Physical characterization of PEGylated exosome constructs: Size, charge, and morphology changes in non-specific alkylating N-terminal reactions

Small extracellular vesicles, commonly referred to as exosomes, withhold a promising future in the pharmaceutical industry as carriers for targeted drug delivery due to their high specificity and bioavailability when compared to synthetic-based vectors. They, however, present some limitations for systematic administration because of natural organism defenses and their high-water solubility, ultimately making it difficult for them to reach the intended target. To improve the delivery capacity of these nanoparticles, the possibility for the construction of PEGylated versions was explored in this work. This process was performed, analyzed, and characterized using N-terminal specific PEGylation reactions targeted to the protein contents in the exosomal membrane. For this, two different mono-methoxy polyethylene glycols (mPEG) of 5 and 20 kDa were reacted with exosomes under alkylating conditions. The resulting 5k and 20k PEGylated exosome constructs were characterized and compared with unmodified exosomes, using size, morphology, and zeta potential as comparison parameters. Results after analysis showed an absorbance reduction of approximately 65% and 34% (for the 5 and 20 kDa conjugates respectively), a reduction of 10 to 20% in peak resolution, particle size increase corresponding to the polymer sizes used, and a slight reduction in electric distribution of about 2 to 3 mV less than the unmodified vesicles. The data obtained may provide insights for the optimization of exosome PEGylation strategies for therapeutic use.