Biomaterials 35 (2014) 8694e8702
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
DNA-mediated biomineralization of rare-earth nanoparticles for simultaneous imaging and stimuli-responsive drug delivery Li Zhou, Zhaowei Chen, Kai Dong, Meili Yin, Jinsong Ren*, Xiaogang Qu* State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2014 Accepted 17 June 2014 Available online 4 July 2014
A DNA-guided method for surface engineering of NaGdF4:Ce/Tb hybrid nanoparticle has been proposed. In this study, the DNA molecules that retained after one-pot NaGdF4:Ce/Tb synthesis is directly utilized as biotemplate for CaP heterogeneous nucleation, thus the dual-purpose function of DNA is realized in the current study which could afford a new type of pH-responsive theranostic platform to enhance the therapeutic efficiency while minimizing side effects. The introduction of another layer of aptamer molecules on CaP facilitated cellular uptake of the resulting nanocomposite into specific target cells via receptor-mediated endocytosis. After been taken by the target tumor cells, the NaGdF4:Ce/Tb@CaP was found to be mostly accumulated in lysosome, which facilitated the dissolving of CaP coatings as non-toxic ions to initiate drug release and efficient cancer cell destruction. We envision that the hybrid nanocarrier may serve as practical and multifunctional probe for cancer therapy and the presented synthesis approach here may also benefit the preparation of many other types of multifunctional inorganicbiomolecular hybrid nanostructures based on the DNA nanotechnology. © 2014 Elsevier Ltd. All rights reserved.
Keywords: NaGdF4:Ce/Tb DNA Biomineralization Imaging Controlled drug delivery
1. Introduction With the rapid advances of nano-biotechnology, there has been an explosion of interest in the use of biomolecules possessing sophisticated structures and outstanding functions as building blocks for the development of multifunctional nanomaterial [1e5]. This biomolecules-based strategy opens up a new avenue for rationally control the structures as well as physical and chemical features of the materials. Due to the conformational polymorphism, sequencespecific recognition and robust physicochemical nature, DNA has been extensively investigated in nanotechnology and material science [1,6]. The use of DNA as template appears to be one of the most promising avenues available for fabricating a variety of metallicnanomaterials with potential applications ranging from electronics to biology [7e15]. DNA is rich of phosphate groups, amino groups and heterocyclic nitrogen atoms, it offers nucleation sites for metallic nanoparticles and provides control over the material growth and stability [6e15]. Meanwhile, the DNA attached on the prepared material could also be employed as recognition unit for the biological application or to organize the nanoparticles into
* Corresponding authors. Tel./fax: þ86 431 85262625. E-mail addresses: [email protected], [email protected] (J. Ren), [email protected] (X. Qu). http://dx.doi.org/10.1016/j.biomaterials.2014.06.034 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
periodic or discrete one-, two-, and three-dimensional architectures for material purpose [1,6e8]. For example, Kelley et al. recently reported that a chimeric DNA molecule could program both the growth and the biofunctionalization of the CdTe nanocrystals for bioimaging applications [1,7]. Although these DNAbased hybrid materials hold great promise in nanotechnology and nanomedicine, the exploring of further functions of the DNA anchored on the material surface still remains a big challenge in this field. Porous inorganic nanoparticles with high specific surface area have emerged as appealing material recently for the development of delivery systems, where various guest molecules could be absorbed into the pores and later released into various solutions [16e19]. Meanwhile, due to the unique optical and magnetic behavior, the lanthanide ions doped-porous nanocrystals have been extensively employed for biomedical applications, including our recent development of DNA-based porous lanthanide doped NaGdF4 nanocarriers for cell-specific drug delivery [20e25]. Compared with organic fluorophores that modified on the surface of material, the doped luminescent lanthanide cations provide complementary properties such as resistance to photobleaching, long luminescence lifetimes, absence of reabsorption and sharp emission bands in the visible and the near-infrared [26e30]. Moreover, it could be employed as a probe that combines therapy
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functions with multi-mode imaging methodologies which could provide complementary information in biomedical studies [31e35]. While the Ln3þ doped-porous nanoparticles (Ln3þ-pNP) have been adopted as promising nanomaterials for biological application, these delivery platforms might significantly compromise their efficiency because the entrapped drug may start leaking out of the carrier immediately after administration due to the lack of capping/blocking agent. Therefore, the ability to maintain optimum therapeutic efficacy, i.e. no premature release in blood circulation whilst having a rapid drug release in tumor tissues, remains a significant challenge for the development of Ln3þ-pNP based multifunctional delivery system. During the past several decades, nature's strategies to the formation of a wide range of specially designed organic-inorganic hybrid materials such as bone, teeth, and shells have attracted increasing attention [36]. The mild condition and high efficiency of these preparation processes have inspired research to mimic them in vitro and a great deal of biominerals with tunable morphologies and properties has been prepared [37,38]. In terms of materials design, bio-inspired calcium phosphate (CaP) hybrid materials have attracted tremendous interest; this is mainly due to the fact that these hybrids have a wide range of very useful properties and potential applications [39e48]. For example, Epple et al. proposed multi-shell CaP-DNA nanoparticles and CaP-DNA/siRNA nanohybrids for effective transfection of cells [44]. Adair et al. developed CaP nanoparticles that encapsulated both fluorophores and chemotherapeutics for in vitro imaging and anticancer drug delivery [47,48]. Here inspired by the high binding affinity of CaP toward DNA, we propose to engineer the Ln3þ-pNP surface with a layer of natural CaP to overcome the big challenges in Ln3þ-pNP applications mentioned above. As shown in Scheme 1, followed by our previous report, porous NaGdF4:Ce/Tb nanoparticles were prepared by using DNA as superior ligands. Then the hydrophilic DNA molecules that attached on the NaGdF4:Ce/Tb nanoparticles were utilized as the nucleation sites for CaP heterogeneous nucleation. Thereafter, by precisely controlling the following growing process, CaP mineral nanoshells which possessed pH-dependent
Ca2+
DOX- NaGdF4:Ce/Tb
HPO42-
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biodegradability could be formed (designed as NaGdF4:Ce/ Tb@CaP). Thus the dual-purpose function of DNA was realized in the current study which could afford a new type of pH-responsive theranostic platform to enhance the therapeutic efficiency while minimizing side effects. Moreover, an additional layer of aptamer (Apt) was attached to the mineral surfaces via chelation interactions, which produced a colloidal stabilized nanocomposite with biospecific properties (designed as NaGdF4:Ce/Tb@CaP-Apt). 2. Materials and methods 2.1. Materials Ln(NO3)3 and doxorubicin (DOX) were purchased from Sangon (ShangHai, China). NaF and dimethyl sulfoxide (DMSO) were purchased from Shanghai Chemical Factory (Shanghai, China). Fish sperm DNA was obtained from SigmaeAldrich. Ultrapure water (18.2 MU; Millpore Co., USA) was used throughout the experiment. The oligonucleotide used in this article was synthesized by Sangon Biotechnology Inc. (Shanghai, China). The sequence was as follows: 50 -GGTGGTGGTGGTTGTGGTGGTGGTGGT-30 Aptamer (Apt) 50 -TTAGGG TTAGGGTTAGGGTTAGGG TTA-30 control DNA with randomized sequence (Rdm) 2.2. Measurements and characterizations FT-IR analyze was carried out on a Bruker Vertex 70 FT-IR Spectrometer. SEM images were obtained with a Hitachi S-4800 FE-SEM. The UVeVis absorption spectra and fluorescence spectra were recorded using a JASCO V-550 UVeVisible and a JASCO FP6500 spectrophotometer (JASCO International Co.,LTD., Tokyo, Japan). TEM images were recorded using an FEI TECNAI G2 20 high-resolution transmission electron microscope operating at 200 kV. The samples were degassed at 90 С for 5 h. The magnetic properties of samples were collected on a MPM5-XL-5 superconducting quantum interference device (SQUID) magnetometer. N2 adsorptionedesorption isotherms were obtained on a Micromeritics ASAP 2020M automated sorption analyzer. The specific surface areas were calculated from the adsorption data in the low pressure range using the BET model and pore size was determined following the BJH method. The zeta potential of the nanomaterials in HEPES was measured in a Zetasizer 3000HS analyzer. Cell imaging was performed with a fluorescent microscope (Olympus, BX51). 2.3. Preparation of NaGdF4:Ce/Tb nanoparticle 280 mL Gd(NO3)3 (0.2 M), 35 mL Ce(NO3)3 (0.2 M) and 15 mL of Tb(NO3)3 (0.2 M) were added to 100 mL beaker and diluted to 15 mL with water. Then 5 mL of the DNA stock solution (6.4 mM) was added quickly under magnetic stirring. Subsequently,
Aptamer
MR imaging Lanthanide luminescence
DOX- NaGdF4:Ce/Tb@CaP
Scheme 1. Schematic illustration of the biomineralization process inspired DOX loaded NaGdF4:Ce/Tb@CaP-Apt hybrid. DOX loaded NaGdF4:Ce/Tb@CaP-Apt was internalized via receptor-mediated endocytosis. The DOX was released through the dissolving of CaP within intracellular endolyso-somal compartment.
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stoichiometric amount of NaF (the molar ratio of NaF to lanthanide ion was set to 5:1) in 40 mL of water was added dropwise to the above solution. The mixture was agitated for about 10 min, then transferred to autoclave, sealed, and hydrothermally treated at 90 C for 12 h. The system was cooled to room temperature naturally. Pure powders could be obtained by purifying the samples with water several times to remove the remnants. 2.4. Synthesis of NaGdF4:Ce/Tb@CaP nanocomposites Mineralization of the NaGdF4:Ce/Tb nanoparticles was carried out in the presence of calcium nitrate and ammonium phosphate using a method similar to that developed by Schmidt et al. [49]. In a typical procedure, 0.03 mL calcium nitrate stock solution (0.1 M) was added to the solution containing the NaGdF4:Ce/Tb nanoparticles (40 mg) and stirred slowly for 1 h. After that, 0.02 mL of the ammonium phosphate solution (0.1 M) was slowly added and stirred for 1 h. This procedure of sequential addition of the stock solutions was repeated 8 times. Then, the suspension was allowed to age for another 12 h without stirring, and followed by centrifugation, washing with phosphate-buffered saline for several times to obtain the NaGdF4:Ce/Tb@CaP product.
weighted and T2-weighted MR imaging. The Eppendorf tubes were then scanned in a 1.5-T Magnetom Espree MIR system after the preparation. 2.7. Cargo loading and release experiment For rhodamine B loading, NaGdF4:Ce/Tb nanoparticles were soaked in a solution of rhodamine B solution (0.5 mg/mL) for 24 h and followed by centrifugation, washing with pure water to remove unloaded and adsorbed molecules. All the washing solutions were collected, and the loading amount was approximately 1.12% (w/w) based on UVeVis spectroscopy. After that, mineralization of the rhodamine B loaded nanoparticle and introduction of Apt molecules to the resulting nanocomposites were carried out as described above. The as-prepared material was then dispersed in 5 mL aqueous buffer solutions (pH 7.4 phosphate buffer and pH 4.5 acetate buffer) at room temperature. Aliquots were taken from the suspension at predetermined time and the delivery of rhodamine B from the nanocomposites to the buffer solution was monitored via the absorbance band of the dye centered at 553 nm. To obtain DOX-NaGdF4:Ce/Tb@CaP nanocomposites, DOX loading and mineralization process were performed as above. The loading amount was approximately 1.2% (w/w) by UVeVis absorbance spectroscopy.
2.5. Attaching of NaGdF4:Ce/Tb@CaP with Apt Before the experiment, AS1411 Apt was first incubated with HEPES buffer (10 mM, 2.5 mM MgCl2, 140 mM KCl, pH 7.4) to form G quadruplex structure. Then NaGdF4:Ce/Tb@CaP (1 mg/mL) solution was incubated with the G quadruplex structure of the AS1411 Apt with different concentration (5, 10 mM, 30 mM, 50 mM, 70 mM) in the 4 С fridge for 12 h. The resulting NaGdF4:Ce/Tb@CaP-Apt was separated by centrifugation and washed with HEPES buffer for several times to remove the free Apt. The supernatant of the dispersions was analyzed by UVeVis spectrophotometry at 260 nm.
2.8. CaP disintegration experiments NaGdF4:Ce/Tb@CaP (10 mg) material was dispersed in 5 mL aqueous buffer solutions (pH 7.4 phosphate and pH 4.5 acetate buffers). Aliquots were extracted at given time intervals and replaced with the same volume of fresh medium. The release rate of calcium ions was monitored by taking a 50 mL sample and diluting it using Arsenazo III solution (40 0 mL, 0.2 mM) in HBS (HEPES-buffered saline where [HEPES]¼ 20 mM and [NaCl] ¼ 150 mM at pH 7.4). The absorbance of Arsenazo III/ Ca2þ complex in the solution was measured at 656 nm, and the concentration of calcium ions was calculated based on the standard curve.
2.6. Magnetic resonance imaging T1-weighted and T2-weighted MR imaging were obtained by using a 1.5-T clinical MRI instrument (Siemens Medical System). Dilutions of NaGdF4:Ce/ Tb@CaP nanocomposite in HEPES buffer containing 1% agarose with different concentrations as contrast agent were placed in 4.0 mL Eppendorf tubes for T1-
2.9. Cell cultures and MTT assay The MCF-7 human breast cancer cell, MDA-231 breast cancer cell, HeLa human cervical epithelioid carcinoma cell and human embryonic kidney 293 cells (HEK) were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with
Fig. 1. Representative SEM and TEM micrographs of (A, C) NaGdF4:Ce/Tb and (B, D) mineralized NaGdF4:Ce/Tb.
L. Zhou et al. / Biomaterials 35 (2014) 8694e8702 10% fetal bovine serum at 37 C with 5% CO2 for 24 h. Cell viability was determined by measuring by MTT assay [31]. 2.10. Fluorescence microscopy To do the test, the concentration of MCF-7 cells was fixed at a density of 105 cells/ well in 24-well assay plates. NaGdF4:Ce/Tb@CaP-Apt and NaGdF4:Ce/Tb@CaP-Rdm nanocomposites were added to the cells and the mixture was incubated at 37 C for 4 h to allow the uptake of the nanoparticles. The cells were then washed several times with PBS. Then 500 mL fixing solution (1% glutaraldehyde and 10% formaldehyde) was added to each well for 30 min and finally the fluorescence intensity was monitored.
3. Results and discussion To perform the experiment, the NaGdF4:Ce/Tb nanoparticles were first synthesized according to our previous report [31]. As shown in Fig. 1A and C, mono-dispersed porous nanoparticles with a diameter of about 120 nm were obtained with the addition of DNA molecules (fish sperm DNA). It was worth to note that the minimal different between the as-prepared material with the previous reports may ascribe to the different type of DNA that used in each study. The successful capping of DNA molecules on the nanoparticles was confirmed by the observation of typical absorbance of DNA at 260 nm after the reaction and the existence of C, O, N and P elements in the NaGdF4:Ce/Tb nanoparticles (Fig. S1, S2). Moreover, the FT-IR spectra of the NaGdF4:Ce/Tb material showed typical absorption bands at 1070 and 988 cm1, which could be ascribed to the OeP stretching mode of phosphate groups (Fig. 2A).
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The N2 adsorption and desorption analysis was introduced to investigate the surface area and pore properties of the product. A typical-type isotherm with H1-hysteresis loops was observed, demonstrating the presence of mesopores (Fig. S3). The BET surface area, total pore volume, and average pore size were calculated to be 105.05 m2/g, 0.274 cm3/g and 6.3 nm, respectively. Next, the prepared NaGdF4:Ce/Tb nanoparticles were mineralized by the sequential addition method, in which CaP deposition on the material was initiated by calcium nitrate to allow for the electrostatic localization of calcium ions at phosphate of the DNA backbone. Thereafter, the addition of sodium phosphate as the source of a phosphate anion induced the growth of nanophase CaP. The successful mineralization of NaGdF4:Ce/Tb nanoparticle was confirmed by various methods. TEM image investigations of NaGdF4:Ce/Tb@CaP (~130 nm) provided direct evidence of the distribution of CaP on the surfaces of NaGdF4:Ce/Tb (Fig. 1D). The nucleation of CaP at the interfaces was also supported by the enhanced peaks in the FT-IR spectrum, which corresponded to the OePeO bending modes appearing around 600 and 560 cm1, PeO asymmetric stretching mode at around 1000 cm1 (Fig. 2A and B). Zeta potential analysis of the NaGdF4:Ce/Tb showed that the surface potential of the nanoparticle increased from 25.7 mV to 7.3 mV in PBS buffer (pH 7.4) after DNA templated mineralization process, suggesting that mineral deposition almost completely shielded anionic phosphate charges (Fig. 2C). To further confirm the presence of calcium and phosphate in the shell, energy-dispersive X-ray spectroscopy (EDX) was used to analyze
Fig. 2. (A), (B) FT-IR spectra of (a, a0 ) NaGdF4:Ce/Tb, (b, b0 ) NaGdF4:Ce/Tb@CaP, (c, c0 ) NaGdF4:Ce/Tb@CaP-Apt. (C) Zeta potential measured at each step of the coating process in deionized water. (D) EDX spectrum of the NaGdF4:Ce/Tb@CaP nanoparticles.
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To investigate the feasibility of using NaGdF4:Ce/Tb@CaP-Apt particles as multifunctional delivery system in bio-related fields, the fluorescence and magnetic properties of NaGdF4:Ce/Tb@CaPApt were examined. The photoluminescence (PL) emission spectra of NaGdF4:Ce/Tb@CaP-Apt solution was first measured at room temperature. As shown in Fig. 3A, the luminescence of the NaGdF4:Ce/Tb@CaP-Apt in water appeared similar to that of the NaGdF4:Ce/Tb samples, indicating that the characteristic luminescent property of the NaGdF4:Ce/Tb nanoparticles was unaffected by the CaP coating. Moreover, the colloidal solutions of the NaGdF4:Ce/ Tb@CaP-Apt (0.5 mg/mL) exhibited bright green luminescence when excited with a UV lamp, indicating that those nanocomposites had good potential for use as luminescent labels in biological imaging (Fig. 3A, inset). The magnetic field dependency of NaGdF4:Ce/Tb and NaGdF4:Ce/Tb@CaP-Apt were also measured to evaluate the potential application of the materials as MRI contrast agents. Room temperature magnetization curve of NaGdF4:Ce/Tb and NaGdF4:Ce/Tb@CaP-Apt demonstrated their paramagnetic character (Fig. 3B). The mass magnetic susceptibility value of NaGdF4:Ce/Tb@CaP-Apt was calculated to be 1.11 104 emu/g Oe. The T1- and T2- weighted MR images evaluated at a 1.5 T human clinical scanner revealed an enhancement of MR signal as increasing of the concentrations of the nanocomposites (Fig. 3B, inset). From the slope of the plot of the relaxation rate (1/T1) as a
Fig. 3. (A) Emission spectrum of NaGdF4:Ce/Tb nanoparticles before (pink line) and after mineralized (indigo line). (B) Room temperature magnetization curve of NaGdF4:Ce/Tb@CaP. Inset: (A) the photographs of dispersed solution containing NaGdF4:Ce/Tb (left) and NaGdF4:Ce/Tb@CaP under UV lamp, (B) T1-weighted and T2weighted MR images of NaGdF4:Ce/Tb@CaP at various concentrations in buffer solution.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the rough surfaces. The significant appearance of Ca element and P element indicated the formation of CaP which was the characteristic product of template-induced mineralization (Fig. 2D). Furthermore, the change of sorption type was expected due to the capping effect of the CaP (Fig. S3). After successful mineralization of NaGdF4:Ce/Tb nanoparticle, the nanocomposites were dispersed in the Apt solution to obtain NaGdF4:Ce/Tb@CaP@Apt. The anchoring amount of Apt increased as an increasing of Apt concentration and the maximum immobilization efficiency was determined to be about 30.3 mmol/g (Fig. S4, S5). After anchoring another layer of Apt molecules, the final zeta potential of the nanocomposites reached 19.18 mV. Owing to the spatial configuration of the Apt was needed for target, the fluorescent probe molecule NMM (Nmethyl mesoporphyrin IX) was used to prove that the G quadruplex structures of Apt were retained after adsorption. Significant fluorescence enhancement was observed upon mixing NaGdF4:Ce/ Tb@CaP@Apt with NMM, indicating the presence of quadruplex structures of Apt on the nanocomposites (Fig. S6). Taken together, these results indicated the successful synthesis of NaGdF4:Ce/ Tb@CaP through the DNA templated mineralization and the attached another layer of Apt could provide the NaGdF4:Ce/Tb@CaP excellent selectivity for the following biological applications.
Fig. 4. (A) Dye release profiles for NaGdF4:Ce/Tb measured at (a) pH 4.5, (b) pH 7.4 and NaGdF4:Ce/Tb@CaP at (c) pH 4.5 and (d) pH 7.4. (B) Release profiles of calcium ions from NaGdF4:Ce/Tb@CaP in different pH buffers. The error bars represent the standard deviation of three measurements.
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function of Gd3þ concentration (Fig. S7), the relaxivity r1 value of the material was determined to be 6.33 mM1S1, which was higher than commercially available Magnevist (4.0 mM1S1). These results clearly suggested that NaGdF4:Ce/Tb@CaP-Apt could serve as an effective label material for MR imaging contrast. To investigate the pH-responsive gating effect of the hybrid nanomaterials, dye loading was accomplished by soaking the NaGdF4:Ce/Tb nanoparticles in a solution of rhodamine B (RhB) before CaP mineralization. The resulting particles were then dispersed in buffer at different values to test their controlled release property. As can be seen in Fig. 4A, a very clear and highly effective pH-operable gating effect was demonstrated by monitoring the absorbance of RhB (553 nm). There was a steady release with stepwise increasing from 13 to 85.6% by adjusting the pH values from 7.4 to 4.5 and the release reached a plateau within 24 h. In contrast, only negligible rhodamine B was released from dye loaded NaGdF4:Ce/Tb@CaP-Apt at physiological pH (pH 7.4). The significant difference indicated that the induced release in lower pH was due to the degradation of the mineral composite at the surface. Moreover, the dye loaded NaGdF4:Ce/Tb showed a fast RhB release rate irrespective of the pH value of the release medium, further supporting the conclusion that the mineral structure had a direct effect on the accessibility of the pores and conferred the superiority merely adsorption of monolayer of organic molecules on NaGdF4:Ce/Tb. In addition, the disintegration of the mineral coating was investigated by monitoring the concentration of the released calcium ions as a function of time. As shown in Fig. 4B, most of the calcium ions were released within 2 h at lysosomal pH (pH 4.5) while insignificant calcium ions were released at pH 7.4. Since the microenvironments of extracellular tumors tissues and intracellular lysosomes and endosomes are acidic, these findings further indicated that upon receptor-mediated endocytosis by cells, the CaP mineral coatings could be readily dissolved in acidic intracellular compartments, facilitating the diffusion based drug release from the nanocarriers.
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Cellular cytotoxicity would be important to be considered for the actual application of a potential carrier in biomedical fields. To explore the applications of NaGdF4:Ce/Tb@CaP-Apt nanoparticles in biomedicine, we first tested their potential toxicity on several types of cells. The standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine the relative viabilities of MCF7 human breast cancer cell, MDA-231 breast cancer cell, HeLa human cervical epithelioid carcinoma cell and human embryonic kidney 293 cells (HEK) after they were incubated with NaGdF4:Ce/ Tb@CaP-Apt at various concentrations for 24 h. No significant cytotoxicity of the nanocomposites was observed for all four types of cells under varying concentration range, manifesting the excellent biocompatibility of the NaGdF4:Ce/Tb@CaP-Apt in all dosages (Fig. S8). To test the targeting specificity of the nanocarrier, flow cytometry experiments were carried out. The DOX loaded NaGdF4:Ce/ Tb@CaP-Apt products were prepared and then incubated separately with three types of cancer cells (MCF-7, MDA-231 and HeLa) and control cells (HEK). The histograms showing numbers of cells exhibiting different fluorescence intensity indicated a clear shift to higher fluorescence on the three target cells treated with DOXNaGdF4:Ce/Tb@CaP-Apt in comparison with that of control cells (Fig. 5). The obvious signal difference suggested that Apt-attached NaGdF4:Ce/Tb@CaP was more easily internalized by the target cells than the control cells. Flow cytometry experiment was also carried out to evaluate the target cell uptake of DOX-NaGdF4:Ce/ Tb@CaP under different surface coverages of the Apt molecules. It was found that cellular uptake of DOX-NaGdF4:Ce/Tb@CaP-Apt increased with increasing amount of Apt on the nanocomposites. This observation indicated that the high internalization of the DOXNaGdF4:Ce/Tb@CaP-Apt by the cancer cell could be attributed to the specific interaction between Apt on the nanocomposites and the receptors overexpressed on the plasma membrane of the cancer cells. All these conclusions demonstrated the efficiency of our system as drug carriers for targeted therapy.
Fig. 5. Flow cytometry analysis to monitor the binding of DOX loaded nanoparticles with HEK cells (A), MCF-7 cells (B), MDA cells (C) and HeLa cells (D). The cells without the treatment of DOX loaded nanoparticles were used as the control samples.
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Fig. 6. Fluorescence microscope images of MCF-7 human breast cancer cells incubated with NaGdF4:Ce/Tb@CaP-Apt (A), NaGdF4:Ce/Tb@CaP-Rdm (B) and PBS (C). Each series can be classified to the NaGdF4:Ce/Tb@CaP, lysosome of cells (being dyed in red by lysotracker red), nuclei of cells (being dyed in blue by DAPI) and the merge of the three channels, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Next, the feasibility of cellular imaging using the NaGdF4:Ce/ Tb@CaP-Apt was investigated by using MCF-7 cells. After incubating 100 mg/mL of NaGdF4:Ce/Tb@CaP-Apt with MCF-7 cells for 4 h, intense Tb3þ green signal was observed from the cells (Fig. 6). In sharp contrast, under the identical experimental conditions, weak luminescence was observed from the MCF-7 cells treated with DOX-NaGdF4:Ce/Tb@CaP-Rdm or PBS, supporting that NaGdF4:Ce/Tb@CaP-Apt was uniquely suited for targeted fluorescence imaging of the living cells in vitro. To determine the location of the nanocomposites, the cells were also counterstained with DAPI and lysotracker dye to show the nucleus and lysosome, respectively. The colocalization between NaGdF4:Ce/Tb@CaP-Apt (green) and lysosome (red) showed that most of the nanocomposites resided in the endolysosomal compartments, which was beneficial for CaP dissolving to allow the release of drug molecules. To verify the feasibility of the NaGdF4:Ce/Tb@CaP-Apt nanosystem for intracellular therapeutic, the MTT assay was used for testing of the viability of MCF-7 and HEK cells in the presence of different concentrations of NaGdF4:Ce/Tb@CaP-Apt, DOX loaded NaGdF4:Ce/Tb@CaP-Apt (DOX-NaGdF4:Ce/Tb@CaP-Apt) and free DOX. As shown in Fig. 7, the NaGdF4:Ce/Tb@CaP-Apt did not show cytotoxicity against the two types of cells. However, growth inhibition of cells was observed after incubation with both free DOX and DOX-NaGdF4:Ce/Tb@CaP-Apt, which presented dosedependent cytotoxicity behavior (Fig. 7A). The remarkably higher cytotoxic efficacy of the DOX loaded NaGdF4:Ce/Tb nanoparticles towards the MCF-7 cells than that of free DOX could ascribe to the presence of Apt on the particle which resulted in an enhanced intracellular uptake and killing efficacy. As for HEK 293T cells, the relatively low killing efficiency of DOX-NaGdF4:Ce/Tb@CaP-Apt can be attributed to the minimal internalization of the nanovector owing to two possible reasons: 1) lack of receptors on the cell surface; 2) negatively charged surfaces of the nanoparticles. It is
known that DOX suppresses the growth of cancer cells via intercalation with cellular DNA, inducing apoptosis. The cell apoptosis in the current study was probed by Annexin V FITC and propidium iodide (PI) assay plus flow cytometry. As illustrated in Fig. 7D, a significantly higher proportion of cell population treated with DOXNaGdF4:Ce/Tb@CaP-Apt was Annexin V FITC positive as compared to the untreated cells (Fig. 7C), suggesting the fact that apoptosis could be induced in MCF-7 cells after DOX-NaGdF4:Ce/Tb@CaP-Apt treatment. 4. Conclusion We have successfully designed and synthesized a natural inorganic CaP covered NaGdF4:Ce/Tb hybrid system by DNA-mediated mineralization, which are capable of releasing guest drugs from the CaP-blocked pore under pH control. In preparation process, the DNA molecules that retained after one-pot NaGdF4:Ce/Tb synthesis was directly utilized as biotemplate for CaP heterogeneous nucleation to entrap guest molecules within the porous NaGdF4:Ce/Tb nanoparticles. This process was cost efficiency and the afforded multifunctional nanocarriers could be readily taken up by cells without premature leakage and visualized through dual-mode imaging. The introduction of another layer of Apt molecules facilitated cellular uptake of the resulting nanocomposite into specific target cells via receptor-mediated endocytosis. After been taken by the target tumor cells, the NaGdF4:Ce/Tb@CaP was found to be mostly accumulated in lysosome, which facilitated the dissolving of CaP coatings as non-toxic ions to initiate drug release and efficient cancer cell destruction. The in vitro efficacy of DOX loaded NaGdF4:Ce/Tb@CaP-Apt was further confirmed by the MTT experiment, which showed that the nanocomposites exhibited a highly efficacious for killing cancer cells while sparing normal cells. Given that the nanocomposites could be readily prepared with low cost, this lanthanide ions doped hybrid nanocarrier may serve as
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Fig. 7. Cytotoxicity of DOX, DOX-NaGdF4:Ce/Tb@CaP-Apt and NaGdF4:Ce/Tb@CaP-Apt on (A) MCF-7 cells and (B) HEK cells after incubation of 24 h. Flow cytogram representing apoptosis assay based on Annexin V-FITC and PI staining (C, D). In the flow cytogram, the cells in Q3 region denotes live cells, Q4: apoptotic, Q2: late apoptotic and Q1: necrotic cells.
practical and multifunctional probe for cancer therapy and the presented synthesis approach here may also benefit the preparation of many other types of multifunctional inorganic-biomolecular hybrid nanostructures based on the DNA nanotechnology. Acknowledgments The authors are grateful for the referee's helpful comments on the manuscript. Financial support was provided by the National Basic Research Program of China (2011CB936004 and 2012CB720602) and the National Natural Science Foundation of China (Grants 91213302, 21210002, 21301169). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.06.034
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
DNA-mediated biomineralization of rare-earth nanoparticles for simultaneous imaging and stimuli-responsive drug delivery Li Zhou, Zhaowei Chen, Kai Dong, Meili Yin, Jinsong Ren*, Xiaogang Qu* State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2014 Accepted 17 June 2014 Available online 4 July 2014
A DNA-guided method for surface engineering of NaGdF4:Ce/Tb hybrid nanoparticle has been proposed. In this study, the DNA molecules that retained after one-pot NaGdF4:Ce/Tb synthesis is directly utilized as biotemplate for CaP heterogeneous nucleation, thus the dual-purpose function of DNA is realized in the current study which could afford a new type of pH-responsive theranostic platform to enhance the therapeutic efficiency while minimizing side effects. The introduction of another layer of aptamer molecules on CaP facilitated cellular uptake of the resulting nanocomposite into specific target cells via receptor-mediated endocytosis. After been taken by the target tumor cells, the NaGdF4:Ce/Tb@CaP was found to be mostly accumulated in lysosome, which facilitated the dissolving of CaP coatings as non-toxic ions to initiate drug release and efficient cancer cell destruction. We envision that the hybrid nanocarrier may serve as practical and multifunctional probe for cancer therapy and the presented synthesis approach here may also benefit the preparation of many other types of multifunctional inorganicbiomolecular hybrid nanostructures based on the DNA nanotechnology. © 2014 Elsevier Ltd. All rights reserved.
Keywords: NaGdF4:Ce/Tb DNA Biomineralization Imaging Controlled drug delivery
1. Introduction With the rapid advances of nano-biotechnology, there has been an explosion of interest in the use of biomolecules possessing sophisticated structures and outstanding functions as building blocks for the development of multifunctional nanomaterial [1e5]. This biomolecules-based strategy opens up a new avenue for rationally control the structures as well as physical and chemical features of the materials. Due to the conformational polymorphism, sequencespecific recognition and robust physicochemical nature, DNA has been extensively investigated in nanotechnology and material science [1,6]. The use of DNA as template appears to be one of the most promising avenues available for fabricating a variety of metallicnanomaterials with potential applications ranging from electronics to biology [7e15]. DNA is rich of phosphate groups, amino groups and heterocyclic nitrogen atoms, it offers nucleation sites for metallic nanoparticles and provides control over the material growth and stability [6e15]. Meanwhile, the DNA attached on the prepared material could also be employed as recognition unit for the biological application or to organize the nanoparticles into
* Corresponding authors. Tel./fax: þ86 431 85262625. E-mail addresses: [email protected], [email protected] (J. Ren), [email protected] (X. Qu). http://dx.doi.org/10.1016/j.biomaterials.2014.06.034 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
periodic or discrete one-, two-, and three-dimensional architectures for material purpose [1,6e8]. For example, Kelley et al. recently reported that a chimeric DNA molecule could program both the growth and the biofunctionalization of the CdTe nanocrystals for bioimaging applications [1,7]. Although these DNAbased hybrid materials hold great promise in nanotechnology and nanomedicine, the exploring of further functions of the DNA anchored on the material surface still remains a big challenge in this field. Porous inorganic nanoparticles with high specific surface area have emerged as appealing material recently for the development of delivery systems, where various guest molecules could be absorbed into the pores and later released into various solutions [16e19]. Meanwhile, due to the unique optical and magnetic behavior, the lanthanide ions doped-porous nanocrystals have been extensively employed for biomedical applications, including our recent development of DNA-based porous lanthanide doped NaGdF4 nanocarriers for cell-specific drug delivery [20e25]. Compared with organic fluorophores that modified on the surface of material, the doped luminescent lanthanide cations provide complementary properties such as resistance to photobleaching, long luminescence lifetimes, absence of reabsorption and sharp emission bands in the visible and the near-infrared [26e30]. Moreover, it could be employed as a probe that combines therapy
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functions with multi-mode imaging methodologies which could provide complementary information in biomedical studies [31e35]. While the Ln3þ doped-porous nanoparticles (Ln3þ-pNP) have been adopted as promising nanomaterials for biological application, these delivery platforms might significantly compromise their efficiency because the entrapped drug may start leaking out of the carrier immediately after administration due to the lack of capping/blocking agent. Therefore, the ability to maintain optimum therapeutic efficacy, i.e. no premature release in blood circulation whilst having a rapid drug release in tumor tissues, remains a significant challenge for the development of Ln3þ-pNP based multifunctional delivery system. During the past several decades, nature's strategies to the formation of a wide range of specially designed organic-inorganic hybrid materials such as bone, teeth, and shells have attracted increasing attention [36]. The mild condition and high efficiency of these preparation processes have inspired research to mimic them in vitro and a great deal of biominerals with tunable morphologies and properties has been prepared [37,38]. In terms of materials design, bio-inspired calcium phosphate (CaP) hybrid materials have attracted tremendous interest; this is mainly due to the fact that these hybrids have a wide range of very useful properties and potential applications [39e48]. For example, Epple et al. proposed multi-shell CaP-DNA nanoparticles and CaP-DNA/siRNA nanohybrids for effective transfection of cells [44]. Adair et al. developed CaP nanoparticles that encapsulated both fluorophores and chemotherapeutics for in vitro imaging and anticancer drug delivery [47,48]. Here inspired by the high binding affinity of CaP toward DNA, we propose to engineer the Ln3þ-pNP surface with a layer of natural CaP to overcome the big challenges in Ln3þ-pNP applications mentioned above. As shown in Scheme 1, followed by our previous report, porous NaGdF4:Ce/Tb nanoparticles were prepared by using DNA as superior ligands. Then the hydrophilic DNA molecules that attached on the NaGdF4:Ce/Tb nanoparticles were utilized as the nucleation sites for CaP heterogeneous nucleation. Thereafter, by precisely controlling the following growing process, CaP mineral nanoshells which possessed pH-dependent
Ca2+
DOX- NaGdF4:Ce/Tb
HPO42-
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biodegradability could be formed (designed as NaGdF4:Ce/ Tb@CaP). Thus the dual-purpose function of DNA was realized in the current study which could afford a new type of pH-responsive theranostic platform to enhance the therapeutic efficiency while minimizing side effects. Moreover, an additional layer of aptamer (Apt) was attached to the mineral surfaces via chelation interactions, which produced a colloidal stabilized nanocomposite with biospecific properties (designed as NaGdF4:Ce/Tb@CaP-Apt). 2. Materials and methods 2.1. Materials Ln(NO3)3 and doxorubicin (DOX) were purchased from Sangon (ShangHai, China). NaF and dimethyl sulfoxide (DMSO) were purchased from Shanghai Chemical Factory (Shanghai, China). Fish sperm DNA was obtained from SigmaeAldrich. Ultrapure water (18.2 MU; Millpore Co., USA) was used throughout the experiment. The oligonucleotide used in this article was synthesized by Sangon Biotechnology Inc. (Shanghai, China). The sequence was as follows: 50 -GGTGGTGGTGGTTGTGGTGGTGGTGGT-30 Aptamer (Apt) 50 -TTAGGG TTAGGGTTAGGGTTAGGG TTA-30 control DNA with randomized sequence (Rdm) 2.2. Measurements and characterizations FT-IR analyze was carried out on a Bruker Vertex 70 FT-IR Spectrometer. SEM images were obtained with a Hitachi S-4800 FE-SEM. The UVeVis absorption spectra and fluorescence spectra were recorded using a JASCO V-550 UVeVisible and a JASCO FP6500 spectrophotometer (JASCO International Co.,LTD., Tokyo, Japan). TEM images were recorded using an FEI TECNAI G2 20 high-resolution transmission electron microscope operating at 200 kV. The samples were degassed at 90 С for 5 h. The magnetic properties of samples were collected on a MPM5-XL-5 superconducting quantum interference device (SQUID) magnetometer. N2 adsorptionedesorption isotherms were obtained on a Micromeritics ASAP 2020M automated sorption analyzer. The specific surface areas were calculated from the adsorption data in the low pressure range using the BET model and pore size was determined following the BJH method. The zeta potential of the nanomaterials in HEPES was measured in a Zetasizer 3000HS analyzer. Cell imaging was performed with a fluorescent microscope (Olympus, BX51). 2.3. Preparation of NaGdF4:Ce/Tb nanoparticle 280 mL Gd(NO3)3 (0.2 M), 35 mL Ce(NO3)3 (0.2 M) and 15 mL of Tb(NO3)3 (0.2 M) were added to 100 mL beaker and diluted to 15 mL with water. Then 5 mL of the DNA stock solution (6.4 mM) was added quickly under magnetic stirring. Subsequently,
Aptamer
MR imaging Lanthanide luminescence
DOX- NaGdF4:Ce/Tb@CaP
Scheme 1. Schematic illustration of the biomineralization process inspired DOX loaded NaGdF4:Ce/Tb@CaP-Apt hybrid. DOX loaded NaGdF4:Ce/Tb@CaP-Apt was internalized via receptor-mediated endocytosis. The DOX was released through the dissolving of CaP within intracellular endolyso-somal compartment.
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stoichiometric amount of NaF (the molar ratio of NaF to lanthanide ion was set to 5:1) in 40 mL of water was added dropwise to the above solution. The mixture was agitated for about 10 min, then transferred to autoclave, sealed, and hydrothermally treated at 90 C for 12 h. The system was cooled to room temperature naturally. Pure powders could be obtained by purifying the samples with water several times to remove the remnants. 2.4. Synthesis of NaGdF4:Ce/Tb@CaP nanocomposites Mineralization of the NaGdF4:Ce/Tb nanoparticles was carried out in the presence of calcium nitrate and ammonium phosphate using a method similar to that developed by Schmidt et al. [49]. In a typical procedure, 0.03 mL calcium nitrate stock solution (0.1 M) was added to the solution containing the NaGdF4:Ce/Tb nanoparticles (40 mg) and stirred slowly for 1 h. After that, 0.02 mL of the ammonium phosphate solution (0.1 M) was slowly added and stirred for 1 h. This procedure of sequential addition of the stock solutions was repeated 8 times. Then, the suspension was allowed to age for another 12 h without stirring, and followed by centrifugation, washing with phosphate-buffered saline for several times to obtain the NaGdF4:Ce/Tb@CaP product.
weighted and T2-weighted MR imaging. The Eppendorf tubes were then scanned in a 1.5-T Magnetom Espree MIR system after the preparation. 2.7. Cargo loading and release experiment For rhodamine B loading, NaGdF4:Ce/Tb nanoparticles were soaked in a solution of rhodamine B solution (0.5 mg/mL) for 24 h and followed by centrifugation, washing with pure water to remove unloaded and adsorbed molecules. All the washing solutions were collected, and the loading amount was approximately 1.12% (w/w) based on UVeVis spectroscopy. After that, mineralization of the rhodamine B loaded nanoparticle and introduction of Apt molecules to the resulting nanocomposites were carried out as described above. The as-prepared material was then dispersed in 5 mL aqueous buffer solutions (pH 7.4 phosphate buffer and pH 4.5 acetate buffer) at room temperature. Aliquots were taken from the suspension at predetermined time and the delivery of rhodamine B from the nanocomposites to the buffer solution was monitored via the absorbance band of the dye centered at 553 nm. To obtain DOX-NaGdF4:Ce/Tb@CaP nanocomposites, DOX loading and mineralization process were performed as above. The loading amount was approximately 1.2% (w/w) by UVeVis absorbance spectroscopy.
2.5. Attaching of NaGdF4:Ce/Tb@CaP with Apt Before the experiment, AS1411 Apt was first incubated with HEPES buffer (10 mM, 2.5 mM MgCl2, 140 mM KCl, pH 7.4) to form G quadruplex structure. Then NaGdF4:Ce/Tb@CaP (1 mg/mL) solution was incubated with the G quadruplex structure of the AS1411 Apt with different concentration (5, 10 mM, 30 mM, 50 mM, 70 mM) in the 4 С fridge for 12 h. The resulting NaGdF4:Ce/Tb@CaP-Apt was separated by centrifugation and washed with HEPES buffer for several times to remove the free Apt. The supernatant of the dispersions was analyzed by UVeVis spectrophotometry at 260 nm.
2.8. CaP disintegration experiments NaGdF4:Ce/Tb@CaP (10 mg) material was dispersed in 5 mL aqueous buffer solutions (pH 7.4 phosphate and pH 4.5 acetate buffers). Aliquots were extracted at given time intervals and replaced with the same volume of fresh medium. The release rate of calcium ions was monitored by taking a 50 mL sample and diluting it using Arsenazo III solution (40 0 mL, 0.2 mM) in HBS (HEPES-buffered saline where [HEPES]¼ 20 mM and [NaCl] ¼ 150 mM at pH 7.4). The absorbance of Arsenazo III/ Ca2þ complex in the solution was measured at 656 nm, and the concentration of calcium ions was calculated based on the standard curve.
2.6. Magnetic resonance imaging T1-weighted and T2-weighted MR imaging were obtained by using a 1.5-T clinical MRI instrument (Siemens Medical System). Dilutions of NaGdF4:Ce/ Tb@CaP nanocomposite in HEPES buffer containing 1% agarose with different concentrations as contrast agent were placed in 4.0 mL Eppendorf tubes for T1-
2.9. Cell cultures and MTT assay The MCF-7 human breast cancer cell, MDA-231 breast cancer cell, HeLa human cervical epithelioid carcinoma cell and human embryonic kidney 293 cells (HEK) were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with
Fig. 1. Representative SEM and TEM micrographs of (A, C) NaGdF4:Ce/Tb and (B, D) mineralized NaGdF4:Ce/Tb.
L. Zhou et al. / Biomaterials 35 (2014) 8694e8702 10% fetal bovine serum at 37 C with 5% CO2 for 24 h. Cell viability was determined by measuring by MTT assay [31]. 2.10. Fluorescence microscopy To do the test, the concentration of MCF-7 cells was fixed at a density of 105 cells/ well in 24-well assay plates. NaGdF4:Ce/Tb@CaP-Apt and NaGdF4:Ce/Tb@CaP-Rdm nanocomposites were added to the cells and the mixture was incubated at 37 C for 4 h to allow the uptake of the nanoparticles. The cells were then washed several times with PBS. Then 500 mL fixing solution (1% glutaraldehyde and 10% formaldehyde) was added to each well for 30 min and finally the fluorescence intensity was monitored.
3. Results and discussion To perform the experiment, the NaGdF4:Ce/Tb nanoparticles were first synthesized according to our previous report [31]. As shown in Fig. 1A and C, mono-dispersed porous nanoparticles with a diameter of about 120 nm were obtained with the addition of DNA molecules (fish sperm DNA). It was worth to note that the minimal different between the as-prepared material with the previous reports may ascribe to the different type of DNA that used in each study. The successful capping of DNA molecules on the nanoparticles was confirmed by the observation of typical absorbance of DNA at 260 nm after the reaction and the existence of C, O, N and P elements in the NaGdF4:Ce/Tb nanoparticles (Fig. S1, S2). Moreover, the FT-IR spectra of the NaGdF4:Ce/Tb material showed typical absorption bands at 1070 and 988 cm1, which could be ascribed to the OeP stretching mode of phosphate groups (Fig. 2A).
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The N2 adsorption and desorption analysis was introduced to investigate the surface area and pore properties of the product. A typical-type isotherm with H1-hysteresis loops was observed, demonstrating the presence of mesopores (Fig. S3). The BET surface area, total pore volume, and average pore size were calculated to be 105.05 m2/g, 0.274 cm3/g and 6.3 nm, respectively. Next, the prepared NaGdF4:Ce/Tb nanoparticles were mineralized by the sequential addition method, in which CaP deposition on the material was initiated by calcium nitrate to allow for the electrostatic localization of calcium ions at phosphate of the DNA backbone. Thereafter, the addition of sodium phosphate as the source of a phosphate anion induced the growth of nanophase CaP. The successful mineralization of NaGdF4:Ce/Tb nanoparticle was confirmed by various methods. TEM image investigations of NaGdF4:Ce/Tb@CaP (~130 nm) provided direct evidence of the distribution of CaP on the surfaces of NaGdF4:Ce/Tb (Fig. 1D). The nucleation of CaP at the interfaces was also supported by the enhanced peaks in the FT-IR spectrum, which corresponded to the OePeO bending modes appearing around 600 and 560 cm1, PeO asymmetric stretching mode at around 1000 cm1 (Fig. 2A and B). Zeta potential analysis of the NaGdF4:Ce/Tb showed that the surface potential of the nanoparticle increased from 25.7 mV to 7.3 mV in PBS buffer (pH 7.4) after DNA templated mineralization process, suggesting that mineral deposition almost completely shielded anionic phosphate charges (Fig. 2C). To further confirm the presence of calcium and phosphate in the shell, energy-dispersive X-ray spectroscopy (EDX) was used to analyze
Fig. 2. (A), (B) FT-IR spectra of (a, a0 ) NaGdF4:Ce/Tb, (b, b0 ) NaGdF4:Ce/Tb@CaP, (c, c0 ) NaGdF4:Ce/Tb@CaP-Apt. (C) Zeta potential measured at each step of the coating process in deionized water. (D) EDX spectrum of the NaGdF4:Ce/Tb@CaP nanoparticles.
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To investigate the feasibility of using NaGdF4:Ce/Tb@CaP-Apt particles as multifunctional delivery system in bio-related fields, the fluorescence and magnetic properties of NaGdF4:Ce/Tb@CaPApt were examined. The photoluminescence (PL) emission spectra of NaGdF4:Ce/Tb@CaP-Apt solution was first measured at room temperature. As shown in Fig. 3A, the luminescence of the NaGdF4:Ce/Tb@CaP-Apt in water appeared similar to that of the NaGdF4:Ce/Tb samples, indicating that the characteristic luminescent property of the NaGdF4:Ce/Tb nanoparticles was unaffected by the CaP coating. Moreover, the colloidal solutions of the NaGdF4:Ce/ Tb@CaP-Apt (0.5 mg/mL) exhibited bright green luminescence when excited with a UV lamp, indicating that those nanocomposites had good potential for use as luminescent labels in biological imaging (Fig. 3A, inset). The magnetic field dependency of NaGdF4:Ce/Tb and NaGdF4:Ce/Tb@CaP-Apt were also measured to evaluate the potential application of the materials as MRI contrast agents. Room temperature magnetization curve of NaGdF4:Ce/Tb and NaGdF4:Ce/Tb@CaP-Apt demonstrated their paramagnetic character (Fig. 3B). The mass magnetic susceptibility value of NaGdF4:Ce/Tb@CaP-Apt was calculated to be 1.11 104 emu/g Oe. The T1- and T2- weighted MR images evaluated at a 1.5 T human clinical scanner revealed an enhancement of MR signal as increasing of the concentrations of the nanocomposites (Fig. 3B, inset). From the slope of the plot of the relaxation rate (1/T1) as a
Fig. 3. (A) Emission spectrum of NaGdF4:Ce/Tb nanoparticles before (pink line) and after mineralized (indigo line). (B) Room temperature magnetization curve of NaGdF4:Ce/Tb@CaP. Inset: (A) the photographs of dispersed solution containing NaGdF4:Ce/Tb (left) and NaGdF4:Ce/Tb@CaP under UV lamp, (B) T1-weighted and T2weighted MR images of NaGdF4:Ce/Tb@CaP at various concentrations in buffer solution.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the rough surfaces. The significant appearance of Ca element and P element indicated the formation of CaP which was the characteristic product of template-induced mineralization (Fig. 2D). Furthermore, the change of sorption type was expected due to the capping effect of the CaP (Fig. S3). After successful mineralization of NaGdF4:Ce/Tb nanoparticle, the nanocomposites were dispersed in the Apt solution to obtain NaGdF4:Ce/Tb@CaP@Apt. The anchoring amount of Apt increased as an increasing of Apt concentration and the maximum immobilization efficiency was determined to be about 30.3 mmol/g (Fig. S4, S5). After anchoring another layer of Apt molecules, the final zeta potential of the nanocomposites reached 19.18 mV. Owing to the spatial configuration of the Apt was needed for target, the fluorescent probe molecule NMM (Nmethyl mesoporphyrin IX) was used to prove that the G quadruplex structures of Apt were retained after adsorption. Significant fluorescence enhancement was observed upon mixing NaGdF4:Ce/ Tb@CaP@Apt with NMM, indicating the presence of quadruplex structures of Apt on the nanocomposites (Fig. S6). Taken together, these results indicated the successful synthesis of NaGdF4:Ce/ Tb@CaP through the DNA templated mineralization and the attached another layer of Apt could provide the NaGdF4:Ce/Tb@CaP excellent selectivity for the following biological applications.
Fig. 4. (A) Dye release profiles for NaGdF4:Ce/Tb measured at (a) pH 4.5, (b) pH 7.4 and NaGdF4:Ce/Tb@CaP at (c) pH 4.5 and (d) pH 7.4. (B) Release profiles of calcium ions from NaGdF4:Ce/Tb@CaP in different pH buffers. The error bars represent the standard deviation of three measurements.
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function of Gd3þ concentration (Fig. S7), the relaxivity r1 value of the material was determined to be 6.33 mM1S1, which was higher than commercially available Magnevist (4.0 mM1S1). These results clearly suggested that NaGdF4:Ce/Tb@CaP-Apt could serve as an effective label material for MR imaging contrast. To investigate the pH-responsive gating effect of the hybrid nanomaterials, dye loading was accomplished by soaking the NaGdF4:Ce/Tb nanoparticles in a solution of rhodamine B (RhB) before CaP mineralization. The resulting particles were then dispersed in buffer at different values to test their controlled release property. As can be seen in Fig. 4A, a very clear and highly effective pH-operable gating effect was demonstrated by monitoring the absorbance of RhB (553 nm). There was a steady release with stepwise increasing from 13 to 85.6% by adjusting the pH values from 7.4 to 4.5 and the release reached a plateau within 24 h. In contrast, only negligible rhodamine B was released from dye loaded NaGdF4:Ce/Tb@CaP-Apt at physiological pH (pH 7.4). The significant difference indicated that the induced release in lower pH was due to the degradation of the mineral composite at the surface. Moreover, the dye loaded NaGdF4:Ce/Tb showed a fast RhB release rate irrespective of the pH value of the release medium, further supporting the conclusion that the mineral structure had a direct effect on the accessibility of the pores and conferred the superiority merely adsorption of monolayer of organic molecules on NaGdF4:Ce/Tb. In addition, the disintegration of the mineral coating was investigated by monitoring the concentration of the released calcium ions as a function of time. As shown in Fig. 4B, most of the calcium ions were released within 2 h at lysosomal pH (pH 4.5) while insignificant calcium ions were released at pH 7.4. Since the microenvironments of extracellular tumors tissues and intracellular lysosomes and endosomes are acidic, these findings further indicated that upon receptor-mediated endocytosis by cells, the CaP mineral coatings could be readily dissolved in acidic intracellular compartments, facilitating the diffusion based drug release from the nanocarriers.
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Cellular cytotoxicity would be important to be considered for the actual application of a potential carrier in biomedical fields. To explore the applications of NaGdF4:Ce/Tb@CaP-Apt nanoparticles in biomedicine, we first tested their potential toxicity on several types of cells. The standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine the relative viabilities of MCF7 human breast cancer cell, MDA-231 breast cancer cell, HeLa human cervical epithelioid carcinoma cell and human embryonic kidney 293 cells (HEK) after they were incubated with NaGdF4:Ce/ Tb@CaP-Apt at various concentrations for 24 h. No significant cytotoxicity of the nanocomposites was observed for all four types of cells under varying concentration range, manifesting the excellent biocompatibility of the NaGdF4:Ce/Tb@CaP-Apt in all dosages (Fig. S8). To test the targeting specificity of the nanocarrier, flow cytometry experiments were carried out. The DOX loaded NaGdF4:Ce/ Tb@CaP-Apt products were prepared and then incubated separately with three types of cancer cells (MCF-7, MDA-231 and HeLa) and control cells (HEK). The histograms showing numbers of cells exhibiting different fluorescence intensity indicated a clear shift to higher fluorescence on the three target cells treated with DOXNaGdF4:Ce/Tb@CaP-Apt in comparison with that of control cells (Fig. 5). The obvious signal difference suggested that Apt-attached NaGdF4:Ce/Tb@CaP was more easily internalized by the target cells than the control cells. Flow cytometry experiment was also carried out to evaluate the target cell uptake of DOX-NaGdF4:Ce/ Tb@CaP under different surface coverages of the Apt molecules. It was found that cellular uptake of DOX-NaGdF4:Ce/Tb@CaP-Apt increased with increasing amount of Apt on the nanocomposites. This observation indicated that the high internalization of the DOXNaGdF4:Ce/Tb@CaP-Apt by the cancer cell could be attributed to the specific interaction between Apt on the nanocomposites and the receptors overexpressed on the plasma membrane of the cancer cells. All these conclusions demonstrated the efficiency of our system as drug carriers for targeted therapy.
Fig. 5. Flow cytometry analysis to monitor the binding of DOX loaded nanoparticles with HEK cells (A), MCF-7 cells (B), MDA cells (C) and HeLa cells (D). The cells without the treatment of DOX loaded nanoparticles were used as the control samples.
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Fig. 6. Fluorescence microscope images of MCF-7 human breast cancer cells incubated with NaGdF4:Ce/Tb@CaP-Apt (A), NaGdF4:Ce/Tb@CaP-Rdm (B) and PBS (C). Each series can be classified to the NaGdF4:Ce/Tb@CaP, lysosome of cells (being dyed in red by lysotracker red), nuclei of cells (being dyed in blue by DAPI) and the merge of the three channels, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Next, the feasibility of cellular imaging using the NaGdF4:Ce/ Tb@CaP-Apt was investigated by using MCF-7 cells. After incubating 100 mg/mL of NaGdF4:Ce/Tb@CaP-Apt with MCF-7 cells for 4 h, intense Tb3þ green signal was observed from the cells (Fig. 6). In sharp contrast, under the identical experimental conditions, weak luminescence was observed from the MCF-7 cells treated with DOX-NaGdF4:Ce/Tb@CaP-Rdm or PBS, supporting that NaGdF4:Ce/Tb@CaP-Apt was uniquely suited for targeted fluorescence imaging of the living cells in vitro. To determine the location of the nanocomposites, the cells were also counterstained with DAPI and lysotracker dye to show the nucleus and lysosome, respectively. The colocalization between NaGdF4:Ce/Tb@CaP-Apt (green) and lysosome (red) showed that most of the nanocomposites resided in the endolysosomal compartments, which was beneficial for CaP dissolving to allow the release of drug molecules. To verify the feasibility of the NaGdF4:Ce/Tb@CaP-Apt nanosystem for intracellular therapeutic, the MTT assay was used for testing of the viability of MCF-7 and HEK cells in the presence of different concentrations of NaGdF4:Ce/Tb@CaP-Apt, DOX loaded NaGdF4:Ce/Tb@CaP-Apt (DOX-NaGdF4:Ce/Tb@CaP-Apt) and free DOX. As shown in Fig. 7, the NaGdF4:Ce/Tb@CaP-Apt did not show cytotoxicity against the two types of cells. However, growth inhibition of cells was observed after incubation with both free DOX and DOX-NaGdF4:Ce/Tb@CaP-Apt, which presented dosedependent cytotoxicity behavior (Fig. 7A). The remarkably higher cytotoxic efficacy of the DOX loaded NaGdF4:Ce/Tb nanoparticles towards the MCF-7 cells than that of free DOX could ascribe to the presence of Apt on the particle which resulted in an enhanced intracellular uptake and killing efficacy. As for HEK 293T cells, the relatively low killing efficiency of DOX-NaGdF4:Ce/Tb@CaP-Apt can be attributed to the minimal internalization of the nanovector owing to two possible reasons: 1) lack of receptors on the cell surface; 2) negatively charged surfaces of the nanoparticles. It is
known that DOX suppresses the growth of cancer cells via intercalation with cellular DNA, inducing apoptosis. The cell apoptosis in the current study was probed by Annexin V FITC and propidium iodide (PI) assay plus flow cytometry. As illustrated in Fig. 7D, a significantly higher proportion of cell population treated with DOXNaGdF4:Ce/Tb@CaP-Apt was Annexin V FITC positive as compared to the untreated cells (Fig. 7C), suggesting the fact that apoptosis could be induced in MCF-7 cells after DOX-NaGdF4:Ce/Tb@CaP-Apt treatment. 4. Conclusion We have successfully designed and synthesized a natural inorganic CaP covered NaGdF4:Ce/Tb hybrid system by DNA-mediated mineralization, which are capable of releasing guest drugs from the CaP-blocked pore under pH control. In preparation process, the DNA molecules that retained after one-pot NaGdF4:Ce/Tb synthesis was directly utilized as biotemplate for CaP heterogeneous nucleation to entrap guest molecules within the porous NaGdF4:Ce/Tb nanoparticles. This process was cost efficiency and the afforded multifunctional nanocarriers could be readily taken up by cells without premature leakage and visualized through dual-mode imaging. The introduction of another layer of Apt molecules facilitated cellular uptake of the resulting nanocomposite into specific target cells via receptor-mediated endocytosis. After been taken by the target tumor cells, the NaGdF4:Ce/Tb@CaP was found to be mostly accumulated in lysosome, which facilitated the dissolving of CaP coatings as non-toxic ions to initiate drug release and efficient cancer cell destruction. The in vitro efficacy of DOX loaded NaGdF4:Ce/Tb@CaP-Apt was further confirmed by the MTT experiment, which showed that the nanocomposites exhibited a highly efficacious for killing cancer cells while sparing normal cells. Given that the nanocomposites could be readily prepared with low cost, this lanthanide ions doped hybrid nanocarrier may serve as
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Fig. 7. Cytotoxicity of DOX, DOX-NaGdF4:Ce/Tb@CaP-Apt and NaGdF4:Ce/Tb@CaP-Apt on (A) MCF-7 cells and (B) HEK cells after incubation of 24 h. Flow cytogram representing apoptosis assay based on Annexin V-FITC and PI staining (C, D). In the flow cytogram, the cells in Q3 region denotes live cells, Q4: apoptotic, Q2: late apoptotic and Q1: necrotic cells.
practical and multifunctional probe for cancer therapy and the presented synthesis approach here may also benefit the preparation of many other types of multifunctional inorganic-biomolecular hybrid nanostructures based on the DNA nanotechnology. Acknowledgments The authors are grateful for the referee's helpful comments on the manuscript. Financial support was provided by the National Basic Research Program of China (2011CB936004 and 2012CB720602) and the National Natural Science Foundation of China (Grants 91213302, 21210002, 21301169). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.06.034
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