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Targeted Imaging of Brain Tumors with a Framework Nucleic Acid Probe Tian Tian, Jiang Li, Cao Xie, Yanhong Sun, Haozhi Lei, Xinyi Liu, Jiaoyun Xia, Jiye Shi, Lihua Wang, Weiyue Lu, and Chunhai Fan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17927 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Applied Materials & Interfaces

Targeted Imaging of Brain Tumors with a Framework Nucleic Acid Probe Tian Tian,†‡ Jiang Li,† Cao Xie,# Yanhong Sun,†* Haozhi Lei,† Xinyi Liu,† Jiaoyun Xia,⊥ Jiye Shi,§ Lihua Wang,† Weiyue Lu,#* Chunhai Fan†* †

Division of Physical Biology & Bioimaging Center, CAS Key Laboratory of Interfacial Physics

and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‡

Institute of Interdisciplinary Integrative Biomedical Research, Shanghai University of

Traditional Chinese Medicine, Shanghai 201203, China #

Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China

⊥

School of Chemistry and Biology Engineering, Changsha University of Science and

Technology, Changsha 410004, China §

UCB Pharma, Slough, SL1 14EN Berkshire, UK

ABSTRACT: Development of agents for delivering drugs and imaging probes across the blood brain barrier (BBB) remains a major challenge. In this study, we designed a biocompatible framework nucleic acid (FNA)-based imaging probe for brain tumor targeting. We employed a typical type of FNAs, tetrahedral DNA nanostructures (TDNs), as the building block, which were modified with angiopep-2 (ANG), a 19-mer peptide derived from human Kunitz domain of aprotinin. This probe exhibited high binding efficiency with low-density lipoprotein receptor-related protein-1 (LRP-1) of BBB and glioma. We found that ANG-TDNs stayed intact 1

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for at least 12 hours in serum, and that ANG modification effectively enhanced cellular uptake of TDNs in brain capillary endothelial cells (bEnd.3) and Uppsala 87 Malignant Glioma cells (U87MG). Remarkably, studies in both in vitro and in vivo models revealed that ANG-TDNs could cross the BBB. Especially, in vivo imaging showed strong fluorescent signals in U87MG human glioblastoma xenograft in nude mice. This study establishes that FNA-based platform provides a new theranostic tool for studying and therapy of brain tumors.

KEYWORDS: Blood Brain Barrier, DNA Tetrahedron, Angiopep-2, Glioma, Imaging

INTRODUCTION In the development of new drugs for the central nervous system (CNS) diseases, the blood brain barrier (BBB) permeability is a key limiting factor for the penetration and distribution of drugs from blood to brain.1-2 It has been reported that 98% of all small molecules and 100% of all big molecules cannot cross the BBB.3 In order to enhance the brain permeability of the drugs, several approaches have been developed (e.g. receptor-mediated transcytosis, nanocarrier-mediated transport, and BBB disruption).4-6 Receptor-mediated transcytosis across the BBB has been explored more actively because of its high specificity. For example, the low density lipoprotein receptors are present on brain capillary endothelial cells and glioma cells and possesses ability to mediate transport of ligands across BBB.4, 7 Angiopep-2, as a ligand of the LRP-1, which is a 19-mer peptide derived from human Kunitz domain of aprotinin, exhibites a much higher BBB transcytosis efficacy than transferrin and aprotinin.3 Hence, angiopep-2 is usually used to mediate drugs across BBB. 2


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Nanoparticles as carriers play an essential role in brain drug delivery, imaging and diagnosis due to their multi-functionality. A number of inorganic nanoparticles (e.g. gold nanoparticles, iron oxide nanoparticles and quantum dots) or high-molecular polymer (e.g. dendrimers, polymeric micelle) were coupled with brain targeting peptides to perform brain tumor imaging or deliver drugs into brain.8-11 For instance, an optical/paramagnetic nanoprobe based on PAMAM dendrimer was designed to noninvasively visualize brain tumors by magnetic resonance imaging and near-infrared fluorescence imaging.11 A triple-modality MRI-Photoacoustic-Raman Nanoparticle was developed using 60 nm gold core, Raman molecular tag, 30 nm silica coating and DOTA-Gd3+ for brain tumor molecule imaging.12 Lipid nanoparticle was used to deliver siRNA to silence neuronal gene expression in the brain, which resulted in effective knockdown of target genes in injection site and surrounding regions.13 Although several types of nanocarriers have proven effective for brain-targeting delivery, the safety and effectiveness of these nanomaterials remains to be examined.14-15 Self-assembled DNA nanostructures open new opportunities for drug delivery as a type of intrinsically biocompatible and biodegradable nanomaterials.16-26 Especially, framework nucleic acids (FNAs) that have well defined frames and cavities hold the promise for developing precisely controlled delivery agents. As a typical type of FNAs, DNA tetrahedral nanostructures (TDNs) self-assembled with four designed DNA strands have been popularly employed for various biological applications.27-28 Importantly, functional moieties and signal molecules can be spatially and stoichiometrically controled in TDNs.29 Previous studies showed that TDN readily internalized by living cells and showed high resistance to degradation.30-32 Because of these properties, TDNs have been applied to design imaging bioprobe for tumor-targeting, dual-modality in vivo imaging by using both NIR and SPECT modalities.29 TDNs have also been 3



used for targeted delivery of siRNA, aptamer, CPG and drugs.29, 33-35 For example, Anderson and coworkers employed TDNs to deliver siRNAs and silence target genes in subcutaneous tumor model mice. They found that the blood circulation time of siRNAs in vivo could be prolonged by conjugation with TDNs.34 In this work, we aimed to develop a DNA tetrahedral nanoparticle for brain targeting imaging and drug delivery (Figure 1). We modified TDNs with fixed numbers of angiopep-2 and NIR dyes (ANG-TDNs) and investigated their brain targeting efficiency in vitro and in vivo.

EXPERIMENTAL SECTION DNA oligonucleotides were purchased from Taraka Biotechnology Co., Ltd. (Dalian, China). Angiopep-2 (TFFYGGSRGKRNNFKTEEY) was obtained from Chinapeptide Co., Ltd. (Suzhou, China). Zeba spin desalting columns and plates were purchased from Thermo Fisher Scientific Inc. (USA). Amicon filters were purchased from Millipore (Billerica, MA, USA). GelRed DNA gel stain solution was purchased from Biotium (USA). All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All oligonucleotide sequences were listed in Table S1. The brain capillary endothelial cells (bEnd.3) were acquired from ATCC (Manassas, VA), and were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. The U87 MG cells were obtained from Shanghai Institute of Cell Biology, and were maintained in Minimum Essential Medium (MEM, Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. BALB/c nude mice and ICR mice, male, weighing (20 ± 2) g, were purchased from Shanghai SLAC Laboratory 4


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Animal Co. LTD (Shanghai, China) and kept under SPF conditions. All TDNs were assembled in an Applied Biosystems Veriti 96 well Thermal Cycler. 2D Fluorescent images were acquired with an in vivo imaging system (Berthold NightOWL LB 983, Germany). 3D Fluorescent images were acquired with IVIS Lumina XRMS Series III Multi-species Optical and X-ray Imaging System (PerkinElmer, USA). All TDNs with or without arms were synthesized by mixing equimolar quantities (1 µM) of oligonucleotides in TM buffer (10 mM Tris base, 5 mM MgCl2, pH 8.0). Detailed procedures for TDN modifications, characterization and intracellular analysis were provided in the supporting information. In vivo and ex vivo intracranial glioblastoma imaging and slice distribution were performed using BALB/c nude mice, which were anesthetized with chloral hydrate and U87 MG cells (8 × 105 cells suspended in 5 μL PBS) were implanted into the right brain (1.8 mm lateral, 0.6 mm anterior to the bregma, and 3 mm of depth) with the help of a stereotactic fixation device. All animal care and experimental procedures were conducted using institutionally approved protocols. All in vivo experiments were conducted under the authority of project and personal licenses granted by the institutionally approved protocols.

RESULTS AND DISCUSSION Synthesis and Characterization of TDNs and ANG-TDNs. The TDNs with or without arms were assembled from four designed DNA single strands (Table S1) as previous methods.28 Angiopep-2 modified single-stranded DNA (ANG-ssDNA) were synthesized by the help of click action kit, the scheme is shown in Figure 2a. The ANG-ssDNA structure was characterized by nondenaturing polyacrylamide gel electrophoresis (PAGE) shown in Figure 2b. Due to the increased molecule weight, the mobility of ANG-ssDNA was decreased 5



compared to ssDNA, suggesting that angiopep-2 has been successfully linked on the single-stranded DNA. The angiopep-2-functionalized TDNs (ANG-TDNs) were prepared by the hybridizing of ANG-ssDNA and three-armed TDNs. The structure of TDNs or ANG-TDNs was characterized by PAGE shown in Figure 2c, the increased retardation by a strand-wise addition of DNA strands could be observed in the PAGE analysis. The sizes of TDNs and ANG-TDNs were further confirmed by dynamic light scattering (DLS) and atomic force microscopy (AFM). From the DLS measurement and the AFM images (Figure 2d, e and Figure S1), we found that TDNs and ANG-TDNs were assembled successfully. The hydrodynamic diameter of TDNs and ANG-TDNs measured by DLS was ~10.5 nm and ~15.7 nm respectively, and the height of TDNs and ANG-TDNs measured by AFM was ~2.0 nm and ~2.4 nm respectively, which is consistent with previous observations of DNA tetrahedral structures.29, 36-37 In vitro Stability and Cytotoxicity of TDNs and ANG-TDNs. After preparing the materials, we investigated their stability and cytotoxicity in vitro. TDNs and ANG-TDNs were incubated with 50% non-inactivated FBS respectively at the same concentration (50 nM) for a period of time (0, 2, 4, 8, 12 and 24 h). After incubation, PAGE analysis was taken to detect the structural changes of TDNs or ANG-TDNs. PAGE results showed that TDNs or ANG-TDNs strip remained almost unchanged for 8 h incubation, reflecting the presence of intact tetrahedral nanostructure in FBS (Figure 2f, g). After incubation for 12 h, the strips of nanostructures still be observed with attenuated intensity, and ANG-TDNs exhibited stronger stability than TDNs, the ANG-TDNs strip dropt to ~75%, but TDNs strip dropt to ~60% (Figure 2g). That because the design of side arms on edges of TDNs could improve DNA nanostructure stability in FBS by steric inhibition of enzyme binding.

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Next, we evaluated the cytotoxicity of TDNs and ANG-TDNs using MTT assay. TDNs and ANG-TDNs were separately incubated with brain capillary endothelial cells (bEnd.3 cells) and Uppsala 87 Malignant Glioma cells (U87MG cells) at different concentrations (0, 25, 50 and 100 nM) for 24 h. We found that neither TDNs nor ANG-TDNs can induce measurable loss in the viability of cells even at a concentration of 100 nM (Figure 2h). This result indicated that both TDNs and ANG-TDNs had no cytotoxicity, and they could be used in vivo research. Cell uptake and In vitro BBB-Penetrating Efficiency. Having determined the stability and the safety of the materials, we then investigated the cellular uptake ability of ANG-TDNs and TDNs in bEnd.3 cells and U87MG cells. Cy3 dye was labeled on one of main strands of TDNs. ANG-TDNs and TDNs were separately incubated with bEnd.3 cells or U87MG cells for 4 h. After incubation for 4 h, we observed intense Cy3 fluorescence in cells with confocal fluorescence microscopy. In Figure S2a, b, the Cy3 fluorescence were predominantly localized in the cytoplasm, indicating that both TDNs and ANG-TDNs could enter into cells. But the fluorescence intensity of ANG-TDNs in cytoplasm was much higher than that of TDNs, suggesting that angiopep-2 modification could enhance the cellular uptake of TDNs. We simultaneously used flow cytometry to evaluate the cellular uptake efficiency. With the incubation time prolonged, the cell uptake rate of the materials also increased (Figure S3). After incubation for 4 h, the mean fluorescent intensity of cells treated with ANG-TDNs was nearly two folds of that of TDNs (Figure S2b, d), suggesting the high cellular uptake efficiency of ANG-TDNs in normal cell and glioma cell of brain. In order to estimate the BBB-penetrating efficiency of the TDNs and ANG-TDNs, an in vitro BBB model was established (Figure 3a). The in vitro BBB was formed after incubation with bEnd.3 cells in the upper chamber of the transwells for 7 days. After the transendothelial

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electrical resistance (TEER) values of the bEnd.3 cell monolayers reached higher than 300 Ω cm2, the in vitro BBB was formed. Then, we moved the upper chambers into the lower chambers which seeded with U87MG cells for the follow penetrating and cascade-targeting study. ANG-TDNs and TDNs labeled with Cy3 were separately added in the upper chamber of transwells for 6 h. After incubation for 6 h, the inserts were moved away, and U87MG cells were further incubated until for 24 h. Then we observed Cy3 fluorescence intensity in U87MG cells which seeded in the acceptor chambers in advance with confocal fluorescence microscopy. In Figure 3b, c, the fluorescence intensity of ANG-TDNs in cytoplasm was much higher than that of TDNs (~ 4 folds), suggesting that the ANG-TDNs passed through the BBB model and then located into the cytoplasm of U87MG cells. Furthermore, we evaluated the receptor targeting specificity of ANG-TDNs. U87MG cells were treated with ANG-TDNs in the presence of the low density lipoprotein receptor-associated protein (RAP), which was utilized as a receptor competitor of LRP receptor.11 Confocal and Flow cytometry results showed that the uptake of ANG-TDNs decreased by ~65% after the preincubation of RAP (Figure 3d, e). The results indicated that angiopep-2 played a predominant role in the mediating cellular uptake of ANG-TDNs. In vivo Imaging. Having established the excellent brain targeting of ANG-TDNs in cells, then we investigated the brain targeting capability of TDNs and ANG-TDNs in vivo. Dlight 755 dyes were labeled on one of main chains of TDNs or ANG-TDNs. We first injected TDNs or ANG-TDNs into normal nude mice via tail vain, and then respectively detected the fluorescent intensity of Dlight 755 at 0, 30, 60 and 90 min post injection by a small animal imager. As shown in Figure 4a, we can observe the in vivo biodistribution of TDNs or ANG-TDNs after injection. Part of the

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fluorescent signal was focused on the brain area of mice in ANG-TDNs group, indicating that ANG-TDNs could be distributed into brain more efficiently than TDNs. What’s more, we investigated the biodistribution of TDNs and ANG-TDNs by detecting the fluorescent intensity of major organs (e.g. heart, liver, spleen, lung, kidney and brain) after injecting DNA nanoparticles for 2 h (Figure 4b, c and Figure S4). The fluorescent images showed that the fluorescent intensity of TDNs and ANG-TDNs both located in liver and kidney. However, the fluorescent intensity of ANG-TDNs was lower than that of TDNs in kidney, whereas slightly higher than that of TDNs in liver. Also the fluorescence of ANG-TDNs could be detected in normal mice brain. These suggest the different in-vivo distribution of TDNs and ANG-TDNs. In addition, the blood was collected at different times to detect the fluorescent intensity. The blood distribution half-life of ANG-TDNs was 8.2±0.7 min, approximately 1.7 fold longer than that of TDNs (4.5±0.5 min) (Figure 4d), indicating that peptide modification could prolong the circulation time in vivo. In order to further investigate the brain tumor targeting ability of ANG-TDNs, we established in situ glioma mouse model. TDNs or ANG-TDNs labeled with Dlight 755 were injected into in situ glioma bearing nude mice via tail vain, and then the three-dimensional animal fluorescent in vivo imaging was performed (Figure S5). As shown in Figure 5a, 90 min after injection, the fluorescence intensity of ANG-TDNs was mainly detected in the brain tumor region of mice, and remarkably higher than that of TDNs, indicating that ANG-TDNs could cross brain blood barrier and concentrate in the glioma site (Video S1 and S2). Then the brains of mice were dissected and imaged. Compared with TDNs group, the fluorescence intensity of brain of mice in ANG-TDNs group was much higher, the value of the fluorescence intensity was approximately fourfold higher than that of TDNs (Figure 5b), and mainly appeared in the location of glioma. In addition,

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the brain sections of mice in ANG-TDNs group had higher fluorescent intensity than that of TDNs (Figure 5c). These results indicated that ANG-TDNs could cross brain blood barrier and obviously concentrate in glioma.

CONCLUSIONS In this study, we developed a FNA-based image probe to target brain tumor. We modified TDNs with angiopep-2 and found it had the ability of brain targeting in vitro and in vivo. Angiopep-2 modification effectively enhanced cellular uptake of TDNs in U87MG cells and bEnd.3 cells, and the uptake was competitively inhibited by the low density lipoprotein receptor-associated protein, suggesting that cell uptake of ANG-TDNs mediated by angiopep-2. Also, compared to TDNs, ANG-TDNs could cross the BBB model in vitro and cascade-target to U87MG cells. In vivo imaging studies indicated that ANG-TDNs not only crossed the BBB of normal mice, but also exhibited stronger fluorescent signal in U87MG human glioblastoma xenograft in nude mice. Especially, ANG-TDNs exhibited excellent stability in serum and no cytotoxicity.

ASSOCIATED CONTENT Supporting Information. Experimental, one table, five figures and two videos are provided in the supporting information. Table S1 presents the information of oligonucleotide sequence in this work. Figure S1 provides 10


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the characterization of TDNs and ANG-TDNs. Figure S2 -S3 presents the cell uptake image and analysis of TDNs and ANG-TDNs. Figure S4 provides the quantitative analysis of the total fluorescent intensity of liver, kidney and brain. Figure S5 and Video S1-S2 are the 3D reconstruction of in vivo imaging of TDNs and ANG-TDNs in mice. AUTHOR INFORMATION Corresponding Authors

* E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] Notes

The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the Ministry of Science and Technology of China (2016YFA0201200, 2016YFA0400902), the National Science Foundation of China (11575278, 21675167, 81690263, 21227804, 21505148, 11405013,31371015), Natural Science Foundation of

Shanghai

(15ZR1448400),

Key

Research

Program

of

Frontier

Sciences

(NO.QYZDJ-SSW-SLH031) and Youth Innovation Promotion Association, CAS.

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FIGURES

Figure 1. Schematics for FNA-based brain-targeting imaging. A brain-targeting imaging probe was designed using tetrahedral DNA nanostructures (TDNs). Angiopep-2 was introduced to mediate the blood brain barrier (BBB) permeability of TNDs, and effectively enhanced cellular uptake of TDNs in brain capillary endothelial cells (bEnd.3) and Uppsala 87 Malignant Glioma cells (U87MG).

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Figure 2. The characterization, in vitro stability and cytotoxicity of TDNs and ANG-TDNs. (a) The synthesis scheme of ANG-ssDNA and ANG-TDNs. (b) PAGE analysis of ANG-ssDNA. Line1: DNA marker, line 2: ssDNA, line 3: ANG-ssDNA. (c) PAGE analysis of DNA nanostructures. Line 1: DNA marker, line 2: strand A, line 3: strand A+B, line 4: strand A+B+C, line 5: TDNs, line 6: TDNs with three arm strands, line 7: ANG-TDNs. (d) DLS measurement statistics of ANG-TDNs. (e) AFM images of ANG-TDNs. (f) PAGE analysis of TDNs and ANG-TDNs after incubating with 50% FBS for 0h, 2h, 4h, 8h, 12h and 24h respectively. (g) Semiquantitative analysis of the relative band intensity of TDNs and ANG-TDNs. (h) Cell viability statistics of bEnd.3 cells and U87MG cells after incubating with TDNs and ANG-TDNs respectively.

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Figure 3. Cell uptake and in vitro BBB-penetrating efficiency of TDNs and ANG-TDNs. (a) Schematic illustration of the in vitro BBB model using a transwell system to evaluate the penetration capability of TDNs and ANG-TDNs. Left, the in vitro BBB model. Right, the bEnd.3 and U87MG cell co-culture model. (b) Confocal images of DNA nanostructures in U87MG cells after transporting across the BBB model, red pointed DNA nanostructures, blue pointed cell nucleus. (c) Fluorescent statistic of U87MG cell uptake efficiency of DNA nanostructures after transporting across the BBB model. (d) Cell uptake of ANG-TDNs after preincubation with RAP in U87 MG cells, blue pointed cell nucleus, red pointed ANG-TDNs. (e) Fluorescent statistic of cell uptake efficiency of ANG-TDNs after the preincubation of RAP. In all panels, **P

Targeted Imaging of Brain Tumors with a ... - ACS Publications

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