Multifunctional Carboxymethyl Cellulose-Based Magnetic Nanovector

Feb 14, 2013 - Katerina Hola , Zdenka Markova , Giorgio Zoppellaro , Jiri Tucek , Radek Zboril. Biotechnology Advances 2015 33 (6), 1162-1176 ...
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Multifunctional Carboxymethyl Cellulose-Based Magnetic Nanovector as a Theragnostic System for Folate Receptor Targeted Chemotherapy, Imaging, and Hyperthermia against Cancer Balasubramanian Sivakumar, Ravindran Girija Aswathy, Yutaka Nagaoka, Masashi Suzuki, Takahiro Fukuda, Yasuhiko Yoshida, Toru Maekawa, and Dasappan Nair Sakthikumar* Bio Nano Electronics Research Center, Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Japan S Supporting Information *

ABSTRACT: A multifunctional biocompatible nanovector based on magnetic nanoparticle and carboxymethyl cellulose (CMC) was developed. The nanoparticles have been characterized using TEM, SEM, DLS, FT-IR spectra, VSM, and TGA studies. We found that the synthesized carboxymethyl cellulose magnetic nanoparticles (CMC MNPs) were spherical in shape with an average size of 150 nm having low aggregation and superparamagnetic properties. We found that the folate-tagged CMC MNPs were delivered to cancer cells by a folate-receptor-mediated endocytosis mechanism. 5-FU was encapsulated as a model drug for delivering cytotoxicity, and we could demonstrate the sustained release of 5-FU. It was also observed that the FITC-labeled CMC MNPs could effectively enter cells, and the fate of nanoparticles was tracked with Lysotracker. The CMC MNPs could induce significant cell death when an alternating magnetic field was applied. These results indicate that the multifunctional CMC MNPs possess a high drug loading efficiency and high biocompatibility and with low cell cytotoxicity and can be considered to be promising candidates for CMC-based targeted drug delivery, cellular imaging, and magnetic hyperthermia (MHT).

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INTRODUCTION Nanoscience is considered to be one of the most prominent research arenas in recent times, and there are several scientific disciplines that benefit from it. Among the different scientific disciplines, biomedicine is one of the most promising research areas owing to the fact that nanobiomedicine developed from nanoscience and technology could revolutionize the diagnosis and therapeutic regime. Considering the biomedical applications of nanoparticles, advances in nanoscience and nanotechnology have driven magnetic nanoparticles (MNPs) to be extensively more exploited than other nanoparticles owing to their magnetic and nanosized features, two advantageous features that, when combined, offer a promising and efficient tool for biomedical research with advanced materials.1,2 MNPs, especially superparamagnetic iron oxide nanoparticles (SPIONS), with proper surface modification and functionalization have been extensively studied and are being commonly used in biomedical applications.3−17 MNP has the unique advantage that it can be employed for both diagnostic and therapeutic applications because it can be used as a contrast agent for magnetic resonance imaging, drug delivery, and magnetic hyperthermia. MNPs can be employed as effective drug carriers to offer targeted delivery and the sustained release of the same to enhance bioavailability, and it can also be steered to arrive at a targeted area of the body by the physical force from an external magnetic field.18 For biomedical © 2013 American Chemical Society

applications, MNPs should possess several physicochemical properties, including their size, high magnetization value, and biocompatible surface coating layer, which should be nontoxic. The choice to modify the surfaces of MNPs is crucial to engineering MNPs for successful application in vivo. Appropriate surface coatings offer a strategy to modify the surface properties of MNPs, including the surface charge and chemical functionality for targeting. One of the major roles of surface coatings is to offer a steric barrier to inhibit the agglomeration of nanoparticles. Another major advantage is that with an effective surface coating MNPs can escape uptake by the reticuloendothelial system (RES), thereby prolonging the blood circulation time. Several natural and synthetic polymers (e.g., polycaprolactone (PCL), polylactide (PLA) and poly(lactide-coglycolide) (PLGA)) have been intensively explored to study the nature of the chemical structure of the polymer, molecular weight and conformation, mode of polymer anchoring or attachment, and degree of particle surface coverage.19 Among the natural polymers, cellulose is the most abundant biopolymer, and cellulose-based derivatives have numerous advantages over several biopolymers such as biodegradability, recyclability, Received: December 20, 2012 Revised: February 10, 2013 Published: February 14, 2013 3453

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Synthesis of Free and Drug-Loaded Folate Conjugated CMC MNPs. The drug-loaded folate-conjugated CMC MNPs were prepared in three steps. Synthesis of Folate-Conjugated CMC Nanoparticles. Folic acid was conjugated to CMC nanoparticles by a previously reported method.20 Briefly, folic acid (1 mg/mL) was prepared and carboxyl groups were functionalized on the folic acid surface with 0.78 mg of N-(3-dimethyl aminopropyl)-N0-ethylcarbodiimidehydrochloride (EDC) and 0.98 mg of N-hydroxysuccinimide (NHS). The mixture was allowed to stir for 2 h at room temperature. Twenty-five milliliters of CMC (5 mg/mL) was added dropwise to the above mixture and was allowed to stir overnight. One percent CaCl2 was added to the folate-conjugated CMC mixture to achieve the ionic cross-linking of the nanoparticles. Synthesis of Drug-Loaded Folate-Conjugated CMC Nanoparticles. Two milligrams of 5-FU in methanol was added drop-bydrop to a folate-conjugated CMC mixture and was kept stirring for 1 h to evaporate the methanol. One percent CaCl2 was added for crosslinking, and the mixture was allowed to stir for 30 min. Synthesis of Drug-Loaded Folate-Conjugated CMC MNPs. Magnetite nanoparticles were prepared by a previously reported method based on the controlled chemical coprecipitation of Fe2+ and Fe3+ (1:2 ratio) in an ammoniacal medium at 80 °C under a nitrogen atmosphere.27,28 In a typical synthesis, 0.02 M of ferrous sulfate (FeSO4·7H2O) and 0.04 M of FeCl3·6H2O were dissolved in 200 mL of deionized water. The mixture was stirred and heated to 80 °C under a nitrogen atmosphere. Twelve milliliters of a 25% ammonia solution was injected into the flask. Stirring was continued for 20 min to allow the growth of the nanoparticles. After 20 min, the solution was cooled to room temperature and the resulting magnetite nanoparticles were centrifuged. The nanoparticles were washed three times with distilled water. The pH of the suspension was brought to neutral by adding dilute HCl, and the particles were rewashed with distilled water for further experiments. The as-synthesized magnetite nanoparticles were added drop-by-drop to folate-conjugated drug-loaded and drug-free CMC nanoparticle suspensions. The resulting reaction mixture was centrifuged, washed twice with deionized water and lyophilized to get a fine brown powder containing CMC MNPs that were used for characterization studies. Synthesis of FITC-Labeled, Drug-Loaded, Folate-Conjugated CMC MNPs. FITC in methanol (1 mg/mL) was added to 10 mg of folate-conjugated, drug-loaded CMC MNPs that had been dissolved in 10 mL of PBS at pH 7.4. The mixture was allowed to stir for 12 h in the dark under ambient conditions. FITC-labeled, folate-conjugated, drug-loaded CMC MNPs were collected with a permanent magnet to remove unconjugated FITC and washed with PBS at pH 7.4 several times. The obtained nanoparticles were used for imaging studies. Characterization of Nanoparticles. TEM images of magnetite nanoparticles and CMC MNPs were acquired with a JEM-2200-FS field-emission transmission electron microscope (JEOL, Japan) at an operating voltage of 200 kV. Hydrophilic treatment was used on TEM grids using a Joel Datum HDT-400 hydrophilic treatment device. TEM samples were prepared by dropping the diluted samples onto carboncoated hydrophilic copper TEM grids. EDS (JEOL JED-2300T) was carried out to analyze the elemental composition of magnetic nanoparticles. The SEM images of CMC nanoparticles and folate-conjugated CMC MNPs were acquired with a scanning electron microscope (JEOL, JSPM-6490, Japan). A nanoparticle suspension was diluted with ultrapure water and dropped onto a silica substrate and dried under vacuum, and the images were acquired. An FTIR spectroscopic analysis of CMC, folic acid, 5-FU, magnetic nanoparticles, and lyophilized samples of folate-conjugated CMC MNPs and 5-FU-loaded folic acidconjugated CMC MNPs was recorded on a Perkin-Elmer spectrophotometer in the spectral range of 4000 to 400 cm−1 at room temperature. The data sets were averaged over 32 runs. The thermogravimetric analysis (TGA) of CMC, magnetic nanoparticles, and CMC MNPs was carried out on a Shimadzu DTG-60H thermal analyzer. TGA of all the samples was performed up to a temperature of 900 °C, starting from room temperature in a nitrogen atmosphere. A heating rate of 10 °C/min was maintained for the samples. VSM analysis of the

reproducibility, cost effectiveness and availability in a wide variety of forms. However, the poor solubility of cellulose and also its poor reactivity make it challenging to engineer directly to other beneficial materials for biological applications. Thus the derivative modification of cellulose can overwhelm these limitations while preserving the advantageous features of cellulose. Carboxymethyl cellulose (CMC) is a water-soluble cellulose derivative with carboxymethyl groups (−CH2−COONa) bonded to some of the hydroxyl groups on the cellulose backbone. CMC has immense applications in the food, pharmaceutical, and cosmetic sectors owing to its viscosity, nontoxicity, and hypoallergenic nature.20,21 CMC nanoparticles offer carboxylic acid functionality to the nanoparticles, and hence they are often used as stabilizers for the syntheses of several other nanoparticles. 5-Fluorouracil (5-FU) is one of the efficient chemotherapeutic anticancer drugs that is normally administered either orally or through intravenous injections.23−24 The metabolism of 5-FU in the anabolic pathway inhibits the methylation reaction of deoxyuridylic acid to thymidylic acid. 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Thus a thymine deficit in the cell aggravates the unbalanced growth and death of the cancer cell. The effects of DNA and RNA deprivation are most obvious on those cells that grow most rapidly, especially cancer cells and hence the uptake of 5-FU at a more rapid rate. However, one of the major limitations of existing cancer chemotherapies is the lack of specificity of anticancer drug delivery, and hence most anticancer drugs have deleterious cytotoxic effects on normal healthy cells.24,25 Therefore, there is an increasing demand for the development of the efficient delivery of drugs to the targeted site to exploit the potency of chemotherapeutic agents. In this study, we introduce CMC as an efficient surface coating material to highlight our efforts in engineering targeted CMC MNPs for their multiple applications in imaging, drug targeting, and the destruction of cancer cells by magnetic hyperthermia (MHT). Because MNPs resist opsonization owing to their hydrophilic surfaces, they are cleared slowly and therefore our choice of coating material was CMC, which is an excellent biocompatible, biodegradable polymer. The CMC MNPs were made specifically to target the folate receptors that are often overexpressed in several cancers by conjugating the folate group to nanoparticles.26 The objectives of the current study are to demonstrate (i) the development and characterization of a biocompatible MNP based on CMC, (ii) the incorporation of drug 5-FU to study the antiproliferative activity of cancer cells in vitro, (iii) the imaging of cancer cells and the study of the uptake and fate of nanoparticles by specifically loading an imaging moiety into CMC MNPs, and (iv) the study of the effect of MHT on cancer cells with CMC MNPs.

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EXPERIMENTAL SECTION

Materials. Ferric chloride hexahydrate (FeCl3·6H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), NaOH, NH4OH, carboxymethyl cellulose, and calcium chloride were purchased from Kanto Chemicals, Japan. Folic acid and FITC were procured from Sigma-Aldrich (St. Louis, MO, USA). N-(3-Dimethyl aminopropyl)-N0-ethylcarbodiimidehydrochloride (EDC), and N-hydroxysuccinimide (NHS) were procured from Tokyo Chemical Industries, Japan. 2,4- Dihydroxy-5-fluoruracil (5-FU) was purchased from Nacalai Tesque, Inc., Japan. Trypan blue, 0.025% trypsin, and Annexin V-FITC apoptosis detection kit were purchased from Sigma-Aldrich. Alamar blue toxicology kit, DAPI, and Lysotracker were purchased from Invitrogen. 3454

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Cell Culture Maintenance. A human breast cancer cell line (MCF7cells), a glial cell line (G1 cells), and a mouse fibroblast cell line (L929 cells) were cultivated for in vitro experimental studies. The cell lines were obtained from the Riken Culture Collection Center, Japan. The MCF7 cell line was routinely grown in minimum essential medium supplemented with 5% heat-inactivated fetal bovine serum, sodium pyruvate, nonessential amino acids, and penicillin/streptomycin (100 units/mL). Mouse fibroblast cell line L929 and glial cell line G1 were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 5% heat-inactivated fetal bovine serum and penicillin/streptomycin (100 units/mL) in T25 flasks. All of the cell lines were cultured at 37 °C in a humidified 5% CO2 atmosphere, and the cells were subcultured every 2 days. The cells were maintained in glass-base dish for confocal studies, in 96 well plates for cytotoxicity studies, and in 35 mm plates for viability studies after MHT. Internalization Studies of MNPs. The internalization of nanoparticles by MCF7, G1, and L929 was studied with cellular imaging and flow cytometry. The labeling of nanoparticles with fluorescent components is one of the most commonly employed methods for analysis as a nonradioactive labeling technique. Fluorescently labeled nanoparticles offer a quick, simple, and effective way to quantify cell- linked nanoparticles by fluorometry.29−31 In our study, FITC was labeled with CMC MNPs to impart fluorescence to the nanoparticles for cellular imaging studies. MCF7 cell line G1 and mouse fibroblast cell line L929 were routinely grown in their respective medium as mentioned above on a glass-base dish for 24 h for imaging studies. The cells were washed with PBS, and 20 μL of nanoparticles suspended in a medium was added. The cells were incubated for different time intervals (1, 2, and 4 h). After incubation, the culture medium was removed and the cells were washed twice with PBS buffer and stained with DAPI. In addition to confirming the endosomal-mediated uptake of the nanoparticles, we performed Lysotracker staining to map the lysosomes of the cells that were viewed under confocal microscopy (Olympus IX 81 in DU897 mode).

samples was performed in a vibrating sample magnetometer (VSM model 7407, Lakeshore Cryotronics, Inc.) Drug Encapsulation Efficiency and Drug Release Studies. Encapsulation efficiencies of as-prepared CMC MNPs were determined by an indirect method. Briefly, the model drug, 5-FU-loaded, folate-conjugated CMC MNPs, in the reaction mixture was centrifuged at 10 000 rpm for 30 min. The pellet was washed with deionized water and resuspended in deionized water. The sample was freeze-dried to get the powdered sample. The supernatant that retained the unencapsulated drug was collected separately, and the absorbance of the same was recorded at 265 nm. The concentration of free drug was calculated from the absorbance value, which was based on the standard curve for 5-FU. Encapsulation efficiency percentage was calculated as follows

encapsulation efficiency (%) =

5-FUT − 5-FUF × 100 5-FUT

where 5-FUT is the total 5-FU and 5-FUF is the free 5-FU present in the supernatant. The in vitro drug release profile of 5-FU from nanoparticles was carried out in PBS at pH 5.5 and 7.4. Ten micrograms of lyophilized nanoparticles was dispersed in 50 mL of PBS at pH 5.5 and 7.4 in a water bath shaker set at 37 °C with a shaking speed of 120 rpm. Three milliliters of the sample supernatant was withdrawn at fixed time intervals to record the absorbance at 265 nm, and at the same time, the suspension was compensated with 3 mL of fresh PBS. drug release (%) =

5-FUR × 100 5-FUT

where 5-FUR is the concentration of 5-FU released at collection time t and 5-FUT is the total amount of 5-FU that was encapsulated in the nanoparticles.

Figure 1. Schematic representation of the synthesis of the multifunctional nanovector illustrating a folate-targeted CMC nanoparticle incorporating MNPs, anticancer drug 5-FU, and imaging moiety FITC. 3455

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The cellular uptake of fluorescent CMC MNP has been studied using flow cytometry (JSAN cell sorter, Bay Bioscience). MCF7, G1, and LAG were plated at a density of 2.5× 106 cells per well in their respective media. After 80% confluence was attained, 200 μg/mL FITC-labeled, folate-conjugated CMC MNPs was added to the plates and incubated for 4 h. After the incubation period, the cells were washed three times with PBS to remove any unbound nanoparticles. The cells were trypsinized and resuspended in 500 μL of PBS. Cells were fixed with 500 μL of 3% paraformaldehyde for 20 min. After fixation, the cells were again washed with PBS and permeabilized using

500 μL of ice-cold methanol and incubated for 10 min. PBS (500 μL) was added to the cells after incubation, and the cells were again washed and finally resuspended in 500 μL of PBS. Flow cytometry was performed in an FITC channel, and data were analyzed by JSAN App San software to study the uptake of nanoparticles by the cell lines. Cytocompatibility Analysis of CMC MNPs. Nanoparticle samples (bare and drug-free CMC MNPs) were prepared and diluted to different concentrations (0.5, 1, 1.5, 2, and 2.5 mg/mL) with PBS (pH 7.4) for treatment in 96-well tissue culture plates for cytotoxicity studies in the above-mentioned three different cell lines. The cell viability was estimated by alamar blue assay. Alamar blue assay signifies the reduction ability of the cells, indicating the active metabolism happening inside the cell. The active metabolism diminishes when the test materials express toxicity that leads to reduced capability. The fluorescence intensity of the alamar blue assay was quantified at 580−610 nm. Antiproliferative Studies on Cancer Cells. Antiproliferative studies of cancer cells when incubated with folate-conjugated, drugloaded CMC MNPs in the absence and presence of an external magnetic field were performed with control cells that were neither treated with nanoparticles nor exposed to a magnetic field. All experiments were performed in triplicate. Antiproliferative Studies of Plain 5-FU and 5-FU-Loaded CMC MNPs. Plain 5-FU at different concentrations and 5-FU-loaded CMC MNPs samples were diluted to different concentrations (1, 2, 3, and 4 mg/mL) with PBS (pH 7.4) for treatment in 96 well tissue culture plates for cytotoxicity studies in MCF7, G1, and L929 cells. The cell viability was estimated by the alamar blue assay. MHT-Based Cytotoxicity Mediated by CMC MNPs and 5-FULoaded CMC MNPs. We studied the effect of magnetic hyperthermia of folate-conjugated CMC MNPs and drug-loaded, folate-conjugated

Figure 2. Suspension of bare (A) MNPs, (B) CMC MNPs, and (C) FITC-labeled CMC MNPs under UV illumination.

Figure 3. TEM images of (a) Fe3O4 and (b) CMC MNP and TEM EDS of (c) Fe3O4 and (d) CMC MNP. 3456

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Figure 4. SEM images of (a) CMC nanoparticles; CMC MNPs (b) before dispersion and (c) after dispersion by sonication and dilution; and (d) SEM-EDS of CMC MNPs. CMC MNPs in two different cancer cell lines, namely, MCF7 (breast cancer cell line) and G1 (glioma cell line). Both cells were seeded at a density of 1.6 × 105 cell/mL in 35 mm tissue culture dishes. After 24 h of incubation, the culture was replenished with a fresh medium containing (2 and 4 mg/mL) folate-conjugated drug-free and drugloaded CMC MNPs at different concentrations. The cells in both concentrations were exposed to the ac magnetic field (H = 18.03 kA/m, B = 166.25A, frequency = 305 kHz) for 60 min. After the exposure, the cells were washed three times with PBS to remove the magnetic suspension. Then, the cells were harvested, trypsinized (0.025% trypsin, 5 min), and counted in the presence of trypan blue to determine the percentage of cell viability. To study the effect of incubation time on the viability of cells after magnetic hyperthermia, the cells were incubated in a CO2 incubator at 37 °C for varying periods (0, 4, 12, and 24 h) of time. The number of cells was counted to determine the change in the viability of cells with different incubation times. The viability of the cells that were exposed to the magnetic field was compared to that of the control cells at 37 °C that were not treated with a nanoparticle suspension or exposed to a magnetic field. The experiments were done in triplicate. Annexin V−PI Staining. Annexin V−PI staining is a commonly employed method of differentiating apoptotic and necrotic cells resulting after MHT. For the simultaneous detection of apoptotic and necrotic cell death, a co-staining technique with FITC-conjugated Annexin V, in tandem with DNA-binding dye PI, was used. Briefly, MCF7 and G1 were grown in a glass-base dish for 24 h with a standard medium, and the cells were then replenished with fresh medium containing drug-loaded CMC MNPs nanoparticles. The cells were exposed to the ac magnetic field (H = 18.03 kA/m, B = 166.25 A, frequency = 305 kHz) for 60 min for MHT. Cells were then stained with an apoptosis kit at three different time intervals (i.e., immediately after hyperthermia treatment and 12 and 24 h after MHT). The cells were washed in cold PBS to remove MNPs and were stained with

Annexin V conjugate and PI. The images were viewed with a confocal microscope (Olympus IX 81 under DU897 mode).

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RESULTS AND DISCUSSION Preparation of Nanoparticles. In this study, we synthesized uniform magnetic nanoparticles with the desired size on the basis of coprecipitation from Fe2+ and Fe3+ solutions under the N2 condition at 80 °C. Figure 1 illustrates the schematic representation of the synthesis of a multifunctional nanovector. A folate group was attached to CMC by an EDSNHS coupled reaction, resulting in the formation of a −CO− NH bond. The addition of 1% CaCl2 resulted in the ionic crosslinking of the nanoparticles. MNPs were added to CMC nanoparticles, and they were incorporated inside CMC nanoparticles to form folate-conjugated CMC MNPs. The colloidal suspension of the resulting ferrofluids is shown in Figure 2, clearly demonstrating that CMC is a good agent for stabilizing aqueous solutions of Fe3O4 nanoparticles. CMCstabilized Fe3O4 nanoparticles show a well-dispersed appearance, and the bare, unmodified Fe3O4 nanoparticles precipitate easily. The CMC MNPs were loaded with FITC and used for cell imaging studies. Fluorescent nanoparticles were stable up to 1 month upon storage in the dark. Characterization of Nanoparticles. A typical TEM micrograph of MNPs (Fe3O4) and CMC MNPs and an EDS spectrum of the samples are shown in Figure 3. The results suggested that MNPs were fairly smooth and spherical in shape with an average size in the range of 10 ± 3 nm. By TEM analysis, MNP-incorporated CMC nanoparticles were spherical. With the inclusion of MNPs, the particles remained spherical 3457

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with MNPs located inside CMC nanoparticles, and the average size of CMC MNPs were in the range of 100−150 nm. TEM EDS confirmed the presence of Fe and O in the sample. The morphology of plain CMC and CMC-modified MNP was characterized by SEM. We observed monodisperse smooth, spherical CMC nanoparticles of size around 100−150 nm (Figure 4a). CMC magnetite nanoparticles with spherical shapes were observed, with some degree of agglomeration (Figure 4b). We could get almost evenly dispersed CMC NPs after high dilution and ultrasonic treatment (Figure 4c). SEM EDS (Figure 4d) confirms the presence of C, Fe, and O in the nanoparticles that indicates the presence of the CMC coating over magnetite nanoparticles. Dynamic light scattering (Zetasizer) was employed to measure the average size distribution of the bare and CMC MNPs, both suspended in distilled water. The results (Figure 5)

Figure 6. Zeta potential of MNPs and CMC MNPs.

depicts the presence of CMC in the CMC nanocomposite and also the presence of negative charge imparted to the nanocomposite by CMC. The magnetic property of MNPs, CMC MNPs, folateconjugated, drug-loaded CMC MNPs, and FITC-loaded MNPs was investigated using a vibrating sample magnetometer at 300 K, and the obtained results are shown in Figure 7.

Figure 7. Magnetization vs magnetic field for the samples: (a) MNPs, (b) FITC-loaded MNPs, (c) CMC MNPs, and (d) folate-conjugated, drug-loaded CMC.

The remanent magnetization (Mr) and coercivity (Hc) for all samples were close to zero, demonstrating the characteristic of superparamagnetism. Though CMC-coated MNPs exhibited a slight decrease in magnetization compared to that of plain MNPs, an external magnet could very easily adsorb CMC MNP nanoparticles and the magnetization value was enough for common bioapplication.32 The observed result again proves that the CMC coating on the magnetic particles does not affect the magnetic behavior of the Fe3O4 core and these conjugates are robust enough to be manipulated by an external magnetic field for cancer cell hyperthermia. Also, it could offer a smooth and efficient approach to delivering a drug to a targeted site under an external magnetic field. The confirmation of the conjugation of the folate group, MNPs, and encapsulation of the drug, 5-FU, in CMC nanoparticles were studied by the changes observed in the FTIR spectrum of the composite. Figure 8 shows the FTIR spectra of (a) plain CMC, (b) folic acid, (c) 5-FU, (d) drug-loaded, folate-conjugated CMC MNPs, (e) folate-conjugated CMC MNPs, and (f) MNPs. Evidence for the conjugation of the folate group with CMC in the composite arises from the peak

Figure 5. Dynamic light scattering (DLS) spectra of (a) bare MNPs and (b) CMC MNPs.

suggest that the average hydrodynamic diameter of MNPs ranged between 45 and 80 nm, and we have observed an increase in the size of CMC MNPs. A broad distribution peak ranging from 80 to 200 nm distinctly shows the CMC coating over MNPs. The average size determined by DLS was slightly greater than the observed diameter of nanoparticles from TEM and SEM. This can be attributed to the aggregation of nanoparticles in the case of MNPs or the swelling of CMC for CMC MNPs. To study the change in the surface nature of MNPs by the CMC coating, we analyzed the zeta potential of the bare and CMC MNPs. The zeta potential typically measures the potential at the interface between a solid surface and liquid medium. Zeta potential studies of CMC-coated MNPs showed an increase in the absolute value of the zeta potential from −0.30 to −30 mV (Figure 6). The clear negative shift in the zeta potential of CMC MNPs compared to that of bare ones 3458

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1677 cm−1.36 The presence of a broad peak at 1434 cm−1 indicates the presence of CMC in the composite.37,38 These results indicate that 5-FU and MNPs were encapsulated in folate-conjugated CMC nanoparticles. The folate peak at 1607 cm−1 was observed in folate-conjugated CMC MNPs, whereas the peak at 1677 cm−1 showed that 5-FU was absent in the sample, indicating that the sample was devoid of drug. The TGA curves of (a) MNP, (b) CMC MNPs, and (c) plain CMC in a nitrogen atmosphere are shown in Figure 9. The rate of weight loss is augmented by the rise in temperature. In the TGA curve of CMC, two distinguishing areas can be observed where weight is being lost significantly. The initial loss in weight is attributed to the loss of moisture from the sample. The second loss may be due to the loss of CO2 from the polysaccharide. The COO− group in CMC could be decarboxylated in that temperature range. Alternatively, a slight weight loss was observed for MNPs in the temperature range of 50 to 150 °C owing to the evaporation of residual moisture that is adsorbed on the surface of the nanoparticles, and the particles displayed excellent thermal stability up to 900 °C. The TGA curve of CMC MNPs also displayed a similar pattern to that of raw CMC, demonstrating the decomposition of CMC at around 250 °C. The sharp weight loss in the second area that is observed in the curve of CMC was absent in the curve of CMC MNPs, which clearly indicates the presence of MNPs in the nanocomposite. From the TGA curves, it is evident that we could develop nanoparticles composed of both CMC and MNPs. Drug Encapsulation and Drug Release Profile Studies. The encapsulation of 5-FU was determined by the analysis of supernatant for free drug using UV−vis spectrophotometry after the recovery of the pellets that retains the encapsulated drug by ultracentrifugation. In our study, we could attain an encapsulation efficiency of 89% for the potent anticancer drug 5-FU when drug was added to folate-conjugated CMC MNPs. To assess the potential of utilizing CMC MNPs as carriers of 5-FU, the release profiles of 5-FU from CMC MNPs were evaluated at 37 °C in PBS at pH 5.5 and 7.4. The experimental conditions were maintained by regularly compensating for the

Figure 8. FTIR spectra of (a) plain CMC, (b) folic acid, (c) 5-FU, (d) drug-loaded, folate-conjugated CMC MNPs, (e) folate-conjugated CMC MNPs, and (f) MNPs.

at 1607 cm−1 in the nanoparticle, which clearly indicates the bending mode of −NH vibrations of the folate group.33 The FTIR spectrum of iron oxide nanoparticles exhibits strong bands in the lower-frequency region below 800 cm −1 due to the iron oxide skeleton, and other regions have weaker bands. The two significant absorption bands at 640 and 589 cm−1 in iron oxide are a consequence of the splitting of the absorption band of the Fe−O bond of bulk Fe3O4 at 570 cm−1 that shifts to a higher wavenumber.33,34 The broad band at 3300−3450 cm−1 was attributed to the −OH stretching vibration that arise from surface hydroxyl groups on the nanoparticles.35 It can be seen that by comparing the spectrum of the composite with that of pure 5-FU the peak at 1693 cm−1 was significant in the 5-FUencapsulated CMC MNPs. This peak can be attributed to the CO group that was seen in the pure 5-FU spectrum at

Figure 9. TGA curves of (a) MNP, (b) CMC MNPs, and (c) plain CMC. 3459

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The percentage of release of 5-FU was based on the normal standard curve of 5-FU. The drug release was performed for up to 60 h. The release kinetics at pH 5.5 and 7.4 within 60 h indicated that the pH strongly influenced the 5-FU release from CMC MNPs. Though the stationary phases were acquired at relatively nearby time intervals, drug release at both pH values showed a different pattern of release. We observed a small initial burst release of 18% in PBS buffer with pH 7.4 and 25% in pH 5.5 buffer in the first 2 h, which can be ascribed to the extra drug molecules that may be very close to the CMC MNPs diffusing quickly from the CMC matrix into buffer in the first few hours. More than 85% of the encapsulated drug was released in a period of 60 h in pH 5.5 buffer whereas 74% of the drug was released in pH 7.4 buffer. Between the two different pH conditions studied, drug release under acidic conditions enables a higher percentage of release of 5-FU that might favor anticancer drug delivery using CMC MNPs. Thus this pH sensitivity could be advantageous for drug release for the targeted tumor regions, so we suggest that our nanoparticle can be administered via different routes of drug delivery where pH is one of the significant parameters for drug release. Hence the slow drug release from the nanoparticles signifies the potential of the nanoparticles as a sustained drug delivery system for cancer therapy. Internalization of Nanoparticles. The cellular uptake and endocytosis of folate-conjugated CMC MNPs were studied by confocal microscopy to determine the intracellular fate of the same. The study was performed in folate-positive MCF7 and G1 cancer cell lines and folate-negative L929 cells as a control.

incubation medium. The suggested mechanism for drug release from nanoparticles may be due to the diffusion of drug from the polymer matrix and polymer matrix degradation.39 We propose that because the size of the drug molecule was much smaller compared the nanoparticle, which has been loaded, the diffusion of the drug molecule from the nanoparticle matrix might have played a major role in the release profile. However, the responsiveness of biopolymers to physiological changes such as pH, temperature, and external stimuli that trigger a sustained controlled release of the therapeutic agent is gaining greater importance.40 The in vitro release profile of CMC 5-FU MNPs is illustrated in Figure 10.

Figure 10. In vitro release of 5-FU from 5-FU-loaded CMC MNPs in PBS at different pH values.

Figure 11. Internalization of CMC MNPs by L929, G1, and MCF7 cells. (a, e, i) Bright-field images of the cells treated with nanoparticles, (b, f, j) nuclear staining with DAPI, (c, g, k) fluorescence images of cells internalized with FITC-loaded nanoparticles, and (d, h, l) exhibition of the lysosomal staining of the cells with Lysotracker red. 3460

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Figure 12. Bright-field and fluorescence confocal image of G1 and MCF7 cells treated with CMC MNPS at different time intervals. Bright-field images of (a, e, i) G1 and (c, g, k) MCF7 at 1, 2, and 4 h and confocal fluorescence of (b, f, j) G1 and (d, h, l) MCF7 treated with nanoparticles for 1, 2, and 4 h.

Figure 13. Flow cytometry analysis of FITC-labeled, folate-conjugated CMC MNPs in L929, MCF7, and G1 cells after 4 h of incubation. The results confirm the specificity of labeled targeted nanoparticles toward cancer cells.

After 4 h of incubation, the cells were washed with PBS and subjected to confocal analysis (Figure 11). The luminescence signal of the internalized nanoparticles was highly discrete. DAPI staining of the nucleus was carried out, and we observed that the cells were viable with discrete nuclear staining. The luminescence from the nanoparticles was seen in the cytoplasm, possibly entrapped in the vesicles around the nucleus, and indicates that there is no nuclear permeability of nanomaterials. To confirm the speculation about the endosome-mediated entry of nanoparticles, lysosomal staining was performed with Lysotracker red. The overlapping signal of the green fluorescent signal from FITC-loaded, folate-conjugated CMC MNPs and the red signal from lysosomes confirm the endosomal gained entry of nanoparticles into the cells.41

However, the efficiency of the nanoparticles for biological diagnostic imaging depends on their specificity directed toward cancer cells, leaving normal, healthy cells. To achieve the specific entry of nanoparticles into cancer cells, we engineered our CMC MNPs with folate as the targeting moiety that targets overexpressed folate receptors in cancer cells. MCF7, G1, and L929 cell lines were treated with fluorescent folateconjugated CMC MNPs for 4 h. We observed the strong green fluorescence of FITC in MCF7 and G1 cells when excited at 488 nm using green filters, whereas L929 cells show a negligible internalization of nanoparticles (Figure 11). The efficient internalization and fluorescence signals of nanoparticles toward cancer cells demonstrated the efficiency of the specific cancertargeting folate moiety on CMC MNPs. 3461

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The uptake of nanoparticles by the cells was also studied at different incubation periods. After 1 h of incubation with folate-conjugated CMC MNPs, the cells were analyzed for the internalization of nanoparticles in folate-positive MCF7 and G1 cells. Although fluorescence signals were observed, the fluorescence intensity was very low after 1 h of incubation (Figure 12). The incubation period was prolonged by extending the incubation of the cells with nanoparticles for 2 and 4 h to study the time-dependent uptake tendency of nanoparticles. Though we could observe an increase in the fluorescence intensity as a function of incubation time (2 and 4 h) in MCF7 and G1 cells, the fluorescence signals remain almost the same as that after 4 h even after prolonging the incubation time. This can be attributed to the saturation of nanoparticles binding to folate receptors on the cancer cells. The enhanced cellular uptake of targeted nanoparticles by different cell lines was also analyzed by flow cytometry. Figure 13 illustrates the results of flow cytometry analysis. The fluorescence intensity of MCF7 and GI cells incubated with folate-targeted CMC MNPs was stronger than that of the control L929 cells that clearly indicates the specificity of nanoparticles toward cancer cells. The shift in the intensity of fluorescence in cancer cells can be attributed to the enhanced uptake of nanoparticles that can be correlated with the confocal images in Figure 11. Cytocompatibility Analysis of Magnetic CMC Nanoparticles. To analyze the cytocompatibility of CMC MNPs, both the bare and carboxymethyl cellulose-coated magnetic nanoparticles were incubated with the MCF7, G1, and L929 cells for 24 h, and an alamar blue assay was carried out (Figures 14 and 15). When the cells were treated with bare

Figure 15. Cytocompatibility of CMC MNPs. Both cancer cells (MCF7 and G1) and control cells (L929) were treated with different concentrations of CMC-coated MNPs, and the viability was determined by an alamar blue assay.

the viability of cancer cells and normal cells; (2) the effect of MHT of drug-free CMC MNPs in two different cancer cell lines; and (3) the combined effect of drug and MHT on cancer cells. In the first case, we studied the cell cytotoxicity imparted by plain drug and drug-loaded CMC MNPs at different concentrations and different period of incubation in different cell lines. When the cells were treated with plain 5-FU, we observed increased cytotoxicity in a concentration-dependent manner in all of the cell lines irrespective of cancer and noncancerous cells (data provided in the Supporting Information, S1). In the case of folate-conjugated, drug-loaded nanoparticles, we observed that the cell viability decreased considerably as a function of the concentration of nanoparticles (Figure 16). In our studies, at the highest concentration of

Figure 14. Alamar blue assay results for the cytotoxicity of bare MNPs on MCF7, G1, and L929 cells at various concentrations. Figure 16. Antiproliferative effect of 5-FU-loaded CMC MNPs on different cell lines after 24 and 48 h of incubation.

MNPs at the highest concentration of 2.5 mg/mL, the viability was found to be 58, 55 and 52% for MCF7, G1, and L929 cells, respectively. The cellular viability was increased to 85, 86, and 85% in MCF7, G1, and L929 cells, respectively, when treated with the highest concentration, 2.5 mg/mL CMC MNPs, which clearly indicates the success of the surface modification of MNPs with CMC. This clearly ensures their safe application for drug delivery and magnetic hyperthermia. In addition, applying a carbohydrate coating to MNPs has not only proven to increase the cytocompatibility of the nanoparticles but also has enhanced the easy internalization of the same when the targeting moiety, folate, was attached to the nanoparticles. Antiproliferative Studies with CMC MNPs. The cell viability studies were studied under three different conditions: (1) the effect of plain drug and drug-loaded nanoparticles on

4 mg/mL, the decline in viability after 24 h of incubation was 59 and 55% for MCF7 and G1 cells, respectively, and the viability of L929 cells was 70% with the alamar blue assay. Because the in vitro drug release profile exhibited a sustainable release of drug over a period of 60 h, we extended the cell viability assay to 48 h. The cell cytotoxicity in cancer cells was greater with a prolonged incubation period that signifies the action of the drug in the cells. The viability was drastically reduced to 34 and 32% in MCF7 and G1, respectively. Because the nanoparticles were tagged with the folate group, the chance of nanoparticle uptake by the cancer cell line could be much greater when compared to that of the normal cell line, L929, because the cancer cells possesses numerous folate receptors. 3462

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Figure 17. MHT setup. Cells in culture plates treated with CMC MNPs were exposed to an alternating magnetic field for HT study.

Figure 18. MHT study of CMC MNPs and drug-loaded CMC MNPs in cancer cells. MCF7 and G1 cells treated with drug-loaded and drug-free CMC MNPs were exposed to an alternating magnetic field for HT treatment, and the viability was acessed by trypan blue staining.

Therefore, a considerable decrease in cell viability has been observed in G1 and MCF7 cells compared to that in L929 cells. We studied the MHT of CMC MNPs using the experimental setup in Figure 17 in the second and third cases. Cancer cells were grown on a culture plate and were exposed to an alternating magnetic field for 60 min at different concentrations of CMC MNPs. In the second case, the effect of MHT on drug-free CMC MNPs was investigated in two different cancer cell lines, MCF7 and G1 cells, and the result is depicted in Figure 18. We performed the studies in three sets: (a) control cells, without a field and without MNPs, (b) cells without nanoparticles but exposed to a field for 60 min, (c, d) cells with 2 and 4 mg/mL nanoparticles, respectively, and a field applied for 60 min. The exposure of the cells to the magnetic field in the absence of CMC MNPs did not show any significant effect on cell viability, which clearly indicates that magnetic field exposure does not cause any severe damage to cells. However, the viability of the cancer cells was found to be reduced drastically when the cells were exposed to the ac magnetic field in the presence of CMC MNPs for a period of 60 min. To confirm the cell viability after HT, we trypsinized the cells under study and immediately began to count cells after trypan blue staining. The cell pellet obtained after centrifugation was diluted with 1 mL of medium for trypan blue staining. Because trypan blue dye cannot enter viable cells and remain excluded by cells, only the dead cells take up trypan blue. The trypan blue-stained cells were quantified via Countess. We observed that the cell cytotoxicity of drug-free CMC MNPs with MHT alone was 59 and 57% in MCF7 and G1 cells, respectively, at a concentration of 4 mg/mL nanoparticles. In the third set of experiments (Figure 18), we studied the combined cytotoxic effect of MHT and drug-loaded CMC MNPs. When cells were treated with 5-FU-loaded CMC MNPs in the presence of a magnetic field for 60 min, the cell viability

decreased significantly to 25 and 23% for MCF7 and G1 cells, respectively. The decrease in cell viability was much greater when compared to that of cells treated with drug-free CMC MNPs. MHT might have triggered the release of drug from the CMC matrix, resulting in increased cell death. These results imply that MHT application along with the targeted drug can work as an effective tool to target heat to cancer-affected areas, thus significantly overcoming the existing challenges associated with traditional HT treatments to kill cancer cells. Cell cytotoxicity induced by MHT at different time intervals of incubation was studied (Figure 19 and Table 1). Drug-free

Figure 19. Effect of magnetic hyperthermia on the viability of MCF7 and G1 cells in the presence and absence of drug-loaded CMC MNPs at different time intervals.

and drug-encapsulated CMC MNPs (4 mg/mL) were used for the study. The observed cell viability was 41 and 43% for MCF7 and G1 cells, respectively, immediately after MHT treatment with drug-free CMC MNPs whereas the viability was only 25 and 23% for MCF7 and G1 cells, respectively, when 3463

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Table 1. Percentage of Cell Viability after Magnetic Hyperthermia with Plain CMC MNPs and 5-FU-Loaded CMC MNPs at Different Time Intervals in Cancer Cells hyperthermia with CMCMNPs

hyperthermia with CMCMNPs + drug

cell lines

control

immediately after MHT

12 h

24 h

immediately after MHT

12 h

24 h

MCF7 G1

100 100

41 43

32 30

23 21

25 23

19 15

6 4

Figure 20. Annexin V−PI staining after MHT. Bright-field images of (a, d, g) G1 cells immediately after hyperthermia, 12 and 24 h, (b, e, h) cells undergoing apoptosis after hyperthermia, and (c, f, i) necrotic cells stained with PI after hyperthermia at different time intervals.

Annexin V−PI Staining. MHT treatment is capable of inducing both necrosis and apoptosis.42−46 Necrosis is cell death induced by external damage, which is normally arbitrated via annihilation of the plasma membrane through the loss of its integrity, and it can occur in a very short period of time. Apoptosis is based on the concept of programmed cell death, which consists of a much slower sequence of events that requires a few hours to several days. It is the mechanism in which a cell follows a programmed series of incidents to “prepare” for its death with minimal disturbance to neighboring cells. The indices of apoptosis, both morphological and biochemical, are distinctive and are entirely different from those of necrosis. One of the early events in apoptosis is the display of phosphatidylserine on the external surface of the plasma membrane that binds to Annexin V, which is displayed as green florescence by FITC. Red fluorescence with propidium iodide is indicative

treated with drug-loaded CMC MNPs. After 12 h of incubation, the cell viability decreased further to 32 and 30% for drug-free CMC MNPs and to 19 and 15% for drug-loaded CMC MNPs. After 24 h of incubation, the cell viability was 23 and 21% for drug-free CMCMNPs and 6 and 4% for drug-loaded CMC MNPs in MCF7 and G1 cells, respectively. Hyperthermia treatment resulted in immediate cell death after exposure to the field, but a remaining small number of cells survived. The surviving injured cells could not recover from the damage induce by hyperthermia owing to the action of the drug present in the CMC matrix. The drug might have diffused upon HT and resulted in cell death upon incubation. We propose that these could be the reasons for the observed cell death during incubation. This result strongly suggest that HT-induced drugloaded CMC NPs have great potential in the cancer treatment scenario. 3464

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part of this study has been supported by a grant for the Programme of the Strategic Research Foundation at Private Universities S1101017, organized by the MEXT, Japan, since April 2012.

of the loss of membrane integrity associated with necrosis (Figure 20) Inside the cell, endosomes and lysosomes are involved in the endocytic pathway, and the lysosomes are the terminal degradation compartment of the phagocytosed material. It is obvious that endocytic and phagocytic vesicles fuse with lysosomes, resulting in secondary lysosomes. The confinement of MNPs within endosomal structures (as observed from confocal images, Figure 11) considerably reduced their contact with intracellular structures. Therefore, the cell death mechanisms can be based on the concept that the MNPs confined in the lysosomes aggravate the disruption of lysosomal membrane when the cells are subjected to alternating magnetic field. The lysosomal inclusion is then released into the cytoplasmatic space, eliciting cell death. It may be pure necrotic cell death or a quick apoptosis and also pure apoptosis, as observed in confocal analysis (Figure 20). The translocation of phosphatidylserine and propidium iodide internalization owing to damage in the plasma membrane resulted in the cell death that is the cumulative effect of both apoptosis and necrosis events in cancer cells, after the application of the external magnetic field. A comprehensive evaluation of the actual mechanism at the cellular level including the study of different markers, such as caspases, proteases, and cytokines, is essential to studying the mechanism of cell death.

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(1) Hao, R.; Xing, R. J.; Xu, Z. C.; Hou, Y. L.; Gao, S.; Sun, S. H. Synthesis, Functionalization, and Biomedical Applications of Multifunctional Magnetic Nanoparticles. Adv. Mater. 2010, 22, 2729−42. (2) Dave, S. R.; Gao, X. Monodispersed Magnetic Nanoparticles for Biodetection, Imaging, and Drug Delivery: A Versatile and Evolving Technology. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2009, 6, 583−609. (3) Koppolu, B.; Rahimi, M.; Nattama, S.; Wadajkar, A.; Nguyen, K. T. Development of Multilayer Polymeric Particles for Targeted and Controlled Drug Delivery. J. Nanomed. Nanotechnol. 2010, 6, 355− 361. (4) Lu, A. H.; Salabas, E. L.; Schuth, F. Magnetic Nanoparticles: Synthesis, Protection, Functionalization, and Application. Angew. Chem., Int. Ed. 2007, 46, 1222−1244. (5) Yanase, M.; Shinkai, M.; Honda, H.; Wakabayashi, T.; Yoshida, J.; Kobayashi, T. Intra-cellular Hyperthermia for Cancer Using Magnetite Cationic Liposemes: An in vivo Study. Jpn. J. Cancer Res. 1998, 89, 463−470. (6) Moroz, P.; Jones, S. K.; Gray, B. N. Magnetically Mediated Hyperthermia: Current Status and Future Direction. Int. J. Hyperther. 2002, 18, 267−284. (7) Zhu, A.; Yuan, L.; Jin, W.; Dai, S.; Wang, Q.; Xue, Z.; Qin, A. Polysacharide Surface Modified Fe3O4 Nanoparticles for Canaptothecin Loading and Delivery. Acta Biomater. 2009, 5, 1489−1498. (8) Kumar, A.; Jena, P. K.; Behera, S.; Lockey, R. F.; Mohapatra, S.; Mohapatra, S. Multifunctional Magnetic Nanoparticles for Targeted Delivery. J. Nanomed. Nanotechnol. 2010, 6, 64−69. (9) Sathe, T. R.; Agrewal, A.; Nie, S. Mesoporous Silica Beads Embedded with Semiconductor Quantum Dots and Ion Oxide Nanocrystals: Dual-Function Microcarriers for Optical Encoding and Magnetic Separation. Anal. Chem. 2006, 78, 5627−5632. (10) Pankhurst, Q. A.; Thanh, N. K. T.; Jones, S. K.; Dobson, J. Progress in Applications of Magnetic Nanoparticles in Biomedicine. J. Phys. D: Appl. Phys. 2009, 42, 224001−224014. (11) Berry, C. C.; Curtis, A. S. G. Functionalization of Magnetic Nanoparticles for Applications in Biomedicine. J. Phys. D: Appl. Phys. 2002, 36, R167−R181. (12) Jain, T. K.; Foy, S. P.; Erokwu, B.; Dimitrijevic, S.; Flask, C. A.; Labhasetwar, V. Magnetic Resonance Imaging of Multifunctional Pluronic Stabilized Iron-Oxide Nanoparticles in Tumor-Bearing Mice. Biomaterials 2009, 30, 6748−6756. (13) Gneveckow, U.; Jordan, A.; Scholz, R.; Bruss, V.; Waldofner, N. Description and Characterization of the Novel Hyperthermia and Thermoablation System MFH 300F for Clinical Magnetic Fluid Hyperthermia. Med. Phys. 2004, 31, 1444−1451. (14) Widder, K. J.; Senyel, A. E.; Scarpelli, G. D. Magnetic Microspheres: A Model System of Site Specific Drug Delivery In Vivo. Pro. Soc. Exp. Biol. Med. 1978, 141−146. (15) Bisht, S.; Feldmann, G.; Sony, S.; Ravi, R.; Karikar, C.; Maitra, A.; Maitra, A. Polymeric Nanoparticle-Encapsulated Curcumin (“Nanocurcumin”): A Novel Strategy for Human Cancer Therapy. J. Nanobiotechnol. 2007, 5, doi:10.1186/1477-3155-5-3. (16) Kropke, R. D.; Wassel, R. A.; Mondalek, F.; Grady, B.; Chen, K.; Liu, J. Z.; Gibson, D.; Dormer, K. J. Magnetic Nanoparticles: Inner Ear Targeted Molecule Delivery and Middle Ear Implant. Audiol. Neurootol. 2006, 11, 123−133. (17) Sou, K.; Inenaga, S.; Takeoka, S.; Tsuchida, E. Loading of Curcumin into Macrophages using Lipid-Based Nanoparticles. Int. J. Pharm. 2008, 352, 287−293. (18) Yoon, T. J.; Kim, J. S.; Kim, B. G.; Yu, K. N.; Cho, M. H.; Lee, J. K. Multifunctional Nanoparticles Possessing A “Magnetic Motor

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CONCLUSIONS In our study, a multifunctional carboxymethyl cellulosebased nanovector tailored for targeting, imaging, and efficient delivery of a therapeutic agent was developed. The nanovector was targeted to a folate-receptor tumor marker that was overexpressed in cancer cells. Our nanovector codelivered superparamagnetic fluorescent nanoparticles and a potential therapeutic agent to cancer cells. Potential anticancer drug 5-FU was encapsulated and was released in a sustainable manner over a period of 60 h. The combined treatment of cancer cells with targeted drug-loaded CMC MNPs and MHT synergistically killed almost 95% of cancer cells. The collective results of (1) the biocompatibility of the CMC MNPs (low cytotoxicity with drug-free CMC MNPs) and (2) the huge decrease in the viability of cancer cells after the simultaneous exposure of drug and the application of an alternating magnetic field suggest that drug-loaded CMC MNPs could be used as an efficient nanovector against cancer. In addition, fluorescent CMC MNPs were also exploited for cancer cell imaging, thus tailoring these nanoparticles to a theragnostic nanovector for effective cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

Cytotoxicity of L929, MCF7, and G1 cells when treated with 5-FU. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Fax: (+81)-(0)366-77-1140. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS B.S. thanks the Rotary Yoneyama Foundation, Japan, for providing a fellowship for conducting his doctoral course. Also, 3465

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