Capturing Circulating Tumor Cells through a Combination of

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Capturing Circulating Tumor Cells through a Combination of Hierarchical Nano-topography and Surface Chemistry Guang Yang, Xilin Li, Yang He, Xiang Xiong, Pu Wang, and Shaobing Zhou ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.7b00683 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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ACS Biomaterials Science & Engineering

Capturing Circulating Tumor Cells through a Combination of Hierarchical Nano-topography and Surface Chemistry Guang Yang†‡, Xilin Li†, Yang He†, Xiang Xiong†, Pu Wang†, Shaobing Zhou†* †

School of Materials Science and Engineering, Key Laboratory of Advanced

Technologies of Material, Minister of Education, Southwest Jiaotong University, Chengdu 610031, Sichuan, P.R. China ‡

College of Medicine, Southwest Jiaotong University, Chengdu 610031, Sichuan, P.R.

China

KEYWORDS: Cell capture, circulating tumor cells, polydopamine, nanotopography, nanofibers

ABSTRACT:

Circulating tumor cells (CTCs) are known as a minimally invasive multifunctional biomarker for earlier diagnosis, prognosis, recurrence risk assessment and therapeutic monitoring in recent years. However, the approach of effectively capturing these CTCs is still difficult to be obtained due to the extremely low abundance of CTCs and the diverse phenotypes of cancer cells. In this study, we present a novel necklace-like polydopamine nanospheres (PDA NSs)/alginate composite nanofiber with a hierarchical nano-topographical structure and a surface chemical signal for capturing the CTCs. The height of the nano-topography, which is 1

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formed by connecting PDA NSs with nanofibers via electrospinning, can be easily adjusted by changing the size of the PDA NSs. Four types of cancer cells are employed to investigate the capture efficiency of the fiber. More importantly, in a blood environment containing rare cancer cells the fiber still has a great ability of capturing these cells. Therefore, this nanofiber is deemed to be applied as a potential device for diagnosis of cancer disease.

1. INTRODUCTION

Detection of circulating tumor cells (CTCs) can help clinicians to collect information about different phases of the metastatic process, further achieving early diagnosis of malignancies and implement accurate and effective treatment to cancer disease. 1 CTCs are a type of cells which are detached from the primary or metastatic tumor sites and enter into the peripheral bloodstream. Because of their usefulness in earlier diagnosis, prognosis, recurrence risk assessment and therapeutic monitoring, 2-4 CTCs have attracted more and more attention as a minimally invasive multifunctional biomarker in recent years.

5

However, the effective capture of CTCs is still a great

challenge, owing to the facts that in blood the amount of CTCs is extremely rare (a few to hundreds per 109 hematologic cells), moreover, they are mixed with normal blood components. 5-6 To capture and detect the CTCs from bloodstream, several kinds of isolation methods, 7-9 based on the physical cell properties or the known cell surface markers, 2,

2


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10-12

have been developed such as microfiltration, 13 density gradient centrifugation, 14

label-free micropatterns,

15

antibody-modified magnetic beads,

16

antibody-modified

substrates (e.g. silicon nanowires, 17 nanofibers, 18 glass substrates, 19 cryogels, 20 and carbon nanotube films21). These approaches can capture the certain type of CTCs in some degree, however, there still remain several issues. 9, 22-23 For example, they have bad effects on the viability or phenotype of captured cells through employing these treatments including stresses from shear force, non-physiologic temperature variation, aggressive reagents (e.g. trypsin), UV exposure. Notably, to date most of the isolation method mainly relied on identifying and binding the epithelial cell adhesion molecule (EpCAM) expressed on the surface of CTCs with anti-EpCAM. Although the using of anti-EpCAM did make a great contribution to increase the capture efficiency of CTCs, there still loss of cancer cells with low EpCAM expression. To address these problems, we

develop

a

polymer

electrospun

nanofibers

with

hierarchical

surface

nano-topography and a specific surface chemistry signal to capture CTCs. The nanofibrous structure, which is similar to the structure of extracellular matrix,

24

can

enhance local topographic interactions between the nanosubstrates and the nanoscaled cellular surface components.

25-27

In addition, the production of nanofibers through

electrospinning is more facile and cheaper than other methods; and several reports have demonstrated that hierarchical structured micro-/nanofibers can be achieved by electrospinning,

28

which are benefit to further enhance the topographic interaction

between cells and substrates. Accordingly, in this study a polymer electrospun necklace-like nanofibers with 3



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hierarchical surface nano-topography and a specific surface chemistry signal is developed. Firstly, the surface nano-topography structure was achieved by introducing the polydopamine nanoparticles into the nanofibers to form hierarchical necklace-like nanofibers. Secondly, the polydopamine molecules provide a specific surface chemistry signal for cell capture, which have been reported that they have not only good biocompatibility but also an ability of promoting cellular adhesion.

29-32

The

alginate, a widely used cyto-compatible material, 33 was employed as the fiber matrix. The combination of the hierarchical surface nano-topography and surface chemistry signal is expected to provide an alternative strategy for highly effectively capturing circulating tumor cells.

2. MATERIALS AND METHODS 2.1. Materials.

Dopamine hydrochloride were purchased from BioKem Reagent Company (China). Ammonia (NH4OH, 25-28%) and ethanol were purchased from Chengdu Kelong Chemical Reagent Company (China). Polyethylene oxide (MW 1000 kDa) were obtained from Xiya Reagent Research Center (China). Sodium alginate from brown algae, Pluronic F127, calcein AM, propidium iodide (PI), fluorescein isothiocyanate (FITC) labeled phalloidin and 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) were purchased from Sigma-Aldrich (USA). Alexa 488-labeled anti-CD45 (marker for WBCs) and Alexa 647-labeled anti-CK18 (marker for epithelial cells) were purchased from Abcam (USA). Other chemicals with reagent 4


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grade or better were acquired from commercial sources and used without further purification. Deionized (DI) water was used through all experiments.

2.2. Cancer cell lines and culture conditions.

HepG2 cells and A549 cells were gifts from Sichuan University (Chengdu, China). U251 cells and PANC-1 cells were purchased from Cell Bank of Chinese Academy of Sciences. A459 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone, USA) with 10% (v/v) newborn calf serum (NBCS, Gibco, USA), all other cancer cells were cultured in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM-H, HyClone, USA) with 10% (v/v) NBCS. All cells were cultured at 37 °C under 5% CO2 and fully humidified conditions.

2.3. Preparation and characterization of polydopamine nanospheres (PDA NSs).

Polydopamine nanospheres (PDA NSs) were synthesized in a simple method as reported previously.34 Taking the synthesis of PDA NSs with diameter of 800 nm as an example, briefly, first of all, 1.5 mL ammonia aqueous solution (NH4OH, 25-28%), ethanol (120 mL) and 270 mL deionized water were mixed together under mild stirring at room temperature for about 30 min; then dopamine hydrochloride (1.5 g) was dissolved in deionizedwater (30 mL) and added into the above mixture solution. After the addition of dopamine hydrochloride solution, the color of whole solution turned to be dark brown gradually. At last, the whole mixed solution was kept stirring at room temperature for another 24 hours. After that, the PDA NSs were gained by centrifugation and washed with water for three times. To control the size of PDA NSs 5



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(from 800 nm to 270 nm), the molar ratio of ammonia to dopamine hydrochloride was tuned from ~5.1 to ~1.4 as shown in Figure S1. The diameters of PDA NSs was measured by a dynamic light scattering (DLS) measurement device (Malvern Zetasizer Nano-ZS100 apparatus) and repeated at least three times. The morphologies of the micelles were observed with a field emission scanning electron microscope (FE-SEM) (JSM-7001F, JEOL, Ltd. Japan). The FE-SEM sample was prepared by added a drop of PDA NSs dispersion onto a clean silicon wafer. After dried, the sample was coating by platinum.

2.4. Fabrication and characterization of PDA NSs/alginate composite nanofibers.

To fabricate the PDA NSs/alginate composite nanofibers with different sizes of PDA NSs, a calculated amount of PDA NSs (with diameter ranging from 270 nm to 800 nm) suspension in water was added into the sodium alginate / Pluronic F127 / PEO mixture solution to achieve the desired PDA NSs: fiber matrix weight ratio, including: 1:10, 1:6, 1:4, 1:2 for particle size of 270 nm, 400 nm, 650 nm and 800 nm respectively. The final content of sodium alginate / Pluronic F127 / PEO was 2 wt% : 2 wt% : 2 wt%. The blend solution of sodium alginate / Pluronic F127 / PEO (2 wt% : 2 wt% : 2 wt%) without PDA NSs was also prepared. 35, 36 Then the homogeneous blend solution was transferred into a 5 mL plastic syringe connected with a stainless steel needle (inner diameter: 0.33 mm). The flow rates of were 0.3 mL/h to 0.5 mL/h by a microinjection pump (LSP02-1B, LongerPump, China). The distance between the needles and the collector (aluminum foil wrapped

6


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onto a grounded rotor with speed of 140 rpm) was 14 cm and the applied voltage was 19 kV. The relative humidity was kept below 40%. The composite nanofibers were observed by FE-SEM (JSM-7001F, JEOL, Ltd. Japan) and transmission electron microscopy (TEM) (JEOL 2100F, JEOL Ltd., Japan). The SEM samples were prepared by coating a thin layer of platinum onto the nanofibers. The TEM samples were collected onto a copper grid covered with a carbon film. The ImageJ software (1.46 h, NIH, USA) was employed to analyze the SEM images and the average diameter of nanoﬁbers was counted. Before the further investigation, composite nanofibers were crosslinked in advanced as previous report. 37 In general, nanofibers were soaked in 95% ethanol for 5 min and wash with 2 wt% CaCl2 solution in ethanol for 10 min. Then the nanofibers were immersed in CaCl2 solution (2 wt%) for at least 1 h, followed with immersion in DI water for 1 h to remove excess CaCl2.

2.5. Cell capture on the PDA NSs / alginate composite nanofibers.

Four types of cancer cells were employed in the cell capture experiments, including HepG2 cells, A549 cells, U251 cells and PANC-1 cells. Samples of cell capture experiments were fabricated by collecting composite nanofibers onto the glass wafer (diameter: 15.6 mm) for about 10 min during electrospinning and crosslinking these nanofibers with CaCl2 as described above. After the samples were sterilized by 75% alcohol and Ultraviolet irradiation for 8 h, they were rinsed with PBS for three times and placed in 24 well cell culture plate (Corning, USA). Then 1 mL of cancer

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cell suspension (about 1×105) was added onto each sample and incubated at 37℃ and 5% CO2 for a certain capture time (10 min, 20 min, 45 min, 60 min and 120 min). At each preset time point, samples were rinsed gently with PBS for three times, fixed by 2.5% glutaraldehyde in PBS for about 1 h, stained by DAPI and finally observed by fluorescence microscope (IX51, Olympus, Japan). At least, ten randomly selected areas of each sample were imaged and the images were then analyzed by ImageJ software (1.46 h, NIH, USA) to count the average captured cell numbers. All CTCs capture experiments of different groups at different time points, have repeated for at least three times. According to the analysis of the fluorescence images, the actual cell density captured on the nanofibers was calculated by the average cell number on an image (Nactual)/the image area (Aimage). While the theoretical cell density was the ratio of the total cell number (Ntotal) added to each well and the area of each well (Awell). So the cell capture efficiency can be calculated as following formula:

38

Cell capture

efficiency = (Nactual / Aimage) / (Ntotal / Awell) × 100%, in our experiments, Aimage was 0.26 mm2; Ntotal was 1×105; and Awell was 191.13 mm2. 2.6. Observation of captured cells’ morphologies.

At 1 h post the cell capture experiment, the captured cells on the nanofibers were fixed by glutaraldehyde (2.5% in PBS) for at least 2h and then observed by FE-SEM (JSM-7001F, JEOL Ltd., Japan) and Confocal laser scanning microscopy (CLSM, TCS SP8, Leica, Germany).

8


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For FE-SEM observation, the fixed samples were immersed in a series of alcohol aqueous solution (30%, 50%, 75%, 85%, 95%, and 100%) sequentially to for dehydration. After dried in the vacuum oven over night, the samples were coated with platinum, then checked with the FE-SEM. For CLSM observation, after fixed by glutaraldehyde, cells on nanofibers were stained by DAPI (for staining nuclei) and fluorescein isothiocyanate (FITC) labeled phalloidin (for staining actin cytoskeleton) in sequence.

2.7. Cell viability assay.

To evaluate the cyto-compatibility of the nanofibers, cells captured on the nanofibers were incubated for a relative long term and investigated by a Live/Dead staining method. Briefly, after cell capture experiments for 1 h, samples with captured cells were gently rinsed with PBS for three times and fresh culture medium were added. After a certain period of incubation (0 h, 24 h, and 48 h), the cells were stained with calcein AM and PI, then observed with fluorescence microscope (IX51, Olympus, Japan) at each time point. The cell viability assay of each group has repeated for three times.

2.8. Cell capture and identification from the whole blood.

A549 cells and PANC-1 cells were spiked into the healthy human blood (collected from venous blood of healthy volunteers in Sichuan Academy of medical sciences & Sichuan Provincial People's Hospital) to achieved an artificial patient blood samples, with concentrations of 10, 50 and 100 cells/mL, repectively. After 1 h 9



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cell capture experiments on the nanofibers, three-color immunostaining method was used to identify the captured cancer cells and white blood cells (WBCs). Briefly, the captured cells were fixed by glutaraldehyde (2.5% v/v in PBS) for at least 2 h and gently rinsed by PBS. Then 20 μL of 0.5% Triton-X-100 in PBS were added for 10 min to increase cellular permeability, followed a PBS rinse. At last, 20 μL of anti-CD45 (maker for WBCs) 39 solution (50 μL of antibody stock solution in 1 mL of PBS)(staining for 30 min), 20 μL of anti-CK (marker for epithelial cells) solution (50 μL of antibody stock solution in 1 mL of PBS) (staining for 30 min) and 20 μL of DAPI solution (staining for 15 min) were dropped onto the samples sequentially and each addition of dye was followed by a PBS rinse. After staining, samples were observed by using CLSM (TCS SP8, Leica, Germany). Each CTCs capture experiment has repeated for at least three times.

2.9. Statistics Analysis.

Each experiment was performed in triplicate or more specimens. Results were shown as: mean ± standard deviation. To determine statistical significance of the data, single factorial analysis of variance (ANOVA) was performed.

3. RESULTS AND DISCUSSION 3.1. Monodispersed Polydopamine Nanospheres.

The monodispersed polydopamine nanospheres (PDA NSs) were successfully fabricated according to previous report.

34

Simply by adjusting the molar ratio of

10


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ammonia to dopamine hydrochloride, PDA NSs with different sizes are obtained as shown in Figure S1 in the Supporting Information (SI). From the scanning electron microscope (SEM) images in Figure 1, we can find that these PDA NSs were all uniformly spherical in shape with the average diameters ranging from 270 nm, 400 nm, 650 nm to 800 nm while the molar ratio of ammonia to dopamine hydrochloride decreased from ~5.1 to ~1.4 (Figure S1).

3.2. Fabrication and characterization of PDA NSs/alginate composite nanofibers.

Based on the monodispersed PDA NSs with different sizes, the hierarchical PDA NSs/alginate composite nanofibers were successfully fabricated by an electrospinning technology as shown in Figure 2. To obtain the necklace-like PDA NSs-loaded alginate composite nanofibers, the introduction of a series of weight ratio of PDA NSs with different diameters into nanofiber matrix is optimized (data are not shown); and the optimal weight ratios of PDA NSs to nanofiber matrix are achieved as following 1:10 (270 nm), 1:6 (400 nm), 1:4 (650 nm), 1:2 (800 nm). The concentration of PDA NSs in nanofiber matrix can be employed to adjust surface nano-topography structure and surface chemistry signal for binding tumor cells. Besides the particles concentration, the particle size also plays an important role in affecting the formation of necklace-like nanofibers. It has been reported that the particle diameter, around or a little lager than the nanofiber diameter, is one of the ideal conditions to form the necklace-like fibers during electrospinning.40 After electrospun, all composite nanofibers were further crosslinked by CaCl2 in order to make the fiber insoluble and

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immobilize the nanoparticles in fiber matrix. From the SEM images (Figure 2a), it can be found that the crosslinking did not affect the necklace-like structure; however, the diameters of fiber matrix were shrunk as shown in Table S1, which made more “beads” expose on the individual fiber, leading to a more rough surface of the nanofibers film. To confirm that the PDA NSs were successfully encapsulated into the fiber matrix, transmission electron microscopy (TEM) and energy dispersive x-ray spectroscopy (EDX) elemental analysis were employed to verify the structure. As shown in Figure 2b, spherical structure in darker color than the nearby nanofiber matrix can be distinguished and the nitrogen element (only contained in dopamine) is mainly appeared in the corresponding round area in the elemental mapping, which are direct evidences that the PDA NSs have been successfully encapsulated. In addition, the average height of protrusion of each group of composite nanofibers (Figure 2c) was measured according to the SEM images of composite nanofibers. With the size of particles in fiber matrix increases, the average height of surface protrusion increases from 67.39 nm to 272.37 nm for un-crosslinked fibers and from 81.66 nm to 292.12 nm for crosslinked fibers, respectively. It’s easy to understand that composite nanofibers containing larger particles display higher protrusions.

3.3. Cell capture on the PDA NSs / alginate composite nanofibers.

To investigate the cell capture capacity of the composite nanofibers, four types of cancer cells were employed, including human hepatoma cell line (HepG2), human

12


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lung epithelial tumor cell line (A549), human glioma cell line (U251) and human pancreatic carcinoma cell line (PANC-1). A set of cell capture experiments with different incubation time were applied by using different nanofibers, including PDA NSs/alginate composite nanofibers with different nano-topography structures, and the alginate nanofibers without PDA NSs. One alginate smooth plane was used as control. The capture efficiencies of different samples against different incubation time were plotted in Figure 3. We can find that after 120 min incubation, for all four types of cells, the alginate plane showed barely no capture effect; the pure alginate nanofibers performed a little capture effect (13.70 ± 5.72% for HepG2 cells, 6.83 ± 2.67% for A549, 7.20 ± 3.94% for PANC-1 cells and 12.13 ± 8.84% for U251 cells); while the composite nanofibers had much better capture efficiencies, especially the one containing 270 nm PDA NSs, which displayed the maximal cell capture efficiency (72.85 ± 11.67% for HepG2 cells, 52.36 ± 15.13% for A549, 62.85 ± 14.66% for PANC-1 cells and 50.76 ± 12.99% for U251 cells). These results demonstrate that the introduction of PDA NSs can effectively enhance the capture efficacy of nanofibers for CTCs. Additionally, in terms of these particles-loaded composite nanofibers, it seems that the smaller PDA NSs in the composite nanofibers, the higher capture efficiency of the composite nanofibers would achieve. Furthermore, for all the composite nanofibers with different cells, we can see that there are no significant difference between each groups and the capture efficiencies increase slowly in the first 20 min, while after 45 min, the efficiencies of 270 nm and 400 nm groups increased obviously faster than the groups of 650 nm and 800 nm, and after 60 min, 13



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the increase of efficiencies of all groups turned to be smooth. Additionally, since there still exists normal cells, like free endothelial cells (ECs) or smooth muscle cells in blood circulation, the ECs were also employed to test the capture capacity of the composite nanofibers containing 270 nm or 400 nm PDA NSs (Figure S2). After 120 min incubation, the capture efficiency of nanofibers containing 270 nm PDA NSs, is 47.45 ± 14.12%, and the capture efficiency of nanofibers containing 400 nm PDA NSs is 38.08 ± 9.37%. Though the capture efficiency of nanofibers for ECs were a little lower than for the CTCs, it seems that the composite nanofibers have similar capture effect to the ECs, which may due to that the promotion effect of cell adhesion of polydopamine is nonspecific.

3.4. Morphologies of captured cells.

To further understand the capture mechanism and distinguish the contribution of the hierarchical nano-topography of nanofibers and surface chemistry signal of PDA NSs to the cell capture, confocal laser scanning microscopy (CLSM) were employed to observe the morphologies and the actin cytoskeleton distribution of cancer cells on the composite nanofibers (Figure 4a, Figure S3-S5 in SI). After 60 min incubation, the four types of cancer cells on all nanofibers still displayed spherical shape and some amount of filopodia are spread out around the cellular bodies. However, the amount and shapes of the filopodia of the same cells on each samples are different. It seemed that the captured cells on the 270 nm PDA NSs-loaded composite nanofibers displayed more filopodia, leading a better attachment with composite nanofibers,

14


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which is also agreed with the capture efficiency shown in Figure 3. To directly visualize the interaction between cells and composite nanofibers, PANC-1 cells captured on the nanofiber samples was further observed by FE-SEM (Figure 4b). The spherical cellular bodies with many filopodia, which is agreed with CLSM results (Figure 4a), were found to attach closely with the composite nanofibers by anchoring onto the PDA NSs encapsulated in the nanofiber matrix through the filopodia. These results indicate that instead of a weak interaction like electrostatic adsorption, there is an enough strong interaction between the captured cells and the nanofibers, which are consistent with the previous reports about topographical interaction between cancer cells and nanostructures.

38, 41

From the SEM images (Figure 4b), we can further find

that the filopodia have a better attachment with the smaller PDA NSs, especially on the 270 nm PDA NSs contained composite nanofibers, which may be owing to that smaller PDA NSs lead to higher density of NSs in the individual fiber, resulting in more binding sites of the fibrous film. That is, the hierarchical nano-topography structure of the necklace-like nanofiber gives more contribution to the interaction between the cells and nanofibers, leading to an increase of the capture efficiency. In addition, the partially exposed surface of the PDA NSs embedded in the fiber matrix give some chemical signal for capturing CTCs, which has been confirmed that on the one hand the PDA can promotes the adsorption of serum proteins which are benefit to cell adhesion, due to the presence of some functional groups (e.g. hydroxyl groups, carboxyl groups, catechol groups and so on) on the PDA surface;

29-32

on the other

hand, the catechol groups on the PDA NSs surface possess a high binding affinity to 15



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diverse nucleophiles (e.g. amines, thiol, and imidazole), which can be anchored to peptides and proteins on cell surface.

42-45

Therefore, it can be concluded that the

combination of the hierarchical nano-topography structure and surface chemical signal of PDA promotes the capture of CTCs.

3.5. Cell viability assay.

The cytocompatibility of the composite nanofibers were also assessed. The alginate nanofibers and alginate planes were used as controls. After 1 h cell capture experiments, cells were cultured on different samples for a longer time (24 h or 48 h). Then at preset time points (0 h, 24 h, and 48 h), Live/Dead staining was performed and cells on the samples were observed by fluorescence microscope as shown in Figure S6-S9. We can find that after 24 h incubation, four types of cells all spread and attached well on the composite nanofibers or alginate nanofibers, and some cells proliferated after 48 h incubation. While cells on the alginate planes were still stay in spherical shape and without proliferation even after 48 h. The statistical results (Figure S10) from the fluorescence images (Live/Dead staining) showed that the viabilities of almost all cells captured on the composite nanofibers remained over 80%. These results indicate that these PDA NSs-loaded alginate nanofibers are cytocompatible.

3.6. The whole blood test.

To simulate a more realistic situation, we spiked the PANC-1 cells and A549 cells into human whole blood at 10, 50 and 100 cells/mL, respectively. After 1 h cell 16


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capture experiments, a three-color immunostaining method

39

was applied to

distinguish the captured cancer cells from the captured white blood cells (WBCs). As shown in Figure 5, the cancer cells of PANC-1 and A549 cells, (CK+/CD45-/DAPI+, 10 µm < cell sizes < 40 µm) can be easily identified from the WBCs (CK-/CD45+/DAPI+, cell sizes < 15 µm), indicating that the PDA NSs/alginate composite nanofibers, especially the one containing 270 nm PDA NSs, have a considerable potential in capturing rare circulating tumor cells from real human whole blood.

4. CONCLUSION

In summary, we have successfully developed a necklace-like composite nanofiber with hierarchical surface nano-topography and a specific surface chemistry signal via electrospinning for capturing circulating tumor cells. PDA nanoparticles are connected by nanofibers to form the hierarchical topography structure; the partially exposed PDA provides the chemical signal. The combination of the topography and chemical signal increases the cancer cell capture efficiency by offering more complex interface and more binding sites. Furthermore, the necklace-like composite nanofiber has good cyto-compatibility and considerable potential in capturing rare circulating tumor cells from real human whole blood. Therefore, this work provides a simple and effective strategy for capturing cancer cells and it has great potential for application in diagnosis of malignancies.

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ASSOCIATED CONTENT Supporting Information. Additional figures including Table S1 and Figure S1-S10 as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Average diameters of the fibrous part of different nanofibers; the size of PDA NSs versus the molar ratio of ammonia to dopamine hydrochloride; endothelial cells capture efficiency; confocal laser scanning microscopy images of HepG2, A549 and U251 cells; live/dead staining of captured HepG2, A549, U251 and PANC-1 cells; viability of four types of CTCs captured on different samples. AUTHOR INFORMATION Corresponding Author * Shaobing Zhou E-mail: [email protected]; [email protected] ORCID Shaobing Zhou: 0000-0002-6155-4010 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was partially supported by National Natural Science Foundation of China (Nos. 51373138, 21574105, 51703189, 51725303), Sichuan Province Youth Science and Technology Innovation Team (Grant No.2016TD0026), Construction Program for Innovative Research Team of University in Sichuan Province (14TD0050) 18


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and the Fundamental Research Funds for the Central Universities (No. 2682017CX040).

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Moon, S.; Chung, H.; et al. Tissue Adhesive Catechol-Modified Hyaluronic Acid Hydrogel for Effective, Minimally Invasive Cell Therapy. Adv. Funct. Mater. 2015, 25, 3814-3824.

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FIGURES

Scheme 1. The schematic illustrations of the capture of CTCs by hierarchical electrospun PDA NSs / alginate composite nanofibers.

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Figure 1. The SEM images of polydopamine nanospheres (PDA NSs) with different sizes and corresponding diameter distribution.

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Figure 2. a) The SEM images of PDA NSs / alginate composite nanofibers with PDA NSs diameter ranging from 270 nm to 800 nm before and after the crosslinking of alginate. The optimal weight ratio of PDA NSs to nanofiber matrix was as following: 1:10 (270 nm), 1:6 (400 nm), 1:4 (650 nm), and 1:2 (800 nm). b) The typical TEM image of the PDA NSs / alginate composite nanofibers with PDA NSs diameter of 800 nm, and the corresponding EDX elemental mapping for carbon (C), nitrogen (N) and oxygen (O). c) The average height of protrusion of each group of composite nanofibers.

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Figure 3. Evaluation of cancer cells capture efficiency on different samples for different culture time, including a) HepG2 cells, b) A549 cells, c) U251 cells and d) PANC-1 cells.

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Figure 4. The topographical interaction between captured PANC-1 cells and different samples. a) The confocal laser scanning microscopy images of captured PANC-1 cells in 60 min. The nucleus (blue) and the actin (green) of captured cells were stained with DAPI and FITC labeled phalloidin. b) The SEM images of the captured PANC-1 cells in 60 min.

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Figure 5. Three-color immunocytochemistry method for distinguishing the spiked PANC-1 cells and A549 cells from white blood cells (WBCs) in real blood based on Alexa Fluor 647-labbeled anti-CK (marker for epithelial cells), Alexa Fluor 488-labeled anti-CD45 (marker for WBCs), and DAPI (stain DNA content of the nucleus). PANC-1 cells or A549 cells were spiked into the whole blood at 20 cells / mL respectively. Then the cell capture experiments were performed on the 270 nm PDA NS-loaded nanofibers and the incubation time is 60 min.

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TOC Figure

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1130x396mm (96 x 96 DPI)


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Capturing Circulating Tumor Cells through a Combination of

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