Stimuli Response of Cystamine-Core Dendrimer Revealed by

ARTICLE pubs.acs.org/JPCB

Stimuli Response of Cystamine-Core Dendrimer Revealed by Diffusion and NOE NMR Studies Xueyan Feng,‡ Yiyun Cheng,*,† Qinglin Wu,† Jiahai Zhang,§ and Tongwen Xu*,‡ †

School of Life Sciences, East China Normal University, Shanghai, 200062, People’s Republic of China CAS Key Laboratory of Soft Matter Chemistry, School of Chemistry and Material Science, §Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230026 and 230027, People’s Republic of China

‡

bS Supporting Information ABSTRACT: Stimuli response behaviors of cystamine-core dendrimer in the presence of several reducing agents including vitamin C, sodium bisulfite, and DL-Dithiothreitol are described. A competitive redox cleavage and supramolecular aggregate formation model is proposed based on PFG NMR and 1H NMR titration experiments. Furthermore, reduction-responsive release of guest molecules from interior pockets of the cystaminecore dendrimer is confirmed by NOE studies. The results suggest that cystamine-core dendrimer is a versatile scaffold or precursor in the design of reduction-sensitive polymeric nanocapsules for biomedical purposes.

1. INTRODUCTION Stimuli responsive polymers are intelligent materials that respond with dramatic property changes to specific environmental changes (pH, ionic strength, temperature, reduction potential, ultraviolet or infrared light, and enzyme).1
3 These smart polymers have gained increasing interest especially in the field of controlled and self-regulated drug and gene delivery systems in the past decade.4,5 Because of the difference in redox potential between the oxidizing extracellular and the reducing intracellular environments, reduction-sensitive polymers are preferred for intracellular triggered drug delivery rather than other stimuli-responsive polymers.6
8 These polymers usually contain disulfide linkages that are stable in blood circulation systems and extracellular media, and degraded through thiol-disulfide exchange reactions at a time scale from minutes to hours.7,9 Dendrimers are hyperbranched polymers with spherical or ellipsoidal shapes, extremely low polydispersity, and well-defined molecular weights and numbers of surface functionalities.10
14 Bioactive compounds can either be encapsulated in the hydrophobic pockets of dendrimer through hydrophobic interactions or attached to the globular surface via ionic interactions or covalent conjugates.15
21 These physicochemical properties give dendrimers the ability to act as versatile scaffolds in the design of multifunctional drug delivery systems.12,22
30 Cystamine-core poly(amidoamine) (PAMAM) dendrimers with reduction-sensitive property were first synthesized by Tomalia and co-workers in 2003.31 The dendrimer may exhibit a highly sensitive and rapid response to reducing agents due to the fact that only one disulfide bond in the macromolecular structure should be reduced to r 2011 American Chemical Society

cleave the dendrimer. The combination of dendritic structure and reduction-sensitive property in cystamine-core dendrimer makes it a versatile precursor in the design of reduction-sensitive delivery systems. In addition, the thiol-core dendrons produced by the reduction of cystamine-core dendrimers were widely used to dendronize gold nanoparticles,32 iron oxide nanoparticles,33 quantum dots,32,34 biotin,35,36 peptide,37 and DNA38,39 for biomedical purposes. Therefore, the redox responsive behavior of the cystamine-core dendrimer is of great importance in understanding the physicochemical properties of reduction-sensitive dendrimers and the design of reducing agent triggered polymeric drug delivery systems. Here we use NMR techniques including pulsed-field gradient (PFG) NMR, 1H NMR titration, and Nuclear Overhauser enhancement (NOE) to monitor the redox reactions between sulfide linkages in the central core of dendrimer and reducing agents including vitamin C (VC), sodium bisulfite (NaHSO3), and DLDithiothreitol (DTT).

2. EXPERIMENTAL SECTION 2.1. Materials. Generation 3 (G3) cystamine-core and amineterminated PAMAM dendrimer was purchased from SigmaAldrich Co. (St. Louis, MO). The dendrimer stored in methanol was distilled to remove the solvent, resulting in a white gel. Dendrimer stock solutions (7.14  10
4 M) were prepared by Received: January 13, 2011 Revised: February 24, 2011 Published: March 18, 2011 3777

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The Journal of Physical Chemistry B dissolving the white gel into deuterated water (D2O) for NMR studies. Vitamin C (VC) and DL-Dithiothreitol (DTT) were gifts from School of Life Sciences, University of Science and Technology of China. Sodium deoxycholate was obtained from Shanghai BBI Co. Ltd. (Shanghai, China). Sodium bisulfite (NaHSO3) and 1,

Figure 1. Diffusion coefficients of G3 cystamine-core PAMAM dendrimer in the presence of different reducing agents as a function of reducing agent/ dendrimer molar ratio, (a) VC, (b) NaHSO3, and (c) DTT.

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4-dioxane (99.99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1,4-Dioxane was used as an internal standard used in the 1H NMR titration and PFG NMR studies. The NaHSO3, VC, and DTT solutions were freshly prepared before NMR studies. Deuterated solvents were obtained from Beijing Chongxi High-Tech Incubator Co. (Beijing, China). 2.2. 1H NMR Titration. 1H NMR titration experiments were conducted on a Bruker Avance 500.132 MHz instrument at 298.2 ( 0.2 K. The molar equivalent of reducing agents to G3 dendrimer in the NMR samples ranges from 0 to 2 (1
9: 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2). G3 dendrimer concentration in the 1H NMR titration experiments is fixed at 1.08  10
4 M. 2.3. PFG NMR Studies. Diffusion behaviors of dendrimer in the samples are measured by PFG NMR studies.40 1,4-Dioxane was used as an internal standard to monitor the variations of viscosity of the samples. The PFG experiments were performed using a standard Bruker pulse program on the same NMR instrument at 298.2 ( 0.2 K. The heating and cooling unit was switched on to reach and stabilize a desired temperature, which avoids the influence of temperature variation on diffusion measurement. The time interval (Δ) between gradient pulses was chosen as 600 ms, while the duration of the gradient pulses (δ) was 3 ms. The pulse gradients (g) were increased from 10%

Scheme 1. Proposed Redox Cleavage and Aggregate Models for Systems Containing Reduction-Sensitive Dendrimer and Reducing Agents (VC, NaHSO3, and DTT)

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Figure 2. Expanded region of the 1H NMR spectra for G3 cystamine-core PAMAM dendrimer/reducing agent/D2O solution, (a) VC, and (b) NaHSO3, the ratios of reducing agent/dendrimer in 1H NMR 1
9 are 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, and 2.

to 80% of the maximum gradient strength (50 G/cm) in 8 steps to attenuate the spin
echo signal. The self-diffusion coefficient (D) was obtained by fitting the spin
echo signal and gradient strength by the following equation (Figure S1 of the Supporting Information, SI): In ¼ I0 exp½
γ2 Dδ2 ðΔ
δ=3Þg 2 

ð1Þ

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). The diffusion coefficient of a particle can be converted into the hydrodynamic radius (rs) of the particle by Stokes
Einstein eq 2: D¼

kT 6πηrs

ð2Þ

3. RESULTS AND DISCUSSION

where k is the Boltzmann constant, T is the systematic temperature, and η is the viscosity of the solution. Therefore, the hydrodynamic radius of the dendritic nanostructure can be calculated using eq 3: rs ¼ r0  10ðlgD0
lgDs Þ

dioxane, 2.12 Å) and D0 and Ds are self-diffusion coefficients of 1, 4-dioxane and dendrimer, respectively. 2.4. 2D-NOESY Studies. 2D-NOESY spectra of G3 cystamine-core PAMAM dendrimer/sodium deoxycholate complexes in the absence and presence of VC were recorded on a 600.13 MHz Bruker NMR instrument. The concentration of G3 dendrimer was kept constant at 2.86  10
4 M and sodium deoxycholate at a concentration of 4.58  10
3 M. The molar ratio of VC/G3 cystamine-core dendrimer is 0.5. The NOESY experiments were performed with a 1.5 s relaxation delay, 205 ms acquisition time, 11.4 μs 1H 90
pulse width, and 300 ms mixing time. Sixteen transients were averaged for 1024 complex t1 points. All of the data were processed with NMRpipe software on a Linux workstation with standard Lorentz
Gauss window function and zero-filling in both dimensions.41,42

ð3Þ

where r0 is the hydrodynamic radius of the internal standard (1,4-

3.1. Stimuli Response Behaviors of Cystamine-Core PAMAM Dendrimer in the Presence of Different Reducing Agents. NMR techniques have been demonstrated to be facile,

powerful, sensitive, and noninvasive tools in the qualitative and quantitative analysis of chemical reactions and intra/intermolecular interactions.20,43,44 PFG NMR relies on the fact that the diffusion coefficient of a target decreases if it aggregates into 3779

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Figure 3. Expanded region of the 1H
1H NOESY spectra of cystamine-core G3 PAMAM dendrimer/sodium deoxycholate/D2O solution (2.86  10
4 M G3 dendrimer and 4.58  10
3 M sodium deoxycholate in 500 μL D2O) in the absence (a) and presence (c) of VC (VC/dendrimer molar ratio is 0.5) at a mixing time of 300 ms, showing much weaker NOE cross-peaks between the protons of deoxycholate and dendrimer after the addition of reducing agents. The cross-peaks between dendrimer and deoxycholate are indicated by labels.

Scheme 2. Molecular Structure of Sodium Deoxycholate with Atom Labeling

larger species or increases if it cleaves into smaller compounds,40,41 thereby allowing us to characterize the hydrodynamic size of the cystamine-core dendrimer and investigate the possibility of aggregate formation during the redox reactions. Figure 1a shows diffusion coefficients of G3 cystamine-core PAMAM dendrimer with increasing VC concentrations. Diffusion coefficient of the cystamine-core

dendrimer increased from 1.15  10
10 to 1.63  10
10 m2/s and radius of the dendrimer decreases from 2.0 to 1.4 nm (Figure S2 of the SI) when 0.5 mol equiv of VC were added into the dendrimer solution. The dramatic decrease in dendrimer size reveals the cleavage of disulfide linkages at the central core of dendrimers (Scheme 1a). Since VC is an intracellular nutrient that protects cells from oxidant stress, the responsive characteristic of cystamine-core dendrimer to VC indicates its promising use as a versatile scaffold in the design of intracellular triggered delivery systems. Surprisingly, the dendrimer size gradually reverts to its original value (∼2.0 nm) in the absence of VC as the concentration of reducing agent increases from 0.5 to 1 mol equiv. The VC molecule has several hydroxyl groups that can act as hydrogen-bond donors and mediate the assembly of two thiol-core dendrons through hydrogen-bond interactions41,45 (Scheme S1 of the SI). Upfield shifts of dendrimer surface methylene protons were observed in 1H NMR titrations of cystamine-core dendrimer with VC (Figure 2a and Scheme S2 of the SI), confirming the critical role of hydrogen-bond interactions in the formation of aggregates.40,46 The competitive effect of redox cleavage and hydrogen-bond mediated aggregate explains the appearance of a second maximum diffusion coefficient peak at 1.5 mol equiv of VC. The diffusion coefficient of the dendrimer in the presence of 2 mol equiv of VC (1.15  10
10 m2/s) is very close to the value of the G3 3780

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Scheme 3. Reducing Agent-Induced Release of Sodium Deoxycholate from the Interior Pockets of Cystamine-Core PAMAM Dendrimer

cystamine-core dendrimer in D2O, suggesting the formation of stable aggregates. Therefore, the conformation of the reduction-sensitive dendrimer changes from “cystamine-core and amine-surface” to “VC-core and thiol-surface” after the addition of more VC molecules (Scheme 1a). In the case of NaHSO3, the diffusion coefficient of G3 dendrimer exhibits a significant increase until the addition of 1 mol equiv of NaHSO3 and a decrease in the range of 1 to 1.75 mol equiv of NaHSO3 (Figure 1b). The postponed appearance of the first maximum diffusion coefficient peak is attributed to the much weaker reducing ability of NaHSO3 than VC. The slight increase of the diffusion rate of the dendrimer at 2 mol equiv of NaHSO3 reveals that redox reaction is still active at this stage and that redox reaction and aggregate formation together contribute to the observed diffusion coefficient. Supramolecular aggregate is formed on the basis that SO32
and SO42
ions in the system act as junction bridges between amine-terminated dendrimers or dendrons via ionic interactions. Downfield shift of the surface methylene protons in the 1H NMR spectra of cystamine-core dendrimer titrated with NaHSO3 (Figure 2b) validates the ionic interaction-mediated molecular assembly model as proposed in Scheme 1b.46 The fast exchange behavior of ionic interaction driven aggregates (G3/NaHSO3) causes a much higher observed diffusion coefficient than that of hydrogen-bond driven assemblies (G3/VC). The reducing ability of DTT is between that of VC and NaHSO3 (the maximum diffusion coefficient appears at 0.75 mol equiv of DTT). The diffusion behaviors of dendrimer with increasing DTT are more complicated in Figure 1c and the second maximum diffusion coefficient peak appears at 1.25
1.5 mol equiv of DTT (Scheme 1c). Redox splitting is predominant in the presence of 1.25 and 1.5 molar equivalents of DTT, while supramolecular aggregates play important roles at 1 and 1.75 mol equiv of DTT. Slight shifts of the surface protons of dendrimer after the addition of DTT in Figure S3 of the SI

further confirm the formation of supramolecular aggregates between DTT and dendrimer. 3.2. Reduction-Sensitive Release of Guest Molecules Encapsulated in Cystamine-Core PAMAM Dendrimers. In order to confirm the potential of cystamine-core PAMAM dendrimer as a reduction-sensitive polymeric scaffold for drug delivery, we determined the release behavior of sodium deoxycholate from cystamine-core dendrimer in the absence and presence of VC. NOE and 1H NMR titration analysis in our previous studies have demonstrated that sodium deoxycholate localizes in the interior pockets rather than on the surface of PAMAM dendrimers, which is helpful for us to analyze the stimuli-responsive behavior of dendrimer.45 NOE analysis reveals spatial distances between specific protons in a molecule or a complex of molecules.42,47,48 The intensity of cross-peaks between two types of protons depends on the proton distance and the number of protons (ÆNOEæ ≈ N 3 r
6, ÆNOEæ is the intensity of the cross-peak in the NOE spectroscopy, N is the number of equivalent nuclei, and r is the spatial distance between the protons).20,43,49,50 Therefore, the decrease of cross-peak intensity between a guest molecule and the dendrimer scaffold can be recognized as the escape of guests from the dendritic cage.17,20,45 As shown in Figure 3a, strong NOE interactions are observed between cystamine-core PAMAM dendrimer (protons a
d, Scheme S2 of the SI) and sodium deoxycholate (protons 1, 4b, 5b, 6, 15, 16b, 22a, 22b, 23b, Scheme 2). After the addition of 0.5 mol equiv of VC into the dendrimer/sodium deoxycholate inclusion, most of the crosspeaks between protons (b, d) of dendrimer and protons of deoxycholate disappeared (Figure 3b), also, significant decreases in the intensities of cross-peaks between protons (a, c) of dendrimer and protons (1, 4b, 5b, 6, 16b, 22a, 22b) of deoxycholate were observed, confirming a burst release of deoxycholate molecules from dendritic matrix in the presence of VC molecules. As demonstrated in PFG studies, cystamine-core 3781

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4. CONCLUSIONS In summary, cystamine-core dendrimer exhibits a reductioncleavable characteristic in the presence of VC, DTT, and NaHSO3. A competitive model including reducing cleavage and hydrogen-bond/ ionic interaction mediated cross-linking was proposed from the PFG NMR and 1H NMR titration results. The cleavage of full generation dendrimers to thiol-core dendrons after drug/gene release may maintain the high payload efficiency and reduce the toxicity issue of high generation dendrimers. The modifiable surface and wellestablished bioconjugate chemistry of PAMAM dendrimer allow us to design a satisfactory nanodevice that can exclude the interferential stimuli in complicated in vivo circulation and secretion systems. Although the reduction-sensitive dendrimer investigated here remains far from practical use, its stimuli-responsive behavior is helpful for the design of smart polymers for drug delivery and disease diagnosis, i.e., ox-red sensors for living cell imaging based on the surface engineered reduction-sensitive dendrimer. Besides, NMR techniques have been proved to be powerful tools in the investigation of stimuli-responsive polymeric systems and provided new insights into the physicochemical properties of cystamine-core dendrimers. ’ ASSOCIATED CONTENT

bS

Supporting Information. Further information on the sizes of dendrimers in the presence of reducing agents calculated from diffusion NMR studies, and the 1H NMR titration spectra of the cystamine-core dendrimer by different reducing agents. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (Y.C.); [email protected] (T.X.).

’ ACKNOWLEDGMENT We are thankful for financial support including the Talent Program of East China Normal University (Grant No. 77202201), the “Dawn” Program of Shanghai Education Commission (Grant No. 10SG27), and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20093402110041) to this program. ’ REFERENCES (1) Jeong, B.; Gutowska, A. Trends Biotechnol. 2002, 20, 305. (2) Guo, D. S.; Chen, S.; Qian, H.; Zhang, H. Q.; Liu, Y. Chem. Commun. 2010, 46, 2620. (3) Fernandez-Trillo, F.; van Hest, J. C. M.; Thies, J. C.; Michon, T.; Weberskirch, R.; Cameron, N. R. Chem. Commun. 2008, 2230. (4) Du, F. S.; Wang, Y.; Zhang, R.; Li, Z. C. Soft Matter 2010, 6, 835. (5) Bawa, P.; Pillay, V.; Choonara, Y. E.; du Toit, L. C. Biomed. Mater. 2009, 4, 022001.

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