Vascular Endothelial Growth Factor Incorporated Multilayer Film

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Tissue Engineering and Regenerative Medicine

Vascular Endothelial Growth Factor-Incorporated Multilayer Film Induces Pre-Angiogenesis in Endothelial Cells Daheui Choi, Misaki Komeda, Jiwoong Heo, Jinkee Hong, Michiya Matsusaki, and Mitsuru Akashi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00100 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ACS Biomaterials Science & Engineering

Vascular Endothelial Growth Factor-Incorporated Multilayer Film Induces Pre-Angiogenesis in Endothelial Cells Daheui Choi1, Misaki Komeda2, Jiwoong Heo1, Jinkee Hong1,*, Michiya Matsusaki2 and Mitsuru Akashi2,3,* 1

Department of Chemical and Biomolecular Engineering, Yensei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea

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Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan 3

Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoaka, Suita, Osaka, 565-0871, Japan

Keywords: Layer-by-Layer assembly, Vascular endothelial growth factor, Multilayer film, Proliferation, pre-angiogenesis

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Abstract Scaffolds featuring chemically immobilized growth factors have been developed to enhance cellular functions and maintain growth factor bioactivity. However, problems including cytotoxicity and growth factor structural deformation may occur during growth factor conjugation, which can negatively affect the cells. Therefore, we designed a method to improve the long-term storage of growth factors and the target cells’ ability to undergo angiogenesis by incorporating the primary pro-angiogenic growth factor vascular endothelial growth factor (VEGF) into a multilayer film. Using the layer-by-layer (LbL) assembly technique with fibronectin, heparin, and tannic acid, we prepared a VEGF-incorporated multilayer film (VEGF film) that is smooth and stable and increases cell proliferation by up to 2.5 times that of the control group cells. In addition, we prepared the VEGF film directly onto the endothelial cells to maximize the efficacy of VEGF, and we observed cells floating in the growth medium owing to the stiffness of the multilayer film. Although the cells were hard to attach to the culture plate surface due to film stiffness, cell survival and proliferation were maintained. To evaluate the extent of the pre-angiogenesis undertaken by the endothelial cells after VEGF film coating, we examined the expression of the angiogenic marker CD31. CD31 expression was increased after applying the VEGF film, and the cells adopted an elongated morphology, forming tight connections to make clusters. Thus, we conclude that the VEGF-incorporated multilayer film induced endothelial cells to undergo pre-angiogenesis, suggesting its potential use in tissue engineering applications.

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Introduction For several decades, growth factors have been used to control cellular behaviors, such as growth, proliferation, healing, and differentiation.1-4 Growth factors facilitate many cellular processes and precisely control cell signaling.5 Therefore, growth factors have been added directly to culture media, which is considered the easiest method for growth factor treatment. However, most growth factors have a short half-life when reconstituted in aqueous solution;6-8 therefore, media that contains growth factors must be changed every 2 to 3 days. To prevent protein degradation or denaturation during cultivation, growth factors are immobilized by covalent bonding to substrates or long-chain polymers.9-11 In addition, growth factors can be incorporated into hydrogels or microspheres for sustained release and long-term storage.6, 12-14 However, these methods frequently involve crosslinking, which requires chemical crosslinks and can have unexpected effects on the cells or tissues.15 In addition, specific protein binding sites may be disrupted or blocked during synthesis.16 To design a new growth factor delivery system, the layer-by-layer (LbL) self-assembly method, which builds a multilayer film from the molecular level, has been the primary approach.17-19 In this method, various “building blocks” that exhibit complementary interactions, such as electrostatic interactions, hydrogen bonding, or biological recognition at the molecular level, are repeatedly deposited on the desired surface.20-24 Using LbL assembly, the structure and morphology of the constructed film can be optimized by modulating the building block type, pH, salt concentration, and number of deposition cycles.25, 26 By taking advantage of LbL assembly, 3



diverse types of growth factors, including basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP-2), and vascular endothelial growth factor (VEGF), can be immobilized by electrostatic interactions and delivered to target cells and tissues.20, 27-29 VEGF is an angiogenic growth factor that stimulates pre-angiogenic processes, including elongation, migration, and proliferation.30, 31 Angiogenesis is essential for both embryonic development and normal tissue formation at an injury site,31 and it comprises a series of highly organized cellular events, including vascular initiation, formation, maturation, remodeling, and regression. Its progression can be manipulated by both tissue type and environmental stimuli.32 Threedimensional (3D) tissue constructs have recently been created in vitro; however, promoting angiogenesis and vascularization to deliver sufficient oxygen and nutrients to the inner cells are key challenges.33, 34 Therefore, to increase the vascularization potential of the endothelial cells (ECs), researchers have used an LbL nanofilm assembly method to prepare VEGF multilayer films.20, 27, 35 In addition, VEGF exhibits a binding affinity for heparin (Hep),36 which aids in the long-term storage and immobilization of VEGF and is mainly used for VEGF delivery.37, 38 For example, Wang et al. developed a Hep/VEGF multilayer LbL film on a titanium disk to promote EC proliferation and blood compatibility.38 Another research group also demonstrated that VEGF could be deposited using the LbL assembly method, by developing a VEGF and BMP-2 dualrelease film to deliver multiple growth factors to a bone defect site.20 Recently, LbL films that were formed directly onto mammalian cells (e.g., fibroblasts or stem cells) have been used in tissue engineering and other applications in the biomedical field.24, 39, 40 In our previous study, we designed Arg-Gly-Asp (RGD)-incorporated LbL films formed on mesenchymal stem cells, which promoted cytoprotection under high-stress conditions30, 31 and indicated that coating cells 4


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with specialized LbL films can precisely control cellular behavior and functions. In addition, we have coated various types of mammalian cells, including fibroblasts and cardiomyocytes, with LbL multilayer films to construct 3D model tissues for pharmaceutical assays.41 To our knowledge, no reports have described the application of a growth factor to the cell surface using this technology to control cellular functions and facilitate prolonged growth factor storage. In this study, we directly applied a multilayer film, consisting of VEGF and natural polymers, to human umbilical vein ECs (HUVECs) to immobilize VEGF within the LbL films and directly promote pre-angiogenesis in film-coated vascular ECs. We used the LbL assembly technique to deposit VEGF within the multilayer film, as this technique has been demonstrated to produce stable and biocompatible multilayer films. We selected fibronectin (FN), Hep, and tannic acid (TA) natural polymers, which are widely used in biomedical applications, as building blocks to incorporate VEGF via biological recognition and hydrophobic interactions. VEGF-incorporated LbL film (VEGF film) is biocompatible and highly stable under varying physiological conditions and can induce cell proliferation. Furthermore, VEGF film-coated HUVECs prepared via a filtration-LbL method exhibit higher expression of the angiogenic marker cluster of differentiation 31 (CD31) than control cells.42 After film preparation, cells are elongated and tightly bound together, suggesting that applying the VEGF film may induce pre-angiogenesis in endothelial cells.

Materials and Methods Materials 5



All materials were used without further purification. FN from bovine plasma, heparin sodium salt from porcine intestinal mucosa (Hep), and TA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human recombinant VEGF-A165, H2SO4 (95%), H2O2 (36%), and PBS (10×, without Mg2+ and Ca2+) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Preparation of LbL film on flat substrates We used the LbL dipping method to prepare multilayer films on flat substrates. To provide surfaces with a negative charge, the substrates were subjected to piranha treatment (H2O2:H2SO4, 1:3) for 5 min and washed with deionized water. The substrate was immersed (1 min or 5 min for VEGF) into each polymer/protein solution (0.1 mg/mL FN, 1 mg/mL Hep, 1 mg/mL TA, and/or 1 µg/mL VEGF in PBS) then rinsed with PBS to remove weakly bound molecules. Each counter polymer layer was sequentially deposited following the previous layer. A QCM (USI, Fukuoka, Japan) was used to verify VEGF film formation as indicated by the number of attached building blocks. The AT-cut quartz crystal electrode had a frequency of 9 MHz (USI, Fukuoka, Japan). The film preparation on the crystal electrode was the same as that on the wafers. The filmdeposited QCM electrode was placed onto the QCM sensor to measure the resulting frequency change (i.e., decrease or increase). LbL film characterization A field emission-scanning electron microscope (FE-SEM, Carl Zeiss, Oberkochen, Germany) and a non-contact mode of atomic force microscope (AFM, X-10, Park Systems, Santa Clara, CA, USA) were used to evaluate the surface morphology of the VEGF film. Film thicknesses were verified using a profilometer (Dektak 150, Veeco, Plainview, NY, USA). To assess the 6


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stability of the VEGF film under physiological conditions, the 5 tetralayers of the VEGF film that had been prepared on the QCM electrode were immersed into 1× PBS and stored in an incubator for 7 days. At the determined time point, the film was removed from the PBS and washed with distilled water to measure the film decomposition rate. The film was dried with N2 gas and the frequency changes were measured with the QCM. Cell culture HUVECs (Lonza, Basel, Switzerland) were seeded in a 100-mm tissue culture dish (IWAKI, Tokyo, Japan) and maintained in endothelial cell based medium-2 (EBM-2) (Lonza) supplemented with EGM-2MV SingleQuot Kit Supplements & Growth Factors (Lonza). All assays were performed before the HUVECs reached 5 passages. Cytotoxicity and proliferation test To assess cell viability after the film application, VEGF and/or FN/Hep were prepared on 24well plates (IWAKI) as shown in Figure 4A (upper panel). The 24-well plate was formed from tissue culture polystyrene, a modified surface that is produced by exposure to oxygen plasma or sulfuric acid, which allows cells to attach to the plate surface.43 The prepared films were deposited directly onto the well plate without pretreatment. Briefly, polymer (or VEGF) solution was applied to each well (1 or 5 min) to allow the molecules to attach to the bottom of the well plate. After sufficient deposition, the solution was removed, and the well plate was rinsed with PBS. The same film preparation conditions and procedures were employed as those used for the dipping LbL method on the flat substrate. After 5 tetralayers of VEGF film (FN/Hep/VEGF/TA)5) and five bilayers of FN/Hep film ((FN/Hep)5) were prepared on the well plates, HUVECs were 7



seeded (density, 1×104 cells) on either the films or on bare (control) well plates. (The subscript in the film composition description indicates the number of repetitions of each indicated tetra- or bilayer.) To conduct the cytotoxicity test, cells were cultured (1 day) on films or bare well plates, and a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay was performed (n=3). Each plate was incubated (2 h) with a 10% CCK-8 solution, then the proportion of viable cells was calculated by measuring the resulting signal with a plate reader (BioTek Instruments, Winooski, VT, USA), at a wavelength of 450 nm. To conduct a cell proliferation assay, HUVECs were seeded (density, 3×103) on films or bare well plates (n=3), and incubated (5 days) without a medium change. At each indicated time point (1, 3, and 5 d), the cells were detached from the substrate by trypsinization and counted. Preparation and application of VEGF and FN/Hep films to HUVECs and subsequent proliferation assay We used the filtration-LbL method to prepare and apply multilayer films to the HUVECs (Figure 4A, bottom row). Trypsinized HUVECs were placed onto a 3-µm pore insert, and the insert was then placed into a 6-well plate (Corning, Corning, NY, USA). Polymer solutions and PBS were pre-dispensed into the appropriate wells. The HUVEC inserts were placed into a well containing FN solution for 1 min and rinsed in a well containing PBS. The subsequent Hep, VEGF, and TA layers were applied in the same manner. The solution concentrations were limited to 0.5 µg/mL VEGF and 50 µg/mL TA, and the dipping time for the VEGF layer was limited to 3 min to reduce cytotoxicity during film preparation. Cell proliferation was analyzed via a CCK-8 assay. Film-prepared HUVECs were seeded (density, 5×103 cells) in 24-well plates and cultured for 7 days without a medium change. At the indicated time points, the cells were incubated for 2 h 8


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with a 10% CCK-8 solution, and the rate of cell proliferation was measured by determining the absorbance of the cells at a wavelength of 450 nm, using a plate reader. Immunostaining After the film preparation and application, HUVECs were seeded (density, 5×104 cells/well) on a 24-well plate and cultured (7 days) without a medium change. To visualize cell nuclei and CD31 expression, the cells were fixed (15 min at room temperature) with 4% paraformaldehyde, permeabilized (15 min) with 0.2% Triton X-100 in PBS, then washed 3 times with PBS. The cells were then blocked (60 min) with 1% bovine serum albumin (BSA) in PBS and incubated (60 min) in a polyclonal rabbit anti-CD31 antibody (reactive against human and mouse CD31; diluted 1:15 in BSA-PBS, Abcam). A secondary goat anti-rabbit Alexa Fluor 546-conjugated IgG antibody (Invitrogen, Waltham, MA, USA) was then applied for 60 min at room temperature to allow binding to the primary antibody. After washing with PBS, cells were incubated (30 min) with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) to observe their nuclei before being examined using a fluorescence microscope (EVOS, Thermo Fisher Scientific, Waltham, MA, USA). To calculate the strength of the CD31 fluorescence signal, approximately 1,000 cells were selected to represent each group, and the intensity of their fluorescence was measured using ImageJ software.

Results and Discussion In the present study, we selected FN, Hep, VEGF, and TA as building blocks to produce a VEGF film, as each of these materials is known to exhibit advantageous and specific molecular 9



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interactions (Figure 1). For example, FN is a high molecular weight glycoprotein that occurs in the extracellular matrix (ECM), and the RGD tripeptide found in FN is a motif that binds the transmembrane receptor integrin (Figure 1A).44 Hep is a heterogeneous glycosaminoglycan45 that is biocompatible, has anticoagulation properties (Figure 1C)46,

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and is widely used in

biomedical applications. Hep is able to bind various types of proteins, including growth factors and chemokines,48 and binds very tightly with FN at the micro- and nano-levels via an interdomain interaction with a dissociation constant (Kd) of approximately 10-8 M (Figure 1A).36 By exploiting the binding affinity between FN and Hep, we prepared and applied FN/Hep multilayer films onto individual cell membranes to generate cell aggregates via a cell–cell crosslinking process.49 In the present study, we incorporated VEGF into a multilayer structure to promote neovascularization of HUVECs. The VEGF family consists of 5 members, including VEGF-A, which promotes the migration of ECs to facilitate angiogenesis.50 For this reason, we selected VEGF-A as a building block for our multilayer film. The various VEGF-A isoforms that result from alternative splicing of the VEGFA exons (Figure 1B) exhibit different heparin-binding and solubility properties depending on the splice site. VEGFA exons 6 and 7 encode heparin-binding domains; thus, we selected the VEGF-A165 isoform to use as a building block to deposit with Hep since it is a predominant angiogenesis factor that includes exon 7 in its mRNA structure.50, 51 The last material used was the TA polyphenol (Figure 1D), the main component of green tea and wine, which exhibits antibacterial and anticancer properties without cytotoxicity.52, 53 TA is also known to attract proteins via hydrophobic interactions and hydrogen bonding,54 which may improve the robustness of the VEGF film by allowing noncovalent bonding between VEGF and TA as well as electrostatic interaction. In this study, we have prepared VEGF incorporated multilayer film with 10


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high stability by using TA, FN and Hep. Particularly, the TA is responsible for allowing VEGF to stably be incorporated into the layers by reason of hydrophobic interaction and electrostatic interaction. The film deposition mechanism, facilitated by attaching the building block molecules, is illustrated in Figure 2A. We assessed the frequency of the multilayer VEGF film and found that it decreased in a linear, stepwise manner, indicating the formation of a stable multilayer film on the flat substrate. In addition, we measured the thickness of films composed of varying numbers of tetralayers (Figure 2B), and the resulting growth curve exhibited the same trend as that observed for the quartz crystal microbalance (QCM)-measured frequency data. Although FN has a negative charge under physiological conditions, due to its isoelectric point (~5.5), the FN layer was successfully adsorbed onto the negatively charged surfaces via hydrogen bonding or van der Waals interactions. Each molecule exerted a strong binding affinity for the counter molecules, which enabled a stable, linear growth kinetic (Figure 2A, B). The parameters of the building materials, as measured using QCM data, are shown in Figure 2C. The proportion of the deposited FN to Hep substrate was 15.46, consistent with that of the FN/Hep film we previously reported. Low adsorption of Hep is caused by desorption of FN during the Hep-coating step.49 The amount of VEGF adsorbed in the 5 tetralayers was approximately 80 ng/cm2, which is sufficient to induce pre-angiogenesis. In the present study, we designed a VEGF multilayer film using both FN/Hep and TA to stabilize each layer. Accordingly, the amounts of FN and TA attached were higher than those of the other molecules, allowing the stable VEGF layers to be continuously adsorbed. To confirm the influence of the FN/Hep and TA layers on LbL film formation, we prepared VEGF-incorporated multilayer films that lacked FN/Hep and TA layers. Stepwise 11



decreases in the frequency of a TA/VEGF 5-bilayer and an FN/Hep/VEGF/Hep 5-tetralayer film that formed without FN/Hep (Figure S1A) or TA layers (Figure S1B), respectively, are shown in Figure S1. In both cases, the observed decreasing frequency showed an overall increase; however, the materials were unable to sufficiently attach to the corresponding electrode, with frequency charges of 358 Hz and 673 Hz for the 5-bilayer and 5-tetralayer films, respectively. Thus, the FN/Hep and TA layers stabilized the multilayer film by enabling the posterior layers of the film to be continuously adsorbed. In a previous study, Kozlovskaya et al. demonstrated that a TA multilayer film, in which hydrogen-bonded TA multilayer capsules were prepared with poly(Nvinylpyrrolidone), was highly robust under a wide range of pH conditions, varying from pH 2 to 10.55 Therefore, the FN/Hep/VEGF/TA-repeated multilayer composition was selected to build the VEGF film to improve HUVEC function. We observed the surface morphologies of the VEGF films via SEM and AFM, and we found the surface of the 5-tetralayer VEGF film to be even and smooth, with a surface roughness (root mean square, Rq) of approximately 6.6 nm, despite the residual attachment of phosphate-buffered saline (PBS) salts (Figure 3). In addition, the VEGF film exhibited a high surface coverage rate. Given its surface topography and the multilayer growth curve, we concluded that the VEGF film had been successfully constructed. As discussed in a previous report on film growth, we predicted that the VEGF film would be stable under physiological conditions because of its composition. To confirm the stability of the VEGF film, we prepared it on a QCM electrode, immersed it in PBS, and then incubated it at 37ºC for 7 days. At the indicated time points, the resulting frequency changes were measured. The normalized frequency changes of the VEGF film, dependent upon the immersion period, indicated that the VEGF film underwent only 27.7% decomposition during the 7-day incubation 12


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period (Figure 4A). This indicates that the VEGF film is a stable multilayer film under physiological conditions. We also evaluated the rate of VEGF release by the multilayer film after the 7-day incubation via an enzyme-linked immunosorbent assay. The results revealed that VEGF was released at a rate of 8.13 pg/cm2 (data not shown), indicating that the VEGF film was not degraded by PBS and that VEGF could be immobilized within the multilayer film for an extended period of time. Measurement of morphology changes after PBS incubation revealed that PBS exposure resulted in a surface that was 1.63 times rougher (Figure 4B) than that of the unexposed VEGF film (Figure 3C). This change to the film surface resulted from aggregated residue during long-term storage in PBS; however, it did not appear to affect the stability of the VEGF film. To determine the VEGF film’s ability to induce HUVEC proliferation, we prepared multilayer films on well plates (Figure 5A, upper panel; see Experimental Section) by sequentially depositing the various polymer solutions to produce a multilayer film. We produced a VEGFincorporated film and additional multilayer films consisting of only FN and Hep (FN/Hep film), and the HUVECs were seeded immediately after film construction. We tested the viability of the cells seeded with the VEGF compared to the FN/Hep film (Figure 5B) and found that the VEGF film did not exert any cytotoxicity on the HUVECs during the 24 h of exposure. We hypothesized that this was because the highly stable VEGF film did not induce the HUVECs to detach from the substrate or cause deformation of their cellular morphologies during the culture period. Furthermore, TA has been widely used in biomedical applications due to its established biocompatibility,56, 57 indicating that the VEGF film is unlikely to have any toxic effects on cells. Given the known effects of VEGF on EC proliferation, migration, and tube formation,27, 51 we 13



analyzed the effect of the VEGF film on the proliferation of HUVECs cultured with films. As shown in Figure 5C, the VEGF film induced an increase in cell proliferation of up to 46% compared with the FN/Hep film at day 5 of the culture period (see Figure S2). At the molecular level, binding of VEGF to the VEGF receptor, VEGFR-2, at the EC surface phosphorylates VEGFR-2. This activates the phospholipase Cg–protein kinase C–ERK1/2 pathway, leading to cell proliferation, which is the first stage of tube formation.58 Thus, the VEGF-functionalized multilayer film is likely to induce cell proliferation pathways in HUVECs.59 Finally, we deposited the VEGF film directly onto the HUVECs using the filtration-LbL method. In our previous study,41 we compared cell loss and the leakage of lactate dehydrogenase during film deposition via the filtration-LbL and the centrifugation–LbL methods, since the latter is the conventional film preparation method used on curved surfaces such as particles and cellular membranes.41 During centrifugation, cells continually suffer various stresses, such as gravitational force and shear stress, which may lead to necrosis.24, 41 Thus, in the present study, we used the filtration-LbL method rather than the centrifugation-LbL method to produce a multilayer film (Figure 5A, bottom panel). Using this method, we easily generated film-covered cells without the lengthy preparation necessary for centrifugation-LbL. During the deposition of the film on the cellular membrane, each building block was directly applied to the cells, as indicated in the film preparation diagram (Figure 5A), which could induce unexpected effects on the cells due to a high concentration of the given material in aqueous solution. For example, a TA solution can induce cell toxicity at a concentration within only microgram ranges.56 VEGF has also been reported to exert toxic effects on a neuronal cell line at high doses (>400 ng/mL).59 We tested the toxicity of the building materials using a CCK-8 assay prior to directly depositing the 14


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film onto the HUVECs to determine the optimized concentrations for each (data not shown). As demonstrated in a previous study in which an FN/Hep multilayer film was constructed and applied to mammalian cells,49 a high concentration of either FN (0.1 mg/mL) or Hep (1 mg/mL) did not inhibit cellular functions or cause cytotoxicity. Therefore, we only adjusted the concentrations of the TA and VEGF solutions to 50 µg/mL and 0.5 µg/mL, respectively, to minimize the cytotoxic effects. After applying the VEGF film via the filtration-LbL method, the HUVECs were seeded on a culture plate to assess whether they could undergo tube formation. We assessed cellular morphology after 8 h of culturing and found that while control and FN/Hep film-covered HUVECs attached completely to the substrate, VEGF film-covered HUVECs remained floating in the culture medium (Figure S3, 6A). However, the floating cells were approximately 83% more viable than the control HUVECs, indicating that the VEGF film is not cytotoxic (Figure 6B). We demonstrated in our previous study that a multilayer film composed of natural polymers, such as collagen and hyaluronic acid, does not fully cover the cell surface and inhibits cellular attachment and migration.60 However, if the polymer film is rigid or stiff, cellular adhesion may be inhibited.61 TA is likely to increase the mechanical strength and stiffness of the film due to its structure.62 During the culture period, unattached HUVECs can activate apoptosis due to their inability to adhere to a substrate;40 however, in the present study, HUVECs still proliferated throughout the 7-day culture period. The proliferation index of the cells coated with the FN/Hep film was higher than the control 7 days after application of the film, suggesting that FN and/or Hep may promote cell proliferation. This hypothesis is supported by previous studies that revealed that Hep can regulate cell growth in normal vessels and promote endothelial cell proliferation,63 whereas FN can promote cell attachment, spreading, and 15



differentiation, via its RGD sequence.64 VEGF is a growth factor that acts with bFGF and is critical for vascular development.65 To confirm the induction of pre-angiogenesis in VEGF film-coated HUVECs, we cultured the cells for 7 days, then assessed their morphology (Figure 7A). Matrigel is commonly used to facilitate vessel formation by ECs. This structure, which is predominantly composed of laminin or hydrogel, has been used because it provides ECs with a 3D environment that mimics the in vivo environment.66 Amano et al. previously demonstrated vascularization of normal human cardiac microvascular ECs in a 3D co-culture with fibroblasts, and they continuously secreted VEGF.41 Prior to the 3D culture of VEGF film-coated HUVECs, we cultivated the cells as a monolayer to maximize their pre-angiogenesis potential. As shown in Figure 7A, CD31 staining was used to observe cellular morphology after film application. CD31 is a vascular marker that plays a critical role in forming tight cell–cell junction complexes and is also an endothelial marker protein that is constitutively expressed on the surfaces of control HUVECs (Figure 7A).67 The cellular morphology and expression of CD31 by FN/Hep film-coated HUVECs was indistinguishable from those of the control group. By contrast, VEGF film-coated HUVECs were elongated and closely associated at cellular junctions.30 Coating the HUVECs with the VEGF film caused them to form tight intercellular adhesions, thus inducing the formation of cellular clusters, consistent with the results of previous research.68 In addition, Hep can mediate the functions of various growth factors, as demonstrated by its ability to mimic peptide nanofibers and promote HUVEC angiogenesis.69 In the present study, we also measured CD31 expression by assaying fluorescence intensity and found that VEGF film-coated cells exhibited a CD31 expression level that was twice as high as that of the control cells (Figure 7B). CD31 is important 16


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for both the transmigration of leukocytes and endothelial cell–cell adherence, both of which are essential processes required for neovascularization.67 Therefore, we hypothesized that coating with VEGF film could potentially induce pre-angiogenesis in ECs (Figure 7C). In addition, given the short half-life of VEGF,51, 70 we predicted that a stable VEGF film would protect VEGF from environmental degradation, thus enabling HUVEC proliferation and neovascularization.

Conclusion In this study, we prepared a multilayer, LbL-assembled film that incorporated VEGF and stimulated cellular proliferation and neovascularization. The VEGF film can be built on both a flat substrate and a cellular membrane because of its biological interactions with both platforms. Furthermore, TA, one of the materials used to produce the film, stabilized the VEGF film under physiological conditions via noncovalent binding with VEGF. The VEGF film induced a 2.8-fold increase in HUVEC proliferation compared with the control cells. By using filtration-LbL assembly, which reduces cytotoxicity and cell loss during deposition, we fabricated the VEGF film onto the HUVECs. However, the VEGF film-covered HUVECs exhibited reduced adhesion owing to the stiffness of the TA. Despite the reduced adhesion, the HUVECs were able to survive and proliferate during the 7-d experimental period. VEGF induces angiogenesis in cells; therefore, the VEGF film-covered HUVECs were elongated, interacted closely with each other, and increased their expression of CD31. All of these characteristics are critical for inducing neovascularization. We conclude that the VEGF LbL-film enhances endothelial functions, and thus has great potential for use in tissue engineering applications. 17



Figures

Figure 1. The structures of the building blocks used in the present study. (A) FN molecular domains. (B) Gene (mRNA) structure of VEGF and VEGF-A165. (C, D) Molecular structures of the major repeated units of Hep and TA.

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Figure 2. (A) Stepwise decreases in the frequency of the VEGF films with increasing numbers of tetralayers as measured using a QCM (n=3). (B) The thickness growth curve of the VEGF films with increasing numbers of tetralayers. (C) Parameters of each building block as measured using a QCM.

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Figure 3. (A) SEM and (B) AFM images of 5 tetralayers of VEGF film.

Figure 4. (A) Degradation rate of VEGF film over 7 d as measured by the observed change in frequency. (B) SEM and AFM images of VEGF film after 5 d of incubation in PBS. 20


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Figure 5. (A) Schematic illustration of LbL film preparation either using the dipping (Dip-LbL) method on a 6-well plate (upper row) or the filtration-LbL method on a cellular membrane (lower row). (B) Cytotoxicity of the VEGF film against HUVECs prepared on a well plate. (C) Effect of the VEGF film on HUVEC proliferation over 5 d of treatment.

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Figure 6. (A) Schematic diagram of HUVEC behavior after VEGF film application. (B) HUVEC proliferation after application (7 d) of a VEGF or an FN/Hep film.

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Figure 7. (A) Fluorescence microscopy images of DAPI (blue) and CD31 (red)-stained HUVECs with and without the indicated film coating. (B) Normalized CD31 fluorescence intensity per cell as calculated using ImageJ software. (C) Schematic illustration of the effectiveness of VEGF film application on HUVEC behavior. The VEGF-A ligands bind to their receptors (VEGFR-1, 2), and cell proliferation and neovascularization are induced in the HUVECs by the VEGF film.

Author information Corresponding author *E-mail: [email protected], [email protected] 23



Notes The authors declare no competing financial interest.

Acknowledgement This research was supported by the Bio &Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (No. 2012M3A9C6050104). This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (No. HI14C-3266), JST, PREST O (15655131) and a Grant-in-Aid for Scientific Research (B) (26282138 and 17H02099).

Supporting Information Film growth curves and cell images are displayed on Supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table of Contents

Vascular Endothelial Growth Factor-Incorporated Multilayer Film Induces Pre-Angiogenesis in Endothelial Cells Daheui Choi1, Misaki Komeda2, Jiwoong Heo1, Jinkee Hong1,*, Michiya Matsusaki2 and Mitsuru Akashi2,3,*

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Vascular Endothelial Growth Factor Incorporated Multilayer Film

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