Fabrication of affinity-based drug delivery systems based on electrospun chitosan sulfate/poly(vinyl alcohol) nanofibrous mats.

Benign electrospinning of chitosan in aqueous medium is an open challenge mainly due to its insolubility in neutral pH and inter- and intramolecular hydrogen bonding interactions. Here, we developed a simple and widely-used methodology to improve the chitosan electrospinnability through the sulfation of chitosan and its further mixing with poly(vinyl alcohol) for the first time. The FTIR, 1H NMR and elemental analyses showed the successful sulfation of chitosan. Furthermore, the viscosity and electrical conductivity measurements revealed the high solubility of chitosan sulfate (CS) in aqueous media. In the next step, a uniform electrospun nanofibrous mat of CS/PVA was fabricated with a fiber diameter ranging from 90 to 340 nm. The crosslinked CS/PVA (50/50) nanofibrous mat as the optimum sample showed a swelling ratio of 290 ± 4 % and a high Young's modulus of 3.75 ± 0.10 GPa. Finally, malachite green (MG) as a cationic drug model was loaded into different samples of chitosan film, CS film, and CS/PVA (50/50) nanofibrous mat and its release behavior was studied. The results of these analyses revealed that the CS/PVA (50/50) nanofibrous mat can successfully load higher contents of the MG and also release it in a sustained manner.

Regioselective sulfated chitosan produces a biocompatible and antibacterial wound dressing with low inflammatory response.

The aim of current study is to tailor chitosan derivate which is water-soluble while presents original biological features of chitosan. For this purpose, the 6-O chitosan sulfate (CS) with naked amine groups was synthesized via regioselective modification of chitosan (C) during which both crosslinking capacity and antibacterial properties of the C were remained intact. This was achieved by sulfation the C under controlled acidic conditions using chlorosulfonic acid/sulfuric acid mixture. Subsequently, a chemically crosslinked hydrogel of the CS was used as a wound dressing substrate. The modified sulfate groups retained the biocompatibility of C and showed antibacterial effects against gram-positive and gram-negative bacteria. In addition, the presence of sulfate groups in the CS chemical structure improved its anticoagulant activity compared to the unmodified C. Both in vitro and in vivo enzyme-linked immunosorbent assay (ELISA) measurements showed that CS had a higher potential to bind and scavenger anti-inflammatory cytokines, including IL-6 and transforming growth factor-ß (TGF-ß), both of which play critical roles in the early stage of the wound healing process. After treatment of full-thickness wounds with CS hydrogels, the macrophage cells (c.a. 6 × 104 cells) expressed significantly more M2 phenotype markers compared to the C group (4.2 × 104 cells). Furthermore, the CS hydrogel induced better re-epithelialization and vascularization of full-thickness wounds in mice compared to the C hydrogel during 30 days.

Sulfation of chitosan from Sepia kobiensis as potential anticoagulant and antibacterial molecule.

The present work aimed to synthesis of chitin, chitosan and sulfation of chitosan from cuttlebone of cuttlefish Sepia kobiensis. Principally chitin was extracted through sequential processes of demineralisation and deproteinzation. Then chitosan was synthesized by a deacetylation and finally sulfated at semi-heterogeneous condition using chlorosulfonic acid in N,N-dimethylformamide. The synthesized macromolecules were characterized for its structural, physical and thermal (CHN, DDA, FT-IR, NMR, XRD, Viscometric analysis, SEM and DSC) properties. Apart from anticoagulant potential of the sulfated chitosan was tested using human plasma by means of activated partial thromboplastin time (APTT) and prothrombin time (PT). Further sulfated chitosan was tested for antibacterial potential by well diffusion method against eleven human pathogenic clinical isolates of both Gram positive and Gram-negative strains and minimum inhibitory concentrations (MIC) was calculated accordingly. The results of this study revealed the effectiveness of the sulfated chitosan at semi-heterogeneous conditions as a potent antibacterial and anticoagulant molecule.

Chitosan sulfate-lysozyme hybrid hydrogels as platforms with fine-tuned degradability and sustained inherent antibiotic and antioxidant activities.

The control of the properties and biological activities of chitosan-lysozyme hybrid hydrogels to exploit their interesting biomedical applications depends largely on the chitosan acetylation pattern, a difficult parameter to control. Herein, we have prepared sulfated chitosan-lysozyme hydrogels as versatile platforms with fine-tuned degradability and persistent bactericidal and antioxidant properties. The use of chitosan sulfates instead of chitosan has the advantage that the rate and mechanisms of lysozyme release, as well as antibacterial and antioxidant activities, depend on the sulfation profile, a structural parameter that is easily controlled by simple chemical modifications. Thus, while 6-O-sulfated chitosan hydrogels allow the release of loaded lysozyme in a short time (60% in 24 h), due to a high rate of degradation that allows rapid antibiotic and antioxidant activities, in 3-O-sulfated systems there is a slow release of lysozyme (80% in 21 days), resulting in long-lasting antibiotic and antioxidant activities.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.

Effects of selenide chitosan sulfate on glutathione system in hepatocytes and specific pathogen-free chickens.

This study aimed to investigate the effects of selenide chitosan sulfate (Se-CTS-S) on glutathione (GSH) system in hepatocytes and chickens. Chitosan, sodium selenite (Na2SeO3), selenide chitosan, chitosan sulfate (CTS-S), and Se-CTS-S were added to the culture medium and the basal diets; glutathione peroxidase (GSH-Px) activity, GSH content, total antioxidant capacity (T-AOC), and mRNA levels of cellular GPx (GPx-1) and phospholipid hydroperoxide GPx (GPx-4) in vivo and in vitro were determined. The results showed that Se-CTS-S increased (P < 0.05) GPx-1 and GPx-4 mRNA levels in hepatocytes and livers, and GSH-Px activity, GSH content, and T-AOC in the medium, hepatocytes, plasma, and livers compared with the control and chitosan treatments. Compared with CTS-S, Se-CTS-S treatments increased (P < 0.05) GPx-1 and GPx-4 mRNA levels in hepatocytes and livers, and GSH-Px activity, GSH content, and T-AOC capacity in the medium, hepatocytes, and livers. Compared with Na2SeO3 and CTS-Se, Se-CTS-S increased (P < 0.05) GPx-1 mRNA levels in hepatocytes and livers, GPx-4 mRNA levels in hepatocytes and livers, GSH-Px activity in the medium, hepatocytes, and livers, GSH contents in plasma and livers, and T-AOC in the medium, plasma, and livers. Thus, Se-CTS-S showed better biological activity that mainly benefited from the synergistic effects of Se and sulfate on GSH system.

Antithrombin III-mediated blood coagulation inhibitory activity of chitosan sulfate derivatized with different functional groups.

Acylated chitosan sulfate (ChS1), a sulfated polysaccharide with high anticoagulant activity, was chemically synthesized and structurally characterized using FT-IR analysis. The beneficial structural properties and high availability of the sulfate group in ChS1 led to greater anticoagulant activity through both the intrinsic and common pathways with antithrombin III (AT III)-mediated inhibition, particularly involving coagulation factors FXa and FIIa. The analysis of the binding affinities using surface plasma resonance found that the equilibrium dissociation constant (KD) of ChS1 for FXa and FIIa in the presence of AT III was 67.4 nM and 112.6 nM, respectively, indicating the stronger interaction of the AT III/ChS1 complex with the ligands and the inhibition of activated FX and FII. The results of amidolytic assays further demonstrated the stronger inhibition of the proteolytic conversion of factor X by the intrinsic FXase complex and of FII by the prothrombinase complex. Molecular docking analysis further validated the protein-ligand interactions of ChS1 with AT III and their binding affinity.

Modification of chitosan grafted with collagen peptide by enzyme crosslinking.

Free radicals are closely related to the occurrence and development of aging, cancer and inflammation. In this paper, the microbial transglutaminase (MTGase) was used as a catalyst to graft the collagen peptide (COP) molecules on the amino group of carboxymethyl chitosan sulfate (CMCS) to improve the antioxidant effects. FT-IR and NMR spectroscopy were used to confirm the successful grafting of COP to CMCS. Degree of substitution (DS) of CMCS-COP could be controlled by adjusting the reaction conditions. With the increase of concentration, the ability of each sample on scavenging capacity and reducibility tends to increase obviously. The results of anticoagulant experiments showed that the ability of CMCS and CMCS-COP with three different degrees of substitution on activated partial thrombin time (APTT) and prothrombin time (PT) values were all increased to compare with the control group. No relevant cytotoxicity against NIH-3T3 mouse fibroblasts was found for the copolymers. These results suggested that CMCS-COP would appear to be a promising candidate for wound dressing application.

Osteochondral Defects Healing Using Extracellular Matrix Mimetic Phosphate/Sulfate Decorated GAGs-Agarose Gel and Quantitative Micro-CT Evaluation.

Tissue engineering has a major emphasis in creating tissue specific extracellular ambiance by altering chemical functionalities of scaffold materials. Heterogeneity of osteochondral tissue necessitates tailorable bone and cartilage specific extracellular environment. Carboxylate- and sulfate-functionalized glycosaminoglycans (GAGs) in cartilage extracellular matrix (ECM) create an acidic ambience to support chondrogenic activity, whereas phosphate-rich environment in bone enables chelation of calcium leading to the formation of mineralized matrix along with an alkaline environment to support osteogenesis. In this study, chitosan, a naturally occurring GAGs, was functionalized with phosphate/sulfate groups analogous to bone/cartilage ECM and incorporated in thermogelling agarose hydrogel for delivery to osteochondral defects. In vitro studies revealed significantly higher adhesion and proliferation of adipose derived mesenchymal stem cells (ADMSCs) with blended hydrogels as compared to that of native agarose. Cell differentiation and RT-PCR studies of the phosphorylated hydrogels revealed higher osteogenic potential, while sulfated hydrogels demonstrated enhanced chondrogenic activity in comparison to agarose. Recovery of osteochondral defects after delivery of the thermoresponsive agarose-based hydrogels decorated with phosphorylated derivatives showed significantly higher bone formation. On the other hand, cartilage formation was significant with chitosan sulfate decorated hydrogels. The study highlights the role of chitosan derivatives in osteochondral defect healing, especially phosphorylated ones as bone promoter, whereas sulfated ones act as cartilage enhancer, which was quantitatively distinguished through micro-CT-based noninvasive imaging and analysis.

Preparation and characterization of carboxymethyl chitosan sulfate/oxidized konjac glucomannan hydrogels.

Hydrogel wound dressing is a new type of biomaterial with performance that is better than traditional and biological dressings. It has been extensively researched and the application in the field of biomedicine is common. In this study, we developed a simple and nontoxic method for preparing a new type of composite hydrogel, which formed through the Schiff-base reaction between the aldehyde of oxidized konjac glucomannan (OKGM) and the amino of carboxymethyl chitosan sulfate (CMSS). The chemical structures of this composite hydrogel were characterized by transform infrared spectroscopy (FT-IR). The micro-morphology of hydrogels were analyzed by scanning electron microscopy (SEM). Meanwhile, the properties of composite hydrogels including gelation time, swelling ability, water evaporation rate, hemolytic potential and biological compatibility were also investigated in different means. The results gained from these studies show that this composite hydrogels have a series of properties such as short gelation time, good swelling ability, appropriate water evaporation rate, excellent hemocompatibility and well biological compatibility. Considering these excellent performance, this composite hydrogels can be used as a wound dressing to treat injured skin.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics.

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.

Preparation of chitosan sulfate and vesicle formation with a conventional cationic surfactant.

Chitosan of high molecular weight and 85% deacetylation was used to prepare chitosan sulfate (CHS) by employing an industrial recognized green and highly reactive sulfating agent gas SO3. FT-IR and solid-state CP-MAS 13C NMR spectra confirmed that sulfate groups were successfully introduced into chitosan chains with a sulfur content of 16.50% and the substitution degree of 1.75 according to the results of elemental analysis. The aggregation behavior of the mixture of chitosan sulfate polyelectrolyte and oppositely charged surfactant cetyltrimethylammonium bromide (CTAB) was characterized by surface tension, steady-state fluorescent, turbidity, ζ potential and transmission electron microscopy. The results indicate that the CHS/CTAB mixture has pretty high surface activity and low critical aggregation concentration. The CHS/CTAB mixture successively forms spherical aggregates, precipitation, vesicles and micelle aggregates coated by CHS chains by increasing surfactant concentration due to the cooperative hydrophobic and electrostatic interactions.

Gold nanoparticles in the engineering of antibacterial and anticoagulant surfaces.

Simultaneous antibacterial and anticoagulant surfaces have been prepared by immobilization of engineered gold nanoparticles onto different kinds of surfaces. The gold nanoparticle core is surrounded by a hemocompatible, anticoagulant polysaccharide, 6-O chitosan sulfate, which serves as reduction and stabilizing agent for the generation of gold nanoparticles in a microwave mediated reaction. The particle suspension shows anticoagulant activity, which is investigated by aPTT and PT testing on citrated blood samples of three patients suffering from congenital or acquired bleeding disorders. The amount of nanoparticles deposited on the surfaces is quantified by a quartz crystal microbalance with dissipation unit. All gold containing surfaces exhibit excellent antimicrobial properties against the chosen model organism, Escherichia coli MG 1655 [R1-16]. Moreover, blood plasma coagulation times of the surfaces are increased after deposition of the engineered nanoparticles as demonstrated by QCM-D.

Assessment of palmitoyl and sulphate conjugated glycol chitosan for development of polymeric micelles.

INTRODUCTION: Amphiphilic copolymers are capable of forming core shell-like structures at the critical micellar concentration (CMC); hence, they can serve as drug carriers. Thus, in the present work, polymeric micelles based on novel chitosan derivative were synthesized. METHODS: Block copolymer of palmitoyl glycol chitosan sulfate (PGCS) was prepared by grafting palmitoyl and sulfate groups serving as hydrophobic and hydrophilic fractions, respectively. Then, fourier transform infrared spectra (FTIR) and spectral changes in iodine/iodide mixture were carried out. RESULTS: FTIR studies confirmed the formation of palmitoyl glycol chitosan sulfate (PGCS) and spectral changes in iodine/iodide mixture indicated CMC which lies in the range of 0.003-0.2 mg/ml. CONCLUSION: Therefore, our study indicated that polymeric micelles based on palmitoyl glycol chitosan sulphate could be used as a prospective carrier for water insoluble drugs.