Purnendu Parhi

Role of Proteins and Water in the Initial Attachment of Mammalian Cells to Biomedical Surfaces: A Review

Anchorage-dependent mammalian cells are typically grown in vitro on hydrophilic glass and plastic substrata in a medium supplemented with 5–20% v/v blood-serum proteins. Inoculated single cells gravitate from suspension to within close proximity of substrata surfaces whereupon initial contact and attachment occurs followed by progressive cell adhesion, spreading, and ultimately proliferation. A critical examination of the role of proteins and water in the initial attachment phase concludes that the cell attachment phase is not mediated by biological recognition of surface-adsorbed ligands by cell membrane receptors as frequently depicted in various textbook explanations of cell adhesion. This conclusion is based on extensive experimental evidence showing that blood proteins do not adsorb on hydrophilic surfaces that are most conducive to cell growth but do adsorb on hydrophobic surfaces that are not conducive to cell growth. As a consequence, the conventional idea that initial cell attachment is mediated by various adhesin factors adsorbed from serum-protein solutions is viewed as untenable. Rather, it is concluded that the initial contact-and-attachment of cells to hydrophilic surfaces is controlled by physicochemical interactions unrelated to biological recognition. The general physics of these interactions is known but an adequate descriptive theory that can be tested against experimentally measured cell adhesion kinetics has yet to be developed. The role of these physicochemical interactions in stimulating biological machinery within cells to fully adhere and proliferate on surfaces of biotechnical interest is unknown but is of great significance to the science underlying various biomedical and biotechnical applications of materials.

Volumetric interpretation of protein adsorption: kinetics of protein-adsorption competition from binary solution.

The standard solution-depletion method is implemented with SDS-gel electrophoresis as a multiplexing, separation-and-quantification tool to measure competition between two proteins (i and j) for adsorption to the same hydrophobic adsorbent particles (either octyl sepharose or silanized glass) immersed in binary-protein solutions. Adsorption kinetics reveals an unanticipated slow protein-size-dependent competition that controls steady-state adsorption selectivity. Two sequential pseudo-steady-state adsorption regimes (State 1 and State 2) are frequently observed depending on i, j solution concentrations. State 1 and State 2 are connected by a smooth transition, giving rise to sigmoidally-shaped adsorption-kinetic profiles with a downward inflection near 60 min of solution/adsorbent contact. Mass ratio of adsorbed i, j proteins (m(i)/m(j)) remains nearly constant between States 1 and 2, even though both m(i) and m(j) decrease in the transition between states. State 2 is shown to be stable for 24 h of continuous-adsorbent contact with stagnant solution whereas State 2 is eliminated by continuous mixing of adsorbent with solution. In sharp contrast to binary-competition results, adsorption to hydrophobic adsorbent particles from single-protein solutions (pure i or j) exhibits no detectable kinetics within the timeframe of experiment from either stagnant or continuously mixed solution, quickly achieving a single steady-state value in proportion to solution concentration. Comparison of binary competition between dissimilarly-sized protein pairs chosen to span a broad molecular-weight (MW) range demonstrates that selectivity between i and j scales with MW ratio that is proportional to protein-volume ratio (ubiquitin, Ub, MW=10.7 kDa; human serum albumin, HSA, MW=66.3 kDa; prothrombin, FII, 72 kDa; immunoglobulin G, IgG, MW=160 kDa; fibrinogen, Fib, MW=341 kDa). Results are interpreted in terms of a kinetic model of adsorption that has protein molecules rapidly diffusing into an inflating interphase that is spontaneously formed by bringing a protein solution into contact with a physical surface (State 1). State 2 follows by rearrangement of proteins within this interphase to achieve the maximum interphase concentration (dictated by energetics of interphase dehydration) within the thinnest (lowest volume) interphase possible by ejection of interphase water and initially-adsorbed proteins. Implications for understanding biocompatibility are discussed using a computational example relevant to the problem of blood-plasma coagulation.

Surface-Energy Dependent Contact Activation of Blood Factor XII

Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution exhibits a parabolic profile when scaled as a function of silanized-glass-particle activator surface energy (measured as advancing water adhesion tension tau(a)(o)=gamma(lv)(o)cos theta in dyne/cm, where gamma(lv)(o) is water interfacial tension in dyne/cm and theta is the advancing contact angle). Nearly equal activation is observed at the extremes of activator water-wetting properties -36<tau(a)(o)<72 dyne/cm (0 degrees <or=theta<120 degrees), falling sharply through a broad minimum within the 20<tau(a)(o)<40 dyne/cm (55 degrees <theta<75 degrees) range over which activation yield (putatively FXIIa) rises just above detection limits. Activation is very rapid upon contact with all activators tested and did not significantly vary over 30 min of continuous FXII-procoagulant contact. Results suggest that materials falling within the 20<tau(a)(o)<40 dyne/cm surface-energy range should exhibit minimal activation of blood-plasma coagulation through the intrinsic pathway. Surface chemistries falling within this range are, however, a perplexingly difficult target for surface engineering because of the critical balance that must be struck between hydrophobicity and hydrophilicity. Results are interpreted within the context of blood plasma coagulation and the role of water and proteins at procoagulant surfaces.

Volumetric interpretation of protein adsorption: Interfacial packing of protein adsorbed to hydrophobic surfaces from surface-saturating solution concentrations

The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square or hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square or hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM.

Contact activation of blood plasma and factor XII by ion-exchange resins.

Sepharose ion-exchange particles bearing strong Lewis acid/base functional groups (sulfopropyl, carboxymethyl, quaternary ammonium, dimethyl aminoethyl, and iminodiacetic acid) exhibiting high plasma protein adsorbent capacities are shown to be more efficient activators of blood factor XII in neat-buffer solution than either hydrophilic clean-glass particles or hydrophobic octyl sepharose particles (FXII (activator)→(surface) FXIIa; a.k.a autoactivation, where FXII is the zymogen and FXIIa is a procoagulant protease). In sharp contrast to the clean-glass standard of comparison, ion-exchange activators are shown to be inefficient activators of blood plasma coagulation. These contrasting activation properties are proposed to be due to the moderating effect of plasma-protein adsorption on plasma coagulation. Efficient adsorption of blood-plasma proteins unrelated to the coagulation cascade impedes FXII contacts with ion-exchange particles immersed in plasma, reducing autoactivation, and causing sluggish plasma coagulation. By contrast, plasma proteins do not adsorb to hydrophilic clean glass and efficient autoactivation leads directly to efficient activation of plasma coagulation. It is also shown that competitive-protein adsorption can displace FXIIa adsorbed to the surface of ion-exchange resins. As a consequence of highly-efficient autoactivation and FXIIa displacement by plasma proteins, ion-exchange particles are slightly more efficient activators of plasma coagulation than hydrophobic octyl sepharose particles that do not bear strong Lewis acid/base surface functionalities but to which plasma proteins adsorb efficiently. Plasma proteins thus play a dual role in moderating contact activation of the plasma coagulation cascade. The principal role is impeding FXII contact with activating surfaces, but this same effect can displace FXIIa from an activating surface into solution where the protease can potentiate subsequent steps of the plasma coagulation cascade.

Experimental and theoretical studies on localized surface plasmon resonance based fiber optic sensor using graphene oxide coated silver nanoparticles

An optical fiber based refractive index sensor using graphene oxide (GO) encapsulated silver nanoparticles (AgNPs) is reported. The AgNPs are encapsulated with a very thin layer of GO as it controls the inter-particle distance thereby preventing aggregation. The encapsulation also enhances the colloidal stability and prevents the oxidation of the AgNPs by separating them from direct contact with the aqueous medium. High-resolution transmission electron microscopy results support the formation of 1 nm thick GO around AgNPs of an average size of 35 nm. A Raman spectrometer and a UV–VIS spectrometer have been used to characterize and study the synthesized nanoparticles along with GO. Further, Raman spectra support a 64.72% increase in D-peak intensity and a 52.91% increase in G-peak intensity of the GO-encapsulated AgNPs (GOE-AgNPs) with respect to GO. Further, the GOE-AgNPs are immobilized on the core of functionalized plastic-cladded silica fiber. FESEM confirms the immobilization of the GOE-AgNPs on the fiber core. We observed that the peak absorbance changes by 87.55% with a 0.05 change in the refractive index. The sensitivity of the proposed fiber sensor is found to be 0.9406 ΔA/RIU along with a resolution of 12.8 × RIU. MATLAB is used to calculate the absorbance of the AgNPs by considering the bound and free electron contribution along with the size-dependent dispersion of the nanoparticles. We found that the simulation results are in good agreement with the experimental results.

Nano ceria supported nitrogen doped graphene as a highly stable and methanol tolerant electrocatalyst for oxygen reduction

Ceria (CeO2) nanoparticles with ellipsoid shape are coupled on a nitrogen doped reduced graphene oxide sheet through a single step solvothermal procedure. This non-precious-metal based nanocomposite material displayed enhanced electrochemical oxygen reduction activity with a 4e− process similar to commercial Pt/C but with much higher stability and methanol tolerant properties.

Electrophoretic Implementation of the Solution-Depletion Method for Measuring Protein Adsorption, Adsorption Kinetics, and Adsorption Competition Among Multiple Proteins in Solution

The venerable solution-depletion method is perhaps the most unambiguous method of measuring solute adsorption from solution to solid particles, requiring neither complex instrumentation nor associated interpretive theory. We describe herein an SDS-gel electrophoresis implementation of the solution--depletion method for measuring protein adsorption and protein-adsorption kinetics. Silanized-glass particles with different surface chemistry/energy and hydrophobic sepharose-based chromatographic media are used as example adsorbents. Electrophoretic separation enables quantification of adsorption competition among multiple proteins in solution for the same adsorbent surface, demonstrated herein by adsorption--competition kinetics from binary solution.

Nanoceria-supported NrGO as highly stable electrocatalyst for oxygen reduction

Today, the world is facing a severe challenge due to depletion of traditional fossil fuels. Scientists across the globe are working for solution that involves a dramatic shift to practical and environmentally sustainable energy sources. High-capacity energy systems, such as metal–air batteries, fuel cells, are highly desirable to meet the urgent requirement of sustainable energies. Among the fuel cells Direct methanol fuel cells (DMFCs) are recognized as an ideal power source for mobile applications and have received considerable attention in recent past. In this advanced electrochemical energy conversion technologies Oxygen Reduction Reaction (ORR) is of utmost importance. However, the poor kinetics of cathodic ORR in DMFCs significantly hampers their possibilities of commercialization. The oxygen is reduced in alkaline medium either through a 4-electron (equation i) or a 2-electron (equation ii) reduction pathway at the cathode as given below. O2 + 2H2O + 4e.......... 4OH...............................(i) O2 + H2O + 2e.......... OH+ HO2-........................(ii) The sluggish kinetics of ORR, demands high loading of precious metal-containing catalysts (e.g., Pt), which unfavorably increases the cost of these electrochemical energy conversion devices. Therefore, synthesis of active electrocatalyst with increase in ORR performance is need of the hour. In the recent literature there are many reports on transition metal oxide (TMO) based ORR catalysts (Co, Mn, Cu, Fe oxides) for their high activity. It was found that 2D graphene layer having high electrical conductivity, large surface area and excellent chemical stability, appeared to be an ultimate choice as support material to enhance the catalytic performance of bare TMOs. TMOs are also having drawbacks like low electrical conductivity, which seriously affects the electron transfer process during ORR. Also during the practical test for some of the synthesized electrocatalysts methanol crosses over the electrolyte membrane and contaminate the cathode compartment which abruptly disturbs the ORR process. So methanol tolerant capability of ORR catalyst is another important factor needs consideration prior to practical application. Here we are reporting the synthesis of CeO2/NrGO nanocomposite via single step microwave solvothermal method which displayed very good oxygen reduction activity with nearly four electron transfer pathway in alkaline medium, which is similar to commercial Pt/C. Furthermore, the CeO2/NrGO exhibit superior electrochemical stability and methanol tolerance capability to that of commercial Pt/C. Growth of CeO2 on graphene surface has been discussed. 1. Soren, S. et.al. (2016) RSC Adv. 6, 77100-77104. 2. Huang, X. et.al. (2015) Science 348, 1230-1234. 3. Suntivich, J. (2013) Nat. Chem. 3, 546–550.