Lateral Control of Protein Adsorption on Charged Polymer Gradients

Abstract

This work describes the fabrication, characterization, and protein adsorption behavior of charged polymer gradients. The thin gradient films were fabricated by a two-step technique using UV-initiated free-radical polymerization in a reactor with a moving shutter. A homogeneous layer of cationic poly(2-aminoethyl methacrylate hydrochloride) was first formed, followed by a layer of oppositely charged poly(2-carboxyethyl acrylate) with a continuously increasing thickness. Adsorption from protein solutions as well as human blood plasma was investigated by imaging surface plasmon resonance and infrared microscopy. The results showed excessive protein adsorption in the areas where one of the polymers dominated the composition, while there was a clear minimum at an intermediate position of the gradient. The charge of the surface was estimated by direct force measurements and found to correlate well with the protein adsorption, showing the lowest net charge in the same area as the protein adsorption minimum. We therefore hypothesize that a combination of the charged polymers, in the right proportions, can result in a protein-resistant surface due to balanced charges.

pH-control of the protein resistance of thin hydrogel gradient films

“…Between the areas where pepsin and lysozyme adsorb, there is at every pH a region of very low adsorption, which shifts via the pH. In conjunction with the data which is presented and discussed above, this confirms previous suggestions that this region is charge-neutral, 16,38 and that the protein resistance in this area stems from the elimination of electrostatic interaction, possibly in combination with strong hydration of ionizable groups of the hydrogels.…”

“…From the sequential adsorption of the two proteins, it is evident that pepsin and lysozyme adsorb to the left and right sides of the gradient in Figure 6, respectively (this is not explicitly shown here, but see Figure 4 in ref 16 for an example). This indicates the presence of charges of opposite sign on the gradient at pH 4.3, though the amount of adsorbed lysozyme was lower, probably due to the lower degree of ionization of the PCEA, as concluded from both the swelling profiles and the force measurements.…”

“…16,32 is plotted over the gradient surface, to indicate the amount of protein adsorption. Figure 6a shows the protein adsorption on the P(AEMA-CEA) gradient as the pH is varied from 4.3 to 7.5.…”

“…16,38 Further, in Figure 2, PCEA has a larger pH-dependent swelling compared to PAEMA which results in a dilute negative charge volume, possibly allowing diffusion of lysozyme into the polymer to a greater extent at high pH, which then may prevent desorption as the pH is decreased. This would be in agreement also with the result that pepsin appears to desorb from the PAEMA region in figure 6a, which showed the least degree of swelling.…”

“…In previous work we have demonstrated that such a hydrogel gradient fabricated from two oppositely charged polymers grafted in sequence, displays a region of high protein resistance. 16 This region is characterized by charge-balance and reduced swelling due to the absence of charge-charge repulsion, whereas on either side of this charge-balanced region on the gradient there was net surface charge, significant swelling, and protein adsorption was considerable. The use of a gradient surface with continuously varying properties, instead of a polymer bilayer of fixed composition, permits parallel studies of both protein-resistant regions and nearby, less resistant regions of related composition and chemistry.…”