Effect of Hydrophobicity and Electric Force on Protein Adsorption as a Function of Temperature

As mentioned above, the main driving forces for protein adsorption are electrostatic force and hydrophobic interaction. These are strongly dependent on tem-

Temperature (°C)

FIG. 6 Reduced amount (NS/NSMAX) of HIV-1 capsid P24 protein absorbed onto thermally sensitive polystyrene core cationic cross-linked poly(NIPAM) shell as a function of temperature (at pH 6.1, 10 mM phosphate buffer).

perature for thermally sensitive particles. Therefore, many studies were done on the temperature dependence of protein adsorption on thermally sensitive particles referring to the conditions affecting the microenvironment and surface properties of the particles.

For example, the high adsorption of human serum albumin (HSA) on poly-(NIPAM) particles at 40°C and low adsorption at 20°C were explained as the effect of temperature-dependent hydrophobicity of the particles, as already mentioned [11,28]. On the other hand, the effect of pH adsorption was attributed to the electrostatic effect. That is, at pH 4.7, which was close to the IEP of HSA (4.9), the adsorption was maximal and decreased with increasing pH, becoming negligible at pH 8.6. This suggested that the electrostatic force between protein molecules and particles was the determining factor for the adsorption.

The electrostatic force depends on the ionic strength. Therefore, it is expected that the adsorption would be controlled with ionic strength. The effect of ionic strength on adsorption was determined in the solution at pH 4.7 and 8.6. The effect was opposite between two pHs, i.e., adsorption decreased with increasing ionic strength at pH 4.7 and increased at pH 8.6. These results reflected a screening effect by free ions. The above results were obtained at 40°C. However, this was not the case at 20°C where the effect of ionic strength was small. This was because charges were already buried in swollen particles at 20°C [29].

Sugiyama et al. examined protein adsorption on thermally sensitive particles that are not composed of poly(NIPAM) [30]. Several copolymers of hydroxy-

propyl methacrylamide (HPMA) and alkyl methacrylate [RMA, typically methyl methacrylate (MMA)] exhibit a clear response to temperature change if the ratio of two comonomers is in a suitable range. Some kinds of polymers were prepared using different initiators in the presence or absence of a third comonomer, (methacryloyloxy)ethylphosphorylcholine (MPC). The surface potential was controlled by the kind of initiators and addition of MPC. The equilibrium degree of hydration of one of the copolymers prepared was 35% at 28°C but 7% at 43°C. The adsorption experiments were carried out using two proteins, bovine serum albumin (Alb, at pH 5.6) and human serum gamma globulin (Glo, at pH 6.4) at 24°C and 43°C.

Their results are shown in Fig. 7, which indicates that the particles at 42°C adsorb more proteins than those at 25°C, except for the case in which globulin was adsorbed on positively charged particles. The major results support the above-mentioned general concept that particles become hydrophobic at a temperature higher than the transition temperature and susceptible to adsorption due to hydrophobic interaction with proteins. The exception was supposed to result

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FIG. 7 Schematic representation of albumin and globulin on poly(HPMA-MMA) part. The thickness of bars between a protein molecule and a particle indicates the amount of protein absorbed. Boldface + and - indicate a more strongly effective charge than the plain letters. Gray particles indicate higher hydrophobicity and hardness than white ones.

FIG. 7 Schematic representation of albumin and globulin on poly(HPMA-MMA) part. The thickness of bars between a protein molecule and a particle indicates the amount of protein absorbed. Boldface + and - indicate a more strongly effective charge than the plain letters. Gray particles indicate higher hydrophobicity and hardness than white ones.

from the electrorepulsive force between slightly positive protein and positive particles.

The second point that Fig. 7 shows us is that the higher the surface charge, the more proteins were adsorbed regardless of the charge being positive or negative without exceptions. This fact indicates that electric force contributes to the adsorption more or less positively. This might mean that the distribution of ionic groups or dissociation of proteins changes when the protein meets the charged particles to generate electrostatic force between them.

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