Insights into the Interactions between Dendrimers and Bioactive

Sep 3, 2009 - 2D-NOESY analysis indicated the localization of bile salt in the interior pockets of dendrimers. The orientation of the guest in the poc...
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Insights into the Interactions between Dendrimers and Bioactive Surfactants: 3. Size-Dependent and Hydrophobic Property-Dependent Encapsulation of Bile Salts Qinglin Wu,† Yiyun Cheng,*,†,‡ Jingjing Hu,‡ Libo Zhao,‡ and Tongwen Xu*,‡ Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, UniVersity of Science and Technology of China, Hefei, Anhui 230027, P. R. China, Laboratory of Functional Membranes, Department of Chemistry, UniVersity of Science and Technology of China, Hefei, Anhui 230026, P. R. China ReceiVed: July 14, 2009; ReVised Manuscript ReceiVed: August 11, 2009

The supramolecular structures of dendrimer-bile salt complexes have been investigated by multidimensional and multinuclear NMR techniques, such as 1H NMR, COSY, TOCSY, NOESY, and DOSY. 2D-NOESY analysis indicated the localization of bile salt in the interior pockets of dendrimers. The orientation of the guest in the pockets was predicted by the NOE cross-peaks based on NOESY spectrum. 1H NMR experiments suggested that no electrostatic interactions between the amine groups of dendrimers and the negatively charged group of bile salts occur in the complexes. DOSY studies further confirmed the inclusion structures based on the diffusion coefficient information. The supramolecular structures of dendrimer-bile salt complexes were mainly formed by hydrophobic interactions/hydrogen-bond interactions in the interior pockets of dendrimers. In addition, size- and hydrophobic property-dependent encapsulation of bile salts and bile derivates in the cavities was observed. These results suggest a new interaction model of dendrimer-surfactant aggregates and provide new insight into the interactions between dendrimers and bioactive surfactants. 1. Introduction Bile salts, the main products of cholesterol metabolism in animals, play an important role in the digestion of lipids. These naturally occurring steroid molecules with amphipathic properties possess hydrophobic and hydrophilic regions and form aggregates or micelles in aqueous solutions such as a synthetic surfactant.1 Structurally, the hydrophobic region of bile salts is made up of a steroidal core with the methyl groups at the C-18, C-19, and C-21 positions, which is called the convex side or the β plane;1 whereas the hydrophilic region consists of two or three hydroxyl groups at the C-3, C-7, and C-12 positions as well as a short and flexible aliphatic tail with hydrophilic groups, such as carboxylate, at the end, often called as the concave side or the R plane. The physicochemical properties of bile salts, such as the aggregation and micellization, are of great importance to understanding their physiological functions.2,3 Therefore, it is not surprising that aggregate behavior of bile salts and their interactions with lipids, biocompatible polymers, surfactants, and other bioactive molecules has been extensively studied in the past decades.3-5 Dendrimers are synthetic monodisperse macromolecules with a well-defined branching structure that radiates from a central core.6 They have globular or ellipsoidal shapes, nanoscale sizes, relative nonpolar pockets in the interior, and well-defined numbers of functionalities on the surface.7 Since their successful synthesis in the mid-1980s, dendrimers have attracted considerable attention because of the wide and limitless variations in their chemistry; availability of numerous synthetic methodologies; and their unique structures, versatility, and so forth, which render them a reliable alternative to traditional polymers in a wide range of applications.8-15 Up to now, dendrimers have been * To whom correspondence should be addressed. E-mails: (Y.C.)yycheng@ mail.ustc.edu.cn, (T.X.)[email protected]. † Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences. ‡ Laboratory of Functional Membranes, Department of Chemistry.

widely used as synthetic hosts in host-guest systems,16-19 drug capsules,20-25 gene delivery vehicles,9,26 cancer targeting platforms,13,27-33 proteinandotherbiomacromoleculemimicries,6,8,9 nanomaterial synthesis templates,34 and catalyst scaffolds.35 The study of dendrimer-based host-guest systems is of central importance to the chemical, biomedical, and pharmaceutical applications of dendrimers.36-48 Recently, we have investigated the host-guest chemistry of dendrimer-based drug formulations and aggregations by NMR techniques including 1D (1H and 13C) and 2D NMR (nuclear Overhauser enhancement spectroscopy, NOESY) studies.16,18,36-38,49 NMR is a noninvasive, sensitive, and powerful method for quantitative determination of an analyte, especially for the noncovalent interactions between a ligand and a receptor.35,50-53 It can give information on the formation of aggregates, ion pairing, encapsulation, molecular orientation, and size variations.52 On the basis of NMR studies, different interaction mechanisms between dendrimers and guest molecules have been proposed, and the effects of dendrimer generation, surface functionality, and guest size, pKa value, hydrophobic property, and spatial steric hindrance on the host-guest interactions have been discussed.18,36 In addition to the drug molecules, ionic surfactants with amphiphilic properties make ideal guests for dendrimers because of their long hydrophobic tails and the negatively or positively charged heads in their structures.18,54-59 The molecular interactions between biocompatible polymers such as dendrimers and bioactive surfactants such as bile salts are of great physicochemical and physiological significance to understand the fate of these polymers under physiological conditions,48,53,59 such as the alimentary tract and the circulating blood. The structure, nature, and interaction mechanisms of the dendrimer and bioactive surfactant aggregates are also helpful to interpret many biological phenomena, such as the fluidity of proteins on the surface of cell membranes, the transport of dendrimers across cell membranes,48 and the isolation of membrane proteins. In addition, it is reported that mixed micelles have advanced

10.1021/jp906661z CCC: $40.75  2009 American Chemical Society Published on Web 09/03/2009

Interactions of Dendrimers, Bioactive Surfactants SCHEME 1: Molecular Structure of the Bile Salts (Sodium Deoxycholate and CHAPS)

properties in comparison with single-component micelles.5 Dendrimers are also ideal amphiphilic surfactants that have been applied in many research and industry fields. Therefore, the aggregates of dendrimers and bile salts are also a kind of mixed micelles with distinct properties compared to traditional surfactants with hydrophilic head and hydrophobic tails. Furthermore, it is well-known that loss of bile salts is the beginning of several biological responses with a final result in the decrease of plasma cholesterol level in the blood. A class of polymers named bile salt sorbents were synthesized for the electrostatic/ hydrophobic binding of bile salts and the removal from the alimentary canal and the circulating blood by excretion.60,61 Dendrimers with large numbers of cationic groups on the surface and relative hydrophobic pockets in the interior make them excellent candidates as a new class of bile salt sorbents. To the best of our knowledge, it is the first report of interactions between dendrimers and bile salts. Herein, we investigated the interactions between dendrimers and bile salts by NMR studies. The effect of guest size (sodium deoxycholate with a molecular weight of 392 Da, and 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate,CHAPS, a bile salt derivate with a molecular weight of 614 Da, Scheme 1) on the electrostatic attachment and hydrophobic encapsulation behaviors were discussed. Homonuclear correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) experiments were conducted to confirm the proton peak assignments of bile salts. 1H NMR and 2D-NOESY techniques were employed to analyze the ion-binding and hydrophobic encapsulation of bile salts both on the surface and in the interior of dendrimers. Diffusion-ordered spectroscopy (DOSY) studies were further used to characterize the structure of the dendrimer-bile salt aggregates.

J. Phys. Chem. B, Vol. 113, No. 39, 2009 12935 2. Experimental Section 2.1. Materials. Generation 5 (G5) ethylenediamine (EDA)cored poly(amidoamine) (PAMAM) dendrimer used as a host model in this study was purchased from Dendritech, Inc. (Midland, MI). Sodium deoxycholate was obtained from Shanghai BBI Co. Ltd. (Shanghai, China). 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) was purchased from Sigma-Aldrich Co. (St. Louis, MO). Deuterium oxide (D2O) was purchased from Beijing Chongxi High-Tech Incubator co., Ltd. (Beijing, China). All the chemicals were used as received. G5 PAMAM dendrimer solution (2.0 mg/mL), 73.6 mg/mL sodium deoxycholate, and 115.3 mg/mL CHAPS solutions were prepared and used as stock solutions. 2.2. 1H NMR Studies. 1H NMR experiments were obtained on a Bruker Advance 500.132 MHz NMR spectrometer at 298.2 ( 0.2 K for different concentrations of sodium deoxycholate and CHAPS in D2O, as well as for G5 PAMAM dendrimer/ bile salts with different molar ratios and a fixed dendrimer concentration of 2.0 mg/mL. 2.3. 2D-COSY and TOCSY Studies. The 2D-COSY spectrum of sodium deoxycholate solution was obtained by the standard pulse program using the Bruker 500.132 MHz NMR spectrometer, with 1024 × 2048 data points. The relaxation delay was 2 s. Sixteen scans were averaged. A sine-bell-squared window function and zero-filling were applied to both dimensions. The TOCSY spectrum was obtained using an 8.4 µs, 90° pulse width with a relaxation delay of 2 s. The mixing time was 55 ms. A spin-lock pulse was applied for the mixing period with a spin-lock field of 10-12 kHz with DIPSI modulation. Eight scans were collected for each of the 512 increments in the indirect dimension. Both dimensions were processed using the sine-bell window function and zero-filling to a 1024 × 2048 matrix before Fourier transformation. 2.4. 2D-NOESY Studies. 2D-NOESY spectra of sodium deoxycholate/G5 dendrimer solution (2.0 mg G5 dendrimer and 2.0 mg sodium deoxycholate dissolved in 1 mL D2O) and CHAPS/G5 dendrimer solution (2.0 mg G5 and 3.1 mg CHAPS dissolved in 1 mL D2O) were acquired at 500.132 MHz, using a 300 ms mixing time. The mixing time of 300 ms was chosen for the optimization of the cross-peak intensities with minimum distortions. The experiments were performed with a 2 s relaxation delay and 8.4 µs 1H 90° pulse width. Thirty-two transients were averaged for 1024 complex t1 points. All data were processed with NMRPipe software on a Linux workstation with standard Lorentz-Gauss window function and zero-filling in both dimensions. 2.5. DOSY Studies. DOSY spectra of G5 dendrimer-sodium deoxycholate complex and free G5 dendrimer were performed at the same Bruker 500.132 MHz instrument, using standard Bruker pulse program, a stimulated echo pulse sequence with bipolar gradients (STEBPGP1s). Bipolar spoil gradients were used with a total duration of about 3 ms. Gradient recovery delays were 0.1-1 ms. The gradient field was linearly increased in 64 steps. Individual rows of the 2D diffusion data were phased and baseline. All the data were processed by Bruker Xwinnmr 3.1 (Bruker Biospin). 3. Results and Discussion 3.1. Chemical Shift Assignments and Ionic Interactions of Bile Salts and Dendrimers. The proton chemical shift assignments of sodium deoxycholate were carried out on the basis of 1H NMR spectrum, a homonuclear COSY spectrum, a TOCSY spectrum, and the reference data.62 A 1H NMR spectrum of sodium deoxycholate in D2O is shown in Figure 1.

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Figure 1. 1H NMR spectrum and proton chemical shift assignments of sodium deoxycholate in D2O. The numbers represent the protons of sodium deoxycholate, and the zoomed regions are the proton peaks for the methyl groups and other methylene and methine groups, respectively.

Apart from the protons of methyl groups (CH3) and the protons close to the hydroxyl groups (OH), other protons in sodium deoxycholate are located in a limited chemical shift range of 1.0-2.5 ppm. The extensive overlap of the methylene (CH2) and methine (CH) groups prevent the full assignment of protons for a bile salt. Therefore, more efficient approaches to the proton peak assignments of sodium deoxycholate are conducted by homonuclear COSY and TOCSY techniques. The COSY spectrum reveals J-coupled hydrogen groups (through bonds) via cross-correlation peaks off the diagonal.63 Figure 2 shows some cross-peaks that have been assigned by COSY. It allows a straightforward and unambiguous assignment of the chemical shifts obtained from a single COSY experiment. The TOCSY spectrum creates correlations among all protons within a given spin system, not limited to geminal or vicinal protons, as in the COSY spectrum. The TOCSY spectrum of sodium deoxycholate is shown in Figure S1 of the Supporting Information. The H3 and H12 methine protons, which are located on the C3 and C12 geminal to the hydroxyl groups in the bile salt, exhibit single peaks on the contour plot centered at 3.57 and 3.99 ppm, respectively. H18, H19, and H21 methyl protons show peaks at 0.65, 0.86, and 0.89 ppm, respectively. Other proton signal assignments of deoxycholate are labeled in Figures 1 and 2. Our proton assignments are in good agreement with the reference data for sodium deoxycholate.62 For the G5 amine-terminated PAMAM dendrimer, the 1H NMR spectrum should have six proton peaks corresponding to four kinds of methylene groups (∼a-d) in interior pockets and two kinds of methylene groups (b′ and d′) in the outermost layer of the dendrimer. The peaks for protons d and d′ are overlapped in the dendrimer solutions without pH adjusting (pH 9-10), whereas the protons b′ exhibit a significant downfield shift and finally have a similar chemical shift with protons c due to the presence of primary amine groups. Therefore, only four broad peaks can be found in the 1H NMR spectrum of amineterminated PAMAM dendrimers (Figure S2). However, the number of proton peaks in the spectrum depends much on the protonation of the amine groups as well as the formation of ion pairs between cationic amine groups and negatively charged guest molecules. Both cases will cause a significant downfield shift of protons b′ and d′, which are vicinal to the amine groups on the surface of dendrimers. At this stage, six peaks of dendrimers (∼a-d, b′, and d′) can be observed in the 1H NMR

spectrum. A further decrease in the pH value in the dendrimer solution will cause the splitting of peaks for interior pocket protons (∼a-d) due to the protonation of tertiary amine groups and the formation of quaternary ammonium groups in the relative hydrophobic pockets. The chemical shift variations are helpful to characterize the status of protonation and the formation of ion pairs of both interior pocket ternary amine groups and surface primary amine groups.36,38,64 After the assignment of chemical shifts of the host molecule (G5 PAMAM dendrimer) and the guest molecule (sodium deoxycholate), we can easily employ the 1H NMR titration technique to investigate the host-guest chemistry of the dendrimer-bile salt complex. As shown in Figure 3, when different amounts of sodium deoxycholate were titrated into the G5 dendrimer solution, the chemical shift of protons ∼a-d, b′, and d′ is scarcely changed, indicating a lack of electrostatic attachment of bile salts on the surface of the dendrimers. Similar results were obtained (Figure 4) when CHAPS was titrated into a G5 solution. In our previous results, negatively charged drug molecules, including phenobarbital,16,17 phenylbutazone,65 sulfamethoxazole,16 mycophenolic acid,36 methotrexate, and Congo red, were observed to cause a significant downfield shift of protons b′ and d′ and a weak upfield shift of protons ∼a-d, which is due to the ionic binding of these guests on the surface of dendrimers, and hydrophobic/hydrogen-bonding interactions in the relative nonpolar pockets of dendrimers. Similarly, positively charged guests, such as amantadine and propranolol were able to cause an upfield shift of the methylene protons (∼a-d and ∼a′-d′) of a carboxylate-terminated dendrimer (G4.5).38 In the case of surfactants such as sodium dodecyl sulfate (SDS),18,37 a downfield shift of protons b′ and d′ is also observed when the mass ratio of SDS and G5 dendrimer reaches 1. There are some possibilities that reduce the electrostatic attachment of sodium deoxycholate on the surface of the dendrimer. First, the G5 dendrimer or sodium deoxycholate/ CHPAS may be in its noncharged form, which will not form ion pairs with the other one in solution. This phenomenon is found when venlafaxine hydrochloride is titrated into a G4.5 dendrimer solution, which is due to the deprotonation of venlafaxine and protonation of the carboxylate groups on the surface of the G4.5 dendrimer.38 For the G5 dendrimer-sodium deoxycholate complex, the primary amine groups of G5 have a

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Figure 2. Contour plots of the 1H-1H COSY spectrum of sodium deoxycholate in D2O (a). The expanded regions of the 1H-1H COSY spectrum are further shown in panels b and c.

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Figure 3. Expanded region of the 1H NMR titration spectra of the G5 dendrimer with sodium deoxycholate. The concentration of G5 dendrimer was kept constant at 6.947 × 10-5 M. (a) G5 dendrimer; (b) G5 dendrimer + 17.36 equiv of sodium deoxycholate; (c) G5 dendrimer + 26.04 equiv of sodium deoxycholate; (d) G5 dendrimer + 43.4 equiv of sodium deoxycholate; (e) G5 dendrimer + 52.08 equiv of sodium deoxycholate; (f) G5 dendrimer + 60.76 equiv of sodium deoxycholate; (g) G5 dendrimer + 69.44 equiv of sodium deoxycholate; (h) free sodium deoxycholate.

Figure 4. Expanded region of the 1H NMR titration spectra of G5 dendrimer with CHAPS. The concentration of G5 dendrimer was kept constant at 6.947 × 10-5 M. (a) G5 dendrimer; (b) G5 dendrimer + 11.7 equiv of CHAPS; (c) G5 dendrimer + 17.56 equiv of CHAPS; (d) G5 dendrimer + 23.41 equiv of CHAPS; (e) G5 dendrimer + 29.26 equiv of CHAPS; (f) G5 dendrimer + 35.11 equiv of CHAPS; (g) G5 dendrimer + 46.82 equiv of CHAPS; (h) CHAPS alone.

pKa value around 10.5, whereas the carboxylic acid of cholic acid and deoxycholic acid have a pKa value around 4.2 The pD values of the G5-bile salt solutions range from 9 to 10 in the titration experiments. The bile salt should not be protonated in the solutions, and the amine groups of G5 dendrimer should be at least partially charged. For the G5-CHAPS solution, the negatively charged SO3- group in CHAPS could not be protonated in neutral or base solutions. Therefore, the nonshift of dendrimer proton peaks in the 1H NMR spectrum of G5-deoxycholate or G5-CHAPS complexes cannot be interpreted by the noncharged form of the host or guest molecules. Second, spatial steric hindrance between the charged groups of host and guest may reduce the formation of stable ion pairs. Previous studies have shown that the ionic interaction strength between quaternary ammonium and the G4.5 dendrimer is weak because of the serious steric hindrance of the three methyl

groups around the nitrogen atom.38 However, in the G5deoxycholate or G5-CHAPS system, the steric hindrance in the ion pairs of -NH3+/COO- or -NH3+/SO3- will not be a problem to prevent the formation of complexes due to the presence of an aliphatic tail linked to the -COO- or -SO3groups. A reasonable explanation for the lack of electrostatic interactions on the surface of dendrimer is the extremely hydrophobic property of the steroidal core of deoxycholate/ CHAPS. The aqueous solubility of the deoxycholate was low when the hydrophilic carboxylate group of deoxycholate was bound to the surface of a dendrimer. In our experiment, a white precipitation was observed during the early stage of the complex formation, indicating the presence of electrostatic binding of bile salts on the surface of dendrimers in the insoluble aggregates. This phenomenon was also observed in titration of SDS monomers into a dendrimer solution.18 However, the

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Figure 5. The 1H-1H NOESY spectra of G5 dendrimer/sodium deoxycholate/D2O solution (2 mg of G5 dendrimer in 1 mL of D2O, G5/sodium deoxycholate mass ratio ) 1) at mixing time of 300 ms (a); the expanded region showing much stronger cross-peaks between the protons of deoxycholate and protons (a and c) of dendrimer than those between the protons of deoxycholate and protons (b and d) of dendrimer (b). The cross-peaks are indicated by rectangles.

addition of more SDS monomers will dissolve the insoluble aggregate, followed by the formation of a new complex with SDS bilayers or micelles adsorbed on the surface of dendrimers. In the meantime, a significant downfield shift of protons b′ and d′ is observed. This is much different from the dendrimerdeoxycholate system in this study because the formation of SDS bilayers or micelles is helpful for the aqueous solubility of the complex, whereas the formation of deoxycholate-based micelles is difficult, although a much higher concentration of bile salt was added. It is worth noticing that the disappearance of the insoluble aggregates in D2O under sonication was observed, suggesting the formation of a more stable dendrimerdeoxycholate complex. The relationship of deoxycholate with dendrimer and the structure of the stable complex need to be further characterized by multidimensional NMR techniques, such as NOESY and DOSY analysis. 3.2. Localization of Bile Salts in the Interior Pockets of PAMAM Dendrimers by NOESY Analysis. The NOE is a manifestation of cross-relaxation between two nuclear spins that

are close to each other in space.52 NOESY analysis is the most widely used tool for revealing spatial relationships among protons in a molecule or in a complex of molecules, such as dendrimer-surfactant aggregates.66-69 It has been proved to be effective for providing the evidence for both inter- and intramolecular interactions in a complex.1,35,50,68 The intensity (volume) of cross-peaks between two specific nuclei in the NOESY spectrum predominantly depends on the nucleus distance (/N ∼ r-6, is the volume of the cross-peaks, r is the spatial distance between two nuclei, and N is the number of equivalent nuclei). If a guest molecule binds to a host molecule, there should be cross-peaks between guest nuclei and host nuclei in the corresponding spectral region. In other words, the absence of a cross-peak in the region can be used to rule out the host-guest interactions. In addition, NOESY experiments allow the asymmetrical guest orientation within the host molecule to be determined, since sites of the guest in close contact with the host might be identified by NOE.52 Herein,

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NOESY analysis was used to give evidence of the localization of bile salts in the interior pockets of dendrimers. The 1H-1H NOESY spectrum of G5-deoxycholate complex at a mixing time of 300 ms is shown in Figure 5. The mixing time of 300 ms was chosen for the optimization of the crosspeak intensities with minimum distortions. Previous studies have shown that spin diffusion will not give rise to pseudo-crosspeaks at mixing times up to 500 ms for G5 dendrimer-SDS aggregates and G5 dendrimer-drug complexes.18,36 For G5 dendrimer-deoxycholate/CHAPS complexes in this study, the molecular weight of the aggregates is similar to that of previous G5 dendrimer-SDS/drug complexes. Therefore, the cross-peaks appeared in the NOESY spectrum of these complexes are mostly ascribed to primary NOE interactions at a mixing time of 300 ms. In Figure 5, strong positive cross-peaks are observed among the three methyl protons (H18, H19, and H21) of deoxycholate and interior pocket protons (Ha, Hb, Hc, and Hd) of G5 dendrimer. Medium-sized NOE peaks are observed among the methine groups (H3R and H12R) directly bonded to the two hydroxyl groups, methylene protons (H2, H4, and H5) next to the two hydroxyl groups in deoxycholate, and the pocket protons (Ha∼Hd). The spectra do not show any cross-peak between other protons of bile salts and protons ∼Ha-Hd of the dendrimer. Generally, the presence of a NOE cross-peak between two nuclei at a sufficiently short mixing time means the protons are close, within a distance of 5 Å.66 The presence of cross-peaks between protons of bile salt and dendrimers indicated the close proximity between the host and guest. One may assume that the cross-peaks were generated by the adsorption of bile salts on the surface of a dendrimer by a certain interaction force. However, this assumption cannot interpret the observed crosspeaks due to the following reasons: (1) the spatial distances among protons H18, H19, H21, H3R, H12R, H2, H4, and H5 of deoxycholate and protons Ha∼Hd of dendrimer cannot be within the limit of 5 Å at the same time, (2) the intensities of crosspeak for protons Hb, Hd of a G5 dendrimer and protons of deoxycholate should be much stronger than those between protons Ha, Hc of the dendrimer and the protons of the bile salt because protons Hb, Hd localize much closer to the surface of a dendrimer than protons Ha, Hc. This is difficult for us to believe when considering the reverse cross-peak intensities of them in Figure 5. Moreover, no cross-peak between protons H3R, H2 of deoxycholate and protons Hb and Hd of the dendrimer was observed, but cross-peaks between H3R, H2 and Ha, Hc are clearly seen, indicating distant relationships between these protons is spatial, and (3) we have concluded from the 1H NMR titration experiments that no electrostatic interaction between deoxycholate/CHAPS and dendrimers occurs on the dendrimer surface. If the hydrogen-bonding interactions between the -NH2 groups and the -COO-/-SO3- groups on the surface of a dendrimer lead to the presence of cross-peaks, strong NOE interactions should be observed between H22, H23 of the bile salt and the methylene protons of the dendrimer. However, these protons did not show any interactions with the dendrimer protons. On the basis of these discussions, we can deduce that the presence of cross-peaks was caused by the hydrophobic encapsulation of the deoxycholate by the dendrimer. Since the intensity of a cross-peak between two protons in the NOESY spectrum predominantly depends on the proton distance, we can obtain the relative orientation of the deoxycholate in the relative nonpolar pockets of a G5 dendrimer. The close spatial proximity of methyl groups and the methylene groups have been previously found for many

Wu et al. SCHEME 2: The Possible Localization Model of Deoxycholate at the Cavities of Dendrimers

host-guest systems (dendrimers were used as hosts; SDS, phenylbutazone, phenobarbital, and mycophenolic acid were used as guests). The methyl groups localized near the branches (protons Ha and Hc) of dendrimers are essential for the formation of stable host-guest complexes. In addition, we found that the protons (such as H3R, H12R) near the two hydroxyl groups have strong NOE interactions with the dendrimer scaffolds, indicating that the hydroxyl groups play an important role in the formation of dendrimer-deoxycholate inclusions. There are large numbers of amide groups (-CO-NH-, N ) 2n+3 - 4, n is the dendrimer generation) groups and ternary amine groups (NR3, N ) 2n+2 - 2) in a PAMAM dendrimer. The amide groups and ternary amine groups can act as hydrogen-bond donors and receptors of hydroxyl groups of deoxycholate (Scheme 2). Therefore, the inclusion of deoxycholate in the dendrimer pockets may be hydroxyl group-centered (hydrogen-bonding interactions) and methyl group-assisted (hydrophobic interactions). These results are further confirmed by the cross sections taken at the positions of protons ∼Ha-Hd of the dendrimer (Figure 6) and protons H18, H19, H21, H3R, and H12R of the deoxycholate (Figure S3, available in the Supporting Information). Interestingly, clear cross-peaks between protons ∼H1-H23 in bile salt in the presence of G5 dendrimer were observed in Figure 5. The intensities of these cross-peaks are much increased compared with that in the NOESY spectrum of free deoxycholate conducted under the same experimental conditions (Figure 7). Some new cross-peaks between the protons of the bile salt appear in Figure 5 due to the presence of the dendrimer, suggesting that the conformation of deoxycholate in solution is different from that in the dendrimer-deoxycholate complex. These new cross-peaks are attributed to either inter- or intramolecular NOE interactions of deoxycholate. It is rational that the intramolecular NOE interactions cause the presence of these cross-peaks because the strong hydrophobic interactions between the hydrophobic pockets of dendrimer and steroidal core of deoxycholate can result in a bent structure of the bile salt to reduce its size and increase the hydrophobic interaction strength in the congested and nonpolar microenvironment of a dendrimer pocket. In the case of the G5/CHAPS complex at the same molar concentration, no cross-peaks were observed between dendrimer protons and the CHAPS protons in the NOESY spectrum (Figure 8). This phenomenon can be explained by the molecular property

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Figure 7. The 1H-1H NOESY spectrum of free sodium deoxycholate (2 mg sodium deoxycholate dissolved in 1 mL D2O) under the same experiment conditions as used in the NOESY spectrum of the G5 dendrimer-sodium deoxycholate complex in Figure 5.

Figure 6. Cross sections (∼a-d) taken at the position of the protons (∼a-d) of the dendrimer along the ω1 from the 1H-1H NOESY spectrum of the G5/sodium deoxycholate/D2O solution.

of the CHAPS. Although CHAPS and sodium deoxycholate possess the same steroidal core, the size of CHAPS (614 Da) is much larger than that of deoxycholate (392 Da), which may exceed the size encapsulation limit of a G5 dendrimer. In addition, the longer hydrophilic tails with zwitterion in the chain

significantly increase the hydrophilic property of CHAPS. It is well-known that the interior pocket of a PAMAM dendrimer is relatively nonpolar, which may reduce the encapsulation of polar guests. Therefore, the encapsulation of bile salts by dendrimers is size-dependent and hydrophobic property-dependent. In our previous studies, the size of a drug molecule or a surfactant monomer did not show a size-dependent encapsulation/ electrostatic binding behavior because the molecular weight of the guests range in the size of 100-300 Da.16,18 In this study, completely different interaction models between dendrimers and bile salts were deduced from the NMR studies. The use of bile salts with increased size and hydrophobic property as guests of dendrimers exhibited distinct host-guest behaviors compared with previous systems. 3.3. Size of the Dendrimer-bile Salt Complex by DOSY Analysis. Diffusion experiments can be used to evaluate the average hydrodynamic size of a complex or aggregate.70 The popularity of diffusion NMR has been boosted by the development of DOSY, in which the components of a mixture are separated by different diffusion coefficients (D) and the data can be displayed as a pseudo-2D NMR spectrum with chemical shifts and diffusion coefficient in two dimensions, respectively.52 From DOSY analysis, it is possible to clearly understand the interaction model deduced from 1H NMR titration experiments and NOESY analysis.35,70 A typical DOSY spectrum for G5 dendrimer-deoxycholate is shown in Figure 9. A rapid chemical exchange between the free and bound states of deoxycholate is observed. Compared with the DOSY spectrum of free G5 dendrimer (Figure S4 in the Supporting Information), the diffusion coefficient of dendrimer is slightly increased in the presence of deoxycholate. Dendrimers as well as dendrimer/ bile salt aggregates can be regarded as spherical molecules and complexes. The size of the dendrimer and aggregate can be estimated from the Stokes-Einstein equation (D ) kBT/6πηrH; kB is the Boltzmann constant, T is the absolute temperature, η is the solution viscosity, and rH is the hydrodynamic radius of the molecule or complex). Considering the solution viscosity is approximately the same for pure dendrimer and dendrimer-bile

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Figure 8. The 1H-1H NOESY spectra of G5 dendrimer/CHAPS/D2O solution (2 mg of G5 dendrimer and 3.1 mg of CHAPS dissolved in 1 mL of D2O).

Figure 9. 2D-DOSY spectrum of the G5-sodium deoxycholate complex. The four signals on the top line represent lgD for the G5 dendrimer; the signals below represent the weighted average of lgD for the free deoxycholate molecule and the bound deoxycholate.

salt solution,52 the increase in the diffusion coefficient for the dendrimer upon the addition of bile salt means that the size of the G5 dendrimer-deoxycholate complex is smaller than that of the G5 dendrimer in solution.64 This is in good accordance with our 1H NMR titration and NOESY results that deoxycholate localizes only in the interior pockets of dendrimers. Strong hydrophobic interactions between the bile salt and dendrimer scaffolds in the interior of dendrimer can decrease the hydrodynamic diameter of dendrimer in solutions, which was previously reported by Astruc et al.64

On the basis of the 1H NMR titration, NOESY, and DOSY results, we establish a new model of dendrimer-bile salt aggregates and provide new insights into the host-guest chemistry of dendrimer-drug/surfactant complexes. The nature and structure of dendrimer-bile salt complexes are essential for the analysis of the fate of dendrimers in physiological conditions, such as circulating blood, the design of new classes of bile salt sorbents for therapeutic purposes,60,61 and the understanding of host behaviors of dendrimers in different host-guest systems.

Interactions of Dendrimers, Bioactive Surfactants 4. Conclusions For the first time, the interactions between dendrimers and bioactive surfactants such as bile salts were investigated. The NMR analysis has shown that the bile salts localize only in the interior pockets of dendrimers. The aggregation formation between dendrimers and bile salts is driven by hydrophobic interaction and hydrogen-bond interaction. The hydroxyl groups and the methyl groups in bile salts play an important role in dendrimer-bile salt interactions. The encapsulation of bile salts by dendrimers is guest size-dependent and hydrophobic propertydependent. The formation of dendrimer-bile salt inclusions decreased the dendrimer size due to strong hydrophobic interactions between dendrimer scaffolds and the steroidal core of the bile salts. The NMR studies of the dendrimer-bile salt system provide new insight into the interactions between dendrimers and bioactive surfactants. In our future works, the precise localization of surfactants such as bile salts in dendrimer pockets will be analyzed by a combination of dynamic simulations and NMR techniques. Acknowledgment. We thank the Natural Science Foundation of Anhui Province (No. 070413112) and the Innovation Foundation from Hefei National Laboratory for Physical Sciences at Microscale (C07-06) for financial support of this program. Supporting Information Available: Additional 1H NMR, NOESY, and DOSY spectra. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Funasaki, N.; Fukuba, M.; Kitagawa, T.; Nomura, M.; Ishikawa, S.; Hirota, S.; Neya, S. J. Phys. Chem. B 2004, 108, 438. (2) Cabral, D. J.; Hamilton, J. A.; Small, D. M. J. Lipid Res. 1986, 27, 334. (3) Coello, A.; Meijide, F.; Nunez, E. R.; Tato, J. V. J. Pharm. Sci. 1996, 85, 9. (4) Lee, Y. K.; Nam, J. H.; Shin, H. C.; Byun, Y. Circulation 2001, 104, 3116. (5) Jana, P. K.; Moulik, S. P. J. Phys. Chem. 1991, 95, 9525. (6) Tomalia, D. A. Prog. Polym. Sci. 2005, 30, 294. (7) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M. G. K.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117. (8) Svenson, S. Eur. J. Pharm. Biopharm. 2009, 71, 445. (9) Svenson, S.; Tomalia, D. A. AdV. Drug DeliVery ReV. 2005, 57, 2106. (10) Cheng, Y. Y.; Xu, Z. H.; Ma, M. L.; Xu, T. W. J. Pharm. Sci. 2008, 97, 123. (11) Cheng, Y. Y.; Wang, J. R.; Rao, T. L.; He, X. X.; Xu, T. W. Front. Biosci. 2008, 13, 1447. (12) Cheng, Y. Y.; Xu, T. W. Eur. J. Med. Chem. 2008, 43, 2291. (13) Svenson, S.; Chauhan, A. S. Nanomedicine 2008, 3, 679. (14) D’Emanuele, A.; Jevprasesphant, R.; Penny, J.; Attwood, D. J. Controlled Release 2004, 95, 447. (15) Najlah, M.; Freeman, S.; Attwood, D.; D’Emanuele, A. Int. J. Pharm. 2006, 308, 175. (16) Cheng, Y. Y.; Wu, Q. L.; Li, Y. W.; Xu, T. W. J. Phys. Chem. B 2008, 112, 8884. (17) Cheng, Y. Y.; Li, Y. W.; Wu, Q. L.; Zhang, J. H.; Xu, T. W. Eur. J. Med. Chem. 2009, 44, 2219. (18) Cheng, Y. Y.; Li, Y. W.; Wu, Q. L.; Xu, T. W. J. Phys. Chem. B 2008, 112, 12674. (19) Reinhold, F.; Kolb, U.; Lieberwirth, I.; Grohn, F. Langmuir 2009, 25, 1345. (20) D’Emanuele, A.; Attwood, D. AdV. Drug DeliVery ReV. 2005, 57, 2147. (21) Morgan, M. T.; Nakanishi, Y.; Kroll, D. J.; Griset, A. P.; Carnahan, M. A.; Wathier, M.; Oberlies, N. H.; Manikumar, G.; Wani, M. C.; Grinstaff, M. W. Cancer Res. 2006, 66, 11913. (22) Duncan, R.; Izzo, L. AdV. Drug DeliVery ReV. 2005, 57, 2215. (23) Boas, U.; Heegaard, P. M. H. Chem. Soc. ReV. 2004, 33, 43. (24) Gupta, U.; Agashe, H. B.; Asthana, A.; Jain, N. K. Biomacromolecules 2006, 7, 649. (25) Chauhan, A. S.; Jain, N. K.; Diwan, P. V.; Khopade, A. J. J. Drug Targeting 2004, 12, 575. (26) Dufes, C.; Uchegbu, I. F.; Schatzlein, A. G. AdV. Drug DeliVery ReV. 2005, 57, 2177. (27) Gillies, E. R.; Frechet, J. M. J. Drug DiscoVery Today 2005, 10, 35.

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