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Curcumin-loaded amine-functionalized mesoporous silica nanoparticles inhibit the #-synuclein fibrillation and reduce its cytotoxicity-associated effects Nayere Taebnia, Dina Morshedi, Soheila Yaghmaei, Farhang Aliakbari, Fatemeh Rahimi, and Ayyoob Arpanaei Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02935 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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Curcumin-loaded amine-functionalized mesoporous silica nanoparticles inhibit the α-synuclein fibrillation and reduce its cytotoxicity-associated effects Nayere Taebnia, †, ‡ Dina Morshedi, †,* Soheila Yaghmaei, ‡ Farhang Aliakbari, † Fatemeh Rahimi, † Ayyoob Arpanaei†,* †

Department of Industrial and Environmental Biotechnology, National Institute of Genetic

Engineering and Biotechnology (NIGEB), Tehran - Karaj Highway, Tehran, Iran ‡

Department of Chemical and Petroleum Engineering, Sharif University of Technology, Azadi

Ave., Tehran, Iran. KEYWORDS. α-synuclein fibrillation; Curcumin; Cytotoxicity; Mesoporous silica nanoparticles

ABSTRACT. This study aimed to develop a drug carrier based on amine-functionalized mesoporous silica nanoparticles (AAS-MSNPs) for a poorly water-soluble drug, curcumin, and to study its effects on the α-synuclein fibrillation and cytotoxicity properties. Here, we show that AAS-MSNPs possess high values of loading efficiency and capacity (33.5% and 0.45 mg drug/mg MSNPs, respectively) for curcumin. It is also revealed that α-synuclein species interact strongly

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with curcumin-loaded AAS-MSNPs, leading to a significant inhibition in the fibrillation process. Furthermore, these samples reduce toxic effects of curcumin. However, drug-loaded AAS-MSNPs cannot affect the cytotoxic properties of the formed fibrils considerably. In addition, loaded curcumin onto AAS-MSNPs show enhanced stability in comparison to that of the free drug. These remarkable properties introduce AAS-MSNPs as a promising tool for the formulation of poorly water-soluble drugs such as curcumin.

INTRODUCTION The majority of innovative drugs display very poor aqueous solubility characteristics, resulting in their poor bioavailability. To overcome this hurdle, different strategies for nanomaterials-based drug delivery carriers have been developed such as liposomes, polymers and inorganic nanoparticles [1, 2]. Since their first appearance in drug delivery by Vallet-Regi et al. in 2001 [3], there has been ever increasing attention in synthesis and application of mesoporous silica nanoparticles (MSNPs) due to their particular characteristics [4-7]. Silica-based nanoparticles have attracted considerable attention as drug carriers because of their unique features, such as large surface area, tunable pore size, facile surface multi-functionalization, chemical inertness, excellent biocompatibility and biodegradability as well as high chemical and physical stability. These characteristics have made MSNPs ideal for biomedical applications [7, 8]. However, due to the hydrophilic surface, the loading of hydrophobic drugs onto MSNPs is usually very low [7]. Chemical modification of the surfaces of MSNPs with proper functional groups provides different binding sites and therefore significant amount of drug can be loaded onto the particle matrix [4, 9]. Various drugs can be loaded onto functionalized MSNPs either by incorporation into the matrix or adsorption onto the surface of nanoparticles [10].


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The aim of this work is to develop functionalized MSNPs for loading a poorly water-soluble drug in order to enhance its effects. To achieve this, we selected a well-known polyphenol drug, curcumin (CUR) (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6- heptadiene-3,5-dione), as the model drug which has been shown to exhibit therapeutic effects against neurological disorders such as Parkinson’s disease. CUR is a natural and low molecular weight polyphenol derived from the root of the herb Curcuma longa [11-14]. CUR possesses a variety of pharmacological applications, including antioxidant, anti-amyloid and anti-inflammation; and according to the recent researches, it displays low toxicity to normal cells at low concentrations [12, 13-16]. Spillantini et al. showed that CUR potentially inhibits the formation of amyloid fibrils and toxic oligomeric forms of αsynuclein (α-Syn) which is found to accumulate in amyloid-rich Lewy bodies and consequently causes toxic effects in the brain of Parkinson’s patients and finally lead to neuronal death [17]. In spite of the wide range of its therapeutic activities, the application of CUR is limited due to its hydrophobic nature and consequently low aqueous solubility and bioavailability [8, 18, 19]. Studies to date revealed extremely low levels of CUR in serum (22 – 41 ng/ml) even after high amounts of oral administration (8 g/day) [19-21]. In order to enhance water solubility and thus bioavailability of CUR through loading on a carrier, various types of nanoparticles such as liposomes, hydrogels, and cyclodextrins have been used for this hydrophobic drug [21-23]. Reports have been published previously on the influence of amine-functionalized MSNPs on CUR and other poor water soluble drugs loading, release and bioavailability for cancer cells [24,25]. However, the novel approach of the present study is using AAS-MSNPs as nanocarriers for CUR delivery in order to reduce α-Syn fibrillation and its cytotoxicity. The surface functionalization of MSNPs using 3-(2-aminoethyl amino) propyltrimethoxysilane (AAS) molecules was employed to increase the loading capacity and efficiency of CUR onto MSNPs. Characterization of the


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synthesized nanoparticles, study of the adsorption isotherms of CUR onto the MSNPs, and the release profile of CUR in the medium were also undertaken. Finally, the effect of CUR-AASMSNPs on the fibrillation of α-Syn and their toxic effects on rat pheochromocytoma (PC-12) cells were evaluated. EXPERIMENTAL SECTION Materials. Tetraethylorthosilicate (TEOS), ethanol (99.9%), cetyltrimethylammonium bromide (CTAB), 3-(2-aminoethyl amino) propyltrimethoxysilane (AAS, 97 vol%), hydrochloric acid, glacial acetic acid, and ethylene diamine tetraacetic acid (EDTA) were purchased from Merck (New York, USA). Trypsin solution and 3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). CUR was purchased from Acros organics (Geel, Belgium). Penicillin–streptomycin, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Gibco BRL (Gaithersberg, MD, USA). All the chemicals and solvents were used without further purification. Water was deionized using Q-check controller system (OES Co., USA). PC12 cells and recombinant human α-Syn were acquired from the Pasteur Institute (Tehran, Iran) and National Institute of Genetic Engineering and Biotechnology (NIGEB), respectively. Preparation and characterization of AAS-MSNPs. MSNPs were synthesized according to the template removing procedure described in our previous work [26]. Afterwards, in order to modify the surface of MSNPs, grafting procedure was employed. For that, 100 mg of the obtained MSNPs was dispersed in ethanol for 10 minutes followed by addition of deionized water for hydrolysis and glacial acetic acid as the catalyst. Then, AAS was added into the reaction mixture, and the solution was stirred for 1 h at 1000 rpm. Once the reaction was completed, AAS-MSNPs were washed with ethanol and deionized water. Hitachi S5500 electron microscopy operating at 30 kV


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was used to acquire images of MSNPs. The pore characteristics of the samples were studied by determining the nitrogen adsorption using a surface area and pore size analyzer (SA3100, Beckman coulter, USA) at −196 °C. Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) plots were used to determine the surface area and pore characteristics of MSNPs. Zeta (ζ) potential values of the particles before and after drug loading were measured using a Malvern Zetasizer Nano instrument (S90, UK). Preparation of CUR-loaded Nanoparticles. The drug loading was performed by soaking AASMSNPs in 1 mL ethanol solutions of CUR at a ratio of 1:1 w/w and the mixture was stirred gently at 80 rpm for 24 h under ambient conditions. CUR-loaded nanoparticles, denoted as CUR-AASMSNPs, were separated by centrifugation and carefully washed with ethanol to remove any physico-sorbed and loosely bounded CUR. The supernatants were collected, and the drug content was quantified by a colorimetric assay [27] using UV-Vis spectroscopy at 450 nm. A standard curve was formulated from known concentrations of curcumin in ethanol and drug content was determined by comparing the absorbance of the sample, measured at 450 nm, with the standard curve. Then, the drug loading efficiency and capacity were calculated using the following equations: Loading efficiency (%) = [

(Total mass of applied curcumin – mass of nonadsorbed curcumin)

Loading capacity (mg/mg) = [

Total mass of applied curcumin

] × 100

(Total mass of applied curcumin – mass of nonadsorbed curcumin) Total mass of MSNPs

]

(1) (2)

Study of adsorption isotherms. Langmuir, Freundlich and Langmuir-Freundlich isotherm equations were used to analyze the isotherm of CUR adsorption onto MSNPs and obtain equilibrium parameters. Langmuir equation is as follows: 𝑞𝑒 =

𝑞𝑚 𝐾𝐿 𝐶𝑒

(3)

1+𝐾𝐿 𝐶𝑒


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where, 𝑞𝑒 is the equilibrium adsorption capacity (mg adsorbate/mg adsorbent), 𝐶𝑒 is the equilibrium liquid-phase concentration (mg/L), 𝑞𝑚 is the monolayer adsorption capacity (mg adsorbate/mg adsorbent), and 𝐾𝐿 is the adsorption equilibrium constant (L/mg). This model is based on the formation of a monomolecular layer of adsorbate on the surface of the adsorbent. In this model, all active sites are identical and energetically equivalent, and there is a finite capacity for the adsorbate. On the other hand, the Freundlich model is not restricted to the formation of monolayer, and it is used to describe adsorption on heterogeneous surfaces, with different values of adsorption energy. This fact results in the first occupation of the most active sites with maximum energy. [28]. The Freundlich equation is as follows: ⁄𝑛

𝑞𝑒 = 𝐾𝐹 𝐶𝑒1

(4)

where, 𝐾𝐹 is Freundlich constant with multilayer adsorption related to bond strength and is the amount adsorbed at unit concentration, which is, at 1 mg/L; and n is the adsorption intensity and indicates bond energies between the adsorbate and the adsorbent [28]. If the Langmuir isotherm is enhanced by considering the intermolecular interactions, LangmuirFreundlich model is derived as follows: 1⁄𝑛

𝑞𝑒 =

𝑞𝑚 𝐾𝐶 𝐶𝑒

(5)

1⁄𝑛

1+𝐾𝐶 𝐶𝑒

Where, 𝐾𝐶 is Langmuir-Freundlich constant [29]. In vitro drug release study. Five milligrams of CUR-AAS-MSNPs were dispersed in 25 mL of Tris-HCl buffer (25 mM Tris, 150 mM NaCl, and pH 7.4). The samples were incubated at 37 °C for 48 h, and continuous stirring (120 rpm) was kept on to avoid limitation by external diffusion constrains. At predetermined time intervals, 1 mL of solution was withdrawn from the release


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medium and the same volume of fresh buffer was added to continue the release test. Then, the withdrawn samples were centrifuged at 10,000 rpm for 15 min and the supernatant was measured by UV-Vis spectroscopy at 450 nm (Cecil CE750, Cambridge, UK). Then, the cumulative release percentage was calculated. Effect of AAS-MSNPs and CUR-AAS-MSNPs on the aggregation of α-Syn. The aggregation study was performed in 1.5 ml eppendorf tubes containing α-Syn monomers at the concentration of 2 mg/mL in Tris-Cl buffer (25 mM, pH 7.4) in the absence and presence of AAS-MSNPs and CUR-AAS-MSNPs, before and after the burst release of CUR. The solution was microfuged (10000 rpm) for 10 minutes at 4 ºC to remove any traces of pre-existing oligomers or aggregates before incubation with AAS-MSNPs and CUR-AAS-MSNPs, [26]. Each sample with a total volume of 150 µL—containing fixed protein concentration of 2 mg/mL and nanoparticles concentration of 100 µg/mL—was shaken in a 1.5-mL Eppendorf tube at 80 rpm and 37 °C in water bath for 24 h to complete the fibrillation process. As a control, 150 µL of α-Syn was also incubated in the absence of drug-loaded nanoparticles. Three independent experiments were performed for each sample [26]. Thioflavin T (ThT) fluorescence assay. ThT is a dye that specifically binds to amyloid aggregates, and thus allowing quantitative assessment of the presence of fibrillar species [29]. In order to analyze the fibril formation using ThT assay, 10 µL of α-Syn solution was added to 490 µL of 10 µM ThT solution containing 10 mM Tris (pH 8.0) and was mixed gently. The fluorescence signals were acquired in the emission range of 450-550 nm by exciting the samples at 440 nm. Each experimental point was an average of the fluorescence signal of three samples containing aliquots of the same solution. A Cary Eclipse VARIAN fluorescence


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spectrophotometer (Mulgrave, Australia) was used for the fluorescence assays at room temperature. Fluorescent staining of aggregates (fluorescence microscopy). By using the amyloid-specific fluorophore, ThT, fluorescence microscopy images of protein aggregates can be observed [30]. For that, 15 µL of the incubated samples were added to 15 µL of ThT (500 µM). Samples were incubated for 5 min at room temperature, and then were spread onto a microscopic slide. Finally, Fluorescence microscopy images were recorded by Ceti inverso TC100 microscope (Medline scientific, Oxon, UK). Circular Dichroism (CD) Analysis. CD spectra of samples were obtained in the Far-UV region (190–260 nm) using an AVIV 215 spectropolarimeter (Aviv Associates, Lakewood, NJ, USA) in 0.1-cm circular cuvettes at room temperature. Cell viability study. In vitro cytotoxicity experiments were performed using MTT reduction [31]. PC12 cells were seeded in 96-well plates at density of 3×104 cells/well in 100 µL of DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Then, samples were incubated at 37 °C, in a 5% CO2 atmosphere with 95% relative humidity. After 24 h, the old media were replaced with the fresh media containing different concentrations of CUR, bare and AAS-MSNPs, and CUR-AAS-MSNPs in the presence and absence of α-Syn. As CUR is sparingly soluble in aqueous solutions, the solubility and stability of this molecule was enhanced by heat treatment. The mixture was heated, and then filtered to get a homogenous solution [32,33]. After a further period of 24 h, the old media were replaced with fresh media containing 10% MTT (5 mg/mL), and the plate was incubated for additional 4 h at 37 °C. Finally, the medium of each well was replaced with 100 µL of DMSO, and the plate was shaken slowly for 10 min in darkness in order to ensure formed formazan crystals being entirely dissolved. Absorption values at 570 nm


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were determined using an automatic multi-well assay plate reader (Expert 96, Asys Hitchech, Austria). Cells grown in the medium without further treatment were taken as the control value at 100% viability. Cell viability was calculated as follows: 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =

𝐴𝑏𝑠570 (𝑇𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠) 𝐴𝑏𝑠570 (𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠)

× 100

(6)

RESULTS AND DISCUSSION Preparation and characterization of AAS-MSNPs. Scanning electron microscopy (SEM) and bright field scanning transmission electron microscopy (BF-STEM) images confirm the spherical shapes and monodispersity of synthesized bare and AAS-MSNPs (Figure 1). It is also obvious that the coating layer of AAS molecules does not notably change the size and morphology of MSNPs.

(a)

(b)

Figure 1. SEM (right) and BF-STEM (left) images of (a) bare MSNP (b) AAS-MSNP

The size of bare and amine functionalized MSNPs estimated using STEM images indicated no significant difference (83±6 nm and 84±2, respectively, p < 0.05). However, as it was shown in


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our previous work [26], hydrodynamic sizes of the prepared particles measured by dynamic light scattering (DLS) technique are considerably larger, most likely due to the aggregation of MSNPs. The nitrogen adsorption/desorption isotherms of bare and functionalized MSNPs were typical type IV isotherms according to the IUPAC classification (Figure 2). The pore characteristics of bare and functionalized MSNPs were determined according to the BET and BJH procedures from the adsorption branches of the isotherms.

(a)

(b)

Figure 2. Nitrogen adsorption/desorption isotherms of (a) bare MSNPs and (b) AAS-MSNPs at −196 °C.

The values for the BET specific surface area (SBET), the total pore volume (VT) and the pore diameter (dBET and dBJH) of bare and AAS-MSNPs and CUR-AAS-MSNPs are given in Table 1. Table 1. Nanoparticles characterization Sample

SBET (m2/g)

VT (cm3/g)

dBET (nm)

dBJH (nm)

ζ (mV)

Bare MSNPs

882.1±0.9

1.08±0.1

4.1±0.4

3.8±0.1

-16.1±0.6

AAS-MSNPs

575.7±0.7

0.81±0.2

2.9±0.8

2.6±0.4

+24.5±1.3

CUR-AAS-MSNPs

571.4±0.6

0.77±0.3

2.5±0.3

2.2±0.2

+18.7±0.9


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It can be seen that all the MSPNs possess high SBET and VT, indicating their potential application as a carrier in storing more drug molecules in the drug-release system [34]. Furthermore, the results show that SBET, VT and dBJH were reduced after functionalization and drug loading due to the attachment of AAS and CUR molecules to the pore surfaces. Despite the reduction in the adsorbed amount of nitrogen for AAS-MSNPs because of decrease in the pore size, the shape of the hysteresis loop remained unchanged. This means that the pore shape is not significantly changed by the post-synthesis approach of surface modification. The results of ζ potential, presented in table 1, reveal that ζ potential value of bare MSNPs move toward positive values as the surface modification using AAS takes place most likely due to the amine groups presented in these molecules. These results clearly state that AAS molecules are successfully attached on the MSNPs surface. AAS-MSNPs show higher loading capacity and efficiency for Curcumin. After investigation of morphological characteristics, bare and AAS-MSNPs were probed for their ability to load CUR. No significant changes in the size of nanoparticles were observed following loading the CUR onto MSNPs (data not shown), but the zeta potential value was decreased for both types of MSNPs, possibly due to the existing negatively charged groups such as hydroxyl, carbonyl and methoxy, in CUR molecules [35]. The results revealed that both, bare and AAS-MSNPs, can be loaded with CUR but with different loading capacities (Figure 3). By increasing the initial mass ratio of CUR to MSNPs from 1:2 to 2:1, the drug loading efficiencies were increased from 5.3 to 13.9% and 22.4 to 33.5% for bare and AAS-MSNPs, respectively (Figure 3a). While, under the same condition, the drug loading capacity (mg CUR/mg MSNPs) values were decreased from 0.11 to 0.07 and 0.45 to 0.17 for bare and AAS-MSNPs, respectively (Figure 3b). These amounts of drug loading capacity are considerably higher than the previously reported results [25], mainly due to


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different functional molecules. Previous studies have utilized APTES molecules, containing one amine group, while in the present work MSNPs were amino- functionalized using EDS molecules with two amine groups.

CUR:MSNP=1:2 80

1

(a)

CUR:MSNP=2:1

Drug Loading Capacity

100

Drug Loadind Efficiency (%)

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CUR:MSNP=1:1 CUR:MSNP=2:1

60 40 20 0

Bare-MSNPs

0.8 0.6

(b)

CUR:MSNP=1:1 CUR:MSNP=1:2

0.4 0.2 0

AAS-MSNPs

Bare-MSNPs

AAS-MSNPs

(d)

(c)

Figure 3. The effect of surface modification and initial mass ratios of CUR/MSNPs on (a) drug loading efficiency (b) drug loading capacity. Adsorption Isotherms for CUR on (c) bare MSNPs and (d) AASMSNPs.

The comparison of the above-mentioned results shows that CUR loading efficiency and capacity are considerably higher for AAS-MSNPs in comparison to those of the bare MSNPs. These high values can be attributed to the mesoporous structure of MSNPs. Furthermore, this also may be related to the possible hydrophobic interactions between the drug molecule and the functional groups existing on the surface of AAS-MSNPs. CUR molecules possess a hydrophobic molecular


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structure consisting of two phenyl and two vinyl groups. Because of such hydrophobic structure, CUR is not soluble in water. The hydrophobic interactions between CUR and hydrophobic organic backbone of AAS molecules enhance the drug adsorption onto AAS-MSNPs. Moreover, electrostatic attractive forces between hydroxyl and carbonyl groups of CUR and positively charged amine groups existing on the surface of AAS-MSNPs may also play an important role in the increased loading capacity of these particles in comparison to that of the bare MSNPs. On the other hand, the amine groups of AAS molecules retain the hydrophilic properties of the nanoparticles and results in their well-dispersion in the aqueous solution. As similar enhancement in aqueous dispersion demonstrated in several previous nanoparticle-mediated CUR delivery studies, [36,37] CUR-AAS-MSNPs and CUR-MSNPs remained completely dispersed in aqueous media in the current work. The adsorption process was conducted for 24 h in ethanol to ensure that the equilibrium concentration is achieved at different initial concentrations of CUR (𝐶𝑒 ). The CUR loading capacity is reported as 𝑞𝑒 (mg of CURR/mg of MSNPs). Langmuir, Freundlich and LangmuirFreundlich models were used to fit the experimental results. The results of adsorption isotherms for CUR on bare and AAS-MSNPs and the equilibrium parameters are presented in Figure 3 and Table 2. Regression coefficients show that Langmuir-Freundlich model fits experimental results with more accuracy indicating that the adsorption process of CUR onto MSNPs deviates from ideal, and the intermolecular interaction of CUR and its interaction with the nanoparticles are involved in the adsorption process [38]. The main reason for such intermolecular interactions can be based on hydrophobic attractive forces.


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Table 2. Isotherm constants using nonlinear fitting for adsorption of CUR on bare and AAS-MSNPs

Langmuir 𝑞𝑚 Bare MSNPs AASMSNPs

𝐾𝐿

Freundlich R2

Langmuir-Freundlich

n

𝐾𝐹

R2

0.24

0.87

0.268

4.322 0.95

2.152

0.437

4.205 0.96

2.138 0.389 0.89

𝑞𝑚

𝐾𝐶

0.122 63.81

n

R2

0.49

0.99

0.432 48.00 0.515 0.998

In vitro release of Curcumin. The in vitro release of CUR form AAS-MSNPs was followed over a period of 48 h at 37°C. Suitable drug delivery systems should not only exhibit proper drugloading efficiency and capacity but should also release the drug in a controlled manner in order to provide sustained delivery [39]. Figure 4 represents the cumulative release of CUR from AASMSNPs. It shows that the release profile of CUR from AAS-MSNPs is sustained, starting with an initial burst release, during which a significant amount of drug release (~58%). This initial phase is followed by a slower rate release, and after 12 h, the release profile reaches the plateau. The fraction of drug, released over 3 h, is possibly corresponding to CUR adsorbed on the AAS-MSNPs surface and the later slow release may occur due to desorption of CUR electrostatically attached to the amine groups of the AAS molecules existing in the pores of MSNPs. Over a period of 48 h approximately 75.1% of the loaded drug was released in the media and the rest is expected to be constantly attached onto the AAS-MSNPs (Figure 4).


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100

Cumulative Release (%)

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

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80 60

a

40

c

b

20 0 0

50

100

150

200

Time (hr)

Figure 4. The cumulative release profile of CUR in the medium of Tris-HCl buffer (pH 7.4). The samples were incubated at 37 °C and 120 rpm for 7 days. The inset illustrates: a) AAS-MSNPs, b) CUR-AASMSNPs immediately after drug loading and c) CUR-AAS-MSNPs after burst release (12 h).

The effect of AAS-MSNPs and CUR-AAS-MSNPs on the α-Syn aggregation. The effects of AAS- and CUR-AAS-MSNPs and the released CUR on the α-Syn fibrillation were monitored by ThT fluorescence assay, Far-UV CD spectroscopy and fluorescence microscopy. α-Syn was exposed to the nanoparticles at a constant concentration of 100 µg/mL. In our previous work, it was shown that AAS-MSNPs present a considerably high inhibiting impact on the α-Syn fibrillation [26]. This can be understood by considering the isoelectric point of α-Syn (pI 4.74) [40]. Due to the solution condition used in this work (pH 7.4), electrostatic attractive forces between positively charged AAS-MSNPs and α-Syn monomers leads to the adsorption of monomers on the particle, and hence results in the protein concentration depletion and inhibition of protein fibrillation [26]. Similar results obtained in the current study. But herein, we also demonstrate that the treatment with CUR and CUR-AAS-MSNPs decreases the fibrillation of αSyn by a more considerable extent in comparison to that of the AAS-MSNPs (Figure 5). ThT assay results indicate that the free CUR results in larger inhibition properties against α-Syn fibrillation in comparison to that of the CUR-AAS-MSNPs (Figure 5a).


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Figure 5b represents the Far-UV CD spectra of α-Syn protein incubated in the absence and presence of AAS-, CUR-AAS-MSNPs or free CUR. Monomeric α-Syn in aqueous solutions usually has a disorder structure which is characterized by a deep negative minimum at around 200 nm. In the absence of CUR, conversion of random coil structure to the ordered crossed β-sheet aggregate results in the appearance of deep negative minimum at 218 nm (Figure 5b). Taken together, these results suggest that both free CUR and CUR-AAS-MSNPs remarkably prevents the fibrillation and oligomer formation of α-Syn. The obtained CD data clearly showed that the addition of positively charged AAS-MSNPs results in a reduced β-structure content. This is more severe for CUR and CUR-AAS-MSNPs with higher inhibitory effects, implying that they can completely perturb the formation of β structures and causes the protein fibrils to exhibit only a smooth negative peak around 200 nm. These results indicate that released CUR notably prevent αSyn aggregates formation and keep the monomers in their natural soluble forms. All to gather, it can be interpreted that the presence of AAS-MSNPs, CUR-AAS-MSNPs as well as free CUR molecules leads to interactions with α-Syn monomeric form and consequently to make them remain in the natural conformation and not undergo the fibrillation process. Similar effects have been observed in the interaction of α-Syn with positively charged polyethylenimine-coated human serum albumin nanoparticles, leading to a significant alteration in the α-Syn structure [41]. However, a different mechanism has been reported to describe the effect of CUR on α-Syn fibrillation. Curcumin binds strongly to monomeric α-Syn without making any significant alteration in the unfolded state of that [42,43]. Therefore, it prevents the α-Syn molecules to form high molecular weight aggregates. It has also been reported that CUR can disrupt preformed aggregates and increase the soluble forms of α-Syn in this way too [42, 43].


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(a)

Negative Control No MSNPs AAS-MSNPs CUR-AAS-MSNPs CUR

160 140 120

10

(b)

5 0 -5

100

-10

∆e

Intensity (a.u.)

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80

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α-Syn Fibrill AAS-MSNP CUR-AAS-MSNP CUR

-20

40

-25

20

-30

0

-35

450

470

490 510 Wavelength (nm)

530

550

195

205

215

225

235

Wavelength (nm)

(c)

Figure 5. (a) ThT assay results of α-Syn fibrils formed in the absence and presence of 100 µg/mL AASMSNPs and CUR-AAS-MSNPs. Negative control does not contain neither fibrils nor MSNPs (b) Far-UV CD spectrophotometer analysis of α-Syn incubated in the absence and presence of AAS- and CUR-AASMSNPs at 100 µg/mL (c) Fluorescence microscopy images of α-Syn aggregates formed in the absence and presence of AAS-MSNPs, free CUR and CUR-AAS-MSNPs (from left to right, respectively).

To further confirm, fluorescence microscopy was utilized to investigate the effect of CUR and MSNPs on the fibril formation. The protein aggregates, causing induced emission of fluorescence through interaction with ThT, can be seen as highly bright particles in the obtained images (Figure 5c). Antifibrilation activity of released and unreleased CUR. According to the results of drug release profile (Figure 4) significant amount of CUR remains adsorbed onto the AAS-MSNPs even after 7 days. Therefore, the effect of CUR-AAS-MSNPs after burst release and the supernatant


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containing the released CUR on the fibrillation process were also evaluated through ThT assay and the obtained results were compared with those of freshly prepared CUR-AAS-MSNPs. These results indicate the fact that even though the burst release phase has passed, CUR-AAS-MSNPs maintain their inhibitory effects on α-Syn fibrillation (Figure 6). For further confirmation, AFM images were provided to clearly compare the effects of CUR-AAS-MSNPs before and after burst release on α-Syn fibrillation. As shown in figure 6c, in both cases fibrillar aggregates no longer exist and it seems that both samples prevent the fibrillation process through the same mechanism. 400

(a)

400

300 200

Intensity (a.u.)

(b)

Intensity (a.u.)

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300

200

100

100

0

0

(c)

Figure 6. (a) ThT assay results of α-Syn fibrils formed in the presence of 100 µg/mL CUR-AAS-MSNPs before and after burst release (b) ThT assay results of α-Syn fibrils formed in the presence of 100 µg/mL CUR-AAS-MSNPs and the supernatant after 3 and 7 days (c) AFM images of α-Syn fibrils in the absence and presence of CUR-AAS-MSNPs before and after burst release, after 24 h incubation (from left to right, respectively).


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In continue, the effect of CUR-AAS-MSNPs samples and the corresponding supernatants after 3 and 7 days of releasing process on the fibrillation was evaluated by ThT assay. It can be inferred from the obtaining results that the amount of CUR attached onto the AAS-MSNPs remains active and possesses its inhibitory effects on α-Syn fibrillation, while the released CUR existing in the supernatant samples exhibit very lower inhibitory effects on the protein aggregation (Figure 6b). In other words, the released CUR loses its activity after 3 or 7 days, while the stabilized CUR on AAS-MSNPs remains active during this time. In this regard, two possible mechanisms can be considered (Scheme 1). First, the α-Syn molecules attach to CUR molecules already adsorbed on the surface of CUR-AAS-MSNPs and this leads to the α-Syn concentration depletion in the solution. Second, the presence of protein in the vicinity of CUR-AAS-MSNPs may change the hydrophobicity of the small environment and facilitate the release of CUR from the surface followed by attachment of CUR molecules to the α-Syn monomers and preventing the protein molecules aggregation. In addition, the cumulative effect of positively charged AAS-MSNPs and CUR on the fibrillation of α-Syn not only enhances the inhibitory effects on the fibril formation, but also reduces the toxic effects of free CUR.


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Scheme 1. Two possible mechanisms for the reaction of maintaining CUR with protein monomers

Cytotoxicity properties of CUR-loaded MSNPs and α-Syn species. To evaluate toxic effects of CUR-AAS-MSNPs and α-Syn species, in vitro cytotoxicity was assessed on PC-12 cells. The results show a significant cell viability reduction over 100 µM of free CUR while, these neuronal cells retained about 85% of their cell viability at low concentrations of the AAS-MSNPs and CURAAS-MSNPs (≤100 μg/mL) indicating the cell protecting effects of nanocarriers from the toxic effects of CUR. This is possibly due to the controlled release of the compound from CUR-AASMSNPs samples (Figure 7a). At the same time, it is expected that CUR reduces the cytotoxicity effects of α-Syn species through inhibition of the fibril formation. But as our results show (Figure 7b) free CUR possess considerably high cytotoxicity effects, and such effects are not observed for the loaded CUR onto AAS-MSNPs. CUR-AAS-MSNPs showed a considerably high weakening effect on the cytotoxicity properties of α-Syn species, which possibly is a result of the cumulative effects of CUR and AAS-MSNPs. In other words, cytotoxicity effects of the free CUR are hampered through its immobilization onto the MSNPs.


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120

120

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(b)

100

Cell viability (%)

100 10 µg/mL 50 µg/mL 100 µg/mL 250 µg/mL 500 µg/mL

80 60 40

80 60 40 20

20

0 0

120

(c) Cell viability (%)

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100 80 60 40 20 0

Figure 7. Viabilities of PC-12 cells determined by MTT assay, after 24 h incubation with (a) CUR, AAS, and CUR-AAS-MSNPs at different concentrations in the absence of α-Syn, (b) CUR, AAS-, and CURAAS-MSNPs at concentration of 100 µg/mL in the presence of α-Syn, (c) 7h protein aggregates in the presence of CUR-AAS-MSNPs, before and after the burst release.

According to the fact that there are usually toxic aggregates in central neuronal system of patients and they seem to be responsible for transmission and development of such diseases, if drug loaded carriers could prevent toxic effects of pre-instructed aggregates, they would become very promising tools. To evaluate this, PC12 cells were treated with 7-h protein aggregates alone or with CUR-loaded MSNPs before and after burst release (Figure 7c) and then, their cytotoxicity effects were evaluated. Results reveal the fact that the effect of drug loaded nanoparticles on toxic


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aggregates is quite lower than their effect on the monomeric form of protein molecules. This reveals that CUR-AAS-MSNPs can efficiently prevent the formation of protein fibrils whereas are not considerably capable of affecting the primary formed aggregates and inhibit their cytotoxicity effects. SUMMARY AND CONCLUSIONS In the present work, we have designed and prepared amine-functionalized MSNPs intended to improve the bioavailability and increase the drug loading capacity of a model poorly water-soluble drug, CUR. AAS-MSNPs present very high values of loading capacity and efficiency for CUR possibly owing to hydrophobic and electrostatic interactions between organic molecules existing on the surface of AAS-MSNPs and CUR molecules. Moreover, the stability of CUR molecules is enhanced through the loading process. The as-prepared drug-loaded nanoparticles considerably prevent the fibrillation of α-Syn and reduce its cytotoxicity-associated effects. Interestingly, the observed inhibitory effect exists even for the drug permanently attached onto AAS-MSNPs after a burst release phase. However, they only affect monomeric form of the protein and are not effective while exposed to the pre-instructed aggregates. In addition, CUR-AAS-MSNPs present remarkably lower cytotoxicity effects in comparison to that of the free CUR. According to this experimental evidence and previously published studies which support the therapeutic effects of CUR, it is suggested that new strategies for the formulation of CUR should be further explored as a potential therapeutic compound for Parkinson’s and the related disorders. As our results indicate a suitable approach for the formulation of CUR is its loading onto the AAS-MSNPs. AAS-MSNPs are introduced as a suitable drug carrier, possessing the ability to improve CUR bioavailability and stability, and to reduce its cytotoxicity effect on cells. However, further investigations are still


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required to completely and precisely identify biocompatibility and safety of such a nano-system for biomedical applications. AUTHOR INFORMATION Corresponding Author * Address correspondence to: [email protected], [email protected] ACKNOWLEDGMENT This work is financially supported by National Institute of Genetic Engineering and Biotechnology (NIGEB) and Iran National Science Foundation (INSF). The authors thank NTNU Nanolab for providing instrumentation facilities. The Research Council of Norway is acknowledged for the support to the Norwegian Micro-and Nano-Fabrication Facility, NorFab (197411/V30). ABBREVIATIONS MSNPs, Mesoporous Silica Nanoparticles; AAS, 3-(2-aminoethyl amino) propyltrimethoxysilane; α-Syn, α-Synuclein; CUR, Curcumin. REFERENCES (1) Lipinski, C. Poor aqueous solubility- an industry wide problem in drug discovery. Am. Pharm. Rev. 2002, 5, 82-85. (2) Shi, J.; Votruba, A.R.; Farokhzad, O. C.; Langer, R. Nanotechnology in drug delivery and tissue engineering: from discovery to applications. Nano Lett. 2010, 10, 3223-3230. (3) Vallet-Regi, M.; Ramila, A.; Del Real, R. P.; Perez-Pariente, J. A new property of MCM-41: drug delivery system. Chem. Mater. 2001, 13, 308–311.


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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 29

(4) Salonen, J.; Lehto, V. P. Fabrication and chemical surface modification of mesoporous silicon for biomedical applications. Chem. Eng. J. 2010, 137, 162-172. (5) Hu, Y.; Zhi, Z.; Zhao, Q.; Wu, C.; Zhao, P.; Jiang, H.; et al. 3D cubic mesoporous silica microsphere as a carrier of poorly soluble drug Carvedilol. Micropor. Mesopor. Mat. J. 2012, 147, 94-101. (6) Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C. W.; Lin, V. S. Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carrier. Adv. Drug. Deliv. Rev. 2008, 60, 1278-1288. (7) Tsai, C. H.; Vivero-Escoto, J. L.; Slowing, I. I.; Fang, I. J.; Trewyn, B. J.; Lin, V.S.Y. Surfactant-assisted controlled release of hydrophobic drugs using anionic surfactant templated mesoporous silica nanoparticles. Biomaterials. 2011, 32, 6234-6244. (8) Jin. D.; Park, K. W.; Lee, J. H.; Song, K.; Kim, J. G.; Seoa, M.L.; et al. The selective immobilization of curcumin onto the internal surface of mesoporous hollow silica particles by covalent bonding and its controlled release. J. Mater. Chem. 2011, 21, 3641-3645. (9) Hoffmann, F.; Cornelius, M.; Morell, J.; Fröba, M. Silica-based mesoporous organic-inorganic hybrid materials. Angew. Chem. Int. Ed. 2006, 45, 3216-3251. (10) Sadeghi, R.; Moosavi-Movahedi, A. A.; Emam-jomeh, Z.; Kalbasi, A.; Razavi, S. H.; Karimi, M.; et al. The effect of different desolvating agents on BSA nanoparticle properties and encapsulation of curcumin. J. Nanopart. Res. 2014, 16, 1–14. (11) Tsai, Y. M.; Jan, W. C.; Chien, C. F.; Lee, W. C.; Lin, L. C.; Tsai, T. H. Optimized nanoformulation on the bioavailability of hydrophobic polyphenol, curcumin, in freely-moving rats. Food Chem. 2011, 3, 918–925.


24

Page 25 of 29

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

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(12) Brouet, I.; Ohshima, H. Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem. Biophys. Res. Commun. 1995, 206, 533–540. (13) Ammon, H. P.; Wahl, M. A. Pharmacology of Curcuma longa. Planta. Med. 1991, 57, 1–7. (14) Maheshwari, R. K.; Singh, A. K.; Gaddipati, J.; Srimal, R. C. Multiple biological activities of curcumin: a short review. Life. Sci. 2006, 78, 2081–2087. (15) Bar-Sela, G.; Epelbaum, R.; Schaffer, M. Curcumin as an anti-cancer agent: review of the gap between basic and clinical applications. Curr. Med. Chem. 2010, 17, 190–197. (16) Jurenka, J.S. Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: a review of preclinical and clinical research. Altern. Med. Rev. 2009, 14, 141–153. (17) Spillantini, M. G.; Schmidt, M. L.; Lee, V. M.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Alpha-Synuclein in Lewy bodies. Nature. 1997, 388, 839–840. (18) Cui, J.; Yu, B.; Zhao, Y.; Zhu, W.; Li, H. L.; Zhai, G. Enhancement of oral absorption of curcumin by self-microemulsifying drug delivery systems. Int. J. Pharm. 2009, 371, 148–155. (19) Lan, L.; Fadi, S. B.; Razelle, K. Liposome-encapsulated curcumin. Cancer. 2005, 104, 13221331. (20) Kaminaga, Y.; Nagatsu, A.; Akiyama, T.; Sugimoto, N.; Yamazaki, T.; Maitani, T.; et al. Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of catharanthus roseus. FEBS Lett. 2003, 555, 311-316. (21) Priyadarsini, K. I. Photophysics, photochemistry and photobiology of curcumin: Studies from organic solutions, bio-mimetics and living cells. J. Photochem. Photobiol. C: Photochem. Rev. 2009, 10, 81-95.


25

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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 29

(22) Sou, K.; Inenaga, S.; Takeoka, S.; Tsuchida, E. Loading of curcumin into macrophages using lipid-based nanoparticles. Int. J. Pharm. 2008, 352, 287–293. (23) Tiyaboonchai, W.; Tungpradit, W.; Plianbangchang, P. Formulation and characterization of curcuminoids loaded solid lipid nanoparticles. Int. J. Pharm. 2007, 337, 299–306. (24) Jambhrunkar, S.; Qu, Z.; Popat, A.; Yang, J.; Noonan, O.; Acauan, L.; et al. Effect of surface functionality of silica nanoparticles on cellular uptake and cytotoxicity. Mol. Pharmaceutics. 2014, 11, 3642-3655. (25) Oliveira, L. F.; Bouchmella. K; Gonçalves, K. A.; Bettini. J; Kobarg, J.; Cardoso, M. B. Functionalized silica nanoparticles as an alternative platform for targeted drug-delivery of water insoluble drugs. Langmuir. 2016, 32, 3217-3225. (26) Taebnia, N.; Morshedi, D.; Doostkam, M.; Yaghmaei, S.; Ali-Akbari, F.; Singh, G.; Arpanaei, A. The effect of mesoporous silica nanoparticles surface chemistry and concentration on the alpha-synuclein fibrillation. RSC. Adv. 2015, 5, 60966-60974. (27) Thangapazham, R. L.; Puri, A.; Tele, S.; Blumenthal, R.; Maheshwari, R. K. Evaluation of a nanotechnology based carrier for delivery of curcumin in prostate cancer cells. Int. J. Oncol. 2008, 32, 1119-1123. (28) Sheet, I.; Kabbani, A.; Holail, H. Removal of heavy metals using nanostructured graphite oxide, silica nanoparticles and silica/graphite oxide composite. Energy Procedia. 2014, 50, 130–138. (29) Cabaleiro-Lago, C.; Szczepankiewicz, O.; Linse, S. The Effect of Nanoparticles on Amyloid Aggregation Depends on the Protein Stability and Intrinsic Aggregation Rate. Langmuir. 2012, 28, 1852-1857


26

Page 27 of 29

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

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(30) Nilsson, M. R. Techniques to study amyloid fibril formation in vitro. Methods. 2004, 34, 151160. (31) Aranda, A.; Sequedo, L.; Tolosa, L.; Quintas, G.; Burello, E.; Castell J. V.; et al. Dichloro-

dihydrofluorescein diacetate (DCFH-DA) assay: a quantitative method for oxidative stress assessment of nanoparticle treated cells. Toxicol. In Vitro. 2013, 27, 954–963. (32) Jagannathan, R.; Abraham, P. M.; Poddar, P. Temperature-Dependent Spectroscopic Evidences of Curcumin in Aqueous Medium: A Mechanistic Study of Its Solubility and Stability. J. Phys. Chem. B. 2012, 116, 14533 -14540. (33) Kurien, B. T.; Singh, A.; Matsumoto, H.; Scofield, R. H. Improving the solubility and pharmacological efficacy of curcumin by heat treatment. Assay. Drug. Dev. Technol. 2007, 5, 567 -576. (34) Yang, P. P.; Quan, Z. W.; Lu, L. L.; Huang, S. S.; Lin, J. Luminescence functionalization of

mesoporous silica with different morphologies and applications as drug delivery systems. Biomaterials. 2008, 29, 692–702. (35) Alam, S.; Panda, J. J.; Chauhan, V. S. Novel dipeptide nanoparticles for effective curcumin

delivery. Int. J. Nanomedicine. 2012, 7, 4207-4222. (36) Mohanty, C.; Sahoo, S. K. The in vitro stability and in vivo pharmacokinetics of curcumin

prepared as an aqueous nanoparticulate formulation. Biomaterials. 2010, 31, 6597–6611. (37) Das, R. K.; Kasoju, N.; Bora, U. Encapsulation of curcumin in alginate-chitosan-pluronic

composite nanoparticles for delivery to cancer cells. Nanomedicine. 2010, 6, 153–160. (38) Li, S.; Hu, J.; Liu, B. A study on the adsorption behavior of protein onto functional

microspheres. J. Chem. Technol. Biotechnol. 2005, 80, 531–536.


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Page 28 of 29

(39) Duan, J.; Zhang, Y.; Han, S.; Chen, Y.; Li, B.; Liao, M.; et al. Synthesis and in vitro/in vivo

anti-cancer evaluation of curcumin-loaded chitosan/polybutylcyanoacrylate nanoparticles. Int. J. Pharm. 2010, 400, 211–220. (40) Vigneswara, V.; Lowenson, J. D.; Powell, C. D.; Thakur, M.; Bailey, K.; Clarke, S.; Ray, D.

E.; Carter, W. G. Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme. J. Biol. Chem. 2006, 281, 32619–325629. (41) Mohammad-Beigi, H.; Shojaosadati, S. A.; Tayaranian Marvian, A.; Pedersen, J. N.; Klausen,

L. H.; et al. Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HAS NPs) alter α-synuclein conformation and aggregation kinetics, Nanoscale. 2015, 8, 41-54. (42) Pandey, N.; Strider, J.; Nolan, W. C.; Yan, S. X.; Galvin, J. E. Curcumin inhibits aggregation

of alpha-synuclein. Acta. Neuropathol. 2008, 115, 479–489. (43) Singh, P. K.; Kotia, V.; Ghosh, D.; Mohite, G. M.; Kumar, A.; Maji, S. K. Curcumin modulates

alpha-synuclein aggregation and toxicity. ACS. Chem. Neurosci. 2013, 4, 393-407.


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