(K-436) Controlling Protein Coronas of Polymersomes in Response to Ions

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row K - Poster # 436

Presenting Author(s)

OT

Owen Tabah

Student
Clemson University, United States
DN

Daniel Nichols

Student
Clemson University
Clemson, South Carolina, United States

Primary Investigator(s)

Jessica Larsen (she/her/hers)

Carol and John Cromer ’63 Family Endowed Associate Professor
Clemson University, United States

Introduction:: Improving targeted drug delivery has become a primary concern for the treatment of diseases in which traditional drug treatments fall short due to biological barriers, solicited immunogenic responses, and low bioavailability. Self-assembled polymeric vesicles, such as polymersomes, have attracted interest in nanomedicine due to their potential to encapsulate and deliver both hydrophobic and hydrophilic drugs with higher specificity, as well as longer circulation time and minimized immune responses in comparison to their lipid counterparts. Our research seeks to develop an understanding of polyethylene-glycol (PEG) brushed polymersome protein adsorption (protein corona formation) through dialysis against various concentrations of different salts, exposure to a common serum, and analysis via gel electrophoresis. As a result of dialysis, we hypothesize that the external PEG brush of Polyethylene Glycol-b-Polylactic Acid (PEG-PLA) polymersomes will contain ions associated with the concentrations and compositions of the bulk dialysate. These ions lead to effects following the Hofmeister Series, which suggests that specific salts will cause variations in the stability of proteins and polyelectrolytes due to salting-in and salting-out phenomena. This will alter the protein conformation near the polymersomes and therefore change protein absorptive behavior. We aim to analyze the full effects of ions on the PEG brush, exploring possible conformational changes and differences in counterion evaporation to develop a more precise method of targeted drug delivery by recruiting specific targeting ligand proteins and forming a tissue-targeting protein corona.

Materials and Methods:: Polymersomes were formed by dissolving PEG-PLA into dimethyl sulfoxide (DMSO). That solution was then injected into 1 mL of MilliQ water at a constant rate (5 µL/min) with constant stirring (100 rpm). Excess DMSO was removed by dialyzing the polymersomes in milli-Q water with Spectra-Por Float-A-Lyzer-G2. Finally, the polymersomes were dialyzed against ionic solutions of NaCl (0 mM, 50 mM, 100 mM, and 200 mM) and CaCl₂ (0 mM, 25 mM, 50 mM, and 100 mM). Dialyzed polymersomes were sterilized with syringe filters, injected and incubated in fetal bovine serum, and then pelleted by centrifugation. The polymersomes were resuspended in 2x Leammli sample buffer and heated to remove proteins from the nanoparticles. Differences in protein corona formation were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Results, Conclusions, and Discussions::

Our results consist of visual characterization of the protein corona formed onto our PEG-PLA polymersomes after SDS-PAGE, with confirmation of proteins via western blot. We used FBS during the incubation of our polymersomes so that many different proteins were able to interact simultaneously. This allowed for a complex corona to form, simulating common biological fluids. By using various salts, NaCl and CaCl₂, we were able to differentiate the protein coronas based on the composition and concentration of salt. Our results indicate a few possible mechanisms for this differentiation. Firstly, in the case of NaCl shown in Figure 1A, we have seen little to no variation in the types of proteins forming the corona but see variation in the amounts of proteins based on the salt concentration. This potentially indicates variations in the outer PEG brush conformation caused by the Hofmeister effects of the ions on the polymer. It is shown through literature that differences in PEG conformation can attribute to different amounts of proteins absorbed to PEGylated surfaces (Li et al., 2021). Secondly, in the case of CaCl₂, we observed differences in the molecular weights of proteins comprising the corona. This can be seen with the red markers in Figure 1B, where two new protein bands not readily found on the unionized polymersomes or NaCl ionized polymersomes are observable. Additionally, band strength increased across CaCl₂ concentration for some proteins with a molecular weight around 150 kDa, and new bands appeared at higher CaCl₂ concentrations representing proteins around 25 kDa. This could be due to Ca being a more strongly hydrated ion which changes the counter ion exchange of protein adsorption.

Acknowledgements (Optional): :

References (Optional): :

Li, M., Jiang, S., Simon, J., Paßlick, D., Frey, M. L., Wagner, M., Mailander, V., Crespy, D., & Landfester, K. (2021). Brush conformation of polyethylene glycol determines the stealth effect of nanocarriers in the low protein adsorption regime. Nano Letters, 21(4), 1591–1598. https://doi.org/10.1021/acs.nanolett.0c03756