Nanoparticles integrating natural and synthetic polymers for in vivo insulin delivery.

The aims of the current study were to develop insulin-loaded nanoparticles comprised of various polymers at different compositions, and to evaluate their ability to lower blood glucose levels in diabetic rats following subcutaneous and oral administrations. Several combinations of natural and synthetic polymers have been utilized for preparation of nanoparticles including, chitosan, alginate, albumin and Pluronic. Nanosized (170 nm-800 nm) spherical particles of high encapsulation efficiency (15-52%) have been prepared. Composition and ratios between the integrated polymers played a pivotal role in determining size, zeta potential, and in vivo hypoglycemic activity of particles. After subcutaneous and oral administration in diabetic rats, some of the insulin-loaded nanoparticles were able to induce much higher hypoglycemic effect as compared to the unloaded free insulin. For instance, subcutaneous injection of nanoparticles comprised of chitosan combined with sodium tripolyphosphate, Pluronic or alginate/calcium chloride, resulted in comparable hypoglycemic effects to free insulin, at two-fold lower dose. Nanoparticles were well-tolerated after oral administration in rats, as evidenced by by measuring levels of alanine aminotransferase, aspartate aminotransferases, albumin, creatinine and urea. This study indicates that characteristics and delivery efficiency of nanomaterials can be controlled via utilizing several natural/synthetic polymers and by fine-tuning of combination ratio between polymers.

3D printing of an integrated triphasic MBG-alginate scaffold with enhanced interface bonding for hard tissue applications.

Osteochondral defects affect both of cartilage and subchondral areas, thus it poses a significant challenge to simultaneously regenerate two parts in orthopedics. Tissue engineering strategy is currently regarded as the most promising way to repair osteochondral defects. This study focuses on developing a multilayered scaffold with enhanced interface bonding through 3D printing. One-shot printing process enables control over material composition, pore structure, and size in each region of the scaffold, while realizes seamlessly integrated construct as well. The scaffold was designed to be triphasic: a porous bone layer composed of alginate sodium (SA) and mesoporous bioactive glasses (MBG), an intermediate dense layer also composed of SA and MBG and a cartilaginous layer composed of SA. The mechanical strength including the interface adhesion strength between layers were characterized. The results indicated that SA crosslinking after 3D printing anchored different materials together and integrated all regions. Additional scaffold soaking in simulated body fluid (SBF) and cell culture medium induced apatite deposition and had weakened the compressive and tensile strengths, while no layer dislocation or delamination occurred.

Alginate-Mediated Synthesis of Hetero-Shaped Silver Nanoparticles and Their Hydrogen Peroxide Sensing Ability.

A new method for the simple synthesis of stable heterostructured biopolymer (sodium alginate)-capped silver nanoparticles (Ag-NPs) based on green chemistry is reported. The as-prepared nanoparticles were characterized using the ultraviolet-visible (UV-Vis) absorption spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS) techniques. The results showed that the as-prepared Ag-NPs have a heterostructured morphology with particle size in the range 30 ± 18-60 ± 25 nm, showing a zeta potential of -62 mV. The silver nanoparticle formation was confirmed from UV-Vis spectra showing 424 nm as maximum absorption. The particle size and crystallinity of the as-synthesized nanoparticles were analyzed using TEM and XRD measurements, respectively. FTIR spectra confirmed the presence of alginate as capping agent to stabilize the nanoparticles. The Ag-NPs also showed excellent sensing capability, with a linear response to hydrogen peroxide spanning a wide range of concentrations from 10-1 to 10-7 M, which indicates their high potential for water treatment applications, such as pollution detection and nanofiltration composites.

Development and characterization of novel ambroxol sustained-release oral suspensions based on drug-polymeric complexation and polymeric raft formation.

The aim was to develop sustained-release aqueous suspensions of ambroxol utilizing drug-polymer complexation and raft-forming formulations. Ambroxol-carrageenan (ABX-CRG) complexation was studied for the optimum binding capacity, which was used to prepare the complex by kneading and coprecipitation. The prepared complex was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and powder X-ray diffractometry. The complex was formulated as suspensions in aqueous raft-forming vehicle of sodium alginate (NA) and calcium carbonate (CC). The suspensions differed in the molecular weight and concentration of NA, in addition to CC level and inclusion of CRG in excess of drug-polymer complexation. In 0.1 M HCl as simulated gastric fluid, the suspensions were observed for their ability to form rafts and studied for drug-release. The optimum sustained-release, raft forming and pourable formulation using high molecular weight NA, NA concentration of 18 mg/ml and CC concentration of 9 mg/ml was reached. Another optimum suspension was obtained by replacement of CC with excess CRG. However, pH dissolution profiles of the optimum suspensions revealed less pH sensitivity of the release consequent to this replacement as well as more stable ABX release upon aging. Relative to Gaviscon liquid, the optimum suspensions formed rafts of similar strength and higher resilience.

Exploring Endolysin-Loaded Alginate-Chitosan Nanoparticles as Future Remedy for Staphylococcal Infections.

Endolysins are a novel class of antibacterials with proven efficacy in combating various bacterial infections, in vitro and in vivo. LysMR-5, an endolysin derived from phage MR-5, demonstrated high lytic activity in our laboratory against multidrug-resistant S. aureus (MRSA) and S. epidermidis strains. However, endolysin and proteins in general are associated with instability and short in vivo half-life, consequently limiting their usage as pharmaceutical preparation to treat bacterial infections. Nanoencapsulation of endolysins could help to achieve better therapeutic outcome, by protecting the proteins from degradation, providing sustained release, thus could increase their stability, shelf life, and therapeutic efficacy. Hence, in this study, the feasibility of alginate-chitosan nanoparticles (Alg-Chi NPs) to serve as drug delivery platform for LysMR-5 was evaluated. LysMR-5-loaded nanoparticles were prepared by calcium ion-induced pre-gelation of alginate core and its complexation with chitosan. The formation of nanoparticles was confirmed on the basis of DLS, zeta potential, and electron microscopy imaging. The LysMR-5-loaded nanoparticles presented a hydrodynamic diameter of 276.5 ± 42, a PDI of 0.342 ± 0.02, a zeta potential - 25 mV, and an entrapment efficiency of 62 ± 3.1%. The potential ionic interaction between alginate, chitosan, and LysMR-5 was investigated by FT-IR and SEM-EDX analysis. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), nano-sized particles with characteristic morphology were seen. Different antibacterial assays and SDS-PAGE analysis showed no change in endolysin's structural integrity and bioactivity after entrapment. A direct antibacterial effect of blank Alg-Chi Nps, showing enhanced bactericidal activity upon LysMR-5 loading, was observed against S. aureus. At physiological pH (7.2), the release profile of LysMR-5 from Alg-Chi NPs showed a biphasic release and followed a non-Fickian release mechanism. The biocompatible nature as revealed by cytocompatibility and hemocompatibility studies endorsed their use as drug delivery system for in vivo studies. Collectively, these results demonstrate the potential of Alg-Chi NPs as nano-delivery vehicle for endolysin LysMR-5 and other therapeutic proteins for their use in various biomedical applications.

Acetylated Nanocellulose for Single-Component Bioinks and Cell Proliferation on 3D-Printed Scaffolds.

Nanocellulose has been demonstrated as a suitable material for cell culturing, given its similarity to extracellular matrices. Taking advantage of the shear thinning behavior, nanocellulose suits three-dimensional (3D) printing into scaffolds that support cell attachment and proliferation. Here, we propose aqueous suspensions of acetylated nanocellulose of a low degree of substitution for direct ink writing (DIW). This benefits from the heterogeneous acetylation of precursor cellulosic fibers, which eases their deconstruction and confers the characteristics required for extrusion in DIW. Accordingly, the morphology of related 3D-printed architectures and their performance during drying and rewetting as well as interactions with living cells are compared with those produced from typical unmodified and TEMPO-oxidized nanocelluloses. We find that a significantly lower concentration of acetylated nanofibrils is needed to obtain bioinks of similar performance, affording more porous structures. Together with their high surface charge and axial aspect, acetylated nanocellulose produces dimensionally stable monolithic scaffolds that support drying and rewetting, required for packaging and sterilization. Considering their potential uses in cardiac devices, we discuss the interactions of the scaffolds with cardiac myoblast cells. Attachment, proliferation, and viability for 21 days are demonstrated. Overall, the performance of acetylated nanocellulose bioinks opens the possibility for reliable and scale-up fabrication of scaffolds appropriate for studies on cellular processes and for tissue engineering.

Exploring a glycosylation methodology for the synthesis of hydroxamate-modified alginate building blocks.

Alginate, an anionic polysaccharide, is an important industrial biomaterial naturally harvested from seaweed. Many of its important physicochemical properties derive from the presence of charged carboxylate groups presented as uronic acids within the polysaccharide backbone. An ability to modify these carboxylates with alternate functional groups would enable the design and implementation of new alginate systems possessing different physicochemical properties. We present herein our approach to the chemical synthesis of alginate disaccharides, modified at the carboxylate C6 position with bioisosteric hydroxamate residues. The synthesis and utilisation of C6-hydroxamate donor and acceptor building blocks enables a first access to protected α- and ß-linked hydroxamate-containing disaccharides, additionally equipped with a functional tether at the reducing terminus. The evaluation of these building blocks for chemical glycosylation demonstrates the incorporation of bioisosteric functional groups into an alginate disaccharide backbone and highlights the important contribution of both acceptor and donor reactivity underpinning uronate glycosylations.

Synthesis of compounds having antimicrobial activity from alginate.

Compounds having antimicrobial activity were synthesized from sodium alginate, the main constituent of brown algae. Sodium alginate was oxidized with sodium periodate to get alginate dialdehyde (ADA). FTIR spectrum of the ADA gave very small peak characteristic for aldehyde groups at 1720 cm-1, indicating that the aldehyde group is masked somehow. It may be hydrated, involving at hemiacetal formation or hemialdol, similar to cellulose dialdehyde. Two methods were used for the condensation of ADA with o-phenylenediamine analogs to obtain the final products. The first method was stirring at room temperature and the second method was heating in microwave. The microwave method gave higher yield and shorter reaction time than the other method. The condensation reaction is considered as a shiff-base formation and the proposed mechanism was suggested. The condensation products were characterized by FTIR and UV spectra. The antimicrobial potency for five of these products in addition to the used alginate and to the precursor amines was evaluated against four pathogenic fungi and six pathogenic bacteria species.

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.

Oxidized Alginate-Based Hydrogels for Tissue Engineering Applications: A Review.

Oxidized alginate (OA)-based hydrogels have drawn considerable attention as biodegradable materials for tissue engineering applications. OA possesses a faster degradation rate and contains more reactive groups compared to native alginate. This review summarizes the research publications reporting the development of OA-based hydrogels for tissue engineering applications including bone, cartilage, blood vessel, cornea, and other soft tissues, highlighting OA key properties and processing approaches.

High-Performance Filaments from Fractionated Alginate by Polyvalent Cross-Linking: A Theoretical and Practical Approach.

A series of alginate fractions with significant differences in molecular weight and uronic acid compositions were produced by consecutive fractionation and converted to thin and strong cross-linked polymer filaments via extrusion into calcium, aluminum, or polyaluminum (PolyAl) polyvalent solutions followed by drawing and drying. Models were elaborated to relate the alginate uronic acid composition to the tensile performance in both the wet gel filament and the dry filament states. The wet gel model was compared to the theory of the unidirectional elongation of charged polyelectrolyte gels based on the classical rubber elasticity of dilated polymer networks, extended to include the contributions of non-Gaussian chain extensions and the effect of electrostatic interactions. The theory of equilibrium swelling pressure was applied to describe the observed shrinkage of the alginate gels following immersion in a polyvalent solution. Congruent with the theoretical model of charged gels, the tensile performance of the gel filaments prepared from CaCl2 depended on the compositional ratio of guluronic acid dyads in the alginate fraction multiplied by the alginate concentration, while the tensile behavior of wet gel filaments prepared by AlCl3 instead resembled that of elastic solid materials and depended only on the alginate concentration. The dry filament tensile properties were greatly dependent on the preparation conditions, particularly the ratio of stress to alginate concentration and the nature of the ions present during filament drawing. The PolyAl solution effectively caused shrinkage of alginate to a strong extent, and the resulting filaments behaved as highly stiff materials able to withstand stresses of approximately 500 MPa and having elastic moduli as high as 28 GPa.

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.

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.

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).

Formulation and preparation of stable cross-linked alginate-zinc nanoparticles in the presence of a monovalent salt.

Polysaccharide-based nanoparticles can be formed, under the right conditions, when a counterion is added to a dilute polysaccharide solution. In this study, the possibility of preparing stable alginate nanoparticles cross-linked with zinc was investigated. The effects of the ionic strength of the solvent and the concentration of zinc were studied. The nanoparticles were characterized by dynamic light scattering, zeta potential and pH measurements. The results showed that an increase in the ionic strength of the solvent provided nanoparticles with considerably narrower size distributions compared to pure water, and a small size. The zinc content was shown to be an important factor for the formation of the nanoparticles. In fact, a critical zinc concentration was needed to obtain nanoparticles, and below this concentration particles were not formed. A stepwise increase in the amount of zinc revealed the process of formation of the nanoparticles. The stages of the nanoparticle formation process were identified, and differences according to the ionic strength of the solvent were also reported. Furthermore, the stability test of the most promising formulation showed a stability of over ten weeks.

Synthesis and evaluation of hydroponically alginate nanoparticles as novel carrier for intravenous delivery of propofol.

Commercial lipid emulsion of propofol (CLE) has several drawbacks including pain on injection and emulsion instability. In this paper, a novel nanocarrier system is introduced to improve stability and solubility of the poorly soluble anesthetic drug, propofol, for intravenous administration. In this paper, alginate is modified using a facile method in which the carboxylic group of alginate is grafted to octanol. The octanol-grafted alginate (Alg-C8) is then employed to prepare nanoparticles which are subsequently used for encapsulation of propofol. The nanoparticles are analyzed for their pH, osmolarity, particle size, stability, morphology and sleep recovery and the results are compared with CLE as control. It is revealed that nanoparticles have the average particle size of 180 nm ± 1.2 and spherical morphology which is less than CLE while their pH, osmolarity and profile of release of formulated nanoparticles are similar to those of CLE. In addition, the results show good chemical and physical storage stability for the nanoparticles at room temperature for at least 6 months compared to CLE as control. The animal sleep recovery test on rats shows no significant difference in time of unconsciousness and recovery of the righting reflex between nanoparticles and CLE. It is concluded that encapsulated nanoparticles introduced here could be a promising clinical intravenous system for delivery of poorly soluble anesthetic propofol. In addition, this study provides an efficient and facile method for preparing a carrier system for water insoluble drugs.

Development of Pasteurella multocida-loaded microparticles for hemorrhagic septicemia vaccine.

In this study, Pasteurella multocida-loaded alginate microparticles (MPs) for subcutaneous vaccination was developed by emulsification-cross-linking technique. Formulation parameter was varied as a ratio of polymer and bacterin. Optical microscopy revealed spherical particles with uniformly distribution. A mean particle size of approximately 6 µm has been successfully constructed using simple mixer and ultrasonic probe. The zeta potential of the MPs showed negatively charge of approximately -23 mV determined by Zeta Pals® analyzer. The entrapment efficiency and the in vitro bacterin released profile could be controlled by varying the amount of alginate. The high entrapment efficiency up to 69% was achieved with low concentration of alginate. The MPs possessed a slow bacterin release profile, up to 30 days. In vivo safety and potency tests were proved that the alginate MPs were safe and induced protective immunity in mice. In addition, after storage for 6 months at either 4 °C or room temperature, the protective immunity in mice was maintained.

Acceptor reactivity in the total synthesis of alginate fragments containing α-L-guluronic acid and ß-D-mannuronic acid.

The total synthesis of mixed-sequence alginate oligosaccharides, featuring both ß-D-mannuronic acid (M) and α-L-guluronic acid (G), is reported for the first time. A set of GM, GMG, GMGM, GMGMG, GMGMGM, GMGMGMG, and GMGGMG alginates was assembled using GM building blocks, having a guluronic acid acceptor part and a mannuronic acid donor side to allow the fully stereoselective construction of the cis-glycosidic linkages. It was found that the nature of the reducing-end anomeric center, which is ten atoms away from the reacting alcohol group in the key disaccharide acceptor, had a tremendous effect on the efficiency with which the building blocks were united. This chiral center determines the overall shape of the acceptor and it is revealed that the conformational flexibility of the acceptor is an all-important factor in determining the outcome of a glycosylation reaction.

Microfluidic production of perfluorocarbon-alginate core-shell microparticles for ultrasound therapeutic applications.

The fabrication of micrometer-sized core-shell particles for ultrasound-triggered delivery offers a variety of applications in medical research. In this work, we report the design and development of a glass capillary microfluidic system containing three concentric glass capillary tubes for the development of core-shell particles. The setup enables the preparation of perfluorocarbon-alginate core-shell microspheres in a single process, avoiding the requirement for further extensive purification steps. Core-shell microspheres in the range of 110-130 µm are prepared and are demonstrated to be stable up to 21 days upon immersion in calcium chloride solution or water. The mechanical stability of the particles is tested by injecting them through a 23 gauge needle into a polyacrylamide gel to mimic the tissue matrix. The integrity of the particles is maintained after the injection process and is disrupted after ultrasound exposure for 15 min. The results suggest that the perfluorcarbon-alginate microparticles could be a promising system for the delivery of compounds, such as proteins, peptides, and small-molecule drugs in ultrasound-based therapies.

Multifunctional interpenetrating polymer network hydrogels based on methacrylated alginate for the delivery of small molecule drugs and sustained release of protein.

Multifunctional injectable thermo-/pH-responsive hydrogels as release systems for the oral delivery of small molecule drugs and the local delivery of protein are presented. The injectable interpenetrating polymer network (IPN) hydrogels based on poly(ethylene glycol) methacrylate, N-isopropylacrylamide, and methacrylated alginate were prepared by using ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) as a redox initiator system at body temperature, and the obtained hydrogels overcame the instability of calcium cross-linked alginate hydrogels under physiological conditions. The hydrogels showed good mechanical strength by rheometer and exhibited temperature and pH sensitivity by a swelling test. Diclofenac sodium (DCS) as a model for small molecule water-soluble anti-inflammatory drugs and bovine serum albumin (BSA) as a model for protein drugs were encapsulated in situ in the hydrogel. The DCS and BSA release results indicated that these hydrogels, as carriers, have great potential for use in the oral delivery of small molecule drugs and for long-term localized protein release. Furthermore, the cytotoxicity of these hydrogels was studied via live/dead viability and alamarBlue assays using adipose tissue-derived mesenchymal stem cells.