Promoting Multivalent AntibodyâAntigen Interactions by Tethering

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Promoting Multivalent Antibody-Antigen Interactions by Tethering Antibody Molecules on a PEGylated Dendrimer-Supported Lipid Bilayer Po Ying Yeh, Yih-Ruey Chen, Chien-Fang Wang, and Ying-Chih Chang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01515 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Promoting Multivalent Antibody-Antigen Interactions by Tethering Antibody Molecules on a PEGylated Dendrimer-Supported Lipid Bilayer Po-Ying Yeh1,2, Yih-Ruey Chen1, Chien-Fang Wang1, Ying-Chih Chang1,2* 1

Genomics Research Center, Academia Sinica, 128, Sec 2, Academic Rd., Nankang, Taipei 115, Taiwan 2

Department of Chemical Engineering, Stanford University, Stanford, CA 94305

Keywords (4-6 words): dendrimer, supported lipid bilayer, microfluidics, circulating tumor cell, circulating tumor emboli

ACS Paragon Plus Environment

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Abstract

To efficiently isolate maximal quantity of circulating tumor cells (CTCs) and circulating tumor cell microembolis (CTMs) from patient blood by antibody coated microfluidics, a multifunctional, pegylated polyamidoamine-dendrimers conjugated supported lipid bilayer surface construct was proposed to enhance accessibility of antibody molecules to the antigen molecules on target CTCs. The combination of a hydrated, stretchable dendrimer and a laterally-mobile supported lipid bilayer (SLB) provide attached antibody molecules with 2.5-dimensional chain movement, achieving multivalency between the surface antibody and cell antigen molecules. An over 170% enhancement is distinctive for Panc-1 cells that expresses low antigen level. Of seven pancreatic ductal adenocarcinoma patients, an average 440 single CTCs and 90 CTMs were collected in 2mL peripheral blood, which were 1.6 times and 2.3 times more, than those captured by the SLB-only microfluidics. In summary, we have demonstrated a material design to enhance multivalent antibody-antigen interaction, which is useful for rare cell enrichment and cancer detection.


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1. Introduction Circulating tumor cells (CTCs) and its cell clusters can cause metastasis, a primary cause of cancer-related death for patients with solid-tumor cancers

1,2

. CTCs can be enumerated via non-

invasive liquid biopsy of blood and used as a biomarker to reveal the metastatic status of a patient. Analysis of blood samples has found a propensity for increased CTC counts as cancer progressed in individuals 3. The ability to monitor disease progression over time could facilitate appropriate modification to a patient's therapy, potentially improving their prognosis and quality of life. Owing to the importance of CTCs, various approaches have been exploited to isolate and capture CTCs from blood. It is technically challenging in that a large population of cells, such as red blood cells, platelets, white blood cells (WBCs), stem cells, and endothelial cells could all interfere with CTC capture, as CTC only represents a very small population, in the order of 10~5000 per million blood cells per mL 4, 5. To enrich CTCs from blood cells, one may utilize physical principles, such as filtration, centrifuge, or microfluidics devices to separate cells based on differential cell sizes, rigidity, density or surface charges

3,8-11

; or one may utilize biological principles, using antibody or

aptamer coated magnetic beads or microfluidics devices to positively or negatively select target CTCs

3, 10, 11

. We are particularly interested in developing CTC capture microfluidic platforms

based on biological principle in that potentially different antibody/aptamer coatings may target different subpopulation of CTCs, which would be useful for different clinical applications and discovery. Previously, antibody of epithelial cell antigen molecule (anti-EpCAM) was widely used to target EpCAM antigen that is widely expressed on epithelial tumor cells 6. Anti-vimentin was used to identify CTCs undergoing epithelial-mesenchymal transition 7. Antibody of human


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epidermal receptor 2 (HER2) was used to capture HER2 positive CTCs, which is therapeutically relevant in identifying breast cancer subtype 12. Anti-CD44 was used to capture CTC subset with stem cell signature 13. Conversely, cocktails of various antibody were designed to collect a more comprehensive CTC population

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. Regardless of the antibody selection, the design of antibody

interfacial molecular organization is pivotal in determining the capture performance. Conventionally, antibody molecules were conjugated on the surfaces of microfluidics or microbeads via covalent linkers, as a result, the total number of antibody-antigen pairs may be limited by the fixed number of antibody molecules per surface area underneath a cell (Scheme 1A). Polymer brushes or dendrimers as the long flexible linkers may circumvent the shortcomings of short chain linkers by promoting local, short-range antibody-antigen clustering 15

. We had previously developed an antibody conjugated supported lipid bilayer (SLB) coating

(Scheme 1B). The fluidic nature of SLB was utilized to manipulate the clustering state of the surface-bound ligands with respect to the specific cell location to promote antibody-antigen cluster. The antibody-SLB microfluidics has provided a strategy for capturing and purifying viable CTCs of colorectal cancer, pancreatic cancer, and prostate cancer. Its clinical utility has further proven as the quantity of CTCs collected by the antibody-SLB microfluidics is well correlated with the disease progression 16-19. Both dendrimer based, and SLB based coatings have respectively shown to be effective in capturing CTCs on microfluidics in comparison with the fixed density antibody coating. In this work, we propose a surface construct containing fully-hydrated, stretchable dendrimer on a laterally-mobile SLB. By conjugated antibody molecules on a dendrimer-SLB surface (Scheme 1C), antibody molecules may move not only on the SLB surface plane, but also in the vertical direction away from the surface, a so-called “2.5-dimensional” chain movement. We anticipate


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that this design could lead to an entropic favored spatial arrangement to accommodate more antibody-antigen pairs formation. The chemical composition and chain length of spacer arms, and the surface charge of dendrimer will be designed and synthesized to modulate the antibody surface density, non-fouling property, and antibody chain flexibility. To confirm our hypothesis and the feasibility of surface design and their resulting surface properties, quartz crystal microbalance with dissipation (QCM-D), zeta-potential, and atomic force microscopy were applied. The effects of these surface properties to the CTC isolation will be evaluated by integrating these surface constructs to the microfluidics for both control cell lines and clinical blood samples. Finally, to validate the effect of surface organization toward the capture sensitivity, three different cancer cell lines ranking from high to low antigen expression levels or different antigen density: colon carcinoma cells (HCT116, high EpCAM density), non-small cell lung cancer cells (H1975, medium EpCAM density), and human pancreatic carcinoma cells (Panc-1, low EpCAM density), were tested in buffer and blood conditions. Pancreatic adenocarcinoma (PDAC) patient samples were further tested to confirm its feasibility in applying to clinical usages.


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A.

B.

C.

Scheme 1. Schematic illustration of three surface design strategies for antibody immobilization and the hypothetical interaction with antigens of a target cell. Antibody molecules were coated (A) via fixed linkers, resulting in restricted accessibility to each cell antigen. (B) via lipid molecules in SLB with 2-dimensional lateral mobility, resulting in antibody-antigen clustering on SLB plane. (C) via long spacer arm dendrimer-SLB with both lateral and vertical mobility for entropic favored spatial arrangement, resulting in maximal antibody-antigen pair formation.


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2. Materials and Methods 2.1. Chemical Reagents 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-(cap-biotinyl) sodium salt (b-PE) dissolved in chloroform were purchased

from

Avanti

Polar

Lipids.

Texas

Red-1,2-dihexadecanoyl-sn-glycero-3-

phosphoethanolamine triethylammonium salt (TR-DHPE) dissolved in chloroform was purchased from Thermo Scientific. All three components were used without further purification. NeutrAvidinTM (NA) biotin-binding protein (Life Technologies) was used after being solubilized in a 10 mM phosphate buffered saline solution (PBS) containing 150 mM sodium chloride aqueous solution with pH adjusted to 7.4. Bovine serum albumin (BSA) (Sigma-Aldrich) was used for non-specific binding tests. Green 5-chloromethylfluorescein diacetate (CMFDA) cell tracker dyes were used for cell staining. Accutase® cell detachment solution was purchased from Millipore (SCR005). ProLong® antifade mountants with 40,6-diamidino-2-phenylindole (DAPI, Life Technologies) was used to mount the membrane with label cells. Vacutainer tubes (10 mL, BD Biosciences) coated with 18 mg of ethylenediaminetetraacetic acid (EDTA) were used for blood collection. The water used in all experiments was obtained from a Milli-Q RO system (Millipore) with a product resistivity of 18.2 MΩ. Generation-7 polyamidoamine dendrimer (PAMAM G7) dissolved in methanol (7.5 wt.%) was purchased (Sigma-Aldrich) and used without further purification. Succinic anhydride was purchased from Alfa-Aesar.

2.2. Biotinylation of EpCAM antibody and PAMAM dendrimer EpCAM antibody. Monoclonal anti-EpCAM, EpAb4-1, were prepared as described previously 20

. The antibodies were biotinylated (b-Ab) using sulfo-succinimidyl-6-(biotin-amido) hexanoate


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(sulfo-NHS-LC-biotin, Life Technologies)

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. Briefly, EpAb4-1 in PBS (0.65 mg/mL, as

determined by Nanodrop 1000 spectrophotometer (Thermo Scientific)), was prepared to react with 10 mM sulfo-NHS-LC-biotin (with molar ratio of 50, dissolved in Milli-Q DDI water) for 30 min at room temperature. Excess biotin was removed by dialysis in PBS for 24 h at 4 oC with a buffer change after 12 h. About ten biotin molecules were conjugated with one EpAb antibody as determined with a biotin quantitation kit (Life Technologies). PAMAM Dendrimer. Either ɑ-biotinyl-ω-NHS polyethylene glycol (NHS-PEG60-biotin, 3000 Da, RAPP Polymer, Figure 1A) or sulfo-NHS-LC-biotin (557 Da, spacer arm length 2.24 nm, Figure S1A) were conjugated the PAMAM G7 dendrimers (Sigma-Aldrich) (Figure 1B) for dendrimer biotinylation. The PAMAM solution of 0.15 g was dried in a vacuum chamber to remove methanol. For conjugating NHS-PEG60-biotin with PAMAM to form amine-capped DPEG60 (AD-PEG60, Figure 1C), the dried PAMAM was dissolved in 0.8 mL/0.2 mL DMF/DMSO and allowed to react overnight with 7 or 22 mM NHS-PEG60-biotin (molar ratio of 15 or 46). For conjugating sulfo-NHS-LC-biotin with PAMAM to form amine-capped D-C5 (AD-C5, Figure S1B), the dried PAMAM was dissolved with 0.8 mL double deionized water (DDI) and allowed to react with 0.2 mL sulfo-NHS-LC-biotin of 12 mM (molar ratio of 25) on ice for 4 h. In both cases, after biotinylation, excess biotin spacer arms were removed by dialysis in DDI for 24 h at 4 oC. The products were freeze dried overnight and then were reconstituted with 0.8 mL PBS. Subsequently, 20 mg of succinic anhydride dissolved in 0.2 mL PBS (molar ratio of 2000) was reacted with biotinylated PAMAM solution (0.8 mL) for 4 h to cap the amine with carboxyl functionalities. Excess succinic anhydride was removed by dialysis in PBS for 24 h at 4 oC to yield 1 mL of a 117 µM solution of the functionalized dendrimer (D-PEG60 or D-C5).


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Size and zeta potential measurements of PAMAM with or without succinic anhydride modification were determined by Zetasizer Nano ZS in pH 7.4 PBS.

2.3. Surface Preparation Lipid Vesicles. The compositions of lipid vesicles for SLB formation included 85 mol% POPC and 15 mol% b-PE. Fluorescent lipid vesicles included 84.5 mol% POPC, 15 mol% b-PE, and 0.5 mol% TR-DHPE. Chloroform solutions of lipids were mixed in clean glass tubes and the chloroform was gently removed under a slow stream of nitrogen and then completely removed overnight in a vacuum chamber. The dried POPC/b-PE was then hydrated with PBS buffer at a concentration of 3 mg/mL while being mixed vigorously, and then extruded through 100 nm, followed by 50 nm, Nuclepore track-etched polycarbonate membranes (Whatman) for a minimum of 10 times under 150 psi at room temp using a LIPEXTM Extruder (Northern Lipids, Inc.) for the homogenous population of unilamellar vesicles. The POPC/b-PE vesicle size of 65 ± 3 nm (n=5) was determined by Zetasizer Nano ZS in pH 7.4 PBS. Surface Construction. To construct an antibody coated SLB (Surface S), the following steps were used (Figure 2): First, lipid vesicles of 0.15 mg/mL at 50 oC were injected with the solid substrates (glasses, or PMMA) pre-treated with O2 plasma and incubated for 30 min to form a complete SLB coating. A rinse with PBS was used to remove excess vesicles and non-bonded lipids. Second, NA solution of 0.1 mg/mL was injected to incubate for ~1h at room temp followed by PBS rinse. The surface was treated with 0.05 mg/mL b-Ab for 1h at room temp, followed by PBS rinse. To construct an antibody coated carboxylated, PEGylated dendrimerSLB (Surface DS), the following steps were used (Figure 2): First, the SLB was coated and treated with NA as mentioned previously. Then the amine-capped dendrimer (AD-PEG60) could


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be allowed to incubate with the surfaces. Next, the remaining amine groups on the dendrimer surfaces were replaced with carboxyl groups by reacting with succinic anhydride (1 mg/mL in PBS). Following the PBS rinse, a second treatment with 0.1 mg/mL of NA was performed. Finally, b-Ab was conjugated with NA, followed by rinsing with PBS. As a comparison for the spacer effect, an antibody coated carboxylated, AD-C5 dendrimer-SLB surface (Surface DS-L) was prepared. The steps are similar with that of Surface DS, except that AD-C5 (Figure S2) was used instead of AD-PEG60.

2.4. Platform Preparation and Operation Platform Fabrication. The chaotic mixing designed previously developed CTC isolation microfluidics

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microfluidics was adapted by the

, except the plastic cover was injection

molded as shown in Figure S3. Operation using Syringe Pump. A syringe installed on a syringe pump (Harvard PHD 2000) was connected to the outlet of a microfluidics, the pump was operated in the withdraw mode. All reagents were added from the inlet of a microfluidics at a determined flow rate. To construct Surfaces S, DS, or DS-L on the inner surfaces of microfluidics, reagents of same concentration as mentioned previously were flowed into a microfluidics to allow stepwise conjugation for Platforms S, DS, or DS-L.

2.5 Evaluation of Binding Efficiency and Overall Efficiency Cell culture. HCT116 and Panc-1 cells seeded in culture dish were maintained and grown in Dulbecco’s modified Eagle medium (DMEM, Gibco-RBL life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AA, Gibco-RBL life


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Technologies), at 37 oC with 5% CO2 atmosphere in a humidified incubator. Similarly, H1975 cells were maintained and grown in RPMI1640 (Gibco-RBL life Technologies) supplemented with 10% FBS and 1% AA, at 37 oC with 5% CO2 atmosphere in a humidified incubator. Spiked method. Cultured cells were incubated with Accutase® at 37 oC for 5 min to detach cells from culture dish. The cell suspension was then mixed with cell culture medium to stop the dissociation activity. After centrifuged at 300 g for 5 min, the supernatant was withdrawn, cells were then re-dispensed using cell culture medium. To visualize cells, cancer cells were prestained with CMFDA dye at 37 oC for 30 min. The concentration of cancer cell suspension was measured using a handheld automated cell counter (Scepter™ 2.0 Cell Counter, Millpore). Cell suspension of 2000 cells/mL was prepared by serial dilution. In culture medium. To investigate the tested cancer cell binding efficiency of platform in cell culture medium, cell suspension of 100 µL was injected into platforms at 1.5 mL/h, following by 200 µL PBS at 1.5 mL/h, then 500 µL PBS at 9 mL/h. In whole blood. To investigate the tested cancer cell binding efficiency of platform in whole blood, H1975 cell suspension of 100 µL was spiked into 1.9 mL blood. Sample loading operates 1.5 mL/h, following by 400 µL PBS at 1.5 mL/h, then 1 mL PBS at 9 mL/h. Blood samples from non-cancer volunteers were drawn and collected into 10 mL Vacutainer® tubes containing the anticoagulant EDTA (BD Biosciences) and used immediately. The human blood tests were approved by Institutional Review Board (IRB) of Academia Sinica (ASIRBOI-12040), Academia Sinica. In WBC mixture. To acquire WBCs from the whole blood samples, Ficoll Paque plus (GE Healthcare) was applied to separate red blood cells, WBCs, and plasma. The Ficoll Paque PLUS was filled into a 15 mL centrifuge tube, the blood sample of same volume of Ficoll Paque was


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then slowly added on top of the Ficoll Paque. The tube was centrifuged immediately at 400 g for 30 min. After centrifuge, the plasma on the very top was drawn and kept separately. The buffy coat containing WBCs was withdrawn. The WBC solution of determined concentration was reconstituted using plasma drawn earlier. Around 200 HCT116 cells in 100 µL medium were spiked with 200 µL WBC solution of pre-determined concentration. Sample loading operates 1.5 mL/h, following by 200 µL PBS at 1.5 mL/h, then 500 µL PBS at 9 mL/h. Binding Efficiency and Overall Efficiency Definition. Along with spiking cancer cells in the inlet of microfluidics, three separate 100 µL cell suspension were dispensed in three separate home-made glass-bottomed wells. Cell numbers counted in those three wells were averaged to represent the cell number flowed through the platform. The cells captured in the platform were counted using fluorescence microscope. The binding efficiency is defined as the cell number captured in microfluidic platform after washing divided by the spiked cell number. Binding cells were released from the platforms follows a similar protocol reported previously 24. Briefly, the air foam solution was produced by a mixture of air and 5% BSA and gently vortexed for 1 min for foam creation. Around 250 µL foamy solution was flowed through the microfluidic at 9 mL/h. The eluted solution was filtered with an IsoporeTM membrane filter (2 µm pores, Millipore) to isolate the released cells. After subsequent PBS washing and 4% formaldehyde fixation, the membrane was mounted using ProLong® antifade mountants containing 4',6diamidino-2-phenylindole (DAPI, for staining cell nucleus) for subsequent cell counting. The overall efficiency is defined as the cell number on membrane divided by the spiked cell number. The release efficiency is the overall efficiency divided by binding efficiency.

2.6. Clinical Samples and Immunostaining


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Clinical Samples. The peripheral blood samples were obtained from 7 Stages 0 to IV PDAC patients and 2 non-cancer volunteers. Eligible patients had histologically or histopathologically confirmed PDAC. The blood samples of patients were collected for analysis before treatment from June to December 2015 in National Taiwan University Hospital. All samples were processed in Academia Sinica, Taipei, Taiwan. The study protocol including experimental design and performance of this study were clearly described and was reviewed and approved by IRBs of Academia Sinica (ASIRBOI-12040) and National Taiwan University Hospital (IRB02-104047). Immunostaining. The polyclonal antibody against wide-spectrum cytokeratin (panCK, Abcam, ab9377, 1:200, staining at 4oC for overnight) and immunoglobulin G-Alexa Fluor 647 (IgGAF647, Life Technologies, 1:500, staining at room temperature for 1h) were used for epithelial origin cancer cell identification. The antibody of CD45-FITC (DAKO, f0861, 1:10, staining at room temp for 1h) was used for the staining of the WBCs. PBS was used to wash out nonbonded antibodies after each staining step. The protocol to count elute cells was described previously. Cells with panCK+(red)/CD45-(green)/DAPI+(blue) staining were enumerated as CTCs. Enumeration of CTMs including both CTC-only clusters and CTC-WBC company clusters. Clusters containing at least two panCK+/CD45-/DAPI+ cells with distinct nuclei larger than 15 µm in diameter with or without WBC attached or containing at least one panCK+/CD45/DAPI+ cells with WBC attached were classified as a CTM.

2.7 Instrumentation Dynamic light scattering characterization (Zetasizer Nano ZS, Malvern). The size, electrophoretic mobility, and zeta potential of dendrimer is determined by Zetasizer Nano ZS


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using non-invasive backscatter optics for size measurement and phase analysis light scattering for zeta potential measurement. Viable light Spectrophotometry (NanoPhotometer P 300, IMPLEN). The absorbance of wavelength 500nm is measured by NanoPhotometer P 300 at single wavelength mode. The results window shows the amount of light of 500 nm passed through sample relative to the reference. MALDI-TOF MS (Ultraflex II TOF, Bruker). The molecular weight of dendrimer is determined by MALDI-TOF MS (Matrix-assisted laser desorption/ionization (MALDI) coupled with timeof-flight (TOF) mass spectrometry). Quartz Crystal Microbalance-Dissipation (QCM-D, Q-Sense E4, Biolin Scientific). Quartz crystal chips (AT-cut quartz crystals, f0 = 5 MHz, Q-Sense E4) coated with silicon dioxide (SiO2) were cleaned in 0.1 M sodium dodecyl sulfate, followed by rinsing with Milli-Q water, drying under nitrogen, and exposing to oxygen plasma for 20 s. For QCM-D measurement, the chamber was stabilized to 25 oC. All measurements were recorded at the third overtone (15 MHz). The surface coating steps were identical to those mentioned in previous section. Atomic Force Microscopy (AFM, MFP-3D-BIO, Asylum Research). To acquire the surface morphology, all studied surfaces were immersed in PBS and scanned in the tapping mode with an AFM equipped with silicon probes (BS-Multi 75 Al, Budget Sensor). Each sample was imaged three times at different locations on the substrate to ensure reproducibility. Surface polarization of studied surfaces was measured in the air by AFM operating in the scanning Kelvin probe mode using chromium/platinum-coated conductive probes (Tap 300E-G, Budget Sensor).


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Fluorescence Recovery After Photobleaching (FRAP, TCS-SP5-MP-SMD, Leica). The lipid mobility was examined with a confocal microscope and single molecule detection system. Microscopy (Eclipse Ti-E, Nikon). All fluorescent images were captured and analysized by Nikon Eclipse Ti-E inverted microscope. Flow Cytometry (FACS CantoTM II, BD Biosciences). EpCAM expression of the cancer cells was measured with a flow cytometer system in the following manner: The cells were detached from the cell culture dish using trypsin, and the cell suspension was centrifuged at 300 g for 5 min. After withdrawing supernatant, the cells were re-suspended in anti-mouse anti-EpCAM solution (200-titration) for 15 min. After washing non-bonded anti-EpCAM with PBS, a second antibody, anti-mouse IgG 488, was incubated with cell solution for 1 h. Any non-bonded antimouse IgG 488 was washed using PBS, the labeled cell solution was kept at 4 oC until analysis.

3. Results 3.1 Dendrimer Modification and Characterization To create the dendrimer-containing CTC capture surface, first the biotinylated spacer arms, NHS-PEG60-biotin, was bonded to the PAMAM G7 dendrimer to create AD-PEG60, as shown in Figure 1. The NHS-PEG60-biotin spacer arm consisted of an N-hydroxysuccinimide group, a PEG60 chain, and biotin. To conjugate the PEG60 spacer arm to the PAMAM dendrimer, PAMAM G7 was incubated with a 15- or 46-fold concentrated NHS-PEG-biotin solution, yielding modified dendrimer AD-PEG60, resulting in 3 or 5 PEG60 spacer arms conjugated with a PAMAM molecule, as determined with a biotin quantitation kit.


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Figure 1. Functionalization of the PAMAM dendrimer prior to inclusion in the CTC capture platforms. A. The biotinylated spacer arm used in the study. B. The PAMAM G7 dendrimer. C. Conjugation of the NHS-PEG60-biotin with the PAMAM G7 dendrimer to produce AD-PEG60.


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3.2 Surface Construct Formation and Characterization The modified dendrimers AD-PEG60 was conjugated to the top of the SLB and then capped with carboxylates by succinic anhydride (D-PEG60), the EpCAM antibody was immobilized to the top of D-PEG60 to create the Surface DS, as shown in Figure 2. Surface S, which does not contain a D-PEG60 dendrimer (Figure 2), was the control compared to Surface DS. The stepwise fabrication of surface construct was confirmed using QCM-D (Figure 3). The Roman numerals in Figure 3 correspond to the insertion of reactants at the corresponding Roman numeral in Figure 2.


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Figure 2. Illustration of the coating construction of Surface S and DS for the capture of CTCs. Surface DS includes antibody functionalized dendrimers, AD-PEG60, on an SLB, as well as the subsequent conversion of surface charges from –NH3+ to –COO- using succinic anhydride.


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Coating of Surface S consists of an antibody conjugated SLB without dendrimer spacer. Steps IVI correspond to distinct points in the QCM-D analyses in Figure 3.

A typical QCM-D response for the Surface S, shown in Figure 3A, represents a serial coating on a SiO2-coated quartz crystal. First, a lipid vesicle solution of 0.15 mg/mL was filled into the QCM-D chamber at point I in Figure 3A, resulting in an abrupt frequency decrease because of the adsorption of lipid vesicles following the frequency increase because of the rupture of vesicles to form a stable SLB. The normalized frequency change ∆F and dissipation shift ∆D were 24 Hz and 0.24 x 10-6, which were characteristic of a highly uniformed SLB

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. The NA

solution was dispensed at point II and the conjugation of NA to the SLB resulted in a 62 Hz frequency shift. To complete the Surface S, the NA-SLB surface was washed with buffer and then at point II-1, 0.25 mg/mL solution of b-Ab was dispensed into the chamber to bind with the biotin molecules on SLB via NA linkages. The binding of the b-Ab to the surface resulted in a frequency change of 29 Hz.


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A.

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II-1

I

Surface S

B.

II III

IV

V

VI

* I

Surface DS

Figure 3. The stepwise construct of A. Surface S and B. Surface DS monitored by normalized frequency (f) using QCM-D. The Roman numerals relate to the addition of reactants in the reaction scheme shown in Figure 2. * denotes the PBS washing step.

Similarly, the QCM-D measurement of the construction of Surface DS is shown in Figure 3B. The key difference of Surface DS compared to Surface S was the inclusion of dendrimers. At step III, a 4.7µM solution of dendrimer (AD-PEG60) was injected, resulting in a frequency shift. Succinic anhydride solution (1 mg/mL in PBS) was added at point IV to replace the surface amine groups of AD-PEG60 with carboxyl groups, forming dendrimer D-PEG60. PBS was injected to clean the loosely-bound substances at the point denoted with a *. At point VI another NA solution was injected to specifically bind with the D-PEG60. At step VI a solution of b-Ab was injected to complete the construction of the Surface DS.


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The ∆F measured by QCM-D can refer to the estimated number of molecules per projected 100 nm2 surface area. Based on Sauerbrey Equation, ∆m = -∆F/nC, where C is the mass sensitivity constant (5.72 m2Hz/mg at f = 5 MHz) and n is the overtone number (n=3 for QCM data in Figure 2)

25

, the frequency shifts are proportional to the adsorbed mass. Given the

molecular weight of added molecules, the surface density of immobilized molecules can be estimated. For example, there are on average five PEG60 spacer arms per dendrimer (incubated in 46-fold concentrated NHS-PEG-biotin solution), as determined with a biotin quantitation kit, resulting in ~110k Da molecular weight of D-PEG60. The molecular weights of NA and b-Ab are ~60k and 150k Da, respectively. In addition, the sizes of b-Ab and D-PEG60 are ~7 and ~10 nm measured by zetasizer. The estimated number of molecules per projected 100 nm2 surface area after each of the major steps in the construction of Surfaces DS and S was summarized in Table 1. On Surface S, ~1 b-Ab molecule was conjugated on every five NA on surfaces, most likely due to the physical hindrance effect. Similarly, about 1 D-PEG60 dendrimer was conjugated on every five NA on surfaces. By choosing a long spacer (PEG60, M.W. 3000 Da), there are only ~5 PEG spacer arms per dendrimer due to the physical hindrance. Consequently, there are much fewer accessible biotin sites for the subsequent NA conjugation, as shown by the reduction of 2nd NA surface density (1.4 NA per 100 nm2). Consequently, the resulting surface b-Ab density on the Surface DS (0.8 b-Ab per 100 nm2) is approximately equivalent to that on the Surface S (0.7 b-Ab per 100 nm2). It is anticipated that by reducing the spacer arm molecular weight, more antibody binding sites could be available hence, increasing the surface antibody density. In this study, it is desirable to keep the antibody surface density constant for both Surfaces S and DS, such that we may focus on the effects of chain mobility to the binding efficiency of CTCs. As the


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comparison, the surface b-Ab density on Surface DS-L (1 b-Ab per 100 nm2) is more than that on Surfaces S and DS. The Surface DS-L is a AD-C5 dendrimer-SLB surface as mentioned in section 2.3.

Table 1. Summary of quartz resonance frequency changes (∆F) and estimated corresponding number of molecules per projected 100 nm2 after surfaces were conjugated with the first treatment with 1st NA (step II in Figure 2), dendrimer (step III), 2nd NA (step V), and b-Ab (step VI).

∆F (Hz), (No. of Molecules per 100 nm2)

Surface SLB

1st NA

Dendrimer

2nd NA

b-Ab

S

-25

-62, (3.6)

--

--

-29, (0.7)

DS

-26

-57, (3.3)

-21, (0.7)

-24, (1.4)

-33, (0.8)

DS-L

-25

-57, (3.3)

-41, (1.4)

-60, (3.5)

-42, (1)


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The 2D and 3D surface morphology were profiled by an AFM operating in tapping mode, with the results shown in Figure 4. The AFM analysis showed that the dendrimer coating on Surface DS has an increased surface roughness (RMS, 5 nm) compared to Surface S (2 nm), suggesting that the hydrophilic PEG60-biotin spacer arm resulted in an expanded dendrimer layer.

Surface DS

Surface S 5 4

15

2

0

nm

3

1 0

-15

0

1

2

3

4

5

0

1

2

µm

3

4

5

µm

RMS: 2 nm

RMS: 5 nm 30 30 15

0

1

2

µm

3

4

5

0

1

2

µm

3

4

5 0

nm

µm

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

Biomacromolecules

0

Figure 4. 2D (top row) and 3D (bottom row) surface morphology of Surface S and DS profiled with AFM. The surfaces were measured in pH 7.4 PBS. The scanned area is 5µm by 5µm. The zaxis range is +/- 15 nm. The y-axis labels and scale bar apply to all images in a row. The root mean square (RMS) roughness for each surface is shown between their 2D and 3D images.


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The lipid mobility on the Platforms S and DS were further examined with FRAP. The fluorescent lipid solution was prepared for forming fluorescent SLB coating. As shown in Figure S4, the time to recover half florescence intensity (t0.5) for Platforms DS and S were 34s and 12s, respectively. Although the diffusivity of lipid bilayer was smaller in the case of the Platform DS, the fluid nature of the SLB to promote lateral mobility for antibody-antigen clustering was retained. In step IV (Figure 2) of the Surface DS construction, the surface amine groups of the dendrimers were converted to carboxyl groups by reacting the surface with a concentrated succinic anhydride solution 14, which had a ~4-fold excess carboxyl groups compared to the total number of amine groups. We analyzed this reaction with the dendrimers in solution, and used dynamic light scattering and electrophoretic light scattering to monitor the size and zeta potential, respectively, of PAMAM G7 before and after treatment with succinic anhydride. As shown in Table 2, the size of the carboxyl-modified PAMAM G7 increased from 8 ± 1 nm to 9 ± 2 nm, while the zeta potential changed from 21 ± 1 mV to -13 mV, which were similar to values in a previous report

15

. This confirmed that the surface amine groups can be passivated by

carboxyl groups by reacting with succinic anhydride.

Table 2. The size and zeta potential of PAMAM G7 dendrimer before and after surface functional group modification

PAMAM G7

PAMAM G7 after carboxylation*

Size (nm)

8+/-1

9+/-2

Zeta potential (mV)

21+/-1.0

-13**


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* PAMAM G7 was reacted with succinic anhydride to replace surface amine with carboxyl functional groups. **Standard deviation of 3 measurements is less than 0.5 mV.

We further adjust the carboxylated D-PEG60 dendrimer concentration from 4.7, 8.7 to 16.1 µM in PBS and used QCM-D to observe the corresponding number of conjugated dendrimers on SLB. The frequency drop after D-PEG60 coating was similar when incubating with dendrimer concentration from 4.7, 8.7 to 16.1 µM, indicated similar D-PEG60 immobilization surface density (Table 3). Thus, the D-PEG60 dendrimer solution of 4.7µM is concentrated enough to immobilize D-PEG60 dendrimer on SLB surface.

Table 3. The frequency changes of Surface DS after conjugating with D-PEG60 ∆Frequency (Hz) D-PEG60 Conc. (µM) D-PEG60 4.7

22

8.7

21

16.1

21

3.3 Evaluation of EpCAM Expression Level of Various Cancer Cell Lines CTC isolation techniques that rely on EpCAM antibody-antigen affinity can have limited efficacy when cancer cells have low EpCAM expression. It would be ideal to have a CTC isolation platform that would work well with CTCs cross a wide range of EpCAM expression level. We used flow cytometry to identify the EpCAM expression level of three cancer cell lines as shown in Figure 5. Where HCT116 has high EpCAM expression, H1976 has moderately high


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EpCAM expression, and Panc-1 has low EpCAM expression. The average relative fluorescence intensities of 10000 cells of HCT116, H1975, and Panc-1 cell lines were ~42000, 35000, and 13000 (arbitrary units), respectively.

A.

B.

C.

Figure 5. Flow cytometry evaluation of three cancer cell lines. A. HCT116, B. H1975, and C. Panc-1. The number indicated in the figures (41901 for HCT116, 35132 for H1975, 13111 for Panc-1) is the average FITC intensity of cell population (99.8% for HCT116, 97.7% for H1975, 99.7% for Panc-1) gated by the horizontal bar in each data set.

Beyond EpCAM expression, the affinity between the antibody and the antigen might be related to the receptor density on the cell membrane if the surfaces were immobilized with similar antibody concentration. The average cell diameters of HCT116, H1975, and Panc-1 measured with a Millipore hand held impedance-based cell counter (particle allowed range 6~36 µm) were


26

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determined to be 12, 15, and 17 µm, respectively. It was reported that the binding strength of a cell will be stronger with the increased ligand density immobilized on the surface

26

. Likewise,

with similar ligand density, binding strength might be stronger with the increased receptor density. The relative receptor (EpCAM) density on the cancer cell lines in this study was defined as the fluorescence intensity measured by flow cytometry (Figure. 5) divided by the average surface area of cells (4πR2). The EpCAM density of HCT116, H1975, and Panc-1 was calculated to be 100, 53, and 15 Int/µm2. It is expected that under similar shear stress of flow and distance of cells from surface, the binding affinity would be HCT116 > H1975 > Panc-1 if the surface ligand density is similar 27.

3.4 Binding and Release of Cancer Cell Using Microfluidic Platforms To investigate the binding efficiency of the CTC capture platforms, dispersions of HCT116, H1975, or Panc-1, all pre-stained with CMFDA, were flowed through the microfluidic platforms and the captured cells were subsequently numerated under Nikon Eclipse Ti inverted microscope. The image of the microfluidic platform was shown in Figure S3. The dimension details and the pattern design of the microfluidic platform were reported previously

23

. Approximately 200

suspended cancer cells per 100 µL solution was prepared by cell culture medium. For HCT116 and Panc-1, cells were dispersed in DMEM medium and for H1975, cells were dispersed in RPMI medium to prepared cell solution at predetermined concentration. The 100 µL cell solution was flowed through respective microfluidic platforms at 1.5 mL/h. After washing with PBS at 9 mL/h to remove non-bonded cells, the CTCs captured on the microfluidic surface were counted using a fluorescence microscope to determine the binding efficiency of each platform for each


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type of cancer cell line. After counting, the cells were detached by air foam and collected to an Eppendorf then isolated on 2 µm IsoporeTM membrane filter for cell counting to determine the overall efficiency. The binding efficiency of the different cancer types were shown in Figure 6A. HCT116, which has the highest EpCAM density, has the highest binding efficiency. Conversely, Panc-1 has the lowest binding efficiency. Given that HCT116 and H1975 have a similar expression level of EpCAM expression per cell, as shown in Figure 5, the higher binding efficiency of HCT116 for each capture platform must be related to the higher EpCAM density on HCT116 membrane surface relative to H1975. Moreover, the smaller size of HCT116 means it would experience a lower drag force, and therefore would be less likely to be pulled away during the washing step once it is adhered to the antibody coating surface.

A. Cancer cell binding and overall efficiency in medium


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B. Cancer cell binding and overall efficiency in whole blood

Figure 6 A. The binding efficiency and overall efficiency of the Platforms S and DS for cancer cells spiked in a medium. The cancer cells used were HCT116 colon cancer cell ( lung cancer cell (

), and Panc-1 pancreatic cancer cells (

), H1975

). B. The binding efficiency and

overall efficiency of H1975 lung cancer cells of Platforms S and DS in whole blood. For each condition, approximately 200 cancer cells (in 100 µL medium) were spiked in 1.9 ml blood from a healthy donor.

In terms of the binding efficiencies of two platforms, two trends were apparent in the results shown in Figure 6A. First, for all cancer types in this study, the binding efficiencies of the Platform DS containing dendrimer (D-PEG60) was consistently better than that of Platform S. Second, while both had a very high binding efficiency of HCT116, the Platform DS was most consistent for all cancer types. For example, while the binding efficiencies of Platforms DS was similar compared to Platforms S for HCT116 (98 ± 6% vs. 95 ± 3%) and H1975 (78 ± 14% vs. 66 ± 5%), Platform DS had a significantly higher binding efficiency than Platform S for Panc-1 (78 ± 6% vs. 39 ± 16%). This improvement in binding efficiency of Panc-1, a cancer cell line with lowest EpCAM expression level, occurred even though antibody density was similar on


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Platform DS (0.8 b-Ab molecule per 100 nm2 area) and Platform S (0.7 b-Ab molecule per 100 nm2 area), suggesting that the extended flexible arm providing benefit to access cell antigen molecules. To assess the ability of the platforms to release captured cancer cells, the captured cancer cells were eluted from the chip via disruption of the SLB assembly by a gentle flush with air foam. As in previous work with the Platform S

16

, the SLB assembly could be disintegrated easily by

introducing a hydrophobic component as simple as air bubbles. By injecting continuous air foam to the microfluidic assembly, the adhesive cancer cells can be gently released without disrupting the antibody-antigen bonds. Unlike attempts using mechanical forces 28, 29 or enzymatic cleavage 30, 31

, releasing cells with air foam requires less than 15 dyne/cm2

18

, which minimizes cell

damage and enables downstream molecular analysis and cell culture. The air foam containing released cancer cells after leaving the microfluidics was filtered by a membrane to collect the cells. The cancer cells on the membrane were labeled with DAPI, and the cells that were doublepositive with CellTracker and DAPI were identified as released cancer cells. The overall efficiency shown in Figure 6A is defined as the count of released cancer cells divided by the total number of cells initially inserted into the cancer cell capture system. Like the results with binding efficiency, the Platform DS had a more consistent overall efficiency over the range of cell lines in this study compared to the Platform S. As shown in Figure 6A, the overall efficiency of HCT116, H1975, and Panc-1 cells in the Platform DS was 72 ± 3, 49 ± 18, and 49 ± 14 %, respectively. For comparison, the overall efficiency of HCT116, H1975, and Panc-1 cells in the Platform S was 46 ± 1, 36 ± 20, and 26 ± 3 %, respectively. To investigate the performance of the microfluidic platforms when the cancer cells were in whole blood, samples were prepared in which ~200 H1975 cells in 100 µL of medium were


30

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spiked into a 1.9 mL pre-collected whole blood sample from a healthy volunteer. The whole blood sample were flowed through the platform at 1.5 mL/h, followed by three rinses of 500 µL PBS flowing at 9 mL/h. The release and mounting protocols were similar with the samples shown in Figure 6A. The cells that were double-positive with CellTracker and DAPI were identified as H1975 cells, and single positive DAPI-only cells were identified as WBCs that were non-specifically bound on the surface of platform. As shown in Figure 6B, the binding efficiency of H1975 in the whole blood was 52 ± 4% and 25 ± 13 %, while the overall efficiency was 20 ± 13 % and 10 ± 6 % in the case of Platform DS and Platform S, respectively, where H1975 binding efficiency of Platform DS is two times higher than that of Platform S. The cancer cell binding efficiency of Platforms DS is even superior when interfering blood cells are present, suggesting the increased chain movement could increases the targeted cancer cell binding from interfering blood cells. We had showed that the binding efficiency of cancer cells decreased when interfering blood cells were present. To investigate the competition effect of WBCs in the binding efficiency of H1975, dispersions of H1975 cells spiked with WBCs at pre-determined concentrations were prepared. WBC solutions at predetermined concentration were prepared by spiking concentrated WBC (from buffy coat) to the plasma (same blood sample) acquired by Ficoll Paque separation protocol at a 1:1 volume ratio. About 200 H1975 cells in 100 µL RPMI medium were then spiked in 200 µL of the WBC solutions. The concentrations of WBC solutions were 0, 3.5 x 104, 3.5 x 105, and 3.5 x 106 WBCs per 1 mL plasma. As the comparison, a WBC concentration of a healthy human is ~3.5 x 106/mL. The binding efficiencies of Platform DS for H1975 decreased monotonically with increasing WBC concentration, shown as blue hollow circle in Figure 7, showing that the competition of WBCs had a negative effect on binding efficiency. As the


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comparison, the binding efficiency of Platform DS (blue solid circle) and Platform S (red hollow inverted triangle) in medium and in whole blood was indicated in the Figure 7.

Figure 7. The effect of WBC concentration on the binding efficiency of the Platform DS for H1975 lung cancer cells ( ). For comparison, the binding efficiency of Platform DS ( ) and Platform S ( ) in medium (*) and whole blood (**), as marked in the figure.

3.5 Efficiency in Isolating CTCs and CTMs from Patient’s Blood In most cancers studied, CTCs were rarely detectable in the early stages of disease, including PDAC. The heterogeneity of CTMs regarding epithelial versus mesenchymal cell phenotypes has been demonstrated in small cell lung cancer 32. The lack of proliferation of CTMs, compared to proliferating a single CTC, would theoretically make cancer cells relatively resistant to chemotherapy. Furthermore, it implies that CTMs are cell clusters breaking off from the primary tumor, intravasating via leaky and chaotic tumor vessels and appearing in the blood resulting in collective migration. CTMs have been demonstrated to possess increased metastatic potential compared to single CTC

33

. CTMs derived from multicellular groupings of tumor cells hold


32

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together through intercellular adhesion greatly contribute to the metastatic spread of cancer. Previously we have shown that an SLB coated platform (Platform S) can isolate both CTCs and CTMs from colorectal cancer patients 18. We compared the isolation efficiency of CTCs and CTMs from blood samples of 7 PDAC patients and 2 non-cancer volunteers using Platform DS and S. The procedure to capture the cells was mentioned previously. The numbers of isolated CTCs and CTMs were summarized in Table 4. The images of representative CTCs, WBC, and CTMs obtained from Platform DS and S are similar with those reported previously 17. As indicated in Table 4, a use of the Platform DS resulted in isolating a total of 1.6 times more CTCs and 2.3 times more CTMs compared to the Platform S. Moreover, more CTCs and CTMs were isolated using Platform DS than the Platform S from each patient blood sample. Very few CTCs and CTMs were isolated from blood samples of non-cancer volunteers using the Platform DS, showing that the increase in sensitivity of the using Platform DS was achieved without increasing false positive background signals. We had reported that Platform S is excellent in comparison with other platforms

17, 19

. The

CTC isolated from Platform S is positively correlated to the prognosis. However, Platform DS seems to be even more sensitive, the captured CTC numbers are in general positively correlated with Platform S. As a result, it could be also useful for prognosis and early cancer detection.


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Table 4. Counting of captured CTC and CTM in 2 mL peripheral blood samples of PDAC patients and non-cancer volunteers isolated from Platform DS or S

*CTM classification: CTM counts is based on the summation of clusters containing 1+X (1 CTC + WBCs), 2+X (2 CTCs + WBCs), 3+X (3 CTCs + WBCs), and >5 (more than 4 CTCs + WBCs).

4. Discussion To promote local, short-range clustering to increase binding efficiency of cancer cell lines in a non-competitive condition using dendrimer is demonstrated previously

15

. Here, we found the

non-fouling dendrimer is the key parameter to achieve high binding efficiency and overall efficiency of rare cells when mixing in blood, in which non-specific physical adsorption is prevalent. To design a less non-fouling dendrimer, we prepared the dendrimer AD-C5 (Figure S1B) with the hydrophobic nature of space arm (Figure S1A). The dendrimer, AD-C5, had ~20 C5 spacer arms conjugated with each PAMAM molecule, the molecular weight is ~103,000 Da as determined by TOF-MOLDI (Figure S5). The carboxylated dendrimers D-C5 was conjugated to the top of the SLB and then capped with b-Ab to create the Surface DS-L, as shown in Figure S2. The QCM-D measurement of the serial


34

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construction of Surface DS-L and AFM measurement of Surface DS-L surface morphology was shown in Figure 8A and Figure 8B. The molecular weight of sulfo-NHS-LC-biotin (557 Da) is much less than ɑ-biotinyl-ω-NHS polyethylene glycol (3000 Da), resulting in a better conjugation efficiency with dendrimer. Compared to Surface DS, 2 times more dendrimers could immobilize on SLB surface in the case of Surface DS-L from QCM measurement and Sauerbrey equation. One possible explanation is that the number of biotinylated sites of D-C5 is 4 times more than that of D-PEG60, so more dendrimer could attach to the SLB. Subsequently, 2.5 times more NA and 1.3 times more b-Ab were attached on the Surface DS-L than on the Surface DS. The 2D and 3D surface morphology of Surface DS-L profiled by AFM showed that the dendrimer coating on Surface DS-L has an increased surface roughness (RMS, 4 nm) compared to Surface S (Figure 4). The roughness of Surface DS-L was slightly smaller than DS (4 nm v. 5 nm, Figure 4), suggesting that the hydrophilic PEG60-biotin spacer arm resulted in an expanded dendrimer layer; while in the case of Surface DS-L, the hydrophobic LC-biotin spacer arms wrapped around the dendrimer unites, forming a relatively packed surface. The binding and overall efficiencies of Platform DS-L coated with Surface DS-L were investigated as shown in Figure 8C. For HCT116 and H1975 in medium, the binding efficiencies of the Platform DS-L were better than that of Platform DS (Figure 6A). However, the binding efficiency in Panc-1 of Platform DS is better than that of Platform DS-L, even the b-Ab on surfaces of Platform DS is less dense on that of Platform DS-L. This suggests again that the extended long flexible arm providing benefit to access cell antigen molecules compared to the shorter space arm.


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Additionally, the overall efficiencies of the Platform DS were always higher than the Platform DS-L, indicating better release efficiency of the Platform DS. The lower release efficiency of the Platform DS-L might occur because of the more hydrophobic nature of C5biotin spacer arms resulting in stronger non-specific adsorption of the cells toward the surface, which in turn would lead to damaged cells during the release process. This hydrophobic nature might deteriorate the binding of targeted cells when the interfering cells are present. As shown in Figure 8C, the binding efficiency of H1975 in whole blood significantly decreased compared to that in medium leading to the low overall efficiency. Compared to the number of WBCs as shown in Figure S6 released from the Platform DS (6059 ± 2978 cells), the increased number of WBCs were released from the Platform DS-L (9906 ± 629 cells) also implied that Platform DS-L is less resistant to non-specific WBC adsorption than the Platform DS.


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A. II III

IV

V

VI

* I

B. 5 4

µm

RMS: 4 nm

15

3 2

0 nm

1

-15

0

1

2

µm

3

4

5

30

30

nm

15 nm 0

0

0 0

1

2

3

4

5

µm

C.

Figure 8 A. The stepwise construct of Surface DS-L monitored by normalized frequency (f) using QCM-D. The Roman numerals relate to the addition of reactants in the reaction scheme shown in Figure S2. * denotes the PBS washing step. B. 2D (top row) and 3D (bottom row)


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surface morphology of Surfaces DS-L profiled with AFM. C. The binding efficiency and overall efficiency of the Platforms DS-L for cancer cells spiked in a medium or blood. The cancer cells used were HCT116 colon cancer cell, H1975 lung cancer cell, and Panc-1 pancreatic cancer cells. In the case of spiking in medium, for each condition, around 200 HCT116 or Panc-1 cells spiked in 100 µl DMEM medium or H1975 cells spiked in 100 µl RPMI medium flowed through platform at 1.5 mL/h. In the case of spiking in blood, approximately 200 cancer cells (in 100 µL medium) were spiked in 1.9 ml blood from a healthy donor and then flowed through the platform at 1.5 mL/h.

To further investigate the resistance to non-specific adsorption, 1% BSA was incubated on QCM quartz chips coated with either the Surface S, DS-L, or DS-L with amine capped AD-C5 dendrimer. After incubation for ~2 h, the surfaces were washed using PBS. The frequency shift was monitored by QCM-D and is shown in Figure S7. It was found that the frequency shifted during BSA incubation on the three surfaces, but that the frequency of the Surface S almost returned to original value after PBS washing. After the second BSA incubation and PBS washing, a ~2 Hz shift was measured corresponding to 7 ng (Table S1) of BSA adsorption on Surface S. In the case of the Surface DS-L surface and Surface DS-L with AD-C5 dendrimer, the frequency shifts were -1 and -4 Hz after the first BSA incubation and PBS washing, and ~5 and ~10 Hz shifts after the second BSA incubation, respectively. The difference in frequency shifts showed that the relative amount of non-specific BSA adsorption on the three surfaces was Surface DS-L with AD-C5 > Surface DS-L > Surface S. It is well known that adhesion of blood cells is mediated by non-specific binding, surface adsorbed plasma proteins, and cell-cell interaction. The reduction in non-specific adsorption and


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improvement in affinity are critical parameters in designing surface construction. We had shown that dense b-Ab immobilized on a more hydrophobic D-C5-SLB coating can in general improve the binding efficiency compared to SLB-only surface coating. However, the non-fouling DPEG60 on SLB coating can consistently improve binding efficiency and overall efficiency of investigated cancer cell lines of wide range EpCAM expression level from medium and whole blood.

5. Conclusion The pegylated dendrimer-lipid surface coating provided additional extended hydrophilic flexible arm to access cell antigen molecules to increase local multivalency, resulting in improved binding efficiency compared to lipid-only surface coating with similar anti-EpCAM immobilization density. The binding efficiency was improved especially in those that cancer cells express lower level of EpCAM, such as Panc-1. The pegylated dendrimer-lipid surface coating microfluidics also showed consistent improved efficiency over a range of cancer cell types, which is beneficial in terms of having one platform that can work well for a range of different cancer cells. The hydrophilic nature of pegylated dendrimer reduced non-specific adsorption results in a better overall efficiency especially when interfering blood cells are present. When the pegylated dendrimer-lipid surface coating microfluidics was used to analyze blood from seven PDAC patients, a total of 1.6 times more CTCs and 2.3 times more CTMs were isolated from peripheral blood compared to the same samples analyzed with the lipid-only coating microfluidics.


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Supporting Information The file of “supporting information.doc” including details of synthesis of D-C5 molecule; construction of Surface DS-L measured by QCM and profiled by AFM; photo of microfluidics; SLB fluid nature measured using fluorescence recovery after photobleaching (FRAP) method; quantitation of biotin spacer arms per D-C5 molecule; binding and overall efficiency of Platform DS-L in three cancer cells; comparison in adhered WBCs released from Platform S, DS and DSL; investigation in non-specific BSA adsorption on Surface S, DS-L, and DS-L with AD-C5 dendrimer is available free of charge.

Acknowledgments The authors thank Dr. Han-Chung Wu (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan) for providing anti-EpCAM. The authors also thank M.D. Ming-Chu Chang, YuTing Chang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan), and Yu-Wen Tien (Department of Surgery, National Taiwan University Hospital, Taiwan) for collecting and providing clinical blood samples of PDAC patients.

Corresponding Author Contact Information: Ying-Chih Chang TEL: 886-2-27871277 FAX: 886-2-27899931 Email: [email protected]


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Funding Sources We thank Ministry of Science and Technology (MOST), Taiwan, for the funding support through Grant Nos. MOST 105-0210-01-09-02, 104-2113-M-001-015-MY3. We thank Academia Sinica, Taipei, Taiwan, for the funding support VAT105-V1-3-3. PYY is supported by Academia Sinica Postdoctoral Fellowship 2015-2017.

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