Self-Organized Nanoparticles of Random and Block Copolymers of Sodium 2-(Acrylamido)-2-methyl-1-propanesulfonate and Sodium 11-(Acrylamido)undecanoate as Safe and Effective Zika Virus Inhibitors

Abstract

A series of anionic homopolymers, poly(sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and amphiphilic copolymers of AMPS and sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS₇₅-b-PAaU_n), and random (P(AMPS_m-co-AaU_n)), were synthesized and their antiviral activity against Zika virus (ZIKV) was evaluated. Interestingly, while the homopolymers showed limited antiviral activity, the copolymers are very efficient antivirals. This observation was explained considering that under the conditions relevant to the biological experiments (pH 7.4 PBS buffer) the macromolecules of these copolymers exist as negatively charged (zeta potential about -25 mV) nanoparticles (4-12 nm) due to their self-organization. They inhibit the ZIKV replication cycle by binding to the cell surface and thereby blocking virus attachment to host cells. Considering good solubility in aqueous media, low toxicity, and high selectivity index (SI) of the PAMPS-b-PAaU copolymers, they can be considered promising agents against ZIKV infections.

Keywords: Zika virus; amphiphilic copolymers; sodium 11-(acrylamido)undecanoate; sodium 2-(acrylamido)-2-methyl-1-propanesulfonate.

Figure 1

Chemical structures of PAMPS homopolymer, diblock PAMPS₇₅-b-PAaU_n copolymers and random P(AMPS₅₀-co-AaU₅₀) copolymer.

Figure 2

PAMPS-b-PAaU block copolymers form interpolymer micelles. Spin-spin relaxation time (T₂) of ¹H NMR signal at 1.25 ppm for PAMPS₇₅-b-PAaU₃ (black line), PAMPS₇₅-b-PAaU₁₂ (red line), PAMPS₇₅-b-PAaU₂₈ (green line), PAMPS₇₅-b-PAaU₃₉ (blue line) and P(AMPS₅₀-co-AaU₅₀) (grey line) as a function of pH in D₂O containing 0.1 M NaCl at C_p = 10 g/L (A). Hydrodynamic radius (R_h) for PAMPS₇₅-b-PAaU₃ (black line), PAMPS₇₅-b-PAaU₁₂ (red line), PAMPS₇₅-b-PAaU₂₈ (green line), PAMPS₇₅-b-PAaU₃₉ (blue line), and P(AMPS₅₀-co-AaU₅₀) (grey line) as a function of pH in 0.1 M NaCl aqueous solutions at C_p = 10 g/L and 25 °C (B). Values of hydrodynamic radius (R_h), the surface area of the polymeric spherical nanoparticle, dispersity index (Ɖ) and zeta potentials for PAMPS-PAaU copolymers (C_p =1 mg/mL in PBS, pH= 7.4, T = 25 °C) (C).

Figure 3

PAMPS-PAaU copolymers are not toxic towards Vero cells. Cytotoxicity of PAMPS_m homopolymers (A), PAMPS₇₅-b-PAaU_n and P(AMPS₅₀-co-AaU₅₀) (B) copolymers of various M.W.s at 2000, 1000, 500 and 100 µg/mL. Results of XTT assay of the tested polymers on Vero cells. All experiments were performed in triplicate. Average values with standard error of the mean (error bars) are presented. (C) Three-dimensional fluorescent images of Vero cells incubated with 50 µg/mL of PAMPS₇₅-b-PAaU₂₈-b-F for 1 h. Cell nuclei are denoted in blue, actin is denoted in red and fluorescent polymer is green.

Figure 4

PAMPS₇₅-b-PAaU_n block copolymers are potent inhibitors of Zika infection in vitro. (A) The inhibition of the ZIKV by PAMPS polymers. (B) PAMPS₇₅-b-PAaU_n block copolymers and P(AMPS₅₀-co-AaU₅₀) copolymer at various concentrations in Vero cells. Inhibition of the infection was evaluated by RT-qPCR. Vero cells were infected in the presence of appropriate polymer concentrations for 3 days. Data are shown as logarithmic reduction values (LRV) of ZIKV RNA copy number per milliliter with SEM (error bars). (C) CC₅₀, IC₅₀ and selectivity index (SI) values for PAMPS-PAaU block and random copolymers. (D) Titration results of supernatants of Vero cells infected in the presence or absence of PAMPS₇₅-b-PAaU_n block copolymers and P(AMPS₅₀-co-AaU₅₀) random copolymer in different concentrations. Data are shown as logarithmic reduction values of supernatant infectivity (TCID₅₀) per milliliter of at least three replications. (E) Fluorescence images of cells infected with ZIKV (TCID₅₀ = 2000/mL) in the presence or absence of the polymers at 25 µg/mL for 48 h. Cell nuclei are shown in blue, actin is shown in red and ZIKV E protein is denoted in green. Scale bar = 50 µm.

Figure 5

PAMPS₇₅-b-PAaU₃₉ inhibits the ZIKV replication cycle by blocking virus attachment. Inhibitory activity of copolymers was tested in Vero cells, as described in the Materials and Methods section: virus inactivation assay (A); cell protection assay (B); virus entry assay (C); virus replication, assembly, and egress assay (D). RT-qPCR was used to assess the number of viral RNA copies and to evaluate the inhibition of the infection. The experiments were carried out in triplicate. The data are shown as average values with SEM (error bars). * (p < 0.05) denotes values that are significantly different from the control. Representative fluorescent images of ZIKV particles on the cell surface (E). Vero cells were incubated with ZIKV in the presence or absence of PAMPS₇₅-b-PAaU₃₉ (25 µg/mL) at 4 °C. Cell nuclei are denoted in blue, and ZIKV E protein is denoted in green. The number of ZIKV virions attached to the cells (F). The individual counts, including the statistical error of the mean, are shown.