Preparation of Poly(L,L-Lactide) Microparticles via Pickering Emulsions Using Chitin Nanocrystals

Demina, T. S.; Sotnikova, Yu. S.; Istomin, A. V.; Grandfils, Ch.; Akopova, T. A.; Zelenetskii, A. N.

doi:https://doi.org/10.1155/2018/8518016

Advances in Materials Science and Engineering

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Abstract Introduction Experimental Results Conclusions Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2018 | Article ID 8518016 | https://doi.org/10.1155/2018/8518016

Preparation of Poly(L,L-Lactide) Microparticles via Pickering Emulsions Using Chitin Nanocrystals

T. S. Demina,^1,2Yu. S. Sotnikova,^1,3A. V. Istomin,¹Ch. Grandfils,⁴T. A. Akopova,¹and A. N. Zelenetskii¹

Academic Editor: Renal Backov

Received30 Nov 2017

Accepted13 Mar 2018

Published08 Apr 2018

Abstract

The aim of the present study was to investigate an ability of chitin nanocrystals to be used as stabilizing components for fabrication of poly(L,L-lactide) microparticles via the Pickering oil/water emulsion solvent evaporation technique. The anisometric chitin nanocrystals were extracted from two different samples of crab shell chitin via acetic hydrolysis and analyzed using atomic force microscopy, dynamic light scattering, and FTIR spectroscopy. The extracted nanocrystals showed no difference in the chemical structure but possessed different morphology and aspect ratios as a function of raw chitin used. The effect of chitin nanocrystals characteristics and concentration in the aqueous phase on the total yield, size distribution, and shape and surface morphology of the prepared polylactide microparticles was evaluated.

1. Introduction

Polylactide micro- and nanoparticles are widely used as drug delivery systems and cell microcarriers [1, 2]. To control the cell attachment, biocompatibility, and drug kinetic release, the particles could be coated with functional polysaccharides during or after a preparation procedure [3–6]. As an alternative, the surface of the particles could be modified using especially synthesized amphiphilic copolymers as functional macromolecular emulsifiers [7]. Another approach to fabrication of core-shell microparticles is based on the application of solid nanoparticles as stabilizing components, that is, preparation via the Pickering emulsions.

The Pickering emulsions, that is, emulsions stabilized by solid nanoparticles instead of molecular emulsifiers, provide a number of benefits, such as high resistance to coalescence and low amount of nanoparticles needed to stabilize an interface [8–10]. The Pickering emulsions were successfully used for preparation of a so-called magnetic polymer microspheres consisted of polystyrene core and Fe₃O₄ nanoparticle shells [11]. A range of polymeric nanoparticles made of polymer brushes or polymer-grafted particles of various natures could be also used for the Pickering emulsions, which allows one to synthetize new nanocomposites dedicated to specific applications [12–18]. Usage of biocompatible nanoparticles, such as hydroxyapatite, SiO₂, proteins, lignin, flavonoids, and various polysaccharides, for the Pickering emulsion stabilization is especially interesting for food or biomedical applications [19–22]. Polysaccharides could be transformed into nanodimensional forms using controlled aggregation of macromolecules [23, 24] or extraction of nanocrystals via hydrolysis of amorphous regions of raw polysaccharides [25–27]. Nanocrystals extracted from cellulose, chitin, and starch are highly crystalline rigid nanoparticles, which morphology and size could significantly vary as a function of raw polysaccharide used [26]. The most widely used cellulose and chitin nanocrystals have a rod-like anisometric morphology with a length and diameter varied from several nanometers to micrometers. They are proposed as reinforcing fillers, templates for preparation of mesoporous materials, for fabrication of semiconductor and smart materials, including mechanically adaptive and self-healing ones. The use of polysaccharide nanocrystals as stabilizing agents in the Pickering emulsions was previously described mainly for food industry [28–31]. Efficiency of nanocrystals as stabilizing components for preparation of polymeric microparticles via the emulsion solvent evaporation technique was studied mostly on cellulose nanocrystals [32, 33].

This work was aimed at evaluating the effect of chitin source on characteristics of the obtained chitin nanocrystals and on a possibility to use them as stabilizing agents for fabrication of core-shell poly(L,L-lactide)/chitin microparticles via the Pickering emulsion solvent evaporation technique.

2. Experimental

2.1. Materials

Crab shell chitins were purchased from “Kombio” (Russia) and “Xiamen Fine Chemical” (China) and were used as raw materials for preparation of chitin nanocrystals, which were subsequently marked as ChN-K and ChN-X, respectively. The ash contents of raw chitins were 0.16 wt.% and 1.76 wt.% for “Kombio” and “Xiamen Fine Chemical,” respectively. The nanocrystals were obtained by acidic hydrolysis of the chitin samples according to the procedure described in [34, 35]. Poly(L,L-lactide) (PLLA) with an average molecular weight of 160 kDa was purchased from Sigma-Aldrich and used as a core material for the microparticle fabrication. Polyvinyl alcohol (PVA) marked as Mowiol 10–98 (Germany) was used as received.

2.2. Atomic Force Microscopy (AFM)

The size and morphology of the prepared ChN were evaluated using atomic force microscopy NtegraPrima (NT-MDT, Russia) in tapping mode. The samples for AFM analysis were prepared as follows: 0.1 wt.% aqueous dispersions were dropped on a cover glass and dried in a dust-free chamber [35]. The AFM images were analyzed using Image Analysis 2.0 (NT-MDT, Russia) software.

2.3. Dynamic Light Scattering

The mean size and size distribution of ChN in aqueous dispersions at concentrations of 0.1, 0.5 and 1 wt.% were evaluated by dynamic light scattering (DLS) using a Zetatrac particle size analyzer (Microtrac, Inc., USA) with the aid of Microtrac application software program (V.10.5.3).

2.4. FTIR

The chitin nanocrystals in a form of films cast from 1 wt.% ChN aqueous dispersions were characterized by FTIR using a PerkinElmer Spectrum 100 FTIR spectrometer (PerkinElmer Inc., Wellesley, MA) equipped with universal ATR accessory with 8 scans at 4 cm⁻¹ resolution.

2.5. Preparation of Poly(L,L-Lactide) Microparticles Stabilized with Chitin Nanocrystals

PLLA-ChN core-shell microparticles were prepared via Pickering emulsion solvent evaporation technique by mixing of 6 wt.% solutions of PLLA in CH₂Cl₂ : acetone (9 : 1 v/v) with 0.1–1 wt.% aqueous dispersions of ChN. The scheme of the microparticle fabrication is shown in Figure 1. Briefly, the oil phase, that is PLLA solution, was rapidly added to ChN dispersion to achieve oil/water phase ratio as 1/9 v/v. Microparticles stabilized with polyvinyl alcohol (PVA) solution (2.5 wt.%) as emulsifier in aqueous phase were prepared as model samples. The mixing of the phases was carried out using a four-blade stirrer at a rotation speed of 700 rpm for 3 hours. The emulsions were kept in a water bath through the mixing at 15°C during the first 15 min, and, afterwards, the temperature was increased up to 30°C. After the evaporation of the organic solvents, the obtained PLLA-ChN core-shell microparticles were collected, washed several times with deionized water and fractionated using sieves with apertures of 400, 315, 200, and 100 m, then, freeze-dried and weighted.

2.6. Scanning Electron Microscopy

The surface morphology of the fabricated microparticles was studied by scanning electron microscopy (SEM) using PhenomProX (PhenomWorld, Netherlands) at 10–15 kV.

3. Results and Discussions

3.1. Chitin Nanocrystals Characterization

According to AFM, both types of the chitin nanocrystals had anisometric morphology that was strongly dependent on the chitin source (Figure 2). As could be seen from the calculation of the length and diameter of the nanocrystals, both types of samples had a rather wide size distribution, which was more pronounced for ChN-K nanocrystals. The mean lengths of the nanocrystals were 181 ± 63 nm and 131 ± 94 nm; diameters of 65 ± 14 nm and 93 ± 42 nm for ChN-X and ChN-K, respectively. The calculation of the nanocrystals dimensions showed that the ChN-X had more anisometric morphology than ChN-K: aspect ratios were of 2.8 and 1.4, respectively. Thus, in spite of both raw chitins were from crab shells, the structure of the polysaccharide could significantly vary as a function of animal source (species, parts, etc.) as well as conditions and quality of demineralization and deproteinization stages [36]. An X-ray analysis showed that a crystallinity of the extracted nanocrystals was higher than that of corresponding raw polysaccharides, while the positions of characteristic peaks were unchanged [35].

(a)

(b)

(c)

(d)

(e)

(f)

FTIR spectra of the extracted ChN showed no significant differences between the samples and contained a full set of typical characteristic bands of chitin, such as C-O stretching vibrations of pyranose cycle at 1070 cm⁻¹, asymmetric bridge oxygen stretching at 1155 cm⁻¹, the vibration of a C=O group bonded to hydroxyl at 1619 cm⁻¹, CH bending and symmetric CH₃ deformations at 1376 cm⁻¹ as well as a set of amide bands: Amide I at 1654 cm⁻¹, Amide II at 1553 cm⁻¹ and Amide III at 1309 cm⁻¹ [37].

Since the fabricated ChN were intended to be used as stabilizing components for oil/water Pickering emulsions, their water dispersions were analyzed by DLS. As could be seen in Figure 3 the ChN-X dispersions were less sensitive to the concentration change, while ChN-K showed a wide-range size distribution at 1 wt.% concentration.

(a)

(b)

Thus, the extraction of ChN allowed to prepare nanodimensional forms of chitins having chemical structure of native polysaccharides and anisometric morphology, which was varied in a range of 1.4–2.8 as a function of chitin source.

3.2. Microparticle Fabrication

As could be seen in Figure 4, the ChN could effectively stabilize a simple CH₂Cl₂/water (1/9 v/v) emulsion. The size distribution of oil droplets depends on mixing parameters and effectiveness of oil/water interface stabilization, which is controlled by the ChN hydrophilic-hydrophobic balance and concentration in the aqueous phase.

(a)

(b)

Total yield, size distribution, and shape and surface morphology of the fabricated PLLA microparticles are additionally regulated by an arrangement of nanocrystals at the interface during the evaporation of the organic solvent and decreasing of the oil/water surface area. As could be seen in Figure 5, the total yield of the PLLA microparticles stabilized with ChN increased with increase in nanocrystals concentration in the aqueous phase and reached up to 58 wt.% in the case of 1 wt.% of ChN-X. This yield was higher than that obtained using PVA as the emulsifier in spite of lower amount of ChN in comparison with PVA concentration used: 1 and 2.5 wt.%, respectively.

The comparative stabilization effectiveness of 0.1 wt.% ChN-K was higher than that of 0.1 wt.% ChN-X, but the impact of nanocrystals concentration in the aqueous phase was more pronounced for ChN-X as compared with microparticles stabilized by ChN-K. Increase of the ChN-X content in the aqueous phase led to a clear increase in stabilization effectiveness, that is, increase of total yield and decrease of mean microparticle size, while this dependence was not so obvious in the case of ChN-K. This difference could be caused by the narrower size distribution of ChN-K nanocrystals and their better stability in the aqueous phase at the concentrations used (Figure 3).

SEM observations of all ChN-stabilized microparticles showed that they had more rough surface morphology than that of the ones stabilized using PVA (Figure 6). However, the shape of the fabricated PLLA-ChN microparticles was strongly varied as a function of origin and concentration of ChN. The microparticles stabilized with ChN-X and the ones stabilized with ChN-K at the lowest concentration (0.1 wt.%) had the spherical form, while the microparticles obtained using ChN-K at higher concentrations (0.5 and 1 wt.%) had an irregular shape.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

It could be assumed that the ChN particles were attached so firmly at the oil/water interface that their rearrangement during the solvent evaporation process was impaired. The excess of ChN entrapped at the interface was not so critical in the case of usage of low nanocrystals concentration (0.1 wt.%), and the microparticles were able to remain in the spherical form (Figures 6(b) and 6(c)), while a presence of significant excess of ChN at the higher concentration in the aqueous phase could lead to the development of “raisin” effect. The difference in the shape of ChN-K- and ChN-X-stabilized microparticles could be caused by different aspect ratios of the nanocrystals: 1.4 and 2.8, respectively. The ChN-X entrapped at the interface could turn its orientation to the interface from lengthwise to crosswise, thus, to decrease the covered surface area. From another point of view, the higher stability of ChN-X size at various concentrations (see DLS data) could be a reason of such behavior as well.

As a summary, chitin nanocrystals could effectively serve as a stabilizing component of oil/water emulsions during the preparation of polylactide-ChN core-shell microparticles, which total yield, size distribution, and shape were strongly depended on concentration, size, and size distribution of the nanocrystals used. These microparticles could be used as biodegradable cell microcarriers with improved cell affinity.

4. Conclusions

A promising approach to prepare poly(L,L-lactide) microparticles in a course of solvent evaporation from the Pickering oil/water emulsions stabilized with chitin nanocrystals has been discussed in terms of nanocrystals characteristics and processing parameters. Chitin nanocrystals extracted from two commercially available crab shell chitins showed different morphology and anisometric aspect ratios as well as an ability to be used as stabilizing agents in an aqueous phase during the preparation of polylactide microparticles via the solvent evaporation technique. The nanocrystals with higher aspect ratio allowed us to achieve a higher total yield of the fabricated spherical microparticles than that in a case of classical emulsifier application, that is, polyvinyl alcohol. The increase of nanocrystals concentration in an aqueous phase led to an increase in total yield and to decrease of mean microparticles size. The use of chitin nanocrystals with two fold lower aspect ratio at higher concentration in an aqueous phase caused the formation of microparticles with an irregular shape that could be explained by a lower ability of the nanocrystals to be rearranged at an interface during the solvent evaporation.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors are grateful to Dr. P. S. Timashev and his colleagues from the Institute of Photonic Technologies of “Crystallography and Photonics” of the Russian Academy of Sciences and Institute for Regenerative Medicine of Sechenov University for the access to the equipment.

References

B. Tyler, D. Gullotti, A. Mangraviti, T. Utsuki, and H. Brem, “Polylactic acid (PLA) controlled delivery carriers for biomedical applications,” Advanced Drug Delivery Reviews, vol. 107, pp. 163–175, 2016.
View at: Publisher Site | Google Scholar
R. Dorati, A. DeTrizio, I. Genta et al., “An experimental design approach to the preparation of pegylated polylactide-co-glicolide gentamicin loaded microparticles for local antibiotic delivery,” Materials Science and Engineering: C, vol. 58, pp. 909–917, 2016.
View at: Publisher Site | Google Scholar
Y. Hong, C. Gao, Y. Xie, Y. Gong, and J. Shen, “Collagen-coated polylactide microspheres as chondrocyte microcarriers,” Biomaterials, vol. 26, no. 32, pp. 6305–6313, 2005.
View at: Publisher Site | Google Scholar
J. Wu, T. Kong, K. W. K. Yeung et al., “Fabrication and characterization of monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells for controlled drug release,” Acta Biomaterialia, vol. 9, no. 7, pp. 7410–7419, 2013.
View at: Publisher Site | Google Scholar
L. Lao, H. Tan, Y. Wang, and C. Gao, “Chitosan modified poly(L-lactide) microspheres as cell microcarriers for cartilage tissue engineering,” Colloids and Surfaces B: Biointerfaces, vol. 66, no. 2, pp. 218–225, 2008.
View at: Publisher Site | Google Scholar
A. Privalova, E. Markvicheva, Ch. Sevrin et al., “Biodegradable polyester-based microcarriers with modified surface tailored for tissue engineering,” Journal of Biomedical Materials Research Part A, vol. 103, no. 3, pp. 939–948, 2015.
View at: Publisher Site | Google Scholar
T. S. Demina, T. A. Akopova, L. V. Vladimirov, A. N. Zelenetskii, E. A. Markvicheva, and Ch. Grandfils, “Polylactide-based microspheres prepared using solid-state copolymerized chitosan and D,L-lactide,” Materials Science and Engineering: C, vol. 59, pp. 333–338, 2016.
View at: Publisher Site | Google Scholar
Y. Chevalier and M.-A. Bolzinger, “Emulsions stabilized with solid nanoparticles: Pickering emulsions,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 439, pp. 23–34, 2013.
View at: Publisher Site | Google Scholar
J. Wu and G.-H. Ma, “Recent studies of Pickering emulsions: particles make the difference,” Small, vol. 12, no. 34, pp. 4633–4648, 2016.
View at: Publisher Site | Google Scholar
C. Linke and S. Drusch, “Pickering emulsions in foods–opportunities and limitations,” Critical Reviews in Food Science and Nutrition, pp. 1–15, 2017, In press.
View at: Publisher Site | Google Scholar
C. Wang, C. Zhang, Y. Li, Y. Chen, and Z. Tong, “Facile fabrication of nanocomposite microspheres with polymer cores and magnetic shells by Pickering suspension polymerization,” Reactive & Functional Polymers, vol. 69, no. 10, pp. 750–754, 2009.
View at: Publisher Site | Google Scholar
J. O. Zoppe, R. A. Venditti, and O. J. Rojas, “Pickering emulsions stabilized by cellulose nanocrystals grafted with thermo-responsive polymer brushes,” Journal of Colloid and Interface Science, vol. 369, no. 1, pp. 202–209, 2012.
View at: Publisher Site | Google Scholar
T. Saigal, H. Dong, K. Matyjaszewski, and R. D. Tilton, “Pickering emulsions stabilized by nanoparticles with thermally responsive grafted polymer brushes,” Langmuir, vol. 26, no. 19, pp. 15200–15209, 2010.
View at: Publisher Site | Google Scholar
N. Popadyuk, A. Popadyuk, I. Tarnavchyk et al., “Synthesis of covalently cross-linked colloidosomes from peroxidized Pickering emulsions,” Coatings, vol. 6, pp. 1–14, 2015.
View at: Publisher Site | Google Scholar
H. Guo, D. Yang, M. Yang, Y. Gao, Y. Liu, and H. Li, “Dual responsive Pickering emulsions stabilized by constructed core crosslinked polymer nanoparticles via reversible covalent bonds,” Soft Matter, vol. 12, no. 48, pp. 9683–9691, 2016.
View at: Publisher Site | Google Scholar
K. S. Silmore, C. Gupta, and N. R. Washburn, “Tunable Pickering emulsions with polymer-grafted lignin nanoparticles,” Journal of Colloid and Interface Science, vol. 466, pp. 91–100, 2016.
View at: Publisher Site | Google Scholar
J. Lu, W. Zhou, J. Chen, Y. Jin, K. B. Walters, and S. Ding, “Pickering emulsions stabilized by palygorskite particles grafted with pH-responsive polymer brushes,” RSC Advances, vol. 5, no. 13, pp. 9416–9424, 2015.
View at: Publisher Site | Google Scholar
Y. Zhu, J. Sun, C. Yi, W. Wei, and X. Liu, “One-step formation of multiple Pickering emulsions stabilized by self-assembled poly(dodecyl acrylate-co-acrylic acid) nanoparticles,” Soft Matter, vol. 12, no. 36, pp. 7577–7584, 2016.
View at: Publisher Site | Google Scholar
Z. Wei, C. Wang, H. Liu, S. Zou, and Z. Tong, “Facile fabrication of biocompatible PLGA drug-carrying microspheres by O/W Pickering emulsions,” Colloids and Surfaces B: Biointerfaces, vol. 91, pp. 97–105, 2012.
View at: Publisher Site | Google Scholar
F. Ye, M. Miao, B. Jiang et al., “Elucidation of stabilizing oil-in-water Pickering emulsion with different modified maize starch-based nanoparticles,” Food Chemistry, vol. 229, pp. 152–158, 2017.
View at: Publisher Site | Google Scholar
S. Ge, L. Xiong, M. Li et al., “Characterizations of Pickering emulsions stabilized by starch nanoparticles: influence of starch variety and particle size,” Food Chemistry, vol. 234, pp. 339–347, 2017.
View at: Publisher Site | Google Scholar
T. Zeng, Z.-L. Wu, J.-Y. Zhu et al., “Development of antioxidant Pickering high internal phase emulsions (HIPEs) stabilized by protein/polysaccharide hybrid particles as potential alternative for PHOs,” Food Chemistry, vol. 231, pp. 122–130, 2017.
View at: Publisher Site | Google Scholar
X.-Y. Wang and M.-C. Heuzey, “Chitosan-based conventional and Pickering emulsions with long-term stability,” Langmuir, vol. 32, no. 4, pp. 929–936, 2016.
View at: Publisher Site | Google Scholar
W. W. Mwangi, K.-W. Ho, B.-T. Tey, and E.-S. Chan, “Effect of environmental factors on the physical stability of Pickering-emulsions stabilized by chitosan particles,” Food Hydrocolloids, vol. 60, pp. 543–550, 2016.
View at: Publisher Site | Google Scholar
A. M. Salaberria, J. Labidi, and S. C. M. Fernandes, “Different routes to turn chitin into stunning nano-objects,” European Polymer Journal, vol. 68, pp. 503–515, 2015.
View at: Publisher Site | Google Scholar
J. Huang, P. R. Chang, and N. Lin, Polysaccharide-Based Nanocrystals: Chemistry and Applications, Willey, Hoboken, NJ, USA, 2015.
T. Abitbol, A. Rivkin, Y. Cao et al., “Nanocellulose, a tiny fiber with huge applications,” Current in Opinion on Biotechnology, vol. 39, pp. 76–88, 2016.
View at: Publisher Site | Google Scholar
X. Zhai, D. Lin, D. Liu, and X. Yang, “Emulsions stabilized by nanofibers from bacterial cellulose: new potential food-grade Pickering emulsions,” Food Research International, vol. 103, pp. 12–20, 2018.
View at: Publisher Site | Google Scholar
M. V. Tzoumaki, T. Moschakis, V. Kiosseoglou, and C. G. Biliaderis, “Oil-in-water emulsions stabilized by chitin nanocrystal particles,” Food Hydrocolloids, vol. 25, no. 6, pp. 1521–1529, 2011.
View at: Publisher Site | Google Scholar
M. V. Tzoumaki, T. Moschakis, E. Scholten, and C. G. Biliaderis, “In vitro lipid digestion of chitin nanocrystal stabilized o/w emulsions,” Food & Function, vol. 4, no. 1, pp. 121–129, 2013.
View at: Publisher Site | Google Scholar
T. Angkuratipakorn, A. Sriprai, S. Trantrawong, W. Chaiyasit, and J. Singkhonrat, “Fabrication and characterization of rice bran oil-in-water Pickering emulsion stabilized by cellulose nanocrystals,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 522, pp. 310–319, 2017.
View at: Publisher Site | Google Scholar
I. Kalashnikova, H. Bizot, B. Cathala, and I. Capron, “New Pickering emulsions stabilized by bacterial cellulose nanocrystals,” Langmuir, vol. 27, no. 12, pp. 7471–7479, 2011.
View at: Publisher Site | Google Scholar
N. Rescignano, E. Fortunati, I. Armentano et al., “Use of alginate, chitosan and cellulose nanocrystals as emulsion stabilizers in the synthesis of biodegradable polymeric nanoparticles,” Journal of Colloid and Interface Science, vol. 445, pp. 31–39, 2015.
View at: Publisher Site | Google Scholar
A. V. Istomin, T. S. Demina, E. N. Subcheva, T. A. Akopova, and A. N. Zelenetskii, “Nanocrystalline cellulose from flax stalks: preparation, structure, and use,” Fibre Chemistry, vol. 48, no. 3, pp. 199–201, 2016.
View at: Publisher Site | Google Scholar
Yu. S. Sotnikova, T. S. Demina, A. V. Istomin et al., “Materials based on guar and hydroxypropylguar filled with nanocrystalline polysaccharides,” Fibre Chemistry, vol. 49, no. 3, pp. 188–194, 2017.
View at: Publisher Site | Google Scholar
I. Younes and M. Rinaudo, “Chitin and chitosan preparation from marine sources. Structure, properties and applications,” Marine Drugs, vol. 13, no. 3, pp. 1133–1174, 2015.
View at: Publisher Site | Google Scholar
M. L. Duarte, M. C. Ferreira, M. R. Marvao, and J. Rocha Duarte, “An optimised method to determine the degree of acetylation of chitin and chitosan by FTIR spectroscopy,” International Journal of Biological Macromolecules, vol. 31, no. 1–3, pp. 1–8, 2002.
View at: Publisher Site | Google Scholar

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Copyright © 2018 T. S. Demina et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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