Photothermal Treatment of Human Pancreatic Cancer Using PEGylated Multi-Walled Carbon Nanotubes Induces Apoptosis by Triggering Mitochondrial Membrane Depolarization Mechanism

Abstract

Pancreatic cancer (PC) is one of the most lethal solid tumor in humans, with an overall 5-year survival rate of less than 5%. Thermally active carbon nanotubes have already brought to light promising results in PC research and treatment. We report here the construct of a nano-biosystem based on multi-walled carbon nanotubes and polyethylene glycol (PEG) molecules validated through AFM, UV-Vis and DLS. We next studied the photothermal effect of these PEG-ylated multi-walled carbon nanotubes (5, 10 and 50 μg/mL, respectively) on pancreatic cancer cells (PANC-1) and further analyzed the molecular and cellular events involved in cell death occurrence. Using cell proliferation, apoptosis, membrane polarization and oxidative stress assays for ELISA, fluorescence microscopy and flow cytometry we show here that hyperthermia following MWCNTs-PEG laser mediated treatment (808 nm, 2W) leads to mitochondrial membrane depolarization that activates the flux of free radicals within the cell and the oxidative state mediate cellular damage in PC cells via apoptotic pathway. Our results are of decisive importance especially in regard with the development of novel nano-biosystems capable to target mitochondria and to synergically act both as cytotoxic drug as well as thermally active agents in order to overcome one of the most common problem met in oncology, that of intrinsic resistance to chemotherapeutics.

Keywords: PEG functionalization; apoptosis; carbon nanotubes; mitochondrial therapy.; pancreatic cancer; photothermal ablation.

Figure 1

Schematic illustration of cellular mechanisms involved in nanophotothermolysis of pancreatic cancer cells mediated by MWCNTs-PEG. Following cellular exposure to various concentrations of PEG-ylated MWCNTs PANC-1 cells were irradiated using 808 nm, 2W Laser beam.

Figure 2

A: UV-Vis spectra of MWCNTs-PEG aqueous solution versus PEG solution B: DLS analysis of PEG-MWCNTs complexes. C: AFM image of a PEG functionalized MWCNT D: MTT-based determination of cell growth/viability. Control samples were exposed to normal cell culture conditions. Test groups were exposed to 5, 10 or 50μg/mL MWCNT-PEG (1 hr, 37˚C), followed by Laser irradiation (3 min, 808 nm, 2W/cm2). Abbreviations: UV-VIS-UltraViolet-Visible Spectroscopy, MWCNTs-multi-walled carbon nanotubes, AFM-atomic force microscopy, DLS- dynamic light scattering, MTTMethylthiazol Tetrazolium Assay.

Figure 3

Dark field Light Microscopy Images of MWCNTs-PEG uptake into PANC1 cell. A: Control sample (no MWCNTs-PEG exposure); B: Exposure to 5μg/mL MWCNTs-PEG (1 hr, 37˚C); C: Exposure to 10μg/mL MWCNTs-PEG (1 hr, 37˚C); D: Exposure to 50μg/mL MWCNTs-PEG (1 hr, 37˚C); scale bar: 50 μm.

Figure 4

Detection of apoptosis using Annexin V-cy3. Control sample followed standard cell culture conditions. Test cells were treated with MWCNTs-PEG with different concentrations (5, 10 and 50 μg/mL, respectively) and LASER irradiated. Consequently, all samples were stained with annexin-cy3 fo 5 min (RT, dark). Nucleus staining was also performed using DAPI blue-fluorescence. A: Control sample (no MWCNTs-PEG exposure, no irradiation). Red fluorescence appears in a reduced number of cells, granular aspect coming from limited number of PS groups exposed on the outer surface of the membrane, B: Exposure to 5μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2 ). Reduced number of apoptotic cells, slight increase in the number of PS groups exposed on the surface of the membrane, C: Exposure to 10μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2), Increased number of early-apoptosis entrance cells. Red fluorescence aspect is diffuse, suggesting intense PS translocation process. D: Exposure to 50μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2). The majority of cells present intense, diffuse, red Cy3 fluorescence covering the entire outer surface of the membrane suggesting a highly intense pro-apoptotic effect.

Figure 5

Detection of Mitochondrial Membrane Potential. Control sample followed standard cell culture conditions. Test cells were treated with MWCNTs-PEG with different concentrations (5, 10 and 50 μg/mL, respectively) and LASER irradiated. Consequently, all samples were stained with Mitochondrial Dual Detection Reagent (15 min, RT, dark) with green fluorescence staining the cells showing mitochondrial membrane depolarizarion. Nucleus staining was also performed using DAPI blue-fluorescence. A: Control sample (no MWCNTs-PEG exposure, no irradiation) No green fluorescence is visible, suggesting membrane potential electrical equillibrium, B: Exposure to 5μg/mL MWCNT-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2). Reduced green fluorescence staining; C: Exposure to 10μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2), intense, inhomogenuous, granular aspect of green fluorescence staining; D: Exposure to 50μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2).The majority of cells present green fluorescent staining as a result of mitochondrial potential drop. Aspect is intense and widely spread, with fluorescence overlap between adjacent cells.

Figure 6

Flowcytometric analysis of total ROS accumulation. Forward scatter and FL1- height parameters. A: Control sample (standard cell culture conditions) B: Exposure to 5μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3 min, 808 nm, 2W/cm2). C: Exposure to 10μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3min, 808nm, 2W/cm2 D: Exposure to 50 μg/mL MWCNTs-PEG (1 hr, 37˚C), followed by Laser excitation (3min, 808 nm, 2W/cm2).