pH-Sensitive Micelles Self-Assembled from Amphiphilic Copolymer

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Biomacromolecules 2011, 12, 116–122

pH-Sensitive Micelles Self-Assembled from Amphiphilic Copolymer Brush for Delivery of Poorly Water-Soluble Drugs You Qiang Yang, Ling Shan Zheng, Xin Dong Guo, Yu Qian, and Li Juan Zhang* School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China

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Received September 9, 2010; Revised Manuscript Received November 9, 2010

A novel pH-sensitive amphiphilic copolymer brush poly(methyl methacrylate-co-methacrylic acid)-b-poly(poly(ethylene glycol) methyl ether monomethacrylate) [P(MMA-co-MAA)-b-PPEGMA] was defined and synthesized by atom transfer radical polymerization (ATRP) technique. The molecular structures and characteristics of this copolymer and its precursors were confirmed by 1H NMR, FT-IR, and GPC. The CMC of P(MMA-co-MAA)b-PPEGMA in aqueous medium was determined to be 1-4 mg/L. This copolymer could self-assemble into micelles in aqueous solution with an average size of 120-250 nm determined by DLS. The morphologies of the micelles were found to be spherical by SEM and TEM. Ibuprofen (IBU), a poorly water-soluble drug, was selected as the model drug and wrapped into the core of micelles via dialysis method. Drug entrapment efficiency reached to 90%. The in vitro release behavior of IBU from these micelles was pH-dependent. The cumulative release percent of IBU was less than 20% of the initial drug content in simulated gastric fluid (SGF, pH 1.2) over 12 h, but 90% was released in simulated intestinal fluid (SIF, pH 7.4) within 6 h. The release profiles showed that the P(MMAco-MAA)-b-PPEGMA micelles could inhibit the premature burst drug release under the intestinal conditions. All the results indicate that the P(MMA-co-MAA)-b-PPEGMA micelle may be a potential oral drug delivery carrier for poorly water-soluble drugs.

1. Introduction Core/shell polymeric micelles self-assembled from amphiphilic block copolymers in aqueous solution have been extensively investigated and applied in drug delivery system (DDS).1-12 The inner hydrophobic core of the micelles can enhance the loading efficiency of hydrophobic drugs. The outer hydrophilic shell can provide a stable interface between the hydrophobic core and the aqueous medium, which counterchecks the aggregation of micelles, protects drugs from inactivating under the biological environment, and decreases side effects of drugs on healthy cells and tissues. By selecting suitable hydrophilic segments, which are resistible toward blood or tissues as the outer shell, the polymeric micelles cannot be recognized by certain proteins or phagocytic cells and finally achieve long-circulation in the blood or tissues.13 Oral administration is thought to be more convenient and better patient compliant compared to parenteral administration for less pain and possible infections caused by injection. For an ideal polymeric micellar oral DDS, it should keep stable in the stomach (pH 1.0-2.5) and does not release the entrapped drug or releases in a very slow rate, while releasing the drug in a relatively short time (8-10 h)14,15 after reaching the intestine (pH 5.1-7.8).16-18 The pH-sensitive polymeric micelles may be favorable for oral delivery of poorly water-soluble drugs as they can reduce drug leakage in stomach, prevent burst drug release, and avoid precipitation in the upper area of intestinal tract.19 Upon oral administration, these drug-loaded polymeric micelles could maintain in a close form at low pH of stomach and reduce drug release. The micelles will start to dissociate or swell at higher pH (pH > 5.0) present in the small intestine thereby releasing the entrapped drug. Poly(acrylic acid) (PAA) * To whom correspondence should be addressed. Tel./Fax: +86-2087112046. E-mail: [email protected].

and poly(methacrylic acid) (PMAA), bearing the carboxylic group with pKa around 5-6, are the most representative and frequently pH-responsive polymers used in pH-sensitive oral DDS.13,19-22 They are commonly accepted as bioadhesive and safe polymers, as the ionizable carboxyls of PAA or PMAA in the micellar corona can facilitate mucoadhesion. This enhances the residence time of the drug-entrapped micelles in the gastrointestinal tract and ensures complete drug dissolution, and eventually maximizes drug absorption in the small intestine. As described above, time-controlled, pH-sensitive, and sustained (no burst release behavior) release are the most important parts for a successful oral micelle DDS and much attention has been paid to achieve this goal. Kim et al.22,23 developed a pHsensitive micelle for oral delivery of paclitaxel (PTX). The pHresponsive acrylic acid (AA) and hydrotrope contents N,Ndiethylnicotinamide (DENA) were fabricated by copolymerization to achieve time-controlled rapid delivery (within 12 h). Shen et al.24 manufactured a multifunctional three-layered polymer brush nanomicelle for parenteral administration of anticancer drug. The nanomicelles were found inhibiting the premature burst drug release and promoting endocytosis for fast cellular internalization in the acidic interstitium of solid tumors, which made it an efficient carrier for cancer cytosolic drug delivery. However, lots of the expressed oral DDS of poorly water-soluble drugs have still not reached satisfied performances, such as, the high CMC value of polymers makes the carriers unstable during circulation, burst drug release attributes to local high concentration of drug which is toxic to normal cell, and imprecise stimuresponse property results in uncontrolled releasing. Designing and exploiting novel polymers with reasonable structure could be an effective way to improve the carrier performances. In the current work, a novel pH-sensitive multifunctional amphiphilic polymer brush poly(methyl methacrylate-comethacrylic acid)-b-poly(poly(ethylene glycol) methyl ether

10.1021/bm101058w  2011 American Chemical Society Published on Web 12/01/2010

pH-Sensitive Amphiphilic Copolymer Brush

Biomacromolecules, Vol. 12, No. 1, 2011

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Figure 1. Schematic representation of micellization of P(MMA-co-MAA)-PPEGMA and pH-dependent drug release.

monomethacrylate) [P(MMA-co-MAA)-b-PPEGMA] was designed and synthesized via ATRP method. This amphiphilic copolymer brush could self-assemble into core/shell micelles in aqueous solution. The hydrophilic PEGMA is known to be nonimmunogenic, nonantigenic, nontoxicity, and good antifouling effect to a wide variety of proteins, and so on.25,26 The PPEGMA brushes with short side chains distribute on the surface of micelles, providing a compact steric protective layer to maintain the stability of micelles during biological circulation. The dense outer shell formed by PPEGMA can avoid the aggregation between micelles and alleviate the burst release behavior. The hydrophobic MMA block and pH-sensitive MAA block were designed to be a random copolymer structure, which could spread the pH-sensitive area of micelles. When the polymer self-assembles into micelles, MMA and MAA form the micelle core and make the core more sensitive to the pH change of the environment than normal structure.22 The micelle can swell or even dissociate as the pH increases from the strong acid stomach conditions to the neutral intestine conditions and provide the entrapped contents a fast release rate in the intestinal tract. Ibuprofen (IBU), a prominent nonsteroidal anti-inflammatory drug used extensively in the treatment of various musculoskeletal disorders and painful conditions, is a good candidate for the development of oral controlled release formulations.27 It was used as a model drug and encapsulated into P(MMA-co-MAA)-b-PPEGMA assembled micelles. The sensitivity of the IBU-loaded micelles responding to the pH change could be enhanced and drug release rate could be expedited to a large extent as the MAA block length increased. Figure 1 shows the schematic micellization and pH-dependent drug release process of P(MMA-co-MAA)-b-PPEGMA in aqueous solution.

2. Experimental Section 2.1. Materials. Methyl methacrylate (MMA, Alfa Aesar) and tertbutyl methacrylate (tBMA, TCI-EP) were washed with sodium hydroxide solution (10%), distilled from calcium hydride, and stored under argon at -20 °C. CuBr was purified by stirring in acetic acid and washing with ethanol and ether in turn, and then drying in vacumn. PEGMA (Mn ) 300 Da, Aldrich) was purified by passing through a column filled with neutral alumina to remove inhibitor. Toluene was distilled from calcium hydride. Pyrene (Aldrich, 99%), ethyl 2-bromoisobutyrate (EBriB, Aldrich, 99%), N,N,N,N′,N′-pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%), trifluoroacetic acid (TFA), n-hexane, dichloromethane (DCM), methanol, tetrahydrofuran (THF), acetone, and all other reagents were used as received. 2.2. Characterization. FT-IR spectra of P(MMA-co-MAA)-bPPEGMA and related copolymers were obtained in a transmission mode on a Nicolet Nexus for Euro FT-IR spectrophotometer under ambient condition. Samples were ground with KBr and then compressed into pellets. The spectra were taken from 400 to 4000 cm-1. Typically, 32 scans at a resolution of 8 cm-1 were accumulated to obtain one spectrum.

The number average molecular weight (Mn) and polydispersity index (Mw/Mn) were determined by gel permeation chromatography (GPC) adopting an Agilent 1200 series GPC system equipped with a LC quant pump, PL gel 5 µm 500 Å column and RI detector. The column system was calibrated with a set of monodisperse polystyrene standards using HPLC grade THF as mobile phase with a flow rate of 1.0 mL/min at 30 °C. 1 H NMR spectra measurements were executed on a VARIAN Mercury-Plus 300 NMR spectrometer operating at 250 MHz, using deuterated chloroform (CDCl3-d) or deuterated dimethyl sulfoxide (DMSO-d6) as solvent and tetramethylsilane (TMS) as an internal standard. The temperature was 25 °C. The micelle size and distribution (PDI) were determined by dynamic light scattering (DLS) with a Malvern Zetasizer Nano S. The dialyzation solutions (pH 6.8) were filtered through 0.45 µm pore size filters and the measurements were conducted in a 1.0 mL quartz cuvette, using a diode laser of 800 nm at 25 °C and the scattering angle was fixed at 90°. Morphologies of blank and drug-loaded micelles were investigated by scanning electron microscopy (SEM, JEOL JSM-6490LA, Japan) and transmission electron microscopy (TEM, Hitachi JEM-100CXII, Japan). For SEM measurement, the sample was fixed on an aluminum stub as a thin film and coated with gold before observation. SEM was operated at an accelerating voltage of 10 kV and a magnification of 20000× in the transmission electron mode. For TEM measurement, an aqueous droplet of micellar solution with a polymer concentration of 1 mg/mL was placed on a copper-coated grid. The grid was held horizontally for 30 s to allow the colloidal aggregates to settle. A drop of 1% solution of phosphotungstic acid (PTA) in PBS (pH 7.4) was then added to provide the negative stain. After 1 min, the excess fluid was removed by filter paper and the samples were air-dried. TEM images were obtained at a magnification of 19000 or 100000× at 100 kV. 2.3. Synthesis of P(MMA-co-tBMA)-Br Macroinitiator. All synthetic procedures were carried out under an argon atmosphere and complied with a standard ATRP instruction. In a typical experiment, 143.5 mg of CuBr was added to a predried Schlenk flask and sealed with a rubber septum. The flask was degassed and backfilled three times with argon and then left under argon. MMA (3.180 mL), tBMA (5.715 mL), and PMDETA (0.210 mL) were introduced into the flask via syringe in turn and stirred 10 min until the solution became homogeneous and the Cu/PMDETA catalyst complex formed. Then three “freeze-pump-thaw” cycles were performed to remove any oxygen from the polymerization solution. The flask was filled with argon and immersed into a thermostatted oil bath at 85 °C and the initiator EBriB (0.147 mL) was added. After 20 min, the flask was removed from the oil bath and cooled to room temperature. THF was added into the flask to dissolve the solid polymer. The mixture was then passed through a neutral alumina column to remove the catalyst. Finally, poly(methyl methacrylate-co-tert-butyl methacrylate) [P(MMA-co-tBMA)-Br] was recovered by being precipitated into 10-fold excess of water/methanol (1:1, v/v) mixture, filtered, and dried under vacuum for 24 h. 2.4. Synthesis of P(MMA-co-tBMA)-b-PPEGMA. The synthetic procedure of poly(methyl methacrylate-co-tert-butyl methacrylate)-b-

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poly (poly(ethylene glycol)methyl ether monomethacrylate) [P(MMAco-tBMA)-b-PPEGMA] is similar to P(MMA-co-tBMA)-Br. In brief, a ration of P(MMA-co-tBMA)-Br and CuBr was added into a Schlenk flask and sealed up. Three degassed and backfilled cycles with argon, solvent (toluene), and macromonomer (PEGMA) were introduced with syringe and the mixture was conducted with three “freeze-pump-thaw” cycles. A fixed quantity of PMDETA was then injected into the reaction flask. After stirring for 15 min, the flask was transferred into a thermostatted oil bath at 60 °C, and the polymerization proceeded for 24 h. After cooled to room temperature, the polymer solution was diluted with THF. The THF diluted solution was passed through neutral alumina column and precipitated by n-hexane. The final white powder products were obtained after vacuum drying for 24 h. 2.5. Hydrolysis of the tert-Butyl Methacrylate (tBMA). The tertbutyl groups of P(MMA-co-tBMA)-b-PPEGMA were removed by hydrolysis with DCM as solvent and TFA as hydrolytic acid. In a typical experiment, 500 mg of P(MMA-co-tBMA)-b-PPEGMA was dissolved in 5 mL of DCM. After cooling to 0 °C with ice/water bath, 0.89 mL of TFA (10-fold molar of tert-butyl groups) was slowly added with vigorous stirring. The mixture was stirred at 0 °C for 30 min and then at room temperature for 24 h. After evaporating all the solvents, the residues were dissolved in 5 mL of THF and precipitated into 50 mL of n-hexane (10-fold of THF). The resulting polymer brush P(MMAco-MAA)-b-PPEGMA was collected by filtration and dried in a vacuum oven at 40 °C for 48 h. 2.6. CMC Measurement. The CMC of P(MMA-co-MAA)-bPPEGMA was determined by the fluorescence probe technique using pyrene as a fluorescence probe. When polymeric micelles formed, pyrene was preferentially distributed in the hydrophobic micelle core instead of in the hydrophilic outer shell, thus the environment of pyrene turned from polar to nonpolar. The red shift of the third peak indicates that pyrene molecules have been transferred to the less polar domains of micelle core. In a typical experiment, a solution of pyrene (12 × 10-5 M) in acetone was prepared in advance. The polymer was first dissolved into acetone and then confected to a concentration of 0.1 mg/mL with deionized water. After the acetone was evaporated by stirring for 24 h, the polymer solution was diluted to a series of concentrations from 0.0001 to 0.1 mg/mL with deionized water. Pyrene solution (0.1 mL) was added to a vial and the acetone was allowed to evaporate to form a thin film at the bottom of the vial. Then polymer solutions at different concentrations were added to the vials, respectively, and the final concentration of pyrene was 6 × 10-7 M in water. The combined solution of pyrene and copolymer was equilibrated at room temperature in dark for 24 h before measurement. Fluorescence spectra were obtained using a fluorescence spectrophotometer (F-4500, Hitachi) and the excitation spectra of polymer/ pyrene solutions were scanned from 300 to 350 nm at room temperature, with an emission wavelength of 373 nm and a bandwidth of 0.2 nm. The intensity ratios of I337 to I333 were plotted as a function of logarithm of polymer concentrations. The CMC value was taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations. 2.7. Preparation of Blank and IBU-Loaded Micelles. The core/ shell micelles of P(MMA-co-MAA)-b-PPEGMA were formed using the diafiltration method. In a typical experiment, 10 mg of P(MMAco-MAA)-b-PPEGMA was dissolved in 10 mL of DMF, and 2 mL of deionized water was added dropwise into the mixture with vigorous stirring. The polymer solution was then dialyzed against deionized water for 48 h at 20 °C using a preswollen cellulose membrane bag (molecular weight cutoff, MWCO 3500-4000). The deionized water was replaced every 2 h for the first 12 h and then replaced every 6 h. Subsequently, the solution was lyophilized after being filtered through a 0.45 µm syringe filter, and dried micelle products were obtained in powder. IBU-loaded micelles were prepared using the same procedure. Briefly, IBU (5 mg) and P(MMA-co-MAA)-b-PPEGMA (20 mg) were dissolved in 20 mL of DMF and stirred for 3 h at room temperature, followed by dialysis against deionized water for 24 h. The dialysate

Yang et al. was filtered by a membrane filter (0.45 µm pore) to remove aggregated particles. The filtrate was lyophilized and the received white powder was stored at -20 °C until further experiments. The size and morphology of the blank and IBU-entrapped micelles were monitored by DLS, SEM, and TEM. IBU loading content (LC) and entrapment efficiency (EE) were determined by UV-vis spectrophotometer (UV-2450, Shimadzu, Japan) at 219 nm. A total of 1 mg of the IBU-loaded micelle powder was dissolved in 10 mL of ethanol under vigorous vortexing. The concentration of IBU was calculated according to a standard curve of pure IBU/ ethanol solution. The LC was defined as the weight ratio of loaded drug to the drug-entrapped micelles. EE of IBU was obtained from the weight ratio between the drug incorporated in assembled micelles and that used in the fabrication. 2.8. In Vitro Release of IBU from P(MMA-co-MAA)-b-PPEGMA Micelles. The in vitro IBU release properties from the P(MMAco-MAA)-b-PPEGMA assembled micelles were determined as follows: 5 mg of IBU-entrapped micelle was suspended in 5 mL of simulated gastric fluid (SGF, 0.15 M HCl, 0.05 M KCl, pH 1.2) or simulated intestinal fluid (SIF, 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, 1 L water, pH 7.4) and then placed in a dialysis bag (MWCO 3500-4000). The whole bag was placed into 25 mL of SGF or SIF buffer and shaken (100 rpm) at 37 °C (Dissolution Tester RCZ-8B, TDTF, China). At specified time intervals, a 2 mL (Ve) sample was taken and the same volume of fresh buffer was added to maintain the total volume. The concentrations of IBU in different samples were determined by UV-vis spectrophotometer. The cumulative drug release percent (Er) was calculated using eq 1. The in vitro experiments were repeated three times to get the final release curves.

n-1

Ve Er(%) )

∑C + V C i

1

mIBU

0 n

× 100

(1)

where mIBU represents the amount of IBU in the micelle, V0 is the whole volume of the release media (V0 ) 30 mL), and Ci represents the concentration of IBU in the ith sample.

3. Results and Discussion 3.1. Synthesis and Characterization of the P(MMA-coMAA)-b-PPEGMA Brush. P(MMA-co-MAA)-b-PPEGMA brush was synthesized via twice ATRP followed by selective hydrolysis of the tert-butyl groups of tBMA. As shown in Scheme 1, EBriB was utilized as initiator and Cu/PMDETA complex as the catalyst system for the polymerization of P(MMA-co-tBMA)-Br macroinitiator at 85 °C in bulk.28,29 Subsequently, P(MMA-co-tBMA)-b-PPEGMA was synthesized using the same catalyst complex and the polymerization was carried out in toluene at 60 °C. The final polymer brush was obtained by selective hydrolysis conducted in DCM employing TFA as acid agent. The molecular weight ratio of MMA and MAA was set to 50:50 or 40:60 in the final brush, while a series of P(MMAco-tBMA)-Br macroinitiators were fabricated with different block lengths of MMA and tBMA. Table 1 shows the details of polymer products. For the macroinitiators, the rates of copolymerization in bulk were reasonably fast and the Mw/Mn were below 1.2 (entries 1 and 2). The results indicate that ATRP is a highly active polymerization technique with favorable molecular weight and distribution control.28,30 The macroinitiators were further purified by thrice precipitation and then applied to the following polymerization of macromonomer PEGMA.



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Scheme 1. Synthetic Route of P(MMA-co-MAA)-b-PPEGMA Copolymer Brush

Table 1. Conditions and GPC Results of Copolymer Products

d

entry

sample

MMA/MAAa

solvent

T (°C)

1 2 3 4 5 6 7 8 9

P(MMA3k-co-tBMA5k)-Br P(MMA3k-co-tBMA7.5k)-Br P(MMA3k-co-tBMA5k)-b-PPEGMA5k P(MMA3k-co-tBMA5k)-b-PPEGMA5k P(MMA3k-co-tBMA7.5k)-b-PPEGMA5k P(MMA3k-co-tBMA7.5k)-b-PPEGMA5k P(MMA3k-co-tBMA5k)-b-PPEGMA5k P(MMA3k-co-MAA3k)-b-PPEGMA5kd P(MMA3k-co-MAA4.5k)-b-PPEGMA5kd

50/50 40/60 50/50 50/50 40/60 40/60 50/50 50/50 40/60

bulk bulk toluene THF toluene THF acetone DCM DCM

85 85 60 60 60 60 60 25 25

a The block length ratio of MMA/MAA in polymers. Hydrolysis of tert-butyl group with TFA/DCM.

b

Calculated by theory analysis.

P(MMA-co-tBMA)-b-PPEGMA brushes were prepared by solvent-ATRP. The solvents, such as toluene, THF, or acetone, can be served as reaction medium. The Mw/Mn were a bit increased (entries 3-7) after PEGMA polymerization. In most cases, toluene is better than other solvent systems for its appropriate polarity and favorable dissolving capacity of macroinitiator and monomer, although it is not a good solvent for the catalyst complex because it makes the polymerization heterogeneous. This can be seen in entries 3, 4, and 7, which used the same macroinitiator. The reaction temperature can range from room temperature to 90 °C, expressed by refs 26 and 31. In the current work, we found that 60 °C was the best temperature. The polymerization proceeded too slowly at room temperature and the Mw/Mn increased at high temperature. After passed through a neutral alumina column, the polymer was purified by precipitation (n-hexane as precipitant). The hydrolysis was executed under mild condition utilizing TFA/DCM system.32,33 With the hydrolyzation performing, the number of tert-butyl groups of P(MMA-co-tBMA)-b-PPEGMA were gradually reduced along and the solution became turbid (slight yellow) in respect that P(MMA-co-MAA)-b-PPEGMA is insoluble in DCM. The favorite reaction time of hydrolysis was 12 h. Entries 8 and 9 show the Mn and Mw/Mn of the final polymer brush. GPC was employed to evaluate Mn and Mw/Mn of the macroinitiator and copolymer brushes. Figure 2 (entries 1, 3, and 8 in Table 1) shows the GPC traces of the serials polymer products using the same macroinitiator. All the results appear as a monomodal symmetric distribution, indicating a wellcontrolled polymerization process and the effectiveness of purification. It can also be seen in Table 1 that, after the polymerization of PEGMA, the Mn increased from 8765 (P(MMA3k-co-tBMA5k)-Br) to 14593 g/mol (P(MMA3k-cotBMA5k)-b-PPEGMA5k). The Mw/Mn of the final brush P(MMA3kco-MAA3k)-b-PPEGMA5k rose up a little (1.21, entry 8 in Table 1), more than the macroinitiator (1.12, entry 1 in Table 1), but

c

time 20 20 24 24 24 24 24 12 12

min min h h h h h h h

Mnb

Mnc

Mw/Mnc

8000 10500 13000 13000 15500 15500 13000 11000 12500

8765 11836 14593 13359 17257 15151 13262 10344 11799

1.12 1.15 1.17 1.23 1.20 1.28 1.36 1.21 1.19

Measured by GPC in THF, calibrated against PS standards.

Figure 2. GPC traces of P(MMA3k-co-tBMA5k)-Br (a, entry 1), P(MMA3k-co-tBMA5k)-b-PPEGMA5k (b, entry 3), and P(MMA3k-coMAA3k)-b- PPEGMA5k (c, entry 8).

still lower than the theoretical value of 1.5 for the controlled/ “living” free radical polymerization.34-36 The similar molecular weight change was found during the synthesis of P(MMA3kco-MAA4.5k)-b-PPEGMA5k of entry 9. The chemical structures of the obtained P(MMA-co-tBMA)Br, P(MMA-co-tBMA)-b-PPEGMA, and P(MMA-co-MAA)b-PPEGMA were confirmed by 1H NMR and FT-IR spectroscopy. Representative 1H NMR spectra were depicted in Figure 3. Figure 3a is the 1H NMR spectrum of P(MMA3k-co-tBMA5k)Br (entry 1 in Table 1). The signals at 0.8-1.2, 1.7-2.0, and 3.60 ppm are ascribed, respectively, to -CCH3, -CH2-, and -OCH3 of the MMA unit, while the signal at 1.42 ppm is the characteristic resonance of -C(CH3)3 in the tBMA unit. The characteristic PEGMA peaks of P(MMA3k-co-tBMA5k)b-PPEGMA5k (entry 3 in Table 1) and P(MMA3k-co-MAA3k)b-PPEGMA5k (entry 8 in Table 1) at 3.38 and 3.66 ppm due to -OCH3 and -OCH2-CH2- protons, respectively, can be clearly seen in the 1H NMR spectrum shown in Figure 3b. After

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Yang et al.

Figure 5. Graph of intensity ratios (I337/I335) as a function of logarithm of P(MMA3k-co-MAA3k)-b-PPEGMA5k (a) and P(MMA3k-co-MAA4.5k)b-PPEGMA5k (b) concentrations in aqueous solution.

Figure 3. 1H NMR spectra of P(MMA3k-co-tBMA5k)-Br (a), P(MMA3kco-tBMA5k)-b-PPEGMA5k (b) in CDCl3-d, and P(MMA3k-co-MAA3k)-bPPEGMA5k (c) in DMSO-d6.

Figure 6. The sizes and PDIs of blank (a) and IBU-loaded (b) P(MMA3k-co-MAA3k)-b-PPEGMA5k micelles, and blank (c) and IBUloaded (d) P(MMA3k-co-MAA4.5k)-b-PPEGMA5k micelles determined by DLS. Figure 4. FT-IR spectra of P(MMA3k-co-tBMA5k)-b-PPEGMA5k (a) and P(MMA3k-co-MAA3k)-b-PPEGMA5k (b).

hydrolysis, the tert-butyl group (1.42 ppm) disappears while the carboxyl group (12.30 ppm) emerges, which can be seen in Figure 3c. Figure 4 reveals the representative FT-IR spectra of P(MMA3kco-tBMA5k)-b-PPEGMA5k (a, entry 3 in Table 1) and P(MMA3kco-MAA3k)-b-PPEGMA5k (b, entry 8 in Table 1). The bands of 1734 and 1113 cm-1 are assigned, respectively, to the CdO and C-O stretching vibrations for carbonyl group of carboxylic ester existed in all repeated units. The bands of 2871 and 1470-1380 cm-1 represent the stretching and bending vibrations of -CH2- in the copolymer backbone. The -COOH of P(MMAco-MAA)-b-PPEGMA has been confirmed by 1H NMR and is also obvious in the FT-IR spectrum, as Figure 4b shows. The broad absorbance characteristic from 2800 to 3600 cm-1 is the stretching vibration of -OH in a carboxylic acid group.37,38 3.2. CMC Value of P(MMA-co-MAA)-b-PPEGMA. To confirm the formation of micelles self-assembled from P(MMAco-MAA)-b-PPEGMA brush, CMC values of P(MMA-coMAA)-b-PPEGMA brushes were measured by fluorescence spectroscopy based on selective partition of fluorescence probe in hydrophobic phase against aqueous phase. It has been reported that fluorescence spectra of pyrene solutions contain a vibrational

band exhibiting high sensitivity to the polarity of the pyrene environment.13,39 As concentrations of P(MMA-co-MAA)-bPPEGMA increased, the fluorescence intensity increased and the third peak shifted from 335 to 337 nm in the excitation spectra of pyrene. The ratios of I337 to I335 were plotted against polymer concentrations. As shown in Figure 5, the CMC values of P(MMA3k-co-MAA3k)-b-PPEGMA5k and P(MMA3k-coMAA4.5k)-b-PPEGMA5k were determined to be 1.3 and 3.8 mg/L, respectively. Due to the extremely low CMC values, it is suggested that the micelles formed from P(MMA-co-MAA)b-PPEGMA brush would provide good stability in solution, even after extreme dilution by the larger volume of systemic circulation in the body. 3.3. Characterization of the Blank and IBU-Loaded Micelles. The particle sizes, PDIs, and morphologies of micelles may be different due to selecting different solvents and operating techniques. In this work, two polymer materials (entries 8 and 9 in Table 1) were used for comparison and a Malvern Zetasizer was utilized to measure the average hydrodynamic diameters of the micelles. Figure 6 shows the average diameters and PDIs of blank and IBU-loaded micelles prepared from P(MMA-coMAA)-b-PPEGMA. The IBU-loaded micelles showed a larger size than the plain micelles. It suggested that IBU was incorporated into the micelles effectively. The diameter of



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Table 2. Physical Properties of Blank and IBU-Loaded Assembled Micelles polymer

micelle

Dh (DLS, nm)

PDI

zeta potential (mV)

P(MMA3k-co-MAA3k)-b-PPEGMA5k P(MMA3k-co-MAA3k)-b-PPEGMA5k P(MMA3k-co-MAA4.5k)-b-PPEGMA5k P(MMA3k-co-MAA4.5k)-b-PPEGMA5k

blank IBU-loaded blank IBU-loaded

122 249 138 220

0.093 0.127 0.092 0.045

-29.6 -20.9 -31.7 -26.0

P(MMA3k-co-MAA3k)-b-PPEGMA5k micelles without any drug entrapped was 122 nm (Figure 6a), and the mean size of IBUloaded micelles increased to 249 nm (Figure 6b). For P(MMA3kco-MAA4.5k)-b-PPEGMA5k micelles (Figure 6c,d), the diameter increased from 138 to 220 nm after being entrapped with IBU. The nanoscale size and narrow unimodal PDI indicate that the P(MMA-co-MAA)-b-PPEGMA assembled micelles possess good physical performance as nanocarriers for poorly watersoluble drug. The zeta-potentials of self-assembled micelles with different MAA block length were measured to be both highly negative charges (approximately -30 mV at pH ) 6.8). Although screened by the PPEGMA brush in the micelle shell, the carboxyl acid groups of MAA could be ionized at a pH above 5. The zeta-potentials of micelles decreased a bit but still above -20 mV after entrapped IBU. The highly negative zetapotentials suggest that the P(MMA-co-MAA)-b-PPEGMA micelles are mucoadhesive which can provide adhesive interactions with gastrointestinal mucus and cellular linings, traverse the mucosal absorptive epithelium, maintain the micelles in the intestinal tract for extended periods of time and facilitate the bioadhesion between the micelle carriers and intestinal epithelial cells.40 The characteristics of blank and IBU-loaded micelles were summarized in Table 2. Figure 7 presents the SEM and TEM images of blank and IBU-loaded P(MMA3k-co-MAA4.5k)-b-PPEGMA5k micelles. It can be seen that the micelles took a spherical morphology. The average diameters calculated from SEM and TEM images were nearly consistent with the DLS results. Interestingly, both micelles have similar morphology and narrow PDI, suggesting that the small drug molecules changed hardly the self-assembly process and morphologies of the P(MMA-co-MAA)-b-PPEGMA micelles. The SEM and TEM images also confirmed the size increasing of micelles during drug entrapment, the average size increased about 100 nm after loaded with IBU.

Figure 7. SEM and TEM images of blank (a, c) and IBU-loaded (b, d) P(MMA3k-co-MAA4.5k)-b-PPEGMA5k assembled micelles.

Table 3. Loading Contents and Entrapment Efficiencies of IBU-Loaded Assembled Micelles polymer

weight (mg)

IBU (mg)

LC (%)

EE (%)

P(MMA3k-co-MAA3k)-b-PPEGMA5k P(MMA3k-co-MAA3k)-b-PPEGMA5k P(MMA3k-co-MAA3k)-b-PPEGMA5k P(MMA3k-co-MAA4.5k)-b-PPEGMA5k P(MMA3k-co-MAA4.5k)-b-PPEGMA5k P(MMA3k-co-MAA4.5k)-b-PPEGMA5k

20 20 20 20 20 20

3 5 6 3 5 6

9.5 16.8 17.1 11.5 18.7 19.2

70.0 80.8 68.8 86.6 92.0 79.4

The freeze-dried IBU-loaded micelles were used for LC and EE measurements and in vitro drug release. Table 3 illustrates the LC and EE values of IBU-loaded micelles. It can be seen that weight ratios of drug to polymer had a great influences on the LC and EE and 5/20 mg/mg was the best ratio in the current work.19 Both self-assembled micelles with MMA/MAA block length ratio of 50/50 or 40/60 exhibited the same trend. The maximum of IBU LC and EE was 16.8% and 80.8% for P(MMA3k-co-MAA3k)-b-PPEGMA5k micelles, and that was 18.7% and 92.0% for P(MMA3k-co-MAA4.5k)-b-PPEGMA5k micelles, respectively. This may be that the larger core of long MAA block polymer micelles could accommodate even more drug. 3.4. In Vitro Release of IBU from P(MMA-co-MAA)-bPPEGMA Micelles. In vitro release profiles of two kinds of IBU-entrapped micelles with different MAA block lengths were evaluated at 37 °C in SGF (pH 1.2) and SIF (pH 7.4), respectively, and shown in Figure 8. At pH 1.2, the release rates of IBU were slow, with only 14% of IBU released in 10 h and 17% after 24 h. The P(MMA-co-MAA)-b-PPEGMA micelles were tight in SGF, and IBU was released mainly by diffusion.19,41 In the case of pH 7.4, the release rates of IBU were greatly accelerated without obviously burst release. The IBU release rate was also associated with the block length ratio of MMA and MAA. As MMA and MAA were defined as random copolymer form, the sensitivity of the micelle core responding to the pH change was enhanced as the MAA ratio increased. So the release rate of IBU was speeded up by improving the

Figure 8. In vitro drug release profiles of IBU-loaded P(MMA-coMAA)-b-PPEGMA assembled micelles.

122


MAA block ratio in the P(MMA-co-MAA)-b-PPEGMA. This was confirmed by the in vitro release results. As for P(MMA3kco-MAA3k)-b-PPEGMA5k, 58% of IBU was released in 8 h and 66% after 24 h, with the rest of IBU released completely after 72 h (not shown in the release curve). This implied that the micelles had not swelled enough to promote the drug release. When MAA block length was longer for P(MMA3k-co-MAA4.5k)b-PPEGMA5k, more than 90% of IBU had released in 8 h and all the loaded IBU was released within 12 h, indicating that the micelle structure had swollen enough or even dissociated which made IBU release expeditiously. These pH-sensitive drug release behaviors could attribute to three factors. The first one is the higher solubility of IBU in neutral or alkaline media.14,42 And the second one is that the electrostatic repulsion between negatively charged carboxyl groups of IBU and MAA in SIF. Most importantly, at pH 7.4, the ionization of carboxyl groups of MAA contributes to the swell or even dissociation of micelle structure (if the amount of carboxyl groups is sufficient), then the entrapped IBU could completely released in intestinal tract.43 The in vitro release results show that the P(MMA-co-MAA)-b-PPEGMA micelles can enable the entrapped drug’s release or absorption to take place preferentially in the intestine while avoiding or reducing drug leakage in the stomach. Thus, the micelles self-assembled from pH-sensitive P(MMA-co-MAA)-b-PPEGMA brush can be potentially used as an oral carrier for poorly water-soluble drugs.

4. Conclusions A novel amphiphilic copolymer brush P(MMA-co-MAA)b-PPEGMA was successfully synthesized via twice ATRP followed by hydrolysis of tert-butyl groups and characterized by GPC, 1H NMR, and FT-IR. The results indicated that welldefined products with specific block length ratio and favorable Mw/Mn (=1.2) were acquired. The CMC values of P(MMAco-MAA)-b-PPEGMA in aqueous medium were found to be very low, suggesting a extremely high stability of the assembled micelles. The average size of IBU-incorporated micelles was examined to be 120-250 nm with spherical shape. The in vitro release behaviors of IBU from P(MMA-co-MAA)-b-PPEGMA micelles exhibited pH-dependence. Less than 20% of loaded IBU released in simulated gastric fluid over 24 h, but in simulated intestinal fluid, nearly all the initial drug content released within 12 h. The overall results demonstrate that P(MMA-co-MAA)-b-PPEGMA micelles can deliver hydrophobic drugs to the intestine with little drug leakage in the stomach, and thus, could be employed as a promising vehicle for orally administered poorly water-soluble drug delivery system. Acknowledgment. This work was financially supported by National Natural Science Foundation of China (Nos. 20536020 and 20776049), Guangdong Province Science Foundation (No. 9251064101000009), Guangzhou Municipal Bureau of Science and Technology (No. 2009J1-C511-2), and State Key Laboratory of Chemical Engineering (No. SKL-ChE-09A04).

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BM101058W

pH-Sensitive Micelles Self-Assembled from Amphiphilic Copolymer

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