Chaotropic Salts Influence Protein Corona Formation on Polyethylene (PEG)-b-polylactic acid (PLA) Polymersomes

Saturday, October 11, 2025

9:50 AM - 9:55 AM PDT

Location: Room 29B

Presenting Author(s)

Ashley Blake

Undergraduate Student
Clemson University, United States

Co-Author(s)

GW

Grace Witt

Undergraduate Biomedical Engineering Student
Clemson University
Central, South Carolina, United States
OT

Owen Tabah

PhD Student
University of Maryland, United States
DN

Daniel M. Nichols

Graduate Student
University of Virginia
Mt. Pleasant, South Carolina, United States

Primary Investigator(s)

Jessica Larsen

Associate Professor
Clemson University, South Carolina, United States

Introduction: : Polymersomes (PS) have gained attention as versatile drug carriers because they combine adjustable membrane characteristics with strong colloidal stability and the capacity to deliver both water-soluble and hydrophobic drugs. These nanoscale vesicles, formed from self-assembling amphiphilic block copolymers into bilayer membranes, hold great promise for various therapeutic uses. However, their clinical application is complicated by the formation of the protein corona, a layer of proteins that adsorbs on the nanoparticle surface upon exposure to biological fluids.
The composition of the protein corona plays a critical role in determining PS interactions with biological systems. However, the protein corona is highly dynamic and depends on factors such as particle size, surface chemistry, and the surrounding environment. Because protein adsorption on the PS surface is unavoidable and strongly impacts their biological behavior, researchers are actively exploring ways to control the corona to improve therapeutic outcomes.
In this study, we examined the effects of ionic environments on the protein corona of poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) PS incubated in bovine and human serum. Using chaotropic salts Na⁺, Ca²⁺, and Mg²⁺, arranged by Hofmeister Series strength, we investigated how ionic strength and cation type influence corona formation and enable specific protein binding. This work clarifies key parameters governing protein-PS interactions and provides foundational insight toward engineering PS with tailored corona profiles for personalized drug delivery.

Materials and

Methods: : Polymersomes (PS) were formed by dissolving PEG-b-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 PS in Milli-Q water with Spectra-Por Float-A-Lyzer-G2. Finally, the PS were dialyzed against ionic solutions of NaCl (0 mM, 50 mM, 100 mM, and 200 mM), CaCl₂ (0 mM, 25 mM, 50 mM, and 100 mM), and MgCl₂ (0 mM, 25 mM, 50 mM, and 100 mM). Dialyzed PSs were transferred through syringe filters to autoclaved tubes along with FBS or human serum in a sterile environment. The samples were incubated at physiological temperature and then pelleted by centrifugation. The supernatant was pipetted out. The PS were resuspended in 1x Laemmli 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) and Liquid Chromatography-tandem mass spectrometry (LC-MS/MS).

Results, Conclusions, and Discussions:: The results suggest that the ionic environment significantly influences the protein corona composition on PEG-b-PLA polymersomes (PS). SDS-PAGE analysis and LC-MS/MS data showed distinct changes in protein profiles between salted and unsalted PS samples incubated in FBS. Normalizing salted samples to unsalted controls demonstrated that NaCl minimally affected the corona, with only a modest 1.65-fold increase in apolipoprotein A-II at high salt concentration. In contrast, MgCl₂ induced larger changes, including an approximately 5-fold increase in tubulin-tyrosine ligase, a magnesium-binding enzyme, and several other proteins exhibiting 3-fold changes, consistent with Mg²⁺ adsorption onto the PS surface and zeta potential shifts. Most notably, CaCl₂ incubation caused dramatic enrichment of adiponectin, up to 28-fold, making it one of the top 20 most abundant proteins.
Jaccard similarity analysis showed that protein coronas formed with divalent cations (Ca²⁺ and Mg²⁺) were more similar to each other than those formed with monovalent Na⁺, correlating with stronger PEG binding observed for divalent ions in the literature. This is further supported by zeta potential measurements demonstrating that divalent cations bind more strongly to PEG chains and shift the PS surface charge toward more positive values, enhancing protein adsorption. Calcium caused the most significant protein corona and shape changes, differing from magnesium despite both being divalent, suggesting biologically specific interactions beyond charge effects.
So far, PSs incubated in human serum show similar trends in SDS-PAGE analysis. The CaCl₂ samples exhibit the most pronounced differences, with a band near 275 kDa in the 0 mM control becoming more intense and shifting higher on the gel ladder as Ca²⁺ concentration increases. In comparison, the NaCl samples display increased band intensity around 250 kDa, and the MgCl₂ samples show noticeable intensity increases near 75 kDa and 5 kDa; however, the differences in the NaCl and MgCl₂ gels were far less prominent.
Overall, the findings establish guiding principles for engineering PS protein coronas by demonstrating that divalent ions, especially calcium, strongly shape corona composition through charge-based and biologically specific interactions. These results emphasize the critical importance of considering the ionic environment when designing PS systems to control biological fate and optimize drug delivery.

Acknowledgements and/or References (Optional): :