Zeolite Nanoparticles Inhibit AÎ²âFibrinogen Interaction and Formation

Subscriber access provided by RYERSON UNIV

Article

Zeolite Nanoparticles Inhibit A#-Fibrinogen Interaction and Formation of Consequent Abnormal Structural Clot Hossein Derakhshankhah, Mohammad Javad Hajipour, Ebrahim Barzegari, Alireza Lotfabadi, Maryam Ferdousi, Ali Akbar Saboury, Eng-Poh Ng, Mohammad Raoufi, Hussein Awala, Svetlana Mintova, Rassoul Dinarvand, and Morteza Mahmoudi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10941 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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.

ACS Applied Materials & Interfaces 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 40

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

ACS Applied Materials & Interfaces

Zeolite Nanoparticles Inhibit Aβ-Fibrinogen Interaction and Formation of Consequent Abnormal Structural Clot Hossein Derakhshankhah†∥‡, Mohammad Javad Hajipour ‡∥⊥◆, Ebrahim Barzegari #, Alireza Lotfabadi†∥‡, Maryam Ferdousi ∇, Ali Akbar Saboury #, Eng Poh Ng §, Mohammad Raoufi‡, Hussein Awala¶, Svetlana Mintova¶*, Rassoul Dinarvand‡∥* and Morteza Mahmoudi ‡∥▲* †

Department of Pharmaceutical Biomaterials, Faculty of Pharmacy, Tehran University of Medical Sciences,

Tehran, Iran. ‡

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 13169-

43551, Iran. ∥ Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences,

Tehran 13169-43551, Iran. ⊥ Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute,

Bushehr University of Medical Sciences, Bushehr 75147, Iran. ◆

Non-Communicable Diseases Research Center, Endocrinology and Metabolism Population Sciences Institute,

Tehran University of Medical Sciences, Tehran, Iran #

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.

∇ School of Biology College of Science, University of Tehran, Tehran, Iran. § ¶

School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Malaysia. Laboratory of Catalysis and Spectroscopy, ENSICAEN, University of Caen, CNRS, 6 Boulevard du Maréchal Juin,

14050 Caen, France. ▲ Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

02115, United States.

* Corresponding authors: (RD) email: [email protected]; (SM) email: [email protected]; (MM) email: [email protected]

1 ACS Paragon Plus Environment


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

Abstract EMT-type zeolite nanoparticles (EMT NPs) with particle size of 10-20 nm and external surface area of 200 m2/g have shown high selective affinity toward plasma protein (fibrinogen). Besides, the EMT NPs have demonstrated no adverse effect on blood coagulation hemostasis. Therefore, it was envisioned that the EMT NPs could inhibit possible β-Amyloid (Aβ)-fibrinogen interactions that result in the formation of structurally abnormal clots, which are resistant to lysis, in cerebral vessels of patients with Alzheimer disease (AD). To evaluate this hypothesis, the clot formation and degradation of Aβ-fibrinogen in the presence and absence of the EMT zeolite NPs were assessed. The results clearly showed that the delay in clot dissolution was significantly reduced in the presence of zeolite NPs. By formation of protein corona, the EMT NPs showed a negligible reduction in their inhibitory strength. Docking of small molecules (Aβ-fibrinogen) introduced novel potential inhibitory candidate. The zeolite NPs showed similar inhibitory effects on binding of fibrinogen to both Aβ (25-35) and/or Aβ (1-42). This indicates that the inhibitory strength of these NPs is independent on Aβ sequence and it is suggested that the zeolite NPs adsorb fibrinogen and specifically obstruct their Aβ binding sites. Therefore, the zeolite NPs can be the safe and effective inhibitors in preventing Aβ-fibrinogen interaction and consequent cognitive damage.

Keywords: fibrinogen, clot, EMT zeolite nanoparticles, Alzheimer disease, beta amyloid


Page 2 of 40

Page 3 of 40

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 Alzheimer disease (AD) is the most common neurodegenerative disorder associated with the progressive decline in memory and cerebral function 1-2. The memory loss in AD is linked to the extracellular plaques consisting of a large fibrillar form of beta amyloid (Aβ) causing neuron malfunction (amyloid cascade hypothesis) 3. Up to now, scientists have used many therapeutic approaches based on targeting the Aβ fibrils as a hallmark of AD 4. Many NPs, small molecules, and peptides have capability to inhibit Aβ fibril formation5-9. The fibrillation process is mainly inhibited/delayed via blocking Aβ oligomers and capturing Aβ monomers leading to undesired condition for Aβ nucleation and reduction of free Aβ monomer, respectively9-10. For example, polyoxometalates, graphene oxide, gold, and superparamagnetic iron oxide NPs interfered with fibril formation through binding to Aβ monomers6-8, 10. In some cases, NPs provide large surface area for Aβ monomer/oligomer localization and subsequent fibril formation11. Depending on their physicochemical characteristics (size, charge, shape and composition), NPs show inhibitory or acceleratory effects on amyloidogenesis process6, 11. Few inhibitors have been reported that can destabilize preformed Aβ fibrils. Qu group12 showed that Thioflavins S functionalized graphene oxide specifically bind to and destabilize the preformed Aβ fibrils under laser irradiation. Despite of intensive research in the field, the current therapeutic approaches have not substantial positive effects of treatment of AD. Failure of these strategies in solving the Alzheimer conundrum changed their target 4. In this regard, some epidemiological studies were conducted and revealed that cerebrovascular risk factors determine the severity of cognitive impairment in AD patients 4, 13. Indeed, the cerebrovascular malfunction and cerebral blood flow reduction which occur through AD amplify the disease pathology 14.



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

Page 4 of 40

In recent years, fibrinogen protein was found in the brain of AD patients 15. This finding can be explained by blood brain barrier (BBB) malfunction occurring through AD. Fibrinogen, is a 340 kDa soluble plasma glycoprotein, which plays critical roles in blood clotting, thrombosis and inflammation

16-17

. A new milestone in the study of AD etiology is the discovery of abnormal

structural clots resulted from Aβ-fibrinogen interaction

18

. The fibrin clots formed in a normal

state can easily be lysed by clot lysis enzymes (plasminogen/plasmin), while those formed in AD brains are resistant to degradation, and they are known as one of the main causes of AD progression 19. Based on the newly proposed process of AD development, targeting the Aβ-fibrinogen interaction has been hypothesized as an effective therapeutic approach for prevention and/or treatment of AD

19

. To achieve this goal, different methods have been proposed. The present

study considers two approaches that are described below. The biological impact of nanoparticles (NPs) is strongly influenced by the proteins adsorbed on their surfaces, which is called protein corona

20-23

. The protein types/concentrations of the

corona is dependent on some parameters, including the physicochemical properties of NPs, incubation condition and plasma/serum changes 24-26. Zeolite NPs are crystalline aluminosilicates with uniformed micropores size and shape that can be extensively used for diagnostic and therapeutic purposes 27-28. According to our recent findings, zeolite NPs have no cytotoxic effect against HeLa cells, while they have a high affinity toward fibrinogen

29-30

. Thus, they can be

potential candidates to interact with the pathologic Aβ-fibrinogen. In this study, the inhibitory role of these NPs on Aβ-fibrinogen interaction is investigated by using the EMT-type zeolite NPs in both plain and corona coated forms.


Page 5 of 40

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


An alternative method to target the Aβ-fibrinogen interactions is the use of small chemical compounds to suppress the binding site of Aβ to fibrinogen 19. Recently, Ahn et al. showed that RU-505 small molecule effectively decreases vascular problems and cognitive decline via preventing Aβ-fibrinogen interaction

31

. More effective compounds can be further identified

using computational methods, including virtual screening, docking and drug design. The Aβ-fibrinogen interaction is now known as the main reason of developing the Alzheimer disease. The present investigation is aimed to suppress these Aβ-fibrinogen interactions (binding). Additionally, computational approach is used for describing the possible molecular mechanism of Aβ-fibrinogen/small molecule interactions. The knowledge obtained through these investigations can be applied to design pharmaceutical drugs against the Alzheimer disease.

2. Experimental section 2.1 Synthesis and characterization of EMT-type zeolite NPs The EMT zeolite NPs were prepared according to the method developed by our group

27

.

Briefly, the aluminate solution was first prepared by dissolving 9.074 g of sodium aluminate (Strem Chemicals) and 1.61 g of sodium hydroxide (Prolabo, 97%) in 100 g of double distilled water. 44.00 g of sodium hydroxide was then added to the solution, and a clear suspension was obtained (solution A). The silicate solution was prepared by mixing 57.692 g of sodium silicate (Prolabo, 27% SiO2, 8% Na2O), 20.00 g of sodium hydroxide and 80.00 g of double distilled water in a 250-mL bottle. The solution was stirred until an entirely transparent solution was obtained (Solution B). Both solutions were cooled in an ice bath (4 °C). Solution A was then slowly poured into Solution B under vigorously stirring, forming a turbid suspension with a molar composition of 1Al2O3:5.15SiO2:18.45Na2O:240H2O. The resulting suspension was



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

stirred for additional 5 min before it was crystallized at 30 °C for 36 h. The crystallized EMT nanocrystals were centrifuged at 20000 rpm for 1 h and the purified EMT zeolites were resuspended in double distilled water. This purification step was repeated for several times until the final pH of suspension reached 7. The XRD analysis of solids was performed by a PANalytical X’Pert PRO diffractometer and the XRD pattern was matched with the reference EMT zeolite reported in JCPDS in our previous work27. The mean diameter and morphology of synthesized EMT-type zeolite NPs were characterized using transmission electron microscopy (TEM) (JEOL Model 2010 FEG system, 200 kV) and scanning electron microscopy (SEM) (Philips XL-30, 30 kV). The hydrodynamic size of NPs was measured in water suspension (5 wt%, pH 7.5) using a Malvern Zetasizer Nano Series (DLS). Furthermore, the zeta potential values of NPs at different concentrations (0.1, 1, 3, 6, 10, 15, 20 wt%) were obtained using the same instrument. The surface charge density, σ, was calculated using the Grahame equation: σ = Sqrt(8c0εε0kBT) × sinh(eΨ0/2kBT) where c0 is the concentration in m-3, εε0 is the dielectric permittivity of zeolite (1.3547 × 10-11 AsV-1 m-1 for EMT), kB is the Boltzmann constant (1.381 × 10-23 J K-1), Ψ0 is the surface potential or zeta potential of the zeolite suspension, e is the electronic charge (1.602 × 10-19 C), and T is the absolute temperature (298 K). The surface charge of zeolite NPs, Q, was computed by using the equation as follows: Q = (σ×SBET) / (Si/Al ratio) where SBET is the specific surface area (m2 g-1) and Si/Al ratio is the silicon to aluminum ratio of the zeolite NPs. The chemical composition of the samples was characterized using X-ray fluorescence (XRF) spectroscopy (MagiX PHILIPS PW2540). The BET surface area, pore volume and pore


Page 6 of 40

Page 7 of 40

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


diameters were determined using a Micromeritics ASAP 2010 volumetric adsorption analyzer. Prior to analysis, the samples were degassed at 250 °C for 24 h under vacuum.

2.2. Preparation of hard corona In order to prepare the hard corona coated NPs, the EMT zeolite NPs, with a final concentration of 100 µg/mL, were incubated with 10% and 100% plasmas at 37 °C for 1 h (total volume of solution was 1000 µL). After 1 h of incubation, the solution was immediately centrifuged at 18000 rpm for 20 min. The supernatant was then removed and the pellet containing corona coated NPs were dissolved in a phosphate buffer solution (PBS) and centrifuged at 18000 rpm for 15 min. The washing step was repeated for three times to detach the weakly bound proteins from the NP surface. The resulted hard corona coated NPs were resuspended in PBS (500 µL) and immediately used for different analyses. 2.3. Preparation of amyloid peptides fibrils The formation of Aβ fibril was assessed using thioflavin T (ThT) assay and TEM imaging. The ThT assay was performed as described in previous studies 32. Aβ peptide 1-42 (concentration of 10 µM) and ThT (concentration of 200 µM) were dissolved in 13 mM sodium phosphate buffer (pH: 7.4) and incubated at 37 °C for 24 h 7. The ThT fluorescence was measured after 12 and 24 h. After 24 h of incubation, the fibril formation was confirmed using TEM imaging. Similar recipe, with slight modification, was also repeated for fibrillation of Aβ peptide 25-35. Aβ 25-35 (with a final concentration of 10 µM) and ThT were dissolved in deionized water and incubated at 37 °C for 24 h. After formation of mature fibrils, they were further used for clot formation and degradation analyses.



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

2.4. Clot formation/lysis assays In order to assess the clot formation, fibrinogen (50 µM) and thrombin (30 nM) were dissolved in deionized water to reach the total reaction volume of 200 µL, and the solution turbidity was measured at 450 nm using Elisa plate reader (GEN5/BioTek Power Wave Xs2) for 20 min. This analysis was performed in the presence and absence of Aβ1-42/Aβ25-35 (10 µM). To measure the time required for clot formation/lysis, tissue type plasminogen activator (tPA) (100 nM) and plasminogen (1.5 µM) were also added into this solution and the solution turbidity was then assessed. To evaluate the inhibitory efficacy of zeolite NPs on Aβ-fibrinogen interaction, two concentrations (100 and 200 µg/mL) of bare and corona coated EMT zeolite NPs were incubated with this solution and the time of clot formation/degradation was measured.

2.5. Docking, virtual screening and quantitative structure-activity relationship (QSAR) studies Previous research has placed the Aβ binding site of fibrinogen near the C-terminus of its βchain 31. A molecular docking between C-terminus of the fibrinogen β-chain (SI, Fig. S2-S4) and Aβ (SI, Fig. S5) was performed to characterize the residues that contribute to their interaction (SI, Fig. S6-S10, Table S1). We also docked RU-505 against Aβ protein to discover the binding mechanism of this potential drug (SI, Fig. S11-S13). The structure of RU-505 was drawn in the HyperChem software (HyperCube, Inc.) and geometrically optimized using AM1 semi-empirical method. AutoDock-Vina was used for automated docking to find the lowest-energy poses of the small molecule against the β-Amyloid

33

. A Lamarckian genetic algorithm and an empirical

binding free energy function were also employed to perform the same task using the


Page 8 of 40

Page 9 of 40

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


AutoDockTools 4.2 (ADT) software

34

. LigPlot+ was applied to generate 2D views of

interactions 35. Regarding the difficulty in studying the interaction between C-terminus of the fibrinogen βchain and Aβ even considering the dynamic properties, a high-throughput screening among millions of possible inhibitors seems to be a more practical approach to suppress the fibrinogen and Aβ binding. The ZINC database of over 21 million compounds was used for this purpose (SI, S5)

36

. Structures with >70% similarity to RU-505 were searched in this database (SI, Fig.

S16). The search hits were docked against Aβ to find the strongest inhibitors (SI, Table S2). The quantitative structure-activity relationship (QSAR) method was then applied to build a mathematical model for identifying the structural determinants in the inhibitory potency of Aβ inhibitors (SI, section S6). The VCCLAB E-Dragon was employed to extract structural descriptors 37. By dimension reduction, the number of descriptors decreased from 250 to 11 (SI, Table S3). Finally, a binary logistic regression (BLR) models (SI, Table S4-S9) were built to find the most efficient structural factors in the inhibition potency of inhibitory compounds, as well as to specify the degree of effectiveness of each descriptor in this context (SI, Table S10).

3. Results and discussion 3.1. Characterization of EMT zeolite NPs The colloidal stable EMT zeolite NPs were prepared according to the procedures developed [19]. The fully crystalline samples exhibit Bragg peaks characteristics for the EMT-type zeolite structures (see supplementary information (SI) Fig. S1A); the clear broadening of the peaks is due to the small particle size. The morphology and particle size distribution of the samples are determined by TEM and DLS, respectively. As shown in Fig. 1, the DLS curve of pure EMT type



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

Page 10 of 40

zeolite suspension shows a monomodal particle size distribution. The mean crystal size of the EMT zeolite NPs is ranging from 8-20 nm. The TEM images reveal that the EMT zeolite exhibits well-defined crystalline faces. Crystalline fringes can be clearly seen in the HRTEM images. The distance of 0.73 nm of lattice fringes can be indexed to the (001) plane of the EMT zeolite nanocrystals. The physicochemical properties of the zeolite NPs are summarized in Table 1. This zeolite belongs to the large pore molecular sieves (12-membered ring) with cage-like porous system. EMT zeolite has two types of cages: hypocages (0.75 nm × 0.65 nm in diameter) and hypercages (0.73 nm × 0.73 nm in diameter). The micropore size, BET surface area and external surface area of the EMT zeolite are measured by adsorption of nitrogen at -196 °C after degassing of samples at 250 °C under vacuum for 24 h. The N2 sorption/desorption isotherm is shown in Fig. S1B. The chemical composition of zeolites (Si, Al and Na) was determined by XRF spectroscopy analysis. The Si/Al ratio of the EMT NPs is 1.17, and the high concentration of Na cations is needed to counter balance the negatively charged zeolite crystals, originated from the (Al–O–Si)– groups (Table 1). Zeta potential analysis was performed to examine the concentration effect of zeolite NPs on their colloidal stability before they were applied in blood coagulation study. The EMT suspensions have been prepared with different solid concentrations (0.1, 1, 3, 6, 10, 15 and 20 wt%) prior to zeta potential measurements and the results are shown in Fig. 2 and Table 1. The EMT suspension with a concentration 20 wt% displays zeta potential of –38.7 mV, viz. a value that indicates a highly stable colloidal system with non-agglomerated negatively charged crystals. The negative surface charged NPs in these solutions do not sediment even after several weeks at ambient condition. The colloidal stability of the suspensions is further enhanced with


Page 11 of 40

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


decreasing the concentration of zeolite crystals. The lowest zeta potential value of the suspension is measured to be –51.3 mV at a concentration of 0.1 wt% of EMT zeolites. The surface charge of zeolite nanocrystals has a significant impact on the interactions with human plasma proteins. Thus, charge density and surface charge of the EMT zeolite (0.1 wt%) were calculated using relevant equations (see Methods section). The EMT NPs have surface charge density of –0.608 mC/m2, which is related to its small crystallite size and low zeta potential value (Table 1). Nevertheless, its surface charge is –353.3 mC/g due to its high surface area and low alumina content in the zeolite framework. The surface charge of the EMT zeolite nanocrystals are in good agreement with their hydrophilic properties.

3.2. Clot formation and degradation in the presence of Aβ fibril In order to estimate the clot formation and/or degradation level, the solution turbidity is measured, and expressed as an index. It is found that the solution turbidity changes significantly after oligomerization of fibrinogen to fibrin; this process is mediated by thrombin and/or lysis of fibrin caused by tPA activated plasminogen. Because the Aβ fibrillation is enhanced in the presence of fibrinogen, a structurally abnormal fibrin clot triggered by bound Aβ fibrils is mainly found 31. Therefore, it is more reliable to use the fibrillar form of Aβ, rather than its monomeric and oligomeric forms, to assess the real effects of Aβ on the clot formation/degradation. Aβ peptides were incubated at 37 °C for 24 h. The formation of matured fibrils was assessed by thioflavin T (ThT) fluorescence assay and TEM imaging, which are considered as the most common fibrillation assessment methods (Fig. 3). Figure 3A shows the formation of Aβ fibril in matured form after 12 and 24 h incubation. The matured fibril form of Aβ fibril was also confirmed by TEM imaging (Fig. 3B). The clot formation and degradation were assessed in the



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

Page 12 of 40

presence and absence of preformed Aβ (25-35) and Aβ (1-42) fibrils. The clot formed in the presence of Aβ (25-35) and Aβ (1-42) fibrils showed more resistance to lysis and it took a longer time to degrade (Fig. 4A, B and C). Thus, the result is in agreement with the previous studies, indicating that the interaction between Aβ and fibrinogen causes abnormal clot formation and deposition 31, 38.

3.3. Zeolite NPs inhibit the Aβ-Fibrinogen interaction The regular clot formation and degradation were assessed in the presence of zeolite NPs. Different concentrations (100 and 200 µg/mL) of the EMT NPs showed no significant effect on the clot formation and lysis hemostasis (Fig. 5A and B). Also, the corona coated NPs obtained from 10% and 100% plasmas did not affect general clot formation and degradation (Fig. 5 C-F). Therefore, EMT NPs are biologically safe and exhibit no adverse effect on blood hemostasis. The clot formation and degradation were further evaluated in the presence of Aβ and different concentrations of EMT NPs (100 and 200 µg/mL) to assess their inhibitory efficacy. As seen in Fig. 6A and B, the most stable fibrin was achieved in the presence of Aβ and in the absence of NPs. The EMT NPs could significantly decrease the cunctation in fibrin lysis. Therefore, they can be as effective Aβ-fibrinogen interaction inhibitors in preventing abnormal fibrin formation and undesired thrombosis. The potential candidates for inhibition of Aβfibrinogen interaction should specifically bind to Aβ or fibrinogen and block their binding sites. Thanks to high affinity of the EMT zeolite NPs toward fibrinogen, their inhibitory mechanism can be explained. The fibrinogen molecule has a rod-like shape with dimensions of 9 × 47.5 × 6 nm3. Thus, the molecules are unable to diffuse and adsorb in the pores (0.74 nm3) of EMT zeolite NPs. In this case, we believe that the adsorption only occurs at the external surface of zeolite


Page 13 of 40

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


NPs. Therefore, the selective adsorption of plasma protein is contributed from the textural porosity of nanozeolites but not from their micropores. The EMT zeolite NPs nanoparticles have high charge to surface area ratio (charge density) due to their small crystal size27. These NPs not only tend to affect the receptor binding affinity but also affect the tertiary structure and the charge distribution of the entire fibrinogen protein. As a result, such actions may alter the fibrinogen chain folding through adsorption, and decrease the number of free fibrinogen molecules in the solution. In addition, size-specific electrostatic interaction and other chemical interactions (e.g. hydrogen bonding) between the EMT zeolite NPs and Aβ can also occur through the positively charged arginine or lysine residues of Aβ10. Therefore, the Aβ protein has low chance to interact with fibrinogens in the presence of the EMT NPs. The previous studies demonstrated that the Aβ fibrillation significantly enhances in the presence of fibrinogen

31

. In addition, the Aβ-fibrinogen interaction increases vascular Aβ

deposition which in turn affects blood-brain barrier function

38-39

. All of these events/disorders

have a direct correlation with the cognitive impairment level. Therefore, not only structurally abnormal clot but also vascular amyloid deposition resulted from Aβ-fibrinogen interaction can lead to progressive cognitive impairment. The Aβ-fibrinogen interaction inhibitors such as the EMT zeolite NPs can prevent the Aβ fibrillation, vascular amyloid deposition and abnormal clot formation.

3.4. Effects of corona coated EMT NPs on the Aβ-fibrinogen interaction The biological behavior of corona-coated NPs is dependent on their protein composition and content

20-21, 40

. Very recently, we have identified and quantified the proteins adsorbed on the

EMT NPs after incubation with 10% and 100% plasmas using Liquid chromatography-tandem



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

Page 14 of 40

mass spectrometry (LC-MS/MS) 30. Some plasma proteins such as Ig gamma-4, -3/lambda -2,-3 and Apolipoprotein C-III specifically associated within the protein corona formed in 10% human plasma

30

. All conditions exerted for protein corona formation regarding to concentration and

physicochemical properties of NPs, incubation conditions and plasma concentration were similar to those in our recent study. To investigate the real impacts of EMT NPs on the general coagulation process in human body, the effect of corona-coated NPs on the clot formation/lysis was assessed. The corona-coated EMT NPs considerably reduced the delay in fibrin lysis (Fig. 7A-D). This type of zeolite NPs inhibits the Aβ-fibrinogen interaction even in the presence of protein corona cover. It means that the formation of protein corona does not affect the affinity of EMT NPs for fibrinogen. Therefore, it can be suggested that EMT NPs can be as promising candidate to inhibit the Aβ-fibrinogen interaction in vivo.

3.5. Mechanistic insights on interaction of Aβ with fibrinogen and small molecules As Aβ peptides strengthen the clot network and limit plasmin’s disposal to fibrin, the clots formed in the presence of Aβ are resistant to lysis 41. This implies that the Aβ peptides attach to regions playing an essential role in fibrin degradation. Ahn et al.

31

demonstrated that Aβ

specifically binds to the C-terminus of the fibrinogen β-chain (β366–β414) (see the sequence and structure of Aβ and C-terminus of β-chain in SI, Fig. S2-S5). The nature and regions of interactions between Aβ and fibrinogen were investigated through protein-protein docking experiments (Section S3). Cumulative counts of different interchain bond types in top three docks between Aβ and C-terminus of the fibrinogen β-chain are presented in Table 2 (SI, Fig. S6-S8). According to these results, hydrogen bonds and hydrophobic interactions play the dominant role in binding of Aβ to the C-terminus of the fibrinogen β-chain. Most contributing


Page 15 of 40

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


residues in the interaction between Aβ and C-terminus of the fibrinogen β-chain were identified based on the frequencies of amino acids appearing in the contacts between these molecules (Table 3) (SI, Fig. S9 and S10, Table S1). The locations of the key residues docked against each other are shown in Figure 8. What is remarkable in the case of the C-terminus of the fibrinogen β-chain is the involvement of the two E397 and D398 residues playing critical role in clot formation 42. Many of the other key residues are also located in proximity of this region so called b-hole region (Fig. 8A and B). In the case of Aβ, the majority of key residues are located on the alpha helix and the inner side of the Aβ peptide (Fig. 8C and D). To inhibit Aβ-fibrinogen interaction, it is required to specifically block the binding sites of each of Aβ and/or fibrinogen. Docking small molecules against Aβ was performed to recognize the possible mechanism underlying inhibition of Aβ-fibrinogen interaction. Based on docking analyses, RU-505 binds to Aβ peptides and blocks their fibrinogen-binding site (Fig. 9A and B). RU-505 showed high affinity for small/large helices of Aβ. In addition, four out of the top-5 poses of RU-505 bind to the N-terminal random coil of Aβ (SI, Fig. S11-S13). Thus, RU-505 has two binding regions on the Aβ peptide. Attachment of the inhibitory compound to the coil region of Aβ, which has less contribution to the Aβ-fibrinogen interaction, implies that it may induce conformational changes in Aβ and hence prevent typical interaction with fibrinogen. The docking experiment also shows that the hydrophobic interaction is the only force keeping these molecules attached. Through virtual screening and QSAR studies, we introduced a small molecule that can be a potential candidate for inhibition of Aβ-fibrinogen interaction (SI, sections S5 and S6). T-777 is a new small molecule having higher affinity for Aβ compared to RU-505 (SI, Fig. S16). The attachment of T-777 to Aβ results from two hydrogen bonds and three hydrophobic contacts. The lowest-energy binding pose and its interaction scheme are illustrated in Fig. 9C and D (SI, Fig.



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

Page 16 of 40

S14 and S15). In contrast to the EMT zeolite NPs that block the Aβ binding sites of fibrinogen, the T-777 and RU-505 small molecules block the fibrinogen-binding site of Aβ. EMT NP and T777 small molecule introduced in this study inhibit Aβ-fibrinogen interaction via blocking the binding sites of fibrinogen and Aβ, respectively.

3.6. Effects of EMT NP on preformed abnormal clot We investigated the effects of bare and corona-coated EMT NPs on preformed abnormal clot. After formation of abnormal clot, tissue type plasminogen activator (tPA), plasminogen, and bare/corona-coated EMT NPs were added into the solution and the fibrinolysis time was measured. The data obtained from this analysis showed that EMT NPs have no significant effect on the preformed abnormal clot (Figure 10 A-D). The structurally abnormal clot was resistant to fibrinolysis even in the presence of increasing concentration of EMT NP. This means EMT NP cannot interact with and capture fibrinogen molecules associated within the abnormal clot. Therefore, it can be suggested that EMT zeolite and Aβ 25-35/ Aβ 1-42 have common binding site on fibrinogen

3.7. Role of amino acid sequence of Aβ in abnormal clot formation The residues contributing in the binding of Aβ to fibrinogen were identified using docking analysis. To evaluate the role of amino acid sequence of Aβ in abnormal clot formation, the clot formation/degradation was assessed in the presence of Aβ consisted of residues 25-35. This peptide comprises residues 30-35, which is known as a hot spot region triggering fibrillation 43. Aβ (25-35) contains the most contributing residues (Leu 34 and Met 35) in the interaction of Aβ and fibrinogen. The clot formation and degradation were assessed in the presence and absence of


Page 17 of 40

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


matured Aβ (25-35) fibrils. A significant delay in fibrin clot dissolution was observed in the presence of Aβ (25-35) (Fig. 4C). This finding indicates that the structurally abnormal clot forms in the presence of a shorter version of Aβ. These experimental results validated the theoretical data obtained from docking analysis. The same experimental conditions were exerted to evaluate the inhibitory effect of EMT NPs on Aβ (25-35)/Aβ (1-42)-fibrinogen interactions. The EMT NPs with different concentrations showed inhibitory effect on the interaction of fibrinogen with both

Aβ

(25-35)

and

Aβ

(1-42),

and

considerably

reduced

the

delay in

clot

formation/degradation (Fig. 11A). The corona-coated EMT NPs also inhibited Aβ (25-35)fibrinogen interaction (Fig. 11B and C). Therefore, the inhibitory effect of the EMT NPs on Aβfibrinogen interaction is not related to the sequence of Aβ. These findings confirmed our hypothesis suggesting that the EMT zeolite NPs inhibit the Aβ-fibrinogen interaction via blocking the Aβ binding sites of fibrinogen. Aβ-fibrinogen interaction inhibitors bind directly to Aβ or fibrinogen and block their binding sites. It is well-recognized that Aβ monomer has physiological (e. g. neuroprotective and antioxidative) function in brain44. Neurons secret Aβ as compensatory response to excessive oxidative stress45. It can be as either cause or effect in AD and hence, Aβ targeting cannot be as effective therapeutic approach. On the other hand, fibrinogen enters central nervous system (CNS) under pathological conditions leading to blood brain barrier disruption/leakage and deposit as insoluble fibrin46-47. Fibrinogen/fibrin accumulation in brain induces inflammation and immune responses resulting in neural degeneration/death48-49. Reducing the number of free fibrinogen molecule in brain led to decrease in immune response, cerebral amyloid angiopathy and memory loss18. Therefore, targeting/capturing fibrinogen in brain is more effective compared to targeting/capturing Aβ. This means EMT NP has higher therapeutic efficacy compared to T-



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

Page 18 of 40

777 small molecule. Drug delivery to brain is known as the most challenging issue associated with CNS drug development50. To date, few inhibitors have been reported that can cross BBB51. In recent years, several nanocarriers have been used for delivery of drugs into the brain. These NPs were functionalized with ligands actively target receptors (e. g. transferrin, albumin, apolipoprotein, insulin, ceruloplasmin) expressed at the BBB52-57. In this work, we evaluated the inhibitory efficacy of zeolite NPs on Aβ-fibrinogen interaction. In this work, we evaluated the inhibitory efficacy of zeolite NPs on Aβ-fibrinogen interaction. The future studies should be focused on development of safe strategies to transport bare/functionalized zeolite NPs across BBB.

4. Conclusions The present research reports on different ways to suppress the interaction between Aβ and fibrinogen in the brain, which results in formation of lysis-resistant clots and the consequent development of Alzheimer disease. This approach comprehended various methods from designing small chemical compounds to manufacturing and assaying the inhibitory NPs. EMT zeolite NPs bind the fibrinogen molecules associated with the Aβ proteins thus suppresses the Aβ-fibrinogen interaction within abnormal clots. The template-free zeolite NPs have the advantages of being non-toxic and compatible with the physiological conditions. The inhibition of Aβ-fibrinogen interaction by zeolite NPs was investigated using both Aβ (1-42) and Aβ (2535), and it was revealed that the effect of zeolite NPs does not depend on Aβ sequences. Besides, it is shown that the protein corona does not affect the inhibitory feature of EMT zeolite. The computational and experimental results are in a good agreement, both revealing the major


Page 19 of 40

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


residues contributing in the interaction of Aβ with fibrinogen. Binding of small inhibitory molecules was also investigated; the new compound T-777 with greater inhibition efficacy is found. In summary, the results point toward promising pharmaceutical drugs for overcoming pathological complications associated with the Alzheimer disease. Supporting Information. Detailed results of XRD and computational docking analyses are provided in Supporting Information.



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

Page 20 of 40

References 1. Lashuel, H. A.; Hartley, D.; Petre, B. M.; Walz, T.; Lansbury, P. T., Neurodegenerative Disease: Amyloid Pores from Pathogenic Mutations. Nature 2002, 418 (6895), 291. 2. Mattson, M. P., Pathways Towards and Away from Alzheimer's Disease. Nature 2004, 430 (7000), 631-639. 3. Hardy, J. A.; Higgins, G. A., Alzheimer's Disease: the Amyloid Cascade Hypothesis. Science 1992, 256 (5054), 184. 4. de la Torre, J. C., Is Alzheimer's Disease a Neurodegenerative or a Vascular Disorder? Data, Dogma, and Dialectics. The Lancet Neurol 2004, 3 (3), 184-190. 5. 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 (4), 279-287. 6. Mahmoudi, M.; Quinlan-Pluck, F.; Monopoli, M. P.; Sheibani, S.; Vali, H.; Dawson, K. A.; Lynch, I., Influence of the Physiochemical Properties of Superparamagnetic Iiron Oxide Nanoparticles on Amyloid β Protein Fibrillation in Solution. ACS chem neurosci 2013, 4 (3), 475-485. 7. Mirsadeghi, S.; Dinarvand, R.; Ghahremani, M. H.; Hormozi-Nezhad, M. R.; Mahmoudi, Z.; Hajipour, M. J.; Atyabi, F.; Ghavami, M.; Mahmoudi, M., Protein Corona Composition of Gold Nanoparticles/Nanorods Affects Amyloid Beta Fibrillation Process. Nanoscale 2015, 7 (11), 5004-5013. 8. Mahmoudi, M.; Akhavan, O.; Ghavami, M.; Rezaee, F.; Ghiasi, S. M. A., Graphene Oxide Strongly Inhibits Amyloid Beta Fibrillation. Nanoscale 2012, 4 (23), 7322-7325. 9. Mishra, R.; Bulic, B.; Sellin, D.; Jha, S.; Waldmann, H.; Winter, R., Small‐Molecule Inhibitors of Islet Amyloid Polypeptide Fibril Formation. Angew Chem Int Ed 2008, 47 (25), 4679-4682. 10. Geng, J.; Li, M.; Ren, J.; Wang, E.; Qu, X., Polyoxometalates as Inhibitors of the Aggregation of Amyloid β Peptides Associated with Alzheimer’s Disease. Angew Chem 2011, 123 (18), 4270-4274. 11. 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 (21), 8691-8696. 12. Li, M.; Yang, X.; Ren, J.; Qu, K.; Qu, X., Using Graphene Oxide High Near‐Infrared Absorbance for Photothermal Treatment of Alzheimer's Disease. Adv. Mater. 2012, 24 (13), 1722-1728. 13. Chen, Y.; Gurol, M.; Rosand, J.; Viswanathan, A.; Rakich, S.; Groover, T.; Greenberg, S.; Smith, E., Progression of White Matter Lesions and Hemorrhages in Cerebral Amyloid Angiopathy. Neurol 2006, 67 (1), 83-87. 14. Humpel, C., Chronic Mild Cerebrovascular Dysfunction as a Cause for Alzheimer's Disease? Exp Gerontol 2011, 46 (4), 225-232. 15. Paul, J.; Strickland, S.; Melchor, J. P., Fibrin Deposition Accelerates Neurovascular Damage and Neuroinflammation in Mouse Models of Alzheimer's Disease. J Exp Med 2007, 204 (8), 1999-2008. 16. Mosesson, M., Fibrinogen and Fibrin Structure and Functions. J Thromb Haemost 2005, 3 (8), 1894-1904. 17. Weisel, J. W., Fibrinogen and Fibrin. Adv Prot Chem 2005, 70, 247-299. 20 ACS Paragon Plus Environment

Page 21 of 40

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


18. Cortes-Canteli, M.; Paul, J.; Norris, E. H.; Bronstein, R.; Ahn, H. J.; Zamolodchikov, D.; Bhuvanendran, S.; Fenz, K. M.; Strickland, S., Fibrinogen and β-amyloid Association Alters Thrombosis and Fibrinolysis: a Possible Contributing Factor to Alzheimer's Disease. Neuron 2010, 66 (5), 695-709. 19. Cortes-Canteli, M.; Zamolodchikov, D.; Ahn, H. J.; Strickland, S.; Norris, E. H., Fibrinogen and Altered Hemostasis in Alzheimer's Disease. J Alz Dis 2012, 32 (3), 599-608. 20. Hajipour, M. J.; Akhavan, O.; Meidanchi, A.; Laurent, S.; Mahmoudi, M., HyperthermiaInduced Protein Corona Improves the Therapeutic Effects of Zinc Ferrite Spinel-graphene Sheets Against Cancer. RSC Adv 2014, 4 (107), 62557-62565. 21. Hajipour, M. J.; Raheb, J.; Akhavan, O.; Arjmand, S.; Mashinchian, O.; Rahman, M.; Abdolahad, M.; Serpooshan, V.; Laurent, S.; Mahmoudi, M., Personalized Disease-specific Protein Corona Influences the Therapeutic Impact of Graphene Oxide. Nanoscale 2015, 7 (19), 8978-8994. 22. Shanehsazzadeh, S.; Lahooti, A.; Hajipour, M. J.; Ghavami, M.; Azhdarzadeh, M., External Magnetic Fields Affect the Biological Impacts of Superparamagnetic Iron Nanoparticles. Col Surf B: Biointerfaces 2015, 136, 1107-1112. 23. Monopoli, M. P.; Åberg, C.; Salvati, A.; Dawson, K. A., Biomolecular Coronas Provide the Biological Identity of Nanosized Materials. Nature Nanotechnol 2012, 7 (12), 779-786. 24. Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S., Protein− Nanoparticle Interactions: Opportunities and Challenges. Chem Rev 2011, 111 (9), 5610-5637. 25. Mahmoudi, M.; Abdelmonem, A. M.; Behzadi, S.; Clement, J. H.; Dutz, S.; Ejtehadi, M. R.; Hartmann, R.; Kantner, K.; Linne, U.; Maffre, P., Temperature: the “Ignored” Factor at the Nanobio Interface. ACS Nano 2013, 7 (8), 6555-6562. 26. Hajipour, M. J.; Laurent, S.; Aghaie, A.; Rezaee, F.; Mahmoudi, M., Personalized Protein Coronas: a “Key” Factor at the Nanobiointerface. Biomater Sci 2014, 2 (9), 1210-1221. 27. Ng, E.-P.; Chateigner, D.; Bein, T.; Valtchev, V.; Mintova, S., Capturing Ultrasmall EMT Zeolite from Template-Free Systems. Science 2012, 335 (6064), 70-73. 28. Mintova, S.; Olson, N. H.; Valtchev, V.; Bein, T., Mechanism of Zeolite A Nanocrystal Growth from Colloids at Room Temperature. Science 1999, 283 (5404), 958-960. 29. Laurent, S.; Ng, E.-P.; Thirifays, C.; Lakiss, L.; Goupil, G.-M.; Mintova, S.; Burtea, C.; Oveisi, E.; Hébert, C.; De Vries, M., Corona Protein Composition and Cytotoxicity Evaluation of Ultra-Small Zeolites Synthesized from Template Free Precursor Suspensions. Toxicol Res 2013, 2 (4), 270-279. 30. Rahimi, M.; Ng, E.-P.; Bakhtiari, K.; Vinciguerra, M.; Ahmad, H. A.; Awala, H.; Mintova, S.; Daghighi, M.; Rostami, F. B.; de Vries, M., Zeolite Nanoparticles for Selective Sorption of Plasma Proteins. Sci Reports 2015, 5. 31. Ahn, H. J.; Zamolodchikov, D.; Cortes-Canteli, M.; Norris, E. H.; Glickman, J. F.; Strickland, S., Alzheimer's Disease Peptide β-amyloid Interacts with Fibrinogen and Induces its Oligomerization. Proc Natl Acad Sci 2010, 107 (50), 21812-21817. 32. Hudson, S. A.; Ecroyd, H.; Kee, T. W.; Carver, J. A., The Thioflavin T Fluorescence Assay for Amyloid Fibril Detection Can be Biased by the Presence of Exogenous Compounds. FEBS J 2009, 276, 5960–5972. 33. Trott, O.; Olson, A. J., AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J Comput Chem 2010, 31, 455-461. 21 ACS Paragon Plus Environment


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

Page 22 of 40

34. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J., AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J Comput Chem 2009, 30 (16), 2785-2791. 35. Laskowski, R. A.; Swindells, M. B., LigPlot+: Multiple Ligand–Protein Interaction Diagrams for Drug Discovery. J Chem Inf Model 2011, 51 (10), 2778-2786. 36. Irwin, J. J.; Sterling, T.; Mysinger, M. M.; Bolstad, E. S.; Coleman, R. G., ZINC: a Free Tool to Discover Chemistry for Biology. J Chem Inf Model 2012, 52 (7), 1757-1768. 37. Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V. A.; Radchenko, E. V.; Zefirov, N. S.; Makarenko, A. S., Virtual Computational Chemistry Laboratory–Design and Description. J Comput Aid Mol Des 2005, 19 (6), 453-463. 38. Ahn, H. J.; Glickman, J. F.; Poon, K. L.; Zamolodchikov, D.; Jno-Charles, O. C.; Norris, E. H.; Strickland, S., A novel Aβ-fibrinogen Interaction Inhibitor Rescues Altered Thrombosis and Cognitive Decline in Alzheimer’s Disease Mice. J Exp Med 2014, 211 (6), 1049-1062. 39. McGowan, E.; Pickford, F.; Kim, J.; Onstead, L.; Eriksen, J.; Yu, C.; Skipper, L.; Murphy, M. P.; Beard, J.; Das, P., Aβ42 Is Essential for Parenchymal and Vascular Amyloid Deposition in Mice. Neuron 2005, 47 (2), 191-199. 40. Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C., Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nature Nanotechnol 2013, 8 (10), 772-781. 41. Zamolodchikov, D.; Strickland, S., Aβ Delays Fibrin Clot Lysis by Altering Fibrin Structure and Attenuating Plasminogen Binding to Fibrin. Blood 2012, 119 (14), 3342-3351. 42. Kostelansky, M. S.; Bolliger-Stucki, B.; Betts, L.; Gorkun, O. V.; Lord, S. T., BβGlu397 and BβAsp398 but Not BβAsp432 Are Required for “B:b” Interactions. Biochem 2004, 43 (9), 2465– 2474. 43. Frozza, R. L.; Horn, A. P.; Hoppe, J. B.; Simão, F.; Gerhardt, D.; Comiran, R. A.; Salbego, C. G., A Comparative Study of β-amyloid Peptides Aβ1-42 and Aβ25-35 Toxicity in Organotypic Hippocampal Slice Cultures. Neurochem Res 2009, 34 (2), 295-303. 44. Atwood, C. S.; Obrenovich, M. E.; Liu, T.; Chan, H.; Perry, G.; Smith, M. A.; Martins, R. N., Amyloid-β: a Chameleon Walking in Two Worlds: a Review of the Trophic and Toxic Properties of Amyloid-β. Brain Res Rev 2003, 43 (1), 1-16. 45. Nunomura, A.; Castellani, R. J.; Zhu, X.; Moreira, P. I.; Perry, G.; Smith, M. A., Involvement of Oxidative Stress in Alzheimer Disease. J Neuropathol Exp Neurol 2006, 65 (7), 631-641. 46. Ryu, J. K.; Davalos, D.; Akassoglou, K., Fibrinogen Signal Transduction in the Nervous System. J Thromb Haemost 2009, 7 (s1), 151-154. 47. Schachtrup, C.; Ryu, J. K.; Helmrick, M. J.; Vagena, E.; Galanakis, D. K.; Degen, J. L.; Margolis, R. U.; Akassoglou, K., Fibrinogen Triggers Astrocyte Scar Formation by Promoting the Availability of Active TGF-β after Vascular Damage. J. Neurosci. 2010, 30 (17), 5843-5854. 48. Vidal, B.; Serrano, A. L.; Tjwa, M.; Suelves, M.; Ardite, E.; De Mori, R.; Baeza-Raja, B.; de Lagrán, M. M.; Lafuste, P.; Ruiz-Bonilla, V., Fibrinogen Drives Dystrophic Muscle Fibrosis via a TGFβ/alternative Macrophage Activation Pathway. Gene dev 2008, 22 (13), 1747-1752. 49. Rybarczyk, B. J.; Lawrence, S. O.; Simpson-Haidaris, P. J., Matrix-fibrinogen Enhances Wound Closure by Increasing both Cell Proliferation and Migration. Blood 2003, 102 (12), 40354043. 50. Krol, S.; Macrez, R.; Docagne, F.; Defer, G.; Laurent, S.; Rahman, M.; Hajipour, M. J.; Kehoe, P. G.; Mahmoudi, M., Therapeutic Benefits from Nanoparticles: the Potential Significance of Nanoscience in Diseases with Compromise to the Blood Brain Barrier. Chem rev 22 ACS Paragon Plus Environment

Page 23 of 40

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


2012, 113 (3), 1877-1903. 51. Gao, N.; Sun, H.; Dong, K.; Ren, J.; Duan, T.; Xu, C.; Qu, X., Transition-Metal-Substituted Polyoxometalate Derivatives as Functional Anti-Amyloid Agents for Alzheimer’s Disease. Nat commun 2014, 5. 52. Ulbrich, K.; Hekmatara, T.; Herbert, E.; Kreuter, J., Transferrin-and Transferrin-ReceptorAntibody-modified Nanoparticles Enable Drug Delivery Across the Blood–Brain Barrier (BBB). Eur. J. Pharm. Biopharm. 2009, 71 (2), 251-256. 53. Lockman, P.; Mumper, R.; Khan, M.; Allen, D., Nanoparticle Technology for Drug Delivery Across the Blood-Brain Barrier. Drug Dev Ind Pharm 2002, 28 (1), 1-13. 54. Ulbrich, K.; Knobloch, T.; Kreuter, J., Targeting the Insulin Receptor: Nanoparticles for Drug Delivery Across the Blood–Brain Barrier (BBB). J Drug Target 2011, 19 (2), 125-132. 55. Roney, C.; Kulkarni, P.; Arora, V.; Antich, P.; Bonte, F.; Wu, A.; Mallikarjuana, N.; Manohar, S.; Liang, H.-F.; Kulkarni, A. R., Targeted Nanoparticles for Drug Delivery Through the Blood– Brain Barrier for Alzheimer's Disease. J control release 2005, 108 (2), 193-214. 56. Kreuter, J., Nanoparticulate Systems for Brain Delivery of Drugs. Adv Drug Deliver Rev 2001, 47 (1), 65-81. 57. Barbu, E.; Molnàr, É.; Tsibouklis, J.; Górecki, D. C., The Potential for Nanoparticle-Based Drug Delivery to the Brain: Overcoming the Blood–Brain Barrier. Expert Opin Drug Deliv 2009, 6 (6), 553-565.



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

Page 24 of 40

Table captions Table 1. Physicochemical properties of EMT zeolite NPs. Table 2. Interchain bonds between Aβ and C terminus of the fibrinogen β-chain. Table 3. Most contributing residues in the interaction of Aβ and C terminus of the fibrinogen βchain.

Figures captions Figure 1. DLS curve and TEM image of EMT zeolite NPs. Figure 2. Zeta potential curve of EMT zeolite suspension at different concentrations. Figure 3. (A) Kinetics of Aβ fibrillation at 37 °C after 12 and 24 h monitored by ThT fluorescence intensity. (B) TEM image of mature Aβ (1-42) fibril formed after 24 h. Figure 4. (A and B) Total time for clot formation and degradation in the presence of Aβ 1-42 and (C) Aβ 25-35. (Column graphs indicate mean ± SEM of 3 experiments; asterisk means significant change compared to control at p < 0.05). Figure 5. (A and B) Normal clot formation and lysis in the presence of bare and corona coated EMT zeolite NPs obtained from (C and D) 10% and (E and F) 100% plasmas. (Column graphs indicate mean ± SEM of 3 experiments; asterisk means significant change compared to control at p < 0.05). Figure 6. (A and B) Total time for clot formation and lysis in the presence of Aβ 1-42 and EMT zeolite NPs with different concentrations. (Column graphs indicate mean ± SEM of 3 experiments; asterisk means significant change compared to Aβ42 (10µM) at p < 0.05). Figure 7. Total time for clot formation and degradation in the presence of Aβ 1-42 and corona coated EMT zeolite NPs obtained from (A and B) 10% and (C and D) 100% plasmas. (Column


Page 25 of 40

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


graphs indicate mean ± SEM of 3 experiments; asterisk means significant change compared to Aβ42 (10µM) at p < 0.05). Figure 8. Locations of the top 15 interacting residues on the C terminus of the fibrinogen βchain and the β-Amyloid protein. (A) Front view and (B) side view of C terminus of the fibrinogen β-chain. (C) Front view and (D) side view of Aβ protein. Figure 9. (A and B) The lowest-energy pose of RU-505 on the N-terminal random coil of Aβ peptide and its corresponding scheme of interactions. Radiating lines depict the hydrophobic interactions toward corresponding atoms in ligand. (C and D) The lowest-energy pose, which T777 takes against Aβ and the scheme of interactions for this pose. H-bonds are depicted as green dashed lines. Figure 10. Effects of bare and corona-coated EMT zeolite NPs on abnormal clot formed in the presence of Aβ1-42 (A and B) and Aβ25-35 (C and D) Figure 11. Total time for clot formation and degradation in the presence of (A) Aβ 25-35, bare EMT zeolite NPs, and corona coated EMT zeolite NPs obtained from (B) 10%, and (C) 100% plasmas. (Column graphs indicate mean ± SEM of 3 experiments; asterisk means significant change compared to Aβ42 (10µM) at p < 0.05).



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

Page 26 of 40

Table 1

Zeolite

EMT a

Porosity propertiesa

Mean

Si/Al

Chemical

ratio

composition

SBET

Sext

Vmicro

Vmeso

Vtotal

dmicrob

1.17

Na88(AlO2)88(SiO2)104

680

200

0.24

0.88

1.12

0.74

2

-1

2

-1

dmesoc 20

b

Determined by BJH method.

c

Determined by DFT method.

d

Determined by DLS and TEM (nm).

e

Determined at 0.1 wt% concentration (mC m-2).

f

Determined at 0.1 wt% concentration (mC g-1).


-1

sized 13.8

Charge

Surface

densitye

chargef

-0.608

-353.3

SBET: BET specific surface area (m g ); Sext: external surface area (m g ); Vmicro: micropore volume (cm g ); Vmeso: mesopore volume (cm g-1);

Vtotal: total pore volume (cm3 g-1); dmicro: micropore diameter (nm); dmeso: mesopore diameter (nm).

3

paricle

3

Page 27 of 40

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


Table 2

Cumulative number of

Types of interaction

contacts

Total H-bonds

164

Main chain–Main chain H-bonds

30

Main chain–Side chain H-bonds

74

Side chain–Side chain H-bonds

60

Hydrophobic interactions

154

Ionic interactions (Salt bridges)

21

Cation-π interactions

10

Aromatic-aromatic interactions

7

Aromatic-sulfur interactions

5



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

Page 28 of 40

Table 3 (β366–β414) residues Frequency Aβ residues Frequency Phe374

37

Leu34

34

Trp385

26

Met35

31

Arg380

25

Ala42

28

Trp403

24

Ile41

26

Asp398

21

Phe20

24

Asn371

20

Ala21

22

Asp379

17

Leu17

19

Asp381

17

His14

18

Glu397

15

Lys28

17

Ile369

14

Val24

15

Phe375

12

Lys16

14

Ala409

12

Asp23

13

Met373

11

Val39

13

Leu386

10

His13

12

Trp402

9

Glu11

10


Page 29 of 40

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



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 2


Page 30 of 40

Page 31 of 40

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



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 4


Page 32 of 40

Page 33 of 40

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 5



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


Page 34 of 40

Page 35 of 40

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


Figure 7



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 8


Page 36 of 40

Page 37 of 40

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 9



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 10


Page 38 of 40

Page 39 of 40

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 11



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

TOC


Page 40 of 40

Zeolite Nanoparticles Inhibit AÎ²âFibrinogen Interaction and Formation

Recommend Documents

Zeolite Nanoparticles Inhibit AÎ²âFibrinogen Interaction and Formation