Synthesis of Thermo-Sensitive Micellar Aggregates Self-Assembled

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Synthesis of Thermo-Sensitive Micellar Aggregates Self-Assembled from Biotinylated PNAS-b-PNIPAAm-b-PCL Triblock Copolymers for Tumor Targeting Chang-Yun Quan, De-Qun Wu, Cong Chang, Guo-Bing Zhang, Si-Xue Cheng, Xian-Zheng Zhang,* and Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan UniVersity, Wuhan 430072, China ReceiVed: March 24, 2009; ReVised Manuscript ReceiVed: May 10, 2009

The stimuli-sensitive diblock copolymer poly(N-acroyloxysuccinimide)-b-poly(N-isopropylacrylamide) (PNASb-PNIPAAm) was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. By using the terminal carboxyl group of the diblock copolymer to initiate the ring-opening polymerization of ε-caprolactone (CL), the amphiphilic PNAS-b-PNIPAAm-b-PCL triblock copolymer was further synthesized. The triblock copolymer was characterized by NMR, IR, and SEC-MALLS. To enhance the internalization to tumor cells, biotin was introduced into the triblock copolymer. The LCST of biotinylated PNAS-b-PNIPAAmb-PCL was about 35.3 °C. The formation of micellar aggregates (MAs) self-assembled from biotinylated PNAS-b-PNIPAAm-b-PCL was confirmed by CMC and TEM. The cell viability study demonstrated that the MAs have a low cytotoxicity. The antitumor drug doxorubicin (Dox) was loaded in the MAs, and in vitro release behavior of Dox showed the MAs exhibited thermo-sensitive drug release. The confocal microscopy studies confirmed that, with pretreatment of biotin-transferrin, the self-assembled MAs could specifically bind to tumor cells, indicating that the multifunctional MAs could be used as a promising drug carrier for tumor targeting. Introduction Stimuli-sensitive amphiphilic copolymers with characteristics that changed in response to an external environment, such as temperature, electric potential, and pH, etc., have attracted increasingly attention.1 Being a promising type of drug carrier, micelle self-assembled from amphiphilic copolymers has been extensively investigated in biomedical applications.2–4 One of important advantages of drug carriers is that they can enhance the water-solubility of hydrophobic drugs for improved bioavailability.5 And the other advantage is the small size of micelles makes them susceptible to the uptake by tumor cells.6 Besides, a specific targeting ligand can also be attached to the water-exposed segment of hydrophilic blocks to realize “active targeting”.7 Mediated by the targeting ligand receptor on the surface of tumor cells, the antitumor drugs loaded in the targetligand conjugated micelles could be directly delivered into cellular media and released from the micelles,8 which could greatly enhance the therapeutic efficacy of anticancer drugs and minimize adverse side effects.9,10 In the past decade, many synthesis strategies, such as conventional radical polymerization (CRP),11,12 atom transfer radical polymerization (ATRP),13,14 and reversible addition-fragmentation chain transfer (RAFT) polymerization,15,16 have been used to prepare amphiphilic copolymers. Compared with ATRP, RAFT is a metal-free process and could avoid the contamination caused by transition metal catalyst, which is vitally important for biomedical applications.17 Furthermore, RAFT polymerization provides a more straightforward route to prepare polymers than CRP and can also be utilized to precisely control the polymer architectures, molecular weight (Mw), and incorporation * To whom correspondence should be addressed. Phone/fax: 86-2768754509. E-mail: [email protected].

of appropriate functional groups for targeted delivery of therapeutic agents.18,19 In this study, a multifunctional PNAS-b-PNIPAAm-b-PCL triblock copolymer was designed and synthesized. The flexible PNAS segment was introduced to offer an optimized accessibility for compounds containing an amino group such as cystamine (used as a cross-linker in the further research) and biotin,20 which could bind strongly with avidin.21 And it was found that the biotinylated PNAS-b-PNIPAAm-b-PCL copolymers could be self-assembled into shell-core micellar aggregates (MAs) in aqueous media. The hydrophilic PNIPAAm segment, a thermo-sensitive block,22 was utilized to form the shell, and the PCL segment, having good biocompatibility,23 served as a hydrophobic block to form the core of the micelles. Additionally, the degradation of the PCL segment and its influence on drug release are also under study. With the pretreatment of biotin-transferrin, the resulting MAs could specifically bind to tumor cells, and the targeting ligand conjugated MAs could be used for tumor targeting carriers as schematically illustrated in Figure 1. Experimental Methods Materials. N-Isopropylacrylamide (NIPAAm) was purchased from Acros and used as received. ε-Caprolactone (CL) and tetrahydrofuran (THF) were obtained from Acros and Shanghai Chemical Reagent Company, respectively, and used after distillation. N,N′-Azobisisobutyronitrile (AIBN) was purchased from Shanghai Chemical Reagent Company and used after recrystallization with 95% ethanol. Biotin and fluorescein isothiocynate labeled avidin (FITC-avidin) were purchased from Pierce and used as received. Biotinylated transferrin (biotintransferrin) obtained from Sigma-Aldrich was used as received. Dulbecco’s modified eagle medium (DMEM) and dimethyl

10.1021/jp902637n CCC: $40.75  2009 American Chemical Society Published on Web 06/04/2009

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J. Phys. Chem. C, Vol. 113, No. 26, 2009 11263 SCHEME 1: Synthesis of Biotinylated PNAS-b-PNIPAAm-b-PCL Triblock Copolymer

Figure 1. Schematic illustration of the targeting of the thermo-sensitive MAs self-assembled from biotinylated PNAS-b-PNIPAAm-b-PCL copolymer.

sulfoxide (DMSO) were obtained from GIBCO Invitrogen Corporation. Doxorubicin hydrochloride (Dox · HCl) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. (China). N-Hydroxysuccinimide (NHS) was purchased from Shanghai Chemical Reagent Company and N-acroyloxysuccinimide (NAS) was synthesized according to the reported procedure.24 All other reagents and solvents were used without further purification. Synthesis of Chain Transfer Agent (CTA). 2-(Phenylcarbonothioylthio)acetic acid (CTA) was synthesized by the following method. In brief, bromobenzene (24.96 g) was used to prepare phenylmagnesium bromide in THF at 80 °C. After the stirring with methanedithione (40.19 g) for 24 h, magnesium phenylcarbonothioyl hypobromothioite was obtained. Finally, bromoacetic acid (22.0 g) was added dropwise to the resulting solution and the reaction was carried out for 24 h to yield the crude product, 2-(phenylcarbonothioylthio)acetic acid (15.2 g). After purification, crimson solid was obtained. 1H NMR (in DMSO) δ 7.48 (2H), 7.95 (2H), 7.64 (1H), 4.286 (2H). Synthesis of PNIPAAm-Based Macromolecules CTA (macroCTA) by RAFT. MacroCTA was synthesized according to the literature25 with some modifications. NIPAAm (3.42 g, 30 mmol), AIBN (1.64 mg, 0.01 mmol), and CTA (21.2 mg, 0.1 mmol) were dissolved in 10 mL of THF. The solution was degassed by bubbling with nitrogen for 1 h at room temperature. Then the polymerization was carried out at 70 °C for 48 h. The product was precipitated in an excess of diethyl ether twice and dried under vacuum after being filtered. The product was further purified by dissolving it in distilled water and dialyzing against distilled water for 1 week, using a dialysis membrane with a molecular weight cutoff (MWCO) of 8000-10000 g/mol (Shanghai Chemical Reagent Co., China). The final product NIPAAm macroCTA was harvested by freeze-drying. Synthesis of PNAS-b-PNIPAAm Copolymer by RAFT. The synthesis of PNAS-b-PNIPAAm copolymer was conducted at 70 °C in THF. In detail, NIPAM macroCTA (2.5 g, 0.0625 mmol), NAS (1.25 g, 7 mmol), and AIBN (4.1 mg, 0.025 mmol) were dissolved in 8 mL of THF. The solution was purged with nitrogen for 1 h at room temperature, then the polymerization was carried out at 70 °C for 48 h. The product was purified by repeated precipitation and dialysis as mentioned above, and the final product PNAS-b-PNIPAAm was harvested by freezedrying. Synthesis of PNAS-b-PNIPAAm-b-PCL Triblock Copolymer. PNAS-b-PNIPAAm-b-PCL was synthesized by ringopening polymerization of CL. PNAS-b-PNIPAAm with -COOH terminal groups actually served as the macroinitiator in the

TABLE 1: Molecular Weights of Polymers Measured by SEC-MALLS polymer

Mw

Mw/Mn

PNIPAAm PNAS-b-PNIPAAm PNAS-b-PNIPAAm-b-PCL

51 110 52 180 59 570

1.739 1.611 1.622

polymerization. The typical polymerization procedures are as follows (Scheme 1). PNAS-b-PNIPAAm (0.5 g) and anhydrous toluene (40 mL) were placed in a flask. Moisture was removed by azeotropic drying through evaporation of about 20 mL of toluene at 140 °C. After cooling, a solution of CL (0.5 g) was placed into the flask and the reaction was carried out at 120 °C for 48 h. The product was purified by repeated precipitation and dialysis as mentioned above, and the final product PNASb-PNIPAAm-b-PCL was obtained by freeze-drying. Characterization of PNAS-b-PNIPAAm-b-PCL Triblock. a. 1H NMR. 1H NMR spectra were recorded on a Mercury VX300 spectrometer at 300 Hz, using DMSO as a solvent. b. FT-IR Spectra. FT-IR spectra were recorded on an AVATAR 360 spectrometer. Samples were pressed into potassium bromide (KBr) pellets. c. SEC-MALLS of Polymer. The size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) were used to determine the molecular weights of polymers. A dual detector system, consisting of a MALLS device (DAWN EOS, Wyatt Technology) and an interferometric refractometer (Optilab DSP, Wyatt Technology), was used. The columns used were styragel HR1 and HR4. The concentration of each copolymer was kept constant at 10 mg/ mL and THF (chromatographic grade) was used as the eluent at a flow rate of 0.3 mL/min. The MALLS detector was operated at a laser wavelength of 690 nm. The molecular weight of copolymers was shown in Table 1. Conjugation of Triblock by Biotin. Biotin was introduced into the PNAS-b-PNIPAAm-b-PCL (0.3 g) copolymer by using an amide condensation reaction between amino groups in biotin (7 mg) and NAS in distilled water (12 mL) at 25 °C for 24 h. The purification process for macromonomer followed the same process.

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Characterization of Biotinylated PNAS-b-PNIPAAm-bPCL. 1H NMR spectra was recorded on a Mercury VX-300 spectrometer at 300 Hz, using DMSO and D2O as a solvent. Cytotoxicity. In vitro cytotoxicity was evaluated by MTT assay. 6.0 × 103 HeLa cells were incubated in each well of a 96-well plate. After incubation for 24 h in an incubator (37 °C, 5% CO2), the cells were then incubated in culture media containing biotinylated PNAS-b-PNIPAAm-b-PCL at various concentrations for 48 h. The polymer containing DMEM was replaced by fresh DMEM and 20 µL of MTT solution (5 mg/ mL) was added. After incubation for 4 h, the MTT medium was removed from each well and 200 µL of DMSO was added, and then the mixture was stirred at room temperature. The optical density (OD) was measured at 570 nm with a Microplate Reader Model 550 (BIO-RAD, USA). The cell viable rate was calculated by the following equation: viable cell (%) ) (ODtreated/ ODcontrol) × 100, where ODcontrol was obtained in the absence of triblock copolymers and ODtreated was obtained in the presence of particular triblock copolymers. Measurement of Critical Micelle Concentration (CMC). Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer) and pyrene was used as a hydrophobic fluorescent probe.26 Aliquots of pyrene solutions (6 × 10-6 M in acetone, 1 mL) were added to the containers, and the acetone was allowed to evaporate. Ten millimeters of aqueous solutions at different concentrations was then added to the containers containing the pyrene residue. It should be noted that all the aqueous sample solutions contained excess pyrene residue at the same concentration of 6 × 10-7 M. The micelle solutions of biotinylated PNAS-b-PNIPAAm-b-PCL copolymer with various concentrations from 2 × 10-3 to 2.0 g/L were added to each flask. Emission wavelength was carried out at 390 nm, and excitation spectra were recorded ranging from 300 to 360 nm. Both excitation and emission bandwidths were 5 nm. From the pyrene excitation spectra, the intensity ratio I340/I337 was analyzed as a function of the polymer concentration. A CMC value was determined from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentration.27 Transmission Electron Microscopy (TEM). At room temperature, a drop of MA suspension (∼1.0 mg/mL) stained by a drop of 1% (w/v) phosphotungstic acid was placed on a copper grid with Formvar film and dried before observation on a JEM100CXa TEM at an acceleration voltage of 80 keV. The size of MA at the same temperatures was measured by using a ζ-sizer nano series 3600 (Malvern Instruments). A Peltier device integrated into the cuvette holder was used to control the solution temperature. Prior to measurement, the suspensions (∼1.0 mg/mL) were allowed to thermally equilibrate for 3 min. The hydrodynamic radius of the MAs was calculated from diffusion coefficients, using the Stokes-Einstein equation. It is worth pointing out that no aggregation of MAs was observed during the measurements. Determination of LCST. Optical absorbance of polymers in aqueous solutions (1.0 mg/mL, distilled water was used as the solvent) at various temperatures was measured at 500 nm with a Lambda Bio40 UV-vis spectrometer (Perkin-Elmer) to determine its LCST. The sample cell was thermostated in a refrigerated circulator bath at different temperatures prior to measurements. The LCST is defined as the temperature exhibiting a 50% increase of the total increase in optical absorbance. Drug Loading and in Vitro Drug Release. Before loading doxorubicin to the mixed MAs, Dox · HCl (2 mg) was stirred with 1 mL of TEA in 3 mL of DMSO overnight to obtain the

Quan et al. Dox.28 And then biotinylated PNAS-b-PNIPAAm-b-PCL copolymer (40 mg) was dissolved in the solution. The solution was put into a dialysis tube (MWCO: 8000-10000 g/mol, Shanghai Chemical Reagent Co., China) and subjected to dialysis against 1000 mL of distilled water at 25 °C for 24 h. The UV absorbance of the dialysis solution was used to determine the amount of unloaded Dox, which was used further to calculate the encapsulation efficiency (EE%). The total amount of Dox fed initially in PBS solution was 2 mg. The EE% is defined as:

EE% ) (total amount of Dox) - (un-loaded amount of Dox) × total amount of Dox 100% The EE% was found to be 45%. For in vitro drug release study, the resulting drug-loaded MAs were added into 7 mL of PBS solution. These suspensions were transferred into dialysis membranes (molecular weight cutoff: 8000-12000), which were then immersed into 10 mL of PBS 7.4 solution at 25 and 37 °C, respectively. At predetermined time intervals, 10 mL of each dialysis medium was removed for measurement and the same amount of fresh PBS solution was added. The concentration of drug was determined by UV-vis spectroscopy at 497 nm.29 The amount of drug released from copolymers was determined by measuring the difference in concentration of the PBS solution before and after incubating with the drug-loaded MAs. Each batch was analyzed three times and data were given as the average values based on three independent measurements. Confocal Microscopy. Biotinylated PNAS-b-PNIPAAm-bPCL copolymer (2 mg) and FITC-avidin (25 µg) were dissolved in 1.0 mL of DMEM (Dulbecco’s Modified Eagle Medium), respectively. Then the two solutions were mixed and shaken at room temperature for 1 h to form FITC-avidin-MA complex. HeLa cells were seeded into LabTek chamber slide dishes (6.0 × 104 cells/plate) containing 1 mL of DMEM to grow to ∼70% confluence after 24 h of incubation. A 200 µL sample of biotintransferrin (200 mg/mL) was then added to each well and incubated with the cells for 2 h at 37 °C. Following the incubation period, medium was removed and cells were washed with PBS three times. Then, 1 mL of DMEM containing 200 µL of FITC-avidin-MA suspensions was added to each well and incubated with cells for another 5 h at 37 °C. After that, medium was again removed and cells were washed with PBS three times. In addition, another plate of HeLa sample was administrated without the biotin-transferrin step for comparison. The fluorescent images of cells were analyzed by using laser scanning confocal microscopy (Leica TCS SP2AOBS, Germany). Results and Discussion Synthesis and Characterization of PNAS-b-PNIPAAm-bPCL. The chemical structure of PNAS-b-PNIPAAm-b-PCL was confirmed by 1H NMR (in Figure 2). As demonstrated in spectra B and C in Figure 2, the characteristic peak of PNIPAAm and PNAS chain appear at 3.820 (a) and 2.811 ppm (f), respectively. After the ring-opening reaction (Figure 2A), the signal of CL was the following: 1.248 (h), 1.734 (g), 2.447 (i), and 3.574 ppm (j). With respect to the integrity ratio of the block in PNASb-PNIPAAm-b-PCL copolymer, the integrity ratio of the peak is C(a):C(f):C(j) ) 1.53:0.08:0.44, which indicates that the ratio

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Figure 4. 1H NMR spectra of biotinylated PNAS-b-PNIPAAm-bPCLtriblock copolymer (A) in DMSO and (B) in D2O.

Figure 2. 1H NMR spectra of (A) PNAS-b-PNIPAAm-b-PCL, (B) PNAS-b-PNIPAAm, and (C) PNIPAAm in DMSO.

Figure 5. Cytotoxicity of biotinylated PNAS-b-PNIPAAm-b-PCL triblock copolymer.

Figure 3. FR-IR spectra of (A) PNIPAAm, (B) PNAS-b-PNIPAAm, and (C) PNAS-b-PNIPAAm-b-PCL.

of the three moieties is n(NIPAAm):n(NAS):n(CL) ) 75:1:11. According to the SEC-MALLS data (as shown in Table 1), the molar ratio of the block in as-prepared triblock copolymer is n(NIPAAm):n(NAS):n(CL) ) 75:1:10, which is in good accordance with the data obtained from NMR. The structures of PNIPAAm, PNAS-b-PNIPAAm, and PNASb-PNIPAAm-b-PCL were characterized by FT-IR. As exhibited in the FT-IR spectra (Figure 3A-C), absorbance of amide carbonyl groups in PNIPAAm occurs at 1647 cm-1 and the bending frequency of amide N-H appears at 1546 cm-1. Stretch vibration for CdO in PNAS-b-PNIPAAm (Figure 3B) and PNAS-b-PNIPAAm-b-PCL (Figure 3C) appears at 1739 cm-1. Besides this, it has been found that the absorbance of CdO in the PNAS-b-PNIPAAm-b-PCL (Figure 3C) became much stronger than that in the PNAS-b-PNIPAAm (Figure 3B), which supports the formation of the triblock copolymers. All of the results indicate that the triblock copolymer is successfully synthesized.

Synthesis and Characterization of Biotinylated PNAS-bPNIPAAm-b-PCL. The successful conjugation of biotin was confirmed from NMR as shown in Figure 4. From the variety of the characteristic peak from NAS, we are able to determine the amount of biotin molecules conjugated to the copolymer. Comparing Figure 2A with Figure 4A, the characteristic peak of NAS, at 2.811 ppm, becomes weak and the integrity ratio of C(f):C(a) decreases from 0.04 (Figure 2A) to 0.01 (Figure 4A), which means that the biotin has been successfully conjugated with the triblock copolymer. The degree of substitution of biotin calculated based on the substituted NAS units in PNAS segment is around 75% according to 1H NMR spectra. The formation of micelle was detected by NMR in D2O and the data were shown in Figure 4B. Due to the hydrophobicity of PCL, the formation of core-shell micelle isolates the PCL segments in the inner core and produces the disappearance of NMR signals from PCL segments. Additionally, as shown in the amplified inserted figure, the weakened peak at 2.811 ppm also demonstrated that biotin has been successfully conjugated with NAS in the triblock copolymer. Cytotoxicity. The effect of the concentration of biotinylated PNAS-b-PNIPAAm-b-PCL on the proliferation of HeLa cells was used to investigate the cytotoxicity of copolymer and the data are shown in Figure 5. It is evident that the viability values of HeLa cells in the presence of copolymers are all above 90% and increase with increasing concentration of copolymer when the concentration is below 1.0 mg/mL, which demonstrates that the copolymer does not exhibit an apparent inhibition effect. It can also be seen from Figure 5 that the viability values of the cells increase with increasing concentration of the copolymer, which is probably attributed to the existence of the PCL segment in the triblock copolymer.

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Quan et al.

Figure 6. (A) Excitation spectra of pyrene at λem ) 390 nm with increasing concentrations of biotinylated PNAS-b-PNIPAAm-b-PCL triblock copolymer. (B) Plot of the intensity ratio I340/I337 vs. log C for biotinylated PNAS-b-PNIPAAm-b-PCL triblock copolymer.

Figure 7. (A) TEM and (B) size distribution of MAs self-assembled from biotinylated PNAS-b-PNIPAAm-b-PCL copolymer.

CMC. The formation of MAs from an amphiphilic biotinylated PNAS-b-PNIPAAm-b-PCL triblock copolymer was also verified by fluorescence probe technique with use of pyrene. The hydrophilic biotinylated PNAS-b-PNIPAAm segment served as the hydrophilic shell stabilizing the nanoparticle, and the hydrophobic PCL segment formed the core of the micelle. Excitation spectra of pyrene in the biotinylated PNAS-bPNIPAAm-b-PCL solutions are shown in Figure 6A. As can be seen, the fluorescence intensity increases with increasing concentration of biotinylated PNAS-b-PNIPAAm-b-PCL copolymer. Concomitant with the increase in fluorescence intensity, a red-shift from 337 to 340 nm takes place. This is ascribed to the micellization of biotinylated PNAS-b-PNIPAAm-b-PCL because pyrene is preferentially partitioned into the hydrophobic core of the micelles with a change of the photophysical properties. When the intensity ratio I340/I337 is plotted against the logarithm of polymer concentration in Figure 6B, the CMC value is obtained and the value is around 72 mg/L. The high CMC value might be ascribed mainly to the high degree of hydrophilic PNIPAAm units. TEM Studies. The morphology of the MAs was visualized by TEM as shown in Figure 7A. It is evident that the selfassembled MAs are well dispersed as individual particles with a regularly spherical shape. Under LCST, the biotinylated PNAS-b-PNIPAAm segment is hydrophilic, forming the shell of MAs self-assembled from biotinylated PNAS-b-PNIPAAmb-PCL. The long hydrophilic chain would prevent the aggregation of traditional amphiphilic PNIPAAm-based micelles and could make MAs more stable and soluble in water. As shown in Figure 7B, MAs self-assembled from biotinylated PNAS-bPNIPAAm-b-PCL exhibit a narrow size distribution with an average diameter of around 261 nm (peak 279 nm, PDI 0.13). The discrepancy in size of the MAs between the diameter obtained by TEM and that obtained by particle-size analyzer has also been reported in previous literature.30

Figure 8. Absorbance of PNIPAAm (a), PNAS-b-PNIPAAm (b), PNAS-b-PNIPAAm-b-PCL (c), and biotinylated PNAS-b-PNIPAAmb-PCL (d) at different temperatures.

LCST. The LCST of resulting copolymers are determined by monitoring the turbidity change of polymer aqueous solutions upon temperature change. As shown in Figure 8, the LCST of PNIPAAm, PNAS-b-PNIPAAm, PNAS-b-PNIPAAm-b-PCL, and biotinylated PNAS-b-PNIPAAm-b-PCL solutions are about 33.4, 34, 33.7, and 35.3 °C, respectively. Obviously, the presence of the hydrophilic NAS in PNAS-b-PNIPAAm copolymers (curve b) leads to a higher LCST than that of PNIPAAm (curve a). And after the conjugation of hydrophobic PCL, the LCST of PNAS-b-PNIPAAm-b-PCL (curve c) shifts to a lower temperature. Comparing curves c and d, the LCST of biotinylated PNAS-b-PNIPAAm-b-PCL shifts to a high temperature. This is attributed to the presence of hydrophilic biotin that makes the PNAS segment more hydrophilic by replacing -CO-O-N- groups in the NAS moiety by -CO-NH- groups. In Vitro Drug Release. The drug release from the thermosensitive MAs was investigated in PBS pH 7.4 and the data were shown in Figure 9. Dox was selected as a hydrophobic antitumor model drug for in vitro drug release. The drug release profile from MAs shows great changes with temperature alterations around the LCST. Below the LCST (25 °C), the highly hydrated large PNIPAAm blocks would stabilize the hydrophobic/hydrophilic core-shell structure of micelles. Therefore, about 42% drug still remains in the core of the MAs. But when the temperature is raised above the LCST (37 °C), the PNIPAAm shell becomes hydrophobic and the micellar core-shell structure is deformed. Due to the high degree of hydrophobic blocks and the repulsion between the large blocks, the hydrophobic chains of the copolymers are unable to contact tightly, leading to a loosely packed hydrophobic core. Consequently, the loaded drug molecules were squeezed out of the core and about 97% drug is released from MAs. In addition, it was observed that the drug-loaded MAs were well dispersed in

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Figure 9. Drug release of MAs self-assembled from biotinylated PNAS-b-PNIPAAm-b-PCL copolymer in PBS 7.4.

In this study, a multifunctional micellar aggregate selfassembled from biotinylated PNAS-b-PNIPAAm-b-PCL was designed and synthesized via the combination of RAFT and ring-opening polymerization. The conformation of micellar aggregates in aqueous media was confirmed by 1H NMR spectra, fluorescence spectra, and TEM. Due to the temperature-induced structural changes of the micellar shell-core structure, the drug release profile of Dox-loaded MAs showed a thermo-sensitive switching behavior. The internalization studies showed that, with pretreatment of biotin-transferrin, the resulting MAs could specifically bind to tumor cells, indicating that the multifunctional MAs could be used as a promising drug carrier for tumor targeting chemotherapy. Acknowledgment. This work was supported by the National Key Basic Research Program of China (2005CB623903), the National Natural Science Foundation of China (50633020), and the Ministry of Education of China (Cultivation Fund of Key Scientific and Technical Innovation Project 707043). References and Notes

Figure 10. Confocal microscopy images of HeLa cells incubated with FITC-avidin-MA complex (A) with biotin-transferrin pretreating and (B) without biotin-transferrin pretreating: (A1, B1) fluorescence image, (A2, B2) bright field image, and (A3, B3) overlapped image. (The scale bar represents 50 µm.)

the distilled water at 25 °C. But when the temperature reached 37 °C, drug-loaded MAs began to aggregate and settle to the bottom of the dialysis tube. This change demonstrates that temperature-induced deformation and precipitation of the micellar core-shell structure can accelerate the drug release. Confocal Images. The internalization of the FITC-avidinMA complex by HeLa cells was visualized by confocal microscopy (Figure 10). The different distributions of green fluorescence reflect different distributions of the FITC-avidinMA complex in cellular media, further demonstrating different targeting abilities of the resulting MAs. After pretreated by biotin-transferrin, the shape of the cell membrane as well as the cell cytoplasm is clearly identified and there is bright fluorescence (Figure 10A) after 5 h of incubation with the FITCavidin-MA complex. In contrast, as presented in Figure 10B, only a slightly fluorescent signal was detected in the cytoplasm free of biotin-transferrin pretreatment (Figure 10B). The different fluorescence intensity means that (1) mediated by transferrin receptors on the surface of HeLa cells, biotinylated transferrin could be selectively and effectively conjugated with the plasmalemma and (2) the avidin molecules in the FITC-avidin-MA complex could recognize and bind with biotin molecules on the surface of the cell membrane. That is, the biotinylated MAs could be internalized by biotin receptor-mediated endocytosis in a short time period. The facilitation of the cell targeting and internalization of the FITC-avidin-MA complex for the pretargeting approach suggest that the multifunctional MA drug carriers have great potentials for tumor targeting.

(1) Torchilin, V. Eur. J. Pharm. Biopharm. 2009, 71, 431. (2) Jiang, M.; Wu, Y.; He, Y.; Nie, J. Polym. Int. 2009, 58, 31. (3) Huang, C. K.; Lo, C. L.; Chen, H. H.; Hsiue, G. H. AdV. Funct. Mater. 2007, 17, 2291. (4) Convertine, A. J.; Benoit, D. S. W.; Duvall, C. L.; Hoffman, A. S.; Stayton, P. S. J. Controlled Release 2009, 133, 221. (5) Torchilin, V. P. AdV. Drug DeliVery ReV. 2008, 60, 548. (6) Pan, J.; Feng, S. S. Biomaterials 2009, 30, 1176. (7) Byrne, J. D.; Betancourt, T.; Brannon-Peppas, L. AdV. Drug DeliVery ReV. 2008, 60, 1615. (8) Accardo, A.; Tesauro, D.; Pozzo, L. D.; Mangiapia, G.; Paduano, L.; Morelli, G. J. Pept. Sci. 2008, 14, 903. (9) Barz, M.; Tarantola, M.; Fischer, K.; Schmidt, M.; Luxenhofer, R.; Janshoff, A.; Theato, P.; Zentel, R. Biomacromolecules 2008, 9, 3114. (10) Quan, C. Y.; Sun, Y. X.; Cheng, H.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Nanotechnology 2008, 19, 275102. (11) Xu, P. S.; Tang, H. D.; Li, S. Y.; Ren, J.; Kirk, E. V.; Murdoch, W. J.; Radosz, M.; Shen, Y. Q. Biomacromolecules 2004, 5, 1736. (12) You, Y. Z.; Hong, C. Y.; Wang, W. P.; Lu, W. Q.; Pan, C. Y. Macromolecules 2004, 37, 9761. (13) Karanam, S.; Goossens, H.; Klumperman, B.; Lemstra, P. Macromolecules 2003, 36, 3051. (14) Li, C. M.; Buurma, N. J.; Haq, I.; Turner, C.; Armes, S. P.; Castelletto, V.; Hamley, I. W.; Lewis, A. L. Langmuir 2005, 21, 11026. (15) Sun, X. Y.; Luo, Y. W.; Wang, R.; Li, B. G.; Liu, B.; Zhu, S. P. Macromolecules 2007, 40, 849. (16) Krasia, T. C.; Patrickios, C. S. Macromolecules 2006, 39, 2467. (17) Hu, Y. Q.; Kim, M. S.; Kim, B. S.; Lee, D. S. Polymer 2007, 48, 3437. (18) Xu, S. P.; Liu, W. Q. J. Fluorine Chem. 2008, 129, 125. (19) York, A. W.; Kirkland, S. E.; McCormick, C. L. AdV. Drug DeliVery ReV. 2008, 60, 1018. (20) Cheng, C.; Wei, H.; Shi, B. X.; Cheng, H.; Li, C.; Gu, Z. W.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Biomaterials 2008, 29, 497. (21) Lee, E. S.; Na, K.; Bae, Y. H. Nano Lett. 2005, 5, 325. (22) Wei, H.; Zhang, X. Z.; Zhou, Y.; Cheng, S. X.; Zhuo, R. X. Biomaterials 2006, 27, 2028. (23) Wu, D. Q.; Sun, Y. X.; Xu, X. D.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Biomacromolecules 2008, 9, 1155. (24) Liu, F.; Tao, G. L.; Zhuo, R. X. Polym. J. 1993, 25, 561. (25) Chang, C.; Wei, H.; Quan, C. Y.; Li, Y. Y.; Liu, J.; Wang, Z. C.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. J. Polym. Sci. Polym. Chem. 2008, 46, 3048. (26) Hu, X. L.; Liu, S.; Chen, X. S.; Mo, G. J.; Xie, Z. G.; Jing, X. B. Biomacromolecules 2008, 9, 553. (27) Liu, Y. H.; Cao, X. H.; Luo, M. B.; Le, Z. G.; Xu, W. Y. J. Colloid Interface Sci. 2009, 329, 244. (28) Lee, E. S.; Na, K.; Bae, Y. H. J. Controlled Release 2005, 103, 405. (29) Oh, J. K.; Siegwart, D. J.; Lee, H.; Sherwood, G.; Peteanu, L.; Hollinger, J. O.; Kataoka, K.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 5939. (30) Li, Y. Y.; Zhang, X. Z.; Cheng, H.; Zhu, J. L.; Li, U. N.; Cheng, S. X.; Zhuo, R. X. Nanotechnology 2007, 18, 505101.

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