Does arginine aggregate formation in aqueous solutions follow a two-step mechanism?

The formation of aggregates was studied in arginine aqueous solutions using light scattering. The main driving force for aggregate formation is hydrogen bonding between the arginine (Arg) amino acids, which is partially verified using density functional theory calculations. The measurement of energy loss during this process, coupled with Cryo-EM morphology data, indicates that these aggregates are in the solid state. The aggregation occurs in two steps, with a liquid intermediate stage. The investigation of the effect of pH and solute concentration on aggregate formation for other amino acid aqueous solutions verifies that aggregate formation is amino-acid specific, while small-sized clusters formed by weak interactions lead to large-sized aggregation. The water structure around amino acid molecules sheds light on the prediction of their aggregate formation. Homochirality is observed in the aggregates; its existence sheds light on the origin of protein homochirality.

Tailored Sticky Solutions: 3D-Printed Miconazole Buccal Films for Pediatric Oral Candidiasis.

In this research, 3D-printed antifungal buccal films (BFs) were manufactured as a potential alternative to commercially available antifungal oral gels addressing key considerations such as ease of manufacturing, convenience of administration, enhanced drug efficacy and suitability of paediatric patients. The fabrication process involved the use of a semi-solid extrusion method to create BFs from zein-Poly-Vinyl-Pyrrolidone (zein-PVP) polymer blend, which served as a carrier for drug (miconazole) and taste enhancers. After manufacturing, it was determined that the disintegration time for all films was less than 10 min. However, these films are designed to adhere to buccal tissue, ensuring sustained drug release. Approximately 80% of the miconazole was released gradually over 2 h from the zein/PVP matrix of the 3D printed films. Moreover, a detailed physicochemical characterization including spectroscopic and thermal methods was conducted to assess solid state and thermal stability of film constituents. Mucoadhesive properties and mechanical evaluation were also studied, while permeability studies revealed the extent to which film-loaded miconazole permeates through buccal tissue compared to commercially available oral gel formulation. Histological evaluation of the treated tissues was followed. Furthermore, in vitro antifungal activity was assessed for the developed films and the commercial oral gel. Finally, films underwent a two-month drug stability test to ascertain the suitability of the BFs for clinical application. The results demonstrate that 3D-printed films are a promising alternative for local administration of miconazole in the oral cavity.

Food protein glycation: A review focusing on stability and in vitro digestive characteristics of oil/water emulsions.

Recently, increasing studies have shown that the functional properties of proteins, including emulsifying properties, antioxidant properties, solubility, and thermal stability, can be improved through glycation reaction under controlled reaction conditions. The use of glycated proteins to stabilize hydrophobic active substances and to explore the gastrointestinal fate of the stabilized hydrophobic substances has also become the hot spot. Therefore, in this review, the effects of glycation on the structure and function of food proteins and the physical stability and oxidative stability of protein-stabilized oil/water emulsions were comprehensively summarized and discussed. Also, this review sheds lights on the in vitro digestion characteristics and edible safety of emulsion stabilized by glycated protein. It can further serve as a research basis for understanding the role of structural features in the emulsification and stabilization of glycated proteins, as well as their utilization as emulsifiers in the food industry.

Inkjet printing of a thermolabile model drug onto FDM-printed substrates: formulation and evaluation.

OBJECTIVE: The inkjet printing (IP) and fused deposition modeling (FDM) technologies have emerged in the pharmaceutical field as novel and personalized formulation approaches. Specific manufacturing factors must be considered in each adopted methodology, i.e. the development of suitable substrates for IP and the incorporation of highly thermostable active pharmaceutical compounds (APIs) for FDM. In this study, IP and FDM printing technologies were investigated for the fabrication of hydroxypropyl methylcellulose-based mucoadhesive films for the buccal delivery of a thermolabile model drug. Significance: This proof-of-concept approach was expected to provide an alternative formulation methodology for personalized mucoadhesive buccal films. METHODS: Mucoadhesive substrates were prepared by FDM and were subjected to sequential IP of an ibuprofen-loaded liquid ink. The interactions between these processes and the performance of the films were evaluated by various analytical and spectroscopic techniques, as well as by in vitro and ex vivo studies. RESULTS: The model drug was efficiently deposited by sequential IP passes onto the FDM-printed substrates. Significant variations were revealed on the morphological, physicochemical and mechanical properties of the prepared films, and linked to the number of IP passes. The mechanism of drug release, the mucoadhesion and the permeation of the drug through the buccal epithelium were evaluated, in view of the extent of ink deposition onto the buccal films, as well as the distribution of the API. CONCLUSIONS: The presented methodology provided a proof-of-concept formulation approach for the development of personalized mucoadhesive films.

Pectin-mucin interactions: Insights from fluorimetry, thermodynamics and dual (static and dynamic) quenching mechanisms.

Binary systems of citrus peel pectin (a major food carbohydrate) and mucin (a principal oral-gastrointestinal glycoprotein) are studied, as to understand the interactions and thermodynamics between food and biofluids during oral processing and digestion. The fluorimetry emission spectra of mucin were quenched by pectin addition at 293, 301, 310 and 318 K, indicating direct contact between the two macromolecular populations. A red shift, suggesting pectin-induced alterations on mucin conformation, has been observed at 318 K. Intensity-based Stern - Volmer plots fitted second-order polynomial equations, suggesting the coexistence of both static and dynamic quenching, while the increase of the slopes with temperature points to the predominance of dynamic phenomena. Time-resolved fluorescence measurements also point to dynamic quenching related to transient interactions, rather than to specific bonding. Thermodynamic analysis yields negative free energy changes in all cases, with positive changes for enthalpy and large positive values for TΔS. These are in agreement with the Stern - Volmer analysis, suggesting the predominance of transient, dynamic (here entropic) interactions. These provide an image of mucin interacting with pectin macromolecules during the oral processing and digestion of foods, and can relate to the texture, flavor (e.g. astringency) and bioavailability of polysaccharide-based foods.

Interactions between xanthan gum and phenolic acids.

The molecular and colloidal-level interactions between two major phenolic acids, gallic and caffeic acid, with a major food polysaccharide, xanthan gum, were studied in binary systems aiming to correlate the stability of the binary systems as a function of pH and xanthan-polyphenol concentrations. Global stability diagrams were built, acting as roadmaps for examining the phase separation regimes followed by the fluorimetry-based thermodynamics of the interactions. The effects of noncovalent interactions on the macroscopic behavior of the binary systems were studied, using shear and extensional rheometry. The collected data for caffeic acid - xanthan gum mixtures showed that the main interactions were pH-independent volume exclusions, while gallic acid interacts with xanthan gum, especially at pH 7 with other mechanisms as well, improving the colloidal dispersion stability. A combination of fluorimetry, extensional rheology and stability measurements highlight the effect of gallic acid-induced aggregation of xanthan gum, both in structuring and de-structuring the binary systems. The above provide a coherent framework of the physicochemical aspect of binary systems, shedding light on the role of xanthan gum in its oral functions, such as in inducing texture, in model complex systems containing phenolic acids.

Fabrication, characterization, and oxidation resistance of gelatin/egg white protein cryogel-templated oleogels through apple polyphenol crosslinking.

Cryogel-templated oleogels (CTO) were fabricated via a facile polyphenol crosslinking strategy, where apple polyphenol was utilized to crosslink the gelatin/egg white protein conjugates without forming hydrogels. After freeze-drying, cryogel templates were obtained and used to construct CTO by oil absorption. Apple polyphenol crosslinking improved the emulsion-related properties with appearance changes on samples, and infrared spectroscopy further confirmed the interactions between proteins and apple polyphenol. The crosslinked cryogels presented porous microstructures (porosity of over 96 %), enhanced thermal/mechanical stabilities, and could absorb a high content of oil (14.41 g/g) with a considerable oil holding capacity (90.98 %). Apple polyphenol crosslinking also influenced the rheological performances of CTO, where the highly crosslinked samples owned the best thixotropic recovery of 85.88 %. Moreover, after the rapid oxidation of oleogels, the generation of oxidation products was effectively inhibited by crosslinking (POV: 0.48 nmol/g, and TBARS: 0.53 mg/L). The polyphenol crosslinking strategy successfully involved egg white protein and gelatin to fabricate CTO with desired physical/chemical properties. Apple polyphenol acted as both a crosslinker and an antioxidant, which provided a good reference for fabricating pure protein-based CTO.

Development of mucoadhesive 3D-printed Carbopol/Eudragit/SNAC tablets for the oral delivery of enoxaparin: In vitro and ex vivo evaluation.

3D-printed dosage forms comprised of Carbopol and Eudragit were fabricated through semi-solid extrusion, combining Enoxaparin (Enox) and the permeation enhancer SNAC in a single-step process without subsequent post-processing. Inks were characterized using rheology and Fourier-transform infrared (FTIR) spectroscopy. The stability of Enox in the fabricated dosage forms was assessed by means of Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) analysis. In vitro release studies revealed the release of Enox in a sustained manner, whereas ex vivo experiments demonstrated the mucoadhesive properties of the 3D-printed dosage forms and their ability to enhance Enox permeability across intestinal mucosa. Cellular assays (CCK-8 assay) revealed a dose- and time-dependent response following incubation with the 3D-printed dosage forms. The encapsulation of SNAC in the 3D-printed dosage forms demonstrated their capacity to increase the transcellularly transport of macromolecule across Caco-2 monolayer in a reversible manner, as confirmed by Transepithelial Resistance (TEER) measurements.

Analytical rheology as a tool for the structural investigation of citrus pectin.

Rheological analysis of citrus pectin at pH 3 and 7 elucidates its structural dynamics, revealing distinct behaviors influenced by pH. At pH 3, pectin exhibits shear-thinning, with solvent-independent unified rheological profiles identifying three concentration regimes: 0.5%-1.5%, 2%-3%, and 3.5%-4%. These regimes, alongside Cox-Merz superpositions, outline the semi-dilute (c*) and concentrated (c**) transitions at 1.5%-2% and 3%-3.5%, respectively. Moreover, a Morris equation exponent of 0.65 indicates flexible, mobility-restricted macromolecules. Conversely, at pH 7, increased viscosities and Morris plot linearity for p = .1 suggest rigid chain behavior due to electrostatic repulsion among ionized acidic groups. This rigidity leads to concentration-dependent self-assembly structures that diverge from expected unified rheological profiles, a deviation amplified by heating-cooling cycles. This study clarifies the impact of pH on citrus pectin's rheology and emphasizes the intricate relationship between polymeric chain rigidity, self-assembly, and viscosity. By providing a refined understanding of these mechanisms, our findings contribute to the broader field of polysaccharide research, offering insights critical for developing and optimizing pectin-based applications in various industries.

Antioxidant polysaccharide-enriched fractions obtained from olive leaves by ultrasound-assisted extraction with α-amylase inhibition, and antiproliferative activities.

Polysaccharide-rich materials were extracted from the alcohol-insoluble solids of Olea europaea l. **leaves. Structural characteristics were determined by colorimetric techniques, FT-IR, GC-MS, SEC/MALS/VD/DRI, and NMR (1H,13C). The extract and its main macromolecular components were characterized to assess their ability toward antioxidant, α-amylase inhibition, and antiproliferative activities. Results revealed that the ultrasound olive leave extract comprises polysaccharides with uronic acid, galactose, arabinose, and glucose in molar percentages of 11.7%, 11.3%, 7.5%, and 4.9% respectively, constituting 41% of the total mass. In addition, polyphenols (21%) and proteins (9%) are associated with these polysaccharides. Further, the extract showed noticeable ORAC and free radical scavenging abilities, in addition to high in vitro antiproliferative activity against Caco-2 colon carcinoma cell lines. Similarly, the extract exhibited a strong, uncompetitive inhibition of α-amylase by 75% in the presence of the extract with 0.75 µg/mL of concentration. This research concludes that ultrasound extraction method can be used for the extraction of polysaccharide-polyphenol-protein complexes. These conjugates exhibit the potential for combined biological activities resulting from a synergistic effect of its compounds, making them promising ingredients for the development of functional food.

Yogurt Products Fortified with Microwave-Extracted Peach Polyphenols.

Pectin and polyphenols have been obtained from choice peach flesh using microwave extraction, with the resulting extracts used in functionalizing strained yogurt gels. A Box-Behnken design was utilized in order to co-optimize the extraction process. Soluble solid content, total phenolic content, and particle size distributions were measured in the extracts. Extraction at pH 1 yielded the highest phenolic content, while increases in the liquid-to-solid ratio resulted in a decrease in soluble solids and an increase in particle diameter. Selected extracts were then incorporated into strained yogurt, and the resulting gel products were assessed for color and texture over a two-week period. All samples were darker and had more red tones than the control set yogurt, while exhibiting less yellow tones. The cohesiveness of all samples remained stable over the gels' aging of two weeks (break-up times always remaining within 6 s and 9 s), which is close to the expected shelf-life of such products. The work required for the deformation of most samples increases with time, indicating that the products became firmer due to the macromolecular rearrangements in the gel matrix. The extracts obtained with the highest microwave power (700 W) give less firm samples. This was due to the microwave-induced loss of conformation and self-assembly of the extracted pectins. The hardness of all samples increased over time, gaining from 20 to 50% of the initial hardness due to the rearrangement of the pectin and yogurt proteins over time. The products with pectin extracted at 700 W were again exceptions, losing hardness or remaining stable after some time. Overall, this work combines the sourcing of polyphenols and pectin from choice fruit; it uses MAE for isolating the materials of interest; it mechanically examines the resulting gels; and it performs all the above under a specifically-set experimental design aiming towards optimizing the overall process.

Capturing the onset of oral processing: Merging of a model food emulsion drop with saliva.

The events occurring before and during the merging of a model liquid food emulsion with saliva have been captured ex vivo using confocal microscopy. In the order of a few seconds, millimeter-sized drops of liquid food and saliva touch and are deformed; the two surfaces eventually collapse, resulting in the merging of the two phases, in a process reminiscent of emulsion droplets coalescing. The model droplets then surge into saliva. Based on this, two distinct stages can be distinguished for the insertion of a liquid food into the oral cavity: A first phase where two intact phases co-exist, and the individual viscosities and saliva-liquid food tribology should be important to texture perception; and a second stage, dominated by the rheological properties of the liquid food-saliva mixture. The importance of the surface properties of saliva and liquid food are highlighted, as they may influence the merging of the two phases.

Pediatric and Geriatric-Friendly Buccal Foams: Enhancing Omeprazole Delivery for Patients Encountering Swallowing Difficulties.

Buccal foams containing omeprazole (OME) have been developed as potential drug delivery systems for individuals encountering swallowing difficulties, particularly pediatric and geriatric patients. The buccal foams were formulated from lyophilized aqueous gels of maltodextrin, used as a sweetener, combined with various polymers (alginate, chitosan, gelatin, tragacanth) to fine tune their structural, mechanical, and physicochemical properties. Consistent with the requirements for efficient drug delivery across buccal epithelium, the foam comprised of hydroxypropyl methylcellulose and alginate (HPMC-Alg-OME), exhibited moderate hardness and high mucoadhesion resulting to prolonged residence and increased transport of the active across porcine epithelium. The HPMC-Alg-OME foam induced a 30-fold increase in the drug's apparent permeability across porcine buccal tissue, compared to the drug suspension. The developed buccal foams exhibited excellent stability, as evidenced by the unchanged omeprazole content even after six months of storage under ambient conditions (20 °C and 45% RH). Results indicate that buccal foams of omeprazole may address the stability and ease of administration issues related to oral administration of the drug, particularly for children and elderly patients who have difficulty swallowing solid dosage forms.

Fabrication of 3D Printed Hollow Microneedles by Digital Light Processing for the Buccal Delivery of Actives.

In the present study, two different microneedle devices were produced using digital light processing (DLP). These devices hold promise as drug delivery systems to the buccal tissue as they increase the permeability of actives with molecular weights between 600 and 4000 Da. The attached reservoirs were designed and printed along with the arrays as a whole device. Light microscopy was used to quality control the printability of the designs, confirming that the actual dimensions are in agreement with the digital design. Non-destructive volume imaging by means of microfocus computed tomography was employed for dimensional and defect characterization of the DLP-printed devices, demonstrating the actual volumes of the reservoirs and the malformations that occurred during printing. The penetration test and finite element analysis showed that the maximum stress experienced by the needles during the insertion process (10 N) was below their ultimate compressive strength (240-310 N). Permeation studies showed the increased permeability of three model drugs when delivered with the MN devices. Size-exclusion chromatography validated the stability of all the actives throughout the permeability tests. The safety of these printed devices for buccal administration was confirmed by histological evaluation and cell viability studies using the TR146 cell line, which indicated no toxic effects.

The influence of KCl concentration on the gelation of myofibrillar protein giant squid (Dosidicus gigas) due to molecular conformation change.

Introduction: Protein gelation process is of importance in food industry. The objective of this study is to investigate the influence of salt concentration variation, which induced protein conformation change, on protein's intermolecular interactions and its gelation process. Methods: Paramyosin has been separated and purified from myofibrillar protein extracted from giant squid. Then Giant squid's paramyosin molecular mass and intermolecular interactions were quantified by means of light scattering techniques. Finally, the micro-rheology study via diffusing wave spectroscopy (DWS) technique revealed that this conformation change dramatically affected myofibrillar protein gelation process. Results: The obtained apparent molecular weight (ca 2 × 105 g/mol) suggested that protein molecules existed as dimers, while the second virial coefficient A2 significantly reduced from -3.98456 × 10-5 to -5.07575 × 10-4 ml mol/g2 when KCl concentrated from 0.15 to 1 mol/L. Light scattering data also suggest that paramyosin dimers are stiff, with a persistence length of 120 nm, almost the length of a molecule and independent of salt concentration. Mean-square displacement (MSD) of tracer particles at 5 temperatures with 4 salt concentrations displayed that this conformation change had dramatic effect. Therefore, G' and G" were remarkably altered with at least one order of magnitude difference owing to this event occurrence. Conclusions: Paramyosin conformation change due to KCl concentrated enhances attractive interactions with apparent molecular mass increase, which resulted in majority paramyosin molecules (> 99%) in dimeric form and promoted aggregates formation. DWS technique revealed that the conformation change dramatic affected this process characterized by the correlation functions, MSD, and G' and G". This study brings forward data on understanding the effect of a major salt supplement, KCl, on the chemical physics of a major muscle protein.

Quality control evaluation of paediatric chocolate-based dosage forms: 3D printing vs mold-casting method.

Pharmaceutical compounding is a core activity in the preparation of patient-specific dosage forms. In the current study we aimed to investigate whether 3D printing could be employed for the preparation of pediatric-friendly personalized dosage forms that fulfil the acceptance criteria specified in the pharmacopoeias for conventional dosage forms. We then compared the 3D printed dosage forms with the same formulations prepared with mold-casting, a method frequently applied during pharmaceutical compounding. The molded dosage forms failed to pass most of the quality control tests, including the mass uniformity and content uniformity tests, as well as dose accuracy, contrary to the 3D printed, which not only passed all tests but also enabled precision overdose adjustment. Hence, 3D printing of chocolate-based dosage forms may effectively serve as an acceptable alternative method to mold casting in compounding patient-specific medication at the point-of-care.

Stability and rheology of plant-derived hydrocolloid-mucin mixtures.

Mixtures of mucin with pectin were investigated in a range of pectin to mucin ratios and pH values. The phase stability was first studied as absorbance measured at 500 nm (turbidity). Co-existence of the two materials did not result in co-sedimentation or relevant phase separations, while lower pH enhanced aggregation and partial sedimentation of individual components, especially for mucin. The above are in line with the recorded zeta potential values, which are negative for both components at neutral pH and drop down to almost zero at acidic values. The sizes of the particles, as recorded by dynamic light scattering, show a similar trend to the absorbance values, indicating that phase separations are in line with events at the scale of a few hundred nm. Such interactions reflect in shear rheology: The viscosity corresponding to 50 s-1 decreases upon substitution of pectin with mucin at pH 7 and 3, suggesting a flow dominated by changes in the space occupancy by the two components and by changes in the size of the self-assembled structures. The results were compared with those of more complex and typical hydrocolloids extracted from olive compost: The overall shape of the stability diagram of the two ingredients match, suggesting similar modes of action in the presence of mucin for other natural materials. These data throw some light in the norms during the co-existence of food polysaccharides and mucin in oral and gastrointestinal environments.

The role of glycerol on the thermal gelation of myofibrillar protein from giant squid (Dosidicus gigas) mince.

This work studies the effect of glycerol on the chemical physics of the thermal gelatin of protein from giant squid minced meat. The presence of glycerol induced changes in the nano protein particles (NPP) self-assembled structures. These nanoscale events resulted in dramatic changes on the interactions between proteins when forming gels, with the contribution of ionic interactions increasing by 17% upon gelation, that of hydrogen bonds reducing by 50%, that of hydrophobic interactions decreasing by 45%, and that of disulphide bonding increasing by 18%. Glycerol also induced cluster formation in myofibrillar solutions. As a result, incorporation of glycerol increased springiness, resilience, and adhesiveness of the formed gels by 13%, 25%, and 370% respectively. The heating gelation of myofibrillar proteins was monitored at various temperatures via recording the elastic and storage moduli. Rheology and micro-rheology studies revealed that the presence of glycerol increased G' and G″ of thermally-gelled giant squid meat.

Semi-solid extrusion 3D printing of starch-based soft dosage forms for the treatment of paediatric latent tuberculosis infection.

OBJECTIVES: The development of age-appropriate dosage forms is essential for effective pharmacotherapy, especially when long-term drug treatment is required, as in the case of latent tuberculosis infection treatment with up to 9 months of daily isoniazid (ISO). Herein, we describe the fabrication of starch-based soft dosage forms of ISO using semi-solid extrusion (SSE) 3D printing. METHODS: Corn starch was used for ink preparation using ISO as model drug. The inks were characterized physicochemically and their viscoelastic properties were assessed with rheological analysis. The morphology of the printed dosage forms was visualized with scanning electron microscopy and their textural properties were evaluated using texture analysis. Dose accuracy was verified before in-vitro swelling and dissolution studies in simulated gastric fluid (SGF). KEY FINDINGS: Starch inks were printed with good resolution and high drug dose accuracy. The printed dosage forms had a soft texture to ease administration in paediatric patients and a highly porous microstructure facilitating water penetration and ISO diffusion in SGF, resulting in almost total drug release within 45 min. CONCLUSIONS: The ease of preparation and fabrication combined with the cost-effectiveness of the starting materials constitutes SSE 3D printing of starch-based soft dosage forms a viable approach for paediatric-friendly formulations in low-resource settings.

In vitro digestion of tofu with different textures using an artificial gastric digestive system.

Two kinds of tofu with obvious differences in texture ["GDL" and "CaSO4", standing for tofus made with the application of either glucono-δ-lactone (GDL) or calcium sulfate, with measured hardness 23.1 ± 3.3 g and 105.2 ± 25.1 g, respectively] were used as to investigate the in vitro progress and extent of tofu digestion, using an independently-developed artificial gastric digestion system (AGDS). The particle size distributions of both CaSO4 and GDL tofu shifted towards smaller particles as the digestion time increased, while the viscosity of the gastric digesta also increased. Tofu proteins were hydrolyzed in the simulated stomach, with GDL tofu showing a higher hydrolysis rate, based on the temporal evolution of SDS-PAGE bands, and had a higher amino acids accumulation than CaSO4 tofu at the end of gastric digestion. In the absence of peptic enzymes, the protein was acidically-hydrolyzed, but the degree of hydrolysis was much lower than in the presence of enzymes; these findings are in accord with the changes in microstructure observed by scanning electron microscopy. The results indicated that the in vitro extent of tofu digestion is related to its hardness, which is in turn related to its microstructure; they also indicated the potential of our developed in vitro dynamic stomach in studying semi-solid foods.