(157p) Polymer Amphiphiles for Directed Self-Assembly Around Hepatitis B Virus Particles | AIChE

(157p) Polymer Amphiphiles for Directed Self-Assembly Around Hepatitis B Virus Particles


Pitman, M. - Presenter, Clemson University
Maghak, L., Clemson University
Larsen, J., Clemson University
Background: Hepatitis B virus (HBV) is a global health issue with over 250 million chronic cases world-wide, and is attributed to an estimated 800,000 deaths annually due to liver failure, or complications leading to hepatocellular carcinoma or cirrhosis1. Current therapies are typically life-long1 and can lead to issues with patient compliance and cost. Chronicity of infection is due in part to the generation of non-infectious subviral particles, composed solely of the surface antigens, which are produced 1,000 to 10,000 times more than infectious particles during HBV replication1,2. These surface antigens contribute to the downregulation of a number of immune responses1–3 which have been shown to recover after the antigen is removed3. Current HBV therapies suppress viral replication, but do not remove existing virus particles. The goal of this project is to create a system to stably encapsulate infectious and subviral HBV particles in vivo by the directed assembly of polymer nanoparticles. We hypothesize that this would recover proper immune function and prevent further hepatocyte infection. To create amphiphiles for directed self-assembly, antibody fragments were prepared to act as targeting ligands and conjugated to block copolymer amphiphiles.

Methods: Poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA) and poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) were added to human blood components by solvent injection and imaged using transmission electron microscopy (TEM) to determine the effect of free polymer in the blood. Beads coated with Hepatitis B surface antigen (HBsAg) were produced to be used in place of HBV particles. Carboxylated polystyrene beads were sized by dynamic light scattering, then covalently coated with HBsAg and re-measured to confirm successful coating. Antibody fragments were prepared by digestion with pepsin and subsequently reduced with cysteamine, then purified by centrifugal filtration. Size was measured with non-reducing SDS-PAGE. The antibody fragments were conjugated to maleimide-PEG-b-PLA or maleimide-PEG-b-PLGA by thiol-maleimide reaction. An indirect enzyme-linked immunosorbent assay (ELISA) was used to measure binding activity of the antibody fragment alone and fragment-conjugated polymer to confirm binding ability of the conjugated polymer. For a non-specific binding control, un-modified PEG-b-PLA was measured by solvent injection of the polymer into a dilute solution of antigen coated beads, with size measurements taken by dynamic light scattering before solvent injection and immediately, three hours, and 24 hours after.

Results: PEG-b-PLA had no negative effect on various blood components, but PEG-b-PLGA led to disruption of plasma and platelets, indicating that it would not be suitable for injection into the blood. SDS-PAGE showed successful fragmentation of the antibody and indirect confirmation of successful antibody-polymer conjugation. The signal produced by ELISA was lower for the fragment-conjugated polymer than the unconjugated antibody fragment, but remained significantly above background levels indicating that binding still occurs. DLS results demonstrate the successful coating of antigen onto the polystyrene beads, but was determined not to be reliable for measuring non-specific encapsulation. Plans for future work include TEM imaging of the non-specific encapsulation, size-exclusion chromatography to confirm antibody-polymer conjugation, and specific encapsulation of the coated beads by solvent injection. We expect that the unconjugated polymer will show minimal encapsulation of the coated beads, instead predominantly forming empty polymersomes. In contrast, we expect that the specific encapsulation will show polymersome formation around the beads.

1 M.-F. Yuen, D.-S. Chen, G. M. Dusheiko, H. L. A. Janssen, D. T. Y. Lau, S. A. Locarnini, M. G. Peters and C.-L. Lai, Nat. Rev. Dis. Prim., 2018, 4, 1–20.

2 M. Dandri and J. Petersen, Clin. Infect. Dis., 2016, 62, S281–S288.

3 A. Bertoletti and C. Ferrari, Gut, 2012, 61, 1754–1764.