Immobilization of Hydrophilic Low Molecular-Weight Molecules in

Article pubs.acs.org/JPCB

Immobilization of Hydrophilic Low Molecular-Weight Molecules in Nanoparticles of Chitosan/Poly(sodium 4‑styrenesulfonate) Assisted by Aromatic−Aromatic Interactions Juan Pablo Fuenzalida,† Mario E. Flores,‡ Inés Móniz,§ Miguel Feijoo,§ Francisco Goycoolea,† Hiroyuki Nishide,∥ and Ignacio Moreno-Villoslada*,‡ †

IBBP, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, 48149 - Münster, Germany Instituto de Ciencias Químicas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Los Rios, Chile § Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, España ∥ Department of Applied Chemistry, School of Science and Engineering, Waseda University, Tokyo 169-8555, Japan ‡

S Supporting Information *

ABSTRACT: The immobilization of the hydrophilic low molecular-weight cationic molecules rhodamine 6G, methylene blue, and citidine in nanoparticles composed of two opposite charged polyelectrolytes, poly(sodium 4-styrenesulfonate) and chitosan, is studied, and the results correlated with their physicochemical properties. Nanoparticles containing both polyelectrolytes have been synthesized showing hydrodynamic diameters of around 200 nm and tunable zeta potential. It was found that the strength of binding of the cationic molecules to the polyanion bearing charged aromatic groups poly(sodium 4-styrenesulfonate) by means of short-range aromatic−aromatic interactions increases with their hydrophobicity and polarizability, as seen by 1H NMR and UV−vis spectroscopies, and diafiltration. Consequently, association efficiencies of 45, 21, and 12% have been found for the three molecules, respectively, revealing the different ability of the molecules to be immobilized in the nanoparticles. These results provide a proof of concept on a new strategy of immobilization of hydrophilic low molecular-weight molecules based on aromatic−aromatic interactions between polyelectrolytes and their aromatic counterions. nanogels, or nanoprecipitates.9 The former are suitable to encapsulate lipophilic molecules. Polysaccharide nanogels are produced by ionic gelation and are suitable to encapsulate hydrophilic high molecular-weight molecules, since these molecules present low diffusion constants and are retained by the nanoporous structure of the nanogels. Examples of nanoand microgels are found in the literature made of alginate and chitosan, cross-linked, respectively, with calcium and tripolyphosphate.10,11 However, these materials fail when trying to encapsulate hydrophilic LMWM, since they present high diffusion constants, and easily escape from the matrices.12 The nanoprecipitation technique involves several methods. Apart from the use of self-assembling of lipids forming the socalled solid−lipid nanoparticles,13 nanoprecipitates composed of water-insoluble polymers, but soluble in water-soluble organic solvents, such as poly(lactic-co-glycolic acid), may be achieved by the solvent displacement technique.14,15 Lipophilic LMWM may be efficiently encapsulated in solid−lipid

1. INTRODUCTION Immobilization of hydrophilic low molecular-weight molecules (LMWM) in different materials is important to achieve improved properties of such materials. Hydrophilic LMWM may provide luminescent, redox, acid−base, and other interesting properties that may be useful in materials performance, such as in antioxidant devices for food industry, or in sensing and stimuli responsive materials.1−3 For pharmaceutical uses, adequate means of immobilization may provide a control on the diffusion and release of drugs from different matrices. Besides, it may help improving the targeting to malign cells and tissues, by immobilizing the drugs on adequate carriers.4,5 This is especially important in pathologies such as HIV where the therapeutic arsenal is partly composed of low molecular-weight hydrophilic drugs such as nucleoside reverse transcriptase inhibitors.6 Among different useful matrices, those of submicron size are attractive since they may act as effective carriers in blood plasma, or may provide homogeneous dispersion in formulations in the food or pharmacy industries, among other applications.4,7,8 Different organic nanostructures may comprise nanocapsules containing a lipophilic core, polysaccharide © 2014 American Chemical Society

Received: April 16, 2014 Revised: July 23, 2014 Published: July 23, 2014 9782

dx.doi.org/10.1021/jp5037553 | J. Phys. Chem. B 2014, 118, 9782−9791

The Journal of Physical Chemistry B

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Figure 1. Molecular structures of the LMWM and the polyelectrolytes.

producing the so-called territorial binding.23−25 In order to immobilize hydrophilic LMWM in nanoparticles, site-specific binding to the nanoparticle components is desirable. Aromatic−aromatic interactions are secondary short-range site-specific interactions, which involve the release of water from the hydration shell of the interacting groups.26−29 When occurring between polyelectrolytes containing charged aromatic rings and aromatic counterions, they provide higher binding constants between the two species, and resistance to the cleaving effect of the presence of electrolytes such as NaCl. These interactions have shown to be of potential use for tuning molecular properties of the counterions, such as luminescent, redox, and acid−base properties.30−32 Specifically, systems undergoing aromatic−aromatic interactions have been investigated for drug delivery and chemical catalysis.33,34 We have shown that the extent of binding and the state of aggregation of different aromatic counterions in the presence of polyelectrolytes strongly depends on the structure of the polymer as a whole, and variables such as functional groups, localization of the charge, flexibility, hydrophobicity, linear charge density, and linear aromatic density seem to determine the behavior of the system.35−37 As these interactions are short-range site-specific interactions, ion pairs are formed, providing local hydrophobicity. In this respect, we have observed that the amphiphilia of the polyelectrolyte also plays an important role in order to retain the LMWM confined in the polyelectrolyte domain.38,39 In this paper, we will first show the formation of chitosan/ poly(sodium 4-styrenesulfonate) (CS/PSS) submicron particles, which are analyzed by dynamic light scattering (DLS) and turbidimetry. CS is a natural occurring polycation, while PSS is a synthetic polymer that bears negatively charged aromatic groups. The occurrence of aromatic−aromatic interactions between PSS and different cationic LMWM, such as rhodamine 6G (R6G), methylene blue (MB), and the nucleoside citidine (CTD) will be then comparatively evaluated by UV−vis spectroscopy, 1H NMR, and diafiltration (DF). Finally, as an absolute novelty of this paper, we will explore the use of these interactions as a tool to immobilize the three

nanoparticles or nanoprecipitates made by the solvent displacement technique, but hydrophilic LMWM are not normally retained in the nanoprecipitates, since in the formation process they are released to the aqueous phase. In order to overcome the problem of the diffusion of hydrophilic LMWM out of the nanosystems, different drugs have been covalently bound to different polymers, forming the so-called polymer conjugates. The new chemical entities formed may be efficiently incorporated in pharmaceutical applications, but the high cost related to synthetic efforts, environmental implications, and regulatory processes regarding unknown toxicities is a drawback of their use.16 Current techniques to encapsulate hydrophilic LMWM within polymeric micro- and nanostructures involve double emulsion methods17 and layer-by-layer (LbL) assembly.18,19 Nanoprecipitates may be also achieved by mixing two complementary charged polyelectrolytes at both definite absolute and relative concentrations, so that nanointerpolymer complexes are formed.20−22 The composition of the system must guarantee that one of the components is present in a sufficient excess to provide the resulting nanosolid with enough surface charge in order to prevent aggregation. Besides, vigorous stirring and careful reactant addition is necessary in order to obtain low polydisperse populations of nanoparticles. The formation and stability of the resulting nanoprecipitates is kinetically controlled due to the high number of charges in each single molecule. On the other hand, the interaction of the polyelectrolytes with charged hydrophilic low molecular-weight counterions is ruled by equilibrium processes. In this context, in the process of nanoparticle formation, the hydrophilic LMWM interacting with one of the nanoparticle components is ejected from the system, as the relative low charge of the LMWM can not compete with the high charge of the polyelectrolyte bearing the same charge. The interaction between polyelectrolytes and their counterions are mainly of long-range electrostatic nature. These interactions produce nonsite-specific binding between the molecules: the interacting reactants keep their hydration shell, and the counterions are able to move along the polymer chains, 9783



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the UV−vis spectra of the molecules change in the presence of PSS that may be in the supernatant, an excess of the polymer (10−2 M) was added to the samples to be analyzed, and the concentration of the analytes was determined with the aid of a calibration curve obtained in the presence of the same constant amount of PSS. In order to verify interactions between the LMWM and PSS, DF experiments have been done. Details for DF procedures can be found elsewhere.40,41 Prior to use, PSS was fractionated over a poly(ether sulfone) membrane with a molecular-weight cutoff of 10 000 Da, and the highest molecular-weight fraction was chosen for DF experiments so that no macromolecule is able to traverse the 5000 Da DF membrane. The main magnitudes managed in DF analyses are the filtration factor (F), defined as the ratio between the volume in the filtrate and the constant volume in the DF cell, the concentration in the filtrate of the LMWM under study (cLMWMfiltrate), the concentration of free LMWM in the cell solution (cLMWMfree), the concentration of LMWM reversibly bound to the polyelectrolyte (cLMWMrev‑bound), the apparent dissociation constant (KLMWMdiss), defined as the ratio cLMWMfree/cLMWMrev‑bound, the DF parameters km, j, u, and v, and the polymer concentration in mole per liter of monomeric units (cP). km and j parameters (the absolute value of the slope of the curve ln cLMWMfiltrate versus F in the absence and in the presence of the polyelectrolyte, respectively) are related to the strength of the interaction, while v and u are related to the amounts of LMWS reversibly or irreversibly bound to the polymer, respectively. By irreversibly bound we consider molecules bound in processes that may be reversible with an apparent dissociation constant that tends to zero at the conditions of the experiment, or molecules confined in matrices with release kinetics slower than the DF kinetics.

different hydrophilic low molecular-weight aromatic molecules in CS/PSS complexes, and the corresponding relative association efficiencies will be related to the relative strength of interaction with PSS, and ultimately, to the hydrophilic LMWM physicochemical properties, namely, hydrophobicity, polarizability, and hydrogen bond formation ability.

2. EXPERIMENTAL SECTION 2.1. Reagents. Commercially available PSS (Aldrich, synthesized from the para-substituted monomer), R6G (Acros), MB (Synth), and CTD (Aldrich) were used to prepare the solutions in deionized distilled water. Ultrapure CS in its hydrochloride salt was purchased from FMC Biopolymers (Protasan CL113 Batch No. FP-503−03; the degree of acetylation was certified by the supplier to be 14%, with a Mw of 119 kDa). The pH was adjusted with NaOH and HCl. NaCl (Scharlau) was used to adjust the ionic strength. D2O (Acros, 99.8% D) was used as the solvent for NMR studies. The molecular weight of the polymers was estimated as 230.7 and 206.2 g/mol of ionizable groups (n+ and n−) for CS and PSS, respectively. 2.2. Equipment. The unit used for DF studies consisted of a filtration cell (Amicon 8010, 10 mL capacity) with a magnetic stirrer, a regenerated cellulose membrane with a molecularweight cutoff of 5000 Da (Ultracel PLCC, 25 mm diameter), a reservoir, a selector, and a pressure source. Distilled water was deionized in a Simplicity Millipore deionizer. The pH was controlled on an UltraBasic Denver Instrument pH meter. UV−vis measurements were performed in a Heλios γ spectrophotometer. 1H NMR measurements were made with a JNM-Lambda500 (JEOL, 500 MHz) spectrometer. The size of the nanoparticles was determined by photon correlation spectroscopy, and the value of the zeta potential was determined by laser Doppler electrophoresis using a Malvern Zetasizer NanoZS (ZEN 3600, Malvern Instrument) analyzer fitted with a red laser light beam (λ = 632.8 nm). Association efficiencies have been obtained using a Hitachi CT15E centrifuge. 2.3. Procedures. Conventional and well-known procedures have been followed for DF, 1H NMR, and UV−vis spectroscopies. Particular experimental conditions are provided in the Figure captions. UV−vis measurements were recorded using optical path lengths ranging between 1.0 and 10−2 cm in order to have absorbances in the range of 0.1−1.0. Polyelectrolyte complexations were performed at room temperature mixing definite volumes of 1 × 10−2 M CS and 1 × 10−2 M PSS aqueous solutions. The final apparent concentration of both polyelectrolytes was defined in order to obtain different molar mixing ratios (CS/PSS, corresponding to the same cationic and anionic unit ratio n+/n−), and constant total amount of polymeric charges (CS + PSS, corresponding to n+ + n−) of 2 × 10−3 M. Turbidity of the colloidal dispersions was analyzed in the UV−vis spectrometer, observing the position of the baseline of the corresponding dispersions at a wavelength where no absorption by any of the molecules is observed. Inclusion of the LMWM in the nanoparticles was assayed by mixing the LMWM with PSS prior to nanoparticle formation. The association efficiency was determined by analyzing the concentration of CTDN, MB, and R6G in the supernatant after centrifugation of the formed nanoparticles at 10000g for 40 min. DF could not be used, since nanoparticles interact with the DF membrane forming a film over it during the DF process. The final concentration of the LMWM is 1.5 × 10−4 M. Since

3. RESULTS AND DISCUSSION 3.1. LMWM. The three LMWM chosen for this study are cationic at sufficiently low pH. R6G and MB are charged in a wide range of pH, while CTD is charged at pH lower than 3.9. They present different characteristics concerning hydrophobicity and polarizability. Both parameters where calculated for these molecules with the Marvinsketch 5.4.1.1 2011 software, a widely used tool for physicochemical and electronic properties prediction.42−46 The results are shown in Table 1. It Table 1. Some Physicochemical Properties of the LMWM

molecule

theoretical partition coefficient (log P)

number of hydrogen bond donors

number of hydrogen bond acceptors

polarizability (Å3)

R6G MB CTD

5.1 3.04 −2.1

2 0 4

4 3 6

51.61 33.12 22.58

can be seen that the most hydrophobic molecule is R6G, followed by MB, while CTD is the most hydrophilic, as deduced from the theoretical values of the log P. The calculated values coincide with those reported in the literature.47−49 Additionally, a larger number of hydrogen bonds may be formed with CTD as deduced from the larger number of donor and acceptor atoms, a fact that can reinforce a strong hydration shell of the drug. On the other hand, the polarizability of the molecules increases in the order CTD < MB < R6G, and this property may influence the ability of the molecules to undergo 9784



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be related to the rigidity of both CS and PSS, so that extended, highly hydrated structures could be formed even near the neutralization point, decreasing the electrophoretical mobility and preventing macroprecipitation.51 In addition, CS deprotonation may occur to some extent upon binding to PSS since this polysaccharide is prone to deprotonation depending on environmental conditions.52,53 Changes on the acid−base properties of macromolecules such as proteins54 or LMWM confined in different media have been reported.55 3.3. LMWM/PSS Interaction. 3.3.1. UV−Vis. R6G presents two bands in its UV−vis spectrum centered at 527 nm (monomer band) and 500 nm (dimer band), as can be seen in Figure 3A. At increasing concentrations, the intensity of the dimer band increases at the expense of the monomer band due to a higher probability to undergo face-to-face self-contacts.35 At the concentration used in our experiments, 1 × 10−4 M, the relative intensity of the dimer band is high. The interaction with 10-fold PSS produces an increase on the intensity of the dimer band, so that dimer formation is enhanced. Additionally, the monomer band is shifted 7 nm to lower energies, up to 534 nm, as a consequence of aromatic−aromatic interactions between the dye and the polymeric aromatic functional groups.35 Other nonaromatic polyelectrolytes produce dye aggregates on their environment, but not a displacement of the monomer band to lower energies, a fact that evidence the different mechanism of binding to these polymers, based on long-range electrostatic interactions between the dye and the polymer, and aromatic− aromatic interactions between dyes.35,37 In the presence of polymers bearing charged aromatic rings, ion-pairs are formed due to short-range aromatic−aromatic interactions, and at moderate excess of the polymeric aromatic functional groups, these ion-pairs tend to aggregate, as is the case of the complex R6G/PSS. The aggregation of these ion pairs is also influenced by factors such as the linear charge density, linear aromatic density, hydrophobicity, and flexibility of the polyelectrolytes. Due to the high hydrophobicity of R6G, the ion-pairs produced with the benzenesulfonate groups are highly hydrophobic, and the aggregates are stabilized in a hydrophobic environment. For this reason, the interaction between PSS and R6G is more resistant to the cleaving effect of high concentrations of NaCl compared to the interactions between this dye and other nonaromatic polyanions,35 and a low influence on the spectrum of the dye is observed upon adding 0.1 M NaCl, as can be seen in Figure 3A. As in the case of R6G, the other xanthene dye, MB, shows two bands around 610 and 666 nm in its UV−vis spectrum in water (see Figure 3B).56 Due to the high concentration of the dye in our experiments, the dimer band, appearing at 611 nm, is

aromatic−aromatic interactions, dispersion forces, and shortrange electrostatic interactions. 3.2. CS/PSS Nanoparticle Formation. CS/PSS nanoparticles were made as described in the Experimental Section. The total apparent concentration of polymeric functional groups (CS + PSS) was set to 2 × 10−3 M. Nanocomplex formation was explored as a function of the CS/PSS ratio at pH 4.5. The results concerning nanocomplex size, zeta potential, and turbidity are shown in Figure 2. It can be seen that

Figure 2. Size (hydrodynamic diameter) (●), turbidity (▲), and zeta potential (■) of the nanocomplexes as a function of the CS/PSS ratio. Error bars correspond to the standard deviation of the measurements (n = 9, 3 measurements × 3 samples).

turbidity and size correlate very well, and a maximum in the CS/PSS ratio range of 0.8−1.0 is found. All the formulations showed the formation of nanoprecipitates of apparent size lower than 200 nm, with PDI values in the range of 0.20 ± 0.03 with the exception of the formulation formed at a CS/PSS ratio of 0.8 that showed an apparent size of 340 ± 40 nm with a PDI value of 0.50 ± 0.10. Assays reported in the literature show the formation of polydisperse structures and macroprecipitates for other polycation/polyanion systems in a wide range of relative concentrations, and similar or lower absolute concentrations of both polyelectrolytes.11,50 The zeta potential of the CS/PSS nanoparticles turns from negative to positive in the range of the relative concentrations studied, a fact that correlates with the charge of the polymer that is in excess at every composition. The positive zeta potential achieved under an excess of CS is low by comparison to other systems described in the literature using CS of the same degree of acetylation.11 These facts may

Figure 3. UV−vis spectra of solutions containing 1 × 10−4 M of R6G (A), 1 × 10−4 M of MB (B), and 1 × 10−4 M of CTD (C), in the absence of any additive (a), and in the presence of 1 × 10−3 M of PSS (b), and 1 × 10−3 M of PSS and 0.1 M of NaCl (c). 9785



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also relatively intense, indicating a higher probability of the dyes to undergo face-to-face self-contacts.36,37 In fact, the intensity of this band relative to the intensity of the corresponding monomer band is higher than in the case of R6G. This could be due to the higher planarity of this dye, which may favor the self-contacts. In the presence of 10-fold PSS, the dimer band is centered at 607 nm and broadens. This shift to higher energies and broadening arises as a consequence of the formation of higher order aggregates in the environment of PSS, according to the exciton theory, since a higher local concentration of the dyes is induced by the electrostatic attraction of the cationic dyes and the negative charges of the polyelectrolyte.37,57 Note that in the case of R6G, the presence of the polymer produces an increase on the dimerization, but not polymerization, as in the case of MB. This different behavior comparing both dyes may be due to a combination of factors, such as the higher planarity of MB, its smaller size, a lower strength of binding to the aromatic benzenesulfonate groups associated with its lower polarizability, and a lower tendency to stay confined in hydrophobic environments due to its lower hydrophobicity. At lower concentration of the dye (10−5 M) and the same relative concentration of the polymer (10-fold) we observe, as in the case of R6G, the formation of MB dimers in the presence of PSS, appearing an intense band at around 610 nm.37 The addition of NaCl to the samples produces a minor change in the corresponding spectrum, revealing the resistance of the interaction to the cleaving effect of added electrolytes. The CTD UV−vis band is dependent on the pH, and its maximum shifts from 280 nm at pH lower than 3.0 to 271 nm at pH higher than 5.5, as can be seen in Figure 4. The pKa of

Figure 5. Maxima of absorbance of 1 × 10−4 M of CTD as a function of the pH in the absence of any additive (■), and in the presence of 1 × 10−3 M of PSS (●), and 1 × 10−3 M of PSS and 0.1 M of NaCl (▲).

value of the standard deviation found is assigned to the isosbestic point.

Figure 6. Standard deviation of the absorbances corresponding to the spectra of 1 × 10−4 M of CTD at eight different pH values between 2.5 and 9.0, as a function of the wavelength, in the absence of any additive (■), and in the presence of 1 × 10−3 M of PSS (●), and 1 · 10−3 M of PSS and 0.1 M of NaCl (▲).

PSS shows two absorption maxima at 227 and 264 nm, the former more intense than the latter.30 The spectrum of the polymer was subtracted in each case from the spectra of CTD in the presence of the polymer for further analysis. It can be seen in Figure 3C that the CTD absorbance maximum is shifted 3 nm to lower energies in the presence of 10-fold PSS at pH 3. This shift is partially reversed by the addition of 0.1 M NaCl to the solutions, indicating a high long-range electrostatic nature of the interaction, which is easily cleaved under high ionic strength conditions. Additionally, in Figure 5 it can be seen that the presence of 10-fold PSS produces a shift on the pKa of the acid−base equilibrium of CTD from 3.91 to 4.31, since the polyanion stabilizes the acid form of the nucleoside. Changes in the pKa of different molecules in the presence of polyelectrolytes have been also reported in the literature.32,55 In the presence of 0.1 M of NaCl, the pKa is only shifted to 4.13. On the other hand, the isosbestic point related to the spectral

Figure 4. UV−vis spectra of 1 × 10−4 M of CTD at pH 2.5 (a), 3.0 (b), 3.5 (c), 4.0 (d), 4.5 (e), 5.5 (f), 6.5 (g), and 9.0 (h).

the nucleoside, calculated following the shift of the UV−vis band, is found to be 3.91 ± 0.05, as can be seen in Figure 5. The acid−base properties of this molecule are related to protonation of its aromatic ring, as can be seen in Figure 1. A isosbestic point appears at around 265 nm upon interconversion between the acid and the basic forms of the nucleoside when changing the pH, as can be seen in Figure 4. The exact wavelength of the isosbestic point is experimentally obtained evaluating the standard deviation of the spectral data at every pH for each wavelength, evaluated near 265 nm, as can be seen in Figure 6. The wavelength corresponding to the minimum 9786



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the case of R6G, the signals broaden and tend to disappear. This is due to a decrease on the molecular mobility, due to the formation and aggregation of ion-pairs. These ion-pair aggregates are stabilized by the hydrophobic environment furnished by the polymer. On the contrary, MB, although it also undergoes aromatic−aromatic interaction with the benzenesulfonate groups of PSS and consequent ion-pair formation and aggregation, is more mobile due to its lower size and higher planarity, and undergoes faster chemical exchange, so that clear signals in 1H NMR spectra in the presence of the polymer are observed. The interaction of CTD and PSS has been observed at pH 2 in order to enhance electrostatic interactions upon protonation of the nucleoside. The chemical shifts of CTD protons corresponding to 1 × 10−3 M of CTD in the absence and in the presence of 10-fold PSS at different conditions are shown in Table 2. Aromatic protons H1 and H2 are the most upfield shifted achieving increments of around 0.2 ppm in the presence of the polymer. Particularly, H2 is upfield shifted 0.2562 ppm, higher than the corresponding to H1 (0.1720 ppm). The rest of the protons are upfield shifted less than 0.1 ppm. This may indicate that the approach of CTD to the benzenesulfonate group occurs by the less hindered part of the nucleoside, i.e., opposite to the ribose ring, which may remain hydrated and orientated toward the bulk, allowing the benzene aromatic currents to mainly influence proton H2. The upfield shift of the aromatic protons is, however, lower than that obtained for R6G and MB aromatic protons in the presence of the same polymer,35,36 achieving values up to 0.6 ppm, and indicating less proximity or less affinity between the polymer and CTD. The upfield shifts of the 1H NMR signals are ascribed to aromatic−aromatic interactions, as can be deduced by comparison to the spectrum obtained in the presence of poly(sodium vinylsulfonate) (PVS), which does not contain aromatic groups. It can be seen in Figure 8 that, in the presence of this polyelectrolyte, the changes on the CTD spectrum are nearly negligible, indicating that the interaction does not produce close binding, and is mainly ruled by long-range electrostatic interactions. Upon adding 0.1 M of NaCl to the CTD/PSS complex, the corresponding upfield shifts are prevented, as can be also seen in Figure 8, indicating the quenching of the interaction, in accordance to an interaction mainly driven by long-range electrostatic interactions. In contrast, as observed in previous experiments, the interaction between PSS and R6G or MB is not sensitive to the addition of NaCl, (and, however, decreasing the linear aromatic density of the polymer by inserting maleate units in the polymeric backbone, sensitivity to the addition of NaCl increasingly appears), indicating a stronger influence of the aromatic−aromatic interaction in the overall free energy.35−37 The sensitivity of the CTD/PSS interaction to the addition of increasing amounts of NaCl is analyzed in Table 2 and Figure 9. It can be seen that 1H NMR signals of CTD protons go back to their original positions when increasing the NaCl concentration, showing an exponential pattern. This exponential decay phenomenon is normally correlated to the affinity of both interacting species.61,62 3.3.3. Diafiltration Studies. In order to better understand the relative ability of PSS to bind the LMWM, DF provides a direct measurement, since it is a separation technique. The experimental conditions must be tuned in order to adjust the results to the narrow sensitivity window of this technique. In all cases, the polymer/LMWM ratio was fixed to 10-fold. The absolute concentrations were tuned in order to avoid self-

changes between the acid and the basic forms of CTD is also shifted to lower energies, from 265 to 267 nm in the presence of 10-fold PSS, and is not shifted at all in the presence of 10fold PSS and 0.1 M of NaCl, as can be seen in Figure 6. UV−vis experiments show a higher dependence of the sensitivity of the interaction on the addition of NaCl in the case of CTD, comparing to the xanthene dyes, a fact that may be related to its higher hydrofilia, lower polarizability, and its reinforced hydration sphere by means of a higher number of hydrogen bonds with water, which minimizes the contribution of short-range aromatic−aromatic interactions in the overall interaction.36 3.3.2. 1H NMR. 1H NMR is a useful technique to evaluate changes on the molecular environment when aromatic− aromatic interactions occur. When aromatic rings stack on each other, one aromatic ring places in the shielding cone of the other, resulting in upfield shifts of 1H-resonances.58−60 The 1H NMR spectra corresponding to 1 × 10−3 M of the LMWM in the absence and in the presence of 10-fold PSS are shown in Figure 7. The assignment of the signals is related to the structures shown in Figure 1. It can be seen that the signals corresponding to aromatic protons of all the LMWM are upfield shifted in the presence of PSS, as an evidence of the close proximity between the benzenesulfonate groups and the LMWM. However, several contrasting results are obtained. In

Figure 7. 1H NMR spectra of a 10−2 M PSS solution in D2O (a), and 10−3 M solution of R6G (b,c), MB (d,e), and CTD (f,g), in the absence of PSS (c,e,g), and in the presence of 10−2 M of PSS (b,d,f). 9787



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Table 2. Chemical Shifts of Protons H1−H8 of CTD 10−3 M at pH 2 and Different Conditions experiment

[PSS] (M)

CTD CTD/PSS-0 CTD/PSS-0.005 CTD/PSS-0.01 CTD/PSS-0.02 CTD/PSS-0.05 CTD/PSS-0.1

0 10−2 10−2 10−2 10−2 10−2 10−2

[NaCl] (M)

5 1 2 5 1

0 0 × 10−3 × 10−2 × 10−2 × 10−2 × 10−1

H1

H2

H3

H4

H5

H6

H7

H8

8.009 7.837 7.878 7.880 7.910 7.975 7.990

6.113 5.857 5.912 5.923 5.965 6.046 6.077

5.792 5.709 5.726 5.732 5.746 5.768 5.787

4.234 4.135 4.155 4.161 4.178 4.207 4.227

4.107 4.048 4.059 4.064 4.074 4.091 4.107

4.064 3.994 4.009 4.013 4.025 4.045 4.063

3.840 3.736 3.757 3.764 3.780 3.809 3.828

3.711 3.630 3.646 3.651 3.665 3.686 3.704

ments showed that, in the absence of added NaCl, very low dissociation constants are found upon interaction between PSS and the three LMWM, and the obtained data fell beyond the sensitivity of the diafiltration technique for analysis.37,63 However, NaCl can be added to cleave long-range electrostatic interactions and favor the dissociation of the electrostatic complexes. Control experiments undertaken in the absence of the polymer, showed that a fraction of 20−30% of the LMWM, specifically R6G and MB, remained retained in the diafiltration system. This is due to the tendency of these molecules to produce self-aggregates on hydrophobic or negatively charged surfaces. Thus, this loss of analyte during its permanence in contact with the cell components, and passage through the highly porous DF membrane is unavoidable, and is considered in the interaction analysis. In the presence of the polymer, once the long-range electrostatic interactions are cleaved under a high NaCl concentration, the remaining interaction can be considered to be due to other interactions such as hydrogen bond formation, aromatic−aromatic interactions, including hydrophobic interactions and short-range electrostatic interactions. The most resistant to the cleaving effect of NaCl was the complex R6G/PSS, as can be seen in Figure 10 and Table 3.

Figure 8. 1H NMR spectra of a 10−2 M PSS solution in D2O (a), and 10−3 M solution of CTD in the presence of 10−2 M of PSS and 0.1 M of NaCl (b), in the presence of 10−2 M of PVS (c), and in the absence of any polyelectrolyte (d).

Figure 10. DF profiles [ln(cLMWMfiltrate) versus F] of 1 × 10−5 M of R6G (a), 2 × 10−5 M of MB (b), 1 × 10−3 M of CTD (c), in the absence of any polyelectrolyte and in the presence of NaCl 0.1 M (■), in the presence of 10-fold PSS and NaCl 0.1 M (▲), and in the presence of 10-fold PSS and NaCl 0.01 M (●). Other experimental conditions are shown in Table 2.

In the presence of 0.1 M of this salt, a KR6Gdiss of 0.31 ± 0.13 was obtained. Note that the absolute concentrations of both the dye and the polymer are the lowest of this series of experiments, which eventually should favor the dissociation of the complex. As expected, the complex MB/PSS was also resistant to the cleaving effect of the addition of NaCl, but the KMBdiss obtained (1.2 ± 0.3) achieved a higher value due to a higher hydrophilia of this dye, and its lower polarizability. Note that the absolute concentrations of both the dye and the polymer double those of the previous experiment. On the other hand, in the case of the more polar, less polarizable CTD, despite that an absolute concentration 2 orders of magnitude higher than in the case of the dyes must be used, in the

Figure 9. Chemical shift increment of CTD protons in 1 × 10−3 M CTD solutions in D2O in the presence of 1 × 10−2 M of PSS and different NaCl concentration, normalized by the corresponding value in the absence of PSS or NaCl, and exponential adjustment of the corresponding mean values. The analytical function is y = 0.886·e−24.4x (y = mean chemical shift, and x = NaCl concentration).

aggregation of the dyes, and provide with enough sensitivity for concentration analyses of the filtration fractions. The apparent dissociation constants may be calculated for such specific experimental conditions, considering that the equilibrium kinetics are faster than the diafiltration kinetics, so that the steady-state approximation can be assumed. Our own experi9788



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Table 3. Results of DF of R6G, MB, and CTD at pH 3−5 in the Presence of Different Additivesa experiment

[LMWM] (M)

CTD-NaCl CTD/PSS-NaCl-001 CTD/PSS-NaCl-01 MB-NaCl MB/PSS-NaCl R6G-NaCl R6G/PSS-NaCl

1 1 1 2 2 1 1

× × × × × × ×

−3

10 10−3 10−3 10−5 10−5 10−5 10−5

[PSS] (M)

[NaCl] (M)

linear adjustment

R2

vb

uc

j

km

KLMWMdissd

1 × 10−2 1 × 10−2

0.01 0.01 0.1 0.1 0.1 0.1 0.1

y = −1.02x −6.3 y = −0.59x −7.3 y = −0.88x −6.6 y = −0.81x −11.2 y = −0.51x −11.5 y = −0.90x −6.6 y = −0.22x - 13.1

0.99 0.99 0.99 0.99 0.97 0.97 0.99

1.00 0.99 1.00 0.7 0.8 0.7 0.78

0 0.01 0 0.3 0.2 0.3 0.22

0.59 0.88 0.51 0.22

1.0 0.81

1.4 ± 0.1 7.3 ± 0.1 1.2 ± 0.3

2 × 10−4 1 × 10−4 1 × 10−4

“init” applies for initial values. y = ln⟨cLMWM ⟩, x = F, R lineal regression coefficient. v = −((⟨cLMWM exp(jΔF)])). cu = 1 − v. dKLMWMdiss was calculated considering (j/1 − j) ≤ KLMWMdiss ≤ (j/km − j). a

filtrate

b

2

blank R6G MB CTD

155 185 223 169

± ± ± ±

02 25 15 03

PDI 0.22 0.27 0.45 0.32

± ± ± ±

0.02 0.02 0.06 0.04

zeta potential (mV) −30.9 −30.1 −26.2 −31.6

± ± ± ±

0.3 0.1 1.8 0.9

ΔF)/(cLMWMcell−init[1 −

■

CONCLUSIONS Physicochemical properties such as hydrophobicity and polarizability of three LMWM have been compared, and they decrease in the order R6G > MB > CTD. These molecules bear positively charged aromatic groups, so they are susceptible to undergo aromatic−aromatic interactions with the negatively charged polyelectrolyte containing aromatic groups PSS. The relative strength of these interactions also followed the order R6G > MB > CTD, as found by UV−vis and 1H NMR spectroscopies, and diafiltration. As an absolute novelty of this paper, immobilization of these molecules in CS/PSS nanoparticles showing a negative zeta potential has been achieved. The relative association efficiencies were 45, 21, and 12%, for R6G, MB, and CTD, respectively, as corresponding to the respective strength of interaction with PSS and decreasing hydrophobicity and polarizability. The CS/PSS nanoparticles synthesized showed hydrodynamic diameters of around 200 nm and tunable zeta potential, depending on the polyelectrolyte in excess. These results provide a proof of concept of a new

Table 4. Physicochemical Properties of CS/PSS Nanoparticles in the Absence and the Presence of 1.5 × 10−4 M of the LMWM Prepared at a CS/PSS Ratio of 0.6−0.54 (Mean ± Standard Deviation, n = 3) and Corresponding Association Efficiencies size (nm)

0.31 ± 0.13

component on the overall interaction, and consequent ability to produce and stabilize ion-pairs. Thus, the most tightly hydrated, less polarizable, but most polar CTD exhibits the highest tendency to be displaced by the polycation CS in the nanoparticle formation process. Intermediate behavior, consistent with the results found by DF and 1H NMR and UV−vis spectroscopies, is found for MB, which, despite its aromatic− aromatic interactions, is more mobile and hydrophilic. On the contrary, R6G shows the highest tendency to undergo sitespecific aromatic−aromatic interactions, as deduced by the results found by DF and 1H NMR and UV−vis spectroscopies, which produce the highest association efficiency, and that can be correlated with its highest hydrophobicity and polarizability. The size of the nanopartices formed in the presence of the LMWM ranges between 166 and 238 nm with PDI values ranging between 0.27 and 0.45. These values are higher than those obtained for the CS/PSS nanoparticles at a ratio 0.6. Finally, the values of the zeta potential range between −26 and −32 mV, consistent with the nanocomplexes presenting excess of the polyanion. These results provide a proof of concept of a new strategy of immobilization of hydrophilic LMWM not only in polyelectrolyte complexes, but also in any other form of nanovehicles such as those obtained by double emulsion and layer-by-layer methods. The new strategy is based on the occurrence of aromatic−aromatic interactions between polyelectrolytes and their aromatic counterions. Additionally, recently released promising results concerning PSS biocompatibility and in vivo target selectivity support the further study of these systems in nanomedicine.65

presence of 0.1 M of NaCl, the interaction is almost completely cleaved, and a KCTDdiss higher than 7.0 ± 0.5 is obtained. Decreasing the amount of added NaCl 1 order of magnitude, the cleaving effect is less significant, and a KCTDdiss similar to that found in the case of the MB/PSS complex in the presence of 0.1 M of NaCl is obtained, achieving a value of 1.4 ± 0.1. These results highlight a correlation between the hydrophobicity and polarizability of the LMWM in our studies with the strength of binding to PSS and the stability of the resulting complexes.64 3.4. Inmobilization Studies. Assays to immobilize the three LMWM in CS/PSS nanoparticles have been done. The formulations chosen contained a CS/PSS ratio of 0.54, thus bearing an excess of the polyanion. The concentration of the polyanion was 2.5 × 10−3 M, while the concentration of the LMWM was 17-fold lower (0.15 × 10−3 M). The concentration of CS was 1.35 × 10−3 M, so that the maximum concentration of positive charges in the particles, assuming that all the LMWM can be encapsulated, is 1.5 × 10−3 M. Thus, the maximum ratio between positive and negative charges is 0.6. Subtracting the concentration of CS from that of PSS, an excess of 1.15 × 10−3 M of negative charges remains in the formulation, which constitutes an almost 8-fold excess over the LMWM concentration. As expected, the ability of PSS to bind the LMWM dramatically decreases upon interaction and complex formation with the positively charged CS, despite the excess of negative charges. Nevertheless, the binding is still significant. The retention of the LMWM in the nanoparticles achieved different values that correlate with the strength of binding to PSS, and ultimately to their hydrophobicity and polarizability, as can be seen in Table 4. Thus, 45, 21, and 12% of the initial R6G, MB, and CTD, respectively, was retained in the nanoparticles. The differences on the association efficiency are ascribed to the relative contribution of the aromatic−aromatic interaction

LMWM

⟩

filtrate init

0.90

association efficiencya (%) − 44.84 ± 0.21 20.87 ± 0.06 12.32 ± 1.62

a

Association efficiency (%) was calculated as [(Total LMWM amount − Free LMWM amount)/Total LMWM amount] × 100. 9789



Article

Aromatic-Aromatic Interactions with Poly(sodium 4-styrenesulfonate). React. Funct. Polym. 2014, 81, 14−21. (13) Uner, M.; Yener, G. Importance of Solid Lipid Nanoparticles (SLN) in Various Administration Routes and Future Perspectives. Int. J. Nanomed. 2007, 2, 289−300. (14) Schubert, S.; Delaney, J. T.; Schubert, U. S. Nanoprecipitation and Nanoformulation of Polymers: From History to Powerful Possibilities Beyond Poly(lactic acid). Soft Matter 2011, 7, 1581−1588. (15) Zhang, C.; Pansare, V. J.; Prud’homme, R. K.; Priestley, R. D. Flash Nanoprecipitation of Polystyrene Nanoparticles. Soft Matter 2012, 8, 86−93. (16) Bekkara-Aounallah, F.; Gref, R.; Othman, M.; Reddy, L. H.; Pili, B.; Allain, V.; Bourgaux, C.; Hillaireau, H.; Lepêtre-Mouelhi, S.; Desmaële, D.; et al. Novel Pegylated Nanoassemblies Made of SelfAssembled Squalenoyl Nucleoside Analogues. Adv. Funct. Mater. 2008, 18, 3715−3725. (17) Cohen-Sela, E.; Chorny, M.; Koroukhov, N.; Danenberg, H. D.; Golomb, G. A New Double Emulsion Solvent Diffusion Technique for Encapsulating Hydrophilic Molecules in PLGA Nanoparticles. J. Controlled Release 2009, 133, 90−95. (18) Mora-Huertas, C. E.; Fessi, H.; Elaissari, A. Polymer-Based Nanocapsules for Drug Delivery. Int. J. Pharm. 2010, 385, 113−142. (19) Yan, S.; Zhu, J.; Wang, Z.; Yin, J.; Zheng, Y.; Chen, X. Layer-byLayer Assembly of Poly(L-glutamic acid)/Chitosan Microcapsules for High Loading and Sustained Release of 5-Fluorouracil. Eur. J. Pharm. Biopharm. 2011, 78, 336−345. (20) Al-Qadi, S.; Alatorre-Meda, M.; Zaghloul, E. M.; Taboada, P.; Remunán-López, C. Chitosan−Hyaluronic Acid Nanoparticles for Gene Silencing: The Role of Hyaluronic Acid on the Nanoparticles’ Formation and Activity. Colloids Surf., B 2013, 103, 615−623. (21) Chen, Y.; Mohanraj, V.; Wang, F.; Benson, H. E. Designing Chitosan−Dextran Sulfate Nanoparticles Using Charge Ratios. AAPS PharmSciTech 2007, 8, 131−139. (22) Hu, C. S.; Chiang, C. H.; Hong, P. D.; Yeh, M. K. Influence of Charge on FITC-BSA-Loaded Chondroitin Sulfate−Chitosan Nanoparticles Upon Cell Uptake in Human Caco-2 Cell Monolayers. Int. J. Nanomed 2012, 7, 4861−4872. (23) Manning, G. S. The Molecular Theory of Polyelectrolyte Solutions with Applications to the Electrostatic Properties of Polynucleotides. Q. Rev. Biophys. 1978, 11, 179−246. (24) Manning, G. S. Limiting Laws and Counterion Condensation in Polyelectrolyte Solutions. 8. Mixtures of Counterions, Species Selectivity, and Valence Selectivity. J. Phys. Chem. 1984, 88, 6654− 6661. (25) Manning, G. S. Counterion Condensation Theory of Attraction between Like Charges in the Absence of Multivalent Counterions. Eur. Phys. J. E 2011, 34, 1−18. (26) Hunter, C. A.; Lawson, K. R.; Perkins, J.; Urch, C. J. Aromatic Interactions. J. Chem. Soc., Perkin Trans. 2 2001, 651−669. (27) Meyer, E. A.; Castellano, R. K.; Diederich, F. Interactions with Aromatic Rings in Chemical and Biological Recognition. Angew. Chem., Int. Ed. 2003, 42, 1210−1250. (28) Mignon, P.; Loverix, S.; Steyaert, J.; Geerlings, P. Influence of the π−π Interaction on the Hydrogen Bonding Capacity of Stacked DNA/RNA Bases. Nucleic Acids Res. 2005, 33, 1779−89. (29) Willerich, I.; Ritter, H.; Gröhn, F. Structure and Thermodynamics of Ionic Dendrimer−Dye Assemblies. J. Phys. Chem. B 2009, 113, 3339−3354. (30) Moreno-Villoslada, I.; González, F.; Rivera, L.; Hess, S.; Rivas, B. L.; Shibue, T.; Nishide, H. Aromatic−Aromatic Interaction between 2,3,5-Triphenyl-2H-tetrazolium Chloride and Poly(sodium 4-styrenesulfonate). J. Phys. Chem. B 2007, 111, 6146−6150. (31) Moreno-Villoslada, I.; Jofré, M.; Miranda, V.; González, R.; Sotelo, T.; Hess, S.; Rivas, B. L. pH Dependence of the Interaction between Rhodamine B and the Water-Soluble Poly(sodium 4styrenesulfonate). J. Phys. Chem. B 2006, 110, 11809−11812. (32) Moreno-Villoslada, I.; Murakami, T.; Nishide, H. Comment On “J- and H-Aggregates of 5,10,15,20-Tetrakis-(4-sulfonatophenyl)-

strategy of immobilization of hydrophilic low molecular-weight molecules based on aromatic−aromatic interactions between polyelectrolytes and their aromatic counterions.

■

ASSOCIATED CONTENT

S Supporting Information *

Complete lists of authors for refs 16 and 47. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*FAX: 56-63-2293520; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Fondecyt (Grants Nos. 1090341 and 1120514, Chile), Grant-in-Aid for Scientific Research (No. 24225003, MEXT, Japan), and DFG (Project GRK 1549 IRTG “Molecular and Cellular GlycoSciences”, Germany) for financial support.

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REFERENCES

(1) Kostova, D. Triphenyltetrazolium Chloride as a New Analytical Reagent for Molybdenum(VI): Application to Plant Analysis. J. Anal. Chem. 2011, 66, 384−388. (2) McHedlov-Petrossyan, N. O.; Vodolazkaya, N. A.; Doroshenko, A. O. Ionic Equilibria of Fluorophores in Organized Solutions: The Influence of Micellar Microenvironment on Protolytic and Photophysical Properties of Rhodamine B. J. Fluoresc. 2003, 13, 235−248. (3) Tolentino, C.; dos Santos, M. V.; Ribeiro, S.; Barud, H.; de Araujo, C. B.; Gomes, A.; de Melo, L. In Biopolymer Random Laser Consisting of Rhodamine 6g and Silica Nanoparticles Incorporated to Bacterial Cellulose, Conference on Lasers and Electro-Optics 2012, San Jose, California, 2012/05/06; Optical Society of America: San Jose, CA, 2012; p JW4A.51. (4) Freitas, J. R. A. What Is Nanomedicine? Nanomed.: Nanotechnol., Biol. Med. 2005, 1, 2−9. (5) Vinogradov, S. V.; Poluektova, L. Y.; Makarov, E.; Gerson, T.; Senanayake, M. T. Nano-NRTIs: Efficient Inhibitors of HIV Type-1 in Macrophages with a Reduced Mitochondrial Toxicity. Antivir. Chem. Chemother. 2010, 21, 1−14. (6) Richman, D. D. HIV Chemotherapy. Nature 2001, 410, 995− 1001. (7) Wang, S.; Su, R.; Nie, S.; Sun, M.; Zhang, J.; Wu, D.; MoustaidMoussa, N. Application of Nanotechnology in Improving Bioavailability and Bioactivity of Diet-Derived Phytochemicals. J. Nutr. Biochem. 2014, 25, 363−376. (8) Sharma, P.; Garg, S. Pure Drug and Polymer Based Nanotechnologies for the Improved Solubility, Stability, Bioavailability and Targeting of Anti-HIV Drugs. Adv. Drug Delivery Rev. 2010, 62, 491− 502. (9) Vrignaud, S.; Benoit, J.-P.; Saulnier, P. Strategies for the Nanoencapsulation of Hydrophilic Molecules in Polymer-Based Nanoparticles. Biomaterials 2011, 32, 8593−8604. (10) Alonoso, J. M.; Goycoolea, F. M. Chitosan−Polysaccharide Blended Nanoparticles for Controlled Drug Delivery. In Natural-Based Polymers for Biomedical Applications; Reis, R. L., Neves, N. M., Ed.; Woodhead International, Ltd: Cambridge, U.K., 2008; pp 544−679. (11) Goycoolea, F. M.; Lollo, G.; Remuñań -López, C.; Quaglia, F.; Alonso, M. a. J. Chitosan−Alginate Blended Nanoparticles as Carriers for the Transmucosal Delivery of Macromolecules. Biomacromolecules 2009, 10, 1736−1743. (12) Araya-Hermosilla, E.; Muñoz, D.; Orellana, S.; Yáñez, A.; Olea, A. F.; Oyarzun-Ampuero, F.; Moreno-Villoslada, I. Immobilization of Rhodamine 6G in Calcium Alginate Microcapsules Based on 9790



Article

porphyrin and Interconversion in PEG-b-P4VP Micelles”. Biomacromolecules 2009, 10, 3341−3342. (33) Frühbeißer, S.; Gröhn, F. Catalytic Activity of Macroion− Porphyrin Nanoassemblies. J. Am. Chem. Soc. 2012, 134, 14267− 14270. (34) Sharma, S. K.; Kumar, R.; Kumar, S.; Mosurkal, R.; Parmar, V. S.; Samuelson, L. A.; Watterson, A. C.; Kumar, J. Influence of EDA-π Interactions in Drug Encapsulation Using Nanospheres. Chem. Commun. 2004, 2689−2691. (35) Moreno-Villoslada, I.; Fuenzalida, J. P.; Tripailaf, G.; ArayaHermosilla, R.; Pizarro, G. D. C.; Marambio, O. G.; Nishide, H. Comparative Study of the Self-Aggregation of Rhodamine 6G in the Presence of Poly(sodium 4-styrenesulfonate), Poly(N-phenylmaleimide-co-acrylic acid), Poly(styrene-alt-maleic acid), and Poly(sodium acrylate). J. Phys. Chem. B 2010, 114, 11983−11992. (36) Moreno-Villoslada, I.; Torres, C.; Gonzalez, F.; Shibue, T.; Nishide, H. Binding of Methylene Blue to Polyelectrolytes Containing Sulfonate Groups. Macromol. Chem. Phys. 2009, 210, 1167−1175. (37) Moreno-Villoslada, I.; Torres-Gallegos, C.; Araya-Hermosilla, R.; Nishide, H. Influence of the Linear Aromatic Density on Methylene Blue Aggregation around Polyanions Containing Sulfonate Groups. J. Phys. Chem. B 2010, 114, 4151−4158. (38) Toncelli, C.; Pino-Pinto, J. P.; Sano, N.; Picchioni, F.; Broekhuis, A. A.; Nishide, H.; Moreno-Villoslada, I. Controlling the Aggregation of 5,10,15,20-Tetrakis-(4-sulfonatophenyl)-porphyrin by the Use of Polycations Derived from Polyketones Bearing Charged Aromatic Groups. Dyes Pigm. 2013, 98, 51−63. (39) Gómez-Tardajos, M.; Pino-Pinto, J. P.; Díaz-Soto, C.; Flores, M. E.; Gallardo, A.; Elvira, C.; Reinecke, H.; Nishide, H.; MorenoVilloslada, I. Confinement of 5,10,15,20-Tetrakis-(4-sulfonatophenyl)porphyrin in Novel Poly(vinylpyrrolidone)s Modified with Aromatic Amines. Dyes Pigm. 2013, 99, 759−770. (40) Rivas, B. L.; Pereira, E. D.; Moreno-Villoslada, I. Water-Soluble Polymer−Metal Ion Interactions. Prog. Polym. Sci. 2003, 28, 173−208. (41) Moreno-Villoslada, I.; Miranda, V.; Jofré, M.; Chandía, P.; Villatoro, J. M.; Bulnes, J. L.; Cortés, M.; Hess, S.; Rivas, B. L. Simultaneous Interactions between a Low Molecular-Weight Species and Two High Molecular-Weight Species Studied by Diafiltration. J. Membr. Sci. 2006, 272, 137−142. (42) Nikolic, K.; Agababa, D. Prediction of Hepatic Microsomal Intrinsic Clearance and Human Clearance Values for Drugs. J. Mol. Graphics Modell. 2009, 28, 245−252. (43) Schomburg, K. T.; Wetzer, L.; Rarey, M. Interactive Design of Generic Chemical Patterns. Drug Discovery Today 2013, 18, 651−658. (44) Tóth, G.; Mazák, K.; Hosztafi, S.; Kökösi, J.; Noszál, B. SpeciesSpecific Lipophilicity of Thyroid Hormones and Their Precursors in View of Their Membrane Transport Properties. J. Pharm. Biomed. Anal. 2013, 76, 112−118. (45) Aggarwal, N.; Kumar, R.; Dureja, P.; Khurana, J. M. Synthesis, Antimicrobial Evaluation and QSAR Analysis of Novel Nalidixic Acid Based 1,2,4-Triazole Derivatives. Eur. J. Med. Chem. 2011, 46, 4089− 4099. (46) Augustijns, P.; Wuyts, B.; Hens, B.; Annaert, P.; Butler, J.; Brouwers, J. A Review of Drug Solubility in Human Intestinal Fluids: Implications for the Prediction of Oral Absorption. Eur. J. Pharm. Sci. 2014, 57, 322−332. (47) Knox, C.; Law, V.; Jewison, T.; Liu, P.; Ly, S.; Frolkis, A.; Pon, A.; Banco, K.; Mak, C.; Neveu, V.; et al. Drugbank 3.0: A Comprehensive Resource for ‘Omics’ Research on Drugs. Nucleic Acids Res. 2011, 39, D1035−41. (48) Wishart, D. S. Drugbank and Its Relevance to Pharmacogenomics. Pharmacogenomics 2008, 9, 1155−62. (49) Wishart, D. S.; Knox, C.; Guo, A. C.; Shrivastava, S.; Hassanali, M.; Stothard, P.; Chang, Z.; Woolsey, J. Drugbank: A Comprehensive Resource for in Silico Drug Discovery and Exploration. Nucleic Acids Res. 2006, 34, D668−72. (50) Schatz, C.; Domard, A.; Viton, C.; Pichot, C.; Delair, T. Versatile and Efficient Formation of Colloids of Biopolymer-Based Polyelectrolyte Complexes. Biomacromolecules 2004, 5, 1882−1892.

(51) De Greef, T. F. A.; Smulders, M. M. J.; Wolffs, M.; Schenning, A. P. H. J.; Sijbesma, R. P.; Meijer, E. W. Supramolecular Polymerization. Chem. Rev. 2009, 109, 5687−5754. (52) Mertins, O.; Dimova, R. Insights on the Interactions of Chitosan with Phospholipid Vesicles. Part I: Effect of Polymer Deprotonation. Langmuir 2013, 29, 14545−51. (53) Claesson, P. M.; Ninham, B. W. pH-Dependent Interactions between Adsorbed Chitosan Layers. Langmuir 1992, 8, 1406−1412. (54) Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; García-Moreno, E.; Large Shifts, B. in pKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5260−5265. (55) Moreno-Villoslada, I.; González, F.; Rivas, B. L.; Shibue, T.; Nishide, H. Tuning the pKa of the Antihistaminic Drug Chlorpheniramine Maleate by Supramolecular Interactions with Water-Soluble Polymers. Polymer 2007, 48, 799−804. (56) Rabinowitch, E.; Epstein, L. F. Polymerization of Dyestuffs in Solution. Thionine and Methylene Blue1. J. Am. Chem. Soc. 1941, 63, 69−78. (57) Kemnitz, K.; Tamai, N.; Yamazaki, I.; Nakashima, N.; Yoshihara, K. Fluorescence Decays and Spectral Properties of Rhodamine B in Submono-, Mono-, and Multilayer Systems. J. Phys. Chem. 1986, 90, 5094−5101. (58) Haigh, C. W.; Mallion, R. B. New Tables of ‘Ring Current’ Shielding in Proton Magnetic Resonance. Org. Magn. Reson. 1972, 4, 203−228. (59) Yajima, T.; Maccarrone, G.; Takani, M.; Contino, A.; Arena, G.; Takamido, R.; Hanaki, M.; Funahashi, Y.; Odani, A.; Yamauchi, O. Combined Effects of Electrostatic and π−π Stacking Interactions: Selective Binding of Nucleotides and Aromatic Carboxylates by Platinum(II)−Aromatic Ligand Complexes. Chem.Eur. J. 2003, 9, 3341−3352. (60) Poudel, P. P.; Chen, J.; Cammers, A. Intramolecular π-Stacking in Isostructural Conformational Probes Depends Strongly on Charge Separation, a Proton NMR Study. Eur. J. Org. Chem. 2008, 2008, 5511−5517. (61) Chill, J. H.; Louis, J. M.; Miller, C.; Bax, A. NMR Study of the Tetrameric KCSA Potassium Channel in Detergent Micelles. Protein Sci. 2006, 15, 684−698. (62) Tosha, T.; Yoshioka, S.; Takahashi, S.; Ishimori, K.; Shimada, H.; Morishima, I. NMR Study on the Structural Changes of Cytochrome P450cam Upon the Complex Formation with Putidaredoxin. Functional Significance of the Putidaredoxin-Induced Structural Changes. J. Biol. Chem. 2003, 278, 39809−39821. (63) Araya-Hermosilla, R.; Araya-Hermosilla, E.; Torres-Gallegos, C.; Alarcón-Alarcón, C.; Moreno-Villoslada, I. Sensing Cu2+ by Controlling the Aggregation Properties of the Fluorescent Dye Rhodamine 6G with the Aid of Polyelectrolytes Bearing Different Linear Aromatic Density. React. Funct. Polym. 2013, 73, 1455−1463. (64) Lee, C.-L.; Kuo, L.-J.; Wang, H.-L.; Hsieh, P.-C. Effects of Ionic Strength on the Binding of Phenanthrene and Pyrene to Humic Substances: Three-Stage Variation Model. Water Res. 2003, 37, 4250− 4258. (65) Voigt, J.; Christensen, J.; Shastri, V. P. Differential Uptake of Nanoparticles by Endothelial Cells through Polyelectrolytes with Affinity for Caveolae. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 2942− 2947.

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Immobilization of Hydrophilic Low Molecular-Weight Molecules in

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