Soft Matter

PAPER pH-dependent nano-capturing of tartaric acid using dendrimers

b bc bd Oana G. Schramm,*a Xaviera Lopez-Cort e s, Leonardo S. Santos, V. Felipe Laurie, e a af a Fernando Danilo Gonza lez Nilo, Michal Krolik, Rainer Fischer and Stefano Di Fiore

Cite this: Soft Matter, 2014, 10, 600

The ability of dendrimers to bind to various target molecules through non-covalent interactions was used to capture water soluble organic reagents, such as tartaric acid (TA), from dierent matrices, i.e. aqueous solutions and wine samples. The inuence of the pH, dendrimer type, generation and feeding concentration on the hostguest complexation of TA was investigated. The maximum binding capacity of TA in aqueous solutions was achieved by amine end-capped dendrimers at pH 5. At extreme pH values of 2 and 11, the binding of TA dropped strikingly, demonstrating the pH-dependency underlying the hostguest interactions. The linear correlation between the maximum binding capacity of TA at pH 5 and the number of primary amine groups on the surface of PAMAM and PPI dendrimers strongly indicated that hostguest complex formation between TA and dendrimers is largely dependent on
Received 23rd August 2013 Accepted 5th November 2013 DOI: 10.1039/c3sm52255e www.rsc.org/softmatter

electrostatic interactions. Molecular simulations conrmed the predominant electrostatic nature of the interactions between TA and the amine end-capped dendrimers and also provided important information on the spatial distribution of TA within the PAMAM G5 dendrimer. All these results designate dendrimers as potential nano-capturing systems for the removal/recovery of TA from complex matrices such as wine, industrial waste or fruit juices.

Introduction
The unique properties of dendrimers such as high surface density of functional groups, their well-dened and relatively monodisperse nanostructure, high loading capacity, tunable solubility and biocompatibility and their ability to respond to external stimuli designate these molecules as smart nanocarriers.13 The ability of dendrimers to encapsulate dierent target molecules, initially reported for model compounds such as pyrene and rose Bengal4 and later applied to dierent interest

Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany. E-mail: oana.schramm@ime.fraunhofer.de; Fax: +49 241 6085 10000; Tel: +49 241 6085 12153

Nanobiotechnology Division at University of Talca, Fraunhofer Chile Research Foundation Center for Systems Biotechnology (FCR-CSB), Talca, Chile. E-mail: xaviera.lopez@fraunhofer.cl

c Laboratory of Asymmetric Synthesis, Institute of Chemistry and Natural Resources, University of Talca, Talca, Chile. E-mail: lssantos@utalca.cl; Fax: +56 71 200 448; Tel: +56 71 200 448 d School of Agricultural Sciences, University of Talca, Talca, Chile. E-mail: aurie@ utalca.cl; Fax: +56 71 200 212; Tel: +56 71 200 214 e

Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Biological Sciences, University Andres Bello, Santiago, Chile. E-mail: fernando.gonzalez@unab.cl; Tel: +56 2 2770 3606

f Institute of Molecular Biotechnology RWTH Aachen University, Germany. E-mail: scher@molbiotech.rwth-aachen.de; Fax: +49 241 871 062; Tel: +49 (0)241 802 663

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areas, such as catalysis,5,6 drug711/gene delivery12,13 and imaging,10,14 can be eectively exploited for removing valuable reagents such as tartaric acid (TA) from various beverages, including wine, fruit juices or waste uids from industrial processes. TA is widely used in food, cosmetic and pharmaceutical industries. It naturally occurs in some plants, with particular prevalence in grapes. TA and malic acid represent more than 90% of the total titratable acidity of wine and grape juices, whereby TA is the dominant component in most of the cases. TA plays a major role in the sensorial perception of acidity, indirectly aecting the color, taste as well as chemical and biological stability of wine.15 Therefore, TA is a valuable commodity in wine production used mainly for the purpose of modulating products with decient acidity. Because grapes cultivated in warm regions tend to have a low content of acidity, must and wine produced from these grapes require acidication. In contrast, vines cultivated under cool climates may produce grapes that are too acidic, and wines obtained from these grapes may need physical, chemical or microbiological deacidication.16 Moreover, wines would naturally form tartrate salts with potassium or calcium ions that may grow as crystals large enough to represent an esthetic problem.17 Besides a few alternatives to avoid the formation of these salts,18 the main processes for tartrate removal include cold stabilization at 04  C for two to three weeks,19,20 chemical precipitation21 that also requires low temperature and relatively long incubation times, solvent extraction,22,23

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membrane separation24 and evaporation. These methods are the most widely adopted processes for stabilization of wine and for the recovery of TA from industrial waste. However, when it comes to recovering TA the long processing times, large solvent consumption, extreme temperatures and metatartrate formation are drawbacks which could be addressed.17,20 A simple alternative approach for the removal and recovery of TA could be the capturing of TA by dendrimers through non-covalent interactions. The use of dendrimers for trapping hydrophobic drugs, dyes,710 and metal ions2529 has been extensively investigated. These studies showed that the high anity between dendrimers and guest molecules is based on metalligand coordination or non-covalent bonding, including electrostatic interactions, hydrogen bonding and hydrophobic interactions. These interactions are oen reversible and pH-dependent. Diallo et al.27,28 demonstrated the pH dependency of dendrimermetal supramolecular assemblies with a maximum binding up to 74 mole of copper per mole of PAMAM G4 at pH 9 and no complex formation at pH 5. However, the complexation of water soluble organic molecules such as organic acids30 for the purpose of removal and recovery has been less investigated. To the best of our knowledge, this is the rst report describing the trapping of the water-soluble organic acid, TA, by dendrimers as an eective nanotechnology-based approach for the removal and/or recovery of TA. Thorough investigation revealed the inuence of the dendrimer generation, feeding concentration and pH on binding capacity, thus providing useful information about preferred binding sites and the mechanisms involved in the trapping and release of TA. All-atom molecular dynamics analysis fully conrmed the experimental data and facilitated a closer view into the TAdendrimer supramolecular systems.

TA binding studies Ultraltration-based method. The binding of TA from aqueous solutions to dendrimers was determined by using an ultraltration method. Several dendrimers with dierent surface groups, cores and generations were screened for their ability to bind to TA. Briey, dendrimer aqueous solutions (0.2 mM) were mixed in a 1 : 1 v/v ratio with 20 mM TA aqueous solution to give a nal TA/dendrimer molar ratio of 100 : 1. The dendrimerTA mixtures were shaken for 1 h at room temperature. Prolonged interaction times, i.e. 24 h, did not show signicant improvement in the extent of binding (data not shown). The samples were then transferred to a 96-well lter plate with a molecular weight cut o (MWCO) of 3.5 kDa. The lter plate was placed onto a 96-well collector plate and centrifuged at 3 400 rpm for 1 h at 4  C. The concentration of TA in the ltrate was determined by UV/vis spectrometry using the kit for enzymatic TA detection. The amount of TA bound to dendrimers (mmol TA per mmol dendrimer) was estimated using the equation: (ciTA cfTA)/cdendr where ciTA is the initial concentration of TA (in the TAdendrimer mixture, before centrifugation), cfTA is the concentration of TA in the ltrate, aer centrifugation, and cdendr is the concentration of dendrimers (kept at 0.1 mM), aer mixing with TA solution. Further studies based on ultraltration were carried out with selected dendrimers in aqueous solutions according to the protocol described above. In this case, the concentration of the dendrimers was kept constant at 0.2 mM and the concentration of TA was varied to achieve several TA/dendrimer molar ratios (200 : 1, 100 : 1, 50 : 1 and 25 : 1). Furthermore, the pH of the TAdendrimer solutions was adjusted to the desired pH values with 1 N HCl or 1 N NaOH solutions. The mixtures were shaken for 1 h at room temperature and then centrifuged as described. The amount of tartaric acid bound to the dendrimer (mmol TA per mmol dendrimer) was estimated using the same equation described above. The extent of binding (EOB, %) refers to the binding capacity of dendrimers to TA, where the initial feeding concentration ratio of TA to dendrimer was 100 : 1 and was estimated using the equation: ((ciTA cfTA)/ciTA) 100.

Experimental part
Materials Ethylenediamine (EDA)-cored and amine terminated poly(amidoamine) (PAMAM) generation 4, 5 (G4, G5) dendrimers, diaminododecane (DAD)-cored and tris(hydroxymethyl)amidomethane end-capped poly(amidoamine) (PAMAM-tris-OH) generation 3 (G3), diaminohexane (DAH)-cored and tris(hydroxymethyl)amidomethane end-capped poly(amidoamine) (PAMAM-tris-OH) generation 4 (G4) dendrimers, diaminobutane (DAB)-cored and sodium carboxylate-terminated poly(amidoamine) (PAMAM) generation 3.5 and 4.5 (G3.5, G4.5) dendrimers, DAB-cored and succinamic acid end-capped poly(amidoamine) (PAMAM-Suc) generation 3, 4 (G3, G4) dendrimers, TA 0.1 M aqueous solution and Pur-A-lyzer dialysis bags with MWCO of 1 and 3.5 kDa were purchased from Sigma Aldrich. Dialysis membrane, MWCO 0.5 kDa, was purchased from Spectrum Laboratories Inc. Poly(propylene imine) (PPI) dendrimers, generation 3, 4, 5 (G3, G4, G5), with a DAB core and amine surface groups were obtained from SyMO-Chem. Poly(amidoamine) (PAMAM-Me) dendrimers with a EDA core and methyl carboxylate end-groups were synthesized according to the literature.3 A kit for measuring TA concentration was purchased from Megazyme. Acroprep Advance 96-well lter plates were obtained from Pall Corporation.

Reverse dialysis-based method. The binding studies of dendrimers with a MW lower than 3.5 kDa to TA in aqueous solutions, or in red wine samples (Chilean merlot wine, TA content 16.0 mM), were carried out by using a reverse dialysis method. Briey, 10 mL of TA sample (10 mM aqueous solutions) or 10 mL red wine were placed in a 50 mL falcon tube together with a dialysis bag containing 0.5 mL dendrimer solution (2 mM for aqueous solution samples or 3.2 mM for the wine samples) to give a TA/dendrimer molar ratio of 100 : 1. The falcon tubes were shaken for 24 h at room temperature. The TA content of the sample outside of the dialysis bag was then determined

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enzymatically using a TA kit described above. The amount of TA bound to dendrimer (mmol TA per mmol dendrimer) was estimated using the formula: (xiTA/1.05 xfTA)/xdendr where xiTA is the initial amount of TA (mmol) in the aqueous solution or in the wine sample, xfTA is the nal amount of TA (mmol) outside the dialysis bag and xdendr the amount of dendrimer (mmol) in the dialysis bag; 1.05 is a correction coecient compensating the change of volume within the dialysis system. Computational methods PAMAM G5 which presented the highest binding capacity for TA at pH 5 was chosen for detailed molecular dynamics (MD) investigation and was parameterized according to the rules for all-atom CHARMM27. The force eld31 was not included in conventional force eld due to the dendritic structures. Charges, missing bond, angle, dihedral parameters of the PAMAM core, as well as intermediate dendrons and the outer surface were estimated from similar terms within the force eld. An atomistic model for PAMAM G5 was built at pH 5 where 183 amines in total are protonated (128 terminal amines plus 55 tertiary amines from the fourth generation). The molecular parameters for TA were obtained from the database of the ParamChem interface (https://www.paramchem.org), using the CGenFF force eld32 at pH 5, where both carboxylic groups of tartaric acid were considered to be fully deprotonated (charge of TA 2). The PAMAM G5 structure and TA molecules parameterized at pH 5 were implemented for two models, the rst one containing 100 molecules of TA and 1 molecule of PAMAM (system 100 TA1 PAMAM) and the second one comprising 200 molecules of TA and 1 molecule of PAMAM (system 200 TA1 PAMAM) according to the experimental conditions of the binding studies. For these models, PAMAM G5 was set to the origin of the coordinate system. Subsequently the dendrimer 3) of was solvated in a water periodic box (192 184 177 A 33 TIP3 water molecules, then the corresponding number of TA molecules were added in a random way along the water box. In order to neutralize the system, 17 and 217 sodium ions were added to the rst and second systems respectively. Both systems were minimized for 30 000 steps and equilibrated through MD simulations. The MD simulations were performed at 298 K in the isobaricisothermal ensemble for 34 ns. The temperature was maintained with the Langevin thermostat with a damping coecient of 1 ps1 whereas the pressure (1 atm) was kept constant by using the Langevin piston method.34 The equations of motion were integrated with a 2 fs time step. The van der Waals (vdW) cuto was set to 12 and the long-range electrostatic forces were computed using the particle-mesh Ewald approach. The MD simulation was performed using the NAMD2.9 (ref. 35) program and CHARMM27 force eld31 for standard ions and water molecules. All analyses were done with VMD36 soware. For the analysis of the molecular dynamics, the data collected along the trajectories were used to calculate the radius

of gyration Rg of the dendrimer and dierent concepts were applied such as radial pair distribution function (RDF) of TA molecules. Furthermore, several scripts were implemented in order to get relevant information from MD simulations, such as with respect to any atom capture of TA within a distance of 8.5 A for of the dendrimer for the rst TAdendrimer system and 15 A the second TAdendrimer system, the number of hydrogen bond interactions, vdW and electrostatic interaction energy between the dendrimer and the entrapped TA molecules.

Results and discussion

TA binding studies aqueous solutions Several dendrimers with dierent surface groups, cores and generations were screened for their ability to capture TA (Table 1). These preliminary binding studies were carried out by an ultraltration based method without adjusting the pH of the hostguest solutions and at 100 : 1 TA/dendrimer feeding molar ratio (intrinsic pH range 35). The results of these experiments revealed that the surface functional groups of the dendrimers induced striking dierences in the TA uptake (Fig. 1). The binding capacity of the dendrimers with the same density of end-groups decreased as follows: PAMAM z PPI > PAMAM-trisOH > PAMAM-COOMe > PAMAM-COONa > PAMAM-Suc. As expected, the amine end-capped dendrimers showed the highest binding anities, most likely due to electrostatic interactions of the dendrimers' terminal amine groups with the carboxylic acid groups of TA. In the case of hydroxyl end-capped PAMAM dendrimers, the extent of binding was moderate. We assumed that these dendrimers bound to TA mainly by hydrogen bonding rather than electrostatic interactions between TA and dendrimer surface groups. Previous reports also evidenced only low contributions of surface electrostatic interactions of PAMAM-OH with other acidic molecules, such as indomethacin9,37 or carbon dioxide.38 The lack of surface electrostatic interactions and the diminished hydrogen bonding contributed to the low binding anity of methyl ester endcapped PAMAM dendrimers. However, the capacity of these dendrimers was slightly higher than that of sodium carboxylate
Table 1

Dendrimers used for the TA binding studies Core EDA EDA DAB DAB DAB DAD DAH EDA EDA EDA EDA DAB DAB Generation 4 5 3 4 5 3 4 2.5 3.5 3.5 4.5 3 4 End-group NH2 NH2 NH2 NH2 NH2 Tris(hydroxymethyl)amidomethane Tris(hydroxymethyl)amidomethane COOMe COOMe COONa COONa Succinamic acid Succinamic acid

Dendrimer PAMAM-G4 PAMAM-G5 PPI-G3 PPI-G4 PPI-G5 PAMAM-tris-OHG3 PAMAM-tris-OHG4 PAMAM-G2.5-Me PAMAM-G3.5-Me PAMAM-G3.5 PAMAM-G4.5 PAMAM-Suc-G3 PAMAM-Suc-G4

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Fig. 1 Extent of binding of various dendrimers to TA in aqueous solutions at 100 : 1 TA/dendrimer loading molar ratio.

end-capped PAMAM-G3.5 and succinamic acid terminated dendrimers since electrostatic repulsions between the negatively charged carboxylic groups of TA and the surface carboxylic groups of the dendrimers might have occurred. The binding capacity of dendrimers with the same end functional groups and cores increased with the generation, so it seems that the higher generation dendrimers could bind to more molecules of TA as they possess more surface groups and larger cavities. Based on the results of the preliminary screening, PAMAM amine end-capped G4 and G5 dendrimers were selected for further investigations. The binding capacity of the dendrimers of dierent generations was thoroughly investigated at specic pH values (including the pH of wine, 3.23.5) and at various TA/ dendrimer feeding molar ratios in order to evaluate the eect of the dendrimer generation and the inuence of the pH on the extent of binding (Fig. 2). The maximum binding capacities of amine end-capped PAMAM G4 and G5 were achieved at pH 5 with little dierences compared to the results observed at pH 3.23.5. In fact, compared to pH 3.23.5, at pH 5 both carboxylic groups of TA are slightly more dissociated (pH > pKa1 z 2.95 and pKa2 z 4.25), therefore some more TA molecules can bind electrostatically to the dendrimers. At this pH the binding capacity of PPI dendrimers was also investigated and compared to that of PAMAM dendrimers. The binding capacity of both type of dendrimers increased almost linearly with the feeding concentration of TA up to a certain level when the saturation with TA was achieved. There was no signicant dierence between the binding ability of the PAMAM and PPI dendrimers with the same number of amine surface groups. Interestingly, the highest extent of binding correlated fairly well with the number of amino surface groups of the dendrimers, i.e. a molecule of PPI-G4 with 32 amino groups could bind on average to 31.1 TA molecules and a molecule of PAMAM G4 with 64

Fig. 2 Binding capacity of PAMAM dendrimers to TA at dierent pH and several tartaric acid/dendrimer feeding molar ratios, A PAMAMG4 and B PAMAM-G5 (error bars represent standard deviation of at least three independent experiments).

surface groups could bind to approximately 63 TA molecules (Fig. 3), suggesting that each amino surface group can bind up to one molecule of TA. A good linear correlation (R2 > 0.99, slope > 0.99) was found for both PAMAM and PPI dendrimers up to the generation corresponding to 64 amino end-groups (G4 for PAMAM and G5 for PPI). For higher generations, i.e. PAMAM G5 with 128 surface groups, the binding capacity slightly deviated from linearity (R2 > 0.98, slope 0.89) most likely due to the increased steric hindrance on the surface of the dendrimer.

Fig. 3

Correlation between the maximum binding capacities of PAMAM and PPI dendrimers to TA and the number of amino endgroups.

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In the pH range of 3.23.5, the pH range of wine, the TA carboxylic groups are less dissociated, thus the binding capacities of dendrimers were slightly, although not signicantly, lower than at pH 5. At pH 2 when both carboxylic acid groups of TA are undissociated and no electrostatic interactions with positively charged amino groups of dendrimers can occur, almost no binding to TA could be detected. Moreover, according to Goddard et al.39 PAMAM dendrimers undergo pH-dependent conformational changes and at low pH values the shell of the dendrimers is preferentially protonated and therefore relatively dense packed so that the available void spaces are drastically reduced and consequently the binding capacity of dendrimers might drop down. At pH 7.5 the amino groups of the PAMAM dendrimers are partially neutral (pKa of tertiary amines 6.36.85 and pKa of amino terminal groups 9.010.77 (ref. 28)). Consequently, at this pH the anity of the dendrimers to TA decreased. When all amino groups were completely neutral (pH 11) almost no binding to TA could be detected. The considerable dierences in binding anity determined at various pH values (i.e. from almost 90% binding capacity at pH 5 to almost 0% binding capacity at pH 2 and 11) demonstrated the high pH-responsiveness of amine end-capped dendrimers indicating also their ability to release guest molecules upon pH shi. Similar binding experiments were carried out with potassium sorbate (PS), an additive of large use in the food industry, including production of some types of wines. In this case although similar electrostatic interactions between the carboxylate anion and amino surface groups of PAMAM dendrimers can take place, the low extent of hydrogen bonding limited the binding capacity of dendrimers (Fig. 4). On the other hand, hydrogen bonding alone could not induce strong anities between hydroxyl end-capped PAMAM dendrimers and TA, as shown above (Fig. 1). All these results indicated that for amine end-capped dendrimers electrostatic interactions are crucial to achieve high binding capacity to organic acids and these interactions synergistically cumulate with hydrogen bonding.

The inuence of the surface functional groups on the binding capacity of the dendrimers (see comparative studies with TA and PS), considered together with the linear correlation between maximum binding capacity and the number of the amino end-groups, strongly suggests that guest molecules are located in close proximity to the amino end-groups, most likely at the periphery of the dendrimers. These ndings are in accordance with a recent report by Cheng et al.40 who investigated the interactions between PPI dendrimers and amino acids by NMR techniques. NOESY spectra evidenced that glutamic acid molecules are encapsulated by PPI-G4 by electrostatic interactions. Furthermore it could be shown that glutamic molecules are located at the periphery of the dendrimer since no cross peak between PPI's core and glutamic acid could be detected. However, upon addition of glutamic acid distinct chemical shi of about 0.1 ppm could be assigned to the protons of the PPI's core. Electrostatic interactions also contributed to the high binding capacity of amino-terminated dendrimers to carboxylic hydrophobic drugs, such as ibuprofen, with a maximum binding capacity of 78 molecules ibuprofen per PAMAM G4 molecule and 32 molecules ibuprofen per PAMAM G3 molecule, whereas only 24 molecules of ibuprofen could be encapsulated by PAMAM-OH G5.41 In this case, besides the electrostatic attractions, another driving force for the encapsulation of guest molecules can be attributed to the hydrophobic interactions. However, fewer indications about the binding position of ibuprofen inside the PAMAM dendrimers could be evidenced. TA binding studies using wine samples The ability of dendrimers to bind to TA was also investigated in complex matrices, such as in white wine (French wine) by using the ultraltration-based method and in red wine (Chilean merlot wine) by using the reversed dialysis-based method (Fig. 5). It is noteworthy that the red wine samples could not be ltered through the lter with MWCO 3.5 kDa even without addition of dendrimers, most likely due to the presence of colloidal fractions. Interestingly, the binding capacity of the dendrimers to TA did not drastically decrease for the wine samples and was comparable with the binding capacity of TA from aqueous solutions at pH 3.23.5 (Fig. 6).

Fig. 4 Binding capacity of PAMAM G4 and G5 to PS and TA at 200 : 1 acid to dendrimer feeding molar ratio and unadjusted pH values.

Fig. 5

Schematic representation of the reverse dialysis method for nano-capturing TA using dendrimers.

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Fig. 6

Extent of binding of dendrimers to TA in various matrices (aqueous TA solution, white wine and red wine).

The presence of several competitor molecules, such as other organic acids, phenolic compounds, salts, etc., did not appear to disturb the interactions between dendrimers and TA molecules. A reasonable explanation for the relatively low decrease of the TA binding capacity of approx. 15% for the white wine and approx. 25% for the red wine as compared to aqueous solutions might be that the main acidic competitive components in wine, such as malic acid, citric acid, lactic acid, are less concentrated and have slightly higher pKa values than TA. TA binding studies computational approach Computational methods have been widely used for complementing and/or conrming experimental data on the hostguest interactions of dendrimers with dierent target molecules. A comprehensive review in this eld has been recently published by Lamm et al.42 In order to gain a deeper understanding of the interactions between dendrimers and TA molecules and the mechanisms underlying the entrapment we carried out molecular dynamics

simulations of TAdendrimer systems. Several scripts were implemented for the analysis of the data obtained from the molecular simulations. We rstly calculated the number of TA with respect to any atom of molecules within a distance of 8.5 A the dendrimer for the rst TAdendrimer system, 100 TA1 PAMAM (feeding molar ratio 100 mmol TA to 1 mmol of dendrimer), and for the second TAdendrimer system, 200 TA1 PAMAM (200 mmol TA to 1 mmol of dendrimer, Fig. 7). These data made estimation of the number of TA molecules captured by the dendrimer (PAMAM G5) with a high degree of accuracy possible. The MD results corresponded to 86.5 1.4 molecules of TA per molecule of dendrimer, for the system 100 TA1 PAMAM, and as expected higher values, 100.2 1.7 molecules of TA per molecule of dendrimer, for the system 200 TA1 PAMAM. These results are in very good agreement with the experimental data of the binding studies, 87.73 2.94 molecules of TA per molecule of dendrimer, at 100 : 1 TA/dendrimer feeding molar ratio and 106.3 9.3 molecules of TA per molecule of dendrimer, at 200 : 1 TA/dendrimer feeding molar ratio (see Fig. 2B and 3). The MD simulations also provided indication that the electrostatic energy between the dendrimer and TA molecules of the dendrimer is stronger than the vdW interwithin 8.5 A actions (Fig. S1). The number of hydrogen bonds between the dendrimer and the TA molecules was also taken into account and MD simulations revealed that about 110 hydrogen bonds are formed between the amide groups and the terminal amines of the dendrimer with the carboxylic and hydroxyl groups of TA (Fig. S2). Consequently, both electrostatic and hydrogen bond

Fig. 7 Number of TA molecules captured by PAMAM G5 for 100 TA1 PAMAM (black line) and 200 TA1 PAMAM (grey line).

Fig. 8 Radial pair distribution function between the core of PAMAM and TA molecules (grey line) and RDF between the core of PAMAM and the amino end groups of PAMAM (black line), system 100 TA1 PAMAM.

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Fig. 9 Number of TA molecules captured by PAMAM G5 within 18 A distance from the core of PAMAM G5 as estimated by molecular dynamics simulations, system 100 TA1 PAMAM (black line), system 200 TA1 PAMAM (grey line).

interactions play a key role in the entrapment of TA molecules. This nding is reinforced by the result of a radial pair distribution function between the core of PAMAM and TA molecules. This analysis indicated that there are two distinct shells of TA molecules, one of them being located between 2.5 and 18 A from the dendrimer core and the second shell beyond 18 A (Fig. 8). Furthermore, the analysis of the radial pair distribution function revealed that the most TA molecules are located distance from the core of PAMAM G5 (i.e. approx. beyond 18 A 81.6% of total TA captured molecules for the 100 TA1 PAMAM system) and provided evidence that the TA molecules captured by the dendrimer are in contact with the surface of the dendrimers (Fig. 9). It is noteworthy that the radial pair distribution function between the PAMAM core and the terminal amino groups of the dendrimer follows a similar radial distribution pattern,

from the featuring two main prominent shells, one 2.518 A dendrimer core and the other beyond 18 A. This strongly suggests that the amino end groups of the dendrimer lie in close proximity to the TA molecules (Fig. 8). This result is in agreement with the experimental data that indicated that one amino end group can accommodate up to one molecule of TA. Moreover the radial pair distribution function between the positively charged amino terminal groups of PAMAM and carboxylic groups of TA strongly conrmed the hypothesis of the close proximity between these two groups (Fig. 10). Interestingly when all charged amino groups of PAMAM G5 (183 amino groups) were taken into account, the MD simulations revealed that by increasing the TA/dendrimer feeding molar ratios from 30 : 1 to 100 : 1 and 200 : 1, one TA molecule bound to 1.91, 1.71 and 0.93 amino charged groups, respectively. In order to establish the location of the second shell of TA molecules with respect to the dendrimer surface, the radius of gyration of PAMAM G5 was calculated from the molecular dynamics simulation (Fig. 11). The results show a contraction of the dendrimer from 3.5 to 2.4 nm among the simulation. The Rg value for PAMAM G5 is 2.4 0.29 nm; this value is in agreement with the experimental data reported in the literature.43 It was possible to estimate which type of interactions are the most preponderant by calculating the radial pair distribution functions between the amino end-groups and the carboxylic groups of TA and between the amino end-groups and the hydroxyl groups of TA. The results showed that the interactions between amino terminal groups and carboxylic groups of TA are most predominant (Fig. 10). Fig. 12 represents a snapshot of the TAdendrimer system 100 TA1 PAMAM revealing as well the two shells distribution pattern of TA molecules within PAMAM, one close to the periphery where most of the TA molecules (depicted in cyan) are located and a second shell inside of dendrimer less populated (TA molecules depicted in orange).

Fig. 10 RDF between the amine end-groups of PAMAM and the carboxyl group of TA (black line) and RDF between the NH3+ of PAMAM and the hydroxyl group of TA (grey line), system 100 TA1 PAMAM.

Fig. 11 Radii of gyration (Rg) for the PAMAM dendrimer among the reaction coordinate. The value of Rg is 2.4 0.29 nm for the 4 last ns of simulation.

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assigned to the two hydroxyl groups of TA and electrostatic/ hydrogen bonding assigned to the other TA carboxylic group.

Conclusions
The present study showed that amine end-capped PAMAM and PPI dendrimers are able to eectively bind to the water soluble acid, TA, even in complex matrices such as white or red wine. The binding capacity of these dendrimers is dependent on the feed concentration of TA and increases linearly until reaching saturation. The linear correlation between the number of amino end-groups of dendrimers of dierent generations and the maximum binding capacity of TA strongly indicates that the dendrimers can accommodate up to one molecule of TA per amino end-group, suggesting a peripheral location of the TA molecules in the dendrimerTA supramolecular assemblies. Moreover, the comparison between the binding anities of dendrimers displaying dierent end functional groups to TA or PS could provide more insights into the nature of the hostguest architectures, conrming that surface electrostatic interactions between the positively charged amino surface groups and negative carboxylic groups of the organic acids are crucial for the high binding interactions and they synergistically cumulate with hydrogen bonding. These ndings were strongly conrmed by MD simulations that facilitated a precise overview on the TAdendrimer supramolecular systems. Although MD investigation showed that a relatively low percentage of TA molecules (up to 19% for the system 100 TA1 PAMAM and up to 15% for the system 200 TA1 PAMAM) are located inside of the dendrimers, these TA molecules are in close proximity to amino end-groups that are backfolded inside of the dendrimers conrming the experimental nding regarding the one-to-one correlation between the amino surface-groups and TA captured molecules. Owing to the main electrostatic character of the TA dendrimer interactions, the formation of the TAdendrimer complexes can be simply tuned by changing the pH, with a maximum complex formation at pH 5 and a minimum at extreme pH values 2 and 11, when the electrostatic attractions were drastically reduced. Interestingly, the binding capacity of the dendrimers to TA did not drastically decrease for the wine samples and was comparable with the binding capacity of TA from aqueous solutions. In conclusion, the experimental investigation as well as the molecular dynamics simulations evidenced amine end-capped dendrimers as eective nano-capturing systems for removal/ recovery of TA or for simplifying the analysis of TA in complex matrices such as pomace, juices, wines, vinasses and wine industrial waste.

Fig. 12

Snapshot of the system 100 TA1 PAMAM.

The amino end-groups present a similar distribution pattern, some of them being extended also inward by backfolding into the dendrimer as also evidenced in Fig. 13. Here only the TA molecules from the inner shell are depicted in order to evidence the spatial proximity of the amino terminal groups buried inside the dendrimer and the TA molecules. These interactions are based on the electrostatic attractions as well as on hydrogen bonding. Considering all the results from the MD simulations and the experimental data it can be concluded that TA molecule binds electrostatically with one carboxylic group to one terminal amino group of the dendrimer, whereas the rest of the TA molecule is located inside the dendrimer. This binding conguration is favored by the additional hydrogen bonding

Notes and references

1 D. Astruc, E. Boisselier and C. Ornelas, Chem. Rev., 2010, 110, 18571959. 2 D. A. Tomalia, So Matter, 2010, 6, 456474. 3 D. A. Tomalia, A. M. Naylor and W. A. Goddard, Angew. Chem., Int. Ed., 1990, 29, 138175.
Soft Matter, 2014, 10, 600608 | 607

Fig. 13 Snapshot of the system 100 TA1 PAMAM (only some TA molecules from the inner shell are depicted).

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Soft Matter

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