A Novel AS1411 Aptamer-Based Three-Way Junction Pocket DNA

Apr 18, 2018 - Active targeting of nanostructures containing chemotherapeutic agents can improve cancer treatment. Here, a three-way junction pocket D...
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Article Cite This: Mol. Pharmaceutics 2018, 15, 1972−1978

A Novel AS1411 Aptamer-Based Three-Way Junction Pocket DNA Nanostructure Loaded with Doxorubicin for Targeting Cancer Cells in Vitro and in Vivo Seyed Mohammad Taghdisi,†,‡ Noor Mohammad Danesh,§ Mohammad Ramezani,∥ Rezvan Yazdian-Robati,‡ and Khalil Abnous*,∥,⊥

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†

Targeted Drug Delivery Research Center and ∥Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad 91778-99191, Iran ‡ Department of Pharmaceutical Biotechnology and ⊥Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad 91778-99191, Iran § Research Institute of Sciences and New Technology, Mashhad 91778-99191, Iran ABSTRACT: Active targeting of nanostructures containing chemotherapeutic agents can improve cancer treatment. Here, a three-way junction pocket DNA nanostructure was developed for efficient doxorubicin (Dox) delivery into cancer cells. The three-way junction pocket DNA nanostructure is composed of three strands of AS1411 aptamer as both a therapeutic aptamer and nucleolin target, the potential biomarker of prostate (PC-3 cells) and breast (4T1 cells) cancers. The properties of the Dox-loaded three-way junction pocket DNA nanostructure were characterized and verified to have several advantages, including high serum stability and a pH-responsive property. Cellular uptake studies showed that the Dox-loaded DNA nanostructure was preferably internalized into target cancer cells (PC-3 and 4T1 cells). MTT cell viability assay demonstrated that the Dox-loaded DNA nanostructure had significantly higher cytotoxicity for PC-3 and 4T1 cells compared to that of nontarget cells (CHO cells, Chinese hamster ovary cell). The in vivo antitumor effect showed that the Dox-loaded DNA nanostructure was more effective in prohibition of the tumor growth compared to free Dox. These findings showed that the Dox-loaded three-way junction pocket DNA nanostructure could significantly reduce the cytotoxic effects of Dox against nontarget cells. KEYWORDS: targeted delivery, doxorubicin, three-way junction pocket DNA nanostructure, tumor growth, internalization, cytotoxicity

1. INTRODUCTION Prostate and breast cancers are the most frequent male and female malignancies, respectively.1,2 They are categorized among the main leading causes of cancer death in men and women worldwide, respectively.3,4 Chemotherapy is one of the most common types of cancer treatment. Its incapability to specifically target cancer tissues causes severe side effects of chemotherapy and limits its efficacy.5,6 Targeted drug delivery systems using nanoparticles and sensing agents can overcome this drawback.7,8 Doxorubicin (Dox), an antineoplastic drug, has been broadly applied in clinical chemotherapy of different cancers, including breast and prostate cancers. However, its short half-life and cardiotoxicity have restricted application of Dox in the clinic.2,9 Aptamers have emerged as attractive targeting agents in the development of targeted drug delivery systems for diagnostic and therapeutic applications. They are single-stranded oligonucleotide segments that are isolated by SELEX (systematic evolution of ligands by exponential enrichment).10,11 Aptamers can tightly bind to various targets, such as small molecules, peptides, and even intact cells.12,13 Aptamers have diverse advantages over antibodies, including facile chemical © 2018 American Chemical Society

modification, cost-effective synthesis, thermal stability, and lack of toxicity and immunogenicity.14−16 Also compared to antibodies, aptamers are physically smaller, leading to more and faster internalization of aptamers into cancer tissues.17,18 The AS1411 aptamer is a guanosine-rich oligonucleotide that binds to nucleolin.19,20 Nucleolin is a multifunctional protein that is abundant in the nucleus of normal cells. Also, this protein is overexpressed in the cell membrane of tumor cells, such as prostate, lung, and breast cancers.17,21 Nucleolin is implicated in transporting molecules from the cell surface to the nucleus, cell proliferation, and DNA replication.22,23 The AS1411 aptamer has shown antiproliferative activity and cytotoxic effects against cancer cells, owing to methuosis, a novel type of nonapoptotic cell death.24,25 In the present study, a three-way junction pocket DNA nanostructure was developed for targeted delivery of Dox to tumor cells in vitro and in vivo (Figure 1). The three-way Received: Revised: Accepted: Published: 1972

February 5, 2018 March 18, 2018 April 18, 2018 April 18, 2018 DOI: 10.1021/acs.molpharmaceut.8b00124 Mol. Pharmaceutics 2018, 15, 1972−1978

Article

Molecular Pharmaceutics

for 1 h at room temperature. For the in vivo assay after the reaction, the resultant DNA nanostructure was centrifuged and concentrated using a 10K centrifugal device. The prepared DNA nanostructure was analyzed with agarose gel electrophoresis (2.5%, running time 25 min) and dynamic light scattering (DLS) (Malvern, U.K.). The control complex was prepared like the three-way junction pocket DNA nanostructure, but the sequence of AS1411 was replaced with a control sequence. 2.4. Serum Stability of the DNA Nanostructure. The stability of three-way junction pocket DNA nanostructure in serum was investigated by agarose gel electrophoresis (2.5%). The DNA nanostructure (final concentration of 15 μM) was added to human serum for 6 h. Thereafter, phenol−chloroform was utilized to extract DNA nanostructures, and then, this DNA nanostructure was analyzed with 2.5% agarose gel electrophoresis for 50 min. 2.5. Preparation of Dox-Load DNA Nanostructure. The Dox-load DNA nanostructure was prepared through incubation of different concentrations of the DNA nanostructure (0−4 μM) with a fixed concentration of Dox (5 μM), and fluorescence spectra of Dox were recorded (λEx= 480 nm) by a Synergy H4 microplate reader (BioTeK, U.S.A.). 2.6. Dox Release Study. An in vitro Dox release study was performed using centrifugal devices. The Dox-loaded DNA nanostructure was added to citrate−phosphate buffer (pH 7.4 or 5.5). At each predetermined time interval, the Dox-loaded DNA nanostructure was separated from the buffer using the 10K centrifugal device. The fluorescence intensity of the solution at the bottom of the centrifugal device was analyzed (λEx= 480 nm and λEm= 600 nm) to specify the concentration of dissociated Dox. 2.7. Evaluation of Cellular Uptake. 4T1, PC-3, and CHO cells were seeded into 12-well plates at a density of 2 × 105 cells/well. Afterward, the cells were incubated with 5 μM Dox and the Dox-loaded three-way junction pocket DNA nanostructure (5 μM Dox). After 3 h, the cells were washed and trypsinized for 7 min and suspended in phosphate buffer saline (PBS). Then, the mean fluorescence intensity of Dox in the cells was measured by flow cytometry (BD Accuri C6 flow cytometer, BD Biosciences, U.S.A.). The data were investigated by FlowJo 7.6.1 software. Also, fluorescence microscopy was applied to study the cancer cell uptake of the Dox-loaded DNA nanostructure. The PC-3 and CHO cells (1 × 105 cells) were incubated in RPMI medium in 6-well plates with collagen coated coverslips (0.1% collagen in acetic acid). After 20 h of incubation, the culture medium was replaced with fresh RPMI. The cells were treated with the Dox-loaded three-way junction pocket DNA nanostructure (5 μM Dox) for 3 h at 37 °C, followed by washing the cells with PBS three times. Next, 4%

Figure 1. Schematic description of the Dox-loaded three-way junction pocket DNA nanostructure. The DNA nanostructure was composed of three strands of AS1411 aptamer modified with PEG.

junction pocket DNA nanostructure was composed of three strands of AS1411 aptamer. Dox as an antineoplastic agent was loaded into the DNA nanostructure. Compared to previously developed DNA-nanostructure-based targeted delivery systems,14,26,27 this delivery system is very simple, and its preparation is fast. In vitro and in vivo studies were investigated in nucleolin-overexpressing PC-3 and 4T1 tumor cells, and CHO cells were applied as nontarget cells.

2. EXPERIMENTAL SECTION 2.1. Chemicals. All of the PEGylated oligonucleotides for the preparation of the three-way junction pocket DNA nanostructure were supplied from Bioneer (South Korea) (Table 1). The 10K centrifugal device was provided by PALL (U.S.A.). Doxorubicin was purchased from Sigma-Aldrich (U.S.A.). 2.2. Cell Culture. Human prostate cancer cell (PC-3, C427), mouse breast cancer cell (4T1, C604), and Chinese hamster ovary cell (CHO, C111) were obtained from the Pasteur Institute of Iran. Cells were grown and cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma-Aldrich). 2.3. Preparation of the Three-Way Junction Pocket DNA Nanostructure and Control Complex. The three-way junction pocket DNA nanostructure was prepared in two steps. First, 100 μL of Apt1 (100 μM) was incubated with 100 μL of Apt2 (100 μM) for 1 h at room temperature. Second, 100 μL of Apt3 (100 μM) was added to the above solution and incubated Table 1. Oligonucleotide Sequences Used in This Studya

a

oligonucleotide

sequence (from 5′ to 3′)

aptamer1 (Apt1) aptamer2 (Apt2) aptamer3 (Apt3) control sequence 1 control sequence 2 control sequence 3

5-TATGGTGAAGGGAAAGGTGGTGGTGGTTGTGGTGGTGGTGGAAACACCAAACCCAA-PEG2000−3 5-TTGGGTTTGGTGAAAGGTGGTGGTGGTTGTGGTGGTGGTGGAAACCTCCTTTCCTT-PEG2000−3 5-AAGGAAAGGAGGAAAGGTGGTGGTGGTTGTGGTGGTGGTGGAAACCCTTCACCATA-PEG2000−3 5-TATGGTGAAGGGAAACTTCCTGGTGGATGTCCTAGTGGTTCAAACACCAAACCCAA-PEG2000−3 5-TTGGGTTTGGTGAAACTTCCTGGTGGATGTCCTAGTGGTTCAAACCTCCTTTCCTT-PEG2000−3 5-AAGGAAAGGAGGAAACTTCCTGGTGGATGTCCTAGTGGTTCAAACCCTTCACCATA-PEG2000−3

The underlined sequences are AS1411 aptamers. 1973

DOI: 10.1021/acs.molpharmaceut.8b00124 Mol. Pharmaceutics 2018, 15, 1972−1978

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Molecular Pharmaceutics paraformaldehyde was applied to fix the cells for 15 min. After that, the cells were imaged using a fluorescence microscope (CETI, U.K.). 2.8. In Vitro Cytotoxicity. An escalating-dose study of Dox indicated that the IC50 values of Dox for 4T1, CHO ,and PC-3 cells were 2.2, 3, and 1.8 μM, respectively. The in vitro cytotoxicity was investigated by measuring the activity of the mitochondrial dehydrogenase enzyme using an MTT assay. The CHO, PC-3, and 4T1 cells were seeded in 96-well plates (5 × 103 cells/well). After incubation for 20 h, the cells were incubated with Dox (based on the results of escalating-dose study), the three-way junction pocket DNA nanostructure, the Dox-loaded control complex, and the Dox-loaded three-way junction pocket DNA nanostructure (with the same concentration of Dox) for 3 h at 37 °C. Thereafter, the culture medium was replaced with fresh RPMI, and the cells were incubated for 72 h at 37 °C. Then, 10 μL of MTT solution (5 mg/mL) was added to each well for 4 h. After that, the solution was removed, and subsequently, 100 μL of DMSO was added to each well. The plates were shaken for 5 min, and the absorbance was measured (545 nm) with the microplate reader. 2.9. Animal Models. The animal experiment was performed in compliance with the guidelines of the Institutional Ethics and Research Advisory committees of Mashhad University of Medical Sciences. BALB/c mice, 4−6 weeks old, were obtained from the Pasteur Institute of Iran. For the tumor models, subcutaneous tumors were established by injection of 3 × 105 4T1 cells into the right flanks of mice. The size of the tumor was measured using calipers. When the tumor volume reached nearly 40 mm3, the mice were randomly divided into three groups: Dox (equal to 1.2 mg/kg Dox per mouse), the Dox-loaded DNA nanostructure (equal to 1.2 mg/ kg Dox per mouse), the DNA nanostructure and PBS, all injected via the tail vein. The tumor growth was monitored for 20 days. 2.10. Statistics. For all experiments, the data are shown as mean ± SD, n = 4, which indicated the number of repeats. Student’s t test was used for analysis of data. P < 0.05 was considered statistically significant.

3. RESULTS 3.1. DNA Nanostructure Characterization. Agarose gel electrophoresis was applied to verify the formation of the DNA nanostructure. As shown in Figure 2a, when Apt2 was incubated with Apt1, the mobility of the band of Apt1 was retarded, and only one band appeared (lane 2), confirming the hybridization of them to each other. Also, a new band with slower mobility appeared (lane 3) when Apt3 was added to the mixture of Apt1 and Apt2, indicating the successful formation of the DNA nanostructure. Stability analysis exhibited that the band of the DNA nanostructure was sharp after 6 h of treatment with serum (Figure 2b), verifying the stability of the DNA nanostructure in serum. The particle size for DNA nanostructure as obtained by DLS was 10.3 ± 1.3 nm. 3.2. Dox Loading in the DNA Nanostructure. The formation of the Dox-loaded DNA nanostructure was investigated by fluorometric analysis. The fluorescence intensity of Dox is quenched following its intercalation with DNA.28,29 Figure 3 displays the quenching profile of Dox fluorescence spectra following the addition of different concentrations of the DNA nanostructure (0−4 μM) to a fixed concentration of Dox (5 μM). Results confirmed that the maximum quenching of

Figure 2. (a) Agarose gel electrophoresis of the DNA nanostructure to confirm the formation of the DNA nanostructure. Lane 1: Apt1, lane 2: Apt1 + Apt2, lane 3: Apt1 + Apt2 + Apt3 (DNA nanostructure). (b) Serum stability of DNA nanostructure. Lane 1: DNA nanostructure and lane 2: DNA nanostructure after incubation with serum for 6 h.

Dox happened at a 1:2.5 mol ratio of the DNA nanostructure to Dox, and this ratio was used for the next experiments. 3.3. pH-Triggered Dox Release from the Dox-Loaded DNA nanostructure. The in vitro release profile of Dox from the Dox-loaded DNA nanostructure was evaluated at different pHs. At pH 7.4, the release of Dox was less than 32% after 72 h, while pH 5.5 (simulating the pH of lysosome and cancer cell environment) significantly increased Dox release over 70% (Figure 4). 3.4. Internalization Assay. The fluorescence FL2 histograms of CHO, 4T1, and PC-3 cells after treatments with 5 μM Dox and the Dox-loaded DNA nanostructure (5 μM Dox) have been shown in Figure 5. The FL2 log intensities for 4T1 cells after treatments with Dox and the Dox-loaded DNA nanostructure were 999 ± 105 and 934 ± 79, respectively. The FL2 log intensities for PC-3 cells after treatments with Dox and the Dox-loaded DNA nanostructure were 1184 ± 96 and 1974

DOI: 10.1021/acs.molpharmaceut.8b00124 Mol. Pharmaceutics 2018, 15, 1972−1978

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Molecular Pharmaceutics

Figure 3. Fluorescence spectra of Dox (5 μM) after treatment with increasing concentrations of the DNA nanostructure (from top to bottom 0, 0.3, 0.5, 1, 2, 4 μM). Results indicated that the maximum quenching of Dox happened at a 1:2.5 mol ratio of the DNA nanostructure to Dox.

Figure 4. Profile of Dox release from the Dox-loaded DNA nanostructure in citrate−phosphate buffer at 37 °C at pH 7.4 (bottom) and at pH 5.5 (top). The Dox-loaded DNA nanostructure showed a larger and much faster Dox release at pH 5.5.

1112 ± 121, respectively. The FL2 log intensities for CHO cells after treatments with Dox and the Dox-loaded DNA nanostructure were 739 ± 34 and 351 ± 42, respectively. The fluorescence microscopy images of PC-3 and CHO cells treated with the Dox-loaded DNA nanostructure have been shown in Figure 6. As shown in Figure 6, slight fluorescence was found in the CHO cells (nontarget) treated with the Doxloaded DNA nanostructure, while PC-3 cells (target cells) treated with the Dox-loaded DNA nanostructure exhibited strong fluorescence. 3.5. In Vitro Antitumor Effect. To assess the antitumor effect in vitro, the cytotoxicity of the Dox-loaded DNA nanostructure was examined on CHO, 4T1, and PC-3 cells (Figure 7). The 4T1 cell viabilities after treatments with Dox, the DNA nanostructure, the Dox-loaded control complex, and the Dox-loaded DNA nanostructure were 47.4 ± 1.8, 80.3 ± 1.6, 82.9 ± 6, and 25.6 ± 2%, respectively. The CHO cell viabilities after treatments with Dox, the DNA nanostructure, the Dox-loaded control complex, and the Dox-loaded DNA nanostructure were 51 ± 4.2, 95.6 ± 7.9, 84.8 ± 4.2, and 79.6 ± 3.8%, respectively. The PC-3 cell viabilities after treatments with Dox, the DNA nanostructure, the Dox-loaded control

Figure 5. (a) Flow cytometry histogram of 4T1 cells after incubation with the Dox-loaded DNA nanostructure (blue), Dox (red), and nontreated cells (green). (b) Flow cytometry histogram of PC-3 cells after incubation with the Dox-loaded DNA nanostructure (blue), Dox (red), and nontreated cells (green). (c) Flow cytometry histogram of CHO cells after incubation with the Dox-loaded DNA nanostructure (blue), Dox (red), and nontreated cells (green).

complex, and the Dox-loaded DNA nanostructure were 46.8 ± 2.7, 85.7 ± 9, 80.1 ± 3.5, and 33.2 ± 1.5%, respectively. 3.6. In Vivo Antitumor Effect. The in vivo therapeutic efficacy of the Dox-loaded DNA nanostructure in mice bearing subcutaneous 4T1 tumors was investigated. The tumor volumes for Dox, PBS, the DNA nanostructure, and the Doxloaded DNA nanostructure treatments were 845 ± 50, 1049 ± 120, 907 ± 103, and 424 ± 39 mm3, respectively (Figure 8). 1975

DOI: 10.1021/acs.molpharmaceut.8b00124 Mol. Pharmaceutics 2018, 15, 1972−1978

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Molecular Pharmaceutics

Figure 6. (a) Fluorescent image of CHO cells after treatment with the Dox-loaded DNA nanostructure and the image merged to show noninternalization of the DNA nanostructure. (b) Fluorescent image of PC-3 cells after treatment with the Dox-loaded DNA nanostructure and the image merged to show internalization of the DNA nanostructure.

Figure 7. Effects of the Dox-loaded DNA nanostructure, Dox, and the DNA nanostructure on nontarget (CHO) and target (PC-3 and 4T1) cell viability. Cells were treated with the Dox-loaded DNA nanostructure, Dox, Dox-loaded control complex, and the DNA nanostructure for 3 h. Then after 72 h, the viability of the cells was analyzed using an MTT assay.

4. DISCUSSION Presence of the AS1411 aptamer, as a therapeutic aptamer and targeting agent, in the building blocks of the DNA nanostructure, appropriate Dox loading, excellent serum stability, and simple and rapid preparation of the developed Dox-loaded three-way junction pocket DNA nanostructure are some of the advantages of the designed targeting platform. It has been proven that the modification of aptamers, especially at their 3′-ends, can increase the stability of aptamers to endogenous serum nucleases.30 So, the 3′-end of all three strands of the AS1411 aptamer were capped using PEG as a nontoxic and nonimmunogenic polymer. Also, PEG could enhance the size of the DNA nanostructure, leading to an increase in the half-life of DNA nanostructure by preventing exclusion from renal filtration. Moreover, the PEGylation could

improve the function of the designed delivery system by a decrease of its degradation by metabolic enzymes, escape of the PEGylated DNA nanostructure from the reticuloendothelial system, and reduction of adsorption of other biomaterials on the surface of the DNA nanostructure.31−33 Dox is loaded in the DNA nanostructure via its intercalation with the AS1411 aptamer and dsDNA areas in the DNA nanostructure. The interaction of Dox with the DNA nanostructure was investigated by recording the quenching profile of Dox fluorescence following incubation with the DNA nanostructure. In the presence of the 2 μM DNA nanostructure, the fluorescence intensity of Dox (5 μM) was about 15% (Figure 3), verifying the formation of the Doxloaded three-way junction pocket DNA nanostructure. The drug loading of the designed targeted delivery platform (1:2.5 1976

DOI: 10.1021/acs.molpharmaceut.8b00124 Mol. Pharmaceutics 2018, 15, 1972−1978

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Molecular Pharmaceutics

Cytotoxicity was analyzed using cell viability test and cytotoxicity of the Dox-loaded DNA nanostructure was compared with free Dox in different cell lines. As shown in Figure 7, CHO cells treated with the Dox-loaded DNA nanostructure displayed significantly higher cell viability compared to the CHO cells treated with free Dox (p < 0.05), indicating the reduced toxicity of the Dox-loaded DNA nanostructure for cells that do not overexpress nucleolin. Also, PC-3 and 4T1 cells treated with the Dox-loaded DNA nanostructure showed significantly lower cell viability in comparison with CHO cells treated with the Dox-loaded DNA nanostructure (p < 0.05), verifying the importance of the internalization of the Dox-loaded DNA nanostructure into cells via the AS1411 aptamer−nucleolin mechanism. The antitumor efficacy of the Dox-loaded DNA nanostructure was examined in 4T1 tumor-bearing mice. As indicated in Figure 8, the tumor growth trend of the Dox-loaded DNA nanostructure treatment was significantly slower than that of free Dox group, confirming the higher internalization of the Dox-loaded DNA nanostructure into tumor cells compared to free Dox and the good function of the developed targeted delivery system.

Figure 8. In vivo antitumor efficacy of the Dox-loaded DNA nanostructure against mice bearing 4T1 cells after intravenous administration of PBS, Dox, the DNA nanostructure, and the Doxloaded DNA nanostructure via the tail vein for 20 days. The tumor growth trend of the Dox-loaded DNA nanostructure treatment was significantly slower than other treatments.

5. CONCLUSION In summary, a novel Dox-loaded three-way junction pocket DNA nanostructure containing three strands of AS1411 aptamer was developed for treatment of 4T1 and PC-3 cells (target cells). The Dox-loaded DNA nanostructure is a simple, cancer targeting, pH-responsive platform with high serum stability. The presented Dox-loaded DNA nanostructure was efficiently internalized into target cells and reduced tumor cell viability, while it was not internalized into CHO cells (nontarget). Furthermore, the tumor growth was remarkably prohibited when the tumor−allograft mice were treated with the Dox-loaded DNA nanostructure.

mol ratio of the DNA nanostructure to Dox) was much more than the payload that was already reported for anthracycline chemotherapy agents using aptamers and DNA nanostructures for delivery, like the sgc8 aptamer−daunorubicin delivery system (1.3:1 mol ratio of aptamer to daunorubicin),34 the polyvalent aptamer−epirubicin delivery platform (1:2 mol ratio of polyvalent aptamer to epirubicin),27 and the PSMA aptamer−Dox conjugate (1.2:1 mol ratio of aptamer to Dox).35 As shown in Figure 4, when the Dox-loaded DNA nanostructure was incubated at pH 5.5, a much faster and larger Dox release was achieved through the protonation of the −NH2 group of Dox under acidic conditions, which reduced the interaction between Dox and the DNA nanostructure. So, the presented targeted delivery system could release Dox under the mild acidic microenvironment of cancer cells as a pHresponsive targeting platform. In tumor cells, the release of Dox from the Dox-loaded three-way junction pocket DNA nanostructure should be much more than this amount due to the presence of nuclease enzymes in tumor cells, which could facilitate the release of Dox from the DNA nanostructure by destruction of the DNA-based vector. A flow cytometry assay was used to monitor the internalization of the Dox-loaded DNA nanostructure into target cells. As shown in Figure 5, obvious fluorescence signals were observed for 4T1 and PC-3 cells (target cells) treated with the Dox-loaded DNA nanostructure, whereas a low detectable fluorescence was found for CHO cells (nontarget) treated with the Dox-loaded DNA nanostructure (p < 0.05), indicating that the designed targeted delivery system was efficiently internalized into the cells (4T1 and PC-3), which overexpressed nucleolin as the target of the AS1411 aptamer, via a ligand− receptor mechanism. Moreover, the Dox-loaded DNA nanostructure could be internalized into target cells as well as free Dox. The same conclusion was also obtained from fluorescence microscopy analysis (Figure 6), in which bright green cells were detected in the Dox-loaded DNA nanostructure-treated PC-3 cells.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel.: +98 513 1801535; Fax.: +98 513 882 3251 (K.A.) ORCID

Seyed Mohammad Taghdisi: 0000-0001-9836-2189 Khalil Abnous: 0000-0001-6314-0164 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of this study was provided by Mashhad University of Medical Sciences. REFERENCES

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Molecular Pharmaceutics

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DOI: 10.1021/acs.molpharmaceut.8b00124 Mol. Pharmaceutics 2018, 15, 1972−1978