Protein delivery based on uncoated and chitosan-coated mesoporous silicon microparticles

Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60-90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems

In Vitro and Sensory Evaluation of Capsaicin-Loaded Nanoformulations

Capsaicin has known health beneficial and therapeutic properties. It is also able to enhance the permeability of drugs across epithelial tissues. Unfortunately, due to its pungency the oral administration of capsaicin is limited. To this end, we assessed the effect of nanoencapsulation of capsaicin, under the hypothesis that this would reduce its pungency. Core-shell nanocapsules with an oily core and stabilized with phospholipids were used. This system was used with or without chitosan coating. In this work, we investigated the in vitro release behavior of capsaicin-loaded formulations in different physiological media (including simulated saliva fluid). We also evaluated the influence of encapsulation of capsaicin on the cell viability of buccal cells (TR146). To study the changes in pungency after encapsulation we carried out a sensory analysis with a trained panel of 24 students. The in vitro release study showed that the systems discharged capsaicin slowly in a monotonic manner and that the chitosan coating had an effect on the release profile. The cytotoxic response of TR146 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, was reduced following its encapsulation. The sensory study revealed that a chitosan coating results in a lower threshold of perception of the formulation. The nanoencapsulation of capsaicin resulted in attenuation of the sensation of pungency significantly. However, the presence of a chitosan shell around the nanoformulations did not mask the pungency, when compared with uncoated systems

An investigation of the interactions between an E. coli bacterial quorum sensing biosensor and chitosan-based nanocapsules

We examined the interaction between chitosan-based nanocapsules (NC), with average hydrodynamic diameter ∼114–155 nm, polydispersity ∼0.127, and ζ-potential ∼+50 mV, and an E. coli bacterial quorum sensing reporter strain. Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) allowed full characterization and assessment of the absolute concentration of NC per unit volume in suspension. By centrifugation, DLS, and NTA, we determined experimentally a “stoichiometric” ratio of ∼80 NC/bacterium. By SEM it was possible to image the aggregation between NC and bacteria. Moreover, we developed a custom in silico platform to simulate the behavior of particles with diameters of 150 nm and ζ-potential of +50 mV on the bacterial surface. We computed the detailed force interactions between NC-NC and NC-bacteria and found that a maximum number of 145 particles might interact at the bacterial surface. Additionally, we found that the “stoichiometric” ratio of NC and bacteria has a strong influence on the bacterial behavior and influences the quorum sensing response, particularly due to the aggregation driven by NC

Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers

We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem’s influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers

Advances in Chitin/Chitosan Characterization and Applications

Functional advanced biopolymers have received far less attention than renewable biomass (cellulose, rubber, etc.) used for energy production. Among the most advanced biopolymers known is chitosan. The term chitosan refers to a family of polysaccharides obtained by partial de-N-acetylation from chitin, one of the most abundant renewable resources in the biosphere. Chitosan has been firmly established as having unique material properties as well as biological activities. Either in its native form or as a chemical derivative, chitosan is amenable to being processed—typically under mild conditions—into soft materials such as hydrogels, colloidal nanoparticles, or nanofibers. Given its multiple biological properties, including biodegradability, antimicrobial effects, gene transfectability, and metal adsorption—to name but a few—chitosan is regarded as a widely versatile building block in various sectors (e.g., agriculture, food, cosmetics, pharmacy) and for various applications (medical devices, metal adsorption, catalysis, etc.). This Special Issue presents an updated account addressing some of the major applications, including also chemical and enzymatic modifications of oligos and polymers. A better understanding of the properties that underpin the use of chitin and chitosan in different fields is key for boosting their more extensive industrial utilization, as well as to aid regulatory agencies in establishing specifications, guidelines, and standards for the different types of products and applications

On the Fractionation and Physicochemical Characterisation of Self-Assembled Chitosan–DNA Polyelectrolyte Complexes

Chitosan is extensively studied as a carrier for gene delivery and is an attractive non-viral gene vector owing to its polycationic, biodegradable, and biocompatible nature. Thus, it is essential to understand the chemistry of self-assembled chitosan–DNA complexation and their structural and functional properties, enabling the formation of an effective non-viral gene delivery system. In this study, two parent chitosans (samples NAS-032 and NAS-075; Mw range ~118–164 kDa) and their depolymerised derivatives (deploy nas-032 and deploy nas-075; Mw range 6–14 kDa) with degrees of acetylation 43.4 and 4.7%, respectively, were used to form polyelectrolyte complexes (PECs) with DNA at varying [–NH3+]/[–PO4−] (N/P) molar charge ratios. We investigated the formation of the PECs using ζ-potential, asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS), refractive index (RI), ultraviolet (UV) and dynamic light scattering (DLS) detectors, and TEM imaging. PEC formation was confirmed by ζ-potential measurements that shifted from negative to positive values at N/P ratio ~2. The radius of gyration (Rg) was determined for the eluting fractions by AF4-MALS-RI-UV, while the corresponding hydrodynamic radius (Rh), by the DLS data. We studied the influence of different cross-flow rates on AF4 elution patterns for PECs obtained at N/P ratios 5, 10, and 20. The determined rho shape factor (ρ = Rg/Rh) values for the various PECs corresponded with a sphere morphology (ρ ~0.77–0.85), which was consistent with TEM images. The results of this study represent a further step towards the characterisation of chitosan–DNA PECs by the use of multi-detection AF4 as an important tool to fractionate and infer aspects of their morphology

A comparative study of the digestion behavior and functionality of protein from chia (Salvia hispanica L.) ingredients and protein fractions

Protein derived from chia (Salvia hispanica L.), characterized by a balanced amino acid composition, represents a potentially healthier and environmentally friendly alternative poised for innovation within the plant-based food sector. It was hypothesized that the growing location of chia seeds and processing techniques used might influence protein digestion patterns, which in turn could affect the biological functions of the digestion products. To examine this hypothesis, we assessed the gastrointestinal fate of degummed-defatted flour (DDF), protein concentrate (PC), and isolated albumin (Alb) and globulin (Glo) fractions. Furthermore, we compared the antioxidant and anti-inflammatory activities of the resulting digesta by means of in vitro and cellular assays. Post-gastrointestinal digestion, the PC exhibited elevated levels of soluble protein (7.6 and 6.3 % for Mexican and British PC, respectively) and peptides (24.8 and 27.9 %, respectively) of larger molecular sizes compared to DDF, Alb, and Glo. This can be attributed to differences in the extraction/fractionation processes. Leucine was found to be the most prevalent amino acids in all chia digesta. Such variations in the digestive outcomes of chia protein components significantly influenced the bioactivity of the intestinal digestates. During gastrointestinal transit, British Glo exhibited the best reactive oxygen species (ROS) inhibition activity in oxidative-stressed RAW264.7 macrophages, while Mexican digesta outperformed British samples in terms of ROS inhibition within the oxidative-stressed Caco-2 cells. Additionally, both Mexican and British Alb showed effectively anti-inflammatory potential, with keratinocyte chemoattractant (KC) inhibition rate of 82 and 91 %, respectively. Additionally, Mexican PC and Alb generally demonstrated an enhanced capacity to mitigate oxidative stress and inflammatory conditions in vitro. These findings highlight the substantial potential of chia seeds as functional food ingredients, resonating with the shifting preferences of health-conscious consumers

Mesoscopic gelation of chitosan and genipin at below critical concentrations

Genipin is increasingly used as a crosslinking agent for chitosans due to its low cytotoxicity as a naturally occurring extract of the plant Gardenia jasminoides. Genipin reacts with the primary amino groups of chitosan to form blue hydrogels. We studied the gelation kinetics of different chitosans varying in their properties (molar mass 34 000-213 000 g mol-1, degree of acetylation 9-20%) and genipin in detail. We found that critical sol-gel transition times obtained from dynamic light scattering were in good agreement with the results obtained by small deformation oscillatory rheometry and microviscosimetry at high concentrations of chitosan. However, at below critical concentrations, we found a second regime of gelation that followed the same Ross-Murphy\u27s gelation kinetics. The macroscopic appearance of these samples was a suspension of weak gel-like particles that were sensitive to mechanical forces. We believe that the material is a mesoscopic gel, as described for other polymers. To the best of our knowledge, this is the first time that this phenomenon has been described for the gelling system of chitosan and genipin

A quality by design approach for optimization of Lecithin/Span® 80 based nanoemulsions loaded with hydrophobic drugs

Lately, nanoemulsions loaded with hydrophobic drugs have been successfully developed to improve the treatment of several global diseases. On this subject, a detailed study of the crucial role of the excipients and the experimental conditions used for these nanosystems is still required. Thus, the aim of this work was the development of nanoemulsions of Benzidazole (Class I, log P = 0.91), Praziquantel (Class II, log P = 2.44), Pyrimethamine (Class II/IV, log P = 2.69), Niclosamide (Class II/IV, log P = 4.5), and Triclabendazole (Class II/IV, log P = 5.9) using Span® 80, soybean lecithin and Miglyol® 812 as excipients. A Placket-Burman design was selected to identify the main parameters that influence in the desirable characteristics of such formulations. Then, a full factorial design was built to analyze the effect of the factors identified in the screening phase. Plackett-Burman design indicated that Miglyol® 812 and lecithin were the two most influencing factors on the hydrodynamic diameter of the systems. In addition, the association efficiency was influenced by the log P of each drug while the response stability in PBS was modified by Span® 80 and log P. The results of the full factorial design revealed that concentration of Miglyol® 812 and log P values of each drug have a remarkable impact on the stability of the nanosystems. The optimal conditions for the preparation of nanoemulsions were verified by other independent experiment and the results were in agreement with the predicted optimum values. Thus, this methodology could serve as an attracttive platform to deliver other hydrophobics compounds in stable nanoemulsions.Fil: Real, Daniel Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Farmacia; ArgentinaFil: Hoffmann, Stefan. Westfälische Wilhelms Universität; AlemaniaFil: Leonardi, Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Farmacia; ArgentinaFil: Goycoolea, Francisco M.. Westfälische Wilhelms Universität; Alemania. University of Leeds; Reino UnidoFil: Salomon, Claudio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Farmacia; Argentin

Electrostatic Self-Assembled Chitosan-Pectin Nano- and Microparticles for Insulin Delivery

A polyelectrolyte complex system of chitosan-pectin nano- and microparticles was developed to encapsulate the hormone insulin. The aim of this work was to obtain small particles for oral insulin delivery without chemical crosslinkers based on natural and biodegradable polysaccharides. The nano- and microparticles were developed using chitosans (with different degrees of acetylation: 15.0% and 28.8%) and pectin solutions at various charge ratios (n+/n− given by the chitosan/pectin mass ratio) and total charge. Nano- and microparticles were characterized regarding particle size, zeta potential, production yield, encapsulation efficiency, stability in different media, transmission electron microscopy and cytotoxicity assays using Caco-2 cells. The insulin release was evaluated in vitro in simulated gastric and intestinal media. Small-sized particles (~240–~1900 nm) with a maximum production yield of ~34.0% were obtained. The highest encapsulation efficiency (~62.0%) of the system was observed at a charge ratio (n+/n−) 5.00. The system was stable in various media, particularly in simulated gastric fluid (pH 1.2). Transmission electron microscopy (TEM) analysis showed spherical shape particles when insulin was added to the system. In simulated intestinal fluid (pH 6.8), controlled insulin release occurred over 2 h. In vitro tests indicated that the proposed system presents potential as a drug delivery for oral administration of bioactive peptides