Uniform, Polycrystalline, and Thermostable Piperine-Coated Gold

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Uniform, polycrystalline and thermostable piperinecoated gold nanoparticles to target insulin fibril assembly Bibin Gnanadhason Anand, Dolat Singh Shekhawat, Kriti Dubey, and Karunakar Kar ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 02 May 2017 Downloaded from http://pubs.acs.org on May 6, 2017

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ACS Biomaterials Science & Engineering

Uniform, polycrystalline and thermostable piperinecoated gold nanoparticles to target insulin fibril assembly Bibin G. Anand2, Dolat S. Shekhawat2, Kriti Dubey2 and Karunakar Kar1* 1

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

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Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India-342011

KEYWORDS. Piperine, Gold nanoparticles, Insulin, Amyloids, Thioflavin T, Hemocompatible

ACS Paragon Plus Environment

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ABSTRACT. Since the process of insulin fibril assembly is linked to a multitude of medical problems, finding effective and biocompatible inhibitors against such aggregation process could be beneficial. Targeting the aggregation prone residues of insulin may perhaps work as an effective strategy to prevent the onset of insulin fibril assembly. In this work, we have synthesized uniform sized, thermostable gold nanoparticles (AuNPspiperine) surface-functionalized with piperine to target amyloid-prone residues of insulin. We found that the process of both spontaneous and seedinduced amyloid formation of insulin was strongly inhibited in the presence of AuNPspiperine. Surface functionalization of piperine was found to be critical to its inhibition effect because no such effect was observed for free piperine as well as for uncoated control gold nanoparticles. Fluorescence quenching data revealed binding of AuNPspiperine with insulin’s native structure which was further validated by docking studies that predicted viable H-bond and CH-π interactions between piperine and key aggregation-prone residues of insulin’s B-chain. Our hemolysis assay studies further confirmed that these piperine coated nanoparticles were hemocompatible. Data obtained from both experimental and computational studies suggest that the retention of native structure of insulin and the ability of the piperine molecule to interact with the aggregation-prone residues of insulin are the key factors for the inhibition mechanism. The findings of this work may help in the development of nanoparticle-based formulations to prevent medical problems linked to insulin aggregation.


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1. INTRODUCTION: The process of amyloid fibril formation of proteins is considered as one of the foundational events that trigger the onset of a number of pathologies including several neurodegenerative diseases (1, 2). About 40 amyloidogenic proteins have been identified to be responsible for several diseases such as Aβ-linked Alzheimer’s disease, huntingtin-linked Huntington’s disease and α-synuclein-linked Parkinson’s disease (3). Aggregation of normal functional proteins such as insulin into amyloid fibrils has also been reported to be linked to many medical severities (4-6). Insulin fibril assembly is known to be directly linked to type 2 diabetes (7). The storage of insulin as therapeutic agent is also a big concern because of the tendency of insulin molecules to form toxic amyloid aggregates (8, 9). Formation of amyloid like aggregates of insulin has been reported in the artery walls located at the site of injection (6). Hence, one of the effective strategies to target these aggregation linked severities could be the prevention of the process of protein aggregation. Successful designing of effective inhibitors of protein aggregation process has gained much attention in recent years. Several candidates including single molecules, amino acids, natural compounds, peptides and proteins have been reported to act as inhibitors of amyloid fibril formation of different proteins (8, 10-15). Over the past decade much research has also focused on the surface functionalization of metallic nanoparticles with potential compounds to target amyloid formation of proteins (15-18). Various properties of the nanoparticles, such as their size, thermal stability as well as their biocompatibility are considered as critical factors for their inhibition effect against amyloid fibril formation of proteins (19-22). Few studies of our group under in vitro conditions have found that the anti-amyloid activity of inhibitors is greatly enhanced when these inhibitor molecules are surface functionalized with the nanoparticles (15, 23).


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Based on these information, we chose piperine, a natural compound known for its multiple health benefits (see supplementary Table S1), to target the process of insulin fibril assembly. The piperine molecule has a unique structure (Figure 1a), which is already known to interact with proteins and DNA molecules through viable H-bonds mediated through its C=O and –O– functional groups (24-27). Formation of strong H-bonds between piperine and valine residues of protein molecules has already been reported (25). Recent studies have revealed that “LVEALYL” segment of the B-chain of insulin plays a key role to begin the fibril formation process (28, 29). Peptides containing “LVEALYL” sequence have been reported to influence the aggregation properties of insulin. Based on these evidences, we hypothesized that piperine could be an effective molecule to interact with “LVEALYL” segment of insulin’s B chain, preferably with the valine residue and we postulated that such piperine-insulin interaction would restrict the process of insulin fibril assembly. Since surface functionalization of inhibitor molecules on nanoparticles have been reported to greatly enhance their inhibition efficacies against the process of protein aggregation (15, 23), we synthesized gold nanoparticles surface-functionalized with piperine to target aggregation prone segment of insulin.

We have successfully synthesized uniform sized (~10 nm) gold nanoparticles which are surface functionalized with piperine and have tested their inhibition effect on amyloid fibril formation of insulin under in vitro conditions. We have examined the inhibition effect of piperine coated gold nanoparticles on both spontaneous and seed-induced fibril assembly process of insulin. Using both experimental and computational approaches we have also explored the possible molecular interaction between piperine and insulin. Finally, the hemocompatibility of piperine coated nanoparticles have been tested.


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2. EXPERIMENTAL SECTION:

2.1. Materials: Piperine (from natural source with purity of ≥ 97 %) was obtained from Sigma-Aldrich. AuCl4, lyophilized powder of type I collagen (calf skin), bovine serum albumin (BSA) were procured from Sigma-Aldrich. Type I Collagen extracted from RTT (rat tail tendon) was obtained from Dr. Balaraman’s laboratory at CLRI India. The preparation of RTT collagen sample for fibril assembly studies was done following the established protocol (30, 31) (see the methods in the supplementary information). Insulin was obtained from HIMEDIA (India). All other reagents and chemicals used in this work were purchased either from HIMEDIA or SigmaAldrich. Extinction coefficients used for insulin was 6080 M-1cm-1 at 278 nm and 43824 M-1cm-1 at 280 nm for BSA. 2.2. Synthesis of gold nanoparticles: The synthesis of piperine functionalized gold nanoparticles was carried out by following established protocol (15) in which approximately 0.2 mM of piperine was boiled under stirring condition with 1 mM KOH. To this boiling sample, ~0.2 mM of AuCl4 was added and the mixed sample was then kept under stirring condition till the end of the reaction as indicated by the change in of the color of the sample towards ruby red (absorbance peak within 510-520 nm). The control gold nanoparticles used in our experiment were prepared by citrate-capped method (16). A sample containing 0.2 mM HAuCl4 was boiled and stirred under the reflux condition for 30 min and 7.76 mM of freshly prepared aqueous Tri Sodium citrate (Na3C6H5O7) was added directly into the boiling solution of HAuCl4. The reaction was allowed to continue for ~20 to 30 min to attain the maximum reduction. These synthesized gold nanoparticles were aged for 7 days at room temperature to make sure that all the piperine molecules present in the solution get conjugated to nanoparticles. Initial characterization of both the control and piperine coated gold nanoparticles were examined by


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measuring the absorption spectra using UV Spectrophotometer (Shimadzu Japan UV1800). This method assumes that 100% reduction reaction during the synthesis of the gold nanoparticle in the presence of piperine. Thus the concentration of piperine that was used to prepare the gold nanoparticle was considered for adding to the aggregating insulin sample at different molar ratios. To get rid of any free piperine, if present in the solution, the synthesized nanoparticles were centrifuged at ~16162 rcf after which the sample was resuspended in the vial and stored at room temperature. For inhibition studies the molar ratio values maintained for aggregation reactions in the presence of coated nanoparticles were determined considering the concentration of the conjugated piperine molecules. 2.3. Fluorescence spectroscopy: A Perkin Elmer LS-55 fluorescence spectrometer was used for all fluorescence related experiments reported in the current study. For monitoring the quenching of insulin in the presence of AuNPspiperine, we measured the fluorescence emission of the insulin sample in the presence of different concentrations of AuNPspiperine. The λex and λem values used for insulin were fixed at 276 nm and 305 nm respectively. The slit width for both excitation and emission was set at 10 nm throughout the measurements. Detailed methods for quenching studies in the current study are available in the supplementary information. To study the amyloid fibril formation of protein samples, Thioflavin T binding assay was used following the established protocol (32, 33). For ThT measurements, the final sample volume was maintained at 1ml, which contained 100 µl of the protein sample undergoing aggregation reaction (at 43 µM), 50 µl of the ThT solution from a stock of 1.25 mM and 850 µl of 1X PBS. The sample containing ThT was then excited at 440 nm with a slit width of 5 nm and the emission was observed at 490 nm. For every reading mentioned in the text, ThT reading of the sample containing everything except the protein aggregates was always recorded which was


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used as blank for the same sample. It was confirmed that Thioflavin T signal was not altered in the presence of gold nanoparticles, and piperine coated gold nanoparticles. Hence, in this study, the decrease in the ThT-signal in the inhibited aggregation reactions points to the inhibition of amyloid fibril formation in the presence of piperine coated gold nanoparticles. For seed induced aggregation studies ~15% (w/w) preformed fibrils were used as seeds as reported in previous studies (32, 34, 35). 2.4. Circular dichroism (CD) spectroscopy. We performed CD experiments by using JASCO CD spectrometer (model J-815-150 L) with attached Peltier temperature controller. The pathlength of the cuvette was 2 mm and the band width was set at 5 nm. We recorded CD spectra of insulin samples undergoing aggregation in the presence and in the absence of AuNPspiperine. All the CD measurements have been carried out at room temperature and the scan speed for recording the signal was maintained at 50 nm per minute. CD signal of the PBS buffer was used as the reference (for blank) for the insulin sample in the absence of inhibitors. However, to record the CD signal of insulin sample from an inhibited reaction, [AuNPspiperine + PBS buffer] was used as the reference (for using as blank). Hence, the reported CD spectra described in this study are all base-line corrected ones. 2.5. Amyloid fibril formation of proteins. Amyloid fibril formation was initiated by incubating the protein monomer samples in PBS (pH 7.4) at ~70°C (15) in the presence and in the absence of AuNPspiperine, free piperine and AuNPscontrol. For conducting seed-induced aggregation of proteins, we followed the established protocol (15, 35) in which preformed amyloid fibrils (~15 % weight/weight) of the respective proteins were used as seeds. 2.6 Collagen fibril formation. Collagen stock solution was prepared by dissolving the lyophilized powder of collagen in 20% acetic acid solution (maintaining collagen concentration


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at 2 mg.ml-1) at 4°C, under stirring condition at least for three days. For studying fibril formation, final sample volume was maintained at 1 ml which had 100 µL of 10X PBS buffer make it 1X PBS at pH 7.4. This sample was then immediately transferred to the cuvette for recording the absorbance reading (at 37°C). The process of collagen fibril formation was studied by monitoring the absorbance of collagen sample at 313 nm using Spectrophotometer (Thermo Scientific NanoDrop 2000c) (30). The concentration of collagen was maintained at 0.3 mg ml−1 (in PBS, pH 7.4), and measurements were recorded at 37 °C. The reference solution for every experiment was prepared from water, AuNPspiperine and 10x PBS buffer, without collagen for the baseline correction. 2.7. Lysozyme activity: The activity of lysozyme was determined against M. lysodeikticus using the turbidometric method as reported earlier (11). The decrease in turbidity of a 1 ml bacterial-cell suspension (0.3 mg.ml-1) in buffer (50 mM phosphate buffer maintained at pH 6.5) was monitored after the addition of 100 µl of lysozyme from stock solution of 1 mg.ml-1 (~70µM). To the reference cell, 100 µl of lysozyme solution was added. The decrease in absorbance at 450 nm was monitored every 1s during a total incubation of 3 min. Activity was measured in the presence of AuNPspiperine at 1:10 and 1:20 molar ratio values of protein:ligand. The lysozyme sample (100 µl from 70µM stock) was incubated with AuNPspiperine ( ~350 µl and 700 µl from a ~200µM nanoparticle stock solution to maintain the molar ratios of 1:10 and 1:20 respectively. The same amount of AuNPspiperine was added to the reference cell. For the preparation of all the above samples the final volume of the reaction mixture was kept constant at 1 ml. 2.8. Scanning Electron Microscopy. A Carl Zeiss EVO18 SEM was used to see the morphology of collagen fibrils as well as RBCs. The samples were first casted over the silver


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stubs and were then allowed to dry in air. Then these spots were washed repeatedly with ultrapure water and again kept for air drying. The samples were then sputtered with gold/palladium for 180 seconds (which gives a buildup thickness of ~ 2-6 nm) under vacuum condition. The voltage of the SEM was fixed at 20.00KV. 2.9. Atomic force microscopy (AFM). Atomic force microscopy measurements were performed using XE-70 Park Systems. For AFM measurements, the aggregated samples were diluted (10 folds) from which an aliquot of 20 µl was kept on freshly cleaved mica and then it was allowed to dry at room temperature. Images were taken immediately using tapping mode (NC-AFM) with a resonance frequency of 300 Hz and a set point value of 11.3 nm. All AFM images were captured under ambient condition. 2.10. Fluorescence Microscopy: The protein samples for fluorescence imaging studies were prepared on a glass slide. A small aliquot (~20 µl) of the sample was added on a clean glass slide and then air dried. The air dried aggregated sample was then stained with ThT solution (concentration 1.25mM) and then the sample was visualized using FLoid cell imaging station Life Science. The fibrils were detected using green filter of the instrument. 2.11. Dynamic Light Scattering and zeta potential measurements: The hydrodynamic size (diameter) measurements of the synthesized nanoparticles were performed on a Malvern Zetasizer Nano ZS (Malvern Instruments, Southborough, Massachusetts) equipped with a backscattering detector (173 degrees). Samples were filtered through a pre-rinsed 0.2-µm filter before a minimum of three measurements per sample were made. Zeta potential measurements were carried out using a Nano ZS (Malvern) and a titrator MPT-2. An aqueous suspension of gold nanoparticles was filtered through a 0.45 µm PTFE membrane before measurement.


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2.12. ATR-FTIR studies. A Bruker Vertor 70 spectrometer (equipped with silicon carbide source and MCT detector) was used for obtaining FTIR spectra of only piperine and AuNPspiperine. OPUS 6.5 software (Bruker Co., Germany) was used for data processing. All original spectra of piperine and AuNPspiperine were processed for baseline correction between 4000 cm-1and 700 cm-1 for further analysis. All spectra shown in the data are average of two acquisitions. 2.13. Hemolysis assay. The blood sample was collected from a healthy volunteer donor in vacutainers. Established protocol (36) was followed to study the effect of piperine coated nanoparticles on Red Blood Cells (RBCs). RBCs were separated by centrifuging the blood sample at 252 rcf for 10 minutes at room temperature. The pellets of RBCs were collected and washed thrice with sterile phosphate buffer saline (PBS). The pellet was then resuspended in PBS to make a diluted (~3 times) stock solution, from which an aliquot of ~ 100µl was added to AuNPspiperine for conducting the lysis assays. The suspension was slightly vortexed and stored under static condition for four hours at 37°C. The samples were then vortexed and were centrifuged at 252 rpm for 10 min. The absorbance spectrum of the supernatant obtained from sample, after centrifugation step, was recorded using UV-visible spectrophotometer. 2.14. Transmission Electron Microscopy: Transmission electron microscope (HR-TEM JEOL JEM- 2100 and JEOL 1010) was used to examine AuNPspiperine. AuNPspiperine were spotted on a carbon-coated grid for 2 min and the samples were then washed with water and air dried. For amyloid aggregate samples were spotted on a carbon coated grid for ~2 min and then washed two times with water. The samples were then stained with 1 % (w/v) aqueous uranyl acetate solution for ~2 min followed by another washing step. Air-dried grids were then examined.


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2.15. Native Gel Electrophoresis: Native (non- denaturing) polyacrylamide gel (12.5% gel) electrophoresis was performed at a constant voltage of 20 mA with a mini-PROTEIN II BioRad electrophoresis system using a Tris-HCl polyacrylamide gel. The gels were stained with silver stain as reported earlier (23). The Native PAGE experiments were conducted two times in duplicates and the data obtained from repeat experiments are shown as supplementary Figure S9. 2.16. Molecular docking studies. Molecular docking studies were performed using Discovery studio 4.0 (DS4). The ligand (piperine) structure was obtained from PubChem (CID 8082) and was prepared using ‘prepare ligand wizard’ of DS4. X-ray crystal structure of insulin (PDB ID: 4I5Z) (37) was obtained from Protein Data Bank (PDB) and prepared through ‘prepare protein wizard’ of DS4. The structures were cleaned by removing water and heteroatoms leaving behind nascent molecules. The pre-processing and protonation were carried out using CHARMm force fields (38). The ligand and the protein molecule were then docked using a blind approach (undefined active site) with 100 conformations to choose and 100 orientations to refine by following CDocker protocol (39). More detailed information on the protocol for the docking studies is given in the supplementary information. 2.17. Data analysis: All plots and graphs displayed in this article were obtained by using Origin-2015 graphics and analysis tool. Linear fit analysis for calculating Stern-Volmer constant and associated binding constant parameter was also performed using Origin-2015 software. 3. RESULTS AND DISCUSSION: 3.1. Synthesis and characterization of piperine coated gold nanoparticles We synthesized gold nanoparticles which are surface functionalized with piperine (AuNPspiperine) by following the established protocol, as schematically represented in the panel a


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of Figure 1(15). The UV-Visible spectra of AuNPspiperine sample showed a distinct SPR peak at ~530 nm (Figure 1e). Dynamic light scattering data of AuNPspiperine sample clearly showed a homogeneous population of nanoparticles with average hydrodynamic radius of ~10 nm (Figure 1f). The value of the surface charge obtained for these AuNPspiperine was found to be -4.46 mV, as evident from our Zeta potential measurements (Supplementary information Figure S1). Transmission electron microscopy (TEM) images further confirmed the formation of homogenous species of spherical AuNPspiperine (Figure 1b). To understand the chemistry of surface functionalization of piperine molecules with the nanoparticles, we conducted ATR-FTIR measurements for both uncoated and coated piperine samples. The FTIR signatures obtained for both control piperine (black curve) and surface-functionalized piperine (AuNPspiperine) samples (red curve) are shown in Figure 1g. The piperine molecule consists of a piperidine moiety, a methylene dioxy phenyl ring or dioxol group and a CH stretch (40). The FTIR-spectra of isolated piperine (black curve, Figure 1g) sample showed the presence of several characteristic peaks as reported earlier (40). The FTIR spectra of AuNPspiperine sample showed disappearance of the characteristic peak linked to = C – O group (red curve, Figure 1g). This result suggests that the gold nanoparticles may be surface-functionalized with the dioxol group. Further, we have characterized the thermal stability of AuNPspiperine sample by exposing these samples to 70˚C, the temperature at which all the fibril assembly reactions have been carried out. The SPR peaks of the samples remained unaltered even after several days of incubation at 70˚C (Supplementary information Figure S7). This signifies that these nanoparticles are very thermostable which may act as good candidates for preventing temperature induced insulin aggregation. To further characterize the intrinsic structural properties of the AuNPs piperine nanoparticles, high-resolution TEM (HRTEM) was performed which confirmed the


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polycrystalline nature of the nanoparticles (Figure 1c). To throw light into the nature of alignment of crystalline fringes, we analyzed the HRTEM image using FFT ( Gatan digital micrograph software ) and the data displayed fringe patterns with d spacing of 2.1 Å ( suggesting (111) FCC crystal) and 2.3Å ( suggesting (200) index in FCC crystals) (inset of Figure 1c). We then examined SAED (the selected area electron diffraction ) pattern of these AuNPspiperine nanoparticles using image J software and the ring patterns of the obtained data revealed (111), (222), (200), (311), and (420) indexed face-centered cubic crystal (fcc) structure for the nanoparticles (Figure 1d). To perform control experiments, we generated control gold nanoparticles without piperine by using citrate mediated protocol(16). The SPR peak for these nanoparticles was observed near 535 nm (Supplementary material, Figure S2) and the hydrodynamic radius of these particles was found to vary within 10-40 nm (Supplementary Figure S2).The surface charge of these citrate capped gold nanoparticles was found to -19.5 mV (See supplementary Figure S2). TEM images of these nanoparticles also revealed the presence of spherical nanoparticles of 10-20 nm of size (Supplementary material, Figure S2). Crystallinity of the citrate capped gold nanoparticles showed fringe patterns of fringe with d spacing of 2.3Å, indicating (200) index in FCC crystal. The SAED analysis of the citrate capped gold nanoparticle revealed the existence of (111) and (420) planes with a d-spacing of 2.3 Å and 9.1Å respectively. 3.2. Effect of piperine coated nanoparticle on insulin fibril assembly: Amyloid fibril formation of insulin was initiated by incubating the protein monomers at 70°C in PBS at pH 7 (15, 32, 35) and the aggregation reaction was monitored by reading the


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fluorescence signal of Thioflavin T, a well-known amyloid specific dye (41). Though amyloid aggregation of insulin can be triggered under different conditions including altered solvent environments (42, 43), here, in this study we selected temperature induced amyloid assembly of insulin in PBS buffer which has been already established by our lab and by other research groups. For carrying out the aggregation reaction, concentration of the insulin was maintained at ~43 µM, a value that was chosen based on our earlier studies on insulin amyloid fibril formation (15, 26). Thioflavin T assay data, as shown in Figure 2a, display the effects of both piperine, AuNPspiperine on the process of insulin fibril assembly. Only insulin sample, without any additives, followed a typical aggregation kinetics pattern (Figure 2a, ) as reported in previous studies (15, 35). However, in the presence of AuNPspiperine, strong suppression of the aggregation process was observed, in a dose dependent manner (Figure 2a, ,,). Since the concentration of the stock solution of piperine coated nanoparticles was ~ 200 µM, the maximum dose that we could test in the current investigation was at 1:3 molar ratio of protein:inhibitor. Isolated piperine (Figure 2a,,,), at similar concentrations, showed slight suppression of insulin aggregation, which suggests the significance of surface functionalization of nanoparticles with piperine. Further, our experiments on the effect of control gold nanoparticles plus free piperine molecules did not show such suppression effect (Figure 2a, supplementary Figure S8,). To examine the morphology of the insulin fibrils obtained from both inhibited and uninhibited aggregation reaction, we looked at the TEM images of the samples after completion of the aggregation reactions. The morphology of the fibrils looked similar to each other, as evident from TEM data (Figure 2c, 2d). The fluorescence images of the Thioflavin T stained fibrils (Figure S11) obtained from both inhibited and uninhibited aggregation reactions correlated well with the


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aggregation data (Figure 2a) as well as with the data reported recently (38). Although it is difficult to extract any quantitative information from the TEM and fluorescence images, it appears that the insulin fibrils formed in the presence of AuNPspiperine are of amyloid nature and have similar morphology like the control insulin fibrils. These inhibitors may perhaps delay the aggregation process without interfering with the aggregation pathway. To further understand the effect of AuNPspiperine on the morphology of the aggregating species formed during inhibition, we looked at AFM images of aggregating insulin sample (taken from both inhibited and uninhibited reactions) at 3 h time point, a time point where ThT signals show a substantial difference between the kinetics of the control insulin sample (Figure 2a,, supplementary Figure S12) and the [insulin+AuNPspiperine at 1:3 molar ratio] sample (Figure 2a, ). The AFM data, as shown in Figure 2f (supplementary Figure S13) indicate the occurrence of spheroidal oligomers(35) formed in an aggregating insulin sample in the absence of inhibitor. However, we also observed the presence of relatively smaller oligomers of insulin in an inhibited reaction, as evident from Figure 2g (supplementary Figure S14). Considering the large difference between the ThT signals at 3h time point, we predict that most of the insulin oligomers that are formed in the presence of AuNPspiperine may be of non-amyloid and amorphous in nature as suggested by Verma et al. recently(44). This result suggests the possible interference of AuNPsPiperine with the oligomerization process of insulin during its fibril assembly reaction. Similar data on the formation of non-specific oligomers of insulin in the presence of inhibitors have been recently reported (40, 43). Since preformed seeds are known to modulate insulin aggregation kinetics (45), We further examined the effect of these AuNPspiperine on seed-induced fibril assembly of insulin. The results, as shown in Figure 2h, clearly showed suppression of seed-induced fibril formation (15, 35) of insulin in the presence of AuNPspiperine. We further extended these


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aggregation experiments to another globular protein, serum albumin (bovine serum albumin), and the results are summarized in the supplementary Figure S3. The data shown in Figure S3 indicated suppression of amyloid fibril formation of serum albumin in the presence of AuNPspiperine. However, in case of serum albumin, slightly higher molar ratio of protein:inhibitor was required to observe the suppression of serum albumin aggregation. To see whether AuNPspiperine can retain the native insulin structure during aggregation, we conducted native PAGE experiments on insulin samples undergoing fibril assembly. Figure 2e shows the respective bands obtained for aggregating insulin samples in the presence and in the absence of inhibitors at 3 h time point. The results clearly indicate the retention of native structures of insulin in the presence of AuNPspiperine (Figure 2e). However, we observed a smear like band for the sample obtained from inhibited aggregation reaction. It is possible that the on pathway small molecular weight oligomers of protofibrils are being arrested from growing into the mature amyloid fibrils. The data also suggest that in the presence of highest dose (1:3 molar ratio of insulin:inhibitor) of AuNPspiperine complete suppression of fibril assembly may not be achieved and in such cases it is possible that few monomers would still follow normal aggregation pathway with a delayed kinetics. It is also possible that the aggregation process is delayed by interfering with the growth of smaller aggregated species resulting in a smear like band in the native gel. Though further experiments are required to understand this observation, it is clearly evident from the native PAGE data that the intensity of the monomer band from the inhibited reaction was higher than the rest, suggesting the retention of native conformation of insulin in the presence of AuNPspiperine . To further confirm this result we conducted circular dichroism measurements on these samples and the obtained data (Figure 2b) also confirmed the retention of


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native structures of insulin in the presence of AuNPspiperine. Both the CD and native PAGE data clearly agree to the retention of native protein species at the monomeric as well as in multimeric forms of insulin in the presence of inhibitors. Since the nature of CD curve obtained from insulin sample in the presence of AuNPspiperine looked largely similar to the curve obtained for native insulin sample, it is possible that the oligomeric species as represented by the smear in native PAGE and AFM (Figure 2e, 2g) may still retain native like secondary structures. 3.3. Quenching studies on the interaction between insulin and AuNPspiperine It is has been reported that stabilization of the native protein structure is one of the critical factors to prevent the process of amyloid aggregation of proteins (46). Our results from CD (Figure 2b) and Native PAGE experiments (Figure 2e) also pointed to the retention of native insulin species in the presence of AuNPspiperine. Hence, to gain more insights into piperine-insulin interaction, we conducted both computational and fluorescence quenching experiments. We examined the quenching effect of AuNPspiperine on insulin native monomers (see methods in the supplementary material) and the data are shown in Figure 3a. The intensity of fluorescence emission of insulin was efficiently quenched in the presence of AuNPspiperine in a dose dependent manner. The concentration of insulin was reduced to ~9 µM to avoid the saturation of the emission signal from the instrument. Using the obtained quenching data, we calculated the SternVolmer constant (Ksv) (panel b, Figure 3) and the value was found to be ~4.48x104 M-1. Further analysis of the obtained fluorescence quenching data was performed to predict the binding constant (Ka) and the binding site parameter (n) (see methods in supplementary material). Binding constant (Ka) parameter, obtained from the plot of log (F0-F)/F vs log [Q] (27), was found to be 4.23 (±1.5) x 102 M-1 (Figure 3c). The calculated binding site parameter (n) was found to be ~ 0.6, indicating a single binding site within the insulin molecule.


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3.4. Molecular docking of native insulin structure with piperine: Next, we performed molecular docking studies to further understand the piperine-insulin interaction, particularly to examine whether piperine has an inherent affinity for the aggregationprone residues of insulin. Using Discovery Studio (DS 4.0), blind docking studies were carried out (15) and the obtained data are shown in Figure 3d. The value of CDocker energy obtained for insulin-piperine interaction was found to be -19.4 kcal mol-1. The structure of the insulinpiperine complex, as shown in Figure 3d, predicts interaction of the piperine molecule with the B-chain residues of insulin mediated by four hydrogen bonds and one pi-alkyl interaction (inset Table, Figure 3d). It is well known that the B-chain segment “LVEALYL” plays a foundational role to begin the fibril formation of insulin (28). Notably, as evident from our docking data, piperine showed strong interactions with the valine (V) and the glutamic acid (E) residues of the aggregation prone segment (LVEALYL) of B-chain of insulin. Prediction of aggregation propensity of insulin residues by AGGRECAN-3D tool (supplementary Figure S5 and S6) also suggests the binding of piperine with the aggregation prone residues of insulin. Hence, in this study, the binding of piperine, in its conjugated form, with aggregation prone residues of insulin appears to be an important factor for the inhibition effect. Recent studies have already reported similar mechanism of inhibition of protein aggregation due to the binding of surface functionalized nanoparticles to the amyloidogenic core of the aggregating protein (15, 23). Though aggregation of insulin was studied at 70˚C, a temperature that promotes the formation of partially folded intermediate states (I), the docking study between native-insulin (N) and piperine is still important. Because of the existence of thermodynamically driven equilibrium of N I at the transition temperature of insulin, it is predicted that half of the total population would still retain the native conformation. Hence, insulin sample, having


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AuNPspiperine in it, when incubated at 70˚C, it is possible that such inhibitor molecules may interact with the native insulin species. Such interaction between piperine and native-insulin is predicted to shift the N I equilibrium towards the native state, reducing the population of aggregation prone I states. FTIR data (Figure 1g) suggest that the gold nanoparticles may be surface-functionalized with the dioxol group of piperine. Such conjugation would predict that the piperidine moiety, the carbonyl amide linkage and side chain of piperine molecule are free for interaction. The possible conformational alternations happened to the conjugated piperine molecule when it is surfacefunctionalized was difficult to predict. However we assumed that the functional groups other than the one engaged in conjugation would still be available for making non covalent contacts with the neighboring functional groups present within the binding site of the protein. This is completely based on assumption, as it was difficult on our end with limitations in the docking studies that allowed us to test the docking of insulin with the free piperine rather than with the conjugated one. Although the molecular docking was performed with free piperine, it provides valuable information, at least in pointing out the critical moieties of the conjugated-piperine capable of interacting with the aggregation prone regions in the B-chain of insulin molecule. 3.5. Hemocompatibility of AuNPspiperine inhibitors After establishing the suppression-effect of the AuNPspiperine against insulin aggregation, we further examined the biocompatibility of these nanoparticle based inhibitors. Here, we examined the hemocompatibility of piperine coated nanoparticles on human RBCs to understand the significance of the piperine functionalized gold nanoparticles in medical applications. We tested the toxicity effect of these nanoparticles on the intact red blood cells by following the established protocol for hemolysis assay (35, 36). Data generated from our hemolysis assay


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experiments clearly indicate that no lysis of intact RBCs was observed in the presence of AuNPspiperine (Figure 4a, 4b). UV-vis spectra (Figure 4a) did not show any indication of lysis of RBCs in the presence of AuNPspiperine and this hemocompatibilty nature of the gold nanoparticles was further confirmed by scanning electron microscopy (SEM) images (Figure 4b). 3.5. Effect of AuNPspiperine lysozyme activity To test the effect of piperine coated gold nanoparticles on another biological system, we further tested the effect of AuNPspiperine on biological activity of proteins, considering lysozyme as a convenient model enzyme. Protein ligand interaction may perhaps stabilize the native structures and it is important to know whether such stabilization has any impact on the bioactivity of the protein molecules. About ~ 25% enhancement of the catalytic efficiency of lysozyme was observed at pH 6.5 in the presence of AuNPspiperine (Supplementary Figure S10). Such an enhancement of lysozyme activity was not observed in the presence of isolated piperine under similar conditions, which further suggests the importance of surface functionalization of piperine for enhancing lysozyme activity. 3.6. Possible mechanism of the inhibition effect of AuNPspiperine : Piperine (1-[5-(1,3-Benzodioxol-5-yl)-1-oxo-2,4-pentadienyl] piperidine (40) has a unique structure comprising of piperidine moiety attached through a carbonyl amide linkage and a side chain that has a methylene dioxyphenyl ring. Piperine is known to strongly interact with different biomolecules including proteins and DNA (24-27). The C=O group and both the oxygen atoms of the dioxalane ring of piperine are known to actively take part in H-bond interactions between piperine and DNA(26). Formation of H-bond interaction between valine residue of serum albumin and –O- (para) group of piperine molecule has been reported (25).


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Further, binding of piperine with Androgenic Receptor (AR) protein has also been reported to involve two H-bond interactions: first interaction is between –O– (para) group of piperine with arginine residue (-NH2 group), and the second one is between C=O of piperine with the valine residue (27). Piperine is also known to directly interact with the arginine residue of human tear lipocalin protein via H-bonding interactions (25). Our computational data reveal that valine and glutamic acid of insulin’s B-chain are the key residues which interact with the piperine molecule (Figure 3d). Both CD and Native PAGE data clearly suggest that the AuNPspiperine inhibitors are capable of retaining the native conformation of insulin structure (Figure 2b, 2e). Based on these evidences, we predict that the stabilization of the native insulin conformation, through native insulin-AuNPspiperine interaction, would reduce the population of the temperature induced partially folded aggregation-prone conformers of insulin, leading to suppression of fibril assembly process. It is possible that AuNPspiperine inhibitors may interfere with the intermolecular hydrophobic interactions between partially folded aggregation prone species. To address the issue of piperine’s ability to interfere with the hydrophobic interactions between temperatureinduced partially folded species required to drive self-association of protein species, we examined the effect of AuNPspiperine on fibril formation of type I collagen extracted from rat tail tendon (RTT). Type I collagen, the most abundant form among collagens, is known to undergo a process of fibril formation at 37˚C in PBS buffer (pH 7.4) (30) and such process of self-assembly of collagen triple helical molecules is known to be driven by hydrophobic interactions (47-49). Our turbidity data indicated that AuNPspiperine inhibitors have the potential to supress the fibril formation of type I collagen (panel a of Supplementary Figure S4). To generalize this inhibition effect of AuNPspiperine on the fibril formation of type I collagen, we also tested its inhibition effect on the fibril formation of calf-skin collagen and similar results were obtained (panel b,


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supplementary Figure S4). The morphologies of the collagen fibrils obtained from inhibited reactions (panel d and panel e, supplementary Figure S4) looked similar to the typical morphology observed for in vitro grown collagen fibrils (50). Suppression of amyloid fibril formation as well as collagen fibril formation suggests that piperine may have the potential to interfere with hydrophobic interactions necessary to initiate the self-assembly of protein molecules. Inhibition effect on protein aggregation process can also be enhanced if the inhibitors molecules were able to interfere with the secondary nucleation process during insulin fibril assembly. Insulin fibril assembly undergoes an amyloid fibril formation through secondary nucleation pathway(43), where the preformed fibrils enhance addition of monomers to the growing ends, displaying a rapid growth phase during aggregation. Our data on the seed-induced aggregation of insulin, as shown in figure 2h, display an aggregation curve without a lag phase suggesting the ability of the preformed insulin fibrils to trap the monomers into an aggregation pathway primarily driven by secondary nucleation (35) . However a substantial suppression of such process was observed in the presence of piperine coated gold nanoparticles suggesting its direct interference with the secondary nucleation of insulin fibril assembly. It is important to notice that, isolated piperine and control gold nanoparticles did not show any such suppression effect on insulin fibril assembly (Figure 2a) and on type I collagen fibril formation (panel c, supplementary Figure S4), confirming the significance of surface functionalization of the piperine molecule. Conformational constraints and the hydrophobic nature of the inhibitor molecule are considered as two key factors to achieve the inhibition of amyloid fibril formation of proteins (51). We believe that the conjugation of piperine molecule with the gold nanoparticles may favour proper orientation of the C = O group, and both the


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oxygen atoms of its dioxalane moiety for an effective interaction with the respective functional groups of the amino acids located in the binding site of insulin. We also predict that both the size and the homogeneity of these nanoparticles are critical properties for insulin-piperine interaction leading to inhibition of the aggregation process. Recently, it was found that selected quinones have the potential to interfere with the aggregation of insulin (52). The same study also pointed out that these inhibitors molecules attenuate the oligomerization of insulin species (52). Insulin aggregation has also been known to be suppressed in the presence of polyphenols (35, 51, 53). Recent reports have also targeted insulin aggregation through designed peptide conjugates (44) On the issue of biocompatibility, we found that AuNPspiperine inhibitors are hemocompatible. These particles were also observed to enhance the activity of lysozyme. It is also important to notice that these nanoparticles are very stable at 70˚C for a prolonged time (more than 4 weeks). Such thermostable nature may be vital for the effective use of these inhibitors against temperature induced aggregation of protein species during their storage as therapeutic agents, particularly against insulin fibril assembly during its storage (8, 9). 4. CONCLUSIONS: This study confirms that gold nanoparticles surface functionalized with piperine have the potential to suppress amyloid fibril formation of insulin under in vitro conditions. Surface functionalization of the gold nanoparticles with piperine seems to be critical for the suppression effect because such strong suppression of insulin fibril assembly was not observed in the presence of either isolated piperine or control gold nanoparticles. In addition to the anti-amyloid activity, these piperine coated nanoparticles were able to prevent fibril formation of type I collagen. Stabilization of native structure of the protein, particularly through binding of the


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conjugated-inhibitor molecule to the aggregation prone residues of the insulin, appears to be one of the critical factors for the prevention of insulin fibril assembly. Though this work is an in vitro investigation, the information gained may provide critical foundational knowledge to clinical researchers targeting medical complications related to insulin fibril-assembly. ASSOCIATED CONTENT Supporting Information. Supplementary information: Tables S1; Figures S1-S11; Methods; “This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author Correspondence and requests for materials should be addressed to K.K. email: [email protected], [email protected] Author Contributions The manuscript was written through contributions of all authors. BGA synthesized and characterized the nanoparticles and conducted the computational docking studies. BGA, DSS and KD carried out all biophysical experiments including aggregation and quenching experiments. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by BRNS grant (KK) (Grant No.37 (1)/14/38/2014-BRNS), DSTPURSE and UGC-SAP and LRE-JNU funding support. Notes The authors declare no competing financial interests.


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Figure legends: piperine Figure 1. Characterization of piperine coated gold nanoparticles (AuNPs ) (a) Schematic representation of the surface functionalized gold nanoparticles. (b) TEM images piperine (c) HR TEM data showing a fringe displaying the evenly sized (~10nm) spherical AuNPs spacing of 2.1 Å and 2.3Å. Inset shows splitting of spots in the FFT image. (d) A selected area piperine electron diffraction pattern (SAED) of AuNPs . The Scherrer ring patterns indicate the FCC piperine gold of nanocrystalline nature. (e) UV-Vis spectrum of AuNPs with absorption maxima around 530 nm due to surface plasma resonance. (f) Dynamic Light Scattering (DLS) peak of piperine showing an average diameter of ~10 to 20 nm. (g) FTIR Spectra of (—) piperine AuNPs piperine and (—) AuNPs nanoparticles. Figure 2. Suppression of amyloid fibril formation of insulin in the presence of AuNPs (a) Thioflavin T assay data showing aggregation curves for 43 µM of insulin sample in PBS at pH 7: () insulin only; () insulin + piperine (1:1); () insulin + piperine piperine piperine (1:2); () insulin + piperine (1:3); () insulin + AuNPs (1:1); () insulin + AuNPs piperine control (1:2); () insulin + AuNPs (1:3); ()insulin + piperine + AuNPs (1:3); () insulin + control piperine + AuNPs (1:3:3). (b) CD spectra of insulin sample (43 µM) undergoing piperine aggregation in the presence and in the absence of AuNPs : (--) insulin at 0 h, ; (--) insulin at piperine 3 h, (--) insulin + AuNPs (1:3 molar ratio) at 3 h. CD curve for inhibited reaction was obtained after baseline subtraction with the CD signal of AuNPspiperine sample in PBS. (c) and (d) Transmission electron microscopic images of final insulin fibrils obtained from both inhibited and uninhibited aggregation reactions, as labeled. Scale bar 200 nm. (d) Native PAGE of insulin piperine samples in the presence and in the absence of AuNPs during aggregation: (1) soluble insulin at 0 h; (2) aggregating insulin sample at 3 h, (3) aggregating [insulin+piperine] sample at piperine 3 h (4) aggregating [insulin+ AuNPs ] sample at 3 h (5) aggregating [insulin+ control AuNPs] sample at 3h. Molar ratio of protein to inhibitor was maintained at 1:3 for all Native PAGE experiments. Uncropped native PAGE data are shown in Figure S9 of the supplementary information. (e) AFM image of oligomers of control insulin sample (at 43 µM) at 3 h time point. (f) AFM image of insulin oligomers of insulin formed in the presence of AuNPspiperine (1:3 molar ratio) at 3 h time point. (g) Inhibition of seed-induced aggregation of 43 µM insulin in the piperine presence of AuNPs at 1:3 molar ration of protein: inhibitor. ~15% w/w preformed fibrils were used as seed. piperine.

Figure 3. Interaction of piperine coated gold nanoparticles with insulin monomers. (a) Fluorescence emission spectra of ~9 µM insulin sample with increasing concentration of piperine piperine AuNPs . Excitation wavelength was 276 nm. Concentrations of AuNPs were: (---) 0 µM; (---) 0.5 µM; (---) 1 µM; (---) 1.2 µM; (---) 2.0 µM; (---) 3.0 µM; (---) 4.0 µM; (---) piperine 6.0 µM. (b) Stern-Volmer plot of insulin and AuNPs . (c) The plot of log ((F0-F)/F) versus piperine

log [Q] for insulin binding with AuNPs . All the fluorescence measurements were carried out at room temperature in PBS buffer at pH 7. (d) Docked complex of insulin (PDB ID: 4I5Z) with piperine represents five interactions: four H-bonds (GLU21:OE1-piperine:H28, VAL18:O-


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piperine:H39, ARG22:O3-piperine H21, ARG22:O3-piperine:HE) and one pi-alkyl interaction (ARG:22-piperine Figure 4. Biological compatibility of piperine coated gold nanoparticles. (a) UVvisible spectra obtained from hemolysis assay: (—) RBCs in PBS buffer (positive control); (—) piperine

RBCs + 160 µM piperine; (—) RBCs + 160 µM AuNPs ;(—) RBCs + 160µM AuNPs; (—) RBCs in water (negative control). (b) SEM images of intact RBC sample in the presence of piperine AuNPs .Scale bar is 2µm


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KOH, Boiling/ Stirring

FIGURE 1


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FIGURE 2


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FIGURE 3


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FIGURE 4


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For Table of Contents Use only.

Uniform, polycrystalline and thermostable piperinecoated gold nanoparticles to target insulin fibril assembly Bibin G. Anand, Dolat S. Shekhawat, Kriti Dubey and Karunakar Kar*


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Inhibition of insulin fibril assembly 136x81mm (150 x 150 DPI)


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Uniform, Polycrystalline, and Thermostable Piperine-Coated Gold

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