Spontaneous Adsorption Of Proteins Research Articles

In Ukrainian

The aim of the research is the development of compositions and technology for obtaining bioactive glass-ceramic calcium phosphate-silicate coatings on titanium for dental prosthetics, to find the relationship between the structural parameters of the calcium phosphate-silicate coatings surface on titanium with their bioactivity. The structure of the material was studied by means of petrographic analysis (microscope Mi-2e) and X-ray phase analysis (DRON-3M). Solubility and bioactivity of coatings were evaluated in solutions: distilled water (GOST R 31576-2012), SBF (ISO 23317:2012), 10 % albumin solution. The surface free energy was determined by the Owens-Wendet-Rebel-Kaebel method. The surface microrelief of the coatings was assessed by the mean arithmetic deviation of the profile Ra (Surtronic 3+ profilometer). The surface layer structure was investigated by X-ray fluorescence (SPRUT spectrometer-analyzer) and X-ray spectral (PEM Tesla 3 LMU + oxygen spectrometer Oxford X-max 80 mm) methods. The behavior of stem cells on the coatings surface was investigated by fluorescence microscopy (Carl Zeiss Axio Observer Z1). Criteria were selected of model glass synthesis and the model glasses were melted. Glass-ceramic coatings for titanium were applied by slip technology and synthesized under conditions of short-term low-temperature heat treatment. It has been found that the model glasses, after heat treatment, are characterized by behaviour of a volumetric fine-disperced (crystals ≤ 1 μm) crystallization of calcium phosphate - HAP and FAP (Σ ≈ 24÷44 % by volume). The possibility of spontaneous adsorption of proteins on the coatings surface has been confirmed due to the provision of coating parameters: SEM ≈ 65 mJ/m 2 , R a ≈ 3.5 μm and the presence of through pores with a diameter of 10÷100 μm. The changes in the structure and composition of the coatings surface after ageing in SBF have been studied for 1 month period. It has been found that the provision for these model glasses of a set of indicators, namely: solubility in distilled water (30 days) - 0.89 wt. %; ion yield of Ca 2+ is 0.26 wt. % and groups [PO 4 ] 3 – -0.162 wt. %, pH ~ 7.3, is sufficient for the formation of precursors of hydroxyapatite on the coatings surface in vitro. Gradual displacement (build-up) of the coatings surface layers (due to the dissolution of the coating and deposition of the SBF components) was found within the boundaries up to 6.7 μm and the apatite-like layer was formed in 28 days of soaking. These structural and chemical transformations of the surface of glass-ceramic coating form a durable apatite-like layer for 35 days period under in vitro conditions. The formation of this layer creates conditions for increasing the area of mesenchymal stem cell spacing on the coatings surface and allows their use in the creation of bioengineering design with stem cells (in vivo).

Steering protein and salt ad- and desorption by an electrical switch applied to polymer-coated electrodes

Although solid-phase chromatography is a well-established method for protein separation, chemically intensive and often costly regeneration steps are needed to make reuse of the adsorbent possible. Here, we demonstrate the use of electrochemical principles as sustainable alternative. We make use of spontaneous adsorption of proteins to solid electrodes and reverse this process by applying an electric potential to regenerate the interface. This allows for adsorption of proteins to take place at 0 V difference between the electrodes, due to electrostatic interactions between the protein and the electrode surface. The desorption is then triggered by applying a potential difference (−1.2 V) between the electrodes.It is demonstrated that the incorporation of negatively charged polystyrene sulfonate (PSS) or positively charged polydiallyldimethylammonium chloride (PDMAC) in or on top of the respective activated carbon electrodes increases the amount of exchanged protein from 1 to 10 mg g−1, as compared to simple activated carbon electrodes. Interestingly, salt ad- and desorption occurs in opposite cycles compared to protein ad- and desorption, resulting in simultaneous concentration and desalting of the protein when 0 V is applied. On top of that, we also found that an enrichment in β-lactoglobulin could be achieved starting from whey protein isolate. These results clearly demonstrate that electrochemical technologies can be used not only for protein separation (including removal of salt), but also for protein fractionation, while not requiring solvent use.

Carboxylic acid mediated antimicrobial activity of silicone elastomers

Due to their high biocompatibility silicone elastomers are the material of choice in many sensitive health care applications. However, the inherent hydrophobicity of the polymer makes silicones more susceptible to spontaneous protein adsorption and subsequent biofilm formation than more hydrophilic abiotic materials. Hence, the development of antimicrobial silicone elastomers could help to reduce potential biofilm-associated infections when using silicone based medical devices. In this study, we describe carboxylic-acid-modified silicone elastomers that are biocompatible and exhibit a specific antimicrobial activity against clinically relevant pathogens even after being stored in common packaging materials.

Effect of particle functionalization and solution properties on the adsorption of bovine serum albumin and lysozyme onto silica nanoparticles

Silica nanoparticles present an enormous potential as controlled drug delivery systems with high selectivity towards diseased cells. This application is directly related to the phenomenon of protein corona, characterized by the spontaneous adsorption of proteins on the nanoparticle surface, which is not fully understood. Here, we report an investigation on the influence of pH, ionic strength and temperature on the thermodynamics of interaction of bovine serum albumin protein (BSA) with non-functionalized silica nanoparticles (SiO2NPs). Complementary, we also investigated the ability of polyethylene glycol (PEG) and zwitterionic sulfobetaine (SBS) surface-modified nanoparticles to prevent the adsorption of BSA (protein negatively charged at physiological pH) and lysozyme (protein positively charged at physiological pH). We showed that BSA interaction with SiO2NPs is enthalpically governed. On the other hand, functionalization of silica nanoparticles with PEG and SBS completely prevented BSA adsorption. However, these functionalized nanoparticles presented a negative zeta potential and were not able to suppress lysozyme anchoring due to strong nanoparticle-protein electrostatic attraction. Due to the similarity of BSA with Human Serum Albumin, this investigation bears a resemblance to processes involved in the phenomenon of protein corona in human blood, producing information that is relevant for the future biomedical use of functionalized nanoparticles.

Utilizing corona on functionalized selenium nanoparticles for loading and release of doxorubicin payload

Incorporation of nanoparticles in serum containing-media leads to spontaneous adsorption of proteins on the nanoparticle surface thus forming “protein corona”. The current study focuses on the variation in corona formation across differently functionalized (CTAB, SDS and Brij-58) SeNPs surfaces and the application of these coronated-SeNPs as reservoirs for holding positively charged doxorubicin (dox). Protein quantification and densitometry study reveal that cationic SeNPs (CTAB-SeNPs) promotes maximum corona formation, and also has a higher affinity towards predominantly negatively charged serum albumin. Further, protein coronation had a significant impact on the mean hydrodynamic diameter of the functionalized SeNPs. Parameters such as surface functionalization, total adsorbed protein and bound albumin content determine the loading capacity and release of dox payload. Protein densitometry reveals, that CTAB-SeNPs has the highest amount of bound albumin which in turn aided in enhanced loading of dox on the cationic NPs. The coronated-nanocarriers were further subjected to different pH-trigger, wherein all the coronated SeNPs had an enhanced dox release at pH- 4.6. The study also highlights that protein corona helps in impeding the burst release of dox from the conjugated system. Additionally, payload leakage study suggests, that bound corona proteins help in retaining dox and prevents significant desorption of the therapeutic molecule over time. However, the presence of positively charged dox disrupts the stability of the bound corona proteins making them prone to leakage. Finally, the cellular toxicity of the functionalized NPs were assessed, wherein dox-loaded protein coronated SeNPs induced significant cell death. The feasibility of employing protein-coronated-SeNPs as a new mode of drug delivery has been highlighted in this study.

Fluorescence alteration of MPA capped CdSe quantum dots by spontaneous biomarker protein adsorption

Quantum dots (QDs) have significant potentials in biomedical applications of bioimaging and biosensing. Spontaneous adsorption of proteins on QDs surface is a common phenomenon, which occurred to serum proteins in biological samples, and has been observed to enhance QDs fluorescence. In this study, fluorescence alteration of 3-mercaptopropionic acid (MPA) capped CdSe quantum dots by four individual biomarker proteins was investigated. By monitoring the fluorescence emission of QDs, the biomarker protein adsorbed spontaneously on QDs surface was recognized and quantified. When alpha fetoprotein (AFP) or heat shock protein 90 alpha (HSP90α) were present, the QDs became brighter. The presence of cytochrome C (CytoC) or lysozyme (Lyz) made the QDs dimmer first, and then brighter. Within five minutes response time all four biomarker proteins were detected individually with the estimated detection limit in the range of 1–10 ng/mL and good linear dynamic ranges. The results suggested that the fluorescence of QDs was responsive to not only serum proteins but also biomarker proteins. The fluorescence response was able to correlate quantitatively with the amount of biomarker proteins in relatively low concentrations. These results provide more information to understand QDs and support their applications in biomedical fields.

Inorganic nanoparticles as potential regulators of immune response in dendritic cells.

The spontaneous adsorption of proteins on nanoparticles (NPs) in biological media is exploited to prepare complexes of NPs and proteins from cancer cells' lysates for application in cancer immunotherapy. Gold (Au) and silica NPs were synthesized, incubated with cancer cells' lysates and characterized. Dendritic cells (DCs) were challenged with protein-coated NPs, their maturation, viability and morphology were evaluated and lymphocytes T proliferation was determined. Silica and Au NPs bound different pools of biomolecules from lysates, and are therefore promising selective carriers for antigens. When incubated with immature DCs, NPs were efficiently endocytosed without cytotoxicity. Finally, protein-coated AuNPs promoted DC maturation and DC-mediated lymphocyte proliferation, at variance with lysate alone and protein-coated silica NPs, that did not promote DCs maturation. These results demonstrate that the spontaneous formation of protein coronas on NPs represents a possible approach to fast, easy, cost-effective DCs stimulation.

Spontaneous Protein Adsorption on Graphene Oxide Nanosheets Allowing Efficient Intracellular Vaccine Protein Delivery.

Nanomaterials hold potential of altering the interaction between therapeutic molecules and target cells or tissues. High aspect ratio nanomaterials in particular have been reported to possess unprecedented properties and are intensively investigated for their interaction with biological systems. Graphene oxide (GOx) is a water-soluble graphene derivative that combines high aspect ratio dimension with functional groups that can be exploited for bioconjugation. Here, we demonstrate that GOx nanosheets can spontaneously adsorb proteins by a combination of interactions. This property is then explored for intracellular protein vaccine delivery, in view of the potential of GOx nanosheets to destabilize lipid membranes such as those of intracellular vesicles. Using a series of in vitro experiments, we show that GOx nanosheet adsorbed proteins are efficiently internalized by dendritic cells (DCs: the most potent class of antigen presenting cells of the immune system) and promote antigen cross-presentation to CD8 T cells. The latter is a hallmark in the induction of potent cellular antigen-specific immune responses against intracellular pathogens and cancer.

Kinetics of protein adsorption on gold nanoparticle with variable protein structure and nanoparticle size

The spontaneous protein adsorption on nanomaterial surfaces and the formation of a protein corona around nanoparticles are poorly understood physical phenomena, with high biological relevance. The complexity arises mainly due to the poor knowledge of the structural orientation of the adsorbed proteins onto the nanoparticle surface and difficulties in correlating the protein nanoparticle interaction to the protein corona in real time scale. Here, we provide quantitative insights into the kinetics, number, and binding orientation of a few common blood proteins when they interact with citrate and cetyltriethylammoniumbromide stabilized spherical gold nanoparticles with variable sizes. The kinetics of the protein adsorption was studied experimentally by monitoring the change in hydrodynamic diameter and zeta potential of the nanoparticle-protein complex. To understand the competitive binding of human serum albumin and hemoglobin, time dependent fluorescence quenching was studied using dual fluorophore tags. We have performed molecular docking of three different proteins--human serum albumin, bovine serum albumin, and hemoglobin--on different nanoparticle surfaces to elucidate the possible structural orientation of the adsorbed protein. Our data show that the growth kinetics of a protein corona is exclusively dependent on both protein structure and surface chemistry of the nanoparticles. The study quantitatively suggests that a general physical law of protein adsorption is unlikely to exist as the interaction is unique and specific for a given pair.

Gel Electrophoresis Analysis of the Hard Coronas of Human Serum Albumin on Silica Nanoparticles: Size Dependence of Corona Formation

The rapid and spontaneous adsorption of proteins on nanoparticle (NP) surfaces in biological fluids such as blood is an important phenomenon as it possibly determines "what the cells see" and, thus, the fates of NPs in living organisms. In order to quantitatively understand protein coronas at the molecular level, we investigated human serum albumin (HSA) coronas that were produced on silica NPs of 20 nm and 50 nm diameters using conventional gel electrophoresis. Analysis of the concentration dependence of protein adsorption showed that HSA coronas preferentially formed a monolayer on silica NPs and revealed the presence of hard protein coronas. HSA adsorption was clearly dependent on NP size, and this might be due to the different surface curvatures of NPs of different sizes.

Do Clustering Monoclonal Antibody Solutions Really Have a Concentration Dependence of Viscosity?

Protein solution rheology data in the biophysics literature have incompletely identified factors that govern hydrodynamics. Whereas spontaneous protein adsorption at the air/water (A/W) interface increases the apparent viscosity of surfactant-free globular protein solutions, it is demonstrated here that irreversible clusters also increase system viscosity in the zero shear limit. Solution rheology measured with double gap geometry in a stress-controlled rheometer on a surfactant-free Immunoglobulin solution demonstrated that both irreversible clusters and the A/W interface increased the apparent low shear rate viscosity. Interfacial shear rheology data showed that the A/W interface yields, i.e., shows solid-like behavior. The A/W interface contribution was smaller, yet nonnegligible, in double gap compared to cone-plate geometry. Apparent nonmonotonic composition dependence of viscosity at low shear rates due to irreversible (nonequilibrium) clusters was resolved by filtration to recover a monotonically increasing viscosity-concentration curve, as expected. Although smaller equilibrium clusters also existed, their size and effective volume fraction were unaffected by filtration, rendering their contribution to viscosity invariant. Surfactant-free antibody systems containing clusters have complex hydrodynamic response, reflecting distinct bulk and interface-adsorbed protein as well as irreversible cluster contributions. Literature models for solution viscosity lack the appropriate physics to describe the bulk shear viscosity of unstable surfactant-free antibody solutions.

Effects of Pressure on the Adsorption of Proteins at Aqueous-Solid Interfaces

The spontaneous adsorption of proteins at aqueous-solid interfaces plays an important role in nature, medicine, and biotechnology. A sound understanding of the molecular mechanisms and interactions upon protein adsorption is required for the development of new materials coatings and for the choice of solvent conditions. So far, protein adsorption studies have been carried out mainly as a function of protein solution concentration, pH-value, and surface chemistry. In addition, several temperature-dependent studies have shed some light on the thermodynamics underlying protein adsorption. In contrast, effects of pressure on this process are widely unknown. Applying pressure to molecular systems generally offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. In the case of protein adsorption, using pressure as a thermodynamic variable allows for the determination of volume changes of adsorption, volume changes of unfolding in the adsorbed state, and changes of protein-interface interactions that determine the degree of protein adsorption. We have designed new high pressure cells for total internal reflection fluorescence (TIRF) spectroscopy and neutron reflectometry (NR) for pressures up to 2500 bar in order to access these quantities. The results obtained so far indicate a pressure-induced increase of the degree of protein adsorption at both the water-silica and water-poly(styrene) interfaces. Moreover, a drastic decrease of the volume change of unfolding has been found when proteins are adsorbed at the water-silica interface. Apparently, pressure exerts a distinct influence on the process of protein adsorption and provides a new complemental view on the underlying mechanism.

Electrochemically Triggered Selective Adsorption of Biotemplated Nanoparticles on Self‐Assembled Organometallic Diblock Copolymer Thin Films

AbstractThe controlled adsorption of the iron‐containing cage protein ferritin at the nanoscale using stimuli‐responsive self‐assembled diblock copolymer thin‐film templates is reported. The diblock copolymer used study consists of a cylinder‐forming polystyrene‐block‐polyferrocenylsilane (PS‐b‐PFS), with PFS as the minor block, and shows reversible redox properties. To prevent any spontaneous protein adsorption on either block, the electrolyte pH is selected to leave the ferritin negatively charged, and the protein concentration and solution ionic strength are carefully tuned. Selective adsorption of ferritin on the PFS domains of the self‐assembled thin films is then triggered in situ by applying a positive potential, simultaneously oxidizing the PFS and attracting the ferritin electrostatically.

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly( l-lysine)- g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL- g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.