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

Enikolopov Institute of Synthetic PolymerMaterials, Russian Academy of Sciences, 70 Profsoyuznaya St., 117393Moscow, Russia Institute for Regenerative Medicine, Sechenov University, 8-2 Trubetskaya St., Moscow 119991, Russia Moscow Aviation Institute (National Research University), 4 Volokolamskoe Shosse, 125993 Moscow, Russia Interfaculty Research Centre on Biomaterials (CEIB), Chemistry Institute, University of Liège, B6C Allée du 6 Août 11, B-4000 Liege (Sart-Tilman), Belgium


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][4][5][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.
e 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][9][10].e Pickering emulsions were successfully used for preparation of a so-called magnetic polymer microspheres consisted of polystyrene core and Fe 3 O 4 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][13][14][15][16][17][18].Usage of biocompatible nanoparticles, such as hydroxyapatite, SiO 2 , proteins, lignin, flavonoids, and various polysaccharides, for the Pickering emulsion stabilization is especially interesting for food or biomedical applications [19][20][21][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][26][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].
e most widely used cellulose and chitin nanocrystals have a rod-like anisometric morphology with a length and diameter varied from several nanometers to micrometers.
ey are proposed as reinforcing llers, templates for preparation of mesoporous materials, for fabrication of semiconductor and smart materials, including mechanically adaptive and self-healing ones.e use of polysaccharide nanocrystals as stabilizing agents in the Pickering emulsions was previously described mainly for food industry [28][29][30][31].E ciency of nanocrystals as stabilizing components for preparation of polymeric microparticles via the emulsion solvent evaporation technique was studied mostly on cellulose nanocrystals [32,33].
is work was aimed at evaluating the e ect 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.

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.e ash contents of raw chitins were 0.16 wt.% and 1.76 wt.% for "Kombio" and "Xiamen Fine Chemical," respectively.e 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.

Atomic Force Microscopy (AFM).
e size and morphology of the prepared ChN were evaluated using atomic force microscopy NtegraPrima (NT-MDT, Russia) in tapping mode.e 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].e AFM images were analyzed using Image Analysis 2.0 (NT-MDT, Russia) software.

Dynamic Light Scattering.
e 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).

FTIR.
e chitin nanocrystals in a form of lms 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.

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 CH 2 Cl 2 : acetone (9 : 1 v/v) with 0.1-1 wt.% aqueous dispersions of ChN. e scheme of the microparticle fabrication is shown in Figure 1.Brie y, 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 emulsi er in aqueous phase were prepared as model samples.e mixing of the phases was carried out using a fourblade stirrer at a rotation speed of 700 rpm for 3 hours.e emulsions were kept in a water bath through the mixing at 15 °C during the rst 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 μm, then, freeze-dried and weighted.Advances in Materials Science and Engineering

Scanning Electron Microscopy.
e surface morphology of the fabricated microparticles was studied by scanning electron microscopy (SEM) using PhenomProX (Phenom-World, Netherlands) at 10-15 kV.

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.
e 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.e 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.us, in spite of both raw chitins were from crab shells, the structure of the polysaccharide could signi cantly 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].
FTIR spectra of the extracted ChN showed no signi cant di erences 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 CH 3 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 ChN-K 0.1 wt.% ChN-K 0.  4 Advances in Materials Science and Engineering the concentration change, while ChN-K showed a widerange size distribution at 1 wt.% concentration.us, 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.

Microparticle Fabrication.
As could be seen in Figure 4, the ChN could e ectively stabilize a simple CH 2 Cl 2 /water (1/9 v/v) emulsion.e size distribution of oil droplets depends on mixing parameters and e ectiveness of oil/water interface stabilization, which is controlled by the ChN hydrophilichydrophobic balance and concentration in the aqueous phase.
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. is yield was higher than that obtained using PVA as the emulsi er in spite of lower amount of ChN in comparison with PVA concentration used: 1 and 2.5 wt.%, respectively.e comparative stabilization e ectiveness 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 e ectiveness, 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. is di erence 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. e 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.
It could be assumed that the ChN particles were attached so rmly at the oil/water interface that their rearrangement during the solvent evaporation process was impaired.e 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 signi cant excess of ChN at the higher concentration in the aqueous phase could lead to the development of "raisin" e ect.e di erence in the shape of ChN-K-and ChN-X-stabilized microparticles could be caused by di erent aspect ratios of the nanocrystals: 1.4 and 2.8, respectively.e 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 e ectively 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.
ese microparticles could be used as biodegradable cell microcarriers with improved cell a nity.

Conclusions
A promising approach to prepare poly(L,L-lactide) microparticles in a course of solvent evaporation from the Pickering oil/water 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 di erent 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.e 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 emulsi er application, that is, polyvinyl alcohol.e increase of nanocrystals concentration in an aqueous phase led to an increase in total yield and to decrease of mean microparticles size.e 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.

Figure 3 :Figure 4 :
Figure 3: Number-weighted size distribution of ChN-K (a) and ChN-X (b) in their aqueous dispersions at various concentrations.

PVA 0. 1 Figure 5 :
Figure 5: e total yield and microparticle size distribution as a function of origin and concentration of chitin nanocrystals in the aqueous phase.