A novel 3D breast-cancer-on-chip platform for therapeutic evaluation of

drug delivery systems

Yongli Chena,b, Dan Gaoa,c,, Yanwei Wanga,c, Shuo Linb, and Yuyang Jianga,d

a State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Biology,

Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China

b Key Lab of Chemical Genomics, School of Chemical Biology & Biotechnology, Graduate

School at Shenzhen, Peking University, Shenzhen 518055, China

c Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua

University, Shenzhen 518055, China

d School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China

Corresponding authors. E-mail address: gao.dan@sz.tsinghua.edu.cn1

Synthesis of CDs, CDs-PEG and CDs-PEG-FA

CDs were synthesized by the hydrothermal method, which was a simple, convenient and one-

step method [1]. Briefly, 0.2 g sodium citrate and 1.5 g NH4HCO3 were dissolved in 10 mL

water, and the mixture was sealed into a Teflon equipped stainless steel autoclave. The

autoclave was placed in a drying oven for hydrothermal treatment at 180 °C for 4 h and then

cooled down to room temperature. The purification of the CDs was conducted against DI

water through a dialysis tube (3000 Da, molecular weight cutoff) for 48 h. Then, the CDs

were passivated by PEG. Briefly, 1.0 mg ml-1 of CDs in DI water was sonicated with 10 mg

ml-1 of poly (ethylene glycol) diamine (sigma, St. Louis, MO, USA) for 5 min. 1-(3-

Dimethylaminopropyl)-3-ethylcarbodiimide (EDC, Sigma, St. Louis, MO, USA) was added

to reach the final concentration of 5 mM, and the solution was sonicated for another 60 min.

Then, 2.5 mM N-hydroxysuccinimide (NHS, Sigma, St. Louis, MO, USA) was added and the

mixture was stirred for 24 h. After centrifugation (15,000 rpm) for 1 h in PBS, the supernatant

was collected to obtain CDs-PEG. The FA-conjugated CDs-PEG was prepared by conjugating

the CDs-PEG with activated FA. In brief, 35 mg of FA was mixed with 15 mg of EDC and 25

mg of NHS in 1 mL PBS for 15 min at room temperature, followed by adding 20 mg of CDs-

PEG, then the mixture was stirred for 24 h and the product was dried under vacuum.

Characterization of the synthesized CDs-PEG-FA

CDs exhibited a broad absorption band for UV-Vis measurement, demonstrating the typical

absorption of an aromatic system and the sp2-carbon network. Moreover, the successful

functionalization of FA onto the CDs surface could be proved from the peak at 285 nm, which

was consistent with the previous research [2] (Figure S1). In addition, the CDs showed a clear

fluorescence emission peak centered at 470 nm when excited at 405 nm. After PEGylation,

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the CDs-PEG were well dispersed in water with high optical transparency and exhibited a

slight decrease in fluorescence intensity. And the CDs-PEG functionalized with FA resulted in

a significant decrease in fluorescence intensity.

The changes of chemical functional groups on the synthesized CDs upon surface

passivation were characterized by FTIR spectroscopy and XPS measurements. In detail, the

FTIR spectrum of CDs exhibited characteristic absorption peaks of C-H bending vibration at

1346 cm-1, C=O stretching vibration at 1732 cm-1, as well as a broad band in the range of

2850-3485 cm-1 corresponded to carboxylic acid and hydroxyl groups, respectively (Figure.

S1C). Upon surface passivation, the new bands of N-H and C-N stretching vibrations at 3450

and 1153 cm-1 were presented, demonstrating the successful formation of amide groups by

chemical conjugation of carboxylic acid of CDs surface and the amine-terminated PEG. When

passivation with FA, CDs-PEG-FA exhibited characteristic peaks at 1480, 1596 and 1680 cm−1

which belonged to FA [3]. In addition, XPS spectra were also presented to confirm the

composition of the respective CDs and the successful surface passivation. Specifically, CDs

have sp3 carbon (284.52 eV), sp2 carbon (285.34 eV), C-O groups (286.12 eV), C=O groups

(288.10 eV) and COOH groups (288.92 eV), indicating that the CDs are abundant in

hydroxyl, carbonyl and carboxylic acid groups on the surfaces (Figure S1D). After

modification with amine terminated PEG, the intensity of C-O peak increased due to C-O

groups in PEG. And the amount of carbonyl group decreased with the appearance of the new

peak of C-N group, which indicated carbonyl group in CDs and amine group in PEG were

successfully reacted to amide groups (Figure S1E). Further, the intensities of C-N, C=O and

COOH groups were increased for CDs-PEG-FA, which also confirmed the formation of more

amide groups between the free amine terminated PEG and FA after modification with FA

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(Figure S1F). In addition, the size and morphology of the passivated CDs were investigated

by TEM, which showed that these CDs were mono-dispersion and photochemical stable, and

their sizes were distributed from 2 to 9 nm (Figure S1G). The interlayer spacing of CDs was

shown as 0.22 nm, representing the graphitic nature of the CDs [4]. Interestingly, the sizes of

CDs-PEG-FA were larger than those of CDs, with an average diameter of 12 nm, which

would be benefit for high permeation and accumulation in tissue [5] (Figure S1H) . This small

size nanoparticle could be quickly removed from the body through the kidney after delivering

the drug which could be regarded as an attractive nanocarrier for their applications in drug

delivery [6].

LC-MS/MS conditions for DOX quantification

A Quattro Premier XE mass spectrometer (Waters, USA) was connected to the LC system

through an electrospray ionization (ESI) interface. The ESI source was operated in positive

ionization model. Quantification was performed using multiple reaction monitoring (MRM)

method with the transition of the parent ion to the product ion of m/z 544.5 →397.1 for DOX.

The MS parameters were as follows. Capillary voltage was set at 2.8 kV in positive ion mode.

Source temperature was maintained at 120 ºC while the desolvation temperature at 200 ºC. N2

was used for desolvation gas (flow rate at 800 L h-1) and cone gas (flow rate at 23 L h-1) while

Ar for collision gas (flow rate at 0.15 mL min-1). Chromatographic analysis was performed

using the HPLC Waters 2695 system (Waters, USA) equipped with a quaternary pump, an

online degasser, a column heater and an autosampler. The chromatographic separations were

achieved on an X BridgeTM C18 column (4.6 mm × 150 mm, 5μm, Waters Corp., Milford,

MA, USA) column by gradient elution using mobile phases of (A) 0.1% formic acid-water

and (B) acetonitrile at a flow rate of 0.5 mL/min. The gradient conditions were as follows: In

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the first half minute, an isocratic run by 70% (v/v) A and 30% (v/v) B; from 0.5 to 6.0 min, a

linear gradient from 70% A to 50% A; from 6.0 to 6.5 min, an isocratic run at 50% A; from

6.5 to 8.0 min, a linear gradient from 50% A to 70% A; from 8.0 to 12.0 min, an isocratic run

at 70% A. The column temperature was room temperature and the injection volume was 10

μL. Data were collected and analyzed by using Masslynx V4.1 software (Waters, USA).

Western blotting analysis

BT549, T47D and HUVECs cells in culture dishes were harvested and lysed with ice-cold

lysis buffer. Proteins were extracted from the cells, separated in 15% SDS olyacrylamide gel,

and then transferred to PVDF membranes. The membranes were blocked in TBST buffer

(0.125 M NaCl, 25 mM Tris, pH 8.0) containing 5% defatted milk for 2 h and then probed

with specific first antibody against human folate receptor-α (FOLR1, 1:1000; R&D Systems,

Minneapolis, MN, USA) overnight at 4 ºC. Followed by a HRP-conjugated second antibodies

(1:3000) hybridization, the protein bands were visualized by an imaging system (Bio-Rad,

Munich, Germany) using the SuperSignal West Pico Chemiluminescent Substrate kit.

Microfluidic device fabrication

Microfluidic device was fabricated from PDMS using standard soft lithography and replica

molding techniques. The master copies for PDMS molding were fabricated with SU-8 2007

and SU-8 2050 negative photoresists (Microchem, Newton, CA) respectively. Briefly, the

negative photoresist (Microchem, Newton, CA) was spin-coated onto the cleaned silicon

wafer. After spinning, the wafer was prebaked at 95 °C for 5 min, and then exposed to UV

light through the transparent mask. After post baking and development, the master was hard-

baked for 5 min at 95 °C. A 10:1 weight mixture of PDMS prepolymer and curing agent was

poured onto the silicon master and cured in an oven at 80 °C for 2 h. After cooling, the PDMS

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replicas were peeled off from the master and the connection holes were pierced before

sealing. The PDMS replicas were irreversibly sealed with glass slides after oxygen plasma

(PDC-32G, Harrick Plasma, Ithaca, NY, USA) treatment for 90 s. The devices were cured at

60 °C for 2 h to reinforce the bonding.

Surface tension research in cell culture chambers

When BME gel was introduced into the chambers, the gas−liquid interface would form

naturally and the flow could not go through the minor connecting channel. The vital factor

was the surface tension of the mixture at the end positions of the minor channels where the

gas-liquid interface existed. To ensure that the designed channels had the ability to prevent

cell-agarose mixture from going through the minor channel, we made a theoretical analysis.

As shown in Fig. S4, the mixture was assumed to flow uniformly and steadily, thus

10\* MERGEFORMAT ()

where P0 is the atmospheric pressure, Ps is the surface tension induced pressure, and ΔPf and

ΔPf′ are the pressure drops in the cell culture chambers and the holes, respectively.

In accordance with the Fanning equation

(2)

(3)

where λ is the Friction coefficient, l is the length between the minor channel and the end of

the chambers (as shown in Fig. S6), d is the equivalent diameter, μ and μ′ are the line

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velocities in the chamber and outlet hole respectively. d is determined by eq 3:

(4)

h and w are the height and width of the chamber, respectively. The constants, friction

coefficient and Reynold’s number are

(5)

(6)

where T is the viscosity coefficient of the solution. When combined eqs 2, 4, 5 and 6

(7)

Although the cross-sectional area of the outlet hole is different from that of the chamber, the

volumetric flow rate (v) is the same. Thus

(8)

When we combined eqs 3, 5, 6, and 8

(9)

Then, we combined eqs 8, and 9 to get the surface tension induced pressure

(10)

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Fluorescein sodium diffusion on chip

Because the drug solutions must transport across two microchannels to the tumor spheroids

chamber, the dimensions of the channels had significant influence on the diffusion rate. In our

experiment, fluorescein sodium was used as a detectable indicator to investigate the feasibility

for drug delivery. Firstly, a portion of Basement Membrane Extract (BME, Trevigen,

Gaithersburg, MD, USA) was injected to the connecting microchannel and allowed to gel at

room temperature for 1 h. 5 μM fluorescein sodium was finally injected into channel a, and

the injection was immediately stopped after the channels were completely filled. The images

were taken under an inverted fluorescence microscope (Olympus IX51, Olympus Co.

Ltd., Tokyo, Japan), and the fluorescent signals were quantified using ImageJ software (NIH,

USA).

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Figure S1. (A) UV-Vis absorbance spectra of CDs, CDs-PEG, and CDs-PEG-FA. Inset shows the CDs (left), and CDs-PEG (right) under UV illumination at 365 nm. (B) Fluorescence spectra of CDs, CDs-PEG, and CDs-PEG-FA with the excitation wavelengths of 405 nm. (C) FTIR spectra of CDs, CDs-PEG, and CDs-PEG-FA. The high-resolution XPS C 1s peaks of (D) CDs, (E) CDs-PEG, and (F) CDs-PEG-FA. (G) TEM images of CDs with a corresponding size distribution histogram. (H) TEM images of CDs-PEG.

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pH7.4pH6.4

Dru

gre

leas

e(%

)0 10 20 30 40 50

0

10

20

30

40

50

60

70

80

Time (h)

Figure S2 In vitro DOX release profile from CDs-PEG-FA in PBS at pH 6.4 and pH 7.4 at 37 ºC, respectively.

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Figure S3 The images of the microfluidic devices with different dimensions of U-shape microchambers, and the corresponding COMSOL simulations of flow velocity. (A) The image of the microfluidic device and the flow velocity field in a microchamber with the dimension of 400 μm (length) × 200 μm (width) × 100 μm (height), the width of entrance was set as 100 μm. (B) The image of the microfluidic device and the flow velocity field in a microchamber with the dimension of 300 μm (length) × 200 μm (width) × 100 μm (height), the width of entrance was set as 150 μm.

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Figure S4 Design and mathmodeling for the surface tension research. a) Three-dimensional (3D) structure of the ECM and capillary channels. b) The horizontal section of the area in the rectangle with a red line in Figure a are the pressures at different positions of the ECM channel and the gas-liquid interface when the channel was filled with the BME. c) The pressures at the bottom and the top of the outlet hole when filled with the BME. P2, P1, P0, and (Ps + P0) are the pressures of the corresponding positions; ΔPf and ΔPf’ are the pressure drops in the ECM channel and the holes, respectively. The line velocities in the ECM channel and outlet hole are μ and μ′, respectively.

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Figure S5 A fluorescein sodium diffusion assay was performed to evaluate the diffusion among the three microchannels on the microfluidic device. (A)-(D) Microscope fluorescence images obtained from 0-180 min after injecting of fluorescein sodium into channel a. (E) The fluorescence intensity of fluorescein sodium solutions in the channel c were compared with that of channel a, and their ratios were quantified by imageJ software.

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100 μm50 μm

A B

Figure S6 Adhesion of HUVECs on the PDMS microchannel (A) and the BME interface (B) without any treatment.

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200 μm

Figure S7 A bright field image of BT549 MCTS generated on the microfluidic device with uniform diameters.

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0.5 1.0 1.5 2.0 2.5 3.0

0.0

0.5

1.0

Time (h)

Fllu

ores

cen

inte

nsity

ratio

of r

ight

to le

ft ch

anne

l

(B)

Figure S8 The relative quantitative results of the fluorescence signal changes in the left and right channels from channel a to b from 0.5 h to 3 h.

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