Insights into the Interactions between Dendrimers and Multiple

May 12, 2010 - School of Life Sciences, East China Normal University. ... (25) These properties are of central importance in the fundamental studies ...
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J. Phys. Chem. B 2010, 114, 7265–7273

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Insights into the Interactions between Dendrimers and Multiple Surfactants: 5. Formation of Miscellaneous Mixed Micelles Revealed by a Combination of 1H NMR, Diffusion, and NOE Analysis Kun Yang,† Yiyun Cheng,*,†,‡ Xueyan Feng,† Jiahai Zhang,§ Qinglin Wu,§ and Tongwen Xu*,† CAS Key Laboratory of Soft Matter Chemistry, School of Chemistry and Material Science, UniVersity of Science and Technology of China, Hefei, Anhui, 230026, People’s Republic of China, School of Life Sciences, East China Normal UniVersity, Shanghai, 200062, People’s Republic of China, and Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, UniVersity of Science and Technology of China, Hefei, Anhui, 230027, People’s Republic of China ReceiVed: March 24, 2010; ReVised Manuscript ReceiVed: April 21, 2010

We studied the formation and growth of miscellaneous mixed micelles in dendrimer and surfactant mixtures. NMR techniques including 1H NMR titration, diffusion (PGSE and DOSY) measurement, and NOE analysis were used to investigate the shape, size, interaction mode, spatial localization, and molecular orientation of the formed dendrimer/surfactant aggregates at different stages. The results suggest the formation of the following supramolecular aggregates when an equal molar concentration of sodium dodecylsulfate (SDS) and sodium deoxycholate (SDC) were added into a generation 4 (G4) cationic dendrimer: (1) the encapsulation of the two surfactants in the interior pockets of dendrimer at extremely low surfactant concentrations; (2) the binding of SDS on the surface of G4 dendrimer above the saturated encapsulation concentration; (3) the formation of globular SDS micelles and SDC dimer in the aqueous solution above the CMC of each surfactant; (4) the accumulation of SDS molecules on the surface of dendrimer in a bilayer fashion at high surfactant concentrations; (5) the interactions of dendrimer with the globular SDS micelles; and (6) the encapsulation of SDC monomers or dimers in the globular SDS micelles. The competitive binding/encapsulation of the two surfactants at different stages was evaluated. The results provide a new insight into the interactions of dendrimers with mixed surfactant systems. 1. Introduction Mixed micelles, usually composed of two or more kinds of surfactants,1 have considerable advantages over individual surfactant because of synergistic effects between the mixed components.2-7 The performance of these mixed micelles can be easily modulated by altering the mixed surfactant ratio and components. Mixed micelles have been widely used in biological and industrial processes8,9 including detergency, dispersion/flocculation, flotation, emulsifying, cosmetics, drug delivery, corrosion inhibition, enhanced oil recovery, and nanolithography.10,11 Mixed micelle systems make an extensive contribution to the field of both academic and commercial applications of surfactants and surfactant-containing systems,12-16 such as artificial cell membranes, lipid-surfactant microemulsions, and polymer-surfactant aggregates. In a polymer-surfactant system, the surfactant exhibits decreased critical micellization concentration (CMC) and lower surface tension.17-23 In addition, the polymer itself can benefit from binding/interaction with the surfactants, such as enhanced viscosity, drug loading efficiency, improved rheology, and decreased cytotoxicity.24 The interactions of polymers with mixed micelles combine the advantages of both polymer/ surfactant and mixed micelle systems. There are several reasons * To whom correspondence should be addressed. E-mail: yycheng@ mail.ustc.edu.cn (Y.C.) and [email protected] (T.X.). † CAS Key Laboratory of Soft Matter Chemistry, School of Chemistry and Material Science, University of Science and Technology of China. ‡ School of Life Sciences, East China Normal University. § Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China.

for the increasing interests in such systems in the past decades. First, unexpected physicochemical properties of such systems might be obtained when the advantages of the two systems are gathering together, such as phase separation and self-assembly.25 These properties are of central importance in the fundamental studies of the polymer/surfactant interaction systems. Second, though the dynamic natures for the mixed micelles, these globular micelles can be viewed as colloidal particles and their interactions with polymers are controlled by the surface charge density and geometry of the micelles.26 Therefore, such interactions can be used for the biomimicry of protein/DNA or protein/ protein interactions when the polymer is linear or globular polymer, respectively.27,28 Third, due to the formation of miscellaneous aggregate structures in the polymer/mixed micelle systems, the investigation of such systems by various techniques is helpful for the development of methodology in providing information on detailed interactions in the complicated systems.29 As a new class of artificial macromolecules, dendrimer possesses well-defined numbers (generation-dependent) of interior hydrophobic pockets and surface charged/noncharged functionalities,30-35 which make it a perfect candidate as a host molecule in the host-guest systems, such as dendrimer/ surfactant systems,27,28,36 dendrimer-based drug delivery systems,37-48 dendrimer/siRNA systems for gene interference,49,50 dendrimer/catalyst systems,51 and dendrimer/dye systems.52 The structure, aggregate nature, and interaction model of the dendrimer/surfactant systems have been studied by different techniques, including fluorescence probe,19,53,54 turbidimetry,

10.1021/jp1026493  2010 American Chemical Society Published on Web 05/12/2010

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SCHEME 1: Molecular Structures of SDS and SDC Molecules

small angle neutron scattering (SANS),17 dynamic light scattering (DLS),55 electron paramagnetic resonance (EPR),20,56 transmission electron microscopy (TEM),57,58 viscosity,59,60 isothermal titration calorimetry (ITC),17,61 Krafft temperature,62,63 atom force microscopy (AFM),28,64 and nuclear magnetic resonance (NMR).27,28,36 Up to now, the interactions of linear polymers with oppositely charged mixed micelles29,65,66 and the interactions of dendrimers with a single component surfactant21,27,28,36,55,61,67,68 have been investigated by many research groups, but to the best of our knowledge, no report on the physicochemical properties of interaction systems containing dendrimers/dendritic polymers and mixed micelles is found in the references. The interactions of dendrimers with mixed micelles are helpful to interpret many biological phenomena, such as the fluidity of membrane proteins on the lipid bilayers of a cell, the gene delivery mechanism when dendrimer is used as a vector, and the interactions of liposomes with a hydrophobic or hydrophilic protein. In addition, the dendrimers with amphiphilic property,27 hydrophobic interior, and hydrophilic surface can be viewed as a globular micelle. The interaction in the dendrimer/mixed micelle is essential for understanding the ionic/hydrophobic interactions in triple-component surfactant systems. Furthermore, the competitive binding/encapsulation of two ionic surfactants on the surface or in the interior of dendrimers is useful in the design and optimization of dendrimer-based host-guest systems.45 Consequently, it is of great interest and necessity to investigate the formation and growth of different aggregate structures in the dendrimer/mixed micelle systems. In this study, we investigated the binding behavior of the mixed micelles composed of the two surfactants sodium deoxycholate (SDC) and sodium dodecylsulfate (SDS) with generation 4 (G4) poly(amidoamine) (PAMAM) dendrimers using NMR techniques including 1H NMR, diffusion, and nuclear Overhauser enhancement (NOE) analysis. The molecular structures of SDS and SDC are shown in Scheme 1. The SDS molecule is a widely commercially used surfactant containing a long hydrophobic aliphatic chain and a hydrophilic head, while SDC consists of a bulky hydrophobic steroidal core and a relatively shorter chain linked with a hydrophilic carboxylate group. PAMAM dendrimers constitute the first dendrimer family to be synthesized and commercialized. Up to now, they have been the most investigated and characterized dendrimers. NMR, as a noninvasive, sensitive, and powerful method, has been

Yang et al. proved to be an effective tool in the analysis of ion-pairing, inclusion, aggregates, and self-assembly structures,69 especially in the host-guest systems.70 Herein, 1H NMR experiments were carried out to point out the interaction force, exchange rate for the bound-state/free-state in the dendrimer/mixed micelle aggregates, diffusion analysis (including pulsed gradient spin-echo, PGSE, and diffusion-ordered spectroscopy, DOSY) was used to characterize the size and bound-state/free-state ratio in the aggregates, while NOE measurements were used to validate the encapsulation and the localization of the SDS and SDC molecules in the interior pockets of dendrimers. 2. Experimental Section 2.1. Materials. G4 ethylenediamine (EDA)-cored and primary amine-terminated PAMAM dendrimer was purchased from Dendritech Inc. (Midland, MI). The average number of primary amine groups of G4 dendrimer is 62.7 measured by a ninhydrin assay. The molecular weight of G4 dendrimer is 14195 by MALDI-TOF mass spectrum, and the polydispersity index (PDI) of the dendrimer is 1.01 characterized by gel permeation chromatography. SDS and sodium deoxycholate were obtained from Shanghai BBI Co., Ltd. (Shanghai, China). Deuterium oxide (D2O) was purchased from Beijing Chongxi High-Tech Incubator Co., Ltd. (Beijing, China). PAMAM dendrimer stored in methanol solutions was distilled to remove the solvents before the NMR studies. Other chemicals were used as received. 2.2. Sample Preparation. The G4 PAMAM dendrimer was dissolved in D2O at a concentration of 10 mg/mL, which was used as the stock solution in the NMR studies. The dendrimer/ surfactant complex solutions were prepared by dissolving the appropriate amount of SDS and SDC in 2 mg/mL G4 dendrimer/ D2O solution. The solutions were transferred to 5-mm NMR tubes and stored at room temperature. The samples in NMR tubes were sonicated for 30 min before NMR studies. For all the NMR studies described below, a certain amount of pyridine was added into each NMR tube and used as an internal standard. Pyridine was obtained from the Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China) with a purity of 99.5%. 2.3. 1H NMR Studies. 1H NMR experiments were conducted on a Bruker Advance 500.132 MHz NMR spectrometer at 298.2 K for dendrimer/SDS/SDC/D2O solutions with different molar ratios of SDS(SDC)/G4 dendrimer at 0:1, 1:1, 2:1, 4:1, 8:1, 16: 1, 32:1, 64:1, 128:1, and 256:1 at a fixed G4 dendrimer concentration of 2 mg/mL. The SDS and SDC concentrations are equal to each other in the mixed micelles. The temperature was kept constant within (0.2 K with the use of a Bruker temperature control unit. 2.4. Diffusion Studies. The diffusion behavior of compounds in a mixture can be investigated from PGSE or the DOSY experiments. The PGSE NMR experiments were performed with a standard Bruker pulse program. The time interval between gradient pulses is chosen as 600 ms, while the duration of the gradient pulses is 2.2 ms. The pulse gradients were increased from 10% to 80% of the maximum gradient strength (50 G/cm) in 16 steps to attenuate the spin-echo signal. DOSY spectra of SDS/SDC/G4 dendrimer at molar ratios of 4:4:1 and 128:128:1 were performed with the same Bruker 500.132 MHz instrument, using the standard Bruker pulse program, a stimulated echo pulse sequence with bipolar gradients (STEBPGP1s).27 The pulse sequences include a 3 ms delay to allow residual eddy current to decay and a sine-shaped gradient pulse was utilized to further minimize eddy currents. The pulsed gradients were increased in a linear ramp (64 steps). All data were processed by Bruker Xwinnmr 3.1 (Bruker Biospin).

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Figure 1. The 1H NMR spectrum and the chemical shift assignments of the mixed SDS/SDC solution.

2.5. NOE Measurements. NOESY spectra of SDS/SDC/G4 dendrimer/D2O solutions at molar ratio of 16:16:1 and 64:64:1 (the dendrimer concentration was fixed at 2 mg/mL) were acquired at 500.132 MHz, using a mixing time of 300 ms.36 The experiments were performed with a 2 s relaxation delay and 8.4 us 1H 90° pulse width. A total of 32 transients were averaged for 1024 complex t1 points. All data were processed with NMRpipe software on a Linux workstation with standard Lorents-Gauss window function and zero-filling in both dimensions. 3. Results and Discussion 3.1. 1H NMR Studies. 1H NMR spectrum has been well developed as a powerful approach for the evaluation of intermolecular interactions/bindings in recent years. Changes of the chemical shift for a certain nucleus in a 1H NMR spectrum provide information on the increase or decrease of the electron cloud intensity around the nucleus, which is helpful in the analysis of the existence, the types, and the relationships of interactions between two molecules.71 In addition, the variations of half-peak width, peak splitting, and number of peaks reveal the exchange rate between the bound-state and the free-state, which is critical in the analysis of the dynamics of the binding process and the stability of formed aggregate/inclusion in the mixed systems.69 Figure 1 and Scheme 1 show the chemical shift assignments of protons in the two guest molecules SDS and SDC in the absence of dendrimers. The assignments were carried out on the basis of homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and previous proton assignments for SDS and SDC.27,28 It can be observed that the chemical shifts of most protons of SDS and SDC range from 0.7 to 2.2 ppm, and these protons have no overlapping with the dendrimer (G4 PAMAM dendrimer) protons, whose chemical shifts range from 2.2 to 3.4 ppm. This makes it accessible for us to explore the possible host-guest or ionic interactions in the dendrimer/ SDS/SDC systems. Theoretically, G4 PAMAM dendrimer has six peaks corresponding to the four kinds of methylene protons (a-d) in the inner scaffold and two kinds of methylene protons (b′ and d′) adjacent to the surface functionalities. However, the peak for protons d overlaps with the peak for protons d′, while the signal for protons b′ overlaps with that for protons c. Therefore, only four broad peaks are observed in the 1H NMR spectrum of PAMAM dendrimer if no interaction occurs between dendrimer and guest molecules. In cationic dendrimer/anionic surfactant systems, the positively charged/noncharged amine groups (depending on the pH condition of the mixing solution) on the surface of the dendrimer have been proved to provide binding sites for the anionic

Figure 2. Expanded region of the 1H NMR titration spectra of G4 dendrimer with mixed SDS and SDC. The concentration of G4 dendrimer was kept constant at 2 mg/mL. The molar ratio of SDS/ SDC/G4 dendrimer ranges from 0:0:1 to 256:256:1 (1-10).

Figure 3. Chemical shift variations of dendrimer protons in the presence of different molar ratios of SDS/SDC/dendrimer (G4 dendrimer concentration being a constant of 2 mg/mL).

surfactants, forming an ion pair and/or hydrogen bond with the surfactant.9,17,20,36,61,72 The primary driving force during the formation of dendrimer/surfactant aggregates was reported to be ionic interaction, which occurs between the positive charged groups on the surface of the dendrimer and the negative charged group of the surfactant.61,73 The evidence for the formation of an ion pair/hydrogen bond between surfactant and dendrimer is the significant downfield-shift of protons b′ and protons d′ of PAMAM dendrimer due to the decrease of electron cloud density around these protons.43,44 As shown in Figure 2 as well as Figure S1 (Supporting Information), the splitting of the overlapped b′/c, d/d′ broad peaks of the dendrimer during the addition of mixed SDS/SDC solutions (equal molar concentration of SDS and SDC during the experiment, Scheme S1 (Supporting Information)) in the spectrum can be used to identify the potential interactions between dendrimer and mixed surfactants. The surfactant (SDS and SDC) concentration dependence of the dendrimer chemical shift (protons c, b′, d, and d′) is further illustrated in Figure 3. At low surfactant concentrations (molar ratio of SDS/SDC/dendrimer ranges from 1:1:1 to 8:8: 1), the chemical shifts of protons b′ and d′ are scarcely changed, and the pH value (10.5-11.5) of the dendrimer/SDS/SDC solution is above the pKa value of primary amine groups on the surface of the dendrimer (pKa ≈ 10.0, most of these amine groups are noncharged),46,74 suggesting that ionic binding/ hydrogen bonding of the SDS and SDC molecules on the surface

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of the dendrimers is not the predominant force during the formation of dendrimer/surfactant aggregates at this stage. Two possible reasons can be proposed to explain the results in Figures 2 and 3. The first one is that no interaction occurs between surfactant and dendrimer at low surfactant concentrations. This can be easily ruled out because of the significantly decreased diffusion coefficients of the two surfactants below their critical micellization concentrations (CMC, the CMC of SDS in aqueous solution is around 8 mM17 and that of SDC is around 4.1 mM75) obtained from the PGSE NMR results which will be discussed in detail in Section 3.2. Another explanation is that most of the added SDS and SDC molecules are encapsulated in the interior pockets during this period. Our previous work has concluded that SDC molecules do not bind on the surface of PAMAM dendrimers as no shift of the peaks for protons b′ and protons d′ can be observed even at high SDC concentrations.27 This is probably due to the extremely hydrophobic property of the steroidal core of SDC. The aqueous solubility of SDC is low when the carboxylate group of SDC is bound to the dendrimer surface, which reduces the formation of stable ion pairs between dendrimer and SDC in aqueous solutions. As a result, the SDC molecules can only locate in the interior pockets by hydrophobic interaction.27 For SDS, the amphiphilic surfactant can coexist on the surface and in the interior of the dendrimer.36 These facts suggest that the SDS and SDC molecules penetrate into the globular dendrimer, locate at its hydrophobic scaffold, and form a stable supramolecular inclusion with dendrimer in the mixed system at low surfactant concentrations. The structure of the supramolecular inclusion consisting of three components will be further discussed in Section 3.3, where 1H-1H NOE spectroscopy is used to calculate the average spatial distance between specific protons of the host and guests. When the molar ratio of SDS/SDC/dendrimer ranges from 16:16:1 to 128:128:1, a progressively increased downfield shift of the peaks for protons b′ and protons d′ was observed, suggesting a gradually increased tendency of ionic/hydrogen bond interactions between the dendrimer and surfactants during this period. Obviously, the saturation of supramolecular inclusion of SDC and SDS by dendrimer is reached. Though SDC molecules do not bind with the dendrimer surface, the SDS molecules were reported to interact with the terminal amine groups of the dendrimer by ionic, ion-dipole, or hydrogenbonding interactions. When the molar ratio of SDS/SDC/ dendrimer is above 64:64:1, concentrations of the two surfactants have exceeded their critical micellization concentrations (CMC, the CMC of SDS is according to 57:57:1 and the CMC of SDC is according to 29:29:1, respectively), which means that SDS and SDC form single-component or multicomponent (mixed) micelles. The SDS molecule with a long lipophilic/flexible tail and a hydrophilic sulfate head has a tendency to form a sphere micelle in water (hydrophobic core and hydrophilic and negatively charged surface), while SDC with an extreme hydrophobic steroidal core consisting of three aliphatic rings and a short/nonflexible tail is unable to build a sphere micelle in aqueous solutions.27,36 In the references, the formation of dimers/oligomers driven by hydrophobic interactions between the steroidal cores of two or more SDC monomers was proposed by several groups.76,77 Though the presence of spatial hindrance between two globular structures, the formed micelles can still react with the dendrimer surface by ionic/ionic-dipole/hydrogenbond interactions. It is worth noticing that bilayer-structured SDS micelles are probably present on the surface of the dendrimer and interact with the dendrimer by cooperative interactions. Such a compact binding model can increase the

Yang et al. chance of interactions between dendrimer and surfactants and reduce the spatial hindrance effect, which has been proposed in several dendrimer/surfactant systems,20,28,36,56 such as the cationic dendrimer/SDS system and the anionic dendrimer/ dodecyltrimethylammonium bromide (DTAB) system. Interestingly, a significant upfield shift of the peaks for protons b′ and protons d′ is observed when the ratio of surfactant and dendrimer ranges from 128:128:1 to 256:256:1. For the dendrimer/surfactant complex, if the exchange rate between the free-state and bound-state is fast, the spectrum contains only one signal for both free- and bound-state, and the observed chemical shift is a weighted average of the chemical shifts for free-state and bound-state.69 As only one peak for protons b′ or protons d′ is observed in the 1H NMR spectrum (Figure 2), there should be a transition of the interaction mode between the two concentrations (128:128:1 and 256:256:1). The upfield shift of the peaks (protons b′ and d′) indicates that a larger percent of amine groups on the dendrimer surface participate in the interactions of surfactants with the dendrimer at the ratio of 128:128:1 than that at the ratio of 256:256:1. At higher surfactant concentrations, the concentrations of single-component micelles or mixed micelles reach their “critical aggregation concentration” (“CAC”) in the dendrimer/micelle interacting systems. As a result, the bilayer-structured surfactants on the dendrimer surface reorganize into globular-structured micelles, followed by the adsorption of the anionic and globular structure on the cationic and polar surface of dendrimer by ion-dipole/hydrogenbond interactions. This model explains well the chemical shift variations when increasing amounts of surfactants are added into the dendrimer solution (Figure 2) and this process will be further discussed in Section 3.2 by diffusion analysis. 3.2. Diffusion Measurements. The variation of molecular size is a crucial parameter in the evaluation of aggregate or complex formation.70 On the basis of the Stokes-Einstein equation, the connection between molecular size and diffusion coefficient can be used to analyze the structural properties of the formed aggregates or complexes.78 The observed diffusion coefficient is associated with the fractions and sizes of the freestate and bound-state in the mixture as well as the viscosity of the solution.

Dobs ) Df(Cf /Ct) + Db(Cb /Ct)

(1)

where Dobs is the observed diffusion coefficient, Df and Db are the diffusion coefficients of free-state and bound-state, respectively, Cf and Cb are the concentrations of free-state and boundstate, respectively, and Cf + Cb ) Ct. Recently, techniques such as diffusion NMR (PGSE and DOSY)79,80 and dynamic light scattering (DLS)81 were used to measure the diffusion coefficient. Diffusion NMR is proved to be a powerful method in multiple-component mixture analysis,78 study of aggregation formation, ligand and receptor recognition, and high-throughput screening of drugs. The concept behind these applications is based on the fact that the diffusion coefficient of a ligand will be changed during its addition into a target solution if there is a specific interaction between the target and ligand.70,78 Here, PGSE NMR and DOSY were used to probe the host-guest interactions in the SDS/SDC/dendrimer system. The terminal methyl group of SDS (SDS-4, Scheme 1) and the methyl protons of SDC (SDC-18, Scheme 1) were chosen as representatives for the surfactant SDS and SDC in the diffusion measurement experiments, respectively. The diffusion coefficients of SDS, SDC, and G4 dendrimer are shown in Figures 4-6, respectively. The logarithms of the

Interactions of Dendrimers and Multiple Surfactants

Figure 4. Diffusion coefficient of SDS in the mixture of SDS/SDC/ G4 dendrimer as a function of the surfactant/dendrimer molar ratio (G4 dendrimer concentration being a constant of 2 mg/mL).

Figure 5. Diffusion coefficient of SDC in the mixture of SDS/SDC/ G4 dendrimer as a function of the surfactant/dendrimer molar ratio (G4 dendrimer concentration being a constant of 2 mg/mL).

diffusion coefficients for SDS, SDC, and G4 dendrimer are plotted versus the surfactant/dendrimer molar ratios. The dendrimer concentration was kept constant at 2 mg/mL during the whole titration experiment. The molar ratio of SDS/SDC/G4 dendrimer ranges from 0:0:1 to 256:256:1. As shown in Figures 4 and 5, the diffusion coefficients of SDS and SDC significantly decreased when a mixture of SDS and SDC (equal molar concentration, the ratio of SDS/SDC/dendrimer ranges from 1:1:1 to 4:4:1) was added into the dendrimer solution. The diffusion coefficient of the internal standard (pyridine) in the SDS/SDC/dendrimer solution is not changed during the addition of surfactants indicating that there is no change in solution viscosity in the titration experiment (Figure S2 in the Supporting Information). The decreased diffusion coefficient of the surfactants means increased molecular size and decreased molecular mobility (aggregates and/or supramolecular inclusions). As mentioned in Section 3.1, the concentrations of SDS and SDC during this period are below their CMCs, and hence the decreased diffusion coefficient of SDS or SDC is not caused by the formation of micelles, which have much slower mobility in solutions than surfactant monomers. Also, the ionic binding of surfactants on the surface of dendrimer cannot explain the decreased diffusion behavior of the surfactants because of the following reasons: (1) SDC cannot bind on the surface of dendrimer but has the same diffusion behavior with SDS27 and (2) the pH value (10.5-11.5) of the SDS/SDC/dendrimer solutions rules out the presence of ionic interactions on dendrimer surface. The PAMAM dendrimer has a relative nonpolar interior, while the SDS and SDC molecules are amphiphilic guests with proper sizes which can be encapsulated

J. Phys. Chem. B, Vol. 114, No. 21, 2010 7269 in the pockets. Therefore, the encapsulations of SDS and SDC in the interior pockets of dendrimers and the formation of supramolecular inclusions are proposed to explain the diffusion behaviors of SDS and SDC. This is confirmed by the diffusion behavior of G4 dendrimer (Figure 6) as well as the NOE spectroscopy analysis of the resulting complexes (Section 3.3) during the same period. The diffusion coefficient of G4 dendrimer slightly increases in the range of 1:1:1 to 4:4:1, suggesting the decrease of the dendrimer size. If the SDS/SDC molecules are bound on the surface of dendrimers, the molecular size of the dendrimer will be increased rather than decreased. However, if the SDS or SDC monomers are encapsulated in the pockets of the dendrimer, the hydrophobic interactions between the dendritic scaffold and the hydrophobic region of surfactants can cause the shrinkage of the dendrimer, which explains well the diffusion behaviors of both host molecule and guest molecules. When more surfactants were added into the dendrimer solution (8:8:1 ≈ CMCSDC), significant decrease of dendrimer diffusion coefficient and increase of SDS diffusion coefficient are observed in Figures 4-6. The increased molecular size of the dendrimer is caused by the surface binding of the SDS molecules, which is evident from the 1H NMR studies as discussed in Section 3.1. On the other hand, the increased diffusion coefficient of SDS can be explained by the coexistence of free-state and bound-state surfactants. According to eq 1, the increased fraction of free SDS or SDC surfactants will increase the observed diffusion coefficient of the surfactants. The binding of SDS monomers on the surface of dendrimers is driven by ion-dipole or hydrogen-bond interactions as discussed above. In this period, a transition from the slow-exchange model in the formation of supramolecular inclusions to the fast-exchange model in the formation of surface-binding aggregates is proposed. When SDS or SDC monomers are trapped in the dendrimer pockets, the congested dendritic structure, the spatial hindrance in the interior, and the strong hydrophobic interactions reduce the escape of the surfactants from the dendrimer (slowexchange). However, the ion-dipole/hydrogen-bond interaction mediated aggregate is not as stable as the inclusion structure and the surfactants can enter in or depart from the dendrimer surface (fast-exchange) if the surfactant molecules are bound on the surface of dendrimers. The transition from interior encapsulation to surface binding also provides a semiquantitative method for the determination of the encapsulation capacity of the dendrimer toward each surfactant because the existence of surface binding indicates the saturation of interior encapsulation of the surfactants. Hence, we can deduce that the saturated number of SDS and SDC molecules entered into a G4 dendrimer is 4-8 in the mixing system. The fast-exchange model also explains well the scarcely changed diffusion coefficients of SDS and SDC as more surfactants are added into the system in the ratio range of 8:8:1 to 32:32:1. More surfactants means more concentrations of free-state and bound-state surfactants, but the observed surfactant diffusion coefficient depends on the fraction of free-state and bound-state rather than the molar concentration of each component. Therefore, the diffusion coefficients of the surfactants are slightly changed during this period. In the ratio range of 64:64:1 to 128:128:1, the concentrations of SDS and SDC have exceeded their CMCs. The SDS globular micelles and SDC dimers/oligomers form in the dendrimer solutions. In this process, the diffusion coefficients of SDS and SDC are equal to each other within NMR errors as presented in Figures 4 and 5, suggesting that the SDS and SDC micelles are bound together in the solution. This result directly proved

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Figure 6. Diffusion coefficient of G4 dendrimer in the presence SDS/ SDC as a function of the surfactant/G4 dendrimer molar ratio (G4 dendrimer concentration being a constant of 2 mg/mL).

the formation of binary mixed micelles consisting of SDS and SDC. Since SDS is more flexible than SDC, and SDC is more hydrophobic than SDS, the SDC dimers or oligomers should locate in the hydrophobic core of SDS globular/bilayer micelles. The formed mixed micelles can also interact with dendrimers by ion-dipole/hydrogen-bond interactions (ternary mixed micelles), which will further decrease the diffusion coefficient of the surfactants and dendrimers. The observed diffusion coefficient of the surfactant is the weighted average of the coefficients of the sites:70

Dobs ) Df(Cf /Ct) + Db(Cb /Ct) + Dm(Cm /Ct)

Figure 7. The 2D NOESY spectrum of SDS/SDC/G4 dendrimer mixtures at a molar ratio of 16:16:1 and a mixing time of 300 ms. The dendrimer concentration was fixed at 2 mg/mL.

(2)

where Dm is the diffusion coefficient of micelle-state surfactant, Cm is the concentration of micelle-state surfactant, and Cf + Cb + C m ) C t. At later stages, a competition on the binding of bilayerstructured mixed micelles and globular-structured mixed micelles (SDS and SDC) on the surface of dendrimer occurs in the mixing system. As discussed in the 1H NMR studies, the bilayer-structured micelles bind dendrimer at a surfactant/ dendrimer ratio of 128:128:1, while globular-structured micelles interact with dendrimer at a ratio of 256:256:1. At higher surfactant concentrations, the concentration of formed micelle may exceed its CAC in the presence of the dendrimer and directly react with the dendrimer surface. Although there is a structure transition in the dendrimer/surfactant complexes between these two surfactant concentrations, the observed diffusion coefficient is scarcely changed in Figures 5 and 6 because of the multiple contributions (both fraction and the structure of each formed aggregates/inclusions) on the observed diffusion coefficient as described in eq 2. Similar results were obtained in Figures S3 and S4 (Supporting Information) when DOSY was used to describe the diffusion behaviors of the dendrimer and surfactants. 3.3. NOE Spectroscopy. NOESY provides precise information on the relative spatial arrangement of specific protons through NOE interactions (cross-peaks) in the spectrum.70,78 The NOE interaction arises from dipole-dipole interaction between two protons within a distance limit of 5 Å. The intensity of the NOE interaction (volume of the cross-peak) reveals the numbers of related protons and the spatial distance between them. The presence of cross-peaks in the NOE spectroscopy at a short mixing time means the target protons are close to each other, otherwise, they are distant from each other, which can be used to rule out a specific interaction between the protons.36,39,82

Figure 8. The 2D NOESY spectrum of SDS/SDC/G4 dendrimer mixtures at a molar ratio of 64:64:1 and a mixing time of 300 ms. The dendrimer concentration was fixed at 2 mg/mL.

NOESY is proved to be an effective tool in the study of intramolecular and intermolecular interactions in multicomponent systems, especially for the host-guest interactions.83-86 In addition, NOESY can be used to analyze the molecular orientation within a host molecule.27 Herein, NOESY was used to give evidence of the encapsulation of SDS and SDC molecules in the interior pockets of dendrimers. The NOESY spectra of SDS/SDC/G4 dendrimer solutions at molar ratios of 16:16:1 and 64:64:1 at a mixing time of 300 ms are shown in Figures 7 and 8. According to our previous studies on dendrimer/SDS and dendrimer/SDC systems, the mixing time of 300 ms was chosen for the optimization of the

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SCHEME 2: Proposed Aggregate Structures for SDS/SDC/Dendrimer Complexes at Different SDS/SDC/Dendrimer Molar Ratios

cross-peak intensities with minimum distortions. Strong negative cross-peaks between aliphatic chain protons of SDS (SDS-3) and scaffold protons a, b, c, and d located in the interior pockets of dendrimer are observed in Figure 7 as well as Figure S5 (Supporting Information). Medium cross-peaks between SDC protons (SDC-18) and dendrimer protons a and c are observed (Figure S5, Supporting Information). If the surfactant molecules are bound on the surface of dendrimers, the mentioned crosspeaks should not be observed because the spatial distances between these protons are larger than 5 Å.36 In addition, protons a and c of the dendrimer seem to be the binding center in the complex since all the observed cross-peaks relate to these protons. If the cross-peaks are caused by the surface binding of surfactants, protons b′ and d′ located adjacent to the surface functionalities should be the nearest protons to the surface-bound surfactants. Therefore, the observed NOE cross-peaks in Figure 7 prove the entrance of SDS and SDC molecules into the interior hydrophobic region of the dendrimer. The association of protons

(SDC-18) of SDC with the scaffold of dendrimer indicates that the inclusion of SDC in the dendrimer pockets is hydroxyl group-centered (hydrogen-bond interactions between the hydroxyl groups and the amide groups of dendrimer scaffold) and methyl group-assisted (hydrophobic interactions). At a higher surfactant concentration (64:64:1), NOE cross-peaks between SDS protons (SDS-2 and SDS-3) and SDC protons (SDC-3) are observed in Figure 8, which confirms the formation of the SDS/SDC mixed micelles in the solution. Although cross-peaks between dendrimer protons and surfactant (SDS and SDC) protons are still observed in Figure 8 as well as Figure S6 (Supporting Information), the intensities of these cross-peaks are decreased. It seems that the amount of surfactants located in the interior of dendrimer decreases, which is probably due to the increased ion concentrations in the dendrimer pockets at higher surfactant concentrations. By a combination of analysis from 1H NMR titration, diffusion NMR, and NOESY studies, the detailed interaction

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models among dendrimer, SDS and SDC monomers, SDS and SDC micelles, and SDS/SDC mixed micelles are proposed in Scheme 2. First, SDS and SDC molecules penetrate into the interior cavities of dendrimers by hydrophobic and/or hydrogenbond interactions (Scheme 2a, the ratio of SDS/SDC/dendrimer ranges from 1:1:1 to 4:4:1). After the saturated point for surfactant encapsulation (4:4:1 to 8:8:1), SDS molecules are associated with the surface amine groups of the dendrimers by ion-dipole/hydrogen-bond interactions, while SDC molecules are dispersed in the solution in the pattern of monomers (Scheme 2b, 8:8:1 ≈ CMCSDC). SDC molecules aggregate into dimers above the CMC of SDC in aqueous solutions (Scheme 2c, CMCSDC ≈ CMCSDS). SDS molecules aggregate into globular micelles above the CMC of SDS (Scheme 2d, CMCSDS ≈ CACmixed micelle). Then, the mixed micelles of SDS/SDC form and interact with the dendrimers (Scheme 2d, 64:64:1), and the surfactant micelles interact with dendrimers in a bilayer fashion (Scheme 2e, 128:128:1). Finally, the globular SDS micelles or SDS/SDC mixed micelles bind on the surface of the dendrimer (Scheme 2f, 256:256:1). 4. Conclusions In the present work, NMR techniques including 1H NMR titration, diffusion measurements, and NOESY analysis were employed in exploring the aggregate structures consisting of dendrimer, SDS, and SDC. SDS and SDC molecules penetrate into the dendritic pockets at low surfactant concentrations. After the saturation of molecular inclusion, SDS molecules can interact with the dendrimer surface while SDC molecules cannot, which is caused by the structural difference of the two surfactants. Slow-exchange and fast-exchange models and miscellaneous mixed micelles are proposed at different surfactant concentrations. The NMR studies in the analysis of dendrimer-multiple surfactant systems provide a new insight to the interactions between dendrimer and mixed surfactants (mixed micelles). Acknowledgment. We thank the Specialized Research Fund fortheDoctoralProgramofHigherEducation(No.20093402110041) for financial support to this program. Supporting Information Available: Further information on the structure of the G4 dendrimer and 1H NMR and DOSY analysis of the SDS/SDC/dendrimer complexes. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Thomas, H. G.; Lomakin, A.; Blankschtein, D.; Benedek, G. B. Langmuir 1997, 13, 209. (2) Shiloach, A.; Blankschtein, D. Langmuir 1998, 14, 7166. (3) Fainerman, V. B.; Aksenenko, E. V.; Mys, A. V.; Petkov, J. T.; Yorke, J.; Miller, R. Langmuir 2010, 26, 2424. (4) Reynolds, P. A.; Gilbert, E. P.; Henderson, M. J.; White, J. W. J. Phys. Chem. B 2009, 113, 12243. (5) Ziserman, L.; Abezgauz, L.; Ramon, O.; Raghavan, S. R.; Danino, D. Langmuir 2009, 25, 10483. (6) Xin, J.; Liu, D.; Zhong, C. J. Phys. Chem. B 2009, 113, 9364. (7) Jiang, Y.; Lu, X. Y.; Chen, H.; Mao, S. Z.; Liu, M. L.; Luo, P. Y.; Du, Y. R. J. Phys. Chem. B 2009, 113, 8357. (8) Shen, H. H.; Thomas, R. K.; Taylor, P. Langmuir 2010, 26, 320. (9) Dong, S.; Li, X.; Xu, G.; Hoffmann, H. J. Phys. Chem. B 2007, 111, 5903. (10) Zhang, W.; Hao, J. G.; Shi, Y.; Li, Y. J.; Wu, J.; Sha, X. Y.; Fang, X. L. Int. J. Pharm. 2009, 376, 176. (11) Singh, J.; Unlu, Z.; Ranganathan, R.; Griffiths, P. J. Phys. Chem. B 2008, 112, 3997. (12) Mir, M. A.; Gull, N.; Khan, J. M.; Khan, R. H.; Dar, A. A.; Rather, G. M. J. Phys. Chem. B 2010, 114, 3194. (13) Rozner, S.; Shalev, D. E.; Shames, A. I.; Ottaviani, M. F.; Aserin, A.; Garti, N. Colloids Surf., B 2010, 77, 22.

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