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Cells Cultured on Core−Shell Photonic Crystal Barcodes for Drug Screening Fanfan Fu,† Luoran Shang,† Fuyin Zheng, Zhuoyue Chen, Huan Wang, Jie Wang, Zhongze Gu, and Yuanjin Zhao* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China S Supporting Information *
ABSTRACT: The development of effective drug screening platforms is an important task for biomedical engineering. Here, a novel methacrylated gelatin (GelMA) hydrogelencapsulated core−shell photonic crystal (PhC) barcode particle was developed for three-dimensional cell aggregation culture and drug screening. The GelMA shells of the barcode particles enable creation of a three-dimensional extracellular matrix (ECM) microenvironment for cell adhesion and growth, while the PhC cores of the barcode particles provide stable diffraction peaks that can encode different cell spheroids during culture and distinguish their biological response during drug testing. The applicability of this cell spheroids-on-barcodes platform was investigated by testing the cytotoxic effect of tegafur (TF), a prodrug of 5-fluorouracil (5-FU), on barcode particle-loaded liver HepG2 and HCT-116 colonic tumor cell spheroids. The cytotoxicity of TF against the HCT-116 tumor cell spheroids was enhanced in systems using cocultures of HepG2 and NIH-3T3 cells, indicating the effectiveness of this multiple cell spheroids-on-barcodes platform for drug screening. KEYWORDS: colloidal crystal, barcode, cell spheroid-on-a-chip, drug screening, microfluidics
1. INTRODUCTION
drug research platform with distinct advantages is still anticipated. In this paper, we present a novel cell spheroids-on-barcodes platform for drug screening, as indicated in Figure 1. Barcode particles, which encode information about their specific compositions and enable simple identification, are attracting a great deal of interest in biomedical fields.20−24 Among various barcode strategies,25−28 photonic crystal (PhC) barcode particles, whose encoding information is carried in their characteristic reflection peaks originating from their photonic band gap (PBG) structure, have distinct advantages of minimal spectral width, remarkable encoding stability, freedom from any fluorescent background, and controllable size.29,30 Thus, they hold significant promise for multiplex bioassays.31−34 However, the potential value of such barcode particles for cell cultures and drug screening remains unexplored, and the creation of completely biocompatible barcode particles is a key factor to enable this goal.35−40 Here, we employed bioactive methacrylated gelatin (GelMA) hydrogel-encapsulated core−shell PhC barcode particles to culture three-dimensional (3D) cell aggregation and for use in drug screening. The hydrogel shells surrounding the barcode particles enable creation of a 3D extracellular matrix (ECM) microenvironment for cell adhesion
Current drug development processes rely heavily on in vitro cell culture protocols and in vivo animal testing. However, simple cell culture assays lack the complexity of living systems and cannot easily mimic the physiology process of drug metabolism or clarify the combined effects on different organs or tissues for drug discovery. Moreover, animal models are difficult to analyze and have inherent complexity, involving many ethical issues. The notion of reconstructing physiological functions at the cellular or organ level on a chip is attracting increasing interest in the fields of drug discovery and biological research.1,2 Through the combination of the multidisciplinary advantages of biomimetics,3 microfabrication,4,5 and microfluidics,6,7 several kinds of organ-on-a-chip microsystems have been developed for screening drug toxicity and for other applications.8−17 However, most of these microsystems are based on a single organ cell source so that they are incapable of modeling situations of organ−organ or tissue−tissue communication and are unable to predict complex drug metabolism and the effect of metabolite activity on nontarget tissues. Although some multiorgan microsystems have been suggested as alternatives,18,19 they require complicated chip integration or cell array preparations. In addition, because of inefficient interactions between the cells of different organs, these multiple organ-on-a-chip microsystems still have difficulty in mimicking actual body situations. Therefore, the development of a new © XXXX American Chemical Society
Received: April 26, 2016 Accepted: May 23, 2016
A
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Schematic diagram of the cell spheroids-on-barcodes platform for drug screening. The GelMA hydrogel-encapsulated green, red, and blue PhC barcode particles were first cultured with HCT-116, NIH-3T3, and HepG2, respectively. Then, they were mixed together and cultured in different drug solutions. The cytotoxic effects of the drugs were tested by screening the cell viabilities on different barcode particles. colloidal crystal beads were calcined at 800 °C for 3 h to improve their mechanical strength. The synthesis process for the GelMa hydrogel was conducted according to a previous report.10 The porcine gelatin (10 g) was first dissolved in 100 mL of PBS buffer at 60 °C for 1 h. Then, methacrylic anhydride (8 mL) was added drop by drop to the solutions under vigorous magnetic stirring at 60 °C for 3 h. The precursor GelMa solution was diluted by preheated PBS buffer (400 mL, 40−50 °C) and rotated at 60 °C for 0.5 h. Finally, the solution was dialyzed against water (9 times, 5 L) for 5 days using a dialysis membrane with an MWCO of 8 000. When the dialysis liquid was lyophilized, the GelMa product was obtained. To synthesize the hydrogel hybrid PhC barcode particles, the PhC barcode particles were first immersed in pregel solutions of different concentrations (0.15, 0.3, and 0.45 g/mL) at 45 °C for 6 h. Then, the solution was polymerized to form a hydrogel by exposure to ultraviolet light (365 nm, 80 W, 30 s). Finally, the hydrogel containing the template spherical silica colloidal crystal cluster (SCCC) was immersed in a buffer solution for swelling. The hydrogel-encapsulated core−shell PhC particles were obtained by selectively etching the nanoparticles in the outer layer of the hydrogel hybrid PhC barcode particles using HF (0.05 vol %) for varying times (30, 40, 50, and 70 min). 2.3. Cell Culture. HepG2, NIH-3T3, and HCT-116 cells were regularly cultured and passaged with DMEM or McCoy’s 5A medium supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37 °C with 5% CO2. The core−shell PhC particles were disinfected by exposure to ultraviolet light for 6 h and washed with a sterile PBS solution 3 times before cell culture. The cells that were loaded on the surface of the core−shell PhC particles were treated using traditional procedures. Briefly, HepG2, NIH-3T3, and HCT-116 cells were seeded on the bottom surface of a 12-well tissue culture plate (2 × 105 cells per well) covered with core−shell PhC particles with different characteristic diffraction peaks for 24 h. Then, the cell-laden particles were transferred into fresh DMEM or McCoy’s 5A medium and cultured for another 48 h. After this process, three kinds of cell-laden particles were achieved: HepG2, HCT-116, and NIH-3T3 cell-laden particles. Then, the particle-loaded liver HepG2 and HCT-116 colonic tumor cell spheroids and normal NIH-3T3 fibroblasts were selectively mixed and transferred into a complex medium (7/3 VDMEM/VMcCoy’s 5A) to form three different coculture systems (mixtures of HCT-116 and NIH-3T3, HCT-116 and HepG2, and all three kinds of cells) for cytotoxicity testing with TF or 5-FU for 48 h. The concentrations of both TF and 5-FU were 100 and 200 μM. HepG2, NIH-3T3, and HCT-116 cell-laden particles were also separately cultured as control experiments under the same conditions.
and growth, while the PhC cores of the barcode particles offer stable diffraction peaks to encode different 3D cell aggregation types during culture and distinguish the biological responses of these cells during drug testing. The applicability of this platform was confirmed by investigating the cytotoxic effect of tegafur (TF), a prodrug of 5-fluorouracil (5-FU), on barcode particles loaded with liver and tumor cell spheroids. These features make our cell spheroids-on-barcodes platform quite promising for drug development.
2. EXPERIMENTAL SECTION 2.1. Materials. Six kinds of SiO2 nanoparticles with lengths of 218, 230, 255, 275, and 300 nm were purchased from Nanjing Dongjian Biological Technology Co., Ltd. GelMa hydrogel was self-prepared. Gelatin from porcine skin, methacrylic anhydride, dimethyl sulfoxide (DMSO), 5-fluorouracil, Hoechst 33342, and 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Tegafur was purchased from Shanghai Yanyi Biotechnology Corporation (Shanghai, China). Calcein-AM (molecular prober) was purchased from Life Technologies (Waltham, MA), and glutaraldehyde was purchased from Aladdin (Shanghai, China). HepG2 cells (a human hepatocellular carcinoma cell line), HCT-116 cells (the colon cancer cell line), and NIH-3T3 cells (normal fibroblast cells) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (China), and McCoy’s 5A medium was purchased from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). Penicillin-streptomycin was obtained from Gibco (Waltham, MA). Cellulose dialysis membranes (molecular weight cutoff (MWCO) = 8 000−14 000) were acquired from Shanghai Yuanye Biotechnology Corporation (Shanghai, China). Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 MΩ cm. 2.2. Preparation of Core−Shell PhC Barcode Particles. The barcode particles, which were assembled by silica nanoparticles with different diameters, were fabricated by the droplet template method according to our previous work.3 Briefly, SiO2 nanoparticles (20 wt %) in a variety of particle sizes (218, 230, 255, 275, and 300 nm) and with a good monodispersity were dispersed in water. The monodispersed emulsion droplets were generated using single-emulsion glass capillary microfluidics. The flow rates of the continuous and dispersed phases were 10 and 0.5 mL/h, respectively. Then, the immature barcode particles were formed after being washed and dried. Finally, the silica B
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. Generation of core−shell PhC particles. (a) Schematic diagram of the core−shell PhC particle generation. SEM images of (b) the SCCC template surface, (c) the hydrogel hybrid SCCC surface, (d) the core−shell particle surface, and (e−g) cross sections of the core−shell particles under different magnifications. The insets of b−d are SEM images of (b) the SCCC template particle, (c) the hydrogel hybrid SCCC particle, and (d) the core−shell particle. Scale bars are 500 nm in b−d, 100 μm in e, and 2 μm in f and g. 2.4. Cytotoxicity Assays and Cell Morphology Observations. The cell mixtures of HCT-116 and NIH-3T3, HCT-116 and HepG2, and HCT-116, HepG2, and NIH-3T3 were cultured in the complex medium supplemented with TF or 5-FU at concentrations of 100 or 200 μM for 48 h. After incubation for 48 h, the different cell-laden particles were separated, and the MTT/PBS solution (5 μg/mL) was added. The different cell particles were separated depending on the photonic crystal structural colors under an ordinary microscope. During this process, the particles were flowed into a microchannel with several cross subchannels. The particles were separated into these cross channels depending on their color using a fluid pulse that was opposite to these cross channels. There were 10 cell-laden particles in each well. MTT assays of the different cell-laden particles were carried out according to the manufacturer’s instructions. The mean value and standard deviation of five paralleled assays for each sample were recorded. To test the different cell availabilities of these coculture systems under the same conditions, the HepG2, NIH-3T3, and HCT116 cell-laden particles that were separately cultured without TF or 5FU were set as control experiments. The HepG2, HepG2 and HCT116 mixture, and three-cell mixture systems were used for P450 (CYP3A4) activity assays. The P450 (CYP3A4) activity assays were performed according to the manufacturer’s protocol (Promega). The morphology of the cell-laden microsystems was also observed. The cell-laden particles were transferred to a new culture dish after treatment with 100 μM TF or 5-FU for 48 h. Then, the cell-laden particles were counterstained with calcein AM (2 μg/mL, 2 mL per well) for 20 min at 37 °C followed by being washed twice with PBS and treated with glutaraldehyde (2.5%, 2 mL per well) for 6 h at 4 °C. The cell-laden particles were also counterstained with Hoechst 33342 (1 μg/mL, 2 mL per well) for 20 min at 37 °C. Finally, the cells were observed using an inverted fluorescence microscope. To characterize the cell morphology, the cell-loaded PhC particles were washed repeatedly and dehydrated with gradient ethanol (20%, 40%, 60%, 80%, and 100%) before SEM characterization. 2.5. Characterization. Reflection spectra were obtained at a fixed glancing angle using an optical microscope equipped with a fiber optic spectrometer (Ocean Optics, USB2000-FLG). Morphologies of the particles were explored by SEM images using a field emission scanning
electron microscope (FESEM, Ultra Plus, Zeiss). SEM images of the cells laden on the surface of the PhC particles were taken by scanning electron microscopy (SEM, Hitachi S-3000N). Microscope images of the beads were obtained with an optical microscope (Olympus BX51) equipped with a CCD camera (Media Cybernetics Evolution MP5.0). 2.6. Statistical Analysis. The one-way analysis of variance (ANOVA) statistical method was employed to evaluate the significance of the experimental data. A value of 0.05 was selected as the significance level, and the data were labeled with a single asterisk for p < 0.05, two asterisks for p < 0.01, and three asterisks for p < 0.001.
3. RESULTS AND DISCUSSION 3.1. Preparation of the Core−Shell PhC Particles. In a typical experiment, the hydrogel-encapsulated core−shell PhC barcode particles were fabricated by replicating SCCC templates, as indicated in Figure 2a. These SCCC templates in well-controlled sizes of several hundred micrometers were prepared by the self-assembly of silica nanoparticles in droplets, which became closely packed and formed an ordered spherical cluster structure during dehydration (Figure 2b). This ordered packing of the nanoparticles endowed the clusters with interconnected nanopores throughout the templates that enabled infiltration of the GelMA pregel solution. After the pregel solution had penetrated the nanopores and filled all voids in the templates by capillary action, the solution was polymerized to form a hydrogel by exposure to ultraviolet light. Then, the hydrogel containing the template SCCC was immersed in a buffer solution for swelling. Because the hydrogel within the nanopores of the SCCCs was difficult to swell consistently with the outer hydrogel due to the fixed template space, the surface layer became ruptured. Thus, when the hydrogel was mechanically disrupted, the hybrid SCCC particles at the site of the rupture fell off and became separate (Figure 2c). Finally, the hydrogel-encapsulated core−shell PhC C
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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(Figure S1a). Thus, reflection spectra with two distinguishable peaks, one from the close-packed silica nanoparticle core and one from the inversely opaline hydrogel shell, could be observed from these core−shell PhC particles (Figure S1b). Although the numbers of encoding PhC particles could be increased in theory by using two peak spectra, the peaks from the hydrogel change when cells are cultured on their surfaces: this would result in erroneous decoding. In contrast, low concentrations (0.15 g/mL) of the hydrogel could impart the particles with a transparent shell layer (Figure 4), and only one stable reflection peak would be observed from the particles. However, if the hydrogel concentration was too low, then this would decrease the stability of the shell layer structures of the particles during cell culture. Therefore, core−shell PhC particles with an optimized GelMA hydrogel concentration of 0.3 g/mL were fabricated for the following experiments. 3.3. Cell Spheroid Construction on the Core−Shell PhC Particles. To investigate the biological performance of the core−shell PhC particles, those with different shell thicknesses were employed for HepG2 (hepatocellular carcinoma) cell cultures, as shown in Figure 5. However, only small cells could attach to the surface of the SCCC templates (Figure 5a), and these fell off the templates easily during washing and replacement of the medium because of weak adhesion between the cells and the silica nanoparticles. The effect of the GelMA hydrogel in the nanopores of the hybrid SCCCs improved cell attachment (Figure 5b), and when the particles were treated to form core−shell structures, they achieved effective cell adhesion and growth (Figure 5c). This can be ascribed to the formation of a 3D ECM-like microenvironment by the GelMA hydrogel shell layers, which was more beneficial for the cell cultures than the twodimensional (2D) structures on the hybrid SCCC surfaces. There were no obvious differences in cell culture efficacy among the various core−shell particles with different shell layer thicknesses (Figures 5c−e). This was confirmed by the results of the MTT assays of the HepG2 cells (Figure S2), which is the most common method for quantifying cell viability on different particle substrates. Thus, given the ease of fabrication, the core−shell PhC particles with thin GelMA hydrogel shell layers were suitable for constructing the cell spheroids-on-barcodes platform. This was also confirmed by confocal laser scanning microscopy (CLSM) images of the HepG2 cells cultured on PhC core−shell particles with little etching, as shown in Figure S3. To implement the concept of cell spheroids-on-barcodes, different kinds of cell spheroids were cultured on distinguishable barcode particles. Then, the cell spheroid-laden barcode particles were mixed and incubated in a drug solution. Performance of the drugs can be screened by contrasting and
particles were obtained by selectively etching the outer layer of the silica nanoparticles, leaving an inverse opaline hydrogel shell structure of the hybrid SCCCs (Figures 2d−g). 3.2. Optical Characterization of the Core−Shell PhC Barcode Particles. Because of the orderly arranged structure of the composed silica nanoparticles, the SCCC templates and their resulting particles were all imparted with a unique photonic band gap (PBG) property. This leads to light with certain wavelengths or frequencies located in the PBG being prohibited from propagating through the materials and therefore reflected. Thus, the SCCC templates, hybrid SCCCs, and core−shell PhC particles all showed vivid colors and possessed characteristic reflection peaks (Figure 3). Under
Figure 3. (a−c) Optical microscopy images of the SCCC templates, hydrogel hybrid SCCCs, and core−shell PhC particles, respectively. Scale bar is 200 μm. (d) Optical images and reflection spectra of six kinds of core−shell PhC barcode particles.
normal conditions, the main reflection peak position λ of the core−shell PhC particles can be estimated by Bragg’s equation, λ = 1.633dnaverage, where d is the center-to-center distance between two neighboring silica nanoparticles and naverage is the average refractive index of the particles. Therefore, when different sizes of silica nanoparticles are employed, a series of core−shell PhC particles with different diffraction peaks and structural colors can be obtained, which can then be used for encoding (Figure 3d). Note that the light reflection spectra of the core−shell PhC particles are also affected by the concentration of the infiltrated hydrogels and the thickness of the etched shell layers. As the hydrogel concentration (0.45 g/mL) and shell layer thickness increased, the transparency of the shell decreased, and iridescent colors appeared in the shell layers of the particles
Figure 4. Optical images of the core−shell PhC particles with a transparent shell layer structure. The etching times were (a) 30, (b) 40, and (c) 50 min. Scale bar is 200 μm. D
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 5. Comparison of cell attachment on different particles. Hoechst 33342 fluorescent images (first column), calcein AM fluorescent images (second column), and optical microscopy images (third column) of HepG2 cells cultured on different particles for 48 h. Cells were cultured on (a) the SCCC templates, (b) the GelMa hydrogel hybrid SCCCs, (c) the core−shell PhC particles with little etching, (d) the core−shell PhC particles with medium etching, and (e) the core−shell PhC particles with thick etching. The purpose of using Hoechst and calcein staining was to confirm that these cells were attached to the surface of the particles. Scale bar is 200 μm.
analyzing the growth rates of cell spheroids on each kind of barcode particle. An important requirement for carrying out this process is the need for stability of the barcodes during cell culture. Although general encoding strategies that use fluorescent dyes, quantum dots, or photopatterning can achieve a large number of distinct codes, they might become difficult to read when cell spheroids are cultured on their surfaces or give inaccurate outcomes due to cell staining. Here, we overcame this restriction by employing the PhC reflection peaks of the GelMA hydrogel-encapsulated core−shell particles and reading the cell information from the surface cell statistical results. When cell spheroids were cultured or stained on the barcode particles, they interacted with only the surface hydrogel and did not affect the periodic structure or refractive index of the PhC core. Thus, the encoded reflection peak positions of our barcode particles remained constant during cell adhesion and growth, as shown in Figure S4. This indicates the high encoding
accuracy of the core−shell PhC particles for cell spheroids-onbarcodes applications. 3.4. Cell Spheroids-on-Barcodes Platform for Drug Screening. To demonstrate the reliability of this platform for drug screening, HepG2 and HCT-116 cells and NIH-3T3 fibroblast cells were cultured on three kinds of GelMAencapsulated core−shell PhC particles (encoded reflection peaks at 606, 560, and 480 nm) to mimic liver, colon tumor, and normal tissue cell spheroids, respectively. All PhC barcode particles were suitable for the three kinds of cell culture and proliferation (Figure S5). More importantly, all cell types cultured could maintain high activity and excellent phenotype configuration (Figure 6). These outcomes are important for reproducing the key functional units of native organs using simple cell spheroids. After growth of mature liver, colon tumor, and normal tissue cell spheroids was achieved, the laden barcode particles were selectively mixed and employed for E
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 6. SEM images of different cell spheroids cultured on the particles. (a) HepG2, (b) NIH-3T3, and (c) HCT-116 cells. (d−f) Surface amplification images of a−c, respectively. Scale bars are 100 μm in a−c and 30 μm in d−f.
Figure 7. TF and 5-FU drug screening on the organs-on-barcodes platform. Hoechst 33342 fluorescent images, calcein AM fluorescent images, and optical microscopy images of the HCT-116 and HepG2 coculture systems (a) before and (b) after TF drug treatment for 48 h. The concentration of TF was 100 μM. Scale bar is 200 μm. Results of the cell MTT assays of the (c) HCT-116 and (d) HepG2 coculture systems before and after the TF or 5-FU drug treatments for 48 h. Results of the cell MTT assays of the (e) HCT-116 and (f) HepG2 and NIH-3T3 coculture systems before and after the TF or 5-FU drug treatments. There were 10 replicates for each group. HepG2 and HCT-116 cell-laden particles, which were separately cultured without TF or 5-FU, were set as control experiments, and the cellular activity of these cells was set as 100%. Error bars represent standard deviations.
F
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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these cells during drug testing. On the basis of these core−shell PhC barcode particles, HepG2, HCT-116, and NIH-3T3 were cocultured as liver and tumor cell spheroids to test the cytotoxic effect of TF. The system appeared to reproduce the hepatic function of synthesizing P450 enzymes, which could convert the noncytotoxic TF to cytotoxic 5-FU and reveal its cytotoxicity. These features indicate the effectiveness of our multiple cell spheroids-on-barcodes platform and make them highly promising for new drug development. As in traditional organs-on-chips microsystems, our cell spheroids-on-barcodes platform was also aimed at creating physiologically relevant models of human tissues and organs to investigate the basic mechanisms of organ physiology and disease. However, organs-on-chips microsystems have some fundamental limitations, such as being incapable of modeling situations of organ−organ or tissue−tissue communication due to the single organ cell source, requiring complicated chip integration or cell array preparations for the multiorgan microsystems construction, and having difficulty in mimicking actual body situations because of inefficient interactions between the cells of different organs. Conversely, our platform was flexibly composed of barcode particle-laden cell spheroids and thus avoided many of the limitations of organs-on-chips microsystems. In the principle work, we focused on a hepatictumorous model and applied the platform to two assays of cytotoxicity testing, TF and 5-FU, that are commonly used in the pharmaceutical industry. On the basis of this work, we have demonstrated the advantages of our cell spheroids-on-barcodes platform in facilitating construction, effectively modeling body situations, and accurately predicting complex drug metabolism. In the future, we propose that the cell spheroids-on-barcodes platform be expanded to conduct a large number of additional applications, such as the panel of preclinical assays of other drugs. Likewise, other cell spheroids may be compatible with this platform, formed by incorporating different barcode particle-laden cell spheroids. Thus, we conclude that the cell spheroids-on-barcodes platform is a promising strategy for drug screening.
testing of the cytotoxic effects of TF and 5-FU. 5-FU is an anticancer drug that has been widely used to treat colon cancers. TF is an oral prodrug of 5-FU and is not immediately cytotoxic to tumor cells but becomes so after conversion to 5FU by P450 enzymes in the liver. Thus, we expected to be able to screen the pharmacokinetic effects of these drugs based on our cell spheroids-on-barcodes platform. For this purpose, the HCT-116 and NIH-3T3 cell spheroidloaded barcode particles were first cultured separately or together in a TF solution for 48 h. No cytotoxicity of this agent to the NIH-3T3 or HCT-116 cells was obvious (Figure S6), and the cells retained their high activity during the assays (>90% in the MTT viability assay, Figure S7). This indicated the low direct cytotoxicity of TF to cells, consistent with the description above. However, when the cell spheroids-onbarcodes platforms loaded with HCT-116 and HepG2 cell spheroids were tested, TF caused significant decreases in cell viability, as shown in Figure 7 and Figure S8. The viabilities of the HCT-116 and HepG2 cells decreased to 83.89 ± 0.80% and 83.02 ± 0.95%, respectively (Figures 7c and d). These data are consistent with those seen when isolated cells were cultured directly with 5-FU with cell viabilities of 71.37 ± 1.42% and 75.51 ± 1.20% for the HCT-116 and HepG2 cells on the bead surfaces, respectively (Figures 7c and d and Figure S9), and 73.29 ± 2.42% and 76.93 ± 1.81% for the HCT-116 and HepG2 cells on the plate surfaces, respectively. From these results, we conclude that barcode particles loaded with HepG2 cell spheroids can effectively reproduce some functions of the liver such as the synthesis of P450 enzymes that convert noncytotoxic TF to cytotoxic 5-FU. To further evaluate the function of these multiple cell spheroids-on-barcodes platforms, barcode particles loaded with normal NIH-3T3 cell spheroids were also cocultured with HCT-116 and HepG2 cell spheroid-loaded barcode particles. The presence of NIH-3T3 cells in conventional cultures can improve the viability of many organ cell types, such as HepG2 cells. Thus, we expected that this multiple cell spheroids-onbarcodes system would also show a better performance. The results are shown in Figure S10 and Figures 7e and f. Treatment with TF led to a decrease in viability in all three kinds of cells. Compared with those of the HCT-116/HepG2 or HepG2/NIH-3T3 cell spheroid coculture systems (Figure S11), the effects of TF were enhanced slightly, and the viabilities of the HCT-116 and HepG2 cells further decreased to 81.35 ± 1.24% and 76.43 ± 1.40%, respectively. These results were consistent with our expectations and indicated that NIH-3T3 cells contribute to maintaining higher activity and higher cytochrome P450 expression by HepG2 cells (Figure S12). Thus, our multiple cell spheroids-on-barcodes system could effectively reproduce some basic features of different organs and could be employed as a potential platform for drug screening.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04966. Optical characterization of the core−shell PhC particles, results of the cell MTT assays based on the core−shell PhC particles, CLSM images of the HepG2 cells, reflection spectra of the core−shell PhC particles, images of different cells cultured on the core−shell PhC particles, images of different cells, MTT assays of different cells, optical microscopy images of the HCT116 and HepG2 coculture systems, images of HCT-116, HepG2, and NIH-3T3 coculture systems, and cytochrome P450 (CYP3A4) activity in HepG2 cell-laden PhC core−shell particles (PDF)
4. CONCLUSION In summary, we developed novel GelMA hydrogel-encapsulated core−shell PhC barcode particles for use in different organ cell cultures and drug screening. The GelMA hydrogel shells of the barcode particles enabled the creation of a 3D ECM microenvironment and were suitable for the adhesion and growth of different cell types with high viability. The PhC cores of the barcode particles gave stable diffraction peaks for encoding different types of cell spheroids on their shell layers during culture and distinguishing the biological responses of
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions †
F.F. and L.S. contributed equally to this work.
Notes
The authors declare no competing financial interest. G
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
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(17) Sung, J. H.; Shuler, M. L. A Micro Cell Culture Analog (μCCA) With 3-D Hydrogel Culture of Multiple Cell Lines to Assess Metabolism-Dependent Cytotoxicity of Anti-Cancer Drugs. Lab Chip 2009, 9, 1385−1394. (18) Liu, X. L.; Wang, S. T. Three-Dimensional Nano-Biointerface As a New Platform for Guiding Cell Fate. Chem. Soc. Rev. 2014, 43, 2385−2401. (19) Stevens, M. M.; George, J. H. Exploring and Engineering the Cell Surface Interface. Science 2005, 310, 1135−1138. (20) Kim, S. H.; Shim, J. W.; Yang, S. M. Microfluidic Multicolor Encoding of Microspheres with Nanoscopic Surface Complexity for Multiplex Immunoassays. Angew. Chem., Int. Ed. 2011, 50, 1171−1174. (21) Ye, B. F.; Ding, H. B.; Cheng, Y.; Gu, H. C.; Zhao, Y. J.; Xie, Z. Y.; Gu, Z. Z. Photonic Crystal Microcapsules for Label-Free Multiplex Detection. Adv. Mater. 2014, 26, 3270−3274. (22) Kim, S. H.; Shum, H. C.; Kim, J. W.; Cho, J. C.; Weitz, D. A. Multiple Polymersomes for Programmed Release of Multiple Components. J. Am. Chem. Soc. 2011, 133, 15165−15171. (23) Liu, W.; Shang, L. R.; Zheng, F. Y.; Qian, J. L.; Lu, J.; Zhao, Y. J.; Gu, Z. Z. Photonic Crystal Encoded Microcarriers for Biomaterial Evaluation. Small 2014, 10, 88−90. (24) Hou, J.; Zhang, H. H.; Yang, Q.; Li, M. Z.; Jiang, L.; Song, Y. L. Hydrophilic-Hydrophobic Patterned Molecularly Imprinted Photonic Crystal Sensors for High-Sensitive Colorimetric Detection of Tetracycline. Small 2015, 11, 2738−2742. (25) Kuang, M.; Wang, D.; Bao, H.; Gao, M.; Möhwald, H.; Jiang, M. Fabrication of Multicolor-Encoded Microspheres by Tagging Semiconductor Nanocrystals to Hydrogel Spheres. Adv. Mater. 2005, 17, 267−270. (26) Lee, H.; Kim, J.; Kim, H.; Kim, J.; Kwon, S. Colour-Barcoded Magnetic Microparticles for Multiplexed Bioassays. Nat. Mater. 2010, 9, 745−749. (27) Liu, H.; Li, Y.; Sun, K.; Fan, J.; Zhang, P.; Meng, J.; Wang, S.; Jiang, L. Dual-Responsive Surfaces Modified with Phenylboronic AcidContaining Polymer Brush to Reversibly Capture and Release Cancer Cells. J. Am. Chem. Soc. 2013, 135, 7603−7609. (28) Bettinger, C. J.; Langer, R.; Borenstein, J. T. Engineering Substrate Topography at The Micro- and Nanoscale to Control Cell Function. Angew. Chem., Int. Ed. 2009, 48, 5406−5415. (29) Lee, H. S.; Kim, J. H.; Lee, J. S.; Sim, J. Y.; Seo, J. Y.; Oh, Y. K.; Yang, S. M.; Kim, S. H. Magnetoresponsive Discoidal Photonic Crystals Toward Active Color Pigments. Adv. Mater. 2014, 26, 5801− 5807. (30) Zhao, Y. J.; Shang, L. R.; Cheng, Y.; Gu, Z. Z. Spherical Colloidal Photonic Crystals. Acc. Chem. Res. 2014, 47, 3632−3642. (31) Ge, J. P.; Yin, Y. D. Responsive Photonic Crystals. Angew. Chem., Int. Ed. 2011, 50, 1492−1522. (32) Zhao, Y. J.; Cheng, Y.; Shang, L. R.; Wang, J.; Xie, Z. Y.; Gu, Z. Z. Microfluidic Synthesis of Barcode Particles for Multiplex Assays. Small 2015, 11, 151−174. (33) Shang, L. R.; Fu, F. F.; Cheng, Y.; Wang, H.; Liu, Y. X.; Zhao, Y. J.; Gu, Z. Z. Photonic Crystal Microbubbles as Suspension Barcodes. J. Am. Chem. Soc. 2015, 137, 15533−15539. (34) Ruan, S. B.; Zhang, L.; Chen, J. T.; Cao, T. W.; Yang, Y. T.; Liu, Y. Y.; He, Q.; Gao, F. B.; Gao, H. L. Targeting Delivery and Deep Penetration using Multistage Nanoparticles for Triple-Negative Breast Cancer. RSC Adv. 2015, 5, 64303−64317. (35) Lee, J.; Bisso, P. W.; Srinivas, R. L.; Kim, J. J.; Swiston, A. J.; Doyle, P. S. Universal Process-Inert Encoding Architecture for Polymer Microparticles. Nat. Mater. 2014, 13, 524−529. (36) Gou, M.; Qu, X.; Zhu, W.; Xiang, M. L.; Yang, J.; Zhang, K.; Wei, Y. Q.; Chen, S. C. Bio-Inspired Detoxification using 3D-Printed Hydrogel Nanocomposites. Nat. Commun. 2014, 5, 3774. (37) Wang, S. T.; Liu, K.; Liu, J.; Yu, Z. T. F.; Xu, X. W.; Zhao, L.; Lee, T.; Lee, E. K.; Reiss, J.; Lee, Y. K.; Chung, L. W. K.; Huang, J. T.; Rettig, M.; Seligson, D.; Duraiswamy, K. N.; Shen, C. K. F.; Tseng, H. R. Highly Efficient Capture of Circulating Tumor Cells by Using Nanostructured Silicon Substrates with Integrated Chaotic Micromixers. Angew. Chem., Int. Ed. 2011, 50, 3084−3088.
ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (Grants 21473029 and 51522302), the NSAF Foundation of China (Grant U1530260), the National Science Foundation of Jiangsu (Grant BK20140028), the Research Fund for the Doctoral Program of Higher Education of China (Grant 20120092130006), the Program for New Century Excellent Talents in University, and the Scientific Research Foundation of Southeast University.
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REFERENCES
(1) Lee, W.; Park, J. The Design of a Heterocellular 3D Architecture and Its Application to Monitoring the Behavior of Cancer Cells in Response to The Spatial Distribution of Endothelial Cells. Adv. Mater. 2012, 24, 5339−5344. (2) Goral, V. N.; Au, S. H.; Faris, R. A.; Yuen, P. K. Methods for Advanced Hepatocyte Cell Culture in Microwells Utilizing Air Bubbles. Lab Chip 2015, 15, 1032−1037. (3) Zheng, F. Y.; Cheng, Y.; Wang, J.; Lu, J.; Zhang, B.; Zhao, Y. J.; Gu, Z. Z. Aptamer-Functionalized Barcode Particles for The Capture and Detection of Multiple Types of Circulating Tumor Cells. Adv. Mater. 2014, 26, 7333−7338. (4) Feng, Q.; Zhang, L.; Liu, C.; Li, X. Y.; Hu, G. Q.; Sun, J. S.; Jiang, X. Y. Microfluidic Based High Throughput Synthesis of Lipid-Polymer Hybrid Nanoparticles with Tunable Diameters. Biomicrofluidics 2015, 9, 052604. (5) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Cells on Chips. Nature 2006, 442, 403−411. (6) Park, T. H.; Shuler, M. L. Integration of Cell Culture and Microfabrication Technology. Biotechnol. Prog. 2003, 19, 243−253. (7) Li, Y.; Wang, S. W.; Huang, R.; Huang, Z.; Hu, B. F.; Zheng, W. F.; Yang, G.; Jiang, X. Evaluation of The Effect of The Structure of Bacterial Cellulose on Full Thickness Skin Wound Repair on a Microfluidic Chip. Biomacromolecules 2015, 16, 780−789. (8) Frey, O.; Misun, P. M.; Fluri, D. A.; Hengstler, J. G.; Hierlemann, A. Reconfigurable Microfluidic Hanging Drop Network for Multi-Tissue Interaction and Analysis. Nat. Commun. 2014, 5, 4250. (9) Cheng, Y.; Zheng, F. Y.; Lu, J.; Shang, R. L.; Xie, Z. Y.; Zhao, Y. J.; Chen, Y. P.; Gu, Z. Z. Bioinspired Multicompartmental Microfibers from Microfluidics. Adv. Mater. 2014, 26, 5184−5190. (10) Yue, K.; Trujillo de Santiago, G.; Alvarez, M. M.; Tamayol, A.; Annabi, N.; Khademhosseini, A. Synthesis, Properties, and Biomedical Applications of Gelatin Methacryloyl (GelMA) Hydrogels. Biomaterials 2015, 73, 254−271. (11) Allazetta, S.; Kolb, L.; Zerbib, S.; Bardy, J.; Lutolf, M. P. CellInstructive Microgels with Tailor-Made Physicochemical Properties. Small 2015, 11, 5647−5656. (12) Zhang, B.; Montgomery, M.; Chamberlain, M. D.; Ogawa, S.; Korolj, A.; Pahnke, A.; Wells, L. A.; Massé, S.; Kim, J.; Reis, L.; Momen, A.; Nunes, S. S.; Wheeler, A. R.; Nanthakumar, K.; Keller, Go.; Sefton, M. V.; Radisic, M. Biodegradable Scaffold with Built-in Vasculature for Organ-On-a-Chip Engineering and Direct Surgical Anastomosis. Nat. Mater. 2016, DOI: 10.1038/nmat4570. (13) Ma, C.; Zhao, L.; Zhou, E. M.; Xu, J.; Shen, S. F.; Wang, J. Y. On-Chip Construction of Liver Lobule-like Microtissue and Its Application for Adverse Drug Reaction Assay. Anal. Chem. 2016, 88, 1719−1727. (14) Pati, F.; Gantelius, J.; Svahn, H. A. 3D Bioprinting of Tissue/ Organ Models. Angew. Chem., Int. Ed. 2016, 55, 2−18. (15) Ma, M. L.; Chiu, A.; Sahay, G.; Doloff, J. C.; Dholakia, N.; Thakrar, R.; Cohen, J.; Vegas, A.; Chen, D.; Bratlie, K. M.; Dang, T.; York, R. L.; Hollister-Lock, J.; Weir, G. C.; Anderson, D. G. Core− Shell Hydrogel Microcapsules for Improved Islets Encapsulation. Adv. Healthcare Mater. 2013, 2, 667−672. (16) Khetani, S. R.; Bhatia, S. N. Microscale Culture of Human Liver Cells for Drug Development. Nat. Biotechnol. 2007, 26, 120−126. H
DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces (38) Yu, L.; Liu, X. K.; Yuan, W. C.; Brown, L. J.; Wang, D. Y. Confined Flocculation of Ionic Pollutants by Poly (L-dopa)-Based Polyelectrolyte Complexes in Hydrogel Beads for Three-Dimensional, Quantitative, Efficient Water Decontamination. Langmuir 2015, 31, 6351−6366. (39) Pan, G. Q.; Guo, B. B.; Ma, Y.; Cui, W. G.; He, F.; Li, B.; Yang, H. L.; Shea, K. J. Dynamic Introduction of Cell Adhesive Factor via Reversible Multicovalent Phenylboronic Acid/cis-diol Polymeric Complexes. J. Am. Chem. Soc. 2014, 136, 6203−6206. (40) Bai, S.; Nguyen, T. L.; Mulvaney, P.; Wang, D. Y. Using Hydrogels to Accommodate Hydrophobic Nanoparticles in Aqueous Media via Solvent Exchange. Adv. Mater. 2010, 22, 3247−3250.
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DOI: 10.1021/acsami.6b04966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
