Curcumin-Poly(sodium 4-styrenesulfonate) Conjugates as Potent Zika Virus Entry Inhibitors

Curcumin, a natural product with recognized antiviral properties, is limited in its application largely due to its poor solubility. This study presents the synthesis of water-soluble curcumin-poly(sodium 4-styrenesulfonate) (Cur-PSSNan) covalent conjugates. The antiflaviviral activity of conjugates was validated in vitro by using the Zika virus as a model. In the development of these water-soluble curcumin-containing derivatives, we used the macromolecules reported by us to also hamper viral infections. Mechanistic investigations indicated that the conjugates exhibited excellent stability and bioavailability. The curcumin and macromolecules in concerted action interact directly with virus particles and block their attachment to host cells, hampering the infection process.


CMC determination
The critical micelle concentration for Cur-PSSNa n was determined with fluorescence spectroscopy

Figure S5 .
Figure S5.GPC chromatograms of Cur-PSSNa n conjugates obtained using 0.1 M NaCl aqueous solution containing 20% v/v acetonitrile as an eluent.

Figure S6 .
Figure S6.Positive ion ESI mass spectra of Cur-PSSNa 12 showing curcumin molecular ions (as H + and Na + adducts) in end group of conjugate (top).The plot with expanded Y axis reveals fragmentation of the polymer chain, since the differences between indicated signals correspond to the molar mass of the repeating unit, i.e., 206 Da (middle).An expanded view of the m/z 2896-2905 region is given to show the heaviest observed ions (bottom).

Figure S7 .
Figure S7.Positive ion ESI mass spectra of Cur-PSSNa 26 showing curcumin molecular ions as H + and Na + adducts (top).The plot with expanded Y axis reveals fragmentation of the polymer chain, since the differences between indicated signals correspond to the combined molar mass of the repeating unit and a methylene group, i.e., 220 Da (middle).An expanded view of the m/z 2896-2905 region is given to show the heaviest observed ions (bottom).

Figure S8 .
Figure S8.Positive ion ESI mass spectra of Cur-PSSNa 40 showing curcumin molecular ions (as H + and Na + adducts).The plot with expanded Y axis reveals fragmentation of the polymer chain, since the differences between indicated signals correspond to the combined molar mass of the repeating unit and a methylene group, i.e., 220 Da (middle).An expanded view of the m/z 2896-2905 region is given to show the heaviest observed ions (bottom).

Figure S9 .
Figure S9.Positive ion ESI mass spectrum in the MS/MS mode showing fragmentation of the m/z 369 molecular ion of curcumin (collision energy 20 eV).
using diphenyl-1,3,5-hexatriene (DPH) as a fluorescence probe.Stock solutions of the fluorescent probe concentration of 1•10 -3 M in THF were prepared, and then diluted with PBS to obtain the final concentration of DPH equal to 5.0•10 -6 M. For the CMC determination a series of conjugate solutions were prepared at the concentrations ranging from 0.001 to 0.5 mM.The samples were incubated for 24 h in the dark at room temperature before the fluorescence was measured.The fluorescence anisotropies were determined using vertically polarized light at λ = 350 nm for excitation and both vertically (I VV ) and horizontally (I VH ) polarized light at λ = 428 nm for emission.The steady-state anisotropy ( ) was  calculated according to the following equation: =   -    + 2 where: is defined as the intensity of light when both the excitation and emission polarizers are fixed   vertically, and is the intensity of light when the excitation polarizer is fixed vertically and the   emission polarizer is mounted horizontally; is an instrument correction factor ( and  =   /    are fluorescence intensities for horizontally fixed excitation polarizer, while the emission polarizer   was fixed horizontally and vertically, respectively).CMC was determined using first derivative of fluorescence anisotropy.

Figure S12 .
Figure S12.The fluorescence anisotropy of DPH (c = 5.0•10 -6 M, λ ex = 350 nm) as a function of conjugates concentration in PBS (A), and the first derivative of anisotropy versus the conjugate

Figure S13 .Figure S14 .Figure S15 .BFigure S16 .
Figure S13.Cytotoxicity of Cur in Vero and U251 cells.The assay was performed in triplicate.Average values with standard deviations (error bars) are presented.

Figure S17 .
Figure S17.ITC measurements; raw data, calorimetric isotherms of the binding of Cur-PSSNa n conjugates to HSA.Experiments were carried out in PBS at 37 C.The lines represent the best fit of the one class binding sites model to the experimental data.