Droplet-Based Microfluidics as a Platform to Design Food-Grade Delivery Systems Based on the Entrapped Compound Type.

Microfluidic technology has emerged as a powerful tool for several applications, including chemistry, physics, biology, and engineering. Due to the laminar regime, droplet-based microfluidics enable the development of diverse delivery systems based on food-grade emulsions, such as multiple emulsions, microgels, microcapsules, solid lipid microparticles, and giant liposomes. Additionally, by precisely manipulating fluids on the low-energy-demand micrometer scale, it becomes possible to control the size, shape, and dispersity of generated droplets, which makes microfluidic emulsification an excellent approach for tailoring delivery system properties based on the nature of the entrapped compounds. Thus, this review points out the most current advances in droplet-based microfluidic processes, which successfully use food-grade emulsions to develop simple and complex delivery systems. In this context, we summarized the principles of droplet-based microfluidics, introducing the most common microdevice geometries, the materials used in the manufacture, and the forces involved in the different droplet-generation processes into the microchannels. Subsequently, the encapsulated compound type, classified as lipophilic or hydrophilic functional compounds, was used as a starting point to present current advances in delivery systems using food-grade emulsions and their assembly using microfluidic technologies. Finally, we discuss the limitations and perspectives of scale-up in droplet-based microfluidic approaches, including the challenges that have limited the transition of microfluidic processes from the lab-scale to the industrial-scale.

Biomimetic Nanovesicles-Sources, Design, Production Methods, and Applications.

Despite all the progress in the field of liposomes and nanoparticles for applications as drug and gene delivery systems, the specific targeting and immune system escape capabilities of these systems are still limited. Biomimetic nanovesicles emerged as a strategy to overcome these and other limitations associated with synthetic carriers, such as short circulation time, cytotoxicity, and difficulty in crossing biological barriers, since many of the desirable abilities of drug delivery systems are innate characteristics of biological vesicles. Thus, the question arises: would biomimetic nanovesicles be responsible for addressing these advances? It is currently known that biomimetic nanovesicles (BNV) can combine the intrinsic advantages of natural materials with the well-known production methods and controllability of synthetic systems. Besides, the development of the biotechnology and nanotechnology fields has provided a better understanding of the functionalities of biological vesicles and the means for the design and production of biomimetic nanovesicles (BNV). Based on this, this work will focus on tracking the main research on biomimetic nanovesicles (BNV) applied as drug and gene delivery systems, and for vaccines applications. In addition, it will describe the different sources of natural vesicles, the technical perspectives on obtaining them, and the possibility of their hybridization with synthetic liposomes.

Trends in hydrogel-based encapsulation technologies for advanced cell therapies applied to limb ischemia.

Ischemia occurs when blood flow is reduced or restricted, leading to a lack of oxygen and nutrient supply and removal of metabolites in a body part. Critical limb ischemia (CLI) is a severe clinical manifestation of peripheral arterial disease. Atherosclerosis serves as the main cause of CLI, which arises from the deposition of lipids in the artery wall, forming atheroma and causing inflammation. Although several therapies exist for the treatment of CLI, pharmacotherapy still has low efficacy, and vascular surgery often cannot be performed due to the pathophysiological heterogeneity of each patient. Gene and cell therapies have emerged as alternative treatments for the treatment of CLI by promoting angiogenesis. However, the delivery of autologous, heterologous or genetically modified cells into the ischemic tissue remains challenging, as these cells can die at the injection site and/or leak into other tissues. The encapsulation of these cells within hydrogels for local delivery is probably one of the promising options today. Hydrogels, three-dimensional (3D) cross-linked polymer networks, enable manipulation of physical and chemical properties to mimic the extracellular matrix. Thus, specific biostructures can be developed by adjusting prepolymer properties and encapsulation process variables, such as viscosity and flow rate of fluids, depending on the final biomedical application. Electrostatic droplet extrusion, micromolding, microfluidics, and 3D printing have been the most commonly used technologies for cell encapsulation due to their versatility in producing different hydrogel-based systems (e.g., microgels, fibers, vascularized architectures and perfusable single vessels) with great potential to treat ischemic diseases. This review discusses the cell encapsulation technologies associated with hydrogels which are currently used for advanced therapies applied to limb ischemia, describing their principles, advantages, disadvantages, potentials, and innovative therapeutic ideas.

Stabilization mechanisms of O/W emulsions by cellulose nanocrystals and sunflower protein.

Oil-in-water (O/W) emulsions stabilized by cellulose nanocrystals (CNC) and/or sunflower proteins (SFP) were produced, aiming to study the effects of each and the mixture of these stabilizers on the interfacial behavior and physicochemical properties of O/W emulsions. The presence of CNC (non-surface activity compound) did not affect SFP solutions' adsorption kinetics since there were no differences in the interfacial tension curves of SFP and mixtures of stabilizers over time. However, either stabilizer provided alone high resistance against droplet coalescence over time (no evidence of oiling-off and no difference in the mean droplet size values), even systems with less viscoelastic interface (2 % CNC). Although droplet coalescence was prevented by steric hindrance and reduction of interfacial tension between the oil-water phases provided by CNC and SFP, respectively, these emulsions were unstable to the creaming phenomenon. Only the mixture of these stabilizers was able to prevent both destabilization mechanisms, initially by adsorption and anchoring of SFP on the interface, followed by adsorption of CNC in the free interface spaces, and finally by the interaction of non-adsorbed CNC particles in the continuous phase, which led to an increase in system viscosity. Thus, based on the results of interfacial properties and emulsions characteristics, we had a better understanding of stabilization mechanisms of O/W emulsions by a food-grade particle and a plant-derived protein.

Interactions of ß-carotene with WPI/Tween 80 mixture and oil phase: Effect on the behavior of O/W emulsions during in vitro digestion.

This study investigated the impact of adding ß-carotene on the structure of fresh O/W emulsions with different oil phase (sunflower oil-LCT or NEOBEE®1053-MCT) and emulsifiers (WPI, Tween 80 - T80 or WPI/T80 mixture). In this sense, the behavior of emulsions through the gastrointestinal tract, the stability and bioaccessibility of ß-carotene were also assessed. The ß-carotene reduced the interfacial tension of the LCT/MCT-water systems. The addition of ß-carotene promoted an increase of viscoelasticity of LCT/MCT-T80 (0.5%WPI/0.5%T80 and 1%T80 w/w) interfaces, but an increase of WPI content reduced the viscoelasticity of interfacial layers (LCT/MCT-1% WPI). These changes in the interface properties influenced the mean droplet size and ζ-potential of the fresh emulsions. LCT systems presented similar bioaccessibility/stability of ß-carotene. However, ß-carotene entrapped within protein-coated MCT droplets was more stable than within T80-MCT systems. Our results show that ß-carotene interacted with other ingredients of emulsions changing their properties and behavior under gastrointestinal tract as well as the stability/bioaccessibility of ß-carotene.

Impact of whey protein/surfactant mixture and oil type on the gastrointestinal fate of emulsions: Ingredient engineering.

The engineering of ingredients emerges as a strategy to design emulsified products aiming to control the lipid hydrolysis. In this context, oil-in-water (O/W) emulsions composed of different oil phases (Sunflower oil - LCT or NEOBEE® 1053 - MCT) and stabilized by whey protein isolate - WPI (1% w/w), Tween 80 - T80 (1% w/w) or varied ratios of WPI/T80 (0.9975%WPI/0.0025%T80; 0.75%WPI/0.25%T80; 0.5%WPI/0.5%T80 w/w) were produced and submitted to simulated gastrointestinal conditions. The lipolysis of LCT was influenced by the fatty acid chain length and emulsifier composition, while only the fatty acid chain length affected the lipolysis of MCT. The emulsions produced with LCT and 1%WPI or 09975%WPI/00025%T80 showed the highest release rate of free fatty acids (FFAs), but similar result was observed for the 0.5%WPI/0.5%T80 system. In the 0.5%WPI/0.5%T80 mixture, WPI and T80 worked together and achieved an improved performance during the gastric (stability similar as 1%T80 emulsion) and small intestinal phases (lipolysis similar as 1%WPI emulsion). The rational selection of ingredients is useful to design emulsions with improved performance as a delivery system since the emulsion structural stability during digestion, the oil type and interaction between lipase-interface had a marked impact on the efficiency of lipid digestion.

Cellulose nanocrystals from ultrasound process stabilizing O/W Pickering emulsion.

Cellulose nanocrystals (CNC) are bio-based solid particles arisen as promising stabilizers for Pickering emulsions in food, pharmaceutical and cosmetics industries. This study aimed to understand the stabilization mechanism of oil-in-water emulsion using CNC as stabilizing particles. CNC were obtained from cellulose microcrystalline after acid hydrolysis, dialysis, ultrasound treatment and vacuum filtration. Atomic force microscopy (AFM) showed needle-shaped CNC. The CNC presented good stability against agglomeration due to the high electrostatic repulsion between particles, making them feasible to be used in O/W emulsions. O/W emulsions were stabilized by CNC and prepared using rotor-stator and ultrasound as mechanical processes. Emulsions stabilized by CNC were opaque, homogeneous and kinetically stable during few days. Small droplets generated during the ultrasound process, could be covered by cellulose nanoparticles that acted as an effective mechanical barrier against droplets coalescence in a Pickering mechanism. The mechanism of droplets stabilization was associated with electrostatic and steric repulsion between droplets. Emulsions were evaluated varying the proportion between flaxseed oil and cellulose nanocrystals (CNC). Emulsions with a lower proportion of CNC showed better kinetic stability compared to emulsions with higher CNC proportion. After 7 days of storage, the viscosity of emulsions with a higher proportion of CNC particles decreased, which was associated to the emulsion destabilization. Our results improved the understanding of the relationship between the proportions of oil and particles for emulsion properties by evaluating the potential application of CNC as a food emulsifier.

The stabilizing effect of cellulose crystals in O/W emulsions obtained by ultrasound process.

The encapsulation of lipophilic bioactive compounds, such as flaxseed oil, is usually done using O/W emulsions as carrier matrix. The aim of this study was to understand the stabilization mechanism of micro-nano cellulose crystals produced from acid hydrolysis in O/W emulsion. Effects of emulsification process conditions using ultrasound on the cellulose particles properties were evaluated varying the proportion of oil-cellulose particles in the emulsion formulation. Cellulose structure did not change using different conditions of emulsification and X-ray diffraction showed major presence of cellulose I. Particle size distribution of cellulose was bimodal and mean particle size reduced after hydrolysis. Emulsions stabilized by cellulose were opaque, homogeneous and showed good kinetic stability. The largest microcrystals were displayed between the oil droplets, preventing the flocculation of the droplets while smaller particles were adsorbed on the oil-water interface. The mechanism of droplets stabilization was not associated to the reduction of interfacial tension. Stabilization was associated to significant effect of electrostatic repulsion and increase in viscosity. Moreover, the flaxseed oil droplets were completely surrounded by cellulose nanocrystals, showing also Pickering-type stabilization. Therefore, emulsions with cellulose crystals were stabilized by different mechanisms and have interesting properties and characteristics for the protection of lipophilic compounds that could be applied in food and cosmetics products.

Modulating in vitro digestibility of Pickering emulsions stabilized by food-grade polysaccharides particles.

An in vitro digestibility protocol was used to elucidate the role of different emulsifying polysaccharides particles on the lipid digestion rate of oil-in-water Pickering emulsions. Emulsions stabilized by cellulose crystals (CCrys), cellulose nanofibers (CNFs), chitosan particles and a conventional emulsifier (Tween 80) were evaluated concerning microstructure, droplet size, zeta potential and free fatty acids released during digestion. After gastric step, the high positive charge of chitosan-stabilized emulsions favored the droplets disaggregation resulting in a mild effect of bridging flocculation by particles sharing and displacement of the size curve distribution toward lower size. After passing through the intestinal condition, these emulsions presented few droplets and chitosan aggregates with a monomodal size distribution and high mean droplet size (D4,3 = 197 ±â€¯8 µm). On the other hand, Tween 80, CCrys and CNFs were able to inhibit lipid digestion and no changes on mean droplet size were observed following intestinal step. CNFs-stabilized emulsion showed the lowest lipid digestion, whereas the strong adherence of the CCrys particles onto the droplet interface became them resistant to displacement by surface-active components (i.e. bile salts and lipase enzyme). On the other hand, a slow lipid hydrolysis could be observed in chitosan-stabilized emulsions promoted by competition between chitosan aggregates and intestinal fluids by the oil droplet interface. Studying the emulsions stabilized using different polysaccharides particles on gastrointestinal conditions we could elucidate important features for their potential application as control systems of lipid digestion rate, as well as, as delivery systems of lipophilic compounds.

Coupling of high-intensity ultrasound and mechanical stirring for producing food emulsions at low-energy densities.

In this study, coupling of ultrasound (US) device and rotor-stator (RS), operating at low-energy densities, was studied as an alternative process to individual US and RS to produce modified starch-stabilized oil-in-water emulsions, as well as its potential use to encapsulate eugenol. To this aim, a full factorial design was employed to evaluate the effects of the US nominal power (0, 360 and 720 W) and RS nominal power (0, 150 and 300 W) on the physical properties, encapsulation efficiency and kinetic stability of emulsions produced. Firstly, the action of modified starch and eugenol onto interface oil-water was evaluated. The emulsifier was rapidly adsorbed on the interface water-sunflower oil reducing the interfacial tension from 25 to 16 mN/m, while eugenol did not show surface activity. The increase of energy density, in general, resulted in droplet size reduction, indicating the relevant role of the forces involved in the droplet breakup on emulsion stability. Coupling was more efficient on the droplets breakup producing smaller droplet size with narrower size distribution. While the coupled system work during 5 min for an energy density of 583 J/mL, the corresponding emulsification time for operating singly US and RS were 7.09 min and 17.04 min, respectively. Therefore, the main advantage associate to coupled process is the reduction of processing time to produce an emulsion with better kinetic stability.

Cellulose nanofibers from banana peels as a Pickering emulsifier: High-energy emulsification processes.

Cellulose nanofibers (CNFs) from banana peels was evaluated as promising stabilizer for oil-in-water emulsions. CNFs were treated using ultrasound and high-pressure homogenizer. Changes on the size, crystallinity index and zeta potential of CNFs were associated with the intense effects of cavitation phenomenon and shear forces promoted by mechanical treatments. CNFs-stabilized emulsions were produced under the same process conditions as the particles. Coalescence phenomenon was observed in the emulsions produced using high-pressure homogenizer, whereas droplets flocculation occurred in emulsions processed by ultrasound. In the latter, coalescence stability was associated with effects of cavitation forces acting on the CNFs breakup. Thus, smaller droplets created during the ultrasonication process could be recovered by particles that acted as an effective barrier against droplets coalescence. Our results improved understanding about the relationship between the choice of emulsification process and their effects on the CNFs properties influencing the potential application of CNFs as a food emulsifier.

Impact of oil type and WPI/Tween 80 ratio at the oil-water interface: Adsorption, interfacial rheology and emulsion features.

The relationship between the composition and structure of food emulsions was evaluated from the effect of a mixture of emulsifiers Whey protein (WPI) - Tween 80 (T80) and the oil phase features, such as chain length and unsaturation degree (sunflower oil, a long chain triacylglycerol - LCT or NEOBEE® 1053, a medium chain triacylglycerol - MCT). Emulsions with LCT showed higher droplet size than MCT as a consequence of its higher viscosity. All emulsions exhibited shear thinning behavior, but the viscosity was influenced by their interface composition. An occurrence of the destabilization mechanism by creaming was observed in turbidimetric measurements, but no visual phase separation could be observed, indicating a good kinetic stability after a 7-day storage. The initial interfacial tension of the water-LCT or water-MCT oil was about 25 mN/m, but the WPI addition (1% w/w) reduced the initial interfacial tension to approximately 20 mN/m. The increase of T80 concentration led to a decrease of the interfacial tension, reaching a value around 10 mN/m in systems with pure T80. The curves of interfacial tension of systems with LCT or MCT showed differences in the decay rate of tension over time. These differences were attributed to characteristics of the oil phase (hydrophobicity, unsaturation degree, presence of impurities) and the different proportions of each emulsifier within the mixture of emulsifiers. Finally, a higher viscoelastic interface was observed in LCT emulsions, which were mainly stabilized by WPI molecules. Such molecules presented a higher resistance to the displacement due to the competitive adsorption phenomenon, since the LCT is a more hydrophobic oil. On the other hand, the interface with MCT and a higher T80 concentration was less viscoelastic due to an easier displacement of WPI from the interface and the replacement by T80. The results indicate that T80 can be used in combination with WPI to produce emulsions with good stability and lower concentration of synthetic compounds. Lastly, the interfacial layer composition is not only dependent on the WPI-T80 ratio in the bulk phase, but also on the oily phase features. These results provide a potential strategy for designing emulsified foods based on the choice of ingredients and knowledge of the interaction between them.