Article pubs.acs.org/JPCB
Nanoscopic Structural Investigation of Physically Cross-Linked Nanogels Formed from Self-Associating Polymers Yurina Sekine,*,† Hitoshi Endo,‡,§ Hiroki Iwase,∥ Shigeo Takeda,⊥ Sada-atsu Mukai,⊥,# Hiroshi Fukazawa,† Kenneth C. Littrell,∇ Yoshihiro Sasaki,⊥,# and Kazunari Akiyoshi⊥,# †
Materials Sciences Research Center, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195, Japan Neutron Science Laboratory, High Energy Accelerator Research Organization, 203-1 Shirakata, Tokai, Ibaraki 319-1106, Japan § Department of Materials Structure Science, The Graduate University for Advanced Studies (SOKENDAI), 203-1 Shirakata, Tokai, Ibaraki 319-1106, Japan ∥ Comprehensive Research Organization for Science and Society, 162-1 Shirakata, Tokai, Ibaraki 319-1106, Japan ⊥ Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan # ERATO Bio-Nanotransporter Project, Japan Science and Technology Agency (JST), Kyoto University, Kastura, Nishikyo-ku, Kyoto 615-8510, Japan ∇ Neutron Science Directorate, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6393, United States ‡
S Supporting Information *
ABSTRACT: The detailed structure of a nanogel formed by self-association of cholesterol-bearing pullulans (CHPs) was determined by contrast variation small-angle neutron scattering. The decomposition of scattering intensities into partial scattering functions of each CHP nanogel component, i.e., pullulan, cholesterol, and the cross-term between the pullulan and the cholesterol, allows us to investigate the internal structure of the nanogel. The effective spherical radius of the skeleton formed by pullulan chains was found to be 8.1 ± 0.3 nm. In the CHP nanogel, there are about 19 cross-linking points where a cross-linking point is formed by aggregation of trimer cholesterol molecules, and the spatially inhomogeneous distribution of the cross-linking points in the nanogel can be represented by the mass fractal dimension of 2.6. The average radius of gyration of the partial chains can also be determined to be 1.7 ± 0.1 nm by analyzing the extracted cross-correlation between the cross-linker and the tethered polymer chain quantitatively, and the size agrees with the value assuming random distribution of the cross-linkers on the chains. As the result, the complex structure of the nanogels is coherently revealed at the nanoscopic level.
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delivery.9,18,19 Although the spatial distribution of cross-linking points in the nanogels would be highly related to their properties, no clear nanoscopic information has been obtained so far due to the complex structure with the multicomponents. In this paper, the detailed structural analyses with small-angle neutron scattering (SANS) of physically cross-linked nanogels are reported. We investigated a size-controlled spherical cholesterol-bearing pullulan (CHP) nanogel (Figure 1), in which the main driving force for the self-association is the hydrophobic interaction of the cholesterol molecules, originally developed by Akiyoshi et al.20 CHP nanogels are known to possess several superior properties, for instance, a high loading capacity for proteins and assistance with the refolding of
INTRODUCTION
Hydrogels are capable of absorbing large amounts of water in their three-dimensional polymeric networks.1 Because of their high water absorptivity and good biocompatibility, they have been widely applied in biomedical engineering and biotechnology.2−5 To optimize their physical and chemical properties for the applications, structural regulations at the nanoscopic level are essential.6−8 Recently, nanometer-sized particulate hydrogels (termed nanogels) which have characteristic properties of both hydrogels and nanoparticles are attracting growing interest particularly for use in drug delivery systems, tissue engineering, and diagnostics.9−13 Owing to their unique internal space, nanogels are able to trap various bioactive compounds such as proteins and nucleic acids stably.14−17 Furthermore, nanoscaled dimensions of nanogels contribute rapid stimulus response, which is attractive for application in triggered drug © XXXX American Chemical Society
Received: July 6, 2016 Revised: October 19, 2016
A
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
I(Q ) = Δρp2 Spp(Q ) + 2Δρp Δρc Scp(Q ) + Δρc 2 Scc(Q ) (1)
where Spp(Q) and Scc(Q) are the self-terms representing the self-correlations of the pullulan chains and the cholesterol molecules, respectively, and Scp(Q) is the cross-term reflecting the cross-correlation between the cholesterol molecules and the pullulan chains in the CHP nanogel. Δρi (with i = p and c for pullulan and cholesterol, respectively) represents the scattering contrast between the component i and the solvent, and Q is the momentum transfer defined as Q = 4π sin(θ/2)/λ with scattering angle θ and neutron wavelength λ. Note that the dimension of the partial scattering function is length cubed. In order to extract the partial scattering functions, the scattering length density of the solvent was varied by mixing deuterated and protonated water. In the case that the scattering intensities are obtained with m different contrasts, the observed scattering curves for each Q value yield a set of linear equations expressed as (2)
I = M·s
where I and s represent vectors of intensities and the partial scattering functions, respectively, i.e., ⎛ I1(Q ) ⎞ ⎜ ⎟ I = ⎜ ⋮ ⎟, ⎜ ⎟ ⎝ Im(Q )⎠
Figure 1. (a) Schematic of cholesterol-bearing pullulan (CHP) nanogel simulated by a simple random-flight chain model. (b) Chemical structure of CHP. Degree of substitution of cholesterol was estimated to be 2.1 per 100 anhydroglucoside units of pullulan.
⎛ Spp(Q )⎞ ⎟ ⎜ s = ⎜ Scp(Q ) ⎟ ⎟ ⎜ ⎟ ⎜ S ( Q ) ⎠ ⎝ cc
(3)
and a m × 3 matrix M consisting scattering contrasts, that is, ⎛ 1Δρ 2 21Δρ 1Δρ c p ⎜ p ⎜ ⋮ ⋮ M=⎜ ⋮ ⎜ ⋮ ⎜⎜ m 2 m m ⎝ Δρp 2 Δρc Δρp
14
denatured proteins (termed chaperone activity). Fluorescence quenching method has been previously used to characterize the hydrophobic interaction in the CHP nanogels, probing the aggregation behavior of the cross-linkers but without any spatial information.20 Simple insight into the size and shape of the particles was later provided by conventional SANS experiments.21 Here we quantitatively reveal the detailed spatial structure of the cross-linking points in the CHP nanogels by contrast variation (CV) SANS. The contrast variation technique can be easily applied with neutrons by exploiting the difference in nuclear scattering arising from hydrogen/ deuterium replacement in the system.22−27 As described below in detail, our sophisticated application of CV-SANS enabled us to separate individual scattering signals from each component, that is, the polymer networks, the cross-linkers, and the crosscorrelation between the polymer networks and the cross-linkers in the CHP nanogel. We further determine the spatial distribution of the cross-linking points in the particle on the nanometer scale by both theoretical consideration and coherent fitting analyses. The length scale of the polymer chain lying between two cross-linking points (termed partial chains) is also evaluated by analyzing the extracted cross-correlation between the networks and the cross-linkers, something that is usually not possible to extract experimentally. As a result, the detail of the structure of CHP nanogel is revealed for the first time.
Δρc 2 ⎞ ⎟ ⎟ ⋮ ⎟ ⋮ ⎟ ⎟ m 2 Δρc (Q )⎟⎠ 1
(4)
with the scattering contrasts of the jth contrast, Δρi. On the basis of eqs 1 and 2, the intensities can be decomposed into the partial scattering functions by calculating the orthogonal matrix MT, where MT·M = E, i.e., s = MT·I. The detailed procedure is described elsewhere.28 Pullulan Scattering Function. The distribution of pullulan in the CHP nanogel is assumed to be spherically symmetric and almost homogeneous on the basis of the observation by atomic force microscopy13 and transmission electron microscopy.29 In this condition, the corresponding scattering intensity may be proportional to the scattering amplitude of a sphere with the radius Rp, which is given by j
Φ(QR p) =
3[sin(QR p) − QR p cos(QR p)] 4 πR p 3 3 (QR p)3
(5)
To allow for size polydispersity, a Gaussian distribution of Rp, with the standard deviation σR, is also taken into account. Then the form factor becomes
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SCATTERING THEORY Partial Scattering Functions and Contrast Variation. Since a CHP nanogel consists of pullulan, cholesterol, and water, the scattering intensity from the CHP nanogel can be described using partial scattering functions Sij(Q) as follows:
Pp(Q ) =
1 2π σR
⎧ (x − R p)2 ⎫ ⎪ ⎪ ⎬ dx Φ2(Qx) exp⎨ − 2 ⎪ ⎪ −∞ 2σR ⎩ ⎭
∫
∞
(6)
To allow for the interaction between pullulan chains, an appropriate structure factor should also be included. We B
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B applied the Percus−Yevick (PY) model30−33 by assuming that the excluded volume effect is dominant; that is, the effect is determined by the total volume fraction and the individual volume of the particles. This assumption may be valid in the case of low concentration of the particles (refer to Figure S1 in the Supporting Information). The details of the structure factor based on the PY model, SPY(Q), are given elsewhere.33 Therefore, the partial scattering function of pullulan, Spp(Q), can be described as Spp(Q ) = n p[Pp(Q ) + A p2 (Q ){SPY(Q ) − 1}]
1 Np
gcp(Q ) =
=
∫0
Np
⎛ b2 Q 2 ⎞ exp⎜ − x ⎟ dx 6 ⎠ ⎝
⎛ b2Q 2Np ⎞ 1 − exp⎜ − 6 ⎟ ⎝ ⎠ b2Q 2Np/6
(13)
with the polymerization degree of the partial chain, Np, and the bond length, b, by assuming the concept of ideal chain. Figure 2 shows a schematic illustration of Np and b in a polymer chain.
(7)
where A p (Q ) =
1 2π σR
⎧ (x − R p)2 ⎫ ⎪ ⎪ ⎬ dx Φ(Qx) exp⎨ − 2 ⎪ ⎪ −∞ 2σR ⎩ ⎭
∫
∞
(8)
with the number density np of the nanogel particle. Cholesterol Scattering Function. The partial scattering function of cholesterol directly reflects the distribution and the individual volume of the cross-linking points in the particle. As described in detail in Results and Discussion, the partial scattering function of cholesterol, Scc(Q), slightly increases at low Q, suggesting nonuniform distribution of the cholesterol cross-linkers. Therefore, the spatial configuration of the crosslinking points in the particle might be as a mass fractal with the fractal dimension, df, and the upper-cutoff length, ξ. The corresponding structure factor, Sfractal(Q), is given by Teixeira34 as follows:
Figure 2. Schematic illustration of a partial chain. Np represents a Npth segment, and b is the bond length.
The distribution of Np is further considered, that is, gcp(Q ) =
Sfractal(Q ) = 1 + df Γ(df − 1) 1 sin[(df − 1) tan−1(Qξ)] (QR c)d f [1 + 1/(Q 2ξ 2)](d f − 1)/2
Npvp
(9)
where Rc is the characteristic length of (assumed to be spherical) cholesterol aggregates. Then, Scc(Q) is described as Scc(Q ) = ncPc(Q ) Sfractal(Q ) SPY(Q )
Pc(Q ) = Vc exp( −R c Q )
∫0
∞
⎛ x2 ⎞ x 2 exp⎜ − ⎟ dx = ⎝ 2R c ⎠
2
2
b Q x /6
) dx =
2R g 2Q 2
(14)
2
(15)
with the number of the partial chains in the single particle, nchain. The other parameters and functions are the same as what are defined by Scc(Q) and Spp(Q).
(11)
8π 3 R c 3
x
b2Q 2x 6
Scp(Q ) = ncVcnchain gcp (Q ) SPY(Q ) Sc(Q ) exp( −R c 2Q 2/2)
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In eq 11, Rc corresponds the standard deviation of the normal distribution. In this case, the volume of the cross-linker, Vc, is given by Vc = 4π
(
1 − exp −
where the Rg = Npb /6 is the average radius of gyration of the partial chain, and vp is the volume of the one segmental unit. Finally, the pullulan-cholesterol cross-correlation function, Scp(Q), is defined as
(10)
2
2Np
∫0
2Np
exp( − 2R g 2Q 2) − 1 + 2R g 2Q 2 2
Here, nc is the number density of the cross-linker. Pc(Q) reflects the density profile of the single aggregate of cholesterols. By assuming normal distribution of the density profile, Pc(Q) is given by the Fourier transformation of a Gaussian, that is, 2
vp
EXPERIMENTAL SECTION Preparation of CHP Nanogels. CHP was synthesized by a reaction between the hydroxyl groups of pullulan (Hayashibara Biochemical Laboratory, Inc., Japan) and the isocyanate groups of cholesteryl N-6(-isocyanatohexyl)carbamate in dehydrated dimethyl sulfoxide as reported previously.20 The average molecular weight of pullulan is 5.5 × 104 g mol−1, which was determined by size exclusion chromatography.20 The polydispersity which is the ratio of the weight-average molecular weight (Mw) and the number-average molecular weight (Mn), Mw/Mn, of cholesterol bearing pullulan chains is about 1.17.35 The degree of substitution (DS) of the cholesterol was determined by 1H nuclear magnetic resonance spectroscopy to be 2.1 per 100 anhydroglucoside units of pullulan (refer to Figure S3 in Supporting Information). The scattering signal from the cholesterol and the scattering contrast between
(12)
Vc is related to the aggregation number of the cholesterol molecules. The interparticle interaction of the CHP particles can be included by SPY(Q) because the center of gravity for the CHP particle agrees with those of pullulan and cholesterol (refer to Figure S2 in Supporting Information). Pullulan−Cholesterol Cross-Correlation. The crosscorrelation between pullulan and cholesterol represents the pair-correlation between the cross-linker and the tethered partial chains; therefore, the cross-correlation function is given by C
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B pullulan and cholesterol in the CHP increase as the value of DS increases. Therefore, the CHP with DS of 2.1 was synthesized in this experiment. CHP powder was suspended in pure H2O and D2O and stirred for 24 h at 40 °C. An optically clear suspension was obtained by sonication using a sonicator (W-113MKII, Honda Electronics Co. Ltd., Japan) for 60 min. The suspension was purified by dialysis and filtration (0.1 mm PVDF filter, MillexGV, Millipore Co., USA). The CHP nanogel samples for SANS experiments, with a deuterium oxide (D2O) volume fraction, ϕD2O = 1.0, 0.9, 0.8, 0.7 and 0, were prepared by mixing the dispersions. The hydrodynamic radii of the synthesized CHP nanogels in both H2O and D2O were measured by dynamic light scattering (DLS) using a Malvern Zetasizer (Marvern Instruments Ltd., Worcestershire, U.K.), and the identical value (10 nm) was evaluated (refer to Figure S4 in Supporting Information). The weight and volume fractions of the CHP nanogels in the solution were 3.0% and 1.9%. The CHP nanogel particles interact with each other and form a macrogel at high concentration;36 thus the CHP nanogel dispersion at this concentration is very close to the particle aggregation concentration. The values of weight and volume fractions of each component in the CHP nanogel solution including water, pullulan, and cholesterol were summarized in Table 1.
Figure 3. Scattering length density of pullulan (red solid line), cholesterol (green broken line), and water (blue dotted line) as a function of D2O volume fraction in water, ϕD2O. The experimental conditions are marked by black filled circles on the dotted line.
used mass density is 1.59 g cm−3 for the pullulan by assuming the value is same as that of sucrose,37 1.05 g cm−3 for the cholesterol,38 1.00 g cm−3 for the protonated water, and 1.11 g cm−3 for the deuterated water, respectively. For the pullulan, the scattering length density was calculated on the basis of the presence of 10 hydrated water molecules39−41 and the exchanging at three hydroxyl groups per anhydroglucoside unit. The used density of the hydrated water is 1.20 g cm−3.42 The data were analyzed with a homemade program developed by using the IGOR pro software (Wavemetrics, Inc. USA).
Table 1. Weight and Corresponding Calculated Volume Fraction of Each Component of the CHP Nanogel Solution water w/w %a free water v/v %b
95.7
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CHP nanogels
97 hydrated water
pullulan
cholesterol
2.4
1.8
0.1
RESULTS AND DISCUSSION Figure 4 shows the scattering intensities of the CHP nanogels dispersed in water with ϕD2O = 1.0, 0.9, 0.8, 0.7, and 0. The
3.0
a
The weight fraction was measured. bThe volume fraction was calculated on the basis of their chemical structures, mass densities, and the number of hydrated water molecules per anhydroglucoside unit.37−42 The values used are described in SANS Experiments.
SANS Experiments. SANS experiments were performed using a time-of-flight diffractometer, TAIKAN43 at J-PARC, Japan, in the Q range of 0.078−19 nm−1 at room temperature with neutron wavelengths between 0.07 and 0.76 nm. All data were normalized to an absolute intensity by the coherent scattering of a glassy carbon43 as a standard sample after the necessary data corrections such as air scattering, cell scattering, and incoherent scattering subtraction. The scattering intensities from the sample cell and the solvent were subtracted by using measured transmission and the volume fraction of the solvent. In addition, the incoherent scattering intensities from the solute were also subtracted as constant values. Because the scattering intensities at low Q (Q < 0.13 nm−1) and high Q (Q > 2.0 nm−1) were still affected by these excess scattering, we used intensity data in Q range from 0.13 nm−1 to 2.0 nm−1 for the analyses in this study. For CV-SANS, we measured a series of CHP nanogel samples dispersed in water with a deuterium oxide (D2O) volume fraction, ϕD2O = 1.0, 0.9, 0.8, 0.7, and 0. Figure 3 shows the variation of the scattering length densities of pullulan, cholesterol, and water as a function of ϕD2O, which were calculated on the basis of their chemical structures and mass densities. The scattering intensity at around matching point of cholesterol, i.e., ϕD2O = 0.15, is too weak to achieve sufficient statistics, so we measured the samples at ϕD2O = 1.0, 0.9, 0.8, 0.7, and 0 with enough intensity for CV-SANS analysis. The
Figure 4. Scattering intensities of CHP nanogels dispersed in solvents with a deuterium oxide (D2O) fraction, ϕD2O = 1.0 (open red circles), 0.9 (open blue circles), 0.8 (open green circles), 0.7 (open yellow circles), and 0 (open purple circles) with error bars in the momentum transfer range, Q, of 0.13−2.0 nm−1. Solid curves represent reconstructed intensities from partial scattering functions.
scattering intensities vary with D2O fraction in the water. The highest total scattering intensity is obtained at ϕD2O = 1.0. The intensities decrease with increase in H2O fraction and increase again at ϕD2O = 0. In addition, the shape of the scattering intensities of the CHP nanogels at ϕD2O = 1.0, 0.7, and 0 changes significantly. This is characteristic of multicomponent systems because the contrast matching points for pullulan and cholesterol against water are different. D
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
given as a function of the radius, which is one of the great advantages of the absolute intensity of SANS experiments. A few negative values are observed for Spp(Q) at Q > 0.6 nm−1 due to bad statistics of the data. We verified the effect and confirmed that the determined parameters are hardly affected (refer to Figure S5 in Supporting Information). Next, we analyzed the partial scattering function of cholesterol, Scc(Q). The amplitude of Scc(Q) reflects the distribution of cholesterol molecules in the CHP nanogel. Scc(Q) shows a power-law decay at low Q and a steeper decrease at high Q. Because the analysis of Spp(Q) confirms that the particles are dissolved in solution without particle aggregation, the power-law decay at low Q originates from the spatial distribution of the cholesterol molecules in the particles. On the basis of the facts that the particle size determined by Spp(Q) is roughly 8 nm in radius and the number of the cholesterol molecules in the solo particle is very limited, the origin of the power law can be concluded to arise from the inhomogeneous spatial distribution of the crosslinkers consisting of the cholesterol molecules. The intensity at high Q reflects the shape and size of the cross-linkers. In this case, a mass fractal distribution with correlation length, ξ, is assumed to apply to reproduce the scattering intensity, where ξ is related to the upper cutoff length of the structure (eq 9).34 Note that the mass fractal distributions are not always related to the self-similar structures such as the diffusion limited aggregate45 and the surface fractal structures,46 so the powerlaw decay over wide Q range is not essential. The red solid line in Figure 5 shows the fitting result for Scc(Q). From the analysis, a mean radius of the cross-linkers, Rc, and fractal dimension, df, of the cholesterol molecules were obtained as Rc = 0.5 ± 0.1 nm and df = 2.6 ± 0.1 with ξ = 3.7 ± 0.1 nm. Because the volume of the single cross-linking point can also be evaluated by Rc, the aggregation number of cholesterol molecules in a cross-linking point was calculated to be 3.4 from the van der Waals volume of a single cholesterol molecule in water equating to 0.61 nm3. In this study, we used the cholesterol-modified pullulan with molecular weight of 5.5 × 104 g mol−1 which was substituted by 2.1 cholesterols per 100 anhydroglucoside units. Because 10 pullulan chains are aggregated in a CHP nanogel,20 the number of cholesterols in a CHP nanogel is estimated to be approximately 65. Approximately 19 cross-linking points thus are formed from an average of 3.4 cholesterols in a CHP nanogel. The upper-cutoff correlation length of the cholesterol distribution in the CHP nanogel, ξ = 3.7 ± 0.1 nm, is significantly smaller than the nanogel radius, Rp = 8.1 ± 0.3 nm, which suggests that most cholesterols are trapped deep inside the nanogel. It is emphasized that ξ is the characteristic length of exponential decay, so a certain number of the cross-linkers may exist in the area beyond ξ. The value of the fractal dimension of the spatial distribution of cholesterol aggregations in the CHP nanogel (df = 2.6) is a characteristic of an internal structure between a isotropical sphere (df = 3.0) and loosely aggregated macromolecules in aqueous solution (df = 1.7−2.0).47−49 A df = 2.6 is typically observed by weakly segregated 3D networks,50 so this result suggests attractive interaction between the cross-linkers. Akiyoshi et al.20 proposed how the cholesterol groups aggregate and act as a physical cross-linking point in the CHP nanogel, and this is well-supported by our results here. This unique structural characteristic of the CHP nanogel is likely what contributes to the specific interaction between bioactive
To analyze the structure of each component in the CHP nanogel, the scattering intensities should be decomposed into each partial scattering function according to eq 1 and the related description. Figure 5 shows the partial scattering
Figure 5. Partial scattering functions of pullulan, Spp(Q) (open blue circles), cholesterol, Scc(Q) (open red squares), and pullulancholesterol cross-term, Scp(Q) (open yellow triangles) with error bars. Blue, red, and yellow solid lines represent the fitting results for Spp(Q), Scc(Q), and Scp(Q), respectively.
functions of pullulan, Spp(Q), cholesterol, Scc(Q), and the pullulan−cholesterol cross-term, Scp(Q), which reflect the spatial correlations of each component and allow us to elucidate the fine structural information. Spp(Q) is larger than Scc(Q) for low Q (0.13 nm−1 ≤ Q ≤ 0.42 nm−1) because the volume fraction of the pullulan is larger than that of the cholesterol in the CHP nanogel. The cross-term Scp(Q) is positive and its magnitude lies between that of Spp(Q) and Scc(Q) at low Q. If there is no correlation or repulsion between the pullulan and the cholesterol, the intensity of Scp(Q) is null or negative, respectively. Therefore, a positive Scp(Q) confirms a noticeable spatial cross-correlation between the pullulan and the cholesterol. This evidence experimentally confirms the existence of cholesterol-bearing pullulan network-structure in the CHP nanogel. The scattering intensities of the CHP nanogel were fairly well reconstructed based on eq 1 (solid lines in Figure 4), which indicate that the partial scattering functions are obtained with sufficient accuracy. The reconstructed intensity with ϕD2O = 0.7 does not reproduce the experimental values very well at low Q. This occurs due to the lowest intensity compared to the others (more than 1 order of magnitude lower than the most intense one). We will further discuss the partial scattering functions in detail below. At first, the partial scattering function of pullulan, Spp(Q) was evaluated by the sphere model (eq 7) with PY structure factor representing the simple hard-core excluded volume effect. This simple model shown by the blue solid curve in Figure 5 can reproduce Spp(Q) with two tuning parameters, that is, the radius of the pullulan chain aggregate, Rp, found to be 8.1 ± 0.3 nm and the standard distribution of Rp, σR, which is 1.1 ± 0.1 nm. The slightly larger hydrodynamic radius, Rh was measured by dynamic light scattering, which is 10 nm as described in Experimental Section. Since Rh is assumed to be the radius of a hard sphere, the difference may be caused by the additional hydrodynamic effects such as permeability of the solvent, surface drag of CHP, etc., and such differences are typically observed polymeric aggregations.44 The result indicates that the pullulan chains forms a CHP nanogel skeleton and the nanogel particles can exist stably in water solution. Note that the fitting parameters were only the radius and its standard deviation, and the other values (the volume and the number density) were E
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B
without any contradictions. Finally, our coherent manner for the structural analyses with CV-SANS leads us to determine the fine structure of the CHP nanogel consistently.
compounds such as drugs and proteins and nanogels at the nanometer scale. The cross-correlation between the cross-linking points and the partial chains, Scp(Q), was evaluated with eq 15. Since the parameters obtained by Scc(Q) and Spp(Q) can be fixed, the fitting parameters were two, namely, the average radius of gyration of the partial chains, Rg, and the forward scattering intensity, I0, which were obtained as Rg = 1.7 ± 0.1 nm and I0 = 38 ± 0.03 nm3. As shown in Figure 5, the fitting curve can reproduce Scp(Q) fairly well. The converted value for Rg based on a former SANS result21 and the molecular weight ratio is 1.8 nm; therefore the obtained Rg is very reasonable. Moreover, the forward scattering intensity, I0, can be related to the average number of bond segments in the single partial chain, which is approximately 17. Note that I0 is the product of nc, Vc, nchain, Np, and vp (see eq 15). This means that about three monomers compose one segment with the bond length 1 nm. These are convincing evidence of the accuracy of the experiments and the applied theoretical models. The fitting curve can reproduce Scp(Q) adequately at low Q, while the curve deviates at high Q, which may be due to the neglect of the excluded volume effect of the chains in the theory. All the achieved fitting parameters are summarized in Table 2. Other parameters were determined by the other methods and fixed.
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S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b06795. Scattering profiles, NMR spectrum, size distribution data, and effect of background subtraction (PDF)
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*E-mail:
[email protected]. Phone: +81 292843871. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work (author Y.S.) was supported partially by JSPS KAKENHI Grant JP25790087. The neutron scattering experiment at J-PARC was approved by the Neutron Science Proposal Review Committee of J-PARC/MLF (Proposal 2013A0098).
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σR (nm)
8.1 ± 0.3
Spp(Q) Rc (nm) Scc(Q) Scp(Q)
df (nm)
0.5 ± 0.1
2.6 ± 0.1
3.7 ± 0.1 I0 (nm3)
1.7 ± 0.1
38 ± 0.03
REFERENCES
(1) Osada, Y.; Kajiwara, K.; Fushimi, T.; Irasa, O.; Hirokawa, Y.; Matsunaga, T.; Shimomura, T.; Wang, L.; Ishida, H. Gels Handbook; Elsevier Science & Technology: Amsterdam, 2000. (2) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2012, 64, 18−23. (3) Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science 2012, 336 (6085), 1124−1128. (4) Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Hydrogels in pharmaceutical formulations. Eur. J. Pharm. Biopharm. 2000, 50 (1), 27−46. (5) Haque, A.; Kurokawa, T.; Gong, J. P. Super tough double network hydrogels and their application as biomaterials. Polymer 2012, 53 (9), 1805−1822. (6) Tamura, M.; Ichinohe, S.; Tamura, A.; Ikeda, Y.; Nagasaki, Y. In vitro and in vivo characteristics of core-shell type nanogel particles: Optimization of core cross-linking density and surface poly (ethylene glycol) density in PEGylated nanogels. Acta Biomater. 2011, 7 (9), 3354−3361. (7) Seiffert, S. Impact of polymer network inhomogeneities on the volume phase transition of thermoresponsive microgels. Macromol. Rapid Commun. 2012, 33 (13), 1135−1142. (8) Sanson, N.; Rieger, J. Synthesis of nanogels/microgels by conventional and controleed rdical crosslinking copolymerization. Polym. Chem. 2010, 1 (7), 965−977. (9) Raemdonck, K.; Demeester, J.; De Smedt, S. Advanced nanogel engineering for drug delivery. Soft Matter 2009, 5 (4), 707−715. (10) Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; et al. Emerging applications of stimuli-responsive polymer materials. Nat. Mater. 2010, 9 (2), 101−113. (11) Soni, K. S.; Desale, S. S.; Bronich, T. K. Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation. J. Controlled Release 2016, 240 (28), 109−126. (12) Li, Y.; Maciel, D.; Rodrigues, J.; Shi, X.; Tomas, H. Biodegradable polymer nanogels for drug/nucleic acid delivery. Chem. Rev. 2015, 115 (16), 8564−8608. (13) Sasaki, Y.; Akiyoshi, K. Nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications. Chem. Rec. 2010, 10 (6), 366−376.
1.1 ± 0.1 ξ (nm)
Rg (nm)
AUTHOR INFORMATION
Corresponding Author
Table 2. Evaluated Fitting Parametersa Rp (nm)
ASSOCIATED CONTENT
a
Rp, the mean radius of the polymer chain aggregate forming a nanogel; σR the standard distribution of the Rp; Rc, the mean radius of a cross-linker; df and ξ, the fractal dimension and the correlation length of the cross-linkers in a nanogel; Rg the average radius of gyration of the partial chains; I0 the forward scattering intensity of Scp(Q).
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CONCLUSIONS We investigated the nanoscale structure of a CHP nanogel by CV-SANS, which allowed for the decomposition of scattering intensities into partial scattering functions of each CHP nanogel component, i.e., pullulan, cholesterol, and water. From an analysis of the partial scattering functions of pullulan, Spp(Q), the effective spherical radius of the skeleton formed by pullulan chains was found to be 8.1 ± 0.3 nm, which is slightly smaller than the hydrodynamic radius evaluated by dynamic light scattering. The partial scattering function of cholesterol, Scc(Q), provides information on the cross-linking points of the CHP nanogel. In the CHP nanogel, the cross-linking points consist of 3.4 cholesterol molecules dispersed inhomogeneously within the interior space of the nanogel, as represented by a mass fractal distribution with a dimension of 2.6. The unique internal space of the nanometer-scaled CHP nanogel may contribute to the specific functions of the nanogel such as chaperone-like activity. The cross-term between the polymer networks and the cross-linkers, Scp(Q), directly reflects the structure of the partial chains in the networks, and our theoretical consideration leads us to extract proper values F
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
saccharide nanogel-integrated system. Colloid Polym. Sci. 2014, 292 (2), 325−331. (37) Yeung, C. C.; Hersey, J. A. Ordered powder mixing of coarse and fine particulate systems. Powder Technol. 1979, 22 (1), 127−131. (38) Myers, R. L. The 100 Most Important Chemical Compounds: A Reference Guide; Greenwood Publishing Group: Westport, CT, U.S., 2007. (39) Paolantoni, M.; Sassi, P.; Morresi, A.; Santini, S. Hydrogen bond dynamics and water structure in glucose-water solutions by depolarized Rayleigh scattering and low-frequency Raman spectroscopy. J. Chem. Phys. 2007, 127 (2), 024504. (40) Lee, S. L.; Debenedetti, P. G.; Errington, J. R. A computational study of hydration, solution structure in dilute carbohydrate solutions. J. Chem. Phys. 2005, 122 (20), 204511. (41) Mason, P. E.; Neilson, G. W.; Enderby, J. E.; Saboungi, M. L.; Brady, J. W. Structure of aqueous glucose solutions as determined by neutron diffraction with isotope substitution experiments and molecular dynamics calculations. J. Phys. Chem. B 2005, 109 (27), 13104−13111. (42) Merzel, F.; Smith, J. C. Is the first hydration shell of lysozyme of higher density than bulk water? Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (8), 5378−5383. (43) Takata, S.; et al. The design and q resolution of the small and wide angle neutron scattering instrument (TAIKAN) in J-PARC. JPS Conf. Proc. 2015, 8, 036020. (44) Mortensen, K.; Brown, W.; Almdal, K.; Alami, E.; Jada, A. Structural properties of seld-assembled polymeric aggregates in aqueous solutions. Langmuir 1997, 13 (14), 3635−3645. (45) Witten, T. A.; Sander, L. M. Diffusion-limited aggregation, a kinetic critical phenomenon. Phys. Rev. Lett. 1981, 47 (19), 1400− 1403. (46) Bale, H. D.; Schmidt, P. W. Small-angle X-ray-scattering investigation of supermicroscopic porosity with fractal properties. Phys. Rev. Lett. 1984, 53 (6), 596−599. (47) Chen, S.; Teixeira, J. Structure and fractal dimension of proteindetergent complexes. Phys. Rev. Lett. 1986, 57 (20), 2583−2586. (48) Hanselmann, R.; Burchard, W.; Ehrat, M.; Widmer, H. Structural properties of fractionated starch polymers and their dependence on the dissolution process. Macromolecules 1996, 29 (9), 3277−3282. (49) Stauffer, D.; Aharony, A. Introduction to Percolation Theory, 2nd ed.; Taylor & Francis: Oxfordshire, U.K., 1994. (50) Freltoft, T.; Kjems, J. K.; Sinha, S. K. Power-law correlations and finite-size effects in silica particle aggregates studied by small-angle neutron scattering. Phys. Rev. B: Condens. Matter Mater. Phys. 1986, 33 (1), 269−275.
(14) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecular complexation between bovine serum albumin and the self-assembly hydrogel nanoparticle of hydrophobized polysaccharide. J. Am. Chem. Soc. 1996, 118 (95), 6110−6115. (15) Ayame, H.; Morimoto, N.; Akiyoshi, K. Self-assembled cationic nanogels for intracellular protein delivery. Bioconjugate Chem. 2008, 19 (4), 882−890. (16) Blackburn, W. H.; Dickerson, E. B.; Smith, M. H.; McDonald, J. F.; Lyon, L. A. Peptide-functionalized nanogels for targeted siRNA delivery. Bioconjugate Chem. 2009, 20 (5), 960−968. (17) Wu, C.; Bottcher, C.; Haag, R. Enzymatically crosslinked dendritic polyglycerol nanogels for encapsulation of catalytically active proteins. Soft Matter 2015, 11 (5), 972−980. (18) Zha, L.; Banik, B.; Alexis, F. Stimulus responsive nanogels for drug delivery. Soft Matter 2011, 7 (13), 5908−5916. (19) Morimoto, N.; Qiu, X. P.; Winnik, F. M.; Akiyoshi, K. Selfassembled pH-sensitive cholesteryl pullulan nanogel as a protein delivery vehicle. Macromolecules 2008, 41 (16), 5985−5987. (20) Akiyoshi, K.; Deguchi, S.; Tajima, H.; Nishikawa, T.; Sunamoto, J. Microscopic structure and thermoresponsiveness of a hydrogel nanoparticle by self-assembly of a hydrophobized polysaccharide. Macromolecules 1997, 30 (4), 857−861. (21) Inomoto, N.; Osaka, N.; Suzuki, T.; Hasegawa, U.; Ozawa, Y.; Endo, H.; Akiyoshi, K.; Shibayama, M. Interaction of nanogel with cyclodextrin or protein: Study by dynamic light scattering and smallangle neutron scattering. Polymer 2009, 50 (2), 541−546. (22) Higgins, J. S.; Benoit, H. C. Polymers and Neutron Scattering; Oxford University Press: Oxford, 1997. (23) Lindner, P., Zemb, T., Eds. Neutron, X-ray and Light Scattering: Introduction to an Investigate Tool for Colloidal and Polymeric Systems; Elsevier: Amsterdam, 1991. (24) Doe, C.; Jang, H.; Kline, S.; Choi, S. SANS investigation of selectively distributed single-walled carbon nanotubes in a polymeric lamellar phase. Macromolecules 2010, 43 (12), 5411−5416. (25) Wu, B.; et al. Spatial distribution of intra-molecular water and polymeric components in polyelectrolyte dendrimers revealed by small angle scattering investigations. J. Chem. Phys. 2011, 135 (14), 144903. (26) Endo, H.; Miyazaki, S.; Haraguchi, K.; Shibayama, M. Structure of nanocomposite hydrogel investigated by means of contrast variation small-angle neutron scattering. Macromolecules 2008, 41 (14), 5406− 5411. (27) Bouchoux, A.; Ventureira, J.; Gésan-Guiziou, G.; GarnierLambrouin, F.; Qu, P.; Pasquier, C.; Pezennec, S.; Schweins, R.; Cabane, B. Structural heterogeneity of milk casein micelles: a SANS contrast variation study. Soft Matter 2015, 11 (2), 389−399. (28) Endo, H. Study on multicomponent systems by means of contrast variation SANS. Phys. B 2006, 385−386 (1), 682−684. (29) Sekine, Y.; Moritani, Y.; Ikeda-Fukazawa, T.; Sasaki, Y.; Akiyoshi, K. A hybrid hydrogel biomaterial by nanogel engineering: bottom-up design with nanogel and liposome building blocks to develop a multidrug delivery system. Adv. Healthcare Mater. 2012, 1 (6), 722−728. (30) Percus, J.; Yevick, G. Analysis of classic statistical mechanics by means of collective coordinates. Phys. Rev. 1958, 110 (1), 1−13. (31) Thiele, E. Equation of state for hard sphere. J. Chem. Phys. 1963, 39 (2), 474−479. (32) Wertheim, M. Exact solution of the Percuss-Yevick integral equation for hard spheres. Phys. Rev. Lett. 1963, 10 (8), 321−323. (33) Kinning, D.; Thomas, E. Hard-sphere interactions between spherical domains in diblock copolymers. Macromolecules 1984, 17 (9), 1712−1718. (34) Teixeira, J. Small-angle scattering by fractal systems. J. Appl. Crystallogr. 1988, 21 (6), 781−785. (35) Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Self-aggregates of hydrophobized polysaccharides in water. Formation and characteristics of nanoparticles. Macromolecules 1993, 26 (12), 3062−3068. (36) Sekine, Y.; Okazaki, K.; Ikeda-Fukazawa, T.; Ichikawa, M.; Yoshikawa, K.; Mukai, S.; Akiyoshi, K. Microrheology of polyG
DOI: 10.1021/acs.jpcb.6b06795 J. Phys. Chem. B XXXX, XXX, XXX−XXX
