One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79 °C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ‐receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs’ presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs’ influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs’ impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs’ influence on treated skeletal myoblasts, not interfering with basic cell functions.

Bioengineered spider silk is a biomaterial that has exquisite mechanical properties, biocompatibility, and biodegradability. Iron oxide nanoparticles can be applied for the detection and analysis of biomolecules, target drug delivery, as MRI contrast agents and as therapeutic agents for hyperthermia-based cancer treatments. In this study, we investigated three bioengineered silks, MS1, MS2 and EMS2, and their potential to form a composite material with magnetic iron oxide nanoparticles (IONPs). The presence of IONPs did not impede the self-assembly properties of MS1, MS2, and EMS2 silks, and spheres formed. The EMS2 spheres had the highest content of IONPs, and the presence of magnetite IONPs in these carriers was confirmed by several methods such as SEM, EDXS, SQUID, MIP-OES and zeta potential measurement. The interaction of EMS2 and IONPs did not modify the superparamagnetic properties of the IONPs, but it influenced the secondary structure of the spheres. The composite particles exhibited a more than two-fold higher loading efficiency for doxorubicin than the plain EMS2 spheres. For both the EMS2 and EMS2/IONP spheres, the drug revealed a pH-dependent release profile with advantageous kinetics for carriers made of the composite material. The composite spheres can be potentially applied for a combined cancer treatment via hyperthermia and drug delivery.

Multifunctional core/shell type nanomaterials composed of nanocrystalline, lanthanide doped fluorides and gold nanoparticles (Au NPs) were successfully prepared. The products were synthesized to combine luminescence properties of the core NPs, i.e., LnF 3 /SiO 2 −NH 2 and KLn 3 F 10 / SiO 2 −NH 2 , and plasmonic activity of the shell Au NPs within a single nanomaterial. The luminescent lanthanide NPs (10 or 150−200 nm) were separated from the gold NPs (6−30 nm) using an amine modified silica shell (thickness ≈30 nm). The synthesized products exhibited bright green (Tb 3+ ) and red (Eu 3+ ) emission under UV light irradiation. Surface modification with Au NPs influenced the product emission and luminescence decay characteristics. The luminescent-plasmonic nanomaterials were used as platforms for surface enhanced Raman scattering (SERS) measurements. 4-Mercaptobenzoic acid, choline, and T4 bacteriophages were utilized as SERS probes. For all synthesized nanomaterials, the SERS spectra for all probes studied exhibited higher intensity in comparison with the spectra measured using a commercial SERS substrate. Cytotoxicity of the products was evaluated in fibroblast cells. The results obtained showed biocompatibility of the synthesized nanomaterials in a dose-dependent manner.

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