Poly(ethylene glycol) -block- poly(sodium 4-styrenesulfonate) Copolymers as Efficient Zika Virus Inhibitors: In Vitro Studies

Abstract

A series of poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) (PEG-b-PSSNa) copolymers were synthesized, and their antiviral activity against Zika virus (ZIKV) was determined. The polymers inhibit ZIKV replication in vitro in mammalian cells at nontoxic concentrations. The mechanistic analysis revealed that the PEG-b-PSSNa copolymers interact directly with viral particles in a zipper-like mechanism, hindering their interaction with the permissive cell. The antiviral activity of the copolymers is well-correlated with the length of the PSSNa block, indicating that the copolymers' ionic blocks are biologically active. The blocks of PEG present in copolymers studied do not hinder that interaction. Considering the practical application of PEG-b-PSSNa and the electrostatic nature of the inhibition, the interaction between the copolymers and human serum albumin (HSA) was evaluated. The formation of PEG-b-PSSNa-HSA complexes in the form of negatively charged nanoparticles well-dispersed in buffer solution was observed. That observation is promising, given the possible practical application of the copolymers.

Figure 1

(A) Reaction scheme. (B) ¹H NMR spectrum of PEG₂₂₀-b-PSSNa₈₁ copolymer in D₂O. (C) GPC chromatograms (RALS response) of PEG-b-PSSNa copolymers collected at a 0.8 mL/min flow rate. A 0.1 M NaNO₃ aqueous solution containing 20% v/v acetonitrile was used as an eluent.

Figure 2

PEG-b-PSSNa and PSSNa are not cytotoxic and hamper ZIKV replication in vitro. (A) Cytotoxicity of PEG-b-PSSNa of various molecular weights at 1000, 500, and 100 μg/mL. Results of XTT assay of the tested polymers on Vero and U251 cells. All experiments were performed in triplicate. Average values with standard deviations (error bars) are presented. (B) Inhibition of ZIKV H/PF/2013 replication cycle by PEG-b-PSSNa and PSSNa. The assay was carried out in the Vero and U251 cells infected with the ZIKV H/PF/2013 virus in the presence of different polymers at given concentrations. Inhibition of the infection was evaluated using RT-qPCR. Data are shown as the average logarithmic reduction values (LRV) of ZIKV RNA copy number per milliliter with SEM (error bars). All experiments were performed in triplicate. (C) Experimental values of half-maximal inhibitory concentration (IC50) of PEG-b-PSSNa in Vero and U251 cells. (D) Virus inactivation assay results after ZIKV incubation with polymers (100 μg/mL).

Figure 3

Inhibition of the ZIKV replication by PEG-b-PSSNa and PSSNa. U251 cells were infected with different virus strains in the presence or absence of different polymers and incubated for four days. Inhibition of the infection was evaluated using RT-qPCR. The results are presented as average values of three replications with SEM (error bar).

Figure 4

Representative calorimetric isotherms of the binding of PEG-b-PSSNa copolymers to HSA. Experiments were carried out in PBS at 37 °C. The lines represent the best fit of the one class of binding sites model to the experimental data.