Multifunctional Nanofibers for Specific Purification and Release of

During cell capture at 37 °C, the polymer coated surface appears to be hydrophobic, which will promote highly efficient cell capture including CTCs b...
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Multifunctional Nanofibers for Specific Purification and Release of CTCs Zhili Wang, Na Sun, Min Liu, Yi Cao, Kewei Wang, Jine Wang, and Renjun Pei ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00048 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 7, 2017

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Multifunctional Nanofibers for Specific Purification and Release of CTCs Zhili Wang†, Na Sun†, Min Liu, Yi Cao, Kewei Wang, Jine Wang* and Renjun Pei* CAS Key Laboratory for Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, China School of Nano Technology and Nano Bionics, University of Science and Technology of China, Hefei 230026, China Email: [email protected], [email protected] † These authors contributed equally to this work Keywords: purification, chitosan nanofiber, CTCs, non-destructive release, thermo-responsive ABSTRACT: Recovering pure and viable circulating tumor cells (CTCs) from blood has been still a challenging task for the following molecular characterization and functional analysis, which has attracted wide attention these days. Herein, we fabricate a thermo-responsive chitosan nanofiber substrate to effectively capture, purify and release the target cancer cells, assisting by PNIPAAm brushes and DNA hybridization. The PNIPAAm brushes are designed to enable WBCs to detach from aptamer-PNIPAAm-chitosan-nanofibers (aptamer-P-CNFs) surfaces during the conformational transition. Meanwhile these specific captured CTCs are retained at a high purity. Moreover, effective and intact release of CTCs from the substrates without any foreign agents is realized by complementary sequences efficiently hybridizing with aptamers, and the specific cell release makes CTCs further purified. The present work provides a new strategy in the design of bio-interface for recovering target CTCs from whole blood samples with high purity.

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In the last decade, series of nanostructured materials have been successfully incorporated to CTC detection devices to increase capture efficiency,1 such as silicon nanopillars/nanowires,2-5 graphene oxide,6-11

nanoparticles,12-15

nanofibers,16-19

nanotubes,20

fractal

structures21-22

and

nanorough-featured surfaces23-24. However, it has been reported that the enhanced topographical interaction between cells and nanostructures could increase nonspecific adhesion from blood cells, especially from WBCs, which results in a decrease in capture selectivity of CTCs.25 To improve the selectivity and purity of CTC capture, some interesting attempts have been made including bio-interface design to hinder nonspecific cell adhesion8 and in-situ culture of the captured CTCs to weed out blood cells.14 Nevertheless, recovering viable and dissociated CTCs is still a challenge. The existing techniques such as enzymatic cleavage of DNA26-27 and electrical release28-29 are able to achieve effective CTC release, while extensive foreign agents are concurrently introduced into CTC samples, or it may lead to a reduction in the viability of released CTCs. Recently, another technique based on thermal responsive polymers has attracted attention on cell capture and release.19, 30-32 Poly(N-isopropylacrylamide) (PNIPAAm) is well known for its sharp conformational transition from expanded coil to compact globule as the temperature is changed around its lower critical solution temperature (LCST).33 So far, PNIPAAm and its derivatives have been used in the fabrication of temperature-responsive interfaces, nanofibers and hydrogels for cell adhesion and detachment, and the response has been proved to be fast and effective. For example, the thermal-triggered wettability change generated by PNIPAAm has been reported to achieve reversibly capture and release combined with a hydrophobic anchor.30 Moreover, direct regulation from PNIPAAm brushes to induce surface hydrophobic-to-hybrophilic switch was employed to capture and release CTCs.2, 31 The realization of cell release from thermal-responsive interface is likely due to a force of upward thrust resulting from the conformational transition, pushing cells off the polymer brushes. Unfortunately, thousands of blood cells trapped on PNIPAAm coated surface are also released simultaneously34 during the sharp conformational transition, which leads to a decrease in CTC purity. We are interested in the upward thrust from the conformational transition of PNIPAAm

brushes

and

wondering

whether

it

could

be

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regulated

to

differentiate

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adhesion/detachment behaviors between blood cells and target cells answering to the thermal response. Therefore, it is crucial to confirm a certain reaction time for PNIPAAm, with an appropriate force of upward thrust from the polymer brushes, to detach these blood cells trapped nonspecifically on the surface but not the target CTCs captured by specific affinity binding, which could help a great deal to achieve CTC samples with high purity.

Figure 1. Schematic illustration of thermoresponsive chitosan nanofibers for cell capture, purification and release assisted by PNIPAAm brushes and aptamers.

As reported, nanofibers could well mimic extracellular matrix to provide a matchable nanostructure for high efficiency CTC capture16, 18. In our recent work, we studied cell capture/release performance on chitosan nanofibers grafted with antifouling molecule and cell-capture agent, on which cancer cell morphology could be fine-controlled.35 Herein, we report a thermo-responsive PNIPAAm coated chitosan nanofiber substrate that can capture, purify and release targeted cancer cells, as shown in Figure 1. The idea is to fabricate an exactly suitable PNIPAAm brush layer by atom transfer radical polymerization (ATRP) to provide a regulatable upward thrust during its conformational transition, and poly methacrylic acid (PMAA) was then introduced as an anchor to provide functional groups for the immobilization of capture agent (an aptamer selected against EpCAM36). During cell capture at 37oC, the polymer coated surface exhibits to be hydrophobic which will promote highly efficient cell capture including CTCs by aptamer affinity and blood cells by hydrophobic interaction. When the temperature is reduced to 20oC, the regulated polymer brushes elongate under its conformational transition, and the blood cells without constraint from capture ACS Paragon Plus Environment

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agents are detached from the hydrophilic surfaces, enabling the purification of captured CTCs. Moreover, the elongated polymer brushes provide adequate acting space for complementary sequences (CS) to efficiently hybridize with the aptamers, leading to an effective and intact release of CTCs from the interface without any foreign agents. The cell release based on DNA hybridization is also specific, which makes the released CTCs further purified. RESULTS AND DISCUSSION Capture, purification and release performance on chitosan nanofibers

Figure 2. a) Synthetic approach employed to the ATRP and carbodiimide chemistry to covalently graft DNA-aptamers onto chitosan nanofibers for specific capture, purification and release of target cells. b) SEM images of chitosan nanofibers by electrospinning.

To achieve specific capture, purification and release of target cells, chitosan nanofibers were modified by PNIPAAm brushes and DNA aptamers, as shown in Figure 2a). Chitosan nanofibers with a diameter of 225±50 nm were fabricated by electrospinning, exhibiting excellent transparent performance in water (Figure 2b)). They were treated with NH3•H2O-methanol solution before any modification to stabilize the structure of nanofibers. The initiator for ATRP was grafted through covalent amide linkage onto chitosan nanofibers, and surface initiated ATRP was carried out with N-isopropylacrylamide to yield polymer brushes. To introduce DNA aptamers in the bio-interface for ACS Paragon Plus Environment

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specific cell capture, PMAA was grafted onto Br-terminated PNIPAAm brushes in another ATRP to provide multivalent functional groups (-COOH). Finally, effective immobilization of NH2-aptamers was realized via simple EDC/NHS coupling chemistry under mild aqueous conditions at 4oC with unfolding PNIPAAm brushes. The morphologies of fresh-electrospining chitosan nanofibers, water treated chitosan nanofibers, PNIPAAm grafted chitosan nanofibers after ATRP and aptamer-P-CNFs after cell capture tests were shown in Figure S1.

Figure 3. a) Quantitative evaluations of cell capture/purify performance on aptamer-P-CNFs with different polymerization time of NIPAAm. b) Quantitative evaluation of capture and purify specificity on aptamer-P-CNFs using EpCAM-positive cancer-cell lines of MCF-7, EpCAM-negative cancer-cell lines of CCRF-CEM and human white blood cells. c) Influence of incubation time on cell-capture/release efficiency of aptamer-P-CNFs. (MCF-7 cells) d) The viability of released cells observed for MCF-7 cell capture/release studies using aptamer-P-CNFs. The original cells were directly harvested from a cell culture flask. (e) Images of live and dead cells. The cells were treated with a mixture of calcein AM (green: live) and PI (red: dead) using a live/dead cell staining kit. f) Comparison of MCF-7 cell capture/purify and release on three control substrates in parallel: (i) chitosan nanofibers(CNFs), (ii) P-CNFs, and (iii) aptamer-P-CNFs.

To ensure the temperature sensitivity of PNIPAAm brushes, the graft chain length of PMAA was controlled here by its molar ratio to NIPAAm and reaction time. More importantly, to realize differentiated detachment behaviors between blood cells and target cells to the thermal response, it is absolutely essential to retain the validity of CTC capture during the conformational transition. Therefore, we performed series of cell-capture/purify experiments on aptamer-PNIPAAm modified chitosan nanofibers (aptamer-P-CNFs) to verify a suitable length of PNIPAAm brushes, which was ACS Paragon Plus Environment

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standardized here by polymerization time of NIPAAm, for the following purifying treatment. The aptamer-P-CNFs substrates were incubated with 1 mL MCF-7 cell suspension (105cells mL−1, pre-stained with DiI) at 37°C for 30 min, and washed 3-5 times gently by PBS at 37°C to weed cells unbound off. Then, the substrates were transferred to 20°C for purifying treatment with an incubation of 20 min, being washed 3-5 times gently. The results were summarized in Figure 3a), Figure S4. We can find that the capture efficiency of target cells decreased with prolonging of polymerization time, especially at 4 h. It was probably due to the embedding of active site (-Br) for the following polymerization of MAA when the length of PNIPAAm brushes was too long, leading to a decrease in grafting density of aptamers. On the other hand, it was shown that the discrepance of capture efficiency between pre-purify (capture) and post-purify (after purification) increased when the polymerization time of NIPAAm prolonged from 1 h to 6 h, which indicated that more captured cells were forced to deviate from the interface during purifying treatment with the lengthening of PNIPAAm brushes. The primal number of captured cells on aptamer-P-CNFs was up to 457±32 cells/mm2 at 1 h of NIPAAm polymerization time, and cells of 441±36 cells/mm2 were still on after purifying treatment, indicating little influence on the specific capture from conformational transition of PNIPAAm then. As a result, it is an appropriate polymerization time of 1 h for NIPAAm to ensure the validity of CTC capture during purifying treatment. To validate the differentiated capture as well as detachment behaviors of non-target cells from CTCs to the thermal response, the EpCAM-negative cancer-cell lines (CCRF-CEM cells) and freshly isolated human white blood cells (WBCs) were used to test the capture and purification performance, as shown in Figure 3b, Figure S5. There were approximately 173±17 cells/mm2 of CCRF-CEM and 111±8 cells/mm2 of WBCs on aptamer-P-CNFs after capture with an incubation time of 30 min at 37°C, resulting from the hydrophobic interaction between cells and PNIPAAm. Moreover, extremely low capture efficiency, only 4 cells/mm2 (0.8±0.2%) and 1.6 cells/mm2 (0.25±0.1%) of CCRF-CEM and WBCs respectively, was achieved after purification at 20°C, which demonstrated an excellent purifying effectiveness of aptamer-P-CNFs for non-target cells through the conformational transition of PNIPAAm. The aptamer-P-CNFs substrate is of satisfactory ability to capture CTCs with high ACS Paragon Plus Environment

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efficiency and detach non-target blood cells from the interface assisted by aptamers and PNIPAAm. To obtain effective release with maximum capture yield, we then examined the effect of incubation time on cell capture efficiency after purification and cell-release performance on aptamer-P-CNFs surfaces (Figure 3c, Figure S6). Cell release tests were performed by a co-incubation with complementary strands at 4°C for 20 min. It was shown clearly that captured cells after purification nearly reached saturation at 30 min of incubation, with a capture efficiency of 88.2±7.2%. Over 95% of captured cells could be released when the incubation time was no more than 30 min, and the release efficiency tended to decrease at 45 min, due to the increasing non-specific interaction between captured cells and aptamer-P-CNFs. The results demonstrated that high efficient cell-capture and effective cell-release using complementary strands from the aptamer-P-CNFs surfaces could be achieved with an incubation time of 30 min for capture. A live/dead cell assay was employed to evaluate the viability of recovered cells released from the aptamer-P-CNFs surfaces. The image of live/dead staining was shown in Figure 3d,e, and we can see little difference of the viability between original cells and released cells. The percentage of viable cells was 99%±1% and 95%±3% in original group and release group, respectively.

To validate the effect of PNIPAAm brushes on non-specific cell adhesion as well as aptamers on specific capture and release, cell experiments were performed on three kinds of CNFs substrates: (i) bare CNFs: chitosan nanofibers without any modification; (ii) P-CNFs: chitosan nanofibers grafting with PNIPAAm brushes; and (iii) aptamer-P-CNFs: chitosan nanofibers modified with PNIPAAm brushes and aptamers. The results were summarized in Figure 3f, Figure S7. Compared with bare CNFs, the captured cells on P-CNFs could be detached through conformational transition at 20°C. The phenomenon confirmed the purification effect from PNIPAAm brushes for non-specific cell adhesion. Meanwhile, the captured cells with a high efficiency on aptamer-P-CNFs after purifying treatment at 20°C, and then were specific released by a complementary strand. It demonstrated the effectiveness of the introduction of aptamers, which successfully induced specific cell binding to PNIPAAm brushes on CNFs surfaces. ACS Paragon Plus Environment

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Specific capture and release of mixture samples on aptamer-P-CNFs surfaces

Figure 4. a) Cell capture and release of MCF-7 cells mixtured with pre-stained WBCs at a constant ratio of 1:1 on aptamer-P-CNFs substrates by respectively spiking 105 cells/mL. b) Fluorescence images of MCF-7 cells (red) and WBCs (green) on aptamer-P-CNFs surfaces after they were captured, purified and released.

To further evaluate the specificity of cell capture and release on the aptamer-P-CNFs surfaces, a mixture sample of MCF-7 cells (prestained with DiI) and human white blood cells (WBCs, pre-stained by DiO) were used for a specific cell-capture contrast experiment at a constant ratio of 1:1 by spiking 105 MCF-7 cells, as shown in Figure 4. A considerable capture efficiency of 94% of MCF-7 cells was obtained at 37°C, and a high purity of captured MCF-7 cells was achieved after purification at 20°C with 91% of capture efficiency for MCF-7 and 0.26% for WBCs. Furthermore, 90.2% of spiked MCF-7 cells, which means 99.1% for captured MCF-7 cells, and only 0.08% of original WBCs were finally obtained after the co-incubation with complementary strands at 4oC, in an increased purity of 99.9% on target MCF-7 cells. The results demonstrated that high capture efficiency and purity of target cells can be achieved on aptamer-P-CNFs surface with the cooperation of PNIPAAm and DNA aptamer, and that the specific release was realized by a complementary strand which also provided a further purification for target CTCs. ACS Paragon Plus Environment

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Capture sensitivity of rare number CTCs in WBCs samples

Figure 5. a) The capture performance of mixture samples with different spiked MCF-7 cell numbers in 106 WBCs. b) Fluorescence images of a mixture sample with 200 MCF-7 cells before and after purification on the aptamer-P-CNFs substrates. The green fluorescence labeled WBCs and red fluorescence labeled MCF-7 cells.

To evaluate capture purity and capture sensitivity from blood cells on the aptamer-P-CNFs surfaces, a series of studies were performed using artificial cell mixture samples by spiking 50, 100, 200 MCF-7 cells (pre-stained by DiI dye) into 1 ml WBCs solution (106 cells/ml, pre-stained by DiO), respectively. The simplified blood system will also help to understand the effect on target cell detection from WBCs. The results summarized in Figure 5a showed that 61%-86% of target cells could be captured from cell mixture samples, while less than 0.06-0.08% of WBCs were trapped on the substrates. As shown in Figure 5b, the aptamer-P-CNFs surfaces exhibited excellent capture purity of target cells. Isolation of rare CTCs in fresh whole blood samples

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Figure 6. a) Cell capture and release efficiencies at different target cell number in blood samples on aptamer-P-CNFs. The statistics b) and fluorescence c) of WBCs after purification on aptamer-P-CNFs ( Prestained MCF-7 cells and trapped WBCs were stained by DAPI after purification ).

Finally, the isolation efficiency of whole blood samples was studied to explore the potential clinical applications of the aptamer-P-CNFs substrates. To mimic clinical blood samples of cancer patients, 10, 50, 100 and 1000 MCF-7 cells (prestained by DIi, red) were spiked in 1 mL human whole blood, respectively. 50%-70% of MCF-7 cells were detected from the whole blood samples, and over 90% of detected cells could be successfully released from the aptamer-P-CNFs substrates. For example, when 10 MCF-7 cells were spiked into 1 mL whole blood, 7 cells were detected on the aptamer-P-CNFs surface, and 6 cells were finally recovered, as shown in Figure 6a. There were about 4200 WBCs captured on the aptamer-P-CNFs substrates after purification, and less than 2000 WBCs were released into the obtained MCF-7 cell samples. The results demonstrated that the aptamer-P-CNFs surfaces also exhibited excellent detection purity of target cells, which was shown in Figure 6b. CONCLUSION ACS Paragon Plus Environment

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In summary, we fabricated a thermo-responsive chitosan nanofiber substrate that can capture, purify and release target cancer cells with the assisting of PNIPAAm brushes and DNA hybridization. The influence of PNIPAAm brushes was investigated, and an appropriate length of the brushes was confirmed to ensure high efficient capture of target CTCs and to enable WBCs detaching from the aptamer-P-CNFs surfaces through the conformational transition, resulting in the purification of captured CTCs. Moreover, effective and intact release of CTCs was achieved by complementary sequences efficiently hybridizing with aptamers without any foreign agents, and the specific cell release makes CTCs further purified. The present work provides a new strategy in the design of bio-interface for recovering targeted CTCs with high purity from patient blood. EXPERIMENTAL SECTION Materials：Chitosan ( Mw=190-310 kDa) with a degree of deacetylation of 85%, Poly(ethylene oxide) (PEO, Mw=1000 kDa), Copper(I) bromide (CuBr, 98%), α-Bromoisobutyryl bromide (BIBB, 98%), 1-Ethyl-3-(3’-dimethylaminopropyl) 2,2’-Bipyridyl,

carbodiimide

(EDC),

3-3’-Dioctadecyloxa-carbocyanine

N-Hydroxysuccinimide perchlor

(DiO),

(NHS), and

1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlo-rate (DiI) were purchased from Sigma-Aldrich. Methacrylic acid (MAA) and N-Isopropylacrylamide (NIPAM, 99%) were purchased from TCI (Shanghai) Development Co., Ltd. Pentamethyldiethylenetriamine (PMDETA) was obtained from Energy Chemical (Shanghai, China). Aptamer 5’-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3’ was purchased from Sangon Biotech Co., Ltd. (Shanghai, China), which was 5’-modified with amino groups and HPLC-purified by the manufacturer. Methanol, dichloromethane, triethylamine, acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. Preparation of Chitosan Nanofibers (CNFs): Chitosan/PEO solution (Chitosan: PEO=9:1) was prepared at 3 wt% concentration in 90 wt% aqueous acetic acid under magnetic stirring for 8 h at room temperature. The spinneret needle was maintained at a high electric potential by a high voltage power supply (DW-N303-1AC D8, Dongwen High Voltage Supplier, Tianjin, China) for ACS Paragon Plus Environment

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electrospinning, and an aluminum plate was used as the collector. The capillary spinneret needle was connected to a plastic syringe filled with a spinning solution. The syringe used in the experiment had 5# or 7# needle and a DC voltage of 14-18 kV was applied between the syringe tip and aluminum foil. Chitosan nanofibers were deposited on the glass slides which were placed onto the aluminum foil, and the nanofiber density on glasses were controlled by the spinning time. The electrospun samples were dried under vacuum at 60oC overnight to remove acetic acid, and then were cut into 1 cm× 1 cm or 1 cm× 2 cm. Synthesis of PNIPAAm Modified Chitosan Nanofibers (P-CNFs): A solution of triethylamine (1.0 mL, 7.2 mmol) in dichloromethane was prepared, and the glasses coated with CNFs were immersed in the solution. Then, α-Bromoisobutyryl bromide (0.91 mL, 7.2 mmol) was added dropwise at 0 oC. The CNFs was left in solution for overnight at room temperature and then was washed with dichloromethane and water, followed by drying using compressed nitrogen. Next, the CNFs substrates and CuBr (42 mg, 0.3 mmol) were added to a 50 mL Schlenk tube, which was then sealed, evacuated and back-filled with Ar for three times. NIPAM (1.0 g, 8.84 mmol) and PMDETA (185 µL, 0.9 mmol) were dissolved in 20 mL methanol/H2O (50 to 50) solvent and purged with argon for 30 min. Finally, the monomer solution was transferred into the tube via a syringe and the reaction was kept at 30oC for 1 h, and then immersed in DI water overnight. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) (IR Prestige-21, Shimadzu) was used to confirm the presence of grafted PNIPAAm on the CNFs (Figure S2). Preparation of PMAA-Modified P-CNFs (PMAA-P-CNFs): Briefly, MAAS (0.88 mmol), CuBr (10 mg), and bpy (23 mg) were added into 15 mL water. The reaction mixture was sonicated for 2 min and degassed with high pure argon for 30 min. The P-CNFs substrates were put into the Schlenk tube and the tube was then thoroughly purged with argon. The reaction solution was injected into the tube by a syringe. The polymerization was terminated after 30 min, and the PMAA-P-CNFs substrates were then immersed in DI water overnight. The terminal-initiation, Br-terminated PNIPAAm chains as macromolecular initiators for polymerization of MAA was reported ACS Paragon Plus Environment

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previously.37 Cell experiments: Before cell experiment, the PMAA-P-CNFs substrates (1 cm× 1 cm) were treated with EDC (0.1 M) and NHS (0.1 M). Then, the 5’-amino-modified DNA aptamer (1 ×10−6 M in 1 × PBS) was placed onto the substrate and incubated at room temperature overnight before washing with PBS. The substrates were placed into a 24-well plate, and 1 mL of cell suspensions (105 cells/mL) was loaded. After incubation for 15-60 min at 37oC, 5% CO2, the substrates were gently washed with PBS (37oC) 3-5 times and 1 mL 2.5% glutaraldehyde solution in 1 × PBS was injected in to fix the captured cells. Subsequently, we imaged and counted cells using the fluorescence microscope. For the purification experiment of non-specific cells, the substrates (1 cm× 2 cm) were transferred to a 4-well Lab-TekTM Chamber Slide (1 cm × 2 cm well−1) to count the captured cell totally, and 1 mL cell suspensions (105 MCF-7 cells/mL and WBCs of 105 cells/mL freshly isolated from healthy blood) were loaded. After incubation, the substrates were put at 20oC for 20 min and then gently washed with PBS (20oC) 3-5 times. The artificial CTC-containing samples were prepared by spiking prestained MCF-7 cells into 1 mL whole blood or white blood cell suspension (106 cells/mL) freshly isolated from healthy blood, with the final MCF-7 cell concentration of 50, 100 and 200 cells/mL respectively, which were tested also in a 4-well Lab-TekTM Chamber Slide. SUPPORTING INFORMATION Supporting Information Available: The following files are available free of charge: SEM images, ATR-FTIR spectrums, Fluorescence images. ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21575154), the Science Foundation of Jiangsu Province (No. BE2016680), the Jiangsu Province Six Talent Peaks program and the CAS/SAFEA International Innovation Teams program. Z. W. and N.S. contributed equally to this work.

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The table of contents A thermo-responsive chitosan nanofiber substrate is fabricated here to capture, purify and release CTCs assisted by PNIPAAm and DNA hybridization. The influence of PNIPAAm brushes is investigated to ensure specific capture of CTCs during conformational transition, and enable WBCs detaching from the aptamer-P-CNFs surfaces. Moreover, specific release of CTCs is achieved by complementary sequences, which makes CTCs further purified.

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