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Temperature-Induced Phase Separation in Molecular Assembly of Nanotubes Comprising Amphiphilic Polypeptide with Poly(N-Ethyl Glycine) in Water by a Hydrophilic-Region Driven Type Mechanism Tetsuya Hattori, Toru Itagaki, Hirotaka Uji, and Shunsaku Kimura J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03419 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 24, 2018

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The Journal of Physical Chemistry

Temperature-Induced Phase Separation in Molecular Assembly of Nanotubes Comprising Amphiphilic Polypeptide with Poly(N-ethyl glycine) in Water by a Hydrophilic-Region Driven Type Mechanism

Tetsuya Hattori, Toru Itagaki, Hirotaka Uji, and Shunsaku Kimura* Department of Material Chemistry, Graduate School of Engineering, Kyoto University Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto, Japan 615-8510

ABSTRACT Two kinds of amphiphilic polypeptides having different types of hydrophilic polypeptoids, poly(sarcosine)-b-(L-Leu-Aib)6 (ML12) and poly(N-ethyl glycine)-b-(L-Leu-Aib)6 (EL12), were self-assembled via two paths to phase-separated nanotubes. One path was via sticking ML12 nanotubes with EL12 nanotubes, and the other was a preparation from a mixture of ML12 and EL12 in solution. In either case, nanotubes showed temperature-induced phase separation along the long axis, which was observed by two methods of labeling one phase with gold nanoparticles and fluorescence resonance energy transfer between the components. The phase-separation was ascribed to aggregation of poly(N-ethyl glycine) blocks over the cloud point temperature. The addition of 5% trifluoroethanol was needed for the phase separation, because the tight association of the helices in the hydrophobic region should be loosened to allow lateral diffusion of the components to be separated. The phase-separation in molecular assemblies in water based on the hydrophilic-region driven type mechanism therefore requires sophisticated balances of association forces exerting among the hydrophilic and hydrophobic regions of the amphiphilic polypeptoids.

Introduction Phase-separated molecular assemblies are attracting attention in a wide range from biological activities arising from phase-separated cell membranes1 to hierarchical nanoand micro-scale molecular architectures.2 The sizes of the phase-separated molecular assemblies are thus widely dispersed from the nano-3-4 to mesoscale5 even if limited to lipid membranes. The phase separation in lipid membranes can be induced possibly by 1

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two mechanisms based on the hydrophobic or hydrophilic region exerting primary influence on phase separation. The former typical example is lipid “rafts” where a cholesterol-rich liquid-ordered domain is segregated from a fluid liquid-disordered region. The latter one is rare to find out, but the related example is perineuronal nets made by a chondroitin sulfate rich region anchored by semaphorin3A.6 In either case, the phase-separated structure is deeply related with the biological activities. Even though nature is rich in such phase-separated molecular assemblies, the molecular assemblies with chimeric morphologies due to phase separation in membrane are still limited in the reports using artificial synthetic molecules. We have previously reported a molecular assembly displaying a chimeric morphology of a round-bottomed flask shape based on phase separation of a mixture of amphiphilic polypeptides having hydrophobic blocks of right- and left-handed helices, poly(sarcosine)-b-(L-Leu-Aib)6 and poly(sarcosine)-b-(D-Leu-Aib)6.7 An equimolar mixture of the two polypeptides generated vesicles, whilst the single component alone self-assembled into nanotubes. Two kinds of the right- and left-handed helices tightly associate together to form a stereocomplex structure. Accordingly, when poly(sarcosine)-b-(L-Leu-Aib)6 and poly(sarcosine)-b-(D-Leu-Aib)6 were mixed at an uneven molar ratio, the excessive component was segregated from the stereocomplex region. As a result, the membrane region of the excessive component took a nanotube morphology and the membrane region of the stereocomplex took a vesicular morphology, where two different-shaped membranes coexisted to afford the round-bottomed flask molecular assembly. The mechanism behind the round-bottomed flask morphology is therefore categorized into the hydrophobic-region driven type. On the other hand, phase separation based on the hydrophilic-region driven type mechanism is considered to be more difficult to demonstrate in water, because hydrophobic interaction arising naturally from the hydrophobic region is a prevailing force to self-assemble into well-defined morphologies in water. In the aim of expanding the phase-separated molecular assemblies from the hydrophobic-region driven type to the hydrophilic-region driven type, the hydrophilic blocks of amphiphilic polypeptides are newly designed here to induce phase separation. Polypeptoids are a class of polypeptides composed of poly(N-substituted glycine).8 Poly(N-methyl glycine) (poly(sarcosine)) and poly(N-ethyl glycine) were reported to be highly water-soluble, but polypeptoids with N-substitution of longer alkyl chains were water insoluble.9 Random copolymers of poly(N-ethyl glycine-co-N-butyl glycine) were thermally responsive to exhibit reversible phase transitions with tunable cloud point temperature by changing the composition in aqueous solution.10 We consider a 2


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possibility of poly(N-ethyl glycine) showing a cloud point temperature below 100 oC when poly(N-ethyl glycine) would be densely incorporated into a surface region of a molecular assembly. A similar idea using a molecular assembly to display thermo-insensitive chromophores (isobutylamide) densely on surface in order to endow the molecular assembly with a thermosensitive property was previously reported by Kono et al.11 We therefore design a molecular assembly of nanotube composed of a mixture of poly(sarcosine)-b-(L-Leu-Aib)6 (ML12) and poly(N-ethyl glycine)-b-(L-Leu-Aib)6 (EL12) (Figure 1). ML12 and EL12 have a common hydrophobic helix, which favors self-assembling into nanotubes.12 Phase separation in nanotubes induced by aggregation of EL12 over a cloud point temperature according to the hydrophilic-region driven type mechanism is herein studied.

Methods Syntheses of amphiphilic polypeptoids Amphiphilic polypeptoids (Figure 1) were synthesized by the conventional liquid phase method similarly to the previous reports.7, 12-14 The degrees of polymerization of poly(sarcosine) and poly(N-ethyl glycine) blocks were determined from 1H NMR and MALDI-TOF-MS (Supporting Information (SI) Figures S1–S5). Details of syntheses of polypeptides containing N-ethyl glycine were described in SI. Preparation of nanotubes A typical preparation method of nanotubes was as follows. Amphiphilic polypeptoids in ethanol (50 mg/mL) were mixed at a specific molar ratio, and an aliquot (ca. 4 µL) was injected into 5% ethanol aqueous solution (500 µL) at 4°C, stirred for 15 minutes, and heated at 45°C for 8 h. In some cases, the solution was treated with further heating at 85°C for 3 h. The experiments of joining nanotubes were carried out with heat treatment at 50°C for 24 h in the presence of 5% trifluoroethanol. Morphology observation by transmission electron microscopy (TEM) TEM images were analyzed by JEOL JEM-2000EXII at an accelerating voltage of 100 kV. The peptide nanotube solutions were applied on a carbon-coated Cu grid. The samples were negatively stained by 2% uranyl acetate, followed by suction of the excess solution with a filter paper. In the case of AuNPs staining, the peptide aqueous solutions were applied on a carbon-coated Cu grid, and AuNPs were added on the grid to label 3



nanotubes. The excess amounts of AuNPs were washed with Milli-Q water. This labeling steps were repeated twice. The samples were negatively stained by 2% uranyl acetate, followed by suction of the excess solution with a filter paper. Fluorescence resonance energy transfer (FRET) measurement Fluorescence spectra were recorded by JASCO FP-6600 fluorescence spectrophotometer. The optical path length was 1 cm. The peptide concentrations were ca. 0.17 mg/mL. The fluorescence spectra were measured with excitation at 280 nm.

Results and discussion Nanotube formation ML12 and EL12 were respectively self-assembled in 5% ethanol aqueous solution with a heat treatment at 45 oC for 8 h followed by 85 oC for 3 h (Figure 2). TEM observations revealed that nanotubes were spontaneously generated as low as 45 oC, which is in contrast to the case of the nanotubes prepared from poly(sarcosine)-b-(L-Leu-Aib)6 (the degree of polymerization (n) of poly(sarcosine) was 25), which required a heat treatment at 90 oC. ML12 has a poly(sarcosine) block of n = 18, which should make the amphiphilic polypeptoid less hydrophilic to promote molecular assembling in water resulting in nanotube formation at low temperature. With further heating at 85 oC for 3 h, nanotubes elongated their lengths partly over 1 µm with keeping a diameter of ca. 80 nm. The average nanotube lengths before and after heating at 85 oC increased from 370 nm to 680 nm for ML12 nanotubes and from 630 nm to 1180 nm for EL12 nanotubes. The elongation rate was more remarkable with EL12 nanotubes, which may reflect a less hydrophilic property of poly(N-ethyl glycine) than poly(sarcosine). Cloud point Thermal response of the elongated nanotubes after a heat treating at 85 oC was studied by UV absorption fluctuation at 250 nm of the nanotube solutions (Figure 3). EL12 nanotubes showed large fluctuations in the time response of absorption over 70 oC, which is ascribable to aggregation of the elongated nanotubes. Optical images of the solution actually showed some aggregates floating at 65 oC and 85 oC (SI Figure S6). This behavior is in contrast with ML12 nanotubes showing no fluctuation at 70 oC. It is striking that poly(N-ethyl glycine) (n = 16) was not thermal responsive (SI Figure S7), 4


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but became to show the cloud point temperature around 70 oC when poly(N-ethyl glycine) was displayed densely on molecular assemblies of nanotubes. Joining nanotubes Elongation of nanotubes by sticking ML12 nanotubes with EL12 nanotubes was examined. We found out that the addition of trifluoroethanol to ML12 and EL12 nanotube dispersions was effective to join them resulting in increase of fractions over 2 µm in lengths under the conditions of 50 oC for 24 h incubation (Figure 4). Since two kinds of nanotubes were prepared in advance and mixed together, it is most likely that the nanotubes joined together. The average nanotube lengths after heating at 50 oC for 24 h in the presence of 5% trifluoroethanol were 970 nm for ML12 nanotubes and 1810 nm for EL12 nanotubes, each of which was longer than the average nanotube length of the corresponding nanotube with a heat treatment at 50 oC for 24 h in the absence of trifluoroethanol (Figure 2). A mixture of ML12 and EL12 nanotubes afforded the longest nanotube of an average length of 2180 nm. Previously, we reported that ML12 with injection into water self-assembled into nanosheets of a few hundreds nm, which determined the nanotube length and diameter upon morphology conversion from nanosheet to nanotube with heating at 90 oC. The size of the nanosheets was speculated to be set by the balance between the suppressive effect of hydrophilic chains to conceal the hydrophobic edge of the nanosheet and the promoting effect of the hydrophobic edge to grow larger on the size of the molecular assembly. Trifluoroethanol has been frequently used as a powerful solvent to solubilize peptides with promoting helical structures in most cases. Trifluoroethanol is also a good solvent for the hydrophobic region of the polypeptoids, (L-Leu-Aib)6, with keeping the helical conformation. Elongation of nanotubes observed here is thus speculated to be due to the enhancement of the hydrophobic edge effect making the open edges of the nanotubes more hydrophobic by distribution of trifluoroethanol in this region leading to the nanotube elongation. However, we cannot exclude the possibility that trifluoroethanol may stabilize the helical conformation of the hydrophobic block to enhance the association force among the helices, resulting in the shift of the thermodynamically stable state into the elongated nanotubes. Phase-separated nanotubes evidenced by labeling with gold nanoparticles In order to evaluate distributions of ML12 and EL12 in their mixed nanotubes, lipoic acid-derivatized ML12 (LML, Figure 1) was mixed in ML12 by 20% to prepare LML/ML12 nanotubes. Lipoic acid-derivatized polypeptides can adsorb gold 5



nanoparticles (AuNPs) via Au-S linkages, and have been used to clarify the distribution of the corresponding polypeptide in the molecular assemblies by TEM observations.7, 15 A mixture of two kinds of nanotubes of LML/ML12 and EL12 was incubated at 45 oC for 8 h followed by incubation at 50 oC for 24 h in the presence of trifluoroethanol. After staining with gold nanoparticles (AuNPs), the distribution of AuNPs along nanotubes in TEM images was evaluated and classified into three classes; 1) nearly no AuNP adsorption (homogeneous nanotube), 2) AuNP adsorption unevenly to nanotubes along the long axis (phase separated nanotube), and 3) AuNP adsorption whole to nanotubes (homogeneous nanotube). The definition of class 2) is the most important for evaluation of phase-separated nanotubes. Since the criterion of AuNPs clustering on nanotube surfaces may depend on the total number of AuNPs adsorbing on surfaces, the nanotubes were also categorized into two groups of nanotubes labeled by more than 50 AuNPs (relatively longer nanotubes; NT>50AuNP) and those less than 50 AuNPs (relatively shorter nanotubes; NT NA) to obtain NA/lA and NB/lB as a surface density of the number of AuNPs/100 nm (N/l). When a nanotube has regions of (NB/lB)/(NA/lA) > 3, the nanotube was classified into class 2). When all the regions were labeled by less than 2 AuNPs/100 nm (N/l < 2), the nanotube was classified into class 1). With incubation at 50 oC for 8 h, the ratio of class 2) was less than 10% about both nanotubes of NT>50AuNP and NT50AuNP nanotubes are mostly (nearly 90%) classified into class 3), indicating that LML distributed homogeneously to the whole nanotubes after joining LML/ML12 and EL12 nanotubes at 50 oC in the presence of 5% trifluoroethanol. LML, ML12, and EL12 components should be therefore allowed to diffuse laterally in nanotube membranes at 50 oC in the presence of 5% trifluoroethanol. Remarkable changes in AuNP distributions were observed with further incubation at 90 oC for 1 h to increase the ration of class 2) over 20% on NT50AuNP nanotubes (Figure 5). The temperature-induced phase separation is therefore observed with incubation at 90 oC, which is over the cloud point temperature of ca. 70 oC of EL12 nanotubes. Taken together, EL12 components were segregated from other components owing to temperature-induced aggregation of poly(N-ethyl glycine) blocks in nanotubes. The mechanism for the phase separation is thus the hydrophilic-region driven type.

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Phase-separation in nanotubes prepared from a solution of LML, ML12, and EL12 The phase separation in nanotubes is tested by another path for nanotube preparation. All the three components of LML, ML12, and EL12 were mixed together at a molar ratio of 0.1:0.4:0.5. An aliquot of the mixture was injected into 5% ethanol aqueous solution and incubated at 45 oC for 8 h. After labeling nanotubes with AuNPs, the AuNP distributions in TEM images are classified into three classes (Figure 6). In this case, it is clearly shown that the class 2) (phase-separated nanotube) was negligible for any nanotubes regardless of AuNP labeling rates. With incubation at 90 oC for 1 h, however, any phase-separated nanotubes were not observed (Figure 6), which is in contrast to the temperature-induced phase separation in nanotubes after joining LML/ML12 and EL12 nanotubes. Temperature-induced rearrangement of the three components in nanotubes therefore was not allowed under the conditions. Successively, the same experiment was carried out in the presence of 5% trifluoroethanol (Figure 7). With incubation of nanotubes prepared from a mixture of the three components at 50 oC for 24 h in the presence of 5% trifluoroethanol, the ratio of class 2) (phase-separated nanotube) was negligible. With further incubation at 90 oC for 1 h, however, the ratio of class 2) raised up to 40 % in the total nanotubes. It is therefore concluded that the temperature-induced phase separation in nanotubes occurred above the cloud point temperature of EL12 nanotubes with help of trifluoroethanol, which should allow lateral diffusion of the three components in the nanotube membranes probably because the molecular packing of the hydrophobic helices in the hydrophobic region of nanotube membranes should be loosened by the presence of trifluoroethanol. Phase-separation evidenced by fluorescence resonance energy transfer In addition to the AuNP labeling method, the phase separation was confirmed by the fluorescence resonance energy transfer (FRET) method. The methyl esters at C terminals of ML12 and EL12 were replaced with N-ethylcarbazolyl (MLE) and naphthyl groups (ELN), respectively (Figure 1). An aliquot of a mixture of ML12, MLE, EL12, and ELN at a molar ratio of 85:15:90:10 was injected into 5% ethanol aqueous solution and incubated at 45 oC for 8h followed by at 50 oC for 24 h. The fluorescence spectra obtained by excitation at 280 nm showed emissions from around 330 and 340 nm (naphthalene) and around 360 and 370 nm (N-ethylcarbazole) (Figure 8) as the peaks were assigned on the basis of the individual fluorescence spectrum (SI Figure S8). With excitation of the naphthyl groups at 280 nm, the fluorescence intensity from the naphthyl groups will diminish in accordance with the degree of FRET from a naphthyl group to N-ethylcarbazolyl groups nearby. The intensity ratio of these two emissions 7



(I361/I338) from nanotubes prepared from a mixture of ML12, MLE, EL12, and ELN was the highest after incubation at 50 oC for 24 h in the presence of 5% trifluoroethanol reflecting a good fluorescence resonance energy transfer occurring from naphthyl to N-ethylcarbazolyl groups due to the homogeneous mixing of these four components. With successive incubation at 90 oC for 1 h, the intensity ratio became lower at 90 oC, and the lower value was maintained even after the temperature decreased below the cloud point temperature, indicating that the phase separation occurred in nanotubes over the cloud point temperature to suppress the energy transfer and the phase-separated nanotubes were maintained at a lower temperature than the cloud point temperature due to the dissipation of trifluoroethanol during heating at 90 oC. Conclusion The temperature-induced phase separation in nanotubes was demonstrated experimentally by two ways. One experiment was to stick one-type nanotube with the other-type nanotube, where the nanotubes were prepared in advance. The other experiment was to prepare nanotubes from a mixed solution of the two kinds of polypeptoids. These two different experiments resulted in the same phase-separated nanotubes, meaning that the phase-separation occurred in accordance with a thermodynamic control. The phase-separation in nanotubes is induced by the hydrophilic-region driven mechanism. Importantly, the lateral diffusion of the components should be allowed for the reorganization of disposition of the components in nanotubes. Amphiphilic polypeptides have been reported to be able to diffuse laterally in membranes, for example, as follows.14 Two kinds of nanotubes of poly(sarcosine)-b-(L-Leu-Aib)6 and poly(sarcosine)-b-(D-Leu-Aib)6 stuck together to convert the nanotube morphology into a flat sheet morphology. During this morphology change, we observed a spherical morphology at the joining region of the two nanotubes as a transient morphology, which can be rationally explained by lateral diffusion of the components in the membrane, and cannot be explained by exchanging components between nanotubes. As described before, trifluoroethanol is considered to exert the effect on the lateral diffusion of the components and joining nanotubes together owing to the property as a good solvent for the hydrophobic helical block. In the present case, the molecular packing of (L-Leu-Aib)6 helices in the hydrophobic region was so tight that the molecular packing was needed to be loosened by trifluoroethanol to reorganize the disposition of the components, which was driven by aggregation of hydrophilic chains of poly(N-ethyl glycine) over the cloud point 8


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temperature. The trifluoroethanol content in the system, however, decreased gradually during incubation at 90 oC for 1 h, which recovered the tight molecular packing of helices to fix the phase-separated state induced at 90 oC even at room temperature. At room temperature below of the cloud point, there is no segregation force exerting between poly(sarcosine) and poly(N-ethyl glycine), which means that the homogeneously mixed nanotubes would be a thermodynamically stable state. But, the lateral diffusion of the polypeptoids in the phase-separated nanotubes was not allowed in the absence of trifluoroethanol due to the tight molecular packing to keep the phase-separated state at room temperature, which was attainable above the cloud point. The sophisticated balance between the association forces exerting among hydrophobic and hydrophilic blocks therefore should be taken in account for generation of the phase-separated molecular assemblies as demonstrated here. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxx. Synthetic scheme of N-ethyl glycine-NCA; Synthetic scheme of ELN; 1H NMR spectrum and MALDI TOF mass spectrum of ML12; 1H NMR spectrum and MALDI TOF mass spectrum of EL12; 1H NMR spectrum and MALDI TOF mass spectrum of LML; 1H NMR spectrum and MALDI TOF mass spectrum of MLE; 1H NMR spectrum and MALDI TOF mass spectrum of ELN; Optical images of dispersion of EL12 nanotubes heated at various temperatures; Time responses of UV absorptions at 250 nm of poly(N-ethyl glycine) with varying temperatures; Fluorescence spectra of ELN and MLE in a methanol solution.

Corresponding Author [email protected] ORCID Shunsaku Kimura: 0000-0003-0777-9697 Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported partially by JSPS KAKENHI Grant Number JP16H02279 and Analysis and Development System for Advanced Materials, Research Institute for Sustainable Humanosphere, Kyoto University.

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Figure 1 Chemical structures of amphiphilic polypeptides.

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Figure 2 TEM images and nanotube length histograms of (a, e) ML12 at 45 oC for 8h, (b, f) ML12 at 85 oC for 3 h, (c, g) EL12 at 45 oC for 8 h, and (d, h) EL12 at 85 oC for 3 h. The vertical axis of counts represents the number of nanotubes in the range of the corresponding length in TEM images. Bars in TEM images indicate 1000 nm.

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Figure 3 Time responses of UV absorptions at 250 nm with varying temperatures of (a) ML12 nanotubes and (b) EL12 nanotubes.

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Figure 4 TEM images and nanotube length histograms of (a, d) ML12 nanotubes, (b, e) EL12 nanotubes, and (c, f) a mixture of ML12 and EL12 nanotubes after heating at 50 o

C for 24 h in the presence of 5% trifluoroethanol. Bars in TEM images indicate 1000

nm (a, b) and 500 nm (c).

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Figure 5 AuNP distribution along nanotube long axis by TEM images of a mixture of LML/ML and EL12 nanotubes after (a) heating at 50 oC for 24 h in the presence of 5% trifluoroethanol followed by (b) heating at 90 oC for 1 h. Three classes of nanotube histograms about AuNP distribution along nanotube long axis of (c) NT50AnNP (total number of nanotubes; 27 at 50 oC, 12 at 90 oC). Blue bars are under the condition of (a) and red bars are under the condition of (b). Three classes are class 1; nearly no AuNP adsorption (homogeneous nanotube), class 2; AuNP adsorption unevenly to nanotubes along the long axis (phase separated nanotube), and class 3; AuNP adsorption whole to nanotubes (homogeneous nanotube). The vertical axes of (c) and (d) represent ratios of the number of the nanotubes corresponding to the class against the total number of nanotubes analyzed in the TEM images. Bars in TEM images indicate 500 nm.

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Figure 6 AuNP distribution along nanotube long axis by TEM images of nanotubes prepared from a mixture of LML, ML12, and EL12 (0.1:0.4:0.5) in solution after (a) heating at 45 oC for 8 h followed by (b) heating at 90 oC for 1 h. Three classes of nanotube histograms about AuNP distribution along nanotube long axis of (c) NT50AnNP (total number of nanotubes; 1 at 50 oC, 18 at 90 oC). Blue bars are under the condition of (a) and red bars are under the condition of (b). The definitions of the vertical and horizontal axes of (c) and (d) are the same as Figure 5. Bars in TEM images indicate 500 nm.

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Figure 7 AuNP distribution along nanotube long axis by TEM images of nanotubes prepared from a mixture of LML, ML12, and EL12 (0.1:0.4:0.5) in solution after (a) heating at 45 oC for 8 h followed by heating at 50 oC for 24 h in the presence of 5% trifluoroethanol and (b) successive heating at 90 oC for 1 h. Three classes of nanotube histograms about AuNP distribution along nanotube long axis of (c) NT50AnNP (total number of nanotubes; 47 at 50 oC, 19 at 90 oC). Blue bars are under the condition of (a) and red bars are under the condition of (b). The definitions of the vertical and horizontal axes of (c) and (d) are the same as Figure 5. Bars in TEM images indicate 1000 nm (a) and 500 nm (b).

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Figure 8 (a) Fluorescence spectra of nanotubes prepared from a mixture of ML12, MLE, EL12, and ELN at a molar ratio of 85:15:90:10 with incubation at 50 oC for 24 h in the presence of 5% trifluoroethanol (black) followed by incubation at 90 oC for 1 h (red), and the temperature decreased to room temoperature (blue). The excitation wavelength was 280 nm. (b)The fluorescence intensity ratios, I361/I338, of these three fluorescence 19



spectra. TOC Graphic

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Temperature-Induced Phase Separation in ... - ACS Publications

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