Chemically tunable electrochemical dissolution of noncontinuous polyelectrolyte assemblies: an in situ study using ecAFM.

The electrochemically triggered dissolution of noncontinuous polyelectrolyte assemblies presenting distinct nanomorphologies and its tuning by chemical cross-linking were monitored locally, in situ, by electrochemical atomic force microscopy. Poly-l-lysine and hyaluronic acid deposited layer-by-layer on indium tin oxide electrodes at specific experimental conditions formed well-defined nanostructures whose morphologies could be easily and precisely followed along the dissolution process. In addition to shrinkage of polyelectrolyte nanodroplets, ecAFM images revealed the faster dissolution of coalesced structures compared to droplet-like complexes, and the readsorption of dissolved polyelectrolytes onto slower dissolving neighboring structures. Covalently cross-linked PLL/HA assemblies dissolved only partially, and exhibited slower dissolution rates compared to native multilayers, with a clear dependence on the cross-link density. Tuning the electrochemical dissolution of polyelectrolyte multilayers through chemical cross-linking opens new prospects for future biomedical applications, such as the development of advanced drug or gene delivery platforms allowing for tightly controlled releases of different compounds at specific rates.

Biological properties of photocrosslinked alginate microcapsules.

An alternative form of gene therapy using recombinant cell lines delivering therapeutic products encapsulated in alginate hydrogel has proven effective in treating many murine models. The lack of long-term capsule stability has led to a new strategy to reinforce the microcapsules with a photopolymerized interpenetrating covalent network of N-vinylpyrrolidone (NVP) and sodium acrylate. Here the properties for potential application in gene therapy are reported. In assessing potential toxicity of the unpolymerized residues, HPLC showed that even after 1 week of washing, no toxic monomers could be detected. Their ability to sustain cell growth was monitored with growth of the encapsulated cells in vitro and in vivo. Although the initial photopolymerization caused significant cell damage, the cells were able to recover normal growth rates thereafter. After implanting into mice, the NVP-modified capsules showed a high level of biocompatibility as measured by hematological and biochemical functional tests. There was also no difference in the amount and type of plasma proteins adsorbing to the NVP-modified and the classical alginate capsules, thus indicating their similar biological compatibility. Both in vitro and in vivo tests confirmed that the NVP-modified capsules were more resistant to osmotic stress than the alginate microcapsules. Furthermore, when applied to the treatment of a murine model of human cancer by delivering encapsulated cells secreting angiostatin, the NVP-modified microcapsules suppressed tumor growth as successfully as the regular alginate microcapsules. In conclusion, the covalently modified microcapsules have shown a high level of biocompatibility, safety, increase in stability, and clinical efficacy for use as immunoisolation devices in gene therapy.

Biodegradable water absorbent synthesized from bacterial poly(amino acid)s.

Biodegradable hydrogels prepared by gamma-irradiation from microbial poly(amino acid)s have been studied. pH-Sensitive hydrogels were prepared by the gamma-irradiation of poly(gamma-glutamic acid) (PGA) produced by Bacillus subtilis and poly(epsilon-lysine) (PL) produced by Streptomyces albulus in aqueous solutions. When the gamma-irradiation dose was 19 kGy or more, and the concentration of PGA in water was 2 wt.-% or more, transparent hydrogels could be produced. For the 19 kGy dose, the produced hydrogel was very weak, however, the specific water content (wt. of absorbed water/wt. of dry hydrogel) of this PGA hydrogel was approximately 3,500. The specific water content decreased to 200, increasing when the gamma-irradiation dose was over 100 kGy. Under acid conditions or upon the addition of electrolytes, the PGA hydrogels shrunk. The PGA hydrogel was pH-sensitive and the change in the volume of the hydrogel depended on the pH value outside the hydrogel in the swelling medium. This PGA hydrogel was hydrodegradable and biodegradable. A new novel purifier reagent (coagulant), made from the PGA hydrogels, for contaminated turbid water has been found and developed by Japanese companies. A very small amount of this coagulant (only 2 ppm in turbid water) with poly(aluminum chloride) can be used for the purification of turbid water. A PL aqueous solution also can change into a hydrogel by gamma-irradiation. The specific water content of the PL hydrogel ranged from 20 to 160 depending on the preparation conditions. Under acid conditions, the PL hydrogel swelled because of the ionic repulsion of the protonated amino groups in the PL molecules. The rate of enzymatic degradation of the respective PL hydrogels by a neutral protease was much faster than the rate of simple hydrolytic degradation.

Sunlight induces N epsilon-(carboxymethyl)lysine formation from glycated polylysine-iron(III) complex.

Sunlight was found to strongly induce the formation of N epsilon-(carboxymethyl)lysine (CML) from glycated polylysine in the presence of Fe(III) ion. The initial step of this Fe(III)-catalyzed CML formation was noted to be similar to that of blueprint photography as was confirmed by the production of Turnbull's blue in sunlight-exposed glycated human serum albumin ferricyanide solution in the presence of Fe(III). Based on this, photoinduced oxidative C-C bond cleavage of the Amadori compound was assumed to be initiated by photochemical single electron transfer front ligand to Fe(III) in the Fe(III)-Amadori compound complex affording the Fe(II)-Amadori compound radical intermediate, which eventually yields either CML or active oxygen species. CML is thus a useful oxidative stress marker. The mechanism proposed here would explain the high accumulation of CML in lens protein and skin actinic elastosis.

Raman spectroscopic evidence for structural changes in poly-L-lysine induced by an approximately 50 mT static magnetic field.

We have explored the mechanism of coupling of an approximately 50 mT static magnetic field with the alpha helices of poly-L-lysine. Structural changes in poly-L-lysine were determined by Raman spectroscopy. Our testable hypothesis is that static magnetic fields of this magnitude can couple with the alpha-helical segments of the polypeptide, and, as a result, the structure of the polypeptide is significantly altered. Our model further suggests that a static magnetic field can promote protein unfolding and can prevent refolding.

Investigation of light-induced conformation changes in spiropyran-modified succinylated poly(L-lysine).

To determine the maximum range of coupling between side-chain photochromism and polypeptide conformation change, we modified the carboxylate side chains of succinylated poly(L-lysine) with a spiropyran to form polypeptide I. The extent of modification was determined to be 35.5%. The spacer group length between the polypeptide alpha-carbon and the dye was 12 atoms, providing minimum polypeptide-dye interaction. Conformation changes were monitored by circular dichroism as a function of light adaptation and solvent composition (hexafluoroisopropanol [HFIP] vs trifluoroethanol [TFE]). Under all solvent compositions, the dark-adapted dye was in the merocyanine form. Light adaptation by visible light converted the dye to the spiropyran form. When dissolved in TFE, I adopted a helical conformation insensitive to light adaptation. With increasing percentage HFIP, a solvent-induced helix-to-coil transition was observed around 80% (vol/vol) HFIP. At 100% HFIP, both light- and dark-adapted forms of I were in the coil state. Near the midpoint of the solvent-induced helix-to-coil transition, light adaptation caused conformation changes. Applying helix-to-coil transition theory, we measured a statistically significant difference in coil segment-HFIP binding constant for light- vs dark-adapted solutions (6.38 +/- 0.03 M-1 vs 6.56 +/- 0.03 M-1), but not for the nucleation parameter sigma (1.2 +/- 0.4 10(-3) vs 1.3 +/- 0.3 x 10(-3). The small binding constant difference translated to a light-induced binding energy difference of 17 cal/mol/monomer. Near the midpoint of the helix-to-coil transition, collective interactions between monomer units made possible the translation of a small energy difference (less than RT) into large macromolecular conformation changes.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for detection of hydroxyl radicals in the vicinity of biomolecules using radiation-induced fluorescence of coumarin.

A novel method is described to quantitate radiation-induced hydroxyl radicals in the vicinity of biomolecules in aqueous solutions. Coumarin-3-carboxylic acid (CCA) is a non-fluorescent molecule that, upon interaction with radiation in aqueous solution, produces fluorescent products. CCA was derivatized to its succinimidyl ester (SECCA) and coupled to free primary amines of albumin, avidin, histone-H1, polylysine, and an oligonucleotide. When SECCA-biomolecule conjugates were irradiated, the relationship between induced fluorescence and dose was linear in the dose range examined (0.01-10 Gy). The fluorescence excitation spectrum of irradiated SECCA-biomolecule conjugates was very similar to that of 7-hydroxy-SECCA-biomolecule conjugates, indicating the conversion of SECCA to 7-hydroxy-SECCA following irradiation. Control studies in environments that excluded certain radiation-induced water radicals for both the conjugated and unconjugated forms of irradiated SECCA demonstrated that: (1) the induction of fluorescence is mediated by the hydroxyl radical; (2) the presence of oxygen enhances induced fluorescence by a factor of about 1.4, and (3) other primary water radicals and secondary radicals caused by interaction of primary water radicals with biomolecules do not significantly influence the induced fluorescence. The data indicate that the induction of fluorescence on SECCA-biomolecule conjugates records specifically the presence of the hydroxyl radical in the immediate vicinity of the irradiated biomolecule. The method is rapid and sensitive, uses standard instrumentation, and the sample remains available for further studies.

Evidence for light-induced lysine conformational changes during the primary event of the bacteriorhodopsin photocycle.

Fourier transform infrared difference spectroscopy is used to examine the role of lysine in the primary event of the bacteriorhodopsin photocycle. Isotopically labeled lysine is used to tentatively assign the lysine modes in the BR and K species. The results suggest that the lysine side-chain undergoes conformational changes in concert with the known light-induced chromophore structural alterations.

Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping.

The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2-nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission.

Radiation effects of poly(L-glutamic acid) and poly(L-lysine) in the helix-coil transitional state.

The degradation of poly(L-glutamic acid) and poly(L-lysine) by X-rays and ultraviolet radiation has been studied at various pH values, including the helix-coil transition region. It was found that X-rays cause maximum degradation in the transition region for both polypeptides, indicating that the efficiency of degradation is affected not only by the secondary structure but also by some other factors related to the transition. On the contrary, the degradation by monochromatic ultraviolet 2537 A radiation showed a strong dependence on helix content of PLGA, suggesting that degradation occurs more effectively in the helical form than in the coil form. Theoretical calculations were made to approximate the pH dependence of degradation, assuming different cross sections for the peptide bonds in helix, coil, and junctions between helical and coil regions.