Photo-cross-linked hydrogels with polysaccharide-poly(amino acid) structure: new biomaterials for pharmaceutical applications

The aim of this work has been the preparation and characterization of novel hydrogels with polysaccharide-poly(amino acid) structure having suitable physicochemical properties for pharmaceutical applications. In the first step, hyaluronic acid (HA) and alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) have been derivatized with methacrylic anhydride (AMA), thus obtaining HA-AMA and PHM derivatives, respectively. In the second step, aqueous solutions of both these derivatives have been irradiated at 313 nm to obtain chemical hydrogels. The hydrogel obtained by irradiating for 15 min an aqueous solution containing 4% w/v of HA-AMA and 4% w/v of PHM resulted in the highest yield. Its swelling ability was dependent on the pH and nature of the external medium. Besides, this hydrogel undergoes a partial hydrolysis, especially in the presence of enzymes, such as esterase or hyaluronidase, but the entity of this degradation is lower than that observed for a hydrogel based on HA-AMA alone. The ability of this hydrogel to entrap drug molecules has been evaluated by using thrombin as a model drug. In vitro release studies and a platelet aggregation test demonstrated that the HA-AMA/PHM hydrogel is able to release thrombin in the active form, thus suggesting its suitability for the treatment of hemorrhages.     

Sodium cholate sorption on cationic dextran hydrogel microspheres. 1. Influence of the chemical structure of functional groups

New hydrogel microspheres based on crosslinked dextran, containing pendant quaternary ammonium groups with different chemical structures have been synthesized and tested as possible bile acid sorbents The in vitro sodium cholate sorption by these hydrogels has been followed in the absence or in the presence of competing anions The sorption results have indicated a strong influence of the chemical structure of functional groups on both the affinity and selectivity towards cholate ions. The best sorption performances were obtained with hydrogels having in the structure of functional groups an alkyl substituent with the length higher than C(8).     

碳化二亚胺对透明质酸进行化学修饰的研究

目的：用水溶性的碳化二亚胺(EDC)作为交联剂对透明质酸(HA)进行化学修饰,研究交联产物(HA-EDC)的物化性质和微观结构。方法：制备HA与EDC的交联产物,对该交联产物进行流变性和热学性能的研究,并对其进行红外光谱、拉曼光谱以及核磁共振的微观结构研究。结果：反应时间和交联剂添加量的不同可以改变交联反应的交联度。不同摩尔比的EDC和HA的交联产物有不同的溶胀性。光谱分析得到交联产物的N-酰脲的结构。结论：可以用碳化二亚胺来对透明质酸进行化学修饰,得到具有很强吸水性并且吸水性可以控制的交联产物,交联反应过程中,产物的结构从不稳定的O-异酰脲转变为稳定的N-酰脲。     

Hemoglobin recognition by imprinting in semi-interpenetrating polymer network hydrogel based on polyacrylamide and chitosan

Semi-interpenetrating polymer network (semi-IPN) hydrogel was prepared to recognize hemoglobin, by molecularly imprinted method, in the mild aqueous media of chitosan and acrylamide in the presence of N,N'-methylenebisacrylamide as the cross-linking agent. The hydrogel obtained has been investigated by using thermal analysis, X-ray diffraction, differential scanning calorimetry (DSC), and environmental scanning electron microscope (ESEM). Langmuir analysis showed that an equal class of adsorption was formed in the hydrogel, and the adsorption equilibrium constant and the maximum adsorption capacity were evaluated to be 4.27 g/mL and 36.53 mg/g wet hydrogel, respectively. The imprinted semi-IPN hydrogel has a much higher adsorption capacity for hemoglobin than the nonimprinted hydrogel with the same chemical composition and also has a higher selectivity for the imprinted molecule.     

Structural Relaxation During Drying and Rehydration of Food Materials��the Water Effect and the Origin of Hysteresis

The state of water in foodstuffs is a guiding principle in food design, and the equilibrium concept of water activity (Aw) is ubiquitous. It is regarded as a primary variable or “hurdle” in preservation technology, and a key variable influencing chemical reaction during storage. However, the amount of water in any system differs as function of water activity depending whether it is determined by water sorption or desorption. Even though this hysteresis behaviour has already been described in the literature, no physical interpretation of its origin has yet been proposed with respect to detailed molecular organisation. This work shows, for two different food powders, gluten and a milk-based product that the hysteresis disappears when either go through their glass transition. A more complete DSC analysis for gluten during different sorption/desorption cycles demonstrates that the hysteresis is dependent on the ageing of the material, which evolves in the glassy state and is induced by structural relaxation.     

Hydration of polymeric components of cartilage--an infrared spectroscopic study on hyaluronic acid and chondroitin sulfate

Hydrated polysaccharides are major constituents of cartilage and play an important role in its water-binding properties. Infrared (IR) spectroscopy and sorption isotherms have been used to investigate the hydration behavior of the glycosaminoglycans hyaluronic acid and chondroitin sulfate. IR-dichroism of the vibrational modes of the pyranose ring is found at relative humidities (RH) smaller than 84%. The IR-dichroism data for the vibrational modes of the pyranose ring have been analyzed with respect to the helical structure of these polysaccharides. The orientation vanishes at higher relative humidities (>84%), because a strong increase in the water uptake occurs in the observed sorption isotherms. Differences in the IR-absorbance of the O-H stretching mode of sorbed water between hyaluronic acid and chondroitin sulfate are shown to be caused by the additional hydration of the sulfate groups. The corresponding H-bonds are weaker than those of the hydration shell of the pyranose rings.     

Compatibility of cells of the nervous system with structured biodegradable chitosan-based hydrogel matrices

Hydrogel matrices for cell cultivation have been generated by two-photon laser polymerization of unsaturated chitosan derivatives and methacrylated hyaluronic acid. The adhesive and toxic properties of the matrices have been assessed, and the matrices have been shown to have a good compatibility with primary hippocampal cell cultures. The formation of morphologically normal neural networks by cells of the nervous system cultured on the surface of hydrogel matrices has been observed. The metabolic status of dissociated hippocampal cells cultured on the matrices was similar to that of the control cultures, as shown by the results of MTT reductase activity assay. Thus, matrices based on unsaturated polysaccharide derivatives crosslinked by laser irradiation showed good compatibility with differentiated cells of the nervous system and considerable potential for use in neurotransplantation.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpyridium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractionated by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and a proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma.

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpirdium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractioned by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin‐based bioink through microextrusion and visible light‐initiated crosslinking

Bioprinting has a great potential to fabricate three‐dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris‐bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC‐laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine.     

Rheological properties of sulfoacetate derivatives of cellulose

Water-soluble cellulose acetate sulfate derivatives (CAS) have been prepared through chemical reaction involving sulfuric acid as a catalyst. These CAS have been obtained from cellulosic materials of different origins (pure cellulose, wheat bran, maize bran) and their rheological behavior in salt-free aqueous solution has been estimated in dilute and semi-dilute regime using dynamic viscoelastic and viscosity measurements. Influence of concentration, temperature of solubilization and temperature of measurement has been investigated. Weak gel-like properties were exhibited at elevated concentration (typically above 7-8 g/L). These systems also exhibited thixotropic properties: the structure was partly broken down upon shearing and recovered at rest. They also displayed thermoreversibility with large hysteresis, the melting temperature being approximately 15 degrees C higher than the temperature at which gelation took place. These overall observations clearly indicate that these distinctive properties arise from intermolecular association of the macromolecular chains of the cellulose derivative.     

Moisture Sorption Isotherms of Various Tobaccos

The equilibrium moisture contents of sun-cured (Kroumougrad), flue-cured (Bright Yellow—4) and air-cured (Burley-21 and Matsukawa) tobaccos were measured over a relative humidity range from 5 to 80% at 20°C. The moisture sorption isotherms of tobaccos were of sigmoid type, and classified into two groups. In a lower humidity range below ca. 40% RH, the A group (Kroumougrad and BY-4) had a smaller moisture sorption capacity than B group (Burley-21 and Matsukawa), while in a higher humidity range above ca. 50% RH the former had a larger moisture sorption capacity than the latter. By extracting with water, the moisture content of BY-4 was increased in the lower humidity range, while it decreased in the higher humidity range. However, the moisture content of Matsukawa was scarecely changed by extracting it with water. These results suggest that the differences in equilibrium moisture content with the type of curing were due to the differences in contents of water soluble com- ponents. To control the hygroscopic properties of a tobacco, therefore, the influences of the addition of sucrose and glycerol on the equilibrium moisture content were quantitatively analysed. The moisture sorption capacity of tobacco was greatly different from its nitrogen sorption capacity. The specific surface area of tobacco calculated from moisture sorption isotherm was ca. 110 times larger than the specific surface area calculated from the nitrogen sorption isotherm. Both the nitrogen and moisture sorption data should be necessary for better understanding of the complicated sorption-desorption phenomena in tobaccos.     

Carbon nanotubes as structural nanofibers for hyaluronic acid hydrogel scaffolds

We have successfully dispersed functionalized single-walled carbon nanotubes (SWNTs) within hyaluronic acid-water solutions. Hybrid hyaluronic acid (HA) hydrogels with SWNTs were then formed by cross-linking with divinyl sulfone. We have found a considerable change in the morphology of the lyophilized hybrid hydrogels compared to HA hydrogels. The high water uptake capacity, an important property of HA hydrogels, remained almost unchanged after 2 wt % SWNT (vs HA) incorporation, despite a dramatic enhancement in the dynamic mechanical properties of the hybrid hydrogels compared to native ones. We have found a 300% enhancement in the storage modulus of hybrid hydrogel with only 2 wt % of SWNTs vs HA (0.06 wt % vs total weight including water content). This apparent contradiction can be explained by a networking effect between SWNTs, mediated by HA chains. As in biological tissue, HA plays a dual role of matrix and linker for the rigid reinforcing nanofibers.     

Antimicrobial properties of fibrous cellulose and other polymeric materials modified with chlorhexidine]

Since fibrous cellulose and other polymeric materials are widely used as dressings, it was of interest to study the mechanism of the antimicrobial action of the products from such materials physically and chemically modified by chlorhexidine, a broad spectrum antiseptic. As vehicles and prolongation agents the following products were used: dressing gauze and cation exchange derivatives of cellulose and starch i.e. monocarboxycellulose (MCC), phosphate cellulose (PC), carboxymethyl cellulose (CMC), monocarboxyl starch (MCS) and alginic acid (AA), a natural compound. It was shown that chemical attachment of chlorhexidine provided a 2-4-fold increase in the antimicrobial effect of the preparations as compared to the use of physical sorption. The antimicrobial effect of the polymeric form of chlorhexidine based on MCC and PC was much higher than that based on MCC, MCS and AA.     

Heparin interacting protein mediated assembly of nano-fibrous hydrogel scaffolds for guided stem cell differentiation

A new methodology is developed to conjugate hyaluronic acid (HA) hydrogel with novel nano-fibrous architectures via non-covalent assembly that specifically allows for targeted adipose-derived stem cells (ASCs) differentiation and soft tissue engineering. The assembly of non-covalently associated hydrogel network produced via the interaction of a low molecular weight heparin (LMWH) modified HA derivative and heparin interacting protein (HIP). The multifunctional star poly(ethylene glycol) (PEG) and HIP copolymer has the capability to mediate the non-covalent assembly of nano-fibrous HA hydrogel networks via affinity interactions with LMWH. The effect of the HIP mediation on in vitro gelation, rheological characteristics, degradation, equilibrium swelling, adipose-derived stem cells (ASCs) proliferation and differentiation of nano-fibrous hydrogel is examined. The results suggest the potential utility of this unique design of the bioactive nano-fibrous HA hydrogel in directing the differentiation of ASCs and adipogenesis in ECM-mimetic scaffolds in vitro. These studies demonstrate that this nano-fibrous HA hydrogel can render the formulation of a therapeutically effective platform for in vitro adipogenesis applications.     

A novel nanocomposite film prepared from crosslinked cellulosic whiskers

Cellulose whiskers are increasingly being used as a reinforcing phase in polymer systems and their use is a growing area of importance in bionanocomposite research. Although the reinforcing effect of cellulose whiskers has been studied in various polymers, the impact of crosslinking cellulose whiskers has not been explored so far. This work deals with the development of novel cellulose nanocomposites, wherein the cellulose nanowhiskers are crosslinked with poly(methyl vinyl ether-co-maleic acid) and poly(ethylene glycol). The morphology of the nanocomposite was studied using atomic force microscopy (AFM), which revealed a network structure embedded in a continuous phase. The water sorption studies demonstrated that the crosslinked nanocomposites are capable of absorbing up to ～900% water and have potential to be used as hydrogels.     

Disulfide cross-linked hyaluronan hydrogels

A new disulfide cross-linking strategy was developed to prepare hyaluronic acid (HA) hydrogel from thiol-modified HA. First, dithiobis(propanoic dihydrazide) (DTP) and dithiobis(butyric dihydrazide) (DTB) were synthesized and then coupled to HA with carbodiimide chemistry. Next, disulfide bonds of the initially formed gel were reduced using dithiothreitol (DTT) to give, after exhaustive dialysis, the corresponding thiol-modified macromolecular derivatives HA-DTPH and HA-DTBH. The degree of substitution of HA-DTPH and HA-DTBH could be controlled from 20% to 70% of available glucuronate carboxylic acid groups. The pK(a) values of the HA-thiol derivatives were determined spectrophotometrically to be pK(a) = 8.87 (HA-DTPH) and pK(a) = 9.01 (HA-DTBH). The thiol groups could be oxidized in air to reform disulfide linkages, which resulted in HA-DTPH and HA-DTBH hydrogel films. Further oxidation of these hydrogels with dilute H(2)O(2) created additional cross-links and afforded poorly swellable films. The disulfide cross-linking was reversible, and films could be again reduced to sols with DTT. Release of blue dextran from cross-linked films was used as a model for drug release. The rapid gelation of the HA-DTPH solution under physiological conditions was also achieved, which demonstrated the capacity for in situ cell encapsulation. Thus, L-929 murine fibroblasts were encapsulated in HA-DTPH hydrogel; these cells remained viable and proliferated during 3 days of culture in vitro.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Effect of cellulose physical characteristics, especially the water sorption value, on the efficiency of its hydrolysis catalyzed by free or immobilized cellulase

Cellulase, an enzymatic complex that synergically promotes the degradation of cellulose to glucose and cellobiose, free or adsorbed onto Si/SiO₂ wafers at 60 °C has been employed as catalyst in the hydrolysis of microcrystalline cellulose (Avicel), microcrystalline cellulose pre-treated with hot phosphoric acid (CP), cotton cellulose (CC) and eucalyptus cellulose (EC). The physical characteristics such as index of crystallinity (I_C), degree of polymerization (DP) and water sorption values were determined for all samples. The largest conversion rates of cellulose into the above-mentioned products using free cellulase were observed for samples with the largest water sorption values; conversion rates showed no correlation with either I_C or DP of the biopolymer. Cellulose with large water sorption value possesses large pore volumes, hence higher accessibility. The catalytic efficiency of immobilized cellulase could not be correlated with the physical characteristics of cellulose samples. The hydrolysis rates of the same cellulose samples with immobilized cellulase were lower than those by the free enzyme, due to the diffusion barrier (biopolymer chains approaching to the immobilized enzyme) and less effective contact between the enzyme active site and its substrate. Immobilized cellulase, unlike its free counterpart, can be recycled at least six times without loss of catalytic activity, leading to higher overall cellulose conversion.     

Characterization of very dense mineral oxide–gel composites for fluidized-bed adsorption of biomolecules

Efficient design of fluidized-bed biomolecule adsorption from crude feed stock requires particles with elevated density, large adsorption capacity and broad chemical stability. Moreover, combinations of small particle diameters with high densities allow for high fluidization velocities while preserving a rapid mass transfer.This approach has been implemented by combining stable porous mineral oxide of high density (2.2, 4.7, 5.7, 9.4 g/ml) with functionalized hydrogels. The cross-linked hydrogel derivative fills the internal porosity of the beads and provides a high equilibrium binding capacity.Various porous mineral oxides (silica, titania, zirconia and hafnia) have been characterized in term of fluidization behavior, surface reactivity and chemical resistance to harsh CIP procedures. Porous zirconia particles were also modified into ion-exchangers by suitable surface modification and intraparticle polymerization of functionalized stable derivatives of acrylic monomers. Back-mixings in fluidized bed columns were analyzed by residence time distribution analysis of inert tracers. 328 and 218 mixing plates per meter were found for respectively, bed expansions of 1.7 and 2.9. The dynamic protein adsorption behaviors of zirconia-based polymeric anion-exchange sorbents were obtained in fluidized-bed, using BSA as model protein. A dynamic binding capacity of 62 mg/ml was observed at a fluidizing velocity of 320 cm/h. These investigations substantiate the favorable physical and chemical characteristics anticipated for dense composite beads for use as fluidized bed adsorbents.