Development of phenol-grafted polyglucuronic acid and its application to extrusion-based bioprinting inks.

In this present work, we developed a phenol grafted polyglucuronic acid (PGU) and investigated the usefulness in tissue engineering field by using this derivative as a bioink component allowing gelation in extrusion-based 3D bioprinting. The PGU derivative was obtained by conjugating with tyramine, and the aqueous solution of the derivative was curable through a horseradish peroxidase (HRP)-catalyzed reaction. From 2.0 w/v% solution of the derivative containing 5 U/mL HRP, hydrogel constructs were successfully obtained with a good shape fidelity to blueprints. Mouse fibroblasts and human hepatoma cells enclosed in the printed constructs showed about 95% viability the day after printing and survived for 11 days of study without a remarkable decrease in viability. These results demonstrate the great potential of the PGU derivative in tissue engineering field especially as an ink component of extrusion-based 3D bioprinting.

Efficient Synthesis of Muramic and Glucuronic Acid Glycodendrimers as Dengue Virus Antagonists.

Carbohydrates are involved in many important pathological processes, such as bacterial and viral infections, by means of carbohydrate-protein interactions. Glycoconjugates with multiple carbohydrates are involved in multivalent interactions, thus increasing their binding strengths to proteins. In this work, we report the efficient synthesis of novel muramic and glucuronic acid glycodendrimers as potential Dengue virus antagonists. Aromatic scaffolds functionalized with a terminal ethynyl groups were coupled to muramic and glucuronic acid azides by click chemistry through optimized synthetic strategies to afford the desired glycodendrimers with high yields. Surface Plasmon Resonance studies have demonstrated that the compounds reported bind efficiently to the Dengue virus envelope protein. Molecular modelling studies were carried out to simulate and explain the binding observed. These studies confirm that efficient chemical synthesis of glycodendrimers can be brought about easily offering a versatile strategy to find new active compounds against Dengue virus.

Interpenetrating polymer network scaffold of sodium hyaluronate and sodium alginate combined with berberine for osteochondral defect regeneration.

Degradation of the articular cartilage and structural remodeling of the subchondral bone are regarded as the two major pathological characteristics of osteoarthritis. This study aimed to investigate the effect of an interpenetrating polymer network (IPN) of a sodium hyaluronate and sodium alginate (HA/SA) scaffold combined with berberine (BER) on osteochondral repair. We first developed an IPN scaffold of HA/SA and evaluated its characteristics. Then, we analyzed the effect of the HA/SA scaffold combined with BER on the healing of osteochondral defects in vivo. Finally, we explored the mechanism of this system in osteochondral repair. The results showed that the system could simultaneously regenerate not only the cartilage but also the subchondral bone. Our results also revealed that the subchondral bone was partially repaired by activating the Wnt signaling pathway and the cartilage was protected from degeneration by the upregulation of autophagy. This study demonstrated that the combination of the IPN scaffold of HA/SA and BER is a promising strategy for the osteochondral defect regeneration.

Preparation of Well-Dispersed Chitosan/Alginate Hollow Multilayered Microcapsules for Enhanced Cellular Internalization.

Hollow multilayered capsules have shown massive potential for being used in the biomedical and biotechnology fields, in applications such as cellular internalization, intracellular trafficking, drug delivery, or tissue engineering. In particular, hollow microcapsules, developed by resorting to porous calcium carbonate sacrificial templates, natural-origin building blocks and the prominent Layer-by-Layer (LbL) technology, have attracted increasing attention owing to their key features. However, these microcapsules revealed a great tendency to aggregate, which represents a major hurdle when aiming for cellular internalization and intracellular therapeutics delivery. Herein, we report the preparation of well-dispersed polysaccharide-based hollow multilayered microcapsules by combining the LbL technique with an optimized purification process. Cationic chitosan (CHT) and anionic alginate (ALG) were chosen as the marine origin polysaccharides due to their biocompatibility and structural similarity to the extracellular matrices of living tissues. Moreover, the inexpensive and highly versatile LbL technology was used to fabricate core-shell microparticles and hollow multilayered microcapsules, with precise control over their composition and physicochemical properties, by repeating the alternate deposition of both materials. The microcapsules' synthesis procedure was optimized to extensively reduce their natural aggregation tendency, as shown by the morphological analysis monitored by advanced microscopy techniques. The well-dispersed microcapsules showed an enhanced uptake by fibroblasts, opening new perspectives for cellular internalization.

Synthesis of chitosan-alginate microspheres with high antimicrobial and antibiofilm activity against multi-drug resistant microbial pathogens.

The successful treatment of multi-drug resistant microbial pathogens represents a major challenge for public health management. Here, chitosan-alginate (CS/ALG) microspheres with narrow size distribution were fabricated by ionically cross linking method using Ca2+ ions as agents for polymer solidification. The physicochemical properties of CS/ALG microspheres, such as surface morphology and size, were studied by SEM. The functional group interactions were confirmed by Fourier transform infrared (FTIR) spectroscopy. SEM revealed that the CS/ALG microspheres were spherical in shape with smooth surfaces, size was 50-100 µm. The synthesized CS/ALG microspheres showed antibacterial and antibiofilm activity on bacteria of public health relevance. CS/ALG microspheres exhibited antibacterial activity at the concentration of 5-20 µg, with significant inhibitory zones on multiple antibiotic resistant pathogens, including Gram positive Staphylococcus aureus, Enterococcus faecalis, and Gram negative Pseudomonas aeruginosa and Proteus vulgaris. Furthermore, in situ light microscopy and confocal laser scanning microscopy (CLSM) showed that CS/ALG microspheres inhibited the bacterial biofilm formation in S. aureus, E. faecalis P. aeruginosa and P. vulgaris after a single treatment with 40 µg. Overall, our findings underlined that chemically synthesized CS/ALG biomaterial has high antibacterial and antibiofilm activity against a number of microbial pathogens of interest for human health, thus this synthesis route can be further exploited for drug development in current biomedical science.

Tunable injectable alginate-based hydrogel for cell therapy in Type 1 Diabetes Mellitus.

Islet transplantation has the potential of reestablishing naturally-regulated insulin production in Type 1 diabetic patients. Nevertheless, this procedure is limited due to the low islet survival after transplantation and the lifelong immunosuppression to avoid rejection. Islet embedding within a biocompatible matrix provides mechanical protection and a physical barrier against the immune system thus, increasing islet survival. Alginate is the preferred biomaterial used for embedding insulin-producing cells because of its biocompatibility, low toxicity and ease of gelation. However, alginate gelation is poorly controlled, affecting its physicochemical properties as an injectable biomaterial. Including different concentrations of the phosphate salt Na2HPO4 in alginate hydrogels, we can modulate their gelation time, tuning their physicochemical properties like stiffness and porosity while maintaining an appropriate injectability. Moreover, these hydrogels showed good biocompatibility when embedding a rat insulinoma cell line, especially at low Na2HPO4 concentrations, indicating that these hydrogels have potential as injectable biomaterials for Type 1 Diabetes Mellitus treatment.

Synthesis and evaluation of anti-fungal activities of sodium alginate-amphotericin B conjugates.

Sodium alginate (SA) was oxidized using periodate and amphotericin B (AmB) was conjugated via imine and amine linkages to the oxidized alginate. Oxidization drastically reduced the molecular weight (MW) of the alginate. The conjugates were highly water-soluble to the extent of 1000mg/mL making them useful for therapeutic applications. SA-AmB conjugates derived from 20 and 50% oxidized alginate were non-toxic to HEK 293T and RAW 264.7 cell line at 100µg/mL and was also non-hemolytic to human blood at 100µg/mL. In vitro release of AmB into phosphate buffer from the imine conjugates was negligible with less than 0.2% of the drug released in 48h. Capping of residual aldehyde handles using 2-ethanolamine or glycine resulted in increased release of the drug in vitro. Injectable gels of gelatin crosslinked with oxidized alginate incorporating the SA-AmB conjugates as well as AmB were also fabricated and drug release was examined. In vitro release from the gel discs showed that AmB was released to the extent of 15-20% in 2days. The SA-AmB conjugates showed potent anti-fungal activity against C. albicans, C. neoformans and C. parapsilosis. The injectable gels seem to have potential for prolonged release of AmB when implanted.

Mass Production of Cell-Laden Calcium Alginate Particles with Centrifugal Force.

This paper describes a centrifuge-based device for oil-free and mass production of calcium-alginate (Ca-alginate) particles. The device is composed of four components: a tank with a glass capillary for forming sodium alginate droplets, a collecting bath with calcium chloride (CaCl2 ) solution, a waste liquid box, and a bypass channel bridged between the collecting bath and the waste liquid box. When the device is centrifuged, extra CaCl2 solution in the collecting bath is delivered to the waste liquid box to maintain the appropriate liquid level of CaCl2 solution for the production of monodisperse Ca-alginate particles. The proposed device enables oil-free production of over 45 000 uniformly sized Ca-alginate particles in a single 240 s process, whereas using the conventional method with only a glass capillary, ≈1000 particles are formed within the same processing time. Because of the high biocompatibility of the oil-free process, the device is applicable to cell encapsulation in Ca-alginate particles with high cell viability, as well as the formation of a macroscopic 3D cellular structure using Ca-alginate particles covered with cells as assembly modules. These results suggest that the device can be a useful tool for preparing experimental platforms in biomedical and tissue engineering fields.

Preparation and characterization of electrospun alginate/PLA nanofibers as tissue engineering material by emulsion eletrospinning.

Scaffolds made by biomaterials offer favorite environment for cell grow and show a wide potential application in tissue engineering. Novel biocompatibility materials polylatic acid (PLA) nanofiber membranes with favorable biocompatibility and good mechanical strength could serve as an innovative tissue engineering scaffold. Sodium alginate (SA) could be used in biomedical areas because of its anti-bacterial property, hydrophilicity and biocompatibility. In this article, we chose PLA as continuous phase and SA as dispersion phase to prepare a W/O emulsion and then electrospun it to get a SA/PLA composite nanofiber membranes. The CLSM images illustrated that the existence of SA was located on the surface of composite fibers and the FTIR results confirmed the result. A calcium ion replacement step was used as an after-treatment for SA/PLA nanofiber membranes in order to anchor the alginic ion in a form of gelated calcium alginate (CA). The single fiber tensile test shows a good mechanical property of CA/PLA nanofiber membranes, and the nanofiber membranes are beneficial for cell proliferation and differentiation owing to MTT array as well as Alizarin red S (ARS) staining test.

Synthesis and Analysis of Glucuronic Acid-Conjugated Metabolites of 4-Bromo-2,5-Dimethoxyphenethylamine.

In the study reported here, two glucuronic acid-conjugated metabolites of 4-bromo-2,5-dimethoxyphenethylamine (2C-B)-a ring-substituted psychoactive phenethylamine-were chemically synthesized for the first time and a method for analyzing them in urine was developed. ß-D-Glucuronide of 4-bromo-2,5-dimethoxyphenylethylalcohol was successfully synthesized using methyl 2,3,4-tri-Ο-acetyl-1-O-(trichloroacetimidoyl)-α-D-glucuronate as a glucuronyl donor and boron trifluoride diethylether complex as a Lewis acid catalyst. ß-D-Glucuronide of 4-bromo-2,5-dimethoxyphenylacetic acid was synthesized by condensing 4-bromo-2,5-dimethoxyphenylacetic acid and benzyl D-glucuronate followed by benzyl group deprotection based on catalytic hydrogenation. Two glucuronic acid-conjugated metabolites of 2C-B in urine were qualitatively and semiquantitatively evaluated via direct liquid chromatography/mass spectrometry (LC/MS) analysis of a diluted urine sample. The simple method proposed is expected to be useful for studying the metabolic fate of 2C-B.

Usefulness of Alginate Lyases Derived from Marine Organisms for the Preparation of Alginate Oligomers with Various Bioactivities.

Alginate-degrading enzyme, alginate lyase, catalyzes the cleavage of glycosidic 1-4 O-linkages between uronic acid residues of alginate by a ß-elimination reaction leaving a 4-deoxy-l-erythro-hex-4-ene pyranosyluronate as nonreducing terminal end. The enzymes from a wide variety of sources such as marine molluscs, seaweeds, and marine bacteria have been discovered and studied not only from a point of view of enzymological interest of enzyme itself but also for elucidation of fine chemical structure of alginate, structure-activity relationship of alginate, and biological activities and physicochemical features of the enzymatic digestion products. Based on the substrate specificities, alginate lyases are classified into three groups: poly(ß-d-mannuronate) lyase, poly(α-l-guluronate) lyase, and bifunctional alginate lyase, which are specific to mannuronate, guluronate, and both uronic acid residues, respectively. We have studied enzymological aspects of these three types of alginate lyases, and bioactivities of enzymatically digested alginate oligomers. In this chapter, we described the purification and characterization of three types of alginate lyases from different marine origins and overviewed the bioactivities of alginate oligomers.

In situ thiolated alginate hydrogel: Instant formation and its application in hemostasis.

An in situ formed hydrogel was synthesized by sodium alginate and cysteine methyl ester, which turned the sodium alginate into thiolated alginate (SA-SH). SA-SH can in situ formed into hydrogel (SA-SS-SA) with a large amount of water through covalent bond in less than 20 s. The structure characterization showed that the mechanism of SA-SH gelation was thiol-disulfide transformation. The rheology and cytotoxicity experiments of SA-SS-SA hydrogel were also investigated, which indicated that SA-SS-SA hydrogel had an appropriate mechanical strength as well as an excellent biocompatibility. The SA-SS-SA hydrogel would degrade under certain conditions after a few days and its mechanism was disulfide alkaline reduction. Finally, the hemostatic property of SA-SH was tested by rat tail amputation experiment. The time to hemostasis of rat reduced from 8.26 min to 3.24 min, which proved that SA-SH had an excellent hemostatic property.

Enhanced bioadhesivity of dopamine-functionalized polysaccharidic membranes for general surgery applications.

UNLABELLED: An emerging strategy to improve adhesiveness of biomaterials in wet conditions takes inspiration from the adhesive features of marine mussel, which reside in the chemical reactivity of catechols. In this work, a catechol-bearing molecule (dopamine) was chemically grafted onto alginate to develop a polysaccharide-based membrane with improved adhesive properties. The dopamine-modified alginates were characterized by NMR, UV spectroscopy and in vitro biocompatibility. Mechanical tests and in vitro adhesion studies pointed out the effects of the grafted dopamine within the membranes. The release of HA from these resorbable membranes was shown to stimulate fibroblasts activities (in vitro). Finally, a preliminary in vivo test was performed to evaluate the adhesiveness of the membrane on porcine intestine (serosa). Overall, this functionalized membrane was shown to be biocompatible and to possess considerable adhesive properties owing to the presence of dopamine residues grafted on the alginate backbone. STATEMENT OF SIGNIFICANCE: This article describes the development of a mussels-inspired strategy for the development of an adhesive polysaccharide-based membrane for wound healing applications. Bioadhesion was achieved by grafting dopamine moieties on the structural component on the membrane (alginate): this novel biomaterial showed improved adhesiveness to the intestinal tissue, which was demonstrated by both in vitro and in vivo studies. Overall, this study points out how this nature-inspired strategy may be successfully exploited for the development of novel engineered biomaterials with enhanced bioadhesion, thus opening for novel applications in the field of general surgery.

Fabrication, Characterization, and Evaluation of Bionanocomposites Based on Natural Polymers and Antibiotics for Wound Healing Applications.

The aim of our research activity was to obtain a biocompatible nanostructured composite based on naturally derived biopolymers (chitin and sodium alginate) loaded with commercial antibiotics (either Cefuroxime or Cefepime) with dual functions, namely promoting wound healing and assuring the local delivery of the loaded antibiotic. Compositional, structural, and morphological evaluations were performed by using the thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and fourier transform infrared spectroscopy (FTIR) analytical techniques. In order to quantitatively and qualitatively evaluate the biocompatibility of the obtained composites, we performed the tetrazolium-salt (MTT) and agar diffusion in vitro assays on the L929 cell line. The evaluation of antimicrobial potential was evaluated by the viable cell count assay on strains belonging to two clinically relevant bacterial species (i.e., Escherichia coli and Staphylococcus aureus).

Cationic carbon quantum dots derived from alginate for gene delivery: One-step synthesis and cellular uptake.

UNLABELLED: Carbon quantum dots (CQDs), unlike semiconductor quantum dots, possess fine biocompatibility, excellent upconversion properties, high photostability and low toxicity. Here, we report multifunctional CQDs which were developed using alginate, 3% hydrogen peroxide and double distilled water through a facile, eco-friendly and inexpensive one-step hydrothermal carbonization route. In this reaction, the alginate served as both the carbon source and the cationization agent. The resulting CQDs exhibited strong and stable fluorescence with water-dispersible and positively-charged properties which could serve as an excellent DNA condensation. As non-viral gene vector being used for the first time, the CQDs showed considerably high transfection efficiency (comparable to Lipofectamine2000 and significantly higher than PEI, p<0.05) and negligible toxicity. The photoluminescence properties of CQDs also permitted easy tracking of the cellular-uptake. The findings showed that both caveolae- and clathrin-mediated endocytosis pathways were involved in the internalization process of CQDs/pDNA complexes. Taken together, the alginate-derived photoluminescent CQDs hold great potential in biomedical applications due to their dual role as efficient non-viral gene vectors and bioimaging probes. STATEMENT OF SIGNIFICANCE: This manuscript describes a facile and simple one-step hydrothermal carbonization route for preparing optically tunable photoluminescent carbon quantum dots (CQDs) from a novel raw material, alginate. These CQDs enjoy low cytotoxicity, positive zeta potential, excellent ability to condense macromolecular DNA, and most importantly, notably high transfection efficiency. The interesting finding is that the negatively-charged alginate can convert into positively charged CQDs without adding any cationic reagents. The significance of this study is that the cationic carbon quantum dots play dual roles as both non-viral gene vectors and bioimaging probes at the same time, which are most desirable in many fields of applications such as gene therapy, drug delivery, and bioimaging.

Controllable Multicompartmental Capsules with Distinct Cores and Shells for Synergistic Release.

A facile and flexible approach is developed for controllable fabrication of novel multiple-compartmental calcium alginate capsules from all-aqueous droplet templates with combined coextrusion minifluidic devices for isolated coencapsulation and synergistic release of diverse incompatible components. The multicompartmental capsules exhibit distinct compartments, each of which is covered by a distinct part of a heterogeneous shell. The volume and number of multiple compartments can be well-controlled by adjusting flow rates and device numbers for isolated and optimized encapsulation of different components, while the composition of different part of the heterogeneous shell can be individually tailored by changing the composition of droplet template for flexibly tuning the release behavior of each component. Two combined devices are first used to fabricate dual-compartmental capsules and then scaled up to fabricate more complex triple-compartmental capsules for coencapsulation. The synergistic release properties are demonstrated by using dual-compartmental capsules, which contain one-half shell with a constant release rate and the other half shell with a temperature-dependent release rate. Such a heterogeneous shell provides more flexibilities for synergistic release with controllable release sequence and release rates to achieve advanced and optimized synergistic efficacy. The multicompartmental capsules show high potential for applications such as drug codelivery, confined reactions, enzyme immobilizations, and cell cultures.

A robust super-tough biodegradable elastomer engineered by supramolecular ionic interactions.

Alginate-based supramolecular ionic polyurethanes (ASPUs) as mechanically tunable biomaterials with high strength and toughness in both dry and hydrated states are developed under metal-free conditions. The Young's modulus and tensile strength of ASPUs are tuned from 30 to 100 MPa, and 20 to 50 MPa, respectively. Interestingly, the ASPUs exhibit a small hysteresis loop, minimal loss of tensile strength and minimal creep deformation after 100 repetitive cycles which makes them of use for engineering of load-bearing tissues. This is the first report that describes a linear PU can resist a large number of cyclic stresses without significant stretching. These bio-based elastomers engineered by ionic interactions are biocompatible and biodegradable. The ASPUs demonstrate a similar in vivo degradation rate compared to polycaprolactone (PCL). These biomaterials also demonstrate a rapid self-healing and recovery after rupture, and have a linear biodegradation profile. Furthermore, histological examination of subcutaneous transplanted ASPUs after five months reveals low immunological response and low fibrosis.

Preparation and characterization of a novel sodium alginate incorporated self-assembled Fmoc-FF composite hydrogel.

Dipeptides and their derivatives have attracted tremendous attention owning to their excellent abilities of self-assemble assembling into various structures which have great potentials for applications in biology and/or nanotechnology. In the present study, we dedicate to fabricate a rigid and structure controllable Fmoc-FF/SA composite hydrogel. We found that the modified dipeptide, fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Phe-Phe) can self-assemble into rigid hydrogels with structures of nanowires, layered thin films or honeycombs as the change of sodium alginate (SA) concentration. Meanwhile, CD-spectroscopy demonstrated that SA appeared to control the process, but it did not change the arrangement of the Fmoc-FF peptide. Our results demonstrated that the formed hydrogel showed physical and chemical stability as well as possessing good biocompatibility. Rheological measurements showed that the addition of SA could improve the stability of the hydrogel. Cell viability assay revealed that the Fmoc-FF and Fmoc-FF/SA hydrogels are both beneficial for cell proliferation in-vitro. Our results indicated that the fabricated Fmoc-FF/SA composite hydrogels could be used in tissue engineering and drug delivery in the future.

Development and evaluation of alginate-chitosan gastric floating beads loading with oxymatrine solid dispersion.

Oxymatrine (OM) can be metabolized to matrine in gastrointestinal ileocecal valve after oral administration, which affects pharmacological activity and reduce bioavailability of OM. A type of multiple-unit alginate-chitosan (Alg-Cs) floating beads was prepared by the ionotropic gelation method for gastroretention delivery of OM. A solid dispersion technique was applied and incorporated into beads to enhance the OM encapsulation efficiency (EE) and sustain the drug release. The surface morphology and internal hollow structure of beads were evaluated using optical microscopy and scanning electron microscopy (SEM). The developed Alg-Cs beads were spherical in shape with hollow internal structure and had particle size of 3.49 ± 0.09 mm and 1.33 ± 0.09 mm for wet and dried beads. Over 84% of the optimized OM solid dispersion-loaded Alg-Cs beads were able to continuously float over the simulated gastric fluid for 12 h in vitro. The OM solid dispersion-loaded Alg-Cs beads showed drug EE of 67.07%, which was much higher than that of beads loading with pure OM. Compared with the immediate release of OM capsules and pure OM-loaded beads, the release of OM from solid dispersion-loaded Alg-Cs beads was in a sustained-release manner for 12 h. Prolonged gastric retention time of over 8.5 h was achieved for OM solid dispersion-loaded Alg-Cs floating beads in healthy rabbit in in vivo floating ability evaluated by X-ray imaging. The developed Alg-Cs beads loading with OM solid dispersion displayed excellent performance features characterized by excellent gastric floating ability, high drug EE and sustained-release pattern. The study illustrated the potential use of Alg-Cs floating beads combined with the solid dispersion technique for prolonging gastric retention and sustaining release of OM, which could provide a promising drug delivery system for gastric-specific delivery of OM for bioavailability enhancement.