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This paper reports the deposition of active protein thin films by a novel laser-based approach termed matrix-assisted pulsed laser evaporation (MAPLE). We have deposited uniform 10 nm to nearly 1 μm thin films of insulin and horseradish peroxidase (HRP). We performed several experiments to characterize the chemical integrity of the deposited films. Matrix assisted laser desorption/ionization and liquid chromatography/electrospray ionization mass spectrometry experiments performed on MAPLE-deposited insulin films indicate that the laser−material interaction involved in this deposition technique does not modify the protein's mass. Fourier transform infrared spectroscopy experiments show that the chemical functionality and secondary structure of MAPLE-deposited HRP are nearly identical to those of the native protein. We also find that deposited HRP films retain their ability to catalyze the reduction of 3,3‘-diaminobenzidine (DAB), suggesting that the active site of transferred proteins is unaffected by the MAPLE process. We also produced patterns and multilayers with feature sizes from 20 to 250 μm by depositing different biomaterials through a shadow mask. Patterns of physisorbed HRP were then protected from dissolution in an aqueous environment by a semipermeable polymer overlayer that was deposited in situ using pulsed laser deposition. This polymer membrane protects the protein pattern when it is exposed to DAB solution and enables the optical observation of HRP activity for spots as small as 2000 μm². These results demonstrate that MAPLE is a preferred technique for depositing active biomolecules for applications ranging from microfluidic sensor devices to gene and protein recognition microarrays.

The release of transforming growth factor-β (TGF-β) from a lipid microstructure has been demonstrated. Lipid microcylinders, with dimensions of 100 × 0.5 μm and composed of a diacetylenic lipid, have been loaded with 25 ng TGF-β/mg lipid. Physical and bioactive release characteristics of TGF-β from these microcylinders and from microcylinders embedded in an agarose hydrogel are reported. Release of TGF-β from lipid microcylinders follows typical diffusionlimited characteristics, where 10-12% of the TGF is released in the first 10h at 37°C. The release rate is shown to be temperature controlled and dependent on the integrity of the lipid microcylinder. Immobilization of the lipid microcylinder in a hydrogel matrix composed of agarose and gelatin does not impair the diffusion of TGF-β from the lipid microcylinders. The utilization of microcylinders as release vehicles in wound repair is discussed.

Specimens of kidney, heart, lung, eye, skin, conjunctiva, choroid plexus and brain were obtained from a patient with systemic lupus erythematosus (SLE) post-mortem. The tissues were examined for immune-complex deposition by direct immunofluorescence utilizing antisera specific for IgG, IgA, IgE, IgM and the complement components Clq and C3. Tissues were also examined for C-type viral antigen by indirect immunofluorescence with an antisera raised to HEL-12 virus. Immune complexes were detected along basement membranes and/or in vessel walls of kidney, heart, lung, skin, conjunctiva and eye. HEL-12 viral antigen was detected in the same distribution as immunoglobulins and complement in all tissues. Preimmune serum did not mediate immunofluorescence and the reaction mediated by the anti-HEL-12 virus serum could be blocked by absorption with purified viral antigens. Two renal biopsies were obtained from a second SLE patient during clinical exacerbation and remission. Immunofluorescence analysis of the biopsy obtained during exacerbation showed 3 + deposition of immunoglobulin, complement and HEL-12 viral antigen. In contrast, the biopsy obtained 6 months later during remission showed trace -1 + immunofluorescence for HEL-12 viral antigen, immunoglobulin and complement. We conclude that HEL-12 viral antigen is a component of widely disseminated immune complexes in SLE, and that the disappearance of HEL-12 viral antigen occurs concomitantly with the loss of immune deposits.

Certain sugars, particularly trehalose, dramatically alter physical properties of dry phospholipids in ways that mimic the presence of water. As a result, these sugars are capable of preserving the integrity of dry liposomes and membranes. Since these effects could conceivably be due to the presence of small amounts of water in the dry preparations of sugar and lipid, we have done careful measurements of the residual water contents in the dry samples and report the results here. Lyophilized liposomes composed of palmitoyloleoylphosphatidylcholine and phosphatidylserine (9:1) contain at most 0.2 mol of H2O per mol of lipid. When the trehalose concentration in the dry mixtures is increased, there is no increase in the apparent water content of the samples over a wide range of sugar concentrations. Over the same range of trehalose contents the maximal effect of trehalose on physical properties of the lipids and on stabilization of liposomes is achieved. We conclude that the stabilization does not require retention of residual amounts of water in the dry trehalose-phospholipid preparations. Similar studies with other sugars show a relationship between the amount of sugar interacting with the lipid and the ability of the same sugar to stabilize dry liposomes.

Chemically modified glass substrates were used to demonstrate differential adhesion, growth, and differentiation of endothelial cells. Endothelial cells were examined for adhesion and growth on glass, glass treated with N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA), or EDA with a subsequent treatment with physically adsorbed extracellular matrix components human fibronectin and heparin sulfate. EDA and EDA/human fibronectin showed similar abilities to support adhesion, spreading, and proliferation of endothelial cells. In contrast, heparin sulfate inhibited endothelial cell adhesion to EDA. Differentiation of endothelial cells resulting in precapillary cord formation was triggered by addition of basic fibroblast growth factor (bFGF). On EDA and EDA/human fibronectin bFGF causes confluent endothelial cell monolayers to differentiate and form cords, which resulted in a large-scale spatial redistribution of cells on the surface. Formation of organized neovascular assemblies was demonstrated on coplanar molecular patterns of EDA and a nonadhesive perfluorinated alkylsilane (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchloros ilane (13F). Endothelial cells preferentially adhered to the EDA lines and after 24-48 hr, microfilaments aligned with the long axes of the patterned EDA region. Finally, endothelial cells that became confluent within the confines of the EDA region (bound by the nonadhesive, 13F domains) were observed to differentiate into neovascular cords in long-term culture (7-10 days) with bFGF.

The persistence of numerous pathogenic bacteria important in disease states, such as tuberculosis, in humans and domestic animals has been ascribed to an inhibition of fusion between the phagosomal vesicles containing the bacteria and lysosomes in the host cells [Elsbach, P. & Weiss, J. (1988) Biochim. Biophys. Acta 974, 29-52; Thoen, C. O. (1988) J. Am. Vet. Med. Assoc. 193, 1045-1048]. In tuberculosis this effect has been indirectly attributed to the production of cord factor (alpha,alpha-trehalose 6,6'-dimycolate). We show here that cord factor is extraordinarily effective at inhibiting Ca2(+)-induced fusion between phospholipid vesicles and suggest a mechanism by which cord factor confers this effect. These findings are likely to be important in our understanding of the pathogenesis and treatment of many diseases of bacterial etiology.