- Nano Express
- Open Access
Gold Nanoparticles and Microwave Irradiation Inhibit Beta-Amyloid Amyloidogenesis
© to the authors 2008
- Received: 1 August 2008
- Accepted: 11 September 2008
- Published: 11 October 2008
Peptide-Gold nanoparticles selectively attached to β-amyloid protein (Aβ) amyloidogenic aggregates were irradiated with microwave. This treatment produces dramatic effects on the Aβ aggregates, inhibiting both the amyloidogenesis and the restoration of the amyloidogenic potential. This novel approach offers a new strategy to inhibit, locally and remotely, the amyloidogenic process, which could have application in Alzheimer’s disease therapy. We have studied the irradiation effect on the amyloidogenic process in the presence of conjugates peptide-nanoparticle by transmission electronic microscopy observations and by Thioflavine T assays to quantify the amount of fibrils in suspension. The amyloidogenic aggregates rather than the amyloid fibrils seem to be better targets for the treatment of the disease. Our results could contribute to the development of a new therapeutic strategy to inhibit the amyloidogenic process in Alzheimer’s disease.
- Alzheimer’s disease
Protein misfolding and aggregation in general and amyloidogenesis in particular are of growing interest as scientists recognize their role in devastating degenerative diseases such as Alzheimer’s and Parkinson’s disease. Protein and peptide aggregation into mature amyloid fibrils is a multistep process initiated by conformational changes with samplings of prefibrillar intermediate amyloidogenic aggregates (PIAA), such as oligomers, protofibrils, pores, amylospheroids, and short fibrils . Alzheimer′s disease is a neurodegenerative disorder characterized by the presence of extracellular deposits of amyloid protein and plaques in the brain, composed primarily of toxic aggregates (PIAA and amyloid fibrils) of β-amyloid protein (Aβ) . In a recent report, we demonstrated the feasibility of remote deposit redissolving by using the local heat dissipated by gold nanoparticles (AuNP) selectively attached to the Aβ fibrils when irradiated with microwaves (MW) [3–5]. Although the mature fibril was once assumed to be the biologically toxic species, it has recently been hypothesized that soluble intermediates such as PIAA are most damaging [6–8]. Recently, several authors have hypothesized that the key to an early pathogenic event in the onset of Alzheimer’s disease is likely the formation of amyloidogenic species rather than the amyloid fibrils. A strategy for the treatment of the disease could be reducing the amyloidogenicity of this species [9, 10]. In an early stage, this inhibition is of major importance to develop a potential strategy for treating the Alzheimer’s disease. Thus, the current study intends to demonstrate that the inhibition of the aggregation process of β-amyloid in vitro by applying weak microwave fields (0.1 W) is possible in the presence of AuNP.
Could MW and AuNP Treatment Halt the Amyloidogenic Process of PIAA?
Medical application of MW began in the 1970s . In animals and humans, local MW exposure stimulates tissue repair and regeneration, alleviates stress reactions, and facilitates recovery in a wide range of diseases . MW also modulates the effect of X-rays at both cellular and organism levels. Diseases reported to be successfully treated with MW are gastric, duodenal ulcers, cardiovascular diseases, respiratory sickness, tuberculosis, skin diseases, etc. . MW irradiation with low-power density also stimulates the immune potential of macrophages and T cells . On the other side, more intense MW fields produce effects such as conformational changes and denaturation processes on proteins . The use of MW and AuNP to produce local and remote heating is a powerful tool for the development of new strategies to manipulate the aggregation state of toxic proteins [3, 4]. Irradiation has been extensively explored as a means of remote heating of biological tissues mediated by inorganic nanoparticles . In this study, we selectively bound AuNP to Aβ1–42 PIAA (Aβ PIAA) and investigated the effect of MW irradiation on the amyloidogenic process. To allow selective attachment to PIAA, AuNPs were linked to peptide CLPFFD, which contains the LPFFD sequence that attaches selectively to the amyloidogenic Aβ1–42 structures, forming the conjugate AuNP-CLPFFD. LPFFD recognizes a particular (hydrophobic) domain of the β-sheet structure .
CLPFFD was synthesized following fluorenylmethyloxycarbonyl (Fmoc) strategy and solid phase synthesis where the peptide is C-termed with an amide (CLPFFD-NH2). Fmoc-protected amino acids were purchased from Novabiochem (Laufelfingen, Switzerland) and Perseptive Biosystems (Framingham, Massachusetts). O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), Fmoc-AM handle, and resin MBHA were also obtained from Novabiochem. Chemical reagentsN,N′-diisopropylcarbodiimide (DIPCI), 1-Hydroxy-1H-Benzotriazole (HOBt), triethylsilane, and dimethylaminopyridine (DMAP) were from Fluka (Buchs, Switzerland). Manual synthesis included the following steps: (i) resin washing with DMF (5 × 30 s); (ii) Fmoc removal with 20% piperidine/DMF (1 × 1 min + 2 × 7 min); (iii) washing with DMF (5 × 30 s); (iv) washing with DMF (5 × 30 s) and CH2Cl2(5 × 30 s); (v) Kaiser’s test (with a peptide-resin sample); (vi) DMF washing (5 × 30 s). Cleavage of the peptide was carried out by acidolysis with trifluoroacetic acid (TFA) using triethylsilane and water as scavengers (94:3:3, v/v/v) for 60–90 min. TFA was removed with N2stream and the oily residue precipitated with dry tert-butyl ether. Peptide crude was recovered by centrifugation and decantation of the tert-butyl ether phase. The solid was redissolved in (water:acetonitrile 1:1) and lyophilized. The peptide was analyzed by RP-HPLC [Waters 996 photodiode array detector (λ = 443 nm) equipped with a Waters 2695 separation module (Milford, MA), a Symmetry column (C18, 5 μm, 4.6 × 150 mm), and Millennium software; flow rate = 1 mL/min, gradient = 5–100% B over 15 min (A = 0.045% TFA in H2O, and B = 0.036% TFA in acetonitrile)]. The peptide was purified by semipreparative RP-HPLC [Waters 2487 Dual Absorbance Detector equipped with a Waters 2700 Sample Manager, a Waters 600 Controller, a Waters Fraction Collector, a Symmetry column (C18, 5 μm, 30 × 100 mm2), and Millennium software]. The peptide was finally characterized by amino acid analysis with a Beckman 6300 analyzer and by MALDI-TOF with a Bruker model Biflex III. The result of the amino acid analysis of CLPFFD-NH2was Asp 1.0 (1), Pro 0.97 (1), Leu 1.0 (1), Phe 2.03 (2), and in the mass spectrum MALDI-TOF of CLPFFD-NH2, the peaks [M+H+] = 740 and [M+Na+] = 762 were found.
Citrate-coated AuNP (12.5 ± 1.7 nm) were prepared by citrate reduction of HAuCl4 in accordance with Ref. . An aqueous solution of HAuCl4 (100 mL, 1 mM) was refluxed for 5–10 min, and a warm (50–60 °C) aqueous solution of sodium citrate (10 mL, 38.8 mM) was added quickly . Reflux was continued for another 30 min until a deep red solution appeared. The solution was filtered through 0.45 μm Millipore syringe filters to remove any precipitate, the pH was adjusted to 7.4 using dilute NaOH solution, and the filtrate was stored at 4 °C. AuNPs were observed by Transmission Electronic Microscopy (TEM) using a JEOL JEM-1010 microscope. The specimen was prepared by dropping AuNP on formvar carbon-coated copper microgrids and letting them dry.
Conjugation of CLPFFD with AuNP
After dialysis, 3 mL of AuNP-CLPFFD was centrifuged at 16,000g for 30 min (AuNP-CLPFFD sediments) and the supernatant was evaporated to dryness, and an analysis of amino acids and HPLC ES-MS were carried out. In both cases, the presence of the peptide was not detected.
The AuNP-CLPFFD pellet obtained after dialysis and centrifugation was washed twice with 300 μL of 1% TFA. For washing, the pellet was redissolved and centrifuged at 16,000g for 30 min (this treatment allows the detachment of non-covalent molecules that could be retained and non-covalently attached to AuNP-CLPFFD pellet). In the supernatant, free peptide was not detected by HPLC ES-MS, which indicates that after dialysis the free peptide was completely eliminated. It is important to mention that washing with a dilute solution of trifluoroacetic acid, 1% TFA, does not provoke the cleavage of CLPFFD from the AuNP-CLPFFD conjugate.
Characterization of Conjugates AuNP-CLPFFD
X-ray photoelectron spectroscopy (XPS) experiments were performed in a PHI 5500 multitechnique System (from Physical Electronics) with a monochromatic X-ray source (Aluminum Kalfa line of 1486.6 eV energy and 350 W), placed perpendicular to the analyzer axis and calibrated using the 3d5/2 line of Ag with a full width at half maximum (FWHM) of 0.8 eV. The analyzed area was a circle of 0.8 mm diameter, and the selected resolution for the spectra was 187.5 eV of Pass Energy and 0.8 eV/step for the general spectra and 23.5 eV of Pass Energy and 0.1 eV/step for the spectra of the different elements. Some measurements were done after some cleaning by sputtering the surface with an Ar+ion source (4 keV energy). All measurements were made in an ultra high vacuum chamber pressure between 5 × 10−9and 2 × 10−8 Torr. In AuNP-CLPFFD, the expected peaks from S 2p, S 2s, and Au 4f core levels were detected. High-resolution data have also been recorded in the S 2p, S 2s, and Au 4f, spectral regions. The S 2p signal consists of a broad band with a maximum at 162.2 eV that corresponds to the chemisorptions of sulfur grafted onto gold. The experimental curve fitted with the signals S 2p3/2 and S 2p1/2 (162.1 and 163.3 eV signals, respectively) that correspond to a doublet for sulfur atoms bound to gold (supplementary data). According to our calculations, the signal of 164 eV was not found in this peak, which led us to conclude that unbound S species are not present in the samples. In addition, the S 2s photoelectron peak was observed. This peak is a particularly good parameter since it is a singlet, thus making the interpretation straightforward. The S 2s photoelectron binding energy (BE) from bulk peptide with the free thiol and AuNP peptide thiolate are found at 228.2 and 227.3, respectively. The accuracy of these BE values is estimated to be ±0.2 eV at the worst. In the case of bare AuNP and capped AuNP, the signal corresponding to Au 4f7/2 is positioned at 84.2 eV. The fact that the measured Au 4f7/2 photoelectron BE is not detected by the sulfur chemisorption is probably because the peak separation is too small.
Estimation of the Number of Peptide Attached Molecules per AuNP
The amount of peptide molecule per NP was estimated by analysis of amino acids and absorption spectrophotometry. The concentration of AuNP in the solutions was obtained taking into account the molar coefficient of extinction of the 12 nm diameter (5.7 × 107 M−1 cm−1) AuNP and an analysis of amino acids of the pellet obtained after centrifugation of the conjugates at 13,500 rpm for 30 min (in such conditions, the NP sediment). In the amino acid analysis, a hydrolysis of the peptide conjugated to the AuNP (the non-conjugated peptide was eliminated in Section “Conjugation of CLPFFD with AuNP”) was performed; thus it was possible to determine the concentration of attached peptide molecules. The number of peptide molecules per AuNP was obtained by dividing the number of peptide molecules per mL of solution by the number of particles per mL of solution. This ratio was obtained in triplicate in three independent syntheses and conjugations. The degree of conjugation of AuNP-CLPFFD is 460 ± 30 peptide molecules per AuNP.
Preparation of Aβ PIAA Solutions
Aβ1–42 was purchased from r-Peptide (USA). About 1 mg peptide was suspended in water (1 mL) and this suspension was divided in 10 aliquots. Peptide aliquots were lyophilized in glass vials and stored at −20 °C. To obtain a homogeneous Aβ1–42 solution free from aggregates, the peptide was treated with 200 μL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for 30 min at room temperature. The HFIP was then lyophilized and the peptide was dissolved in water to obtain a 400 μΜ solution of Aβ PIAA .
Aβ PIAA/AuNP-CLPFFD and Controls for Irradiation
Sodium citrate, AuNP, AuNP-CLPFFD, or CLPFFD were added to the Aβ PIAA and the samples were incubated for 25 min at room temperature. We irradiated two samples with different ratio of Aβ/AuNP-CLPFFD (10 µM:1 nM and 10 µM:0.2 nM) and the controls.
Aβ PIAA/1 nM AuNP-CLPFFD Samples were prepared by mixing 12.5 μL of 400 μM Aβ, 100 μL of 5 nM AuNP-CLPFFD, and 388 μL of sodium citrate 1.2 mM.
Aβ PIAA/0.2 nM AuNP-CLPFFD Samples were prepared by mixing 12.5 μL of 400 μM Aβ PIAA, 20 μL of 5 nM AuNP-CLPFFD, and 468 μL of sodium citrate 1.2 mM.
For the irradiation process we used three controls: (1) Aβ PIAA, a solution of PIAA (12.5 μL of 400 μM Aβ) was mixed with 487.5 μL of sodium citrate 1.2 mM; (2) Aβ PIAA + AuNP, a solution of PIAA (12.5 μL of 400 μM Aβ) was mixed with 100 μL of 5 nM AuNP and 388 μL of sodium citrate 1.2 mM; (3) Aβ PIAA + CLPFFD, a solution of Aβ PIAA (12.5 μL of 400 μM Aβ) was mixed with 26 μL of 7.8 μM CLPFFD and 461.5 μL of sodium citrate 1.2 mM. The ratio CLPFFD to Aβ (1:24.5) is not enough to inhibit the fibril growth.
We used an HP HP83651B signal generator, working in a frequency range from 10 MHz to 50 GHz with a signal modulator HP8510C for determining the working frequency and power. Power was 100 mW. We apply the microwaves in a resonating chamber under mild magnetic stirring. The resonating chamber was a copper cavity of 6 cm diameter by 12 cm height with 0.5 cm thick walls. Microwaves arrived from the top through a waveguide while another waveguide read the signal inside the chamber. A resonant peak was chosen. Samples were introduced, and inside each vial a magnetic stirrer was introduced and the samples stirred for homogeneity. Samples and controls were irradiated for 10 and 30 min and then were centrifuged at 16,000g for 1 min and finally observed by TEM. Irradiated samples were incubated at room temperature for 48 h, and the fibril formation was determined by ThT test and by TEM.
Incubation of Irradiated Samples and Controls for Fibril Formation
The irradiated samples and controls and non-irradiated samples and controls were incubated at room temperature for 48 h to form amyloid fibrils.
Determination of Fibril Formation
Glycine 0.1 M buffer pH = 8.4 was introduced in 384 Nunc well fluorescence microliter plates and samples were added and mixed. Then Thioflavin T (100 µM) was added and mixed. The final peptide and Thioflavine T concentration is 4 µM. The fluorescence signal was measured (excitation wavelength 440 ± 10 nm) in a FL600 Microplate Fluorescence reader with KC4 version 2.7 software Biotek instruments, INC.
Transmission Electronic Microscopy
Aliquots of 10 μL (10 μM de Aβ) of the preparations were adsorbed for 1 min onto glow-discharged carbon-coated collodium films on 200-mesh copper grids. The TEM grids were then blotted and washed twice in distilled H2O before staining with uranyl acetate 2% for 2 min for visualization by TEM (JEOL JEM-1010).
Could, However, the Irradiated Aβ PIAA/AuNP-CLPFFD Complex be Incorporated During Fibril Growth of Fresh Aβ PIAA?
Summing up, MW and AuNP linked to a peptide that selectively attaches to amyloidogenic Aβ1–42structures inhibit irreversibly their normal aggregation. The resulting irradiated products are not amyloidogenic. Our approach provides a viable means to inhibit irreversibly the amyloidogenic process of Aβ. Further investigations in our laboratory include an assessment of the irradiation effect on Aβ structure and potential toxicity. This tool could be used for therapeutical purposes by inhibiting locally and remotely the amyloidogenic process of proteins.
We acknowledge Elisenda Coll of Servei Cientific-Tecnics (Universitat de Barcelona) for assistance in TEM observations and Aurora Morales for XPS asignations. This work was supported by FONDECYT 1061142, FONDAP 11980002 (17 07 0002), and AECI A/010967/07.
- Murphy RM: Biochim. Biophys. Acta Biomembranes. 2007,1768(8):1923. COI number [1:CAS:528:DC%2BD2sXosF2gt7w%3D] 10.1016/j.bbamem.2006.12.014View ArticleGoogle Scholar
- Arimon M, Diez-Perez I, Kogan MJ, Durany N, Giralt E, Sanz F, et al.: FASEB J.. 2005, 19: 1344. COI number [1:CAS:528:DC%2BD2MXntlaqsLY%3D]Google Scholar
- Kogan MJ, Bastus NG, Amigo R, Grillo-Bosch D, Araya E, Turiel A, Labarta A, Giralt E, Puntes VF: Nano Lett.. 2006, 6: 110. COI number [1:CAS:528:DC%2BD2MXhtlajsL3I] 10.1021/nl0516862View ArticleGoogle Scholar
- Bastús NG, Kogan MJ, Amigó R, Grilló-Bosch D, Araya E, Turiel A, et al.: Mater. Sci. Eng. C. 2007, 27: 1236. COI number [1:CAS:528:DC%2BD2sXovFGhu7w%3D] 10.1016/j.msec.2006.08.003View ArticleGoogle Scholar
- Olmedo I, Araya E, Sanz F, Medina E, Arbiol J, Toledo P, Álvarez-Lueje A, Giralt E, Kogan MJ: Bioconjugate Chem.. 2008,19(6):1154. COI number [1:CAS:528:DC%2BD1cXmsVOgs70%3D] 10.1021/bc800016yView ArticleGoogle Scholar
- Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Glabe CG: Science. 2003, 300: 486. COI number [1:CAS:528:DC%2BD3sXivFyms7k%3D] 10.1126/science.1079469View ArticleGoogle Scholar
- Walsh D, Selkoe DJ: J. Neurochem.. 2007, 101: 1172. COI number [1:CAS:528:DC%2BD2sXmsFensrc%3D] 10.1111/j.1471-4159.2006.04426.xView ArticleGoogle Scholar
- Masliah E: Nature. 2008, 451: 638. COI number [1:CAS:528:DC%2BD1cXhs1Kntrw%3D] 10.1038/451638aView ArticleGoogle Scholar
- Estrada LD, Soto C: Curr. Top. Med. Chem.. 2007, 7: 115. COI number [1:CAS:528:DC%2BD2sXht1Wjs7c%3D] 10.2174/156802607779318262View ArticleGoogle Scholar
- A. Rauk, Dalton Trans. 1273 (2007)Google Scholar
- Rojavin MA, Ziskin MC: Q. J. Med.. 1998, 91: 57. COI number [1:STN:280:DyaK1c7nvFOgtQ%3D%3D]View ArticleGoogle Scholar
- Banik S, Bandyopadhyay S, Ganguly S: Bioresour. Technol.. 2003, 87: 155. COI number [1:CAS:528:DC%2BD38XpsVeltLk%3D] 10.1016/S0960-8524(02)00169-4View ArticleGoogle Scholar
- Pakhomov AG, Akyel Y, Pakhomova ON, Stuck BE, Murphy MR: Bioelectromagnetics. 2001, 19: 393. 10.1002/(SICI)1521-186X(1998)19:7<393::AID-BEM1>3.0.CO;2-XView ArticleGoogle Scholar
- Novoselova EG, Fesenko EE, Makar VR, Sadovniko VB: Bioelectrochem. Bioenerg.. 1999, 49: 37. COI number [1:CAS:528:DyaK1MXnsl2lurw%3D] 10.1016/S0302-4598(99)00059-8View ArticleGoogle Scholar
- Bohr H, Bohr J: Phys. Rev.. 2000, 61: 4310. COI number [1:CAS:528:DC%2BD3cXitlWis7s%3D]Google Scholar
- Alexiou C, Arnold W, Klein RJ, Parak FG, Hulin P, Bergemann C, et al.: Cancer Res.. 2000, 60: 6641. COI number [1:CAS:528:DC%2BD3cXosl2isbc%3D]Google Scholar
- Permanne B, Adessi C, Saborio GP, Fraga S, Frossard MJ, Van Dorpe J, et al.: FASEB J.. 2002, 16: 860. COI number [1:CAS:528:DC%2BD38Xktl2ktbc%3D]Google Scholar
- Bieschke J, Siegel SJ, Fu Y, Kelly JW: Biochemistry. 2008, 47: 50. COI number [1:CAS:528:DC%2BD2sXhsVamsbrO] 10.1021/bi701757vView ArticleGoogle Scholar
- LeVine H: Protein Sci.. 1993, 2: 404. COI number [1:CAS:528:DyaK3sXksVCntLo%3D] 10.1002/pro.5560020312View ArticleGoogle Scholar