Visible-light-initiated thiol-acrylate photopolymerization of heparin-based hydrogels.

An in situ heparin-based forming hydrogel that cures under visible-light is formulated using eosin Y as a photoinitiator with triethanolamine as an electron donor to initiate reaction of thiolated-heparin with acrylate-ended poly(ethylene glycol). Formulations and irradiation conditions are presented for control of heparin content (1.6 to 3.3% w/v), modulus (100-10,000 Pa), and gelation time (30-600 s). Encapsulation of 3T3 fibroblasts in the hydrogel gave over 96% viability for all conditions examined. In vitro characterization of epidermal growth factor released from the hydrogel confirmed that the growth factor remains bioactive. The ability to deliver growth factors, fast gelation kinetics under visible light, and independent control of physical and biochemical properties makes this system a promising candidate for use in regenerative medicine. In particular, irradiation conditions that achieve gelation in 150s are compatible with the stringent light exposure limits of the retina, which affords a wide safety margin for use with other tissues.

In vitro haemocompatibility and stability of two types of heparin-immobilized silicon surfaces.

Heparin was covalently immobilized onto a silicon surface by two different methods, carbodiimide-based immobilization and photo-immobilization. In the former method, a (3-aminopropyl) trimethoxysilane (APTMS) self-assembled monolayer (SAM) or multilayer was first coated onto the silicon surface as the bridging layer, and heparin was then attached to the surface in the presence of water-soluble carbodiimide. In the latter method, an octadecyltrichlorosilane (OTS) SAM was coated on the silicon surface as the bridging layer, and heparin was modified by attaching photosensitive aryl azide groups. Upon UV illumination, the modified heparin was then covalently immobilized onto the surface. The hydrophilicity of the silicon surface changed after each coating step, and heparin aggregates on APTMS SAM and OTS SAM were observed by atomic force microscopy (AFM). In vitro haemocompatibility assays demonstrated that the deposition of APTMS SAM, APTMS multilayer and OTS SAM enhanced the silicon's haemocompatibility, which was further enhanced by the heparin immobilization. There is no evident distinction regarding the haemocompatibility between the heparin-immobilized surfaces by both methods. However, heparin on silicon with APTMS SAM and multilayer as the bridging layers is very unstable when tested in vitro with a saline solution at 37 degrees C, due to the instability of APTMS SAM and multilayer on silicon. Meanwhile, photo-immobilized heparin on silicon with OTS SAM as the bridging layer showed superb stability.

Photoinduced dissociation of heparin-derived oligosaccharides controlled by charge location.

The development of strategies based on mass spectrometry to help for deep structural analysis of acidic oligosaccharides remains topical. We thus examined the dissociation behavior of deprotonated ions of heparin-derived di- to tetra-saccharides under UV irradiation at 220 nm. Depending on the ionization state of the carboxylic groups, an oxidized species issued from electron photodetachment was observed in complement to photoinduced fragmentation of precursor ions. The influence of the charge location in the oligosaccharide dianions on the balance between photodissociation and electron photodetachment is examined and a way to direct the relaxation pathways, (i.e., dissociation versus electron detachment), is proposed using sodium adducts. The oxidized species was subjected to activated-electron photodetachment (activated-EPD) leading to complementary informative fragment ions to those issued from photodissociation. Directed photoinduced dissociation at 220 nm and activated-EPD should complement the more conventional CAD and IRMPD activation modes for deeper structural analysis of acidic oligosaccharides-derived anions.

Wavelength-tunable ultraviolet photodissociation (UVPD) of heparin-derived disaccharides in a linear ion trap.

A set of three heparin-derived disaccharide deprotonated ions was isolated in a linear ion trap and subjected to UV laser irradiation in the 220-290 nm wavelength range. The dissociation yields of the deprotonated molecular ions were recorded as a function of laser wavelength. They revealed maximum absorption at 220 nm for the nonsulfated disaccharide, but centered at 240 nm for the sulfated species. The comparison of the fragmentation patterns between ultraviolet photodissociation (UVPD) at 240 nm and CID modes showed roughly the same distribution of fragment ions resulting from glycosidic bond cleavages. Interestingly, UVPD favored additional cross ring cleavages of A and X type ion series enabling easier sulfate group location. It also reduced small neutral losses (H(2)O).

Interaction of ethidium bromide with heparin.

The reactions of hydrated electrons produced during pulse radiolysis have been utilized to investigate the binding of ethidium bromide to heparin. Complexes of ethidium bromide and heparin can be dissociated with salt. Divalent cations are more effective than monovalent cations in this respect. Pulse-radiolysis investigations at different temperatures indicate that the thermodynamic parameters governing the interaction of ethidium bromide with heparin are deltaH' = 11-6 kcal mole-1 and deltaS' = 42-6 cal deg-1 mole-1.

Effect of gamma radiation on the molecular weight and the anticoagulant activity of heparin.

4 g% aqueous solutions of heparin were irradiated with the gamma radiation doses of 4.6 x 10(5) or 9.2 x 10(5) rads. The irradiated and also the non-irradiated heparin samples were fractionated using a Sephadex G-200 column. With radiation, the peak of the molecular weight distribution curves shifted toward the lower molecular weight. Also, the number average molecular weight decreased by 8.2 and 11.5% with the doses of 4.6 x 10(5) and 9.2 x 10(5) rads, respectively. The anticoagulant activity depended on the molecular weight of the heparin fractions. For the heparin fractions with molecular weights below 7,900, the anticoagulant activity decreased with radiation. Thus, for a heparin fraction with a molecular weight of 6,200, the anticoagulant activity decreased from 211 to 198 IU/mg after 4 h of irradiation.

[Effect of laser irradiation on the serological and morphological indices of experimental immune reactivity (a preliminary report)]. / Vliianie na lazernoto obluchvane vurkhu niakoi serologichni i morfologichni pokazateli na imunnata reaktivnost v eksperiment (predvaritelno suobshtenie).

The authors examined guinea pigs, irradiated for a period of six days with helium-neon lazer according to a scheme, proposed by them. They determined the changes in the complement serum activity, blood level of heparine and histamine as well as histologic changes in adrenals, liver and spleen. When they compared the control animals (nonirradiated) with the experimental animals, they established a statisticaly significant elevation of the complement activity in serum, a tendency to lowering of histamine with an increase of heparine and hyperplastic reaction in zona glomerulosa of the adrenals. The obtained results suggested stimulation of immune mechanisms with antiallergic and antinflammatory effect.

The role of ultraviolet irradiation and heparin-binding epidermal growth factor-like growth factor in the pathogenesis of pterygium.

Ultraviolet (UV) light is one of the major factors implicated in the pathogenesis of pterygium. The mechanism by which UV light induces this disease remains elusive. The aim of this study was to evaluate the effects of UVB irradiation on the expression of growth factors in cultured pterygium epithelial cells and to demonstrate their distribution within pterygium. We cultured pterygial epithelial cells from pterygium explants and these cells were exposed to 20 mJ/cm(2) of UVB. Total RNA was extracted at 0, 6, and 12 hours after irradiation. (32)P-labeled cDNA was synthesized and analyzed using microarray technology to determine the differential expression of 268 growth factor and cytokine related genes. Semiquantitative reverse transcriptase-polymerase chain reaction was used to corroborate this data. Conditioned media derived from cells exposed to UVB irradiation was analyzed for protein expression by enzyme-linked immunosorbent assay. Immunohistochemistry was used to evaluate the distribution of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pterygium tissue. Analysis of the hybridization signals revealed that the genes encoding HB-EGF, fibroblast growth factor 3, and cytotoxic trail ligand receptor were consistently elevated at 6 and 12 hours after UVB treatment. HB-EGF mRNA was elevated 6.8-fold at 6 hours after irradiation and was augmented in culture supernatants after the same treatment. Furthermore, HB-EGF reactivity was identified in the epithelium and vasculature of pterygium by immunohistochemistry. HB-EGF was present in normal limbal epithelium, although it was not induced in cultured limbal epithelial cells by UV irradiation. HB-EGF is a potent mitogen, localized in pterygium tissue, and significantly induced by UVB in pterygium-derived epithelial cells. We postulate that this growth factor is a major driving force in the development of pterygia and a means by which UV irradiation causes the pathogenesis of pterygium.

Free electron laser induces specific immobilization of heparin on polysulfone films.

Covalent immobilization of heparin has been developed to reduce the amount of heparin administered systematically during long-term dialysis. Recently, it was doubted partially because of the complexion during immobilization process. In this study, we investigated a novel method for specific immobilization of heparin on polysulfone (PSF) via free electron laser (FEL) irradiation. Laser wavelengths of 6.18 or 6.31 microm, the typical absorption bands of carboxyl groups of heparin and aromatic rings in PSF, respectively, were chosen to irradiate the thin heparin membrane formed on PSF surfaces. The amount of heparin immobilized on PSF was measured by the toluidine blue method. The binding of heparin on PSF was analyzed by X-ray photoelectron spectroscopy (XPS). The immobilization of heparin resulted in a hydrophilic surface on which decreased platelet adhesion was observed. The efficiency differences, depending on laser wavelengths, were discussed from the point of view of structural and environmental differences of light-absorbing groups.