Functionalized Thermoresponsive Micelles Self-Assembled from

DSP (8.85 × 10−5 mol) in 3 mL of DMF was then added to the reaction flask to obtain biotin−PEG dimer and biotin−PEG was connected by disulfide ...
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Bioconjugate Chem. 2008, 19, 1194–1201

Functionalized Thermoresponsive Micelles Self-Assembled from Biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA for Tumor Cell Target Cheng Cheng, Hua Wei, Jing-Ling Zhu, Cong Chang, Han Cheng, Cao Li, Si-Xue Cheng, Xian-Zheng Zhang,* and Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan 430072, China. Received January 4, 2008; Revised Manuscript Received March 30, 2008

Novel micelles, comprising hydrophilic PEG shells, hydrophobic PMMA cores, and thermosensitive P(NIPAAmco-HMAAm) segments were self-assembled from the biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA triblock copolymer. The thermosensitive micelles exhibited superior stability and showed thermotriggered drug release behavior upon temperature alterations. The fluorescence spectroscopy and confocal microscopy studies confirmed that the self-assembled biotinylated micelles can be specifically and efficiently bonded to cancer cells with the administration of biotin-transferrin, suggesting that the multifunctional micelles have great potential as drug carriers for tumor targeting chemotherapy.

INTRODUCTION Traditional cancer chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by anticancer drugs. However, the nonspecific actions of the anticancer agents lead to systemic toxicity, causing undesirable severe side effects such as damage to liver, kidney, hair loss, and bone marrow (1). To reduce the side effects and to increase the local drug concentration at the targeting site, biotin-avidin systems (BAS) have been introduced to drug delivery systems to realize pretargeting in tumor chemotherapy (2, 3). Due to the considerable binding affinity between biotin and avidin, BAS can serve as a universal linkage between biotinylated polymeric drug carriers and other proteins. In the pretargeting approach, the biotinylated therapeutic carrier is administered after an appropriate delay, followed by the administration of active targeting ligands, such as avidin-conjugated monoclonal antibodies (4). In the past decade, amphiphilic block copolymers have been extensively investigated with respect to fundamental research as well as biomedical applications (5–14). The unique character of the amphiphilic block copolymers enables them to selfassemble in an aqueous solution to form micelles (15–20). Due to the small size and the hydrophilic surface, polymeric micelles are not easily recognized and captured by the reticuloendothelial systems (RES) (21, 22). Accordingly, the polymeric micelles have a relatively long circulation time after intravenous administration and, as a result, they enrich in tumor tissues due to the enhanced permeation and retention effect (EPR effect) (23). Very recently, intelligent polymeric materials have been developed to be drug carriers for controlled release, such as stimuliresponsive polymeric micelles (24–27) and bioactive site-specific targeting carriers (4, 28). To date, as far as we know, very few studies deal with multifunctional drug carriers especially selfassembled polymeric micelles with both environmental sensitivity and specific targeting function (29, 30). In this study, we designed and prepared a new type of multifunctional micelle as the drug carrier which not only has thermosensitivity, but also can be functionalized with a variety of ligands. The micelles were self-assembled by biotinylated * Corresponding author. Tel./fax: +86 27 68754509. E-mail address: [email protected] (X. Zhang).

Scheme 1. Schematic Illustration of the Thermally Induced Structure Change of Micelles Self-Assembled from the Triblock Copolymer in an Aqueous Solution

amphiphilic biotin-poly(ethylene glycol)-block-poly(N-isopropylacrylamide-co-N-hydroxylmethylacrylamide)-block-poly(methyl methacrylate) (biotin-PEG-b- P(NIPAAm-co-HMAAm)b-PMMA) triblock copolymer. The self-assembly and thermally induced change of the triblock copolymer micelle are schematically illustrated in Scheme 1. In the triblock copolymer, the PEG block was introduced as a hydrophilic segment due to its good biocompatibility and ability to stabilize the micelles and to protect them from being cleared up by the RES (31). Since the PEG shell was hydrophilic regardless of the temperature, the micelles self-assembled from the triblock copolymer would be more stable in water due to the fact that the PEG shell would prevent the aggregation of conventional PNIPAAm-based micelles once reaching the temperature above the LCST (20, 32, 33). Besides, the flexible PEG spacer offered an optimized accessibility for ligands (28). The PMMA block was used as a hydrophobic segment. The purpose to incorporate the poly(Nisopropylacrylamide-co-N-hydroxylmethylacrylamide) block was to endow the copolymer with thermosensitivity. Poly(N-isopropylacrylamide) (PNIPAAm) is known for exhibiting a reversible thermoresponsive phase transition in an aqueous solution at around 32 °C, termed lower critical solution temperature (LCST). However, such a LCST is not always favorable for biomedical applications since the physiologic temperature is around 37 °C. Herein, the hydrophilic N-hydroxylmethylacrylamide (HMAAm) was employed to adjust the LCST of the copolymer, since HMAAm is more hydrophilic, and an increase in its content would lead to a higher LCST. The

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Functionalized Micelles for Tumor Cell Target

P(NIPAAm-co-HMAAm) block was hydrophilic when the temperature was lower than the LCST, and became hydrophobic when the temperature was higher than the LCST. Furthermore, the biotin molecule was introduced to the micelle drug delivery system and BAS can be further used as reagents for pretargeting method in chemotherapy to increase the local concentration of drug at the target site resulting from the fact that active recognizing ligands can be incorporated into the drug delivery system through biotin-avidin interactions. In the pretargeting approach, the biotinylated drug carrier is administered after an appropriate delay, following the recognizing ligands conjugated with avidin, instead of coupling with them directly which may cause the denaturation of these ligands during the synthesis process in organic solvent. In addition, the delay allows for the antibody to localize and concentrate in the specific tissues, and then the thermoresponsive drug carrier can perform the controlled thermotriggered release by changing the environmental temperature.

MATERIALS AND METHODS Materials. N-Isopropylacrylamide (NIPAAm), 3-mercaptopropionic acid (MPA), and 2-amino-ethanethiol hydrochloride (AET · HCl) purchased from Acros were used as received. Rhodamine B, N-hydroxymethylacrylamide (HMAAm), Nhydroxysuccinimide (NHS), and dicyclohexylcarbodiimide (DCC) were obtained from Tianjin Chemical Reagent Co. (Tianjin, China) and used as received. Biotin, dithiobis (succinimidyl propionate) (DSP), and fluorescein isothiocyanate-labeled avidin (FITC-avidin) were purchased from Pierce, and used as received. 1,1-Carbonyldiimidazole (CDI) and biotinylated transferrin (biotin-trasferrin) obtained from Sigma-Aldrich were used as received. N,N′-Dimethylformamide (DMF), methyl methacrylate (MMA), tetrahydrofuran (THF), and 1,4-dioxane obtained from Shanghai Chemical Reagent Co. were used after distillation. Diamine poly(ethylene glycol) (H2N-PEG-NH2) with a molecular weight of 3000 g/mol (in THF) and 4,4′-azobis-4-cyanovaleric acid (ACVA) were purchased from Fluka and used as received. Dithiothreitol (DTT) was obtained from Shanghai BoAo Biotechnical Co. Ltd. and used as received. N,N′Azobisisobutyronitrile (AIBN) provided by Shanghai Chemical Reagent Co. (Shanghai, China) was used after recrystallization from 95% ethanol. MTX was kindly gifted by SuRi Biochem Co. Ltd. (Suzhou, China). All other reagents and solvents were used without further purification. Preparation of Biotin-PEG-b-P(NIPAAm-co-HMAAm)-bPMMA. Amino-terminated biotinylated poly(ethylene glycol) (biotin-PEG-NH2) was synthesized according to the literature (24). Biotin-PEG-NH2 (1.77 × 10-4 mol) was dissolved in 3 mL DMSO. DSP (8.85 × 10-5 mol) in 3 mL of DMF was then added to the reaction flask to obtain biotin-PEG dimer and biotin-PEG was connected by disulfide bonds. The reaction mixture was stirred for 24 h at room temperature. The dimer was reduced by adding DTT (2.66 × 10-4 mol) to the reaction mixture and the reaction was carried out at room temperature for 24 h to obtain sulfhydryl-terminated biotinylated PEG (biotin-PEG-SH). Carboxyl-terminated biotin-PEG-b-P(NIPAAm-co-HMAAm) block was synthesized by copolymerizing NIPAAm and HMAAm (the molar ratio of NIPAAm and HMAAm is 10:1) by using ACVA as an initiator (30, 31) and biotin-PEG-SH as a macromolecular chain transfer reagent. Amino-terminated PMMA (PMMA-NH2) was prepared by radical polymerization by using AET · HCl as a chain transfer agent. MMA (4.8 × 10-2 mol), AET · HCl (1.92 × 10-3 mol), and AIBN (2.4 × 10-4 mol) were dissolved in 20 mL DMF. The solution was degassed by bubbling with nitrogen for 30 min. Polymerization was carried

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out at 60 °C for 6 h. After reaction, the product was purified by repeated precipitation in water, and then the product was dried in vacuum. The resulting carboxyl-terminated biotin-PEG-b-P(NIPAAmco-HMAAm) (0.10 g), PMMA-NH2 (0.10 g), and NHS (4.0 × 10-5 mol) were dissolved in 4 mL dioxane. DCC (4.0 × 10-5 mol) in 1 mL dioxane was added dropwise to the solution under nitrogen atmosphere. After 24 h reaction at room temperature, the product was precipitated in excess diethyl ether and dried in vacuum after filtration. GPC Measurements. Number-average molecular weight (Mn) of PMMA-NH2, biotin-PEG-b-P(NIPAAm-co-HMAAm)COOH, and biotin-PEG-b-P(NIPAAm -co-HMAAm)-b-PMMA were determined by gel permeation chromatographic (GPC) system equipped with a Waters 2690D separations module and a Waters 2410 refractive index detector. THF was used as the eluent at a flow rate of 0.3 mL/min. Waters millennium module software was used to calculate molecular weight on the basis of a universal calibration curve generated by polystyrene standard with narrow molecular weight distribution. Optical Absorption Measurements. Optical absorbance of the biotin-PEG-b-P(NIPAAm-co-HMAAm)-COOH and biotinPEG-b-P(NIPAAm-co- HMAAm)-b-PMMA copolymer aqueous solution (600 mg/L) at various temperatures was measured at 542 nm with a Lambda Bio40 UV-vis spectrometer (PerkinElmer). Sample cell was thermostatted in a refrigerated circulator bath at a predefined temperature prior to measurements. The LCST of the polymer solution was defined as the temperature producing a half-increase of the total increase in optical absorbance. In Vitro Cytotoxicity Study. For each well in a 96-well plate, 200 µL of human vein endothelial cell line (ECV304) in RPMI1640, with a concentration of 2.5 × 105 cells/mL, was added. After incubation for 24 h in incubator (37 °C, 5% CO2), The culture medium was changed to 200 µL of RPMI1640 containing biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA copolymer with a particular concentration and the mixture was further incubated for 48 h. Then, RPMI1640 with polymer was replaced by fresh RPMI1640 and 20 µL of MTT solution (5 mg/mL) and was added to the ECV304. After incubation for 4 h, 200 µL of DMSO was added and shaken at room temperature. The optical density (OD) was measured at 570 nm with a Microplate Reader (model 550). Micelle Formation. Membrane-dialysis method was used to prepare micelles through self-assembly of biotin-PEG-bP(NIPAAm-co-HMAAm)-b-PMMA copolymer. In brief, the triblock copolymer (10 mg) was dissolved in 2 mL DMF. The solution was put into a dialysis tube (molecular weight cutoff: 8000-10 000 g/mol) and subjected to dialysis against 1000 mL distilled water for 24 h, then lyophilized before further examinations. FT-IR and 1H NMR Characterizations. FT-IR spectra were recorded on an AVATAR 360 spectrometer. Samples were pressed into potassium bromide (KBr) pellets. 1H NMR spectra were recorded on a Mercury VX-300 spectrometer at 300 Hz using CDCl3 and D2O as the solvents. Fluorescence Measurements. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer) and pyrene was used as a hydrophobic fluorescent probe (20). Aliquots of pyrene solutions (6 × 10-6 M in acetone,1 mL) were added to containers, and the acetone was allowed to evaporate. Ten-milliliter aqueous polymer solutions at different concentrations were 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 solutions were kept at room temperature for 24 h to reach the solubilization equilibrium of

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pyrene in the aqueous phase. Excitation was carried out at 340 nm, and emission spectra were recorded ranging from 350 to 600 nm. Both excitation and emission bandwidths were 10 nm. From the pyrene emission spectra, the intensities (peak height) of I373nm were analyzed as a function of the polymer concentration. Transmission Electron Microscopy (TEM). A drop of micelle suspension 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 JEM-100CXa transmission electron microscope (TEM) at an acceleration voltage of 80 kV. Size Distribution Measurements. The size distribution of micelles was determined by a Nano Series Nano-ZS (MALVERN Instrument) zetasizer. The micelle aqueous solution (100 mg/L) was passed through a 0.45 µm pore-sized syringe filter and kept in the thermostat of the apparatus at 37 °C ((0.05 °C) for 20 min to reach the equilibrium prior to the measurements. Drug Loading and In Vitro Drug Release. Biotin-PEG-bP(NIPAAm-co- HMAAm)-b-PMMA triblock copolymer (10 mg) and MTX (5 mg) were dissolved in 2 mL DMF. The solution was put into a dialysis tube and subjected to dialysis against 1000 mL distilled water at 25 °C for 24 h. To determine the EE, the drug-loaded micelles solution was lyophilized, and then dissolved in DMF and analyzed by UV absorbance at 303 nm. After dialysis, the dialysis tube was directly immersed into 400 mL distilled water. Aliquots of 4 mL were withdrawn from the solution periodically. The volume of solution was held constant by adding 4 mL distilled water after each sampling. The amount of MTX released from micelles was measured at different temperatures using UV absorbance at 303 nm. Fluorescence Dye Loading. Biotin-PEG-b-P(NIPAAm-coHMAAm)-b-PMMA triblock copolymer (10 mg) and rhodamine B (5 mg) were dissolved in 2 mL DMF. The solution was put into a dialysis tube and subjected to dialysis against 1000 mL distilled water at 25 °C for 24 h. Then, the dye-loaded micelles were lyophilized before further measurements. Cell Uptake Study. The dye-loaded freeze-dried micelles (200 µg) and FITC-avidin 100 µg were dissolved in 1.0 mL 0.01 M phosphate buffer saline (PBS, pH 7.4), respectively. Then the two solutions were mixed and shaken at room temperature for 30 min. The solution was centrifuged (10 00012 000 r/min) for 15 min, and then the sediment was washed with cold PBS three times, and suspended again in 2.0 mL PBS as the FITC-labeled micelle samples. HeLa, A549, and ECV304 cells were seeded into a 24-well plate (1 × 105 cells/well) containing 1 mL of DMEM media to grow to ∼70% confluence after 24 h incubation. 100 µL biotin-transferrin (200 µg/mL) was then added in each well and incubated with cells for 2 h at 37 °C. After the incubation, the medium was removed and cells were washed with cold PBS three times. Then, 1 mL DEME medium containing 100 µL FITC-avidin-rhodamine B loaded micelle complex was added in each well and incubated with cells for another 2 h at 37 °C. After that, medium was again removed and cells were washed with cold PBS three times. In addition, another plate of HeLa sample was administrated without the biotin-transferrin step for comparison. Finally, 1 mL of DMSO was added in each well to lyse cells. Fluorescence emission intensities of FITC at 490 nm and rhodamine B at 570 nm (LS55 luminescence spectrometer, Perkin-Elmer) provided means for the measurement of internalized FITC and rhodamine B levels. Confocal Microscopy Observation. Cells were seeded into specific confocal investigation plates containing 1 mL DMEM media (1 × 105 cells/plate). Then the samples were treated in the same way of fluorescent spectroscopy analysis. The fluo-

Cheng et al. Scheme 2. Synthesis of Biotin-PEG-b-P(NIPAAm-co-HMAAm)-bPMMA Triblock Copolymer

Table 1. GPC Data of Biotin-PEG-b-P(NIPAAmco-HMAAm)-COOH, PMMA-NH2, and Biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA sample

Mn

Mw/Mn

biotin-PEG-b-P(NIPAAm-co-HMAAm)-COOH PMMA-NH2 biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA

11 032 5 473 15 484

1.6 1.7 1.8

rescent images of cells were analyzed using laser scanning confocal microscopy (Leica TCS SP2AOBS, Germany).

RESULTS AND DISCUSSION Synthesis of Biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA. Carboxyl-terminated biotin-PEG-b-P(NIPAAm-co-HMAAm) block (biotin-PEG-b-P(NIPAAm-co-HMAAm)-COOH) was synthesized by copolymerizing NIPAAm and HMAAm by using ACVA as an initiator and biotin-PEG-SH as a macromolecular chain transfer reagent. And amino-terminated PMMA (PMMA-NH2) was prepared by radical polymerization by using AET · HCl as a chain transfer agent. Finally, the triblock copolymers of biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA were obtained by a condensation reaction between the carboxylic end groups of biotin-PEG-b-P(NIPAAm-coHMAAm) and amino end groups of PMMA. The detailed synthesis procedure is illustrated in Scheme 2. The block coupling was confirmed by molecular weights determined by GPC (Table 1) and 1H NMR spectrum of biotin-PEG-bP(NIPAAm-co-HMAAm)-b-PMMA in CDCl3 (Figure 4a), which reveals the presence of signals from PEG, P(NIPAAmco-HMAAm), and PMMA blocks. In addition, it can be seen from Figure 1 that the LCSTs of biotin-PEG-b-P(NIPAAm-co-HMAAm)-COOH and biotinPEG-b-P(NIPAAm- co-HMAAm)-b-PMMA were determined to be 43.1 and 40.8 °C, respectively. Cytotoxicity Study. Cytotoxicity study was performed to evaluate the cytotoxicity of the biotinylated triblock copolymer.

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Figure 1. Optical absorbance of biotin-PEG-b-P(NIPAAm-co-HMAAm)COOH and biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA triblock copolymer in aqueous solutions/suspensions at various temperatures. (Polymer concentration was 600 mg/L). Figure 3. FT-IR spectra of (a) biotin-PEG-b-P(NIPAAm-co-HMAAm)COOH; (b) PMMA-NH2; (c) biotin-PEG-b-P(NIPAAm-co-HMAAm)b-PMMA block copolymer; and (d) biotin-PEG-b-P(NIPAAm-coHMAAm)-b-PMMA micelles.

Figure 2. Cytotoxicity study of biotin-PEG-b-P(NIPAAm-co-HMAAm)b-PMMA triblock copolymer.

The effect of the polymer concentration on the proliferation of human vein endothelial cells (ECV304) was studied and it can be seen from Figure 2 that the cell viability was above 60% even when the copolymer concentration was as high as 3.3 g/L, suggesting that the copolymer exhibits no apparent cytotoxicity. MicelleFormation.Thestructuresofbiotin-PEG-b-P(NIPAAmco- HMAAm)-COOH, PMMA-NH2, biotin-PEG-b-P(NIPAAmco-HMAAm)-b-PMMA block copolymer, and the resulting micelles were characterized by FT-IR. As exhibited in the FTIR spectra, the absorbance of amide carbonyl groups in P(NIPAAm-co-HMAAm) occurs at 1650 cm-1, and bending frequency of amide N-H appears at 1550 cm-1 (Figure 3a). Stretch vibration for CdO in ester in PMMA-NH2 appears at 1730 cm-1 (Figure 3b). All the peaks mentioned above can be seen from the spectrum of biotin-PEG-b-P(NIPAAm-coHMAAm)-b-PMMA polymer (Figure 3c), which supports the formation of the triblock copolymers. Furthermore, it has been found that the absorbance of CdO in the ester segments is quite weak in the spectrum of biotin-PEG-b-P(NIPAAm-co-HMAAm)b-PMMA micelles (Figure 3d), as compared with that in the spectrum of copolymer. However, the typical peaks at 1550 and 1650 cm-1 from the P(NIPAAm-co-HMAAm) segment in the spectrum of micelle (Figure 3d) are as strong as those in the spectrum of biotin-PEG-b-P(NIPAAm-co-HMAAm)-COOH regardless of the formation of micelles (Figure 3a). According to our previous study (33), the reason is that block copolymers form core-shell micellar structure with completely isolated hydrophobic inner cores and hydrophilic outer shells. Consequently, the peak for stretch vibration of CdO in the ester segments of micelles is apparently weakened. 1 H NMR analyses can provide another insight into the selfassembly process since aggregation may result in a decreased mobility of the core-forming block and a resultant suppression of the NMR signal intensities. The blocks in amphiphilic biotin-

PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA possess disparate solubilities in selective solvents. Consequently, the NMR signals originating from all three blocks can be recorded when using CDCl3. In contrast, the use of water as NMR solvent results in insufficient mobility and a suppression of the signals pertinent to PMMA block. In fact, as shown in Figure 4b, the use of water results in the total absence of characteristic NMR signals originating from PMMA block. This result indicates the formation of the well-defined core-shell structure of the block copolymer in aqueous solution, which exhibits a shell of biotinPEG-b-P(NIPAAm-co-HMAAm) and a core of PMMA. The formation of micelles from the triblock copolymers was also verified by a fluorescence probe technique using pyrene. In the emission spectra (Figure 5a), fluorescence intensity increases with increasing copolymer concentration, due to the fact that pyrene in water has a very small absorption, which increases when it is transferred into a hydrophobic environment. This effect also supports the proposed micelle formation. From the plot of fluorescence intensity versus polymer concentration (Figure 5b), fluorescence intensity is essentially constant at low concentration. Above a certain concentration, intensity increases dramatically, indicating formation of micelles and the transfer of pyrene into the hydrophobic core of the micelles. This concentration can be defined as the critical micellar concentration (cmc). The cmc value of the triblock copolymer obtained in this study is a low concentration of 8.5 mg/L, implying that the micelles could be used in very dilute aqueous media such as body fluids. In a word, the results of fluorescence are in good agreement with those of FT-IR and 1H NMR. From these results, we conclude that the triblock copolymers do form micelles in the aqueous media, which further conforms that the triblock copolymer was successfully synthesized because a physical mixture of the three polymers would lead to the separation in water. Characterization of Polymeric Particles. As can be visualized by transmission electron microscopy (TEM) (Figure 6a), the self-assembled micelles are well-dispersed as individual particles with a regularly spherical shape. As shown in Figure 6b, the micelles exhibited a narrow size distribution with an average diameter of 195 nm (PDI 0.115). Compared with the size determined by the particle-size analyzer, the particle size observed by TEM is smaller. The variation in particle size measured by TEM and particle-size analyzer is attributed to the fact that dynamic light scattering (DLS) measurement of

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Figure 4. 1HNMR spectra of biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA in (a) CDCl3 and (b) D2O, respectively.

Figure 5. (a) Fluorescence emission spectra of pyrene with increasing concentration of biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA; (b) the intensity of I373 nm in the emission spectra as a function of logarithm of copolymer concentration.

the particle-size analyzer gives the hydrodynamic diameter rather than the actual diameter of the dried particles. Similar discrepancy in size was also reported in the literature (34, 37, 38). In order to provide direct evidence of the stability of the micelles formed by the triblock copolymers, we kept the micelles aqueous solution for 40 days at room temperature. No obvious change in the morphology of the micelles was observed after 40 days (Figure 6c), indicating the micelles have superior stability in aqueous solution at room temperature. Consistent with the TEM observation, it was found that the size (average diameter 186 nm) and the size distribution (PDI 0.070) were very similar to that obtained 40 days before (Figure 6d). This suggests that the superior stability of the micelles would be favorable for the practical biomedical applications. In Vitro Drug Release. The thermotriggered drug release behavior of the biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-

PMMA micelles was also investigated in this study. Methotrexate (MTX), a poorly water-soluble anticancer drug, was loaded in the micelles by the membrane-dialysis method at room temperature with entrapment efficiency (EE) of around 10%. The in vitro drug release property of the biotin-PEG-bP(NIPAAm-co-HMAAm)-b-PMMA micelles was evaluated at temperatures below (37 °C) and above (43 °C) the LCST of biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA micelle. The drug release profiles show drastic changes with temperature alterations as presented in Figure 7. Compared with the release at 37 °C, the drug release is accelerated dramatically at 43 °C due to the temperature-induced structural changes of the micelles. When the temperature increased above the LCST, the P(NIPAAm-co-HMAAm) segment becomes hydrophobic, which makes the hydrophilic shell of the micelle structure become thinner. As a result, the drug diffuses out quickly and about 92% of the drug is released from the micelles during 96 h at 43 °C. Cell Uptake Study. Cervical cancer HeLa cells have been extensively characterized for intracellular delivery through receptor-mediated endocytosis using the iron-carrying protein transferrin for targeting (39–41). In the two-step pretargeting protocol, we used HeLa cells to study the intracellular uptake of the triblock copolymer micelles in vitro. Rhodamine B, a versatile fluorescence dye, was initially entrapped in the micelles during the dialysis at room temperature as a probe. FITC-labeled avidin was then associated with the rhodamine B loaded biotinylated micelles. Then after the preincubation of biotintransferrin and HeLa cells, FITC-avidin-rhodamine B loaded micelle complex was infused to “chase” biotin-transferrin through the biotin-avidin interaction. Based on the fluorescence emission intensities of rhodamine B at 570 nm and FITC at 490 nm, the cell uptake of the FITC-avidin-rhodamine B loaded micelle was calculated as follows (42): Uptake ) [(concentration of the dye internalized into cells in each well)/(concentration of the dye added in each well)] × 100% Human lung cancer cells (A549) and vein endothelial cells (ECV304) with the same administration procedure as well as the HeLa samples free of the preincubating with biotinylated transferrin were also studied for comparison. The cell uptake calculated by both fluorescence probes is presented in Figure 8. Based on the fluorescence probe of FITC, with the presence of biotin-transferrin, cell uptake values are 30.6% for HeLa, 9.4% for A549, and 8.7% for ECV304, respectively. On the

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Figure 6. (a) TEM image and (b) size distribution of biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA micelles; (c) TEM image and (d) size distribution of biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA micelles self-assembled in an aqueous medium after 40 days.

Figure 7. Drug release from the thermoresponsive biotin-PEG-bP(NIPAAm-co-HMAAm)-b-PMMA micelles loaded with MTX at different temperatures (37 and 43 °C).

Figure 8. The rate of double fluorescence probes labeled triblock copolymer micelle drug carriers uptaken by different cells at 37 °C. HeLa* means Hela cells without the biotin-transferrin preincubating was used as the control.

contrary, in the absence of biotin-transferrin, the HeLa cell uptake is merely around 9.4%. And the above four results calculated from rhodamine B are 28.7%, 6.1%, 4.9%, 7.0%, respectively. These results were in close accord with that of the other fluorescence probe.

Figure 9. Confocal microscopy images of HeLa cells with pretargeting treatment incubated with FITC-avidin and rhodamine B loaded micelles under different observation conditions (a-c). Confocal microscopy images of HeLa cells without pretargeting treatment incubated with FITC-avidin and Rhodamine B loaded micelles under different observation conditions (d-f). The green fluorescence in images (a) and (d) represent the existence of FITC (FITC labeled avidin), and the red fluorescence in images (b) and (e) represent the existence of rhodamine B (rhodamine B loaded biotinylated micelle). (c) and (f) are the coalescent of bright-field and double fluorescence images, and the yellow fluorescence in them indicate the coexistence of green (FITC) and red fluorescence (rhodamine B).

Confocal Microscopy Observation. The internalization of the triblock copolymer micelles into HeLa cells was also visualized by confocal microscopy. After 2 h incubation with FITC-avidin-rhodamine B loaded micelle complex at 37 °C, HeLa cells pretreated with biotin-transferrin (Figure 9a,b,c) show remarkably more intense fluorescence in both cell membrane and cell cytoplasm than those free of biotin-transferrin pretreatment (Figure 9d,e,f), confirming the facilitation of the cell binding and internalization for the pretagerting approach. In the confocal images, the green fluorescence belongs to FITC (FITC labeled avidin), while the red fluorescence belongs to rhodamine B (rhodamine B loaded biotinylated micelle). Furthermore, the yellow fluorescence in Figure 9c shows the coexistence of green (FITC) and red fluorescence (rhodamine

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B) indicating the intracellular delivery of dye (as poorly soluble drug model) by triblock copolymer micelles as well as the biotin molecule does exist in the copolymer since the micelle conjugate with FITC labeled avidin through the action of biotin. In addition, the confocal microscopy investigations of A549 and ECV304 are in a similar situation to Figure 9d,e,f. Overall, the qualitative results from double-fluorescence probe microscopy studies are in good agreement with the quantitative results obtained from fluorescence spectroscopy, substantiating the involvement of transferrin-mediated internalization in the cellular uptake of triblock copolymer micellar drug carrier.

CONCLUSIONS In summary, novel micelles, comprising hydrophilic PEG shells,hydrophobicPMMAcores,andthermosensitiveP(NIPAAmco-HMAAm) segments were self-assembled from the biotinPEG-b-P(NIPAAm-co-HMAAm)-b-PMMA triblock copolymer. The TEM observation and size distribution analysis illustrated the superior stability of the thermosensitive micelles. The polymeric micelles showed a thermotriggered drug release behavior upon temperature alterations ascribed to the thermoinduced structural change of the micellar structure. The pretargeting delivery property of the triblock copolymer micelles was investigated by fluorescence spectroscopy as well as confocal microscopy, and the results confirmed that the self-assembled micelles can be specifically and efficiently bonded to cancer cells with the administration of biotin-transferrin, suggesting that the multifunctional micelles have great potential as drug carriers for the tumor targeting chemotherapy.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (50633020) and National Key Basic Research Program of China (2005CB623903).

LITERATURE CITED (1) Jaracz, S., Chen, J., Kuznetsova, V. L., and Ojima, I. (2005) Recent advances in tumor-targeting anticancer drug conjugates. Bioorgan. Med. Chem. 13, 5043–54. (2) Ouchi, T., Yamabe, E., Hara, K., Hirai, M., and Ohya, Y. (2004) Design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide. J. Controlled Release 94, 281–91. (3) Hong, C. Y., and Pan, C. Y. (2006) Direct synthesis of biotinylated stimuli-responsive polymer and diblock copolymer by RAFT polymerization using biotinylated trithiocarbonate as RAFT agent. Macromolecules 39, 3517–24. (4) Nobs, L., Buchegger, F., Gurny, R., and Allemann, E. (2006) Biodegradable nanoparticles for direct or two-step tumor immunotargeting. Bioconjugate Chem. 17, 139–45. (5) O’Reilly, R. K., Joralemon, M. J., Wooley, K. L., and Hawker, C. J. (2005) Functionalization of micelles and shell cross-linked nanoparticles using click chemistry. Chem. Mater. 17, 5976–88. (6) O’Reilly, R. K., Hawker, C. J., and Wooley, K. L. (2006) Crosslinked block copolymer micelles: functional nanostructures of great potential and versatility. Chem. Soc. ReV. 35, 1068–83. (7) Webber, G. B., Wanless, E. J., Armes, S. P., Tang, Y. Q., Li, Y. T., and Biggs, S. (2004) Nano-anemones: stimulus-responsive copolymer-micelle surfaces. AdV. Mater. 16, 1794–8. (8) Liu, F. T., and Liu, G. J. (2001) Poly(solketal methacrylate)block- poly(2-cinnamnoyloxyethyl methacrylate)-block-poly(allyl methacrylate): synthesis and micelle formation. Macromolecules 34, 1302–7. (9) Brigger, I., Dubernet, C., and Couvreur, P. (2002) Nanoparticles in cancer therapy and diagnosis. AdV. Drug DeliVery ReV. 54, 631–51.

Cheng et al. (10) Rosler, A., Vandermeulen, G. W. M., and Klok, H. A. (2001) Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. AdV. Drug DeliVery ReV. 53, 95–108. (11) Allen, C., Maysinger, D., and Eisenberg, A. (1999) Nanoengineering block copolymer aggregates for drug delivery. Colloids Surf., B 16, 3–27. (12) Kwon, G. S. (2003) Polymeric micelles for delivery of poorly water-soluble compounds. Crit. ReV. Ther. Drug Carrier Syst. 20, 357–403. (13) Li, J., Li, X., Ni, X., and Leong, K. W. (2003) Synthesis and characterization of new biodegradable amphiphilic poly(ethylene oxide)-b-poly[(R)-3-hydroxybutyrate]-b-poly(ethylene oxide) triblock copolymers. Macromolecules 36, 2661–7. (14) Liu, K. L., Goh, S. H., and Li, J. (2008) Controlled synthesis and characterizations of amphiphilic poly[(R,S)-3-hydroxybutyrate]-poly(ethylene glycol)-poly[(R,S)-3-hydroxybuty rate] triblock copolymers. Polymer 49, 732–41. (15) Li, J., Ni, X., Li, X., Tan, N. K., Lim, C. T., Ramakrishna, S., and Leong, K. W. (2005) Micellization phenomena of biodegradable amphiphilic triblock copolymers consisting of poly(β-hydroxyalkanoic acid) and poly(ethylene oxide). Langmuir 21, 8681–5. (16) Li, X., Mya, K. Y., Ni, X., He, C., Leong, K. W., and Li, J. (2006) Dynamic and static light scattering studies on selfaggregation behavior of biodegradable amphiphilic poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) triblock copolymers in aqueous solution. J. Phys. Chem. B 110, 5920–6. (17) Cammas-Marion, S., Okano, T., and Kataoka, K. (1999) Functional and site-specific macromolecular micelles as high potential drug carriers. Colloids Surf., B 16, 207–15. (18) Chen, X. R., Ding, X. B., and Zheng, Z. H. (2005) Intelligent crew-cut aggregates formed by thermosensitive block copolymers and their multiple morphologies. Macromol. Biosci. 5, 157–63. (19) Zhang, J. X., Qiu, L. Y., Jin, Y., and Zhu, K. J. (2005) Physicochemical characterization of polymeric micelles constructed from novel amphiphilic polyphosphazene with poly(Nisopropylacrylamide) and ethyl 4-aminobenzoate as side groups. Colloids Surf., B 43, 123–30. (20) Chung, J. E., Yokoyama, M., Aoyagi, T., Sakurai, Y., and Okano, T. (1999) Thermo-responsive drug delivery from polymeric micelles constructed using block copolymers of poly(Nisopropylacrylamide) and poly(butylmethacrylate). J. Controlled Release 62, 115–27. (21) Jones, M. C., and Leroux, J. C. (1999) Polymeric micelles-a new generation of colloidal drug carriers. Eur. J. Pharmaceut. Biopharmaceut. 48, 101–11. (22) Kataoka, K., Harada, A., and Nagasaki, Y. (2001) Block copolymer micelles for drug delivery: design, characterization and biological significance. AdV. Drug DeliVery ReV. 47, 113– 31. (23) Maeda, H., Wu, J., Sawa, T., Matsumura, Y., and Hori, K. (2000) Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Controlled Release 65, 271–84. (24) Liu, X. M., Pramoda, K., Yang, Y. Y., Chow, S. Y., and He, C. B. (2004) Cholesteryl-grafted functional amphiphilic poly(Nisopropylacrylamide-co-N-hydroxylmethylacrylamide): synthesis, temperature sensitivity, self-assembly and encapsulation of a hydrophobic agent. Biomaterials 5, 2619–28. (25) Gillies, E. R., Jonsson, T. B., and Frechet, J. M. J. (2004) Stimuli-responsive supramolecular assemblies of linear-dendritic copolymers. J. Am. Chem. Soc. 126, 11936–43. (26) Hay, D. N. T., Rickert, P. G., Seifert, S., and Firestone, M. A. (2004) Thermoresponsive nanostructures by self-assembly of a poly(N-isopropylacrylamide)-lipid conjugate. J. Am. Chem. Soc. 126, 2290–1. (27) Topp, M. D. C., Dijkstra, P. J., Talsma, H., and Feijen, J. (1997) Thermosensitive micelle-forming block copolymers of poly(ethylene glycol) and poly(N-isopropylacrylamide). Macromolecules 30, 8518–20.

Functionalized Micelles for Tumor Cell Target (28) Gref, R., Couvreur, P., Barratt, G., and Mysiakine, E. (2003) Surface-engineered nanoparticles for multiple ligand coupling. Biomaterials 24, 4529–37. (29) Lee, E. S., Na, K., and Bae, Y. H. (2005) Super pH-sensitive multifunctional polymeric micelle. Nano Lett. 5, 325–9. (30) Sawant, R. M., Hurley, J. P., Salmaso, S., Kale, A., Tolcheva, E., Levchenko, T. S., and Torchilin, V. P. (2006) “SMART” drug delivery systems: double-targeted pH-responsive pharmaceutical nanocarriers. Bioconjugate Chem. 17, 943–9. (31) Tan, J. F., Ravi, P., Too, H. P., Hatton, T. A., and Tam, K. C. (2005) Association behavior of biotinylated and non-biotinylated poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate). Biomacromolecules 6, 498–506. (32) Li, Y. Y., Zhang, X. Z., Kim, G. C., Cheng, H., Cheng, S. X., and Zhuo, R. X. (2006) Thermosensitive Y-shaped micelles of poly(oleic acid-Y-N-isopropylacrylamide) for drug delivery. Small 2, 917–23. (33) Wei, H., Zhang, X. Z., Zhou, Y., Cheng, S. X., and Zhuo, R. X. (2006) Self-assembled thermoresponsive micelles of poly(N-isopropylacrylamide-b-methyl methacrylate. Biomaterials 27, 2028–34. (34) Li, Y. Y., Zhang, X. Z., Zhu, J. L., Cheng, H., Cheng, S. X., and Zhuo, R. X. (2007) Self-assembled, thermoresponsive micelles based on triblock PMMA-b-PNIPAAm-b-PMMA copolymer for drug delivery. Nanotechnology 18 Art. No. 215605. (35) Chen, G., and Hoffman, A. S. (1995) Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH. Nature 373, 49–52.

Bioconjugate Chem., Vol. 19, No. 6, 2008 1201 (36) Wei, H., Zhang, X. Z., Cheng, C., Cheng, S. X., and Zhuo, R. X. (2007) Self-assembled, thermosensitive micelles of a star block copolymer based on PMMA and PNIPAAm for controlled drug delivery. Biomaterials 28, 99–107. (37) Morita, T., Horikiri, Y., Suzuki, T., and Yoshino, T. (2001) Preparation of gelatin microparticles by co-lyophilization with poly(ethylene glycol): characterization and application to entrapment into biodegradable microspheres. Int. J. Pharm. 219, 127– 37. (38) Ye, Q., Zhang, Z. C., Jia, H. T., He, W. D., and Ge, X. W. (2002) Formation of monodisperse polyacrylamide particles by radiation-induced dispersion polymerization: Particle size and size distribution. J. Colloid Interface Sci. 253, 279–84. (39) Chan, W. C., and Nie, S. (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281, 2016–2018. (40) Das, M., Mardyani, S., Chan, W. C. W., and Kumacheva, E. (2006) Biofunctionalized pH-responsive microgels for cancer cell targeting: rational design. AdV. Mater. 18, 80–3. (41) Zhang, H., Mardyani, S., and Chan, W. C. W. (2006) Design of biocompatible chitosan microgels for targeted pH-mediated intracellular release of cancer therapeutics. Biomacromolecules 7, 1568–72. (42) Xiong, X. B., Mahmud, A., Uludag, H., and Lavasanifar, A. (2007) Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells. Biomacromolecules 8, 874–84. BC8000062