Hydroxypropyl-β-Cyclodextrin Depletes Membrane Cholesterol and Inhibits SARS-CoV-2 Entry into HEK293T-ACEhi Cells

Vaccination has drastically decreased mortality due to coronavirus disease 19 (COVID-19), but not the rate of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alternative strategies such as inhibition of virus entry by interference with angiotensin-I-converting enzyme 2 (ACE2) receptors could be warranted. Cyclodextrins (CDs) are cyclic oligosaccharides that are able to deplete cholesterol from membrane lipid rafts, causing ACE2 receptors to relocate to areas devoid of lipid rafts. To explore the possibility of reducing SARS-CoV-2 entry, we tested hydroxypropyl-β-cyclodextrin (HPβCD) in a HEK293T-ACE2hi cell line stably overexpressing human ACE2 and Spike-pseudotyped SARS-CoV-2 lentiviral particles. We showed that HPβCD is not toxic to the cells at concentrations up to 5 mM, and that this concentration had no significant effect on cell cycle parameters in any experimental condition tested. Exposure of HEK293T-ACEhi cells to concentrations of HPβCD starting from 2.5 mM to 10 mM showed a concentration-dependent reduction of approximately 50% of the membrane cholesterol content. In addition, incubation of HEK293T-ACEhi cells with HIV-S-CoV-2 pseudotyped particles in the presence of increasing concentrations of HPβCD (from 0.1 to 10 mM) displayed a concentration-dependent effect on SARS-CoV-2 entry efficiency. Significant effects were detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects. These data indicate that HPβCD is a candidate for use as a SARS-CoV-2 prophylactic agent.


Introduction
Coronavirus disease 19 (COVID-19) is a severe infectious disease caused by a recently discovered coronavirus family member, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) [1]. Since its first identification in 2019 in China, it has spread worldwide, resulting in the COVID-19 pandemic.

Cytotoxicity Assay
Cell viability was evaluated using the MTT assay to measure cellular metabolic activity as an indicator of cell viability. HEK293T-ACE hi cells were seeded at a concentration of 10 4 cells/well in 100 µL of complete medium with 10% FBS into 96-well plates. One day post-seeding, a vehicle or HPβCD (concentration range 0.01-40 mM) were added and tested for overnight incubation, followed by a 4 h incubation with a Thiazolyl Blue Tetrazolium Bromide (MTT; Merck KGaA, Darmstadt, Germany) solution (5 mg/mL) at 37 • C in a humidified 5% CO 2 /air atmosphere. The formazan formed was dissolved in 150 µL acid isopropanol (0.1 N HCl in isopropanol) added to all wells. The absorbance was measured by a multiplate reader at a 570 nm wavelength with a 620 nm reference wavelength. All experiments were performed twice (n = 8 for each experiment) in independent cultures. The results were expressed as a percentage of the controls.

Cell Cycle Analysis
The analysis of the cell cycle using flow cytometry was performed to investigate the potential cytotoxicity of HPβCD based on the quantification of cellular DNA content using a fluorescent DNA-selective stain, propidium iodide (PI), that exhibits emission signals proportional to DNA mass. HEK293T-ACE hi cells were seeded in a 24-well plate (75,000 cells/well) in a complete medium at 37 • C in 5% CO 2 . After 24 h post-seeding, cells were treated overnight with 5 or 20 mM HPβCD or a vehicle. The cell cycle analysis was carried out immediately after 24 h, or 72 h after the overnight HPβCD treatment to evaluate whether HPβCD can affect the cell cycle stages over time.
After the treatment or recovery, cells were collected by trypsinization, pelleted by centrifugation for 5 min at 200× g, and then resuspended in ice-cold PBS. To permeabilize the samples, 50 µg/mL of RNase A and 50 µg/mL PI staining solution with 0.1% Triton X-100 were added for 20 min at 4 • C in the dark. After incubation, flow cytometry was performed to determine the cell cycle distribution using an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Monza, Italy).

Plasma Membrane Cholesterol Content
Plasma membrane oxidase-sensitive cholesterol was quantified using a radioisotopic assay as previously described [12]. Briefly, HEK293T-ACE2 hi cells, 48 h after seeding, were incubated with 3 µCi/mL (1-2,3) of cholesterol (Perkin Elmer, MA, USA) for 24 h in the presence of 2 µg/mL of an acyl-coenzyme A, a cholesterol acyltransferase (ACAT) inhibitor (Sandoz 58035; Merck, Germany), to ensure that all cholesterol was in the free form. Cells were then treated for 18 h with increasing concentrations of HPβCD (from 0.01 mM to 15 mM) or a vehicle, along with 2 µg/mL of ACAT inhibitor. The culture medium was then removed, and cholesterol oxidase enzyme from Streptomyces (Merck, Darmstadt, Germany) dissolved in PBS was added at a concentration of 1 U/mL for 2 h at 37 • C. The PBS was then removed, lipids were extracted from the cell monolayers after 18 h of incubation with 2-propanol, and thin layer chromatography (TLC) was performed to separate radioactive free, esterified, and oxidated cholesterol within the extracted lipid Pathogens 2023, 12, 647 4 of 11 fraction (the mobile phase was composed of 90 mL hexane, 10 mL ethyl ether, and 1 mL methanol). The plasma membrane cholesterol content was expressed as the percentage of oxidated cholesterol over the total cholesterol. In parallel, the total protein content was quantified in cell monolayers using the bicinchoninic acid assay (BCA; Thermo Fisher Scientific, MA, USA) following the manufacturer's instructions. All experiments were performed twice (n = 5-9) in independent cultures.

Pseudotyped Lentiviral Particles Production
Lentivector particles were produced by calcium phosphate DNA transfection of HEK293T cells with the HIV-1 packaging construct pSPAX2, the miniviral genome bearing the expression cassette encoding GFP (FUGW-GFP), and the plasmid encoding either the SARS-CoV-2 Spike Envelope glycoproteins or the VSVg envelope expressing plasmid pMD2.G (addgene). Lentivector particles were also produced without the viral entry glycoprotein as a negative control. For vector production, packaging, transfer, and envelope encoding, plasmids were transfected at a ratio of 8:8:4 µg for 3.5 × 10 6 cells plated 1 day before transfection in 100 mm dishes. Lentivectors were recovered from the cell culture supernatant 48 h after transfection, centrifuged at 3000× g for 5 min, filtrated (0.45 µm filter, Millipore, Burlington, MA, USA), aliquoted, and stored at −80 • C until use.

Single-Cycle Infectivity Assay
HEK293T-ACE hi cells (75,000 cells/well) were seeded in a 24-well plate and cultured in complete medium (w/o antibiotics) at 37 • C in 5% CO 2 overnight. Twenty-four hours post-seeding, cells (untreated or treated with HPβCD; concentration range from 0.1 to 10 mM) were incubated with 200 µL of pseudotyped viral particles overnight. Over the next three days, cells were washed twice with PBS and maintained in culture with 1 mL of complete medium at 37 • C in 5% CO 2 . After 72 h post-infection, GFP expression was qualitatively evaluated using EVOS Cell Imaging Systems (Thermo Fisher Scientific) and quantitatively evaluated using cytofluorimetry with an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific) to determine infection efficiency. Three independent experiments were performed (n = 5-9).

Curve Fit and Statistical Analysis
The CD concentrations were converted to Log10 values, and curve fit and IC50 values were calculated using the Nonlinear regression One site-Fit logIC50 function of the Prism software, version 7 (GraphPad Software, San Diego, CA, USA). Statistical significance was assessed by one-way analysis of variance (ANOVA) and post-hoc Dunnett's test for multiple comparisons against the control (vehicle-exposed) group. A value of p < 0.05 was considered statistically significant. All data are presented as mean ± standard error of mean (SEM). Statistical analysis was performed using Prism statistical analysis software, version 7 (GraphPad Software, CA, USA) or SPSS for Windows v.28 (SPSS Inc., Chicago, IL, USA).

HPβCD Concentration-Dependent Effects on Cell Viability and Cell Cycle
Although HPβCD has been reported to exhibit high biocompatibility, we first evaluated the cytotoxic effects of overnight exposure to HPβCD (concentration range from 0.01 to 40 mM) or a vehicle (Ctrl) on HEK293T-ACE hi cells. The analysis revealed a concentrationdependent effect of HPβCD exposure on cell viability in HEK293T-ACE hi cells (one-way ANOVA: F(9151) = 73.007, p < 0.001, followed by post-hoc Dunnett's test). HPβCD was not found to be toxic up to a concentration of 5 mM (p = 0.964). Higher concentrations were increasingly toxic, with a 40 mM concentration of HPβCD resulting in extreme toxicity (reduction of cell viability by~90%; p < 0.001 vs. Ctrl) ( Figure 1). version 7 (GraphPad Software, CA, USA) or SPSS for Windows v.28 (SPSS Inc., Chicago, IL, USA).

HPβCD Concentration-Dependent Effects on Cell Viability and Cell Cycle
Although HPβCD has been reported to exhibit high biocompatibility, we first evaluated the cytotoxic effects of overnight exposure to HPβCD (concentration range from 0.01 to 40 mM) or a vehicle (Ctrl) on HEK293T-ACE hi cells. The analysis revealed a concentration-dependent effect of HPβCD exposure on cell viability in HEK293T-ACE hi cells (oneway ANOVA: F(9151) = 73.007, p < 0.001, followed by post-hoc Dunnett's test). HPβCD was not found to be toxic up to a concentration of 5 mM (p = 0.964). Higher concentrations were increasingly toxic, with a 40 mM concentration of HPβCD resulting in extreme toxicity (reduction of cell viability by ~90%; p < 0.001 vs. Ctrl) ( Figure 1). It has been reported that HPβCD is less toxic than other CDs (including the parent βcyclodextrins, βCDs) (9). We used the same experimental conditions to evaluate the cytotoxic effects induced by αCD, which is able to extract cholesterol from membranes, although to a lesser extent than HPβCD. We demonstrated that overnight exposure to αCD at concentrations of 2.5 or 5 mM resulting in different effects than those observed for HPβCD (see above). The significant reduction in cell viability was demonstrated through a one-way ANOVA, which showed F(2,23) = 17.974, p < 0.001 vs. control, followed by a post-hoc Dunnett's test ( Figure S1).
In leukemic cell lines, HPβCD inhibits cell growth by reducing intracellular cholesterol and inducing G2/M cell-cycle arrest [15], showing that this CD can affect the cell cycle. We then evaluated HPβCD-induced effects on the cell cycle of HEK293T-ACE hi immediately, 24 h, or 72 h after overnight exposure (concentrations: 5 or 20 mM). One-way ANOVA analysis revealed a significant effect on the percentage of cells in the G0/G1 phase (F(2,25) = 16.951, p < 0.001) and the G2/M phase (F(2,25) = 3.790, p = 0.038) immediately after overnight exposure only at the highest concentration of HPβCD tested (20 mM). This concentration increased the percentage of cells in the G2/M phase (p = 0.044, post-hoc Dunnett's test), while decreasing the percentage of cells in G0/G1 (p < 0.001, post-hoc Dunnett's test). In the same experimental conditions, the proportion of S-phase cells was not significantly affected by HPβCD (Figure 2a). The effect of 20 mM HPβCD on the HEK293T- It has been reported that HPβCD is less toxic than other CDs (including the parent β-cyclodextrins, βCDs) (9). We used the same experimental conditions to evaluate the cytotoxic effects induced by αCD, which is able to extract cholesterol from membranes, although to a lesser extent than HPβCD. We demonstrated that overnight exposure to αCD at concentrations of 2.5 or 5 mM resulting in different effects than those observed for HPβCD (see above). The significant reduction in cell viability was demonstrated through a one-way ANOVA, which showed F(2,23) = 17.974, p < 0.001 vs. control, followed by a post-hoc Dunnett's test ( Figure S1).
In leukemic cell lines, HPβCD inhibits cell growth by reducing intracellular cholesterol and inducing G2/M cell-cycle arrest [15], showing that this CD can affect the cell cycle. We then evaluated HPβCD-induced effects on the cell cycle of HEK293T-ACE hi immediately, 24 h, or 72 h after overnight exposure (concentrations: 5 or 20 mM). One-way ANOVA analysis revealed a significant effect on the percentage of cells in the G0/G1 phase (F(2,25) = 16.951, p < 0.001) and the G2/M phase (F(2,25) = 3.790, p = 0.038) immediately after overnight exposure only at the highest concentration of HPβCD tested (20 mM). This concentration increased the percentage of cells in the G2/M phase (p = 0.044, post-hoc Dunnett's test), while decreasing the percentage of cells in G0/G1 (p < 0.001, post-hoc Dunnett's test). In the same experimental conditions, the proportion of S-phase cells was not significantly affected by HPβCD (Figure 2a). The effect of 20 mM HPβCD on the HEK293T-ACE hi cell cycle was reversible since it had no effect on the HEK293T-ACE hi cell cycle 24 or 72 h after overnight exposure (Figure 2b,c). A concentration of 5 mM of HPβCD had no significant effect on the cell cycle parameters in any experimental condition tested ACE hi cell cycle was reversible since it had no effect on the HEK293T-ACE hi cell cycle 24 or 72 h after overnight exposure (Figure 2b,c). A concentration of 5 mM of HPβCD had no significant effect on the cell cycle parameters in any experimental condition tested ( Figure  2a-c). The percentage of apoptotic cells was not significantly affected by HPβCD treatments at 24 or 72 h or immediately after an overnight exposure ( Figure 2). Overall, our experiments did not show evidence of HPβCD-induced cell toxicity or cell cycle alterations for concentrations up to 5 mM.

HPβCD Effects on Membrane Cholesterol Content
Cholesterol, and in particular its distribution in membrane rafts, is essential for viral interaction with ACE2 receptors and entry into the cell [16]. Therefore, we evaluated the modulating effect of increasing concentrations of HPβCD with overnight exposure on the membrane cholesterol content of HEK293T-ACE hi cells. The analysis revealed a concentration-dependent reduction of approximately 50% of the initial membrane 3 H-cholesterol content induced by HPβCD exposure in HEK293T-ACE hi cells (one-way ANOVA: F(7,58) = 18.627; p < 0.001, followed by post-hoc Dunnett's test, Figure 3a). In particular, a significant reduction in membrane 3 H-cholesterol content was induced by HPβCD exposure starting from 2.5 mM and reaching the maximum inhibitory effect at 10 mM. The concentration-response inhibition curve showed an IC50 value of 1.99 mM (Figure 3b). Overall, our experiments did not show evidence of HPβCD-induced cell toxicity or cell cycle alterations for concentrations up to 5 mM.

HPβCD Effects on Membrane Cholesterol Content
Cholesterol, and in particular its distribution in membrane rafts, is essential for viral interaction with ACE2 receptors and entry into the cell [16]. Therefore, we evaluated the modulating effect of increasing concentrations of HPβCD with overnight exposure on the membrane cholesterol content of HEK293T-ACE hi cells. The analysis revealed a concentrationdependent reduction of approximately 50% of the initial membrane 3 H-cholesterol content induced by HPβCD exposure in HEK293T-ACE hi cells (one-way ANOVA: F(7,58) = 18.627; p < 0.001, followed by post-hoc Dunnett's test, Figure 3a). In particular, a significant reduction in membrane 3 H-cholesterol content was induced by HPβCD exposure starting from 2.5 mM and reaching the maximum inhibitory effect at 10 mM. The concentration-response inhibition curve showed an IC50 value of 1.99 mM (Figure 3b).

HPβCD Effects on Pseudotyped SARS-CoV-2 Particle Entry into HEK293T-ACE2 hi Cells
Lentiviral particles that were pseudotyped with SARS-CoV-2 Spike Envelope glycoprotein (HIV-S-CoV2) have been repeatedly used to investigate SARS-CoV-2 entry mechanisms [17,18]. HEK293T-ACE hi cells were incubated overnight with HIV-S-CoV2 pseudotyped particles in the presence of increasing concentrations of HPβCD (from 0.1 to 10 mM) or a vehicle and maintained in culture for 72 h. Three days post-incubation, GFP expression was qualitatively evaluated using EVOS Cell Imaging Systems and quantitatively evaluated using cytofluorimetry to determine the viral particles' entry efficiency. The analysis revealed a concentration-dependent effect of HPβCD exposure on entry efficiency (one-way ANOVA: F(6,41) = 222.178, p < 0.001, followed by post-hoc Dunnett's test) (Figure 4a-c), with clearly significant effects detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects (see above). The concentration-response inhibition curve showed an IC50 value of 0.78 mM. , the concentration-response curve was fitted using the Nonlinear regression One site-Fit logIC50 function of the Prism software to obtain the IC50 value. Data are expressed as means ± SEM (n = 4 per group). A one-way ANOVA (### p < 0.001) followed by the post-hoc Dunnett's test (*** p < 0.001 vs. vehicle-treated cells) was used to analyze the differences between the experimental conditions.

HPβCD Effects on Pseudotyped SARS-CoV-2 Particle Entry into HEK293T-ACE2 hi Cells
Lentiviral particles that were pseudotyped with SARS-CoV-2 Spike Envelope glycoprotein (HIV-S-CoV2) have been repeatedly used to investigate SARS-CoV-2 entry mechanisms [17,18]. HEK293T-ACE hi cells were incubated overnight with HIV-S-CoV2 pseudotyped particles in the presence of increasing concentrations of HPβCD (from 0.1 to 10 mM) or a vehicle and maintained in culture for 72 h. Three days post-incubation, GFP expression was qualitatively evaluated using EVOS Cell Imaging Systems and quantitatively evaluated using cytofluorimetry to determine the viral particles' entry efficiency. The analysis revealed a concentration-dependent effect of HPβCD exposure on entry efficiency (one-way ANOVA: F(6,41) = 222.178, p < 0.001, followed by post-hoc Dunnett's test) (Figure 4a-c), with clearly significant effects detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects (see above). The concentration-response inhibition curve showed an IC50 value of 0.78 mM.
As a positive control for viral entry, we performed experiments with HIV-VSVg pseudo-particles. No significant effect on their entry was observed after the modestly cytotoxic 10 mM HPβCD dose (one-way ANOVA: F(2,37) = 21.582, p < 0.001, followed by post-hoc Dunnett's test) (see Figure S2).  ACE hi cells were incubated with pseudotyped viral particles containing a GFP expression cassette overnight in the presence of different concentrations (0.1 to 10 mM) of HPβCD or a vehicle. Cells were also incubated overnight with lentivector particles without viral entry glycoprotein containing a GFP expression cassette as a negative control (Ctrl-). Three days post-transduction, (a) GFP expression was measured by flow cytometry and (b) the concentration-response curve was fitted using the Nonlinear regression One site-Fit logIC50 function of the Prism software to obtain the IC50 value. Qualitative analysis (c) was performed using EVOS Cell Imaging Systems Panel on live-HEK293T-ACE hi cells before proceeding to the quantitative analysis. Scale bar = 500 µm. Data are expressed as means ± SEM (n = 3-11, three independent experiments). A one-way ANOVA (### p < 0.001) followed by the post-hoc Dunnett's test (*** p < 0.001 vs. vehicle treated cells) was used to analyze the differences between the experimental conditions.
As a positive control for viral entry, we performed experiments with HIV-VSVg pseudo-particles. No significant effect on their entry was observed after the modestly cytotoxic 10 mM HPβCD dose (one-way ANOVA: F(2,37) = 21.582, p < 0.001, followed by post-hoc Dunnett's test) (see Figure S2).

Discussion
The main finding of this study is that HPβCD, at concentrations that are neither cytotoxic nor interfere with cell cycle, markedly reduces (down to approximately 22%) the entry of pseudotyped SARS-CoV-2 particles into HEK293T cells stably overexpressing human ACE2 (HEK293T-ACE hi cells). Pseudotyped HIV-1 lentiviral particles with the SARS-CoV-2 Spike Envelope glycoprotein are widely used to study molecular and cellular mechanisms of SARS-CoV-2 entry [17,18], allowing recognition of the principal membrane receptors and associated proteins necessary for virus entry.
We also demonstrated that HPβCD can decrease membrane 3 H-cholesterol content by approximately 50%. Remarkably, the IC50 values of the HPβCD-induced decrease in HIV pseudotyped SARS-CoV-2 Spike particle entry and the membrane cholesterol content were close to each other, both at approximately 1 mM. The first effective concentration of HPβCD was close to one order of magnitude below its lowest cytotoxic concentration. Therefore, our hypothesis is that HPβCD-induced decrease in membrane cholesterol content leads to a disruption in membrane organization; more specifically, the cholesterol-rich lipid rafts where many known SARS-CoV-2 receptors are located [16], thus hindering the processes of viral entry into the cell.
Lipid rafts are microdomains of the cell membrane that are rich in cholesterol, sphingolipids, and proteins. This lipid composition causes the membrane to acquire greater rigidity, making it an ideal platform for several receptors including ACE2 [16,19,20]. These structures are critical for coronaviruses, including SARS-CoV-1, to enter into cells and maintain infectivity [8]. Cholesterol depletion in the cell membrane, regardless of the method used, causes profound changes in the membrane's physical properties [21] and induces the re-localization of receptors localized in the lipid rafts, including ACE2, to areas without lipid drafts [16]. Of further interest is the fact that not only ACE2, but also several other co-receptors that are relevant for SARS-CoV-2 binding and internalization such as heparan sulfate proteoglycan [22], Syndecan-1/4 [23], Neuropilin-1 [24], L-SIGN [25], HDL scavenger receptor B type 1 [26], CD147 [27], and human Toll-like receptors [28] are abundant in the lipid rafts [16]. This means that HPβCD can perturb SARS-CoV-2 binding not only to ACE2 but also to all the other co-localized co-receptors, thus strengthening the inhibitory effect of HPβCD on virus entry.
Another relevant consequence of this putative mechanism of action is that it is variantindependent. This is because the interference with the binding and internalization of SARS-CoV-2 is primarily related to a physical disturbance of the cellular membrane. This is particularly relevant in view of the extremely rapid appearance of SARS-CoV-2 variants with the accumulation of a huge number of mutations that can weaken vaccine protection [29,30].
The motivation for our study was to search for a substance that could be used as a prophylaxis for SARS-CoV-2 infection. This substance would need to be easily delivered to the site of entry of the virus, i.e., the nasal and oropharyngeal mucosa. CDs were identified as ideal candidates due to their longstanding experience in intranasal delivery as carriers of scarcely soluble drugs, showing their safety and ease of use [31,32]. All types of CDs, including αCDs, βCDs, and γ-cyclodextrins (γCDs), can extract cholesterol from cell membranes and disrupt lipid rafts, although with different efficiencies depending on the size and hydrophobicity of their inner cavity [33]. However, αCDs are more efficient in extracting phospholipids than cholesterol [34] and are toxic to cells even at low concentrations, and γCDs are not sufficiently hydrophobic [34,35]. βCDs have the best characteristics to exert this protective action. Among the βCDs, methylβCD (MβCD) and HPβCD are those that have been most studied. Their in vitro activity for cholesterol depletion is similar, as well as their solubility, due to hydrophilic modifications (such as methylation and hydroxypropylation).
Lu et al. were among the first to test MβCD in the context of SARS-CoV infection [8], while Li et al. evaluated the role of MβCD to inhibit SARS-CoV-2 infection in vitro [17]. We used a similar approach, but chose to evaluate HPβCD instead. For the purpose of using CDs as SARS-CoV-2 prophylaxis, MβCD did not meet our requirements. This was because, in the direct comparison between HPβCD and MβCD, the latter was shown to be significantly more toxic both in vitro, even at very low concentrations [36,37], and in vivo [38]. More specifically, the latter study showed that the outer hair cells (OHC) in the organ of Corti appeared histologically normal after treatment with 13 mM of HPβCD, while a higher concentration (27 mM) only caused sporadic damage. Instead, 13 mM of MβCD caused severe damage to the OHC [37]. Notably, in our study, the most effective concentration of HPβCD that was able to inhibit pseudotyped SARS-CoV-2 particle infection (5 mM) was approximately five times lower than the mildly toxic concentration tested in [38]. During the course of our study, a relevant study by Bezerra et al. [39] was published. These authors used experimental models that were partially different from ours (native VERO cells and VERO E6 expressing Transmembrane Protease Serine 2 and Human Angiotensin-Converting Enzyme 2; Calu3 and, as in our study, ACE2 transfected 293T) and showed that HPβCD primarily reduces virus replication and the release of infectious SARS-CoV-2 particles rather than inhibiting entry into the cells. Treatment of human primary monocytes with HPBCD also reduced inflammatory cytokines. Although some relevant experimental conditions were different (e.g., HPβCD was used at much higher concentrations than in the present study), these data are complementary to our findings and indicate an interesting therapeutic potential for this compound.

Conclusions
In conclusion, data on the ability of HPβCD to interfere with SARS-CoV-2 infection is accumulating, making it a strong candidate as a prophylactic agent.