2.1.

Cell Lines

P388D1 murine lymphocytic monocytes (Ma) (ATCC# CCL-46) and human hypopharyngeal squamous carcinoma cells (FaDu) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Corp., Carlsbad, California) supplemented with 10% fetal bovine serum, 25 mM HEPES buffer ($\mathrm{pH}\approx 7.4$), $100\mathrm{U}/\mathrm{mL}$ of penicillin, and $100\mathrm{\mu g}/\mathrm{mL}$ of streptomycin. FaDu is an adherent cell line and was maintained as a monolayer, while the Ma line was grown in suspension. Both cell lines were maintained under physiological conditions in an incubator (37°C, 5.0% ${\mathrm{CO}}_2$, and 95% humidity).

2.2.

Nanoparticles

AuNS (AuroShell^™) were purchased from Nanospectra Biosciences Inc. (Houston, Texas). NS were coated with polyethylene glycol (PEG) in order to prevent aggregation. AuNS were supplied as a suspension in deionized water ($2.82\times {10}^{11}\text{particles}/\mathrm{mL}$) and consisted of a 120-nm-diameter silica core encased in a gold shell of 15 nm thickness giving a total particle diameter of 150 nm.

2.3.

${\mathit{Ma}}^{\mathit{NS}}$ Incubation

We seeded $5\times {10}^6$ P388D1 Ma in 35 mm cell culture dishes in 2 mL of culture medium. The dishes were incubated overnight to allow the cells to settle. Culture medium was exchanged for $100\mu \mathrm{l}$ of PEGylated ($2.8\times {10}^{11}\text{particles}/\mathrm{mL}$) AuNS colloid in 1.9 mL of culture medium. The $\mathrm{Ma}+\mathrm{AuNS}$ were incubated for 24 h at 37°C and then treated with mitomycin C for 1 h to prevent cell division. After the Ma had ingested the AuNS, they no longer remained in suspension culture and strongly adhered to the plastic surface. This was in contrast to their normal suspension culturing. The ${\mathrm{Ma}}^{\mathrm{NS}}$ were washed three times with Hanks’ balanced salt solution with calcium chloride and magnesium chloride (Gibco, Carlsbad, California) to wash away the excess of noningested NS and mitomycin C residues. ${\mathrm{Ma}}^{\mathrm{NS}}$ were then detached with trypsin and a rubber spatula, washed, and counted.

The concentration of AuNS in the Ma was studied using a UV-Vis-NIR spectrophotometer (Varian UV-Vis-NIR spectrophotometer Cary 6000i, Varian) as previously described in detail.²⁴ Briefly, in order to calculate the % uptake of AuNS in Ma, reference solutions containing AuNS in the absence of Ma were used. Consequently, Ma incubated under similar conditions but in the absence of AuNS was similarly treated, and their absorbance value subtracted from those of the ${\mathrm{Ma}}^{\mathrm{NS}}$ groups to give the pure absorbance of the incorporated nanoparticles. % uptake of AuNS was calculated by the absorbance of incorporated AuNS at $\lambda =819\mathrm{nm}$ divided by the absorption of the AuNS alone $\times 100$.

2.4.

Two-Photon Microscopy

We plated out $1\times {10}^3$ empty Ma or ${\mathrm{Ma}}^{\mathrm{NS}}$ on 35-mm glass-bottomed imaging dishes. The cells were incubated overnight in clear buffer. Two-photon micrographs were generated using an inverted Zeiss laser-scanning microscope (LSM 410, Carl Zeiss, Jena, Germany). This system allows the differential visualization of cells and nanoparticles using confocal and two-photon microscopy. The reflectance from the ingested AuNS was pseudo-colored green to allow their identification.

2.5.

Cell Proliferation Assay

The CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, Wisconsin) was used for determining the number of viable cells. Following treatment, MTS reagent ($30\mu \mathrm{L}$) was added to each well (96-well plate) containing cells and $150\mu \mathrm{L}$ DMEM. The plates were incubated for 1 h and transferred to an Infinite^® M1000 PRO microplate reader (TECAN Group Ltd., Mannedorf, Germany) for absorbance reading at 490 nm. MTS values obtained from methanol treated cultures (100% dead) were considered background and subtracted from all experimental results. The absorption at 490 nm, due to the presence of AuNS and dead cells, was therefore compensated for.

2.6.

Drug Toxicity

FaDu cells and ${\mathrm{Ma}}^{\mathrm{NS}}$ ($2\times {10}^3$ FaDu and $1\times {10}^3$ ${\mathrm{Ma}}^{\mathrm{NS}}$) were pipetted into the wells of a 96-well plate. A total of three well plates were prepared, one for each chemotherapeutic drug. Drugs were added to DMEM medium to obtain the following concentrations: 0.1 to $10\mathrm{\mu g}{\mathrm{mL}}^{-1}$ of bleomycin, 0.001 to $0.05\mathrm{\mu g}{\mathrm{mL}}^{-1}$ of doxorubicin, and 1 to $75\mathrm{\mu g}{\mathrm{mL}}^{-1}$ of cisplatin. In all cases, cell suspensions and chemotherapeutic agents were incubated for five days at 37°C, after which time MTS reagent ($30\mu \mathrm{L}$) was added to each well and the incubation continued for an additional 60 min. The MTS assay was read in a microplate reader (ELx Bio-Tek Instruments). The pharmacological potency resulting in $\sim 70$ to 90% survival was used in subsequent combined therapy studies.

2.7.

Moderate Photothermal Therapy Toxicity

A fiber-coupled diode laser (Intense, North Brunswick, New Jersey) was used to irradiate cells at a wavelength of 810 nm. Each well was irradiated separately using an irradiance of 7, 14, or $28\mathrm{W}{\mathrm{cm}}^{-2}$ for 7.5 min. Additionally, in order to establish the time to reach temperature equilibrium, three irradiation times were investigated (5, 7.5, and 10 min) using a laser irradiance of $7\mathrm{W}{\mathrm{cm}}^{-2}$ corresponding to radiant exposures of 2.10, 3.15, and $4.20\mathrm{kJ}{\mathrm{cm}}^{-2}$, respectively. The radiant exposure resulting in $\sim 90\%$ survival was used in subsequent combined therapy studies. All irradiations were performed at a constant temperature of 37°C. After irradiation, the plates were incubated for five days. Following incubation, MTS assays were performed as previously described. The experiments were performed in triplicate.

2.8.

Combined Chemotherapy and Moderate Photothermal Therapy

The setup for combined chemotherapy and NIR light treatment is shown in Fig. 1. FaDu and ${\mathrm{Ma}}^{\mathrm{NS}}$ were combined at a ratio of $\mathrm{FaDu}:{\mathrm{Ma}}^{\mathrm{NS}}$ of 2:1. Based on the toxicity results for each of the three drugs, concentrations resulting in 70 to 90% survival were chosen. Therefore, $5\mu \mathrm{g}{\mathrm{mL}}^{-1}$ bleomycin, $10\mathrm{\mu g}{\mathrm{mL}}^{-1}$ cisplatin, and $0.005\mu \mathrm{g}{\mathrm{mL}}^{-1}$ doxorubicin were added to the combined FaDu cells and Ma in the 96-well plates prior to irradiation. Each photothermal-treated well was irradiated separately using an irradiance of $7\mathrm{W}{\mathrm{cm}}^{-2}$ (8 mm beam diameter) for 7.5 min resulting in a radiant exposure of $3.15\mathrm{kJ}{\mathrm{cm}}^{-2}$. Otherwise, the irradiation protocol using the 810 nm fiber-coupled diode laser was identical to that previously described in Sec. 2.7. Following treatment, the plates were incubated for five days and MTS assays were performed.

Fig. 1

Basic concept of combined drug and ${\mathrm{Ma}}^{\mathrm{NS}}$ mediated MPTT: 1, Ma incubated with AuNS for 24 h; 2, FaDu tumor cells combined with ${\mathrm{Ma}}^{\mathrm{NS}}$ for 24 h; 3, addition of chemotherapeutic agent; 4, MPTT: NIR laser irradiation ($\lambda =810\mathrm{nm}$; 7.5 min exposure; $7\mathrm{W}{\mathrm{cm}}^{-2}$ irradiance); 5, MTS viability assay.

2.9.

Statistical Analysis

Data analysis was performed in Microsoft Excel. All data were normalized to the control group of each individual experiment and all error bars represent standard deviations. GraphPad QuickCalcs (GraphPad Software, Inc., La Jolla, CA) was used for Student’s $t$ test; $p$ values less than 0.05 were considered significant. In order to determine the degree of interaction between MPTT and the chemo-agents, the following equation was used:²⁸

The numerator includes the product of the surviving fraction (SF) of the individual treatments separately and the denominator includes the SF of the combined treatments. A value of $\alpha =1$ indicates an additive effect, while values of $\alpha <1$ or $\alpha >1$ indicate antagonistic or synergistic effect, respectively.

3.1.

Macrophages Endocytosis of Polyethylene Glycolylated Gold Nanoshell

We incubated $5\times {10}^6$ P388D1 cells with $1.4\times {10}^{11}$ PEGylated AuNS for 24 h. Ma showed significant uptake of AuNS as measured by UV/Vis spectrophotometry at $\lambda =819\mathrm{nm}$. The % uptake of PEGylated AuNS in the Ma was 4% corresponding to approximately $(0.8\text{to}1.2)\times {10}^3\mathrm{NS}/\mathrm{Ma}$. The uncertainty in AuNS concentration was due to cell division during incubation and was estimated based on the doubling time of the Ma cell line (32 h).²⁹ Aggregates of AuNS in the cytoplasm of Ma could be visualized with two-photon microscopy for empty [Figs. 2(a) and 2(b)] and loaded [Figs. 2(c) and 2(d)] Ma. In previous reports, TEM images of ${\mathrm{Ma}}^{\mathrm{NS}}$ revealed extensive intracellular presence of the nanoparticles, in small aggregates, inside vacuoles, which were dispersed throughout the cellular cytoplasm.^23,24

Fig. 2

Two-photon fluorescence images of Ma: (a) and (b) empty Ma, and (c) and (d) ${\mathrm{Ma}}^{\mathrm{NS}}$. The scale bar in (a) denotes $6\mu \mathrm{m}$. Nanoshell aggregates inside Ma are shown by green reflectance dispersed throughout the cell.

3.2.

Drug Toxicity

The bleomycin titration results [Fig. 3(a)] show that the ${\mathrm{LD}}_{50}$ is in the range of 5 to $10\mathrm{\mu g}{\mathrm{mL}}^{-1}$. As shown in Fig. 3(b), doxorubicin was the most potent of the three drugs tested (${\mathrm{LD}}_{50}\sim 0.01\mu \mathrm{g}{\mathrm{mL}}^{-1}$), while cisplatin (${\mathrm{LD}}_{50}\sim 25\mu \mathrm{g}{\mathrm{mL}}^{-1}$) was the least toxic [Fig. 3(c)]. Based on the previously established criterion of 70 to 90% survival, the results in Figs. 3(a)–3(c) suggest that doxorubicin, bleomycin, and cisplatin, concentrations of 0.05, 5, and $10\mu \mathrm{g}{\mathrm{mL}}^{-1}$, respectively, should be used in the combined treatments.

Fig. 3

Effects of drug on cell survival. Dose response curves for (a) bleomycin, (b) doxorubicin, and (c) cisplatin. In all cases, the surviving fraction was normalized to the no drug controls. Each data point represents $\text{mean}\pm \text{standard deviation}$ of three trials.

3.3.

Photothermal Toxicity

The effects of increasing NIR laser irradiance on cell viability were determined for 7, 14, and $28\mathrm{W}{\mathrm{cm}}^{-2}$. Irradiation time was 7.5 min and the $\mathrm{FaDu}:{\mathrm{Ma}}^{\mathrm{NS}}$ ratio was 2:1. As illustrated in Fig. 4(a), $7\mathrm{W}{\mathrm{cm}}^{-2}$ irradiance had limited effect on cell viability (89% survival) compared to nontreated controls. At irradiances of 14 or $28\mathrm{W}{\mathrm{cm}}^{-2}$, significant MPTT toxicity was observed: 71 and 20%, respectively. The effects of MPTT treatment times at $7\mathrm{W}{\mathrm{cm}}^{-2}$ were also investigated [Fig. 3(b)]. Irradiation times of 5 min resulted in minimal cell toxicity, while 7.5 and 10 min irradiations produced 89% cell survival. The observation that there was no increase in cell lethality between 7.5 and 10 min irradiation times indicated that the cell hybrid cultures had achieved a thermal steady state. Based on these results, an irradiation time of 7.5 min and an irradiance of $7\mathrm{W}{\mathrm{cm}}^{-2}$ were chosen for the combined MPTT and chemotherapy studies.

Fig. 4

Surviving fractions as a function of irradiance and irradiation time. In all cases, the $\mathrm{FaDu}:{\mathrm{Ma}}^{\mathrm{NS}}$ ratio was 2:1 and cells were irradiated with $\lambda =810\mathrm{nm}$ light. (a) Cells were irradiated for 7.5 min and (b) irradiance of $7\mathrm{W}{\mathrm{cm}}^{-2}$ was used. Each data point represents $\text{mean}\pm \text{standard deviation}$ of three trials.

3.4.

Combined Chemotherapy and Moderate Photothermal Therapy Induced Hyperthermia

In order to investigate the degree of interaction between the chemotherapeutic agents and NIR-induced MHT, suboptimal levels of both drug concentrations and MPTT levels, as determined in the above experiments, were used. The effects on drug efficacy of Ma-mediated MPTT MHT for all three drugs are shown in Fig. 5. The cell survival values for controls (drug-only) were compared to the results obtained by MPTT-induced MHT. Student’s $t$ tests were used to verify significant differences between these values and are indicated in Fig. 5 by an asterisk (*). When cell survival was compared to drug-only controls, there was a significant decrease in cell survival for all three drugs by MPTT-induced MHT ($p<0.001$). The corresponding alpha values, calculated from Eq. (1), are shown in Table 1 and indicate additive effects for the combined MPTT/doxorubicin and the combined MPTT/bleomycin groups, with a synergistic effect noted for the combined MPTT/cisplatin treatment.

Fig. 5

Combined chemotherapy and MPTT. Student’s $t$ tests were used to verify significant differences between controls and MPTT-induced MHT. Significant differences ($p<0.05$) are denoted by asterisks. The surviving fraction was normalized to the no-treatment controls. Each data point corresponds to the $\text{mean}\pm \text{standard deviation}$ of three trials.

Table 1

Degree of interaction between NIR-induced MHT and chemotherapy.