Electrostatic Forces Mediated by Choline Dihydrogen Phosphate

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Electrostatic Forces Mediated by Choline Dihydrogen Phosphate Stabilize Collagen Ami Mehta, Jonnalagadda Raghava Rao, and Nishter Nishad Fathima J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07055 • Publication Date (Web): 19 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015

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The Journal of Physical Chemistry

Electrostatic Forces Mediated by Choline Dihydrogen Phosphate Stabilize Collagen Ami Mehta, J Raghava Rao, N Nishad Fathima

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Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India

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Author to whom correspondence should be made Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India, Tel: +91 44 24411630; Fax: +91 44 24911589 E-mail: [email protected]; [email protected] 1

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ABSTRACT: Crosslinkers aid in improving biostability of collagen via different mechanisms. Choline dihydrogen phosphate (cDHP), a biocompatible ionic liquid has been reported as a potential crosslinker for collagen. However, its mechanism is yet unclear. This study explores the effect of cDHP on the physico-chemical stability of collagen and nature of its interaction. Dielectric behavior of collagen-cDHP composites signifies that cDHP enhances intermolecular forces. This was demonstrated by increase in crosslinked groups and high denaturation temperature of collagen-cDHP composites. XRD measurements reveal minor conformational change in helices. Molecular modeling studies illustrate that the force existing between collagen and cDHP is electrostatic in nature. Herein, it is postulated that dihydrogen phosphate anion attaches to cationic functional groups of collagen, resulting in closer vicinity of various side chains of collagen, forming physical and chemical crosslinks within collagen, contributing to its structural stability. Our study suggests that dihydrogen phosphate anions can be employed for developing a new class of biocompatible crosslinkers. Keywords: Collagen; choline dihydrogen phosphate; crosslinking; electrostatic force; dielectric spectroscopy

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1. INTRODUCTION Ionic liquids (ILs) are new age materials currently employed in the field of proteomics for their unique property of solubilisation and stabilization1. The interaction of imidazoliumbased ILs in particular, 1-butyl, 3-methylimidazolium chloride (Bmim Cl), has been well studied for various proteins2-4, including type I collagen at different hierarchical orders5. Bmim Cl does not influence collagen at the molecular level, however, at the skin matrix level, it has demonstrated opening up of pores and dissolution of the matrix at higher temperatures6-7. Imidazolium-based ILs show stabilizing effect for certain proteins, while it destabilizes others. In addition to this, imidazolium-based ILs is reported to have cytotoxicity concerns in human cell lines and aquatic organisms8-9. In view of preparing biomaterials, there is a dire need to explore other classes of ILs, which can stabilize protein, lack cytotoxicity concerns and are similar to nutrients present in our body. Therefore, whole new classes of ILs are being developed that have compounds with biocompatible cations, viz., amino acid ILs10 and choline salts11. Hydrophilic phenylalanine ILs have been reported to reduce aggregation of globular protein, myoglobin at low concentrations through protein-protein interactions12. In case of choline salts, choline dihydrogen phosphate (cDHP) has been studied for various proteins like cytochrome c, collagen, lysozyme and interleukin-213-16. cDHP also known as (2-hydroxy-ethyl) trimethylammonium dihydrogen phosphate has cation choline (C5H14NO+) and anion, (H2PO4¯) bonded mainly via two hydrogen bonds and van der Waals’ forces. In addition to this, choline based ionic liquids have also been reported for pharmaceutical applications, providing improved and stabilized solvent for protein therapeutics17. This signifies that choline cation can be employed for preferring biocompatibility over other cytotoxic ionic liquids such as imidazolium.

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The advantage of biocompatible ILs over other classes of ILs is that they have immense potential to design biomaterials applicable for tissue engineering and wound healing. Collagen, triple helical in structure is stabilized by inter- and intra-chain hydrogen bonds and structural water molecules18. Collagen-based biomaterials have always been of focus in reconstructive medicine, especially in tissue engineering19. However, biodegradation rate and mechanical stability of biomaterials is not up to mark for many in vitro and in vivo applications. Strengthening of collagen for biomedical applications can be achieved by addition of various crosslinking agents, which provides biostability, i.e., stability towards conformation, thermal and enzymatic activity. Several crosslinking agents like iron20, zirconium21, aldehydes like formaldehyde and glutaraldehye22, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)23 and biocompatible crosslinker, genipin24. Glutaraldehyde is the most convenient and traditional crosslinker. However, due to cytotoxicity concerns, the use of glutaraldehyde has been discontinued and genipin has been preferred for crosslinking25. Most of these crosslinking agents react with the carboxyl groups of amino acids like glutamic acid and aspartic acid and other amine groups to form amide bonds26. Genipin offers spontaneous crosslinking reactivity through dimerization27. Irrespective of crosslinking method, these crosslinking agents produce a two point connection with collagen, bearing crosslinking products with certain structural similarities. It is likely that cDHP stabilizes the triple helical structure of collagen through protein-protein interactions or crosslinking. However, no clear mechanism has been illustrated yet. This paper aims at understanding the mechanism of action of biocompatible IL, choline dihydrogen phosphate with type I collagen. This study also compares another biocompatible crosslinker, genipin with choline dihydrogen phosphate, in order to understand the effect of stabilization provided by the biocompatible IL. 4


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2. EXPERIMENTAL 2.1 Materials. Ionic liquid, choline dihydrogen phosphate (cDHP) was purchased from Io-li-Tech, GmBH, Germany and used without further purification. Ninhydrin, collagenase from Clostridium histolyticum, picrylsulfonic acid (2,4,6 – trinitrobenzenesulfonic acid or TNBS) solution were purchased from Sigma Aldrich India. Millipore water was used for all experimental assays. 2.2 Isolation of Acid-Soluble Type I Collagen. Type I collagen was obtained by teasing rat tail tendons (RTT) of 6 month old male albino rats (Wistar strain) at 4 ºC. Collagen was extracted as described earlier5. Briefly, RTTs were washed thoroughly with distilled water and solubilized using 0.5 M acetic acid and then dialysed extensively against 50 mM phosphate buffer. The concentration of collagen was determined by the amount of hydroxyproline content released using Woessner method28. Purity of collagen solution was confirmed using SDS-PAGE electrophoresis. The stock collagen solution was diluted using 50 mM acetate buffer (pH 4.0) for experimental assays. The collagen solution (MW 300 kDa) was stored at 4 ºC. 2.3 Preparation of Collagen-cDHP Composites. Four different concentrations of choline dihydrogen phosphate, 0.05% (w/v) - C-CH1; 0.5% (w/v) - C-CH2; 5% (w/v) - C-CH3; 10% (w/v) - C-CH4 were considered in this study to determine the influence of cDHP on 0.15 mg/mL (or 0.5 µM), type I collagen. A total volume of 20 mL was prepared by adding varied volumes of choline dihydrogen phosphate drop wise to collagen and mixed homogenously by stirring continuously for 2 h. The final concentration of collagen was 0.15 mg/mL at pH 4.0. 0.05% (w/v) genipin added to 0.15 mg/mL collagen (CG – collagen-genipin composite) and considered as positive control for this study. The temperature was maintained at 4 °C constant throughout the preparation by cooling system, therefore preventing denaturation of collagen due 5



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to heat generated during mixing. The solution was then incubated at 4 °C overnight, for completion of reaction. For SEM and XRD measurements, collagen-cDHP composites were lyophilized under the pressure of 48 mTorr at -40 °C. For measuring the hydrothermal temperature, collagen-cDHP and collagen-genipin composites were poured on polyethylene plates and air dried by placing in laminar air flow hood. The membranes were peeled easily and used for measuring the denaturation temperature using microshrinkage tester29. 2.4 Physico-Chemical Characterization Degree of Crosslinking of Collagen - TNBS Assay. The degree of crosslinking of genipin and choline dihydrogen phosphate with collagen was measured using TNBS acid to determine the ε-amino groups present in collagen. The lysine content before and after crosslinking was estimated to calculate crosslinking efficiency. Collagen-cDHP composites were prepared 24 h in prior for crosslinking to take place. To one mL of this solution, 1 mL of 4% sodium bicarbonate solution and 1 mL of 0.5% (v/v) TNBS solution was added. The tubes were then placed in hot water bath at 60 ºC in dark for 4 h. 3 mL of 6 N HCl was added to the solution and treated at 40 ºC for 90 min. Absorbance of the resulting brown solution was measured at 346 nm after dilution. The non-crosslinked sample was treated in a similar manner using TNBS solution. The degree of crosslinking was calculated by the difference in absorbance divided by the absorbance of the native. Degree of Biodegradation - Collagenolytic Assay. To one mL of collagen-cDHP composites taken in test tubes, 1% of collagenase enzyme solution (50:1 collagen: enzyme ratio) prepared in 25 mM collagenase buffer (0.04 M CaCl2, 0.05 M Tris-HCl, pH 7.2) was added. After 6 h, 0.2% ethanol solution of ninhydrin was added to the mixture and the tubes were placed in water bath at 100 °C for 20 min. Subsequently, the solution was diluted three times (with 9 6


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mL Millipore water). The absorbance was then measured at 570 nm and the degree of biodegradation was calculated as follows,

% collagen degradation = 100 - [

* 100]

(Equation 1)

Dielectric Behavior. AC impedance analysis was performed using CH Instrumental (USA) electrochemical analyzer CH- model 660B with a classical three-electrode system, glassy carbon electrode was used as a working electrode, platinum as the reference and a saturated calomel electrode as counter electrode was used. Prior to measurements, N2 was purged to the samples for 10 min to remove air bubbles. The experimental operating conditions were initial E (V) = 0.09, high frequency (Hz) = 105, low frequency (Hz) = 0.01, amplitude (V) = 0.005, quiet time (sec) = 2, cycles (0.1-1 Hz) = 1, cycles (0.01-0.1 Hz) = 1, cycles (0.001-0.01 Hz) = 1. Impedance Z (Ω) is measured as the voltage drop30. Dielectric data has been represented in terms of admittance Y (S), which is a conjugated quantity. The measurements were performed in triplicate. The impedance (unit Ω) is given by the expression

(Equation 2)

where, Z is the total (complex) impedance, Z' is the real part, Z″ is the imaginary part of the complex impedance, R is the resistance, and X is the reactance response to an alternating signal. The magnitude of the complex impedance and the phase angle (θ) include the complex impedance expressed as Equation 2.3

(Equation 3)

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(Equation 4)

The dielectric loss tangent/ dissipation factor for collagen-cDHP composites with different additive concentrations has been calculated using this Equation (5),

(Equation 5)

The admittance Y (unit S) (inverse of impedance) is written as (Equation 6)

Where Y' is the real component describing the energy stored and Y″ is the imaginary component depicting the energy dissipated by the system. The relaxation time is related to fmax, the frequency that gives the maximum magnitude of Y’’ as follows,

(Equation 7)

XRD Measurements. XRD measurements of lyophilized collagen composites with/without choline dihydrogen phosphate were recorded between 5° to 40° (2θ value) at a scan rate of 2 °min-1. XRD measurements were carried out on powder XRD (PANalytical) using Cu Kα X-rays of wavelength 1.54 Å. The voltage and current applied were 40 kV and 40 mA respectively. The obtained data were also compared with standard data of collagen. Intense peaks for collagen were obtained at 2θ = 7.5 and 20.531.

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2.5. Statistical Analysis. All experiments were performed in triplicates (n = 3) and the means were compared using Students’t-test using GraphPad Instat Version 3.06 2.6 Molecular Modeling Studies. Preparation of Protein. As collagen is a macromolecule, the number of repeating units was restricted for the purpose of modeling and simulation studies. Non-protein species such as ligands and excess water molecules causing steric hindrance to protein-ligand interaction were removed. Charge of 2.977 was added to protein by Kollman scheme. Kollman united atom charges and polar hydrogens were added to the protein PDB using Autodock tools. The peptide sequence for collagen used for modeling in this study is, GEYGPPGPPGPPGPPGPAGAKGPAGNYGADGQY GEYGPPGPPGPPGPPGPAGAKGPAGNYGADGQY GEVGLYGLSGPPGPPGPVGPPGPPGNAGPNGLY This proposed sequence was adapted from PDB ID: 1CAG32 and developed by Object Technology Framework (OTF) using the GENCOLLAGEN package33. This sequence was chosen for modeling, as it contains the amino acid residues, which usually play a significant role in interacting with the additives and alter the microenvironment of collagen. The purpose of conducting molecular modeling studies was to look into the interaction between collagen and cDHP by determining the exact location of binding to amino acid, in case if any binding occurs. Preparation of Ligand. The pdb file of choline dihydrogen phosphate was acquired from ChemSpider, Royal Society of Chemistry. Six active rotatable torsions from choline dihydrogen phosphate were observed that can readily interact with protein. 9



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Docking Setup. Docking was performed using Autodock v. 4.2.6. An excellent feature of Autodock is to combine energy evaluation using pre-calculated grids of affinity potential for protein-ligand interaction using various search algorithms. Flexible docking was initiated by keeping all rotatable bonds in the ligands free. Grid size was set to 40 × 40 × 40 grid points (x, y and z), with spacing between grid points kept as 0.375 Å. The Lamarckian genetic algorithm was chosen to search for the best conformers. Standard docking protocol was applied34. Twenty independent docking runs were carried out by employing genetic algorithm searches.

3. RESULTS AND DISCUSSION Rheology. The rheological behavior, i.e., viscosity of liquid is given by the resistivity of the flow due to high shear strength. This reflects the strength of inter- and intra-molecular forces present in the protein molecule. Proteins in liquid medium tend to get closer resulting in entanglement, thus giving high shear strength35. The dependence of rheological behavior of collagen on cDHP concentration is given in Figure 1.

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Figure 1. Rheological behaviour of collagen - cDHP composites at 20°C. Absolute viscosity showed an increasing trend. Concentration of collagen = 0.15 mg/mL, CG – collagen:0.05 genipin and C-CH1-4 = collagen: cDHP = 1:0.05-10. Data represents mean ± SEM. Student’s ttest was used for statistical analysis, where n = 3 and p < 0.05 The absolute viscosity determined using viscometer for native collagen was found to be 1.4 cP. The viscosity of collagen treated cDHP composites was found to significantly increase linearly with concentration of cDHP (p < 0.05, n = 3). The viscosity of C-CH1 is similar to CG. Such rheological behavior of collagen due to the presence of additive suggests aggregation of collagen with additive. This kind of aggregation is generally attributed to the increase in intra- as well as inter-molecular forces, such as H-bonding, induced dipole interactions, close proximity of functional groups of collagen, electrostatic charge-charge repulsion between collagen, cDHP and surrounding water molecules, therefore restricting the mobility of collagen-cDHP composites, resulting in increased viscosity and integrity. 11



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Electronic

Absorption

and

Emission

Spectral

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Studies

of

Collagen-cDHP

Composites. In the presence of additive, minute alterations are likely to occur in the structure of collagen, which can be preliminary screened by UV-visible spectroscopy. Collagen has two absorption maxima, at 200 nm and 278 nm. A strong absorption peak at 200 nm reflects the change in the aggregation or folding of collagen in the presence of additive, whereas the weak peak at 278 nm indicates the microenvironment changes, π-π* electron transition of aromatic amino acids36. At 278 nm, the absorbance is primarily due to tyrosine residues, as tryptophan is absent in type I collagen and the absorbance of phenylalanine is negligible.

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Figure 2A. UV-Vis spectra of collagen-cDHP composites, inset showing the trend of absorbance by tyrosine residues in collagen at 278 nm due to presence of additive, and B. Fluorescence spectra of collagen-cDHP composites at 300 - 400 nm and C. at 450 - 600 nm with concentration of collagen = 0.15 mg/mL, CG – collagen:0.05 genipin and C-CH1-4 = collagen: cDHP = 1:0.05-10.. Data in Fig 2A (inset) represents mean ± SEM. Student’s t-test was used for statistical analysis, where n = 3 and p < 0.0018 Figure 2A shows that the electronic absorption spectra of native collagen and cDHP are similar and the amount of absorption varies only at 200 nm. No signature peak was observed on adding cDHP to collagen. At 200 nm, increase in absorbance of collagen-cDHP composites was found to be concentration dependent of cDHP. Molecular collision does not demonstrate change in absorption spectra whereas complex formation often results in change in absorption spectra37. Therefore, increased absorbance can be attributed mainly to helix-coil transition owing to increased inter- and intra-molecular forces between collagen and cDHP. In an intact helical structure of type I collagen, a majority of tyrosine residues (~12) are present in telopeptide region and ~2 residues are present in the helical region38. These tyrosine residues play a significant role in crosslinking of collagen by forming complexes either between telopeptides of two collagen molecules or between telopeptide of one collagen and helical domain of another. Although, they cannot be classified as a crosslinker marker, but it aids in predicting the microenvironment changes that occur in type I collagen in the presence of additive. Lysine and hydroxylysine have negligible UV absorption; hence they were not considered a part of this study. Changes in the absorbance of tyrosine residue detected at 278 nm are shown in Figure 2A (inset). It is expected that the absorbance of tyrosine residues is low, due to minimum number of residues present in collagen. Pooled analysis shown in Figure 2A inset demonstrated a significant 13



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~65% increase in intensity of tyrosine due to addition of cDHP (p < 0.0018, n = 3). The addition of cDHP results in alterations in the microenvironment occurring due to the chromophores of collagen. It is likely that in the presence of cDHP, tyrosine residues interact with the additive resulting in subtle disturbance in the microenvironment of polypeptide chain preferably through crosslinking or exposure of tyrosine residues from the hydrophilic to hydrophobic core in order to interact with various functional groups of neighboring collagen molecule. This also explains the aggregation observed at 200 nm. Increased motility of tyrosine residue was also observed in the presence of genipin, a biocompatible crosslinker, indicating that genipin induces microenvironmental changes such as crosslinking within collagen. Collagen contains one intrinsic fluorophore, tyrosine residues. The environment surrounding the fluorophore responds to the excited state of tyrosine by stabilizing the excited dipoles and resulting in red shifted emission spectra. The electronic emission spectra of tyrosine amino acids present in collagen is shown in figure 2B. The emission intensity of tyrosine residues at 305 nm of collagen-cDHP composites (C-CH1 to C-CH-4) shows fluorescence quenching and red shift (305-309 nm) in the presence of cDHP. Compared to native, intensity of CG composite is also reduced. This indicates that additives induce alteration, which can be attributed to microenvironment changes within collagen as a result of increased molecular vibration and rotations. There was low or lack of emission intensity for cDHP ionic liquid. Fluorescence quenching of collagen-cDHP composites is ascribed to various molecular interactions, namely, conformational changes, molecular collisions and complex formations through crosslinking. The decrease in fluorescence intensity suggests significant exciton deconfinement of tyrosine residues in collagen. This is owing to the collisional deactivation of the fluorescence state due to short-range specific interactions like H-bonding. H-bonding at N-H 14


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site could result in alteration of intra-molecular charge transfer by hindering the formation of electron density on nitrogen heteroatom39-40. This leads to deactivation of fluorescence emission owing to the changes in the surrounding collagen fluorophores inducing dissolution or aggregation of collagen. Such microenvironment changes result in bringing the amino acids of collagen molecule closer, thereby promoting increased inter- and intra-molecular forces within collagen. Furthermore, at higher wavelengths of emission, Figure 2C reveals increased intensity of collagen-cDHP composites compared to native. At higher wavelength, complex products, which are formed through intermolecular crosslinks through interaction of tyrosine, lysine and histidine residues of collagen with that of neighboring molecule in the presence of additive. This concentration-dependent intensity can be generally attributed to the increased formation of crosslinked products in the presence of cDHP, indicating that cDHP promotes formation of interand intra-molecular crosslinks. Degree of Biodegradation, Crosslinking Efficiency and Hydrothermal Stability of Collagen-cDHP Composites. Collagen is enzymatically cleaved using collagenase enzyme under in vitro conditions. Bacterial collagenase enzyme was considered in this study, as the objective of this study was to determine the influence of choline dihydrogen phosphate on degradation of collagen. The biodegradation of collagen can be studied spectrophotometrically using ninhydrin which labels free primary and secondary amines. The degree of biodegradation of collagen-cDHP composites was also measured.

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Figure 3. Crosslinking, thermal and enzymatic studies of collagen-cDHP composites A. Collagen with additives show concentration-dependent increased degree of crosslinking efficiency using TNBS assay. B. Increase in ~20 °C was observed in hydrothermal denaturation temperature, Td for collagen-cDHP composites. C. Degree of biodegradation by collagenolytic hydrolysis of collagen-cDHP composites. Data represents mean ± SEM. Student’s t-test was used for statistical analysis, where n = 3 and 0.001 < p > 0.5

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TNBS colorimetric assay determines the residual amine groups of collagen where crosslinking occurs. It can be observed from figure 3A that addition of genipin results in crosslinking of about 30% of functional side chain groups of collagen. Furthermore, increase in crosslinking of collagen molecule was observed to be concentration dependent of cDHP. There is a remarkable three-fold increase in percentage of crosslinked groups (p < 0.001, n = 3). About, 89% crosslinked groups for 5% and 10% cDHP (C-CH3 and C-CH4) were observed. Indeed, crosslinking levels off for high concentration of cDHP. This could be because higher crosslinking may result in entanglement/folding of the helical domain leading to compactness. This may mask the functional groups of amino acid residues such as tyrosine, lysine and histidine, which are capable to form complex crosslinked products within collagen or with neighboring collagen molecule. Corresponding to increase in crosslinking groups, increase in the denaturation temperature was also observed for collagen-cDHP membranes (Figure 3B). The hydrothermal temperature for native collagen is about ~63 °C. A significant increase in about 20 °C was observed after the addition of cDHP (p < 0.05). This increase in hydrothermal temperature is attributed to the strengthening of the inter- and intra-molecular forces between collagen and cDHP. Higher the H-bonding, higher will be the compactness of protein and higher the shrinkage temperature. Figure 3C explicits that native collagen shows high percentage of biodegradation (~99%) indicating collagen being completely degraded by collagenase enzyme. This is expected, as under in vitro conditions, the collagen loses its mechanical integrity in the absence of crosslinkers or any other such additives. However, in the presence of additives, genipin and choline dihydrogen phosphate, the degree of biodegradation was reduced to ~ 65%. Further addition of cDHP, has reduced significantly to ~ 7.7% (p < 0.001, n = 3). This signifies that the 17



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cDHP enhances the physical and chemical crosslinks at higher concentration, resulting in strengthening of the collagen molecule in the presence of higher concentrations of cDHP. From these observations, it can be observed that in the presence of cDHP, different forces come into interplay between collagen, cDHP and the surrounding water molecules. As a result of which, both physical crosslinks (H-bonding and other weak inter- and intra-molecular forces) as well as in chemical crosslinks (increased crosslinking efficiency) were observed. Increased H-bonding is caused primarily due to alteration in the surrounding medium, such as alteration in dynamics of water, conformational change of the poly-proline type II structure of collagen in the presence of additive. Such alteration in H-bonding is observed in the presence of water molecules. To further understand the nature of these intermolecular studies, other characterizations like bioimpedance spectroscopy and XRD analysis were performed. Bioimpedance Spectroscopy of Collagen-cDHP Composites. Dielectric relaxation phenomenon measured by dielectric spectroscopy is a highly sensitive tool for exploring the dynamics of water surrounding collagen, confirming the structural stability of protein. The changes in the dielectric behavior of collagen-cDHP composites provide deeper insights on the solute-solvent interaction and hydration behavior of collagen. The polar as well as non-polar functional groups of the macromolecules are drifted and displaced apart in response to an applied electric field, inducing conduction and polarization effect41. Presence of choline dihydrogen phosphate at the interface will affect the reorientation of polar and non-polar functional groups, thereby influencing number and strength of the electric dipoles of collagen-cDHP composites. It has been reported that the presence of protons as charge carriers greatly influences the polarization and conduction processes42-43. In that case, choline dihydrogen phosphate will have the highest permittivity (Y’’) as it has electron rich, N+ group present in the choline cation. The 18


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change in electric dipole reflects the mobility of the functional groups, which in turn predicts structural stability of the collagen.

Figure 4. Dielectric behaviour collagen-cDHP composites at 25 °C, respectively. A. Nyquist plot: real part admittance (Y’) versus imaginary part admittance (Y”). B. Cole-Cole plot: real part impedance (Z’) versus imaginary part imedance (-Z”). C and D. Bode plot: changes in the log (Z/ohm) and –phase as a function of log (Freq/Hz). Dielectric relaxation profile of collagen-choline dihydrogen phosphate composites can be seen in Figure 4. Figure 4A shows the Nyquist plot (real Y’ vs. imaginary Y’’) for determining the admittance of biological materials and figure 4B shows the Cole-Cole plot depicting the 19



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complex dielectric impedance plane plotted using real Z’ vs. imaginary Z’’. From the Nyquist and Cole-Cole plot, it can be observed that native collagen has the lowest permittivity. All collagen-CDHP composites show concentration dependent increased permittivity at low frequencies compared to collagen alone. This increase in Y’’ with an increase in the concentration of additive can be attributed to the greater number of proton charge carriers and sites available for the protein to accumulate and pass the current. Increased flow of electric current implies increased dipole sites, which is typical of proton polarization and conduction processes. These sites are created by newly formed intra- and inter-molecular crosslinks such as H-bonds, H-bond water, van der Waal’s forces and hydrophobic forces that enhances the stability of triple helical collagen. The difference in Y’’ of native collagen and collagencDHP/collagen-genipin composites, due to increased flow of electric current in collagen-cDHP composites compared to native is owing to proton accumulation at the phase interface. This indicates that collagen-cDHP composites have greater ability for proton transport through the newly formed intra- and inter-molecular forces and also to store polarizable charges at the phase interface. This signifies physical/chemical crosslinking of collagen44. It can be observed that collagen-genipin (CG) composites show dielectric behavior similar to that of C-CH1, suggesting that dimer formation of genipin crosslinking collagen brings in equivalent alteration in dipoles surrounding collagen. Bode plot (Figure 4C) of collagen shows a phase angle at high frequency of about 103 Hz. In the presence of additives, phase angle is increased to higher frequencies from 103 – 105 Hz. Shift to higher phase angle signifies that the permittivity component of their admittance dominates. Higher phase angle, θ exhibited by C-CH3 and C-CH4 corresponds to decreased tan δ (see equation 4 and 5), which indicates increased dielectric loss or high dielectric absorption by 20


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collagen-cDHP composites. This change in phase angle with frequency owing to addition of cDHP is possibly due to electrostatic interaction of charged functional groups of collagen with cDHP, resulting in increased physical crosslinks and/or covalent-like bonds. A plot of log Z vs. Freq (Hz) shows that collagen-cDHP composites have increased admittance at low frequencies (Figure 4D). Increased permittivity results in lower impedance, i.e., polarity. This indicates that the dielectric behavior of collagen-cDHP is primarily due to charge hopping between discrete sites of the functional groups of collagen and phosphate anion of cDHP, which mediates charge movements. This can be attributed to electrostatic or other non-covalent interactions due to which there is a high probability of charge transfer between two adjacent amino acids, leading to high coupling strength. The charge transfer could be due to electrostatic interaction of phosphate group with the arginine residues of collagen resulting in formation of H-bonds, alteration in the hydration shell around collagen. In short, cDHP induces electrostatic interactions, generating increased H-bonding network within the helices of collagen. Relaxation time, τ was calculated from the maximum frequency obtained for collagencDHP composites (see equation 7). Relaxation time for any composite is the time required for the molecule to relax after electric field is applied. The primary factor that determines the relaxation time is the density of hydrogen bonding partners, i.e., additives present in the solution along with macromolecule.

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Figure 5. Relaxation time profile of collagen-cDHP composites calculated from the maximum frequency obtained for concentration of collagen = 0.15 mg/mL, CG – collagen:0.05 genipin and C-CH1-4 = collagen: cDHP = 1:0.05-10. Data represents mean ± SEM. Student’s t-test was used for statistical analysis, where n = 3 and p < 0.036 Figure 5 presents the relaxation time profile of collagen-cDHP composites. It can be observed that there is an increase in relaxation time in collagen-cDHP composites C-CH1 and CCH2 compared to native. With the further addition of cDHP, there is a remarkable decrease by five fold in the relaxation time profile of C-CH3 and C-CH4 (p < 0.036, n = 3). This kind of exquisite behavior indicates that the collagen is restricted in translational and rotational motion at 0.5% cDHP (C-CH2). With an increase in concentration of the additive, the decrease in relaxation time can be attributed to alteration in the hydrogen bonding density by the increased hydroxyl groups from the choline cation. This alteration brings about reorientation in the dipole moments of collagen, which in turn alters the polarity.

22


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The changing polarity of collagen-cDHP composites is suggested from the refractive indices measured in Table 1. It can be observed that the refractive index of collagen-cDHP composites increases with an increase in concentration of cDHP, with C-CH4 having the maximum refractive index. This trend statistically suggests that the polarity is being shifted from hydrophilic to hydrophobic, resulting in compactness of collagen macromolecule (p < 0.0004). Refractive indices of collagen-cDHP composites at lower concentration correlated with their relaxation times, which suggest compactness of collagen at 0.5% cDHP con

centration, i.e.,

C-CH2. Table 1. Refractive Indices of Collagen-cDHP Composites Using Refractometer Composites

Weight Ratio (Collagen: additive)

Refractive Index at 25 °C

1:0 Native 1.3319 ± 10-4 1: 0.05 CG 1.3321 ± 10-4 1:0.05 C-CH1 1.3322 ± 10-4 1:0.5 C-CH2 1.3324 ± 10-4 1:5 C-CH3 1.3327 ± 10-4 1:10 C-CH4 1.3330 ± 10-4 Data in Table 1 represents mean ± SEM. Student’s t-test was used for statistical analysis, where n = 3 and p < 0.0004. Conformational Studies: X-ray Diffraction of Collagen-cDHP Composites. XRD analysis and its diffraction pattern of collagen composites can be determined as a function of scattering angle (2θ). Characteristic peaks of collagen31 are found at two diffraction angles (2θ), viz., a sharp peak at 7.5° and a wide peak at 20.5°. The first peak corresponds to the diameter of triple helix of collagen and the second peak corresponds to single left handed helix chain.

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Figure 6. XRD measurements of collagen-cDHP composites as a function of 2θ (degree) for collagen-cDHP composites. A shift in 2θ value of collagen was observed with addition of cDHP. Concentration of collagen = 0.15 mg/mL, CG – collagen:0.05 genipin and C-CH1-4 = collagen: cDHP = 1:0.05-10. Figure 6 reveals the diffraction pattern of native collagen showing characteristic peaks at 7.5° and at 19.4°. Little variation in the second peak can be generally attributed to variability in 24


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assembly of fibrils at the molecular level45. Also, the structural property of collagen-cDHP composites is amorphous in nature. As a result of which, broad and scattered intensity is observed. In the presence of additive, cDHP, a slight alteration in the characteristic peaks was observed. The collagen-cDHP composites show peaks at 7.5° and 20° corresponding to collagen as well as at 11° and 16° corresponding to cDHP. With the addition of cDHP, the second peak was observed to show shift from 19.4° to 21.3° for C-CH4 as compared to native. The diameter of helix corresponding to peak at 7.5° demonstrated a shift to 9.2°, in the case of C-CH4, which indicates that at higher concentrations of cDHP, increased crosslinks are formed within collagen as a result of which the widening of diameter is observed. Collagen-genipin composite also showed a shift from 7.5° to 12°, and a peak at 17.3°. The lack of peaks in the range of 11-17° in native could suggest that these peaks correspond to crosslinking of collagen with additive resulting in formation of complex crosslinked products such as dimer formation, inter- and intramolecular crosslinks within collagen and helix-helix interaction. Addition of cDHP results in minor conformational changes, such as widening of diameter, minor alterations in the single left handed helix. This could be attributed to the attachment of phosphate anion to the functional groups of collagen. However, no major alteration in the helicity of collagen in the presence of cDHP was observed, suggesting that the helicity is intact. Conformational Studies: Changes in Secondary Structure of Collagen-cDHP Composites Using ATR-FTIR Spectroscopy. FTIR spectroscopy is a sensitive technique to investigate the conformational changes at the secondary structure level. Changes in functional groups of side chain amino acids in collagen due to additives can be monitored by analysis of the vibration of characteristic peptide bonds, namely, amide I (C=O in acetamide group of amide groups), amide II (N–H strongly coupled to the C–N stretching) and amide III (C–N stretching 25



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and N–H in plane bending) which are positioned at 1651, 1549, and 1238 cm−1, respectively. It is known that amide I and amide II band are the signatures of collagen triple helix46. Table 2 shows that the hydration shell surrounding native collagen is represented by a wide band from 3300-3400 cm-1, indicating the water bridges stabilizing collagen. Our results of native collagen are concordant with those reported in previous findings47. Compared to native, treatment of collagen with choline dihydrogen phosphate shows a red shift (from 1642 cm-1 in native to 1659 cm-1) in amide I band (Supplementary Figure S1). A similar red shift is observed for amide II band (from 1540 cm-1 to 1551 cm-1). Such shift indicates changes in dipole moment of the collagen functional groups bringing them in close proximity. Shift in amide I band suggests increased H-bonding leading to minor alteration in the conformational structure of collagen due to presence of choline dihydrogen phosphate. Compared to native, amide A band that labels -OH stretching shifts remarkable from 3370 cm-1 to 3421 cm-1 indicating interaction between free hydroxyl groups of choline dihydrogen phosphate and side chain functional groups of collagen and surrounding water molecules. This indicates increased formation of physical crosslinks (Hbonding) in the presence of choline dihydrogen phosphate. In summary, the preservation of amide I and amide II bands suggest that the type II polyproline structure of collagen is retained intact in the presence of choline dihydrogen phosphate.

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Table 2. Various Band Positions of Collagen – CDHP Composites Using ATR-FTIR Spectroscopy Band position (cm-1) Bands A B I II

Control (1:0)

CG (1:0)

C-CH1 (1:0.05)

C-CH2 (1:0.5)

C-CH3 (1:5)

C-CH4 (1:10)

3370

3370

3470

3457

3385

3421

-

-

-

-

-

-

1642

1643

1647

1650

1657

1659

1540

1543

1544

1545

1547

1551

III

1240 1241 1240 1241 1240 1241 Data in Table 2 shows the band positions of FTIR spectra of collagen-cDHP composites. Amide I, II and III are characteristic peaks determining the secondary structure of collagen. Morphological Studies: SEM Imaging of Collagen-cDHP Composites. The surface morphology of collagen-cDHP composites were observed using Scanning Electron Microscopy (SEM). Figure 7 shows sheet-like morphology of native collagen with diminutive interfibrillar space. Collagen-genipin composites reveal a porous morphology with an average interfibrillar space of about 7 ± 0.05 µm.

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Figure 7. SEM micrographs of collagen-cDHP composites at pH = 4.0 (scale bar = 20 µm). Fibril formation for composites was initiated on a glass slide at pH 7.4 (scale bar = 10 µm). Change in porosity and amount of fibrils can be observed. Concentration of collagen = 0.15 mg/mL, CG – collagen:0.05 genipin and C-CH1-4 = collagen: cDHP = 1:0.05-10. Porosity is generated primarily due to crosslinking of genipin with functional side groups of collagen, resulting in reorientation of the hydration dynamics of collagen. The small porous morphology was also observed for collagen-cDHP composite, C-CH1 with an average interfibrillar space of about 10 ± 0.02 µm. In addition to this, the texture of C-CH1 composites was observed to be slightly amorphous in nature. It is interesting to note that the surface of CCH4 composites is smooth, continuous and striated compared to native and C-CH1. The average interfibrillar space in C-CH4 was measured to be 1.45 ± 0.05 µm. This kind of unique 28


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morphology at maximum concentration of cDHP suggests that, the additive might aid in facilitating alignment of collagen fibrils, thereby decreasing the interfibrillar space to form a compact matrix. The self-assembly of tropocollagen molecules in the presence of additive was also performed and observed under SEM. It can be observed from Figure 7 that there is an increased population of fibrils for native collagen at pH 7.0. However, in the presence of additive C-CH4 there is dramatic decrease in the formation of fibrils. This was accompanied by lengthening of fibrils. Addition of cDHP is likely to result in alteration of hydration shell surrounding collagen molecule, thus reducing population and strengthening the fibrils. Molecular Modeling Studies. To investigate the nature of interaction between collagen (protein) and choline dihydrogen phosphate (as ligand) that results on enhanced intermolecular forces, viz., physical and chemical crosslinks, molecular modeling studies were carried out. The initial step was to determine the active conformations of protein and ligand binding site by systematic conformational search. For the molecular modeling studies in this study, we have not included water molecules so that clear insights on the interaction between collagen and cDHP can be obtained. Several conformation of ligand was docked on favorable sites of protein by the Autodock procedures. Only dihydrogen phosphate group of ligand interacted with protein. No interaction was observed between choline cation and collagen, reinforcing that the anions of ionic liquids play an influential role in solubilizing and stabilization of macromolecules48. The recognition site of protein were identified, examined and analyzed.

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Figure 8. Molecular modeling studies exhibiting the electrostatic interaction between collagen and choline dihydrogen phosphate A. light blue regions show electrostatic force exerted by collagen in the presence of cDHP. B. shows the electrostatic force in other regions of collagen where cDHP can potentially bind. C. shows the electrostatic force exerted by the dihydrogen phosphate molecule at the point of interaction. It was observed that on binding, no hydrogen or any other covalent bonds occurred between ligand and protein. The ligand is capable to bind only the light blue regions of collagen where electrostatic force was high (Figure 8A). Red regions demonstrated no electrostatic force. 30


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This indicates that the interaction between choline dihydrogen phosphate and collagen was purely electrostatic in nature. The energy of the ligand-protein complex docked on protein was evaluated to measure the electrostatic force exerted (Figure 8B and 8C). It was found to be 2.753 x 104 kJ/mol. With this range of electrostatic force, there is increased probability that salt bridges are formed between the ion pairs, suggesting increased inter- and intra-molecular forces between collagen and choline dihydrogen phosphate as well as within collagen itself. Electrostatic interactions on the surface of collagen have previously shown to promote stability likely due to charge-pair interactions between acidic and basic amino acid residues of collagen49. It can be postulated that highly charged kosmotropic dihydrogen phosphate (H2PO42-) anion interacts with amino acids of triple helical collagen by exerting an electrostatic force. The phosphate anion attaches primarily to arginine residues bringing about a minor conformational change, henceforth bringing the functional side groups of collagen closer. It is also equally likely that choline cations interact by forming H-bonds with collagen. However, this kind of conformational change in collagen was not observed in molecular modeling studies due to their limitations.

4. CONCLUSIONS In summary, this study investigates the interaction of collagen with choline dihydrogen phosphate that results in enhanced inter- and intra-molecular forces. It was demonstrated that the dielectric behavior of collagen-cDHP composites signifies increased formation of intra- and inter-molecular forces within the helices of collagen due to electrostatic interaction between collagen and cDHP. Conformational changes in the helices of collagen, increased crosslinking 31



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and high denaturation temperature was also observed as a function of concentration of cDHP. In addition to this, degree of biodegradation by collagenase was also reduced for collagen-cDHP composites. These results suggest that addition of cDHP to collagen leads to formation of physical crosslinks as well as chemical crosslinks. In addition to this, molecular modeling studies show that the force existing between collagen and choline dihydrogen phosphate is electrostatic in nature. Choline dihydrogen phosphate, a potential crosslinker stabilizes collagen by exerting electrostatic force that results in attachment of phosphate anion to functional amino acid side chains of collagen. This further leads to phosphorylation of the side chains and minor conformational changes in collagen, thus developing new H-bonds and chemical crosslink complexes thereby bringing alteration of structural conformation and strengthening of collagen molecule through various inter- and intra-molecular forces leading to formation of physical crosslinks, viz., H-bonding between hydrogen atoms of HPO4- and collagen and between hydroxyl atoms of collagen and hydroxyl atoms of choline. Chemical crosslinks are generated by various side functional groups of collagen interacting with the neighboring molecules. cDHP is an excellent compound promoting both physical and chemical crosslinks and offering biostability to collagen. This study contributes the significance of whole new class of biocompatible crosslinkers for type I collagen. Choline dihydrogen phosphate as an ionic liquid crosslinker has immense potential to be implemented for regenerative medicine, especially in bioimplants, as it serves a purpose in strengthening collagen macromolecule in terms of mechanical support, resistivity towards enzymatic and thermal degradation.

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ACKNOWLEDGEMENTS The authors thank CSIR 12th five year plan project - Research Initiatives for Waterless Tanning (RIWT- CSC0202) for financial support. We thank Ankur Sood and E. Divya Niveditha for their immense support in technical assistance.

Supporting Information Available: This study proves that choline dihydrogen phosphate can be a potential biocompatible crosslinker for type I collagen as it stabilizes collagen even at low concentrations and structural and molecular dynamics studies show that the interaction between them is purely electrostatic in nature. This material is available free of charge via the Internet at http://pubs.acs.org.

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Electrostatic

Interactions.


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Electrostatic Forces Mediated by Choline Dihydrogen Phosphate

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