Preclinical efficacy and safety of novel SNAT against SARS-CoV-2 using a hamster model

Abstract

To address the unprecedented global public health crisis due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we designed and developed a novel antiviral nano-drug, called SNAT (Smart Nano-Enabled Antiviral Therapeutic), comprised of taxoid (Tx)-decorated amino (NH₂)-functionalized near-atomic size positively charged silver nanoparticles (Tx-[NH₂-AgNPs]) that are stable for over 3 years. Using a hamster model, we tested the preclinical efficacy of inhaled SNAT on the body weight, virus titer, and histopathology of lungs in SARS-CoV-2-infected hamsters, including biocompatibility in human lung epithelium and dermal fibroblasts using lactase dehydrogenase (LDH) and malondialdehyde (MDA) assays. Our results showed SNAT could effectively reverse the body weight loss, reduce the virus load in oral swabs, and improve lung health in hamsters. Furthermore, LDH assay showed SNAT is noncytotoxic, and MDA assay demonstrated SNAT to be an antioxidant, potentially quenching lipid peroxidation, in both the human cells. Overall, these promising pilot preclinical findings suggest SNAT as a novel, safer antiviral drug lead against SARS-CoV-2 infection and may find applications as a platform technology against other respiratory viruses of epidemic and pandemic potential.

Keywords: Antiviral drug candidate; COVID-19; Inhaler; Nanotechnology; SARS-CoV-2; SNAT.

Fig. 1

A schematic depicting the study design used for testing the efficacy of SNAT on SARS-CoV-2-infected golden Syrian hamsters

Fig. 2

Detail characterization of SNAT. Representative high resolution-transmission electron microscopy (HR-TEM) image of the “seed” amino-functionalized silver nanoparticles (NH₂-AgNPs) showing spherical particle morphology of elemental/metallic silver (Ag⁰) with mean particle diameter of 5.8 nm ± 2.8 nm A and B. S/TEM micrographs of SNAT: surface docetaxel (Tx)-decorated (red arrow) amino-functionalized silver nanoparticles (Tx–[NH₂-AgNPs]) showing two or more individual NH₂-AgNPs (blue arrow), each ~ 5 nm diameter, self-assembled forming a somewhat triangular architecture collectively embedded within Tx molecules C. Inset in C shows the molecular structure of Tx. UV–Vis spectra of docetaxel (Tx)-decorated amino-functionalized silver nanoparticles (Tx–[NH₂-AgNPs]) (green spectrum/red arrow; (blue) green suspension), NH₂-AgNPs alone (yellow spectrum, yellow suspension), and Tx alone (in sterile Milli-Q water) (orange spectrum showing a flat line; inset to the right showing molecular structure of Tx) D. The “seed” of NH₂-AgNPs alone had a maximum absorbance (λ_max) at 406 nm, which red-shifted to 420 nm (a change of 14 nm) and blue shifted to 366 nm (a change of 40 nm) upon direct surface binding with Tx molecules. Representative EDS spectra and elemental composition (mass/atom %) of SNAT E and F

Fig. 3

SNAT is safer to human cells. To determine the cytotoxic effect of SNAT on human lung epithelial and dermal fibroblast cells, at 24 h post SNAT treatment, lactate dehydrogenase (LDH) release as an indicator of percentage of cell death was monitored. Known inducer of cell death, 1 mg/mL G418, was used as a positive control in this study A. Effect of SNAT on oxidative stress. In vitro oxidative stress response assessment of SNAT in primary human lung epithelial cells B and dermal fibroblast cells C, showing no malondialdehyde (MDA) lipid peroxidation in both the cell assays. Average of three experiments is depicted in the plot. “**” denotes significantly lower compared to negative control (p < 0.05); “***” denotes significantly higher compared to negative control (p < 0.001); and NS denotes each treatment group is not significantly different from negative control (p > 0.05). Negative control denotes diluent buffer (sterile water), and positive control denotes hydrogen peroxide (200 µM)

Fig. 4

SNAT treatment significantly lowers SARS-CoV-2 titers in the oral secretions. Oral swab suspension collected on different days were tested for virus yield in Vero cells and the TCID₅₀ was calculated using the Reed and Muench formula as per standard protocols. RNA was extracted from 140 µL of oral swab suspension using QIAmp viral RNA extraction kit (Qiagen) as per standard protocols. The RNA concentrations were measured with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The virus concentration in the specimens were detected by qPCR monitoring nucleocapsid N gene using the SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay (Integrated DNA technologies). The limit of detection for this assay is 50 copies. For panels A and B, each point represents mean ± S.D. of three individual experiments. ANOVA was used to compare between group means. Between groups differences are significant at p < 0.05, denoted by *; day 5 through day 13 were significantly different at p < 0.01, denoted by **

Fig. 5

SNAT treatment protects SARS-CoV-2-infected hamsters from body weight loss. SNAT treatment significantly protects SARS-CoV-2-infected hamsters from weight loss. Although weight loss began day 1 post infection (PI), the highest weight loss occurred during the first week of infection for the SARS-CoV-2-infected group compared to the control-uninfected group. Percent weight loss was normalized to the control group. ANOVA was used to compare between group means. Each scatter dot indicates mean ± S.E.. Between group differences are significant at p < 0.0001, denoted by ***; day 3 through day 5 were significantly different at p = 0.05, denoted by *

Fig. 6

SNAT improves lung health in SARS-CoV-2-infected hamster. Hematoxylin and eosin (H&E) staining of the lungs of hamsters challenged with SARS-CoV-2 with or without SNAT treatment at 3 dPI. Panels 1.0 and 1.1; 2.0 and 2.1; and 3.0 and 3.1 denote lungs from uninfected control, SARS-CoV-2 infected, and SARS-CoV-2 infected and treated with SNAT at 2.0X and 20X magnifications, respectively. Blue arrow denotes focal hyaline membrane formation