Novel Chemo-Photothermal Therapy in Breast Cancer Using Methotrexate-Loaded Folic Acid Conjugated Au@SiO2 Nanoparticles

Abstract

Low level laser therapy (LLLT) is known as a safe type of phototherapy to target tumor tissue/cells. Besides, using targeted nanoparticles increases the successfulness of cancer therapy. This study was designed for investigating the combined effect of folate (FA)/Methotrexate (MTX) loaded silica coated gold (Au@SiO₂) nanoparticles (NPs) and LLLT on the fight against breast cancer.NPs were synthesized and characterized using FTIR, TEM and DLS-Zeta. The NPs had spherical morphology with mean diameter of around 25 nm and positive charge (+13.3 mV) while after conjugation with FA and MTX their net charge reduced to around -19.7 mV.Our findings in cell uptake studies clearly showed enhanced cellular uptake of NPs after FA and MTX loaded NPs in both breast cancer cell lines especially on MDA-MB-231 due to high expression of folate receptors. The results indicated that LLLT had a proliferative effect on both breast cancer cell lines but in the presence of engineered breast cancer targeted nanoparticle, the efficacy of combination chemo-photothermal therapy was significantly increased using MTT assay (p<0.05), DAPI staining, and cell cycle findings. The highest apoptotic effect on breast cancer cell lines was observed in the cells exposed to a combination of MTX-FA loaded Au@SiO₂ NP and LLLT proved by DAPI staining and cell cycle(by increasing the cell arrest in subG0/G1). Taken together a combination of chemotherapy and LLLT improves the potential of breast cancer therapy with minimum side effects.

Keywords: Au@SiO2 nanoparticles; Breast cancer; Folic acid; Low level laser therapy; Methotrexate.

Fig. 1

The stepwise synthetic scheme for the preparation of folate and methotrexate loaded biocompatible Au@SiO₂ NPs nanoparticles

Fig. 2

a) FTIR spectra of Au@SiO₂ nanoparticles, b) size distribution of FA-MTX conjugated Au@SiO₂ NPs measured by dynamic light scattering (DLS) c) The zeta potential of Au@SiO₂ and FA-MTX conjugated Au@SiO₂ NPs measured by dynamic light scattering (DLS) at pH=7.4 and T=25 °C, d) Chromatogram of unloaded MTX and FA separated from FA-MTX conjugated Au@SiO₂ NP measured simultaneously by HPLC method

Fig. 3

TEM image of Au@SiO₂ nanoparticles

Fig. 4

Quantitative cell uptake assay of rhodamine B-labelled Au@SiO₂ nanoparticles (NP) or rhodamine B-labelled MTX-FA loaded Au@SiO₂ nanoparticles (NPD) in MCF-7 (a) and MDA-MB-231(b) cell lines for exposure durations of 0.5 h, 1.5 h and 3h obtained by flow cytometry. Non-treated cells of both cell lines were employed as negative control. c Comparison of mean fluorescent intensity of rhodamine B-labelled Au@SiO₂ nanoparticles (NP) or rhodamine B-labelled MTX-FA loaded Au@SiO₂ nanoparticles (NPD) for exposure durations of 0.5 h, 1.5 h and 3h obtained by flow cytometry

Fig. 5

A Qualitative cell uptake assay using Rhodamine B-labelled Au@SiO₂ nanoparticles (NP) in MCF7 with exposure durations of 30 (a), 90 (b) and 180 (c) min or rhodamine B-labelled MTX-FA loaded Au@SiO₂ nanoparticles (NPD) with exposure durations of 30 (d), 90 (e) and 180 (f) min and (B) Qualitative cell uptake assay using Rhodamine B-labelled Au@SiO₂ nanoparticles (NP) in MDA-MB-231 with exposure durations of 30 (a), 90 (b) and 180 (c) min or rhodamine B-labelled MTX-FA loaded Au@SiO₂ nanoparticles (NPD) with exposure durations of 30 (d), 90 (e) and 180 (f) min captured by florescent microscopy

Fig. 6

a MCF-7 and (b) MDA-MB-231 cells growth inhibition rates after treatment with different concentration of NP, MTX and FA-MTX loaded Au@SiO₂ nanoparticles (NPD) after exposure time of 24, 48 and 72h

Fig. 7

A comparison of cell growth inhibition rates exposed to different laser powers (30, 60, 75, 90 and 105 J/cm²) for treatment groups of laser alone, laser + Au@SiO2 nanoparticles and laser + MTX-FA loaded Au@SiO2 nanoparticles directed for two cell line MCF-7 (a) and MDA-MB-231(b) with subsequent checking after 24h

Fig. 8

Apoptosis assay using DAPI staining for MCF-7 or MDA-MB-231 cells, images captured using an inverted microscope. The untreated cells as the negative control (a), cells treated with laser (75 J/cm²) as positive control (b) cells treated with Au@SiO₂ nanoparticles (NP (c), cells treated with laser (75 J/cm²) and Au@SiO₂ nanoparticles (NP) (d), cells treated with MTX without laser irradiation (e), cells treated with MTX and laser irradiation (f), cells treated with MTX-FA-loaded Au@SiO₂ nanoparticles (NPD) without laser exposure (g), and cells treated with MTX-FA-loaded Au@SiO₂ nanoparticles (NPD) with Laser exposure (h)

Fig. 9

Cell cycle distributions investigated for MCF-7 (A) or MDA-MB-231 (B) cells. The untreated cells as negative control (a), cells treated with laser (75 J/cm²) as positive control (b) cells treated with Au@SiO₂ nanoparticles (NP) (c), cells treated with laser (75 J/cm²) and Au@SiO₂ nanoparticles (NP) (d), cells treated with MTX without laser irradiation (e), cells treated with MTX and laser irradiation (f), cells treated with MTX-FA-loaded Au@SiO₂ nanoparticles (NPD) without laser exposure (g), and cells treated with MTX-FA-loaded Au@SiO₂ nanoparticles (NPD) with Laser exposure (h), C) Quantitative results of cell cycle arrest and its distribution