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Article pubs.acs.

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Modifying the HydrophilicHydrophobic Interface of PEG-b-PCL To Increase Micelle Stability: Preparation of PEG-b-PBO-b-PCL Triblock Copolymers, Micelle Formation, and Hydrolysis Kinetics
Xiaobo Zhu, Michael Fryd, Benjamin D. Tran, Marc A. Ilies, and Bradford B. Wayland*,

Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, United States Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, Pennsylvania 19140, United States
S * Supporting Information

ABSTRACT: A set of PEG-b-PBO-b-PCL triblock copolymers were prepared for comparative studies of micelle formation and relative stability compared to the parent PEG-b-PCL diblock copolymers. Block copolymers that were characterized by 1H NMR and GPC were self-assembled in water by nanoprecipitation from organic solvents. Spherical micelles produced in near-quantitative yield were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The initial step in degradation of the block copolymers assembled in spherical micelles is shown by GPC and 1H NMR in acidic media to occur by hydrolysis of the interface ester group which cleaves the PCL segment in the core from the -PBOPEG and -PEG segments. Kinetics of hydrolysis of the ester groups that bind the hydrophobic PCL segment with either PEG or PBO units were followed by 1H NMR and demonstrate that the triblock has a reduced rate of hydrolysis. Inserting a short block of PBO between the PEG and PCL segments provides increased protection from hydrolysis for the ester group at the hydrophilichydrophobic interface.

INTRODUCTION Amphiphilic diblock copolymers self-assemble into a variety of supramolecular aggregate structures including coreshell micelles,15 filomicelles,68 and polymersomes911 that are finding applications as drug and gene delivery systems.1216 In particular, micelles formed from block copolymers that contain poly(ethylene glycol) (PEG) and poly(-caprolactone) (PCL) segments are widely applied in the design of drug delivery systems because the hydrophilic PEG block is biocompatible and the hydrophobic PCL block is biodegradable.1721 The PEG-b-PCL block copolymers that were designed for drug delivery have a hydrolytically sensitive ester2225 link between the hydrophilic and hydrophobic segments. This ester group is normally located at the aqueous/hydrophobic interface in coreshell micelles and thus is readily accessed for reactions with the aqueous medium. Hydrolysis of the interface ester leads to shedding of the PEG block and destabilization of the micelle.2629 This paper reports on an initial effort to tune the stability of micelles by moving the hydrolytically accessible ester linkage away from the coreshell interface. This is achieved by inserting a short nonbiodegradable hydrophobic segment of poly(1,2-butylene oxide) (PBO) between the PCL core and PEG corona. Our working hypothesis is that the rate of PEG shedding can be retarded by separating the interface between the biodegradable PCL and nondegradable PEG blocks from the hydrophilichydrophobic interface and thus extends micelle stability without affecting the self-assembly process. This article reports on the preparation of low-polydispersity
XXXX American Chemical Society

RESULTS AND DISCUSSION Synthesis and Characterization of PEG45-b-PCL62 and PEG45-b-PBO9-b-PCL61 Block Copolymers. The block copolymers of PEG45-b-PCL62 and PEG45-b-PBO9PCL61 were synthesized as shown in Scheme 1. Molecular weights of the polymers were determined by 1H NMR analysis (Table 1 and Figure S2), and the molecular weight distribution was evaluated by GPC (Figure S1). Amphiphilic block copolymer PEG45-b-PCL62 (PDI = 1.08) was synthesized utilizing methoxy poly(ethylene glycol) as the macroinitiator for the controlled ring-opening polymerization (ROP) of caprolactone (CL) in the presence of stannous octoate (Sn(Oct)2) as a catalyst.14,21 The labeled NMR spectrum is shown in Figure S2. The degree of polymerization (DP) of CL was evaluated to be 62 by comparing the integration of signals at 3.62 ppm for CH2 of PEG (labeled b) and triplet at 2.30 ppm for CH2 of PCL (labeled d). Copolymer was synthesized by
Received: November 18, 2011 Revised: December 22, 2011

PEG-b-PCL diblock and PEG-b-PBO-b-PCL triblock polymers that self-assemble into spherical micelles. Kinetics of acidcatalyzed hydrolysis of the interface ester groups are shown to occur substantially slower in the triblock copolymer micelles when compared to micelles of the parent diblock copolymer.

dx.doi.org/10.1021/ma202530v | Macromolecules XXXX, XXX, XXXXXX

Macromolecules Scheme 1. Synthesis of PEG45-b-PCL62 and PEG45-b-PBO9-b-PCL61 Block Copolymers

Article

Table 1. Synthetic Results for PEG45-b-PCL62 and PEG45b-PBO9-b-PCL61 Block Copolymers


sample PEG45-b-PBO9 PEG45-b-PBO9-PCL61 PEG45-b-PCL62
a

Mn(theory)a 2830 9260 8600

Mn(NMR)b 2650 9610 9080

W(PEG)c 0.76 0.21 0.22

PDId 1.08 1.10 1.08

Number-average molecular weight of block copolymers based on a standard conversion of 36.0 (3.0)% for each monomer polymerization. b Number-average molecular weight of block copolymers determined by integration of 1H NMR resonances unique to each segment. c Weight fraction of PEG in the block copolymers based on the Mn from 1H NMR. dPolydispersity (Mw/Mn) evaluated by GPC.

terminating the reaction at only moderate CL conversion (36.0 (3.0)%) to ensure narrow molecular weight distributions. The triblock copolymer of PEG45-b-PBO9-PCL61 (PDI = 1.10) was analogously synthesized by ROP of CL with PEG45b-PBO9 as macroinitiator and Sn(Oct)2 as catalyst. Synthesis of PEG45-b-PBO9 (PDI = 1.08) was accomplished by anionic polymerization of 1,2-butylene oxide (BO) with PEG as a macroinitiator.3033 The DP of the PBO was determined by comparing the integration of signals at 3.62 ppm for CH2 of PEG (labeled b) and triplet at 0.93 ppm for CH3 of BO methyl group (labeled e). Preparation and Characterization of PEG45-b-PCL62 and PEG45-b-PBO9-b-PCL61 Block Copolymer Micelles. Samples of 1.0 mg/mL of PEG45-b-PCL62 and PEG45-bPBO9-PCL61 copolymer micelles were prepared by adding water to a stock solution of polymer in acetone under vigorous stirring.24,34 Results from dynamic light scattering (DLS) studies for these micelle solutions are shown in Figure 1. The single monomodal particle distributions are indicative of highly efficient micelle formation and provide that the average micelle sizes of 22 nm for PEG45-b-PBO9-b-PCL61 and 18 nm for PEG45-b-PCL62. The slightly larger average size for the
B

triblock copolymer from the DLS is consistent with the larger hydrophobichydrophilic ratio. TEM images of the micelles from diblock and triblock copolymers revealed only spherically shaped particles with average sizes comparable to those obtained by DLS (Figure 1). Neither DLS nor TEM observations produced evidence for larger particles such as filomicelles and polymerosomes even at concentrations of polymer higher than the 1.0 mg/mL used in this study. Selective formation of spherical micelles probably results from the strong self-assembling property of the PCL hydrophobic core. Both the di- and triblock copolymer micelles have similar size and narrow size distributions, which justifies comparing results of acid catalyzed hydrolysis studies. Acid-Catalyzed Degradation of PEG45-b-PCL62 and PEG45-b-PBO9-b-PCL61 Block Copolymer Micelles. The hydrolytic degradation of PEG45-b-PCL62 and PEG45-bPBO9-b-PCL61 block copolymer micelles was evaluated in aqueous HCl (pH = 1.0) at 25 C by terminating the hydrolysis with NaOH at 3.0 h time intervals and extracting the polymeric materials with CH2Cl2 for 1H NMR and GPC studies. GPC traces for the polymer products from degradation of PEG45-bPCL62 micelles in water at a series of time intervals are shown in Figure 2A,B. The GPC results clearly show that the PEG45b-PCL62 diblock copolymer is cleaved into two individual polymer units with the low-molecular-weight peak occurring precisely at the same position as the pure PEG block shown in Figure S1. The average molecular weight decreased and the width increased regularly for the PEG45-b-PCL62 diblock copolymer GPC peak as the relative intensity of peak for PEG increased with the time of degradation. The 1H NMR chemical shift of the unique CH2 group that is connected to the hydrophilichydrophobic interface ester group (Scheme 2) occurs at 4.23 ppm in the PEG-b-PCL block copolymer and shifts to 3.62 ppm when the interface ester group hydrolyzes and the PEG segment cleaves off. The
dx.doi.org/10.1021/ma202530v | Macromolecules XXXX, XXX, XXXXXX

Macromolecules

Article

Figure 1. TEM images of PEG45-b-PCL62 (A) and PEG45-b-PBO9-b-PCL61 (B) micelles. Size distribution of PEG45-b-PCL62 (C) and PEG45-bPBO9-b-PCL61 (D) micelles determined by DLS.

Figure 2. GPC traces for degradation products of PEG45-b-PCL62 micelles before normalization of block copolymer peak (A), after normalization (B), PEG45-b-PBO9-b-PCL61 micelles before normalization (C), and after normalization (D).

peak intensity of this special CH2 group relative to the intensity of the end methyl group of PEG, which is set as constant, was monitored by the time evolution of the 1H NMR (Figure S3). The 1H NMR peak intensity of the CH2 group decreases with time as hydrolysis proceeds. Results from DLS and TEM studies show that diblock copolymers of PEG45-b-PCL62 self-assemble into narrowly size distributed micelles with a coreshell structure. The hydrophilic corona permits relatively facile access of water to the junction between the hydrophilic and hydrophobic polymer segments. The ester group at the hydrophilichydrophobic interface is exposed to the aqueous solution for hydrolysis. The initial event for acid-catalyzed ester hydrolysis is observed to
C

occur exclusively at the unique interface ester group as shown by appearance of the PEG homopolymer segment on the GPC traces and a decrease of 1H NMR peak intensity of the CH2 group that connects to ester group at the junction of the PEG PCL on 1H NMR traces. These observations are in agreement with the results of previous hydrolytic degradation studies of PEG-b-PCL which concluded that an initial stage of interfacial erosion was followed by bulk degradation in the micellar core.24,27,35,36 Short segments of hydrophobic and hydrolytically stable PBO were inserted between the PEG and PCL blocks as a strategy to tune access of water to the interface ester linkage and thus alter the rate of hydrolysis and ultimately the micelle
dx.doi.org/10.1021/ma202530v | Macromolecules XXXX, XXX, XXXXXX

Macromolecules Scheme 2. Hydrolysis of the Interface Ester Groups in Spherical Micelles

Article

stability. The working hypothesis is that water can only freely reach the PEGPBO junction in the triblock copolymer micelles and thus access by water to the ester group at the PBOPCL interface will be retarded by the insulating PBO segment. The GPC and 1H NMR results for the interface ester hydrolysis of the PEG45-b-PBO9-b-PCL61 triblock copolymer micelles (Figure 2C,D and Figure S3) are analogous with those for the PEG45-b-PCL62 diblock copolymer micelles. The GPC results show that the PEG45-b-PBO9-b-PCL61 triblock copolymer micelle is cleaved into two individual polymer units with the low-molecular-weight peak occurring at the same position as the PEG45-b-PBO9 block shown in Figure S1. The average molecular weight decreased and the width of the distribution increased regularly for the PEG45-b-PBO9-bPCL61 triblock copolymer GPC peak as the relative peak intensity of PEG45-b-PBO9 increased with the time of hydrolysis. The time evolution of the 1H NMR for the triblock copolymer micelle ester hydrolysis products is shown in Figure S3(B). The 1H NMR intensity of the unique CH group (Scheme 2) bonded to the ester group at the PEGPBOPCL junction decreases with time relative to the end methyl group of PEG as hydrolysis proceeds, which is very similar to results for the PEG45-b-PCL62 diblock copolymer. However, the rate of the disappearance of the CH 1H NMR resonance resulting from the interface ester hydrolysis for the triblock copolymer micelle proceeds slowly relative to that of the diblock copolymer micelle. Kinetic Comparison of Acid Hydrolysis of PEG45-bPCL62 and PEG45-b-PBO9-b-PCL61 Block Copolymer Micelles. Quantitative comparison of the rate for acidcatalyzed (pH = 1.0) hydrolysis of the ester group at the PEGPCL and PEGPBOPCL junctions were obtained by following the time evolution of the 1H NMR. First-order kinetic plots for the intact di- and triblock copolymers as a function of time are shown in Figure 3. The kinetic plots demonstrate that the rate of interface ester hydrolysis for the PEG45-b-PCL62 diblock copolymer micelles (0.11 h1) is faster than that of the PEG45-b-PBO9-b-PCL61 triblock copolymer micelles (0.041 h1). The half-life of the interface ester for diblock copolymer micelles is 6.3 h compared to a 16.9 h half-life for triblock copolymer micelles. Insertion of a short hydrophobic PBO segment between PEG and PCL is thus observed to provide
D

Figure 3. First-order kinetic plots for the acid-catalyzed hydrolysis of interface ester groups that occur at the PEGPCL and PEGPBO PCL junctions. [P]t is defined as the relative molar concentration of the intact block copolymer at time t.

Narrow polydispersity PEG45-b-PBO9-b-PCL61 triblock and PEG45-b-PCL62 diblock copolymers were prepared for comparative studies of micelle formation and relative stability in acidic media. DLS results indicate that both polymers form near-quantitative yields of micelles that are shown to be spherical by TEM. GPC and 1H NMR results for the degradation of block copolymer spherical micelles proved that the initial hydrolysis of the micelles occurs at the interface ester group that binds the PCL segment with PBOPEG or PEG segments. Kinetics of hydrolysis of this unique ester group was followed by 1H NMR and showed that insertion of a short chain PBO segment between the nondegradable PEG block and the biodegradable PCL units reduced the rate of hydrolysis of the interface ester group for the diblock copolymer micelle by a factor of 2.7, which is attributed to the PBO segment
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increased kinetic stability for the PEG-b-PBO-b-PCL triblock copolymer micelles relative to the PEG-b-PCL diblock copolymer micelles. Systematic variation of the chemical nature and size of the spacer segments in a series of triblock copolymers is currently being used to evaluate their influence on micelle formation and stability.

CONCLUSIONS

Macromolecules providing a kinetic barrier for water to reach the ester group at the PEGPBOPCL junction.

Article

Materials. Tetrahydrofuran (THF) and toluene were distilled just prior to use. Methoxy poly(ethylene glycol) (PEG, Mn = 2000, Mw/Mn = 1.06) was purified by precipitating from petroleum ether prior to use. -Caprolactone (CL) was dried by CaH2 and distilled under vacuum. 1,2-Butylene oxide (BO) was also dried by CaH2 and distilled before use. 18-Crown-6 ether (18C6) and stannous octoate (Sn(Oct)2) were used as received. Potassium naphthalenideTHF solution (0.89 M) was prepared by mixing of naphthalene (3.0 g), potassium (0.92 g), and THF (26 mL). All chemicals were purchased from Sigma-Aldrich. Characterization. 1H NMR spectra were obtained using Bruker 400 and 500 MHz spectrometers with CDCl3 as solvent and internal standard. Gel permeation chromatography (GPC) measurements were carried out on a Shimadzu LC-20AV liquid chromatography system equipped with PolarGel-M 300 7.5 mm column and SPD-20AV UV/vis detector. DMF was used as the eluent at a flow rate of 1.0 mL/ min at 50 C. Calibration was based on polystyrene standards. Synthesis of PEG45-b-PBO9 Diblock Copolymer. Dried PEG (8.0 g, 4.0 mmol) was dissolved in 80 mL of anhydrous THF in a 250 mL dry flask under an inert atmosphere. Potassium naphthalenide (9.0 mL, 0.89 M in THF) and 18C6 (1.5 g, 5.7 mmol)THF solution were added into the solution via syringe. After stirring the mixture for 15 min while the dark green color persisted, BO (10 mL, 0.12 mol) was added into the reaction mixture via syringe. The solution was stirred for 1 h at room temperature, the reaction was terminated by the adding of HCl (0.80 mL, 12.2 M), and the green solution became colorless again. The undissolved inorganic salt was removed by filtration, and the PEG-b-PBO block copolymer was precipitated from petroleum ether. The white precipitates were recovered by filtration, redissolved in toluene, and precipitated from the petroleum ether again. The precipitates were collected and dried under vacuum at 40 C for 48 h. Synthesis of PEG45-b-PBO9-b-PCL61 Triblock Copolymer. Dried PEG45-b-PBO9 (0.90 g, 0.34 mmol) was dissolved in dry toluene (16 mL) in a 50 mL dry flask under an inert atmosphere. CL (6 mL, 0.054 mol) and Sn(Oct)2 (0.02 g) were added into the solution via syringe. The mixture was gently refluxed under nitrogen for at 120 C. After stirring the solution for 5 h, excess cold methanol was poured into the solution to terminate and precipitate the product. The white precipitate was collected by filtration, redissolved in dichloromethane, and precipitated from the methanol again. The precipitate was collected and dried under vacuum at 40 C for 48 h. Micelle Preparation and Characterization. The nanoprecipitation method was used to prepare the PEG45-b-PCL62 and PEG45PBO9-PCL61 micelles. Polymer stock solutions (10 mg/mL) of PEG45-PCL62 and PEG45-PBO9-PCL61 were prepared in acetone. Polymer stock solutions (0.8 mL) were introduced into vials, and filtered deionized water (8 mL) was subsequently added to the stirred polymer solutions at the rate of 1.0 mL/min. The remaining acetone in resulting micelle suspensions was removed at room temperature under a flow of nitrogen, applied for 36 h. The near-quantitative yield of micelle suspensions were filtered through a PVDF 100 nm pore size membrane filter to ensure removal of occasional small quantities of nondispersed polymer aggregates. Dynamic Light Scattering (DLS). Hydrodynamic diameter of micelles was measured by a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) at 25 C, using 1 cm polystyrene cuvettes. The mean diameter was obtained from the instruments DTS software using the volume reading. Transmission Electron Microscopes (TEM). Micelle morphology was evaluated by a JEOL JEM-1400 TEM operating at an acceleration voltage of 80 kV. The 1.0 mg/mL of micelle solution was diluted by a factor of 10 to reduce the aggregation during sample drying process. One drop of micelle solution was deposited on carboncoated copper grid (Ted Pella Inc., Redding, CA). The droplet was
E

EXPERIMENTAL SECTION

then blotted and allowed to evaporate under ambient conditions.37,38 The average diameters of the spherical micelles were analyzed by a digital micrograph. Degradation of PEG45-b-PCL62 and PEG45-b-PBO9-b-PCL61 Block Copolymer Micelles. The hydrolytic degradation of PEG45PCL62 and PEG45-b-PBO9-b-PCL61 micelles was studied in HCl (pH = 1.0) at 25 C. The acid-catalyzed degradation experiment was done in a series of vials. In each 5 mL vial, 0.7 mL of micelle suspension (1.0 mg/mL) was added. Subsequently, 0.7 mL of HCl (2 M) solution was added into the vials. The reactions were terminated by adding 1.4 mL of NaOH (1 M) solution every 3 h. The final degradation products were extracted from water solution by CH2Cl2 for 1H NMR and GPC evaluation.

ASSOCIATED CONTENT

S * Supporting Information

Experimental details on preparation of PEG45-b-PCL62 diblock copolymer; GPC traces and 1H NMR spectra for PEG-b-PCL, PEG-b-PBO, and PEG-b-PBO-b-PCL block copolymers; 1H NMR time evolution of ester hydrolysis for di- and triblock copolymers. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: bwayland@temple.edu.

ACKNOWLEDGMENTS Partial support of this research by NSF (CHE 0809395) and funding of a JEOL JEM-1400 TEM by the NSF (CHE0923077) are gratefully acknowledged. The authors are also grateful to Dr. Hongwen Zhou for TEM experiments. REFERENCES
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dx.doi.org/10.1021/ma202530v | Macromolecules XXXX, XXX, XXXXXX