Luteolin-Loaded Elastic Liposomes for Transdermal Delivery to Control Breast Cancer: In Vitro and Ex Vivo Evaluations

Abstract

The study aimed to prepare and optimize luteolin (LUT)-loaded transdermal elastic liposomes (LEL1-LEL12), followed by in vitro and ex vivo evaluations of their ability to control breast cancer. Various surfactants (Span 60, Span 80, and Brij 35), and phosphatidyl choline (PC) as a lipid, were used to tailor various formulation as dictated by "Design Expert^® software (DOE). These were characterized for size, polydispersity index (PDI), and zeta potential. The optimized formulation (OLEL1) was selected for comparative investigations (in vitro and ex vivo) against lipo (conventional liposomes) and drug suspension (DS). Moreover, the in vitro anticancer activity of OLEL1 was compared against a control using MCF-7 cell lines. Preliminary selection of the suitable PC: surfactant ratio for formulations F1-F9 showed relative advantages of Span 80. DOE suggested two block factorial designs with four center points to identify the design space and significant factors. OLEL1 was the most robust with high functional desirability (0.95), minimum size (202 nm), relatively high drug release, increased drug entrapment (92%), and improved permeation rate (~3270 µg/cm²) as compared with liposomes (~1536 µg/cm²) over 24 h. OLEL1 exhibited a 6.2- to 2.9-fold increase in permeation rate as compared with DS (drug solution). The permeation flux values of OLEL1, and lipo were found to be 136.3, 64 and 24.3 µg/h/cm², respectively. The drug disposition values were 670 µg, 473 µg and 148 µg, for OLEL1, lipo and DS, respectively. Thus, ex vivo parameters were significantly better for OLEL1 compared with lipo and DS which is attributed to the flexibility and deformability of the optimized formulation. Furthermore, OLEL1 was evaluated for anticancer activity and showed maximized inhibition as compared with DS. Thus, elastic liposomes may be a promising approach for improved transdermal delivery of luteolin, as well as enhancing its therapeutic efficacy in controlling breast cancer.

Keywords: cytotoxicity against MCF-7; design expert-based optimization; elastic liposomes; ex vivo permeation and drug deposition; luteolin.

Figure 1

Chemical structures of (A) luteolin, and (B) phosphatidylcholine of Phospholipon 90G.

Figure 2

Design Experiment 3-dimensional response surface plots of the vesicle size (Y₁), zeta potential (Y₂), and % EE (Y₃). (A) Three-dimensional response surface plot of (Y₁) depicting minimal change in size with increase in PC content at high surfactant level. However, the size increases with decrease in Span 80 content at any PC levels. (B) Three-dimensional response surface plot of (Y₂) depicting changes in zeta potential with change in PC and Span 80 levels. Here, the lowest value observed at low levels of both factors. (C) Three-dimensional response surface plot of (Y₃) which revealed a significant increase in the % EE of LUT at lower level of PC and higher level of Span 80.

Figure 3

Design experiment factor interaction plots of (a) the vesicle size (Y₁), (b) zeta potential (Y₂), and (c) % EE (Y₃). Green dots represent center points.

Figure 4

The desirability function of the optimized proposed formulations (OLEL1).

Figure 5

(A) Morphological study of OLEL1, using TEM, (B) elasticity of developed LUT-loaded elastic liposomes (LEL1-LEL12) and comparison against liposomes.

Figure 6

In vitro drug release pattern of the optimized elastic liposome formulations (OLEL1) as compared with conventional liposome (lipo) and drug solution (DS) over period of 12 h. OLEL1 and lipo showed significant difference (p < 0.05). Data presented are mean ± s.d (n = 2).

Figure 7

(A) Ex vivo LUT release pattern of the optimized elastic liposomes formulations (OLEL1) as compared with conventional liposome (lipo) and drug solution (DS) over a period of 24 h. Data presented are mean ±s.d (n = 2), and (B) drug deposition study of OLEL1, lipo and DS into the skin after 24 h of permeation study. Data presented are mean ± s.d (n = 2).

Figure 8

Effect of different concentrations of luteolin standard and luteolin formulation (OLEL1) on viability of MCF7 cells evaluated by MTT assay. Data are presented in percent (%) in comparison with control as 100%. Tukey test was utilized to analyze statistically significant difference between different concentration exposures and control. Difference was considered significant if p value was found to be <0.05. NS = not significant when compared with control; *** = p < 0.001 when compared with control; NS = not significant when compared with same concentration group of luteolin standard; ### = p < 0.001 when compared with same concentration group of luteolin standard.