Ketal Cross-Linked Poly(ethylene glycol)-Poly(amino acid)s

Feb 23, 2011 - The hydrolysis kinetics study showed that the hydrolysis half-life (t1/2) of ketal cross-links was estimated to be 52 h at pH 7.4, wher...
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Ketal Cross-Linked Poly(ethylene glycol)-Poly(amino acid)s Copolymer Micelles for Efficient Intracellular Delivery of Doxorubicin Sang Jin Lee,†,‡ Kyung Hyun Min,†,‡ Hong Jae Lee,§ Ahn Na Koo,§ Hwa Pyeong Rim,‡ Byeong Jin Jeon,‡ Seo Young Jeong,*,‡ Jung Sun Heo,§ and Sang Cheon Lee*,§ ‡

Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Korea Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 1 Hoegidong, Dongdaemun-gu, Seoul 130-701, Korea

§

bS Supporting Information ABSTRACT: A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(Laspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) selfassembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (KSV). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t1/2) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.

’ INTRODUCTION Recent advances in functional self-assembled polymer micelles based on amphiphilic block copolymers have led to the development of smart nanocarriers that can enhance the delivery efficiency of anticancer drugs, genetic drugs, and imaging agents.1-6 Polymer micelles have expressed many benefits for cancer chemotherapy due to the effective resistance to rapid renal clearance and nonspecific uptake by the reticuloendothelial system (RES).7,8 This unique property enables the micellar passive targeting and enhanced permeability and retention (EPR) effect for effective tumor therapy.9-11 For the polymer micelles to be an ideal carrier, they should meet the following three requisites: high stability in blood, minimized drug loss before reaching target tissues, and the facilitated release of loaded drugs within target cells. However, most polymer micelles suffer from low structural stability, and furthermore, the drug release is initiated upon intravenous administration, resulting in drug loss at unwanted sites.12,13 Recently, increasing the micellar stability is an issue of a great interest, because the polymer micelle tends to lose its carrier properties by disintegration in vivo conditions, r 2011 American Chemical Society

caused by the dilution to below critical micelle concentration or adverse shift in the micelle-unimer equilibrium. This resulted in the low delivery efficiency to target sites, and furthermore, the EPR effect cannot be expected. To date, cross-linking of micellar shells or cores has been recognized as a useful approach to stabilize micelle structure, because the cross-linking holds micelles in a thermodynamically frozen state.14-19 However, within target cells, the cross-links should be cleaved to release entrapped drugs, because the maintenance of cross-links can act as a barrier to drug release. For this reason, shell or core cross-linked polymer micelles (CLMs) satisfying not only enhanced stability but also specific intracellular drug-releasing property are an important target. Our recent research has been focused on disulfide cross-linked micelles and mineral-reinforced micelles, and we demonstrated that they are good examples for enhanced robustness and Received: December 15, 2010 Revised: February 4, 2011 Published: February 23, 2011 1224

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Scheme 1. Illustration of Shell Cross-Linking of DOX-Loaded Polymer Micelles with a pH-Labile Ketal Cross-Linker and Intracellular Release of Dox Triggered by Endosomal pH

the facilitated release of entrapped anticancer drugs at cytoplasm.20,21 It is well-known that intracellular endosomes (pH 5.0-5.5) and lysosomes (pH 4.0-4.5) have mildly acidic pH.21-23 These environments with lowered pH than extracellular compartments have provided an opportunity for designing specific intracellular drug delivery systems. Therefore, the incorporation of pHbreakable shell cross-links into polymer micelles may enable the design of the useful intracellular nanocarriers. Herein, we describe the synthesis of CLMs using a crosslinking agent containing pH-hydrolyzable ketal groups to produce a biocompatible, robust, and smart nanocarrier that can preferentially release doxorubicin (DOX) in response to endosomal pH. Recently, a fundamental study of pH-sensitive shell cross-linked nanoparticles with hydrolytically labile cross-links was reported, and the research scope focused on the feasibility of shell cross-linking and pH-dependent breakage of cross-links.24 As another example, an acid-labile core cross-linked micelle was reported for pH-triggered release of antitumor drugs.25 As most CLMs, these systems were based on nondegradable polyacrylate or polystyrene with limited biocompatibility.14-19 For CLMs to be practically useful, the first consideration is to tailor the polymer design for nontoxic biodegradable CLMs. Another point of view is the design of micelle structures suitable for intravenous administration. For long-circulation for enhanced EPR effects, the use of PEG as micellar outer corona was recommended.26,27 Based on these consideration, we selected poly(ethylene glycol) (PEG) and poly(amino acid)s as building blocks for CLMs due to their low toxicity and immunogenicity.28,29 In contrast to current CLM systems based on nondegradable polyacrylate and polystyrenes, our system can be eliminated from body through renal excretion as nontoxic PEG and degraded amino acid molecules (L-aspartic acid and Lphenylalanine) after expressing its roles for drug delivery. As a polymer micelle for shell cross-linking, a ABC triblock copolymer

with rational sequence of poly(ethylene glycol)-b-poly(L-aspartic acid)-b-poly(L-phenylalanine) (PEG-PAsp-PPhe) was utilized. PEG-PAsp-PPhe is expected to form polymer micelles featuring the PEG corona, the PAsp middle shell, and the PPhe inner core. The three domains of the core, the shell, and the corona, are each expected to play a role in generating a useful CLM nanocarrier. The PEG outer corona can keep the micelles stable by protecting against the formation of intermicellar bridges during the shell cross-linking process, which is a problem associated with coreshell type micelles of AB diblock copolymers.30 In addition, the PEG corona enables the prolonged circulation of CLMs for effective EPR effects. The PPhe core serves as a reservoir of liphophilic drugs. The PAsp middle shells with carboxylic acid groups provide a region for shell cross-linking with a ketalcontaining cross-linker. The cross-links in the middle shells not only can enhance micellar stability against micelle-destabilizing conditions but also can protect drug release efficiently at extracellular environments (pH 7.4). Upon endocytosis, the hydrolysis of ketal linkages in cross-links can be promoted by an acidic endosomal pH (∼5.0) to trigger intracellular drug release. Scheme 1 illustrates the key idea and working principle of our ketal cross-linked micelles as an intracellular carrier of DOX. In this study, we describe the rational synthetic approach of ketal cross-linked micelles (KCLMs) and the cross-linking effect on the micellar robustness. The hydrolysis kinetics of ketal crosslinks was investigated at endosomal and physiological pH. The inhibited DOX release at pH 7.4 and the facilitated DOX release in response to endosomal pH were demonstrated. The intracellular release of DOX from KCLMs was visualized using breast cancer cells (MCF-7) and correlated with the inhibitory effect of cancer cell proliferation. The ketal cross-linking is designed to occur by the reaction of ketal-containing cross-linkers with Asp moieties within middle shells, and thus, the maintenance of PEG outer corona may lead to the enhanced accumulation of micelles at target tissues. 1225

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’ EXPERIMENTAL SECTION Materials. R-Methoxy-ω-amino-poly(ethylene glycol) (CH3OPEG-NH2) with number average molecular weight (Mn) of 2000 g/ mol and polydispersity index (PDI) of 1.06 (GPC) was purchased from SunBio Inc. (Seoul, Korea) and used as received. β-Benzyl L-aspartate (BAsp), L-phenylalanine (Phe), doxorubicin hydrochloride (DOX 3 HCl), pyridinium p-toluenesulfonate (PPTS), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide (EDC), triphosgene, pyrene, 1-dodecylpyridinium chloride (DPC), N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide (HETFA), 2-methoxypropene, and 2,20 -(ethylenedioxy)bis(ethylamine) were purchased from Aldrich Co. (Milwaukee, WI) and used as received. Tetrahydrofuran (THF) was distilled from Na/benzophenone under N2, prior to use. N, N-Dimethylformamide (DMF) was dried and distilled over calcium hydride. β-Benzyl L-aspartate N-carboxyanhydride (BAsp-NCA) and Lphenylalanine N-carboxyanhydride (Phe-NCA) of high purity were synthesized by the Fuchs-Farthing method using triphosgene.31 Anal. Calcd for BAsp-NCA (C12H11NO5): C, 57.83; H, 4.45; N, 5.62. Found: C, 57.93; H, 4.49; N, 5.64. Anal. Calcd for Phe-NCA (C10H9NO3): C, 62.82; H, 4.74; N, 7.33. Found: C, 62.29; H, 4.90; N, 7.28. Instrumentation. Nuclear Magnetic Resonance Spectroscopy (NMR). 1H NMR spectra were recorded at 400 MHz on a Varian INOVA400 NMR spectrometer with a sample spinning rate of 5 kHz at 25 °C. Samples for NMR measurement were prepared by dissolving 10 mg of samples in 0.5 mL of DMSO-d6. Gel Permeation Chromatography (GPC). Molecular weight distributions were determined using a GPC equipped with a Waters 2414 refractive index detector, 515 HPLC pump, and three consecutive Styragel columns (HR1, HR2, and HR4). The eluent was DMF with a flow rate of 1 mL/min. The molecular weights were calibrated with polystyrene standards. Elemental Analysis. Elemental analysis was performed on a Perkin Elmer Series II CHNS/O Analyzer 2400. Zeta Potential Measurement. The zeta potential (ζ) was measured in a phosphate buffered saline (PBS) solution (10 mM, pH 7.4) using a 90 PLUS (Brookhaven Instruments Cooperation, New York, U.S.A.) particle size analyzer. Fluorescence Measurements. Pyrene fluorescence was recorded on a JASCO FP-6500 spectrofluorometer for determination of critical micelle concentration (cmc). For micellar solutions, doubly distilled water (20 mL) was added dropwise to a vigorously stirred THF solution of the block copolymer. After THF was evaporated in vacuo, the micellar solution was diluted to obtain a concentration range from 2 to 1  10-4 g/L. An aqueous pyrene solution (12  10-7 M) was mixed with micellar solutions to obtain copolymer concentrations from 1 to 5  10-5 g/L. The pyrene concentration in the samples was 6.0  10-7 M. All the samples were sonicated for 10 min and were allowed to stand for 1 day at 25 °C before measurements.32 The cmc was calculated based on a red shift of (0,0) band from 332 to 336 nm in pyrene excitation spectra.32 Light Scattering Measurements. Dynamic light scattering measurements were performed using a 90 Plus particle size analyzer (Brookhaven Instruments Corporation). The sample solutions were purified by passing through a Millipore 0.45 μm filter. The scattered light of a vertically polarized He-Ne laser (632.8 nm) was measured at an angle of 90° and was collected on an autocorrelator. The hydrodynamic diameters (d) of micelles were calculated by using the Stokes-Einstein equation.33 The polydispersity factor of micelles, represented as μ2/Γ2, where μ2 is the second cumulant of the decay function and Γ is the average characteristic line width, was calculated from the cumulant method.33 Transmission Electron Microscopy. Transmission electron microscopy (TEM) was performed on a JEM-2000EX (JEOL Tokyo, Japan),

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operating at an acceleration voltage of 200 kV. For the observation of size and distribution of micellar particles, a drop of sample solution (concentration = 0.5 g/L) was placed onto a 200-mesh copper grid coated with carbon. About 2 min after deposition, the grid was tapped with a filter paper to remove surface water, followed by air-drying. Negative staining was performed using a droplet of a 5 wt % uranyl acetate solution. Synthesis of a Ketal-Containing Cross-Linker. The ketalcontaining bifunctional cross-linker was synthesized by a two-step literature process.34 In brief, to a THF solution (250 mL) of HETFA (10.9 g, 69.3 mmol), PPTS (0.7 g, 2.8 mmol) was added. Molecular sieves (5 Å) was then added, and the reaction mixture was stirred. After 20 min, 2-methoxypropene (2 g, 27.7 mmol) was added, and the reaction was maintained for 24 h at room temperature. The reaction product was purified by column chromatography. Yield: 55%. 1H NMR (DMSO-d6): δ 1.30 (s, 6H), 3.5 (s, 8H), 6.7 (s, 2H). The reaction product (3 g, 8.48 mmol) was dissolved in aqueous NaOH (6 M, 20 mL) solution and stirred for 3 h. Finally, the ketal cross-linker was isolated by extraction with CH2Cl2 (3  200 mL) and dried with anhydrous MgSO4, followed by evaporation under vacuum. Yield: 45%. 1H NMR (DMSO-d6): δ 1.38 (s, 6H), 1.65 (s, 4H,), 2.86 (t, 4H), 3.47 (t, 4H). Synthesis of a PEG-PAsp-PPhe Triblock Copolymer. PEG45PBAsp8-PPhe19 that has PEG units of 45, BAsp units of 8, and Phe units of 19 was synthesized by a procedure established in our laboratory.21 To a stirred solution of CH3O-PEG-NH2 (5 g, 0.0025 mol) in dry DMF (50 mL) was added BAsp-NCA (6.23 g, 0.025 mol) at 35 °C under nitrogen. After 24 h, Phe-NCA (9.56 g, 0.050 mol) and dry DMF (100 mL) were added to the reaction mixture, and the reaction was maintained for an additional 24 h. PEG45-PAsp8-PPhe19 was isolated by repeated precipitation from DMF into diethyl ether. Yield: 90%. The deprotection of PEG45-PBAsp8-PPhe19 was performed by treating the block copolymer (5 g) with 0.1 N NaOH (200 mL) to remove benzyl groups. The aqueous solution was then dialyzed using a membrane (molecular weight cutoff (MWCO): 1000) for 24 h, followed by freeze-drying. Shell Cross-Linking of PEG-PAsp-PPhe Micelles. Polymer micelles of PEG45-PAsp8-b-PPhe19, consisting of PEG coronas, PAsp shells, and PPhe cores, were prepared by dialyzing the polymer solution in DMSO against doubly distilled water. Micelles were immersed into acidic aqueous solution (pH 4.0, 5 mg/4.8 mL) of EDC (21 mM) and NHS (21 mM) for 3 h. Shell cross-linking was carried out by adding a solution of the amine-terminated bifunctional ketal cross-linker (10.5 mM, 0.2 mL, water/THF (1:1, v/v)) to a NHS-activated micellar solution (1 g/L) at pH 9.0. The reaction mixture was stirred for 10 h at room temperature and then dialyzed against doubly distilled water (pH 9.0) for 3 h to remove unreacted cross-linkers. The dialyzate was lyophilized to obtain the KCLMs. Steady-State Fluorescence Quenching. Fluorescence quenching experiments were performed by adding aqueous solutions of DPC (0.75 mL) with a various concentration (0.0016-0.5 mM) to a pyreneloaded noncross-linked micelles (NCLMs) and KCLMs (pyrene concentration = 1  10-6 M, polymer concentration = 0.75 g/L, 0.75 mL). In a Stern-Volmer equation (I0/I - 1 = kqτ0[Q] = KSV[Q]), I0 and I represent the integrated fluorescence intensity of pyrene in the absence and in the presence of quencher, respectively. [Q] is the molar concentration of quencher and τ0 is the fluorescence lifetime in the absence of the quencher. Time-resolved fluorescence measurement in our previous study showed that pyrene had a lifetime (τ0) of 128 ns in aqueous phase.21 The sample solution containing pyrene was excited at 338 nm. The pyrene fluorescence was detected at 393 nm. Stability of Cross-Linked Micelles. Kinetic stability of KCLM was investigated by adding SDS. The effect of SDS on KCLMs in aqueous media was estimated by dynamic light scattering analysis and fluorescence spectroscopy. A SDS solution (1 mL, 7.5 g/L) was added to 1226

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Figure 1. Synthetic route for the PEG45-PAsp8-PPhe19 triblock copolymer. the KCLM solution (2 mL, 0.75 g/L), and the solution was stirred at 500 rpm. At predetermined time periods, scattered light intensity (SLI) was monitored and compared to the initial scattered light intensity (SLI0) by dynamic light scattering analysis. For time-dependent stability of DOX-loaded micelles, fluorescence spectroscopy was utilized to monitor the change of DOX fluorescence intensity ratio (FI/FI0) in the presence of SDS, where FI is the intensity at predetermined times, and FI0 is the intensity at initial time. For comparison, the SDS treatment of the NCLM solution was conducted under the same condition. Hydrolysis Kinetics of Ketal Cross-Links in KCLMs. Hydrolysis kinetics of ketal cross-links in KCLMs was estimated using 1H NMR spectroscopy. The KCLMs (1 mL, 10 mg/mL) were dissolved in a mixture of D2O (0.5 mL) and each buffer solution (0.5 mL, pH 7.4 phosphate buffer, pH 5.0 acetate buffer). At predetermined time intervals, the hydrolysis kinetics was monitored by time-dependent quantification of the decrease in ketal integration at 1.34 ppm. Halflives of ketal hydrolysis at different pH were calculated using the Arrhenius equation.

Preparation of DOX-Loaded Non-Cross-Linked Polymer Micelles (DOX-NCLMs). DOX was loaded into PEG45-PAsp8-PPhe19 micelles by the dialysis method. Before loading DOX to the PEG45PAsp8-PPhe19 micelles, DOX 3 HCl (2 mg, 0.0034 mmol) was stirred with TEA (0.6 μL, 0.0044 mmol) in DMF (0.6 mL) overnight in the dark. The triblock copolymer (20 mg) was dissolved in 4 mL of DMF for 3 h at 70 °C, and then the DOX solution was subsequently added and stirred in the dark at room temperature. The solution was dialyzed using a membrane (Spectrapor, MWCO: 1000) for 12 h and followed by lyophilization in the dark. To determine the drug loading content and loading efficiency, DOX-loaded polymer micelles were dissolved in DMF, and then measured by fluorescence emission intensity at 588 nm (excitation at 480 nm). The drug loading content and loading efficiency, based on the standard curve of DOX in DMF, were calculated to be 8.5 wt % and 94.1%, respectively.

Preparation of DOX-Loaded Ketal Cross-Linked Polymer Micelles (DOX-KCLMs). DOX-KCLMs were prepared based on an identical process used for shell cross-linking of DOX-free PEG45-PAsp8PPhe19 micelles, except that the DOX-loaded micelles were used as a template for ketal cross-linking instead of DOX-free micelles.

Preparation of DOX-Loaded Shell Cross-Linked Micelles with Nondegradable Cross-Links (DOX-CLMs-ND). DOXCLMs-ND were prepared following the identical process used for

DOX-KCLMs, except that 2,20 -(ethylenedioxy)bis(ethylamine) was used as a nondegradable cross-linker instead of the ketal cross-linker. Controlled DOX Release from DOX-Loaded KCLMs. In vitro release profiles of DOX from DOX-loaded NCLMs and KCLMs were investigated in the aqueous buffer solutions (pH 7.4 phosphate buffer and pH 5.0 acetate buffer). Various DOX-loaded micelles were dispersed in aqueous buffer solutions (1 mL, 0.75 g/L), and transferred to a dialysis membrane bag (MWCO: 1000). The release experiment was initiated by placing the dialysis bag in 10 mL of release media. The release medium was shaken at a speed of 150 rpm at 37 °C. At predetermined time intervals, samples (10 mL) were withdrawn and replaced with an equal volume of the fresh medium. The concentration of released DOX in the samples was determined by measurement of fluorescence emission intensity at 588 nm (excitation at 480 nm) based on the standard curve obtained using DOX in pH 7.4 phosphate buffer and pH 5.0 acetate buffer. Cell Culture. Human breast cancer MCF-7 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul). Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco BRL), and 1% (v/v) penicillin-streptomycin (Gibco BRL). Cells were cultured in a humidified incubator at 37 °C with 5% CO2. The culture medium was replaced every two days. Biocompatibility of NCLMs and KCLMs. MCF-7 cells were seeded into 96-well flat-bottomed tissue-culture plate at 2000 cells/well, and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. The micelles solution was diluted with culture medium to obtain a concentration range from 1 to 300 μg/mL. After the incubation for 24 and 48 h, medium was removed, and cells were washed with PBS, and the medium was replaced with cell counting kit-8 (CCK-8) solutions (Dojindo Laboratories, Kumamoto). The absorbance of individual wells was measured at 450 nm by a microplate reader (Biorad Elizer, PA). Cytotoxicity. In vitro cytotoxicity of DOX-loaded KCLMs were evaluated by measuring the half maximal inhibitory concentration (IC50) using the CCK assay. Briefly, MCF-7 cells were seeded onto a 96-well plate at a density of 5  103 cells per well in 200 μL of medium and incubated at 37 °C and 5% CO2. After 24 h, the medium of each well was then replaced by 200 μL of fresh medium containing free DOX, DOX-loaded NCLMs, DOX-loaded KCLMs at various drug concentrations from 0.01 to 50 μg/mL, and then the plates were incubated (37 °C, 1227

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Figure 2. 1H NMR spectra of CH3O-PEG-NH2 (a), PEG45-PBAsp8 (b), PEG45-PBAsp8-PPhe19 (c), and PEG45-PAsp8-PPhe19 (d) in DMSO-d6. Arrows in (c) and (d) show the complete deprotection of benzyl groups of PBAsp blocks. 5% CO2). After 24 h, the medium was removed, and cells were washed with the fresh medium, followed by the CCK assay. IC50 was calculated as the concentration of DOX yielding 50% inhibition of cell proliferation, compared to the untreated control. Intracellular Distribution of DOX-Loaded KCLMs. MCF-7 cells (5  103 cells/mL per well) were seeded onto a cover glass-bottom dish in 2 mL of DMEM medium supplemented with 10% FBS, 1% antibiotics (penicillin 100 U/mM, streptomycin 0.1 mg/mL). After incubation for 24 h (37 °C, 5% CO2), the medium was carefully aspirated and replaced with 1 mL of medium containing 5 μg/mL DOX equivalent of DOX-KCLM and LysoTracker (50 nM). The cells were incubated for 1 and 5 h, then washed three times with PBS. Formaldehyde (1 mL, 3.7%) was added, and the cells were fixed for 5 min. Finally, the solution was aspirated, and the cells were rinsed three times with PBS, and then the coverslips were transferred to glass slides. The confocal laser scanning microscopy (CLSM) images of LysoTracker labeled MCF-7 cells treated with DOX-KCLMs were obtained using a confocal laser scanning microscope (C1si, Nikon, Japan) by green-fluorescing (λex = 470-490 nm) and red-fluorescing (λex = 520-550 nm).

’ RESULTS AND DISCUSSION Preparation and Characterization of PEG-PAsp-PPhe Micelles. The PEG45-PAsp8-PPhe19 copolymer was synthesized via

the one-pot two-step polymerization of β-benzyl L-aspartate Ncarboxyanhydride (BAsp-NCA) and L-phenylalanine N-carboxyanhydride (Phe-NCA) in the presence of a CH3O-PEG-NH2 macroinitiator and a subsequent deprotection process (Figure 1). The composition ratio of PEG-PAsp-PPhe was calculated by the peak integration ratio of -OCH2CH2- protons of PEG at 3.49 ppm, PhCH2- of PBAsp at 5.05 ppm, and phenyl protons in of PPhe at 7.16 ppm (Figure 2). The molar composition ratio of monomeric repeating units in PEG, PAsp, and PPhe

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was 45:8:19. The conversion of monomeric BAsp and Phe to polymeric PBAsp and PPhe was found to be 80 and 95%, respectively (Table 1). The Mn of PEG45-PAsp8-PPhe19, calculated by 1H NMR, was 5570 g/mol. GPC analyses showed a narrow molecular weight distribution (Mw/Mn = 1.08; Table 1). To produce the block copolymer with narrow molecular weight distribution, we used PEG45 with an low polydispersity index (PDI) of 1.06 (GPC). This initial low molecular weight distribution may contribute to the low PDI of resulting PEG45PAsp8-PPhe19. Besides, N-carboxyanhydride (NCA)-based polymerization for poly(amino acid) blocks was known to follow pseudoliving polymerization, in case the ratio of the monomer to the amine-based initiator ([M]0/[I]0) is below about 20.35 The choice of starting PEG and the inherent feature of NCA polymerization may afford such a narrow molecular weight distribution of PEG45-PAsp8-PPhe19. We considered the specific composition for a design of PEG45-PAsp8-PPhe19. First, the choice of PEG45 with Mn of 2000 g/mol has been acceptable as nanocarriers for intravenous administration.21,26 In addition, PEG45 has a small hydrodynamic diameter and, thus, can be easily excreted through renal filtration after carrying out its role.36 Second, the PAsp composition of 8 units is selected, since this length would be enough for shell cross-linking reaction. Third, the 19 units of Phe can induce the self-assembly by hydrophobic interaction to form micelles. In case the length of hydrophobic PPhe is not enough, the micellar structure cannot be formed, because the block copolymer exists in a water-soluble state. In contrast, too much composition of hydrophobic PPhe makes the block copolymer lose its dispersibility and resulted in precipitation in water. For this reason, we reasonably selected the block composition of PEG, PAsp, and PPhe for PEG45-PAsp8-PPhe19. Rationally designed PEG45-PAsp8-PPhe19 self-assembled to form core-shell-corona polymer micelles with three welldefined, distinct domains: the inner hydrophobic PPhe core, the anionic PAsp middle shell, and the solvated PEG outer corona. The mean hydrodynamic diameter was 55.3 nm. The cmc of the block copolymer, estimated by pyrene excitation spectra,27 was 15.3 mg/L (Table 1). The zeta potential (ζ) of PEG45-PAsp8-PPhe19 micelles was reasonably negative (ζ = 29.8 mV). Formation of Ketal Cross-Linked Polymer Micelles. Ketal cross-linking of PAsp middle shells was performed by adding the ketal cross-linkers to an aqueous solution of PEG45-PAsp8PPha19, in which Asp groups were preactivated with 2-fold excess EDC at pH 4.0. Bifunctional ketal cross-linkers with the primary amines reacted with O-acylurea to form intramicellar crosslinking bridges via amide bond formation. The feed molar ratio of the ketal cross-linker to Asp repeating units of the PAsp middle shells was 2:1. It is noteworthy that the zeta potential of KCLMs are -19.2 mV, which is much higher than that of NCLMs (ζ = 29.8 mV; Table 2). The increased zeta potential reflects the conversion of some carboxylic acid groups of middle PAsp shells to amide linkages formed during shell cross-linking reaction. KCLMs maintain a reasonable size of precursor polymer micelles. TEM images show the spherical nature of polymer micelles, and that the shell cross-linking reaction did not induce the intermicellar aggregation, which is consistent with dynamic light scattering results (Figure 3). This indicates that the PEG outer corona confines the shell cross-linking reaction within the PAsp middle shells, preventing the formation of intermicellar aggregates. 1228

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Figure 3. Size distributions and morphology of NCLMs (a and c) and KCLMs (b and d) estimated by dynamic light scattering and TEM. Scale bars = 100 nm.

Table 1. Characteristics of the PEG45-PAsp8-PPhe19 Copolymer composition ratioa

feed ratio

a

copolymer

([EG]/[BAsp]/[Phe])

([EG]/[Asp]/[Phe])

PEG45-PAsp8-PPhe19

45:10:20

45:8:19

conversion of BAspa

conversion of Phea

(%) 80

Mw/

(%)

Mna

b

Mn

95

5570

1.08

cmc c (mg/L) 15.8

Calculated by 1H NMR spectra b Estimated by GPC. c Critical micelle concentration at 25 °C.

Table 2. Micelle Sizes, Zeta Potentials, and Polydispersity Factors of Various Micelles micelles

da (nm)

zeta potentialb (mV)

μ2/Γ2c

NCLM

55.3

-29.8

0.15

KCLM

54.5

-19.2

0.20

DOX-NCLM

54.7

-34.6

0.18

DOX-KCLM

52.5

-18.1

0.15

a Mean hydrodynamic diameters at 25 °C. b Estimated at pH 7.4 at 25 °C. c Polydispersity factor estimated by dynamic light scattering.

Steady-State Fluorescence Quenching. Shell cross-links of KCLMs can act as a diffusion-inhibiting barrier. This indicates that the diffusion of small molecules across the cross-linked networks within middle shells would be prevented to a certain degree. Steady-state quenching analysis is a useful tool to estimate the accessability of small molecules to micellar hydrophobic core domains.37 We monitored the access of DPC, a quencher molecule to a hydrophobic fluorescent pyrene loaded in KCLMs. For KCLMs, it is expected that the collisional fluorescence quenching of pyrene by DPC would be inhibited,

because the shell cross-links may inhibit the penetration of quenchers to micellar core to quench the pyrene fluorescence. The diffusion rate across the cross-linked shells was estimated using fluorescence quenching of pyrene loaded within micellar cores by DPC.38 As shown in Figure 4, the quenching constant (KSV) and the bimolecular rate constant (kq), calculated by a Stern-Volmer kinetic model of steady-state quenching experiments: I0/I - 1 = kqτ0[Q] = KSV[Q], substantially decreased for KCLMs with cross-linked shells. The KSV value of KCLMs was lowered by 54% over NCLMs, reflecting that the shell cross-links act as a diffusion barrier for low molecular weight compounds. Normally, the drug release behavior from the micellar core is dependent on the diffusion-controlled process. Therefore, this result indicates that shell cross-linking may be effective in reducing the drug release at pH 7.4, where the ketal cross-links are intact. Stability of Cross-Linked Micelles. The kinetic stability of KCLMs and NCLMs was monitored using light scattering analysis in the presence of SDS, a strong micelle-disrupting agent.21,39 For NCLMs, the relative scattering intensity was dramatically decreased to 70% after 1 h, which indicates SDSinduced dissociation of micelles (Figure 5(a)). On the other 1229

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Figure 4. Stern-Volmer plots for micelle-entrapped pyrene (6.0  10-6 M) quenched by DPC. Figure 6. Arrhenius plots for hydrolysis kinetics of ketal cross-links within KCLMs at pH 7.4 and 5.0. (A and A0 represent the integrations of (CH3)2 peaks of ketal cross-links after a prefixed period of incubation time and before incubation, respectively).

Figure 7. 1H NMR spectra of KCLMs in D2O at pH 5.0 at various incubation period and the postulated corresoponding structure of ketal cross-linked shell domains. Arrows indicate the gradual disappearance of ketal peaks.

Figure 5. (a) Kinetic changes in relative scattered light intensity (SLI/ SLI0) for NCLMs and KCLMs, and (b) the relative fluorescence intensity (FI/FI0) of DOX-NCLMs and DOX-KCLMs in the presence of SDS (2.5 g/L).

hand, the scattered intensity of KCLM solutions was minimally decreased. Even after 3 h, KCLMs maintained the initial scattering intensity over 90% (Figure 5(a)). This indicates that KCLMs could remain their assembled structure even in the harsh condition for micelle dissociation, because shell cross-linkers enable polymer micelles to be resistant to SDS effects. The stability of polymer micelles in a drug-loaded state was estimated by monitoring DOX fluorescence. This study was based on the photophysical property of DOX. The DOX fluorescence is selfquenched when DOX are in a densely packed status in the core of micelles, whearas the DOX fluorescence was recovered upon DOX releasing form the micelles. As shown in Figure 5(b), NCLMs treated with SDS exhibited abrupt increase in DOX

fluorescence, and, within 1 h, increased up to 40% of the initial fluorescence intensity (FI/FI0). On the other hand, KCLMs treated with SDS exhibited slowed increase, and, within 2 h, increased only to 20% of initial fluorescence intensity. This indicats that NCLMs released the more amount of DOX than KCLMs in an identical time period. These results indicate that shell cross-linking is effective not only in enhancing micellar stability but also in inhibiting the DOX release from the micelles. Hydrolysis Kinetic of Ketal Cross-Links within KCLMs. Hydrolysis kinetics of the ketal cross-links within KCLMs at physiological pH (7.4) and endosomal pH (5.0) was investigated by incubating KCLMs in a mixture of D2O and each buffer solution (phosphate buffer and acetate buffer). The hydrolysis kinetics was monitored by the integration ratio of the integration at a certain period of incubation of the ketal peak ((CH3)2CO2-, 1.34 ppm) to the integration before hydrolysis. The half-lives of ketal hydrolysis at different pH were calculated using the Arrhenius equation. As shown in Figure 6, the hydrolysis rate was much faster at pH 5.0 than at pH 7.4. The half-life of ketal 1230

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Figure 8. pH-Controlled DOX release profiles from DOX-NCLMs and DOX-KCLMs (n = 3).

hydrolysis at pH 5.0 was calculated to be 0.7 h, whereas the halflife at pH 7.4 was 52 h. This indicates the 74-fold faster hydrolysis of cross-links at endosomal pH. Figure 7 shows the correlation between the postulated structure changes at pH 5.0 within crosslinked shells and the disappearance of ketal peaks in NMR spectra. After 1 min incubation, the hydrolysis process already began to occur, but the most ketal linkages at 1.34 ppm remained intact. As the incubation time increased, the ketal integration gradually decreased, whereas the peak intensity of acetone peaks at 2.12 ppm increased. After 3 h, the ketal resonance peak almost completely disappeared. This pH-dependent hydolysis analysis indicates that the ketal cross-links can maintain its structure to strengthen the micelle structure, reduce the drug loss at bloodstream, and finally undergo the cleavage at endosomes to facilitate the loaded anticancer drugs. Controlled Drug Release from DOX-Loaded KCLMs. To estimate controlled DOX-releasing properties, DOX-NCPMs and DOX-KCLMs with identical drug loading contents (8.5 wt %) were used. Figure 8 shows that the pH-dependent DOX release rate from NCLMs and KCLMs. The DOX release from NCLMS did not notably depend on the differences between extracellular (pH 7.4) and endosomal pH (pH 5.0). On the other hand, DOX-KCLMs showed a dramatic pH-dependent DOX release behavior. At pH 7.4, DOX-KCLMs effectively inhibited the DOX release in the extracellular media at 48 h by 43.8% over polymer micelles without ketal cross-links (NCLMs). Although shell cross-linking did not totally block the release of DOX from KCLMs, it displayed the inhibitory effect to some extent. In contrast, at pH 5.0, the DOX release from KCLMs was accelerated by 68.7% at 48 h, compared with the DOX release from KCLMs at pH 7.4. As described in hydrolysis kinetics study, the structural integrity of ketal cross-links within micellar shells could be maintained at pH 7.4, and thus the diffusion-dependent DOX release could be inhibited to a certain degree. On the other hand, the fast breakage of shell cross-links at pH 5.0 would facilitate the diffusion of DOX molecules across the middle shells of micelles. The percentage of DOX release from KCLM at pH 5.0 is around 35% at 48 h, and the continuous DOX release is expected over longer period of times. In our experiments, the DOX concentration in the release medium at each collection time was below 10 ug/mL, irrespective of medium pH. It was reported that the aqueous solubility of DOX at pH 7.4 was reported to be 62 ug/mL. At pH 5.0, the degree of DOX protonation increases as does its solubility, and

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Figure 9. In vitro cytotoxicity of DOX, DOX-NCLMs, and DOXKCLMs with MCF-7 cells after 24 h (n = 3).

the reported solubility was 370 ug/mL.40 This indicates that sink conditions were maintained during the release experiments. One may have question of whether the enhanced rate of the DOX release from KCLMs at pH 5.0 over at pH 7.4 is caused mainly by the enhanced DOX solubility. For DOX-NCLMs, the pH-dependent DOX release behavior was observed. As shown in Figure 8, the release amount at pH 5.0 from DOX-NCLMs was higher by 2.2% at 24 h, over at pH 7.4. In order to examine the pH effect on the DOX release from the shell cross-linked micelles, the DOX release from DOX-loaded micelles with nondegradable cross-links (DOX-CLMs-ND) was examined (Figure S1 in the Supporting Information). The release amount at pH 5.0 was higher as low as 0.3% at 24 h, over at pH 7.4, and the difference was not significant. This shows that the pH effect on the DOX release was negligible, in case the cross-linked structure was maintained. This strongly supports that the enhanced release rate of DOX at pH 5.0 from KCLMs was primarily due to the hydrolysis of ketal cross-links, not due to the increased solubility of DOX. In Vitro Cytotoxicity. The cytotoxicities of DOX-free NCLMs and KCLMs were evaluated using a MCF-7 human breast cancer cell line with various concentrations. NCLMs and KCLMs showed no noticeable cytotoxicity up to 300 μg/mL (Figure S2 in the Supporting Information). In vitro cytotoxicities of DOX, DOX-NCLMs and DOX-KCLMs for MCF-7 cells were estimated. As shown in Figure 9, DOX-KCLMs showed the effective inhibitory effect on the proliferation of cancer cells. The somewhat lower toxicity of DOX-KCLMs (IC50 = 6.96 μg/mL) compared to free DOX (IC50 = 0.48 μg/mL) and DOX-NCLMs (IC50 = 2.55 μg/mL) was probably due to the gradual release of DOX within the endosomes. DOX-NCLMs without ketal crosslinks began releasing DOX immediately upon placing in the culture media. For this reason, the released DOX could be accumulated within cell nucleus in a short time period. In contrast, the DOX release rate from DOX-KCLMs was slower because the DOX release was accelerated after hydrolysis of ketal cross-links within cellular endosomes. The potential not only for minimizing drug loss in blood but also for selective accumulation in tumor tissue by the EPR effect may enhance its overall therapeutic efficacy in vivo relative to free DOX and DOXNCLMs. Intracellular Distribution of DOX-Loaded KCLMs. The endocytosis of DOX-KCLMs by MCF-7 cells was monitored by confocal laser scanning microscopy (CLSM). The acidic cellular endosome was labeled with green-fluorescent 1231

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disrupt the cross-links, and finally triggered the facilitated DOX release to inhibit proliferation of cancer cells.

Figure 10. CLSM images of MCF-7 cells incubated with Lysotracker (50 nM), free DOX (5 μg/mL), DOX-KCLMs (DOX = 5 μg/mL), and DOX-CLMs-ND (DOX = 5 μg/mL) for 5 h exposure. (a) Free DOX for 1 h exposure, (b) DOX-KCLMs for 1 h exposure, (c) DOX-KCLMs for 5 h exposure, (d) DOX-CLMs-ND for 1 h exposure, and (e) DOXCLMs-ND for 5 h exposure (green fluorescence is associated with LysoTracker and the red fluorescence is expressed by free DOX and released DOX). Scale bar = 20 μm.

LysoTracker as an indicator. We monitored first the intracellular DOX release from NCLMs and localization of DOX within cells using CLSM. As shown in Figure S3 (Supporting Information), even after 1 h incubation with MCF-7 cells, DOX released from NCLMs showed the strong red florescence within the endosomes and also a larger population of DOX was found within the nuclei. This indicates that DOX-NCLMs began to release DOX right upon addition into the cell culture media. In other words, DOX-NCLMs tended to release DOX in the extracellular area, and the released DOX was localized within the endosomes and nuclei in a short time period. After 5 h incubation, the DOX population within nuclei was more pronounced than within endosomes. To verify that facilitated DOX release in cell culture experiments is indeed related to the degradation of cross-links at endosomal pH, we performed the cell experiments using DOX-KCLMs with degradable cross-links and also with DOXCLMs-ND with nondegradable cross-links. In Figure 10, the DOX distribution was visualized for DOX-KCLMs and DOXCLMs-ND. For DOX-KCLMs, after 1 h incubation, DOX was released from KCLMs with low florescence intensity within the endosomes, and the small population of DOX was found within nuclei (Figure 10b). After a 5 h incubation, DOX of a strong fluorescence was released from DOX-KCLMs. DOX not only spread out in endosomes but also accumulated in the nuclei (Figure 10c). For DOX-CLMs-ND, the DOX fluorescence was confined within the endosome at 1 h incubation (Figure 10d). After a 5 h incubation, it is noteworthy that the DOX fluorescence intensity within nuclei was much lower than DOX-KCLMs (Figure 10e). This reflects that the nondegradable cross-links of DOX-CLMs-ND acted as barriers of DOX release even within acidic endosomes and thereby could not trigger the enhanced DOX release. This is consistent with the negligible enhancement of DOX release rate at endosomal pH (Figure S1 in the Supporting Information). These results strongly supports that the facilitated DOX release within cells was primarily related to the hydrolysis of ketal cross-links of DOX-KCLMs. Based on in vitro results, we confirmed that DOX-KCLMs was taken up by the cells via endocytosis, and transferred into endosomes to

’ CONCLUSIONS We have developed a novel, robust, biocompatible shell crosslinked micelles that could preferentially release entrapped DOX under intracellular endosomal compartments The introduction of acid-labile ketal cross-links into PEG-poly(amino acid)s micelles may meet the major requirements of targeted nanocarriers with a high delivery efficiency: (i) high stability in the bloodstream, (ii) prolonged blood circulation due to the PEG outer corona (minimized uptake by RES), (iii) ability to minimize drug loss before reaching target disease tissues, (iv) passive targeting and cellular uptake, and (v) preferential drug release within target cells. This new shell cross-linked micelle can serve as a useful intracellular nanocarrier of many poorly water-soluble anticancer drugs. ’ ASSOCIATED CONTENT

bS

Supporting Information. Cell viability of DOX-free NCLMs and KCLMs, pH-dependent DOX release from DOXCLMs-ND, and the confocal laser scanning microscope images of intracellular DOX release from NCLMs. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: þ82-2-961-2317. Fax: þ82-2-960-1457. E-mail: schlee@ khu.ac.kr; [email protected]. Author Contributions †

These authors contributed equally to this work.

’ ACKNOWLEDGMENT This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0007721) and the Fundamental R&D Program for Core Technology of Materials (K0006028) by Ministry of Knowledge Economy, Korea. ’ REFERENCES (1) Kim, S. H.; Jeong, J. H.; Lee, S. H.; Kim, S. W.; Park, T. G. J. Controlled Release 2008, 129, 107–116. (2) Oh, K. T.; Yin, H.; Lee, E. S.; Bae, Y. H. J. Mater. Chem. 2007, 17, 3987–4001. (3) Lee, Y.; Fukushima, S.; Bae, Y.; Hiki, S.; Ishii, T.; Kataoka, K. J. Am. Chem. Soc. 2007, 129, 5362–5363. (4) Convertine, A. J.; Diab, C.; Prieve, M.; Paschal, A.; Hoffman, A. S.; Johnson, P. H.; Stayton, P. S. Biomacromolecules 2010, 11, 2904–2911. (5) Mikhail, A. S; Allen, C. Biomacromolecules 2010, 11, 1273–1280. (6) Shiraishi, K.; Kawano, K.; Minowa, T.; Maitani, Y.; Yokoyama, M. J. Controlled Release 2009, 136, 14–20. (7) Du, Y.-Z.; Weng, Q.; Yuan, H.; Hu, F.-Q. ACS Nano 2010, 4, 6894–6902. (8) Talelli, M.; Rijcken, C. J. F.; van Nostrum, C. F.; Storm, G.; Hennink, W. E. Adv. Drug Delivery Rev. 2010, 62, 231–239. (9) Kawaguchi, T.; Honda, T.; Nishihara, M.; Yamamoto, T.; Yokoyama, M. J. Controlled Release 2009, 136, 240–246. 1232

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