Are aqueous solutions of choline-based ionic liquid biocompatible cross-linkers for collagen?

Journal Pre-proof Are aqueous solutions of choline-based ionic liquid biocompatible cross-linkers for collagen?

Aafiya Tarannum, Nitin P. Lobo, J. Raghava Rao, N. Nishad Fathima PII:

S0167-7322(19)35295-X

DOI:

https://doi.org/10.1016/j.molliq.2020.112654

Reference:

MOLLIQ 112654

To appear in:

Journal of Molecular Liquids

Received date:

22 September 2019

Revised date:

8 January 2020

Accepted date:

6 February 2020

Please cite this article as: A. Tarannum, N.P. Lobo, J.R. Rao, et al., Are aqueous solutions of choline-based ionic liquid biocompatible cross-linkers for collagen?, Journal of Molecular Liquids(2020), https://doi.org/10.1016/j.molliq.2020.112654

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© 2020 Published by Elsevier.

Journal Pre-proof

Are aqueous solutions of choline-based ionic liquid biocompatible cross-linkers for collagen? Aafiya Tarannum, Nitin P. Lobo, J Raghava Rao, N. Nishad Fathima*

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Inorganic and Physical Chemistry Laboratory, CSIR-Central Leather Research Institute, Adyar,

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Chennai-600020, India

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KEYWORDS. Biocompatible crosslinker, Collagen-based biomaterial, T2 Relaxation,

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Hemocompatibility, Choline-based ionic liquid

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ABSTRACT. Identification of biocompatible cross-linkers is extremely important in the field of

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biomaterials, as it aids in improving stability to protein but also elicits less immune response. Towards this, aqueous solution of choline-based ILs (CILs) has been keyed out as biocompatible

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cross-linkers with an aim to understand their impact and effect on stabilization of collagen. The

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changes in the hydration dynamics of collagen treated with aqueous solution of CILs has been probed using transverse NMR relaxation and impedance measurements. Upon treatment of aqueous solution of CILs with collagen, there is a reorientation in the surrounding water milieu suggesting compactness in the structure of collagen, which is indicative of crosslinking. Further, the conformational and thermal stability of collagen have been substantiated. It is ascertained that the helicity of collagen remains intact as witnessed through the Rpn values measured through circular dichroic measurements and shifts in frequencies of amide bands as measured through FT-IR measurements. A significant increase in the melting and denaturation temperature suggesting stability of collagen has been observed through DSC measurements. Aqueous

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Journal Pre-proof solution of CILs treated collagen scaffolds have been further appraised for crosslinking efficiency, enzymatic stability, hemolytic and platelet attachment assays. The efficacy of aqueous solution of CILs treated scaffolds has been evaluated for biocompatibility with NIH-3T3 fibroblasts cells and further confirmed by the phase-contrast microscopic images of these cells. Interaction between the collagen and aqueous solution of choline-based ILs is typically anchored by non-covalent interactions like electrostatic, hydrogen, and van der Waals interactions.

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Therefore, the current study implies that the aqueous solution of CILs can be explored as

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biocompatible cross-linkers for collagen and thus can be employed for biomedical applications.

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1. Introduction

Ionic liquids, well known as “task-specific”, “designer”, and “new age” material are currently

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explored in the field of proteomics owing to their excellent stability and solubility1. The varying

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classes of ionic liquids viz., imidazolium, phosphonium, ammonium and choline salts with

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varying proteins are thoroughly being researched2-5. They have been reported to influence the stabilization, denaturation and aggregation events in proteins6-7.

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Conventional ILs like imidazolium and phosphonium has been well studied and it has shown

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stabilizing effect for a range of proteins, albeit these ILs have reported the cytotoxic behavior with cell lines and aquatic organisms8-9. In lieu of preparation of biomaterials, it is imperative to re-explore varying classes of ILs, which will have stabilizing characteristics, sans toxicity and akin to the nutrients present in our body. In this context, choline-based ILs (CILs) have been identified and reported to be biocompatible. Choline (N, N, N- trimethylethanolammonium) refers to the class of quaternary ammonium salts and have been reported as precursors for neurotransmitters (acetylcholine receptors), and are involved in many functions including memory and muscle control10. As established by Food and Nutrition board of the National

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Journal Pre-proof Institute of Medicine has set a value for adequate intake (AI) of choline for different age groups and gender (300-500 mg/day)11. Choline-based ILs has been reported as a promising media for the stabilization of proteins and they provide great potential advances in the field of liquid formulation for therapeutic proteins12. Recently, these ILs have been reported to enhance the stability of Herceptin, demonstrating a promising additive in developing stable therapeutic antibody formulations13. This led to the present study to explore CILs to design collagen-based

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biomaterials for tissue engineering and wound healing applications.

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Type I collagen, a primary structural protein is triple helical in structure and is stabilized by

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inter- and intra-chain hydrogen bonds and water molecules14. Collagen-based biomaterials have

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been utilized as scaffolds/sponges for burns and wounds, grafts, sutures, bone substitutes, hemostatic agents and blood vessel prostheses due to its excellent biocompatibility,

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biodegradability and low immunogenicity15. However, thermal, mechanical, biodegradability and

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other properties of collagen-based biomaterials are not up to the mark. In order to improve its thermal and enzymatic stability, various chemical cross-linkers viz., iron16, glutaraldehyde17, 1-

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ethyl-3-(3-(dimethylamino)-propyl) carbodiimide18 (EDC) and epoxy compounds19 are being

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studied. Also, natural cross-linkers such as genipin, citric, ferulic and tannic acid are being explored and investigated20-22. Most of these crosslinking agents react with the carboxyl groups of amino acids and other amine groups to form amide bonds23. Glutaraldehyde has been widely reported as crosslinkers for collagen17, however owing to its toxicity concerns the use has been discontinued. Hence, there is a dire need to identify and develop a class of biocompatible crosslinkers for collagen. Choline based salts have been utilized as a substituent for glutaraldehyde, which exhibited good cell viability and adhesion properties as required for

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Journal Pre-proof biomedical implant applications24. Further, the influence of choline-based ILs has demonstrated to stabilize collagen by exerting an electrostatic force25. Herein, aqueous solution of choline-based ILs viz., choline acetate, choline citrate and choline sulfate have been chosen to understand the stabilizing effect on collagen. The choice of cations and anions was based on kosmotropic and chaotropic behavior of ions as mentioned in the Hofmeister series26. Further, this work aims at providing significant and mechanistic insights

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using various biophysical studies. The reorganization of water molecules around the hydration

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shell of collagen on addition of aqueous solution of CILs has been studied using NMR and

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dielectric measurements. The conformational stability of the collagen solution upon treatment

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with aqueous solution of CILs has been studied using circular dichroic studies. Aqueous solution of CILs treated collagen scaffolds have been further appraised for crosslinking efficiency,

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enzymatic stability, hemolytic and platelet attachment assays. The efficacy of aqueous solution

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of CILs treated scaffolds was evaluated for biocompatibility with NIH-3T3 fibroblast cells and it

2. Experimental

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2.1 Materials

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has been confirmed by the phase-contrast microscopic images of these cells.

Rat tail tendons were teased from six month old albino rats (Wistar strain) and it was used for the extraction of type I collagen. Choline acetate, choline citrate and choline sulfate were procured from Ionic Liquid Technologies GmBH (IoLiTec, Germany). Ninhydrin, 2, 2-diphenyl-1picrylhydrazyl (DPPH), Collagenase from Clostridium histolyticum, and was procured from Sigma Aldrich. NIH-3T3 fibroblast cells were procured from ATCC. Methanol was purchased from Thermo Fischer Scientific and the above materials were used as received without further purification. Millipore water was used for all the studies.

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Journal Pre-proof 2.2 Isolation of type I collagen Rat tails tendons (RTT) from six month old abino rats (Wistar strain) were teased for the extraction of type I collagen. The excised collagen fibers were thoroughly washed using 0.9% NaCl under cold conditions. At 4 °C, collagen fibers were dissolved in 0.5 M acetic acid and it was continuously stirred till the solution was obtained. Purification was carried out using 5% NaCl and the precipitate was collected through centrifugation, followed by dialysis against 50

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mM phosphate buffer with a pH of 6.5 for three consecutive days. Then, collagen was re-

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dissolved in 0.5 M acetic acid and the final dialysis was carried out extensively against 0.05 M

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acetic acid (pH 4). The concentration of collagen was determined through hydroxyproline

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content by method of Woessner27. SDS-PAGE was carried out to confirm the purity of collagen (supplementary figure S1). The collagen solution was stored at 4 °C and the further experiments

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were carried out.

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2.3 Preparation of collagen-CILs solution/scaffold Preparation of collagen and aqueous solutions of CILs treated RTT fibers

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RTT collagen fibers were freshly teased from six month old abino rats (Wistar strain) and

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washed with (0.9%) saline. It was incubated at 25 °C in aqueous solution of CILs (acetate, citrate and sulfate) overnight and the experiments were carried out. Preparation of collagen and CILs (aqueous) treated collagen solution Collagen-CILs (aqueous) solution (choline acetate, choline citrate, choline sulfate) of varying concentrations (1:0.05%, 1:0.5% and 1:1% (volume/volume percentage (v/v))) was blended in acetate buffer under stirring for 3 h at 4 °C throughout to prevent denaturation of collagen due to heat generated during mixing. The working concentration of collagen was 2.7 µM (0.8 mg/ml) at pH 4.0. Collagen concentration was kept constant and the CILs concentration was varied.

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Journal Pre-proof Preparation of collagen and aqueous solution of CILs treated collagen scaffold For preparation of collagen and aqueous solution of CILs scaffold, 10 wt. (percentage%) of collagen solution was taken and varying aqueous solution of CILs (0.05, 0.5 and 1%) was added followed by stirring for 3 h at 4 °C. The prepared samples were poured in a mold of 2.5 cm diameter. It was kept at ̶ 50 °C for 2 hours. The frozen samples were lyophilized for 24 hours to make it as a scaffold.

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2.4 NMR measurements

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The NMR experiments were performed on aqueous solution of CILs treated RTT fibers using a

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Bruker Avance-III HD 400 WB NMR spectrometer (9.4 T). The operating frequency for proton

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was 400.07 MHz. The experiments were carried out under static condition at room temperature using a double resonance 4 mm MAS probe. The excess of water from the surface of the

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collagen fibers was gently removed with tissue paper and then packed in a 4-mm-diameter

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zirconia rotor with a Kel-F cap in random orientation. The Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence 90°− τ − (180° − 2τ)n, was used to measure proton transverse relaxation time in

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each sample. Here, proton 90° pulse length of 5 µs, relaxation delay of 6.5 s and τ of 0.6 ms was

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employed. The values of T2 were obtained by fitting to a multi exponential function and the best fit was obtained for three exponentials namely, T2short, T2intermediate, and T2long. The reported values for each sample were obtained by taking average from three measurements. 2.5 Impedance measurements Impedance measurements for native collagen and CILs (aqueous) treated collagen solution were carried out using CH Instrumental analyzer CH-model 660. The effect of CILs on the resultant dipole of the collagen responding to an alternating electric field was assessed using the three classical electrode systems, wherein, the glassy carbon, a platinum and saturated calomel

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Journal Pre-proof electrode serves as a working electrode, counter electrode and as reference electrode, respectively. Dielectric data can be described in terms of the imaginary part Y″ of the complex admittance in units of Ω-1, 𝑌 ∗ = 𝑌 ′ + 𝑗𝑌 "

(1)

Where the real component Y′ describes the energy stored and Y″ relates to the energy dissipated

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by the system.

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2.6 Circular dichroism studies

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Circular dichroic studies for native collagen and CILs (aqueous) treated collagen solutions were

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carried out using Jasco 815 Circular Dichroism Spectropolarimeter. The spectra can be detected under nitrogen atmosphere in the far UV region ranging from 190 to 260 nm using

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Approximately, 400 µl of native collagen and collagen-CILs (aqueous) solutions was scanned

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with 0.2 nm intervals with a path length of 1 mm with computer averaged three scans for each sample. The data were obtained in milli degrees and further converted to molar ellipticity (deg.

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cm2. dmol-1). Molar ellipticity was plotted against wavelength in nanometers (nm). Also,

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influence of aqueous solution of CILs on the temperature induced conformational changes of collagen was studied at pH 4 with the temperature range of 25-50 ºC and an interval of 1 ºC under N2 atmosphere. 2.7 Viscosity measurements Viscosity for native collagen and CILs (aqueous) treated collagen solutions was performed using a Brookfield DV-II+Pro viscometer at 25 C to study the influence of CILs on the rheological property of collagen. Native collagen was used as a control.

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Journal Pre-proof 2.8 Scanning electron microscopy The cross sectional morphology of native collagen and aqueous solution of CILs treated collagen scaffolds were observed under a scanning electron microscope (Phenom Pro) at an accelerating voltage of 10 kV. The samples were sputter coated with gold, prior to scanning. 2.9 FT-IR studies Native collagen and aqueous solution of CILs treated collagen scaffolds were lyophilized and

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samples were studied using Jasco FT/IR-4200 (Fourier Transform Infrared Spectrometer) by

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KBr pellet method at 25 °C with 40 scans in the range of 4000-400 cm-1 with a resolution of 4

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cm-1.

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2.10 Differential Scanning Calorimetry

The denaturation temperature (Td) of native collagen and aqueous solution of CILs treated

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collagen scaffolds was effectuated using differential scanning calorimeter (NETZSCH) from 20

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to 100 ºC at the rate of 1 ºC min-1. Samples were blot-dried and the predetermined weights of

2.11 Ninhydrin Assay

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samples were sealed in the aluminium pans.

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For Ninhydrin assay, 5 mg of native collagen and aqueous solution of CILs treated collagen scaffolds were weighed followed by the addition of 2 ml of 0.2 % of ninhydrin solution and was heated to 100 ºC for 20 min and cooled down to room temperature. Then, 10 ml of 50% isopropanol was added and absorbance was measured by UV-Visible spectrophotometer at 570 nm. The amount of free amino groups is proportional to the value of Abs570, and glycine at varying known concentrations was used to create standard curve of glycine concentration vs absorbance. The degree of crosslinking is calculated by the given formula: 𝐷𝑒𝑔𝑟𝑒𝑒 𝑜𝑓 𝑐𝑟𝑜𝑠𝑠𝑙𝑖𝑛𝑘𝑖𝑛𝑔 (%) =

𝐴𝑚𝑖𝑛𝑜𝑜 −𝐴𝑚𝑖𝑛𝑜𝑐 ∗ 100 𝐴𝑚𝑖𝑛𝑜𝑜

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Journal Pre-proof Where, Aminoo is the free NH2 concentration in non-crosslinked samples, and Aminoc is the free NH2 concentration in crosslinked samples 2.12 Enzymatic stability In vitro degradation tests of native collagen and aqueous solution of CILs treated collagen scaffolds were performed using bacterial collagenase obtained from Clostridium histolyticum. 5 mg of non-crosslinked and crosslinked collagen scaffolds were incubated in 10 ml of collagenase

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solution (0.6 µg/ml) with the loss of samples as a function of time at 37 ºC was investigated.

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟 𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 ∗ 100 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡

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𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 (%) =

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The percentage of degradation was calculated using the given formula,

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2.13 Swelling studies

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5 mg of native collagen and aqueous solution of CILs treated collagen scaffolds was weighed and immersed in a beaker containing distilled water and the samples were taken and weighed at

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subsequent time intervals and the swollen weight was noted. The percentage of swelling of the

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non-cross-linked and cross-linked collagen scaffolds was calculated using the formula given as

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below,

𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 (%) =

𝑊𝑆 −𝑊𝐷 ∗ 100 𝑊𝐷

Where, WD is the weight of the dry samples and WD is the weight of the swollen samples. 2.14 Hemolytic Assay Hemocompatibility evaluation of native collagen and aqueous solution of CILs treated collagen scaffolds was investigated using hemolytic assay. 5ml of blood was freshly withdrawn from human body and sodium citrate (anticoagulant) was added followed by immediate centrifugation for 15 min to pack erythrocytes. 50 µl of RBC and 950 µl of PBS were added to non-cross-

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Journal Pre-proof linked and cross-linked collagen scaffolds and it was incubated in the dark for an hour at room temperature. Then, the samples were centrifuged for 10 min at 6000 rpm. The absorbance was measured at 540 nm using UV-Visible spectrophotometer. The obtained results were compared with the positive control (50 µl RBC + 950 µl of water) and the negative control (50 µl RBC + 950 µl of PBS). 𝑂𝐷 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ∗ 100 𝑂𝐷 𝑜𝑓 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝑂𝐷 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

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𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 (%) =

2.15 Platelet Adhesion Assay

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The hemocompatibility of native collagen and aqueous solution of CILs treated collagen

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scaffolds was studied using platelet adhesion assay. Here, the whole was collected and

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centrifuged at 3000 rpm for 20 min to obtain platelet rich plasma (PRP). Prior to incubation with PRP, the scaffolds were pre-equilibrated with PBS for 30 min at room temperature followed by

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incubation with PRP for two hours. Then, they were washed with PBS to remove unattached

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platelets followed by fixing with 2.5% glutaraldehyde solution. Platelet adhesion on varying

(SEM).

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concentration of scaffolds was evaluated by observing under scanning electron microscopy

2.16 DPPH Assay

Antioxidant properties of native collagen and aqueous solution of CILs treated collagen scaffolds were assessed by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. 5 mg of sample of sample was weighed and 3ml of 100 µM DPPH solution in methanol. The reaction mixture was then incubated in the dark for 30 minutes followed by measuring the absorbance at 517 nm. 𝐷𝑃𝑃𝐻 𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 (%) =

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑏𝑙𝑎𝑛𝑘 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 100 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑏𝑙𝑎𝑛𝑘

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Journal Pre-proof 2.17 Cytotoxicity studies The cytotoxicity of native collagen and aqueous solution of CILs treated collagen scaffolds has been evaluated using MTT assay (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) at different culture peroids up to 48 hrs in triplicates. The scaffolds were UV sterilized before seeding it with NIH-3T3 osteoblasts like cells. The scaffolds with NIH-3T3 were incubated at 37 ºC under 5% CO2. MTT was added to each well and it was incubated for 3 hrs.

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The obtained formazan complex was dissolved in dimethyl sulfoxide and the OD was measured

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with the microplate reader at 570 nm. Percentage of cell viability was calculated from the OD of

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samples and control. In addition to, the morphology of the cells present in native collagen and

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aqueous solution of CILs treated collagen scaffolds incubated conditioned media was observed under phase contrast microscopy.

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3. Results and Discussion

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3.1 Changes in hydration dynamics of native collagen and aqueous solution of CILs treated collagen RTT fibers assessed via T2 relaxation and impedance measurements

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3.1.1 T2 relaxation measurements

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Nuclear magnetic resonance measurement depicts the hydrodynamic behavior of water molecules in native RTT collagen fibres. CPMG decays for the native and aqueous solution of CILs treated RTT collagen fibres have been reported in Figure 1 and it signifies the relaxation behavior of water when treated with varying aqueous solution of CILs, as T2 describes the decay of the excited magnetization perpendicular to its magnetic field. The relaxation times viz, T 2short, T2intermediate and T2long outline the behavior of water in different states adhering to the bound water, weakly bound water and free water, respectively. On interaction of aqueous solution of CILs with the RTT collagen fibres, there was a significant change in the dynamics of water molecule

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Journal Pre-proof as examined from the changes given in Table 1. As seen from the Table 1, T2short value corresponds to the bound water component signifies the hydration water bound to the protein surface. Weakly bound water (T2intermediate) intends forming bonds with charged or polar molecules by stabilizing the structure and by mediating contacts between the proteins and ligands. Free lattice water (T2long) has more molecular mobility in solution. However, a water molecule shares the property of a weakly bound and hydration water by strongly interacting with

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proteins and stabilizing them28. Our finding has been consistent with the three relaxation times of

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native collagen fibres in water with the existing literatures29-30 and the impact of aqueous

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solution of CILs on collagen fibres reflects the interactions between them and it is evident from

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Table 1. There was a slight decrease in the shorter and intermediate relaxation time for CA, CC and CS-treated collagen fibres, whereas a significant change was observed for long relaxation

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times when compared to native collagen fibres. T2long relaxation time essentially reflects fast

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dynamics of water i.e. free water and such dynamics are treated as dynamically averaged ones usually occuring on picosecond time scales, which include reorientation and translational

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dynamics of free water31-32. Therefore, the observed T2 long values may be treated in a

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qualitative sense rather than quantitative one. The influence of choline-based ionic liquid on water dynamics (without collagen) was also studied and it was found that the CILs are not influential to relaxation dynamics of water (Table S1). The changes detected in the mobility of water directly correlates to the structural changes of the collagen. Increased crosslinking efficacy of collagen usually happens during aging and it also decreases the rotational and translational water mobility33-34. It was noticed that there was a marked decrease in long relaxation times for the CS, CC, CA-treated collagen fibres when compared to native collagen fibres. Slight decrease in the T2 short and intermediate relaxation times are likely due to the formation of crosslinks

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Journal Pre-proof formed between aqueous solution of CILs and functional side groups of collagen, thereby stabilizing collagen. From the above stated results, it has been ascertained that the decrease in the three relaxation times is a clear demonstration that aqueous solution of CILs stabilizes collagen. To further understand the dynamics of collagen, impedance measurements was performed. Table 1. 1H T2 Relaxation times of water in native and aqueous solution of choline acetate (CA),

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choline citrate (CC) and choline sulfate (CS) treated collagen RTT fibres

T2short [ms]

T2intermediate [ms]

Native

3.07±0.22

10.13±0.59

67.84±8.95

CA treated

2.40±0.15

9.00±0.38

36.93±2.80

CC treated

2.89±0.10

10.51±0.35

44.76±3.94

CS treated

2.65±0.12

9.63±0.36

27.53±1.42

T2long [ms]

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Sample codes

Figure 1. 1H T2 relaxation decays of native, aqueous solution of choline acetate (CA), choline citrate (CC) and choline sulfate (CS) treated collagen fibres. The continuous lines are the fit results. 3.1.2 Impedance measurements Dielectric measurement is a significant tool for probing the dynamics of water surrounding collagen, which in turn confirms the stability of collagen. It provides the significant insights about the hydration network surrounding the biomacromolecules. Dielectric profile of native and

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Journal Pre-proof CILs (aqueous) treated collagen solutions has been depicted by means of impedance and admittance plotted by exerting real Y′ vs. imaginary Y″ (Nyquist plot) respectively. Figure 2 evidences the Nyquist plot and Bode plot for determining the total impedance35. Nyquist plot for neat 1% CIL (CA, CC, CS) is given in the supplementary figure (S2). An electric field is applied causing the drift and displacement in the free and bound charges, which in turn induces the conduction and polarization of currents. As seen from Figure 2, aqueous solution of CILs

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showed highest permittivity and this might be due to the greater number of proton charge carriers

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and sites available for the protein to accumulate and pass the current. These sites are created by

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newly formed intra- and intermolecular crosslinks that enhance the stability of triple helical

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structure36. Aqueous CIL solution influences the hydrophilic domains with the collagen, which in turn influences the hydration network of collagen. Mobility in the functional groups aqueous

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CILs treated collagen solutions reflects the change in electric dipole confirming the stability in

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structure of collagen. Aqueous solution of CILs showed the highest permittivity, whereas the native collagen exhibited the lowest permittivity. All CILs (aqueous) treated collagen solutions

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show concentration dependent permittivity and the increase or decrease in permittivity shows the

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interaction of an external electric field with the electric dipole moment of the sample. Bode plot usually expresses the phase shift and it has been plotted against the logarithmic frequency axis. As seen from the Figure 2, it was shown that there was a shift in phase angle to higher frequencies for CILs (aqueous) treated collagen solutions from 103-105 Hz. This is due to the fact that the collagen is a protein with varying functional groups leading to its charged behavior. Changes in the phase angle with frequency indicate the electrostatic interactions between the functional side chain of collagen and anionic moieties. This in turn alters the hydration environment by reorienting and reorganizing the charges of polar functional groups on the

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Journal Pre-proof surface of collagen. Factors which influences the degree of hydration comprises of molecular asymmetry, polar nature, the hydrogen bonding capacity and the strength of forming ion pairs between the amino and carboxyl group of collagen to that of the functional groups. Anions (acetate, citrate, sulfate) of choline-based IL (aqueous solutions) might form hydrogen bonds with collagen, and they will also undergo electrostatic interaction with amine functional groups of collagen36. Usually the hydrophilic group resides on the surface of the collagen and these

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moieties interfere with the rearrangement of hydrogen-bonded network and causing the

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reorientation around the peptide bonds37. These results complement the hydration dynamics of

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collagen treated CILs (aqueous) solutions. To further understand the nature of these

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intermolecular interactions, the conformational changes in the native collagen and CILs (aqueous) treated collagen solutions can be validated using viz., circular dichroism and viscosity

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Nyquist Plot

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C

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A

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studies.

Bode Plot

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E

F

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Journal Pre-proof Figure 2. Impedance measurements (Nyquist Plot and Bode Plot) for native collagen and aqueous CIL solution A, D- choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), B, E- choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), C, F- choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen solutions, (Conditions- pH 4, Temperature- 25 ºC, Concentration of collagen- 2.7 µM (0.8 mg/ml)) 3.2 Changes in secondary structure of native collagen and aqueous CILs treated collagen

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solutions assessed via CD measurements

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Circular dichroic spectroscopic studies investigates the alteration in secondary structure of

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collagen due to its interaction with aqueous solution of CILs and changes the molar ellipticity

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value at 222 nm was monitored. CD spectra of type I collagen evidences peak at 197 and 222 nm, which is distinctive of peptide linkages corresponding to back-bone configuration and

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polyproline II conformation, which accords to the secondary structure of collagen respectively38.

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Changes in the molar ellipticity at positive peak (222 nm) demonstrate the changes in the polyproline II conformation, whereas the negative peak (197 nm) attributes to the conformational

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changes in the peptide chain. The parameter Rpn, a characteristic ratio for assessing the triple

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helicity of collagen signifies the ratio for positive peak intensity over negative peak intensity39. Figure 3 reveals the molar ellipticity for collagen treated with aqueous solution of choline based ILs (acetate, citrate and sulfate) at 222 nm with varying ratios (1:0.05, 0.5 and 1%) at various temperatures. Melting temperature of native collagen is 37 ºC as reported in the literature and the effect of aqueous solution of CILs on collagen has been probed. As seen from figure 3 that the choline acetate and sulfate (CA & CS) treated collagen demonstrates the highest thermal stability at the ratio of 1:1 (Collagen- CA & CS) of 43 ºC, when compared to native collagen and this might be due to strong electrostatic interaction of acetate and sulfate group with the amine

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Journal Pre-proof functional groups of collagen resulting in the alteration of hydration shell around collagen. There was a slight increase in thermal stability for CC (choline citrate) treated collagen, when compared to native collagen, which was around 41 ºC. Concentration dependent increase in thermal stability was witnessed for aqueous solution of choline-based ILs treated collagen. However, at higher concentrations of aqueous solution of CILs, crosslinking increases the thermal stability of choline treated collagen. Figure 3d displays the Rpn (ratio of the positive to

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negative peak heights) of collagen and aqueous solution of CILs as a function of temperature.

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They can be used as a measure of triple-helix content, with the values closer to 0.12 affirming a

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fully triple-helical structure40. Rpn values are in fully triple-helical range for aqueous solution of

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CILs treated collagen confirming the helicity of polyproline II conformation at the higher concentration of aqueous solution of CILs treated collagen, albeit with the increase in

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temperature there was an increase/decrease in the Rpn values indicating denaturation at higher

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temperatures. Thus, these findings indicate the increase in thermal stability of collagen when treated with aqueous solution of choline-based ILs (CILs) with the changes in the dynamics of

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protein and this has been driven by electrostatic interactions.

A

B D

C 17

Journal Pre-proof Figure 3. Variation of molar ellipticity for native collagen and aqueous solutions of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen solutions, d. Rpn values for CA, CC, CS (Conditions- pH 4, Temperature- 25-50 ºC, Concentration of collagen- 2.7 µM (0.8 mg/ml)) 3.3 Changes in rate of flow of native collagen and aqueous CILs treated collagen solutions

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assessed via viscosity measurements In order to understand the influence of aqueous solution of CILs on rheological behavior of

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collagen, viscosity measurements were performed. Figure 4 unveils the absolute viscosity for

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native and aqueous solution of CILs treated collagen. It was observed that there was an increase

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in the viscosity of aqueous solution of CILs treated collagen and this reflects the strength of

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inter- and intra- molecular forces41. There was a negligible difference in acetate, citrate and sulfate ions when treated with collagen. Concentration dependent increase in the rheology of

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aqueous solution of CILs was witnessed. This might be due to the electrostatic interactions

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between anions of choline-based IL (acetate, citrate, sulfate) and cationic functional groups of

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collagen suggesting the microenvironmental changes leading to the increased viscosity.

Figure 4. Rheological measurements for native collagen, a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS))

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Journal Pre-proof treated collagen solutions, (Conditions- pH 4, Temperature- 25 ºC, Concentration of collagen2.7 µM (0.8 mg/ml)) 3.4 Characterization of native and aqueous solution of CILs treated collagen scaffolds 3.4.1 Changes in the morphology of native and aqueous solution of CILs treated collagen scaffolds assessed via scanning electron microscopy The cross-sectional morphology of native and aqueous solution of CILs treated collagen

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scaffolds was descried using scanning electron microscopy. An ideal biomaterial should be

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highly porous, as it helps in encapsulating drugs efficiently and it also provides an environment

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for cell attachment and proliferation. As seen from Figure 5 native collagen scaffold discloses a

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sheet-like morphology having minute interfibrillar space, whereas the aqueous solution of CILs treated collagen scaffolds showed a uniform porous morphology. On a comparative note, it was

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interesting to note that the surface of the C: CIL (1:0.5) exhibited to be continuous and smooth,

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thereby forming a compact matrix. The interconnection between pores could be due to the crosslinking network formation between aqueous solution of CILs and functional side groups of

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collagen, which resulted in reorientation of the hydration network of collagen. Further, the shifts

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in the frequency of functional groups and changes in thermal stability of native collagen and aqueous solution of CILs treated collagen scaffolds has been evaluated using FT-IR and DSC studies respectively. CONTROL

CA1

CA2

CA3

CC1

CC2

CC3

CS1

CS2

CS3 19

Journal Pre-proof Figure 5. Scanning electron micrographs for native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1% (C:CA)), b. choline citrate (1:0.05, 1:0.5, 1:1% (C:CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1% (C: CS)) treated collagen scaffolds 3.4.2 Changes in the functional groups of native and aqueous solution of CILs treated collagen scaffolds assessed via FT-IR measurements Fourier transform infrared spectroscopy is a sensitive and potential tool for examining the

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changes in conformation of protein at the secondary structure level. The interaction between the

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chemical groups at the molecular level has been studied and it is due to the change in molecular

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vibration of the chemical bonds. The energy of the molecular vibration is localized to the specific

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group leading to the concept of characteristic group frequencies. It helps to analyze the interactions between collagen and aqueous solution of choline-based ILs (CILs). Collagen

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exhibits its characteristic vibration bands at amide A, amide B, amide I, amide II, amide III

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corresponding to N-H and O-H stretching at 3400 cm-1, C-H stretching at 2930 cm-1, carbonyl stretching (C=O) at 1640 cm-1, N-H stretching vibration strongly coupled with C-N stretching

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vibration at 1560 cm-1, C-N stretching and N-H plane bending at 1240 cm-1, respectively42. The

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characteristic bands for CILs (choline acetate, choline citrate and choline acetate) are given in supplementary section (figure S3). The characteristic bands for choline were recorded around 3373, 3073 and 949 cm-1, which corresponds to hydroxyl groups, quaternary ammonium and CN symmetric and asymmetric stretching respectively43. For acetate, the band at 1411 cm-1 attributes to C-O symmetric and asymmetric stretching44. The bands for citrate were found to be around 1395 and 1086 cm-1 corresponding to asymmetric stretching of COO¯ and C-H bending45, the band at 1126 and 616 cm-1 attributes to sulfate ions46, thus confirming that the CILs does not interferes with the collagen bands.

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Journal Pre-proof Table 2 depicts the conformational changes in the secondary structure of collagen. Shifts in frequencies of the bands are related to collagen, thus indicating no significant change in its secondary structure and are concordant to the reported values. A wide band was centered around 3300-3400 cm-1, which is assigned to hydration water surrounding collagen signifying the stabilization of protein47. Significant changes for the aqueous solution of CILs treated samples was observed with the witnessing changes in the frequencies of the band amide I, amide II and

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amide III band red shifted from 1642 cm-1 to 1658 cm-1, 1560 cm-1 to 1575 cm-1 and 1240 cm-1 to

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1258 cm-1 respectively. However, for collagen treated aqueous solution of CILs at lower

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concentration (0.05 and 0.5%), concentration dependent shifts in the band frequencies was

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observed. These shifts in frequencies might be due to the interaction between the hydroxyl moieties of choline and collagen. There is a high probability of bonding between the functional

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groups of collagen with the anions of aqueous solution of CILs (acetate, citrate and sulfate).

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Thus, this study suggests the hydrogen bonding and electrostatic interaction between collagen and aqueous solution of choline-based ILs. It is in coherence with the hydration studies, which

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confirms the crosslinking between collagen and aqueous solution of CILs.

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Table 2. FT-IR for native collagen scaffolds and aqueous solution of choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- pH 4, Temperature- 25 ºC) Bands/Codes

Amide A

Amide B

Amide I

Amide II

Amide III

Native collagen

3285

3173

1640

1557

1240

1:0.05%

3285

3170

1640

1559

1240

1:0.5%

3288

3170

1643

1561

1243

1:1%

3295

3170

1649

1565

1248

C:CA

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Journal Pre-proof

C:CC

3285

3170

1642

1560

1240

1:0.5%

3294

3179

1645

1561

1247

1:1%

3299

3184

1653

1571

1250

1:0.05%

3285

3170

1640

1560

1240

1:0.5%

3289

3170

1646

1560

1245

1:1%

3289

3170

1656

1564

1252

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C:CS

1:0.05%

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3.4.3 Changes in thermal stability of native collagen and aqueous solution of CILs treated

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collagen scaffolds assessed via differential scanning calorimetry

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The thermal stability for the native collagen and aqueous solution of CILs treated collagen scaffold thermograms has been evaluated using differential scanning calorimetry (DSC). The

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denaturation temperature (Td) for native collagen scaffolds was found to be around 63 ºC48. As

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seen from figure 6, there was a significant increase in denaturation temperature for CILs treated collagen scaffolds and this might be due to the fact that the aqueous solution of CILs manifests

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the rise in crosslinking efficacy. It might be owing to the electrostatic interaction and hydrogen

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bonding between the carboxyl groups of collagen with choline and the amine functional groups to that of acetate, citrate and sulfate ions. The increased thermal stability of the aqueous solution of CILs treated collagen scaffolds accentuates the effect of aqueous CILs on the scaffolds. Further, the native collagen and aqueous solution of CILs treated collagen scaffolds were subjected to the biological evaluation to study its crosslinking efficiency, enzymatic stability, hemocompatibility and cytotoxicity of the prepared scaffolds.

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B

C

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A

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Figure 6. DSC thermograms for native collagen scaffolds and aqueous solution of a. choline

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acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline

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sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- Temperature- 20-100 ºC)

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3.5 Biological evaluation of native collagen and aqueous solution of CILs treated collagen

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scaffolds

3.5.1 Changes in the crosslinking efficiency for aqueous solution of CILs treated collagen

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scaffolds assessed via Ninhydrin Assay

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Ninhydrin assay is a sensitive technique used to quantify the free ε-amino groups present in aqueous solution of CILs treated collagen scaffolds49. The available free ε-amino groups for aqueous solution of CILs treated collagen have been measured. The results in figure 7 indicate the % loss of free amino groups by ascertaining the degree of crosslinking and it is due to each amino group, which participates in intra- or inter-molecular crosslinking of collagen. In the absence of aqueous solution of CILs in collagen, it was considered to be zero percent crosslinked. For aqueous solution of CILs treated collagen scaffolds, concentration dependent loss (in %) of free ε-amine groups was evidenced and this might be owing to the fact that the free ε-

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Journal Pre-proof amino groups of collagen would have involved in bond formation. It was found that the 80% of free ε-amino groups have cross-linked at aqueous solution of C-CIL (1:1%), whereas for C-CIL (1:0.05%) and C-CIL (1:0.05%), it was found to be around 50 and 30% respectively. Higher degree of crosslinking at higher concentration was witnessed for aqueous solution of CILs treated collagen scaffolds and the free amine (-NH3+) groups of collagen impedes CILs. As the aqueous solution of CILs concentration increases, the loss of ε-amino groups reaches up to 80%.

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As it is apparent from the above results that the addition of aqueous solution of CILs causes loss

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of free ε-amino groups and it might be owing to the interaction of amine functional groups of

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collagen and thereby forming intra- and inter-molecular crosslinks. Thus, the obtained results for

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crosslinking efficiency were in concordant and complementary to NMR measurements, wherein

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na

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the decreased relaxation times indicated crosslinking of collagen upon treatment with CILs.

Figure 7. Crosslinking efficiency for native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- pH 4, Temperature25 ºC)

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Journal Pre-proof 3.5.2 Changes in the enzymatic degradation of native collagen and aqueous solution of CILs treated collagen scaffolds assessed via collagenolytic assay Stability of aqueous solution of CILs treated collagen scaffolds against enzymatic degradation was studied by examining the rate of degradation of aqueous solution of CILs treated collagen scaffolds in 15 ml of bacterial collagenases. The percentage of collagen degradation was calculated as a function of time at varying concentrations and can be seen from figure 850.

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Aqueous solution of CILs treated collagen scaffolds exhibits the stabilizing effect as the collagen

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degraded around 25% even after incubation for 48 hrs, whereas the native collagen got degraded

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completely within 24 hrs (Figure 8). It was noted that the aqueous solution of CILs treated

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collagen scaffolds displayed a higher degree of stability when compared to untreated collagen scaffolds. The enzymatic stability of the aqueous solution of CILs treated collagen scaffolds

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increased with the increase in concentration of CILs and this might be due to the enhanced

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crosslinks formed at higher concentration, which results in the strengthening of collagen

A

B

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molecules at higher concentrations of aqueous solution of choline-based ILs (CILs).

Figure 8. Enzymatic degradation of native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline

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Journal Pre-proof sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- Temperature- 25 ºC, Time- 24 and 48 hrs) 3.5.3 Changes in the water uptake of native collagen and aqueous solution of CILs treated collagen scaffolds assessed via swelling studies The ability of a scaffold to uptake and hold water is an important characteristic for tissue engineering applications, since it reflects the blood-absorbing property of the hemostat and it is

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said to be ideal parameter in evaluating hemostat and dressing materials. It also impacts the cell

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behavior viz., cell differentiation, growth and adhesion51. The swelling of native collagen and

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aqueous solution of CILs treated collagen scaffolds is shown in figure 9. The different

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concentration of aqueous solution of CILs treated collagen scaffolds was plotted as a function of time (1-6 hours) and degree of swelling was measured. The swelling ratio for the native collagen

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and CILs treated collagen scaffolds (lower and higher concentration) was found to be around

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1300% and 1100% and 900% respectively, confirming that the water uptake has been reduced with the increasing concentration of CILs and this might be due to the low availability of

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hydrophilic groups of collagen to interact with aqueous solution of CILs, as an outcome of the

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higher degree of crosslinking with the collagen matrix52. It was interesting to note that the maximum water uptake was seen in the first couple of hours. As known from the existing literatures, that the swelling decreases as a result of the stabilization of the collagen matrix and this might be owing to the reduction in hydrophilic functional groups of collagen (amino groups). It was noticed that the aqueous solution of CILs treated collagen scaffolds exhibited sufficient swelling, which in turn might aid and assist the diffusion of bioactive molecules during healing process.

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Journal Pre-proof

B

A

C

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Figure 9. Swelling studies for native collagen scaffolds, a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS))

-p

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treated collagen scaffolds, (Conditions- Temperature- 25 ºC, Time- 1-6 hrs) 3.5.4 Changes in the hemocompatibility of native collagen and aqueous solution of CILs

lP

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treated collagen scaffolds assessed via hemolytic assay

For an artificial skin substitute to be flourishing it should be compatible with blood cells and

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hemolytic assay is an evidential parameter to evaluate the blood compatibility of the prepared native collagen and aqueous solution of CILs cross-linked collagen scaffolds. As apparent from

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the figure 10, no significant damage to the erythrocytes was observed indicating the

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hemocompatibility of the aqueous solution of CILs treated collagen scaffolds. All scaffolds showed hemolysis ratio under the permissible limit (5%) of the ASTM F756-00, 2000 (standard), validating the hemocompatibility of scaffolds53. The presence of aqueous solution of CILs in the scaffolds didn’t compromise with the hemocompatibility of scaffolds.

27

Journal Pre-proof CA1 CA2 CA3 CC1

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C

CS3

PC

CC3

NC

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CS1 CS2

CC2

-p

Figure 10. Hemolysis ratio for native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline

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sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- Temperature- 25 ºC,

lP

Time- 1-6 hrs)

3.5.5 Changes in the attachment of platelets in native collagen and aqueous solution of

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CILs treated collagen scaffolds assessed via platelet attachment assay

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For an artificial skin substitute to be successful it should facilitate the process of wound healing

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and activation of platelets is a significant step in the healing process. Post incubation of native collagen and aqueous solution of CILs treated collagen scaffolds with platelet rich plasma (PRP), they were observed under SEM to appraise the attachment of platelets on scaffolds. From the figure 11, it was witnessed that the attachment of platelets to the aqueous solution of CILs treated collagen scaffolds were on par with native collagen scaffolds. Aqueous solution of CILs treated collagen scaffolds displayed platelets with pseudopodia, thus confirming the platelet activation. As it is well-known that the platelet activation is a key step in the healing process for the release of chemical signals, which is required in the succeeding steps54. From the platelet attachment assay, it is apprehended that the aqueous solution of CILs in the scaffolds didn’t

28

Journal Pre-proof affect the hemocompatibility of the collagen and these results are in complementary to the hemolysis ratio, which is below the permissible limits of the ASTM standards for evaluation of

CA1

CA2

CA3

CC2

CC3

CC1

-p

ro

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CONTROL

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scaffolds.

lP

CS1

CS2

CS3

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Figure 11. Platelet attachment for native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline

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sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- Temperature- 25 ºC)

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3.5.6 Changes in the scavenging activity of native and aqueous solution of CILs treated collagen assessed via DPPH assay Presence of free radicals will cause damage to the proliferating cells. So, it is very significant for a biomaterial to have antioxidant property, which will aid in faster wound healing. From the DPPH assay (figure 12), it was observed that the native collagen possess 12% of free radical scavenging activity. This free radical scavenging activity of the collagen could be owing to the presence of Gly-Pro sequences55. From the results, it’s clearly evident that the antioxidant activity of the scaffolds was increased with the increasing concentrations of the aqueous solution

29

Journal Pre-proof of choline-based IL treated collagen scaffolds (CILs). It has been reported in the literature that these CILs have potential antioxidant activity. The presence of –NH and –OH groups in the CILs were predicted to involve in the free radical scavenging activity47. All these aqueous solution of CILs (choline-based IL scaffolds) showed better antioxidant activity at higher concentrations (1:1). Choline acetate treated collagen scaffolds (CA) exhibited 45% of free radical scavenging activity, whereas choline citrate (CC) and choline sulfate (CS) treated collagen scaffolds evinced

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thus can be employed for various biomedical applications.

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38.6 and 43.5 %, respectively, which is comparably higher to that native collagen scaffolds and

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Figure 12. Scavenging activity of native collagen scaffolds and aqueous solution of a. choline

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acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Conditions- Temperature- 25 ºC) 3.5.7 Changes in the cytotoxicity of native and aqueous solution of CILs treated collagen assessed via MTT assay Biocompatibility is one of the major prerequisites for the preparation and development of biomaterials for its potential use in biomedical applications such as drug delivery and wound management56. It is requisite to assess the cytotoxicity of the prepared CILs treated scaffolds and it was ascribed using MTT assay. NIH-3T3 fibroblast cells were used for evaluating the

30

Journal Pre-proof biocompatibility of aqueous solution of CILs treated collagen scaffolds. As seen from figure 13, it evinced no significant changes among aqueous solution of CILs treated collagen scaffolds compared to native collagen scaffolds. The cells were found to be viable even after 48 hrs and the incorporation of aqueous solution of CILs into collagen scaffolds didn’t affect the viability of cells, thus demonstrating a non-toxic behavior when compared to native collagen scaffolds. In addition to, the cells were found to be unaffected and viable even after the incorporation of

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aqueous solution of CILs to the scaffolds and it was observed from figure 14. It was observed

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that the cells showed better adhesion on the aqueous solution of CILs treated collagen scaffolds.

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The presence of moderate hydrophilic groups provides better cell adhesion and thus the cells

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adhere, spread and grow on hydrophilic surfaces than on hydrophobic ones. From the above results, it is clear that aqueous solution of CILs treated collagen scaffolds might render adhesion

lP

to cells, which in turn might enhance cell proliferation. Since, they haven’t exhibited any

B

Jo

ur

A

na

reduction in cell viability; they can be employed for biomedical applications.

Figure 13. Biocompatibility studies for native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C: CC)), c.

31

Journal Pre-proof choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds, (Concentration- 0.8 µM, Time- 24 and 48 hrs) CA1

CC2

CC3

CA2

CA3

CC1

CS2

CS3

-p

ro

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CONTROL

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CS1

lP

Figure 14. Morphology of NIH 3T3 fibroblast cells on native collagen scaffolds and aqueous solution of a. choline acetate (1:0.05, 1:0.5, 1:1 (C: CA)), b. choline citrate (1:0.05, 1:0.5, 1:1 (C:

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CC)), c. choline sulfate (1:0.05, 1:0.5, 1:1 (C: CS)) treated collagen scaffolds

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Proposed mechanism behind the study:

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The interaction between the collagen and aqueous solution of choline-based ILs (CILs) implicates the non-covalent interactions comprising of electrostatic and hydrogen bonding. It is likely that the hydrogen bonds might form between the hydroxyl molecules of choline and hydroxyl groups of collagen (OH…OH). It is postulated that the hydrogen bonds can be formed between the hydroxyproline residues of collagen and anions of CILs (acetate, citrate and sulfate) (OH…OH), whereas electrostatic forces prevails between amine functional group of collagen and anions of choline-based ILs. There can be probable interactions between the arginine and histidine residues of collagen (positively charged residues) to that of ILs are high, as they are

32

Journal Pre-proof readily available for bonding4. Therefore, the minor conformational changes in the helices of collagen has been reported due to the electrostatic interactions on the surface of collagen that stabilizes protein. This might be owing to charge-pair interactions between acidic and basic amino acid moieties of collagen, thus facilitating the intra- and intermolecular interactions by reorientating the surrounding water milieu, thereby providing stability to the collagen by forming crosslinks.

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Conclusions

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This study examines the effect of aqueous solution of choline-based ILs (CILs) on collagen

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signifying the electrostatic interactions. NMR measurements brought out the changes in the

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hydration network surrounding collagen indicating stability in structure and the dielectric studies revealed the reorientation in surrounding water milieu complementing the NMR measurements.

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Witnessing increase in the melting and denaturation temperature of collagen as a function of

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concentration of aqueous solution of CILs also indicated stability and compactness in structure of collagen. In addition to this, collagen-CILs (aqueous) solutions evidenced increased cross-

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linking, increased enzymatic stability and non-cytotoxicity. These properties are significant and

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ideal for a cross-linker, which can be readily used in bioimplants and thus, contributing to the identification and development of whole new class of biocompatible cross-linkers for collagen. ACKNOWLEDGEMENT One of the authors, AT acknowledges Council of Scientific and Industrial Research (CSIR) for Senior Research Fellowship. Authors also thank Dr. B. V. N. Phani Kumar and R. Ravikanth Reddy, CSIR-CLRI for 2H NMR measurements and discussions. CSIR-CLRI communication code: 1310.

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Journal Pre-proof Author Information Corresponding Author Tel.: +91 44 24437137, +91 44 24437188 E-mail addresses: [email protected], [email protected] (N.N. Fathima) ABBREVIATIONS CIL, Choline-based Ionic Liquid; CD, Circular Dichroism; CA, Choline Acetate; CC, Choline

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Citrate; CS, Choline Sulfate

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2. Tarannum, A.; Rao, J. R.; Fathima, N. N. Stability of Collagen in Ionic Liquids: Ion Specific Hofmeister Series Effect. Spectrochim Acta A 2019, 212, 343-348.

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3. Tarannum, A.; Muvva, C.; Mehta, A.; Rao, J. R.; Fathima, N. N. Phosphonium based Ionic Liquids- Stabilizing or Destabilizing Agents for Collagen? RSC Adv. 2016, 6, 4022-

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4. Tarannum, A.; Muvva, C.; Mehta, A.; Rao, J. R.; Fathima, N. N. Role of Preferential Ions of Ammonium Ionic Liquid in the Destabilization of Collagen. J. Phys. Chem. B 2016, 120, 6515-6524. 5. Tarannum, A.; Rao, J. R.; Fathima, N. N. Choline-Based Amino Acid ILs-Collagen Interaction: Enunciating their Role in Stabilization/Destabilization Phenomena. J. Phys. Chem. B 2018, 122, 1145-1151. 6. Meng, Z.; Zheng, X.; Tang, K.; Liu, J.; Ma, Z.; Zhao, Q. Dissolution and Regeneration of Collagen Fibers using Ionic Liquid. Int. J. of Biol. Macromol. 2012, 51, 440-448.

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9. Bernot, R. J.; Brueseke, M. A.; Evans-White, M. A.; Lamberti, G. A. Acute and Chronic

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10. Zeisel, S. H.; Da Costa, K. A.; Franklin, P. D.; Alexander, E. A.; Lamont, J. T.; Sheard,

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Beiser, A. Choline, an Essential nutrient for Humans. Faseb J. 1991, 5, 2093-2098.

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11. Zeisel, S. H.; Da Costa, K. A. Choline: an Essential Nutrient for Public Health. Nut. Res.

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Declaration of competing interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Journal Pre-proof CRediT authorship contribution statement

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Aafiya Tarannum: Conceptualization, Methodology, Investigation, Writing - original draft, Writing – Review and Editing Nitin P. Lobo: Supervision, Investigation, Validation J. Raghava Rao: Supervision, Methodology, Resources N. Nishad Fathima: Supervision, Methodology, Resources, Writing- Review & editing, Validation

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Journal Pre-proof Highlights  Identification of choline-based ILs as biocompatible cross-linkers for collagen  Choline-based ionic liquids treated collagen solutions indicated stability and compactness in structure.  Choline-based ionic liquid treated collagen scaffolds showed high crosslinking efficiency and enzymatic stability  The scaffolds were found to be hemo- and cytocompatible.  These properties are significant and ideal for a cross-linker and can be readily used in

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bioimplants.

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