Interaction Between Nano-sized Spherical Polyelectrolyte Brushes And Proteins

In recent years, interaction between polyelectrolyte (PE) and biomacromolecules has attracted many interests in biotechnology, which lead to multiple applications in protein separation, drug delivery, wound healing, and biosensing. PE modified nanoparticles (NPs) can be ideal candidate for selective adsorption of protein, enzyme, nucleic acid, polysaccharide, and lipid. Since NPs are small enough to interact with cellular machinery and potentially to reach previously inaccessible targets, such as the brain and blood. Spherical polyelectrolyte brushes (SPBs) as colloidal NPs can be used as carriers for protein immobilization and separation. Through adjusting surrounding conditions, SPBs can adsorb and desorb proteins tunably. Therefore, SPB should be ideal candidate for protein immobilization, purification, separation, and applied to targeting drug delivery, high-performance diagnostic assays, and nano-bioreactor.SPBs were synthesized by photo-emulsion polymerization, consisting of a polystyrene core with a diameter around100nm and a polyelectrolyte shell with a thickness from30to150nm densely grafted on the core surface. The polyelectrolyte shell consists of either weak anionic poly(acrylic acid)(PAA) or weak cationic poly(2-aminoethyl methacrylate hydrochloride)(PAEMH). Magnetic SPBs consist of magnetic nanoparticles in the polystyrene core and PAA chains. In this paper, the interactions between proteins and anionic SPBs as a function of pH and ionic strength have been systematically compared with that for cationic SPB in both qualitative and quantitative ways. Such studies provide valuable insight into the interaction mechanism between proteins and SPBs and effects on interaction. SPB and magnetic SPB were used as carrier for enzyme immobilization and as nano-sized bioreactor for catalytic reaction, and test enzymatic activity before and after the immobilization in SPB. Main work and conclusions as follow:1. Bovine serum albumin (BSA) is employed as model protein to investigate their interaction with PAA-SPBs. The pH dependence of phase state, architecture, interaction behavior between proteins and PAA-SPBs were examined by turbidimetric titration, dynamic light scattering (DLS), and zeta potential measurement. Results reveal the existence of three pH regions, corresponding to adsorption, aggregation, and desorption of BSA from SPB upon decreasing pH. Isothermal titration calorimetry (ITC) was applied to determine the amount of adsorption, binding affinity, and thermodynamics of BSA adsorption onto SPBs in quantitative way. Small angle X-ray scattering was employed to investigate the subtle change of shell structure of PAA-SPBs before and after adsorption of BSA, which demonstrated that BSA molecules were distributed inside the shell of SPBs. The interaction between proteins and SPBs is caused mainly by electrostatic interaction. Both pH and ionic strength of system influence the adsorption amount of BSA in PAA-SPBs simultaneously.2. Cationic spherical polyelectrolyte brushes were employed as carrier for BSA immobilization. In this section, we continue to investigate the interaction in both qualitative and quantitative ways, following the characterization methods as above. We also found that adsorption, aggregation, and desorption of BSA by cationic SPBs could be tuned by increasing pH. However, the extent of pH range for BSA and cationic SPBs was wider than anionic SPBs. In addition to pH and ionic strength, protein and SPBs stoichiometry, and SPB thickness can influence pH region for adsorption and adsorbed amount as well. Therefore, through modulating pH, ionic strength, bulk stoichiometry of system, and SPB thickness, cationic SPBs can adsorb and desorb BSA effectively under optimized conditions.3. Turbidimetric titration, DLS, zeta potential measurement, ITC, and adsorption measurement were used to investigate the interaction between various proteins and anionic/cationic SPBs. Results reveal that interaction behavior between anionic SPBs and BSA is very similar to BLG, while pH window for BSA adsorption by cationic SPBs is significantly different from that of BLG. Therefore, we find that it is difficult to use PAA-SPBs to discriminate these two proteins with similar pls. However, selective adsorption between BSA and BLG can be achieved with cationic SPBs by proper selection of pH, ionic strength, bulk stoichiometry, and SPB thickness. This may arise from the different electrostatic interaction behaviors between SPB and protein "charge patches" or "charge regulation". The larger negative charge patch of BLG distributes centrally. Furthermore, positive and negative charge patches of BLG display dipolar distribution. While, smaller negative charge patches of BSA distribute discretely. We also study the interaction between basic papain and cationic SPBs. Compared to acidic proteins, basic protein adsorption by cationic SPB is in a narrow pH region and shifts to higher pH value. Cationic SPBs adsorb basic protein much weaker than acidic proteins. The sequence of binding affinity and stoichiometry of proteins onto cationic SPBs was observed by ITC as BLG>BSA>papain, which resulted from the size, isoelectric point, and charge anisotropy of proteins. Therefore, through adjusting pH, ionic strength, bulk stoichiometry, and SPB thickness, cationic SPBs have potential applications in separation and selective binding of BSA, BLG, and papain under optimized conditions. While, anionic SPBs provide mild conditions for BSA and BLG co-immobilization. 4. Spherical poly(acrylic acid) brushes (PAA-SPBs) and magnetic PAA-SPBs were employed as carriers for glucoamylase (GA) to catalyze amylolysis. Firstly, turbidimetric titration, ITC, and adsorption experiment were used to investigate the pH and ionic strength dependent interaction between GA and PAA-SPBs/magnetic PAA-SPBs in both qualitative and quantitative ways. Then, GA immobilization in PAA-SPBs and magnetic PAA-SPBs carried out under optimal conditions. We study the dynamics of amyloysis and activity of GA before and after immobilized in PAA-SPBs and magnetic PAA-SPBs. Experimental results demonstrate that immobilization of GA in PAA-SPBs and magnetic PAA-SPBs does not lead to the loss of activity of GA. The electrostatic attraction and hydrogen bonding between GA and PAA chains grafted on SPBs enhances the enzymatic activity, and SPBs provide stable surrounding such as pH and ionic strength for GA immobilization, so the GA immobilized in PAA-SPBs and magnetic PAA-SPBs displays higher catalytic activity than free GA.