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This study examines the capabilities of an actively targeting superparamagnetic nanoparticle to specifically deliver therapeutic and magnetic resonance imaging contrast agents to cancer cells.
Iron oxide nanoparticles were synthesized and conjugated to both a chemotherapeutic agent, methotrexate, and a targeting ligand, chlorotoxin, through a poly(ethylene glycol) linker. Cytotoxicity of this nanoparticle conjugate was evaluated by Alamar Blue cell viability assays, while tumor cell specificity was examined in vitro and in vivo by magnetic resonance imaging.
Characterization of these multifunctional nanoparticles confirms the successful attachment of both drug and targeting ligands. The targeting nanoparticle demonstrated preferential accumulation and increased cytotoxicity in tumor cells. Furthermore, prolonged retention of these nanoparticles was observed within tumors in vivo.
The improved specificity, extended particle retention, and increased cytotoxicity toward tumor cells demonstrated by this multifunctional nanoparticle system suggest that it possesses potential for applications in cancer diagnosis and treatment.
The development of tumor specific multifunctional nanoparticles is currently an area of intense research with the potential to revolutionize the diagnosis and treatment of cancer. These particle systems, also referred to as nanovectors, have been envisioned as novel contrast agents for non-invasive molecular imaging and targeted carriers for drug delivery [1, 2]. As a major class of these materials, superparamagnetic iron oxide nanoparticles have been examined extensively for applications in cancer diagnosis and therapeutics due to their biocompatibility and magnetic properties [3, 4]. Early forms of these nanoparticles, which exploit the body’s natural clearance pathways (e.g. reticuloendothelial system (RES)-mediated passive targeting), are currently in clinical use as magnetic resonance imaging (MRI) contrast agents for the detection of hepatic lesions  and are in late-stage clinical trials for imaging of lymph node metastasis .
In addition, the magnetic properties of these iron oxide nanoparticles have also been examined as a means of remotely directing therapeutic agents specifically to a disease site . Nanoparticles possess a high surface area-to-volume ratio allowing them to be loaded with large amounts of a therapeutic or cytotoxic drug. As with other drug delivery systems, specific accumulation in malignant tissues results in reduced dosages necessary for a therapeutic effect and less deleterious side-effects associated with non-specific uptake of cytotoxic drugs by healthy tissue. Additional advantages of drug carrier systems over traditional systemic chemotherapy include the ability to improve the solubility of hydrophobic drugs, stabilization and protection of drugs from premature degradation, and improved blood half-life of the therapeutic agent. However, shortcomings of magnetic drug targeting, such as poor penetration depth and diffusion of the released drug from the disease site, have limited their use to the treatment of cancer cells at or near the surface of the skin [8, 9]. Furthermore, like passive targeting strategies, nanoparticles delivered to tumors by these processes may not necessarily penetration into tumor cells themselves.
The next step in achieving specific accumulation within tumor cells for broader and more effective applications in various cancer types involves the use of active targeting strategies which exploit highly specific receptor-ligand type interactions at the cell surface . Advances in the understanding of the molecular biology of cancers have identified unique cell surface markers that can be targeted by molecules such as vitamins [11, 12], nucleic acids , peptides , and monoclonal antibodies (mAb) [15, 16]. Although few of these selective tumor targeting nanoparticle systems have yet made their way into the clinic use, these platforms have emerged as one of the most promising forms of nanomedicine [1, 17].
A unique targeting agent currently under investigation is chlorotoxin (CTX), a peptide originally isolated from Leiurus quinquestriatus scorpion venom . Exhibiting a high affinity for tumors of the neuroetodermal origin [19–21], a synthetic version of this peptide covalently linked to iodine 131 is currently in clinical trials as a means of targeting radiation to brain tumor cells . In our previous work, we demonstrated the targeting specificity of CTX conjugated iron oxide nanoparticles for glioma tumors [23, 24]. Recently, specific binding of CTX to cancer cells was found to be facilitated by matrix metalloproteinase-2 (MMP-2) . A near-infrared labeled CTX (CTX:Cy5.5) has demonstrated preferential accumulation in a wide variety of tumors, including prostate cancer, intestinal cancer, and sarcoma, suggesting a greater applicability of this targeting agent for other forms of cancer .
Here we present a multifunctional nanoparticle system comprised of iron oxide nanoparticles conjugated to CTX and a conventional chemotherapeutic agent, methotrexate (MTX) to serve both as a diagnostic and therapeutic tumor targeted nanovector (NP-MTX-CTX). In this nanoparticle system, a poly(ethylene glycol) (PEG) layer was also integrated to serve both as a biocompatible coating and linking molecule for the covalent attachment of the functional ligands to the iron oxide core. Extensive physiochemical characterized by techniques such as transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR) were used to ensure each stage of the synthesis process was properly executed. The tumor cell specific targeting of the NP-MTX-CTX conjugate was evaluated in vitro by comparing MRI contrast enhancement corresponding to nanoparticle uptake by tumor cells in comparison to normal healthy cells. The efficacy of this nanoparticle as a drug delivery system was examined by in vitro cell viability assay. Finally, MRI experiments were carried out with mice bearing xenograft tumors to evaluate the preferential accumulation of NP-MTX-CTX in tumor cells in vivo.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Iron oxide nanoparticles were synthesized through co-precipitation of a 1:2 molar ratio of Fe(II) to Fe(III) with sodium hydroxide in the presence of PEG. Specifically, 0.31 g FeCl2(H2O)4 and 0.52 g FeCl3 were dissolved in 100 mL of D.I. water (HPLC grade) containing 0.85 mL of 12 M HCl. 60.0 g PEG (600MW) was then stirred into the solution. The mixture was titrated with 100 mL 1.5 M NaOH under an inert nitrogen atmosphere at 40°C with constant mechanical stirring and sonication. The resulting colloidal nanoparticles were isolated with a permanent magnet and the supernatant decanted. The remaining pellet was then washed twice with 200 mL of D.I. water to remove excess PEG and reaction byproducts. Physically adsorbed PEG was removed from the nanoparticles through washing with 2 M HNO3 for 10 min under sonication. A covalently linked amine terminated PEG coating was then immobilized on the surface of the nanoparticles as described previously [26, 27]. This amine was then utilized for the attachment of MTX via a succinimidyl ester. 25 mg of MTX was dissolved in 0.250 mL of dimethyl sulfoxide (DMSO) and combined with 0.875 mL MES buffer pH 6.0. 1.25 mL of PEG-amine modified nanoparticles suspended in 3mM sodium citrate and 40mM sodium bicarbonate (~8 mg Fe/mL) were then combined with the MTX mixture. 32.5 mg of of 1-Ethyl-3- (3-dimethylamino-propyl) carbodiimide (EDC) and 21.75 mg of N-hydroxysuccinimide were then dissolved in 0.250 mL MES buffer pH 6.0 and added to the nanoparticle/MTX mixture. After 15 min at 25°C, the pH of the solution was adjusted to ~8.0 and the solution was allowed to react on a rocker for 2 hrs at 25°C. MTX-bound nanoparticles (NP-MTX) were then purified by gel chromatography using S-200 resin (GE Healthcare, Piscataway, NJ, USA). CTX was then conjugated using a procedure reported previously .
TEM samples were prepared by dipping 300 mesh silicon-monoxide support films (Ted Pella, Inc., Redding, CA, USA) in aqueous nanoparticle suspensions. The grids were then dried under vacuum for 2 hrs and observed on a Phillips 400 TEM (Philips Eindhoven, The Netherlands) operating at 100 KV. Powder X-ray diffraction (XRD) patterns were acquired from dried nanoparticle samples with a Philips 1820 X-ray diffractometer using Cu-Kα radiation (λ = 1.541 Å) at 40 kV and 20 mA.
Magnetic characterization was performed on a dried nanoparticle sample using a MPMS-5S SQUID magnetometer (Quantum Design, San Diego, CA). R2 relaxation measurements were performed on a 4.7-T Bruker magnet (Bruker Medical Systems, Karlsruhe, Germany) equipped with Varian Inova spectrometer (Varian, Inc., Palo Alto, CA, USA). Samples of the PEG-coated nanoparticles and Feridex I.V. (Advanced Magnetics, Inc., Cambridge, MA, USA) were suspended 1% agarose at concentrations of 0.02, 0.2, 2, and 20 μg Fe/mL. A 5 cm volume coil and spin echo imaging sequence was utilized to acquire T2 values of the nanoparticle samples. Images were acquired with a repetition time (TR) of 3000 ms and echo times (TE) of 13.7, 16, 20, 40, 60, 90, 120 and 170 ms. The spatial resolution parameters were: acquisition matrix of 256× 128, field-of-view (FoV) of 40 × 40 mm, section thickness of 1 mm, and 2 averages.
FTIR spectra were acquired using a Nicolet 5-DXB FTIR spectrometer (Thermo Scientific, Boston, MA, USA) at a resolution of 4 cm−1. FTIR samples were prepared with 2 mg of dried nanoparticles, CTX, and MTX, with each added to 200 mg of KBr and mixed in a mortar and pestle and pressed into a pellet for analysis. XPS experiments were performed by the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) at the University of Washington (Seattle, Washington). XPS spectra of dried nanoparticle samples were obtained using a Surface Science Instrument X-probe spectrophotometer with a monochromatized Al X-ray source and 5 eV flood gun for charge neutralization. X-ray spot size for the acquisition was on the order of 800 μm and take-off angle was 55°. Pressure in the analytical chamber during the acquisition was less than 5 × 10−9 Torr.
Rat cardiomyocytes (rCM) (Cell Applications, San diego, CA, USA) were grown on fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) coated substrates in rat cardiomyocyte cell culture media (Cell Applications). Rat 9L/lacZ glioma (9L) and human D283 medulloblastoma (D283) cells (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) formulated with high glucose and supplemented with sodium pyruvate (Invitrogen), 1% streptomycin/penicillin (Invitrogen) and 10% FBS. To evaluate nanoparticle uptake, 106 cells of each cell type were seeded in 75 cm2 flasks and grown for 48 hrs until cells reached 70–80% confluence. Cells were then washed with 10 mL of phosphate buffered saline (PBS) twice. Cell culture medium containing 100 μg Fe/mL nanoparticles was added to the flasks and the cells incubated at 37°C with 5% CO2. Following incubation, cells were washed three times with PBS and detached using trypsin/EDTA (Invitrogen). Cells were resuspended in PBS containing 10% FBS. Gel phantoms for MR imaging were prepared by suspending 106 cells in 50 μL of 1% low-melting agarose (BioRad, Hercules, CA, USA) and loading them into a pre-fabricated 12-well agarose sample holder. MR images were acquired as described in the previous section. TR of 3000 ms and variable TEs of 15–90 ms were used.
Cytotoxicity of the nanoparticle conjugates was evaluated by measuring cell viability using an Alamar Blue assay (Invitrogen). 9L cells were seeded in 96-well plates at a concentration of 20,000 cells/well and were grown for 8 hrs. Cells were then incubated for 2 hrs with either NP-MTX-CTX or NP-MTX (100 μg Fe/mL) in DMEM. Cells were then washed three times with PBS and the Alamar Blue assay was performed as described by the manufacture’s protocol. Cell viability was measured at time points of 3, 12, 24, 48, and 72 hrs.
In vivo mouse studies were performed in accordance with the University of Washington Institutional Animal Care and Use Committee. 9L flank xenograft tumors were prepared in athymic (nu/nu) mice as described previously . Targeting NP-MTX-CTX and non-targeting NP-MTX (6 mg Fe/kg) were administered via intravenous injection. MR imaging was conducted on a 4.7-T magnet (Bruker Medical Systems) and spectrometer (Varian, Inc.) as described in the previous sections. A multi-echo multi-slice spin echo imaging sequence was used to acquire images and measure T2 of tumor and muscle tissue. The parameters used to acquire axial images were: TR of 3000 ms and TE of 20, 40, 60, and 80 ms; FoV of 40 × 40 mm2; slice thickness of 1.5 mm; 10 slices; matrix size of 256 × 128; and 2 averages. The imaging parameters for coronal images were: TR of 2000 ms and TE of 20, 40, 60, and 80 ms; FoV of 70 × 40 mm2; matrix size of 256 × 128; slice thickness of 1 mm; 20–24 slices; and 2 averages. A multi-mouse holder was used to image 2 mice during each scan.
The physiochemical properties of a nanoparticle system are critical factors in their successful application in vivo. Particle size must be small enough to avoid rapid filtration by clearance organs, such as the liver and spleen, to allow them to reach target tissues . The size and morphology of the nanoparticles synthesized in this study were evaluated by TEM (Figure 1A). The nanoparticles possess a roughly spherical shape with a diameter in the range of 5–8 nm. Assuming a polymer coating of approximately 3–4 nm, these particles remain below the size necessary to extravasate through blood vessel walls . The chemical composition and crystal structure of the nanoparticles was determined by XRD. The XRD pattern of the nanoparticles (Figure 1B) is consistent with that of the magnetite (Fe3O4) reference (PDF# 019-0629). Possessing an inverse spinel crystal structure with oxygen ions forming a close-packed cubic lattice and iron ions located at interstices, the magnetic properties of these iron oxides arises from electron hopping between the Fe2+ and Fe3+ ions that coexist at the octahedral sites.
To determine the efficacy of this nanoparticle to serve as a MRI contrast agent, its magnetic properties were examined using a superconducting quantum interference device (SQUID). At both 5K and 300K, these particles demonstrated superparamagnetic behavior displaying a magnetization curve with no hysteresis loop (Figure 1C). The saturation magnetization (Ms) was found to be approximately 80 e.m.u./g nanoparticle. Typically used for their ability to shorten transverse relaxation times (T2), iron oxide nanoparticles provide negative (hypointense) contrast enhancement using T2-weighted pulse sequences. The relaxivity, r2, is an indicator of the effectiveness of these nanoparticles as a contrast agent. Here, we evaluated the r2 of this nanoparticle system in comparison to commercially available Feridex I.V. at 4.7 T by acquiring phantom MR images of agarose gels containing various concentrations of the contrast agents. R2 relaxation rates (1/T2) were measured and plotted as a function of iron concentration (Figure 1D). The r2 of each contrast agent was given by the slope of the linear correlation of R2 with iron concentration. An r2 of 185.6 s−1mM−1 was measured for Feridex I.V. and 637.8 s−1mM−1 for the iron oxide nanoparticles synthesized in this study, which corresponds to an approximate 3.4-fold greater contrast enhancement effect at this field strength.
In addition to particle size, the surface chemistry of the nanoparticle system plays a significant role in their behavior in vivo. Bare nanoparticles tend to agglomerate due to their large surface area-to-volume ratio and loss of electrostatic repulsion as a result of ions present under physiological conditions. These aggregates are then subjected to the adsorption of plasma proteins and subsequent clearance by the RES. As a result, they are quickly eliminated from the bloodstream by macrophages. To prevent nanoparticle agglomeration and increase their blood circulation time, a variety of polymeric coatings have been applied to nanoparticles. Of these polymers, PEG has been identified as being particularly effective [11, 29]. In addition to preventing plasma protein adsorption, PEG also aids in particle dispersion by forming steric barriers to the agglomeration. As reported previously, a heterobifunctional PEG was used to covalently graft the polymer to the iron oxide nanoparticle surface through siloxane bonds [26, 27]. The surface modification of these nanoparticles was evaluated by XPS. High-resolution XPS spectra of the O1s region (Figure 2A) display peaks characteristic of the oxide surface with both bound water and hydroxyl groups used to react with the silane functional group of the heterobifunctional PEG molecule. XPS spectra of the C1s region (Figure 2B) display peaks characteristic of the chemical bonds present in the ethylene glycol monomer units (C–C and C–O) and amide linkages formed in this particular molecule.
MTX was covalently linked to the PEG coating using a succinimidyl ester reaction [30, 31], while CTX was subsequently conjugated through a thiol-ether linkage . To confirm the successful conjugation of both ligands, NP-MTX-CTX conjugate was characterized by FTIR (Figure 2C). Spectra of NP-MTX-CTX showed the standard MTX characteristic absorbance peaks at 1644 and 1603 cm−1. In addition, the tertiary amino stretch found in the MTX molecule located at 1260 cm−1 was also observed on the spectra for NP-MTX-CTX confirming the successful immobilization of the drug. The presence of CTX in the nanoparticle conjugate was observed by the presence of amide and carboxylate peaks found at 1650 and 1550/1397 cm−1, respectively. CTX attachment was further verified by the methyl symmetric/asymmetric stretch located at 2922 and 2863 cm−1 of the alanine residues of CTX. A notable peak at 1095 cm−1 in the NP-MTX-CTX spectra is attributed to the siloxane bond formed between the PEG coating and the nanoparticle surface.
To evaluate the targeting specificity of the NP-MTX-CTX conjugate for cancer cells, an in vitro nanoparticle uptake assay was performed using MRI. T2 measurements were acquired for cells suspended in agarose, similar to those described for nanoparticles alone in the previous section, to quantify nanoparticle uptake. Two cancer cell lines, D283 and 9L, and rCM, representing healthy tissue, were incubated with NP-MTX-CTX for 2 hrs at a concentration of 100 μg.Fe/mL. After purifying unbound nanoparticles, cell samples were encased alongside with control cells and imaged. A T2-map was then generated from a series of MR images (Figure 3A). The amount of nanoparticles bound or internalized by the cells is correlated to the reduction in T2 from the values obtained for control cells (Figure 3B). An approximately 2–3 times higher uptake was observed for NP-MTX-CTX treated D283 and 9L cells in comparison with the normal healthy cells. Slight non-specific uptake by the rCMs is likely due to the PEG coating of the nanoparticles facilitating internalization of the particles across the cell membrane .
To further confirm target specificity, 9L cells incubated with NP-MTX or NP-MTX-CTX conjugates were evaluated under the same conditions described above; a significant difference in MRI contrast was observed between 9L cells incubated with NP-MTX and NP-MTX-CTX (Figure 3C). T2 quantification further showed that an approximately 3-fold difference in T2 change from control cell levels was observed for 9L cells treated with NP-MTX-CTX in comparison to non-targeted NP-MTX (Figure 3D). This result shows that the CTX on nanoparticles substantially increases the internalization of the nanoparticles and, concurrently, the number of MTX molecules delivered per cell. In our previous work, we showed that CTX-bound nanoparticles were internalized, likely through receptor-mediated endocytosis, and trafficked to lysosomes in cytoplasm of the cell .
MTX is a widely utilized chemotherapeutic for the treatment of leukemia, osteosarcoma, and head and neck cancer . Its primary mechanism of action involves the inhibition of the enzyme dihydrofolate reductase (DHFR) resulting in cytotoxicity by inhibiting synthesis of dTMP and purine precursors for DNA synthesis . However, the efficacy of this drug is limited by its short blood half-life and toxic dose-related side effects . Targeted delivery via nanoparticle-based drug delivery systems may serve to overcome these limitations.
To evaluate the ability of the NP-MTX-CTX conjugate to serve as a therapeutic agent, an in vitro cytotoxicity assay was performed with 9L cells exposed to either NP-MTX or NP-MTX-CTX. Cells were treated with the nanoparticle conjugates at a concentration of 100 μg.Fe/mL for 2 hrs after which their viability was monitored for 72 hrs by the Alamar Blue assay (Figure 4). Both NP-MTX and NP-MTX-CTX demonstrated the ability to inhibit cell growth over the first 12 hrs after treatment. However, cellular viability at this time point was significantly lower for the cells that had been exposed to NP-MTX-CTX (42%) than those exposed to the non-targeting NP-MTX (63%). At 24 hrs, the viability for cells that were treated with NP-MTX-CTX reached a minimum at 25.6% and maintained this level to for additional 24 hrs, while cells treated with NP-MTX began to recover with increase proliferation reaching normal untreated levels by 72 hrs. The increased cytotoxicity of the targeting conjugate is likely due to the increased uptake of these nanoparticles as observed in the previous section as well as prolonged retention in target cells.
MRI has become one of the primary imaging modalities used in the detection and diagnosis of tumors due to its excellent soft tissue contrast. The use of target-specific contrast agents has the potential to greatly improve the ability of MRI to detect lesions allowing for earlier detection and more accurate delineation of tumor boundaries. Additionally, the application of combined therapeutic and imaging nanoparticles may provide an opportunity for real-time monitoring of drug delivery [30, 31].
The efficacy of the NP-MTX-CTX conjugate to target tumor tissue was evaluated in athymic (nu/nu) mice with 9L xenografts. Tumor bearing mice were injected with either NP-MTX or NP-MTX-CTX at a dose of 6 mg Fe/kg. MR images were acquired with a multi-echo multi-slice spin echo imaging sequence prior to the administration of the nanoparticle conjugates and at 1 and 3 days post-injection. T2-maps were generated and overlays of the tumor region superimposed on proton-density weight images to visualize nanoparticle accumulation quantitatively (Figure 5). Pre-injection T2 values in the tumors were measured at approximately 70 ms (orange/red on the colorized scale). Images acquired one day post-injection showed a decrease in T2 in the tumors of the mice receiving either NP-MTX or NP-MTX-CTX. However, at this time point the tumor of the mouse receiving the targeting nanoparticles was more thoroughly highlighted in yellow signifying a T2 decrease to 40–50 ms. At 3 days post-injection, the tumor T2 values for the mouse receiving NP-MTX-CTX remained at the decreased level (40–50 ms), while that of the mouse receiving NP-MTX recovered to its pre-injection level. These results suggest that the targeting NP-MTX-CTX remained in the tumor for prolonged time, likely due to enhanced internalization of the nanoparticles in the tumor cells, while NP-MTX may have cleared from the tissue over time, which is consistent to the in vitro results shown above. Initial accumulation of the non-targeting nanoparticle conjugate in the tumor may have resulted from the enhanced permeation and retention (EPR) effect rather than specific accumulation [10, 35].
A longitudinal in vivo MRI study was also performed to monitor the retention of the NP-MTX-CTX in tumor as compared to muscle tissue. T2 measurements during 24 hrs post-injection (Figure 6A) showed a significant T2 drop in the tumor in the first 0.5 hrs, which recovered slightly to approximately 40 ms after 7 hrs with little change within 24 hrs. A similar trend was observed for muscle; however, the change in T2 from pre-injection levels was minimal. T2 measurements acquired at one week intervals post-injection showed that the T2 of the tumor remained at the decreased level for a prolonged time of nearly three weeks, thereafter it recovered nearly to the pre-injection level (Figure 6B). Variation in the T2 of muscle over this period was also minimal in comparison to the pre-injection values. These results suggest that the NP-MTX-CTX conjugates were retained in the tumor for at least 2 weeks. Such prolonged retention provides a wider time window for MR imaging, and may be useful in the sustained delivery of therapeutics to tumors.
We have developed a tumor targeted iron oxide nanoparticle conjugate that is capable of delivering MTX specifically to tumor cells. In vitro MR imaging experiments confirm the preferential accumulation of the NP-MTX-CTX conjugate in glioma and medulloblastoma cell lines as compared the normal healthy cells. Cell viability studies have demonstrated that the NP-MTX-CTX is more effective than NP-MTX in inducing cytotoxicity in cancer cells. In vivo MRI experiments performed with 9L xenograft tumor bearing mice displayed decreased T2 in tumor regions of mice receiving NP-MTX-CTX at both 1 and 3 day imaging time points post-injection suggesting persistent accumulation and binding of these nanoparticles in tumors. Mice receiving NP-MTX also showed a slight initial decrease in T2 of the tumor (e.g., in the first day post-injection), but the T2 value recovered to the pre-injection level at 3 days post-injection suggesting clearance of the non-targeting nanoparticles. Long-term evaluation of T2 values between tumor and muscle suggests that the NP-MTX-CTX conjugate is capable of delivering bound chemotherapeutic agents specifically to tumor cells and are retained in the tumor for at least 2 weeks.
Findings obtained from this study warrant further examination of this nanoparticle system. The evaluation of the efficacy of NP-MTX-CTX to reduce tumor size in vivo is currently underway. In addition, thorough evaluation of toxicity and biodistribution are also necessary for the clinical use of this nanoparticle system.
This work was supported by NIH grants (R01CA119408, R01 EB006043, R01CA134213).