Poly(amidoamine) - American Chemical Society

Article pubs.acs.org/JPCB

Poly(amidoamine) and Poly(propyleneimine) Dendrimers Show Distinct Binding Behaviors with Sodium Dodecyl Sulfate: Insights from SAXS and NMR Analysis Tianfu Li,† Naimin Shao,‡ Yuntao Liu,† Jingjing Hu,§ Yu Wang,† Li Zhang,† Hongli Wang,† Dongfeng Chen,*,† and Yiyun Cheng*,‡,∥ †

China Institute of Atomic Energy, Beijing 102413, People’s Republic of China Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200062, People’s Republic of China § Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, People’s Republic of China ∥ Shanghai Key Laboratory of Magnetic Resonance, Department of Physics, East China Normal University, Shanghai 200062, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: We investigate the interactions of generation 3 (G3) poly(amidoamine) (PAMAM) and G3 poly(propylenimine) (PPI) dendrimers with sodium dodecyl sulfate (SDS) in aqueous solution. Size and structure of the dendrimer−SDS aggregates as a function of SDS/dendrimer molar ratio were revealed by SAXS and NMR. G3 PAMAM has a relatively open and dense-core structure, while G3 PPI with the same number of surface amine groups possesses a compact and uniform structure. Upon addition of SDS, much more SDS monomers were encapsulated in the interior of PPI rather than in PAMAM. More significant size increase in PAMAM−SDS aggregate is observed at low SDS concentrations, due to the binding of SDS on PAMAM surface and further assembly into larger supramolecular structures. Both noncooperative and cooperative binding of SDS on G3 PPI surface are observed, while only noncooperative binding is proposed on G3 PAMAM, due to its open surface and large surface group distance. The size of the PPI−SDS complex is larger than that of PAMAM−SDS at higher SDS concentrations. Within the investigated SDS concentrations, SDS exhibits much stronger interactions with G3 PPI than with G3 PAMAM. These results provide new insights into dendrimer− surfactant interactions and explain why PPI is much more cytotoxic than PAMAM.

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INTRODUCTION

and show synergistic effects compared to single dendrimers and surfactants.12,13 To date, the interactions of dendrimers and surfactants were investigated by techniques such as nuclear magnetic resonance (NMR),6 electron paramagnetic resonance (EPR),14 isothermal titration calorimetry (ITC),11 electromotive force (EMF),11 small-angle X-ray or neutron scattering (SAXS or SANS),11,15 fluorescent spectroscopy,16 transmission electron microscopy (TEM),7 atomic force microscopy (AFM),9 and quartz crystal microbalance (QCM).17 Dendrimer−surfactant interactions are mainly driven by ionic interactions or hydrophobic interactions or a combination of the two interactions.18,19 The interactions lead to the formation of miscellaneous aggregates.20−22 In the presence of dendrimers, surfactant usually shows lower critical aggregation concentration (CAC) compared to its critical micelle

Dendrimers are promising macromolecules with well-defined tree-like spherical structures.1,2 They comprise three main components: a central core, repeat units with branched structures, and surface functionalities.3 Because of the unique properties like monodispersity, nanoscale size, large numbers of surface functionalities, and interior cavities, dendrimers have gained great interest in fields ranging from polymer chemistry and material chemistry to industrial and biomedical applications during the past decades.4,5 Among these researches, the host− guest behaviors of dendrimers toward a list of guests such as drugs, dyes, surfactants, catalysts, biomacromolecules, and inorganic nanoparticles have held great scientific interest due to their key roles in the broad applications of dendrimers.6 Surfactants are ideal candidates as guests in dendrimer-based host−guest systems due to their amphiphilic nature, charged or noncharged polar head, and nanoscale size.7−11 Their complexes with dendrimers can be used as templates to guide the synthesis of nanoparticles and as vehicles of drugs or genes © 2014 American Chemical Society

Received: December 27, 2013 Revised: February 24, 2014 Published: March 7, 2014 3074

dx.doi.org/10.1021/jp412660p | J. Phys. Chem. B 2014, 118, 3074−3084

The Journal of Physical Chemistry B

Article

Scheme 1. Molecular Structures of G3 PAMAM and G3 PPI Dendrimers

concentration (CMC).11,23 The effect of dendrimer core, generation and surface functionality, surfactant chain length and charge property, environmental pH condition and salt concentration on the interaction mechanisms, the surfactant binding capacity of dendrimers, the CAC values, and the aggregate sizes and structures were investigated.6,11 Since the pioneer reports on dendrimer synthesis in the early 1980s, more than a hundred families of dendrimer were synthesized.24,25 Among these dendrimers, poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI) dendrimers are the mostly studied and characterized ones which are both synthesized by a divergent strategy and available on the market.3,26 PPI dendrimer has a shorter repeated unit (−CH 2 CH 2 CH 2 −) compared to PAMAM dendrimer (−CH2CH2COCH2CH2−), Scheme 1), thus PPI dendrimer is smaller than PAMAM with the same number of surface functionality.27 Besides the size difference, the interior of a PPI dendrimer is much more hydrophobic and congested than that of PAMAM dendrimer due to the presence of amide groups in PAMAM interior. The structural differences of PPI and PAMAM dendrimers may have interesting physicochemical behaviors when acting as hosts. The more hydrophobic nature of PPI dendrimer interior indicates higher capacity for hydrophobic guest loading. A recent study reported that a PPI dendrimer is much more toxic than a PAMAM dendrimer with the same number of surface functionality.28 To reveal the exact reason behind these interesting phenomena, we need to reveal the binding behaviors of PAMAM and PPI dendrimers with surfactants. In the current study, we investigated the binding behaviors of generation 3 (G3) PAMAM and G3 PPI dendrimers with an anionic surfactant−sodium dodecyl sulfate (SDS). Our definition of dendrimer generation is in accordance with the

generation definition initially applied for PAMAM dendrimers by Tomalia.29 Both G3 PAMAM and G3 PPI dendrimers have 32 surface amine groups on the surface (Scheme 1). In previous studies,14,18,20,21,30−32 the binding behaviors of dendrimers with surfactants at different pH values, dendrimer generations, salt concentrations, and solution temperatures were investigated in detail. These parameters significantly affect the interactions between dendrimers and surfactants. Here, we focus on the effect of dendrimer structure (dendrimer chemistry, interior hydrophobicity, and surface group density) on the binding behaviors at native states (pH values of the dendrimer/SDS solutions were monitored at different surfactant/dendrimer molar concentrations, and the interactions between dendrimers and SDS were measured in water without salts). We investigated the solution structures of the two dendrimers and their binding behaviors with SDS by a combination of NMR and SAXS. NMR and SAXS are powerful and sensitive tools in characterizing dendrimer−surfactant interactions. NMR provides atomic-level and molecular-level insights into the interaction mechanisms, molecular mobility, and spatial distances between atoms, 6 while SAXS directly gives information on structure and size of the yielding dendrimer− surfactant aggregates.33 A combination of two techniques can provide detailed information on interactions of SDS with the two dendrimers. To the best of our knowledge, this is the first report on the distinct binding behaviors of PAMAM and PPI dendrimers with surfactants.

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EXPERIMENTAL SECTION Materials. Diaminobutane (DAB)-cored and amine-terminated G3 PPI dendrimer with a molecular weight of 3513 Da was purchased from Sigma-Aldrich (St. Louis, MO). Ethylenediamine (EDA)-cored and amine-terminated G3 PAMAM 3075



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micelles in our investigated systems. The analytic formulation has been developed as38

dendrimer was purchased from Dendritech, Inc. (Midland, MI). SDS was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Deuterium oxide was purchased from Sigma-Aldrich (St. Louis, MO). G3 PAMAM dendrimer was stored in methanol, and the solvent was distilled before use. All other chemicals were used as received without further purification. SAXS Studies. SAXS experiments were performed on the SAXS instrument at beamline 1W2A of the Beijing Synchrotron Radiation Facility (297.0 ± 1.0 K).34 The X-ray wavelength was 1.54 Å, the selected sample-to-detector distance was chose to be 184 cm, and the corresponding wave vector Q was ranging from 0.02 to 0.29 Å−1. The dendrimer concentration of all the samples was kept at 5 mg/mL, while the molar ratios of SDS and dendrimer are 0, 4, 8, 12, 16, 32, and 64. The samples were loaded in 1 mm path length cells and measured at room temperature. Buffer scattering have also been measured before and after measuring each sample. The measured intensity has been integrated to 1D I(Q) which was then normalized to the monitored transmitted beam intensity and then subtracted the averaged buffer scattering. The reduced data were then analyzed by both model independent approach and modeling method. Model Independent Approach: Indirect Fourier Transform (IFT) Analysis. The intensity I(Q) measured in a SAXS experiment is given by the Fourier transform of the pair distance distribution function P(r) of the particle.35,36 I(Q ) = 4π

∫0

∞

P(r )

sin(Qr ) dr Qr

2 ⎡ 3Vc(ρ − ρ )j (QR c) 3Vs(ρs − ρsolv )j1 (QR s) ⎤ c s 1 ⎥ P(Q ) = scale⎢ + QR c QR s ⎣ ⎦ (3)

where j1(x) = (sin x − x cos x)/ x2, Rs = Rc + T, and Vi = (4π/ 3)Ri3. ρc, ρs, and ρsolv are the electron density of the core, the shell, and the solvent, respectively, Rc radius of the core, Rs radius of the shell, and T the thickness of the shell. In reality, the dendrimer/SDS complex may not be perfectly spherical core−shell structured. However, because the scattering intensity is statistically and orientationally averaged for all particles in the measured samples, and also the low resolution of the technique, the core−shell model serves as a good model in fitting the SAXS data. Therefore, the quantitive modeling results provide instructive and helpful information. At low molar ratios of SDS and dendrimer, the contribution of the interdendrimer correlation to I(Q) is found to be negligible in our diluted samples when the Coulomb interaction between dendrimers can be ignored. As increasing SDS concentration, the SDS/dendrimer complexes became effectively charged. Therefore, significant interparticle interference peak in SAXS data at low Q range was found due to the strong Coulomb interaction. The S(Q) was calculated using the method developed by Hansen et al. and included in the modeling,39 and we used the software package developed by Kline40 to conduct the modeling. The adjusted model parameters are the effective charge, ionic strength, and volume fraction. Therefore, the charging status of the complex can be monitored based on the modeling results. 1 H NMR Studies. All the NMR spectra were acquired at 298.2 ± 0.1 K on a Varian 699.804 MHz NMR spectrometer, equipped with a 5 mm standard probe. The 1H NMR spectra were obtained with 32 scans and a 2 s relaxation delay. The samples were maintained at least 2 min to avoid the fluctuation of temperature before each acquisition. The concentration of PPI dendrimer and PAMAM dendrimer for each sample is 2 mg/mL. The molar ratios of SDS and dendrimer are 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 32, 64, and 128. Dioxane was added as an internal standard. Pulse Gradient Spin Echo (PGSE) NMR Studies. The self-diffusion coefficients of dendrimer−SDS complexes were measured by a standard PGSE sequence on the same NMR instrument at 298.2 ± 0.1 K. The heater and cooling unit was switched on to reach and stabilize the desired temperature, which avoids the influence of temperature variation on diffusion measurement. The time interval (Δ) between gradient pulses is 100 ms and 400 ms for dioxane and dendrimer−SDS complexes, respectively. The duration time of gradient pulses (δ) is 3 ms. The recycle time is 10 s. The pulse gradients (g) linearly increased in 16 steps to attenuate the spin-echo signal, and the maximum gradient strength is 70 G/cm. The gradient pulse was calibrated on a mixture of D2O and H2O (10% D2O and 90% H2O) under the same experimental conditions. The diffusion coefficients (D) of each sample were obtained by fitting the spin-echo signal and gradient strength by the equation

(1)

The P(r) function gives direct information about the structure in real space. The function is a measure of the probability of all distances between a pair of points within the particles weighted by the excess electron density at the points. From the function, the maximum dimension of the particle can be directly determined where P(r) goes to zero, and the variation in the shapes or internal structures of the particles can be easily detected. However, due to the finite experimental range of scattering vectors, the influence of instrumental smearing effects, and insufficiently corrected background scattering, it is not possible to derive P(r) by direct inverse Fourier transformation. Nevertheless, the IFT method, first introduced by Glatter, has been well developed and can be applied to calculate the P(r) function. In particular, we used the program GNOM by Svergun for the calculation of P(r) from the SAXS data.36 SAXS Data Modeling. For data modeling, the theoretical result of a given model can be calculated and therefore fitted to the experimental data using the nonlinear least-squares method. For the investigated system, the SAXS intensity distribution I(Q) can be given by the analytical expression37 I(Q ) = AP(Q )S(Q ) + Ibgd

(2)

where A is the scattering amplitude which is a function of the particle number density and its volume and average excess electron density, P(Q) the intramolecular structure factor, S(Q) the interdendrimer structure factor, and Ibgd the background. For the intramolecular structure factor P(Q), the spherical “core + shell” model was applied to fit the experimental data successfully, noticing the spherical shape and electron density variations in the radial direction of the dendrimers or SDS

In = I0 exp[−γ 2Dδ 2(Δ − δ /3)g 2] 3076

(4)



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Figure 1. SAXS data for G3 PAMAM and G3 PPI dendrimers fitted using a core−shell model.

where In and I0 are the intensities of spin-echo signal when the sine-shaped field gradient is present and absent, respectively, and γ is the proton magnetogyric ratio (2.68 × 108 s−1 T−1). Two-Dimensional Nuclear Overhauser Spectroscopy (2D NOESY) Studies. The 2D NOESY experiment was conducted to confirm if the SDS molecules are encapsulated within the interior pockets of PPI or PAMAM dendrimers. The NOESY experiments for PPI−SDS and PAMAM−SDS complexes were conducted on the same NMR instrument at 298.2 ± 0.1 K using standard pulse sequences. A water suppression pulse was added to improve signal sensitivity. 1 s relaxation delay, 146.63 ms acquisition time, a 6.5 μs 90°pulse width, and a 300 ms mixing time were chosen. 32 transients were averaged for 512 × 1024 complex points. All the data were processed with NMRpipe software on a Linux workstation.

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RESULTS AND DISCUSSION Structural Differences of G3 PPI and G3 PAMAM Dendrimers. The SAXS data for G3 PAMAM and G3 PPI dendrimers in Figure 1 were fitted using the core−shell model. The overall radius of G3 PPI is calculated to be 15.7 Å, which is much smaller than that of G3 PAMAM (23.5 Å, Table 1). The

Figure 2. Pair distance distribution function P(r) determined by IFT from the SAXS data for G3 PAMAM and G3 PPI dendrimers. The functions calculated from homogeneous spheres with the same size and molecular weight are also given (the same SAXS intensity at Q = 0 determined by IFT, which is proportional to the integrated electron density over the whole dendrimer volume).

Table 1. Structures of G3 PAMAM and G3 PPI Dendrimers dendrimer G3 PAMAM G3 PPI

overall radius (Å) (SAXS)

surface group number

23.5 ± 0.2

32

15.7 ± 0.4

32

surface group area (Å2) 217 96.8

homogeneous branching density distribution throughout the dendrimer. The conformation of dendrimer in solution has been extensively studied both theoretically and experimentally, and the dense-core model, rather than the dense-shell model, is by now accepted as the correct one.42,43 Usually, a structure transition from an open surface to a closed and congested surface for PAMAM dendrimers is observed above generation 4 or 4.5.44 The fuzzier surface and core−shell structure of G3 PAMAM dendrimer in our SAXS analysis is in accordance with this rule. The dendrimer structure also depends on its repeated unit and surface charge. Considering the shorter and more hydrophobic repeated unit of PPI compared to PAMAM dendrimer, it is not hard to understand that G3 PPI has a rather uniform branch density distribution. In contrast, the longer and less hydrophobic repeated unit of G3 PAMAM provides more freedom for branch back-folding or turning and stronger interactions with solvent water. Therefore, a fuzzy surface sphere with flexible branching unit, less hydrophobic interior and lower surface group density for G3 PAMAM compared to rather homogeneous sphere with stiff branching unit, hydrophobic interior, and higher surface group density for G3 PPI can be concluded (Table 1). The structural differences motivate us to investigate the binding behaviors of G3 PAMAM and G3 PPI dendrimers with surfactants.

surface group distance (Å) 14.7 ± 0.2 9.8 ± 0.3

fitting results suggest that G3 PPI dendrimer has a uniform density profile throughout the dendrimer (shell thickness is zero), while G3 PAMAM has a shell thickness around 11.1 Å with electron density closer to that of solvent water. The core− shell structure of PAMAM dendrimer is in accordance with earlier studies.41 Similar results are observed from the pair distance distribution functions P(r) for both dendrimers. As shown in Figure 2, compared to the P(r) calculated for homogeneous spheres of the same size and molecular weight, the P(r) for G3 PAMAM and G3 PPI shift to smaller distances. In addition, the P(r) for G3 PAMAM has smaller values at large distances than that of homogeneous sphere. This is due to the lower branching density at the surface region of G3 PAMAM; in other words, G3 PAMAM dendrimer has a fuzzier surface. In comparison, the P(r) for G3 PPI is rather close to that of homogeneous sphere at large distances, indicating the 3077



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Binding Behaviors of G3 PAMAM and G3 PPI Dendrimers with SDS. As shown in Figures 3 and 4, G3 PAMAM and G3 PPI show distinct binding behaviors with SDS within the SDS/dendrimer molar ratio of 0−4. At a SDS/ dendrimer molar ratio of 0.5, the relative diffusion coefficient of SDS in G3 PAMAM−SDS complex determined by PGSE NMR is about 3 times larger than that in the G3 PPI−SDS complex. The diffusion coefficient of SDS is similar to that of G3 PPI in the G3 PPI−SDS complex, suggesting that SDS is tightly bound with the PPI dendrimer. With increasing SDS/ dendrimer molar ratios (0−4), the relative diffusion coefficient of G3 PPI dendrimer increases while that of G3 PAMAM decreases. This phenomenon can be explained by the encapsulation of SDS molecules within G3 PPI dendrimer and the ionic binding of SDS molecules on the surface of G3 PAMAM dendrimer (Scheme 2). Hydrophobic interactions between the aliphatic chain of SDS and hydrophobic interior of G3 PPI dendrimer lead to a shrunk structure of the complex compared to free G3 PPI dendrimer.22 On the other hand, SDS molecules binding on the surface of G3 PAMAM dendrimer causes increased dendrimer size with increasing SDS/ dendrimer molar ratios. This model well explains why SDS in the G3 PPI−SDS complex has a similar diffusion coefficient with G3 PPI (slow exchange of free and bound state) but shows much higher diffusion coefficient in the G3 PAMAM−SDS complex than the dendrimer (fast exchange of free and bound state). We further conducted a 1H−1H 2D NOESY experiment to confirm the inclusion structure of G3 PPI−SDS complexes.45−47 As shown in Figure 5a, strong NOE interactions between the protons H3 of SDS protons and G3 PPI protons and medium interactions between protons (H1 and H4) of SDS and dendrimer protons are observed, indicating encapsulation of SDS molecules within the interior pockets of G3 PPI dendrimer.19 Since peak HA of G3 PPI is overlapped with peak H2 of SDS, the cross-peaks at related regions are not discussed. However, for the G3 PAMAM−SDS complex, only medium NOE interactions between protons H3 of SDS and dendrimer protons (Ha−d) are observed in Figure 5b. Considering that G3 PAMAM has a larger molecular weight (6900 Da) than G3 PPI (3513 Da), the weaker NOE cross-peaks for the G3 PAMAM− SDS complex in Figure 5 at the same mixing time suggest that

Figure 3. Diffusion coefficients of G3 PAMAM and G3 PPI dendrimers relative to those of dioxane in the presence of different amounts of SDS. The molar ratio of SDS and dendrimer ranges from 0.5 to 128. Dioxane was used as an internal standard to rule out the influence of solvent viscosity on the diffusion coefficients. The protons on PAMAM (Ha−d) and PPI (HA−C) dendrimers in this study are assigned according to reference 28.

Figure 4. Diffusion coefficients of SDS relative to those of dioxane in G3 PAMAM−SDS and G3 PPI−SDS complexes. The molar ratio of SDS and dendrimer ranges from 0.5 to 128. The protons on SDS (H1−4) in this study are assigned according to reference 28.

Scheme 2. Binding of SDS Molecules in the Interior and on the Surface of G3 PPI (a) and G3 PAMAM (b) Dendrimers

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Figure 6. 1H NMR of G3 PPI (a) and G3 PAMAM (b) dendrimers titrated with different SDS/dendrimer molar ratios. The red arrows in (a) represent the coexistence of non-cooperative and cooperative binding SDS on G3 PPI dendrimer.

methylene protons (HC) located on the dendrimer surface exhibit a downfield shift due to the ionic bindings of SDS molecules.27 Especially at SDS/G3 PPI molar ratios of 6 and 8, three independent peaks for HC were observed. This phenomenon can be explained by a coexistence of noncooperative and cooperative binding SDS on G3 PPI surface, which is already discussed in our previous study for G4 PAMAM−SDS complexes.22 In comparison, only noncooperative binding of SDS on G3 PAMAM dendrimer is observed in Figure 6b. Tomalia et al. demonstrated that the noncooperative or cooperative binding of surfactants on dendrimer surface depends much on dendrimer generation.44 Surfactant interacts with low generation dendrimers (G ≤ 3.5) via noncooperative ionic bindings but binds with high generation dendrimers (G ≥ 4.5) through cooperative bindings. This distinct behavior was attributed to the transition of dendrimer morphology from an open structure of low generation dendrimers to a congested structure of high generation ones. High surface charge density of the latter generations leads to the observed cooperative binding. Most recently, Wagner and Li found that the cooperative binding strength increases quadratically with the polyelectrolyte’s charge density and in proportion to the surfactant’s hydrophobicity in a polyelectrolyte−surfactant system.48 As revealed in our SAXS analysis, G3 PPI dendrimer has dense surface groups while G3 PAMAM dendrimer has a fuzzy and open surface. Considering both

Figure 5. 2D NOESY spectra of G3 PPI−SDS (a) and G3 PAMAM− SDS (b) complexes at a SDS/dendrimer molar ratio of 4. The mixing time for both spectra is 300 ms.

much fewer SDS molecules are encapsulated within the G3 PAMAM dendrimer. This distinct encapsulation behavior is mainly due to the hydrophobic nature of the G3 PPI interior and the relative polar microenvironment of the G3 PAMAM interior. At SDS/dendrimer molar ratios of 4−12, the diffusion coefficient of G3 PPI also significantly decreases (Figure 3). This is due to the saturation of SDS encapsulation within PPI dendrimer interior and binding of SDS molecules on its surface. During this period, a SDS bilayer structure is proposed to form on the surface of G3 PPI dendrimer. This is evident from the 1 H NMR spectra of G3 PPI−SDS complexes in Figure 6a. The 3079



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dendrimer has 32 surface amine groups and G3 PPI is smaller than G3 PAMAM, the group distance on G3 PPI dendrimer is much smaller than that on G3 PAMAM dendrimer (Table 1). The differences in surface group density and dendrimer structure of the two dendrimers well explain the distinct binding behavior of SDS on G3 PPI and G3 PAMAM surface during this period. At SDS/dendrimer molar ratios above 12, G3 PPI and SDS form a larger aggregate than the G3 PAMAM−SDS complex. During this period, SDS forms micelles in the interacting solution. G3 PAMAM dendrimer interacts with these SDS micelles in a fast-exchange fashion. In comparison, the SDS bilayer formed on G3 PPI dendrimer by cooperative bindings connects two PPI dendrimers, forming a dumbbell-like structure. Such a dumbbell structure is proposed by Ottaviani et al. using EPR and can explain why the G3 PPI−SDS complex is larger than the G3 PAMAM complex at high SDS concentrations.20,21 The interactions of SDS with G3 PAMAM and G3 PPI dendrimer are further investigated by SAXS. The SAXS data for dendrimer−SDS complexes at different molar ratios were analyzed by the IFT method. As shown in Figures 7 and 8, significant increases in the scattering intensity upon addition of SDS are observed, indicating the binding or association of SDS

Figure 8. Top: SAXS data and IFT analysis for G3 PPI−SDS at different molar ratios. Bottom: P(r) functions for the G3 PPI−SDS complexes determined by IFT analysis.

molecules to both dendrimers. The maximum distance in the aggregates, as determined from the P(r), is much larger than that of dendrimer in the absence of SDS. At high SDS/ dendrimer molar ratios, P(r) functions for both dendrimer complexes showed similar behavior to that of pure SDS micelle, indicating the formation of SDS micelles. The appearance of negative values at the middle distances are due to the well assembled SDS which has lower electron density of the hydrophobic tail and higher electron density of the hydrophilic head than solvent water. It is interesting to note that, for G3 PPI dendrimer, negative values of P(r) can be found at all measured molar ratios, while for the G3 PAMAM dendrimer, negative P(r) values only start to appear until molar ratio reaches 64. This means that PPI has more significant effect on the assembly of SDS into micelles. This phenomenon is in accordance with the results from NMR analysis that a SDS bilayer structure is formed on the surface of PPI dendrimer via cooperative bindings. In Figures 7 and 8, it is also found that at high molar ratios of SDS and dendrimer the theoretical results do not agree with experimental data in the low Q range which is due to the ignorance of the interparticle structure factor S(Q) in the IFT analysis. To accounting for the S(Q), we have also performed the data modeling analysis (Figure 9 and Table S1). The SDS/ dendrimers complexes were effectively charged at high molar ratios due to the formation of SDS micelles. The variations of

Figure 7. Top: SAXS data and IFT analysis for G3 PAMAM−SDS at different molar ratios. The dots are the experimental SAXS data, and the solid lines are the fitted results. Bottom: P(r) functions for the G3 PAMAM−SDS complexes determined by IFT analysis. 3080



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Figure 9. Fitting the SAXS data of G3 PAMAM−SDS (a) and G3 PPI−SDS (b) complexes using a core−shell model.

the overall aggregate size for both dendrimers at different SDS/ dendrimer molar ratios are shown in Figure 10. For G3

PAMAM dendrimer, the maximum distance and overall aggregate size increases to very large values (>80 Å) at SDS/ dendrimer molar ratios of 4 and 8, indicating formation of large aggregates. During this molar ratio range, SDS monomer binds with G3 PAMAM dendrimer surface and several G3 PAMAM dendrimers may associate with each other via hydrophobic interactions between the bound SDS molecules, yielding a larger aggregate structure (Scheme 3). Further addition of SDS molecules disassembles this aggregate structure and the final size of the complex decreases to 55 Å at SDS/dendrimer molar ratios above 12. This is because SDS form micelles on the dendrimer surface and the aggregates carrying net negative charges repulse with each other, preventing the formation of aggregates. Interestingly, this assembly and disassembly process is not observed in the diffusion coefficient analysis in Figure 3, in which the size of dendrimer aggregate increases with increasing SDS/dendrimer molar ratios and finally achieves a constant size at molar ratios above 64. This is because we analyze the diffusion coefficient of dendrimer or SDS in PGSE

Figure 10. Size variations of the dendrimer−SDS complexes at different SDS/dendrimer molar ratios based on the SAXS data modeling.

Scheme 3. SDS Molecules Bound on the Surface of G3 PAMAM Dendrimer Causes the Formation of Larger Aggregates at a SDS/G3 PAMAM Molar Ratio of 4

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interactions between PPI dendrimer and cell membrane may induce pore formation on the cell membrane and followed by leakage of intracellular proteins.55 Such a result well explains why G3 PPI dendrimer is much more toxic (>50-fold) than G3 PAMAM dendrimer.28 As demonstrated by previous studies, the interactions between dendrimers and surfactants depend much on dendrimer generation, dendrimer surface functionality, surfactant property (i.e., length and number of hydrophobic tails and charge property of hydrophilic head), solution pH value, ionic strength, and temperature.56 In future studies, we will compare the binding behaviors of PAMAM and PPI dendrimers of different generations with biosurfactants such as DMPC to investigate the exact reason behind the distinct toxicity profiles of both dendrimers and the above parameters will be considered.

NMR but the whole aggregate size in the SAXS analysis. The G3 PAMAM dendrimer−surfactant aggregate is formed by noncooperative interactions and the SDS molecules are in a fast exchange of free and bound state. In this case, the dimension of the loosely bounded aggregates is not simply inversely proportional to the diffusion coefficient. And at high molar ratios, the binding of more SDS and the appearance of interparticle repulsion would further suppress the diffusion of dendrimers. For G3 PPI dendrimer, the encapsulation of SDS monomers within the dendrimer interior pockets at low SDS/ dendrimer ratios is followed by the cooperative binding of SDS on dendrimer surface at high SDS/dendrimer ratios. Therefore, no large aggregate structure was examined in the SAXS measurements. The size of the G3 PPI−SDS complex gradually increases to a stable value of 60 Å, which is larger than the size of G3 PAMAM−SDS complex. This result is in accordance with the diffusion coefficient variations of both dendrimers in the presence of SDS in Figure 3. Within the investigated SDS/dendrimer molar ratio (0− 128), SDS interacts much more strongly with G3 PPI dendrimer than with G3 PAMAM dendrimer. This is evident in the PGSE NMR analysis in Figure 4. At all SDS/dendrimer molar ratios, SDS in the G3 PPI−SDS system shows much lower diffusion coefficients than that in the G3 PAMAM−SDS system. This is attributed to the encapsulations of SDS within G3 PPI dendrimer at low SDS/dendrimer molar ratios and binding of SDS on its surface via cooperative interactions at higher ratios. The distinct binding behaviors of SDS in the interior and on the surface of both dendrimers are not attributed to different pH values of the dendrimer−SDS mixture solutions due to the following reasons. (1) The pKa values of tertiary amines in the interior of PAMAM and PPI dendrimers are around 6.0,49,50 and the pH values of all the complex solutions are above 8.45 (Table S2 in the Supporting Information). The tertiary amine groups in the interior of both dendrimers are not protonated during the addition of SDS molecules. In this case, the stronger binding of SDS within PPI dendrimer interior is due to the more hydrophobic nature of PPI dendrimer interior rather than different protonation states of the tertiary amine groups. (2) The pH values of G3 PPI/ SDS solutions (9.88−12.07) are higher than that of G3 PAMAM/SDS solutions (8.45−10.98) at equal SDS/dendrimer molar ratios (Table S2). In this case, a smaller percent of surface amine groups on G3 PPI dendrimers are protonated than on G3 PAMAM dendrimers during the addition of SDS molecules (pKa values of the surface amine groups of PAMAM and PPI dendrimers are around 10.0).51,52 Therefore, the stronger interactions of SDS on PPI dendrimer surface than on PAMAM dendrimer is mainly due to distinct structures of both dendrimers as revealed by SAXS analysis rather than different pH values or protonation states. At high SDS/dendrimer molar ratios (32:1 to 128:1), the pH values of PPI/SDS solutions are around 12.0, and most of the surface amine groups are not protonated. The cooperative interactions between SDS and the PPI dendrimer surface should be ion-dipole interactions rather than ionic interactions during this period. The stronger interaction of surfactants such as SDS with G3 PPI than with G3 PAMAM provides insights into the cytotoxicity profiles of both dendrimers. Phospholipids such as dimyristoylphosphatidylcholine (DMPC) are also surfactants with hydrophobic tails and a hydrophilic and charged head.53,54 Thus, G3 PPI dendrimer should interact strongly with these biosurfactants, which are main components of cell membrane. The strong

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CONCLUSIONS

Dendrimer−surfactant interactions have held great scientific interest because of synergistic effect of the dendrimer− surfactant complex and their potentials for broad applications in industry, pharmaceutical sciences, and biological systems.6 Although many studies have investigated such interactions in aqueous solutions and at interface of phases during the past two decades, no report on the behaviors of PAMAM dendrimer versus PPI dendrimer in their interactions with surfactants is available. The comparison of PAMAM and PPI dendrimers can provide insights into their distinct behaviors in biological systems such as cytotoxicity and cellular uptake process. In this study, we investigated the structure differences between PAMAM and PPI dendrimers and compared their binding behaviors with an anionic surfactant by a combination of NMR and SAXS analysis. G3 PAMAM dendrimer shows a relatively loose, open, and dense-core structure and G3 PPI has a compact, closed, and uniform one. At low SDS/dendrimer molar ratios, G3 PPI mainly encapsulates SDS monomers within its interior pockets while G3 PAMAM interacts with SDS molecules on its surface by noncooperative interactions (supported by PGSE and NOESY). This process continues until the SDS encapsulation within G3 PPI is saturated. Then SDS also binds on G3 PPI surface via noncooperative ionic interactions (supported by PGSE) and followed by a gradual transition from a noncooperative binding to a cooperative one at higher SDS/G3 PPI molar ratios (supported by 1H NMR titration). G3 PAMAM dendrimer forms larger aggregates with SDS at low SDS/dendrimer molar ratios (4 or 8) and followed by disassembly of the aggregate at higher ratios (supported by SAXS analysis). The G3 PAMAM−SDS complex size is larger than the size of the G3 PPI−SDS complex at low SDS/ dendrimer molar ratios; as the binding proceeds, a reverse trend is observed at high molar ratios (supported by both PGSE NMR and SAXS). At all SDS/dendrimer molar ratios, SDS interacts much more strongly with G3 PPI rather than with G3 PAMAM. These binding behaviors are due to distinct interior characters and surface structures of G3 PAMAM and G3 PPI dendrimers. Such results are helpful for us to understand the distinct toxicity profiles of PAMAM and PPI dendrimers and provide new insights into the design of biocompatible dendrimers for drug and gene delivery. 3082



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ASSOCIATED CONTENT

S Supporting Information *

Further information on SAXS analysis and pH values of the dendrimer/SDS complex solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (D.C.). *E-mail [email protected] (Y.C.). Author Contributions

T.L. and N.S. contributed equally to this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the support of the BSRF in providing the SAXS research facility and thank Dr. Guang Mo, Prof. Zhihong Li, and Prof. Zhonghua Wu for their kind help in conducting the SAXS measurement. We thank the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20120076110021), the National Science Foundation of China (No. 11005159, No.595 1105232), and the “973 program” (No. 2010CB833105) for financial support of this work.

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