Hybrid laser activated phycocyanin/capecitabine treatment of cancerous MCF7 cells

Abstract

Laser-induced fluorescence is recently used as an efficient technique in cancer diagnosis and non-invasive treatment. Here, the synergic therapeutical efficacies of the Capecitabine (CAP) chemodrug, photosensitive Phycocyanin (PC) and graphene oxide (GO) under laser irradiation were investigated. The therapeutical efficacies of diverse concentrations of CAP (0.001-10 mg/ml) and PC (0.5-10 mg/ml) alone and with laser irradiation on human breast adenocarcinoma (MCF-7) cells were examined. The interactional effects of 100 mW SHG Nd:YAG laser at 532nm and GaAs laser at 808 nm ranging power of 150 mW- 2.2W were considered. The contribution of graphene oxide (GO) in biocompatible concentrations of 2.5-20 ng/ml and thermal characteristics of laser exposure at 808 nm on GO + fluorophores have been studied. The effects of the bare and laser-excited CAP + PC on cell mortality have been obtained. Despite the laser irradiation could not hold up the cell proliferation in the absence of drug interaction considerably; however, the viability of the treated cells (by a combination of fluorophores) under laser exposure at 808 nm was significantly reduced. The laser at 532 nm excited the fluorescent PC in (CAP + PC) to trigger the photodynamic processes via oxygen generation. Through the in-vitro experiments of laser-induced fluorescence (LIF) spectroscopy of PC + CAP, the PC/CAP concentrations of the maximum fluorescence signal and spectral shifts have been characterized. The synergic effects of the laser exposures and (CAP + PC) treatment at different concentrations were confirmed. It has been shown here that the laser activation of (CAP + PC) can induce the mortality of the malignant cells by reducing the chemotherapeutic dose of CAP to avoid its non-desirable side effects and by approaching the minimally invasive treatment. Elevation of the laser intensity/exposure time could contribute to the therapeutic efficacy. Survival of the treated cells with a combination of GO and fluorophores could be reduced under laser exposure at 808 nm compared to the same combination therapy in the absence of GO. This survey could benefit the forthcoming clinical protocols based on laser spectroscopy for in-situ imaging/diagnosis/treatment of adenocarcinoma utilizing PC + CAP + GO.

Fig. 1.

(a) The photothermal interaction of GO and photo activation of PC using two laser beam exposures at different wavelengths of 808 and 532 nm respectively (b) The experimental schematic of LIF spectroscopy arrangement to collect the fluorescence emission of PC + CAP from the MCF7 cells in microplates.

Fig. 2.

(a) LIF spectra of different concentrations of (CAP + PC) at C_CAP= 2 mg/ml and several PC concentrations of 1, 2, 5 and 10 mg/ml (b) LIF peak intensity versus CAP concentration ranging 0.5-10 mg/ml. This indicates that maximum fluorescence signal takes place at C_PC= 10 mg/ml and C_CAP= 3 mg/ml. Note that only PC is excited by laser at 532 nm.

Fig. 3.

(a) Fluorescence peak wavelength in terms of various PC concentrations in CAP + PC solution at C_CAP= 1, 2 and 5 mg/ml (b) LIF peak wavelength of CAP + PC solution in terms of C_CAP: 1-10 mg/ml and for typical C_PC of 10 mg/ml. Note that PC undergoes red shift at dense solution. Similarly, a lucid blue shift takes place in terms of CAP concentration.

Fig. 4.

MCF7 cell viability versus different laser fluences ranging 4.8-70 W/cm² at 808 nm for 3 minutes exposure time and 48 h incubation.

Fig. 5.

MCF7 cell viability after (a) 24 h and 48 h chemotherapy by CAP in various concentrations ranging 0.001-10 mg/ml. According to the statistical analysis, the measured typical P-Value is ≤ 0.044.

Fig. 6.

MCF7 cell viability after (a) 48 h treatment in terms of PC concentration ranging 0.5-10 mg/ml without (with) laser exposure at 532 due to photoactivation of anticancer drug (b) 24 (48) h treatment by 5 and 10 mg/ml PC concentrations (c) after 48 h laser exposure at 808 nm, 150 mW at 532 nm, 30-100 mW power with (without) PC ≈ 5 mg/ml treatment (d) Emitted LIF peak intensity versus time by the treated cells using 5 mg/ml of PC. Inset up: LIF peak wavelength versus time representing a lucid blue shift. Inset down: Collected LIF emission from treated cells using 5 mg/ml of PC after T = 0 and 24 h incubation. According to the statistical analysis, the measured typical P-Value is ≤ 0.003.

Fig. 7.

Viability of MCF7 cells under CAP + PC treatment in terms of various CAP concentrations (0.25-5 mg/ml) (a) for different concentrations of PC at ▪ 1, ●2.5 and▴5 mg/ml under laser exposures at 532 nm, 100 mW (b) for certain PC concentration of 5 mg/ml without/under the laser exposures at 532 and 808 nm. According to the statistical analysis, the measured typical P-Value is ≤ 0.03.

Fig. 8.

MCF7 cell viability (a) after 48 and 72 h treatment in terms of GO concentrations of 10 and 20 ng/ml (b) versus GO concentration between 2.5-20 ng/ml with 48 h treatment by ▴GO without laser exposure, ▪GO + laser 532 nm/100 mW exposure, ●GO+ laser 808 nm/1.5 W exposure. According to the statistical analysis, the measured typical P-Value is ≤ 0.01.

Fig. 9.

MCF7 cell viability after 48 h incubation in vicinity of (from left to right): 2.5 µg/ml of CAP treatment GOI with 1 ng/ml concentrations+ laser at 808 nm, 2W with 3 min exposure time and laser 808 nm, 1.5W with 3 and 6 min exposure time; 2.5 µg/ml of CAP + GOI(II) with (2.5 ng/ml) concentrations; 2.5 µg/ml of CAP +1.5 mg/ml of PC + GOI(II) with 1 (2.5 ng/ml) concentration; CAP with 1 ng/ml; GOI + PC with 0.3 mg/ml under 532 nm laser exposure with 3 min exposure time and 100 mW power. Inset: Microscopic image of MCF7 cells with 20 times magnitude -Up: before treatment, Down: After 48 h incubation under the treatment by CAP + PC + GOI. According to the statistical analysis, the measured typical P-Value is ≤ 0.013.