Nanoemulsions Embedded in Alginate Beads as Bioadhesive Nanocomposites for Intestinal Delivery of the Anti-Inflammatory Drug Tofacitinib.

Oral administration of nanoparticles (NPs) is a promising strategy to overcome solubility and stability issues of many active compounds. However, this route faces major obstacles related to the hostile gastrointestinal (GI) environment, which impairs the efficacy of orally administered nanomedicines. Here, we propose nanocomposites as a promising approach to increase the retention time of NPs in the intestinal tract by using bio- and mucoadhesive matrixes able to protect the cargo until it reaches the targeted area. A microfluidic-based approach has been applied for the production of tailored nanoemulsions (NEs) of about 110 nm, used for the encapsulation of small hydrophobic drugs such as the anti-inflammatory JAK-inhibitor tofacitinib. These NEs proved to be efficiently internalized into a mucus-secreting human intestinal monolayer of Caco-2/HT29-MTX cells and to deliver tofacitinib to subepithelial human THP-1 macrophage-like cells, reducing their inflammatory response. NEs were then successfully encapsulated into alginate hydrogel microbeads of around 300 µm, which were characterized by rheological experiments and dried to create a long-term stable system for pharmaceutical applications. Finally, ex vivo experiments on excised segments of rats' intestine proved the bioadhesive ability of NEs embedded in alginate hydrogels compared to free NEs, showing the advantage that this hybrid system can offer for the treatment of intestinal pathologies.

Development and Survival of Human Ovarian Cells in Chitosan Hydrogel Micro-Bioreactor.

Background and Objectives: To test the long-term ability of human ovarian cortex cells to develop in unconventional culture conditions. Materials and Methods. Ovarian cortex cells from fetuses aged 23 to 39 weeks gestation were cultured for 90 days in hollow chitosan hydrogel micro-bioreactors and concurrently in traditional wells. Various cell-type counts were considered. Results: With intact follicles as a denominator, the percentage of growing intact follicles at Day 0 varied widely between ovaries (0 to 31.7%). This percentage tended to increase or stay relatively constant in bioreactor as in control cultures; it tended more toward an increase over time in bioreactor vs. control cultures. Modeled percentages showed differences (though not significant) in favor of bioreactor cultures (16.12% difference at D50 but only 0.12% difference at D90). With all follicles present as a denominator, the percentage of growing primary and secondary follicles at D0 varied widely between ovaries (0 to 29.3%). This percentage tended to increase over time in bioreactor cultures but to decrease in control cultures. Modeled percentages showed significant differences in favor of bioreactor cultures (8.9% difference at D50 and 11.1% difference at D90). At D50 and D90, there were only few and sparse apoptotic cells in bioreactor cultures vs. no apoptotic cells in control cultures. Conclusions: Over three months, bioreactor folliculogenesis outperformed slightly traditional culture. This is an interesting perspective for follicle preservation and long-term toxicological studies.

Lubrication and Wear Protection of Micro-Structured Hydrogels Using Bioinspired Fluids.

We report the fabrication and the use of a bioinspired synovial fluid acting as a lubricant fluid and antiwear agent at soft and porous chitosan hydrogel tribopairs. This synthetic synovial fluid is composed of sodium hyaluronate (HA) and a bottle-brush polymer (BB) having a polycationic attachment group and polyzwitterionic pendant chains. The 2.5%w/w chitosan hydrogel plugs are organized in a bilayered structure exposing a thin and dense superficial zone (SZ), covering a porous deep zone (DZ), and exhibiting microchannels perpendicularly aligned to the SZ. Using a low-load tribometer, the addition of HA lubricating solution at the hydrogel-hydrogel rubbing contact drastically decreased the coefficient of friction (CoF) from µ = 0.20 ± 0.01 to 0.04 ± 0.01 on the DZ configuration and from µ = 0.31 ± 0.01 to 0.08 ± 0.01 on the SZ surface when increasing the HA concentration from 0 to 1000 µg/mL and its molecular mass from 10 to 1500 kDa, similar to what was found when using the BB polymer alone. When combining the BB polymer and the 1500 kDa HA, the CoF remained stable at µ = 0.04 ± 0.01 for both studied contact configurations, highlighting the synergistic interaction of the two macromolecules. Hydrogel wear was characterized by assessing the final gel surface roughness by the means of an interferometer. Increasing HA concentration and molecular weight plus the addition of the BB polymer led to a dramatic surface wear protection with a final gel surface roughness of the hydrogels similar to the untested gels. In brief, the BB polymer in combination with high molecular weight HA is a potential lubricating fluid as well as a wear resistant agent for soft materials lubrication and wear protection.

Bioinspired microstructures of chitosan hydrogel provide enhanced wear protection.

We describe the fabrication of physical chitosan hydrogels exhibiting a layered structure. This bilayered structure, as shown by SEM and confocal microscopy, is composed of a thin dense superficial zone (SZ), covering a deeper zone (DZ) containing microchannels orientated perpendicularly to the SZ. We show that such structure favors diffusion of macromolecules within the hydrogel matrix up to a critical pressure, σc, above which channels were constricted. Moreover, we found that the SZ provided a higher wear resistance than the DZ which was severely damaged at a pressure equal to the elastic modulus of the gel. The coefficient of friction (CoF) of the SZ remained independent of the applied load with µSZ = 0.38 ± 0.02, while CoF measured at DZ exhibited two regimes: an initial CoF close to the value found on the SZ, and a CoF that decreased to µDZ = 0.18 ± 0.01 at pressures higher than the critical pressure σc. Overall, our results show that internal structuring is a promising avenue in controlling and improving the wear resistance of soft materials such as hydrogels.

3-D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization.

Bone substitute fabrication is of interest to meet the worldwide incidence of bone disorders. Physical chitosan hydrogels with intertwined apatite particles were chosen to meet the bio-physical and mechanical properties required by a potential bone substitute. A set up for 3-D printing by robocasting was found adequate to fabricate scaffolds. Inks consisted of suspensions of calcium phosphate particles in chitosan acidic aqueous solution. The inks are shear-thinning and consist of a suspension of dispersed platelet aggregates of dicalcium phosphate dihydrate in a continuous chitosan phase. The rheological properties of the inks were studied, including their shear-thinning characteristics and yield stress. Scaffolds were printed in basic water/ethanol baths to induce transformation of chitosan-calcium phosphates suspension into physical hydrogel of chitosan mineralized with apatite. Scaffolds consisted of a chitosan polymeric matrix intertwined with poorly crystalline apatite particles. Results indicate that ink rheological properties could be tuned by controlling ink composition: in particular, more printable inks are obtained with higher chitosan concentration (0.19 mol·L-1).

Dynamic Structuration of Physical Chitosan Hydrogels.

We studied the microstructure of physical chitosan hydrogels formed by the neutralization of chitosan aqueous solutions highlighting the structural gradients within thick gels (up to a thickness of 16 mm). We explored a high polymer concentrations range (Cp ≥ 1.0% w/w) with different molar masses of chitosan and different concentrations of the coagulation agent. The effect of these processing parameters on the morphology was evaluated mainly through small-angle light scattering (SALS) measurements and confocal laser scanning microscopy (CLSM) observations. As a result, we reported that the microstructure is continuously evolving from the surface to the bulk, with mainly two structural transitions zones separating three types of hydrogels. The first zone (zone I) is located close to the surface of the hydrogel and constitutes a hard (entangled) layer formed under fast neutralization conditions. It is followed by a second zone (zone II) with a larger thickness (∼3-4 mm), where in some cases large pores or capillaries (diameter ∼10 µm) oriented parallel to the direction of the gel front are present. Deeper in the hydrogel (zone III), a finer oriented microstructure, with characteristic sizes lower than 2-3 µm, gradually replace the capillary morphology. However, this last bulk morphology cannot be regarded as structurally uniform because the size of small micrometer-range-oriented pores continuously increases as the distance to the surface of the hydrogel increases. These results could be rationalized through the effect of coagulation kinetics impacting the morphology obtained during neutralization.

Highly stretchable hydrogels from complex coacervation of natural polyelectrolytes.

The controlled complex coacervation of oppositely charged hyaluronic acid (Mw ≈ 800-1000 kg mol-1) and chitosan (Mw ≈ 160 kg mol-1, degree of acetylation = 15%) led to hydrogels with controllable properties in terms of elasticity and strength. In this work, we performed desalting by dialysis of high ionic strength solutions of mixed polyelectrolytes and showed that the control of the pH during the polyelectrolyte assembly greatly impacts the mechanical properties of the hydrogel. First, for pHs from 5.5 to 7.5, a slight coacervation was observed due to low chitosan protonation and poor polyelectrolyte associations. Then, for pHs from 3.0 to 5.5, coacervation and syneresis led to free-standing and easy to handle hydrogels. Finally, for pHs from 2.0 to 3.0 (close to the pKa of the hyaluronic acid), we observed the unusual stretchability of these hydrogels that could arise from the pre-folding of hyaluronic acid chains while physical crosslinking was achieved by hyaluronic acid/chitosan polyelectrolyte complexation.

Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation.

The formation of chitosan hydrogels without any external cross-linking agent was successfully achieved by inducing the gelation of a viscous chitosan solution with aqueous NaOH or gaseous NH3. The hydrogels produced from high molecular weight (Mw ≈ 640 000 g mol(-1)) and extensively deacetylated chitosan (DA ≈ 2.8%) at polymer concentrations above ∼2.0% exhibited improved mechanical properties due to the increase of the chain entanglements and intermolecular junctions. The results also show that the physicochemical and mechanical properties of chitosan hydrogels can be controlled by varying their polymer concentration and by controlling the gelation conditions, that is, by using different gelation routes. The biological evaluation of such hydrogels for regeneration of infarcted myocardium revealed that chitosan hydrogels prepared from 1.5% polymer solutions were perfectly incorporated onto the epicardial surface of the heart and presented partial degradation accompanied by mononuclear cell infiltration.

Stretchable hydrogels of chitosan/hyaluronic acid induced by polyelectrolyte complexation around neutral pH.

In this work, we propose the formation of stretchable hydrogels at neutral pH from the physical crosslinking of chitosan (CS) and hyaluronic acid (HA) by polyelectrolyte complexation. A mixture of CS (Mw ≈ 600 kg/mol, degree of acetylation ≈ 50 %) solution and HA (Mw ≈ 77 kg/mol) solution was prepared with an excess of salts screening the electrostatic interactions CS/HA. In a controlled manner, the polyelectrolyte complexation was induced through the progressive dialysis of the salted polymer mixture against a sodium acetate solution (AcONa, 0.01 M) for 7 days. Depending on [HA], various materials were obtained: viscous solutions at [HA] = 0.75 % (w/v); hydrogels at [HA] = 1.50-2.24 % (w/v) with Young modulus of 14 kPa and stretchable to 200 %. The small angle X-ray scattering characterization of the hydrogels revealed a multiscale organization related to the conformation of the polymers induced by the physical interactions. The dialysis process with AcONa was optimized by adding a dialysis step against a zinc acetate solution containing Zn2+. The combination of polyelectrolyte complexation between CS/HA and metal complexation between Zn2+ and the polymers led to an enhancement of the hydrogel stretchability up to 400 %.

Electrodeposition of a biopolymeric hydrogel: potential for one-step protein electroaddressing.

The electrodeposition of hydrogels provides a programmable means to assemble soft matter for various technological applications. We report an anodic method to deposit hydrogel films of the aminopolysaccharide chitosan. Evidence suggests the deposition mechanism involves the electrolysis of chloride to generate reactive chlorine species (e.g., HOCl) that partially oxidize chitosan to generate aldehydes that can couple covalently with amines (presumably through Schiff base linkages). Chitosan's anodic deposition is controllable spatially and temporally. Consistent with a covalent cross-linking mechanism, the deposited chitosan undergoes repeated swelling/deswelling in response to pH changes. Consistent with a covalent conjugation mechanism, proteins could be codeposited and retained within the chitosan film even after detergent washing. As a proof-of-concept, we electroaddressed glucose oxidase to a side-wall electrode of a microfabricated fluidic channel and demonstrated this enzyme could perform electrochemical biosensing functions. Thus, anodic chitosan deposition provides a reagentless, single-step method to electroaddress a stimuli-responsive and biofunctionalized hydrogel film.

Chitosan Based MicroRNA Nanocarriers.

Vectorization of microRNAs has shown to be a smart approach for their potential delivery to treat many diseases (i.e., cancer, osteopathy, vascular, and infectious diseases). However, there are barriers to genetic in vivo delivery regarding stability, targeting, specificity, and internalization. Polymeric nanoparticles can be very promising candidates to overcome these challenges. One of the most suitable polymers for this purpose is chitosan. Chitosan (CS), a biodegradable biocompatible natural polysaccharide, has always been of interest for drug and gene delivery. Being cationic, chitosan can easily form particles with anionic polymers to encapsulate microRNA or even complex readily forming polyplexes. However, fine tuning of chitosan characteristics is necessary for a successful formulation. In this review, we cover all chitosan miRNA formulations investigated in the last 10 years, to the best of our knowledge, so that we can distinguish their differences in terms of materials, formulation processes, and intended applications. The factors that make some optimized systems superior to their predecessors are also discussed to reach the highest potential of chitosan microRNA nanocarriers.

Macrophage polarization in vitro and in vivo modified by contact with fragmented chitosan hydrogel.

We have previously shown that implantation of a fragmented chitosan hydrogel suspension (chitosan-FPHS) into a traumatic spinal cord lesion in adult rats led to significant axon regrowth and functional recovery, which was associated to a modulation of inflammation. Using an in vitro culture system, we show here that polarization of bone marrow-derived macrophages is indeed modified by direct contact with chitosan-FPHS. Reducing the degree of acetylation (DA) and raising the concentration of chitosan (Cp, from 1.5% to 3%), favors macrophage polarization toward anti-inflammatory subtypes. These latter also migrate and adhere efficiently on low, but not high DA chitosan-FPHS, both in vitro and in vivo, while inflammatory macrophages rarely invade a chitosan-FPHS implant in vivo, no matter the DA. Our in vitro model setup should prove a valuable tool for screening diverse biomaterial formulations and combinations thereof for their inflammatory potential prior to implantation in vivo.

Cytocompatibility / Antibacterial Activity Trade-off for Knittable Wet-Spun Chitosan Monofilaments Functionalized by the In Situ Incorporation of Cu2+ and Zn2.

The wet spinning of cytocompatible, bioresorbable, and knittable chitosan (CTS) monofilaments would be advantageous for a variety of surgical applications. The complexation capacity of chitosan with Cu2+ or Zn2+ can be leveraged to enhance its antibacterial activity, but not at the expense of cytocompatibility. In this work, a wet-spinning process was adapted for the in situ incorporation of Cu2+ or Zn2+ with chitosan dopes to produce monofilaments at different drawing ratios (τtot) with various cation/glucosamine molar ratios, evaluated in the fibers (rCu,f and rZn,f). Cytocompatibility and antibacterial activity of wet-spun monofilaments were, respectively, quantified by in vitro live-dead assays on balb 3T3 and by different evaluations of the proliferation inhibition of Staphylococcus epidermidis (Gram+) and Escherichia coli (Gram-). Knittability was tested by a specific tensile test using a knitting needle and evaluated with an industrial knitting machine. It was found that rCu,f = 0.01 and rZn,f = 0.03 significantly increase the antibacterial activity without compromising cytocompatibility. Wet spinning with τtot = 1.6 allowed the production of knittable CTS-Cu monofilaments, as confirmed by knitting assays under industrial conditions.

Controlled Polyelectrolyte Association of Chitosan and Carboxylated Nano-Fibrillated Cellulose by Desalting.

We prepared chitosan (CHI) hydrogels reinforced with highly charged cellulose nanofibrils (CNF) by the desalting method. To this end, the screening of electrostatic interactions between CHI polycation and CNF polyanion was performed by adding NaCl at 0.4 mol/L to the chitosan acetate solution and to the cellulose nanofibrils suspension. The polyelectrolyte complexation between CHI polycation and CNF polyanion was then triggered by desalting the CHI/CNF aqueous mixture by multistep dialysis, in large excess of chitosan. Further gelation of non-complexed CHI was performed by alkaline neutralization of the polymer, yielding high reinforcement effects as probed by the viscoelastic properties of the final hydrogel. The results showed that polyelectrolyte association by desalting can be achieved with a polyanionic nanoparticle partner. Beyond obtaining hydrogel with improved mechanical performance, these composite hydrogels may serve as precursor for dried solid forms with high mechanical properties.

Nanocomposite sponges for enhancing intestinal residence time following oral administration.

In this work, nanocomposites that combine mucopenetrating and mucoadhesive properties in a single system are proposed as innovative strategy to increase drug residence time in the intestine following oral administration. To this aim, novel mucoadhesive chitosan (CH) sponges loaded with mucopenetrating nanoemulsions (NE) were developed via freeze-casting technique. The NE mucopenetration ability was determined studying the surface affinity and thermodynamic binding of the nanosystem with mucins. The ability of nanoparticles to penetrate across a preformed mucins layer was validated by 3D-time laps Confocal Laser Scanning Microscopy imaging. Microscopy observations (Scanning Electron Microscopy and Optical Microscopy) showed that NE participated in the structure of the sponge affecting its stability and in vitro release kinetics. When incubated with HCT 116 and Caco-2 cell lines, the NE proved to be cytocompatible over a wide concentration range. Finally, the in vivo biodistribution of the nanocomposite was evaluated after oral gavage in healthy mice. The intestinal retention of NE was highly enhanced when loaded in the sponge compared to the NE suspension. Overall, our results demonstrated that the developed nanocomposite sponge is a promising system for sustained drug intestinal delivery.

Development of Bioinspired Functional Chitosan/Cellulose Nanofiber 3D Hydrogel Constructs by 3D Printing for Application in the Engineering of Mechanically Demanding Tissues.

Soft tissues are commonly fiber-reinforced hydrogel composite structures, distinguishable from hard tissues by their low mineral and high water content. In this work, we proposed the development of 3D printed hydrogel constructs of the biopolymers chitosan (CHI) and cellulose nanofibers (CNFs), both without any chemical modification, which processing did not incorporate any chemical crosslinking. The unique mechanical properties of native cellulose nanofibers offer new strategies for the design of environmentally friendly high mechanical performance composites. In the here proposed 3D printed bioinspired CNF-filled CHI hydrogel biomaterials, the chitosan serves as a biocompatible matrix promoting cell growth with balanced hydrophilic properties, while the CNFs provide mechanical reinforcement to the CHI-based hydrogel. By means of extrusion-based printing (EBB), the design and development of 3D functional hydrogel scaffolds was achieved by using low concentrations of chitosan (2.0-3.0% (w/v)) and cellulose nanofibers (0.2-0.4% (w/v)). CHI/CNF printed hydrogels with good mechanical performance (Young's modulus 3.0 MPa, stress at break 1.5 MPa, and strain at break 75%), anisotropic microstructure and suitable biological response, were achieved. The CHI/CNF composition and processing parameters were optimized in terms of 3D printability, resolution, and quality of the constructs (microstructure and mechanical properties), resulting in good cell viability. This work allows expanding the library of the so far used biopolymer compositions for 3D printing of mechanically performant hydrogel constructs, purely based in the natural polymers chitosan and cellulose, offering new perspectives in the engineering of mechanically demanding hydrogel tissues like intervertebral disc (IVD), cartilage, meniscus, among others.

Efficacy of epicardial implantation of acellular chitosan hydrogels in ischemic and nonischemic heart failure: impact of the acetylation degree of chitosan.

This work explores the epicardial implantation of acellular chitosan hydrogels in two murine models of cardiomyopathy, focusing on their potential to restore the functional capacity of the heart. Different chitosan hydrogels were generated using polymers of four degrees of acetylation, ranging from 2.5% to 38%, because the degree of acetylation affects their degradation and biological activity. The hydrogels were adjusted to a 3% final polymer concentration. After complete macromolecular characterization of the chitosans and study of the mechanical properties of the resulting hydrogels, they were sutured onto the surface of the myocardium, first in rat after four-weeks of coronary ligation (n=58) then in mice with cardiomyopathy induced by a cardiac-specific invalidation of serum response factor (n=20). The implantation of the hydrogels was associated with a reversion of cardiac function loss with maximal effects for the acetylation degree of 24%. The extent of fibrosis, the cardiomyocyte length-to-width ratio, as well as the genes involved in fibrosis and stress were repressed after implantation. Our study demonstrated the beneficial effects of chitosan hydrogels, particularly with polymers of high degrees of acetylation, on cardiac remodeling in two cardiomyopathy models. Our findings indicate they have great potential as a reliable therapeutic approach to heart failure.

Feasibility study and direct extraction of endogenous free metallic cations combining hemodialysis and chelating polymer.

In this article, we report the conception and the use of dialysis-based medical device for the extraction of metals. The medical device is obtained by addition in the dialysate of a functionalized chitosan that can chelate endogenous metals like iron or copper. This water-soluble functionalized chitosan is obtained after controlled reacetylation and grafting of DOTAGA. Due to the high mass of chitosan, the polymer cannot cross through the membrane and the metals are trapped in the dialysate during hemodialysis. Copper extraction has been evaluated in vitro using an hemodialysis protocol. Feasibility study has been performed on healthy sheep showing no acute toxicity througout the entire dialysis procedure and first insights of metallic extraction even on healthy animals.

Micron range morphology of physical chitosan hydrogels.

Physical chitosan hydrogels are potential biomaterials for several biomedical applications, such as wound healing, tissue repair, and drug delivery. Controlling the microstructural organization of chitosan gels is one of the keys for monitoring the physical, mechanical, and biological properties. As a result, the main objective of the present work was to explore the microstructural organization of chitosan hydrogels in relation with the processing conditions of gelation. For this purpose, different gelation routes were studied, that is, chitosan solution neutralization of an aqueous or hydroalcoholic solution and neutralization of an alco-gel. Overall, the resulting morphology after processing was determined by the medium viscosity during neutralization and the nature and concentration of the base. The effect of these processing parameters on the morphology was evaluated mainly through small angle light scattering (SALS) measurements including in situ measurements during chitosan neutralization. As a result, we reported different bulk microstructures consisting in 200-400 nm aggregates (primary particles) agglomerated into micrometer range clusters or arranged into more organized structures, that is, forming microchannels (4-6 µm). We thus established a qualitative and quantitative relation between supramolecular morphology and gelation conditions of chitosan hydrogels.

Prilling and characterization of hydrogels and derived porous spheres from chitosan solutions with various organic acids.

This work emphazises the importance of the solubilizing conditions for the elaboration of chitosan hydrogel beads, which were produced using electromagnetic laminar jet breakup technology, resulting in dried porous beads by further freeze-drying. Paramaters such as the acid nature and concentration (acetic, formic, citric, lactic, maleic and malic, 0.1 to 0.5 mol·L-1), the chitosan concentration (2 to 5 wt%) and composition of the gelation bath (NaOH, with or without EtOH) were studied. Viscosity versus strain rate measurements were carried out on chitosan acidic solutions and the viscoelastic behaviour was studied on hydrogels. The solutions exhibiting the highest viscosities led to the stiffest macrohydrogels, as a result of chitosan carboxylate interactions. Specific surface areas of the freeze-dried beads were determined in the range from 12 to 107 m2·g-1. Their internal texture was observed by Scanning Electron Microscopy. Water uptake was also measured for further use in the field of water purification.