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. 2021 Jul 16:12:682337.
doi: 10.3389/fphar.2021.682337. eCollection 2021.

Development, Optimization and Evaluation of 2-Methoxy-Estradiol Loaded Nanocarrier for Prostate Cancer

Nabil A Alhakamy  1   2   3 , Osama A Ahmed  1   2 , Usama A Fahmy  1   2 , Hani Z Asfour  4 , Adel F Alghaith  5 , Wael A Mahdi  5 , Sultan Alshehri  5 , Shadab Md  1   2
Affiliations

Development, Optimization and Evaluation of 2-Methoxy-Estradiol Loaded Nanocarrier for Prostate Cancer

Nabil A Alhakamy et al. Front Pharmacol. .

Abstract

The therapeutic efficacy of antineoplastic agents possessing a selective target to the nucleus of the cancer cells could be enhanced through novel formulation approaches. Thus, toward the improvement of the anticancer potential of 2-methoxy estradiol (2 ME) on prostate cancer, the drug was entrapped into the hydrophobic micelles core formulated with Phospholipon 90G and d-α-tocopheryl polyethylene glycol succinate (TPGS). Optimization of the formulation was done by Box-Behnken statistical design using Statgraphics software to standardize percentages of TPGS and phospholipid to obtain the smallest particle size. The optimized formulation was found to be spherical with nanometer size of 152 ± 5.2 nm, and low PDI (0.234). The entrapment efficiency of the micelles was 88.67 ± 3.21% with >93% release of 2 ME within 24 h. There was a 16-fold increase in apoptosis and an 8-fold increase in necrosis of the PC-3 cells when incubated with 2 ME micellar delivery compared to control cells (2.8 ± 0.2%). This increased apoptosis was further correlated with increased BAX expression (11.6 ± 0.7) and decreased BCL-2 expression (0.29 ± 0.05) in 2 ME micelles treated cells when compared to the control group. Further, loss of mitochondrial membrane potential (∼50-fold) by the drug-loaded micelles and free drug compared to control cells was found to be due to the generation of ROS. Findings on cell cycle analysis revealed the significant arrest of the G2-M phase of the PC-3 cells when incubated with the optimized formulation. Simultaneously, a significantly increased number of cells in pre-G1 revealed the maximum apoptotic potential of the drug when delivered via micellar formulation. Finally, upregulation of caspase-9, p53, and NO, with downregulation of TNF-α, NF-κβ, and inflammatory mediators of the PC-3 cells established the superiority of the micellar approach against prostate cancer. In summary, the acquired results highlighted the potentiality of the 2 ME-micellar delivery tool for controlling the growth of prostate cancer cells for improved efficacy.

Keywords: 2-methoxyestradiol; cell cycle; mitochondrial membrane potential; mixed micelles; molecular markers.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) Main plot effect on the interaction of phospholipid, TPGS contents and stirring time on the particle size of 2 ME-loaded micelles formulation. (B) Pareto chart on the interaction of phospholipid, TPGS content and stirring time on the particle size of 2 ME-loaded micelles formulation. (C) Contour plot on the interaction of phospholipid, TPGS content and stirring time on the particle size of 2 ME-loaded micelles formulation.
FIGURE 2
FIGURE 2
Optimized 2 ME-loaded mixed micelles investigated by TEM.
FIGURE 3
FIGURE 3
The cumulative release pattern of 2 ME from the optimized 2 ME-loaded mixed micelles in phosphate buffer (pH 7.4). Values are expressed as mean ± SD (n = 3).
FIGURE 4
FIGURE 4
Cell viability of the cells treated with 2 ME-loaded mixed micelles, free drug (2 ME) and blank micelles were presented. Control group is 100% cell viability. Values are expressed as mean ± SD (n = 3). * represents significant difference between 2 ME-loaded mixed micelles (p < 0.05) vs. blank micelles and free 2 ME [except for 0.4 μg/ml for (p > 0.05). # represents significant difference between free 2 ME vs. blank micelles (p < 0.05)].
FIGURE 5
FIGURE 5
Apoptotic and necrotic assessment of blank-micelles, free 2 ME, and 2 ME-loaded micelles in PC-3 cell line. The cells were exposed to the samples for 24 h and stained with Annexin-V/FITC and propidium iodide, control (A) (i), blank micelles (ii), free 2 ME (iii), and 2 ME-loaded micelles (iv). (B) Representation of PC-3 cell death following apoptotic and necrotic assay by cytometric analysis after annexin V staining. Values are expressed as mean ± SD (n = 3). * represents significant difference from control group (p < 0.05). # represents significant difference between 2 ME-loaded micelles vs other groups.
FIGURE 6
FIGURE 6
Flow cytometric analysis of blank micelles, free-2ME, and 2 ME-loaded micelles on the cell cycle distribution of PC-3 cells. * represents significant difference from control group (p < 0.05). Values are expressed as mean ± SD (n = 3).
FIGURE 7
FIGURE 7
A. BAX and B. BCL-2 expression levels of the blank micelles, free-2ME, and 2 ME-loaded micelles exposed PC-3 cells and vehicle control cells after 24 h. BAX and BCL-2 expression levels were normalized with regard to control cells. * represents significant difference between 2 ME-loaded micelles vs other formulations group (p < 0.05). Values are expressed as mean ± SD (n = 3).
FIGURE 8
FIGURE 8
Loss of mitochondrial membrane potential (MMP) of the PC-3 cells following incubation with blank micelles, free-2 ME and 2 ME-loaded micelles and vehicle control cells. * represents significant difference from control group (p < 0.05). Values are expressed as mean ± SD (n = 3).
FIGURE 9
FIGURE 9
Change in the expression levels of caspase-9 (A), p53 (B), NO (C), and NF-κβ (D) on the PC-3 cells following incubation with blank micelles, free-2 ME, and 2 ME-loaded micelles and vehicle control cells. * represents significant difference between 2 ME-loaded micelles vs other formulations group (p < 0.05). Values are expressed as mean ± SD (n = 3).
FIGURE 10
FIGURE 10
Change in the expression levels of TNF-α (A), COX-2 (B), and IL-6 (C) on the PC-3 cells following incubation with blank micelles, free-2 ME and 2 ME-loaded micelles, and vehicle control cells. * represents significant difference between 2 ME-loaded micelles vs other formulations group (p < 0.05). Values are expressed as mean ± SD (n = 3).
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