key: cord-0974456-w7oqe3d0
authors: Rejinold, N. Sanoj; Piao, Huiyan; Jin, Geun-woo; Choi, Goeun; Choy, Jin-Ho
title: Injectable niclosamide nanohybrid as an anti-SARS-CoV-2 strategy
date: 2021-08-24
journal: Colloids Surf B Biointerfaces
DOI: 10.1016/j.colsurfb.2021.112063
sha: 18699890f44f29b79af413b2a6c6f119b7189fff
doc_id: 974456
cord_uid: w7oqe3d0

COVID-19 is a rapidly evolving emergency, which necessitates scientific community to come up with novel formulations that could find quick relief to the millions affected around the globe. Remdesivir being the only injectable drug by FDA for COVID-19, it initially showed some promising results, however, failed later and rejected by the WHO. Therefore, it is important to develop injectable formulation that are effective and affordable. Here in this work, we formulated poly ethylene glycol (PEG) coated bovine serum albumin (BSA) stabilized Niclosamide (NIC) nanoparticles (NPs) (∼ BSA-NIC-PEG NPs) as an effective injectable formulation. Here, serum albumin mediated strategy was proposed as an effective strategy to specifically target SARS-CoV-2, the virus that causes COVID-19. The in-vitro results showed readily water dispersible formulation having particle size <120 nm size and they were well stable even after 3 weeks. Even though the in-vitro studies showed promising results, the in-vivo pharmaco-kinetic (PK) study using rats showed the need of conducting further experiments to specifically target the SARS-CoV-2 in the virus infected model. We expect that this present formulation would be highly preferred for targeting hypoalbuminemia conditions, which was often reported in elderly COVID-19 patients. Such studies are on the way to summarize its potential applications in the near future.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

COVID-19 has been affecting 204 M individual all around the world leaving 4.32 M into fatal deaths as of Aug 12-2021 . Additionally, the new genetic variant of COVID-19 making the current situation worse than ever before ever since it was first reported in the UK [1] . Therefore, it is urgent to discover new anti-viral medicines, which could be combined along with the ongoing global vaccinations. Remdesivir is the only injectable formulation for COVID-19 [2] [3] [4] [5] [6] [7] [8] , which initially showed promising results, however, the drug had controversial results since it showed no or negligible results in the COVID-19 patient studies, thereby WHO had finally declared its ineffectiveness [9] . Therefore, developing novel formulations are very urgent. On the other hand, FDA approved anthelmintic NIC [10] has showed anti-viral properties among other FDA approved drug molecules in the in-vitro experiments on SARS-CoV-2 infected VERO cells. It was reported that even a small dosage of 0.16 µM of NIC (57.14 ng/mL) could induce potential anti-viral effects on SARS-CoV-2 infected VERO cells [11] . However, the major limitation of NIC is associated with its poor water solubility, thereby low bioavailability [12] .

NIC has been extensively studied for anti-cancer [13, 14] , anti-inflammatory [15] and anti-viral potency [16] . It has shown significant anti-viral effects on wide variety of viral pathologies including MERS [17] , SARS [18] , the similar family of COVID-19. The major mechanisms involved in NIC antiviral potency against SARS-CoV-2 are as follows: 1) inhibiting endocytosis;

2) inhibiting autophagy and 3) inhibiting the viral replications. (Scheme 1) [19] .

Previously there have been various physical and chemical modifications for NIC to improve its solubility; however, most of such studies related to anti-cancer therapy. For example, poly ethylene glycol (PEG) was chemically conjugated with NIC in order to improve its bioavailability [12] .

However, it is important to mention that there were not so many published work related to injectable NIC formulations in the literature. In addition, most of the in-vitro experiments give a clarity neither on formulation nor on the NIC dissolving procedures.

In the present study, we formulated NIC with bovine serum albumin (BSA) and the resulting nanoparticles (NPs) were coated physically with PEG. The major reason for selecting BSA was J o u r n a l P r e -p r o o f due to its immense bio-compatibility [20, 21] and stabilizing capability [22] . Various biomedical applications including drug delivery [23] [24] [25] [26] [27] [28] [29] , tissue engineering [30] [31] [32] [33] and bio imaging, etc. extensively utilize BSA. It has also been reported that BSA modification could improve anti-viral potency [34] against Ebola pseudo viruses and virus like particles [35] . In addition, albumin plays a very important role in SARS-CoV-2 infection. Hypoalbuminemia is associated with severely affected COVID-19 patients and in such conditions, the virus can bind on serum albumin, dysregulating the cellular nutrient transportation and leading to systemic symptoms observed in SARS-CoV-2 infection and sepsis. When albumin bind to SARS-CoV-2 virions, this could lead to the damage of endothelial glycocalyx by inhibition of albumin transport binding sites. This vascular endothelial glycocalyx (VEGLX) damage has also been reported as targeting regimen to treat COVID-19 [36] . In this context, rationally modified antiviral drug loaded serum albumin therapy might be beneficial for improved COVID-19 therapy [37] . Additionally there have been several reports for hypoalbuminemia as an early diagnostic rational [38] and a targeting strategy towards COVID-19 [39] .

On the other hand, PEGylation has been reported to improve the anti-viral effects [40] , by enhancing the blood circulation of antiviral agents in-vivo. PEG has been extensively studied for drug delivery applications. Therefore, NIC would improve its therapeutic properties once they are modified with BSA and further coated with PEG for improved blood circulation, which might enhance their in-vivo efficacy [41] .

In the present work, were are trying to address the following questions. 1) How effectively NIC molecules could be loaded into the BSA/PEG hybrids? 2) How nano-hybridization would affect the overall solubility of NIC? 3) What would be the future perspectives of these NIC-Nanohybrids based on the in-vivo pharmaco-kinetics? The research hypotheses are as follows: 1) NIC being a neutral molecule, it could be stabilized using BSA via weak forces such as hydrogen bonding; 2)

We expect that the solubility of NIC would be increased mainly because of the BSA stabilized structure of NIC; 3) We propose that PEG-BSA-Stabilized NIC NPs would be ideal in order to target SARS-CoV-2 for better efficacy either through extracellular or intracellular pathways (Scheme 1).

J o u r n a l P r e -p r o o f

NIC (C13H8Cl2N2O4) was obtained from DERIVADOS QUIMICOS, Murcia, Spain. Anhydrous Ethanol (99.9%), and isopropyl alcohol (IPA) were purchased from Daejung, Gyeonggi-do, South

Korea. BSA and (β-cyclodextrin were purchased from Sigma Aldrich, USA. PEG, weight average molecular weight, Mn ~ 10,000, was purchased from Sigma, Milwaukee W1 53233, USA.

For NIC NPs, 5 mg of NIC was dissolved in 1 mL IPA. Thus, prepared solution of ~ 250 µL was directly added in to 9.75 mL of distilled water, where the particles were formed immediately with white opalescent coloration. 

The PEG-BSA-NIC NPs were synthesized as follows; initially, NIC (5 mg/mL) was dissolved in IPA, from which 250 µL solution was drop wisely added in to 9.5 mL of distilled water containing BSA (50 mg/mL) until it formed an opalescent yellow coloration. Finally, 250µL PEG (5 mg/mL) was added, followed by 15-30 min rotary evaporation to remove any remnant IPA. Detailed information is given in the Fig. S1 .

PEG-BSA-NIC NPs were synthesized as follows. Initially, NIC (5 mg/mL) was dissolved in IPA solution, from which 250 µL solution was drop wisely added in to 9.5 mL of distilled water containing BSA (50 mg/mL) until it formed an opalescent yellow coloration. Finally, 250 µL PEG (5 mg/mL) was added, followed by 15-30 min rotary evaporation to remove any remnant IPA. The resulting solution was freeze dried for 48 h in order to get the yellow powder, which was directly added in to 10 mL distilled water followed by probe sonication for 4 min. The uniform suspension was further syringe filter using 0.45µm PVDF filter (Fig. S2 ).

J o u r n a l P r e -p r o o f

The in-vitro drug release was done using dissolution apparatus. For the analysis, initially calibration curve was made in deionized water (DI water) (Fig. S3) . In order to understand how NIC could be released from BSA-NIC and PEG-BSA-NIC NPs with and without serum conditions, two buffer solutions were made with a pH 7.4, of which one had 3% fetal bovine serum (FBS) added in it, mimicking the severe hypoalbuminemia condition ( albumin < 2.5 g/dL) [42] . The pH of the dissolution media was chosen as 7.4 mainly because it mimics the blood pH [46] . 50 mg of samples were added in to ~ 310 mL solution in dissolution chamber. At pre-determined time intervals, 2.5 mL aliquots were withdrawn, and syringe filtered using 0.45 µm PVDF filters. The absorbance was measured at 340 nm using DI water as the solvent.

Stability studies were conducted at room temperature, refrigerator conditions (4°C) and under serum condition (3%) respectively. The size and poly dispersity indices (PDI) were measured using Zeta Sizer Otsuka Electronics DLS/Zeta EL-SZ-2000 for 3 weeks.

A pharmacokinetic (PK) study was performed in male Sprague-Dawley rats to the injection. In order to prepare for a control formulation, we made NIC solution using minimal additives. The two raw materials used to make NIC nanoparticles are ethanol, which is a relatively bio-compatible solvent and the other is aqueous solution of β-cyclodextrin, which is widely used to dissolve hydrophobic drugs in water [43] . Accordingly, the control formulation was prepared by dissolving NIC in 0.24 % β-cyclodextrin solution (ethanol-water mixture; 2:8 v/v) to make a final concentration of 2 mg/mL. The hydrodynamic size of the prepared NIC solution was less than 10 nm, implying that the NIC was well dissolved in the solution having no particle properties ( Fig. 1 ). 2 mg/kg of control formulation was also injected intravenously to rats. Then the blood samples were collected by centrifuging for 2 min at 13,000 rpm in order to separate the plasma that was immediately frozen until further analysis using HPLC-mass spectrometry (HPLC-MS).

J o u r n a l P r e -p r o o f

The blood (~ 600 µL) was drawn from the jugular vein for analysis. A plasma calibration curve was prepared by using NIC standards at concentrations of 0, 5, 10, 50, 100, 500, 1000, and 2000 ng/mL. Samples were prepared by adding 100 µL of the internal standard, topiramate to 20 μL aliquot of rat plasma sample. In general, internal standard like topiramte drug is used to have a precised and accurate data, where volume errors are difficult to predict and control [44] . Then, they were vortexed for 5 sec, followed by centrifugation at 13,000 g for 5 min at 4 °C. The 20 µL of supernatant was mixed with 180 µL of 50 % methanol. Then, 150 µL of aliquot of the samples were transferred to sample vial for the analysis. Samples were analyzed by Acquity I-Class UPLC system (Waters) with mass spectrometer (Waters Xevo TQ-S). The LC analytical column was a Thermo Hypersil Gold (2.1 x 50 mm, 1.9 µm) and was maintained at 50°C during the measurements. Mobile phase was 5 mM ammonium acetate/water solution, and methanol with a flow rate of 0.4 mL/min.

The 

The statistical significances of the differences were evaluated with a two-tailed Student's t-test. P value < 0.05 was considered as statistically significant.

NIC nanohybrids such as BSA-NIC NPs and PEG-BSA-NIC NPs were synthesised using a very simple co-acervation technique [45] . The interaction beteen NIC, BSA, PEG was confirmed using FT-IR analysis. As shown in the Fig. 1 , the characteristic peaks for NIC was found at 3577 and 1210 cm -1 respectively. These NIC characteristic peaks were assigned for -NH strtetching and -OH in plane bending respectively. Interestingly, these two main peaks of NIC were absent in both BSA-NIC and PEG-BSA-NIC NPs, possibly due to their involvement in hydrogen bonding interaction [46] . In addition, the methylene peak in the NIC was absent in both BSA-NIC and PEG-BSA-NIC NPs confirming that hydrogen bonding could be achieved through these functional groups [47] .

On the other hand, BSA stabilization of NIC was further confirmed by noticing the characteristic peak shifts compared to the control BSA alone. As per reported literature, BSA has characteristic peaks at ~ 1656 (Amide I, mostly due to the carbonyl (C=O) stretching vibration of the peptide linkages and stretching vibration bands of -OH groups), 1537 (Amide II, mostly from the in plane bending vibration of N-H and stretching vibrations of C-N bond), and ~ 3300 cm −1 , amide A (mostly stretching vibrations of -NH bond), respectively) [48] . It was noted that all the Amide-I and II bands were perturbed to either lower or higher wavelength (Fig.1) , indicating the changes in their secondary protein structure, owing to the well stabilization of NIC by BSA molecules as indicated in Fig. 1c .

PEG coating on BSA-NIC NPs was finally confirmed by observing the charactersitic peak shifts of PEG bands. From the FTIR, it was clear that PEG had the characteristic bands at 2929 cm -1 , The band at 1242 cm −1 in PEG corresponds to CH2 twisting, and it has got less intensified in PEG coated BSA-NIC NPs, possibly due to a hydrogen bonding characteristic property [50] .

Further, the FT-IR analyses were validated with the proton NMR data ( Fig. 1d, 

The DLS analysis showed that both BSA-NIC and PEG-BSA-NIC NPs had optimum size in the range of < 150 nm. However, after PEG coating, BSA-NIC NPs reduced significantly low particle size from 153 ± 2.61 to 110 ± 3.67 nm (Fig. 2a, 2b) . This might be due to the PEG coating, wherein it can well stabilize the particle. In fact, it is well known that PEG coating can reduce the particle size mainly due to the capping nature of PEG. Thanks to the hydroxyl groups in PEG which can reduce the agglomeration via capping effect [53, 54] . On the other hand, the β-cyclodextrin NIC nano formulation had shown ultra small particle size (Fig. S5) .

The zeta potential for control NIC NPs were found to be around -27.03 ± 3.69 mV, whereas the BSA-NIC NPs showed almost similar zeta potential as that of control NIC NPs (-32.19 ± 1.39 mV). This could be mainly because of the interaction between NIC and BSA, indicating that either BSA must have coated on the surface or NIC could be get inside the drug sockets of BSA. On the other hand, PEG coating on the BSA-NIC NPs shifted the zeta potential significantly towards a lower values (-16.55 ± 0.75 mV) (Fig. 2c ). This indicated a proper coating of PEG on the surface of BSA-NIC NPs. All the NPs were stable right after they were prepared freshly, as shown in the Fig. 2e with a good Tyndall effect on laser exposure (Fig. 2f) .

Further, UV analysis was done on various samples including NIC, BSA-NIC and PEG-BSA-NIC NPs in order to understand whether there was any characteristic peak shift for NIC, before and after modification with BSA, and PEG. UV spectra showed a major characteristic band for BSA at 277 nm, originated from the aromatic amino acids (Trp, Tyr, and Phe) and backbone (n → π* transition, peptide bond) of BSA. NIC showed its characteristic peak at ~ 345.5 nm in water, whereas, BSA showed its specific peak at 277 nm. On modification with BSA, the BSA-NIC hybrids were able to maintain their individual peaks at 279 (BSA) and 340 nm (NIC). The slight blue shift of NIC absorption from 340 to 345.5 nm indicated their encapsulation in the BSA.

Similarly, the NIC peak (340 nm) was blue shifted to 342 in PEG-BSA-NIC NPs as well. Moreover, the BSA bands were slightly shifted on modification with BSA and PEG (Fig. S6) , indicating their secondary bonding might have affected on NIC loading through H-bonding.

It should be noted that BSA is globulin molecule having large molecular size with loose structure, entailing its hydrophobic cavities for drug loading or encapsulation. When poorly water-soluble J o u r n a l P r e -p r o o f NIC molecules were added, BSA could interact with NIC through the hydrogen bonding, resulting in compact molecular self-assembled structure. On the other hand, when NIC molecules get into the drug sockets of BSA, its total conformation would have changed. This protein-disrupted structure of BSA suggests that the conformation of BSA might have changed through a strong interaction between BSA and NIC [55] .

Further, we analyzed the morphology of developed BSA-NIC and PEG-BSA-NIC NPs by FE-SEM. The particle size obtained from DLS measurements were almost matched with the FE-SEM results shown in the supporting Fig. S7 with very small average particle size. The rod shaped morphology could be due to a thermodynamically more stable structure of the PEG-BSA-NIC NPs [56] . The elongated NIC structures were cut down into smaller pieces of <150 nm, indicating that BSA and PEG modification would enhance their solubility for better in-vitro and in-vivo therapeutic outcome. It should also be noted that SARS-CoV-2 viral particles were reported to possess a size < 100 nm. Therefore, in order to adsorb such small sized virions, it is important to match with their size in the optimum range. We expect that our rod shaped, PEG-BSA-NIC NPs might be a good strategy in this context. In addition, the rod shaped NPs reported to possess better blood circulation than the spherical NPs [57] .

The major problem associated with NIC has been its solubility. Therefore, our most important priority was to somehow improve its solubility by modifying with BSA and PEG moieties. Here, compared to the NIC NPs alone, the powdered BSA-NIC NPs and PEG-BSA-NIC NPs were readily solubilized in water as shown in the Fig. 3 . In addition, a real time video footage was given in the supporting information as Video S1 and Video S2, which clearly shows the readily water soluble characteristic property after modifying with BSA and PEG.

Further, we have checked a long-term stability of NIC-nanohybrids at three different conditions such as room temperature (RT), refrigerator (4°C) and with 3% serum. As observed in the Fig. 4 , both BSA-NIC and PEG-BSA-NIC NPs could retain the particle size pretty well over 3 weeks.

Whereas the NIC-NPs showed irregular size as per DLS along with very high poly dispersity indices (Fig. S8) . It is worthy to mention that even after 3 weeks, all the samples showed very good stability in terms of PDI (poly dispersity index) and Tyndall effect as shown in Supporting J o u r n a l P r e -p r o o f However, the particle size for the NIC NPs were unstable especially in the room temperature and refrigerator conditions after two weeks whereas, the serum conditions were stable, with large particle size (<500 nm). In addition, the PDI values for control NIC NPs were also irregular indicating that NIC NPs could be destabilized in the solution form when it was in an intact form, necessitating a proper coating, as we demonstrated in the present study. On the other hand, even though, both BSA-NIC and PEG-BSA-NIC NPs were stable at three different conditions as mentioned, the particle size seemed increasing from week 1. Therefore, it is advised to store the formulation as a freeze-dried powdered form rather than in a solution form. In a clinical aspect, therefore, we recommend to keep these formulations in powder form until it can be reconstituted in the desirable intravenously injectable solution (IV solution). Table S1 shows the details of NIC contents in various injectable samples. After understanding the drug contents, we conducted in-vitro drug release studies using two samples such as BSA-NIC NPs and PEG-BSA-NIC NPs with two different ratios such as 2:5 and 1:40 (NIC:BSA ratio).

These two formulations with different concentrations of BSA were tested in order to understand how BSA concentration would account for the drug release pattern. As shown in Fig. 5 , it was obvious that both NPs were stable in serum conditions and thereby better release pattern under serum conditions than that at non-serum buffer (pH 7.4). It was noted that the BSA concentration had slightly improved the release pattern under serum conditions (Fig. 5b) . This might be attributed for the high stability of NIC hybrids with higher amount of BSA, which was further improved under serum conditions. Approximately ~ 80% NIC was released under the serum conditions from 1:40 ratio sample with PEG coating, indicating that NIC molecules could be better solubilized in the presence of BSA and PEG. Previously there have been such reports for better drug release profile by BSA stabilization [58] . In addition, BSA [59] as well as PEG modified NPs [60] can show a controlled release pattern as well.

Finally, the as made PEG-BSA-NIC NPs were tested for in-vivo bioavailability using rats. The samples were injected into female Sprague-Dawley rats at 2 mg/kg as mentioned previously. On the other hand, the control NIC formulation cleared very quickly from the body within 2 h indicating the poor stability thereby low absorption of NIC than the PEG-BSA-NIC NPs. The sustained circulation until 6 h could be due to the physical coating of PEG molecules. It is well known that PEG modification could effectively improve the blood circulation by inhibiting the opsonin adhesion, thereby escaping the phagocytic capture [61] .

As shown in Fig. 6 , the mean area under the concentration-time curve (AUC-last) was calculated to be 375.49 ± 63.27 (ng·h/mL). The detailed PK parameters were given in the Table 1 . Table 1 , shows that PEG-BSA-NIC NPs were able to maintain the plasma concentration of NIC quite longer time in comparison with the control NIC formulation. Though the AUC was slightly higher for the β-cyclodextrin-NIC control formulation, their t1/2 was very low and negligible and therapeutically irrelevant. On the other hand, it is worthy to say that PEG-BSA-NIC NPs could improve almost ~ 8.35 fold higher t1/2 than the control NIC formulation. The AUC value for control NIC formulation was quite higher 552.40 ± 303.76 ng·h/mL, while they showed very low t1/2 (0.15 ± 0.02 h). On the other hand, the both PEG-BSA-NIC NPs and BSA-NIC NPs exhibited same tmax of 0.25 h with high Cmax for the PEG-BSA-NIC NPs (904.76 ± 117.52 ng/mL). It should be noted that a maximum NIC concentration from PEG-BSA-NIC NPs was achieved in a sustained manner as the t1/2 was very high (1.28 ± 0.41 h). This sustained plasma NIC concentration might be beneficial for achieving better anti-COVID therapeutic outcome than the control sample.

The present pandemic situation is an emergency as the daily COVID-19 cases are surging worldwide. Despite of the worldwide vaccination strategies, where it can only prevent the diseases, there are millions still suffering with COVID-19, especially with the newly mutated delta [62] [63] [64] and lambda variants. In particular, old aged group with existing health issues such as diabetes and cardiovascular diseases are vulnerable. Therefore, a proper anti-COVID-19 therapeutic strategy is of great scope. However, finding a completely new medication has always been a challenge. On the other hand, there have been several potential drug candidates, with negligible bio-availability owing to their poor water solubility. The present drug candidate, NIC, in our study is such a candidate. Here our main challenge is to improve NIC solubility and eventually achieve anti-viral effects.

Firstly, the major reason for choosing BSA was to improve the drug solubility of NIC. Previously, Chen et al., (2018) reported stabilization of poorly water soluble curcumin by BSA [65] . Secondly, to utilize BSA as a specific targeting agent for the infected sites under hypoalbuminemia condition due to the SARS-CoV-2, which has been well reported, particularly in severely affected patients [66] . For example, found that hypoalbuminemia could result in poor prognosis in COVID-19 patients [67] .

In this aspect, BSA itself could be selectively taken up by the infected cells. Hypoalbuminemia majorly observed in patient with diabetes [68] and cardiovascular diseases [69] , and these groups are highly prone to COVID-19, leading to mortality. Hypoalbuminemia condition leads to a poor prognosis in 80% of the non-surviving COVID-19 positive patients. This could be due to the fact that serum albumin maintains plasma oncotic pressure and function as a vehicle for different endogenous/exogenous compounds. Even though hypoalbuminemia is not considered as a cause of underlying pathology, it is more like a condition, therefore, albumin based strategical therapeutic approaches would be highly advantageous for COVID-19.

In the present study, it was very clear that poorly soluble NIC drug was able to be readily watersoluble after albumin coating, and PEGylation, indicating its successful i.v. formulation, for the first time (Video S1 & S2). However, in the previously reported oral formulation (200 mg/kg) for anti-cancer therapy, the in-vivo PK performance was determined to be below 100 ng/mL [70] . In fact we achieved almost the similar PK profile with the readily water-soluble i.v. formulation at J o u r n a l P r e -p r o o f the 2 mg/kg dosage, and therefore, this might be advantageous to have better anti-COVID outcome in clinical model.

Another highlight in our case was the smaller particle size ~120 nm, particularly for the PEG-BSA-NIC NPs, and this could be beneficial for easy adsorption on the virus with similar size, thereby better internalization within the host cells. The SARS-CoV-2 has less than 200 nm particle size [71] . The comparatively low availability of our intravenously injected PEG-BSA-NIC NPs might be associated with non-hypoalbuminemia condition in the normal rats.

It should be noted that, injectable formulations were hardly explored for NIC. The major studies reported were PEG covalently modified with NIC, which was developed for cancer therapy to treat SKOV-3 tumor xenograft in NOD/SCID mouse [72] . Even though, their PK studies showed less than 50-100 ng/mL of NIC plasma concentration, the NIC formulation (by electro spraying) was able to induce antitumor efficacy in SKOV-3 xenograft model indicating that, the present PEG-BSA-NIC NPs might be therapeutically beneficial for viral infected cells too.

In many of the reported studies related to screening FDA approved drug candidates for SARS-CoV-2 virus, the methodology related to drug formulation was not mentioned clearly (Fig. S10) .

In that context, a readily water/biological fluid (Culture media) soluble NIC formulation is of great relevance.

In the future studies, we will be analyzing how these readily soluble NIC nanohybrids could target the SARS-CoV-2 infected sites under hypoalbuminemia condition? We hypothesize that a damaged glycocalyx could lead to hypoalbuminemia condition there by higher chance of cellular intake of albumin coated NPs. In addition, we hypothesize that when albumin coated NIC nanohybrids bind with the viral particles, it could easily enter the infected cells allowing intra cellular anti-viral mechanism.

Under an injury or sepsis condition associated with COVID-19, glycocalyx damage was reported to be dominant, meaning that viral particles could easily get into host cells as the barrier is already damaged. In addition, the hypoalbuminemia condition could lead to an enhanced cellular uptake of serum albumin coated NIC nanohybrids. Our future studies on the present nanohybrids will be oriented on this aspect with the SARS-CoV-2 infected model (scheme S1).

J o u r n a l P r e -p r o o f

We successfully developed highly water soluble and injectable PEG-BSA-NIC NPs, having optimum particle size <150 nm. The in-vitro characterizations such as FT-IR, NMR, DLS and zeta analyses clearly showed the successful PEG coating on BSA-NIC NPs. The present injectable formulations were stable for 3 weeks under ambient condition at room temperature and at 4°C in refrigerator, and in serum. Further, the in-vitro NIC release from PEG-BSA-NIC NPs was significantly higher even in the serum condition. Compared to the previous studies related to intraperitoneal NIC formulation [12] , we were able to make an i.v. one, for the first time, which was readily water soluble, resulting in improved bioavailability. In addition, the average particle size of PEG-BSA-NIC NPs lower than 150 nm, as determined by the DLS method (Fig. 2) , could be considered as an ideal one for injectable solution. Their thickness could also be estimated from the SEM study (~20 nm in diameter in Fig. S6 ), which was significantly smaller than the previous reports on the NIC formulation with a size of ~ 100 nm in diameter and ~ 500 nm in length [72] .

According to the PK studies for the present PEG-BSA-NIC nanohybrid drug and the control NIC formulation, the plasma concentration of NIC for the former was determined to be less than 100 ng/mL, whereas its t1/2 value was greatly improved to be 1.28 h, which was ~ 8.35 fold higher than that of the latter, the control NIC (0.15 h). In such a way the NIC concentration could be well sustained in the systemic circulation and would be useful for treating COVID-19. We still believe that our readily water-soluble NIC nanohybrids would be very effective in clinical model of SARS-CoV-2 virus and we are on the way to understand how these NIC nanohybrids would enhance the therapeutic outcome in a hypoalbuminemia model sooner.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Table 1 . PK parameters of PEG-BSA-NIC NPs and control NIC formulation after a single intravenous administration into rats. * AUC-Area under the curve; ** Cmaxmaximum plasma concentration; *** tmax -time required to reach Cmax; **** t1/2 -elimination half-life.

Covid-19: New coronavirus variant is identified in UK

an updated systematic review and meta-analysis

Theoretical Description for Anti-COVID-19 Drug Remdesivir Electrochemical Determination, Assisted by Squaraine Dye-Ag2O2 Composite

Remdesivir for COVID-19: Why Not Dose Higher?

Remdesivir for COVID-19: Why Not Dose Higher?

Remdesivir potently inhibits carboxylesterase-2 through covalent modifications: signifying strong drug-drug interactions

Comparison of Time to Clinical Improvement With vs Without Remdesivir Treatment in Hospitalized Patients With COVID-19

Significant Inhibition of Porcine Epidemic Diarrhea Virus In Vitro by Remdesivir, Its Parent Nucleoside and beta-d-N-4-hydroxycytidine

Covid-19: Remdesivir has little or no impact on survival, WHO trial shows

Amorphous Solid Dispersions and the Contribution of Nanoparticles to In Vitro Dissolution and In Vivo Testing: Niclosamide as a Case Study

FDA-Approved Drugs with Potent In Vitro Antiviral Activity against Severe Acute Respiratory Syndrome Coronavirus

Injectable pegylated niclosamide (polyethylene glycol-modified niclosamide) for cancer therapy

Niclosamide reverses cisplatin resistance by inhibiting Bcl-2 and Stat3 in HER2-positive breast cancer

Redox-sensitive nanoparticles based on xylanlipoic acid conjugate for tumor targeted drug delivery of niclosamide in cancer therapy

Niclosamide inhibits the inflammatory and angiogenic activation of human umbilical vein endothelial cells

Niclosamide: Beyond an antihelminthic drug

SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide

Plausible mechanisms of Niclosamide as an antiviral agent against COVID-19

Biocompatibility study of modified injectable hyaluronic acid hydrogel with mannitol/BSA to alveolar bone cells

In vitro and in vivo biodegradation and biocompatibility of an MMT/BSA composite coating upon magnesium alloy AZ31

Comparison of stability of bovine serum albuminstabilized emulsions prepared by microchannel emulsification and homogenization

Evaluation of folic acid functionalized BSA-CaFe(2)O(4 )nanohybrid carrier for the controlled delivery of natural cytotoxic drugs hesperidin and eugenol

Polyelectrolytic BSA nanoparticles containing silicon dihydroxide phthalocyanine as a promising candidate for drug delivery systems for anticancer photodynamic therapy

Modulation of Entrapment Efficiency and In Vitro Release Properties of BSA-Loaded Chitosan Microparticles Cross-Linked with Citric Acid as a Potential Protein-Drug Delivery System

A. Nag, Synthesis, Detailed Characterization, and Dual Drug Delivery Application of BSA Loaded Aquasomes

BSA modified, disulfide-bridged mesoporous silica with low biotoxicity for dual-responsive drug delivery

Curcumin nanocapsules stabilized by bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) for drug delivery and theranosis

Conversion of amine groups on chitosan-coated SPIONs into carbocyclic acid and investigation of its interaction with BSA in drug delivery systems

BSA loaded bead-on-string nanofiber scaffold with coreshell structure applied in tissue engineering

Covalently polysaccharide-based alginate/chitosan hydrogel embedded alginate microspheres for BSA encapsulation and soft tissue engineering

Engineering BSA-dextran particles encapsulated bead-onstring nanofiber scaffold for tissue engineering applications

Synthesis and Characterization of Collagen Grafted Poly(hydroxybutyrate-valerate) (PHBV) Scaffold for Loading of Bovine Serum Albumin Capped Silver (Ag/BSA) Nanoparticles in the Potential Use of

Bovine serum albumin modified the intracellular distribution and improved the antiviral activity of an oligonucleotide

Chemically Modified Human Serum Albumin Potently Blocks Entry of Ebola Pseudoviruses and Viruslike Particles

Vascular Endothelial Glycocalyx Damage in COVID-19

SARS-CoV-2 Bound Human Serum Albumin and Systemic Septic Shock

The association of low serum albumin level with severe COVID-19: a systematic review and meta-analysis

Serum Albumin Is Associated With Higher Inflammation and Carotid Atherosclerosis in Treated Human Immunodeficiency Virus Infection

Pegylation of IFNalpha and antiviral activity

Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver

Risk factors and prognosis of hypoalbuminemia in surgical septic patients

Cyclodextrins in Parenteral Formulations

Precision of Internal Standard and External Standard Methods in High Performance Liquid Chromatography, Special Issues

Preparation of BMP-2 Containing Bovine Serum Albumin (BSA) Nanoparticles Stabilized by Polymer Coating

Formulation and evaluation of cyclodextrin complexes for improved anticancer activity of repurposed drug: Niclosamide, Carbohydrate Polymers

Niclosamide Blocks Rice Leaf Blight by Inhibiting Biofilm Formation of Xanthomonas oryzae

Bovine Serum Albumin Amplified Reactive Oxygen Species Generation from Anthrarufin-Derived Carbon Dot and Concomitant Nanoassembly for Combination Antibiotic-Photodynamic Therapy Application

Synthesis of Monodispersed Gold Nanoparticles with Exceptional Colloidal Stability with Grafted Polyethylene Glycol-<i>g</i>-polyvinyl Alcohol

MgO nanoparticles coated with Polyethylene Glycol as carrier for 2-Methoxyestradiol anticancer drug, bioRxiv

Preferential Accumulation of Phospholipid-PEG and Cholesterol-PEG Decorated Gold Nanorods into Human Skin Layers and Their Photothermal-Based Antibacterial Activity

PLGA-PEG Nanoparticles Coated with Anti-CD45RO and Loaded with HDAC Plus Protease Inhibitors Activate Latent HIV and Inhibit Viral Spread

Engineering Poly(ethylene glycol) Particles for Improved Biodistribution

Role of capping agents in the application of nanoparticles in biomedicine and environmental remediation: recent trends and future prospects

Discriminating changes in protein structure using tyrosine conjugation

Gold nanorod distribution in mouse tissues after intravenous injection monitored with optoacoustic tomography

A comparison between sphere and rod nanoparticles regarding their in vivo biological behavior and pharmacokinetics

BSA nanoparticles as controlled release carriers for isophethalaldoxime palladacycle complex; synthesis, characterization, in vitro evaluation, cytotoxicity and release kinetics analysis

Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear

Formulation of tunable size PLGA-PEG nanoparticles for drug delivery using microfluidic technology

Stealth Coating of Nanoparticles in Drug-Delivery Systems, Nanomaterials (Basel)

Covid-19: How effective are vaccines against the delta variant?

The reproductive number of the Delta variant of SARS-CoV-2 is far higher compared to the ancestral SARS-CoV-2 virus

Predominance of Delta variant among the COVID-19 vaccinated and unvaccinated individuals

Curcumin nanocapsules stabilized by bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) for drug delivery and theranosis

Decreased serum albumin level indicates poor prognosis of COVID-19 patients: hepatic injury analysis from <sc>2,623 hospitalized cases</sc>

Hypoalbuminemia predicts the outcome of COVID-19 independent of age and co-morbidity

COVID-19 and diabetes mellitus: from pathophysiology to clinical management

Cardiovascular disease and COVID-19

Antihelminth Compound Niclosamide Downregulates Wnt Signaling and Elicits Antitumor Responses in Tumors with Activating APC Mutations

Size distribution of virus laden droplets from expiratory ejecta of infected subjects

Preclinical evaluation of a nanoformulated antihelminthic, niclosamide, in ovarian cancer