Cell-Capture and Release Platform Based on Peptide-Aptamer

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A Cell-Capture and Release Platform Based on Peptide-Aptamer-modified Nanowires Jingying Li, Cui Qi, Zheng Lian, Qiusen Han, Xinhuan Wang, Shuangfei Cai, Rong Yang, and Chen Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09407 • Publication Date (Web): 08 Jan 2016 Downloaded from http://pubs.acs.org on January 15, 2016

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ACS Applied Materials & Interfaces

A Cell-Capture and Release Platform Based on Peptide-Aptamer-modified Nanowires Jingying Li,[-] Cui Qi,[-] Zheng Lian, Qiusen Han, Xinhuan Wang, Shuangfei Cai, Rong Yang,* Chen Wang* CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China

* Corresponding Author: [email protected]; [email protected] [-]

These authors are equally contributed.

ABSTRACT

Nanowires have been attracted much attention due to their potential

bio-applications, such as delivery of drugs or sensing devices. Here we report the development of a unique cell-capture and release platform based on nanowires. The combination of nanowires, surface-binding peptides, and cell-targeting aptamers leads to specific and efficient capture of cancer cells. Moreover, the binding processes are reversible, which is not only useful for downstream analysis but also for reusability of the substrate. Our work provides a new method in the design of the cell-capture and release platform, which may open up new opportunities of developing cell-separation and diagnosis systems based on cell-capture techniques. KEYWORDS: peptides, aptamers, GaN, nanowires, cell-capture

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INTRODUCTION During recent years, nanowires (NWs) have obtained much attention for their potential bio-applications.1-11 Semiconductor NWs have been widely used in bio-devices due to their high-aspect-ratio, high rigidity and excellent electro-optical properties. Controllable immobilization of recognition molecules to devices’ surface is important for the specificity and efficiency of cell-capture. For conjugation of targeting molecules with device surfaces, two kinds of methods have been reported: covalent

12-15

and non-convalent.16,17 For covalent approach,

Wang et al. introduced streptavidin onto the substrates by N-hydroxysuccinimide (NHS)/maleimide chemistry, and then biotinylated targeting molecules such as antibodies or aptamers were linked onto the surfaces through biotin–strepavidin interactions;

12

Chen et al. reported aptamers could be attached on the SiNWAs

surface by click chemistry;

14

And Tan et al. announced aptamers could be modified

on particles by thiol chemistry.

15

Non-covalent functionalization is facilitated by

coating the substrates with bio-molecules (for example, avidin or bovine serum albumin) via physical adsorption;16,17 then, biotinylated probes were connected to surfaces. In addition to these functionalization methods, an alternative approach based on engineered peptides which are biologically friendly has been developed. Inspired from Nature, peptides that have a strong affinity to a particular surface have been obtained by combinatorial biology.

17-27

These solid surface-binding peptides have

unique properties: (i) They contain short sequences of amino acids and can be flexibly designed for multi-functionality; (ii) They can specifically bind to the desired places


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of multi-component surfaces; (iii) They can work in conditions of biological friendliness and desirable for various biomedical applications. Compared with covalent methods, affinity-based peptide conjugation is easier to use since it does not need special and extensive chemical reactions. Using these engineered peptides as molecular linkers to functionalize biomedical devices is still largely unexplored. Here, integrating an engineered gallium nitride (GaN) affinitive peptide P118-20 with tumor cell specific binding DNA aptamer S2.228,29 and nanowire surfaces, we construct a unique sensing platform that can efficiently recognize and capture cancer cells. The S2.2 aptamer is a 25-base oligonucleotide that has high affinity and specificity to mucin 1 (MUC1) protein.28,29 MUC1 protein is a transmembrane glycoprotein of the

mucin

family.

MUC1

is

overexpressed

in

most

adenocarcinomas.

Adenocarcinoma is an important type of cancers in the glands of the body, including breast, prostate, lung and ovarian cancer, etc. MUC1 is a good target for cancer-therapy. 28-31 GaN is one of the well-known III-V semiconductors. It has excellent optical and electrical properties,32-35 good chemical stability, and biocompatibility.36-38 GaN-based biosensors have been used for detection of antibodies of different cancers,

39,40

action

potentials of cardiac cells, 41 and sodium flux of neuronal cells ,42 etc. In previous work we reported that GaN nanowires could be used to regulate protein-adsorption and cell-adhesion by topography modification and UV- light irradiation.43-45 Here combining the GaN nanowires with GaN affinitive peptide P1 and the MUC1 aptamer S2.2, we achieved great enhancement of the recognition of the surface to



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MUC1-positive cancer cells. Our work provides a new method in the design of the cell-capture and release platform, which has great potential in cancer-cell separation and diagnosis.

RESULTS AND DISCUSSION The multifunctional 3D nanostructured GaN platform for cell capture was prepared as shown in Figure 1. Firstly, we synthesized GaN nanowires using chemical vapor deposition method.

43-45

After that, we introduced a GaN binding peptide

biotin-P1 onto the surfaces. Subsequently, biotinylated aptamer S2.2 is connected to bio-P1 through strepavidin. Thus, combining the P1-S2.2 conjugate and NW surface, we obtained a platform which can recognize and capture MUC1-positive cancer cells. Secondly, the captured cells can be released from the GaN surface by DNAase. The released-cells show excellent viability and they can be further cultured for biochemical analysis. Thirdly, the peptide P1 could desorb from the surface after NaCl solution treatment.

20

The reversibility of the binding of the peptide with the

surface makes the substrate reusable. Finally, the functionalized surface can be regenerated by repeating the above process.


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Figure 1. Illustration of the peptide-aptamer modified nanowire surfaces for capture and release of targeted cancer cells. The efficient capture of cancer cells can be achieved on the GaN surfaces by integrating the specific cancer-cell recognition agents, the surface binding peptides and the nanowire surfaces. The surfaces can be regenerated.

The morphology of the GaN NW surfaces was examined using scanning electron microscopy (SEM). A typical SEM image (Figure 2A) exhibits a network of NWs with diameters around 50 nm on the whole substrate. For comparison, Figure 2B shows the flat GaN surface prepared by metal-organic chemical vapor deposition (MOCVD). Larger scale SEM images of these two surfaces were shown in Figure S1 (Supporting Information). The successful conjugation of peptide P1 and aptamer S2.2 was confirmed by XPS spectra (Figure S2, Supporting Information), water contact angle measurement (Figure S3) and Raman spectra (Figure S4). To study the performance of cell-capture on the GaN surfaces, the MCF-7 cells (MUC1-positive cell line) have been incubated on the GaN NW and flat surfaces for 45 min, respectively. Figure 2C,2D showed the fluorescence microscopy images of MCF-7 cells captured on these two surfaces. One can see that after modification with



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dual-functional biomolecules, the GaN NW surface could capture much more tumor cells than flat surface. Since the 3D GaN NW surfaces consisted of a network of NWs which have high surface areas, the NW surface could adsorb much more binding biomolecules and result in the enhancement of cell-capture efficiency. Insets of Figure 2C, 2D were cartons of cell-capture and detailed confocal microscope images of cells. More images were shown in Figure S5 (Supporting Information). We also compared cell-capture experiments with and without serum on P1-S2.2-modified GaN NW surfaces. The results were shown in Figure S6 (Supporting Information). The serum affected the numbers of MCF-7 cells captured on the surface a little bit, but not much.

Figure 2. ( A) and (B) are SEM images of the GaN nanowire and flat surface; (C) and (D) are Fluorescence microscope images of captured MCF-7 cells on bio-functionalized GaN nanowire and flat surface, respectively. Insets are cartons of cell-capture and detailed confocal microscope images of cells (actin stained by FITC-phalloidin, nucleus stained by DAPI).


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To further show how our designed platform can be regenerated, we studied the adsorption of peptide P1 on GaN NW surfaces (Figure 3). Strepavidin conjugated dynabeads were used to monitor the adsorption of peptide biotin-P1 on the substrate. From the optical microscopy images in Figure 3, one can see that there are lots of dynabeads bind to the surface, which indicates that the peptide P1 has a high binding affinity for the GaN surface.18,19 After 1M NaCl treatment,20 almost no dynabeads left on the surface. The changed numbers of beads indicated the adsorption and desorption of peptide P1 on the surface. The process could be repeated as shown in the middle of section of Figure 3 and the functionalized surface can be regenerated.

Figure 3. Optical microscopy images of cycles of adsorption and desorption of bio-P1 peptide on the GaN NW surfaces monitored by the changed numbers of strepavidin-dynabeads. The scale bar is 50 µm.

To monitor the adsorption of aptamer S2.2, fluorescein isothiocyanate (FITC) molecules were conjugated to one side of bio-S2.2. Figure 4A and 4B are optical



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microscopy images of bio-S2.2 conjugated to the bio-P1 modified GaN NW surface through strepavidin before and after NaCl treatment. The strong fluorescence intensity on Figure 4A indicates a higher level adsorption of aptamer S2.2. The adsorbed aptamer S2.2 will act as targeting molecules to recognize and capture MUC1-positive cancer cells. After treatment of NaCl solution, the loss of bindings between biotin-P1 and GaN surfaces causes desorption of aptamer S2.2 from the surfaces, indicated by the reduced fluorescence intensity (Figure 4B). To verify the importance of peptide P1 in the cell-capture processes, we studied cell-capture experiments on GaN NW surfaces with and without biotin-P1. As shown in Figure 4C, the numbers of cells captured with biotin-P1 is 16-fold more than that without biotin-P1. This is because there are much higher adsorption of aptamer S2.2 with biotin-P1 than that without biotin-P1 (Figure 4D).

Figure 4. (A) and (B): The optical microscopy images of P1-S2.2 conjugates absorbed on GaN NW surfaces before and after NaCl treatment. (C) Comparison of capture of MCF-7 cells with/without biotin-P1. (D) Comparison of adsorption of aptamer S2.2 with/without biotin-P1.


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To optimize the cell-capture conditions, we examined both of the effects of S2.2 aptamer concentration and MCF7-cell incubation time on the cell-capture performance. The results are shown in Figure S7. After that we studied the specificity of the designed GaN platform. For comparison, we use a MUC1-positive cell line (MCF7 breast-cancer cell) and a MUC1-negative cancer cell line (Ramos cell) to do the cell-capture experiments. We designed a parallel experiment with four different GaN surfaces: (1) P1-S2.2 modified NW surface; (2) P1-Random DNA modified NW surface; (3) P1-S2.2 modified flat surface; (4) P1-Random DNA modified flat surface. The results suggest that combining bioconjugate-modification and the NWs can achieve high cell-capture efficiency as shown in Figure 5. We also compared the number of captured cells between A549 cells (another MUC1-positive cancer-cell line) and Jurkat cells (another MUC1-negative cancer-cell line) using P1-S2.2 functionalized GaN NW surfaces. The results also showed that our designed surface could specifically recognize the A549 cells (Figure S8, Supporting Information).



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Figure 5. Densities of captured MCF-7 cells (positive) and Ramos cells (negative) on different GaN substrates (Navy: P1-S2.2 modified NW surface; red: P1-Random DNA modified NW surface; Cyan: P1-S2.2 modified flat surface; Magenta: P1-Random DNA modified flat surface).

To further demonstrate the ability of the dual-functional P1-S2.2 modified GaN NW surface to recognize and isolate targeting cancer cells, we used a mixture cell suspension of MCF-7 cells (red) and Ramos cells (green) to do the cell-capture experiment. In our study, 1 ml mixed cell suspension having the same number of MCF-7 cells and Ramos cells (105 cells/ml) was prepared as shown in Figure 6A. And then the mixed cells were seeded on the GaN NW surface. After incubation for 30 min, MCF-7 cells were captured selectively on the GaN surface (Figure 6B). The ratio of MCF-7 cells in the captured cells is higher than 90% (Figure 6C). The results indicate that the GaN cell-captured platform is capable of selectively recognize, isolate and capture the targeting tumor cells from the mixture of different cells. One can further use this platform to capture targeting cancer cells from whole blood samples. For example, Zhang et al

46

developed a cell–capture system combining

horizontal TiO2 nanofibers with epithelial cell adhesion molecule antibody to isolate and detect circulating tumor cells (CTC). They successfully captured cancer cells from the whole blood samples of colorectal cancer patients as well as gastric cancer patients.


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Figure 6. (A) and (B) are confocal fluorescent images of a mixed cell suspension containing 1:1 MCF-7 cells (red) and Ramos cells (green) before and after selectively captured by peptide-aptamer modified GaN NW substrate. (C) Statistics of each cell ratio.

Controlled cell-release without damage is important for subsequent cell-culture or single-cell analysis. In our study, enzymolysis of S2.2 aptamers was taken place by DNAase treatment. After incubation with MCF-7 cells for 30 min, the GaN NW surface captured a large number of MCF-7 cells. The time dependent effect of enzymolysis of S2.2 aptamers was recorded by fluorescence microscope (Figure S9, Supporting Information). The released cells could be further cultured. As shown in Figure 7, when 80 U/ml DNAase was applied for 40 min, about 98% of the captured MCF7 cells (Figure 7A) were released from the surface. We collected the released MCF-7 cells and cultured for several days. The cells for the third generation (after 36 h) still have excellent viability as shown in Figure 7C, which provides great potential



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for further molecular and cell analysis. Insets of Figure 7C, 7D were confocal microscope images of MCF-7 cells. More fluorescence and confocal microscopy images of the cells that released and then cultured were shown in Figure S10, S11 (Supporting Information).

Figure 7. Fluorescence microscopy images of MCF-7 cells on GaN NW surfaces: (A) before and (B) after treatment of DNAase; (C) The cells cultured for the third generation after released from the surfaces. Insets are cartons and detailed confocal microscope images of cells (actin stained by FITC- phalloidin, nucleus stained by DAPI).


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CONCLUSIONS In summary, a dual-functional peptide-aptamer conjugate-modified nanowire platform for capture, isolation and release cells has been successfully constructed. The device surface was functionalized using an engineered peptide P1. The cell-capture efficiency was owing to the high surface area of GaN nanowires and the selectivity of P1-S2.2 bioconjugates with the MUC1 protein expressed on cell membrane. The functionalized nanowire substrate can also specifically isolated MCF-7 cells from a mixture of cells. Furthermore, the reversibility of the binding of peptide P1 with the GaN surface makes the substrate reusable. Moreover, the surface can efficiently release the captured cancel cells. The released cells show excellent viability and they can be further cultured for biochemical analysis. The approach of engineered peptide-functionalized nanowires with the tumor affinitive aptamers can be adapted for other substrates and capture different types of cells. Our work describe here supplies a general method to develop multi-functional systems of capture and release targeting cells, and will have potential applications such as cell-separation and detection.

MATERIALS AND METHODS Materials. Gallium oxide (99.9%) and gallium (99.99%) were purchased from Sigma Aldrich. Streptavidin was obtained from Heowns Biochem Technologies (USA). DNAase was purchased from Solarbio Science & Technology (Beijing) CO., Ltd. Acridine orange (AO) was provided by Sigma Aldrich; Cell Tracker Green



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CMFDA and Cell Tracker Orange CMTMR were purchased from Invitrogen. Dynabeads (M-280 Streptavidin) were supplied by Invitrogen. Biotinylated P1 peptide (bio-SVSVGMKPSPRP-NH2) was synthesized by GL Biochem (Shanghai) Ltd. All aptamers including biotin modification were synthesized by Sangon Biotech (Shanghai) Co., Ltd. S2.2 aptamer: biotin-5’-GCA GTT GAT CCT TTG GAT ACC CTG G-3’; Random DNA aptamer (CtrDNA): biotin-5’-TTT TCT TTT CTT TTC TTT TCT TTT C-3’.

Fabrication of GaN Nanowire Arrays. GaN nanowire surfaces were fabricated by CVD method. Briefly, the source materials (Ga2O3, Ga and graphite) were together put in a quartz boat which was placed in the middle of a furnace. The gold coated silicon substrate was annealed for 30 min and then placed downstream from the source. The ammonia (NH3) was the reaction gas and flew at 40 sccm. The N2 was the carrier gas. The furnace temperature was increased at 10℃/min and kept for 30 min after reaching 950℃. Then the furnace was cooled down to room temperature with N2 gas. GaN Surface Functionalization and Characterization. The GaN flat and nanowire surfaces were cleaned using acetone and ethanol, rinsed with deionized water and then dried with N2 gas. The samples were immersed into 0.1 mM peptide bio-P1 solution for 3 h. After washed three times by 0.1% phosphate buffered saline (PBS), the surfaces were treated with 20 µg/ml of streptavidin for 60 min, and then flushed with PBS solution to remove excess streptavidin. Finally, the substrate was


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treated with biotin-S2.2 aptamer or biotin-control DNA solution for another 1 h and then washed with PBS for 3 times. For the adsorption of aptamer S2.2 without biotin-P1, the GaN NW surfaces were directly treated with the FITC conjugated biotin-S2.2 aptamer solution for 1h and then washed with PBS for 3 times. The GaN-P1-S2.2 Surfaces were characterized with scanning electron microscopy (SEM, Hitachi S-4800), X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi), contact angle goniometry (DSA100) and Raman spectroscopy (Renishaw).

Cell Culture. MCF-7 cells (human breast adenocarcinoma) and A549 cells (lung epithelial cells) were cultured in DMEM medium (Hyclone) with 100 IU/ml penicillin, 100µg/ml streptomycin, 1% glutamine and supplemented with 10% (V/V) heat-inactivated calf serum (Gibco). Jurkat Clone E6-1 cells (human T lymphocyte) and Ramos cells (B-cell, human Burkitt’s lymphoma) were cultured in 1640 medium and IMEM medium (Hyclone) respectively, with 100 IU/ml penicillin, 100 µg/ml streptomycin, 1% glutamine (Glu) and with 10% (V/V) heat-inactivated calf serum (Gibco) , 1% Glu and supplemented with 10% (V/V) streptomycin.

Cell Capture and Release Experiments. First, MCF-7 and Ramos cells were stained by 10 µg/ml AO for 20 min. Second, the functionalized GaN surfaces were placed into a commercial 48-well cell-culture plates and then 0.5 ml cell suspension (105 cells/ml) was added. Third, after incubation at 37℃ and 5% CO2 for 45 min, the cells on GaN surfaces were gently washed with medium for 3 times and studied under



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fluorescent microscope. For cell-release experiments, the captured cells were released by treated with DNAase (80 U/ml) in PBS at 37℃ for 30 min. To further confirm the selectivity of S2.2 aptamer for MCF-7 cells, the MCF-7 and Ramos cells were stained by CMTMR and CMFDA respectively, then mixed in equal numbers and seeded on a functionalized GaN NW substrates for 30 min. After that, the GaN substrate was rinsed with medium for 3 times and studied by confocal fluorescence microscope (Carl Zeiss, Germany). After cell-capture process or cell-culture for three generations of the released MCF-7 cells, the samples were fixed with 4% paraformaldehyde for 20 min and washed several times using PBS. Then the samples were permeabilized with PBST (0.1% Triton X-100 in PBS) for 10 min, washed with PBS for 3 times. After that the fixed cells were stained for actin and nucleus. Actin was stained with phalloidin (PA) conjugated

with

FITC

(Sigma

Aldrich).

The

nucleus was

stained

with

4',6-diamidino-2-phenylindole (DAPI, Sigma Aldrich). The concentration for both PA and DAPI was 10 µg/ml. Cells were then imaged on the confocal fluorescence microscope.

ACKNOWLEDGMENTS We gratefully thank the National Natural Science Foundation of China (21261130090, 21405027, 21573050, 21073047, 21503053), Chinese Academy of Sciences (XDA09030303) for financial support.


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Supporting Information: More characterization of GaN NW surfaces, additional confocal microscopy and fluorescence microscopy images of the captured cells, and more cell-capture data. This material is available free of charge via the Internet at http://pubs.acs.org.

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A unique platform integrating the specific cancer-cell recognition aptamers, the surface binding peptides and the nanowire surfaces has been constructed to achieve the efficient capture of cancer cells.


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Cell-Capture and Release Platform Based on Peptide-Aptamer

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