Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and C-H stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed 'spectroscopic protein-to-lipid ratio', combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05 mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with -turns and unordered motifs at the expense of intermolecular -sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research.

Blood serum fractions are hotly debated adjuvants in bone replacement therapies. In the present experiment, we coated demineralized bone matrices (DBM) with serum albumin and investigated stem cell attachment in vitro and bone formation in a rat calvaria defect model. In the in vitro experiments, we observed that significantly more cells adhere to the serum albumin coated DBMs at every time point. In vivo bone formation with albumin coated and uncoated DBM was monitored biweekly by computed tomography until 11 weeks postoperatively while empty defects served as controls. By the seventh week, the bone defect in the albumin group was almost completely closed (remaining defect 3.0 ± 2.3%), while uncoated DBM and unfilled control groups still had significant defects (uncoated: 40.2 ± 9.1%, control: 52.4 ± 8.9%). Higher density values were also observed in the albumin coated DBM group. In addition, the serum albumin enhanced group showed significantly higher volume of newly formed bone in the microCT analysis and produced significantly higher breaking force and stiffness compared to the uncoated grafts (peak breaking force: uncoated: 15.7 ± 4 N, albumin 46.1 ± 11 N). In conclusion, this investigation shows that implanting serum albumin coated DBM significantly reduces healing period in nonhealing defects and results in mechanically stronger bone. These results also support the idea that serum albumin coating provides a convenient milieu for stem cell function, and a much improved bone grafting success can be achieved without the use of exogenous stem cells.

Drug binding induces the disorder-to-order conformational transition of the natively unfolded antimicrobial peptide CM15.

Extracellular vesicles (EVs) are currently in scientific focus, as they have great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, and thus, additional insight into their molecular-level properties, membrane-protein interactions, and membrane rigidity is still needed. We here demonstrate the use of red-blood-cell-derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular-level structural information on these systems. Flow-linear dichroism (flow-LD) measurements show that EVs can be oriented by shear force and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage, this interaction ceases; this is coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight into vesicle rigidity, which decreases in time parallel to changes in protein conformation. Overall, we propose that both linear dichroism and circular dichroism spectroscopy can provide simple, rapid, yet efficient ways to track changes in the membrane-protein interactions of EV components at the molecular level, which may also give insight into processes occurring during vesiculation.

A new, simple and "green" method was developed for the surface modification of 20 nm diameter Stöber silica particles with 3-aminopropyl(diethoxy)methylsilane in ethanol. The bulk polycondensation of the reagent was inhibited and the stability of the sol preserved by adding a small amount of glacial acetic acid after appropriate reaction time. Centrifugation, ultrafiltration and dialysis were compared in order to choose a convenient purification technique that allows the separation of unreacted silylating agent from the nanoparticles without destabilizing the sol. The exchange of the solvent to acidic water during the purification yielded a stable colloid, as well. Structural and morphological analysis of the obtained aminopropyl silica was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements, Fourier-transform infrared (FTIR), 13 C and 29 Si MAS nuclear magnetic resonance (NMR) spectroscopies, as well as small angle X-ray scattering (SAXS). Our investigations revealed that the silica nanoparticle surfaces were partially covered with aminopropyl groups, and multilayer adsorption followed by polycondensation of the silylating reagent was successfully avoided. The resulting stable aminopropyl silica sol (ethanolic or aqueous) is suitable for biomedical uses due to its purity.

The protein mediated approach is a common method for the synthesis of photoluminescent gold quantum clusters (GQCs), where proteins, acting as reducing and stabilizing agents, react with gold salts through cysteine residues. For the better understanding of the phenomenon, the aqueous phase reaction of HAuCl4 and L-cysteine has been investigated at the supramolecular level by various experimental techniques and molecular mechanics simulations. We have observed the formation of a novel photoluminescent product, (AuCys)n β , which shows emission in the orange region of the spectrum. Small-and wide-angle X-ray scattering (SWAXS) measurements have revealed the presence of nanosized lamellae, which have an internal multilayer superlattice structure with a characteristic periodic distance of 1.3 nm. Based on the results, the layers are built up by zigzag shaped (AuCys)n polymer chains connected through aurophilic bonds. The aurophilic network is stabilized via salt bridges and hydrogen bonds, which are also responsible for the interlayer interactions. Here, the evolution of the multilayer structure has been monitored by the combined application of photoluminescence spectroscopy and time-resolved SAXS. It has been concluded that there is a strong correlation between the emission and the scattering intensity, which suggests that the two-and three-dimensional aggregation of the building blocks to form sheets and multilayers are simultaneous processes. Furthermore, we have revealed that the formation and behavior of (AuCys)n β show significant differences to that of Au-L-glutathione compounds desrcibed earlier despite the similarity of L-cysteine and L-glutathione. These results evidence that L-cysteine and gold species form building blocks that can be applied expansively in supramolecular and cluster chemistry.

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Besides the outstanding potential in biomedical applications, extracellular vesicles (EVs) are also promising candidates to expand our knowledge on interactions between vesicular surface proteins and small-molecules which exert biomembrane-related functions. Here we provide mechanistic details on interactions between membrane active peptides with antimicrobial effect (MAPs) and red blood cell derived EVs (REVs) and we demonstrate that they have the capacity to remove members of the protein corona from REVs even at lower than 5 µM concentrations. In case of REVs, the Soret-band arising from the membrane associated hemoglobins allowed to follow the detachment process by flow-Linear Dichroism (flow-LD). Further on, the significant change on the vesicle surfaces was confirmed by transmission electron microscopy (TEM). Since membrane active peptides, such as melittin have the affinity to disrupt vesicles, a combination of techniques, fluorescent antibody labeling, microfluidic resistive pulse sensing, and flow-LD were employed to distinguish between membrane destruction and surface protein detachment. The removal of protein corona members is a newly identified role for the investigated peptides, which indicates complexity of their in vivo function, but may also be exploited in synthetic and natural nanoparticle engineering. Furthermore, results also promote that EVs can be used as improved model systems for biophysical studies providing insight to areas with so far limited knowledge.