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dextran matrixes for controlled protein release
European Polymer Journal 114 (2019) 197–205
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Study of nanostructured fibroin/dextran matrixes for controlled protein release Jose Manuel Ageitos, Amelia Pulgar, Noemi Csaba, Marcos Garcia-Fuentes
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Centre for Research in Molecular Medicine and Chronic Diseases (CiMUS), Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Universidade de Santiago de Compostela, Avda. Barcelona s/n, 15782 Santiago de Compostela, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Silk fibroin Dextran sulphate Protein release Nanostructure Porogen Water-annealing
Microporous films are structures with attractive features as high porosity and high intrinsic surface area. Unfortunately, most biocompatible materials useful for the preparation of such microporous films are suboptimal for drug delivery due to poor processability and/or drug encapsulation properties. Silk fibroin (SF) is a biocompatible protein with good drug release properties and suitable characteristics for pharmaceutical processing. Dextran sulphate (DSS) is an anionic polysaccharide with the capacity to interact and stabilize many proteins. In the current study, we evaluate the effect of DSS on the physical properties of SF/DSS blend films, how it affects the molecular and microscopic structure of the system and its capacity for providing controlled release of a model protein. Using this system, micro- and nanostructured films could be prepared through a green, water-only process. It was found that DSS acted both as a modifier of SF secondary structure and as a functional porogen, where the size of nano- and micropores varied with the blending ratios. High ratios showed higher swelling, porosity and crystallinity, resulting in modified protein release kinetics, as compared to pure SF films. Considering both their mild preparation method and their physical and pharmaceutical properties, SF/DSS films stand out as ideal systems for sustained protein delivery applications.
1. Introduction Silks are proteinaceous polymers produced by different arthropods, such as silkworms, spiders, scorpions, mites, or bees. Among them, the silk from silkworms (Bombyx mori) is the most studied, and it is widely used for multiple applications, due to its ease of manipulation and availability from the sericulture industry [1]. Silk fibroin (SF) is a high molecular weight fibrous protein (∼390 kDa) and the main component of silkworm silk, conjointly with sericin, a hydrophilic protein that envelops and glues fibroin fibres [2]. SF is composed by repetitive sequences rich in glycine, alanine and serine that allows the formation of β-sheet structures [3] responsible of its physicochemical properties such as insolubility, high tensile strength, and toughness. SF can be extracted from silkworm cocoons and converted into a soluble form by several methods [4–8], including solubilization in concentrated salt [LiBr, LiCNS, CaCl2-ethanol, Ca(CNS)2, ZnCl2, NH4CNS, CuSO4 + NH4OH, Ca(NO3)2] or concentrated acid (phosphoric, formic, sulfuric, hydrochloric) solutions [8]. Among them, solubilization with 9.3 M LiBr is the prevailing method [5] due to the enhanced stability of SF solutions during processing [7]. Then, soluble SF can be casted in different forms such as films, hydrogels, sponges, fibres, microparticles,
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and others [9]. These devices are biocompatible, biodegradable and have the capacity to load and release biomolecules. SF films have been employed for wound dressing, tissue engineering, and controlled drug delivery due to their permeability to oxygen and vapor, biodegradability, high mechanical performance and amenability to easy processing [10]. In addition, in order to modulate its properties, SF has been blended with other polymers such as sericin [11], elastin [10], collagen, chitosan [12], hyaluronic acid [13,14], alginate [15], pectin [16], telechelic-type polyalanine [17] and others [10,18]. In general, these blends are employed to improve the mechanical properties of SF (e.g. elasticity, toughness), while others improve other properties such as water uptake, swelling, cell adhesion and proliferation. Another strategy resides in combining SF with sodium chloride, sugar, paraffin and poly(ethylene oxide), which can be employed as porogens due to their immiscibility with either organic or aqueous SF solutions [9,19]. These compounds produce discrete domains inside the casted SF matrix that can be leached with an appropriate solvent, leading to porous structures that can be used for modified drug release and tissue engineering [20]. Unfortunately, many of these compounds need to be removed with toxic, polluting organic solvents (e.g. hexafluoro isopropanol (HFIP), methanol or hexane), which can limit their
Corresponding author. E-mail address: [email protected] (M. Garcia-Fuentes).
https://doi.org/10.1016/j.eurpolymj.2019.02.028 Received 30 September 2018; Received in revised form 15 February 2019; Accepted 19 February 2019 Available online 20 February 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
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overnight in a fume hood. After drying, films with an average thickness of 23 µm were obtained. SF/DSS films were water-annealed in a sealed chamber saturated with water-vapor (∼99% of relative humidity) for 12 h at 20 °C to induce β-sheet formation [36,37].
biocompatibility [21]. Despite these inconveniences, microporous materials have interesting properties such as high surface area, loading capacity and gas permeability [22,23], as well as improved degradability due to their increased contact area with the media [23]. Dextran sulphate (DSS) is a complex, branched anionic polymer of anhydroglucose employed as anticoagulant, antilipaemic, and antiviral agent, which has the ability to form complexes with cationic polymers and to increase the loading efficiency of certain drugs, such as doxorubicin or mitomycin [24–26]. DSS and SF have been described as enzyme stabilization agents [27–31], therefore, their combination could be interesting for the encapsulation of labile drugs such as proteins. Indeed, SF fibres have already been chemically modified with dextran and showed attractive characteristics for wound dressing [32]. In the current report, we prepare physically blended SF/DSS films and we characterize in detail the effect of both compounds on their physical and micro-structural properties, central characteristics for the controlled release of grown factors [33], anesthetic drugs [34], and antibacterial compounds [35]. Finally, we have studied the effect of DSS on the controlled release of a model protein, horseradish peroxidase II (HRP), loaded SF/DSS blend films.
2.3. Characterization of SF/DSS blend films 2.3.1. Fourier transform infrared spectroscopy (FTIR) FTIR absorption spectra from 400 to 4000 cm−1 wavenumber range were collected using a VARIAN FT-IR 670 (Varian Inc. Scientific Instr., Palo Alto, CA) equipped with a diamond attenuated total reflection (ATR) attachment (Pike, GladiATR, Pike Technology, Madison, WI) at 4 cm−1 resolution, using 64 scans. FTIR spectra were processed using OPUS Spectroscopy Software 5.0 (Bruker, Bremen, Germany) and were normalized to the maximum intensity of the amide I region. Peak position and bandwidth of the component bands of amide I region were obtained using a Fourier deconvolution method (a Lorentzian distribution was assumed) [38] and second-derivative spectra with OriginPro 2017 (Origin lab corporation, Northampton, MA). The average composition (%) of SF secondary structure was obtained by integrating the area of each deconvoluted curve and then normalizing to the total area of the amide I region of at least three different spectra [38].
2. Experimental 2.1. Materials
2.3.2. Scanning electron microscopy The surface and section morphology of the films were studied by field emission scanning electron microscopy (FESEM, UltraPLus, Carl Zeiss, Germany). Lyophilized and air-dried films were coated with Iridium previously to observation. Thickness of the films was directly measured from the observed sections.
Bombyx mori cocoons were kindly donated by Dr. Jose Luis Cenís (IMIDA, Murcia, Spain). Sodium carbonate, lithium bromide, polypropylene glycol (PEG MW: 10,000), dextran sulphate sodium salt from Leuconostoc spp. (DSS, average molecular weight: 15,420 Da, sulphur content: 17.9%), dimethyl sulfoxide (DMSO), polyethylene (20) glycol sorbitan monolaurate (Tween 20) and horseradish peroxidase II (HRP) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium phosphate saline buffer (PBS; 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4) was purchased from Fisher Scientific Co. (Fair Lawn, NJ). All the chemicals were used without any further purification.
2.4. Study of the physicochemical properties of the SF/DSS films and degradation test Swelling ratio and water absorption measurements: To evaluate SF/ DSS films swelling, individual samples (4 cm2, ≈32 mg) were weighed (WI), immersed in 5 ml of 10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween-20, pH 7.5 (PBS-T) and incubated at 37 °C for different time points (5–240 min) under agitation. After each time point, nonabsorbed liquid was removed with filter paper and the swollen weight (WS) was measured. Swollen samples were dried at room temperature for 3 days in a fume hood and weighed (WF). The samples were measured by triplicate per group. Weight loss in dissolution: Mass loss tests were made by immersing different SF/DSS films samples (3.5 cm2, ≈26 mg) in 5 ml of PBS-T and incubated at 37 °C for different time points (1–20 days) under agitation. Samples were dried, and the weight variation was determined gravimetrically (WI, WF). The samples were measured by triplicate per group. Swelling ratio (SR), % of water uptake (WU) and % of weight loss (WL) were calculated according the following equations: SR (w/w): [(WS − WF)/WF]; WU (%): [(WS − WF)/WS] × 100;[39] WL (%): 100 − [(WI − WF)/WI] × 100.
2.2. Methods 2.2.1. Silk fibroin purification and concentration Silk degumming and fibroin purification were conducted as previously described in the literature [5,6,13]. B. mori cocoons were cut in 1 cm2 pieces, rinsed with ultrapure water, and boiled in 2 l of 0.02 M sodium carbonate for 30 min. Then, degummed silk was incubated in 1 l of ultrapure water for 20 min with gentle agitation. Excess of water was removed, and the rinsing process was repeated twice. Degummed silk was rinsed again in ultrapure water, spread out on a clean piece of aluminium foil and dried in a fume hood during 24 h. Degummed silk was cut in small pieces and 4 volumes (v/w) of 9.3 M lithium bromide were added to this sample. Degummed silk was incubated at 60 °C in a water batch during 5 h, until the complete melting of fibres was observed. 20% (w/v) SF solution was dialyzed against 1 l Milli-Q (MQ) water in a Slide-A-Lyzer cassette 3500 for 48 h. Dialyzed SF was centrifuged at 7000g, 4 °C, for 40 min, and the resulting transparent solution that was stored at 4 °C. Concentration of SF was conducted by dialysis against a 10% solution of PEG. SF concentration was determined gravimetrically. Final SF concentration (7.4%) was adjusted by adding MQ water.
2.5. Characterization of model protein release in SF/DSS blend films 2.5.1. Model protein loading and release HPR was labelled with fluorescein isothiocyanate (FITC) following the protocol described by Lu et al. [40]. Briefly, FITC-V (EMP-Biotech GmbH, Berlin, Germany) was dissolved in DMSO (10 mg/ml) and gradually added to a solution of HRP (10 mg/ml) in sodium carbonate solution (pH 8.2, 100 mM). HRP and FTIR solution was incubated overnight at 4 °C, free FITC was removed by diafiltration (7000g, 4 °C, 15 min) with PBS on an Amicon ultra-4 centrifugal filter ultracel 10 K (Merck KGaA, Darmstadt, Germany). FITC-HRP was frozen at −20 °C until use. Protein/fluorescence ratio was determined using a NanoDrop 2000/ 2000c UV–Vis (Thermo Scientific) and a fluorimeter (EnVision 2104
2.2.2. Preparation of SF/DSS blend films DSS was weighed according to the amounts required for SF:DSS ratios (w:w) of 100:0, 89:11, 80:20 and 67:33 and dissolved in the previously described 7.4% aqueous SF solution. Blends were stirred at room temperature during 1 h for complete dissolution of DSS. The mix was then aliquoted with a SF density of 8 mg/cm2 in 3.5 cm polystyrene plates or 6 cm passivated glass plates. SF solutions were left to dry 198
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and smooth appearance. SF/DSS(80:20) and SF/DSS(67:33) films present cavities that are larger in the latter case. The presence of unevenness and cavities suggested that discrete DSS domains can be found in the blend at the highest ratios assayed. Films were water-annealed for the induction of β-sheet formation in SF. After water annealing, the films became wrinkled, especially at low DSS content, and lost transparency. These changes suggest an increase in β-sheet content in the films [42] since similar changes have been described for SF immersed in methanol, the traditional annealing method [5]. Micrographs of waterannealed films (Fig. 1) showed that the cavities in high DSS ratio films became deeper after this treatment, resembling pores. However, the cross-sections of the films did not show such pores but distinguishable microdomains in the films with high DSS ratio [SF/DSS(80:20): 0.6 ± 0.2 µm and SF/DSS(67:33): 2.3 ± 0.5 µm] (Fig. 1). This suggests that in SF/DSS(80:20) and SF/DSS(67:33), the DSS content surpassed the critical ratio of the polymer mixture above which the blend exhibits heterogeneous properties [43]. The formation of dextran domains in SF films has been previously described in methanol treated SF/ dextran(80:20) films by Hines and Kaplan [44]. They found that the size of this domains encapsulated within the silk depended on the molecular weight of the dextran and the film treatment; in this sense, the current results showed that the size of such domains can also be modulated by the loading ratio of DSS.
Multilabel Reader, Perkin Elmer, Waltham, MA) (λEx: 490 nm; λEm. 525 nm) [41]. FITC-HRP was mixed with an appropriate concentration of DSS according to the protein loading (0.33, 0.99 and 2% of SF content) and the SF/DSS ratio of the film (100:0, 89:11, 80:20 and 67:33) and mixed for 30 min. SF solution (7.4%) was added to the different DSS/FITC-HRP solutions and incubated for 1 h under agitation. SF/DSS/FITC-HRP solutions were casted onto 96-well plate and film blends were water-annealed as described above. For release studies, 200 µl of PBS-T was added to each well and the plate was incubated at 37 °C with rotatory agitation. The supernatants of the wells were removed and replaced by fresh PBS-T at different time points (1 h, 1, 3, 5, 10, 15, 20, 25 days). Released FITC-HRP in the supernatant was measured at 525 nm, and the release (%) was determined with respect to the initial loading of FITC-HRP in the films. 2.5.2. Modelling of release profile The mechanism of HRP release from the SF blend films was investigated by fitting several release kinetic models to the in vitro fractional release vs. time data. The formulas and basic principles of the model are available in method section of supplementary material. 3. Results and discussion 3.1. Characterization of SF/DSS films
3.1.1. Structural study of SF/DSS films The effect of DSS in the blends was also studied by FTIR (Fig. 2). FTIR spectra of the SF/DSS films were similar, except for the DSS signals (3469, 1219, 980, 801 and 577 cm−1), which increased in intensity with DSS content in the film (Fig. 2). Before water-annealing, all films showed a maximum peak in 1639 cm−1 in the amide I region which is attributable to SF random coil structure [45]. The percentage of random coil of SF/DSS 100:0 films were similar to the ones reported by Wei et al. [46] for pure SF films, although higher than the ones reported
The effect of DSS content in SF/DSS blend films was studied by evaluating morphological, structural, and physicochemical changes in the films. From a macroscopic point of view, all the assayed bends yield films with uniform flat shape; however, as the concentration of DSS in the blend increased, the films became less transparent and acquired a whitish colour. Upon microscopic observation of the surface, the morphology of the films changed with increasing DSS in the blend (Fig. 1). The surface of SF/DSS(100:0) and SF/DSS(89:11) films has a uniform
Fig. 1. Scanning electron micrographs of surface and cross-sections of SF/DSS blend films before and after water-annealing process. 199
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between SF and the polysaccharide [48]. SF is an amphoteric protein with negative and positive domains, however, the C-terminal region is markedly positive, with a ratio 9:1 positive/negative charges [49], this could explain the interaction with the anionic polymer DSS. In the same way, a reduction in the intensity of SF signal at 1515 cm−1 was also observed with increased DSS content, which suggested an increase in the conformational freedom of SF [50]. This band at 1515 cm−1 has been assigned to the CH bending mode and the ring CC stretching of the side chain of tyrosine [50], and it is characteristic for silk II conformation [51]. After water annealing, a shift in amide I region from 1639 to 1620 cm−1 was observed, together with a reduction of the shoulder at 1533 cm−1. These signals (1639, 1533 cm−1) are characteristic of random coil structure in SF [45], while 1620 cm−1 is associated with aggregated β-sheet structure [37]. The presence of the shoulder at 1650 cm−1 is indicative of silk I conformation [52], and this band is usually assigned as random coil [42,45,53], suggesting the coexistence of random coil and β-sheet structures. Although the overall β-sheet content increased after water-annealing for all prototypes, the differences observed among groups were maintained around similar values to those observed before water-annealing (Table 1). 3.2. Study of SF/DSS films in solution Films before water-annealing dissolved immediately when immersed in water. After water-annealing, all blend films kept their shape, absorbed water, and became flexible. Films quickly lost weight during the initial 30 min in solution (Fig. 3), and then, this process progressively slowed down until reaching a plateau after three days. The percentage of mass loss was proportional to the DSS content in the film. It has been described that dextran is released from SF films proportionally to its molecular weight [44]. Given that the DSS employed in this study was a mixture of different molecular weight polymers (9–20 kDa), the initial burst loss can be attributed to the low molecular weight DSS. After the initial weight loss phase, the films mass decreased linearly with time, being possible to observe differences between the SF/DSS ratios employed. In this second phase, contrary to the previous one, the percentage of mass loss per day (ΔW) decreased with increasing DSS content: SF/DSS(100:0)ΔW = −0.38%/d (R2: 0.995); SF/DSS (88:11)ΔW = −0.26%/d (R2: 0.958); SF/DSS(80:20)ΔW = −0.20%/d (R2: 0.922); SF/DSS(66:33)ΔW = −0.17%/d (R2: 0.839). The slower mass loss in this second phase can be related to the increase in β-sheet content observed for high DSS content films (Table 1).
Fig. 2. FTIR spectra of air-dried SF/DSS blend films. A. Detailed view amide I and II bands of SF/DSS blend films before and after water-annealing process (W.A.). B. FTIR spectra of SF/DSS blend films after water-annealing and dextran sodium sulphate (DSS), depicting the characteristic bands of DSS.
3.2.1. Study of SF/DSS films after their immersion in solution: molecular structure The molecular structure of the blend films after immersion in PBS-T
Table 1 Structural comparison of SF/DSS blend films before and after water-annealing treatment (WA). Sample
Treatment
β sheet (%)
Random coil (%)
SF/DSS(100:0)
– WA
34.2 ± 4.2 48.3 ± 4.0
34.3 ± 1.8 28.8 ± 5.6
SF/DSS(89:11)
– WA
38.4 ± 4.5 49.9 ± 6.6
31.1 ± 2.8 27.7 ± 3.5
SF/DSS(80:20)
– WA
38.7 ± 4.1 51.3 ± 6.5
27.2 ± 1.7 26.3 ± 6.3
SF/DSS(67:33)
– WA
41.0 ± 0.7 55.0 ± 6.4
29.1 ± 2.3 22.7 ± 5.4
by Hu et al. [37]. Amide I deconvolution showed that as the DSS content increased also did the β-sheet content of the films, while the random coil content decreased (Table 1). Similar results have been reported for SF blended with chitosan, alginate, hyaluronic acid, and cellulose [13,14,47]. Chen et al. proposed that the polymer-induced conformation transition is produced by the strong hydrogen bonding
Fig. 3. Time course of the weight loss of SF/DSS blend films in solution. The rectangle insert is a zoom of the initial points of the study. 200
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Fig. 4. FTIR spectra of freeze-dried SF/DSS blend films after incubation in PBS-T buffer for different days (D). (A) General view depicting characteristic bands of Dextran. (B) Magnification of amide I region of SF/DSS blend films.
buffer was studied by FTIR (Fig. 4). After 1 day it was observed that the characteristic signals of DSS (1219, 980, 801 and 577 cm−1) disappear and FTIR spectra of all blend films become similar (Fig. 4A). However, some differences can still be detected in the 3600–3400 cm−1 region that correspond to dextran ν(OH) vibrations [54]. Here, we could observe that the DSS shoulder at ∼3469 cm−1 in SF/DSS(67:33) films decreased from day 0 (D0) to day 1 and decreased further again at day 5, suggesting that in this blend the DSS release follows sustained kinetics. Regarding the amide I region (Fig. 4B), a decrease in the shoulder at 1646 cm−1 in the amide I region could be observed for all blends. This band is assigned to random coil SF [55], and its reduction until day 5 indicates that the initial weight loss phase is not only attributable to DSS release, but also solubilisation of the amorphous domains of silk I, as α-helix content of silk I requires more time for this process [45]. After 20 days, the band at 1620 cm−1 disappeared from SF/DSS(88:11) and SF/DSS(80:20), with the peak top of amide I shifting to 1626 and 1632 cm−1, respectively. This suggest that the proportion of intermolecular β-sheet decreased and intramolecular β-sheet became predominant [37,56,57]. The β-sheet content of SF/DSS(88:11) and SF/ DSS(80:20) decreased after 20 days in solution (34% and 24%, respectively), while SF/DSS(100:0) and SF/DSS(67:33) became more crystalline by the solubilization of random coil structures (57% and 58%, respectively). As matter of fact, both SF/DSS(100:0) and SF/DSS (67:33) films showed a similar spectra during the all the study except for a relative increase of the band at 1515 cm−1 at days 5 and 20 for the latter composition. This indicates that SF/DSS(67:33) was losing conformational freedom, becoming more crystalline [50].
3.2.2. Study of SF/DSS films after their immersion in solution: macromolecular structure The effect of incubation in liquid on the microscopic structure of the films was further evaluated by SEM (Fig. 5). The thickness of DSS containing films increased after 1 day in liquid, especially in SF/DSS (80:20) and SF/DSS(67:33) films, nearly triplicating its original value
Fig. 5. Scanning electron micrograph of freeze-dried SF/DSS blend film sections after 1 and 20 days immersed in PBS-T buffer media.
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blends were determined in vitro (Fig. 6). Water-annealed films showed lower SR and WU values than those reported for methanol treated SF films [13,59]. However, the WU of SF/DSS(100:0) was higher than the one reported by Karve et al. for water-annealed SF films [60]. SF/DSS films absorbed liquid quickly upon immersion since the maximum SR and WU were observed at 5 min with the exception of SF/DSS(67:33). In this latter case SR and WU values increased up to 30 min and were higher as compared to the other films (Fig. 6B). The increase in the average SR of SF/DSS blends was proportional to the DSS ratio (7, 33 and 66%, respectively) and similar to the values reported for SF chemically linked to DSS (46%) [32]. However, all SF films showed similar behaviour after achieving the maximum values, when SR and WU gradually decreased and stabilized after 105 min. Swelling behaviour seems to be related with the mass loss process (Fig. 3): as DSS is released from the film, the pores formed (Fig. 5) increase the relative surface area of the SF/DSS films, allowing higher WU and SR, and hence increasing the thickness of blend films. As observed by SEM imaging (Fig. 5), SF/DSS(67:33) films were able to keep their swollen state even after 20 days in aqueous media, while the other blends collapsed after long incubation times. These results are consistent with the efficient preservation of β-sheet signals in the FTIR spectra of SF/DSS (67/33) as compared to other compositions (Fig. 4B).
(Figs. 1 and 5). As previously observed in the dry state (Fig. 1), SF/DSS (100:0) and SF/DSS(89:11) were similar, while SF/DSS(80:20) and SF/ DSS(67:33) presented different morphology (Fig. 5). SF/DSS(80:20) films presented pores with an average size of 0.5 ± 0.2 µm, similar to the encapsulated domains observed in dry state (0.6 ± 0.1 µm). Interestingly, the increase in thickness after incubation in water did not induce increase in the size of pores. This can be explained by the preferential retention of the solvent in the amorphous regions of SF [8]. As DSS increases the crystallinity of SF, it is expected that the inner surface of the pores is more crystalline than other areas. This would explain the low volume variability of the pores as compared with the rest of the film in those preparations where amorphous SF is more prevalent. After 1 day in PBS-T buffer solution, SF/DSS(67:33) films presented an inner structure resembling a sponge. Mainly, two population of pores with diameters 1.6 ± 0.5 µm and 4.2 ± 0.7 µm were found (Fig. 5), being more numerous than the DSS encapsulated domains (2.3 ± 0.5 µm) observed in dry state (Fig. 1). These results indicate that DSS acts as a porogen for SF films, and the pore size is dependent on DSS concentration. Similar behaviour has been previously described for SF/PEG blends [19,58], where micro-phase separation induced the formation of pores upon extraction of PEG when incubated in water. Nevertheless, DSS showed higher miscibility with SF than PEG as 10% PEG was found sufficient for induction of the phase separation, while the formation of bundles of SF globules was not observed [19] even at SF/DSS(50:50) (data not shown).
3.3. Study of protein association and release For these studies, we encapsulated horseradish peroxidase II conjugated to fluorescein (FITC-HRP) in the different SF/DSS films. This model protein was selected because it has a molecular weight and isoelectric point similar to many proteins of therapeutic interest (such as the bone morphogenetic protein or fibroblast growth factor) [61,62], thus providing a relevant model for drug delivery characterization studies. In order to evaluate the cargo effect in the release profile three different loadings of FITC-HRP were employed in the blend films: 0.33, 0.99 and 2.00% w/w in relation to the SF content. A first observation of this study was that irrespective of the film composition, the percentage of protein released (%) at the end of the study was inversely proportional to the loading (Fig. 7). Therefore, in all blends 0.33% FITC-HRP showed the highest percentage of released protein. SF/DSS(100:0) films achieved nearly complete protein release after 25 days when loaded with 0.33% HRP (Fig. 7A), showing a 20% of burst release followed by a continuous release phase for up to 25 days. This release profile for SF/DSS(100:0) was different to the previously reported by Hofmann et al. [63] for water-annealed SF films loaded with HRP. These authors have observed a two-phase release, with an initial burst followed by a lag phase of 2 days, and a plateau by day 10. However, the total accumulative release (∼55%) was similar to the one reported in the current study for the 2% loaded formulation. The differences can be explained based on the different β-sheet content between the two films, since the SF/DSS(100:0) films investigated in this work have similar β-sheet content (Table 1) to methanol-treated SF films reported by these authors. Indeed, in the study by Hofmann et al., the methanol treated SF films presented a longer lag-phase (5 days) followed by a linear release with a slope similar to the results from the present study. In summary, our pure SF shows an initial release typical of water-annealed films, but a follow-up sustained release more similar to previous data on methanol-treated films [64]. This difference could be mediated by the differences in the protocols for water-annealing [36] that might result in higher β-sheet content in our samples [65]. The effect of the addition of DSS in the SF produced prototypes with clearly different kinetics. At low DSS content, i.e. SF/DSS(89:11), the protein release kinetics was modified to lower burst effects and more sustained release rates than the pure SF films (Fig. 7B). These changes suggest that inclusion of DSS might improve the compatibility of HRP with the film matrix and avoid accumulation of loosely bound protein on the material surface, which is assumed to be responsible for burst release [44]. With high levels of DSS, SF/DSS(80:20) and (67:33), the
3.2.3. Swelling properties of SF/DSS films The swelling ratio (SR) and the water uptake (WU) of the SF/DSS
Fig. 6. Time course of the swelling ratio (A) and water uptake (B) of SF/DSS blend films in solution. 202
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Fig. 7. Cumulative release profile of FTIC-HRP loaded SF/DSS films (● 0.33; ▲0.99; ■ 2.00). A. SF/DSS(100:0). B. SF/DSS(89:11). C. SF/DSS(80:20). D. SF/DSS (67:30).
burst increased to values higher than those observed with the pure SF system (Fig. 7C and 7D). This high burst might be caused by protein being released together with DSS in the first minutes by the newly formed pores described previously. However, once this burst phase is over, these systems provided a slower protein release rate than the pure SF films, and as a matter of fact, no prototype reached levels close to 100% release during the time course of the study. This slower release rate might be related to the higher β-sheet content of the blends with DSS, since this parameter is known to influence the release profiles from SF matrices [63].
catalyse the formation of β-sheets in SF, leading to water-annealed films with crystalline contents similar to methanol processed silk. The different structural characteristics of the films integrating increasing amounts of DSS lead to different protein release kinetics. For example, devices with low burst and diffusion-controlled release kinetics extended for over 25 days have been prepared based on these compositions. In summary SF/DSS represent a highly flexible system for sustained protein delivery that can be prepared by a completely green, water-only process, indicating a technology of large interest for drug delivery applications.
3.3.1. Modelling of HRP release The empirical accumulative release profiles SF/DSS blend films (Fig. 7) were fitted to zero order, first order, Higuchi, and KorsmeyerPeppas models (Table S1). By comparing the R2 for the different models, it was observed that SF/DSS(100:0) and SF/DSS(89:11) seem to follow a Fickian diffusion, as the regression for Higuchi model were nearest to the unit and higher than R2 values for Zero-order and Firstorder kinetic models. This mechanism was confirmed with the Korsmeyer-Peppas model, as the diffusion exponent n was close to 0.5 [66]. Similar results have been obtained for the release of DSS [44] and rhodamine B [46] from SF films. In the case of SF/DSS(80:20) and SF/ DSS(67:33) films, the n value was lower than 0.5, which suggest that they follow a quasi-Fickian diffusion after the initial burst release. This mechanism has been described in diffusion processes where the preferential passage of the solvent occurs by interconnected pores [67] such as those observed in the microstructure of SF/DSS(80:20) and SF/ DSS(67:33) films (Fig. 5) and in SF blends with high swelling ratio [68]. These results show that it is possible to modulate protein release kinetics in SF film matrices by changing their DSS content. SF and DSS have been described as materials with the capacity to protect loaded proteins from degradation. Concretely, SF offers an inert microcrystalline matrix were proteins can be dispersed and incorporated; DSS can protect the conformation structure of many proteins by forming ionic complexes with them. SF/DSS films should present both protein stabilization mechanisms. Altogether, SF/DSS films can be prepared through a water-only process compatible with simple and efficient protein loading and show control release and protein protection characteristics. This combination of features is ideal for formulations used in hormone supplementation and tissue engineering therapies.
Data availability The raw/processed data required to reproduce these findings can be obtained on request by contacting the corresponding author. Acknowledgments Funding: This work was financially supported by Ministerio de Economía y Competitividad (MAT2017-84361-R, FEDER Funds, granted to M.G.-F.) and Xunta de Galicia (Grupos de Referencia Competitiva, FEDER Funds). Author Contributions: J.M.A. and A.P. performed all the experiments. J.M.A. designed the experimental process and analysed the data. N.C. and M.G.-F. organized the experiments, provided a research strategy and discussed the results. All authors wrote the paper. All authors have given approval to the definitive version of the manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.02.028. References [1] Y. Qi, H. Wang, K. Wei, Y. Yang, R.Y. Zheng, I.S. Kim, K.Q. Zhang, A review of structure construction of silk fibroin biomaterials from single structures to multilevel structures, Int. J. Mol. Sci. 18 (2017), https://doi.org/10.3390/ijms18030237. [2] K. Yazawa, K. Ishida, H. Masunaga, T. Hikima, K. Numata, Influence of water content on the β-sheet formation, thermal stability, water removal, and mechanical properties of silk materials, Biomacromolecules 17 (2016) 1057–1066, https://doi. org/10.1021/acs.biomac.5b01685. [3] J.M. Ageitos, K. Yazawa, A. Tateishi, K. Tsuchiya, K. Numata, The benzyl ester group of amino acid monomers enhances substrate affinity and broadens the substrate specificity of the enzyme catalyst in chemoenzymatic copolymerization, Biomacromolecules 17 (2016) 314–323, https://doi.org/10.1021/acs.biomac. 5b01430. [4] H. Yamada, H. Nakao, Y. Takasu, K. Tsubouchi, Preparation of undegraded native molecular fibroin solution from silkworm cocoons, Mater. Sci. Eng. C 14 (2001) 41–46, https://doi.org/10.1016/S0928-4931(01)00207-7. [5] D.N. Rockwood, R.C. Preda, T. Yücel, X. Wang, M.L. Lovett, D.L. Kaplan, Materials fabrication from Bombyx mori silk fibroin, Nat. Protoc. 6 (2011) 1612–1631, https://doi.org/10.1038/nprot.2011.379. [6] R.C. Preda, G. Leisk, F. Omenetto, D.L. Kaplan, Bioengineered silk proteins to control cell and tissue functions, in: J.A. Gerrard (Ed.), Protein Nanotechnol. Protoc. Instrumentation, Appl. Methods Mol. Biol., New York, 2013, pp. 19–41, doi: 10.
4. Conclusions SF/DSS blends represent an interesting biomaterial composition with added value as compared to pure SF, since DSS can interact specifically with certain protein and stabilize them. Herein, we provide a detailed study characterizing the effect of DSS on SF. Our results show that DSS is miscible with SF at low ratios, but phase separation between the polymers is observed with high DSS content. The resulting DSS microdomains act as porogens upon exposure to aqueous media and 203
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Study of nanostructured fibroin/dextran matrixes for controlled protein release Jose Manuel Ageitos, Amelia Pulgar, Noemi Csaba, Marcos Garcia-Fuentes
T
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Centre for Research in Molecular Medicine and Chronic Diseases (CiMUS), Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Universidade de Santiago de Compostela, Avda. Barcelona s/n, 15782 Santiago de Compostela, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Silk fibroin Dextran sulphate Protein release Nanostructure Porogen Water-annealing
Microporous films are structures with attractive features as high porosity and high intrinsic surface area. Unfortunately, most biocompatible materials useful for the preparation of such microporous films are suboptimal for drug delivery due to poor processability and/or drug encapsulation properties. Silk fibroin (SF) is a biocompatible protein with good drug release properties and suitable characteristics for pharmaceutical processing. Dextran sulphate (DSS) is an anionic polysaccharide with the capacity to interact and stabilize many proteins. In the current study, we evaluate the effect of DSS on the physical properties of SF/DSS blend films, how it affects the molecular and microscopic structure of the system and its capacity for providing controlled release of a model protein. Using this system, micro- and nanostructured films could be prepared through a green, water-only process. It was found that DSS acted both as a modifier of SF secondary structure and as a functional porogen, where the size of nano- and micropores varied with the blending ratios. High ratios showed higher swelling, porosity and crystallinity, resulting in modified protein release kinetics, as compared to pure SF films. Considering both their mild preparation method and their physical and pharmaceutical properties, SF/DSS films stand out as ideal systems for sustained protein delivery applications.
1. Introduction Silks are proteinaceous polymers produced by different arthropods, such as silkworms, spiders, scorpions, mites, or bees. Among them, the silk from silkworms (Bombyx mori) is the most studied, and it is widely used for multiple applications, due to its ease of manipulation and availability from the sericulture industry [1]. Silk fibroin (SF) is a high molecular weight fibrous protein (∼390 kDa) and the main component of silkworm silk, conjointly with sericin, a hydrophilic protein that envelops and glues fibroin fibres [2]. SF is composed by repetitive sequences rich in glycine, alanine and serine that allows the formation of β-sheet structures [3] responsible of its physicochemical properties such as insolubility, high tensile strength, and toughness. SF can be extracted from silkworm cocoons and converted into a soluble form by several methods [4–8], including solubilization in concentrated salt [LiBr, LiCNS, CaCl2-ethanol, Ca(CNS)2, ZnCl2, NH4CNS, CuSO4 + NH4OH, Ca(NO3)2] or concentrated acid (phosphoric, formic, sulfuric, hydrochloric) solutions [8]. Among them, solubilization with 9.3 M LiBr is the prevailing method [5] due to the enhanced stability of SF solutions during processing [7]. Then, soluble SF can be casted in different forms such as films, hydrogels, sponges, fibres, microparticles,
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and others [9]. These devices are biocompatible, biodegradable and have the capacity to load and release biomolecules. SF films have been employed for wound dressing, tissue engineering, and controlled drug delivery due to their permeability to oxygen and vapor, biodegradability, high mechanical performance and amenability to easy processing [10]. In addition, in order to modulate its properties, SF has been blended with other polymers such as sericin [11], elastin [10], collagen, chitosan [12], hyaluronic acid [13,14], alginate [15], pectin [16], telechelic-type polyalanine [17] and others [10,18]. In general, these blends are employed to improve the mechanical properties of SF (e.g. elasticity, toughness), while others improve other properties such as water uptake, swelling, cell adhesion and proliferation. Another strategy resides in combining SF with sodium chloride, sugar, paraffin and poly(ethylene oxide), which can be employed as porogens due to their immiscibility with either organic or aqueous SF solutions [9,19]. These compounds produce discrete domains inside the casted SF matrix that can be leached with an appropriate solvent, leading to porous structures that can be used for modified drug release and tissue engineering [20]. Unfortunately, many of these compounds need to be removed with toxic, polluting organic solvents (e.g. hexafluoro isopropanol (HFIP), methanol or hexane), which can limit their
Corresponding author. E-mail address: [email protected] (M. Garcia-Fuentes).
https://doi.org/10.1016/j.eurpolymj.2019.02.028 Received 30 September 2018; Received in revised form 15 February 2019; Accepted 19 February 2019 Available online 20 February 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
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overnight in a fume hood. After drying, films with an average thickness of 23 µm were obtained. SF/DSS films were water-annealed in a sealed chamber saturated with water-vapor (∼99% of relative humidity) for 12 h at 20 °C to induce β-sheet formation [36,37].
biocompatibility [21]. Despite these inconveniences, microporous materials have interesting properties such as high surface area, loading capacity and gas permeability [22,23], as well as improved degradability due to their increased contact area with the media [23]. Dextran sulphate (DSS) is a complex, branched anionic polymer of anhydroglucose employed as anticoagulant, antilipaemic, and antiviral agent, which has the ability to form complexes with cationic polymers and to increase the loading efficiency of certain drugs, such as doxorubicin or mitomycin [24–26]. DSS and SF have been described as enzyme stabilization agents [27–31], therefore, their combination could be interesting for the encapsulation of labile drugs such as proteins. Indeed, SF fibres have already been chemically modified with dextran and showed attractive characteristics for wound dressing [32]. In the current report, we prepare physically blended SF/DSS films and we characterize in detail the effect of both compounds on their physical and micro-structural properties, central characteristics for the controlled release of grown factors [33], anesthetic drugs [34], and antibacterial compounds [35]. Finally, we have studied the effect of DSS on the controlled release of a model protein, horseradish peroxidase II (HRP), loaded SF/DSS blend films.
2.3. Characterization of SF/DSS blend films 2.3.1. Fourier transform infrared spectroscopy (FTIR) FTIR absorption spectra from 400 to 4000 cm−1 wavenumber range were collected using a VARIAN FT-IR 670 (Varian Inc. Scientific Instr., Palo Alto, CA) equipped with a diamond attenuated total reflection (ATR) attachment (Pike, GladiATR, Pike Technology, Madison, WI) at 4 cm−1 resolution, using 64 scans. FTIR spectra were processed using OPUS Spectroscopy Software 5.0 (Bruker, Bremen, Germany) and were normalized to the maximum intensity of the amide I region. Peak position and bandwidth of the component bands of amide I region were obtained using a Fourier deconvolution method (a Lorentzian distribution was assumed) [38] and second-derivative spectra with OriginPro 2017 (Origin lab corporation, Northampton, MA). The average composition (%) of SF secondary structure was obtained by integrating the area of each deconvoluted curve and then normalizing to the total area of the amide I region of at least three different spectra [38].
2. Experimental 2.1. Materials
2.3.2. Scanning electron microscopy The surface and section morphology of the films were studied by field emission scanning electron microscopy (FESEM, UltraPLus, Carl Zeiss, Germany). Lyophilized and air-dried films were coated with Iridium previously to observation. Thickness of the films was directly measured from the observed sections.
Bombyx mori cocoons were kindly donated by Dr. Jose Luis Cenís (IMIDA, Murcia, Spain). Sodium carbonate, lithium bromide, polypropylene glycol (PEG MW: 10,000), dextran sulphate sodium salt from Leuconostoc spp. (DSS, average molecular weight: 15,420 Da, sulphur content: 17.9%), dimethyl sulfoxide (DMSO), polyethylene (20) glycol sorbitan monolaurate (Tween 20) and horseradish peroxidase II (HRP) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium phosphate saline buffer (PBS; 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4) was purchased from Fisher Scientific Co. (Fair Lawn, NJ). All the chemicals were used without any further purification.
2.4. Study of the physicochemical properties of the SF/DSS films and degradation test Swelling ratio and water absorption measurements: To evaluate SF/ DSS films swelling, individual samples (4 cm2, ≈32 mg) were weighed (WI), immersed in 5 ml of 10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween-20, pH 7.5 (PBS-T) and incubated at 37 °C for different time points (5–240 min) under agitation. After each time point, nonabsorbed liquid was removed with filter paper and the swollen weight (WS) was measured. Swollen samples were dried at room temperature for 3 days in a fume hood and weighed (WF). The samples were measured by triplicate per group. Weight loss in dissolution: Mass loss tests were made by immersing different SF/DSS films samples (3.5 cm2, ≈26 mg) in 5 ml of PBS-T and incubated at 37 °C for different time points (1–20 days) under agitation. Samples were dried, and the weight variation was determined gravimetrically (WI, WF). The samples were measured by triplicate per group. Swelling ratio (SR), % of water uptake (WU) and % of weight loss (WL) were calculated according the following equations: SR (w/w): [(WS − WF)/WF]; WU (%): [(WS − WF)/WS] × 100;[39] WL (%): 100 − [(WI − WF)/WI] × 100.
2.2. Methods 2.2.1. Silk fibroin purification and concentration Silk degumming and fibroin purification were conducted as previously described in the literature [5,6,13]. B. mori cocoons were cut in 1 cm2 pieces, rinsed with ultrapure water, and boiled in 2 l of 0.02 M sodium carbonate for 30 min. Then, degummed silk was incubated in 1 l of ultrapure water for 20 min with gentle agitation. Excess of water was removed, and the rinsing process was repeated twice. Degummed silk was rinsed again in ultrapure water, spread out on a clean piece of aluminium foil and dried in a fume hood during 24 h. Degummed silk was cut in small pieces and 4 volumes (v/w) of 9.3 M lithium bromide were added to this sample. Degummed silk was incubated at 60 °C in a water batch during 5 h, until the complete melting of fibres was observed. 20% (w/v) SF solution was dialyzed against 1 l Milli-Q (MQ) water in a Slide-A-Lyzer cassette 3500 for 48 h. Dialyzed SF was centrifuged at 7000g, 4 °C, for 40 min, and the resulting transparent solution that was stored at 4 °C. Concentration of SF was conducted by dialysis against a 10% solution of PEG. SF concentration was determined gravimetrically. Final SF concentration (7.4%) was adjusted by adding MQ water.
2.5. Characterization of model protein release in SF/DSS blend films 2.5.1. Model protein loading and release HPR was labelled with fluorescein isothiocyanate (FITC) following the protocol described by Lu et al. [40]. Briefly, FITC-V (EMP-Biotech GmbH, Berlin, Germany) was dissolved in DMSO (10 mg/ml) and gradually added to a solution of HRP (10 mg/ml) in sodium carbonate solution (pH 8.2, 100 mM). HRP and FTIR solution was incubated overnight at 4 °C, free FITC was removed by diafiltration (7000g, 4 °C, 15 min) with PBS on an Amicon ultra-4 centrifugal filter ultracel 10 K (Merck KGaA, Darmstadt, Germany). FITC-HRP was frozen at −20 °C until use. Protein/fluorescence ratio was determined using a NanoDrop 2000/ 2000c UV–Vis (Thermo Scientific) and a fluorimeter (EnVision 2104
2.2.2. Preparation of SF/DSS blend films DSS was weighed according to the amounts required for SF:DSS ratios (w:w) of 100:0, 89:11, 80:20 and 67:33 and dissolved in the previously described 7.4% aqueous SF solution. Blends were stirred at room temperature during 1 h for complete dissolution of DSS. The mix was then aliquoted with a SF density of 8 mg/cm2 in 3.5 cm polystyrene plates or 6 cm passivated glass plates. SF solutions were left to dry 198
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and smooth appearance. SF/DSS(80:20) and SF/DSS(67:33) films present cavities that are larger in the latter case. The presence of unevenness and cavities suggested that discrete DSS domains can be found in the blend at the highest ratios assayed. Films were water-annealed for the induction of β-sheet formation in SF. After water annealing, the films became wrinkled, especially at low DSS content, and lost transparency. These changes suggest an increase in β-sheet content in the films [42] since similar changes have been described for SF immersed in methanol, the traditional annealing method [5]. Micrographs of waterannealed films (Fig. 1) showed that the cavities in high DSS ratio films became deeper after this treatment, resembling pores. However, the cross-sections of the films did not show such pores but distinguishable microdomains in the films with high DSS ratio [SF/DSS(80:20): 0.6 ± 0.2 µm and SF/DSS(67:33): 2.3 ± 0.5 µm] (Fig. 1). This suggests that in SF/DSS(80:20) and SF/DSS(67:33), the DSS content surpassed the critical ratio of the polymer mixture above which the blend exhibits heterogeneous properties [43]. The formation of dextran domains in SF films has been previously described in methanol treated SF/ dextran(80:20) films by Hines and Kaplan [44]. They found that the size of this domains encapsulated within the silk depended on the molecular weight of the dextran and the film treatment; in this sense, the current results showed that the size of such domains can also be modulated by the loading ratio of DSS.
Multilabel Reader, Perkin Elmer, Waltham, MA) (λEx: 490 nm; λEm. 525 nm) [41]. FITC-HRP was mixed with an appropriate concentration of DSS according to the protein loading (0.33, 0.99 and 2% of SF content) and the SF/DSS ratio of the film (100:0, 89:11, 80:20 and 67:33) and mixed for 30 min. SF solution (7.4%) was added to the different DSS/FITC-HRP solutions and incubated for 1 h under agitation. SF/DSS/FITC-HRP solutions were casted onto 96-well plate and film blends were water-annealed as described above. For release studies, 200 µl of PBS-T was added to each well and the plate was incubated at 37 °C with rotatory agitation. The supernatants of the wells were removed and replaced by fresh PBS-T at different time points (1 h, 1, 3, 5, 10, 15, 20, 25 days). Released FITC-HRP in the supernatant was measured at 525 nm, and the release (%) was determined with respect to the initial loading of FITC-HRP in the films. 2.5.2. Modelling of release profile The mechanism of HRP release from the SF blend films was investigated by fitting several release kinetic models to the in vitro fractional release vs. time data. The formulas and basic principles of the model are available in method section of supplementary material. 3. Results and discussion 3.1. Characterization of SF/DSS films
3.1.1. Structural study of SF/DSS films The effect of DSS in the blends was also studied by FTIR (Fig. 2). FTIR spectra of the SF/DSS films were similar, except for the DSS signals (3469, 1219, 980, 801 and 577 cm−1), which increased in intensity with DSS content in the film (Fig. 2). Before water-annealing, all films showed a maximum peak in 1639 cm−1 in the amide I region which is attributable to SF random coil structure [45]. The percentage of random coil of SF/DSS 100:0 films were similar to the ones reported by Wei et al. [46] for pure SF films, although higher than the ones reported
The effect of DSS content in SF/DSS blend films was studied by evaluating morphological, structural, and physicochemical changes in the films. From a macroscopic point of view, all the assayed bends yield films with uniform flat shape; however, as the concentration of DSS in the blend increased, the films became less transparent and acquired a whitish colour. Upon microscopic observation of the surface, the morphology of the films changed with increasing DSS in the blend (Fig. 1). The surface of SF/DSS(100:0) and SF/DSS(89:11) films has a uniform
Fig. 1. Scanning electron micrographs of surface and cross-sections of SF/DSS blend films before and after water-annealing process. 199
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between SF and the polysaccharide [48]. SF is an amphoteric protein with negative and positive domains, however, the C-terminal region is markedly positive, with a ratio 9:1 positive/negative charges [49], this could explain the interaction with the anionic polymer DSS. In the same way, a reduction in the intensity of SF signal at 1515 cm−1 was also observed with increased DSS content, which suggested an increase in the conformational freedom of SF [50]. This band at 1515 cm−1 has been assigned to the CH bending mode and the ring CC stretching of the side chain of tyrosine [50], and it is characteristic for silk II conformation [51]. After water annealing, a shift in amide I region from 1639 to 1620 cm−1 was observed, together with a reduction of the shoulder at 1533 cm−1. These signals (1639, 1533 cm−1) are characteristic of random coil structure in SF [45], while 1620 cm−1 is associated with aggregated β-sheet structure [37]. The presence of the shoulder at 1650 cm−1 is indicative of silk I conformation [52], and this band is usually assigned as random coil [42,45,53], suggesting the coexistence of random coil and β-sheet structures. Although the overall β-sheet content increased after water-annealing for all prototypes, the differences observed among groups were maintained around similar values to those observed before water-annealing (Table 1). 3.2. Study of SF/DSS films in solution Films before water-annealing dissolved immediately when immersed in water. After water-annealing, all blend films kept their shape, absorbed water, and became flexible. Films quickly lost weight during the initial 30 min in solution (Fig. 3), and then, this process progressively slowed down until reaching a plateau after three days. The percentage of mass loss was proportional to the DSS content in the film. It has been described that dextran is released from SF films proportionally to its molecular weight [44]. Given that the DSS employed in this study was a mixture of different molecular weight polymers (9–20 kDa), the initial burst loss can be attributed to the low molecular weight DSS. After the initial weight loss phase, the films mass decreased linearly with time, being possible to observe differences between the SF/DSS ratios employed. In this second phase, contrary to the previous one, the percentage of mass loss per day (ΔW) decreased with increasing DSS content: SF/DSS(100:0)ΔW = −0.38%/d (R2: 0.995); SF/DSS (88:11)ΔW = −0.26%/d (R2: 0.958); SF/DSS(80:20)ΔW = −0.20%/d (R2: 0.922); SF/DSS(66:33)ΔW = −0.17%/d (R2: 0.839). The slower mass loss in this second phase can be related to the increase in β-sheet content observed for high DSS content films (Table 1).
Fig. 2. FTIR spectra of air-dried SF/DSS blend films. A. Detailed view amide I and II bands of SF/DSS blend films before and after water-annealing process (W.A.). B. FTIR spectra of SF/DSS blend films after water-annealing and dextran sodium sulphate (DSS), depicting the characteristic bands of DSS.
3.2.1. Study of SF/DSS films after their immersion in solution: molecular structure The molecular structure of the blend films after immersion in PBS-T
Table 1 Structural comparison of SF/DSS blend films before and after water-annealing treatment (WA). Sample
Treatment
β sheet (%)
Random coil (%)
SF/DSS(100:0)
– WA
34.2 ± 4.2 48.3 ± 4.0
34.3 ± 1.8 28.8 ± 5.6
SF/DSS(89:11)
– WA
38.4 ± 4.5 49.9 ± 6.6
31.1 ± 2.8 27.7 ± 3.5
SF/DSS(80:20)
– WA
38.7 ± 4.1 51.3 ± 6.5
27.2 ± 1.7 26.3 ± 6.3
SF/DSS(67:33)
– WA
41.0 ± 0.7 55.0 ± 6.4
29.1 ± 2.3 22.7 ± 5.4
by Hu et al. [37]. Amide I deconvolution showed that as the DSS content increased also did the β-sheet content of the films, while the random coil content decreased (Table 1). Similar results have been reported for SF blended with chitosan, alginate, hyaluronic acid, and cellulose [13,14,47]. Chen et al. proposed that the polymer-induced conformation transition is produced by the strong hydrogen bonding
Fig. 3. Time course of the weight loss of SF/DSS blend films in solution. The rectangle insert is a zoom of the initial points of the study. 200
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Fig. 4. FTIR spectra of freeze-dried SF/DSS blend films after incubation in PBS-T buffer for different days (D). (A) General view depicting characteristic bands of Dextran. (B) Magnification of amide I region of SF/DSS blend films.
buffer was studied by FTIR (Fig. 4). After 1 day it was observed that the characteristic signals of DSS (1219, 980, 801 and 577 cm−1) disappear and FTIR spectra of all blend films become similar (Fig. 4A). However, some differences can still be detected in the 3600–3400 cm−1 region that correspond to dextran ν(OH) vibrations [54]. Here, we could observe that the DSS shoulder at ∼3469 cm−1 in SF/DSS(67:33) films decreased from day 0 (D0) to day 1 and decreased further again at day 5, suggesting that in this blend the DSS release follows sustained kinetics. Regarding the amide I region (Fig. 4B), a decrease in the shoulder at 1646 cm−1 in the amide I region could be observed for all blends. This band is assigned to random coil SF [55], and its reduction until day 5 indicates that the initial weight loss phase is not only attributable to DSS release, but also solubilisation of the amorphous domains of silk I, as α-helix content of silk I requires more time for this process [45]. After 20 days, the band at 1620 cm−1 disappeared from SF/DSS(88:11) and SF/DSS(80:20), with the peak top of amide I shifting to 1626 and 1632 cm−1, respectively. This suggest that the proportion of intermolecular β-sheet decreased and intramolecular β-sheet became predominant [37,56,57]. The β-sheet content of SF/DSS(88:11) and SF/ DSS(80:20) decreased after 20 days in solution (34% and 24%, respectively), while SF/DSS(100:0) and SF/DSS(67:33) became more crystalline by the solubilization of random coil structures (57% and 58%, respectively). As matter of fact, both SF/DSS(100:0) and SF/DSS (67:33) films showed a similar spectra during the all the study except for a relative increase of the band at 1515 cm−1 at days 5 and 20 for the latter composition. This indicates that SF/DSS(67:33) was losing conformational freedom, becoming more crystalline [50].
3.2.2. Study of SF/DSS films after their immersion in solution: macromolecular structure The effect of incubation in liquid on the microscopic structure of the films was further evaluated by SEM (Fig. 5). The thickness of DSS containing films increased after 1 day in liquid, especially in SF/DSS (80:20) and SF/DSS(67:33) films, nearly triplicating its original value
Fig. 5. Scanning electron micrograph of freeze-dried SF/DSS blend film sections after 1 and 20 days immersed in PBS-T buffer media.
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blends were determined in vitro (Fig. 6). Water-annealed films showed lower SR and WU values than those reported for methanol treated SF films [13,59]. However, the WU of SF/DSS(100:0) was higher than the one reported by Karve et al. for water-annealed SF films [60]. SF/DSS films absorbed liquid quickly upon immersion since the maximum SR and WU were observed at 5 min with the exception of SF/DSS(67:33). In this latter case SR and WU values increased up to 30 min and were higher as compared to the other films (Fig. 6B). The increase in the average SR of SF/DSS blends was proportional to the DSS ratio (7, 33 and 66%, respectively) and similar to the values reported for SF chemically linked to DSS (46%) [32]. However, all SF films showed similar behaviour after achieving the maximum values, when SR and WU gradually decreased and stabilized after 105 min. Swelling behaviour seems to be related with the mass loss process (Fig. 3): as DSS is released from the film, the pores formed (Fig. 5) increase the relative surface area of the SF/DSS films, allowing higher WU and SR, and hence increasing the thickness of blend films. As observed by SEM imaging (Fig. 5), SF/DSS(67:33) films were able to keep their swollen state even after 20 days in aqueous media, while the other blends collapsed after long incubation times. These results are consistent with the efficient preservation of β-sheet signals in the FTIR spectra of SF/DSS (67/33) as compared to other compositions (Fig. 4B).
(Figs. 1 and 5). As previously observed in the dry state (Fig. 1), SF/DSS (100:0) and SF/DSS(89:11) were similar, while SF/DSS(80:20) and SF/ DSS(67:33) presented different morphology (Fig. 5). SF/DSS(80:20) films presented pores with an average size of 0.5 ± 0.2 µm, similar to the encapsulated domains observed in dry state (0.6 ± 0.1 µm). Interestingly, the increase in thickness after incubation in water did not induce increase in the size of pores. This can be explained by the preferential retention of the solvent in the amorphous regions of SF [8]. As DSS increases the crystallinity of SF, it is expected that the inner surface of the pores is more crystalline than other areas. This would explain the low volume variability of the pores as compared with the rest of the film in those preparations where amorphous SF is more prevalent. After 1 day in PBS-T buffer solution, SF/DSS(67:33) films presented an inner structure resembling a sponge. Mainly, two population of pores with diameters 1.6 ± 0.5 µm and 4.2 ± 0.7 µm were found (Fig. 5), being more numerous than the DSS encapsulated domains (2.3 ± 0.5 µm) observed in dry state (Fig. 1). These results indicate that DSS acts as a porogen for SF films, and the pore size is dependent on DSS concentration. Similar behaviour has been previously described for SF/PEG blends [19,58], where micro-phase separation induced the formation of pores upon extraction of PEG when incubated in water. Nevertheless, DSS showed higher miscibility with SF than PEG as 10% PEG was found sufficient for induction of the phase separation, while the formation of bundles of SF globules was not observed [19] even at SF/DSS(50:50) (data not shown).
3.3. Study of protein association and release For these studies, we encapsulated horseradish peroxidase II conjugated to fluorescein (FITC-HRP) in the different SF/DSS films. This model protein was selected because it has a molecular weight and isoelectric point similar to many proteins of therapeutic interest (such as the bone morphogenetic protein or fibroblast growth factor) [61,62], thus providing a relevant model for drug delivery characterization studies. In order to evaluate the cargo effect in the release profile three different loadings of FITC-HRP were employed in the blend films: 0.33, 0.99 and 2.00% w/w in relation to the SF content. A first observation of this study was that irrespective of the film composition, the percentage of protein released (%) at the end of the study was inversely proportional to the loading (Fig. 7). Therefore, in all blends 0.33% FITC-HRP showed the highest percentage of released protein. SF/DSS(100:0) films achieved nearly complete protein release after 25 days when loaded with 0.33% HRP (Fig. 7A), showing a 20% of burst release followed by a continuous release phase for up to 25 days. This release profile for SF/DSS(100:0) was different to the previously reported by Hofmann et al. [63] for water-annealed SF films loaded with HRP. These authors have observed a two-phase release, with an initial burst followed by a lag phase of 2 days, and a plateau by day 10. However, the total accumulative release (∼55%) was similar to the one reported in the current study for the 2% loaded formulation. The differences can be explained based on the different β-sheet content between the two films, since the SF/DSS(100:0) films investigated in this work have similar β-sheet content (Table 1) to methanol-treated SF films reported by these authors. Indeed, in the study by Hofmann et al., the methanol treated SF films presented a longer lag-phase (5 days) followed by a linear release with a slope similar to the results from the present study. In summary, our pure SF shows an initial release typical of water-annealed films, but a follow-up sustained release more similar to previous data on methanol-treated films [64]. This difference could be mediated by the differences in the protocols for water-annealing [36] that might result in higher β-sheet content in our samples [65]. The effect of the addition of DSS in the SF produced prototypes with clearly different kinetics. At low DSS content, i.e. SF/DSS(89:11), the protein release kinetics was modified to lower burst effects and more sustained release rates than the pure SF films (Fig. 7B). These changes suggest that inclusion of DSS might improve the compatibility of HRP with the film matrix and avoid accumulation of loosely bound protein on the material surface, which is assumed to be responsible for burst release [44]. With high levels of DSS, SF/DSS(80:20) and (67:33), the
3.2.3. Swelling properties of SF/DSS films The swelling ratio (SR) and the water uptake (WU) of the SF/DSS
Fig. 6. Time course of the swelling ratio (A) and water uptake (B) of SF/DSS blend films in solution. 202
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Fig. 7. Cumulative release profile of FTIC-HRP loaded SF/DSS films (● 0.33; ▲0.99; ■ 2.00). A. SF/DSS(100:0). B. SF/DSS(89:11). C. SF/DSS(80:20). D. SF/DSS (67:30).
burst increased to values higher than those observed with the pure SF system (Fig. 7C and 7D). This high burst might be caused by protein being released together with DSS in the first minutes by the newly formed pores described previously. However, once this burst phase is over, these systems provided a slower protein release rate than the pure SF films, and as a matter of fact, no prototype reached levels close to 100% release during the time course of the study. This slower release rate might be related to the higher β-sheet content of the blends with DSS, since this parameter is known to influence the release profiles from SF matrices [63].
catalyse the formation of β-sheets in SF, leading to water-annealed films with crystalline contents similar to methanol processed silk. The different structural characteristics of the films integrating increasing amounts of DSS lead to different protein release kinetics. For example, devices with low burst and diffusion-controlled release kinetics extended for over 25 days have been prepared based on these compositions. In summary SF/DSS represent a highly flexible system for sustained protein delivery that can be prepared by a completely green, water-only process, indicating a technology of large interest for drug delivery applications.
3.3.1. Modelling of HRP release The empirical accumulative release profiles SF/DSS blend films (Fig. 7) were fitted to zero order, first order, Higuchi, and KorsmeyerPeppas models (Table S1). By comparing the R2 for the different models, it was observed that SF/DSS(100:0) and SF/DSS(89:11) seem to follow a Fickian diffusion, as the regression for Higuchi model were nearest to the unit and higher than R2 values for Zero-order and Firstorder kinetic models. This mechanism was confirmed with the Korsmeyer-Peppas model, as the diffusion exponent n was close to 0.5 [66]. Similar results have been obtained for the release of DSS [44] and rhodamine B [46] from SF films. In the case of SF/DSS(80:20) and SF/ DSS(67:33) films, the n value was lower than 0.5, which suggest that they follow a quasi-Fickian diffusion after the initial burst release. This mechanism has been described in diffusion processes where the preferential passage of the solvent occurs by interconnected pores [67] such as those observed in the microstructure of SF/DSS(80:20) and SF/ DSS(67:33) films (Fig. 5) and in SF blends with high swelling ratio [68]. These results show that it is possible to modulate protein release kinetics in SF film matrices by changing their DSS content. SF and DSS have been described as materials with the capacity to protect loaded proteins from degradation. Concretely, SF offers an inert microcrystalline matrix were proteins can be dispersed and incorporated; DSS can protect the conformation structure of many proteins by forming ionic complexes with them. SF/DSS films should present both protein stabilization mechanisms. Altogether, SF/DSS films can be prepared through a water-only process compatible with simple and efficient protein loading and show control release and protein protection characteristics. This combination of features is ideal for formulations used in hormone supplementation and tissue engineering therapies.
Data availability The raw/processed data required to reproduce these findings can be obtained on request by contacting the corresponding author. Acknowledgments Funding: This work was financially supported by Ministerio de Economía y Competitividad (MAT2017-84361-R, FEDER Funds, granted to M.G.-F.) and Xunta de Galicia (Grupos de Referencia Competitiva, FEDER Funds). Author Contributions: J.M.A. and A.P. performed all the experiments. J.M.A. designed the experimental process and analysed the data. N.C. and M.G.-F. organized the experiments, provided a research strategy and discussed the results. All authors wrote the paper. All authors have given approval to the definitive version of the manuscript. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.02.028. References [1] Y. Qi, H. Wang, K. Wei, Y. Yang, R.Y. Zheng, I.S. Kim, K.Q. Zhang, A review of structure construction of silk fibroin biomaterials from single structures to multilevel structures, Int. J. Mol. Sci. 18 (2017), https://doi.org/10.3390/ijms18030237. [2] K. Yazawa, K. Ishida, H. Masunaga, T. Hikima, K. Numata, Influence of water content on the β-sheet formation, thermal stability, water removal, and mechanical properties of silk materials, Biomacromolecules 17 (2016) 1057–1066, https://doi. org/10.1021/acs.biomac.5b01685. [3] J.M. Ageitos, K. Yazawa, A. Tateishi, K. Tsuchiya, K. Numata, The benzyl ester group of amino acid monomers enhances substrate affinity and broadens the substrate specificity of the enzyme catalyst in chemoenzymatic copolymerization, Biomacromolecules 17 (2016) 314–323, https://doi.org/10.1021/acs.biomac. 5b01430. [4] H. Yamada, H. Nakao, Y. Takasu, K. Tsubouchi, Preparation of undegraded native molecular fibroin solution from silkworm cocoons, Mater. Sci. Eng. C 14 (2001) 41–46, https://doi.org/10.1016/S0928-4931(01)00207-7. [5] D.N. Rockwood, R.C. Preda, T. Yücel, X. Wang, M.L. Lovett, D.L. Kaplan, Materials fabrication from Bombyx mori silk fibroin, Nat. Protoc. 6 (2011) 1612–1631, https://doi.org/10.1038/nprot.2011.379. [6] R.C. Preda, G. Leisk, F. Omenetto, D.L. Kaplan, Bioengineered silk proteins to control cell and tissue functions, in: J.A. Gerrard (Ed.), Protein Nanotechnol. Protoc. Instrumentation, Appl. Methods Mol. Biol., New York, 2013, pp. 19–41, doi: 10.
4. Conclusions SF/DSS blends represent an interesting biomaterial composition with added value as compared to pure SF, since DSS can interact specifically with certain protein and stabilize them. Herein, we provide a detailed study characterizing the effect of DSS on SF. Our results show that DSS is miscible with SF at low ratios, but phase separation between the polymers is observed with high DSS content. The resulting DSS microdomains act as porogens upon exposure to aqueous media and 203
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