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Biological and Medical Applications of Materials and Interfaces

Biodegradable Anisotropic Microparticles for Stepwise Cell Adhesion and Preparation of Janus Cell Microparticles Honghao Zheng, Wang Du, Yiyuan Duan, Keyu Geng, Jun Deng, and Changyou Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14884 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 7, 2018

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ACS Applied Materials & Interfaces

Biodegradable Anisotropic Microparticles for Stepwise Cell Adhesion and Preparation of Janus Cell Microparticles Honghao Zheng1, Wang Du1, Yiyuan Duan1,2, Keyu Geng1, Jun Deng1, Changyou Gao1,2* 1 MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China 2 Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University, Hangzhou 310058, China *Corresponding author E-mail: [email protected] KEYWORDS: microparticles, biomaterials, anisotropic morphology, Janus particles, cell adhesion

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ABSTRACT: The biomimetic anisotropic particles have different physicochemical properties on the opposite two sides, enabling diverse applications in emulsion, photonic display, and diagnosis. However, the traditional anisotropic particles have a very small size, ranging from sub-microns to a few microns. The design and fabrication of anisotropic macron-sized particles with new structures and properties is still challenging. In this study, anisotropic polycaprolactone (PCL) microparticles well separated with each other were prepared by crystallization from the dilute PCL solution in a porous 3D gelatin template. They had fuzzy and smooth surfaces on each side, and a size as large as 70 µm. The fuzzy surface of the particle adsorbed significantly larger amount of proteins, and were more cell-attractive regardless of the cell types. The particles showed stronger affinity to fibroblasts over hepatocytes, which paved a new way for cell isolation merely based on the surface morphology. After a successive seeding process, Janus cell microparticles with fibroblasts and endothelial cells (ECs) on each side were designed and obtained by making use of the anisotropic surface morphology, which showed significant difference in ECs functions in terms of prostacyclin (PGl2) secretion, demonstrating the unique and appealing functions of this type of anisotropic microspheres.


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1. INTRODUCTION Asymmetry exists ubiquitously in nature, especially in biology systems. Most biological molecules (amino acids, glucose molecule and lipids), self-organized superstructures (DNAs and proteins), and tissues are highly asymmetric in chemistry and physics to guide special functions and allow complex biological processes. A well-known case is that the asymmetric structure of lipids gives rise to selforganization into bilayers to protect cells from the external environment, which contributes to the formation of organisms. This phenomenon provides the very significant inspiration for developing asymmetric materials, in which Janus particle is one of the most representative examples. 1-4When placed in a specific external environment, the Janus particles with the asymmetric chemical composition exhibit some unique functions such as emulsification, self-assembly and molecular recognition. 5,6 These features enable a broad range of applications, for instance, amphiphilic surfactants, 7-12 photonic display materials, 13, 14

and self-propulsion motors.

15-17

After being decorated by bioactive molecules,

18-19

the Janus

particles can be endowed with complex functionalities in specific fields with ingenious biochemical properties, making them promising for diverse applications disease diagnosis,

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imaging

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and therapy.

26, 27

20, 21

throughout the whole processes of

The Janus particles with amphiphilic properties are

more easily adsorbed onto oil–water interfaces than the corresponding isotropic particles, and their larger size enables them to segregate to and remain at interfaces more strongly than molecular surfactants.28,29 Recently, magnetic Janus particles are reported with a convex hydrophilic surface/concave oleophilic surface by emulsion interfacial polymerization and selective surface assembly, realizing the rapid and efficient separation of tiny oil droplets from water.30 The potential of Janus particles in biomedical applications has also been widely reported in synergistic drug delivery, cell targeting, multimodal bioimaging, biosensing, and combinations of these capacities.31 Unlike uniform particles, Janus particles allow diverse or incompatible functions to be combined in one structural entity. A single Janus particle with multiple compartments can serve as an ideal carrier for


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drugs of different water solubilities. This type of particles offers a promising strategy for enhancing the efficacy of combination therapy, which requires the combined delivery of hydrophobic chemotherapeutic drugs and highly charged small interfering RNAs (siRNAs) that serve to prevent drug resistance.32 In addition to Janus grains with asymmetric chemical properties, a variety of novel material systems containing asymmetric shape,

33, 34

size

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and structure

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are conceived and fabricated. The typical

examples are patchy particles, multi-compartment particles and complex particles. 37 However, the design and fabrication of asymmetric material systems with new structures and properties is still challenging. Moreover, endorsement of novel functions is of both scientific and technical significance to promote the development and application of asymmetric materials.38-42 The traditional anisotropic particles have a very small size, ranging from sub-microns to a few microns. 43

The small anisotropic particles are usually used as carrier for loading and delivery of functional

molecules. However, they are apparently too small to function as cell carriers, and to construct Janus cell particles. In this work, well dispersed Janus microparticles with a size as large as 70 µm and asymmetric surface morphology are fabricated by using biodegradable polycaprolactone (PCL). Each particle has a distinct fuzzy and a smooth surface on the opposite side, which is composed of the pure PCL polymers. It is known that the surface morphology of a material has a significant impact on cell behaviors such as adhesion and differentiation. For example, the rough surface is advantageous for the adhesion of osteoblasts,

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differentiation.

endothelial cells and stem cells, which influences further the cell proliferation and 45

Some deliberately designed topological features can induce cell orientation and

directional migration.

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With the advantage of large size, the adhesion behaviors of cells can be

compared directly on one anisotropic PCL microparticle. Finally, different types of cells are attached onto the two faces, forming a Janus cell microparticle which is a simplified micro-organ model to mimic a real heterogeneous tissue.


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2. EXPERIMENT SECTION 2.1 Materials PCL (Mn = 80 kDa), rhodamin B isothiocyanate (RBITC), and fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) were purchased from Sigma-Aldrich. Fibronectin (Fn) was purchased from Gibco Company. Tetrahydrofuran (THF), ethanol and tert-butanol were obtained from Chinese Medicine Company and purified by standard methods. Gelatin was purchased from Chinese Medicine Company and sieved to collect the particles with a size of 280~450 µm. The water used in all the experiments was treated in a Millipore Milli-Q Reference purification system.

2.2 Preparation of Anisotropic PCL Particles The gelatin template fabricated from the gelatin particles

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was immersed into 5% PCL/THF dilute

solution and maintained under a low pressure of 0.07~0.08 MPa. After the pressure was released, the PCL/THF solution-filled gelatin template was kept at 37 oC for 5 min, coarsened in a -20 oC refrigerator overnight to allow sufficient phase separation, and finally freeze-dried. The gelatin template was dissolved in Millipore water at 37 oC to obtain the anisotropic PCL particles. Their shape and size were observed by an optical microscope (IX81, Olympus, Japan), and the surface structure was imaged by scanning electron microscopy (SEM, HITACHI S-4800). The chemical composition of the particles was determined by elemental analysis (EA, Vario MICRO cube) and SEM-EDX (HITACHI S-4800). As a comparison, the anisotropic PCL particles were also fabricated on a solid substrate. Briefly, 10 µL PCL/tetrahydrofuran (THF) solution (5 wt% and 10 wt%, 37 oC) was dropped on a 6×6 mm glass slide, which was then placed in the -20 oC refrigerator for 8 h to obtain the 2-D particle arrays with the fuzzy surface upwards. The 2-D particle arrays were transferred to another glass slide pre-coated by 10 wt% PVP, yielding the arrays with the smooth surface upwards.

2.3 Protein Adsorption


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FITC-BSA was dissolved in PBS to obtain 1 mg/mL solution. 150 µL fibronectin (200 µg/mL Fn) was incubated with 50 µL 1 mg/mL RBITC/dimethyl sulfoxide (DMSO) at room temperature overnight to obtain the fluorescent-labeled Fn (RBITC-Fn) after exhaustive dialysis. Finally, the RBITC-Fn was diluted to 30 µg/mL. The anisotropic PCL particles (1 mg) were respectively incubated in 1 mg /mL FITC-BSA and 30 µg /mL RBITC-Fn solution at 37 oC for 3 h, and then washed with PBS to rinse off the non-adsorbed proteins. The fluorescence microscopy (Axiovert200, Carl Zeiss) and confocal laser scanning microscopy (CLSM, LSM510, Carl Zeiss) images were captured to characterize the anisotropic distribution of proteins on the particles. The same parameters were used to measure the same batch of samples. SEM with affiliated EDX was applied to quantify the nitrogen amount on the fuzzy and smooth surfaces of PCL particles after RBITC-Fn adsorption.

2.4 Cell Culture The 2-D arrays of PCL particles exposed with the fuzzy or smooth surfaces in a 48-well culture plate were sterilized in the clean bench with a UV light with a power of 30 w for 30 min, and 5×104 NIH3T3 fibroblasts were seeded and cultured for 8 h. Meanwhile, 1 mg suspended anisotropic PCL particles were sterilized by 75% ethanol solution. After washed by PBS, they were placed into wells of a 48-well culture plate, which were previously treated with pluronic127 to avoid cell adhesion. On each well 5×104 NIH3T3 fibroblasts or HepG2 cells were seeded. The final concentration of the microspheres for all the samples was controlled as 1 mg/mL. In order to accelerate cell adhesion onto the microspheres, the medium was pipetted every 15 min in the first 2 h, and then every 2 h. The number of adhered cells was determined after being seeded for 8 h. The samples were prefixed with 4% paraformaldehyde in PBS for at least 5 h. After dehydrated by a graded series of ethanol and tert-butanol, each for 15 min, the samples were freeze-dried. The shape and morphology of different cells adhering on the particles was imaged by SEM. The cell number on different surfaces was counted according to the SEM images. As a co-culture cell system model, NIH3T3 and HepG2 cells were pre-stained with 20 mM Dil (red) and Dio (green),


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respectively, and then mixed at a 1:1 ratio and incubated with the anisotropic PCL particles for 8 h (3×104 NIH3T3 fibroblasts, 3×104 HepG2 cells, 1 mg PCL particles). CLSM was used to investigate the adhering state of the two different cells on the particles.

2.5 Preparation of Janus cell microspheres and detection of PGI2 Fibroblasts were seeded on the anisotropic PCL particles as mentioned above (5×104 fibroblasts, 0.5 mg PCL particles, 96-well culture plate) in the first 8 h. After further static incubation for 20 h, the particles were seeded with 5×104 endothelial cells for 12 h, with pipetting occasionally in the first 8 h. In this way, the fibroblasts and the endothelial cells were adhered on the fuzzy and smooth surfaces (Fib/EC Janus cell microspheres), respectively. As a control, the particles seeded with fibroblasts were cultured to the same time. Finally, SEM was used to characterize the cell morphology and numbers on different sides of the particles. The Janus cell microspheres could be obtained by seeding endothelial cells first, following with fibroblasts seeding too. In brief, 5×104 endothelial cells were seeded on the anisotropic PCL particles, and then 5×104 fibroblasts were added and cultured for 12 h as mentioned above. In this way, the endothelial cells and the fibroblasts were adhered on the fuzzy and smooth surfaces (EC/Fib Janus cell microspheres), respectively. As a control, the particles seeded with endothelial cells (5×104) only were cultured to the same time. Finally, SEM was used to characterize the cell morphology and numbers on different sides of the particles. The same numbers of ECs and PCL microspheres were used for the PGI2 secretion study with three different cell seeding manners. Firstly, the 96-well plate was treated by Pluronic to block cell adhesion, into which 0.5 mg PCL particles were placed in each well. The endothelial cells (5×104) were then seeded in three manners. (1) ECs were seeded, followed by seeding of fibroblasts (5×104) to obtain the Janus cell microspheres, with the ECs on the fuzzy surface and the fibroblasts on the smooth surface. (2) ECs


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were seeded on the microspheres alone. (3) The mixture of ECs (5×104) and fibroblasts (5×104) were seeded simultaneously. Most of the cells would adhere on the PCL particles due to the anti-adhesion effect of the substrate. These cells were further cultured for 24 h before the prostacyclin (PGI2) secretion amount was assayed through PGI2 ELISA Kit 49.

2.6 Real-time PCR Analysis The expression of adhesion- and cell cycle-correlated genes including cylin D1, vinculin, integrin β1, cylin D1, P27, and P21, was quantified by real-time quantitative PCR (qRT-PCR) analysis. Briefly, after being cultured on the 2-D arrays of PCL particles exposed with the fuzzy or smooth surfaces for 8 h, the particles with the adhered cells were harvested and placed in another well. In this way, only the cells attached to the fuzzy or smooth surface would be analyzed. Moreover, due to the physical hindrance the cells would not adhere onto the opposite side, avoiding any possible interference. In the case of transferred 2-D arrays on a PVP-coated substrate, the slow dissolution of PVP, if any, would not deter the adhesion of cells onto the particles due to fast sedimentation of cells. Indeed, SEM observation did not find any cells on the fuzzy surface after harvesting the cell-laden microspheres. The cells cultured on TCPS were harvested as a control. Then Single Cell Sequence Specific Amplification Kit (Vazyme, China) was applied for the One-step mRNA reverse transcription. The obtained cDNA was adopted as a template for the subsequent PCR amplification. The qRT-PCR reactions were performed by CFX96 (Bio-Rad, USA) and the SYBR Premix EX TaqTM kit (Takara, China). The relative gene expression values were calculated based on the comparative DDCT (threshold cycle) method, and normalized according to the housekeeping gene 18s. The primers used in this study were designed by Primer Premier 6 software (Premier Biosoft, USA) and listed in Table S1.

2.7 Statistical analysis


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Data are expressed as mean ± standard deviation (SD). Statistical analysis was determined by one-way analysis of variance (ANOVA) with the Origin software. Means comparison was performed with the Tukey’s method. A p value < 0.05 is considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Separated Anisotropic PCL Particles Phase-separation of polymer solution is a feasible and robust method to prepare 2-D and 3-D porous materials. For example, phase separation of concentrated PCL/THF solution (≥10%) filled in a porous gelatin template can yield a 3-D porous scaffold.

48,50

The skeleton of the scaffold is consisted of

aggregated PCL microparticles, which are tightly bonded with each other and thus are hardly separated to obtain single microparticles. Fabricating uniform 0-D polymeric microspheres with desirable surface features is still challenging due to various limitations of the available methods. The aggregation of PCL particles might be alleviated or even avoided at a critical low concentration of polymers. To explore the possibility, the polymer solution was cast onto glass slides to allow the crystallization and convenient monitoring. At a lower concentration of 5%, the obtained PCL particles were well separated, exposing the fuzzy surface upwards (Figure S1a, c). By contrast, severe bridging and aggregation of the particles occurred at a concentration of 10% (Figure S1b, d). Transfer of the sparse particles to conductive paste exposed the smooth opposite side of the particles, revealing the anisotropic nature of the crystallized PCL microparticles (Figure S1e). To prepare large amount of well dispersed single anisotropic PCL microparticles, 5 wt% PCL/THF PCL solution was filled into a porous gelatin template to allow phase separation and guide crystallization, followed by dissolution of the template in water. The obtained particles showed difference in transparency under an optical microscope (Figure 1a1, a2, Figure S2a), and some were bright (Figure 1a1)


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and some were dark (Figure 1a2) due to the different orientation of the fuzzy and smooth surfaces. The elimination of concentric rings on the bright particle could be clearly observed (Figure S2b1, b2), suggesting that the particle is an individual and integral polymer crystal with a crystal form of ringbanded spherulite. The particle size was typically around 70 µm in the long axis and 40 µm in the minor axis. SEM observation confirmed that each particle had distinct morphology in two faces: one smooth and one fuzzy on the opposite side (Figure 1b1, b2). Elemental analysis (Figure S2c) did not find detectable nitrogen element, and the ratio of carbon/oxygen was identical to the theoretical value of PCL. SEM-EDX further shows that both the fuzzy and smooth surfaces had a similar elemental composition of carbon and oxygen elements (Figure 1c1, c2, insets) with neglectable nitrogen (Figure S2d1, d2), suggesting the same PCL composition and neglectable gelatin remnant. To sum up, the anisotropic PCL microparticles can be successfully prepared on a solid substrate with the fuzzy surface upwards, or be obtained with large amount in a separated state by adjusting the phase separation environment.

3.2 Protein Adsorption When the materials come into contact with a biological system, their surface will adsorb different types and amounts of proteins, resulting in a surface-bound protein layer known as protein corona which triggers the subsequent cell behaviors through interactions with cell membrane surface receptors.

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It is

well known that many properties such as surface roughness influence on the protein adsorption. 52 Herein, fluorescent FITC-BSA was used as a representative to observe directly the difference in protein adsorption on the anisotropic PCL microparticles. Figure 2a2 and Figure S3a show that the fluorescence emitted from the particle surfaces was unevenly distributed through the entire particle, and its intensity depended strongly on their textures by comparing the transmission mode (Figure 2a1): very strong emission from the fuzzy side, while very weak or even no fluorescence from the smooth side.


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This observation was further consolidated by CLSM characterization via the Z-scanning (Figure 2b, Movie S1a). The top surface showed a wrinkle-like pattern with strong fluorescence (Figure 2b1, b2), which gradually became invisible in the centre (Figure 2b3) except of the edge (Figure 2b4). The 3D reconstruction images (Figure 2c, d) and rotation video (Movie S1b) display a hemisphere distribution of the fluorescence from the fuzzy side of the anisotropic PCL microparticles. Fibronectin is a classical glycoprotein in the extracellular matrix that binds to membrane-spanning receptor proteins integrin, playing an important role in cell adhesion. This important protein was selected as a second representative after labeled with RBITC. Figure 2e2 and Figure S3b indicate very strong fluorescence emission on the fuzzy surface, while very weak or even no fluorescence on the smooth surface. The anisotropic hemispherical fluorescence distribution on the single particle was further confirmed by CLSM (Figure 2 f, g, h). Moreover, the SEM-EDX results (Figure 2 i1, i2) demonstrate that the intensity of nitrogen signal, which is solely derived from adsorbed proteins, on the fuzzy surface was stronger than that on the smooth surface after RBITC-Fn adsorption. It has to mention that for the EDX analysis, the real area of X-ray contact on the fuzzy surface is much smaller than that on the smooth surface, due to the narrow focus of the X-ray beam. Even in this case, the determined intensity of nitrogen is still stronger than that on the smooth surfaces. A similar phenomenon was observed when the anisotropic PCL microparticles were incubated in RBITC-labeled serum (RBITC-FBS) that contains multi-types of proteins (Figure S 4 a, b, and Movie S1c). Again, the nitrogen signal on the fuzzy surface was stronger than that on the smooth surface, and was further evidenced by the SEM-EDX results (Figure S4 c1，c2). The statistical fluorescence intensity of the fuzzy surface was stronger than that on the smooth surface as well (Figure S4 d1-d3). In summary, the fuzzy face adsorbs significantly larger amount of proteins compared with the smooth face, which is attributed to its nano-micron structure and larger surface area. 53

3.3 Cell Adhesion


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Asymmetries in cell types and cell quantities are one of the most important features in the biological systems. For example, the blood vessel consists of three different layers, and the cell types gradually change from fibroblasts in the outer layer to smooth muscle cells in the medium layer and endothelial cells in the lumen layer. Similar asymmetries also exist at the tendon-to-bone insertion and the multilayer structure of skin.54 Therefore, the design of interfaces of an asymmetric structure is of both scientific and technological significance to better mimic the biological structure and achieve the better construction of engineered tissues and organs. So far a series of methodologies based on the anisotropic variation of surface properties such as surface chemistry, matrix stiffness, and surface topology have been applied to generate cell gradients to mimic the biological structures.

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However, there is no way to prepare Janus

cell microspheres with two different types of cells on the opposite surfaces of a microsphere. The cell adhesion on a substrate is the first cellular event that further regulates the subsequent cell responses such as cell proliferation, migration, and differentiation. 56 The anisotropic PCL particles offer a unique platform to prepare Janus cell microspheres. In the first step, a single type of NIH 3T3 fibroblasts was used to explore the difference in cell adhesion onto the fuzzy and smooth surfaces. For this purpose, the 2-D PCL particles arrays with fuzzy and smooth (by transfer to a PVP-covered substrate) surfaces upwards (Figure S5) were used to allow cell adhesion onto the fuzzy and smooth surfaces, respectively. On the 2-D arrays with fuzzy surface upwards, a large number of cells adhered, which showed good spreading with a large number of filopodia attaching onto the featured surface (Figure 3a). On the 2-D arrays with smooth surface upwards, only a few numbers of cells could adhere, and nearly all the cells remained a spherical morphology, demonstrating poorer cell adhesion (Figure 3b). The statistical cell number on fuzzy surfaces was obviously larger than that on the smooth surfaces (Figure 3c). The cell-substrate interactions dominate the cellular behaviors.57-58 Vinculin

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and integrin β160, 61 are

involved in focal adhesion formation. Cylin D1 is required for progression through the G1 phase of cell cycle after cells adhere to a substrate62. These three genes were significantly up-regulated on the fuzzy


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surface compared to those on the smooth surface (Figure 3d). By contrast, the cell cycle inhibitors P27 63 and P21 64 were significantly up-regulated on the smooth surface compared to those on the fuzzy surface, suggesting that the smooth surface is not conducive to cell spreading and proliferation. Next, the adhesion of fibroblasts onto the dispersed anisotropic PCL microparticles was studied. After a sufficient contacting process, the cell morphology and numbers were observed and quantified by SEM (Figure 4a). Figure 4b shows that the majority of NIH3T3 fibroblasts attached and spread very well on the particles, preferentially on the fuzzy surface. Plenty pseudopodia were found to bind strongly on the fuzzy region, but not on the smooth area (Figure 4b3, inset). Different from the well spreading morphology of fibroblasts, HepG2 cells exhibited spherical shape no matter on the smooth face or on the fuzzy face (Figure 4c). Quantitative analysis reveals that the numbers of cells were several folds larger on the fuzzy face than on the smooth face regardless of the cell type (Figure 4d), demonstrating that the fuzzy face is more cell-attractive. Moreover, the results in Figure 4b-d disclose a fact that different types of cells do respond differently to the anisotropic PCL microparticles, and the fibroblasts are more intensively adhered than the hepatocytes. This fact suggests the performance of cell-selectivity, which was further revealed by the simultaneous coculture of fibroblasts and hepatocytes (Figure 4e). Besides of the absolute majority number of the red Dilstained NIH3T3 fibroblasts over the green Dio-stained HepG2 cells (Movie S2a-d), the fibroblasts were more elongated while the hepatocytes were more spherical (Movie S2a-d), which are in good agreement with the results of culture of single types of cells (Figure 4b-d). These results convey the possibility for the anisotropic PCL particles to effectively capture a target type of cells based on their surface microstructure. To clarify if the difference in cell size has an influence on the cell adhesion, both the fibroblasts and hepatocytes were measured in suspension by optical microscopy (Figure S6a, b). They exhibited similar spherical morphology and size. The statistical analysis proves that there is no significant difference in cell


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diameter (Figure S6c). Therefore, one can safely conclude that the two types of cells had a similar cell size when they firstly contacted with the PCL particles. After the cell adhered and spread on the particles, the fibroblasts exhibited a well spreading morphology with significantly larger size (Figure 4b1-b3) than HepG2 (Figure 4c1-c3). This is further confirmed by the statistical results of the cell spreading areas (Figure S6d). Therefore, the possible influence of cell size on the adhesion of cells onto the PCL anisotropic particles can be excluded. The larger adhesion of fibroblasts over hepatocytes is mainly attributed to the surface texture of the PCL microparticles and the nature of cells used. The topography may influence on cytoskeletal organization, and cell adhesion, polarization, migration, proliferation, and differentiation. The influence of polymer topography on cell behaviors is highly dependent on the polymeric materials and cell types utilized. Therefore, altering the surface roughness of these materials has been pursued. It was found that the fibroblast adhesion at initial time increases with texture as 400 nm > 700 nm > smooth.65 The textured PLGA substrates were fabricated by a liquid–liquid phase separation process, and the smooth surface was applied as a control. There is no significant difference for HepG2 cell adhesion on the two different surfaces at initial stage of culture.66 However, the fibroblasts show more sensitive to the surface topology than HepG2 cells at the initial culture time, which might result in more fibroblasts adhesion on particles than HepG2 cells, and the well spreading morphology of fibroblasts than HepG2 cells on particles. Finally, Janus cell particles, e.g. two different types of cells are located on the opposite surfaces of anisotropic PCL particles, were designed and fabricated by taking into account the advantages of the anisotropic surface textures (Figure 5a). The fibroblasts were seeded on the anisotropic particles firstly, which preferably attached onto the fuzzy surface. A further incubation with endothelial cells (ECs) enabled the smooth surface covered with ECs, resulting in the Janus cell particles which are a simplified biomimetic tissue. In this process, the ECs could only contact and adhere on the smooth surfaces, because the fuzzy surface was occupied by the fibroblasts, which prevented the ECs from contacting the fuzzy surfaces. Figure 5b1, c1 reveal that the NIH3T3 fibroblasts occupied the fuzzy surface in the first culture,


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leaving the smooth surface almost uncovered. After the second ECs seeding, the smooth surface was covered by ECs too (Figure 5b2), with a similar number to that on the fuzzy surface, while the cell number on the fuzzy surface did not change (Figure 5c2). In this way, the fuzzy and smooth surfaces were covered by fibroblasts and ECs, respectively, leading to the formation of Janus cell microspheres for the first time. In contrast, the endothelial cells could be also seeded on the particles firstly, which adhered onto the fuzzy surfaces (Figure 6a1, b1). Then, the smooth surfaces were covered with the fibroblasts after a second cell seeding (Figure 6a2, b2). The reversed Janus cell microspheres with the ECs on the fuzzy surfaces and the fibroblasts on the smooth surfaces were thus obtained. It has to mention that the formation of Janus cell microspheres should be manipulated by optimizing the experimental parameters. For example, when the density of ECs was doubled as 10×104, both sides of the anisotropic PCL microparticles would be covered by the ECs without statistical difference (Figure S7a, b). Moreover, the PCL particles did not show apparent toxicity to ECs (Figure S7c). Vascular endothelium cells could produce multiple functional cues with antithrombotic and anticoagulant capabilities to keep vascular homeostasis. For example, prostacyclin (PGI2) is an antithrombotic substance that has received special attention in recent years, which is mainly synthesized by endothelial cells.67 On one hand, the secretion of PGI2 could inhibit platelet aggregation and promote vasodilatation, and finally prevent thrombosis. On the other hand, the reduction of PGI2 production could accelerate platelet aggregation and activate the platelet function, contributing to the haemostasis and wound healing.68 The PGI2 secretion of the Janus cell microspheres with the ECs on the fuzzy surfaces and the fibroblasts on the smooth surfaces was significantly reduced than the mixed cells (ECs and fibroblasts) adhering on the PCL particles (Figure 6c). In the cell co-culture system, the direct cell–cell contact can involve adherens junctions between the cells, indicating that the adherens junctions may play an important role in the communication of cells.69 In addition, the indirect cell co-culture strategy is usually explored and compared, which can also regulate cell behaviors and functions.70 This unique Janus cell loading methodology can avoid the direct contacts


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between the two different types of cells, which in turn affects the cell differentiation, showing its unique feature in the multiple cell loading. So far the microspheres and porous scaffolds are most commonly used as cell carriers for cell payload and delivery. Compared with these traditional carriers, the Janus cell microparticles have the feasibility to construct more complex tissues and even organs in terms of structure and function by a certain organized form similar to the self-assembly process of Janus particles. Moreover, special types of functional cells can be placed on each side of the anisotropic particles, achieving some more appealing and unique functions.

4 CONCLUSIONS Separated PCL microparticles having fuzzy and smooth surfaces on the opposite sides were fabricated. They had a size of 70 µm and were well dispersed in medium. This type of anisotropic microparticles showed huge difference in biological applications, exemplified by the protein adsorption and cell adhesion. The fuzzy surface adsorbed significantly larger amount of proteins, and were more cellattractive regardless of the cell types investigated. They showed the excellent isolation performance to cells due to their strong affinity to fibroblasts over hepatocytes. The cell adhesion- and cell cycle-related vinculin and integrin β1, and cylin D1 were significantly up-regulated, whereas the cell cycle inhibitors P27 and P21 were significantly down-regulated on the fuzzy surface. By using a successive seeding protocol, Janus cell microspheres with fibroblasts and endothelial cells on each side were designed and obtained by making use of the anisotropic surface morphology. It showed significant difference in ECs functions in terms of prostacyclin (PGl2) secretion, demonstrating the unique and appealing functions of this type of microspheres.


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ASSOCIATED CONTENT Supporting Information: SEM images and optical microscopy images of anisotropic PCL particles formed on glass slides. The additional characterization data of suspended anisotropic PCL particles, including optical microscopy images, polarized optical image, element analysis, SEM-EDX spectra measured on fuzzy and smooth surfaces. SEM images of 2D arrays of PCL particles with the fuzzy and smooth surfaces upwards in large scale. Characterizations of anisotropic PCL particles after being adsorbed with RBITC-FBS: transmission, fluorescent images, and 3D reconstruction images. SEM-EDX results of nitrogen on fuzzy surface and smooth surface of the particles after RBITC-FBS adsorption. Fluorescent images of fuzzy surface and smooth surface, and corresponding statistical fluorescence intensity on PCL particle arrays. Optical microscopy images of NIH3T3 fibroblasts and HepG2 cells in solution, and the statistical cell diameters of these two types of cells. The statistical spreading areas of NIH3T3 fibroblasts and HepG2 cells after being seeded onto the anisotropic PCL particles for 8 h. Fluorescent images, Z-scanning CLSM video and 3D reconstruction rotation videos of anisotropic PCL particles after being adsorbed with FITC-BSA and RBITC-FBS. Z-scanning CLSM videos and 3D reconstruction rotation videos of NIH3T3 fibroblasts and HepG2 cells adhering on anisotropic PCL particles. The primers of the genes.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACKNOWLEDGEMENTS This study is financially supported by the National Key Research and Development Program of China (2016YFC1100403), the Natural Science Foundation of China (21434006), the 111 Project of China


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(B16042), and the Fundamental Research Funds for the Central Universities (2017XZZX001-03B and 2017XZZX008-05).

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Figure captions: Figure 1. Characterizations of anisotropic PCL microparticles well isolated with each other. Optical microscopy images (a) and SEM images (b) of single microparticles. SEM-EDX results of fuzzy surface (c1) and smooth surface (c2) of the microparticles, respectively. Figure 2. The fuzzy and smooth surfaces of anisotropic PCL particles adsorb different amount of proteins. FITC-BSA adsorption (a-d) and RBITC-Fn adsorption (e-h): transmission (a1,e1) and fluorescent (a2,e2) images, and series of Z-scanning CLSM images (b1~b4, f1-f4) and 3D reconstruction images (c,d and g,h). Scale bar: 50 µm. SEM-EDX characterization of nitrogen to reveal RBITC-Fn adsorption on fuzzy (i1) and smooth (i2) surfaces, respectively. Insets are corresponding SEM images. Figure 3. SEM images of NIH 3T3 fibroblasts adhering on the fuzzy surface (a) and smooth surface (b, pointed out by arrows). The statistical cell numbers on fuzzy and smooth surfaces (c, box plot, n=30), and relative gene expression (d) of cells adhering on fuzzy and smooth surfaces. The corresponding expression values of cells on TCPS were used as controls. * and ** indicates statistical significant difference at p

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