Potent anti‐angiogenesis and anti‐tumour activity of pegaptanib‐loaded tetrahedral DNA nanostructure

Abstract Objectives Pegaptanib might be a promising anti‐tumour drug targeting VEGF to inhibit tumour vascular endothelial cell proliferation. However, the poor biostability limited its application. In this study, we took tetrahedron DNA nanostructures (TDNs) as drug nanocarrier for pegaptanib to explore the potent anti‐angiogenesis and anti‐tumour activity of this drug delivery system. Materials and methods The successful synthesis of TDNs and pegaptanib‐TDNs was determined by 8% polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis and dynamic light scattering (DLS). The cytotoxicity of pegaptanib alone and pegaptanib‐TDNs on HUVECs and Cal27 was evaluated by the cell count kit‐8 (CCK‐8) assay. The effect of pegaptanib and pegaptanib‐TDNs on proliferation, migration and tube formation of HUVECs induced by VEGF was examined by CCK‐8 assay, wound healing assay and tubule formation experiment. The cell binding capacity and serum stability were detected by flow cytometry and PAGE, respectively. Results Pegaptanib‐TDNs had stronger killing ability than pegaptanib alone, and the inhibiting effect was in a concentration‐dependent manner. What's more, pegaptanib‐loaded TDNs could effectively enhance the ability of pegaptanib to inhibit proliferation, migration and tube formation of HUVECs induced by VEGF. These might attribute to the stronger binding affinity to the cell membrane and greater serum stability of pegaptanib‐TDNs. Conclusions These results suggested that pegaptanib‐TDNs might be a novel strategy to improve anti‐angiogenesis and anti‐tumour ability of pegaptanib.


| INTRODUC TI ON
Aptamers are DNA or RNA oligonucleotides, which can be synthesized and have high affinity and specificity to a number of biochemical targets. [1][2][3] Aptamers have many advantages over antibodies such as cell-free chemically synthesis, high tissue penetration, non-immunogenicity, adaptable modification, low cost and thermostable. 4 Therefore, aptamers have attracted extensive attention in terms of targeted therapy. 5 Pegaptanib is an RNA aptamer that is specific to VEGF165, a subgroup of the VEGF family. In December 2004, the US Xueping Xie and Yuxin Zhang contribute equally to this work. FDA approved pegaptanib for the treatment of all types of AMD. 2 As we all know, tumour blood vessels play an important role in tumour growth, providing essential oxygen and nutrients for tumour metabolism and metastasis. 6,7 VEGF is a very important regulator of endothelial cell growth and survival. 8 So inhibiting VEGF may be a viable way to treat cancer. 9,10 Therefore, pegaptanib might be a promising candidate for VEGF-targeting drugs for cancer therapy. There are few studies on the anti-tumour effect of pegaptanib.
Pegaptanib is delivered through the bloodstream to the site of the tumour, which is different from intravitreal injection. 11 The relatively poor biostability in vivo, such as the susceptibility to nucleases and removed from the circulation rapidly limit the use of pegaptanib in cancer treatment. 12 It is important to introduce an effective aptamer delivery system to improve the biostability and half-time in vivo. [13][14][15][16] Heo et al generated an aptamer-antibody hybrid complex by reacting an anti-continine antibody with the continnine-conjugated pegaptanib aptamer, which suggested a novel aptamer delivery system for pegaptanib. 5 DNA nanomaterials have attracted extensive attention in recent years due to their nanometer size, molecular recognition and controllability. [17][18][19] TDNs, self-assembled by four single-stranded DNAs (ssD-NAs) based on their highly specific Watson-Crick base pairing, is one of the hot topics in the research field of the DNA nanomaterials. 20,21 In our previous study, we investigated the applications of TDNs in molecular regulation, disease therapy and drug delivery. [22][23][24][25][26][27] Zhang et al successfully transported antisense peptide nucleic acids (asPNAs) into methicillin-resistant Staphylococcus aureus cells by TDNs to effectively inhibit bacterial. 26 Hyukjin Lee et al showed TDNs could be regarded as siRNA nanocarrier to silence target genes in tumours. 28 More interestingly, Ma et al synthesized an intelligent DNA nanorobot based on TDNs which enhance protein lysosomal degradation of HER2 in vitro. 29 What's more, some chemotherapeutic drugs loaded TDNs could overcome drug-resistant cancers. 27,30 In this study, we took TDNs as the nanocarrier of pegaptanib ( Figure 1A) to investigate their effects of anti-angiogenesis and anti-tumour compared with pegaptanib alone.

| Characterization of TDNs and Pegaptanib-TDNs
The successful synthesis of TDNs and pegaptanib-TDNs was examined by 8% polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. [32][33][34] Capillary electrophoresis was directed by Qsep100 TM automatic nucleic acid protein analysis system. The F I G U R E 1 A, Sketch map of pegaptanib-TDNs. B, Native PAGE to verify the assembly of TDNs and pegaptanib-TDNs. C, The peak chart of marker and each molecule detected by capillary electrophoresis. D, Typical size distribution graphs of pegaptanib-S4 and pegaptanib-TDNs hydrodynamic sizes of TDNs and pegaptanib-TDNs were measured by A Zetasizer Nano-ZS (Malvern Instruments).

| Wound healing assay
This section was prepared on the basis of the previously reported methods. 35,36 HUVECs were seeded in 6-well plates and cultured for 24 hours. After serum-free starvation overnight, we used the sterilizer tip to scrape a two-way wound at the bottom and washed the cells three times with PBS. The cells were treated with VEGF (25 ng/mL), VEGF (25ng/mL) +TDNs (375 nmol/L), VEGF (25 ng/mL) +pegaptanib (375 nmol/L) and VEGF (25 ng/mL) +pegaptanib-TDNs (375 nmol/L). Wound closure was imaged after cultivation for 0 and 24 hours, respectively.
The cell pellets were resuspended into 500 μL PBS. Subsequently, cell suspensions were measured by flow cytometry.

| Statistical analysis
One-way ANOVA (analysis of variance) or Student-Newman-Keuls test was used to perform statistical analysis of data and P < 0.05 indicated that group means were significantly different. All quantitative results were presented as mean ± standard deviation (SD).
Capillary electrophoresis was also utilized to examine the synthesis of these materials. As shown in Figure 1C, the peak of pegaptanib-S4 and pegaptanib-TDNs shifted to the right compared with S4 and TDNs. Size of TDNs and pegaptanib-TDNs which, respectively, was about 10nm and 22nm was measured by DLS ( Figure 1D). All the results proved that pegaptanib was successfully loaded onto TDNs.

| Cell proliferation of HUVECs and Cal27
The cell proliferation was evaluated by CCK-8 assay. As shown in Figure 2A

| Pegaptanib-TDNs can inhibit proliferation, migration and tube formation of HUVECs induced by VEGF
VEGF, an angiogenic factor, is crucial to promote the prolifera- In Figure 3A, pegaptanib (250 nmol/L) and pegaptanib-TDNs (250 nmol/L) could not inhibit the proliferation of HUVECs induced by VEGF. In Figure 3B, 375 nmol/L pegaptanib-TDNs had a stronger inhibitory effect compared with 375 nmol/L pegaptanib alone.
HUVECs migration is a necessary process of angiogenesis.
VEGF is a chemokine of HUVECs, which can promote HUVECs migration by activating cytoskeleton remodelling signalling pathways. 9,37 A wound healing assay was applied to assess the inhibition of pegaptanib and pegaptanib-TDNs on VEGF-induced migration of HUVECs. After incubation with 375 nmol/L pegaptanib for 24 hours, wound closure was 39%, whereas it was just 23% when cell cultured in 375 nmol/L pegaptanib-TDNs for 24 hours. At the same time, wound closure in control and VEGF group was 38% and 62% ( Figure 3C,D). Both pegaptanib and pegaptanib-TDNs could inhibit migration of HUVECs induced by VEGF. But pegaptanib-TDNs showed remarkably stronger inhibition compared with pegaptanib alone which was statistically significant.
These results suggested that pegaptanib loaded onto TDNs could effectively enhance the ability of pegaptanib to inhibit proliferation, migration and tube formation of HUVECs.

| The serum stability of pegaptanib and pegaptanib-TDNs
Subsequently, the serum stability of pegaptanib and pegaptanib-TDNs was evaluated. The result of gel electrophoresis demonstrated that pegaptanib-TDNs was almost completely degraded after incubated with 10% (v/v) FBS at 37°C in 5% CO 2 for 24 hours, but pegaptanib occurred obvious degradation at 2 hours and complete degradation <6 hours ( Figure 5C). Statistical analysis in Figure 5D showed that pegaptanib was degraded more rapidly than pegaptanib-TDNs.

| D ISCUSS I ON
In this study, we synthesized pegaptanib-TDNs and investigated the effect of anti-tumour and anti-angiogenesis. Pegaptanib, an RNA aptamer, was successfully linked to a vertex of TDN ( Figure 1).

Pegaptanib-TDNs could inhibit proliferation of HUVECs and Cal27
in a concentration-dependent manner. When these two cell lines were cultured with 375 nmol/L pegaptanib-TDNs, a significant inhibition of cell proliferation was observed. However, pegaptanib had no apparent restraint on cell proliferation in the same concentration ( Figure 2). Cal27 with VEGF high expression is a human tongue squamous cell carcinoma cell line. 38 Abundant blood vessels provide essential conditions for its growth and metastasis. 39

| CON CLUS IONS
Taken together, we put forward a novel drug carrier for pegaptanib.
TDNs could help pegaptanib overcome the limitations of aptamer and broaden its application in VEGF-targeting cancer therapy. Our results demonstrated again that TDNs might be a vehicle of potential value for disease therapy.

ACK N OWLED G EM ENTS
This study was funded by the National Natural Science Foundation of China (81771125, 81471803).

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

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