Synthesis and characterization of fluoropolymeric substrata with immobilized minimal peptide sequences for cell adhesion studies. I.

In this work, poly(tetrafluoroethylene-co-hexafluoropropylene) (also known as fluorinated ethylene propylene; FEP) was functionalized at the surface using a radio frequency glow discharge plasma. This particular surface modification produced controlled densities of hydroxyl functionality on the FEP surface. These surface hydroxyl groups provided sites for the covalent attachment of minimal peptide sequences, that are specific for neuronal attachment. FSCA, ATR-FTIR, ToF-SIMS, and fluorescence spectroscopy were used to evaluate peptide reaction efficiencies and to verify that intact peptide sequences were covalently attached to the FEP surfaces. These modified substrata were then used to study the cell attachment and response to covalently bound minimal peptide sequences. Cell attachment and differentiation results using NG108-15 and PC12 neuronal cell lines are presented in the adjoining paper by Ranieri et al.

Neuronal cell attachment to fluorinated ethylene propylene films with covalently immobilized laminin oligopeptides YIGSR and IKVAV. II.

Material surfaces that can mediate cellular interactions by the coupling of specific cell membrane receptors may allow for the design of a biomaterial that can control cell attachment, differentiation, and tissue organization. Cell adhesion proteins have been shown to contain minimum oligopeptide sequences that are recognized by cell surface receptors and can be covalently immobilized on material surfaces. In this study, cell attachment to fluorinated ethylene propylene (FEP) films functionalized with the laminin-derived oligopeptides, YIGSR and a 19-mer IKVAV-containing sequence, was assessed using NG108-15 neuroblastoma and PC12 cells. A radiofrequency glow discharge (RFGD) process that replaces the FEP surface fluorine atoms with reactive hydroxyl functionalities was used to activate the film surfaces. The oligopeptides were then covalently coupled to the surface by their C-terminus using a standard nucleophilic substitution reaction. The covalent attachment of the oligopeptides to the FEP surface was verified using electron spectroscopy for chemical analysis (ESCA). Receptor-mediated NG108-15 cell attachment on the YIGSR-modified films was determined using competitive binding assays. Average cell attachment on the oligopeptide immobilized films in medium containing soluble CDPGYIGSR was reduced by approximately a factor of 2, compared to cell attachment in serum-free medium alone. No significant decrease in cell attachment was noted in medium containing the mock oligopeptide sequence CDPGYIGSK. FEP films immobilized with the 19-mer IKVAV sequence demonstrated a higher percentage of receptor mediated cell attachment on the film surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamics surrounding Cys-34 in native, chemically denatured, and silica-adsorbed bovine serum albumin.

We report the steady-state and time-resolved fluorescence of 6-acryloyl(dimethylamino)naphthalene (acrylodan) covalently attached to Cys-34 in bovine serum albumin (BSA). For this conceptually simple system, complicated fluorescence intensity and anisotropy decay kinetics are observed. The steady-state and time-resolved results demonstrate the presence of an excited-state reaction for the BSA-acrylodan system. Additional analysis shows that dipolar relaxation of the environment surrounding acrylodan within BSA is responsible for most of the observed time-dependent evolution of the emission spectrum. The effects of temperature, chemical denaturation, and protein adsorption to a bare silica substrate are also investigated. These results demonstrate the complexity of the changes within a protein/biorecognition element that affect the signal from a single fluorescent reporter group.

Spatial control of neuronal cell attachment and differentiation on covalently patterned laminin oligopeptide substrates.

The spatial control of neuronal cell attachment and differentiation via specific receptor mediated interactions, may provide an effective means for the in vitro reconstruction of neuronal cell architecture. In this study, receptor-specific oligopeptide sequences derived from the extracellular matrix (ECM) molecule laminin, a potent neural cell attachment and differentiation promoter were covalently bound on fluorinated ethylene propylene (FEP) films. The degree of receptor-specific cell attachment and the ability to spatially control neurite outgrowth by covalently patterning the oligopeptide sequences on the FEP film surface were assessed. FEP films were first chemically activated with a Radio Frequency Glow Discharge (RFGD) process that covalently replaces the surface fluorine atoms with reactive hydroxyl groups. Oligopeptides containing the YIGSR sequence from the B1 chain of laminin and the water soluble oligopeptide containing the IKVAV sequence (CSRARKQAASIKVAVSADR) from the A chain were covalently bound to the hydroxylated FEP films. Electron Spectroscopy for Chemical Analysis (ESCA) verified the covalent attachment of the oligopeptides to the material surface. The degree of receptor mediated NG108-15 cell attachment on immobilized CDPGYIGSR films was determined using competitive binding media. A 78% reduction in cell attachment was observed on films containing CDPGYIGSR in the cell plating medium. Only a 23% reduction in cell attachment was noted on films plated in medium containing a mock CDPGYIGSK sequence. FEP films immobilized with the IKVAV oligopeptide sequence were shown to mediate PC12 cell attachment and a competitive binding medium also significantly attenuated cell attachment on the immobilized films. The spatial patterning of these oligopeptide sequences to the FEP surface was shown to localize cell attachment and neurite extension on the patterned pathways. The surrounding unmodified FEP surface was inhibitory in serum containing medium and prevented cellular interactions outside the oligopeptide modifications. The spatial immobilization of laminin oligopeptides on FEP films provides a means to organize the attachment and differentiation of neuronal cells in a receptor-specific manner.

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma.

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 microns column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10-1000 ng/ml (0.012-1.17 microM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.