Strategically designed antifibrotic gold nanoparticles to prevent

Oct 26, 2017 - Here, to target such Hyp-mediated interactions, we have synthesized uniform, thermostable and hemocompatible Hyp coated gold ...
0 downloads 0 Views 5MB Size
Download PDF
Article Cite This: Langmuir XXXX, XXX, XXX-XXX

pubs.acs.org/Langmuir

Strategically Designed Antifibrotic Gold Nanoparticles to Prevent Collagen Fibril Formation Bibin Ganadhason Anand,‡ Kriti Dubey,‡ Dolat Singh Shekhawat,‡ Kailash Prasad Prajapati,† and Karunakar Kar*,† †

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan-342011, India

‡

S Supporting Information *

ABSTRACT: Because uncontrolled accumulation of collagen fibrils has been implicated in a series of pathologies, inhibition of collagen fibril formation has become one of the necessary strategies to target such collagen-linked complications. The presence of hydroxyproline (Hyp) at the Y position in (Gly-XY)n sequence pattern of collagen is known to facilitate crucial hydrophobic and hydration-linked interactions that promote collagen fibril formation. Here, to target such Hyp-mediated interactions, we have synthesized uniform, thermostable, and hemocompatible Hyp coated gold nanoparticles (AuNPsHYP) and have examined their inhibition effect on the fibril formation of type I collagen. We found that collagen fibril formation is strongly suppressed in the presence of AuNPsHYP and no such suppression effect was observed in the presence of free Hyp and control Gly-coated nanoparticles at similar concentrations. Both isothermal titration calorimetric studies and bioinformatics analysis reveal possible interaction between Hyp and (Gly-Pro-Hyp) stretches of collagen triple-helical model peptides. Further, gold nanoparticles coated with proline (AuNPsPRO) and tryptophan (AuNPsTRP) also suppressed collagen fibril formation, suggesting their ability to interfere with aromatic-proline as well as hydrophobic interactions between collagen molecules. The Hyp molecules, when surface functionalized, are predicted to interfere with the Hyp-mediated forces that drive collagen selfassembly, and such inhibition effect may help in targeting collagen linked pathologies.

■

INTRODUCTION

Since accumulation of unregulated collagen is linked to many diseases, it is essential to find potential inhibitors against collagen fibril formation. Such a strategy to target collagen fibril formation may perhaps be useful in treating collagen linked diseases.13 Targeting the driving forces that are known to promote the onset of collagen fibril formation may possibly become an effective strategy for inhibition of collagen selfassembly.13,14 Aromatic-proline interactions15,16 and hydrophobic interactions17 are considered as the critical factors for promoting self-assembly of triple helical molecules into higher order structures. Studies have also revealed that hydration and hydroxyproline mediated intermolecular hydrogen bonding interactions are vital to stabilize triple-helical molecules and to drive self-assembly of collagen molecules into higher order structures.18−23 Brodsky’s laboratory, using collagen model peptides, has shown that hydroxyproline plays an important role in driving self-assembly of triple-helical collagen peptides24,25 into higher order structures. The sequence of type I collagen has frequent occurrence of (Gly-Pro-Hyp)

Collagen is an important structural protein that provides a crucial mechanical framework which becomes vital to many structural and functional roles of tissues including the extracellular matrix (ECM).1,2 In tissues, triple helical3 collagen molecules undergo a process of self-assembly to form supramolecular structures such as D-periodic fibrils of type I collagen,1 networks of type-IV collagen,4 and arrays of type VIII collagen.5 Though the self-assembly process of collagen triplehelical molecules is considered as a fundamental process in biology, unregulated collagen fibril formation has been implicated in many medical complications such as thrombosis, restenosis, and tissue stiffness related heart diseases. Exposed collagen is known to trigger thrombosis which is directly linked to myocardial infarction and stroke.6 Abnormalities linked to type VIII collagen are known to induce atherosclerosis lesions and plaque formation,7 and rupture of such plaques causes cardiac arrest as well as stroke. Uncontrolled accumulation of collagen at the sites where stents are deployed after angioplasty is also known to cause blockage of cardiac arteries.8,9 Collagen is also linked to tissue fibrosis severities10,11 including hypertensive heart diseases.12 © XXXX American Chemical Society

Received: May 3, 2017 Revised: October 24, 2017 Published: October 26, 2017 A

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Proline, Hydroxyproline) of amino acid solutions were boiled separately under strong stirring condition in the presence of 1 mM of KOH. Approximately 0.2 mM of AuCl4 was added to such boiling solution followed by continuous stirring until the end of the reaction (appearance of red wine or ruby red color, absorbance peak within ∼510−530 nm). The synthesized gold nanoparticles were aged for 7 days and then purified by centrifuging it at 10,000 rpm for 10 min to remove the free unreacted amino acid. The obtained pellet was then resuspended in water, and washed twice before use. Molar ratio values mentioned for AuNPsamino acids, in the current study, represent the concentration of the conjugated amino acids. For this, we assumed that 100% of the amino acid molecules are surface-functionalized. Since the nanoparticle suspensions were aged for a week of time followed by a centrifugation step to separate and to resuspend the pellet containing the nanoparticles, we assumed that all the inhibitor molecules are the conjugated ones. Hence, the molar ratio value was maintained only considering the concentration of the coated-amino acids rather than the concentration of the nanoparticles. Characterization of Nanoparticles. The initial characterization of the amino acid functionalized gold nanoparticles were measured by Shimadzu UV1800 to record the absorbance spectra of the nanoparticle samples. Then the samples were analyzed by Fourier Transform Infrared spectroscopy (FTIR) by using KBr pellet method. An aliquot of about 100 μL of the gold nanoparticles were added to the pellet obtained from hydraulic press. The samples were then examined using FTIR 8400 S (Shimadzu Japan) that recorded % transmittance against wavenumber. The hydrodynamic size (diameter) measurements of the functionalized gold nanoparticles were measured using a Malvern Zetasizer Nano ZS (Malvern Instruments, Southborough, Massachusetts) equipped with a backscattering detector (173°). Samples were filtered through a prerinsed 0.2 μm syringe filter followed by equilibration of ∼5 min at 25 °C. The surface charge zeta potential measurements were carried out using a Nano ZS (Malvern) and a titrator MPT-2. All the measurements were repeated at least two times. The morphology of the gold nanoparticles was characterized by transmission electron microscopy (TEM). Samples of the gold nanoparticles were prepared by placing a small drop on carbon-coated copper grids and allowing the solvent to evaporate in air. The air-dried grids were then examined by transmission electron microscope (HRTEM JEOL JEM-2100). TEM measurements were performed on instrument operated at an accelerating voltage between 80 and 200 kV. The obtained images were processed by GATAN microscopy software. The crystalline morphology and lattice of the nanoparticles were analyzed from the ED pattern and the HR-TEM images by using ImageJ and GATAN microscopy software. Circular Dichroism (CD) Spectroscopy. We performed CD experiments by using JASCO CD spectrometer (model J-815-150 L) with attached Peltier temperature controller. The path length of the cuvette was 2 mm. All the CD measurements have been carried out at room temperature and the scan speed for recording the signal was maintained at 50 nm per minute. CD signal of the PBS buffer was used as the reference sample (for blank) for the control collagen sample in the absence of inhibitors. However, to record the CD signal of collagen sample from an inhibited reaction, [AuNPsHYP + PBS buffer] was used as the reference (for using as blank). Hence, the reported CD spectra described in the text are the baseline subtracted ones. Collagen Fibril Formation. Collagen stock solution was prepared by dissolving the lyophilized powder of collagen in 20% acetic acid solution (maintaining collagen concentration at 1 mg.mL−1) at 4 °C, under stirring condition at least for 3 days. For studying fibril formation, final sample volume was maintained at 1 mL, with appropriate (100 μL) addition of 10× PBS buffer to the sample to make it 1× PBS at pH 7.4. This sample was then immediately transferred to the cuvette for recording the absorbance reading. The collagen fibril formation was studied by monitoring the absorbance of collagen sample at 313 nm using Spectrophotometer (Thermo Scientific NanoDrop 2000c).35 The concentration of collagen was maintained at 0.3 mg mL−1 (in PBS, pH 7.4), and measurements were recorded at 37 °C. The reference solution for every experiment was

triplet stretches along with many (Gly-Xaa-Hyp) regions (SI Figure S1) suggesting the significance of Hyp residue at Y position15 in the (G-X-Y)n repeating patterns within the collagen sequence. Fertala’s laboratory has already identified a Triple helical Telopeptide (T-TBR) (776GIAGQRGVVGLOGQRGFOGERG796) binding region within the sequence of α1 chain of type I collagen that binds to a nonhelical telopeptide region of another collagen triple helix, promoting collagen fibril assembly process.13,14 Here, we hypothesized that specifically targeting hydroxyproline mediated interactions between collagen molecules possibly will lead to prevention of the process of collagen fibril formation. Based on this hypothesis, in this work, we synthesized gold nanoparticles surface-functionalized with Hyp (AuNPsHYP). Surface functionalized gold nanoparticles have recently been shown to interfere with the self-assembly processes of amyloidogenic proteins that is believed to be mainly driven by hydrophobic interactions.26,27 Successful synthesis of amino acid coated gold nanoparticles has been reported where the gold nanoparticles are functionalized with amino acids through their amino group, allowing the carbonyl and the side chain groups of the conjugated amino acid to interact with the binding site residues of the protein.26,28,29 Some studies have also proposed that inhibition efficacies of some compounds are greatly enhanced when the inhibitor molecules are surface functionalized.26,27 Here, we have studied the inhibition effect of AuNPsHYP nanoparticles on the fibril formation of type I collagen (both from rat tail tendon and calf skin sources) under in vitro conditions, maintaining physiological pH at 7.4 (PBS buffer) and temperature at 37 °C. Type I collagen is the most abundant in the human body.30 In vitro fibril formation of type I collagen has been studied by several researchers.31−33 Hence, we chose type I collagen as a model convenient system for studying the process of collagen fibril formation and its inhibition in the presence Hyp coated gold nanoparticles. In addition to AuNPsHYP, gold nanoparticles coated with proline (AuNPsPRO), tryptophan (AuNPsTRP) and glycine (AuNPsGLY) were also examined for comparative studies. Studies have also been conducted to analyze the binding affinity of AuNPsHYP with a collagen model peptide, (Pro-Hyp-Gly)10, using isothermal titration calorimetry (ITC). Further, molecular interaction between collagen triple helical model peptides and hydroxyproline molecule has been studied using computational docking tools. Finally, we have also checked the hemocompatibility of these amino acid coated nanoparticles.

■

EXPERIMENTAL SECTION

Materials. Gold(III) chloride hydrate (HAuCl4·xH2O), hydroxyproline, potassium hydroxide, sodium hydroxide, trisodium citrate, Thioflavin T, lyophilized powder of type I collagen (calf skin), and silver nitrate were purchased from Sigma Aldrich, USA. Phosphate buffer saline (PBS), glycine, tyrosine, tryptophan, proline, and phenylalanine were procured from HIMEDIA. The collagen model peptide (Pro-Hyp-Gly)10 was purchased from Peptides international USA. Type I collagen (rat tail tendon) was received as a gift from Dr. Balaraman’s laboratory at CLRI India. The rat tail tendon collagen sample used for fibril assembly studies was prepared following the established protocol34(see the methods in the Supporting Information). Leishman stain, glutaraldehyde, ethanol, and HPLC grade water (LiChrosolv) were obtained from Merck. Synthesis of Gold Nanoparticles. The rapid synthesis of amino acid functionalized gold nanoparticles was carried out by following KOH reduction method.26,29 Briefly, 0.2 mM (Glycine, Tryptophan, B

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 1. Biophysical characterization of hydroxyproline coated gold nanoparticles (AuNPsHYP): (a) Schematic representation of the surface functionalized gold nanoparticles. (b) TEM images displaying the evenly sized (∼30 nm) spherical AuNPsHYP. (c) HR TEM data showing a fringe spacing of 2.01 and 2.4 Å. Inset shows splitting of spots in the FFT image. (d) Selected area electron diffraction pattern (SAED) of AuNPsHYP. The Scherrer ring patterns indicate the polycrystalline nature of FCC gold. (e) UV−vis spectrum of AuNPsHYP with absorption maxima around ∼530 nm due to surface plasma resonance. (f) SEM (scale bar 3 μM), optical and dark field microscopy images of intact RBC sample in the presence of AuNPsHYP. (g) Dynamic light scattering (DLS) data of AuNPsHYP, showing an average diameter of ∼20−30 nm. (h) Zeta potential measurements of AuNPsHYP sample. prepared from water, free amino acids, surface-functionalized amino acids, and buffer, without collagen for the baseline correction. The turbidity curves shown in the figures are baseline subtracted for each time point. The reference solution contained same amount of AuNPsHYP in the PBS buffer. Hence the rise in the turbidity signal was assumed only because of the collagen fibril formation in the cuvette. The method for extraction of type I collagen has been described in the Supporting Information. Scanning Electron Microscopy. Carl Zeiss EVO18 SEM was used to see the morphology of collagen fibrils as well as RBCs from the hemolysis experiments. The sample suspensions were dropped onto silver stubs after which it was dried and sputtered with gold before viewing under scanning electron microscope. Transmission Electron Microscopy (TEM). Transmission electron microscopy measurements were performed using (HR-TEM JEOL JEM-2100) equipment. The generated collagen fibril samples were casted over the carbon grids and dried. The dried samples were then stained with 2% uranyl acetate after which the grids were washed and air-dried. The images were acquired at an accelerating voltage of 120 kV. Hemolysis Assay. The blood sample was collected from a volunteer donor and the RBCs were separated out from the serum by spinning the blood at 1500 rpm for 10 min.36,37 The pellets of RBCs were then washed five times in PBS buffer and were then diluted with 1× PBS (25% v/v). From this diluted stock of RBCs, an aliquot of ∼100 μL was added to the AuNPsHYP sample (concentration ∼0.18 mM). All the samples were incubated at 37 °C for 4 h. RBC sample incubated with deionized water was considered as the positive control

and the sample in PBS buffer was considered as the negative control. After incubation the samples are vortexed again and centrifuged at 1600 rpm for 10 min. The obtained pellet was stored for microscopic observation. The supernatant that contained the lysate was carefully separated out to check its absorbance, in order to quantify the percentage of lysis. The detailed methods for calculation of percent lysis and for the staining protocols for microscopic visualization of RBC-morphology have been given in the Supporting Information. Molecular Docking Studies. Molecular docking studies were performed using AutoDock Vina wizard of PyRx (v 0.8).38 The ligand (Hyp) structure was obtained from HIC-Up and X-ray crystal structures of collagen model peptides were obtained from RCSB (PDB IDs: 1CAG and 1Q7D).20,39 Prior to docking, preprocessing and protonation of receptor molecules (collagen peptides) were performed by removing the unwanted ligand and water molecules and by adding polar hydrogen atoms. Then these PDB files were converted to PDBQT files. For docking, AutoDock Vinna used a search space of grid box size (x = 84.83 Å, y = 16.42 Å, z = 15.49 Å) for 1CAG and (x = 15.95 Å, y = 43.64 Å, z = 14.12 Å) for 1Q7D. The results obtained were analyzed based on the binding affinity energy (kcal mol−1) parameters linked to the respective protein−ligand complexes. The protein−ligand docked complex with the lowest energy was chosen for further analysis using Discovery Studio visualizer (v 16.1.015350). Isothermal Titration Calorimetry (ITC). The binding studies of AuNPsHYP inhibitors with soluble triple-helical collagen model peptides of (Pro-Hyp-Gly)10 were performed using NanoITC (TA Instruments, USA) at 298 K.36,40 ITC titration of 10 μM of peptide sample with 50 μM nanoparticle was performed. All solutions were C

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir degassed under vacuum to eliminate air bubble formation inside the calorimeter cell. The reference cell was filled with double-distilled and degassed water. During titration 1 μL of the AuNPsHYP (50 μM) was injected to (Pro-Hyp-Gly)10 containing sample cell at an interval of 300 s. To correct the heat effects of dilution and mixing, control experiments were performed at the same concentrations of the collagen peptide and the nanoparticle sample and the obtained data were subtracted from the respective (Pro-Hyp-Gly)10 peptideAuNPsHYP titrations. The data were analyzed and best-fitted using Nano Analyze 3.6 software to extract the thermodynamic parameters of the protein−ligand interaction.36 Data Analysis. All plots and graphs displayed in this article were obtained by using Origin-2015 graphics and analysis tool.

■

RESULTS Synthesis and Characterization of AuNPsHYP. Gold nanoparticles coated with hydroxyproline (AuNPsHYP) were synthesized by following the established protocol (Figure 1a). The UV−visible spectra of these nanoparticles showed a SPR peak around ∼530 nm confirming the formation of nanoparticles (Figure 1e). Dynamic light scattering data of AuNPsHYP samples clearly showed a homogeneous population of nanoparticles with average hydrodynamic radius of ∼30 nm (Figure 1g). The surface charge value obtained from zeta potential measurements for AuNPsHYP was found to be −72.6 mV, as evident from Figure 1h. Transmission electron microscopy (TEM) images further confirmed the formation of spherical uniform nanoparticles for AuNPsHYP (Figure 1b). To elucidate the chemistry of surface functionalization of hydroxyproline with the nanoparticles, FTIR measurements were conducted and the obtained result, as shown in SI Figure S2, revealed that the carbonyl groups are exposed outside due to the presence of its carbonyl stretching frequency around 1740 cm−1 for AuNPsHYP.26,29 To further characterize the intrinsic structural properties of the AuNPsHYP nanoparticles, high-resolution TEM (HRTEM) was performed which confirmed the polycrystalline nature of the nanoparticles (Figure 1c). We further examined SAED (the selected area electron diffraction) pattern of these AuNPsHYP sample using ImageJ software and the ring patterns of the obtained data show the presence of (220), (200), and (111) indexed facecentered cubic crystal (FCC) structure for AuNPsHYP (Figure 1d). Effect of AuNPsHYP on Collagen Fibril Formation. Collagen fibril formation was initiated by incubating the acidsoluble type 1 collagen solution in PBS buffer (pH 7.4) at 37 °C and the optical density of the sample at 313 nm was monitored as a function of time to record the onset of collagen self-assembly. The turbidity curve obtained for control collagen sample showed a typical pattern18,35,41 consisting of a negligible lag phase, a growth phase, and a plateau phase (pink curve, Figure 2a). However, in the presence of AuNPSHYP, gradual suppression of such fibril formation was observed in a dose dependent manner (Figure 2a). Though the lag time was not observed to be significantly affected by the presence of these inhibitors, the extent of the fibril formation was substantially reduced. Such inhibition effect, without any delay in the lag phase, was recently observed in a study on inhibition of collagen fibril formation.35 No such inhibition effect on collagen fibril formation was observed in the presence of free hydroxyproline (Figure 2a, orange curve) and control gold nanoparticles surface functionalized with glycine (AuNPsGLY) at similar concentrations (Figure 2a, black curve). The characteristic properties of the AuNPsGLY sample have been given in the

Figure 2. Inhibition effect of AuNPsHYP on fibril formation process of type I collagen. (a) Turbidity data showing fibril formation of calf skin collagen (0.3 mg.mL−1) in the presence and in the absence of inhibitors under physiological temperature 37 °C and in PBS buffer. Molar ratio of collagen:inhibitor is given in the parentheses: pink, control collagen; orange, collagen + Hyp (at 1:100); dark green, collagen + AuNPsHYP (at 1:25); dark blue, collagen + AuNPsHYP (at 1:50); red, collagen + AuNPsHYP (at 1:100); black, collagen + AuNPsGLY (at 1:100); (b) CD profiles collagen after fibril formation. blue, control collagen; red, collagen + free Hyp (1:100 molar ratio); light green, collagen + AuNPsHYP (1:100 molar ratio). All CD spectra shown here were baseline subtracted. (c) TEM images showing the fibril morphology of collagen fibrils in the presence and in the absence of inhibitor, as labeled. Double headed arrows indicate the presence of D-periodicity. Red arrows indicate the gold nanoparticles. Scale bar 100 nm. (d) Inhibition of fibril formation of type I collagen from rat tail tendon: red, control collagen; dark blue, collagen + AuNPsHYP (at 1:100); dark green, collagen + AuNPsGLY (at 1:100).

Supporting Information (Figure S6).This result suggests the significance of surface-functionalized hydroxyproline residues to interfere with the collagen fibril formation. Conformational changes occur in the collagen molecule during their assembly process into fibrils,42 and under such fibril forming condition, the triple helical structure of the semiflexible collagen molecule is believed to be perfected during the lag phase, facilitating the nucleation event and intramolecular interaction.43 It is possible that the presence of AuNPsHYP at higher concentration (1:100) can interfere with both the nucleation phase and the growth phase of the fibril formation process. It is also interesting to see that similar slight-delay in the lag phase was observed in the presence of control gold nanoparticles (AuNPsGLY at 1:100). However, the fibril formation process was not suppressed in the presence of AuNPsGLY (Figure 2a, black curve). It is possible that AuNPsGLY (at 1:100) can influence the nucleation event to some extent which may not be sufficient to block the intermolecular interactions leading to collagen-fibril assembly. In another control experiment involving gold nanoparticles capped with citrate (AuNPsCYT), we observed that these nanoparticles did not show any inhibition effect on fibril formation of RTT collagen (Figure S12). To generalize this inhibition effect on type I collagen, we examined the effect of AuNPsHYP on fibril formation of type I collagen obtained from rat tail tendon (RTT) (see methods in D

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 3. Interaction of hydroxyproline with the triple-helical collagen model peptides. (a) Docking studies of the collagen model peptide (POGPOGPOGPOGPOAPOGPOGPOGPOGPOG) (PDB ID 1CAG) showing three viable H-bonding interactions as labeled. Binding affinity for this interaction was −3.1 kcal.mol−1. (b) Molecular docking studies of the collagen model peptide (PDB ID 1Q7D) (XGPOGPOGFOGERGPOGPOGPOX) showing four strong hydrogen bond interactions as labeled. Binding affinity for this interaction was −3.2 kcal.mol−1. (c) Sequence of the peptides are given in the SI file.

control collagen fibrils. Such a difference in the signal intensity may be attributed to the lower fibril concentration in an inhibited reaction. Both CD and TEM data suggest that the inhibition effect mediated by AuNPsHYP may be achieved without altering the normal collagen fibril formation pathway. Hemocompatibility of AuNPsHYP Inhibitors. To understand the relevance of the hydroxyproline coated gold nanoparticles in biomedical applications, we examined the hemocompatibility of these nanoparticle-based inhibitors on human RBCs. We tested the toxicity effect of these nanoparticles on the intact red blood cells by following the established protocol for hemolysis assay.36,37 Data generated from our hemolysis assay experiments showed no indication of RBC-lysis in the presence of AuNPs HYP (Figure 1f). Hemocompatibility of these nanoparticle based inhibitors was also confirmed by scanning electron microscopy (SEM), optical microscopy, and dark field microscopy data as shown in Figure 1f.

Supporting Information). The turbidity data as shown in Figure 2d clearly shows that a similar inhibition effect was seen for the calf-skin collagen sample (Figure 2a). Next, we looked at the morphology of the samples of collagen fibrils formed both in the absence and in the presence of AuNPsHYP inhibitors. Characteristic features of the fibrils, obtained from both sources, remained unchanged, as evident from the TEM images shown in Figure 2c. The morphology of the collagen fibrils obtained from both inhibited and uninhibited reactions displayed the typical 67 nm D-periodic pattern in our TEM images (Figure 2c). The AFM images also showed similar fibril morphology for collagen samples obtained from both inhibited and uninhibited reactions (SI Figure S3). We further looked at the CD signal of the fibril samples, and the CD profiles showed a typical positive peak at ∼222 nm for all the samples, confirming the presence of triple helical conformation in the fibrils (Figure 2b). However, the intensity of the triple-helix peak at 222 nm for collagen fibrils obtained from the inhibited reaction was observed to be lower than the peaks obtained for E

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir Molecular Docking of Collagen Peptides with Hydroxyproline. To further understand the molecular interaction between hydroxyproline and triple helical domains of collagen, we performed molecular docking studies using selected triple helical collagen model peptides (SI Figure S1). Using AutoDock Vina wizard, blind docking studies were carried out and the obtained data are shown in Figure 3. The structures of the triple helix-hydroxyproline docked complexes predict formation of strong H-bonding interactions mediated by CO and hydroxyl groups of the Hyp (insets of Figure 3). The value of binding affinity energy obtained for peptide−Hyp interactions was found to be −3.1 kcal.mol−1 for 1CAG peptide and was observed to be −3.2 kcal.mol−1 for 1Q7D peptide. The IQ7D peptide has the sequence GFOGER, that is derived from the triple helical telopeptide binding region (T-TBR) of α-1 chain of the type I collagen.14 Such sequence stretch with GFOGER span is also known to occur in the integrin binding region of the collagen sequence.39 It is well-known that the telopeptide binding region (T-TBR), (776GIAGQRGVVGLOGQRGFOGERG796) plays a foundational role to begin the fibril formation of type I collagen by specifically interacting with the nonhelical telopeptide sequence residues of the interacting collagen molecule.13,14 Notably, as evident from our docking data (Figure 3), viable contacts with the Arg, Gly, and Hyp (belonging to the same chain) of the triple-helical GFOGER domain were observed. Such data suggest binding of Hyp to the T-TBR region of type I collagen could be one of the factors necessary for the inhibition effect. Isothermal Titration Calorimetry (ITC) Results on AuNPsHYP−Collagen Interaction. Since type I collagen triple-helical molecules in PBS tend to aggregate, it was difficult to directly perform ITC experiments on RTT type I collagen. Hence, we obtained a well-studied collagen model peptide (Pro-Hyp-Gly)1024 which is known to attain a very stable triple helical conformation in PBS buffer. Before ITC measurements, the peptide was dissolved in PBS buffer and kept at low temperature at least for 3 days to allow the formation of triplehelical structures. We examined the triple-helical conformation of the (Pro-Hyp-Gly)10 peptide by monitoring the CD signal that displayed a positive peak at 222 nm (SI Figure S8). Titration of Hyp-coated gold nanoparticles into a soluble sample containing (Pro-Hyp-Gly)10 triple helical molecules showed affinity of AuNPsHYP for the triple helical structures. The obtained results, as shown in Figure 4, indicate affinity of AuNPsHYP for (Pro-Hyp-Gly)10 triple helices, revealing important thermodynamic parameters of hydroxyproline-(ProHyp-Gly)10 interaction, as shown in the inset of Figure 4b (Ka = 2.4 × 106 M−1, Kd = 4.01 × 10−7 M, ΔH = −25.0 kcal.mol−1, ΔS = −54.5 cal.mol−1 K−1, ΔG = −8.9 kcal.mol−1).

Figure 4. Interaction of triple-helical collagen peptide with AuNPsHYP by Isothermal Titration Calorimetry (ITC). (a) Baseline subtracted raw data from ITC for titration of triple helical collagen model peptide (Pro-Hyp-Gly)10 (10 μM) with ligand AuNPsHYP (50 μM) at 298 K, showing the calorimetric response peaks in μcal.s−1 during successive injections of AuNPsHYP to the peptide sample in the cell. (b) Integrated heat profile of data generated from ITC data. The solid line represents the minimized independent fitting by a model of one site per monomer (using Nano Analyze 2.1 software from Thermal Analysis). Inset of panel b displays the binding parameters obtained for AuNPs−peptide interaction.

role of the functionalized-Hyp during suppression of collagen assembly. The binding data on the interaction between conjugated-Hyp with collagen model peptide (Pro-Hyp-Gly)10 clearly suggest the affinity of Hyp residues for the triple helical (Gly-Pro-Hyp) domains. Such experimental data on contacts between AuNPsHYP and (Pro-Hyp-Gly)10 was further supported by our computational docking studies where we clearly observed the potential of the Hyp to interact with (Gly-Xaa-Yaa) triple helical conformers. Though it was difficult for us to conduct docking studies using conjugated-Hyp as a ligand due to limitations of docking protocols, it was assumed that the geometrical alternations, if any, due to the conjugation of −NH moiety of the Hyp molecule with the nanoparticles (after synthesis), would still allow its −COOH group and 4′-OH group to interact with the binding regions in the triple helical molecule. We conjugated one gold atom to the −NH moiety of the Hyp molecule using Discovery Studio 4.0 to test the binding efficacy of such modified Hyp with the collagen model peptides. This modified ligand (Hyp with one gold atom at its −NH group) was then docked with collagen model peptide 1CAG (see methods in SI) and the docking results displayed five viable H-bonds (SI Figure S11), with a binding affinity of −16.44 kcal.mol−1. It is interesting to see that such a value of binding affinity is higher than the binding affinity of free Hyp molecule with 1CAG peptide. It is possible that the conformational constraints due to conjugation of the gold atom may enhance the interaction between the peptide and the ligand. Further extensive computational experiments would be required to understand the key factors that enable the conjugated ligand to interact with the protein moiety more

■

DISCUSSION This study has revealed that gold nanoparticles surface functionalized with hydroxyproline can effectively suppress the fibril formation of type I collagen under in vitro conditions. Since no suppression of collagen fibril formation was observed in the presence of free Hyp (uncoated), it appears that surfacefunctionalization of Hyp was critical for its available functional groups to interfere with the collagen−collagen interactions, causing the inhibition of fibril assembly. This assumption is supported by recent reports on the enhanced efficacies of the surface functionalized inhibitors due to gain of structural constraint.27,44 The observation that Gly-coated nanoparticles did not suppress collagen fibril formation also suggests specific F

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 5. Schematic representation of targeting collagen fibril formation through hydroxyproline coated gold nanoparticles.

already known to promote collagen−collagen contacts during fibril assembly.17,25 Interestingly, both of these nanoparticles, namely, AuNPsPRO and AuNPsTRP showed similar inhibition effects on fibril formation of type I collagen (SI Figure S7, panel a). However, these amino acids (at similar concentrations) in their free forms did not show any such inhibition effect (Figure S7, panel b), confirming the significance of their surface functionalization to show the inhibition effect. These results point to the ability of proline and aromatic molecules to interfere with the intermolecular aromatic−proline and aromatic−aromatic interactions necessary for collagen selfassembly. Generation of D-periodic fibrils of type I collagen is considered to be driven by both nonspecific and specific interactions. Presence of Hyp may help in nonspecific interactions,22,45 whereas the interactions driven by hydrophobic16,17,25 and charged residues46,47 are known to bring specific interactions between triple helical molecules. Hence, in addition to Hyp mediated driving forces, targeting intermolecular interactions mediated by charged residues and aromatic groups could also prove to be an effective strategy for inhibition of collagen fibril assembly. The physiochemical properties of the nanoparticles such as size and charge have been considered as critical factors for their antifibrotic properties.48,49 Here, in our study, both the control gold nanoparticles (AuNPsGLY) and the Hyp coated nanoparticles (AuNPsHYP) were found to be of similar size (∼20 nm). The zeta potential values were also found to be −55.9 mV for the AuNPsGLY and −72 mV for AuNPsHYP. Nanoparticles coated with Hyp, Pro, and Trp could suppress the collagen fibril formation whereas Gly coated nanoparticles did not show such inhibition effect. Recently, we observed that piperine coated gold nanoparticles (size ∼10 nm, −zeta potential −4.5 mV) showed inhibition effect on fibril formation of type I collagen (at 1:30 molar ratio of collagen to inhibitor).50 The control nanoparticles, AuNPsCYT (size ∼10− 30 nm, −zeta potential −19.5 mV) in the same study50 did not

efficiently. Our docking studies on the triple helix of the collagen model peptide (PDB ID 1Q7D) shows hydroxyproline’s affinity for the GFOGER sequence. More importantly, the Triple helical Telopeptide binding region (T-TBR) in the collagen molecule contains a −GFOGER− stretch.13,14 Hence, it appears that the binding of the AuNPsHYP with T-TBR may be one of the reasons behind the inhibition effect. Recently, our group has shown that tyrosine coated nanoparticles (functionalized through −NH2)26 showed inhibition effect against the amyloid fibril formation of insulin. The same study also revealed that reversing the orientation of the coated tyrosine residue mediated by its functionalization through the CO moiety29 did not significantly alter the inhibition effect.26 The question of which region of the collagen sequence is interacting with Hyp is very important because the longer sequence of type I collagen comprises imino acids, hydrophobic residues, and charged residues, all of which are believed to be playing critical roles for driving the fibril-assembly process. Considering our ITC data on (Pro-Hyp-Gly)10 peptide that displayed a single binding site for the ligand (surfacefunctionalized hydroxyproline), we predict the possibility of multiple binding sites in type I collagen sequence to accommodate AuNPsHYP ligands (Figure 5). This assumption is further supported by the sequence analysis of type I collagen which displays regular occurrence of (Gly-Pro-Hyp) triplets spanning from C-terminal to N-terminal zone (SI Figure S1). Also, a significant suppression effect on collagen fibril formation was observed only at 1:100 molar ratio of collagen:inhibitor, whereas slight inhibition was observed at 1:25 molar ratio. To explore the possibility of the effect of other amino acids on collagen fibril formation we synthesized stable gold nanoparticles coated with Pro and Trp. The characterization of these nanoparticles is shown in SI Figure S4 and Figure S5. Pro shares a similar structure with Hyp, without 4′-OH group, and the presence of aromatic residues such as Trp and Tyr is G

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir show any suppression effect on collagen fibril formation. A recent biophysical study on the fibril formation of Alzheimer’s linked Aβ peptide has revealed that gold nanoparticles can effectively involve Aβ aggregation. However, the resultant amyloid plaques were observed to have less β-structures and more random conformations, unlike the typical β-sheet rich amyloid plaques.49 In our study, the control gold nanoparticles (AuNPsGLY, AuNPsCYT) did not alter the turbidity curve indicating their noninhibitory nature. It is possible that such noninhibitory control gold nanoparticles can coaggregate with collagen resulting in the formation of nonhelical fibrillar entities, unlike the control collagen fibrils. It is also possible that the control nanoparticles facilitate the collagen fibril growth without incorporating themselves into the fibrillar structures. Though further experiments are certainly required to know how nanoparticles of different sizes can alter the fibril formation process of collagen, in the current study, we predict that the functionalization of the amino acids is playing a dominant role during suppression of the collagen fibril formation. Hyp-mediated osmolytic effect has been reported to influence both the conformational and aggregation properties of macromolecules, preferably through alterations in the solvation effect.51,52 Sugar molecules have been reported to inhibit fibril formation of type 1 collagen, preferably through reordering of water molecules leading to solvent mediated inhibition effect.18 However, such solvent mediated effect is observed only when these molecules are present in very high concentrations (in the range of 2 to 3 M). In our study the maximum concentration for hydroxyproline was maintained at ∼100 μM. Hence, we believe that the Hyp-induced solvation effects on collagen triple-helical molecules, if any, due to the presence of Hyp in the solution may not be sufficient to cause the prevention of collagen fibril formation. Again, only surface functionalized Hyp, not the free Hyp, is capable of suppressing the fibril formation, suggesting direct interaction of AuNPsHYP with the collagen rather than the solvent mediated indirect interactions. Though the pathophysiology of lethal collagen linked fibrotic growth in the body system is considered as a heterocomponent assembly which includes different collagen types (type III, type V, and type I), targeting fibril assembly of type I collagen is believed to be an effective strategy to reduce fibrosis complications.14,53 Chung et al. using a mouse model has already established the concept for reducing formation of fibrotic deposits by inhibiting self-assembly of collagen molecules into fibrils.14 Prevention of uncontrolled growth of collagen could benefit the treatment of severe pathologies including tissue fibrosis disorders and restenosis linked complications. For example, curcumin coated stents have been reported to be effective in reducing restenosis complications54 and studies have also revealed that curcumin can influence collagen metabolism in model systems. Targeted drug delivery devices such as the design of magnetic nanoparticles coated with Hyp residues or making receptor specific microspheres loaded with Hyp coated gold nanoparticles can help in the prevention of the onset of these collagen-activated diseases. Considering the vital role of Hyp mediated driving forces behind the collagen self-assembly process, the results of this work establish the protective effect of AuNPsHYP against collagen fibril assembly. Crystal structures of collagen model peptides55,56 have already revealed the Hyp mediated water bridges between triple helical molecules, and

we believe that AuNPsHYP can effectively interfere with these fundamental forces. Though this work is an in vitro investigation, the results obtained could be beneficial to the researchers specifically targeting collagen activated diseases such as tissue fibrosis, in cell and animal models.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b01504. Analysis of collagen sequence; FTIR spectra; AFM and TEM images; characterization of proline, tryptophan, and glycine coated gold nanoparticles; circular dichroism signals; materials and methods section (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. ORCID

Bibin Ganadhason Anand: 0000-0001-7339-6657 Karunakar Kar: 0000-0001-7047-6539 Author Contributions

B.G.A., K.D., D.S.S., K.P.P., and K.K. designed and conducted experiments, analyzed the data, and wrote the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Jawaharlal Nehru University (AIRF and CIF-School of Life Sciences) and IIT Jodhpur for providing us the required facilities. This work was supported by BRNS grant (KK) (Grant No. 37 (1)/14/38/2014-BRNS). Authors thank DSTPURSE II, UGC RN, UGC DRS SAP-I for support.

■

REFERENCES

(1) Kielty, C. M.; Grant, M. E.The Collagen Family: Structure Assembly and organization in the Extracellular Matrix, In Connective Tissue and its Heritable Disorders; Wiley-Liss: New York, 2002; pp 159−222. (2) Brodsky, B.; Ramshaw, J. A. The collagen triple-helix structure. Matrix Biol. 1997, 15, 545−554. (3) Ramachandran, G. N.; Kartha, G. Structure of collagen. Nature 1955, 176, 593−595. (4) Yurchenco, P. D.; Ruben, G. C. Type IV collagen lateral associations in the EHS tumor matrix. Comparison with amniotic and in vitro networks. American journal of pathology 1988, 132, 278−291. (5) Stephan, S.; Sherratt, M. J.; Hodson, N.; Shuttleworth, C. A.; Kielty, C. M. Expression and supramolecular assembly of recombinant alpha1(viii) and alpha2(viii) collagen homotrimers. J. Biol. Chem. 2004, 279, 21469−21477. (6) Farndale, R. W.; Sixma, J. J.; Barnes, M. J.; de Groot, P. G. The role of collagen in thrombosis and hemostasis. J. Thromb. Haemostasis 2004, 2, 561−573. (7) Plenz, G. A.; Deng, M. C.; Robenek, H.; Volker, W. Vascular collagens: spotlight on the role of type VIII collagen in atherogenesis. Atherosclerosis 2003, 166, 1−11. (8) Osherov, A. B.; Gotha, L.; Cheema, A. N.; Qiang, B.; Strauss, B. H. Proteins mediating collagen biosynthesis and accumulation in arterial repair: novel targets for anti-restenosis therapy. Cardiovasc. Res. 2011, 91, 16−26. (9) Lafont, A.; Durand, E.; Samuel, J. L.; Besse, B.; Addad, F.; Levy, B. I.; Desnos, M.; Guerot, C.; Boulanger, C. M. Endothelial dysfunction and collagen accumulation: two independent factors for

H

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir restenosis and constrictive remodeling after experimental angioplasty. Circulation 1999, 100, 1109−1115. (10) Burke, J. P.; Mulsow, J. J.; O’Keane, C.; Docherty, N. G.; Watson, R. W.; O’Connell, P. R. Fibrogenesis in Crohn’s disease. Am. J. Gastroenterol. 2007, 102, 439−448. (11) Bailey, J. R.; Bland, P. W.; Tarlton, J. F.; Peters, I.; Moorghen, M.; Sylvester, P. A.; Probert, C. S.; Whiting, C. V. IL-13 promotes collagen accumulation in Crohn’s disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells? PLoS One 2012, 7, e52332. (12) Cecelja, M.; Chowienczyk, P. Role of arterial stiffness in cardiovascular disease. JRSM cardiovascular disease 2012, 1, 1. (13) Steplewski, A.; Fertala, A. Inhibition of collagen fibril formation. Fibrog. Tissue Repair 2012, 5, S29. (14) Chung, H. J.; Steplewski, A.; Chung, K. Y.; Uitto, J.; Fertala, A. Collagen fibril formation. A new target to limit fibrosis,. J. Biol. Chem. 2008, 283, 25879−25886. (15) Persikov, A. V.; Ramshaw, J. A.; Brodsky, B. Prediction of collagen stability from amino acid sequence. J. Biol. Chem. 2005, 280, 19343−19349. (16) Kar, K.; Ibrar, S.; Nanda, V.; Getz, T. M.; Kunapuli, S. P.; Brodsky, B. Aromatic interactions promote self-association of collagen triple-helical peptides to higher-order structures. Biochemistry 2009, 48, 7959−7968. (17) Cejas, M. A.; Kinney, W. A.; Chen, C.; Vinter, J. G.; Almond, H. R.; Balss, K. M.; Maryanoff, C. A.; Schmidt, U.; Breslav, M.; Mahan, A.; Lacy, E.; Maryanoff, B. E. Thrombogenic collagen-mimetic peptides: Self-assembly of triple helix-based fibrils driven by hydrophobic interactions. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 8513−8518. (18) Kuznetsova, N.; Chi, S. L.; Leikin, S. Sugars and polyols inhibit fibrillogenesis of type I collagen by disrupting hydrogen-bonded water bridges between the helices. Biochemistry 1998, 37, 11888−11895. (19) Kuznetsova, N.; Leikin, S. Does the triple helical domain of type I collagen encode molecular recognition and fiber assembly while telopeptides serve as catalytic domains? Effect of proteolytic cleavage on fibrillogenesis and on collagen-collagen interaction in fibers. J. Biol. Chem. 1999, 274, 36083−36088. (20) Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution. Science 1994, 266, 75−81. (21) Privalov, P. L. Stability of proteins. Proteins which do not present a single cooperative system. Adv. Protein Chem. 1982, 35, 1− 104. (22) Leikin, S.; Rau, D. C.; Parsegian, V. A. Temperature-favoured assembly of collagen is driven by hydrophilic not hydrophobic interactions. Nat. Struct. Mol. Biol. 1995, 2, 205−210. (23) Vitagliano, L.; Berisio, R.; Mazzarella, L.; Zagari, A. Structural bases of collagen stabilization induced by proline hydroxylation. Biopolymers 2001, 58, 459−464. (24) Kar, K.; Amin, P.; Bryan, M. A.; Persikov, A. V.; Mohs, A.; Wang, Y. H.; Brodsky, B. Self-association of collagen triple helic peptides into higher order structures. J. Biol. Chem. 2006, 281, 33283− 33290. (25) Kar, K.; Wang, Y. H.; Brodsky, B. Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures. Protein Sci. 2008, 17, 1086−1095. (26) Dubey, K.; Anand, B. G.; Badhwar, R.; Bagler, G.; Navya, P. N.; Daima, H. K.; Kar, K. Tyrosine- and tryptophan-coated gold nanoparticles inhibit amyloid aggregation of insulin. Amino Acids 2015, 47, 2551−2560. (27) Anand, B. G.; Dubey, K.; Shekhawat, D. S.; Kar, K. CapsaicinCoated Silver Nanoparticles Inhibit Amyloid Fibril Formation of Serum Albumin. Biochemistry 2016, 55, 3345−3348. (28) Daima, H. K.; Selvakannan, P. R.; Shukla, R.; Bhargava, S. K.; Bansal, V. Fine-tuning the antimicrobial profile of biocompatible gold nanoparticles by sequential surface functionalization using polyoxometalates and lysine. PLoS One 2013, 8, e79676. (29) Selvakannan, P.; Ramanathan, R.; Plowman, B. J.; Sabri, Y. M.; Daima, H. K.; O’Mullane, A. P.; Bansal, V.; Bhargava, S. K. Probing the

effect of charge transfer enhancement in off resonance mode SERS via conjugation of the probe dye between silver nanoparticles and metal substrates. Phys. Chem. Chem. Phys. 2013, 15, 12920−12929. (30) Di Lullo, G. A.; Sweeney, S. M.; Körkkö, J.; Ala-Kokko, L.; San Antonio, J. D. Mapping the Ligand-binding Sites and Diseaseassociated Mutations on the Most Abundant Protein in the Human, Type I Collagen. J. Biol. Chem. 2002, 277, 4223−4231. (31) Williams, B. R.; Gelman, R. A.; Poppke, D. C.; Piez, K. A. Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results. J. Biol. Chem. 1978, 253, 6578−6585. (32) Piez, K. A.; Carrillo, A. L. Helix Formation by Single- and Double-Chain Gelatins from Rat Skin Collagen. Biochemistry 1964, 3, 908−914. (33) Kleinman, H. K.; Wilkes, C. M.; Martin, G. R. Interaction of fibronectin with collagen fibrils. Biochemistry 1981, 20, 2325−2330. (34) Chandrakasan, G.; Torchia, D. A.; Piez, K. A. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution. J. Biol. Chem. 1976, 251, 6062−6067. (35) Perumal, S.; Dubey, K.; Badhwar, R.; George, K. J.; Sharma, R. K.; Bagler, G.; Madhan, B.; Kar, K. Capsaicin inhibits collagen fibril formation and increases the stability of collagen fibers. Eur. Biophys. J. 2015, 44, 69−76. (36) Dubey, K.; Anand, B. G.; Shekhawat, D. S.; Kar, K. Eugenol prevents amyloid fibril formation of proteins and inhibits amyloid induced hemolysis. Sci. Rep. 2017, 7, 40744. (37) Mattson, M. P.; Begley, J. G.; Mark, R. J.; Furukawa, K. Abeta25−35 induces rapid lysis of red blood cells: contrast with Abeta1−42 and examination of underlying mechanisms. Brain Res. 1997, 771, 147−153. (38) Dallakyan, S.; Olson, A. J. Small-molecule library screening by docking with PyRx. Methods Mol. Biol. 2015, 1263, 243−250. (39) Emsley, J.; Knight, C. G.; Farndale, R. W.; Barnes, M. J. Structure of the integrin alpha2beta1-binding collagen peptide. J. Mol. Biol. 2004, 335, 1019−1028. (40) Zeng, Z.; Patel, J.; Lee, S. H.; McCallum, M.; Tyagi, A.; Yan, M.; Shea, K. J. Synthetic polymer nanoparticle-polysaccharide interactions: a systematic study. J. Am. Chem. Soc. 2012, 134, 2681−2690. (41) Dubey, K.; Kar, K. Type I collagen prevents amyloid aggregation of hen egg white lysozyme. Biochem. Biophys. Res. Commun. 2014, 448, 480−484. (42) George, A.; Veis, A. FTIRS in water demonstrates that collagen monomers undergo a conformational transition prior to thermal selfassembly in vitro. Biochemistry 1991, 30, 2372−2377. (43) Kadler, K. E.; Holmes, D. F.; Trotter, J. A.; Chapman, J. A. Collagen fibril formation. Biochem. J. 1996, 316, 1−11. (44) Porat, Y.; Abramowitz, A.; Gazit, E. Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem. Biol. Drug Des. 2006, 67, 27−37. (45) Kar, K.; Amin, P.; Bryan, M. A.; Persikov, A. V.; Mohs, A.; Wang, Y. H.; Brodsky, B. Self-assembly of triple helical peptides into higher order structures. J. Biol. Chem. 2006, 281, 33283−33290. (46) Rele, S.; Song, Y.; Apkarian, R. P.; Qu, Z.; Conticello, V. P.; Chaikof, E. L. D-periodic collagen-mimetic microfibers. J. Am. Chem. Soc. 2007, 129, 14780−14787. (47) Keshwani, N.; Banerjee, S.; Brodsky, B.; Makhatadze, G. I. The role of cross-chain ionic interactions for the stability of collagen model peptides,. Biophys. J. 2013, 105, 1681−1688. (48) Kim, Y.; Park, J.-H.; Lee, H.; Nam, J.-M. How Do the Size, Charge and Shape of Nanoparticles Affect Amyloid β Aggregation on Brain Lipid Bilayer? Sci. Rep. 2016, 6, 1 DOI: 10.1038/srep19548. (49) Lee, H. E.; Lee, H. K.; Chang, H.; Ahn, H. Y.; Erdene, N.; Lee, H. Y.; Lee, Y. S.; Jeong, D. H.; Chung, J.; Nam, K. T. Virus templated gold nanocube chain for SERS nanoprobe. Small 2014, 10, 3007− 3011. (50) Anand, B. G.; Shekhawat, D. S.; Dubey, K.; Kar, K. Uniform, Polycrystalline, and Thermostable Piperine-Coated Gold NanoI

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir particles to Target Insulin Fibril Assembly. ACS Biomater. Sci. Eng. 2017, 3, 1136−1145. (51) Kar, K.; Kishore, N. Enhancement of thermal stability and inhibition of protein aggregation by osmolytic effect of hydroxyproline. Biopolymers 2007, 87, 339−351. (52) Keswani, N.; Kar, K.; Kishore, N. Thermodynamic properties of aqueous 4-hydroxyproline at different temperatures. J. Chem. Thermodyn. 2010, 42, 597−604. (53) Romanic, A. M.; Adachi, E.; Kadler, K. E.; Hojima, Y.; Prockop, D. J. Copolymerization of pNcollagen III and collagen I. pNcollagen III decreases the rate of incorporation of collagen I into fibrils, the amount of collagen I incorporated, and the diameter of the fibrils formed. J. Biol. Chem. 1991, 266, 12703−12709. (54) Jang, H.-S.; Nam, H. Y.; Kim, J.-M.; Hahm, D.-H.; Nam, S. H.; Kim, K. L.; Joo, J.-R.; Suh, W.; Park, J.-S.; Kim, D. K.; Gwon, H.-C. Effects of curcumin for preventing restenosis in a hypercholesterolemic rabbit iliac artery stent model. Catheterization and Cardiovascular Interventions 2009, 74, 881−888. (55) Bella, J.; Brodsky, B.; Berman, H. M. Hydration structure of a collagen peptide. Structure 1995, 3, 893−906. (56) Brodsky, B.; Persikov, A. V. Molecular structure of the collagen triple helix. Adv. Protein Chem. 2005, 70, 301−339.

J

DOI: 10.1021/acs.langmuir.7b01504 Langmuir XXXX, XXX, XXX−XXX