Folic Acid-Functionalized Hybrid Photonic ... - ACS Publications

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Biological and Medical Applications of Materials and Interfaces

Folic Acid-Functionalized Hybrid Photonic Barcodes for Capture and Release of Circulating Tumor Cells Chengxin Luan, Huan Wang, Qi Han, Xiaoyan Ma, Dagan Zhang, Yueshuang Xu, Baoan Chen, Minli Li, and Yuanjin Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06882 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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ACS Applied Materials & Interfaces

Folic Acid-Functionalized Hybrid Photonic Barcodes for Capture and Release of Circulating Tumor Cells Chengxin Luan1, Huan Wang2, Qi Han1, Xiaoyan Ma1, Dagan Zhang2, Yueshuang Xu1, Baoan Chen1,*,Minli Li2*, Yuanjin Zhao1,2* 1

Department of Hematology and Oncology (Key Department of Jiangsu Medicine), Zhongda

Hospital, School of Medicine, Southeast University, Nanjing 210009, China. 2

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China. * Corresponding authors: Southeast University, Nanjing 210096, China. E-mail addresses: [email protected] (B.A.C); [email protected] (M. L.L); [email protected] (Y.J.Z)

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： Abstract：

Recovery of circulating tumor cells (CTCs) from cancer patients by an efficient CTCs capture and release method can greatly increase their application in diagnostics and treatment of cancers. In this paper, we presented a folic acid (FA)-functionalized hybrid photonic barcode for capture and release of CTCs. The hybrid photonic barcodes were formed by two nano-ordered parts, poly (ethylene glycol) diacrylate (PEGDA) inverse opal structure for sustaining integrity and methacrylated gelatin (GelMA) gel filler for conjugating FA molecules to mediate cell capture. The nano-ordered structures of the hybrid photonic barcodes not only increased contact area, but also decreased steric hindrance among FA molecules. Thus, the topographic interaction between the barcodes and CTCs was greatly enhanced. In addition, GelMA gel was soft and extracellular matrix (ECM) alike, which was beneficial to decrease impairment to CTCs during the CTCsbarcode interaction as well as preserving their viability. Demonstrated by four CTCs types, Hela (epithelial tissue, folate receptor positive, FR+), A02 (bone marrow, FR+), Raji (lymphoid tissue, FR+) and A549 (epithelial tissue, folate receptor negative, FR-), FR+ CTCs could be captured efficiently with reliability and specificity. The captured cells could be controllably released with high viability just by quick trypsinization. The whole processes were simple and efficient. These features indicated that the FA-functionalized hybrid photonic barcodes were promising for full recovery of CTCs from cancer patients, which was important for diagnosis and treatment of cancer.

Keywords: folate receptor; inverse opal; hydrogel; circulating tumor cells; tumor


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1. Introduction Cancer is a leading cause of death around the world, whose prevention needs an early, efficient, accurate and patient-friendly detection method.1-3 Circulating tumor cells (CTCs) have recently emerged as a liquid biopsy of tumors promising for early detection, diagnosis, staging, curative effect evaluation, progression prediction and prognosis evaluation in various cancers.4 CTCs fall off from their primary tumor into circulation and travel to different sites at early stage, which contributes to cancer metastasis. Therefore, measuring CTCs from peripheral blood can provide more direct and precise tumor information with less invasive procedures and less rate of false positives than other methods.

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Due to their extremely low abundance, about one in 109

hematologic cells, it is a tremendously challenge to separate and measure them.

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The current

methods are various and the primary method is to utilize capture molecules such as aptamer to certain types of CTCs, antibody against epithelial cell adhesion molecule (EpCAM) or cytokeratin immobilized on certain carriers to separate and analyze them. Up to now, the only product on the market approved by the U.S. food and drug administration (FDA) for detecting ®

CTCs is CellSearch (Veridex, Raritan, NJ), which uses antibody of EpCAM.4,7,9-15 However, besides complex procedures, these capture molecules are expensive and they may lose efficacy because of possible epithelial-to-mesenchymal transition of CTCs during metastasis process.11,16,17 In addition, these capture molecules may only be effective in certain types of tumor with defined genomic profiles or show limit affinity to CTCs.18,19 Therefore, other selective capture molecules and the platform that can easily capture, release and measure CTCs at early stage are still needed. 20 Recently, folate receptors (FR-α, β, γ), which are required for uptaking of folate and commonly overexpressed in cancer cells while expressed at low to negligible levels in most


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normal tissues, have been utilized for many targeting applications such as cancer imaging, CTCs detection and tumor targeting therapy because they are targeting ligands of folic acid (FA) with high affinity. 18,19,21,22 For CTCs analysis methods, carriers are generally needed, which includes glass wafer, nanometer materials such as nano-substrates and silicon dioxide nano pillar, hydrogel etc.6,7,23,24 Although with many advantages, the cell-friendly detection and noninvasive recovery of CTCs by these carriers may be challenging due to their limit biocompatibility, which is essential to comprehensive and accurate analysis of the CTCs for cancer diagnosis and treatment.9,25 In addition, these methods may be expensive and complex to fabricate. As an alternative, inverse opal structured photonic barcodes with good biocompatibility, high surfaceto-volume ratio due to their homogeneous nanostructure, easy fabrication and inexpensiveness have been increasingly studied as carriers in various applications such as biomolecule detection, intelligent actuator, microorganism and cell capture.26-33 Thus, we herein presented FA-functionalized hybrid photonic barcodes for capture and release of CTCs with good cell recovery. The hybrid photonic barcodes were composed by two nanoorderd building blocks, poly (ethylene glycol) diacrylate (PEGDA) and methacrylated gelatin (GelMA) gel. PEGDA gel with their good mechanical strength kept the stability and integrity of the inverse opal structure during the capture and release processes. GelMA with their abundant amino group could provide sufficient binding sites for FA coupling and their soft texture could decrease impairment to CTCs during the CTCs-barcode interaction.34 The GelMA blocks were spaced by the PEGDA blocks uniformly and formed a particular nano topography, which not only increased contact area, but also decreased steric hindrance among FA molecules, therefore, the topographic interaction between the barcodes and CTCs was greatly enhanced. The process to immobilize FA onto the barcodes was very simple, just by one step, the amide interaction


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between the carboxyl group of FA and amino group of GelMA. The capture efficiency was benefit from surface characteristic of the barcode, the high affinity between FR and FA. More attractively, ferroferric oxide magnetic nanoparticles (FOMNPs) endowed the photonic barcodes with response to magnetic field, which could greatly facilitate separating process after cell capture. The release process was also simple and efficient, just by trypsinization within a short time (Fig.1). It was demonstrated that three different tissue sourced FR positive (FR+) CTCs, Hela (epithelial tissue), A02 (bone marrow) and Raji (lymphoid tissue) could be captured efficiently with good sensitivity, reliability and specificity from normal cells or FR negative (FR) CTCs (A549, epithelial tissue). The captured cells could be controllably released with high viability. These features make the FA-functionalized hybrid photonic barcodes promising for capturing, detecting, and releasing CTCs, which could be helpful to early detection, diagnosis and treatment of cancer.

Figure 1. Schematic illustration of the capture and release of CTCs by FA-functionalized hybrid photonic barcode. (a) FA modification. (b) CTCs capture process. (c) CTCs release process.


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2. Experimental section 2.1. Materials SiO2 nanoparticles (Nanjing Nanorainbow Biotechnology Co., Ltd.); Ferroferric oxide magnetic nanoparticles (FOMNPs) (Nanjing Nanoeast Biotech co., Ltd.); GelMA hydrogel was synthesized from gelatin; Gelatin and methacrylic anhydride (Sigma-Aldrich, St. Louis, MO); Dimethyl sulfoxide (DMSO), N-Hydroxysuccinimide (NHS), Poly (ethylene glycol) diacrylate (PEGDA) with molecular weights of 700, 2-hydroxy-2-methylpropiophenone (HMPP) photo initiator (Sigma-Aldrich, Shanghai, China); 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide (EDC) (Alfa Aesar Chemicals Co., Ltd., China); Hydrofluoric acid (HF) (Sinopharm Chemical Reagent Co., Ltd.); Human cell line A02 (Tianjin Hematonosis Hospital); Raji cell line (Shanghai Cell Bank of Chinese Academy of Sciences); Hela and A549 cell lines (ATCC, American type Culture Association); The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or roswell Park Memorial Institute 1640 medium(RPMI-1640)(Hyclone, Utah, USA) supplemented with 10% Fetal bovine serum (FBS) (Biological Industries, Israel); Phosphate buffer (PBS, 0.01 and 0.1 M), 0.25% Trypsin-EDTA(Gibco, Canada); PBS Tween-20 (PBST, 0.05% Tween-20 in 0.1 M PBS) was self-prepared; Penicillin-streptomycin (Beyotime Biotechnology, China); Cell Counting Kit (CCK-8) (Dojindo, China); Folic acid (FA) (Aladdin, Shanghai, China); Fluorescein isothiocyanate-polyethylene glycol2000-FA(FITC-PEG2000-FA) (Ponsure Biotechnology, Shanghai, China); FITC linked polyclonal rabbit antibody to FA (Cloud Clone Corp, USA); Ficoll 400 (tbdscience , Tianjin, China); Centrifuge tube, 96 and 6-well plates and cell culture flasks (Coring, China); Water used in all experiments was purified by a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA). 2.2. Fabrication of the hybrid photonic barcode


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Silica colloidal crystal beads (SCCBs) with reflection peaks at 560 nm were generated by selfassembly of SiO2 nanoparticles using a T-shaped microfluidic device as previously reported.26 The SCCBs were used as sacrificed templates to generate inverse opal barcodes from the first pregel solution with 80% PEGDA, 19% water and 1% HMPP. Briefly, the hydrophilic treated and dried SCCBs were put into the first pregel solution for 3 hours, during which the pregel solution could fully penetrated the voids between the silica nanoparticles of the SCCBs through capillary attraction. After polymerization by ultraviolet light, the hybrid barcodes were stripped from the outside hydrogel. Then, the inverse opal barcodes were harvested after removing the template SCCBs by HF for 24 hours. After hydrophilic treated and dried, the inverse opal barcodes were filled by the second pregel solution with 0.08gGelMA, 320 µL water, 10 µL FOMNPs and 4 µL HMPP. The hybrid photonic barcodes were lastly harvested after polymerization and stripping. 2.3. FA modification. The linkage between the GelMA and FA occurred within EDC/NHS/DMSO solution, with FA, EDC and NHS following about 1:2:3 mol/L ratio. Briefly, 30 mg FA was fully dissolved in 5 mL DMSO at 37°C for 2 hours. Then 30 mg EDC and 50 mg NHS were added with sufficient mixing. After incubation within the solution at 37°C for 12 hours, the hybrid photonic barcodes were modified with FA by the amide interaction between the carboxyl group of FA and amino group of GelMA. Lastly, the FA modified hybrid photonic barcodes were collected and washed 2 times with 0.1 M PBS buffer. To verify the FA modification, two methods were utilized. For the first one, the FA modified hybrid photonic barcodes and non FA modified hybrid photonic barcodes were dried shielded from light and pestled separately to test infrared spectrum by Fourier transform infrared


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spectroscopy. For the second one, a FITC linked polyclonal rabbit antibody to FA was incubated with the FA modified hybrid photonic barcodes and non FA modified hybrid photonic barcodes for 3 hours. After washing by PBST and 0.1 M PBS three times respectively, they were observed by fluorescence microscopy. 2.4. Cell capture assay and its capture ability exploration To verify the cell capture ability of the FA modified hybrid photonic barcodes, FR high expression cell line, Hela cell was cultured in DMEM supplemented with 10% FBS. Immediately before experiments, Hela cells were digested, medium changed and diluted to cell suspension with concentration of 1×106 cells/mL. The capture experiments were performed using a 96-well plate and divided into two groups, the FA modified hybrid photonic barcodes were the experimental group and the non FA modified hybrid photonic barcodes were used as control group, 20 barcodes of the experimental group and the control group were placed into the wells of 96-well plate respectively. Every well was pipetted with 150 µL cell suspension and put in an incubator for 3 hours, during which the well was gently shaken every 30 min. Afterwards, the suspension was sucked out and the barcodes were carefully washed with 0.01 M PBS three times. The barcodes were inspected by inverted microscope and the cells captured by the barcodes were estimated with help of a fine needle to turn over the barcodes. The barcodes were also carefully transferred to a cell-culture dish for imaging by confocal laser scanning microscope. 2.5. Optimum capture time exploration To figure out the optimal capture time for the four cell lines, Hela and A549 were cultured in DMEM supplemented with 10% FBS, A02 and Raji were cultured in RPMI-1640 supplemented with 10% FBS. Similar to section 2.4, the cells were digested, medium changed and diluted to


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cell suspension with concentration of 1×106 cells/mL. 20 FA modified hybrid photonic barcodes were placed into the wells of 96-well plate and 150 µL cell suspension of the each four cell lines was pipetted into the wells respectively. Then the wells were put in an incubator and observed by inverted microscope every 15 min respectively. The cells captured by the barcodes were estimated with help of a fine needle to turn over the barcodes. 2.6. FR expression screening of the four cell lines. The four cells were rinsed, medium changed, diluted to cell suspension and pipetted into a 6well plate. Then same amount of FITC-PEG2000-FA was added to the four cells respectively and incubated at incubator for 30 min, with or without the presence of free FA as blocking reagent. The cells were then washed and analyzed by flow cytometry, FITC-PEG2000-FA was associated with the FL1-H gate. 2.7. Cell release and viability characterization. After CTCs capture, the FA modified hybrid photonic barcodes were washed gently by 0.01 M PBS and gathered by a magnet. 0.25% Trypsin-EDTA was used to release the captured cells. To find out the proper trypsinization time, the FA modified hybrid photonic barcodes were trypsinized 20 s, 30 s and 40 s respectively, and inspected by confocal laser scanning microscope. To test cell viability of the obtained cells after capture and release processes, we performed viability studies by cell counting kit-8 assay (CCK-8 assay). Briefly, after totally release of the captured cells, the released cells were re-suspended by culture medium and cell counted. The corresponding original cells were suspended with the same count same to the released cells, as control group. Then they were performed CCK-8 assay at 60, 90, 120, 150, 180, 210 min respectively.


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2.8. Cell capture and post-staining in clinical tumor model In a typical experiment using Hela cells as example, 10 mL healthy donor’s blood was R blood collection tube, mononuclear cells were enriched and red collected by BD Vacutainer○

blood cells were removed by Ficoll 400. Then the mononuclear cells were washed by 0.01 M PBS two times and re-suspended by 4 ml culture medium, counted and divided into two equal groups, 2 mL respectively. After that, Hela cells were pipetted into one group to reach about 1000 CTCs/mL, and the other one as control group. Then the cell capture process was carried out for 90 min as previously described with 10 FA modified hybrid photonic barcodes in each well. After capture process, the cells captured by the barcodes were estimated with help of a fine needle to turn over the barcodes observed under inverted microscope. Then the capture efficiency was calculated by the following equation: Capture efficiency = NC/NT × 100%. Where NC is the average number of cells captured by the barcodes; NT is the total number of cells in each well. After that FITC-PEG2000-FA was used to stain the cells for 30 min. Then the barcodes were carefully washed with 0.01 M PBS three times and gathered by a magnet. Finally the barcodes were observed by confocal laser scanning microscope.

3. Results and discussion 3.1. Fabrication of the hybrid photonic barcode The inverse opal barcodes were made of PEGDA gel by replicating silica colloidal crystal beads (SCCBs) templates, which was showed in Fig.2 a-c. The dried SCCBs with their closely packed and nano ordered structure through themselves formed nanochannels, which were ready for infiltration of the pregel solution by capillary force. After polymerization by ultraviolet light, stripping and HF etching, inverse opal barcodes were obtained. The polymerized PEGDA


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hydrogel was firm and stable, which could keep stability and integrity of the inverse opal structure during the capture and release processes. Similarly, the inverse opal barcodes were dried, infiltrated by GelMA-FOMNPs pregel, polymerized and stripped, then the hybrid photonic barcodes were obtained (Fig. 2 d). Compared with PEGDA gel, GelMA gel was softer, which could greatly decrease impairment to CTCs during the CTCs-barcodes interaction. Abundant amino groups of GelMA gel could provide sufficient functional groups for FA modification through amide interaction. The scanning electron microscope (SEM) images (Fig. 2 e-g) showed the structures of SCCBs, the inverse opal barcodes and the hybrid photonic barcodes respectively. The hexagonal alignment of the nanoparticles on the surface of the SCCBs was convex (Fig. 2 e) and the inverse opal barcodes was concave (Fig. 2 f). After the concave void of the inverse opal barcodes was filled by GelMA gel, a particular nano topography of the hybrid photonic barcode was composed by PEGDA and GelMA gel (Fig. 2 g). The GelMA blocks were spaced uniformly by the PEGDA blocks at nano scale, which not only increased contact area, but also decreased steric hindrance among FA molecules. Therefore, the topographic interaction between the barcodes and CTCs was greatly enhanced. Their nano structure was also confirmed by their vivid barcode color, which showed red shift compared with SCCBs (Fig.3 a-c). The hybrid photonic barcode also possessed magnetic response endowed by FOMNPs, which shown in Fig.3 d, e. In addition, the diameter of the hybrid photonic barcode was about 334 µm, which was easy to manipulate during the CTCs capture and release process. The magnetic response feature could greatly facilitate barcodes gathering process, simply realized by an external magnetic field.


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Figure 2. The fabrication process of hybrid photonic barcode and SEM images of related structures. (a-d) The fabrication processes of hybrid photonic barcode:(a) SCCB, (b) hybrid SCCB, (c) inverse opal barcode, (d) hybrid photonic barcode. (e-g) SEM images of related structures: (e) the surface of a SCCB, (f) the surface of an inverse opal barcode, (g) the surface of a hybrid photonic barcode. The scale bars are 200 nm.

Figure 3. Reflection images of the related barcodes (a-c) and magnetic response feature of the hybrid photonic barcodes (d, e). (a) template SCCBs, (b) inverse opal barcodes,(c) hybrid


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photonic barcode. (d-e) The hybrid photonic barcodes with external magnetic field (d) and without external magnetic field (e). The scale bars are 200 µm. 3.2. FA modification and characterization To characterize the FA on the hybrid photonic barcodes, the infrared spectrum detection and antibody antigen reaction were carried out. After activation by EDC/NHS/DMSO, the carboxyl group of FA was activated and could react with amino group of GelMA, thus amide bond formed, which could be affirmed by infrared spectrum.

As shown in Fig.S1, after FA

conjugation, there was an extra absorption peak at wave number around 1540 compared with non FA modified barcodes. To further confirm the FA modification of the hybrid photonic barcodes, a FITC linked polyclonal rabbit antibody to FA was utilized. As shown in Fig.S2, the fluorescence intensity of the FA modified hybrid photonic barcodes was much stronger than the non FA modified hybrid photonic barcodes. GelMA gel is a gelatin derivative with abundant amino groups, and the nano structure of the hybrid photonic barcodes rendered the amino groups more easily to react with carboxyl groups of FA molecules. 3.3. Cell capture assay and optimum capture time exploration To verify the cell capture ability of the FA modified hybrid photonic barcodes, Hela cells were used. The Hela cells have high FRα expression so that they could be captured by immobilized FA of the hybrid photonic barcodes. In Fig.4, Hela cells were captured by the FA modified hybrid photonic barcodes but not by the non FA modified hybrid photonic barcodes, which indicated that Hela cells were captured by the FA modified on the hybrid photonic barcodes instead of the PEGDA-GelMA gel. Thus, the capture ability of the FA modified hybrid photonic barcodes was confirmed.


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The optimum capture time must be figured out to achieve high CTCs-capture efficiency by the FA modified hybrid photonic barcodes (Fig.S3). In this study, we explored the optimum capture time of four kinds of CTCs, Hela cells, A02 cells, Raji cells and A549 cells with different FR expression level, respectively. With the increasing of incubation time, the number of captured cells initially increased, then reached a plateau at 75 min for Hela cells, 90 min for A02 cells and 105 min for Raji cells. While, the number of captured A549 cells remained very low, just roughly equal to the cell density of the cell suspension even at 120 min. This result indicated that the FA modified hybrid photonic barcodes captured FR+ CTCs selectively; The FR types or FR density of Hela, A02 and Raji cells were different, however, the three types cell lines, no matter from epithelial tissue (Hela), bone marrow (A02) or lymphoid tissue (Raji), could be captured by the FA modified hybrid photonic barcode. This could be ascribed to 1) The strong affinity between FA and FR. 2) Particular nano topography composed by PEGDA and GelMA gel of the hybrid photonic barcode could not only increase contact area, but also decrease steric hindrance among FA molecules, therefore, the topographic interaction between the barcodes and CTCs was greatly enhanced. 3) The radial diffusion of the barcode particles and the target cells in solution led to high flexibility and fast reaction kinetics.


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Figure 4. Confocal laser scanning microscope images of Hela cells captured by the hybrid photonic barcodes. (a) with FA modification and (b) without FA modification. The scale bars are 200 µm. 3.4. The characterization of FR function of the four cell lines To verify the function of FR in the four types of cell lines, the cells were incubated with FITCPEG2000-FA and flow cytometry was carried out. As shown in Fig.5(a-d), Hela cells exhibited high affinity for FITC-PEG2000-FA, A02 and Raji cells showed medium and mild affinity, while A549 cells showed extremely low affinity. We also studied the effect of competitive substrate to the affinity. The free FA molecules were used as a competitive blocking reagent and added into the incubation solution with FITC-PEG2000-FA simultaneously. The results in Fig.5 (e-h) indicated that the amount of FITC-PEG2000-FA bonding with the cells significantly decreased when the free FA existed. These results indicated that the bonding behavior of FITCPEG2000-FA to the cells was specific and FR dependent. Though the FR expressions level of A02 and Raji were not very high, they could still be captured by FA modified hybrid photonic barcode, which could be ascribed to the structure advantages of the barcodes, discussed by section 3.3.


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Figure 5. Flow cytometry analyses of the function of FR in the four types of cell lines by FITCPEG2000-FA as FL1-H gate. (a-d) Without free FA. (e-h)With free FA. 3.5. Cell release and viability characterization. After CTCs capture, the capture cells were released by trypsinization, which is the most used way to break down the bonds between the cells and cultivate interface for its simpleness, efficiency as well as controllability. As shown in Fig.S4, after trypsinized about 40s, the cells were totally separated from the FA modified hybrid photonic barcodes (using Hela cells as example). The trypsinization time was less than the time required by general cell passage time, which indicated it was safe to the captured CTCs. To test the cell viability of the obtained cells after capture and release processes, viability studies by cell counting kit-8 assay (CCK-8 assay) was carried out. As shown in Fig.S5, the cell viability of the three types of released FR+ CTCs emulated their corresponding original cells after 180 min, which suggested that the released cells had good recovery capacity and viability. This might because that the cell-barcodes interaction was mediated by GelMA gel, which was


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cell friendly because it was soft and similar to ECM. Moreover, the less trypsinization time compared with general cell passage time also decreased the impairment to the CTCs. 3.6. Application in clinical tumor model To explore the clinical utility of the FA modified hybrid photonic barcodes, clinical tumor model was built to carry out cell capture studies. The clinical tumor model was prepared by mixing cancer cells into healthy donor’s blood and using healthy donor’s blood without cancer cells as control group. After the capture process, the cells captured by the barcodes were estimated and the capture efficiency was calculated, which exceeded 85%. Then the cells were stained with FITC-PEG2000-FA and inspected by confocal laser scanning microscope. As shown in Fig.6 using Hela cells as example, after incubated in the group mixed with Hela cells, the barcodes captured cells and stained by the fluorescent dye, while the control group was negative. This result indicated that the FA modified hybrid photonic barcodes could selectively capture the FR+ cells in complex environment, which showed potential in clinical application.


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Figure 6. Clinical utility of the FA modified hybrid photonic barcodes. (a,c) Confocal laser scanning microscopy images of the FA modified hybrid photonic barcodes incubated in the healthy donor’s blood with Hela cells at bright field (a) and fluorescent field (c). (b,d) Confocal laser scanning microscopy images of the FA modified hybrid photonic barcodes incubated in the healthy donor’s blood without Hela cells at bright field (b) and fluorescent field (d). The scale bars are 200 µm.

4. Conclusion In conclusion, we developed a novel barcode with FA modification for capture and release of CTCs from a complex tumor model. The barcode was formed by two nano-ordered parts, PEGDA inverse opal structure for sustaining integrity and GelMA gel filler as functional part. GelMA gel was similar to ECM and was soft, which was beneficial to interact with cells as well as preserving their viability. GelMA gel also had abundant amino groups to conjugate FA molecules for the FA-FR mediated cell capture. After photo polymerization, PEGDA gel was stable enough to keep the integrity of the hybrid barcodes in complex testing environment. The particular nano structure could greatly enhance the topographic interaction between the barcodes and CTCs because the structure not only increased contact area, but also decreased steric hindrance among FA molecules. These features made the barcode ideal for capture and release of FR+ CTCs with simple processes, specificity, controllability and high efficiency, from epithelial tissue or bone marrow or lymphoid tissue, even from complex clinical tumor model. Though the FA-functionalized hybrid photonic barcodes based method in this paper is only suited to FR+ CTCs, it still has great practical values because most of cancer cells are FR overexpressed. Moreover, after necessary corollary equipment is developed to replace manual operation and


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further adjustments for clinical samples, its potential will be further unlocked. Therefore, the FAfunctionalized hybrid photonic barcode was worth to be further developed and a promising tool for diagnosis and treatment of cancer.

Supporting Information Infrared spectrum images to verify FA modification; fluorescent images of a FITC linked polyclonal rabbit antibody to FA for verification of FA modification; optimization of the CTCs capture time by the FA modified hybrid photonic barcodes for four cell lines; images of cell release by trypsinization; viability characterization of the obtained cells after capture and release processes by CCK-8 assay compared with the corresponding original cells as control group.

Author Contributions C.L. carried out the experiments, analyzed the data and wrote the paper; Y. Z. conceived the idea, designed the experiment and revised the paper; H.W., Q.H., X.M., D.Z., and Y.X. helped carry out the experiments, analyze data and write the paper; B.C. and M.L. contributed to scientific discussion of the article.

Acknowledgement This work was supported by the National Science Foundation of China (Grant Nos. 21473029 and 51522302), the Key Medical Projects of Jiangsu Province (Grant No. BL2014078), Key Discipline of Jiangsu Province (2016-2020), and the Scientific Research Foundation of Southeast University.


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Disclosure The authors declare no conflicts of interest in this work.

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