Newly Designed Magnetic and Non-Magnetic Nanoparticles for Potential Diagnostics and Therapy of Alzheimer's Disease

chnology te & io B io B f m o Skaat and Margel, J Biotechnol Biomater 2013, 3:2 a

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n DOI: 10.4172/2155-952X.1000156

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o J Journal of Biotechnology & Biomaterials ISSN: 2155-952X

Review Article Open Access Newly Designed Magnetic and Non-Magnetic Nanoparticles for Potential Diagnostics and Therapy of Alzheimer’s Disease Hadas Skaat and Shlomo Margel* Department of Chemistry, Institute of Nanotechnology and Advanced Materials, Ramat-Gan 52900, Israel

Abstract The pathogenesis of many neurodegenerative diseases, including Alzheimer’s disease (AD) is characterized by protein aggregation into amyloid fibrils. In AD, the fibrils are of the amyloid-β (Aβ) peptide. The development of new approaches based on nanotechnology for early detection and potential treatment of AD is of high current interest. This review describes a pioneering approach involving the design, synthesis and utilization of new engineered magnetic and non-magnetic nanoparticles for inhibition and acceleration of conformational changes of the fibril-

forming proteins, e.g. insulin and amyloid-β 40 (Aβ40) proteins. A novel method for detection of the location and removal of precursor protofibrils and fibrils by their selective marking by functional magnetic iron oxide nanoparticles

was also demonstrated. These non-fluorescent and fluorescent iron oxide (maghemite, γ-Fe2O3) nanoparticles of narrow size distribution were synthesized by controlled nucleation and growth mechanism. Surface coatings of these nanoparticles with a functional fluorinated polymer and peptides, e.g. Leu-Pro-Phe-Phe-Asp (LPFFD) and

Aβ40, through various activation methods, were performed. New uniform biocompatible α-amino acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains were also synthesized. The effect of these nanoparticles on amyloid fibril formation kinetics was elucidated. These engineered nanoparticles are effective in the study and control of the process of amyloid fibril formation, and as selective biomarkers of amyloid plaques for multimodal imaging. This study may contribute to the mechanistic understanding of the protein aggregation processes, leading to development of new diagnostic and therapeutic strategies against amyloid-related diseases.

difficulty of crossing the blood brain barrier (BBB), and have greaterin Keywords: Neurodegenerative diseases; Fluorescent γ-Fe2O3 nanoparticles; Poly(N-acryloyl amino acid) nanoparticles; Amyloid β vivo stability [18,19]. peptide; Protein folding The neuronal damage of AD is irreversible, and eventually leads Introduction to dementia and ultimately death. Therefore, there is an urgent need for early diagnosis by in vivo imaging agents that specifically detect the Amyloid aggregate formation is a pathological process that occurs in many diseases, including Parkinson’s, Huntington’s, prion diseases and location and density of amyloid plaques in the living human brain [20- Alzheimer’s disease (AD) [1-3]. The proteins undergo transition from 22]. Compounds such as Thioflavin T (ThT) derivatives and Congo red the normally soluble form into amyloid fibrils organized mainly into (CR) have been evaluated as potential probes for fluorescence imaging cross β-sheets, which accumulate in the extracellular space of various of Aβ plaques [20-22]. Peptides such as radiolabeled Aβ derivatives tissues [4-6]. The extracellular plaques that form in AD are composed of and tritium-LPFFD have also been investigated as Aβ plaque-selective fibrils of the Aβ peptide, a small peptide with 39-43 amino acids [7]. Aβ imaging agents. The core structures of these reagents contribute to their is continuously secreted by normal cells in culture, and is a component high binding affinity to Aβ aggregates [23,24]. of plasma and cerebrospinal fluid of healthy individuals in the soluble form [8,9]. In AD, this Aβ self-assembles to form neurological toxic In this review, we describe the development of a new, pioneering aggregates with various morphologies such as soluble oligomers and nanotechnology-based approach for potential diagnostics and therapy insoluble protofibrils and fibrils. According to the literature, the soluble of neurodegenerative diseases, particularly AD. We describe the oligomers are the most toxic species that cause the death of brain cells design, synthesis and use of various new engineered magnetic and [10,11]. Currently, there is no cure for these diseases and treatment non-magnetic nanoparticles for controlling (enhance or delay) the options are extremely limited [12]. A pharmacological approach for conformational changes of proteins that form amyloid fibrils, e.g. preventing amyloid aggregation is interfering with the pre-fibrillar Aβ protein involved in AD, and for specific marking and removal of species, and/or destabilizing the β-sheet conformations by agents that 40 specifically stabilize the soluble form of the protein [13]. precursor protofibrils and amyloid fibrils via a magnetic field [25-29]. Recent literature indicates increasing interest in developing nanoparticles to detect, prevent and treat protein-misfolding diseases *Corresponding author: Shlomo Margel, Department of Chemistry, Institute of [14-16]. Their potential influence on protein fibrillation is a function Nanotechnology and Advanced Materials, Ramat-Gan 52900, Israel, Tel: 972-3- of the nanoparticle surface physical and chemical properties. The 5318861; Fax: 972-3-6355208; E-mail: [email protected] large surface area to volume ratio of nanoparticles allows modification Received April 03, 2013; Accepted April 29, 2013; Published May 03, 2013 of surface properties, so as to control the adsorption and interaction Citation: Skaat H, Margel S (2013) Newly Designed Magnetic and Non-Magnetic processes. Increased local protein concentration on the nanoparticle Nanoparticles for Potential Diagnostics and Therapy of Alzheimer’s Disease. J surface, and/or changes in protein conformation upon binding could Biotechnol Biomater 3: 156. doi:10.4172/2155-952X.1000156 promote aggregation, while trapping of early intermediates may inhibit Copyright: © 2013 Skaat H, et al. This is an open-access article distributed under further aggregation [17]. Moreover, in vitro and in vivo studies have the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and demonstrated that nanoparticles can be exploited for overcoming the source are credited.

J Biotechnol Biomater ISSN:2155-952X JBTBM an open access journal Volume 3 • Issue 2 • 1000156 Citation: Skaat H, Margel S (2013) Newly Designed Magnetic and Non-Magnetic Nanoparticles for Potential Diagnostics and Therapy of Alzheimer’s Disease. J Biotechnol Biomater 3: 156. doi:10.4172/2155-952X.1000156

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Selective Marking of Amyloid Fibrils by Dualmodal Maghemite Nanoparticles Biocompatible, non-toxic and biodegradable magnetic iron oxide nanoparticles have been developed, and their use has been demonstrated in various biomedical applications, such as hyperthermia, diagnosis, cell-labeling and sorting, DNA separation, MRI and drug delivery [30-36]. The addition of fluorescent moieties may also provide new multimodal nanomaterials, with a broad range of both potential diagnostic and therapeutic applications [37]. In our study, we developed a novel method for selective marking of amyloid fibrils, e.g. insulin and Aβ40, by both fluorescent and non- fluorecent γ-Fe2O3 nanoparticles [25,27]. We first chose insulin as a model amyloidogenic protein. Insulin amyloid fibril deposits had been Figure 2: AFM images of the human insulin amyloid fibril/γ-Fe2O3 nanoparticle observed in patients with type II diabetes, and after insulin infusion assemblies prepared by fibrillation of human insulin in the presence of6 (w/w)% of γ-Fe2O3 nanoparticles for different time intervals: 3 (A), 3.5 (B1,B2), and repeated injection. Then, we extended our study to Aβ40 protein 4 (C) and 5 (D1,D2) h. B2 and D2 represent higher magnification of the circled involved in AD. γ-Fe2O3 nanoparticles of narrow size distribution were regions shown in B1 and D1, respectively. synthesized by nucleation, followed by controlled growth of maghemite thin films onto gelatin-iron oxide nuclei [25,34]. The diameter of the

γ-Fe2O3 nanoparticles was measured to be 15.0 ± 1.2 nm, and their stability against agglomeration was observed by transmission electron microscopy (TEM) (Figure 1). The γ-Fe2O3 nanoparticles containing the fluorescent probe rhodamine (R-γ-Fe2O3) or fluorescein (F-γ-Fe2O3) were prepared similar to the non-fluorescent γ-Fe2O3 nanoparticles, substituting the gelatin for gelatin covalently bound to rhodamine isothiocyanate (RITC), or fluorescein isothiocyanate (FITC), respectively [27,28]. While many fluorescent nanoparticles have their fluorescent moieties bound to the surface, our nanoparticles contain fluorescent dye, covalently encapsulated within the nanoparticles. There is, therefore, retention of the surface properties, including the zeta potential and surface bound ligand capacity. The encapsulation also increases the photostability of the dye [38].

Magnetic human amyloid fibril/γ-Fe2O3 nanoparticle assemblies were prepared by interaction of the γ-Fe2O3 nanoparticles with the insulin or Aβ40 amyloid fibrils, during or after their formation [34]. The nanoparticles selectively labeled the insulin and Aβ40 amyloid fibrils at both stages, even under competitive conditions, e.g. in the presence of 4% HSA. Figure 3: Fluorescence microscope images of Aβ40 protofibrils and fibrils labeled with the R-γ-Fe O nanoparticles. Single staining by adding 8% (w/ Different stages of growth of the human insulin fibril/γ-Fe2O3 2 3 w ) of the γ-Fe O -R nanoparticles to the protofibrils (A1) and fibrils (B2) nanoparticle assemblies during the insulin fibrillation process are Aβ40 2 3 formed after 88 h and 120 h, respectively; double staining by adding 8% (w/ depicted in the atomic force microscopy (AFM) images (Figure 2). wAβ40) of the R- γ-Fe2O3 nanoparticles and ThT to the fibrils formed after 120 The samples were prepared by dispersing fresh monomeric insulin h (C1). A2, B2 and C2 corresponding to the optical images of A1, B1 and C1, in an aqueous continuous phase, in the presence of 6 (w/w)% of the respectively.

γ-Fe2O3 nanoparticles. 3 h after the initiation of the fibrillation process (Figure 2A) the appearance of small spherical-like aggregate structures is revealed, indicating the onset of the fibril growth. Figures 2B-2D illustrates the gradual growth of the insulin fibrils. The initial fibrils are small and linear, while the more developed fibrils are longer and

partially twisted. The selective binding of the γ-Fe2O3 nanoparticles onto the insulin fibrils, with almost no unassociated nanoparticles in the background, is also shown in figure 2.

Selective fluorescent marking of Aβ40 protofibrils and fibrils with

8% (w/wAβ40) of the R-γ-Fe2O3 nanoparticles was also illustrated (Figure 3). By adding the nanoparticles before or after the completion

of the fibrillation process, the resulting Aβ40 protofibrils (Figures 3A1 and 3A2) and fibrils (Figures 3B1 and 3B2) are single-stained with red fluorescence. As mentioned above, ThT is a fluorescent dye that binds Figure 1: TEM image of the γ-Fe2O3 nanoparticles. specifically to amyloid fibrils. Adding ThT to the Aβ40 fibril/R-γ-Fe2O3

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1.4 1.2 A B 1.0

0.8

0.6

0.4 Absorbance 600 nm

0.2

0.0

2.0

1.5

1.0 C (mg/mL)

0.5 A B 0.0 Figure 4: Magnetic Aβ fibrils dispersed in aqueous continuous phase, 40 0 10 20 30 40 50 60 70 80 90 before (A) and after (B) applying a magnetic field. The Aβ40 fibril/nanoparticle t (h) assemblies were prepared by addition of 8% (w/wAβ40) of the γ-Fe2O3 nanoparticles to Aβ fibrils dispersed in 1 mL 0.1M PBS (pH 7.4). 40 Figure 6: Kinetics of the insulin fibril formation in aqueous continuous phase

at pH 1.6 and 65°C in the absence (A) and the presence (B) of 6% (w/winsulin)

of the γ-Fe2O3/PHFBA nanoparticles. Kinetics was measured using absorption at 600 and 280 nm.

before fibrillation 3.0 h 20 A 4.0 h

10 5.0 h 23.0 h 0 ) -1 -10 dmol

Figure 5: HRTEM images of the γ-Fe O nanoparticles (A) and the γ-Fe O / 2 2 3 2 3 -20 PHFBA core-shell nanoparticles (B). before fibrillation 3.0 h 9.0 h B /deg cm 20 23.0 h nanoparticle assemblies, followed by removal of excess ThT, resulted in 3 30.0 h θ (10 double-stained Aβ40 fibrils, with red and yellow fluorescence (Figures 10 92.0 h 3C1 and 3C2). This result confirms the selective marking of the 40Aβ fibrils by the R-γ-Fe2O3 nanoparticles. The magnetic properties of the 0

γ-Fe2O3 nanoparticles bound to the insulin or Aβ40 amyloid fibrils are also advantageous. The obtained magnetic amyloid fibril assemblies can -10 easily be completely removed from the aqueous phase using a simple magnet (Figure 4). -20 200 220 240 260 Effect of Fluorinated Magnetic Core-Shell Nanoparticles Wavelength (nm) on the Kinetics of Insulin Amyloid Fibril Formation Figure 7: CD spectra of the formed insulin fibrils in the absence (A) and the

presence (B) of 6% (w/winsulin) of the γ-Fe2O3/PHFBA nanoparticles at different Fluorinated alcohols such as trifluoroethanol and times during the fibrillation process. hexafluoroisopropanol have been reported to induce α-helical conformation in fibril-forming peptides [39,40]. Their strong containing groups, however, do not induce α-helix formation, and hydrophobic character probably causes alterations of the hydration may even enhance the rate of fibril growth. Fluorinated nanoparticles shell of the amyloidic sequence. Based on these results, Rocha et al. [40] are therefore, potential candidates for the inhibition and reverse of demonstrated the utilization of fluorinated nanoparticles, composed of conformational changes of amyloid proteins [40,41]. a complex of polyampholytes and dodecanoic and perfluorododecanoic In our study, newly designed fluorinated γ-Fe2O3 core-shell acid, in the induction of an α-helix-rich structure in the B18 peptide magnetic nanoparticles were shown to induce a significant direct slow (a short peptide sequence with a strong tendency to self assemble and transition from α-helix to β-sheets that occurs during insulin fibril form amyloid fibrils). Their alkylated analogues, in absence of fluorine- formation [26]. These uniform magnetic γ-Fe2O3/Poly(2,2,3,3,4,4,4-

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heptafluorobutyl acrylate) (γ-Fe2O3/PHFBA) core-shell nanoparticles Without the presence of nanoparticles (Figure 7A), before the initiation were prepared by emulsion polymerization of the fluorinated monomer of the fibrillation process, the CD spectrum of the insulin aqueous 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA), in the presence of the solution shows a band at 195 nm and a double minimum at 208 and

γ-Fe2O3 nanoparticles. The high-resolution TEM (HRTEM) images 222 nm, indicating native protein dominated by α-helix conformation. (Figure 5) show that the core comprised crystalline iron oxide, with By heating the insulin solution for 5 h, the CD spectrum of the insulin perfect arrangement of the atomic layers, while the 3-4 nm PHFBA shows the disappearance of the 195 and 222 nm bands, as well as shell was amorphous. appearance of a minimum ellipticity at 216 nm, indicating the presence of extensive β-sheet structures. Similar CD spectra were also observed in The effect of γ-Fe O /PHFBA nanoparticles on the kinetics of 2 3 the presence of 6% (w/w ) of the non-coated γ-Fe O nanoparticles. the insulin fibril formation was investigated. Without the presence insulin 2 3 In the presence of 6% (w/w ) of the γ-Fe O /PHFBA nanoparticles of nanoparticles (Figure 6A), the main growth of the insulin fibrils insulin 2 3 (Figure 7B), the α-helical structure remains almost constant for up to 22 occurred approximately 3 h after initiation of the fibrillation process, h of heating, beyond which transformation to β-sheet structure begins. and completed after approximately 5 h. Similar behavior was also observed in the presence of 6% (w/winsulin) of the uncoated γ-Fe2O3 The inhibition of the insulin fibril formation, in the presence of the nanoparticles [25]. However, in the presence of 6% (w/winsulin) γ-Fe2O3/ γ-Fe2O3/PHFBA nanoparticles, was also confirmed by TEM images PHFBA nanoparticles (Figure 6B), insulin fibril growth was significantly (Figure 8). After 9 h of the fibrillation process, in the absence (Figures delayed, and initiation occured only after 22 h. The presence of the 8A1 and 8A2) or presence of 6% (w/winsulin) of the non-coated γ-Fe2O3 perfluorinated carbon groups (-CF2 and -CF3) on the surface of the nanoparticles (Figure 8B), the length of the fibrils was up to several

γ-Fe2O3 nanoparticles probably stabilizes the α-helix structure by micrometers. The non-coated γ-Fe2O3 nanoparticles also appear to hydrophobic interactions, delaying the α-helix to β-sheet transition selectively bind with the axial external surface of the fibrils, with almost during the insulin fibril formation [39,40]. The loss of the ordered no unassociated nanoparticles (Figure 8B). However, at the same time water coating around the peptide, due to the perfluorinated carbon point, different behavior was observed in the presence of 6% (w/winsulin) groups, causes the –NH and –C=O groups of the backbone to interact of the γ-Fe2O3/PHFBA core-shell nanoparticles, revealing only free on short distances with each other, favoring the α-helix conformation. nanoparticles, indicating that the fibrils were not yet formed (Figure Short-range interactions between nearby amino-acid residues stabilize 8C). Figures 8D1 and 8D2 show that 50 h after the initiation of the the α-helix structure, in contrast to long-range interactions stabilizing fibrillation process, the obtained fibrils were still much shorter than the β-sheet structure [39]. An opposite correlation between the insulin those observed in the presence of the non-coated γ-Fe2O3, and were not fibril formation and the insulin monomeric concentration is obtained, marked at all by the core-shell nanoparticles. as observed by the increase and decrease of the measured absorbances Effect of Peptide-Conjugated Fluorescent-Maghemite of fibrils (600 nm) and monomeric insulin (280 nm), respectively. In the absence of nanoparticles, complete fibril formation is observed Nanoparticles on the Kinetics of Aβ40 Fibrillation after 5 h, while the concentration of the insulin is almost zero. This Process indicates that after 5 h, all the monomeric insulin had been converted Several reserch groups have been involved in the identification to insulin fibrils. This correlation was also observed in the presence of of the specific peptide sequence that is critical in amyloid aggregate the γ-Fe2O3/PHFBA nanoparticles, but only after 92 h. formation [42-46]. The FF residues, the hydrophobic core of residues Circular dichroism (CD) analyses were performed to determine 19-20 of the Aβ protein, are well known as crucial sequences for the secondary structure changes during the insulin fibril formation, in the β-sheet formation that trigger the fibrillation process [42-46]. It is possible that the Aβ fibrillation process is partially driven by the absence and the presence of the core-shell nanoparticles (Figure 7). hydrophobic interactions, via recognition between the FF pairs [42-46]. Short peptides homologous to the hydrophobic core of Aβ, for example LPFFD, were designed to bind specifically to the entire Aβ, and used as inhibitors of its fibrillation process via the FF recognition motif [47- 49]. Although peptide or protein analogues with specific FF binding sites may be of primary importance for studying neurodegenerative disorders, no therapeutic agents using this strategy have been developed. This is because of difficulties with penetrating the BBB, the complexity of their synthesis, and that their stability and efficacy has been found to be low in vivo [50]. Various nanoparticles have been reported to promote the protein nucleation process leading amyloid fibril formation in vitro, for example, copolymer particles of N-isopropylacrylamide/N-tert- butylacrylamide, cerium oxide, quantum dots (QDs), carbon nanotubes and titanium oxide [51,52]. Only few studies have reported the inhibitory effect of nanoparticles on the Aβ fibrillation process. Very

Figure 8: TEM images showing the effect of the γ-Fe2O3/PHFBA nanoparticles recently, Cabaleiro-Lago et al. [53] reported the inhibition of the Aβ40 on the insulin fibril formation. In the absence (A1, A2) and the presence of: 6% fibril formation by copolymer nanoparticles of variable hydrophobicity, (w/winsulin) of the non-coated γ-Fe O nanoparticles, 9 h after initiation of the 2 3 and also demonstrated the dual effect of commercial polystyrene (PS) fibrillation process (B), or 6% (w/winsulin) of the γ-Fe2O3/PHFBA nanoparticles, 9 h (C) and 50 h (D1 and D2) after the initiation of the fibrillation process. A2 nanoparticles, with amino modification toward the Aβ40 and Aβ42 fibril and D2 represents higher magnification of the circled region shown in A1 and formation [54]. Yoo et al. [55] have shown an inhibition effect of CdTe D1, respectively. QDs on Aβ40 fibrillation. Fluorinated nanoparticles [41] and sulfonated

Page 5 of 8 and sulfated PS nanoparticles [56], have also been reported as potential candidates for the inhibition of Aβ fibril formation. 1.0 Our study was extended by surface modification of the fluorescent- A B C γ-Fe2O3 nanoparticles with different peptides (Aβ40 and LPFFD), which 0.8 led to a significant increase in their binding affinity towards the Aβ40 fibrils, and affected the kinetics of the Aβ fibrillation process [28]. 40 0.6 For this purpose, F-γ-Fe2O3 nanoparticles were coated with human serum albumin (HSA), via a precipitation process. A succinimidyl polyethylene glycol succinimidyl ester (NHS-PEG-NHS) spacer 0.4 arm was covalently conjugated to the F-γ-Fe2O3~HSA nanoparticles. This was used for the covalent conjugation of Aβ or LPFFD to their 40 0.2

surface [28]. The effect of these conjugated proteins on the kinetics T Fluorescence (a.u.) Normalized Th of the Aβ40 fibrillation process was elucidated. The Aβ40-conjugated nanoparticles (F-γ-Fe O ~HSA-PEG-Aβ ) were found to accelerate the 0.0 2 3 40 0 20 40 60 80 100 120 140 160 180 200 Aβ40 fibrillation process, while the LPFFD-conjugated nanoparticles (F t (h) γ-Fe2O3~HSA-PEG-LPFFD) caused inhibition. Figure 10: Kinetics of the Aβ40 fibrils formation in PBS at 37°C in the absence Fibril formation kinetics of the Aβ40 was monitored by the temporal (A) and presence of 10 (B) and 100 (C)% (w/wAβ40) of the F-γ-Fe2O3~HSA- development of ThT binding [57], at different concentrations of the F-γ- PEG-LPFFD nanoparticles.

Fe2O3~HSA-PEG-Aβ40 nanoparticles (Figure 9). In the absence of the nanoparticles (Figure 9A), the main growth of the Aβ40 fibrils occurred Fibril formation kinetics of Aβ40 in the absence (A) and the approximately 60 h after initiation of the fibrillation process, and presence (B,C) of the F-γ-Fe2O3~HSA-PEG-LPFFD nanoparticles was completed after approximately 120 h. This sigmoidal behavior was also also investigated (Figure 10). The observed behavior was opposite to observed in the presence of different concentrations of the control F-γ- the behavior obtained in the presence of similar concentrations of Fe2O3~HSA nanoparticles [28]. However, the presence of increasing the F-γ-Fe2O3~HSA-PEG-Aβ40 nanoparticles. The Aβ40 fibril growth concentrations of the F-γ-Fe2O3~HSA-PEG-Aβ40 nanoparticles kinetics were delayed in the presence of 10 and 100% (w/wAβ40) of the gradually accelerates the kinetics of the Aβ fibril growth. Instead of 40 γ-Fe2O3-F~HSA-PEG-LPFFD nanoparticles, and initiated after only initiating the fibrillation process in the absence of the F-γ-Fe2O3~HSA- 78 and 124 h, respectively. Despite this contrasting behavior, the same

PEG-Aβ40 nanoparticles after 60 h, the addition of increasing selective marking of the fibrils with the Aβ40-conjugated nanoparticles concentrations of the F-γ-Fe2O3~HSA-PEG-Aβ40 nanoparticles of 1 (B), or LPFFD-conjugated nanoparticles was observed [28].

10 (C), and 100 (D)% (w/wAβ40), accelerates the initiation of the fibril formation to be after 51, 26, and 1.5 h, respectively. This indicates that Effect of Amino Acid-Based Polymer Nanoparticles on the Kinetics of Aβ Fibrillation Process the Aβ40 conjugated nanoparticles substantially decrease the fibrillation 40 lag time, and thereby, enhance the kinetics the Aβ40 fibril formation. Poly (amino acid) nanoparticles have recently attracted great The promoting effect probably results from the presence of the surface attention due to their potential non-toxicity, biocompatibility, non- bound Aβ40, which is known for its high affinity towards Aβ40 prefibril immunogenicity and biofunctionality. These nanoparticles containing monomers and oligomers [58-60]. This high binding affinity leads to amino acid moieties are potentially useful at various biomedical the enhanced adsorption of the Aβ40 prefibril aggregates to the surface applications, such as drug or gene delivery agents, tissue engineering of the nanoparticles, resulting in their rapid association to form fibrils scaffolds and chiral recognition [62-65]. The synthesis and radical [61]. polymerization of different acryl monomers that their side chain composed of amino acid moieties have been reported [66]. Their corresponding polymers, poly (N-acryloyl amino acids), are expected

1.0 to be functional materials, and studies were mainly focused on their C unique polymerization behavior, structures and properties [67-69]. D B A Yet, there was no report describing the preparation of polymeric 0.8 nanoparticles composed of N-acryloyl amino acid monomers. In our study, we designed for the first time new uniform 0.6 biocompatible amino acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains [29]. The dipeptide 0.4 residues were designed similarly to the hydrophobic core sequence of Aβ. The rational of our design is to engineer polymer nanoparticles 0.2 containing extremely high concentrations of the FF motif. This motif

Normalized Th T Fluorescence (a.u.) Normalized Th in the nanoparticles, therefore, should act as a recognition motif within Aβ and interferes in its fibril formation. 0.0 0 20 40 60 80 100 120 140 160 Poly (N-acryloyl-L-phenylalanyl-L-phenylalanine methyl ester) t (h) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-methoxy Figure 9: Kinetics of the Aβ40 fibrils formation in PBS at 37°C in the absence (A) and presence of 1 (B), 10 (C) and 100 (D)% (w/wAβ40) of the F-γ-Fe2O3~HSA- ethanol, followed by precipitation of the obtained polymer in aqueous PEG-Aβ nanoparticles. 40 solution [29], as illustrated in figure 11. Cell viability assay confirmed

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h after initiation of the fibrillation process, and was completed after

NH2 I NH2,HCl II O O approximately 120 h. However, the kinetics of the Aβ40 fibril growth,

O Cl O Cl A NH in the presence of increasing concentrations of the polyA-FF-ME NH

CH3OH, OºC DMAP, TEA DCM, OºC nanoparticles, was significantly delayed (Figures 12B-12D). The

HO O H3CO O presence of 10 (B), 20 (C), and 100 (D)% (w/wAβ40) of the polyA-FF- FF FF-ME ME nanoparticles inhibits the initiation of the fibrils formation to be only after 85, 99, and 233 h, respectively. This indicates that the

n polyA-FF-ME nanoparticles substantially increase the fibrillation lag

O NH O NH time, and thereby, inhibit the kinetics of the Aβ40 fibril formation. O III O IV This inhibitory effect might be explained by the presence of the pairs BP, PVP H2O NH NH HOCH CH OCH , 75ºC of FF residues of these nanoparticles, which are known for their high 2 2 3

H CO O H CO O 3 3 affinity to the corresponding residues 40 ofAβ prefibril aggregates

A-FF-ME Soluble polyA-FF-ME polyA-FF-ME nanoparticles through hydrophobic interactions. This high binding affinity disturbs the monomer-critical nuclei equilibrium by trapping the monomers, and/or blocking the growing oligomers ends on the surface of the nanoparticles, thereby decreasing their solution concentration and B interfering with their elongation to form fibrils. However, the ability to distinguish whether these nanoparticles adsorbed the monomers, or oligomers, or both, remains to be investigated. Our assumption that the observed inhibitory effect, in the presence of the polyA-FF-ME nanoparticles, is derived from specific hydrophobic interactions between the nanoparticles and the Aβ prefibril aggregates,

500 nm via the FF recognition motif, was examined by replacing the polyA- FF-ME nanoparticles by polyA-AA-ME nanoparticles. An opposite Figure 11: Synthetic route (A) and HRSEM image (B) of the polyA-FF-ME behavior was observed compared the presence of similar concentrations nanoparticles. of the polyA-FF-ME nanoparticles. The kinetics of the Aβ40 fibril growth

in the presence of 10, 20, and 100 % (w/wAβ40) were accelerated, and initiated after 53, 38, and 18 h, respectively (Figure 13). This promoting effect is probably due to the absence of the FF interfering moieties in 1.0 these nanoparticles. Therefore, the increased local concentration of the

Aβ40 prefibrils adsorbed on the polyA-AA-ME nanoparticle surface promoted their growth into fibrils. The promoting effect achieved in the 0.8 A C D presence of the polyA-AA-ME nanoparticles may also be considered as B a desired process. This is because that the polyA-AA-ME nanoparticles 0.6 decrease the fibrillation lag time, thereby shorting the half life time of

the Aβ40 prefibrils, thereby decreasing their toxicity in solution. 0.4 Summary and Conclusions

0.2 This review describes the design and synthesis of new uniform Normalized Th T Fluorescence (a.u.) Normalized Th

0.0 0 50 100 150 200 250 300 1.0

t (h) 0.8 D Figure 12: Kinetics of the Aβ40 fibrils formation in PBS at 37°C in the absence (A) and presence of 10 (B), 20 (C) and 100 (D)% (w/w ) of the polyA-FF-ME Aβ40 C A nanoparticles. 0.6 B that no significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g. PC-12 and SH-SY5Y, was observed. In the 0.4 presence of these nanoparticles, a significant slow secondary structure transition from random coil to β-sheets during Aβ40 fibril formation was 0.2 observed, resulting in significant inhibition of Aβ40 fibrillation kinetics. T Fluorescence (a.u.) Normalized Th However, the polyA-FF-ME analogous nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide in the polymer side groups, polyA- 0.0 0 50 100 150 AA-ME nanoparticles, accelerate the Aβ40 fibrillation kinetics. t (h)

Kinetics of the Aβ40 fibril formation in the absence and presence of increasing concentrations of the polyA-FF-ME nanoparticles Figure 13: Kinetics of the Aβ40 fibrils formation in PBS at 37°C in the absence (A) and presence of 10 (B), 20 (C) and 100 (D)% (w/w ) of the polyA-AA-ME are shown in figure 12. In the absence of the nanoparticles (Figure Aβ40 nanoparticles. 12A), the main growth of the Aβ40 fibrils occurred approximately 60

Page 7 of 8 functional magnetic iron oxide nanoparticles and amino acid-based 11. Kumar S, Udgaonkar JB (2010) Mechanisms of amyloid fibril formation by polymer nanoparticles. These engineered nanoparticles affect the proteins. Curr Sci 98: 639-656. kinetics of the amyloid fibrillation process differentially. The fluorinated 12. Karran E, Mercken M, De Strooper B (2011) The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics. Nat maghemite core-shell nanoparticles, γ-Fe2O3/PHFBA, inhibit the insulin Rev Drug Discov 10: 698-712. amyloid fibrillation process. The presence of the perfluorinated carbon 13. Härd T, Lendel C (2012) Inhibition of amyloid formation. J Mol Biol 421: 441- groups on the nanoparticles’ surface probably stabilizes the α-helix 465. structure via hydrophobic interactions, delaying the α-helix to β-sheet 14. Spuch C, Saida O, Navarro C (2012) Advances in the treatment of transition during the insulin fibril formation. The LPFFD-conjugated neurodegenerative disorders employing nanoparticles. Recent Pat Drug Deliv maghemite nanoparticles and the polyA-FF-ME nanoparticles inhibit Formul 6: 2-18. the Aβ40 fibrillation process. This inhibition probably results from 15. Sahni JK, Doggui S, Ali J, Baboota S, Dao L, et al. (2011) Neurotherapeutic the intermolecular hydrophobic interactions between the pairs of FF applications of nanoparticles in Alzheimer’s disease. J Control Release 152: residues of the nanoparticles, with the corresponding residues of the 208-231.

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