Development, Characterization and Cell Viability Inhibition of PVA Spheres Loaded with Doxorubicin and 4'-Amino-1-Naphthyl-Chalcone (D14) for Osteosarcoma

Abstract

Chalcones (1,3-diaryl-2-propen-1-ones) are naturally occurring polyphenols with known anticancer activity against a variety of tumor cell lines, including osteosarcoma (OS). In this paper, we present the preparation and characterization of spheres (~2 mm) from polyvinyl alcohol (PVA) containing a combination of 4'-Amino-1-Naphthyl-Chalcone (D14) and doxorubicin, to act as a new polymeric dual-drug anticancer delivery. D14 is a potent inhibitor of osteosarcoma progression and, when combined with doxorubicin, presents a synergetic effect; hence, physically crosslinked PVA spheres loaded with D14 and doxorubicin were prepared using liquid nitrogen and six freeze-thawing cycles. Physical-chemical characterization using a scanning electron microscope (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) presented that the drugs were incorporated into the spheres via weak interactions between the drugs and the polymeric chains, resulting in overall good drug stability. The cytotoxicity activity of the PVA spheres co-encapsulating both drugs was tested against the U2OS human osteosarcoma cell line by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay, and compared to the spheres carrying either D14 or doxorubicin alone. The co-delivery showed a cytotoxic effect 2.6-fold greater than doxorubicin alone, revealing a significant synergistic effect with a coefficient of drug interaction (CDI) of 0.49. The obtained results suggest this developed PVA sphere as a potential dual-drug delivery system that could be used for the prominent synergistic anticancer activity of co-delivering D14 and doxorubicin, providing a new potential strategy for improved osteosarcoma treatment.

Keywords: PVA; chalcone; doxorubicin; freeze–thaw; osteosarcoma.

Figure A1

FTIR of the studied samples exhibiting the whole scanned region.

Figure A2

FTIR spectra of doxorubicin detailing a large number of important bands around 1800–500 cm⁻¹.

Figure A3

Detailed FTIR spectra of pure D14, PVA and the conjugated D14.

Figure A4

Deconvolution of the PVA majority peak into amorphous and crystallinity peaks in order to calculate the % crystallinity. Only two peaks were used for deconvolution in order to represent an average value of the whole hydrogel.

Figure A5

Cell viability inhibition after exposure to doxo, D14 and the combination of both drugs. Data is shown as mean and standard deviation.

Figure 1

Macroscopic images of the PVA spheres (a,b) and its morphology evaluated by SEM from the surface (c,e,g,i) and internal (d,f,h,j) structure of (c,d) pure PVA, (e,f) doxo-PVA, (g,h) D14-PVA, (i,j) D14-doxo-PVA.

Figure 2

Fourier transform infrared spectroscopy (FTIR) analysis of PVA sphere, doxo-loaded PVA sphere, D14-loaded PVA sphere and D14 and doxo-loaded PVA sphere. In the figure, the symbols represent ν—stretching, δ—bending, ρ—rocking, ω—wagging. Region (i) is water absorption and (ii) represents aromatic ketone—C-CO aryl skeletal vibration.

Figure 3

Differential scanning calorimetry thermograms of PVA spheres, D14-loaded PVA sphere, doxo-loaded PVA sphere and D14 and doxo-loaded PVA sphere. The y-axis represents watts/sample g (W/g). A statistically significant difference for D14 group is indicated by (*).

Figure 4

X-ray diffraction pattern of PVA sphere and loaded with D14, doxo and D14-Doxo.

Figure 5

Interaction between the PVA and the drugs. (A) Interaction between PVA and D14. The PVA and the drug are presented as sticks highlighted in green and blue, respectively. (B) Interaction between PVA and doxo. The PVA and the drug are presented as sticks highlighted in green and pink, respectively. The non-polar hydrogens were removed for better visualization of the interactions. The figures were made with PyMOL software (v.1.1.2, Warren Lyford DeLano, open-source software available at [53].

Figure 6

MTT assay to measure cell viability of U2OS cells treated for 24 h with PVA sphere containing only D14 (~8 µg/mL) or doxo (~4 µg/mL), PVA sphere loaded with both drugs D14 + doxo and PVA empty spheres. The cell viability % is obtained by the ratio of the treated cells to the control and data is shown as mean +- standard deviation. Statistical analysis was performed by using one-way ANOVA followed by Tukey post-test and significant results were considered when *, *** < 0.001 and **** < 0.0001.