| Abstract |
Globular proteins are unique polymers with specific functions and desirable properties, including high catalytic activity, toughness, and large substrate binding affinity, that are difficult to achieve synthetically. Due to their functional diversity, they are promising candidates for numerous applications. The incredible diversity of structure and function of the proteins combined with the stability, chemical diversity and prosessability of synthetic polymers, can give access to materials with the advantages of both components. These hybrid materials can be used as therapeutics or agents for drug delivery, biocatalysis, protein separation and purification and biosensors.
One recently developed subclass of these bioconjugates is called giant amphiphiles. These amphiphilic substances are nanosized self-assembled systems which consist of an enzyme headgroup and a single covalently connected hydrophobic polymeric tail. Several methods have been developed for the synthesis of giant amphiphiles. A technique that is commonly used for this synthesis is the atom transfer radical polymerization (ATRP), which allows great control over the molecular weight distribution.
In the present work, the size of giant amphiphiles in solution during the first and the last stage of their synthesis was investigated with Dynamic Light Scattering. The protein used as the hydrophilic part was Bovine Serum Albumin (BSA), and the polymers that were conjugated with BSA were polystyrene and poly(N-isopropylacrylamide). During the course of the research, aggregates were observed in the first step of the synthesis, where the protein is conjugated with the initiator dissolved in dimethyl sulfoxide (DMSO). Looking into this issue, two different solvents were used for the initiator, acetonitrile (MeCN) and tetrahydrofuran (THF).
At first, the effect of the different organic co-solvents on the protein was studied. For this purpose, the protein is dissolved in water together with the three co-solvents. The DMSO co-solvent shows somewhat lower hydrodynamic radius for the protein than the other two co-solvents, indicating that BSA prefers the MeCN and THF co-solvents. Afterwards, these measurements were repeated, using an aqueous phosphate buffer (20mM) with ph=7.4 as a solvent. The buffer is a better solvent for the protein than the water, and thus BSA adopts a more compact conformation in water than in the buffer where it is more swollen. Also, there is no important aggregation in the case of the buffer, and DMSO was again the worst co-solvent.
Then the bioconjugation reaction is performed and the effect of the co-solvents on the bio-initator was studied. THF could not dissolve the initiator so the synthesis was performed with MeCN and DMSO. After the reaction, the bioconjugates were purified with dialysis. A different situation is observed in all these cases comparing to the results before the conjugation. In all cases the presence of two populations is obvious, indicating the existence of large aggregates further than the single protein chains. What differs between the cases of the different co-solvents is the relative amount of the small vs the large sizes. When MeCN is used the amount of aggregates is the lowest. Furthermore, it was noted that the conditions of the dialysis (time, volume, solvent used) seem to affect the aggregation of the bio-initiator.
Lastly, the study of the conformations of the giant amphiphiles is presented. The bioconjugate with PS shows a large particle size, which is attributed possibly to the formation of vesicles in the solution. The next amphiphile was synthesized with PNIPAM as the hydrophobic group. The unexpected aggregation observed in this case led to the investigation of the first steps of the synthesis.
|
Μελέτη του μηχανισμού σχηματισμού δομών από γιγάντια αμφίφιλα πολυμερούς - πρωτεΐνης
