Hydrogen Peroxide-Responsive Nanoprobe Assists Circulating Tumor

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A hydrogen peroxide-responsive nanoprobe assists circulating tumor cell identification and colorectal cancer diagnosis Chunting Li, Ruijun PAN, Peiyong Li, Qinghua Guan, Junping Ao, Kai Wang, Li Xu, Xiaofei Liang, Xin Jin, Chuan Zhang, and Xinyuan Zhu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017

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Analytical Chemistry

A hydrogen peroxide-responsive nanoprobe assists circulating tumor cell identification and colorectal cancer diagnosis Chunting Li,† Ruijun Pan,

∥

Peiyong Li,*,‡ Qinghua Guan,† Junping Ao,§

Kai Wang,§ Li Xu,† Xiaofei Liang,§ Xin Jin,† Chuan Zhang,*,† and Xinyuan Zhu*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal

Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China. ‡

Department of Nuclear Medicine Ruijin Hospital, School of Medicine, Shanghai Jiao

Tong University, Shanghai 200025, People’s Republic of China. §

State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute,

Shanghai Jiao Tong University, School of Medicine, Shanghai 200032, People’s Republic of China. ∥

Department of Gastrointestinal Surgery, Ruijin Hospital, School of Medicine,

Shanghai Jiao Tong University, Shanghai 200025, People’s Republic of China.

1

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: In clinic, numeration of circulating tumor cells (CTCs) plays a critical role in cancer diagnosis and treatment but conventional CTC identification and counting that rely on specific antibodies to characterize cell’s surface antigens are costive and with limitations. Importantly, false positive or negative results may be occurred due to the high heterogeneity and epithelial-mesenchymal transition (EMT) of CTCs. Herein we demonstrated a novel and effective CTC detecting nanoprobe that could rapidly response to the high level of endogenous H2O2 of CTCs and report the signal through fluorescence emission. Briefly, hydrophobic coumarin-benzene boronic acid pinacol ester (Cou-Bpin) was grafted onto hydrophilic glycol chitosan (GC) to form an amphiphilic molecule, which further assembled into micellar nanoparticles in aqueous solution. This new nanoprobe was highly sensitive to H2O2 with a detection limit of 0.1 µM and could rapidly enter the cells within 30 min. Upon exposing to intracellular H2O2, the nanoprobe exhibited remarkable one-photon and two-photon luminescent characteristics, which was suitable to image endogenous H2O2 of various human colorectal cancer cells and assist the identification of CTCs. Compared to conventional CTC counting assay, the nanoprobe-based CTC numeration could overcome the false-negative findings due to the low expression of cytokeratin 19 (CK19). In a clinic test, CTC counting results based on the new nanoprobe match better to the postoperative pathological results of four clinic patients who had colorectal cancer at different stages.

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Circulating tumor cells (CTCs) have been regarded as predictive biomarkers in cancer diagnosis and treatment.1,2 In clinic, capture and analysis of CTCs that are being estimated as an essential “liquid biopsy”3,4 hold great promise in the stratification of cancer patients for adopting specific adjuvant therapies, monitoring response to treatments, and the identification of cancer patients at high risk of relapse.5 However, CTC counting and identification are challenging due to the extremely low counts in the whole blood stream. As such, enrichment is generally applied first based on their morphologic criteria (cell size or density) or tumor-specific markers before the analysis.6,7 After enrichment, thus far, identification and molecular analysis of CTCs majorly rely on the genetic and surface antigen analysis, such as genomic DNA sequencing,8 determination of DNA and RNA methylation,9 expression of CK19, HER2, MAGE-A3, PBGD on cell membrane, etc.10,11 Although these methods show many merits, their drawbacks cannot be ignored. For instance, in genetic analysis, direct DNA sequencing of rare CTC is difficult unless DNA amplification is performed.12 When identified by surface antigen analysis, false-negative findings of CTCs often occurred because of EMT effect that results in the decreased expressions of epithelial markers.13,14 Moreover, CTCs can nonspecifically absorb proteins and other blood cells during the process of enrichment, which potentially produces false-positive results. All these limitations may lead to incorrect CTC counts, which could be dangerous when therapeutic implications are applied based on these results.15,16 Therefore, developing new method that can rapidly and precisely identify CTCs is highly desirable for practical use. Compared to normal 3



cells, it is well-known that cancer cells often show exuberant metabolism and have high levels of endogenous H2O2.17-19 CTCs, as a specific type of cancer cells, presumably exhibit the same feature.20 Herein, we assumed that the high level of H2O2 could be considered as another unique characteristic of CTCs and subsequently used for their identification, aiming to overcome the shortcomings that exist in aforementioned methods. To directly detect and image intracellular H2O2 in tumor cells, thus far, many small molecules based one-photon/two-photon fluorescent probes have been developed.21,22 However, most of these molecular probes exhibit hydrophobic feature and their applications are restricted because of the low stability in physical condition and difficulty of cell interalization.23-28 To construct a new probe that is suitable for CTC identification, in this study, we incorporated a H2O2-responsive and two-photon fluorophore with a biocompatible polymer to form the nanosized probe, which could offer advantages over the small molecule probes, including improved water solubility, excellent ability to enter cells, low cytotoxicity, etc. In detail, coumarin derivative Cou-Bpin with excellent two-photon properties29,30 was grafted onto GC via amidation reaction, in which GC was an excellent biocompatible and biodegradable polymer to serve as an ideal carrier.31-33 After conjugation, the Cou-Bpin-grafted GC (GC-Cou-Bpin) exhibited amphiphilic feature and could assemble into nanosized micelles in aqueous solution. Based on its H2O2-responsive and excellent one-photon/two-photon optical properties, this nanoprobe was further employed for assisting the identification of CTCs derived from human colorectal cancer (CRC), 4


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which is one of the most commonly diagnosed malignancies and causes the major death in virtue of metastasis rather than the primary tumor.34-37 Noticeably, the responsive nanoprobe demonstrated the capability of rapid cellular internalization and effectively respond to the endogenous H2O2 in varied cancer cell lines (HCT 116, HT 29, SW620). The high level of H2O2 in tumor cells initiated the deprotection of phenylboronic acid pinacol ester group on nanoprobes and enabled the emission of blue fluorescent light, which assisted us to conveniently identify the CTC through fluorescence imaging. With relatively low detection limit and wide dynamic range that matched the intracellular H2O2 level of tumor cells, the responsive nanoprobe was utilized as a complementary method for a more precise CTC identification that could overcome the false-negative findings due to the lack of CK19 expression in both in vitro experiment and clinic test, implying its significant potential as a portable tool for practical use.

EXPERIMENTAL SECTION Synthesis of GC-Cou-Bpin, GC-Cou Conjugates, and the nanoprobes. The synthetic procedure of Cou-Bpin was described in Supporting Information (Scheme S1). Then, GC (73 mg, 0.284 mM glucosamine residues) was completely dissolved in deionized water (20 mL) and the obtained solution was vigorously stirred for 1 h. At the meantime, Cou-Bpin (30 mg, 0.071 mmol), EDC (16.3 mg, 0.085 mmol) and NHS (9.8 mg, 0.085 mmol) dissolved in dimethyl sulfoxide (DMSO, 5 mL) were mixed a 20 mL glass vail and stirred for 30 min. After that, the activated Cou-Bpin solution was added into aqueous GC solution. After continuous stirring at ambient 5



temperature for 24 h, the resulting crude product was dialyzed against the solvent of DMSO/H2O (1:4 v/v) for 1 day and deionized water for another 1 day (MWCO: 14 kDa). Then the aqueous solution was lyophilized to obtain the pure GC-Cou-Bpin. Similarly, 7-hydroxycoumarin-3-carboxylic acid was conjugated to glycol chitosan to form GC-Cou as a reference nanoprobe for control experiments (Scheme S2). Prior to their use, both responsive and control nanoprobes were prepared by dissolving the materials in DMSO and then dialysis against H2O to form micellular nanoparticles. Cell Viability. The relative cytotoxicity of GC-Cou-Bpin nanoprobe in vitro was assessed using MTT assay against HT 29 and HEK 293 cell lines. Briefly, the cells were seeded into 96-well plates at 10 × 103 cells per well and incubated for 24 h. Then different concentrations of GC-Cou-Bpin nanoprobe from 0 to 1000 µg/mL were added into cells and incubated for another 24 h. Finally, 20 µL of MTT assay stock solution (5 mg/mL) was added to each well. After incubation for another 4 h, the entire medium containing unreacted MTT was removed. Then, 200 µL per well of DMSO was added to dissolve the obtained blue formazan crystals followed by 15 min shaking. The absorbance of formazan was measured in a BioTek® Synergy H4 hybrid reader at a wavelength of 490 nm. Flow Cytometry. HEK 293, BRL-3A, SW620, HT 29, and HCT 116 cells were seeded in six-well plates at 5 × 105 cells per well in 2 mL culture medium separately. After incubation for 24 h, the fresh medium containing GC-Cou-Bpin nanoprobe was added with the final concentration of 400 µg/mL. These cells were incubated at 37 oC for 30, 60, and 120 min, respectively. Then all above cells were collected and 6


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measured on a BD LSRFortessa flow cytometer. Data for 1.0 × 104 gated events were collected and analyzed by FlowJo software. Followed the same procedure, the cell uptake ability of control GC-Cou nanoprobe by those three human colon cancer cells was tested. Confocal Laser Scanning Microscopy (CLSM). Two kinds of normal cells (HEK 293, BRL-3A) and three kinds of human colon cancer cells (SW620, HT 29 and HCT 116) were seeded in 6-well glass-bottomed plates with a density of 1 × 105 cells per well in 2 mL culture medium and cultured for 24 h. Next, the GC-Cou-Bpin nanoprobe (400 µg/mL) in fresh culture mediums was added into each well. The incubation time for these cells was set to 30, 60, and 120 min at 37 oC, respectively. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde following a standard protocol. To stain the nuclei of cells, PI solution was added and incubated for 10 min followed by washing with PBS twice. Finally, the slides were mounted and observed with one-photon (Leica TCS SP8) and two-photon (Leica TCS SP8 STED 3X) confocal laser scanning microscope. Capture and H2O2 Imaging of Spiked Cancer Cells from Human Blood. To expand the application of GC-Cou-Bpin nanoprobe in the area of CTC identification, HCT 116 cells were used as a model cell line to mimick the capture and identification of CTCs derived from patients with colorectal carcinoma. The spiked cancer cells were treated with trypsin and resuspended in human normal whole blood with a density of 100 cells. Then, rare HCT 116 cells were incubated with the mixture of anti-epithelial cell adhesion molecule (EpCAM) decorated magnetic beads (80 µL) 7



and GC-Cou-Bpin nanoprobe (100 µg/mL) for 30 min to capture the suspending spiked cancer cells. After the removal of blood and washing with PBS, the spiked cancer cells were concentrated and stained with 10 µL of phycoerythrin (PE) labeled anti-CD45 and fluorescein isothiocyanate (FITC) labeled anti-CK19 antibodies for 10 min. Finally, the spiked HCT 116 cancer cells were washed with deionized water twice and fixed on glass slide for two-photon CLSM analysis. Blood Sample Collection，，CTC Enrichment and Identification. Four patients with CRC prior to surgical or any other therapeutic intervention were enrolled in the study and their clinicopathological features were listed in Table 1. Peripheral blood samples were also collected from 4 healthy volunteers (2 men and 2 women) and analyzed as control. The Local Ethics Committee of the Shanghai Jiao Tong University approved this study. As a comparative study, CellSearch method was used for CTC numeration,38-40 in which CTCs were enriched via anti-EpCAM magnetic beads. Briefly, red blood cells in the whole blood samples from CRC patients were discarded by centrifugation at 1500 rpm for 15 min. CTCs were then immunomagnetically enriched from supernatants using anti-EpCAM magnetic beads and washed twice with PBS by magnetic separation. Then the cells were stained and fixed with the same procedure used for spiked cancer cell imaging. The slides were mounted and imaged by two-photon confocal laser scanning microscopy (Leica TCS SP8). Finally, numerations of CTCs were analyzed based on identifying CTC as EpCAM-isolated intact cells with CK19 positive and CD45 negative staining (the specific marker of hematopoietic cells). Meanwhile, for nanoprobe based CTC 8


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identification and numeration, the captured cells were resuspended in 50 µL PBS solution and stained by the responsive nanoprobe (100 µg/mL) for 30 min and followed with fluorescence imaging. Fluorescence In Situ Hybridization (FISH) Analysis. The isolated CTCs were resuspended in PBS solution and incubated with the responsive nanoprobe (100 µg/mL) for 30 min and washed twice with PBS by centrifugation. Then CTCs were fixed on glass slides with 4% paraformaldehyde and dehydrated sequentially in 75%, 85% and 100% ethanol at 3 min each. The FISH probe-Vysis Spectrum Orange labeled centromere probe 8 (CEP8) was added onto the slides and processed according to the manufacturer’s instructions. After that, the cells were incubated with Alexa Fluor 594 conjugated anti-human CD45 at room temperature for 1 h, washed with mounting media for CLSM observation. CTC was defined as more than two CEP8 signals in nuclei, which was identified as a hyperdiploid cell.41,42

RESULTS AND DISCUSSION Synthesis and Characterization of GC-Cou-Bpin and Self-Assembled Nanoprobe. To construct the nanoprobe for CTC identification, H2O2-responsive two-photon fluorophore Cou-Bpin was synthesized first. As shown in Figure S1, precursors of the fluorophore ethyl 7-hydroxy-coumarin-3-carboxylate (molecule 1) and

4-methylbenzeneboronic

acid

pinacol

ester-ethyl-coumarin-3-carboxylate

(molecule 2) were successfully synthesized according to the literature,43 which was confirmed by NMR analysis (Figure S3 and Figure S4). Then, Cou-Bpin (molecule 3) was synthesized through hydrolysis and purified by recrystallization based on a 9



previously reported method with minor modifications (see details in Supporting Information).44 The chemical structure of Cou-Bpin was further confirmed by NMR spectroscopy (Figure S5)

Figure 1. Synthetic scheme of GC-Cou-Bpin conjugate based H2O2-responsive nanoprobe and its responsiveness to intracellular H2O2 of CTCs. The synthesized Cou-Bpin was then conjugated to glycol chitosan through amidation reaction in the presence of EDC and NHS (Figure 1). After conjugation, the small molecules were removed by dialysis to obtain the purified GC-Cou-Bpin. 1H NMR spectrum revealed that the final product contained aromatic protons which appear at 7.3-7.9 ppm (Figure S5c). The FTIR spectra also confirmed the success of Cou-Bpin grafting on GC molecule (Figure S6). Compared to pristine GC, the intensity of the characteristic peak lying at 1597 cm-1 attributed to the NH2 deformation vibration weakened and a new peak at 1626 cm-1 corresponding to amide bond appeared. In this study, we define the grafting ratio as the number of Cou-Bpin groups per 100 glucosamine units of GC, which can be determined by the ICP

10


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measurement of boracium content. Based on the ICP results, the content of boracium was 0.86 wt% and the grafting ratio was calculated to be 17.04%. After conjugation, the resulting GC-Cou-Bpin exhibits as an amphiphile comprised of hydrophobic Cou-Bpin in the side chain and hydrophilic glycol chitosan in the backbone. As such, nanosized micelles were assembled by transferring the responsive amphiphile from nonselective solvent into aqueous buffer solution. The nanoprobe was then characterized by means of dynamic light scattering (DLS), transmission electron microscopy (TEM), and zeta potential analysis. The DLS result shows that the average hydrodynamic diameter of nanoprobe is around 50 nm (Figure 2a). At the meantime, spherical nanoparticles with an average diameter about 40 nm can be observed under TEM imaging (Figure 2b), which is consistent with the DLS measurement.

Owing

to

the

amphiphilic

feature

of

GC-Cou-Bpin,

its

amine-containing hydrophilic part would locate at the corona after self-assembly, leading to a positive charged surface of the micelle structure.45 As shown in Figure 2c, the responsive nanoprobe indeed exhibits a highly positive charged surface with a zeta potential of 34.5 ± 1.0 mV. It has been widely reported that the positive charged surface of nanoparticles can facilitate their cellular internalization.46 Therefore, it could be expected that the nanoprobe can rapidly response to the intracellular H2O2 for CTC identification.

11



Figure 2. Characterizations of GC-Cou-Bpin and the self-assembled nanoprobe. (a) DLS analysis of the responsive nanoprobe assembled by GC-Cou-Bpin; (b) TEM image of the self-assembled nanoprobe; (c) zeta potential of the self-assembled responsive nanoprobe; (d) One-photon excitation and emission spectra of GC-Cou-Bpin in the absence and presence of H2O2. (e) two-photon emission spectrum of GC-Cou-Bpin responsive nanoprobe (400 µg/mL) when incubated with H2O2 at the concentrations from 0 µM to 100 µM for 30 min. The two-photon spectrum was excited at 800 nm; (f) The plot of two-photon fluorescence intensity versus H2O2 concentration (0 µM to 100 µM) when incubated the responsive nanoprobe with H2O2 for 30 min (slope: 0.93, R2= 0.9986). The error bars represent the standard deviations of the results obtained from 3 parallel measurements. Fluorescent Properties of GC-Cou-Bpin Nanoprobe. The optical properties of GC-Cou-Bpin nanoprobe were examined in PBS buffer. As shown in Figure 2, the fluorescence spectrum of the intact nanoprobe exhibited an excitation maximum at λex = 350 nm. When incubated with excess H2O2 for 30 min, the excitation peak shifted to 400 nm. At the meantime, the addition of H2O2 resulted in an augment of blue fluorescence with the emission at 450 nm upon exciting the nanoprobe at 400 nm (Figure 2d). Furthermore, a kinetic study revealed that the fluorescence increased rapidly within 2000 s and tended to reach the equilibrium at ~ 6000 s (Figure S7a) 12


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while incubating the responsive nanoprobe with large excess H2O2 (1mM), giving a

pseudo-first-order rate constant of 5.79 × 10-4 /s (Figure S7b). This result demonstrated that the responsive nanoprobe was highly sensitive towards H2O2. Moreover, when incubating the responsive nanoprobe with varied concentrations of H2O2 (from 0 µM to 100 µM) for 30 min, the plot of log(F-F0) vs. log([H2O2]) exhibited a slope of 0.92, indicating an approximate first-order dependence on H2O2 concentration (Figure S8a and 8b) and thereby giving a detection limit of 0.1 µM. Besides the one-photon fluorescence, Cou-Bpin is also a two-photon (TP) fluorophore which endows the nanoprobe with additional advantages for imaging purposes, including relatively deep tissue penetration, low photo damage to cells, and high signal to noise ratio by minimizing background fluorescence.47,48 Herein, its two-photon fluorescent property was also investigated. The nanoprobe was excited at 800 nm and the blue fluorescence was recorded at 450 nm. Similar to one-photon fluorescence spectrum, the intensity of two-photon fluorescence increases correspondingly along with the addition of H2O2 (Figure 2e). A plot of log(F-F0) vs. log([H2O2]) exhibited a slope of 0.93, also indicating an approximate first-order dependence on concentration of H2O2 (Figure 2f). Moreover, the influence of excitation power on nanoprobe’s two-photon fluorescence was further investigated. As shown in Figure S8c and S8d, a quadratic relationship between the fluorescence intensity and the intensity of excitation light is verified when the power of laser increased from 90 mW to 350 mW, indicating an excellent two-photon property of our nanoprobe. 13



Cell Viability Assay. As a cellular nanoprobe, cytotoxicity is another important parameter that has to be evaluated before its biomedical applications. Therefore, the inherent cytotoxicity of responsive nanoprobe was tested by the means of a standard MTT assay. The HT 29 cells and HEK 293 cells were selected as typical human cancer cells and normal cells respectively. Cell viabilities of the HT 29 and HEK 293 cells treated with different concentrations of materials and different incubation time (1.5 h and 24 h) demonstrate that the nanoprobe has excellent biocompatibility with both normal and cancer cells, even when the nanoprobe reaches up to 1000 µg/mL (Figure 3a and Figure S15).

Figure 3. (a) Cell viability of HT 29 (green) and HEK 293 (blue) against different concentrations of the responsive nanoprobe for 24 h (error bars represent standard deviations, n=6). (b) The fluorescent intensity of three kinds of human colon cancer cells incubated with the responsive nanoprobe and control GC-Cou nanoprobe (400 14


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µg/mL) for 30 min (error bars represent standard deviations, n=3). Flow cytometry histogram profiles of (c) HCT 116 and (d) HEK 293 cells incubated with nanoprobe (400 µg/mL) for different intervals at 37 oC. (e) Two-photon CLSM images of HCT 116 cells treated with the responsive nanoprobe (400 µg/mL) for (i) 0 h, (ii) 30 min, (iii) 1 h, and (iv) 2 h (upper panel), the cell nuclei were stained with PI (middle panel), and the merged images (lower panel). Cellular Uptake and H2O2 Imaging with Nanoprobe in Living Cells. Based on its rapid response to H2O2, we employed this nanoprobe for intracellular H2O2 detection and imaging in the living cells. In the experiment, two normal cell lines HEK 293, BRL-3A and three cancer cell lines HCT 116, HT 29, SW620 were incubated with the nanoprobe since they potentially had different levels of endogenous H2O2. At the meantime, to illustrate the cellular internalization behavior of the nanoprobe, a control GC-Cou nanoprobe was also incubated with these cells. The control nanoprobe did not contain the protection group (a phenylboronic acid pinacol ester) and therefore can perform as a fluorescent probe to track the cell uptake without the presence of H2O2. Flow cytometry and fluorescence microscopy revealed that the control nanoprobe could be rapidly internalized the tested cells within 30 min (Figure 3b), which might be attributed to its positively charged surface. Notably, when incubating with normal cells (HEK 293 and BRL-3A), there was no marked augment of fluorescence even the incubation time was prolonged to 2 h (Figure 3d, Figure S9c and Figure S11). In contrast, for cancer cells (HCT 116, HT 29, SW620), strong blue fluorescence could be seen both under the one-photon and two-photon microscope in 30 min (Figure 3c, Figure S9a, S9b, Figure 3e, Figure S10, and Figure S12), demonstrating its capability to fast distinguish the normal and cancer cell. Importantly, under the same condition, flow cytometry revealed that the fluorescence profiles of 15



three used cancer cells incubated with responsive and control nanoprobe was distinct from one to another (Figure 3b, 3c and Figure S9a, S9b). As shown in Figure 3b, the HT 29 had the highest fluorescence than those of other two cell lines when control nanoprobe was used. In contrast, HCT 116 exhibited the strongest fluorescence when incubated with the responsive nanoprobe. As the fluorescent intensity of responsive nanoprobe is linearly related to the concentration of H2O2, the varied fluorescence aroused by responsive probe implies that these tumor cells have different levels of intracellular H2O2. The Cell Spike Isolation and Detection in Whole Blood. In general, CTCs are defined as nucleated EpCAM-positive cells, expressing cytoplasmic cytokeratins 8, 18, and 19 but lacking CD45. HCT 116 cells, as a model cancer cell line, were enriched by magnetic beads and stained by GC-Cou-Bpin nanoprobe, FITC-labeled anti-CK19, and PE-labeled anti-CD45. As shown in two-photon CLSM images of spiked cancer cells, the strong blue fluorescence was switched by the high level of intracellular H2O2 in HCT 116 cells (Figure 4a). Meanwhile, HCT 116 cells are CK19 positive and CD45 negative, so they are easily stained with strong green fluorescence yet without red fluorescence. As a control, U-87 MG cancer cells which cannot express CK19, can only be stained by the responsive nanoprobe with blue fluorescence (Figure 4b). This result exhibits a great potential using H2O2-responsive nanoprobe to reduce the false-negative results in CTC identification.

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Figure 4. Two-photon CLSM images of (a) spiked cancer cells (HCT 116 cells) captured by EpCAM labeled magnetic beads and (b) U-87 MG cells stained with the responsive nanoprobe, FITC-labeled anti-CK19, and PE-labeled anti-CD45. Nanoprobe-Assisted CTC Identification in Clinical Test. Owing to its fast responsiveness and the capability of effectively discriminating the cancer and normal cells, the responsive nanoprobe was further employed to assist the identification of CTC in a clinic test. In the experiment, CTCs captured and identified by commercialized CellSearch assay and characterized by the responsive nanoprobe were conducted as a comparison study. For the CellSearch assay, CTC counting relies on the anti-CK19 positive staining (green fluorescence) associated with anti-CD45 negative staining (red fluorescence) and DAPI staining (blue fluorescence) after capturing by EpCAM beads. In the latter case, the responsive nanoprobe assists the CTC identification based on the blue fluorescence emission trigged by endogenous H2O2 of the captured cells. The threshold of fluorescence intensity to determine a CTC is three times higher than the average background fluorescence generated by the nonfluorescent cells. As expected, no CTCs were enriched and detected in the blood samples of four healthy persons using above two CTC numeration methods. In contrast, when the whole blood samples from four patients were collected and analyzed, positive CTC read outs were obtained. More importantly, these two methods rely on totally different detection principle. Thus, they can simultaneously 17



work on a same individual CTC. As demonstrated in Figure 5, the surface of CTC was stained with green fluorescence (FITC-labeled anti-CK19). At the meantime, its cytoplasm was shown as blue because of the fluorescence emission generated by responsive nanoprobe under high level of endogenous H2O2. Besides these typical CTCs with both strong green and blue fluorescence (Figure 6 and Figure S14), it could be noticed that some of the captured CTCs were stained with strong blue fluorescence, but with weak or even without green fluorescence (Figure 6ii, vi, viii and Figure 14a-d). Therefore, the results of CTC numeration based on the responsive nanoprobe were normally higher than those counted by CellSearch assay even though we have ignored the cells with relatively weak blue fluorescence, which might be aroused by dead or apoptosis CTCs. Similar to the case of U-87 MG demonstrated above, the higher CTC counts by the responsive nanoprobe may be attributed to the high heterogeneity and EMT of CTCs, which result in the lack of CK19 expression. Yet, these CTCs still have a relatively high level of H2O2 and can be identified by the responsive nanoprobe.

Figure 5. The three-dimensional two-photon CLSM images of CTCs derived from CRC patient 1.

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Table 1. Summary of clinicopathological features, CTCs counts and relative fluorescent intensity in whole blood derived from patients with colorectal cancer. Tumor

Intestinal

TNM

size

lymph

stage

(cm)

node

Patient 1

3x3x2.5

0/12

T2N0M0

Patient 2

2x1.5x1

0/12

Patient 3

4x4.5x1

Patient 4

5x4x1.5

Sample

CTC Pathology

Counts a

b

Adenomatous type, low grade dysplasia

26

19

T1N0M0

Adenomatous type, high grade dysplasia

5

2

3/12

T4N1M0

Ulcerative type. moderately differentiated

8

6

2/12

T4N1M0

Ulcerative type. poorly differentiated

6

4

a. the number of CTCs was counted according to the blue fluorescence of H2O2 responsive nanoprobe. b. the number of CTCs was counted according to the green fluorescence of FITC-labeled anti-CK19. To further confirm the blue fluorescent cells were real CTCs, they were stained with both H2O2-responsive nanoprobe and FISH probe as CTC was hyperdiploid cell and would show more than two CEP8 signals in nuclei when stained with FISH probe. Figure 7 demonstrated that the blue fluorescent cells stained by responsive nanoprobe really have more than two signals of orange fluorescence emitted by FISH probe. Finally, the CTC counts based on conventional numeration method and responsive nanoprobe were listed in Table 1. At the meantime, postoperative pathological results of four participating patients were also shown in the table. In clinic, total CTC number of 5 is a typical criterion for evaluating the clinical prognosis of treated cancer patients. Examining the CTC numeration and postoperative pathological results, it could be found that CTC counts of all tumor-positive patients were no less than 5 when the responsive probe was applied. Instead, traditional CTC counting assay 19



reported that only two patients have CTCs higher than 5. The measured CTCs of other two patients were less than the criterion number, which might potentially mislead their diagnosis and proper treatment. This comparison study demonstrates that the responsive nanoprobe can decrease false-negative findings in CTC numeration and provide a more reliable diagnosis. Therefore, it may replace expensive antibodies, such as anti-CK19, to effectively quantify the CTC counts and help guiding the clinical therapy of cancer patients.

Figure 6. Two-photon CLSM images of CTCs derived from CRC patients with different malignancy and carcinogenic types. (i) and (ii) Patient 1, (iii) and (iv) Patient 2, (v) and (vi) Patient 3, (vii) and (viii) Patient 4. CTCs were stained with the responsive nanoprobe, FITC-labeled anti-CK19, and PE-labeled anti-CD45. 20


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Figure 7. Identification of CTCs by FISH probe and the responsive nanoprobe staining. CONCLUSIONS In summary, a biocompatible and sensitive H2O2-responsive nanoprobe composed of hydrophobic coumarin-derivative fluorophore and hydrophilic glycol chitosan had been successfully constructed by grafting Cou-Bpin onto GC molecules for endogenous H2O2 imaging in CTCs. The synthesized nanoprobe exhibited hallmark luminescent characteristics with both one-photon and two-photon light excitation. Importantly, the nanoprobe was highly selective and sensitive to H2O2 which could be used for intracellular H2O2 detection and imaging with the detection limit as low as 0.1 µM. In vitro experiments demonstrated that the responsive nanoprobe could sense different concentrations of endogenous H2O2 in various types of human colon cancer cells. Based on its excellent fluorescent properties and fast response to intracellular H2O2, we further employed them for CTC staining and numeration. Compared with the current methods in CTCs’ identification in Table S1, the responsive nanoprobe based CTC numeration can overcome the false-negative findings aroused by the lack 21



of surface biomarkers (e.g. CK-19) and provide a more reliable diagnosis, indicating that the nanoprobe can be utilized as a new “liquid biopsy” tool for clinic CTC analysis. Moreover, it may also help to establish the relationship between H2O2 level and clinical features of the tumor to assist the clinical treatments of cancer patients.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI：

AUTHOR INFORMATION Corresponding Authors * Tel.: +86-021-34188822. E-mail: [email protected] * Tel.: +86-021-54740717. E-mail: [email protected] * Tel.: +86-021-54746215. E-mail: [email protected] Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program (2015CB931801), National Natural Science Foundation of China (21374062, 51690151, 51473093), the Recruitment Program of Global Experts (15Z127060012). The authors also thank the PhD financial support from Prof. Jianwei Pan and the FISH kit from Cyttel Biosciences INC. 22


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For TOC only:

A hydrogen peroxide-responsive nanoprobe assists circulating tumor cell identification and colorectal cancer diagnosis

Chunting Li, Ruijun Pan, Peiyong Li,* Qinghua Guan, Junping Ao, Kai Wang, Li Xu, Xiaofei Liang, Xin Jin, Chuan Zhang,* and Xinyuan Zhu*

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Hydrogen Peroxide-Responsive Nanoprobe Assists Circulating Tumor

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