Multilayered Controlled Drug Release Silk Fibroin Nanofilm by

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Multilayered controlled drug release silk fibroin nano-film by manipulating secondary structure Moonhyun Choi, Daheui Choi, and Jinkee Hong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00687 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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Multilayered controlled drug release silk fibroin nano-film by manipulating secondary structure Moonhyun Choi, Daheui Choi and Jinkee Hong* Department of Chemical & Biomolecular Engineering, College of Engineering, Yonsei University 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea *Corresponding author: Jinkee Hong E-mail address: [email protected], Tel: +82-2-2123-5748

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ABSTRACT Many studies of drug delivery nano-platforms have explored drug loading affinity or controlled release. The nano-platforms can be influenced by their inherent building blocks. Natural polypeptide silk fibroin (SF) is an excellent nano-platform material because of its high biocompatibility and unique structural properties. SF secondary structures have different properties that can be changed by external stimuli. Thus, the characterization of SF-containing platforms is strongly affected by secondary structure transformations. Structural changes can occur spontaneously, which hinders the control of structural variation in aqueous conditions. Herein, we successfully prepared a controllable secondary structure composed of SF/heparin (HEP) layer-by-layer assembled nanofilms using simple solvents (glycerol and methanol). SF in the SF/HEP nanofilms takes up than 90%, which means configurations of SF have a strong effect on the characters of the nanofilms. We investigated the degradation profiles of SF/HEP nanofilms depending on their β-sheet contents and demonstrated an immediate correlation between the transformation of secondary structures inside the nanofilms and the degree of degradation of nanofilms. Finally, SF/HEP nanofilms were used as a delivery platform for incorporating the anti-cancer drug epirubicin (EPI). We could control the loading efficiency and release profile of EPI with various β-sheet contents of the nanofilms.

KEYWORDS: silk fibroin, nanofilms, controlled release, layer-by-layer (LbL) assembly


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Introduction In recent years, many studies have been undertaken to design and develop different kinds of nanoscale drug delivery platforms to incorporate therapeutic molecules and deliver them with various modifications or using various functional materials.1-6 The design and fabrication of novel drug delivery platforms is a promising and rapidly developing biomedical application.7-12 There are crucial factors for versatile and controllable drug delivery platforms with desirable release kinetics and accurate loading. First, the availability of appropriate materials—these should be absolutely harmless to the host, and must have the necessary physicochemical and biomedical properties, including degradability in biological media. Second, the platform should be able to stabilize drugs or molecules. In accordance with maintaining the activity of therapeutic molecules and the period of release, the maintaining the activity of materials is crucial for suitable therapy. Third, a tunable controlled drug loading and release system is needed for controlling the loading and release behavior according to the disease status and the patient's condition. Therefore, development of versatile and controllable platforms for desirable release kinetics and accurate loading amounts should be undertaken in biomedical fields. The optimal approach to achieving these goals is to fabricate tunable drug delivery platforms using a nanoscale, layer-by-layer (LbL) assembly.13 LbL assembly of nanofilms is driven by physical molecular interactions in the absence of chemical reactions or precursors, which produces a highly biocompatible and nontoxic system.14-21 In addition, within the consolidated nanofilms, all of the materials, including highly sensitive biomolecules (growth factors, proteins, antibodies, etc.), are stably solidified, thus minimizing the loss of biological activity.22-25 Without this stabilization, biological molecules in aqueous solutions can easily be destroyed by molecular


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motion. Moreover, within the nanofilms, the quantity, structures, and proportions of the multilayer components can be readily adjusted at the nanolevel. The features of LbL nanofilms depend on the inherent properties of the building blocks. Therefore, it is important to appropriately decide what kinds of materials will form the building blocks. Silk fibroin (SF) is a PDA-proved natural polypeptide extracted from Bombyx mori silkworms.26 A variety of biomaterial-related studies have demonstrated the suitability of SF for biomedical applications because of its biocompatibility, biodegradability, low immunogenicity, material versatility, cost-effectiveness, ease of processing, and advantageous tensile properties.2734

SF has been variously utilized in nanoparticles,35,36 scaffolds,37,38 hydrogels,39,40 and films,41

for biomedical applications that include wound healing,42 cancer therapy,43 osteogenesis,44 antiaging therapy,45 antioxidant activity,46 cartilage regeneration,47 and stabilization of bioactive molecules.48 These superior performances can be realized because SF is able to undergo diverse structural transformations at the molecular scale. These structural transformations are involved in ratio changes of secondary structures such as random coils, alpha (α)-helices, beta (β)-sheets, and β-turns. The permeability, release kinetics, mechanical properties, and binding affinities of the secondary structures are affected by solvents, stretching, annealing, and sonication. The exquisite control of SF secondary structures is very important for appropriate use. Herein, we prepared multilayered nanofilms via LbL assembly of SF and heparin (HEP) as a drug-loaded nano-platform. The assembly of the multilayered nanofilms involved molecular interactions of the functional groups of SF and HEP. The SF/HEP LbL-assembled nanofilms were fabricated by controlling the β-sheet content. The physiological stability and degree of degradation of these nanofilms were directly related to the stacking of the β-sheets. Furthermore,


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the hydrophobic character of the β-sheets permitted interactions with hydrophobic drugs. Finally, the β-sheet content in nanofilms was associated with drug loading affinity and release profile.

Experimental section Materials SF with a molecular weight of approximately 100 kDa, derived from the domesticated B. mori silkworm, was purchased from Advanced BioMatrix (Carlsbad, CA, USA). The hydrophobic molecule 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), epirubicin hydrochloride (EPI), HEP sodium salt from porcine intestinal mucosa, and glycerol solutions (86–89%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Fabrication of SF/HEP LbL-assembled films with controlled β-sheet content Silicon wafer and quartz glass as a substrate were treated with O2 plasma (CUTE-1B) for 2 min to bestow an overall negative charge and hydrophilic character with oxygen-containing functional groups. The purified substrates were first immersed in SF, SF + glycerol 10% (w/w), or SF + glycerol 100% (w/w) solutions (1 mg/mL, pH 3) for 20 min followed by three washing steps in deionized water (pH 3) for 2, 1, and 1 min, respectively. Glycerol percentage means weight of glycerol against weight of SF. Therefore, SF+glycerol 10% and 100% indicate that glycerol is added 0.1 mg/mL and 1 mg/mL in SF solution (1 mg/mL), respectively. Washing is essential in the LbL assembly to remove weakly-bound materials. The SF-coated substrates were dipped in an HEP solution (1 mg/mL, pH 3) for 20 min followed by three rinses with deionized


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water (pH 3) for 2, 1, and 1 min, respectively. The SF/HEP multilayer-coated substrates were dried using N2 gas. The dried SF/HEP LbL nanofilms on the substrates were solvent-treated by immersion in 50% methanol (MeOH) or 50% glycerol for 12 h. Glycerol and MeOH treated SF/HEP LbL nanofilms were rinsed by dipping DI water for 5 min and dried using N2 gas.

Characterization of SF LbL-assembled nanofilms The growth and thickness of the SF/HEP LbL nanofilms in dried state were measured with a Dektak 150 surface stylus profilometer (Veeco Instruments Inc.) comprising a 5 µm stylus tip and a stylus force of 3 mg. Adsorption and deposition of SF/HEP LbL nanofilms were evaluated using a model QCM200 quartz crystal microbalance (QCM; Stanford Research Systems, Sunnyvale, CA, USA). After deposition of SF or HEP layer on the most outer surface, the SF/HEP nanofilms on the QCM electrode were completely dried by N2 gas. The secondary structure of SF in SF/HEP LbL nanofilms was analyzed using Fourier transform infrared (FTIR) spectroscopy with a model 4700 device (Jasco, Easton, MD, USA) and circular dichroism (CD) with a model J-815 device (Jasco). CD wavelength scans were conducted three times at 25 °C between 190 and 250 nm using a bandwidth of 1 nm.

Stability test of SF/HEP LbL-assembled nanofilms For characterization of SF/HEP LbL-assembled nanofilms degradation, the nanofilms were immersed into 1 PBS (pH 7.4) and stored in incubator (37°C). The changes in thickness of the nanofilms were monitored by profilometer after each time point in PBS. The degree of


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degradation is expressed in later thickness (T)/initial thickness (To). All degradation studies of the SF/HEP nanofilms were performed in triplicate.

EPI loading of SF/HEP LbL-assembled nanofilms To incorporate EPI in the SF/HEP LbL assembled nanofilms, the samples were incubated with 5 ml of EPI solution (5 µg/ml) under dynamic conditions at room temperature for 60 min. After the nanofilms in EPI solution, the nanofilms were rinsed by dipping 5 min in DI water. The loading efficiency of EPI with SF/HEP nanofilms was measured using ultraviolet-visible (UV-VIS) spectroscopy with a model V-670 apparatus (Jasco). The intensity of the absorption at 485 nm was dependent on the β-sheet content in the nanofilms. Photoluminescence (PL) spectroscopy using a model FP-8300 device (Jasco) was used to gauge the quantity of EPI, with the hydrophobic regions in the nanofilms assessed using 1,8-ANS (8 mM) as a hydrophobic fluorescent

probe.

In vitro release kinetics of EPI from SF/HEP LbL-assembled nanofilms To determine the binding kinetics of EPI to SF/HEP LbL-assembled nanofilms depending on the proportions of the secondary structures, the nanofilms were incubated with 2 mL of an EPI solution (20 µg/mL) at room temperature. The incorporation of EPI into the SF/HEP nanofilms was monitored at 485 nm by UV-VIS absorption spectroscopy. To monitor the release of EPI from the nanofilms, samples were incubated with 2 mL of phosphate buffered saline (PBS) and cumulative EPI release experiments were conducted by measuring EPI-associated fluorescence (excitation wavelength 485 nm, emission wavelength 545 nm) at physiological conditions of pH


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°C.

In vitro cytotoxicity with SF/HEP LbL-assembled nanofilms To assess the anticancer effect of the EPI-loaded SF/HEP LbL-assembled nanofilms, the HeLa cells were cultured on 100 mm dishes in a 5% CO2 incubator for 2–3 days to reach 95% confluency. The cell culture medium was composed of 90% Dulbecco's modified Eagle medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin-glutamine. All products were purchased from Gibco Life Technologies (Waltham, MA, USA). HeLa cells were seeded in wells of 24-well plates (SPL Life Science, Pocheon-si, Korea) at a density of 7×104 cells per well. One day after seeding, the EPI-loaded SF/HEP LbL-assembled nanofilms, which had the same size (1 × 0.5 cm) were used to treat cells, with the replacement of the medium with fresh culture medium (1.5 mL per well). Each treatment was performed in triplicate. For comparison of the wafer effect on cells, non-treated silicon wafers were also used. The cells treated with films were incubated for 1 or 2 days. Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT solution (5 mg/mL PBS) was used to treat cells for 2 h in an incubation. The absorbance of the color developed in each well was determined at 540 nm using a SpectraMax 340 PC plate reader (Molecular Devices, Sunnyvale, CA, USA). For statistical Analysis, the cell viability data were expressed from the experiment repeated at least three times. The data shown on graphs represent the mean ± s.d. All experiments were performed in triplicate and compared with the control group using the one-way analysis of


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variation (ANOVA) and Scheffe method (IBM SPSS Statistics Data Editor). Error bars represent standard deviation. * shows statistical signiﬁcance of p < 0.05.

Results and discussion We prepared LbL-assembled nanofilms with by using both natural SF and HEP. Surface charge density of SF can be controlled depending on the pH. SF is a polypeptide comprised of a repeating sequence of amino acids (Gly-Ala-Gly-Ala-Gly-Ser) with an isoelectric point (pI) of 4. SF was dispersed in deionized water (pH 3) to expose positively charged functional groups on the surface. HEP was also dissolved in deionized water having the same pH 3 to create a partially charged state. The positively charged amine group (NH3+) of SF and the negatively charged carboxylic acid (COO-) of HEP can physically interact, mainly via electrostatic interactions and partially via hydrogen bonding. Figure 1a illustrates the chemical structures of SF/HEP LbLassembled films by the physically alternating adsorption of SF and HEP through the driving forces of electrostatic interaction and hydrogen bonding. The fabrication of SF/HEP LbL nanofilms was demonstrated by profilometer measurements. Figure 1b presents the thickness growth curve of 5, 10, 15, 30, and 60 bilayers of (SF/HEP)n nanofilms. The increase in the thickness was linear and a per-bilayer thickness of 14.19 ± 6.27 nm was achieved. Additionally, we have measured AFM for confirming the morphology of SF/HEP LbL nanofilms depending on adding glycerol or solvent treatments. AFM images of SF/HEP LbL nanofilms were obtained by scanning 20 µm x 20 µm areas. Figure R1 displays that each SF/HEP LbL nanofilms is fully coated on the silicon wafer (Figure S1, Supporting Information). The deposition of SF/HEP LbL nanofilms was monitored using QCM


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measurements. Figure 1c displays the changes in frequency during deposition of SF and HEP layers on the QCM electrode. After the adsorption of SF and HEP molecules, the frequency

decreased, indicating SF/HEP LbL nanofilms were successfully prepared. Furthermore, we measured the mass of SF and HEP in nanofilms, and the ratio of SF/HEP in QCM analysis (Table S1, Supporting Information). The SF composed more than 90% of the total mass of SF/HEP LbL nanofilms, indicating that the LbL nanofilms can differ markedly in structure and function according to the structures or properties of SF. Figure 1. (a) Schematic illustration of SF/HEP LbL-assembled nanofilms. The driving force for assembly is the electrostatic interaction between positively charged amine groups of SF and negatively charged carboxylic acid and sulfate of HEP, and hydrogen bonding between SF hydroxyl groups and HEP carboxylic acid residues. (b) Thickness growth curve of SF/HEP LbL-assembled nanofilms measured using a mechanical profilometer. (c) Frequency changes in SF (black squares) and HEP (red circles) LbLassembled nanofilms by quartz crystal microbalance according to the number of deposited layers.


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Changes in the secondary structure of SF can be achieved using various methods, such as solvent treatment, sonication, solvent vapor exposure, physical stretching, and others. However, the precise control of β-sheet content or quality using these approaches is difficult and can increase the cost of SF-containing products. The structural control of the silk protein with physical crosslinks (β-sheet) by treatments results in robust and stable coating.49-51 To optimize the secondary structure control, we explored the use of different ratios of two solvents (glycerol and MeOH) with SF and glycerol. Numerous researches have been previously conducted for investigating molecular structures of SF with using MeOH and glycerol solvents.52 Additionally, content of β-sheet can be controlled by concentration of glycerol.53,54 Depending on the treatment or the addition of glycerol, the controlled fabrication of β-sheet content was possible in SF/HEP nanofilms (Figure 2a).

Figure 2. (a) Illustration of the control of β-sheet by the addition of glycerol and solvent treatment. Fourier-transform infrared spectroscopy (FT-IR) of (b) SF/HEP LbL film, (c) SF + glycerol 10%/HEP LbL film, (d) SF/HEP LbL film with glycerol treatment, and (e) SF/HEP LbL film with MeOH treatment.


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FTIR spectroscopy was used to analyze the secondary structure of SF in the nanofilms. The technique has been used to reveal protein secondary structure. The nanofilms that were fabricated included SF/HEP, SF + glycerol 10%/HEP, SF/HEP with glycerol treatment, and SF/HEP with MeOH treatment. All the nanofilms that contained SF were characterized at the airsolid surface. We focused on the amide I region between 1600 and 1700 cm-1, which provides the most information on the secondary structure of proteins. For our purposes, the amide I mode associated with random coil conformations results in bands in the range of 1638–1655 cm-1. The curve that had absorption bands at the frequency range of 1656–1662 cm-1 corresponded to αhelices. The absorption bands at the frequency range of 1616–1637 cm-1 were associated with C=O stretching vibrations relating to the β-sheet conformational backbone in nanofilms. For untreated SF/HEP nanofilms, the ratios of the absorption bands at 1638–1655 cm-1 and 1656–1662 cm-1 wavelengths were higher than the ratio at 1616–1637 cm-1 (low β-sheet state). The results indicated the predominance of the random coil and α-helix structure in the nanofilms. Because LbL assembly involved SF solution at pH 3, the untreated SF/HEP nanofilms would have more β-sheet content than general SF-containing products fabricated at a neutral pH (Figure 2b). With the addition of glycerol (0.1 mg/mL) in SF solutions, the absorption bands representing random coil and α-helix configurations were relatively decreased and the absorption band representing β-sheet configuration was relatively increased (medium β-sheet state) (Figure 2c). In case of SF/HEP nanofilms with glycerol and MeOH treatments, Figure 2d and 2e illustrate that solvent-treated SF/HEP nanofilms possessed the greatest β-sheet configuration (high β-sheet state), with MeOH-treated SF/HEP nanofilms displaying slightly more β-sheet structures than glycerol-treated SF/HEP nanofilms. The secondary structures (a-helix, random coil and b-sheet) of the SF/HEP nanofilms were evaluated using Fourier self-deconvolution


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(FSD) of the infrared absorbance spectra for quantification of the FTIR bands (Supporting information, Figure S2). And then, the area of each graph was calculated to compare the 2nd structure contents (Supporting information, Table S2). As a result, 2nd structures ratio in the nontreated SF/HEP nanofilms are a-helix (38.24%), random coil (34.33%), and b-sheet (27.43%). 2nd structures ratio in the SF+glycerol 10%/HEP nanofilms are a-helix (33.9%), random coil (27.94%), and b-sheet (38.15%). In case of the glycerol treated SF/HEP nanofilms, a-helix, random coil and b-sheet are 29.51%, 25.17% and 45.32%, respectively. In case of the MeOH treated SF/HEP nanofilms, a-helix, random coil and b-sheet are 30.49%, 19.29% and 50.21%, respectively. Additionally, we clearly confirmed the difference in β-sheet content in accordance with treatments or the addition of glycerol as monitored by CD spectra analysis (Supporting information, Figure S3). In CD spectra, negative ellipticity at 215 nm means the conformational transformation of SF from random coil to β-sheet. The negative ellipticity at 215 nm are gradually increase depending on β-sheet contents. The proportion of SF secondary structures in nanofilms can affect the physiological stability associated with the stacking of the β-sheet in nanofilms due to hydrophobic interaction. Adding glycerol and solvents can induce the replacement of α-helices or random coils with β-sheets. As more glycerol is added, multiple β-sheet configurations will form inside the nanofilms. The probability of contact between β-sheets will increase and the resulting intramolecular or intermolecular interactions with the β-sheets would promote the formation of tertiary structures. These β-stacking-induced formations are very physiologically stable in the aqueous condition. Figure 3a displays the degree of degradation of SF/HEP nanofilms depending on the added amounts of glycerol or solvent treatment. The degree of degradation of nanofilms was monitored by measuring thickness variations at the physiological conditions of pH 7.4 and 37 °C. Without


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any treatments, the thickness of the SF/HEP nanofilms decreased to 45.19% of the original thickness for 10 min and then to 38.55% for 30 min. For nanofilms fabricated with 10% glycerol, the thickness of the SF + glycerol 10%/HEP nanofilms decreased to 77.92% after immersion in PBS for 10 min. The thickness of the SF + glycerol 100%/HEP nanofilms decreased to 87.56% and 84.04% after immersion in PBS for 10 min and 30 min, respectively. For solvent-treated SF/HEP nanofilms, the thickness of glycerol-treated nanofilms was only slightly decreased to 93.61% of the original thickness. For MeOH-treated nanofilms, no difference in thickness was evident in PBS (Figure 3a). It means that more 60% components of the the nontreated SF/HEP nanofilms disappeared and physiological stability of the nontreated SF/HEP nanofilms is very

low, comparing MeOH treated SF/HEP nanofilms. Figure 3. (a) Degree of degradation (T/To) of SF/HEP LbL films depending on the ratio of the added glycerol and solvent treatment. FTIR spectra of (b) SF/HEP LbL films, (c) SF + glycerol 10%/HEP LbL film, and (d) SF/HEP LbL film treated MeOH before (black line) and after (red line) immersion in PBS. To demonstrate whether physiological stability was directly affected by the changes of secondary structure, we carefully observed the FTIR variation of the nanofilms before and after a 60-min immersion in PBS. The FTIR peak of the untreated SF/HEP nanofilms decreased after


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immersion in PBS, and the absorption bands representing α-helix and random coil also reduced (Figure 3b). FTIR analysis of SF + glycerol 10%/HEP nanofilms revealed essentially unchanged absorption bands of the β-sheet structure. However, declines in the absorption bands of α-helix and random coil were evident (Figure 3c). In case of MeOH-treated SF/HEP nanofilms, almost all the absorption bands were maintained. From these results, we can conclude that β-sheet structure formation in the nanofilms is directly related to physiological stability. In addition, the changes in thickness before and after immersion in PBS were evident to the naked eye. The colors of the nanofilms, due to reflective index, are dependent on the thickness. Different refractive indices result in reflection of light at wavelengths proportional to the thickness of the nanofilms for the material with higher refractive index. Color changes were apparent in the nanofilms with low and medium β-sheet content, whereas there were no color changes in the glycerol- or MeOH-treated nanofilms (high β-sheet) (Supporting information, Figure S4). We examined whether the SF/HEP nanofilms could serve as a drug delivery nano-platform. The hydrophobic drug EPI was incorporated into the SF/HEP nanofilms followed by incubation at room temperature. To systematically study EPI-SF interactions, the SF/HEP nanofilms with different β-sheet contents, and hence different loading efficiency, were used. EPI could interact with the SF/HEP nanofilms by electrostatic and hydrophobic interactions. The electrostatic interaction of the carboxyl group (COO-) of HEP with the amine group (NH3+) of EPI occurred on the surface of the nanofilms. EPI incorporation inside the nanofilms was driven by hydrophobic interaction (Figure 4a). The EPI adsorption by electrostatic interaction similarly occurred on the surface of each nanofilm. Thus, the β-sheet content of nanofilms was an important determinant of drug loading efficiency. We assessed the capacity of the nanofilms to incorporate hydrophobic molecules to investigate whether they could be employed for drug


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delivery. A hydrophobic fluorescent probe (1,8-ANS) was introduced into the four types of the SF/HEP nanofilms. The probe fluoresces in a hydrophobic environment and has been routinely used to measure the incorporation of hydrophobic drugs into nanofilms (Figure S6b, Supporting Information). The fluorescence intensities for the SF/HEP nanofilms was the trend of b-sheet content in the SF/HEP nanofilms. It means that the hydrophobicity of SF/HEP nanofilms is different depending adding glycerol or solvent treatments (hydrophobicity; SF/HEP_MeOH treated > SF/HEP_glycerol treated > SF+ glycerol 100%/HEP > SF/HEP_non treated).

Figure 4. (a) EPI loading mechanism of SF/HEP films involving electrostatic interaction between the NH3+ of EPI and COO-of HEP, and hydrophobic interaction involving the aromatic ring of EPI and β-sheet of SF. (b) Fluorescent intensity of EPI-loaded SF/HEP LbL films depending on the ratio of added glycerol and solvent treatment (EPI excitation and emission wavelength of 474 and 551, respectively). (c) EPI total and (d) normalized release profile from SF/HEP LbL nanofilms. (e) In vitro cytotoxicity of EPI loaded SF/HEP LbL nanofilms for HeLa


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cells. HeLa cell viability (%) when exposed to SF/HEP LbL nanofilms having different β-sheet content. * shows statistical signiﬁcance of p < 0.05. To confirm the affinity between β-sheet contents and EPI, we compared the degree of EPI incorporation by analyzing the PL and UV-VIS spectra. Figure 4b shows the fluorescence intensity of nanofilms loaded with EPI. SF/HEP nanofilms with a high β-sheet content displayed higher fluorescent intensity. Moreover, EPI adsorption of the SF/HEP nanofilms at 480 nm differed according to the types of sample (Figure S6c and S6d, Supporting Information), with the drug loading efficiency differing depending on β-sheet content (Figure 4c). The EPI loaded in non-treated SF/HEP, SF + glycerol 10%/HEP, SF + glycerol 100%/HEP, glycerol-treated SF/HEP, and MeOH-treated nanofilms was 2.05 ± 0.35, 2.22 ± 0.41, 2.65 ± 0.2, 2.86 ± 0.08, and 3.13 ± 0.23 µg/cm2, respectively (Figure S6a, Supporting Information). As shown in Figure 4d, normalized data was obtained by calculating (EPI release amounts each time)/(final EPI release amounts). This normalization helps to understand and analyze kinetics of EPI release from each SF/HEP nanofilms. After 1h, EPI from glycerol and MeOH treated SF/HEP nanofilms were released 47.62% and 39.48%. On the other hand, EPI from nontreated SF/HEP nanofilms was released almost 80%. It indicates that higher β-sheet structure in the nanofilms was associated with a more sustained released of EPI release. Finally, the in vitro cytotoxicity of the nanofilms was determined against HeLa cells using the MTT assay. As shown in Figure 4e, the β-sheet content of the nanofilms does not affect HeLa cell viability after a 1-day treatment. However, after 2 days, nanofilms with a higher β-sheet content were more effective in killing the HeLa cells. There is little statistical significance (p value < 0.05) between cell viability on nontreated SF/HEP nanofilms and cell viability on MeOH treated SF/HEP nanofilms. It means that there is


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definitely a difference of killing effect on cancer and that EPI loaded MeOH treated SF/HEP nanofilms is more effective for anti-cancer.

Conclusion We have successfully prepared SF/HEP LbL-assembled nanofilms with controlling secondary structure of SF. The β-sheet content in the SF/HEP nanofilms could be controlled with the use of solvents or treatments. The secondary structure of the controlled SF/HEP nanofilms influenced the physiological stability depending on β-sheet contents. We understood mechanism and casual relationship between SF configuration of the nanofilms and degree of degradation. FTIR spectroscopy revealed that the SF secondary structure was the main influencer of the nanofilm properties. A higher β-sheet content (maximum 50.21%) in SF/HEP nanofilms was associated with greater nanofilm stability under physiological conditions. By manipulating the structural properties of materials in water-based solutions with glycerol or MeOH, we have analyzed the hydrophobicity of the SF/HEP nanofilms by using hydrophobic probe 1,8 ANS. And then, the loading efficiency and release profile of EPI in the nanofilms could be regulated by configurations of SF in the EPI-loaded nanofilms. EPI loaded SF/HEP nanofilms with highest βsheet content is more effective for anti-cancer. . ASSOCIATED CONTENT Supporting Information.


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Characterization of SF/HEP nanofilms: QCM mass analysis, CD spectra, optical microscopy Confirmation of loading EPI and adsorption of 1,8 ANS by PL and UV-vis spectra AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Jinkee Hong: 0000-0003-3243-8536 Author Contributions J.H supervised the overall experiments. M.C. developed the SF/HEP nanofilms and wrote the paper D.C conducted cell viability test. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. (match statement to author names with a symbol) Notes The authors also declare no competing financial interest.

ACKNOWLEDGMENT 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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Multilayered Controlled Drug Release Silk Fibroin Nanofilm by

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