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Thrombin is the final protease produced in the clotting pathways. Thrombin has been used in the clinic more than six decades for topical hemostasis and wound management. In human plasma the half‐life of thrombin is shorter than 15 seconds due to close control by inhibitors. In order to stabilize thrombin, this enzyme was conjugated covalently and physically to γ‐Fe2O3 magnetic nanoparticles. The physical conjugation was accomplished through adsorption of thrombin to BSA coating on the nanoparticles. The coagulant activity of the covalently bound thrombin was significantly lower than that of the physically adsorbed thrombin. Leakage of the physically bound thrombin into PBS containing 4% HSA was negligible. The physical conjugation of thrombin onto the nanoparticles stabilized the thrombin against its major inhibitor antithrombin III and improved its storage stability. At optimal CaCl2 concentration, the clotting time by the bound thrombin is shorter than that of the free enzyme. This novel conjugated thrombin may be an efficient candidate for topical hemostasis and wound healing.

Nanoparticles with innovative optical, chemical, and magnetic properties combined in a single nanoparticle may be useful as biosensors, targeting agents, and therapeutic agents in the biomedical field. This study describes new magnetic nanoparticles (MNPs) containing the fluorescent dye rhodamine isothiocyanate (RITC) covalently encapsulated within the nanoparticles. These nanoparticles have been prepared by nucleation followed by controlled growth of iron oxide layers onto iron oxide/gelatin-RITC nuclei. The formed RITC labeled MNPs (R-MNPs) are of narrow size distribution, exhibit the fluorescent spectrum of RITC, yet are more photostable. Because of the covalent encapsulation of RITC within the MNPs no detectable leakage of the fluorescent dye into the aqueous continuous phase was observed. This manuscript also demonstrates that the surface of the R-MNPs retains similar ligand binding efficiency as the equivalent nonfluorescent MNPs. Specific cell labeling was obtained by incubating glia cells with R-MNPs conjugated to glial cell line-derived neurotrophic factor (GDNF) protein. We further showed that the R-MNPs may be used for pH sensing between the pH range of 5 and 9. This feature may enable the use of the R-MNPs as a pH sensor of animal tissues and cell compartments. Thus, these functional narrow size distribution R-MNPs with both magnetic and fluorescent properties may provide an important research tool for biological sensing.

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