Copolymer-Green-Synthesized Copper Oxide Nanoparticles Enhance Folate-Targeting in Cervical Cancer Cells In Vitro

Abstract

Cervical cancer is fast becoming a global health crisis, accounting for most female deaths in low- and middle-income countries. It is the fourth most frequent cancer affecting women, and due to its complexity, conventional treatment options are limited. Nanomedicine has found a niche in gene therapy, with inorganic nanoparticles becoming attractive tools for gene delivery strategies. Of the many metallic nanoparticles (NPs) available, copper oxide NPs (CuONPs) have been the least investigated in gene delivery. In this study, CuONPs were biologically synthesized using Melia azedarach leaf extract, functionalized with chitosan and polyethylene glycol (PEG), and conjugated to the targeting ligand folate. A peak at 568 nm from UV-visible spectroscopy and the characteristic bands for the functional groups using Fourier-transform infrared (FTIR) spectroscopy confirmed the successful synthesis and modification of the CuONPs. Spherical NPs within the nanometer range were evident from transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The NPs portrayed exceptional binding and protection of the reporter gene, pCMV-Luc-DNA. In vitro cytotoxicity studies revealed cell viability >70% in human embryonic kidney (HEK293), breast adenocarcinoma (MCF-7), and cervical cancer (HeLa) cells, with significant transgene expression, obtained using the luciferase reporter gene assay. Overall, these NPs showed favorable properties and efficient gene delivery, suggesting their potential role in gene therapy.

Keywords: cervical cancer; copper oxide; cytotoxicity; folate targeting; gene delivery; green synthesis.

Figure 1

Photograph of Melia azedarach.

Figure 2

Schematic representation of the synthesis of CuO and Cs-CuONPs.

Figure 3

UV-vis spectra of CuONPs, their functionalized counterparts, and an amplified section of the UV-vis spectra showing the slight peak variations among the nanoparticles.

Figure 4

FTIR analysis of (A) CuONPs, (B) Cs-CuONPs, (C) PEG-Cs-CuONPs, (D) F-Cs-CuONPs, and (E) PEG-F-Cs-CuONPs.

Figure 5

TEM micrographs of the nanoparticles. Bar scale = 100 nm. (A) CuONPs, (B) Cs-CuONPs, (B.i) Cs-CuONPs (zoomed), (C) F-Cs-CuONPs, (D) PEG-Cs-CuONPs, and (E) PEG-F-Cs-CuONPs.

Figure 6

Electrophoretic mobility shift assay of the nanocomplexes. Lane 1 represents the positive control, consisting of naked pDNA (0.25 µg/mL). (A) Cs-CuONPs, lanes 1–5 (0, 0.2, 0.4, 0.6, 0.8 µg), (B) F-Cs-CuONPs, lanes 1–5 (0, 0.2, 0.4, 0.6, 0.8 µg), (C) PEG-Cs-CuONPs, lanes 1–5 (0, 0.1, 0.2, 0.3, 0.4, 0.5 µg), and (D) PEG-F-Cs-CuONPs, lanes 1–5 (0, 0.1, 0.2, 0.3, 0.4 µg). White arrows/yellow numbers indicate optimum binding ratios, preceded by the sub-optimum ratio and followed by the supra-optimum ratio.

Figure 7

The serum nuclease protection assay. (A) Lane 1 contains the negative control (pDNA treated with FBS), while lane 2 contains the positive control of only naked pDNA (0.25 µg). (B) Lane 1–3: Cs-CuONPs (0.4, 0.6, 0.8 µg); lanes 4–6: F-Cs-CuONPs (0.2, 0.4, 0.6 µg). (C) Lanes 1–3: PEG-Cs-CuONPs (0.2, 0.3, 0.4 µg). (D) Lanes 1–3: PEG-F-Cs-CuONPs (0.2, 0.3, 0.4 µg). All nanocomplexes were complexed to pDNA (0.25 µg) and treated with 10% FBS.

Figure 8

Ethidium bromide intercalation assay of the nanocomplexes. Incubation mixtures contained pCMV-Luc DNA (2.5 µg) in 100 µL HBS, with the addition of increasing amounts of the (A) Cs-CuONPs and F-Cs-CuONPs, and (B) PEG-Cs-CuONPs and PEG-F-Cs-CuONPs.

Figure 9

MTT assay portraying the cell viability of (A) CuONPs (0.153 µg; 0.115 µg; 0.077 µg), together with the nanoparticles in (B) HEK293, (C) HeLa, and (D) MCF-7 cells. The ratios are as follows: Cs-CuO (0.4:1; 0.6:1; 0.8:1), F-Cs-CuO (0.2:1; 0.4:1; 0.6:1), PEG-Cs-CuO (0.3:1; 0.4:1; 0.5:1), and PEG-F-Cs-CuO (0.2:1; 0.3:1; 0.4:1). Data are represented as means ± SD (n = 3). *** p < 0.001. was considered to be statistically significant.

Figure 10

In vitro luciferase activity and competition studies in (A) HEK293, (B) HeLa, and (C) MCF-7 cells. Each column represents the mean ± SD (n = 3). Luciferase expression was measured in RLU/mg protein. *** p < 0.001 was considered to be statistically significant. The ratios were represented as follows: Cs-CuO (0.4:1; 0.6:1; 0.8:1), F-Cs-CuO (0.2:1; 0.4:1; 0.6:1), PEG-Cs-CuO (0.2:1; 0.3:1, 0.4:1), and PEG-F-Cs-CuO (0.2:1, 0.3:1, 0.4:1).