Morphological Effects of CuO Nanostructures on Fibrillation of Human

Subscriber access provided by READING UNIV

Article

Morphological Effects of CuO Nanostructures on Fibrillation of Human Serum Albumin Suraj Konar, Shubhatam Sen, and Amita Pathak J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b08432 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 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 B 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 41 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

Morphological Effects of CuO Nanostructures on Fibrillation of Human Serum Albumin Suraj Konar,1 Shubhatam Sen,2 Amita Pathak1* 1

Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India 2

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

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 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: The influence of different morphologies of nanostructures on amyloid fibrillation has been investigated by monitoring the fibrillation of human serum albumin (HSA) in presence of rod, sphere, flower and star shaped copper oxide (CuO) nanostructures. The different morphologies of CuO have been synthesized from aqueous solution based precipitation method using various organic acids viz. acetic acid, citric acid and tartaric acid. The fibrillation process of HSA has been examined using various biophysical techniques e.g. Thioflavin T fluorescence, Congo red binding studies through UV spectroscopy, circular dichroism spectroscopy and fluorescence microscopy. The monolayer protein coverage on the CuO nanostructures has been established through DLS studies and the well-fitted Langmuir isotherm model has been used to interpret the differential adsorption behaviour of HSA molecules on the CuO nanostructures. The nanostar-shaped CuO, by virtue of their higher specific surface area (94.45 m2 g−1), presence of high indexed facets {211} and high positive surface charge potential (+16.2 mV at pH 7.0) were found to show the highest adsorption of the HSA monomers and thus were more competent to inhibit the formation of HSA fibrils compared to the other nanostructures of CuO.


Page 2 of 41

Page 3 of 41 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

Development of amyloid fibrils from unfolded and partially folded proteins results in numerous neurodegenerative disorders1-3. Such types of disorders are mainly associated with the accumulation of intracellular toxic amyloid fibrils with cross β-sheet rich content 4. The fibrillation process involves the self-assembly of protein monomers into the short-lived oligomers, which in turn transforms into long-lived oligomers that produces the fibrils

5

eventually. Consequently, inhibition or disturbance of this fibrillation process is regarded as one of the emergent propositions in the prevention of all amyloid-related diseases. In recent times, nanomaterial-modulated fibrillations of various amyloidogenic proteins have gained attention as they are found to play a pivotal role in promoting, hindering, or disintegrating the fibrillation process 6-8. Nanomaterials are reported to have strong interactions with the protein monomers or growing oligomers leading to interference in the nucleation as well as in the elongation step 9-10 of the protein fibrillation process. Owing to the dimensional similarity of the nanomaterials with the protein molecules, they are also known to offer significant therapeutic potential by regulating the fibrillation process. The physicochemical properties of the nanomaterials including their chemical composition

11

, surface charge12 and surface chirality

13

have been recognized as key

factors for controlling the propensity of various proteins to form fibrils. Few researchers have also studied the effect of size and shape of the engineered nanoparticles on the modulation of amyloid protein aggregation 14-16 however, the mechanistic details of the influence of the various morphologies of the nanostructures on the fibrillogenesis are still elusive. Human serum albumin (HSA), a physiologically important serum protein (owing to its crucial role in the transportation of fatty acids, metal ions and other essential compounds like vitamins, hormones, amino acids and drugs 17), is considered as a model protein for studying the 3 ACS Paragon Plus Environment


fibrillogenesis due to its propensity to aggregate in vitro under suitable condition

Page 4 of 41

18-19

. Their

fibrillation can be easily accomplished by disturbing the ambient physiological conditions of pH, temperature and ionic strength

19-21

. Besides, the effects of various additives such as – alcohol,

sugar, surfactants and metal ions on HSA aggregation have been well studied22-25. On the contrary, only a few reports are available on nanomaterials modulated fibrillation of HSA

26-27

while, the morphological effects of the nanomaterials on fibrillation of HSA is yet to be elucidated. Copper, a necessary trace element i.e. micronutrient, plays a vital role in formation of red blood cells, human metabolism and subsists in the body bound to amyloidogenic proteins

28-30

.

Cu nanoparticles have been reported to exhibit the ability to inhibit proteins aggregation 31-32. In addition, Cu (II) ions have been found to significantly affect fibrillation of peptides and proteins 33-34

and their interaction as well as their effect on HSA fibrillation has also been investigated in

earlier reports

23,35

. It may also be noted that besides Cu (II) ion and Cu nanoparticles, CuO

nanoparticles have also been studied in various biological studies e.g. magnetic resonance imaging, interaction study with HSA etc. 36-37. Thus, to gain better insight on the morphological effect of nanostructures on the amyloid fibrillation process CuO has been chosen as a platform for the present study. Herein, the rod, sphere, flower and star shaped morphologies of CuO nanostructures have been synthesized using aqueous based chemical precipitation technique and their potency to inhibit the fibrillation of HSA have been investigated. The influence of synthesized nanostructures on the HSA fibrillation process has been monitored through the Thioflavin T (ThT) fluorescence study, Congo red (CR)-binding assay, circular dichroism (CD) spectroscopy and fluorescence microscopy. Further, the adsorption capacity of the nanostructures with HSA


Page 5 of 41 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


molecules has also been investigated through DLS studies and the Langmuir isotherm model has been used to interpret the differential adsorption behaviour of HSA molecules on the CuO nanostructures. The adsorption ability of various morphologies of nanostructures to HSA has been explained on the basis of their specific surface area, facet directed growth and surface charge potential.

2.

MATERIALS AND METHODS 2.1.

Materials. All chemicals were of analytical grade and were used without further

purification. Copper acetate monohydrate (Cu(CH3COO)2.H2O, Merck Ltd, Mumbai, 98%), Acetic acid (Merck Ltd, Mumbai, 99%), Tartaric acid (Merck Ltd, Mumbai, 98%), Citric acid (Merck Ltd, Mumbai, 98%), HPLC grade ethanol (Merck Ltd, Mumbai) and Sodium hydroxide (NaOH, Merck Ltd, Mumbai, ≥ 97%) were used in this experiment. Human serum albumin (HSA), Congo red (CR) and Thioflavin T (ThT) were purchased from Sigma Chemical Co. (St. Louis) and were used as received. 2.2.

Synthesis and Characterization of CuO Nanostructures. CuO nanostructures

were synthesized through previously reported method by our research group 38. Briefly, aqueous solution of copper acetate (0.1 M) was mixed with excess of NaOH at 100 °C by maintaining the solution pH at 10. Then, the solution was cooled to room temperature and black precipitate of CuO was washed with water and separated by centrifugation. The residue was dried in a vacuum oven at 120 °C for 6 h to obtain the dry powder of CuO (referred as CuOR in the text). The above procedure for obtaining CuO was repeated with the addition of acetic acid, tartaric acid and citric acid in the initial aqueous solution of copper acetate and the thus-obtained black powder have been referred to as CuOP, CuOF and CuOS respectively in the following text. 5 ACS Paragon Plus Environment


The crystalline phase of CuO nanostructures in the synthesized samples were ascertained through powder X-ray diffraction (XRD) studies, carried out on a Bruker AXS Diffractometer D8 powder XRD, Germany, using Cu-Kα radiation (λ = 1.5418 Ǻ) at the applied voltage of 40 kV and at a scan rate of 3° min-1 between the 2θ range of 30 - 80°. Presence of surface functional groups in the synthesized samples were established through Fourier transformed infrared (FTIR) spectroscopy, using Perkin-Elmer Spectrum RX-II, Model no. 73713, USA, within a spectral range of 4000–400 cm-1. The morphology of the synthesized CuO nanostructures were analyzed by Carl Zeiss MERLIN scanning electron microscope (SEM), Germany, outfitted with a field emission gun at an accelerating voltage of 5 kV. Diluted CuO samples were drop casted on aluminum foil and coated with gold prior to microscopic study. Zeta potential measurement was performed to determine the surface charge of the samples using a Malvern Nano ZS instrument, U.K. The isoelectric point (IEP) of CuO was determined using aqueous dispersion of the samples in a series of pH values ranging between 2 and 10. Each measurement was done in triplicate. The specific surface areas of the degassed (at 200 °C for 6 h) CuO samples have been calculated from the nitrogen adsorption-desorption isotherms, carried out on Micromeritics 3Flex Surface Characterization Analyzer, USA, using the standard Brunauer–Emmett–Teller (BET) adsorption model. 2.3.

HSA Fibrillation Study

2.3.1

Fibril Formation. The HSA stock solution was freshly prepared by dissolving

HSA in Milli-Q water and stored at 4 °C. Accurate HSA concentration was calculated by UVVis spectrophotometer (Shimadzu UV-2450) using molar extinction coefficient of 35,219 M−1 cm−1 at 280 nm39. The stock solutions of each of the CuO nanostructures were prepared by dispersing 0.25 mg of the respective CuO samples in 1 ml of phosphate buffer [of pH 7.0 (20


Page 6 of 41

Page 7 of 41 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


mM)] and subjected to ultrasonication for 2 min prior to use. To study the influence of the various CuO nanostructures on the inhibition of HAS fibrillation, 200 µL of the each of the CuO stock solutions were separately added into 50 µM HSA at pH 7.0 (20 mM phosphate buffer) in presence of 60% (v/v) ethanol and incubation at 37 ºC for 24 h. As a control, the HSA solution (without the CuO) was incubated in phosphate buffer of pH 7.0 (20 mM) under the same experimental condition24. The experimental techniques used for studying the inhibition of fibrillation of HSA in the presence of CuO nanostructures (such as – ThT fluorescence, CR binding study, CD spectroscopy and ThT fluorescence imaging analysis) have been carried out using only the supernatant, after separation of CuO from the protein solution through centrifugation. 2.3.2. ThT Fluorescence. The fibrillation of HSA solutions in presence and absence of CuO nanostructures was monitored using Thioflavin-T (ThT) fluorescence study. The stock solution of ThT dye was prepared in phosphate buffer and the concentration was determined spectrophotometrically using the molar extinction coefficient at 416 nm of 26600 M−1cm−1.40 To monitor the ThT binding, the aliquots withdrawn from various sets of solutions at definite time intervals and incubated with ThT for 5 min at room temperature. The final concentrations of protein and ThT dye were kept 2 µM 10 µM respectively. The fluorescence of the aliquots were measured at 25 °C using a Horiba Jobin Yvon Fluoromax 4 spectrofluorimeter with an excitation wavelength of 450 nm and the emission spectra were observed within the wavelength range of 470 nm to 600 nm. The slit width and integration time were kept at 5 nm and 0.3 s respectively. All spectra were corrected with respect to the corresponding blank. Each measurement was done at least three times and error bars represent the standard deviation from the mean value. The possibility of reduction of fluorescence due to the absorption of incident and emitted light, the



Page 8 of 41

inner filter effect has been taken into account and all the fluorescence spectra were corrected by the following relation 41: = × 10

( )

(1)

Where and are the corrected and observed fluorescence intensities of the samples respectively, and are the absorbance of the samples at the excitation and emission wavelength respectively. 2.3.3. Congo Red (CR) Binding Study. The CR difference absorption spectra of various HSA fibrillar solutions in the presence and absence of different CuO nanostructures was measured using an UV−Vis absorption spectrophotometer, Shimadzu UV–2450 within the scanning range of 450 to 625 nm using a quartz cuvette of 1 cm path length. The final protein and dye concentrations were kept at 4 µM and 10 µM respectively in the final solutions. The solutions were diluted by using 20 mM phosphate buffer (pH 7.0). Each spectrum was corrected with respect to the corresponding blank i.e. the difference absorption spectrum of the CR + HSA solution (i.e., solution of CR bound HSA) was acquired after subtracting the absorbance of the CR solution (i.e., aqueous solutions of free CR). Similarly, the difference spectra of the CuO + CR + HSA solutions (i.e., solution of CuO and CR bound HSA) were generated after subtracting the absorbance of the aqueous suspension of the respective CuO nanostructures (i.e., aqueous suspension of the free CuO of the respective morphology) and that of the aqueous solutions of the CR solution (i.e., aqueous solutions of free CR). 2.3.4. Circular Dichroism (CD) Spectroscopy. Far-UV CD spectra of the samples were observed on a JASCO-810 automatic recording spectrophotometer under a constant nitrogen flow at 25 °C. The CD spectra were acquired in a strain free quartz cuvette of 0.1 cm path length, in the wavelength range of 190 to 240 nm at a scan rate of 50 nm per min. The aliquots of native


Page 9 of 41 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


HSA, HSA fibrils, HSA fibrillar solutions containing different CuO nanostructures were diluted with 20 mM phosphate buffer (pH 7.0) to achieve final HSA concentration of 2 µM for each solution. The changes in secondary structural contents of HSA was determined by using the online DICHROWEB server

42

. Each spectrum was corrected with respect to the appropriate

blank. Each experiment has been performed in triplicate and error bars represent the standard deviation from the mean value. 2.3.5. Fluorescence Microscopy. 10 µL of HSA fibrillar solutions in absence and presence of CuO nanostructures were incubated with 5 µL of 1 mM ThT to achieve the required staining. Then the solutions were transferred to a glass slide and covered with a cover slip. The microscopic images were captured using a Leica DM 2500M microscope equipped with a fluorescence attachment. 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) 2.3.6. Adsorption Studies. In order to get further insight, the adsorption studies of HSA with CuO nanostructures were performed by gentle swirling different concentrations of native HSA (10, 20, 30, 40, 50, 60 and 70 µM) into a fixed amount of dispersed CuO nanostructures (200 µL of 0.25 mg/mL = 50 ppm) separately in 10 mM phosphate buffer (pH 7.0) for 2 h in a rotary shaker at 200 rpm at 25 °C. The solution was left to settle for 30 min to achieve the equilibrium. Then the solutions were centrifuged at 10,000 rpm for 5 min and the supernatant solutions were transferred into new eppendorfs for analysis. The residue containing nanostructures-HSA aggregates further dispersed in phosphate buffer and hydrodynamic diameters were measured through dynamic light scattering method (DLS, HORIBA Scientific, Nanoparticle Analyzer SZ-100). The adsorption capacity of CuO nanostructures with HSA molecules was estimated from the amount of unreacted HSA in supernatant solutions by



Page 10 of 41

measuring their absorbance at 280 nm through UV-Vis spectrophotometer (Shimadzu UV-2450). For the control experiment, different concentrations of native HSA (10, 20, 30, 40, 50, 60 and 70 µM) in the absence of the nanostructures were gently swirled for 2 h in a rotary shaker at 200 rpm at 25 °C, settled for 30 min and measured their absorbance at 280 nm through UV-Visible spectrophotometer.

3.

RESULTS AND DISCUSSIONS 3.1.

Characterization of Nanoparticles. The indexed XRD pattern (Figure 1A) of

various CuO nanostructures, are nearly equivalent to each other and match with the JCPDS file number 80-1916 indicating monoclinic structure with the space group of C2/c. The FTIR spectra of CuO nanostructures are shown in Figure 1B, where the characteristic peak positioned at ~430 cm-1 can be ascribed to Cu-O stretching band along (202) direction, peaks at ~520 and ∼610 cm-1 can be assigned to Cu-O stretching along (202) direction

43

. The absorption peaks positioned at

around 1540 and 1410 cm-1 in CuOP, 1615 and 1365 cm-1 in CuOF can be assigned to antisymmetric and symmetric C=O stretching modes of carboxylate groups respectively, probably due to the adsorbed acetate and tartarate anions on CuO surface

44

. Similarly, in the FTIR

spectrum of CuOS, the peaks at ∼1580 and ∼1375 cm-1 may be attributed to stretching frequencies of C=O resulting from the citrate group adsorbed on CuOS surface

45

. In the FTIR

spectrum of CuOS sample, the absence of peaks in between 1680 and 1750 cm-1 indicates the absence of unreacted acids, which has been used as chelating and capping agent. The XRD and FTIR spectra of all the samples confirm the purity of the CuO samples.


Page 11 of 41 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.

(A) Indexed XRD and (B) FTIR spectra of (a) CuOR, (b) CuOP, (c) CuOF and

(d) CuOS nanostructures. The surface morphology of the synthesized CuO nanostructures is visualized through the SEM, as shown in Figure 2. The SEM images of the CuO nanostructures demonstrate the homogeneous distribution of rod, sphere, flower and star shaped morphologies (Figure 2A, B, C and D respectively). The rod shaped particles of CuOR possess lengths in between 130-160 nm, the spherical particles of CuOP have diameters in between 15-25 nm and flower shaped CuOF consists of four to six branches with particle size in between 130-160 nm. The SEM image of CuOS sample shows the particles are star-shaped in morphology with the conical tip (quadpod, pentapod or hexapod) lengths varying between 80 – 100 nm (Figure 2D).



Figure 2.

SEM images of (A) CuOR, (B) CuOP, (C) CuOF and (D) CuOS nanostructures.

The scale bars represent 200 nm. The surface charge of CuO samples are determined from the zeta potential measurements by dispersing the samples at different pH solutions and the graphs are shown in Figure 3. The isoelectric points (IEP) of CuOR, CuOP, CuOF and CuOS nanostructures are found to be 6.4, 8.1, 9.2 and 9.5 respectively.


Page 12 of 41

Page 13 of 41 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 3.

Surface charge potentials of CuO nanostructures at different pH values. Error bars

represent the standard deviation from the mean value estimated from at least three individual measurements. The specific surface areas of degassed (at 200 °C for 6 h) samples are calculated from the nitrogen adsorption – desorption isotherms as shown in Figure 4, using the Brunauer–Emmett– Teller (BET) adsorption model. The specific surface area values for CuOR, CuOP and CuOF are found to be 37.75, 55.68 and 70.24 m2 g−1 whereas, the value is increased to 94.45 m2 g−1 for star-shaped CuOS. The higher value of specific surface area for CuOS may be ascribed to the presence of many sharp conical tips on their surface.



Figure 4.

Nitrogen adsorption-desorption isotherms of (A) CuOR, (B) CuOP, (C) CuOF and

(D) CuOS nanostructures. 3.2.

HSA Fibrillation Study

3.2.1. ThT Binding Study. Thioflavin T (ThT), a fluorescent dye, exclusively binds to cross β-sheet structures of amyloid fibrils and shows significant increase in fluorescence intensity at 485 nm when excited at 450 nm, allowing one to quantify the amount of amyloid fibrils

46

. The effect of the CuO nanostructures on the HSA fibrillation process has been

investigated by the ThT binding fluorescence study. It is important to note that, ThT does not


Page 14 of 41

Page 15 of 41 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


exhibit discernible change in fluorescence intensity in the control sets comprising native HSA and CuO nanostructures separately, when excited at 450 nm. The representative ThT fluorescence spectra of HSA solutions in absence and presence of various CuO nanostructures after incubation at 37 ºC for 24 h is presented in Figure 5A. Co-incubation of CuOR, CuOP and CuOF with HSA causes ~21%, ~43% and ~53% reduction in the ThT fluorescence intensity respectively while CuOS results in the maximum reduction (~61%) in ThT fluorescence intensity, compared to the control HSA fibrillar solution. As the decrease in ThT fluorescence intensity is indicative of reduced formation of amyloid fibrils, it may be suggested that the star shaped CuOS is more efficient than other CuO nanostructures in inhibition of HSA fibrillation.

Figure 5.

(A) Representative ThT fluorescence spectra of various HSA solutions in the

absence and presence of CuO nanostructures after incubation at 37 °C for 24 h, and (B) Normalized ThT fluorescence intensity at 485 nm of the same HSA fibrillar solutions at different time intervals. The continuous lines through the data points are obtained through the fitting to the kinetic model using equation 2. Error bars represent the standard deviation from the mean value estimated from at least three individual measurements.



Page 16 of 41

The development of HSA fibrils with time in the absence and presence of various CuO nanostructures is monitored by the ThT fluorescence kinetics study (Figure 5 B), with extraction of aliquots at definite time intervals. In general, the fibrillation of proteins proceeds through a lag phase, which involves formation of “critical” nucleus by the association of protein monomers or oligomers, followed by a rapid elongation of fibrils. During the incubation of HSA in absence of nanostructures, the kinetics curve shows a sigmoidal nature with a very short lag phase followed by a growth phase, related to elongation of fibrils, and finally reaching the equilibrium plateau which corresponds to the final state of fibrillation process. 13 The similar types of curves are also obtained in case of HSA-CuOR and HSA-CuOP, indicating that CuOR and CuOP were not able to inhibit the fibrillation process in HSA appreciably. However, for the HSA-CuOF and HSACuOS solutions, a noticeable extended lag phase followed by a sigmoidal growth is observed. This finding indicates that the presence of flower and star shaped CuO nanostructures results in extension of the lag phase (where ThT-detectable species are unavailable) followed by exponential growth to the final plateau region. To gain quantitative information about the ThT fluorescence kinetics of HSA fibrillation, the experimental data are fitted to the following equation. 27,47 = ! +

#$ % #&'&(&$) *+

(2)

,((,(- )/ .

Where y is the fluorescence intensity at definite time t,

!

and &

are the initial and

maximum fluorescence intensities respectively, 0*. is the time required to reach half the 1

maximum fluorescence intensity, and 2 is the apparent first-order fibrillation constant. The lag time is calculated by using the following equation 3. 1

345 0678 = 0*. − :

(3)

1


Page 17 of 41 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 the absence of the nanostructures, the lag time of the fibrillation process of native HSA is found to be 0.19 ± 0.05 h. However, the lag time in the presence of CuOR and CuOP nanostructures is increased to 1.26 ± 0.10 h and 2.04 ± 0.12 h respectively. However, the lag time in presence of CuOF and CuOS is significantly increased to 6.42 ± 0.16 h and 13.23 ± 0.23 h respectively. The much higher value of the lag time in case of CuOS indicates that the nucleation process is significantly delayed in presence of CuOS. The results suggest that CuOS shows more efficiency than other CuO nanostructures in restricting the formation of critical nuclei, causing hindrances in the preliminary stage of fibrillation process and thereby inhibiting the formation of HSA fibrils. Further, to validate the findings of ThT fluorescence assay, the inhibition of HSA fibrillation by CuO nanostructures is also investigated by the Congo red binding assay. Table 1: ThT Fluorescence Kinetic Data of the of HSA Solutions in the Absence and Presence of CuO Nanostructures [shown in Fig. 5B] Lag time (h) Sample Plateau value (a.u.) t1/2 (h) HSA 29.2 ± 1.3 7.9 ± 0.12 0.19 ± 0.05 CuOR + HSA 23.8 ± 1.5 9.2 ± 0.15 1.26 ± 0.10 CuOP + HSA 18.1 ± 1.1 10.5 ± 0.32 2.04 ± 0.12 CuOF + HSA 14.2 ± 0.9 15.3 ± 0.29 6.42 ± 0.16 CuOS + HSA 11.9 ± 1.1 17.2 ± 0.40 13.23 ± 0.23

3.2.2

Congo Red (CR) Binding Study. Congo red (CR), a hydrophobic azo dye being

used as a probe to identify the formation of the cross β-sheet rich amyloid fibrils. CR binds with the amyloid fibrils due to the electrostatic interaction in between positively charged amino acids of protein and negatively charged sulfonic groups in CR. CR, in its free state, gives the maximum absorption peak at 498 nm; However, in presence of amyloid fibrils, a characteristic red shift of 35-40 nm in the maxima of CR absorption spectrum is observed 48. The CR binding assay has been performed to evaluate the inhibitory action of CuO nanostructures towards the



Page 18 of 41

formation of HSA fibrils and the corresponding difference spectra are presented in Figure 6. In the absence of nanostructures the native HSA fibrils shows a characteristic spectral maximum at ∼541 nm, signifying the presence of well-defined amyloid fibrils. In case of HSA-CuOR and HSA-CuOP nanostructures, the spectral maxima are found at ∼540 nm and ∼538 nm respectively indicating the formation of increased cross β-sheet rich structures, whereas in case of HSACuOF and HSA-CuOS the absorption maxima are found at ∼533 nm and ∼530 nm respectively. This result indicates that all the solutions except HSA-CuOF and HSA-CuOS have produced well-ordered amyloid fibrillar species. It is important to note that, the CuO nanostructures alone at the experimental concentrations did not alter the absorbance of CR (data not shown). In the CR difference absorption spectra (as shown in Figure 6) the shifts in the position of the peak or changes in the extinction coefficient of the dye on addition of the protein, or vice versa, are only indicative of changes in the electronic environment of the molecule and hence is only a reflection 49

of the interaction of the CR dye molecules with the HSA fibrils . Comparison of the spectral shifts in the CR absorption studies thus only qualitatively signifies the relative amounts of HSA fibrils contained in the different solution mixtures. Quantification of the amount of HSA fibrils in the different samples have therefore been carried out and reported through the analysis of the secondary structural contents of HSA protein probed through CD spectroscopy.


Page 19 of 41 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 6.

The Congo red difference absorption spectra for various HSA fibrillar solutions

incubated with CuOR, CuOP, CuOF and CuOS at 37 °C for 24 h. 3.2.3

Circular Dichroism Study. Circular dichroism (CD) spectroscopy is a sensitive

technique to quantify the changes in various secondary structural contents of a protein during their fibrillation process. In present study, CD spectroscopy is used to probe the effect of CuO nanostructures on the conformational changes of HSA before and after inducing the fibrillation condition. Figure 7A represents the far–UV CD spectra of native HSA and HSA fibrillar solutions in absence and presence of CuO nanostructures. Two negative minima (at 208 and 222 nm) observes in the CD spectrum of native HSA, indicate the α-helicity of the protein50. The negative bands at 208 nm and 222 nm respectively correspond to the π → π* and n → π* transitions of the carbonyl groups in the polypeptide chains. Fibrillation of native HSA is marked by significantly decreased intensities (i.e., lower negative mdeg values) of these two bands in the CD spectrum, suggesting substantial loss of α-helicity along with enhancement of β- sheet structure. The HSA solution incubated in presence of the CuO nanostructures, show a noticeable



increase in the mdeg values (i.e., more negative mdeg values) for both the bands (at 208 and 222 nm) compared to the control HSA fibrillar solution which suggests their ability to retain the αhelicity of HSA. The effect is however observed to be more pronounced in presence of CuOS compared to the other samples of CuO.

Figure 7.

(A) Far-UV CD spectra and (B) corresponding histogram of % secondary

structure content of native HSA and HSA fibrillar solution in absence and presence of CuOR, CuOP, CuOF and CuOS after incubation at 37 °C for 24 h in the presence of 60% (v/v) ethanol. Error bars represent standard deviation from the mean value estimated from at least three individual measurements. The quantification of the amount of secondary structures in the native HSA as well as in the HSA fibrils (in presence and absence of the CuO nanostructures) has been carried out using the online DICHROWEB server and the corresponding histogram is represented in Figure 7B. Native HSA was found to contain ~54% α-helix and only ~7% β-sheet, while HSA fibrillar solution (in the absence of CuO nanostructures) showed ~24% α-helix and ~25% β-sheets. This indicates the conversion of the α-helix to β-sheet during fibrils formation, which is the hallmark


Page 20 of 41

Page 21 of 41 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 amyloid fibrillation. Conversely, a substantial variation in the secondary structural content is observed in the presence of CuO nanostructures where the amount of β-sheet is found to decrease along with an increase in the amount of α-helix with respect to the control HSA fibrils, as is evident from Fig. 7B. From the observations it can be inferred that the presence of CuO nanostructures hinders the α-helix to β-sheet conversion. The inhibition in the α-helix to β-sheet conversion is found to follow in the order: CuOR < CuOP < CuOF < CuOS where, CuOS showed maximum inhibition ability towards the conversion. This observation from the CD studies is well consistent with the result achieved from ThT fluorescence and CR binding assays. For studying the influence of surface directing agents, ThT fluorescence and CD spectroscopic studies for HSA fibrillation have also been carried out in presence of the various free acids (i.e., acetic/tartaric/citric), as a control. It may be mentioned that no noticeable changes in the ThT fluorescence spectra and CD spectra of HSA fibrillar solution were observed in presence of the acids at the concentration level used in this work. This reiterates that the observed changes in ThT fluorescence, CR binding study and CD spectroscopic studies could only be due to the presence of the various morphologies of CuO nanostructures in the HSA fibrillar solution. 3.2.3. Fluorescence Microscopic Study. Fluorescence microscopic technique is employed to observe the morphological development of HSA fibrils in presence and absence of CuO nanostructures after the incubation for fibrillation. The fluorescence microscopic image of HSA fibrils without CuO nanostructures (Figure 8A) shows an intense ThT fluorescence indicating the presence of HSA fibrillar moieties in large quantity. Nevertheless, in presence of CuO nanostructures, the ThT fluorescence intensity of HSA fibrillar solutions is found to be reduced appreciably and the extent of ThT fluorescence intensity of HSA in presence of CuO



shows the following order CuOR > CuOP > CuOF (Figure 8 B shows the fluorescence microscopic image of HSA-CuOR, other images are not shown). The diminished fluorescence intensity may be attributed to the absence of the large amount of fibrillar species in the solutions. However, the fluorescence intensity is found to be significantly lower in presence of CuOS (Figure 8 C), as compared to other CuO nanostructures, suggesting that CuOS exhibits higher inhibitory potency than others. Thus, the results from all the biophysical techniques discussed above indicate that the star shaped CuO exhibits significantly higher efficiency in inhibition of HSA fibrillation.

Figure 8.

Fluorescence microscopic images of HSA solutions in the absence and presence

of CuOR and CuOS after incubation at 37 °C for 24 h in the presence of 60% (v/v) ethanol at pH 7.0 respectively. The scale bars represent 100 µm. 3.2.4 Adsorption Study. Fibrillation process is established to be affected by adsorption of proteins on the surface of the nanostructures which in turn depends on the physicochemical parameters of the nanostructures such as size, shape, surface charge, composition and surface modification 12,14,16,51. Jiang et al. had noticed that the plasma protein HSA was adsorbed on the surface of FePt nanoparticles in a monolayer fashion forming nanoparticle-HSA corona52. Catlano et al. showed that bovine serum albumin (BSA) can be used as a dispersing agent for


Page 22 of 41

Page 23 of 41 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


large aggregates formed by two types of nanoparticles, namely – the pyrolytic SiO2 and colloidal SiO2, in PBS buffer and a model was proposed based on the external area of the agglomerates obtained from DLS study53. They found that multilayers and monolayer of irreversibly adsorbed BSA molecules formed with the pyrolytic SiO2 and the colloidal SiO2 respectively. Following course, the present study also investigates the nature of the CuO nanostructure-HSA adsorption with the help of DLS studies.

Figure 9.

Hydrodynamic diameter values of CuO nanostructures suspended in PBS buffer

(0 µM HSA), then added to HSA solutions within the concentration range of 10-70 µM, centrifuged and then resuspended in phosphate buffer. Thus, DLS data refers to the fraction of HSA irreversibly adsorbed on CuO agglomerates. The hydrodynamic diameters (DH) of CuO nanostructures were measured after removal of the CuO nanostructures adsorbed with the HSA molecules and resuspended in phosphate buffer (10 mM, pH = 7.0) and the same has been shown in Figure 9. The DH of the CuO nanoparticles in phosphate buffer solution reflected agglomeration and the diameters were found to be much higher than those observed from TEM or SEM analysis that were carried out using an aqueous 23 ACS Paragon Plus Environment


Page 24 of 41

dispersions of CuO38. Interestingly, the DH of the CuO nanoparticles in phosphate buffer solution was found to gradually decrease with the addition of HSA (as is depicted in Figure 9). This suggests that the diffusion of protein molecules takes place throughout the interparticle spaces and remain adsorbed the surface and do not reach the core of nanostructure agglomerates53. The protein (HSA) coverage of CuO nanostructures were calculated based on the external area of the agglomerates by assuming the agglomerates are of same size and shape, spherical in nature with diameters equal to the DH measured in the phosphate buffer. The values of DH and the details of the calculations are tabulated in Table S1 and corresponding surface coverage values are shown in Figure S1 in the Supporting Information. The results of these calculations indicate that the proteins form monolayers on the surfaces of three morphologies of CuO nanostructures (CuOR, CuOP and CuOS) with the surface coverage (θ) value of 1. For CuOF, the surface coverage was found to be slightly higher (θ = 1.07) than the theoretical ‘side-on’ monolayer however, they too have been assumed to have monolayer cover of the protein molecules within the experimental limits. Thus, from DLS studies, the HSA molecules can be inferred to be adsorbed onto various morphologies of CuO nanostructures in monolayer fashion. However, to understand the fibrillation process of HSA in presence of the different morphologies of CuO nanostructures, it is essential to examine their adsorption capacity towards native HSA, because the un adsorbed / free protein are known to initiate the formation of fibrils

54

. The present study employs the

Langmuir adsorption model for estimating the adsorption capacity of the different CuO nanostructures towards HSA, where a fixed amount of each of respective CuO nanostructures were mixed with varying concentrations of HSA and the amount of unreacted HSA in supernatant solutions were estimated by measuring their respective absorbance at 280 nm


Page 25 of 41 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


through UV-Vis spectrophotometer. Although the Langmuir adsorption model is indeed more appropriate for quantifying the amount of adsorbate adsorbed on the adsorbent surface either in gas phase or in liquid phase (at a given temperature) and is effectively more relevant to small adsorbate molecules than to the complex molecules such as proteins. However, it is also one of the most versatile models that have been applied to accurately interpret the adsorption isotherms for a large variety of systems. Moreover, there are quite a number of literature reports that have applied the model to interpret protein adsorption on solid surfaces51,55-56. Recently, Maleki et al. used the Langmuir isotherm model for measuring the adsorption ability of BSA by gold nanoparticles57. They obtained well fitted Langmuir isotherm and interpreted the typical protein adsorption process accordingly even though they violate each one of the four requirements for the Langmuir model

58

[i.e., (a) homogeneous adsorption sites, (b) monolayer adsorption of

protein molecules, (c) reversible equilibrium between adsorbed proteins with unbound proteins during the experimental period, and (d) absence of interaction force between protein molecules on the surface to alter their adsorption behavior58]. With the formation of HSA monolayer upon CuO nanostructures having been established through DLS studies in the present investigation, and that being one of the primary requirements for the validity of the Langmuir isotherm model, justifies the interpretation of the CuO-protein (HSA) adsorption studies through Langmuir adsorption model. The well-known equation for Langmuir isotherm model is in the following form 59: ?

;< = *+ =

… (4)

$ >?

The linear form of Langmuir isotherm is represented as follows >? ?

= < + =

*

… (5)

$ CuOF > CuOP > CuOR.

4.

CONCLUSIONS

The fibrillation process is established to be strongly dependent on the morphology of CuO nanostructures. The inhibiting efficiency of star shaped CuO nanostructures is found to be higher than the rod, spherical and flower shaped CuO nanostructures towards HSA fibrillation. The ThT binding study shows ~61% reduction of ThT fluorescence intensity and the CD binding study indicates a ~45% relative decrease in β-sheet content in presence of CuO nanostars as compared to control HSA fibrillar solution. The ThT kinetic study and CR binding studies suggest that star shaped CuO nanostructure efficiently reduce the formation of HSA fibrils by extending the lag phase, thus affecting the nucleation and growth phase. With the help of DLS study the protein coverage was calculated which indicates that HSA forms monolayer on the agglomerates of CuO nanostructures in phosphate buffer solution. Langmuir adsorption isotherm reveals that the 30 ACS Paragon Plus Environment

Page 30 of 41

Page 31 of 41 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


adsorption capability of nanostars is higher than the other shaped morphologies which facilitates trapping of HSA monomers and reduce the free HSA monomers in solution. The presence of high indexed facets and relatively large value of BET specific surface area explain the superior adsorption ability of nanostars than others. Zeta potential measurements of nanostructures clearly show that the electrostatic interactions are major driving force for the interaction between the star-shaped CuO nanostructures and HSA. We believe that this study provides fruitful information regarding the effect of morphological variation of nanostructures on fibrillation of proteins.

Author Information Corresponding Author * Email: [email protected]; Telephone: +91 3222 281922, +91 94340 38730

Notes The authors declare no competing financial interest.

Acknowledgements The authors would like to acknowledge the Central Research Facility (CRF), IIT Kharagpur for providing infrastructural facilities. S. Konar thanks the Indian Institute of Technology Kharagpur for financial support.



REFERENCES 1.

Dobson, C. M. Protein folding and misfolding. Nature 2003, 426, 884-890.

2.

Stefani, M.; Dobson, C. M. Protein aggregation and aggregate toxicity: New insights into

protein folding, misfolding diseases and biological evolution. J. Mol. Med. 2003, 81, 678-699. 3.

Selkoe, D. J. Folding proteins in fatal ways. Nature 2004, 428, 445-445.

4.

Kagan, B. L.; Thundimadathil, J. Amyloid Peptide Pores and the Beta Sheet

Conformation. Adv. Exp. Med. Biol. 2010, 677, 150-167. 5.

Mulaj, M.; Foley, J.; Muschol, M. Amyloid Oligomers and Protofibrils, but Not

Filaments, Self-Replicate from Native Lysozyme. J. Am. Chem. Soc. 2014, 136, 8947-8956. 6.

Brancolini, G.; Toroz, D.; Corni, S. Can small hydrophobic gold nanoparticles inhibit β2-

microglobulin fibrillation? Nanoscale 2014, 6, 7903-7911. 7.

Yoo, S. I.; Yang, M.; Brender, J. R.; Subramanian, V.; Sun, K.; Joo, N. E.; Jeong, S. H.;

Ramamoorthy, A.; Kotov, N. A. Inhibition of Amyloid Peptide Fibrillation by Inorganic Nanoparticles: Functional Similarities with Proteins. Angew. Chem. Int. Ed. 2011, 50, 51105115. 8.

Zhang, D. M.; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.;

Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J. Gold Nanoparticles Can Induce the Formation of Protein-based Aggregates at Physiological pH. Nano Lett. 2009, 9, 666-671. 9.

Linse, S.; Cabaleiro-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S.

E.; Dawson, K. A. Nucleation of protein fibrillation by nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 8691-8696.


Page 32 of 41

Page 33 of 41 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


10.

Choi, T. S.; Lee, H. H.; Ko, Y. H.; Jeong, K. S.; Kim, K.; Kim, H. I. Nanoscale Control

of Amyloid Self Assembly Using Protein Phase Transfer by Host-Guest Chemistry. Sci.Rep. 2017, 7, 5710. 11.

Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Dawson, K. A.; Linse, S. Dual Effect

of Amino Modified Polystyrene Nanoparticles on Amyloid β Protein Fibrillation. ACS Chem. Neurosci. 2010, 1, 279-287. 12.

Moores, B.; Drolle, E.; Attwood, S. J.; Simons, J.; Leonenko, Z. Effect of Surfaces on

Amyloid Fibril Formation. PLoS One 2011, 6, e25954. 13.

Sen, S.; Dasgupta, S.; DasGupta, S. Does Surface Chirality of Gold Nanoparticles Affect

Fibrillation of HSA? J. Phys. Chem. C 2017, DOI: 10.1021/acs.jpcc.7b05354. 14.

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? Scientific Reports 2016, 6, 19548. 15.

Moore, K. A.; Pate, K. M.; Soto-Ortega, D. D.; Lohse, S.; van der Munnik, N.; Lim, M.;

Jackson, K. S.; Lyles, V. D.; Jones, L.; Glassgow, N.; et al. Influence of gold nanoparticle surface chemistry and diameter upon Alzheimer's disease amyloid-β protein aggregation. J. Biol. Eng. 2017, 11, DOI 10.1186/s13036-017-0047-6. 16.

Goy-Lopez, S.; Juarez, J.; Alatorre-Meda, M.; Casals, E.; Puntes, V. F.; Taboada, P.;

Mosquera, V. Physicochemical Characteristics of Protein-NP Bioconjugates: The Role of Particle Curvature and Solution Conditions on Human Serum Albumin Conformation and Fibrillogenesis Inhibition. Langmuir 2012, 28, 9113-9126. 17.

Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.; Ascenzi, P. Human serum

albumin: from bench to bedside. Mol. Aspects Med. 2012, 33, 209-290.



18.

Taboada, P.; Barbosa, S.; Castro, E.; Mosquera, V. Amyloid fibril formation and other

aggregate species formed by human serum albumin association. J Phys Chem B 2006, 110, 20733-20736. 19.

Juarez, J.; Taboada, P.; Mosquera, V. Existence of Different Structural Intermediates on

the Fibrillation Pathway of Human Serum Albumin. Biophys. J. 2009, 96, 2353-2370. 20.

Juarez, J.; Taboada, P.; Goy-Lopez, S.; Cambon, A.; Madec, M. B.; Yeates, S. G.;

Mosquera, V. Additional Supra-Self-Assembly of Human Serum Albumin under Amyloid-LikeForming Solution Conditions. J. Phys. Chem. B 2009, 113, 12391-12399. 21.

Taboada, P.; Barbosa, S.; Castro, E.; Gutierrez-Pichel, M.; Mosquera, V. Effect of

solvation on the structure conformation of human serum albumin in aqueous-alcohol mixed solvents. Chem. Phys. 2007, 340, 59-68. 22.

Pandey, N. K.; Ghosh, S.; Dasgupta, S. Fructose restrains fibrillogenesis in human serum

albumin. Int. J. Biol. Macromol. 2013, 61, 424-432. 23.

Pandey, N. K.; Ghosh, S.; Nagy, N. V.; Dasgupta, S. Fibrillation of human serum

albumin shows nonspecific coordination on stoichiometric increment of Copper(II). J. Biomol. Struct. Dyn. 2014, 32, 1366-1378. 24.

Pandey, N. K.; Ghosh, S.; Dasgupta, S. Effect of surfactants on preformed fibrils of

human serum albumin. Int. J. Biol. Macromol. 2013, 59, 39-45. 25.

Stirpe, A.; Pantusa, M.; Rizzuti, B.; Sportelli, L.; Bartucci, R.; Guzzi, R. Early stage

aggregation of human serum albumin in the presence of metal ions. Int. J. Biol. Macromol. 2011, 49, 337-342.


Page 34 of 41

Page 35 of 41 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


26.

Sen, S.; Konar, S.; Pathak, A.; Dasgupta, S.; DasGupta, S. Effect of Functionalized

Magnetic MnFe2O4 Nanoparticles on Fibrillation of Human Serum Albumin. J. Phys. Chem. B 2014, 118, 11667-11676. 27.

Sen, S.; Konar, S.; Das, B.; Pathak, A.; Dhara, S.; Dasgupta, S.; DasGupta, S. Inhibition

of fibrillation of human serum albumin through interaction with chitosan-based biocompatible silver nanoparticles. RSC Adv. 2016, 6, 43104-43115. 28.

Lovell, M. A.; Robertson, J. D.; Teesdale, W. J.; Campbell, J. L.; Markesbery, W. R.

Copper, iron and zinc in Alzheimer's disease senile plaques. J. Neurol. Sci. 1998, 158, 47-52. 29.

Wright, J. A.; Wang, X. Y.; Brown, D. R. Unique copper-induced oligomers mediate

alpha-synuclein toxicity. FASEB J. 2009, 23, 2384-2393. 30.

Bonomo, R. P.; Deflora, A.; Rizzarelli, E.; Santoro, A. M.; Tabbi, G.; Tonetti, M.

Copper(II) Complexes Encapsulated in Human - Red-Blood-Cells. J. Inorg. Biochem. 1995, 59, 773-784. 31.

Ishtikhar, M.; Rahisuddin; Khan, M. V.; Khan, R. H. Anti-aggregation property of

thymoquinone induce by copper-nanoperticles: A biophysical approach. Int. J. Biol. Macromol. 2016, 93, 1174-1182. 32.

Ishtikhar, M.; Usmani, S. S.; Gull, N.; Badr, G.; Mahmoud, M. H.; Khan, R. H. Inhibitory

effect of copper nanoparticles on rosin modified surfactant induced aggregation of lysozyme. Int. J. Biol. Macromol. 2015, 78, 379-388. 33.

Ghosh, S.; Pandey, N. K.; Bhattacharya, S.; Roy, A.; Nagy, N. V.; Dasgupta, S. Evidence

of two oxidation states of copper during aggregation of hen egg white lysozyme (HEWL). Int. J. Biol. Macromol. 2015, 76, 1-9.



34.

Jin, L.; Wu, W. H.; Li, Q. Y.; Zhao, Y. F.; Li, Y. M. Copper inducing A beta 42 rather

than A beta 40 nanoscale oligomer formation is the key process for Aβ neurotoxicity. Nanoscale 2011, 3, 4746-4751. 35.

Pandey, N. K.; Ghosh, S.; Dasgupta, S. Fibrillation in Human Serum Albumin Is

Enhanced in the Presence of Copper(II). J. Phys. Chem. B 2010, 114, 10228-10233. 36.

Chibber, S.; Ahmad, I. Molecular docking, a tool to determine interaction of CuO and

TiO2 nanoparticles with human serum albumin. Biochem. and Biophys. Rep. 2016, 6, 63-67. 37.

Perlman, O.; Weitz, I. S.; Azhari, H. Copper oxide nanoparticles as contrast agents for

MRI and ultrasound dual-modality imaging. Phys. Med. Biol. 2015, 60, 5767-5783. 38.

Konar, S.; Kalita, H.; Puvvada, N.; Tantubay, S.; Mahto, M. K.; Biswas, S.; Pathak, A.

Shape-dependent catalytic activity of CuO nanostructures. Journal of Catalysis 2016, 336, 1122. 39.

Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to Measure and Predict the

Molar Absorption-Coefficient of a Protein. Protein Sci. 1995, 4, 2411-2423. 40.

Darghal, N.; Garnier-Suillerot, A.; Salerno, M. Mechanism of thioflavin T accumulation

inside cells overexpressing P-glycoprotein or multidrug resistance-associated protein: Role of lipophilicity and positive charge. Biochem. Biophys. Res. Commun. 2006, 343, 623-629. 41.

Peng, W.; Ding, F.; Jiang, Y. T.; Sun, Y.; Peng, Y. K. Evaluation of the biointeraction of

colorant flavazin with human serum albumin: insights from multiple spectroscopic studies, in silico docking and molecular dynamics simulation. Food Funct. 2014, 5, 1203-1217. 42.

Whitmore, L.; Wallace, B. A. DICHROWEB, an online server for protein secondary

structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 2004, 32, W668-W673.


Page 36 of 41

Page 37 of 41 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


43.

Dar, M. A.; Kim, Y. S.; Kim, W. B.; Sohn, J. M.; Shin, H. S. Structural and magnetic

properties of CuO nanoneedles synthesized by hydrothermal method. Appl. Surf. Sci. 2008, 254, 7477-7481. 44.

An, H. R.; Cai, F. S.; Wang, X. W.; Yuan, Z. H. A simple method for controlling the

morphology of CuO nanostructures by droplet. Adv. Eng. Mater. II, 2012, 535, 280-283. 45.

Yang, L. X.; Liu, B.; Li, G. H. Morphology-controlled fabrication of CuO architectures.

Adv. Mater. Res.2012, 581, 544-547. 46.

LeVine, H. Quantification of beta-sheet amyloid fibril structures with thioflavin T.

Methods Enzymol. 1999, 309, 274-284. 47.

Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.;

Fink, A. L. Effect of environmental factors on the kinetics of insulin fibril formation: Elucidation of the molecular mechanism. Biochemistry 2001, 40, 6036-6046. 48.

Wu, J. W.; Liu, K. N.; How, S. C.; Chen, W. A.; Lai, C. M.; Liu, H. S.; Hu, C. J.; Wang,

S. S. S. Carnosine's Effect on Amyloid Fibril Formation and Induced Cytotoxicity of Lysozyme. PLoS ONE 2013, 8, e81982. 49.

Kamaljeet; Bansal, S.; SenGupta, U. A Study of the Interaction of Bovine Hemoglobin

with Synthetic Dyes Using Spectroscopic Techniques and Molecular Docking. Front. Chem. 2017, 4, DOI: 10.3389/fchem.2016.00050. 50.

Bhattacharya, A.; Das, S.; Mukherjee, T. K. Insights into the Thermodynamics of

Polymer Nanodot-Human Serum Albumin Association: A Spectroscopic and Calorimetric Approach. Langmuir 2016, 32, 12067-12077.



51.

Scopelliti, P. E.; Borgonovo, A.; Indrieri, M.; Giorgetti, L.; Bongiorno, G.; Carbone, R.;

Podesta, A.; Milani, P. The Effect of Surface Nanometre-Scale Morphology on Protein Adsorption. PLoS ONE 2010, 5, e11862. 52.

Jiang, X.; Weise, S.; Hafner, M.; Rocker, C.; Zhang, F.; Parak, W. J.; Nienhaus, G. U.

Quantitative analysis of the protein corona on FePt nanoparticles formed by transferrin binding. J. R. Soc. Interface 2010, 7, S5-S13. 53.

Catalano, F.; Alberto, G.; Ivanchenko, P.; Dovbeshko, G.; Martra, G. Effect of Silica

Surface Properties on the Formation of Multilayer or Submonolayer Protein Hard Corona: Albumin Adsorption on Pyrolytic and Colloidal SiO2 Nanoparticles. J. Phys. Chem. C 2015, 119, 26493-26505. 54.

McMasters, M. J.; Hammer, R. P.; McCarley, R. L. Surface-induced aggregation of beta

amyloid peptide by ω-substituted alkanethiol monolayers supported on gold. Langmuir 2005, 21, 4464-4470. 55.

Mohsen-Nia, M.; Bidgoli, M. M.; Behrashi, M.; Nia, A. M. Human Serum Protein

Adsorption onto Synthesis Nano-Hydroxyapatite. Protein J. 2012, 31, 150-157. 56.

Roach, P.; Farrar, D.; Perry, C. C. Surface tailoring for controlled protein adsorption:

Effect of topography at the nanometer scale and chemistry. J. Am. Chem. Soc. 2006, 128, 39393945. 57.

Maleki, M. S.; Moradi, O.; Tahmasebi, S. Adsorption of albumin by gold nanoparticles:

Equilibrium and thermodynamics studies. Arabian J. Chem. 2017, 10, S491-S502. 58.

Latour, R. A. The Langmuir isotherm: A commonly applied but misleading approach for

the analysis of protein adsorption behavior. J. Biomed. Mat. Res. Part A 2015, 103, 949-958.


Page 38 of 41

Page 39 of 41 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


59.

Langmuir, I. The Adsorption Of Gases On Plane Surfaces Of Glass, Mica And Platinum.

J. Am. Chem. Soc. 1918, 40, 1361-1403. 60.

Arya, S.; Kumari, A.; Dalal, V.; Bhattacharya, M.; Mukhopadhyay, S. Appearance of

annular ring-like intermediates during amyloid fibril formation from human serum albumin. Phys. Chem. Chem. Phys. 2015, 17, 22862-22871. 61.

Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle

size and surface properties determine the protein corona with possible implications for biological impacts. P. Natl. Acad. Sci. U.S.A. 2008, 105, 14265-14270. 62.

Kulkarni, M.; Mazare, A.; Park, J.; Gongadze, E.; Killian, M. S.; Kralj, S.; von der Mark,

K.; Iglic, A.; Schmuki, P. Protein interactions with layers of TiO2 nanotube and nanopore arrays: Morphology and surface charge influence. Acta Biomater. 2016, 45, 357-366. 63.

Yang, S. T.; Liu, Y.; Wang, Y. W.; Cao, A. N. Biosafety and Bioapplication of

Nanomaterials by Designing ProteinNanoparticle Interactions. Small 2013, 9, 1635-1653. 64.

Su, D. W.; Xie, X. Q.; Dou, S. X.; Wang, G. X. CuO single crystal with exposed {001}

facets - A highly efficient material for gas sensing and Li-ion battery applications. Sci. Rep. 2014, 4, 5753. 65.

Rabe, M.; Verdes, D.; Seeger, S. Understanding protein adsorption phenomena at solid

surfaces. Adv. Colloid Interface Sci. 2011, 162, 87-106. 66.

Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Factors affecting the clearance

and biodistribution of polymeric nanoparticles. Mol. Pharmaceutics 2008, 5, 505-515.



TOC GRAPHIC


Page 40 of 41

Page 41 of 41 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


52x44mm (150 x 150 DPI)

ACS Paragon Plus Environment

Morphological Effects of CuO Nanostructures on Fibrillation of Human

Recommend Documents