Protein nanoparticles with ligand-binding and enzymatic activities

Olga V Morozova,1,2 Elizaveta R Pavlova,1,3 Dmitry V Bagrov,1,4 Nikolay A Barinov,1 Kirill A Prusakov,1,3 Elena I Isaeva,2 Victor V Podgorsky,1 Dmitry V Basmanov,1 Dmitry V Klinov1 1Department of Biophysics, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency (FRCC PCM), Moscow, Russia; 2Ivanovsky Institute of Virology of the National Research Center of Epidemiology and Microbiology of N.F. Gamaleya of the Russian Ministry of Health, Moscow, Russia; 3Moscow Institute of Physics and Technology, Moscow, Russia; 4Department of Bioengineering, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia Purpose: To develop a general method for NP fabrication from various proteins with maintenance of biological activity. Methods: A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay. Results: Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20–250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls. Conclusion: Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities. Keywords: protein nanostructures, nanoprecipitation, atomic force and electron microscopy, energy-dispersive X-ray spectroscopy, ultraviolet and circular dichroism spectroscopy, ELISA, lysozyme activity assa