2.1.

Chemicals and Plasmids

The following chemicals and fluorophore probes were used: PL-PEG- $\mathrm{N}{\mathrm{H}}_2$ (Avanti Polar Lipids, Mt. Eden, AL), folate (Sigma, St. Louis, MO), fluoroscein isothiocyanate (FITC, Sigma), cell-counting kit 8 (CCK8, Dojindo Laboratories, Kumamoto, Japan), and an in situ cell death detection kit (TUNEL) (R&D Systems, Minneapolis, MN).

2.2.

CoMoCAT^® Single-Walled Carbon Nanotubes

The CoMoCAT^® method produces single-walled carbon nanotubes using a silica-supported bimetallic cobalt-molybdate catalyst.²⁰ The material is composed of a narrow distribution of nanotube types, with the (6,5) semiconducting chirality dominating and an average diameter of $0.81\mathrm{nm}$ .¹⁰ This type of nanotube possesses an intense absorption band at approximately $980\mathrm{nm}$ .¹⁰

2.3.

Absorption Spectra of the SWNT Suspension

The absorption spectra of SWNT suspension were obtained using an ultraviolet-visible (UV/VIS) spectrometer (Lambda 35, Perkin-Elmer, USA), with a $2\text{-}\mathrm{nm}$ slit width at a scan speed of $200\mathrm{nm}∕\min$ .

2.4.

Temperature Measurement during NIR Radiation of SWNTs

For ex vitro experiments, SWNT solutions (nanotube concentration of $3.5\mu \mathrm{g}∕\mathrm{mL}$ ) were irradiated by the $980\text{-}\mathrm{nm}$ laser at $1\mathrm{W}∕{\mathrm{cm}}^2$ . Temperature was measured in $10\text{-}\mathrm{s}$ intervals with a thermocouple placed inside the solution for a total of $2\min$ . The thermocouple was placed outside the path of the laser beam to avoid direct exposure of the thermocouple to the laser light. For in vivo measurement, tumors with SWNTs were irradiated by the $980\text{-}\mathrm{nm}$ laser at $1\mathrm{W}∕{\mathrm{cm}}^2$ . Surface temperatures of the tumors were measured in $30\text{-}\mathrm{s}$ intervals with an infrared thermal camera (A40, FLIR, Boston, MA) for a total of $5\min$ . All the experiments were conducted at room temperature.

2.5.

Cell culture

Murine mammary tumor line EMT6 cells were cultured in RPMI 1640 (GIBCO) supplemented with 15% fetal calf serum (FCS), penicillin $(100\text{units}∕\mathrm{mL})$ , and streptomycin $(100\mu \mathrm{g}∕\mathrm{mL})$ in 5% $\mathrm{C}{\mathrm{O}}^2$ and 95% air at $37°\mathrm{C}$ in a humidified incubator.

2.6.

SWNTs Functionalized by Various Phospholipids

Solutions of SWNTs were functionalized with one or two phospholipid-poly (ethyleneglycol) PL-PEG molecules, PL-PEG-FA, or PL-PEG-FITC, following the procedures described by Kam ⁹

2.7.

FR+ Cells and FR− Cells

EMT6 cells were cultured in FA-free RPMI-1640 medium (GIBCO). It is known that the FA-starved cells overexpress FRs on the cell surfaces. EMT6 cells were passaged for at least four rounds in the FA-free medium before use to ensure overexpression of FR on the surface of the cells (FR+ cells). FR− cells were harvested by culturing them in RPMI 1640 with abundant FA to give few available free FRs on the cell surfaces.⁹

2.8.

Tumor Model

EMT6 cells $(2\times {10}^6)$ in a $100\text{-}\mu \mathrm{l}$ solution were injected into the flank region in female Balb/c mice. Animals were used in experiments on days 7 to 10 after the inoculation of cells, when tumors were $5\text{to}7\mathrm{mm}$ in diameter.

2.9.

Laser Irradiation of Tumor Cells in Tissue Culture

Cells ( $1\times {10}^4$ per well) growing in $35\text{-}\mathrm{mm}$ Petri dishes were incubated with FA-SWNT suspension for $2\mathrm{h}$ , rinsed with phosphate buffered saline (PBS), and exposed to light at a fluence of $60\text{to}120\mathrm{J}∕{\mathrm{cm}}^2$ ( $0.5\text{to}1\mathrm{W}∕{\mathrm{cm}}^2$ for $2\min$ ). The light source was a $980\text{-}\mathrm{nm}$ semiconductor laser.

2.J.

Laser Irradiation of Mouse Tumors

SWNT or FA-SWNT was injected directly into the center of each tumor at a dose of $1\mathrm{mg}∕\mathrm{kg}$ , $6\mathrm{h}$ before illumination with the $980\text{-}\mathrm{nm}$ light (day 7 after inoculation with tumor cells). The light was delivered to the tumors on day 8 after tumor cell inoculation using a fiber optic delivery system. The power density at the illumination area, which encompassed the tumor and $0.5\text{to}1\mathrm{cm}$ of the surrounding skin, was $1\mathrm{W}∕{\mathrm{cm}}^2$ . The total light dose delivered to each tumor was $300\mathrm{J}∕{\mathrm{cm}}^2$ . During the laser irradiation, mice were anesthetized with an intraperitoneal injection of pentobarbital sodium and were restrained in a specially designed holder.

2.K.

Confocal Microscopy

The cells were imaged by a commercial laser scanning microscope (LSM 510/ConfoCor 2) combination system (Zeiss, Jena, Germany) equipped with a Plan-Neofluar $40\times ∕1.3$ NA oil differential interference contrast microscope (DIC) objective. FITC excited at $488\mathrm{nm}$ with an Ar-ion laser (reflected by a HFT $488\text{-}\mathrm{nm}$ beamsplitter HFT) and the fluorescence emission was recorded through a $500\text{to}550\text{-}\mathrm{nm}$ infrared bandpass filter.

FITC fluorescence in vivo was measured on the stage of a stereo microscope (Lumar V12, Zeiss, Jena, Germany) with a hydrargyrum lamp (HBO100) as the light source. Six hours after the SWNT-FITC or FA-SWNT-FITC injection, the mice were anesthetized with pentobarbital sodium and were restrained in a specially designed holder for imaging analysis with a fluorescence stereo microscope. The light beam through a $470∕40\mathrm{nm}$ bandpass filter was used to excite the fluorescent probes. The fluorescent emission was recorded through the $525∕50\text{-}\mathrm{nm}$ bandpass channel.

2.L.

Determination of Cell Cytotoxicity

Cell cytotoxicity assay was performed with a colorimetric tetrazolium salt-based assay. To determine the cytotoxicity of SWNTs, tumor cells ( $1\times {10}^3$ per well) were cultured in a 96-well microplate for $24\mathrm{h}$ and then incubated with SWNTs of different concentrations for $12\mathrm{h}$ , rinsed with PBS, and incubated for another $72\mathrm{h}$ in complete medium. To detect photothermal cytotoxicity, tumor cells were incubated with FA-SWNT incubation for $2\mathrm{h}$ , followed by irradiation of the $980\text{-}\mathrm{nm}$ laser at a fluence of $60\text{to}120\mathrm{J}∕{\mathrm{cm}}^2$ ( $0.5\text{to}1\mathrm{W}∕{\mathrm{cm}}^2$ for $2\min$ ). Cell cytotoxicity was assessed $12\mathrm{h}$ after the laser irradiation with CCK8. OD450, the absorbance value at $450\mathrm{nm}$ , was read with a 96-well plate reader (INFINITE M200, Tecan, Switzerland) to determine the viability of the cells.

2.M.

TUNEL Staining

Individual tumors were obtained $3\mathrm{h}$ after laser treatment and were placed immediately in Tris-buffered zinc fixative [ $0.1\mathrm{M}$ Tris HCl buffer (pH 7.4) containing $3.2\mathrm{mM}$ calcium acetate, $22.8\mathrm{mM}$ zinc acetate, and $36.7\mathrm{mM}$ zinc chloride] for $6\text{to}18\mathrm{h}$ , transferred to 70% ethanol, dehydrated, and embedded in paraffin. Several cryostat sections, $10\text{-}\mu \mathrm{m}$ thick, were cut from each tumor. DNA fragmentation of some tumor samples was detected by the terminal deoxynucleotide transferase-based, in situ cell death detection kit (TUNEL) according to the manufacturer’s instructions. Biotinylated nucleotides incorporated into the DNA fragments were detected using a streptavidin-fluoresceine conjugate, which was excited with the $488\text{-}\mathrm{nm}$ line of an Ar laser. Fluorescence was recorded using a $500\text{to}550\text{-}\mathrm{nm}$ bandpass filter.

3.1.

Absorption of Single-Walled Carbon Nanotubes Solubilized in PEG

A stable SWNT-PEG suspension was obtained after the final centrifugation of the solution. The NIR absorption spectra of SWNT-PEG exhibited a strong band at approximately $980\mathrm{nm}$ , significantly higher than the water absorption in the same region [Fig. 1a and 1b ], which is typical for CoMoCAT^® samples.¹⁰ To detect the effects of $980\text{-}\mathrm{nm}$ optical excitation of SWNTs, we carried out a control experiment by radiating an aqueous solution of SWNTs ex vitro. We observed that irradiation of a SWNT solution (nanotube concentration of $3.5\mu \mathrm{g}∕\mathrm{mL}$ ) by a $1\text{-}\mathrm{W}∕{\mathrm{cm}}^2$ $\left(980\text{-}\mathrm{nm}\right)$ laser for $2\min$ caused its temperature to elevate up to about $70°\mathrm{C}$ . However, the aqueous solution without SWNTs caused a temperature elevation to $55°\mathrm{C}$ [Fig. 1c]. These findings clearly demonstrat the enhanced absorption of the $980\text{-}\mathrm{nm}$ light by the SWNTs.

Fig. 1

Absorption spectra of CoMoCAT^® single-walled carbon nanotubes solubilized in PEG. (a) UV-vis-NIR absorption spectra of the SWNTs suspension and water. (b) Absorbance of SWNT suspension of different concentrations at $980\mathrm{nm}$ (optical $\text{path}=0.5\mathrm{cm}$ ). The solid line is a linear fit. (c) Temperature measurements of an ex vitro control experiment using a SWNT solution $(3.5\mu \mathrm{g}∕\mathrm{mL})$ and water during irradiation by a $980\text{-}\mathrm{nm}$ laser at $1\mathrm{W}∕{\mathrm{cm}}^2$ for $2\min$ at room temperature. The results demonstrated the enhanced laser energy absorption by the SWNTs. (d) Cytotoxicity of SWNTs. Cells were incubated in the SWNT solution of different concentrations $(1.75\mu \mathrm{g}∕\mathrm{mL}\text{to}7\mu \mathrm{g}∕\mathrm{mL})$ for $12\mathrm{h}$ , then washed and incubated in complete medium for $72\mathrm{h}$ before assessing cell viability. Cells not incubated with SWNTs were used as control. Bars (means+SD, $\mathrm{n}=4$ ).

3.2.

Cytotoxicity of Single-Walled Carbon Nanotubes Solubilized in PEG

We investigated the cytotoxic effects of SWNTs using EMT6 tumor cells with CCK8 assay. EMT6 cells were cultured for $12\mathrm{h}$ with SWNTs of different concentrations (from $1.75\mu \mathrm{g}∕\mathrm{mL}\text{to}7.5\mu \mathrm{g}∕\mathrm{mL}$ ), washed, and then incubated for another $72\mathrm{h}$ in complete medium before detection. No cytotoxicity was observed, as shown in Fig. 1d.

3.3.

Selective Targeting of Cancer Cells by FA-SWNT

We use folate to determine whether SWNTs could be selectively internalized into cancer cells with specific tumor markers. To exploit this system, we obtained highly water-soluble individualized SWNTs noncovalently functionalized by PL-PEG-FA [Fig. 2a ].⁹ The FR-positive EMT6 cells (FR+ cells) with overexpressed FRs on the cell surfaces and the FR− cells without surface FRs were used (see Sec 2). Both FR+ and FR− cells were exposed to FA-SWNT for $2\mathrm{h}$ , washed, and then irradiated by a $980\text{-}\mathrm{nm}$ laser $(0.5\mathrm{W}∕{\mathrm{cm}}^2)$ for $2\min$ . After the irradiation, extensive cell death was observed in the FR+ cells, evidenced by drastic cell morphology changes [Fig. 2b, upper panel], whereas the FR− cells remained intact [Fig. 2b, lower panel]. These results show that FR+ cancer cells could selectively internalize FA-SWNT and could be selectively destroyed by irradiation of the $980\text{-}\mathrm{nm}$ laser light.

Fig. 2

Selective targeting and killing of cancer cells. (a) Chemical structure of PL-PEG-FA and PL-PEG-FITC synthesized by conjugating PL-PEG- $\mathrm{N}{\mathrm{H}}_2$ with FA or FITC, respectively, for solubilizing individual SWNTs. (b) Upper panel: Confocal images of EMT6 cells of FR+ or FR− after incubation in a solution of SWNTs with two cargoes (PL-PEG-FA and PL-PEG-FITC). The strong green FITC fluorescence inside the FR+ cells confirms the SWNT uptake by FA and FITC cargoes. The small amount of green fluorescence inside the FR− cells, confirmed the lack of SWNT uptake by FA and FITC cargoes $(\times 40)$ . (Inset) High-magnification images show the details of fluorescence in the cells. Lower panel: Images of dead FR+ cells with rounded cell morphology and unharmed FR− cells after irradiation ( $980\text{-}\mathrm{nm}$ laser radiation at $0.5\mathrm{W}∕{\mathrm{cm}}^2$ for $2\min$ ). (Inset) High-magnification images show the details of the dead cells (FR+) and the live cells (FR−). (Color online only.)

3.4.

Laser Treatment of Tumor Cells with Different Concentrations of FA-SWNT—In Vitro Results

We used CCK 8 assay investigate the effects of SWNTs on tumor cells during laser treatment. The tumor cells were incubated with FA-SWNT solution for $2\mathrm{h}$ , followed by irradiation with a $980\text{-}\mathrm{nm}$ laser. The tumor cytotoxicity depended on both the SWNT concentration and the laser dose [Fig. 3a ]. At a fluence of $60\mathrm{J}∕{\mathrm{cm}}^2$ , tumor cells treated by the laser without the presence of FA-SWNT remained essentially intact, while noticeable photothermal cellular cytotoxicity was observed with the presence of FA-SWNT. With a higher fluence $(120\mathrm{J}∕{\mathrm{cm}}^2)$ , the laser energy alone resulted in a marked cytotoxicity (37%). However, FA-SWNT significantly enhanced the photocytotoxicity. The FA-SWNT at $1.75\mu \mathrm{g}∕\mathrm{mL}$ and $3.5\mu \mathrm{g}∕\mathrm{mL}$ yielded cytotoxicity rates of 60% and 85% at $120\mathrm{J}∕{\mathrm{cm}}^2$ , as shown in Fig. 3a. Figure 3b shows the effective destruction of tumor cells using laser irradiation $(120\mathrm{J}∕{\mathrm{cm}}^2)$ with and without FA-SWNT (at a dose of $3.5\mu \mathrm{g}∕\mathrm{mL}$ ). Apparently, both the FA-SWNT concentration and the laser dose are controlling factors for thermally induced cytotoxicity.

Fig. 3

Viability of tumor cells under different treatments. (a) Cells tumor cells treated with laser only or with laser-FA-SWNT. The treated cells were incubated in complete medium for $12\mathrm{h}$ before assessing cell viability. Cells without treatment were used as control. Bars (means+SD, $\mathrm{n}=4$ ). (b) Optical images of tumor cells under different treatments as indicated.

3.5.

Laser Treatment of Mouse Tumors with FA-SWNT—In Vivo Results

We used the mammary tumor model with EMT6 cells in the female Balb/c mice to investigate the in vivo effects of FA-SWNT. The mouse tumors with or without FA-SWNT were treated by the $980\text{-}\mathrm{nm}$ laser. To determine the effects of NIR optical excitation of SWNTs inside tumors, we measured the temperature on the tumor surface during the irradiation by the $980\text{-}\mathrm{nm}$ laser with an infrared thermal camera. In one experimental mouse, irradiation of tumors with a power density of $1\mathrm{W}∕{\mathrm{cm}}^2$ with FA-SWNT $(1\mathrm{mg}∕\mathrm{kg})$ for $5\min$ caused a surface temperature elevation of $63°\mathrm{C}$ [Fig. 4a ]. Without FA-SWNT, the tumor irradiated at the same light dose caused a surface temperature elevation of $54°\mathrm{C}$ [Fig. 4a]. Experiments with other animals yielded similar results. These findings clearly show that FA-SWNT could effectively enhance the tumor photothermal therapy.

Fig. 4

Laser treatment induced cell death in vivo. (a) Temperature on the surface of a mouse tumor during irradiation by the $980\text{-}\mathrm{nm}$ laser at $1\mathrm{W}∕{\mathrm{cm}}^2$ for $5\min$ with and without FA-SWNT $(1\mathrm{mg}∕\mathrm{kg})$ . This result clearly demonstrated the selective thermal effect caused by SWNT absorption of $980\text{-}\mathrm{nm}$ light in the tumor. (b) Fluorescent emissions of FITC from a mouse tumor sample and normal tissue around the tumor, $6\mathrm{h}$ after injection, observed with a fluorescence stereo microscope. The normal tissue surrounding the tumor injected with SWNT-FITC shows more intense fluorescent emission than that surrounding the tumor injected with FA-SWNT-FITC. The control sample was injected with PBS. These results indicated that FA-SWNT has higher tumor selectivity than SWNT. (c) Images of tumor tissue (upper panel) and normal tissue (lower panel) sections after different treatments. TUNEL staining analysis of tissue sections from a mouse was performed $3\mathrm{h}$ after treatment. Samples were selected from the tumor and the normal tissue within the laser beam but $0.5\mathrm{cm}$ away from the tumor. This result clearly demonstrated that FA-SWNT enhanced the thermal damages of the tumor while contributing limited damage to the normal tissue around the tumors. (d) Cell death rates of the tumor section, after different treatments (100 cells/group $\times 5$ ). (e) Cell death rates of the normal section after different treatments (100 cells/group $\times 5$ ).

To investigate the SWNT distribution in tumors, we directly injected SWNT-FITC or FA-SWNT-FITC solution into the tumors, and the target tumors were observed with a fluorescence stereo microscope $6\mathrm{h}$ after the injection. The fluorescence of FA-SWNT was detected inside the tumors, but not in the normal tissue around the tumors [Fig. 4b]. However, the fluorescence of SWNT was detected in both the injected tumors and in surrounding normal tissue [Fig. 4b]. This indicates that the FA-SWNT has a greater higher affinity with tumor cells due to the tumor-specific antibodies, while SWNT alone could easily reach the surrounding tissue through intracellular diffusion.

To investigate the therapy effect of SWNT on tumors during laser treatment, we examined tissue sections from different samples by TUNEL staining. The tumors were directly injected with SWNT or FA-SWNT solution for $6\mathrm{h}$ , followed by irradiation with a $980\text{-}\mathrm{nm}$ laser ( $1\mathrm{W}∕{\mathrm{cm}}^2$ for $5\min$ ). The tissue sections were obtained $3\mathrm{h}$ after treatment. TUNEL staining showed morphologically the treatment-induced cell death at different levels under different treatments. Tumors treated by the laser alone resulted in a marked cell death rate (56%), while a more significant cell death rate was observed with the presence of SWNT (78%) and FA-SWNT (88%), as shown in Figs. 4c and 4d.

For the tissue section selected from normal tissue within the laser beam but about $0.5\mathrm{cm}$ away from the tumor, laser energy alone resulted in a low cell death rate (16%), as shown in Fig. 4e, with SWNT enhancement, the cell death rate of normal tissue around the tumor was about 36%. However, the FA-SWNT administered to the mouse showed no impact on the normal tissue around the tumors with a cell death rate of 17.5%, similar to that of laser-only treatment, as shown in Figs. 4c and 4e. These results clearly indicate that FA-SWNT could specifically target the tumor cells and enhance thermal damages to the target cells while not affecting normal tissues around the tumor.