Neutron Reflectometry Elucidates Protein Adsorption from Human Blood Serum onto PEG Brushes.

Poly(ethylene glycol) (PEG) brushes are reputed for their ability to prevent undesired protein adsorption to material surfaces exposed to biological fluids. Here, protein adsorption out of human blood serum onto PEG brushes anchored to solid-supported lipid monolayers was characterized by neutron reflectometry, yielding volume fraction profiles of lipid headgroups, PEG, and adsorbed proteins at subnanometer resolution. For both PEGylated and non-PEGylated lipid surfaces, serum proteins adsorb as a thin layer of approximately 10 Å, overlapping with the headgroup region. This layer corresponds to primary adsorption at the grafting surface and resists rinsing. A second diffuse protein layer overlaps with the periphery of the PEG brush and is attributed to ternary adsorption due to protein-PEG attraction. This second layer disappears upon rinsing, thus providing a first observation of the structural effect of rinsing on protein adsorption to PEG brushes.

General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires.

Recent reports on the hitherto underestimated antigenicity of poly(ethylene glycol) (PEG), which is widely used for pharmaceutical applications, highlight the need for efficient testing of polymer antigenicity and for a better understanding of its molecular origins. With this goal in mind, we have used the phage-display technique to screen large, recombinant antibody repertoires of human origin in vitro for antibodies that bind poly(vinylpyrrolidone) (PVP). PVP is a neutral synthetic polymer of industrial and clinical interest that is also a well-known model antigen in animal studies, thus allowing the comparison of in vitro and in vivo responses. We have identified 44 distinct antibodies that bind specifically to PVP. Competitive binding assays show that the PVP-antibody binding constant is proportional to the polymerization degree of PVP and that specific binding is detected down to the vinylpyrrolidone (VP) monomer level. Statistical analysis of anti-PVP antibody sequences identifies an amino-acid motif that is shared by many phage-display-selected anti-PVP antibodies that are similar to a previously described natural anti-PVP antibody. This suggests a role for this motif in specific antibody/PVP interactions. Interestingly, sequence analysis also suggests that only a single antibody chain containing this shared motif is responsible for antibody binding to PVP, as confirmed upon systematic deletion of either antibody chain for 90% of selected anti-PVP antibodies. Overall, a large number of antibodies in the human repertoires we have screened bind specifically to PVP through a small number of shared amino acid motifs, and preliminary comparison points to significant correlations between the sequences of phage-display-selected anti-PVP antibodies and their natural counterparts isolated from immunized mice in previous studies. This study pioneers the use of antibody phage-display to explore the antigenicity of biotechnologically relevant polymers. It also paves the way for a fast, cost-effective, and systematic in vitro analysis, thus reducing the need for animal immunization experiments. Moreover, identifying the encoding DNA sequence of polymer-binding antibodies via phage-display enables future applications of a molecular biology approach to protein-polymer conjugation, based on protein-antibody fusion.

Neutron reflectometry elucidates density profiles of deuterated proteins adsorbed onto surfaces displaying poly(ethylene glycol) brushes: evidence for primary adsorption.

The concentration profile of deuterated myoglobin (Mb) adsorbed onto polystyrene substrates displaying poly(ethylene glycol) (PEG) brushes is characterized by neutron reflectometry (NR). The method allows to directly distinguish among primary adsorption at the grafting surface, ternary adsorption within the brush, and secondary adsorption at the brush outer edge. It complements depth-insensitive standard techniques, such as ellipsometry, radioactive labeling, and quartz crystal microbalance. The study explores the effect of the PEG polymerization degree, N, and the grafting density, σ, on Mb adsorption. In the studied systems there is no indication of secondary or ternary adsorption, but there is evidence of primary adsorption involving a dense inner layer at the polystyrene surface. For sparsely grafted brushes the primary adsorption involves an additional dilute outer protein layer on top of the inner layer. The amount of protein adsorbed in the inner layer is independent of N but varies with σ, while for the outer layer it is correlated to the amount of grafted PEG and is thus sensitive to both N and σ. The use of deuterated proteins enhances the sensitivity of NR and enables monitoring exchange between deuterated and hydrogenated species.

Thermoresponsive cell culture substrates based on PNIPAM brushes functionalized with adhesion peptides: theoretical considerations of mechanism and design.

Thermoresponsive tissue culture substrates based on PNIPAM brushes are used to harvest confluent cell sheets for tissue engineering. The prospect of clinical use imposes the utilization of culture medium free of bovine serum, thus suggesting conjugation with adhesion peptides containing the RGD minimal recognition sequence. The optimum position of the RGD along the chain should ensure both cell adhesion at 37 °C and cell detachment at T(L) below the lower critical solution temperature of PNIPAM. Design guidelines are formulated from considerations of brush confinement by the cells: (i) Cell adhesion at 37 °C is controlled by the RGDs accessible without brush compression. (ii) Cell detachment at T(L) is driven by a disjoining force due to confinement of the swollen brush by cells retaining integrin-RGD bonds formed at 37 °C. These suggest placing the RGDs at the grafting surface or its vicinity. Randomly placed RGDs do not enable efficient detachment because a large fraction of the integrin-RGD bonds are not sufficiently tensioned at T(L), in line with experimental observations (Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Immobilization of celladhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest. Tissue Eng. 2004, 10, 1125-1135). The theory framework enables analysis of culture media based on polymer brushes conjugated with adhesion peptides in general.

Neutron reflectometry from poly (ethylene-glycol) brushes binding anti-PEG antibodies: evidence of ternary adsorption.

Neutron reflectometry provides evidence of ternary protein adsorption within polyethylene glycol (PEG) brushes. Anti-PEG Immunoglobulin G antibodies (Abs) binding the methoxy terminated PEG chain segment specifically adsorb onto PEG brushes grafted to lipid monolayers on a solid support. The Abs adsorb at the outer edge of the brush. The thickness and density of the adsorbed Ab layer, as well as its distance from the grafting surface grow with increasing brush density. At high densities most of the protein is excluded from the brush. The results are consistent with an inverted "Y" configuration with the two FAB segments facing the brush. They suggest that increasing the grafting density favors narrowing of the angle between the FAB segments as well as overall orientation of the bound Abs perpendicular to the surface.

Theoretical considerations on mechanisms of harvesting cells cultured on thermoresponsive polymer brushes.

Poly (N-isopropylacrylamide) (PNIPAM) brushes and hydrogels serve as temperature-responsive cell culture substrates. The cells adhere at 37 °C and are detached by cooling to below the lower critical solution temperature T(LCST) ≈ 32 °C, an effect hitherto attributed to change in PNIPAM hydration. The article proposes a mechanism coupling the change of hydration to integrin mediated environmental sensing for cell culture on brushes and hydrogels in serum containing medium. Hydration is associated with swelling and higher osmotic pressure leading to two effects: (i) The lower osmotic pressure in the collapsed brush/hydrogel favors the adsorption of serum borne extracellular matrix (ECM) proteins enabling cell adhesion; (ii) Brush/hydrogel swelling at T < T(LCST) gives rise to a disjoining force f(cell) due to confinement by the ventral membrane of a cell adhering via integrin-ECM bonds. f(cell) places the integrin-ECM bonds under tension thus accelerating their dissociation and promoting desorption of ECM proteins. Self consistent field theory of PNIPAM brushes quantifies the effect of the polymerization degree N, the area per chain Σ, and the temperature, T on ECM adsorption, f(cell) and the dissociation rate of integrin-ECM bonds. It suggests guidelines for tuning Σ and N to optimize adhesion at 37 °C and detachment at T < T(LCST). The mechanism rationalizes existing experimental results on the influence of the dry thickness and the RGD fraction on adhesion and detachment.