A Smart Magnetic Nanoaptamer: Construction, Subcellular Distribution

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A Smart Magnetic Nanoaptamer: Construction, Subcellular Distribution and Silencing HIF for Cancer Gene Therapy Rui Yang, Wei-Yu Mu, Qiu-Yun Chen, Qiang Wang, and Jing Gao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00204 • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

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ACS Biomaterials Science & Engineering

A Smart

Magnetic

Nano-aptamer:

Construction,

Subcellular

Distribution and Silencing HIF for Cancer Gene Therapy Rui Yang†, Wei-Yu Mu†, Qiu-Yun Chen†,* Qiang Wang‡, Jing Gao‡

†

School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang,

Jingkou District, Xuefu Road 212013, People’s Republic of China. ‡

School of Pharmacy, Jiangsu University; Zhenjiang, Jingkou District, Xuefu Road

212013, People’s Republic of China. Contact information of the corresponding author is as follows: Qiu-Yun Chen: School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jingkou District, Xuefu Road 212013, People’s Republic of China. *E-mail address: [email protected] (Q.Y. Chen).

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ABSTRACT：：Attenuating the expression of HIF-1α (hypoxic inducible factor) by siRNA has effect on the proliferation of hypoxia cancers. Mitochondria targeting siRNA may silence the level of HIF-1α for cancer gene therapy. A GAG-rich DNA was conjugated to GC-rich DNA for the synthesis of functional magnetic nano-aptamer（DNA-Fe3O4）to keep the innate character of targeting aptamer. The DNA-Fe3O4 can load the hydrophobic dye (BODIPY-OCH3) by the GC-rich sequences resulting in fluorescent nano-aptamer (BFe@DNA). Self-assembly of BFe@DNA with target aptamer resulted in formation of BFe@DNAH. Subcellular fluorescence imaging results confirm that BFe@DNAH can accumulate in MCF-7 cells and selectively target mitochondrion. In particular, BFe@DNAH can transport siRNA to breast cancer cells or tissues for the attenuation of HIF-1α and ATP and the inhibition on growth of cancer cells in vivo. Therefore, BFe@DNAH is a smart nanoaptamer platform for the development of subcellular imaging agents and gene therapy. KEYWORDS: nano-aptamer, mitochondria, siRNA, gene therapy


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INTRODUCTION Hypoxia is common in most solid tumors. Tumor hypoxia is related to the activation of the hypoxia inducible factor-1α (HIF-1α).1 HIF attenuates the tumor cells resistance to radiation therapy and chemotherapy.2 Moreover, HIF can trigger a transformation from mitochondrial oxidative phosphorylation to anaerobic glycolysis in mitochondrion.3,4 Therefore, HIF-1 has been reported as a cancer therapeutic target. Attenuating the activity of HIF-1α has been a pathway to cancer chemotherapy.5 A growing number of compounds, such as PX-478, rapamycin, YC-1, 17-AAG and mimics of catalase were reported to inhibit the expression of HIF-1α in cell or animal models.6 However, because of their side effects, less inhibitors have been used as pharmacologic attenuators of HIF-1α. MicroRNAs (miRNAs) regulate protein expression. For example, miR-20b and miR-199a can attenuate the expression of HIF-1α protein in cancer cells.7,8 Small interfering RNA (siRNA) and aptamers have been reported to interfere with protein expression.9 To enforce the migration of nucleic acids to the site of actions, positive charged iron oxide nanoparticles were widely used as deliveries of DNA for gene therapy.10 Aptamer is capable of specifically binding to proteins or small molecules.11 Several aptamer conjugated magnetic nanoparticles have been reported as biosensors for MR imaging or drug delivery.12,13 Generally, aptamer conjugates to the surface of nanoparticles by C-S bond, S-S bond or amide bond.14-17 Fluorescence imaging show increased uptake of aptamer conjugated nanoprobe by cancer cells. However, it is hard to keep the innate character of targeting aptamer after the chemical modification. The self-assembly of aptamer by base-match can keep the innate properties of aptamers. Integrating aptamer with magnetic nanoparticles by the self-assembly of aptamers and metals would produce targeting nanoaptamers for gene delivery. Moreover, mitochondria targeting siRNA can be attenuators of HIF-1 for cancer gene therapy. Herein, we report a two unit nano-aptamer, which can bind with targeting aptamer by base-match principle and load the organic dye through the rich-GC chain forming hydrophobic layers (Shceme 1). Moreover, the smart nano-aptamer can deliver siRNA for the attenuation of ATP and HIF-1α.

MATERIALS AND METHODS Materials. All reagents were of analytical-reagent grade and directly used without further



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purification. Aptamers (obtained from Sangon Biotech) were presented as below: DNA1: 5’-GAGGAGACAACAACAGCGCGCGC-3’. The other DNAs were presented as: DNA2: 5’-GCGCGCGCACAACAACAGAGGAG-3’; GLUT: 5’-TGTTGTTGTCCCACCCACCCTCCCAAGTATGTGGAGCAACTGTGTGG-3’; HIF: 5’-TGTTGTTGTCTACGTGCT-3’. siRNA： 5’-GAUGAAAGAAUUACCGAAUTT-3’ and 5’-AUUCGGUAAUUCUUUCAUCTT-3’. The BODIPY-OCH3 [8-(4-(methyloxy) phenyl)-4, 4-difluoro-1, 3, 5, 7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene)] was prepared as reported.18, 19 Synthesis of DNA-Fe3O4. DNA1 (64 µL, 100 µM) was mixed with FeCl3 (200 µL, 10 mM) in citrate buffer (750 µL, 10 mM) for 2 min at 40℃ resulting in solution A. DNA2 (64 µL, 100 µM) was mixed with (NH4)2Fe(SO4)2 (111 µL, 10 mM) and Vitamin C (20 µL, 10 mM) for 2 min at 40℃ to form solution B. Later, solution A and solution B were mixed and then NH3·H2O (20 µL) was added.20, 21 The mixture was cooled to room temperature (23±2℃) resulting in DNA-Fe3O4 after heating to 69℃ and maintaining temperature for 30 min on a shaker under nitrogen conditions. The DNA-Fe3O4 was purified by magnetic precipitation and the DNA-Fe3O4 dissolved in ultrapure water was stored at 4℃ for further use. Synthesis of BODIPY-OCH3@DNA-Fe3O4 (Labelled as BFe@DNA). BODIPY-OCH3 (96 µL, 1 mM) in DMSO was added to DNA-Fe3O4 (2000 µL, C shaken at 4℃ for 3 h. The BFe@DNA (200 µL, C

dsDNA=60

dsDNA =6.4

µM) The solution was

µM) was obtained after purification

with semipermeable membrane (MWCO: 3500) to remove free dyes and the BFe@DNA solution was stored at 4 °C before further use. The loaded BODIPY-OCH3 was measured based on the absorption at 499 nm. Synthesis of BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA. BFe@DNA (200 µL, C


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dsDNA =60

µM) was mixed with HIF (or GLUT) (128 µL, 100 µM) in ultrapure water, and shaken

at 4℃ for 24 h to get BFe@DNAH (BFe@DNAG), which was mixed with siRNA (59 µL, 20 µM) in DEPC water at 4℃ for 48 h to form BFe@DNAH-siRNA. The samples were purified by magnetic precipitation. The BFe@DNAH, BFe@DNAG or BFe@DNAH-siRNA solution was stored at 4 ℃ before further use. The solid powder can be obtained after vacuum freeze-drying at -80 ℃.

RESULTS AND DISCUSSION Synthesis and characterization of BFe@DNA. FeII/III-DNA2 was synthesized using DNA2 (or DNA1) as template, among which GAGGAG as bind section for the Fe2+ ion (or Fe3+).22 FT-MS data show that there is one FeII ions combined with DNA2 and one FeIII ion combined with DNA1 (Figure S1-S4). The interaction of Fe2+ with GAG containing aptamer (DNA2) was further confirmed by CD spectra. Guanine (G) and adenine (A) bases contain imidazole rings that coordinate to metal ions. 23-25 The peaks at 250 and 280 nm decreased and the peak at 220 nm shifted through titration of Fe2+ into the solution of DNA2. The FeII-DNA2 converted into Fe3O4@DNA by co-precipitation with FeIII-DNA1 in base condition, which changed the CD spectra (Figure 1A). Intercalation of BODIPY-OCH3 with rich GC section resulted BFe@DNA (Scheme 1). The ratio of BODIPY-OCH3 to dsDNA in BFe@DNA is 6:1. Further assembly of BFe@DNA with HIF (or GLUT) by the linker sequence ‘ACAACAACA’ pairing with TGTTGT sequence produces BFe@DNAH (or BFe@DNAG), which assembled into a nanosphere due to the hydrophobic-hydrophobic interaction of BODIPY-OCH3 in water. The ratio of BODIPY-OCH3 to HIF (or GLUT) is 6:1. Next the assembly of BFe@DNAH with siRNA by charge attractive interaction produces BFe@DNAH-siRNA.



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The UV absorption peaks at 260 nm, 500 nm and 400-700 nm of BFe@DNA are the characteristic absorptions of DNA, BODIPY-OCH3 and Fe3O4, respectively (Figure S5, Figure 1 B and C). The Raman spectrum exhibits mainly three bands at 310, 543 and 662 cm-1, corresponding to the T2g, Eg and A1g, which are Raman bands of magnetite (Fe3O4) (Figure 1D).26 This means that the compositions of BFe@DNA contain magnetite (Fe3O4). The size of Fe3O4@DNA, BFe@DNA, BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA are almost 2-8 nm (Figure 1E).

Fe3+ DNA1 GC

ACA

NH3 H2O GAG DNA-Fe 3O4

DNA2 Fe

2+

GLUT

HIF BFe@DNA H(G)

BFe@DNA Targ eting

siRNA

Fluorescence

OCH3

Hypoxic tumors cell

siR N

BFe@DNAH(G)-siRNA

NBN F F HIF-1a BODIPY-OCH 3

A

Mitochondria

Scheme 1. Formation of BFe@DNA, BFe@DNAH(G) and BFe@DNAH(G)-siRNA.


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Figure 1. Characterization of BFe@DNA. (A) Circular dichroism (CD) spectra of dsDNA (CDNA, 2 µM), dsDNA-Fe2+ (CDNA, 2 µM) and dsDNA-Fe3O4 (CDNA, 2 µM) in ultrapure water. (B) UV-vis spectra of BFe@DNA in water (CdsDNA, 1 µM, CssDNA, 2 µM). (C) UV-vis and fluorescence spectra of BODIPY-OCH3 in CH2Cl2 (CDNA, 2 µM ): (a) UV-vis spectra of BODIPY-OCH3, (b) excitation fluorescence spectra and (c) emission fluorescence spectra (λ

ex/em

= 501/510 nm). (D) Raman spectra of BFe@DNA (30 µM) in water. (E) TEM images of



nanoparticles (DNA-Fe3O4 (A), BFe@DNA (B), BFe@DNAH (C), BFe@DNAG (D) and BFe@DNAH-siRNA (E)).

Stability of nano-aptamers. The stability of nano-aptamers in phosphate-buffered solution (PBS) with different pH (pH=5.0, 6.0, 7.4 and 9.4) was carried out by comparing the UV-vis absorption of nano-aptamers at 555 nm (the absorption of Fe3O4) after 48 h at room temperature with the initial UV-vis absorption of nano-aptamers in ultrapure water at 555 nm (Figure S6(A)). BFe@DNA, BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA showed less reduction in the absorption peak of Fe3O4 (555 nm) at neutral, indicating the Fe3O4 of BFe@DNA, BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA are more stable at the neutral pH condition while they are less stable in acid condition (pH=5.0). In the same way, as shown in Figure S6 (B), BFe@DNA, BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA exhibited the greatest reduction in the absorption peak of BODIPY-OCH3 (500 nm) at pH=5.0, indicating that the BODIPY-OCH3 was released from BFe@DNA (or BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA) in acid condition, while less free BODIPY-OCH3 can be monitored in neutral condition. In summary, results demonstrate that the BFe@DNA, BFe@DNAH, BFe@DNAG and BFe@DNAH-siRNA nano-aptamers are more stable in neutral condition. Cell imaging and mitochondria targeting. To confirm the nano-aptamers cancer targeting properties and subcellular location, the fluorescence imaging of MCF-7 (breast cancer) and PATU-8988 (pancreas cancer) cells was carried out. The fluorescent signal of MCF-7 cells showed green fluorescence at 500 nm when incubated with BFe@DNAH (CDNA=2 µM). There was a strong fluorescent signal in MCF-7 cells when incubated with BFe@DNAH, proving that the


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probe BFe@DNAH can enter living MCF-7 cancer cells (Figure 2A). Therefore, BFe@DNAH could be potent probe for cellular imaging.

Figure 2A. Images of living MCF-7 cancer cells treated with BFe@DNAH (CDNA, 2 µM). (A) Image of MCF-7 cells. (B) Image of MCF-7 cancer cells treated with BFe@DNAH (CDNA, 2 µM). After incubated with Mito Tracker RED, a mitochondria-targeted dye, the fluorescent signal of MCF-7 cells showed red fluorescence at 579 nm (Figure 2B). It can be seen clearly that the green fluorescence of c1 was obvious when compared with the overlay image c. Due to the high overlap between BFe@DNAH and Mito Tracker RED, BFe@DNAH could be a mitochondria-targeted probe. Through the comparison of c and c1, the green fluorescence of c1 was stronger than that of c, which indicated BFe@DNAH has a stronger targeting effect on mitochondria of MCF-7 cancer cells than BFe@DNA because of the targeting ability of targeting aptamer (HIF). To figure out the effect of aptamer, two kinds of aptamers (HIF and GLUT)

27-29

were assembled into BFe@DNA

resulting BFe@DNAH (or BFe@DNAG). It was found that both the cell absorption and mitochondria-target property of nanoparticles BFe@DNAH (or BFe@DNAG) were improved when compared with BFe@DNA. The green image of BFe@DNAG is brighter than that of BFe@DNAH, indicating more BFe@DNAG could enter into cells, because of the high expression of GLUT (glucose transporter 1) receptors on the surface of MCF-7 cells.30, 31 Also, compared



with subcellular dyes of nucleus and cell membrane, it could be obviously concluded that the BFe@DNAH (or BFe@DNAG) has better mitochondria-target property, indicating that encapsulated BODIPY-OCH3 has some effect on the subcellular cite of the two nanoparticles and both BFe@DNAH and BFe@DNAG could be a mitochondria-targeted dyes in biological imaging. Due to the over-expression of HIF-1 in the pancreatic cancer cell than the breast cancer cell,32, 33 the green image of BFe@DNAH in PATU-8988 cells is brighter than that of BFe@DNAH in MCF-7 cells (Figure 2C). Our results demonstrate that both BFe@DNAH and BFe@DNAG with targeting aptamers (HIF and GLUT) can be targeting aptamers enhanced imaging agent for MCF-7 cells and PATU-8988 cells.

Figure 2B. (A): Visualization of MCF-7 cancer cells that treated with BFe@DNA (CDNA, 2 µM), BFe@DNAH (CDNA, 2 µM) and Mito Tracker RED at 37°C. (a) or (a1): Image of MCF-7 cells when incubated with BFe@DNA or BFe@DNAH, respectively; (b) and (b1): images of MCF-7


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cells when incubated with Mito Tracker RED. (c) or (c1): the overlaid image of (a) and (b) (or (a1) and (b1)). (B): The influence of BFe@DNA, BFe@DNAH and Mito Tracker RED on MCF-7 cancer cells fluorescence area. (C): The influence of BFe@DNA, BFe@DNAH and Mito Tracker RED on MCF-7 cancer cells fluorescence IOD. *, p < 0.05 versus control (Mito Tracker RED).

Figure 2C. Visualization of MCF-7 cells and PATU-8988 cells that incubated with BFe@DNA (CDNA, 2 µM), BFe@DNAH (CDNA, 2 µM), BFe@DNAG (CDNA, 2 µM), Mito Tracker RED and Hoechst 33258/ Dil at 37°C, respectively.

Silencing HIF-1 and ATP by siRNA. Small interfering RNA (siRNA sequence-1. 5’-GAUGAAAGAAUUACCGAAUTT-3’, sequence-2. 5’-AUUCGGUAAUUCUUUCAUCTT-3’) is a double RNA with the function of regulating gene expression of HIF-134, which is a potential candidate for therapy of cancer cells. However, less siRNA can enter cells by itself. So, we expected BFe@DNAH can carry siRNA by the positive and negative charge force to regulate gene expression and energy metabolism of cancer cells and further kill cancer cells with no side effect



to normal tissue. As shown in Figure 3, BFe@DNAH and BFe@DNAH+vector did not interfere with the HIF-1α protein, whereas the BFe@DNAH-siRNA silenced HIF-1α, which indicated that BFe@DNAH can take siRNA into MCF-7 cells. It has been mentioned that HIF-1 regulates the energy metabolism.3, 4 The effect for BFe@DNAH-siRNA on ATP was assayed. As seen in Figure 4, the concentration of ATP in MCF-7 cells incubated with BFe@DNAH-siRNA decreased gradually with time. The relative concentration of ATP was 60.54% and 16.65%, respectively. Results demonstrated that BFe@DNA-siRNA can be both attenuator of ATP and HIF-1α.

Figure 3. Effect of BFe@DNAH, BFe@DNAH+vector and BFe@DNAH-siRNA on the level of HIF-1α in MCF-7 cells treated with BFe@DNAH (CDNA, 1µM) and BFe@DNAH-siRNA (CDNA, 1µM) for 48 h.

Figure 4. Effect of BFe@DNAH-siRNA (C

siRNA,

1 µM) on ATP concentration in MCF-7 cells


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with incubation for 0 h, 2 h, 4 h, 12 h, 24 h, 48 h. The results are represented as mean ± SD (n = 3). *, p < 0.05 versus control; * *, p < 0.01 versus control.

Cytotoxicity. After incubated with BFe@DNAH-siRNA and BFe@DNAH for different time, the morphology of MCF-7 cells was recorded. The morphology of MCF-7 cells extremely shrunk when cells were incubated with BFe@DNAH-siRNA for 24 and 48 h (Figure 5A (E) and Figure 5A (F)), but almost have no change when incubated with BFe@DNAH (Figure 5A (E1) and Figure 5A (F1)), indicating BFe@DNAH-siRNA can inhibit the growth of cancer cells (Figure 5B).

Figure 5A. Effect of BFe@DNAH-siRNA (1 µM) (Fig.7 A to F) and BFe@DNAH (1 µM) (A1 to F1) on the morphology of MCF-7 cells. The incubation time from A to F (or A1 to F1): 0, 2, 4, 12, 24 and 48 h, respectively.

Figure 5B. Cell viability of MCF-7 cancer cells when incubated with BFe@DNAH-siRNA (1 µM)



for 0, 24 and 48 h. *, p < 0.05 versus control; * *, p < 0.01 versus control. In vivo antitumor efficiency. The efficiency of BFe@DNAH-siRNA therapy in vivo was investigated using 4T1 tumors xenograft model in BALB/c mice. After dosing, the tumor volume of all BALB/c mice were measured every 2 days and a volume change chart was plotted. It can be seen that BFe@DNAH-siRNA and 5-Fluorouracil all can inhibit the growth of breast cancer compared with the saline experimental group.35 BFe@DNAH-siRNA (0.1207 mg·mL-1) could inhibit the growth of tumor compared to the control (Figure 6). At the same time high concentration of BFe@DNAH-siRNA (0.2413 mg·mL-1) showed more efficient inhibition indicating that the inhibition is concentration dependent. Results demonstrated the antitumor effect of BFe@DNAH-siRNA. BFe@DNAH-siRNA had a negligible change of body weight, implying that nanoparticles have a good biocompatibility (Figure 6D). By comparing the organ sections of the blank group (saline) and the BFe@DNAH-siRNA (0.2414 mg·mL-1) administration group, the histological examination and images of the major organs (i.e., spleen, thymus, liver) were taken. Results suggested great biosafety of BFe@DNAH-siRNA compared to the control (Figure 6F and Figure S7 A, B, C).36, 37 Therefore, it demonstrated that the HIF-1α siRNA could be used to the attenuator of tumors. BFe@DNAH-siRNA can be the gene therapy with less side effects.


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Figure 6. (A) Gross solid tumor images of mice injected with (a) saline, (b) 5-Fluorouracil, (c) BFe@DNAH-siRNA (0.1207 mg·mL-1), and (d) BFe@DNAH-siRNA (0.2413 mg·mL-1), at 15 d post-treatment, respectively. (B) The weight of thymuses, livers, spleens and tumors of mice. (C) The tumor growth curves with different treatments. *, p < 0.05; * *, p < 0.01. Error bars indicate SD (n = 7). (D) Change of body weight of the tumor-bearing mice. (E) The representative of the tumor sections examined by H&E staining. The scale bars are 100 µm. (F) Histochemical study of the organs harvested from mice injected with BFe@DNAH-siRNA (0.2413 mg·mL-1) and physiological saline only (control group) in 15 d after treatment. The scale bars are 100 µm.



CONCLUSION In summary, we constructed a new kind of fluorescence nano-aptamer (BFe@DNA). BFe@DNA could further assemble with targeting aptamer (such as, HIF and GLUT) resulting in mitochondria targeting fluorescence nano-aptamer BFe@DNAH (or BFe@DNAG). In particular, BFe@DNAH can transfer siRNA into cancer cells silencing the expression of HIF-1α，decreasing the level of ATP and enhancing the anti-tumor activity with less side effect. Silencing HIF-1α is a potential strategy for future cancer therapy. Therefore, BFe@DNAH-siRNA can be used to the HIF-1α based inhibition of hypoxia tumors. Moreover, the BFe@DNA is a smart nano-aptamer platform for the development of cellular fluorescence imaging agents and a carrier of siRNA for the precision diagnosis and therapy of cancer cells. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: A table (Table S1) showing the DNA sequences, figures showing the MS spectrum of DNA1

(Figure S1), the FT-MS spectrum of DNA1-Fe3+ (Figure S2), the MS spectrum of DNA2 (Figure S3), the FT-MS spectrum of DNA2-Fe2+ (Figure S4), the UV-vis spectra of ssDNA1, ssDNA2 and dsDNA-Fe3O4, UV-vis spectra of Fe@DNA, BFe@DNAH, BFe@DNAG, and BFe@DNAH-siRNA in phosphate-buffered solution (PBS) with different pH; figures showing the UV-vis absorption change (A/A0) of BFe@DNA (a), BFe@DNAH (b), BFe@DNAG (c) and BFe@DNAH-siRNA (d) at 555 nm after 48 h at different pH values (Figure S6 (A)) and the UV-vis absorption change (A/A0) of BFe@DNA BFe@DNA (a), BFe@DNAH (b), BFe@DNAG (c) and BFe@DNAH-siRNA


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(d) at 500 nm after 48 h at different pH values (Figure S6 (B)) and the thymuses, livers, and spleens images of mice injected with (1) saline, (2) 5-Fluorouracil, (3) BFe@DNAH-siRNA (0.1207 mg·mL-1), and (4) BFe@DNAH-siRNA (0.2413 mg·mL-1), at 15 d post-treatment, respectively (Figure S7); experimental and methods including characterization, stability assay, cell image, mitochondria image, western blot analysis, ATP measurement, antitumor in vivo and dose calculation.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID 0000000204378663 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by the National Science Foundation of China (21571085, 21271090), the Open Fund of Beijing National Laboratory for Molecular Sciences (BNLMS20150123) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (no. KYCX17_1804). REFERENCES (1) Wilson, W. R.; Hay, M. P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer. 2011, 11, 393-410. DOI: 10.1038/nrc3064



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TABLE OF CONTENTS GRAPHIC

For Table of Contents Use Only. Manuscript Title: “A Smart Magnetic Nano-aptamer: Construction, Subcellular Distribution and Silencing HIF for Cancer Gene Therapy” Authors: Rui Yang, Wei-Yu Mu, Qiu-Yun Chen,* Qiang Wang, Jing Gao


A Smart Magnetic Nanoaptamer: Construction, Subcellular Distribution

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