Engineered Decomposable Multifunctional Nanobioprobes for

Article pubs.acs.org/ac

Engineered Decomposable Multifunctional Nanobioprobes for Capture and Release of Rare Cancer Cells Min Xie,†,§ Ning-Ning Lu,†,§ Shi-Bo Cheng,† Xue-Ying Wang,† Ming Wang,‡ Shan Guo,† Cong-Ying Wen,† Jiao Hu,† Dai-Wen Pang,† and Wei-Hua Huang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China ‡ Renmin Hospital of Wuhan University, Wuhan 430060, China S Supporting Information *

ABSTRACT: Early detection and isolation of circulating tumor cells (CTCs) can provide helpful information for diagnosis, and functional readouts of CTCs can give deep insight into tumor biology. In this work, we presented a new strategy for simple isolation and release of CTCs using engineered nanobioprobes. The nanobioprobes were constructed by Ca2+-assisted layer-by-layer assembly of alginate onto the surface of fluorescent-magnetic nanospheres, followed by immobilization of biotin-labeled anti-EpCAM. As-prepared anti-EpCAM-functionalized nanobioprobes were characterized with integrated features of anti-EpCAMdirected specific recognition, fluorescent magnetic-driven cell capture, and EDTAassisted cell release, which can specifically recognize 102 SK-BR-3 cells spiked in 1 mL of lysed blood or human whole blood samples with 89% and 86% capture efficiency, respectively. Our proof-of-concept experiments demonstrated that 65% of captured SK-BR-3 cells were released after EDTA treatment, and nearly 70% of released SK-BR-3 cells kept their viability, which may facilitate molecular profiling and functional readouts of CTCs.

C

CTC-derived molecular signatures and functional readouts provide more valuable and significant discernment into tumor biology, which may facilitate personalized therapy.29,30 Therefore, it is an urgent need to develop a new CTC assay that can capture CTCs with high efficiency, but also release CTCs without disruption of its viability and biological functions. Recently, various affinity nanosubstrates or microfluidic chips modified by thermoresponsive polymer31,32 or aptamer33−36 have been constructed for capture and release of cancer cells; however, realization of capturing and releasing CTCs through nanobioprobes has rarely been reported. Therefore, the development of engineered nanobioprobes that can capture and release CTCs simply, sensitively, and efficiently would give an alternative choice to early cancer diagnosis. In this work, we fabricated anti-EpCAM functionalized nanobioprobes (anti-EpCAM-Alg-FMNB), which can be formed and decomposed reversibly to capture and release cancer cells. Here, Ca2+-initiated layer-by-layer (LBL) selfassembly was employed to deposit alginate coating on fluorescent-magnetic nanospheres (FMNS) to fabricate alginate-encapsulated FMNS (Alg-FMNS). Moreover, Ca2+ further bridged anti-EpCAM anchored onto the surface of Alg-FMNS to construct anti-EpCAM-Alg-FMNB, which can be easily destroyed by EDTA treatment.37 The experimental results

irculating tumor cells (CTCs) are traveling cells in physiological fluids that are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases.1 In routine cancer diagnostics, it remains unclear whether early tumor spread has taken place until the manifestation of overt metastasis. Therefore, detection of CTCs from peripheral blood, as a “liquid biopsy”, represents a reliable potential alternative to routine cancer diagnostics, and is expected to be able to complement routine tissue biopsies of metastatic tumors for therapy guidance.2 To exploit CTCs as a new cancer “biomarker” for disease progression and guide implementation of therapy, several strategies have been put forward to develop diagnostic assays capable of detecting and enumerating CTCs, such as filterbased microdevice3,4 and density-based centrifugation method,5 because of the physical differences between CTCs and hematologic cells. Compared with normal or blood cells, CTCs specifically express epithelial cell adhesion molecule (EpCAM) which is often used as a biotarget in CTC sensing methodologies,6 such as anti-EpCAM-functionalized microfluidic chips7−10 or anti-EpCAM-coated nanostructured substrates for cell-affinity assay.11,12 Besides, nanobioprobes are often equipped with magnetism-manipulable, fluorescencetrackable, and biotargeting properties for recognition, capture, isolation, and imaging of cancer cells,13−26 which are regarded as promising tools for CTC enumeration.27,28 Although the capturing and enumeration of CTCs provides helpful information for diagnosis, it is conceivable that the © 2014 American Chemical Society

Received: March 3, 2014 Accepted: April 9, 2014 Published: April 9, 2014 4618

dx.doi.org/10.1021/ac500820p | Anal. Chem. 2014, 86, 4618−4626

Analytical Chemistry

Article

Characterization of First Alginate Layer Formation on NS-PEI-QDs. To get the alginate coating, sodium alginate was first incubated with NS-PEI-QDs. To avoid the influence of PEI in the chemical shift detection, pure MAA-CdTe QDs instead of NS-PEI-QDs were used for incubation with sodium alginate. Subsequently, the chemical shift of elemental Cd in alginatetreated CdTe QDs and MAA-CdTe QDs was examined using X-ray photoelectron spectrometry (XPS) (Kratos, Model XSAM800). The infrared spectra of sodium alginate, NS-PEIQDs, and NS-PEI-QDs-Alg were obtained on a Thermo Nicolet 360 FT-IR spectrophotometer. The dynamic weight loss tests of sodium alginate, NS-PEI-QDs, and NS-PEI-QDsAlg were conducted on a SETARAM SETSYS-TGA TG/DSC Thermoanalyzer for qualitative and quantitative analysis of the alginate on the NS-PEI-QDs surface. All tests were performed in an air purge using sample weights of 5−10 mg over a temperature range of 25−800 °C at a scan rate of 20 °C/min. Characterization of Formation and Disintegration of Alginate Shell on NS-PEI-QDs. To clearly check whether or not alginate shell can be formed or decomposed reversibly on NS-PEI-QDs, FITC-alginate derivative (FITC-Alg) was synthesized (see the Supporting Information) and used as a reporter.42 In this experiment, CdTe QDs was not assembled on NS-PEI, since the fluorescence of FITC was completely concealed by CdTe QDs. FITC-Alg was used to get NS-PEIAlg, NS-PEI-Alg-Ca2+-Alg, NS-PEI-Alg-(Ca2+-Alg) × 2, NSPEI-Alg-(Ca2+-Alg) × 3. The fluorescence spectra of assynthesized samples were obtained using a Shimadzu Model RF-5301 fluorescence spectrophotometer. Subsequently, the assynthesized samples were mixed 1:1 (v/v) with 50 mM EDTA and rotated for 5 min. The fluorescent spectra of supernatant and reconstituted samples were also detected. Fabrication and Characterization of SA-Alg-FMNS and Anti-EpCAM-Alg-FMNB. To have anti-EpCAM-Alg-FMNS, SA, and biotin conjugation system was used. First, streptavidinalginate conjugate (SA-Alg) was synthesized (see the Supporting Information). Subsequently, Alg-FMNS were activated by Ca2+, and incubated in sequence with SA-Alg (namely, SA-Alg-FMNS) and biotin-labeled anti-EpCAM to get anti-EpCAM-Alg-FMNB. FITC-biotin was used to confirm whether or not SA was successfully conjugated onto the surface of Alg-FMNS through Ca2+ bridge. Simply, 50 μL of 5 mg/mL SA-Alg-FMNS or AlgFMNS were incubated with 2 μL of 50 μg/mL FITC-biotin for 30 min, respectively. After incubation, the samples were washed three times and finally dispersed in ultrapure water for microscopic observation. Similarly, 2 μL of 100 μg/mL FITC-labeled goat antimouse secondary antibody was used to incubate with 50 μL of 5 mg/mL anti-EpCAM-Alg-FMNB or SA-Alg-FMNS for 30 min, respectively. After removal of excess FITC-labeled goat antimouse antibody, the reconstituted antiEpCAM-Alg-FMNB or SA-Alg-FMNS were observed under fluorescent microscope. Meanwhile, FITC-biotin labeled SAAlg-FMNS or FITC-labeled secondary antibody stained antiEPCAM-Alg-FMNB were treated with 50 mM EDTA for 5 min, followed by microscopic observation. Recognition of SK-BR-3 Cells Using Anti-EpCAM-AlgFMNB. About 2 × 106 SK-BR-3 cells were first cultured in a flask and then detached using trypsin−EDTA solution, and collected by centrifugation at 1000 rpm for 6 min at room temperature. Subsequently, the cells were washed and resuspended in 1 × PBS. Then, 100 μL of 5 mg/mL antiEpCAM-Alg-FMNB were added to Epppendorf tubes contain-

demonstrated that anti-EpCAM-Alg-FMNB can specifically recognize, capture, and isolate SK-BR-3 cells (EpCAM-positive cell line)38 but not Hela cells (EpCAM-negative cell line).39 After EDTA treatment, alginate shell on anti-EpCAM-AlgFMNB was disintegrated, allowing detachment of FMNS from the surface of SK-BR-3 cells. Namely, the capture and release of EpCAM-postive cancer cells were successfully achieved by using anti-EpCAM-Alg-FMNB. The nanobioprobes were further applied for isolation of 102 cancer cells spiked in 1 mL of lysed blood or human whole blood, more than 85% of capture efficiency were obtained. The captured cells were suitable for routine immunostaining, indicating that this smart nanobioprobe might be a good candidate for CTC detection.

■

MATERIALS AND METHODS Materials. Streptavidin, poly(ethylenimine) (PEI; molecular weight (MW) = 25 kDa and 750 kDa), biotin (5-fluorescein) conjugate (FITC-biotin), fluorescein 5(6)-isothiocyanate, 1ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4′,6-diamidino-2phenylindole (DAPI), FITC-labeled goat antimouse secondary antibody, calcein-AM, and propidium iodide (PI) were purchased from Sigma−Aldrich. Biotin-labeled mouse antihuman anti-EpCAM monoclonal antibody (mAb) was obtained from eBioscience. PE-labeled mouse antihuman anticytokeratin (PE-CK) and FITC-labeled mouse antihuman CD45 (FITCCD45) were purchased from BD Biosciences. Reactive oxygen species (ROS) assay and apoptosis detection kits were obtained from Beyotime Institute of Biotechnology. SK-BR-3 cells (a human breast cancer cell line) and Hela cells (a human cervix cancer cell line) were purchased from China Type Culture Collection. All the media used for cell culture were obtained from Gibco Corp. Sodium alginate and chemicals for synthesis of CdTe QDs and NS-COOH were supplied by Shanghai Chemical Reagent Company. The viscosity of 10 g/L sodium alginate at 20 °C was ≥0.02 mm2/s. Fabrication of FMNS with Alginate Shell. PEI-coated magnetic nanospheres (NS-PEI) were prepared according to previous reports.14,40 NS-PEI dispersed in ultrapure water was further incubated 1:1 (v/v) with water-soluble mercaptoacetic acid-capped CdTe (MAA-CdTe) QDs (5 × 10−6 M, emission at 565 nm), which were prepared via a one-pot method through refluxing and hydrothermal processes.41 After 30 min of incubation, an excess of CdTe QDs were removed by washing twice to obtain NS-PEI-QDs (namely, FMNS). Alginate was assembled onto the surface of NS-PEI-QDs to get NS-PEI-QDs-Alg-Ca2+-Alg using Ca2+ as the activator (see the Supporting Information). To characterize the fluorescent property and dispersibility of NS-PEI-QDs-Alg-Ca2+-Alg, microscopic images were obtained using a Zeiss microscope with a 100× objective lens (AxioObserver Z1, Zeiss, Germany) and transmission electron microscopy (TEM) images, together with EDX analysis, were obtained via high-resolution transmission electron microscopy (HRTEM) (Model JEM2010FEF, UHR). Alginate and Ca2+ assembly processes can be repeated several times to make multiple layers of Ca2+-Alg. Generally, four layers of alginate were assembled onto the surface of NS-PEI-QDs, namely, NS-PEI-QDs-Alg-(Ca2+-Alg) × 3 were finally formed. Dynamic light scattering (DLS) and ζpotential measurement (Malvern, Zetasizer Nano) were employed to check the stepwise assembly of QDs, alginate, and Ca2+. 4619



Article

Scheme 1. Ca2+-Assisted Construction and Disintegration of Anti-EpCAM-Alg-FMNBa

a

Mechanism steps are described as follows: (1) NS-PEI was used as fundamental material to support QDs for fabrication of FMNS; (2) alginate was incubated with FMNS to construct FMNS with first alginate layer; (3, 4) Ca2+ was used to activate alginate-coated FMNS, followed by incubation with alginate to deposit second and third alginate layer on FMNS, forming Alg-FMNS; (5) Alg-FMNS were activated by Ca2+ to conjugate with SAAlg for fabrication of SA-Alg-FMNS; (6) SA-Alg-FMNS were incubated with biotin-labeled anti-EpCAM to construct anti-EpCAM-Alg-FMNS; and (7) anti-EpCAM-Alg-FMNS were decomposed by EDTA treatment, allowing disconnection between anti-EpCAM and FMNS.

ing 1 × 105 SK-BR-3 cells in 400 μL of 1 × PBS and incubated for 30 min at room temperature. The 100 μL of 5 mg/mL SAAlg-FMNS without anti-EpCAM were added to 400 μL of dispersed cells as the control. Meanwhile, 100 μL of 5 mg/mL anti-EpCAM-Alg-FMNB were mixed with 1 × 105 Hela cells in 400 μL 1 × PBS and incubated for 30 min as another control. Anti-EpCAM-Alg-FMNB and SA-Alg-FMNS were subsequently separated with a magnetic scaffold after incubation and then observed using a Zeiss microscope. In addition, SAAlg-FMNS incubated with SK-BR-3 cells and anti-EpCAM-AlgFMNB incubated with Hela cells without magnetic attraction were also seen via observation with a Zeiss microscope. Isolation of Cancer Cells from Whole Blood. The healthy human whole blood was obtained from Renmin Hospital of Wuhan University, with the anticoagulant sodium heparin. Lysed blood was obtained by treating whole blood with red blood cell lysing buffer (Boster Company), according to the manufacturer’s instructions. DAPI-prestained SK-BR-3 cells (102) were spiked in 1 mL of lysed blood, which were investigated by anti-EpCAM-Alg-FMNB at a concentration of 1 mg/mL. Anti-EpCAM-Alg-FMNB captured cells were kept in 96-well plate, which were observed and counted using a Zeiss microscope. In addition, various density (102, 103, 104, 105 cells/mL) of DAPI-prestained SK-BR-3 cells spiked in human whole blood were investigated by anti-EpCAM-Alg-FMNB at a concentration of 1 mg/mL. Anti-EpCAM-Alg-FMNB captured

cells were kept in 96-well plate, which were observed and counted using a Zeiss microscope. The captured cells can be used for common three-color immunocytochemistry (ICC) that cells were fixed with 4% paraformaldehyde (30 min), permeabilized with 0.1% Triton-X 100 (30 min), and stained with 10 μg/mL DAPI, PE-CK, and FITC-CD45 (following the kit instruction) for 2 h at 4 °C. After washing, the captured cells were put into a 96-well tissue culture plate for fluorescent microscopy imaging. Capture, Release and Recover of SK-BR-3 Cells. Hemocytometry was taken to examine the capture and release efficiency of anti-EpCAM-Alg-FMNB to SK-BR-3 cells. SK-BR3 cells cultured in a flask were detached and collected as mentioned above. Subsequently, the cells were washed and resuspended in 1 × PBS, and the cell concentration was determined by hemocytometry. Then, 100 μL of 5 mg/mL anti-EpCAM-Alg-FMNB were mixed with 1 × 105 SK-BR-3 cells in 400 μL of 1 × PBS, incubated for 30 min at room temperature, and then separated with a magnetic scaffold for 5 min. Consecutively, the uncaptured SK-BR-3 cells in solution were counted again by hemocytometry. The capture efficiency was defined as the ratio of the number of target cells captured to the number of target cells initially introduced. To release SKBR-3 cells, the captured SK-BR-3 cells attracted by magnetic scaffold were further incubated 1:1 (v/v) with 50 mM EDTA solution for 5 min. After EDTA treatment, the FMNS detached from SK-BR-3 cells were removed by magnetic scaffold and SK4620



Article

Characterization of Alg-FMNS. LBL assembly is often used to fabricate multifunctional nanomaterials or nanobiodevices,45−48 where electrostatic interaction is the main force to promote assembly. Notably, Parak et al. constructed functional capsules doped with metal nanoparticles in polyelectrolyte-multilayer shells, which are sensitive to laser beams, allowing the encapsulated material to leave the interior of the capsules under laser exposure.45,49,50 To fabricate smart decomposable multifunctional nanobiorobes, we put forward Ca2+-initiated LBL assembly to combine multiple alginate layers and biotargeting molecules on nanocarrier. Here, Ca2+ was used as the bridge to sandwich two negatively charged alginate polymers, which could be snatched by EDTA, allowing reversible integration and decomposition of the alginate shell on a nanosupporter. To confirm the reliability of Ca2+-assisted assembly, FITCAlg was synthesized and used to directly monitor the assembly process. As shown in Figure 1a, the first FITC-Alg layer on NS-

BR-3 cells remained in solution were counted again to obtain release efficiency (calculated as the ratio of the number of target cells released to the number of target cells captured). EDTA can be repeated used to increase release efficiency. The viability of released SK-BR-3 cells was examined by ROS assay kit where SK-BR-3 cells were incubated with DCFH-DA for 20 min at 37 °C and observed using a Zeiss microscope. SKBR-3 cells incubated with DCFH-DA and further loaded with Rosup (positive control of ROS) were also imaged. For quantitative analysis, SK-BR-3 cells that did not show green fluorescence were regarded as live cells with good viability. In addition, annexin V-FITC and PI were used for fluorescent staining of released SK-BR-3 cells to evaluate their viability. SKBR-3 cells that did not show fluorescence of annexin V-FITC and PI were counted as live cells. Finally, the released SK-BR-3 cells were further cultured under CO2 incubator to detect their proliferation ability and the cells without capture and release treatment were also cultured in the same condition as control. In addition, SK-BR-3 cells cultured for 24 h with or without UV exposure for 1 h were stained by calcein-AM (2 μg/mL) and PI (3 μg/mL). SK-BR-3 cells that were only labeled by calcein-AM were identified as live cells.

■

RESULTS AND DISCUSSION Fabrication of Engineered Anti-EpCAM-Alg-FMNB. We have demonstrated that FMNS can be prepared by either encapsulation14 or LBL assembly method,40 which can be further equipped with biotargeting properties for recognition, capture, and isolation of cancer cells.14,16,18,20 However, the release of captured cancer cells has not been achieved by these conventional FMNS. It was reported that alginate incorporated into a microfluidic chip can form hydrogel by Ca 2+ addition,37,43,44 which can be destroyed by EDTA,37 facilitating reversible capture and release of target cells. Inspired by these works, we proposed to fabricate anti-EpCAM-Alg-FMNB, which can be attached to and detached from the cancer cells reversibly. Scheme 1 demonstrates the processes to fabricate smart anti-EpCAM-Alg-FMNB. First, PEI-coated magnetic nanospheres (NS-PEI) were prepared and electrostatically interacted with MAA-CdTe QDs to obtain fluorescentmagnetic NS-PEI-QDs. After that, alginate was incubated with FMNS to deposit the first alginate layer on FMNS, which were verified by XPS, Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA) (see Figure SI-1 in the Supporting Information). Subsequently, Ca2+ was used for activation of alginate-coated FMNS, enabling further assembly of alginate. By using this strategy, Ca2+ and alginate can be stepwise assembled onto the surface of FMNS to fabricate Alg-FMNS, which were examined by DLS and ζpotential measurement (see Figure SI-2 in the Supporting Information), as shown and discussed in the Supporting Information. Generally, three layers of alginate were deposited on the FMNS, as shown in Scheme 1. To fabricate functionalized nanobioprobes for capture and release of cancer cells, Ca2+activated Alg-FMNS were incubated with SA-Alg conjugate, and then with biotin-labeled anti-EpCAM to obtain antiEpCAM-Alg-FMNB. It is well-known that EDTA can compete with alginate to interact with Ca2+, forming EDTA−Ca2+ chelate.37 Therefore, the alginate shell of anti-EpCAM-AlgFMNB could be destroyed by EDTA treatment, resulting in disconnection between anti-EpCAM and FMNS, which made reversible capture and release of target cells possible.

Figure 1. Characterization of Alg-FMNS: (a) growth (dashed line) and detachment (solid line) of alginate shell on NS-PEI using FITCAlg as reporter; (b) fluorescent spectra of released FITC-Alg in supernatant after FITC-Alg-coated NS-PEI being incubated with EDTA; (c) TEM image of Alg-FMNS; and (d) EDX spectrum of AlgFMNS.

PEI only gave slight fluorescence. With FITC-Alg layer growth, the fluorescence of FITC was increased correspondingly (dashed line in Figure 1a), which was consistent with the DLS and ζ-potential measurement, confirming that the Ca2+assisted alginate assembly was reliable and reproducible. To test if the alginate-Ca2+ layer on NS-PEI can be constructed and destroyed reversibly, NS-PEI with different alginate-Ca2+ layers were incubated with EDTA. The FITC fluorescence of NS-PEI was dramatically dropped after EDTA treatment (solid line in Figure 1a) and the detached FITC-Alg in supernatant was also detected (Figure 1b). Taken together, it was clearly illustrated that the alginate-Ca2+ layer can be reversibly formed and decomposed. The microscopic and TEM images were obtained to characterize the fluorescent property and dispersibility of AlgFMNS. As displayed in Figure SI-2c in the Supporting Information, Alg-FMNS were dispersed well. In addition, each nanosphere shown in bright field had a corresponding 4621



Article

fluorescent dot, indicating that the assembly process was rather uniform. The TEM image in Figure 1c (where the aggregation of Alg-FMNS was possibly caused by magnetic-field-induced magnetic fusion in TEM imaging) demonstrated that small nanoparticles were deposited on NS-PEI, which was confirmed by EDX analysis (Figure 1d), where elemental Fe, O, Cd, and Te were clearly detected. We could also find elemental Ca in the EDX analysis, which played an important role in alginate shell formation. Therefore, we concluded that Alg-FMNS with strong fluorescence and good dispersibility were successfully constructed. Characterization of Anti-EpCAM-Alg-FMNB. FITC-biotin was used as a reporter to test whether SA was successfully immobilized onto the surface of Alg-FMNS. Compared with Alg-FMNS (see Figure SI-3e in the Supporting Information), SA-Alg-FMNS showed strong FITC fluorescence after incubation with FITC-biotin (see Figure SI-3c in the Supporting Information). Next, FITC-biotin-labeled SA-AlgFMNS were further incubated with EDTA, and FITC fluorescence of FMNS was rarely observed (see Figure SI-3g in the Supporting Information). Thus far, we knew that SA can be reversibly attached to and detached from the surface of AlgFMNS. Subsequently, FITC-labeled secondary antibody was employed to report the conjugation of anti-EpCAM onto the surface of SA-Alg-FMNS. As demonstrated in Figure 2c, antiEpCAM-Alg-FMNB had strong FITC fluorescence (no clear FITC fluorescence was observed on SA-Alg-FMNS; see Figure

2e), implying that anti-EpCAM was successfully conjugated onto the surface of SA-Alg-FMNS. In addition, the antiEpCAM-Alg-FMNB in bright field and fluorescent field were well-matched, confirming that anti-EpCAM-Alg-FMNB were successfully formed (see Figures 2b and 2c). We have demonstrated that SA can be detached from Alg-FMNS via EDTA treatment. Similarly, the fluorescence of FITCsecondary antibody-labeled anti-EpCAM-Alg-FMNB was diminished after EDTA addition, confirming the release of antiEpCAM from anti-EpCAM-Alg-FMNB (see Figure 2g). Taken together, we concluded that anti-EpCAM can be easily attached onto the surface of Alg-FMNS through alginate-Ca 2+ connection and then conveniently be detached by EDTA addition. These characters enabled anti-EpCAM-Alg-FMNB applicable for capture and release of EpCAM-positive cancer cells. Specificity of Anti-EpCAM-Alg-FMNB to SK-BR-3 Cells. EpCAM is overexpressed on SK-BR-3 cells,38 which are employed as model CTCs to investigate the specificity of anti-EpCAM-Alg-FMNB in capture and isolation of EpCAMpositive cancer cells. SK-BR-3 cells were incubated with antiEpCAM-Alg-FMNB, followed by magnetic attraction. Meanwhile, SK-BR-3 cells mixed with SA-Alg-FMNS (without antiEpCAM) and Hela cells (EpCAM-negative) incubated with anti-EpCAM-Alg-FMNB were used as negative controls. As demonstrated in Figure 3, strong fluorescence of FMNB was

Figure 3. Specific recognition and isolation of (a−c) SK-BR-3 cells by anti-EpCAM-Alg-FMNB incubated for 30 min with subsequent magnetic scaffold separation; (d−f) SK-BR-3 cells incubated with SA-Alg-FMNS without magnetic separation (Control 1); and (g−i) Hela cells incubated with anti-EpCAM-Alg-FMNB without magnetic separation (Control 2). (Panels a, d, and g (top row) show bright-field images; panels b, e, and h (middle row) show fluorescence images; and panels c, f, and i (bottom row) show merged images.)

observed on the surface of SK-BR-3 cells (see Figures 3b and 3c), indicating that EpCAM positive cancer cells can be recognized and isolated by anti-EpCAM-Alg-FMNB. In contrast, control experiments demonstrated that SA-AlgFMNS did not capture SK-BR-3 cells (see Figures 3e and 3f, as well as Figures SI-4a and SI-4b in the Supporting Information), meanwhile, anti-EpCAM-Alg-FMNB could not recognize EpCAM-negative Hela cells (see Figures 3h and 3i, as well as Figures SI-4c and SI-4d in the Supporting Information). Hence, our multifunctional nanobioprobes could specifically

Figure 2. Ca2+-enabled conjugation and release of anti-EpCAM on Alg-FMNS: (a) schematic graph showing the principle of confirming conjugation of anti-EpCAM on SA-Alg-FMNS; (b, c) incubation of FITC-labeled goat antimouse antibody with anti-EpCAM-Alg-FMNB; (d, e) incubation of FITC-labeled goat antimouse antibody with SAAlg-FMNS; (f, g) release of anti-EpCAM from anti-EpCAM-AlgFMNB via EDTA treatment reported by FITC-labeled goat antimouse antibody. (Panels b, d, and f show bright-field images; panels c, e, and g show fluorescence images.) 4622



Article

Figure 4. Capture of SK-BR-3 cells from whole blood: (a) calibration plot of cancer cells captured from whole blood with different cell concentrations (error bars represent standard deviations (n = 3)); (b−e) microscopic images of cells captured from mimic clinical blood samples and identified with three-color ICC identification.

Figure 5. Microscopic images of SK-BR-3 cells separated by anti-EpCAM-Alg-FMNB (a, b) before EDTA treatment and (c, d) after EDTA treatment. (Panels a and c show bright-field images; panels b and d show fluorescence images.) (e) Quantitative analysis of fluorescence intensity of captured SK-BR-3 cells before and after EDTA treatment. (f) Capture and release efficiency of anti-EpCAM-Alg-FMNB to SK-BR-3 cells. Error bars represent standard deviations (n = 3).

were identified by double staining with DAPI and anti-EpCAMAlg-FMNB. To test detection limit of anti-EpCAM-Alg-FMNB to EpCAM-positive cells, cell spike numbers from 104 to 102 were further investigated and >85% capture efficiency was obtained (Figure 4a). The captured cancer cells can be used for ICC identification by PE-CK (a marker for epithelial cells), FITC-CD45 (a marker for WBCs), and DAPI nuclear staining. As shown in Figures 4b−e, the SK-BR-3 cell was DAPI+/CK +/CD45− and WBCs were DAPI+/CK−/CD45+. These results showed that cancer cells can be isolated by antiEpCAM-Alg-FMNS from whole blood. Release of Captured Cancer Cells. Our experimental results revealed that EDTA could snatch Ca2+ (the essential component of anti-EpCAM-Alg-FMNB) to form EDTA−Ca2+ chelate, cutting the connection between anti-EpCAM and FMNS, which implied that FMNS could be detached from SKBR-3 cells after EDTA treatment. The results for capture and release of SK-BR-3 cells using anti-EpCAM-Alg-FMNB were displayed in Figures 5a−f. Before EDTA treatment, the SK-BR3 cells captured by anti-EpCAM-Alg-FMNB showed strong fluorescence (Figure 5b). While, after EDTA addition, the SK-

recognize and isolate EpCAM-positive cells, suggesting that anti-EpCAM-Alg-FMNB might be potentially used for the recognition and isolation of CTCs. Capture and Isolation of Rare Cancer Cells from Whole Blood. To test if anti-EpCAM-Alg-FMNB can specifically recognize and isolate rare cancer cells, antiEpCAM-Alg-FMNB were used for sorting DAPI-stained SKBR-3 cells spiked in lysed blood. The results showed antiEpCAM-Alg-FMNB did not have specific interaction with white blood cells (WBCs) (see Figure SI-5 in the Supporting Information). As few as 102 SK-BR-3 cells were effectively isolated and detected from 1 mL of lysed blood with 89% ± 10% capture efficiency (n = 3), demonstrating that antiEpCAM-Alg-FMNB were applicable in the isolation and detection in the CTCs. Thereafter, we further applied anti-EpCAM-Alg-FMNB into mimic CTC blood samples, to explore the clinical utility of antiEpCAM-Alg-FMNB. We first evaluated the isolation of 105 SKBR-3 cells (prestained by DAPI) spiked in 1 mL of whole blood. As shown in Figure SI-6 in the Supporting Information, SK-BR-3 cells were successfully isolated and captured which 4623



Article

BR-3 cells demonstrated vague fluorescence (Figure 5d), indicating that anti-EpCAM-Alg-FMNB were discomposed and FMNS were detached from the surface of SK-BR-3 cells. Quantitative analysis showed that the fluorescent intensity of SK-BR-3 cells after EDTA treatment was ∼20% of that of fresh captured SK-BR-3 cells, further confirming that FMNS were successfully detached from SK-BR-3 cells (see Figure 5e). Thereafter, hemocytometry was used to examine the capture and release efficiency of anti-EpCAM-Alg-FMNB to SK-BR-3 cells. As described in Figure 5f, the capture efficiency was 90% ± 2% when using anti-EpCAM-Alg-FMNB with a concentration of 1.0 mg/mL on samples containing 1 × 105 SK-BR-3 cells in 0.5 mL PBS buffer. Meanwhile, ∼65% ± 6% of captured cells could be released by EDTA treatment, suggesting that anti-EpCAM-Alg-FMNB can be used for the capture and release of SK-BR-3 cells. Viability of Released Cancer Cells. The cellular ROS level is related to cell apoptosis. Therefore, the viability of released SK-BR-3 cells was examined by ROS assay, using DCFH-DA as an indicator. DCFH-DA was hydrolyzed by esterases to dichlorofluorescin (DCFH), which was trapped within the cell. If cells had ROS, nonfluorescent DCFH can be oxidized by ROS to dichlorofluorescin (DCF), showing green fluorescence. Therefore, we could tell whether the cells sustained oxidative stress based on the level of green fluorescence. As shown in Figures 6a−c, the majority of SK-

In addition, annexin V-FITC and PI were used for double fluorescent staining of released SK-BR-3 cells (see Figure SI-7 in the Supporting Information). The results showed that 70% of cells had good viability (no fluorescence at all), 9% of cells went through early apoptosis (only stained by annexin VFITC), and 21% of cells almost lost their viability (stained by annexin V-FITC and PI, or PI only). Combining results from ROS assay and fluorescent staining, we concluded that ∼70% of the released SK-BR-3 cells kept their viability, which was comparable to the previously reported results.37 Finally, the released SK-BR-3 cells were cultured in a CO2 incubator for 24 h, followed by calcein-AM and PI staining. As demonstrated in Figure SI-8 in the Supporting Information, SK-BR-3 cells spread well on the substrate. Fluorescent images showed that SK-BR-3 cells were obviously stained by calcein-AM (Figure SI8b in the Supporting Information) but not PI (Figure SI-8c in the Supporting Information), suggesting that the viability of SK-BR-3 cells was well-maintained after release. In contrast, if SK-BR-3 cells were forced to go through the apoptosis process (exposed under UV light for 1 h, as negative control), we could find that the majority of SK-BR-3 cells were stained by PI (see Figure SI-8g in the Supporting Information). In fact, the released SK-BR-3 cells retained good proliferation ability. As shown in Figure 6g, the released SK-BR-3 cells were sparsely seeded onto the tissue culture plate. After overnight incubation, cells spread well and showed clear growth tendencies. The cells were amplified and reached almost 100% confluency after culture (Figure 6h), which could be used for further cell passage. After subculture, the cells grew well (Figure 6i), exhibiting similar growth behavior of cells without capture and release treatment. These combined results from ROS assay, apoptosis detection and proliferation experiment indicated that our nanobioprobes were biocompatible, which did not show obvious activity damage to cancer cells during capture and release procedures. Taken together, we found that released cells retained their viability, facilitating further molecular profiling and genetic analysis.

■

CONCLUSIONS In this work, we developed a cancer cell capture and release nanobioprobe with integrated features of captureagentdirected specific recognition, FMNS-driven cell capture, and EDTA-assisted cell releasebased on Ca2+-alginate chemistry. As-prepared anti-EpCAM-Alg-FMNB can specifically capture EpCAM-positive SK-BR-3 cells but not EpCAM-negative Hela cells and WBCs, and even detect 102 spiked SK-BR-3 cells in 1 mL of whole blood with 86% efficiency, demonstrating the novel specificity and sensitivity of this nanobioprobe. Furthermore, we showed that 65% of captured cells were released if anti-EpCAM-Alg-FMNB were disintegrated by EDTA treatment. ROS assay, together with annexin V-FITC and PI-based fluorescent assays, showed that ∼70% of the released cells maintained their viability and proliferation ability. Although affinity nanosubstrates or microfluidic chips have been fabricated for the capture and release of EpCAMexpressed cancer cells, we believe our anti-EpCAM-AlgFMNB represents an additional promising choice for the capture and release of cancer cells, because of its simple, sensitive, efficient, and fast yet low-cost characteristics. The smart nanobioprobe offers a potential opportunity for the detection and analysis of cancer cells at nascent stages which

Figure 6. ROS assay and proliferation detection of released SK-BR-3 cells: (a−c) SK-BR-3 cells incubated with ROS reporter DCFH-DA only; (d−f) SK-BR-3 cells incubated with DCFH-DA and Rosup (positive control of ROS). Proliferation of the released SK-BR-3 cells also is depicted ((g) seeding after release, (h) reaching confluency, and (i) subpassage). (Panels a, d, g, h, and i show bright-field images; panels b and e show fluorescence images; and panels c and f show merged images. Scale bar = 20 μm.)

BR-3 cells did not show green fluorescence. In contrast, if SKBR-3 cells were further loaded with Rosup (positive control of ROS), almost all of the SK-BR-3 cells exhibited green fluorescence (Figures 6d−f). Quantitative analysis demonstrated that 68% ± 7% of SK-BR-3 cells did not show fluorescence indicating that a majority of the cells maintained good viability after release. 4624



Article

(15) Yoon, T.-J.; Yu, K. N.; Kim, E.; Kim, J. S.; Kim, B. G.; Yun, S.H.; Sohn, B.-H.; Cho, M.-H.; Lee, J.-K.; Park, S. B. Small 2006, 2, 209−215. (16) Xie, H.-Y.; Xie, M.; Zhang, Z.-L.; Long, Y.-M.; Liu, X.; Tang, M.L.; Pang, D.-W.; Tan, Z.; Dickinson, C.; Zhou, W. Bioconjugate Chem. 2007, 18, 1749−1755. (17) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. ACS Nano 2008, 2, 889−896. (18) Xie, M.; Hu, J.; Long, Y.-M.; Zhang, Z.-L.; Xie, H.-Y.; Pang, D.W. Biosens. Bioelectron. 2009, 24, 1311−1317. (19) Medley, C. D.; Bamrungsap, S.; Tan, W.; Smith, J. E. Anal. Chem. 2011, 83, 727−734. (20) Song, E.-Q.; Hu, J.; Wen, C.-Y.; Tian, Z.-Q.; Yu, X.; Zhang, Z.L.; Shi, Y.-B.; Pang, D.-W. ACS Nano 2011, 5, 761−770. (21) Di Corato, R.; Bigall, N. C.; Ragusa, A.; Dorfs, D.; Genovese, A.; Marotta, R.; Manna, L.; Pellegrino, T. ACS Nano 2011, 5, 1109−1121. (22) Durgadas, C. V.; Sharma, C. P.; Sreenivasan, K. Nanoscale 2011, 3, 4780−4787. (23) Lien, Z.-Y.; Hsu, T.-C.; Liu, K.-K.; Liao, W.-S.; Hwang, K.-C.; Chao, J.-I. Biomaterials 2012, 33, 6172−6185. (24) Fu, A.; Wilson, R. J.; Smith, B. R.; Mullenix, J.; Earhart, C.; Akin, D.; Guccione, S.; Wang, S. X.; Gambhir, S. S. ACS Nano 2012, 6, 6862−6869. (25) Maltez-da Costa, M.; de la Escosura-Muñiz, A.; Nogués, C.; Barrios, L.; Ibáñez, E.; Merkoçi, A. Nano Lett. 2012, 12, 4164−4171. (26) Wu, C.-H.; Huang, Y.-Y.; Chen, P.; Hoshino, K.; Liu, H.; Frenkel, E. P.; Zhang, J. X. J.; Sokolov, K. V. ACS Nano 2013, 7, 8816−8823. (27) Wang, X.; Qian, X.; Beitler, J. J.; Chen, Z. G.; Khuri, F. R.; Lewis, M. M.; Shin, H. J. C.; Nie, S.; Shin, D. M. Cancer Res. 2011, 71, 1526−1532. (28) Kim, S.; Han, S.-I.; Park, M.-J.; Jeon, C.-W.; Joo, Y.-D.; Choi, I.H.; Han, K.-H. Anal. Chem. 2013, 85, 2779−2786. (29) Lianidou, E. S.; Markou, A.; Strati, A. Cancer Metastasis Rev. 2012, 31, 663−671. (30) Doyen, J.; Alix-Panabières, C.; Hofman, P.; Parks, S. K.; Chamorey, E.; Naman, H.; Hannoun-Lévi, J.-M. Crit. Rev. Oncol./ Hematol. 2012, 81, 241−256. (31) Liu, H.; Liu, X.; Meng, J.; Zhang, P.; Yang, G.; Su, B.; Sun, K.; Chen, L.; Han, D.; Wang, S.; Jiang, L. Adv. Mater. 2013, 25, 922−927. (32) Hou, S.; Zhao, H.; Zhao, L.; Shen, Q.; Wei, K. S.; Suh, D. Y.; Nakao, A.; Garcia, M. A.; Song, M.; Lee, T.; Xiong, B.; Luo, S.-C.; Tseng, H.-R.; Yu, H.-h. Adv. Mater. 2013, 25, 1547−1551. (33) Shen, Q.; Xu, L.; Zhao, L.; Wu, D.; Fan, Y.; Zhou, Y.; OuYang, W.-H.; Xu, X.; Zhang, Z.; Song, M.; Lee, T.; Garcia, M. A.; Xiong, B.; Hou, S.; Tseng, H.-R.; Fang, X. Adv. Mater. 2013, 25, 2368−2373. (34) Li, S.; Chen, N.; Zhang, Z.; Wang, Y. Biomaterials 2013, 34, 460−469. (35) Zhao, W.; Cui, C. H.; Bose, S.; Guo, D.; Shen, C.; Wong, W. P.; Halvorsen, K.; Farokhzad, O. C.; Teo, G. S. L.; Phillips, J. A.; Dorfman, D. M.; Karnik, R.; Karp, J. M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 19626−19631. (36) Wan, Y.; Liu, Y.; Allen, P. B.; Asghar, W.; Mahmood, M. A. I.; Tan, J.; Duhon, H.; Kim, Y.-t.; Ellington, A. D.; Iqbal, S. M. Lab Chip 2012, 12, 4693−4701. (37) Hatch, A.; Pesko, D. M.; Murthy, S. K. Anal. Chem. 2012, 84, 4618−4621. (38) Kurkuri, M. D.; Al-Ejeh, F.; Shi, J. Y.; Palms, D.; Prestidge, C.; Griesser, H. J.; Brown, M. P.; Thierry, B. J. Mater. Chem. 2011, 21, 8841−8848. (39) Zhang, N.; Deng, Y.; Tai, Q.; Cheng, B.; Zhao, L.; Shen, Q.; He, R.; Hong, L.; Liu, W.; Guo, S.; Liu, K.; Tseng, H.-R.; Xiong, B.; Zhao, X.-Z. Adv. Mater. 2012, 24, 2756−2760. (40) Xie, M.; Hu, J.; Wen, C.-Y.; Zhang, Z.-L.; Xie, H.-Y.; Pang, D.W. Nanotechnology 2012, 23, 035602. (41) Wu, S.; Dou, J.; Zhang, J.; Zhang, S. J. Mater. Chem. 2012, 22, 14573−14578. (42) Zhu, H.; Srivastava, R.; McShane, M. J. Biomacromolecules 2005, 6, 2221−2228.

may provide helpful information for diagnosis and significant directions for personalized therapy.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional Figures SI-1 to SI-8. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

These two authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 81071227, 21375099), the Science Fund for Creative Research Groups (No. 20921062), and the Program for New Century Excellent Talents in University (No. NCET-10-0611), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1030).

■

REFERENCES

(1) Balic, M.; Williams, A.; Lin, H.; Datar, R.; Cote, R. J. Annu. Rev. Med. 2013, 64, 31−44. (2) Alix-Panabières, C.; Pantel, K. Clin. Chem. 2013, 59, 110−118. (3) Hosokawa, M.; Hayata, T.; Fukuda, Y.; Arakaki, A.; Yoshino, T.; Tanaka, T.; Matsunaga, T. Anal. Chem. 2010, 82, 6629−6635. (4) Hofman, V. J.; Ilie, M. I.; Bonnetaud, C.; Selva, E.; Long, E.; Molina, T.; Vignaud, J. M.; Fléjou, J. F.; Lantuejoul, S.; Piaton, E.; Butori, C.; Mourad, N.; Poudenx, M.; Bahadoran, P.; Sibon, S.; Guevara, N.; Santini, J.; Vénissac, N.; Mouroux, J.; Vielh, P.; Hofman, P. M. Am. J. Clin. Pathol. 2011, 135, 146−156. (5) Park, J.-M.; Lee, J.-Y.; Lee, J.-G.; Jeong, H.; Oh, J.-M.; Kim, Y. J.; Park, D.; Kim, M. S.; Lee, H. J.; Oh, J. H.; Lee, S. S.; Lee, W.-Y.; Huh, N. Anal. Chem. 2012, 84, 7400−7407. (6) Joosse, S. A.; Pantel, K. Cancer Res. 2013, 73, 8−11. (7) Nagrath, S.; Sequist, L. V.; Maheswaran, S.; Bell, D. W.; Irimia, D.; Ulkus, L.; Smith, M. R.; Kwak, E. L.; Digumarthy, S.; Muzikansky, A.; Ryan, P.; Balis, U. J.; Tompkins, R. G.; Haber, D. A.; Toner, M. Nature 2007, 450, 1235−1239. (8) Adams, A. A.; Okagbare, P. I.; Feng, J.; Hupert, M. L.; Patterson, D.; Göttert, J.; McCarley, R. L.; Nikitopoulos, D.; Murphy, M. C.; Soper, S. A. J. Am. Chem. Soc. 2008, 130, 8633−8641. (9) Stott, S. L.; Hsu, C.-H.; Tsukrov, D. I.; Yu, M.; Miyamoto, D. T.; Waltman, B. A.; Rothenberg, S. M.; Shah, A. M.; Smas, M. E.; Korir, G. K.; Floyd, F. P., Jr.; Gilman, A. J.; Lord, J. B.; Winokur, D.; Springer, S.; Irimia, D.; Nagrath, S.; Sequist, L. V.; Lee, R. J.; Isselbacher, K. J.; Maheswaran, S.; Haber, D. A.; Toner, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 18392−18397. (10) Dharmasiri, U.; Njoroge, S. K.; Witek, M. A.; Adebiyi, M. G.; Kamande, J. W.; Hupert, M. L.; Barany, F.; Soper, S. A. Anal. Chem. 2011, 83, 2301−2309. (11) Wang, S.; Wang, H.; Jiao, J.; Chen, K.-J.; Owens, G. E.; Kamei, K.-i.; Sun, J.; Sherman, D. J.; Behrenbruch, C. P.; Wu, H.; Tseng, H.-R. Angew. Chem., Int. Ed. 2009, 48, 8970−8973. (12) Wang, S.; Liu, K.; Liu, J.; Yu, Z. T.-F.; Xu, X.; Zhao, L.; Lee, T.; Lee, E. K.; Reiss, J.; Lee, Y.-K.; Chung, L. W. K.; Huang, J.; Rettig, M.; Seligson, D.; Duraiswamy, K. N.; Shen, C. K.-F.; Tseng, H.-R. Angew. Chem., Int. Ed. 2011, 50, 3084−3088. (13) Wang, D.; He, J.; Rosenzweig, N.; Rosenzweig, Z. Nano Lett. 2004, 4, 409−413. (14) Xie, H.-Y.; Zuo, C.; Liu, Y.; Zhang, Z.-L.; Pang, D.-W.; Li, X.-L.; Gong, J.-P.; Dickinson, C.; Zhou, W. Small 2005, 1, 506−509. 4625



Article

(43) Hatch, A.; Hansmann, G.; Murthy, S. K. Langmuir 2011, 27, 4257−4264. (44) Shah, A. M.; Yu, M.; Nakamura, Z.; Ciciliano, J.; Ulman, M.; Kotz, K.; Stott, S. L.; Maheswaran, S.; Haber, D. A.; Toner, M. Anal. Chem. 2012, 84, 3682−3688. (45) Skirtach, A. G.; Javier, A. M.; Kreft, O.; Köhler, K.; Alberola, A. P.; Möhwald, H.; Parak, W. J.; Sukhorukov, G. B. Angew. Chem., Int. Ed. 2006, 45, 4612−4617. (46) Gregersen, K. A. D.; Hill, Z. B.; Gadd, J. C.; Fujimoto, B. S.; Maly, D. J.; Chiu, D. T. ACS Nano 2010, 4, 7603−7611. (47) Javier, A. M.; del Pino, P.; Bedard, M. F.; Ho, D.; Skirtach, A. G.; Sukhorukov, G. B.; Plank, C.; Parak, W. J. Langmuir 2008, 24, 12517− 12520. (48) Pavlov, A. M.; Sapelkin, A. V.; Huang, X.; P’ng, K. M. Y.; Bushby, A. J.; Sukhorukov, G. B.; Skirtach, A. G. Macromol. Biosci. 2011, 11, 848−854. (49) Rivera-Gil, P.; Nazarenus, M.; Ashraf, S.; Parak, W. J. Small 2012, 8, 943−948. (50) Ochs, M.; Carregal-Romero, S.; Rejman, J.; Braeckmans, K.; De Smedt, S. C.; Parak, W. J. Angew. Chem., Int. Ed. 2013, 52, 695−699.

4626


Engineered Decomposable Multifunctional Nanobioprobes for

Recommend Documents