Quantum Dots Conjugated with Fe3O4-Filled Carbon Nanotubes for

Nov 6, 2012 - A novel and specific nanoplatform for in vitro simultaneous cancer-targeted optical imaging and magnetically guided drug delivery is dev...
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Quantum Dots Conjugated with Fe3O4‑Filled Carbon Nanotubes for Cancer-Targeted Imaging and Magnetically Guided Drug Delivery Mei-Ling Chen, Ye-Ju He, Xu-Wei Chen,* and Jian-Hua Wang* Research Center for Analytical Sciences, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China S Supporting Information *

ABSTRACT: A novel and specific nanoplatform for in vitro simultaneous cancer-targeted optical imaging and magnetically guided drug delivery is developed by conjugating CdTe quantum dots with Fe3O4-filled carbon nanotubes (CNTs) for the first time. Fe3O4 is filled into the interior of the CNTs, which facilitates magnetically guided delivery and improves the synergetic targeting efficiency. In comparison with that immobilized on the external surface of CNTs, the magnetite nanocrystals inside the CNTs protect it from agglomeration, enhance its chemical stability, and improve the drug loading capacity. It also avoids magnetic nanocrystals-induced quenching of fluorescence of the quantum dots. The SiO2-coated quantum dots (HQDs) attached on the surface of CNTs exhibit favorable fluorescence as the hybrid SiO2 shells on the QDs surface prevent its fluorescence quenching caused by the CNTs. In addition, the hybrid SiO2 shells also mitigate the toxicity of the CdTe QDs. By coating transferrin on the surface of the herein modified CNTs, it provides a dualtargeted drug delivery system to transport the doxorubicin hydrochloride (DOX) into Hela cells by means of an external magnetic field. The nanocarrier based on the multifunctional nanoplatform exhibits an excellent drug loading capability of ca. 110%, in addition to cancer-targeted optical imaging as well as magnetically guided drug delivery.

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INTRODUCTION Magnetic nanoparticles (MNPs) have received extensive attention in biomedical communities1 due to their potentials in medical applications.2−6 These applications associate closely with the unique magnetic properties of MNPs with comparable size to biologically important objects. The biological objects could be tagged on the MNPs, which are monitored and manipulated in the presence of external magnetic fields. However, MNPs in biomedical applications are so far not widely available due to their intrinsic instability, as the highly active MNPs are easily oxidized in air and tend to agglomerate for reducing the energy associated with high surface/volume ratio, which results in the loss of magnetism and dispersibility of naked MNPs.7 The highly dispersible magnetic carbon nanostructures, for example, hybrids with carbon nanostructures and controllable magnetic nanoparticles, exhibit favorable chemical reactivity and minimal cytotoxicity. This offers promising opportunities in biomedical applications.8 Recently, efforts have been made for fabricating magnetic carbon nanostructures by conjugating MNPs with carbon nanotubes (CNTs).9 The large surface area of CNTs facilitates loading of nanoparticles, biological species, and drugs, endowing multiple functions in nanoscale.10,11 Fe3O4-CNTs hybrid was reported by attaching Fe3O4 on the exterior surface of CNTs for drug delivery with an ca. 62% gemcitabine loading efficiency.12 CNTs were also decorated by Fe2O3 and Fe, which offers an ca. 3.2% doxorubicin loading efficiency.13 It is obvious © 2012 American Chemical Society

that these materials provide limited drug loading capability, because the exterior surface of CNTs is partly occupied by the MNPs. Therefore, magnetic carbon nanotubes (MCNTs) with better drug loading capability are highly desired for improving cancer therapeutic effect. For biomedicinal applications, fluorescence probe has been conjugated to MCNTs for visualizing the biological process. Among various fluorophores, QDs were frequently used for in vivo bioimaging due to their photostability and sharper spectra. A nanosystem by decorating the exterior surface of CNTs with iron oxide nanoparticles and CdTe QDs was reported for cell imaging.14 However, the attachment of iron oxide nanoparticles and QDs on the active surface of CNTs substantially reduced the CNTs capacity for drug storage and further functionalization.15,16 In addition, direct contact of QDs with iron oxide nanoparticles on CNTs surface causes fuorescence quenching and diminishes the magnetic-fluorescent dual functionality.17,18 These drawbacks might be eliminated by encapsulating MNPs into the interior cavity of CNTs. This could obviously avoid aggregation and corrosion of the MNPs, and minimize the potential toxic side-effects by exposure of free MNPs to biological environment.18 To the best of our knowledge, there Received: October 5, 2012 Revised: November 5, 2012 Published: November 6, 2012 16469

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Scheme 1. Preparation of Water-Dispersible DOX-Fe3O4@CNT-HQDs-Trf Conjugatesa

a

(A) Fe3O4@CNT; (B) PSS-coated Fe3O4@CNT; (C) Fe3O4@CNT-HQDs; (D) Fe3O4@CNT-HQDs-Trf; (E) DOX-Fe3O4@CNT-HQDs-Trf. Pro MPD diffractometer (PW3040/60, PAnalytical B.V, Holland). Surface charge property of the materials was investigated by measuring zeta potential with a Nano Zetasizer (Malvern, England). TEM images were obtained with a field-emission transmission electron microscope at an accelerating voltage of 200 kV (JEM-2100F, JEOL, Ltd., Japan). The hysteresis loops were obtained with a vibrating sample magnetometer (VSM 7407, LakeShore). Cell viability was assessed by MTT assay with multidetection microplate reader (μQuant, Biotek) at 570 nm. Cell image after taking up Fe3O4@CNT-HQDs was recorded on an inverted fluorescent microscope (Leica, blue and green excitation). Filling Fe3O4 into the Interior Cavity of CNTs. 1.5 g of CNTs was treated with 150 mL of nitric acid (3 mol L−1) under vigorous stirring in a round-bottom flask equipped with a condenser for 24 h. Next, the treated CNTs were collected by filtration and rinsed with DI water until neutral washout was achieved, followed by drying at 60 °C in vacuum for 10 h. The purified CNTs were cut into small pieces by suspending in a mixture of sulfuric acid/nitric acid (3:1, v/v) and sonicating at 65 °C for 24 h. The cut CNTs were finally obtained after filtration and thoroughly rinsed by ethanol and DI water, and dried at 60 °C in a vacuum for 10 h. 50 mg of the previously treated CNTs was put in a 4 mL centrifuge tube. The tube was suspended in a filter flask, which was then covered with a rubber stopper and evacuated (Figure S1). A saturated ferric nitrate solution in ethanol was added drop-wise into the centrifuge tube by an injector, followed by quickly opening the rubber stopper. A pressure difference between the internal and external of CNTs pushes the Fe3+ cations filling into the interior cavity of CNTs with the aid of sonication and stirring. After filtration and thorough washing with DI water and drying, the CNTs were put in a porcelain ark and calcined in nitrogen at 800 °C for 6 h (heating ramp: 3 °C/min) to convert the filled Fe3+ into Fe3O4 MNPs. The final product was collected by cooling to room temperature. Preparation of PSS-Modified Magnetic CNTs and HQDs Nanoparticles. Thirty milligrams of magnetic CNTs was mixed with 10 mL of PSS solution (1%, m/v) for 24 h under sonication. PSS was adsorbed onto the surface of CNTs via π−π interactions. After filtration and thorough washing with 0.1 M NaCl and DI water, CNTs were dispersed in water and allowed to stand for 24 h at room temperature to obtain a stable suspension. HQDs nanoparticles were obtained by a previously reported procedure.19 2.5 mL of thioglycollic-capped CdTe nanocrystals (4 × 10−4 mol L−1) was precipitated by 2-propanol and redispersed in 50 mL of aqueous solution of Cd2+ (1.65 mL, 0.1 mol L−1) and 36 μL of TGA (pH 11.2). 0.5 mL of diluted ammonia (6.25%, m/v) and 0.49 mL of TEOS were mixed with the redispersed CdTe colloidal solution in a beaker, which is sealed to minimize ammonia evaporation. After being stirred for 3 h, the CdTe nanoparticles were coated with a thin

is no report about magnetic−fluorescent nanohybrids by MNPs encapsulation with CNTs. We report herein the preparation (Scheme 1) of a highly versatile multicomponent nanosystem by filling the hollow cavity of CNTs with Fe3O4 MNPs and decorating the exterior surface with transferrin (Trf) and hybrid SiO2-coated QDs (HQDs). The Fe3O4@CNT-HQDs-Trf magnetic−fluorescent nanocomposites offer a number of advantages: (1) encapsulation of Fe3O4 in the interior of CNTs avoids agglomeration of the MNPs; (2) the hybrid provides a large effective surface area producing an improved drug loading capacity; and (3) the biocompatible and chemically inactive hybrid SiO2 shells provide a cover layer to mitigate the toxicity of QDs and to protect its fluorescence from being quenched by CNTs.19 In an external magnetic field, MNPs in the interior and Trf on the exterior surface of CNTs serve as a dual-targeted drug delivery system to penetrate cell membranes and thereby shuttle the drugs into cells, followed by targeting the Trf-receptors overexpressed on Hela cells. Meanwhile, the magneticfluorescent CNTs nanocarriers serve as a fluorescence imaging probe for observing the drug delivery efficiency and controlled drug release.

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MATERIALS AND METHODS

CNTs (i.d./o.d. = 6−8 nm/10−20 nm) were purchased from Chengdu Organic Chemicals, poly(allylamine) (PAH, Mw 15 000), poly (sodium 4-styrenesulfonate) (PSS, Mw 70 000), dimethyl sulfoxide (DMSO), and Transferrin (Trf, T3309) were obtained from Aldrich (Milwaukee, MI). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was the product of Shanghai Aladdin Reagents. N-Hydroxysuccinimide (NHS) was received from Acros Organics (Thermo Fisher Scientific). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit was the product of Nanjing KeyGEN Biotech. Doxorubicin hydrochloride (DOX) was received from Shanghai Sangon Biotech. DMEM (high glucose) cell culture medium, penicillin, trypsin, and streptomycin were purchased from Gibco Invitrogen. Sodium chloride, tetraethyl orthosilicate (TEOS), ferric chloride, and thioglycolic acid (TGA) were obtained from National Medicines Co., China. DI water of 18 MΩ cm was used throughout. UV−vis spectra were recorded on a U-3900 UV−vis spectrophotometer (Hitachi, Japan). Fluorescence spectra were measured on a F7000 fluorescence spectrometer (Hitachi, Japan). Magnetic properties were measured using a 7407 vibrating specimen magnetometer (LakeShore, U.S.). X-ray diffraction was conducted with an X’Pert 16470

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SiO2 layer containing Cd2+ and TGA. The mixture was refluxed to facilitate nucleation of the CdS-like clusters and growth in the SiO2 shell. The product was finally filtered through a 0.22 μm filter to remove the composite fibers created during the refluxing process. PAH Capping HQDs and Their Conjugation with Magnetic CNTs. Two milliliters of HQDs nanoparticles was precipitated by 2propanol and sonicated in PAH solution (1 mL, 10 mg mL−1) for 20 min. The PAH capped HQDs (PAH-HQDs) were collected and purified with 2-propanol by ultracentrifugation to remove excessive free PAH, and then redispersed in aqueous solution for future use. One milliliter of PAH-HQDs solution was mixed with 1 mL of PSSmodified magnetic CNTs (60 μg mL−1) and 100 μL of NaCl (0.2 mol L−1) solutions. The mixture was incubated at 37 °C for 2 h to facilitate conjugation of PAH-HQDs with magnetic CNTs. Fe3O4@CNTHQDs was then collected by centrifuging at 6000 rpm for 5 min followed by rinsing with ultrapure water three times. Finally, Fe3O4@ CNT-HQDs was resuspended in DI water. Preparation of the Fe3O4@CNT-HQDs-Trf Conjugate. Ten microliters of Trf (10 mg mL−1) reacted with 10 μL of EDC (10 mg mL−1) in dark at pH 5.5 in PBS for 10 min at room temperature. Ten microliters of NHS (10 mg mL−1) was then added, and the reaction continued for 20 min. The formed amine-reactive Trf-NHS ester reacts further with Fe3O4@CNT-HQDs in the dark at pH 7.4 in PBS for 3 h to form amide bonds, which finally produce Fe3O4@CNTHQDs-Trf conjugate. The excessive reagents were removed by discarding the supernatant after centrifugation, and the Fe3O4@ CNT-HQDs-Trf conjugate was washed three times with DI water and finally redispersed in fresh PBS and stored at 4 °C for future use. DOX Loading onto Fe3O4@CNT-HQDs-Trf and the Ensuing Drug Release Behaviors. DOX (1 mmol L−1) was mixed with 70 mg mL−1 of Fe3O4@CNT-HQDs-Trf and Fe3O4@CNT-HQDs in PBS at various pH values for 24 h in the dark at room temperature. The products were denoted as DOX-Fe3O4@CNT-HQDs-Trf and DOX-Fe3O4@CNT-HQDs and collected by repeated ultracentrifugation with PBS until the supernatant became free of reddish color (corresponding to free of DOX), and then suspended in PBS and stored at 4 °C. The amount of free DOX in the supernatant was determined by measuring its absorbance at 490 nm, on the basis of which the drug loading efficiency can be estimated. DOX-Fe3O4@CNT-HQDs-Trf was dispersed into 2 mL of PBS buffer in the dark at various pH at 37 °C. After different time intervals, the magnetic CNTs nanoassemblies were separated by ultracentrifugation, and the released amount of DOX in the supernatant was estimated by UV−vis spectroscopy. Cell Culture and Cell Viability Assay. Hela cells and HEK293 human kidney cells were maintained in a DMEM medium containing 10% fetal bovine serum, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin. Cell culture was cultivated in a complete medium at 37 °C in a humidified environment of 5% CO2. The cells were harvested from subconfluent cultures by using trypsin/EDTA solution (5%) and resuspended in fresh complete medium before plating. The dependence of in vitro viability of cells on Fe3O4@CNTHQDs was assessed by MTT assay. Cells were plated in 100 μL of DMEM at a density of 1000 cells in a 96-well plate. After incubation overnight to allow the cells to attach on the plate, 10 μL portions of DOX-Fe 3O4@CNT-HQDs-Trf, Fe3 O4@CNT-HQDs-Trf, DOXFe3O4@CNT-HQDs, and DOX with various concentrations were added to each well, and incubation was carried out for 24, 48, and 72 h at 37 °C in 5% CO2. Afterward, 50 μL of MTT (1 mg mL−1 in PBS) was added to the culture in each well followed by incubation for 4 h. The medium was then replaced with 150 μL of dimethyl sulfoxide, and the absorbance was monitored with a microplate reader at 570 nm. A linear relationship between cell number and optical density was established, and the cell viability ratio was calculated according to A570 nm/A570 nm(control). In Vitro Cellular Studies. After being cultured for 24 h, Hela cells were washed with PBS (pH 7.4) and then incubated with DOXFe3O4@CNT-HQDs-Trf and Fe3O4@CNT-HQDs-Trf at 37 °C for 1, 4, 12, and 24 h in DMEM medium. The cells were washed repeatedly with sterilized PBS before analysis. To further evaluate the role of Trf

in cellular uptake of DOX-Fe3O4@CNT-HQDs-Trf, HEK293 cells were cultured with DOX-Fe3O4@CNT-HQDs-Trf for 4 h and then washed with sterilized PBS and analyzed by an inverted fluorescence microscopy (blue excitation for Fe3O4@CNT-HQDs imaging, green excitation for DOX imaging).

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RESULTS AND DISCUSSION Preparation and Characterization of DOX-Fe3O4@ CNT-HQDs-Trf Conjugates. XRD patterns of CNTs and Fe3O4@CNT are illustrated in Figure 1. A strong diffraction

Figure 1. XRD patterns of (a) CNTs and (b) Fe3O4@CNT.

peak (2θ) at 25.98 and two small peaks at 42.78 and 44.35 were attributed to the graphite structure (002), (100), and (101) planes of the CNTs (Figure 1a). After Fe3O4 was filled into the interior of the CNTs, the characteristic peaks of CNTs remain unchanged, while new diffraction peaks were identified at 30.29, 35.67, 43.43, 57.20, and 62.81 (Figure 1b), corresponding to the (220), (311), (400), (511), and (440) planes of the standard XRD data for Fe3O4, which are in good agreement with the theoretical values (JCPDS card: 72-2303). These observations well indicate the formation of Fe3O4@CNT heterostructure. Figure 2 illustrated TEM images of bare CNTs, Fe3O4@ CNT, and Fe3O4@CNT-HQDs. It is obvious that Fe3O4 nanoparticles are uniformly distributed in the hollow cavity of CNTs (Figure 2B), while no changes were found in the exterior surface, which is as smooth as that of bare CNTs (Figure 2A). The size of Fe3O4 nanoparticles inside the hollow cavity of CNTs is approximately 6−8 nm, which is in agreement with the inner diameter of CNTs. After the formation of Fe3O4@ CNT-HQDs by conjugating Fe3O4@CNT with PAH-HQDs (Figure 2C), HQDs are clearly seen to coat on the surface of CNTs. It also illustrated that the diameter of Fe3O4@CNTHQDs is obviously increased as compared to that of Fe3O4@ CNT, due to the coating of PSS and HQDs on the surface of magnetic CNTs. UV−vis spectra of Trf, Fe3O4@CNT-HQDs, Fe3O4@CNTHQDs-Trf, DOX, and DOX-Fe3O4@CNT-HQDs-Trf are shown in Figure 3. The absorption at 280 nm due to Trf is clearly observed for Fe3O4@CNT-HQDs-Trf, proving successful conjugation of Trf on Fe 3 O 4 @CNT-HQDs. After adsorption on Fe3O4@CNT-HQDs-Trf, slight red-shifts were recorded for the absorptions of DOX at 490 and 232 nm,20 indicating interactions between Fe3O4@CNT-HQDs-Trf and DOX. In the case of DOX-Fe3O4@CNT-HQDs-Trf, the absorption of Trf at 280 nm is not clearly identified as it emerged by the strong absorption of DOX within UV region. 16471

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Figure 2. TEM images of (A) bare CNTs, (B) Fe3O4@CNT, and (C) Fe3O4@CNT-HQDs.

Fe3O4@CNT-HQDs-Trf. This indicated that DOX is adsorbed on the surface of Fe3O4@CNT-HQDs-Trf by π−π stacking. Magnetic and Optical Properties. Magnetic properties of the CNTs nanocomposites are evaluated by vibrating specimen magnetometer (VSM). Figure 4A shows that both Fe3O4@ CNT and Fe3O4@CNT-HQDs exhibit ferromagnetic behavior with a hysteresis loop at 300 K. Fe3O4@CNT exhibits saturation of magnetization (Ms) at 5.8 emu/g. This value is much larger than that of pristine CNTs (0.24 emu/g) from the magnetic catalyst residues, which are very difficult to remove completely even after purification by acid treatment. This is indirect evidence for the encapsulation of Fe3O4 in CNTs. After conjugation with HQDs on Fe3O4@CNT surface, the Ms value of Fe3O4@CNT-HQDs is droped to 3.4 emu/g, which might be explained by the diamagnetic contribution of HQDs surrounding the MCNTs.23 The inset in Figure 4A indicated that Fe3O4@CNT-HQDs dispersed in water can be well separated from the dispersion using an external magnet, suggesting potential applications of the nanocomposites in magnetic guiding and separation. The fluorescence spectra of Fe3O4@CNT and Fe3O4@CNTHQDs clearly illustrate that the conjugation of magnetic CNTs with HQDs endows it with excellent fluorescent properties (Figure 4B). In comparison, the fluorescence of its analogue by directly conjugating the QDs with Fe3O4@CNT, that is, Fe3O4@CNT-QDs, is almost completely quenched at identical conditions. This indicated that SiO2 shells coated on QDs play an important role in maintaining the fluorescence property of HQDs and endow Fe3O4@CNT with attractive luminescent property with a PL quantum yield of 18% (Table S1). The optical images in the inset illustrate well-dispersed Fe3O4@ CNT-HQDs in water with no aggregation, and very strong light emission from Fe3O4@CNT-HQDs is observed under UV irradiation. Figure 5A clearly indicated that the size of Fe3O4@CNTHQDs is smaller than 200 nm and the particle size of Fe3O4@ CNT-HQDs in aqueous solution remains virtually unchanged with the variation of its concentration in a certain range. Further studies indicated that Fe3O4@CNT-HQDs is insensitive to the variation of pH, and a constant PL intensity is recorded within a wide range of pH 3−10 (Figure 5B). This is of particular interest for serving as an intracellular imaging probe and drug carrier, as most intracellular organelles, for example, endosomes, are acidic at pH 4−6. The excellent stability of Fe3O4@CNT-HQDs might be explained in that the biocompatible hybrid SiO2 shells make QDs less sensitive to the surrounding chemicals, 19 and CNTs shell coating sequestrates the MNPs, avoiding its aggregation. Further studies show that Fe3O4@CNT-HQDs exhibit favorable stability in the biological media of NaCl, PBS, BSA, and DMEM (Figure 5C), and strong light emission is recorded for Fe3O4@CNT-HQDs under UV radiation in these media (Figure 5D), indicating no suppression on the emission of

Figure 3. UV−vis spectra of Trf, Fe3O4@CNT-HQDs, Fe3O4@CNTHQDs-Trf, DOX, and DOX-Fe3O4@CNT-HQDs-Trf. Inset: Photographs of Fe3O4@CNT-HQDs-Trf solutions without (a) and with (b) bound DOX.

The reddish color of DOX-Fe3O4@CNT-HQDs-Trf provides further evidence for the formation of DOX-CNT conjugate reflected by its characteristic absorption at 490 nm (Figure 3, inset b). Zeta potentials can provide further evidence for the identification of CNTs and their composite materials (Table 1). PSS is used to modify the magnetic CNTs to improve their Table 1. Zeta Potential Values of the Magnetic CNTs and Its Composite Materialsa material

zeta potential (mV)

HQDs PAH-HQDs Fe3O4@CNT Fe3O4@CNT-PSS Fe3O4@CNT-HQDs Fe3O4@CNT-HQDs-Trf DOX-Fe3O4@CNT-HQDs-Trf a

−39.0 +40.0 +1.0 −44.6 +44.0 +9.9 +10.1

± ± ± ± ± ± ±

2.1 3.2 0.2 1.8 4.6 0.4 0.3

The data are averaged from two measurements.

solubility, which results in a negative potential due to the presence of HSO3− groups on the surface. The potential was converted to positive after conjugation with PAH-HQDs, indicating the adsorption of PAH-HQDs onto the Fe3O4@ CNT-PSS. Although Trf is an acidic protein carrying a net negative charge at a pH that exceeds its pI value (pITrf = 5.9),21 Fe3O4@CNT-HQDs-Trf in aqueous solution (pH 7) shows a zeta potential of +(9.9 ± 0.4) mV. The positive value is probably due to the presence of free amine groups of PAH. In a neutral medium, the hydrophobicity of DOX is increased and its protonation decreased.22 Thus, after DOX loading, the zeta potential shows no obvious change as compared to that of 16472

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Figure 4. (A) Magnetic hysteresis curves of (a) CNTs, (b) Fe3O4@CNT, and (c) Fe3O4@CNT-HQDs. (B) Fe3O4@CNT-HQDs dispersed in water with a magnetic field. (C) Fluorescence spectra of Fe3O4@CNT, HQDs, Fe3O4@CNT-HQDs, and Fe3O4@CNT-QDs. (D) Photographs of Fe3O4@CNT-HQDs solutions ((a) in ambient condition, (b) under UV irradiation).

Figure 5. (A) Dependence of particle size of Fe3O4@CNT-HQDs on its concentration. (B) Dependence of fluorescence intensity of Fe3O4@CNTHQDs on pH. (C,D) Digital photographs for the dispersion status of Fe3O4@CNT-HQDs in (1) NaCl, (2) PBS, (3) BSA, and (4) DMEM (10% serum-containing medium) for 4 h incubation at 37 °C ((C) ambient condition, (D) UV irradiation).

in the endosomes/lysosomes, that is, pH 5.5, a significant increase of DOX release is observed. After incubating the drug loaded composites at pH 5.5 for 24 h, ca. 50% of the DOX originally loaded on Fe3O4@CNT-HQDs-Trf is released into the buffer medium. This pH-sensitive drug release behavior is of particular interest for the intracellular delivery of DOX in a biological system. Cellular Uptake of Magnetic/Fluorescent CNTs Nanoparticles. The uptake and intracellular distribution of the magnetic/fluorescent CNTs nanoparticles were evaluated by fluorescence microscope and TEM images. It is clearly seen from Figure 7A,B that cellular uptake of Fe3O4@CNT-HQDsTrf by Hela cells is significantly increased as compared to that of naked Fe3O4@CNT-HQDs. As a comparison, only slight

Fe3O4@CNT-HQDs in these biological media. This means that Fe3O4@CNT-HQDs can potentially be used as a novel fluorescent nanoprobe for intracellular imaging. DOX Loading on Magnetic CNTs Nanoassembly and Its Subsequent Release. The loading of DOX on Fe3O4@ CNT-HQDs-Trf is found to be effective; that is, a 110% loading efficiency is observed at pH 8.5 (the pH-dependent loading of DOX is illustrated in Figure 6a). Afterward, the drug loaded MNPs are suspended in a PBS buffer media with various pH values, that is, pH 5.5, pH 7.4, and pH 8.5, to evaluate the drug release behaviors. Figure 6b shows that the profile of DOX release from Fe3O4@CNT-HQDs-Trf is highly pH-dependent, and a very limited amount of DOX is released at pH 8.5 and 7.4. However, when the pH value is adjusted to be close to that 16473

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Figure 6. pH-dependent loading and release of DOX with Fe3O4@CNT-HQDs-Trf as drug carrier.

Figure 8. TEM images of (A) bare HeLa cells, and (B,C) Fe3O4@ CNT-HQDs-Trf treated HeLa cells for 12 h. The white arrows point to the aggregated Fe3O4@CNT-HQDs-Trf inside Hela cells, and the black arrows point to Fe3O4@CNT-HQDs-Trf outside Hela cells.

it could be a favorable intercellular imaging probe with the ability of cell penetration. Cellular Uptake of Drug Loaded DOX-Fe3O4@CNTHQDs-Trf. Cancer-targeted drug delivery and optical imaging are demonstrated by using the magnetic/fluorescent CNTs nanoparticles. Figure 9 illustrated the fluorescence images of

Figure 7. Fluorescence images of Hela cells incubated for 4 h with (A) Fe3O4@CNT-HQDs; (B) Fe3O4@CNT-HQDs-Trf; and (C) Fe3O4@ CNT-HQDs-Trf under magnetic field. HEK293 cells directly labeled by (D) Fe3O4@CNT-HQDs-Trf for 4 h. Left, bright-field images; middle, fluorescent images; right, merged images of the left and middle ones.

emission is observed when incubating the Trf negative HEK293 cells with Fe3O4@CNT-HQDs-Trf at the same conditions (Figure 7D). This demonstrates the targeting of Fe3O4@CNTHQDs-Trf to Hela cells through the interaction between Trf and receptors of the Hela cells. It is noticeable that the uptake of Fe3O4@CNT-HQDs-Trf is further remarkably improved in the presence of an external magnetic field (Figure 7C). This is attributed to the increase of Fe3O4@CNT-HQDs-Trf concentration in a confined area. The presence of Trf in Fe3O4@CNTHQDs-Trf and the use of a magnetic field exhibit a synergetic effect on targeting the magnetic/fluorescent CNTs nanoparticles in the Hela cells. TEM images are used to investigate the Fe3O4@CNTHQDs-Trf treated Hela cells to elucidate that the magnetic/ fluorescent CNTs nanoparticles are internalized in Hela cells rather than being bound to the surface of the cells. Unlike the bare Hela cells (Figure 8a), some black patches, that is, the aggregates of Fe3O4@CNT-HQDs-Trf, are observed inside the endosomes (Figure 8b,c). The cell morphology results indicated that the majority of internalized Fe3O4@CNTHQDs-Trf accumulates inside the endosomes, indicating that

Figure 9. Fluorescent microscopic images of Hela cells directly labeled by DOX-Fe3O4@CNT-HQDs-Trf for 4 h ((A) bright-field images; (B) fluorescent images by blue light excitation; (C) the merged images of (A) and (B); and (D) fluorescent images by green light excitation).

Hela cells incubated with DOX-Fe3O4@CNT-HQDs-Trf in the presence of a magnetic field. The yellow emission comes from HQDs, and the red emission is attributed to DOX. Figure 9A indicated that the drug molecules rapidly accumulate inside the cells and are ultimately targeted to the cell nuclei after 1 h incubation, while Fe3O4@CNT-HQDs-Trf itself does not seem to cross the nuclear membrane and exist in the cytoplasm. These results demonstrate that DOX releases at low pH 16474

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Figure 10. Concentration-dependent survival curves of Hela cells treated by magnetic/fluorescent CNTs nanoparticles for 24 h. The therapeutic effect of DOX, DOX-Fe3O4@CNT-HQDs, and DOX-Fe3O4@CNT-HQDs-Trf in the absence and presence of magnetic field is evaluated by MTT assay ((B) 24 h; (C) 48 h; (D) 72 h).

in addition to attractive fluorescence and extremely high drug loading efficiency. Fe3O4@CNT-HQDs exhibits low cytotoxicity, and only a minute amount is required for effective diagnostic and therapeutic applications. Fluorescence and TEM images indicated internalization of DOX-Fe3O4@CNT-HQDsTrf by endosomes where DOX is efficiently released and entered the cell nucleus and induced Hela cells death. This well indicated the effectiveness and selectivity of the drug carrier system, demonstrating the potentials of using Fe3O4@CNTHQDs for highly target-specific diagnosis and therapy. In addition, by encapsulating other magnetic crystals and conjugating with various fluorescent therapeutic agents, similar multifunctional nanocarriers may be developed for biomedical applications.

environment of the endosomes and migrates into the nucleus to bind DNA. Cytotoxicity Test. Figure 10 presents in vitro cytotoxicity studies of the magnetic/fluorescent CNTs nanoparticles in Hela cells by MTT assay. Hela cells are incubated with free DOX, DOX-Fe3O4@CNT-HQDs, and DOX-Fe3O4@CNTHQDs-Trf in the absence and presence of magnetic field for 24, 48, and 72 h, respectively. Fe3O4@CNT-HQDs-Trf exhibited almost no toxicity to Hela cells, whereas drug loaded DOX-Fe3O4@CNT-HQDs-Trf is more cytotoxic than free DOX and DOX-Fe3O4@CNT-HQDs, indicating that DOXFe3O4@CNT-HQDs-Trf is more readily internalized through the receptor-mediated endocytosis mechanism, while free DOX is transported into cells by a passive diffusion mechanism.24 In addition, the cytotoxicity is further enhanced in the presence of an external magnetic field due to the enrichment of DOXFe3O4@CNT-HQDs-Trf in a confined area near the Hela cells, resulting in more contacts of DOX-Fe3O4@CNT-HQDs-Trf with the cancer cells. This clearly demonstrated that the presence of Trf in Fe3O4@CNT-HQDs-Trf and the use of a magnetic field synergistically improved the growth inhibition effect on Hela cells.

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ASSOCIATED CONTENT

S Supporting Information *

Scheme of Fe3+ cations pushed to fill the CNTs interior cavity under vacuum by controlling the pressure difference, and quantum yield of the Fe3O4@CNT-HQDs. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSIONS Quantum dots conjugated with carbon nanotubes filled with Fe3O4, that is, Fe3O4@CNT-HQDs, create a new fluorescent nanocomposite that facilitates simultaneous imaging/mapping of cancer cells, rendering double-targeted drug delivery. The encapsulating of Fe3O4 in the interior cavity of CNTs and attaching of HQDs on the exterior surface combines the advantages of HQDs and CNTs and displays excellent stability,

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (X.-W.C.); jianhuajrz@ mail.neu.edu.cn (J.-H.W.). Notes

The authors declare no competing financial interest. 16475

dx.doi.org/10.1021/la303957y | Langmuir 2012, 28, 16469−16476

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ACKNOWLEDGMENTS This work is financially supported by the Natural Science Foundation of China (21075013, 21275027, 21235001, 21105008), the Program of New Century Excellent Talents in University (NCET-11-0071), and the Fundamental Research Funds for the Central Universities (N110605001, N110705002, and N110805001).

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dx.doi.org/10.1021/la303957y | Langmuir 2012, 28, 16469−16476