Progress in Colloid Delivery Systems for Protection and Delivery of Phenolic Bioactive Compounds: Two Study Cases-Hydroxytyrosol and Curcumin.

Insufficient intake of beneficial food components into the human body is a major issue for many people. Among the strategies proposed to overcome this complication, colloid systems have been proven to offer successful solutions in many cases. The scientific community agrees that the production of colloid delivery systems is a good way to adequately protect and deliver nutritional components. In this review, we present the recent advances on bioactive phenolic compounds delivery mediated by colloid systems. As we are aware that this field is constantly evolving, we have focused our attention on the progress made in recent years in this specific field. To achieve this goal, structural and dynamic aspects of different colloid delivery systems, and the various interactions with two bioactive constituents, are presented and discussed. The choice of the appropriate delivery system for a given molecule depends on whether the drug is incorporated in an aqueous or hydrophobic environment. With this in mind, the aim of this evaluation was focused on two case studies, one representative of hydrophobic phenolic compounds and the other of hydrophilic ones. In particular, hydroxytyrosol was selected as a bioactive phenol with a hydrophilic character, while curcumin was selected as typical representative hydrophobic molecules.

Strain Hardening in Highly Acetylated Chitosan Gels.

Strain hardening has recently emerged as a near-universal response of biological tissues to mechanical stimulation as well as a powerful regulator of cell fate. Understanding the mechanistic basis for this nonlinear elasticity is crucial for developing bioinspired materials that mimic extracellular matrix mechanics. Here, we show that covalent networks built from highly acetylated chitosans exhibit strain hardening at physiological pH and osmolarity. While varying the chitosan physical-chemical composition and network connectivity, we provide evidence that temporary nodes arising from the entangling of chains between stable cross-links are at the root of nonlinear elasticity. The contour length (Lc) of the said chains revealed that the larger the chain length between the cross-links, the greater is the entanglement over disentanglement upon network stretching. To this end, we calculated that the minimum number of Khun's segments in Lc that contributes to the onset of strain hardening is 15. Furthermore, we identified a relationship between critical strain marking nonlinear elasticity and the network connectivity, being similar to that found for the cytoskeletal collagen matrix, indicating the potential use of semiflexible (neutral pH-soluble) chitosans in assembling extracellular matrix mimics.

Ionotropic Gelation of Chitosan Flat Structures and Potential Applications.

The capability of some polymers, such as chitosan, to form low cost gels under mild conditions is of great application interest. Ionotropic gelation of chitosan has been used predominantly for the preparation of gel beads for biomedical application. Only in the last few years has the use of this method been extended to the fabrication of chitosan-based flat structures. Herein, after an initial analysis of the major applications of chitosan flat membranes and films and their usual methods of synthesis, the process of ionotropic gelation of chitosan and some recently proposed novel procedures for the synthesis of flat structures are presented.

Long lasting mucoadhesive membrane based on alginate and chitosan for intravaginal drug delivery.

The intravaginal route of administration can be exploited to treat local diseases and for systemic delivery. In this work, we developed an alginate/chitosan membrane sufficiently stable in a simulated vaginal fluid and able to dissolve over time at a very slow and linear rate. The membrane demonstrated good mechanical properties both in its swollen and dry form. As a study case, we evaluated the viability of this potential drug delivery system for the treatment of bacterial vaginosis, a common disease affecting women in their reproductive age. Metronidazole was effectively included in the alginate/chitosan membrane and its bactericide effect was demonstrated against Staphylococcus aureus and Gardnerella vaginalis, simultaneously showing good biocompatibility with a cervix epithelial cell line. Since this alginate/chitosan membrane is stable in a simulated vaginal environment, is easy to fabricate and can be used for the controlled release of a model drug, it represents a promising drug delivery system for local intravaginal applications.

On the Mechanism of Genipin Binding to Primary Amines in Lactose-Modified Chitosan at Neutral pH.

The present manuscript deals with the elucidation of the mechanism of genipin binding by primary amines at neutral pH. UV-VIS and CD measurements both in the presence of oxygen and in oxygen-depleted conditions, combined with computational analyses, led to propose a novel mechanism for the formation of genipin derivatives. The indications collected with chiral and achiral primary amines allowed interpreting the genipin binding to a lactose-modified chitosan (CTL or Chitlac), which is soluble at all pH values. Two types of reaction and their kinetics were found in the presence of oxygen: (i) an interchain reticulation, which involves two genipin molecules and two polysaccharide chains, and (ii) a binding of one genipin molecule to the polymer chain without chain-chain reticulation. The latter evolves in additional interchain cross-links, leading to the formation of the well-known blue iridoid-derivatives.

On the Formation and Stability of Chitosan/Hyaluronan-Based Complex Coacervates.

This contribution is aimed at extending our previous findings on the formation and stability of chitosan/hyaluronan-based complex coacervates. Colloids are herewith formed by harnessing electrostatic interactions between the two polyelectrolytes. The presence of tiny amounts of the multivalent anion tripolyphosphate (TPP) in the protocol synthesis serves as an adjuvant "point-like" cross-linker for chitosan. Hydrochloride chitosans at different viscosity average molar mass, , in the range 10,000-400,000 g/mol, and fraction of acetylated units, FA, (0.16, 0.46 and 0.63) were selected to fabricate a large library of formulations. Concepts such as coacervate size, surface charge and homogeneity in relation to chitosan variables are herein disclosed. The stability of coacervates in Phosphate Buffered Saline (PBS) was verified by means of scattering techniques, i.e., Dynamic Light Scattering (DLS) and Small-Angle X-ray Scattering (SAXS). The conclusions from this set of experiments are the following: (i) a subtle equilibrium between chitosan FA and does exist in ensuring colloidal stability; (ii) once diluted in PBS, osmotic swelling-driven forces trigger the enlargement of the polymeric mesh with an ensuing increase of coacervate size and porosity.

Glycosylated-Chitosan Derivatives: A Systematic Review.

Chitosan derivatives, and more specifically, glycosylated derivatives, are nowadays attracting much attention within the scientific community due to the fact that this set of engineered polysaccharides finds application in different sectors, spanning from food to the biomedical field. Overcoming chitosan (physical) limitations or grafting biological relevant molecules, to mention a few, represent two cardinal strategies to modify parent biopolymer; thereby, synthetizing high added value polysaccharides. The present review is focused on the introduction of oligosaccharide side chains on the backbone of chitosan. The synthetic aspects and the effect on physical-chemical properties of such modifications are discussed. Finally, examples of potential applications in biomaterials design and drug delivery of these novel modified chitosans are disclosed.

pH-Assisted Gelation of Lactose-Modified Chitosan.

We report on a controlled process allowing for the gelation of a diol-rich chitosan-derivative named CTL (lactose-modified chitosan) in the presence of boric acid as the cross-linker. A two-step approach is described, namely (i) the mixing of CTL and boric acid at pH = 5, a condition where the inorganic component is mildly reactive; (ii) the addition of sodium bicarbonate (NaHCO3) as a trigger, allowing for the gradual and slow pH increase. The goal was to convert gradually the almost inert neutral boric acid into the much more reactive borate anion, the latter promoting the formation of borate esters with CTL diols. Gelling kinetics as well as mechanical behavior at small and large deformations was investigated by rheometry. CTL-boric acid gels behaved essentially as transient networks, hence continuously assembling and dissociating in a highly dynamic fashion. The present gelling mechanism preserves the strain-hardening behavior in the nonlinear region of stress-strain response, corroborating the already suggested potential applications of such gels as mimics of biological soft tissues.

Lactose-Modified Chitosan Gold(III)-PEGylated Complex-Bioconjugates: From Synthesis to Interaction with Targeted Galectin-1 Protein.

Galectins (Gal) are a family of glycan-binding proteins characterized by their affinity for ß-galactosides. Galectin-1 (Gal-1), a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Coordination chemistry has been engaged to develop versatile multivalent neoglycoconjugates for binding Gal-1. In this study we report a fast and original method to synthesize hybrid gold nanoparticles in which a hydrochloride lactose-modified chitosan, named CTL, is mixed with dicarboxylic acid-terminated polyethylene glycol (PEG), leading to shell-like hybrid polymer-sugar-metal nanoparticles (CTL-PEG-AuNPs). The aim of this paper is to preliminarily study the interaction of the CTL-PEG-AuNPs with a target protein, namely, Gal-1, under specific conditions. The molecular interaction has been measured by Transmission Electron Microscopy (TEM), UV-vis, and Raman Spectroscopy on a large range of Gal-1 concentrations (from 0 to 10-12 M). We observed that the interaction was strongly dependent on the Gal-1 concentration at the surface of the gold nanoparticles.

Complex Coacervates between a Lactose-Modified Chitosan and Hyaluronic Acid as Radical-Scavenging Drug Carriers.

Complex coacervation of two oppositely charged polysaccharides, namely a lactose-modified chitosan (CTL) and hyaluronan (HA), was investigated in this study. Coacervates of the two polysaccharides were prepared by drop-by-drop injection of HA into CTL. Transmittance and dynamic light scattering (DLS) measurements in combination with TEM analyses demonstrated the formation of spheroidal colloids in the nano-/microsize range showing good homogeneity. Strikingly, the presence of 150 mM supporting NaCl did not hamper the colloid formation. Stability studies on selected formulations demonstrated that HA/CTL coacervates were stable up to 3 weeks at 37 °C and behaved as pH-responsive colloids since transition from entangled to disentangled chains was attained for a proper pH range. The possibility of freeze-drying the coacervates for storage purposes and the ability of encapsulating selected payloads were investigated as well, for two values of the fraction of the lactitol side-chain substitution (FL). Finally, biological tests using human neutrophils were undertaken at acidic pH value (pH = 6.0): under such experimental conditions, akin to those frequently occurring in the inflammatory microenvironment, coacervates scavenged reactive oxygen species (ROS) generated by these cells in basal conditions. Given the well documented bioactivity of CTL with respect to chitosan toward cartilage regeneration, these findings point to a possible application of HA/CTL-based colloids as scavenging and bioactive carriers for the delivery of therapeutic molecules at confined inflamed sites such as knee joints.

Boric Acid Induced Transient Cross-Links in Lactose-Modified Chitosan (Chitlac).

The present paper explores the effect of boric acid on Chitlac, a lactose-modified chitosan which had previously shown interesting biological and physical-chemical features. The herewith-reported experimental evidences demonstrated that boric acid binds to Chitlac, producing conformational and association effects on the chitosan derivative. The thermodynamics of boric acid binding to Chitlac was explored by means of 11B NMR, circular dichroism (CD), and UV-vis spectroscopy, while macromolecular effects were investigated by means of viscometry and dynamic light scattering (DLS). The experimental results revealed a chain-chain association when limited amounts of boric acid were added to Chitlac. However, upon exceeding a critical boric acid limit dependent on the polysaccharide concentration, the soluble aggregates disentangle. The rheological behavior of Chitlac upon treatment with boric acid was explored showing a dilatant behavior in conditions of steady flow. An uncommonly high dependence in the scaling law between the zero-shear viscosity and the concentration of Chitlac was found, i.e., η0 ∝ CCTL5.8, pointing to interesting potential implications of the present system in biomaterials development.

Silver-containing antimicrobial membrane based on chitosan-TPP hydrogel for the treatment of wounds.

Treatment of non-healing wounds represents hitherto a severe dilemma because of their failure to heal caused by repeated tissue insults, bacteria contamination and altered physiological condition. This leads to face huge costs for the healthcare worldwide. To this end, the development of innovative biomaterials capable of preventing bacterial infection, of draining exudates and of favoring wound healing is very challenging. In this study, we exploit a novel technique based on the slow diffusion of tripolyphosphate for the preparation of macroscopic chitosan hydrogels to obtain soft pliable membranes which include antimicrobial silver nanoparticles (AgNPs) stabilized by a lactose-modified chitosan (Chitlac). UV-Vis and TEM analyses demonstrated the time stability and the uniform distribution of AgNPs in the gelling mixture, while swelling studies indicated the hydrophilic behavior of membrane. A thorough investigation on bactericidal properties of the material pointed out the synergistic activity of chitosan and AgNPs to reduce the growth of S. aureus, E. coli, S. epidermidis, P. aeruginosa strains and to break apart mature biofilms. Finally, biocompatibility assays on keratinocytes and fibroblasts did not prove any harmful effects on the viability of cells. This novel technique enables the production of bioactive membranes with great potential for the treatment of non-healing wounds.

Polysaccharide-based networks from homogeneous chitosan-tripolyphosphate hydrogels: synthesis and characterization.

Polysaccharide networks, in the form of hydrogels and dried membranes based on chitosan and on the cross-linker tripolyphosphate (TPP), were developed using a novel approach. TPP was incorporated into chitosan by slow diffusion to favor a controlled gelation. By varying chitosan, TPP, and NaCl concentration, transition from inhomogeneous to homogeneous systems was achieved. Rheology and uniaxial compression tests enabled to identify the best performing hydrogel composition with respect to mechanical properties. FTIR, (31)P NMR, and spectrophotometric methods were used to investigate the interaction chitosan-TPP, the kinetics of phosphates diffusion during the dialysis and the amount of TPP in the hydrogel. A freeze-drying procedure enabled the preparation of soft pliable membranes. The lactate dehydrogenase assay demonstrated the biocompatibility of the membranes toward fibroblasts. Overall, we devised a novel approach to prepare homogeneous macroscopic chitosan/TPP-based biomaterials with tunable mechanical properties and good biocompatibility that show good potential as novel polysaccharide derivatives.

Silk Fibroin-Enriched Bioink Promotes Cell Proliferation in 3D-Bioprinted Constructs.

Three-dimensional (3D) bioprinting technology enables the controlled deposition of cells and biomaterials (i.e., bioink) to easily create complex 3D biological microenvironments. Silk fibroin (SF) has recently emerged as a compelling bioink component due to its advantageous mechanical and biological properties. This study reports on the development and optimization of a novel bioink for extrusion-based 3D bioprinting and compares different bioink formulations based on mixtures of alginate methacrylate (ALMA), gelatin and SF. The rheological parameters of the bioink were investigated to predict printability and stability, and the optimal concentration of SF was selected. The bioink containing a low amount of SF (0.002% w/V) was found to be the best formulation. Light-assisted gelation of ALMA was exploited to obtain the final hydrogel matrix. Rheological analyses showed that SF-enriched hydrogels exhibited greater elasticity than SF-free hydrogels and were more tolerant to temperature fluctuations. Finally, MG-63 cells were successfully bioprinted and their viability and proliferation over time were analyzed. The SF-enriched bioink represents an excellent biomaterial in terms of printability and allows high cell proliferation over a period of up to 3 weeks. These data confirm the possibility of using the selected formulation for the successful bioprinting of cells into extracellular matrix-like microenvironments.

Ionic Strength Impacts the Physical Properties of Agarose Hydrogels.

Agarose is a natural polysaccharide known for its ability to form thermoreversible hydrogels. While the effects of curing temperature and polysaccharide concentration on mechanical properties have been discussed in the literature, the role of ionic strength has been less studied. In the present manuscript, we investigate the effects of supporting salt concentration and the role of cation (i.e. Na+ or Li+, neighbors in the Hofmeister series), on the setting and performance of agarose hydrogels. Compressive and rheological measurements show that the supporting salts reduce the immediate elastic response of agarose hydrogels, with Li+ showing a stronger effect than Na+ at high ionic strength, while they significantly increase the extent of linear stress-strain response (i.e., linear elasticity). The presence of increasing amounts of added supporting salt also leads to a reduction in hysteresis during mechanical deformation due to loading and unloading cycles, which is more pronounced with Li+ than with Na+. The combination of rheological measurements and NMR relaxometry shows a mesh size in agarose hydrogels in the order of 6-17 nm, with a thickness of the water layer bound to the biopolymer of about 3 nm. Of note, the different structuring of the water within the hydrogel network due to the different alkali seems to play a role for the final performance of the hydrogels.

Immediate stress dissipation in dual cross-link hydrogels controls osteogenic commitment of mesenchymal stem cells.

In vitro studies of mesenchymal stem cells (MSCs) differentiation have been predominantly performed with non-physiologically elastic materials. Here we report the effect of different viscoplastic ECM mimics on the osteogenic engagement of MSCs in 2D. We have developed soft hydrogels, composed of a lactose-modified chitosan, using a combination of permanent and temporary cross-links. The presence of temporary cross-links has a minor effect on the shear modulus of the hydrogels, but causes an immediate relaxation (dissipation) of the applied stress. This material property leads to early osteogenic commitment of MSCs, as evidenced by gene expression of runt-related transcription factor 2 (RUNX2), type 1 collagen (COL1A1), osteocalcin (OCN), alkaline phosphatase enzyme activity (ALP) and calcium deposit formation. In contrast, cells cultured on purely elastic hydrogels with only permanent cross-link begin to differentiate only after a longer period of time, indicating a dissipation-mediated mechano-sensing in the osteogenic commitment of MSCs.

Controlled Quenching of Agarose Defines Hydrogels with Tunable Structural, Bulk Mechanical, Surface Nanomechanical, and Cell Response in 2D Cultures.

The scaffolding of agarose hydrogel networks depends critically on the rate of cooling (quenching) after heating. Efforts are made to understand the kinetics and evolution of biopolymer self-assembly upon cooling, but information is lacking on whether quenching might affect the final hydrogel structure and performance. Here, a material strategy for the fine modulation of quenching that involves temperature-curing steps of agarose is reported. Combining microscopy techniques, standard and advanced macro/nanomechanical tools, it is revealed that agarose accumulates on the surface when the curing temperature is set at 121 °C. The inhomogeneity can be mostly recovered when it is reduced to 42 °C. This has a drastic effect on the stiffness of the surface, but not on the viscoelasticity, roughness, and wettability. When hydrogels are strained at small/large deformations, the curing temperature has no effect on the viscoelastic response of the hydrogel bulk but does play a role in the onset of the non-linear region. Cells cultured on these hydrogels exhibit surface stiffness-sensing that affects cell adhesion, spreading, F-actin fiber tension, and assembly of vinculin-rich focal adhesions. Collectively, the results indicate that the temperature curing of agarose is an efficient strategy to produce networks with tunable mechanics and is suitable for mechanobiology studies.

Influence of Temperature and Polymer Concentration on the Nonlinear Response of Highly Acetylated Chitosan-Genipin Hydrogels.

Strain hardening, i.e., the nonlinear elastic response of materials under load, is a physiological response of biological tissues to mechanical stimulation. It has recently been shown to play a central role in regulating cell fate. In this paper, we investigate the effect of temperature and polymer concentrations on the strain hardening of covalent hydrogels composed of pH-neutral soluble chitosans crosslinked with genipin. A series of highly acetylated chitosans with a fraction of acetylated units, FA, in the range of 0.4-0.6 was synthesized by the homogeneous re-N-acetylation of a partially acetylated chitosan or the heterogeneous deacetylation of chitin. A chitosan sample with an FA = 0.44 was used to prepare hydrogels with genipin as a crosslinker at a neutral pH. Time and frequency sweep experiments were then performed to obtain information on the gelling kinetics and mechanical response of the resulting hydrogels under small amplitude oscillatory shear. While the shear modulus depends on the chitosan concentration and is almost independent of the gel temperature, we show that the extent of hardening can be modulated when the gelling temperature is varied and is almost independent of the experimental conditions used to build the hydrogels (ex situ or in situ gelation). The overall effect is attributed to a subtle balance between the physical (weak) entanglements and covalent (strong) crosslinks that determine the mechanical response of highly acetylated chitosan hydrogels at large deformations.

Sulfated lactose-modified chitosan. A novel synthetic glycosaminoglycan-like polysaccharide inducing chondrocyte aggregation.

Lactose-modified chitosan (CTL) is sulfated using SO3·py or SO3·DMF as sulfating agents. The two products are characterized by elemental analysis, FT-IR, 1H,13C-DEPT-HSQC and 1H,13C-HSQC-TOCSY experiments which allow the extent and selectivity of chemical sulfation to be determined. Dynamic Light Scattering shows a pH-dependent association of the sulfated polysaccharides which are described as flexible by the Smidsrød's B parameter and the intrinsic viscosity at infinite ionic strength. Shear viscosity and intrinsic viscosity show that sulfation protocols lead to chain scission which is more pronounced when SO3·DMF is used. The sulfated samples are able to induce aggregation of human bone marrow mesenchymal stem cells, resulting in the formation of smaller nodules compared to the unmodified CTL sample. Over time, the sample with the higher degree of sulfation allows further aggregation between cell clusters while the sample with the lower degree of sulfation shows dissolution of the aggregates.