Preparation of poly(vinyl alcohol) nanofibers containing disulfiram-copper complex by electrospinning: a potential delivery system against melanoma

Abstract

Background: Melanoma poses a significant threat to human health, making the development of a safe and effective treatment a crucial challenge. Disulfiram (DS) is a proven anticancer drug that has shown effectiveness when used in combination with copper (DS-Cu complex).

Objectives: This study focuses on encapsulation of DS-copper complex into nanofiber scaffold from polyvinyl alcohol (PVA) (DS-Cu@PVA). In order to increase bioavailability towards melanoma cell lines and decrease its toxicity.

Methods: The scaffold was fabricated through an electrospinning process using an aqueous solution, and subsequently analyzed using ART-Fourier transform infrared spectroscopy (ART-FTIR), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). Additionally, cellular cytotoxicity, flow cytometry analysis, and determination of caspase 3 activity were conducted to further characterize the scaffold.

Results: The results confirmed that encapsulation of DS-Cu complex into PVA was successful via different characterization. The scanning electron microscopy (SEM) analysis revealed that the diameter of the nanofibers remained consistent despite the addition of DS-Cu. Additionally, ATR-FTIR confirmed that the incorporation of DS-Cu into PVA did not significantly alter the characteristic peaks of PVA. Furthermore, the cytotoxicity assessment of the DS-Cu@PVA nanofibrous scaffold using human normal skin cells (HFB4) demonstrated its superior biocompatibility compared to DS-Cu-free counterparts. Notably, the presence of DS-Cu maintained its effectiveness in promoting apoptosis by increasing cellular reactive oxygen species, proapoptotic gene expression, and caspase 3 activity, while simultaneously reducing glutathione levels and oncogene expression in human and mouse melanoma cell lines (A375 and B16F10, respectively). Overall, these findings suggest that the addition of DS-Cu to PVA nanofibers enhances their biocompatibility and cytotoxic effects on melanoma cells, making them a promising candidate for biomedical applications.

Conclusion: The findings indicate that the targeted delivery of DS-Cu onto a PVA nanofiber scaffold holds potential approach to enhance the efficacy of DS-Cu in combating melanoma.

Keywords: Anticancer; Disulfiram; Electrospinning; Melanoma; Polyvinyl alcohol.

Fig. 1

Morphology of electrospun PVA and DS-Cu@PVA nanofiber mixed nanofibers, nanofiber distribution (a-e), Edx analysis (f). [PVA (13%) in ethanol: distilled water (1:1) and DS-Cu @PVA ((b) 1 µM, c 2 µM, d 4 µM, and (e) 6 µM)]

Fig. 2

FTIR spectra for electrospun DS-Cu@PVA nanofiber scaffolds (a) PVA; b DS-Cu; c DS-Cu@PVA (2 µm); and (d) DS-Cu@PVA (6 µm). [PVA (13%) in ethanol: distilled water (1:1) and DS-Cu@PVA ((c) 2 µM, d 6 µM)]

Fig. 3

Cytotoxicity of DS-Cu-free and DS-Cu@PVA nanofibers scaffolds against normal dermal cells and melanoma cell lines. A Cytotoxicity of serial concentrations of DS-Cu-free and DS-Cu@PVA nanofibers scaffolds on human normal dermal fibroblast (HFB4) cells. B, C Cytotoxicity of serial concentrations of DS-Cu-free and DS-Cu@PVA nanofibers scaffolds against human and murine melanoma cell lines (A375 and H16-F10, respectively). D Morphological alterations of normal dermal cells and both melanoma cell lines after 72 h treatment with 2 μM of DS-Cu-free and DS-Cu@PVA nanofibers scaffolds using phase contrast microscope (Magnification × 100). Data are demonstrated as mean ± SEM. [PVA (13%) in ethanol: distilled water (1:1) and DS-Cu@PVA (1 μM, 2 μM, 4 μM, and 6 μM)]

Fig. 4

Flow cytometric analysis of apoptosis induction in DS-Cu-free and DS-Cu@PVA-treated melanoma cells at 2 μM. Flow cytometry illustration for apoptotic activity, including (I) flow charts of the untreated, DS-Cu-free and DS-Cu@PVA-treated human A375 cells after staining with annexin V/PI and (II) percentages of total apoptotic cell populations in the untreated and treated A375. Data are expressed as mean ± SEM. [PVA (13%) in ethanol: distilled water (1:1) and DS-Cu@PVA (2 μM)]

Fig. 5

Relative fold change in cellular reactive oxygen species (ROS) and glutathione (GSH) in DS-Cu-free and DS-Cu@PVA-treated melanoma cells at 2 μM. A Relative fold increment in ROS content and (B) relative fold decrement in GSH in the treated human and murine melanoma cell lines (A375 and H16-F10, respectively). Data are expressed as mean ± SEM. [PVA (13%) in ethanol: distilled water (1:1) and DS-Cu@PVA (2 μM)]

Fig. 6

QPCR detection of key gene expressions and caspase activity in DS-Cu-free and DS-Cu@PVA-treated melanoma cells. A Relative fold change in the expression of p53, p21, β-catenin, BCL-2, and cyclin D after 72 h incubation of A375 cells with DS-Cu-free and DS-Cu@PVA at 2 μM. B Relative fold increment in caspase 3 activity in the treated human and mouse melanoma cells (A375 and H16-F10, respectively) relative to the untreated cells. Data are expressed as mean ± SEM. [PVA (13%) in ethanol: distilled water (1:1) and DS-Cu@PVA (2 μM)]