Preparation, characterization, and cytotoxicity of CPT/Fe₂O₃-embedded PLGA ultrafine composite fibers: a synergistic approach to develop promising anticancer material

Abstract

The aim of this study was to fabricate camptothecin/iron(III) oxide (CPT/Fe₂O₃)-loaded poly(D,L-lactide-co-glycolide) (PLGA) composite mats to modulate the CPT release and to improve the structural integrity and antitumor activity of the released drug. The CPT/Fe₂O₃-loaded PLGA ultrafine fibers were prepared for the first time by electrospinning a composite solution of CPT/Fe₂O₃ and neat PLGA (4 weight percent). The physicochemical characterization of the electrospun composite mat was carried out by scanning electron microscopy, energy dispersive X-ray spectroscopy, electron probe microanalysis, thermogravimetry, transmission electron microscopy, ultraviolet-visible spectroscopy, and X-ray diffraction pattern. The medicated composite fibers were evaluated for their cytotoxicity on C2C12 cells using Cell Counting Kit-8 assay (Sigma-Aldrich Corporation, St Louis, MO). The in vitro studies indicated a slow and prolonged release over a period of 96 hours with mild initial burst. Scanning electron microscopy, thermogravimetry, and X-ray diffraction studies confirmed the interaction of CPT/Fe₂O₃ with the PLGA matrix and showed that the crystallinity of CPT decreased after loading. Incorporation of CPT in the polymer media affected both the morphology and the size of the CPT/Fe₂O₃-loaded PLGA composite fibers. Electron probe microanalysis and energy dispersive X-ray spectroscopy results confirmed well-oriented composite ultrafine fibers with good incorporation of CPT/Fe₂O₃. The cytotoxicity results illustrate that the pristine PLGA did not exhibit noteworthy cytotoxicity; conversely, the CPT/Fe₂O₃ composite fibers inhibited C2C12 cells significantly. Thus, the current work demonstrates that the CPT/Fe₂O₃-loaded PLGA composite fibers represent a promising chemotherapeutic system for enhancing anticancer drug efficacy and selectively targeting cancer cells in order to treat diverse cancers.

Keywords: C2C12 cells; Fe2O3 nanoparticles; camptothecin; cytotoxicity; electrospinning.

Figure 1

Illustration for the preparation of camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite fibers by electrospinning process. Abbreviations: CPT, camptothecin; Cu, copper; Fe₂O₃, iron(III) oxide; PLGA, poly(D,L-lactide-co-glycolide).

Figure 2

Scanning electron microscopic images of (A and B) pristine poly(D,L-lactide-co-glycolide), (C and D) camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite ultrafine fibers, and (E) iron(III) oxide nanoparticles at different magnifications (yellow circles represent point bonding). (F) Energy dispersive X-ray spectrum of camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite. The inset photographic images represent the (i) pristine and (ii) composite nano-fibrous mats under ultraviolet fluorescence (λ_max 256 nm).

Figure 3

(A) Representative transmission electron microscopic image of the camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite. The inset image shows the uniform distribution of iron(III) oxide nanoparticles. Red and blue arrows demonstrate the distinct camptothecin layer and iron(III) oxide nanoparticles, respectively. (B) Electron probe microanalysis mapping result of the camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite. The purple square represents the selected area. Abbreviations: C, carbon; Fe, iron; N, nitrogen; O, oxygen.

Figure 4

X-ray diffraction patterns of (A) free camptothecin, (B) iron(III) oxide nanoparticles, (C) electrospun pristine poly(D,L-lactide-co-glycolide), and (D) camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite fibers. Note: The red dashed square represents the iron(III) oxide nanoparticle signals.

Figure 5

Thermogravimetric analysis graphs of (A) camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite and (B) pristine poly(D,L-lactide- co-glycolide) ultrafine fibers. Note: The inset graph represents the corresponding first derivatives in nitrogen atmosphere.

Figure 6

Fourier transform infrared spectra of (A) camptothecin/iron(III) oxideembedded poly(D,L-lactide-co-glycolide) composite and (B) pristine camptothecin/ iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) ultrafine fibers.

Figure 7

In vitro release profile of (A) camptothecin from electrospun camptothecin/ iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite fibers and (B) A355/A368 ratios of camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co- glycolide) composite mat against incubation time in phosphate buffered saline. Note: The inset scanning electron microscopic image shows the camptothecin/ iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite after incubation in phosphate buffered saline at 37°C for 30 days.

Figure 8

In vitro cytotoxicity of (A) free camptothecin and camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite. Untreated C2C12 cells and cells treated with pristine poly(D,L-lactide-co-glycolide) were used as a control. Representative phase contrast images of C2C12 cell lines (B) unexposed to, (C) exposed to 5 μg, and (D) exposed to 10 μg camptothecin/iron(III) oxide-embedded poly(D,L-lactide-co-glycolide) composite. Note: Magnification 40×. Abbreviations: CPT, camptothecin; PLGA, poly(D,L-lactide-co-glycolide).