Efficient CD44-targeted magnetic resonance imaging (MRI) of breast cancer cells using hyaluronic acid (HA)-modified MnFe2O4 nanocrystals
© Lee et al.; licensee Springer. 2013
Received: 2 December 2012
Accepted: 31 January 2013
Published: 2 April 2013
Targeted molecular imaging with hyaluronic acid (HA) has been highlighted in the diagnosis and treatment of CD44-overexpressing cancer. CD44, a receptor for HA, is closely related to the growth of cancer including proliferation, metastasis, invasion, and angiogenesis. For the efficient detection of CD44, we fabricated a few kinds of HA-modified MnFe2O4 nanocrystals (MNCs) to serve as specific magnetic resonance (MR) contrast agents (HA-MRCAs) and compared physicochemical properties, biocompatibility, and the CD44 targeting efficiency. Hydrophobic MNCs were efficiently phase-transferred using aminated polysorbate 80 (P80) synthesized by introducing spermine molecules on the hydroxyl groups of P80. Subsequently, a few kinds of HA-MRCAs were fabricated, conjugating different ratios of HA on the equal amount of phase-transferred MNCs. The optimized conjugation ratio of HA against magnetic content was identified to exhibit not only effective CD44 finding ability but also high cell viability through in vitro experiments. The results of this study demonstrate that the suggested HA-MRCA shows strong potential to be used for accurate tumor diagnosis.
KeywordsColloidal nanoparticles Nanomedicine Nanobioimaging Hyaluronic acid Magnetic resonance imaging
CD44 is a cell-surface glycoprotein antigen of breast cancer cells that is well known for its specific binding with hyaluronic acid (HA) [1–3]. It is the multifunctional cell-surface molecule involved in pathologic properties of cancer cells such as cell proliferation, differentiation, migration, angiogenesis, and chemokines [4–6]. Therefore, detecting CD44 is vital for accurate diagnosis as well as identifying effective anticancer drugs. Especially, when HA binds with CD44, this binding-mediated signals trigger cytological activities such as structural changes in the membrane and tumor cell migration [7–10]. Hence, HA has been frequently utilized as a targeting moiety to detect CD44 in the diagnosis and treatment of specific cancers directly associated with CD44 [11–14].
For sensitive and specific detection of cancer via the CD44 receptor, molecular imaging has been strongly considered due to the accurate acquisition of highly sensitive images and deeper insight into in vivo conditions [15–17]. Of the various molecular imaging techniques, magnetic resonance (MR) imaging has been widely recommended because it is non-invasive and provides high-resolution and tomographic real-time images at the cellular and molecular levels [18–20]. Therefore, CD44-targeted MR imaging has been applied in the treatment of cancer such as monitoring therapeutic efficacy and determining the progonosis of cancer.
To facilitate better interpretation of the MR images, recently developed MnFe2O4 nanocrystals (MNCs), synthesized by the thermal decomposition method in the organic phase, are well suited because of their fine crystalline structure and high magnetic sensitivity with an excellent size and composition control [21, 22]. However, these MNCs are insoluble in aqueous phase and could not be used directly in biomedical applications, so appropriate modification is necessary for phase transfer as well as further conjugation of specific targeting moieties such as HA [23, 24]. In addition, identification of specific amounts of targeting moieties on the MNCs resulted in the most efficient cellular uptake and imaging in vitro. Therefore, finding the optimal HA density on MNCs is needed for the most effective diagnosis and treatment for CD44-overexpressed breast cancer.
Polysorbate 80 (polyoxyethylene sorbitan monooleate, P80), spermine, 1,10-carbonyldiimidaziole (CDI), 1,4-dioxane (99.8%), iron(III) acetylacetonate, manganese(II) acetylacetonate, 1,2-hexadecanediol, dodecanoic acid, dodecylamine, benzyl ether, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Hyaluronic acid (20 kDa) was obtained from Lifecore Biomedicals (Chaska, MN, USA). Phosphate-buffered saline (PBS; 10 mM, pH 7.4), Dulbecco's modified Eagle's medium, Roswell Park Memorial Institute medium (RPMI), and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Both MDA-MB-231 and MCF-7 cells, breast carcinoma cell lines [26–28], were obtained from the American Type Culture Collection (Manassas, VA, USA). Sulfo-N-hydroxysuccinimide (sulfo-NHS) and 2,4,6-trinitrobenzene sulfonic acid (TNBSA) solution were purchased from Pierce (Thermo Scientific, Waltham, MA, USA). All other chemicals and reagents were of analytical grade.
Synthesis of MNCs
Monodispered magnetic nanocrystals, soluble in hydrophobic solvent, were synthesized using the thermal decomposition method . First, iron(III) acetylacetonate (2 mmol), manganese (II) acetylacetonate (1 mmol), 1,2-hexadecanediol (10 mmol), dodecanoic acid (6 mmol), and dodecylamine (6 mmol) were dissolved in 20 mL of benzyl ether under a blanket of nitrogen. The mixture was reacted for 2 h at 200°C and then further heated at 300°C for 1 h. All processes were under nitrogen atmosphere. After the mixtures were cooled at room temperature, the products were purified twice with 20 mL of pure ethanol. Finally, MNCs were grown to approximately 12 nm by the seed-mediated growth method.
Preparation of A-MNCs and HA-MRCAs
A-MNCs were fabricated using the nano-emulsion method . First, 10 mg of MNCs was dissolved in 4 mL of n-hexane (organic phase). The organic phase was injected into 30 mL of de-ionized water (aqueous phase) containing 100 mg of aminated P80. After mutual saturation, the solution was emulsified for 20 min under ultrasonification (ULH700S, Ulssohitech, Cheongwon-gun, South Korea) at 450 W. The mixture was kept overnight at room temperature to remove the volatile organic solvent. The products were purified using a centrifugal filter (Centriprep YM-3, 3-kDa molecular weight cutoff (MWCO), Amicon, Millipore Corporation, Billerica, MA, USA) in triplicate at 3,000 rpm for 30 min.
HA-MRCAs with different molar ratios of HA were fabricated by EDC-NHS chemistry. First, the pH of the A-MNC solution was adjusted to neutral condition by the addition of 0.1 N HCl solution. Then, various amounts of HA (0.43, 1.7, and 6.8 μmol) were dissolved in the 40 mL of de-ionized water followed by the addition of EDC and sulfo-NHS. Each HA solution was added to A-MNC solution containing 5 mg of MNCs. The HA and A-MNCs were reacted for 2 h at room temperature. Finally, EDC, sulfo-NHS, and unbound HA were removed using dialysis (MWCO, 25, 000) against excess de-ionized water.
Characterization of A-MNCs and HA-MRCAs
The size distributions and zeta potential values of A-MNCs and HA-MRCAs were measured using laser scattering (ELS-Z, Otsuka Electronics, Osaka, Japan). The inorganic ratios (%) and the crystallinities of magnetic nanocrystals in A-MNCs and HA-MRCAs were analyzed using a thermo-gravimetric analyzer (SDT-Q600, TA Instruments, Newcastle, DE, USA) and X-ray diffraction (X-ray diffractometer Ultima3, Rigaku, Tokyo, Japan) at 25°C, respectively. The magnetic properties of A-MNCs and HA-MRCAs were also detected by a vibration sample magnetometer (model 9407, Lake Shore Cryotronics, Inc., Westerville, OH, USA) at 25°C.
Cell viability assay for A-MNCs and HA-MRCAs
The cytotoxic effect of A-MNCs and HA-MRCAs against MDA-MB-231 cells (CD44-abundant cancer cell line) was analyzed by measuring the inhibition of cell growth using an assay for WST-1 ((4-(3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate)). MDA-MB-231 cells were maintained in RPMI containing 10% FBS and 1% antibiotics at 37°C in a humidified atmosphere with 5% CO2. MDA-MB-231 cells were harvested at a density of 1.0 × 104 cells/100 μL in a 96-well plate and incubated at 37°C in 5% CO2 atmosphere overnight. The cells were then treated with various concentrations of A-MNCs and HA-MRCAs for 24 h. After incubation, the cells were rinsed with 100 μL PBS (pH 7.4, 1 mM), and then 10 μL of WST-1 solution was added to each well. The absorbance was measured at 450 nm with a reference wavelength of 600 nm. The relative percentage of cell viability was determined as the ratio of formazan intensity in viable cells treated with A-MNCs and HA-MRCAs to the intensity in non-treated (control) cells [29–31].
Cellular targeting efficiency of HA-MRCAs
The targeting efficiency of HA-MRCAs was examined by MR imaging of breast carcinoma cell line MDA-MB-231 cells (high CD44 expression) and MCF-7 cells (low CD44 expression). First, target cells (1.0 × 107 cells) were harvested and washed three times with blocking buffer (FBS (0.2%) and NaN3 (0.02%) in phosphate-buffered solution (pH 7.4, 10 mM)) to inhibit non-specific binding effects. The solutions containing HA-MRCAs were applied to each cell line (1 and 0.5 μg, respectively) at 4°C for 30 min. The cells were then washed with blocking buffer three times to remove non-binding HA-MRCAs. Next, 200 μL of 4% paraformaldehyde was added to re-suspend the cells. After targeting efficiency was analyzed via MRI, the cells were dissolved in nitric acid for 2 h at 180°C, and the concentrations of magnetic nanocrystals (Fe + Mn) were measured using inductively coupled plasma atomic emission spectrometry (ICP-AES).
MR imaging procedures
We performed in vitro MR imaging experiments with a 1.5-T clinical MRI instrument with a micro-47 surface coil (Intera, Philips Medical Systems, Best, The Netherlands). The T 2 weights of the A-MNC- and HA-MRCA-treated cells (MDA-MB-231 and MCF-7 cells) were measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence at room temperature with the following parameters: TR = 10 s, 32 echoes with 12-ms even echo space, number of acquisitions = 1, point resolution of 156 × 156 μm, and section thickness of 0.6 mm. For acquisition of T 2-weighted MR images of A-MNC- and HA-MRCA-treated cells, the following parameters were adopted: resolution of 234 × 234 μm, section thickness of 2.0 mm, TE = 60 ms, TR = 4,000 ms, and number of acquisitions = 1.
Results and discussion
Characterization of aminated P80
MNCs, soluble in non-polar organic solvent with high monodispersity, were made using the thermal decomposition method to serve as MR contrast agents. For the identification of optimal HA density for efficient CD44-overexpressed breast cancer cell imaging and phase transference of hydrophobic MNCs into aqueous phase, the tri-hydroxyl groups of polysorbate 80 (P80) were modified with amine groups using spermine and the cross linker, 1,1′-carbonyldiimidazole (CDI) . CDI was used to activate hydroxyl groups of P80 and generate reactive imidazole carbamate intermediates. When the amine group of spermine attacked the intermediate, imidazoles were released, and stable tri-urethane (N-alkyl carbamate) linkages were fabricated. After conjugation, the characteristic bands of aminated P80 were verified by FT-IR spectra, which represented N-H stretching of an amine group (3,550 cm−1), C-N stretching of an amide group (3,400 cm−1), and N-H bending of an amine group (1,600 cm−1) (Additional file 1: Figure S1). On the contrary, the O-H stretching of hydroxyl groups (3,500 cm−1) of P80 was not detected in aminated P80 because all hydroxyl groups of P80 reacted with excess CDI. Furthermore, the chemical structures of aminated P80 were analyzed by 1H-NMR to show δ values of 7.11 (−CONH-), 4.29 (−NH2), 3.22 (−OCH2-), 2.72, 1.77 (−CH2-), and 2.17 (−NH-) ppm (Additional file 1: Figure S2). To quantify the primary amine groups (−NH2) in aminated P80, a TNBSA assay was used since primary amine groups replace sulfonic acid groups in TNBS molecules. Therefore, this substitution produces a chromogenic complex for which the absorbance at 355 nm is proportional to the number of amine groups (Additional file 1: Figure S3) . A standard curve was created using glycine because this amino acid molecule possesses one primary amine group per molecule. The absorbance of aminated P80 confirmed that the number of primary amine groups in aminated P80 was approximately 2.4-fold higher than that of glycine. These results showed that all hydroxyl groups of P80 were modified with amine groups, and the MNCs could be modified with HA through the generation of an amide bond.
Synthesis and characterization of A-MNCs and HA-MRCAs
Subsequently, A-MNCs were fabricated with pre-synthesized aminated P80 through the nano-emulsion method. The HA, CD44-targeting polysaccharide, was conjugated to the A-MNCs by EDC/NHS chemistry to provide breast cancer cell affinity. Carboxylic acid groups in HA were activated by EDC, and then sulfo-NHS was reacted to generate sulfo-NHS ester. Amine groups as nucleophiles on the A-MNCs were conjugated with these activated ester groups, and the NHS group rapidly left the intermediates, thereby creating stable amide linkages between A-MNCs and HA to form HA-MRCAs .
Relaxivity of HA-MRCAs and A-MNCs
To assess the MR contrast effect of HA-MRCAs, we performed MR imaging using HA-MRCAs, with A-MNCs used as a control. The relaxivity coefficients were measured and calculated (A-MNCs 361.6 mM−1 s−1, HA-MRCAs (i) 380.0 mM−1 s−1, HA-MRCAs (ii) 366.0 mM−1 s−1, and HA-MRCAs (iii) 407.3 mM−1 s−1), and representative T 2-weighted MR images were collected (Additional file 1: Figure S5). HA-MRCAs exhibited MR contrast effects that were remarkably higher than those of commercial MR imaging contrast agents (ferumoxide 190.5 mM−1 s−1) based on the fact that HA-MRCAs induced better contrast in MR imaging than ferumoxide . The high relaxivity coefficients of HA-MRCAs were achieved not only by the substitution of one of the Fe ions with a Mn ion, but also by the high crystallinity and monodispersity of the MNCs synthesized by the thermal decomposition method [42–44]. Further, relaxation rate (R 2) and the concentration of MNCs had a linear relationship in all HA-MRCAs. Therefore, the targeting efficiency of HA-MRCAs could be determined based on the concentration of the MR contrast agent in the tumor, which should be directly proportional to the relaxivity. Interestingly, HA-MRCAs exhibited similar or better relaxivity compared with A-MNC. This might be attributed to the HA domain of HA-MRCAs. HA can form many hydrogen bonds with surrounding water molecules owing to its abundant functional groups, such as hydroxyl and carboxylic groups. Hydrogen bonding between HA in the coating layer of HA-MRCAs and water molecules formed the hierarchical structures. In this structure, the mobility of water molecules in the diffusing layer is confined, and the residence time of water increases due to hydrogen bonding. These phenomena result in the enhancement of the transverse relaxation rate [45–51]. Therefore, HA-MRCAs possessed similar relaxivity, even after HA modification.
Cell viability assay with A-MNCs and HA-MRCAs
Targeting efficiency of HA-MRCAs against CD44-overexpressing cancer cells
HA-MRCAs with various ratios of HA were fabricated to determine the most efficient conditions for achieving accurate detection of CD44-overexpressing cancer. With HA conjugation, the surface charge changed from positive to negative, resulting in an increase in cell viability. Then, we confirmed that HA-MRCAs exhibited similar relaxivity in spite of the HA modification, which allowed the comparison of targeting efficiency via MR imaging. Varying the HA ratio could control the targeting ability of each HA-MRCA. Especially, HA-MRCAs (ii) and HA-MRCAs (iii) represented a sufficiently high MR imaging sensitivity to diagnose CD44-overexpressing cancer from in vitro studies. HA was modified four more times in the fabrication of HA-MRCAs (iii) compared to HA-MRCAs (ii); however, both HA-MRCAs (ii) and HA-MRCAs (iii) revealed similar targeting ability. Based on these results, HA-MRCAs (ii) appear to be appropriate for diagnosis of CD44-overexpressing cancer and can be utilized as drug carriers as well as in theragnosis systems using HA .
HA-modified MR contrast agents
Magnetic resonance imaging
This work was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A085136); grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (No. 1020340); and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011–0018360).
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