Structure of a multilayer nanofilm to increase the encapsulation

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Structure of a multilayer nanofilm to increase the encapsulation efficiency of basic fibroblast growth factor Uiyoung Han, and Jinkee Hong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01099 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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Molecular Pharmaceutics

Structure of a multilayer nanofilm to increase the encapsulation efficiency of basic fibroblast growth factor Uiyoung Han1 and Jinkee Hong*,1 1

School of Chemical Engineering and Material Science, Chung-Ang University, 84, Heukseokro, Dongjak-gu, Seoul 06974, Republic of Korea KEYWORDS

Multilayer nanofilm, basic fibroblast growth factor, encapsulation, electrostatic interaction, molecular adsorption

ABSTRACT

In this study, we established the structure of a multilayer nanofilm that more efficiently encapsulates basic fibroblast growth factor (bFGF). First, a positively charged layer material was selected from biocompatible polymers such as collagen (Col), poly(beta-amino ester) (Poly2), and chitosan (Chi), while considering the film thickness. We then investigated the change in bFGF encapsulation efficiency when the multilayer structure was changed from a tetra-layer to a tri-layer. As a result, we obtained a highly improved bFGF encapsulation efficiency in the nanofilm using a positively charged layer formed by a blend of Col and Poly2 and a negatively

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charged poly(acrylic acid) (PAA) layer within a tri-layered structure. In particular, we found that a significant amount of adsorbed bFGF was desorbed again during the film fabrication process of a tetra-layered nanofilm. In the conventional nanofilm, bFGF was regarded as a polycation and formed a multilayer nanofilm that was composed of a tetra-layered structure and was represented as (polycation/polyanion/bFGF/polyanion)n where n = number of repeated tetra-layers. This study suggested that bFGF should not be considered a polycation, rather it should be considered as a small quantity of molecule that exists between the polyanion and polycation layers, such that the

nanofilm

is

composed

of

repeating

units

of

(polycation/polyanion/bFGF/polycation/polyanion), when the amount of bFGF adsorbed is considerably lower than that of other building blocks.

INTRODUCTION

Basic fibroblast growth factor (bFGF) plays a crucial function in tissue regeneration and self renewal of stem cells and is especially involved in biomedical fields, such as tissue and stem cell engineering. bFGF is used excessively in in vitro and in vivo experiments; however, its unstable structure may be responsible for obtaining results that differ from those expected. Hence, moderate amounts of the signaling molecule in the active state must be delivered precisely to the site of action to ensure that the effect of inherent signaling for a given cell type is fully utilized.1 Although most growth factors are introduced via simple carrier-based delivery systems in the current tissue and stem cell engineering protocols, the carrier molecules have restricted modulation over the growth factor-release kinetics.2 To overcome this limitation, many research groups have studied delivery carriers that incorporate and continuously release bFGF.3-5


2

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A molecular adsorption-based multilayer structure nanofilm is a highly versatile method for multi-functional coating on various surfaces.6-8 Delivery systems using multilayer films containing drugs or biomolecules for biomedical application have been investigated because of their facile fabrication and application.9-10 In particular, multilayer nanofilms for growth factor delivery are ideal because film properties, such as the release rate, are adjustable.11-12 For these reasons, multilayer-structured nanofilms formed by continuous molecular adsorption have been used to encapsulate bFGF for wound healing and stem cell engineering applications.13-15 In the conventional nanofilm-based bFGF incorporation process, biocompatible polyelectrolytes offer a surface that has binding affinity for bFGF. bFGF adsorbed on the polyelectrolyte and encapsulated into a multilayer nanofilm is protected from direct contact with external stress that could lead to structural denaturation, and thus, the activity of bFGF is protected.16-17 In previous studies about multilayer nanofilm for bFGF encapsulation, its content not exceed a few microgram.

18-19

Besides, the difficulty of producing recombinant bFGF also causes reducing the

amount of bFGF used in nanofilm fabrication. Thus, the concentration of the bFGF solution used to compose the multilayer nanofilm is conventionally between 1 to 10 µg/mL, which is 100 to 1000 times lower than that of the other components. For this reason, the amount of bFGF adsorbed is significantly smaller than that of the other building blocks, resulting in a lower encapsulation efficiency of bFGF. This insufficient adsorption of bFGF raises questions regarding the excessive iteration process in film preparation. Considering the short half-life of bFGF, reducing the process time is preferable for efficient bFGF utilization. Also, since large amounts of a polyelectrolyte disassembled from the nanofilm may cause cytotoxicity, the proportion of bFGF in the nanofilm should be increased by enhancing the adsorption and


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inhibiting the desorption of bFGF. For these reasons, in the present study, we aimed to develop a multilayer nanofilm with increased bFGF encapsulation efficiency.

In this study, we investigated the effect of the nanofilm properties on the encapsulation and release of bFGF. Various multilayer nanofilms comprising biocompatible polyelectrolytes such as collagen (Col), poly(beta-amino ester) (Poly2), chitosan (Chi), heparin (Hep), and poly(acrylic acid) (PAA) were used as bFGF reservoirs and delivery carriers. The formation of each layer was based on electrostatic attraction, such that the positively charged layer comprised Col, Poly2, or Chi, and the negatively charged layer comprised Hep or PAA. The bFGF which have partially positive charge was adsorbed on negatively charged PAA or Heparin layer. The composition of the positively charged layer was selected in consideration of the growth in film thickness, which is related to the surface area available for bFGF adsorption. We also investigated the loading and release of bFGF in nanofilms with different architectures, one that possessed a tetra-layered structure (polycation/polyanion/bFGF/polycation) or another that possessed a tri-layered structure (polycation/polyanion/bFGF). We hypothesized that depending on this order of multilayer assembly, the amount of bFGF adsorbed would be different. The remarkably small amount of the building blocks used to form a nanofilm based on electrostatic attraction may cause different surface charge alterations compared to those observed in the conventional adsorption/desorption process.

MATERIALS AND METHODS

Materials


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PAA (MW = 1800 g/mol, Sigma Aldrich, Saint Louis, MI, USA), heparin sodium salt (Hep; >180 USP, Sigma Aldrich), and Chi (200–800 cP, Sigma Aldrich) dipping solutions were prepared by dissolving each reagent in filtered (using 0.2 µm filters) 0.1 M sodium acetate buffer (SAB; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 1 mg/mL (pH 5.4). The bFGF (MW = 17.2 kDa, Gibco) solution was prepared by dissolving bFGF in 0.1 M SAB (pH 5.4) to obtain a concentration of 1.0 µg/mL. Poly2 (MW = 10,000 g/mol) was synthesized as previously described.20 The Col+Poly2 blend dipping solution was prepared by mixing Col (type I collagen from rat tail tendon, Santa Cruz Biotechnology, Santa Cruz, CA, USA) with Poly2, which was diluted to 1.0 mg/mL using SAB (pH 5.4).

Formation and characterization of the multilayer nanofilm

Silicon wafer were oxygen plasma-prepared using the CUTE-1B surface treatment (Femto Science, Gyeonggi-Do, Korea). These negatively charged substrates were sequentially dipped in the solutions described in the materials section to construct the following multilayer structures: (polycation/polyanion/bFGF/polyanion)n and (polycation/polyanion/bFGF)n, where “n” indicates the number of tri-layers or tetra-layers. Briefly, substrates were dipped in a polycation solution consisting of Col, Poly2, Chi, or Col+Poly2 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. In the same manner, the surface of polytetrafluoroethylene (PTFE) porous membrane (pore size: 400 nm) and polyethylene terephthalate (PET) film was coated with the nanofilm. Additionally, polystyrene nanoparticles (PSNPs; Sigma Aldrich) were coated using the above protocol to measure the surface zeta potential. In this case, dispersed PSNPs in each solution were collected by centrifugation at 8,000 rpm. The thickness of nanofilms was measured using a profilometer


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(Detak 150, Veeco, NY, USA) and the surface zeta potential of the nanofilm-coated PSNPs was obtained using a nanoparticle analyzer (SZ-100, Horiba, Japan).

Characterization of bFGF release

To determine the release profile of bFGF in each sample, the film (approximately 1 cm × 1 cm) was incubated in 10 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 bFGF released was determined using an enzyme-linked immunosorbent assay (ELISA), following the protocol of the ELISA kit (R&D systems, Minneapolis, MN, USA).

RESULTS & DISCUSSION

Thickness and degradation of multilayer nanofilms

Before bFGF incorporation, we measured the thickness of each multilayer film comprising different polycation layers to determine the components of the nanofilm. Many previous studies on controlled drug delivery using multi-layered structural nanofilms have adjusted the number of layers to control the loading and release of the drug.21-22 Although various factors, such as binding energy, surface energy, and molecular size, are related to drug incorporation into a nanofilm, the thickness of the film is determined by the surface properties of the adsorbed layer, which affect the contact and adsorption areas of the growth factor. Hence, changes in the film thickness, including its growth and degradation, are important film characteristics for bFGF incorporation.


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We prepared nine-bilayer nanofilms, represented as (polycation/polyanion)n, n = number of bilayers, with several combinations of polycation layers; the polycation layer of each nanofilm comprised Col, Col+Poly2 (8:2, 6:4, 5:5, mass ratio), Poly2, and Chi. Although these biocompatible materials are suitable as bFGF delivery and carrier agents, their intrinsic characteristics and structures are different. We investigated the film thickness at the interval of two bilayers to observe its growth tendency. As seen in Figure 1a, two nanofilms using Poly2 or Chi polycation layer exhibited relatively low film growth, such that the thickness of the ninebilayer nanofilm was under 100 nm. When the polycation layer comprised Col or Col+Poly2(5:5), although the film thickness was twice that obtained using Poly2 or Chi, the growth rate of the film gradually decreased, suggesting restricted growth of the nanofilm. The thickness curve of the nanofilm composed of Col+Poly2 (8:2) or (6:4) showed a higher final thickness and continuous film growth compared with the curve of the other nanofilms. A nanofilm that is too thin or that has a limited growth rate would be restricted for its application for varied specific targets. Therefore, considering the result of the film growth curve, we expected that nanofilms composed of Col+Poly2 (8:2) or (6:4) would be more suitable in controlling and increasing the bFGF encapsulation efficiency. The thickness growth curve of such nanofilm also shows the possibility of continuous thickness growth, indicating that the thickness of the nanofilm can be adjusted through the number of layers.

To estimate the film degradation rate, the film thickness was plotted against the physiological incubation time. The Figure 1b shows that the thickness decrease was relatively rapid in nanofilms using a Poly2 polycation layer. In contrast, the thickness of nanofilms comprising Col or Chi showed slight decrease over 3 days of incubation. In three of the nanofilms made from different ratios of Col and Poly2, as the Poly2 ratio increased, the film degradation rate also


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increased. Most of the nanofilm comprising Col+Poly2 (5:5) was degraded within 1 day, like the nanofilm with the Poly2 polycation layer. The purpose of bFGF encapsulation is to maintain its activity and to prevent its structural degradation; therefore, a nanofilm that degrades within 1 day is unfit as a reservoir to encapsulate bFGF. As a result, we hypothesized that the suitable nanofilm components to form the polycation layer to incorporate and release bFGF is a mixture between Col and Poly2 at ratios of 8:2 and 6:4.

bFGF release from tetra-layer nanofilms with different polycation layers

We investigated the bFGF release from each nanofilm with a tetra-layer structure incorporating bFGF, as (polycation/polyanion/bFGF/polyanion)n (n = number of tetra-layers), to demonstrate our assumption based on the film thickness results. The bFGF release was expressed as the concentration of active bFGF in PBS buffer (in 10 mL of PBS) in the presence of the nanofilm (1 cm × 1 cm) over 3 days. The bFGF was released from the nanofilm by disassembly between bFGF and nanofilm components, which is accelerated in physiological condition due to temperature and salt. Considering the short half-life of bFGF, the concentration of active bFGF in solution at specific points is more meaningful in practical applications than cumulative bFGF release, which does not reflect bFGF denaturation. In Figure 2, despite the low thickness of the nanofilm consisting of a Poly2 polycation layer (Poly2 nanofilm, expressed as ‘polycation nanofilm’), a maximum of 550 pg/mL of bFGF was released from the Poly2 nanofilm, which was higher than that released from Chi, Col, Col+Poly2 (5:5), and Col+Poly2 (8:2) nanofilms. However, the concentration of active bFGF rapidly decreased after 24 hours because most of the bFGF released from the Poly2 nanofilm, which decomposed over 1 day, was exposed to physiological conditions, resulting in its denaturation. As mentioned above, we expected the


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Col+Poly2 (8:2) and (6:4) nanofilms to be efficient for bFGF encapsulation; however, only the Col+Poly2 (6:4) nanofilm showed sustained bFGF release over 3 days. The graphs in Figure 2 show that a small amount of bFGF was released from each nanofilm. When the nanofilm had a low thickness (Chi) or had limited growth (Col), as expected, bFGF encapsulation into the nanofilm was not efficient. Although the Col+Poly2 (5:5) nanofilm had degradation properties similar to the Poly2 nanofilm, almost no bFGF was encapsulated into the Col+Poly2 (5:5) nanofilm. Based on this result, we conjectured that bFGF adsorption was inhibited on the nanofilm surface due to the addition of Col into the polycation component. Col has both positively and negatively charged amino acid residues resulting in an isoelectric point (pI) value of 7.8 for Col type I.23 In contrast, Poly2 comprises only positively charged groups and has a high surface charge density.24 The different surface charge densities between Col and Poly2 might affect the formation of the polyanion layer that forms the surface for bFGF adsorption. In the electrostatic adsorption process of a multilayer nanofilm, considerable differences in charge density of the two oppositely charged building blocks cause a convergence of net charge.25 When the surface charge is fixed as negative, a negatively charged material is attempting to adsorb onto a negatively charged surface, causing the sequential adsorption process to stop.25 In this case, the net charge of a Col/Hep film may converge to a negative charge because of the highly negatively charged Hep, resulting in a decreased film growth rate. The film growth result (Figure 1a) showed a gradual decrease in the thickness of the Col/Hep nanofilm with increase in the number of bilayers. Therefore, when collagen is dominant in the polycation layer, the bFGF encapsulation efficiency decreases because the Hep layer, in which bFGF will be adsorbed, is not adsorbed well on the Col layer. Therefore, the nanofilm shows slow thickness growth and rapid degradation. However, among the Col+Poly2 nanofilms with different ratios, the Col+Poly2


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(6:4) nanofilm showed the maximum amount (250 pg/ml) of bFGF release. This result indicated that when the ratio of Col and Poly2 was 6:4, the rapid degradability of Poly2 was complemented by Col, and the low level of bFGF encapsulation by Col was improved by Poly2. Hence, we set the polycation layer composition to Col+Poly2 (6:4) and used this in further experiments.

Comparison of bFGF release between tetra-layer and tri-layer nanofilms

In conventional multilayer structural nanofilms for bFGF delivery, bFGF is regarded as a polycation because of its positively charged amino acid residues; thus, the bFGF layer was located between the polyanion layers as polyanion/bFGF/polyanion. However, usually 1 µg/mL of bFGF is used for the dipping solution for nanofilms because of its high cost and low dose in in vivo and in vitro applications. Figure 2 shows that less than nanogram quantities of bFGF are encapsulated into the nanofilm. In contrast, other layers of the nanofilm were formed from 1 mg/mL of dipping solutions; moreover, our previous research also showed that the amount of adsorbed Col+Poly2 or Hep on the nanofilm was more than 1 µg.15 Therefore, we speculated whether bFGF might be considered as a minor material that only exists between the polycation and polyanion layers in low amounts and should not be considered as a polycation layer. If bFGF cannot make a significant alteration to the surface charge because of its remarkably low adsorbed amount, it is important that a polycation, rather than a polyanion, is adsorbed on the bFGF layer. To determine if the bFGF encapsulation efficiency is altered by the order of the multilayer structure, we investigated the bFGF release from following tri-layer or tetra-layer nanofilms: (Col(6)+Poly2(4))/Hep/bFGF)10, (Col(6)+Poly2(4))/Hep/bFGF/PAA)10, (Col(6)+Poly2(4))/ PAA /bFGF)10, and (Col(6)+Poly2(4))/Hep/bFGF/ PAA)10. We also used poly(acrylic acid) (PAA)


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instead of Hep to determine if a difference in the polyanion material would affect bFGF encapsulation.

The

maximum

concentration

(Col(6)+Poly2(4)/Hep/bFGF)10,

of

bFGF

released

from

each

nanofilm,

(Col(6)+Poly2(4)/Hep/bFGF/PAA)10,

(Col(6)+Poly2(4)/PAA/bFGF)10, and (Col(6)+Poly2(4)/PAA/bFGF/PAA)10 was 1000 pg/mL, 100 pg/mL, 3300 pg/mL, and 500 pg/mL, respectively (Figure 3a-d). Interestingly six to ten times more bFGF concentration was detected in the tri-layer nanofilms than in the tetra-layer nanofilms, indicating higher bFGF encapsulation efficiency of tri-layer structured nanofilms. In addition, when PAA was used as the polyanion instead of Hep, the amount of bFGF released increased. In our previous research, the thickness and roughness of a nanofilm using PAA was higher than that of the nanofilm using Hep, resulting in a larger bFGF adsorption area.15 Thus, PAA, which has a linear polymer chain, is more effective in increasing the adsorption and sustained release of bFGF in a multilayer nanofilm system than the polymeric carbohydrate molecule, heparin. Although the only difference between the formation of the tri-layer and tetralayer nanofilms was the additional PAA layer adsorption process in tetra-layer nanofilm formation, the bFGF encapsulation efficiency was significantly different. Based on this result, we predicted that bFGF would be disassembled from the nanofilm in the PAA adsorption process.

Therefore, we investigated the concentration of bFGF in each dipping solution after film preparation to determine the amount of bFGF isolated from the nanofilm during the multilayer fabrication. We also measured the surface charge of nanofilm-coated polystyrene nanoparticles to determine the surface charge alterations caused by adsorption of each layer. In the


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(Col(6)+Poly2(4)/Hep/bFGF/PAA)10 nanofilm, the concentration of bFGF in the PAA dipping solution (38 ng/mL) was remarkably higher than that in the other dipping solutions (1.3 ng/mL and 3.2 ng/mL in Col+Poly2 and Hep solution, respectively) (Figure 4a). This demonstrated that the bFGF adsorbed on the Hep layer detached when the nanofilm was immersed into the PAA dipping solution. We also hypothesized that the small amount of bFGF adsorbed on the polyanion layer would not change the net charge from negative to positive. Figure 4b shows the surface charge of the nanofilm at each layer adsorbed, in which the values changed sequentially from 18.3 mV to -31.45 mV, -25.24 mV, and -30.42 mV in the nanofilms using the PAA polyanion layer, and changed from 18.3 mV to -44.34 mV, -41.62 mV, and -43.43 mV in the nanofilms using the Hep polyanion layer. In other words, the surface charge of the bFGF adsorbed layer showed a negative charge as the PAA or Hep layer, indicating that the bFGF layer did not act as a polycation layer. However, when bFGF was adsorbed onto the polyanion layer, the surface charge showed a slight increase. This result showed indirectly that positively charged bFGF is partially adsorbed on the polyanion layer. We hypothesized that bFGF adsorbed onto the polyanion layer was disassembled by the attraction between with polyanions in the dipping solution. In conventional multilayer nanofilms, the polyelectrolyte adsorbed on the other layer is not detached during washing or subsequent adsorption processes. However, the fundamental experiment on how a small amount of a polycation (polyanion) adsorbed on a polyanion (polycation) layer is affected by continuous adsorption of polyanions (polycations) has not been reported. In order to help to fill this knowledge gap, we investigated how the film structure affect to bFGF encapsulation. Eventually, we showed that the tri-layer structure (polycation/polyanion/bFGF) is more suitable to increase the encapsulation efficiency of bFGF


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compared to the tetra-layered structure. In addition, we have previously reported the result of biological experiments using such tri-layer structured nanofilm for sustained bFGF release.15

Meanwhile, the process of film preparation via continuative molecular adsorption in solutionphase has facilitated the fabrication of functional nanofilm with low-cost and large-area. Hence, we attempted to coat polymeric membrane with the nanofilm to identify feasibility of tri-layered nanofilm for biomedical application. We used porous PTFE membrane and flexible PET film as a substrate for coating our nanofilm. As a result, the nanofilm was also well formed on this two substrates, as well as silicon wafer which was used in this study, in which the nanofilm-coated surface was slightly opaque (Figure 5a). As observed in the result of film growth (Figure 1a), the thickness of the nanofilm was increased proportionally as the number of layer increases. Likewise, the adsorption amount of nanofilm components onto the substrate also increased as the adsorption process was repeated. The growth of nanofilm on the substrate causes decrease of transparency and the opacity also increases with the growth of the nanofilm. As the number of tri-layer increase from 8 to 14, the opacity of the nanofilm-coated PET film was clearly different (Figure 5c). These result showed that the adjustable multilayer nanofilm coating was formed successfully on the PTFE and PET substrates. It also shows the feasibility of the application of nanofilm for controlled growth factor release that most important function of drug delivery system. Meanwhile, The thickness of (Col(6)+Poly2(4)/Hep/bFGF)8 nanofilm coated inside pore was around 200 nm which was similar to the result using silicon wafer (Figure 5b). Likewise, the thickness of same nanofilm coated on PET film was also around 200 nm Figure 5d). This result showed that our multilayer nanofilm with tri-layered structure is successfully adsorbed on various substrate. We anticipate that this study will provide a protocol for a rapid preparation


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process of multilayer nanofilms for bFGF delivery and presents a novel film structure for the encapsulation of low amounts of macromolecular drugs.

CONCLUSION

Multilayer nanofilms have significant potential in bio-applications, not only because of the controllability of the amount and the release rate of bFGF, but also because of their use as versatile coating materials and substrates. However, in conventional multilayer films, supernumerary polymer building blocks and bFGF have been used because of the low bFGF encapsulation efficiency. In this study, we attempted to improve the bFGF encapsulation efficiency of multilayer nanofilms in two ways. First, we fabricated various nanofilms with different polycation layers using Col, Poly2, Chi, and their mixtures. We also prepared nanofilms with

different

adsorption

order

such

as

(polycation/polyanion/bFGF/polyanion)

and

(polycation/polyanion/bFGF). Quantification of the bFGF release from these nanofilms showed that the tri-layer structured nanofilm consisting of a Col+Poly2 (6:4) polycation layer was the most suitable for the encapsulation and release of bFGF. The thickness and degradation of the nanofilm partially affect the bFGF encapsulation efficiency; however, the components of the polycation layer and the adsorption order were more important factors for bFGF adsorption. In particular, six to ten times more bFGF was released from the tri-layered nanofilms compared to the tetra-layered nanofilms; the only difference between the two was the material that was adsorbed

onto

the

adsorbed

bFGF

layer:

(polyanion/bFGF/polycation)

or

(polyanion/bFGF/polyanion). Finally, we showed that disassembly of bFGF from the nanofilm occurred when the polyanion layer was consecutively adsorbed onto the adsorbed bFGF layer.


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The results of this study will provide an improved platform for the delivery of signaling molecules, including growth factors.

FIGURES

Figure 1. Variation in the thickness of nanofilms composed of different polycation layers was plotted against the increase in (a) the number of layers and (b) incubation time. Col, collagen; Hep, heparin; Poly2, poly(beta-amino ester); Chi, chitosan. The legend of Figure 1b is the same as Figure 1a.


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Figure 2. Concentration of bFGF released from nanofilms consisting of the following polycation layers: Poly2, Chi, Col, Col+Poly2 (5:5), Col+Poly2 (8:2), or Col+Poly2 (6:4). Col, collagen; Hep, heparin; Poly2, poly(beta-amino ester); Chi, chitosan; bFGF, basic fibroblast growth factor.


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Figure 3. Concentration of bFGF released from nanofilms having the following structures: (a) (Col(6)+Poly2(4)/Hep/bFGF)10 (tri-layered), (b) (Col(6)+Poly2(4)/Hep/bFGF/PAA)10 (tetralayered),

(c)

(Col(6)+Poly2(4)/PAA/bFGF)10

(tri-layered),

and

(d)

(Col(6)+Poly2(4)/PAA/bFGF/PAA)10 (tetra-layered). Col, collagen; Hep, heparin; Poly2, poly(beta-amino ester); PAA, poly(acrylic acid); bFGF, basic fibroblast growth factor.


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Figure 4. (a) Concentration of bFGF in each dipping solution after nanofilm fabrication of (Col(6)+poly2(4)/Hep/bFGF/PAA)10, indicating the amount of detached bFGF from the nanofilm during adsorption process of other layer. (b) The zeta-potential of polystyrene nanoparticles coated

with

(Col(6)+poly2(4)/PAA/bFGF/PAA)

and

(Col(6)+poly2(4)/Hep/bFGF/PAA)

nanofilms. The surface charge was plotted against the alterations in the outermost layer. Col, collagen; Hep, heparin; Poly2, poly(beta-amino ester); PAA, poly(acrylic acid); bFGF, basic fibroblast growth factor.


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Figure 5. (a) The image of non-coated PTFE membrane (left) and that coated with (Col(6)+Poly2(4)/Hep/bFGF)8 nanofilm (right). (b) The SEM image of the surface of the nanofilm-coated membrane and non-coated membrane (inserts). (c) The image of non-coated flexible PET film (left), that coated with (Col(6)+Poly2(4)/Hep/bFGF)8 nanofilm (middle) and with (Col(6)+Poly2(4)/Hep/bFGF)14 nanofilm. (d) The cross-section SEM image of PET film coated with (Col(6)+Poly2(4)/Hep/bFGF)8 nanofilm.

ASSOCIATED CONTENT Corresponding Author *Tel.: (+82) 02-820-5561, Fax: (+82) 02-824-3495, e-mail: [email protected] (J. Hong)


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Notes The authors declare that there are no competing financial interests. ACKNOWLEDGEMENTS This research was supported by the Chung-Ang University Graduate Research Scholarship in 2015. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF2017R1E1A1A01074343).

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Structure of a multilayer nanofilm to increase the encapsulation

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