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Efficient encapsulation and sustained release of basic fibroblast growth factor in nanofilm: extension of the feeding cycle of human induced pluripotent stem cell culture Uiyoung Han, Hee Ho Park, Yu Jin Kim, Tai Hyun Park, Ju Hyun Park, and Jinkee Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05519 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 9, 2017

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ACS Applied Materials & Interfaces

Efficient Encapsulation and Sustained Release of Basic Fibroblast Growth Factor in Nanofilm: Extension of the Feeding Cycle of Human Induced Pluripotent Stem Cell Culture Uiyoung Han1,§, Hee Ho Park2,§, Yu Jin Kim3, Tai Hyun Park2, Ju Hyun Park3,* and Jinkee Hong1,* 1

School of Chemical Engineering & Materials Science, College of Engineering, Chung-Ang

University, Seoul, 06974 Korea 2

School of Chemical and Biological Engineering, Seoul National University, Seoul, 08826

Korea 3

Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon,

Gangwon-do 24341 Korea §

These authors contributed equally

KEYWORDS controlled release, basic fibroblast growth factor, induced pluripotent stem cell culture, multitrilayer film, layer-by-layer assembly ABSTRACT Basic fibroblast growth factor (bFGF) has an established pivotal function in biomedical engineering, especially for the human pluripotent stem cells (iPSCs). However, the limitation of

ACS Paragon Plus Environment

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bFGF is the ease of denaturation under normal physiological conditions, inducing loss of its activity. In this study, we designed multi-trilayered nanofilm composed of a repeating polycation/polyanion/bFGF structure, which has high loading efficiency and short build-up time. We also investigated that the loading and release of bFGF from the nanofilm with two parameters (counter-polyanion and film architectures). Then, we prepared the optimized nanofilm which maintains sustained bFGF level in physiological condition to apply the nanofilm to human induced pluripotent stem cells (iPSCs) culture. The amount of bFGF release from 12 trilayer nanofilm was 36.4 ng/cm2 and activity of bFGF encapsulated into the nanofilm was maintained (60%) until 72 hours during incubation at 37 °C. As a result, the iPSCs grown in the presence of the nanofilm with tridaily replacement of growth medium maintained undifferentiated morphology and expression levels of pluripotency marker proteins.

INTRODUCTION Fibroblast growth factor-2, also known as basic fibroblast growth factor (bFGF), is a therapeutic protein that plays a crucial role in various cellular functions. A bFGF is involved in human induced pluripotent stem cells (iPSCs) self-renewal, embryonic development, angiogenesis, tissue regeneration, bone regeneration, development and maintenance of the nervous system, and wound healing.1-7 bFGF also stimulates the proliferation, differentiation, and migration of various cell types.8-9 Although the therapeutic effectiveness of bFGF has not yet been realized and the exact mechanism by which functions in the maintenance of pluripotency is not fully understood, bFGF plays a vital role in maintaining the self-renewing activity and pluripotency of iPSCs in culture.10-13 Furthermore, for the successful use of iPSCs in regenerative medicine, it is essential that the self-renewal ability be maintained and that sudden


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differentiation, into other cell types, be reduced to allow for differentiation into specific cell lineages. However, the use of bFGF has been limited as it is highly unstable under normal cell culture conditions.5, 14-15 Poor bFGF stability and the conventional schedule of daily medium changes during iPSCs culture lead to fluctuations in bFGF levels and the loss of iPSCs characteristics, while sustained release of bFGF into the culture medium improved both the maintenance of pluripotency and self-renewal properties.16 In this context, various approaches are used to maintain bFGF concentration in cell culture media, including the use of microspheres16, coacervates17, hydrogels18, and coatings19 with biodegradable and biocompatible polymers. By encapsulating and immobilizing bFGF, these methods prevent exposure to external stressors such as heat and protein-solvent interaction, which would lead to bFGF denaturation.20 Notably, the layer-by-layer (LbL) assembled nanofilm is suitable for bFGF delivery and features a controllable release of bFGF. The conventional LbL assembled

nanofilm

based

on

electrostatic

interaction

for

bFGF

delivery

had

(polycation/polyanion/bFGF/polyanion)n (where, n = number of tetralayers) multi-tetralayer architecture because the bFGF exhibits a net positive charge under the physiological (isoelectric point (pI) of bFGF is 9.6). In that case, the adsorption of positively charged bFGF was regarded as a polycation layer which induced the following polyanion adsorption.21-22 In this study, we set the concentration of bFGF in the dipping solution as 1 µg/ml because the bFGF requirement in normal iPSCs culture conditions (< 100 ng/ml) is considerably lower than the concentration of other components (~1 mg/ml) used in formation of LbL assembled nanofilm. Especially, we used the sodium acetate buffer for solvent of dipping solution because COL exhibits positive charge at pH 5.1. To use the same solvent for LbL process, the bFGF was also dispersed

in

the

same

buffer.

Then,

we

have

encapsulated

the

bFGF

in


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(polycation/polyanion/bFGF)n (where, n = number of trilayers) multi-trilayer nanofilms which have short build-up time and high encapsulation efficiency compared with conventional multitetralayer nanofilm (Figure 1). The multi-bilayer structure using bFGF-mixed solution is more efficient to reduce the produced time of LbL assembled nanofilm; however, it decreases the encapsulation efficiency of bFGF. If the bFGF-mixed polycation is use to LbL dipping solution, bFGF is difficult to be adsorbed on polyanion layer. This is because the polycation is adsorbed faster than bFGF onto polyanion layer due to relatively high concentration, and adsorbed polycation disrupts the adsorption of bFGF. In the bFGF-mixed polyanion, the positively charged bFGF binds with polyanion in the solution-state before polyanion is adsorbed onto polycation layer. It brings a disturbance in adsorption of polyanion, and the adsorption of bFGF is also decreased. Therefore, we expect this multi-trilayer structure to be a potential breakthrough in limitation of protein delivery using LbL assembled nanofilms, such as long processing time and low loading efficiency. The nanofilm was composed of biocompatible and functional materials such as poly(β-amino ester) (Poly2), collagen (COL), poly(acrylic acid) (PAA), and heparin (HEP). The blending of Poly2 and COL composed the polycation layer and HEP or PAA was used for formation of polyanion layer. Then, we investigated the adsorption of each material and measured the loading and release of bFGF in various film arrangements such the trilayer film and the tetralayer film. Finally, we optimized the nanofilm structure to maintain the bFGF level in physiological condition and applied the nanofilm for induced pluripotent stem cells (iPSCs) culture. The nanofilm, coated on flexible substrate, was designed as “inner-bracelet”, which was then inserted into culture dish (Figure 2). This biofunctional “inner-bracelet” reduced the frequency of medium replacement by providing sustained bFGF release into the culture medium. This


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approach minimized bFGF concentration fluctuations and improved in vitro human iPSC culture. Furthermore, this method greatly reduces the costs of replacement medium and the labor required for the maintenance of iPSCs and ESCs.

MATERIALS AND METHODS Materials Poly(acrylic acid) (PAA; MW = 1800, Sigma Aldrich, Saint Louis, MI, USA) and heparin sodium salt (HEP; >180 USP, Sigma Aldrich) solutions were prepared by dissolving each reagent in filtered (0.2 µm) 0.1 M sodium acetate buffer (SAB; Gibco of Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 1 mg/ml. Basic fibroblast growth factor solution (bFGF; MW=17.2 kDa, Gibco) was prepared by dissolving bFGF in 0.1 M SAB at a concentration of 1.0 µg/ml. Poly(β-amino ester) (Poly2; MW=10,000) was synthesized as previously described.23 Poly2+collagen blending solution was prepared by mixing collagen type 1 (COL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) with Poly2, which was diluted to 1.0 mg/ml in SAB. The blending ratio of Poly2/COL solution was 40/60 (w/w). We used polystyrene microbeads (PSMBs; Sigma Aldrich, diameter = 1 µm) to measure the zeta potential of outermost layer in LbL assembled nanofilm. LbL assembled nanofilm construction and characterization Silicon substrates were oxygen plasma prepared by CUTE-1B surface treatment (Femto science, KR). These negatively charged substrates were sequentially dipped in the solutions described above to construct the following multilayer structures (Figure 1): (Poly2+COL/PAA/bFGF)n, (Poly2+COL/PAA/bFGF/PAA)n,

(Poly2+COL/HEP/bFGF)n,

and

(Poly2+COL/HEP/bFGF/HEP)n, where “n” indicates the number of film trilayers or tetralayers.


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These LbL assembled nanofilms are respectively designated as PAA trilayer, PAA tetralayer, HEP trilayer, and HEP tetralayer films. Briefly, substrates were dipped in Poly2+COL blending solution for 10 min, washed twice in deionized water for 2 min, and then the dipping and washing steps were repeated with PAA, HEP, and bFGF solutions. To prepare the nanofilms for cell culture, we repeated this process with polyethylene terephthalate (PET) substrates (0.5 × 12 cm, coated area: 0.5 × 4 cm). The PET substrates were coated with a PAA trilayer film and a HEP trilayer film was subsequently deposited above the PAA trilayer film. These nanofilms were composed of seven PAA trilayers and five HEP trilayers following the structure: (Poly2+COL/PAA/bFGF)7(Poly2+COL/HEP/bFGF)5 and designated bFGF+HEP multi-trilayer nanofilm (bFGF+HEP MNF). Additionally, polystyrene microbeads (PSMBs) were coated using the above protocol for measurement of the surface zeta potential. In this case, dispersed PSMBs in each solution were collected by centrifugation at 8,000 rpm. The thickness of nanofilms was measured by a profilometer (Detak 150, Veeco, NY, USA) and the surface zeta potential of the nanofilm-coated PSMBs was obtained using a nanoparticle analyzer (SZ-100, Horiba, Japan). The surface morphology was analyzed by atomic force microscopy (AFM; NX-10, Park Systems, CA, USA) and field-emission scanning electron microscopy (FE-SEM; LIBRA 120 microscope, Carl Zeiss, Germany). The amount of each layer deposited via the LbL assembly was calculated using quartz crystal microbalance analysis (QCM; QCM 200, Stanford Research Systems, CA, USA). To perform quantitative analysis of the deposition in LbL assembly, the frequency change of the QCM-electrode was measured by QCM-device, and it was converted to mass change by Sauerbrey equation.24

Characterization of bFGF release


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To determine the release profile of bFGF in each sample, the film (ca. 1 × 1 cm) was incubated in 1 ml of phosphate buffered saline (PBS) at 37°C. At different time points, the supernatant was collected and replaced with 1 ml of fresh PBS. All collected supernatants were stored at -20°C until bFGF measurement. The amount of released bFGF was determined by enzyme-linked immunosorbent assay (ELISA), following the protocol of the ELISA kit (R&D systems, MN, USA). The inactivation rate of bFGF released from the bFGF+HEP MNF was determined by a similar methodology, using 0.1 ml of supernatant collected from 10 ml of PBS (replacing 0.1 ml of fresh PBS). Human induced pluripotent stem cells (iPSCs) and cell culture Human iPSCs were obtained from The National Center for Stem Cell and Regenerative Medicine in Korea for use in this study. Briefly, the cell line was generated by introducing four transcription factors (Oct4, Sox2, cMyc, and Klf4) into human dermal fibroblasts using a Sendai Virus system. iPSCs were maintained in ES medium (DMEM/F12 containing 20% knock-out serum replacement (KSR; Gibco of Thermo Fisher Scientific, Waltham, MA, USA), 1× nonessential amino acids (NEAA; Gibco), 1 mM β-mercaptoethanol (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), and 10 ng/ml bFGF (BioVision, Milpitas, CA, USA)) on feeder layers of a mitomycin-C-treated mouse embryonic fibroblast cell line (STO). On day 0, same concentration was used to seed same amount of iPSCs in a 12 (or 6)-well plate (Nunc LabTek,

Rochester,

NY,

USA),

on

feeder

cells,

in

ES

medium

containing

bFGF.

Immunocytochemical analysis was performed in a 12-well plate, and AP staining and immunoblot analysis were performed in a 6-well plate. On day 1, the medium was replaced with fresh ES medium containing bFGF, and the nanofilm was added to the side of the well. On day 4, the medium was replaced with fresh ES medium and the nanofilm was replaced with a new


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nanofilm. For control experiments, ES medium was replaced daily with or without bFGF, or was replaced with bFGF containing ES medium on day 1 and day 4. On day 7, iPSCs were either analyzed or passaged to verify the prolonged effect of the nanofilm on iPSCs. Passaging was performed at the end of each week (day 7 and day 14) using collagenase type IV. Cells were dissociated into small clumps by gentle pipetting and then re-seeded to a new feeder layer of mitomycin-C-treated STO cells. Immunocytochemical analysis Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed in PBS. The cells were then permeabilized with 0.05% Tween-20 in PBS (PBST) for 2 h and blocked in PBS containing 3% w/v BSA, and 0.1% Tween-20. For primary antibody solutions, 1:500 dilutions of Oct4 and Nanog rabbit polyclonal antibody (Santa Cruz Biotechnology, CA, USA) and SSEA-4 mouse monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA) were prepared and incubated with the cells for 12 hours at 4°C. Following the incubation, stained cells were washed five times with PBST. Then, the cells were incubated and counterstained in a 1:500 dilution of Alexa 594-labeled goat anti-rabbit and Alexa 488-labeled goat anti-mouse antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The plates were washed five times in PBST and then analyzed by fluorescence microscopy (Olympus IX71, Olympus Optical Co. Ltd, Tokyo, Japan). Alkaline phosphatase (AP) staining AP staining was performed according to the manufacturer’s protocol (Sigma-Aldrich, MO, USA). Briefly, culture medium was removed and iPSCs were fixed with 4% paraformaldehyde for 15 min. After fixation, iPSCs were washed with PBS. Then, alkaline phosphatase staining solution was added to cover each well and incubated in the dark at room temperature for 15 min.


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After incubation, the solution was aspirated and washed with PBS. Finally, PBS was added to prevent cells from drying and AP expressing colonies (stained colonies) were observed. Immunoblot analysis Cell culture medium was removed and treated with collagenase type IV solution for 20 min to detach iPSC colonies. The iPSCs were allowed to settle to the bottom of a 15-ml conical tube for 5-10 min after which the feeder cell-containing supernatant was removed. The cell lysate was prepared with RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and protease inhibitor cocktail) at 4°C for 1 h. After centrifugation at 12,000 rpm, the cell lysate was analyzed for total protein content using the PierceTM BCA protein assay kit (ThermoFisher Scientific, USA), according to the manufacturer’s protocol. Equal amounts of total protein from each iPSC lysate were resolved by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, CA, USA). A 1:1000 dilution of anti-Oct4 rabbit polyclonal antibody (Abcam, MA, USA) and a 1:1000 dilution of anti-Actin mouse monoclonal antibody (SigmaAldrich, MO, USA) were used primary antibody solutions and incubated at 4°C for 16 h. Following the incubation, the membrane was washed ten times in TBST and incubated in a 1:2500 dilution of HRP-conjugated goat anti-rabbit or goat anti-mouse antibody (Santa Cruz Biotechnology, CA, USA) at room temperature for 1 h. After washing ten times in TBST, the membrane was developed with ECL substrate solution (GE Healthcare, Uppsala, Sweden). Bands were analyzed using the Image J program (National Institutes of Health, MD, USA) to quantify the protein.

RESULTS AND DISCUSSION LbL assembly of multi-trilayer nanofilm


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A blended solution of Poly2 and collagen (Poly2+COL) was used to form a polycation layer. When the polycation layer was composed of only COL, the thickness growth rate gradually decreased and the final film had limited thickness (Figure S1a). Because COL has both positively and negatively charged groups, it exhibits lower charge density than HEP or PAA, which may induce the charge imbalance in repetitive deposition of oppositely charged building blocks. Then, this would lead to decreased net surface charge density, and which would also further prevent adsorption in LbL assembly. Moreover, when only Poly2 was used the bulk of the nanofilm degraded within 24 h (Figure S1b). These results show that the Poly2+COL blended solution is more suitable for the formation of a sustained release LbL assembled nanofilm. We hypothesized that if the amount of bFGF deposited on a polyanion layer of a trilayer film was kept relatively low, then the outermost layer would maintain a negatively charged state. This would allow the polycation layer to sufficiently adsorb onto the bFGF-deposited layer by electrostatic interactions. We coated the polystyrene microbeads (PSMBs) with a multi-trilayer nanofilm and measured the zeta-potentials of the PSMBs after the deposition of each layer (Figure 3). The zeta-potential of bare PSMBs was approximately -45 mV. When the first Poly2+COL layer was deposited on PSMBs, the surface of PSMBs became positively charged (+16 mV). The surface charge then changed from positive to negative (-30, -47 mV) after the deposition of a PAA or HEP layer. However, despite the deposition of bFGF on top of the PAA or HEP layer, the charge was only slightly increased by +4 or +6 mV, respectively and remained in a negatively charged state (-26, -41 mV). Zeta potential changed again from negative to positive (+24 mV) after the second Poly2+COL layer deposition, and a repetitive charge reversal was observed during continuing depositions of the trilayer film layers. Taken together, these


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results show that the new adsorption of a polycation or polyanion layer overwhelms, or masks, the charge of the previous deposited layer and allows for the stepwise growth of a multilayer film by electrostatic LbL assembly.25-26 Since we confirmed the electrostatic adsorption in our multi-trilayer nanofilm, we then examined the increasing thickness of (Poly2+COL/PAA/bFGF)n and (Poly2+COL/HEP/bFGF)n films, designated “PAA trilayer films” and “HEP trilayer films,” respectively (Figure 4). PAA and HEP trilayer films continuously increased in thickness as trilayers were added. It exhibits the potential in which the amount of bFGF encapsulated in the nanofilm can be controlled by the number of trilayers. Additionally, the growth curve of the increase in thickness of both trilayer films could be divided into two phases. Exponential increase in thickness was observed in the films composed of fewer than 7-8 trilayers. This phenomenon was caused by the interdiffusion of polyelectrolyte, as reported in previous investigation on polyelectrolyte LbL assembly.27-28 During the LbL assembly of the trilayer film, Poly2, PAA, and HEP diffused along the film surface during adsorption, and partially diffused out during the adsorption of other layers. This resulted in the formation of Poly2/PAA and Poly2/HEP coupled layers on the surface of the PAA and HEP trilayer films, respectively (Figure S2a and b). Consistent with previously reported results, these coupled layers increased the roughness and total surface area of each film, contributing to the exponential increase in thickness observed.29 However, the growth curve for the increased thickness of the trilayer film with the higher proportion of collagen was more linear (Figure S1a), indicating Poly2 supported the exponential film growth. After the initial exponential growth, the thicknesses of both trilayer films steadily increased by 100-200 nm per trilayer, accounting for the linear growth phase. During the linear growth phase, LbL building blocks were transferred to the film-solution interface through the thick outermost


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layer. The diffusion rate was finite over a constant diffusion time resulting in the outermost layer having a consistent thickness and an end to the exponential phase of film growth. Therefore, the film growth rate became constant when the outermost layer had reached maximum thickness. The exponential-to-linear growth transition of LbL multilayer film assembly has been well documented.30-31 Notably, a multi-trilayer nanofilm with this pattern of thickness growth would be suitable for bFGF delivery because of the short construction, which helps to maintain bFGF activity. PAA and HEP trilayers exhibited different thickness growth rates, with PAA being more rapid than HEP (Figure 4). Generally, salt screens the charge repulsion in the polymer chain, and it brings conformation change from loose polymer to clumped polymer. So, we anticipated that the ions in sodium acetate buffer more increased the thickness of the layer composed of PAA. Furthermore, the rq values of PAA and HEP trilayer films (n = 12) were 135.36 and 33.95, respectively, indicating that the PAA trilayer film has a rougher surface (Figure S3). The amount of adsorption in LbL assembled nanofilm is affected by the roughness and thickness of the film due to their effect on total surface area. Therefore, we expect bFGF to be more easily deposited onto the growth surface of a PAA trilayer film than onto that of a HEP trilayer film. Optimization of encapsulation and release of bFGF To measure the amount of adsorption of each layer in the LbL assembled nanofilm, we coated a quartz crystal microbalance (QCM) electrode with the nanofilms and analyzed the change in QCM electrode frequency. The mass change was calculated by Sauerbrey equation.32 Four different

nanofilms

were

constructed:

(Pol2+COL/PAA/bFGF/PAA)n,

(Poly2+COL/HEP/bFGF/HEP)n, (Poly2+COL/PAA/bFGF)n, and (Poly2+COL/HEP/bFGF)n, and designated PAA tetralayer, HEP tetralayer, PAA trilayer, and HEP trilayer films, respectively.


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The frequency (-∆F) and mass (∆m) changes increased in magnitude with increasing number of layers (Figure 5a and b). There were no significant differences between the cumulative -∆F caused by adsorption of PAA or HEP polyanion layers in the tetralayer versus trilayer film configuration. However, the -∆F of PAA was higher than that of HEP in both the tetralayer (n = 3, PAA: 148.4 Hz, HEP: 44.9 Hz) and trilayer films (n = 3, PAA: 138.3, HEP: 52.8 Hz). These results show that, consistent with the thickness growth results, more PAA was deposited on the nanofilm than heparin. In tetralayer film construction, PAA and heparin were significantly more adsorbed on the Poly2+COL layer than on the bFGF layer. This potentially indicates that the bFGF layer did not provide enough negative charge for adsorption of a polycation layer. This may be because of the low concentration of adsorbed bFGF, as indicated by the results of the zeta potential measurements. The values of -∆F and -∆m after adsorption of the bFGF layer were negative on all occasions, indicating that the mass degraded from the adsorbed nanofilm on the QCM electrode was more than mass of bFGF adsorbed. For these reasons, the adsorption of bFGF was difficult to measure as an isolated variable. The cumulative -∆F caused by adsorption of Poly2+COL in the PAA tetralayer (n = 3) and the PAA trilayer film (n = 3) were 238.8 and 151 Hz, respectively. The -∆F values of the HEP tetralayer and HEP trilayer films were 188.4 and 137.6 Hz, respectively. The results indicate that more Poly2+COL was deposited in the tetralayer film than on the trilayer film. Furthermore, this shows that Poly2+COL was more efficiently deposited on the polyanion layer than on the bFGF layer. As a result, the deposition of positively charged bFGF slightly decreases the negative charge density on outermost layer and partially prevents the adsorption of Poly2+COL layer,


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which indirectly shows the presence of bFGF in the nanofilms. Therefore, we examined the amount of encapsulated bFGF by enzyme linked immunosorbent assay (ELISA). We measured the cumulative amount of bFGF released from each nanofilm (ca. 1×1 cm, n = 12) during incubation at 37°C in PBS buffer for 72 h (Figure 6a). The cumulative amount of bFGF released from the PAA trilayer, PAA tetralayer, HEP trilayer, and HEP tetralayer films were 50.73, 8.42, 10.56, and 0.54 ng/cm2, respectively. As expected, the amount of bFGF released from the PAA trilayer film was more than that released from the HEP trilayer film and the result was observed in the tetralayer films. Taken together, the data indicate that more bFGF was deposited on the PAA layer than on the HEP layer. The nanofilm consisting of a multitrilayer structure also had higher encapsulation efficiency than that of the multi-tetralayer nanofilm. The key difference in the process of formatting tetralayer and trilayer films was the degree of additional adsorption of the polyanion layer onto the bFGF layer. The observed difference was because of the loss of bFGF from the nanofilm during the adsorption of the fourth polyanion layer in the polycation/polyanion/bFGF/polyanion tetralayer film. Furthermore, the amount of cumulative bFGF released from the HEP trilayer film was 10-fold more than that released from the HEP tetralayer film and approximately 6-fold difference was observed between the PAA trilayer and tetralayer films. Therefore, the difference between bFGF encapsulation in the tetralayer and trilayer film was more significant in the HEP films, and resulted in considerable loss of bFGF during the adsorption of the forth heparin layer in the HEP tetralayer film. Given these results, we assumed that the bFGF deposited on the heparin layer was more easily detached by diffusion or surface erosion, and that bFGF would be more rapidly released from HEP films than from PAA films.


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Previously, it has been shown that in an LbL assembled nanofilm, consisting of poly(β-amino ester), drug release was induced by diffusion and surface erosion.33 A linear release profile was observed when the dominant mechanism of release was surface erosion. However, a curved release profile, more rapid than the linear profile, was observed when there was an additional driving force of release, such as diffusion. We examined the release of bFGF from PAA trilayer (n = 12) and HEP trilayer (n = 12) films (Figure 6b). Both release profiles were curved and a more rapid release occurred in the HEP trilayer film. bFGF release from PAA trilayer and HEP trilayer films reached 90% after 20 and 4 h, respectively. These results indicate that bFGF was released by both surface erosion and diffusion in our multi-trilayer film, and that the diffusion of bFGF was more rapid in the HEP trilayer film than in the PAA trilayer film. These results correspond with the expected tendency of bFGF release in Figure 6a. The morphology of the film surface changed after storage in PBS buffer, and was more noticeable in the PAA trilayer film (Figure S2c and d). Therefore, the rapid release seen in the HEP trilayer film was likely induced by diffusion and not surface erosion. We hypothesized that the rapid diffusion of bFGF in the HEP trilayer film was because of weak electrostatic interactions between heparin and bFGF. The PAA trilayer film showed high encapsulation efficiency, and was more suitable for application in sustained bFGF release. However, the iPSCs grown in the presence of the PAA trilayer film (n = 12) had decreased colony size, indicating that the PAA trilayer film (n = 12) induced the growth inhibition of iPSCs (Figure S4). We expected that the cell growth inhibition was induced by PAA disassembled from the nanofilm in iPSC media. So, we investigated the effect of PAA trilayer film on cell proliferation in human dermal fibroblast cell (HDFC) culture (Figure S5a). As a result, the proliferation of HDFC cultured with presence of PAA trilayer film


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was lower than that of HDFC cultured with 20 ng/ml of bFGF, and similar to that of control HDFC. Meanwhile, heparin is a critical material in the culture of iPSCs. Heparin enhanced the binding between bFGF and the FGF receptor (FGFR) on iPSCs and promoted the mitogenic response by inducing FGFR dimerization.34 For this reason, we developed a nanofilm composed of PAA and HEP trilayer films for sustained bFGF release, to incorporate the beneficial effects of heparin (bFGF+heparin multi-trilayer nanofilm, bFGF+HEP MNF for short). The optimized nanofilm has the following structure: (Poly2+COL/PAA/bFGF)7(Poly2+COL/HEP/bFGF)5. We measured the change in activity of bFGF released from bFGF+HEP MNF (Figure 6c). Only 20% of bFGF remained active after 72 h incubation in PBS buffer at 37°C, however 60% of bFGF encapsulated in bFGF+HEP MNF remained active under the same incubation conditions. The maximum amount of active bFGF released from bFGF+HEP MNF was 36.4 ng/cm2. Therefore, we concluded that the maintenance of active bFGF by sustained release from bFGF+HEP MNF was effective for continuous cell signaling by bFGF-FGFR binding. First, we confirmed the effect of bFGF+HEP MNF on cell proliferation in HDFC culture (Figure S5b). After 3 days of culturing, HDFC counts were the most elevated when the culture included bFGF+HEP MNF, indicating the effect of sustained bFGF release. Following these promising results, we tried using bFGF+HEP MNF to extend the feeding cycle of iPSCs in culture. Maintenance of pluripotency in iPSCs using bFGF+heparin multi-trilayer nanofilm (bFGF+HEP MNF) Human iPSCs exhibit a tightly packed and flat human ESC-like morphology, and are characterized by large nuclei and limited cytoplasm.35-37 Both iPSCs and ESCs require a continuous supply of bFGF to maintain the undifferentiated state.38 The bFGF growth factor sustains strong extracellular-signal-regulated kinase (ERK) phosphorylation, thereby maintaining


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pluripotency and survival.31 Figure S6 shows a schematic representation of tridaily media changes with bFGF+Heparin multi-trilayer nanofilm (bFGF+Hep MNF). Culture medium was carefully replaced with fresh ES medium containing bFGF at days 1 and 4. Then, the nanofilm was added directly into the culture medium at the outer edge of the well. At day 7, pluripotency status was evaluated by observing morphology, iPSC colony development, and the expression of pluripotency markers as determined by immunocytochemical analysis. Human iPSCs cultured in bFGF-containing ES medium with daily medium replacement maintained large sized, tightly packed, iPSC colonies with endothelial morphology (Figure 7a). We then cultured iPSCs in ES medium with or without 10 ng/ml bFGF, but no nanofilm, with tridaily replacement. The absence of bFGF resulted in large areas of colonies with flat and epithelial morphology, and no expression of the SSEA-4 and Oct4 pluripotency markers (straight arrows; Figure 7b).39 This highlighted the importance of a continuous supply of active bFGF to maintain pluripotency in iPSCs. Therefore, no further analysis was performed with this control group. Additionally, iPSCs grown in ES medium containing bFGF with tridaily replacement appeared to exhibit some cell growth inhibition, detachment, and cell death. Although some iPSCs expressed of SSEA-4 and Oct4, areas of differentiated cells were observed in the colonies (Straight arrows; Figure 7c). In the presence of bFGF+HEP MNF, iPSC colonies maintained a tightly packed, endothelial morphology and high SSEA-4 and Oct4 expression levels, even with tridaily medium replacement (Figure 7d). Previously, bFGF+heparin promoted similar cell growth by the same signal transduction pathway as native bFGF, through transmembrane tyrosine kinase receptor (FGFR).15 This demonstrates that bFGF from bFGF+HEP MNF is effective in maintaining the morphology and pluripotency of iPSCs.


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The pluripotency status of iPSCs in each experimental group was further examined by alkaline phosphatase (AP) staining, to identify an early stage pluripotency marker.40 Daily medium replacement with bFGF-containing medium showed a large number of stained colonies, indicating that AP activity was maintained in iPSCs (Figure 8a). When bFGF-containing medium was replaced on a tridaily schedule, significant cell damage, growth inhibition, detachment of colonies, and differentiation were observed. This demonstrated that tridaily replacement with bFGF-containing medium provided a harsh environment for iPSCs. However, tridaily medium replacement with bFGF+HEP MNF enabled the iPSC colonies to maintain a similar number of AP-stained colonies as the control group, suggesting that controlled bFGF release is as effective as daily replacement of bFGF-containing medium. From a morphological stand point, judging whether the individual colony is differentiated or undifferentiated is difficult, because this depends on the degree of differentiated areas. Therefore, in order to quantify the pluripotency of these iPSCs, we analyzed the expression of the Oct4 pluripotency marker by immunoblot analysis (Figure 8b and c), which is a representative transcription factor with crucial role in pluripotency.39 Normalization to the total protein was performed to quantitatively represent the pluripotency in each experimental group. The results showed significantly less Oct4 in culture groups with bFGF-containing medium that was replaced tridaily than in those in which the medium was replaced daily. These results support the claim that bFGF is important for the maintenance of Oct4 protein levels in iPSCs. Tridaily replacement of bFGF+HEP MNF medium produced iPSC colonies that maintained Oct4 protein levels similar to those of the control group. This demonstrates the effectiveness of controlled release of bFGF from bFGF+HEP MNF.


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To check the long-term cell growth and pluripotency of iPSCs cultured with the nanofilm, the culture period was extended to 21 days, including passages every 7 days. iPSC colonies were examined at day 21. Human iPSCs cultured with ES medium containing bFGF and with daily medium replacement maintained large sized, tightly packed, and iPSC colonies with endothelial morphology as expected from a conventional iPSC culture procedure (Figure 9a). The effect of tridaily changes of reduced bFGF-containing ES medium was evaluated. Cell growth inhibition and detachment of iPSC colonies were observed in this group, so further passaging was only performed on the surviving iPSC colonies. By this stage, most of the surviving iPSCs had undergone differentiation to a flat and epithelial morphology but SSEA-4 and Oct4 expression were still observed in some iPSC colonies (Figure 9b, straight arrows). In the case of tridaily medium replacement with bFGF+HEP MNF, SSEA-4 and Oct4 expression were observed without any differentiation to a flat and epithelial morphology, suggesting that bFGF+HEP MNF is feasible for long-term use (Figure 9c). No genome/epigenome modifications were performed on iPSCs, and observation of correct morphology and pluripotency markers (SSEA-4, Oct4) suggest that these iPSCs can be used to form embryoid bodies (EBs) and differentiate into different germ layers.16 Our results demonstrate that human iPSCs can grow in an undifferentiated state and maintain their pluripotency with the addition of bFGF+HEP MNF. The use of nanofilm has several unique advantages when compared with other sustained release systems. The nanofilm can be coated onto the side or bottom of culture wells providing an easy and convenient human stem cell culture system. These results demonstrate that bFGF+HEP MNF is biocompatible and allows human iPSCs to maintain pluripotency, and to grow in an undifferentiated state with less frequent replacement of the culture medium.


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CONCLUSIONS Although LbL assembled nanofilm for drug delivery has acknowledged advantages such as widespread application and tunable drug release, its use for growth factors delivery, which requires maintenance of their biological activity, thus far has been limited because its fabrication is a time-consuming process inducing loss of the activity. Therefore a nanofilm to deliver bFGF to biological condition needs high encapsulation efficiency and sustained-release property. In this study, we investigated how film structure and components influence adsorption and release of bFGF in electrostatic LbL assembly. In tetralayer film structure, the deposition of bFGF was considered formation of one layer as building block for LbL assembly and bFGF layers were located between two polyanion layers due to its positively charged surface. On the other hand, the bFGF layers were located between polyanion and polycation layers in trilayer film structure because the amount of deposited bFGF was relatively low, which cannot change the surface charge of polyanion layer. As a result, we discovered that the trilayer-structured film has a higher bFGF encapsulation efficiency than tetralayer-structured film, indicating that when the deposited bFGF had no significant change in the surface charge, the adsorption of polyanion on bFGF layer incurred loss of bFGF. We also designed a nanofilm (bFGF+HEP MNF) consisting of PAA and Hep trilayer film to maintain active bFGF level in physiological condition until 3 days and the effect was verified by applying the nanofilm to iPSC culture with tridaily feeding. Consequently, human iPSCs grown in the presence of bFGF+HEP MNF maintained their undifferentiated morphology and expression levels of SSEA-4 and Oct4 pluripotency marker proteins, as well as AP activity even with tridaily medium exchange. It is anticipated that this technology will provide a


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beneficial tool for maintaining the pluripotency of iPSCs and ESCs in culture and enable significant savings associated with less frequent medium replacement. This study provides a protocol for expeditious LbL assembly of a nanofilm for growth factor delivery and presents a novel platform for the culture of iPSCs with less frequent feeding.

FIGURE


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Figure 1. (a) Schematic illustration of nanofilm preparation with a repeating trilayer and tetralayer structure. (b) Chemical structure of the nanofilm components. The first layer consists of a blend of poly(β-amino ester) and collagen type I (Poly2+COL), it is positively charged and is deposited directly on the substrate. Heparin or PAA, which have negatively charged functional


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groups, adsorb onto the Poly2+COL layer by electrostatic interaction. The positively charged protein, bFGF, is then bound to a PAA or HEP layer.

Figure 2. Schematic illustration of extension of feeding cycle in the iPSCs culture from daily to tridaily by inserting bFGF loaded nanofilm like “inner-bracelet”. The nanofilm for sustained release and efficient encapsulation of bFGF allows bFGF in culture media to maintain constant level.


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Figure 3. The zeta-potential of the PAA trilayer (black) and HEP trilayer coated PSMBs as a function of the outermost layer. Each layer was repeatedly deposited onto PSMBs. Error bars represent standard deviation from the mean (n=3).


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Figure 4. Dependence of the thickness of PAA trilayer film (black) and HEP trilayer film (red) on the number of trilayers. Error bars represent standard deviation from the mean (n=5).

Figure 5. QCM frequency variations at each step of the assembly of the building blocks in (a) PAA tetralayer film (black), HEP tetralayer film (red), (b) PAA trilayer film (black) and HEP trilayer film (red).


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Figure 6. (a) Amount of bFGF released from PAA trilayer, PAA tetralayer, HEP trilayer, and HEP tetralayer films. (b) Cumulative bFGF release and (inserts) normalized bFGF release of PAA trilayer film (black) and HEP trilayer film (red), n=12 (c) Activity of bFGF and bFGF in bFGF+HEP MNF after storage in PBS at 37°C for 72 h.


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Figure 7. Effect of bFGF+HEP MNF on the morphology and expression of pluripotency markers after 7 days of culture. Expression of SSEA-4 (green) and Oct4 (red) in iPSCs cultured in medium (a) containing bFGF with daily replacement (b) absence of bFGF with tridaily replacement (c) containing bFGF with tridaily replacement, and (d) tridaily replacement with bFGF+HEP MNF. Arrows indicate differentiated cells with flat, epithelial morphology.


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Figure 8. Effect of bFGF+HEP MNF on the expression of pluripotency markers after 7 days of culture. iPSCs were cultured in medium containing bFGF with daily replacement, in medium containing bFGF with tridaily replacement, and in medium containing bFGF+HEP MNF with tridaily replacement, and were analyzed by (a) AP staining (b) Immunoblot analysis of Oct4 expression and (c) ImageJ for quantitative assay. Normalized Oct4 protein is represented as one arbitrary unit. The loading volumes for each lane were normalized by the amount of total protein, as determined using the PierceTM BCA protein assay kit. Equal amounts of total protein were loaded. Red-colored colonies indicate pluripotent iPSCs with AP activity.


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Figure 9. Effect of bFGF+HEP MNF on the morphology and expression of pluripotency markers after 21 days of culture. Fluorescence microscopy images show the expression of SSEA4 (green) and Oct4 (red) in iPSCs cultured in medium (a) containing bFGF with daily replacement (b) containing bFGF with tridaily replacement, and (c) tridaily replacement with bFGF+HEP MNF. Arrows indicate differentiated cells with flat, epithelial morphology.

ASSOCIATED CONTENT Supporting Information The film growth curve and degradation of (Poly2+COL/heparin)n nanofilm is shown with different

Poly2

and

collagen

ratios.

The

surface

morphology

and

roughness

of

(Poly2+COL/PAA/bFGF)12 and (Poly2+COL/HEP/bFGF)12 nanofilms can be seen in the SEM and AFM images. The image and cell counts of human dermal fibroblast cells and results of


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proliferation assays are provided. The schematic protocol of the process and the timeline for human iPSC culture with multilayer nanofilm are presented. ASSOCIATED CONTENT Corresponding Author *Tel.: (+82) 02-820-5561, Fax: (+82) 02-824-3495, e-mail: [email protected] (J. Hong) *Tel.: (+82) 033-250-6566, Fax: (+82) 033-259-5645, e-mail: [email protected] (J. H. Park) Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS This research was supported by the National Research Foundation of Korea (NRF), funded by the

Ministry of

Science,

ICT

and

Future

Planning

of

the

Korean

government

2012M3A9C6050104, 2016M3A9C6917405. Additionally, this research was also supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI14C-3266, HI15C-1653).

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