key: cord-0871877-cyvan79u
authors: Park, Hee Ho; Kim, Hyelim; Lee, Han Sol; Seo, Eun U.; Kim, Ji-Eun; Lee, Jee-Hyun; Mun, Yong-Hyeon; Yoo, So-Yeol; An, Jiseon; Yun, Mi-Young; Kang, Nae-Won; Kim, Dae-Duk; Na, Dong Hee; Hong, Kyung Soo; Jang, Jong Geol; Ahn, June Hong; Bae, Jong-Sup; Song, Gyu Yong; Lee, Jae-Young; Kim, Hong Nam; Lee, Wonhwa
title: PEGylated Nanoparticle Albumin-Bound Steroidal Ginsenoside Derivatives Ameliorate SARS-CoV-2-Mediated Hyper-Inflammatory Responses
date: 2021-04-14
journal: Biomaterials
DOI: 10.1016/j.biomaterials.2021.120827
sha: f8901f2ab3f2093b01ebbe13d62bd5f00b9fac51
doc_id: 871877
cord_uid: cyvan79u

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.

F-CCTTCCTGTGCCTCTCCTTTA, R-AGGCATCATCCAGTCAAACCA 12 2. hNOX2: 13 F-AACGAATTGTACGTGGGCAGA, R-GAGGGTTTCCAGCAAACTGAG 14 3. hNLRP3: 15 F-TGCCGGGGCCTCTTTTCAGT, R-CCACAGCGCCCCAACCACAA 16 4. hMCP-1: 17 F-CGCTCAGCCAGATGCAATCAATGC, R-GGTTTGCTTGTCCAGGTGGTCCA 18 5. hVCAM1: 19 F-TGTCAATGTTGCCCCCAGAGATACA, R-GGCTGTAGCTCCCCGTTAGGGA 20 6 . hICAM1: 21 F-GTGTCCTGTATGGCCCCCGACT, R-ACCTTGCGGGTGACCTCCCC 22 23 2.13. Caspases detection assay. Caspase-Glo® 1 inflammasome assay kit (G9951, Promega, 24

Madison, WI, USA) and Caspase-Glo® 3/7 Assay System (G8090, Promega, Madison, WI, 25 USA) were used to measure the activation of the caspases in PBMCs. 26 27 2.14. Synthesis of BSA-mPEG. mPEG-MAL (20 mg) was slowly added to BSA (100 mg) 28 solution, and the mixture was stirred for 18 h at room temperature. The resulting solution was 29 dialyzed against double-deionized water (DDW) using a dialysis membrane with a molecular 30 weight cut-off of 6-8 kDa (Cellu-Sep T2; Membrane Filtration Products, Seguin, Texas, 1 USA). The dialysis product (i.e., BSA-mPEG) was lyophilized and stored at -70°C until use. Tokyo, Japan). The formulations were placed onto the surface of a 200-mesh carbon-coated 2 copper grid and negatively stained with uranyl acetate (UA). For the measurement of EE, an 3 aliquot (10 µL) of NAB-or PNAB-ginsenosides was vortex-mixed with acetonitrile (ACN; 4 990 µL) containing 0.1% (v/v) formic acid for 5 min. The mixture was sonicated for 2 min 5 and centrifuged at 13,000 × g for 5 min. The supernatant (50 µL) was then mixed with ACN 6 (950 µL) containing 0.1% (v/v) formic acid and internal standard (TC, 50 ng/mL), which was 7 the analytical sample for high-performance liquid chromatography (HPLC) analysis. 8

Chromatographic separation was performed using an Agilent 1260 Infinity HPLC system 9 (Agilent Technologies, Palo Alto, CA, USA). An aliquot (20 µL) of the analytical sample 10 was injected into a Kinetex 2.6 μm C18 100 Å column (100 × 4.6 mm; Phenomenex, CA, 11 USA) with a C18 guard column (4 × 2.0 mm; Phenomenex, CA, USA) at 25°C. The elution 12 was carried out under an isocratic condition at a flow rate of 0.35 mL/min, and the total run 13 time was 5 min. The mobile phase consisted of ACN and DDW with 0.1% (v/v) formic acid 14 (80:20, v/v). Mass spectrometric detection was achieved using API 3200 LC-MS/MS (SCIEX,  15 Framingham, Massachusetts, USA). The optimized ionization source settings of ion spray 16 source temperature, curtain gas pressure, gas 1, and gas 2, were 250°C, 25 psi, 50 psi, and 50 17 psi, respectively. The ion spray voltage, declustering potential, entrance potential, collision 18 energy, and collision gas were set at 5500V, 150 V, 1 V, 52 the permeability assay was performed as previously described. [45] In brief, the engineered 19 blood vessel was fabricated by using microneedles as templates. The collagen microchannels 20 were fabricated by inserting in the sol-state type I rat tail collagen (Corning, Bedford, MA, 21 USA) and subsequently removing them after the gelation. The human umbilical vein into the perfusable blood vessel and monitoring the molecular transport using a confocal 2 microscope (LSM700, Zeiss, Jena, Germany). The transendothelial permeability was 3 quantified by using a custom-written MATLAB code (The Mathworks Inc., Natick, USA). 4

To induce SARS-CoV-2-mediated inflammation in the engineered blood vessel, the SARS-5

CoV-2 patients' plasma was introduced in the blood vessel and incubated for 2 h. Since the 6 plasma of SARS-CoV-2 patients is coagulated, it was diluted in 1:2 ratio (plasma: media) 7 before the injection to ensure sufficient fluidity. For the immunofluorescence imaging, previously described [46] . Briefly, a 2-cm midline incision was made to expose the cecum and 24 adjoining intestine. The cecum was then tightly ligated using a 3.0-silk suture 5.0 mm from 25 the cecal tip, punctured with a 22-gauge needle, and then gently squeezed to extrude feces 1 from the perforation site. The cecum was then returned to the peritoneal cavity and the 2 laparotomy site sutured using 4.0-silk. For sham operations, the cecum of animals was 3 surgically exposed but not ligated or punctured and then returned to the abdominal cavity. For infectious diseases such as SARS-CoV-2 pneumonia, apparent clinical signatures are 9 commonly observed in the patient blood, including blood coagulation, [49, 50] NETosis, [51] 10 cytokine storm, [1] and inflammation. [24, 52] Those clinical signatures become severe in ICU 11 or deceased cases. Considering that there is no approved therapeutics to treat SARS-CoV- 2 12 infection, timely regulation of the clinical signatures is the primary strategy to improve 13 patient care. During the analysis of SARS-CoV-2 patient blood samples, we found that 14 histone H4 level was highly upregulated in the plasma of ICU patients. Histone H4, one of 15 the five main histone proteins composing chromatin structures in eukaryotic cells, [53] has 16 been implicated as an important risk factor in sepsis. [31] It is released into the blood 17 circulation, causing abnormal blood coagulation and NETosis. [54, 55] In a recent study, 18 abnormal coagulation (confirmed by D-dimer concentration) [56, 57] and NETosis (i.e., 19 cfDNA, NET, MPO activity, and NE) [58] [59] [60] [61] were also confirmed in the SARS-CoV-2 ICU 20 patient plasma, and the correlation between elevated histone H4 level and blood coagulation 21 [54, 62] /NETosis [63, 64] was validated. According to previous studies, albumin exhibits the 22 potential to inhibit the histone H4-induced platelet aggregation, [29] suggesting albumin as a 23 therapeutic candidate for managing blood coagulation. 24 We analyzed the plasma of SARS-CoV-2 patients using enzyme-linked immunosorbent 1 assay (ELISA) and found that the level of histone H4 was highly upregulated in the SARS-2

CoV-2 patient plasma ( Figure S1, Supporting Information) . From the clinical outcome data, 3 the level of histone H4 was higher in ICU patients than non-ICU cases ( Figure S1a,  4 Supporting Information) and in deceased cases than survival cases ( Figure S1b showing a significantly higher lung inflammation in the high-histone H4 cases than low-7 histone H4 ones ( Figure S1c , Supporting Information). 8 9

Through the analysis of COVID-19 patient plasma samples, we found that (1) serum albumin 11 level was decreased, (2) blood coagulation and NETosis were increased in association with 12 the histone H4 increase, and (3) To prepare PNAB-ginsenosides, bovine serum albumin (BSA) was adopted as a core 3 material and modified using monomethoxyPEG maleimide (mPEG-MAL), which can 4 selectively react with thiol groups on BSA surface (Figure 1a) . inflammatory interstitial fluid (pH 6.6) and plasma (pH 7.4) (Figure 1g) . Notably, both 7 formulations displayed not only sustained (up to 72 h) but also complete Rg6 release patterns. 8

As expected from the isoelectric point of BSA (approximately 4.7), the pH dependency was 9 barely observed, implying these formulations can efficiently release Rg6 in the inflammatory 10 tissues, as well as blood vessels. Meanwhile, each release profile was fitted using 11 mathematical models, including zero-order, first-order, Higuchi, and Baker-Lonsdale models, 12 and corresponding release constants (k 0 , k 1 , k H , and k BL , respectively) were estimated by the 13 least-squares regression (Figure 1h ). Among them, the first-order model exhibited the highest 14 correlation coefficients (R 2 ) compared to the others, which is in good accordance with the 15 fact that the drug-protein dissociation model is putatively expressed as first-order 16 kinetics. [ 

patients 20

Increased level of lactate dehydrogenase (LDH), which is an enzymatic indicator reflecting 21 the tissue damage, in ICU patients' plasma support the clinical data of increased histone H4 22 level and lung inflammation confirmed by CT images ( Table 1 ). The level of D-dimer was 23 highly upregulated in SARS-CoV-2 ICU patients, showing a 10-fold increase compared to 24 the non-ICU case ( Table 1 ). The high level of D-dimer indicates the existence of blood 25 coagulation in response to SARS-CoV-2 viral infection. Furthermore, histone H4 is also 1 known to promote neutrophil extracellular traps (NETs) formation. [ 

In addition, we also found that the blood albumin level was reduced as the severity of 3 symptoms increases ( Table 1 ). The decreased level of serum albumin in deceased individuals 4 was also recently reported in Wuhan, China, [74] supporting the patients' outcomes in Daegu, 5

South Korea case as shown in this study. The dramatically enhanced serum cytokine level, 6 which is associated with cytokine storm, was also observed in the peripheral blood As shown in Figure 2a , the portion of BSA-Rg6-and RSA-Rgx365-bound histone H4 was 19 more than two-fold higher than unbound (suspended in the supernatant) portion. This result 20

shows that the PNAB-Rg6 and PNAB-Rgx365 can capture serum histone H4. 21

The effect of albumin-ginsenoside in the suppression of NETosis was validated by 22 treating PNAB-Rg6 and PNAB-Rgx365 to SARS-CoV-2 ICU patient-derived neutrophils. 23

Those drugs reduced the levels of cell-free DNA (cfDNA) (Figure 2b) , implying the potential 24 capability of those drugs in the treatment of sepsis. Furthermore, the protein levels related to 25 NETosis were also recovered to normal range, including NET, myeloperoxidase (MPO) 1 activity, and neutrophil elastase (NE) (Figure 2c-e) . These results demonstrate that PNAB-2 steroidal ginsenosides can effectively suppress the histone H4-mediated NET. 3 4

Excessive level of serum cytokine, termed cytokine storm, is a symptom that is commonly 6 observed in SARS-CoV-2 patients. [76] Through the cytokine array analysis, we also 7 confirmed the substantial increase of inflammatory cytokines in SARS-CoV-2 patients' 8 plasma (Figure 3a) . However, upon the treatment of PNAB-Rg6 and PNAB-Rgx365 in the 9 SARS-CoV-2 patients' plasma, the level of inflammatory cytokines was significantly reduced 10 ( Figure 3a) . 11

The PNAB-steroidal ginsenoside nanotherapeutics were also effective in the PBMCs 12 

Another critical signature in SARS-CoV-2 patients is the cytokine storm. In this study, we 3 evaluated the cytokine storm issue through two signaling pathways, NF-κB and SREBP2. In 4 the PBMCs derived from SARS-CoV-2 ICU patients, the NF-κB signaling was upregulated, 5 in which NF-κB signaling is known to strongly correlate with cytokine production. [77] We 6 also confirmed that the activated NF-κB signaling correlated with cytokine storm (Figure 3) . 7 SREBP2, which belongs to the downstream signaling of NF-κB, regulates the inflammasome 8 formation upon the infection. [78] 9

The viability of SARS-CoV-2 ICU patients' PBMCs decreased as a function of time due 10 to the over-activation of inflammatory signaling pathways (Figure 4a ). We evaluated the 11 effect of PNAB-steroidal ginsenoside in the rescue of PBMC viability. The PNAB-Rg6 and 12 PNAB-Rgx365 could rescue the viability of PBMCs derived from ICU patients (Figure 4a) . 13

According to the analysis, the level of SREBP2 and caspase activities were reduced when 14 PNAB-Rg6 was treated and further reduced when PNAB-Rgx365 was treated (Figure 4b-d) . 15

The SREBP2 is known to be related to inflammasome formation, which is subsequently 16 known to activate caspase-1 activity and promote the production of IL-1β. [79] In addition, 17 the activities of caspase 1 and 3/7 were rescued, which is known to be activated by SREBP2. We also evaluated the reactive oxygen species (ROS) level after the administration of PNAB-25

Rg6 and PNAB-Rgx365 in the CLP-operated mouse model. We found that the administration 1 of PNAB-Rg6 and PNAB-Rgx365 could suppress the ROS level. The previous studies also 2 suggested that the Rgx365 can prevent pulmonary injury upon particulate matter exposure by 3 inhibiting the ROS generation [33] . Collectively, ROS assay in this study in conjunction with 4 the previous studies suggests that the PNAB-ginsenoside can prevent tissue damage in the 5 septic mouse model by suppressing ROS generation. The efficacy validation of PNAB-Rg6 6 and PNAB-Rgx365 using animal models clearly displayed the capability of those drugs for 7 the prevention of infection-mediated sepsis. 8 9

It has been reported that in response to the decreased serum albumin level, the insulin-11 induced gene-1 protein (INSIG1) dissociates from the SREBP2-SCAP complex, and the 12 SREBP2-SCAP complex translocates to Golgi apparatus. [81] Then the sphingosine-1-13 phosphate (S1P) and S2P dissociates C-term and N-term of SREBP2, and the SREBP2 N-14 term translocates to the nucleus and activates the inflammasome. [81] It is supposed that 15 steroidal ginsenosides, Rg6 and Rgx365, interrupt such cleavage of SREBP2 into C-and N-16 term and ultimately inhibit inflammasome and sepsis ( Figure 4f ). Structurally, Rg6 has a 17 similar molecular structure with betulin, which is known as an SREBP2 suppressor ( Figure  18 S2, Supporting Information). [82] Rgx365 is composed of Rg4 and Rg6, of which Rg4 is the 19 main constituent, therefore we can consider that the Rgx365 component has a similar 20 molecular structure with botulin ( Figure S2 , Supporting Information). [33, 82] 21 Previously, it was shown that convalescent plasma direct neutralization of the virus, 22

control the overactive immune systems, such as cytokine storm, Th1/Th17 ratio, and 23 complement activation, and immunomodulate hypercoagulable state. [25] In particular, the 24 efficacy of treatment in COVID-19 patients injected with albumin and convalescent plasma 25 mice model confirmed the prescribed in vitro model-based results, showing rescued survival 1 rate, prevention of tissue damage, and suppressed cytokine storm. The confirmation through 2 the in vitro and in vivo models support the promising capability of these drugs for the 3 therapeutics for the severe SARS-CoV-2 patients. 4 5

In this study, we demonstrated the suppression of NETosis and cytokine storm in SARS-7

CoV-2 patients' plasma and patients-derived PBMCs by using PNAB-steroidal ginsenoside 8 nanotherapeutics. The drugs could suppress NETosis via the modulation of serum histone H4 9 level and cytokine storm through downregulation of NF-κB and SREBP2 signaling pathways. Declaration of Interests Statement 20

The authors declare that there is no conflict of interest. 21

Data availability 23

The raw/processed data required to reproduce these findings cannot be shared at this time due 24 to legal or ethical reasons. 25 

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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 mean intensity-weighted 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

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

The release constants (k 0 , k 1 , k H , and k BL ) were

Figure 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 PNABginsenoside complex. PNAB-Rg6 and PNAB-Rgx365 (50 μg/ml) were introduced in SARS

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

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

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

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

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

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

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)

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

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 CLPoperated septic mice models after the treatment of PNAB-Rg6 and PNAB-Rgx365. PNAB-Rg6 and PNAB-Rgx365 (5 mg/kg) were delivered

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

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). declare the following financial interests/personal relationships

was demonstrated, in which albumin was shown to inhibit platelet aggregation by binding to 1 histone. [29] It is supposed that another role of albumin is inhibition of platelet aggregation, 2 pulmonary hemorrhage, and endothelial necrosis by binding with extracellular histones 3 secreted by a viral infection in severe COVID-19 patients. [40] Overall, we could modulate 4 the apparent clinical signatures of SARS-CoV-2 patients using PNAB-steroidal ginsenoside 5 nanotherapeutics. 6In this study, we fabricated steroidal ginsenoside saponin-bound albumin nanoparticles 7 for the following reasons: 1) To make a long-acting steroidal ginsenoside for greater 8 immunosuppressive effect, 2) To control organ damage and blood clotting mechanism due to 9 excessive inflammatory reaction by removing endotoxins such as histone and inflammatory 10 cytokines released in infectious diseases, 3) To prevent severe patients from dying of 11 hypotension as a result of septic shock. The development of albumin formulated 12 nanotherapeutics is of great value as a symptomatic treatment and is essential for the 13 improvement of morbidity and mortality prior to the antiviral agent development in the 14 current SARS-CoV-2 pandemic situation. 15Numerous studies highlight the importance of materials science and nanotechnology in 16 the field of antiviral research, proposing insightful approaches for the treatment of COVID-17 19 by means of innovative methodologies [83] [84] [85] . In this study, we presented a novel 18 ginsenoside delivery system via NAB technology. The PNAB-Rg6 and PNAB- Rgx365 19 showed marked efficacy in both in vitro and in vivo models. First, the PBMCs isolated from 20 severe SARS-CoV-2 patients demonstrated the capability for suppression of histone H4-21 induced NET, cytokine storm, and inflammasome. Second, the engineered blood vessel 22 model exhibited the effect of those drugs for the suppression of blood clot formation and 23 vascular inflammation. The reduced blood clot formation is presumably due to the reduced 24 fibronectin expression after the drug treatment. (Figure 5 ) Third, the CLP-operated septic 25