Synthesis and physicochemical and dynamic mechanical properties of a water-soluble chitosan derivative as a biomaterial

The physicochemical and rheological properties of a water-soluble chitosan (WSC) derivative were characterized in order to facilitate its use as a novel material for biomedical applications. The WSC was prepared by conjugating glycidyltrimethylammonium chloride (GTMAC) onto chitosan chains. Varying the molar ratio of GTMAC to chitosan from 3:1 to 6:1 produced WSCs with a degree of substitution (DS) that ranged from 56% to 74%. The WSC with the highest DS was soluble in water up to concentrations of 25 g/dL at room temperature. An increase in the polymer concentration gradually increased both the pH and conductivity of the WSC solutions. The rheological properties of the WSC solutions were found to be dependent on the salt and polymer concentrations as well as the DS value. In the absence of salt, the rheological behavior of the WSC was found to be typical of that for a polyelectrolyte in the dilute solution regime. However, the addition of salt decreased the viscosity of the polymer solution due to the reduction of electrostatic repulsions by the positively charged trimethylated ammonium groups of the WSC. In the concentrated regime, the viscosity of the WSCs was found to follow a power-law expression. The lowest DS WSC had the more favorable viscoelastic properties that were attributed to its high molecular weight, as confirmed by the stress relaxation spectra and intrinsic viscosity measurements. The effect of DS on the degree of interaction between WSC and the lipid egg phosphatidylcholine was investigated by FTIR analysis. Overall, the lower DS WSC had enhanced rheological properties and was capable of engaging in stronger intermolecular physical interactions.     

Vortex shedding as a mechanism for free emboli formation in mechanical heart valves

The high incidence of thromboembolic complications of mechanical heart valves (MHV) limits their success as permanent implants. The thrombogenicity of all MHV is primarily due to platelet activation by contact with foreign surfaces and by nonphysiological flow patterns. The latter include elevated flow stresses and regions of recirculation of blood that are induced by valve design characteristics. A numerical simulation of unsteady turbulent flow through a bileaflet MHV was conducted, using the Wilcox k-omega turbulence model for internal low-Reynolds-number flows, and compared to quantitative flow visualization performed in a pulse duplicator system using Digital Particle Image Velocimetry (DPIV). The wake of the valve leaflet during the deceleration phase revealed an intricate pattern of interacting shed vortices. Particle paths showed that platelets that were exposed to the highest flow stresses around the leaflets were entrapped within the shed vortices. Potentially activated, such platelets may tend to aggregate and form free emboli. Once formed, such free emboli would be convected downstream by the shed vortices, increasing the risk of systemic emboli.     

Triplet exciton formation as a novel photoprotection mechanism in chlorosomes of Chlorobium tepidum

Chlorosomes comprise thousands of bacteriochlorophylls (BChl c, d, or e) in a closely packed structure surrounded by a lipid-protein envelope and additionally contain considerable amounts of carotenoids, quinones, and BChl a. It has been suggested that carotenoids in chlorosomes provide photoprotection by rapidly quenching triplet excited states of BChl via a triplet-triplet energy transfer mechanism that prevents energy transfer to oxygen and the formation of harmful singlet oxygen. In this work we studied triplet energy transfer kinetics and photodegradation of chlorosomes isolated from wild-type Chlorobium tepidum and from genetically modified species with different types of carotenoids and from a carotenoid-free mutant. Supporting a photoprotective function of carotenoids, carotenoid-free chlorosomes photodegrade approximately 3 times faster than wild-type chlorosomes. However, a significant fraction of the BChls forms a long-lived, triplet-like state that does not interact with carotenoids or with oxygen. We propose that these states are triplet excitons that form due to triplet-triplet interaction between the closely packed BChls. Numerical exciton simulations predict that the energy of these triplet excitons may fall below that of singlet oxygen and triplet carotenoids; this would prevent energy transfer from triplet BChl. Thus, the formation of triplet excitons in chlorosomes serves as an alternative photoprotection mechanism.     

NO induced novel DNA lesions: formation mechanism.

2'-Deoxyguanosine was treated with NO/O2 mixture at pH 7.0-7.8, and as well as the known major products such as 2'-deoxyxanthosine and 2'-deoxyoxanosine, some unidentified products were detected by RP-HPLC. In the present study, one of them has been characterized as a novel lesion, N2-nitro-2'-deoxyguanosine by spectrometric and chromatographic analysis. The mechanism for the production is also discussed.     

Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri = 200) and is complete for Ri = 50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.     

Methacrylated glycol chitosan as a photopolymerizable biomaterial

Glycol chitosan is a derivative of chitosan that is soluble at neutral pH and possesses potentially useful biological properties. With the goal of obtaining biocompatible hydrogels for use as tissue engineering scaffolds or drug delivery depots, glycol chitosan was converted to a photopolymerizable prepolymer through graft methacrylation using glycidyl methacrylate in aqueous media at pH 9. N-Methacrylation was verified by both (1)H NMR and (13)C NMR. The degree of N-methacrylation, measured via (1)H NMR, was easily varied from 1.5% to approximately 25% by varying the molar ratio of glycidyl methacrylate to glycol chitosan and the reaction time. Using a chondrocyte cell line, the N-methacrylated glycol chitosan was found to be noncytotoxic up to a concentration of 1 mg/mL. The prepolymer was cross-linked in solution using UV light and Irgacure 2959 photoinitiator under various conditions to yield gels of low sol content ( approximately 5%), high equilibrium water content (85-95%), and thicknesses of up to 6 mm. Cross-polarization magic-angle spinning (13)C solid state NMR verified the complete conversion of the double bonds in the gel. Chondrocytes seeded directly onto the gel surface, populated the entirety of the gel and remained viable for up to one week. The hydrogels degraded slowly in vitro in the presence of lysozyme at a rate that increased as the cross-link density of the gels decreased.     

Calpain as a novel regulator of autophagosome formation

Ubiquitously expressed micro- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene. The micro- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Therefore we investigated the involvement of calpains in autophagy using MEFs derived from Capns1 knockout mice and Capns1 depleted human cells as model systems. We found that autophagy is impaired in Capns1-deficient cells by immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments. Accordingly, the enhancement of lysosomal activity and long-lived proteins degradation, normally occurring upon starvation, are also reduced. In Capns1-depleted cells ectopic LC3 accumulates in early endosome-like vesicles that might represent a salvage pathway for protein degradation when autophagy is defective.     

Hsp70 chaperone-based gel composition as a novel immunotherapeutic anti-tumor tool

The recent advances in designing Hsp70-based anti-cancer vaccines and the ability of the chaperone to penetrate inside a living cell prompted us to develop a non-invasive method for the treatment of surface tumors. We designed hydrogel-containing gel-forming substances and human recombinant Hsp70 and applied them on the surface of a 7-day-old B16F10 melanoma tumor. According to the results of histochemistry, Hsp70 diffused through skin layer inside the B16 tumor, and this transport was proved by biochemical data. The application of Hsp70 gel reduced the rate of tumor growth by 64 % and prolonged the life of animals by 46 %. Increased survival was correlated with the enhancement of B16-specific cytotoxicity and up-regulation of gamma–interferon production. Taken together, the data confirm the anti-tumor effect of pure recombinant Hsp70 delivered intratumorally and demonstrate the relevance of a novel non-invasive technology of Hsp70-based therapy.     

Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.     

Photoresponsive PEG-anthracene grafted hyaluronan as a controlled-delivery biomaterial

Ophthalmic drug delivery to the posterior segment of the eye could benefit from a responsive controlled drug delivery system with light or laser inducible changes. For example, the delivery of age-related macular degeneration drugs requires invasive monthly injections making long-term photoresponsive drug delivery a desirable option. The feasibility of this may be facilitated by both the transparency of the eye and the advanced technology in ophthalmic lasers. Hyaluronic acid photogels that are compatible with retinal pigment epithelial cell lines are shown here to deliver a variety of small and large model drugs over the long term (months). Varying UV exposures resulted in decreases/increases or the turning off and on of delivery, potentially allowing the therapy to be tailored to suit the patient and the disease.     

Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers

Elastin and silk spidroins are fibrous, structural proteins with elastomeric properties of extension and recoil. While elastin is highly extensible and has excellent recovery of elastic energy, silks are particularly strong and tough. This study describes the biophysical characterization of recombinant polypeptides designed by combining spider wrapping silk and elastin‐like sequences as a strategy to rationally increase the strength of elastin‐based materials while maintaining extensibility. We demonstrate a thermo‐responsive phase separation and spontaneous colloid‐like droplet formation from silk‐elastin block copolymers, and from a 34 residue disordered region of Argiope trifasciata wrapping silk alone, and measure a comprehensive suite of tensile mechanical properties from cross‐linked materials. Silk‐elastin materials exhibited significantly increased strength, toughness, and stiffness compared to an elastin‐only material, while retaining high failure strains and low energy loss upon recoil. These data demonstrate the mechanical tunability of protein polymer biomaterials through modular, chimeric recombination, and provide structural insights into mechanical design. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 693–703, 2016.     

Chalcone embedded polyurethanes as a biomaterial: Synthesis, characterization and antibacterial adhesion

An antibacterial dimethylamino-chalcone embedded multiblock copolymer (PCL-PEG) was synthesized and characterized using FT-IR, ¹H NMR, SEM and SEC and the compound was characterized using FT-IR, ¹H NMR, and ¹³C NMR. A 10% copolymer composite was prepared and casted as film to be used as a biomaterial and the copolymer films without the compound acted as control. TGA, DSC, AFM, SEM and EDAX analysis were performed for the above samples. Surface roughness (R_a) of the copolymer composite film was less when compared to the copolymer film which indicated the proper distribution of chalcone in the composite film. copolymer composite film was hydrophilic compared to copolymer film. Antibacterial adhesion studies were performed for copolymer composite polymer film and evaluated using CFU measurement and SEM analysis. Copolymer composite film shows promising antibacterial adhesion compared to the copolymer film. Hence the copolymer composite film can be used as a new biomaterial endowed with antibacterial properties.     

Abeta-polyacrolein aggregates: novel mechanism of plastic formation in senile plaques

High levels of acrolein (H2C=HC-CH=O) occur in Alzheimer's brain. Amyloid-beta (Abeta) peptide co-localizes with acrolein presumably due to Abeta-induced lipid peroxidation. Focal production of acrolein may yield a transient elevation in the concentration of acrolein that may be susceptible to polymerization via basic latex polymer chemistry. Following incubation of Abeta with acrolein (16-750 mM), we observed the formation of thin plastic fragments that were extensively punctuated. Planar aggregates stained for protein and for cross-beta structures suggesting an Abeta-polyacrolein colloidal mixture. Depending on acrolein concentration and incubation time, we observed uniformly sized planar aggregates (approximately 10 microm2) or monolayers (>100 mm2) of thin polyacrolein films embedded with Abeta oligomers. The ability of Abeta to catalyze the polymerization of acrolein is likely due to Abeta's surfactant and redox properties. These observations suggest that plastic in the form of Abeta-polyacrolein latexes may exist in neural tissue contributing to the pathogenesis of Alzheimer's disease.     

Fish skin as a biomaterial for halal collagen and gelatin

Around 40% of the total catch weight of fish is regarded as byproducts, consisting of skin, fins, bones, scales, viscera, etc. The utilization of these byproducts is important to increase their commercial values as well as to prevent environmental pollution. Meanwhile, nowadays, it is getting a global trend to provide foods and other industrial materials which have been accredited as halal products for Moslem communities. As a way of processing fish byproducts to meet the halal criteria, preparation of collagen and gelatin would be useful to fulfill the market demand. As a result of screening studies on fishery byproducts, fish skin has been found to be the good source for halal collagen and gelatin, which show satisfactory quality compared with those from bovine sources which could cause bovine spongiform encephalopathy (BSE).     

Sol–gel synthesis,characterization and luminescence properties of SrMgAl2SiO7:Eu2+ as a novel nanocrystalline phosphor

In this research, a new SrMgAl₂SiO₇:Eu²⁺ phosphor was synthesized via the sol–gel method. The phase‐forming processes were studied by thermogravimetric–differential thermal analysis and X‐ray diffraction technique. Scanning electron microscopy showed that there is uniform morphology and microstructure owing to the sol–gel route. Spectrophotometry and colorimetry analyses illustrated that, under short ultraviolet excitation, the main emission peak occurred at 421 nm and also a relatively pure blue color was observed that was ascribed to the 4f⁶5d¹(²D) → 4f⁷(⁸S_7/2) transition of Eu²⁺ with color coordination of x = 0.187, y = 0.077. Finally, it was found that the color and phase purity of the synthesized powder increased as calcinations time increased. Copyright © 2010 John Wiley & Sons, Ltd.     

Ibuprofen as a chemesthetic stimulus: evidence of a novel mechanism of throat irritation

This paper reports a study of the oral and pharyngeal chemesthetic effects of the non-steroidal anti-inflammatory drug (NSAID) ibuprofen [2-(4-isobutylphenyl)propanoic acid], which pilot experiments had indicated produces an unusual sensory irritation of the throat. In experiment 1 subjects swallowed aqueous solutions of ibuprofen prepared with different buffering agents and gave ratings of irritation and taste in the mouth and throat. The results showed that ibuprofen irritates the throat much more than the mouth, and that its quality in the throat is characterized primarily as sting/prick, itch and tickle (often leading to cough). Based upon the results obtained with the different buffering agents, we hypothesized that the sting/prick/itch qualities of throat irritation were pH-dependent. Parametric manipulation of solution pH in experiment 2 confirmed this hypothesis. The same experiment revealed that, in contrast to other oral irritants (e.g. capsaicin and menthol), repeated stimulation caused neither sensitization nor desensitization of throat irritation. In the final experiment we found that ibuprofen's throat irritation could not be modulated by temperature, as it should be if stimulation occurred via capsaicin-sensitive receptors. We therefore conclude that ibuprofen has novel chemesthetic properties, which are not mediated by capsaicin-sensitive (vanilloid) receptors, and that a major component of the throat irritation it produces occurs via a pH-dependent receptor mechanism.     

Determination of the number of cross-links in a protein gel from its mechanical and swelling properties

The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.