Modulating hierarchical self-assembly behavior of a peptide amphiphile/nonionic surfactant mixed system

Abstract

The self-assembly behavior of a nonionic surfactant (n-dodecyl tetraethylene monoether, C₁₂E₄) and a peptide amphiphile (PA, C₁₆-GK-3) mixed system was investigated using a combination of microscopic, scattering and spectroscopic techniques, including transmission electron microscopy (TEM), field emission-scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), polarized optical microscopy (POM) observations, small-angle X-ray scattering (SAXS), Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) and rheological measurements. The change of the contents of C₁₆-GK-3 and C₁₂E₄ induced the transitions in the nanostructures and simultaneously led to changes in macroscopic properties, i.e., mixtures of C₁₂E₄ with C₁₆-GK-3 can be hierarchically self-assembled into various helical nanofibers and then further assembled to dandelion-like and dendrite nanostructures by changing the content of C₁₆-GK-3 and C₁₂E₄, which resulted in transitions from solution to two phase, sol and hydrogel states that were noted on increasing the concentration of C₁₆-GK-3 at a fixed concentration of C₁₂E₄ or varying C₁₂E₄ concentration at a fixed concentration of C₁₆-GK-3. On the basis of a series of characterizations, we proposed a possible mechanism of the self-assembly, for which the hydrogen bonding interaction between the headgroups of C₁₆-GK-3 and between C₁₆-GK-3 and C₁₂E₄, as well as hydrophobic interaction between the alkyl chains of C₁₆-GK-3 and C₁₂E₄, were the main driving forces.

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Introduction

Under an environment of vigorous promotion of environmental protection and development of green chemistry, the study of peptide amphiphiles (PAs) has become a hot topic, and they have been attracting much attention.^1–4 PAs are a class of molecules that normally consist of a hydrophobic alkyl tail or lipid chain with the functions of a bioactive hydrophilic peptide sequence, which induces them to have amphiphilicity and promotes their self-assembly behavior.^5–8 Thus, PAs are known to assemble into a variety of nanostructures, such as micelles, vesicles, nanotubes, nanorods, nanobelts, nanoribbons, and nanofibers, in different solvents or under certain conditions such as pH, temperature and ionic strength.^9–15 The self-assembly process is controlled by many factors, including hydrophobic interactions, electrostatic interactions, inter or intramolecular hydrogen bonds, and van der Waals forces.^16–18 Because of their rich self-assembling behavior and biocompatibility, PAs have wide applications in the field of biomaterials and nanomedicine, including tissue engineering, 3D cell culture, regenerative medicine, bacteria inhibition and drug delivery.^19–25

In general, peptide amphiphiles have biologically relevant lipid chain lengths, in particular palmitoyl (hexadecyl, C₁₆).²⁶ For example, Hest et al. have investigated the influence of mono-alkyl chain length on the self-assembly of PAs containing an octapeptide derived from a protein of the malaria parasite P. falciparum. The results indicated that short chains (C₆–C₁₂) can only produce random coil structures, whereas β-sheet ordering structure was only observed for C₁₄ and C₁₆ derivatives and the C₁₆ variant showed the most extended thermal stability range for a β-sheet structure.²⁷ Hotta et al. have discussed the effects of the salt concentrations using sodium dihydrogen orthophosphate (NaH₂PO₄) on the sol–gel transition behaviors, especially the gelation speed and the gel characteristics, of the designed PA (C₁₆-W3K) hydrogels in an aqueous solution. Their results indicated that the solution exhibited higher gelation speeds and higher mechanical properties at higher salt concentrations, and concurrently, the density, length of wormlike micelles, and the conformational ratio of β-sheets to α-helices in the equilibrium C₁₆-W3K solutions all increased with the increase in the salt concentrations.²⁸ Moreover, the study of the interaction between traditional surfactants and PAs can significantly broaden the research field which can show various phase behaviors and particular self-assembly structures. For example, Hamley et al. have investigated the self-assembly of Pluronic copolymer P123 with the PA C₁₆-KTTKS. The results revealed that the β-sheet structure of C₁₆-KTTKS is retained even with 20 wt% added P123 and the nanotapes comprising peptide bilayers observed for C₁₆-KTTKS itself in an aqueous solution are progressively replaced by cylindrical fibrils upon addition of more P123.²⁹ They have also studied the interaction between the classical anionic surfactant sodium dodecyl sulfate (SDS) and C₁₆-KTTKS. The results indicated that the addition of SDS leads to a transition from tapes to fibrils via intermediate states that include twisted ribbons, and the addition of SDS is also shown to enhance the development of remarkable lateral “stripes” on the nanostructures, which is ascribed to counterion condensation.³⁰

In this article, the binding of a nonionic surfactant (n-dodecyl tetraethylene monoether, C₁₂E₄) to a PA, C₁₆-GK-3, containing a cationic peptide headgroup and its effects on the self-assembly behavior of C₁₂E₄/C₁₆-GK-3 mixed systems were investigated. Transmission electron microscopy (TEM), field emission-scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), polarized optical microscopy (POM) observations, small-angle X-ray scattering (SAXS), Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) and rheological measurements were used to characterize their physicochemical properties. Our aim is to examine the influence of the uncharged surfactant C₁₂E₄ on the aggregation of C₁₆-GK-3 in aqueous solutions to increase and enrich the examples of peptides/surfactants systems.

Experimental

Chemicals

Nonionic surfactant (C₁₂E₄) was purchased from Acros Organics (USA) and the purity was greater than 99%. C₁₆-GK-3 (98%) was purchased from GL Biochem (Shanghai) Ltd. and used without further purification. The C₁₆-GK-3 (pI = 8.94) molecule is a 3-amino acid chain composed of glycine, histidine, and lysine attached to a 16-carbon alkyl tail (Fig. 1). All the abovementioned reagents were used without further purification. Water used in the experiments was triply distilled using a quartz water purification system.

Fig. 1 Chemical structures of (a) C₁₆-GK-3 and (b) C₁₂E₄ molecules.

Methods and characterization

For transmission electron microscopy (TEM) observations, about 5 μL of solution was placed on a carbon-coated copper grid (400 mesh) and the excess solution was wicked away with a filter paper. The copper grids were freeze-dried and observed on a JEOL JEM-100 CXII (Japan) at an accelerating voltage of 80 kV with a Gatan Multiscan CCD for collecting images. FE-SEM observations were carried out on a JSM-6700F. For atomic force microscopy (AFM) observations, a drop of gel solution was placed on a silica wafer, which was freeze-dried at −60 °C for 12 hours and then observed using Dimension Icon (American) with Scan Asyst. A SCANASYST-AIR silicon nitride probe was employed.

The FT-IR spectrum was obtained on a VERTEX-70/70v spectrometer (Bruker Optics, Germany). Circular dichroism (CD) spectra were obtained using a JASCO J-810 spectropolarimeter, which was flushed with nitrogen during operation. Wavelength scans were recorded at 0.1 nm intervals from 300 to 180 nm. The hydrogels were determined using a 0.1 mm path length quartz cuvette; the micellar solution used a 1 mm path length quartz cuvette. Small-angle X-ray scattering (SAXS) observations were carried out on a HMBG-SAX X-ray small-angle scattering system (Austria) with a Ni-filtered Cu Kα radiation (0.154 nm) source operating at 50 kV and 40 mA. The distance between the sample and detector was 27.8 cm. Polarized microscopy observations were carried out on an AXIOSKOP 40/40 FL (ZEISS, Germany) microscope and a Nikon Eclipse E400 microscope equipped with a LINKAM THMS 600 heating/cooling stage.

The rheological measurements were carried out on a HAAKE RS75 rheometer with a cone-plate system (Ti, diameter, 35 mm; cone angle, 1°). For the shear-dependent behavior, the viscosity measurements were carried out at shear rates ranging from 0 to 1000 s⁻¹. In oscillatory measurements, an amplitude sweep at a fixed frequency of 1 Hz was performed prior to the following frequency sweep to ensure that the selected stress was in the linear viscoelastic region. The viscoelastic properties of the samples were determined by oscillatory measurements in the frequency range of 0.01–10 Hz. The samples were measured at 20.0 ± 0.1 °C with the help of a cyclic water bath.

Results and discussion

Fig. 2 shows the phase diagrams of the C₁₆-GK-3/C₁₂E₄ system at the fixed concentration of C₁₂E₄ (50 g L⁻¹) with increasing amount of C₁₆-GK-3 (Fig. 2A) and the images for five typical samples (Fig. 2B–F), respectively. It can be observed that the color of a 50 g L⁻¹ solution of C₁₂E₄ is translucent. On the addition of C₁₆-GK-3, the sample becomes a two-phase state (20–40 g L⁻¹ added C₁₆-GK-3, the upper phase is translucent and the bottom phase is transparent) and then transfer to homogeneous transparent solution, sol and hydrogel states, respectively. This points to a solubilizing effect of the added C₁₆-GK-3, which appears to break up the larger sized vesicles of C₁₂E₄.

Fig. 2 Phase transition with the addition of C₁₆-GK-3 to 50 g L⁻¹ C₁₂E₄ solutions (A) and sample photographs of typical samples: c_C12E4 = 50 g L⁻¹ and c_C16-GK-3 varies as (B) 0, (D) 20, (D) 30, (E) 50, and (F) 600 g L⁻¹. T = 20.0 ± 0.1 °C.

Moreover, Fig. 3 shows the phase diagrams of the C₁₆-GK-3/C₁₂E₄ system at the fixed concentration of C₁₆-GK-3 (50 g L⁻¹) with increasing the amount of C₁₂E₄ (Fig. 3A) and the images for five typical samples (Fig. 3B–F). It can be observed that the color of a 50 g L⁻¹ solution of C₁₆-GK-3 is transparent. Thus, through these experimental results, it can be speculated that the rich phase behavior of the C₁₆-GK-3/C₁₂E₄ mixed system can be adjusted by the composition of C₁₂E₄ and C₁₆-GK-3, and this is the result of the interaction between C₁₂E₄ and C₁₆-GK-3.

Fig. 3 Phase transition with the addition of C₁₂E₄ to 50 g L⁻¹ C₁₆-GK-3 solutions (A) and sample images of typical samples: c_C16-GK-3 = 50 g L⁻¹ and c_C12E4 varies as (B) 50, (C) 70, (D) 200, (E) 300, and (F) 400 g L⁻¹. T = 20.0 ± 0.1 °C.

Microstructures of the self-assembled aggregates

The microstructures of the self-assembled aggregates were investigated by TEM, FE-SEM and AFM observations. TEM images of the nanostructures formed with constant c_C12E4 (50 g L⁻¹) and varying c_C16-GK-3 are shown in Fig. 4. It was clearly observed that increasing additions of C₁₆-GK-3 to C₁₂E₄ solution induced a morphological transition in the self-assembled state, ultimately changing from vesicle (Fig. 4A) to globular dandelion-like nanostructures (Fig. 4B, c_C16-GK-3 = 100 g L⁻¹) and then to dendrite structures composed of micron-sized fibers (Fig. 4C and D, c_C16-GK-3 = 200 g L⁻¹ and 300 g L⁻¹).

Fig. 4 TEM images of the nanostructures formed with constant c_C12E4 (50 g L⁻¹) and varying c_C16-GK-3. (A) 0 g L⁻¹ C₁₆-GK-3; (B) 100 g L⁻¹ C₁₆-GK-3; (C) 200 g L⁻¹ C₁₆-GK-3; and (D) 300 g L⁻¹ C₁₆-GK-3.

The delicate structure of 50 g L⁻¹ C₁₆-GK-3/100 g L⁻¹ C₁₂E₄ was further characterized by SEM and AFM (Fig. 5). The dandelion-like morphology consists of left-handed helical nanotapes (Fig. 5C), and some nanotapes further coiled to form a larger-sized superhelix. The appearance of the assemblies shown in AFM images may be more compact than the TEM. It is speculated that the dandelion-like structures with a relative height of 1.35 μm compared to the peripheric surface could come in contact with the tip, leading the nanotapes at the surface somewhat destroyed and even separated from the assemblies; consequently, the remaining structures at the surface could not be supported and collapsed. The AFM images of the dendrite structures formed with c_C12E4 = 50 g L⁻¹ and c_C16-GK-3 = 200 g L⁻¹ are shown in Fig. 6. The assemblies are exclusively composed of helical nanotapes with left-handed bias (Fig. 5B). Furthermore, some of the nanohelices aggregated into bundles. The measured helical pitch is about 170 nm. From the abovementioned results, a hierarchically self-assembled process can be achieved in which helical nanotapes with left handed direction induced by the chirality of the peptide molecules were first observed, followed by the smaller-sized nanohelices aggregating into larger assemblies. The schematic of the formation of the C₁₆-GK-3/C₁₂E₄ nanostructure with the increase in concentration of C₁₆-GK-3 is shown in Scheme 1.

Fig. 5 SEM (A, B, C) and AFM (D, E, F) images of the dandelion-like nanostructures formed with c_C12E4 = 50 g L⁻¹ and c_C16-GK-3 = 100 g L⁻¹. (A–C): images were taken under different magnifications. Inset of (B) is a real dandelion image. (D) is the AFM height images; (E) is the AFM peak force images; and (F) is the three-dimensional (3D) morphology of the sample.

Fig. 6 AFM images of the dendrite nanostructures formed with c_C12E4 = 50 g L⁻¹ and c_C16-GK-3 = 200 g L⁻¹. (A) is the AFM height images; (B) is the AFM peak force images; and (C) is the three-dimensional (3D) morphology of the sample.

Scheme 1 The schematic of the formation of C₁₆-GK-3/C₁₂E₄ nanostructure with the increase in concentration of C₁₆-GK-3.

If the concentration of c_C16-GK-3 is fixed at 50 g L⁻¹ and the concentration of c_C12E4 is changed, the microstructures of the C₁₆-GK-3/C₁₂E₄ mixed system were also changed from vesicle to dendrite structures, as shown in Fig. 7. If we further increase the concentration of C₁₂E₄ to 300 g L⁻¹ and 400 g L⁻¹, the sample can form liquid crystals (LCs), and it is hard to use TEM to observe its morphology. Thus, POM was used to observe its polarization properties, and the results are shown in Fig. 8. It can be observed that for the pure 200 g L⁻¹ C₁₂E₄, it cannot form LCs and there is no polarized texture. However, when 50 g L⁻¹ C₁₆-GK-3 was added, it could form LCs and a polarized texture can be observed. For 300 g L⁻¹ and 400 g L⁻¹ C₁₂E₄, the systems are lamellar liquid crystals (LLCs), which showed Maltese cross textures. When 50 g L⁻¹ C₁₆-GK-3 was added, although the lamellar structures of the samples were kept, it changed to an oily texture, indicating the influence of C₁₆-GK-3 on the properties of C₁₂E₄ liquid crystals.

Fig. 7 TEM images of the nanostructures formed with constant c_C16-GK-3 (50 g L⁻¹) and varying c_C12E4: (A) 50 g L⁻¹ C₁₂E₄; (B) 100 g L⁻¹ C₁₂E₄; (C) 200 g L⁻¹ C₁₂E₄.

Fig. 8 POM images of C₁₂E₄ LLC matrix without C₁₆-GK-3: (A) 200 g L⁻¹ C₁₂E₄, (B) 300 g L⁻¹ C₁₂E₄, (C) 400 g L⁻¹ C₁₂E₄. POM image of C₁₂E₄ LLC matrix with C₁₆-GK-3: (a) 200 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, (b) 300 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, (c) 400 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3.

To further obtain the details about the phase behaviors of C₁₆-GK-3/C₁₂E₄ LLC composites, SAXS measurements were carried out. Fig. 9 shows the variation of SAXS results of 50 g L⁻¹ C₁₆-GK-3 as a function of the concentration of C₁₂E₄ (200–400 g L⁻¹). The SAXS results show two scattering peaks, indicative of the highly ordered supramolecular structures. All the ratios of the two peaks are 1 : 2, suggesting a typical lamellar structure of the supra-molecular structures. Moreover, the position of the peaks slightly moved to the left with the increase of C₁₂E₄. The d-spacing of the lamellar lattice was calculated by d = 2π/q₁ (q₁ was the value of the first peak). The calculated inter-planar distances (d) of the typical lamellar structures are 12.1, 9.1, and 6.6 nm for 200 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, 300 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, and 400 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, respectively. Comparing these values with the values of d-spacing for the pure 300 g L⁻¹ (d = 10.55 nm) and 400 g L⁻¹ (d = 8.32 nm) C₁₂E₄, it can be observed that the addition of C₁₆-GK-3 decreased the d-spacing of the lamellar lattice, which indicated that the molecular arrangement is more closely packed in C₁₆-GK-3/C₁₂E₄ LLC composites.

Fig. 9 The results of SAXS of (A) pure C₁₂E₄ LLC and (B) C₁₆-GK-3/C₁₂E₄ LLC composites. (C) The ball-and-stick models (obtained by Material Studio software) of C₁₂E₄ and C₁₆-GK-3 molecules. Color code for atoms: blue, nitrogen; red, oxygen; dark gray, carbon; light gray, hydrogen.

Rheological measurements can give macro properties of the system, and it is an important method to investigate LLC materials.^31–33 The results of the rheological properties of liquid crystal composites as a function of the concentration of C₁₂E₄ when c_C16-GK-3 = 50 g L⁻¹ are shown in Fig. 10. It can be observed that the increase of c_C12E4 greatly increased the range of the linear viscoelastic region of the LLC phase, and the shear modulus increased with the increase of c_C12E4. For example, the elastic modulus (G′) of the weak lamellar phase of 200 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3 was 4.7 Pa, and the value of G′ increased progressively from 120 Pa to 413 Pa when c_C12E4 increased from 30 g L⁻¹ to 40 g L⁻¹. For 200 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, G′ and the viscous modulus (G′′) are slightly dependent on oscillatory frequency with the same values of about 3.4 Pa at f = 5 Hz, indicating the weak elasticity. When c_C12E4 increases to 300 and 400 g L⁻¹, G′ and G′′ are nearly independent of oscillatory frequency but increase to 7.49 and 4.87 Pa for 300 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3 and 9.11 and 6.03 Pa for 400 g L⁻¹ C₁₂E₄/50 g L⁻¹ C₁₆-GK-3, respectively, exhibiting the obvious viscoelasticity and the dominant elastic property. The η* of both systems decreased with the increase of frequency, indicating a shear-thinning behavior (Fig. 10C). Moreover, the value of η* increased with the increase of c_C12E4, which indicated that the increase of c_C12E4 enhanced the mechanical properties of C₁₆-GK-3/C₁₂E₄ LLC composites.

Fig. 10 Rheological properties of C₁₆-GK-3/C₁₂E₄ LLC composites as a function of the concentration of C₁₂E₄ when c_C16-GK-3 = 50 g L⁻¹: (A) G′ and G′′ as a function of the applied stress (τ) at f = 1.0 Hz, (B) G′ and G′′ and (C) η* as a function of f.

To further characterize the features of the formed nanostructures at various concentrations of C₁₂E₄ and C₁₆-GK-3, the FTIR and CD spectra were measured, as shown in Fig. 11. From the IR spectra, it can be observed that for pure C₁₆-GK-3, no β-sheet secondary structure was noticed. The spectra of the C₁₂E₄/C₁₆-GK-3 mixtures, however, changed. The spectra displayed a strong peak at 1619 cm⁻¹, which is consistent with the presence of β-sheets, and a second one at 1679 cm⁻¹, which is characteristic of an antiparallel arrangement of the β-sheets.³⁴ An additional contribution at 1650 cm⁻¹ indicates that the peptide region also adopts random coil characteristic.³⁵ The wide peak (∼3400 cm⁻¹) is well-known for symmetric and antisymmetric O–H stretching modes, the stretching vibration of N–H locates at 3300 cm⁻¹ and the peaks at 2920 cm⁻¹ and 2852 cm⁻¹ are the asymmetric and symmetric stretching vibration of the CH₂ of C₁₂E₄ and C₁₆-GK-3, respectively. For CD analysis, it can be observed that the pure C₁₆-GK-3 solution lacks characteristic structure, whereas no CD signals can be observed in the micelle solution of C₁₂E₄. After the interaction between C₁₂E₄ and C₁₆-GK-3, the formation of helical dandelion-like or dendrite structures resulting in a remarkable CD signal (Fig. 11d), β-sheets and random coil structures were proved to coexist from the peaks at 215 and 200 nm confirming the helical structure and higher ordered molecular arrangement of C₁₂E₄/C₁₆-GK-3.³⁶ It was obvious that the peak for the β-sheet structure was greater than that of the random coil structure, which indicated that more β-sheet structures than random coil structures formed in the obtained nanostructures.

Fig. 11 (A) FT-IR spectra of samples: (a) C₁₆-GK-3, (b) 50 g L⁻¹ C₁₂E₄/100 g L⁻¹ C₁₆-GK-3, (c) 50 g L⁻¹ C₁₂E₄/200 g L⁻¹ C₁₆-GK-3. (B) CD spectra of (a) 100 g L⁻¹ C₁₆-GK-3, (b) 50 g L⁻¹ C₁₂E₄, (c) 50 g L⁻¹ C₁₂E₄/100 g L⁻¹ C₁₆-GK-3, (d) 50 g L⁻¹ C₁₂E₄/200 g L⁻¹ C₁₆-GK-3.

Mechanism of the interaction between C₁₆-GK-3 and C₁₂E₄

All the abovementioned results indicate that there is strong interaction between C₁₆-GK-3 and C₁₂E₄. Because C₁₆-GK-3 possesses both a hydrophobic tail and many hydrogen-bond sites, such as three amide groups and one carboxylic acid, the hydrogen bonding interaction between the amide groups of C₁₆-GK-3 and polyoxyethylene groups of C₁₂E₄ and hydrophobic interaction between the alkyl chains of C₁₆-GK-3 and C₁₂E₄ during self-assembly led to the formation of the bilayer structures, which serve as basic structural units for subsequent hierarchical self-assembly.³⁷ Due to the chiral nature of C₁₆-GK-3 molecules, the hydrogen bond between the headgroup of C₁₆-GK-3 and C₁₂E₄ leads the bilayer structure to roll into the helical fibers. It is suggested that a subtle balance among various hydrogen bonds played an important role in the construction of the nanostructures. Increasing additions of C₁₆-GK-3 to C₁₂E₄ solution induced the formation of the strong hydrogen bonds between the amide groups of C₁₆-GK-3 and stabilized the bilayer structures, which eventually formed dandelion-like and dendrite nanostructures composed of micron-sized helical fibers.

Conclusion

The aggregation behavior of the mixture of a peptide amphiphile (C₁₆-GK-3) and a nonionic surfactant (C₁₂E₄) in an aqueous solution was systematically investigated by TEM, HR-TEM, AFM, SAXS, FT-IR, CD and rheological measurements. By fixing the concentration of C₁₂E₄, while changing the concentration of C₁₆-GK-3 or fixing the concentration of C₁₆-GK-3 while increasing the concentration of C₁₂E₄, the transitions from solution to two phase, sol and hydrogel states were obtained. A hierarchical self-assembly process was observed in which left-handed nanofibers induced by the chirality of peptide molecules were first formed, and the resulting nanohelices aggregated into larger dandelion-like and dendrite nanostructures. The assembly process was driven by the hydrophobic interactions of the alkyl chains and hydrogen bonding interaction between the headgroups of C₁₆-GK-3 and between C₁₆-GK-3 and C₁₂E₄. Understanding of the self-assembled supramolecular structures not only offers insights into how rationally designed chiral nanostructures form from simple peptide building blocks but is also of significant practical value for the development of both biological and chemical studies.

Acknowledgements

We gratefully acknowledge financial support from the National Natural Science Foundation of China (21573130, 21173128, 21203109) and the Natural Science Foundation for Distinguished Young Scholars of Shandong Province (JQ201303).

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