Article pubs.acs.org/Langmuir
Self-Assembly of a Diblock Copolymer with Pendant Disulfide Bonds and Chromophore Groups: A New Platform for Fast Release Liang Yuan,† Jingchuan Liu,† Jianguo Wen,‡ and Hanying Zhao*,† †
Key Laboratory of Functional Polymer Materials, Ministry of Education, Department of Chemistry, Nankai University, Tianjin 300071, P. R. China ‡ Institute of Polymer Science and Engineering, Hebei University of Technology, Tianjin 300130, P. R. China S Supporting Information *
ABSTRACT: An amphiphilic block copolymer comprising poly(ethylene glycol) (PEG) and poly(2-(methacryloyl)oxyethyl-2′-hydroxyethyl disulfide) (PMAOHD) blocks was synthesized by atom transfer radical polymerization (ATRP). Pyrenebutyric acid was conjugated to the block copolymer by esterification, and a block copolymer with pendant disulfide bonds and pyrenyl groups (PEG-b-P(MAOHD-g-Py)) was obtained. 1H NMR and gel permeation chromatography (GPC) results demonstrated the successful synthesis of the block copolymer. The cleavage of the disulfide bonds and the degrafting of the pyrenyl groups were investigated in THF and a THF/methanol mixture. Fluorescence spectroscopy, GPC, and 1H NMR results demonstrated fast cleavage of the disulfide bonds by Bu3P in THF. Fluorescence results showed the ratio of the intensity of the excimer peak to the monomer peak decreased rapidly within 20 min. GPC traces of the block copolymer moved to a long retention time region after addition of Bu3P, indicating the cleavage of the disulfide bonds and the degrafting of the pyrenyl groups. PEG-b-P(MAOHD-g-Py) can self-assemble into micelles with poly(MAOHD-g-Py) cores and PEG coronae in a mixture of methanol and THF (9:1 by volume). The dissociation of the micelles in the presence of Bu3P was investigated. After cleavage of the disulfide bonds in the micellar cores, a pyrene-containing small molecular compound and a block copolymer with pendant thiol groups were produced. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and 1H NMR were employed to track the dissociation of the polymeric micelles. All the techniques demonstrated the dissociation of the micelles and the fast release of pyrenyl groups from the micelles.
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INTRODUCTION Amphiphilic block copolymers in aqueous solution can selfassemble into micellar structures. The polymeric micelles can be used as nanocarriers for drugs and many insoluable compounds. Currently, polymeric micelles as promising nanosized antitumor drug carriers are being extensively studied.1−3 Polymeric micelles offer many unique advantages, such as passive accumulation in tumors, prolonged circulation time in blood, and the enhanced uptake by tumors.4 To enhance the anticancer efficacy, rapid drug release is anticipated when the drug-loaded micelles are within the cancer cells.5 Degradable micelles, in response to external triggers, such as pH,6,7 temperature,8,9 light,10,11 and reductive,12,13 oxidative,14 and enzymatic reactions,15 were prepared to meet the requirement of rapid release. Sheddable polymeric micelles are a type of micelles formed by amphiphilic block copolymers with cleavable linkages between the hydrophobic and the hydrophilic blocks.16−19 Upon exposure to light, low pH, or a reducing environment, the linkages are cleaved and the hydrophilic blocks escape from the micelles into the aqueous media. In research reported by Oh and co-workers, a disulfide-linked diblock copolymer of © 2012 American Chemical Society
polylactide and poly(oligo(ethylene glycol) monomethyl ether methacrylate) (PLA-SS-POEOMA) was prepared by successive ring-opening polymerization (ROP) of LA and atom transfer radical polymerization (ATRP) of OEOMA from a disulfidelabeled initiator.16 The disulfide linkages were cleaved and the hydrophobic cores composed of PLA precipitated from the aqueous solution upon introduction of DL-dithioerythritol to the solution. Block copolymers with photocleavable nitrobenzyl groups at the junctions of the blocks were also synthesized. For example, Fustin and co-workers anchored a cleavable onitrobenzyl ester derivative at the junctions of various amphiphilic block copolymers through ATRP and click chemistry.20 However, the sheddable micelles usually result in aggregates of the hydrophobic cores after cleavage reactions, and the loaded hydrophobic drugs are not effectively released from the cores where they are encapsulated. In another approach, cleavable linkages are incorporated into the side chains of the hydrophobic blocks of block Received: May 21, 2012 Revised: July 2, 2012 Published: July 5, 2012 11232
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Scheme 1. Synthesis of the Disulfide-Functionalized Monomer MAOHD and Pyrene-Conjugated Diblock Copolymer (PEG-bP(MAOHD-g-Py))
Scheme 2. Schematic Illustration of the Cleavage of the Disulfide Bonds and the Fast Release of Pyrenyl Groups from the Cores of the Micelles Formed by PEG-b-P(MAOHD-g-Py)
copolymers.21,22 Upon exposure to external stimuli, with partial or complete cleavage of the links, the properties of the hydrophobic cores change abruptly and the cores dissociate within a relatively short period of time. The drugs, either conjugated covalently to the backbones through cleavable
bonds or encapsulated in the hydrophobic cores, are then released in a burst-like manner. Zhao and co-workers developed light-breakable polymeric micelles, in which photolabile chromophores were grafted to the hydrophobic blocks.23 The hydrophobic blocks were transformed into hydrophilic blocks 11233
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Institute of Chemical Agents was dried in the presence of LiAlH4 and distilled before use. All the solvents were distilled before use. Synthesis of the Disulfide-Functionalized Monomer 2(Methacryloyl)oxyethyl-2′-hydroxyethyl Disulfide (MAOHD). BHEDS (6.16 g, 40.0 mmol) was dissolved in 50 mL of THF in a round-bottomed flask, and the solution was cooled to 0 °C. TEA (3.30 mL, 22.5 mmol) was added into the solution, and the solution was stirred for 20 min. Methacryloyl chloride (2.00 mL, 22.5 mmol) dissolved in 15 mL of dry THF was added into the solution dropwise at 0 °C. The mixture was stirred at room temperature for 24 h. After filtration, the solution was washed with HCl solution (0.3 mol/L), NaOH solution (0.3 mol/L), and water, respectively. The organic layer was dried over MgSO4 and concentrated by rotary evaporation. The crude product was purified by column chromatography (petroleum ether:CH2Cl2:methanol = 70:10:1 by volume), and the final product was obtained (2.8 g, yield: 61%). The 1H NMR spectrum of MAOHD is shown in Figure S1 in Supporting Information. 1H NMR δ (400 MHz, CDCl3, TMS, ppm): 5.62, 6.16 (CH2CCH3, s, 2H), 4.46 (CH2CH2OH, t, 2H), 3.92 (COOCH2, t, 2H), 3.00 (CH2CH2OH, t, 2H), 2.92 (COOCH2CH2, t, 2H), 1.98 (CH2 CCH3, s, 3H). Synthesis of Diblock Copolymer with Pendant Disulfide Groups. The diblock copolymer was synthesized by ATRP of MAOHD using PEG-Br as the macroinitiator. CuBr (0.0173 g, 0.120 mmol) and dNbpy (0.0979 g, 0.240 mmol) were dissolved in 3.5 mL of DMF in a 25 mL two-neck flask, and the solution was degassed with three freeze−pump−thaw cycles. A degassed solution of PEG-Br (0.60 g, 0.12 mmol) and MAOHD (0.535 g, 2.40 mmol) in 3.5 mL of DMF was transferred into a solution of CuBr/dNbpy through a syringe, and the mixture was degassed with two freeze−pump−thaw cycles. The polymerization was carried out at 45 °C for 24 h under nitrogen atmosphere. DMF was removed by rotary evaporation, and the polymer was redissolved in THF. The polymer solution was passed through a neutral Al2O3 column to remove the copper ions. Polymer was obtained after pouring the polymer solution into excess cold diethyl ether, isolating by centrifugation, and drying under vacuum. Based on GPC, the number average molecular weight and the molecular weight distribution of PEG-b-PMAOHD block copolymer are 11.7K and 1.20, respectively. Synthesis of Pyrene-Conjugated Diblock Copolymer PEG-bP(MAOHD-g-Py). PEG-b-PMAOHD (0.3 g), PyBA (0.292 g, 1.02 mmol), and DMAP (6.7 mg, 0.051 mmol) were dissolved in 35 mL of CH2Cl2 and stirred in a water−ice mixture for 30 min, and EDC·HCl (0.195 g, 1.02 mmol) was added into the solution. The solution was stirred at 40 °C for 48 h and concentrated by rotary evaporation. After the reaction, the polymer was precipitated in cold ether. The product was extracted with ether using a Soxhlet extractor to remove the possible free PyBA. The yield of the pyrene-conjugated block copolymer was 85%. Preparation of Polymeric Micelles. A predetermined amount of PEG-b-P(MAOHD-g-Py) was molecularly dissolved in 1 mL of THF (depending on the content of pyrenyl groups), and 9 mL of methanol was added into the above solution dropwise. The observation of a cloudy solution indicated the formation of polymeric micelles in the mixture. The final solution was allowed for equilibration overnight before performing the measurements. In this research, the concentration of the polymeric micelles is expressed in the molar concentration of pyrene moieties based on the 1H NMR result.
upon exposure to UV light, and the polymeric micelles dissociated within 15 min. Recently, Zhong and co-workers designed acid-activatable doxorubicin (DOX) prodrug nanogels. DOX was grafted to the polymer backbone via a hydrazone bond, which was readily cleavable at endosomal pH.24 The formation of disulfides from thiols is a redox-sensitive process, and the disulfides are readily reduced to thiols within a reducing environment.25−31 In previous research, disulfide bonds were introduced into nanocarrier systems and applied in drug release. For example, Thayumanavan and co-workers demonstrated that polymer chains could be intermolecularly cross-linked through disulfide bond formation and the structures disassembled in the presence of the peptide glutathione.32,33 In this research, pyrenyl groups used as a model compound were grafted to the backbones of an amphiphilic block copolymer via disulfide bonds, and the fast release of the model compound from the self-assembled structures within a reducing environment was studied. Because the fluorescence properties of pyrenyl groups have been well studied and the fast release process and the dissociation of the self-assembled structures can be tracked by fluorescence and other techniques, the pyrenyl group was used as the model compound in this research. As shown in Scheme 1, a disulfidefunctionalized monomer was synthesized, and ATRP of the monomer initiated by bromine-terminated poly(ethylene glycol) (PEG-Br) yields an amphiphilic diblock copolymer.34,35 Pyrenyl groups were conjugated to the polymer backbones through esterification. The cleavages of the disulfide bonds in a good solvent and a selective solvent were investigated. In a selective solvent, pyrene-conjugated diblock copolymer was able to self-assemble into micelles with PEG blocks in the coronae and pyrenyl groups inside the cores. The hydrophobic cores could undergo fast dissociation in the presence of Bu3P (Scheme 2). The cleavage of the disulfide bonds and the fast release of pyrenyl groups from the micellar cores were investigated. The advantages of this approach are high loading efficiency of the model compound and the introduction of cleavable bonds in the macromolecular structure. To our knowledge, the use of redox-sensitive disulfide bonds in the preparation of core-degradable micelles has not yet been reported. This research system can be used as a novel platform to study the rapid release of drugs based on a redox-sensitive process, and the results obtained in this research can be applied in the design of new controlled release systems.
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EXPERIMENTAL SECTION
Materials. Poly(ethylene glycol) monomethyl ether (CH3O-PEGOH, Mn = 5000) was purchased from Fluka and used as received. PEG-Br was prepared by reaction of CH3O-PEG-OH and 2bromoisobutyryl bromide (Supporting Information). Methacryloyl chloride was prepared by reaction of methacrylic acid with oxalyl chloride in the presence of a catalytic amount of DMF. CuBr (99.5%) purchased from Guo Yao Chemical Company was purified by washing with glacial acetic acid and drying in a vacuum oven at 100 °C. 2Bromoisobutyryl bromide (98%, Aldrich), 4-(dimethylamino)pyridine (DMAP, 99%, Alfa Aesar), 4,4′-di-5-nonyl-2,2′-bipyridine (dNbpy, 98%, Alfa Aesar), N,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, Fluka), 1-pyrenebutyric acid (PyBA, 97%, Alfa Aesar), and tributylphosphine (Bu3P, 95%, Tianjin Institute of Chemical Agents) were used as received. Bis(2-hydroxyethyl) disulfide (BHEDS, technical grade, Alfa Aesar) was purified by distillation under reduced pressure. Triethylamine (TEA, 99%) purchased from Tianjin
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RESULTS AND DISCUSSION As illustrated in Scheme 1, PEG-Br macroinitiator was prepared by a reaction of 2-bromoisobutyryl bromide and CH3O-PEGOH.36 1H NMR results show that the average degree of polymerization of the macroinitiator is about 109. Disulfidefunctionalized monomer MAOHD was synthesized by esterification of bis(2-hydroxyethyl) disulfide. 1H NMR spectra of MAOHD and PEG-Br can be found in the Supporting Information. ATRP of MAOHD was carried out at 45 °C with CuBr/dNbpy as catalyst and PEG-Br as macroinitiator. Figure 1 11234
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and the signal representing the methylene protons next to the hydroxyl groups shifts from 4.28 to 4.18 ppm (peak e in Figure 2) after esterification. On the basis of 1H NMR result, the esterification of the hydroxyl groups on PMAOHD blocks is complete. In this paper, the block copolymer after esterification is denominated as PEG109-b-P(MAOHD14-g-Py). GPC curves of PEG109 -b-P(MAOHD 14-g-Py) and its precursors are shown in Figure 3. GPC traces show clear shifts
Figure 1. 1H NMR spectrum of poly(ethylene glycol)-block-poly(2(methacryloyl)oxyethyl-2′-hydroxyethyl disulfide) diblock copolymer (PEG109-b-PMAOHD14).
shows the 1H NMR spectrum of the block copolymer. In Figure 1, signals at 3.65 ppm (peak f) corresponding to the oxymethylene protons (OCH2CH2) in PEG and at 3.39 ppm (peak g) representing the methyl protons (OCH3) on terminal groups could be observed. Other signals at 4.28 ppm (peak a) representing the methylene protons next to the hydroxyl group, at 3.93 ppm (peak d) representing the methylene protons next to the ester group, and at 2.98 (peak b) and 2.93 ppm (peak c) representing the methylene protons next to the disulfide groups were also observed. Based on the integral ratio of peak a to peak g, the average repeating unit number of PMAOHD block was determined to be 14. In this paper, the block copolymer is referred to as PEG109-b-PMAOHD14. Pyrene-conjugated macromolecules are frequently used in the studies of polymer configuration and self-assemblies.37,38 In this research, the pyrenyl group grafted to the diblock copolymer chains through the disulfide bond was used as a model compound, and the fast release of the chromophore groups from the self-assembled structures in a reducing environment was investigated. Pyrenyl groups were grafted to the polymer chains by esterification between PyBA and PMAOHD blocks. The reaction assisted by EDC/DMAP was conveniently performed in CH2Cl2, and 2 equiv of PyBA was used to drive the reaction to completion. The 1H NMR spectrum of the pyrene-conjugated block copolymer is shown in Figure 2. In the spectrum, broad peaks at 7.3−8.5 ppm representing the protons on the pyrenyl groups are observed,
toward a high molecular weight region after ATRP and esterification. In comparison to the macroinitiator, the apparent number-average molecular weight of PEG109-b-PMAOHD14 increased from 6.9 to 11.7K after ATRP and further increased to 22.4K after esterification. The molecular weight distribution of PEG109-b-PMAOHD14 was 1.20. However, after esterification, the polydispersity of the block copolymer reached 1.40. The increase in the polydispersity is probably attributed to the π−π stacking interactions between the pendant pyrenyl groups. To investigate the effect of the polymer structure on the fluorescence properties of the block copolymer, the photophysical properties of PEG109-b-P(MAOHD14-g-Py) were studied in THF, a good solvent for the block copolymer. In this research, the molar concentration of pyrene moieties in the solution was used to represent the polymer concentration. Figure 4 shows the fluorescence emission spectra of the block copolymer in THF at different concentrations. The emission spectra were excited at a wavelength of 342 nm. In the spectra, not only monomer emission peaks but also a structureless
Figure 2. 1H NMR spectrum of PEG109-b-P(MAOHD14-g-Py) diblock copolymer measured in CDCl3.
Figure 4. Fluorescence emission spectra of PEG109-b-P(MAOHD14-gPy) in THF at different concentrations (mol/L). In the measurements, the excitation and emission slits were set at 3.0 and 1.5 nm, respectively.
Figure 3. GPC curves of PEG-Br (dark), PEG109-b-PMAOHD14 (red), and PEG109-b-P(MAOHD14-g-Py) (blue).
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150 min while the relative intensities of the monomer peak at 377, 383, 388, 397, and 418 nm increased with the decrease of the excimer peak. The changes in the emission spectra can be explained by the cleavage of the disulfide bonds and the degrafting of the pyrenyl groups from the polymer backbones. The density of the chromophore groups on the polymer backbones decreases with the degrafting of the pyrenyl groups, and the possibility for the encounter of the excited pyrenyl molecules with those in the ground state is much less, so the intensity of the excimer peak decreased upon addition of Bu3P. The ratio of the intensity of the excimer peak (IE, at 476 nm) to the monomer peak (IM, at 397 nm) can be used to measure the efficiency of the formation of the excimer. Figure 6 shows the
excimer emission band at 476 nm was observed at all concentrations, even at a concentration as low as 5.0 × 10−7 M. For pyrene molecules, the broad excimer emission band in the range from 450 to 550 nm originates from the encounter of pyrene molecules in the excited singlet state with those in the ground state, which is usually observed when the concentration of pyrenyl groups is high enough (10−4 to 10−3 M). To demonstrate the effect of the pendant structure on the fluorescence emissions, a controlled experiment was conducted. Figure S3 in the Supporting Information shows the fluorescence emission spectra of PEG109-b-P(MAOHD14-g-Py) solution at a concentration of 1.0 × 10−7 M and a pyrene solution at a concentration of 5.0 × 10−7 M. The polymer solution presents a strong excimer emission at 476 nm; however, no excimer emission band is observed for the pyrene solution. Therefore, the pendant structure of the chromophore groups in the block copolymer plays a key role in the excimer formation. In Figure 4, it is obvious that the intensity of the fluorescence emission spectra increases with concentration at lower concentration range (
