PEGylated nanoparticle albumin-bound steroidal ginsenoside derivatives ameliorate SARS-CoV-2-mediated hyper-inflammatory responses

Abstract

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on a global scale urges prompt and effective countermeasures. Recently, a study has reported that coronavirus disease-19 (COVID-19), the disease caused by SARS-CoV-2 infection, is associated with a decrease in albumin level, an increase in NETosis, blood coagulation, and cytokine level. Here, we present drug-loaded albumin nanoparticles as a therapeutic agent to resolve the clinical outcomes observed in severe SARS-CoV-2 patients. PEGylated nanoparticle albumin-bound (PNAB) was used to promote prolonged bioactivity of steroidal ginsenoside saponins, PNAB-Rg6 and PNAB-Rgx365. Our data indicate that the application of PNAB-steroidal ginsenoside can effectively reduce histone H4 and NETosis-related factors in the plasma, and alleviate SREBP2-mediated systemic inflammation in the PBMCs of SARS-CoV-2 ICU patients. The engineered blood vessel model confirmed that these drugs are effective in suppressing blood clot formation and vascular inflammation. Moreover, the animal model experiment showed that these drugs are effective in promoting the survival rate by alleviating tissue damage and cytokine storm. Altogether, our findings suggest that these PNAB-steroidal ginsenoside drugs have potential applications in the treatment of symptoms associated with severe SARS-CoV-2 patients, such as coagulation and cytokine storm.

Keywords: Albumin; COVID-19; Cytokine storm; Ginsenoside; NETosis.

Fig. 1

Characterizations of PNAB-Ginsenosides. (a) Scheme illustration of PNAB-ginsenosides. (b) MALDI-TOF mass spectra for BSA (left) and PEGylated Nanoparticle Albumin-Bound (PNAB) (right) at feed ratios of BSA:PEG of 1:2.5. (c) Acrylamide gels stained with Coomassie blue stain (left) and barium iodide stains for PEG (right). (d) Characteristics of PNAB-Rg6 and PNAB-Rgx365 including intensity-weighted mean hydrodynamic diameter, polydispersity index, zeta potentials, and encapsulation efficiency (EE) of PNAB-Rg6 and PNAB-Rgx365. (e) The number-weighted size distribution (inset: intensity-weighted size distribution with modal values of each peak) and (f) transmission electron microscopy (TEM) images of PNAB-Rg6 and PNAB-Rgx365 (scale bar: 500 nm). (g) The controlled-release pattern of PNAB-Rg6 and PNAB-Rgx365 under pathophysiological pH conditions (pH 6.6 for acidic inflammatory interstitial fluid and pH 7.4 for plasma) and (h) mathematical model fitting of the release profiles (zero-order, first-order, Higuchi, and Baker-Lonsdale models). The release constants (k₀, k₁, k_H, and k_BL) were estimated using the least-squares regression analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

PNAB-Rg6 and PNAB-Rgx365 suppressed histone H4-mediated neutrophil extracellular trap (NET) by binding to histone H4. (a) Binding assay of histone H4 to PNAB-ginsenoside complex. PNAB-Rg6 and PNAB-Rgx365 (50 μg/mL) were introduced in SARS-CoV-2 patients' plasma and subsequently precipitated. The histone H4 level in the precipitate (bound to BSA) and supernatant (unbound) was quantified. (b–e) Effect of PNAB-Rg6 and PNAB-Rgx365 (50 μg/mL) in the suppression of NETosis. The suppression of NETosis was validated via changes of (b) cfDNA, (c) NET, (d) MPO activity, and (e) NE (**p < 0.01).

Fig. 3

PNAB-Rg6 and PNAB-Rgx365 ameliorate SARS-CoV-2-mediated cytokine storm via down-regulation of NF-κB activation. (a) Inhibition of cytokine storm in SARS-CoV-2 patients' plasma after the treatment of PNAB-Rg6 and PNAB-Rgx365 (50 μg/mL, 6 h). (b) Decreased NF-κB activation in SARS-CoV-2 ICU patients' PBMCs upon the PNAB-Rg6 and PNAB-Rgx365 treatment (50 μg/mL, 6 h) (*p < 0.05 and **p < 0.01). (c–h) Decreased cytokine levels in SARS-CoV-2 ICU patients' PBMCs upon the PNAB-Rg6 and PNAB-Rgx365 (50 μg/mL, 6 h) treatment including (c) IL-1β, (d) IL-4, (e) IL-6, (f) IL-8, (g) interferon (IFN)-γ, and (h) tumor necrosis factor (TNF)-α (*p < 0.05 and **p < 0.01). (i) Schematic illustration on the effect of Rgx365 in the suppression of NETosis and cytokine storm.

Fig. 4

PNAB-Rg6 and PNAB-Rgx365 inhibit SREBP2-mediated inflammasome in PBMCs of SARS-CoV-2 ICU patients. (a) Time-course monitoring of SARS-CoV-2 ICU patients' PBMCs upon the treatment of PNAB-Rg6 and PNAB-Rgx365 (50 μg/mL, 6 h) (***p < 0.001). (b–d) Effect of PNAB-Rg6 and PNAB-Rgx365 (50 μg/mL, 6 h) in the activation level of (b) SREBP2, (c) caspase-1, and (d) caspase-3/7 in SARS-CoV-2 ICU patients' PBMC. (e) Real-time PCR analysis of NETosis- and inflammation-related mRNAs including SREBP2, NOX2, NRLP3, MCP1, VCAM1, ICAM1, and ELAM1 (*p < 0.05 and **<0.01). (f) Potential mechanism of Rg6 and Rgx365 in the suppression of SREBP2-mediated inflammasome.

Fig. 5

PNAB-Rg6 and PNAB-Rgx365 alleviate blood clot formation and vascular inflammation. (a) Schematic illustration of the efficacy test of PNAB-Rg6 and PNAB-Rgx365 for the recovery of the vascular function using a 3D engineered blood vessel model. SARS-CoV-2 patients' plasma was introduced for vascular disruption. After 2 h of incubation with the SARS-CoV-2 patients' plasma, PNAB-Rg6 and PNAB-Rgx365 were treated for 24 h. (b) Molecular transport of 40 kDa FITC-Dextran out of the engineered blood vessel. (c) Quantified transendothelial permeability after the treatment of PNAB-Rg6 and PNAB-Rgx365 (**p < 0.01). (d) Immunofluorescence images of an engineered blood vessel after the treatment of PNAB-Rg6 and PNAB-Rgx365. Red: F-actin, green: ICAM-1, and blue: fibrinogen. (e) The effect of PNAB-Rg6 and PNAB-Rgx365 in the alleviation of blood clot formation and vascular inflammation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

PNAB-Rg6 and PNAB-Rgx365 rescue the survival rate of septic mice models and prevent the cytokine storm and tissue damage. (a) The time-course survival rate of CLP-operated septic mice models after the treatment of PNAB-Rg6 and PNAB-Rgx365. PNAB-Rg6 and PNAB-Rgx365 (5 mg/kg) were delivered intravenously 24 h after the CLP-operation. (b) Histological analysis of mice lung tissue after the treatment of PNAB-Rg6 and PNAB-Rgx365. (White scale bar = 75 μm). (c) Pathology-related signatures including vascular permeability, lung ICAM-1 expression, and leukocyte and neutrophil migration in bronchoalveolar lavage (BAL), and NF-κB and SREBP2 activities in lung tissue. (*p < 0.05). (d) Expression of tissue damage markers including C-reactive protein (CRP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (*p < 0.05). (e) Expression of cytokines such as IL-1β, IL-6, IL-8, and INF-γ (*p < 0.05). (f) Quantification of ROS level in mouse lung endothelial cells (**p < 0.01).