Fundamental advances in hydrogels for the development of the next generation of smart delivery systems as biopharmaceuticals.

Recent advances in developing and applying therapeutic peptides for anticancer, antimicrobial and immunomodulatory remedies have opened a new era in therapeutics. This development has resulted in the engineering of new biologics as part of a concerted effort by the pharmaceutical industry. Many alternative routes of administration and delivery vehicles, targeting better patient compliance and optimal therapeutic bioavailability, have emerged. However, the design of drug delivery systems to protect a range of unstable macromolecules, including peptides and proteins, from high temperatures, acidic environments, and enzymatic degradation remains a priority. Herein, we give chronological insights in the development of controlled-release drug delivery systems that occurred in the last 70 years or so. Subsequently, we summarise the key physicochemical characteristics of hydrogels contributing to the development of protective delivery systems concerning drug-targeted delivery in the chronospatial domain for biopharmaceuticals. Furthermore, we shed some light on promising hydrogels that can be utilised for systemic bioactive administration.

MALDI-TOF-MS and in-depth dynamic simulations on the molecular forces determining the stability of the 4-hydroxybenzoic acid - ß-Casein complex following UHT-like treatment.

Previous work has suggested the ability of 4-hydroxybenzoic acid (4HBA) to bind to ß -casein following ultra high temperature-like processing (UHT) in model aqueous systems. The present work confirmed directly, using MALDI-TOF-MS, the presence of covalently bound 4HBA following UHT-like treatment. In subsequent molecular dynamics simulations, the 3D structure of the ß -casein molecule was modified so that the meta-C of 4HBA ring and the side chain amino group of lys32 were linked covalently. Such simulations further indicated that the covalent addition of the phenolic compound had impacted the protein density and solvent accessibility. Hydrogen bond analysis between lys32 and the remainder of the protein structure revealed that the covalent complexation supported the formation of additional hydrogen bonds. These increased from potentially 9, in the single protein molecule, to 51 in the complex with 4HBA. However, the persistence of hydrogen bonds was reduced, leading overall to decreased stability and increased protein flexibility.

The effect of trisodium phosphate crosslinking on the diffusion kinetics of caffeine from chitosan networks.

This work examines the relationship between microstructural properties of hot-moulded chitosan networks, crosslinked with trisodium phosphate, and diffusive behaviour from these networks. Analysis through infrared spectroscopy (FTIR) confirmed successful crosslinking of the polymer chains and bioactive entrapment, while X-ray diffraction (WAXD) and dynamic oscillation in-shear elucidated the higher order structural properties of each matrix, as they transitioned from solutions to amorphous gels to semi-crystalline matrices. The picture of molecular motion observed in these systems and consequent application of the Flory-Rehner theory further indicated that different extents of chitosan crosslinking yielded a distinct water infusion functionality seen in the levels of swelling. Diffusion of caffeine from these delivery vehicles showed that network structural properties (governed by crosslinker concentration) had a significant effect on the release kinetics of the entrapped bioactive. The relationship between network mesh characteristics and diffusion properties were further confirmed by correlating caffeine release rates and molecular pore size.

Binding parameters and molecular dynamics of ß-lactoglobulin-vanillic acid complexation as a function of pH - part B: Neutral pH.

Interactions between the dimeric form of ß-lactoglobulin and vanillic acid were investigated at pH 7.2, using a variety of spectroscopic techniques and molecular dynamics (MD) simulations. FTIR and CD studies showed alterations in the secondary structure of the protein upon its interaction with the ligand. Fluorescence measurements indicated that the dimeric complex with the phenolic acid produced a large dissociation constant (KD) compared to the monomeric counterpart at acidic pH (part A of this series). Stoichiometry of 1:1 was identified for the ß-lactoglobulin-vanillic acid complex by Job plot analysis at neutral pH suggesting two ligand molecules can participate in binding with the dimer. Molecular docking and MD simulations suggested that the top-ranked binding sites of the ligand were located at the entrance of each ß-barrel structure of the dimer. These simulations also allowed identification of the contribution of water molecules, in the form of protein-water-ligand bridging interactions, to the complexes.

Green and clean modification of cassava starch - effects on composition, structure, properties and digestibility.

There is a growing need for clean and green labeling of food products among consumers globally. Therefore, development of green modified starches, to boost functionality, palatability and health benefits while reducing the negative processing impacts on the environment and reinforcing consumer safety is in high demand. Starch modification started in mid-1500s due to the inherent limitations of native starch restricting its commercial applications, with chemical modification being most common. However, with the recent push for "chemical-free" labeling, methods of physical and enzymatic modification have gained immense popularity. These methods have been successfully used in numerous studies to alter the composition, structure, functionality and digestibility of starch and in this review, studies reported on green modification of cassava starch, one of the most common utilized starches, within the last ten years have been critically reviewed. Recent research has introduced starch as an abundant, natural substrate for producing resistant starches through biophysical technologies that act as dietary fiber in the human body. It is evident that different techniques and processing parameters result in varying degrees of modification impacting the techno-functionality and digestibility of the resultant starch. This can be exploited by researchers and industrialists in order to customize starch functionality in accordance with application.

Maturation Process, Nutritional Profile, Bioactivities and Utilisation in Food Products of Red Pitaya Fruits: A Review.

Red pitaya (Hylocereus polyrhizus, red pulp with pink peel), also known as dragon fruit, is a well-known species of pitaya fruit. Pitaya seeds and peels have been reported to exhibit higher concentrations of total polyphenols, beta-cyanins and amino acid than pulp, while anthocyanins (i.e., cyanidin 3-glucoside, delphinidin 3-glucoside and pelargonidin 3-glucoside) were only detected in the pulp extracts. Beta-cyanins, phenolics and flavonoids were found to increase gradually during fruit maturation and pigmentation appeared earlier in the pulp than peel. The phytochemicals were extracted and purified by various techniques and broadly used as natural, low-cost, and beneficial healthy compounds in foods, including bakery, wine, dairy, meat and confectionery products. These bioactive components also exhibit regulative influences on the human gut microbiota, glycaemic response, lipid accumulation, inflammation, growth of microbials and mutagenicity, but the mechanisms are yet to be understood. The objective of this study was to systematically summarise the effect of red pitaya's maturation process on the nutritional profile and techno-functionality in a variety of food products. The findings of this review provide valuable suggestions for the red pitaya fruit processing industry, leading to novel formulations supported by molecular research.

Binding parameters and molecular dynamics of ß-lactoglobulin-vanillic acid complexation as a function of pH - Part A: Acidic pH.

Protein-phenolic compound interactions are commonly investigated with inappropriate linear equations for the analysis of binding strength and stoichiometry. This work utilises more appropriate protocols for the investigation of molecular interactions between vanillic acid and ß-lactoglobulin at pH 2.4, where the protein predominately exists as a monomer. Non-linear binding and Job plot analysis were conducted on fluorescence data to effectively determine the interaction's dissociation constant (KD, 2.93 × 10-5 M) and stoichiometry (1:1). Furthermore, spectroscopic techniques revealed statistically significant alterations to the conformational characteristics of ß-lactoglobulin upon complexation. Molecular dynamics (MD) simulations support a 1:1 interaction stoichiometry and reveal that the stabilisation of vanillic acid was dynamic in nature but mainly supported by four π-alkyl interactions and one hydrogen bond, located within the ß-barrel of the monomer. Water molecules, which are generally not accounted for in MD simulation analysis, were shown to be an important factor in the ligand stabilization via bridging interactions.

Manipulation of the Glass Transition Properties of a High-Solid System Made of Acrylic Acid-N,N'-Methylenebisacrylamide Copolymer Grafted on Hydroxypropyl Methyl Cellulose.

Crosslinking of hydroxypropyl methyl cellulose (HPMC) and acrylic acid (AAc) was carried out at various compositions to develop a high-solid matrix with variable glass transition properties. The matrix was synthesized by the copolymerisation of two monomers, AAc and N,N'-methylenebisacrylamide (MBA) and their grafting onto HMPC. Potassium persulfate (K2S2O8) was used to initiate the free radical polymerization reaction and tetramethylethylenediamine (TEMED) to accelerate radical polymerisation. Structural properties of the network were investigated with Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), modulated differential scanning calorimetry (MDSC), small-deformation dynamic oscillation in-shear, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The results show the formation of a cohesive macromolecular entity that is highly amorphous. There is a considerable manipulation of the rheological and calorimetric glass transition temperatures as a function of the amount of added acrylic acid, which is followed upon heating by an extensive rubbery plateau. Complementary TGA work demonstrates that the initial composition of all the HPMC-AAc networks is maintained up to 200 °C, an outcome that bodes well for applications of targeted bioactive compound delivery.

Decoupling diffusion and macromolecular relaxation in the release of vitamin B6 from genipin-crosslinked whey protein networks.

This study examined the release of vitamin B6 from a hydrogel made of whey protein isolate (WPI). Work was carried out at ambient temperature without preheating the whey protein. Native-state macromolecules were crosslinked with a nontoxic compound, genipin. Experimentation included a ninhydrin assay with UV-vis absorbance, FTIR, 13C NMR, compression testing, SEM imaging, WPI matrix swelling and vitamin release protocols. It was confirmed that geninin crosslinked effectively the protein chains whose network strength was reinforced with increasing crosslinker concentrations. The modified Flory-Rehner theory predicted the molecular weight between crosslinks, network mesh size and crosslinking density in the swollen WPI gels as a function of added crosslinker. Transport patterns of vitamin B6 through the polymeric matrix were monitored over prolonged periods of observation. These were examined with the generalised Fick's equation and the Peppas-Sahlin equation to unveil the interplay between diffusion and relaxation dynamics in the anomalous transport of the bioactive compound.

Physicochemical Properties and Effects of Honeys on Key Biomarkers of Oxidative Stress and Cholesterol Homeostasis in HepG2 Cells.

Manuka honey and newly developed honeys (arjuna, guggul, jiaogulan and olive) were examined for their physicochemical, biochemical properties and effects on oxidative stress and cholesterol homeostasis in fatty acid-induced HepG2 cells. The honeys exhibited standard moisture content (<20%), electrical conductivity (<0.8 mS/cm), acidic pH, and monosaccharides (>60%), except olive honey (<60% total monosaccharides). They all expressed non-Newtonian behavior and 05 typical regions of the FTIR spectra as those of natural ones. Guggul and arjuna, manuka honeys showed the highest phenolic contents, correlating with their significant antioxidant activities. Arjuna, guggul and manuka honeys demonstrated the agreement of total cholesterol reduction and the transcriptional levels of AMPK, SREBP2, HCMGR, LDLR, LXRα. Jiaogulan honey showed the least antioxidant content and activity, but it was the most cytotoxic. Both jiaogulan and olive honeys modulated the tested gene in the pattern that should lead to a lower TC content, but this reduction did not occur after 24 h. All 2% concentrations of tested honeys elicited a clearer effect on NQO1 gene expression. In conclusion, the new honeys complied with international norms for natural honeys and we provide partial evidence for the protective effects of manuka, arjuna and guggul honeys amongst the tested ones on key biomarkers of oxidative stress and cholesterol homeostasis, pending further studies to better understand their modes of action.

Swelling behaviour and glass transition in genipin-crosslinked chitosan systems.

High-solid chitosan matrices were prepared to investigate the effect of their swelling on structural relaxation and glass transition. Degree of crosslinking in genipin-crosslinked chitosan networks was measured with a ninhydrin assay and a suitable crosslinker concentration was determined for gels used in swelling and thermomechanical analysis. Fourier transform infrared spectroscopy and wide angle X-ray diffraction examined the intermolecular interactions, crystallinity and amorphicity of the biopolymer networks. Swelling characteristics following immersion in water included approximate equilibrium values of the average molecular weight between crosslinks and network mesh size, e.g. 902 g mol-1 and 110 nm for the preparation with an initial solids content of 60% (w/w) after 80 min of swelling, which were quantified with the modified Flory-Rehner theory. Thermal glass transition temperature was observed in the condensed crosslinked networks (≥ 70% w/w total solids) during differential scanning calorimetry experiments. Time-temperature superposition of rheological measurements, obtained with dynamic oscillation in-shear, generated a master curve describing the viscoelastic behaviour of the system, moving through the rubbery, glass transition region and glassy state. Combined analysis using the modified Arrhenius and William-Landel-Ferry theories determined mechanical glass transition temperatures in the range of -68 to -8 °C for the crosslinked biopolymer at intermediate-solid concentrations (40 to 60% w/w solids). An understanding of the effect of swelling on molecular network characteristics was achieved, which is crucial for greater control in the design of systems for the targeted delivery of bioactive compounds.

Molecular Functionality of Plant Proteins from Low- to High-Solid Systems with Ligand and Co-Solute.

In the food industry, proteins are regarded as multifunctional systems whose bioactive hetero-polymeric properties are affected by physicochemical interactions with the surrounding components in formulations. Due to their nutritional value, plant proteins are increasingly considered by the new product developer to provide three-dimensional assemblies of required structure, texture, solubility and interfacial/bulk stability with physical, chemical or enzymatic treatment. This molecular flexibility allows them to form systems for the preservation of fresh food, retention of good nutrition and interaction with a range of microconstituents. While, animal- and milk-based proteins have been widely discussed in the literature, the role of plant proteins in the development of functional foods with enhanced nutritional profile and targeted physiological effects can be further explored. This review aims to look into the molecular functionality of plant proteins in relation to the transport of bioactive ingredients and interaction with other ligands and proteins. In doing so, it will consider preparations from low- to high-solids and the effect of structural transformation via gelation, phase separation and vitrification on protein functionality as a delivery vehicle or heterologous complex. Applications for the design of novel functional foods and nutraceuticals will also be discussed.

Morphology of genipin-crosslinked BSA networks yields a measurable effect on the controlled release of vitamin B6.

We examined the morphology of a network made with native BSA molecules being crosslinked with genipin at ambient temperature. Ninhydrin assay, FTIR, WAXD, SEM and mechanical tests documented successful crosslinking that enhanced the structural properties of the three dimensional structure. Its hydrophilic nature allows swelling with water absorption, which can be monitored with the modified Flory-Rehner theory to predict the molecular weight between adjacent crosslinks, network mesh size and crosslinking density as a function of crosslinker addition. Characterisation studies were carried out with a view to developing a delivery vehicle for the controlled release of vitamin B6 over a prolonged period of observation. Moving boundaries associated with swelling of the protein matrix resulted in vitamin transport that could be described with the interplay of diffusional and relaxational kinetics via the Peppas-Sahlin equation. Combination of diffusion and swelling equilibrium theories unveils a measurable effect of network characteristics on vitamin B6 release.

Structural-rheological characteristics of Chaplin E peptide at the air/water interface; a comparison with ß-lactoglobulin and ß-casein.

The Chaplin E peptide is a surface-active agent that can adsorb to the air/water interface and form interfacial films that display distinct interfacial properties as a function of pH. The ~2 nm thick homogeneous Chaplin E film formed under acidic conditions contains ordered structures that give a high dilatational elasticity. In contrast, the heterogeneous film formed under basic conditions contained fibrils resulting in a rough ~17 nm thick film with predominantly viscoelastic properties, probably due to the reduced intermolecular interactions. These fibrils were also susceptible to breakage, fragmenting into shorter fibrils, which gave a greater elasticity. The fibrils also lead to a greater shear viscosity compared to the ordered structures aligned within the Chaplin E film at pH 3.0. A higher stability was observed for the foam formed by the Chaplin E compared to the foam formed by ß-lactoglobulin, consistent with the greater rheological properties observed for the Chaplin E film at the interface. Our findings suggest that Chaplin E has potential to provide long time stability to dispersions in food, consumer goods or pharmaceutical applications, forming films with greater rheological properties and at least similar thickness to those formed by other surface-active proteins such as ß-casein and ß-lactoglobulin.

Quantitative analysis of the phase volume of agarose-canola oil gels in comparison to blending law predictions using 3D imaging based on confocal laser scanning microscopy.

Studying the phase behaviour of composite gels facilitates understanding of their structural and textural properties at low and intermediate levels of solids. In this work, the phase behaviour of a model system of agarose including various concentrations of canola oil was studied. This was pursued using a variety of techniques including SEM, FTIR, microDSC and dynamic oscillation in-shear. The structural studies recorded strong, continuous agarose networks supporting soft, discontinuous canola oil inclusions, with increasing levels of canola oil strengthening the composite system. A novel confocal laser scanning microscopy (CLSM) method for quantitative in situ examination of the oil phase volume was developed using three-dimensional (3D) imaging and image analysis software - FIJI and Imaris. Microscopic observations were assessed in relation to theoretical predictions from rheology-based blending-law analysis. Quantitative outcomes from the combined 3D imaging and image analysis are in close agreement with the volume predictions for the oil phase obtained from the isostrain blending law indicating the suitability of this approach in quantifying the phase behaviour of composite materials. The results of this work indicate that the developed microscopic method shows promise and could be used in the determination of phase volume in more complex and industrially relevant systems.

Effect of low-frequency ultrasound on the particle size, solubility and surface charge of reconstituted sodium caseinate.

Low-frequency sonication (20 kHz) was applied to sodium caseinate suspensions (4%, 7% and 10% protein concentrations) at pH 4.0, 4.6, 6.7 and 9.0. Particle size, zeta potential and solubility analysis were used to evaluate the physical changes of the sodium caseinate suspensions before and after the application of ultrasound. At pH 6.7 the particle size remained between 5 and 7 µm for all concentrations before and after sonication (15-400 J/mL), resulting in no significant change (p > 0.05). Similarly, sonication did not significantly (p > 0.05) affect the solubility at pH 6.7. At this pH, the initial solubility was high at 94-98% (w/w) before sonication. At pH 9.0 for 4% and 7% concentrations, suspensions became more negatively charged and the initial particle size increased to 78-82 µm. In the presence of larger suspensions, the application of ≥15 J/mL reduced the particle size to less than 2 µm. By contrast to pH 6.7, the solubility at pH 9.0 for 4% and 7% protein suspensions reached 99% before and after sonication. Viscosity was the highest (80 mPa.s at 15 sec-1) for a 10% protein concentration at pH 9.0. As the protein concentration of the sodium caseinate suspensions decreased from 10% to 4% at pH 9.0, the viscosity of the suspensions also decreased. However, application of low-frequency ultrasound had no effect on the viscosity of the sodium caseinate suspensions. Due to the absence of large insoluble aggregates in reconstituted sodium caseinate suspensions, the overall effect of low-frequency sonication were largely insignificant at native pH and only became evident at outlier pH values when the casein proteins associate.

Structural relaxation and glass transition in high-solid gelatin systems crosslinked with genipin.

Structural relaxation and glass transition were examined in the swelling behaviour of a high-solid biopolymer matrix; genipin-crosslinked gelatin. Degree of swelling was quantified by the Flory-Rehner theory that furnishes estimates of average molecular weight between crosslinks and network mesh size. Fourier transform infrared spectroscopy and wide angle X-ray diffraction described intermolecular interactions and the extent of amorphicity in the crosslinked matrix. Micro differential scanning calorimetry provided evidence of the changing thermodynamic characteristics of the gelatin network in the presence of the crosslinker. Modulated differential scanning calorimetry and small deformation oscillatory rheology unveiled the vitrification properties of the system. Experimental measurements were treated with the time-temperature superposition principle to unveil an extensive master curve through the rubbery, glass transition and glassy states. Viscoelastic behaviour was modelled with the combined predictions of the modified Arrhenius and William-Landel-Ferry theories to pinpoint the mechanical glass transition temperature that was compared with predictions from calorimetry. Comprehensive understanding of polymeric behaviour during swelling affords greater control in the design of targeted delivery matrices for drugs and other bioactive compounds.

Classification of hydrocolloids based on small amplitude oscillatory shear, large amplitude oscillatory shear, and textural properties.

Dynamic rheological and mechanical properties of seven commercial (xanthan [XG], guar [GG], high methoxylated pectin [HMP], κ-carrageenan [κ-Car], agar [AG], alginate [ALG], and carboxymethylcellulose [CMC]) and four emerging hydrocolloids (basil seed gum [BSG], sage seed gum [SSG], Balangu-Shirazi seed gum [BSSG], and cress seed gum [CSG]) were investigated and the classification of the hydrocolloids were carried out based on them. AG belonged to the first class with 0.81 membership function (MF), κ-Car and HMP grouped in the second class with 0.68 and 0.71 MFs, respectively, XG, BSG, and SSG were depended to the third class with 0.61-0.70 MFs, finally, CMC, GG, BSSG, ALG, and CSG related to the fourth class, as the most populated class, with MF > 0.61. The first class contained the highest amount of hardness parameter (43.40 ± 2.76 g), the second class included the highest pseudoplasticity parameter (shear-thinning ratio = -0.54 ± 0.03) and relaxation time (66.25 ± 2.61 s) and the fourth cluster comprised the highest frequency dependency of viscous modulus (exponent of power-law model for viscous modulus vs. frequency = 0.30 ± 0.05). In addition, the results of this study showed that there was a distinct relationship between nonlinear harmonics in the stress wave and fundamental characteristics of hydrogel networks. The investigation of the rheo-mechanical properties of biopolymers in large deformation under shear and normal forces can have an important role in the prediction of the behavior of the material in real processes and application conditions, especially in the food industry. Due to the inconvenience of large deformation mechanical tests, such as Weissenberg effect, the complication of the results analyzing and sampling difficulty of semi-dilute samples; herein, we determined the correlation between large deformation (LAOS and texture analysis) and small deformation (SAOS) tests properties. The studied rheo-mechanical parameters showed high correlation with the four mentioned network parameters (more than 65% similarity index). Using these results, other scientists could rationally design the experiments and avoid experiments with similar parameters.

Structural variation in gelatin networks from low to high-solid systems effected by honey addition.

Honey is a biologically active material functioning antibacterial, anti-inflammation and immune responses that enhance wellbeing. This research aims to record and rationalise the structural properties of honey as part of a convenient delivery system in the presence of gelatin that provides the structuring matrix. In doing so, we employ dynamic oscillation in-shear, micro and modulated DSC, WAXD, FTIR and ESEM. A wide range of solids was employed from 10% (w/w) gelatin to mixtures with up to 75% (w/w) honey. Increasing addition of co-solute created thermally stable gelatin networks, which at high levels of total solids undergo a glass transition. This allows deconvolution of the total heat flow into the reversing and non-reversing thermograms. In addition, mechanical spectra can be treated by the combined free volume/reaction rate theory to predict the molecular dynamics of the gelatin-honey system. Molecular interactions between the two components and the relative contribution of honey to the crystalline or amorphous part of the binary preparation are elucidated guiding future applications for orally and topically treated ailments.

Modeling counterion partition in composite gels of BSA with gelatin following high pressure treatment.

We examine the morphology of hydrogels made of bovine serum albumin and gelatin following high pressure processing at 300 MPa for 15 min at 10 and 80 °C. Emphasis is on the distribution of added calcium counterions between the polymeric phases seen in changes in the structural properties of the composite gel. Protocol includes thermal and HPP treatments, dynamic oscillation rheology, ESEM, and modeling from the "synthetic polymer approach" to rationalize results. Pressurization at 10 °C produced continuous gelatin networks with dispersed BSA inclusions whereas pressurization at 80 °C yielded an inverse dispersion of BSA as the continuous phase supporting liquid gelatin inclusions. Lewis and Nielsen equations were adapted to predict the counterion distribution between the polymeric phases that profoundly affected the structural properties of the pressurized gels. The concept of counterion partition (pc) is introduced to the literature to follow the phase behavior of the composites in the presence of added calcium counterions.