Interaction between PEO–PPO–PEO Copolymers and a Hexapeptide

Dec 20, 2011 - Interaction between PEO–PPO–PEO copolymers and a hexapeptide, growth hormone releasing peptide-6 (GHRP-6), was investigated by NMR ...
0 downloads 0 Views 813KB Size
Download PDF
Article pubs.acs.org/Langmuir

Interaction between PEO−PPO−PEO Copolymers and a Hexapeptide in Aqueous Solutions Lianwei Jia,†,‡ Chen Guo,*,† Junfeng Xiang,§ Ning Wang,†,‡ Liangrong Yang,† Yalin Tang,§ and Huizhou Liu*,† †

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People's Republic of China ‡ Graduate University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China § Beijing National Laboratory for Molecular Sciences, Center for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China S Supporting Information *

ABSTRACT: Interaction between PEO−PPO−PEO copolymers and a hexapeptide, growth hormone releasing peptide-6 (GHRP-6), was investigated by NMR to study the potential use of the copolymers in peptide drug delivery. 1H NMR and nuclear Overhauser effect spectroscopy (NOESY) measurements determined that PO methyl protons interacted with methyl protons of the Ala moiety, aromatic protons of the Trp moiety, and some of the Phe aromatic protons. The Lys moiety and part of the Phe moiety entered the hydrophilic EO environment via hydrogen bonding. PEO−PPO−PEO copolymers and the peptide formed a complex in 1:1 stoichiometry. Binding constants between copolymers and GHRP-6 were determined, and it was indicated that the copolymers containing more EO and PO units will lead to greater affinity with the peptide. Isothermal titration calorimetry (ITC) measurements confirmed the results of NMR experiments. This study indicates that PEO−PPO−PEO copolymers have great potential in delivering peptide drugs.

■

INTRODUCTION Many proteins and peptides are promising anticancer drugs because of their incredible selectivity and low side effects.1−3 The structure and conformation of proteins and peptides have a close relationship to their biological activities. Bioavailability of these drugs, however, suffers severely from degradation via enzymatic hydrolysis, precipitation, inefficient cell entry, absorption onto nontarget sites, and some other factors.4,5 Many approaches have been developed for prolonging the biological activity and improving the bioavailability of the peptide and protein drugs. PEGylation, employing poly(ethylene glycol) (PEG) as a modifying polymer, plays an important role in enhancing the potentials of peptides and proteins as therapeutic agents.6−9 Liposomes, vesicles formed by concentric spherical phospholipid bilayers, are used to incorporate the therapeutic agents into their inner aqueous compartment.4,10 Polymeric hydrogels11−14 and micelles,9,15,16 which are more controllable and stable polymeric delivery systems, effectively protect the vulnerable biodrugs from hostile environments and control the release of the drugs. Among numerous polymer products, poly(ethylene oxide)− poly(propylene oxide)−poly(ethylene oxide) (PEO−PPO− PEO) block copolymers, commercially known as Pluronic or Poloxamer, have been widely examined in experimental medicine and pharmaceutical sciences.17−21 Composed of hydrophilic ethylene oxide and hydrophobic propylene oxide units, the amphiphilic copolymers self-assemble into micelles © 2011 American Chemical Society

with their concentrations exceeding critical micelle concentration (CMC) or the temperature above critical micelle temperature (CMT).22 The biocompatible EO shell effectively prevents the micelles from undesired interactions with proteins and protects the core through steric stabilization.15 The hydrophobic core, formed by PO units, is the “microreservoir” to incorporate water-insoluble drugs.17,21,23 Incorporation into Pluronic micelles increases solubility and stability of the drugs. Moreover, it improves the pharmacokinetics and biodistribution of the drugs, enhancing their accumulation in the tumors.17 Besides, growing evidence shows that Pluronic block copolymers possess biological modifying properties, such as sensitizing multidrug-resistant cancer (MDR) to various anticancer agents and enhancing drug transport across cellular barriers.18,21,24,25 The applications of PEO−PPO−PEO copolymers in pharmaceutical formulations are mainly focused on delivering and controlling release of hydrophobic drugs. There are, however, few reports on their delivery of hydrophilic peptide and protein drugs. The PEO−PPO−PEO copolymers have the potential to interact with peptides and be used to delivery peptide drugs. PO units of the copolymers can interact with hydrophobic moieties of peptides, such as aromatic and methyl Received: September 21, 2011 Revised: November 18, 2011 Published: December 20, 2011 1725

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

Scheme 1. Structure of GHRP-6

redissolved in D2O at a phosphate concentration of 10 mM. The buffer was used to prepare stock solutions of GHRP-6 and PEO− PPO−PEO copolymers. The solutions used for the Job plot measurements were prepared from separate stock solutions of 2 mM PEO−PPO−PEO copolymer and 2 mM GHRP-6. The mole fraction of GHRP-6 ranged from 0 to 1, while the total concentration of PEO− PPO−PEO copolymer plus GHRP-6 was kept at 2 mM for each solution. Solutions for determination of binding constants contained 2 mM GHRP-6 and various concentrations of PEO−PPO−PEO copolymers. Solutions for determination of changes in amide protons were prepared by solving GHRP-6, PEO−PPO−PEO copolymers, poly(propylene glycol), and poly(ethylene glycol) in H2O/D2O (90:10) buffer solutions. NMR Experiments. NMR experiments were conducted on a Bruker Avance 600 spectrometer equipped with a microprocessorcontrolled gradient unit and an inverse-detection multinuclear BBI probe with an actively shielded z-gradient coil. The temperature is controlled by a Bruker BVT 3200 temperature control unit, which has an accuracy of ±0.1 °C. 1H−13C HSQC, COSY, and TOCSY experiments were carried out for the assignment of GHRP-6 protons. For all the experiments, the samples were equilibrated at the desired temperature for at least 10 min before measurement. DSS−D2O solution was sealed in a capillary which was added into the sample solutions to eliminate temperature-induced shifts as a reference. In the Job plot measurements, the temperatures were regulated to keep the copolymers in the micelle region (310 K for P84, 298 K for P103 and F127) and in the unimer region (298 K for P84, 290 K for P103, and 293 K for F127).22 A diffusion-ordered spectroscopy (DOSY) experiment was performed by using Hahn spin−echo-based PFG pulse sequence.36 The pulse gradients were incremented from 2% to 95% of the maximum gradient strength in a linear ramp (32 steps). Interaction sites were determined by a nuclear Overhauser effect spectroscopy (NOESY) experiment. Isothermal Titration Microcalorimetry (ITC). The calorimetric measurements were conducted with a TAM 2277-201 microcalorimetric system (Thermometric AB, Järfälla, Sweden) with a stainless steel sample cell of 1 mL at 298.15 ± 0.01 K. The cell was initially loaded with 0.6 mL of PBS buffer or PEO−PPO−PEO copolymers in PBS buffer (1 mM). GHRP-6 solutions (15 mM) were injected into the sample cell via a 500 μL Hamilton syringe controlled by a 612 Thermometric Lund pump. The system was stirred at 50 rpm with a gold propeller. The observed enthalpy (ΔHobs) was obtained by integration over the peak for each injection in the plot of heat flow P against time t.

protons, through hydrophobic interaction; EO units can interact with hydrophilic parts of peptides, such as the amide bond, through hydrogen bonding. Recent studies reported that PEO−PPO−PEO copolymers can entrap nisin (a 34 amino acid peptide) and reduced glutathione by the EO units.26,27 Therefore, it is necessary to investigate interaction between PEO−PPO−PEO copolymers and peptides. Furthermore, a systematic investigation of the interaction between PEO− PPO−PEO copolymers and peptides concerns not only applications of the copolymers in pharmaceutical and biological fields but also their applications in coating,26 separation of biomolecules,28,29 and designing hierarchically ordered nanostructures through peptide−polymer systems.30,31 In this paper, growth hormone releasing peptide-6 (GHRP-6) is used as a model peptide to investigate the interaction of PEO− PPO−PEO copolymers with peptide drugs. Growth hormone releasing peptide-6 is a synthetic hexapeptide that stimulates growth hormone release in vivo and in vitro.32,33 It is also reported to play a neuroprotective role and has the potential use for treating neurodegenerative diseases.34,35 Interaction between PEO−PPO− PEO copolymers and GHRP-6, including interaction site, stoichiometry, and association affinity, is studied using NMR methods.

■

MATERIALS AND METHODS

Materials. Growth hormone releasing peptide-6 (GHRP-6) acetate (99%) with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was purchased from TASH Biotechnology Co. Ltd. (Shanghai, China). Scheme 1 shows the structure of the peptide. PEO−PPO−PEO block copolymers were kindly donated by BASF. The average composition and molecular weight of the copolymers were determined by the manufacturer (list in Table 1). Poly(ethylene glycol) (Mw ≈ 4000) and

Table 1. Composition of PEO−PPO−PEO Block Copolymers and Their Binding Constants with GHRP-6

polymer

molecular weight

average no. of PO units

P84 P103 F127

4200 4950 12600

43 60 65

a

average no. of EO units

Ka (M−1)a

Ka (M−1)b

2 × 19 2 × 17 2 × 100

70.7 ± 1.1 80.6 ± 1.2 374.0 ± 1.1

42.0 ± 1.2 409.1 ± 1.1 1076.5 ± 1.1

■

Calculated from the data of H13. bCalculated from the data of H14.

RESULTS AND DISCUSSION

Influence of PEO−PPO−PEO Copolymer on GHRP-6. The structure determination and signal assignment of GHRP-6 were conducted by COSY, TOCSY, and 1H−13C HSQC measurements (see Figures S1−S3 in Supporting Information).

poly(propylene glycol) (Mw ≈ 1000) were purchased from Alfa Aesar. Deuterated water was bought from CIL Corp. Sample Preparation. A buffer was prepared by dissolving monobasic and dibasic phosphate in D2O and then lyophilized and 1726

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

Figure 1. 1H NMR spectra of GHRP-6 in D2O (upper) and P103 solution (down).

Figure 2. 1H NMR spectra of 2 mM GHRP-6 in various concentrations of P84.

Figure 3. Chemical shift changes of some protons in GHRP-6 with an increase in the concentration of (A) the peptide and (B) P84.

protons in Phe (H14−H18) and Lys (H19−H23) moieties shift linearly upfield, while protons in His (H1−H4) and Ala (H12 and H13) moieties show downfield shifts. When different concentrations of P84 are added to GHRP-6 solutions, H14, H15, H16, and Lys protons (H19−H23) obviously demonstrate a downfield shift, while the chemical shifts of other protons show the same trend as those with increased peptide concentration.

Figure 1 gives 1H NMR spectra of GHRP-6 in D2O and P103 solutions. 1H NMR spectral changes of GHRP-6 with various concentrations of peptide are shown in Figure S4 (Supporting Information), and 1H NMR spectral changes of GHRP-6 with various concentrations of P84 are shown in Figure 2. Chemical shift changes of some protons are demonstrated in Figure 3. When increasing the peptide’s concentration from 1 mM to 10 mM, aromatic protons in Trp (H8−H11) and 1727

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

The chemical shift changes of GHRP-6 with increasing concentration are attributed to the aggregate of the peptide.37,38 The downfield shift of H14, H15, H16 and Lys protons (H19−H23) indicate that these protons enter a more hydrophilic environment with the addition of P84. The shifts of other protons suggest that the peptide aggregates upon the addition of copolymer. The formation of copolymer−peptide complex was studied by pulsed field-gradient (PFG) NMR spectroscopy. This method, also referred to as diffusion-ordered spectroscopy (DOSY), is a powerful tool for studying molecular aggregation and interaction.39−41 The basis of this method relies on the differences in the self-diffusion coefficient of diffusing species; the molecule in aggregate or bound state diffuses slower than that in free state.39 In Figure 4, the diffusion coefficient of

Figure 5. NOESY spectrum of GHRP-6 in F127 solution at 298 K. The correlated region of PO−CH3 and H13 protons is shown.

environment. It is deduced that PO−CH3 protons interact with these protons via hydrophobic interaction, and the interaction with H13 is the strongest among them. Because PO−CH3 groups stay at the inner space of the micelle, 43,44 there should be some interaction between EO units and the hydrophilic part of the peptide. From the NOESY spectrum (see Figure S5), the cross-peak between EO−CH2− protons and H23 is observed, though its intensity is weak. In Figure 6, NHd shifts slightly upfield with an elevation in peptide concentration. However, it obviously shifts downfield with addition of P84 and poly(ethylene glycol) (see Figure S6 in Supporting Information), and it changes little upon the addition of poly(propylene glycol). It is inferred that NHd forms hydrogen bonds with EO units. As H14, H15, H16, and protons in Lys moieties shift downfield with increasing concentration of the copolymers, it is suggested that H14, H15, H16, and the Lys moiety enter the hydrophilic EO environment. Hydrogen bonding is responsible for their interaction with EO units, and the hydrogen bonds formed with EO units occur at NH2 adjacent to H23 and the amide group near H14. The PO units are reported to enable the copolymers to bind to hydrophobic sites on biopolymer molecules through hydrophobic interaction,17 and they are responsible for their interaction with and insertion into the lipid monolayer.45,46 Herein, PO methyl protons interact with methyl protons and some of the aromatic protons in GHRP-6, and the interaction with H13 is the strongest among them. PEO are reported to prevent the absorption of proteins.47−49 Recent studies, however, show that EO units entrap nisin (a 34 amino acid peptide)26 and reduced glutathione.27 Hydrogen bonding is responsible for the interaction of EO units with hydrophilic parts of peptides. In this study, 1H NMR and NOESY spectra infer that the Lys moiety and part of the Phe moiety interact with EO units via hydrogen bonding. Association Stoichiometry. The stoichiometry of the copolymer−peptide complex in the solutions is studied by the method of continuous variations. This method, often called Job’s method, keeps the sum of the total substrate and ligand

Figure 4. Diffusion-ordered spectrum (DOSY) of 5 mM GHRP-6 in D2O and in 5 mM F127 solutions at 298 K.

GHRP-6 decreases drastically in the presence of F127 (from about 2.7 × 10−10 m2/s in D2O solution to about 5.5 × 10−11 m2/s in F127 solution), confirming the formation of the copolymer−peptide complex. The broad range of F127 signals, in the top of Figure 4, is due to the polydispersity of PEO− PPO−PEO copolymers.42 Interaction Site Determination. The interaction sites between GHRP-6 and PEO−PPO−PEO copolymer were determined by nuclear Overhauser effect spectroscopy (NOESY) measurement. The NOESY spectrum provides distance information between pairs of hydrogen atoms separated by less than 0.5 nm. In Figure 5, the cross-peak for PO−CH3 protons and H13 is observed, showing that H13 protons are spatially close to PO−CH3 protons via hydrophobic interaction. Chemical shift changes of amide protons are shown in Figure 6. Amide protons on both sides of H13 (NHb and NHc) shift upfield with increasing P84, whereas they shift downfield with an elevation in peptide concentration. It is suggested that the hydrogen bonds in these amide groups break upon the addition of P84. This also confirms that the Ala moiety enters a more hydrophobic environment. In the NOESY spectrum (see Figure S5 in Supporting Information), cross-peaks between PO−CH3 protons and H17, H18 and aromatic protons are observed, although their intensities are weak. 1H NMR spectra demonstrate that H17, H18 and aromatic protons in the Trp moiety shift upfield upon the addition of P84, showing that they enter a more hydrophobic 1728

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

Figure 6. 1H NMR spectra of NH signals in GHRP-6 as a function of (A) the peptide concentration and (B) P84 concentration (GHRP-6 concentration is 2 mM). The spectra were recorded in H2O/D2O (90:10) solutions at 298 K.

Figure 7. Job plot derived from the chemical shift of H14 for the GHRP-6/copolymer titrations. (A) Copolymers in the micelle state. (B) copolymers in the unimer state.

complex at equilibrium. The observed chemical shifts (δobs) of ligand protons in the mixed solutions are the mole fraction weighted average of the shifts observed in free (δfree) and bound (δbound) states,51

concentrations constant and measures some property of which the value changes when the substrate and ligand form a complex.50,51 The maximum in the plot relates to the stoichiometry. Because signals near HDO signal were severely affected by it in H2O/D2O (90:10) solutions, experiments for association stoichiometry and binding constants were carried out in D2O solutions, and chemical shifts of H14 were monitored instead of NHd. In this study, the total concentration of PEO−PPO−PEO copolymer plus GHRP-6 was kept constant for each solution, while the mole fraction of GHRP-6 ranged from 0 to 1. The H13 signal becomes difficult to discriminate when the ratio of GHRP-6/copolymer (with a large amount of PO units) is too low from the overlap of the methyl protons, so the chemical shift of H14 is monitored as the GHRP-6 mole fraction is varied. In Figure 7, the resulting plots demonstrate a maximum at 0.5, indicating that GHRP-6 interacts with PEO−PPO−PEO copolymers according to a 1:1 stoichiometry, irrespective of the copolymers in the form of unimer or micelle. Binding Constants. The 1H NMR and DOSY spectra suggest that GHRP-6 is in fast exchange between free and bound states. In the 1:1 association model, the substrate (S) and ligand (L) are in equilibrium.

δobs = χfreeδfree + χboundδ bound where χfree and χbound are the mole fractions of ligand in the free state and bound state, respectively. In this study, the binding constants were obtained by using a WinEQNMR2 software,52 which utilized a nonlinear least-squares fitting on the observed chemical shift data. The fitting curves with the experimental data are shown in Figure 8. These fittings match the experiment data well, except for H14 with P84 (Figure 8B), which has a little deviation. H13 signal is overlapped by the PO−CH3 peak when the concentrations of P103 and F127 exceed 10 mM. The binding constants of GHRP-6 with P84, P103, and F127 are listed in Table 1. The Ka values from H13 and H14 both increase significantly from P84, P103 to F127. For P103 and F127, the binding constants from H14 are much larger than those from H13. This difference is related to the different interaction mechanisms on the two sites. For the Ka derived from H13, the value increases a little from P84 to P103 and increases significantly from 80.6 ± 1.2 M−1 of P103 to 374.0 ± 1.1 M−1 of F127. However, the number of PO units increases from 43 of P84 to 60 of P103, and to 65 of F127. In the case of H14, Ka demonstrates a salient increase from 42.0 ± 1.2 M−1 of P84 to 409.1 ± 1.1 M−1 of P103, whereas the number of EO units changes from 38 of P84 to 34 of P103. Binding constants for the peptide with poly(propylene glycol) (PPG 1000) and poly(ethylene glycol) (PEG 4000) are determined to be 30.7 ± 1.2 M−1 and 35.6 ± 1.1 M−1, which are smaller than the values

S + L ⇌ SL The binding constant, Ka, is defined as

Ka =

[SL] [S][L]

where [S] and [L] are concentrations of substrate and ligand at equilibrium, respectively, and [SL] is the concentration of 1729

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

Figure 8. Chemical shifts of H13 (A) and H14 (B) protons of GHRP-6 as a function of concentrations of the copolymers, 298 K. The concentration of GHRP-6 was constant at 2 mM in the solutions.

It shows that PEO−PPO−PEO copolymers could form a 1:1 complex with GHRP-6 in aqueous solutions. Moreover, the study indicates that copolymers with more EO and PO units (F127 for example) will lead to greater affinity with the peptide. PO units enable the copolymers to bind to hydrophobic sites of the peptide, and EO units form hydrogen bonds with hydrophilic moieties of peptide. It is reported that hydrogen bonding can preserve the integrity of biological structures.13 The critical micellization concentrations (CMC) of PEO− PPO−PEO copolymers at 37 °C range from millimole to micromole; for the copolymers with large amount of PO units, such as P123 and F127, the CMC values are on the scale of micromoles.17 This makes the copolymers stay as stable micelles against dilution when entering the body. Although the peptide forms a complex with monomeric Pluronics, the micelles can protect the peptide more effectively than the monomeric copolymers. Furthermore, the micelle systems offer long blood circulation time with slow release of the free drug in the body.17 The biological modifying properties make it one of the most potent drug targeting systems, as increasing evidence demonstrates that PEO−PPO−PEO copolymers are capable of enhancing drug transport across cellular barriers and sensitizing multidrug-resistant cells.18,21,24 The results of this study indicate that PEO−PPO−PEO copolymers can be a promising candidate for delivering and controlling release of peptide drugs, especially for the oligopeptides containing hydrophobic moieties. Unlike electrostatics interaction of ionic polymers with peptides, nonionic PEO−PPO−PEO copolymers interact with peptides via hydrophobic interaction and hydrogen bonding. For effective delivery of peptide drugs, these temperatureresponsive copolymers can be modified with functional groups to develop intelligent delivery systems, such as modifications with pH-responsive polymers,54−56 or anchored to magnetic nanoparticles, which can be targeted and controlled release by an external magnetic field.57 The interaction between copolymers and peptides can also be utilized to design hierarchically ordered nanostructures. Besides, the interaction between the copolymers and peptides has to be considered when PEO−PPO−PEO copolymers are used for coatings.

from PEO−PPO−PEO copolymers. These results infer that both EO and PO units contribute to the binding affinities of the two separate sites. Besides, copolymers with more EO and PO units lead to greater affinity with the peptide. ITC is a quantitative technique that directly measures the binding affinity, enthalpy changes (ΔH) and binding stoichiometry (n) of the interaction between molecules in solution.53 Figure 9 shows the variation of ΔHobs for GHRP-6

Figure 9. Calorimetric titration curves for GHRP-6 into PBS buffer and 1 mM solutions of PEO−PPO−PEO copolymers against the final concentration of GHRP-6 at 298.15 K.

titrated into PBS buffer and 1 mM solutions of PEO−PPO− PEO copolymers. From the calorimetric titration curves, it is obvious that the interaction of GHRP-6 with F127 is pronouncedly stronger than those with P84 and P103. This is in accordance with the result obtained from NMR measurement. The interaction enthalpy curve of peptide and F127 was fitted using the computer program of TAM (Digitam 4.1 from Thermometric AB). The fitted data show that the formation of the peptide−F127 complex is in an approximate 1:1 stoichiometry, and the binding constant is 426.3 ± 119.2 M−1. Considering that the different methods (microcalorimetry and NMR) might lead to the experimental deviation, the difference in binding constant values obtained from the two methods is acceptable. Former studies on PEO−PPO−PEO copolymers in the pharmaceutical field mainly focused on the delivery and controlled release of hydrophobic drugs. This study investigates the potential use of the copolymers in delivering peptide drugs.

■

CONCLUSION Interaction between PEO−PPO−PEO copolymers and GHRP-6 was investigated to study the potential use of PEO−PPO−PEO copolymers in peptide drug delivery. In 1H NMR spectra, H14, H15, H16, and protons in the Lys moiety demonstrate downfield shifts with increasing concentrations of PEO−PPO−PEO 1730

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

(11) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860−862. (12) Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur. J. Pharm. Biopharm. 2000, 50, 27−46. (13) Lee, K. Y.; Yuk, S. H. Prog. Polym. Sci. 2007, 32, 669−697. (14) Bromberg, L. E.; Ron, E. S. Adv. Drug Delivery Rev. 1998, 31, 197−221. (15) Haag, R. Angew. Chem., Int. Ed. 2004, 43, 278−282. (16) Peer, D.; Karp, J. M.; Hong, S.; FaroKhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotechnol. 2007, 2, 751−760. (17) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189−212. (18) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. Adv. Drug Delivery Rev. 2002, 54, 759−779. (19) Kakizawa, Y.; Kataoka, K. Adv. Drug Delivery Rev. 2002, 54, 203−222. (20) Kabanov, A.; Zhu, J. A.; Alakhov, V. Adv. Genet. 2005, 53, 231− 261. (21) Batrakova, E. V.; Kabanov, A. V. J. Controlled Release 2008, 130, 98−106. (22) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414−2425. (23) Ma, J. H.; Guo, C.; Tang, Y. L.; Zhang, H.; Liu, H. Z. J. Phys. Chem. B 2007, 111, 13371−13378. (24) Kabanov, A. V.; Alakhov, V. Y. Crit. Rev. Ther. Drug Carrier Syst. 2002, 19, 1−72. (25) Kabanov, A. V.; Batrakova, E. V.; Miller, D. W. Adv. Drug Delivery Rev. 2003, 55, 151−164. (26) Tai, Y. C.; Joshi, P.; McGuire, J.; Neff, J. A. J. Colloid Interface Sci. 2008, 322, 112−118. (27) Jia, L. W.; Guo, C.; Yang, L. R.; Xiang, J. F.; Tang, Y. L.; Liu, H. Z. J. Phys. Chem. B 2011, 115, 2228−2233. (28) Liang, D. H.; Chu, B. Electrophoresis 1998, 19, 2447−2453. (29) Svensson, M.; Berggren, K.; Veide, A.; Tjerneld, F. J. Chromatogr., A 1999, 839, 71−83. (30) Whitesides, G. M. Small 2005, 1, 172−179. (31) Hentschel, J.; Borner, H. G. J. Am. Chem. Soc. 2006, 128, 14142−14149. (32) Argente, J.; GarciaSegura, L. M.; Pozo, J.; Chowen, J. A. Horm. Res. 1996, 46, 155−159. (33) Ghigo, E.; Arvat, E.; Muccioli, G.; Camanni, F. Eur. J. Endocrinol. 1997, 136, 445−460. (34) Delgado-Rubin de Celix, A.; Chowen, J. A.; Argente, J.; Frago, L. M. J. Neurochem. 2006, 99, 839−849. (35) del Barco, D. G.; Perez-Saad, H.; Rodriguez, V.; Marin, J.; Falcon, V.; Martin, J.; Cibrian, D.; Berlanga, J. Neurotox. Res. 2011, 19, 195−209. (36) Price, W. S. Concepts Magn. Reson. 1998, 10, 197−237. (37) Zhang, S. G. Nat. Biotechnol. 2003, 21, 1171−1178. (38) Aoudia, M.; Rodgers, M. A. J. Langmuir 2005, 21, 10355− 10361. (39) Price, W. S. In Encyclopedia of nuclear magnetic resonance; Grant, D. M., Harris, R. K., Eds.; Wiley: New York, 2002; Vol. 9, pp 364−374. (40) Johnson, C. S. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, 203− 256. (41) Cohen, Y.; Avram, L.; Frish, L. Angew. Chem., Int. Ed. 2005, 44, 520−554. (42) Nilsson, M.; Hakansson, B.; Soderman, O.; Topgaard, D. Macromolecules 2007, 40, 8250−8258. (43) Mortensen, K.; Pedersen, J. S. Macromolecules 1993, 26, 805− 812. (44) Su, Y. L.; Wang, J.; Liu, H. Z. Macromolecules 2002, 35, 6426− 6431. (45) Frey, S. L.; Zhang, D.; Carignano, M. A.; Szleifer, I.; Lee, K. Y. C. J. Chem. Phys. 2007, 127. (46) Amado, E.; Kerth, A.; Blume, A.; Kressler, J. Langmuir 2008, 24, 10041−10053. (47) Amiji, M.; Park, K. Biomaterials 1992, 13, 682−692.

copolymers, while H17, H18, and aromatic protons in the Trp moiety shift upfield. NOESY measurement determines that PO methyl protons interact with H13, H17, H18, and Trp aromatic protons via hydrophobic interaction, and the interaction with H13 is the strongest among them. It is inferred that H14, H15, H16, and protons in the Lys moiety enter the hydrophilic EO environment via hydrogen bonding from NOESY and 1H NMR spectra. The formation of a peptide−copolymer complex is confirmed by DOSY experiment, and the complex is formed in 1:1 stoichiometry. The association constants of GHRP-6 with P84, P103, and F127 are determined from the chemical shift data. It is inferred that copolymers with more EO and PO units will lead to greater affinity with the peptide. ITC measurements confirm the results of NMR experiments. This study indicates that PEO−PPO−PEO copolymers are capable of interacting with oligopeptides, and they show great potential for delivering peptide drugs. Systematic investigation into interaction mechanism of PEO−PPO−PEO copolymers with peptides is necessary for their further applications.

■

ASSOCIATED CONTENT

S Supporting Information *

COSY, TOCSY, and 1H−13C HSQC spectra of GHRP-6 in D2O (Figures S1−S3), 1H NMR spectral changes of GHRP-6 with increasing peptide concentration (Figure S4), NOESY spectrum of GHRP-6 in P127 solution (Figure S5), 1H NMR spectra of NH protons in GHRP-6 with addition of PPO and PEG (Figure S6), and spectra obtained from ITC measurements (Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.G.) or [email protected] (H.Z.L.). Tel.: (86) 10 6264 2032. Fax: (86) 10 6255 4264.

■

ACKNOWLEDGMENTS This work is supported by the National Key Natural Science Foundation of China (No. 21136009), Innovative Research Group Science Fund (No. 20821092), and State Key Laboratory of Chemical Engineering (SKL-ChE-11A04). The authors thank Yuchun Han and Prof. Yilin Wang (Institute of Chemistry, Chinese Academy of Sciences) for the help with ITC experiments and data analysis.

■

REFERENCES

(1) Frokjaer, S.; Otzen, D. E. Nat. Rev. Drug Discovery 2005, 4, 298− 306. (2) Hoskin, D. W.; Ramamoorthy, A. Biochim. Biophys. Acta 2008, 1778, 357−375. (3) Janin, Y. L. Amino Acids 2003, 25, 1−40. (4) Torchilin, V. P.; Lukyanov, A. N. Drug Discovery Today 2003, 8, 259−266. (5) Hamman, J. H.; Enslin, G. M.; Kotze, A. F. Biodrugs 2005, 19, 165−177. (6) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40, 539−551. (7) Roberts, M. J.; Bentley, M. D.; Harris, J. M. Adv. Drug Delivery Rev. 2002, 54, 459−476. (8) Veronese, F. M.; Pasut, G. Drug Discovery Today 2005, 10, 1451− 1458. (9) Haag, R.; Kratz, F. Angew. Chem., Int. Ed. 2006, 45, 1198−1215. (10) Torchilin, V. P. Nat. Rev. Drug Discovery 2005, 4, 145−160. 1731

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732

Langmuir

Article

(48) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176−186. (49) Chen, H.; Brook, M. A.; Chen, Y.; Sheardown, H. J. Biomater. Sci., Polym. Ed. 2005, 16, 531−548. (50) Connors, K. A. Binding Constants: The Measurement of Molecular Complex Stability; John Wiley & Sons: New York, 1987. (51) Fielding, L. Tetrahedron 2000, 56, 6151−6170. (52) Hynes, M. J. J. Chem. Soc., Dalton Trans. 1993, 311−312. (53) Buurma, N. J.; Haq, I. Methods 2007, 42, 162−172. (54) Bromberg, L.; Alakhov, V. Y.; Hatton, T. A. Curr. Opin. Colloid Interface Sci. 2006, 11, 217−223. (55) Niu, G.; Zhang, H.; Song, L.; Cui, X.; Cao, H.; Zheng, Y.; Zhu, S.; Yang, Z.; Yang, H. Biomacromolecules 2008, 9, 2621−2628. (56) Yang, L. R.; Guo, C.; Jia, L. W.; Liang, X. F.; Liu, C.; Liu, H. Z. J. Colloid Interface Sci. 2010, 350, 22−29. (57) Chen, S.; Li, Y.; Guo, C.; Wang, J.; Ma, J. H.; Liang, X. F.; Yang, L. R.; Liu, H. Z. Langmuir 2007, 23, 12669−12676.

1732

dx.doi.org/10.1021/la203693c | Langmuir 2012, 28, 1725−1732