Rationale and Objective:We wanted to test the hypothesis that linear extended polymers are able to translocate across compromised endothelia to a much higher degree than polymers that are in a coiled configuration. The basis for this hypothesis is the concept of polymer reptation, a process allowing linear polymers to move around fixed obstacles and through small pores. Materials and Methods:The conjugation of polylysine with DTPA was varied from 55% to 95% of lysine moieties for a fixed polymer degree of polymerization. The variable conjugation constructs were assessed in their ability to enhance tumor signals in an animal model. The blood circulation time was also assessed, as was the proton relaxivity of the constructs. Results:The relaxivity values of the variable conjugation constructs indicated that the conformation of the highly conjugated polymers was consistent with an extended conformation. Images of the highly conjugated constructs with cryoelectron microscopy showed structures also consistent with an extended peptide chain conformation. These constructs gave tumor signal enhancements 10 times larger than observed for constructs of lower conjugation at the same injection dose. Conclusions:The conformation of polymer agents is of decisive importance in the imaging efficacy of tumors. Polylysine conjugates of Gd-DTPA, at conjugation levels of 90% or higher, assume an extended linear conformation, which enables them to translocate across tumor endothelium more effectively than less highly conjugated polymers.

The structure of Gd-DTPA-polylysine, Gd-DOTA-polylysine, Gd-SCN-Bz-DOTA-polylysine, and Gd-DTPA-poly(glu:lys) was investigated with circular dichroism, gel permeation chromatography, low angle light scattering, and proton longitudinal relaxivity. Molecular modeling calculations were performed and predicted helical secondary structure for charged Gd-chelator residues, i.e., Gd-DTPA, when the DTPA conjugation levels reached 90% and higher. This helical secondary structure was observed with circular dichroism. The conformational transition from coiled to extended linear was observed also by gel permeation chromatography and by proton relaxivity measurements. The helical secondary structure was not observed when the chelator was changed to DOTA. The residue charge interactions were eliminated in this case since the Gd-DOTA complex had no net charge. For this construct, the gel permeation and relaxivity measurements indicated a coiled conformation. An extended linear conformation was regained when the chelator complex was changed to Gd-SCN-Bz-DOTA, which had a net negative charge. The functional aspects of these structures were investigated by MR imaging of an animal tumor model. The linear extended polymer constructs gave 10-fold higher tumor signals then the coiled-collapsed constructs, indicating a much higher degree of trans-endothelial transport in the tumors.

Phospholipid bilayers, supported on UV irradiated carbon shadowed nitrocellulose electron microscope grids, have been used to induce two-dimensional crystal growth of IgE and IgG anti-DNP monoclonal antibodies. The UV irradiation renders the grids hydrophilic in a very uniform fashion and allows for the transfer of phospholipid monolayers from an air/water interface in a sequential dipping procedure. The surface coverage achieved was nearly 100% as measured by antibody binding and by the formation of protein arrays on the bilayer covered grids. The supported bilayers appear to be stably held and are appropriate for slow binding conditions and long incubation times with low concentrations of binding protein.

Background fluorescence from serum chromophores is substantially reduced by a laser photobleaching method. Human and bovine serum samples were illuminated with 337-nm light from a pulsed N2 laser for a short period of time. The serum emission in the region of 440 to 550 nm was reduced by an order of magnitude with no evident damage to serum proteins as judged by the unchanged activity of alkaline phosphatase and aspartate aminotransferase.

A wide-angle, crossed-beam laser Doppler scattering configuration is described. This configuration is optimized for measurement of electrophoretic mobility of blood cells. It is easy to align and maintain aligned and is stable against microphonic disturbance. The optimum scattering angle for blood cells is shown to be in the range of 25°–45°, a condition that is easy to implement with this configuration and within the geometrical constraints imposed by the use of narrow gap rectangular electrodes for applying an electric field.

The principal electrode problems associated with laser Doppler electrophoresis in high salt conditions and at high current densities (pH drift, colloidal particle ejection, and gas bubbling) have been solved through the use of hybrid electrodes that are platinized Ag/AgCl or by the use of platinized Ag. For salt conditions well below physiological saline an agarose gel overcoating on either of these electrode types has been useful for containing the increased tendency for the ejection of colloidal particles.

The specific combination of antigen with antibody was detected rapidly and with high sensitivity by an assay based on particle electrophoresis and laser light scattering. Antibody attachment to dilute suspensions of submicroscopic polystyrene spheres coated with antigen was detected as changes in the Doppler shift of scattered laser light when the suspended particles were subjected to an electric field. Antibody or antigen concentrations in the range of 5–10 nanograms per ml can be detected in 30 min. The principal experimental features of the technique are described and include: a) choice of particles; b) coating procedures; c) treatment for non-specificity; d) choice of measurement medium.

We describe a simple method for coating platinized Pt electrodes with bovine serum albumin. The coating does not alter the electrical properties of the electrodes in dilute salt solutions, seems to be durable enough for extended use, and, most importantly, prevents a specific type of surface reaction from occurring between the Pt surface and a sucrose buffer. These electrodes are then used for studies of cellular electrokinetic phenomena by the technique of laser Doppler spectroscopy.

We have observed that the intensity fluctuations of light scattered from solutions of hemoglobin contain a term independent of the scattering vector. We have interpreted these fluctuations as arising from the hemoglobin association-dissociation reaction. In support of this interpretation we find that variations in these fluctuations with solution conditions agree with the known variations in the reaction.