Photochemical Cross-linking Research Articles

Effects of photochemical crosslinking on the microstructure of collagen and a feasibility study on controlled protein release

Photochemical crosslinking is an emerging technique able to modify the physicochemical properties of collagen. However, whether this technique can be used to modify collagen-based structures for drug delivery has not been studied. This study demonstrated that the microporous structure of photochemically crosslinked collagen was affected by rose Bengal and laser energy level. Using the optimized process parameters, the authors fabricated photochemically crosslinked collagen structures encapsulated with sample proteins and demonstrated that photochemical crosslinking reduced the initial burst effect and protein release without compromising the protein bioactivity. The fiber meshwork in collagen structures was also characterized, and it was found that photochemical crosslinking did not significantly alter the mesh size. This study reports the effects of photochemical crosslinking on the microstructure of collagen structures and suggests the feasibility of using photochemically crosslinked collagen structures for controlled protein release.

Aliphatic β-nitro alcohols for non-enzymatic collagen cross-linking of scleral tissue

The success of riboflavin photochemical cross-linking of the cornea in treating keratoconus and post-surgical keratectasia has prompted interest in cross-linking scleral tissue with a potential application to stabilize myopic progression. Applying an UVA light source to the sclera is difficult, particularly in the posterior region. An alternate pharmacologic approach to scleral cross-linking may be possible. The present study was undertaken in order to identify nitrite related compounds capable of inducing scleral tissue cross-linking and to gain information regarding the possible chemical mechanisms involved. 8 × 4 mm strips of porcine and human sclera were incubated in various concentrations of nitrite related agents (1–100 mM) at 37 °C. pH 7.4 was used for all experiments except those involving NaNO 2. Following a 24–96 h incubation period, the samples were tested for cross-linking effects using thermal shrinkage temperature ( T s) analysis. Several compounds were studied including NaNO 2, 2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol, 2-nitrophenol, 2-nitroethane, 2-aminoethanol, isopentyl nitrite, DPTA/NO, DETA/NO, and urea, a nitrous acid trap. The results indicate that short chain aliphatic β-nitro alcohols (2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-2-pentanol) are particularly effective cross-linking agents at pH 7.4, showing both time and concentration dependent effects. Furthermore, nitrosation does not appear to induce tissue cross-linking. In conclusion, aliphatic β-nitro alcohols can cross-link scleral tissue at physiologic pH and temperature. Since β-nitro alcohols are known to have reasonable toxicity profiles, these agents could find utility as pharmacologic cross-linking agents for scleral thinning disease.

Fabrication of nano-fibrous collagen microspheres for protein delivery and effects of photochemical crosslinking on release kinetics

Protein compatibility is important for protein drug delivery using microsphere-based devices. Collagen has excellent protein compatibility but has poor mechanical stability for microsphere fabrication and open meshwork for controlled release. In this study, a protein-compatible fabrication method for injectable collagen microspheres has been developed. The surface morphology, interior microstructure and protein release characteristics of collagen microspheres were investigated. Moreover, effects of photochemical crosslinking on these characteristics were also studied. Finally, the mechanisms governing the protein release and the retention of protein bioactivity were studied. Stable and injectable collagen microspheres consisting of nano-fibrous meshwork were successfully fabricated under ambient conditions in an organic solvent and crosslinking reagent-free manner. These microspheres have open meshwork and showed large initial burst and rapid release of proteins. Photochemical crosslinking significantly reduced the initial burst effect and controlled the protein release in a photosensitizer dose-dependent manner without significantly altering the mesh size. We further demonstrated that there was significantly higher protein retention within the photochemically crosslinked collagen microspheres as compared with the uncrosslinked, suggesting a secondary retention mechanism. Lastly, both surfactant treatment and photochemical crosslinking did not compromise the bioactivity of the encapsulated proteins. In summary, this study reports a novel collagen microsphere-based protein delivery system and demonstrates the possibility to use photochemical crosslinking as the secondary retention mechanism for proteins.

Autosensitized oxidation of glycated bovine lens proteins irradiated with UVA-visible light at low oxygen concentration

Advanced glycation endproducts (AGEs) are generated in lens proteins following ascorbic acid glycation. Autosensitized photo-processes induced by these AGEs exposed to UVA-visible light at low oxygen concentrations, such as those found in the eye lens, were studied in order to establish their photo-damaging potential. Ascorbic acid glycated bovine lens proteins, irradiated with UVA and/or visible light at 5% oxygen concentration showed an increase in the amount of carbonyl groups and an increase in the emission of chemiluminescence. Both are indicators of the occurrence of amino acid oxidation within the proteins. A good correlation was found between the chemiluminescence intensity and the extent of the glycation process. No modifications in the SDS-PAGE patterns were observed when 6-day glycated proteins were irradiated with UVA-visible light at low oxygen pressure, excluding the occurrence of photochemical crosslinking. When the same samples were derivatized with 2,4-dinitrophenylhydrazine and analyzed by Western blot, using polyclonal antibodies to the DNP moiety, an increase in the carbonyl content restricted to the AGE-crosslinked proteins fractions was observed. These results indicate that photo-oxidations are limited to the nearby environment of the sensitizers. On the contrary, when native lens proteins were irradiated in the presence of small molecular weight models of AGEs, prepared by incubating ascorbate with Nalpha-acetylated lysine and arginine, the occurrence of both photochemically induced crosslinking and oxidative processes were observed, emphasizing the importance of the localization of the sensitizer. Taken together, these results provide information with respect to the actual photosensitizing ability of endogenous AGEs to induce photo-oxidative processes in lens proteins.

Photochemical molecular imprinting of cholesterol

Cinnamoylated photocrosslinkable cyclodextrin derivatives (BCC) were synthesized by the substitution of β-cyclodextrin (β-CD) with cinnamoyl chloride (CC) and crosslinked with either hexamethylenediisocyanate (HMDI) or toluenediisocyanate (TDI). Cyclodextrin rings were substituted with one or two cinnamoyl moieties, as found from mass spectrometry. The polymeric matrix with cholesterol molecular imprint was obtained on irradiation of molecular assembly formed by the cinnamoyl-functionalized β-cyclodextrin-cholesterol with light at 275 nm, absorbed exclusively by the cinnamoyl chromophores. Irradiation induced crosslinking due to the photodimerization of the cinnamoyl moieties. To determine the adsorption properties of the produced material imprinting was performed in the presence of tritiated cholesterol and the intensity of β radiation from the material was measured. The materials obtained by the adsorption of tritiated cholesterol by nonirradiated polymer were used as controls. It was found that the polymer photocrosslinked in the presence of cholesterol have shown a considerable higher adsorption capacity for cholesterol than the control materials. This confirmed successful formation of molecularly imprinted polymer (MIP) by photochemical crosslinking. The selectivity of imprinting was also confirmed using compounds of similar structures, i.e. ergosterol, dehydroergosterol, and Vitamin D.

PH-Sensitive hydrogels of dextran: Synthesis, characterization and in vivo studies

pH-Sensitive hydrogels of dextran were synthesized by photochemical cross-linking reaction of methacrylate dextran (DEX-MA) at different derivatization degree, functionalized with acidic residues through reaction with phthalic anhydride. The hydrogels were characterized by FT-IR spectra, swelling measurements, experiments of chemical and enzymatic hydrolyses. The swelling data agreed with the formation of networks having pH-sensitive behaviour. This property was confirmed by the morphological examination performed by scanning electron microscopy on samples maintained in media at different pH. (S)-4-Isobutyl-2-phenylpropionic acid (ibuprofen) was loaded into the polymeric matrices. The analysis of the release profiles of the drug from the three networks showed that all the matrices were able to retain ibuprofen during the transit through the stomach, releasing it in a sustained way in the intestinal tract at a rate strictly dependent on the derivatization degree in methacrylic groups. In vivo studies verified the biocompatibility of the materials. Moreover, when the matrix loaded with ibuprofen was administered to rats, it was able to protect them from the ulcerogenic effects of the drug.

Transcription Factor E Is a Part of Transcription Elongation Complexes

A homologue of the N-terminal domain of the alpha subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helix-turn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions -9 and -11. In complexes stalled at +20, TFE cross-linked specifically to positions +9, +11, and +16 of the non-template strand. Analyses of transcription complexes stalled at position +20 revealed a TFE-dependent increase of the resumption efficiency of stalled RNA polymerase and a TFE-induced enhanced permanganate sensitivity of thymine residues in the transcription bubble. These results demonstrate the presence of TFE in early elongation complexes and suggest a role of TFE in stabilization of the transcription bubble during elongation.

Non-destructive mechanical characterisation of UVA/riboflavin crosslinked collagen hydrogels

Aims:To establish a non-destructive method of characterising the mechanical properties of collagen hydrogels to model corneal tissue and to examine the effect of photochemical crosslinking on their mechanical properties.Methods:Collagen hydrogels...

(Photo)crosslinkable Smectic LC Main‐Chain Polymers

AbstractThis paper presents the synthetic route to SmA LC main‐chain polymers, that can be (photo)crosslinked without solvent in the bulk phase. They are based on soluble polymalonates, in which higher ordered phases can be suppressed by copolymerization with a laterally brominated biphenyl. Two routes were developed to incorporate the crosslinkable groups into the polyester backbone. The first consists in the incorporation of phenols into the polyester. These phenols are not reactive enough to participate in the transesterification reaction used to build up the polymer, but they can be esterified afterwards with acrylates. Thermally or photochemically created radicals then start the crosslinking. The second route is based on the incorporation of benzophenone as side group. It allows a photochemical crosslinking. Crosslinked fibers (monodomains) show the potential of the smectic LC main‐chain elastomers as actuators.magnified image

Archaeal Transcription: Function of an Alternative Transcription Factor B from Pyrococcus furiosus

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.

Photoinduced Surface Alignment for Optical Thin Films and Liquid Crystal Displays

Over the past few years, photoinduced surface alignment of liquid crystal layers has evolved into a mature technology which enables the production of high-quality liquid crystal displays and functional optical thin films. Owing to its versatile possibilities to control the azimuthal and polar orientation of liquid crystals without mechanical intervention, photoalignment (PA) brings along considerable advantages over alternative alignment methods and opens up a plethora of novel applications. Improved optical performances, high production throughputs and yield increases are additional benefits of this technology. PA materials undergoing anisotropic photochemical crosslinking reactions have now reached technological maturity and are entering the market on a large commercial scale.

3535 POSTER Phase II trial investigating the efficacy and safety of sunitinib in patients with unresectable hepatocellular carcinoma (HCC)

Intra-disc injection of mesenchymal stem cells (MSCs) to treat disc degeneration may lead to unfavorable complications, particularly osteophyte formation. Development of an effective method to block the injection portal, prevent the leakage of injected cells and materials and, hence, prevent osteophyte formation is of the utmost importance before MSC-based therapies can be applied in a clinical setting. Here we seek to alleviate the cell leakage problem and the associated complication osteophyte formation by developing an injectable annulus plug to block the injection portal during intra-disc delivery. Specifically, we fabricated a needle-shaped collagen plug by photochemical crosslinking and successfully delivered it intra-discally, in association with MSCs in collagen microsphere carriers, using a custom-made delivery device. The mechanical performance of the plug and its effectiveness in reducing cell leakage were evaluated ex vivo under compression and in torsion push-out tests. The results demonstrate that the plug survived physiologically relevant loadings and significantly reduced leakage and enhanced retention of the injected materials. Finally, a pilot in vivo study in rabbits was conducted to evaluate the performance of the plug. Microcomputed tomography imaging and histology revealed that the plug significantly reduced osteophyte formation. This work suggests the potential of the annulus plug as an adjunct or annulus closure device for intra-disc delivery of cells and materials.

Study of Bacteriophage T4-encoded Dam DNA (Adenine-N6)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein–DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.

Comparison of Femtosecond Laser and Continuous Wave UV Sources for Protein–Nucleic Acid Crosslinking

Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.

Correlation between heparin release and polymerization degree of organically modified silica xerogels from 3-methacryloxypropylpolysilsesquioxane

This work aimed to investigate the use of an organically modified porous silica matrix (poly(methacryloxypropyl)–poly(silsesquioxane); P-MA–PS) as a release system for heparin. The matrices were obtained from methacryloxypropyltrimethoxysilane (MAS) via the sol–gel process under acidic conditions following photochemical polymerization and cross-linking of the organic matrix. Modulation of the polymerization degree of the organic matrix in the range 0–71% allowed adjusting the release kinetics of heparin according to therapeutic needs. It was demonstrated that higher drug loads and a decreasing polymerization degree resulted in a faster release profile of heparin, which followed a square root of time kinetic according to the Higuchi model. The hydrolytic degradation of hybrid xerogel was found to follow a zero-order kinetic whereas the heparin concentration did not show an influence on the degradation rate of the matrix. Since the released heparin retained its biological activity, the P-MA–PS matrices are of clinically interest, e.g. as coating on drug eluting coronary stents.

Surface Imprinting in Layer‐by‐Layer Nanostructured Films

AbstractIn this Full Paper, we develop a novel approach for the generation of stable molecularly imprinted sites in polymeric films by combining the layer‐by‐layer (LbL) technique and photochemical crosslinking of the layered structure. After photo‐crosslinking, the imprinted films show high reproducibility and rapid loading and unloading of imprinted sites by the template molecules. Moreover, the competitive adsorption of template molecules and redox labels into the imprinted film using electrochemical methods indicates that the imprinted film has higher affinity for template molecules. We believe this approach may have some advantages over traditional ways of preparing imprinted sites in polymer matrices and it may open a new avenue for the functionalization of LbL films.

Photochemical Cross-Linking for Collagen-Based Scaffolds: A Study on Optical Properties, Mechanical Properties, Stability, and Hematocompatibility

Photochemical Cross-Linking for Collagen-Based Scaffolds: A Study on Optical Properties, Mechanical Properties, Stability, and Hematocompatibility

Crosslinked sulfonated poly(ether ether ketone) proton exchange membranes for direct methanol fuel cell applications

In the present study, a series of the crosslinked sulfonated poly(ether ether ketone) (SPEEK) proton exchange membranes were prepared. The photochemical crosslinking of the SPEEK membranes was carried out by dissolving benzophenone and triethylamine photo-initiator system in the membrane casting solution and then exposing the resulting membranes after solvent evaporation to UV light. The physical and transport properties of crosslinked membranes were investigated. The membrane performance can be controlled by adjusting the photoirradiation time. The experimental results showed that the crosslinked SPEEK membranes with photoirradiation 10 min had the optimum performance for proton exchange membranes (PEMs). Compared with the non-crosslinked SPEEK membranes, the crosslinked SPEEK membranes with photoirradiation 10 min markedly improved thermal stabilities and mechanical properties as well as hydrolytic and oxidative stabilities, greatly reduced water uptake and methanol diffusion coefficients with only slight sacrifice in proton conductivities. Therefore, the crosslinked SPEEK membranes with photoirradiation 10 min were particularly promising as proton exchange membranes for direct methanol fuel cell (DMFC) applications.

Photochemical Cross-Linking for Collagen-Based Scaffolds: A Study on Optical Properties, Mechanical Properties, Stability, and Hematocompatibility

Collagen presents an attractive biomaterial for tissue engineering because of its excellent biocompatibility and negligible immunogenicity. However, some intrinsic features related to the mechanical stability and thrombogenicity limit its applications in orthopedic and vascular tissue engineering. Photochemical cross-linking is an emerging technique able to stabilize tissue grafts and improve the physicochemical properties of collagen-based structures. However, other important properties of collagen-based structures and the effect of processing parameters on these properties have not been explored. In this study, we aim to investigate the dose dependence of tensile and swelling properties on two parameters, namely, laser energy fluence and rose Bengal photosensitizer concentration. We also study the compression properties using cyclic compression test, long-term stability using subcutaneous implantation, and hematocompatibility using platelets adhesion test, of cross-linked collagen structures. Moreover, because limited optical penetration in turbid media is the major obstacle for light-based techniques, we also characterize the optical properties, which partially determine the effective optical penetration depth in collagen gel samples, during photochemical cross-linking. Laser energy fluence and rose Bengal concentration are important parameters affecting the cross-linking efficiency, which was characterized as the mechanical and the swelling properties, in a dose-dependent manner. Under the experimental conditions in this study, the peak fluence was 12.5 J/cm2 and the minimal rose Bengal concentration for effective cross-linking was >0.00008% (0.786 micromol). Photochemical cross-linking also enhanced the compression strength and long-term stability of collagen structures without compromising the tissue compatibility. Furthermore, photochemical cross-linking reduced platelet adhesion and abolished fibrin mesh formation, thereby improving the hematocompatibility of collagen structures. These results suggest the feasibility of using the photochemically cross-linked collagen structures for orthopedic and vascular tissue engineering. Finally, the effective optical penetration depth in collagen gel samples is wavelength and rose Bengal concentration dependent, and was approximately 12 mm at 514 nm at 0.001% (9.825 micromol), the rose Bengal concentration mostly used in this study.