Transferrin-Conjugated Micelles: Enhanced Accumulation and

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Transferrin-Conjugated Micelles: Enhanced Accumulation and Antitumor Effect for Transferrin-Receptor-Overexpressing Cancer Models Jun Yue,†,‡ Shi Liu,† Rui Wang,†,‡ Xiuli Hu,† Zhigang Xie,† Yubin Huang,† and Xiabin Jing*,† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ Graduate School of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: As the transport protein for iron, transferrin can trigger cellular endocytosis once binding to its receptor (TfR) on the cell membrane. Using this property, we conjugated transferrin onto the surface of biodegradable polymeric micelles constructed from amphiphilic block copolymers. The core of micelle was either labeled with a near-infrared dye (NIR) or conjugated with a chemotherapeutic drug paclitaxel (PTX) to study the biodistribution or antitumor effect in nude mice bearing subcutaneous TfR-overexpressing cancers. DLS and TEM showed that the sizes of Tf-conjugated and Tf-free micelles were in the range of 85−110 nm. Confocal laser scanning microscopy and flow cytometry experiments indicated that the uptake efficiency of the micelles by the TfR-overexpressing cells was enhanced by Tf conjugation. Semiquantitative analysis of the NIR signals collected from the tumor site showed that the maximum accumulation was achieved at 28 h in the M(NIR) group, while at 22 h in Tf−M(NIR) groups; and the area under the intensity curve in the Tf−M(NIR) groups was more than that in M(NIR) group. Finally, the tumor inhibition effects of targeting micelles were studied with the gastric carcinoma model which overexpressed TfR. The analysis of tumor volumes and the observation of H&E-stained tumor sections showed that Tf−M(PTX) had the best antitumor effect compared with the control groups (saline, PTX, and M(PTX)). The results of this study demonstrated the potential application of Tf-conjugated polymeric micelles in the treatment of TfRoverexpressing cancers. KEYWORDS: transferrin, TfR, micelle, antitumor effect, paclitaxel

1. INTRODUCTION Although much progress has been made toward cancer therapy, cancer is still a leading cause of death around the world. Along with the appearance of novel chemotherapeutic drugs (e.g., paclitaxel, doxorubicin, camptothecin, and cisplatin), chemotherapy has been considered to be effective to inhibit tumor growth and tumor metastasis.1−3 However, the applications of current drugs are limited due to (1) the poor targeting efficiency, which can cause serious toxicity to the normal tissues of the body; (2) the multidrug resistance,4,5 which has been correlated with the presence of some molecular “pumps” in tumor-cell membranes that can expel drugs from the interior. To solve these problems, various nanoscale drug delivery systems6−9 have been developed as a promising class of drug dosage form for cancer therapy. The antitumor drugs loaded in the nanocarriers (with the name “nanomedicine”) have several advantages: (1) the accumulation of nanomedicines in tumor can be increased due to the enhanced permeability and retention (EPR) effect;10 (2) the circulation of drugs in blood can be prolonged because of the shielding effect of nanacarriers, especially when the nanocarriers are PEGylated;11,12 (3) some © 2012 American Chemical Society

hydrophobic drugs can be dissolved in water with the help of amphiphilic carriers; and (4) tumor microenvironment (vascular abnormalities, oxygenation, perfusion, pH, and metabolic states) that differs from normal tissues allows researchers to elaborate therapeutic strategies to increase the drug delivery to the tumor tissues.13−17 The form of “nanocarriers”18−21 can be nanospheres, nanogels, nanowires, nanorods, etc. In-depth studies have been given to the influence of the shape and size of nanomedicines on drug delivery and cellular endocytosis.22,23 Among these nanocarriers, nanospheres, including liposomes, polymeric micelles, and vesicles have been used as the main carrier form of drugs for their good fluidity and ease of preparation. Once into the tumor tissues, the transport of nanomedicines through the cell membranes becomes necessary. Although PEGylation can prolong the circulation of nanomedicine in Received: Revised: Accepted: Published: 1919

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blood, the cell uptake efficiency may decrease. Some strategies24 have been developed to overcome the PEG dilemma. One efficient way is to design a system which is capable of reversing its surface from negative to positive at tumor extracellular pH (∼6.8). By virtue of electrostatic interactions between positively charged nanoparticle surface and the negatively charged cell membrane, the nanomedicine can be easily internalized.25,26 Another way is coupling target ligands on the nanoparticle surface.27−30 The ligand can recognize its specific receptor on the cell membrane and trigger receptor-mediated endocytosis, which is rapid and selective, and thus can effectively enhance the cellular uptake of nanoparticles. Currently, many attempts are being made to explore this potential ligand−receptor-mediated delivery system. Transferrin (Tf), a well-known blood plasma protein, is responsible for the transport of iron into the cells by binding to the transferrin-receptor (TfR) on the cell membrane.31 Because TfRs are overexpressed in many human cancers, TfR is expected to be a good cellular marker for delivery of anticancer drugs as well as for diagnosis of malignancies in patients whose individual tumors express elevated TfRs.32,33 In the past few decades, Tf−TfR mediated drug delivery has been widely studied in the treatment of breast cancer, prostate cancer, myeloid leukemia, etc.34−37 And a lot of effort, using Tf or antiTfR antibodies as the target moiety in designing Tf−TfR mediated drug delivery systems,33,38−40 has been directed toward delivery of drugs across the blood−brain barrier for the treatment of brain tumors (such as glioma). The form of these drug delivery systems includes not only Tf−drug conjugates41,42 but also Tfcarrying nanocarrier such as Tf−liposome or Tf−polymersome systems, but Tf−micelle systems are seldom reported. Compared with other types of nanocarriers (e.g., liposomes or vesicles), polymeric micelles own its advantages, such as easily controllable size and size distribution, versatility in chemical structure and composition, ease in preparation, and so on. In our previous studies,28,43,44 we have prepared several kinds of active targeting micellar systems using small targeting ligands (such as folate acid, lactose, etc.) which exhibited better endocytosis efficiency by target cell line or increased antitumor effect to the target tumor model. However, these targeting ligands, at the same time, are the nutritional components in the human body and therefore may greatly compromise the targeting effect of micelles in vivo. In contrast, using Tf as the target ligand can resolve this problem to some extent, because the Tf−TfR interaction is more efficient and specific and only a small amount of Tf conjugated on the micelle can realize better endocytosis efficiency.45 Therefore, in this study, Tf-carrying micelles which contained a near-infrared dye (NIR) or chemotherapeutic drug paclitaxel (PTX) were prepared. The former was used to study the biodistribution of targeting micelle in vivo, while the latter was used as the model drug to study the antitumor effect in TfRoverexpressed cancer model (SGC-7901 gastric carcinoma). Both PTX and Tf were covalently combined to the carrier polymers to ensure the stability of the drugs during circulation in the blood and to realize the constant release of the drugs once they enter the cancer cells. The results showed that Tfconjugation could enhance the uptake of micelles by target cells and Tf−M(PTX) exhibited the best antitumor effect, compared with the control groups.

the literature.46,47 Tin(II) 2-ethylhexanoate (Sn(Oct)2, Strem Chemicals, 90% in 2-ethylhexanoic acid), β-mercaptoethylamine (Aldrich, 95%), 3-mercaptopropionic acid (Alfa Aesar, 99%), 2iminothiolane hydrochloride (Traut’s Reagent, Sigma, 98%), dicyclohexylcarbodiimide (DCC, Aldrich, 99%), 4-dimethylaminopyridine (DMAP, Aldrich, 99%), and monomethoxyl poly(ethylene glycol) (mPEG, average Mn ∼5000, Aldrich) were used as received. 2-Mercaptoethanol (Aldrich, 95%), β-alanine (99%), and maleic anhydride (98%) were purchased from Shanghai Jingchun Chemical Reagent Co., Ltd. (Shanghai, China). L-Lactide (LA) was prepared in our own laboratory and recrystallized from ethyl acetate three times before use. Human holo-transferrin (Tf, Cat. No. T0665) was purchased from Sigma. Methylene chloride (CH2Cl2) and triethylamine (TEA) were dried over CaH2 and distilled before use. 2.2. Synthesis of mPEG-b-P(LA-co-MHC/NIR) and mPEG-b-P(LA-co-MCC/PTX). First, block copolymer mPEGb-P(LA-co-MAC) with allyl groups on the side chain was synthesized according to ref 48. Then radical-mediated thiol− ene reactions were carried out to convert the allyl groups to hydroxyl or carboxyl groups using 2-mercaptoethanol or 3mercaptopropionic acid, respectively. As an example, the conversion of allyl groups to hydroxyl groups is shown here. Briefly, 1.0 g of copolymer mPEG-b-P(LA-co-MAC) (0.14 mmol) and 0.11 g of 2-mercaptoethanol (1.4 mmol) were dissolved in 15 mL of THF in a 50 mL round-bottom quartz flask, followed by N2 bubbling with a gentle flow for 30 min to eliminate dissolved oxygen. Then the mixture was stirred at room temperature under UV light (254 nm, 1.29 mW/cm2). Two hours later, the light source was turned off and the reaction mixture was poured into a large amount of cold diethyl ether. The precipitates were collected, redissolved in a small amount of chloroform, and reprecipitated into diethyl ether. The copolymer was collected and dried in vacuo for 8 h to give final product mPEG-b-P(LA-co-MHC). The carboxyl groups containing copolymer mPEG-b-P(LA-co-MCC) was synthesized in a similar way with 3-mercaptopropionic acid instead of 2-mercaptoethanol. Second, a near-infrared dye (NIR, offered by Dalian University of Technology, the chemical structure is shown in Scheme S1 in the Supporting Information) was conjugated with mPEG-bP(LA-co-MHC) via a DCC-catalyzed condensation reaction. Briefly, 0.2 g of mPEG-b-P(LA-co-MHC) (0.0125 mmol) was dissolved in 10 mL of dry CH2Cl2. Then 18 mg of NIR (0.025 mmol), 10 mg of DCC (0.05 mmol), and 6 mg of DMAP (0.05 mmol) were added successively to the solution. The mixture was cooled to 0 °C and stirred under argon atmosphere for 24 h. Finally, dicyclohexylurea (DCU) formed was filtrated out and the filtrate was precipitated in a large amount of diethyl ether. The conjugate mPEG-b-P(LA-co-DHC/NIR) was collected and dried in vacuo for 4 h. Purification process was conducted by dissolving the conjugate in DMF (10 mg/mL) and dialyzing against THF for 24 h to remove unreacted NIR. Similarly, the antitumor drug PTX was conjugated to the copolymer mPEG-bP(LA-co-MCC) using the same procedure to give the final conjugate mPEG-b-P(LA-co-MCC/PTX). 2.3. Synthesis of mal-PEG-b-PLA. The block copolymer mal-PEG-b-PLA (with a maleimide group at the end of PEG segment) was synthesized through the reaction between aminoPEG-b-PLA (with an amino group at the end of PEG segment) and succinimidyl 3-maleimidopropanoate (SMP). The synthesis of amino-PEG-b-PLA has been reported by our group previously,28 so it is not described here.

2. MATERIALS AND METHODS 2.1. Materials. Carbonate monomer 2-methyl-2-allyloxycarbonyl-propylene carbonate (MAC) and α-allyloxyl, ω-hydroxyl poly(ethylene glycol) (allyl-PEG) were prepared according to 1920

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mL. Finally, the Tf-conjugated micelles (Tf−M(NIR) or Tf− M(PTX)) were harvested by lyophilization of the collected micellar solution. The amount of Tf conjugated on the surface of hybrid micelles was measured by a Micro BCA Protein Assay Kit (Product No. 23225). Before the measurement, four concentrations (0.5, 0.1, 0.05, and 0.01 mg/mL) of each sample as well as a series of standard concentrations of Tf solutions (100, 50, 25, 10, 5, 2.5, and 1 μg/mL) were prepared. The measurement followed the procedures described in the set. The results were expressed as Tfx−M(NIR) or Tfx−M(PTX), where x stands for the number of Tf molecules on each micelle. 2.7. The in Vitro Release of PTX from M(PTX) and Tf− M(PTX). The in vitro release of PTX from M(PTX) and Tf− M(PTX) was studied in PBS (pH = 7.4) buffer containing 10% ethanol. Briefly, 20 mL of M(PTX) or Tf−M(PTX) (concentration: 1 mg/mL) was placed in a dialysis bag (cutoff size: 3500 Da), followed by immersing the dialysis bag in 100 mL of PBS. At desired time intervals (day 1, 3, 5, 9, 12), 2 mL of the micellar solution in the dialysis bag was taken out and freeze-dried for analysis by 1H NMR in DMSO-d6. At the same time, the release medium was exchanged with 100 mL of fresh medium. For each sample at a specific time point (t), the relative molar content of PTX remaining in the polymer (Nt) was determined by 1H NMR according to the known Mn of PEG (5K), which was expected to be stable during the experimental period. And thus the release ratio (R %) could be calculated with the fomula R % = (1 − Nt/ N0) × 100%, where N0 represents the molar content of PTX in the original polymer before release experiments were conducted. 2.8. Detection of TfR Expression on Three Cell Lines. Three human cell lines, SGC-7901 (gastric carcinoma), SKOV-3 (ovarian carcinoma), and MCF-7 (breast carcinoma), were selected to study the TfR (CD71) expression on the membrane. One milliliter of each cell suspension (1 × 106/mL) was seeded in a 12-well culture plate. After 12 h growth, the cells were incubated with 1 μg of FITC-labeled anti-human CD71 (BioLegend, Cat. No. 334103), 1 μg of FITC-labeled mouse IgG2a (BioLegend, Cat. No. 400207), and PBS, respectively, at 0 °C for 30 min. A single cell suspension was prepared consecutively by trypsinization, washing with PBS, and filtration through 200 nylon mesh. Thereafter, 10,000 cells were lifted using a cell stripper (Media Tech. Inc.), and analyzed using a FACS Calibur flow cytometer (BD Biosciences) for FITC. 2.9. Cell Uptake of Tf-Conjugated Micelles. The cell uptake of targeting micelles as well as the nontargeting micelles was studied by confocal laser scanning microscopy (CLSM) and flow cytometry (FC) analysis. For CLSM observations, three types of cells (SGC-7901, SKOV-3, and MCF-7) were seeded separately in 6-well plates with a clean coverslip put in each well. After growing at 37 °C overnight, the cells were incubated with M(RhB) or Tf−M(RhB) at 37 °C for 2 h at an equivalent RhB concentration of 1.5 μg/mL. Thereafter, the cells were washed with PBS three times, fixed with 4 wt % formaldehyde (1 mL/ well) at room temperature for 10 min, and then washed with PBS three times. For nucleus labeling, fixed cells were incubated with 1 mL of DAPI solution (containing 1 μg of DAPI) in each well for 10 min and then washed with PBS five times. The coverslips were taken out and placed on a glass microscope slide for CLSM analysis. For flow cytometry analysis, 1 × 105 SGC-7901, SKOV-3, or MCF-7 cells were separately seeded in a 6-well plate and allowed to grow overnight. The cells were then treated with Tf−M(RhB) or M(RhB) at 37 °C for 2 h (the equivalent concentration of RhB was 1.0 μg/mL). Then, a single cell suspension was prepared

SMP was synthesized according to ref 49 with some modifications. Briefly, 1.84 g of β-alanine (20 mmol) and 2.03 g of maleic anhydride (20 mmol) were dissolved in 25 mL of degassed DMF, and the mixture was stirred at room temperature for 2 h. Then the solution was cooled in an ice bath, followed by addition of 8.5 g of DCC (40 mmol) and 2.8 g of NHS (25 mmol). Five minutes later, the reactor was withdrawn from the ice bath and the solution was stirred at room temperature for 12 h. The resulting suspension was filtrated to remove the dicyclohexylurea formed. 100 mL of water was added to the filtrate, and the product was extracted with CH2Cl2 (4 × 50 mL). The organic phase was dried over anhydrous Na2SO4, and then filtration was carried out to get light yellow solution. After concentrating, the solution was precipitated with petroleum ether to give a white solid. The solid was collected and dried in vacuo for 4 h (yield: 40%). To prepare mal-PEG-b-PLA, 0.5 g of amino-PEG-b-PLA (69 μmol) was dissolved in 10 mL of CH2Cl2, followed by addition of 10 μL of TEA. After two minutes’ stirring at room temperature, 88 mg of SMP (0.345 mmol) was added, and the reaction was continued for 12 h. Finally, the solvent was evaporated, and the residues were dissolved in 5 mL of CHCl3 and then precipitated with diethyl ether. The precipitates were dried in vacuo for 8 h. 2.4. Characterization of the Block Copolymers. 1H NMR spectra were recorded on a Bruker AV300 M in CDCl3 at 25 °C. Chemical shifts were given in parts per million from that of tetramethylsilane (TMS) as an internal reference. Gel permeation chromatography (GPC) measurements were conducted with a Waters 410 GPC instrument equipped with a Waters Styragel column (HT3) and a differential refractometer detector. CHCl3 was used as eluent at a flow rate of 1 mL/min at 35 °C. 2.5. Preparation of Hybrid Micelles M(NIR) and M(PTX). Hybrid micelles were prepared through selective-solvent evaporation methods. The preparation of hybid micelles M(NIR) composed of mPEG-b-P(LA-co-MHC/NIR) and malPEG-b-PLA was shown here as an example. Briefly, 90 mg of mPEG-b-P(LA-co-MHC/NIR) and 10 mg of mal-PEG-b-PLA were dissolved in 10 mL of acetone, and then 20 mL of Milli-Q water was added dropwise to the above solution in the dark. After all the water was added, the suspension was stirred under ambient conditions until most of the acetone was evaporated. Residual acetone was removed by dialysis of the suspension against water for 24 h. The final suspension in the dialysis bag was freeze-dried to give spongelike micelles. The hybrid micelles M(PTX) composed of 90 wt % of mPEG-b-P(LA-co-MCC/ PTX) and 10 wt % of mal-PEG-b-PLA were prepared in the same way. 2.6. Transferrin Conjugation. The conjugation of Tf to the hybrid micelles followed two steps: First, 5 mg of Tf was dissolved in 1 mL of sodium borate/EDTA buffer (0.15 M/0.1 mM, pH = 8.0), followed by addition of 40 times molar excess Traust’s reagent. After oscillation at 37 °C for 1 h, the buffer of Tf solution was replaced with 10 mM HEPES (pH = 7.0) containing 0.15 M NaCl and 0.01 M EDTA using a Microcon YM-3 concentrator tube (Millipore), and the thiolated Tf (Tf-SH) was concentrated to a volume of 100 μL. Second, 50 mg of hybrid micelles (M(NIR) or M(PTX)) and the above Tf-SH were mixed with 10 mL of HEPES buffer (10 mM, pH = 7.0). The conjugation was performed overnight in an oscillator thermostated at 25 °C. Unreacted Tf was removed through a Sepharose CL-6B column with PBS buffer (10 mM, pH = 7.4) as the eluent. Before elution, the mixture was concentrated to a volume of 1 1921

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Scheme 1. Synthesis of mPEG-b-P(LA-co-MHC/NIR), mPEG-b-P(LA-co-MCC/PTX), and mal-PEG-b-PLA

consecutively by trypsinization, washing with PBS, and filtration through 200 nylon mesh. Finally, 10,000 cells were lifted using a cell stripper (Media Tech. Inc.) and analyzed using a FACS Calibur flow cytometer (BD Biosciences) for RhB. In order to avoid the interference of autofluorescence emitted from the cell itself, blank cells without addition of any RhB-labeled micelles were analyzed and their fluorescent intensity was designated as the threshold value. Only the fluorescent intensity that exceeded the threshold value can be considered as the uptake signal. 2.10. Cytotoxicity of M(PTX) and Tf−M(PTX). The cytotoxicity of PTX conjugates was measured by the MTT method with free PTX as a control. Briefly, SGC-7901 cells were seeded in two 96-well plates at 5,000 cells/well 12 h prior to incubation with samples. Each well was filled with 100 μL of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/L streptomycin. Then PTX, M(PTX), and Tf−M(PTX) at five final PTX concentrations (150, 83, 15, 1.5, 0.15 μg/mL) were added to the wells, respectively (4 parallel wells for each sample at a specific PTX concentration). After coincubation at 37 °C for 48 h (or 72 h), 20 μL of MTT solution (5 mg/mL in PBS) was added and the plate was incubated for another 4 h. Then, the solution of each well was carefully removed and 150 μL of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the optical density at 490 nm (OD490nm) of each well was measured by a microplate reader. The survival rate of cells coincubated with PTX, M(PTX), or Tf−M(PTX) was calculated as the ratio of their OD490nm over the control cells treated only with PBS. 2.11. Subcutaneous Gastric Cancer Xenografts. Sixty female Balb/c nude mice, weighing 18−23 g, were purchased

from Nanjing Qingzilan Technolygy Co. Ltd. and handled under protocols approved by the Animal Center of Jilin University. For each mouse, 1 × 106 SGC-7901 gastric cancer cells in 0.1 mL of PBS buffer were implanted subcutaneously in the armpit of the left anterior limb. Twenty days later, tumor nodules with a size of 50−150 mm3 were observed in 51 mice. These tumor-bearing mice were divided into two parts: 12 mice for fluorescent imaging and the others for antitumor efficacy study. 2.12. In Vivo Imaging of Free NIR, M(NIR), and Tf− M(NIR). Twelve mice bearing SGC-7901 xenografts were injected with free NIR, M(NIR), Tf170−M(NIR), and Tf80− M(NIR) from the tail vein (three mice for each drug), respectively. At specific time intervals, the mice were anesthetized and exposed to the CRI Maestro In-vivo Imaging System from Cambridge Research & Instrumentation, Inc., MA, USA, which consisted of a light-tight box equipped with a 150 W halogen lamp and an excitation filter (671−705 nm) to excite NIR. Fluorescence was detected by a CCD camera equipped with a C-mount lens and an emission filter (750 nm long pass). A spectral data “cube” was created by acquiring a series of images at different wavelengths. In this cube, a spectrum is associated with every pixel. The resulting data can be used to identify, separate, and remove the contribution of autofluorescence in analyzed images by the commercial software (Maestro 2.4). 2.13. Antitumor Efficacy. Thirty-six mice bearing tumor nodules were randomly divided into four groups: (1) normal saline (blank control); (2) PTX; (3) M(PTX); and (4) Tf80− M(PTX). Before injection, all the mice were marked and weighed, and the length and width of the tumor were measured as the initial size. The day of starting injection was designated as day 1. For groups 2, 3, and 4, an equivalent PTX dose of 9 mg/kg 1922

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Figure 1. TEM images (upper, scale bar: 500 nm) of M(NIR) (A), Tf170−M(NIR) (B), M(PTX) (C), and Tf170−M(PTX) (D) and their particle sizes (lower) determined by DLS.

mouse was intravenously injected via tail vein at day 1, day 5, and day 10, separately. For group 1, the mice were injected with an equivalent volume of normal saline at day 1, day 5, and day 10 similarly. The tumor size was measured every other day, and the tumor volume (V) was calculated by the formula V = 1/2ab2, where a and b were the length and width of tumor, respectively. For histology observation, the tumor tissues were carefully excised from the body at the 30th day and quickly fixed with 10% paraformaldehyde solution for 48 h. After that, the tumor tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Finally, a series of micrographs of H&E-stained tumor sections were taken under a 4× objective and merged together using the Nikon NIS-Elements software to give a full-scale scene. 2.14. Statistical Analysis. The data were expressed as mean ± standard deviation (SD). Student’s t test was used to determine the statistical difference between various experimental and control groups. Differences at a level of p < 0.05 were considered statistically significant.

Table 1. Information about the Properties of Micelles micelle M(NIR) M(PTX) Tf170− M(NIR)e Tf80− M(NIR)e Tf80− M(PTX)e

feed wt ratio Tf:micelle

diametera (nm)

PDIb

CMCc (mg/L)

Rwd (μg/mg micelle)

0.1

85 92 110

0.24 0.21 0.19

2.6 0.9 2.8

41 ± 2.5

0.04

94

0.23

2.2

23 ± 1.9

0.04

112

0.20

1.1

18 ± 2.2

a

Determined by DLS. bThe polydispersity index of micelle (PDI) determined by DLS. cCritical micelle concentration (CMC) determined by pyrene fluorescence probe spectrometry (note: the CMCs of NIR-labeled micelles were speculated from their precursor copolymers without NIR-labeling). dThe amount of Tf in micelles determined by MicroBCA method. eNumbers after Tf are the average number of Tf per micelle which was calculated from the formula NA = (RwMn(micelle) × 10−3)/Mn(Tf), where Mn(Tf) = 80 kg/mol; Mn(micelle) = 4πR3NA/3υ (R is the radius of micelle; NA is the Avogadro constant; υ = 0.95 mL/g).

3. RESULTS AND DISCUSSION 3.1. Preparation of Hybrid Micelles and Their Conjugation with Tf. Using radical-mediated thiol−ene reactions,50 hydroxyl-containing block copolymer mPEG-b-P(LA-coMHC) and carboxyl-containing block copolymer mPEG-bP(LA-co-MCC) were synthesized from the same precursor copolymer mPEG-b-P(LA-co-MAC) (Scheme 1A). The former was used to conjugate with near-infrared dye (NIR), while the latter was used to conjugate with paclitaxel (PTX). 1H NMR (Figure S1 in the Supporting Information) was used to characterize the prepared copolymers, and the relative content of PTX in polymer was calculated from the ratio of integral area of benzene ring to that of the known Mn of PEG, while for NIR conjugates, the content of NIR was calculated from the standard Abs760nm vs concentration curve of NIR in DMSO. In this study, the content of PTX and NIR on their conjugates was ∼20 wt % (about 2.5 PTX per chain) and ∼9 wt % (about one NIR per chain), respectively. In addition, to facilitate Tf coupling on the micelle surface, another amphiphilic block copolymer, mal-PEG-

b-PLA, with a maleimide group at the end of the PEG segment, was synthesized (Scheme 1B). By varying the molar ratio of monomer to PEG in feed, the Mn of block copolymers could be controlled (Table S1 in the Supporting Information). The hybrid micelles, which contained maleimide groups at the external shell and NIR (or PTX) in the internal core, were prepared using the solvent-evaporation method. To facilitate coassembling, the hydrophobic segments of the two block copolymers were designed with the same length (3,000) and the hydrophilic PEG segment was fixed to 5,000. Since the size of micelle is an important parameter that may influence the accumulation of micelles in the tumor, the proper size of micelles for targeted tumor delivery is generally controlled between 70 and 200 nm.51 In this study, spherical morphologies of prepared hybrid micelles were observed by TEM experiments and the average diameter of M(NIR) and M(PTX) determined by DLS was 85 and 90 nm, respectively (Figures 1A and 1C). Formation of the micelles was also confirmed by 1H NMR analysis of 1923

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Figure 2. XPS spectra of M(PTX) (A), Tf30−M(PTX) (B), and Tf80−M(PTX) (C).

Figure 3. The release of PTX from M(PTX) and Tf−M(PTX) (A) and the change of Mn of PLA at specific release time points (B) determined by 1H NMR.

Figure 3A, the release of PTX exhibited the constant release property, while the Mn of PLA did not change much during the release time (Figure 3B), indicating that most of the released PTX was in the native form. 3.3. In Vitro Cellular Uptake. Three human carcinoma cell lines, MCF-7, SGC-7901, and SKOV-3, were chosen to study the uptake of fluorescence-labeled micelles. Before measurement, the TfR expression on the three cell membranes was assayed via fluorescence-activated cell sorting (FACS) analysis, using a TfR specific antibody (anti-CD71). As a control, the TfR-nonspecific antibody (mouse IgG2a) was investigated simultaneously. In order to avoid endocytosis of antibody during incubation, the temperature was set at 4 °C. As shown in Figure S4 in the Supporting Information, the TfR-specific antibody bound on the membrane of MCF-7 and SGC-7901 cell lines was far more than the control, while the SKOV-3 cell line did not make difference between the two antibodies. This result reflected that MCF-7 and SGC-7901 cell lines express more TfR than SKOV-3. Afterward, the uptake of Tf-conjugated and Tf-free micelles by MCF-7, SGC7901, and SKOV3 was studied by CLSM. To adapt the instrumental limition of excitation filter, rhodamine B (RhB) was temporarily used to label the micelle instead of NIR. As shown in Figure 4A, Tf-conjugated micelles displayed higher RhB fluorescence in TfR-overexpressing cells (MCF-7 and SGC7901) than the Tf-free mecelles, while for the SKOV-3 cell line, there was little difference of uptake between the Tf-conjugated micelles and Tf-free micelles. This implied that the enhanced uptake of Tf-conjugated micelles by MCF-7 and SGC-7901 was due to the Tf−TfR mediated endocytosis. To further investigate the influence of Tf content on the uptake of Tf-containing micelles by TfR-overexpressing cells, three targeting micelles

micelles in D2O (Figure S2 in the Supporting Information). For M(NIR) and M(PTX), only the signal of PEG appeared at 3.6 ppm; all signals belonging to LA and MHC (or MCC) units disappeared. This indicated that the P(LA-co-MHC/NIR) and PLA or P(LA-co-MCC/PTX) and PLA blocks aggregated into the solid core of the micelle in the aqueous environment because of their hydrophobic character. Transferrin was conjugated to the surface of hybrid micelles via the mild Michael addition reaction. Unreacted Tf was removed through a Sepharose CL-6B column, and the amount of Tf on the surface of micelles was quantitated using the MicroBCA method. After conjugation, the morphology of micelles did not change and the average diameter of Tf−M(NIR) and Tf−M(PTX) increased by 15 and 25 nm, respectively (Figures 1B and 1D). By varying the ratio of Tf to micelles in feed, the amount of Tf on the surface of micelles could be controlled. The detailed information about the micelles was shown in Table 1. Moreover, the existence of Tf on the surface of micelles was verified by XPS analysis. As shown in Figure 2, the nitrogen signal of Tf-free hybrid micelles was hardly detected, while that of the Tf-conjugated micelles showed the nitrogen signal at 399 eV and the signal intensity increased with the increase of Tf on the surface. 3.2. In Vitro Release of PTX. The release of PTX from Tf− M(PTX) and M(PTX) was studied in PBS buffer containing 10% (v:v) of ethanol. At a specific release time point, the micelle sample was analyzed by 1H NMR spectra in DMSO-d6 (Figure S3 in the Supporting Information). Because the PEG is expected to be stable during the release of PTX, the molar content of PTX remaining in the polymers can be calculated by comparing the integral area of resonances at 7.0−8.0 ppm (15 H of phenyl in PTX) with that at 3.5 ppm (PEG main chain). As shown in 1924

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Figure 4. (A) CLSM images of human MCF-7 (A and A′), SGC-7901 (B and B′), and SKOV3 (C and C′) cells incubated with Tf−M(RhB) (A, B, and C) or M(RhB) (A′, B′, and C′). Upper left: fluorescence of DAPI. Upper right: fluorescence of RhB. Lower left: bright field image. Lower right: overlay of the above three images. (B) CLSM images of SGC-7901 cells incubated with Tf30−M(RhB) (I), Tf80−M(RhB) (II), and Tf170−M(RhB) (III), respectively, at an equivalent RhB content of 1.5 μg/mL for 2 h. (1) DAPI-stained nuclei; (2) RhB fluorescence from micelles; (3) bright field image; (4) overlapping of 1 and 2.

with different surface Tf content were incubated with SGC-7901 cells. As shown in Figure 4B, the fluorescence observed is approximately proportional to the Tf numbers on the micelles, indicating again the contribution of Tf−TfR mediated endocytosis to the cell uptake.

In order to semiquantitatively test the uptake efficiency of Tfcontaining micelles and Tf-free micelles, flow cytometry (FC) experiments were carried out. As shown in Figure 5, the average fluorescent intensity of MCF-7 and SGC-7901cells treated with Tf30−M(RhB) was nearly 3−5 times more than that of those cells 1925

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Figure 5. Flow cytometry analysis of human MCF-7, SGC-7901, and SKOV3 cells incubated with targeting micelles and nontargeting micelles for 2 h (A); and SGC-7901 cells incubated with targeting micelles containing different Tf on the surface for 2 h (B). The equivalent RhB concentration for each test was 1.5 μg/mL.

Figure 6. Cell viability of SGC-7901 treated with PTX, M(PTX), Tf170−M(PTX), and Tf80−M(PTX) at 37 °C for 48 h (A) and 72 h (B), respectively (C(equ. PTX): equivalent concentration of PTX). (C) Cell viability of SGC-7901 treated with blank micelles without PTX.

treated with M(RhB) (from 1 × 103 to 3 × 103 for MCF-7 and from 2 × 102 to 1 × 103 for SGC-7901). However, SKOV-3 did not make such a difference between Tf170−M(RhB) and M(RhB). From Tf30−M(RhB) to Tf170−M(RhB), the intensity peak of the treated SGC-7901 cells moved from 1 × 103 to more than 2 × 103. All these FC results demonstrated the Tf−TfR

interaction mediated endocytosis of the RhB-labeled micelles by the TfR-overexpressing cells. 3.4. In Vitro Cytotoxicity. In order to test the cytotoxicity of free PTX, M(PTX), Tf170−M(PTX), and Tf80−M(PTX) to SGC-7901 cells, MTT experiments were carried out. As shown in Figure 6A, PTX shows the most cytotoxicity after 48 h 1926

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Table 2. IC50a of PTX, M(PTX), Tf170−M(PTX), and Tf80− M(PTX) to SGC-7901 Cells (μg/mL) sample

PTX

M(PTX)

Tf170−M(PTX)

Tf80−M(PTX)

48 h 72 h

33.7 ± 0.45 16.5 ± 0.11

88.2 ± 0.093 30.5 ± 0.32

65.1 ± 0.18 9.1 ± 0.44

78.5 ± 0.43 10 ± 0.087

flow conditions. However, along with time elapsing, the amount of free NIR in the tumor site decreased rapidly. It seems that the previously accumulated NIR had come out of the tumor tissues, while in micellar groups, the amount of NIR was retained over a long time. Compared with the Tf-free micelles, more Tfconjugated micelles were observed in the tumor site. In order to get the tumor uptake−time relationship, the NIR signal of the tumor site was plotted as a function of time. Figure 9 shows that the accumulation of NIR reached its maximum at 6 h in the free NIR group and then the signal decreased quickly; in the M(NIR) group, the signal increased relatively slowly at the early stage and achieved maximum at ∼28 h; in Tf170−M(NIR) and Tf80− M(NIR) groups, the signal increased constantly and achieved maximum at ∼22 h; the maximum intensities and the areas under the intensity curves of Tf170−M(NIR) and Tf80−M(NIR) groups were more than those of the M(NIR) group and the free NIR group within the observed time period. Increasing the amount of Tf on the micelle surface from 80 to 170 led to a slight increase of NIR uptake by tumor tissue. Thus in the following studies of the antitumor effect of PTX-conjugated micelles, Tf80−M(PTX) was used. 3.6. In Vivo Antitumor Efficacy. To test if increased accumulation of micelles in the tumor site after Tf-conjugation could better inhibit tumor growth, the in vivo antitumor effect of Tf-conjugated and PTX loaded micelles was studied with SGC7901 implanted nude mice. When the tumor grew to a size of 50−150 mm3, the mice were randomly divided into four groups with 9 mice in each group. The equivalent dose of PTX (27 mg/ kg) was divided into three times (at day 1, 5, and 10) for injection, and the tumor sizes were measured every other day. Because the initial tumor volume of individual mouse (V0) was not uniform, the parameter Vt/V0 at day t, instead of the tumor volume (Vt), was used to investigate the antitumor effect. Figure 10A shows the relative tumor size as a function of time. It was notable that the administration of Tf−M(PTX) was much more efficacious in tumor suppression compared with other groups, which was consistent with the enhanced uptake by tumor tissues mentioned above. More direct proof is shown in the picture of excised tumors from the mice on the 30th day. As shown in Figure 11, the average volume of the Tf−M(PTX) group was much smaller than that of any other groups. In addition, the suppression of tumor growth for drug administration groups was dependent on the initial tumor volume. Figure 10B shows that the PTX-induced antitumor effect is more efficacious for the mice with smaller initial tumor size than those with larger initial tumor size. This phenomenon implies the importance of chemotherapy at the early stage of tumor growth. Finally, tumor slices from the mice of the experimental groups 30 days postinjection were prepared and stained with hematoxylin and eosin (H&E). In order to objectively evaluate the inhibitory effect of PTX-conjugated micelles, a series of micrographs of H&E-stained tumor sections were taken under a 4× objective and merged together to give a full-scale scene. As shown in Figure 12, for saline and PTX groups, a large amount of living cells with only a small necrotic region distributed in the tumors, while high extents of necrotic region could be seen in the tumors from the M(PTX) and Tf−M(PTX) groups. Notablely for the Tf− M(PTX) group, there were only a few viable tumor remnants, mostly located in the peripheral region of tumor whereas the bulk of the tumor tissue was in the degeneration or necrosis state, which corroborates the notably enhanced anticancer efficacy of Tf−M(PTX) once more.

a

The half inhibitory concentration (IC50) determined by the MTT method. The data were expressed as mean ± SD.

Figure 7. Fluorescent imaging of nude mice treated with free NIR, M(NIR), Tf80−M(NIR), and Tf170−M(NIR) at different time points.

coincubation with SGC-7901, which may be caused by fast diffusion of free PTX into the cells. After 72 h (Figure 6B), however, the difference of cytotoxicity between PTX and PTXloaded micelles disappeared. Compared with M(PTX) (IC50 = 30.5 μg/mL), the cytotoxicities of Tf170−M(PTX) (IC50 = 9.1 μg/mL) and Tf80−M(PTX) (IC50 = 10 μg/mL) at 72 h were much higher (Table 2). This enhanced cytotoxicity of Tf− M(PTX) can be attributed to the efficient endocytosis mediated by Tf−TfR interactions and subsequent delivery of PTX inside the SGC-7901 cells. In addition, the blank micelles without PTX exhibited very little toxicity to SGC-7901 cells (Figure 6C). 3.5. In Vivo Real-Time Imaging. In order to observe the in vivo biodistribution of targeting micelles and nontargeting micelles after iv injection, twelve nude mice with subcutaneously implanted TfR-overexpressed cancer model (gastric cancer SGC-7901) were injected with free NIR, M(NIR), Tf80− M(NIR), and Tf170−M(NIR), respectively. At specific time intervals, the mice were anesthetized and imaged with CRI Maestro 500FL system. As shown in Figure 7, some distribution characters were obtained for the four groups. Compared with the micellar NIR, clearance of free NIR from the body was much faster. The signals of the micellar groups were still strong after 46 h, which offered the chance of redistribution of micelles into the tumor site. In order to semiquantitatively illustrate the biodistribution information, the photon numbers per unit area (average signals) of four different parts of the mouse body (as shown in the top right corner picture in Figure 7) were collected at specific time points. Figure 8 shows the average signals of the four parts at 6, 14, 22, and 35 h. As the main clearance organs, liver, kidney, and spleen (LKS) intercepted a lot of injected samples. This can be explained by the efficient elimination of particles from the blood by the reticuloendothelial system (RES).52 At 6 h, the amount of NIR in tumor tissues in the free NIR group was more than that in the micellar groups, which may be caused by the rapid diffusion rate of free NIR under the blood 1927

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Figure 8. Average signals collected from the four parts of the mouse body at different time points (LKS, liver, kidney, and spleen; H&L, heart and lung).

gastric cancer model may also be applicable to the other TfRoverexpressing cancers due to the similar targeting mechanisms. Furthermore, for the endeavors concerning the design of nanoscale drug delivery systems to cross the blood−brain barrier (BBB) for the treatment of nervous system diseases (i.e., Alzheimer’s disease, Parkinson’s disease, etc.), the current study implies referential importance because the brain capillaries have been identified to highly express TfR.53

4. CONCLUSIONS Surface maleimide-functionalized hybrid micelles containing NIR or PTX were prepared by mixing mal-PEG-b-PLA with mPEG-b-P(LA-co-MHC/NIR) or mPEG-b-P(LA-co-MCC/ PTX), respectively. Tf was successfully linked onto the micellar surface via mild Michael addition reaction. CLSM and flow cytometry experiments showed that the cellular uptake efficiency of micelles by TfR-overexpressing cell lines could be enhanced after Tf conjugation. In vivo near-infrared imaging showed that the maximum accumulation of micelles was achieved at 28 h for the M(NIR) group, while at 22 h for Tf−M(NIR) groups. Although the maximum accumulation of free NIR was achieved earlier (6 h) than for the micellar groups, the clearance of NIR from the body was much faster than that of the micellar groups. The maximum intensities and the areas under the intensity curves of Tf−M(NIR) groups were more than those of the

Figure 9. Average NIR signals collected from the tumor site as a function of time.

Taking all the above results together, we can see that the enhanced antitumor effect of Tf−M(PTX) has a close relationship with the following factors: constant release of drug (PTX) from the polymer micelles, enhanced endocytosis efficiency of micelles by target cell lines, and increment of time-dependent accumulation of micelles in the tumor site after Tf conjugation. From the applicability aspect, the results with the 1928

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Figure 10. (A) Relative tumor sizes of SGC-7901 subcutaneous model as a function of time (the arrows represent the iv injection time); *p < 0.01 vs saline group, #p < 0.01 vs PTX group, &p < 0.05 vs M(PTX). (B) Relative tumor volumes on the 30th day calculated in two groups according to the initial tumor volumes being more or less than 100 mm3. *#&p < 0.05.

Figure 11. Picture of excised tumors on the 30th day (dividing rule: cm).

Figure 12. H&E-stained tumor slices from mice on the 30th day for saline (A), PTX (B), M(PTX) (C), and Tf−M(PTX) (D) group. For each group, the full-scale image of one tumor slice was obtained by stitching a series of pictures which were taken under a 4× objective. The white arrows represent the necrotic regions in the interior of the tumor. The white rectangle indicates the location of the enlarged image.

M(NIR) group and free NIR group within the observed time period. Finally, the tumor inhibition effects of targeting micelles were studied with the gastric carcinoma models which

overexpressed TfR. The analysis of tumor volumes and the observation of H&E-stained tumor sections indicated that Tf− M(PTX) had the best antitumor effect compared with the 1929

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control groups (saline, PTX, and M(PTX)). Therefore, Tfconjugated micelles might be used as the effective drug delivery system for the treatment of TfR-overexprssing carcinoma.

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ASSOCIATED CONTENT

S Supporting Information *

A depiction of the structure of NIR used in this study; figures depicting 1H NMR spectra of amphiphilic block copolymers and their conjugates, M(NIR) and M(PTX), and micelle samples and flow cytometry detection of TfR expression; and table of information about the amphiphilic block copolymers. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Changchun Institute of Applied Chemistry, State Key Laboratory of Polymer Physics and Chemistry, 5625 Renmin Street, Changchun 130022, P. R. China. Tel/fax: +86-43185262775. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by the National Natural Science Foundation of China (Project No. 21004062, 20674084), by “100 Talents Program” of the Chinese Academy of Sciences (No. KGCX2-YW-802), and by the Ministry of Science and Technology of China (“973 Project”, No. 2009CB930102).

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