DNA-Programmed Quantum Dot Polymerization for Ultrasensitive

Sep 20, 2016 - Abstract: Development of smart DNA nanostructures is of great value in cancer studies. Here, by integrating rolling circle amplificatio...
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DNA-Programmed Quantum Dot Polymerization For Ultrasensitive Molecular Imaging of Cancer Cells Zhi Li, Xuewen He, Xucheng Luo, Li Wang, and Nan Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02864 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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DNA-Programmed Quantum Dot Polymerization For Ultrasensitive Molecular Imaging of Cancer Cells Zhi Li, Xuewen He, Xucheng Luo, Li Wang, Nan Ma* The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China ABSTRACT: Inorganic nanocrystals, such as quantum dots (QDs), hold great promise as molecular imaging contrast agents because of their superior optical properties. However, the molecular imaging sensitivity of these probes is far from optimized due to the lack of efficient and general method for molecular engineering of nanocrystal into effective bio-probes for signal-amplified imaging. Herein, we develop a strategy to boost the molecular imaging sensitivity of QDs over the limit by co-polymerizing QDs and cell-binding aptamers into linear QD-aptamer polymers (QAPs) through DNA-programmed hybridization chain reaction. We show that the cancer cells treated with QAPs exhibit much stronger photoluminescence (PL) signal than those treated with QDaptamer monomers (QAMs) because of multivalent binding and multi-QD-based signal amplification. The enhanced cell binding and imaging capacity of QAPs significantly improves imaging-based discrimination between different cancer cell types. This approach adds a new dimension for engineering inorganic nanoparticles into effective bio-probes for biomedical applications.

Molecular imaging is essential for cancer diagnosis, drug evaluation, and imaging-guided surgery.1,2 It is crucial to optimize the imaging sensitivity to improve the diagnostic effectiveness and accuracy especially at early disease state.3 QDs hold great promise for cancer molecular imaging because of their superior optical properties including large extinction coefficient, high quantum yield, and resistance to photobleaching.4-6 Bio-functionalized QDs could specifically target cell surface biomarkers to identify and localize cancer cells.7-10 While a variety of QD probes have been constructed for the imaging of different cancer cell types, QD probes capable of signal-amplified imaging of cancer cell surface biomarkers remain underdeveloped. The imaging signal of the regular biotargeting scheme is bottlenecked by the abundance and binding affinity of cell surface receptors since the conventional molecular imaging probes are designed on a one-to-one binding basis. The moderate signal-to-background ratio of individual cells may compromise diagnostic accuracy and sensitivity for cancer early diagnosis. DNA molecules have proven to be versatile tools for nanostructure assembly,11-17 biomolecular recognition,18 and biomineralization.19 The base pairing rules of DNA molecules enables precise and programmable self-assembly of nanomaterials into higher-order nanostructures with defined pattern.20 DNA aptamers, which are generated by in vitro selection, are superior bio-targeting moieties because of their small size, high stability, and potential generalization toward most cancer cell types.21 On the other hand, DNA molecules could serve as unique template for one-step growth of DNAfunctionalized QDs with precisely tailored valency, thus providing an unprecedented means for assembling QDs with pre-defined geometry.22,23 While DNA-templated monomeric QD-aptamer probes have been constructed for specific imaging of cancer cells,22 the imaging sensitivity and discrimina-

tion capability between different cell types remain to be optimized for single cell analysis. We envision that the imaging sensitivity of QDs could be significantly enhanced if the bio-functionalized QDs are constructed in a polymer form, which enables both multi-dentate targeting and multi-QD-based signal amplification. Herein, we report the use of DNA molecules to program the polymerization of QDs and cell-binding aptamers into linear QD-aptamer polymers for ultrasensitive imaging of specific cancer cell types. Sgc8c aptamer with high specificity towards CCRFCEM cells is selected for investigation.24 In order to obtain highly ordered polymers with repeating QD-aptamer subunits, we employ hybridization chain reaction (HCR)25-27 for bottomup construction of polymers from two monomers – QD monomer (M1) and aptamer monomer (M2). As shown in Figure 1, each monomer is prepared by linking the functional unit (QD or aptamer) with the reactive hairpin unit (H1 or H2) through overhangs respectively. Polymerization is triggered by a single-stranded DNA initiator (I) that could open up the hairpin of M1 through DNA strand displacement. Subsequently, the exposed single-stranded region of the opened hairpin (M1) could hybridize with M2 and generate a single-stranded region that in turn reacts with M1. The polymer chain will continue growing until most of the monomers are exhausted. DNAcapped CdTe QDs are prepared using ps-po chimeric DNA molecules as templates following a previously reported protocol.16 The QDs feature an absorption peak at 572 nm and an emission peak at 623 nm (Figure 2a). The quantum yield of asprepared QDs is determined to be 18.4%. DNA hybridization and polymerization are monitored by native polyacrylamide gel electrophoresis (PAGE) (Figure 2b). Hybridization between DNA-QD and H1 overhangs results in a shift of the QD PL band (lane 1 and 2). Also, hybridization between aptamer and H2 overhangs results in a shift of the aptamer band containing pre-stained DNA (lane 3 and 4). Polymerization of M1

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Figure 2. Characterization of DNA-QD and QAPs. (a) Absorption and photoluminescence spectra of DNA-capped CdTe QDs. (b) Native polyacrylamide gel electrophoresis (PAGE) characterization of the polymerization reaction. (Aptamer (lane 3) and aptamer+H2 (lane 4) are pre-stained with GelRed before loading on the gel)

Figure 1. Schematic illustration of QD-aptamer polymers (QAPs) for signal-amplified imaging of cancer cell surface biomarkers. (a) Preparation of QD monomer (M1) and aptamer monomer (M2) by hybridizing DNA-QD and aptamer with H1 and H2 through overhangs respectively. (b) Copolymerization of QD monomer and aptamer monomer into QAPs through DNA-programmed hybridization chain reaction. (c) Schematic illustration of multivalent binding of QAP with cell surface receptors of a single cancer cell. and M2 is triggered by the initiator (0.1× equivalent) and yields large product with pronounced retarded mobility in the gel (lane 5). In contrast, M1 and M2 do not react with each other in the absence of the initiator (lane 6). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are conducted for nano-characterization of the polymer (Figure 3 and Figure S1). DNA-QDs are spherical and near monodisperse with a mean diameter of 3.5 nm. QAPs are mostly linear with evenly distributed QDs. The average number of QDs within the polymers is estimated to be 21.6 ± 6.0 according to high-resolution TEM images. The QAPs remain stable in buffer solution during 24 hours incubation at 37 °C (SI Figure S2). Next, we apply QAPs for molecular imaging of human acute lymphoblastic leukemia cells (CCRF-CEM) by targeting the cell surface receptor protein tyrosine kinase 7 (PTK7). In order to evaluate the imaging sensitivity of the polymer, QD-

aptamer monomers (QAMs) are also prepared for comparison. CCRF-CEM cells are incubated with QAPs or QAMs at 37 °C for 1 hour and cell PL images are acquired on an inverted fluorescence microscope (see experimental section for more details). As shown in Figure 3a, CCRF-CEM cells treated with QAPs exhibit much stronger PL than that treated with QAMs at various QD concentrations ranging from 5 nM to 50 nM. The control Ramos cells treated with either polymers or monomers exhibit minimal background PL (Supporting Information Figure S3), confirming specificity of both QAP and QAM toward CCRF-CEM cells. Flow cytometry measurements also show that CCRF-CEM cells treated with QAPs are much more luminescent than that treated with QAMs (50 nM QDs) (Supporting Information Figure S4), which is consistent with fluorescence microscopy results. As shown in Figure 3b, QAPs impart 10.5-fold (5 nM QDs), 6.2-fold (10 nM QDs), 4.5-fold (20 nM QDs), and 3.8-fold (50 nM QDs) enhancement of mean PL intensities over QAMs at various QD concentrations. In particular, QAPs could offer strong cell imaging signals at very low QD concentration (5 nM), which could not be achieved by QAMs even when reaching the binding plateau (> 50 nM). The equilibrium dissociation constants of QAPs (Kd[p]) and QAMs (Kd[m]) are calculated to be 0.189 ± 0.025 nM and 8.13 ± 1.54 nM respectively, inferring enhanced binding affinity of QAP with CCRF-CEM cells due to multivalent binding. The discrimination factors of QAPs between CCRF-CEM and Ramos cells are 32.5 (20 nM QDs), 12.0 (50 nM QDs), and 10.5 (100 nM QDs) respectively, which are significantly higher than QAMs (5.3 (20 nM QDs), 3.6 (50 nM QDs), and 3.2 (100 nM QDs)) (Figure 3c). It is noteworthy that the highest discrimination factor is achieved at relatively low QD concentration (20 nM), which is likely due to reduced non-specific binding at low QD concentrations. The cytotoxicity of QAPs with CCRF-CEM cells is evaluated using CCK-8 assay. Minimal effects on cell viability are observed at various QD concentrations (5 nM-200 nM) (Figure S5). To further investigate the dependence of imaging sensitivity on polymer lengths, we prepare three classes of QAPs with different lengths by varying the initiator concentration for polymerization reaction. As shown in Figure 4a and 4b, in comparison with the long polymer (0.1× equivalent I) used for above experiments, QAPs prepared with higher initiator concentration (0.5× and 0.9× equivalent I) exhibit accelerated

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Figure 3. Low magnification (left) and high-resolution (right) TEM images of DNA-QD and QAPs.

Figure 4. Molecular imaging of CCRF-CEM cells with QAP and QAM. (a) Fluorescence microscopy images of CCRFCEM cells treated with QAP and QAM at various QD concentrations. (b) Binding curves of QAP and QAM with CCRFCEM cells. (c) Discrimination factors between CCRF-CEM and Ramos cells for QAP and QAM at various QD concentrations.

mobility in agarose gel and smaller hydrodynamic sizes as shown by dynamic light scattering. The as-prepared medium (0.5× equivalent I) and short (0.9× equivalent I) polymers contain 7.0 ± 1.9 and 3.5 ± 1.2 QD subunits on average respectively (Figure 4c). The three classes of QAPs and QAMs are then applied for targeted imaging of CCRF-CEM cells. As shown in Figure 4d, the mean PL intensity of CCRF-CEM cells gradually increases with increasing polymer length. This is likely because longer QAPs contain more QDs for signal amplification and are more favorable for multi-dentate binding with cell surface receptors. The QAPs were further applied for analysis of clinical samples. Peripheral blood samples from acute lymphoblastic leukemia patients were treated with QAPs and QAMs (50 nM QDs) respectively and the cancer cells were detected by fluorescence microscopy. The mean PL intensity of cancer cells stained with QAPs is about 4-fold higher than that of QAMs, indicating significant improvement in molecular imaging sensitivity (SI Figure S6). In summary, we have synthesized a new class of QDaptamer co-polymers for signal-amplified imaging of cancer cell surface biomarkers. The imaging sensitivity of QAPs is significantly improved over QAMs, and strong cell imaging signal could be achieved at very low QD concentration (as low as 5 nM QDs). More importantly, QAPs could offer much higher cell discrimination factor than QAMs, thus highlighting their potential for accurate and sensitive detection of single cancer cells such as circulating tumor cells (CTC). The reported polymeric QD-aptamer conjugates pave the way for engineering inorganic nanoparticle-based probes with enhanced imaging sensitivity or therapeutic potency. For example, it could be extended to magnetic nanoparticles to improve the sensitivity of magnetic resonance imaging, and to gold nanoparticles to enhance photothermal therapy efficacy.

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AUTHOR INFORMATION Corresponding Author *[email protected]

ACKNOWLEDGMENT We thank Yuan Chen at Fujian Medical University for kindly providing peripheral blood samples from acute lymphoblastic leukemia patients. This work was supported in part by the NSFC (21175147, 91313302, 21475093, 21522506), the National HighTech R&D Program (2014AA020518), 1000-Young Talents Plan, PAPD, and startup funds from Soochow University.

REFERENCES

Figure 5. QAP with different polymer lengths for CCRFCEM cell imaging. (a) Agarose gel electrophoresis characterization of QAPs prepared with different initiator concentrations (0.1×, 0.5×, and 0.9× equivalent). (b) Dynamic light scattering (DLS) measurements of QAPs with different lengths. (c) Statistics of the number of QDs in each type of QAP. (d) Fluorescence microscopy images and mean PL intensity of CCRFCEM cells treated with QAM and QAP with different lengths (QD concentration = 10 nM).

ASSOCIATED CONTENT Supporting Information Experimental Section, AFM image of QAPs (Figure S1), stability measurements of QAPs (Figure S2), fluorescence microscopy images of Ramos cells treated with QAPs and QAMs at various QD concentrations (Figure S3), flow cytometry analysis of CCRF-CEM cells treated with QAPs and QAMs (Figure S4), cytotoxicity of QAPs with CCRF-CEM cells (Figure S5), Cancer cell detection of peripheral blood samples from acute lymphoblastic leukemia patients (Figure S6). The Supporting Information is available free of charge on the ACS Publications website.

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