Development of a Bifunctional Aptamer Targeting the Transferrin

Dec 23, 2016 - The treatment of brain disorders is greatly hindered by the presence of the blood–brain barrier, which restricts the overwhelming maj...
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Development of a Bifunctional Aptamer Targeting the Transferrin Receptor and Epithelial Cell Adhesion Molecule (EpCAM) for the Treatment of Brain Cancer Metastases Joanna Macdonald,† Justin Henri,† Lynda Goodman,† Dongxi Xiang,† Wei Duan,†,‡ and Sarah Shigdar*,†,‡ †

School of Medicine and ‡Centre for Molecular and Medical Research, Deakin University, Geelong, Victoria 3128, Australia S Supporting Information *

ABSTRACT: The treatment of brain disorders is greatly hindered by the presence of the blood−brain barrier, which restricts the overwhelming majority of small molecules from entering the brain. A novel approach by which to overcome this barrier is to target receptor mediated transport mechanisms present on the endothelial cell membranes. Therefore, we fused an aptamer that binds to epithelial cell adhesion molecule-expressing cancer cells to an aptamer targeting the transferrin receptor. This generated a proof of concept bifunctional aptamer that can overcome the blood−brain barrier and potentially specifically target brain disorders. The initial fusion of the two sequences enhanced the binding affinity of both aptamers while maintaining specificity. Additionally, mutations were introduced into both binding loops to determine their effect on aptamer specificity. The ability of the aptamer to transcytose the blood−brain barrier was then confirmed in vivo following a 1 nmol injection. This study has shown that through the fusion of two aptamer sequences, a bifunctional aptamer can be generated that has the potential to be developed for the specific treatment of brain disorders. KEYWORDS: Aptamer, bifunctional, blood−brain barrier, epithelial cell adhesion molecule (EpCAM), receptor mediated transcytosis, transferrin receptor

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INTRODUCTION Defined as the control center of the human body, the brain requires a precise microenvironment in order to function optimally. This environment is provided and maintained through the defined function of the blood−brain barrier (BBB). Playing a key role in brain homeostasis, this barrier segregates the brain from the peripheral blood circulation, impeding the influx of blood-borne molecules greater than 500 Da.1,2 While this barrier prevents neurotoxic substances from entering and harming the brain, it also results in the prevention of the overwhelming majority (98%) of small molecules from crossing into the brain, resulting in limited treatment options for neurological disorders.3 Numerous strategies have been proposed to evade this formidable barrier, including transcranial delivery, barrier disruption via intracarotid arterial infusion of vasoactive agents and transnasal delivery.4 While these strategies potentially allow drugs to avoid or bypass the BBB, they have a number of limitations, including drug diffusion, crossing additional membranes, and allowing the influx of other neurotoxic substances.4−6 In addition to these limitations, these methods carry great risk to the patients.7,8 A promising new approach toward bypassing this barrier, is to hijack active transport mechanisms present on the endothelial cell membranes. The transferrin receptor (TfR), a membrane glycoprotein responsible for iron homeostasis, has been identified as © XXXX American Chemical Society

an ideal target for this purpose given its high expression on the surface of the BBB.9,10 This has been explored by Yu and colleagues, who developed a low affinity bispecific antibody capable of exploiting the TfR and targeting an enzyme associated with Alzheimer’s disease.11 Reduced amyloid-β production following antibody administration, demonstrated that through hijacking this transport pathway, a therapeutically relevant concentration of antibody can be delivered across the BBB in vivo.11 Given the severe side effects and immunological risk antibodies pose, a novel therapeutic strategy is required. Aptamers, also known as chemical antibodies, are small single stranded RNA or DNA molecules that bind to their target through shape recognition, similar to that of conventional antibodies.12 Through functioning by molecular recognition, aptamers can be developed for therapeutic applications with the same intended function as antibodies, thus giving them the ability to be employed in a range of pharmacological areas, including drug discovery, diagnostics, drug delivery vehicles, and protein inhibitors. Though analogous to their protein counterpart in regards to target recognition and application, aptamers have a number of key advantages, the main being Received: November 1, 2016 Accepted: December 23, 2016 Published: December 23, 2016 A

DOI: 10.1021/acschemneuro.6b00369 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Sensitivity and Specificity of Bifunctional Aptamers. While monofunctional aptamers have been proven as effective therapeutics, generating an aptamer that is only capable of crossing the BBB would be an ineffective method of treating neurological disorders. Therapeutics developed in this manner would result in nonspecific drug delivery, consequently leading to neurotoxic side effects. To overcome this, we sought to generate a bifunctional aptamer that was capable of crossing the BBB and targeting specific populations associated with neurological disorders. Using structure prediction software RNA fold, the EpCAM aptamer, EpA, and the previously described TfR aptamer were fused together.19,22 To maintain the suggested 2D structure, the addition of a TA linker was inserted to join the two aptamers together. Following the initial fusion, modifications were introduced into both binding regions to generate aptamers of varying affinity. Introducing modifications into the stem region or binding loop of aptamers can lead to a loss of specificity or sensitivity or both.23 Therefore, following mutation, it is essential to reconfirm sensitivity and specificity. Similar to the characterization of the EpCAM aptamers, human and mouse cell lines were employed to ensure the aptamers still retained specificity and sensitivity to the native conformation of the proteins. The cell lines used in these biological assays included a cell line expressing the TfR (bEnd.3), a cell line expressing EpCAM (MDA-MB-231), and a cell line expressing neither protein (HEK293T). Through the initial fusion of the aptamers, a bifunctional aptamer was generated, TEPP (Figure 1), which displayed a moderate affinity toward the TfR (Kd = 110.3 ± 22.04 nM) (Figure 2, Table 1) and EpCAM (85.61 ± 28.49 nM) (Figure 2, Table 1). Following this fusion, the affinity of the TfR portion of the bifunctional aptamer increased compared to that of the single TfR aptamer (TfR4 Kd = 365 ± 83 nM).19 Considering that through truncation of the SYL3C aptamer, the aim was to generate smaller aptamers with higher affinities, this was an interesting observation. When all seven nucleotides within the binding region of the EpCAM targeting portion of the aptamer were mutated (G22T, G23T, T24C, T25C, G26T, C27T, and G28T), generating TEPN (Figure 1), no binding toward the MDA-MB-231 cells was observed (Kd > 1000 nM), suggesting that the mutations had resulted in a complete loss of sensitivity toward the EpCAM protein. In contrast to this, as a result of these mutations, an increase in affinity toward the TfR was observed (Kd = 65.22 ± 22.93 nM) (Figure 2, Table 1). Following the mutation of all four nucleotides within the binding region of the TfR portion of TEPP (G6T, T7G, A8C, and C9A), generating TENP, no binding toward the bEnd.3 cells was observed (Kd > 1000 nM), suggesting a complete loss of sensitivity toward the TfR. In addition to this, the introduction of these mutations resulted in a decrease in affinity toward the EpCAM protein (382.5 ± 96.71 nM). As expected, the fusion of the two mutated binding regions of TEPN and TENP resulted in the generation of an aptamer that demonstrated no sensitivity toward the TfR receptor (Kd > 1000 nM) or EpCAM (Kd > 1000 nM). It has been previously recognized that introducing mutations within the binding region of aptamers can cause an increase in affinity but can also generate nonbinding sequences.24,25 Considering that all the nucleotides within the EpCAM and TfR binding portion of the bifunctional aptamer were mutated, it is not surprising that a complete loss of sensitivity was observed. While the single stranded loop is the site of binding, it is the tertiary structure of the aptamer that is critical for determining aptamer target interaction.26 As an increase

their size, production process, increased stability, and lack of immunogenicity.13−15 The therapeutic potential of monofunctional nucleic acid aptamers can be further enhanced through the production of bifunctional aptamers, developed for different mechanistic actions. The first entails the fusion of two aptamers whose binding activity is independent of each other and the second involves the fusion of two sequences in which the binding of the first aptamer influences the binding of the second aptamer.16 Synthesis via the second pathway opens up the possibility of specifically delivering drug payloads to sites within the body not accessible by drugs alone, for example, the brain, by targeting the TfR and then a specific neurological disorder. Recently, through the rational truncation of the 64 nucleotide GS24 DNA aptamer generated by Chen et al., which selectively recognizes the extracellular domain of the mouse TfR, we developed an aptamer 14 nucleotides in length.17 Given this success, we sought to explore the effect of completing similar truncations to a 48 base pair SYL3C DNA aptamer, which specifically targets epithelial cell adhesion molecule (EpCAM), a cell surface marker overexpressed on a number of solid tumors.18 Here, we describe the truncation of this large DNA aptamer (SYL3C), specifically targeting EpCAM, from 48 nucleotides to a mere 17 nucleotides, without altering aptamer specificity and sensitivity. This aptamer was then fused to the 14 nucleotide TfR DNA aptamer previously described, to develop a bifunctional therapeutic delivery vehicle capable of crossing the BBB via transcytosis and specifically targeting brain cancer metastases derived from EpCAM positive cancer cells.19 This model serves as a proof of concept for the potential development of this aptamer for the treatment of brain disorders by interchanging the EpCAM targeting portion with other aptamers or other therapeutic agents.

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RESULTS AND DISCUSSION The treatment of brain disorders is greatly hindered by their location. Situated in the safe sanctuary provided by the BBB, the delivery of therapeutics is greatly restricted.3 Emerging strategies to overcome this barrier have included the development of therapeutic modalities capable of crossing into the brain via receptor-mediated transcytosis.9,20,21 While this allows entrance into the brain, these modalities lack specificity and may ultimately cause neurotoxicity. We have recently described the truncation of a TfR aptamer that has a moderate binding affinity to the TfR, a property shown to be optimal for transcytosis across the BBB.11,19 While this aptamer can transcytose the barrier, once in the brain, it would freely diffuse and potentially cause neurotoxicity. Therefore, we sought to further functionalize this aptamer by combining it with an aptamer targeting a biomarker overexpressed on cancer cells known to metastasize to the brain to direct the aptamer to a specific population of cells once in the brain in order to limit neurotoxicity as a proof of concept for further development. Given its overexpression on a number of epithelial carcinomas with a high incidence of metastasising to the brain, EpCAM was the biomarker chosen.18 Through the rational truncation of the 48 nucleotide SYL3C aptamer into its 3 corresponding hair pin loops, we generated 3 aptamers with enhanced sensitivity and specificity when compared to the parent sequence (Figures S1, S2, and S3 Supporting Information). Guided by the degree of cellular internalization in combination with the calculated binding affinity, EpA was chosen to be combined with the TfR aptamer to form the bifunctional aptamer. B

DOI: 10.1021/acschemneuro.6b00369 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Table 1. Binding Affinity of Bifunctional Aptamers to Transferrin Receptor and EpCAM aptamer TEPP TEPN TENP TENN

bEnd.3 (Kd, nM) MDA-MB-231 (Kd, nM) HEK293T (Kd, nM) 110.3 ± 22.04 65.22 ± 29.93 >1000 >1000

85.61 ± 28.49 >1000 382.5 ± 96.71 >1000

>1000 >1000 >1000 >1000

stabilized the tertiary structure of the whole aptamer, thus increasing the binding affinity. While the introduction of these mutations caused a loss of sensitivity in one or both ends of the bifunctional aptamer, they produced control aptamers that could be employed for further characterization of the double positive aptamer. Internalization of Bifunctional Aptamers Suggests Therapeutic Potential. As discussed previously, to be developed for therapeutic applications, aptamers must be internalized into the cells rather than merely attaching to the cell surface. With the knowledge that the single TfR and EpCAM aptamers were internalized intracellularly, it was imperative to ensure that the fusion of the two sequences had no negative influence on this ability. To do this, 400 nM of each aptamer was incubated with bEnd.3, MDA-MB-231, and HEK293T cells for 60 min followed by visualization using confocal microscopy. TEPP and TEPN were internalized to a varying degree into the TfR positive cells (bEnd.3 cells), shown by the punctate pattern, which is indicative of endocytosis, while TENN and TENP showed no internalization (Figure 3).27 TEPP and TENP were internalized into the MDA-MBA-231 cells, while TENN and TEPN showed no internalization (Figure 3). As no aptamer internalization was apparent in the HEK293T cells, internalization appeared to be specific. Determination of Aptamer Permeability Across an in Vitro Blood−Brain Barrier Model. In the last 30 years, in vitro BBB model systems have been effectively used to study modulation of BBB permeability in pathological, physiological, and pharmacological conditions.28 In order to establish easy, adequately tight, and reproducible in vitro BBB models, immortalized brain endothelial cell lines have been created in growing

Figure 1. Bifunctional aptamer structures: (A) TEPP; (B) TEPN; (C) TENP; and (D) TENN.

in affinity toward the TfR was observed for TEPN following the introduction of mutations within the EpCAM binding portion, it is possible that mutations in the binding loop altered or

Figure 2. Specificity of the bifunctional aptamers. TYE665 labeled aptamers were incubated with the bEnd.3, MDA-MB-231, or HEK293T cell line and analyzed by flow cytometry. The mean fluorescence intensity (MFI) was plotted against varying concentrations of EpCAM aptamers (1−200 nM) at a cell density of 5 × 105 cells/mL. Representative binding curves of aptamers with bEnd.3 and MDA-MB-231 cells: (A) TEPP; (B) TEPN; (C) TENP; and (D) TENN. Data shown are mean ± SEM (n = 3). C

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specificity in target accuracy, cancer cells were not seeded in isolation, but instead, examined alongside cells devoid of EpCAM expression. Therefore, both HEK293T and MDA-MB231 cells were seeded in the lower compartment at a ratio of 1:1. Through incubation with an anti-EpCAM antibody, the cell lines were able to be differentiated and were subsequently visualized via confocal microscopy (Figure 4). As the results

Figure 3. Internalization of bifunctional aptamers. Each bifunctional aptamer was incubated with mouse TfR positive (bEnd.3), EpCAM positive (MDA-MB-231), and negative (HEK293T) cell lines for 60 min at 37 °C, followed by visualization using laser scanning confocal microscopy. Data are representative of three independent experiments. Representative images of TEPP; TEPN; TENP; and TENN. Red, bifunctional aptamer; blue, nuclear stain (n = 3). Figure 4. Internalization of bifunctional aptamers with an in vitro BBB model. Each bifunctional aptamer was incubated with the in vitro BBB for 3 h at 37 °C, prior to trypsinization and visualization using laser scanning confocal microscopy. An anti-EpCAM antibody was used to differentiate EpCAM positive MDA-MB-231 cells. Red, bifunctional aptamer; blue, nuclear stain; green, anti-EpCAM antibody. Scale bar: 10 μM.

number in recent years. One of the most widely used and characterized cerebral endothelial cell lines, applied in permeability studies, is the commercially available bEnd.3-5 of murine origin.29 In order for the bifunctional aptamers to be successfully applied in targeting brain metastases, they must first be able to cross the BBB. To assess if the aptamers were capable of penetrating the BBB, an in vitro model of this physiological system was used as a proof of concept. With the intention of producing a tight monolayer of endothelial cells, bEnd.3 cells were subcultured and seeded on transwell inserts. The integrity of this barrier was assessed daily, for 7 days, through measurement of the transendothelial electrical resistance (TEER). The TEER continued to increase, with highest measurements recorded at day five. Our model consistently maintained an average TEER of 140 Ω cm2 on the fifth day, reflecting sufficient tightness to study permeability.30 Creating an aptamer that is not only capable of crossing the BBB but able to then target specific populations of cells is essential in the pursuit of targeting brain disorders or diseases. In order to investigate the ability of the aptamers to not only transverse through an endothelial monolayer but target EpCAM positive cancer cells, MDA-MB-231 cells were seeded in the lower compartment of the in vitro BBB model. To further test aptamer

demonstrate, and consistent with our previous findings, TEPP was able to successfully cross the BBB and specifically target only MDA-MB-231 cells. Again, TENN failed to internalize into any cell line, and TENP was not observed to be internalized into the EpCAM expressing cells due to its inability to pass through the in vitro model. In contrast, while TEPN was successfully internalized into bEnd.3 cells, in theory if transcytosis were to occur across the BBB, it would not have the ability to further target and be internalized into any cells. Indeed, this is what was observed and confirmed through the MDA-MB-231 cells being devoid of internalization. Again, as no aptamer internalization was apparent in the HEK293T cells, this adds further support in the highly selective ability of the bifunctional aptamers. Although interest has peaked into the use of antibodies to cross the BBB for targeted therapy, researchers have strived to engineer bifunctional antibodies D

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Figure 5. Biodistribution of bifunctional aptamers. NOD/SCID mice received a single iv injection of 40 nmol/kg of aptamer. The concentration of aptamers, expressed as % of injected dose (ID) per gram of tissue, was determined at 30 and 60 min after the agent administration using ELISA. (A) Whole body biodistribution; (B) brain biodistribution. Data are means ± SEM (n = 3).

was targeting both the TfR on the BBB and throughout the body, which may explain the high accumulation throughout the body. Therefore, through future work utilizing EpCAM positive tumor bearing mice, the biodistribution of this bifunctional system will be investigated to gain an understanding of how the system will function in a patient suffering from EpCAM positive brain metastases. Taking the favorable accumulation of the TEPP aptamer into account and its retention profile over the 60 min period, this aptamer has the potential to be developed as an effective modality for overcoming the BBB, opening up a new window for targeted drug delivery to the brain. The concept of developing aptamers for the treatment of brain disorders is not new. There have been numerous reports of aptamers generated for the targeted treatment of specific brain diseases such as glioblastoma and Alzheimer’s disease.34−36 While these aptamers have been shown to be highly specific for their target and demonstrated efficient cellular uptake, they are incapable of crossing the BBB. To overcome this restrictive barrier, through numerous rounds of in vivo selection, Cheng and colleagues generated an RNA brain penetrating aptamer known as A15.37 Through in vitro and in vivo characterization, it was confirmed that the A15 aptamer possessed the ability to enter brain endothelial cells under physiological conditions and in addition to this, could enter the brain parenchyma.37 While the ability of this aptamer to enter the brain parenchyma is noteworthy, the exact target of the aptamer is unknown and upon entering the brain, the aptamer has no targeted delivery system. Compared to these disease specific aptamers and the brain penetrating aptamer, TEPP is a mechanistic aptamer, with the ability of specifically transcytosing the BBB via a known target and targeting a disease of choice.

with the ability to successfully enter the brain and be applied for use as potential drug delivery carriers. However, antibodies are still struggling to penetrate the shield of the BBB with ease and success. In vivo studies have observed limited brain uptake with TfR antibodies and poor safety profiles.31,32 The results so far show our aptamers are not only capable of crossing the BBB in vitro but are able to target EpCAM expressing cancer cells exclusively. While in vitro models of the BBB serve as valuable tools in assessing the permeability of a substance across the BBB, they still have limitations. Due to the simplistic nature of an in vitro model, its inability to mimic the extensive variety of different cell types involved in BBB regulation remain a major drawback.33 Therefore, our next step was to investigate the ability of our aptamers to penetrate the BBB in vivo. Transcytosis across the BBB in Vivo. Confirmation of transcytosis across the BBB in vivo is the pinnacle characteristic for this therapeutic modality. While in vitro characterization is essential to confirm aptamer sensitivity and specificity, if this modality is incapable of transcytosing the BBB in vivo, pursuing further therapeutic development is pointless. To establish if the aptamers were capable of crossing the BBB, healthy mice received a single iv injection of aptamer (40 nmol/kg), and biodistribution analyses within the whole body were conducted at 30 and 60 min. Illustrated in Figure 5A, accumulation of all four aptamer structures was observed within the brain. The detected uptake of the transferrin negative aptamers suggests that these aptamers may potentially be crossing the BBB via a nonspecific transport mechanism. Given that the percentage of injected dose of TEPP is 12-fold higher than that of the TENN aptamer at 30 min and 10.8 at 60 min, we can conclude that in addition to the minimal nonspecific uptake observed, this aptamer is specifically taken up by active transport. Furthermore, the significantly higher uptake of TEPP and TEPN compared to TENN and TENP confirms that the specific brain uptake is a result of the TfR portion of the bifunctional aptamer. As shown by the whole body biodistribution (Figure 5B), the aptamers accumulated rapidly in highly perfused organs, such as the liver, heart, kidneys, lungs, and spleen, 30 min after administration. In comparison to this, 60 min after administration, the accumulated dose of the aptamers in these organs was markedly lower than that observed at 30 min. The difference in retention within these organs at these two time points indicates that the high levels are most likely the result of blood perfusion and not due to aptamer binding. While in the highly perfused organs there was a higher percentage of injected dose of TEPP compared to the control aptamers, it is important to recognize that these animals were healthy, carrying no EpCAM positive tumors. This means that the double binding targeted system

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CONCLUSION This study has shown that the truncation of an aptamer generated against the membrane glycoprotein EpCAM and its subsequent fusion with an aptamer against the mouse TfR successfully generated an aptamer with both specificity and sensitivity. From previous studies targeting the TfR, great insight has been gained into the characteristics essential to ensure a good safety profile as well as to also maximize brain uptake when targeting this receptor.11,32 Using therapeutic antibodies, it was discovered that a lower binding affinity was more favorable than that of a high affinity antibody, with the highest uptake observed utilizing an antibody with a binding affinity of 111 ± 16 nM to the TfR.11 In this study, we have shown the ability of the TEPN aptamer, which possesses a moderate binding affinity (65.22 ± 29.93 nM), to successfully transcytose E

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as for flow cytometry. Following removal of media, cells were incubated in binding buffer (PBS with 1 mM MgCl2, 0.1 mg/mL tRNA, 1 mg/mL bovine serum albumin, and 10% FCS) at 37 °C for 30 min and washed twice in binding buffer prior to incubation with 200 or 400 nM aptamer for 60 min at 37 °C. Bisbenzimide Hoechst 33342 (3 μg/mL) (Sigma) was added to the cells during the final 10 min of incubation. The aptamer solution was removed, and the cells were washed 3 times for 5 min each in PBS prior to visualization using a FluoView FV10i laser scanning confocal microscope (Olympus). In Vitro Blood−Brain Barrier Transcytosis. Transwell inserts (polyethylene terephthalate (PET) with 0.4 μm diameter pores) within a 24-well plate (Corning) were incubated with 100 μL of 50% Collagen IV for 2 h at 37 °C. Wells were filled with 500 μL of DMEM media, supplemented with 10% FCS, and transwell inserts were placed on top. BEnd.3 cells were then seeded onto the luminal side of the filter at a density of 1.34 × 105 cells per filter and were allowed to grow for 7 days. Media in the luminal and abluminal compartments was replaced on day two and day four, in order to supplement the growth of the monolayer. On day seven, MDA-MB-231 and HEK293T cells were seeded into the lower compartment at a density of 1.5 × 105 cells per cell line per well and allowed to settle for 2 h. Media was then removed from the upper compartments, and 100 μL of aptamer at a concentration of 2 μM was pipetted on top of the transwell membrane. In order to differentiate the cell lines, an anti-EpCAM antibody conjugated to the fluorophore FITC (ab8666) was added to the lower compartment in the last hour of aptamer incubation at a concentration of 10 μg/mL. Following a 3 h incubation at 37 °C, media in both compartments was removed. Cells were then resuspended in 100 μL of PBS, and 2 μL of Hoechst nuclear stain (10 mg/mL) was added and allowed to incubate for 10 min. Cells were then trypsinized and washed three times in 100 μL of PBS before viewing under a FluoView FV10i laser scanning confocal microscope (Olympus). Determination of Membrane Integrity. In order to evaluate the integrity of the membrane, the transendothelial electrical resistance (TEER) of the transwells was measured. The electrodes of an EVOM2 Epithelial Voltohmmeter (World Precision Instruments) were inserted into both the luminal and abluminal compartments of the BBB model, and the resistance was recorded in ohms. This value was then multiplied by the area of the transwell. The same was then performed on a blank transwell insert and subtracted from the previously recorded values in order to obtain the TEER in Ω cm2. In Vivo Blood−Brain Barrier Transcytosis and Biodistribution. Healthy NOD/SCID mice received a single intravenous injection (40 nmol/kg) of aptamer. At 30 and 60 min postinjection, mice were sacrificed, and the brains were excised. Fifty microliters of monoclonal anti-FITC antibody (6.7 μg/mL, Sigma, Cat. No. F5636) in PBS containing 0.1 mg/mL tRNA and 1 mg/mL BSA was added to goat anti-mouse IgG precoated wells (Sapphire Bioscience, Cat. No. 600-11050). After 1 h incubation at room temperature, anti-FITC antibody was removed, followed by thorough washes with PBS. The treated wells were blocked with 50 μL of 1× SuperBlock blocking buffer (Thermo Scientific) at room temperature for 1 h, followed by 3 washes with PBS, 3 min per time. The brain homogenized samples containing biotin-labeled aptamers (100 μL/well) were added and incubated for 1 h at room temperature. After extensive washing, 50 μL of 1:5000 diluted Pierce High Sensitivity streptavidin HRP conjugate (Thermo Scientific, Cat. No. 21140) was added to each well to bind biotin-conjugated aptamer. Following 1 h incubation at room temperature and extensive washing, the bound aptamer was detected with a Quanta Blu fluorogenic peroxidase substrate system (Thermo Scientific, Cat. No. 15169) and measured at a wavelength of 325/ 420 nm using the VICTOR TM X5 Plate Reader (PerkinElmer Life Sciences). Statistical Analysis. Results are expressed as mean ± SEM. Significance (P < 0.05) was assessed using an unpaired t test using Graph Pad Prism 6.0 (San Diego, CA). Unless otherwise specified, all results were averaged from biological triplicates.

the BBB in vivo. Through the simple fusion of the TfR aptamer with EpA, we have developed a system that we hypothesize, upon transcytosing through the BBB, will specifically deliver cytotoxic payloads to the EpCAM expressing brain metastases. This bifunctional aptamer is currently being investigated further as a novel therapeutic for brain disorders.

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METHODS

Cell Lines and Cell Culture. The cell lines of human and mouse origin used in this study were purchased from the American Type Culture Collection. They are mouse brain endothelial cells (bEnd.3), human chronic myelogenous leukemia (K562), human embryonic kidney cells (HEK293T), human ovarian adenocarcinoma (HEY), human colorectal cancer cells (HT29), and human breast adenocarcinoma cells (MDA-MB-231). All cells were grown and maintained in culture with Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Victoria, Australia) supplemented with 10% fetal calf serum (FCS). All cells were maintained at 37 °C in a 5% CO2 atmosphere. Aptamers. All aptamers used in this project were purchased from Integrated DNA Technologies (IDT, Coralville, IA). The SYL3C aptamer was truncated to 17 base pairs and fused to the truncated GS24 aptamer to generate the bifunctional aptamer. Through the introduction of modifications within the binding region of the aptamer, three mutated versions were generated to serve as controls. All oligonucleotide sequences were HPLC purified and labeled with a TYE665 fluorophore on the 5′ end. The sequences were as follows:

SYL3C

5′-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3′ EpA 5′-AC AGA GG TTG CGT CTG T-3′ EpB 5′-CCC ACG TTG TCA TGG C- 3′ EpC 5′-GGG GTT GGC CCC-3′ Scrambled 5′-CGC GCG CCG CAT TCC TTT TGC GGC GCG CG-3′ TEPP 5′-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3′ TENP 5′-GC GCG TGCA CGC GC TA ACG GA GGTTGCG TCC GT-3′ TEPN 5′-GC GCG GTAC CGC GC TA ACG GA TTCCTTT TCC GT-3′ TENN 5′-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3′ Determination of Aptamer Affinity. The dissociation constant (Kd) of the aptamers was determined by measuring binding to native protein targets using flow cytometry. The truncated SYL3C aptamer was incubated with EpCAM positive cells (HT29 and HEY) and EpCAM negative cells (HEK293T and K562). Similarly, the bifunctional aptamers were incubated with TfR positive cells (bEnd.3), EpCAM positive cells (MDA-MB-231), and a cell line expressing neither protein (HEK293T). Aptamers were allowed to fold into their most accessible and stable three-dimensional structure using a thermocycler heating protocol (PerkinElmer) (85 °C for 5 min, slow cooling to 22 °C over 10 min, and 37 °C for 15 min). Cells (5 × 105) were first incubated with binding buffer for 30 min (PBS with 1 mM MgCl2, 0.1 mg/mL tRNA, 1 mg/mL bovine serum albumin, and 10% FCS) followed by two washes with binding buffer prior to incubation with serial concentrations of TYE665-labeled aptamer in a 100 μL volume of binding buffer for 30 min at 37 °C. The cells were washed three times with PBS, resuspended in 50 μL of PBS, and subjected to flow cytometric analyses (FACS Canto II flow cytometer, Becton Dickinson). Unless otherwise specified, aptamer Kd was calculated from the normalized median values for fluorescent intensity, from biological triplicates (n = 3). Confocal Microscopy. Twenty-four hours prior to labeling, cells were seeded at a density of 75 000 cells/cm2 in an 8-chamber slide (Lab-Tek II, Nunc). Each aptamer was prepared in the same manner F

DOI: 10.1021/acschemneuro.6b00369 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00369. Rational truncation of SYL3C EpCAM aptamer, the truncation of aptamers enhancing sensitivity and specificity, and truncated aptamers retaining the ability to be internalized (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. ORCID

Sarah Shigdar: 0000-0001-8084-8447 Author Contributions

S.S. conceived the study; J.M. oversaw the study; J.M., L.G., J.H., and D.X. performed the work; W.D. and S.S. reviewed the manuscript. Funding

The authors acknowledge Deakin University as the funding source for this project. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acschemneuro.6b00369 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience Gadkar, K., Prabhu, S., Ordonia, B. A., Nguyen, Q., Lin, Y., Lin, Z., Balazs, M., Scearce-Levie, K., Ernst, J. A., Dennis, M. S., and Watts, R. J. (2013) Addressing Safety Liabilities of TfR Bispecific Antibodies That Cross the Blood-Brain Barrier. Sci. Transl. Med. 5, 183ra157. (33) Xu, G., Mahajan, S., Roy, I., and Yong, K. T. (2013) Theranostic quantum dots for crossing blood-brain barrier in vitro and providing therapy of HIV-associated encephalopathy. Front. Pharmacol. 4, 140. (34) Aptekar, S., Arora, M., Lawrence, C. L., Lea, R. W., Ashton, K., Dawson, T., Alder, J. E., and Shaw, L. (2015) Selective Targeting to Glioma with Nucleic Acid Aptamers. PLoS One 10, e0134957. (35) Ylera, F., Lurz, R., Erdmann, V. A., and Fürste, J. P. (2002) Selection of RNA Aptamers to the Alzheimer’s Disease Amyloid Peptide. Biochem. Biophys. Res. Commun. 290, 1583−1588. (36) Tannenberg, R. K., Shamaileh, H. A., Lauridsen, L. H., Kanwar, J. R., Dodd, P. R., and Veedu, R. N. (2013) Nucleic acid aptamers as novel class of therapeutics to mitigate Alzheimer’s disease pathology. Curr. Alzheimer Res. 10, 442−448. (37) Cheng, C., Chen, Y. H., Lennox, K. A., Behlke, M. A., and Davidson, B. L. (2013) In vivo SELEX for Identification of Brainpenetrating Aptamers. Mol. Ther.–Nucleic Acids 2, e67.

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DOI: 10.1021/acschemneuro.6b00369 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX