Folic Acid-Decorated pH-Responsive Nanoniosomes With Enhanced Endocytosis for Breast Cancer Therapy: In Vitro Studies

Abstract

Breast cancer is the most common invasive cancer in women and the second leading cause of cancer death in women after lung cancer. The purpose of this study is a targeted delivery toward in vitro (on MCF7 and 4T1 breast cancer cell lines) through niosomes-based nanocarriers. To this end, different bioactive molecules, including hyaluronic acid (HA), folic acid (FA), and polyethylene glycol (PEG), were used and compared for surface modification of niosomes to enhance endocytosis. FA-functionalized niosomes (Nio/5-FU/FA) were able to increase cell cytotoxicity and reduce cell migration and invasion compared to PEG-functionalized niosomes (Nio/5-FU/PEG), and HA-functionalized niosomes (Nio/5-FU/HA) groups in MCF-7 and 4T1 cell lines. Although the Nio/5-FU/PEG and Nio/5-FU/HA demonstrated MCF7 cell uptake, the Nio/5-FU/FA exhibited the most preponderant endocytosis in pH 5.4. Remarkably, in this study 5-FU loaded niosomes (nonionic surfactant-based vesicles) were decorated with various bioactive molecules (FA, PEG, or HA) to compare their ability for breast cancer therapy. The fabricated nanoformulations were readily taken up by breast cancer cells (in vitro) and demonstrated sustained drug release characteristics, inducing cell apoptosis. Overall, the comprehensive comparison between different bioactive molecules-decorated nanoniosomes exhibited promising results in finding the best nano formulated candidates for targeted delivery of drugs for breast cancer therapy.

Keywords: 5-FU; breast cancer; endocytosis; folic acid; hyaluronic acid; niosome.

FIGURE 1

The preparation and characterization of functionalized niosomes by the thin layer hydration method. MLV: multilamellar vesicles; SUV: small uni-lamellar vesicles.

FIGURE 2

3D plots of the results derived from the central composite design for size (A), polydispersity index [PDI; (B)], encapsulation efficacy [EE; (C)] and release (D) as a function of the parameters (concentrations of Span^® 60 and cholesterol).

FIGURE 3

Optimized responses were obtained by coated and uncoated formulations under the optimal conditions. (A) Vesicle size. (B) Polydispersity. (C) Entrapment efficacy. (D) Release percentage. (E) Zeta Potential. Data represent means ± standard deviations (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05.

FIGURE 4

(A–D) The particle size of synthesized niosomes as determined by dynamic light scattering. (E) Transmission electron micrograph and size distribution of Nio/5-FU. (F) Transmission electron micrograph and size distribution of Nio/5-FU/PEG. (G) Transmission electron micrograph and size distribution of Nio/5-FU/FA. (H) Transmission electron micrograph and size distribution of Nio/5-FU/HA. (I) In vitro release of 5-FU from different niosomal formulations at pH 7.4: 5-FU-loaded pristine niosomes (Nio/5-FU) and those that had been decorated with folic acid (FA), polyethylene glycol (PEG) or hyaluronic acid (HA). (Nio/5-FU/FA). (J) In vitro release of 5-UL from different niosomal formulations at pH 5.4. (K) Fourier transform infrared FTIR spectra of a) Span 60, b) Cholesterol, c) niosome, d) 5-FU, e) Nio/5-FU, f) Nio/5-FU/PEG, g) Nio/5-FU/HA, and h) Nio/5-FU/FA.

FIGURE 5

(A) Percentage cell viability of different dilutions of niosomes on non-malignant MCF10A cells. (B) The effects of 5-FU, Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA on the viability of MCF7 cells. (C) The effects of 5-FU, Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA on the viability of 4T1 cells. (D) Half-maximum inhibitory concentration (IC₅₀) values after 48 h of exposure of MCF7 breast cancer cells to 5-FU, Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA. (E) IC₅₀ values after 48 h treatment of malignant 4T1 cells to 5-FU, Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA. Data represent means ± standard deviations (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05.

FIGURE 6

The effects of control, 5-FU and different niosome formulations (Nio), 5-FU, Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA of (A) MCF7 and (B) 4T1 breast cancer cells that became apoptotic after 48 h of treatment. Data represent means ± standard deviations (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05. (C,D) Flow cytometric analysis of (C) MCF7 and (D) 4T1 cells after treatment with IC50 concentration of vehicle (Nio), 5-FU, Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA formulations. The upper left square (Q1) shows the percentage of necrotic cells, and the upper right square (Q2) exhibits the percentage of late apoptotic cells, (Q3) exhibits the percentage of early apoptotic cells, and (Q4) shows the percentage of live cells.

FIGURE 7

Changes in intracellular ROS content, indicated by the fluorescence of 2′, 7′-dichlorodihydrofluorescein (DCF), are summarized in (A) for MCF7 cells and (B) for 4T1 cells. Data represent means ± standard deviations (n = 3). For all charts, ***: p < 0.001; **: p < 0.01; *: p < 0.05.

FIGURE 8

The uptake of niosomes in MCF7 cells was investigated with confocal laser scanning microscopy. The niosomes investigated were: Nio/5-FU, Nio/5-FU/PEG, Nio/5-FU/HA and Nio/5-FU/FA. (A) Representative CLSM images of MCF7 cells stained with coumarin 6 and Nile-red. (B) Schematic depicting the effect of pH on the release of contents from a niosome.