Transferrin-Modified c[RGDfK]-Paclitaxel Loaded Hybrid Micelle for

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Transferrin-Modified c[RGDfK]-Paclitaxel Loaded Hybrid Micelle for Sequential Blood-Brain Barrier Penetration and Glioma Targeting Therapy Pengcheng Zhang,†,§ Luojuan Hu,‡,§ Qi Yin,† Linyin Feng,*,‡ and Yaping Li*,† †

Center of Pharmaceutics and ‡State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ABSTRACT: The effective chemotherapy for glioblastoma multiform (GBM) requires a nanomedicine that can both penetrate the blood-brain barrier (BBB) and target the glioma cells subsequently. In this study, Transferrin (Tf) modified cyclo-[Arg-Gly-Asp-D-Phe-Lys] (c[RGDfK])-paclitaxel conjugate (RP) loaded micelle (TRPM) was prepared and evaluated for its targeting efficiency, antiglioma activity, and toxicity in vitro and in vivo. Tf modification significantly enhanced the cellular uptake of TRPM by primary brain microvascular endothelial cells (BMEC) to 2.4-fold of RP loaded micelle (RPM) through Tf receptor mediated endocytosis, resulting in a high drug accumulation in the brain after intravenous injection.The c[RGDfK] modified paclitaxel (PTX) was released from micelle subsequently and targeted to integrin overexpressed glioma cells in vitro, and showed significantly prolonged retention in glioma tumor and peritumoral tissue. Most importantly, TRPM exhibited the strongest antiglioma activity, as the mean survival time of mice bearing intracranial U-87 MG glioma treated with TRPM (42.8 days) was significantly longer than those treated with Tf modified PTX loaded micelle (TPM) (39.5 days), PTX loaded micelle (PM) (34.8 days), Taxol (33.6 days), and saline (34.5 days). Noteworthy, TRPM did not lead to body weight loss compared with saline and was less toxic than TPM. These results indicated that TRPM could be a promising nanomedicine for glioma chemotherapy. KEYWORDS: glioma, blood-brain barrier, micelle, sequential targeting

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INTRODUCTION Glioblastoma multiform (GBM) is one of the most aggressive and lethal cancers, with a median survival of about 14 months and a 5 year survival rate of less than 5%.1 Chemotherapeutic drugs such as carmustine, lomustine, and Temozolomide have shown some promise, but the patients’ survival improvement is usually limited mainly due to overexpression of the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT), which repairs the DNA injury caused by the above-mentioned alkylator.2 Several widely used chemotherapeutic agents with distinct antitumor mechanisms such as paclitaxel (PTX), doxorubicin and topotecan are unable to penetrate the bloodbrain barrier (BBB), which is still functional at the outer rim of the tumor and the malignant cells infiltrated peritumoral tissue.3,4 In the past decade, several brain targeting ligands were developed with the improvement of brain drug accumulation,5 but targeting GBM tumor or infiltrated glioma cells after penetrating BBB remains challenging. Recently, dual-targeting nanocarriers were developed by modifying nanocarriers with ligands such as wheat germ agglutinin (WGA),6 angiopep,7 and lactoferrin (Lf),8 the receptors of which are expressed on both BBB and glioma cells. However, these receptors are also highly expressed throughout the brain,9,10 and the degradation of ligands during transportation across BBB would further reduce the selectivity of nanocarriers.11 The nonspecific distribution of © 2012 American Chemical Society

drugs in the whole brain after BBB penetration may cause neurotoxicity, which affects all cognitive function.12 To solve this issue, we designed and constructed a novel sequential-targeting nanomedicine by encapsulating a drug conjugate that can target glioma cells into a BBB-penetrating micelle. Transferrin (Tf), an endogenous protein that can be effectively transcytosed across the BBB and can direct carrier systems with encapsulated drugs across the BBB by receptor mediated transcytosis (RMT),13,14 was grafted onto the micelle. cyclo-[Arg-Gly-Asp-D-Phe-Lys] (c[RGDfK]), a peptide that specifically binds to integrin overexpressed glioma cells,15,16 was conjugated to PTX to obtain c[RGDfK]-paclitaxel conjugate (RP) (Scheme 1).The targeting efficiency of Tf modified RP loaded hybrid micelle (TRPM) was evaluated in the aspects of internalization by primary brain microvascular endothelial cells (BMECs) and U-87 MG cells in vitro and the brain distribution in vivo. The general toxicity and antiglioma activity of TRPM was evaluated on nude mice bearing intracranial U-87 MG gliomablastoma. Received: Revised: Accepted: Published: 1590

November 24, 2011 April 6, 2012 April 12, 2012 April 12, 2012 dx.doi.org/10.1021/mp200600t | Mol. Pharmaceutics 2012, 9, 1590−1598

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Scheme 1. Synthesis Scheme of RPa

4-dimethylamino-pyridine (93 mg, 0.762 mmol), succinic anhydride (76 mg, 0.762 mmol), and triethylamine (77 mg, 0.762 mmol) were dissolved in 15 mL anhydrous dioxane and stirred overnight at room temperature under an N2 atmosphere. The reaction solution was evaporated and redissolved in chloroform. The solution was sequentially washed with 10 mM HCl and saturated brine solution, dried over Na2SO4, and evaporated to yield PTXS as an off-white solid. PTXS was activated by dicyclohexylcarbodiimide/N-hydroxysuccinimide (1:1.2:1.2, mol/mol) in anhydrous tetrahydrofuran (THF) for 12 h at room temperature under an N2 atmosphere. The reaction solution was filtered, and the filtrate was evaporated to yield PTXS succinimide ester (PTXSSE). PTXSSE, c[RGDfK] and diisopropylethylamine (1:1.2:1.2, mol/mol) were dissolved in anhydrous N,N-dimethylformamide and stirred overnight at room temperature under an N2 atmosphere. The reaction solution was evaporated under vacuum to yield crude RP. Purification of crude RP was carried out on a HPLC system (Waters 1525 chromatography system with a 2489 UV−vis detector) with the following conditions: Waters XBridge BEH130 Prep C18 (5 μm, 10 × 150 mm); the flow rate was 3 mL/min with the mobile phase starting from 55% solvent A (0.05% trifluoroacetic acid in water) and 45% solvent B (acetronitrile) (0−4 min) to 90% solvent A and 10% solvent B at 10 min; the monitored wavelength was 237 nm. The fractions containing RP were collected and lyophilized (RD85, Millrock). Analytical HPLC was performed on an Agilent 1100 with the following conditions: Agilent HC-C18 column (5 μm, 4.6 × 250 mm); the flow rate was 1 mL/min with the mobile phase starting from 70% solvent A (0.05% trifluoroacetic acid in water) and 30% solvent B (acetronitrile) (0−5 min) to 30% solvent A and 70% solvent B at 40 min; the monitored wavelength was 237 nm. The retention time of RP on analytical HPLC was 23.1 min. The retention time of PTX under the same condition was 29.3 min. RP was also determined using a 300 MHz 1H NMR spectrometer (Varian, USA) and an electrospray ionization time-of-flight (ESI-TOF) mass spectrometer (Finnigan MAT-9, Thermo), respectively. Preparation of RP and PTX Loaded Micelles. RP loaded micelle (RPM) was prepared using a blend of PCL-PEEP and Mal-PEG-PCL by a dialysis method. Briefly, 4 mg RP, 45 mg PCL-PEEP, and 5 mg Mal-PEG-PCL were dissolved in 250 μL THF, and then 1.25 mL deionized water was added dropwise over 10 min. The solution was stirred gently for 30 min followed with dialysis against 2 L of deionized water overnight to remove THF. The RPM was then purified and concentrated using Amicon Ultra-15 (MWCO: 100 kDa, Millipore). For Tf conjugation, Tf was first thiolated and characterized as previously described.20 The thiolated Tf was then incubated with RPM at a thiolated Tf to maleimide group molar ratio of 1:10 for 8 h to obtain Tf conjugated RPM (TRPM). The TRPM was then purified and concentrated using Amicon Ultra15 (MWCO: 100 kDa, Millipore). PTX loaded micelle (PM) and Tf conjugated PM (TPM) were prepared using the same procedure as described above and were used as control. Finally, all of the micelles were sterilized by 0.22 μm filtration. Hydrolysis. The RP solution was added to a phosphate buffer solution (PBS) at pH 5.0, 7.4, and 8.4 to evaluate the effect of pH on RP hydrolysis (final concentration was 10 μM). The solutions were incubated (37 °C, 150 rpm) and sampled at scheduled times for HPLC analysis to determine RP content. Each experiment was repeated in triplicate. To evaluate the stability of RP in plasma, RP solution or RPM were added to

a

DMAP, 4-(dimethylamino)pyridine; DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide; DMF, dimethylformamide.

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EXPERIMENTAL SECTION Materials. Paclitaxel (PTX) was purchased from Shanghai Sunve Pharmaceutical Co., Ltd. (China). cyclo-[Arg-Gly-Asp-DPhe-Lys] (c[RGDfK]) (purity >98%) was synthesized by GL Biochem Ltd. (China). Transferrin (Tf), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-iminothiolane hydrochloride (ITH), thiazolylblue tetrazoliumbromide (MTT), and Ellman’s reagent were obtained from SigmaAldrich (USA). BODIPY TR-X SE was purchased from Invitrogen (USA). Taxol was purchased from Bristol-MyersSquibb Company (USA). Poly(ε-caprolactone)-block-poly(ethyl ethylene phosphate) (PCL-PEEP) was synthesized and characterized as previously described,17 and the degrees of polymerization (DP) of PCL and PEEP were 31 and 35, respectively. Maleimide-poly(ethylene glycol)-block-poly(ε-caprolactone) (Mal-PEG-PCL) was synthesized by ring-opening polymerization using maleimide-poly(ethylene glycol)-OH as initiator in the presence of stannous octoate,18 and the DP of PEG and PCL were 77 and 28 determined by 1H NMR, respectively. All other reagents were of analytical grade and used as received without further purification. Cells and Animals. U-87 MG cell line was obtained from the American Type Culture Collection (ATCC, USA), and HEK-293 cells stably expressing enhanced green fluorescence protein (HEK-293-EGFP) were kindly provided by National Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute. Both cell lines were grown in DMEM containing 10% fetal bovine serum (FBS). Primary brain microvascular endothelial cell (BMEC) was obtained from Sprague−Dawley rats brain and was grown in EMB-2 (Lonza, Switzerland) containing 10% FBS. All the cells were incubated at 37 °C in a humidified and 5% CO2 incubator. ICR mice (18−22 g, ♂), Sprague−Dawley (SD) rats (100− 120 g, ♂), and BALB/c-nu mice (18−22 g, ♂) were obtained from Shanghai Experiment Animal Center, Chinese Academy of Sciences, and maintained at 25 ± 2 °C on a 12 h light-dark cycle with free access to food and water. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee at Shanghai Institute of Materia Medica, Chinese Academy of Sciences. c[RGDfK]-Paclitaxel Conjugate (RP) Synthesis. To synthesize RP, PTX was first reacted with succinic anhydride to obtain PTX-2′-succinate (PTXS) as previously described with minor modification.19 Briefly, PTX (500 mg, 0.586 mmol), 1591

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Figure 1. HPLC chromatogram (A), 1H NMR spectrum (B), and low- and high-resolution ESI-TOF mass spectrum (C) of RP.

rat plasma (final concentration was 10 μM). The solutions were incubated (37 °C, 150 rpm) and sampled at predetermined intervals for RP content determination using HPLC. In Vitro Release. The release profiles of RP from RPM and TRPM were investigated in 1 M sodium salicylate, which has been reported to aid in solubilizing PTX without destabilizing the PEG-b-poly(phenylalanine) micelle.21 Briefly, dialysis bags (MWCO: 6−8 kDa, Spectrum Lab) containing RPM or TRPM (containing 0.3 μmol RP) were incubated with 5 mL of 1 M sodium salicylate (37 °C, 150 rpm). At scheduled time intervals, the release medium was withdrawn and replaced with a predetermined volume of fresh medium. The concentrations of RP and PTX resulting from RP hydrolysis were measured by HPLC as described above. Cellular Uptake. Primary BMECs were seeded on a 12-well plate and allowed to reach confluence.17 The cells were then treated with 10 μM RP containing RPM or TRPM for 1 h at 37 °C. To prove the transferrin receptor (TfR)-mediated clathrin-dependent endocytosis, 5 mg mL−1 free Tf or 10 μM chlorpromazine (Chl) were added 30 min prior to TRPM addition in 3 wells, respectively. The cells were then washed 3 times with ice-cold PBS and lysed with Triton X-100 (1%, v/v) for 30 min. The RP and PTX in cell lysates were extracted with methanol and determined by HPLC. The amount of endocytosed drugs were then normalized with the amount of cellular protein quantified by the Coomassie brilliant blue method. To investigate whether c[RGDfK] conjugate could target the integrin overexpressing U-87 MG cells, a c[RGDfK]-BODIPYTR conjugate (RB, m/z = 1123.4904 corresponding to the [M + H]+) was synthesized and purified following the same procedure as described for RP synthesis. U-87 MG cells (3 × 104 cells/well) and HEK-293-EGFP cells (2 × 104 cells/well) were cocultured on 10 mm2 glass coverslips coated with poly-Llysine (PLL) in 24-well plates for 24 h. The coculture model was incubated for 1 h with BODIPY-TR or RB (5 μM), washed 3 times with ice-cold PBS, fixed with 4% polyformaldehyde,

Figure 2. Sizes (A) and zeta potentials (B) of PM (black line), TPM (red line), RPM (green line), and TRPM (blue line).

stained with DAPI (10 μg mL−1) for 10 min, and washed with PBS twice again. The cells were then mounted on glass slides with 3 μL of MobiGlow (MoBiTec, Germany) and visualized by confocal microscopy (FluoView FV1000, Olympus). Cytotoxicity Assay. U-87 MG cells were seeded in a 96well plate at 5 × 103 cells/well and incubated for 24 h. The medium was replaced with fresh medium containing various concentrations of PTX, RP, PM, and RPM. At 72 h, the cell viability was measured by SRB assay according to the 1592

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manufacturer’s instruction (Sigma, USA), and the dose-effect curves were plotted. Cell Cycle Analysis. U-87 MG cells were seeded in a 6-well plate at 2 × 105 cells/well and incubated for 12 h. The exponentially growing U-87 MG cells were treated with 20 nM PTX, PM, RP, RPM, c[RGDfK], a mixture of PTX and c[RGDfK], or mixture of PM and c[RGDfK] for 24 h, respectively. Then, cells were harvested and fixed in 70% ethanol for 12 h at 4 °C. The cells were collected by centrifugation (1 × 103 rpm, 4 min, 4 °C), washed twice with cold PBS to remove residual ethanol, resuspended in 0.5 mL PBS containing 0.5 mg/mL RNase A, 0.02 mg/mL PI, and 0.1% Triton X-100, and incubated at 37 °C for 30 min. Cell cycle profiles of treated U-87 MG cells were studied using a FACS calibur flow cytometer (Becton Dickinson, USA). The data were analyzed through Mod-Fit 2 software (Becton Dickinson, USA). Western Blot and Antibodies. The mice bearing intracranial U-87 MG glioma were established as follows: U-87 MG cells (1 × 105 cells in 1 μL PBS) were injected into the right striatum (1.8 mm lateral to the bregma, 0.6 mm anterior to the bregma, and 3 mm of depth) of male BALB/c-nu mice (18−22 g) at 1 μL/min using a stereotactic fixation device with mouse adaptor. Four weeks after implantation, three mice were anaesthetized and sequentially perfused with 0.9% saline to remove the blood. The glioma, peritumoral, and normal brain tissue were collected. The tissues were weighed and homogenized in ice-cold RIPA lysis buffer (20 μL/mg tissue). The resulting homogenates were centrifuged under 1.5 × 104 g at 4 °C for 15 min, and the protein concentrations in supernatant were determined using the Coomassie brilliant blue method. Primary antibodies used for Western blot analysis were rabbit anti-integrin αv antibody (1:1000, Cell signaling, USA) and mouse anti-β-actin antibody (1:15000, Sigmaaldrich, USA). Application of Micelles in Animals. Intracranial glioma bearing mice were established as described above. Four weeks after implantation, the mice were randomly assigned into one of the following groups (n = 12 per group): Taxol, PM, TPM, and TRPM, and mice were administrated via tail vein at the equivalent 10 mg kg−1 dose of PTX. At 1, 4, 12, and 24 h after the administration, the blood samples were collected and centrifuged under 3 × 103 g at 4 °C for 5 min immediately to harvest plasma. Then, the mice were killed and tissues including heart, liver, spleen, lungs, kidneys, and tumor, peritumoral, and normal brain tissue were collected, washed with saline, blotted, and weighed. All the samples were stored at −80 °C until HPLC analysis. The tissues were homogenized (Precellys 24, Bertin technology), and drugs in the homogenate and plasma were extracted and analyzed by HPLC. Mice bearing intracranial U-87 MG glioma were established as described above. The mice were randomly assigned into one of the following groups (n = 8): saline, Taxol, PM, TPM, and TRPM, and mice were administrated via tail vein at the equivalent 10 mg kg−1 dose of PTX at 7, 12, 17, 22, and 27 days after tumor implantation. The body weights of mice were monitored twice a week for 4 weeks after tumor implantation. The survival time of mice were recorded. Statistics. Statistical analysis was performed using the Student’s t-test. Survival data were presented using Kaplan− Meier plots and were analyzed using a log-rank test using SPSS program 19.0. The differences were considered significant for P < 0.05 and very significant for P < 0.01.

Figure 3. Stability of free and encapsulated RP in PBS and plasma. The effect of pH (A) and plasma (B) on the stability of RP (n = 3 for each group at each time point). All error bars reflect SD.

Figure 4. Cumulative release profile of RP from RP loaded micelle (RPM) and transferrin modified RPM (TRPM) in 1 M sodium salicylic acid at 37 °C (n = 3 for each group at each time point). All error bars reflect SD.

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RESULTS Synthesis and Characterization of RP. To confer the specificity of PTX targeting glioma cells, we conjugated targeting peptide c[RGDfK] to PTX by derivatizing the 1593

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Figure 5. In vitro targeting efficiency. Cellular uptake of transferrin modified c[RGDfK]-paclitaxel loaded micelle (TRPM) by primary brain microvascular endothelial cells (BMEC) in the presence or absence of free transferrin (Tf) and chlorpromazine (Chl) in the comparison with c[RGDfK]-paclitaxel loaded micelle (RPM) (n = 3) (A). *P < 0.05 compared with RPM; **P < 0.01 compared with TRPM. High resolution ESITOF spectrum of RB (B). Cellular uptake of BODIPY-TR and c[RGDfK]-BODIPY-TR conjugate (RB) by the coculture of U-87 MG and HEK293-EGFP cells after 1 h incubation (C). Red, BODIPY-TR; green, HEK-293-EGFP cells expressing enhanced green fluorescent protein (EGFP); blue, nuclei stained by DAPI. Bar: 50 μm. Outlines of HEK-293-EGFP cells were drawn in white line.

2′-hydroxyl function of PTX with succinic anhydride, activating with dicyclohexylcarbodiimide/N-hydroxysuccinimide, and coupling with c[RGDfK] peptide through the lysine ε-amino group (Scheme 1). The purity of RP was >98% as verified by HPLC (Figure 1A), and the structure of RP was confirmed by 1 H NMR spectroscopy where the characteristic peaks of PTX and c[RGDfK] could be found (Figure 1B). In addition, the low and high resolution ESI-MS spectrum of RP exhibited a peak at m/z = 1539.6589 corresponding to the [M + H]+, which indicated that the molecular formula of RP was C78H94N10O23 (Figure 1C). Micelle Preparation and Characterization. We encapsulated RP into polyphosphoester based hybrid micelle to construct RP loaded micelle (RPM), and then conjugated Tf onto RPM to obtain BBB permeable nanomedicine (transferrin modified RPM, TRPM) using Traut’s reagent.20 The particle sizes of RPM and TRPM determined by dynamic light scattering (DLS) were 38.49 ± 1.97 nm (n = 3) and 98.44 ± 2.67 nm (n = 3) with an acceptable polydispersity index (PDI < 0.22 for both micelles) (Figure 2A). The ζ potential of RPM and TRPM were 1.38 ± 0.27 mV and −8.71 ± 0.78 mV, respectively (Figure 2B). Compared with RPM, TRPM showed increased particle size and decreased ζ potential, owing to the coupling of hydrophilic, nanosized, and negatively charged Tf onto the micelle.22,23 Control formulations, PTX loaded micelle (PM, 33.31 ± 0.61 nm, −2.02 ± 0.46 mV), and transferrin modified PM (TPM, 87.85 ± 2.32 nm, −12.33 ± 1.46 mV), showed comparable size and ζ potential with RPM and TRPM, respectively (Figure 2). The drug loading capacities of RPM and TRPM as determined by HPLC were 3.67 ± 0.12% (n = 3) and 3.18 ± 0.10% (n = 3), respectively. The encapsulation

Figure 6. Cell viability of U-87 MG cells after incubation with various concentration (from 0.1 to 2500 nM) of paclitaxel (PTX), paclitaxel loaded micelle (PM), RP, and RPM for 72 h.

efficiencies of RPM and TRPM were 82.6 ± 3.0% (n = 3) and 82.9 ± 2.9% (n = 3), which were a bit lower than that of PM (91.2 ± 3.5%) and TPM (89.9 ± 3.4%), reflecting the relatively high water solubility of RP compared with PTX. Hydrolysis. To assess whether micelle could protect RP from degradation, we investigated the hydrolysis of RP in PBS and plasma in vitro. According to HPLC analysis, the hydrolysis product of RP was PTX through the hydrolysis of ester bond between PTX and succinate. The hydrolysis of RP was found to accelerate with increasing pH of PBS (Figure 3A), such that 66% of RP was hydrolyzed into PTX at pH 8.4 in 2 h, while it 1594

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Figure 7. Effects of treatment with paclitaxel (PTX), PTX loaded micelle (PM), c[RGDfK]-paclitaxel conjugate (RP), RP loaded micelle (RPM), and a physical mixture of c[RGDfK] and PTX or PM on the cell cycle of U-87 MG cells after 24 h incubation.

took 8 h to degrade 51% of RP at pH 7.4. The hydrolysis of RP was greatly enhanced in plasma due to the presence of plasma esterase but could be significantly inhibited by encapsulating RP into the micelle (Figure 3B). After 8 h of incubation, more than 90% of free RP was degraded, but 92% of encapsulated RP remained intact. These results demonstrate that the micelle can protect the drug from degradation possibly by limiting the interaction of RP with esterase in plasma. In Vitro Release. We investigated the in vitro cumulative release profiles of RP from RPM and TRPM. RPM and TRPM showed similar biphase release behaviors (Figure 4), which indicated that the Tf modification did not affect the release behavior of RP. For the first 12 h, the release of RP was fast, but no burst release was observed. Then, it slowed down, and most of the encapsulated RP was released within 72 h. Cellular Uptake. To assess whether TRPM would be able to specifically deliver RP to glioma cells in vitro, we used primary BMEC to establish the in vitro BBB model. TRPM exhibited enhanced cellular uptake of 2.4-fold (P < 0.05) compared with RPM after 1 h incubation (Figure 5A) and could be significantly inhibited by excess free Tf (P < 0.01). It was reported that the endocytosis of Tf was clathrindependent,24 a process that can be inhibited by chlorpromazine.25 Significant TRPM endocytosis inhibition was observed in the presence of 10 μM chlorpromazine (P < 0.01), indicating that clathrinis was indeed involved in the endocytosis of TRPM. In order to prove our assumption that c[RGDfK] conjugated molecules can target the integrin overexpressed cells, we synthesized a conjugate of c[RGDfK] and BODIPY-TR (RB, m/z = 1123.4904) (Figure 5B), and the cellular uptakes of RB and BODIPY-TR were evaluated on the coculture of U-87 MG and HEK-293-EGFP cells, which express high and low level integrin, respectively.26,27 The cellular uptake of BODIPY-TR

Figure 8. Expression of integrin αv in the normal brain tissue (1), peritumoral brain tissue (2), and glioma (3) of intracranial glioma bearing mice.

showed no difference between U-87 MG and HEK-293-EGFP cells, but RB was found to preferentially accumulate in U-87 MG cells (Figure 5C). This result implies that c[RGDfK] conjugation could increase the drug accumulation in glioma after transport across the BBB thereby decreasing the neurotoxicity of drug. Cytotoxicity. Since the chemical modification of drug molecules can sometimes lead to loss of activity, the cytotoxicity of PTX, RP, PM, and RPM were evaluated on U-87 MG cells. The study showed that the antiproliferative effects of RP and RPM were not significantly different from that of PTX and PM (Figure6), indicating that the conjugation of PTX to c[RGDfK] and encapsulation of the conjugate into polymeric micelle did not affect its cytotoxicity. Cell Cycle. To further investigate whether c[RGDfK] conjugation affects the mechanisms of action of PTX, we analyzed the cell cycle distribution of U-87 MG cells using flow cytometry. Free PTX and PM were shown to induced potent G2/M arrest on U-87 MG cells, consistent with the report that disturbance of microtubule dynamics was the antitumor mechanism of PTX.28 As RP converted to PTX efficiently in the presence of esterase, the antitumor mechanism of RP should be the same as PTX. RPM induced the most potent G2/M arrest in the cell cycle, while cells treated with a mixture of c[RGDfK] and PTX (or PM) did not show any discernible 1595

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Figure 9. Biodistribution of Taxol, paclitaxel loaded micelle (PM), transferrin modified paclitaxel loaded micelle (TPM), and TRPM in intracranial U-87 MG glioma bearing mice at 1, 4, 12, and 24 h after intravenous injection (n = 3 for each group at each time point). *P < 0.05 and **P < 0.01 compared with TPM. †P < 0.05 and ††P < 0.01 compared with PM. All error bars reflect SD.

the main limitations for clinical application of PTX is its toxicity. The BWC of TRPM treated mice was similar to that of saline treated ones. On the contrary, the body weight loss was observed in Taxol, PM, and TPM treated mice since the first dose. Despite the obvious increase of drug accumulation in the liver, lungs, spleen, and kidneys, TRPM displayed substantially less toxicity than Taxol, PM, and TPM.

improvement compared to cells treated with PTX or PM alone (Figure 7). Integrin αv Expression. The expression of integrin αv in the brain of intracranial glioma bearing mice was investigated (Figure 8). U-87 MG glioma tumor were found to express a very high level of integrin αv, which was in accordance with a report by Skuli et al.26 Integrin αv was barely expressed in normal brain tissue, but the amount of integrin αv in peritumoral tissue increased due to the infiltration of U-87 MG cells.29,30 Biodistribution. To evaluate the BBB penetration and glioma targeting efficiency of TRPM in vivo, the distribution of TRPM in tumor and other tissues was investigated. The accumulation of TRPM in glioma, peritumoral, and normal brain tissue was significantly higher compared with TPM at 1 h postadministration (P < 0.01), and a longer retention of TRPM was achieved in tumor and peritumoral tissues where the high level of integrin was expressed when compared with TPM (Figure 9). The clearance of TRPM from other tissues was also the slowest among all of the formulations, indicating that c[RGDfK] conjugation affects the pharmacokinetic properties of PTX. Antiglioma Activity and Toxicity. We next examined whether increased glioma accumulation and specificity could lead to an enhanced antiglioma effect by recording the survival time of intracranial U-87 MG glioma bearing mice (Figure 10A). The mean survival times of TRPM, TPM, PM, Taxol, and saline treated mice were 42.8, 39.5, 34.8, 33.6, and 34.5 days, respectively. Compared with saline, TPM (P < 0.01) and TRPM (P < 0.01) significantly prolonged the survival time, but PM showed no therapeutic effect. TRPM was significantly superior to TPM (P < 0.01), which is rational considering its higher accumulation and longer retention in glioma and peritumoral tissue. Finally, we investigated the toxicity of TRPM in terms of body weight change (BWC) of mice (Figure 10B), as one of

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DISSCUSSION The efficacy of glioblastoma chemotherapy is hindered by low brain drug accumulation and low selectivity. Therefore, we developed a novel micelle that could penetrate the BBB and target the glioblastoma tumor subsequently by encapsulating RP into a Tf modified micelle. The first step of our research was the synthesis of RP. An important goal in drug conjugate design is to retain a suitable balance between biological stability of the drug conjugate and efficient conversion to the active parent drug in vivo.31,32 RP degraded quickly into parent drug PTX in plasma due to the hydrolysis of the ester bond between PTX and succinate (Figure 3B). However, as the linkage with c[RGDfK] is crucial for leading PTX to glioma cells, the hydrolysis would impair the targeting ability. So, we encapsulated RP into polymeric micelles. Our result confirmed the general consideration that nanocarriers could protect encapsulated drug from premature degradation (Figure 3B),33 implying that the targeting capability of RP could be preserved in physiological solution. The release kinetic of RP from micelles was similar to Cho’s report (Figure 4).34 As the hydrolysis of free RP was fast in the presence of esterase, the sustained release of RP from RPM or TRPM could help to retain a relatively high level of RP in biological fluid such as plasma and cerebrospinal fluid, which may favor the targeting of RP to the glioma. According to our result (Figure 5A), the endocytosis of TRPM was TfR-dependent and involved clathrin as the report,13,14 which was more efficient than that of RPM. 1596

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peritumoral tissue could facilitate the accumulation of c[RGDfK] modified drug and inhibit the invasion of glioma subsequently. Compared with other receptors such as LfR and LRP, which are also highly expressed in normal brain tissue,9,10 integrin provids much higher specificity. The pharmacokinetic properties of TRPM were different from TPM (Figure 9), as c[RGDfK] modification not only increases the affinity of drug to integrin overexpressed tumor but may also alter the drug interaction with transporters such as P-glycoprotein (P-gp).35 After being transcytosed across the BBB through a TfR dependent pathway, the released RP could not be exfluxed as efficiently as PTX, leading to its high accumulation in the brain. Chemical modification to PTX also slowed the excretion of the RP from mice (Figure 9), and a similar phenomenon to that observed when the biodistributions of poly-(L-γ-glutamylglutamine)-paclitaxel conjugate and linoleic acid-paclitaxel conjugate were evaluated.36,37 The in vivo antitumor activity of TRPM was the most potent among all the formulations without significant toxicity (Figure 10). Therefore, its efficacy could be improved further by escalating the dosage because it has been proved that a higher dose will result in better clinical effect when Abraxane was used for tumor chemotherapy.38 As no body weight loss was observed after the administration of TRPM, the frequency of administration could also be increased to maintain high drug concentration in the tumor, which should lead to better antitumor activity. In conclusion, we have provided a novel sequential targeting nanoscale formulation of PTX, TRPM, for targeted glioma chemotherapy. Tf conjugation significantly enhanced the ability of a hybrid micelle to transport RP into primary BMECs in vitro and brain in vivo, while c[RGDfK] modification improved the drug selectivity to integrin overexpressed glioma cells in vitro and prolonged the retention of drug in glioma and peritumoral tissue in vivo. Most importantly, TRPM significantly prolonged the survival time of intracranial glioma bearing mice without showing obvious toxicity. These results indicated that this sequential targeting nanomedicine could be promising therapeutics for future glioma treatments.

Figure 10. Antiglioma activity and toxicity of micelles. Kaplan−Meier survival curves of mice treated with saline, Taxol, paclitaxel loaded micelle (PM), transferrin modified PM (TPM), and transferrin modified c[RGDfK]-paclitaxel loaded micelle (TRPM) (n = 8 for each group) (A), and the body weight change of the treated mice (n = 8) (B). All error bars reflect SEM.*P < 0.05 and **P < 0.01 compared with TPM; ††P < 0.01 compared with saline.

However, more drug exposure in the brain will cause higher neurotoxicity if glioblastoma specificity is not achieved. BODIPY-TR was chosen as a fluorescent indicator because of its hydrophobicity and cell membrane permeability, which was much like PTX. The enhanced selectivity of TB could be attributed to the decreased cellular uptake through diffusion and increased endocytosis through integrin-mediated pathway after c[RGDfK] conjugation. RPM showed similar cytotoxicity but more potent cell cycle arrest activity compared with PTX, RP, and PM (Figures 6 and 7). The difference in incubation time may be the most possible explanation. The enhanced cell cycle arrest activity of RP may result from its increased cellular uptake through integrin-mediated endocytosis because the cell cycle arrest activity (RPM > RP > c[RGDfK] + PTX ≈ c[RGDfK] + PM) correlated closely with the content of RP in the medium (RPM > RP > c[RGDfK] + PTX = c[RGDfK] + PM). However, RP was converted into PTX efficiently, and during the 72 h incubation period, the cellular drug accumulation of the four formulations was not significantly different. The expression of integrin in the brain of mice bearing intracranial glioblastoma was shown in Figure 8, indicating a very significant difference in integrin αv expression between normal brain tissue and glioma tumor, so active targeting of RP to glioma was possible. The increased integrin αv expression in

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AUTHOR INFORMATION

Corresponding Author

*Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China. Tel/Fax: +86-21-2023-1979. E-mail: [email protected] (Y.L.); [email protected] (L.F.). Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Basic Research Program of China (2010CB934000 and 2007CB935804), the National Natural Science Foundation of China (30925041 and 30901866), and Shanghai Elitist Program (11XD1406200) are gratefully acknowledged for financial support.

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