pH-Sensitive Micelles with Cross-Linked Cores ... - ACS Publications

ARTICLE pubs.acs.org/Langmuir

pH-Sensitive Micelles with Cross-Linked Cores Formed from Polyaspartamide Derivatives for Drug Delivery Wang Lin and Dukjoon Kim* Department of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon, Kyunggi 440-746, Korea ABSTRACT: A series of polyaspartamide derivatives were synthesized by grafting O-(2-aminoethyl)-O0 -methylpoly(ethylene glycol) 5000 (MPEG), 1-(3-aminopropyl) imidazole (API), and cinnamate onto polysuccinimide (PSI) with the respective degrees of substitution adjusted by the feed molar ratio. The chemical structure of the prepared polymer was confirmed using FT-IR and 1H NMR spectroscopy. A new pH-sensitive polymeric micelle based on the synthesized polymer was prepared and characterized, and its pH-sensitive properties were characterized by the measurement of light transmittance and particle sizes at varying pH values. pH-dependent aggregation and deaggregation behavior was clearly observed in the polymer aqueous dispersion system. Photo-cross-linking of the cinnamate branches cross-linked the core of the micelles. The core cross-linked micelles showed high stability over a wider pH range and displayed obvious pH-dependent swelling
shrinking behavior instead of micelle
unimer transition behavior. This micelle system overcame the drawback of the facile disintegration of normal polymeric micelles and showed obvious delayed paclitaxel release in in vitro drug delivery experiments.

’ INTRODUCTION Micelles formed from synthetic amphiphilic polymers have attracted significant attention as carriers for the delivery of lowsolubility drugs, proteins, genes, and diagnostic imaging agents.1
4 Initially, one of the most intensively studied topics in the developing field of micelles is micelles formed from amphiphilic block copolymers, which provide a simple model for the study of micelle morphology and properties.5 To date, more and more multifunctional micelles, so-called “smart” drug carriers, have been reported.6 Compared to block copolymers, grafted copolymers can introduce a variety of specific pendent groups and thus more easily modify the polymer properties associated with each specific function. For this reason, grafted copolymers are currently of increasing interest for the design of multifunctional drug delivery systems.7
11 A variety of polymers may be chosen to build the core
shell architecture of the polymeric micelles. However, from a pharmacological point of view, poly(amino acid) and its derivatives are of particular interest owing to their superior biocompatible, biodegradable, and nontoxic properties.12
14 Polysuccinimide (PSI) is an intermediate product typically used in the preparation of many poly(amino acid) derivatives. Because the succinimide rings in the PSI readily react with primary amines, many different specific groups can be introduced into the polymer backbone to modify the polymeric properties.15
19 Poly(ethylene glycol) (PEG) is usually used as a source of hydrophilic chains in micelle drug carriers because this polymer is known to be highly hydrated, soluble, nontoxic, nonimmunogenic, and able to serve as an efficient steric protector for microparticulates in biological media.20 Different organs, tissues, and cellular compartments may possess large differences in pH, which makes the pH a suitable r 2011 American Chemical Society

stimulus for drug release. The extracellular pH of tumor tissue is approximately 7.0, which is slightly lower than the normal pH of 7.4. After cellular uptake and en route to the lysosome, the pH inside the endosome changes from 7.0 to 5.0.21 Polymers containing imidazole functionalities such as poly(histidine) and poly(4vinylimidazile) have been examined for potential application as intracellular drug delivery systems for endosomal release. The cationic imidazole ring possesses a lone electron pair on an unsaturated nitrogen and is thus easily protonated to give a pKa value of 6.0. These imidazole rings provide a trigger for endosomal drug release and enable the polymers to mediate endosomal escape through the hypothesized proton sponge effect. In spite of these advantages, poly(histidine) and poly(4-vinylimidazole) have some restrictions in their broad biomedical applications primarily because of the difficulty in synthesizing poly(histidine) and the nonbiodegradability of poly(4-vinylimidazole), respectively.22
30 This laboratory’s previous studies reported the preparation of imidazole-containing pH-sensitive poly(amino acid) derivatives by grafting 1-(3-aminopropyl)imidazole (API) onto PSI for drug delivery application, showing a sharp phase transition behavior at a pH near 7.31,32 However, as a delivery system, all polymeric micelles are burdened with the drawback of disintegration after dilution into body fluids, resulting in premature drug release.33 In this study, to redress this problem, cinnamate, with a photocross-linkable double bond, was chosen as a cross-linker to reinforce the micelle core
shell structure. Cinnamic acid, a naturally occurring aromatic fatty acid of low toxicity, is widely Received: January 11, 2011 Revised: July 13, 2011 Published: August 23, 2011 12090

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Langmuir used in flavors, synthetic indigo, and pharmaceuticals, with even some growth-inhibitory effects on human tumor cell lines.34,35 The photo-cross-linkable cinnamate groups were introduced into the grafted polyaspartamide derivatives to form hydrophobic micelle cores. It was predicted that the resulting cross-linked micelles would maintain their small size and core
shell morphology, whereas their dissociation upon dilution and pH changes would be permanently suppressed. The premature drug release before the acid stimuli would be effectively restrained, and the circulating time of the micelle-loaded drugs would be prolonged. All of the proposed properties of the resulting micelles with a cross-linked core were investigated and confirmed in this work. The pH-sensitive micelle
unimer and shrinking
swelling behavior of the resulting amphiphilic polymer aqueous dispersion system, before and after cross-linking, was investigated by light transmittance and particle size measurements. Hydrophobic antitumor drug paclitaxel was loaded into this pH-sensitive micelle, and the drug-release behavior at different pH values was studied in vitro.

’ EXPERIMENTAL SECTION Materials. L-Aspartic acid, N,N-dimethylformamide (DMF), phosphoric acid (85%), mesitylene, sulforane, dimethyl sulfoxide-d6 (DMSO-d6), 1-(3-aminopropyl)imidazole, ethanolamine, phosphate-buffered saline (PBS 7.4), and cinnamyl chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA), and O-(2-aminoethyl)-O0 -methylpoly(ethylene glycol) 5000 (MPEG5000-NH2) was purchased from Fluka (Switzerland). Paclitaxel (PTX) was supplied by Baosai (China). Polysuccinimide (PSI) was synthesized from L-aspartic acid, following previous work.31 The product was washed with methanol and dried in a vacuum oven at 70 °C for 1 day. The weight-average molecular weight of the prepared PSI was 51 000 g mol
1. Synthesis of MPEG/Imidazole/EA-g-polyaspartamides. To prepare the MPEG-grafted PSI, MPEG5000-NH2 (0.2 mmol) was dissolved in 10 mL of DMF. The MPEG solution was added dropwise to the PSI solution (0.1 g mL
1 DMF) at room temperature. The reaction mixture was stirred continuously under a N2 atmosphere at 70 °C. After 30 h, the desired amount of 1-(3-aminopropyl) imidazole was added to the solution and reacted under the same conditions for another 12 h. Then, excess ethanolamine (EA) was added to the solution over 6 h to aminolyze the residual succinimide unit. The product mixture was dialyzed against distilled water using a dialysis membrane (molecular weight cutoff 10 000
12 000 g mol
1) to remove residual MPEG, 1-(3-aminopropyl) imidazole, and ethanolamine; the product was then freeze dried. 1 H NMR: δ = 7.62 (
N
CHdN
), 7.16 (
N
CHdCH
), 6.88 (
CH
CHdN
), 4.70
4.50 (
NH
CH
CdO), 3.92 (dN
CH2
CH2
), 3.52 (
O
CH2CH2
O
of the MPEG chain), 3.10 (
NH
CH2
CH2
OH), 2.97 (
CH
CH2
CdO
), 1.80 (
CH2
CH2
CH2
). Synthesis of MPEG/Imidazole/Cinnamate-g-polyaspartamides. The prepared copolymer powder was dissolved in 15 mL of dry DMF and reacted overnight with 1.2 molar equiv of cinnamyl chloride in the presence of pyridine at room temperature. The reaction mixture was dialyzed against methanol using a dialysis membrane (molecular weight cutoff 10 000
12 000 g mol
1) to remove pyridine and residual cinnamyl chloride. Then, the purified product was dissolved in methanol, dialyzed against distilled water to change the solvent to pure water, and then freeze dried. 1 H NMR: δ = 7.62 (
N
CHdN
), 7.48 (C6H5
CHdCH
), 7.4 (C6H5
CHdCH
), 7.16 (
N
CHdCH
), 6.88 (
CH
CHdN
), 6.55 (C 6 H5
CHdCH
), 4.70
4.50 (
NH
CH
CdO), 3.92

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(
NH
CH2
CH2
), 3.52 (
O
CH 2CH2
O
of the MPEG chain), 3.10 (
NH
CH2
CH2
OH), 2.97 (
CH
CH2
CdO
), 1.80 (
CH2
CH2
CH2
).

Preparation of PTX-Loaded Micelles with Cross-Linked Cores. PTX was loaded into pH-sensitive polymeric micelles by the pH-induced micellization method. A paclitaxel ethanol solution (1.0 mg in 1 mL) was added to the 10 mL pH-sensitive polymer-containing PBS solution (1 g L
1) at pH 5. At this polymer concentration, 1 mg mL
1, which was much higher than the critical aggregation concentration (CAC), 0.005
0.01 mg mL
1,31,32 the formation of micelles would be expected by an increase in pH. The mixture was vigorously stirred, and the pH was adjusted to 7.4 to achieve PTX-loaded micelles. Thereafter, the mixture was stirred vigorously for 2 h at 40 °C to remove small amounts of ethanol. The amount of PTX loaded into the prepared micelles was determined by reverse-phase HPLC. The prepared micelles were filtered through a 0.45 μm filter into an irradiation cell, bubbled with nitrogen for 20 min, and then exposed to UV irradiation for 2 h to yield a core that was entirely photo-cross-linked. The filtration process was conducted to remove any atmospheric dirt, impurities, and free drug aggregates. Because the solubility of PTX is quite low in water, less than 1 μg mL
1, free drug is easily aggregated in this aqueous PBS medium. In this drug-loading process, hydrophobic PTX was dispersed in hydrophobic PSI cores surrounded by a hydrophilic MPEG corona at pH 7.4.

In Vitro PTX Release from pH-Sensitive Polymeric Micelles. In vitro release profiles of PTX from micelles were investigated in release media comprising PBS solution (pH 7.4 and 5) containing 2.4 wt % Tween 80 and 4 wt % Cremophor EL. This PBS release media is frequently used for PTX release experiments because the solubility of PTX is reported to be enhanced by the presence of Tween 80 and Cremophor EL without a noticeable influence on the polymer micelle structure and size.36,37 The PTX-loaded micelles (3.0 mL) were introduced into a dialysis membrane tube (molecular weight cutoff 3500 g mol
1). The membrane tube was immersed in 30 mL of the release medium, which was thermostated in a shaking water bath (30 strokes min
1) at 37 °C. At predetermined time internals, the membrane tube containing the micelles was removed from the medium tube and placed into another tube containing the same amount of fresh medium under identical release conditions. The released amount of PTX in every release medium tube was measured by HPLC, and the cumulative released amount of PTX was calculated by summing the amount of released PTX in all medium tubes. Characterization. 1H NMR and FT-IR Spectroscopy. 1H NMR and FT-IR spectra were employed to identify the chemical structure of the synthesized polymers. All 1H NMR spectra were measured using a 500 MHz NMR spectrometer (Unity Inova 500, Varian, Santa Clara, CA, USA) for samples dissolved in DMSO-d6. All FT-IR spectra were recorded with a Fourier transform infrared spectrometer (Unicam1000, Mattson, Fremont, CA, USA). The samples were prepared as KBr powers. Light Transmittance. The light transmittance of the polymer solution of PBS (initially 1.0 g L
1) was measured at varying pH values using a UV spectrophotometer at a 500 nm wavelength (U-3210, Hitachi, Japan). The pH value was gradually lowered by adding 0.1 N HCl after adjusting the pH of the initial polymer solution to 9. The percentage relative transmittance was obtained by comparing the transmittance of the polymer solution at a given pH with PBS at 7.4. Particle Size and Swelling Ratio. To investigate the mean particle size and size distribution of the prepared micelles, electrophoretic light scattering (ELS) measurements were performed using an ELS-Z2 (ELS8000, Otsuka Electronics, Japan) with a laser light wavelength of 638 nm and a scattering angle of 165°. The concentration of the polymer solution in PBS for those measurements was approximately 1.0 mg mL
1. Prior to measurement, the polymer solution was filtered through a 0.45 μm filter to remove dirt, impurities, and free drug aggregates, and the pH of the polymer solution was adjusted using 0.1 N HCl or 0.1 N 12091

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Scheme 1. Overall Synthesis Scheme of MPEG/Cinnamoyl/Imidazole-g-polyaspartamide

NaOH standard aqueous solutions. The swelling ratio (SR) was defined as follows SR ¼ V sw ¼ V sh ¼

V sw V sh

ð1Þ

1 6πdsw 3

ð2Þ

1 6πdsh 3

ð3Þ

column (Zorbax 300SB-C18, 5 μm) were used for the analysis of PTX. The mobile phase was acetonitrile
water (60:40 v/v) at a flow rate of 0.5 mL min
1. PTX concentrations were obtained using a calibration curve prepared for PTX dissolved in acetonitrile.

’ RESULTS AND DISCUSSION Synthesis and Characterization of Amphiphilic Polyaspartamide Copolymers. The polymers were synthesized according

where Vsw and Vsh are the mean volumes and dsw and dsh are the mean diameters of the nanospheres in the swelling and shrinking states, respectively. HPLC Analysis of PTX. Reverse-phase HPLC was performed with an NS-4000 system composed of a quaternary gradient pump, a variably programmable UV/vis detector, and data management software (Multichro 2000). UV/vis detection at 227 nm and a reverse-phase

to Scheme 1. The PSI was synthesized by the thermal condensation of L-aspartic acid in the presence of phosphoric acid. The chemical structures of the synthesized polymers were confirmed by FT-IR and 1H NMR. Figure 1 shows the FT-IR spectra of synthesized polymers. According to previous studies, PSI has characteristic absorption peaks of the succinimide ring at 1727, 1393, 1272, and 1263 cm
1.31 After the graft reaction, the peaks relating to succinimide disappeared, with new peaks relating to acylamide and imidazole rings appearing. The spectrum of 12092

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MPEG/API/EA-g-polyaspartamide (a) showed characteristic peaks at 1654 cm
1 (CdO of acylamide), 1542 cm
1 (C
N of acylamide), 1110 and 1080 cm
1 (C
H of imidazole), and

Figure 1. FT-IR spectra of (a) MPEG/API/EA-g-polyaspartamide and (b) MPEG/API/cinnamate-g-polyaspartamide, respectively.

Figure 2. 1H NMR spectra of (a) MPEG/API/EA-g-polyaspartamide and (b) MPEG/API/cinnamate-g-polyaspartamide, respectively.

917 cm
1 (deformational vibrations of the heterocycle). Although the absorption peaks of the benzene ring of cinnamate nearly overlapped with that of the heterocycle, an additional peak at 1710 cm
1 (CdO of cinnamate) was observed in the MPEG/API/cinnamate-g-polyaspartamide spectrum (b). The 1H NMR spectra of the MPEG/API/EA-g-polyaspartamide and MPEG/API/cinnamate-g-polyaspartamide are shown in Figure 2. The 1H NMR spectrum of MPEG/API/EA-gpolyaspartamide showed characteristic signals at 7.60, 7.18, and 6.91 ppm. These three peaks are assigned to protons in the imidazole rings. The spectra of the MPEG/API/cinnamate-gpolyaspartamide showed three additional peaks at 7.4, 7.48, and 6.55 ppm, which were assigned to the cinnamate benzene and alkene protons, respectively. The signals between 5.1 and 5.4 ppm of the methane protons in the PSI rings completely disappeared, and a new signal at 4.5
4.7 ppm appeared in the spectra of the grafted polymers, indicating that all of the succinimide rings were opened by aminolysis. The 1H NMR spectra provided information regarding not only the structures of the synthesized polymers but also the quantitative length ratio of the branches grafted onto the PSI backbone. The synthesis method used in this study has an advantage of permitting the synthesis of amphiphilic copolymers with a different hydrophobic/hydrophilic balance by controlling the degree of substitution (DS) of branches. Table 1 presents the resulting DS of MPEG5000-NH2, 1-(3-aminopropyl) imidazole, and cinnamate according to the feed molar ratio. The DS of MPEG5000-NH2 was defined as the molar ratio of MPEG5000NH2 to all of the repeating succinimide units in the PSI polymer and was calculated by comparing the integral of the peak at σ = 3.52, belonging to the methylene proton of the PEG chain, to that of the methine proton of the polymer backbone peak at σ = 4.50
4.70. In the same way, the DS of 1-(3-aminopropyl)imidazole was determined by comparing the integral of a peak assigned to the methane proton of 1-(3-aminopropyl)imidazole with that of the peak assigned to the methine proton of the main chain of the graft copolymers by 1H NMR. In all cases, the DS of the grafted branches continued to increase with an increase in the feed molar ratio. All polymers were obtained in high yields, exceeding 80%. Control of Particle Size for the EPR Effect. Different degrees of substitution of different branches influenced the properties of the micelles formed from the graft copolymer. Figure 3 shows the different initial particle sizes of the micelles formed from MPEG/ API/cinnamate-g-polyaspartamide (samples P1.5, P2.0, and P2.4). These initial particle sizes increased with increasing DS of the MPEG branches. In the basic buffer solution, with nearly the same DS of API and cinnamate groups when the DS of

Table 1. Degrees of Substitution of MPEG5000-NH2, 1-(3-Aminopropyl)imidazole, and Cinnamate in MPEG/API/Cinnamate-gpolyaspartamide

a

sample

feed molar ratio of

feed molar ratio

degrees of substitution of

degrees of substitution

degrees of substitution of

name

MPEG5000 mol %a

of API mo1 %b

MPEG5000-NH2 mol %c

of API mol %c

cinnamate mol %c

I100

2.5

100

2.0

66

32

I150

2.5

150

2.0

82

16

I175

2.5

175

2.0

90

8

P1.5

2.0

150

1.5

86

12.5

P2.0 P2.4

2.5 3.0

150 150

2.0 2.4

82 79

16 18.6

MPEG5000-NH2/succinimide unit in moles. b 1-(3-Aminopropyl)imidazole/succinimide in moles. c Determined by 1H NMR. 12093

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Figure 3. Effect of the degree of substitution of MPEG on the initial size of particles prepared under basic conditions.

Figure 4. Effect of the degree of substitution of API on the pH dependence of light transmittance of un-cross-linked micelles formed from MPEG/API/cinnamate-g-polyaspartamide.

MPEG5000 was 1.5%, the particle size approached 100 nm. However, when the DS of MPEG5000 was 2.4%, the particle size increased to approximately 250 nm. This was due mainly to an increase in the hydrophilic proportion of the amphiphile, which formed a corona surrounding the hydrophobic core. The higher hydrophilic/hydrophobic ratio of the amphiphile rendered the micelles more loosely packed with a larger mean particle size. Because of enhanced permeability and the retention effect (EPR effect) of the tumor vasculature, appropriate particle sizes of the micelles promote a much better accumulation of the micelleincorporated drugs into tumors than in normal tissues.38 Convenient control of the particle size would provide the micelles an optimized particle size for the passive targeting method in antitumor drug delivery. pH-Dependent Light Transmittance of the Prepared Micelles. The pH sensitivity of the graft copolymer was examined by measuring the change in the light transmittance and particle size of the polymer aqueous dispersion system at varying pH values. Figure 4 illustrates the pH-dependent light transmittance of uncross-linked polymer solutions measured at a UV wavelength of

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Figure 5. Effect of the degree of substitution of API on the pH dependence of the light transmittance of cross-linked micelles formed from MPEG/API/cinnamate-g-polyaspartamide.

500 nm. As shown in Figure 4, the initial micelles formed from the MPEG/API/cinnamate-g-polyaspartamide show a low light transmittance at pH 9. In the experiment, the pH value was gradually lowered by adding 0.1 N HCl to the initial polymeric micelles to adjust the pH of the aqueous solution to pH 4. In the process, the aqueous dispersion of the MPEG/API/cinnamate-gpolyaspartamide showed gradually increasing light transmittance in the basic environment as the micelles of the amphiphilic copolymer were gradually diluted by the HCl solution. However, it drastically increased near pH 7 and maintained a high light transmittance at pH values below 7. This was attributed to the hydrophobic to hydrophilic transition that arose from the protonation of the imidazole groups, whose pKa value was 6.5. In the acidic environment, the light transmittance remained as high as 95%, indicating the total change of the dispersion system into a solution. This is obvious evidence of the pH-dependent micelle
unimer transition of the prepared amphiphile dispersion system. This pH dependence of the phase transition was reversible when the polymer was not cross-linked. The micelles formed from the MPEG/API/cinnamate-g-polyaspartamide were exposed to UV irradiation; subsequently, the core was cross-linked. The pH-dependent transmittance of the core cross-linked micelles was measured in an identical manner. As shown in Figure 5, as 0.1 N HCl was added to achieve pH 9 and initially cross-link the core micelles, the transmittance increased gradually because of the dilution effects of the acid solution. There is also an obvious jump in the transmittance near pH 7 due to the hydrophobic to hydrophilic transition of the imidazole groups. However, no phase transition behavior in the core cross-linked micelles can be observed through changing pH values. As shown in Figure 5, the light transmittance of the micelles with a higher DS of imidazoles (sample I175) increased more drastically than for the samples with a lower DS of imidazoles (sample I100). This is because the higher DS of the pH-sensitive groups brings more ionized imidazoles whereas the higher repulsion of more ionized groups renders the micelles more loosely packed. The different compaction of these cross-linked micelles with different pH sensitivity ushered in the different light transmittance. However, the light transmittance of the core cross-linked micelles never entirely returned to a state of transparency because the amphiphile polymers can never be 12094

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Figure 6. Effect of the degree of substitution of API on the pH dependence of the mean size of uncross-linked micelles formed from MPEG/API/cinnamate-g-polyaspartamide.

Figure 7. Effect of the degree of substitution of API on the pH dependence of the mean size of cross-linked particles formed from MPEG/API/cinnamate-g-polyaspartamide.

resolved into aqueous solutions as a result of the cross-linking of the cinnamate groups in the hydrophobic cores of the micelles. Micelle
Unimer Transition and Swelling
Shrinking Behavior. The particle size change at varying pH values of both uncross-linked and cross-linked samples was checked through the electrophoretic light scattering method. All of the micelles showed very narrow particle size distributions. There was no obvious particle size change before and after the UV cross-linking of the core, which showed that there is no cross-linking of the cinnamate groups among the different particles. This is evidence that high-quality micelles with uniform and independent nanoparticles dispersed in aqueous solution were prepared from MPEG/API/cinnamate-g-polyaspartamide. The change in the mean particle size of the un-cross-linked micelles formed from MPEG/API/cinnamate-g-polyaspartamide according to the pH is shown in Figure 6. Here, the polymer with a DS of MPEG of 2.0% (samples I100, I150, and I175) was chosen as a model. In the basic environment, the amphiphilic polymers formed quite stable micelles with a mean particle size of approximately 150 nm. Usually, the micelles can

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Figure 8. Loaded amount of PTX per unit mass of polymer as a function of the PTX feed amount.

be considered to be particles with a spherical core and a hydrophilic corona surrounding this core. In the basic environment, the cinnamate and imidazole groups formed a hydrophobic core and the PEG groups formed a hydrophilic corona. However, no particle size could be detected in the acidic environment. The reason is that when the pH value was lower than pH 7, protonation of imidazole groups changed these groups from hydrophobic to hydrophilic and therefore increased the solubility of the polymer in the aqueous solution. The electrostatic repulsion between the ionized imidazole groups would also be attributed to the deaggregation of the micelles. The particle size change shown in Figure 6 corresponds well to the transmittance change shown in Figure 4. Both results are obvious evidence for the phase transition of the micelles formed from MPEG/API/cinnamate-g-polyaspartamide. After UV irradiation, cinnamate formed cores of the crosslinked micelles. Figure 7 shows the change in the mean particle sizes of micelles with cross-linked cores, according to changes in pH values. As the pH of the aqueous dispersion system decreased, the micelles did not disintegrate because of the cross-linking of the cinnamate-formed cores. Instead, the mean particle size of the micelles increased as the pH fell below 7. The reason is that the imidazole groups transferred from the hydrophobic core to the hydrophilic corona. The electrostatic repulsion between the ionized imidazole groups also played an important role in the swelling process. Similar to the light transmittance shown in Figure 7, the higher DS of pH-sensitive groups bring about a higher particle swelling ratio. From pH 9 to 5, the swelling ratios of samples I150 (with 82% pH-sensitive groups and 16% cross-linkable groups) and I175 (with 90% pH-sensitive groups and 8% cross-linkable groups) were 1.73 and 3.58, respectively. As for sample I100, because of the high degree of cross-linking and the low ratio of pH-sensitive groups, no obvious swelling behavior was observed. Thus, the swelling ratio had a positive correlation with the DS of pH-sensitive groups and a negative correlation with the DS of cross-linkable groups. Figures 7 and 5 illustrate the pH-dependent swelling
shrinking behavior of the core cross-linked micelles rather than the phase-transition behavior. Drug-Loading Efficiency and Drug-Release Pattern. The ability of the micelles to take hydrophobic drugs was tested by the 12095

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networks as drug carriers. The micelles formed from MPEG/API/ cinnamate-g-polyaspartamide, with a cross-linked core, showed very little premature drug release at high pH values (7.4) and an obvious pH-triggered release at low pH values (5). These results indict the core cross-linked micelles as competent carriers for intraocular drug delivery, especially for endosomal release.

Figure 9. In vitro PTX release patterns from micelles formed from MPEG/API/cinnamate-g-polyaspartamide in a PBS medium at different pH values.

PTX loading experiment. PTX is a significant antineoplastic agent, but its very low water solubility severely limits its bioavailability and clinical applications. The loading efficiency according to the feed amount of PTX is shown in Figure 8. Prior to saturation, the loaded amount of PTX increased linearly with the increasing PTX feed amount. The concentration of the prepared micelles was 1.0 g L
1, and in the saturation state, that of the loaded paclitaxel/polymer could reach as high as nearly 15%, a concentration of approximately 0.15 g L
1. This is a very high drug trap efficiency for the polymeric micelles; this polymeric micelle could potentially be a competent carrier for the delivery of paclitaxel. Figure 9 shows the cumulative PTX release profile from micelles formed from MPEG/API/cinnamate-g-polyaspartamide. PTX was continuously released from the micelles contained in a dialysis membrane tube. The release medium was PBS (pH 5 and 7.4, 37 °C) containing 2.4 wt % Tween 80 and 4 wt % Cremophor EL. As shown in the figure, at pH 5, the PTX in the un-cross-linked micelles burst out. Theoretically, the PTX should be released simultaneously when the micelles are exposed to the acidic medium because of the micelle
unimer transition of the carrier dispersion system. The PTX in cross-linked micelles was released at pH 5 much more slowly than that in un-cross-linked micelles. This is due to the steric blockade of the tightly cross-linked network of micelle cores. Quite slowly, PTX release was detected from cross-linked micelles at pH 7.4. The reason should be the shrunken state of the carrier particles and the blockade of the more tightly cross-linked network of the cores. However, comparatively much more PTX was gradually and continuously released from the un-cross-linked micelles at pH 7.4. Although at pH 7.4 the micelles were in a shrunken state, the un-cross-linked molecular state of the carrier particles lacked enough protection from the PTX in the core and there was a balance of PTX solubility between the micelle cores and the medium. Therefore, as the medium was renewed, the equilibrium was unbalanced and PTX was continuously released from the un-cross-linked micelles, indicating that even in bodily fluids at pH 7.4 the drugs can gradually be released from the un-cross-linked carriers prior to reaching the targeted cells. This premature release from the un-cross-linked micelle carriers in body fluid emphasized the necessity and importance of creating micelles with cross-linked

’ CONCLUSIONS A biodegradable polymer was synthesized by the ring-opening grafting of MPEG-NH2, API, and cinnamate onto PSI. The grafted polymer was used to prepare pH-sensitive micelles. The particle size of the polymer-formed micelles could be conveniently controlled by changing the degree of substitution of MPEG in the copolymer. Before cross-linking, the prepared polymer showed a sharp pH-dependent phase transition near pH 7 that was due to the protonation or deprotonation of the imidazole rings. After UV irradiation, the micelles displayed obvious pHdependent swelling
shrinking behavior instead of micelle
unimer transition behavior, which indicted that the cinnamateformed cores of the micelles were effectively cross-linked. The prepared micelles with such properties have many possible applications over a diverse range of drug delivery fields, especially as highly efficient intracellular drug delivery systems for endosomal release. As an example of hydrophobic antineoplastic agents, paclitaxel was effectively incorporated into the prepared polymeric micelles. The drug-loaded micelles, before being cross-linked in a low-pH environment, released the drug in a single burst because of the micelle
unimer transition of the polymer in buffer solution, which corresponded well to the pH-dependent swelling
shrinking behavior of the core cross-linked micelles. At pH 7.4, very slow drug release was detected from the core cross-linked micelles while at the same time a large amount of drug can gradually be released from the un-cross-linked micelles. These indicate that the cross-linking of the core of the micelles can effectively decrease drug release into bodily fluids, thereby increasing the drug circulation time and preventing premature drug release. This pH-sensitive micelle system overcame the drawback of the easy disintegration of normal polymeric micelles and is a perfect candidate for a drug delivery carrier for intracellular drug delivery and endosomal release, which requires a trigger system at very small pH changes in the physiological environment. ’ AUTHOR INFORMATION Corresponding Author

*Fax: +82 31 290 7272. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) via a grant funded by the Korean government (MEST) (2010-0027955). ’ REFERENCES (1) Kabanov, A. V.; Alakhov, V. Y. Crit. Rev. Ther. Drug Carrier Syst. 2002, 19, 1–72. (2) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Adv. Drug Delivery Rev. 2003, 55, 403–419. (3) Hubbell, J. A. Science 2003, 300, 595–596. 12096

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