Nano Res. 2011, 4(12): 1248–1260

1248

Multifunctional FeCo–Graphitic Carbon Nanocrystals for Combined Imaging, Drug Delivery and Tumor-Specific Photothermal Therapy in Mice

Sarah P. Sherlock and Hongjie Dai () Department of Chemistry, Stanford University, Stanford, CA 94305, USA Received: 25 July 2011 / Revised: 22 September 2011 / Accepted: 27 September 2011 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011

ABSTRACT Ultrasmall FeCo–graphitic carbon shell nanocrystals (FeCo/GC) are promising multifunctional materials capable of highly efficient drug delivery in vitro and magnetic resonance imaging in vivo. In this work, we demonstrate the use of FeCo/GC for highly effective cancer therapy through combined drug delivery, tumor-selective near-infrared photothermal therapy, and cancer imaging of a 4T1 syngeneic breast cancer model. The graphitic carbon shell of the ~4 nm FeCo/GC readily loads doxorubicin (DOX) via π–π stacking and absorbs near-infrared light giving photothermal heating. When used for cancer treatment, intravenously administrated FeCo/GC–DOX led to complete tumor regression in 45% of mice when combined with 20 min of near-infrared laser irradiation selectively heating the tumor to 43–45 °C. In addition, the use of FeCo/GC–DOX results in reduced systemic toxicity compared with free DOX and appears to be safe in mice monitored for over 1 yr. FeCo/GC–DOX is shown to be a highly integrated nanoparticle system for synergistic cancer therapy leading to tumor regression of a highly aggressive tumor model.

KEYWORDS Nanocrystals, photothermal therapy, doxorubicin, hyperthermia, magnetic resonance imaging

1. Introduction

The combination of hyperthermia with chemotherapy has been previously demonstrated as a means of improving cancer therapy. It has been demonstrated that tumors are sensitive to heat, and that exposing cancerous cells to hyperthermic conditions can enhance the cytotoxicity of certain chemotherapy drugs [1–5]. There are two major challenges in the application of combined hyperthermia and chemotherapy, namely, delivery of the drug to the tumor and localized hyperthermia at the tumor region.

Current chemotherapeutic agents have improved cancer prognosis, yet the systemic administration of these agents still leads to severe side effects for the patient, and often chemotherapy alone is insufficient to treat highly aggressive cancers [2, 4, 6]. In an effort to improve therapeutic efficacy of certain agents, hyperthermia has been incorporated in certain chemotherapy regimens and has demonstrated enhanced survival [2, 4, 7, 8]. Unfortunately, achieving controllable delivery of heat only to tissues of interest has been a challenge, with most techniques heating large regions of tissue [4, 9]. Non-specific heating of

Nano Res. 2011, 4(12): 1248–1260 ISSN 1998-0124DOI 10.1007/s12274-011-0176-z CN 11-5974/O4Research Article

Address correspondence to hdai1@stanford.edu

Nano Res. 2011, 4(12): 1248–1260

1249

tissues could add to the systemic toxicity already induced by chemotherapy alone. In an effort to improve delivery of chemotherapeutic agents or the localized delivery of heat, multiple nanoparticle platforms have been employed.

Chemotherapy-loaded nanoparticles passively accu- mulate in the tumor through the enhanced permeability and retention effect [10]. Once extravesated, the nano- particles are retained in the tumor leading to higher intratumor drug concentrations. In a similar manner, near-infrared (NIR) light absorbing nanoparticles accumulate in tumors and have been utilized to convert electromagnetic energy into heat for tumor hyperthermia or tumor ablation [11–13]. Use of NIR light optimizes tissue penetration depth and enables intracellular heating to create a region-specific heat application thereby minimizing systemic toxicities [5, 8, 11].

Drug delivery nanoparticles have been previously combined with NIR light absorbing photosensitizer nanoparticles to create a cooperative nanoparticle system. This involves the co-administration of two nanoparticle systems: a drug-loaded nanoparticle and a separate photosensitizer such as gold nanoparticles or nanorods [11, 12]. These cooperative systems have shown significant enhancement of drug efficacy through localized tumor hyperthermia. All-in-one systems, acting as both a drug delivery vehicle and a photosensitizer have been developed, but often have large diameters (> 70 nm) and have not been evaluated for use in vivo [14–16]. In addition, most of these systems lack the ability to image the drug–photosensitizer complex using a clinically relevant imaging modality.

We have previously reported FeCo–graphitic shelled nanocrystals (FeCo/GC), containing a highly magnetic iron–cobalt core surrounded by a single or double layer of graphitic carbon [17–19]. The core diameter of these nanocrystals can be altered between ~4 nm and ~7 nm depending on the synthetic conditions, making this an ultrasmall multifunctional material [18]. This material has been demonstrated as a highly sensitive magnetic resonance imaging (MRI) contrast agent due to the highly magnetic core, and as a NIR photosensitizer due to light absorption by the graphitic shell. Recently, we demonstrated the highly effective loading of doxorubicin (DOX) onto the surface of

FeCo/GC through π-stacking on the graphitic sidewall leading to a nanoparticle complex capable of drug delivery, photothermal heating, and MRI contrast enhancement in vitro [19]. The cellular toxicity of doxorubicin bound to FeCo/GC is significantly enhanced when combined with 808 nm NIR laser induced photothermal heating to 43 °C. This in vitro toxicity enhancement at 43 °C compared with that at 37 °C is due, in part, to a two-fold enhancement in the cellular uptake of FeCo/GC–DOX when heated to 43 °C for 20 min. The other contributing factor to the toxicity enhancement of FeCo/GC–DOX at 43 °C is an increased sensitivity of cancer cells to DOX under these hyperthermic conditions [3, 19].

In this work, we investigate the use of ~4 nm FeCo/ GC nanocrystals as a multifunctional cancer therapy agent to treat the aggressive mouse breast tumor model 4T1 in vivo. This highly integrated nanoparticle system is capable of treating high risk tumors through delivery of DOX and site-specific heat transfer to nanoparticle-containing tumors through NIR laser- mediated photothermal heating. This is achieved with the additional benefit of being able to track the drug complex in the body using MRI. The use of FeCo/GC–DOX combined with tumor-specific photo- thermal therapy results in complete tumor regression in 45% of mice, a result not observed with other therapeutic strategies assessed in this work. This repre- sents an important advance towards the development of multifunctional nanoparticles for cancer therapy, leading to significantly increased treatment efficacy and high survival rate with little systemic toxicity.

2. Materials and methods

2.1 FeCo/GC synthesis and DOX loading

Preparation of FeCo/GC nanocrystals and DOX loading was conducted as previously described [17–19]. To summarize, iron(Ⅲ) nitrate and cobalt(Ⅱ) nitrate were dissolved in methanol, mixed with silica powder, and dried to make a growth catalyst. Growth was conducted using chemical vapor deposition (CVD) of methane at 800 °C for 5 min. Silica was removed from the resulting nanocrystals by soaking in hydrofluoric acid, followed by washes with ethanol and then pure

Nano Res. 2011, 4(12): 1248–1260

1250

water. Nanocrystals were sonicated with phospholipid- branched polyethylene glycol–carboxylate (PL–brPEG) for 1 h, followed by centrifugation at 15 000 rpm for 6 h.

To load DOX, excess PL–brPEG was removed by washing twice through a 100 kDa centrifuge filter (Millipore). DOX was mixed with FeCo/GC at a DOX concentration of 1.7 mmol/L at pH ~8.5. After incu- bation for ~15 h, unbound DOX was removed through a 100 kDa centrifuge filter, followed by centrifugation for 5 min at 10 000 g to remove any aggregates. The concentration of DOX and FeCo/GC was determined by UV–visible spectroscopy (Cary 300, Varian) using a DOX extinction coefficient of 10 500 mol–1·L·cm–1 at 490 nm. The FeCo/GC concentration was determined using an extinction coefficient of 3.5 × 106 mol–1·L·cm–1 at 808 nm [19]. Concentrations of FeCo/GC–DOX used during experiments are reported as the concentration of DOX in solution, calculated by subtracting the FeCo/GC signal from the UV–visible absorbance curve.

2.2 Cell culture and in vitro toxicity assay

4T1 murine breast cancer cells obtained from American Type Culture Collection were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Cells were grown in a humidified incubator at 37 °C with 5% CO2. For cell toxicity tests, 4T1 cells suspended in cell medium were mixed with 25 µg DOX/mL of either free DOX or FeCo/GC–DOX, an equivalent FeCo/GC concentration or Phosphate buffered saline (PBS) for untreated cells. To ensure equal drug concentrations in samples, cells were not washed prior to irradiation. Once cells were mixed with the respective drug conjugates, cell solutions were irradiated with an 808 nm laser with a target temperature of ~43 °C for samples containing FeCo/GC. Cell solutions were held at ~43 °C for 20 min with continuous temperature monitoring using an infrared thermal camera. Non-irradiated cells remained at room temperature protected from laser exposure. After heating, cells were washed 3 times and plated in a 96-well plate. After 3 days of growth, viability was assessed using CellTiter 96 (Promega). The percentage of viable cells was calculated as the fraction of non-irradiated untreated cells. Values in Fig. 1(d) represent the average viability of ~10 wells

per group. The Student’s t-test was used to calculate statistical significance.

2.3 Animal handling and tumor inoculation

All animal experiments were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) protocols. For tumor inoculation, 4T1 cells were cultured as described above and trypsinized to remove them from the surface. Cells were washed with cell medium and then 3 times with PBS. For subcutaneous tumor inoculation, 1–2 million cells were inoculated through a 25 gauge needle on the shoulder of female mice (~6 weeks old). Mice were anesthetized with 2% isoflurane in oxygen during tumor inoculation, imaging and laser irradiation.

2.4 MRI imaging

4T1 tumor-bearing mice were injected intravenously with 12 mg DOX/kg body weight of FeCo/GC–DOX. Mice were imaged on a 1 T small animal scanner (M2, Aspect, Israel). For T1-weighted images, a steady state gradient-recalled echo sequence (Tr = 30 ms, Te = 4.3 ms) was used 10 h post-injection. For T2-weighted images a fast spin-echo sequence was used (Tr = 3 000 ms, Te = 86.7 ms) 24 h after FeCo/GC–DOX injection. For comparison of FeCo/GC–DOX treated and untreated mice, region-of-interest (ROI) measurements were performed using ImageJ and taken from a slice in the center of the tumor. Muscle signal was taken from a ROI on the leg. The muscle/tumor ratios shown in Fig. 2 are calculated from ROI measurements taken from the exact slices shown, however, general trends showing tumor contrast enhancement were consistent over a minimum of 3 tumors analyzed.

2.5 Fluorescence imaging

FeCo/GC was suspended with a 1: 1 mixture of PL–brPEG and phospholipid–polyethylene glycol (5k)–NH2. Following centrifugation at 15 000 rpm for 6 h, excess surfactant was removed by a washing through a 100 kDa centrifuge filter six times. IR800– N-hydroxysuccinimide (Licor) was reacted with the amine group on the surface of FeCo/GC. Unreacted dye was washed away using a similar filtration procedure following DOX loading as described

Nano Res. 2011, 4(12): 1248–1260

1251

above. Athymic nude mice with 4T1 tumors were intravenously injected with FeCo/GC-DOX-IR800 at a dose of 12 mg DOX/kg of body weight and were subsequently imaged using an IVIS Spectrum imaging system (Xenogen). Mice were imaged out to 2 days post-injection, during which time clear signals in the tumor region were observed.

2.6 In vivo treatment and NIR laser heating

Female Balb/c mice were inoculated with 4T1 tumors subcutaneously on the shoulder ~1 week prior to initiation of treatment. Treatment was started when tumors were approximately 100 mm3 in volume as determined by caliper measurements using the formula:

volume = (length × width2) /2. Mice were randomly assigned to treatment groups in the following numbers: FeCo/GC–DOX + Laser (n = 11), FeCo/GC–DOX (n = 7), FeCo/GC + Laser (n = 7), FeCo/GC (n = 7), free DOX (n = 8), free DOX + FeCo/GC + Laser (n = 6), untreated (n = 8). The exclusion of certain laser-irradiated groups (i.e., free DOX and untreated) was determined by the lack of apparent therapeutic effect determined from prior treatment studies (see Electronic Supplementary Material (ESM), Fig. S-1).

Mice were treated with 6 mg/kg free DOX, 12 mg/kg FeCo/GC–DOX, or an equivalent FeCo/GC con- centration. Mice were dosed twice per week with a maximum of eight doses. Tumor volume and body

Figure 1 Structure and cellular toxicity of FeCo/GC–DOX combined with laser irradiation. (a) Schematic view of DOX π-stacking on the graphitic sidewall of functionalized FeCo/GC. NIR laser light irradiates the graphitic carbon shell of the FeCo/GC–DOX complex andsubsequent vibrational relaxation leads to heating of the surrounding environment. (b) Photographs of solutions of DOX, FeCo/GC–DOX,and FeCo/GC. The DOX concentration in solutions was 450 µmol/L, with a nanocrystal concentration of ~270 nmol/L for the FeCo/GC containing solutions. (c) Transmission electron microscope (TEM) images of FeCo/GC–DOX confirming the presence of uniform individual nanocrystals with an average diameter of ~4 nm. The graphitic-carbon lattice of the shell of a single nanocrystal is resolved (right). (d) Cellular toxicity assay of 4T1 cells incubated with 25 µg DOX/mL of free DOX or FeCo/GC–DOX or equivalent FeCo/GC concentration. Laser irradiation at 808 nm for 20 min raises the temperature to ~43 °C resulting in toxicity enhancement of FeCo/GC containing samples. The most significant toxicity enhancement resulted from combining laser irradiation and FeCo/GC–DOX incubation. The laser had no effect on cells incubated with free DOX or untreated control cells

Nano Res. 2011, 4(12): 1248–1260

1252

Figure 2 Tumor contrast enhancement following FeCo/GC–DOX administration. Positive T1 contrast enhancement of 4T1 tumors (white arrows) was observed for FeCo/GC–DOX injected mice (a) over untreated control (b) mice. T2-weighted images led to negative contrast in tumors for FeCo/GC–DOX (c) treated mice over untreated control (d) mice. The tumor: muscle ratio reflects the tumor contrast alterations for the T1- and T2-weighted imaging sequences

weight was recorded twice per week, and mice were examined for general health and normal behavior four to five times per week. Laser irradiation of animals was performed ~15 h post-injection using an 808 nm laser. The ~15 h delay between dosing and irradiation was chosen to follow FeCo/GC–DOX circulation and permit some DOX release from the surface of FeCo/GC–DOX in the tumor. During 808 nm laser irradiation, reaching the target temperature of 43 °C did not require high laser power, which was generally maintained below 300 mW/cm2. The heating spatial distribution and the temperature of the tumor and surrounding tissues were monitored continually using an infrared thermal camera and a thermoprobe, respectively, to ensure that the tumor temperature did not exceed 45 °C. Importantly, only the tumor region reached the target temperature of 43–45 °C during the 20 min heating period. A 20 min heating time was chosen based on previous in vitro studies that showed an effective FeCo/GC–DOX toxicity enhancement at ~43 °C for 20 min [19]. Immediately following laser irradiation, the mice showed no signs of distress or abnormal behavior.

Tumor volumes and body weights were plotted as relative tumor volumes or percentage body weight as

compared to the initial values at day 0 of the treatment. The survival curve was constructed using non-survival conditions set at either a five-fold increase in tumor volume, or a greater than 10% drop in body weight from day 0 of treatment. A Student’s t-test was used for statistical analysis of results. Tumor growth inhibition was calculated using the formula (1 – T/C) × 100 where T = average volume of treated tumors and C = average volume of untreated control tumors.

2.7 In vivo pharmacokinetics

For blood circulation measurements, mice were intravenously injected with 12 mg DOX/kg of FeCo/ GC–DOX, followed by blood collection from the tail at various time points post-injection. For biodistribution studies, mice were sacrificed at different time points post-injection, and appropriate organs were collected and weighed wet. FeCo/GC–DOX concentrations were determined by monitoring cobalt signals in organs as compared to the injected solution.

Prior to cobalt determination, the graphitic carbon shell of FeCo/GC was removed by calcination at 500 °C for 1 h. Samples were then dissolved in hydrochloric acid, and later diluted with water for cobalt analysis by inductively coupled plasma–weight spectrometry (ICP–MS). Cobalt levels were corrected for native cobalt levels in blood or tissues of untreated mice. Each measurement was based on a minimum of three mice.

For excretion analysis, samples were pooled from multiple mice following treatment with FeCo/GC–DOX (12 mg DOX/kg). Samples from untreated mice were collected for comparison. All samples were processed as described above followed by cobalt detection by ICP–MS. For blood chemistry analysis and complete blood counts, blood was collection from the sub- mandibular vein into a heparinized tube and samples were submitted immediately for analysis.

3. Results and discussion

The fully integrated FeCo/GC nanocrystals used in this work create a single material for drug delivery, MRI contrast and heat delivery to tumors through NIR laser irradiation. Water soluble FeCo/GC suspensions

Nano Res. 2011, 4(12): 1248–1260

1253

were prepared as previously described through sonication of FeCo/GC with PL–brPEG [17, 19]. DOX was loaded non-covalently through π-stacking on the outer graphitic shell of FeCo/GC. The fully assembled complex, as shown in Fig. 1(a), becomes heated when irradiated with an 808 nm NIR laser [18, 19]. The pre- sence of DOX on the FeCo/GC surface was detectable by eye due to the reddish appearance of the FeCo/ GC–DOX solution after removal of unbound DOX (Fig. 1(b)). After DOX loading, FeCo/GC nanocrystals remained singly suspended with an average diameter of ~4 nm (Fig. 1(c)). DOX loading was quantified using UV–visible absorption spectroscopy, demonstrating ~1500 DOX molecules per FeCo/GC nanocrystal.

The toxicity of FeCo/GC–DOX was assessed on 4T1 cells in vitro with and without laser irradiation (Fig. 1(d)). Cells were mixed with FeCo/GC–DOX or free DOX at a concentration of 25 µg DOX/mL, followed laser irradiation at 808 nm for 20 min. The FeCo/GC containing vials were maintained at ~43 °C during laser irradiation, while there was no significant heating in the PBS (untreated) or free DOX vials. After the 20 min incubation, cells were washed and allowed to proliferate for 3 days prior to viability assessment. Cell samples exposed to free DOX showed a ~25% reduction in viability regardless of laser exposure (Fig. 1(d)). Cells incubated with FeCo/GC without drug or laser irradiation showed no reduction in viability as compared to cells incubated with PBS, however, when exposed to the laser and heated to ~43 °C the cells showed a slight reduction in viability (> 10%). FeCo/GC–DOX incubated cells not exposed to the laser showed slightly reduced viability (> 10%), while cells incubated with FeCo/GC–DOX combined with laser irradiation for 20 min to ~43 °C showed a ~40% reduction in viability, making the combination of FeCo/GC–DOX + laser irradiation (FeCo/GC–DOX + Laser) the most lethal combination for 4T1 cancer cells in vitro (Fig. 1(d)). We previously demonstrated that the enhanced toxicity of FeCo/GC–DOX + Laser was due to increased cellular uptake combined with enhanced sensitivity of cells to DOX at 43 °C compared with that at 37 °C [19].

While demonstrating the killing of cells in vitro is necessary during drug development, the performance of agents in vivo is the most important determinant

for assessing future clinical potential. FeCo/GC–DOX combined with laser photothermal therapy has shown promise in vitro as an effective cell killer, an effect that merited investigations into the in vivo behavior of FeCo/GC–DOX. Imaging of FeCo/GC–DOX accu- mulation in syngeneic xenograft 4T1 tumors was used to assess therapeutic potential and determine whether selective NIR laser based photothermal heating of the tumor over surrounding healthy tissues could be achieved.

The high relaxivities of FeCo/GC allow this material to be used as a MRI contrast agent capable of both positive (T1) and negative (T2) image contrast enhan- cement [18]. Following intravenous administration of FeCo/GC, clear T1 contrast enhancement was seen in the aorta and kidneys of mice, indicating nanocrystal blood circulation (Fig. S-2 in the ESM). At longer times post-injection (> 10 h), MRI was used to assess FeCo/ GC–DOX accumulation in tumors. Tumor-bearing mice imaged using a T1-weighted sequence showed positive contrast enhancement within the tumor, indicating nanocrystal accumulation (Figs. 2(a) and 2(b)). The tumor/muscle ratio of the FeCo/GC–DOX treated mice was significantly higher than that of the untreated control mice (1.3 vs. 0.6 in Fig. 2) when imaged using a T1-weighted sequence. When a T2-weighted imaging sequence was used, negative contrast was observed within the tumor of the FeCo/GC–DOX treated mice vs. the untreated control mice (tumor/muscle ratio of 2.4 vs. 4.4 respectively). This result is consistent with T2 contrast due to nanocrystal tumor accumulation.

To further supplement the MRI images and confirm tumor uptake, FeCo/GC–DOX was labeled with a NIR fluorescent dye (IR800) and intravenously injected into 4T1 tumor-bearing mice. At 1 day post-injection, fluorescence imaging of mice showed significant signal at the site of the tumor over surrounding tissues. The fluorescence images in Fig. 3(a) and Fig. S-3 (in the ESM) demonstrate the high uptake of FeCo/GC–DOX at the site of the tumor, enabling selective NIR laser heating of the tumor region over surrounding tissues. This result confirms the MRI measurements, demonstrating passive FeCo/GC–DOX accumulation within tumors and indicating the potential for tumor selective photothermal therapy.

Nano Res. 2011, 4(12): 1248–1260

1254

Figure 3 Fluorescence imaging of FeCo/GC–DOX tumor uptake and tumor selective photothermal heating. (a) FeCo/GC–DOX fluorescently labeled with IR800 showed significant accumulation in 4T1 tumors (black arrow) of mice at 1 day post-injection. (b) NIR laser irradiation of a 4T1 tumor-bearing mouse leads to tumor-specific heating. The black circled region highlights the tumor region, while the white circle indicates the laser irradiation area. Only the direct tumor region of FeCo/GC treated mice reached the target temperature of 43 °C

Upon 808 nm laser irradiation at low laser powers of ~0.1–0.3 W/cm2, FeCo/GC treated mice, with or with DOX attached, showed localized heating in the tumor region as demonstrated by the infrared thermal image in Fig. 3(b). Once selective tumor heating was demonstrated through NIR laser irradiation, in vivo treatment trials on 4T1 tumor-bearing mice were initiated. Mice received free DOX or FeCo/GC–DOX at doses of 6 mg/kg or 12 mg/kg respectively. Doses were given twice per week with a maximum of eight doses throughout the treatment. Free DOX doses were chosen to have acceptable toxicity levels (i.e., body weight loss) in animals throughout the treatment. To determine the effects of laser heating alone, FeCo/GC without DOX attached was given at the same net nano- crystal dose. An additional group consisting of free DOX co-administered with FeCo/GC was included to assess therapeutic benefits of DOX loading on FeCo/GC.

Approximately 15 h after dosing, the laser irradiated mice were exposed to an 808 nm laser with a target tumor temperature of 43–45 °C. The temperature was maintained for 20 min by adjusting the laser power in the range 0.1–0.3 W/cm2, and monitoring the tumor temperature with a thermal probe and an infrared thermal camera. Tissues surrounding the tumor region

did not reach the target temperature of 43–45 °C, demonstrating the tumor-selective heating of this photothermal therapy method. The tumor temperature of untreated control mice, or mice injected with free DOX without FeCo/GC, did not exceed 41 °C during 20 min of laser irradiation.

During the first 2 weeks of treatment, untreated control tumors grew rapidly to a mean volume ~6.5 times the initial mean volume on day 0 of treatment (Fig. 4(a)). Mice receiving FeCo/GC without laser irradiation showed similar tumor growth patterns to the untreated mice, indicating that FeCo/GC without DOX has no tumor therapeutic effect. Mice treated with free DOX, FeCo/GC–DOX or FeCo/GC + laser irradiation (FeCo/GC + Laser) all had ~50% growth inhibition (see Methods). The tumors of mice receiving free DOX with co-administration of FeCo/GC + Laser had 64% tumor growth inhibition. The mice receiving FeCo/GC–DOX + Laser had the smallest tumor volume, with tumor growth inhibited by 78%. As controls, the effect of laser irradiation on untreated mice or on mice receiving free DOX was assessed, and showed no significant effect on tumor growth (see Fig. S-1, in the ESM). This serves as additional confirmation that the heating effect and resulting therapeutic enhancement arises from the presence of FeCo/GC in the tumor, rather than a general non-specific heating effect from laser irradiation.

During treatment the average body weight of mouse cohorts was monitored to assess for systemic toxicity resulting from treatment. Untreated mice, FeCo/GC, and FeCo/GC–DOX treated mice steadily gained weight during the treatment as seen in Fig. 4(b). Mice receiving FeCo/GC + Laser or FeCo/GC–DOX + Laser showed a slight drop in body weight during the first week of treatment, followed by an increase in body weight in the following weeks. Animals treated with free DOX experienced the highest weight loss, and showed continual weight loss throughout the study. Unlike the cases treated with FeCo/GC that showed an initial drop in body weight followed by weight gains, the mice treated with free DOX continued to lose weight up to 21 days at which point the treatment for these groups was ended.

Despite the higher dose of DOX in the FeCo/GC–DOX case (12 mg/kg), this did not result in higher levels of

Nano Res. 2011, 4(12): 1248–1260

1255

systemic toxicity for the mice. Instead, the dose was well tolerated, and could safely be increased if deemed necessary. The significant weight loss in the free DOX (6 mg/kg) treated mice had negative effects on the survival of these groups. To construct the survival curve in Fig. 4(c), non-survival points were set when a tumor reached five times the initial volume, or when mice had lost more than 10% of their initial body weight.

All untreated mice or FeCo/GC treated mice died due to a five-fold increase in tumor volume within the first 2 weeks of treatment (Fig. 4(c)). Most of the free DOX treated mice, with or without concurrent FeCo/GC injection, reached non-survival points during the 4th week of treatment, due to either significant body weight loss or tumor burden. FeCo/GC–DOX treated mice without any laser irradiation showed a slowed

tumor growth rate (Fig. 4(a)) and had a similar survival time to free DOX treated mice (Fig. 4(c)), with eventual mortality due to an increased tumor burden. FeCo/GC + Laser treated mice survived slightly longer, yet most mice did not survive long beyond the 4th week of treatment. The only mice surviving the treatment at the end of 3 months were mice treated with FeCo/ GC–DOX + Laser shown in Fig. 4(c). This result shows the significantly enhanced therapeutic benefit of FeCo/ GC–DOX over free DOX or hyperthermia alone. At the end of the 3 month monitoring period, 55% of the FeCo/GC–DOX + Laser mice were still alive, and 45% of the initial treatment group were alive and remained tumor-free for a monitoring period of over 1 yr. Importantly, the tumor-free mice showed no negative long-term health effects resulting from the treatment

Figure 4 Tumor therapy and long-term survival of mice treated with FeCo/GC–DOX + Laser. (a) Relative 4T1 tumor volumes of different treatment groups (n = 6–11). Treatment was initiated at day 0 when tumor volumes were ~100 mm3. Mice were dosed twice per week with a maximum of eight doses during the treatment period. Laser irradiation was employed ~15 h post-injection for 20 min. Tumortemperatures were maintained between 43 °C and 45 °C during irradiation. Error bars represent the standard error of the mean. (b) Averagepercent body weight of cohorts during the treatment. Individual body weights of animals were compared to their weight at day 0. Error bars represent the standard deviation. (c) Long-term survival of mice in treatment groups monitored for 3 months. Mice were considered non-survival when there was a five-fold increase in tumor volume, or a greater than 10% drop in body weight

Nano Res. 2011, 4(12): 1248–1260

1256

during the ~1 yr monitoring period. FeCo/GC–DOX + Laser proved to be the only

therapeutic strategy capable of completely eradicating the tumor during treatment. The effect of combining laser irradiation and heating at 43 °C for 20 min with FeCo/GC–DOX treatment provided a synergistic enhancement to the treatment (see Methods), rather than a purely additive effect as in the case of free DOX co-administered with FeCo/GC + Laser. It is likely that the high efficacy of FeCo/GC–DOX + Laser is a combination of multiple effects, notably, the sensitivity of 4T1 tumors to heat at ~43 °C, and the enhanced susceptibility of the tumor to DOX at elevated temperatures. The sensitivity of 4T1 cells to heat has been previously demonstrated, whereby the expression of certain heat shock proteins on the cell surface is altered when exposed to non-lethal heat shock [20]. The expression of Hsp72 was increased on heat treated cells, with a concomitant reduction in Hsp25, leading to reduced growth and metastatic potential of 4T1 tumors [20]. This could account in part for the reduced growth rate of the 4T1 tumors in mice treated with FeCo/GC + Laser without any DOX. FeCo/GC–DOX + Laser toxicity enhancement in vitro was established to be a combination of enhanced DOX toxicity at elevated temperatures, and increased cellular uptake at 43 °C over 37 °C. While DOX toxicity enhancement is likely to be a contributing factor to the enhanced efficacy of FeCo/GC–DOX + Laser mice in vivo, the enhancement of cellular uptake of FeCo/GC–DOX within the tumor is a distinct possibility although not yet established. Further systematic studies are needed to fully investigate the effects of photothermal therapy on the intratumor distribution of FeCo/GC–DOX.

While the therapeutic efficacy of free DOX can be enhanced through hyperthermia, improvements in drug delivery are the only way to improve the tolera- bility of this drug. The combined increase in therapeutic efficacy of FeCo/GC–DOX + Laser, the tumor selective heating and improved systemic tolerability of DOX when loaded on FeCo/GC attests to the significance of this material. The development of Doxil has led to increased therapeutic efficacy; however, the side effects from Doxil treatment are still significant and may hinder the use of this drug [21]. In addition, there is no additional benefit of combining photothermal

therapy and imaging with Doxil, due to the lack of imaging and photosensitizing components in the current Doxil formulation.

It is likely that the pH sensitive release of DOX from the surface of FeCo/GC contributes to the reduced systemic toxicity of the nanocrystal–drug complex [19]. The acidic tumor environment could aid in the release of DOX from the nanocrystal surface, while a higher percentage of the drug remains bound and inactive in other tissues. The slow release of DOX from the surface of FeCo/GC may provide continual low levels of DOX which become available to the tumor, and thus another possible reason for the enhanced therapeutic benefit of FeCo/GC–DOX.

A method to quantitatively track the distribution of FeCo/GC in the body was developed to measure the in vivo pharmacokinetic behavior of the FeCo/GC–DOX complex. The method involved the determination of FeCo/GC content in tissues based on cobalt levels measured using ICP–MS. While in the body, the iron and cobalt core is fully protected from the external environment by the surrounding graphitic carbon shell, thereby preventing cobalt from being released into the body. Once tissues were collected, the highly stable graphitic carbon shell was removed by calcination of tissue samples at 500 °C for 1 h. Only after calcination to remove the carbon shell was dissolution of cobalt possible for ICP–MS analysis. The stability of the graphitic carbon shell attests to the safety of this material in vivo by eliminating the risk of metal release into the body.

The ICP–MS method was used to determine the blood circulation and biodistribution of FeCo/GC–DOX following intravenous injection. Due to the slow release of DOX from the FeCo/GC surface at neutral pH, it is believed that DOX remains bound to the nanocrystal surface during circulation [19]. The circulation half-life was determined to be ~75 min, with FeCo/GC–DOX still detectable in the blood more than 10 h after administration (Fig. 5(a)). While FeCo/GC–DOX does not circulate for as long as liposomal DOX delivery vehicles, its circulation half-life is still significantly longer than that of free DOX, reported to be less than 5 min [22]. Biodistribution of FeCo/GC–DOX at 1 day post-injection (Fig. 5(b)) showed high levels of accumulation in the reticuloendothelial system (RES),

Nano Res. 2011, 4(12): 1248–1260

1257

a typical clearance mechanism for nanomaterials. The level of FeCo/GC–DOX in all other organs was low, including the tumor where it was ~1% of the injected dose (ID) per gram of tumor tissue.

Despite the low tumor uptake, FeCo/GC–DOX still showed a significant therapeutic benefit during treatment (Figs. 4(a) and 4(c)). Potential explanations for this are that subsequent doses of FeCo/GC–DOX during treatment may have slightly higher tumor uptake due to hyperthermia-induced vascular per- meability or a tumor priming effect expanding the interstitial space, although these effects have not been

fully investigated [23–25]. In vivo fluorescence imaging shown in Fig. 3(a) showed clear tumor accumulation, that seems to contradict the ~1% ID/gram tumor uptake results in Fig. 5(b). One possible explanation is that the initial dose of FeCo/GC–DOX may accumulate at the edge of the tumor, but after multiple doses, FeCo/GC–DOX accumulation in the tumor becomes more homogenous. Initial accumulation of FeCo/ GC–DOX at the tumor periphery would lead to destruction of tumor cells and vasculature in this region, thereby enhancing therapy by minimizing metastatic spread [24]. It is important to recognize

Figure 5 Pharmacokinetics of FeCo/GC–DOX. (a) Percentage of injected FeCo/GC–DOX per gram of blood following intravenous injection of 12 mg DOX/kg. FeCo/GC–DOX content was evaluated using ICP–MS to measure cobalt content of samples after graphitic-carbon shell removal. Error bars are the standard deviation of blood samples collected from three mice. (b) Biodistribution ofFeCo/GC–DOX following intravenous administration. Mice were sacrificed at 1 day, 1 month or 3 months and FeCo/GC–DOX content was evaluated using the ICP–MS method. Error bars represent the standard deviation of a minimum of three tissue samples fromdifferent mice. FeCo/GC–DOX content in urine (c) and feces (d) samples collected from groups of mice at different timespost-injection. Samples were pooled to maximize sample volume and therefore represent an average value of injected mice

Nano Res. 2011, 4(12): 1248–1260

1258

that the ICP–MS measurements represent an average throughout the entire tumor, while fluorescence signals do not clearly identify the intratumor distribution of FeCo/GC–DOX. Further studies are needed to fully investigate the tumor accumulation and distribution of FeCo/GC–DOX, however, complete assessment is beyond the scope of this work.

Longer-term biodistribution studies showed that during the first month after dosing, FeCo/GC–DOX levels decreased in most organs except the spleen, which showed a slight increase in cobalt levels after 1 month (Fig. 5(b)). At 3 months post-injection FeCo/ GC–DOX levels were significantly reduced in all organs as the material slowly cleared from the body. The method of analysis was not able to distinguish FeCo/GC from FeCo/GC–DOX, therefore the total DOX content of tissues at longer time scales was not fully assessed. It is likely that DOX is slowly released over time from the nanocrystal surface based on previous release tests at neutral pH [19].

The route of clearance of FeCo/GC–DOX from the body was investigated by collecting urine and feces samples from mice at different time points post- injection. An increase in cobalt levels was detected in both the urine and feces of FeCo/GC–DOX treated mice up to 72 h post-injection (Figs. 5(c) and 5(d)). The cobalt signal in urine spiked early and decayed out to the 72 h point, indicating renal excretion of FeCo/GC–DOX occurs shortly after injection, while fecal excretion is the main route of clearance over longer time scales.

Fecal samples with and without calcination to removed the graphitic carbon shell were submitted for ICP–MS analysis to determine the condition of the nanocrystals after excretion. Only samples that were calcined to remove the protective carbon shell showed elevated cobalt levels (see Fig. S-4, in the ESM). These tests confirmed that FeCo/GC is excreted as an intact nanocrystal, not as free cobalt ions in the blood. The lack of cobalt signal in samples that were not subjected to calcination also confirms that the graphitic shell remains fully intact within the body.

In addition to monitoring mice over multiple months, blood tests confirmed that there were no toxic side effects resulting from FeCo/GC–DOX treatment, and that long term retention in the liver and spleen did

not affect organ function (see Table S-1, in the ESM). Previous toxicity tests and histological examination of FeCo/GC treated mice demonstrated that the nanocrystals were safe and did not damage organs over multiple months [17]. The risk of metal ions being released into the body is eliminated through encapsulation in the highly stable graphitic carbon shell. The shell fully protects the metal core from being etched, even when the nanocrystals are soaked in hydrofluoric acid or hydrochloric acid for several days. The lack of toxicity resulting from FeCo/GC with or without DOX attached has been confirmed over the course of multiple studies and supports the future clinical application of this material [17, 18, 26]. Despite the lack of toxicity, future development will necessitate improved tumor uptake and faster clearance from the body. The addition of tumor targeting ligands and improved surface functionalization (e.g., PEG coatings) may aid in increasing the level of FeCo/GC–DOX in the tumor and decrease the uptake in the RES.

Despite the problems with the pharmacokinetic behavior of FeCo/GC–DOX, this material demonstrated significant therapeutic potential in vivo. NIR irradiation of mice treated with FeCo/GC led to selective heating in the tumor region and a significant therapeutic enhancement of both free DOX and FeCo/GC–DOX + Laser. Combination of FeCo/GC–DOX + Laser irra- diation led to 45% complete tumor regression in mice, a result not achieved with other therapies tested in this work. This result is particularly impressive for such a multifunctional system, acting as the drug delivery vehicle, the photosensitizer and an imaging agent. Few other nanoparticle systems, or nanoparticle and photothermal heating combinations, have led to complete tumor regression. Most nanoparticle-based drug delivery with or without laser induced hyper- thermia (< 45 °C) only afforded slowed tumor growth over free drugs, without achieving complete tumor regression or long-term survival in mice [11, 27, 28].

Future applications of this cancer therapy strategy could involve superficial cancers, where light penetration is not an issue, or elimination of tumors within the body through the use of a fiber optic light source. This method could also be applied as an adjuvant therapy after surgical removal of a primary

Nano Res. 2011, 4(12): 1248–1260

1259

tumor to prevent tumor re-growth. Future studies may also be conducted to investigate photothermal enhancement of FeCo/GC–DOX as a method of over- coming drug resistance in DOX-insensitive cell lines. The elimination of a highly aggressive breast tumor model in mice, as demonstrated in this work, reveals the novelty of this multifunctional nanomaterial-based cancer treatment approach. The highly effective cancer treatment enabled by FeCo/GC–DOX, combined with the reduced systemic toxicity associated with traditional DOX therapy, makes FeCo/GC–DOX a promising candidate for future therapeutic applications in the treatment of highly aggressive cancers.

4. Conclusions

FeCo/GC–DOX has been demonstrated to have significantly enhanced therapeutic benefits when com- bined with tumor-specific NIR photothermal heating to 43–45 °C for 20 min. The enhanced therapeutic efficacy, combined with the reduced systemic toxicity of DOX when loaded onto FeCo/GC, makes this an appealing drug delivery system. Treatment with FeCo/ GC–DOX + Laser led to 45% complete regression of 4T1 tumors in mice, a dramatic increase over DOX treatment alone where no tumor regression was observed. This material appears to be non-toxic in mice over the course of many months, and clears from the body by both renal and fecal routes. The use of FeCo/GC–DOX offers a fully integrated cancer therapy system, combining drug delivery, imaging and selective photothermally induced hyperthermia in regions with FeCo/GC–DOX accumulation. The application of FeCo/GC–DOX in this work demon- strates the profound impact that multifunctional nanomaterials could have on future cancer treatment and long-term patient survival.

Acknowledgements

The authors acknowledge the Stanford Center for Innovation in In Vivo Imaging (SCI3) for small animal imaging equipment. The authors acknowledge Guangchao Li at Stanford Environmental Measure- ment 1 for assistance with ICP–MS, Liming Xie for

TEM, and Scott Tabakman, Kevin Welsher and Joshua Robinson for helpful discussions. This work was supported by the National Institute of Health (No. NIH-NCI 5R01CA135109-02).

Electronic Supplementary Material: Supplementary material containing tumor treatment control groups, MRI and fluorescent images of FeCo/GC–DOX treated mice and calcination tests of excreted FeCo/GC–DOX is available in the online version of this article at http://dx.doi.org/10.1007/s12274-011-0176-z and is accessible free of charge.

References

[1] Dewhirst, M. W. Future directions in hyperthermia biology.

Int. J. Hyperthermia 1994, 10, 339–345.

[2] Falk, M. H.; Issels, R. D. Hyperthermia in oncology. Int. J.

Hyperthermia 2001, 17, 1–18.

[3] Hahn, G. M.; Braun, J.; Harkedar, I. Thermochemotherapy:

Synergism between hyperthermia (42–43℃) and adriamycin

(or bleomycin) in mammalian cell inactivation. Proc. Natl.

Acad. Sci. U. S. A. 1975, 72, 937–940.

[4] Hildebrandt, B.; Wust, P.; Ahlers, O.; Dieing, A.; Sreenivasa,

G.; Kerner, T.; Felix, R.; Riess, H. The cellular and molecular

basis of hyperthermia. Crit. Rev. Oncol. Hematolo 2002, 43,

33–56.

[5] Wust, P.; Hildebrandt, B.; Sreenivasa, G.; Rau, B.; Gellermann,

J.; Riess, H.; Felix, R.; Schlag, P. M. Hyperthermia in

combined treatment of cancer. Lancet Oncol. 2002, 3,

487–497.

[6] Vertrees, R. A.; Jordan, J. M.; Zwischenberger, J. B.

Hyperthermia and chemotherapy: The science. In Current

Clinical Oncology: Intraperitoneal Cancer Therapy, Helm,

C. W.; Edwards, R. P., Eds.; Humana Press Inc.: Totowa,

NJ, 2007; pp 71–100.

[7] Helm, C. W.; Edwards, R. P. Current Clinical Oncology:

Intraperitoneal Cancer Therapy; Humana Press Inc.: Totowa,

NJ, 2007.

[8] Hildebrandt, B.; Wust, P. The biologic rationale of

hyperthermia. Cancer Treat. Res. 2007, 134, 171–184.

[9] Purushotham, S.; Chang, P. E. J.; Rumpel, H.; Kee, I. H. C.;

Ng, R. T. H.; Chow, P. K. H.; Tan, C. K.; Ramanujan, R. V.

Thermoresponsive core–shell magnetic nanoparticles for

combined modalities of cancer therapy. Nanotechnology 2009,

20, 305101.

[10] Power, S.; Slattery, M. M.; Lee, M. J. Nanotechnology and

its relationship to interventional radiology. Part II: Drug

Nano Res. 2011, 4(12): 1248–1260

1260

delivery, thermotherapy, and vascular intervention. Cardiovasc.

Intervent. Radiol. 2011, 34, 676–690.

[11] Park, J. H.; von Maltzahn, G.; Ong, L. L.; Centrone, A.;

Hatton, T. A.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J.

Cooperative nanoparticles for tumor detection and photo-

thermally triggered drug delivery. Adv. Mater. 2010, 22,

880–885.

[12] Park, J. H.; von Maltzahn, G.; Xu, M. J.; Fogal, V.;

Kotamraju, V. R.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J.

Cooperative nanomaterial system to sensitize, target, and

treat tumors. Proc. Natl. Acad. Sci. U. S. A. 2010, 107,

981–986.

[13] Robinson, J. T.; Welsher, K.; Tabakman, S. M.; Sherlock, S.

P.; Wang, H.; Luong, R.; Dai, H. J. High performance in vivo

near-IR (> 1 μm) imaging and photothermal cancer therapy

with carbon nanotubes. Nano Res. 2010, 3, 779–793.

[14] Lee, S. M.; Park, H.; Yoo, K. H. Synergistic cancer

therapeutic effects of locally delivered drug and heat

using multifunctional nanoparticles. Adv. Mater. 2010, 22,

4049–4053.

[15] Park, H.; Yang, J.; Lee, J.; Haam, S.; Choi, I. H.; Yoo, K. H.

Multifunctional nanoparticles for combined doxorubicin and

photothermal treatments. ACS Nano 2009, 3, 2919–2926.

[16] Park, H.; Yang, J.; Seo, S.; Kim, K.; Suh, J.; Kim, D.; Haam,

S.; Yoo, K. H. Multifunctional nanoparticles for photo-

thermally controlled drug delivery and magnetic resonance

imaging enhancement. Small 2008, 4, 192–196.

[17] Lee, J. H.; Sherlock, S. P.; Terashima, M.; Kosuge, H.; Suzuki,

Y.; Goodwin, A.; Robinson, J.; Seo, W. S.; Liu, Z.; Luong,

R. et al. High-contrast in vivo visualization of microvessels

using novel FeCo/GC magnetic nanocrystals. Magn. Reson.

Med. 2009, 62, 1497–1509.

[18] Seo, W. S.; Lee, J. H.; Sun, X. M.; Suzuki, Y.; Mann, D.;

Liu, Z.; Terashima, M.; Yang, P. C.; McConnell, M. V.;

Nishimura, D. G. et al. FeCo/graphitic-shell nanocrystals as

advanced magnetic-resonance-imaging and near-infrared

agents. Nat. Mater. 2006, 5, 971–976.

[19] Sherlock, S. P.; Tabakman, S. M.; Xie, L. M.; Dai, H. J.

Photothermally enhanced drug delivery by ultrasmall

multifunctional FeCo/graphitic shell nanocrystals. ACS Nano

2011, 5, 1505–1512.

[20] Bausero, M. A.; Page, D. T.; Osinaga, E.; Asea, A. Surface

expression of Hsp25 and Hsp72 differentially regulates tumor

growth and metastasis. Tumor Biol. 2004, 25, 243–251.

[21] Working, P. K.; Dayan, A. D. Pharmacological-toxicological

expert report: CAELYX. (Stealth liposomal doxorubicin

HCl). Hum. Exp. Toxicol. 1996, 15, 751–785.

[22] Seymour, L. W.; Ulbrich, K.; Strohalm, J.; Kopecek, J.;

Duncan, R. The pharmacokinetics of polymer-bound

adriamycin. Biochem. Pharmacol. 1990, 39, 1125–1131.

[23] Liu, D. L.; Andersson-Engels, S.; Sturesson, C.; Svanberg,

K.; Hakansson, C. H.; Svanberg, S. Tumour vessel damage

resulting from laser-induced hyperthermia alone and in

combination with photodynamic therapy. Cancer Lett. 1997,

111, 157–165.

[24] Liu, P.; Zhang, A.; Xu, Y.; Xu, L. X. Study of non-uniform

nanoparticle liposome extravasation in tumour. Int. J.

Hyperthermia 2005, 21, 259–270.

[25] Lu, D.; Wientjes, M. G.; Lu, Z.; Au, J. L. Tumor priming

enhances delivery and efficacy of nanomedicines. J.

Pharmacol. Exp. Ther. 2007, 322, 80–88.

[26] Kosuge, H.; Sherlock, S. P.; Kitagawa, T.; Terashima, M.;

Barral, J. K.; Nishimura, D. G.; Dai, H. J.; McConnell, M. V.

FeCo/graphite nanocrystals for multi-modality imaging of

experimental vascular inflammation. PLoS One 2011, 6,

e14523.

[27] Liu, Z.; Chen, K.; Davis, C.; Sherlock, S.; Cao, Q. Z.; Chen,

X. Y.; Dai, H. J. Drug delivery with carbon nanotubes for

in vivo cancer treatment. Cancer Res. 2008, 68, 6652–6660.

[28] Liu, Z.; Fan, A. C.; Rakhra, K.; Sherlock, S.; Goodwin, A.;

Chen, X. Y.; Yang, Q. W.; Felsher, D. W.; Dai, H. J.

Supramolecular stacking of doxorubicin on carbon nanotubes

for in vivo cancer therapy. Angew. Chem. Int. Ed. 2009, 48,

7668–7672.