Protein nanoparticles: cellular uptake, intracellular distribution, biodegradation and induction of cytokine gene expression.

Intracellular delivery of protein nanoparticles (NP) is required for nanomedicine. Our research was focused on the quantitative analysis of protein NP intracellular accumulation and biodegradation in dynamics along with host cytokine gene expression. Fluorescent NP fabricated by nanoprecipitation without cross-linking of bovine serum albumin (BSA) and human immunoglobulins (hIgG) pre-labeled with Rhodamine B were non-toxic for human cells. Similar gradual uptake of the NP during 2 days and subsequent slowdown until background values for 5 days for human cell lines and donor blood mononuclear cells revealed that NP internalization was neither cell-type nor protein-specific. NP delivery into cells was inhibited by homologous and heterologous NP but did not depend on the presence of BSA or hIgG in culture media. The protein NP internalization induced interferon α, ß, λ but neither γ nor interleukin 4 and 6 gene expression. Accordingly, cellular uptake of non-toxic protein NP induced Th1 polarized innate response.

Myeloperoxidase-induced fibrinogen unfolding and clotting.

Due to its unique properties and high biomedical relevance fibrinogen is a promising protein for the development of various matrixes and scaffolds for biotechnological applications. Fibrinogen molecules may form extensive clots either upon specific cleavage by thrombin or in thrombin-free environment, for example, in the presence of different salts. Here, we report the novel type of non-conventional fibrinogen clot formation, which is mediated by myeloperoxidase and takes place even at low fibrinogen concentrations (<0.1 mg/ml). We have revealed fibrillar nature of myeloperoxidase-mediated fibrinogen clots, which differ morphologically from fibrin clots. We have shown that fibrinogen clotting is mediated by direct interaction of myeloperoxidase molecules with the outer globular regions of fibrinogen molecules followed by fibrinogen unfolding from its natural trinodular to a fibrillar structure. We have demonstrated a major role of the Debye screening effect in regulating of myeloperoxidase-induced fibrinogen clotting, which is facilitated by small ionic strength. While fibrinogen in an aqueous solution with myeloperoxidase undergoes changes, the enzymatic activity of myeloperoxidase is not inhibited in excess of fibrinogen. The obtained results open new insights into fibrinogen clotting, give new possibilities for the development of fibrinogen-based functional biomaterials, and provide the novel concepts of protein unfolding.

Imaging human keratinocytes grown on electrospun mats by scanning electron microscopy.

Drying is a critical step in preparing cell samples for examination with scanning electron microscopy (SEM). The two commonly used drying procedures are the critical point drying (CPD) and the chemical drying using hexamethyldisilazane (HMDS drying). Here we compared the application of these procedures for the drying of HaCaT human keratinocyte cells grown on electrospun nylon mats. Both drying procedures allowed us to obtain images of the cells and characterize the microvilli on the cell surface. After HMDS drying the membrane was less damaged than after CPD. Both drying procedures could be used to investigate contact guidance-the substrate-induced changes in cell shape. The aspect ratio of HaCaT cells grown on the aligned and random mats was 4.2 ± 2.8 and 1.5 ± 0.3, respectively.

Protein nanoparticles with ligand-binding and enzymatic activities.

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.