Ethics statement

The animal experiments were implemented with the approval of the animal care committee of Xiangya Hospital of Central South University and complied with the rules and regulations and the operating norms of the management of experimental animals and the relevant ethical requirements. The experiments involving clinical samples were approved by the ethics committee of Xiangya Hospital of Central South University. Each individual who provided clinical samples signed an informed consent form.

Cell culture

The human NPC cell line 5-8F (BeNa, Beijing, China) was grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and supplemented with 1% penicillin/streptomycin, and the human normal nasopharyngeal epithelial cell line NP69 (ATCC, Manassas, Virginia, USA) was cultured in keratinocyte serum-free medium (KSFM) under the conditions of 37 °C and 5% CO2.

Aptamer

The aptamer used in this experiment was S3 targeting the CD109 protein on the surface of the 5-8F cells, which was derived from the screening of NPC specific aptamers by Wenting Jia in the same laboratory. The aptamer S3 was screened with NPC cells as positive targets from synthetic DNA libraries by Cell-based Systematic Evolution of Ligands by Exponential enrichment and verified to be the most specific one among the selected sequences.

Human sample collection

NPC tissues were taken from the tissue samples stored in Xiangya hospital, Central South University. The enrolled patients were NPC patients who visited the Otolaryngological Department of Xiangya hospital and underwent biopsy. All the nasopharyngitis tissues were resected from nasopharyngitis patients during biopsy.

Synthesis of DT, S3-DT, DT-Cy5, S3-DT-Cy5, DT-Dox, and S3-DT-Dox

The base sequences of four single-stranded DNA in DT are listed in Table 1.

Table 1 Base sequences of four single-stranded DNA in DNA tetrahedron

For synthesis of DT, 10 μL of A, B, C, and D single-stranded DNA fragments (all at a concentration of 10 μM) were annealed at 95 °C for 10 min in 10 × TAE buffer (10 μL) and ultrapure water (50 μL), followed by natural cooling and storing at room temperature. DT with a final concentration of 1 μM was obtained.

For synthesis of S3-DT, 10 μL of A, B, C, and D single-stranded DNA fragments and S3 fragments (all at a concentration of 10 μM) were annealed at 95 °C for 10 min in 10 × TAE (10 μL) and ultrapure water (40 μL), followed by natural cooling and storing at room temperature. S3-DT with a final concentration of 1 μM was obtained.

For synthesis of DT-Cy5, 10 μL of A, B, C, and D-Cy5 fragments (all at a concentration of 10 μM) were annealed at 95 °C for 10 min in 10 × TAE buffer (10 μL) and ultrapure water (50 μL), followed by natural cooling and storing at room temperature. DT-Cy5 with a final concentration of 1 μM was obtained.

For synthesis of S3-DT-Cy5, 10 μL of A, B, C, and D-Cy5 fragments and S3 DNA fragments (all at a concentration of 10 μM) were annealed at 95 °C for 10 min in 10 × TAE buffer (10 μL) and ultrapure water (40 μL), followed by natural cooling and storing at room temperature. S3-DT-Cy5 with a final concentration of 1 μM was obtained.

For synthesis of DT-Dox, 50 μL of A, B, C, and D fragments (all at a concentration of 400 μM) were annealed at 95 °C for 10 min in 10 × TAE buffer (40 μL) and ultrapure water (160 μL, with a final volume of 400 μL and a final concentration of 50 μM), followed by natural cooling and incubation with 4 μL of 100 mM Dox for 3 h at 37 °C to obtain DT-Dox solution (DT 50 μM, Dox 1 mM), in which the amount of Dox was 0.233 mg.

For synthesis of S3-DT-Dox, 50 μL of A, B, C, and D fragments and S3 fragments (all at a concentration of 400 μM) were annealed at 95 °C for 10 min in 1 × TAE buffer (40 μL) and ultrapure water (110 μL, with a final volume of 400 μL and a final concentration of 50 μM), followed by natural cooling and incubation with 4 μL of 100 mM Dox for 3 h at 37 °C to obtain S3-DT-Dox solution (S3-DT 50 μM, Dox 1 mM), in which the amount of Dox was 0.233 mg.

Agarose gel electrophoresis

2.5% agarose gel was prepared, and the electrophoresis buffer was added into an electrophoresis tank. Next, the prepared gel was placed in the center of the electrophoresis tank, and 50 bp of DNA Ladder, as the reference Marker, was added into the sample well of the gel. A chain, A + B chain, A + B + C chain, DT, and S3-DT were respectively mixed with the loading buffer and added to the sample wells. After that, the lid of the electrophoresis tank was closed, followed by turning on the electrophoresis device. The electrophoresis parameters were set as follows: voltage 120 V, current 400 mA, and electrophoresis time 30 min.

Particle-size analysis

The particle size was measured using a Malvern particle size meter. Briefly, 1 pmol of S3-DT or DT was dissolved in 1 mL of double distilled water and placed in quartz plates for measuring the particle size.

Stability detection of DT and S3-DT

At 37 °C without light exposure, 20 μL of DT or S3-DT (a final concentration of 1 μM) was incubated in 20 μL of McCoy'5A complete medium for 0, 2, 4, 6, 8, 10, 12, and 24 h, and the samples were taken at each time point and then stored at − 20 °C. The collected specimens were subjected to gel electrophoresis to check the bands. Next, 20 μL of DT and S3-DT solutions (a final concentration of 1 μM) were respectively mixed with 20 μL of FBS in a ratio of 1:1 and then incubated at 37 °C for 1, 3, 5, and 7 h. Specimens were taken at the time points and stored at − 20 °C for uniform gel electrophoresis.

Laser confocal analysis

5-8F or NP69 cells (1 × 104) were seeded and incubated in optical dishes for 24 h. Next, cells were washed three times with Dulbecco phosphate-buffered saline (DPBS), and incubated in binding buffer with the addition of Cy5-labeled S3, S3-DT, or DT with a final concentration of 250 nM at 37 °C for 3 h, followed by three washes in DPBS and imaging under a laser confocal microscope (Leica). Photographs were taken at a 63 × lens, with an excitation wavelength of 633 nm and an emission wavelength of 650–750 nm.

Flow cytometry

For analysis of the ability of S3-DT to recognize NPC cells, 5-8F or NP69 cells (4 × 105) were incubated in 12-well plates for 24 h. Cells were rinsed three times with DPBS before the experiment. The binding buffer with Cy5-labeled S3, S3-DT, or DT (250 nM) was added to 5-8F and NP69 cells and incubated at 37 °C for 3 h. After that, cells were washed three times with DPBS again and suspended in 400 μL of DPBS. Cy5 fluorescence in the cells was detected by flow cytometry.

For analysis of the uptake of the synthetic S3-DT-Dox by NPC cells, 5-8F or NP69 cells were scraped down from the culture flasks and washed two times with Hanks buffer. Subsequently, cells were incubated for 1.5 h with Dox, DT-Dox, or S3-DT-Dox at a Dox concentration of 2 μM at 37 °C. Next, the drug-treated cells were removed, and the PBS buffer containing experimental drugs was aspirated. The cells were washed three times with Hanks buffer, added to medium, and continued for 48 h-incubation at 37 °C. The cells were then rinsed two times with Hanks buffer, fixed with 4% formaldehyde for 10 min, and analyzed by flow cytometry.

Fluorescence detection of NPC tissues

Paraffin sections of NPC tissues and nasopharyngitis tissues were heated for 1 h in a 60 °C oven to fully melt the paraffin, followed by dewaxing and hydration, and antigen repair. Then the sections were immersed in antigen repair solution, repaired under high pressure for 2.5 min, and naturally cooled to room temperature, followed by 3 times of washing with PBS (3 min each). Subsequently, the sections were blocked for 60 min at room temperature using aptamer blocking solution, and then cultured at 37 °C for 2 h with biotin-modified S3, S3-DT, or DT at a final concentration of 1 μM. Streptoavidin-modified ZnCdSe/ZnS quantum dot (QD) fluorescent nanoparticles were added and incubated at room temperature for 30 min. ZnCdSe/ZnS quantum dots were purchased from Wuhan Jiayuan QuantumDot Technology (Wuhan, Hubei, China). The solvent was 50 mM Borate buffer solution, pH = 8.4, 0.05% NaN3, concentration 1.0 μM, diluted at 1:100 during use, with excitation wavelength of 488 nm and emission wavelength of 605 nm. After 3 times of washing with PBS (5 min each), the sections were sealed with an anti-fluorescence quencher and photographed under a fluorescence microscope with an excitation wavelength of 488 nm and an emission wavelength of 600 nm.

Optimum loading ratio of Dox in DT

When Dox was intercalated into DNA, its fluorescence would be quenched, which can be used to identify the efficiency of Dox intercalation into DNA structure. Briefly, 50 μL of 2 μM Dox (in TAE) was separately added with 0, 0.1, 0.5, 1, 2, 5, and 10 μL of S3-DT or DT (at a concentration of 1 μM), and added to 100 μL using 1 × TAE), so that the final concentration of S3-DT or DT was 0 nM, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, or 100 nM. The samples were incubated at 37 °C for 3 h, and the fluorescence spectrum of Dox was measured with a fluorometer, with an excitation wavelength of 480 nm and an emission wavelength of 540–700 nm. The drug loading curves of different DNA nanostructures at different molar ratios were plotted. Finally, the optimal drug loading ratio was obtained.

Release efficiency of Dox

10 μL of S3-DT (1 μM), 50 μL of Dox (4 μM), and 10 μL of 1 × TAE (pH = 5) (a total volume of 100 μL) were incubated at 37 °C for 3 h to prepare S3-DT-Dox complexes (100 nM S3-DT and 2 μM Dox). The above solution was mixed with 100 μL of PBS, and then centrifuged (8000 rpm, 3 min) in a 3 K ultrafiltration tube. The filtrate containing the released Dox was obtained at various time points (0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h). The fluorescence intensity of Dox in the filtrate was measured by a fluorometer, and the filtrate was put back into the ultrafiltration tube after the measurement. The drug release rate was calculated by the released Dox/total Dox (the fluorescence intensity of the filtrate after ultrafiltration of 1 μM Dox without S3-DT).

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay

5-8F cells and NP69 cells in the logarithmic growth phase were trypsinized. After the cells were counted, cell suspension was made with a complete medium, and 5 × 103 cells were seeded in each well of a 96-well plate and then cultured at 37 °C for 24 h. Adherent cells were washed two times with Hank’s buffer, followed by addition of an appropriate amount of PBS. Free Dox, DT-Dox, and S3-DT-Dox were added to the wells, and the final concentrations of Dox in each well were 0.01, 0.02, 0.2, 1, 2, 5, 10, 20, and 40 μM, respectively. Drug-treated cells were incubated at 37 °C for 2 h, washed three times with Hank’s buffer, and then cultured with medium containing 1% serum at 37 °C for 48 h. After that, the survival of cells in the 96-well plate was observed with an inverted microscope. After the removal of the culture medium, the cells were rinsed two times with Hank’s buffer. Next, 100 μL of serum-free medium was added to each well of the 96-well plate, followed by addition of 20 μL of the prepared MTS reagent (thawed MTS and PMS mixed at a ratio of 20:1). Light should be avoided during the above process. The 96-well plate supplemented with MTS reagent was incubated at 37 °C for 4 h, and the absorbance was measured at a wavelength of 490 nm with a microplate reader. According to the measured absorbance value, the cell viability was calculated, and the survival curve was plotted.

Western blot

Cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) to obtain protein samples. After the protein concentration was measured with a BCA kit (Beyotime), the corresponding volume of proteins were mixed with the loading buffer (Beyotime) and heated in a boiling water bath for 3 min to denature the protein. Then, the protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto membranes (300 mA), and blocked in blocking solution at room temperature for 60 min or 4 °C overnight. Next, the membranes were incubated for 2–4 h with primary antibodies against CD109 (35717S, 1:1000, CST, Boston, MA, USA) and glyceraldehyde phosphate dehydrogenase (GAPDH) (5174S, 1:1000, CST), followed by 1 h-incubation with a secondary antibody. Lastly, the membranes were added with developer, and the detection was performed using a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).

Animal experiments

Forty-eight BALB/c nude mice (4–6 weeks old, 16 ± 2 g) of specific pathogen free (SPF) grade from the Laboratory Animal Resources of Chinese Academy of Sciences (Shanghai, China) were raised in a SPF grade sterile laminar flow chamber at constant temperature (25–28 °C) and constant humidity (about 50%). They were individually housed with free access to water and food under 12-h light/dark cycles. The padding was replaced every 3 days aseptically.

Fluorescence probe targeting and intra-organ metabolism experiments: 5-8F cells were injected into the armpit of the forelimbs of nude mice with 1 × 107 cells per mouse, and the tumor was formed (5 mm × 4 mm × 4 mm) after 1–2 weeks for subsequent experiments. The nude mice were subjected to injection of 100 μL of DPBS, 100 μL of 50 μM S3-Cy5, 100 μL of 50 μM S3-DT-Cy5, or 100 μL of 50 μM DT-Cy5 via the tail vain, which were grouped as DPBS, S3, S3-DT or DT group, respectively (n = 6 mice/group). Subsequently, the above DNAs circulated in the mouse for 2 h, so that the aptamers were targeted to cancer tissues. The above mice were placed in a box containing isoflurane air for anesthesia. They were then placed in a small animal imager and imaged in the Cy5 fluorescence channel with the exposure time of 10 s. After that, the mice were dissected to obtain the organs, which were washed with DPBS, placed in petri dishes in sequence, and imaged with the small animal imager in the Cy5 fluorescence channel with the exposure time of 10 s.

For tumor xenografts analysis, 1 × 107 tumor cells per mouse were injected into the armpit of the forelimbs of nude mice, and the subsequent experiments were performed when the tumors grew to 50 mm3. Four groups of tumor-bearing mice (DPBS, Free Dox, S3-DT-Dox, and DT-Dox, 6 mice for each group) were taken and respectively injected with 100 μL of DPBS, 100 μL of 1 mM free Dox, 100 μL of 50 μM S3-DT-Dox, or 100 μL of 50 μM DT-Dox (the amount of Dox was 3.5 mg/kg) through the tail vein every three days (1, 4, 7, and 10 days). From the first injection, the tumor volume was measured every two days for 14 days. On day 14, the mice were euthanized, and the tumors were isolated and weighed. Heart, liver, spleen, lung, and kidney tissues were taken and made into paraffin-embedded sections.

Hematoxylin and eosin (HE) staining

The heart, liver, spleen, lung, and kidney tissues were fixed, dehydrated, and then cut into 5 μm thick slices with a microtome. Sections were routinely deparaffinized, stained with hematoxylin solution for 10 min, differentiated with 1% hydrochloric acid alcohol for several seconds, treated with 0.6% ammonia water for several seconds, counterstained with 0.5% eosin solution for 1–3 min, rinsed with tap water for 5 min, and rinsed with deionized water for 1 min, followed by routine dehydration, clearing, drying, and mounting. Finally, images were captured under a microscope (Olympus, Tokyo, Japan).

Statistical analysis

Statistical analysis and graphing of data were performed using SPSS 18.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 7.0. Data were presented as mean ± standard deviation. The comparison between two groups was performed using the t test, and the comparisons among multiple groups were conducted using one-way analysis of variance and Tukey’s post hoc test. A difference with P < 0.05 was considered to be statistically significant.