Poster AA12 - Poly(IC) and Polyethylene Imine-Polyethylene Glycol Polyplexes as an Influenza Prophylactic

Introduction: In the 2022-2023 flu season, the Center for Disease Control (CDC) estimated 21,000 deaths and 31 million symptomatic illnesses in the United States[1]. Current antivirals are grouped into two categories, neuraminidase inhibitors and PA cap-dependent endonuclease inhibitors. However, there is evidence of growing resistance to these drugs, contributing to the need of a new class of antivirals. Polyinosinic polycytidylylic acid (PolyIC) is popularly used as an adjuvant in vaccine delivery through its targeting of Toll like receptor 3 (TLR3). This pathway also results in type 1 and 3 interferon production, which in turn stimulate a range of antiviral mechanisms. Because of this, it has also been investigated as a prophylactic or treatment to various viruses, including hepatitis B virus, human immunodeficiency virus and rhinovirus[2]. Because it affects the host and not the virus, viruses should be unable to develop resistance to it. However, due to stability and toxicity concerns, it hasn't been implemented. Polyethylene Imine-Polyethylene Glycol (PEI-PEG) is a popular polymer used to condense nucleic acids for gene delivery. By combining the two, we can utilize the antiviral capabilities of Poly(IC) while minimizing the dosage concentration to therapeutic levels.
[1] CDC, “Weekly U.S. Influenza Surveillance Report,” Centers for Disease Control and Prevention. Accessed: May 15, 2024. [Online]. Available: https://www.cdc.gov/flu/weekly/index.htm
[2] “Frontiers | Poly-ICLC, a TLR3 Agonist, Induces Transient Innate Immune Responses in Patients With Treated HIV-Infection: A Randomized Double-Blinded Placebo Controlled Trial.” Accessed: May 15, 2024. [Online]. Available: https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.00725/full

Materials and

Methods: Low molecular weight polyIC (Invitrogen) was complexed at different molar ratios of nitrogen to phosphate(N/P) (6,10,15, with either 100% PEI, 50% PEI/50% PEI-PEG, or 100% PEI-PEG. The complexes were formed by dropwise addition of 10 volumes of Poly(IC) to 1 volume of swirling polymer solution, followed by a half hour incubation time, .44um syringe filtration and purification and concentration by 100K Amicon filtration. The hydrodynamic size and zeta potential were determined by nanoparticle tracking analysis (NTA) using a ZetaView. The shape and size were verified by SEM using Tescan xeia3. The strength of the polyplexes were determined using a heparin displacement assay, where the concentration of heparin needed to displace poly(IC) in the polyplex was quantified. The ability to protect from enzyme degradation was also quantified using the T2 ribonuclease. Interferon beta and interferon lamda production were also detected and quantified using the DuoSet from R&D Productions. Briefly, increasing concentrations of poly(IC) were added to MDCK cells and allowed to incubate at 37C,5% CO2 for 5 hours. The supernatants were collected and replaced with fresh media, which was again collected at 48 hours. Similarly, protection from influenza infection was determined by spiking the cells with Udorn (A/Udorn/307/1972(H3N2) immediately after treatment. Toxicity was investigated by crystal violet staining the cells 48 hours after treatment.

Results, Conclusions, and Discussions: 5 polyplex formulations were tested 100%PEI N/P=6, 100% PEI N/P=15, 50% PEI 50% PEI-PEG N/P=10, 100%PEI-PEG N/P=6, and 100%PEI-PEG N/P=15 as well as free poly (IC) as a comparison. The nanoparticle formulations were consistently around 150-215nm. The zeta values varied with the complete PEI-PEG nanoparticles bringing the charge closer to zero in comparison to poly(IC), and the other formulations leading to a positive zeta potential. Consistently, at the same dosage concentrations, the complete PEI formulations lead to significant cell death. All the formulations protected from influenza at low plaque forming unit (PFU) concentrations (120 PFU/mL). However, the formulations with unconjugated PEI showed toxicity at higher concentrations.This work demonstrates that PEI-PEG can be used for efficient delivery of poly(IC). Further studies will elucidate the method of action and uptake

Acknowledgements (Optional): This work was supported by the NIH R01 HL160540.