Core-shell nanofibers for localized melanoma therapy delivering Pioglitazone nanoemulsions and gemcitabine dual loaded system

Abstract

Melanoma is the most aggressive type of skin cancer and has very high rates of mortality. The primary objective of this study was to fabricate core-shell nanofibers as a drug delivery system using the coaxial electrospinning technique, which provides some distinct features. Polycaprolactone (PCL)-chitosan (CS)/polyvinyl alcohol (PVA) core-shell nanofibers embedded with Pioglitazone hydrochloride-loaded Nanoemulsions (PIO NEs), a general anti-diabetic drug, and Gemcitabine hydrochloride (GEM), a chemotherapeutic agent, were prepared to investigate the effects of combination of GEM and PIO against A375 melanoma skin cancer cells in vitro. The prepared PCL-CS/PVA-PIO NEs-GEM core-shell nanofibers exhibited sustained and controlled release profiles of GEM and PIO NEs over 14 days, which was fitted into various kinetic models. The data demonstrated the efficacy of nanoemulsions in improving the solubility and release of the poorly aqueous soluble drug PIO. The maximum amount released from the core-shell nanofibers reached 76.99 ± 1.5% of the GEM and 80.47 ± 2.01% of the PIO in a medium of pH 7.4. The nanofibers' morphology, chemical composition, weight loss, and swelling behavior were evaluated. MTT and flow cytometry analyses demonstrated that the combination of PIO and GEM effectively inhibited the growth of melanoma cancer cell lines by inhibiting proliferation with cell viability of 47.07 ± 2.5%, 45.36 ± 2.8%, and 39.79 ± 1.8% after 24, 48 and 72 h, inducing G₀ /G₁ phase arrest and apoptosis, and exhibited an enhanced combinatorial effect in A375 cells in vitro. Additionally, real-time PCR analysis confirmed the induction of apoptosis by measuring gene expression levels, suggesting that the mechanism is associated with the P53 and PPARγ pathways. The generated core-shell nanofibers exhibit properties that suggest their potential as an innovative local drug delivery system, suitable for direct implantation at the tumor site for melanoma treatment through a unique combination therapy of PIO and GEM.

Keywords: Chitosan; Core-shell nanofibers; Gemcitabine hydrochloride; Melanoma; Nanoemulsions; PCL; PVA; Pioglitazone hydrochloride.

Fig. 1

The schematic of nanofibers preparation.

Fig. 2

PIO nanoemulsions characterization; (a) UV-Vis absorption spectrum of pure Pioglitazone solution and Pioglitazone-loaded Nanoemulsions (PIO NEs), the observed spectral shift and intensity changes in PIO NEs indicate successful drug encapsulation, (b) Particle size distribution profile of PIO-NE determined by dynamic light scattering (DLS), revealing a mean particle size of approximately 199 nm, indicating nanoscale formulation, (c) Scanning Electron Microscopy (SEM) image of PIO-NE, demonstrating spherical morphology and uniform particle distribution, (d) Fluorescence microscopy image of PIO loaded nanoemulsion aggregates (scale bar: 20 μm). Due to the clustering effects in the fluorescently labeled formulation, aggregates are visualized and not individual nanoemulsion droplets. (e) FTIR spectra comparing PIO and PIO-NE, highlighting changes in functional group vibrations and confirming successful formulation of the nanoemulsion.

Fig. 3

Morphological characterization of core-shell nanofibers made through coaxial electrospinning. (a) SEM image of Blank PCL-CS/PVA nanofibers devoid of drugs; (b) SEM image of PCL-CS/PVA nanofibers making up the Pioglitazone Nanoemulsions (PIO NEs) and Gemcitabine (GEM); (c) Magnified view demonstrating the oval surface protrusions corresponding to the encapsulated nanoemulsions; (d) Fluorescence microscopy of PCL-CS/PVA nanofibers, with DAPI labeled nanoemulsions, demonstrates successful core encapsulation; (e) TEM image demonstrating that the Blank nanofibers are in a core-shell structure. Scale bars: 5 and 10 μm for SEM images (a–c), 20 μm for fluorescence image (d), 150 nm for the TEM image (e).

Fig. 4

FTIR (a) Blank (PCL-CS/PVA) NFs, S-1 (PCL-CS/PVA -PIO NEs) NFs, S-2 (PCL-CS/PVA-GEM) NFs, S-3 (PCL-CS/PVA -PIO NEs-GEM) NFs and (b) PCL, CS, PVA, PIO, GEM.

Fig. 5

Water contact angle measurements on the surface of electrospun nanofibers. (a) PCL nanofibers; (b) PCL-CS nanofibers; (c) PCL-CS/PVA core-shell nanofibers; (d) PCL-CS/PVA nanofibers loaded with Pioglitazone Nanoemulsions and Gemcitabine. Contact angle data is averaged ± SD of three measurement attempts from three independent preparations (n = 3). Statistical significance determined by one-way ANOVA and post hoc tests; *p < 0.05, **p < 0.01 to previous group denote increase in hydrophilicity with the addition of PVA and drug-loaded nanoemulsions.

Fig. 6

In vitro cumulative drug release profiles. (a) Release of Pioglitazone (PIO) and Gemcitabine (GEM) from PCL-CS/PVA core-shell nanofibers isolated from Pioglitazone Nanoemulsions and Gemcitabine over 14 days in PBS (pH 7.4) and 37 °C; (b) Release of Pioglitazone from Nanoemulsions only under the same conditions. Data shown as mean ± SD from three independent experiments (n = 3). The error bars and significant markers (*p < 0.05) reflect differences in how at selected time points between each formulation. Data represent mean ± SD of three independent experiments. Error bars are included for all time points.”.

Fig. 7

The viability of A375 melanoma cells treated with the different core-shell nanofibers was measured using an indirect MTT assay after 24, 48 and 72 h. Treatment groups included Control (Cells in culture medium), Blank (PCL-CS/PVA nanofibers without drugs), S-1 (PCL-CS/PVA nanofibers with Pioglitazone Nanoemulsions), S-2 (PCL-CS/PVA nanofibers with Gemcitabine), and S-3 (PCL-CS/PVA nanofibers with both Pioglitazone Nanoemulsions and Gemcitabine). Data shown are mean ± SD (n = 3). All statistical analysis is from one-way ANOVA with post hoc tests; *p < 0.05, **p < 0.01, ***p < 0.001 show significantly different from control and Blank nanofibers.

Fig. 8

Optical microscopy images of A375 melanoma cells after 72 h of treatment. (a) Untreated control cells displaying normal morphology and attachment; (b) S-3 treated cells (PCL-CS/PVA nanofibers with Pioglitazone Nanoemulsions and Gemcitabine) showing a loss of morphology consistent with apoptosis including cell shrinkage, detached cells, and blebs in the cellular membranes. Scale bars = 100 μm.

Fig. 9

Flow cytometry analysis of apoptosis and cell cycle distribution in A375 melanoma cells after 48 h of treatment with core-shell nanofibers. (a-d) Apoptosis detection using Annexin V-FITC/PI staining: (a) Control (Blank nanofibers), (b) S-1 (Pioglitazone NEs), (c) S-2 (Gemcitabine), and (d) S-3 (Pioglitazone NEs and Gemcitabine). Quadrants: Q1 – necrotic cells, Q2 – late apoptotic cells, Q3 – early apoptotic cells, Q4 – viable cells. (e-h) Cell cycle distribution: (e) Control, (f) S-1, (g) S-2, (h) S-3. Treatment with S-3 nanofibers resulted in a significant increase in G0/G1 phase arrest and reduced S phase, indicating cell cycle arrest. Data are representative of three independent experiments. Quantitative results expressed as mean ± SD (n = 3); statistical significance shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

Fig. 10

Relative gene expression of (a) PPARγ and (b) P53 gene expression in A375 melanoma cells after 48 h of treatment with the different core-shell nanofiber formulations. Treatment groups included Blank Control (PCL-CS/PVA nanofibers without drugs), S-1 (PCL-CS/PVA nanofibers with pioglitazone nanoemulsions), S-2 (PCL-CS/PVA nanofibers with Gemcitabine), and S-3 (PCL-CS/PVA nanofibers with Pioglitazone Nanoemulsions and Gemcitabine). Gene expression was normalized and GAPDH was utilized as the housekeeping gene and tested using the ∆∆^CT analysis. Data shown are mean ± SD (n = 3). All statistical analysis is from one-way ANOVA with post hoc tests; *p < 0.05, **p < 0.01, ***p < 0.001 show significantly different from the blank control group.