Amplified Split Aptamer Sensor Delivered Using Block Copolymer

Nov 23, 2018 - Amplified Split Aptamer Sensor Delivered Using Block Copolymer Nanoparticles for Small Molecule Imaging in Living Cells. Chong-Hua Zhan...
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Amplified Split Aptamer Sensor Delivered Using Block Copolymer Nanoparticles for Small Molecule Imaging in Living Cells Chong-Hua Zhang, Hong Wang, Jin-Wen Liu, Ying-Ying Sheng, Jian Chen, Peisheng Zhang, and Jian-Hui Jiang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00670 • Publication Date (Web): 23 Nov 2018 Downloaded from http://pubs.acs.org on November 24, 2018

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Amplified Split Aptamer Sensor Delivered Using Block Copolymer Nanoparticles for Small Molecule Imaging in Living Cells Chong-Hua Zhang a, b, Hong Wang b, Jin-Wen Liu a, Ying-Ying Sheng a, Jian Chen *b, Peisheng Zhang a,b, Jian-Hui Jiang *a

State Key Laboratory of Chemo-Biosensing & Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, China. b Key Laboratory of Theoretical Organic Chemistry and Functional Molecule of Ministry of Education, Hunan Provincial Key Laboratory of Controllable Preparation and Functional Application of Fine Polymers, Hunan Province College Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, Hunan 411201, China, Hunan Provincial Key Lab of Advanced Materials for New Energy Storage and Conversion. a

KEYWORDS : split aptamer, enzyme-free amplification, block copolymer nanoparticles, small molecule imaging, sensor

ABSTRACT: We develop a novel amplified split aptamer sensor for highly sensitive detection and imaging of small molecules in living cells by using cationic block copolymer nanoparticles (BCNs) with entrapped fluorescent conjugated polymer as a delivery agent. The design of split aptamer as the initiator of hybridization chain reaction (HCR) affords the possibility of enhancing the signal-to-background ratio and thus allows high-contrast imaging for small molecules with relatively weak interactions with their aptamers. The novel design of using fluorescent cationic BCNs as the nanocarrier enables efficient and self-tracking transfection of DNA probes. Results reveal that BCNs exhibit high fluorescence brightness allowing direct tracking of the delivery location. The developed amplified split aptamer sensor is shown to have high sensitivity and selectivity for in vitro quantitative detection of ATP with a detection limit of 30 nM. Live cell studies show that the sensor provides a “signal on” approach for specific, high-contrast imaging of ATP. The DNA sensor based HCR system may provide a new generally applicable platform for detection and imaging of low-abundance biomarkers.

Monitoring and visualization of bioactive species in living cells is essential for understanding their physiological functions and pathological effects.1 Development of sensors with selective, sensitive and quantitative signals for live cell studies are in high demand.2 Nucleic acids have proved to be a useful tool for biological analysis in living systems.3 However, their detection sensitivity is one of the major challenges in intracellular applications, especially for those biomolecules which are in very low abundance. Therefore, signal amplification based protocols are drawing increasing attention.4,5 Enzyme-free amplification assay is an emerging technology for ultrasensitive intracellular detection, avoiding cell damage caused by delivering exogenous enzymes or proteins while allowing monitoring targets in low abundance with high signal gain. Among them, hybridization chain reaction (HCR)6 and catalytic hairpin assembly (CHA)7 based amplification methods have been demonstrated for visualization of RNA,8,9 enzyme activity,10 and metal ions11 inside living cells. However, detection of small molecules based on enzyme-free amplification assays in living cells has not been reported. Efficient methods for delivering nucleic acids are another vital factor for live cell analysis. Nanocarriers offer a useful

platform for delivery of nucleic acid probes. Recently, others and our group have explored some delivery systems for constructing intracellular enzyme-free amplification sensors. Although the use of commercially available liposomes has shown some success,8 the delivery efficiency of this method cannot reach a satisfying level for a lot of cell types. We developed an AuNP-peptide based core-shell nanoparticles for electrostatically assembled DNA probes to realize sensitive mRNA detection in living cells.9 Despite the high cellular delivery efficiency, the nanocomplex suffers from complex preparation processes. Other groups developed GO-based delivery system for HCR and CHA probes. However, these methods may involve nonspecific dissociation of the probes and produce false positive signal.10,12 Therefore, to develop nanoparticles that enable facile preparation and high delivery efficiency is highly demanding. Block copolymer nanoparticles, which is formed by assembly of amphiphilic block copolymers, have been established as useful

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Scheme 1. Illustration of (a) synthetic route for block copolymer and (b) split aptamer sensor-based HCR for live cell imaging.

nanocarriers in terms of their hydrophilicity, biocompatibility, low toxicity and easy to function.13 Furthermore, the hydrophobic core enables the nanoparticles to easily entrap hydrophobic drugs or dyes such as fluorescent conjugated polymers by hydrophobic interaction.14,15 In virtue of these advantages, block copolymer nanoparticles have been extensively explored in drug and gene delivery.16-18 However ， the block copolymer nanoparticles remain unexplored for intracellular delivery of nucleic acids sensors for imaging in living systems. Herein, we report a novel amplified split aptamer sensor delivered by block copolymer nanoparticles (BCNs) for small molecule imaging in living cells, as illustrated in Scheme 1. To demonstrate the proof of principle, we choose ATP, a primary energy molecule and cofactor essential for living cells,19 as the model target. Two split aptamer fragment probes, AP1 and AP2, and two HCR hairpin probes, H1 and H2 are designed. The split aptamer fragment probes contain two parts, split aptamer domains acting as the target recognition components,20 and the split initiator components for HCR (as illustrated in Scheme S1 in SI). The signal probe H1 is labelled with a TAMRA fluorophore quenched by a BHQ2 moiety when H1 is in the hairpin conformation, resulting in a low fluorescence background in the absence of ATP. In the presence of ATP target, the two split aptamer fragments can form a stable complex. This complex brings the two tail sequences into close proximity, forming an active initiator for triggering the HCR between H1 and H2. As a result of the formation of the chain-like HCR product, many hairpin signal probes are opened in response to a single ATP target with TAMRA fluorophores separated from BHQ quenchers, thereby activating an amplified fluorescence signal to indicate the concentration of ATP. When ATP is absent in situations, the two split aptamer fragments are not able to trigger HCR because of the too short split initiator sequences, thus no fluorescence signal is activated. To use the amplified

split aptamer sensor in living cells, amino-functionalized fluorescent BCNs are prepared using a synthesized amphiphilic block copolymer PEO113-b-P(St24-co-VBA5). The amphiphilic copolymer can assemble into BCNs with entrapped fluorescent conjugated polymer, poly(9,9dioctylfluorenyl-2,7-diyl) (PFO), using a co-precipitation procedure. The positively charged BCNs enable facile assembly of nucleic acid probes on their surface and allow their efficient delivery into cells. Because use of BCNs as the nanocarrier provides a protective, efficient and self-tracking transfection agent for intracellular DNA sensors, this system offers possibility of high efficiency of delivery and direct visualization of delivery localization. Moreover, the design of split aptamer as the initiator of HCR enables high-contrast imaging for the detection of targets with relatively weak interactions with their aptamers. Therefore, the split aptamer sensor-based HCR system may provide a generally applicable platform for low-abundance biomarker imaging. Firstly, we investigated fluorescence responses of the amplified split aptamer sensor in response to ATP, as shown in Figure 1a. In the absence of ATP, the mixture of two split aptamer probes and two hairpin probes gave very weak fluorescence at 560 nm, implying that the designed probes had a low fluorescence background and no HCR happened between the four probes in the absence of target. In the presence of 1 mM ATP, an intense fluorescence peak was observed at 560 nm. In contrast, control experiments with a mixture of AP1 and H1 plus H2 or a mixture of AP2 and H1 plus H2 both showed negligible fluorescence activation in response to ATP. These observations implied that the ATPmediated assembly of two split aptamer fragments AP1 and AP2 was essential for the activation of fluorescence signal, verifying the design of split aptamer as the initiator of HCR between H1 and H2 in response to the target. A further investigation was performed using gel electrophoresis analysis

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Figure 1. (a) Fluorescence spectral responses obtained from reaction of 1 mM ATP with AP1, AP2, H1 and H2 (1), reaction of AP1, AP2, H1 and H2 (2), reaction of 1 mM ATP with AP1, H1 and H2 (3), reaction of 1 mM ATP with AP2, H1 and H2 (4), (b) Gel electrophoresis images. Lane M, DNA marker; lane 1, H1; lane 2, H2; lane 3, H1 and H2; lane 4, 100 nM initiator, H1 and H2; lane 5, H1, H2 and AP1; lane 6, H1, H2 and AP2; lane 7, H1, H2, AP1 and AP2; lane 8, H1, H2, AP1 and AP2 with 1 mM ATP, (c) Fluorescence responses of the four-probe mixture to ATP of varying concentrations. (d) Specificity evaluation of the four-probe mixture for ATP detection. F0 is fluorescence of the four-probe mixture and F is fluorescence obtained by incubating the four-probe mixture with 1 mM ATP (1), 1 mM CTP (2), 1 mM GTP (3), 1 mM UTP (4), and 1 mM TTP. Error bars are standard deviations of three repetitive experiments. The excitation wavelength used in this assay is 535 nm.

(Figure 1b). No new bands appeared for reactions of two hairpin probes with two aptamer fragments AP1 and AP2 as well as a single fragment AP1 or AP2. In contrast, incubation of 1 mM target ATP with two hairpin probes and two aptamer fragments AP1 and AP2 gave broad bright bands with a maximum size over 3000 base-pairs, which was as large as the HCR product obtained by incubation of a synthetic initiator with two hairpins. This result confirmed the successful HCR triggered by target ATP, validating the proposed design for the activatable amplification approach. The reported split aptamer sensor was found to give activated fluorescence signals dynamically correlated to the concentrations of ATP over a five order of magnitude range (Figure 1c). A quasilinear correlation was obtained for the peak intensities at 560 nm to the logarithmic ATP concentrations in the range from 100 nM to 1 mM with a detection limit estimated to be 30 nM (Figure S1 in SI). This detection limit was much better than existing aptamer-based fluorescence ATP imaging methods,21,22 indicating the advantage of sensitivity enhancement for our amplified split aptamer sensor. Next, the specificity of the split aptamer sensor for ATP was tested. The target ATP led to enhancement of the fluorescence intensity with a high signalto-background ratio of ~7.2-fold (Figure 1d), while no obviously fluorescence enhancement was observed in response to three analogues CTP, GTP and UTP. This result validated the specificity of our aptamer sensor to ATP.

Figure 2. Characterization of BCNs. (a) TEM, (b) DLS, (c) UV absorption (black) and fluorescence emission spectra (blue), (d) zeta potential analysis. BCN (red), BCN-DNA (black)s. Inset: TEM image of BCNs at higher magnification.

To employ the designed DNA sensor for live cell applications, amino-functionalized fluorescent BCNs are prepared. An amphiphilic block copolymer PEO113-b-P(St24co-VBC5) was synthesized by RAFT polymerization starting from a hydrophilic PEO113-TTC macro-RAFT reagent (Scheme S1 in SI).23 Gel permeation chromatography (GPC) analysis showed that the copolymer exhibited relatively narrow polydispersity (Mw/Mn=1.05, Table S2 and Figure S2 in SI). The degrees of polymerization of hydrophobic units of St and functional units of VBC were determined to be 24 and 5 by 1H NMR calculation (Figures S3-S5 in SI). So the diblock copolymer was referred to as PEO113-b-(St24-coVBC5). The chlorine groups in VBC units were then converted by amine groups, which was validated by FTIR spectra (Figure S6 in SI). Subsequently, the PFO-entrapping BCNs were prepared readily using the coprecipitation assay.24 The particle size was controlled by changing the starting concentrations of the copolymer and PFO in the mixture of THF and DMF.25 The as-prepared BCNs with cationic surface exhibited high water-solubility. The as-prepared BCNs was characterized by transmission electron microscope (TEM), as shown in Figure 2a. The nanoparticles displayed uniform spherical shape with a diameter around 25 nm. Dynamic light scattering (DLS) analysis indicated the BCNs had the average hydrodynamic diameters of about 35 nm (Figure 2b). The BCNs had a UV absorption peak at 380 nm and a strong emission with a maximum at 425 nm on excitation at 405 nm (Figure 2c). The fluorescence had no overlapping with that for the HCR probes, implying that the BCNs could be used as a self-tracking agent to visualize the delivery locations of the nanoparticles. These results gave clear evidences for the successful synthesis of the fluorescent BCNs. To deliver DNA probes into the cells, BCNs and DNA probes, two split aptamer fragment probes and two HCR hairpin probes, were simply mixed, which could result in a BCNs-DNA nanocomplex due to electrostatically and π-π stacking

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interactions between DNA and BCNs. The BCN-DNA nanocomplex was confirmed using Zeta potential measurements (Figure 2d), which showed a positively charged surface for the nanoparticles and a negatively charged surface for the BCNs-DNA nanocomplex. The probe loading capacity were obtained by detecting fluorescence intensity of the FAMlabeled hairpin probes before and after incubation with BCNs (Figure S7 in SI). A loading of probes was calculated to be as high as 87.41 ± 4.0%, indicating the loading amount of about 2.66 × 1011 hairpin probes per 1 μg of BCNs, with relative standard deviations (RSDs) of 4.5%, 3.6%, 4.7%, and 4.6% in three repetitive assays for four sets of newly prepared BCNs (Table S3 in SI). Such outstanding reproducibility seemed attributed to the very narrow size distribution and uniform spherical shape of BCNs, which might owe to relatively narrow polydispersity of the synthesized copolymer using RAFT polymerization, enabling block copolymers generated with controllable molecular weight and a predictably narrow PDI.26 The ideal reproducibility of BCNs loading capacity provides a reliable opportunity for semi-quantification detection in living cells. To visualize the cellular uptake, the fluorescence intensity of Hela cells transfected with FAMlabeled probe carried by BCNs and the naked probe as control was analyzed by flow cytometry in a large cell population. As showed in Figure S8 in SI, compared to the control set, the fluorescence intensity in the group incubated with BCNs-DNA complexes were obviously enhanced, indicating a great cellular uptake. Further investigation was made to study the toxicity of BCNs. It was observed that the nanoparticles only exhibited marginal toxicity to HeLa cells at a concentration up to 100 μg mL-1 with the cell viability decreased by ∼12% after 8 h incubation (Figure S9 in SI). These data demonstrated the excellent biocompatibility for the BCNs. The formation of BCN-DNA nanocomplexes allowed the delivery of the amplified split aptamer sensor into cells, which provided the possibility for highly sensitive imaging of ATP in living cells. To test the possibility, HeLa cells were incubated

Figure 3. Images of HeLa cells after incubation with BCNs carrying AP1 and H1 plus H2 (a) BCNs carrying AP2 and H1 plus H2 (b), BCNs carrying AP1 and AP2 plus H1 and H2 (c) for 4 h at 37 °C.

Figure 4. Images of HeLa cells treated with 10 μM oligomycin (a), medium (b), or 5 mM Ca2+ (c) followed by incubation with 100 nM nanocomplex for 4 h at 37 °C.

for 4 h with the mixture of BCNs and a probe set consisting of AP1, AP2, H1 and H2. As shown in Figure 3, we obtained very bright images at the red channel (575-610 nm) for TAMRA and at the green (420-450 nm) channel for BCNs. The bright green images indicated high endocytic efficiency of the BCN-DNA nanocomplexes, and the red images suggested the specific response of the amplified split aptamer sensor to target ATP. In control experiments, HeLa cells were incubated for 4 h with the mixture of BCNs with another probe set consisting of AP1, H1 and H2 or the third probe set consisting of AP2, H1 and H2. While the green channel remained very bright, almost no fluorescence signals were observed in the HeLa cells in the red channel in these two control experiments. This observation not only manifested the efficient delivery of the BCN-DNA nanocomplexes in the cells, but also verified that the activation of fluorescence signal was specific to ATP-mediated assembly of two split aptamer fragments. Furthermore, we demonstrated the potential of the amplified split aptamer sensor for semiquantitative detection of ATP in the cells. The HeLa cells were pretreated with 10 μM oligomycin, a known inhibitor of ATP,27 or 5 mM Ca2+, a commonly used ATP inducer,28 for 30 min, and were subsequently incubated with the mixture of BCNs and a probe set consisting of AP1, AP2, H1 and H2. It was observed that the TAMRA fluorescence decreased dramatically upon treatment with oligomycin (Figure 4), while significant enhancement was obtained in the cells pretreated with 5 mM Ca2+. More importantly, the intensities of the green imagines almost showed negligible changes in these assays. In addition, compared with those non-fluorescence delivery reagents, the bright green fluorescence of the reported BCNs could image the probe delivery efficiency as well as gave us more information about the relative expression levels of ATP (Table S4 in SI) by calculating the ratios of average optical density for red channel to green channel. In order to show the

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ACS Sensors clinical utility of the system, we further spike healthy whole blood samples with Hela and MCF-7 cells and used the new approach for ATP imaging in these cells. After spiked with Hela and MCF-7 cells, the blood samples underwent gradient centrifugation followed by incubated with the reported nanoassembly and FITC-labeled anti-CD45. As showed in Figure S10 in SI, compared with anti-CD45 labeled white blood cells (WBCs), the cancer cells displayed brighter red images, indicating the level of ATP was elevated in cancer cells, which is consistent with previous reports.29 In conclusion, the reported nanoparticles provided a useful tool for living cells analysis. In summary, a novel amplified split aptamer sensor for highly sensitive detection and imaging of small molecules in living cells has been developed. The sensor is rationally designed by combining two split aptamer fragments with HCR amplification in order to minimize the background and improve the signal-to-background ratio. Cationic BCNs with entrapped fluorescent conjugated polymer are used as an efficient self-tracking nanocarrier for the sensor. The amplified split aptamer sensor is shown to have high sensitivity and selectivity for in vitro detection of ATP with a detection limit of 30 nM. Live cell studies show that the sensor provides a “signal on” approach for specific, high-contrast imaging of ATP, and the BCNs allows direct tracking of the delivery location and. The amplified split aptamer sensor may provide a new generally applicable platform for detection and imaging of low-abundance biomarkers.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the ACS Publications website at DOI: . Experimental details; Illustration of design of split aptamer probes; DNA sequences used in this experiment; Fitted curve of fluorescence intensities to ATP concentrations; Molecular weight distribution data of starting linear polymers; Probe loading efficiency of BCNs; Characterizations of polymers; Ratios of average optical density; Flow cytometry analysis of probe uptake efficiency; Toxicity of BCN to HeLa cells; Confocal fluorescent images of healthy blood samples mixed with Hela cells and MCF7 cells.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Jian-Hui Jiang: 0000-0003-1594-4023 Jian Chen: 0000-0002-3329-6384

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by NSFC (21527810, 21705040, 21705041, 51603067, 51773056, 51373002).

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