Biomaterials 63 (2015) 168e176
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Nanoparticle-conjugated aptamer targeting hnRNP A2/B1 can recognize multiple tumor cells and inhibit their proliferation Hui Li a, 1, Lei Guo b, 1, Aixue Huang a, 1, Hua Xu b, Xuemei Liu a, Hongmei Ding a, Jie Dong a, Jie Li a, Chaonan Wang a, Xueting Su a, Xingfeng Ge a, Leqiao Sun a, Chenjun Bai a, Xuelian Shen a, Tao Fang a, Zhanghua Li c, Yong Zhou d, Linsheng Zhan d, Shaohua Li a, *, Jianwei Xie b, *, Ningsheng Shao a, * a
Beijing Institute of Basic Medical Sciences, Beijing, 100850, China State Key Laboratory for Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, No. 27 Taiping Road, Haidian District, 100850, Beijing, China c Deparment of Orthopedics, Wuhan City Hospital No,3&Tongren Hospital of Wuhan University, Wuhan, 430060, China d Beijing Institute of Laboratory of Blood-borne Virus, Beijing, 100850, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2015 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online 14 June 2015
In this study, we further investigated a previously developed aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells. We found that this C6-8 aptamer specifically binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and that it specifically labeled multiple tumor-cell lines as effectively as hnRNP A2/B1 monoclonal antibodies. When conjugated with fluorescent carbon nanodots (CDots) it could freely enter multiple living tumor cell lines (HepG2, MCF-7, H1299, and HeLa), whose growth it inhibited by targeting hnRNP A2/B1. Similar inhibitory effects were observed when the GFP-HepG2 hepatocarcinoma cells treated with C6-8-conjugated CDots were implanted in nude mice. Our work provides a new aptamer for targeting/labeling multiple tumor cell types, and its nanoparticle conjugates bring further advantages that increase its potential for use in cancer diagnosis and therapy. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Tumor cells SELEX Aptamer Heterogeneous nuclear ribonucleoprotein A2/B1 Nanoparticles
1. Introduction The in vitro selection technique known as SELEX (systematic evolution of ligands by exponential enrichment), which was developed in the early 1990s, is a high throughput screening technique that involves the progressive selection of highly specific ligands by repeated rounds of selection and amplification from a large random synthetic nucleic acid library [1,2]. The selected (enriched) oligonucleotide ligands, named aptamers, which are short single-stranded (ss) DNA or RNA molecules, have a high affinity and specificity for targets that are compatible with their specific three-dimensional shapes. Since its initial development, SELEX screening targets have progressed from small organic
* Corresponding authors. E-mail addresses: [email protected] (S. Li), [email protected] (J. Xie), shaonsh@ hotmail.com (N. Shao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.06.013 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
molecules and pure proteins to protein complexes, cells and even tissues [3e8]. As a result, aptamers have attracted increased attention in many fields, especially in cancer research. SELEX-generated aptamers can specifically target tumor cells and provide a new method for biomarker verification. In addition to the comparative binding affinity and specificity to monoclonal antibody, the aptamers have other properties such as easier to be synthesized and modified in vitro, less immunogenic, smaller, and more amenable to long-term preservation. Based on these merits, aptamers have been widely investigated for use in clinical diagnosis and cancer drug screening [9,10]. In recent years, the incidence of cancer has significantly increased, threatening the lives of millions worldwide. Finding good tumor biomarkers and drug targets is a key issue for early diagnosis and effective treatment of cancer. At present, some aptamers targeting tumor cells have been generated via cell SELEX, but in general these only recognize a single tumor type. To our knowledge, there are no reports of aptamers that recognize
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multiple tumor cell types from different origins. In previous work, we used cell SELEX to find an aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells [11]. In this study, we further investigated this C6-8 aptamer, focusing on its capacity to target multiple tumor-cell lines, the identity of the underlying molecular target, and the applicability and benefits of its conjugation with fluorescent carbon nanodots (CDots).
approximately 600 mg of cell (total)-protein extract in 600 mL of binding buffer for 1 h at 37 C. The beads were washed six times, and the binding components were eluted from the beads by heating in 50 mL of SDS PAGE loading buffer and applied to 12% SDSPAGE. The protein band of interest was excised and digested with porcine trypsin for LC-MS/MS analysis, followed by a Mascot database search.
2. Materials and methods
2.6. The binding of the biotinylated C6-8 aptamer to the target molecule
2.1. Random ssDNA library and primers A random ssDNA library (GP30) and primers were synthesized by Invitrogen Co Ltd, Beijing, China. The sequences are listed in the Table 1 of the Supplementary Data. The primers, Plong-1 and P11, were used for standard PCR amplification of double-stranded DNA molecules. Plong-1 and Pstem-loop primers were used to synthesize single-stranded DNA as we have reported previously [6]. 2.2. Cell lines and cultures The rat osteoblast cell line, ROS 17/2.8, was cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and maintained in an incubator at 37 C and 5% CO2. The cervical-cancer cell line, HeLa, the lung cancer cell line, H1299, the hepatoma cell line, HepG2, and the breast-cancer cell line, MCF-7, were all cultured in DMEM supplemented with 10% fetal bovine serum and maintained at 37 C and 5% CO2. 2.3. Fluorescence-microscopy and flow-cytometry analysis of cellaptamer binding After overnight incubation, adherent cells were harvested by trypsinization and fixed in absolute methanol at 20 C for 30 min, and then washed with phosphate-buffered saline (PBS) before the experiment. The FAM-labeled random library (GP30) and the FAM-labeled C6-8 aptamer were each dissolved in 100 mL of incubation buffer at a concentration of 20 mg/mL and heated for 5 min at 95 C before cooling on ice for 10 min. The denatured aptamers were incubated with the tumor cells for 1 h at 37 C in the dark. After washing, binding of the aptamers to tumor cells was determined using fluorescence microscopy and flow cytometry. 2.4. Binding affinity of the C6-8 aptamer to tumor cells The binding affinity of the C6-8 aptamer to HeLa cells was determined as follows. Briefly, 5 105 cells were incubated with varying concentrations of FAM-aptamer in 500 mL of binding buffer at 37 C for 1 h. Cells were washed once with PBS and resuspended in 0.5 mL of PBS. The mean fluorescence intensity of target cells labeled with the aptamer was determined using a FACScan cytometer (BD, USA). The equilibrium dissociation constant (Kd) of the aptamer-cell interaction was calculated via the equation Y ¼ Bmax$X/(Kd þ X), using GraphPad Prism4 software. The affinity assay of antibody to HeLa cells was performed similarly with primary monoclonal antibody and FITC labeled secondary antibody. 2.5. Identification of the molecular target of the C6-8 aptamer The total protein was extracted from HeLa and HepG2 tumor cells. 5 mg of biotinylated C6-8 aptamer or initial library control was dissolved in 500 mL of binding buffer and incubated with 1 ml of streptavidin-coated magnetic beads for 30 min at room temperature. After washing three times, the beads were incubated with
The biotinylated initial library (GP30) and the biotinylated C6-8 aptamer were synthesized by Invitrogen Co Ltd, Beijing, China. In each case, 2 ng of the ssDNA was denatured by heating for 5 min at 95 C, followed by rapid cooling on ice for 10 min, 5 mg of heterogeneous ribonucleoprotein A1 (hnRNP A1) or 5 mg of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) were respectively added to the biotinylated GP30 and C6-8 solutions (final hnRNP concentrations were 500 mg/mL), which were then incubated in 10 mL of incubation buffer for 1 h at 37 C. The products were subjected to 6% natural gel electrophoresis for 1.5 h and transferred to a nylon membrane (70 V for 60 min). After UV crosslinking for 2 min, the membrane was incubated with blocking reagent for 30 min at 37 C and incubation with stabilized streptavidin-HRP conjugate (diluted 1:500) for 30 min at 37 C. After washed three times, the nylon membrane was incubated with equilibration buffer for 10 min at room temperature. The retarded band indicating the binding of the C6-8 aptamer to protein hnRNP A2/B1 or hnRNP A1 and the free aptamer band are developed using the enhanced chemiluminescence Western blotting detection system. 2.7. Fluorescence microscopy analysis of C6-8 aptamer-binding to cancer cells and tissue Directly after fixation, the HeLa cells were incubated at 37 C for 1 h with 100 mL of 20 mg/mL FAM-GP30, 20 mg/mL FAM-C6-8, or 0.5 mg/mL anti-hnRNP A2/B1monoclonal antibodies; in the case of the antibodies, this was followed by another incubation with 1:100-diluted FITC-conjugated goat anti-mouse IgG for 30 min at 37 C. Unbound aptamers and antibodies were washed away with PBS. The cells were counterstained with 0.25% Evans blue and the fluorescence patterns were observed under a fluorescence microscope (Olympus IX71, Tokyo, Japan). Paraffin-embedded tissue sections were antigen retrieved and then permeabilized with 0.1% Tween20 in PBS. After blocking with a solution of 0.1-mg/mL yeast-transfer RNA (ytRNA), 0.1-mg/mL salmon sperm DNA and 1% BSA in PBS, the binding patterns of the sections were determined (as described above) for FAM-GP30, FAM-C6-8 and anti-hnRNPA2/B. 2.8. Synthesis of aptamer-conjugated fluorescent carbon nanodots (Apt-CDots) Citric acid was dissolved in ultrapure water at 0.5 M, and then hydrothermally treated at 20 C for 2 h. The resulting solution, which was light brown and transparent, was filtrated using a 0.22 mm filter, and then subjected to dialysis overnight (molecular weight [MW] cut-off was 1 kDa). The dialysis solution was collected, freeze-dried and weighed. As-prepared carboxyl-terminated CDots were added to ultrapure water at 10 mg/mL. The aptamer (named C6-8), positive control aptamer (named 39 nt, 48 nt length) or negative control aptamer (a scrambled oligonucleotide named control, 48 nt length, (ACGT)12) with the amino-modified 50 end (NH2-aptamer of 6 optical density (OD) was dissolved in 150 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid
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(MES) with 500 mM NaCl at pH 6.5, heated to 95 C for 5 min, and rapidly cooled to 4 C. To this solution, 10 mL of the CDot suspension was added (after sonication for 10 min), and then another 40 mL of the MES/NaCl solution was added and mixed well. After gentle shaking at 37 C for about 30 min, 10 mg of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was rapidly added, and the mixture was shaken at 500 rpm for 48 h at 4 C. The product was then ultrafiltered at 14,000 rpm for 30 min (MW cut-off was 3 kDa) to remove excess reagents. The upper brown layer consisted of the as-synthesized C6-8-Apt-CDots、39 nt-Apt-CDots or control-Apt-CDots and the transparent filtrate was discarded. Ultraviolet (UVeVis) absorption, fluorescence, transmission electron microscopy (TEM), and gel electrophoresis were used to characterize the Apt-CDots. 2.9. Assessment of targeted binding by Apt-CDots Log-phase H1299 cells were seeded in glass bottom dishes and incubated overnight at 37 C and 5% CO2. The culture medium was then replaced with incubation buffer (1 mM MgCl2 in PBS) containing C6-8-Apt-CDots, 39 nt-Apt-CDots, control-Apt-CDots, or undecorated CDots (1 mg/mL). After 1 h, cells were washed twice with PBS and stained with 40 ,6-Diamidino-2-phenylindole (DAPI) at room temperature for 10 min. The fluorescence patterns of each experimental group were examined under a confocal microscope using the same instrument settings. 2.10. Silencing hnRNP A2/B1 expression siRNAs (siB13228164641, siB13228164623) for hnRNP A2/B1 and negative control were synthesized by Guangzhou Ruibo BioCo.,LTD Transfection with the small RNAs was conducted in 6-well plates using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA), as per the manufacturer's instructions. The whole-cell lysate was prepared 48 h after transfection and then subject to a Western Blot. 2.11. In vitro cell viability assay The viability of the cells was determined by the Cell Titer 96® aqueous one-solution cell-proliferation assay, which measures the mitochondrial conversion of MTS to a formazan product via changes in the optical density at 490 nm. Log-phase H1299 cells were plated in a 96-well culture plate (4000 cells per well). After culturing overnight, the cancer cells were either treated with one of the four CDot types (in 1 mM-MgCl2 incubation buffer as described above) at 0.1 mg per well, or they transfected with the small interfering RNA (hnRNP A2/B1 siRNA or control siRNA at 5 pmol/well) using Lipofectamine 2000. After 2 h, the incubation buffer was replaced with complete culture medium and treatment was continued for 0e72 h. At the end of this period, the medium was discarded and 20 mL of MTS reagent, along with 100 mL of fresh culture medium, was added to each well for 1 h. Then, 100 mL of the supernatant were transferred to an ELISA plate and the absorbance in the wells (including blanks) was measured at 490 nm using a microplate reader. 2.12. Animal experiment BALB/c Nude mice were bought from Vital River Laboratory Animal Technology Co. Ltd. All animal operations were performed in accordance with institutional regulations for animal use and care. Log-phase GFP-HepG2 cells were seeded in 6-well culture plate (1.5 106 cells per well). After overnight incubation, the cancer cells were treated with one of the four Cdot types at a
concentration of 1 mg per well in incubation buffer (as above). After 2 h, the cells were trypsinized and counted. A suspension of 1.2 106 GFP-HepG2 cells in 100 mL of PBS was subcutaneously administered into the flank of each nude mouse to test their oncogenicity. Three weeks later, the tumor size of each group was observed using a small-animal imaging instrument and the tumor weight was measured after sacrifice and dissection. 3. Results 3.1. The binding of the C6-8 aptamer to multiple tumor cell lines The C6-8 aptamer was previously shown to target rat osteoblast (ROS 17/2.8) cells. The sequence and secondary structure of the C68 aptamer was obtained using the RNAstructure 4.5 software package as shown in Fig. 1A. In the present study, our fluorescence-microscopy (Fig. 1B) and flow-cytometry (Fig. 1C) results indicate that the C6-8 aptamer bound all the cancer cell lines tested (HepG2, MCF-7, H1299, and HeLa). The binding affinity of the C6-8 aptamer to HeLa cells was determined with a FACScan cytometer and the Kd value was calculated as 2.86 ± 0.72 mM (Fig. 1D). 3.2. Mass-spectrometry assessment of the potential target of the C6-8 aptamer The target protein to which C6-8 binds was determined from total protein extracts of HeLa and Hep-G2 cells, using the streptavidin-biotin system (Fig. 2A). The SDS-PAGE results showed that C6-8 appeared to bind specific bands compared with the initial library control (Fig. 2B). Among them, some may be target candidates and some may be pulled down because of interaction with the candidates. Four bands were chosen (bands 1#-4# in Fig. 2B indicated by arrow) and subjected to mass spectrometry analysis. Among which, three bands pulled down by the C6-8 aptamer were derived from HeLa cells total protein (lane 2 in Fig. 2B) and one band from HepG2 cells total protein (lane 4 in Fig. 2B). The results showed that the candidates of C6-8 targets may be heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), RNA-binding motif protein 3 (RBM3), or proline/glutamine-rich splicing factor (SFPQ). Of these, hnRNP A2/B1 and hnRNP A1 are known to play a significant role in oncogenesis [7,12,13]. It is reported that RBM3 interacts with hnRNP A2/B1 and SFPQ binds to hnRNP A1, therefore hnRNP A1 and hnRNP A2/B1 proteins are purchased and used for the direct binding assay. The EMSA experiment demonstrated C6-8 specifically bound to hnRNP A2/B1 but not to hnRNP A1 proteins (Fig. 2C). 3.3. C6-8 binding to cancer cells and tissues is similar that of hnRNP A2/B1 antibodies Immunofluorescence staining using hnRNP A2/B1 antibodies was compared with staining by FAM-C6-8. Using the same confocal microscopy parameters, the results showed C6-8 specifically bound to the tumor the cell lines (HeLa; Fig. 3A) and tissue (hepatocarcinoma; Fig. 3B), whereas no obvious binding was observed for GP30. Moreover, the location of the fluorescent staining was the same for C6-8 and anti-hnRNP A2/B1; thus, suggesting that the C68 aptamer has potential as a probe for hnRNP A2/B1 and hence cancer cells. To make a comparision of aptamer to antibody in the target affinity, we also determined the dissociation constant value for the monoclonal antibody to the HeLa cells and the Kd value was calculated as 2.21 ± 0.28 nM (Fig. 3C).
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Fig. 1. The C6-8 aptamer specifically bound to multiple tumor cell lines. (A) The sequence of The C6-8 aptamer and its secondary structure; (B) Representative fluorescence micrographs showing binding C6-8 aptamer in multiple tumor cell lines; (C) Flow cytometry analysis showing binding C6-8 aptamer in multiple tumor cell lines; (D) The binding affinity of the C6-8 aptamer to HeLa cells.
Fig. 2. Identification of the target of the C6-8 aptamer. (A) The C6-8 target protein was elucidated from total protein extracts of multiple tumor cell-types using the streptavidin-biotin system; (B) The candidate targets from HeLa total protein (lane 2, 3) and HepG2 total protein (lane 4) that were pulled down by C6-8 aptamer and controls (lane 5, 6) were separated on 12% SDS-PAGE. Four protein bands of interest (number 1# to 4# indicated by arrows) were excised and digested with porcine trypsin for LC-MS/MS analysis; (C) C6-8 was found to specifically bind to hnRNP A2/B1 via an electrophoretic mobility shift assay (EMSA).
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Fig. 3. C6-8-aptamer labeling of tumor cells and tissues was similar to anti-hnRNP-A2/B1 labelling in terms of efficacy. (A) Representative fluorescence micrographs showing HeLa cells labeled with C6-8 aptamer or hnRNP A2/B1 monoclonal antibody; (B) Representative fluorescence micrographs showing cancerous tissue labeled with C6-8 aptamer or anti-hnRNP A2/B1monoclonal antibody; (C) The binding affinity of the hnRNP A2/B1 monoclonal antibody to HeLa cells.
3.4. Characterization of aptamer-conjugated fluorescent carbon nanodots (Apt-CDots) The characterization of Apt-CDots is shown in Fig. 4. The UV spectra of our synthesized Apt-CDot conjugates exhibited
maximum absorption at 260 nm, showing that the aptamers had been well conjugated. They had typical tunable emission characteristics; the emission and excitation maxima were at 458 and 378 nm, respectively. The fluorescence quantum yields for AptCDots were 26% with reference to rhodamine 110. Under UV light,
Fig. 4. Synthesis of aptamer-conjugated fluorescent carbon nanodots (Apt-CDots). (A) The ultravilet (UV) absorption and fluorescent emission spectra of Apt-CDots, in which with increasing longer excitation wavelength (in 20 nm increments starting from 380 nm), inset shows the normalized emission spectra; (B) The solution color of Apt-CDots under natural light and UV light; (C) The TEM of Apt-CDots, and the inset shows their diameter distribution; (D) The 15% natural PAGE on carboxyl-terminated CDots (lane 1) and AptCdots (lane 2) under UV light before(I) and after EB-stainning (II).
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the transparent light-brown solution showed strong blue fluorescence. The mean particle size as determined by TEM was 25 ± 5 nm. The mobility of carboxyl-terminated CDots was greater than that of the Apt-CDot conjugates, and the conjugates were well stained by ethidium bromide (EB) but the carboxyl-terminated CDots faded a little after EB staining. These results clearly show that the AptCDots were successfully synthesized and suitable for cellular imaging applications. 3.5. C6-8-Apt-CDots target the tumor cells The binding of Apt-CDots to H1299 tumor cells, along with their internalization, was studied using confocal microscopy. As shown in Fig. 5, the C6-8-Apt-CDots specifically targeted the tumor cell nuclei (Fig. 5A), while the 39 nt-Apt-CDots targeted the cytoplasm and/or cell membrane in which erythropoietin and its receptor (EPO/EPOR) were overexpressed of these cells (Fig. 5B). This accords with the fact that the target of C6-8, hnRNP A2/B1, is mainly located within the nucleus whereas the target of the 39 nt aptamer, HuEPO-a, is mainly expressed in the cytoplasm and/or cell membrane [14,15]. The control and undecorated nanoparticles showed no binding, or only weak binding, to the tumor cells (Fig. 5C, D). 3.6. C6-8-Apt-CDots inhibit tumor cell proliferation in vitro and in vivo Specifically designed siRNAs targeting the HNRNP A2/B1 gene were transfected into lung-cancer (H1299) cells, and the total cellular protein was extracted from the cells after 48 h of growth, and then analyzed by Western blot. Compared to the negativecontrol siRNA, the expression of hnRNP A2/B1 was downregulated by both siRNA1 and siRNA2, although the siRNA1 was more effective (Fig. 6A). As shown in Fig. 6B, C6-8-Apt-CDots, 39 nt-Apt-CDots and siRNA1 significantly inhibited the growth of the lung-cancer cells at
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each time point compared to the negative-control siRNA, mock, control-Apt-CDots, and undecorated CDots (Fig. 6B). Similar results were observed in nude mice implanted with hepatocarcinoma (GFP-HepG2) cells that were treated with the same aptamer-CDot conjugates and controls (Fig. 6C, D, E). 4. Discussion In this study, it was shown that the C6-8 aptamer we had previously developed by cell SELEX can act as a cancer-specific probe across multiple tumor cell types and that its target is hnRNP A2/B1. Known as an important RNA-binding protein, hnRNP A2/B1 is a major component of the heterogeneous nuclear ribonucleoprotein core complex and amongst the most numerous of the family of 20 major hnRNPs. Although the specificity of C6-8 is similar to the monoclonal antibody of hnRNP A2/B1, the affinity of C6-8 to HeLa cells is much lower than hnRNP A2/B1 monoclonal antibody showed in this manuscript. It is suggested that high affinity aptamers targeting hnRNP A2/B1 protein may be gained through a traditional SELEX to protein instead of cells. The hnRNP A2/B1 proteins participate in alternative splicing of mRNA, RNA transport, post-transcriptional regulation, and maintenance of chromosome telomere length [16e18]. In addition, recent studies suggest that hnRNP A2/B1 and the other members of hnRNP family may play an important role in telomere function, cell proliferation, and carcinogenesis [19e21]. Increased expression of hnRNP A2/B1 (mainly localized in the nucleus) has been reported in many cancer tissues including breast, small-cell lung, ovarian, and colorectal carcinomas [22e25]; therefore, hnRNP A2/B1 may be a specific biomarker for multiple tumor types. Being a smaller molecule, aptamers have more promising prospect in reaching cellular targets compared to antibodies. But most of existing aptamers cannot directly enter cells especially the nucleus. A way to address this problem is to directly or indirectly attach the aptamer to a nanoparticle, thus forming nano-biological
Fig. 5. C6-8-Apt-CDots targeted tumor cells. (A) C6-8-Apt-CDots specifically targeted the tumor cell nuclei; (B) 39 nt-Apt-CDots targeted the cytoplasm of tumor cells; (C, D) The control-Apt-CDots (scrambled aptamers) and CDots (no aptamers) showed no binding, or only weak binding, to the tumor cells.
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Fig. 6. C6-8-Apt-CDotss inhibited tumor-cell proliferation in vitro and in vivo.(A) The expression of hnRNP A2/B1 was downregulated by siRNA1 and siRNA2; (B) MTS assay results showing inhibition of tumor-cell proliferation induced by C6-8-Apt-CDots; (C, D, E) C6-8-Apt-CDots inhibited tumor growth in nude mice.
probes, which can enter targeted cells or tissues via endocytosis, allowing for real-time monitoring cancer cells and markers [26e29]. For anti-cancer efficiency especially for drug delivery vehicles, various aptamer modified nanoparticles facilitates cellular uptake of drugs with the aid of aptamer-guided active targeting through the cell surface target mediated internalization. There is seldom report of aptamer passive internalization by nanoparticle to achieve the aptamer intracellular targeting effect in anti-cancer therapy. We conjugated the C6-8 aptamer with fluorescent CDots to provide a site-specific cellular entry to reach the hnRNP A2/B1 protein, which is mainly located in the nucleus.
Currently, the promising classes of aptamer-conjugated nanomaterials in fields of tumor therapy included aptamer-conjugated polymeric (liposomes [30], micelles [31], and organic polymeric nanoparticles [32] etc.), and inorganic nanoparticles (magnetic nanoparticles [33], silica nanoparticles [34], quantum dots [35], graphene oxide nanosheets [36], carbon tubes, etc.). Aptamerconjugated liposomes, micelles, organic polymeric nanoparticles have good biocompatibility and biodegradability for therapy, well inorganic nanomaterials provide unique optical, electronic, and magnetic properties, and are good indicators for targeting process. But some of aptamer-conjugated nanoparticles have some or
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unknown toxicity, which restricts their applications in vivo. All efficient aptamer-conjugated nanopartiles shows some or all characteristics in one entity, such as, biocompatible, convenient synthesis, easy manipulation or functionalization. Our developed aptamer-conjugated CDots, besides all common characteristics of aptamer-conjugated nanoparticles, also show tunable optical characteristics, and most important, non-toxicity, and thus excellent diagnostic and theranostic capability. The CDots we selected are fluorescent nanoparticles that have shown promise for use as bioimaging probes [37]. Consisting entirely of carbon, they have largely eliminated the major toxicity issues associated with the heavy metals used in conventional fluorescent semiconductor nanocrystals (quantum dots), and compared to these, they exhibit superior brightness and photostability [38]. However, only limited specificity has been achieved in live cells due to the difficulty of achieving functionalization that is optimal for cellular and subcellular targeting. Our synthesized Apt-CDot conjugates, with multivalent presentation of aptamers, were successful in their application to live cells due to the high specificity of the C6-8 aptamer towards the target-cell biomarker, effectively overcoming the issue of nonspecific binding. Non-functionalized carboxyl-terminated CDots and scrambled-ssDNA-CDots (negative controls) can only enter into the cytosol and yield less fluorescence, mainly on account of repulsion between these anionic CDots and the negatively charged cell membranes [39]. 39 nt-Apt-CDots were considered as the positive control: the aptamer 807-39 nt targets overexpressed EPOEPOR located mainly in the cytoplasm and/or cell membranes of tumor cells [40], and they can specifically bind at the cell membrane and some cytoplasmic area with bright, tunable emission characteristics. In contrast, the synthesized C6-8-Apt-CDots were found to specifically target tumor cells and to be localized in the nucleus. The cell proliferation assay showed that the C6-8-Apt-CDots inhibited the growth of tumor cells to a similar extent as hnRNPA2/B1-specific siRNA. Overall, the results of this study demonstrate that the C6-8-Apt-CDots specifically targeted hnRNP A2/B1 protein in the nucleus of multiple tumor cell types and inhibited the growth of these cells. Such conjugates may therefore be of significant diagnostic and therapeutic value in relation to cancer. Acknowledgments We thank Dr. Wenxia Zhou and Dr. Gang Liu for their assistance. This work was supported by the Chinese National High-Tech Research and Development Program (2012AA022501), the National Natural Science Foundation of China (21175152, 81001262), National Science and Technology Major Project of the Ministry of Science and Technology of China (2012ZX09301003-001-010) and the Beijing Municipal National Science Foundation (7132159). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.06.013. References [1] C. Tuerk, L. Gold, Systeatic evolut ion of ligands by exponential enrichment :RNA 1igands to bacteriophage T4 DNA polymerase, Science 249 (4968) (1990) 505e510. [2] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bind specific ligands, Nature 346 (6287) (1990) 818e822. [3] K. Konopka, N.S. Lee, J. Rossi, N. Duzqunes, Rev-binding aptamer and CMV promoter a decoys to inhibit HIV replication, Gene 255 (2) (2000) 235e244. [4] S.W. Gal, S. Amontov, P.T. Urvil, D. Vishnuvardhan, F. Nishikawa, P.K. Kumar, et al., Selection of a RNA aptamer that binds to human activated protein C and
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Nanoparticle-conjugated aptamer targeting hnRNP A2/B1 can recognize multiple tumor cells and inhibit their proliferation Hui Li a, 1, Lei Guo b, 1, Aixue Huang a, 1, Hua Xu b, Xuemei Liu a, Hongmei Ding a, Jie Dong a, Jie Li a, Chaonan Wang a, Xueting Su a, Xingfeng Ge a, Leqiao Sun a, Chenjun Bai a, Xuelian Shen a, Tao Fang a, Zhanghua Li c, Yong Zhou d, Linsheng Zhan d, Shaohua Li a, *, Jianwei Xie b, *, Ningsheng Shao a, * a
Beijing Institute of Basic Medical Sciences, Beijing, 100850, China State Key Laboratory for Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, No. 27 Taiping Road, Haidian District, 100850, Beijing, China c Deparment of Orthopedics, Wuhan City Hospital No,3&Tongren Hospital of Wuhan University, Wuhan, 430060, China d Beijing Institute of Laboratory of Blood-borne Virus, Beijing, 100850, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2015 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online 14 June 2015
In this study, we further investigated a previously developed aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells. We found that this C6-8 aptamer specifically binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and that it specifically labeled multiple tumor-cell lines as effectively as hnRNP A2/B1 monoclonal antibodies. When conjugated with fluorescent carbon nanodots (CDots) it could freely enter multiple living tumor cell lines (HepG2, MCF-7, H1299, and HeLa), whose growth it inhibited by targeting hnRNP A2/B1. Similar inhibitory effects were observed when the GFP-HepG2 hepatocarcinoma cells treated with C6-8-conjugated CDots were implanted in nude mice. Our work provides a new aptamer for targeting/labeling multiple tumor cell types, and its nanoparticle conjugates bring further advantages that increase its potential for use in cancer diagnosis and therapy. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Tumor cells SELEX Aptamer Heterogeneous nuclear ribonucleoprotein A2/B1 Nanoparticles
1. Introduction The in vitro selection technique known as SELEX (systematic evolution of ligands by exponential enrichment), which was developed in the early 1990s, is a high throughput screening technique that involves the progressive selection of highly specific ligands by repeated rounds of selection and amplification from a large random synthetic nucleic acid library [1,2]. The selected (enriched) oligonucleotide ligands, named aptamers, which are short single-stranded (ss) DNA or RNA molecules, have a high affinity and specificity for targets that are compatible with their specific three-dimensional shapes. Since its initial development, SELEX screening targets have progressed from small organic
* Corresponding authors. E-mail addresses: [email protected] (S. Li), [email protected] (J. Xie), shaonsh@ hotmail.com (N. Shao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.06.013 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
molecules and pure proteins to protein complexes, cells and even tissues [3e8]. As a result, aptamers have attracted increased attention in many fields, especially in cancer research. SELEX-generated aptamers can specifically target tumor cells and provide a new method for biomarker verification. In addition to the comparative binding affinity and specificity to monoclonal antibody, the aptamers have other properties such as easier to be synthesized and modified in vitro, less immunogenic, smaller, and more amenable to long-term preservation. Based on these merits, aptamers have been widely investigated for use in clinical diagnosis and cancer drug screening [9,10]. In recent years, the incidence of cancer has significantly increased, threatening the lives of millions worldwide. Finding good tumor biomarkers and drug targets is a key issue for early diagnosis and effective treatment of cancer. At present, some aptamers targeting tumor cells have been generated via cell SELEX, but in general these only recognize a single tumor type. To our knowledge, there are no reports of aptamers that recognize
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multiple tumor cell types from different origins. In previous work, we used cell SELEX to find an aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells [11]. In this study, we further investigated this C6-8 aptamer, focusing on its capacity to target multiple tumor-cell lines, the identity of the underlying molecular target, and the applicability and benefits of its conjugation with fluorescent carbon nanodots (CDots).
approximately 600 mg of cell (total)-protein extract in 600 mL of binding buffer for 1 h at 37 C. The beads were washed six times, and the binding components were eluted from the beads by heating in 50 mL of SDS PAGE loading buffer and applied to 12% SDSPAGE. The protein band of interest was excised and digested with porcine trypsin for LC-MS/MS analysis, followed by a Mascot database search.
2. Materials and methods
2.6. The binding of the biotinylated C6-8 aptamer to the target molecule
2.1. Random ssDNA library and primers A random ssDNA library (GP30) and primers were synthesized by Invitrogen Co Ltd, Beijing, China. The sequences are listed in the Table 1 of the Supplementary Data. The primers, Plong-1 and P11, were used for standard PCR amplification of double-stranded DNA molecules. Plong-1 and Pstem-loop primers were used to synthesize single-stranded DNA as we have reported previously [6]. 2.2. Cell lines and cultures The rat osteoblast cell line, ROS 17/2.8, was cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and maintained in an incubator at 37 C and 5% CO2. The cervical-cancer cell line, HeLa, the lung cancer cell line, H1299, the hepatoma cell line, HepG2, and the breast-cancer cell line, MCF-7, were all cultured in DMEM supplemented with 10% fetal bovine serum and maintained at 37 C and 5% CO2. 2.3. Fluorescence-microscopy and flow-cytometry analysis of cellaptamer binding After overnight incubation, adherent cells were harvested by trypsinization and fixed in absolute methanol at 20 C for 30 min, and then washed with phosphate-buffered saline (PBS) before the experiment. The FAM-labeled random library (GP30) and the FAM-labeled C6-8 aptamer were each dissolved in 100 mL of incubation buffer at a concentration of 20 mg/mL and heated for 5 min at 95 C before cooling on ice for 10 min. The denatured aptamers were incubated with the tumor cells for 1 h at 37 C in the dark. After washing, binding of the aptamers to tumor cells was determined using fluorescence microscopy and flow cytometry. 2.4. Binding affinity of the C6-8 aptamer to tumor cells The binding affinity of the C6-8 aptamer to HeLa cells was determined as follows. Briefly, 5 105 cells were incubated with varying concentrations of FAM-aptamer in 500 mL of binding buffer at 37 C for 1 h. Cells were washed once with PBS and resuspended in 0.5 mL of PBS. The mean fluorescence intensity of target cells labeled with the aptamer was determined using a FACScan cytometer (BD, USA). The equilibrium dissociation constant (Kd) of the aptamer-cell interaction was calculated via the equation Y ¼ Bmax$X/(Kd þ X), using GraphPad Prism4 software. The affinity assay of antibody to HeLa cells was performed similarly with primary monoclonal antibody and FITC labeled secondary antibody. 2.5. Identification of the molecular target of the C6-8 aptamer The total protein was extracted from HeLa and HepG2 tumor cells. 5 mg of biotinylated C6-8 aptamer or initial library control was dissolved in 500 mL of binding buffer and incubated with 1 ml of streptavidin-coated magnetic beads for 30 min at room temperature. After washing three times, the beads were incubated with
The biotinylated initial library (GP30) and the biotinylated C6-8 aptamer were synthesized by Invitrogen Co Ltd, Beijing, China. In each case, 2 ng of the ssDNA was denatured by heating for 5 min at 95 C, followed by rapid cooling on ice for 10 min, 5 mg of heterogeneous ribonucleoprotein A1 (hnRNP A1) or 5 mg of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) were respectively added to the biotinylated GP30 and C6-8 solutions (final hnRNP concentrations were 500 mg/mL), which were then incubated in 10 mL of incubation buffer for 1 h at 37 C. The products were subjected to 6% natural gel electrophoresis for 1.5 h and transferred to a nylon membrane (70 V for 60 min). After UV crosslinking for 2 min, the membrane was incubated with blocking reagent for 30 min at 37 C and incubation with stabilized streptavidin-HRP conjugate (diluted 1:500) for 30 min at 37 C. After washed three times, the nylon membrane was incubated with equilibration buffer for 10 min at room temperature. The retarded band indicating the binding of the C6-8 aptamer to protein hnRNP A2/B1 or hnRNP A1 and the free aptamer band are developed using the enhanced chemiluminescence Western blotting detection system. 2.7. Fluorescence microscopy analysis of C6-8 aptamer-binding to cancer cells and tissue Directly after fixation, the HeLa cells were incubated at 37 C for 1 h with 100 mL of 20 mg/mL FAM-GP30, 20 mg/mL FAM-C6-8, or 0.5 mg/mL anti-hnRNP A2/B1monoclonal antibodies; in the case of the antibodies, this was followed by another incubation with 1:100-diluted FITC-conjugated goat anti-mouse IgG for 30 min at 37 C. Unbound aptamers and antibodies were washed away with PBS. The cells were counterstained with 0.25% Evans blue and the fluorescence patterns were observed under a fluorescence microscope (Olympus IX71, Tokyo, Japan). Paraffin-embedded tissue sections were antigen retrieved and then permeabilized with 0.1% Tween20 in PBS. After blocking with a solution of 0.1-mg/mL yeast-transfer RNA (ytRNA), 0.1-mg/mL salmon sperm DNA and 1% BSA in PBS, the binding patterns of the sections were determined (as described above) for FAM-GP30, FAM-C6-8 and anti-hnRNPA2/B. 2.8. Synthesis of aptamer-conjugated fluorescent carbon nanodots (Apt-CDots) Citric acid was dissolved in ultrapure water at 0.5 M, and then hydrothermally treated at 20 C for 2 h. The resulting solution, which was light brown and transparent, was filtrated using a 0.22 mm filter, and then subjected to dialysis overnight (molecular weight [MW] cut-off was 1 kDa). The dialysis solution was collected, freeze-dried and weighed. As-prepared carboxyl-terminated CDots were added to ultrapure water at 10 mg/mL. The aptamer (named C6-8), positive control aptamer (named 39 nt, 48 nt length) or negative control aptamer (a scrambled oligonucleotide named control, 48 nt length, (ACGT)12) with the amino-modified 50 end (NH2-aptamer of 6 optical density (OD) was dissolved in 150 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid
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(MES) with 500 mM NaCl at pH 6.5, heated to 95 C for 5 min, and rapidly cooled to 4 C. To this solution, 10 mL of the CDot suspension was added (after sonication for 10 min), and then another 40 mL of the MES/NaCl solution was added and mixed well. After gentle shaking at 37 C for about 30 min, 10 mg of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was rapidly added, and the mixture was shaken at 500 rpm for 48 h at 4 C. The product was then ultrafiltered at 14,000 rpm for 30 min (MW cut-off was 3 kDa) to remove excess reagents. The upper brown layer consisted of the as-synthesized C6-8-Apt-CDots、39 nt-Apt-CDots or control-Apt-CDots and the transparent filtrate was discarded. Ultraviolet (UVeVis) absorption, fluorescence, transmission electron microscopy (TEM), and gel electrophoresis were used to characterize the Apt-CDots. 2.9. Assessment of targeted binding by Apt-CDots Log-phase H1299 cells were seeded in glass bottom dishes and incubated overnight at 37 C and 5% CO2. The culture medium was then replaced with incubation buffer (1 mM MgCl2 in PBS) containing C6-8-Apt-CDots, 39 nt-Apt-CDots, control-Apt-CDots, or undecorated CDots (1 mg/mL). After 1 h, cells were washed twice with PBS and stained with 40 ,6-Diamidino-2-phenylindole (DAPI) at room temperature for 10 min. The fluorescence patterns of each experimental group were examined under a confocal microscope using the same instrument settings. 2.10. Silencing hnRNP A2/B1 expression siRNAs (siB13228164641, siB13228164623) for hnRNP A2/B1 and negative control were synthesized by Guangzhou Ruibo BioCo.,LTD Transfection with the small RNAs was conducted in 6-well plates using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA), as per the manufacturer's instructions. The whole-cell lysate was prepared 48 h after transfection and then subject to a Western Blot. 2.11. In vitro cell viability assay The viability of the cells was determined by the Cell Titer 96® aqueous one-solution cell-proliferation assay, which measures the mitochondrial conversion of MTS to a formazan product via changes in the optical density at 490 nm. Log-phase H1299 cells were plated in a 96-well culture plate (4000 cells per well). After culturing overnight, the cancer cells were either treated with one of the four CDot types (in 1 mM-MgCl2 incubation buffer as described above) at 0.1 mg per well, or they transfected with the small interfering RNA (hnRNP A2/B1 siRNA or control siRNA at 5 pmol/well) using Lipofectamine 2000. After 2 h, the incubation buffer was replaced with complete culture medium and treatment was continued for 0e72 h. At the end of this period, the medium was discarded and 20 mL of MTS reagent, along with 100 mL of fresh culture medium, was added to each well for 1 h. Then, 100 mL of the supernatant were transferred to an ELISA plate and the absorbance in the wells (including blanks) was measured at 490 nm using a microplate reader. 2.12. Animal experiment BALB/c Nude mice were bought from Vital River Laboratory Animal Technology Co. Ltd. All animal operations were performed in accordance with institutional regulations for animal use and care. Log-phase GFP-HepG2 cells were seeded in 6-well culture plate (1.5 106 cells per well). After overnight incubation, the cancer cells were treated with one of the four Cdot types at a
concentration of 1 mg per well in incubation buffer (as above). After 2 h, the cells were trypsinized and counted. A suspension of 1.2 106 GFP-HepG2 cells in 100 mL of PBS was subcutaneously administered into the flank of each nude mouse to test their oncogenicity. Three weeks later, the tumor size of each group was observed using a small-animal imaging instrument and the tumor weight was measured after sacrifice and dissection. 3. Results 3.1. The binding of the C6-8 aptamer to multiple tumor cell lines The C6-8 aptamer was previously shown to target rat osteoblast (ROS 17/2.8) cells. The sequence and secondary structure of the C68 aptamer was obtained using the RNAstructure 4.5 software package as shown in Fig. 1A. In the present study, our fluorescence-microscopy (Fig. 1B) and flow-cytometry (Fig. 1C) results indicate that the C6-8 aptamer bound all the cancer cell lines tested (HepG2, MCF-7, H1299, and HeLa). The binding affinity of the C6-8 aptamer to HeLa cells was determined with a FACScan cytometer and the Kd value was calculated as 2.86 ± 0.72 mM (Fig. 1D). 3.2. Mass-spectrometry assessment of the potential target of the C6-8 aptamer The target protein to which C6-8 binds was determined from total protein extracts of HeLa and Hep-G2 cells, using the streptavidin-biotin system (Fig. 2A). The SDS-PAGE results showed that C6-8 appeared to bind specific bands compared with the initial library control (Fig. 2B). Among them, some may be target candidates and some may be pulled down because of interaction with the candidates. Four bands were chosen (bands 1#-4# in Fig. 2B indicated by arrow) and subjected to mass spectrometry analysis. Among which, three bands pulled down by the C6-8 aptamer were derived from HeLa cells total protein (lane 2 in Fig. 2B) and one band from HepG2 cells total protein (lane 4 in Fig. 2B). The results showed that the candidates of C6-8 targets may be heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), RNA-binding motif protein 3 (RBM3), or proline/glutamine-rich splicing factor (SFPQ). Of these, hnRNP A2/B1 and hnRNP A1 are known to play a significant role in oncogenesis [7,12,13]. It is reported that RBM3 interacts with hnRNP A2/B1 and SFPQ binds to hnRNP A1, therefore hnRNP A1 and hnRNP A2/B1 proteins are purchased and used for the direct binding assay. The EMSA experiment demonstrated C6-8 specifically bound to hnRNP A2/B1 but not to hnRNP A1 proteins (Fig. 2C). 3.3. C6-8 binding to cancer cells and tissues is similar that of hnRNP A2/B1 antibodies Immunofluorescence staining using hnRNP A2/B1 antibodies was compared with staining by FAM-C6-8. Using the same confocal microscopy parameters, the results showed C6-8 specifically bound to the tumor the cell lines (HeLa; Fig. 3A) and tissue (hepatocarcinoma; Fig. 3B), whereas no obvious binding was observed for GP30. Moreover, the location of the fluorescent staining was the same for C6-8 and anti-hnRNP A2/B1; thus, suggesting that the C68 aptamer has potential as a probe for hnRNP A2/B1 and hence cancer cells. To make a comparision of aptamer to antibody in the target affinity, we also determined the dissociation constant value for the monoclonal antibody to the HeLa cells and the Kd value was calculated as 2.21 ± 0.28 nM (Fig. 3C).
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Fig. 1. The C6-8 aptamer specifically bound to multiple tumor cell lines. (A) The sequence of The C6-8 aptamer and its secondary structure; (B) Representative fluorescence micrographs showing binding C6-8 aptamer in multiple tumor cell lines; (C) Flow cytometry analysis showing binding C6-8 aptamer in multiple tumor cell lines; (D) The binding affinity of the C6-8 aptamer to HeLa cells.
Fig. 2. Identification of the target of the C6-8 aptamer. (A) The C6-8 target protein was elucidated from total protein extracts of multiple tumor cell-types using the streptavidin-biotin system; (B) The candidate targets from HeLa total protein (lane 2, 3) and HepG2 total protein (lane 4) that were pulled down by C6-8 aptamer and controls (lane 5, 6) were separated on 12% SDS-PAGE. Four protein bands of interest (number 1# to 4# indicated by arrows) were excised and digested with porcine trypsin for LC-MS/MS analysis; (C) C6-8 was found to specifically bind to hnRNP A2/B1 via an electrophoretic mobility shift assay (EMSA).
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Fig. 3. C6-8-aptamer labeling of tumor cells and tissues was similar to anti-hnRNP-A2/B1 labelling in terms of efficacy. (A) Representative fluorescence micrographs showing HeLa cells labeled with C6-8 aptamer or hnRNP A2/B1 monoclonal antibody; (B) Representative fluorescence micrographs showing cancerous tissue labeled with C6-8 aptamer or anti-hnRNP A2/B1monoclonal antibody; (C) The binding affinity of the hnRNP A2/B1 monoclonal antibody to HeLa cells.
3.4. Characterization of aptamer-conjugated fluorescent carbon nanodots (Apt-CDots) The characterization of Apt-CDots is shown in Fig. 4. The UV spectra of our synthesized Apt-CDot conjugates exhibited
maximum absorption at 260 nm, showing that the aptamers had been well conjugated. They had typical tunable emission characteristics; the emission and excitation maxima were at 458 and 378 nm, respectively. The fluorescence quantum yields for AptCDots were 26% with reference to rhodamine 110. Under UV light,
Fig. 4. Synthesis of aptamer-conjugated fluorescent carbon nanodots (Apt-CDots). (A) The ultravilet (UV) absorption and fluorescent emission spectra of Apt-CDots, in which with increasing longer excitation wavelength (in 20 nm increments starting from 380 nm), inset shows the normalized emission spectra; (B) The solution color of Apt-CDots under natural light and UV light; (C) The TEM of Apt-CDots, and the inset shows their diameter distribution; (D) The 15% natural PAGE on carboxyl-terminated CDots (lane 1) and AptCdots (lane 2) under UV light before(I) and after EB-stainning (II).
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the transparent light-brown solution showed strong blue fluorescence. The mean particle size as determined by TEM was 25 ± 5 nm. The mobility of carboxyl-terminated CDots was greater than that of the Apt-CDot conjugates, and the conjugates were well stained by ethidium bromide (EB) but the carboxyl-terminated CDots faded a little after EB staining. These results clearly show that the AptCDots were successfully synthesized and suitable for cellular imaging applications. 3.5. C6-8-Apt-CDots target the tumor cells The binding of Apt-CDots to H1299 tumor cells, along with their internalization, was studied using confocal microscopy. As shown in Fig. 5, the C6-8-Apt-CDots specifically targeted the tumor cell nuclei (Fig. 5A), while the 39 nt-Apt-CDots targeted the cytoplasm and/or cell membrane in which erythropoietin and its receptor (EPO/EPOR) were overexpressed of these cells (Fig. 5B). This accords with the fact that the target of C6-8, hnRNP A2/B1, is mainly located within the nucleus whereas the target of the 39 nt aptamer, HuEPO-a, is mainly expressed in the cytoplasm and/or cell membrane [14,15]. The control and undecorated nanoparticles showed no binding, or only weak binding, to the tumor cells (Fig. 5C, D). 3.6. C6-8-Apt-CDots inhibit tumor cell proliferation in vitro and in vivo Specifically designed siRNAs targeting the HNRNP A2/B1 gene were transfected into lung-cancer (H1299) cells, and the total cellular protein was extracted from the cells after 48 h of growth, and then analyzed by Western blot. Compared to the negativecontrol siRNA, the expression of hnRNP A2/B1 was downregulated by both siRNA1 and siRNA2, although the siRNA1 was more effective (Fig. 6A). As shown in Fig. 6B, C6-8-Apt-CDots, 39 nt-Apt-CDots and siRNA1 significantly inhibited the growth of the lung-cancer cells at
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each time point compared to the negative-control siRNA, mock, control-Apt-CDots, and undecorated CDots (Fig. 6B). Similar results were observed in nude mice implanted with hepatocarcinoma (GFP-HepG2) cells that were treated with the same aptamer-CDot conjugates and controls (Fig. 6C, D, E). 4. Discussion In this study, it was shown that the C6-8 aptamer we had previously developed by cell SELEX can act as a cancer-specific probe across multiple tumor cell types and that its target is hnRNP A2/B1. Known as an important RNA-binding protein, hnRNP A2/B1 is a major component of the heterogeneous nuclear ribonucleoprotein core complex and amongst the most numerous of the family of 20 major hnRNPs. Although the specificity of C6-8 is similar to the monoclonal antibody of hnRNP A2/B1, the affinity of C6-8 to HeLa cells is much lower than hnRNP A2/B1 monoclonal antibody showed in this manuscript. It is suggested that high affinity aptamers targeting hnRNP A2/B1 protein may be gained through a traditional SELEX to protein instead of cells. The hnRNP A2/B1 proteins participate in alternative splicing of mRNA, RNA transport, post-transcriptional regulation, and maintenance of chromosome telomere length [16e18]. In addition, recent studies suggest that hnRNP A2/B1 and the other members of hnRNP family may play an important role in telomere function, cell proliferation, and carcinogenesis [19e21]. Increased expression of hnRNP A2/B1 (mainly localized in the nucleus) has been reported in many cancer tissues including breast, small-cell lung, ovarian, and colorectal carcinomas [22e25]; therefore, hnRNP A2/B1 may be a specific biomarker for multiple tumor types. Being a smaller molecule, aptamers have more promising prospect in reaching cellular targets compared to antibodies. But most of existing aptamers cannot directly enter cells especially the nucleus. A way to address this problem is to directly or indirectly attach the aptamer to a nanoparticle, thus forming nano-biological
Fig. 5. C6-8-Apt-CDots targeted tumor cells. (A) C6-8-Apt-CDots specifically targeted the tumor cell nuclei; (B) 39 nt-Apt-CDots targeted the cytoplasm of tumor cells; (C, D) The control-Apt-CDots (scrambled aptamers) and CDots (no aptamers) showed no binding, or only weak binding, to the tumor cells.
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Fig. 6. C6-8-Apt-CDotss inhibited tumor-cell proliferation in vitro and in vivo.(A) The expression of hnRNP A2/B1 was downregulated by siRNA1 and siRNA2; (B) MTS assay results showing inhibition of tumor-cell proliferation induced by C6-8-Apt-CDots; (C, D, E) C6-8-Apt-CDots inhibited tumor growth in nude mice.
probes, which can enter targeted cells or tissues via endocytosis, allowing for real-time monitoring cancer cells and markers [26e29]. For anti-cancer efficiency especially for drug delivery vehicles, various aptamer modified nanoparticles facilitates cellular uptake of drugs with the aid of aptamer-guided active targeting through the cell surface target mediated internalization. There is seldom report of aptamer passive internalization by nanoparticle to achieve the aptamer intracellular targeting effect in anti-cancer therapy. We conjugated the C6-8 aptamer with fluorescent CDots to provide a site-specific cellular entry to reach the hnRNP A2/B1 protein, which is mainly located in the nucleus.
Currently, the promising classes of aptamer-conjugated nanomaterials in fields of tumor therapy included aptamer-conjugated polymeric (liposomes [30], micelles [31], and organic polymeric nanoparticles [32] etc.), and inorganic nanoparticles (magnetic nanoparticles [33], silica nanoparticles [34], quantum dots [35], graphene oxide nanosheets [36], carbon tubes, etc.). Aptamerconjugated liposomes, micelles, organic polymeric nanoparticles have good biocompatibility and biodegradability for therapy, well inorganic nanomaterials provide unique optical, electronic, and magnetic properties, and are good indicators for targeting process. But some of aptamer-conjugated nanoparticles have some or
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unknown toxicity, which restricts their applications in vivo. All efficient aptamer-conjugated nanopartiles shows some or all characteristics in one entity, such as, biocompatible, convenient synthesis, easy manipulation or functionalization. Our developed aptamer-conjugated CDots, besides all common characteristics of aptamer-conjugated nanoparticles, also show tunable optical characteristics, and most important, non-toxicity, and thus excellent diagnostic and theranostic capability. The CDots we selected are fluorescent nanoparticles that have shown promise for use as bioimaging probes [37]. Consisting entirely of carbon, they have largely eliminated the major toxicity issues associated with the heavy metals used in conventional fluorescent semiconductor nanocrystals (quantum dots), and compared to these, they exhibit superior brightness and photostability [38]. However, only limited specificity has been achieved in live cells due to the difficulty of achieving functionalization that is optimal for cellular and subcellular targeting. Our synthesized Apt-CDot conjugates, with multivalent presentation of aptamers, were successful in their application to live cells due to the high specificity of the C6-8 aptamer towards the target-cell biomarker, effectively overcoming the issue of nonspecific binding. Non-functionalized carboxyl-terminated CDots and scrambled-ssDNA-CDots (negative controls) can only enter into the cytosol and yield less fluorescence, mainly on account of repulsion between these anionic CDots and the negatively charged cell membranes [39]. 39 nt-Apt-CDots were considered as the positive control: the aptamer 807-39 nt targets overexpressed EPOEPOR located mainly in the cytoplasm and/or cell membranes of tumor cells [40], and they can specifically bind at the cell membrane and some cytoplasmic area with bright, tunable emission characteristics. In contrast, the synthesized C6-8-Apt-CDots were found to specifically target tumor cells and to be localized in the nucleus. The cell proliferation assay showed that the C6-8-Apt-CDots inhibited the growth of tumor cells to a similar extent as hnRNPA2/B1-specific siRNA. Overall, the results of this study demonstrate that the C6-8-Apt-CDots specifically targeted hnRNP A2/B1 protein in the nucleus of multiple tumor cell types and inhibited the growth of these cells. Such conjugates may therefore be of significant diagnostic and therapeutic value in relation to cancer. Acknowledgments We thank Dr. Wenxia Zhou and Dr. Gang Liu for their assistance. This work was supported by the Chinese National High-Tech Research and Development Program (2012AA022501), the National Natural Science Foundation of China (21175152, 81001262), National Science and Technology Major Project of the Ministry of Science and Technology of China (2012ZX09301003-001-010) and the Beijing Municipal National Science Foundation (7132159). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.06.013. References [1] C. Tuerk, L. Gold, Systeatic evolut ion of ligands by exponential enrichment :RNA 1igands to bacteriophage T4 DNA polymerase, Science 249 (4968) (1990) 505e510. [2] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bind specific ligands, Nature 346 (6287) (1990) 818e822. [3] K. Konopka, N.S. Lee, J. Rossi, N. Duzqunes, Rev-binding aptamer and CMV promoter a decoys to inhibit HIV replication, Gene 255 (2) (2000) 235e244. [4] S.W. Gal, S. Amontov, P.T. Urvil, D. Vishnuvardhan, F. Nishikawa, P.K. Kumar, et al., Selection of a RNA aptamer that binds to human activated protein C and
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