Salt-Induced Thermoresponsivity of a Cationic Phosphazene Polymer

Oct 2, 2018 - ... P. Moskalets† , Vladimir S. Papkov† , and Alexei R. Khokhlov§ ... of Biochemical Physics, Russian Academy of Sciences, Kosygin ...
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Salt-Induced Thermoresponsivity of a Cationic Phosphazene Polymer in Aqueous Solutions Tatiana V. Burova,† Valerij Y. Grinberg,*,†,‡ Natalia V. Grinberg,† Alexander S. Dubovik,‡ Alexander P. Moskalets,† Vladimir S. Papkov,† and Alexei R. Khokhlov§ †

A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov St. 28, Moscow 119991, Russia N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin St. 4, Moscow 119991, Russia § M.V. Lomonosov Moscow State University, Leninskie Gory 1, Moscow 119991, Russia Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on October 6, 2018 at 04:29:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Poly(ethylaminophosphazene) (PEAP) in salt-free aqueous solution does not reveal thermoresponsive properties but acquires thermoresponsivity upon addition of various poly(protic acid)s. Energetics of the salt-induced phase separation transition of the PEAP solutions upon heating was investigated by highsensitivity differential scanning calorimetry at pH 3.5. At low concentrations of poly(carboxylic acid)s (10−100 mM), the transition temperature and enthalpy vary from 90 to 25 °C and from 1.5 to 35 J g−1, respectively, depending on the pKa1 and the apparent hydrophobicity of the acids. Dependences of the transition parameters (Tt, Δth, and Δtcp) on the citrate buffer concentration were obtained. The binding curve of citrate anions to PEAP was derived. High concentrations of sulfate anions (0.1−0.8 M) induced a marginal phase transition at high temperatures. A mechanism of the PEAP thermoresponsivity is proposed. The Okado−Tanaka model for cooperative hydration of macromolecules is used to quantitatively describe the energetics of the thermoresponsive behavior of PEAP.

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INTRODUCTION Water-soluble thermoresponsive polymers occupy an appreciable position among “smart” functional polymers destined for drug delivery systems.1−4 Thermotropic reversible phase transitions of the functional polymers, polyelectrolytes in particular, provide a unique opportunity for the conformationdependent binding and release of target ligands controlled by the temperature. Despite a growing interest for the synthetic thermoresponsive polymers, their use in drug delivery is a challenge since most of them are not biodegradable while some are toxic.5,6 A combination of the thermoresponsive properties of the polymers with their biodegradability and biocompatibility is of vital significance for biomedical applications.2,7,8 Such a combination is an inherent feature of some polyorganophosphazenesa class of hybrid polymers composed of a backbone of alternating nitrogen and phosphorus atoms and organic side chains.9−15 The polyphosphazene backbone can be disintegrated either hydrolytically or enzymatically in mild physiological conditions.16,17 At a definite chemical composition of the side groups, the polyphosphazenes degrade to harmless and easily excreted low-molecular-weight products.10,12,13,15,18−21 Moreover, it has been shown that the hydrolytic stability of polyphosphazenes can be fine-tuned by selection of the hydrophobicity/ hydrophilicity of the substituent groups.8,10,13,15,19,21 Some polyphosphazenes can acquire polyelectrolyte properties upon protonation of the nitrogen atoms in the backbone22,23 that provide a binding affinity for drugs, proteins, DNA, and other amphiphilic substances of biomedical significance.24−27 © XXXX American Chemical Society

To date, numerous thermoresponsive polyphosphazenes of a diverse chemical composition have been synthesized and investigated.15,23,28−31 The phase transition temperature varies within rather large range from 20 to 100 °C.10,11,13,14,19 It is defined by the polyphosphazene hydrophobic/hydrophilic balance which can be tuned by variations in chemical nature and ratio of the side substituents.23,32−36 The larger the apparent hydrophilicity of the polyphosphazene, the higher the LCST of its aqueous solutions. It has been shown that the phase transition of polyphosphazene solutions can be shifted by changing the solvent composition. The transition temperature is sensitive to the composition of the mixed water−organic solvent (increases with an increase in the hydrophobicity of the organic component),30 to pH (decreases with the increasing pH),15 to concentrations of organic and inorganic salts (decreases in the presence of salting-out salts and increases in the presence of salting-in ones),15 and to the surfactant concentrations (increases notably at low and decreases at high surfactant concentrations).31 Effects of mono-, di-, and polysaccharides on the thermoresponsive polyphosphazenes with methoxypoly(ethylene glycol) and amino acid esters as side groups have been investigated.37 Most of the saccharides decreased the transition temperature of the polymers. The decrease was more prominent for galactose-containing saccharides. The saccharide Received: July 29, 2018 Revised: September 18, 2018

A

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poly(ethylaminophosphazene) in citrate buffer with pH 3.5 undergo thermotropic phase separation transition.22 In this work effects of various poly(protic acid)s on the energetics of the PEAP phase transition have been studied by highsensitivity differential scanning calorimetry. The possible mechanism of the induced thermoresponsivity of the polymer is proposed.

effect was almost independent of the kinds of the amino acid esters and PEG length in the polyphosphazene side groups that implies a key role of a solvent composition in the polyphosphazene phase transition. Thus, numerous available data on a great variety of the synthesized thermoresponsive biodegradable polyphosphazenes and on the possibilities of fine-tuning their LCST imply a remarkable applied potential of these polymers for biomedicine. A particular attention was paid to the design of so-called injectable thermoresponsive hydrogels forming when the polyphosphazene−drug solution entered the body and reached physiological temperatures.38−44 Accordingly, the formation conditions and characteristics of physical polyphosphazene hydrogels, such as rheological and mechanical properties and the affinity for ligands, were investigated in great detail. The polymer concentrations in gels are rather high, which complicates a rigorous quantitative analysis of mechanisms of thermoresponsivity and of possible associated conformational transitions on the molecular level. This in turn hinders our understanding of the key relations between the environment factors and the macromolecular chemical structure responsible for thermoresponsivity. In a certain sense, the empirical information in this field outpaces deep fundamental understanding of the mechanisms defining the polyphosphazene thermoresponsivity. Such an understanding is, however, absolutely necessary for the prediction and control over thermoresponsivity, for the optimization of the transition temperature interval for a specific system, and for the diversification of the thermoresponsive systems without the need to synthesize a large number of test macromolecules. It is believed that thermoresponsive behavior is, first of all, an inherent feature of the macromolecule, while the solvent composition is a secondary factor serving just for the finetuning the transition area to the desired one. Indeed, all the until now investigated polymers possessed a thermoresponsive behavior in salt-free solution. A question arises: will a nonthermoresponsive macromolecule acquire thermoresponsivity upon changing the composition of aqueous solvent? The first positive answer to this question has been reported in our previous study of the energetics of the thermotropic collapse of cross-linked polyphosphazene hydrogels induced by a solvent composition.45 In this paper we report data on the thermoresponsive behavior of poly(ethylaminophosphazene) (Scheme 1) in

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EXPERIMENTAL SECTION

Poly(ethylaminophosphazene) was synthesized by exhaustive aminolysis of a linear poly(dichlorophosphazene) (MW ∼ 13 × 106) with ethylamine as reported early.22,23,27 A complete replacement of chlorine atoms in the precursor polymer by ethylamine was detected by means of element analysis as well as IR and 31P NMR spectroscopy. Poly(ethylaminophosphazene) hydrochloride (PEAP) was prepared by dissolution of poly(ethylaminophosphazene) in 0.01 M HCl with subsequent lyophilization. Poly(carboxylic acid)s, sodium sulfate, and chloride were of reagent grade. PEAP molecular weight of 320 kDa was determined by the sedimentation velocity method in 40 mM glycine buffer in the presence of 0.15 M KCl at pH 3.5 using the partial specific volume of the polymer v = 0.683 cm3 g−1 measured densimetrically. Solutions of PEAP for calorimetric measurements were obtained by dissolution of the lyophilized polymer preparation in poly(carboxylic acid) buffer solutions of pH 3.5. Two procedures were applied for the preparation of the buffer solutions. In the first procedure a poly(carboxylic acid) was dissolved in deionized water with the pH adjustment to pH 3.5 by 0.1 M NaOH. In the second procedure the poly(carboxylic acid) was first neutralized with an equimolar amount of NaOH, and then the pH was adjusted to pH 3.5 with HCl. The first and second procedures resulted in a relatively low and high ionic strength of the buffer solutions of 0.06 ± 0.02 and 0.22 ± 0.01, respectively. Aqueous solutions of the sodium sulfate and sodium chloride were prepared by dissolution of the salts in deionized water with a subsequent adjustment of the pH to 3.5. Calorimetric measurements were performed with a differential adiabatic scanning microcalorimeter DASM-4 (BIOPRIBOR, Russia) within the temperature range 10−120 °C at a heating rate of 1 K min−1 and an excess pressure of 0.25 MPa. The primary data processing and conversion of the apparent partial heat capacity of PEAP into the excess heat capacity function of the phase transition was performed using the NAIRTA 2.0 software (A.N. Nesmeyanov Institute of Organoelement Compounds, Moscow, Russia). The baseline in the transition area was constructed by a spline interpolation of the linear segments of the apparent heat capacity function below and above the transition temperature to the temperature of the peak maximum. The maximum temperature of the excess heat capacity curve was taken as the transition temperature, Tt. The transition enthalpy, Δth, was determined by integration of the excess heat capacity function. The transition heat capacity increment, Δtcp, at the transition temperature was calculated as the difference in the apparent heat capacities of PEAP after and below the transition area, approximated by linear functions and extrapolated to the transition temperature. The transition width ΔtT was determined as a ratio of the transition enthalpy to the maximal value of the excess heat capacity. We have tested thermosensitivity of all poly(protic acid)s used in our work by means of DSC. The heat capacities of 100 mM aqueous solutions of the acids at pH 3.5 were recorded against water within the temperature range 10−100 °C at a heating rate of 1 K min−1. No any characteristic features of thermal transitions (peaks or curvature) were observed on the thermograms. In addition, as checked by visual inspection, the acid solutions remained single phase over this temperature range. Thus, the poly(protic acid)s under consideration do not reveal the thermoresponsivity. Fitting and simulation calculations were performed using the Minerr and LeastSquaresFit functions of Mathcad 14.

Scheme 1. Chemical Structure of Poly(ethylaminophosphazene)

aqueous solutions induced by a definite ionic composition of the solvent. In acidic medium this polymer carries a rather large positive charge (∼1 per 2 monomer units). The polycationic nature of this polymer ensures its beneficial functionality with respect to amphiphilic anionic ligands, drugs in particular. It should be stressed that a salt-free aqueous solution of this polymer does not reveal any phase transition upon temperature variations within the temperature range from 0 to 130 °C. Earlier we reported that solutions of B

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RESULTS AND DISCUSSION Phase Transition in the PEAP Solution Induced by Poly(carboxylic acid)s. We have investigated thermotropic phase behavior of aqueous solutions of PEAP at pH 3.5 in aqueous buffer solutions of different poly(carboxylic acid)s: oxalic, malonic, citric, succinic, glutaric, and adipic acids. They differ by values of the first dissociation exponent, pKa1, and the carbon number, CN, in aliphatic bridges (Table 1). The acids

which is manifested as a high and narrow heat capacity peak within the temperature interval of 20−60 °C (Figure 1a). In contrast, succinic, glutaric, and adipic acids cause a marginal transition of a large width and small height at notably higher temperatures of 50−90 °C (Figure 1b). The nature of the salt-induced transition of PEAP is illustrated in Figure 2. For each carboxylic acid the transition

Table 1. First Dissociation Exponent, pKa1, and the Carbon Number, CN, in Aliphatic Bridges of the Poly(carboxylic acid)s acid

pKa1

CN

oxalic malonic citric succinic glutaric adipic

1.27 2.86 3.13 4.21 4.34 4.42

0 1 2 2 3 4

Figure 2. Calorimetric transition temperature vs cloud point temperature for 2.5 mg mL−1 solutions of PEAP in different solvents at pH 3.5: (1) 100 mM malonic buffer (M, I = 0.087), 100 mM citric buffer (C, I = 0.048), 100 mM adipic buffer (A, I = 0.014), 100 mM oxalic buffer (O, I = 0.141), and 100 mM glutaric buffer (G, I = 0.016); (2) 100 mM malonic, citric, adipic, glutaric, and succinic buffers with I = 0.22 ± 0.02. The Pearson’s correlation coefficient is 0.998. The mean ratio Tt/Tcp is 1.03 ± 0.03.

with pKa1 ∼ 1−3 belong to so-called “strong acids”. These are oxalic, malonic, and citric acids. Accordingly, the acids with pKa1 > 4, such as succinic, glutaric, and adipic, are classified as “weak acids”. Among the investigated acids, oxalic and adipic acids are the least and most hydrophobic, respectively. All the investigated acids, except citric acid, have two carboxylic groups in their structure, thus being classified as dicarboxylic acids. Citric acid has three carboxylic groups in its structure; however, only two of them are apparently dissociated at pH 3.5.46 For this reason, citric acid can also be classified in the frame of our study as a dicarboxylic acid. Further in the text we will keep this name for all carboxylic acids under consideration. Figure 1 shows the apparent partial heat capacity of PEAP in a salt-free aqueous solution and in the buffer solutions of 0.1 M

temperature of PEAP estimated by DSC is plotted against the cloud point temperature, Tcp, determined by visual observation during the heating of the PEAP solutions with a constant rate of 1 K min−1. The data were obtained for the buffer solutions of low and high ionic strength. A perfect linear correlation between Tt and Tcp values with a slope of 1.03 ± 0.03 implies that the calorimetric data refer to the phase separation of the PEAP solutions independently of the ionic strength of buffer. Thermodynamic parameters of the PEAP phase transition induced by various acids are compared in Figure 3.

Figure 1. Apparent partial heat capacity of PEAP at pH 3.5 in different solvents: 1, the salt-free water; 2, 100 mM oxalic buffer (I = 0.141); 3, 100 mM citric buffer (I = 0.048); 4, 100 mM malonic buffer (I = 0.087); 5, 100 mM succinic buffer (I = 0.047); 6, 100 mM glutaric buffer (I = 0.016); 7, 100 mM adipic buffer (I = 0.014). The ionic strength of each buffer is given in the brackets. The polymer concentration is 2.5 mg mL−1.

dicarboxylic acids at pH 3.5. First of all, it is evident that the salt-free aqueous solution of PEAP does not reveal any thermal events, i.e., does not possess thermoresponsive properties. However, in the buffer solutions of dicarboxylic acids endothermic heat capacity transitions appear, indicating a thermoresponsive behavior of PEAP. One can divide the investigated acids into two groups (Figure 1a,b) with respect to their effect on the thermal behavior of PEAP. Oxalic, citric, and malonic acids induce an essentially cooperative transition

Figure 3. Transition temperature (a), enthalpy (b), heat capacity increment (c), and width (d) of the phase separation transition for 2.5 mg mL−1 solutions of PEAP in different 100 mM polycarboxylic buffers with pH 3.5: (O) oxalic, (M) malonic, (C) citric, (S) succinic, (G) glutaric, (A) adipic. Ionic strength of the buffers was varied over the range 0.02−0.1. C

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Macromolecules Three strong acids (oxalic, citric, and malonic) induce the most pronounced transitions of high enthalpy (∼30 J g−1) and narrow width (∼3 °C). These transitions are accompanied by the large negative heat capacity increment. The transitions induced by the weak acids (succinic, glutaric, and adipic) are characterized by notably lower enthalpy and larger width values. The corresponding heat capacity increments are close to zero. The transition temperatures vary significantly within both groups of the dicarboxylic acids. The transition temperature is likely determined by two main factors: electrostatic (related to pKa1) and hydrophobic (determined by a number of CH2 groups in the structure of acid bridge). The lower the pKa1 and the larger the number of CH2 groups, the lower the transition temperature. Mechanism of Binding of Citrate Anions to PEAP. Let us consider in detail the induction effect of anions on the thermotropic phase behavior of the PEAP solutions on the example of citrate anions. Figure 4 shows the excess heat

Figure 5. Dependences of the transition temperature (a), enthalpy (b), heat capacity increment (c), and width (d) for 2.5 mg mL−1 aqueous solution of PEAP on the citrate buffer/PEAP molar ratio at pH 3.5. CCB is a citrate buffer concentration, and C0 is the PEAP monomer unit concentration.

phosphazene) hydrogels induced by salts of poly(protic acid)s, particularly by the phosphates.45 Analyzing our data on the hydrogels, we have suggested that the origin of their induced thermoresponsivity is related to binding of the water structure making anions to the polycationic phosphazene network. The binding leads to the formation of the extended and highly ordered structure of hydration shells of the polyphosphazene subchains which protects them from self-association. The melting of this structure upon heating triggers the hydrophobic interaction of the polyphosphazene nonpolar side chains that leads to self-association of the subchain and, finally, to the gel collapse. Apparently, these concepts could be applied to the phase separation of the PEAP aqueous solutions upon heating induced by salts of poly(carboxylic acid)s, particularly the citrates. In this case, the transition enthalpy (Δth) can be considered as a measure of the binding degree of citrate anions (r) as a ligand to the PEAP macromolecule:

Figure 4. Excess heat capacity functions of 2.5 mg mL−1 solutions of PEAP at pH 3.5 in 10 (1), 20 (2), 30 (3), 40 (4), and 100 mM (5) citrate buffers.

capacity functions of the polymer phase transition at different concentrations of the citrate buffer. At low citrate concentrations (of about 0.01 M), a marginal transition peak of low cooperativity is observed. Upon increasing the citrate concentrations, the transition peak shifts markedly to lower temperatures, increases in height, and becomes narrower. At the citrate concentration of ∼0.1 M, the transition peak approaches near physiological temperatures and becomes distinctly high and narrow reflecting a greatly cooperative event. The dependences of the transition temperature, enthalpy, heat capacity increment, and width of the phase separation transition of the aqueous PEAP solution on the citrate buffer/ PEAP molar ratio are given in Figure 5. The transition temperature decreases gradually by about 30 °C within the range of citrate concentrations 0.01−0.1 M (Figure 5a). The transition enthalpy is outlined by an increasing sigmoidal function reaching a saturation value of 30 J g−1 (Figure 5b). The transition heat capacity increment does not practically depend on the citrate concentration (Figure 5c). The average Δtcp value of −1.04 ± 0.10 J g−1 K−1 points to a significant decrease in the accessibility of nonpolar groups of PEAP to water molecules caused by the phase separation.47 It indicates a clusterlike structure of the polymer in the concentrated phase formed after the phase separation. The transition width decreases substantially, indicating an evident enhancement of cooperative properties of the system. The obtained characteristic changes in the transition parameters of the PEAP solution with the citrate buffer concentration reproduce in general the main features of the thermotropic collapse of poly(methoxyethylamino-

Δt h ∝ r

(1)

where r = Lb/mM; Lb is the concentration of the bound ligand, m is the number of sites in the PEAP macromolecule, and M is the concentration of PEAP. Thus, the apparent binding degree of citrate anions as the ligand to PEAP could be assumed as r = Δt h(L T)/Δt h(L Ts)

(2)

where LT is the total concentration of the ligand, and LsT = 100 mM is the concentration of the citrate buffer at which the dependence Δth(LT) reaches a saturation (Figure 6, 1). According to the model of cooperative binding of small ligands to linear polymer matrix48 r(1 − r ) s × q̃ = (1 − 2r )2 (1 − s)2

(3)

where q̃ is the parameter of cooperativity and s = KLf , where K is the cooperative binding constant and Lf is the free ligand concentration. If M* = mM is the concentration of sites in the system, then it is evident that s = K (L T − rM *)

(4)

It is reasonable to assume that the binding sites are formed by ionized monomer units of PEAP. In this case D

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Figure 7. Excess heat capacity functions of 2.5 mg mL−1 solutions of PEAP in 100 mM succinate buffer with pH 3.5 at different concentrations of sodium chloride, M: 0 (1), 0.025 (2), 0.05 (3), 0.1 (4), and 0.2 (5).

Figure 6. Apparent binding degree of citrate anions as a ligand to PEAP at pH 3.5 vs the total citrate anions concentration: (1) experimental; (2) calculated by the model of cooperative binding of small ligands to the polymer matrix48 at the cooperative binding constant K = 94.1 ± 5.8 M−1 and the cooperativity parameter q̃ = 7.2 ± 1.9. The standard fit error and the Pearson’s correlation coefficient are ±0.03 and 0.997, respectively. The total citrate anions concentration was calculated by the Bate software (ChemBuddy, Poland).

M * = α(c /M 0°)

NaCl. When the NaCl concentration increases, the transition peak shifts notably to the lower temperatures, increases in height, and becomes substantially narrower. These features of the PEAP thermoresponsive behavior induced by NaCl essentially resemble those observed in the presence of a strong acid, for example, citric one (Figure 4). The dependences of the transition parameters for the PEAP−succinate system on the NaCl concentration are given in Figure 8.

(5)

where α is the ionization degree of PEAP; c = 2.5 mg mL−1 and M◦0 = 133 g mol−1 are the weight concentration of PEAP and the molecular weight of its monomeric unit, respectively. The pH titration data for octaethylaminocyclotetraphosphazene49 allow one to estimate α ≃ 0.5 for PEAP at pH 3.5. Equations 3−5 were well fitted to the experimental dependence r(LT) using K and q̃ as adjusted parameters (Figure 6, 2). It was obtained that K = 94.1 ± 5.8 M−1 and q̃ = 7.2 ± 1.9. According to a definition, the intrinsic binding constant is Kint = K/q̃ = 13.0 ± 4.2 M−1. It follows that the standard free energy of a single bond NH+···−OOC− R(COOH)2 in the PEAP−citrate complexes is about of −6.2 ± 0.8 kJ mol−1. This value agrees well with an estimate of the standard free energy of a single ionic bond between biogenic polyammonium cations and carboxylate anions equal to −5.7 kJ mol−1.50 Such an agreement confirms our hypothesis that the origin of the induced thermoresponsivity of PEAP is related to the cooperative binding of citrate anions by the polyphosphazene matrix providing formation of an extended ordered hydration structure of PEAP. In other words, PEAP changes its hydrophobic−hydrophilic balance as a result of formation of the extended complexes with the citrate anions. These complexes acquire a thermoresponsive behavior. Effect of NaCl on the PEAP Phase Transition. In terms of the above proposed hypothesis, the cooperative anion− polycation binding accompanied by ordering of the PEAP hydration shell is the key factor responsible for the induced PEAP thermoresponsivity. If so, the dissociation constants of the polyacids in aqueous solutions may serve as a quantitative measure of the anion binding capacity. Accordingly, factors affecting the dissociation constants in a known way should have an expected impact on the PEAP transition parameters. In particular, it is known that pK1a of poly(carboxylic acid)s decrease with an increasing concentration of NaCl.46,51−53 Consequently, addition of NaCl to the thermoresponsive PEAP solution should facilitate the acid dissociation, increase the anion concentration, and, as a result, enhance the binding effects of the anions. It means that upon addition of NaCl PEAP in solution of a weak acid could behave as in solution of a more strong acid. We have checked this hypothesis on the example of the complexes PEAP−succinate. Figure 7 shows thermograms of the phase transition of PEAP in 0.1 M succinate buffer at different concentrations of

Figure 8. Dependences of the transition temperature (a), enthalpy (b), heat capacity increment (c), and width (d) for 2.5 mg mL−1 solution of PEAP in 100 mM succinate buffer with pH 3.5 on the sodium chloride concentration.

Upon an increase in the NaCl concentration, the transition temperature nearly exponentially decreases, the transition enthalpy increases tending to saturation, the transition heat capacity increment gradually becomes more negative, and the transition width notably decreases. These dependences are quite similar to those obtained for PEAP in solution of the increasing concentrations of the citrate buffer (Figure 5). They are typical of the binding effects. In main features, the effect of NaCl on the PEAP phase transition is consistent with the proposed mechanism of the PEAP thermoresponsivity as a consequence of the polymer hydration shell ordering induced by the cooperative anion binding to cationic sites of PEAP. An alternative mechanism could be proposed which assigns the observed changes in the PEAP transition to the lyotropic effect of NaCl. Kosmotropic salts are known to decrease the transition temperature of thermoresponsive polymers due to the salting-out effect.54,55 It would be of interest to compare the effect of NaCl on poly(N-isopropylacrylamide) (PNIPAM) and PEAP−succinate complex. To do this, we will analyze E

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Macromolecules values of the initial slopes of the dependences of LCST for PNIPAM56 and Tt for PEAP (Figure 8) on the concentration of NaCl. These slopes can serve as a quantitative measure of the lyotropic effect of the salt on the polymer transition. For PNIPAM the dependence Tt(CNaCl) is a linear function with the slope dTt/dCNaCl = −5.3 °C mol−1. The corresponding values for other kosmotropic salts fall within the limits from −25 to −1.5 °C mol−1.56 It means that NaCl is a rather weak lyotropic agent.54 The calorimetric data for PEAP give the initial slope dTt/dCNaCl = −835 °C mol−1 (Figure 8) that exceeds the slope of the dependence Tt(CS) for PNIPAM by more than 2 orders. Evidently, such a steep decrease in the transition temperature cannot be explained by the lyotropic action of a weak salting-out agent such as NaCl. Thus, the changes in the transition parameters of PEAP caused by NaCl support the hypothesis on the cooperative binding of anions possessing the water structuring capacity as the key factor of the induced thermoresponsivity of the polyphosphazene. In the presence of NaCl, a marginal PEAP transition of low cooperativity in solution of the weak acids acquires all features of the energetically pronounced and highly cooperative transition typical for PEAP in solutions of the strong acids. Effect of Sulfate Anions on the Phase Transition of PEAP Solutions. It is well-known that the sulfate anions are considered as the most effective salting-out agent among ions with a water structure making capacity.57 Consequently, it was of principal significance to compare the effects of sulfate and citrate salts on the thermotropic phase transition of the PEAP solutions in view of its mechanism proposed above. Thermograms of PEAP in aqueous solutions at the different concentrations of sodium sulfate are presented in Figure 9.

Figure 10. Dependences of the transition temperature (a), enthalpy (b), heat capacity increment (c), and width (d) for 2.5 mg mL−1 aqueous solution of PEAP on the sodium sulfate concentration at pH 3.5.

concentration corresponding to characteristic changes in the transition parameters: a low-concentration range from 0 to about 0.1−0.2 M and a high-concentration range from 0.2 to 0.8 M. Within the low-concentration range, the changes in the transition temperature (Figure 10a), enthalpy (Figure 10b), and heat capacity increment (Figure 10c) resemble the “binding-type” curves observed for the system PEAP−citrate buffer (Figure 5) although with substantially lower amplitudes. The transition width remains almost unchanged (Figure 10d). In the high-concentration range the transition temperature and enthalpy are linear functions of the sulfate concentration (Figure 10a,b) that is typical of the lyotropic salt effect. An essential feature of the dependence of the transition enthalpy on the sulfate concentration is the absence of a saturation plateau (Figure 10b) which excludes the possibility of calculation of the binding degree of sulfate anions to PEAP. The heat capacity increment becomes substantially more negative (Figure 10c), pointing to an increase in the polymer surface inaccessible to water. The transition width tends to increase (Figure 10d). The broadening of the phase transition implies a decrease in the cooperativity of the system.58 This effect is opposite to the change in the transition width caused by citrate anions (Figures 4 and 5) which enhances the cooperativity of the PEAP transition. It is reasonable to assign the sulfate influence on the PEAP thermoresponsivity to a superposition of two effects: the anion binding and the lyotropic (salting-out) effect.55,59,60 The binding of the doubly charged sulfate anions to a polycation has obviously an anticooperative character, since the primarily bound ligand would retain an uncompensated negative charge and repulse a neighbor ligand. For a more clear illustration of the characteristic features of binding to a polymer matrix, we have performed a simulation of the binding curves for a small ligand in the case of its cooperative and anticooperative binding to a polymer matrix.48 The simulated curves are shown in Figure 11. It appears that the cooperative binding results in a steeply uprising binding curve which reaches saturation at relatively low ligand concentrations. A similar type of binding curve was observed for PEAP in the citrate buffer (Figure 6) as well as for the system PEAP−succinate in the presence of NaCl (Figure 8). The anticooperative binding curve does not reach saturation even at extremely high ligand concentrations.

Figure 9. Excess heat capacity functions of 2.5 mg mL−1 aqueous solutions of PEAP with pH 3.5 at different concentrations of sodium sulfate, M: 0.2 (1), 0.3 (2), 0.4 (3), 0.6 (4), and 0.8 (5).

The appearance of the heat capacity changes typical of the phase separation transition suggests an induction of a thermoresponsive behavior of PEAP by sulfate anions. However, we should note that an “active” range of sulfate concentrations affecting the PEAP transition is 0.1−0.8 M, which exceeds more than 2 orders of magnitude the concentration range of the citrate buffer (Figure 4). Within this large sulfate concentrations range, a notable shift of the PEAP heat capacity peak to the lower temperatures is observed upon increasing the concentration of Na2SO4. However, neither appreciable increase in height nor narrowing of the peak profile is observed in the case of the sulfate anions. Independently of the sulfate concentration, the peak height is markedly less than that one in the case of citrates. The dependences of the transition parameters of the PEAP solution on the sulfate concentration are plotted in Figure 10. For all plots one can discern two ranges of sulfate F

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Simulations of Thermograms of the Phase Transition of PEAP Liganded by Singly Charged Citrate Anions. It was shown by Okado and Tanaka61 that the cooperative hydration is a key factor of the thermoresponsivity of polymers in aqueous solutions. The phase separation of these solutions upon heating is a result of thermal dehydration of macromolecules. According to the Okado−Tanaka model, the hydration degree of macromolecules (θ) is a function of a generalized parameter q and the cooperativity parameters σ:

Figure 11. Simulated binding curves of citrate (1) and sulfate (2) anions as collective ligands to PEAP calculated by the model of cooperative binding of small ligands to a polymer matrix48 with the following model parameters: (1) the intrinsic free energy of binding ΔbGint = −5.7 kJ mol−1,50 the cooperative parameter q = 7.3 (cooperative binding); (2) the intrinsic free energy of binding ΔbGint = −7.0 kJ mol −1 , 50 the cooperative parameter q = 0.24 (anticooperative binding).

θ=

σqw1(q) 1 + σqw01(q)

(6)

É ÅÄÅ ÅÄÅ qφ ÑÉÑ σqw (q) Ñ Ñ λÅÅÅ1 − φ − φ 1 + σqw1 (q) ÑÑÑ expÅÅÅ 1 − φ − qφ ÑÑÑ Å ÑÖ ÅÇ ÑÖ 01 q= Ç 1 + σqw0(q)

where

Besides, the maximal value of the binding degree in this case is notably smaller than that for the cooperative ligand binding. Structural changes in the hydration shell of the PEAP caused by cooperatively and anticooperatively bound anions are schematically illustrated in Scheme 2 on the example of malonate (known as the most effective agent inducing thermoresponsivity) and sulfate anions. In the case of malonate anions the cooperative binding induces an extended ordering of the PEAP hydration shell in which an essential role is played by cooperative van der Waals interactions of the anion hydrophobic bridges and the side chains of PEAP (Scheme 2a). In contrast, in the case of anticooperative binding of sulfate anions, electrostatic repulsion of the bound ligands prevents the formation of the relatively extended hydration clusters (Scheme 2b). In this case the ordering of the hydration shell of PEAP is interrupted, and the system cooperativity is notably lower. For this reason, sulfate anions induce the marginal phase transition in the PEAP solutions. The shift of the PEAP transition temperature upon the increasing sulfate content is just a consequence of changes in solvent quality because of the salting-out effect. In this aspect sulfate anions can be hardly assigned to effective inducers of the polyphosphazene thermoresponsivity.

(7)

with λ and φ are the binding constant of water to polymer and the volume fraction of polymer, respectively. The functions w0(q), w1(q), and w10(q) have the forms n

w0(q) =

∑ qζ − 1 (8)

ζ=1 n

w1(q) =

∑ ζqζ− 1 (9)

ζ=1

w01(q) = w0(q) + w1(q)

(10)

where ζ is the length of a single hydration water sequence and n is the polymerization degree of polymer. The dependence of binding constant on temperature could be given by the van’t Hoff equation:62 ÄÅ É 1 yzÑÑÑÑ ÅÅÅ ΔbH ij 1 j − zzÑÑ λ = expÅÅ− ÅÅÇ R jk T T * {ÑÑÖ (11) where ΔbH is the binding enthalpy and T* is a reference temperature at which λ = 1. We will use further the van’t Hoff transition enthalpy ΔtHvh = −ΔbH for a convenience of using

Scheme 2. Apparent Chemical Structures of the Complexes of PEAP with Anions of Diprotic Acids on the Example of Malonate (a) and Sulfate (b) Anions at pH 3.5a

a

The complexes are formed as a result of the cooperative (a) or anticooperative (b) binding of the anions to PEAP. The red arrows represent ionic bonds between the anions and positively charged nitrogen atoms of the PEAP backbone. The blue shaded rectangles roughly represent the ordered regions of hydrophobic hydration of the PEAP chains. G

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Macromolecules the van’t Hoff equation for simulation of the phase transition caused by dehydration of macromolecules. As a rough approximation, it can be assumed that a heat of the phase separation of aqueous solution of thermoresponsive polymer is equal to the heat of dehydration of the polymer.61 Then the dependence of the current enthalpy of phase transition on temperature could be given in the form typical for thermochemistry: Δh(T ) = Δt h × β(T )

Δt hL = Δt h ×

M 0UL M 0L

(14)

L where MUL 0 and M0 are the apparent average molecular weights of monomer unit of the PEAP in the unliganded and liganded states, respectively. It could be suggested that MUL 0 = (1 − α) L ◦ HCIT M°0 + αMHCl and M = (1 − α)M + αM where α ≃ 0.5 is 0 0 0 0 the ionization degree of PEAP at pH 3.5, M◦0 = 0.133 kDa is the molecular weight of uncharged monomer unit of PEAP, MHCl = 0.1695 kDa is the molecular weight charged monomer 0 = 0.3251 unit of PEAP in the hydrochloride form, and MHCIT 0 kDa is the molecular weight of charged monomer unit of PEAP liganded by the singly charged citrate anion. In that case MUL 0 = 0.151 kDa, ML0 = 0.229 kDa and ΔthL = 20.8 J g−1. The ratio ΔtHvh/ΔthL could be considered as a rough estimate of the apparent molecular weight of hydration site of the PEAP liganded by citric anions. This ratio equals 0.19 ± 0.04 kDa, which is rather comparable with the size of hydration site of the liganded PEAP calculated from its probable chemical structure (ML0 ≃ 0.23 kDa). Thus, we see that in general the energetics of the phase transition of PEAP liganded by singly charged citric anions is adequately simulated by the Okado− Tanaka model of cooperative hydration of thermoresponsive polymers.61

(12)

where Δth is the experimental specific transition enthalpy and β(T) = 1 − θ(T) is the degree of dehydration of polymer in the course of the transition. Consequently, the excess heat capacity function of the transition could be defined as ÄÅ É ÅÅ dβ(T ) ÑÑÑ E Å ÑÑ cp (T ) = Δt hÅÅ ÅÅÇ dT ÑÑÑÖ (13) where a derivative of the degree of dehydration with respect to temperature could be numerically calculated with enough high precision. In the Okado−Tanaka model the macromolecule is considered as an array of identical sites of hydration. We believe that this approximation seems to be completely valid only for PEAP in 100 mM citrate buffer when the sites of citrate binding on the polyphosphazene matrix are close to the saturation. For this reason we examined only this case in terms of the model. We fitted eqs 6−13 to the experimental excess heat capacity function of PEAP for the 100 mM citric buffer with pH 3.5 adjusting the parameters ΔtHvh, T*, and σ at the apparent polymerization degree of PEAP n = 2400. A good fitting was achieved at the following values of the model parameters: ΔtHvh = 4.0 ± 0.6 kJ mol−1, T* = 310.0 ± 0.1 K, and σ = (1.0 ± 0.3) × 10−5 (Figure 12).

■

CONCLUSIONS Salt-free aqueous solution of poly(ethylaminophosphazene) does not undergo thermotropic phase transitions. Addition of small amounts (around the physiological level) of salts of dicarboxylic acids to the aqueous solution of PEAP drastically changes the hydrophobic/hydrophilic balance of the macromolecule, and as a result, the polymer acquires thermoresponsivity. Thus, we demonstrated that thermoresponsive properties could be induced by an appropriate choice of the solvent ionic composition. The induced phase separation transition of the PEAP solution is extremely sensitive to the pKa1 values and concentration of the acids. Analysis of changes in the thermodynamic transition parameters of PEAP in the presence of sodium chloride and sulfate allowed us to exclude the lyotropic effect of salts from the key factors of the induced thermoresponsivity. The key factors of this phenomenon are an effective dissociation of poly(carboxylic acid)s with production of the singly charged and water structure making anions and their cooperative binding to the polycation matrix supported by van der Waals interactions of the hydrophobic groups of the anions. The polyphosphazene chain loses its individuality as a result of the cooperative binding of the anions and behaves in solution as an extended complex possessing a substantially modified hydrophobic−hydrophilic balance. The complexes seem to be stable enough to not dissociate upon heating. The polyphosphazene−anion complex entity is that structure which acquires thermoresponsive properties in solution. Thus, the salt-induced thermoresponsivity of PEAP implies the formation of stable polycation− anion complex particles whose hydration structure is thermoresponsive. In general, energetics of the thermoresponsive behavior of PEAP is adequately simulated by the Okado−Tanaka model of cooperative hydration of thermoresponsive polymers. The possibility of the induction of thermoresponsive properties for a biodegradable polymer with functional groups capable of binding of amphiphilic ligands opens new ways for diversification and fine-tuning of

Figure 12. Excess heat capacity functions of 2.5 mg mL−1 (φ = 1.71 × 10−3) solutions of PEAP in 100 mM citrate buffer with pH 3.5: (1) experimental; (2) fitted by eqs 6−13 at ΔtHvh = 4.0 ± 0.6 kJ mol−1, T* = 310.0 ± 0.1 K, and σ = (1.0 ± 0.3) × 10−5. The standard fit error and the Pearson’s correlation coefficient are ±0.426 kJ mol−1 and 0.995, respectively.

It was of interest to compare the van’t Hoff enthalpy and the experimental specific enthalpy of transition for determination of the size of hydration site for the completely liganded chain of PEAP. In so doing, it is necessary to take into account that the experimental specific enthalpy Δth was determined per 1 g of the unliganded polymer, but the van’t Hoff enthalpy was calculated per 1 mol of monomer units of the liganded polymer. To remove this disagreement, the experimental specific enthalpy was recalculated per 1 g of the liganded polymer: H

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Macromolecules

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smart polymer systems for biomedical purposes without the need for synthesis of new macromolecules.

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AUTHOR INFORMATION

Corresponding Author

*Tel +7-499-135-07-28; fax +7-499-135-50-85; e-mail [email protected] (V.Y.G.). ORCID

Valerij Y. Grinberg: 0000-0002-5948-6004 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported in part by the Russian Foundation for Basic Research under Project 18-03-00172. The authors are deeply grateful to Dr. Fumihiko Tanaka (Department of Physics, Tokyo University) for assisting in the theoretical analysis of the experimental data.

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