Kinetic instability of a chitosan – aspartic acid – water system as a method for obtaining nano- and microparticles

The specific electrical conductivity and dielectric constant of aqueous solutions of ionic aminopolysaccharide chitosan in L-aspartic acid were investigated. An increase of the mobility of charge carriers in these solutions was found in comparison with solutions of an individual acid. The evaluation of the kinetic stability revealed that the viscosity, electrical conductivity and dielectric constant of the chitosan – L-aspartic acid – water system decrease, while the pH value increases. It was shown that the time variation of physicochemical and electrochemical parameters is due to the effects of counterionic association with the transition of macromolecules to the ionomeric state and is accompanied by phase segregation of the polymer phase in the form of nanoand microparticles. The conducted studies carried out have shown the fundamental possibility of controlling the metastable state of this system in order to obtain nanoand microparticles. Keywords


Introduction
Chitosan (CTS), a product of partial or complete deacetylation of chitin, is an ionic copolymer of aminopolysaccharide. Since the temperature of thermal decomposition of CTS is lower than its melting point, the processing of this polymer into the final products involves the step of dissolving in organic or inorganic acids. In an aqueous acidic medium at pH below pKa, the amino groups of CTS are protonated, resulting in the formation of a salt form of the polymer -a positively charged polyelectrolyte (~− NH 3 + ) with ionic conductivity.
The main contribution to the transfer of electricity in an aqueous acidic CTS solution is made by free counterions, as well as excess of hydronium ions [1][2][3]. In this case, the nature of the acid-solvent of CTS significantly affects the quantitative characteristics of electrical conductivity, which determines the conformation of macromolecules in solution and the phase state of the polymer system after removal of the liquid medium [1]. Thus, in strong HCl, a linear decrease in the specific electrical conductivity (aesp) with an increase in the CTS concentration (СCTS) is observed. The conformation of the expanded helix is realized due to the repulsion of charged macrochains. In weak acids (propionic, acetic and formic), the concentration dependence of the specific electrical conductivity is nonlinear, the aesp values grow increase with an increase in CCTS value. There is an attraction and "adhesion" of local sections of macrochains through ionic crosslinking ~− NH 3 + with R − COО − and H-bonds of OHgroups of CTS with the carbonyl oxygen of the acid, as a result of which the macromolecules assume the coiled conformation. CTS solutions in HCl have a weak film-forming ability and give a loose structure with a pore diameter of more than 10 mm when dried on an inert surface. CTS films cast from aqueous solutions of carboxylic acids are characterized by a dense ordered structure. When an electric field is applied in an aqueous acidic CTS solution, internal polarization phenomena can occur, i.e., displacement of charges that create a volumedistributed dipole moment. The relaxation processes of the electric polarization of the CTS salt form are manifested in dielectric spectra in the frequency range below 1010 Hz [4]. Like conductometry, dielectric relaxation spectroscopy is highly sensitive to changes in the electrical conductive properties of aqueous acidic CTS solutions and makes it possible to reliably control the state of counterions (free, bound) in them.
CTS macromolecules are characterized by highly complex behavior in solutions. This is primarily due to the polyelectrolyte swelling of charged macromolecular coils and increased chain rigidity. CTS solutions are also characterized by instability of viscosity properties over time, which is usually explained by the destruction of macromolecules as a result of acid hydrolysis of glycosidic bonds [5][6][7]. However, there are studies that make it possible to exclude destruction as the main reason for the decrease in the viscosity of chitosan solutions in time [8,9] and to explain this effect by conformational rearrangements of macromolecules [10], changes in the supramolecular structure, including aggregation [9,11], and also by phase separation of the system as it is stored [11,12].
CTS is widely used due to a complex of valuable properties, as well as an annually renewable resource base [13][14][15].
Chitosan-containing nanomaterials, in particular nanoparticles, deserve special attention [16,17]. Compared to the original CTS, they acquire additional valuable properties due to their small size, increased surface area, and quantum size effects. Like CTS, chitosan-containing nanoparticles are nontoxic, biocompatible, and biodegradable, which predetermines their use in medicine, as well as in the food, textile, and cosmetic industries [18][19][20][21][22]. For obtaining nanoparticles, the most common used are spray drying under special conditions, or methods of ionotropic gelation and emulsion crosslinking with tripolyphosphate, glutaraldehyde, genipin [16]. In some cases, dibasic and tribasic pharmacopoeial acids (malic, tartaric, citric, and succinic) were used, which simultaneously act as a solvent for the polymer and as a crosslinking reagent [23][24][25]. It should be noted that the use of biologically active acids for the formation of CTS nanoparticles significantly expands the directions of their application in biomedical and pharmacological applications [26][27][28], veterinary medicine [29], and plant growing [30][31][32][33].
The use of L-aspartic acid (AspA), a proteinogenic amino acid that plays an important role in a living organism, seems to be promising [34,35]. Unlike traditional wateracid CTS dissolving media based on monobasic carboxylic acids (HCOOH, AcOH), AspA contains two carboxylic and one amino groups. Therefore, in an aqueous solution, depending on the pH of the medium, its molecules can exist in several ionic forms. At pH = 3.0, the isoelectric point is located, at pH = 4, almost equal proportions of zwitter ions (Н2Asp) and aspartate anions (НAsp − ) are realized, whereas in the range of pH = 4.7-8.0, НAsp − is the predominant ion [36]. The resulting high concentration of hydrogen ions (hydronium), which are the main carriers of electricity, provides prototropic conductivity of an aqueous solution of AspA [37,38].
Previously, we have found that the solubility of AspA in Н2О increases in the presence of CTS [39]. The conditions for the preparation of CTS solutions in AspА -Н2О mixture and some of their properties have also been discussed [40]. Thus, in an aqueous medium, the macromolecules of the salt CTS form exhibit the properties of a weak-ly dissociating polyelectrolyte with a partially compensated charge. The calculated Huggins constants and temperature viscosity coefficients showed an increased rigidity of their macrochains. However, kinetic stability was not evaluated, although the CTS -AspA -H2O system showed all the characteristic features of phase separation. As in the case of synthetic polyelectrolytes [41,42], not only the dissociation of ionogenic groups of CTS aspartate can occur in an aqueous medium, but also the interaction of the polycation with HAspwith the formation of ion pairs, multiplet structures (combination of ion pairs) and the transition of macromolecules to the ionomeric state. At the initial stage of phase separation, these processes should be accompanied by phase segregation of the polymer phase in the form of nano-and microparticles. This assumption served as the basis for this study.
The aim of this work is to study the kinetic stability of the CTS -AspA -H2O system during storage to obtain nanoand microparticles using viscometry, potentiometry, conductometry, and dielectric relaxation spectroscopy as the most informative methods for studying the counterions state (free, bound) in polyelectrolyte solutions [4,[40][41][42]].

Materials
The starting reagents were powdered CTS with a viscosity- The CTS + AspA + Н2О system were stored in a room atmosphere (760 mm Hg, 22±2 °C) for 96 h, regularly taking samples to assess their physicochemical parameters. In all experiments, the conditions for the preparation of the system and the time at which the physicochemical characteristics began to be measured were the same. The stability of the systems was assessed by measuring their conductivity, pH, viscosity and permittivity, as well as by SEM.

Obtaining p articles
To obtain nanoparticles and microparticles, 0.5 μL of a freshly prepared or stored CTS + AspА + Н2О system was sprayed onto a degreased (ethyl alcohol, acetone) glass surface using a mechanical syringe-shaped injector and dried for 1-2 h (the technique is similar [43]). The obtained particles obtained were dried at 22±2 °C under atmospheric pressure to an air-dry state, and analyzed using SEM.

Мethods
Gravimetric measurements were carried out on an Ohaus Discovery analytical balance (USA), weighing accuracy being ±0.0001 g.
pH was measured on a Mettler Toledo Five Easy FE20 pH-meter (MTD, Singapore).
Conductivity was measured on a WTW inoLab Cond 7110 conductometer (Germany) at 25 °C. A thermostated cell with a volume of 25 mL was used. Specific conductiv ity (aesp, S·m −1 ) was calculated using the specific conductivity of water by the formula: where К = 0.1213 m −1 is the cell constant evaluated from 0.01 N KCl solution; R is the resistance of the system, S; aeCTS and aeН2О are the specific conductivity of the chitosancontaining system and water, respectively, S·m −1 .
The viscometric measurements were carried out in an Ubbelohde viscometer (RF) with a capillary diameter of 0.56 mm at 25 °C. The relative viscosity (rel) was calculated from: where t is the flow time of the CTS + AspА + Н2О sistems, s; t0 is that of the AspA aqueous solution, s; СCTS is expressed in g/dL; the accuracy of measuring t and t0 was ±0.1 s.
The frequency dependences of the real (εʹ) and imaginary (εʹʹ) parts of the complex permittivity were measured on an Agilent Microwave Network Analyzer PNA-X N5242A vector network analyzer (USA) using an Agilent 85070E coaxial probe in the frequency range f = 10 7 -10 11 Hz at 25 °С. The dielectric loss tangent was calculated from the ratio tgδ = εʹʹ/εʹ. SEM images were taken on a MIRA\\LMU scanning electron microscope (Tescan, CZ) equipped with an energy dispersive detector (EDX) at a voltage of 30 kV and a conducting current of 400 pA. A 5 nm thick gold layer was deposited on each sample using a K450X Carbon Coater (DE).

Statistical analysis
In each experiment on the study of physicochemical properties, at least 3 parallel experiments were carried out; the arithmetic mean and standard deviation were calculated.

Results and discussion
We have previously shown that the concentration dependence of the reduced specific viscosity of a freshly prepared aqueous solution of CTS aspartate is not linear, has a max or a plateau and a descending branch with a decrease in CTS [40]. This hydrodynamic behavior indicates the implementation of a mixed polyelectrolyte-ionomer mode, when some of the HAspcounterions are in an associated (bound) state with −NH 3 + groups of the macrochain with the formation of ion pairs (Fig. 1a). The revealed features of the counterions state in macromolecular coils of the CTS salt form were proved by studying the electrochemical properties of the polymer system. Thus, the specific electrical conductivity of a CTS solution in AspА -Н2О at СCTS = const increases with an increase in the acid concentration (Fig. 2a, curve 1). A similar dependence is observed for aqueous solutions of individual AspA The evaluation of the dielectric properties also showed an increase in the mobility of the electrically transporting particles in the CTS aspartate solution. It is noteworthy that in the frequency range of f < 10 9 Hz, the real part of the complex dielectric constant of CTS solutions in AspA-H2O mixture is less than εʹ of an aqueous solution of AspA and water (Fig. 3a), and the dielectric loss factor and dielectric loss tangent are larger (Fig. 3b, c). The εʹ values decrease, εʹʹ and tgδ values increase with an increase in СCTS. Consequently, in CTS solutions in AspA-Н2О mixture there is a larger amount of mobile charge carriers providing ohmic current than in AspA-Н2О and aqueous solutions. At f > ~1.6-1.8·10 9 Hz, the εʹ, εʹʹ, and tgδ values of aqueous solutions of CTS aspartate, AspА and water are comparable. This character of the dispersion of εʹ, εʹʹ, and tgδ values of a solution of CTS aspartate is explained by the fact that at high frequencies a "fast" mechanism of ion polarization is realized, the large sizes of macrocoils prevent the movement of macrodipoles (associations of polar groups of a polymer molecule through dipole-dipole interaction) in an alternating electric field and the electric the induction of the system is determined only by the mobility of low molecular weight ions [4]. As f value decreases, the "slow" mechanism of orientational (dipole) polarization occurs, and the possibility of the macrodipoles motion, accompanied by dissipative losses, appears. The smaller f values, the greater are the dielectric losses (εʹʹ, tgδ), since the macrodipoles have more time to orient themselves along the electric field. The high mobility of charged particles in a solution of CTS aspartate was reflected in the kinetic stability of the polymer system. It turned out that exposure at room atmosphere is accompanied by a significant decrease in the relative viscosity of CTS solutions in AspA-Н2О mixture and an increase in the medium acidity (Fig. 4a, curves 1 and 2). These changes are most intense in the first ~ 16-18 hours after preparation of the solution and increase with increasing CTS concentration. So, after 24 hours of storage of solutions with CCTS = 0.002-0.006 monomol/L, the effect of reducing ηrel was 7-10%, and 23% (Fig. 4а, curve 1) for more concentrated solutions, for example, with CCTS = 0.035 monomol/L.
In addition, during storage for ~16 -18 hours in a visually homogeneous solution of CTS in AspA -Н2О mixture, the specific conductivity increases (Fig. 4b, curve 3). The dielectric constant in the frequency range of f < 109 Hz decreases, and in the range of f > 1.8·10 9 Hz it increases with a local maximum (curves 4 and 5). At t > 24 hours, opalescence appears aesp value begins to decrease with the realization of lower values than in a freshly prepared solution; εʹ value decreases with time over the entire range of f. Like ηrel value, in this case, the aesp and εʹ values change to the greater extent, the higher the polymer concentration in the system. After ~66-74 hours the phase separation occurs, which is observed visually as the precipitation of a water-insoluble fine sediment.
Like relative viscosity, specific electrical conductivity and dielectric constant change with time to the greater extent, the higher the polymer concentration, and pH -vice versa. At first glance, a decrease in the viscosity of the system under study with time may be associated with the macromolecules destruction as a result of acid hydrolysis of glycosidic bonds [5][6][7], and an increase in pH is due to deprotonation of AspA with the transition of some of the acid molecules into the zwitterionic H2Asp form. However, upon destruction with a decrease in the molecular weight of CTS, the amount of free counterions and hydronium ions should increase, and the pH value should decrease. As a result, the destructive processes should lead to an increase in ionic conductivity, i.e., electrical conductivity and dielectric constant should increase monotonically, but not very significantly. Since for the CTS -AspA -Н2О polyelectrolyte system under study a different character of the aesp and εʹ kinetics is observed, it seems that its instability in time is associated with the effects of the association of counterions with protonated amino groups of the polycation and their further transformations. Probably, it is thermodynamically not favorable for counterions to be in a free state, and they continue to form ion pairs with -NH 3+ groups of polymer chains (ionomers), while losing in entropy, but gaining in electrostatic energy (Fig. 1b). The transition of counterions from the aqueous medium to the macrocoil volume leads to screening of electrostatic repulsion between the like-charged links of the macrochain, a decrease in aesp, ηrel, and εʹ values of the system and, accordingly, its transition to the ionomeric mode. The resulting effective attraction between monomer units contributes to the dipole-dipole interaction of ion pairs and their combination into multiplet structures (Fig. 1c). Most likely, the stabilization of the latter occurs through complex ion-ion-hydrogen contacts of ionogenic groups of the CTS aspartate macromolecule and the AspA molecule (the possibility of the formation of such CTS-acid contacts has been reported in [1]). Multiplets function as physical crosslinks between different polymer chains, which contributes to the compaction of macrocoils and the formation of nanosized nuclei of a new phase (Fig. 1d). Upon aggregation of the latter to microand macroparticles, the metastable polymer system is divided into two equilibrium phases: the polymer-rich phase precipitates, the polymer-depleted phase is represented by the supernatant liquid. According to [41], phase separation in a polyelectrolyte solution under conditions of ionic association can occur even in a thermodynamically good solvent.
The described associative processes were confirmed and visualized using scanning electron microscopy. For example, in a CTS -AspA -Н2О sample, spherical nanoparticles with a size of d = 40−90 nm (Fig.5a) and ellipsoidal ones with a size of d = 70−260 nm (Fig. 5b) were revealed after 24 and 36 hours, respectively. After ~48-70 hours, the average size of chitosan-containing particles was ~1.4-2.0 μm, width was ~0.6-0.8 μm; their aggregation was also observed.
Elemental analysis data showed that the nanosized polymer phase contains 42.95±1.2% carbon, 21.09±0.9% nitrogen, and 35.96±1.0% oxygen. This suggests that the nanoparticles are represented by the polymer salt form, CTS aspartate.

Conclusions
In conclusion, cooperative interactions between fixed charges of the polycation and low-molecular counterions in the CTS -AspA -Н2О system are accompanied by the formation of ion pairs, multiplet structures, and phase segregation of the polymer substance to form nano-and microparticles. The "fixation" of the nuclei of a new phase at the initial stage of phase separation by injection spraying onto a glass support makes it possible to visualize the formation of particles by the SEM method. Further search for ways to stabilize particles in solution, for example, by creating a "shell" that prevents aggregation, will make it possible to develop methods for their preparation in a preparative amount. Due to the high biological activity of CTS and AspA, chitosan-containing nano-and microparticles can be promising as independent pharmaceutical and veterinary drugs, plant protection products, providing synergy of useful properties of their constituent components, as well as in drug storage and delivery systems, and theranostics.