Probing Paeonol−Pluronic Polymer Interactions by 1H NMR

Oct 25, 2007 - ... of Pluronic micelles for drug delivery after the shell-crosslinking. Changes in self-diffusion coefficients of paeonol with varying...
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J. Phys. Chem. B 2007, 111, 13371-13378

13371

Probing Paeonol-Pluronic Polymer Interactions by 1H NMR Spectroscopy Jun-he Ma,† Chen Guo,*,† Ya-lin Tang,‡ Hong Zhang,‡ and Hui-zhou Liu*,† Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, Beijing 100080, People’s Republic of China, and Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China ReceiVed: July 25, 2007; In Final Form: September 12, 2007

By using a combination of 1H NMR spectroscopy, two-dimensional heteronuclear single-quantum coherenceresolved 1H{13C} and homonuclear rotating-frame Overhauser enhancement NMR correlation experiments with diffusion ordered spectroscopy (DOSY), the location and distribution of a hydrophobic drug, paeonol, have been established with respect to the methyl groups of the poly(ethylene oxide)-poly(propylene oxide) -poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer. The interaction between them is adjustable according to the different temperature-dependent hydrophilicities or hydrophobicities of the triblock copolymer components. On the other hand, such interactions influence the self-assembly properties of the block copolymer amphiphiles in solution. The amount of anhydrous methyl groups of PPO segments shows an increase with increasing paeonol concentration. It was also demonstrated that the shell-crosslinking of the Pluronic polymer has an effect in increasing the amount of anhydrous methyl groups and thus increasing the hydrophobicity of Pluronic micelles. This might be the deeper reason underlying the increase in drug-loading capacity and prolongation in release time of Pluronic micelles for drug delivery after the shell-crosslinking. Changes in self-diffusion coefficients of paeonol with varying copolymer concentrations and types were also determined by the diffusion-based NMR DOSY technique, and values of Ka, ∆G, and n were calculated.

Introduction In recent years, the exploration of polymeric micelles as drug delivery vehicles has attracted considerable interest.1-14 The properties that make polymeric micelles advantageous for drug delivery applications include the fact that they can be made of polymers that are biocompatible and/or biodegradable and that they have a small size and can incorporate and release poorly water soluble, hydrophobic, and/or highly toxic compounds. Among the numerous kinds of polymeric micelles, the poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers (Pluronic), in which PPO acts as the hydrophobic core and PEO as the corona, have attracted much attention.15-22 Recently, a great effort has been made to investigate the Pluronic micelles for neuroleptic targeting and cytotoxic drug delivery23,24 as well as finding ways to modify the molecular structure of PEO-PPO-PEO block copolymers for improving their drug delivery ability.25-28 A most important aspect in using and improving Pluronic micelles as microcontainers for drug delivery is to understand the molecular-level interactions between the micelle and the drug. However, to date, there have been few studies on micelledrug interactions, and the fundamental mechanisms at the molecular level remain unclear.29 NMR spectroscopy has been demonstrated to be a powerful tool in studying the interaction of small molecules with polymers in solution.30 The pulsed field gradient (PFG)-based diffusion NMR techniques, such as affinity NMR, which has shown that * Corresponding authors. Phone: +86-10-62555005; fax: +86-1062554264; e-mail: (C.G.) [email protected] or (H.-z.L.) hzliu@ home.ipe.ac.cn. † Graduate School of Chinese Academy of Sciences. ‡ Institute of Chemistry, Chinese Academy of Sciences.

the diffusion coefficient of a small molecule binding with a receptor in solution is significantly different from the small compound alone observed under PFG conditions,31,32 and the NMR pumping, which allows for unambiguous detection of ligands that bind to a macromolecule,33,34 have recently opened new possibilities for the analysis of interactions in complex systems. PFG-NMR spectroscopy, as a reliable method for obtaining diffusion coefficients of molecules or complexes in solution, can provide information on the interactions between a particular molecule and the components of a solution through the analysis of the changes of the diffusion coefficient measured for that molecule, and much detailed information about intermolecular interactions in solution can thus be obtained by highresolution PFG-NMR experiments.35 The popularity of PFGNMR has been boosted by the development of diffusion ordered spectroscopy (DOSY), in which the components of a mixture are separated as a function of their respective diffusion coefficients36 and the data can be displayed as a pseudo-2-D NMR spectrum with chemical shifts in one dimension and diffusion coefficients in the other. Applications of DOSY include the analysis of polymer mixtures,37 affinity/binding studies,38 and the study of chemical exchange.35 Recently, DOSY has been applied to the study of the solubilizing behavior of drugs in small-molecule micelle systems.39 The poorly water soluble drug, paeonol (2-hydroxyl-4methoxyacetophone, see schematic structure in Figure 1), has been used as a tranquillizer and an anti-hypertensive drug. It tends to exhibit significant analgesic, anti-pyretic, and antibacterial properties as well as use in the treatment of reperfusioninduced myocardium damage.40,41 All of these features of paeonol make it quite meaningful to investigate the interaction between paeonol and Pluronic micelles to develop a paeonol-

10.1021/jp075853t CCC: $37.00 © 2007 American Chemical Society Published on Web 10/25/2007

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Figure 1. 1-D 1H NMR spectra of paeonol in DMSO recorded at 37 °C.

TABLE 1: Composition of PEO-PPO-PEO Block Copolymers polymer

molecular mass

PPO segment mass

no. of PO units

no. of EO units

PPO/PEO

P103 P105 F127 F108

4950 6500 12600 14600

3465 3250 3780 3250

60 56 69 56

2 × 17 2 × 37 2 × 99 2 × 132

1.79 0.76 0.35 0.19

contained Pluronic formulation for further intravenous administration in the body. Thus, paeonol was chosen as a promising candidate for measuring and controlling the distribution of drug molecules in aqueous PEO-PPO-PEO solutions. This paper presents a comprehensive application of NMR techniques, including 1H NMR spectroscopy, diffusion-based NMR DOSY, as well as NMR relaxation in studying the paeonol-Pluronic micelles interaction, the purpose of which is to reveal the molecular mechanism underlying the drugloading and controlled release characters of paeonol in crosslinked and uncrosslinked Pluronic micelles. Experimental Procedures Materials. The PEO-PPO-PEO triblock copolymers were obtained as a gift from BASF. The molecular mass and compositions of these Pluronic polymers are listed in Table 1. Dess-Martin periodinane, 1,4-diaminobutane (DAB), 2,2dimethyl-2-silapentane-5-sulfonate sodium salt (DSS, g97%), and paeonol (g99%) were purchased from Aldrich Chemical Corp. Deuterated dimethyl sulfoxide (DMSO, g99.9 atom % 2H) and deuterated water (D O, g99.9 atom % 2H) were 2 purchased from CIL Corp. All chemicals were used as received. The shell-crosslinked P103 was synthesized according to a method described previously,28 and the detailed preparation and purification process of shell-crosslinked P103 are in the Supporting Information. Sample Preparation. The paeonol-containing samples were prepared by dissolving appropriate amount of polymers and paeonol in D2O solution with gentle agitation, and then the solutions were transferred to 5 mm NMR sample tubes that were sealed immediately with laboratory film. After 15 min of sonication to remove dissolved paramagnetic dioxygen, the sample tubes were stored in a refrigerator before use. NMR Experiments. NMR experiments were conducted on a Bruker Avance 600 spectrometer equipped with a micropro-

cessor-controlled gradient unit and an inverse-detection multinuclear BBI probe with an actively shielded z-gradient coil. The sample temperature was kept constant within (0.1 °C by the use of a Bruker BCU-05 temperature control unit. A 90° pulse calibration was performed for each new sample for DOSY experiments. Pulse sequence from a bipolar pulse pair double stimulated echo pulse sequence (BPPDSTE) was used to avoid convection problems.42 The pulse sequences included a 5 ms delay to allow residual eddy currents to decay. Sine-shaped gradient pulses were utilized to further minimize eddy currents. The pulse gradient duration was chosen for each diffusion time to obtain the minimum residual signal for each component at the maximum gradient strength. The pulse gradients were incremented from 2 to 95% of the maximum gradient strength in a linear ramp (32 steps). The diffusion coefficients were obtained from a single- or double-exponential nonlinear least-squares fitting of the echo attenuation decay. Peak intensities were monitored for all diffusion analyses. Diffusion coefficients were obtained by fitting eq 1 to the results, where K ) γgδ, γ is the gyromagnetic ratio, g is the gradient strength, δ is the gradient pulse width, D is the diffusion coefficient, and ∆ is the diffusion time during which the diffusion is being monitored

I(K) ) I(K ) 0) exp[-K2D(∆ - δ/3)]

(1)

The 1H NMR spectra were measured by a procedure as described elsewhere.43 The spin-lattice relaxation times, T1, were measured by the standard 180:-τ-90°: pulse sequence. At least 16 different delay times were used for each set of measurements. The experiments were repeated 3 times with standard deviations smaller than 0.04 (s). UV-vis Measurements. UV-vis absorption spectra were recorded on a PerkinElmer UV-vis Lambda Bio 40 spectrophotometer with a resolution of 2 nm.

Probing Paeonol-Pluronic Polymer Interactions

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Figure 2. 1H NMR spectra of Pluronic-P103 in the presence of paeonol with molar concentration ratios of paeonol ([Pd]) to P103 ([Ps]) of (a) 0.625, (b) 2.5, and (c) 10, respectively. Spectra were recorded in D2O solution at various temperatures and local expanded regions of the PO-CH3 signals as shown.

Results and Discussion Localization of Paeonol-Pluronic Micelles Interaction Sites: Effect of Paeonol-P103 Interactions on PPO. Changes of the 1H NMR spectra of the PO-CH3 groups with varying molar concentration ratios of paeonol ([Pd]) to P103 ([Ps]) are shown in Figure 2. For aqueous Pluronic solutions with lower [Pd]/[Ps] ratios (Figure 2a, [Pd]/[Ps] ) 0.625), one main signal, ∼1.17 ppm, corresponding to the PO-CH3 protons was observed. Increasing the [Pd]/[Ps] ratio, a new resonance signal, ∼0.93 ppm (see Figure 2b,c), appeared upfield. In fact, this new signal was small at lower [Pd]/[Ps] ratios (see Figure 2a at 9 °C) but became larger and well-resolved at higher [Pd]/[Ps] ratios. Interestingly, this drug-induced new resonance also exhibited a strong temperaturedependent character. It grew progressively larger at the expense of the original signal at ∼1.17 ppm and shifted downfield with increasing temperature. The emergence of this new resonance indicates that the hydrophobic drug molecules have a strong influence on the spin states of the PO methyl protons. A similar phenomenon has been observed by FTIR spectroscopy22 in that the C-H symmetric deformation vibration of the methyl groups of Pluronic polymers in water splits into two components, which are assignable to hydrated and anhydrous methyl species. It can thus be deduced that the two peaks of the PO-CH3 groups of P103 are possibly correlated to the two different states: one is the hydrated state corresponding to the peak around 1.17 ppm (surrounded by water), and the other is the anhydrous state associated with the peak around 0.93 ppm. The possible reason for this phenomenon may be due to the lower chemical exchange rate between the two states at lower temperatures, which will result in two well-resolved signals on the NMR time scale. But, the chemical exchange rate between the two states becomes fast with increasing temperature, which makes the two separate signals finally unresolved at higher temperatures.

Figure 3. Temperature-dependent percentage of hydrated and anhydrous methyl groups of P103 in D2O solution obtained from NMR proton spectra of Figure 2b,c by sum fitting of Lorentz functions. Dotted lines represent the percentage of total integrated peak area of methyl groups at various temperatures to overall integrated peak area at 25 °C. Arrows denote the CMT.

With the assumption of separate hydrated and anhydrous signals, we can determine the fraction of the anhydrous PPO methyl groups since the areas under the resonances are proportional to the amount of methyl protons in the respective states. The areas were determined by fitting a sum of Lorentz functions to the signals and then calculating the relative area percentage under each signal. The results obtained from the NMR proton spectra in Figure 2b,c are presented in Figure 3 as a function of temperature. It can be seen from this figure that the critical micellization temperature (CMT) can be determined from the first inflection of the intersection of two

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Figure 4. Temperature-dependent chemical shifts of hydrated and anhydrous methyl groups of P103 in D2O solution in the presence of paeonol. Data were obtained from NMR proton spectra as displayed in Figure 2c. Arrows denote the CMT.

tangent lines in the percentage of hydrated or anhydrous methyl groups versus temperature plots. As is seen, the proportion of the anhydrous PO methyl groups increased with an increasing temperature, whereas that of the hydrated PO methyl groups exhibited a reverse phenomenon. However, even as high as 50 °C, there was still ca. 5 to ∼10% of the PO methyl groups remaining in the hydrated state. It was also observed that the proportion of the anhydrous PO methyl groups increased with the [Pd]/[Ps] ratio. At the same time, it was found that the overall amount of the anhydrous or hydrated methyl protons remained almost constant when referenced to the integral values of the PO methyl groups at 25 °C (all in the micellar state), suggesting that the changes of the anhydrous and hydrated methyl groups only occurred between the two states themselves, that is to say, the increase of the amount of the anhydrous methyl protons mainly came from the dehydration of the hydrated methyl protons with increasing temperature. Since the driving force for the drug molecules entering the core of the micelles is the hydrophobic effect, and it is also known that the microenvironment around the hydrated methyl groups is hydrophilic (polar) while that around the anhydrous methyl groups is hydrophobic (nonpolar), it is easy to predict that the binding affinity between hydrophobic drug and block copolymer micelles will increase with increasing temperature as the amount of anhydrous methyl groups increases with temperature. The temperature-dependent chemical shifts of hydrated and anhydrous methyl groups of P103 obtained from Figure 2c were plotted in Figure 4. The CMT can also be determined from the first inflection of the chemical shift of anhydrous methyl groups versus the temperature plot. As shown in this figure, the hydrated methyl groups were shifted upfield on increasing the temperature and then showed a nearly linear relationship (in ppm) with temperature after a certain temperature around CMT was reached. However, the anhydrous methyl groups showed a significantly downfield shift, and then the chemical shifts became independent of temperature. It is known that the chemical shifts of NMR resonances are sensitive to the chemical nature of the surroundings of the spins and that the upfield shift could usually be correlated with the formation of the hydrophobic microenvironment.44 Therefore, for the hydrated methyl groups, the small upfield shift can be easily attributed to the dehydration with increasing temperature. But, for the anhydrous methyl groups, things are a little different. Because the

Figure 5. 1H NMR spectra of the PO-CH3 groups of 1% (w/v) uncrosslinked P103 micelles (a) and crosslinked P103 micelles (b) in D2O solution at various temperatures.

increasing amount of the anhydrous methyl groups mainly comes from the hydrated methyl groups, the apparently downfield shift of the anhydrous methyl signal is virtually the result of an upfield shift of the hydrated methyl groups with increasing temperature. The constant chemical shift of the anhydrous methyl at higher temperatures above CMT thus suggests the quite stable chemical nature of the micellar core (mainly consisting of the anhydrous methyl groups). Effect of Shell-Crosslinking on PPO. Quite significantly, it was observed that the 1H resonance for the PO-CH3 groups also resolved into two well-defined peaks of approximately equal area with increasing temperature after the shell-crosslinking (see Figure 5b), which is quite similar to the effect of adding paeonol (see Figure 2b). The resolution of the peaks first increased and then decreased with increasing temperature. This type of behavior has not been previously reported for the simple surfactant systems. According to the previous discussion, it can be assumed that the shell-crosslinking of Pluronic-P103 clearly increased the proportion of the anhydrous PO-CH3 groups at the same condition. We also compared the drug-loading capacity and controlled release of the crosslinked and uncrosslinked P103 micelles (see Figures 2 and 3 in the Supporting Information). It was shown that the drug-loading capacity was increased and that the release time was prolonged after shell-crosslinking, which definitely demonstrates that the shell-crosslinking increased the hydrophobicity and stability of the Pluronic micelles. Effect of Paeonol-P103 Interactions on Paeonol. To investigate the effect of paeonol-P103 interactions on paeonol, the temperature-dependent chemical shifts (δ) of the H-6 of paeonol with varying molar concentration ratios of paeonol ([Pd]) to P103 ([Ps]) are presented in Figure 6a. It was observed that the chemical shifts for all the protons of paeonol (H-5, H-3, and H-7, see Figure S4 in the Supporting Information) exhibit a similar changing trend with increasing temperatures or varying [Pd]/[Ps] ratios. The chemical shift for pure paeonol in D2O shows an increase in ppm values with temperature, but decreases

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Figure 6. Temperature-dependent chemical shifts of the H-6 of paeonol (a) (see Figure 1) and residual HDO signal (b) with varying molar concentration ratios of paeonol ([Pd]) to P103 ([Ps]). The open signal symbol (0) represents the temperature-dependent chemical shifts of pure paeonol in D2O solution.

Figure 7. 2-D HSQC 1H{13C} NMR spectra (a) and 2-D rotating frame nuclear Overhauser effect spectra (ROESY) (b) for 1% P103 in 10 mM paeonol solution at 15 and 37 °C, respectively. Local expanded regions of the PO-CH3 groups as shown.

significantly with increasing the relative concentration of Pluronic micelles. At lower [Pd]/[Ps] ratios, the paeonol protons signals first shift toward downfield and then exhibit an opposite upfield shift when the temperature is above a certain value around CMT. It is generally accepted that the inclusion of a species in a highly hydrophobic environment would result in an upfield shift of the affected protons.45 Therefore, the upfield shift of paeonol indicates that the paeonol molecules might have been trapped in the micellar core. In addition, the penetration of the paeonol into the core of the micelles is clearly temperature-dependent. However, the chemical shift of residual HDO seems to remain unaffected with the increasing addition of

paeonol over the entire temperature range investigated (see Figure 6b), which otherwise validates that the hydrophobic paeonol molecules interact directly with the block copolymer and have no effect on the water structure. Direct Spectral Evidence for Paeonol-P103 Interaction Sites. To locate the paeonol-P103 interaction sites more unambiguously, 2-D 1H{13C} HSQC and ROE spectra for 1% P103 in a 10 mM paeonol solution were measured and are presented in Figure 7a,b, respectively. As shown in Figure 7a, the 13C chemical shift of the anhydrous methyl groups is located ∼2 ppm more downfield than that of hydrated methyl groups, but the 13C chemical shifts for both states together shift

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Figure 8. 2-D DOSY 1H spectra of 10 mM paeonol in D2O (a) and in 10% (w/v) P103 (b) solutions at 37 °C.

Ma et al. of ROESY is inversely proportional to the spacial distance between different nuclei only when the sample is a real solution. But, at 37 °C, the microphase separation has occurred and the complex of anhydrous methyl groups and drugs is in a state of out of water, which may significantly reduce the intensity of the crosspeaks. Determination of Binding Constants (Ka) and Stoichiometry Coefficients (n) Measured by NMR DOSY Spectroscopy. A typical DOSY spectrum displayed for a paeonol-P103 system at 37 °C is shown in Figure 8. The signals of neat paeonol in D2O were labeled by line a, which appeared at a single diffusion coefficient with a high value of ∼10-9 m2 s-1, whereas, in the 10% (w/v) Pluronic-P103 solution (the P103 molecules are mainly in the micellar state16), the effective diffusion coefficients of paeonol labeled by line b decreased by more than one magnitude to ∼10-10 m2 s-1, but the diffusion coefficient of paeonol is still higher than that of P103 micelles, which is less than 10-11 m2 s-1. These results indicate that there is a rapid chemical exchange between the free and the bound states of paeonol molecules on the diffusion time scale (∆). The apparent diffusion coefficient (Dobs) thus reflects the average of the diffusion coefficients of two exchanging species, free (Dfree) and bound (Dbound), as shown in eq 2

Dobs ) (1 - Xfree)Dbound + XfreeDfree

Figure 9. Changes observed in diffusion coefficient (Dobs) of paeonol (10 mM) by varying the copolymer concentration at 37 °C. Solid lines represent the estimated first-order exponential decay curves.

downfield with increasing temperature. Since the 13C NMR chemical shift can provide a measure of the ensemble average of conformers46 and the downfield shift is usually interpreted as an increase in the trans/gauche ratio of the C-C bonds,47,48 it can be deduced that the anhydrous methyl groups remain in a more stretched (trans) conformation and that this nonpolar conformation of the anhydrous methyl groups will probably favor the interaction with the hydrophobic drug molecules. The ROESY spectra for the same sample show that there exist strong positive NOEs between the paeonol protons and the anhydrous methyl groups of P103 (see Figure 7b). As an excellent method for observing dipole-dipole cross-relaxation between nuclei,49 the ROE results present obvious evidence that that the paeonol molecules interact directly with the anhydrous methyl groups of P103 and that this interaction site seems to be unchanged with increasing temperature. However, we noticed that the intensity of the ROESY crosspeaks is much stronger at a lower temperature (15 °C, below the CMT) than that at a higher temperature (37 °C, evidently above the CMT), which may be different from our expentations. This is because the intensity

(2)

In a titration experiment, the receptor and ligand concentration ratios are generally varied either by changing both concentrations or by only changing the receptor or the ligand concentration while holding the other concentration constant.50,51 In the case presented here, only the concentrations of Pluronic polymers were varied. The Dobs of paeonol in different Pluronic solutions, including F108, F127, P105, P103, and the shell-crosslinked P103, are shown in Figure 9. The results show that the Dobs of paeonol decreased significantly with increasing polymer concentration, indicating the decrease of the fraction of paeonol molecules in the free state. The Dfree of paeonol (Table 2) was measured for 10 mM paeonol in D2O solution alone, and the Dbound was then deduced from the y-intercept in Figure 9 when the self-diffusion coefficients were constant following the firstorder exponential decay. According to the model for first-order reversible fast exchange, and assuming n independent and identical sites, a mathematical model was then achieved (eq 3),52 where Ka is the binding constant and [Ps] and [Pd], respectively, represent the total concentrations of the Pluronic micelles and the drug. The stoichiometry coefficient (n) was considered to be a 1:n binding stoichiometry between the Pluronics and the drug, in which n is the average number of drug molecules on each Pluronic molecule. The values of Ka and n were obtained by nonlinear curve fitting of eqs 2 and 3 to the experimental data of Dobs and [Ps]. The corresponding free energy of association (∆G) was also calculated from the values of Ka

xfree ) 1 -

n[Ps] + [Pd] - 1/Ka 2[Pd]

x(

+

)

n[Ps] + [Pd] - 1/Ka 2[Pd]

2

-n

[Ps] [Pd]

(3)

As shown in Table 2, Ka and n of paeonol for a series of Pluronic polymers increased with the increasing PPO/PEO ratio. For an increase in the proportion of the hydrophobic PPO groups in the polymer architecture, there was a corresponding decrease in the free energy of association, suggesting that the hydrophobic

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TABLE 2: Kinetic and Thermodynamic Parametersa Estimated by NMR DOSY Experiments for Paeonol Binding to Various Pluronic Polymers at 37 °C paenol (P)

(Dfree × 1010) ( SD (m2/s)

P(free) P + F108 P + F127 P + P105 P + P103 P + cross P103

10.01 ( 0.03

a

(Dbound × 1010) ( SD (m2s-1)

Ka ( SD (M-1)

∆G ( SD (kcal M-1)

n

2.6 ( 0.05 1.8 ( 0.1 1.3 ( 0.2 1.2 ( 0.2 0.87 ( 0.03

120 ( 15 160 ( 20 190 ( 10 260 ( 15 320 ( 20

-12.34 ( 0.02 -13.08 ( 0.03 -13.53 ( 0.01 -14.23 ( 0.02 -15.10 ( 0.02

10 ( 3 15 ( 4 18 ( 2 24 ( 2 30 ( 2

Means of duplicate results.

TABLE 3: 1H NMR Longitudinal Relaxation Times (s) of 10 mM Paeonol in 10% (w/v) Different Pluronic Polymer Solutions at 37 °C paenol (P)

H-3

H-4

H-5

H-6

H-7

P(free) P + F108 P + F127 P + P105 P + P103 P + cross P103

8.98 1.50 1.43 1.30 1.23 0.653

2.88 0.877 0.829 0.789 0.754 0.623

6.08 1.33 1.31 1.17 1.14 0.899

4.05 1.16 1.19 1.06 1.03 0.902

3.66 1.08 1.02 0.933 0.925 0.703

interactions are predominant in the association of paeonol with Pluronic polymers. Of the five block copolymers studied, the crosslinked P103 had the highest binding affinity (320 M-1), followed by Pluronic-P103 (260 M-1), the most hydrophobic one among the four uncrosslinked Pluronic polymers studied. Yang et al. reported that the crosslinked L121 micelles exhibited an approximately a 10-fold lower cmc as compared to the uncrosslinked ones.28 In our study, we observed that the CMT of 1% P103 in D2O solution decreased from about 22 to 18 °C after shell-crosslinking (see Figure S5 in the Supporting Information). This indicates that the crosslinked Pluronic polymer would aggregate into micelles more easily as compared to the uncrosslinked one at the same condition. The average number of paeonols binding to each Pluronic molecule was found to range from 10 to 30, much smaller than the number of PO units in the molecular architectures studied (∼60), indicating that not all but only part of PO groups bind with the paeonol. Of course, the binding constant and the absolute values of thermodynamic parameters presented here may be different from the real values because the parameters were calculated under the assumption that the Pluronic molecules are all in a micellar state and that no interactions occur between the Pluronic or the paeonol molecules themselves, although in reality, this assumption is not true, as a particular amount of monomer coexists with the micelle and the interaction between Pluronic and paeonol themselves will also be inevitable, especially for the very lipophilic paeonol serving as the ligand. However, it is necessary to calculate Ka for Pluronic molecules, which can give an insight into their contribution to overall binding. The proton longitudinal relaxation times T1 values determined from five distinct 1H peaks of paeonol in D2O solution are presented in Table 3. In bulk paeonol solution, the T1 values for paeonol protons are in the range of 3 to ∼9 s, whereas the T1 values were significantly shortened with the addition of Pluronic micelles. This is consistent with the results reported for Pluronic-F6830 and other high MW surfactants53 with the assumption that the rotational reorientation time of a singlesurfactant molecule lies near the crossover from the extremenarrowing limit to the slow-motion limit. T1 values usually can be used to indicate the local viscosity of micellar domains;54 therefore, the dispersed paeonol molecules in the micelles would experience a greater local viscosity and thus have longer reorientation times and shorter T1 values. It was also observed

that the more hydrophobic the polymer, the shorter the T1 values for drug molecules. Conclusion We have incorporated 1H NMR spectroscopy, 2-D 1H{13C} HSQC, and ROE spectroscopy with diffusion-based NMR DOSY and NMR relaxation techniques to characterize the interaction between paeonol and Pluronic copolymer species. At low temperatured, the two separate resonance signals, which were, respectively, deduced to be associated with hydrated and anhydrous states, for the PO methyl groups were observed when adding paeonol. The anhydrous PO methyl signal grew larger with increasing the mole concentration ratios of paeonol to Pluronic polymers, indicating that the paeonol molecules probably replaced the water molecules around the PO methyl groups and thus induced the formation of the anhydrous PO methyl. The relative peak proportion of the anhydrous PO methyl signal increased with increasing temperature at the expense of the hydrated methyl groups, indicating that increasing the temperature facilitates the dehydration of the PO methyl groups, whereas the two resolved signals became unresolved at higher temperature, suggesting that the chemical exchange rate between the hydrated and the anhydrous states of the PO methyl groups becomes faster at higher temperatures. The temperature-dependent chemical shifts of paeonol in ppm values decrease significantly while those of the HDO signal remain constant with increasing Pluronic concentration, indicating that paeonol molecules could have been encapsulated into the hydrophobic micellar core and interact directly with the micelles. The HSQC results indicate that the anhydrous methyl groups are in a comparative trans conformation and thus have more nonpolar character than the hydrated ones, which would favor the hydrophobic interaction between paeonol and anhydrous methyl groups. The ROE results clearly show that paeonol only interacts with the anhydrous methyl groups. It can thus be concluded that the interaction between the drug molecules and the anhydrous PO-CH3 groups of Pluronic polymers could be the molecular-level reason underlying the solubilization of the hydrophobic drug into the micelles. Therefore, controlling the proportion of the anhydrous methyl groups would be the key factor to reconstruct the Pluronic micelles, which will greatly improve our knowledge on the interaction of drug and Pluronic polymers at a molecular level. The emergence of the anhydrous methyl signal after the shellcrosslinking of the Pluronic polymer indicated that the shellcrosslinking increased the amount of the anhydrous methyl groups and thus resulted in an increase in the hydrophobicity of the Pluronic micelles. Both the increased drug-loading capacity and the prolonged releasing time of Pluronic micelles after the shell-crosslinking indicate an increase in the hydrophobicity of Pluronic micelles. The spin-lattice relaxation time measurements also indicated that the microviscosity of the micellar core greatly increased after the shell-crosslinking.

13378 J. Phys. Chem. B, Vol. 111, No. 47, 2007 The calculated values of Ka, ∆G, and n from the changes in self-diffusion coefficients of paeonol further validated the binding site and also indicated that the binding affinity between drug and micelles increased with the increase of the hydrophobicity of the polymer. Acknowledgment. This work was financially supported by the National Natural Science Foundation of China (20221603, 20676137, and 20490200) and the National High Technology Research and Development Program of China (863 Program) (20060102Z2049). Note Added after ASAP Publication. This article was published ASAP on October 25, 2007. The abstract was revised. The corrected version was reposted on October 31, 2007. Supporting Information Available: 1H NMR spectrum of shell-crosslinked P103; drug-loading and release curves of paeonol in crosslinked and uncrosslinked P103 solutions; temperature-dependent chemical shifts for paeonol with varying P103 molar concentrations; and CMT determination of P103 after shell-crosslinking. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113-131. (2) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science (Washington, DC, U.S.) 1994, 263, 16001603. (3) Bromberg, L.; Magner, E. Langmuir 1999, 15, 6792-6798. (4) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C. M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science (Washington, DC, U.S.) 1999, 284, 1143-1146. (5) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107-6113. (6) Gao, Z.; Lukyanov, A. N.; Singhal, A.; Torchilin, V. P. Nano Lett. 2002, 2, 979-982. (7) Westerlund, F.; Wilhelmsson, L. M.; Norden, B.; Lincoln, P. J. Am. Chem. Soc. 2003, 125, 3773-3779. (8) Kim, E. J.; Shah, D. O. J. Phys. Chem. B 2003, 107, 8689-8693. (9) Lim Soo, P.; Luo, L.; Maysinger, D.; Eisenberg, A. Langmuir 2002, 18, 9996-10004. (10) Qu, X.; Khutoryanskiy, V. V.; Stewart, A.; Rahman, S.; Papahadjopoulos-Sternberg, B.; Dufes, C.; McCarthy, D.; Wilson, C. G.; Lyons, R.; Carter, K. C.; Schatzlein, A.; Uchegbu, I. F. Biomacromolecules 2006, 7, 3452-3459. (11) Cinteza, L. O.; Ohulchanskyy, T. Y.; Sahoo, Y.; Bergey, E. J.; Pandey, R. K.; Prasad, P. N. Mol. Pharmaceutics 2006, 3, 415-423. (12) Barbosa, L. R. S.; Caetano, W.; Itri, R.; Homem-de-Mello, P.; Santiago, P. S.; Tabak, M. J. Phys. Chem. B 2006, 110, 13086-13093. (13) Ristori, S.; Maggiulli, C.; Appell, J.; Marchionni, G.; Martini, G. J. Phys. Chem. B 1997, 101, 4155-4165. (14) Southall, N. T.; Dill, K. A.; Haymet, A. D. J. J. Phys. Chem. B 2002, 106, 521-533. (15) Yang, P. D.; Wirnsberger, G.; Huang, H. C.; Cordero, S. R.; McGehee, M. D.; Scott, B.; Deng, T.; Whitesides, G. M.; Chmelka, B. F.; Buratto, S. K.; Stucky, G. D. Science (Washington, DC, U.S.) 2000, 287, 465-467. (16) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414-2425. (17) Wanka, G.; Hoffmann, H.; Ulbricht, W. Macromolecules 1994, 27, 4145-4159. (18) Zhou, Z. K.; Chu, B. Macromolecules 1988, 21, 2548-2554. (19) Brown, W.; Schillen, K.; Almgren, M.; Hvidt, S.; Bahadur, P. J. Phys. Chem. 1991, 95, 1850-1858.

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