Interaction between PAMAM‐NH2 G0 dendrimer and dissociated sodium chloride in aqueous solution

Journal of Molecular Liquids 171 (2012) 54–59

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Interaction between PAMAM‐NH2 G0 dendrimer and dissociated sodium chloride in aqueous solution Adam Buczkowski a, Pawel Urbaniak b, Joanna Stawowska c, Stanislaw Romanowski d, Bartlomiej Palecz a,⁎ a

Department of Physical Chemistry, University of Lodz, Pomorska 165, Lodz 90-236, Poland Department of Inorganic and Analytical Chemistry, University of Lodz, Tamka 12, 91-403 Lodz, Poland Institute of Applied Radiation Chemistry, Technical University of Lodz, Wroblewskiego 15, 93-590 Lodz, Poland d Department of Theoretical and Structural Chemistry, University of Lodz, Pomorska 163/165, 90-236 Lodz, Poland b c

a r t i c l e

i n f o

Article history: Received 4 April 2012 Received in revised form 16 April 2012 Accepted 18 April 2012 Available online 1 May 2012 Keywords: Isothermal titration calorimetry (ITC) Conductometry Density functional calculations (DFT) PAMAM G0 dendrimer Sodium chloride

a b s t r a c t The protonation constants of poly(amidoamine) dendrimer (PAMAM-NH2 G0) in an aqueous solution of sodium chloride with an ionic strength of 0.05 M were determined by the potentiometric method. The interaction between PAMAM-NH2 and sodium chloride dissociated in water at room temperature was assessed by means of calorimetric and conductometric measurements. Using isothermal calorimetric titration, the number of the moles of dissociated electrolyte combined by a dendrimer mole was estimated. The conductometric measurements indicate a decrease in electrolyte conductivity in the dendrimer solution in relation to the electrolyte solution and confirm that the dendrimer investigated combines electrolyte ions. The results of DFT calculations performed for the protonated structure of PAMAM-NH3+ suggest that the chlorine atom considered is combined with terminal amino groups, which confirms the results of calorimetric and conductometric measurements. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Poly(amidoamine) dendrimers (PAMAM) are polymeric macromolecules that can find their use as nanocarriers of biologically and medically important molecules such as drugs [1–3]. The PAMAM dendrimers of lower generation (G0–G3) have an open structure, in which one cannot distinguish its interior and surface [4,5]. The simpler structure of lower-generation PAMAM dendrimers allows one to treat them as convenient model systems, especially in calculations made by the methods of theoretical chemistry. A molecule of PAMAM-NH2 G0 dendrimer with an ethylenediamine core contains 4 terminal amino groups, two tertiary, core amino groups and 4 amide groups (Fig. 1). Some of the amino groups can be protonated in an aqueous medium. The protonation of amino groups imparts a cationic character to the PAMAM dendrimers of integer generations. Cationic PAMAM dendrimers can combine charged ligands through electrostatic interactions [6,7]. Dissociated sodium chloride is a basic electrolyte of extracellular liquid in mammal organisms. Its 0.9% (abt. 154 mM) aqueous solution is used in medicine and biology as the simplest solution of physiological salt, approximately iso-osmotic with blood plasma. When

⁎ Corresponding author. Fax: + 48 42 635 58 14. E-mail address: [email protected] (B. Palecz). 0167-7322/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2012.04.009

dendrimer with a combined ligand penetrates an organism, it meets in the extracellular liquid an aqueous medium containing many components, including sodium cations (126 mM Na +) and chloride anions (100 mM Cl −). Chloride ions constitute the basic counter-ions for sodium ions in the extracellular fluid [8,9]. Therefore it is of interest to examine the interactions between the cationic PAMAM-NH2 G0 dendrimer and this electrolyte. Some research centers have undertaken studies to characterize the interactions between dendrimers and dissociated sodium chloride. The simulations, performed by the method of molecular dynamics of interactions between charged and neutral dendrimer molecules and ions in a polar solvent treated as a continuous medium, indicate the ion-dendrimer combination [10]. The higher concentration of counter-ions inside the macromolecule than that in the solution reduces the effect of electrolyte concentration on the dendrimer radius. [10] As the equilibrium between the strongly and weakly combined ions is of a dynamic character, a diffusive layer composed of counterions can be formed around the dendrimer macromolecule [11,12]. The calculations made the Monte Carlo method indicate that the number of ions combined by dendrimer decreases with increasing temperature [13]. The aim of this study was to estimate the number of the moles of dissociated sodium chloride combined by a mole of PAMAM-NH2 G0 macromolecules, and that of active sites in the dendrimer structure that combine the ions of dissociated sodium chloride.

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2.2.3. Isothermal titration calorimetry (ITC) Isothermal titration calorimetry (ITC) was conducted using a VP-ITC instrument (MicroCal, USA). Aliquots of 5 μl of 40 mM NaCl in water were injected via a 287.37 μl syringe at intervals of 200 s into 1.4275 ml of 800 μM PAMAM-NH2 G0, stirring at 416 rpm. Titrations were done at 25 °C. All solutions used in the experiments were degassed. For background correction, water (in the cell) was titrated with NaCl in water (in the syringe) at the same concentrations, and the background was subtracted from the final curves.

PAMAM-NH2 G0 dendrimer (m.w. ~ 516 Da, Sigma-Aldrich) with ethylenediamine core, sodium chloride (m.w. = 58.44 Da, FlukaBioChemika, ≥99.5%), water distilled three times and degassed.

2.2.4. Density functional calculations (DFT) All the ground-state geometries of the studied species were obtained by applying the Density Functional Theory (DFT) [16]. The DFT approach utilizes Becke's three-parameter functional [17] with the Vosko et al. local correlation part [18] and the Lee et al. [19] non-local part, abbreviated as B3LYP. The calculations were performed using the standard 6–311 G basis set [20]. Geometry searches were performed for a number of possible isomers to ensure the location of the global minimum on the potential energy surface. No symmetry constraints were imposed during the optimization process. The electron distribution was studied using the Mulliken (electron spin density) electron population analysis schemes. The computations were carried out using the Gaussian09 suite of codes [21].

2.2. Methods

3. Results and discussion

2.2.1. Potentiometric titration Protonation constants were determined by pH-metric titration. Titrations were carried out in a vessel thermostated at 25.0 ± 0.1 °C. The measurements were made under a constant flow of argon. The titrations were carried out at ionic strength 0.05 M and in the presence of extra HCl. The PAMAM-NH2 G0 dendrimer concentration in the samples was 1 mM. The measurements were performed over the range pH = 2–11 with carbonate ion- and oxygen-free NaOH solution. The ionization constant for water pKw was 13.78. Titrations were carried out three times. Each titration consisted of about 100 experimental points. The potentiometric measurements were made by using CERKO microtitrator equipped with an EUROSENSOR (Gliwice, Poland) with the combination pH electrode. The concentration of the sodium hydroxide solution was determined before experiment by titration of a known amount of HCl, according to Gran's method using the computer program GLEE [14]. The Hyperquad [15] computer program was used for calculations of protonation constants from EMF data. Standard deviations quoted refer to random errors only. They are a good indication of the importance of particular species being in equilibrium.

3.1. Potentiometric titration

Fig. 1. The structure of PAMAM-NH2 G0 dendrimer.

2. Materials and methods 2.1. Materials

2.2.2. Conductometric titration During conductometric titration, to 4 ml of 800 μM PAMAM-NH2 G0 solution (in a cell) and 500 μl of 40 mM NaCl solution were injected batchwise 10 μl each 10 s. Next, using the same procedure, the second titration was carried out, in which to 4 ml of pure water NaCl solution was added batchwise. The titration was performed at 25 °C. The values of conductivity corresponding to both titrations were corrected by multiplying the conductivity λ measured (in conventional units) by allowance for dilution P: P¼

V0 þ V1 V0

where: V0 V1

primary volume of the solution titrated, total volume of the titrating solution added.

The protonation constants of dendrimer (Table 1) were determined by the potentiometric-pH-metric method. PAMAM-NH2 G0 can combine 6 protons at most. The analysis of the results obtained clearly shows that the constants determined can be divided into two groups. The first four constants: log K1 to log K4 have similar values. These can be ascribed to the protonation of nitrogen atoms in primary amine groups. The two last constants: log K5 and log K6 concern the addition of protons by the nitrogen atoms of tertiary groups. The nitrogen atoms of amide groups are not protonated within the pH range investigated, i.e. from 2 to 11 logarithmic units. These results are consistent with those of the studies on the protonation reactions of polyamines containing primary and tertiary nitrogen atoms [22]. Very similar results have been obtained for N,N,N′,N′-tetraaminoethyl-1,2-ethylenediamine (PENTEN) or N,N,N′,N′-tetraaminopropyl-1,2-ethylenediamine (PTETRAEN), having similar structures to that of PAMAM-NH2 G0 [22]. The decrease in the basicity of tertiary amines compared to that of primary amines is a well known and widely described phenomenon. The conventional way of explaining this phenomenon motivates it with the obstructed protonation of the nitrogen atom of tertiary amine, which results from the steric compression

Table 1 Successive protonation constants pKi of PAMAM-NH2 G0. Analogous values determined for PENTEN are given for comparison. The meaning of the symbols: A — an unprotonated form of the dendrimer, AHnn + — a protonated form of the dendrimer, which is bonded with n protons. Equilibrium

A + H+ ⇆ AH+ AH+ + H+ ⇆ AH22 + AH22 + + H+ ⇆ AH33 + AH33 + + H+ ⇆ AH44 + AH44 + + H+ ⇆ AH55 + AH55 + + H+ ⇆ AH66 +

PAMAM G0

PENTEN

Log Ki value Lit. log Ki value

Lit. log Ki value

I = 0.05 M NaCl, 298 K

I = 0.2 M KCl, I = 0.1 M KCl, I = 0.1 M KCl, 298 K [25] 298 K [26] 295 K [22]

9.69(2) 9.15(2) 8.69(2) 8.21(2) 6.57(3) 3.19(3)

9.78(2) 9.24(2) 8.84(2) 8.35(1) 6.64(2) 2.77(3)

9.70 9.26 8.74 8.31 6.68 3.15

10.30 9.63 9.26 8.31 3.1–3.5

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around this atom. However more attention is now concentrated on the significance of the hydration of ammonium cation being formed [23]. The field effect influences the increase in amine basicity with the increasing number of alkyl groups on the amine. The hydration effect acts in reverse direction. Tertiary ammonium ions are hydrated or solvated to a considerably lesser extent than primary ions. As a consequence of this tertiary amines are protonated to a lesser extent compared to primary amines despite the stronger field effect. Such a way of explaining the differences in the basicity of protonation centers in dendrimers has been used by Heegaard et al. [24]. On the other hand, the explanation proposed by Krot et al. [25] does not seem to be fully correct. These authors suggest that the phenomenon discussed is an electron-withdrawing effect of amide group and its different interaction with the protonation centers. However in PAMAM-NH2 G0, amide group (\C(O)\NH\) is two methylene groups (\CH2\) away from the protonation centers considered and not in their direct vicinity. Undoubtedly, this weakens the field effect. The comparison of the constants obtained with the values of PENTEN shows that primary nitrogen atoms in dendrimer slightly more difficult undergo protonation (differences from 0.1 to 0.6 logarithmic unit). Considerably greater are the differences in the basicity of internal nitrogen atoms. Probably the stronger protonation of tertiary nitrogen atoms in dendrimer results from the formation of intramolecular hydrogen bonds with the participation of oxygen atoms of amide groups. The diagram shown in Fig. 2 presents the distribution curves of particular protonated forms of PAMAM-NH2 G0. This diagram shows that with the physiological pH = 7.3 PAMAM-NH2 G0 mainly occurs in the form of AH44 +, i.e. with protonated primary nitrogen atoms. In consequence, the potential interaction with metal ions or other positively charged molecules is seriously hindered. Instead, the interactions with anions are favored.

Fig. 3. Dependences of conductivity (in conventional units) on the molar sodium chloride to dendrimer ratio during the titration of the 800 μM PAMAM-NH2 G0 solution with 40 mM sodium chloride solution (■) and during the titration of water with 40 mM sodium chloride solution (◊).

In accordance with the core–shell mechanism [26,27] first are protonated amino groups on the surface of dendrimer and then those inside it. In aqueous solutions at pH close to neutral, all (four) primary amine groups in the molecule of PAMAM-NH2 G0 are protonated, while (two) tertiary internal amine groups remain unprotonated. Probably the four superficial positively charged amino groups formed due to protonation electrostatically combine chloride anions to form adducts of ionic pair character, which reduces the conductivity of the solution of dendrimer and electrolyte compared with the electrolyte solution itself. After exceeding the molar electrolyte to dendrimer ratio equal to about 4/1, each of the superficial amino groups protonated combines chloride ions, thus the increase in electrolyte concentration causes no change in the difference of the conductivities of dendrimer and electrolyte solution as well as the electrolyte solution.

3.2. Conductometric titration 3.3. Isothermal titration calorimetry (ITC) The conductivity values measured (in conventional units) corresponding to the titration of a 800 μM solution of PAMAM-NH2 G0 with sodium chloride solution are clearly lower than the corresponding values of conductivities during the dilution of 40 mM sodium chloride solution in water (Fig. 3). This indicates an interaction of dissociated sodium chloride with dendrimer. The difference in the conductivities (in conventional units) of the 800 μM solution of PAMAM-NH2 G0 titrated with the 40 mM solution of sodium chloride and water titrated with the same NaCl solution (Fig. 4) reaches a plateau with the molar ratio of sodium chloride to dendrimer equal to about 4/1.

Fig. 2. Species distribution curves for PAMAM-NH2 G0 dendrimer vs. pH at 298.1 K, I = 0.05 M NaCl. The meaning of the symbols A and AHn is the same as in Table 1.

The isothermal titration calorimetry (ITC) technique was used to determine the thermal effects of the titration of an 800 μM solution of PAMAM-NH2 G0 (in a cell) with a sodium chloride solution (in a syringe) and the corresponding thermal effects of diluting the electrolyte in water. Their difference was used to calculate the thermal effect of dendrimer-NaCl interaction corrected by the dilution effect (Fig. 5). The diagram (Fig. 5) of this dependence rapidly increases at the molar sodium chloride to dendrimer ratio equal to about 4/1, which is connected with the combination of chloride anions by four superficial protonated amino groups. Above the molar sodium chloride to dendrimer ratio equal to about 6/1, the diagram of this dependence

Fig. 4. The difference in the conductivities (in conventional units) of the 800 μM PAMAM-NH2 G0 solution titrated with a 40 mM sodium chloride solution and water titrated with the same solution.

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Fig. 5. Thermal effect of the interaction between PAMAM-NH2 G0 and sodium chloride corrected with the dilution effect and calculated per one mole of the electrolyte.

reaches a plateau, which corresponds to the total saturation of the macromolecule with electrolyte. The binding and thermodynamic parameters – binding constant (Kb), number of binding centers per one molecule (n), and enthalpy (ΔH) – were computed (Fig. 5) from actual calorimetric data by non-linear fitting using Origin Lab software (USA) for the VP-ITC calorimeter. A molecule of PAMAM-NH2 G0 in aqueous solution can combine up to 4 chloride anions (n = 4.3 ± 0.4). The process of combining these ions by the active sites of dendrimer is exothermal and shows a character of weak electrostatic interactions. 3.4. Density functional calculations (DFT) 3.4.1. Search for minimum energy of the G0-NH3+ and G0-NH3+ with the included Cl atom A complete optimization of the PAMAM-G0-NH3+ molecule structure is shown in Fig. 6. In one of the NH3+groups, hydrogen atom is between the oxygen (O) of the carbonyl group and the nitrogen of the –NH3+ terminal branch. The charge on three H atoms in –NH3+ group is then positive for all the three structures given in Table 2 (Fig. 7). Subsequently, having a completely optimized PAMAM molecule, chlorine atom (No. 86 in Fig. 7) was added to many sites of PAMAM-G0-NH3+ molecule and the resulting systems were again optimized until the complete optimization of the dendrimer structure including Cl. Among these structures there were only three types of dendrimers with a minimal energy. The first type of such an optimized structure is shown as “structure I” in Fig. 7a. Chlorine atom (No. 86) is here situated at “a hole” built by means of the NH3+ group (N atom No. 82), NH2 amine group (N atom No. 25) and NH group (N atom No. 79). The second type of the structures mentioned above is shown in Fig. 7b and it is called “structure II”. Here the chlorine atom is located between two terminal NH2 amines groups (N atom No. 34 and N atom No. 44) and NH3+ group (N atom No. 82). A picture of the third type of structure called “structure III” is shown in Fig. 7c. Chlorine atom takes here the position in the space between the terminal NH3+ group (N atom No. 82), the terminal unprotonated NH2 group (N atom No. 34) and carbon atom of methylene group (C No. 31). The energies of structures I, II and III are as follows: 226.17 kJ/mol, 214.11 kJ/mol and 213.07 kJ/mol, respectively. 3.4.2. Atomic charge distribution The comparison of the charges in Table 2 for the G0-NH3+ and G0NH3+-Cl structures indicates that the structures well describe the atomic

Fig. 6. Full optimization of PAMAM-G0-NH3+ structure.

charges, which suggests that this information could be useful for the effective force field parametrization of higher order dendrimers. We observe the existence of three sites in the G0-NH3+ and G0NH3+-Cl where the distribution of the electron density shows strong Table 2 Mulliken atomic charges. Atom number

Atom type

G0-NH3+

1 8 21 22 23 24 25 26 27 34 35 36 43 44 45 46 59 60 61 62 63 64 65 66 77 78 79 80 81 82 83 84 85 86

N N C C O O N H N N H H H N H H C C O O N H N H H H N H H N H H H Cl

− 0.49 − 0.45 0.59 0.61 − 0.45 − 0.55 − 0.64 0.33 − 0.64 − 0.71 0.31 0.30 0.35 − 0.74 0.31 0.31 0.57 0.61 − 0.56 − 0.43 − 0.66 0.33 − 0.64 0.43 0.18 0.17 − 0.71 0.30 0.30 − 0.79 0.44 0.39 0.38

G0-NH3+-Cl

G0-NH3+-Cl

G0-NH3+-Cl

Structure I

Structure II

Structure III

− 0.37 − 0.43 0.62 0.61 − 0.57 − 0.52 − 0.62 0.37 − 0.64 − 0.72 0.31 0.31 0.36 − 0.74 0.32 0.31 0.61 0.58 − 0.49 − 0.47 − 0.64 0.34 − 0.69 0.41 0.18 0.18 − 0.72 0.30 0.36 − 0.81 0.44 0.41 0.43 − 0.83

− 0.40 − 0.39 0.65 0.58 − 0.48 − 0.53 − 0.64 0.34 − 0.62 − 0.74 0.29 0.39 0.37 − 0.76 0.30 0.36 0.58 0.63 − 0.53 − 0.42 − 0.66 0.34 − 0.67 0.40 0.19 0.18 − 0.72 0.30 0.31 − 0.76 0.42 0.37 0.40 − 0.82

− 0.41 − 0.39 0.64 0.64 − 0.47 − 0.55 − 0.64 0.34 − 0.65 − 0.74 0.29 0.40 0.36 − 0.74 0.32 0.31 0.58 0.61 − 0.53 − 0.43 − 0.66 0.34 − 0.67 0.40 0.19 0.18 − 0.71 0.30 0.31 − 0.76 0.42 0.37 0.41 − 0.82

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Fig. 7. Full optimizations of PAMAM-G0-NH3+ and Cl atom: a) structure I, b) structure II, and c) structure III.

electronegative atom. Neutral G0-NH2 dendrimers have been analyzed by F. Tarazona-Vasques and P.B. Balbuena [28]. In the terminal groups, the electronegative atoms are the N atoms of NH3+ and NH2 groups called the amine site. The next such centers are N in the NH group and O atoms. The distribution of electron density generates an appropriate environment to attract a positive ion or a neutral atom. In our structures, neutral Cl atom is located between the amine sites and has negative charge. The positive atoms are: C atom in the carbonyl groups and all H atoms. 3.4.3. Structural analysis of G0-NH3+ and G0-NH3+ with chlorine atom and the values of bondlength and angles calculated The distances between Cl atom and hydrogen atoms are 2.72 Å for H81, 2.84 Å for H69, 2.83 Å for H58, 2.46 Å for H84 and 2.16 Å for H26 in structure I (Fig. 7a), respectively. The Cl–H distances are: 2.57 Å for H46, 2.38 Å for H36, 2.01 Å for H85 and 2.79 Å for H71 in structure II (Fig. 7b), respectively and 1.99 Å for H85, 2.79 Å for H71, 2.95 Å for H17 and 2.43 for H36 in structure III (Fig. 7c), respectively. The distance between the hydrogen atom in the NH3+ group and the oxygen atom in the carbonyl group is 1.40 Å for H83O61 in the G0-NH3+ structure (Fig. 6). This distance is 1.84 Å for H83O61 and 1.69 Å for H85O23 in structure I, 1.68 Å for H83O61 in structure II and 1.70 Å in structure III. The calculated values for distances between the hydrogen (H66) atom in the NH group and the oxygen atom (O24) in the carbonyl group are 1.69 Å in the G0-NH3+ structure, 1.83 Å in structure I, 1.81 Å in structure II, and 1.85 Å in structure III, respectively. The values of the angle α for the HNC calculated are 117.8° for H64N63C67, 117° for H26N25C28, 117.4° for H26N25C28, 112.3° for H43N27C37 and 114.1° for H66N65C70, in the G0-NH3+. In structure I, the values of angles amount to 117.2°, 114.6°, 111.9 and 113.6°, respectively, in structure II they are equal to 117.9°, 117.2°, 111.1° and 112.6°, respectively, whereas in structure III: 117.9°, 117.1°, 112.0 and 113.2°, respectively. To sum up, the bondlengths and angles calculated are in agreement with the values expected for the corresponding bonds and angles for similar molecules [28–31]. Small variations are detected in the positive charge of the dendrimer. 4. Conclusions The results of potentiometric titration show that in an aqueous medium at pH close to neutral, the superficial primary amine groups of PAMAM-NH2 G0 molecule are protonated. The results of conductometric and calorimetric titrations clearly indicate that the cationic

PAMAM-NH2 G0 dendrimer interacts with sodium chloride in aqueous solution. About four chloride anions are combined by one dendrimer molecule, probably through the terminal protonated amine groups. This process is of exothermal character and may be connected with the formation of ionic-pair adducts by electrostatic interactions. The analysis of many optimized structures of protonated PAMAMG0-NH3+ with chlorine atom allowed us to obtain, as was mentioned previously, three structures with the lowest energy. The presence of Cl atom leads to a flexure of three branches of PAMAM-G0-NH3+ molecule, which indicates a considerable elasticity and readjusting capability of the dendrimer open structure to combine small ligands including ions. The geometry and bondlengths of the structures obtained are quite similar to structures, in which unprotonated amine groups are terminal groups, while the possible small differences in bondlengths can result from the fact that the structure under investigation may protonate one of such superficial groups. Thus, the macromolecule structure under modeling is of cationic character due to this one protonated terminal amine group NH3+. Taking into account the protonation of the macromolecule, we intended to approximate the structure modeled to the form, in which it occurs in aqueous medium. It facilitates the comparison of the calculation results obtained with those of the calorimetric and conductometric titrations. The distribution of Mulliken charges of the structures optimized indicates a considerable polarization of nitrogen–hydrogen bonds in the protonated terminal amine group compared to its unprotonated equivalent. As a result of this protonation, the partial negative charge (in a Mulliken sense) accumulated on the central nitrogen atom of the terminal amine group is considerably decreased (e.g. from −0.72 for N 79 to − 0.81 for N 82 in structure II). This is accompanied by a clear increase in the partial charge of hydrogen atoms connected with the central nitrogen atom under protonation in the terminal amine group (e.g. from + 0.31 for H 35 to +0.43 for H 85 in structure II). One can expect that a similar situation also takes place in the case of the PAMAM macromolecules of integer generations suspended in aqueous solution, in which the preferred site of chloride anion attack should be the hydrogen atoms of the protonated amine groups. As shown by the pH-metric measurements, one can expect the protonation of all the four terminal amine groups in the PAMAM-NH2 G0 molecule in aqueous medium, thereby up to four chloride anions can be combined with a molecule of the dendrimer investigated. The distribution of Mulliken charges of the optimized structures of PAMAM-NH2 G0 indicates three most negative reactivity centers in relation to the neutral chlorine atom considered (Fig. 7, Table 2). One can assume that it is the terminal amine groups that are the attachment sites of chloride anion Cl − in an aqueous medium.

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Acknowledgments Project co-funded by the European Union under the European Social Fund:

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