Feasibility Study of Developing a Saline-Based Antiviral Nanoformulation Containing Lipid-Soluble EGCG: A Potential Nasal Drug to Treat Long COVID

Abstract

A recent estimate indicates that up to 23.7 million Americans suffer from long COVID, and approximately one million workers may be out of the workforce each day due to associated symptoms, leading to a USD 50 billion annual loss of salary. Post-COVID (Long COVID) neurologic symptoms are due to the initial robust replication of SARS-CoV-2 in the nasal neuroepithelial cells, leading to inflammation of the olfactory epithelium (OE) and the central nervous system (CNS), and the OE becoming a persistent infection site. Previously, our group showed that Epigallocatechin-3-gallate-palmitate (EC16) nanoformulations possess strong antiviral activity against human coronavirus, suggesting this green tea-derived compound in nanoparticle formulations could be developed as an intranasally delivered new drug to eliminate the persistent SARS-CoV-2 infection, leading to restored olfactory function and reduced inflammation in the CNS. The objective of the current study was to determine the compatibility of the nanoformulations with human nasal primary epithelial cells (HNpECs). Methods: Nanoparticle size was measured using the ZetaView Nanoparticle Tracking Analysis (NTA) system; contact antiviral activity was determined by TCID50 assay for cytopathic effect on MRC-5 cells; post-infection inhibition activity was determined in HNpECs; and cytotoxicity for these cells was determined using an MTT assay. The rapid inactivation of OC43 (a β-coronavirus) and 229E (α-coronavirus) viruses was further characterized by transmission electron microscopy. Results: A saline-based nanoformulation containing 0.1% w/v EC16 was able to inactivate 99.9999% β-coronavirus OC43 on direct contact within 1 min. After a 10-min incubation of infected HNpECs with a formulation containing drug-grade EC16 (EGCG-4′ mono-palmitate or EC16m), OC43 viral replication was inhibited by 99%. In addition, all nanoformulations tested for their effect on cell viability were comparable to normal saline, a regularly used nasal irrigation solution. A 1-min incubation of an EC16 nanoformulation with either OC43 or 229E showed an altered viral structure. Conclusion: Nanoformulations containing EC16 showed properties compatible with nasal application to rapidly inactivate SARS-CoV-2 residing in the olfactory mucosa and to reduce inflammation in the CNS, pending additional formulation and safety studies.

Evaluation of Novel Nasal Mucoadhesive Nanoformulations Containing Lipid-Soluble EGCG for Long COVID Treatment

“…The current study is the first attempt to validate the suitability of EC16m nanoparticle/saline based mucoadhesive nasal formulations using 3D MucilAir Human Nasal Epithelium Model (MucilAir) and in vitro toxicity/efficacy methods. We previously reported a series test results from EC16 and EC16m nanoformulations, which are from saline-based aqueous suspensions of the nanoparticles such as F18, F18D, F18m and F18Dm [8,9]. These high efficacy nanoformulations lack the mucoadhesive nature, therefore are not suitable for nasal application due to the mucociliary clearance transit time of 20 min [39].…”

“…ZetaView nanoparticle tracking analysis was performed according to a method described previously [9,40]. The particle size distribution and concentration were measured using the Zetaview x20 (Particle Metrix, Meerbusch, Germany) and corre-sponding software.…”

“…In contrast, recently published data showed that engineered EGCG-AgNPs appears cytotoxic to human skin cells (CC50 =30 μg/mL), and the efficacy is poor on human herpes simplex virus type 1 and type 2, with less than log10 2 reduction (<99%) after 60 min incubation [37]. Another study indicated that EGCG-AgNPs have been shown to become cytotoxic at nM levels [38], while EC16 NPs at 1.4 mM is not associated with cytotoxicity [9].…”

“…Based on our recent studies, we hypothesized that epillagocatechin-3-gallate (EGCG)-monopalmitate (EC16m), a compound with multi-mechanisms of antiviral activity, plus anti-inflammatory, antioxidant, and neuroprotective properties, has potential to become a new nasal drug to minimizing Long COVID associated neurologic symptoms such as anosmia [8,9].…”

“…Unlike other engineered EGCG NPs, the EC16/EC16m NPs were made through a "facilitated self-assembling" method (proprietary, patent pending), which does not involve association with metals, monomers, oil, or encapsulation. In addition to the high efficacy of rapid inactivation of human coronavirus OC43 and 229E, the water-based nanoformulations are not associated with cytotoxicity [8,9]. In contrast, recently published data showed that engineered EGCG-AgNPs appears cytotoxic to human skin cells (CC50 =30 μg/mL), and the efficacy is poor on human herpes simplex virus type 1 and type 2, with less than log10 2 reduction (<99%) after 60 min incubation [37].…”