Investigation of the solubility and dispersion degree of calf skin collagen in ionic liquids
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The dissolution of collagen in ionic liquids (ILs) was highly dependent on the polarity of ILs, which was influenced by their sorts and concentrations. Herein, the solubility and dispersion degree of collagen in two sorts of ILs, namely 1-ethyl-methylimidazolium tetrafluoroborate ([EMIM][BF4]) with low polarity and 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]) with high polarity in a concentration range from 10% to 70% at 10 °C were investigated. When 150 mg of collagen was added to 30 mg of ILs, the minimum soluble collagen concentration was 0.02 mg/mL in 70% [EMIM][BF4] with lowest polarity and the maximum was 3.57 mg/mL in 70% [EMIM][Ac] with highest polarity, which indicates that soluble collagen and insoluble collagen fibers were both present. For insoluble collagens, differential scanning calorimetry showed that the thermal-stability was weakened when increasing the ILs concentration and polarity, and the fiber arrangement was looser with a more uniform lyophilized structure, observed by atomic force microscopy and scanning electron microscopy. For soluble collagens, electrophoresis patterns and Fourier transform infrared spectroscopy showed that no polypeptide chain degradation occurred during dissolution, but the thermal denaturation temperature decreased by 0.26 °C~ 7.63 °C with the increase of ILs concentrations, measured by ultra-sensitive differential scanning calorimetry. Moreover, the aggregation of collagen molecules was reduced when ILs polarity was increased as determined by fluorescence measurements and dynamic light scattering, which resulted in an increased loose fiber arrangement observed by atomic force microscopy. If the structural integrity of collagen needs to be retained, then the ILs sorts and concentrations should be considered.
KeywordsIonic liquids concentrations Collagen solubility Structural integrity Dispersion degree Aggregation state
Insoluble collagen fibrils from 10% [EMIM][Ac]
Insoluble collagen fibrils from 30% [EMIM][Ac]
Insoluble collagen fibrils from 50% [EMIM][Ac]
Insoluble collagen fibrils from 70% [EMIM][Ac]
Insoluble collagen fibrils from 70% [EMIM][BF4]
Soluble collagen in 10% [EMIM][Ac]
Soluble collagen in 30% [EMIM][Ac]
Soluble collagen in 50% [EMIM][Ac]
Soluble collagen in 70% [EMIM][Ac]
Ionic liquids (ILs), which are known as “supramolecular solvents”, have appeared as novel solvents that permit the compounding of a series of conventional natural biomasses that are not easily soluble. ILs are molten salts that are composed of bulky organic cations and small inorganic anions [1, 2]. As promising alternatives to conventional organic solvents, ILs possess many unmatched favorable properties, such as miscibility with water and organic solvents, high tunability, low or negligible vapor pressure, and thus, they are classified as “green solvents” [3, 4, 5]. Recently, progress has been rapid in the application of ILs as a novel solvent type. Fukaya et al.  found that 1-ethyl-3-methyl imidazolidic phosphonate ionic liquid had an excellent solubility in cellulose, and the dissolution rate of cellulose was 10% at 30 min and 45 °C. The solubility of chitosan with a molecular weight of 9.7 kDa could reach 12% in [C4mim][CH3COO] at 110 °C as reported by Wu et al.  Philips et al.  studied the solubility of silk fibroin in various ILs, and found that [BMIM]Cl had the highest solubility for protein fibers.
Because many reports have verified the possibility of dissolving natural polymer, such as cellulose, chitosan, and biological protein with ILs, researchers started to consider the application of the high polarity ILs in collagen dissolution. Collagen, as the most abundant fibrous protein in connective tissues of land-based mammals (e.g. calf skin) which are common raw materials in leather industry, has been widely applied in food, pharmaceutical, biochemistry and cosmetic products, because of its unusual combination of good biocompatibility, low antigenicity and controlled biodegradability [9, 10, 11, 12]. These great biological properties of collagen are ascribed mostly to its unique triple helix conformation, which is stabilized by hydrogen bonds [13, 14, 15]. However, the molecular weight of ~ 300 kDa and the large numbers of hydrogen bonds, electrovalent bonds, hydrophobic bonds, and van der Waals forces prevent collagen from being dissolved in general solvents, but it is highly desirable that ILs provide a high transformation and ability to utilize collagen.
The applications of ILs in related collagen research have focused mainly on a solvent medium of blend [16, 17, 18], pretreatment solvent of collagen extraction [10, 19], or conditions of collagen dissolution and changes in regenerated-collagen properties [20, 21, 22]. Wang et al.  studied the dissolution performance of white-hide-powder from leather-making in [C6O2(mim)2][Br]2, and found that when the white hide powder solubility reached 8%, the dissolving time was 55 min at 120 °C. The triple helix structure of white-hide-powder was destroyed partially during [C6O2(mim)2][Br]2 dissolution. As Meng et al.  reported, native skin collagen fibers could dissolve in [BMIM]Cl at 100 °C for 6 h, but the triple helix structure of collagen was partially disturbed during the dissolution and regeneration. The collagen materials used in these studies were usually not highly purified collagen. Hu et al.  dissolved collagen in [EMIM][Ac] with a series of sodium salts at different temperatures (25, 30, 35, 40, and 45 °C) to evaluate [EMIM][Ac]/sodium-salt systems as suitable solvents for collagen. The [EMIM][Ac] solvent system broke most of the hydrogen bonds between the amino hydroxyl and ester oxygen of collagen. A higher temperature appeared to promote the intermolecular distance of collagen significantly and allowed ILs to destroy hydrogen bonds more easily. To prepare the collagen solution efficiently, these studies usually dissolved collagen in ILs at a higher temperature, which was easy to damage the triple-helix structure of collagen. Also, since complete dissolution of collagen in ILs was not achieved, the insoluble part of collagen had not been thoroughly studied. Furthermore, it should be noted that both the sorts and concentrations of ILs, which are closely related to their polarity, have great influences on the properties of collagen [10, 19, 23, 24, 25, 26, 27, 28]. For example, the differences in composition of imidazolium-based ILs endowed by varying anions with different polarity namely acetate, hydrogen sulfate, dicyanamide, dimethyl phosphate, dihydrogen phosphate and sulfate, had resulted in different destabilizing effects on the triple helical structure of collagen ; a notable decreasing trend in denaturation temperature of collagen fibers was discovered with the increase of ILs concentration from 0.1% to 10% (w/v) . Therefore, it will be of importance and significance to study how the ILs sorts and concentrations would affect the structure and properties of collagen in the dissolution process. Moreover, the dispersion degree is a core property of collagen, affecting its functional performances as biomaterials, for instance, the viscoelastic behavior originated by the aggregation of collagen molecules . Thus, the dispersion degree of collagen after the dissolving process should also be investigated.
Herein, we controlled the collagen dissolution at a lower temperature with more safety (~ 10 °C) and investigated the solubility and the dispersion degree of collagen fibrils in two types of 1-ethyl-methylimidazolium ([EMIM]) based ILs containing different anions ([EMIM] tetrafluoroborate ([EMIM][BF4]) and [EMIM] acetate ([EMIM][Ac]), which are liquid state at room temperature), the ions of which exhibited good biocompatibility and mild polarity [22, 29]. [EMIM][Ac] was used in a concentration range of 10%–70%, while [EMIM][BF4] was used solely in 70%, owing to its low polarity. The solubility of collagen in ILs was tested, and insoluble as well as soluble collagens were examined separately to determine their structural integrity and dispersion degree. The obtained results will help to understand the effect of ILs on the solubility and dispersion degree of collagen while maintaining its structural integrity and provide guidance for the application of ILs in collagen research.
Native collagen used in this study was extracted from calf skin by 0.5 M acetic acid that contained 3% pepsin according to the method of Zhang et al. with a slight modification . The ionic liquids (ILs), 1-ethyl-methylimidazolium tetrafluoroborate ([EMIM][BF4]) and 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]) were purchased from Lanzhou Institute of Chemical Physics (Chinese Academy of Sciences, Lanzhou, China) with high purification (≥99%). [EMIM][Ac] was diluted in deionized water at concentrations of 30%, 50%, and 70% (w/w) respectively, while [EMIM][BF4] was diluted in deionized water solely at 70% (w/w). 8-Anilino-1-naphthalenesulfonate (ANS) was from Sigma Chemicals (Sigma-Aldrich, Munich, Germany). Other chemicals were from Chengdu Kelong Industrial (Chengdu, China).
2.2 Processing collagen with ILs
Lyophilized collagen (150 mg) was added to a beaker with 30 g of [EMIM][BF4] (70%) or different concentrations of [EMIM][Ac] (10%, 30%, 50%, 70%) to achieve an ideal concentration of 5 mg/mL. All mixtures were stirred ceaselessly by magnetic stirrer for 48 h in water bath at 10 °C, and then centrifuged at 7900 rpm for 10 min by using a high-speed freezer centrifuge. The insoluble collagen fibrils (Col/ILs-fibrils10%, Col/ILs-fibrils30%, Col/ILs-fibrils50%, Col/ILs-fibrils70%, Col/ILs-fibrils70%B) were washed with distilled water several times, dissolved in 0.5 M acetic acid, and dialyzed against 0.1 M acetic acid for 3 days to remove ILs. The supernatant, which is the soluble collagen (Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50%, Col/ILs-soluble70%) was reserved for the following measurements. The obtained specimen was lyophilized by freeze drying and preserved in a desiccator before used.
2.3 Collagen solubility in ILs
2.4 DSC of insoluble collagen fibrils
The thermal stability of the lyophilized Col/ILs-fibrils70%B, Col/ILs-fibrils10%, Col/ILs-fibrils30%, Col/ILs-fibrils50% and Col/ILs-fibrils70% was detected by using DSC (Netzsch DSC 200PC, Bayern, Germany). The freeze-dried Col and collagen fibrils (~ 3 mg) were weighed into aluminum pans and sealed, then scanned in a nitrogen atmosphere from 35 to 90 °C at 2 °C/min. The measurements were performed in triplicate.
2.5 SEM of insoluble collagen fibrils
The morphologies of the lyophilized Col/ILs-fibrils70%B, Col/ILs-fibrils10%, Col/ILs-fibrils30%, Col/ILs-fibrils50% and Col/ILs-fibrils70% were observed by SEM (S-800, HITACHI, Japan) at 20 kV. Images of each sample were obtained at different spots to confirm the consistency of the morphology observed.
2.6 AFM measurements of insoluble and soluble collagen
The morphologies of the insoluble collagen fibrils (Col/ILs-fibrils70%B, Col/ILs-fibrils10%, Col/ILs-fibrils30%, Col/ILs-fibrils50% and Col/ILs-fibrils70%) and the soluble collagen (Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50% and Col/ILs-soluble70%) were observed by AFM with a pinpoint. The insoluble collagen fibrils were dispersed in 0.5 M acetic acid. The soluble collagen was dispersed in corresponding concentrations of [EMIM][Ac]. The corresponding concentration of collagen was used as a reference. 15 μL of each diluted samples was dropped on mica and then dried at 30 °C for 3 days. Particular attention was given to the fact that 1 day after drying, it was necessary to rinse the mica sheets of Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50%, and Col/ILs-soluble70% slowly with distilled water to remove ILs.
2.7 SDS–PAGE patterns of soluble collagen
SDS-PAGE was performed according to the method of Laemmli  with slight modifications. The lyophilized collagen was dissolved in 0.1 M acetic acid (Col). Col, Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50%, and Col/ILs-soluble70% were mixed with buffer solution and then boiled in water for 5 min. Then, each sample was loaded for each lane and electrophoresed by Mini-PROTEAN 3 Cell (Bio-Rad, California, USA). After electrophoresis, the gel was dyed by 0.25% coomassie brilliant blue R-250 and then destained with 7.5% acetic acid and 5% methanol.
2.8 FTIR of soluble collagen
All collagen samples were homogeneously triturated with 300 mg potassium bromide (KBr) and the mixtures were made into disks under a pressure of 20 MPa. The FTIR spectra were recorded by a Nicolet iS10 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at the wavenumbers ranging from 4000 to 500 cm− 1 with a resolution of 2 cm− 1 at 25 °C.
2.9 US-DSC of soluble collagen
The thermal stability of the soluble collagen Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50%, and Col/ILs-soluble70% was determined by US-DSC (VP-DSC, Microcal, Northampton, USA) and matching concentrations of [EMIM][Ac] and Col were used as references. All specimens were measured at 0.5 mg/mL and performed in triplicate.
2.10 Fluorescence measurements of soluble collagen
Fluorescence measurements of ANS in Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50%, and Col/ILs-soluble70% were measured by a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan), as described by Kamyshny et al.  with a slight modification. The same concentration of soluble collagen (0.5 mg/mL) was prepared to evaluate the aggregation status of collagen molecules. Briefly, the ANS probe was prepared in 0.1 mol/L sodium phosphate buffer (pH 7.0) to obtain a 400 mmol/L transparent solution. Then, 10 mL sample solutions were mixed with 50 μL of ANS solution and the mixtures were homogenized. The fluorescence spectrum was recorded at 20°. The excitation wavelength was fixed at 432 nm and the emission spectra were measured from 450 to 750 nm. The slits were fixed at 5 nm.
2.11 DLS measurements of soluble collagen
A DLS technique that uses a Zetasize (Nano-ZS, Malvern Instruments Ltd., Malvern, UK) was employed for the soluble collagen Col/ILs-soluble10%, Col/ILs-soluble30%, Col/ILs-soluble50%, and Col/ILs-soluble70% to obtain information on the size distribution and average aggregation size. The sample solutions were first diluted to 1.0 mg/mL, then filtered through a 2-μm filter (Millipore, Billerica, USA) and measured in a polystyrene cuvette.
3 Results and discussion
3.1 Solubility of collagen in ILs
The concentration of soluble collagen in different ILs and concentrations
ILs and concentrations
concentration of soluble collagen (mg/mL)
3.2 Thermal analysis of insoluble collagen fibrils
3.3 SEM images of insoluble collagen fibrils
3.4 AFM images of insoluble collagen fibrils
3.5 SDS-PAGE patterns of soluble collagen
3.6 FTIR spectra analysis of soluble collagen
3.7 Thermal analysis of soluble collagen
Thermodynamic parameters of Col and collagen samples dissolved in different concentrations of [EMIM][Ac]a
35.97 ± 0.15
42.73 ± 0.12
35.68 ± 0.09
42.47 ± 0.18
34.09 ± 0.17
42.39 ± 0.10, 39.03 ± 0.14
29.04 ± 0.13
42.31 ± 0.06, 38.55 ± 0.16, 35.36 ± 0.13
27.65 ± 0.11
35.10 ± 0.13
Compared with Col, the thermal denaturation temperature of Col/ILs-soluble70% was reduced by 7.63 °C, which is larger than the decrease of Col-regenerated-35 °C with 4.1 °C reported by Hu et al.  This showed the thermal stability decreased more significantly with [EMIM][Ac] concentration. Hydrogen bonds played an important role in the stabilization of triple-helix structure. The interchain hydrogen bonds were mediated by one water molecule, which likely reinforced the triple-helix at regions that lacked proline . [EMIM][Ac] was deduced to break part of the inter-molecular hydrogen bonds. Therefore, less water oriented hydrogen bonding in collagen molecules in [EMIM][Ac] were formed because of the increased hydrophobic interaction . Therefore, the stability of the collagen helix weakened as the [EMIM][Ac] concentration increased and a continuous decrease in the values of the Tm2 was detected. Together with the results of SDS-PAGE, we can see that although there was no degradation in polypeptide chain during the process of dissolving, the thermal stability of collagen was reduced because of the decreased hydrogen bonds.
3.8 Fluorescence analysis of soluble collagen
3.9 DLS analysis of soluble collagen
3.10 AFM images of soluble collagen
The solubility and dispersion degree of collagen in 70% [EMIM][BF4] and [EMIM][Ac] in concentrations from 10% to 70% were studied. Only insoluble collagen fibers were presented from [EMIM][BF4], due to no dissolution of collagen occurred in [EMIM][BF4]. Though dissolved in [EMIM][Ac], the total dissolution of collagen was not achieved and a phenomenon of partial dissolution resulted with the soluble collagen and insoluble collagen fibers across the entire [EMIM][Ac] concentration range. For the insoluble collagen fibers, although the partial interruption of hydrogen bonds did not make it dissolved, the entire fiber arrangement was looser and the thermal stability decreased with the increase of ILs polarity and concentration. For the soluble collagen, no degradation of polypeptide chain occurred during dissolution. Owing to the fact that more hydrogen bonds were broken with the increase of ILs concentration, the reduction in aggregation states of soluble collagen was more prominent, and resulted in a decreased thermal stability, a looser fiber arrangement, and shorter fiber lengths. Therefore, [EMIM][BF4] is a good dispersion agent for collagen with no destruction to collagen structures, while [EMIM][Ac] is a good solvent and dispersion agent for collagen, depending on the concentration. The information obtained may be useful in the application of ILs in more collagen products with a suitable sort and concentration to retain the structural integrity of the collagen.
QL has equal contribution to the first author. SL designed and performed the experiments, analyzed the data and wrote the manuscript. QL performed the experiments of soluble collagen and was a major contributor in writing the manuscript. GL analyzed the data and improved the manuscript. All authors read and approved the final manuscript.
The research is funded by the National Natural Science Foundation of China (Nos. 21776184 and 21476147).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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