Stabilization of Chitosan Aggregates at the Nanoscale in Solutions in

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Stabilization of Chitosan Aggregates at the Nanoscale in Solutions in Carbonic Acid Marina A. Pigaleva,*,† Ivan V. Portnov,† Andrey A. Rudov,†,‡ Inesa V. Blagodatskikh,§ Timofei E. Grigoriev,§ Marat O. Gallyamov,†,§ and Igor I. Potemkin†,‡ †

Faculty of Physics, Lomonosov Moscow State University, Leninskie gory 1-2, GSP-1, Moscow 119991, Russian Federation DWI - Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, Aachen 52056, Germany § Nesmeyanov Institute of Organoelement Compounds RAS, Moscow, Vavilova 28, GSP-1, Moscow 119991, Russian Federation ‡

ABSTRACT: Chitosan macromolecules with different molecular weights and degrees of acetylation were dissolved in carbonic acid solutions saturated with carbon dioxide at high pressure and were adsorbed from such solutions onto model mica substrate. Their conformation on the substrate was revealed by means of atomic force microscopy. The results of the observations indicate that there are adsorbed nanosized stable aggregates with peculiar, regular, and reproducible geometry, which are formed in carbonic acid solutions. The aggregates appear as rather persistent rod-like structures and consist of a few individual chitosan macromolecules. This effect can be explained by the competition of the Coulomb repulsion of charged monomer units with the attraction between segments of polymer chains. There are several factors contributing to the total intra- and interchain attraction of different monomer units: hydrogen bonding, hydrophobic interaction, and dipole−dipole attraction caused by the formation of ion pairs due to counterion condensation. The uncompensated charges prevent further growth of the aggregates and therefore stabilize their size at the nanoscale range. The experimental data are supported by theoretical calculations and computer simulations.

1. INTRODUCTION

Therefore, the uniformity of the chitosan coating as deposited may be deteriorated. The results of the investigation of polysaccharide solutions typically reveal strong tendency toward aggregation of polysaccharide (including chitosan) molecules in aqueous solvents.11 Conformation of individual chitosan macromolecules in aqueous solutions can be studied reliably only for some specially prepared samples (namely for chitosans with random-type distribution of N-acetyl groups along the chain, i.e., for chitosans, which are reacetylated under homogeneous conditions from previously produced samples of lower DA12) because of nonpredictable and noncontrollable aggregation observed otherwise. Attempts to remove large aggregates, or to influence the aggregation behavior, were made10 by ultracentrifugation, extensive filtering of the solutions, modification of the solvent conditions such as pH, ionic strength, and temperature exposition to an acidic proteinase. Yet, in all cases, strong aggregation could not be prevented. Previously mentioned biomedical applications of chitosan typically require extra purity, which means the absence of any foreign residues of, e.g., solvents or any other chemical agents. Indeed, nowadays there is a huge problem with ever-increasing human hypersensitivity, and it is very important to try to

Chitosan is a polysaccharide, which has numerous applications in biomedical area. For example, it is used in wound dressing, drug delivery, cancer diagnosis, and tissue engineering.1 It is well-known that chitosan is soluble in some aqueous acidic solutions having pH values less than 6.0.2 Such acids as acetic, formic, lactic, propionic, and hydrochloric ones are capable to dissolve chitosan.3,4 Therefore, there is a possibility to obtain thin chitosan coatings as deposited from such solutions. Subsequent neutralization with alkali may be performed in order to convert the chitosan into water-insoluble form. But even dissolved chitosan chains may preserve a certain degree of aggregation, and therefore typically the solutions comprise some intermolecular aggregates along with molecularly dispersed polymer chains. The presence of aggregates or insufficiently solvated residues of chitosan molecules in aqueous solutions was shown repeatedly.5−10 In the literature it is generally believed that even a slight amount of residual N-acetyl groups can cause such an aggregation behavior. Indeed, although aqueous media at pH < 6 serves as a good solvent for deacetylated units of chitosan, it might be a poor solvent for small quantity of residual N-acetyl groups along the chitosan chain. Besides, even the deacetylated units may participate in intra- or interchain links due to e.g. hydrogen bonding. Thus, dissolved chitosan chains even at acidic pH values typically retain some degree of aggregation/compaction. © 2014 American Chemical Society

Received: June 5, 2014 Revised: July 24, 2014 Published: August 7, 2014 5749

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polymer chains in the solvent can influence the stability and mechanical properties of the eventual chitosan coating on the surface of the bioprosthesis. However, there is no information in the literature yet about the conformation of chitosan macromolecules in such peculiar solvent as carbonic acid at high pressure. This is not very surprising because it is rather challenging to reveal the conformation of the macromolecules in solutions, which exist only at high pressure, by means of the commonly used methods for conformational characterization. There are only several works about the possibility to perform the similar characterization of macromolecular conformation for other macromolecules in solutions in supercritical CO2 at high pressure by light,23,24 small-angle neutron,25,26 and smallangle X-ray27,28 scattering. The main challenge in these experiments is the construction of suitable transparent windows sustaining fairly high pressures. Indeed, the solutions of polymers in supercritical CO2 also exist only in the pressurized tightened metal autoclave. In the experiments with the solutions of carbonic acid there is the same problem, but additionally there should be disturbing fluctuations of the dissolved CO2, which are capable to distort the scattering picture. Therefore, using ex situ atomic force microscopy (AFM) as a tool to observe macromolecules as adsorbed on mica from chitosan solution in carbonic acid seems to be an easy-to-perform experiment; at least it can provide direct information on the morphology of chitosan structures, which spontaneously adhered to the substrate during exposure in the autoclave with carbonic acid at high pressure. It was proven previously by Minko et al.29,30 that if there are polycationic macromolecules and the oppositely charged polyanionic mica surface, then the macromolecules demonstrate adsorptionfrozen conformation; i.e., they preserve on the substrate all the main conformational features, which they have had in solution. This means that the conformation of the adsorbed structures on the surface is similar to the macromolecular conformation in the bulk. Therefore, the combination of the method of direct adsorption of single macromolecules on a substrate from highly pressurized solvent with subsequent analysis of their conformation with AFM seems to be very promising. Yet, in the literature one can find only occasional works where such combination of the methods was realized.18,20−22 Previously we have reported results of experiments on visualization of chitosan structures as adsorbed from solutions in carbonic acid on a model mica substrate.18 We succeeded in adsorption of rather smooth, uniform films. Closer look at the films revealed that they are composed by rather persistent elongated rod-like structures. Earlier similar formations were observed using transmission electron microscopy.10 Authors studied chitosans with 1% and 15% of acetylated units. They succeeded in visualization of elongated persistent structures with average lengths between 200 and 1000 nm depending on concentration of chitosan in the initial solution. Therefore, the morphology of the aggregates visualized by Anthonsen et al.10 resembles the elongated rod-like structures reported by us.18 Furthermore, Kulikov et al.31 succeeded in formation of stable nanosized aggregates in aqueous solution but only for oligochitosan. Recently, Kocun et al.32 also studied chitosan adsorbed from solutions in sodium acetate buffer (pH 4.5−5.5) onto a mica substrate. However, from the presented AFM images chitosan macromolecules can be seen as both elongated single individual strands and aggregated bundles. The major discrepancy of this work with our results is the presence of high amount of salt

minimize the probable risks related to it for possible future patients. Therefore, it would be very useful to find a medium, which is absolutely biocompatible and, at the same time, can dissolve chitosan without the formation of large aggregates as well as, hopefully, with the possibility to predict the macromolecular conformation in this medium. Carbonic acid apparently behaves just like this kind of medium. First of all, it is well-known that in solutions of carbon dioxide in water the complex equilibrium system exists. We can describe it in general as follows: CO2 + H 2O ↔ CO2 × H 2O ↔ H 2CO3 ↔ H+ + HCO3−

If we saturate water with pressurized carbon dioxide, the pH will decrease down to the values lower than 3.0 with the increase of pressure up to the hundreds of atmospheres. In the recent studies13−15 the influence of temperature and pressure on a value of pH in the mixtures of water and CO2 was revealed. Thus, as a result, chitosan in carbonic acid is protonated, becomes polyelectrolyte, and dissolves. The approach to dissolve chitosan in water bubbled with CO2 was first proposed by Sakai et al.16 After them, Otake et al.17 investigated the solubility of chitosan in carbonic acid. They reported the achievable concentration of about 70 mg/L at 200 bar pressure and 60 °C temperature after 180 min of mixing for chitosan solution in this acid. As it was discussed before in details,18 carbonic acid combines two important properties thanks to which it appears to be very beneficial solvent for biomedical applications. Those are strong antimicrobial activity at high pressure including the very moment of coating formation on some medical device and remaining absolute biocompatibility later on, when using this device with the adsorbed coating in the human body. Recently, Gorczyca et al.19 showed the possibility to use water bubbled with CO2 instead of organic or inorganic acids for chitosan blend preparation and therefore to obtain stable cross-linked chitosan-based materials with properties relevant for wound healing application. But we believe that the usage of carbonic acid saturated with CO2 at high pressure is even more beneficial. At higher pressure applied the dissolving power of the solution is higher as far as the pH value is lower (below 3.0). Moreover, when changing the dissolving power of “usual” solvents, the inhomogeneity and strong gradients occur. On the contrary, when using carbonic acid at high pressure, one can depressurize the whole volume of the reactor instantly and simultaneously at the same time; i.e., increase of the pH (up to ca. 4.0 and further up to 5.5) everywhere in the solution occurs in a very homogeneous manner. Another example of such a kind of a “physical” solvent would be supercritical CO2, which unfortunately can dissolve chitosan only up to much lower concentrations achievable.20,21 These advantages of the biocompatible solvent and material led to the suggestion of promising method of formation of chitosan coatings from solutions in carbonic acid on bovine pericardium. This biotissue is commonly used for manufacturing the heart valve bioprostheses. Such chitosan coating prevents the calcification of the modified bioprosthesis tissue, and correspondingly, its failure as well as improves the mechanical properties of the biotissue.22 In order to develop the methodological recommendations on the best regime of adsorption of chitosan on a surface of biotissue from the biocompatible solvent, carbonic acid, it is rather important to detect and explain the conformation of the macromolecules in this solvent. Indeed, the conformation of 5750

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saturate water and thus generate carbonic acid. We used water purified on the Millipore Milli-Q synthesis system. Water was purified just before each experiment. The experimental setup is equipped with a pressure generator and the reactor of high pressure, which has inner volume of 10 mL. The system can sustain the pressure of up to 80 MPa (as limited by the reactor). The desired temperature regime is provided by an electronically controlled thermostat. Because of high sensitivity of the method which we have used (AFM detection of single macromolecules), the purity of the reactor was of great importance for the success of the experiments on the adsorption of small amounts of chitosan material. Therefore, before each new experiment we performed the cleaning of the reactor as described in details in the previous report.18 The purity of the reactor was supposed acceptable only if on the AFM micrographs no adsorbed structures were observed after blank exposure of mica to pure carbonic acid without any chitosan in this reactor. Otherwise, the cleaning procedure was repeated. The adsorption of chitosan from water saturated with CO2 under high pressure was performed as described in details in the previous report.18 Briefly, we put in the reactor a certain amount of chitosan powder, 5 mL of freshly prepared Milli-Q water, and a piece of freshly cleaved on both sides mica to be immersed in the water phase. Further we tightened the reactor, injected liquid CO2, and raised the pressure up to the 30 MPa at room temperature (25 °C). The amount of CO2 inside the reactor was around 5 g. At such conditions the aqueous medium inside the tightened reactor slowly became acidic,42 and the chitosan chains became protonated directly inside this reactor in the water phase. As a result, we got a solution of polycation chitosan in carbonic acid solution right inside the reactor. Meanwhile (again in the closed reactor and in the water phase), the spontaneous adsorption of polymer macromolecules from the water phase on the immersed mica surface happened (being driven by electrostatic attractive forces between polycationic chitosan and polyanionic mica surface). We performed an exposition for 7 days. The exposition was ended at room temperature as well by rather slow decompression. Then the sample was retrieved from the reactor and dried in the room atmosphere. Characterization. The method of the macromolecular investigation in our work was atomic force microscopy (AFM). We performed the scanning of a substrate in the tapping mode in air at room conditions using an atomic force microscope Multimode with NanoScope-IIIa controller (Digital Instruments). We used silicon cantilevers NCH (the length of the cantilever is 125 μm, typical tip radius of curvature less than 8 nm, force constant 40 N m−1, resonance frequency 200−400 kHz, manufactured by Nanoworld Instruments, Switzerland). The experimental images were collected with the informational density of 512 × 512 points, scanning frequency of 1 Hz, and the sizes of the images ranged from 12 × 12 μm2 to 1 × 1 μm2. The analysis of the AFM images was performed by means of Nanoscope software (Digital Instruments) and Femtoscan Online software (Advanced Technologies Center, Russia). The molecular masses of chitosan samples (Sigma-Aldrich) were independently measured by means of two different techniques: gel permeation chromatography (GPC) and viscosimetry. Using gel permeation chromatograph (Agilent 1200) calibrated with pullulan standards (from 1.08 to 710 kg mol−1), we determined the apparent molecular weight relative to pullulans of all the six chitosan samples (see Table 1). All measurements were performed on PLaquagel-OH mixed column with the eluent flow rate of 1 mL/min and temperature of the columns 25 °C (aqueous buffer solution, 0.2 M acetic acid, 0.15 M ammonium acetate). The collection and processing of the obtained data were performed by means of Agilent ChemStation and Agilent GPC module software. The viscosity measurements were performed with Ubbelohde capillary viscometer at the temperature of 25 °C. The average viscosity molecular weight for all the six types of the chitosan samples was estimated (Table 1) from the intrinsic viscosity determined in 0.3 M acetic acid/0.2 M sodium acetate using the Mark−Houwink parameters K = 1.38 × 10−4 and α = 0.85.43

(sodium acetate), which can influence the conformation as it is to be discussed further. Moreover, it is well-known that the solubility of chitosan depends not only on pH but also on the nature of the solvent.33 Chitosan is soluble in some acids (aqueous solutions) and insoluble in others. Therefore, the choice of the solvent can influence the chitosan conformational behavior dramatically. There is a heated discussion in the literature about conformational behavior of single weak polyelectrolyte chains in a poor solvent. On the one hand, there is a theory of Dobrynin et al.,34 predicting the pearl-necklace conformation of polyelectrolyte chains. This structure is a result of the Rayleigh instability, which is known to cause a splitting of charged droplets into smaller ones.35 Several computer simulations taking into account Coulomb repulsion between charged monomer units support this model.36−38 Indeed, with the increase of charge of a polymer globule in a poor solvent it becomes unstable and tries to split into smaller globules. But due to the covalent bonds between monomer units of the macromolecule, the globules cannot split completely. They can only maximize distance between them and form structures called pearl-necklace chains. But it is important to note that this approach does not account for the presence of counterions. On the other hand, Khokhlov39 showed that the polyelectrolyte globule can elongate into a cylinder or “cigar-like” globule due to avalanche-type counterion condensation taking place in the poor solvent. Several computer simulations, which came after this theory, predicted that if one takes into account the counterion condensation taking place, one should obtain a larger number of macromolecular conformations depending on the external conditions and chain characteristics such as extended, pearl-necklace, cigar-shape, and collapsed structures.40,41 In our experiments we observe the predicted “cigar-like” shape of the chitosan aggregates. The first effect caused by counterion condensation is the weakening of the electrostatic repulsion between chain segments due to neutralization of the charged groups by condensed counterions. Besides, the formation of ionic pairs between condensed counterions and oppositely charged monomer groups causes an additional interchain attraction due to dipole−dipole interactions. In the present paper, we perform systematic experiments on adsorption of a set of different chitosan samples (with different molecular weights Mw and DA) from solutions in carbonic acid on atomically flat substrate (mica), suitable for the subsequent analysis with AFM. Thus, the present study is aimed at investigation of the dependency of the macromolecular conformation on characteristics of chitosan dissolved in carbonic acid. Such experiments may contribute to better understanding of regularities determining the process of solubilization and aggregation of chitosan in acidic solutions. Also, we perform computer simulations to demonstrate thermodynamic stability of elongated aggregates formed by polyelectrolyte chains in dilute solutions.

2. EXPERIMENTAL PART Materials and Synthesis. We used different samples of chitosan (low molecular weight chitosan #448869, chitosan #419419, medium molecular weight chitosan #448877, chitosan #417963, chitosan #c3646, highly viscous chitosan from crab shells #48165) supplied by Sigma-Aldrich. As a substrate for adsorption of polymer structures we applied mica from Plano GmbH (Germany). In the experiments we utilized CO2 of high purity (>99.997%, Linde Gas Rus, Russia) to 5751

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quantified by the Lennard-Jones intermonomer interaction parameter ϵαβ, α, β = m: ϵmm ≡ ϵ In our simulations, it was varied between ϵ = 1kBT and ϵ = 3kBT, which corresponds to the case of really bad solvent (for comparison, good solvent is described by ϵ = 0). The value of the Lennard-Jones parameter for monomer−counterion (mc) and counterion−counterion (cc) interactions was set to46 ϵmc = ϵcc = 1kBT. Electrostatic interactions between any two monovalent charged particles, qi,j = ±1, separated by a distance rij was given by the Coulomb potential

Table 1. Characteristics of Chitosan Samples (SigmaAldrich)a catalogue no. of chitosan sample

Mw (kg/mol)

Mn (kg/mol)

Mη (kg/mol)

l (nm)

h (nm)

DD (%)

DD1 (%)

448869 419419 448877 417963 c3646 48165

210 1300 1300 990 1400 2300

80 130 240 210 380 680

80 150 270 320 320 790

45 35 50 35 35 45

0.8 0.6 0.9 0.7 0.7 0.9

84 70 74 70 67 74

74 70 72 68 71 70

UCoul(rij) =

a Weight-average Mw and number average Mn molecular weights [kg/ mol] obtained by GPC, viscosity-average Mη molecular mass [kg/mol] as determined by viscosimetry, typical length l [nm] and height h [nm] of rod-like structures as measured directly on the AFM micrographs; the degree of deacetylation DD [%] as detected by pH titration and DD1 [%] as obtained by IR spectroscopy.

203 42 +

msample

rij

The solvent was treated implicitly as a medium with the dielectric constant ε. Each polyelectrolyte chain consisted of N = 30 particles. The fraction of the charged particles in the chain, f, was varied from 20% to 80%. Electric neutrality of the system was maintained by adding of oppositely charged monomer-like counterions. The value of the Bjerrum length was fixed and equal to lB = 3σ, which corresponds to aqueous solutions.47 Connectivity of the particles into polymer chains was realized by the finite extension nonlinear elastic (FENE) potential

The degree of deacetylation of chitosan samples (Sigma-Aldrich) was measured by means of two different techniques: pH titration and IR specroscopy. For the pH titration each chitosan sample (60 mg) was dissolved in a slight excess of HCl (30 mL of H2O + 4 mL of 0.1 M HCl). Using the obtained dependency of pH on the volume of the added NaOH solution, we determined the volume, which was needed for deprotonation of whole chitosan. The degree of deacetylation was calculated using the equation

DD =

kBTlBqiqj

UFENE(rij) = −0.5kspringR 0

2

⎡ ⎛ rij ⎞2 ⎤ ⎢ ln 1 − ⎜ ⎟ ⎥ ⎢⎣ ⎝ R 0 ⎠ ⎥⎦

where kspring is the spring constant set to be equal to 7kBT/σ2 and the maximum extension of the bond is R0 = 2σ. The simulations were performed using the open source software LAMMPS.48 The simulation cell was a cubic box of the size Lx = Ly = Lz = 40σ. The number of polymer chains in the box was 50, which provided the dilute regime. The calculations were carried out in NVT ensemble with periodic boundary conditions. The electrostatic interactions between charged particles of the system were calculated by the Ewald summation method with the accuracy of 10−5. The constant temperature (kBT = 1) was maintained by coupling the system to the Langevin thermostat. At each time step all the particles were subjected to a random force and their velocities lowered by a constant friction. The average magnitude of the random forces and the friction are related in such a way that the “fluctuation-dissipation” theorem is fulfilled. The friction coefficient, ξ, was set to ξ = 0.1 μ/τLJ, where τLJ = σ(μ/ kBT)1/2 is the standart LJ time. The velocity-Verlet algorithm with a time step Δt = 0.01τLJ was used for integration of the equations of motion.

× 100%

CNaOHΔVNaOH

where msample is the weight of the sample [g], CNaOH is an exact concentration of NaOH [mol/dm3], and ΔVNaOH is the volume of the solution of NaOH, required for the titration of amino groups [dm3]. The results of the measurements are shown in Table 1. The FTIR spectra of chitosan were obtained using Thermo Nicolet IS5 FT-IR device with ID5 ATR accessory (diamond crystal). The degree of deacetylation of chitosan samples was determined via comparative analysis of the optical density of 1655 and 3450 cm−1 bands in the FTIR spectra corresponding to the absorbance N-acetyl (1655 cm−1) and hydroxyl groups (3450 cm−1).44

3. COMPUTER SIMULATION We performed molecular dynamics simulations of a solution of the hydrophobic polyelectrolytes with implicit solvent molecules. A coarse-grained approach was used. Both charged and uncharged monomer units of the chains and counterions were modeled as Lennard-Jones particles (beads) of the diameter σ and the same mass μ. The interaction between any pair of the particles was described through the truncated-shifted LennardJones potential:45

4. RESULTS AND DISCUSSION Taking into account the measured heights, lengths and reconstructed widths of the structures obtained by the adsorption of chitosan macromolecules on the substrate from the solutions in carbonic acid, we estimated rather roughly that the volume of the single persistent structure should be in the range of 300−1000 nm3. Comparing it with the expected volume of a single chitosan chain (ca. 300 nm3), we concluded that our elongated structures might be composed by several macromolecules. There were reasons to consider the visualized isolated small persistent structures as monomolecular particles. But this hypothesis required an additional experimental verification. Therefore, we decided to vary the molecular weight of chitosan macromolecules in order to detect its possible influence on the sizes of the elongated structures. If the

⎧ ⎪ FLJ(rij) − FLJ(rcut) if rij ≤ rcut FLJSF(rij) = ⎨ ⎪ if rij > rcut ⎩0 ⎧ ⎪ULJ(rij) − ULJ(rcut) + (rij − rcut)FLJ(rij) if r ≤ rcut ULJSF(rij) = ⎨ ⎪ if r > rcut ⎩0

where ULJ(rij) = 4ϵαβ[(σ/rij)12 − (σ/rij)6], FLJ(rij) = ULJ ′ (rij), rij is the distance between two interacting beads, and rcut is a cutoff radius beyond which the interaction potential is equal to zero. Similarly to the model described in ref 46, we set the cutoff distance rcut = 2.5σ intermonomer interactions, and rcut = 21/6σ for all other pairwise interactions. The solvent quality is 5752

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Figure 1. Microphotographs of chitosan nanostructures on the mica surface, adsorbed from the solution in carbonic acid, chitosan concentration: 0.2 g/L. The samples differ in molecular mass Mn and degree of deacetylation DD: (a) 80 kg/mol, 84%; (b) 130 kg/mol, 70%; (c) 210 kg/mol, 70%; (d) 240 kg/mol, 74%; (e) 380 kg/mol, 67%; and (f) 680 kg/mol, 74%. Scan sizes 2 × 2 μm; scale bar 500 nm; height scale 10 nm.

size would grow with the increase of molecular weight, we would have the clear evidence that the observed structures are monomolecular ones. But if the size would not correlate with the molecular mass, we would have reasons to conclude that the persistent structures are aggregates with the sizes, stabilized by some phenomenon yet to be revealed. Therefore, we performed a set of additional systematic experiments of visualization on six types of different chitosan samples with different molecular masses and degrees of deacetylation. The results of these experiments are shown in Figure 1. In order to determine the molecular mass of different chitosan samples, we have used the GPC and viscosimetry (Table 1). Also, for these six samples we have systematically measured typical lengths and heights of the persistent structures as adsorbed from solutions in carbonic acid on the mica surface and observed with AFM. The results of these measurements are shown in Figures 2 and 3 and summarized in Table 1. From the comparison of the obtained data, we can conclude that the length of the elongated rod-like structures as adsorbed from the carbonic acid on the substrate is independent of the molecular mass. As one can see from Table 1, there is no correlation between the increase of the molecular mass and the values of the length (or height) of the structures. This may be considered as the evidence that the elongated nanostructures, which we observed in the AFM pictures, are not monomolecular formations. But nevertheless, the aggregation number of these formations is equal to several macromolecules because the volume of the aggregates is small (around 300− 1000 nm3). This is opposite to standard conformational behavior of chitosan in aqueous solutions, where much larger aggregates are typically formed.

Figure 2. Typical lengths of persistent rod-like chitosan structures adsorbed from solutions in carbonic acid.

The aggregation of the chitosan macromolecules in aqueous solutions may be related to (a) interaction of hydrophobic groups of chitosan units between each other, (b) formation of inter- and intramolecular hydrogen bonds, and (c) attraction 5753

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Concerning the possible influence of the process of adsorption on the conformation, it may be noted that recently Tiraferri et al.52 showed that adsorption process of chitosan in solutions in hydrochloric acid at pH = 4 is transport-controlled, meaning that all chitosan molecules approaching the surface successfully attach. The major reason for adsorption-frozen conformation of positively charged polyelectrolytes on negatively charged mica is the electrostatic attraction between NH3+ groups on chitosan chain and silicate groups on the mica surface. At the same time, at pH ranging from 2 to 3, the mica surface is only slightly negatively charged, and in this case the surface introduces minimal disturbance in polyelectrolyte molecular conformations. In our case of rather small aggregates consisting only of several macromolecules, this concept of adsorption-frozen conformation seems to be quite applicable as well because some of charged amino groups in chitosan polyelectrolyte chains remain beyond the ion pairs and are situated on the surface of elongated aggregates. Therefore, they remain to be responsible for Coulomb attraction of the aggregate as a whole to the mica surface. With all that said we assume that the substrate does not have any significant influence on the peculiar conformation of the obtained chitosan aggregates. In order to understand the main reason for the formation of aggregates for chitosan macromolecules, we have measured the DD of different chitosan samples by pH titration and IR spectroscopy. The results of the measurements are shown in Table 1. One can notice that the larger sizes of the aggregates reproducibly correspond to the higher degrees of deacetylation (i.e., larger fractions of charged units in the macromolecule in the solution of carbonic acid). The similar behavior with the same dependency was observed before for chitosan dissolved in 0.5 M acetic acid.53 In that article the dissolved objects were transferred from solutions onto a carbon-coated Cu grid and then dried under vacuum. Visualization of the aggregates of different DDs was performed by means of transmission electron microscopy. As a result, the authors detected the inverse relationship between the DD of the chitosan and the length of the aggregates. Therefore, there was the same dependency as we observed in our experiments. Therefore, we suggest the following scheme of the formation of chitosan aggregates in solutions in carbonic acid (see Figure 4). First of all, in solutions in carbonic acid chitosan macromolecules become protonated; i.e., amino groups of chitosan chains become positively charged. Further, negatively charged HCO3− ions in solution in carbonic acid start to form ion pairs with positively charged NH3+ groups of chitosan

Figure 3. Typical heights of persistent rod-like chitosan structures, adsorbed from solutions in carbonic acid, above the substrate level.

caused by the dipole−dipole interactions between ion pairs formed due to counterion condensation on oppositely charged monomers. Let us consider the last cause of aggregation in details. For this reason in our previous paper18 we calculated the permittivity for carbonic acid following the Kirkwood equation (εH2O+CO2 = 72) and after that the Bjerrum length for chitosan in this solvent (lB = 7.2 Å). Knowing the length of average chitosan monomer unit (b = 5.2 Å) and the fraction of charged units ( f = 67−84), we estimated that our conditions correspond to the increased Manning condensation of counterions (ξ ≈ 1.3).49 This means that a lot of counterions for chitosan macromolecule in carbonic acid form ion pairs with the charges of monomer units. It is well-known that ion pair formation promotes aggregation of the macromolecules.50 Indeed, as a result of charges compensation chitosan chain could adopt more compact conformation when besides the repulsion electrostatic forces there is attraction between monomer units because of dipole−dipole interaction of coupled ion pairs. This effect reminds the collapse caused by the additional ionization described in the corresponding article.51 With the increase of degree of ionization the macromolecule swells generally because of the increase of osmotic pressure of counterions. But together with that the possibility of counterion condensation on the polymer chain appears. In our case it should correspond to the formation of the aggregate by means of attraction due to the ion pair formation. Theory predicts that if one takes into account that in the condensed state the permittivity is even lower due to the lower water content, the condensed or aggregated state, with most of polymer chain ions forming ion pairs, can become the most thermodynamically advantageous.

Figure 4. Hypothetical scheme of the “cigar-like” aggregate of several chitosan macromolecules in solution in carbonic acid. 5754

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cluster of the radius R which is formed by m flexible polymer chains, each of N segments. The segment has the length a and the excluded volume v ≈ a3. The polymer volume fraction of the cluster ϕ is controlled by the solvent quality. Let us denote by f ̃ the fraction of bare (uncompensated) charges per chain, which provide long-range Coulomb repulsion. Short-range dipole−dipole attraction of the ionic groups with condensed counterions, hydrogen bonding, and hydrophobic interactions will be quantified by the surface tension coefficient γ. The free energy of the cluster per chain, F/m, can be written as sum of the surface and Coulomb energies:55−59

macromolecules. As a consequence of this process, neutralization of the part of the charges along the chitosan chain and dipole−dipole attraction between the formed ion pairs occurs, thus resulting in aggregation of the chitosan chains. Moreover, the presence of hydrophobic groups along the polymer chains and the ability of chitosan macromolecules to form hydrogen bonds lead to the additional attraction and aggregation of chitosan macromolecules. Nonetheless, some of charged amino groups in chitosan chains remain beyond the ion pairs. Therefore, they remain to be responsible for electrostatic repulsion inside and between the macromolecules as well as for stabilization of the size of the aggregates. As a result of competition of the effects described above one should expect an elongated “cigar-like” shape of chitosan aggregates in solutions in carbonic acid. It is worthwhile to compare our observations with the results of Kocun et al.32 They reported extended and elongated single individual strands as well as aggregated bundles but without any kind of peculiar cigar-shaped morphology for chitosan in acetate buffer at pH 5.1 in the presence of high amount of additional salt (sodium acetate). Yet, the presence of salt at high ionic strength is known to reduce the electrostatic contributions (including attraction of ion pairs), screen ionic sites along the chains, and somewhat suppress Manning condensation. Indeed, according to Fenley et al.54 the amount of coupled counterions decreases along with an increase of the amount of salt. Moreover, if we take into account that under conditions of our research of mild ionic strength the system is only slightly above the limit of Manning condensation, the higher ionic strength of solution as presented in Kocun et al. research should result in being below this limit. These conditions should lead to the suppressed aggregation and ability to visualize single elongated chitosan macromolecules as reported by Kocun et al.32 The narrow size distribution of the chitosan aggregates in the solution in carbonic acid can theoretically be argued. For the case of polymers of simpler primary structureassociating polyelectrolytes comprising strongly associating (insoluble), neutral soluble, and fully dissociated charged monomer units spherical55,56 and nonspherical57 aggregates of optimum size (diameter) can be formed in solutions. The physical reason for that was shown to be a competition between short-range attraction of the insoluble groups and long-range Coulomb repulsion of charged species. As a result, the size of the aggregates does not depend on the length of the chains. It is primarily controlled by the fraction of the charged units: the higher the fraction, the smaller the size of the aggregates.55,56,58 But in these previous works the contribution of dipole−dipole attraction caused by counterion condensation (ion pair formation) was neglected (weakly charged associating polyelectrolytes form swollen clusters). Owing to more complex primary structure of chitosan, where the majority of the monomer units (hydrophobic, the units forming hydrogen bonds and ionic pairs) can be attributed to “insoluble” ones, the model of hydrophobic polyelectrolyte chains34 is more relevant in comparison with the model of strongly associating polyelectrolytes.55−58 However, in contrast to the model by Dobrynin et al.34 predicting stability of single necklace globules in the dilute regime, aggregation of the hydrophobic polyelectrolyte chains and formation of the optimum size clusters is also possible. The aggregation can easily be demonstrated via minimization of the free energy of the cluster per chain.57,59 Let us first consider the spherical

(ef ̃ Nm)2 4πR2γ 3γ Na F = + const = ̅ mkBT mkBT εRmkBT Rϕ 2 4 2 l R + const πNϕf ̃ B 3 v

(1)

where the last equality is obtained with the space-filling condition 4πR3ϕ/3 = vmN. Here, kBT, γ,̅ and lB are the thermal energy, dimensionless surface tension coefficient, γ ̅ = a2γ/kBT, and the Bjerrum length, lB = e2/εkBT, respectively. Minimization of eq 1 with respect to the radius R leads to independence of the cluster size on the 2 2 1/3 length of the polymer chain N, R ∼ a(aγ/l ̅ B f ̃ ϕ ) , and strong dependence of the aggregation number on N, m ∼ (1/N)(aγ/̅ lB f2̃ ϕ). By the definition, m > 1. Therefore, the aggregation of the chains into the cluster is possible, if aγ ̅ > lBNf2̃ ϕ; i.e., the surface tension coefficient has to be high enough and the fraction of bare charged units has to be small. Otherwise (m < 1) the intramolecular “clusters” or necklace globules are formed.34 Similar estimates can be done for the cigar-like (cylindrical) clusters of the length L and thickness R ≪ L. The dominant contribution to the free energy takes the form (ef ̃ Nm)2 2γ Na 2πRLγ F ≈ + const = ̅ mkBT mkBT εLmkBT Rϕ + const ·πNϕf ̃

2 2 lBR

v

(2)

with the space-filling condition πR2Lϕ = vmN. The free energy allows calculation of the thickness of the cluster via minimization with respect to R. The obtained value R ∼ 2 2 1/3 a(aγ/l ̅ B f ̃ ϕ ) is similar to that for the spherical clusters (the difference comes from the numerical factor); i.e., it does not depend on the chain length. Therefore, we can conclude that the competition between the long-range electrostatic repulsion and short-range attraction of the “insoluble” monomer units is responsible for stabilization of the thickness of the cylindrical clusters. The contour length of the clusters L can be estimated from more subtle interplay between surface energy of the cluster’s ends and the energy of thermal motion of the clusters. The first one tends to minimize the overall area of the cluster’s ends via end-to-end aggregation of them into very long cluster. However, this process is unfavorable due to the entropic reasons: few shorter clusters have more degrees of freedom of translational motion than the longer cluster. The optimum length can be calculated if we write down a correction to the free energy (2), δF, |δF| ≪ F, including these contributions 5755

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Figure 5. Snapshots of the polyelectrolyte chains in poor solvent for different values of the fraction of charged monomer units per chain, f, and attraction energy between monomer units, ϵ. Brown squares depict snapshots where most of the clusters have elongated shape.

4πR2γ ϕ̅ 4γ Na δF 1 ≈ + ln = ̅ mkBT mkBT m mNe Lϕ +

ϕ̅ v Nv × ln 2 ϕπR L ϕπR2Le

which were oriented in parallel relative to each other. Depending on parameters of the system (solvents quality and fraction of charged units), the number of the simulation steps varied between 4 × 107 and 108. The typical snapshots of the aggregates are presented in Figure 5 in variables: attraction energy between monomer units ϵ and fraction of charged groups per chain f. Both stability of the single molecules and aggregation of them into the spherical and elongated clusters are possible depending on the parameters of the system. Reliably detectable peculiarities include decrease of the size of the spherical clusters with the increase of the fraction of charged groups (ϵ = 1.1, f = 20%−40%) with ultimate disaggregation at f = 80%. Furthermore, the clusters have very narrow size (aggregation number) distribution; i.e., they are really optimum.55−58 For example, 6 clusters of the aggregation number 6 and 2 clusters of the aggregation number 7 are formed at ϵ = 1.1, f = 30% (Figure 5). Thus, the computer simulations of the spherical cluster formation confirm the simple analytical calculations done after eq 1. Another feature of the diagram in Figure 5 is that the increase of the short-range attraction between monomer units of the chains (ϵ) is responsible for elongation of the clusters. The spherical clusters aggregate with each other forming worms. Brown squares in Figure 5 depict snapshots where most of the clusters have elongated shape. Like in the case of the spherical clusters, the increase of the fraction of charged groups leads to the decrease of the diameter of the worms with their subsequent decomposition into the spherical clusters. For example, the average thickness of the clusters at ϵ = 2, f = 40% corresponds to 3.8 (in units of the bead size), whereas it is 2.2 at ϵ = 2, f = 50% (Figure 5). Furthermore, the thickness of the clusters obtained at ϵ = 2, f = 50% has very narrow distribution: 2.1, 1.9, 2.1, and 2.5, which supports the concept of the optimum thickness of the

(3)

and minimize this correction with respect to L: L=

⎛ 4πR2γ ⎞ ϕ̅ v exp⎜ 2 ̅ ⎟ 2 ϕπR ⎝ a ⎠

(4)

Here ϕ̅ is the average polymer volume fraction in the dilute solution, ϕ̅ ≪ 1. Keeping in mind that the thickness of the cylindrical cluster R does not depend on the chain length N, the length of the cluster is also independent of N. Similar to the spherical clusters, the aggregation number of the cylindrical clusters is inversely proportional to N: m=

⎛ 4πR2γ ⎞ ϕ̅ exp⎜ 2 ̅ ⎟ N ⎝ a ⎠

(5)

We can see from eqs 1 and 2 that the surface contribution to the free energy is lower for the cylindrical clusters (coefficients 3 and 2, respectively) and the Coulomb contributions have similar dependence on the parameters of the system. Therefore, one can expect that increasing the surface tension coefficient can induce transformation of the spherical clusters into the cylindrical ones. Keeping in mind that aggregation of the chitosan chains is caused by strong enough forces (like hydrogen bonding), one can expect high enough value of the surface tension coefficient which is responsible for stability of the cigar-like aggregates. This effect is demonstrated by computer simulations (see description in section 3). The initial structure of the molecules in the simulation box corresponded to an array of fully stretched polymers chains 5756

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cylindrical clusters. On the contrary, the average (contour) length of the worms increases with ϵ (f = 50% in Figure 5). However, they become more compact, “collapsed” at higher values of ϵ (2.5 and 3), which minimizes the area of unfavorable contacts of the worms with the solvent. The results of the computer simulations related to the formation of worm-like aggregates are qualitatively consistent with the experimental images shown in Figure 1.

5. CONCLUSION We address the promising approach to adsorb chitosan coatings from solutions in carbonic acid. We found out that during the adsorption from such solutions on a model substrate chitosan amphiphilic macromolecules self-organize into elongated rodlike structures. These structures are aggregates composed by several macromolecules. The complex of the obtained data allows us to assume that the attraction caused by the insoluble hydrophobic groups, hydrogen bonding, and formation of ion pairs in combination with Coulomb repulsion plays an important role in the formation of aggregates of the chitosan macromolecules in carbonic acid. These data are strongly supported by theoretical calculations and computer simulations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (M.A.P.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The reported studies were supported by the Russian Foundation for Basic Research, projects 13-03-00378-a and 14-03-31416-mol_a. The simulations were performed on multiteraflop supercomputers Lomonosov and Chebyshev at Moscow State University.

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