Femtosecond-Laser-Enhanced Amyloid Fibril Formation of Insulin

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Femtosecond Laser-Enhanced Amyloid Fibril Formation of Insulin Tsung-Han Liu, Ken-ichi Yuyama, Takato Hiramatsu, Naoki Yamamoto, Eri Chatani, Hiroshi Miyasaka, Teruki Sugiyama, and Hiroshi Masuhara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01822 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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Femtosecond Laser-Enhanced Amyloid Fibril Formation of Insulin

Tsung-Han Liu†, Ken-ichi Yuyama†#, Takato Hiramatsu‡, Naoki Yamamoto‡, Eri Chatani*‡, Hiroshi Miyasaka§, Teruki Sugiyama*†∥, Hiroshi Masuhara*†

†

Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan

‡

Department of Chemistry, Graduate School of Science, Kobe University, Kobe, Hyogo

657-8501, Japan

§

Division of Frontier Materials Science and Center for Promotion of Advanced

Interdisciplinary Research, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan

∥

Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma,

Nara 630-0192, Japan

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AUTHOR INFORMATION

*

Corresponding Authors

Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan

E-mail address: [email protected] (H. Masuhara)

E-mail address: [email protected] (T. Sugiyama)

Department of Chemistry, Graduate School of Science, Kobe University, Kobe, Hyogo 657-8501, Japan

E-mail address: [email protected] (E. Chatani)

#

Present Address

Research Institute for Electronic Science, Hokkaido University, N20W10, Kita-Ward, Sapporo 001-0020, Japan (K. Yuyama)


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ABSTRACT Femtosecond (fs) laser-induced crystallization as a novel crystallization technique was proposed for the first time by our group, where crystallization time can be significantly shortened under fs laser irradiation. Similarly, we have further extended our investigation to amyloid fibril formation also known as a nucleation-dependence process. Here we demonstrate that necessary time for amyloid fibril formation can be much shortened by fs laser irradiation leading to favorable enhancement. The enhancement was confirmed by both spectral measurements and direct observations of amyloid fibrils. Thioflavin T fluorescence intensity of laser-irradiated solution increased earlier than that of control solution, and such difference was simultaneously revealed by ellipticity changes. At the same timing before intensity saturation in fluorescence, amount of amyloid fibrils obtained under laser irradiation were generally more than those of control solution. Besides, such enhancement is correlated to laser power threshold of cavitation bubbling. Possible mechanisms are proposed by referring to fs laser-induced crystallization and ultrasonication-induced amyloid fibril formation.

KEYWORDS femtosecond laser, laser ablation, cavitation bubbling, amyloid fibril formation, insulin


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Introduction Femtosecond (fs) laser ablation due to multiphoton absorption of target materials under exposure of amplified fs pulses is considered as a photomechanical phenomenon which can remove tiny part of the target material from its surface or generate physical perturbations to its surroundings without heat accumulation.(1-3) Besides, since wavelength of fs laser is tunable in the near-infrared window, fs laser light can penetrate biological tissues and cells efficiently.(4-5) Accordingly, fs laser ablation has been widely extended to various applications and research fields, for examples, ranging from material science on the scale of nanometers such as nanoparticle preparation and nanoparticle patterning,(6-11) to crystallization and crystal growth on the scale of micrometers,(12-18) to cell manipulation,(19-21) and so on. Especially, fs laser-induced crystallization of protein from its supersaturated aqueous solution was proposed by our group for the first time,(12) and has received much attention since necessary crystallization time can be efficiently shortened and spatio-temporal control of nucleation by few fs laser shots can be realized.(12, 15) Following these results, we further investigated how to significantly enhance crystallization probability, shorten crystallization time and control the morphology of obtained crystals by optimizing fs parameters and laser irradiation position, and eventually we successfully demonstrated single crystal formation by irradiating only one single fs laser pulse at the air/solution interface.(22) Therefore, based on 4 ACS Paragon Plus Environment

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our previous study on crystallization, our interests are extended to amyloid fibril formation also known as a nucleation-dependent process consisting of a nucleation period (or a lag phase) and a growth period.(23-26) Amyloid fibrils, sometimes described as one-dimensional crystals, are ordered but noncrystalline aggregates of peptides or proteins and rich in β-sheets. They are composed of various kinds of peptides or proteins without obvious sequence similarity through self-assembly.(26-29) Amyloid fibril formation in the human body can further cause amyloidosis such as Parkinson’s disease and Alzheimer disease.(30) Therefore, in order to clarify the mechanism, research works related to amyloid fibril formation have received much attention throughout the past few decades. There are several similarities between amyloid fibril formation and protein crystallization; however, quite different from crystallization, generation of denatured protein molecules under strict conditions such as high ionic strength, low pH or high temperature is an indispensable step to trigger amyloid fibril formation from a supersaturated protein solution.(23-25, 31-32) As conventional approaches, amyloid fibril formation can be induced through heating(24, 33) or ultrasonication.(34-36) Interestingly, about the latter method, it is reported that not only local heating but also cavitation bubble formation takes place during ultrasonication irradiation, and thus protein monomers may be captured, conformationally destabilized and unfolded at the surface of ultrasonic cavitation bubbles.(34, 36) Referring to fs laser-induced crystallization in which cavitation bubbles 5 ACS Paragon Plus Environment

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generate during laser irradiation,(12, 15, 22) our proposal related to amyloid fibril formation becomes further promising. Amyloid fibrils are detected by spectroscopic or morphological approaches because of their remarkably small size.(28, 37-38) In the former, amyloid fibril formation can be monitored by light scattering measurements indicating how turbidity or fibril size distribution varies,(23, 39) fluorescence measurements after mixing with amyloid indicator dyes such as thioflavin T (ThT),(26, 31, 38, 40-41) bright-field or polarized light microscopy imaging after staining amyloid fibrils with Congo red,(27) and secondary structure analysis of target proteins by circular dichroism (CD) spectroscopy, or Fourier transform infrared (FTIR) spectroscopy measurements.(23-24, 31, 33, 38, 41) In the latter, atomic force microscopy (AFM),(35, 41) scanning electron microscopy (SEM), or transmission electron microscopy (TEM) is used to directly obtain images of fibrils.(35, 42) In practice, in order to track amyloid fibril formation immediately and quantitatively, spectroscopic measurements are preferred and are considered to be suitable to obtain time courses, whereas morphological images are necessary as direct evidences of fibril formation.(23-24, 31, 33-36, 38, 41-42) Insulin as a commercially available and representative hormone protein has been widely investigated since it can form amyloid fibrils in vitro and also its fibrillation is related to iatrogenic amyloidosis.(27, 38) Insulin molecule is composed of two peptide chains bridged by two disulfide bonds, namely, A-chain (21 amino acid residues) containing two 6 ACS Paragon Plus Environment

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sections of α-helix and B-chain (30 amino acid residues) containing one larger section of α-helix.(33, 43-44) Insulin dimers or monomers are major species in solution under extremely acidic conditions,(43-44) whereas they assemble into hexamers at neutral pH especially in presence of zinc ion.(44-46) To effectively induce amyloid fibril formation of insulin, acidic conditions are experimentally preferable in addition to heating.(33, 43) Therefore, here we report how fs laser enhances amyloid fibril formation of insulin. Time courses were monitored by ThT fluorescence and ellipticity changes, and morphological images of obtained fibrils were revealed by both SEM and AFM observations. Besides, roles of cavitation bubbling were also discussed based on results of laser power dependence experiments. By spatio-temporal control abilities of laser, mechanisms of amyloid fibril formation related to nucleation-growth dynamics and domain formation will be elucidated through microspectroscopy. The present results are also considered to be valuable and promising to provide approaches to locally trigger amyloid fibril formation at desired positions even in vivo, to induce micropatterning at hydrophobic surfaces via selective protein adsorption leading to further contributions in developing biosensors or bioarrays, and so on.

Experimental Section 1. Sample Preparation Solution of insulin (Human, recombinant, Wako Chemical) at 1.72 mM (10 mg/ml) 7 ACS Paragon Plus Environment

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was prepared in 25 mM HCl (pH 1.6) and 100 mM NaCl at room temperature. Prepared insulin solution was further filtrated with a syringe filter (pore size; 0.22 µm, SLGVX13NL, Millipore) to remove large aggregates or dust. For each sample, solution volume was 100 µl, and was preserved in a 1.5-ml glass bottle sealed with a plastic screw cap (Nichiden Rika Glass). Initially all control and experimental samples were pre-incubated in an incubator (EC-40R, AS ONE). Pre-incubation time and temperature were 3 h and 50 °C, respectively. Following pre-incubation, all samples were once cooled down to room temperature for 0.5 h. After that, an experimental sample and a control sample as a pair were simultaneously taken out from the incubator, while only the former was irradiated by laser and the latter was just temporally placed outside the incubator. As one set of experiments, a cycle including taking out a pair of samples from the incubator, laser irradiation, sampling for the following fluorescence measurements and putting samples back to be re-incubated at 50 °C again could be finished in 1 min and was repeated for several times until all experimental samples were irradiated by laser. 2. Optical Setup The optical setup is generally the same as what we have reported in previous works related to fs laser-induced crystallization.(22) Linearly polarized amplified femtosecond (fs) laser (wavelength; 800 nm, pulse, duration; 160 fs, Spitfire Pro, Spectra Physics) as the light 8 ACS Paragon Plus Environment

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source was introduced to an inverted microscope (IX-71, Olympus), and was further focused into a sample bottle from its bottom by an objective lens (10×, N.A. 0.25, PlanN, Olympus). Focal height was set 200 µm above the sample bottle bottom, namely, laser beam was focused inside the solution. By using a variable neutral density filter, a polarizing beam splitter, and a half-wave plate, laser pulse energy throughout the objective lens was fixed to 1-50 µJ/pulse, and was confirmed by a joule meter (842-PE, Spectra Physics). The repetition rate of fs laser pulse train was adjusted to be 10 Hz by a Pockels Cell. Irradiation time was controlled to be 10 s by a mechanical shutter. During laser irradiation, bright-field images obtained by a digital CCD camera (CV-S3200N, JAI) under halogen lamp illumination were used to confirm if cavitation bubbling certainly takes place or not. Note that there was no observable damage on glass bottle surfaces after laser irradiation under above optical conditions. 3. Fluorescence Measurements To track time courses of amyloid fibril formation through fluorescence measurements, ThT as a fluorescence dye was used. Once amyloid fibrils are formed and then ThT molecules attach on them, fluorescence intensity enhancement can be clearly observed.(26, 40) Practically, solution of ThT (Sigma) at 5 µM was prepared in 50 mM Glycine-NaOH buffer (pH 8.6) to avoid fluorescence quenching of ThT at extremely low or high pH.(47) For each fluorescence measurement, firstly 0.5 ml ThT solution and 1.5 µl sample solution were mixed with a vortex mixer for around 5 s, the mixture was put in a quartz cuvette (path length; 2 mm, 9 ACS Paragon Plus Environment

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Starna Scientific), and then after 1-min waiting its fluorescence spectrum was measured by a spectrofluorometer (FluoroMax-4, Horiba). The excitation wavelength and detection region of emission were set to be 445 nm and 460-500 nm, respectively. To monitor amyloid fibril formation, values of fluorescence intensity at 485 nm were continuously recorded and summarized into figures below. 4. CD Measurements To reveal secondary structure changes of proteins, CD in UV region was applied. For sample preparation, 5 µl solution from either control samples or laser-irradiated samples was diluted 60 times with HCl at 25 mM. CD spectra was measured by a CD spectrometer (J-815, Jasco) with a quartz cuvette (110-QS, path length; 1 mm, Hellma Analytics). Detection region, scan rate and accumulation number were 200-250 nm, 200 nm/min and 8 scans, respectively. 5. Field Emission Scanning Electron Microscopy (FE-SEM) Observations For sampling, 10 µl solution from either control samples or laser-irradiated samples was dropped on a freshly-cleaved mica substrate. After waiting for 1 min, 1 ml pure water was used to remove rest solution and also to rinse the mica substrate. Residual water was removed by putting a filter paper at the edge of the mica substrate, and then the substrate dried spontaneously. Mica substrates were mounted on a holder of SEM with double-sided carbon tape after drying spontaneously, and copper tape was used at the edge of substrates to avoid charge accumulation on surfaces. Specimens were slightly coated by platinum through 10 ACS Paragon Plus Environment

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vapor deposition for 40 s from top and for 10 s from sides with an auto fine coater (JFC-1600, JEOL), and were observed under FE-SEM (JSM-7401F, JEOL). SEM imaging mode and accelerating voltage were the secondary electron imaging mode and 3.0-5.0 kV, respectively. 6. Atomic Force Microscope (AFM) Observations Following the same sampling procedures applied in SEM observations, 10 µl solution from either control samples or laser-irradiated samples was dropped on a freshly-cleaved mica substrate. After waiting for 1 min, 1 ml pure water was used to remove rest solution and also to rinse the mica substrate. Residual water was removed by putting a filter paper at the edge of the mica substrate, and then the substrate dried spontaneously. AFM images were obtained using a NanoNavi II/IIe probe station (SII Nanotechnology). The scanning tip used was a micro cantilever (OMCL-AC160TS-C3, Olympus, spring constant; 21-37 N/m, resonance frequency; 270-340 kHz). The number of scanning points was 256 at each axis with a scan rate of 0.5 or 1.0 Hz.

Results and Discussion 1. Spectroscopic Analysis of Amyloid Fibril Formation of Insulin Since ThT has a high sensitivity to recognize amyloid structures, amyloid fibril formation of insulin monitored by fluorescence spectra of ThT mixture was first examined (Figure 1). After 3-h pre-incubation at 50 °C and 0.5-h cooling to room temperature, only 11 ACS Paragon Plus Environment

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experimental samples were irradiated by fs pulses at 50 µJ/pulse (9.5 PW/cm2 per pulse based on our calculation(22)) at repetition rate of 10 Hz for 10 s, and then fluorescence spectra of both experimental and control samples were measured repetitively (Figure 1(a)). The above pre-incubation time, incubation temperature, and insertion of cooling were determined as present experimental conditions after systematic trial experiments, and related discussions are mentioned below. Laser power and repetition rate are referred from previous experiences on fs laser-induced crystallization, in which high laser power and low repetition rate lead to higher frequency to obtain a complete single crystal.(22) Laser exposure time was set to 10 s tentatively for examination. Under this optical condition, it was confirmed by bright-field images that cavitation bubbles generated during laser exposure expand to larger than a few tens of µm, and collapse or are pushed by other bubbles around the focal spot (Figure 2). The lifetime of bubbles was estimated to be at 100-ms timescales. For control samples, when pre-incubation time increased to about 3 h, fluorescence intensity started to remarkably increase. In contrast, for laser-irradiated samples, the lag time was much shortened to around 1 h, that is, it was observed that amyloid fibril formation of insulin can be enhanced by fs laser irradiation (Figure 1(b)). Eventually, fluorescence intensity of both laser-irradiated and control samples reached a similar saturated level after 9-h post-irradiation incubation as simultaneously the solutions became viscous and cloudy. We noticed that nucleation in amyloid fibril formation is considered to be similar to that in 12 ACS Paragon Plus Environment

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crystallization as stochastic processes,(48) although only a set of examples (N=3 under each condition) showing a typical tendency is represented in Figure 1(a). In practice, experiments under the same condition have been repeated for more than 5 times to confirm reproducibility on different days. In addition, to reveal how fractions of secondary structures of insulin varies as amyloid fibril formation proceeds, CD spectra were measured. If amyloid fibril formation takes place, a CD spectrum with two negative bands around 208 and 222 nm ascribed to α-helices gradually merges into a spectrum with only one broader negative band around 216 nm ascribed to β-sheets.(33, 49) Indeed, such interpretable series of spectral shape changes were observed (Figure 3(a)). Accordingly, time courses of α-helix fraction was monitored by ellipticity at 208 nm (Figure 3(b)). As the results, similar to observation in fluorescence measurements, ellipticity of laser-irradiated samples started to change apparently earlier than that of control samples, and ellipticity of both samples reached saturation in the later stage. 2. Morphological Analysis of the Obtained Amyloid Fibrils of Insulin In order to confirm if amyloid fibrils were generated and to clarify if fluorescence intensity correlates closely with the amount of amyloid fibrils, little amount of solutions was taken out for SEM sampling at certain timings during post-irradiation incubation. As post-irradiation incubation time increased from 2 to 4 h, it is observed that the density of amyloid fibrils increased accordingly in both laser-irradiated and control samples as expected 13 ACS Paragon Plus Environment

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(Figure 4). On the other hand, if images of laser-irradiated samples and control samples at the same timings are compared, there are also observable differences in not only fibril density but also length. In other words, in contrast to heating-induced amyloid fibril formation, it takes around 2 less h for amyloid fibrils to grow under fs laser irradiation into the same level. Importantly, SEM images give consistent results with previous fluorescence information, and it is confirmed that necessary time for amyloid fibrils to appear can be certainly shortened by fs laser irradiation. In addition, to analyze fibrils in detail, both SEM and AFM images were obtained from sample solutions taken out at the same timing when ThT fluorescence intensity of laser-irradiated solution was just close to a saturation level while that of control solution was still unsaturated. It is found that fibril density was higher under laser irradiation in addition to incubation than under incubation only (Figure 5(a-d)), and the results are consistent with previous observation shown in Figure 4. To further calculate mean diameter of fibrils obtained under laser irradiation and that under incubation only as controls, 50 amyloid fibrils were randomly selected under each condition and their diameters were measured. From SEM images (Figure 5 (a,c)), the mean diameters are 23.8±4.8 nm and 23.7±5.1 nm, respectively, while it is considered that fibril diameter measured from SEM images may be slightly overestimated because of platinum coating through vapor deposition. On the other hand, from AFM images (Figure 5 (b,d)), the mean heights of fibrils 14 ACS Paragon Plus Environment

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obtained under laser irradiation and that under incubation only as controls are 3.4±0.2 and 4.2±0.2 nm, respectively. It implies fibrils obtained under laser irradiation are slightly thinner than those of controls on average. 3. Laser Power Dependence of Amyloid Fibril Formation of Insulin Since cavitation bubble formation during fs laser irradiation is dependent on laser pulse energy, laser power dependence was examined. According to our previous report on fs laser-induced crystallization of glycine, there is an energy threshold around 2.5 µJ/pulse for cavitation bubble generation described as a nonlinear process.(22) When fs laser was focused into insulin solution, cavitation bubbles were not observable under a CCD camera at 1 µJ/pulse for sure, whereas bubbles were generated by almost every fs laser shot around 2.5 µJ/pulse and were always clearly visible above the threshold as shown in Figure 2. Correspondingly, there is an energy threshold around 2.5 µJ/pulse observed in amyloid fibril formation enhancement by fs laser (Figure 6). The growth curve of laser-irradiated samples at 1 µJ/pulse was indistinguishable from those of control samples. As laser pulse energy increased from 2.5 to 10 µJ/pulse, although growth curves deviated more from those of control samples accordingly, their fluorescence intensity did not increase at the same rate. In other words, enhancement of amyloid fibril formation was achieved under cavitation bubbling, whereas such enhancement reached a saturated level when laser pulse energy became much higher than the energy threshold of cavitation bubble generation. 15 ACS Paragon Plus Environment

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4. Possible Mechanisms for Amyloid Fibril Formation 4.1 Optical Decomposition of Insulin Protein molecules in their supersaturated solution should be partly denatured to initiate amyloid fibril formation.(23-25, 31-32) Accordingly, interactions between fs laser and insulin molecules are firstly considered. Fs laser pulses can transiently generate an intense optical field of 1016-1018 W/cm2 at the focal spot, which is comparable to the intramolecular Coulomb field of a hydrogen molecule. Such intense optical fields result in various novel phenomena, for instances, gold nanoparticles can be produced directly from optical decomposition of AuCl4 when peak optical field was estimated to be 8.8×1018 W/cm2.(7) Compared to our present cases, amyloid fibril formation can be enhanced even under irradiation at 4.75×1014 W/cm2. Accordingly, although direct optical decomposition of insulin may occur based on interactions between an intense optical field and insulin molecules under fs laser irradiation described above, it is considered that direct optical decomposition of insulin in an intense field makes a minor contribution in amyloid fibril formation enhanced by fs laser. 4.2 Multiphoton Excitation of Insulin Insulin is not excited by light at 800 nm through single-photon absorption, whereas multiphoton absorption of insulin likely occurs since generally organic molecules are electronically excited via multiphoton absorption under near-infrared pulsed laser 16 ACS Paragon Plus Environment

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irradiation.(50) Proteins can be photodegraded by the UV absorption of peptide backbone, tryptophan, tyrosine, phenylalanine, and cysteine inside the molecules.(51) Since insulin lacks tryptophan residues, tyrosine play an important role in UV absorption of insulin. When insulin undergoes photoionization processes under continuous UV light exposure (276 nm, ca. 2.2×104 W/cm2) for several h, it is reported that insulin at 17.7 µM in 10 mM phosphate buffer (pH 8.0) undergoes dimerization via dityrosine cross-linking as tyrosine residues are oxidized and electrons are subsequently released, and disulfide bonds further cleave during photolysis.(51) Similarly, under irradiation of gamma rays or accelerated electrons at 10 kGy, insulin at 0.1-1 mM in 50 mM phosphoric buffer (pH 2) is completely degraded. Cleavage, dimerization, and polymerizations of the peptidic chains via linkage of dityrosines can be induced by such irradiation.(52) Interestingly, even when photodimerization of insulin takes place, there is still no evidence to correlate amyloid fibril formation of insulin with such dityrosine formation, and it is also suggested that monomer instead of dimer is preferable to further form amyloid fibrils.(53) Comparably, insulin can be possibly excited through three-photon absorption by fs laser pulses at 800 nm, so it seems that insulin is more likely to be excited than water. However, no other report on photochemical amyloid formation of insulin is available as far as we know. As experimentally confirmed here under a microscope, there was no observable difference between energy thresholds of cavitation bubble generation in pure water and in 17 ACS Paragon Plus Environment

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insulin solution (Figure 2). In other words, in contrast to molecular number of insulin in its solution at 10 mg/ml which is around 3.2×105 times less than that of water, water molecules still have a major possibility to be excited. Accordingly, following discussions related to mechanisms are shifted to water as solvent molecules. 4.3 Multiphoton Decomposition of Water Water has no absorption at 800 nm at room temperature since its band gap is around 6.5 eV, whereas it can be electronically excited through multiphoton absorption if photon density for excitation is high enough.(2, 54-55) Under such intense optical fields, excited water molecules can undergo further photolysis such as ionization or decomposition. Both ionization and decomposition of water take place on the order to 100 fs comparable to the present laser pulse duration, and following species generated as photolysis products, particularly ejected electrons, have µs-ordered lifetime and can further absorb incident laser light.(54) However, the present repetition rate is 10 Hz, in other words, time interval between pulses is 0.1 s and it is still much longer than µs-ordered lifetime. On the other hand, during water ionization, ions and radicals such as H3O+, OH• and hydrated electrons sequentially generate and disappear because of recombination with each other.(55) Especially, under present solution condition at low pH, hydroxyl radicals and hydrated electrons are more likely to be captured by electrophillic species in solution.(56) Accordingly, it is considered that formation of species due to water ionization has very little impact on triggering nucleation. 18 ACS Paragon Plus Environment

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4.4 Cavitation Bubble Formation due to Multiphoton Absorption of Water When laser irradiance (W/cm2) is higher than a certain threshold value, optical breakdown of water accompanying with cavitation bubble generation takes place.(57) Similar phenomena of cavitation bubbling were mentioned in both fs laser-induced crystallization and ultrasonication-induced amyloid fibril formation.(12, 15, 34) For fs laser-induced crystallization, one of present authors directly observed that crystals grow around the bubble surfaces as cavitation bubbles expand or shrink.(15, 58) On the other hand, for ultrasonication-induced amyloid fibril formation, Ohhashi et al. suggested that, in addition to extremely high local temperature elevation, proteins may be destabilized and unfolded at the air-liquid interface of sonication-induced bubbles and thus partially folded conformations are induced.(34) Referring to results of laser power dependence experiments, it implies that cavitation bubble generation also plays an important role in amyloid fibril formation enhanced under fs laser irradiation. Ultrasonication-induced amyloid fibril formation showed lag time of about 1.5 h or even longer under repeated exposure of ultrasonication for 1 min in every 10 min.(34-36) Based on our experimental results, enhancement in fluorescence intensity could not be observable immediately after 10-s fs laser irradiation, that is, nuclei cannot be generated at once and thus still relatively short lag time was necessary. Therefore, at the present stage, it is suggested that conditions for nucleation such as partial dissociation and unfolding are provided during or after cavitation bubble generation under fs laser irradiation 19 ACS Paragon Plus Environment

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to enhance nucleation of amyloid fibril formation. Besides, based on results of laser power dependence experiments, such enhancement saturates when laser power is much higher than the threshold of cavitation bubbling. One possible explanation is reverse effects such as destroying nuclei or formed amyloid fibrils may exist if laser power is too high. Interestingly, such equilibrium between production and breakdown of amyloid fibrils was also reported in ultrasonication-induced amyloid fibril formation.(35) 4.5 Consideration on Photothermal Heating, Transient Temperature Elevation and Incubation Temperature Experimentally, during laser irradiation at 10 Hz for 10 s, a total of 100 laser pulses of 2.5-50 µJ excites the samples. All photon energy roughly equals to 6×10-5-1.2×10-3 cal, which leads to negligible temperature increment of 100-µl sample solution if all photons are assumed to be perfectly absorbed by water. The possibility related to the photothermal effect can be reasonably excluded. In contrast to crystallization experiments commonly at room temperature,(25) amyloid fibril formation cannot be efficiently demonstrated at room temperature, and the length of its lag time is much affected as incubation temperature varies.(36) Therefore, incubation temperature was also adjusted in this work. When incubation temperature was fixed at 40 °C, pre-incubation time before laser irradiation was increased to 4 h and other 20 ACS Paragon Plus Environment

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laser conditions were the same (50 µJ/pulse for 10-s exposure), it took about 2 days for both laser-irradiated samples and control samples to reach saturation of fluorescence intensity (data not shown). Compared to our present demonstrations at 50 °C, apparently all processes can be much shortened as incubation temperature slightly increases. It is reported that local temperature around focal spot is above spinodal temperature of water, ca. 151.5 °C, when cavitation bubbles are generated during optical breakdown of water under fs laser irradiation (wavelength; 800 nm, pulse duration; 100 fs).(57) If such local transient temperature elevation during cavitation bubbling is the main reason to directly trigger amyloid fibril formation, it is considered that the lag time for nucleation processes should be independent of incubation temperature. However, contrary to what we expected, it is experimentally confirmed that the lag time became much longer when the incubation temperature decreased from 50 to 40 °C. In other words, it seems that such transient temperature elevation cannot trigger nucleation directly like heat-induced amyloid fibril formation and thus lag time significantly varies with incubation temperature. Therefore, it is considered that such transient increase in local temperature induced by optical breakdown of water has very less impact on triggering amyloid fibril formation. Once incubation temperature is higher than 70 °C, the whole amyloid fibril formation process finishes in 2 h.(41) At around 80 °C, amyloid fibrillation of bovine insulin is reported to have a maximum growth rate.(31) In addition, referring to preparation of supersaturated 21 ACS Paragon Plus Environment

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solution for crystallization,(59) the solution was slightly heated and then cooled down to room temperature. It was found that, if 0.5-h cooling time was inserted between pre-incubation and incubation after laser irradiation as shown in previous figures, the time gap of fluorescence intensity curves between laser-irradiated samples and control samples enlarged though the lag time of control cases becomes slightly longer. 4.6 Proposed Mechanisms We propose here how to explain amyloid fibrillation enhancement by cavitation bubble formation under fs laser irradiation. We examine formation of denatured states accelerated by physical perturbations, and increased probability of interactions among them. Initially some of well-folded insulin dimers are partially-dissociated and unfolded during 3-h pre-incubation at 50 °C.(24) During the following 0.5-h cooling, although the most parts of those denatured species change back to the native form reversibly, a small amount of aggregation precursors or quasi-aggregates may be newly generated as the insulin solution becomes more supersaturated.(59) Under 10-s laser irradiation, local concentration of such new species around the laser focus is suddenly increased when cavitation bubbles expand, shrink and eventually collapse.(36) The formation of air/solution interfaces (or bubble surfaces) and convection of the surrounding solution accelerate the growth of the precursors or quasi-aggregates by inducing insulin monomer aggregation and its denaturation.(60-61) Finally, again under the incubation at 50 °C after fs laser irradiation, irreversible amyloid 22 ACS Paragon Plus Environment

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fibril formation takes place resulting experimentally in the significant lag time shortening.

Conclusion Amyloid fibril formation of insulin enhanced by fs laser irradiation is demonstrated here for the first time. Fluorescence and CD spectra as spectroscopic analyses, and SEM and AFM images as morphological analysis give consistent results indicating that cavitation bubble formation induced by fs laser irradiation can enhance amyloid fibril formation. The fibrils fabricated by fs laser are a little thinner than those obtained by the control experiment. Besides, based on results of laser power dependence experiments, cavitation bubble formation is directly correlated to such enhancement of amyloid fibril formation similar to fs laser-induced crystallization.(22) Furthermore, incubation temperature and an insertion period of temporary cooling were also adjusted to optimize observed phenomena. It is believed that amyloid fibril formation enhanced by fs laser opens new doors for not only conventional amyloid study but also possible laser applications. Based on this demonstration and previous study on fs laser-induced crystallization, it is promising that amyloid fibril formation can be spatio-temporally triggered by laser irradiation, and even formation of one single assembly of amyloid fibrils may be possibly achieved by irradiating only one single laser shot and generating only one single cavitation bubble as we reported on crystallization of glycine and anthracene.(22, 58) Laser is 23 ACS Paragon Plus Environment

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undoubtedly a powerful tool to initiate reactions spatio-temporally with high resolution. Indeed, recently our group has just demonstrated amyloid fibril assembly formation and even its micropatterning by continuous wave laser trapping. It is believed that the mechanisms can be further clarified by spectroscopic study under microscopes.(42)

Acknowledgements The present work is supported by MOE-ATU Project (National Chiao Tung University) of the Ministry of Education, Taiwan to H.Masuhara, by grants from the Ministry of Science and Technology of Taiwan to H.Masuhara (MOST 105-2811-M-009-022) and to T.S. (MOST 105-2113-M-492-001-), by JSPS KAKENHI Grant Numbers JP16H00772 to E.C., JP17K14427 for Young Scientists (B) to K.Y., and Scientific Research on Innovative Areas “Nano-Material Optical Manipulation” to T.S. (JP16H06507) and “Photosynergetics” to H.Miyasaka (JP26107002). T.-H. Liu was supported by Global Networking Talent 3.0 Plan (National Chiao Tung University) as a visiting research student in Osaka University from Nov. 2015 to Jun. 2016. The authors thank Mr. Jui-Kai Chen, a master student in Department of Applied Chemistry, National Chiao Tung University, for assistance with SEM observations.


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FIGURE LEGENDS

Figure 1

Fluorescence spectra (λex: 445 nm) of insulin amyloid fibril formation under incubation only (black) and under laser irradiation in addition to incubation (red) after mixing with ThT solution (a), and their time courses monitored by peak intensity at 485 nm (b). The error bars represent ±1 standard deviation from the mean fluorescence intensity at 485 nm (N=3). Pulse energy, repetition rate and exposure time were 50 µJ/pulse, 10 Hz and 10 s, respectively.

Figure 2

Bight-field images of cavitation bubbling as single frames captured by the CCD camera under halogen lamp illumination in supersaturated insulin solution (a) and in pure water (b). Brightness of bubbles are strongly affected by heights of bubbles in the focal plane. The scale bars represent 100 µm. Pulse energy and repetition rate were 50 µJ/pulse and 10 Hz, respectively.

Figure 3

Circular dichroism (CD) spectra of insulin amyloid fibril formation under laser irradiation and incubation (red) and under incubation only (black) (a), and their time courses monitored by


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ellipticity at 208 nm as a negative peak value of α-helix (b). Pulse energy, repetition rate and exposure time were 50 µJ/pulse, 10 Hz and 10 s, respectively.

Figure 4

SEM images of insulin amyloid fibrils obtained under laser irradiation and incubation (a-d) and under incubation only as controls (e-h). Sample solutions were taken out from glass bottles and diluted with HCl at 25 mM for sampling after 2-h post-irradiation incubation (a,b,e,f) and after 4-h post-irradiation incubation (c,d,g,h), respectively. The scale bars represent 100 nm (a,c,e,g) or 1 µm (b,d,f,h). Pulse energy, repetition rate and exposure time were 50 µJ/pulse, 10 Hz and 10 s, respectively.

Figure 5

SEM images (a,c) and AFM images (b,d) of insulin amyloid fibrils obtained after 7-h post-irradiation incubation under laser irradiation and incubation (a,b) and under incubation only as controls (c,d). The scale bars represent 1 µm (a,c) or 500 nm (b,d). Pulse energy, repetition rate and exposure time were 50 µJ/pulse, 10 Hz and 10 s, respectively.

Figure 6

Laser power-dependence of amyloid fibril formation of insulin enhanced by fs laser irradiation. Time courses of insulin amyloid fibril formation under incubation only (black


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squares and circles, two independent control samples) and under laser irradiation in addition to incubation (triangles) were monitored by fluorescence peak intensity at 485 nm (λex: 445 nm) after mixing with ThT. Pulse energy, repetition rate and exposure time were 1-10 µJ/pulse, 10 Hz and 10 s, respectively.


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Figure 1


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Figure 2


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Figure 4


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Figure 6


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Table of Contents/Abstract Graphic


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Femtosecond-Laser-Enhanced Amyloid Fibril Formation of Insulin

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