Aptamer‐targeted DNA nanostructures with doxorubicin to treat protein tyrosine kinase 7‐positive tumours

Abstract Objectives Aptamer sgc8c is a short DNA sequence that can target protein tyrosine kinase 7 (PTK7), which was overexpressed on many tumour cells. This study aimed to fabricate a novelty DNA nanostructure drug delivery system target on PTK7‐positive cells—CCRF‐CEM (human T‐cell ALL). Methods Aptamer‐modified tetrahedron DNA was synthesized through one‐step thermal annealing process. The sgc8c‐TDNs (s‐TDNs) loading DOX complexes were applied to investigate the effect to PTK7‐negative and ‐positive cells. Results When s‐TDN:DOX acted on PTK7‐positive and ‐negative cells respectively, the complexes exhibited specific toxic effect on PTK7‐positive cells but not on PTK7‐negative Ramos cells in vitro research. Conclusions In this work, we successfully constructed a PTK7‐targeting aptamer‐guided DNA tetrahedral nanostructure (s‐TDN) as a drug delivery system via a facile one‐pot synthesis method. The results showed that s‐TDN:DOX exhibited enhanced cytotoxicity against PTK7‐positive CCRF‐CEM cells, with a minor effect against PTK7‐negative Ramos cells. Hence, this functionalized TDNs drug delivery system displayed its potential application in targeting PTK7‐positive tumour T‐cell acute lymphoblastic leukaemia.

Aptamers, also known as "chemical antibodies," are small RNA/DNA sequences that are capable of binding to target entities ranging from small molecules to proteins. [15][16][17] In comparison with traditional antibodies, aptamers offer multiple advantages such as high stability, 18 better compatibility, 19 versatile chemical modification, 20 and quick chemical production, 21 making them attractive candidates for targeted cancer therapy. Sgc8c is a DNA sequence with 42 nucleotides that is known to specifically bind to protein tyrosine kinase 7 (PTK-7), a cell membrane protein known to be overexpressed on CCRF-CEM (human T-cell ALL) cells 22,23 and many other tumours 24 such as colon and gastric cancer. Given its potential application as a targeted aptamer, sgc8c may be used to fabricate a drug delivery system, namely, sgc8c-TDN (s-TDN) to selectively target ALL cells.
Considering that DOX has the ability to intercalate into -GC-rich regions of DNA, [25][26][27] we fabricated s-TDN:DOX complexes as a targeted drug delivery system ( Figure 1). Furthermore, we evaluated the behaviour of this nanosystem in vitro. Laser scanning confocal microscopy (LSCM) and flow cytometry (FCM) experiments were performed to investigate the cellular uptake of s-TDNs. In addition, the cellular uptake of DOX was also evaluated by FCM. Cell cytotoxicity tests revealed that s-TDN:DOX was more cytotoxic to target cells as compared with untargeted cells. We conclude that sgc8c-TDN is a feasible targeted drug delivery system and that s-TDN:DOX may serve as an ideal targeted treatment against ALL or other PTK7-positive tumours.

| Cellculture
We obtained PTK7-positive cells (CCRF-CEM, human T-cell ALL) and PTK7-negative cell (Ramos, human Burkitt's lymphoma) from Cell Center of Chinese Academy of Medical Sciences (Beijing, China).

| CellularuptakeofTDNsandaptamer-modifiedTDNs
To investigate the cellular uptake of the nanomaterial, singlestranded DNA S1 was fluorescently tagged with cyanine-5 (Cy5) fluorophore (red fluorescence). FCM was used to quantify the in- In the process of the experiment, cells were washed thrice with PBS at every step and protected from light at all time.

| LoadingofDOXonTDNsandApt-TDNs
As the fluorescence of DOX would be quenched after its embedment into the double-helix structure, 37  to 700 nm. All operations were carried out away from the light.

| CellularuptakeofDOX
From the results of DOX-loading capacity test, we concluded that 1:20 was optimal concentration ratio of TDNs and DOX for the

| Cellcountingkit-8assay
To investigate the targeted toxicity of nanomaterials, cell counting

| Statisticalanalysis
In this study, all tests were conducted in triplicate and verified repeatedly. One-way analysis of variance (ANOV; SPSS 19.0: IBM Corp., Armonk, NY, USA) was employed to analyse the difference between treatment groups and controls, and Student's t-test was applied to analyse the means of each pair of group. A value of P < 0.05 was considered statistically significant all the time.

| SynthesisandcharacterizationoftheDNA nanostructure
Here, a novel targeted DNA nanostructure as a drug delivery system was successfully prepared. As shown in Figure Table 1), so we can calculate that TDNs is about 240 bp.
Moreover, due to the modified with aptamer sgc8c, s-TDNs ought to be about 280 bp. As shown in Figure 2B, the positions of TDNs and s-TDNs matched well with the theoretical values of their molecular weights. 10,33 In addition, DLS was conducted to determine the size of TDNs. Two peaks were displayed on Figure 2C, where in the first peak corresponded to TDNs with a size of about 12 nm, consistent with the results of previous study, 13,31 and the second peak reflected the presence of some large polymers can also be formed during the synthesis process. As reported before, the formation of polymers is a common phenomenon during the reaction process. 10 In addition, the morphology of TDNs was verified by TEM. Displayed on Figure 2D, several triangular nanostructures were observed along with circular structures of 100 nm, which corresponded to the polymers of TDNs in Figure 2C. Together these results suggest that TDNs and s-TDNs were successfully prefabricated.

| AptamermodificationfacilitatesTDNentry intotargetcells
To study the targeting effect of the aptamer, we treated CCRF-CEM and Ramos cells with cy5-TDNs and cy5-s-TDNs. After 12 hours of incubation, cells were collected and analysed by FCM. We found that the uptake efficiency of TDNs was low into both CCRF-CEM and Ramos cells. However, s-TDNs showed higher binding to CCRF-CEM, but not Ramos, suggesting that the aptamer sgc8c could target PTK7 and facilitate TDNs entry into the cell ( Figure 3A,B).
As shown in Figure 3C, CCRF-CEM cells showed up to four times higher uptake than Ramos cells. To demonstrate the phenomenon intuitively, DAPI was performed to make nucleus blue to observe cy5-TDNs and cy5-s-TDNs in these cells. As nucleus of lymphocyte occupies about 90% volume of the whole cell, we can see the red TDNs is closed to the blue nucleus in Figure 4D.

| Drug-loadingcapacityofTDNsands-TDNs
Drug-loading capacity is one of the most important factors to evaluate the efficiency of the drug delivery system. Here, 100 nmol/L TDNs and s-TDNs were individually mixed with DOX at increasing molar ratio for 1 hour at room temperature. As shown in Figure  sgc8c is an isolated single strand, which lacked the ability to bring DOX. TDNs and its derivative exhibit excellent characteristic as a drug delivery vehicle.

| MaximumDoxuptakewasobservedin PTK7-positivecellstreatedwiths-TDNs
Doxorubicin, a widely used antineoplastic drug, can be inserted into the DNA double-stranded structure for the existed of flat aromatic rings in its molecule 38 and is known to inhibit DNA duplication and RNA synthesis. 39 Therefore, the delivery of high concentration of DOX to target cells is essential for the effective killing effect. DOX has multiple side effects for its nonselective feature and hence it is meaningful to decrease its nontargeted cellular uptake. FCM results ( Figure 6A) showed that fluorescent signals of DOX generated from TDNs and s-TDNs were similar in PTK7-negative cells (Ramos); however, PTK7-positive cells treated with s-TDN:DOX showed approximately two times higher signal than those treated with TDNs:DOX.
The value of mean fluorescent intensity of DOX ( Figure 6B,C) was calculated from the FCM data via Flowjo.7.6. We can find that s-TDNs carry more DOX to the target CCRF-CEM cell not into Ramos. Thus, s-TDNs promoted efficient DOX uptake into targeted cells as compared to untargeted TDNs.

| D ISCUSS I ON
Leukaemia is one of the most common therioma and is known to seriously affect the health and lives of patients. Chemotherapy, often used as the priority treatment against leukaemia, has multiple side effects in the body, highlighting the need to develop a targeted delivery system.
In our present study, we found that the emerging DNA material TDNs could freely penetrate the cell plasma membrane without any transfection agent, 11,40,41 suggestive of its potential as a promising drug carrier. The aptamer sgc8c in combination with nanocarriers such as carbon nanotubes 42 and gold nanoparticles 43 has been targeted to PTK7-positive tumours and exhibited promising results in multiple studies; however, these nanocarriers showed more or less toxicity to cells; hence, there is an urgent need to construct a safe and targeted drug delivery system. Here, we fabricated a novel aptamer-modified DNA nanostructure for the targeted delivery of DOX to PTK7-positive cells CCRF-CEM such that it minimizes DOX toxicity to untargeted cells.
We confirmed the ability of sgc8c to specifically target PTK7positive cells CCRF-CEM to facilitate TDNs uptake using FCM and LSCM. As s-TDNs are negatively charged nanomaterial, owing to electrostatic repulsion, it is unlikely that they were taken up via passive processes. 11 In pervious study, Bagalkot et al 38 revealed the uptake of aptamer A10 PSMA is by receptor-mediated endocytic. As sgc8c has the same structure with A10 PSMA; therefore, we can infer that receptor-mediated endocytic is the main mechanism of cellular uptake of s-TDNs. Our in vitro study revealed that s-TDN nanocarrier is a feasible vehicle for targeted drug delivery and that s-TDN:DOX can kill target cells while reducing the cytotoxic effects against PTK7negative cells. TDNs are taken up by caveolin-dependent pathway, 11 which is a major kind of receptor-mediated endocytic. And our results have demonstrated that sgc8c have ability to facilitate TDNs go into targeted cells. With continuously monitored the uptake process by confocal microscopy, a study 11 found TDNs was transported to and trapped within the lysosome, which is an organelle that invading particles are degraded. As the degradation of TDNs, DOX were released from the TDNs subsequently, then diffused to the cytosol and finally to the nucleus to do its work. In conclusion, these findings suggest that the application of s-TDN:DOX may be a potential strategy to curb PTK7-positive tumours such as ALL under clinical settings.
The United States Food and Drug Administration has approved several aptamers for clinical use, including macugen for age-related macular degeneration, 44 as1411 for cancer treatment, 33 and others. 45 In our study, we successfully coupled TDNs to an aptamer, indicating the feasibility of constructing various apt-TDNs as targeted drug delivery systems specific for particular organs and diseases, in the future.

ACK N OWLED G EM ENT
This study was supported by the National Natural Science Foundation of China (81671031, 81470721) and Sichuan Province Youth Science and Technology Innovation Team (2014TD0001).