Rebecca J. Green

Surface plasmon resonance analysis of dynamic biological interactions with biomaterials

Surface plasmon resonance (SPR) is an optical technique that is widely gaining recognition as a valuable tool to investigate biological interactions. SPR offers real time in situ analysis of dynamic surface events and, thus, is capable of defining rates of adsorption and desorption for a range of surface interactions. In this review we highlight the diversity of SPR analysis. Examples of a wide range of applications of SPR are presented, concentrating on work relevant to the analysis of biomaterials. Particular emphasis is given to the use of SPR as a complimentary tool, showing the broad range of techniques that are routinely used alongside SPR analysis.

Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces.

The adsorption of a range of plasma proteins to metal and polymer surfaces has been examined using surface plasmon resonance (SPR). The adsorption of proteins was initially studied on the SPR silver sensor surface, and then on a model polystyrene film spun coated directly onto this substrate. In both cases, reproducible adsorption profiles for albumin were attained which compared well with corresponding atomic force microscopy (AFM) and ellipsometry data on protein monolayer packing and thickness respectively. The SPR data revealed the influence of concentration on both protein adsorption kinetics and the time for formation of a monolayer coating. SPR data also highlighted different adsorption kinetics and final monolayer SPR angle shift values for three plasma proteins which have been interpreted in terms of their molecular dimensions and orientation at the polymer interface. AFM data confirmed the presence of a closely packed protein layer for all three protein systems. These studies are discussed in terms of employing SPR in the study of protein interactions at surfaces which are important in the design and evaluation of novel biomedical polymeric materials.

Interactions of tea tannins and condensed tannins with proteins.

Binding parameters for the interactions of four types of tannins: tea catechins, grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins, and sorghum procyanidins (mDP=17), with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction with gelatin were in the range 10(4) to 10(6) M(-1) and in the order: sorghum procyanidins > grape seed proanthocyanidins > mimosa 5-deoxy proanthocyanidins > tea catechins. Interaction with BSA was generally weaker, with equilibrium binding constants of < or =10(3)M(-1) for grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins and tea catechins, and 10(4)M(-1) for the sorghum procyanidins. In all cases the interactions with proteins were exothermic and involved multiple binding sites on the protein. The data are discussed in relation to the structures and the known nutritional effects of the condensed tannins.

Competitive protein adsorption as observed by surface plasmon resonance

The competitive nature of protein adsorption has been investigated in situ by surface plasmon resonance (SPR) analysis. The adsorption from blood plasma solutions of albumin, fibrinogen and immunoglobulin-G (IgG), to a polystyrene surface was investigated as part of concentration- and time-dependent studies, to observe the sequential adsorption of the three proteins at the surface. Adsorption of plasma solutions at a range of concentrations or incubation times was performed and the resulting surfaces were probed by the addition of an appropriate antibody to the protein surface. The process was repeated for each antigen leading to a surface concentration profile of each protein with respect to plasma concentration and plasma incubation time. The SPR was able to detect changes in the relative surface concentration of each component demonstrating that the proteins residence time at the interface was dependent upon its molecular weight, bulk concentration and surface affinity. All ri,hts reserved

A surface plasmon resonance study of albumin adsorption to PEO–PPO–PEO triblock copolymers

Pluronic surfactants, PEO-PPO-PEO triblock copolymers, have been investigated widely due to their protein-resistant properties in applications as coatings for implants and in controlled drug release systems. We have studied a wide range of these copolymers, varying in both PEO and PPO block size, by adsorbing them to a polystyrene surface and investigating their subsequent resistance to human serum albumin adsorption. This investigation has been carried out in real time, using surface plasmon resonance, with the surfaces subsequently visualized by atomic force microscopy. This approach has allowed determination of the effect of the lengths of the PEO and PPO polymer chains on protein resistivity. For low-molecular-weight Pluronics a significant, yet not complete, reduction in albumin adsorption has been observed whereas higher molecular weight Pluronics appear to completely inhibit adsorption within the time frame of this experiment. An increase in the PPO block size of the copolymer also appears to increase its protein resistance. This work further confirms that the binding strength of the anchoring block to the hydrophobic surface, rather than the length of the protruding hydrophilic PEO chains, determines a copolymers protein resistance capability.

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG.

Aims:  To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco‐2 cells.

Binding of pentagalloyl glucose to two globular proteins occurs via multiple surface sites

The interaction between pentagalloyl glucose (PGG) and two globular proteins, bovine serum albumin (BSA) and ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco), was investigated by isothermal titration calorimetry (ITC). ITC data fit to a binding model consisting of two sets of multiple binding sites, which reveal similarities in the mode of binding of PGG to BSA and rubisco. In both cases, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface, which promotes aggregation and precipitation of the PGG-protein complex. This model was confirmed by turbidimetry analysis of the PGG-BSA interaction. Analysis of tryptophan fluorescence quenching during the interaction of PGG with BSA suggests that binding of PGG leads to some conformational changes that are energetically closer to the unfolded state of the BSA structure, because small red shifts in the resulting emission spectra were observed.

Polymer-drug conjugates for combination anticancer therapy: investigating the mechanism of action.

We developed a family of polymer-drug conjugates carrying the combination of the anticancer agent epirubicin (EPI) and nitric oxide (NO). EPI-PEG-(NO)8, carrying the highest content of NO, displayed greater activity in Caco-2 cells while it decreased toxicity against endothelium cells and cardiomyocytes with respect to free EPI. FACS and confocal microscopy confirmed conjugates internalization. Light scattering showed formation of micelle whose size correlated with internalization rate. EPI-PEG-(NO)8 showed increased bioavailability in mice compared to free EPI.

Competitive adsorption of lysozyme and C12E5 at the air/liquid interface

We have studied the adsorption of lysozyme and pentaethylene glycol monododecyl ether (C12E5) at the air/water interface using neutron reflection and surface tension measurements. The effect of C12E5 concentration was examined at three fixed lysozyme concentrations of 0.01, 1 and 4 g dm−3. The surface tension showed little variation with the addition of C12E5 over the low surfactant concentration region, but with the increase of C12E5 concentration, the surface tension gradually became identical to that corresponding to pure C12E5. These results suggest a progressive replacement of lysozyme by C12E5 and that the observed surface event is dominated by competitive adsorption. The parallel neutron measurements showed that, at low surfactant concentration, the surface was predominantly occupied by lysozyme. At intermediate C12E5 concentrations, the surface layer consisted of both lysozyme and C12E5, with the C12E5 eventually completely replacing the adsorbed lysozyme as the surfactant concentration was further increased. While the neutron results confirm the inference from surface tension measurement, structural analysis clearly showed the partial breakdown of the globular structure of lysozyme induced by the nonionic surfactant. Furthermore, neutron data showed that the adsorbed C12E5 molecules are present at the top surface layer only, suggesting no preferential association or binding between the surfactant and any immersed protein fragments at the interface.

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV).