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Positively charged superparamagnetic iron oxide (SPIO) particles of maghemite were prepared in aqueous solution and subsequently stabilized with poly(ethylene oxide)-block-poly(glutamic acid) (PEOPGA) at a hydrodynamic diameter of 60 nm. Depending on the amount of PEOPGA used, this is accompanied by a switching of their zeta potentials from positive to negative charge (-33 mV). As a prerequisite for in vivo testing, the PEOPGA coated maghemite nanoparticles were evaluated to be colloidally stable in water and in physiological salt solution for longer than six months as well in various buffer systems under physiological pH and salt conditions (AFM, dynamic light scattering). We excluded toxic effects of the PEOPGA coated maghemite nanoparticles. We demonstrated by in vivo MR-imaging and 111In measurements a biodistribution of the nanoparticles into the liver comparable to carboxydextran coated superparamagnetic iron oxide nanoparticles (Resovist®) as a reference nanoscaled MRI contrast medium. This was enforced by a detailed visualization of our nanoparticles by electron microscopy of liver tissue sections. Furthermore, our results indicate that 15% of the injected PEOPGA coated maghemite nanoparticles circulate in the blood compartment for at least 60 min after i.v. application.
Pulmonary surfactant contains two extremely hydrophobic proteins, SP-B and SP-C. We present a novel HPLC method for the preparation of these hydrophobic proteins. It is based on size-exclusion chromatography using the apolar stationary-phase butyl silica gel and isocratic elution with acidified chloroform/methanol. Samples for HPLC were prepared from sheep lung lavage fluid by centrifugation and extraction with chloroform/methanol. Amino acid analyses of the two protein fractions revealed sequences that are consistent with SP-B and SP-C, respectively. MALDI-TOF-MS analyses of the SP-B fraction showed one major peak of dimeric SP-B with m/z 17,361, and additional peaks of monomeric and oligomeric forms, which are predominantly even numbered. The SP-C fraction showed a peak at m/z 4200, consistent with the theoretical mass of the dipalmitoylated form of this protein. The biophysical activity of pure sheep SP-B and SP-C was evaluated by measuring the surface tension using axisymmetric drop shape analysis for captive bubbles. We found distinct surface pressure versus surface area isotherms of SP-B and SP-C indicating different biophysical activities for these surfactant proteins. The new preparative HPLC method is able to replace the established, time-consuming low-pressure liquid chromatography method for the isolation of SP-B and SP-C from lipids.