Fig 1: HPBCD and HPGCD inhibit the SARS-CoV-2 pseudotype in a concentration-dependent manner. A549-ACE2 cells were pre-treated with CD formulations (A) HPBCD, (B) HPGCD, or (C) SBECD for 1 h. Cells were washed 3X with PBS before replacement with a complete cell culture medium. Cells were infected with the SARS-CoV-2 Wuhan (D614G mutant) pseudotype for 8 h. Twenty-four hpi cells were lysed for luciferase readout. The control is the no-treatment comparator. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 2: Cell-treated versus pseudotyped virus-treated effects of HPBCD and HPGCD. (A) Cyclodextrin effect against Wuhan SARS-CoV-2 pseudotype infection compared to the untreated control. rVSV-SARS-CoV-2-Luc (Wuhan variant) infection of A549-ACE2 cells post-treatment with 0–10% w/v formulations of HPBCD and HPGCD; one-way ANOVA *** p < 0.001, **** p < 0.0001. (B) CD treatment of SARS-CoV-2 pseudotyped virus reduces cell infectivity. A total of 20,000 RLU of the rVSV-SARS-CoV-2 Luc pseudotype (Wuhan variant) was incubated with 0.5–10% w/v treatments of HPBCD or HPGCD prior to infection of A549-ACE2 cells. Results plotted as a % of the untreated media control. Data are shown as mean (SD) (n = 3). Statistical analysis was conducted using one-way ANOVA (**** p < 0.0001), where the mean of each column was compared to the no-treatment (NT) group.
Fig 3: Candidate cyclodextrin safety profiles. In vitro cytotoxicity studies were conducted using HPBCD, HPGCD, SBECD, and CRYSMEB. (A) A549-ACE2 cells were treated with hydroxypropyl beta cyclodextrin (HPBCD), hydroxypropyl gamma cyclodextrin (HPGCD), sulfobutylether beta cyclodextrin (SBECD), or KLEPTOSE CRYSMEB, a low methylated beta cyclodextrin (MBCD), for 24 h. CellTox Green dye and an assay buffer were added to make a 2X 1:500 dilution. Fluorescence readout was measured (485–500 nmEx/520–530 nmEm). Cytotoxicity data (n = 3) per CD treatment are shown as individual points on a bar graph. (B) A549-ACE2 cells were exposed to varying % w/v cyclodextrin (HPBCD, HPGCD) treatments for 48 h. (C) Ciliary beat frequency (Hz) pre- and post-treatment and percentage of the active area of ciliary beating. NT, no treatment; PBS, phosphate-buffered saline; CMC, carboxymethyl cellulose.
Fig 4: Beta- and gamma-cyclodextrin effect across SARS-CoV-2 pseudotyped variants. SARS-CoV-2 pseudotype infection of A549-ACE2 cells. Effect of (A) HPBCD and (B) HPGCD against Wuhan D614G, Delta, Epsilon, Beta, Gamma, and Alpha variants represented as percent change compared to the untreated control. Bars above zero indicate an increase in pseudotype infection for those variant/treatment groups.
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