Self-assembled nanoconstructs modified with amplified aptamers

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Self-assembled nanoconstructs modified with amplified aptamers inhibited tumor growth and retinal vascular hyperpermeability via VEGF capturing Jihyun Lee, Byung Joo Lee, Yeong Mi Lee, Hansoo Park, Jeong Hun Kim, and Won Jong Kim Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00949 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 20, 2017

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Molecular Pharmaceutics

Self-assembled nanoconstructs modified with amplified aptamers inhibited tumor growth and retinal vascular hyperpermeability via VEGF capturing Jihyun Lee,†,1,2 Byung Joo Lee,†,3,4 Yeong Mi Lee,1,2 Hansoo Park,5 Jeong Hun Kim,*,3,4,6 and Won Jong Kim*,1,2 1

Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang 37673,

Republic of Korea 2

Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang

37673, Republic of Korea 3

Fight Against Angiogenesis-related Blindness Laboratory, Clinical Research Institute, Seoul

National University Hospital, Seoul 03080, Republic of Korea 4

Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul

03080, Republic of Korea

5

School of Integrative Engineering, Chung-Ang University, Seoul 156-751, Republic of Korea

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Department of Ophthalmology, College of Medicine, Seoul National University, Seoul 03080,

Republic of Korea

KEYWORDS: Anti-VEGF therapy, DNA nanoconstructs, Polymer-DNA conjugates, Anti-tumor therapy, Retinal vascular hyperpermeability

ACS Paragon Plus Environment

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ABSTRACT Here, nanoconstructs consisting of a DNA-amplified aptamer with a biocompatible polymer backbone for capturing target biomolecules are presented. First, the polymer-DNA nanoconstructs were prepared by hybridization of two complementary single-stranded DNAs that were each conjugated to a dextran polymer backbone. The designed polymer-DNA amplified aptamer nanoconstructs (PA-aNCs) were then prepared by utilizing polymer-DNA nanoconstructs conjugated with an aptamer (PA-NCs) using a rolling circle amplification reaction to amplify the aptamer. These PA-aNCs were successfully applied to alleviate tumor growth and VEGF-induced retinal vascular hyperpermeability in vivo through the highly effective capture of human VEGF as a target molecule. These PA-aNCs could be used as therapeutic agent for anti-VEGF therapy by efficiently capturing human VEGF.


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INTRODUCTION As information on the molecular pathogenesis of human diseases has accumulated, therapeutic methods that directly target “disease-inducing” molecules have attracted attention as efficient tools for treatment. After therapeutic monoclonal antibodies were identified as one of the most promising treatments for autoimmune diseases, several reagents for targeting disease-specific pathogenic proteins, such as monoclonal antibodies, receptor fusion proteins, and aptamers, were introduced into clinical use. However, because the majority of monoclonal antibodies have a short half-life of less than 1 month1, patients with chronic diseases need repeated administrations of monoclonal antibodies. Vascular endothelial growth factor (VEGF)-A was first identified as an important survival factor for vascular endothelial cells. In terms of biologic function, VEGF is one of the most potent pro-angiogenic factors and is a key modulator of vascular permeability. VEGF is widely accepted to be the major regulator in pathologic neovascular-related diseases, such as wet-type age-related macular degeneration (AMD) and angiogenic tumor growth. Moreover, as a strong enhancer of vascular permeability, it is also highly involved in diseases associated with pathogenic vascular leakage, including diabetic macular edema. Anti-VEGF treatment is now considered to be the most important treatment modality for wettype AMD, diabetic macular edema, and several cancers. There are various techniques for blocking the VEGF pathway, including using (1) an antibody (bevacizumab, ranibizumab) or (2) aptamer (pegaptanib) to neutralize VEGF or the VEGF receptor (VEGFR), (3) siRNA to target VEGF mRNA, (4) small molecule tyrosine kinase inhibitors (lapatinib, sunitinib) against the VEGF receptor, and (5) soluble VEGF receptors (sVEGFR) to inhibit the interaction between


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VEGF and VEGFR.2,3 Recently, many anti-VEGF drugs have been reported for the treatment of VEGF-related diseases; however, the efficiency, toxicity, and stability of these drugs under physiological conditions in the body are unclear.4,5 Pegaptanib, an RNA aptamer against VEGF165, was the first FDA approved anti-VEGF drug for the treatment of neovascular AMD.6 This aptamer drug was proven to be safe but not very effective for improving vision when compared to other alternative anti-VEGF agents such as ranibizumab (monoclonal antibody against VEGF) and aflibercept (VEGF receptor 1 and 2 fused to the Fc portion of IgG), so it is currently seldom used in clinical practice7,8. Binding affinity against VEGF and in vivo stability are major factors that influence the biological effect of anti-VEGF aptamers. Although it is structurally modified to improve resistance against nuclease, pegaptanib has a relatively short in vivo half-life (9.3 h after intravenous injection and 12 h after subcutaneous injection)9. Herein, we present “polymer-DNA amplified aptamer nanoconstructs (PA-aNCs)” as a selective VEGF capturing agent for the suppression of tumor growth and treatment of macular edema (Scheme 1). The polymer-DNA aptamer nanoconstructs (PA-NCs), as previously reported by our group, were constructed by self-assembly via hybridization of sense DNA and complementary antisense DNA (cDNA) conjugated with oxidized dextran (Dex-CHO).10 The PA-aNCs were prepared by amplification of a VEGF aptamer via an enzymatic reaction named “rolling circle amplification (RCA).” The PA-aNCs are composed of hybridized duplex DNA to allow for the formation of nanoconstructs and amplified aptamer to allow for target of the VEGF molecule respectively. The stability and physico-chemical characteristics of PA-aNCs were evaluated, and their ability to capture VEGF molecules was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, the in vivo suppression of tumor growth and


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treatment of retinal vascular hyperpermeability model by capturing VEGF were demonstrated utilizing PA-aNCs.

Scheme 1. Schematic illustration of PA-aNCs preparation for sensitive and selective capturing of VEGF. PA-aNCs was constructed by self-assembly via DNA hybridization. The efficacy of PAaNCs as a drug was evaluated in tumor-bearing mouse model and retinal vascular hyperpermeability mouse model.


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MATERIALS AND METHODS Materials. Dextran (MW=40 kDa) was obtained from TCI (Tokyo, Japan). Sodium periodate, hydroxylamine hydrochloride, 2,4-dihydroxybenzaldehyde, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) was purchased from sigma-aldrich (St. Louis, USA). N-(ßmaleimidopropionic acid) hydrazide (BMPH) was purchased from Pierce (IL, USA). Amicon ultra centrifugal filters were purchased from Merck Millipore (MA, USA). SYBR Gold I® was purchased from Invitrogen (Eugene, Oregon, USA), and mounting medium for fluorescence with DAPI was purchased from VECTOR (Burlingame, USA). CircLigaseTM ssDNA Ligase (Epicentre Biotechnologies, Madison, WI), Exonuclease I (New England Biolabs, Beverly, MA), Exonuclease III (New England Biolabs, Beverly, MA), Φ29 DNA polymerase (New England Biolabs, Beverly, MA), dNTP mix (Promega, Madison, WI) and recombinant human vascular endothelial growth factor-165 (rhVEGF-165) (BioLegend, San Diego, CA) were also purchased. All DNA sequences were purchased from BIONEER (Daejeon, South Korea) and IDT (mbiotech, Korea). The sequence of human VEGF DNA aptamer was reported previously.11,12 The oligodeoxynucleotides (ODNs) sequences are listed in Table S1.

Synthesis of aldehyde-functionalized dextran (Dex-CHO). Dextran (500 mg, 3.08 mmole of glucose units) was dissolved in 10 mL of deionized water. Sodium periodate (660 mg, 3.08 mmole) also dissolved in 20 mL of deionized water. The prepared sodium periodate solution was added dropwise to the dextran solution, and the mixture was vigorously stirred in dark at 4oC for 12 h. The resulting solution was dialyzed with 3.5 kDa dialysis membrane against deionized


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water in dark at 4oC for 2 days and lyophilized to obtain 477 mg of white powder with 95.4 % yield (Figure S1A). The aldehyde content in Dex-CHO was measured according to the previous report.10 DexCHO (10 mg) was dissolved in hydroxylamine hydrochloride solution (5 mL, 0.25 M) of pH 2.10. The mixture was sonicated for 1 h, and stored in dark at room temperature for 12 h. Degree of periodate cleavage was measured by the titration of the HCl which was generated on the reaction of aldehyde with measured amount of hydroxylamine hydrochloride. The titration was carried out with standard NaOH (0.1 N) solution until the end point reached at pH 2.10. The aldehyde content of the samples was calculated by comparing the titer values of NaOH with that obtained from standard curve by plotting the volume of NaOH against the amount of 2,4dihydroxybenzaldehyde. The average cleavage degree was calculated as 84.6 %. The Dex-CHO, oxidative ring cleavage of dextran, was analyzed by gel permeation chromatography (Shimadzu, Kyoto, Japan) with a refractive index detector (RID-10A, Shimadzu, Kyoto, Japan) using a column (SB-806M, SB-803HQ, ShowaDenko, Tokyo, Japan). Deionized water was utilized as an eluent with flow rate of 1 mL/min, and column temperature was maintained at 40oC. The Dex-CHO was slightly decreased in molecular weight compared to original Dextran (Figure S2A).

Synthesis of hydrazine-modified DNA via sulfide-carbon linkage (DNA-S-C-Hydrazide). To prepare a free thiol group at 5’-end of single stranded DNA, 2 equivalents of TCEP-HCl was added to 1 equivalent of 5’ thiol-blocked DNA in PBS buffer (pH 8.0) and vigorously shaken for 30 min at room temperature. After the deprotection reaction, the reactant was purified using


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centrifugal filteration (Amicon Ultra-4, MWCO 3 kDa). The DNA-S-C-Hydrazide was synthesized by reaction of SH-DNA (1 equivalent) with BMPH (10 equivalents) in dimethylsulfoxide (DMSO)/PBS buffer (pH 8.0) for 12 h at room temperature. After reaction, excess BMPH was removed using centrifugal filteration (Amicon Ultra-4, MWCO 3 kDa), and the resultant solution was lyophilized to obtain DNA-S-C-Hydrazide. This procedures were performed with [Thiol]ODN, [Thiol]cODN, [Thiol]VEGF_aptamer, [Thiol]Scramble and [Thiol]DNA-TAMRA (Figure S1B).

Conjugation of Dex-CHO and DNA-S-C-hydrazide. Dex-S-C-ODN/VEGF_aptamer (POA) conjugate was prepared by reaction of Dex-CHO (1 equivalent, 10 µM) with ODN-S-Chydrazide (5 equivalents, 10 µM) and VEGF_aptamer-S-C-hydrazide (2.5 equivalents, 10 µM) simultaneously in PBS buffer for 24 h at room temperature with shaking. After reaction, the resultant mixture was centrifugal filtered by Amicon Ultra-4 (MWCO 10 kDa) at 3000g for 10 min, and the purified solution was lyophilized. The conjugation of DNAs (ODN, VEGF aptamer) with Dex-CHO was confirmed by gel electrophoresis analysis (Figure S2B). The ratio between ODN (or cODN) and repeating units of Dex-CHO was estimated as 1:45. In addition, the ratio between VEGF_aptamer and repeating units of Dex-CHO was estimated as 1:91. Dex-S-CcODN/VEGF_aptamer (PcOA), Dex-S-C-ODN/Sc (POSc), and Dex-S-C-cODN/Sc (PcOSc) conjugates were also prepared by same procedure (Figure S1C).

Preparation of circularized DNA template. According to the CircLigaseTM protocol, the circularized DNA was obtained. Briefly, phosphorylate modified DNA was mixed with ATP,


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MgCl2, and ligase enzyme in buffer solution. After mixing, the solution was incubated at 60oC for 12 h. After enzyme inactivation process, the resultant solution was treated with 20 U of Exonuclease I and 100 U of Exonuclease III at 37 oC for 30 min. Then enzyme inactivation process was also conducted. The circularized DNA was checked by denatured polyacrylamide gel electrophoresis after purification of DNA PrepMateTM-II, and the concentration was measured by UV absorption at 280 nm. The used circular DNA templates were [Phos]Circ_cVEGF and [Phos]Circ_cSc.

Preparation of polymer-aptamer amplified nanoconstructs (PA-aNCs). The PA-NCs was prepared by hybridization of both POA and PcOA by annealing process. The ‘rolling circle amplification (RCA)’ process for preparation of PA-aNCs was conducted followed by Φ29 polymerase protocol. Briefly, the prepared circularized Circ_cVEGF and the PA-NCs were added into dNTPs, BSA and Φ29 polymerase contained buffer solution. After mixing, the solution was incubated at 30oC for determined time (0.5, 0.75, and 1 h). The resultant solution was incubated at 80oC for enzyme inactivation, and gradually cooled down. The PSc-NCs (polymer-DNA nanoconstructs conjugated with a scrambled DNA) and PSc-aNCs (polymerDNA nanoconstructs conjugated with an amplified scrambled DNA) were also prepared by same procedures (Figure S1D).

Stability test. The prepared aptamer (oligomer), PA-NCs, and PA-aNCs were incubated with fetal bovine serum (FBS) 10 % solution for 4 days at 36.5oC under dark condition. The stability


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of aptamer was confirmed by polyacrylamide gel electrophoresis (PAGE) analysis, and the stability of PA-NCs and PA-aNCs were analyzed by agarose gel electrophoresis (AGE) analysis.

Cytotoxicity test. The cytotoxicity of Dex-CHO, PA-NCs, PA-aNCs were evaluated utilizing the MTT assay. A549 cells were seeded in 96-well plate with a density of 5 x 103 cells/well and incubated for 24 h in CO2 incubator. The prepared samples (Dex-CHO concentration as a standard) with various concentration (0, 100, 200, 300, 400, and 500 nM) were treated in the cell with serum free media (DMEM) for 5 h. These supernatant were eliminated and replaced with fresh serum contained media for another 20 h. Then, these mixtures were replaced with fresh media and MTT solution (5 mg/mL), and incubated for 4 h. The media was removed and 100 uL of DMSO was added into each well to dissolve the internalized purple formazan crystals. The absorption was measured at 570 nm utilizing a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, Perkin Elmer, Wellesley, MA, USA). The relative percentage of the control samples which were added with only media were used as 100 % cell viability.

Transmittance electron microscope (TEM) analysis. Samples were prepared by 10 µM of POA, PA-NCs, and PA-aNCs (concentration of Dex-CHO) onto carbon grids coated with an ultrathin carbon film (400 mesh; Ted Pella, Redding, CA, USA). The grid was dried over 1 day in desiccator before measurement. TEM image was obtained by a JEM-1011 (JEOL, Tokyo, Japan).


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Gel electrophoresis. PAGE and AGE analysis were conducted to confirm the stability of DNA in NCs. The oligomeric DNA was run on the 13 % polyacrylamide gel for 60 min at 100 V using TBE (tris/borate/EDTA) buffer as a running buffer. The DNA was observed by SYBR Gold® staining method, followed by UV illumination. The PA-NCs and PA-aNCs were run on the 1 % agarose gel for 10 min at 100 V using TAE (tris/acetic acid/EDTA) buffer as a running buffer. The DNA was observed by EtBr. The monochrome images were obtained by Davinch Western Imaging System (Davinch-K, Younghwa Science, Korea).

Capturing test of VEGF utilizing PA-aNCs. To confirm the capturing efficiency of rhVEGF by the PA-aNCs, PA-aNCs, each sample was mixed with rhVEGF solution (50 pg) for 4 h at room temperature. The mixture solution was filtered by PD-10 column (Sephadex G-25) (GE Healthcare, Sweden) to purify free rhVEGF (Figure S3). The PA-aNCs and rhVEGF mixture solution was loaded at PD-10 column and eluted for 7 min. The detailed procedure can be referred to manufacturer’s instruction. The filtered solution was concentrated using centrifugal filteration tube (Amicon Ultra-4, MWCO 3 kDa). The amount of non-captured VEGF was calculated and measured by ELISA kit for VEGF (Komabiotech, Seoul) utilizing concentrated solution according to manufacturer’s instruction. The absorbance at 490 nm was measured by VICTOR3 VTM (Multilabel Counter, Perkin Elmer, Wellesley, MA, USA)). The rhVEGF capture ability of PA-NCs, PSc-NCs and PSc-aNCs were measured by same procedure.

Retinal vascular hyperpermeability model induced by VEGF. Six weeks old, specific pathogen free male C57BL/6 mice were purchased from Central Laboratory Animal Inc. (Seoul,


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Korea) and used for experiment after an adaptation period of 1 week. Animal experiments in this study was performed based on the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research. Ahead of experimental procedure, mice were anaesthetized with intraperitoneal injection of Zoletil (30 mg/kg) and Rompun (10 mg/kg) mixture. For induction of retinal vascular hyperpermeability, 40 ng of rhVEGF was injected into the vitreal cavity of every mice. Simultaneous intravitreal injection of P / PA-NCs / PSc-NCs / PA-aNCs / PSc-aNCs was done according to the treatment groups (n = 6 per each group). The assessment of retinal vascular leakage using Evans blue was performed, 24 hrs past the injection of rhVEGF, as previously described by our group13. Under anesthesia, total 200 µL of Evans blue dye solution (dissolved in PBS at the concentration of 20 mg/mL) was injected into the left ventricle of the mice. Mice were sacrificed 2 hrs after dye injection and enucleated. For qualitative analysis, retinas were dissected and flat-mounted, then were photographed by fluorescence microscopy (Nikon). After the measurement of wet weight, retinas were incubated in formamide (18 hrs at 70℃) for the extraction of extravasated Evans blue dye. Then, the retina was removed by centrifugation and the A620 value of each sample was measured with spectrophotometer. Total Evans blue content of each sample was estimated by standard curve and divided by the wet weight14.

In vivo tumor-bearing mice model. A549 cells (1x108) were inoculated subcutaneously into the flank of each female BALB/c-nu/nu mice. The mice were randomly divided into six groups (5 mice per group, n=5) and injected with P / PA-NCs / PSc-NCs / PA-aNCs / PSc-aNCs on day


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0 and day 7. One mouse in each group was sacrificed on day 4 for its immune-histochemical analysis. The anti-tumor effect against A549 growth was assessed by measurement of tumor diameter with a caliper. The measured values were converted to tumor volume using the formula for a prolate ellipsoid as follow equation: Tumor volume = a x b2 x 0.5 (1) where a is the shorter dimension and b is the longer dimension. After injection of each samples, tumor progression in the mice was monitored until day 32, on which time the mice were sacrificed. Change of body weight was also measured. All animal experiments were approved by the POSTECH biotech center ethics committee.

Immunohistochemistry and histology analysis. For immune-histochemical analysis, one mouse in each group was sacrificed on day 4. The tumor were excised and fixed in 10 % neutral buffered formalin (NBF) for 24 h and embedded in paraffin and sequentially sectioned at 4 µm using a Finesse ME microtome. Tumor sections were stained with hematoxylin and eosin (H&E) for the assessment of tumor regression. Images were obtained by a microscopy (Nikon eclipse 80i, USA) with 10x magnification located at the Pohang Center for Evaluation of Biomaterials (Pohang Technopark). For immune-histochemical staining with CD-31, the tumor sections embedded in paraffin cut at 4 µm thick-ness were firstly treated with blocking solution (5 % (w/v) of BSA in DI-water) to avoid non-specific binding. After several times washing with PBS for 15 min, the primary


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antibody was applied for 1 h at room temperature. Secondary antibody conjugated to goat antirabbit lgG-FITC was treated for 1 h. Finally sections on coverslip were mounted in DAPI mounting medium to see the cell nuclei and stored in dark condition at 4oC. Fluorescent images were obtained by utilizing confocal laser scanning microscope (Modified Zeiss Axio Observer, Z1 epi-fluorescence microscope) with 40 x magnification.

RESULTS AND DISCUSSION Synthesis and characterization of PA-aNCs. The PA-NCs, as reported previously, consisted of annealed dsDNA, a VEGF aptamer, and dextran-CHO polymer were prepared utilizing two different dextran-DNA/VEGF aptamer conjugates with hydrazone linkage: Dex-DNA/VEGFapt and Dex-cDNA/VEGFapt.10 The aldehyde functionalized dextran (Dex-CHO) was obtained by oxidization of dextran with sodium periodate, and the ratio of the aldehyde functionalized group in the dextran was calculated to be 84.6 % utilizing a titration curve and the measured amount of 2,4-dihydroxybenzaldehyde. Additionally, the decrease in the MW of oxidized dextran (DexCHO) was analyzed by gel permeation chromatography (GPC) analysis (Figure S2). To conjugate DNA to the aldehyde group in Dex-CHO, the 5ʹ-end of the DNA was modified with a hydrazide group through reaction with N-(ß-maleimidopropionic acid)hydrazide. Utilizing DexCHO, hydrazide functionalized DNA/cDNA, and hydrazide functionalized VEGF aptamer, two different dex-DNA conjugates (Dex-DNA/VEGFapt and Dex-cDNA/VEGFapt) were separately synthesized at a 1:5:2.5 (Dex-CHO:DNA/cDNA:VEGFapt) ratio and purified by ultra-filtration. Then, the PA-NCs were constructed by the simple mixing and annealing of two complementary Dex-DNA/VEGFapt and Dex-cDNA/VEGFapt conjugates. After preparation of the PA-NCs, the


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amplification of the VEGF aptamer conjugated in the PA-NCs was conducted using Φ29 polymerase. Before utilizing the PA-NCs with the amplified VEGF aptamer (PA-aNCs), the PAaNCs solution was purified by ultra-filtration to remove the salts and proteins contained in the reaction buffer and stored at 4°C. The formation of the PA-aNCs was verified by DLS and TEM. The average diameters of the PA-NCs and PA-aNCs measured by DLS were ~ 420 ± 10 nm and ~ 620 ± 30 nm in Dulbecco’s phosphate-buffered saline (DPBS) (Figure 1A). To characterize the size and morphology of PAaNCs, TEM analysis was conducted (Figure 1B). TEM revealed that the sizes of PA-NCs and PA-aNCs in the dry state were ~ 400 nm and ~ 600 nm, respectively. To estimate the stability of PA-NCs and PA-aNCs, an excess amount of cDNA was added to each PA-NC and PA-aNC solution. The addition of cDNA disrupted the formation of PA-NCs, revealing that the hybridization of DNA/cDNA in the PA-NCs was destabilized by the exchange reaction with the added cDNA. In contrast, the formation of PA-aNCs was not disrupted even by the addition of cDNA (Figure S4). This result showed that the PA-aNCs have a higher stability than the PANCs, which might be due to protection of the amplified VEGF aptamer. In order to investigate the protection of the VEGF aptamer by PA-NCs against serum enzymes, including DNA nuclease, the oligomeric VEGF aptamer, PA-NCs, and PA-aNCs were incubated separately in DPBS containing 10 % FBS with shaking at 37°C. As shown in Figure 1C, degradation of the oligomeric VEGF aptamer was observed at 0.5 d, and it was totally degraded after 1 d. However, both PA-NCs and PA-aNCs showed high stability up to 4 d of incubation. These results suggested that the structures of PA-NCs and PA-aNCs were dense enough to protect and shield the VEGF aptamer efficiently from nucleolytic enzymes in the serum.15,16


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Additionally, the PA-aNCs were highly stable for 2 months at room temperature in PBS solution, which suggests the possibility of long-term storage (Figure 1D).

Figure 1. Analysis of PA-aNCs characteristics by using (A) DLS, (B) TEM, (C) PAGE, and (D) AGE. (C) Serum stability test of aptamers analyzed by PAGE. (D) Long-term stability of nanoconstructs analyzed by AGE. The PA-aNCs with freshly prepared (lane 1), after 1 month (lane 2), and after 2 months (lane 3) incubated with PBS at room temperature were tested.

Capture of target VEGF molecules by PA-aNCs. To investigate the potential therapeutic effect of the PA-aNCs, the VEGF capturing efficiency of PA-aNCs was tested. First, the amplification time for the VEGF aptamer was optimized by comparing various RCA times. Briefly, VEGF in intravitreal mimicking solution (10 % FBS, gelatin, chondroitin sulfate sodium salt in DPBS) was mixed with aNCs produced using different amplification times for the aptamer


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(0.5 h, 0.75 h, and 1.0 h). As shown in Figure 2A, the PA-aNCs with a 0.75-h amplification time showed the most efficient VEGF capturing capacity. In addition, the amount of primer (oligomeric aptamer) in PA-aNCs for efficient VEGF capture was optimized using a similar procedure, and 25 pmole primer in PA-aNCs resulted in efficient VEGF capturing characteristics at a given concentration of VEGF (Figure 2B). Therefore, PA-aNCs with 0.75 h of amplification and containing 25 pmole primer per 50 pg of [VEGF] were utilized for further VEGF capturing analyses. Finally, a solution containing VEGF was mixed with each sample of PA-NCs, PScNCs, PA-aNCs, and PSc-aNCs ([primer] = 25 pmole) and shaken for 12 h at 37°C (Figure 2C for intravitreal mimicking solution, and Figure S5 for DPBS). After purification of free-VEGF with a PD-10 column, the amount of unbound VEGF was quantified by a VEGF ELISA kit. Approximately, 97.0 % of VEGF was captured by PSc-aNCs compared to lower VEGF capturing efficiencies for PA-NCs (9.21 %), PSc-NCs (8.18 %), and PSc-aNCs (7.12 %). These results indicated that the amplified VEGF aptamer contributed to an efficient VEGF capture capacity. VEGF is overexpressed in solid tumor tissue and binds to VEGF receptor (VEGFR) tyrosine kinases to activate various signaling pathways, such as angiogenesis and tumorigenesis.12,17 Therefore, tumor growth can be suppressed by anti-VEGF therapy such as the elimination of VEGF with an anti-VEGF agent. In this regard, we hypothesized that PA-aNCs could inhibit tumor growth more efficiently compared to PA-NCs with a short oligomeric aptamer. To study the non-specific cell toxicity of each sample, cytotoxicity tests were carried out utilizing prepared PA-aNCs for a further in vivo analysis (Figure 2D). All samples (P, PA-NCs, and PA-aNCs) showed no significant toxicity in A549 cells, which indicated that they could be


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applied for in vivo tumor regression tests and treatments in a VEGF-induced retinal vascular hyperpermeability model.

Figure 2. ELISA and cytotoxicity test for designed PA-aNCs. (A) For optimizing the amplification time of aptamer, efficiency of VEGF capture was investigated after amplification of aptamer in PA-aNCs on determined incubation time of 0.5, 0.75, and 1 h. Utilizing 25 pmole of VEGF aptamer primer, each PA-aNCs with 0.5 h, 0.75 h, and 1 h of RCA reaction had 550 pmole, 825 pmole and 1125 pmole of VEGF aptamer. (B) The capturing efficiency of VEGF with various concentration of aptamer in PA-aNCs was tested using constant VEGF concentration. (C) The capturing efficiency of PA-NCs, PSc-NCs, PA-aNCs and PSc-aNCs. (D)


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Cell viability analysis of A549 cells treated with various concentrations of each Dex-CHO, PANCs, and PA-aNCs.

In vivo tumor growth inhibition assay. Tumors require new blood vessels, the formation of which is induced by the expression of angiogenic growth factors (e.g., VEGF), to receive nutrients for their growth.18,19 Thus, tumor growth could be suppressed through anti-VEGF therapy, which deactivates VEGF function by the action of anti-VEGF agents. In this respect, the designed PA-aNCs could be a good alternative for the suppression of tumor growth. Therefore, we examined the anti-tumor growth activity of the PA-aNCs through VEGF capture in an A549 xenograft mouse model. As shown in Figure 3A, P, PA-NCs, PSc-NCs PA-aNCs, and PSc-aNCs were injected into tumors (average size on day 0 was 66 mm3) on day 0 and day 7. The nontreated control group showed a 7.4-fold increase in tumor size on day 32. The groups treated with the P, PSc-NCs, and PSc-aNCs rarely showed tumor regression effects (increases of 6.6fold for P, 7.0-fold for PSc-NCs, and 5.9-fold for PSc-aNCs). And the groups treated with the PA-NCs relatively exhibited the tumor regression effect (increases of 6.5-fold for PA-NCs) compared to other P, PSc-NCs, and PSc-aNCs, however the tumor growth inhibition was not sufficient. In contrast, the mice treated with the PA-aNCs showed a 0.74-fold decrease in tumor size on day 32. Because the efficiency of tumor growth inhibition by PA-NCs was 8.8 times lower compared to that of PA-aNCs, the amplification of the aptamer sequence was considered to be a critical factor for the capture of target molecules. We hypothesize that the aptamers in PA-NCs rarely interacted with target molecules because they were not exposed to the surface of the nanoconstruct. As shown in Figure 3B, tumor growth after the injection of PA-aNCs (day 0 and day 7) was suppressed for ~ 5 d, which correlated with the serum stability of PA-aNCs. The


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reason for this result is that the blockage of the VEGF pathway in tumor tissue at the early stage by VEGF capture by PA-aNCs significantly contributed to the inhibition of tumor growth. As shown in the body weight profile, no group exhibited a significant loss of body weight at day 32, revealing that the designed PA-aNCs were not toxic (Figure 3D). The capture or elimination of VEGF from a tumor site would inhibit tumor angiogenesis because the deactivation of VEGF function is critical to the inhibition of tumor angiogenesis. Therefore, we carried out immuno-histochemical staining with anti-CD31 antibody and FITCmodified secondary antibody for each tumor tissue group and assessed tumor angiogenesis (Figure 4). The tumor tissues were obtained on day 4. The tumor tissues stained with CD31 after treatment with P, PA-NCs, PSc-NCs, or PSc-aNCs and those in the non-treated groups showed high FITC fluorescence, whereas fluorescence was rarely observed in the PA-aNC group. In addition, the hematoxylin and eosin (H&E)-stained sections of the tumor sites are represented in figure S6. Nuclei shows loosely packed manner and meronecrosis was observed in case of PAaNCs-treated tumor compared to other NCs treated tumors. These results support that the conclusion that the capture of VEGF molecules by the designed PA-aNCs blocked angiogenesis at the tumor site and subsequently inhibited tumor growth.


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Figure 3. In vivo tumor growth suppression assay. Tumor growth profile of each groups (nontreated control, P, PA-NCs, PSc-NCs PA-aNCs, and PSc-aNCs) from day 0 to day 32 (A) and magnified profile from day 0 to day 14 (B). (C) Ex vivo tumor image of each group at day 32. (D) Body weight profile of each group from day 0 to day 32. The red arrows show the injection date. *P < 0.05.


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Figure 4. Immuno-chemical staining image of tumor tissues for visualizing CD31 (green) at day 4. Nucleus was stained with DAPI (blue). Scale bar represents 50 µm.

In vivo retinal vascular hyperpermeability assay. The tight junctions of the retinal vascular endothelium provide a functional barrier between the neural retina and circulating blood called the inner blood retinal barrier (BRB). Inner BRB dysfunction is common in patients with diabetic retinopathy or retinal vein occlusion (RVO), resulting in the extravasation of plasma into the neural retina, which is called macular edema. An increased intraocular VEGF concentration has been well documented in these patients20, and the disruption of the inner BRB is at least partially mediated by VEGF. In current clinical practice, anti-VEGF therapy is a treatment option for macular edema in diabetic retinopathy or RVO21.


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To assess the VEGF scavenging efficacy of the polymer-DNA aptamer nanoconstructs in vivo, we used a VEGF-induced retinal vascular hyperpermeability model. The degree of Evans blue dye extravasation was significantly decreased in eyes injected with PA-NCs (P = 0.01) or PAaNCs (P < 0.01) compared with that of polymer-injected eyes (Figure 5). In addition, PA-aNCs more effectively suppressed Evans blue dye leakage than PA-NCs (P = 0.03). Six eyes were assigned to each treatment group. Nanoconstructs delivered intravitreally successfully alleviated VEGF-mediated retinal vascular leakage, and nanoconstructs with the amplified aptamer sequence provided even greater improvement of extravasation. In addition, as shown in Figure 1C, PA-aNCs showed a higher stability than the oligomeric aptamer up to 4 d. In summary, the results indicate that the designed PA-aNCs are highly efficient and selective for capturing VEGF and have a long-term nuclease resistant characteristic, which is shortcoming of existing aptamer drugs, demonstrating that they are a good candidate for modified anti-VEGF aptamer drug.


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Figure 5. Intravitreally administrated PA-aNCs reduced VEGF induced retinal vascular hyperpermeability. (A) Representative photographs of the flat-mounted retinas of mice injected with P, PSc-NCs, PSc-aNCs, PA-NCs, and PA-aNCs. Scale bars: 100 µm. (B) Quantitative analysis of Evans blue extravasation in the retinas of each treatment group. Whereas the PScNCs/PSc-aNCs did not affect the amount of Evans blue extravasation induced by VEGF, PA-


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NCs and PA-aNCs significantly ameliorated Evans blue leakage. When compared with PA-NCs injected group, amplification of VEGF binding aptamer conjugated to PA-NCs significantly potentiated this effect. *P < 0.05.


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CONCLUSION In summary, we developed PA-aNCs, a novel DNA-mediated crosslinked nanoconstruct, by DNA hybridization. The PA-aNCs, nanoconstructs consisting of a polymer backbone and an amplified aptamer, showed long-term stability and nuclease resistance. In addition, a high selectivity and sensitivity of PA-aNCs for the target biomolecule was achieved by including the amplified aptamer. The PA-aNCs were applied to treat cancer and VEGF-induced retinal vascular hyperpermeability. After tumor-bearing mice were treated with PA-aNCs, tumor growth was successfully inhibited by the capture of VEGF. Similarly, when injected intravitreously, retinal vascular hyperpermeability was significantly inhibited by PA-aNCs, indicating their potential application for anti-VEGF treatment in macular edema. Our developed PA-aNCs, which have high sensitivity and stability, can be utilized as a new drug for anti-VEGF therapy by capturing the target VEGF.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. DNA sequence information, Synthesis procedures of PA-aNCs, GPC spectra, TEM images, VEGF capture efficiency test.

AUTHOR INFORMATION Corresponding Author


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* Address correspondence to [email protected] * Address correspondence to [email protected] Author Contributions †

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the Institute for Basic Science (IBS) [IBS-R007-D1]. This research was partly supported by Global Ph.D Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012-H1A2A1005715).

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(7) Gragoudas, E. S.; Adamis, A. P.; Cunningham, E. T., Jr.; Feinsod, M.; Guyer, D. R. Pegaptanib for neovascular age-related macular degeneration. The New England journal of medicine 2004, 351, 2805-2816. (8) Schmidt-Erfurth, U.; Chong, V.; Loewenstein, A.; Larsen, M.; Souied, E.; Schlingemann, R.; Eldem, B.; Mones, J.; Richard, G.; Bandello, F. Guidelines for the management of neovascular age-related macular degeneration by the European Society of Retina Specialists (EURETINA). The British journal of ophthalmology 2014, 98, 1144-1167. (9) Tucker, C. E.; Chen, L. S.; Judkins, M. B.; Farmer, J. A.; Gill, S. C.; Drolet, D. W. Detection and plasma pharmacokinetics of an anti-vascular endothelial growth factor oligonucleotide-aptamer (NX1838) in rhesus monkeys. Journal of chromatography. B, Biomedical sciences and applications 1999, 732, 203-212. (10) Namgung, R.; Kim, W. J. A Highly Entangled Polymeric Nanoconstruct Assembled by siRNA and its Reduction-Triggered siRNA Release for Gene Silencing. Small 2012, 8, 3209-3219. (11) Potty, A. S.; Kourentzi, K.; Fang, H.; Jackson, G. W.; Zhang, X.; Legge, G. B.; Willson, R. C. Biophysical characterization of DNA aptamer interactions with vascular endothelial growth factor. Biopolymers 2009, 91, 145-156. (12) Lee, J.; Lee, Y. M.; Kim, W. J. Polymer–DNA Molecular Net for Selective Transportation of Target Biomolecules and Inhibition of Tumor Growth. Chemistry of Materials 2016, 28, 3961-3967. (13) Yun, J. H.; Park, S. W.; Kim, K. J.; Bae, J. S.; Lee, E. H.; Paek, S. H.; Kim, S. U.; Ye, S.; Kim, J. H.; Cho, C. H. Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy. Journal of cellular physiology 2016. (14) Suarez, S.; McCollum, G. W.; Bretz, C. A.; Yang, R.; Capozzi, M. E.; Penn, J. S. Modulation of VEGF-induced retinal vascular permeability by peroxisome proliferator-activated receptor-beta/delta. Investigative ophthalmology & visual science 2014, 55, 8232-8240. (15) Murata, M.; Kaku, W.; Anada, T.; Sato, Y.; Kano, T.; Maeda, M.; Katayama, Y. Novel DNA/Polymer conjugate for intelligent antisense reagent with improved nuclease resistance. Bioorganic & Medicinal Chemistry Letters 2003, 13, 3967-3970. (16) Conway, J. W.; McLaughlin, C. K.; Castor, K. J.; Sleiman, H. DNA nanostructure serum stability: greater than the sum of its parts. Chemical communications (Cambridge, England) 2013, 49, 1172-1174. (17) Kubota, Y. Tumor angiogenesis and anti-angiogenic therapy. The Keio journal of medicine 2012, 61, 47-56. (18) Sitohy, B.; Nagy, J. A.; Dvorak, H. F. Anti-VEGF/VEGFR therapy for cancer: reassessing the target. Cancer research 2012, 72, 1909-1914. (19) Li, N.; Zheng, D.; Wei, X.; Jin, Z.; Zhang, C.; Li, K. Effects of recombinant human endostatin and its synergy with cisplatin on circulating endothelial cells and tumor vascular normalization in A549 xenograft murine model. Journal of Cancer Research and Clinical Oncology 2012, 138, 1131-1144. (20) Aiello, L. P.; Avery, R. L.; Arrigg, P. G.; Keyt, B. A.; Jampel, H. D.; Shah, S. T.; Pasquale, L. R.; Thieme, H.; Iwamoto, M. A.; Park, J. E.; et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. The New England journal of medicine 1994, 331, 1480-1487. (21) Campochiaro, P. A.; Brown, D. M.; Awh, C. C.; Lee, S. Y.; Gray, S.; Saroj, N.;


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Murahashi, W. Y.; Rubio, R. G. Sustained benefits from ranibizumab for macular edema following central retinal vein occlusion: twelve-month outcomes of a phase III study. Ophthalmology 2011, 118, 2041-2049.

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Scheme 1


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Self-assembled nanoconstructs modified with amplified aptamers

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