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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Cancer. 2010 Jul 1;116(13):3285–3293. doi: 10.1002/cncr.25135

Radiofrequency field-induced thermal cytotoxicity in cancer cells treated with fluorescent nanoparticles

Evan S Glazer 1, Steven A Curley 1,2,*
PMCID: PMC2928886  NIHMSID: NIHMS219555  PMID: 20564640

Abstract

Background

Non-ionizing radiation, such as radiofrequency (RF) field and near infrared laser, induces thermal cytotoxicity in cancer cells treated with gold nanoparticles (AuNP). Quantum dots (QD) are fluorescent semiconducting nanoparticles that we hypothesize will induce similar injury following RF field irradiation.

Methods

AuNP and two types of QD (cadmium-selenide and indium-gallium-phosphide) conjugated to cetuximab (C225), a monoclonal antibody against human epidermal growth factor receptor (EGFR-1), demonstrated concentration-dependent heating in a RF field. We investigated the effect of RF field exposure after targeted nanoparticle treatment in a co-culture of two human cancer cell lines that have differential EGFR-1 expression (a high expressing pancreatic carcinoma, Panc-1, and a low expressing breast carcinoma, Cama-1).

Results

RF exposed Panc-1 or Cama-1 cells not containing AuNP or QD had a viability greater than 92%. The viability of Panc-1 cells exposed to the RF field after treatment with 50 nM Au-C225 was 39.4% ± 8.3% without injury to bystander Cama-1 cells (viability was 93.7% ± 1.0%, p ~ 0.0006). Panc-1 cells treated with targeted Cd-Se QD were only 47.5% viable after RF field exposure (p < 0.0001 compared to RF only Panc-1 control cells). Targeted InGaP QD decreased Panc-1 viability to 58.2% ± 3.4% after RF field exposure (p ~ 0.0004 compared to Cama-1 and Panc-1 controls).

Conclusion

We selectively induced RF field cytotoxicity in Panc-1 cells without injury to bystander Cama-1 cells utilizing EGFR-1 targeted nanoparticles, and demonstrated an interesting bifunctionality of fluorescent nanoparticles as agents for both cancer cell imaging and treatment.

Keywords: quantum dot, nanoparticle, noninvasive radiofrequency field treatment, photothermal therapy, hyperthermic cytotoxicity

Introduction

Nanoparticle-based photodynamic therapy for cancer has been primarily focused on near-infrared radiation (NIR) because many nanoparticles absorb and release heat at these wavelengths. 1, 2. These therapies are limited to superficial lesions as NIR transmission through tissue is minimal.3 Other forms of electromagnetic radiation, including non-ionizing radiofrequency (RF) field radiation, penetrates through biologic tissues readily and without significant toxicity.4

We have previously reported on the use of noninvasive RF fields to induce thermal cytotoxicity in human and mammalian cancer cells exposed to targeted solid gold nanoparticles and carbon nanotubes.5, 6 Cancer cells endocytose cell surface receptor-specific antibody-conjugated nanoparticles with subsequent intracellular heat release during shortwave RF field treatment sufficient to produce cellular cytotoxicity.5 The RF treatment device consists of a transmitting plate connected to a RF power generator with a second grounded plate that together capacitively couple samples in a focused RF field. Gold nanoparticles heat in a concentration- and size-dependent linear fashion in this RF field.7

Theranostic nanoparticles, such as fluorescent nanocrystals (i.e. quantum dots), are a category of nanoparticles that are fundamentally changing the way cancer is detected and treated.8 Such nanoparticles can be targeted to malignant cells via antibody or peptide conjugation and can be used for optical imaging or as a marker for other therapeutic cargo.9–11 Quantum dots in the far red region of the visible spectrum permit visualization of in vivo targets.12 Typically, quantum dots consist of a metal core (~ 5 nm diameter) with an inorganic shell to improve quantum yield.13, 14 Common components of these cores include cadmium-selenide (CdSe) or indium-gallium-phosphide (InGaP). Poly (ethylene glycol) (PEG), other polymers, proteins, or chemical hydroxylation is often performed to prolong circulating time in vivo and to decrease potentially toxic metal ion release (i.e., cadmium).15–18

In this work, we investigated an in vitro model of mixed cell populations to determine if targeting selected cells with antibody-conjugated quantum dots or 20 nm gold nanoparticles followed by RF field treatment would kill the targeted cancer cells without injuring bystander cells. Human Panc-1 cells, a pancreatic carcinoma cell line, overexpresses EGFR-1 that can be targeted with cetuximab (C225), a monoclonal antibody raised against this cell surface receptor. A human breast carcinoma cell line, Cama-1, minimally expresses EGFR-15 and it represents the “bystander” or non-targeted cells.

We hypothesized that 1) like gold nanoparticles, quantum dots would heat in a 13.56 MHz RF field and 2) targeting nanoparticles (gold or quantum dot) to cancer cells would result in thermal injury or death to targeted cells after RF field treatment without substantial effect on bystander cells.

Materials and Methods

Cell lines, cell culture, antibodies, quantum dots, gold nanoparticles, and fluorophores

Cell lines Panc-1 and Cama-1 were purchased from American Type Culture Collection (Manassas, VA) and incubated in standard growth conditions (37 °C, 5% CO2). During the experiment, all cultures were maintained in Dulbecco's Modified Eagle’s Medium (DMEM, Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Experiments were performed in standard 60 mm culture dishes or 96 well plates (Corning Inc., Corning, NY). The cell line identities were confirmed by the Characterized Cell Line Core service (STR DNA fingerprinting, M.D. Anderson Cancer Center, Houston, TX, April 2009). Trypsin-EDTA (Mediatech, Inc., Manassas, VA) released cells from the cell culture dishes while 0.5 mg/mL collagenase I (Invitrogen Corp., Carlsbad, CA) was added to the trypsin-EDTA solution to free cells from the 96 well plates.

Cetuximab (C225) was purchased from Bristol-Myers Squibb (New York, NY). Twenty nanometer spherical gold nanoparticles (AuNP) were purchased from Ted Pella, Inc. (Redding, CA). Antibody conjugation kits for CdSe based quantum dots Qdot 605 (QD605) and Qdot705 (QD705), and the fluorophore Alexa Fluor 647 (AF647), were purchased from Invitrogen Corp. (Carlsbad, CA). The InGaP based quantum dot eFluor NC700 (NC700) was purchased from eBioscience, Inc. (San Diego, CA). CdSe based quantum dots QD605 and QD705 emit peak fluorescence at ca. 605 nm and 705 nm, respectively.13 InGaP quantum dot NC700 emit peak fluorescence at ca. 680 nm.19. All contain a zinc-sulfide shell and poly (ethylene glycol) coating with a final hydrodynamic diameter of ~ 20 nm, similar to the gold nanoparticles used.20, 21 Quantum dots act as both the identification and therapeutic agent while a fluorophore (AF647) conjugated to C225 acted as the identifying fluorescent agent for all gold nanoparticle experiments.

All standard laboratory chemicals, including those required for antibody conjugation per instructions in each kit, were purchased from Sigma-Aldrich Corp. (St. Louis, MO) or Thermo Fisher Scientific, Inc. (Waltham, MA) unless otherwise stated.

Conjugation of cetuximab to quantum dots and gold nanoparticles

Cetuximab was conjugated to QD605, QD705, and NC700 nanoparticles according to the manufacturer’s instructions (noted above). Antibody-quantum dot conjugate concentrations were determined as recommended by measuring the absorbance with a Nanodrop spectrophotometer (Thermo-Fisher Scientific, Inc., Waltham, MA). Finally, the conjugates were sterilized under UV light for 15 minutes, a duration not reported to induce significant release of cytotoxic agents.22

Alexa Fluor 647 was conjugated to cetuximab also according to the manufacturer’s instructions (noted above), and that conjugate (C225-AF647) was subsequently conjugated to 20 nm AuNP with a slight modification to a previously published protocol.23 First, the pH of AuNP stock solution was raised to approximately 8.5, the isoelectric point of cetuximab.24 Next, 90 µg of C225-AF647 was slowly added to 10 mL of the modified AuNP solution. This was protected from light to prevent fluorophore bleaching while gently mixing every 15 minutes at room temperature for one hour. Next, it was concentrated 10-fold in a centrifugation filter unit with a 100,000 molecular weight cutoff (Millipore Corp., Billerica, MA) at 3,500x g. Finally, it was sterilized under UV light for 15 minutes.

Generation of RF field and antibody-nanoparticle conjugate heating

Samples were placed in a high voltage, shortwave radiofrequency field (13.56 MHz) produced between two metal electrode plates that capacitively couple the sample plate in an air gap of 10 cm (Therm Med, LLC, Inc., Erie, PA). The radiofrequency field is on the order of ~ 10–15 kV/m at 400–800 W of power.7 Teflon, a material with low RF field absorbance, is used to hold samples in place. Stock solutions of each nanoparticle were diluted with 18 MΩ water to determine heating rates (Millipore Corp., Billerica, MA).

Cells were exposed to the RF field for 5 minutes at a generator power of 200W or for 4 minutes at power for 250W as indicated (total energy generated ~1000 Watt-mins or 60 kJ). The temperature was continuously monitored with an infrared camera (FLIR Systems, Inc., Boston, MA). All media temperatures remained less than 40.5°C.

Panc-1 cells treated with QD605-cetuximab and RF field exposure

An aliquot of Panc-1 cells were exposed to 50 nM QD605-cetuximab for 3 hours while a separate aliquot of Panc-1 cells were exposed to an equal concentration of cetuximab only. After exposure, the cells were washed, combined in equal numbers (2 × 105 cells each) in cell culture dishes (n= 8), and incubated for 24 hours. At that time, half of the dishes were exposed to an RF field for 5 minuets at a generator power of 200 W (n=4). Another group of combined cells (n=4) were not exposed to RF field treatment. Investigation of viability occurred 48 hours later with flow cytometry.

Mixed cell populations treated with cetuximab-conjugated nanoparticles and subsequently exposed to an RF field

An equal number of Panc-1 and Cama-1 cells (3 × 105) were plated together in standard 60 mm cell culture dishes (n = 4). After 24 hours, the cells were treated with 50 nM AuNP-C225-AF647 for three hours. At the same time, an equal number of Panc-1 and Cama-1 cells (2 × 104) were plated together in 96 well plates (replicates of 4). The cells in plates were treated with 50 nM NC700-C225 or 50 nM QD705-C225 for three hours. After treatment, the media was exchanged for fresh complete media without conjugates. Twenty-four hours later, the plates were placed in a RF field for 5 minutes at a generator power of 200 W (total generator power 60 kJ). Forty-eight hours later, flow cytometry was performed. In a similar fashion, mixed cell populations (n = 4) were also treated with conjugate concentrations of 30 nM for three hours, had a fresh media exchange, and the cells were exposed 24 hours later to a RF field for 4 minutes at 250W (total generator power 60 kJ).

Determination of targeted cells

Transmission electron microscopy (TEM), fluorescence microscopy (FM), and flow cytometry were used to discriminate between co-cultured cells containing cetuximab-conjugated nanoparticles and those that did not. TEM was performed with a JEM 1010 microscope (JEOL, Inc. Peabody, MA) and images were taken with an AMT Imaging System (Advanced Microscopy Techniques Crop., Danvers, MA). Fluorescence microscopy was performed with an Eclipse TE2000 microscope system (Nikon Instruments Inc., Melville, NY) with appropriate excitation and emission filters (Chroma Technology Crop., Rockingham, VT).

Determination of cell death in context of target identification after RF therapy

Cell populations were identified with flow cytometry that had high or low fluorescence of QD605, QD705, NC700, or AF647 as an identifier (and in the case of quantum dots, the treatment). Cell death was determined by 7-Amino-actinomycin D (7-AAD, BD Biosciences, Inc., San Jose, CA). Annexin V, a well-characterized protein that binds to cellular inner membrane phospholipid phosphatidylserine exposed during apoptosis, was provided as a kind gift conjugated to the fluorophore Horizon V450 from BD Biosciences, Inc.

Statistical Analysis

Results are expressed as means ± standard errors of the mean unless otherwise noted. Statistical significance, α, was set to p < 0.05. Groups were compared with a two-tailed Student’s t-test, unless otherwise noted. Data were analyzed and plotted with Microsoft Excel for Mac ver. 12 (Microsoft Corp., Redmond, WA), SigmaPlot ver. 10 (Systat Software Inc., San Jose, CA), and FlowJo ver. 8.8.6 (Tree Star, Inc., Ashland, OR).

Results

Production and Internalization of AuNP-C225

After gold nanoparticles (AuNP) were conjugated to cetuximab labeled Alexa Fluor 647 (C225-AF647), there was a small plasmon resonance shift consistent with conjugation and not consistent with gold aggregation (Figure 1). Transmission electron micrographs (TEM) demonstrated gold nanoparticles internalized into Panc-1 cells (Figure 1) but not in Cama-1 cells.

Figure 1.

Figure 1

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Cetuximab-conjugated nanoparticles are preferentially internalized into Panc-1 cells when visualized by TEM (AuNP-C225, A, and NC700-C225, B ). The figure bar represents 2 µm while the bar in the inset images represent 100 nm. Of note, the electron dense cores of NC700 quantum dots are smaller than ribosomes or AuNP. A slight plasmon shift (C) is seen when the antibody is conjugated to the gold nanoparticle. QD705-C225 is predominately seen in Panc-1 cells of the fluorescence microscopy images (D, 200× magnification). The exposure time and settings for each fluorescence microscopic image were the same while the red is false color.

Targeting and identifying quantum dot containing cells

Panc-1 cells demonstrated increased uptake of C225-conjugated quantum dots by fluorescence microscopy, flow cytometry, and TEM (Figure 1). The mixed Panc-1 and Cama-1 co-cultured cells and Panc-1 cells only groups clearly display fluorescence associated with QD705-C225 while the Cama-1 only group does not (Figure 1, red false color). There was a 10 to 100 fold brighter intensity of quantum dot containing cells compared to the negative population. This permitted discrimination between Panc-1 cells with internalized quantum dots or control cells not containing quantum dots.

Heating of quantum dots in an RF field

Deionized water heats in a 200W 13.56 MHz (8.0 KV/m) RF field at 0.28°C/min ± 0.07 °C/min. 100 nM C225 antibody alone in deionized water heats at 1.18°C/min ± 0.13 °C/min in the same field (Figure 2,). All three nanoparticle-C225 conjugates heated significantly faster in a concentration-dependent fashion (p < 0.05, Figure 2,). 100 nM AuNP-C225 heated at 17.4°C/min ± 1.2 °C/min (p < 0.006 compared to C225 or deionized water alone). 100 nM QD705-C225 and 100 nM NC700-C225 heated at 17.3°C/ min ± 0.5°C/min and 8.7°C/min ± 0.2°C/min, respectively (both p < 0.0005 compared to C225 or deionized water alone). Due to the similar components in QD605, the QD605-C225 conjugate heated in a similar manner to QD705-C225.

Figure 2.

Figure 2

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Solutions of cetuximab-conjugated gold nanoparticles (AuNP-C225) and quantum dots (NC700-C225 and QD705-C225) demonstrate a concentration dependent heating rate. All solutions were diluted in 18 MΩ water. Error bars are standard errors of the mean based on triplicate data for each conjugate. DI: deionized water.

RF field-induced thermal cytoxicity in Panc-1 cells

Control Panc-1 cells without quantum dots, gold nanoparticles, or RF treatment had a viability of 99.0% ± 0.1% (Figure 3). After a 5 minute RF exposure without nanoparticles, the viability decreased to 95.2% ± 2.2%, as measured 48 hours after exposure. Treating Panc-1 cells with QD605-C225 conjugates without RF exposure decreased viability to 82.2% ± 3.8%. When QD605-C225 treated cells were subsequently placed in the RF field for 5 minutes, the viability decreased to 47.5% ± 9.8% (p ~ 0.0001). Furthermore, RF field exposure of QD605-bearing Panc-1 cells induced cell necrosis, while QD605-C225 treatment without RF field exposure induced apoptotic cell death in a much smaller fraction of cells, as noted (Figure 3). QD605-C225 was not used in the mixed cell experiments because it had similar heating characteristics similar to QD705-C225.

Figure 3.

Figure 3

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Panc-1 cells positive for QD605 fluorescence had significantly decreased viability compared to QD605 negative cells after RF field treatment (p ~ 0.0001). On the flow cytometry live/dead plot (A), the lower left quadrant gates viable cells, the lower right gates apoptotic cells, the upper left gates necrotic cells, and the upper right gates dead cells of the single cell population. The right panel (B) summarizes the effects of treatment alone with QD605-C225 alone (RF−/QD+), the effects of RF exposure alone (RF+/QD−), and the combination (RF+/QD+) on the viability of Panc-1 cells as measured by flow cytometry compared to controls (RF−/QD−). The percentages in each pie chart represent the percent viable while the p-values are compared to RF−/QD− group. Prior to RF treatment, cells were treated with 50 nM QD605-C225 for three hours while controls were treated with 50 nM C225 alone. Replicates are n=4.

Effects of nanoparticle treatment and RF field exposure on mixed cell populations

All subsequent studies with the remaining three nanoparticles conjugated to C225 (AuNP, QD705, NC700) were performed in Panc-1 and Cama-1 cells in co-culture. The relative positions of two individual cell populations of Cama-1 and Panc-1 cells on a side scatter – AF647 plot were used to identify two distinct cell populations in the experimental groups. Mixed cell population exposed to cetuximab-conjugated quantum dots displayed similar patterns for identification.

Cama-1 cells maintained viability greater than 88% after treatment with cetuximab-conjugated quantum dots and gold nanoparticles as measured 48 hours after RF field exposure (Tables 1, Figure 4). Low-dose treatment (30 nM) of all three cetuximab-nanoparticle conjugates decreased Panc-1 viability to an average of 78% after RF field exposure (p < 0.003). RF field treatment after a 50 nM NC700-C225 yielded a Panc-1 cell viability rate of 58.2% ± 3.4%, while the viability rate was reduced to 56.3% ± 3.3% after 50 nM QD705-C225 treatment followed by RF field exposure (Table 1). 50 nM AuNP-C225-AF647 treatment of mixed cell populations coupled with subsequent RF field exposure reduced Panc-1 viability to 39.4% ± 8.3% (Table 1, p ~ 0.036). QD705-C225 and AuNP-C225 induced three to four times more necrosis than apoptosis in Panc-1 cells after RF exposure (p ~ 0.01 for QD705-C225 and p ~ 0.003 for AuNP-C225) while NC700-C225 induced twice as much apoptosis as necrosis after RF exposure in Panc-1 cells ( p ~ 0.017, Table 2). Cama-1 cell viability after RF field exposure was 93.7% ± 1.0%, 92.8% ± 3.0%, and 92.4% ± 2.6%, respectively, after treatment with 50 nM AuNP-C225-AF647, QD705-C225, and NC700-C225 (Table 1).

Table 1. Viability of mixed cell populations after 50 nM cetuximab conjugate treatment and RF field exposure.

Panc-1 cells demonstrate decreased viability after treatment with a “high” concentration of cetuximab-conjugated nanoparticles (50 nM). Cama-1 cells have a minimal decrease in viability despite nanoparticle conjugate exposure and RF field treatment, due to the minimal internalization of nanoparticles related to the minimal expression of EGFR-1. Significance is compared to Cama-1 RF-within each group. SEM represents replicates of n = 4.

Conjugate (50 nM) RF status Viability ± SEM p-value
NC700-C225 Cama-1 RF− 96.5% ± 0.7% ---
Cama-1 RF+ 92.4% ± 2.58% 0.2295
Panc-1 RF− 82.4% ± 1.9% 0.0024
Panc-1 RF+ 58.2% ± 3.4% 0.0004
QD705-C225 Cama-1 RF− 94.1% ± 0.5% ---
Cama-1 RF+ 92.8% ± 3.0% 0.432
Panc-1 RF− 73.7% ± 0.8% <0.0001
Panc-1 RF+ 56.3% ± 3.3% <0.0001
AuNP-C225 Cama-1 RF− 94.2% ± 2.0% ---
Cama-1 RF+ 93.7% ± 1.0% 0.579
Panc-1 RF− 86.3% ± 0.5% 0.060
Panc-1 RF+ 39.4% ± 8.3% 0.036
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Figure 4.

Figure 4

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The viability of Panc-1 cells, but not Cama-1 cells, decreases after treatment with cetuximab conjugates (50 nM) and RF field exposure (200W for 5 mins.). Cell death is most clearly demonstrated for gold nanoparticles targeted to Panc-1 cells after RF field exposure. RF field exposure after treatment with QD705-C225 and NC700-C225 was also sufficient to produce significant cytotoxicity compared to Cama-1 cells that did not take up the targeted nanoparticles.

Table 2. Distribution of viability after treatment with 50 nM cetuximab conjugates and RF field exposure.

Cetuximab conjugated nanoparticles induce both apoptosis and necrosis of targeted cells (Panc-1) after treatment in an RF field. This table displays the distribution of necrosis, apoptosis, dead, and viable cells of the single cell population after exposure to 50 nM cetuximab conjugates and RF field treatment (200W for 5 mins.). Standard errors of the mean (SEM) represent replicates of n = 4.

Conjugate
(50 nM)		 Necrotic
(% ± SEM %)		 Dead
(% ± SEM %)		 Apoptotic
(% ± SEM %)		 Viable
(% ± SEM %)
NC700-C225 Cama-1
Panc-1		 1.88 ± 0.73
6.62 ± 0.89		 3.41 ± 1.00
21.50 ± 1.05		 2.29 ± 1.04
13.70 ± 1.98		 92.40 ± 2.78
58.20 ± 3.41
QD705-C225 Cama-1
Panc-1		 2.39 ± 0.36
26.50 ± 5.06		 0.82 ± 0.24
9.94 ± 2.20		 4.06 ± 1.05
7.27 ± 1.56		 92.80 ± 1.09
56.30 ± 3.27
AuNP-C225 Cama-1
Panc-1		 1.46 ± 0.43
33.23 ± 3.95		 0.70 ± 0.16
18.75 ± 4.20		 4.08 ± 1.05
8.68 ± 3.07		 93.73 ± 1.04
39.35 ± 8.24
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Discussion

In this model, a non-invasive, non-ionizing shortwave radiofrequency field induced a concentration-dependent cytotoxicity in malignant human cells first treated with antibody-targeted nanoparticles. We demonstrated that the cell specific intracellular hyperthermia caused minimal toxicity to bystander cells that did not take up the EGFR-targeted nanoparticles. Interestingly, the AuNP-C225-AF647 treatment induced the greatest cell death after a single RF field exposure. We suggest that intense heat release during RF field exposure in the “nanoscale” environment immediately around each nanoparticle dominates the cellular killing effect through protein denaturation and disruption of cellular membranes.25 Measuring bulk temperatures only approximates the heat transfer from nanoparticle to the surrounding intracellular environment. At the intracellular level, most likely a relatively low concentration of nanoparticles will be sufficient to induce irreparable injury if structures requisite for cell viability (i.e. nucleus, mitochondria, intact cell membrane, etc.) are severely damaged or destroyed. This is consistent with the interesting finding that QD705-C225 and AuNP-C225 not only heat much faster than NC700-C225, but they also induce a predominately necrotic RF-induced effect, whereas the NC700-C225 conjugates were associated with lower rates of RF-induced heating that predominately induced apoptosis (Table 2). As we have demonstrated, nuances regarding the magnitude of intracellular hyperthermia (media temperatures remained less than 40.5°C at all times here) seem to determine whether cellular death is via apoptotic or necrotic pathways, in general.

Although all of the nanoparticles presented here have a hydrodynamic diameter of ~ 20 nm,20, 21 the surface area of the solid gold nanoparticles has more exposure to the RF field per particle because they are larger than the core of the quantum dots. Although larger semiconducting nanocrystals will fluoresce in the very far red to near infrared region, the semiconducting nature of the particles may permit increased heat dissipation at larger diameters whereas gold nanoparticles tend to heat less when they are greater than ~ 50 nm in diameter.7 In addition, as the underlying theory requires transfer of energy to the surrounding intracellular milieu, different coatings among different nanoparticles may abrogate or enhance the RF-field induced heating. Surface coatings and functionalizing molecules are variable from nanoparticle to nanoparticle and often synthesis-dependent.

The heating rates reported here are not extremely high (< 20°C/minute) due to the low generator powers. This was intentional to prevent any non-specific heating of the media to temperatures above 40.5°C. Although prolonged exposure times are required for cell death between 40.5°C – 43°C, apoptosis begins at these temperatures.4 However, as has been demonstrated here, quantum dots or gold nanoparticles internalized into cancer cells do in fact produce sufficient intracellular hyperthermia in response to the shortwave RF field exposure to induce significant cell death after a single treatment.

The cytotoxicity of quantum dots is being investigated, with some types having more potential toxic effects than others.12, 26–28 Qdot 605 and Qdot 705, for example, contain a well-known cytotoxic agent, cadmium. Although the toxicity associated with free cadmium ions varies with the specific quantum dot and cell under investigation, quantum dot toxicity has been reported at concentrations greater than 0.8 µM.26 In general, in vitro models suggest that cadmium quantum dot concentrations less than 100 nM are safely tolerated while indium based quantum dots (i.e., eFluor NC700) are ten times less toxic.22, 28, 29 Although certain metals (i.e., platinum) have become standard treatment for some cancers, 30 cadmium toxicities to normal cells makes cadmium an unattractive option as an anti-cancer cytotoxic agent. Unfortunately, each type of quantum dot is unique with its own toxicity profile. The type of core, shell, and coating all contribute to variations in release of free ions that may induce cytotoxicity.22

The ideal theranostic agent is one that not only permits diagnosis and treatment of cancer cells, but also permits multimodality treatment without injury to bystander cells. Although multiple agents can be conjugated to a single nanoparticle, the use of quantum dots permits one less conjugation because of the bifunctionality of fluorescence and local nanoscale hyperthermia in an RF field. This is a potentially important new finding and addition to the multifunctionality of quantum dots, nanocrystals, or other fluorescent nanoparticles. These types of nanoparticles have already been used for in vitro and in vivo optical imaging in animal models of malignant cells, including breast, prostate, and pancreatic cancer.31–33 Including other elements in the core or shell structure, such as iron, gadolinium, or gold, may allow the nanoparticles to be used for optical and radiographic imaging with magnetic resonance or computed tomographic scans.33 Some fluorescent nanoparticles can bind to specific proteins, creating the potential to measure circulating serum or intracellular protein levels.34 Plasmon-coupling can be used to observe specific base pair mismatches or mutations in DNA using thiolated DNA probes linked to gold nanoparticles because of specific surface-enhanced Raman-scattering.35 Functionalization and enhancement of circulating half-life of fluorescent nanoparticles can be achieved by linking PEG, proteins, silica, or other biocompatible polymers to the fluorescent core.31, 33, 36 Targeting molecules, including antibodies, peptides, or pharmacologic agents can also be arrayed on the surface to promote cell-specific binding and uptake. This may permit targeted delivery of drugs, silencing RNA, or other therapeutic agents to malignant cells with successful delivery confirmed by noninvasive optical imaging. We have shown in this study that the intrinsic fluorescence of quantum dots and nanocrystals is useful in flow cytometric analysis and identification of specific cell populations. Heat release during RF field exposure in these semi-conducting quantum dots and nanocrystals has not previously been reported, and this opens yet another potential therapeutic use for fluorescence nanoparticles. Cetuximab is an excellent antibody for in vitro models due to the variation in EGFR-1 expression of different cancer cell lines; however, it would be a poor choice in human trials as EGFR-1 is constitutively expressed in many normal tissues, but over expressed in a minority of cancers.37 A major challenge in cancer therapy is to identify cellular targets that are uniquely expressed only on malignant cells. This goal has yet to be fully realized. However, as antibody, drug, and peptide binding to cancer-specific targets identified through genetic and proteomic analyses38, 39 becomes more specific, utilizing bifunctional fluorescent nanoparticles may permit diagnostic and treatment options that are noninvasive and minimally toxic.

Acknowledgments

The Cell Line Characterization Core and High Resolution Transmission Electron Microscopy Core are funded by NCI # CA16672. The authors would like to acknowledge the assistance of Dr. Paul Cherukuri in discussion of nanoparticle properties, as well as Kristine Ash, Katheryn Massey, and Katrina Briggs for administrative assistance and cell culture assistance.

Sources of support: This work was supported by an unrestricted research grant was provided by the Kanzius Research Foundation (Erie, PA). In addition, ESG was supported as a T32 research fellow funded by the National Cancer Institute at the National Institutes of Health (5 T32 CA09599).

Footnotes

The authors declare that they have no conflicts of interest.

Financial disclosure: The authors have no financial disclosures to report.

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