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

SNAT characterization

The amino-functionalized silver nanoparticles (NH2-AgNPs) had a mean TEM diameter of 5.8 nm ± 2.8 nm (S.D.) with a spherical morphology and the core composed of elemental/metallic silver (Ag0) surface functionalized with cationic NH2-functional group (Fig. 2A, B). Upon surface decorating with docetaxel (Tx) led to the formation of Tx–[NH2-AgNPs], i.e., SNAT, whereby two or more individual NH2-AgNPs (blue arrow) self-assembled presenting a near triangular architecture collectively embedded within Tx molecules (red arrow; Fig. 2C). The average hydrodynamic diameters (HDDs) for NH2-AgNPs and SNAT were similar: 4.3 nm and 5.0 nm, respectively, suggesting that Tx binding did not influence the original TEM particle size of seed NH2-AgNPs. Both the NH2-AgNPs and SNAT had high positive mean surface zeta potentials: + 41 mV and + 22 mV, respectively, and are highly stable for over 3 years at room temperature (25 °C) as the standard deviation for HDD was within ± 1 nm and within ± 3 mV for zeta potential over the 3-year period (Supplementary Table S1). Furthermore, no change in λmax over the 3-year period confirmed these DLS results (Supplementary Table S1). Thus, these stability data suggest there is no need for refrigeration during transportation and storage and may serve as an ideal antiviral agent for low resource settings where cold storage chain is unavailable. The blue-shift of 40 nm and red-shift of 14 nm as demonstrated by the UV–Vis spectra suggest a strong electrostatic binding of anionic Tx with cationic NH2-groups on the surface of AgNPs (Fig. 2D). The representative EDS spectra and elemental composition (mass/atom %) for SNAT are presented in Fig. 2E, F, showing that SNAT was composed of Ag, C, O, and N. The dialysis-purified SNAT and NH2-AgNP formulations had an alkaline pH (7.5–8.5) and low electrical conductance of 0.120 mS/cm.

Fig. 2figure 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 (λmax) 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

Determining the optimal dose of SNAT for hamster studies

The optimal dose of SNAT to be used in animal studies was determined testing SNAT in physiologically relevant cells. We tested different doses of SNAT in human lung epithelial cells using an LDH assay. SNAT doses equal to or below 10 µg/mL did not induce any significant cell death (Fig. 3A). Understanding virus-induced oxidative stress is pivotal in the viral life cycle including the pathogenesis of ensuing disease. Reactive oxygen species (ROS) are generated upon virus infection, and in response, a host cell activates an antioxidative defense system for protection [22, 23]. Understanding potential interaction between a virus and a host is critical to developing antivirals that could control viral infection. Many viruses, including SARS-CoV-2, are known to induce oxidative stress to facilitate their replication inside the host [24,25,26]. Lipid peroxidation occurs when excess ROS affects cell membranes and can also lead to oxidation and denaturation of proteins and DNA damage, further inducing inflammatory immune responses and cell death [27]. Our results show no lipid peroxidation (MDA) in human lung epithelial and dermal fibroblast cells upon SNAT treatments as the MDA levels were all below the background concentrations (negative control, dilution buffer). Rather, data revealed a decrease in MDA with up to 10 µg/mL of SNAT treatments in lung cells compared to treatments with the negative control (dilution buffer) or the positive control (hydrogen peroxide) (Fig. 3B, C). Such an ability to quench oxidative stress response makes SNAT an exciting antiviral candidate.

Fig. 3figure 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)

We also performed in vitro neutralization studies against SARS-CoV-2 to determine if Tx alone, NH2-AgNPs alone, and SNAT would neutralize the virus. We found that Tx (1 µg/mL) or NH2-AgNPs (10 µg/mL) alone did not neutralize the virus in vitro, but SNAT (10 µg/mL) did (data not shown). This led us to choose SNAT, over Tx (1 µg/mL) or NH2-AgNPs (10 µg/mL) alone, for the detailed hamster studies.

Based on the above in vitro neutralization effects of SNAT, we further tested doses of 1 µg/mL, 5 µg/mL, and 10 µg/mL of SNAT (2 mL/dose) administered using a nose-only inhalation exposure equipment to hamsters (n = 2/group). The animals were administered SNAT on day 1 and day 2 (24 h after the first dose). The animals were monitored for any signs of distress and body weight. All animals survived the 12-day screening period. There was no significant body weight loss in the animals that received 1, 5, or 10 µg/mL of SNAT (data not shown). Therefore, we decided to use 10 µg/mL of SNAT in the challenge studies.

SNAT reduces SARS-CoV-2 load in hamsters

In COVID-19 patients with acute respiratory illness, the main clinical manifestation is severe lung inflammation [28]. Since SARS-CoV-2 induces severe pathological lesions in the lungs of these Syrian hamsters, we chose to use this animal model [16]. As a direct measure of SARS-CoV-2 infection of hamsters, we monitored virus titer in the oral swabs collected from hamsters on day 01 and every other day post infection. The effect of SNAT inhalation on virus infection of hamsters was monitored by performing virus titration in Vero cells to calculate TCID50 and by determining viral RNA copy numbers using RT-qPCR. Our study demonstrated SNAT to significantly lower virus infection in oral swabs by day 05 as observed by TCID50 (Fig. 4A) and a sharp decline in the viral RNA copy numbers (Fig. 4B). There was a significant decrease in viral titers in oral swabs taken from SARS-CoV-2 infected hamsters exposed to SNAT compared to those that were SARS-CoV-2 infected or those infected but received saline (Fig. 4A). The levels of virus titer reduction (one log or ten-fold) were substantial in animals that were treated with SNAT compared to saline or untreated group (Fig. 4A).

Fig. 4figure 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 **

SNAT protects SARS-CoV-2-infected hamsters from body weight loss

SARS-CoV-2 infection of hamsters resulted in a significant body weight loss by 3 days post infection when compared to uninfected hamsters (Fig. 5). The body weight loss in virus infected hamsters was the greatest during the first week of infection. As a result of viral load reductions in hamsters and improved lung health, hamsters that received SNAT could alleviate the virus-induced body weight loss to a significant extent compared to the group that received saline (Fig. 5). Taken together, the results of this study demonstrate the potential of SNAT to significantly lower SARS-CoV-2 infection and the ensuing lung pathology. Future studies are aimed at deciphering the mechanism by which SNAT inhibits SARS-CoV-2 infection in hamsters.

Fig. 5figure 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 *

SNAT ameliorates lung injury in SARS-CoV-2-infected hamsters

We further examined the early histopathological changes in the lungs of hamsters to understand the effects of SARS-CoV-2 and SNAT treatment (Fig. 6). SARS-CoV-2 infection induced significant changes in lungs (Fig. 6; panels 2.0 and 2.1) compared to lungs from uninfected hamsters at 3 dPI (Fig. 6; panels 1.0 and 1.1). SARS-CoV-2 infection induced patchy evidence of oedema, hemorrhage and vascular congestion, subtle mononuclear infiltration, and focal hyaline membrane formation. The lungs of SARS-CoV-2 infected hamsters that received inhaled SNAT showed significantly lower signs of lung injury with no focal hyaline membrane formation (Fig. 6; panels 3.0 and 3.1), suggesting SNAT’s potential to effectively reduce the lung injury from SARS-CoV-2 infection.

Fig. 6figure 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

Nano-based therapeutics are considered an adaptable alternative to conventional small molecules such as antibodies for the neutralization of viruses. Because variable linkers can be decorated on to the surface of the nanoparticles, multivalent interactions of such nanoparticle drug candidates can be expected with the virus surfaces, potentially leading to virus neutralization [29, 30]. Such approach has been previously described to combat various RNA virus infections like HIV, paramyxovirus, SARS-CoV-2, and others [31,32,33]. The surface-modified multivalent SNAT that we developed has strong potential application as an inhalation antiviral therapeutic while conferring adequate safety to the lung and skin cells [29, 30].

Furthermore, SNAT has favorable physicochemical properties, including small near-atomic size, positive surface charge, aqueous solubility, and stability for over 3 years at room temperature and thus does not require cold storage chain during transportation and storage. The lack of cold storage infrastructure has been identified as one of the reasons impeding vaccine delivery across the low-income nations around the globe, widening the inequality to vaccine access [34]. In this regard, novel inhalation antiviral therapeutics, such as SNAT, that do not require refrigeration or cold storage chain would be ideal to transport from industry in the form of inhaler to patients at home or the clinical setting without the need for refrigeration.

Recently, a trivalent nanobody called ALX-0171 was developed and delivered via a nebulizer in patients infected with respiratory syncytial virus (RSV) in a phase 2b clinical trial [35]. Although RSV clearance was noted, treatment after the infection was established in lower respiratory pathway did not provide clinical benefits to patients, suggesting for treatment intervention during the earlier stage of infection [35]. In our study, early administrations (2 h and 48 h post infection with SARS-CoV-2) of inhaled SNAT conferred significant protection from the weight loss (Fig. 5) and showed improved lung health (Fig. 6; panels 3.0 and 3.1), unlike in hamsters infected with SARS-CoV-2 that showed patchy oedema, hemorrhage and vascular congestion, subtle mononuclear infiltration, and focal hyaline membrane formation in lungs (Fig. 6; panels 2.0 and 2.1).

The latest in the development of antiviral therapy against COVID-19 is Merck and Ridgeback’s experimental oral pill called Molnupiravir (MK-4482/EIDD-2801), a potent ribonucleoside analog that inhibits the replication of SARS-CoV-2. Although initially touted to have 50% protection [36], recent analyses suggest that the drug could only reduce the rate of hospitalization or death by 3% (absolute risk reduction) or 30% (relative risk reduction) among SARS-CoV-2-infected patients (n = 1,433) [37]. In addition, FDA has indicated that Molnupiravir may not be safe to pregnant women owing to its potential for “lethal mutagenesis” and/or off-target effects [38]. Considering these limitations and with the global rise of new variants that are potentially more infectious, and because current therapeutics require intravenous or oral delivery and/or access to COVID healthcare facilities, improving drug access to those underprivileged would require engineering future antiviral therapeutics that are easy to deliver at home without the need for a health care facility, which can be achieved using a ready-to-use inhaler or nebulizer akin to that used for SNAT.

留言 (0)

沒有登入
gif