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Design And Characterisation Of Novel Sorafenib-Loaded Carbon Nanotubes With Distinct Tumour-Suppressive Activity In Hepatocellular Carcinoma

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Design And Characterisation Of Novel Sorafenib-Loaded Carbon Nanotubes With Distinct Tumour-Suppressive Activity In Hepatocellular Carcinoma

Mahmoud Ma Elsayed et al. Int J Nanomedicine.

Abstract

Purpose: Over the past 30 years, no consistent survival benefits have been recorded for anticancer agents of advanced hepatocellular carcinoma (HCC), except for the multikinase inhibitor sorafenib (Nexavar®), which clinically achieves only ~3 months overall survival benefit. This modest benefit is attributed to limited aqueous solubility, slow dissolution rate and, consequently, limited absorption from the gastrointestinal tract. Thus, novel formulation modalities are in demand to improve the bioavailability of the drug to attack HCC in a more efficient manner. In the current study, we aimed to design a novel sorafenib-loaded carbon nanotubes (CNTs) formula that is able to improve the therapeutic efficacy of carried cargo against HCC and subsequently investigate the antitumour activity of this formula.

Materials and methods: Sorafenib was loaded on functionalized CNTs through physical adsorption, and an alginate-based method was subsequently applied to microcapsulate the drug-loaded CNTs (CNTs-SFN). The therapeutic efficacy of the new formula was estimated and compared to that of conventional sorafenib, both in vitro (against HepG2 cells) and in vivo (in a DENA-induced HCC rat model).

Results: The in vitro MTT anti-proliferative assay revealed that the drug-loaded CNTs formula was at least two-fold more cytotoxic towards HepG2 cells than was sorafenib itself. Moreover, the in vivo animal experiments proved that our innovative formula was superior to conventional sorafenib at all assessed end points. Circulating AFP-L3% was significantly decreased in the CNTs-SFN-MCs-treated group (14.0%) in comparison to that of the DENA (40.3%) and sorafenib (38.8%) groups. This superiority was further confirmed by Western blot analysis and immunofluorescence assessment of some HCC-relevant biomarkers.

Conclusion: Our results firmly suggest the distinctive cancer-suppressive nature of CNTs-SFN-MCs, both against HepG2 cells in vitro and in a DENA-induced HCC rat model in vivo, with a preferential superiority over conventional sorafenib.

Keywords: DENA; carbon nanotubes; hepatocellular carcinoma; microcapsules; sorafenib.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic illustration of the synthesis of CNTs-SFN-MCs.
Figure 2
Figure 2
SEM images of pure CNTs (A), CNTs-COOH (B), CNTs-PEG (C), CNTs-SFN (D), CNTs-SFN-MCs (E and F), and plain CNTs-MCs (G and H) at different magnification powers.
Figure 3
Figure 3
TEM images of the pure CNTs (A), CNTs-COOH (B), CNTs-PEG (C), and CNTs-SFN (D).
Figure 4
Figure 4
FT-IR spectra of pristine CNTs (A), purified CNTs (B), pure PEG (C), SFN (D), CNTs-COOH (E), CNTs-COCl (F), CNTs-PEG (G), and CNTs-SFN (H).
Figure 5
Figure 5
TGA of the formulation reaction specimens (A); SFN adsorption into CNTs at different ratios (B).
Figure 6
Figure 6
pH-dependent in vitro release profiles of SFN from the microcapsulated formula (A), and from the PEGylated CNTs-SFN (B). The violet background resembles pH 1.2 and the greenish background resembles pH 7.4.
Figure 7
Figure 7
In vitro anti-proliferative activity of SFN (A), CNTs-SFN (B) and CNTs-PEG (C) against HepG2 cells. Cell viability was measured by MTT assay and IC50 values were calculated by GraphPad Prism software version 6.0. Data are presented as the mean ± SD from three independent experiments. Abbreviations: CNTs-PEG, PEGylated CNTs; SFN, sorafenib; CNTs-SFN, SFN-loaded CNTs.
Figure 8
Figure 8
HPLC chromatogram of (A) SFN, (B) SFN in blank plasma, (C) plasma samples collected after the CNTs-SFN-MCs administration, and (D) the blank plasma.
Figure 9
Figure 9
The mean concentration-time curve of SFN (µg/mL) in plasma after oral administration of SFN and CNTs-SFN-MCs in rats. Data are presented as the mean ± SD (n = 3).
Figure 10
Figure 10
Illustration of the changes in the body weights of the animals in different groups during the experiment.
Figure 11
Figure 11
Effect of the tested compounds on the morphological aspect of livers. Representative photographs of livers from the control (A1–5) group showed normal morphological aspects. Liver from the DENA group (B1–5) showed faint red irregular rough surface and mostly loose consistency. Liver from the SFN-treated group (C1–5) showed firm consistency with irregular rough surface incorporating some scattered micronodules (arrows) of different sizes throughout the liver. Livers in the CNTs-SFN-MCs -treated group (D1–5) showed relatively normal morphological aspects.
Figure 12
Figure 12
Serum AFP-L3% (A); AFU activity (B); representative Western blot analysis of GP73 (C), GPC-3 (D) and HSP70 (E) expression in liver tissues of different groups. (A) Bar chart showing the ratio of AFP-L3 (red) to total AFP (green). In (B), the data are presented as the mean ± SEM (n = 10). *$ and † indicate significant changes from the control, DENA, and SFN groups respectively. *$ and † indicate significant change at p<0.05; **$$ and †† indicate significant changes at p<0.01; ***$$$ and ††† indicate significant changes at p<0.001. Abbreviations: AFP, alpha-fetoprotein; AFP-L3, Lens culinaris agglutinin-reactive fraction of AFP; AFU, alpha l-fucosidase; DENA, N-nitrosodiethylamine; SFN, sorafenib; CNTs-SFN-MCs, sorafenib-loaded microcapsules; GP73, golgi protein 73; GPC-3, glypican-3; HSP70, heat shock protein.
Figure 13
Figure 13
Representative immunofluorescence illustration of liver tissue sections stained for the detection of CTGF, FGF-2, HIF-1α, and mTOR as HCC-relevant biomarkers in control (A), DENA (B), SFN (C), and CNTs-SFN-MCs (D) groups.
Figure 14
Figure 14
Sections of liver tissue obtained from untreated control (A), DENA (B), SFN (C), and CNTs-SFN-MCs-treated rats (D). The white arrows refer to normal hepatic cords, and the black arrows refer to trabeculae formation. The magnification power is x400.

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