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Hyaluronic Acid Immobilized Magnetic Nanoparticles for Active Targeting and Imaging of Macrophages Medha Kamat,† Kheireddine El-Boubbou,† David C. Zhu,‡ Teri Lansdell,† Xiaowei Lu,† Wei Li,§ and Xuefei Huang*,† Department of Chemistry and Departments of Radiology and Psychology, Michigan State University, East Lansing, Michigan 48824, United States, and Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 847 Monroe Avenue, Memphis, Tennessee 38163, United States. Received August 2, 2010; Revised Manuscript Received September 29, 2010
Imaging and targeted delivery to macrophages are promising new approaches to study and treat a variety of inflammatory diseases such as atherosclerosis. In this manuscript, we have designed and synthesized iron oxide based magnetic nanoparticles bearing hyaluronic acid (HA) on the surface to target activated macrophages. The HA-coated nanoparticles were prepared through a co-precipitation procedure followed by postsynthetic functionalization with HA and fluorescein. The nanoparticles were characterized by transmission electron microscopy, thermogravimetric analysis, elemental analysis, dynamic light scattering, and high-resolution magic angle spinning NMR and were biocompatible with cells and colloidally stable in the presence of serum. The HA immobilized on the nanoparticles retained their specific biological recognition with the HA receptor CD44, which is present on activated macrophages in high-affinity forms. Cell uptake studies demonstrated significant uptake of HA nanoparticles by activated macrophage cell line THP-1, which enabled magnetic resonance imaging of THP-1 cells. The uptake of nanoparticles was found to be both HA and CD44 dependent. Interestingly, Prussian blue staining showed that the magnetite cores of the HA-coated nanoparticles were only transiently present inside the cells, thus reducing the potential concerns of nanotoxicity. Furthermore, fluorescein on the nanoparticle was found to be delivered to the cell nucleus. Therefore, with further development, these HA functionalized magnetic nanoparticles can potentially become a useful carrier system for molecular imaging and targeted drug delivery to activated macrophages.
INTRODUCTION Macrophages play central roles in mediating a wide range of infectious and inflammatory diseases, such as tuberculosis, atherosclerosis, rheumatoid arthritis, multiple sclerosis, and graft rejection (1, 2). They are critical in the initiation and maintenance of inflammation, which can lead to tissue destruction. For example, in atherosclerotic plaque development, a large number of macrophages are recruited to the cholesterol-rich site of injured aorta, which damages the fibrous tissue capping the plaques (3, 4). The weakened plaques can rupture, resulting in heart attack and stroke. The presence of a large number of active macrophages in plaques can be viewed as a biomarker for plaque instability (5). Therefore, effective technologies for monitoring macrophages as well as delivery of drugs to modulate macrophage activities can have great diagnostic and therapeutic potentials (1, 6). Recently, the use of nanoparticle systems for biological imaging and drug delivery has attracted increasing attention (1). With their nanometer dimensions and large surface area, nanoparticles present a powerful platform for the attachment of multiple copies of diverse ligands. Furthermore, nanoparticles can have unique magnetic, optic, or fluorescent properties, * Corresponding author. Xuefei Huang, Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA, Tel: (517) 355-9715, ext 329; Fax: (517) 353-1793; E-mail: xuefei@ chemistry.msu.edu. † Department of Chemistry, Michigan State University. ‡ Departments of Radiology and Psychology, Michigan State University. § University of Tennessee Health Science Center.
rendering them suitable for biological imaging (7-9). Although nanoparticles can be nonspecifically taken up by macrophages, the development of nanoparticles bearing specific ligands that are capable of actively targeting macrophages can potentially enhance the efficiency and selectivity of delivery. An attractive targeting ligand is hyaluronic acid (HA1 ), a member of the glycosaminoglycan family (10). HA is composed of tandem disaccharide repeats of β-1,4-D-glucuronic acid-β1,3-D-N-acetylglucosamine. As an important component of the extracellular matrix, HA has been shown to be involved in multiple cellular processes, including cell adhesion, cell migration, innate immunity, and wound healing (10, 11). In addition, HA is highly biocompatible and readily available. These attributes render it uniquely suited for imaging and drug delivery applications (12) which has been studied in cancer (13), cartilage (14), liver cirrhosis (15), and arthritis (16). However, HA has not been utilized for macrophage targeting. Macrophages are known to express HA receptors such as CD44, the major HA receptor in human bodies (17, 18). The interactions between HA and CD44 are crucial for the recruitment of leukocytes and macrophages in atherosclerotic plaque development (19-21). As part of our ongoing program to study atherosclerosis, we have begun to investigate whether the HA immobilized nanoparticles can be used to actively target and 1 Abbreviations: CDMT, 2-chloro-4,6-dimethoxy-1,3,5-triazine; DESPION, dextran coated superparamagnetic iron oxide nanoparticles; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HA, hyaluronic acid; HRMAS-NMR, high resolution magic angle spinning NMR; LPS, lipopolysaccharide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium; PMA, phorbol 12-myristate 13-acetate; TNF-R, tumor necrosis factor R.
10.1021/bc100354m 2010 American Chemical Society Published on Web 10/26/2010
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image macrophages. Herein, we report our results on the uptake and intracellular distribution of HA-coated magnetic nanoparticles in macrophages.
EXPERIMENTAL PROCEDURES Material. All chemicals were reagent grade and were used as received from the manufacturer. Dextran (MW 9-11 kDa), iron(II) chloride, ammonium hydroxide, sodium hydroxide, epichlorohydrin, bovine serum albumin, goat serum, and LPS from E. coli strain 0111:B4 were purchased from Sigma-Aldrich (St. Louis, MO) and used as is. Iron(III) chloride was purchased from Honeywell Riedel de Haen. Hyaluronic acid (MW 30 kDa) was purchased from Lifecore Biomedical. Fluorescein isothiocyanate (FITC), mouse antihuman CD44 monoclonal antibody (clone MEM85), and Alexa Fluor 594 goat antimouse IgG2b secondary antibody were purchased from Invitrogen (Carlsbad, CA). Water used during synthesis was cell culture grade Millipore filtered water. Cell Culture. THP-1 (human monocytic cell line), EA.hy926 (human vascular endothelial cell line), and LNCaP (human prostate cancer cell line) were purchased from American Type Culture Collection (Manassas, VA). THP-1 and LNCaP were maintained in RPMI 1640 supplemented with fetal bovine serum (10%, Sigma-Aldrich, endotoxin free), Penicillin-streptomycinglutamine (1%), HEPES buffer (1%), and sodium pyruvate (1%). EA.hy926 cells were kept in M199 supplemented with fetal bovine serum (10%), penicillin-streptomycin-glutamine (1%), HEPES buffer (1%), and sodium pyruvate (1%). The cells were kept in an 37 °C incubator equipped with 5% CO2. All cell culture related supplies were purchased from GIBCO (Invitrogen, Carlsbad, CA). MTS was purchased from Promega (Madison, WI) and TNF-R ELISA kit was purchased from R&D systems (Minneapolis, MN). Synthesis of HA-DESPIONs and FITC-HA-DESPIONs. Dextran (4.5 g, MW 9-11 kDa) and iron(III) chloride (0.32 g, 1.2 mmol) were dissolved in water (10 mL), and filtered through a 0.22 µm membrane (Millipore). The mixture was cooled to 0 °C and 0.22 µm membrane filtered solution of iron(II) chloride (0.13 g, 0.63 mmol) in water (0.45 mL) was added dropwise. The reaction mixture was then neutralized by addition of icecold ammonium hydroxide solution (0.45 mL). This greenish suspension was heated between 75 and 85 °C for 1.5 h with continuous stirring under a nitrogen atmosphere. Upon completion, reaction was cooled to room temperature, and the excess dextran and ammonium hydroxide were removed using ultrafiltration (Millipore). The reaction mixture was further concentrated to 20 mL total volume using ultrafiltration and filtered through 0.22 µm membrane, followed by washing with water (4 mL) leading to dextran-coated superparamagnetic iron oxide nanoparticles (DESPIONs). To this nanoparticle suspension, an aqueous solution of 5 M NaOH (10 mL) was added followed by dropwise addition of epichlorohydrin (4 mL). The reaction mixture was stirred vigorously for 24 h, after which it was purified using dialysis against DI water with 5 exchanges over 24 h. The reaction was then concentrated to final volume of 30 mL using ultrafiltration. Ammonium hydroxide (8 mL) solution was then added and the reaction was heated at 37 °C for 36 h. The reaction mixture was then purified using dialysis against DI water with 10 water exchanges over 2 days producing NH2DESPIONs. HA was then conjugated to NH2-DESPIONs. HA (100 mg, MW 30 kDa) was dissolved in water and mixed with Amberlite H+ resin until pH ∼3 and stirred for 4 h to convert HA into its carboxylic acid form. Upon completion, the resin was filtered off and water was evaporated to dryness. The protonated HA was then dissolved in a mixture of 4 mL water and 2.5 mL acetonitrile, under nitrogen. To this mixture, N-methyl morpholine (50 µL) was added, followed by addition
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of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 30 mg). The reaction was stirred at RT for 1 h, after which NH2-DESPIONs (26 mg in 10 mL water) was added. The reaction was stirred at RT for 20 h, followed by neutralization with Dowex H+ resin. The resin was filtered off and the product was dialyzed using a 14 kDa cutoff membrane for 2 days with 10 water changes. FITC labeling of HA-nanoparticles (HA-DESPION) was carried out by adding a solution of FITC (5 mg) in DMSO (1 mL) to an aqueous solution of HA-DESPION (30 mg, 6 mL water). As FITC has low aqueous solubility, DMSO (5 mL) was added to the reaction until a homogeneous solution was achieved. The reaction was stirred in the dark for 3 days and purified using ultrafiltration with 100 kDa cutoff membrane. High-Resolution Magic Angle Spinning (HRMAS) NMR. HRMAS NMR experiments were carried out on a Varian Inova500 NMR spectrometer equipped with a 4 mm gHX Nanoprobe (Variannmr Inc., Palo Alto, CA) available at the University of Tennessee Health Science Center (Memphis, TN). The HR-MAS probe with internal lock is capable of performing either direct or indirect (inverse) detection experiments. MAS experiments were performed at spinning rates of up to 2.5 kHz using a 40 µL glass rotor. The HA-DESPIONs were dissolved in D2O solvent and were further diluted at different concentrations with D2O to find out the concentration limit to the NMR signal broadening. HRMAS 1H NMR spectra were obtained using 100-600 scans for each experiment. The sample temperature was regulated with an accuracy of (0.1 °C. Competitive Enzyme Linked Immunosorbent Assay (ELISA). In a 96 well-plate, 100 µL of goat antihuman IgG FC γ (3 µg/well, Millipore, cat no. AP113) in PBS buffer was added to each well. The plate was covered with aluminum foil and incubated at 4 °C overnight. The wells were then washed 3 times with 200 µL PBST buffer (0.5% Tween 20) as follows: the plate was emptied by inversion over a sink, tapped against some layers of soft paper tissue to remove residual liquid, and then washed with buffer repeatedly (insufficient washings may lead to undesired high background). Blocking was completed by adding 5% BSA in PBS (200 µL) to each well followed by incubation at 37 °C for 2 h. The plate was then washed as described above. 100 µL of CD44-FC γ chimera (0.2 µg/well, R&D systems, cat no. 3660-CD) in PBS buffer was then added to each well and incubated at 37 °C for 45 min. The plate was washed 3 times with 200 µL PBST buffer (0.05% Tween 20) as before. 100 µL of b-HA (0.5 µg/well), b-HA + HA 16 KDa (2.5 µg/well), or b-HA + HA-NPs (7.32, 3.66, 2.75, 1.5, 0.88, 0.55, 0.15, 0.088, 0.055 µg HA/well) solutions were then added to each well and incubated at room temperature for 2 h. The plate was then washed 3 times with 200 µL PBST buffer (0.05% Tween 20) and twice with PBS buffer. 100 µL Avidin-HRP in 0.2% BSA-PBS solution was then added to each well and incubated at room temperature for 1 h. The plate was then washed 3 times with 200 µL PBST buffer (0.05% Tween 20) and twice with PBS buffer. 100 µL of chromogenic TMB solution was added to each well, homogenized, and incubated for 15 min, or until a blue color appeared. The plate should be protected against light during this incubation. The reaction was then quenched by adding 0.5 M H2SO4 (50 µL) to each well with positive wells becoming yellow. For quantitative measurements, it was important that each well was incubated for exactly the same length of time. Optical absorbance was directly assessed through the bottom of the microwell plate using an automated ELISA plate reader (Biorad) at 450 nm. Colloidal Stability Studies. The nanoparticles (HA-DESPIONs or FITC-HA-DESPION) were suspended in PBS or RPMI 1640 containing 10% FBS at a concentration of 1 mg/ mL. The change in size was monitored by over a period of six
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days using a Zetasizer Nano zs instrument. The samples were stored at RT and the FITC-HA-DESPIONs were stored in the dark at RT. MTS Cell Viability Assay. THP-1 cells were inoculated in a 96 well plate at cell density of 4 × 105 cells/mL, 200 µL/ well. They were differentiated using phorbol 12-myristate 13acetate (PMA, 10 ng/mL) to induce adhesion. After 16 h, the supernatant was replaced by the desired incubation media (either media alone or serum free media containing 50 µg/mL HADESPION). The incubation was carried out for varying periods of times (see data). The supernatant was removed, cells were washed using serum containing RPMI 1640 twice, and then incubated with serum containing media for 48 h. After 48 h, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS, 20 µL) reagent was added to each well and the plate was further incubated for 3 h. The intensity of brown color was measured using absorbance plate reader at 490 nm (Molecular Devices SpectraMax M5e). All the experiments were performed in triplicates. HA-DESPION Adherence Assay with THP-1 Cells. THP-1 cells were serially diluted from a concentration of 2 million to 8600 cells/mL and plated in a 96 well plate at 200 µL/well, followed by the addition of 20 µL of MTS reagent. The cells were incubated at 37 °C for 3 h and the resulting brown color was read at 490 nm using a plate reader (Molecular Devices SpectraMax M5e). The readings were used to generate a standard curve of absorbance vs number of viable cells. To perform the adherence assay, a 96 well nunc plate was coated with human umbilical cord HA (5 mg/mL) overnight at 4 °C. The plate was washed 3 times with PBS to remove unattached coating. The THP-1 cells were treated with LPS (1 µg/mL) for 2 h, washed with media, counted using Trypan blue. The activated THP-1 cells were added to half the plate with unactivated THP-1 cells on the other half as a control. The plate was then incubated for1.5 h at 37 °C. Upon completion, the plate was washed with cell culture media 6 times to remove unbound cells, followed by resuspension in 200 µL of media in each well. At this time, the MTS reagent (20 µL) was added to each well and the plate was incubated for 3 h and read using a plate reader at 490 nm. The absorbance was correlated to the cell density using the previously generated standard curve. Each set was done in triplicate. Determination of Cellular Uptake by Flow Cytometry. The cells were inoculated in a 6 well plate at a density of 5 × 105 cell/mL, treated with LPS (1 µg/mL) for 2 h at 37 °C, centrifuged to remove the supernatant media, and resuspended in serum free media containing FITC-HA-DESPION or FITCDESPION (2.5 µg of FITC/mL of media) for 2 h at either 37 or 4 °C. Upon completion, the cells were centrifuged, washed with serum containing media twice, resuspended in 300 µL of the media, and analyzed using BD Vantage SE flow cytometer. Viability was assessed using propidium iodide. For the EA.hy926, the same procedure was used except the LPS treatment. After incubation with nanoparticles, the cells were detached using trypsin-EDTA (0.5%) and processed using the above procedure. Cellular Uptake Monitoring by Confocal Microscopy. The cells were suspended in chambered cover glass (Nunc, FisherSci) at a density of 5 × 105 cells/mL. The THP-1 cells were treated with PMA (10 ng/mL) overnight to induce adhesion followed by LPS (1 µg/mL) treatment for 2 h at 37 °C. After one hour, the cells were treated with LysoTracker-594 DND (Invitrogen) at the final concentration of 1 µM, and the samples were incubated at 37 °C for another hour. Upon completion, the supernatants were removed and the cells were washed using PBS, fixed with 10% neutral buffered formalin for 20 min, and treated with 4′,6-diamidino-2-phenylindole (DAPI) at 300 nM concentration to stain the nuclei fluorescent blue. The samples
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were washed with PBS and water. Images were gathered using the Olympus FluoView 1000 LSM confocal microscope. Prussian Blue Staining. The cells were suspended in chambered cover glass (Nunc, FisherSci) at a density of 5 × 105 cells/mL. The THP-1 cells were treated with PMA (10 ng/ mL) overnight to induce adhesion followed by LPS (1 µg/mL) treatment for 2 h at 37 °C. The cells were washed to remove LPS and serum free media containing HA-DESPION or DESPION was added. The cells were incubated with the nanoparticles for half an hour, 1 h, 4 h, and 24 h, respectively, and were then washed repeatedly to remove the unbound particles. The cells were then fixed using 10% neutral buffered formalin for 20 min, washed with PBS buffer, and treated with 1:1 mixture of 4% potassium ferrocyanide and 4% HCl for 25 min at RT. Upon completion, the slides were rinsed with PBS and treated with 0.1 mL of nuclear fast red solution (Sigma) as a counterstain for 5 min, followed by rinsing with PBS. The resulting slides were observed under 40× magnification of an optical microscope. MRI of Cells. THP-1 cells were plated at a density of 5 × 105 cells/mL in a 6 well plate, differentiated using PMA (10 ng/mL) for 16 h followed by treatment with LPS at 1 µg/mL for 2 h at 37 °C. After 2 h, the supernatant was removed and HA-DESPION (100 µg/mL, 10 µg/mL Fe) was added in serum free media to separate wells, and incubated for 2, 4, and 24 h, respectively, at 37 °C. After completion, the supernatant was removed; cells were washed with PBS to remove unbound particles, resuspended in 0.5 mL PBS, and assessed using MRI. MRI measurements were carried out on a GE 3T Signa HDx MR scanner (GE Healthcare, Waukesha, WI) with a wrist coil. Fast, spoiled gradient recalled echo 3D T2*-weighted images were acquired with the following parameters: flip angle ) 15°; echo times (TEs) ) 5.9 ms, 14.5 ms, 23.0 ms, 31.6 ms, 40.2 ms, 48.7 ms, 57.3 ms, and 65.9 ms; time of repetition (TR) ) 72.5 ms; receiver bandwidth (rBW) ) (15.6 kHz; field of view (FOV) ) 6 cm; slice thickness ) 1.5 mm; number of slices ) 16; acquisition matrix ) 256 × 256; number of excitation (NEX) ) 1; and scan time ) 1 min 39 s. Antibody Blocking of THP-1 Cell Uptake of HA-DESPIONs. The THP-1 cells were differentiated using PMA (10 ng/mL) for 16 h at 37 °C, followed by treatment with LPS 1 µg/mL for 2 h at 37 °C. Various concentrations of mouse antihuman CD44 antibody (MEM-85 clone) were added to cells, and the cells continued to be incubated at 37 °C for 1 h. The supernatant was removed and FITC-HA-DESPION (100 µg/mL) was added in serum free media. The cells were incubated at 37 °C for one hour and then washed multiple times to remove unbound particles. Upon completion, the cells were lysed using 1% SDS and the fluorescence of the supernatant was measured using a plate reader. The experiment was performed in 5 sets for each time point.
RESULTS AND DISCUSSION Synthesis and Characterization of HA-DESPIONs. The preparation of HA coated nanoparticles started from formation of the water-soluble nanoparticles by co-precipitating Fe3+ and Fe2+ salts in the presence of dextran (9-11 kDa) (22). The resulting nanoparticles (DESPIONs) were cross-linked by epichlorohydrin to improve the stability of the coating, which was followed by ammonium hydroxide treatment leading to NH2-DESPIONs (22). HA (30 kDa) was then immobilized onto the nanoparticles through amide linkages promoted by CDMT (23) leading to magnetic nanoparticle HA-DESPIONs (Figure 1a). Dynamic light scattering data showed that the DESPION prepared has a hydrodynamic radius of 155 nm with a zeta potential of +43.4 mV. In comparison, HA-DESPION has a hydrodynamic radius of 205 nm with a zeta potential of -33.7
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Figure 1. (a) Synthesis, (b) TEM image, and (c) 1H-HRMAS NMR spectrum of HA-DESPION.
Figure 2. (a) Hydrodynamic radii (nm) of HA-DESPIONs in PBS or in 10% FBS containing media. (b) Percent of viable THP-1 cells upon incubation with HA-DESPIONs (100 µg/mL) as determined by MTS cell viability assays.
mV. The large change of zeta potential is attributed to the negatively charged nature of HA on the external surface of HADESPIONs. TEM revealed that the nanoparticle cores of HADESPIONs are quite homogeneous with an average diameter of 6 nm (Figure 1b). Under dry vacuum conditions encountered in TEM studies, the surface polysaccharides could collapse to form a tight coating, thus explaining the short interparticle distances observed in TEM images. TGA analysis showed that, upon HA incorporation, the percentage of the weight loss due to thermolysis of organics on HA-DESPIONs increased from 78% to 90% (Supporting Information Figure S1) and elemental analysis confirmed the increase of nitrogen upon HA functionalization (Supporting Information Table S1). The presence of HA on HA-DESPIONs was further ascertained by the HRMASNMR technique, which is a powerful method to overcome the field inhomogeneity caused by the superparamagnetic nature of the nanoparticles (24, 25). 1H-HRMAS NMR spectrum of the HA-DESPIONs showed resonances characteristic of HA with the anomeric protons appearing at 4.48 ppm and 4.43 ppm together with a singlet at 1.92 ppm due to the N-acetyl group (Figure 1c). On the basis of 1H NMR integration as well as TGA data, the molar ratio of HA to dextran is determined to be 3:1. In order to test whether HA immobilized on the nanoparticles can be recognized by receptors, we established a competitive ELISA procedure. CD44 was anchored onto microtiter plates in an IgG chimera form. The binding between a biotinylated HA polymer (0.5 µg/mL) and immobilized CD44 was then
tested in the presence of various concentrations of HADESPIONs. HA-DESPIONs effectively inhibited the binding between the HA polymer and CD44 with an IC50 value of 0.5 µg/mL, while the DESPION without HA did not have any effect on HA-CD44 interaction even at a concentration of 50 µg/mL. This proves that HA immobilized on HA-DESPION retains its biological recognition. Colloidal stability of nanoparticles under physiological conditions is an important factor for biological applications. The particle stability in biological media was evaluated by monitoring their hydrodynamic radii by DLS. The HA-DESPIONs maintained their sizes in both PBS buffer and culture media containing 10% fetal bovine serum (FBS) over one week, thus demonstrating that serum proteins do not induce nanoparticle aggregations (Figure 2a). HA-DESPIONs are also biocompatible, which had no effects on cell viabilities as determined through MTS cell viability assays (Figure 2b). In order to facilitate fluorescence monitoring of cellular interactions, fluorescein was attached onto HA-DESPIONs through the reaction with FITC. The resulting FITC-HADESPIONs were characterized in a same manner as HADESPIONs and were found to have similar properties. Fluorescein can also serve as a model drug to evaluate drug delivery. Control reaction of FITC and HA alone under identical conditions gave FITC-HA conjugate with 10% of fluorescence intensity compared with that from FITC-HA-DESPION. This suggests FITC is attached to both HA and the aminated dextran on the nanoparticle surface.
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Figure 3. Percentage of differentiated THP-1 cells adhered on HA coated plates with and without LPS treatment. LPS treatment significantly enhanced THP-1 affinity with HA.
THP-1 Nanoparticle Uptake Kinetics. With the HA coated nanoparticles in hand, we examined their interactions with a human monocytic cell line THP-1. THP-1 cells can be differentiated into adherent and macrophage-like cells by exposure to PMA, which has been used quite extensively as a model for human macrophages (26, 27). The presence of CD44 on THP-1 cells was established by immunohistostaining. Incubation of THP-1 cells with antihuman CD44 IgG mAb followed by Alexafluor 594 labeled anti-IgG secondary antibody gave intense cell surface staining (Supporting Information Figure S2a). Western blot analysis further confirmed the presence of CD44 in THP-1 cells (Supporting Information Figure S2d). It is known that the interactions between HA and CD44 are subject to tight regulations such that the receptor is normally maintained in a quiescent state showing little appreciable HA binding (28). For example, although freshly isolated peripheral blood monocytes and lymphocytes express CD44, they do not bind HA (29). During inflammation, however, the pro-inflammatory cytokine tumor necrosis factor R (TNF-R) induces sulfation and subsequent conformational changes of CD44, which transforms it into a form with much greater HA affinity (21, 28, 30, 31). To establish the THP-1 activation condition, we treated the PMA differentiated THP-1 cells with E. coli LPS, which led to the excretion of a large amount of TNF- R (2500 pg/mL) to the media (Supporting Information Table S2). In comparison, a very low TNF-R level (37.5 pg/ mL) was detected from THP-1 culture without the addition of LPS. The high TNF- R level following LPS should turn CD44 into its high-affinity HA binding form, which was tested in a plate adhesion assay (Figure 3). Upon incubation of cells on HA coated cell culture plates, 66% of the LPS activated THP-1 cells adhered to these plates as compared to 6% of cells not treated with LPS, thus confirming the enhanced affinity of activated THP-1 cells with HA (32). Next, we examined the interactions of HA-DESPIONs with THP-1 cells. LPS activated THP-1 cells were incubated with FITC-HA-DESPIONs for varying periods of time (0.5 h, 1 h, 2 h, 4 h, and 24 h), which was followed by extensive washing to remove unbound particles. The amount of fluorescence retained by the cells was then quantified. Significant amounts of the nanoparticles were rapidly uptaken by THP-1 cells, which did not change much after two hours (Figure 4a). The presence of HA on the nanoparticles was crucial for uptake, as the mean fluorescence intensity of cells incubated with FITC-DESPION without HA coating was only 20% of that with HA coated nanoparticles (Figure 4b). In addition, although other cells such as endothelial cell EA.hy926 also express CD44 (Supporting Information Figure S3), the THP-1 cells have 6-fold higher uptake capacities than EA.hy926 (Figure 4c). This suggests that the HA-DESPIONs can be used as a carrier to enhance delivery selectively to activated macrophages.
Figure 4. (a) Time-dependent uptake of FITC-HA-DESPION (100 µg/ mL) by LPS activated THP-1 cell as measured by the mean fluorescence intensity of the cells. (b) Uptake comparison between FITC-HADESPION and FITC-DESPION as well as FITC-HA-DESPION at 37 °C vs 4 °C by activated THP-1 cells. (c) FITC-HA-DESPION uptake by THP-1 cells compared with that by CD44 expressing endothelial cell EA.hy926.
It is known that extracellular HA is typically endocytosed into lysosomes (33). For effective drug delivery using the HADESPION platform, it is important that the cargo can escape the lysosomes after internalization inside the cells. To monitor the intracellular distribution, we analyzed THP-1 cells by confocal microscopy following incubation with FITC-HADESPION (Figure 5). Interestingly, the green fluorescence was found concentrated in the nuclei (cyan color in Figure 5d) with some in the lysosomes. This points to the potential of using HA-DESPIONs for nuclear drug delivery such as gene therapy (34). To further analyze nanoparticle uptake, we stained the THP-1 cells with Prussian blue stain, which reacts with iron ions present in the magnetite core yielding a characteristic blue color. The cells were incubated with nanoparticles for specific periods of time (0.5 h, 1 h, 4 h, and 24 h) and the unbound particles were washed off. With half-hour HA-DESPION incubation, most Prussian blue stains were found on THP-1 cell exterior surface as evident from the diffuse appearance of the blue color at cell surface (Figure 6a). With one hour incubation, the blue color appeared inside the cells indicating intracellular nanoparticle uptake (Figure 6b). Interestingly, as time went by, the blue color disappeared from inside the cells, suggesting efflux of the core of the nanoparticles from the cellular interior (Figure 6c,d). At 24 h, most of the staining was observed on the exterior cell surface. In contrast, with DESPIONs devoid of HA, the nanoparticles were retained inside the cells even after 24 h (Figure 6e), which was consistent with literature reports (35, 36). An important application of magnetic nanoparticles is in MR imaging, as the magnetite nanoparticles are effective MRI contrast agents (8, 37). The uptake of the HA-DESPIONs by activated THP-1 cells was monitored by MRI upon incubation
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Figure 5. Confocal microscopy images of LPS activated THP-1 cells 1 h after incubation with FITC-HA-DESPION. (a) Fluorescein channel; (b) Lysotracker channel showing location of lysosomes; (c) DAPI channel showing location of nucleus; (d) overlay of a-c; (e) laser image of the cells. The image overlay indicated that the fluorescein was concentrated in the cell nuclei.
Figure 6. Prussian blue staining images of activated THP-1 cells incubated with HA-DESPIONs after (a) 0.5 h (the dispersed blue staining patterns suggested that nanoparticles were bound to the outer cell membrane); (b) 1 h; (c) 4 h; (d) 24 h followed by washing off unbound particles. The magnetite cores were found to be exocytosed out of the cells. (e) Activated THP-1 cells after 24 h incubation with DESPIONS. The intracellular staining suggested the retention of the magnetite core inside the cells. (f) Activated THP-1 cells without nanoparticle incubations.
Figure 7. T2* weighted MR images of activated THP-1 cells (TE ) 40.2 ms). (a) Before incubation with HA-DESPIONs, (b) 2 h, (c) 4 h, and (d) 24 h after incubation with HA-DESPIONs (100 µg/mL) followed by washing off unbound particles. The significant T2* contrast changes upon nanoparticle incubation suggested HA-DESPIONs can be used to monitor macrophages by MRI.
of HA-DESPIONs with cells for set time periods and washing off unbound particles. Significant changes in T2 weighted MR images of THP-1 cells were observed (Figure 7). Despite the fact that most HA-DESPIONs were exocytosed out of the interior of the cells with 24 h incubation (Figure 6d), sufficient amounts of HA-DESPIONs remained bound on the cell surface leading to large contrast change in MRI (Figure 7d). This suggested the potential applicability of these nanoparticles for noninvasive in vivo monitoring of macrophages. Various iron oxide nanoparticles including DESPIONs have been used previously for MR imaging (8, 37). Although, in general, these nanoparticles are found to be biocompatible (38, 39), side effects
Figure 8. Mean fluorescence intensities of activated THP-1 cells incubated with various concentrations of blocking antibody MEM-85, followed by 1 h incubation with FITC-HA- DESPION (100 µg/mL) and washing off unbound particles. MEM-85 significantly reduced cell fluorescence intensities suggesting CD44 was involved in HA-DESPION uptake.
due to iron toxicities and nanotoxicities have been observed both in vitro and in vivo (40-42). For example, internalization of iron oxide nanoparticles resulted in a dose dependent reduction of the viability of growing neuron cells (41). Our observations that the magnetic cores of HA-DESPIONs are only transiently present inside the cells suggest the side effects from administrations of these particles will likely be small. Mechanistic Studies of Nanoparticle Uptake. To better understand HA-DESPION uptake, we examined its temperature dependence. While significant uptake was observed at 37 °C, incubation of HA-DESPION at 4 °C reduced the uptake to the background level (Figure 4b). In addition, the phagocytosis inhibitor cytochalasin D (43) did not significantly affect the uptake (data not shown), which suggested that the uptake was most likely due to an energy-dependent receptor-mediated endocytosis process. In order to test the role of CD44, LPS activated THP-1 cells were incubated with anti-CD44 mAb MEM-85, which has been shown to block the binding of HA by CD44 (44), prior to the introduction with HA-DESPIONs. In the presence of MEM-85, the uptake was significantly decreased highlighting the important role CD44 played (Figure 8). However, other mechanisms should be operating, as there was still 30% residual uptake following MEM-85 blocking. Fluorescein nuclear localization and exocytosis of the magnetite core can be explained by the involvement of CD44. HA-
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DESPIONs bound with cell surface CD44 and were endocytosed into endosomes and lysosomes. It is known that HA can be digested inside the cells, presumably by acid-active lysosomal hyaluronidases (45). Thus, the HA on HA-DESPIONs can be partially cleaved, which would release HA fragments labeled with fluorescein and allow their trafficking to the nucleus. This is supported by the observation from Wight and co-workers that exogenous HA added to permeabilized fibroblast cells could be found in cell nucleus (46). With the large amount of HA on the exterior surface, HA-DESPIONs could remain bound to CD44 in the endosomes, recycle to the cell surface, and be released from the cells accounting for the disappearance of magnetite cores from the intracellular space.
CONCLUSIONS We have synthesized iron oxide magnetic nanoparticles coated with HA, which have excellent colloidal stability and biocompatibility. The HA-DESPIONs retained the native biological recognition of HA by HA receptor CD44. HA-DESPIONs could be efficiently uptaken by activated macrophages, which enabled their imaging by MRI and paved the way for future in vivo studies. Furthermore, the dual labels (fluorescein and magnetite core) on the NPs enabled us to track the magnetite core and the cargo individually. This led to the unique observation that the magnetite core was only present transiently inside the cells. The cargo fluorescein was found to be delivered to the cell nucleus, unveiling the potential of using the HA-DESPION platform for nuclear drug delivery.
ACKNOWLEDGMENT This work was supported by an NSF CAREER award (X.H.), and an American Heart Association Postdoctoral Fellowship (M.N.K.) and Predoctoral Fellowship (K.E.). We would like to thank Prof. Jetze Tepe, Michigan State University, for access to the cell culture facility and the Department of Radiology, Michigan State University, for the usage of the MRI scanner. We would like to thank Melinda Frame for her help with confocal microscopy and Dr. Louis King for FACS studies. Supporting Information Available: TGA and elemental analysis of HA-DESPIONs; anti-TNF-R ELISA procedure and results; CD44 immunohisto-staining and Western blots of THP-1 and EA.hy926 cells. This material is available free of charge via the Internet at http://pubs.acs.org.
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