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Diagnosis–Therapy Integrative Systems based on Magnetic RNA Nanoflowers for Co-drug Delivery and Targeted Therapy Yingshu Guo, Shuang Li, Yujie Wang, and Shusheng Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03346 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Analytical Chemistry

Diagnosis–Therapy Integrative Systems based on Magnetic RNA Nanoflowers for Co-drug Delivery and Targeted Therapy Yingshu Guo,a Shuang Li,a,b Yujie Wang,a,c and Shusheng Zhang*a

a

Shandong Province Key Laboratory of Detection Technology for Tumor Makers, School of

Chemistry and Chemical Engineering, Linyi University, Linyi 276005, China b

Shandong Provincial Key Laboratory of Life-Organic Analysis, College of Chemistry and

Chemical Engineering, Qufu Normal University, Qufu, 273165, China c

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation

Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, P. R. China

Corresponding author E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: This study was to develop a co-drug delivery system for targeting cancer therapy based on magnetic RNA nanoflowers (RNA NF). Compared with traditional nucleic acid structure, convenient separation can be achieved by introducing magnetic nanoparticle (MNP) into RNA NF. Folic acid (FA) modified MNP/RNA NF (FA/MNP/RNA NF) was used as a targeting nanocarrier with excellent biocompatibility to overcome the nonselectivity of MNP/RNA NF. And then, anticancer

drug

doxorubicin

(DOX)

and

photosensitizer

5,

10,

15,

20-tetrakis

(1-methylpyridinium-4-yl) porphyrin (TMPyP4) binding with RNA NF were used as co-drug cargo models. RNA NF was first used for co-drug delivery. So, imaging fluorescent tags, target recognition

element, and drug molecules were all assembled together on the surface of MNP/RNA NF. The experimental results suggested that the treatment efficacy of co-drug delivery platform

(FA/MNP/RNA NF /D/T) was better than single-drug delivery platform (FA/MNP/RNA NF/D). Besides, the FA/MNP/RNA NF was used as a probe for cancer cell detection. The limit of

detection was 50 HeLa cells. In conclusion, the co-drug delivery platform based on FA/MNP/RNA NF was a promising approach for the intracellular quantification of other biomolecules, as well as a diagnosis–therapy integrative system.

Keywords: RNA nanoflowers; RNA nanotechnology; rolling circle transcription; cancer cell; co-drug delivery


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INTRODUCTION Nanotechnology can integrate multiple functionalities such as imaging, drug delivery, and photothermal therapy into a single system, thus offering excellent prospects for noninvasive diagnosis and treatment of cancer. Nanostructure-based diagnosis–therapy integrative systems represent an emerging approach to cancer treatment. A wide range of biocompatible, multifunctional drug carriers such as liposomes,1-3 DNA micelles,4-5 and inorganic nanomaterials 6-8

have great potential by integrating their diagnostic and therapeutic functions. Over the years of

technological developments, diagnosis–therapy integrative systems based on engineered nanoparticles have started to show great promise. Nucleic acids are best known as the carriers of genetic information, but they are also extensively used as versatile materials for designing various self-assembling two or three dimensional nanostructures, because nucleic acid sequences can be designed such that the strands fold into well-defined secondary structures. Several programmed self-assembly of nucleic acid strands have been applied to construct nucleic acid origami with well-defined structure via intramolecular and/or intermolecular interactions. More than three decades ago, Seeman first proposed using deoxyribonucleic acid (DNA) as a material for creating nanostructures,9 this has led to further progress in constructing molecular devices and patterned superstructures for various applications, such as diagnosis, and drug delivery by DNA nanotechnology.10-14 Ribonucleic acid (RNA) has several unique attributes that make it a powerful biomaterial compared to DNA, for example, RNA–RNA interaction is the most stable with lowest free energy among the RNA–RNA, DNA–RNA, and DNA–DNA interactions.15,16 Recently, RNA structures have become increasingly attractive because of the amazing diversity of their structures and functions.17-19



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Various RNA nanostructures, such as RNA nanotube,20 RNA nanosheets21 polygon-shaped RNA22 have been developed. Through several assembly approaches based on RNA nanotechnology, varieties of therapeutic RNA structures harboring multiple therapeutic modules, such as aptamer, ribozymes or small interfering RNA, have been constructed.23-27 However, the rapid development of the RNA nanotechnology has been hindered by major challenges: (i) to produce complex superstructures from simple molecular building blocks and (ii) to target specifically pathways for biomedicine applications. We now present a versatile and simple ‘one-pot’ method based on rolling circle transcription (RCT) by T7 RNA polymerase to produce slender RNA chain24 that be engaged in the composition of the desired RNA nanoflowers (RNA NF) roughly 100 nm in diameter (Scheme 1A). The lengths of RNA by current commercial synthesis are circumscribed and the processes are with expensive ways. In this study, the advantage of RNA NF lies in RCT. Only two DNA sequences (one as a promoter sequence and the other as a template sequence) were used for preparing a long thousands bases-RNA sequence entering into the composition of the RNA NF reducing the costs and circumventing the drawbacks of commercial synthesis. Magnetic nanoparticles (MNPs) were successfully incorporated within RNA NF by biotin-avidin conjugation (MNP/RNA NF) (Scheme 1B). MNP/RNA NF nanocomposites attracted increasing research interest owing to their excellent properties of bioseparation and drug delivery via applying an external magnetic field. To realize the specific targeting delivery, the strategy is to employ active targeting ligands (folic acid, FA) to functionalize the MNP/RNA NF (FA/MNP/RNA NF) for specifically binding folate receptors (FR) overexpressed on the membrane of cancer cells but low-expressed on normal cell. Based on the intercalation, the


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different functional moieties can assemble into MNP/RNA NF conveniently. Both the chemotherapeutic

drug

doxorubicin

(DOX)

and

the

photosensitizer

5,

10,

15,

20-tetrakis(1-methylpyridinium-4-yl) porphyrin (TMPyP4)28 were attached to FA/MNP/RNA NF (FA/MNP/RNA NF/D/T). When exposed to a 650 nm light, reactive oxygen species (ROS) induced by TMPyP4 molecules were generated inside the living cells, followed by cell damage (Scheme 1B). In the presence of DOX, the efficacy toward the target cells was superior to individual drug treatment. Scheme 1 Experimental Section Reagents and materials. Streptavidin-functionalized magnetic nanoparticles (MNP, ~100 nm diameter) were purchased from Tiandz (Beijing, China). Doxorubicin (DOX) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). 5,10,15,20-tetrakis (1-methylpyridinium 4-yl) porphyrin

(TMPyP4)

were

obtained

from

Tokyo

Chemical

Industry

Co.,

Ltd..

1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), N-hydroxysuccinimide ester (NHS), and 2, 7-dichlorofluorescin diacetate (DCFH-DA) was obtained from Sigma-Aldrich. T7 RNA polymerase and ribonucleotide mix were purchased from New England Biolabs. T4 DNA ligase was purchased from Thermo Scientific. Folic acid modified primer 2 was taken from Dalian Takara Biotech. Co., Ltd. (China) (Table S1). Other DNA chains were taken from Shanghai Sangon Biotech. Co., Ltd. (China) (Table S1). The experimental water is taken from pure water meter (18.25 M Ω). For the cellular experiments, all of the reagents, buffers, and culture medium were sterilized by steam autoclave (121°C, 40 min) or filtration (0.22 µm pore size, Millipore), and maintained under a sterile condition.



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Apparatus. Fluorescence spectra were measured by a fluorescence spectrophotometer (F-4600, Hitachi ). Absorption spectra were determinated by a uv-vis spectrophotometer (Cary 60, Agilent). Flow cytometric assays were performed using a flow cytometry (Cytoflex, Beckman Coulter). Zeta potential measurement was performed at 25 °C on a Zeta-size Nano instrument (Zen 3600, Malvern Instruments Ltd.). Magnetic data were collected on a Quantum Design MPMS 3 magnetometer. Transmission electron microscope images (TEM) were performed transmission electron microscope (JEM-2100, JEOL). Confocal fluorescence imaging was performed on a confocal laser scanning microscope (LEICA TCS SP8, Germany). Vivo imaging was performed by an IVIS Lumina II in vivo imaging system. Preparation of RNA NF. All sequences were dissolved and diluted in nuclease-free water. To synthesize circular DNA, 30 µL 2.5 µM of linear template DNA 1 (or linear template DNA 2) and 30 µL 2.5 µM primer 1 (or primer 2) were mixed. The above mixed solutions were heated to 95 °C for 5 min and slowly cooled down to room temperature in one hour for denaturation and annealing. Then, 1 µL 5 U/µL of T4 ligase were added and the mixture was incubated for 12 h at 25 °C to obtain the circularized DNA 1 (or circularized DNA 2). The circularized DNA 1 and the circularized DNA 2 were mixed with 3 µL 25 mM of ribonucleotide triphosphates solution mix, 6 µL 50 U/µL of T7 RNA polymerase. Then, the reaction solution was incubated at 37 °C to form the RNA nanoflowers (RNA NF). Preparation of FA/MNP/RNA NF. 50 µL streptavidin-functionalized magnetic nanoparticles (MNP) were rinsed with DPBS twice and was incubated for 12 h at 37 °C with 30 µL 2.5 µM biotinylated primer 1 (bio-primer 1), then, free DNA were removed by magnetic separation. To synthesize circular DNA, MNP-primer 1 complex were mixed with 30 µL 2.5 µM linear template


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DNA 1. 30 µL 2.5 µM linear template DNA 2 and 30 µL 2.5 µM folic acid modified primer 2 (FA-primer 2) were mixed. The above two kinds of mixed solution were heated to 95 °C for 5 min and slowly cooled down to room temperature in one hour for denaturation and annealing. To ligate the nick in the circularized DNA, 1 µL 5 U/µL of T4 ligase were added and the mixture was incubated for 12 h at 25 °C to obtain the MNP/circularized DNA 1 (or circularized DNA 2). MNP/circularized DNA 1 and circularized DNA 2 were incubated with 3 µL 25 mM of ribonucleotide triphosphates solution mix, 6 µL 50 U/µL of T7 RNA polymerase at 37 °C to obtain FA/MNP/RNA NF. Formed FA/MNP/RNA NF were collected by magnetic separation. Detection procedure for cell. The gold chip was soaked with 5 mM thioglycolic acid solution at room temperature for 12 h and rinsed gently to remove unbound thioglycolic acid molecules. Then, the gold chip was soaked with the solution of 0.2 M EDC and 0.1 M NHS at room temperature for 2 h and rinsed gently following reaction with 20 µL the antibody against the epidermal growth factor receptor (anti-EGFR) for 2 h. Then, 20 µL of 0.1 % bovine serum albumin (BSA) was used to block the remaining actived surface. The gold chip was then rinsed gently and 100 µL cell suspension was added, followed by incubation at 37 °C for 1 h. After removing unbound cells, 20 µL FA/MNP/RNA NF/D was added to the gold chip surface for incubation at 37 °C for 4 h. Then the gold chip was rinsed gently and putted into 200 µL nuclease free water with sonication in ice-bath for 30 min to break the cell. Finally, the solution was detected by fluorescence spectrophotometer. Results and Discussion Characterization. Formation of RNA NF takes place directly in the transcription reaction when DNA templates encoding specifically designed RNAs were introduced. The resulting RNA NF



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was characterized by gel electrophoresis and TEM experiments. To confirm the formation of RNA NF, the gel electrophoresis were used to analysis the synthetic substances (Figure 1A). The results indicated the successful generation of RNA macromolecule through RCT. TEM were used to characterize the morphology of the synthetic substances which were synthesized in 2 h, 6 h, and 10 h by RCT reaction.24 We observed that the fibre-like structure first appeared in the RCT reaction (Figure 1Ba). When the reaction time is up to 6 h, globular structure started to appear (Figure 1Bb). These globular structures began to form a single particle after 10 h (Figure 1Bc). Figure 1 The zeta potential of −15 mV indicated good stability of MNP. After assembling MNP/RNA NF, the zeta potential changed to −21.9 mV (Figure 2A). The multifunctional MNP/RNA NF were also tested through UV−vis spectra (Figure 2B). MNP/RNA NF have a significant absorption intensity of DNA at 260 nm compared to MNPs. This indicated that the RNA NF was successfully decorated to the MNPs surface. The conjugation process was further demonstrated by TEM and EDX results (Figure 2B, C). These characterizations demonstrated the successful assembly of MNP/RNA NF. The magnetic properties of MNP/RNA NF (Figure 2D) were analysed by the magnetization curves.29 The saturation magnetization values of MNP (11.4 emu/g) is a little higher than MNP/RNA NF (9.2 emu/g) attributed to the RNA NF on the MNPs. Those results showed that MNP/RNA NF possessed typical properties of magnetic. So, in the presence of an external magnetic field, the magnetic multifunctional MNP/RNA NF can be easily oriented to separate. Figure 2 To load DOX (or TMPyP4) into FA/MNP/RNA NF, DOX (or TMPyP4) and FA/MNP/RNA NF


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were mixed and stayed at room temperature to allow saturation of drug loading, followed by magnetic separation and removal of free DOX (or TMPyP4) in the supernatant. The amount of removed DOX (or TMPyP4) was determined, and the drug loading capacity of the FA/MNP/RNA NF was calculated accordingly. The loading efficiency of DOX or TMPyP4 was 76.7% and 52%, respectively. Loading efficiency = [(feeding amount of drug - removed amount of drug) /feeding amount of drug] × 100%. Specific Binding and Internalization of FA/MNP/RNA NF. The selective binding ability of FA/MNP/RNA NF/D to FR-positive HeLa cells (human cervix adenocarcinoma cell), but not to FR-negative L02 cells (human hepatocyte cell), was verified through flow-cytometric analysis (Figure 3A and B), providing a basis for the locomotive action of FA moiety guiding FA/MNP/RNA NF/D toward target sites. First, HeLa cells (or L02 cells) were sited with FA/MNP/RNA NF and FA/MNP/RNA NF/D at 37 °C for 4 h, respectively. The signal had changed significantly after FA/MNP/RNA NF/D incubating with HeLa cells. For L02 cells, the signal had no obvious change. The significant difference of fluorescence signal between HeLa cells and L02 cells due to the low-level expression of FR in L02 cells,30 further confirming the specific recognition for FR-positive cells. The same assay for A549 cells (or HepG2 cells) were also shown in (Figure S5A and B). In addition, a process of internalization of FA/MNP/RNA NF/D was imaged through using confocal laser scanning microscopy (CLSM). In the cells’ uptake experiments, CLSM images of HeLa cell after being incubated with FA/MNP/RNA NF/D for 3 h demonstrated the strong DOX fluorescence (Figure 3C up). DOX was located around cell membrane, which can be explained by the limited internalization efficiency within this time period. In contrast, no obvious fluorescence of DOX was appeared in L02 cell (Figure 3D up). All these



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phenomena suggested that the FR expression amount on the cell was a critical factor for enhanced cell uptake of FA/MNP/RNA NF/D. And FA/MNP/RNA NF/D was internalized selectively by HeLa cells. HeLa cells were further incubated with FA/MNP/RNA NF/D for 24 h. Overlay of the dark-field images and bright-field images, the images displayed an obvious DOX fluorescence from cytoplasm to nucleus of HeLa cells (Figure 3C down). Moreover, the cell was stained with DIO and DAPI to co-localization of DOX. The images showed that DOX was conveyed into cell nucleus (Figure 3E). Presumably, DOX unloading from FA/MNP/RNA NF/D is through simple diffusion and facilitated by intracellular factors such as ionic environment, and nuclease degradation.31 However, for L02 cells, no DOX was significant distributed in the nucleus after incubated with FA/MNP/RNA NF/D. The significant difference of fluorescence signal between cancer and normal cells provided a tool for cancer discrimination. All these results confirmed that the FA/MNP/RNA NF have potential application as fluorescently traceable drug carriers. Figure 3 Cytotoxicity Assay. To assess the performance of biomedical applications, the cytotoxicity of FA/MNP/RNA NF, FA/MNP/RNA NF/D and FA/MNP/RNA NF/T for HeLa cells and L02 cells, respectively. FA/MNP/RNA NF, FA/MNP/RNA NF/D and FA/MNP/RNA NF/T were sited with cells respectively. Both HeLa cells and L02 cells exhibited high survival rate (over 92%) after cultured with FA/MNP/RNA NF, indicating that FA/MNP/RNA NF has excellent biocompatibility (Figure 4A). For HeLa and L02 cells (Figure 4B and C), DOX only (or TMPyP4 only) showed dose-dependent cytotoxicity behavior. In HeLa cells, DOX (or TMPyP4) delivered by FA/MNP/RNA NF induced considerably obvious inhibition of cell proliferation, while in L02 cells, FA/MNP/RNA NF/D (or FA/MNP/RNA NF/T) complexes induced slight inhibition of cell


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proliferation (Figure 4D and E). The cytotoxicity of FA/MNP/RNA NF, FA/MNP/RNA NF/D and FA/MNP/RNA NF/T for A549 and HepG2 cells were also tested (Figure S6). This phenomenon is because that the cell surface of L02 cells possessed the low expression of FR, showing the minimized nonspecific uptake. By compared column a with column b, column c with column d (Figure 4 F), it should be found that 650 nm light irradiation showed no considerable damage to cells without TMPyP4. An obvious reduction of cell viability was observed for FA/MNP/RNA NF/D with or without 650 nm light irradiation. Besides, cell viability further decreased obviously for FA/MNP/RNA NF/D/T with 650 nm light irradiation showing a synergistic therapy effect via the combination of TMPyP4 and DOX. So FA/MNP/RNA NF/D/T showed better anticancer effect than FA/MNP/RNA NF/D. Figure 4 To account for the cytotoxicity of FA/MNP/RNA NF/T, the intracellular ROS formation was studied. The cells were incubated with FA/MNP/RNA NF or FA/MNP/RNA NF/T for 4 h and then washed twice in washing buffer (1% BSA). 10 µM of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) prepared in serum-free DMEM medium was added to the cells for 40 min at 37 °C. The cells were under 50 min irradiation (650 nm, 12 mW/cm2). After flow cytometry analysis, no obvious fluorescence change was found in the L02 cell between incubated with FA/MNP/RNA NF and FA/MNP/RNA NF/T (Figure 5B). However an obvious fluorescence change was found in the FA/MNP/RNA NF/T-treated HeLa cells (Figure 5A). Similar phenomena were observed for A549 cells (the lung epithelial cancer cell) and HepG2 cells (Human hepatocellular carcinoma cell) (Figure S5C and D). Combined with the results of Figure 4, the cell death following exposure to the irradiation could be attributed to the accumulation of intracellular ROS induced by



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TMPyP4-containing FA/MNP/RNA NF. Besides, these results indicated that the relative expression levels of FR in single HeLa cells were higher than in L02 cells. Thus, the new method was viable for detecting changes in FR expression levels in tumor cells. The cell morphology was observed under the microscope. Figure 5C and D showed clear morphological changes before or after treated with FA/MNP/RNA NF/T and irradiation. The untreated HeLa cells showed normal morphology. After FA/MNP/RNA NF/T and irradiation treatment, the marked nuclear condensation, membrane breakage, and apoptotic bodies became visibly dominant, indicating cell apoptosis. Figure 5 Detection of folate receptor cells. The fluorescence responses to different concentrations of HeLa cells were measured. This protocol involved a sandwich format, in which the HeLa cells was first captured by the immobilized anti-EGFR on the surface of gold chips, and then can be recognized by FA/MNP/RNA NF/D (Scheme 2). The fluorescence intensity gradually increased with the increasing cell concentration (Figure 6), which confirmed that the molecular recognition mediated cell adhesion can induce the fluorescence intensity change. The fluorescence intensity increased linearly with the cell concentration over the range from 500 to 2000 cells/mL, with a correlation coefficient R of 0.9997. The linear regression equation is Y=0.058X-0.39 (Y is the relative fluorescence intensity; X represents the concentration of target cell, cells/mL; n = 5). The limit of detection was 500 cells/mL. Taking into account that 100 µL of HeLa cell suspension was used for incubation, the presented strategy achieved the limit of detection of only 50 HeLa cells. In addition, the strategy is able to differentiate between HeLa cells and L02 cells based on the high specificity of the over-expressed FR for HeLa cells (Figure S7, Table S2), indicating the wide


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applicability of it for FR over-expressed cell detection. Scheme 2 Figure 6 In vivo tumor effect. The feasibility of nanoprobe for in vivo tumor imaging was investigated in subcutaneous HeLa tumor-bearing mice. The tumor region displayed strong fluorescence and could be distinguished from the normal region (Figure 7A and B). The high contrast for tumor imaging was attributed to FR-targeting and DOX activation. The in vivo treatment effect of FA/MNP/RNA NF/D/T to tumor was assessed by monitoring the change of relative tumor volume (Figure 7C). After the tumor bearing mice were treated with FA/MNP/RNA NF/D or FA/MNP/RNA NF/D/T and then irradiation, tumor growth was slowdown, and the therapeutic effect of FA/MNP/RNA NF/D/T was better than FA/MNP/RNA NF/D by comparing the relative tumor volume. While no therapeutic effect was observed with treatment with only irradiation. Mouse body weight variations before and after

treatments were also examined. Results in Figure 7D indicated that mice treated with only irradiation or untreatment lost significantly more weight than those treated with FA/MNP/RNA NF/D/T or FA/MNP/RNA NF/D, whereas those treated with FA/MNP/RNA NF/D/T or FA/MNP/RNA NF/D showed slight body weight increase. Overall, these results indicated the significance of the designed co-drug platform for in vivo theranostic application. Figure 7 Conclusions A new multifunctional diagnosis–therapy integrative system based on magnetic RNA nanoflowers (MNP/RNA NF) for co-drug delivery and targeted therapy was demonstrated in this paper. Compared with traditional nucleic acid structure, convenient separation can be achieved by



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introducing magnetic nanoparticle (MNP). DOX molecules and TMPyP4 molecules can bind with RNA NF. As proof of concept, a functional/therapeutic strand, folic acid functionalized sequence, was self-assembled to prepare FA/MNP/RNA NF to achieve the targeting delivery of different functionalities into cells. This system combined imaging fluorescent tags, target recognition elements, and targeted delivery molecules into MNP/RNA NF, while possessing high drug loading and specificity. This system also offered an attractive feature of co-delivery of two types of drugs for combination therapy which has the potential to overcome multidrug resistance. In addition, 50 HeLa cells were detected by using FA/MNP/RNA NF as a probe. With the advantage of small size, easy synthesis, good biocompatibility, high binding affinity, and selectivity, this FA/MNP/RNA NF will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and biomolecules detection. ASSOCIATED CONTENT The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Acknowledgments This work was supported by the National Natural Science Foundation of China (21575056, 21535002), the Natural Science Foundation of Shandong Province of China (ZR2016JL010), the Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-13-0845).


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Manoharan, M.; Donahoe, J. S.; Truelove, J.; Nahrendorf, M.; Langer, R.; Anderson, D. G. Nat. Nanotechnol. 2012, 7, 389–393. (11) Kuzuya, A.; Sakai, Y.; Yamazaki, T.; Xu, Y.; Komiyama, M. Nat. Commun. 2011, 2, 449. (12) Zhang, D. Y.; Hariadi, R. F.; Choi, H. M. T.; Winfree, E. Nat. Commun. 2013, 4, 1965. (13) Han, D.; Pal, S.; Nangreave, J.; Deng, Z.; Liu, Y.; Yan, H. Science 2011, 332, 342–346; (14) Lu, C. H.; Cecconello, A.; Willner, I. J. Am. Chem. Soc. 2016, 138, 5172–5185. (15) Shu, D.; Shu, Y.; Haque, F.; Abdelmawla, S.; Guo, P. Nat. Nanotechnol. 2011, 6, 658–667. (16) Lesnik, E. A.; Freier, S. M. Biochemistry 1995, 34, 10807–10815. (17) Guo, P. Nat. Nanotechnol. 2010, 5, 833–842. (18) Dibrov, S. M.; McLean, J.; Parsons, J.; Hermann, T. Proc Natl. Acad. Sci. U S A 2011, 108, 6405–6408. (19) Grabow, W. W.; Jaeger, L. Acc. Chem. Res. 2014, 47, 1871–1880. (20) Yingling, Y. G.; Shapiro, B. A. Nano. Lett. 2007, 7, 2328-2334. (21) Kim, H.; Lee, J. S.; Lee, J. B. Sci. Rep. 2016, 6, 25146. (22) Khisamutdinov, E. F.; Jasinski, D. L.; Guo, P. ACS nano 2014, 8, 4771-4781. (23) Afonin, K. A.; Kireeva, M.; Grabow, W. W.; Kashlev, M.; Jaeger, L.; Shapiro, B. A. Nano Lett. 2012, 12, 5192−5195. (24) Lee, J. B.; Hong, J.; Bonner, D. K.; Poon, Z.; Hammond, P. T. Nat. Mater. 2012, 11, 316-322. (25) Shu, D.; Khisamutdinov, E. F.; Zhang, L.; Guo, P. Nucleic Acids Res. 2014; 42, e10. (26) Jasinski, D. L.; Khisamutdinov, E. F.; Lyubchenko, Y. L.; Guo, P. ACS Nano 2014, 8, 7620-7629.


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Scheme captions Scheme 1. (A) The process of preparation for FA and biotin modified RNA NF. (B) FA and biotin modified RNA NF self-assembled on the surface of MNP via hybridization. The FA/MNP/RNA NF nanocomposite was uptaken by cancer cell, releasing of co-drug for cancer therapy. Scheme 2. Scheme of detection procedure for cell


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Figure captions Figure 1. (A) The polyacrylamide gel electrophoresis results illustrating the formation of RNA NF, line 1: primer 1, line 2: primer 2, line 3: DNA 1, line 4: DNA 2, line 5: primer 1+DNA 1; line 6: primer 2+DNA 2; line 7: RNA macromolecule. (B) TEM images of RCT products by different reaction times for (a-c) 2, 6, 10 hours. Figure 2. (A) The Zeta potential values of MNP and MNP/RNA NF, respectively. (B) UV−vis spectra of MNP, RNA NF and MNP/RNA NF. Inset: TEM images of the MNP and MNP/RNA NF. (C) EDX spectra of the obtained MNP, RNA NF and MNP/RNA NF. (D) Magnetization curves of (a) MNP and (b) MNP/RNA NF. Figure 3. (A) Flow cytometric assay of L02 cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/D (Green). (B) Flow cytometric assay of HeLa cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/D (Green). (C) CLSM images of HeLa cells after incubation with FA/MNP/RNA NF/D at 37 °C for 3 h and 24 h. (D) CLSM images of L02 cells after incubation with FA/MNP/RNA NF/D at 37 °C for 3h and 24 h. (E) CLSM images of HeLa cells after incubation with (a) bright field, (b) DIO, (c) DAPI, (d) FA/MNP/RNA NF/D, and (e) merged images. Figure 4. (A) HeLa and L02 cells viability assays that cells were handled with FA/MNP/RNA NF. (B) HeLa and L02 cells viability assays that cells were handled with free DOX. (C) HeLa and L02 cells viability assays that cells were handled with free TMPyP4. The laser irradiation of 12.0 mW/cm2 on 650 nm was used. (D) HeLa and L02 cells viability assays that cells were handled with FA/MNP/RNA NF/D. (E) HeLa and L02 cells viability assays that cells were handled with FA/MNP/RNA NF/T. The laser irradiation of 12.0 mW/cm2 on 650 nm was used. (F) HeLa cells viability assays that cells were handled with (a) no treatment, (b) laser irradiation of 12.0 mW/cm2



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on 650 nm, (c) FA/MNP/RNA NF/D, (d) FA/MNP/RNA NF/D and laser irradiation of 12.0 mW/cm2 on 650 nm, and (e) FA/MNP/RNA NF/D/T and laser irradiation of 12.0 mW/cm2 on 650 nm. Figure 5. (A) Flow cytometric assay of HeLa cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/T (Green). The laser irradiation of 12.0 mW/cm2 on 650 nm was used. (B) Flow cytometric assay of L02 cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/T (Green). The laser irradiation of 12.0 mW/cm2 on 650 nm was used. (C) HeLa cell morphology before treated with FA/MNP/RNA NF/T and irradiation. (D) HeLa cell morphology after treated with FA/MNP/RNA NF/T and irradiation. Figure 6. fluorescence intensity curves obtained with HeLa cells of (a) 0, (b)500, (c) 1000, (d) 1500 (e) 2000 cells mL−1. Inset: the relationship between relative fluorescence intensity and different concentrations of HeLa cells. The error bars represent the standard deviations calculated from three experiments. Figure 7. In vivo fluorescence images of (A) normal mouse and (B) HeLa tumor-bearing mouse after 24-h post-injection of the FA/MNP/RNA NF/D. (C) Change of relative tumor volume (V/V0) upon different treatments. (D) Change of relative mouse body weight (W/W0) at day 28 compared with day 0 after different treatment.


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Schemes


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COOH COOH COOH COOH

Thioglycollic acid

s

s s

s

1

EDC / NHS

2

anti-EGFR

s

s

s

s

EGFR

Gold Chip

Hela cell

BSA

s

s

s

s

s

s

s

s

Cellular debris Ultrasonic

FA/MNP/RNA NF/D

s s

s s s

Scheme 2. Scheme of detection procedure for cell


s s

s

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Figures

Figure 1. (A) The polyacrylamide gel electrophoresis results illustrating the formation of RNA NF, line 1: primer 1, line 2: primer 2, line 3: DNA 1, line 4: DNA 2, line 5: primer 1+DNA 1; line 6: primer 2+DNA 2; line 7: RNA macromolecule. (B) TEM images of RCT products by different reaction times for (a-c) 2, 6, 10 hours.



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Figure 2. (A) The Zeta potential values of MNP and MNP/RNA NF, respectively. (B) UV−vis spectra of MNP, RNA NF and MNP/RNA NF. Inset: TEM images of the MNP and MNP/RNA NF. (C) EDX spectra of the obtained MNP, RNA NF and MNP/RNA NF. (D) Magnetization curves of (a) MNP and (b) MNP/RNA NF.


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(C) Bright-field

Dark-field

Bright-field

Dark-field

Merged

3h

24 h

(D) Merged

3h

24 h



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Figure 3. (A) Flow cytometric assay of L02 cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/D (Green). (B) Flow cytometric assay of HeLa cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/D (Green). (C) CLSM images of HeLa cells after incubation with FA/MNP/RNA NF/D at 37 °C for 3 h and 24 h. (D) CLSM images of L02 cells after incubation with FA/MNP/RNA NF/D at 37 °C for 3 h and 24 h. (E) CLSM images of HeLa cells after incubation with (a) bright field, (b) DIO, (c) DAPI, (d) FA/MNP/RNA NF/D, and (e) merged images.


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Figure 4. (A) HeLa and L02 cells viability assays that cells were handled with FA/MNP/RNA NF. (B) HeLa and L02 cells viability assays that cells were handled with free DOX. (C) HeLa and L02 cells viability assays that cells were handled with free TMPyP4. The laser irradiation of 12.0 mW/cm2 on 650 nm was used. (D) HeLa and L02 cells viability assays that cells were handled with FA/MNP/RNA NF/D. (E) HeLa and L02 cells viability assays that cells were handled with FA/MNP/RNA NF/T. The laser irradiation of 12.0 mW/cm2 on 650 nm was used. (F) HeLa cells viability assays that cells were handled with (a) no treatment, (b) laser irradiation of 12.0 mW/cm2



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on 650 nm, (c) FA/MNP/RNA NF/D, (d) FA/MNP/RNA NF/D and laser irradiation of 12.0 mW/cm2 on 650 nm, and (e) FA/MNP/RNA NF/D/T and laser irradiation of 12.0 mW/cm2 on 650 nm.


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Figure 5. (A) Flow cytometric assay of HeLa cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/T (Green). The 12.0 mW/cm2 laser irradiation on 650 nm was used. (B) Flow cytometric assay of L02 cells incubated with FA/MNP/RNA NF (Red) or FA/MNP/RNA NF/T (Green). The 12.0 mW/cm2 laser irradiation on 650 nm was used. (C) HeLa cell morphology before treated with FA/MNP/RNA NF/T and irradiation. (D) HeLa cell morphology after treated with FA/MNP/RNA NF/T and irradiation.



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Figure 6. fluorescence intensity curves obtained with HeLa cells of (a) 0, (b)500, (c) 1000, (d) 1500 (e) 2000 cells mL−1. Inset: the relationship between relative fluorescence intensity and different concentrations of HeLa cells. The error bars represent the standard deviations calculated from three experiments.


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Figure 7. In vivo fluorescence images of (A) normal mouse and (B) HeLa tumor-bearing mouse after 24-h post-injection of the FA/MNP/RNA NF/D. (C) Change of relative tumor volume (V/V0) upon different treatments. (D) Change of relative mouse body weight (W/W0) at day 28 compared with day 0 after different treatment.



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For TOC only

FR


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