Does Surface Chirality of Gold Nanoparticles Affect Fibrillation of HSA

Subscriber access provided by University of Rochester | River Campus & Miner Libraries

Article

Does Surface Chirality of Gold Nanoparticles Affect Fibrillation of HSA? Shubhatam Sen, Swagata Dasgupta, and Sunando DasGupta J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b05354 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Does Surface Chirality of Gold Nanoparticles Affect Fibrillation of HSA? Shubhatam Sen,† Swagata Dasgupta,*,§ and Sunando DasGupta*,‡ †

Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

§

Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

‡

Department of Chemical Engineering, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT In order to demonstrate the influence of the surface chirality of the nanoparticles on amyloid fibrillation, the inhibiting effectiveness of the chiral gold nanoparticles, synthesized using the two enantiomeric forms (i.e. D- and L-) of glutamic acid, towards the fibrillation of human serum albumin (HSA) has been investigated. Here the enantiomers of glutamic acid are used as both reducing and stabilizing/capping agent. It is found that the surface chirality is the only major difference between the D-glutamic acid mediated gold (DGAu) and L-glutamic acid mediated gold (LGAu) nanoparticles. The fibrillation process has been monitored using various biophysical techniques e.g. turbidity assay, Thioflavin T fluorescence kinetics, Congo red binding study, circular dichroism spectroscopy, fluorescence microscopy, and transmission electron microscopy. The experimental results illustrate that DGAu is more effective in inhibiting the formation of HSA fibrils than LGAu. Further, the differential inhibiting effect of DGAu and LGAu towards HSA fibrillation has been described on the basis of the dissimilar interaction behavior of the nanoparticles with the HSA molecules, as revealed by the Trp fluorescence quenching, and the isothermal titration calorimetry. It is revealed that the surface chirality of the gold nanoparticles strongly influences the protein adsorption dynamics including the modes of orientation of adsorbed HSA molecules, causing the inhibition of fibrillation to different extents.


Page 2 of 42

Page 3 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Structural transition from the native conformations of proteins into ordered amyloid aggregates is implicated in a large number of neurodegenerative disorders.1, 2 These ordered protein aggregates, known as amyloid fibrils, are characterized by well defined a cross-β sheet motif, featuring β-strands running perpendicular to the fibril axis.3 It is previously established that the amyloid fibril formation is a basic generic property of polypeptide backbones and most of the proteins, if not all, have the potential of undergoing structural transition from their native state to amyloid aggregates under suitable biophysical circumstances.4 In this regard, human serum albumin (HSA) is considered as a convenient model protein for investigating the underlying mechanism of fibrillation process, due to its propensity to easily form amyloid fibrils in vitro.5 HSA, the most abundant protein in blood plasma, contributes significantly to control and regulate the osmotic pressure and various metabolic processes and also act as a multifunctional transport protein in the circulatory system.6, 7 HSA, a natively α-helical (˃60%) protein, comprises a single polypeptide chain of 585 amino acid residues organized in three homologous domains.6 Earlier studies have shown that the formation of amyloid fibrils by destabilization of the native structure of HSA is achievable by controlling specific solution conditions like pH, ionic strength, temperature etc.8-10 Additionally, various external factors like metal ions, sugars, surfactants and other organic molecules have been reported to affect the fibrillation of HSA differently.11-15 Recently, there has been considerable attention on nanoparticles for various biomedical applications due to a variety of their unique attributes, including high surface to volume ratio, size similarity to biological macromolecules, facile surface functionalization etc. The utilization of nanoparticles in the field of amyloid fibrillation is one of those important applications. Various approaches have been explored to modulate the protein fibrillation process by the nanoparticles having different physicochemical properties e.g. 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

size,16 shape,17 and surface properties (e.g. composition,18 surface charge,19 and hydrophobicity20). However, despite the focus on the effects of these physicochemical properties on protein fibrillation as mentioned above, the influence of surface chirality of the nanoparticles in amyloid fibrillation has not been investigated in detail. Chirality is an important and common phenomenon of the biomolecules e.g. most of the amino acids in proteins, sugar molecules in nucleic acids etc. possess chiral properties. There are evidence of utilization of surface molecular chirality in different biological applications e.g. modulating adhesion and differentiation behavior of stem cells,21 protein adsorption dynamics,22 difference in cytotoxicity23 and cell imaging ability24 of quantum dots etc. Recently, cysteine enantiomer modified graphene oxide has been used as a model to show that surface chirality influences the α-helix to β-sheet conformational transition of amyloid-β (1-40) peptide and thus modulates the amyloid fibrillation process differently.25 It has also been shown that the chiral effect requires a combined effect of both the stereoselective interactions between the chiral molecules and the nanoparticle surface. In a separate study, cysteine enantiomer modified selenium nanoparticles have been used to demonstrate that the chiral molecule modified nanoparticles inhibit the formation of amyloid-β (1-40) aggregates in the presence of metal ions by blocking the metal binding sites.26 The different molecular surface chirality is also found to affect the morphology of amyloid-β (1–40) aggregates formed at the liquidsolid interface based on electrostatic interaction- enhanced stereoselective adsorption.27 However, the mechanistic consequences of the surface chirality on fibrillation of proteins still remain unexplored. Therefore, studying the possible influence of surface chirality of nanoparticles on the fibrillation of HSA is worthy of an in-depth investigation. The protein fibrillation phenomenon in the presence of nanoparticles is governed by protein-nanoparticle interactions. Hence, an improved understanding on the surface chirality-induced modulation


Page 4 of 42

Page 5 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the interaction behavior of proteins with nanoparticles is needed to elucidate the dissimilar effects of the surface chirality on protein amyloid fibrillation. Due to the excellent biocompatibility, chemical stability and design flexibility, gold nanoparticles have been extensively utilized in various therapeutic and diagnostic purposes e.g. drug/gene delivery,28 biomedical imaging,29 photothermal therapy,30 sensing31 etc. Moreover, the influence of gold nanoparticles on aggregation of different proteins have also been explored, where the nanoparticles are found to either inhibit or stimulate the fibrillation process depending on their size, charge and/or different surface functional groups.16,

32-35

Thus, AuNPs serve as an ideal platform to investigate the chiral effect on amyloid fibrillation, when their surfaces are modified with chiral molecules. Since there is a growing interest to use the functionalized metal nanoparticles, synthesized by green methods, for various biological applications, amino acids are getting considerable attention as non-toxic mediating agents for successful synthesis of the nanoparticles.36-39 Glutamic acid offers several health benefits by contributing significantly to the central nervous system and is also important for detoxification of ammonia from the body, treatments for a range of neurological disorders, and production of energy for muscle function.40, 41. Hence, D- and L- enantiomeric forms of glutamic acid have been used as both reducing and stabilizing/capping agents, to synthesize the corresponding chiral gold nanoparticles i.e. DGAu and LGAu, respectively. To the best of our knowledge, there is no literature available that has investigated the role and mechanism of the surface chirality of gold nanoparticles as a new tunable property to regulate the fibrillation process of HSA. The objective of the present work is to explore the role of the surface chirality of glutamic acid-mediated gold nanoparticles (DGAu and LGAu) as a new tunable property to regulate the fibrillation process of HSA and also realize the underlying mechanism of the effect of surface chirality on protein-nanoparticle interactions. The potential of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

synthesized nanoparticles to affect the fibrillation process is monitored by the Thioflavin T(ThT) fluorescence study, Congo red (CR)-binding assay, circular dichroism (CD) spectroscopy, fluorescence microscopy, and transmission electron microscopy. Further, to gain additional insights about the modes of interaction behavior of the nanoparticles with the protein, Tryptophan (Trp) fluorescence spectroscopy and isothermal titration calorimetry studies have also been performed.

2. MATERIALS AND METHODS 2.1. Materials. All the chemicals were of analytical grade and purchased from commercial sources without further purification. Human serum albumin (HSA), Thioflavin T (ThT) and Congo red were purchased from Sigma-Aldrich Company. Tetrachloroauric acid (HAuCl4) and D- and L- Glutamic acids were procured from SRL Pvt. Ltd., India. Milli-Q water (resistivity 18.2 MΩ cm at 25 °C) was used to prepare all the solutions. 2.2. Synthesis and Characterization of Chiral Gold Nanoparticles (DGAu and LGAu). The chiral gold nanoparticles were synthesized by reducing HAuCl4 with D- and Lglutamic acid separately to yield D-glutamic acid mediated gold (DGAu) and L- glutamic acid mediated gold (LGAu) nanoparticles respectively. For this, 2 mL aqueous solution of 0.01% (w/v) tetrachloroauric acid was brought to boil under constant stirring, and subsequently, 2 mL of 2.5 mM glutamic acid solution was added under rapid stirring and heated at 95 °C for 15 min. Here, glutamic acid acts as both reducing and stabilizing agent. The reduction of the gold metal ions to yield gold nanoparticles was assessed by the appearance of pink colored solution for both the cases. To remove the free glutamic acid in each case, the resulting nanoparticle solutions were subjected to centrifugation at 8000 rpm for 30 min, washed with Milli-Q water and dried. This procedure was repeated twice. DGAu and LGAu were then subjected to characterization using various physico-chemical 6 ACS Paragon Plus Environment

Page 6 of 42

Page 7 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


techniques. The UV-visible absorption spectra of the samples were recorded on a Shimadzu2450 spectrophotometer to verify the characteristic surface plasmon resonance (SPR) band. X-ray diffraction (XRD) pattern was obtained using Cu−Kα radiation over the 2θ range of 20 −80° with an applied voltage of 40 kV using a Bruker AXS Diffractometer D8 powder XRD. In order to assess the presence of glutamic acids on gold nanoparticles, Fourier transform infrared (FTIR) spectroscopy was carried out using a Perkin-Elmer Spectrum RX-II (Model no. 73713, USA) within the scan range 4000– 800 cm-1. The morphology and size of the particles were obtained by transmission electron microscopy (TEM) using TECNAI G2 20STWIN (Japan) operating at an acceleration voltage of 200 kV. The size distribution histograms were prepared by measuring diameters of more than 100 particles from TEM images using ImageJ software (version 1.33; National Institutes of Health). The zeta potential and the hydrodynamic radius of the samples were determined by a Malvern Nano ZS instrument (Germany). Circular dichroism spectra for the chiral gold nanoparticles (final concentration for each solution kept at 0.2 µM) were recorded on a JASCO-815 automatic recording spectrophotometer at 25 °C between 190 to 260 nm using a quartz cuvette with a 0.1 cm path length. Each CD spectrum, recorded at a scan rate of 50 nm min-1, is an average of three scans. 2.3. Fibril Formation. The stock solution of HSA was prepared in 20 mM phosphate buffer of pH 7.4, and the protein concentration was measured spectrophotometrically at 280 nm using a molar extinction coefficient of 35 219 M−1 cm−1.42 To investigate the effect of the DGAu and LGAu on amyloid fibrillation of HSA, the protein was induced to fibrillation in absence and presence of the nanoparticles following the standard protocol.13, 43 Briefly, HSA (50 µM) was incubated without and with the nanoparticles (5 µM) in the presence of 60% (v/v) ethanol at 37 °C for 30 h. For each analysis, phosphate buffer of pH 7.4 (20 mM) was used for dilution of samples. 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 42

2.4. Turbidimetry Assay. Turbidity measurements of the different samples were performed using a Shimadzu-2450 UV−vis spectrophotometer. The turbidity of control HSA fibrils, as well as in the presence of the nanoparticles, was determined by monitoring the absorbance value at 350 nm. The concentration of the protein in the samples, used for measurements, was kept at 10 µM. Each spectrum was corrected with respect to the corresponding blank and the data are normalized accordingly. Experiments were performed in triplicate. 2.5. ThT Fluorescence Study. ThT fluorescence spectra were recorded in the wavelength range of 470−600 nm on a Horiba Jobin Yvon Fluoromax 4 spectrofluorimeter with the excitation at 450 nm. Aliquots from the protein samples, in the absence and presence of DGAu and LGAu, were withdrawn at definite time intervals and incubated with ThT for 5 min at room temperature. The protein and dye (ThT) concentrations used for measurements were kept at 2 and 10 µM, respectively. Excitation and emission slit widths were 5 nm each with an integration time of 0.3 s. All spectra were subtracted from their appropriate blanks. Each experiment was done in triplicate. As there may be attenuation of fluorescence due to the absorption of the incident and emitted light, inner filter effect has been taken into account and corrected for all the fluorescence spectra by the following relation.44 = × 10( )/

(1)

Where and is the corrected and observed fluorescence intensities respectively, and and are the absorbance at the excitation and emission wavelength respectively. 2.6. Congo Red (CR) Binding Study. CR difference spectra of various HSA solutions in presence and absence of the DGAu and LGAu was obtained by measuring the changes in the absorbance of CR binding assays using a UV−vis-NIR spectrophotometer, Shimadzu-2450 in the scanning range of 450−600 nm. The final protein and dye


Page 9 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentrations were kept at 4 and 10 µM, respectively. Each spectrum was corrected with respect to the corresponding blank. 2.7. Circular Dichroism (CD) Spectroscopy. Far-UV CD spectra (190–240 nm) were recorded at a scan rate of 50 nm min-1 on a JASCO-815 automatic recording spectrophotometer at room temperature, using a quartz cell of path length of 0.1 cm. For the CD measurements, the concentrations of HSA and the nanoparticles were kept at 2 and 0.2 µM, respectively. Each spectrum was corrected with respect to the appropriate blank and an average of three scans. The changes in protein secondary structure content were estimated using an online server, DICHROWEB.45 2.8. Fluorescence Microscopy. The treated protein solutions (10 µL) in the absence and presence of different gold nanoparticles were incubated with 5 µL of 1 mM ThT to achieve the required staining, transferred to a glass slide and then covered with a cover slip. Images were captured using a Leica DM 6000M microscope equipped with a fluorescence attachment and a Leica DFC 450 FX camera. Filter cube no 2 (Leica I3 11, 513, 878, BZ: 01) was used for ThT excitation and emission. All images were acquired at 10X/0.25 (N PLAN EPI). 2.9. Transmission Electron Microscopy (TEM). The treated HSA solutions, diluted 10-fold and negatively stained with uranyl acetate [1% (w/v)], were placed on carbon-coated TEM grids and air dried overnight. Then the samples were examined in a TECNAI G2 20STWIN transmission electron microscope operating at an accelerating voltage of 80 kV. 2.10. Steady State Fluorescence

Quenching Measurement.

Fluorescence

measurements were performed on a Horiba Jobin Yvon Fluoromax-4 spectrofluorimeter at 25 ± 0.1 °C with a 1 cm path length cell. Both excitation and emission slits were set at 5 nm. Samples were excited at 295 nm and emission spectra collected from 310 to 500 nm keeping the slit width and integration time at 2/2 nm and 0.3 s respectively. The concentration of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

nanoparticles (DGAu and LGAu) was varied from 0 to 0.015 µM, while the concentration of HSA was kept fixed at 0.15 µM. Triplicate titrations were conducted for each experiment. Each spectrum was corrected with respect to corresponding blank spectrum. It may be noted that the inner filter effect has been corrected here also, following the method described earlier, for the accuracy of fluorescence measurements. 2.11. Isothermal Titration Calorimetry. The energetics of the interaction of the chiral gold nanoparticles with HSA was studied by isothermal titration calorimetry (ITC) using an isothermal calorimeter (iTC200, Microcal, Northampton, MA. The titration involved successive injections of 0.3 mM HSA (titrant, 2 µL aliquot per injection) at 4 min intervals into the sample cell containing the solution of DGAu/LGAu (concentration, 4.33 µM) at 298 K. The titration cell was stirred continuously at 310 rpm. Reference power and initial delay were set to 5 µcal/s and 2 min, respectively. Each titration data were corrected for the heat of HSA dilution in buffer, which was determined in separate experiment under identical conditions. The data were fitted using a single site theoretical curve in Microcal ORIGIN software as it was found to be the best model to determine the binding stoichiometry (N), binding constant (Ka), and other thermodynamic parameters, as reported earlier.46 The reported thermodynamic quantities were the average of two identical experiments.

3. RESULTS AND DISCUSSION 3.1. Characterization of Nanoparticles. The successful preparation of the gold nanoparticles is initially validated through UV-Vis absorption spectroscopy (Figure S1). The spectra shows the characteristic surface Plasmon resonance (SPR) band, corresponding to the excitation of surface plasmons, centered at about 524 and 525 nm for DGAu and LGAu respectively, confirming the formation of gold nanoparticles.36 XRD analysis for DGAu and LGAu has been performed and similar diffraction patterns are observed for both the two 10 ACS Paragon Plus Environment

Page 10 of 42

Page 11 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


samples. The representative XRD pattern, shown in Figure S2, indicates presence of distinct peaks at 38.1°, 45°, 65° and 78° which can be indexed to corresponding (111), (200), (220) and (311) planes of a face-centered cubic (fcc) gold nanocrystals, in agreement with earlier literature.47 Further, the presence of glutamic acid group on gold surface has been verified by FTIR analysis and no difference in the appearance of the spectra is observed, as expected, between DGAu and LGAu due to the similarity in functional groups. Thus for simplicity, the FTIR spectra for free glutamic acid (GA) and only D-glutamic acid mediated gold nanoparticles (DGAu) are presented in Figure S3. As the nanoparticles are washed several times as stated earlier, therefore it may be assumed that there is no free glutamic acid present in the samples or at least not to the extent that it would affect the results of the present study. IR spectroscopic measurements of both GA and DGAu show bands at 1600-1700 cm-1 due to carboxylic acid C=O stretch and 1500-1550 cm-1 symmetrical N-H bending vibration.48,

49

However, the significant decrease in intensity at ~1515 cm-1 for DGAu, compared to GA, may be attributed of to the attachment of terminal amine to gold surface.47, 50 The decrease in intensity of the broad band at 3100-3600 cm-1, due to asymmetric stretching of amine N-H, again suggests amine-gold surface interaction.48 Thus, attachment of terminal amine groups to gold surface aids in the availability of carboxylic acid groups for binding to HSA. Additionally, to rule out the possibility of any effect of ethanol on synthesized nanoparticles, the FTIR spectra of the samples are acquired after incubating them with 60% ethanol and no change in the spectral pattern is observed. The morphology of the synthesized nanoparticles is determined using TEM and the corresponding images are shown in Figure S4. As evident from the images, mainly spherical gold nanoparticles, with almost uniform size distribution, are formed for both the samples and the mean particle diameters for DGAu (26.5 nm) and LGAu (28 nm) are found to be close to each other. The similar selected area electron diffraction (SAED) patterns for both DGAu and LGAu, shown in the Figure S4, corroborate



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with the XRD results indicating fcc crystal structure of the gold nanoparticles.51 Zeta potential values of DGAu (-13.3 mV) and LGAu (-12.9 mV) at pH 7.4 indicate that both the nanoparticles have a net negative charge on their surfaces due to the presence of carboxylic acid groups. The relatively high zeta potential magnitudes indicate about the fair stability of the nanoparticles. The colloidal stability of both the nanoparticles in phosphate buffer (pH 7.4) is further checked by DLS measurements and existence of any assembled larger particles is not observed even after 7 days (data not shown). Further, to verify chirality of the nanoparticles, DGAu and LGAu are subjected to CD analysis and the corresponding CD profiles are presented in Figure S5. It shows the necessary mirror image spectra, symmetric to X-axis, in the region of 190-260 nm with a strong positive signal for LGAu and a negative signal for DGAu. This indicates that the gold nanoparticles retain the chirality of the corresponding reducing agents, used for their synthesis. Thus, it may be assumed that the surface chirality is the main and only dissimilarity in DGAu and LGAu. 3.2. Turbidity Assay. In the turbidity assay, the presence of the larger aggregated species in the solution due to the self-assembly of proteins is manifested in the high absorbance value at 350 nm indicating the increased turbidity of the solution.12, 52 Thus, the effect of the gold nanoparticles on HSA aggregation is monitored by measuring the absorbance of the samples at 350 nm. The change in turbidity of the solutions of HSA in the absence and presence of the nanoparticles at different time intervals is presented in Figure 1.


Page 12 of 42

Page 13 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Change in turbidity of HSA solutions after incubation in the presence and absence of DGAu and LGAu. ± 1–3% error is associated with each experimental value which is estimated from at least three individual measurements. The turbidity of the HSA solutions is found to increase with time during the incubation and attains a plateau region, where the turbidity ceases to enhance further. As expected the control HSA fibrillar solution shows maximum turbidity indicating the presence of aggregated species in high amount, whereas presence of the nanoparticles shows decreases in the corresponding values. However, compared to LGAu, the presence of DGAu leads to a substantial reduction in the turbidity value compared to the control HSA fibrillar solution throughout the fibrillation process. Now, as the variation in solution turbidity is correlated with the extent of protein aggregation,53 it may be suggested that the self-association process of HSA into the aggregated species is more effectively hindered by DGAu than LGAu. 3.3. Thioflavin-T Fluorescence Spectroscopy. Thioflavin T (ThT) fluorescence assay is carried out to quantify the amyloid formation of HSA and compare the inhibitory effect of the chiral gold nanoparticles on the rate of fibril formation. The ThT dye, which is widely used in detection and quantification of amyloid fibrils, specifically binds to the cross β-sheet structure of amyloid resulting in considerable increase in fluorescence intensity.54 ThT does not fluoresce in its free state, but it displays considerable increase in fluorescence 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

intensity with an emission maximum at around ~485 nm, on excitation at 450 nm, due to binding with amyloid fibrils. Thus, the ThT fluorescence intensity (485 nm) is directly related to the amount of amyloid fibrils present in the solution.53 Kinetics of HSA fibril formation in the absence and presence of different gold nanoparticles is shown in Figure 2. In addition to DGAu and LGAu, the effect of gold nanoparticles (Au), synthesized by the common citrate reduction method,55 on HSA fibrillation is also examined by the ThT fluorescence study. The temporal evolution of HSA fibrils exhibit a typical sigmoidal curve containing a very short lag phase associated with nucleation, a rapid growth phase related to the elongation and propagation of fibrils, and a final equilibrium phase. The ThT fibrillation kinetics of HSA in the presence of Au nanoparticles show a time dependent increase of ThT fluorescence with no significant change in the profile of fibrillation kinetics of HSA in presence of the Au nanoparticles. Notably, the maximum fluorescence value, corresponding to the final state in the fibrillation process is only reduced by ~8%. This observation suggests that the Au nanoparticles are not able to influence the fibrillation of HSA appreciably. However, there occurs a change in the aggregation kinetics of HSA in the presence of the chiral gold nanoparticles indicating their interferences with fibril formation. If DGAu is compared with LGAu, an extended lag phase along with significant attenuation of the ThT fluorescence intensity of the equilibrium phase is observed for DGAu. It may be noted here that the ThT fluorescence intensities with and without the nanoparticles have also been checked to ensure there is no effect of the nanoparticles on ThT fluorescence under the present experimental condition employed herein. Therefore, the reduction of ThT fluorescence in the presence of the nanoparticles does not arise due to the quenching by the nanoparticles, rather due to the presence of amyloid fibrillar species in lower amount. Further, the ThT fluorescence study is also performed in presence of D- and L-glutamic acid separately, at the highest concentration level used in the synthesis of the nanoparticles, to rule out the possibility of any effect of the


Page 14 of 42

Page 15 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


free acids on the fibrillation of HSA (Figure S6). To gain quantitative information about the kinetics of HSA fibrillation, the experimental data are fitted to equation 2:56 =

!("!"#/$)%

(2)

Where y is the fluorescence intensity at time t, and & are the initial and maximum fluorescence intensities, respectively, '

/ is

the time required to reach half the

maximum fluorescence intensity, and k is the apparent first-order fibrillation constant. The lag time is calculated by equation 3: ()* '+,- = '

/

.

(3)

/

Figure 2. Variation of ThT fluorescence intensity (at 485 nm) with time in the absence and presence of different gold nanoparticles. The continuous lines through the data points are obtained through the fitting to the kinetic model. ±1–3% error is associated with each experimental value, estimated from at least three individual measurements. In the absence of the particles, the lag time and the apparent rate constant of the process is found to be 1.05 ± 0.25 h and 0.35 ± 0.03 h-1 respectively. However, the lag time in the presence of particles is increased to 3.62 ± 0.30 h in case of LGAu and to 9.13 ± 0.45 h for DGAu. The much higher value of the lag time in case of DGAu denotes that the nucleation process is significantly delayed in presence of DGAu. Moreover, the lower value 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in apparent rate constant (0.24 ± 0.02 h-1) for DGAu with respect to that of LGAu (0.30 ± 0.02 h-1) suggests that the elongation process is also more affected by the addition of the DGAu than LGAu. Notably, the maximum fluorescence value, corresponding to the final state in the fibrillation process, is reduced significantly for DGAu (~60%) compared to the control HSA fibrillar solution. However, LGAu causes ~26% reduction in the fluorescence value of the plateau. Thus, the results, in accord with the preceding turbidity assay, suggest that DGAu is more efficient than LGAu in inhibiting the formation of HSA fibrils. Further, to corroborate the findings from the ThT fluorescence assay, the modulation of HSA fibrillation by the chiral gold nanoparticles is validated by the Congo red binding study (Figure S7, details in the Supporting information). 3.4. Circular Dichroism Spectroscopy. Circular dichroism is a widely used spectroscopic technique to elucidate the changes in the secondary structure content of proteins during amyloid fibrillation which primarily involves the conversion of α-helix to βsheet.57 The CD spectra of native HSA and HSA fibrillar solutions in the absence and presence of the chiral gold nanoparticles are presented in Figure 3. It must be mentioned here that the final spectra of HSA solutions in presence of the nanoparticles, presented in Figure 3, are obtained by subtraction of the CD spectrum of the individual DGAu or LGAu solution (0.2 µM) from that of the corresponding spectrum for the HSA-D/LGAu solutions. Thus, the influence of the optical signals of the chiral gold nanoparticles on the CD spectra of HSA is avoided. The CD spectrum of native HSA displays the typical profile having two negative minimal peaks at 208 and 222 nm, suggesting the predominance of alpha helical conformation. As expected, the fibrillation of HSA, in absence of the nanoparticles, results in a considerable decrease in the mdeg values (less negative) at those two bands and the overall spectrum is confined to a single negative band at approximately 218 nm, which is a hallmark of β-sheet rich amyloid formation (inset of Figure 3).15 After incubation of HSA in the


Page 16 of 42

Page 17 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


presence of DGAu/LGAu, the CD spectra show noticeable changes in the appearance from that of control HSA fibrils. Nevertheless, it may be noted that the effect of DGAu is more prominent than LGAu in retaining the CD spectral features of α-helical HSA, as evident from the two distinct peaks at 208 and 222 nm with much higher mdeg values (more negative). These observations indicate the effective prevention of structural transition of HSA from αhelix to β-sheets, when DGAu is co-incubated with HSA. Thus, DGAu is believed to stabilize the native folded state of the protein and restrains the unfolding of the same leading to formation of fibrils.

Figure 3. Representative far-UV CD spectra of native HSA and the treated HSA solutions after 30 h incubation in the absence and presence of DGAu and LGAu. Inset showing the magnified CD spectrum of the control HSA fibrillar solution. Further, to gain insight in terms of the conformational stability induced by the nanoparticles, the change in free energy of folding (∆G) of HSA in presence of the different nanoparticles as a function of temperature can be estimated form CD study following the method reported earlier.12,

58

The fraction of folded HSA in the presence of nanoparticles

(0 ) may be represented as follows:



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0 =

(12 13 )

Page 18 of 42

(4)

(14 13 )

Where 5 , 56 and 57 are the mdeg values at 208 nm for HSA-NP solutions, HSA fibrillar solution and native HSA solution respectively. Now, if F and U are the concentrations of folded and unfolded forms of HSA respectively, the folding constant (K) is 8=

9 :

=

;2

(5)

( ;2 )

The free energy of folding (∆=°) is calculated as ∆=° = .?@(A8

(6)

where R is the gas constant (8.314 J K−1 mol−1) and T the incubation temperature (310 K). The negative value of ∆G° (folding) in case of DGAu (-1.89 ± 0.15 kJ/mole) suggests that co-incubation of HSA with DGAu causes stabilization of native HSA. This in turn affects the fibrillation process which is less favored due to the abundance of folded HSA molecules. However, the positive ∆G° (folding) value in presence of LGAu (1.377 ± 0.1 kJ/mole) indicates that it does not impart any stabilization to the native HSA leading to propensity of the HSA molecules to exist in the fibrillar state rather than the folded state under the experimental conditions employed. For further quantitative assessment of the changes in the secondary structure content of HSA, the online DICHROWEB server is used. The control HSA fibrillar solution is found to have a high β-sheet content of ~38% along with only ~10% α-helix. The presence of the DGAu and LGAu causes significant changes in the secondary structure content (Table 1). While LGAu results in ~26% relative reduction in βsheet along with ~53% relative increase in α-helix with respect to the control HSA fibrillar solution, the effect of DGAu is much more prominent as is evident from the respective values of the relative reduction in β-sheet (~60%) and relative increase in α-helix (~70%). These results, in agreement with the previous studies, suggest that conformational transition in HSA


Page 19 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


is more effectively mitigated upon co-incubation with DGAu than LGAu leading to successful modulation of fibrillation. Table 1. Secondary Structure Content (%) of HSA Solutions in the Absence and Presence of the Nanoparticles after Incubation for 30 h at 37 °C Sample α-helix (%) β-sheet (%) β-turn (%) Unordered (%) HSA fibril 10.2 ± 0.8 37.9 ± 1.5 22.8 ± 0.9 29.1 ± 1.7 HSA-DGAu 34.1 ± 1.3 15.2 ± 0.9 24.9 ± 1.6 25.8 ± 1.8 HSA-LGAu 21.8 ± 1.1 27.9 ± 1.8 23.8 ± 1.4 26.5 ± 1.3

3.5. Microscopic Studies: Fluorescence Microscopy And Transmission Electron Microscopy. To further investigate the efficacy of the chiral gold nanoparticles against HSA fibrillation, fluorescence and transmission electron microscopy are performed. ThT-stained fluorescence microscopy is an important tool to visualize the distribution and growth of amyloid fibrils.

Figure 4. Fluorescence microscopic images of HSA solution in the absence and presence of nanoparticles after incubation at 37 °C for 30 h: (a) HSA fibrils (b) HSA-DGAu (c) HSALGAu. Scale bars represent 200 µm. Figure 4a represents the fluorescence microscopic image of control HSA fibrillar solution, showing large amounts fibrillar networks with intense fluorescence intensities. When HSA is co-incubated with LGAu, presence of fibrillar species is clearly visible, although lesser than the control fibrillar solution (Figure 4c). However as compared with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

LGAu, incubation of HSA samples with DGAu results in a reduction in fluorescence intensity with noticeable depletion in the amount of the fibrillar moieties (Figure 4b). Additionally, TEM is utilized to monitor the morphological variation during the HSA fibrillation process. As shown in Figure 5a, the aggregation of HSA alone leads to the formation of long and network fibrils, similar to the earlier reports.9, 43 While co-incubation of HSA with LGAu does not result in effective reduction in fibrillar networks (Figure 5c), the presence of DGAu results in formation of non fibrillar amorphous aggregates leading to inhibition of fibril formation (Figure 5b). Thus, the results obtained from all the spectroscopic and microscopic techniques suggest that although LGAu can hinder the HSA fibrillation to a certain extent, DGAu exerts a significantly greater inhibitory effect on HSA fibril formation than the LGAu. These observations further indicate that surface chirality of the gold nanoparticles is an important factor in modulation of the fibrillation process of HSA.

Figure 5. TEM images of HSA solution after incubation at 37 °C for 30 h in the (a) absence and presence of (b) DGAu and (c) LGAu. Scale bars represent 200 nm. 3.6. Trp Fluorescence Quenching. The intrinsic fluorescence of HSA mainly results from the tryptophan (Trp) residue, located at the 214 position of subdomain IIA on excitation at 295 nm, due to its high quantum yield compared to the other aromatic amino acid residues (e.g. tyrosine and phenylalanine) present in the protein.59, 60 In this study, the Trp residue in native HSA has been selectively excited with an excitation wavelength of 295 nm, and an 20 ACS Paragon Plus Environment

Page 20 of 42

Page 21 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


emission maximum at 345 nm is observed. Fluorescence quenching measurements have been employed earlier for investigating binding and conformation changes of proteins upon interaction with nanoparticles.60, 61 The effect of the chiral gold nanoparticles on the intrinsic Trp fluorescence intensity of HSA is illustrated in Figure 6 showing the fluorescence emission spectra of HSA in the absence and presence of increasing concentration of DGAu and LGAu. It may be noted that the DGAu and LGAu has no fluorescence emission in the concerned region, upon excitation at 295 nm.

Figure 6. Fluorescence spectra of HSA (0.15 µM) with increasing concentration of (a) DGAu and (b) LGAu The result indicates that the gradual decrease in Trp fluorescence intensity of HSA with increase in the concentration of GAu. Quenching of Trp emission of serum albumins by gold nanoparticles has also been observed in earlier studies.16,

62

As the proximity of the

nanoparticle surface (quencher) and the Trp residue of HSA (fluorophore) dictate the efficiency of the fluorescence quenching, analysis of Trp fluorescence quenching of HSA by the DGAu and LGAu provides the insights into the relative accessibility of the chiral gold nanoparticles to the Trp residue.63, 64 Although the intrinsic fluorescence of HSA is found to be quenched for DGAu, there occurs a significant quenching in presence of LGAu. This result suggests that the adsorption of HSA molecules onto the surfaces of DGAu and LGAu



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 42

occur in a manner such that the Trp residue is closer to the LGAu surface in comparison to the DGAu surface. Thus, the close proximity of the Trp residue of HSA to the LGAu surface leads to more efficient quenching compared to DGAu. The reason for the differences in the relative proximity between HSA and the gold nanoparticles has been explained later. In addition, a slight red shift in the emission maxima indicates the less hydrophobic environment of the Trp residue with its more exposure to either the solvent or the nanoparticles during the interactions in both the systems. Further, to elucidate the quenching mechanism, the fluorescence quenching data is fitted to Stern–Volmer equation as follows:60 F0/F = KSV[GAu] +1 = Kqτ0[GAu] +1

(7)

Where F0 and F are the HSA fluorescence intensities in the absence and presence of synthesized gold nanoparticles (GAu) respectively; KSV is the Stern-Volmer quenching constant; Kq is the bimolecular quenching constant and τ0 is the lifetime of fluorophore i.e. tryptophan in the absence of quencher; and [GAu] represents the concentration of the gold nanoparticles. Lifetime (τ0) of HSA tryptophan is 5.6 ns.65 The plots (Figure 7) show a linear relationship between F0/F with [GAu] for both the nanoparticles, indicating the occurrence of static quenching. The much higher values of bimolecular quenching constants (Kq) for both DGAu (0.37 ×1016 M-1s-1) and LGAu (1.09 ×1016 M-1s-1) than the maximum value of the scattering collision quenching constant (2.0×1010 M-1s-1) suggests that the interaction between HSA and D/LGAu occurs thorough static quenching manner. The chiralitydependent interaction behavior of D/LGAu towards HSA is further revealed by the different Stern-Volmer quenching constant (KSV) values, where KSV for LGAu (6.11 ×107 M-1) is found to be almost three times larger than that of DGAu (2.09×107 M-1). Hence, the results suggest that the Trp residue in HSA is in much closer proximity to the surface of LGAu than DGAu.


Page 23 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. The Stern-Volmer plots derived from the quenching of Trp fluorescence of HSA by increasing concentration of (a) DGAu and (b) LGAu. 3.7. Isothermal Titration Calorimetry. In order to quantify the relative strength of the interaction between HSA and the nanoparticles (DGAu and LGAu), isothermal titration calorimetry (ITC) study is performed. ITC offers the most direct and reliable tool to study the energetics of protein-nanoparticle interactions, by quantifying binding affinity constant (Ka), stoichiometry (N), enthalpy changes (∆H) and entropy changes (∆S) during the binding process.66 The ITC responses measured during the titrations of the nanoparticles (DGAu and LGAu) with HSA and the binding curve analyzed with the single set of binding site model are presented in Figure 8. The derived parameters are summarized in Table 2. The results show that the interaction of HSA with both the DGAu and LGAu is associated with favorable enthalpy changes (i.e. ∆H

Does Surface Chirality of Gold Nanoparticles Affect Fibrillation of HSA

Recommend Documents