Synthesis and properties of regenerated cellulose-based hydrogels with high strength and transparency for potential use as an ocular bandage.

Cellulose is a biologically derived material with excellent wound-healing properties. The high strength of cellulose fibers and the ability to synthesize gels with high optical transparency make these materials suitable for ocular applications. In this study, cellulose materials derived from wood pulp, cotton, and bacterial sources were dissolved in lithium chloride/N,N-dimethylacetamide to form regenerated cellulose hydrogels. Material properties of the resulting hydrogels, including water content, optical transparency, and tensile and tear strengths, were evaluated. Synthesis parameters, including activation time, dissolution time, relative humidity, and cellulose concentration, were found to impact the material properties of the resulting hydrogels. Overnight activation time improves the optical transparency of the hydrogels from 77% to 97% at 550 nm, whereas controlling cellulose concentration improves their tear strength by as much as 200%. On the basis of the measured transmittance and strength values of the regenerated hydrogels prepared via the optimized synthesis parameters, Avicel PH 101, Sigma-Aldrich microcrystalline cellulose 435236, and bacterial cellulose types were prioritized for future biocompatibility testing and potential clinical investigation.

Topoisomerase II.etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes.

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.

Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase.

Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is approximately 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug. Conversely, no change in the rate of DNA religation was observed. There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity. The apparent dissociation constant for ellipticine in the mutant topoisomerase II.drug.DNA ternary complex was approximately 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent KD value for the mutant binary (topoisomerase II.drug) complex was approximately 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.

Topoisomerase II binds to ellipticine in the absence or presence of DNA. Characterization of enzyme-drug interactions by fluorescence spectroscopy.

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

Lifetime-based fluorescence energy transfer biosensing of zinc.

A new type of fluorescence transduction method for determining zinc in solution is described. The approach is based upon energy transfer from a fluorescent label on an enzyme, human carbonic anhydrase II, to a colored inhibitor which binds to zinc in the enzyme active site. If zinc is present in solution, it binds to the apoenzyme, which in turn permits the inhibitor to bind to the enzyme; the inhibitor is thus in close proximity to the label on the enzyme and thereby quenches the label's fluorescence by Forster energy transfer with a concomitant reduction of its lifetime, which is quantitated by phase fluorometry.

Fluorescence lifetime-based biosensing of zinc: Origin of the broad dynamic range.

Fluorescence lifetime-based chemical sensors have recently been described for applications in medicine, environmental monitoring, and bioprocess control. These sensors transduce the level of the analyte as a change in the apparent fluorescence lifetime of an indicator phase. We have previously developed a wavelength-ratiometric fluorescence biosensor for zinc based on binding of zinc and dansylamide to apo-carbonic anhydrase which exhibited high sensitivity and selectivity. We demonstrate that the apo-carbonic anhydrase/dansylamide indicator system is very well suited for lifetime-based sensing, with a subnanomolar detection limit and greater than 1000-fold dynamic range. The theoretical basis for the wide dynamic range is discussed.

Entrapment and release characteristics of 2-methoxynaphthalene from cylindrical microstructures formed from phospholipids.

Many natural products that exhibit biocidal activity have poor solubility in water. In order to explore the prolonged delivery of these compounds from microtubules we have utilized 2-methoxynaphthalene as a model to elucidate release characteristics of hydrophobic compounds entrapped in microtubules by spectrophotometric absorbance at 255 nm. Entrapment of this compound in microcylinders was accomplished by addition of 2-methoxynaphthalene to a water-soluble epoxy, or entrapment of the neat compound. Variation of the release rate is possible for 2-methoxynaphthalene based on the mode of entrapment and by variations in the methods used to immobilize the compound within the microcylinders. Unlike conventional microencapsulation techniques which require inclusion of the active agent at the time of formation, the use of microcylinders allows for the inclusion of a variety of active agents and the tailoring of release characteristics after their formation. We report the results of in vitro release rates of 2-methoxynaphthalene from a static diffusion system designed to explore release of hydrophobic compounds into an aqueous environment.

Controlled release from cylindrical microstructures.

Microtubules formed from diacetylenic phosphatidyl-cholines can be made rugged and solvent resistant through electroless deposition of metals (Rudolph et al. 1990). Once dried tubules can capture a range of materials by capillary action when added to a hydrating medium, thus retaining and controlling the release rates of these materials. We report the encapsulation in tubules of a mixture of tetracycline, epoxy monomers and polymers and their in vitro release kinetics in both dynamic and static environments.