Keith M. Faucher

Cytomimetic Biomaterials. 1. In-Situ Polymerization of Phospholipids on an Alkylated Surface

A stabilized, phosphatidylcholine-containing polymeric surface was produced by in-situ polymerization of a self-assembled lipid monolayer on an alkylated substrate. The phospholipid monomer 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphorylcholine was synthesized, prepared as unilamellar vesicles, and fused onto alkylated glass. Free-radical polymerization was carried out in aqueous solution at 70 °C and characterized using either the water-soluble initiator 2,2′-azobis(2methylpropionamidine) dihydrochloride (AAPD) or an oil-soluble initiator 2,2′-azobis(isobutyronitrile) (AIBN). Under optimized conditions, the supported monolayer displayed advancing and receding water contact angles of 64 and 44°, respectively. Angle-dependent ESCA results confirmed the presence of phosphorus and nitrogen and were consistent with theoretical predictions for close-packed monolayer formation with near-normal alignment of lipid chains. In the absence of network formation, polymeric films demonstrated acceptable stability under static conditions in water and air, as well as in the presence of a high shear flow environment. Blood compatibility was assessed in a baboon arteriovenous shunt model, which revealed miminal platelet deposition over a 2 h observation period.

Infrared spectroscopy of aqueous biophysical monolayers

We have used infrared external reflectance spectroscopy at the air/water interface to study: (1) polarized infrared (IR) monolayer spectroscopy at different incoming angles of incidence, and (2) ternary mixed model systems (i.e. two lipids and a peptide) that mimic the composition of pulmonary lung surfactant. Using polarized IR radiation, we have observed a splitting of the symmetric and antisymmetric methylene stretching vibrations in the spectra of long-chain hydrocarbon amphiphiles that has been previously unreported for unpolarized monolayer spectra. The splitting of the C–H bands results in sub-bands identifiable with ordered and disordered chain conformations. The splitting of these bands qualitatively tracks the fractional composition of ordered/disordered states upon monolayer compression. In a separate series of experiments, we have obtained the IR external reflectance spectra of monomolecular films of model mixtures relevant to pulmonary surfactant physiology. Monolayers were composed of 2:1 DPPC-d62:DOPG containing 0, 1 or 2 wt.% of the hydrophobic surfactant proteins SP-B and SP-C (SP-B+C). The CH2 antisymmetric and symmetric stretching bands (∼2920 and 2852 cm−1) along with the analogous CD2 stretching bands (∼2194 and 2089 cm−1) were analyzed, and band heights, integrated intensities and peak frequency positions were plotted as a function of measured surface pressure. These data indicate that the presence of surfactant protein appears the disorder the acyl chains of the DPPC-d62 component in the film, opposite of what is seen for the protiated component, DOPG. Data from these model mixtures indicate that the surfactant protein interacts differently with each of the lipid components.

Thermally induced micro-domain formation in fatty acid Langmuir–Blodgett films observed by attenuated total reflectance infrared spectroscopy

We have utilized attenuated total reflectance infrared (ATR-IR) spectroscopy to study the temperature dependence of conformational transitions and micro-domain structure in single- and binary-component Langmuir � /Blodgett (LB) films. The single-component films such as lignoceric (C24), stearic (C18), and perdeuterated stearic (C18-d35) acids, as well as binary-component films of 4:1 H:D C24:C18-d35 and 4:1 H:D C18:C18-d35 were studied. For C24:C18-d35 binary films, changes in the peak wavenumber of the naCH2 band after heating above the main chain melting temperature (Tm) reflect a thermally induced de-mixing and phase separation of the C24 component from C18-d35. For C18:C18-d35 LB film, heating above Tm shows that C18 and C18-d35 components do not phase segregate and retain a degree of conformational flexibility, even at low temperatures. The carbonyl C� /O stretching region between 1800 and 1600 cm � 1 showed that each LB film contained a mixture of cis � /trans ring dimer conformations at all temperatures below Tm. Above Tm, the LB films converted to the higher energy cis isomer, while upon further cooling the more stable trans isomer predominated. The shorter chain length C18:C18-d35 LB film more easily crystallized the trans ring dimer after heating above Tm. The band splitting of the d CH2 vibration at � /1470 cm � 1 was used to monitor micro-domain

Use of polarized infrared external reflection spectroscopy to study phospholipid monolayers at the air-water interface

The pressure dependence of phospholipid monolayers at the air-water interface has been investigated using polarized infrared external reflection spectroscopy under carefully controlled environmental conditions. Results show that a splitting of the methylene symmetric and antisymmetric stretching vibrations is observed in the polarized spectra that has not been previously reported for unpolarized monolayer spectra. The splitting of the C-H bands results in sub-bands identifiable with ordered and disordered conformations. The intensities of these sub-bands correlate the particular thermodynamic state of the monolayer. The splitting of these bands qualitatively tracks the formation of these domains upon compression as previously seen in epiflourescence microscopy. As the surface pressure of the phospholipid monolayer film increases, the band resulting from the liquid domain structures in the film disappears. Curve fitting based on these results indicates that we can now determine the mole fraction of either ...