key: cord-0948087-4q14173g
authors: Tu, Bin; Wang, Huiyuan; An, Xinran; Qu, Jingkun; Li, Qianqian; Gao, Yanrong; Shi, Mingjie; Qiu, Hong; Huang, Yongzhuo
title: Inhaled heparin polysaccharide nanodecoy against SARS-CoV-2 and variants
date: 2022-02-11
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2022.01.019
sha: b9e5ab9155334f2d6fc98e98997fde82c9951bc1
doc_id: 948087
cord_uid: 4q14173g

The heparin polysaccharide nanoparticles block the interaction between heparan sulfate/S protein and inhibit the infection of both wild-type SARS-CoV-2 pseudovirus and the mutated strains through pulmonary delivery. [Figure: see text]

As of January 27, 2022, there were 363,062,293 cases and 5,645,884 deaths from the COVID-19 pandemic 1 The development of antiviral drugs against infection of SARS-CoV-2 and its variants is still an urgent need. Early treatment is critical for preventing hospitalization and the chronic sequelae of COVID-19, saving lives, and relieving the overburden of medical systems 8 . Notably, for early treatment purposes, a self-administrable dosage form is the priority in drug development.

The blockage of SARS-CoV-2 spike protein against the binding with the receptors on host cells is a promising therapeutic target 9 , and angiotensin-converting enzyme 2 (ACE2) is the most explored virus receptor on host cells 10, 11 . Recent studies have demonstrated that ACE2-expressing exosomes can inhibit SARS-CoV-2 entry to and infect the host cells [12] [13] [14] [15] [16] [17] ; for instance, we previously reported that the intranasal administration of ACE2 + exosomes can inhibit the colonization and infection of SARS-CoV-2 in the nasal epithelium 11 . However, SARS-CoV-2 can also infect the cells with low ACE2 expression, suggesting that there are alternative virus receptors in mediating the infection 18 . Spike protein can bind with some specific glycosaminoglycans (GAGs) on host cell membrane [19] [20] [21] [22] . In particular, S protein can efficiently bind with heparan sulfate (HS), and the binding allows spike protein to maintain an "open" conformation that facilitates the subsequent binding to ACE2 and TMPRSS2, respectively 21, 23 . Therefore, the interaction between spike protein and heparan can be a potential therapeutic target, and the analog heparin is also able J o u r n a l P r e -p r o o f to inhibit SARS-CoV-2 entry to host cells via spike protein mediation 24, 25 .

However, administration of heparin relies on intravenous (i.v.) injection (only injection formulations clinically available), which has two major disadvantages. First, i.v. injection must be handled by a medical professional in clinical settings. Second, i.v. heparin in systemic circulation has a potential bleeding risk 26 . Therefore, to develop a self-administrable and safer heparin delivery system will be much superior for early treatment in COVID-19.

Nanotechnology has played an essential role in COVID-19, represented by the great success of lipid nanoparticle-based mRNA vaccines 27, 28 . We proposed that the heparin NPs for anti-SARS-CoV-2 infection via a non-invasive, self-administrable inhalation delivery can facilitate the lung-specific drug distribution.

Both high-molecular-weight heparin (HMWH) and low-molecular-weight heparin (LMWH) exhibited a dose-dependent inhibition against SARS-CoV-2 in 293T-hACE2 cells, and HMWH had a significantly higher efficacy of anti-infection than LMWH (Fig. 1A and B) . Therefore, the HMWH was employed for the preparation of heparin nanoparticles. Our results are also consistent with a previous finding that longer heparin chains can bind more efficiently to multiple sites and also potentially create stronger steric hindrance to block the spike protein binding with the cell surface viral receptors 24 .

To verify the role of cell surface heparan in mediating SARS-CoV-2 infection, we cells, suggesting that the virus may largely rely on heparan to enter the host cells with low ACE2 expression. More importantly, the Delta-mutated strain was very sensitive to the heparan elimination that caused the most significant inhibition against infection in both 293T and 293T-hACE2 among the tested strains.

The mean diameter and zeta potential of heparin NPs were 247.4 nm (PDI, 0.154) and −25.6 mV, respectively ( Fig. 2A and B ). Cryo-TEM showed the morphology of the NPs with spherical shapes ( Fig. 2A) . Moreover, the heparin NPs had a good colloidal stability (Fig. 2C) . The poly-anionic heparin can crosslink by the cationic chitosan via charge interaction. Chitosan thus serves as a cross-linker in this nanodecoy system. The net charge of the heparin polysaccharide NPs was negative and thereby reduced the risk of positive charge-related side toxicity.

The efficient binding of heparin NPs and pseudovirus was demonstrated by the morphological change, which showed the particle size of the NP/pseudovirus complex was increased to 350 nm ( Fig. 2D-F) . The complex was also observed by cryo-TEM, clearly showing the binding (Fig. 2G ). It should be mentioned that there is a minor peak of 82 nm, which could be the exosomes from the packing cells. Due to the close size of the exosomes and virus, both were collected by ultracentrifugation without further separation.

The 293T and 293T-hACE2 cells were used in this study because 293T cells is a common cell model for SARS-CoV-2 infection via ACE2 or HS receptors 30 . Also, 293T cells are convenient for genetic engineering for stable expression of a target protein (e.g., hACE2). Both HMWP and the heparin NPs efficiently inhibited the cell entry of SARS-CoV-2 pseudovirus in 293T cells and 293T-hACE2 cells as shown in the fluorescence images (Fig. 3A) . The inhibition results were further confirmed by J o u r n a l P r e -p r o o f flow cytometry detection ( Fig. 3B and C) , which shows that the positive cells containing the Dil dye-labeled pseudovirus were dramatically decreased by treatment with HMWP or the heparin NPs. Accordingly, the transfection of pseudovirus in 293T and 293T-hACE2 cells was significantly suppressed by the treatment, as reflected by the decreasing level of luciferase (a reporter gene of pseudovirus) (Fig.   3D and E) . Notably, the heparin NPs had a better inhibition effect than HMWP. This may due to the higher surface area in the nanoparticles. 28 In addition, the NPs can provide multiple ligands to bind with viruses and thus efficiently block the cell entry of viruses.

The Delta variant SARS-CoV-2 has been a main threat in the current global pandemic 31 . Therefore, to develop a broad-spectrum antiviral agent is a pressing need under the current circumstance. It was discovered that the cell entry of Delta pseudovirus was significantly blocked by either HMWH or the heparin NPs, as reflected by the decreased amount of the positive cells containing the Dil-labeled Delta pseudovirus (Fig. 4A) . Furthermore, both HMWH and the heparin NPs were able to inhibit Delta pseudovirus infection (Fig. 4B) . Similarly, the heparin NPs had better efficacy against Delta pseudovirus infection. The result also implys that the heparin NPs could block the infection of Delta pseudovirus.

Another SARS-CoV-2 variant, Delta plus, has also raised public concerns for its increased transmissibility and reduction in monoclonal antibody response. 32 We also tested the inhibition efficacy against Delta plus variant. The results were promising that the heparin NPs were also highly efficient for suppressing the cell entry and infection of the Delta plus variant (Fig. 4C and D) .

The findings demonstrate that the heparin NPs can serve as a potent antiviral agent effectively in various SARS-CoV-2 variants.

Lung is the primary target organ in SARS-CoV-2 infection. Pulmonary drug delivery J o u r n a l P r e -p r o o f is generally considered to be an ideal administration route for anti-Covid-19 therapy due to direct access of drug to the lung 33, 34 . Moreover, inhaled formulations also possess the benefits of self-administration, rapid onset of action, good patient compliance, and reduced systemic side effects 34 . The mouse model was established by introducing the pseudovirus to infect the lung tissue through the bronchus. The inhibition efficacy of the heparin NPs against infection was evaluated by inhaled administration. After treatment with either HMWH or the heparin NPs, the accumulation of the pseudovirus (including the wide-type, Delta, and Delta plus) in the lung was significantly reduced (Fig. 5B-G) . The heparin NPs showed a higher anti-viral efficacy than HMWH. Moreover, the inhibition efficacy of the heparin NPs against transfection by different pseudovirus was evaluated by monitoring the level of the reporter gene luciferase in the lung. The data further demonstrate that the NPs could significantly reduce infection of all the test variants of pseudovirus ( Fig. 5H and J), suggesting that the heparin NPs can serve as a broad-spectrum anti-SARS-CoV-2 drug candidate against various variants.

The mechanism for clearance of the virus after treatment with the heparin NPs was explored. Macrophages are the major scavenger for clearance away pathogens via phagocytosis 35 . For instance, the immune complexes formed by the binding of antibody/virus to the NPs, can be identified and phagocytized by macrophages 36 . Our results show that the viruses neutralized by the heparin NPs were increasingly captured by the macrophages, compared to the control groups (Fig. 6B, D, and F) , suggesting that the thus-formed NP/virus complexes were efficiently identified by the macrophages as an exogenous component and removed away. In addition, similar results were obtained by flow cytometry assay to further confirm the enhanced capture of the viruses by the macrophages after forming the complex with the NPs (Fig. 6C, E and G) .

A previous study revealed that a large number of monocyte-derived macrophages were involved in COVID-19 patients' lungs 37 . Macrophages play an important role J o u r n a l P r e -p r o o f in mitigating respiratory virus infection 38 . Phagocytosis by macrophages is highly dependent on the component sizes; e.g., the macrophages exhibited an enhanced ability to phagocytose the nanoparticles less than 500 nm 39 . Therefore, the NP/virus complex with a relatively large size about 350 nm could facilitate the capture by the macrophages. Nanotechnology has been actively explored for the application against SARS-CoV-2 [44] [45] [46] . In this work, it was demonstrated that the efficacy of the heparin NPs served as nanodecoy for neutralizing SARS-CoV-2 and the Delta-mutated strains. Importantly, heparin can inhibit the enzymatic activity of Furin that cleaves many viral glycoproteins with polybasic residues (e.g., SARS-CoV-2 spike protein), thus activating the S-protein 47 . Notably, a simulation study revealed that the omicron variant has the increased furin-binding ability compared to the wide-type SARS-CoV-2, suggesting that the stronger furin binding could lead to the more efficient fusion at molecular level and higher viral load on the host 48 . Therefore, it is expected that this Heparin nanodecoy strategy is potentially workable in anti-Omicron variant infection.

Besides, heparin can inhibit another protease, Factor Xa, in host cells, which is necessary for processing the spike protein 49 . Therefore, heparin-based neutralization therapy may involve multiple mechanisms against the infection of SARS-CoV-2 and its variants. Yet, further experiments will be needed to confirm the prediction.

It should be noted that a recent clinical trial revealed that LMWH (i.e., Enoxaparin) had a better treatment outcome in hospitalized COVID-19 patients than unfractionated heparin, but the analysis just focused on the impact of the COVID-associated coagulation cascades 50 . Heparin is expected to have multiple synergistic functions when applied to the treatment of COVID-19 51 , apart from the neutralization effect. In terms of neutralization effect, our results suggest that heparin with high molecular weight yields better efficacy of anti-SARS-CoV-2 infection.

Both heparin and chitosan have been widely used in pharmaceutical products.

Specifically, inhaled delivery of heparin and chitosan has been widely explored in either clinical or preclinical studies, and the biosafety has been demonstrated 52-54 . It is expected that the heparin polysaccharide nanodecoy developed in this work will be promising for clinical translation.

It should be mentioned that due to the limitation of pseduovirus and animal models, more work must be carried out to further demonstrate the feasibility of anti-COVID-19. In addition, a PK study will be also helpful for kinetic analysis of the heparin NP-based therapy.

We developed a nanodecoy strategy for anti-COVID-19 by using the heparin NPs to neutralize SARS-CoV-2. The results reveal the superiority of the heparin NPs over heparin. The NPs serve as an effective inhibitor against infection of both wide type virus and Delta mutations in the pseudovirus test. Pulmonary delivery of the heparin NPs can direct access the lung and reduce the unwanted drug exposure to other organs. Heparin is an FDA-approved drug and the heparin NPs are highly biocompatible for human use, and the preparation of the NPs is easy and rapid. Therefore, the heparin NPs have a potential value for clinical translation.

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We are thankful for the support of the National Key Research and Development 

The authors have no conflicts to declare.