AS1411 aptamer modified carbon dots via polyethylenimine‐assisted strategy for efficient targeted cancer cell imaging

Abstract Objectives Carbon dots (CDs), as a fascinating class of fluorescent carbon nanomaterials, have been proven to be powerful tools in the field of bioimaging and biosensing due to their small size, suitable photostability and favourable biocompatibility. However, the cellular uptake of free CDs lacks selectivity and the same negative charges as cell membranes may cause inefficient cell internalization. In this study, an efficient detecting and targeting nanosystem was developed based on the DNA aptamer AS1411 modified CDs with polyethyleneimine (PEI) as connecting bridge. Materials and methods Hydrothermally prepared CDs were assembled with positive‐charged PEI, followed by conjugation with AS1411 through electrostatic interaction to form CDs‐PEI‐AS1411 nanocomplexes. The CDs, CDs‐PEI and CDs‐PEI‐AS1411 were characterized by transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectra, UV–vis spectra, zeta potential measurements and capillary electrophoresis characterizations. The cytotoxicity investigation of the CDs‐PEI‐AS1411 and CDs‐PEI in both MCF‐7 and L929 cells was carried out by the CCK‐8 assay. The cellular uptake of the CDs‐PEI‐AS1411 was studied with confocal microscopy and flow cytometry. Results The as‐prepared nanosystem possessed good photostability and no obvious cytotoxicity. On the basis of the confocal laser scanning microscope observation and the flow cytometry studies, the cellular uptake of CDs‐PEI‐AS1411 nanosystem in MCF‐7 cells was significantly higher than that of L929 cells, which revealed the highly selective detection ability of nucleolin‐positive cells. Conclusions The results of this study indicated that the CDs‐PEI‐AS1411 nanosystem had a potential value in cancer cell targeted imaging.


| INTRODUC TI ON
Carbon dots (CDs) have received great attention in bio-related applications such as biosensing and bioimaging due to their small size, low toxicity, good biocompatibility and good stability. Meanwhile, CDs modified with small oligonucleotide ligands (aptamer), peptides or small molecules have been widely used in targeted fluorescent imaging, drug delivery and cancer therapy. [1][2][3][4][5][6] Among the numerous targeted agent, aptamer possessed chemical stability, non-immunogenicity and easy synthesis, which garnered a lot of attention. [7][8][9][10] It is well known that AS1411 is a common aptamer with 26-mer oligonucleotide, which can bind to the nucleolin that is highly expressed on the surface of most cancer cells. 11 As a result, AS1411 is a promising candidate for precisely targeting. [12][13][14][15][16] However, for AS1411 favoured fluorescence targeting imaging, the relatively complex strategy of chemical coupling is usually adopted to connect imaging agent with AS1411, which may result in poor targeting ability. Besides, the imaging agent conjugated with negative charged aptamer AS1411 always exhibit the same negative charge as cell membrane, which may affect the efficiency of cellular uptake ascribe to the electrostatic repulsion.
Polymer-assisted surface functionalization can easily provide a high surface with abundant functional groups and facilitate attachment of other substances. 17 Polyethylenimine (PEI) is a cationic polymer material containing a large number of amino groups, and widely used in the field of biological transfer due to its high transfer efficiency and low cost. 18,19 In addition, the use of PEI as scaffolds for loading of nanomaterials has been shown as an effective way for facilitating cellular uptake efficiency of these materials, such as quantum dots, Fe 3 O 4 magnetic nanoparticles. [20][21][22] Furthermore, surface functionalization with PEI can give CDs strong positive surface charges, which is beneficial to encapsulating some negatively charged macromolecules, such as DNA. 23,24 Tian et al 25 combined PEI and tetrahedral DNA nanostructures (TDNs) through electrostatic interaction to form novel drug-delivery vehicles with enhanced systemic stability, cell-entry ability and lysosome-escape ability. Furthermore, the high density of amino groups in PEI not only generate plenty of attachment sites, but also render the CDs with good colloidal stability. 26,27 Herein, a fluorescence imaging nanosystem based on AS1411 modified PEI twisted CDs was developed for targeted imaging of nucleolin highly expressed cancer cells (Scheme 1). High brightness CDs were first synthesized from citric acid through hydrothermal process, and then coated with PEI to form the CDs-PEI assembly. Finally, the CDs-PEI assembly was further attached with aptamer AS1411 via electrostatic interaction. To investigate the targeting ability of the developed CDs-PEI-AS1411 nanosystem to cancer cells, the interaction between CDs-PEI-AS1411 and MCF-7 (overexpressed nucleolin) or L929 (low nucleolin expression) cells were studied through cell imaging and flow cytometry.
The results showed that CDs-PEI-AS1411 nanosystem possessed higher cellular uptake efficiency in MCF-7 cells than L929 cells.
For cancer MCF-7 cells, the high affinity between AS1411 and the relevant overexpressed receptor nucleolin led the aptamer release, and the surface charge of nanosystem rise at the same time, resulting in enhanced cellular uptake. By employing the AS1411 aptamer, enhanced selectivity and sensitivity were achieved.

| Preparation of CDs
In a typical synthesis, citric acid (1.05 g) and ethylenediamine (335 µL) were dissolved in 10 mL deionized water. Then the mixed solution was transferred to an autoclave and heated at 200°C for 5 hours. After the product cooled down to room temperature, the product was purified with column chromatography (with water as eluent) to obtain the uniform-sized particles. [28][29][30] Finally, the obtained CDs were light yellow and transparent, which were stored at 4°C for further treatment.

| Preparation of CDs-PEI and CDs-PEI-AS1411
In order to obtain CDs-PEI, 15 µL PEI (2 mg/mL) was mixed with 100 µL CDs solution (4 mg/mL) and 85 µL deionized water, and then stirred overnight at room temperature. For CDs-PEI-AS1411, different volumes of AS1411 (10-50 µL, 100 µmol/L) were mixed with the above CDs-PEI solution with gently stirring at 4°C for 2 hours.
After that, free DNA was removed using centrifugal filtration (MWCO = 30 kDa) at 4000 rpm for 10 minutes, and then the fluorescence intensities were recorded to determine the proper CDs/ aptamer ratios. CDs-PEI-AS1411 were finally collected and kept at 4°C for later experiments.

| Characterizations of CDs, CDs-PEI and CDs-PEI-AS1411
Surface morphology analyses of particles were performed using a Tecnai G2 F20 transmission electron microscope (TEM, FEI). Particle size and zeta potential measurements were carried out with a Nano-ZS90 Zetasizer. The fourier transform infrared (FTIR) spectra were characterized using a 5DX FT-IR spectrometer (Nicolet). The UV-vis spectra were measured by a U-3900H UV-vis spectrophotometer (Hitachi), and the fluorescence spectra were recorded by a RF-5301PC photoluminescence spectrometer (Shimadzu). Capillary electrophoresis analyses were performed on a Qsep100 Bio-Fragment Analyzer (BiOptic). Fluorescence lifetime was recorded using a PPD-850 fluorescence-photon counting detector (HORIBA, Japan). Subsequently, the cell cytoskeleton was stained with FITC-labelled phalloidin and the cells were imaged by a Nikon AIR-MP confocal laser scanning microscope (Japan).

| Flow cytometry analysis
The cellular uptake of the CDs-PEI-AS1411 and the CDs-PEI was further studied with flow cytometry. MCF-7 and L929 cells were cultured overnight at 2 × 10 5 cells per well in 6-well plates. After that, the cells were treated with CDs-PEI-AS1411 or CDs-PEI (the CDs concentration of 200 µg/mL) for 6 or 12 hours. Then, the cells were washed for three times with PBS and centrifuged at 1000 rpm for 5 minutes. After resuspended in 500 µL PBS, the cells were detected by a Coulter FC500 flow cytometer (Beckman).

| Cytotoxicity assay
The cytotoxicity of the CDs-PEI-AS1411 and CDs-PEI was investigated by the CCK-8 assay. MCF-7 cells and L929 cells were seeded (5000 cells per well) in 96-well plates and grew overnight. And then, the cells were replaced with fresh medium containing different concentrations of CDs-PEI-AS1411 or equivalent concentrations of CDs-PEI. After incubation for 24 hours, the CCK-8 reagent (10%) was added to each well to test the cell viability through absorbance measurement at a wavelength of 450 nm with a VariOskanFlas 3001 microplate reader (Thermo).

| Statistical analysis
Statistical significance of the data was analysed through independent sample t test by SPSS Statistics R24.0 software (IBM). Data are presented as mean result ± SD. Probability value of <.05 was considered statistically significant (#P < .05, *P < .05, **P < .01).

| Characterization
Surface morphology of the CDs, CDs-PEI and CDs-PEI-AS1411 were characterized by TEM. As shown in Figure 1A, the CDs were welldispersed and quasi-spherical in shape. While, after treated with PEI, the formed CDs-PEI were aggregated into linear chain ( Figure 1B), which was consistent with the characterization of previous study. 19 Subsequently, with further assembly of AS1411 onto the CDs-PEI, the complexes tended to form larger clusters ( Figure 1C), indicating the successful preparation of CDs-PEI-AS1411 via electrostatic interaction. Moreover, the assembly of CDs-PEI-AS1411 was also demonstrated by dynamic light scattering measurement ( Figure 1D).
The particle sizes of free CDs and CDs-PEI were 26.19 nm and 122.7 nm, respectively. Non-covalent conjugation of AS1411 S C H E M E 1 Predicted mechanism of the different cellular uptake behaviour of CDs-PEI-AS1411 with the nucleolinpositive MCF-7 cancer cells and nucleolinnegative L929 fibroblast cells aptamer to CDs-PEI enlarged its size to 227.1 nm, which was consistent with the above TEM images.
To further confirm the successful preparation of CDs-PEI-AS1411 nanosystem, zeta potentials of the CDs, CDs-PEI and CDs-PEI-AS1411 were first investigated, which was displayed in Figure 2A.
The average zeta potential value of the CDs was −20.3 mV and raised to +15.6 mV after PEI modification, which was attributed to the addition of the positively charged amino groups. Consequently, the positive charge facilitated the assembly of the CDs-PEI with negatively charged aptamer of AS1411 through electrostatic interaction. Additionally, the charge of the CDs-PEI-AS1411 reversed back to −11.8 mV, which further confirmed the successful fabrication of the complexes. Meanwhile, the FTIR spectra provided further understanding on the structure of the formed nanoparticles. As presented in Figure 2B Significantly, the combination between AS1411 and the CDs-PEI was further verified by the measurement of UV/Vis absorption spectra and capillary electrophoresis. As illustrated in Figure 2C, 2 typical UV-vis absorbance peak of the AS1411 at 260 nm could be observed in the CDs-PEI-AS1411. 33 Furthermore, in the capillary electrophoresis ( Figure 2D), a peak at the molecular weight of 28 bp was detected when AS1411 was non-covalently attached to the CDs-PEI, which indicated the successful capping of DNA molecules on the nanoparticles.
To explore the optical properties, the photoluminescence (PL) emission spectra of the CDs, CDs-PEI and CDs-PEI-AS1411 nanoparticles were first investigated, which was shown in Figure 3A. The Previous study showed that aptamer molecules can partially inhibit the fluorescence of CDs. 4 Therefore, in order to maintain their PL property, the fluorescence intensities of CDs-PEI-AS1411 were tested with different CDs/aptamer weight ratios. As shown in Figure   S1, the complexes at CDs/aptamer ratio of 16.7:1 exhibited the highest fluorescence intensity, and we thus chose this ratio for subsequent experiments. Then the fluorescence decay curves ( Figure 3B) of the CDs, CDs-PEI and CDs-PEI-AS1411 were also measured and no significant difference in fluorescence lifetime was found. The high photostability is the key factor of the fluorescent nanomaterials Herein, we studied the photostability of the CDs, CDs-PEI and CDs-PEI-AS1411, which was exposed under excitation of 360 nm for various amounts of time ( Figure 3C). For the CDs and CDs-PEI, the fluorescence intensity decreased only slightly at the beginning (5 min) and then showed a rising tendency in the following irradiation process. As to the CDs-PEI-AS1411, the fluorescence intensity preserved almost 100% of the initial intensity after 60 min irradiation, indicating the high photostability of the resultant complexes.
Meanwhile, the aqueous stability of the CDs-PEI-AS1411 was also investigated. From the fluorescence profile ( Figure 3D), it was observed that the CDs-PEI-AS1411 still exhibited good aqueous stability after storage even for 15 days. The emission wavelength of the CDs-PEI-AS1411 did not shift and the fluorescent intensity changed little. Consequently, our as-prepared nanoparticles with good photostability and aqueous stability were the suitable candidates for the biological imaging applications.

| Cytotoxicity assay
In order to evaluate the cytotoxicity of the obtained nanoparticles CDs-PEI and CDs-PEI-AS1411, the cell viability studies based on CCK-8 assay were performed in normal L929 cells ( Figure 4A) and cancer MCF-7 cells ( Figure 4B) for 24 hours. The CDs-PEI displayed limited effect on cell viability in both cell lines with a viability of above 84% at all studied doses. After the treatment with the CDs-PEI-AS1411, the cell viability of the L929 cells was similar to that of CDs-PEI. While in MCF-7 cells, the CDs-PEI-AS1411 showed enhanced cytotoxicity, and significant differences in the viability between the CDs-PEI and the CDs-PEI-AS1411 emerged with the increased concentration from 400 to 800 nmol/L.

| Intracellular uptake
It is known that AS1411 can bind to nucleolin specifically and then subsequently penetrate into the tumour cell. Nucleolin is known to be highly expressed in MCF-7 cells, but rarely expressed in L929 cells. 13 In this study, we investigated the potential of the CDs-PEI- and 46.4%, respectively, after 6 hours of incubation, and 50.5% and 48.5%, respectively, after 12 hours of incubation ( Figure S3).

| D ISCUSS I ON
In present study, we synthesized a targeting nanoprobe for nucleolin highly expressed cancer cells labelling based on CDs which have been successfully prepared in previous experiments with favourable biocompatibility and fluorescence properties. 34

| CON CLUS IONS
In summary, we developed a surface charge inversion nanosystem for specific targeted imaging using CD nanoparticle probes labelled with DNA aptamer AS1411. In our system, the hydrothermally synthesized CDs were first wrapped with PEI and then modified with AS1411 through electrostatic interaction. The negatively charged CDs-PEI-AS1411 nanocomplexes showed less cellular uptake and good biocompatibility for L929 cells. While for MCF-7 cells, the presence of nucleolin on the membrane made the AS1411 release from CDs-PEI and consequently led the surface charge reverse. The exposed CDs-PEI with rising surface charge exhibited efficient cellular uptake. Due to AS1411 aptamer specific identification and the surface charge inversion, the CDs-PEI-AS1411 nanosystem targeted the MCF-7 cells with high affinity and specificity. We believe our asproposed nanosystem has a powerful potential for highly selective and sensitive detection of nucleolin-positive cells. We would be grateful to Doctor Chenghui Li (Analytical & Testing Center, Sichuan University) for her help of taking laser scanning confocal images.

CO N FLI C T S O F I NTE R E S T
There are no conflicts of interests to declare.

AUTH O R CO NTR I B UTI O N
Ronghui Zhou, Xiaoxiao Cai and Bofeng Zhu designed the experiments. Tingting Kong and Liying Hao performed the experiments.
Yujun Zhang analysed the data. Tingting Kong wrote the manuscript.
Xiaoxiao Cai and Bofeng Zhu critically revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.