Mesoscopic Heterogeneity in Pore Size of Supramolecular Networks

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Interface-Rich Materials and Assemblies

Mesoscopic Heterogeneity in Pore Size of Supramolecular Networks Yuji Matsumoto, Atsuomi Shundo, Masashi Ohno, Nobutomo Tsuruzoe, Masahiro Goto, and Keiji Tanaka Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00641 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Mesoscopic

Heterogeneity

in

Pore

Size

of

Supramolecular Networks Yuji Matsumoto,† Atsuomi Shundo,†,‡,§* Masashi Ohno,¶ Nobutomo Tsuruzoe,¶ Masahiro Goto,†,ǁ and Keiji Tanaka†,‡,§,ǁ*

AUTHOR ADDRESS †

Department of Applied Chemistry, ‡Department of Automotive Science, §International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), and ǁCenter for Future Chemistry, Kyushu University, Fukuoka 819-0395, Japan

¶

Nissan Chemical Industries, Ltd., Tokyo 101-0054, Japan.

KEYWORDS:

self-assembly,

supramolecular

hydrogel,

rheology,

particle

tracking,

heterogeneity, mesh size

ACS Paragon Plus Environment

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ABSTRACT

There has been a considerable interest in developing new types of gels based on a network of fibrous aggregate composed of low-molecular-weight gelators, also known as supramolecular gels (SMGs).

Unlike conventional polymer gels with chemical cross-linking, the network

formation in SMGs does not involve any covalent bonds. Thus, the network in SMGs has been often regarded as homogenous, or less heterogeneous in comparison with that in chemically cross-linked polymer gels. In this study, we have experimentally verified the existence of the network heterogeneity even in SMGs. The thermal motion of probe particles in SMGs, which were prepared from aqueous dispersions of gelators having a different number of peptide residues, PalGH, PalG2H and PalG3H, was tracked. The gels were spatially heterogeneous in terms of the network pore size, as evidenced by the variation in the particle motion depending on the location, at which a particle existed. With varying particle size, it was found that the characteristic length scale of the heterogeneity was in the order of sub- and micrometers and was smaller in the order of the PalG2H, PalG3H and PalGH gels.


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INTRODUCTION Gels are a class of soft materials containing solvent molecules encapsulated in a threedimensional network. A representative gel is polymer which consists of chains chemically crosslinked to one another. In general, the polymer network is inherently heterogeneous in terms of its cross-linking density and pore, or mesh, size.1,2 Such a spatial heterogeneity results mainly from the concentration fluctuation of chains, which is immobilized, or frozen, during the chemical cross-linking reaction.2,3

So far, great efforts have been made to gain a better

understanding of the heterogeneous polymer network and the correlation with macroscopic mechanical properties of polymer gels with chemical cross-linking.1,4,5 In fact, it has been reported that a homogenous polymer network in a poly(ethylene glycol) gel, which was formed from mutually reactive tetra-armed prepolymers of ethylene glycol, provided excellent mechanical properties.4 This has encouraged researchers in the community to study how the network of chemically cross-linked polymer gels can be precisely controlled.1,5 Recently, there has been increasing interest in supramolecular gels (SMGs) as an alternative to the conventional polymer gels.6,7 In SMGs, low molecular-weight molecules, socalled gelator molecules, self-assemble into fibrous aggregates, leading to the formation of a network.8,9 Unlike chemically cross-linked polymer gels, the network formation in SMGs does not involve any covalent bonds.

Thus, one may think that the network in SMGs is

homogeneous, or less heterogeneous, in comparison with that in chemically cross-linked polymer gels.10 However, it has been pointed out that the fibrous aggregate and its network can often be thought of as being in a kinetically-trapped state, rather than a thermodynamically stable state.11,12 In fact, the mechanical properties of SMGs depend on the process by which the selfassembly of gelator molecules occurs to produce the gel state.11 This suggests the possibility that


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the network in SMGs is heterogeneous in its pore size. Such network heterogeneity should be related to the mechanical properties, which play an important role in a wide variety of applications such as injectable gels,13 sprayable materials,14 drug carriers,13,15 cell scaffolds16 and self-healing materials.17 Nevertheless, there have been few studies on the network heterogeneity in SMGs. One of the major reasons for this is the limited number of available techniques for quantitative characterization of the network pore and its size distribution in SMGs. There are several potential approaches for quantifying the network pore size. X-ray and neutron scattering measurements have often been employed for chemically cross-linked polymer gels.1,2,18 However, these may not be applicable to SMGs because a characteristic length scale of the network structure is in the order of sub- and micrometers, which are much larger than that of the polymer network.19,20

Besides, although scanning electron microscopy has excellent

resolution, a specimen to be observed is generally dried, leading often to a collapse of the network.21 By contrast, optical microscopies, including confocal laser scanning microscopy (CLSM), allow us the in-situ observation without this drying problem. However, images must be analyzed with the greatest care taking into account various factors such as contrast, the presence of vertically oriented components and laser-induced heating. Otherwise, the results will lead to the overestimation of the pore size.22,23 Thus, to verify the heterogeneous network in SMGs, an alternative methodology is desired. Here, we focus on a particle tracking technique as a method for studying the network heterogeneity of SMGs.

In this technique, probe particles are dispersed in a medium.

Information on the local properties can be obtained by detecting the thermal motion of a particle because the movement reflects the physical properties of the medium surrounding it.24 Thus, tracking the movement of the particles located at different positions can provide insight into the


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spatial heterogeneity in the medium.25,26 An intriguing advantage of this technique is that the length scale of the observation can be altered by changing the particle size.24,27 This means that the particle motion depends on the size relationship between network pore and particle.28,29 In this study, we report experimental verification of the heterogeneity in the network pore size of SMGs, as schematically illustrated in Figure 1. Probe particle Objective lens (EM)CCD camera

Figure 1 Schematic illustration of the present study using a particle tracking method to address the network pore size and its heterogeneity.

EXPERIMENTAL Materials. N-palmitoyl-(Gly)n-His trifluoroacetate (PalGnH, n = 1, 2 and 3) was synthesized according to a previously reported method.14,30-32 Water used for the gel formation was obtained by distillation with an Autostill WG33 (Yamato Scientific Co., Ltd.) and successive deionization with a Milli-Q Lab system (Merck Millipore Co.). The specific resistance of the purified water was greater than 18·MΩ·cm. For particle tracking measurements, an aqueous dispersion of Fluoresbrite Yellow Green Microspheres, which were polystyrene (PS) particles containing a fluorescent dye, with a concentration of 2.5 wt% was purchased from Polysciences Inc. Diameters (d) of PS particles used were 20 ± 4 nm, 47 ± 5 nm, 200 ± 9 nm, 510 ± 10 nm, 1.1 ± 0.02 µm, 3.1 ± 0.09 µm, and 5.8 ± 0.3 µm (denoted as 20 nm, 50 nm, 200 nm, 500 nm, 1 µm, 3 µm and 6 µm, respectively). Deuterated water (D2O) was purchased from MERCK & Co. Inc.


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and Wako Pure Chemical Industries, Ltd., respectively and were used for Fourier-transform infrared (FT-IR) spectroscopy. For CLSM, sodium 8-anilino-1-naphthalenesulfonate (ANS) was purchased from Tokyo Chemical Industry Co. Ltd. and was used as received.

Rheological measurements. Rheological measurements were made using an Anton Parr MCR 301 rheometer (Anton Paar Japan K.K). In all measurements, a cone-typed plate with a diameter of 50 mm and a tilt angle of 1.0° was used. An aqueous solution of PalGnH pre-warmed at 363 K was mounted on a sample stage. The solution was sandwiched between the cone-typed plate and the flat stage, and left undisturbed at 298 K. After the gelation, viscoelastic functions were examined as functions of frequency and strain.

Fourier-transform infrared spectroscopy.

The gels were prepared from the mixture of

PalGnH and D2O. The use of D2O instead of H2O as a solvent is to subtract the contribution of the solvent from the spectrum. Each sample was sandwiched between CaF2 windows with a 0.5 mm gap. The FT-IR spectra were recorded using a FT/IR-620 spectrometer (JASCO Co.) with a triglycine sulfate detector. All spectra obtained with a resolution of 4 cm-1 and 64 scans at room temperature.

Small angle X-ray scattering measurements.

Small angle X-ray scattering (SAXS)

experiments were carried out at BL40B2 beamline in SPring-8 (Japan). The gel prepared in a quartz capillary with an outside diameter of 2 mm (Hilgenberg GmbH.) was placed in a sample stage. Scattered X-rays were recorded using a Rigaku R-AXIS IV+++ system (300 × 300 mm


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imaging plate) with an exposure time of 300 s. By the circular averaging of a two-dimensional pattern on the image plate, a one-dimensional scattering profile for the sample was obtained.

Atomic force microscopy. A hydrogel was gently placed on a freshly-cleaved mica substrate. After a while, the hydrogel was removed from the substrate and then fibrils not adsorbed on the substrate were rinsed out with a large amount of pure water. The resultant substrate was dried in vacuum at room temperature for 24 hours and was subjected to atomic force microscopic (AFM) observation. AFM images were recorded on an E-sweep equipped with a SPI3800 controller (Hitachi High-Tech Science Co., Ltd.) using an intermittent contact mode at room temperature. The cantilever used was made from silicon, with both sides were uncoated. The spring constant and the nominal tip radius were 14 N·m-1 and 10 nm, respectively.

Particle tracking measurements. PalGnH and a portion of the particle dispersion were welldispersed into pure water in a glass vial. The mixture with a PalGnH concertation of 5 mM was heated at 363 K for 20 min, resulting into a clear solution. An aliquot of the solution was placed in a glass bottom dish (MATSUNAMI GLASS Inc. Ltd.) and it was sealed with a cover glass using vacuum grease. The sample was left undisturbed at room temperature for 24 hours, prior to the particle tracking measurements. Our approach used for particle tracking is based on an inverted microscope, Nikon ECLIPSE Ti, with an NA 1.30 oil-immersion objective lens (Plan Fluor 100×, Nikon), as reported elsewhere.33 For the measurements using larger PS particle with diameters of 500 nm, 1 µm, 3 µm and 6 µm, a halogen lamp was used to illuminate the sample, and a charge-coupled device (CCD) camera (DS-Qi1Mc, Nikon Instech Co., Ltd.) was used to acquire images of


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particles in the samples at a frame rate of 31 Hz. For the measurements using particles with diameters of 20 nm, 50 nm and 200 nm, fluorescence imaging was employed. A mercury lamp through an excitation filter (passband: 426-446 nm) housed in a filter block (CFP, Nikon Instech Co., Ltd.) illuminated the sample. The fluorescence emitted from the particle went through an absorption filter (passband: 460-500 nm) in the filter block. Fluorescence images were acquired by an electron multiplying CCD camera (Ixon ultra 897, Andor Technology Co., Ltd.) at a frame rate of 34 Hz. The imaging software NIS-Elements AR-3.22 (Nikon Instech Co., Ltd.) was employed for the trajectory analysis of particles. A total of 20 particles were individually tracked in each sample. Each particle was monitored 10 times to average the diffusion behavior. The particle tracking measurements were made at 298 K.

Confocal laser scanning microscopy. For CLSM observations, the gels containing the ANS dye were prepared following the previously-reported procedure.26 The concentration of ANS was 60 µM. Each sample was placed in a glass bottom dish and sealed with a cover glass using vacuum grease. Images were obtained using an inverted confocal laser scanning microscope equipped with a 405 nm semiconductor laser (ZEN LSM700, Carl Zeiss Microscopy Co., Ltd.) A pinhole size used corresponds to the thickness of the focal plane of 0.4 µm.

RESULTS AND DISCUSSION Peptide amphiphiles, PalGnH (n = 1, 2, 3), were used as gelators. Figure 2 provides (a) the chemical structure of PalGnH and (b) photographs of the corresponding hydrogels with a gelator concentration of 5 mM, respectively. An aqueous dispersion of PalGnH was heated at 363 K for 20 min, leading to a clear solution. Cooling this solution to room temperature and successive


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aging for 24 hours yielded a hydrogel, which did not flow even after tilting the vial containing it at an angle (see Figure S1 in Supporting Information).

The appearance of the hydrogel

composed of PalGH was nebulous, suggesting that the scattering of visible light occurred due to the presence of molecular aggregations with a relatively-large size. On the other hand, the gels of PalG2H and PalG3H were more transparent than the PalGH gel. Thus, it is most likely that the size of PalG2H and PalG3H aggregations was smaller than that of PalGH.

Figure 2 (a) Chemical structure of N-palmitoyl-(Gly)n-His trifluoroacetate (PalGnH) used as a gelator and (b) photographic images showing the corresponding hydrogels with a gelator concentration of 5 mM.

Figure 3 shows AFM images showing the aggregates in the PalGH, PalG2H and PalG3H gels and the width distribution for 100 different aggregates in the corresponding gels. For all gels, the aggregates were fibrous in shape. Fibrils were composed of molecular assemblies, in which amide moieties of gelator molecules interacted with one another via hydrogen bonding and alkyl chains formed an interdigitated packing structure, as revealed by FT-IR spectroscopy in conjunction with SAXS measurement (see Figures S2 and S3 in Supporting Information).26 The mean width of the fibrils in the PalGH gel was 420 ± 200 nm and the polydisperse length approached several tens of micrometers.34

For PalG2H and PalG3H, the fibril width was

narrower, while the length remained at several micrometers. The mean widths for PalG2H and


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PalG3H were 80 ± 10 and 110 ± 30 nm, respectively. The gel network formed by such fibrils was subjected to study using the particle tracking measurements.

Figure 3 (a) AFM images showing fibrous aggregates in hydrogels composed of PalGH, PalG2H and PalG3H. (b) Probability distribution of width for 100 different aggregates. Insets in panel (b) denote the enlarged distributions.

PS particles containing a fluorescence dye were used as a probe. To alter the probed length scale, PS particles with various diameters (d) ranging from 20 nm to 6 µm were used. The thermal motion of PS particles was tracked at 20 different locations in the same hydrogel. Based on the two-dimensional trajectories of the individual particles, the mean-square-displacement, here symbolically represented as 〈∆r2(t)〉, was obtained from the following equation:24-26

∆r 2 (t ) =

1 N

N

∑ {r (t ) − r (0 )}

2

i

i

(1)

i =1

where ri(t) and ri(0) are positions of a particle i at times of t and 0, and N is the number of particles observed. The 〈∆r2(t)〉 value quantitatively reflects the average travel distance at time t


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from the original position for a particle. Based on the slope (γ) on double logarithmic plots for 〈∆r2(t)〉 against t, the type of the particle diffusion can be discussed.35-37 When γ = 1, that is 〈∆r2(t)〉 is linearly proportional to t, it is apparent that the diffusion of particles follows the random walk statistical model, as is commonly seen for particle diffusion in a liquid.35,36 By contrast, when γ < 1, it can be claimed that particles move in a sub-diffusive manner, which is generally explained in terms of the trapping of particles within the aggregated structure.37 Figure 4 shows double logarithmic plots of 〈∆r2(t)〉 against t for particles embedded in the PalGH hydrogels. Each solid line was obtained by monitoring a single particle 10 times and taking an average.

The slope of the hypotenuse of a right-angled triangle in the figure

corresponds to unity. For particles with a diameter of 6 µm, the slopes for all plots were less than 1, suggesting that the fibrous network trapped the particles.29,35,37 With decreasing particle size, the population of the plot with γ =1 increased. Consequently, γ for all plots drawn with particles having d = 20 nm can be regarded as 1. A reasonable explanation for this is that a particle normally diffused inside a pore of the network.38,39 If that is the case, the pore size of the network should be larger than 20 nm. Here, it is noteworthy that for particles with diameters ranging from 3 µm to 50 nm, there exists the variation in γ for the plots.40 This implies that the particles are not in equivalent environments. Thus, the spatial heterogeneity in the network can be discussed on the basis of the γ variation, which reflects the size relationship between particle and network pore size.28,29


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d = 20 nm

(a)

50 nm

200 nm

500 nm

1 µm

3 µm

6 µm

〈∆r2(t)〉 / µm2

10 2 10 0 10 -2 10 -4 10-6 10-2

10 0 10-2

100 10 -2

100 10 -2 100 10-2 Lag time (t) / s

100 10-2

100 10 -2

10 0

10 0 10-2

100 10 -2

100 10 -2 100 10-2 Lag time (t) / s

100 10-2

100 10 -2

10 0

10 0 10-2

100 10 -2

100 10 -2 100 10-2 Lag time (t) / s

100 10-2

100 10 -2

10 0

〈∆r2(t)〉 / µm2

(b) 102 10 0 10 -2 10 -4 10 -6 -2 10

(c) 〈∆r2(t)〉 / µm2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10 2 10 0 10 -2 10 -4 10 -6 10-2

Figure 4 Double logarithmic plots of 〈∆r2(t)〉 against t for particles with diameters of 20 nm, 50 nm, 200 nm, 500 nm, 1 µm, 3 µm and 6 µm embedded in (a) PalGH, (b) PalG2H and (c) PalG3H hydrogels. The slope of the hypotenuse of triangles corresponds to 1. The γ variation of the 〈∆r2(t)〉 plots was also observed for the PalG2H and PalG3H gels, as shown in Figure 4(b) and (c). However, the particle size showing the γ variation differs from that for the PalGH gel. The sizes, at which the γ variation was maximized, were 3 µm, 500 nm and 1 µm for the PalGH, PalG2H and PalG3H gels, respectively. To clarify such a difference, the non-Gaussian parameter, α2(t), was here adopted. It can be given by the following equation: 41 4 3 ∆r (t ) α 2 (t ) = −1 5 ∆r 2 (t ) 2

(2)

where 〈∆r4(t)〉 is the fourth moment of the particle displacement. The α2(t) value is a measure of the deviation from a Gaussian distribution of the particle displacement and therefore indicates


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the extent of heterogeneity in the system.26,35,36,41 In a perfectly homogenous system, α2(t) is close to 0, while a larger α2(t) indicates the presence of heterogeneity. Figure 5 shows the correlation between 〈 α2(t)〉 and particle diameter in the PalGH, PalG2H and PalG3H gels. 〈α2(t)〉 corresponds to α2(t) averaged over lag times ranging from 0 to 1.6 s, where substantial α2(t) increase with increasing t was not observed. For the PalGH gel, the 〈α2(t)〉 values with smaller particles were nearly zero, meaning that the system looked to be homogeneous. Once d reached 3 µm, the 〈α2(t)〉 was maximized. This makes it clear that the network in the PalGH gel is heterogeneous with a length scale of 3 µm. For the PalG2H and PalG3H gels, a finite value of 〈α2(t)〉 was obtained in a wide range of d: 50 - 500 nm for PalG2H and 500 nm - 1 µm for PalG3H. The d values, at which the maximum of 〈α2(t)〉 was obtained, were 500 nm and 1 µm for PalG2H and PalG3H, respectively. Thus, it can be claimed that the length scale of the heterogeneity was smaller in the order of PalG2H, PalG3H and PalGH.

4 3 α 2(t)

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2

1. 2. 3.

PalGH PalG2H PalG3H

2 3

1

1 0 101

102 103 Diameter (d) / nm

104

Figure 5 Correlation between non-Gaussian parameter, 〈α2(t)〉 and diameter of particles embedded in hydrogels composed of PalGH, PalG2H and PalG3H.


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To discuss on the relationship between heterogeneity and fibril network, direct imaging of the network was tried by CLSM observation with ANS.

The ANS dye emits stronger

fluorescence in a hydrophobic environment than in a hydrophilic one, making it possible to stain a hydrophobic core in a micelle-like assembly.26,42 Figure 6 shows CLSM images for the PalGH, PalG2H and PalG3H hydrogels containing ANS. For all hydrogels, fibrils were observed over the entire region. However, the morphology of the network was not the same among them. The number density of fibrils was higher in the order of PalG2H, PalG3H and PalGH. As observed by AFM, the width of fibrils was smaller in that order. Taking into account that the gelator concentrations, thereby the number densities of the gelator molecules in the gels are all the same for PalGnH, it seems reasonable that the number density of fibrils increased with decreasing fibril width.

Although the quantitative measurements for the pore size and its distribution was

difficult by CLSM observation, or even impossible

22,23

, it seems reasonable to assume that the

pore size of the network is smaller in the order of PalG2H, PalG3H and PalGH. This order is in excellent agreement with the results of the particle tracking measurements.

Figure 6 CLSM images showing fibrous aggregates in hydrogels composed of (a) PalGH, (b) PalG2H and (c) PalG3H. The concentration of ANS, which was used as a probe for CLSM, was 60 µM.


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CONCLUSIONS We examined the diffusion behavior of probe particles embedded in supramolecular hydrogels. The gels used in this study were prepared from aqueous dispersions of gelator molecules having a different number of peptide residues, PalGH, PalG2H and PalG3H, which could self-assemble into fibrous aggregates. The particle tracking experiments with varying particle size revealed that the gels were spatially heterogeneous in terms of network pore size. Notably, it was on the (sub)micrometer scale and was smaller in the order of the PalG2H, PalG3H and PalGH gels. These findings were consistent with the network pore size estimated by the CLSM observation.

The present results illustrate that the supramolecular network was

heterogeneous even though the formation did not involve any covalent cross-linking.

The

knowledge here obtained should be useful for understanding and controlling the mechanical properties of SMGs, thereby leading to the furtherance of their design and functionalization.


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ASSOCIATED CONTENT Supporting Information. See the Supporting Information for characterization data on molecular assembled states and rheological properties.

This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *

Email: [email protected] (A. S.) and

*

Email: [email protected] (K. T.)

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This research was partly supported by JSPS KAKENHI for Young Scientists (A) (no. 15H05496) (A.S.), and for Scientific Research (A) (no. 15H02183) (K.T.). We are also grateful for support from CSTI Impulsing Paradigm Change through Disruptive Technologies (ImPACT) Program (K.T.). The synchrotron radiation facilities experiments were performed at BL40B2 in the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal: 2014A1613).


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TABLE OF CONTENTS


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Figure 1 162x65mm (300 x 300 DPI)


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Figure 2 175x99mm (300 x 300 DPI)


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Figure 3 230x170mm (300 x 300 DPI)


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Figure 4 194x170mm (300 x 300 DPI)


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Figure 5 124x98mm (300 x 300 DPI)


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Figure 6 206x75mm (300 x 300 DPI)


Mesoscopic Heterogeneity in Pore Size of Supramolecular Networks

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