Modulated fluorophore signal recovery buried within tissue mimicking phantoms.

Optically modulated fluorescence from ∼140 nM Cy5 is visualized when embedded up to 6 mm within skin tissue mimicking phantoms, even in the presence of overwhelming background fluorescence and scatter. Experimental and finite element analysis (FEA)-based computational models yield excellent agreement in signal levels and predict biocompatible temperature changes. Using synchronously amplified fluorescence image recovery (SAFIRe), dual-laser excitation (primary laser: λ = 594 nm, 0.29 kW/cm(2); secondary laser: λ = 710 nm, 5.9 kW/cm(2), intensity-modulated at 100 Hz) simultaneously excites fluorescence and dynamically optically reverses the dark state buildup of primary laser-excited Cy5 molecules. As the modulated secondary laser both directly modulates Cy5 emission and is of lower energy than the collected Cy5 fluorescence, modulated Cy5 fluorescence in phantoms is free of obscuring background emission. The modulated fluorescence emission due to the secondary laser was recovered by Fourier transformation, yielding a specific and unique signature of the introduced fluorophores, with largely background-free detection, at excitation intensities close to the maximum permissible exposure (MPE) for skin. Experimental and computational models agree to within 8%, validating the computational model. As modulated fluorescence depends on the presence of both lasers, depth information as a function of focal position is also readily obtained from recovered modulated signal strength.

Single walled carbon nanohorns as photothermal cancer agents.

BACKGROUND: Nanoparticles have significant potential as selective photo-absorbing agents for laser based cancer treatment. This study investigates the use of single walled carbon nanohorns (SWNHs) as thermal enhancers when excited by near infrared (NIR) light for tumor cell destruction. METHODS: Absorption spectra of SWNHs in deionized water at concentrations of 0, 0.01, 0.025, 0.05, 0.085, and 0.1 mg/ml were measured using a spectrophotometer for the wavelength range of 200-1,400 nm. Mass attenuation coefficients were calculated using spectrophotometer transmittance data. Cell culture media containing 0, 0.01, 0.085, and 0.333 mg/ml SWNHs was laser irradiated at 1,064 nm wavelength with an irradiance of 40 W/cm² for 0-5 minutes. Temperature elevations of these solutions during laser irradiation were measured with a thermocouple 8 mm away from the incident laser beam. Cell viability of murine kidney cancer cells (RENCA) was measured 24 hours following laser treatment with the previously mentioned laser parameters alone or with SWNHs. Cell viability as a function of radial position was determined qualitatively using trypan blue staining and bright field microscopy for samples exposed to heating durations of 2 and 6 minutes alone or with 0.085 mg/ml SWNHs. A Beckman Coulter Vi-Cell instrument quantified cell viability of samples treated with varying SWNH concentration (0, 0.01, 0.085, and 0.333 mg/ml) and heating durations of 0-6 minutes. RESULTS: Spectrophotometer measurements indicated inclusion of SWNHs increased light absorption and attenuation across all wavelengths. Utilizing SWNHs with laser irradiation increased temperature elevation compared to laser heating alone. Greater absorption and higher temperature elevations were observed with increasing SWNH concentration. No inherent toxicity was observed with SWNH inclusion. A more rapid and substantial viability decline was observed over time in samples exposed to SWNHs with laser treatment compared with samples experiencing laser heating or SWNH treatment alone. Samples heated for 6 minutes with 0.085 mg/ml SWNHs demonstrated increasing viability as the radial distance from the incident laser beam increased. CONCLUSIONS: The significant increases in absorption, temperature elevation, and cell death with inclusion of SWNHs in laser therapy demonstrate the potential of their use as agents for enhancing photothermal tumor destruction.

Photothermal response of tissue phantoms containing multi-walled carbon nanotubes.

Inclusion of multi-walled carbon nanotubes (MWNTs) into tissue prior to laser therapy has the potential to enhance the selectivity and effectiveness of cancer therapy by providing greater and more controlled thermal deposition. The purpose of this study was to investigate the optical and thermal response of tissue representative phantoms containing MWNTs to optical radiation. Tissue representative phantoms 20 mm in diameter and 1 mm in thickness were created from sodium alginate. Following the inclusion of MWNTs (900 nm in length, 40-60 nm in diameter) in phantoms, the distribution of MWNTs was observed using transmission electron microscopy. A predominantly, evenly dispersed and randomly oriented distribution of MWNTs was observed with a rare presence of MWNT clustering or clumping. In order to characterize the response of MWNT inclusion on optical properties of phantoms, the transmittance and reflectance spectra of phantoms with and without MWNT inclusion were measured with a spectrophotometer over a wavelength range of 200-1400 nm. Inclusion of MWNTs in phantoms dramatically enhanced light absorption across the entire wavelength range as evidenced by a diminished transmittance and reflectance compared with phantoms without MWNTs. In order to evaluate the spatiotemporal temperature distribution associated with laser irradiation of phantoms with and without MWNTs, the temperature was measured at discrete radial distances from the center of the incident laser beam using thermocouples. The rate of temperature increase and peak temperature for phantoms containing MWNTs was much greater compared with phantoms without MWNTs at all measurement locations. In conclusion, MWNT inclusion in tissue phantoms increases the optical absorption and temperature elevation, which may enable more effective photothermal therapies of human disease utilizing lasers.

Optical properties of breast tumor phantoms containing carbon nanotubes and nanohorns.

The degree by which optical properties of tumors are altered following introduction of carbon nanotubes (CNTs) of varying concentration and type is poorly understood, making it difficult to predict the impact of CNT inclusion on the photothermal response to laser therapies. Optical properties were measured of phantoms representative of breast tumor tissue incorporated with multiwalled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), and single-walled carbon nanohorns (SWNHs) of varying concentration (0.01-0.1 mg/ml). Tissue phantoms were made from sodium alginate (3 g/ml) incorporated with polystyrene microbeads (3 µm diam and 1 mg/ml) and talc-France powder (40 mg/ml). Absorption (µ(a)) and reduced scattering (µ's) coefficients of phantoms containing CNTs were determined by the inverse adding-doubling algorithm for the wavelength range of 400-1300 nm. Optical properties of phantoms without CNTs were in the range of µ(a) = 1.04-0.06 mm(-1) and µ's' = 0.05-0.07 mm(-1) at a wavelength of 900 nm, which corresponds with published data for human breast tumor tissue. Incorporating MWNTs, SWNTs, and SWNHs in phantoms with a concentration of 0.1 mg/ml increased (µ(a)) by 20- to 30-fold, 5- to 6-fold, and 9- to 14-fold, respectively, for the wavelength range of 800-1100 nm with minimal change in µ's (1.2- to 1.3-fold). Introduction of CNTs into tissue phantoms increased absorption, providing a means to enhance photothermal therapy.

Measurement of the thermal conductivity of carbon nanotube--tissue phantom composites with the hot wire probe method.

Developing combinatorial treatments involving laser irradiation and nanoparticles require an understanding of the effect of nanoparticle inclusion on tissue thermal properties, such as thermal conductivity. This information will permit a more accurate prediction of temperature distribution and tumor response following therapy, as well as provide additional information to aid in the selection of the appropriate type and concentration of nanoparticles. This study measured the thermal conductivity of tissue representative phantoms containing varying types and concentrations of carbon nanotubes (CNTs). Multi-walled carbon nanotubes (MWNTs, length of 900-1200 nm and diameter of 40-60 nm), single-walled carbon nanotubes (SWNTs, length of 900-1200 nm and diameter <2 nm), and a novel embodiment of SWNTs referred to as single-walled carbon nanohorns (SWNHs, length of 25-50 nm and diameter of 3-5 nm) of varying concentrations (0.1, 0.5, and 1.0 mg/mL) were uniformly dispersed in sodium alginate tissue representative phantoms. The thermal conductivity of phantoms containing CNTs was measured using a hot wire probe method. Increasing CNT concentration from 0 to 1.0 mg/mL caused the thermal conductivity of phantoms containing SWNTs, SWNHs, and MWNTs to increase by 24, 30, and 66%, respectively. For identical CNT concentrations, phantoms containing MWNTs possessed the highest thermal conductivity.

Photothermal response of human and murine cancer cells to multiwalled carbon nanotubes after laser irradiation.

This study demonstrates the capability of multiwalled carbon nanotubes (MWNTs) coupled with laser irradiation to enhance treatment of cancer cells through enhanced and more controlled thermal deposition, increased tumor injury, and diminished heat shock protein (HSP) expression. We also explored the potential promise of MWNTs as drug delivery agents by observing the degree of intracellular uptake of these nanoparticles. To determine the heat generation capability of MWNTs, the absorption spectra and temperature rise during heating were measured. Higher optical absorption was observed for MWNTs in water compared with water alone. For identical laser parameters, MWNT-containing samples produced a significantly greater temperature elevation compared to samples treated with laser alone. Human prostate cancer (PC3) and murine renal carcinoma (RENCA) cells were irradiated with a 1,064-nm laser with an irradiance of 15.3 W/cm(2) for 2 heating durations (1.5 and 5 minutes) alone or in combination with MWNT inclusion. Cytotoxicity and HSP expression following laser heating was used to determine the efficacy of laser treatment alone or in combination with MWNTs. No toxicity was observed for MWNTs alone. Inclusion of MWNTs dramatically decreased cell viability and HSP expression when combined with laser irradiation. MWNT cell internalization was measured using fluorescence and transmission electron microscopy following incubation of MWNTs with cells. With increasing incubation duration, a greater number of MWNTs were observed in cellular vacuoles and nuclei. These findings offer an initial proof of concept for the application of MWNTs in cancer therapy.