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. 2020 Aug 12;12(8):761.
doi: 10.3390/pharmaceutics12080761.

Thymoquinone-Loaded Soy-Phospholipid-Based Phytosomes Exhibit Anticancer Potential against Human Lung Cancer Cells

Nabil A Alhakamy  1   2   3 , Shaimaa M Badr-Eldin  1   4 , Usama A Fahmy  1 , Nabil K Alruwaili  5 , Zuhier A Awan  6 , Giuseppe Caruso  7 , Mohamed A Alfaleh  8 , Ahmed L Alaofi  9 , Faris O Arif  10 , Osama A A Ahmed  1   2 , Adel F Alghaith  9
Affiliations

Thymoquinone-Loaded Soy-Phospholipid-Based Phytosomes Exhibit Anticancer Potential against Human Lung Cancer Cells

Nabil A Alhakamy et al. Pharmaceutics. .

Retraction in

Abstract

Thymoquinone (TQ), a natural polyphenol, has been associated with various pharmacological responses; however, low bioavailability of TQ limits its clinical application. Thus, a novel phytosomal delivery system of TQ-Phospholipon® 90H complex (TQ-phytosome) was developed by refluxing combined with anti-solvent precipitation. This TQ delivery system was optimized by a three-factor, three-level Box-Behnken design. The optimized TQ-phytosome size was (45.59 ± 1.82 nm) and the vesicle size was confirmed by transmission electron microscopy. The in vitro release pattern of the formulation indicated a biphasic release pattern, where an initial burst release was observed within 2 h, followed by a prolonged release. A remarkable increase in dose-dependent cytotoxicity was evident from the significant decrease in IC50 value of TQ-phytosomes (4.31 ± 2.21 µM) against the A549 cell line. The differential effect of TQ-phytosomes in cell cycle analysis was observed, where cancer cells were accumulated on G2-M and pre-G1 phases. Furthermore, increased apoptotic induction and cell necrosis of TQ-phytosomes were revealed with the annexin V staining technique via activation of caspase-3. In reactive oxygen species (ROS) analysis, TQ-phytosomes acted to significantly increase ROS generation in A549 cells. In conclusion, the sustained release profile with significantly-improved anticancer potential could be obtained with TQ by this phytosomal nanocarrier platform.

Keywords: Box-Behnken design; ROS generation; apoptotic potential; biphasic release; cell cycle arrest; cytotoxicity; thymoquinone.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Three-dimensional (A–C) and contour (D–F) plots for the effect of TQ to phospholipid molar ratio (A), temperature process (B), and reflux time on the vesicle size of TQ-phytosomes.
Figure 2
Figure 2
TEM micrographs of the optimized TQ-phytosomes.
Figure 3
Figure 3
Fourier-transform infrared (FTIR) of TQ, Phospholipon® 90H, and TQ-phytosomes.
Figure 4
Figure 4
In vitro release profiles TQ from optimized TQ-phytosomes and suspension in phosphate buffer saline (PBS) (pH 7.4) at 37 ± 0.5 °C (results are presented as mean ± SD, n = 3).
Figure 5
Figure 5
(A) Representation of cytotoxicity of free-TQ, blank-phytosomes, and TQ-phytosomes in A549 cells. (B) Representation of cytotoxicity of free-TQ, blank-phytosomes, and TQ-phytosomes in EA.hy926. # represents significant difference from free-TQ (p < 0.05)) whereas $ represents significant difference from free-TQ (p < 0.05).
Figure 6
Figure 6
Flow cytometric analysis of free-TQ, blank-phytosomes, and TQ-phytosomes on the cell cycle distribution of A549 cells. * represents significant difference from control (p < 0.05), # represents significant difference from free-TQ (p < 0.05) whereas $ represents significant difference from free-TQ (p < 0.05).
Figure 7
Figure 7
(A–D)Apoptotic and necrotic assessment of free-TQ, blank-phytosomes, and TQ-phytosomes in A549 cells. The cells were exposed to the samples for 24 h and stained with annexin-V/FITC and propidium iodide and are plotted. (E) Representation of A549 cell death following apoptotic and necrotic assay by cytometric analysis after annexin V staining. * represents significant difference from control (p < 0.05), # represents significant difference from free-TQ (p < 0.05), whereas $ represents significant difference from free-TQ (p < 0.05).
Figure 8
Figure 8
Analysis ofcaspase-3 content on A549 cells following exposure of free-TQ, blank-phytosomes, and TQ-phytosomes. * represents significant difference from control (p < 0.05), # represents significant difference from free-TQ (p < 0.05), whereas $ represents significant difference from free-TQ (p < 0.05).
Figure 9
Figure 9
Representation of fluorescence intensity following free-TQ, blank-phytosomes, and TQ-phytosome-induced generation of intracellular reactive oxygen species in A549 cells. * represents significant difference from control (p < 0.05), # represents significant difference from free-TQ (p < 0.05), whereas $ represents significant difference from free-TQ (p < 0.05).
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