(K-416) Polysaccharide-Coated Nanoparticle Delivery Systems for Peptide Antigens

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row K - Poster # 416

Presenting Author(s)

Amberlynn L. Demko

Undergraduate Researcher
University of Oklahoma
McKinney, Texas, United States

Co-Author(s)

SB

Sarah Butterfield

Research Assistant
University of Oklahoma, United States
TT

Thao Tran

Undergraduate Researcher
University of Oklahoma, United States
ZC

Zephan Carmody

Undergraduate Researcher
University of Oklahoma, United States
DG

Dixy E. Green

Staff Research Assistant Supervisor
University of Oklahoma Heath Sciences Center, DeAngelis Lab
Oklahoma City, Oklahoma, United States
HY

Hooman Yari

Postdoctoral Research Fellow
University of Oklahoma Heath Sciences Center, Hildebrand Lab, United States
WH

William H. Hildebrand

Primary investigator, Hildebrand Lab
University of Oklahoma Heath Sciences Center, Hildebrand Lab, United States
PD

Paul DeAngelis

Primary investigator, DeAngelis Lab
University of Oklahoma Heath Sciences Center, DeAngelis Lab, Oklahoma, United States

Primary Investigator(s)

SW

Stefan Wilhelm

Associate Professor
University of Oklahoma, United States

Introduction:: Sub-track: Micro/Nano Technologies In Molecular and Cellular Bioengineering, Medicine and Biology
Dendric cell (DC) vaccines may be used to carry antigenic peptides that interact with the major histocompatibility (MHC) Class 2 receptor to trigger CD4+ T cells and antibody production. However, due to safety and efficacy concerns, this type of vaccine needs improvements.1 Controlled drug delivery via nanoparticles has become popular in recent years due to its ability to target specific areas of the body and encapsulate only the necessary components of an antigen or drug. Organic nanoparticles (ONPs) used in the body need a biocompatible coating to ensure colloidal stability. The most popular coating currently is poly(ethylene glycol) (PEG). As the use of PEG poses safety concerns (e.g., allergenicity, tissue accumulation), novel surface modifications for nanomedicines are needed.2 One strategy is the use of sugar coating based on heparosan (HEP), the polysaccharide precursor of heparin, that is naturally present and recycled in the body. We use fluidics to create HEP-coated liposomes that encapsulate the SVG9 peptide to ultimately create a safer and more effective DC West Nile Virus vaccine. To fully quantify the effectiveness of HEP surface coatings, we systematically compared it to both PEG conjugated liposomes and ones lacking any surface modification.

Materials and Methods:: Liposomes were synthesized with a novel fluidics technique using a reconstructed Ender 3 3D printer. A lipid mix of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (thiol-lipid), Cholesterol, 1,2-Disteraroyl-sn-glycero-3-phosphocholine (DSPC) (all Avanti Polar Lipids) was combined with 100% ethanol into one syringe and a West Nile Virus peptide, SVGGVFTSV (SVG9), in an aqueous neutral saline buffer into another syringe. Then a software code was developed that combined the two fluid streams at a controlled rate to create a range of different liposome sizes. The liposomes were characterized using dynamic light scattering (DLS) and static light scattering (SLS). The HEP/PEG coatings were applied to the particles by coupling ortho-pyridyl disulfide (OPSS) activated polymers. (HEP-OPSS synthesized in-house; OPSS-PEG, Laysan Bio) to the thiol group of the thiol-lipid resulting in a disulfide bond. Coated NPs were harvested via centrifugation or ultrafiltration to allow peptide encapsulation efficiency (EE) by mass spectrometry (MS) and evaluation of stability by DLS. Six individual sets of liposomes including HEP- or PEG-coated or plain liposomes (no coating) that either encapsulated SVG9 or buffer alone as control. Cellular uptake studies were performed with THP-1 human monocytes and incubated with equal doses of HEP- or PEG-coated NPs that were stained with DiO (green; liposomes bilayer). Samples were then stained with WGA (red; cell membrane) and finally visualized with confocal microscopy.

Results, Conclusions, and Discussions:: First we determined the optimal molar ratio for the lipids by comparing 3 different lipid molar ratios based on good results in the literature: 55:40:5, 43:29:28 or 1.3:0.9:0.3 (DSPC: Cholesterol: thiol-Lipid). The best molar ratio we found was 1.3:0.9:0.3 based on its lowest polydispersity index (PDI) with a smaller margin of variability. The hydrodynamic diameter was consistently under 100 nm. Next, we added HEP and PEG at a 1:1 molar ratio to the Thiol-Lipid. The HEP-coating protocol added on average 40 nm while PEG-coating added 10 nm to the basal hydrodynamic diameter. Stability tests were performed to assess the colloidal stability of liposomes and to evaluate any potential degradation. The plain liposomes were, as expected, the least stable while PEG liposomes were relatively stable throughout the whole week with only slightly fluctuating size and PDI by the end. HEP-coated liposomes, on the other hand, shrank slightly the first day after synthesis and then had negligible changes throughout the rest of the week. These indicate that HEP processing not only added a more substantial coating (more biocompatible) than PEG but also creates more stable particles. MS analysis data indicated that there was no SVG9 in the controls (expected), and nearly equal amounts of SVG9 in the HEP and PEG liposomes respectively. This data indicates that we have successfully encapsulated the peptide in a stable and biocompatible particle. We can also generally conclude that adding HEP as a surface modifier has no greater effect on EE than PEG suggesting it a reasonable replacement at this stage. Finally, in the preliminary cellular uptake experiments based on confocal laser scanning microscopy, the images indicated qualitatively that there was potentially higher uptake of HEP-coated liposomes than PEG-coated ones as shown by the green (DiO) fluorescent liposomes within the red-stained plasma membrane in the images provided. Since HEP is more biocompatible, stable, and equally functional as its PEG counterpart, this technology platform could potentially provide an opportunity to create a safer and more effective liposomes DC vaccine.

Acknowledgements (Optional): : This project was supported by the Provost’s Summer Undergraduate Research and Creative Activities (UReCA) Fellowship. Its contents, including findings, conclusions, opinions, and recommendations, are solely attributed to the authors and do not necessarily represent the views of the Provost’s Office.
This project was supported by the Presbyterian Health Foundation.

References (Optional): :

< 1. Tesfaye, D. Y.; Gudjonsson, A.; Bogen, B.; Fossum, E. Targeting Conventional Dendritic Cells to Fine-Tune Antibody Responses. Frontiers in Immunology 2019, 10.

< !2. Shi, L.; Zhang, J.; Zhao, M.; Tang, S.; Cheng, X.; Zhang, W.; Li, W.; Liu, X.; Peng, H.; Wang, Q. Effects of Polyethylene Glycol on the Surface of Nanoparticles for Targeted Drug Delivery. Nanoscale 2021, 13 (24), 10748–10764. https://doi.org/10.1039/D1NR02065J.