Mucus-Mimicking Mucin-Based Hydrogels by Tandem Chemical and Physical Crosslinking.

Mucosal tissues represent a major interface between the body and the external environment and are covered by a highly hydrated mucins gel called mucus. Mucus lubricates, protects and modulates the moisture levels of the tissue and is capitalized in transmucosal drug delivery. Pharmaceutical researchers often use freshly excised animal mucosal membranes to assess mucoadhesion and muco-penetration of pharmaceutical formulations which may struggle with limited accessibility, reproducibility, and ethical questions. Aiming to develop a platform for the rationale study of the interaction of drugs and delivery systems with mucosal tissues, in this work mucus-mimicking mucin-based hydrogels are synthesized by the tandem chemical and physical crosslinking of mucin aqueous solutions. Chemical crosslinking is achieved with glutaraldehyde (0.3% and 0.75% w/v), while physical crosslinking by one or two freeze-thawing cycles. Hydrogels after one freeze-thawing cycle show water content of 97.6-98.1%, density of 0.0529-0.0648 g cm⁻3, and storage and loss moduli of ≈40-60 and ≈3-5 Pa, respectively, that resemble the properties of native gastrointestinal mucus. The mechanical stability of the hydrogels increases over the number of freeze-thawing cycles. Overall results highlight the potential of this simple, reproducible, and scalable method to produce artificial mucus-mimicking hydrogels for different applications in pharmaceutical research.

Dual functionality of diacylglycerols in water-in-oil emulsion gel systems.

Distearin (DS) can be used as an emulsifier, due to its surface activity derived from the amphiphilic nature of the molecule, moreover, it can also crystallize and form a 3D crystal network that can induce oil gelation. The current research aimed to examine the ability to combine both emulsifying and oil gelation properties to structure and stabilize water-in-oil emulsion gel system. Different water contents and DS concentrations produce emulsion gels with different textural attributes while incorporating up to 30% of water in a 15% wt. DS-based oleogel resulted in stable white gels. Microscopy imaging confirmed the formation of a water-in-oleogel type emulsion gel characterized by DS crystallization in the continuous phase and at the interface through Pickering mechanism. A positive relation was observed between the G' and hardness values and water content, suggesting gel strengthening resulted from interactions between the DS crystals at the interface and the continuous phase, as suggested by the active filler theory. Thermal analysis revealed two broad melting events at the temperature range of 42.2-44.9 °C and 55.9-58.6 °C for emulsion gels with 10-30% water content, suggesting initial melting of ß' polymorph and transition to ß during melting, which was confirmed by XRD. The results showed that homogenization significantly improved the oil retention of the gels due to increased crystal surface area, while water addition slightly reduced it. Compared with traditional emulsions or oleogels, this water-in-oil gel system demonstrated prolonged stability and enhanced mechanical properties due to the dual functionality of DS at the water/oil interface and bulk.

Isotropic liquid state of cocoa butter.

The complex crystal structure of coca butter (CB) is responsible for the unique melting behavior, surface gloss, and mechanical properties of chocolate. While most studies concentrated on the crystalline state of CB, few studied the isotropic liquid state, which has a major impact on the crystallization process and the characteristics of the resulting crystals. In this study, the molecular organizations of the main CB triacylglycerols (TAGs; 1,3-dipalmitoyl-2-oleoylglycerol, palmitoyl-oleoyl-stearoylglycerol, POS, and 1,3-distearoyl-2-oleoylglycerol) were studied. The findings revealed the tunning-fork (Tf) conformation, commonly found in the crystalline state, is the least abundant in the isotropic liquid state of CB and pure TAGs. Notably, POS was found to interact with itself in CB, while its molecules with Tf conformation, although in small amounts in the mixture, tend to pair with each other at lower temperatures. These results highlight the significance of POS in CB crystallization and provide insights for developing CB alternatives.

Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute.

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

The effect of preparation temperature and composition on bigel performance as fat replacers.

Consumer awareness of the deleterious effect of a diet rich in saturated fat pushes the food industry to find new fat alternatives. Bigels, hybrids of hydrogels and oleogels, are an attractive option for formulating oil-based fat mimetics, particularly lamination fats. This research explored the characteristics of a hydrogel-in-oleogel bigel, made of candelilla wax and xanthan gum. This study investigated the effect of homogenization temperature, hydrogel : oleogel phase ratio, and storage conditions on the bigel melting profile, mechanical and rheological properties, stability, and the structural characteristics involved. The optimal homogenization temperature that resulted in a smooth, firm, margarine-like texture was 42 °C while higher homogenization temperatures produced lumpy unspreadable bigels and lower ones produced soft and smooth texture. The bigel behavior was related to the formation of a low mobility biphasic system, stabilized in a Pickering mechanism by wax crystals that crystallize at 47 °C, above the homogenization temperature. The phase ratios tested, 15 : 85 to 45 : 55 hydrogel : oleogel, appeared to have a limited effect on any of the bigel characteristics. More specifically, no significant differences in melting temperature, texture parameters, flow behavior, and stability, which are reminiscent of margarine, were detected. These results indicated that hydrogel droplets may serve as active fillers, strengthening the bigel matrix when their amount in the bigel increases and the oleogel, which is the dominant phase, decreases. These findings provide an understanding of the way bigel properties depend on the formulation and preparation, which is valuable in the development of bigel fat replacers and other novel food applications.

An experimental and theoretical approach to understand the interaction between particles and mucosal tissues.

Nanonization of poorly water-soluble drugs has shown great potential in improving their oral bioavailability by increasing drug dissolution rate and adhesion to the gastrointestinal mucus. However, the fundamental features that govern the particle-mucus interactions have not been investigated in a systematic way before. In this work, we synthesize mucin hydrogels that mimic those of freshly excised porcine mucin. By using fluorescent pure curcumin particles, we characterize the effect of particle size (200 nm, and 1.2 and 1.3 µm), concentration (18, 35, and 71 µg mL-1), and hydrogel crosslinking density on the diffusion-driven particle penetration in vitro. Next, we derive a phenomenological model that describes the physics behind the diffusion-derived penetration and considers the contributions of the key parameters assessed in vitro. Finally, we challenge our model by assessing the oral pharmacokinetics of an anti-cancer model drug, namely dasatinib, in pristine and nanonized forms and two clinically relevant doses in rats. For a dose of 10 mg kg-1, drug nanonization leads to a significant ∼8- and ∼21-fold increase of the drug oral bioavailability and half-life, respectively, with respect to the unprocessed drug. When the dose of the nanoparticles was increased to 15 mg kg-1, the oral bioavailability increased though not significantly, suggesting the saturation of the mucus penetration sites, as demonstrated by the in vitro model. Our overall results reveal the potential of this approach to pave the way for the development of tools that enable a more rational design of nano-drug delivery systems for mucosal administration. STATEMENT OF SIGNIFICANCE: The development of experimental-theoretical tools to understand and predict the diffusion-driven penetration of particles into mucus is crucial not only to rationalize the design of nanomedicines for mucosal administration but also to anticipate the risks of the exposure of the body to nano-pollutants. However, a systematic study of such tools is still lacking. Here we introduce an experimental-theoretical approach to predict the diffusion-driven penetration of particles into mucus and investigate the effect of three key parameters on this interaction. Then, we challenge the model in a preliminary oral pharmacokinetics study in rats which shows a very good correlation with in vitro results. Overall, this work represents a robust platform for the modelling of the interaction of particles with mucosae under dynamic conditions.

Print-and-Grow within a Novel Support Material for 3D Bioprinting and Post-Printing Tissue Growth.

3D bioprinting holds great promise for tissue engineering, with extrusion bioprinting in suspended hydrogels becoming the leading bioprinting technique in recent years. In this method, living cells are incorporated within bioinks, extruded layer by layer into a granular support material followed by gelation of the bioink through diverse cross-linking mechanisms. This approach offers high fidelity and precise fabrication of complex structures mimicking living tissue properties. However, the transition of cell mass mixed with the bioink into functional native-like tissue requires post-printing cultivation in vitro. An often-overlooked drawback of 3D bioprinting is the nonuniform shrinkage and deformation of printed constructs during the post-printing tissue maturation period, leading to highly variable engineered constructs with unpredictable size and shape. This limitation poses a challenge for the technology to meet applicative requirements. A novel technology of "print-and-grow," involving 3D bioprinting and subsequent cultivation in κ-Carrageenan-based microgels (CarGrow) for days is presented. CarGrow enhances the long-term structural stability of the printed objects by providing mechanical support. Moreover, this technology provides a possibility for live imaging to monitor tissue maturation. The "print-and-grow" method demonstrates accurate bioprinting with high tissue viability and functionality while preserving the construct's shape and size.

Structured edible lipid-based particle systems for oral drug-delivery.

Oral administration is the most popular and patient-compliant route for drug delivery, though it raises great challenges due to the involvement of the gastro-intestine (GI) system and the drug bioavailability. Drug bioavailability is directly related to its ability to dissolve, transport and/or absorb through the physiological environment. A great number of drugs are characterized with low water solubility due to their hydrophobic nature, thus limiting their oral bioavailability and clinical use. Therefore, new strategies aiming to provide a protective shell through the GI system and improve drug solubility and permeability in the intestine were developed to overcome this limitation. Lipid-based systems have been proposed as good candidates for such a task owing to their hydrophobic nature which allows high drug loading, drug micellization ability during intestinal digestion due to the lipid content, and the vehicle physical protective environment. The use of edible lipids with high biocompatibility paves the bench-to-bedside translation. Four main types of structured lipid-based drug delivery systems differing in the physical state of the lipid phase have been described in the literature, namely emulsions, solid lipid nanoparticles, nanostructured lipid carriers, and oleogel-based particles. The current review provides a comprehensive overview of the different structured edible lipid-based oral delivery systems investigated up to date and emphasizes the contribution of each system component to the delivery performance, and the oral delivery path of lipids.

The role of hydrogen bonds in TAG derivative-based oleogel structure and properties.

Glycerol monosterate (GMS) and stearic acid (SA) share a similar carbon chain structure while SA has a carboxyl head group and GMS has two free hydroxyl groups. The current research focuses on the relationship between GMS and SA chemical structure, nano and mesoscale crystal structure, and the oleogel macroscopic characteristics. Molecular analysis revealed the formation of different types of hydrogen bonds, which disappear upon temperature increase at different temperatures. Nano-structural analysis exhibited tight and ordered lamellar structures for SA compared with loosely packed short lamellar structures in GMS oleogel, presumably due to its larger hydrophilic head group. Microstructure imaging revealed ordered anisotropically orientated needle-like crystals in SA and isotopically ordered braid-like crystals in GMS oleogels. Mechanical analysis revealed that gel strength is enhanced when crystal structure is isotropically oriented, similar behavior seen is composite materials, where the structuring agent crystals behave like a reinforcing agent within the oil matrix.

The Effect of the HLB Value of Sucrose Ester on Physiochemical Properties of Bigel Systems.

The current research explored the effect of different sucrose esters (SEs), with different hydrophilic-lipophilic balance (HLB) values, on bigel structure and properties. Bigels consisting of a water phase with glycerol and gelatin and an oil phase with glycerol mono-stearate, lecithin, and SEs with different HLB values were prepared. Rheological and thermal analyses revealed similar gelation-melting transitions governed by glycerol-monostearate crystallization (at ≈55 °C) for all bigel samples. The bigel matrix of the H1 and H2 samples (bigels consisting of SEs with HLBs of 1 and 2, respectively) demonstrated physical gel rheological characteristics of higher elastic and solid-like behavior compared with the H6 sample (bigel consisting SE with HLB 6). A similar trend was observed in the mechanical analysis with respect to hardness, firmness, and spreadability values, which were in the order of H1 > H2 > H6. This behavior was attributed to droplet size observed in the microscopy analysis, revealing significantly smaller droplets in the H1 and H2 samples compared with the H6 sample. These differences in droplet size were attributed to the diffusion kinetics of the low-molecular-weight surfactants. More specifically, the ability of mono-esterified SEs to diffuse faster than fully esterified SEs due to lower molar mass leads to a higher SE content at the oil-in-water (O/W) interface as opposed to the bulk oil phase. The results demonstrate the importance of the interface content in O/W bigel systems, providing an effective way to alter and control the bigel bulk properties.

Controlling lipid intestinal digestibility using various oil structuring mechanisms.

This research demonstrates the ability to direct the rate and extent of lipid hydrolysis of oleogels using a combination of different structuring agents. Combinations of ethyl cellulose (EC) (20 cP and 45 cP) and commercial mixture of mono and di-glycerides (E471), at different ratios, were examined. The results suggest that the combination of E471 and EC significantly affects both gel physical properties and intestinal lipolysis. The gelation profile of the combined system demonstrated the EC sol-gel transition, which is characterized by G' = G'' at high temperatures (∼100 °C) followed by a soft-to-hard gel transition at low temperatures ∼30 °C, which corresponds to E471 crystallization. Such a profile suggests the formation of two gel networks, with the polymer network acting as a platform for E471 crystallization. Mechanical analysis reveals harder gels in the E471 : EC 20 cP mixture compared with the simple addition of each component contribution, suggesting a synergistic effect with a typical maximum at 7 : 3 E471 : EC 20 cP ratio. No significant additive effect was observed for E471 : EC 45 cP mixtures. Maximum lipolysis in the order of EC < E471 : EC < E471 was obtained, implying an effect of the structuring agent used on the lipolysis profile. A first-order kinetics analysis fitted to the lipolysis profiles demonstrated rate constant values in the order of E471 < E471 : EC < EC. Such behavior was attributed to the oil state, liquid vs. solid, and the network strength, both of which limit the lipase activity by hindering liquid TAG accessibility. Overall, the results demonstrate the ability to control gel properties and hydrolysis by manipulating gel composition. Such rational design can be exploited when developing new fat mimetic systems aimed at controlling the lipid digestion profile or the release of hydrophobic components present in the oil phase.

Comparison of Thermal and High-Pressure Gelation of Potato Protein Isolates.

Potato protein isolate (PPI), a commercial by-product of the starch industry, is a promising novel protein for food applications with limited information regarding its techno-functionality. This research focused on the formation of both thermal and high-pressure gels at acidic and neutral pH levels. Our results reveal that physical gels are formed after 30 min by heat at pH 7 and pH 3, while pressure (300-500 MPa) allows the formation of physical gels only at pH 3, and only when the system crosses 30 °C by adiabatic heating during pressurization. Texture profile analysis (TPA) revealed that gel hardness increased with both gelation temperature and pressure, while water-holding capacity was lower for the pressure-induced gels. The proteins released in the water-holding test suggested only partial involvement of patatin in the gel formation. Vitamin C as a model for a thermally liable compound verified the expected better conservation of such compounds in a pressure-induced gel compared to a thermal one of similar textural properties, presenting a possible advantage for pressure-induced gelation.

Shear thinning pectin hydrogels physically cross-linked with chitosan nanogels.

The development of a polymer-nanogel hydrogel based on a pair of polysaccharides is reported for the first time. This new hydrogel exhibits self-healing properties due to physical interactions between soluble pectin chains and chitosan nanogels. The nanogels act as crosslinking agents between pectin chains, leading to the formation of thermos-responsive hydrogel. Due to the dynamic interactions between the chains and the nanogels, the formed network dissociate under applied shear, allowing the hydrogel to flow. Moreover, elimination of the applied shear results in exceptionally fast and comprehensive recovery of the storage modulus, reverting the mixture back into solid form. The viscosity and Young modulus increased with the nanogels concentration while the equilibrium swelling decreased as the nanogels concentration increased suggesting a direct relation between the cross-linking degree and nanogel content. This novel hydrogel displays network recovery suitable for injectable biomedical applications, while benefiting from the advantages of nanogels as carriers.

The Effect of Intramolecular Cross-Linking on Polymer Interactions in Solution.

The conformation of a polymer in a solvent is typically defined by the solvent quality, which is a consequence of the solvent and macromolecule's chemistry. Yet, additional factors can affect the polymer conformation, such as non-covalent interactions to surfaces or other macromolecules, affecting the amount of polymer-solvent interactions. Herein, chemically folded polymers with protein-like architectures are studied and compared to their unfolded linear precursor in good solvents using rheology measurements. The current research reveals that permanent folding by intramolecular chemical cross-linking limits the chain mobility and therefore causes a reduction in polymer-solvent interactions, making a good solvent become theta. This change not only affects the "solvent quality" but also leads to a change in particle-particle interactions as a function of concentration. These findings provide crucial insight into the effects of intramolecular cross-links on macromolecule solubility and self-assembly, which are critical for mimicking structurally similar biological materials.

Tuning the mechanical properties of alginate-peptide hydrogels.

Alginate, a polysaccharide that gels in the presence of divalent ions, has been used in the field of regenerative medicine to facilitate cell growth in impaired tissues by providing an artificial bio-surrounding similar to the natural extra cellular matrix (ECM). Here, we present a systematic investigation of the effect of three arginine-glycine-aspartic acid (RGD)-containing peptides, G6KRGDY, A6KRGDY and V6KRGDY, on the physical properties of alginate-peptide hydrogels. Rheology measurements showed that the storage modulus of the alginate-A6KRGDY and alginate-V6KRGDY gels is an order of magnitude higher than that of the alginate-G6KRGDY gel. Small angle X-ray scattering (SAXS) measurements suggest that the difference in the mechanical properties of the gels is due to the formation of larger peptide junction zones in addition to the ones formed by calcium ions. These findings indicate that the peptides' ability to self-assemble in aqueous solution is a significant factor in tuning the stiffness of the alginate/peptide hybrid hydrogels.

Influencing the crystallization behavior of binary mixtures of stearyl alcohol and stearic acid (SOSA) using ethylcellulose.

In the present study we have characterized the influence of the polymer gelator ethylcellulose (EC) on the crystallization behavior of mixtures of stearyl alcohol and stearic acid (SOSA). The presence of EC led to a more abrupt thermo-reversible crystallization process and an increase in the onset of crystallization temperature from 22.7±0.35°C to 26.5±0.42°C. X-ray analysis indicated that the polymorphism of the mixed SOSA crystals was maintained in the presence of EC; however, changes in the small angle region indicated the presence of the polymer network altered the higher-order organization of the crystal network. Significant changes in the microstructural organization were also observed by light microscopy. A random distribution of needle-like, oriented platelets were observed in SOSA gels, while branched, feather-like structures were apparent in the mixed EC/SOSA system. Temperature-sweep rheological experiments of the combined EC/SOSA system also indicated that prior to crystallizing, SOSA molecules plasticized the polymer chains, resulting in a decrease in the gelation point (cross-over point; G'=G″) from ~110°C to 90°C. This effect was corroborated by DSC experiments, in which it was observed that the glass transition temperature of EC decreased and broadened with increasing SOSA content. Back extrusion flow curves indicated that the addition of EC reduces the brittleness and increases the plasticity of the bulk material, as indicated by the brittleness factor quantified over the steady-state flow regime, even when the combined gelator system was substantially firmer. Although the presence of the EC network resulted in a stress overshoot during initial penetration, by incorporating EC below its critical gelation concentration eliminated the overshoot while still providing plasticity to the SOSA network, such that the flow behavior was shown to be comparable to several commercial margarines. This study has demonstrated the ability of EC to modify the crystallization behavior of a low molecular weight oleogelator, while increasing the plasticity of the polymer network, to form a synergistic oleogelator system.

Ethylcellulose oleogels for lipophilic bioactive delivery - effect of oleogelation on in vitro bioaccessibility and stability of beta-carotene.

The in vitro lipolysis and ß-carotene (BC) transfer from oil to aqueous phase of canola oil ethylcellulose (EC) oleogels were measured using a static monocompartmental model simulating oral, gastric, and duodenal digestive stages. The effects of EC oleogelation on gel in vitro digestibility were examined, using un-structured canola oil as a control. The physicochemical properties of oleogels containing BC were also measured. It was found that oleogels made with 10% 10 cP and 10% 20 cP did not differ significantly in their extent of lipolysis or BC transfer compared to canola oil; however 10% 45 cP and 15.5% 20 cP had a significantly lower extent of lipolysis and BC transfer compared to other formulations. The structure and mechanical strength of the oleogels were both determined to be factors affecting lipolysis and transfer. The presence of BC did not significantly affect the mechanical strength of the gels and EC oleogelation delayed BC degradation under accelerated storage conditions compared to a heated canola oil control. These findings could contribute to the development of new applications for EC oleogels, specifically for the effective delivery of lipophilic molecules.

Modulating the gel properties of soy glycinin by crosslinking with tyrosinase.

The gelation progression and gel properties of enzymatically crosslinked soy glycinin were evaluated in comparison to non-crosslinked glycinin. Glycinin was initially crosslinked using tyrosinase from Bacillus megaterium (TyrBm) and was later used to form gel upon heating. Gelation was evaluated by small deformation rheological measurements and revealed a significant increase in storage modulus (G') obtained in the crosslinked gel. This was confirmed by temperature sweep and frequency sweep measurements that supported the results and proved that the difference in modulus was not frequency dependent. Texture profile analysis showed an increase in hardness and decrease in elasticity of the crosslinked gels. Scanning electron microscopy (SEM) images displayed a more structural network with larger pore size in the crosslinked gel. The less dense structure of the crosslinked glycinin gel network led to a slight decrease in the water holding capacity. Finally, thermal analysis using differential scanning calorimetry (DSC) confirmed no change in the gelation point induced by denaturation, however thermal gravimetric analysis (TGA) did show a difference in the decomposition profile of the crosslinked protein compared with non-crosslinked glycinin. The results suggest that by applying TyrBm mediated crosslinking we may modulate the protein gel properties for tailoring the texture of food products.

Physical and structural characteristics of acrylated poly(ethylene glycol)-alginate conjugates.

Transmucosal delivery of therapeutic agents is a non-invasive approach that utilizes human entry paths such as the nasal, buccal, rectal and vaginal routes. Mucoadhesive polymers have the ability to adhere to the mucus layer covering those surfaces and by that promote drug release, targeting and absorption. We have recently demonstrated that acrylated polymers display enhanced mucoadhesive properties due to their ability to covalently attach to mucus type glycoproteins. We have synthesized an acrylated poly(ethylene glycol)-alginate conjugate (alginate-PEGAc), a molecule which combines the gelation ability of alginate with the mucoadhesion properties arising from both the characteristics of poly(ethylene glycol) and the acrylate functionality. In the current investigation we introduce an in-depth characterization of the thermal, mechanical and structural properties of alginate-PEGAc aimed at gaining a better knowledge of its structure-function relations. The thermal stability, evaluated by thermal gravimetric analysis and differential scanning calorimetry, was compared with that of alginate and the intermediate product thiolated alginate. Dehydration at temperatures up to 200 °C was detected for all samples, followed by distinctive decomposition steps arising from the decomposition of the polymer backbone and side-chains. The nanostructure of the solutions and gels was evaluated from small angle X-ray scattering patterns, to which the "broken rod linked by flexible chain" model was fitted, and from rheology measurements. The maxima arising from electrostatic repulsion between the highly charged alginate chains was diminished for both modified alginate samples, suggesting that modification led to electrostatic screening. Alginate, thiolated alginate and alginate-PEGAc cross-linked with calcium ions demonstrated similar scattering patterns. However, different scattering intensities, gel strengths, and gelation kinetics were observed, suggesting a decrease in the cross-linking density in the order alginate>thiolated alginate>alginate-PEGAc. These results were attributed to the increased size of the grafted side groups, which interfere with the gelation process. Examining the effect of the method of alginate-PEGAc gelation (physical or chemical) has shown that additional UV irradiation of calcium cross-linked gels did not cause a significant change in the network structure and strength. It seems that the concentration of the acrylated end group is not high enough to create a chemically cross-linked network.