Selection of Aptamers for Hydrophobic Drug Docetaxel To Improve Its

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Selection of aptamers for hydrophobic drug - docetaxel to improve its solubility Nandi Chen, Ying Zhu, Zhenzhen Feng, Qing Wang, Shiya Qin, Ke Quan, Jin Huang, Jianbo Liu, Xiaohai Yang, and Kemin Wang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00073 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Applied Bio Materials

Selection of aptamers for hydrophobic drug docetaxel to improve its solubility

Nandi Chen, Ying Zhu, Zhenzhen Feng, Qing Wang, Shiya Qin, Ke Quan, Jin Huang, Jianbo Liu, Xiaohai Yang*, and Kemin Wang*.

AUTHOR

ADDRESS:

State

Key

Laboratory

of

Chemo/Biosensing

and

Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China.

ABSTRACT: With the development of combinatorial chemistry and high-throughput screening, the number of hydrophobic drug candidates keeps increasing. However, the low solubility of hydrophobic drugs could induce erratic absorption patterns and affect the drug efficacy. Aptamers are artificially selected highly water-soluble oligonucleotides that bind to ions, small molecules, proteins, living cells, and even tissues. Herein, to increase the solubility of hydrophobic drug, we screened the aptamer by exploiting DNA library immobilization selection strategy and microfluidic 1 / 25

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

technology. The highly water-soluble aptamer might influence the dissolving capacity of its target. In order to demonstrate the concept, docetaxel (DOC), a second-generation taxoid cytotoxic with significant antitumor agent activity, was chosen as the model. It is generally known that the clinical application of docetaxel is limited greatly owing to its poor water solubility and serious side effects. After 7 rounds of selection, two docetaxel-specific aptamers DOC6-5 and DOC7-38, were successfully obtained and their apparent dissociation constant (Kd) were at nanomolar level. Then, these two 100 mer ssDNA aptamers against docetaxel were truncated to 22 mer ones by utilizing the recognition domain. Moreover, the shorter aptamer exhibited higher binding affinity than 100 mer ssDNA aptamers. By adding the optimized aptamer, the solubility of docetaxel was increased from ca. 14µM to ca. 145µM, and the cytotoxicity of docetaxel did not be reduced in the presence of aptamer. Therefore, the aptamer was used as a solubilizer to improve the solubility of hydrophobic drug (docetaxel) in aqueous phase. This strategy may also be extended to other hydrophobic drugs. Meanwhile, this work could also provide a useful tool for tumor targeting therapy by combining with cell target ligands.

KEYWORDS: Aptamer, hydrophobic drug, docetaxel, SELEX, microfluidic.

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INTRODUCTION

More than 30 % drugs are poorly soluble in aqueous (solubility less than 100 µg/ml) among the US Pharmacopeia. The number of hydrophobic drug candidates keeps increasing by the development of combinatorial chemistry and biologically based high-throughput screening.1 It was estimated that approximately 70 % of the new chemical candidates were hydrophobic.2 Moreover, low solubility might induce erratic absorption patterns, which affect the drug efficacy in clinic. Amounts of pharmaceutical adjuvants were used to increase the solubility, such as castor oil, Tween 80, albumin, which may cause hypersensitivity reactions, skin reactions, and hemolysis.3 In general, strategies to achieve a therapeutical drug level in the circulatory system were usually undesirable due to the possibility of increasing toxicity and decreasing patient compliance. Thus, developing active and safe hydrophobic therapeutic delivery systems does make sense.4

Aptamers, sometimes called chemical antibodies, have shown great potential to play roles similar to antibodies in therapeutics, diagnostics, and drug development.5, 6 One of the important advantages is that aptamers are more stable and resistant to harsh environments. Secondly, aptamers are produced chemically, which are not prone to viral or bacterial contamination. Thirdly, aptamers have shown non-immunogenic and their small size allows more efficient entry into biological compartment. Fan’s group has supposed that the biocompatible and highly water-soluble DNA nanostructures may encapsulate or conjugate poor water-soluble

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drugs, which can overcome many disadvantages such as poor water-solubility, instability and low circulation time.4 Recently, Li et. al 7and Tan et. al

8

conjugated

paclitaxel to nucleic acid through covalent bond. The nucleic acid increased the solubility of paclitaxel significantly. In our previous research, we also proved aptamer could increase the solubility of porphyrin photosensitizer.9-11 However, to conjugate paclitaxel with covalent bond and release paclitaxel in tumor cells, the chemical modification of paclitaxel is very complicated. If the nucleic acid aptamers could bind the hydrophobic therapeutics based on molecular recognition and serve as drug delivery systems, the link and release of hydrophobic therapeutics would be much easier. In the last few decades, around 170 small molecules have got their aptamers, and ~30 of the small molecules are therapeutics.6 However, only 7 of these therapeutics are hydrophobic, which include 17β-estradiol,12 digoxin,13 ibuprofen,14 theophylline,15 dopamine,16 codeine17 and cortisol.18 Nevertheless, none of these aptamers has been used to increase the solubility of these hydrophobic therapeutics.

Herein, a time-effective and facile SELEX method for small molecules was developed by exploiting DNA library immobilization selection strategy and microfluidic technology. The highly water-soluble aptamer was employed to increase the solubility of poorly water-soluble drug. In general, this strategy might overcome some limitations on covalent conjugation aptamers and therapeutic drugs directly19: 1) therapeutics may not provide a suitable functional group for conjugation; 2) conjugation may affect the potency of the drug; 3) the release behavior may need optimization. As a proof of concept, a hydrophobic antitumor drug, docetaxel was 4 / 25


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used as the model. Docetaxel is a second-generation taxoid cytotoxic agent, which has been proven to have significant antitumor activity against various human cancers. The clinical application of docetaxel is limited greatly owing to its poor water solubility and serious side effects. The side effects in clinic, such as hypersensitivity reactions, nail toxicity, myalgia, nasolacrimal duct stenosis and asthenia, are attributable to either docetaxel or the pharmaceutical adjuvant Tween 80.20

RESULTS AND DISCUSSION Isolation of DNA aptamers for docetaxel A DNA library immobilization selection strategy was used here. Generally, 16-18 SELEX rounds selection were required to obtain aptamers in DNA library immobilization selection strategy, thus they led to a long cycles and a high cost. It has been reported that Microfluidic-SELEX technology could accelerate aptamer isolation by controlling highly stringent selection conditions through using very small amounts of target molecules.21 Therefore, the DNA library was immobilized in the channel of microfluidic chip (Design and fabrication of experimental device was described in Supporting Information S2.). A structured DNA library was designed according to the previous work,22 where each molecule includes three functional domains. In order to ensure the library variety, the random sequences usually contain 20-60 bases. We use two 18-base random sequence to ensure the variety of library.23,24 As shown in Figure 1, a Biotin-DNA (BDNA) could hybridize the library and was immobilized on the surface of the microfluidic channel. After injecting the target, docetaxel, aptamer molecules which underwent target-binding-induced conformation change were 5 / 25



separated from the BDNA. Afterward, the target-aptamer complex were collected at the end of microfluidic channel and used for next round selection (The detailed aptamer selection procedures were described in Supporting Information S3.).

The enrichment of the initial ssDNA pool with docetaxel-specific aptamers was monitored by fluorescence method (Selection yield determination was described in Supporting Information S4.). The percentage of ssDNA binding to docetaxel was significantly increased with increasing of the SELEX round. In the 6th and 7th round of selection, the selection yield reached the plateau, and a further two rounds of selection cannot increase the selection yield. The decrease of selection yield after the 7th round selection may be resulted by unspecific elution as the previous work mentioned.25 Hence, the eluted ssDNAs from the 6th and 7th round selection were cloned and sequenced. The repeated aptamer candidates was shown in Figure 1. The DNAMAN 6.0 software separated them into two groups based on the homology. DOC6-5 and DOC7-38 were selected on behalf of each group for further study (The secondary structures of aptamers were shown in Supporting Information S5).

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Figure 1. Overview of the selection process. The microfluidic channel was coated with biotin-BSA at first. Then avidin was immobilized in the microfluidic channel by the biotin-avidin interaction. The hybridization of BDNA-library was captured by avidin on the surface of the microfluidic channel. Library was challenged with target (docetaxel), resulting in elution of aptamers from microfluidic channel surface. The target-aptamer complex could be collected at the end of microfluidic channel, which could be used for next round selection or sequencing. Determination and optimization the affinity of aptamers The affinity and specificity of the sequences was characterized by surface plasmon resonance (SPR) (The method was described in Supporting Information S6.) Dissociation constants (Kd) described the affinity of docetaxel/aptamer interaction. To calculate Kd, the change of resonance angle versus docetaxel concentrations was plotted, and the data points were fitted by the non-linear regression analysis.26 As shown in Figure 2A, the Kd values of two full length aptamers, DOC6-5 and DOC7-38, were 267 ± 6.6 and 372 ± 4.4 nM, respectively. 7 / 25



In order to increase the affinity and decrease the cost, aptamers were optimized based on library design.27 The fixed sequence for immobilization was formed as double helix during aptamer selection, which would be hard to interact with docetaxel. And PCR primers are redundant parts of aptamers, which might also influence the binding between docetaxel and its aptamer. Therefore, aptamer DOC6-5 was split into DOC6-63merA and DOC6-63merB, and aptamer DOC7-38 was split into DOC7-63merA and DOC7-63merB, to confirm whether the four tracts with only one recognition domain could bind to the target respectively. The Kd values of four aptamers, DOC6-63merA, DOC6-63merB, DOC7-63merA and DOC7-63merB, were 0.56, 1.78, 0.48 and 2.11 µM, respectively. DOC6-63merA and DOC7-63merA displayed better binding ability to docetaxel than DOC6-63merB and DOC7-63merB. So, we further cut the forward PCR primer sites of DOC6-63merA and DOC7-63merA and got the shorter aptamers, DOC6-44merA and DOC7-44merA. As shown in Figure 2B, the degree change reached a plateau when the concentration of docetaxel exceed 150 nM, and the Kd values of DOC6-44merA and DOC7-44merA were 66, and 74 nM, respectively. Then, to make sure the central domain do not bind docetaxel, DOC6-44merB and DOC7-44merB were designed by swapping the immobilization sequence in central domain and the binding sequence in BDNA. As shown in Figure 2B, DOC6-44merB and DOC7-44merB could also bind to docetaxel respectively. The Kd values were 41 and 39 nM, which were lower than that of the full length docetaxel binding aptamers. The 18 mer immobilization sequence did not take part in binding to docetaxel. There was a same 22 mer sequence DOC6-22mer in 8 / 25


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both DOC6-44merA and DOC6-44merB, and a same 22 mer sequence DOC7-22mer in both DOC7-44merA and DOC7-44merB. They were comparable to or lower than that of many small molecule-binding aptamers.28-33

Figure 2. The affinity determination for optimized aptamers by SPR. (A):100 mer DNA aptamer; (B): 44 mer DNA aptamer. The binding ability of aptamer DOC6-22mer and aptamer DOC7-22mer Therefore, we supposed aptamer DOC6-22mer and aptamer DOC7-22mer could also exhibit high affinity to docetaxel. However, since the immobilization sequence was no longer existed, a solution-based experiment was designed according to a simple well-known unmodified gold nanoparticle-based colorimetric method (Colorimetric assay and specificity investigation was described in details in Supporting Information S7.). The red-coloured AuNPs, stabilized by the adsorbed citrate anion at the surface, were readily aggregated in the presence of 10 mM NaCl. In the absence of docetaxel, the aptamer could protect the AuNPs from aggregation. And such aggregation shifted the SPR absorption of AuNPs to longer wavelength, resulting in the characteristic red–blue colour change. The absorbance ratios at 670/526 nm were plotted against docetaxel concentrations as shown in Figure 3.

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DOC6-22mer evinced stronger affinity to docetaxel compared to DOC7-22mer. Moreover, unlike some organic targets could adsorb directly onto AuNPs34, docetaxel could not lead AuNPs aggregation significantly (as shown in Figure S7). Therefore, aptamer DOC6-22mer was chosen for further studying.

Normalized A670/A526

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DOC 6-22mer

1.0

DOC 7-22mer

0.8 0.6 0.4

Control 22-mer 0.2 0

100

200

300

400

500

Concentration of docetaxel (nM)

Figure 3. Comparison of the binding ability of DOC6-22mer and DOC7-22mer by using the colorimetric assay, which is represented by the absorbance ratio of AuNPs at 670 nm and 526 nm. The concentration of aptamers were 500 nM. The ratio was normalized to a percentage of the maximum signal coming from 500 nM docetaxel with 500 nM DOC6-22mer. The random probe, Control 22mer, was used as control.

The selectivity of aptamer DOC6-22mer was also demonstrated by the AuNPs colorimetric assay (Figure S8 in Supporting Information showed the selectivity of aptamer DOC7-22mer). Figure 4 showed the specific interaction of aptamer DOC6-22mer with antitumor drugs and other small molecules. Especially, although the structure of docetaxel and paclitaxel were similar, aptamer DOC6-22mer only showed high affinity to docetaxel.

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1.0 Normalizated A670/A526

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0.8 0.6 0.4 0.2 0.0 a b c d e f g h i j k l m n o p q r

Figure 4. Specific interaction of DOC6-22mer with different molecules including docetaxel by using the colorimetric assay. The concentration of aptamer was 500 nM. The concentration of the small molecules were 150 nM. The ratio was normalized to a percentage of the maximum signal coming from 150 nM docetaxel. (From a to r were blank, docetaxel, paditaxel, coralyne, doxorubioin, hematoporphyin, penicillin, amoxicillin, ibuprofen, promazine, triflupromazine, glucose, lactosum, D-mannitol, D-sorbitol, ATP, adenosine, and urea, respectively.) The confirmation changes of aptamer DOC6-22mer in the presence of docetaxel To further understand the affinity interaction, circular dichroism (CD) was utilized to monitor the confirmation change of aptamer DOC6-22mer in the presence of docetaxel. The CD spectra consisted of a positive maximum at 280 nm and a negative band at 250 nm. The negative band signal enhanced with the increasing concentration of docetaxel. Meanwhile, it also blue shifted 3 nm as shown in Figure 5. Therefore, the interaction of aptamers with docetaxel may have an effect on the conformation of structure of aptamers.35-37

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2

0

MiliDegree(o)

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-2

-4

Aptamer 10 µM+ Doc 0 µM Aptamer 10 µM+ Doc 1 µM Aptamer 10 µM+ Doc 2 µM Aptamer 10 µM+ Doc 3 µM Aptamer 10 µM+ Doc 4 µM Doc 4 µM

-6

-8 200

220

240

260

280

300

320

340

Wavelength (nm)

Figure 5. The CD spectra of docetaxel and DOC6-22mer in the presence of different concentration of docetaxel. Verification of docetaxel/DOC6-22mer complex by mass spectrometry In order to observe the docetaxel/DOC6-22mer complex directly, electrospray ionization mass spectrometry (ESI-MS) was employed. There were two mean peaks in Figure 6. Based on the sequence of DOC6-22mer and the molecular weight of docetaxel, we could suppose that the 7425.7 peak was docetaxel/DOC6-22mer complex and the 6618.0 peak was DOC6-22mer. Moreover, according to the IUPAC 2007 standard atomic weights, the docetaxel/DOC6-22mer complex is 7426.17 and the DOC6-22mer is 6618.29. The ESI-MS measured data showed less than 0.03% difference when compared with available calculated mass, which is generally acceptable. The results of ESI-MS showed that aptamer DOC6-22mer could bind docetaxel with high affinity.

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7e+6 6e+6

Aptamer 6618.0

5e+6

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4e+6 3e+6

DOC + Aptamer 7425.7

2e+6 1e+6 0

5000 5500 6000 6500 7000 7500 8000 8500

Mass (Da)

Figure 6. The ESI-MS of the docetaxel/DOC6-22mer complex.

The solubility of docetaxel increased by aptamer DOC6-22mer After verified the close combination between aptamer DOC6-22mer and docetaxel, the aptamer was used to increase the solubility of docetaxel. The docetaxel suspension was first prepared by ultrasonic dispersion method, then the suspension was shaken at room temperature overnight with different concentration of aptamer DOC6-22mer. After that, the suspension was centrifuged with a 30K Amicon ultra tube to separate the dissolved and undissolved drug. Finally the undissolved drug was collected by adding 50% ethanol in Amicon ultra tube, and concentrations of docetaxel were measured by HPLC (The detail method was described in Supporting Information S8.). Briefly, by subtracting the undissolved drug from the suspension, the solubility increased by aptamer was calculated. As shown in Figure 7A, after adding 100 mM aptamer, the solubility of docetaxel kept increasing up to 8h. Moreover, the variation decreased with time extending. Therefore, to achieve a better binding effect at different concentration, an overnight incubation is employed. Figure 7B showed that

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the solubility was increased by adding aptamer DOC6-22mer significantly and it reached a plateau when the concentration of aptamer exceed 500 µM. The docetaxel used in the experiment of solubility increase was superstatured suspension, which means most of docetaxel was undissolved. When 100 µM aptamer was added the dissolved docetaxel is around 60 µM (Figure 7A). Unlike docetaxel solution, particles of docetaxel influence the binding of aptamers. Therefore, after the concentration of aptamer increased more than 500 µM, the dissolved docetaxel stopped increasing obviously. Obviously, the solubility of docetaxel was increased by the highly water-soluble docetaxel binding aptamer in the binding buffer without adding pharmaceutical adjuvant such as Tween 80 and ethanol.

Figure 7. The docetaxel solubility improved by aptamer DOC6-22mer in docetaxel suspension.

The

concentration

of

docetaxel

(DOC)

is

docetaxel

and

docetaxel-aptamer complex which could pass through 30K Amicon ultra tube membrane. A: Optimizing of incubation time. The concentration of aptamer is 100 µM. B: The solubility of docetaxel with adding different concentration of docetaxel. Cytotoxicity of docetaxel/DOC 6-22mer complex 14 / 25


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Furthermore, the cytotoxicity of docetaxel in the presence of aptamer DOC6-22mer was evaluated using the MTT assay (Cell culture and viability assay was described in Supporting Information S9.). We compared the cytotoxicity of free docetaxel with that of docetaxel/aptamer complex on the human breast cancer cells (MCF-7 and MDA-MB-231). As shown in Figure 8, both cells were sensitive to docetaxel, and this result is consistent with previous research.38 The results implied that aptamer DOC6-22mer did not reduce the cytotoxicity of docetaxel. Overall, the docetaxel binding aptamer was a potential drug delivery vehicle in simple salt buffer without adding surfactants and/or solvents to increase the solubility of docetaxel.

Figure 8. Cytotoxicity of docetaxel – loaded aptamer DOC6-22mer. Human breast cancer cell: (A) MCF-7; (B) MDA-MB-231.

CONCLUSIONS

In conclusion, by exploiting the DNA library immobilized strategy and microfluidic technology, a unique 22-mer docetaxel - specific aptamer DOC-22mer was achieved and exhibited high binding affinity and selectivity. Moreover, this 15 / 25



aptamer was used as a solubilizer to improve the solubility hydrophobic drug, i.e. docetaxel, in simple salt buffer. The results also showed that aptamer DOC-22mer did not reduce the cytotoxicity of docetaxel. Although the aptamer cannot be used in clinic directly instead of commercial pharmaceutical adjuvants at the present stage, this work might provide a useful tool for tumor targeting therapy by combining with cell target ligands. Overall, to the best of our knowledge, this is the first research about increasing the solubility of hydrophobic drug by aptamer, and this strategy may also extend to other poorly water- soluble drugs.

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ASSOCIATED CONTENT

Supporting information.

This material is available free of charge via the Internet at http://pubs.acs.org.

Materials and reagents; Design and fabrication of experimental device; Selection of aptamers; Selection yield determination; The secondary structures of aptamers; Affinity determination; Colorimetric assay and specificity investigation; HPLC assay for docetaxel; Cell culture and viability assay.

AUTHOR INFORMATION Corresponding Author *Email: [email protected];

*Email: [email protected]

ORCID Nandi Chen: 0000-0003-0849-014X Jin Huang: 0000-0002-2890-682X Xiaohai Yang: 0000-0001-8122-7140 Kemin Wang: 0000-0001-9390-4938 Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT

This work was supported in part by the National Natural Science Foundation of China (21675047, 21735002, 21521063), the Key Point Research and Invention Program of Hunan Province (2017DK2011) and China Scholarship Council (201606130037).

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REFERENCES

1.

Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N. Strategies to Address Low Drug Solubility in Discovery and Development.

Pharmacological reviews 2013, 1, 315-499. 2.

Lu, Y.; Park, K. Polymeric Micelles and Alternative Nanonized Delivery Vehicles for Poorly Soluble Drugs. Int. J. Pharm. 2013, 453, 198-214.

3.

Shapiro, C. L.; Recht., A. Side Effects of Adjuvant Treatment of Breast Cancer.

New Engl. J. Med. 2001, 26, 1997-2008. 4.

Li, J.; Fan, C.; Pei, H.; Shi, J.; Huang, Q. Smart Drug Delivery Nanocarriers with Self-Assembled DNA Nanostructures. Adv. Mater. 2013, 25, 4386-4396.

5.

Zhou, J.; Rossi, J. Aptamers as Targeted Therapeutics: Current Potential and Challenges. Nat. Rev. Drug Discov. 2016, 16, 181-202.

6.

Dunn, M. R.; Jimenez, R. M.; Chaput, J. C. Analysis of Aptamer Discovery and Technology. Nat. Rev. Chem. 2017, 1, 76.

7.

Li, F.; Lu, J.; Liu, J.; Liang, C.; Wang, M.; Wang, L.; Li, D.; Yao, H.; Zhang, Q.; Wen, J.; Zhang, Z.; Li, J.; Lv, Q.; He, X.; Guo, B.; Guan, D.; Yu, Y.; Dang, L.; Wu, X.; Li, Y.; Chen, G.; Jiang, F.; Sun, S.; Zhang, B.; Lu, A.; Zhang, G. A Water-Soluble Nucleolin Aptamer-Paclitaxel Conjugate for Tumor-Specific Targeting in Ovarian Cancer. Nat. Commun. 2017, 8, 1390.

8

Tan, X.; Lu, X.; Jia, F.; Liu, X.; Sun, Y.; Logan, J. K.; Zhang, K. Blurring the Role of Oligonucleotides: Spherical Nucleic Acids as a Drug Delivery Vehicle. J. 19 / 25



Page 20 of 25

Am. Chem. Soc. 2016, 138, 10834-10837. 9

Yang, X.; Huang, J.; Wang, K.; Li, W.; Cui, L.; Li, X. Angiogenin-Mediated Photosensitizer-Aptamer Conjugate for Photodynamic Therapy. ChemMedChem 2011, 6, 1778-1780.

10

Chen, N.; Yang, X.; Wang, Q.; Jian, L.; Shi, H.; Qin, S.; Wang, K.; Huang, J.; Liu, W. Proof of Concept for Inhibiting Metastasis: Circulating Tumor Cell-Triggered Localized Release of Anticancer Agent via a Structure-Switching Aptamer.

11

Chem. Commun. 2016, 52, 6789-6792.

Chen, N.; Qin, S.; Yang, X.; Wang, Q.; Huang, J.; Wang, K. "Sense-and-Treat" DNA Nanodevice for Synergetic Destruction of Circulating Tumor Cells. ACS

Appl. Mater. Interfaces 2016, 8, 26552-26558. 12

Hea, Y. L.; Man, B. G.; Yeon, S. K.; Ho, S. J.; Matsuura, T.; Kawai, T. Electrochemical

Detection

of

17Beta-Estradiol

Using

DNA

Aptamer

Immobilized Gold Electrode Chip. Biosens & Bioelectron 2007, 22, 2525-2531. 13 Kiani, Z.; Shafiei, M.; Rahimi-Moghaddam, P.; Karkhane, A. A.; Ebrahimi, S. A. In vitro Selection and Characterization of Deoxyribonucleic Acid Aptamers for Digoxin. Anal. Chim. Acta 2012, 748, 67-72. 14

Kim, Y. S.; Hyun, C. J.; Kim, I. A.; Gu, M. B. Isolation and Characterization of Enantioselective DNA Aptamers for Ibuprofen. Bioorg. Med. Chem. 2010, 18, 3467-3473.

15

Jenison, R.; Gill, S.; Pardi, A.; Polisky, B. High-Resolution Molecular Discrimination by RNA. Science 1994, 263, 1425-1429. 20 / 25


Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16

Mannironi, C.; Di Nardo, A.; Fruscoloni, P.; Tocchini-Valentini, G. P. In vitro Selection of Dopamine RNA Ligands. Biochemistry 1997, 36, 9726-9734.

17

Huang, L.; Yang, X.; Qi, C.; Niu, X.; Zhao, C.; Zhao, X.; Shangguan, D.; Yang, Y. A Label-Free Electrochemical Biosensor Based On a DNA Aptamer against Codeine. Anal. Chim. Acta 2013, 787, 203-210.

18

Martin, J. A.; Chavez, J. L.; Chushak, Y.; Chapleau, R. R.; Hagen, J.; Kelley-Loughnane, N. Tunable Stringency Aptamer Selection and Gold Nanoparticle Assay for Detection of Cortisol. Anal. Bioanal. Chem. 2014, 406, 4637-4647.

19

Lao, Y.; Phua, K. K. L.; Leong, K. W. Aptamer Nanomedicine for Cancer Therapeutics: Barriers and Potential for Translation. ACS Nano 2015, 9, 2235-2254.

20

Eckhoff, L.; Nielsen, M.; Moeller, S.; Knoop, A. TAXTOX – a Retrospective Study Regarding the Side Effects of Docetaxel Given as Part of the Adjuvant Treatment to Patients with Primary Breast Cancer in Denmark From 2007 to 2009

21

Acta Oncol. 2011, 50, 1075-1082.

Oh, S. S.; Ahmad, K. M.; Cho, M.; Kim, S.; Xiao, Y.; Soh, H. T. Improving Aptamer Selection Efficiency through Volume Dilution, Magnetic Concentration, and Continuous Washing in Microfluidic Channels. Anal. Chem. 2011, 83, 6883-6889.

22

Oh, S. S.; Plakos, K.; Lou, X.; Xiao, Y.; Soh, H. T. In vitro Selection of Structure-Switching, Self-Reporting Aptamers. Proc. Natl. Acad. Sci. U.S.A. 21 / 25



Page 22 of 25

2010, 107, 14053-14058. 23

Tan, W.; Donovan, M. J.; Jiang, J. Aptamers from Cell-Based Selection for Bioanalytical Applications. Chem. Rev. 2013, 113, 2842-2862.

24

Wang, Q.; Liu, W.; Xing, Y.; Yang, X.; Wang, K.; Jiang, R.; Wang, P.; Zhao, Q. Screening of DNA Aptamers Against Myoglobin Using a Positive and Negative Selection Units Integrated Microfluidic Chip and its Biosensing Application.

Anal. Chem. 2014, 86, 6572-6579. 25

He, J.; Liu, Y.; Fan, M.; Liu, X. Isolation and Identification of the DNA Aptamer Target to Acetamiprid. J. Agric. Food. Chem. 2011, 59, 1582-1586.

26

Niazi, J. H.; Lee, S. J.; Gu, M. B. Single-Stranded DNA Aptamers Specific for Antibiotics Tetracyclines. Bioorg. Med. Chem.2008, 16, 7245-7253.

27

Shangguan, D.; Tang, Z.; Mallikaratchy, P.; Xiao, Z.; Tan, W. Optimization and Modifications

of

Aptamers

Selected

from

Live

Cancer

Cell

Lines.

ChemBioChem 2007, 8, 603-606. 28

Stojanovic, M. N.; de Prada, P.; Landry, D. W. Aptamer-Based Folding Fluorescent Sensor for Cocaine. J. Am. Chem. Soc. 2001, 123, 4928-4931.

29

Yang, J.; Bowser, M. T. Capillary Electrophoresis–SELEX Selection of Catalytic DNA Aptamers for a Small-Molecule Porphyrin Target. Anal. Chem. 2013, 85, 1525-1530.

30

Elshafey, R.; Siaj, M.; Zourob, M. In vitro Selection, Characterization, and Biosensing

Application

of

High-Affinity

Cylindrospermopsin-Targeting

Aptamers. Anal. Chem. 2014, 86, 9196-9203. 22 / 25


Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


31

Eissa, S.; Zourob, M. Selection and Characterization of DNA Aptamers for Electrochemical Biosensing of Carbendazim. Anal. Chem. 2017, 89, 3138-3145.

32

Parashar, A.; Rajput, Y. S.; Sharma, R. Aptamer-Based Sensing of β-Casomorphin-7. J. Agric. Food. Chem. 2015, 63, 2647-2653.

33

Duan, N.; Gong, W.; Wu, S.; Wang, Z. Selection and Application of ssDNA Aptamers Against Clenbuterol Hydrochloride Based on ssDNA Library Immobilized SELEX. J. Agric. Food. Chem. 2017, 65, 1771-1777.

34

Wang, R.; Zhou, X.; Liedberg, B.; Zhu, X.; Memon, A.; Shi, H. Screening Criteria

for

Qualified

Antibiotic

Targets

in

Unmodified

Gold

Nanoparticles-Based Aptasensing. ACS Appl. Mater. Interfaces 2017, 9, 35492-35297. 35

Xing, Y.; Liu, C.; Zhou, X.; Shi, H. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay.

Scientific Reports 2015, 5, 8125. 36

Zhang, L.; Xing, Y.; Liu, C.; Zhou, X.; Shi, H. Label-free colorimetric detection of Cu2+ on the basis of Fenton reaction-assisted signal amplification with unmodified gold nanoparticles as indicator. Sensor Actuat B-Chem. 2015, 215, 561-567.

37

Zhang, L.; Xing, Y.; Liu, L.; Zhou, X.; Shi, H. Fenton reaction-triggered colorimetric detection of phenols in water samples using unmodified gold nanoparticles. Sensor Actuat B-Chem. 2016, 225, 593-599.

38

Hernandez-Vargas, H.; Palacios, J.; Moreno-Bueno, G. Molecular Profiling of 23 / 25



Docetaxel Cytotoxicity in Breast Cancer Cells: Uncoupling of Aberrant Mitosis and Apoptosis. Oncogene 2007, 26, 2902-2913.

24 / 25


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Selection of Aptamers for Hydrophobic Drug Docetaxel To Improve Its

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