Solid Self-Nano Emulsifying Nanoplatform Loaded with Tamoxifen and Resveratrol for Treatment of Breast Cancer

Abstract

The solid self-nanoemulsifying drug delivery system (s-SNEDDS) is a growing platform for the delivery of drugs via oral route. In the present work, tamoxifen (TAM) was loaded in SNEDDS with resveratrol (RES), which is a potent chemotherapeutic, antioxidant, anti-inflammatory and P-gp inhibitor for enhancing bioavailability and to obtain synergistic anti-cancer effect against breast cancer. SNEDDS were developed using capmul MCM as oil, Tween 80 as surfactant and transcutol-HP as co-surfactant and optimized by central composite rotatable design. Neusilin US2 concentration was optimized for adsorption of liquid SNEDDS to prepare s-SNEDDS. The developed formulation was characterized and investigated for various in vitro and cell line comparative studies. Optimized TAM-RES-s-SNEDDS showed spherical droplets of a size less than 200 nm. In all in vitro studies, TAM-RES-s-SNEDDS showed significantly improved (p ˂ 0.05) release and permeation across the dialysis membrane and intestinal lumen. Moreover, TAM-RES-s-SNEDDS possessed significantly greater therapeutic efficacy (p < 0.05) and better internalization on the MCF-7 cell line as compared to the conventional formulation. Additionally, oral bioavailability of TAM from SNEDDS was 1.63 folds significantly higher (p < 0.05) than that of combination suspension and 4.16 folds significantly higher (p < 0.05) than TAM suspension. Thus, findings suggest that TAM- RES-s-SNEDDS can be the future delivery system that potentially delivers both drugs to cancer cells for better treatment.

Keywords: breast cancer; combination therapy; enhanced bioavailability; resveratrol; solid self-nanoemulsifying drug delivery system; synergistic action; tamoxifen.

Figure 1

Parameters of QTPP and CQA for lipid-based drug delivery.

Figure 2

Cytotoxicity of TAM and RES combination in different ratios (1:1; 1:5; 1:10). Data are given as mean ± standard deviation (n = 3) with statistical difference at (**) p < 0.01.

Figure 3

Representation of (a) solubility of TAM and RES in various lipids. Maximum solubility of both drugs was observed in capmul MCM EP. (b) Pseudo ternary phase diagram with different Smix ratios 1:0, 1:1, 2:1, 3:1, 4:1, 5:1, 1:2. (c) Ishikawa diagram for SNEDDS development. (d) 3D surface response plot showing effect of concentration of oil and S_mix on the (A). Droplet size—Droplet size increased with an increase in the concentration of oil whereas, decreased with an increase in the concentration of S_mix, (B). PDI—PDI decreased with an increase in the concentration of oil and increased with an increase in the concentration of S_mix. However, combination of concentration of oil and S_mix provided better homogeneity (C). % Transmittance—% Transmittance increased with decrease in the concentration of oil and increase in concentration of S_mix. Data are given as mean ± standard deviation (n = 3) with statistical difference at (***) p < 0.001.

Figure 3

Representation of (a) solubility of TAM and RES in various lipids. Maximum solubility of both drugs was observed in capmul MCM EP. (b) Pseudo ternary phase diagram with different Smix ratios 1:0, 1:1, 2:1, 3:1, 4:1, 5:1, 1:2. (c) Ishikawa diagram for SNEDDS development. (d) 3D surface response plot showing effect of concentration of oil and S_mix on the (A). Droplet size—Droplet size increased with an increase in the concentration of oil whereas, decreased with an increase in the concentration of S_mix, (B). PDI—PDI decreased with an increase in the concentration of oil and increased with an increase in the concentration of S_mix. However, combination of concentration of oil and S_mix provided better homogeneity (C). % Transmittance—% Transmittance increased with decrease in the concentration of oil and increase in concentration of S_mix. Data are given as mean ± standard deviation (n = 3) with statistical difference at (***) p < 0.001.

Figure 4

Graphical representation of (a) droplet size and (b) zeta potential of TAM-RES-s-SNEDDS.

Figure 5

Surface morphology of (a) TAM, RES, neusilin US2 and s-SNEDDS by SEM. (b) s-SNEDDS by TEM depicting the spherical shape of the droplets.

Figure 6

(a) DSC curve, (b) XRD graph, (c) FTIR spectra of TAM, RES Mixture of TAM and RES, Neusilin US2 and s-SNEDDS.

Figure 6

(a) DSC curve, (b) XRD graph, (c) FTIR spectra of TAM, RES Mixture of TAM and RES, Neusilin US2 and s-SNEDDS.

Figure 7

The in vitro (a) drug release profile of TAM and RES from s-SNEDDS and suspension in SGF (pH 1.2) and SIF (pH 6.8). (b) Permeation of TAM/area per unit time across non-everted gut sac from TAM-RES-s-SNEDDS, suspension and TAM suspension. (c) Lipolysis study showed % FFA release per unit time. (d) Morphology of RBCs after treatment with A. Suspension, B. Triton X100 as negative control, C. PBS as positive control, D. Placebo, E. SNEDDS.

Figure 7

The in vitro (a) drug release profile of TAM and RES from s-SNEDDS and suspension in SGF (pH 1.2) and SIF (pH 6.8). (b) Permeation of TAM/area per unit time across non-everted gut sac from TAM-RES-s-SNEDDS, suspension and TAM suspension. (c) Lipolysis study showed % FFA release per unit time. (d) Morphology of RBCs after treatment with A. Suspension, B. Triton X100 as negative control, C. PBS as positive control, D. Placebo, E. SNEDDS.

Figure 8

Confocal scanning images of drug permeation via (a) suspension and (b) SNEDDS across the intestinal membrane.

Figure 9

In vitro cell line study showed the (a) cytotoxicity of TAM-RES-s-SNEDDS and suspension after incubation with MCF-7 cell. (b) Fluorescent microscopic images of MCF-7 after incubation with A. SNEDDS, B. Suspension. (c) ROS level in MCF-7 after incubation with SNEDDS and suspension using DCFDA technique. (d) The SOD activity in MCF-7 after incubation with SNEDDS, suspension and control. Data are given as mean ± standard deviation (n = 3) with statistical difference at (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001.

Figure 9

In vitro cell line study showed the (a) cytotoxicity of TAM-RES-s-SNEDDS and suspension after incubation with MCF-7 cell. (b) Fluorescent microscopic images of MCF-7 after incubation with A. SNEDDS, B. Suspension. (c) ROS level in MCF-7 after incubation with SNEDDS and suspension using DCFDA technique. (d) The SOD activity in MCF-7 after incubation with SNEDDS, suspension and control. Data are given as mean ± standard deviation (n = 3) with statistical difference at (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001.

Figure 10

Plasma concentration time profile of (a) TAM and (b) RES after oral administration of their SNEDDS and suspension. Data expressed as mean ± SD, n = 6.