Copper Selenide Nanocrystals for Photothermal Therapy

Abstract

Ligand-stabilized copper selenide (Cu_2−xSe) nanocrystals, approximately 16 nm in diameter, were synthesized by a colloidal hot injection method and coated with amphiphilic polymer. The nanocrystals readily disperse in water and exhibit strong near infrared (NIR) optical absorption with a high molar extinction coefficient of 7.7 × 10⁷ cm⁻¹ M⁻¹ at 980 nm. When excited with 800 nm light, the Cu_2−xSe nanocrystals produce significant photothermal heating with a photothermal transduction efficiency of 22%, comparable to nanorods and nanoshells of gold (Au). In vitro photothermal heating of Cu_2−xSe nanocrystals in the presence of human colorectal cancer cell (HCT-116) led to cell destruction after 5 minutes of laser irradiation at 33 W/cm², demonstrating the viabilitiy of Cu_2−xSe nanocrystals for photothermal therapy applications.

There is growing interest in combating cancer with nanoparticle-based therapeutics.¹ Photoinduced heating of nanoparticles using near infrared (NIR) light to destroy cancer cells has been shown to be a potentially effective way to target cell death without damaging surrounding healthy tissue.^2–4 The nanoparticles should be smaller than about 50 nm or so, non-toxic, and have surfaces that can be functionalized with cell recognition moieties. Furthermore, the nanoparticles should respond strongly to light excitation with wavelengths in the range of 650 to 950 nm, due to the high transparency of tissue, blood, and water in this range of wavelengths.⁵

Gold (Au) nanoshells,⁶ nanorods,⁷ and nanocages,^{8, 9} have high optical extinction coefficients in the NIR wavelength range with size- and shape-tunable surface plasmon resonance (SPR) bands that can be photoexcited to generate considerable heat. Therefore, they have been widely studied for optical diagnostic imaging and photothermal therapy. Au nanorods and nanoshells, however, can be considerably large—Au nanorods are typically on the order of ~10 nm in diameter and ~ 50 nm in length,¹⁰ and Au nanoshells are more than 100 nm in diameter.^11–13 The optimum intravenously administered nanoparticles should be between 10 and 50 nm in diameter to increase blood stream circulation time,^14–16 as larger nanoparticles are removed by the reticuloendothelial system, primarily by the liver and spleen, and smaller particles by the renal system.^17–19 Furthermore, the cetyltrimethylammonium bromide (CTAB) coating on Au nanorods is cytotoxic and not easily removed without losing the integrity of the material.¹²

Other nanomaterials including germanium nanocrystals,²⁰ porous silicon,²¹ graphene flakes,²² carbon nanotubes,^{4, 23} copper(II) sulfide nanocrystals,²⁴ and graphitic carbon coated iron cobalt nanocrystals²⁵ have also been shown to generate sufficient photothermal heating by NIR optical illumination to destroy cancer cells. Nonetheless, there is still a need for photothermal nanoparticles in the right size range and adequate cell biocompatibility. Copper-based semiconductors have recently gained recognition as biocompatible alternatives to Cd-containing contrast agents (e.g. CdS, CdSe, CdTe) for in vivo cancer imaging applications.^24,26 CuInS₂@ZnS and CuInSe₂@ZnS core-shell nanocrystals have shown bioimaging efficacy comparable to the Cd-based materials, without the toxicity associated with Cd,^27–29 and recently copper (II) sulfide nanocrystals were explored for photothermal therapy, although with reportedly limited photothermal transduction efficiency.²⁴ In this Letter, we show that Cu_2−xSe nanocrystals are an effective photothermal material, exhibiting marked photo-induced heating when excited by NIR light at 800 nm and similar photothermal transduction efficiency as Au nanorods and Au nanoshells. Human colorectal cancer cell death is also observed by in situ laser-induced photothermal heating of Cu_2−xSe nanocrystals.

Cu_2−xSe nanocrystals were synthesized by arrested precipitation in a hot organic solvent and coated with amphiphilic polymer to be rendered hydrophilic and compatible with biological systems. Based on a modification of methods developed for CuInS₂ and Cu(InxGa_1−x)Se₂ nanocrystals,^{30, 31} two hot reactant solutions of copper chloride and selenourea in oleylamine are combined to form a dark green colloidal dispersion of oleylamine-capped Cu_2−xSe nanocrystals, as in Figure 1 (see Supporting Information for experimental details). Transmission electron microscopy (TEM) images show that the nanocrystals are predominantly spherical in shape with crystalline cores of an average diameter of 16±1 nm. The Cu_2−xSe nanocrystals typically had copper selenide, photothermal therapy (PPT), plasmon resonance, colloidal nanocrystals, amphiphilic polymer, cancer therapy, hyperthermia, gold nanoshells, gold nanorods, photothermal transduction efficiency

Figure 1.

(A) Reaction scheme and a photograph of oleylamine-capped Cu_2−xSe nanocrystals dispersed in chloroform; (B) TEM images of copper selenide nanocrystals. The high resolution TEM image in the inset of (B) shows the crystalline Cu_2−xSe core of the nanocrystals. The average diameter of the nanocrystals is 16±1 nm.

There is growing interest in combating cancer with nanoparticle-based therapeutics.¹ Photoinduced heating of nanoparticles using near infrared (NIR) light to destroy cancer cells has been shown to be a potentially effective way to target cell death without damaging surrounding healthy tissue.^2–4 The nanoparticles should be smaller than about 50 nm or so, non-toxic, and have surfaces that can be functionalized with cell recognition moieties. Furthermore, the nanoparticles should respond strongly to light excitation with wavelengths in the range of 650 to 950 nm, due to the high transparency of tissue, blood, and water in this range of wavelengths.⁵

Gold (Au) nanoshells,⁶ nanorods,⁷ and nanocages,^{8, 9} have high optical extinction coefficients in the NIR wavelength range with size- and shape-tunable surface plasmon resonance (SPR) bands that can be photoexcited to generate considerable heat. Therefore, they have been widely studied for optical diagnostic imaging and photothermal therapy. Au nanorods and nanoshells, however, can be considerably large—Au nanorods are typically on the order of ~10 nm in diameter and ~ 50 nm in length,¹⁰ and Au nanoshells are more than 100 nm in diameter.^11–13 The optimum intravenously administered nanoparticles should be between 10 and 50 nm in diameter to increase blood stream circulation time,^14–16 as larger nanoparticles are removed by the reticuloendothelial system, primarily by the liver and spleen, and smaller particles by the renal system.^17–19 Furthermore, the cetyltrimethylammonium bromide (CTAB) coating on Au nanorods is cytotoxic and not easily removed without losing the integrity of the material.¹²

The hydrophobic oleylamine-capped nanocrystals were coated with amphiphilic polymer composed of a poly(maleic anhydride) backbone with hydrophilic carboxylic acid groups and hydrophobic oleylamine side chains (Figure 2) using techniques described previously.³³ The polymer forms micelles in water, which encapsulate the nanocrystals. The amphiphilic polymer coating enables dispersion of the nanocrystals in aqueous media under physiological conditions i.e., phosphate buffered saline (PBS) at pH 7.4 and 150 mM,³³ and the distal carboxyl groups can be further functionalized with cellular targeting biomolecules such as proteins or antibodies.^{34, 35} With the polymer coating, the Cu_2−xSe nanocrystals have an average hydrodynamic diameter and zeta potential of 39 nm and −40 mV, respectively, which makes them well-suited for in vivo medical applications.

Figure 2.

Amphiphilic polymer encapsulation of Cu_2−xSe nanocrystals. Combining oleylamine passivated Cu_2−xSe nanocrystals and the amphiphilic poly(maleic anhydride)-based polymer leads to encapsulation of the Cu_2−xSe nanocrystals with a hydrophilic exterior. The distal carboxyl groups on the surface facilitate dispersibility in aqueous media.

Figure 3 shows room temperature UV-vis-NIR absorbance spectra of Cu_2−xSe nanocrystals dispersed in water. There is a broad absorbance peak centered at 970 nm with monotonically rising absorbance at wavelengths below 500 nm. Similar absorbance features were observed by Manna and coworkers³² and Garcia and coworkers³⁶ and were assigned to direct and indirect interband transitions for Cu_2−xSe. Bulk Cu_2−xSe has direct and indirect band gap energies of 2.1 to 2.3 eV (540 to 590 nm) and 1.2 to 1.4 eV (1030 to 880 nm), respectively. We also assign the low wavelength (less than approximately 600 nm) absorbance to interband optical transitions; however, we assign the NIR absorbance peak to a surface plasmon resonance. Cu_2−xSe is a p-type semiconductor with a relatively high carrier (holes) concentration and exhibits strong free carrier absorption,³⁷ which in the case of the Cu_2−xSe nanocrystals results in a surface plasmon resonance. Nanocrystals of analogous non-stoichiometric copper sulfides (Cu_2−xS)—in which Cu deficiencies also lead to high densities of holes—have also exhibited NIR absorbance peaks.^38–40 The NIR absorption band from Cu_2−xS was also originally assigned to an indirect interband transition;^{38, 39} however, Burda and coworkers have clarified using Drude theory that the NIR absorption is actually a surface plasmon resonance.⁴⁰ Very recently, Luther and coworkers revealed that Cu_2−xS nanocrystals with vacancy concentrations of ~10²¹ cm⁻³ exhibit surface plasmon resonance bands, thus confiming that substoichiometric copper (I) chalcogenides have absorption characteristics similar to those of metals.⁴¹ The high molar extinction coefficients measured for the Cu_2−xSe nanocrystals (with a value of 7.7 × 10⁷ M⁻¹cm⁻¹ (at 970 nm)) are also consistent with plasmon absorption,⁴¹ and are orders of magnitude higher than expected for an indirect optical transition and considerably higher than strongly absorbing organic dyes, direct bandgap semiconductor quantum dots (See Table 1).

Figure 3.

Absorbance (dotted line) and molar extinction coefficient (solid line) for Cu_2−xSe nanocrystals plotted against wavelength. The absorbance spectrum of polymer coated Cu_2−xSe nanocrystals in water (37 mg/L) reaches a maximum at 970 nm. The molar extinction coefficient was calculated experimentally using Cu_2−xSe solutions of chloroform, and is given per mole of 16 nm Cu_2−xSe nanocrystals (see Supporting Information for calculations). The molar extinction coefficient reaches a maximum of 7.7×10⁷ M⁻¹ cm⁻¹ at 970 nm.

Table 1.

Molar extinction coefficients (per mol of molecules or nanocrystals) of common photoabsorbers, including molecular dyes, direct bandgap semiconductors, and reported photothermal materials.

Photoabsorber	Dimension (nm)	Molar Extinction Coefficient (M−1 cm−1)	Wavelength (nm)
Rhodamine 6G42	Molecular	1.2 × 105	530
Malachite Green42	Molecular	1.5 × 105	617
CdX (X = S, Se, Te)43	r = 2	~ 2 − 5 × 105	At excitonic maximum
Carbon Nanotubes4	r = 0.6, L = 150	7.9 × 106	808
Copper Selenide	r = 8	7.7 × 107	970
Gold Nanospheres44	r = 20	~7.7 × 109	530
Gold Nanorods45	r = 5, L = 27	1.9 × 109	650
Gold Nanoshells46	r1 = 55, r2 = 65	~2 × 1011	800

A significant amount of heat was observed to evolve when the NIR plasmon resonance was optically excited. Photothermal heating of Cu_2−xSe nanocrystals was measured by irradiating an aqueous dispersion with 800 nm light near the plasmon band at low fluence (2 W/cm²) for 5 min. The optical density of the nanocrystal dispersion was adjusted to 1.0 at the excitation wavelength. Figure 5B shows the temperature of the dispersion as a function of irradiation time. Five minutes of light exposure raised the temperature by 22°C, which compares quite favorably with the photothermal heating of Au nanoshells and Au nanorods synthesized in-house (see Supporting Information for experimental details and Figure 5 for TEM characterization)^{13, 47} and obtained commercially. As shown in Figure 4, under similar illumination conditions, with the optical densities of the Au nanorods and nanoshells also adjusted to 1.0 at the excitation wavelength of 800 nm to normalize the photothermal responses of all of the materials, the Au nanoshells increased the temperature by 13°C (synthesized) and 15°C (commercial), and Au nanorods by 20°C (synthesized) and 22°C (commercial) after 5 minutes.

Figure 5.

TEM images of Au nanoshells and nanorods that were purchased from a commercial supplier and synthesized in-house. Synthesized Au nanoshells (A) are 135 nm in diameter, with an approximate shell thickness of 10 nm, while commercial Au nanoshells (B) are 145 nm in diameter, with a 7.5 nm thick Au shell. Synthesized nanorods (C) are 49 × 13 nm (aspect ratio: 3.8) and commercial nanorods (D) are 23 × 7 nm (aspect ratio: 3.3).

Figure 4.

(A) Absorbance spectra of polymer-coated Cu_2−xSe nanocrystals (red solid square), commercial Au nanoshells (blue solid circle) and Au nanorods (blue solid triangle), and synthesized Au nanoshells (black hollow circle) and Au nanorods (black hollow triangle) dispersed in deionized water. All solutions were normalized to an optical density equal to 1.0 at 800 nm (green arrow). (B) The photothermal response of the dispersions in (A) obtained by irradiating 300 μL aliquots of each solution for 5 min with an 800 nm diode laser (6 mm spot size, fluence of 2 W/cm²). The temperature was monitored with an infrared imaging camera. The laser heating of the water contributes approximately 2.5°C to the overall change in temperature in 5 min (green solid square).

The photothermal transduction efficiencies of the Cu_2−xSe nanocrystals and the commercial gold-based nanoparticles were also measured and found to be quite similar. Similar to Roper and coworkers,⁴⁸ nanoparticle dispersions were illuminated until reaching a steady-state temperature increase. The light source was then removed and the temperature decrease was monitored to determine the rate of heat transfer from the system. Figure 6 shows the typical thermal profiles of the different nanoparticles. From an energy balance on the system, the photothermal transduction efficiency could be calculated. The total energy balance for the system is

$${\Sigma }_i{m}_i{C}_{p,i}\frac{\mathit{dT}}{\mathit{dt}}=Q_{\mathit{in},\mathit{np}}+Q_{\mathit{in},\mathit{surr}}-Q_{\mathit{out}}$$ (1)

where m and C_p are the mass and heat capacity of the solvent (water) and T is the solution temperature. Q_in,np is the photothermal energy input from the nanocrystals:

$$Q_{\mathit{in},\mathit{np}}=I(1-{10}^{(-A_{\lambda }})\eta$$ (2)

where I is the laser power (in units of mW), A_λ is the absorbance at the excitation wavelength of 800 nm, and η is the photothermal transduction efficiency, or the fraction of absorbed light energy that is converted to heat. Q_in,surr is the heat input (in units of mW) due to light absorption by the solvent, which was measured independently and found to be 25.1 mW. Q_out is the heat lost to the surroundings:

$$Q_{\mathit{out}}=\mathit{hA}(T-T_{\mathit{surr}})$$ (3)

where h is the heat transfer coefficient, A is the surface area of the container, and T_surr is the ambient surrounding temperature. The lumped quantity hA, was determined by measuring the rate of temperature drop after removing the light source. In the absence of any laser excitation, Eqn (1) becomes

$${\Sigma }_i{m}_i{C}_{p,i}\frac{\mathit{dT}}{\mathit{dt}}=-Q_{\mathit{out}}=-\mathit{hA}(T-T_{\mathit{surr}})$$ (4)

Rearranging Eqn (4),

$$\mathit{dt}=-\frac{m_{H_{2}O}{C}_{p,H_{2}O}}{\mathit{hA}}\frac{\mathit{dT}}{(T-T_{\mathit{surr}})}$$ (5)

and integrating, gives the expression

$$t=-\left(\frac{m_{H_{2}O}{C}_{p,H_{2}O}}{\mathit{hA}}\right)\text{ln}(T-T_{\mathit{surr}})$$ (6)

A characteristic rate constant can then be defined, τ_out = m_H2OC_p,H2O/hA, such that

$$T-T_{\mathit{surr}}=\text{exp}\left(-\frac{\mathit{hA}}{m_{H_{2}O}{C}_{p,H_{2}O}}t\right)=\text{exp}\left(-\frac{t}{{\tau }_{\mathit{out}}}\right)$$ (7)

From the data in Figure 6, τ_out and heat transfer coefficients (hA) were found for each solution during solution heating and cooling, and are tabulated in Table SI 2. At the maximum steady-state temperature, the rate of photothermal heating is then equal to the rate of heat transfer out of the system:

$$Q_{\mathit{in},\mathit{np}}+Q_{\mathit{in},\mathit{surr}}=I(1-{10}^{(-A_{\lambda }})\eta +Q_{\mathit{in},\mathit{surr}}=\mathit{hA}(T_{\text{max}}-T_{\mathit{surr}})$$ (8)

where T_max is the maximum steady-state temperature. Therefore, the photothermal transduction efficiency can be calculated directly from the steady-state temperature increase, since

$$\eta =\frac{\mathit{hA}(T_{\text{max}}-T_{\mathit{surr}})-Q_{\mathit{in},\mathit{surr}}}{I(1-{10}^{(-A_{\lambda }})}$$ (9)

Figure 6B shows the photothermal transduction efficiencies measured for the Cu_2−xSe nanocrystals (22%), and commercial Au nanoshells (13%) and nanorods (21%).

Figure 6.

Steady state heating data (A) for commercial Au nanoshells (black circles), commercial Au nanorods (black triangles) and Cu_2−xSe nanocrystals (red squares). Dispersions of nanocrystals (300 μL) were irradiated with 800 nm light at low fluence (2 W/cm²) using a 6 mm spot size. The thermal time constant τ_out was determined by fitting the temperature fall to Eqn (8). The photothermal transduction efficiency η, was then determined from the steady-state temperature rise using Eqn (9). (B) Plot of the photothermal transduction efficiencies for the Cu_2−xSe nanocrystals, Au nanorods (commercial), and Au nanoshells (commercial).

The photothermal transduction efficiency of the Cu_2−xSe nanocrystals of 22% is nearly equivalent to Au nanorods (21%) and noticeably higher than Au nanoshells (13%). Halas and coworkers have reported similar differences in the photothermal transduction efficiencies between Au nanoshells and nanorods, and have shown the amount of heat generated experimentally is almost three fold less than what is theoretically predicted.⁴⁹ The lower η for Au nanoshells compared to the nanorods and Cu_2−xSe nanocrystals is due to the larger contribution of light scattering to the optical cross-section that does not contribute to heating. Since the nanoparticle dispersions had the same optical density at the excitation wavelength, the same amount of light was attenuated in each measurement (Figure 5B). However, the Au nanoshells are significantly larger (r_nanoshell = 72.5 nm) than the Au nanorods (r_eff,nanorod = 8.7 nm) and Cu_2−xSe nanocrystals (r = 8 nm). The size-dependence of the extinction coefficient and the relative amounts of light scattering and absorption have been extensively studied for Au nanostructures and is well understood.^50–52 El-Sayed and coworkers for example have illustrated how particle size affects the plasmonic properties of gold nanoparticles by normalizing the extinction coefficient to the particle volume (in units of μm³) and considering the relative contributions from light absorption and scattering.⁴⁴ They have observed that Au nanorods have a normalized extinction coefficient (μ_ext) of 1021.05 μm⁻¹, which is the sum of normalized absorption (μ_abs = 986.56 μm⁻¹) and scattering (μ_sca = 34.49 μm⁻¹) coefficients, and that Au nanoshells have a normalized extinction coefficient of 58.39 μm⁻¹, which is the sum of normalized absorption and scattering coefficients of 35.66 μm⁻¹ and 22.73 μm⁻¹, respectively. Therefore, the higher photothermal efficiency of nanorods compared to nanoshells is consistent with their higher absorption compared to scattering (96% vs. 60%, respectively).⁴⁴ Like the Au nanorods, the Cu_2−xSe nanocrystals are small enough that the majority of optical extinction is due to light absorption related to the plasmon resonance.

The biocompatibility of the polymer-coated Cu_2−xSe nanocrystals was tested by conducting a cell viability assay of human colorectal carcinoma HCT-116 cells in the presence of the nanocrystals. Cells were cultured until a confluency of 80% was reached and charged with a combination of new media and Cu_2−xSe dispersed in PBS to give a solution concentration of 39 mg/L (2.8 × 10¹⁵ NCs/L, equivalent concentration to 0.25 OD by absorbance spectroscopy). Cells were incubated for 0.5 hr, 1 hr, 3 hr, and 6 hr at 37°C. Control cells received fresh nanoparticle-free media and were incubated as well for 6 hr. The media was replaced after incubation to remove all unbound nanoparticles and Trypan blue, a membrane permeability stain, was added to assess cell death. Dead cells absorb dye and appear blue, whereas viable cells are clear under a brightfield microscope. Figure 7 shows bright field images of HCT-116 cells after incubation with polymer-coated Cu_2−xSe nanocrystals. The cell viability assay indicates that incubation of HCT-116 cells with the nanocrystals show no signs of cytotoxicity up to 6 hr, and only a slight increase in cell death at 6 hr compared to the control. Additional long-term cytoxoticity studies are underway.

Figure 7.

Bright field optical microscopy images of human colorectal cancer cells (HCT-116) incubated with 39 mg/L polymer coated Cu_2−xSe nanocrystals in PBS for 0.5 hr (A), 1 hr (B), 3 hr (C), and 6 hr (D). A control sample (E) was incubated for 6 hr and did not receive Cu_2−xSe nanocrystals. Cells were incubated for the predetermined time and stained with Trypan blue to visualize cell death.

Cu_2−xSe nanocrystals were added to HCT-116 cells and illuminated with 800 nm light to determine if they would promote photothermal cell death. In photothermal therapy, NIR light is used to excite the nanocrystals and create local temperature increases to destroy diseased cells. Heat-induced protein denaturation occurs above 40°C, which leads to cell death/injury,^{53, 54} and RNA and DNA unfold at temperatures above 85–90°C.⁵⁵ Cells grown in a 12-well plate were combined with media (0.375 mL) and Cu_2−xSe nanocrystals in PBS (0.125 mL) at a solution concentration of 39 mg/L (2.8 × 10¹⁵ NCs/L) and were incubated for 0.5 hr at 37°C. Cells were irradiated for five minutes with and without Cu_2−xSe nanocrystals at 30 W/cm². Bright field imaging of cells stained with Trypan Blue after irradiation (Figure 8 – bottom row) show that all of the cells exposed to nanocrystals exhibited photothermal cell destruction. Exposure of the cells to the NIR laser in the absence of nanocrystals did not compromise cell viability (Figure 8). The power threshold for non-targeted photothermal cell destruction using an 800 nm laser was 30 W/cm² for 5 min. These conditions are slightly more moderate than what is reported for non-targeted in vitro photothermal cell destruction with Au nanoshells (35 W/cm², 7 min) or targeted in vitro photothermal therapy with hollow Au nanoshells (40 W/cm², 5 min).⁵⁶

Figure 8.

Comparison of photothermal destruction of human colorectal cancer cells (HCT-116) without (top row – A and B) and with (bottom row – C and D) the addition of 2.8 × 10¹⁵ Cu_2−xSe nanocrystals/L. Cells irradiated at 30 W/cm² with an 800 nm diode laser for 5 min (circular spot size of 1 mm) were stained with Trypan blue to visualize cell death and imaged with an inverted microscope in bright field mode. Significant cell death is observed with 30 W/cm² irradiation.

Conclusions

Amphiphilic polymer-coated Cu_2−xSe nanocrystals exhibit an intense NIR absorbance peak and significant photothermal heating, comparable to Au nanorods and nanoshells. NIR photoexcitation of the Cu_2−xSe nanocrystals in the presence of human colorectal cancer cells led to significant cell death, verifying that the nanocrystals have the potential for photothermal therapy. The potential for Cu_2−xSe as an in vivo therapeutic is highlighted by its small hydrodynamic diameter that will lead to prolonged blood circulation times when additional non-immunogenic or cellular targeting molecules are attached for targeted photothermal therapy.