Aptamer-Capped Multifunctional Mesoporous Strontium

Nov 9, 2012 - Department of Radiology, The Second Hospital of Jilin University Norman Bethune, Changchun, 130041, P. R. China. •S Supporting ...
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Aptamer-Capped Multifunctional Mesoporous Strontium Hydroxyapatite Nanovehicle for Cancer-Cell-Responsive Drug Delivery and Imaging Zhenhua Li,†,‡ Zhen Liu,†,‡ Meili Yin,† Xinjian Yang,†,‡ Qinghai Yuan,§ Jinsong Ren,*,† and Xiaogang Qu*,† †

State Key Laboratory of Rare Earth Resource Utilization, Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China ‡ Graduate School of the Chinese Academy of Sciences, Beijing, 100039, P. R. China § Department of Radiology, The Second Hospital of Jilin University Norman Bethune, Changchun, 130041, P. R. China S Supporting Information *

ABSTRACT: A novel cancer-cells-triggered controlled-release gadoliniumdoped luminescent and mesoporous strontium hydroxyapatite nanorods (designated as Gd:SrHap nanorods) system using cell-type-specific aptamers as caps has been constructed. Aptamers behave as a dual-functional molecule that acts as not only a lid but also a targeted molecular that can be used in an effective way for therapeutically special cancer cells. After incubated with cancer cells, for example, MCF-7 cells, the doxorubicin-loaded and aptamercapped Gd:SrHap nanorods (designated as Gd:SrHap-Dox-aptamer) can be internalized into MCF-7 cells, resulting in the pore opening and drug releasing. Furthermore, the high biocompatibility and biodegradability Gd:SrHap nanorods with blue autofluorescence and paramagnetism could serve as a good contrast agent of targeting fluorescence and magnetic resonance imaging. We envision that this Gd:SrHap system could play a significant role in developing new generations of site-selective, controlled-release delivery and interactive sensory nanodevices.

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targeted drug delivery.27−30 However, the research for a smart controlled-release system for intracellular drug delivery, in particular, specifically responding to cancer cells, remains a big challenge in this field. Herein we described the design and construction of the first cancer-cell-responsive controlled-release system for intracellular drug delivery based on mesoporous strontium hydroxyapatite nanoparticles by employing aptamer as both a capping and a targeting agent. Strontium plays a significant role in the biomineralization of bone and is used for the treatment of osteoporosis. The strontium-substituted Hap (SrHap) has similar stoichiometry to that of biological apatite and leads to an enhancement of invitro bioactivity.31−33 In particular, it has been demonstrated that the Sr substitute would increase the net positive charges and cationic strength of the Hap nanocarrier, which leads to stable DNA-Hap complex formation and improves gene delivery efficiency.34,35 Taking into account these concepts and being aware of the promising features of mesoporous Hap support as container, we explore the use of DNA as caps on the surface of SrHap for targeted drug delivery. As illustrated in Scheme 1, the SrHap nanocarriers will interact with negatively charged oligonucleotides to form stable complex mainly through strong electrostatic forces, resulting

INTRODUCTION he development of nanosized drug carrier to deliver anticancer agents specifically to cancer tissues has become one of the most promising fields in biomedicine. In particular, the construction of stimuli-responsive controlled-release systems for targeted drug delivery is of crucial importance in cancer therapy.1−5 In recent years, hydroxyapatite (Hap), which is the major inorganic component in tissues of vertebrates, has attracted attention as a promising component of multimodal nanoparticle system.6−11 Mesoporous Hap with various morphology and surface properties has been investigated as a nanocarrier due its biocompatible and osteconductive properties; solubility and less toxicity than silica, quantum dots, carbon nanotubes, or magnetic particles; low production costs; and excellent storage ability.12−16 Despite their promise, however, these existing systems face the difficulty of preventing the leakage of drugs before arriving at the target location and releasing the cargo in a controlled manner. For many practical drug delivery applications, such as chemotherapy, “zero premature release” and “targeted controlled release” of the precious and often toxic pharmaceutical cargo are two important prerequisites that would impact the therapeutic efficacy and cytotoxicity of drug delivery.17−19 In the case of the mesoporous silica nanoparticles-based system, various molecules (including DNA) have been demonstrated for stimuliresponsive drug delivery.20−26 Meanwhile, aptamer-functionalized various nanomaterials have also been reported for

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© 2012 American Chemical Society

Received: October 9, 2012 Revised: November 7, 2012 Published: November 9, 2012 4257

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GGGTCAAGTAGACCAC-3′. The sequence of control aptamer (CA, a scrambled sequence) is: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTTT-3′ Measurements and Characterizations. A field-emission scanning electron microscope (FESEM, S4800, Hitachi) equipped with an energy-dispersive X-ray (EDS) spectrum was applied to determine the morphology and composition of the luminescence Gd-doped mesoporous Gd:SrHap nanorods. TEM was performed on a JEOL 1011 transmission electron microscope at an accelerating voltage of 200 kV. The crystalline structures of the as-prepared samples were evaluated by X-ray diffraction (XRD) analysis on a on a D8 Focus diffractometer (Bruker) using Cu Kα radiation (λ = 0.15405 nm). N2 adsorption−desorption isotherms were recorded on a Micromeritics ASAP 2020 M automated sorption analyzer. The photoluminescence (PL) measurements were performed on a Hitachi F-4500 spectrophotometer equipped with a 150 W xenon lamp as the excitation source. Fluorescence measurements were carried out on Jasco-FP-6500 spectrofluorometer (Jasco International, Tokyo, Japan). UV−vis absorption spectra were recorded using a Varian Cary300 spectrophotometer equipped with a 1 cm path length quartz cell. Thermogravimetric analyses were carried out on a PerkinElmer Pyris Diamond TG/DTA analyzer, using an oxidant atmosphere (air) with a heating program consisting of a dynamic segment (10 °C/min) from 313 to 1173 K. Synthesis of Luminescence Gd-Doped Mesoporous SrHap. The luminescence of 2% Gd-doped mesoporous SrHap nanorod was prepared as follows: Typically, 0.784 g SrCl2·6H2O, 0.027 g Gd(NO3)3·6H2O, 0.5 g CTAB, and 10 mL of ammonia solution (NH3·H2O) were dissolved in deionized water to form 40 mL of solution 1. Solution 1 was stirred for 0.5 h at 37 °C. Solution 2 was obtained by mixing 1.764 g trisodium citrate dehydrate, 0.264 g (NH4)2HPO4, and 20 mL of deionized water. Then, solution 2 was added dropwise to solution 1. After additional agitation for 30 min, the mixing solution 2 was transferred to a Teflon bottle (50 mL) held in a stainless-steel autoclave, sealed, and maintained at 180 °C for 24 h. The obtained precipitate was filtered off and washed several times with deionized water and ethanol. The product was ultrasonically dispersed in 200 mL of acetone, then refluxed at 80 °C for 48 h to remove the residual template CTAB. Finally, samples were collected by centrifugation and dried in vacuum at 70 °C overnight for further characterizations and applications. Typical Drug Loading Experiment. Doxorubicin loading onto Gd:SrHap was done by mixing 50, 100, 150, 200, 250, and 300 μM Dox with Gd:SrHap (0.2 mg/mL) in PBS buffer overnight. The supernatant was collected by centrifugation at 12 000 rpm for 10 min. The supernatant and stock solution was analyzed by UV−vis spectrophotometry at 480 nm, respectively. Adsorption behavior is in line with Lambert−Beer law: A = εCL. By using UV−vis absorption spectroscopy, we can calculate the loading efficiency: E% = WDox/ WGd:SrHap × 100%, where WDox means the mass of Dox loaded in the Gd:SrHap and WGd:SrHap is the mass of Gd:SrHap. The molar extinction coefficient of Dox is 1.06 × 104 L/M·cm. Preparation of Gd:SrHap-Dox-Aptamer Complexes. The AS1411-coated Gd:SrHap-Dox complexes were prepared as follows: AS1411 aptamer was first incubated with PBS buffer (5 mM, 2.5 mM MgCl2, 140 mM KCl, pH 7.4) to form G-quadruplex structure. Gd:SrHap-Dox (2 mg/mL) solution was incubated with the Gquadruplex structure of the AS1411 aptamer (12.6 μM) in a 4 °C fridge for 12 h. The Gd:SrHap-Dox-aptamer complexes were separated by centrifugation (4000 rpm, 10 min). The sediment was washed with buffer for twice. The supernatant of the dispersions was analyzed by UV−vis spectrophotometry at 260 nm. Fluorescence spectra of the Gd:SrHap, Gd:SrHap-Dox-aptamer, and free Dox were measured by using a Jasco-FP-6500 spectro fluorometer. Agarose Gel Electrophoretic Analysis. The agarose gel was prepared by dissolving 1% (w/v) agarose in TAE buffer containing ethidium bromide. Complexes for this assay were prepared at nanoparticle/DNA ratios (w/w) of 100:1, 50:1, 30:1, and 20:1. Drug-Releasing Experiments. For doxorubicin (Dox) releasing from Gd:SrHap, the Gd:SrHap-Dox and Gd:SrHap-Dox-aptamer

Scheme 1. Schematic Representation of Cancer CellsTriggered Release of Drugs from the Pores of Gd:SrHap Capped with Aptamer

in the closing of the mesopores. Importantly, no chemical modification of the Hap nanoparticles or the oligonucleotides is needed, which is superior to previous mesoporous silica nanoparticle-based delivery systems. An anticancer aptamer AS1411 was used as a model system in this work. AS1411 is a 26-mer guanine-rich oligonucleotide DNA aptamer that has been in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma.36 Intriguingly, it shows high binding affinity to nucleolin, which is overexpressed in tumor cells. Previous studies have demonstrated that nucleolin on cancer cell surface is a receptor for AS1411.37−39 Therefore, the incubation of the nanocarriers with cancer cells would result in nucleolin-mediated internalization of the drug-loaded delivery system. The opening protocol will be expected to occur by an effective competitive displacement reaction in the presence of nucleolin, resulting in the complex formation of the nucleolin and aptamer, the uncapping of the pores, and releasing of the entrapped cargos. Furthermore, for effective therapy, the delivered cells must carry long-lived tracking agents for monitoring the position and fate of the injected cells. Several noninvasive imaging techniques for the in vitro and in vivo tracking of targeted nanostructures have been reported. They include the use of fluorescent nanomaterials, such as QDs,40 fluorine-18 (PET),41 iron oxide, and gadolinium magnetic nanoparticles (MRI).42,43 Taking advantage of the self-activated luminescent property of SrHap and the strong magnetic property of Gd-doped nanoparticles, this nanocarrier could be used as predominant targeting contrast agents in fluorescence and magnetic resonance imaging (MRI) simultaneously.

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MATERIAL AND METHODS

Reagents and Materials. Nanopure water (18.2 MΩ; Millipore Co., USA) was used in all experiments and to prepare all buffers. Cetyltrimethylammonium bromide (CTAB) and trisodium citrate dihydrate were obtained from Alfa Aesar. Strontium chloride hexahydrate was obtained from Fluka. Gadolinium(III) nitrate hexahydrate and bovine serum albumin (BSA) were purchased from Aladdin. All chemicals were used as received without further purification. Hemoglobin was obtained from Sigma-Aldrich. Nucleolin peptide was obtained from abcam (ab25315). The AS1411 aptamers and random DNA used were synthesized by Sangon Biotechnology (Shanghai, China). The sequence is as follows: The sequence of AS1411 aptamer is: 5′-GGTGGTGGTGGTTGTGGTGGTGGT4258

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samples were incubated in PBS (5 mM, pH 7.4) or FBS for different periods of time. Dox released from Gd:SrHap was collected by centrifugation at 12 000 rpm for 10 min. The amounts of released Dox in the supernatant solution were measured by UV−vis spectrophotometry. The release of Dox in vitro was monitored by fluorescence microscopy. MCF-7 cells and NIH3T3 were incubated with Gd:SrHap-Dox-aptamer or Gd:SrHap-Dox [Dox = 15 μM] for 3, 6, 12, and 24 h before fluorescence microscopic monitoring. In a typical nucleolin-stimulating experiment, Gd:SrHap-Dox-aptamer (aptamer = 0.5 μM) was incubated in PBS (5 mM, pH 7.4). Then, 40 μL of nucleolin (0.5 mg/mL) was added, and the amounts of released Dox in the supernatant solution were measured by UV−vis spectrophotometry at different periods of time. Cell Culture. The human breast cancer MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco). The cells were kept at 37 °C in a humidified atmosphere containing 5% CO2. The media was changed every 3 days, and the cells were digested by trypsin and resuspended in fresh complete medium before plating. Fluorescence Microscopic Studies for Aptamer Targeted Imaging. The Gd:SrHap (0.2 mg/mL) was added to the AS1411 aptamer (1.26 μM) solution and stirred overnight. The Gd:SrHapaptamer complexes were centrifuged and washed with buffer twice. The Gd:SrHap-aptamer complexes were redispersed in buffer (1 mL) for aptamer-targeted cell imaging. For the cell-imaging test, the concentration of cancer cells was fixed at a density of 105 cells/well in 24-well assay plates. Gd:SrHap and Gd:SrHap-aptamer complexes were added to the cells and incubated at 37 °C for 4 h. The cells were then washed several times with PBS. Then, 500 μL of fixing solution (1% glutaraldehyde and 10% formaldehyde) was added to each well for 30 min; finally, the fluorescence intensity was monitored. Cells were viewed using an Olympus BX-51 optical system microscopy. Pictures were taken with an Olympus digital camera. Cytotoxicity Assays. MTT assays were used to probe cellular viability. Cancer cells were seeded at a density of 5000 cells/well (100 μL total volume/well) in 96-well assay plates. After 24 h of incubation, the as-prepared nanorods, at the indicated concentrations, were added for further incubation of 48 h. To determine toxicity, we added 10 μL of MTT solution (BBI) to each well of the microtiter plate, and the plate was incubated in the CO2 incubator for an additional 4 h. Then, the cells were lysed by the addition of 100 μL of DMSO. Absorbance values of formazan were determined with a Bio-Rad model-680 microplate reader at 490 nm (corrected for background absorbance at 630 nm). Six replicates were done for each treatment group. in Vitro Drug Delivery. For the aptamer-targeted drug delivery experiment, Gd:SrHap-Dox, free Dox, Gd:SrHap-Dox-CA, and Gd:SrHap-Dox-aptamer samples were incubated with both MCF-7 cells and normal NIH3T3 cells for 4 h and then changed to fresh medium after washing. After another incubation of 24 h, the cell viability test was carried out by MTT assay. Magnetic Resonance Imaging. 250 μL of different concentrations of samples dissolved in cell lysis solution was taken in a 96 well plate and imaged using 1.5 T human clinical scanner. The preparation of lysis buffer was: 0.15 M NaCl, 10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 1% Triton X-100, and 1 mM PSMF. The process of collecting cell lysate was as follows: In general, for each 106 cells 100 μL of lysis buffer was added and lysed for 30 min (vortex once every 10 min) in the ice bath. Finally, the supernatant was collected by centrifugation at 10 000 rpm, 4 °C for 10 min, and stored at −20 °C. Targeting cell phantom T1-weighted MR images were done. NIH3T3 and MCF-7 cells were trypsinized, and an aliquot of cell suspension was added to each culture flask to obtain a cell density of 8.0 × 106 cells per flask. The flasks were cultured in DMEM containing 10% (v/v) fetal bovine serum (Gibco) at 37 °C in an atmosphere of 5% (v/v) CO2 in air for 24 h. The media was then removed and replaced with 4 mL of fresh media containing 40 μg/mL Gd:SrHap-aptamer. The flasks were incubated for 4 h and then washed with PBS five times. Finally, the cells were trypsinized, resuspended in

1 mL of PBS, and centrifuged at 3000 rpm for 15 min to obtain cell pellets. Protein Adsorption Experiments. We added 500 μL of BSA (1 mg/mL) or hemoglobin (0.5 mg/mL) to 500 μL of Gd:SrHapaptamer (4 mg/mL) and stirred for 2 h. Then, the Gd:SrHap-aptamerprotein complexes were separated by centrifugation (4000 rpm, 10 min). The supernatant of the dispersions and protein stock solution was analyzed by UV−vis spectrophotometry. Statistical Analysis. All data are expressed as mean result ± standard deviation (SD). All figures shown are obtained from three independent experiments with similar results. The statistical analysis was performed by using Origin 8.0 software.

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RESULTS AND DISCUSSION The monodisperse and uniform gadolinium-doped luminescent mesoporous SrHap nanoparticles were synthesized with average length of 100−120 nm, diameter of ∼20 nm, and aspect ratio of 5.0 (length to diameter), respectively (Figure 1a,b). The highly ordered lattice array over the SrHap

Figure 1. (a) SEM, (b) TEM images, (c) EDS spectrum of Gd:SrHap, and (d) XRD patterns of SrHap (black) and Gd:SrHap (red). The inset in panel b shows the high magnification image.

nanoparticles indicated well-defined mesostructure with average pore size of 2.3 nm (Figure 1b, inset). EDS analysis confirmed the presence of Sr, Gd, O, and P in the nanoparticles (Figure 1c). The wide-angle XRD patterns of pure SrHap and Gd:SrHap, are respectively, shown in Figure 1d. The pattern of the obtained SrHap presents the characteristic broad diffraction peaks, which can be easily indexed to the standard data of hexagonal Sr10(PO4)6(OH)2 (JCPDS 33-1348). Otherwise, the characteristic diffractions of Gd:SrHap present no other phase related to the doped Gd3+, indicating that Gd3+ has been successfully doped into the framework of SrHap and has no more effect on the typical hexagonal phase. N2 adsorption−desorption isotherms (Figure S1a of the SI) showed a typical type-IV curve with a pore volume of 0.326 cm3/g, average pore diameter of 2.3 nm, and a narrow pore distribution, which are consistent with the TEM results. The fluorescence property is illustrated by the PL spectrum. The asprepared nanoparticles give a strong emission peaks at 432 nm (Figure S1b of the SI), and the fluorescence intensity of nanoparticles reduces slightly compared with pure SrHap (date not shown). This result firmly confirms that SrHap has a stable crystal structure and fluorescence intensity changes little after doping Gd3+. The strong autofluorescent, superparamagnetism, and mesoporous nature enable our present Gd:SrHap nano4259

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release of the Gd:SrHap-Dox-aptamer system, we incubated the complex with both MCF-7 cells and NIH3T3 cells, and the behavior of release was monitored by fluorescence microscope. As shown in Figure 2a, blue fluorescence was observed for MCF-7 cells after 3 h of incubation and increased on prolonging the incubation time. Interestingly, the fluorescence of Dox inside cells was very weak originally and increased remarkably over time. In contrast, the fluorescence of neither Gd:SrHap nor Dox was observed even though they were

particles to be used as dual-mode imaging and drug delivery vehicles. Doxorubicin (Dox) was chosen as a model drug owing to its good anticancer activity against a wide spectrum of tumors.44−46 The loading efficiency of Dox is as high as 52.8% in weight (Figure S2a of the SI). After the drug was loaded, the pores of the Gd:SrHap carriers were sealed with AS1411 aptamers. Two mg/mL Gd:SrHap was suspended in buffer containing the aptamers for a final concentration of 12.6 μM. The excess drug was removed by centrifugation and repeated washing. TEM (Figure S2c,d of the SI) confirms that the Gd:SrHap retains the pore structure, and a layer of soft materials (aptamer DNA) about 1.9 nm thick surrounds the Gd:SrHap surface. The size distributions of Gd:SrHap and Gd:SrHap-aptamer nanoparticles are also confirmed. Figure S3 of the SI displays a hydrodynamic diameter of 131 (Gd:SrHap) and 153 nm (Gd:SrHap-aptamer), indicating excellent colloidal dispersity in aqueous solution. Compared with the Gd:SrHap nanorods, the size of Gd:SrHap-aptamer is slightly bigger, which is likely due to aptamer swelling on the nanoparticle surface. The changes in pore volume and diameter were investigated by nitrogen sorption experiments. The decline in surface area and pore volume in the sample Gd:SrHap-aptamer is attributed to partial pore blocking effect induced by the aptamer on the outer shell of the nanoparticle (Table S1 of the SI). Zeta potential evaluation of nanoparticles has been performed at physiological pH (7.4), and the results are shown in Table S2 of the SI. The zeta potential of the nanorods becomes negative after DNA absorption, indicating the formation of Gd:SrHap-aptamer complex. The ability of Gd:SrHap to adsorb aptamer was also tested by agarose gel electrophoresis assay (Figure S4 of the SI). DNA migration in the agarose gel was retarded when complexed with Gd:SrHap nanoparticles. Gd:SrHap nanorods retarded the DNA migration entirely at or above the w/w ratio of 50:1. Quantification of the density of DNA anchored on Gd:SrHap was accomplished by thermogravimetric analysis (TGA), which corresponded to an immobilization efficiency of ∼4.78 μmol g−1 (Figure S2b of the SI). The fluorescent characteristics of Gd:SrHap-Dox-aptamer were demonstrated in Figure S5 of the SI. Nearly 70% of Dox fluorescence is quenched, likely due to the intermolecular interactions between Dox and nanoparticles. In contrast, the fluorescence intensity of Gd:SrHap decreases by only 36% owing to the presence of Dox in the surface of SrHap. The capping properties of the DNA aptamers were then investigated. As illustrated in Figure S6a of the SI, a fast drug release in PBS (pH 7.4) was observed in 12 h for the Gd:SrHap-Dox system due to lacking of the pore-blocking species. However, less than 5% release was observed when the Gd:SrHap-Dox-aptamer nanocarriers were dispersed in PBS (pH 7.4) even for 72 h, and a high stability was shown in neat FBS even incubated for 6 days (Figure S6b of the SI). These results demonstrated a very clear and highly effective aptamer capping effect for encapsulation of drugs against the undesired leaching problem. Owing to the spatial configuration of the aptamer DNA was needed for target, the fluorescent probe molecule NMM (Nmethyl mesoporphyrin IX) was used to prove that the Gquadruplex structures of aptamer were retained after adsorption. As shown in Figure S7 of the SI, strong enhanced fluorescence was observed upon mixing Gd:SrHap-aptamers with NMM attributed to the presence of G-quadruplex structures. To investigate the cancer-cells-triggered controlled

Figure 2. Fluorescence microscope images of MCF-7 cells (a) and NIH3T3 cells (b) incubated with Gd:SrHap-Dox-aptamer for 3, 6, 12 and 24 h at 37 °C. Dox fluorescence (red color) and Gd:SrHap fluorescence (blue color) were recorded in different wavelength ranges, respectively. Scale bar is 20 μm. (c) Release profiles of Dox from Gd:SrHap-Dox-aptamer without nucleolin (black) and until t = 3 h when nucleolin was added (red). 4260

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Figure 3. Cell-viability MTT assay of high-level nucleolin expressed MCF-7 cells (a) and normal NIH3T3 cells (b) treated with Gd:SrHap-Dox, Gd:SrHap-Dox-aptamer, and Gd:SrHap-Dox-CA.

incubated for 24 h in NIH3T3 cells (Figure 2b). MCF-7 cells incubated with Gd:SrHap-Dox were also monitored as a control and showed a fast release of Dox before carriers were uptaken by cells even in 3 h (Figure S8 of the SI). This result further confirmed that our aptamer-capped nanocarrier could achieve the properties of “zero premature release”. Because the aptamer AS1411 binds specifically to nucleolin with high affinity, the opening protocol will be expected to occur by an effective competitive displacement reaction in the presence of nucleolin. The removal of pore blockers aptamers from complex resulted in the opening of the pores and releasing of the entrapped drugs. As a proof of this concept, a partial cargo release of trapped guest could be achieved by the addition of nucleolin to the Gd:SrHap-Dox-aptamer system. A considerable Dox release from the Gd:SrHap-Dox-aptamer system was indeed observed in the presence of nucleolin (Figure 2c). These results confirmed that cancer-cells-triggered release could be achieved with the Gd:SrHap-Dox-aptamer system. After having demonstrated the cell-responsive controlled release from Gd:SrHap-aptamer, we further verified the feasibility for intracellular therapeutic applications. The in vitro cytotoxicity of Gd:SrHap-Dox-aptamer was examined with MCF-7 cells and NIH3T3 cells. For concentrations up to 640 μg/mL, the Gd:SrHap nanoparticles were not cytotoxic (Figure S9 of the SI). The cytotoxicity of the Gd:SrHap-Dox and free Dox at a series of Dox concentrations to both MCF-7 cells (Figure S10a of the SI) and NIH3T3 cells (Figure S10b of the SI) was first performed. The half-maximum inhibitory concentration (IC50) values for Gd:SrHap-Dox incubated with MCF-7 cells (38 μM) and NIH3T3 cells (40 μM) are found to be higher than those of free Dox incubated with MCF7 cells (7 μM) and NIH3T3 cells (10 μM). The reduced toxicity of Gd:SrHap-Dox is probably due to the less efficient cellular uptake of Gd:SrHap-Dox compared with that of free Dox. Delivery of Dox into cancer cells led to growth inhibition and cell death (Figure 3). Compared with Gd:SrHap-Dox, the Gd:SrHap-Dox-aptamer showed a higher cytotoxic efficacy with MCF-7 cells (Figure 3a). In contrast, the cytotoxicity of Gd:SrHap-Dox-aptamer incubated with NIH3T3 cells is low, and the uncapped vehicles are more toxic in NIH3T3 cells (Figure 3b). The higher cytotoxicity of Gd:SrHap-Dox-aptamer complexes with MCF-7 cells is attributed to the specific binding and uptake of the nanocarries to the target cancer cells induced by aptamer. These results indicated that the nucleolin-mediated uptake of aptamers-capped Gd:SrHap-Dox either significantly reduced the side effects of the Dox in normal or improved the damage in cancer cells. Furthermore, a CA with the same length but not responsive to nucleolin was attached to the

Gd:SrHap, in which it could also cap the pores. Although similar amounts of Dox were trapped in the nanoparticles, they did not respond to both MCF-7 cells and NIH3T3 cells, and a very low Dox release was observed. These results further confirmed that the aptamer-capped nanoparticles were capable of controlled delivery of drug molecules in response to specific cancer cells. Compared with the photobleaching and quenching of fluorescent organic molecules47−49 and the toxicity of semiconductor quantum dots,50−52 the Gd-doped SrHap nanoparticles provide the possibility of both optical imaging and MRI of cancer cells due to their excellent optical properties and nontoxicity. To explore the targeting fluorescence imaging, we first incubated MCF-7 cells with Gd:SrHap and Gd:SrHapaptamer complexes, demonstrated in Figure 4b; strong blue fluorescence was observed in MCF-7 cells incubated with Gd:SrHap-aptamer complexes. In contrast, negligible fluorescence was observed upon incubation with Gd:SrHap nanoparticles (Figure 4a). To confirm further the dominant

Figure 4. Selective uptake of Gd:SrHap-aptamer and Gd:SrHap by MCF-7 cells and NIH3T3 cells at 4 h. MCF-7 cells were treated with Gd:SrHap (a), Gd:SrHap-aptamer (b), and Gd:SrHap-CA (d). 3T3 cells were treated with Gd:SrHap-aptamer (c). Scale bar is 10 μm. 4261

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CONCLUSIONS In summary, we have demonstrated, as a proof of concept and for the first time, that the use of aptamers as caps on the surface of mesoporous strontium hydroxyapatite nanocarrier provides a suitable method for the design of delivery system able to selectively release entrapped cargos in responsive to specific cancer cells. The system requires no chemical modification of the Hap nanoparticles or the oligonucleotides. Furthermore, the Gd-doped SrHap-aptamer system could be used as predominant targeting contrast agents in fluorescence and MRI simultaneously, which makes the Gd:SrHap-aptamer nanostructure an ideal candidate for both targeted bioimaging and therapy. Moreover, we have successfully demonstrated that the aptamer-capped nanoparticles showed a remarkably enhanced efficiency in killing cancer cells. The excellent biocompatibility, specific cellular uptake properties, and efficient intracellular drug release provide a basis for in vivo controlled-release biomedical applications. DNA aptamer here served as both a capping and a targeting agent. With aptamer being, in principle, available for any kind of target, this proof of concept might open the door to a new generation of carrier materials and could also provide a general route in the field of versatile controlled delivery nanodevices.

receptor-mediated endocytosis mechanism of the particles, we incubated Gd:SrHap-aptamer complexes with normal NIH3T3 cells and the random DNA-capped Gd:SrHap-CA complexes. As expected, no obvious fluorescence signal was observed for both NIH3T3 cells (Figure 4c) and MCF-7 cells (Figure 4d), and the blue-emitting nanoparticles were randomly distributed all around the cells without any specific interaction with the membrane. This result clearly revealed that there was no interaction between the Gd:SrHap-aptamer and the NIH3T3 cell membrane or the Gd:SrHap-CA with the MCF-7 cell membrane, which resulted in the inhibition of the uptakes. The above results demonstrate that the enhancement of cellular association and uptake is induced by the specific interaction of the Gd:SrHap-aptamer complex with nucleolin at the cellular surface, confirming that the binding capability and specificity of the aptamers are retained after being adsorbed onto the Gd:SrHap. MRI is a premier technique for imaging cancer and other advanced diseases. However, most biological samples exhibit virtually no magnetic background; magnetic nanoparticles have been used for highly sensitive measurements and superior contrast imaging in turbid or otherwise visually obscured samples without purification, allowing for rapid assays.53−55 The potential of using the Gd-doped nanoparticles for MRI imaging was also investigated. The as-prepared nanoparticles were dissolved in cell lysis solution. T1-weighted MR image was evaluated at a 1.5 T human clinical scanner. As shown in Figure 5a, a clear dose-dependent color change was

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ASSOCIATED CONTENT

S Supporting Information *

N2 adsorption−desorption isotherms, PL spectra of Gd:SrHap, quantification of Dox loading at different Dox concentrations, the size, zeta potential, TGA, BET, and TEM images of unmodified and aptamer-modified Gd:SrHap, agarose gel electrophoresis, fluorescence spectra of free Dox and Gd:SrHap-Dox, release profiles of Dox from Gd:SrHap-Dox and Gd:SrHap-Dox-aptamer, fluorescence spectra of NMM treated with different samples, fluorescence microscope images, cytotoxicity experiments, and protein adsorption test. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Figure 5. (a) Phantom NMR images of Gd:SrHap dissolved in cell lysis solution showing T1-weighted bright contrast. (b) T1-weighted images of NIH3T3 (left) and MCF-7 (right) cell pellets.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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observed with increasing the dose due to the relaxation increase in the water proton. To further demonstrate the targeting ability and T1-weighted MR imaging enhancement efficiency of Gd:SrHap-aptamer, MCF-7 and NIH3T3 cell pellets were prepared as described in the Materials and Methods, and the T1-weighted images were acquired (Figure 5b). The MR images clearly displayed an increase in contrast enhancement of MCF-7 cells due to the targeting ability of AS1411 aptamer. In contrast, Gd:SrHap-aptamer incubated with NIH3T3 results in the significant darkening of MR images. These data further support the enhanced uptake of the Gd:SrHap-aptamer material by MCF-7 cells due to the targeting ability of the aptamer group. Finally, because all experiments were done in vitro, we performed protein adsorption test on this nanovehicle to ensure that it could be safe for further in vivo application. As shown in Figures S11a and S11b of the SI, Gd:SrHap-aptamer appeared with a low BSA and hemoglobin adsorption capacity, indicating a better antibiofouling effect.

ACKNOWLEDGMENTS We acknowledge financial support from National Basic Research Program of China (grants 2012CB720602, 2011CB936004) and the National Natural Science Foundation of China (grants 20831003, 21210002, 20833006, 90913007, 21072182, 91213302).

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