Nanotechnology-Driven Strategy Against SARS-CoV-2: Pluronic F127-Based Nanomicelles with or Without Atazanavir Reduce Viral Replication in Calu-3 Cells

Abstract

Despite extensive efforts, no highly effective antiviral molecule exists for treating moderate and severe COVID-19. Nanotechnology has emerged as a promising approach for developing novel drug delivery systems to enhance antiviral efficacy. Among these, polymeric nanomicelles improve the solubility, bioavailability, and cellular uptake of therapeutic agents. In this study, Pluronic F127-based nanomicelles were developed and evaluated for their antiviral activity against SARS-CoV-2. The nanomicelles, formulated using the direct dissolution method, exhibited an average size of 37.4 ± 8.01 nm and a polydispersity index (PDI) of 0.427 ± 0.01. Their antiviral efficacy was assessed in SARS-CoV-2-infected Vero E6 and Calu-3 cell models, where treatment with a 1:2 dilution inhibited viral replication by more than 90%. Cytotoxicity assays confirmed the nanomicelles were non-toxic to both cell lines after 72 h. In SARS-CoV-2-infected Calu-3 cells (human type II pneumocyte model), treatment with Pluronic F127-based nanomicelles containing atazanavir (ATV) significantly reduced viral replication, even under high MOI (2) and after 48 h, while also preventing IL-6 upregulation. To investigate their mechanism, viral pretreatment with nanomicelles showed no inhibitory effect. However, pre-exposure of Calu-3 cells led to significant viral replication reduction (>85% and >75% for 1:2 and 1:4 dilutions, respectively), as confirmed by transmission electron microscopy. These findings highlight Pluronic F127-based nanomicelles as a promising nanotechnology-driven strategy against SARS-CoV-2, reinforcing their potential for future antiviral therapies.

Keywords: Calu-3 cells; Pluronic F127; SARS-CoV-2; antiviral activity; atazanavir (ATV); nanotechnology; poloxamer; polymeric micelles.

Figure 1

Graph for determining poloxamer 407′s critical micellar concentration. As can be seen in the absorbance versus concentration graph (mg/mL), the CMC of poloxamer varied between 0.1 and 0.5 mg/mL. The absorption wavelength used to determine the CMC was 656 nm.

Figure 2

Morphological characterization of micelles and SARS-CoV-2 particles by negative staining technique. Analyses of the micelles (A,B) showed structures with a spherical morphology and with a mean diameter of 50 nanometers. The SARS-CoV-2 particles showed a spherical morphology exhibiting projections on the envelope (spike protein) with a mean diameter of 76 nanometers (C,D). (C) has been colored in Adobe Photoshop.

Figure 3

Viability of Vero E6 and Calu-3 cells in the presence of micelles and micelles inhibition of SARS-CoV-2 replication with different magnitudes in the cell models tested. (A) Vero E6 cells (1 × 10⁴ cells/well) or (B) Calu-3 cells (2 × 10⁴ cells/well) were exposed to micelles in different 1:X ratios (X = 2, 4, 8, 16, 32, 64, 128, 256, or 512) and kept at 37 °C, 5% CO2 for 72 h. The viability of Vero E6 and Calu-3 cells was assessed by LDH dosage; the positive control of the assay was lysed cells (1× lysis solution). (C) Vero E6 cells (1 × 10⁴ cells/well) or (D) Calu-3 cells (2 × 10⁴ cells/well) were infected with SARS-CoV-2 (MOI: 0.01) for 1 h at 37 °C and 5% CO₂. The supernatant was then removed, and the cells were exposed to the micelles in different 1:X ratios (X = 2, 4, 8, 16, 32, 64, 128, 256, or 512) and kept at 37 °C, 5% CO₂ for 24 h. Viral titer was determined by plaque assay. The x-axis refers to the dilution factor applied in the experimental setup. Data are represented as mean ± SD of 6 experiments, and graphs were created using GraphPad Prism 10.1.1 software.

Figure 4

Ultrastructural analysis of Calu-3 cells infected with SARS-CoV-2 (MOI 0.01) and treated with micelle (1:2). (A,B) uninfected and untreated Calu-3 cells (control cell). (C,D) Calu-3 cells infected with SARS-CoV-2 (virus control); note the presence of SARS-CoV-2 particles inside the cytosol (arrows). (E) Calu-3 cell treated with micelle (micelle control). (F–H) Calu-3 cells infected with SARS-CoV-2 and exposed to the micelle; note the presence of defective SARS-CoV-2 particles in the lumen of cytosol vesicles (asterisk). N (Nucleus), SARS-CoV-2 particles (arrows).

Figure 5

Micelles do not exhibit virucidal effects nor seem to affect SARS-CoV-2 adsorption to the host cell. (A) SARS-CoV-2 was exposed to different proportions (1:2 or 1:4) of micelle for 5 and 30 min. After this period, viral infectivity was evaluated in Vero V6 cells. Statistical analysis of SARS-CoV-2 titers in PFU/mL was carried out in comparison with the unexposed virus control (Nil) and pre-exposed virus with different dilutions in two different exposure times. (B) Calu-3 were infected and treated with SARS-CoV-2 and micelles (1:2 or 1:4) at 4 °C. After 1 h, the culture supernatant was exchanged for new medium containing DMEM with 10% fetal bovine serum, and the culture was incubated at 37 °C and 5% CO₂ for 24 h; then the culture supernatant was collected for viral titration by plaque assay on Vero E6 cells. The x-axis refers to the dilution factor applied in the experimental setup. Data are represented as mean ± SD of 6 experiments, and graphs were created using GraphPad Prism 10.1.1 software, analyzed by one-way ANOVA followed by Tukey’s post-test (n = 6), *** p < 0.001.

Figure 6

Pre-treatment of cells with the micelles inhibited SARS-CoV-2 replication. Calu-3 cells (2 × 10⁴ cells/well) were treated with the micelles in different proportions (1:2, 1:4, or 1:8) at 37 °C, 5% CO₂ for 1 h. The medium was then changed to one containing only SARS-CoV-2 (MOI: 0.01), and the cells were incubated at 4 °C for 1 h. After this period, the medium was changed to one containing only 10% SFB. After 24 h, the supernatant was collected, and the viruses were titrated by plaque assay. The x-axis refers to the dilution factor applied in the experimental setup. Data represent the mean ± SD (n = 8).

Figure 7

Ultrastructural analysis of Calu-3 cells pre-exposed to micelle (1:2) and infected with SARS-CoV-2 (MOI 3). (A,B) uninfected and untreated Calu-3 cells (control cell). (C,D) Calu-3 cells 1 hpi with SARS-CoV-2 (infected cell control); note SARS-CoV-2 particles on the cell membrane (arrow). (E,F) Calu-3 cells pre-exposed to micelle (micelle cell control). (G,H) Calu-3 cells pre-exposed to micelle and infected with SARS-CoV-2. N (Nucleus), M (Mitochondria).

Figure 8

Calu-3 cells (6-well plates with 4.0 × 10⁵ cells/well) were infected with SARS-CoV-2 (MOI: 2) for 1 h at 37 °C and 5% CO₂. The supernatant was then removed, and the cells were exposed to fresh medium containing nanomicelles at 1:2 or 1:4 dilutions with or without atazanavir (ATV) at concentrations of 250 µg/mL or 125 µg/mL, respectively, and kept at 37 °C, 5% CO₂ for 48 h. (A) Viral titer was determined by plaque assay. (B) The inhibition percentage was also determined. (C) IL-6 and (D) IL-8 cytokine production was detected using a Human Standard ABTS ELISA Development kit (Peprotech, Thermo Fisher Scientific). Data are represented as mean ± SD of 6 experiments, and graphs were created using GraphPad Prism 10.1.1 software, analyzed by one-way ANOVA followed by Tukey’s post-test (n = 6), * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 9

Diffusion cell used in in vitro release studies (A); in vitro release profile of atazanavir (ATV) encapsulated in polymeric nanomicelles (B); study of ATV kinetics obtained after application of the “Higuchi Model” (C).