A polyol compound is an important component of some NADES.Polyols have been reported to stabilize thetriple helix of collagen to var-ious extents, depending on the number of carbon atoms present in thepolyol molecule. The stabilizing effect is suggested to be achievedthrough the binding of the polyol to the surface of the collagen moleculefollowed by the formation of additional hydrogen bonds [18,19]. Al-though this should indicate a stabilization of collagen in NADES, theproperties of polyols when part of a eutectic mixture could be differentfrom a polyol solution. In NADES, the components are tightly bound in anetwork of hydrogen bonds, which can affect the way the solutes reactswith the surrounding media.NADES have been proposed as potential excipients in pharmaceuti-cal preparations and drug delivery systems, particularly because of theirsolubilizing properties, varying viscosity and antibacterial properties[1,16]. Collagen has good biocompatibility, and both collagen and colla-gen peptides have as mentioned above, beneficial properties for woundhealing. The combination of NADES and collagen has the potential to beincluded in different types of topical formulations, e.g., spray formula-tion, personalized products from 3Dprinting, and wound dressings. Col-lagen to be used in wound products should retain the chemotacticproperties important for wound healing, either as a triple helix or infragmented form as collagen peptides [2,7]. For other purposes like 3Dprinting, the collagen should be able to be crosslinked, either physicallyby pH and temperature, or by a crosslinker. This requires an intact triplehelical structure [20].The aim of the present study was to investigate the physicochemicalproperties of collagen in a selected NADES and aqueous dilutionsthereof to identify potential combinations suitable in pharmaceuticalpreparations. Both pepsin soluble collagen (atelocollagen) and acid sol-uble collagen (telocollagen) were studied. The selected NADEScontained an organic acid (citric acid) and a polyol (xylitol). ThisNADES has shown antibacterial effect combined with unique solventproperties and is, therefore, a candidate excipient in antimicrobial prod-ucts [1]. The unfolding, thermal properties, fragmentation and viscosityof telo- and atelocollagen in NADES were assessed. Freeze-dried colla-gen sheets with NADES were prepared as a potential wound dressing.The structure and mechanical properties of the sheets were evaluated.Our data reveals the potential of the selected NADES and aqueous dilu-tions thereof as excipients in collagen-based products. This is to ourknowledge thefirst study describing solubility and behavior of an extra-cellular matrix protein in NADES.2. Materials and methodsAll experiments were performed at 25 °C unless other stated. Thedata were presented as mean ± highest deviation from three indepen-dent experiments.2.1. MaterialsPepsin soluble collagen (atelocollagen) isolated from industriallyproduced turkey (Meleagris gallopavo) rest raw materials was preparedas described in Grønlien et al. [21]. Acid soluble collagen (telocollagen)from calf skin (Sigma-Aldrich, C9791) and other reagents were of ana-lytical grade and were purchased from either Sigma-Aldrich ChemicalCompany (St. Louis, MO, USA) or Merck KGaA (Darmstadt, Germany).2.2. Preparation of the natural deep eutectic solvent (NADES)The selected NADES was prepared by a solvent evaporation method,according to Wikene et al. [12]. The two components of the NADES weredissolved in warm Milli-Q water (~50 °C) and evaporated at 45 °C for20 min with a rotary evaporator. The liquid obtained was transferredto polypropylene tubes with a tight cap. Water content was determinedby Karl Fischer titration (C20 Coulometric KF Titrator, Mettler ToledoInc., Schwerzenbach, Switzerland). The NADES prepared containedcitric acid/xylitol (molar ratio 1:1) (abbreviated CX). The NADES wasused in the undiluted form or after dilution in Milli-Q water 1:1, 1:10,1:50, 1:100 and 1:200. The pH was measured using a pH 526 MultiCal®pH meter (WTW GmbH, Weilheim, Germany).2.3. Intrinsic viscosity and estimated average molecular weight of collagenCollagen isolated from turkey tendon was dissolved in 0.5 M aceticacid to a concentration of 0.5 mg/ml. The solution was diluted to con-centrations between 0.1 and 0.5 mg/ml. Acetic acid (0.5 M) was usedas negative control. The viscosity of the solutions was determinedwith an Anton Paar Rheometer (Anton Paar Physica MCR301 Rheome-ter, Germany). Samples of 10 ml were measured with a double gap con-centric cylinder (DG 26.7) at 25 °C with a shear rate of 10 s−1.Theintrinsic viscosity of collagen was calculated from the dynamic viscosity[22]. The average molecular weight was then estimated from the intrin-sic viscosity according to the Kuhn-Mark-Houwink-Sakurada equation(Eq.(1)):η½¼KMαð1Þwhereηis the intrinsic viscosity,Mis the average molecular weight,Kandαare values specific for the polymer or protein (1.86 × 10−19and1.8 for collagen, respectively) [23].2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)SDS-gel electrophoresis is a common method for determination ofcollagen polypeptide chains [24,25]. Collagen (both qualities) was solu-bilized in 0.02 M acetic acid or NADES diluted 1:10, 1:50, 1:100 and1:200 with Milli-Q water to afinal concentration of 2.0 mg/ml. Lowerdilutions of NADES (1:1 or undiluted samples) were not tested for prac-tical reasons(i.e., high viscosity). Further, 100μl sample was mixed with50μl buffer to afinal concentration of 0.05% Tris-HCl pH 6.8, 7% glycerol,0.07 M dithiothreitol, 1% (w/v) SDS and 0.001% bromophenol blue. Thesamples were then pre-heated to 50 °C for 10 min and separated bySDS-PAGE by use of 4–12% Bis-Tris gels (Invitrogen, MD, USA),NuPAGE® MOPS SDS running buffer (Invitrogen, MD, USA) and NovexXCell II apparatus (Invitrogen, MD, USA). Protein bands were visualizedby Coomassie Staining and molecular weight determined by use ofBenchmark prestained protein ladder (Novex, Life technologies,10748-010) run simultaneously on the gel.2.5. Preparation of samples for spectroscopyCollagen isolated from turkeyfilet tendon or collagen from calf skinwas dissolved at 1.5 mg/ml in NADES, aqueous dilutions of NADES or0.02 M acetic acid. The samples were gently stirred overnight at roomtemperature (IKA® RO 15, IKA Werke, GmbH, Staufen, Germany,400 rpm) protected from lightandfiltered (5μm Versapor® Membrane,Pall Corporation, MI, USA) prior to the spectroscopic measurements.2.6. UV–Vis spectrophotometryAbsorption spectra were recorded between 190 and 700 nm on aShimadzu UV-2101 PC (Kyoto, Japan) UV–Vis scanning spectrophotom-eter using a quartz cuvette with a 1 cm cell path.2.7. Fluorescence spectroscopyFluorescence measurements were performed on a Photon Technol-ogy International modularfluorescence system (London, Ontario,Canada), Model 101 monochromator with f/4 0.2-m Czerny-Turnerconfiguration. The instrument was equipped with a red-sensitivephotomultiplier. The excitation source was a 75 W xenon lamp. The395K.G. Grønlien et al. / International Journal of Biological Macromolecules 156 (2020) 394–402