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Gold nanoparticles as amyloid-like fibrillogenesis inhibitors
Colloids and Surfaces B: Biointerfaces 112 (2013) 525–529
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Gold nanoparticles as amyloid-like fibrillogenesis inhibitors Shuchen Hsieh ∗ , Chiung-wen Chang, Hsuan-hung Chou Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 4 June 2013 Accepted 20 August 2013 Available online 28 August 2013 Keywords: Nanoparticle Amyloid fibril Insulin Alzheimer’s AFM
a b s t r a c t Amyloid aggregates are one of the likely key factors leading to the development of Alzheimer’s disease (AD) and other amyloidosis associated diseases. Several recent studies have shown that some antidiabetic drugs have a positive therapeutic effect on AD patients by crossing the blood brain barrier (BBB) and preventing or reducing insulin resistance. Nanoparticles (NPs) or nanoscale objects (<600 Da.), are able to cross the BBB at low concentrations, and can specifically target amyloidogenic structures. Thus, NPs are fast becoming indispensable tools for directed drug delivery, particularly when targeting structures or regions in the brain. Here, we have explored the inhibitory effect of gold nanoparticles (AuNPs) on the fibrillogenesis process of insulin fibrils. We found that when AuNPs were co-incubated with insulin, the structural transformation into amyloid-like fibrils was delayed by about a week. Further, the fibrils that formed, exhibited altered structure, shape, and dynamics, which further reduced fibril growth, and the stability of available amyloid-like fibrils with cross- structure for aggregation. Our results demonstrate that AuNPs disrupt insulin amyloid fibrillation resulting in fibrils that are shorter and more compact, and thus may serve a useful role in new therapeutic and diagnostic strategies for amyloid-related disorders. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In living organisms, certain proteins associated with disease can undergo misfolding and conformational changes resulting in fibril rich, highly ordered -sheet structures, or amyloid fibrils [1]. These amyloid fibrils are generally deposited in the brain and/or peripheral tissues, a condition which has been associated with neuropathological diseases such as Alzheimer’s, Parkinson’s, type II diabetes mellitus (T2DM) and more than 20 other amyloid-related disorders [2,3]. In 1939, Harold Himsworth challenged the conventional wisdom that all forms of diabetes could be attributed to insulin deficiency. He postulated that the state of diabetes might result from “inefficient action of insulin” as well as from its deficiency [4]. The role of insulin resistance in diabetes has been a serious topic of debate ever since [5]. Further, de la Monte et al. proved that brain insulin resistance is similar to regular diabetes, and mediates cognitive impairment, particularly Alzheimer’s disease (AD), which has been referred to as “type 3 diabetes” [6,7]. Because researchers hypothesized a common pathological link between T2DM and AD, some currently approved antidiabetic drugs that prevent insulin resistance, and that readily cross the blood brain barrier (BBB), were believed to have therapeutic potential and positive benefits for AD patients [8]. The BBB is a physical
∗ Corresponding author. Tel.: +886 75252000x3931; fax: +886 75253908. E-mail address: [email protected] (S. Hsieh). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.08.029
and physiological barrier for therapeutic drugs being delivered to the brain. It is easily permeable only to molecules with lipophilicity or weight below 600 Da. [9], thus limiting the carrier system to nanoscale objects [10]. Nanoparticles (NPs) have become the topic of intensive research, due in part, to a diverse variety of therapeutic potential in medical applications. NPs exhibit unique physical, chemical, and electronic properties, and in many cases are biocompatible. With their high surface area and tunable surface chemistry, NPs are becoming an indispensable tool in the biological and biomedical fields [11], for such applications as gene transfection [12], drug delivery [13], cell imaging [14] photo-thermal therapy [15], etc. A typical NP used for drug delivery is a polymeric particle modified with biomolecules (e.g., DNA/RNA probes, antibodies, peptides) [16] in the size range of 10–1000 nm [10]. In the treatment of AD, NPs are capable of modulating intracellular tight junctions [17], overcoming the BBB [18], self-assembling an analogue of A proteins [19], and targeting cerebrovascular amyloids [20]. Yoo et al. established a CdTe NP model offered insight into NP systems that exhibit equal to or even better fibrillation inhibition than the best-known proteins [19]. He further noted that biocompatible NP systems with similar characteristics to toxic CdTe NPs may be sought as alternatives for in vivo applications. Moreover, Cabaleiro-Lago et al. proved that NPs can prevent fibrillogenesis as well as temporarily reversing A aggregation [21]. Since various nonpathological proteins and polypeptides have been shown to self-assembly form the amyloid-like fibrils leading to amyloid diseases [3], and NPs have the potential to diagnose
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Fig. 1. TEM images of AuNPs bound to insulin fibrils incubated in a solution at pH 1.6 and 80 ◦ C for 2.5 h, and then stored at room temperature for (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5, and (g) 6 weeks. The lower left panel shows a diagram of AuNPs absorbed on fibrils. The scale bar in each image is 0.5 m.
Fig. 2. AFM topographic images of treated insulin following extended incubation at room temperature for 0 (a, d), 3 (b, e), and 4 (c, f) weeks. The top row (a, b, c) are insulin-only samples and the bottom (d, e, f) are insulin with AuNPs. The AuNPs disrupted the natural fibril formation process.
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Fig. 3. AFM topographic images of AuNP-treated insulin samples that had been co-incubated in solution for 5 weeks (left: a, c and d) and 6 weeks (right: b, e and f) then dropcast onto silicon wafer substrates. The cartoons adjacent to the lower image sets (c, e and e, f) depict typical AuNP seeded intersecting fibril aggregate structures.
or better understand amyloid protein fibrillation, the aim of this work presented here has been to investigate the utility of NPs in preventing or treating amyloid-related disorders. 2. Experimental Insulin powder from bovine pancreas (Sigma–Aldrich, St. Louis, MO, USA) was used without further purification to prepare insulin fibrils. Insulin powder with (or without) AuNPs (10 ± 2 nm) was dissolved in HCl solution at pH 1.6 to make a 1 ppm insulin/AuNP (insulin) solution. AuNPs were prepared by a citrate reduction reaction from a gold colloid solution [22]. The insulin solution was then incubated at 80 ◦ C for 2.5 h using the method described by Jansen et al. [23]. After incubation, the solutions were stored at room temperature for periods of 0–6 weeks. Samples were characterized at various room temperature incubation times using Atomic Force Microscopy (AFM, Asylum Research, MFP-3D), Cryogenic Transmission Electron Microscopy (cryo-TEM, JEM-1400), Circular Dichroism Spectrometer (CD, Jasco, J-810), and UV–vis Spectrophotometer (UV, Jasco, V630). AFM sample substrates were prepared from sections of a silicon (1 0 0) wafer (Tekstarter Co., Ltd.; P-type/Boron dopant). All of the silicon substrates were plasma cleaned (Harrick Scientific Products, Inc.) for 2 min using dry air as the reactive gas to increase the OH concentration at the surface. This process creates a uniformly hydroxylated surface. Insulin or Insulin/AuNP solutions were dropcast .onto clean silicon wafers, allowed to dry, and the imaged with AFM. All AFM images were acquired in AC (tapping) mode. A silicon cantilever (Olympus AC240TS) with a normal spring constant of 2 N m−1 was used for all images, with a scan rate of 1.0 Hz and an image resolution of 512 × 512 pixels.
3. Results and discussion Herein, insulin was chosen as an amyloidogenic protein model to investigate whether AuNPs can inhibit insulin amyloid fibrillation. In our previous study had described that insulin is a practical biotemplate for creating NPs chains, such as AuNPs deposition [24]. Further, literature sources suggest that AuNPs are ideal candidates for new AD therapy due to their selective attachment to A fibrils [25]. They can be synthesized in a variety of sizes and shapes and easily characterized using TEM [26]. To determine the effect of AuNPs on the fibrillation process, we studied insulin fibrils self-assembled into amyloid-like fibrils. Samples were prepared by incubating insulin monomer at pH 1.6 and 80 ◦ C for 2.5 h and then stored at room temperature for periods of 0–6 weeks. The formation of cross- fibrillar aggregates was monitored in the absence and presence of AuNPs, and TEM was used to characterize the morphological variation of the aggregates. Significant aggregation was observed when insulin fibrils were incubated with AuNPs for 1 week at room temperature (Fig. 1), and the aggregate density increased with longer incubation times. The AuNPs had a much higher affinity for the fibrils than for the substrate. The effect of AuNPs on the conformational change of insulin fibrils was also examined using AFM. Two series of AFM images (5 × 5 m2 ) show the morphological transition of insulin fibrils prepared as above (80 ◦ C, 2.5 h, pH 1.6) with further incubation at room temperature for 0 weeks, 3 weeks, and 4 weeks in the absence (Fig. 2a–c) and presence (Fig. 2d–f) of AuNPs, respectively. There is a clear difference in the surface morphology when comparing Fig. 2b and e, with the AuNP-treated insulin fibrils (Fig. 2e) appearing much shorter and more compact than the insulin-only fibrils (Fig. 2b). We also acquired AFM images after 5 and 6 weeks incubation at room temperature and the effect was even more pronounced (Fig. S1 in
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Fig. 4. CD spectra of insulin fibrillation in the absence (a) or presence (b) of AuNPs after incubation for 2.5 h, 1 week to 6 weeks at pH 1.6 and 80 ◦ C. (W = week). Visible absorption spectra of (c) pure intact AuNPs under different pH conditions and (d) AuNPs in a bovine insulin solution (1 mg/1 ml). The schematic illustration (e) corresponds to (c), and (f) corresponds to (d), respectively.
Supplementary Information). In our study, not only the aggregation rate, but also the length, height (thickness), and density of amyloidlike fibril aggregates were affected by the presence of AuNPs. We then acquired smaller scan-size AFM images to observe more detail of the insulin fibril structure after 5–6 weeks incubation (Fig. 3). The AuNPs were mostly located at fibril crossings (the average height of a single fibril was ∼3 nm while crossings were ∼30 nm), where thick intersecting aggregates were formed as shown in the AFM topography images of Fig. 3c and e, and graphically depicted in the adjacent cartoons.
Fig. 4a and b are CD spectra from insulin samples prepared without (a) and with (b) AuNPs. There is no significant difference between the spectra for either type of pre-treated sample after incubating for up to 3 weeks at room temperature. After 4 weeks incubation, the insulin-only sample showed a notable change in the spectra, with attenuation of the feature at 222 nm and a shift in the minima at 208–218 nm. This indicated a structural transformation from the ␣-helix to the -sheet conformation. Very little change was observed in the insulin/AuNP sample spectra after 4 weeks. This suggests that the AuNPs may delay or retard the transition
S. Hsieh et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 525–529
from the ␣-helix to the -sheet form. However, after 5 and 6 weeks incubation, spectra from both samples indicated transformation to the -sheet state with a single feature observed in each at 218 nm. Although AuNPs aggregate under acidic pH conditions (Fig. 4e); this does not appear to influence the aggregation of insulin fibrils when incubated with AuNPs in solution at pH 1.6. In the absorption spectra shown in Fig. 4c, AuNPs under acidic conditions (pH 1.6) exhibit a distinctive red shift of the 520 nm peak to 530 nm accompanied by the formation of a new absorption peak at 680 nm when compared to AuNPs at pH 7. This shift may be attributed to AuNPs aggregation. After adding AuNPs to insulin monomer or to insulin fibrils (Fig. 4d) at pH 1.6, we observed only a slight shift of the 520 nm peak (shown in Fig. 4c, pH 7) to 527 nm and no new absorption peaks were formed. These results suggest a preferential attachment of AuNPs onto insulin fibrils in solution during coincubation thus preventing self-aggregation. Further, due to the presence of the sulphur-containing amino acid ‘cysteine’, insulin can form strong S Au covalent bonds with the AuNPs [27], creating a protective coating around the AuNPs as depicted in Fig. 4f. Summarizing our observations, we propose a simple scheme for describing the mechanism of interaction for insulin fibrils incubated with AuNPs (Fig. S2 in Supplementary Information). Loading the insulin solution with AuNPs during the period of fibrillogenesis, the strong S Au bond frustrates the fibril self-assembly process reducing aggregation and thus inhibiting fibrillation. 4. Conclusions In the present study, we have demonstrated the effect of AuNPs conjugation on the structural evolution of insulin fibrils. We prepared insulin-only and AuNPs-treated insulin solutions (pH 1.6) and incubated them at room temperature for periods of 0–6 weeks. During this fibrillogenesis process, we found that AuNPs inhibited insulin fibrils assembly into protease resistant aggregates. AuNPs delayed the structural transformation into amyloid-like fibrils by about 1 week, and altered the structure, shape, and density of the formed fibrils compared with the insulin-only samples. We previously reported on the use of insulin fibrils as a sacrificial biotemplate for directing AuNPs self-assembly and alignment on surfaces [24,28]. Those results are consistent with our findings in this study where AuNPs exhibited a strong affinity toward insulin fibrils. We further observed here that AuNPs initiated the formation of thick intersecting fibril aggregates after incubation at room temperature for 5–6 weeks. Finally, we have shown that insulin conjugation to AuNPs can affect the protein structure and dynamics, further reducing the growth and stability of available amyloid-like -sheet structure for aggregation. These results suggest that AuNPs may serve a useful role in new therapeutic and diagnostic strategies for amyloid related diseases. Acknowledgments The authors would like to thank the National Science Council of Taiwan (NSC 101-2113-M-110-013-MY3), and the National Sun Yat-sen University Biochip Research Group for financial support of this work. Prof. Hsieh also thanks Dr. David Beck for helpful discussions and proofreading. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.08.029.
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References [1] F. Librizzi, C. Rischel, The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways, Protein Sci. 14 (2005) 3129–3134. [2] J.W. Kelly, The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways, Curr. Opin. Struct. Biol. 8 (1998) 101–106. [3] C.M. Dobson, The structural basis of protein folding and its links with human disease, Philos. Trans. R. Soc. B 356 (2001) 133–145. [4] J.E. Gerich, The genetic basis of type 2 diabetes mellitus: impaired insulin secretion versus impaired insulin sensitivity, Endocr. Rev. 19 (1998) 491–503. [5] G. Reaven, Insulin resistance, type 2 diabetes mellitus, and cardiovascular disease: the end of the beginning, Circulation 112 (2005) 3030–3032. [6] S.M. de la Monte, J.R. Wands, Alzheimer’s disease is type 3 diabetes-evidence reviewed, J. Diab. Sci. Technol. 2 (2008) 1101–1113. [7] S.M. de la Monte, Brain insulin resistance and deficiency as therapeutic targets in Alzheimer’s disease, Curr. Alzheimer Res. 9 (2012) 35–66. [8] K. Akter, E.A. Lanza, S.A. Martin, N. Myronyuk, M. Rua, R.B. Raffa, Diabetes mellitus and Alzheimer’s disease: shared pathology and treatment? Br. J. Clin. Pharmacol. 71 (2011) 365–376. [9] R.J. Boado, Antisense drug-delivery through the blood-brain-barrier, Adv. Drug Deliv. Rev. 15 (1995) 73–107. [10] J.R. Kanwar, X. Sun, V. Punj, B. Sriramoju, R.R. Mohan, S.F. Zhou, A. Chauhan, R.K. Kanwar, Nanoparticles in the treatment and diagnosis of neurological disorders: untamed dragon with fire power to heal, Nanomedicine 8 (2012) 399–414. [11] O. Salata, Applications of nanoparticles in biology and medicine, J. Nanobiotechnol. 2 (2004) 3. [12] S.D. Patil, D.G. Rhodes, D.J. Burgess, DNA-based therapeutics and DNA delivery systems: a comprehensive review, AAPS J. 7 (2005) E61–E77. [13] V.P. Torchilin, Multifunctional nanocarriers, Adv. Drug Deliv. Rev. 58 (2006) 1532–1555. [14] I.H. El-Sayed, X. Huang, M.A. El-Sayed, Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer, Nano Lett. 5 (2005) 829–834. [15] I.H. El-Sayed, X. Huang, M.A. El-Sayed, Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles, Cancer Lett. 239 (2006) 129–135. [16] P. Tiwari, K. Vig, V. Dennis,.S. Singh, Functionalized gold nanoparticles and their biomedical applications, Nanomaterials 1 (2011) 31–63. [17] Z.P. Zhuang, M.P. Kung, C. Hou, D.M. Skovronsky, T.L. Gur, K. Plossl, J.Q. Trojanowski, V.M. Lee, H.F. Kung, Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates, J. Med. Chem. 44 (2001) 1905–1914. [18] Q.R. Smith, A review of blood-brain barrier transport techniques, Methods Mol. Med. 89 (2003) 193–208. [19] S.I. Yoo, M. Yang, J.R. Brender, V. Subramanian, K. Sun, N.E. Joo, S.H. Jeong, A. Ramamoorthy, N.A. Kotov, Inhibition of amyloid peptide fibrillation by inorganic nanoparticles: functional similarities with proteins, Angew. Chem. Int. Ed. 50 (2011) 5110–5115. [20] E.K. Agyare, G.L. Curran, M. Ramakrishnan, C.C. Yu, J.F. Poduslo, K.K. Kandimalla, Development of a smart nano-vehicle to target cerebrovascular amyloid deposits and brain parenchymal plaques observed in Alzheimer’s disease and cerebral amyloid angiopathy, Pharm. Res. 25 (2008) 2674–2684. [21] C. Cabaleiro-Lago, F. Quinlan-Pluck, I. Lynch, S. Lindman, A.M. Minogue, E. Thulin, D.M. Walsh, K.A. Dawson, S. Linse, Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles, J. Am. Chem. Soc. 130 (2008) 15437–15443. [22] S.F. Cheng, L.K. Chau, Colloidal gold-modified optical fiber for chemical and biochemical sensing, Anal. Chem. 75 (2003) 16–21. [23] R. Jansen, W. Dzwolak, R. Winter, Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy, Biophys. J. 88 (2005) 1344–1353. [24] C.W. Hsieh, S. Hsieh, Nanoparticle chain formation on functional surfaces using insulin fibrils as a structure directing agent, J. Mater. Chem. 21 (2011) 16900–16904. [25] M.J. Kogan, N.G. Bastus, R. Amigo, D. Grillo-Bosch, E. Araya, A. Turiel, A. Labarta, E. Giralt, V.F. Puntes, Nanoparticle-mediated local and remote manipulation of protein aggregation, Nano Lett. 6 (2006) 110–115. [26] E. Araya, I. Olmedo, N. Bastus, S. Guerrero, V. Puntes, E. Giralt, M. Kogan, Gold Nnanoparticles and microwave irradiation inhibit beta-amyloid amyloidogenesis, Nanoscale Res. Lett. 3 (2008) 435–443. [27] K.H. Lee, F.M. Ytreberg, Effect of gold nanoparticle conjugation on peptide dynamics and structure, Entropy 14 (2012) 630–641. [28] S.C. Hsieh, C.W. Hsieh, Alignment of gold nanoparticles using insulin fibrils as a sacrificial biotemplate, Chem. Commun. 46 (2010) 7355–7357.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
Gold nanoparticles as amyloid-like fibrillogenesis inhibitors Shuchen Hsieh ∗ , Chiung-wen Chang, Hsuan-hung Chou Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 4 June 2013 Accepted 20 August 2013 Available online 28 August 2013 Keywords: Nanoparticle Amyloid fibril Insulin Alzheimer’s AFM
a b s t r a c t Amyloid aggregates are one of the likely key factors leading to the development of Alzheimer’s disease (AD) and other amyloidosis associated diseases. Several recent studies have shown that some antidiabetic drugs have a positive therapeutic effect on AD patients by crossing the blood brain barrier (BBB) and preventing or reducing insulin resistance. Nanoparticles (NPs) or nanoscale objects (<600 Da.), are able to cross the BBB at low concentrations, and can specifically target amyloidogenic structures. Thus, NPs are fast becoming indispensable tools for directed drug delivery, particularly when targeting structures or regions in the brain. Here, we have explored the inhibitory effect of gold nanoparticles (AuNPs) on the fibrillogenesis process of insulin fibrils. We found that when AuNPs were co-incubated with insulin, the structural transformation into amyloid-like fibrils was delayed by about a week. Further, the fibrils that formed, exhibited altered structure, shape, and dynamics, which further reduced fibril growth, and the stability of available amyloid-like fibrils with cross- structure for aggregation. Our results demonstrate that AuNPs disrupt insulin amyloid fibrillation resulting in fibrils that are shorter and more compact, and thus may serve a useful role in new therapeutic and diagnostic strategies for amyloid-related disorders. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In living organisms, certain proteins associated with disease can undergo misfolding and conformational changes resulting in fibril rich, highly ordered -sheet structures, or amyloid fibrils [1]. These amyloid fibrils are generally deposited in the brain and/or peripheral tissues, a condition which has been associated with neuropathological diseases such as Alzheimer’s, Parkinson’s, type II diabetes mellitus (T2DM) and more than 20 other amyloid-related disorders [2,3]. In 1939, Harold Himsworth challenged the conventional wisdom that all forms of diabetes could be attributed to insulin deficiency. He postulated that the state of diabetes might result from “inefficient action of insulin” as well as from its deficiency [4]. The role of insulin resistance in diabetes has been a serious topic of debate ever since [5]. Further, de la Monte et al. proved that brain insulin resistance is similar to regular diabetes, and mediates cognitive impairment, particularly Alzheimer’s disease (AD), which has been referred to as “type 3 diabetes” [6,7]. Because researchers hypothesized a common pathological link between T2DM and AD, some currently approved antidiabetic drugs that prevent insulin resistance, and that readily cross the blood brain barrier (BBB), were believed to have therapeutic potential and positive benefits for AD patients [8]. The BBB is a physical
∗ Corresponding author. Tel.: +886 75252000x3931; fax: +886 75253908. E-mail address: [email protected] (S. Hsieh). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.08.029
and physiological barrier for therapeutic drugs being delivered to the brain. It is easily permeable only to molecules with lipophilicity or weight below 600 Da. [9], thus limiting the carrier system to nanoscale objects [10]. Nanoparticles (NPs) have become the topic of intensive research, due in part, to a diverse variety of therapeutic potential in medical applications. NPs exhibit unique physical, chemical, and electronic properties, and in many cases are biocompatible. With their high surface area and tunable surface chemistry, NPs are becoming an indispensable tool in the biological and biomedical fields [11], for such applications as gene transfection [12], drug delivery [13], cell imaging [14] photo-thermal therapy [15], etc. A typical NP used for drug delivery is a polymeric particle modified with biomolecules (e.g., DNA/RNA probes, antibodies, peptides) [16] in the size range of 10–1000 nm [10]. In the treatment of AD, NPs are capable of modulating intracellular tight junctions [17], overcoming the BBB [18], self-assembling an analogue of A proteins [19], and targeting cerebrovascular amyloids [20]. Yoo et al. established a CdTe NP model offered insight into NP systems that exhibit equal to or even better fibrillation inhibition than the best-known proteins [19]. He further noted that biocompatible NP systems with similar characteristics to toxic CdTe NPs may be sought as alternatives for in vivo applications. Moreover, Cabaleiro-Lago et al. proved that NPs can prevent fibrillogenesis as well as temporarily reversing A aggregation [21]. Since various nonpathological proteins and polypeptides have been shown to self-assembly form the amyloid-like fibrils leading to amyloid diseases [3], and NPs have the potential to diagnose
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Fig. 1. TEM images of AuNPs bound to insulin fibrils incubated in a solution at pH 1.6 and 80 ◦ C for 2.5 h, and then stored at room temperature for (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5, and (g) 6 weeks. The lower left panel shows a diagram of AuNPs absorbed on fibrils. The scale bar in each image is 0.5 m.
Fig. 2. AFM topographic images of treated insulin following extended incubation at room temperature for 0 (a, d), 3 (b, e), and 4 (c, f) weeks. The top row (a, b, c) are insulin-only samples and the bottom (d, e, f) are insulin with AuNPs. The AuNPs disrupted the natural fibril formation process.
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Fig. 3. AFM topographic images of AuNP-treated insulin samples that had been co-incubated in solution for 5 weeks (left: a, c and d) and 6 weeks (right: b, e and f) then dropcast onto silicon wafer substrates. The cartoons adjacent to the lower image sets (c, e and e, f) depict typical AuNP seeded intersecting fibril aggregate structures.
or better understand amyloid protein fibrillation, the aim of this work presented here has been to investigate the utility of NPs in preventing or treating amyloid-related disorders. 2. Experimental Insulin powder from bovine pancreas (Sigma–Aldrich, St. Louis, MO, USA) was used without further purification to prepare insulin fibrils. Insulin powder with (or without) AuNPs (10 ± 2 nm) was dissolved in HCl solution at pH 1.6 to make a 1 ppm insulin/AuNP (insulin) solution. AuNPs were prepared by a citrate reduction reaction from a gold colloid solution [22]. The insulin solution was then incubated at 80 ◦ C for 2.5 h using the method described by Jansen et al. [23]. After incubation, the solutions were stored at room temperature for periods of 0–6 weeks. Samples were characterized at various room temperature incubation times using Atomic Force Microscopy (AFM, Asylum Research, MFP-3D), Cryogenic Transmission Electron Microscopy (cryo-TEM, JEM-1400), Circular Dichroism Spectrometer (CD, Jasco, J-810), and UV–vis Spectrophotometer (UV, Jasco, V630). AFM sample substrates were prepared from sections of a silicon (1 0 0) wafer (Tekstarter Co., Ltd.; P-type/Boron dopant). All of the silicon substrates were plasma cleaned (Harrick Scientific Products, Inc.) for 2 min using dry air as the reactive gas to increase the OH concentration at the surface. This process creates a uniformly hydroxylated surface. Insulin or Insulin/AuNP solutions were dropcast .onto clean silicon wafers, allowed to dry, and the imaged with AFM. All AFM images were acquired in AC (tapping) mode. A silicon cantilever (Olympus AC240TS) with a normal spring constant of 2 N m−1 was used for all images, with a scan rate of 1.0 Hz and an image resolution of 512 × 512 pixels.
3. Results and discussion Herein, insulin was chosen as an amyloidogenic protein model to investigate whether AuNPs can inhibit insulin amyloid fibrillation. In our previous study had described that insulin is a practical biotemplate for creating NPs chains, such as AuNPs deposition [24]. Further, literature sources suggest that AuNPs are ideal candidates for new AD therapy due to their selective attachment to A fibrils [25]. They can be synthesized in a variety of sizes and shapes and easily characterized using TEM [26]. To determine the effect of AuNPs on the fibrillation process, we studied insulin fibrils self-assembled into amyloid-like fibrils. Samples were prepared by incubating insulin monomer at pH 1.6 and 80 ◦ C for 2.5 h and then stored at room temperature for periods of 0–6 weeks. The formation of cross- fibrillar aggregates was monitored in the absence and presence of AuNPs, and TEM was used to characterize the morphological variation of the aggregates. Significant aggregation was observed when insulin fibrils were incubated with AuNPs for 1 week at room temperature (Fig. 1), and the aggregate density increased with longer incubation times. The AuNPs had a much higher affinity for the fibrils than for the substrate. The effect of AuNPs on the conformational change of insulin fibrils was also examined using AFM. Two series of AFM images (5 × 5 m2 ) show the morphological transition of insulin fibrils prepared as above (80 ◦ C, 2.5 h, pH 1.6) with further incubation at room temperature for 0 weeks, 3 weeks, and 4 weeks in the absence (Fig. 2a–c) and presence (Fig. 2d–f) of AuNPs, respectively. There is a clear difference in the surface morphology when comparing Fig. 2b and e, with the AuNP-treated insulin fibrils (Fig. 2e) appearing much shorter and more compact than the insulin-only fibrils (Fig. 2b). We also acquired AFM images after 5 and 6 weeks incubation at room temperature and the effect was even more pronounced (Fig. S1 in
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Fig. 4. CD spectra of insulin fibrillation in the absence (a) or presence (b) of AuNPs after incubation for 2.5 h, 1 week to 6 weeks at pH 1.6 and 80 ◦ C. (W = week). Visible absorption spectra of (c) pure intact AuNPs under different pH conditions and (d) AuNPs in a bovine insulin solution (1 mg/1 ml). The schematic illustration (e) corresponds to (c), and (f) corresponds to (d), respectively.
Supplementary Information). In our study, not only the aggregation rate, but also the length, height (thickness), and density of amyloidlike fibril aggregates were affected by the presence of AuNPs. We then acquired smaller scan-size AFM images to observe more detail of the insulin fibril structure after 5–6 weeks incubation (Fig. 3). The AuNPs were mostly located at fibril crossings (the average height of a single fibril was ∼3 nm while crossings were ∼30 nm), where thick intersecting aggregates were formed as shown in the AFM topography images of Fig. 3c and e, and graphically depicted in the adjacent cartoons.
Fig. 4a and b are CD spectra from insulin samples prepared without (a) and with (b) AuNPs. There is no significant difference between the spectra for either type of pre-treated sample after incubating for up to 3 weeks at room temperature. After 4 weeks incubation, the insulin-only sample showed a notable change in the spectra, with attenuation of the feature at 222 nm and a shift in the minima at 208–218 nm. This indicated a structural transformation from the ␣-helix to the -sheet conformation. Very little change was observed in the insulin/AuNP sample spectra after 4 weeks. This suggests that the AuNPs may delay or retard the transition
S. Hsieh et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 525–529
from the ␣-helix to the -sheet form. However, after 5 and 6 weeks incubation, spectra from both samples indicated transformation to the -sheet state with a single feature observed in each at 218 nm. Although AuNPs aggregate under acidic pH conditions (Fig. 4e); this does not appear to influence the aggregation of insulin fibrils when incubated with AuNPs in solution at pH 1.6. In the absorption spectra shown in Fig. 4c, AuNPs under acidic conditions (pH 1.6) exhibit a distinctive red shift of the 520 nm peak to 530 nm accompanied by the formation of a new absorption peak at 680 nm when compared to AuNPs at pH 7. This shift may be attributed to AuNPs aggregation. After adding AuNPs to insulin monomer or to insulin fibrils (Fig. 4d) at pH 1.6, we observed only a slight shift of the 520 nm peak (shown in Fig. 4c, pH 7) to 527 nm and no new absorption peaks were formed. These results suggest a preferential attachment of AuNPs onto insulin fibrils in solution during coincubation thus preventing self-aggregation. Further, due to the presence of the sulphur-containing amino acid ‘cysteine’, insulin can form strong S Au covalent bonds with the AuNPs [27], creating a protective coating around the AuNPs as depicted in Fig. 4f. Summarizing our observations, we propose a simple scheme for describing the mechanism of interaction for insulin fibrils incubated with AuNPs (Fig. S2 in Supplementary Information). Loading the insulin solution with AuNPs during the period of fibrillogenesis, the strong S Au bond frustrates the fibril self-assembly process reducing aggregation and thus inhibiting fibrillation. 4. Conclusions In the present study, we have demonstrated the effect of AuNPs conjugation on the structural evolution of insulin fibrils. We prepared insulin-only and AuNPs-treated insulin solutions (pH 1.6) and incubated them at room temperature for periods of 0–6 weeks. During this fibrillogenesis process, we found that AuNPs inhibited insulin fibrils assembly into protease resistant aggregates. AuNPs delayed the structural transformation into amyloid-like fibrils by about 1 week, and altered the structure, shape, and density of the formed fibrils compared with the insulin-only samples. We previously reported on the use of insulin fibrils as a sacrificial biotemplate for directing AuNPs self-assembly and alignment on surfaces [24,28]. Those results are consistent with our findings in this study where AuNPs exhibited a strong affinity toward insulin fibrils. We further observed here that AuNPs initiated the formation of thick intersecting fibril aggregates after incubation at room temperature for 5–6 weeks. Finally, we have shown that insulin conjugation to AuNPs can affect the protein structure and dynamics, further reducing the growth and stability of available amyloid-like -sheet structure for aggregation. These results suggest that AuNPs may serve a useful role in new therapeutic and diagnostic strategies for amyloid related diseases. Acknowledgments The authors would like to thank the National Science Council of Taiwan (NSC 101-2113-M-110-013-MY3), and the National Sun Yat-sen University Biochip Research Group for financial support of this work. Prof. Hsieh also thanks Dr. David Beck for helpful discussions and proofreading. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.08.029.
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References [1] F. Librizzi, C. Rischel, The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways, Protein Sci. 14 (2005) 3129–3134. [2] J.W. Kelly, The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways, Curr. Opin. Struct. Biol. 8 (1998) 101–106. [3] C.M. Dobson, The structural basis of protein folding and its links with human disease, Philos. Trans. R. Soc. B 356 (2001) 133–145. [4] J.E. Gerich, The genetic basis of type 2 diabetes mellitus: impaired insulin secretion versus impaired insulin sensitivity, Endocr. Rev. 19 (1998) 491–503. [5] G. Reaven, Insulin resistance, type 2 diabetes mellitus, and cardiovascular disease: the end of the beginning, Circulation 112 (2005) 3030–3032. [6] S.M. de la Monte, J.R. Wands, Alzheimer’s disease is type 3 diabetes-evidence reviewed, J. Diab. Sci. Technol. 2 (2008) 1101–1113. [7] S.M. de la Monte, Brain insulin resistance and deficiency as therapeutic targets in Alzheimer’s disease, Curr. Alzheimer Res. 9 (2012) 35–66. [8] K. Akter, E.A. Lanza, S.A. Martin, N. Myronyuk, M. Rua, R.B. Raffa, Diabetes mellitus and Alzheimer’s disease: shared pathology and treatment? Br. J. Clin. Pharmacol. 71 (2011) 365–376. [9] R.J. Boado, Antisense drug-delivery through the blood-brain-barrier, Adv. Drug Deliv. Rev. 15 (1995) 73–107. [10] J.R. Kanwar, X. Sun, V. Punj, B. Sriramoju, R.R. Mohan, S.F. Zhou, A. Chauhan, R.K. Kanwar, Nanoparticles in the treatment and diagnosis of neurological disorders: untamed dragon with fire power to heal, Nanomedicine 8 (2012) 399–414. [11] O. Salata, Applications of nanoparticles in biology and medicine, J. Nanobiotechnol. 2 (2004) 3. [12] S.D. Patil, D.G. Rhodes, D.J. Burgess, DNA-based therapeutics and DNA delivery systems: a comprehensive review, AAPS J. 7 (2005) E61–E77. [13] V.P. Torchilin, Multifunctional nanocarriers, Adv. Drug Deliv. Rev. 58 (2006) 1532–1555. [14] I.H. El-Sayed, X. Huang, M.A. El-Sayed, Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer, Nano Lett. 5 (2005) 829–834. [15] I.H. El-Sayed, X. Huang, M.A. El-Sayed, Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles, Cancer Lett. 239 (2006) 129–135. [16] P. Tiwari, K. Vig, V. Dennis,.S. Singh, Functionalized gold nanoparticles and their biomedical applications, Nanomaterials 1 (2011) 31–63. [17] Z.P. Zhuang, M.P. Kung, C. Hou, D.M. Skovronsky, T.L. Gur, K. Plossl, J.Q. Trojanowski, V.M. Lee, H.F. Kung, Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates, J. Med. Chem. 44 (2001) 1905–1914. [18] Q.R. Smith, A review of blood-brain barrier transport techniques, Methods Mol. Med. 89 (2003) 193–208. [19] S.I. Yoo, M. Yang, J.R. Brender, V. Subramanian, K. Sun, N.E. Joo, S.H. Jeong, A. Ramamoorthy, N.A. Kotov, Inhibition of amyloid peptide fibrillation by inorganic nanoparticles: functional similarities with proteins, Angew. Chem. Int. Ed. 50 (2011) 5110–5115. [20] E.K. Agyare, G.L. Curran, M. Ramakrishnan, C.C. Yu, J.F. Poduslo, K.K. Kandimalla, Development of a smart nano-vehicle to target cerebrovascular amyloid deposits and brain parenchymal plaques observed in Alzheimer’s disease and cerebral amyloid angiopathy, Pharm. Res. 25 (2008) 2674–2684. [21] C. Cabaleiro-Lago, F. Quinlan-Pluck, I. Lynch, S. Lindman, A.M. Minogue, E. Thulin, D.M. Walsh, K.A. Dawson, S. Linse, Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles, J. Am. Chem. Soc. 130 (2008) 15437–15443. [22] S.F. Cheng, L.K. Chau, Colloidal gold-modified optical fiber for chemical and biochemical sensing, Anal. Chem. 75 (2003) 16–21. [23] R. Jansen, W. Dzwolak, R. Winter, Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy, Biophys. J. 88 (2005) 1344–1353. [24] C.W. Hsieh, S. Hsieh, Nanoparticle chain formation on functional surfaces using insulin fibrils as a structure directing agent, J. Mater. Chem. 21 (2011) 16900–16904. [25] M.J. Kogan, N.G. Bastus, R. Amigo, D. Grillo-Bosch, E. Araya, A. Turiel, A. Labarta, E. Giralt, V.F. Puntes, Nanoparticle-mediated local and remote manipulation of protein aggregation, Nano Lett. 6 (2006) 110–115. [26] E. Araya, I. Olmedo, N. Bastus, S. Guerrero, V. Puntes, E. Giralt, M. Kogan, Gold Nnanoparticles and microwave irradiation inhibit beta-amyloid amyloidogenesis, Nanoscale Res. Lett. 3 (2008) 435–443. [27] K.H. Lee, F.M. Ytreberg, Effect of gold nanoparticle conjugation on peptide dynamics and structure, Entropy 14 (2012) 630–641. [28] S.C. Hsieh, C.W. Hsieh, Alignment of gold nanoparticles using insulin fibrils as a sacrificial biotemplate, Chem. Commun. 46 (2010) 7355–7357.