There has been extensive research studying the association of proteins and polyelectrolytes especially in regard to protein/peptide drug delivery, protein separation and tissue engineering. Imidazolium quaternized polymers based on poly(chloromethylstyrene) were used in this experiment. The polymers were obtained by the use of nitroxide mediated radical polymerization and post-polymerization functionalization. The quaternization reaction substitutes the chlorine groups with cations in order to ensure that the polymers are soluble and positively charged in water. The polymers that were used include QIm-PCMS1 and QIm-PCMS2. The degree of quaternization of the two polymers was 73% and 100% respectively, which leads to QIm-PCMS1 being more hydrophobic than QIm-PCMS2.
The successful investigation of the complexation with globular proteins has been conducted with small angle scattering methods. Quaternized polymers are anticipated to define their own association state in aqueous media. In any case, it is important to keep in mind that even after quaternization, they still remain hydrophobic polyelectrolytes. The protein chosen is Bovine Serum Albumin (BSA), a negatively charged protein at neutral pH, which serves as a model protein in the small angle neutron scattering complexation studies with the aforementioned cationic polyelectrolytes,
Researchers led by Dr. Stergios Pispas at Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation in Greece in collaboration with Helmholtz-Zentrum Berlin in Germany demonstrated the ability to tune the polyelectrolyte/protein interactions and polyelectrolyte chain–chain associations by modifying the hydrophobic content of the polyelectrolytes.
Quaternized polymer chains are capable of forming aggregates with dimensions much larger than their own size. The dimension scales investigated by small angle neutron scattering (Figure 1a) are in a range that includes the size of the Bovine Serum Albumin. Based on established formulae and equations characteristic parameters for the polyelectrolyte solutions and polyelectrolyte/protein mixtures are deduced. Through these formulae the electrostatic and hydrophobic interactions were found to be of fundamental importance to Bovine Serum Albumin/polymer complexes. Electrostatic interactions are defined by the salt content while hydrophobic interactions are related to the degree of quaternization.
Although different polymer quaternization levels showed no difference in the solutions of the quaternized polymers, they interacted differently with Bovine Serum Albumin. As stated above, they still have different hydrophobic content. At another level however, there was the loss of the alpha-helix structural form of the Bovine Serum Albumin once its process of complexation with the polymers took place (Figure 1b). The alpha-helix form is the basic state in which the secondary structure of the Bovine Serum Albumin exists. Bovine Serum Albumin complexation with the quaternized polymers leads it to be denatured of its secondary structure. This case is similar to the aggregation of casein micelles in milk where the proteins are also met in their denatured state. It can be said that quaternized polymers may act as a medium to associations driven by protein denaturation.
By using light scattering measurements, the polymer structural organization especially at larger length scales is investigated. This organization is mostly affected by the interactions that exist with the Bovine Serum Albumin. Therefore an increase in the Bovine Serum Albumin content leads to a systematic increase in the scattered intensity. In the establishment of high clusters, the associations of polymer chains that were formed at the beginning are strenghtened by the Bovine Serum Albumin molecules. This increase in the mass of clusters ranges in the order of 10. This also implies inter-aggregate associations.
Through the use of small angle neutron scattering, the local interactions that have been presented can be said to be answerable for the assembly at larger length scale. Hence, the interactions that exist between the hydrophobic and positively charged groups that are present in the quaternized polymers, as well as the hydrophobic and negative parts of Bovine Serum Albumin are very conceivable.
The research work is a comprehensive study of protein/hydrophobic polyelectrolyte interactions at a wide range of length scales that demonstrates the tunability of the formed complexes by both hydrophobic and electrostatic effects, and gives insights for the designed construction of polyelectrolyte/protein nanoparticulate structures for biomedical applications.
Figure legend: SANS profiles from QIm-PCMS1 (16 mg ml-1) (blue) and QIm-PCMS1 (16 mg ml-1)/BSA(4 mg ml-1) in D2O with no added salt (red). Lines are fits to the experimental data.
Schematic illustration of the interaction of BSA with QIm-PCMS polymers.
Papagiannopoulos A, Vlassi E, Pispas S, Jafta CJ. Tuning the solution organization of cationic polymers through interactions with bovine serum albumin. Physical Chemistry Chemical Physics. 2017; 19(28):18471-80.
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