Biotinylated thermoresponsive micelle self-assembled from double-hydrophilic block copolymer for drug delivery and tumor target

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Biomaterials 29 (2008) 497–505 www.elsevier.com/locate/biomaterials

Biotinylated thermoresponsive micelle self-assembled from double-hydrophilic block copolymer for drug delivery and tumor target Cheng Chenga, Hua Weia, Bao-Xian Shib, Han Chenga, Cao Lia, Zhong-Wei Guc, Si-Xue Chenga, Xian-Zheng Zhanga,, Ren-Xi Zhuoa a

Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, PR China b Department of Chemistry, Centre of Analysis and Test, Wuhan University, Wuhan 430072, PR China c Engineering Research Center in Biomaterials, Sichuan University, Chengdu 610064, PR China Received 25 July 2007; accepted 1 October 2007 Available online 23 October 2007

Abstract A multifunctional micellar drug carrier formed by the thermosensitive and biotinylated double-hydrophilic block copolymer (DHBC), biotin-poly(ethylene glycol)-block-poly(N-isopropylacrylamide-co-N-hydroxymethylacrylamide) (biotin-PEG-b-P(NIPAAmco-HMAAm)), was designed and prepared. The P(NIPAAm-co-HMAAm) block with an molar feed ratio of NIPAAm and HMAAm (10:1) was identified to exhibit the reversible phase transition at the lower critical solution temperature (LCST) of 36.7 1C. Cytotoxicity study indicated that the biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer did not exhibit obvious cytotoxicity. The block copolymer was capable of self-assembling into micelle in water. Transmission electron microscopy showed that the self-assembled micelles were regularly spherical in shape. The anticancer drug methotrexate (MTX) was loaded in the micelles and the in vitro release behaviors of MTX at different temperatures were investigated. The association of biotin molecule with the copolymer was confirmed by a unique capillary electrophoresis immunoassay (CEIA) method based on enhanced chemiluminescence (CL) detection. The fluorescence spectroscopy analysis as well as confocal microscopy studies confirmed the DHBC drug carriers could specifically and efficiently bind to cancer cells with pretreatment of biotin-transferrin, suggesting that the multifunctionalized DHBC micelle may be a useful drug carrier for tumor targeting. r 2007 Elsevier Ltd. All rights reserved. Keywords: DHBC; Biotinylated thermoresponsive micelle; CEIA-CL; Controlled release; Tumor target

1. Introduction Due to the specific properties which ascribe to the unique core–shell structure, micelles self-assembled from amphiphilic copolymers have attracted growing attention in biomedical applications during the past decade [1], especially as drug carriers for controlled release. Due to the limited passive targeting property, traditional drug loaded micelles exhibit systemic toxicity and cause undesirable severe side effects especially in traditional cancer chemotherapy. The micelles with active targeting property are highly desirable [2]. Recently, biotin–avidin system (BAS) was introduced to drug delivery systems and BAS Corresponding author. Tel.: +86 27 6875 4061; fax: +86 27 6875 4509.

E-mail address: [email protected] (X.-Z. Zhang). 0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.10.004

could be utilized as reagents for pretargeting method in chemotherapy to increase local concentration of drug at the target site resulting from the fact that different ligands can be incorporated into the drug delivery systems through biotin–avidin interactions [3]. In the pretargeting approach, instead of coupling the ligands directly with the drug carriers which may cause the denaturation of these ligands during the synthesis in organic solvent, the recognizing ligands such as biotinylated monoclonal antibodies [4] conjugated with avidin is administered first, then ‘‘chased’’ by the biotinylated therapeutic carriers through the strong BAS interaction after an appropriate delay. The delay allows the antibodies to localize and concentrate in the tumor. Compared with conventional amphiphilic block copolymers, double-hydrophilic block copolymers (DHBCs),

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combining two different hydrophilic blocks, are likely to bring rising applications in biomedical fields [5]. If one of the hydrophilic blocks is thermoresponsive, being able to undergo hydrophilic-to-hydrophobic alteration as temperature increases [6], it would be very promising to control the formation of self-assembled micelles and trigger the release of the loaded drugs. To our knowledge, the investigations of drug release behavior from the environmental stimuliresponsive micelles formed by the DHBCs are limited [7,8]. In this study, we provide a concept to fabricate biotinylated and thermosensitive DHBCs, which are able to self-assemble into multifunctional micelles as drug carriers. Biotin-poly(ethylene glycol)-block-poly(N-isopropylacrylamide-co-N-hydroxymethyl acrylamide) (biotinPEG-b-P(NIPAAm-co-HMAAm)) was designed and prepared. PEG segment was introduced as a highly hydrophilic block due to its good biocompatibility and the ability to stabilize the micelles as well as protect them from being cleared up by the reticuloendothelial system (RES) [9]. The biotin part was utilized for the pretargeting in tumor chemotherapy [4], and the conjugation of biotin moiety with the copolymer was investigated by a unique capillary electrophoresis immunoassay (CEIA) method based on enhanced chemiluminescence (CL) detection. The selfassembling behavior of the biotin-PEG-b-P(NIPAAm-coHMAAm) was investigated and the resulted micelles were further studied as the drug carriers to examine their temperature sensitive properties in controlled release. Moreover, cell interaction of the drug carrier was studied by fluorescence spectroscopy as well as confocal microscopy. 2. Materials and methods 2.1. Materials N-isopropylacrylamide (NIPAAm) and 3-mercaptopropionic acid (MPA) purchased from ACROS were used as received. N-hydroxymethylacrylamide (HMAAm) was obtained from Tianjin Chemical Reagent Co. (Tianjin, China) and used as received. Biotin, streptavidin-horseradish peroxidase (SA-HRP) and fluorescein isothiocynate labeled avidin (FITC–avidin) were purchased from Pierce and used as received. 1,1Carbonyldiimidazole (CDI) and biotinylated transferrin (biotin-trasferrin) obtained from Sigma-Aldrich were used as received. N, N0 -dimethylformamide (DMF), tetrahydrofuran (THF) and 1,4-dioxane obtained from Shanghai Chemical Reagent Co. were used after distillation. Diamine polyethylene glycol (H2N-PEG-NH2) with a molecular weight (Mn) of 3000 g/mol (determined by gel permeation chromatography (GPC) using THF as the eluent) was purchased from Fluka and used as received. N, N0 azobisisobutyronitrile (AIBN) provided by Shanghai Chemical Reagent Co. (Shanghai, China) was used after recrystallization with 95% ethanol. Methotrexate (MTX) was kindly gifted by SuRi Biochem Co. Ltd. (Suzhou, China). Luminol, p-iodophenol (PIP) and 30% hydrogen peroxide solution were obtained from the Chemistry Department of Shanxi Normal University (Shanxi, China) and used as received. All other reagents and solvents were used without further purification.

2.2. Preparation of biotin-PEG-b-P(NIPAAm-co-HMAAm) The biotin-PEG-NH2 was synthesized according to the literature [10]. And P(NIPAAm-co-HMAAm)-COOH blocks were prepared by radical polymerizations with different NIPAAm:HMAAm molar feed ratios of

7:1, 9:1, 10:1, 11:1, 13:1 and 15:1, using MPA as a chain transfer agent [11]. Then P(NIPAAm-co-HMAAm)-COOH with the NIPAAm:HMAAm feed ratio of 10:1 (0.10 g), biotin-PEG-NH2 (0.10 g) and NHS (4.0  105 mol) were dissolved in 4 mL dioxane. DCC (4.0  105 mol) in 1 mL dioxane was added dropwise to the solution under nitrogen atmosphere. After 24 h reaction at room temperature (22 1C), the product was precipitated in an excess of diethyl ether and dried in vacuum after filtered. The product was further purified by dissolving in distilled water and dialyzing against distilled water for 1 week using a dialysis membrane with a molecular weight cut off (MWCO) of 8000–10,000 g/mol (Shanghai Chemical Reagent Co., China) to remove the un-reacted biotin-PEGNH2. The final product biotin-PEG-b-P(NIPAAm-co-HMAAm) was harvested by freeze-drying.

2.3. Gel permeation chromatography (GPC) Number-average Mn of biotin-PEG-NH2, P(NIPAAm-co-HMAAm)COOH with the NIPAAm:HMAAm feed ratio of 10:1, and biotin-PEG-bP(NIPAAm-co-HMAAm) was determined by 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 the polystyrene standard with narrow molecular weight distribution.

2.4. 1H NMR characterization 1 H NMR spectra were recorded on a Mercury VX-300 spectrometer at 300 Hz using D2O as a solvent.

2.5. In vitro cytotoxicity study For each well in a 96-well plate, 200 mL 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 1C, 5% CO2), The culture medium was changed to 200 mL of RPMI1640 containing biotin-PEG-b-P(NIPAAm-co-HMAAm) 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 mL of MTT solution (5 mg/mL) and was added. After incubation for 4 h, the MTT containing medium was removed from each well and 200 mL of DMSO was added and shaken at room temperature. The optical density (OD) was measured at 570 nm with a Microplate Reader (Model 550, Biorad, USA).

2.6. Detection of incorporated biotin The presence of biotin in the copolymer was examined by a unique technique called CEIA-CL [13]. Biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer (5.6 mg) was dissolved in 1.0 mL of phosphate buffer saline (PBS, pH 7.4, 0.01 M). SA-HRP (5 mL, 100 mg/mL) was then added to the copolymer solution, and shaken at room temperature for 30 min to form HRP-SA-biotin-PEG-b-P(NIPAAm-co-HMAAm) complex. The mixture was centrifuged (10,000–12,000 r/min) for 15 min, and then the sediment was washed with PBS three times, and suspended again in 1.0 mL PBS as the final analytical samples. The solutions of 1  107 M SA-HRP standard sample and 1  104 M biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer were also characterized for comparison. In the detection, the applied running voltage was 16 kV, injected voltage was 10 kV (10 s). The migration buffer was phosphate buffer (25 mM, pH 6.0). 20 mM of H2O2, 1.0 mM luminol, and 1.0 mM PIP, pH 11.0 were in the postcolumn cell.

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2.7. Determination 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. Aliquots of pyrene solutions (6  106 M in acetone, 1 mL) were added to containers, and the acetone was allowed to evaporate. Ten millimeters of aqueous 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  107 M. The solutions were kept in a thermostated water bath at 50 1C (70.05 1C) for 24 h to reach the solubilization equilibrium of pyrene. 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 I382 nm were 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.

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shaken at room temperature for 30 min to form FITC–avidin–DHBC complex. The solution was centrifuged (10,000–12,000 r/min) for 15 min, and then the sediment was washed with PBS three times, and suspended again in 2.0 mL PBS. HeLa, A549, and ECV304 cells were seeded into a 24-well plate (1  105 cells/well) containing 1 mL of DMEM (Dulbecco’s Modified Eagle Medium) to grow to 70% confluence after 24 h incubation. 100 mL biotin-transferrin (200 mg/mL) was then added in each well and incubated with cells for 2 h at 37 1C. Following the incubation period, medium was removed and cells were washed with PBS three times. Then, 1 mL DMEM containing 100 mL FITC–avidin–DHBC complex was added in each well and incubated with cells for another 2 h at 37 1C. 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. Finally, 1 mL of DMSO was added in each well to lyse cells. The internalized FITC level was determined by measuring the fluorescence emission intensity of FITC at 490 nm on a LS55 luminescence spectrometer (Perkin-Elmer).

2.13. Confocal microscopy 2.8. Transmission electron microscopy (TEM) A drop of micelles 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 TEM at an acceleration voltage of 80 keV.

Cells were seeded into specific confocal investigation plate containing 1 mL DMEM (1  105 cells/plate). Then, the cell samples were treated in the same way of fluorescent spectroscopy analysis. The fluorescent images of cells were analyzed using laser scanning confocal microscopy (Leica TCS SP2AOBS, Germany).

2.9. Size distribution measurement

3. Results and discussion

The size distribution of micelles was determined by a Nano Series Nano-ZS (MALVERN Instrument) Zeta sizer. The micelle aqueous solution (100 mg/L) was passed through a 0.45 mm pore-sized syringe filter and kept in the thermostat of the apparatus at 40 1C (70.05 1C) for 20 min to reach the equilibrium prior to the measurements.

3.1. Synthesis of biotin-PEG-b-P(NIPAAm-co-HMAAm)

2.10. Determination of LCST Optical absorbance of polymers in aqueous solutions (3.3 g/L, distilled water was used as the solvent) at various temperatures were measured at 542 nm with a Lambda Bio40 UV–vis spectrometer (Perkin-Elmer) to determine its LCST. 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.

2.11. Drug loading and in vitro drug release Biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer (10 mg) and MTX (10 mg) were dissolved in 2 mL DMF. The solution was put into a dialysis tube (MWCO: 8000–10,000 g/mol, Shanghai Chemical Reagent Co., China) and subjected to dialysis against 1000 mL distilled water at 50 1C for 24 h. To determine the encapsulation efficiency (EE), the drug-loaded micelles solution was lyophilized, and then dissolved in DMF and the UV absorbance at 303 nm was measured. The EE was found to be around 3%. For in vitro drug release study, 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 based on the UV absorbance at 303 nm at different temperatures.

2.12. Cell uptake studies Biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer (200 mg) and FITC–avidin (100 mg) were dissolved in 1.0 mL of phosphate buffer saline (PBS, pH 7.4, 0.01 M), respectively. Then the two solutions were mixed and

The synthesis route of biotin-PEG-b-P(NIPAAmco-HMAAm) is shown in Scheme 1. The LCSTs of the P(NIPAAm-co-HMAAm)-COOH blocks with different molar feed ratios of NIPAAm to HMAAm (7:1, 9:1, 10:1, 11:1, 13:1 and 15:1) are 39.4, 39.3, 36.7, 36.2, 33.4 and 32.1 1C, respectively (Fig. 1). The Polymer 3 with NIPAAm to HMAAm feed ratio of 10:1 with the LCST at 36.7 1C was identified to have the proper transition temperature. That is to say, the LCST of P(NIPAAm-co-HMAAm) block was adjusted to be near the physiologic temperature by introducing the hydrophilic HMAAm units since the LCST of PNIPAAm is around 32 1C, which is not favorable for biomedical applications. As a result, Polymer 3 was selected to prepare biotin-PEG-b-P(NIPAAm-coHMAAm) block copolymer for following studies. P(NIPAAm-co-HMAAm)-COOH (Polymer 3, Mn 10,300, PDI 1.84) was connected to the biotin-PEG-NH2 to prepare biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer (Mn 13,700, PDI 1.87). The formation of the biotinPEG-b-P(NIPAAm-co-HMAAm) copolymer could be confirmed by the molecular weight changes. The coupling is also confirmed by 1H NMR spectrum of the copolymer which reveals the typical peaks assigned to PEG segment and P(NIPAAm-co-HMAAm) block, respectively (Fig. 2). 3.2. Biotin conjugation In the current study, a unique technique called CEIA-CL was used to detect the presence of biotin in the copolymer. The schematic illustration of biotin detection by CEIA-CL

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Biotin

+

O

H 2N

x CH 2 CH 2NH 2

CDI DMSO, CH 2Cl 2

O Biotin

x CH 2CH 2NH 2

HC CH2

AIBN, MPA HOOCCH 2CH2S

+

HC

+

CH 2

CONHCH2 OH

CONHCH(CH 3)2 DMF

CH2 CH

m C O

CH 2 CH

n C O

NH CH(CH3 )2

Biotin-PEG-NH2

NH CH2OH

P(NIPAAm-co-HMAAm)-COOH NHS and DCC in dioxane O Biotin

CH 2 CH 2 HNOCCH 2CH2 S

x

CH2 CH

m C O NH

CH 2 CH

CH(CH 3) 2

n C O NH CH2OH

Biotin-PEG-b-P(NIPAAm-co-HMAAm) Scheme 1. Synthesis of biotin-PEG-b-P(NIPAAm-co-HMAAm) block copolymer.

Fig. 2. 1H NMR spectrum of biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer.

Fig. 1. Optical absorbance of P(NIPAAm-co-HMAAm) blocks with different NIPAAm:HMAAm molar feed ratios of 7:1, 9:1, 10:1, 11:1, 13:1 and 15:1 in aqueous solution. ([polymer] ¼ 3.3 g/L, distilled water was used as the solvent).

method is presented in Fig. 3A. Here, the ligand is HRP, which is a kind of well-applied enzyme in biomedical analysis and clinical chemistry fields with the capability of catalyzing the luminol/H2O2/p-iodophenol CL reaction system. And HRP labeled SA can associate with biotin-PEG-b-P(NIPAAm-co-HMAAm) through BAS interaction to form HRP-SA-biotin-PEG-b-P(NIPAAm-coHMAAm) complex. The detection used here is characterized by a simple and low-cost optical system, requiring no light sources, which can avoid the effects of stray light and the instability of light source. Therefore, this unique technique could provide excellent sensitivity with quite

low background disturbing and the cost is much lower as compared with the classical method of detecting conjugated biotin moiety in polymers [14]. As shown in Fig. 3B(a), the electropherogram of the biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer shows no signal, which means that the copolymer has no catalyzing effect on the luminol/H2O2/p-iodophenol reaction system itself. The migration time of the SA-HRP standard is around 9 min (Fig. 3B(b)). For HRP-SA-biotinPEG-b-P(NIPAAm-co-HMAAm) complex, it was found that there was an alternative peak in electropherogram with a migration time of approximately 11 min (Fig. 3B(c)). Through the signals from the HRP-SA-biotin-PEG-bP(NIPAAm-co-HMAAm) complex, we can confirm the presence of biotin moiety on the copolymer as well as the capability of easily functionalizing with ligands for pretargeting approach.

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180

Cell viability (%)

150

120

90

60

0.0

0.5

1.0 1.5 2.0 2.5 3.0 Concentration of copolymer (g/L)

3.5

Fig. 4. Cytotoxicity study of biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer.

Fig. 3. (A) Schematic illustration of detecting biotin by CEIA-CL. (B) Electropherograms of (a) biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer, (b) SA-HRP standard sample and (c) HRP-SA-biotin-PEG-bP(NIPAAm-co-HMAAm) complex.

3.3. In vitro cytotoxicity study Cytotoxicity study was also performed to evaluate the cytotoxicity of biotin-PEG-b-P(NIPAAm-co-HMAAm). The effect of the polymer concentration on the proliferation of ECV304 was studied and the copolymer exhibits no apparent cytotoxicity (Fig. 4). 3.4. CMC In this study, membrane-dialysis method [15] was employed to prepare biotin-PEG-b-P(NIPAAm-coHMAAm) micelles at 50 1C. The P(NIPAAm-coHMAAm) segment, after a hydrophilic-to-hydrophobic alteration at this temperature, formed the hydrophobic core of the micelle, while the hydrophilic biotin-PEG segment formed the corona or outer shell. The CMC of the copolymers obtained in this study is found to be 43.7 mg/L (see Fig. S1 in supplementary information). 3.5. Characterization of polymeric micelles The self-assembly and thermally induced change of biotin-PEG-b-P(NIPAAm -co-HMAAm) micelles are schematically illustrated in Fig. 5a. The morphology of the

micelles was visualized by TEM as shown in Fig. 5b. It is evident that the self-assembled micelles are well dispersed as individual particles with a regularly spherical shape. As the outer hydrophilic shell, the flexible PEG spacer offers an optimized accessibility for ligands [10]. Meanwhile, with respect to the biotin-PEG shell which is hydrophilic regardless of the temperature, the micelles self-assembled from biotin-PEG-b-P(NIPAAm-co-HMAAm) are more stable and soluble in water, and are especially able to prevent the aggregation of traditional amphiphilic PNIPAAm-based micelles when reaching the temperature above the LCST [12,15]. The biotin-PEG-b-P(NIPAAmco-HMAAm) is expected to form micelles in the temperature range from 38 to 50 1C. The size distribution of resulted micelles at 40 1C are shown in Fig. 5c, biotin-PEGb-P(NIPAAm-co-HMAAm) micelles exhibit a narrow size distribution with an average diameter of around 273 nm (peak 303 nm, PDI 0.099). The discrepancy in size of the micelles when comparing the diameter obtained by TEM with that obtained by particle-size analyzer is attributed to the fact that the laser diffraction method (particle-size analyzer) measures the hydrodynamic diameter of micelles in water, whereas the TEM observation reveals the size of the micelles in the solid state. Similar discrepancy in size was also reported in previous literature [16]. In addition, the fairly large size of the micelles self-assembled from biotin-PEG-b-P(NIPAAm-co-HMAAm) is probably attributed to the highly hydrated PEG shell. 3.6. Thermo-responsive drug release behavior In this study, the drug release behavior of the biotinPEG-b-P(NIPAAm-co-HMAAm) micelles was investigated, using MTX, a poorly water-soluble anticancer drug, as a model drug. Because the LCST of biotin-PEG-bP(NIPAAm-co-HMAAm) copolymer micelle was determined to be 41.5 1C (Fig. 6a), the in vitro drug release

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Fig. 5. (a) Schedule illustration of the thermally-induced structure change of micelles self-assembled from biotin-PEG-b-P(NIPAAm-co-HMAAm) in aqueous solution, (b) TEM image and (c) size distribution of biotin-PEG-b-P(NIPAAm-co-HMAAm) micelles.

Fig. 6. (a) Optical absorbance of biotin-PEG-b-P(NIPAAm-co-HMAAm) micelles in aqueous solution([polymer] ¼ 3.3 g/L). (b) Drug release from the self-assembled biotin-PEG-b-P(NIPAAm-co-HMAAm) micelles loaded with MTX at different temperatures.

profile from the biotin-PEG-b-P(NIPAAm-co-HMAAm) micelles was evaluated in distilled water at both the physiologic and a higher temperature (37 and 43 1C). The drug release shows drastic changes with temperature alterations as presented in Fig. 6b. At 43 1C, about 66% of the drug is released from the micelles in 96 h. When the temperature is decreased to 37 1C, the drug release is accelerated dramatically due to the temperature-induced structural changes of the micelles, i.e., the P(NIPAAm-coHMAAm) core turns to be hydrophilic, resulting in the deformation of micellar structure. As a result, the drug diffuses out quickly and about 90% of the drug is released from the micelles in 96 h at 37 1C.

3.7. Cell uptake studies In the two-step pretargeting protocol, we used cervical cancer HeLa cells, human lung cancer cells (A549), and vein endothelial cells (ECV304) to study the intracellular uptake of the biotin-PEG-b-P(NIPAAm-co-HMAAm) micelles in vitro. Firstly, FITC-labeled avidin was associated with biotin-PEG-b-P(NIPAAm-co-HMAAm) micelles to form FITC–avidin–DHBC complex. Then after the preincubating of biotin-transferrin and cells, the FITC–avidin–DHBC complex was infused to ‘‘chase’’ biotin-transferrin through the biotin–avidin interaction. For the comparison, the HeLa cells without preincubating

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with biotinylated transferrin were also studied. Based on the fluorescence emission intensity of FITC, the cell uptake of the FITC-labeled DHBC is calculated using the

following equation [17]: Uptake ð%Þ ¼

Fig. 7. Cell uptake of FITC–avidin–DHBC complex by three kinds of cells (A549, ECV304, HeLa) with biotin-transferrin preincubating and FITC–avidin–DHBC complex by HeLa cells without biotin-transferrin preincubating (Hela*).

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concentration of internalized FITC concentration of FITC in labeled complex added to each well  100.

The cell uptake of complex FITC–avidin–DHBC for different cells and under different conditions is presented in Fig. 7. In the presence of biotin-transferrin, cells association is 12.2% (HeLa), 5.4% (A549), and 5.8% (ECV304), respectively. HeLa cells have been extensively characterized for intracellular delivery through receptormediated endocytosis, using the targeting iron-carrying protein transferrin [18]. The affinity between transferrin and transferrin receptor (TfR) on the surface of Hela cells is much greater than that between transferrin and TfR on the surface of other cells [19]. Therefore, HeLa cells, a kind of cancer cells, are most widely used as the model cells to study the interactions between transferrin and cancer cells. As a result, transferrin resulted in relatively high cellular uptake in HeLa cells, but not in other cell lines. And the mechanism is probably that transferrin can be specifically recognized and taken up by TfR actively expressed on the surface of tumor

Fig. 8. Confocal microscopy images of HeLa cells incubated with FITC–avidin–DHBC complex with biotin-transferrin preincubating. (a) Fluorescence image, (b) bright field image, and (c) overlapped image. Confocal microscopy images of HeLa cells incubated with FITC–avidin–DHBC complex without biotin-transferrin preincubating, (d) fluorescence image, (e) bright field image, and (f) overlapped image. (Scale bar represents 15 mm.)

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cells, especially HeLa cells. Consequently, transferrin may be widely applied either as a carrier or targeting ligand in the active targeting of anti-cancer agents, proteins and genes to primarily proliferating malignant cells that over-express TfR. On the contrary, in the absence of biotin-transferrin, the HeLa cell uptake is merely around 5.5%.

National Key Basic (2005CB623903).

Research

Program

of

China

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials. 2007.10.004.

3.8. Confocal microscopy studies References Internalization of FITC–avidin–DHBC complex by HeLa cells was also investigated by confocal microscopy. After 2 h incubation with the FITC–avidin–DHBC complex at 37 1C, HeLa cells pretreated with biotin-transferrin (Fig. 8a–c) show remarkably more intense fluorescence in both cell membrane and cell cytoplasm than those free of biotin-transferrin pretreatment (Fig. 8d–f), indicating the facilitation of the cell binding and internalization for the pretagerting approach. Additionally, high accumulation of the targeted micelles was also seen in nucleus. The high accumulation of the targeted micelles in nucleus may be attributed to the conventional passive transport, and whether the micelles could be recognized by the nucleus and accumulated in the nucleus without specific recognition remains to be investigated in the further. Overall, the qualitative results from fluorescent microscopy studies are in good agreement with the quantitative results obtained from fluorescence spectroscopy, confirming the involvement of transferrin-mediated internalization in the cellular uptake of DHBC drug carrier. 4. Conclusions Novel micelles, comprised of PEG shells conjugated with biotin and P(NIPAAm-co-HMAAm) cores, were self-assembled from the biotin-PEG-b-P(NIPAAm-coHMAAm) block copolymer at high temperature. The conjugation of biotin with the copolymers was confirmed by CEIA-CL detection. Polymeric micelles loaded with MTX showed a thermosensitive switching behavior for drug release upon temperature alterations ascribed to the thermo-induced structural changes of the micellar core. The pretargeting delivery property of the DHBC micelles was investigated by fluorescence spectroscopy as well as confocal microscopy in vitro and the results confirmed that the self-assembled micelles could be specifically and efficiently bonded to cancer cells with the biotin-transferrin pretreatment, suggesting that the multifunctional micelle drug carriers have great potentials for tumor targeting chemotherapy. Acknowledgments This work was supported by National Natural Science Foundation of China (20504024, 50633020) and

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