogel for controlled release of protein drugs

In this investigation, the fabrication, physico-chemical and biological characterization of a novel smart hydrogel had been evaluated for its potentials in effective controlling protein delivery. The hydrophilic pachyman-based hydrogel was generated facilely by crosslinking hydrosoluble carboxymethyl pachyman (CMP) with epichlorohydrin (ECH). The ECH concentration possessing maximum (99.7%) encapsulation efficiency and the most appropriate swelling characteristics was found to be 1.25% (w/v). The resultant hydrogel exhibited swelling ratios most favorable for drug release in simulated intestinal media. It could release two model protein drugs (bovine serum albumin and lysozyme) in the controlled manner and with full preservation of the protein stability and enzymatic activity. Importantly, the ECH-CMP hydrogel was confirmed to be biocompatible and biodegradable. From these findings, we were able to conclude that the synthesized pachyman-based hydrogel would be a promising delivery carrier candidate for site-specific delivery of protein drugs.     

In situ gelling of alginate/pluronic solutions for ophthalmic delivery of pilocarpine

We prepared a series of alginate and Pluronic-based solutions as the in situ gelling vehicles for ophthalmic delivery of pilocarpine. The rheological properties, in vitro release as well as in vivo pharmacological response of polymer solutions, including alginate, Pluronic solution, and alginate/Pluronic solution, were evaluated. The optimum concentration of alginate solution for the in situ gel-forming delivery systems was 2% (w/w) and that for Pluronic solution was 14% (w/w). The mixture of 0.1% alginate and 14% Pluronic solutions showed a significant increase in gel strength in the physiological condition; this gel mixture was also found to be free flowing at pH 4.0 and 25 degrees C. Both in vitro release and in vivo pharmacological studies indicated that the alginate/Pluronic solution retained pilocarpine better than the alginate or Pluronic solutions alone. The results demonstrated that the alginate/Pluronic mixture can be used as an in situ gelling vehicle to increase ocular bioavailability.     

Inherent charge-shifting polyelectrolyte multilayer blends: a facile route for tunable protein release from surfaces

Recent research has highlighted degradable multilayer films that enable the programmed release of different therapeutics. Multilayers constructed by the layer-by-layer (LbL) deposition that can undergo disassembly have been demonstrated to be of considerable interest, particularly for biomedical surface coatings due to their versatility and mild aqueous processing conditions, enabling the inclusion of biologic drugs with high activity. In this study, we examine the controlled release of a protein using a different mechanism for film disassembly, the gradual dissociation of film interactions under release conditions. Poly(β-amino ester)s and poly(L-lysine) (PLL) were used as the positively charged multilayer components coassembled with a model negatively charged antigen protein, ovalbumin (Ova). The release of the protein from these multilayer films is dominated by the slow shift in the charge of components under physiological pH conditions rather than by hydrolytic degradative release. The time scale of release can be varied over almost 2 orders of magnitude by varying the ratio of the two polyamines in the deposition solution. The highly versatile and tunable properties of these films form a basis for designing controlled and sequential delivery of drug coatings using a variety of polyions.     

Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains from breast-fed infant and investigation of their cholesterol-reducing effects

Hypercholesterolemia has been reported to be the main cause of cardiovascular diseases and the leading cause of death. Therefore, decreasing serum cholesterol level is very important for preventing the cardiovascular diseases. It has been supposed that probiotics in human gastrointestinal tract have the ability to decrease serum cholesterol level by reducing the absorption of cholesterol from the intestinal tract and the bile salt deconjugation. In this study, 28 strains of Lactobacillus spp., isolated from breast-fed infant’s feces, were identified and investigated for their bile salt deconjugation ability. The deconjugation ability of the strains was determined by the release of cholic acid resulting from the deconjugation of conjugated bile salts. Research results showed that four of the strains had bile salt deconjugation ability. The strains with deconjugation ability have been identified in species level by using biochemical test, and molecular techniques, API 50CHL test and 16S rRNA gene sequence analysis respectively. LP1, E3, and E9 strains with deconjugation activity were identified as Lactobacillus rhamnosus and GD2 strain as Lactobacillus plantarum. Even if oxgall decreases the viability of bacteria, the highest amount of cholesterol precipitation (42%) was performed by GD2 strain in the presence of 0.3% (w/v) bile. This study demonstrated that the identified Lactobacillus strains had an excellent ability to survive at low pH, a high bile deconjugation ability, and hypocholesterolemic effect in in vitro conditions.     

The Influence of Recrystallized Caffeine on Water-Swellable Polymethacrylate Mucoadhesive Buccal Films

The aim of this work was to investigate the influence of particles on the properties of polymethacrylate films intended for buccal delivery. A solvent casting method was used with Eudragit RS and RL (ERS and ERL, respectively) as film-forming rate-controlling polymers, with caffeine as a water-soluble model drug. The physicochemical properties of the model films for a series of formulations with increasing concentrations of caffeine were determined in terms of morphology, mechanical and mucoadhesive properties, drug content uniformity, and drug release and associated kinetics. Typically regarded as non-mucoadhesive polymers, ERS and mainly ERL, were found to be good mucoadhesives, with ERL01 exhibiting a work of mucoadhesion (WoA) of 118.9 μJ, which was about five to six times higher than that observed for commonly used mucoadhesives such as Carbopol^® 974P (C974P, 23.9 μJ) and polycarbophil (PCP, 17.4 μJ). The mucoadhesive force for ERL01 was found to be significantly lower yet comparable to C974P and PCP films (211.1 vs. 329.7 and 301.1 mN, respectively). Inspection of cross-sections of the films indicated that increasing the concentration of caffeine was correlated with the appearance of recrystallized agglomerates. In conclusion, caffeine agglomerates had detrimental effects in terms of mucoadhesion, mechanical properties, uniformity, and drug release at large particle sizes. ERL series of films exhibited very rapid release of caffeine while ERS series showed controlled release. Analysis of release profiles revealed that kinetics changed from a diffusion controlled to a first-order release mechanism.     

Biodegradable biocomposite films based on whey protein and zein: barrier, mechanical properties and AFM analysis

Whey and zein protein are byproducts of the food industries and have good film making properties. Single and laminated films were produced from zein protein and whey protein and their film properties were studied. Glycerol and olive oil were used as plasticizer for the single and laminated films. The laminated films exhibited higher ultimate tensile strength (UTS) than the single whey protein films (260% and 200% in the whey-zein-glycerol and whey-zein-olive oil films, respectively). The UTS of the whey protein films increased 2-3-fold after lamination. The laminated films showed higher barrier properties than the single whey protein films (180% in the whey protein-zein-glycerol films and 200% in the whey protein-zein-olive oil films in comparison to single whey protein films) and lower than the single zein films.