Enhanced Intracellular Delivery and Chemotherapy for Glioma Rats by

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Enhanced Intracellular Delivery and Chemotherapy for Glioma Rats by Transferrin-Conjugated Biodegradable Polymersomes Loaded with Doxorubicin Zhiqing Pang,†,‡ Huile Gao,†,‡ Yuan Yu,§ Liangran Guo,†,‡ Jun Chen,†,‡ Shuaiqi Pan,†,‡ Jinfeng Ren,†,‡ Ziyi Wen,†,‡ and Xinguo Jiang*,†,‡ †

Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China, 201203 Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, People’s Republic of China, 201203 § Department of Pharmaceutics, School of Pharmacy, The Second Military Medical University, Shanghai, People’s Republic of China, 200433 ‡

ABSTRACT: A brain drug delivery system for glioma chemotherapy based on transferrin-conjugated biodegradable polymersomes, Tf-PO
DOX, was made and evaluated with doxorubicin (DOX) as a model drug. Biodegradable polymersomes (PO) loaded with doxorubicin (DOX) were prepared by the nanoprecipitation method (PO
DOX) and then conjugated with transferrin (Tf) to yield Tf-PO
DOX with an average diameter of 107 nm and surface Tf molecule number per polymersome of approximately 35. Compared with PO
DOX and free DOX, Tf-PO
DOX demonstrated the strongest cytotoxicity against C6 glioma cells and the greatest intracellular delivery. It was shown in pharmacokinetic and brain distribution experiments that Tf-PO significantly enhanced brain delivery of DOX, especially the delivery of DOX into brain tumor cells. Pharmacodynamics results revealed a significant reduction of tumor volume and a significant increase of median survival time in the group of Tf-PO
DOX compared with those in saline control animals, animals treated with PO
DOX, and free DOX solution. By terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling, Tf-PO
DOX could extensively make tumor cell apoptosis. These results indicated that Tf-PO
DOX could significantly enhance the intracellular delivery of DOX in glioma and the chemotherapeutic effect of DOX for glioma rats.

’ INTRODUCTION Treatment of malignant gliomas represents one of the most formidable challenges in oncology.1 Despite combined therapies of surgery, radiotherapy, and chemotherapy, the prognosis for gliomas remains very poor, especially for glioblastoma, which has a median survival of 12 to 15 months.2 Even with steady advances in drug discovery and targeted therapeutics that improve longterm control in systemic cancers, there has been little impact on the prognosis of patients with malignant gliomas.1 The most notable obstacle underlying the disappointing results in glioma therapeutics is the presence of the blood-brain barrier (BBB), which serves to prevent delivery of potentially active therapeutic compounds.3 Moreover, limited tumor cell drug uptake, intracellular drug metabolism, inherent tumor sensitivity to chemotherapy, and cellular mechanisms of resistance also contribute much to the poor effectiveness of treatment for glioma.4 Therefore, a great deal of effort is presently focused on developing strategies to effectively deliver active drugs to brain tumor cells, improving therapeutic opportunities, efficiency, and patient survival, while decreasing side effects to normal cells. Magnetic resonance imaging (MRI)guided focused ultrasound-induced BBB disruption and convection-enhanced delivery have emerged as leading investigational delivery techniques for the treatment of malignant gliomas.2,5,6 r 2011 American Chemical Society

However, expensive medical devices and potential hazards produced from repeated BBB opening or invasive neurosurgery greatly limit their application in glioma therapy. Thus, targeting drug delivery to glioma cells noninvasively using nanocarrier systems such as micelles, liposomes, nanoparticles (NPs), and dendrimers may be a better approach and has been widely investigated recently.7
9 As a new class of synthetic thin-shelled capsules based on block copolymer chemistry, polymersomes are self-assembled vesicles of amphiphilic block copolymers that currently attract great interest for their structural analogies with living organelles and potential applications as nanosized reactors or drug delivery systems.10
13 Over the past decade, polymersomes were revealed to be promising alternatives to phospholipid-based vesicles regarding their remarkable, attractive, and feasible characteristics such as stealthiness, improved stability, and ease of functionalization.13
15 Compared with liposomes (3
5 nm bilayer), polymersomes are extremely stable and robust with high membrane stability and low membrane permeability, which Received: January 30, 2011 Revised: April 17, 2011 Published: May 02, 2011 1171

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Bioconjugate Chemistry can overcome most of the stability problems (mainly drug leakage and disintegration of membrane) encountered in lipidic vesicles due to the high fluidity of their bilayers.16,17 In addition, it has been evidenced that the physical and chemical properties of polymersomes including particles size, drug loading, surface modification, and even in vivo behavior may be broadly controlled and modulated through rich diversity of block copolymer chemistries (block fraction, block architecture).13 Furthermore, desirable smart polymersomes can be designed to combine features required for drug delivery application, such as biodegradability,18,19 targeting capability,20,21 and responsiveness to biologically relevant stimuli 22 such as pH,23 temperature,24 light,25 and reductive environment.26 Therefore, polymersomes are good candidates for drug delivery carriers, which are currently being developed by many groups. However, the vast majority of present efforts are focused on the fabrication of novel functional polymersomes including biomimetic polymersomes for glioma cells treatment in vitro,27 while in vivo exploitation of biodegradable polymersomes as brain drug delivery systems has rarely been reported. The anthracycline doxorubicin, one of the most powerful anticancer agents, like most small-molecule anticancer agents, has a poor effect on glioma therapy for its inability to cross the BBB and achieve effective concentrations in the glioma cells. Although liposomal doxorubicin has been shown to be a safe treatment with moderate activity that might lead to long-term stabilization in recurrent high-grade glioma patients,28 its poor blood-brain barrier penetration, limited intracellular delivery, and instability are still major limitations in the treatment of malignant glioma. By a secondary targeting strategy,29 liposomes conjugated with two different targeting ligands might possess the ability to traverse the BBB and reach the secondary target (glioma cell), greatly improving gene therapy for glioma rats.30 However, the preparation of two ligand-conjugated liposomes might be rather complicated and uncontrollable. Moreover, the high density of different surface ligands on liposomes might not only increase the possibility to opsonization and accelerate the blood clearance,31 but also aggravate the instability of liposomes. As promising alternatives to liposomes, biodegradable poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) polymersomes functionalized with lactoferrin were proven to cross BBB and accumulate at the glioma site in our previous report.32 However, the large vesicle size above 200 nm might accelerate the blood clearance and minimize the likelihood of crossing the BBB.33 In addition, insufficient intracellular delivery also decreased the doxorubicin concentration in glioma cells. Thus, biodegradable polymersomes with better vesicle properties such as rational size and surface modification are still necessary for effective glioma targeting delivery. In this study, doxorubicin, as a model small-molecule anticancer agent, was encapsulated into biodegradable PEG-PCL polymersomes with small vesicle size. As a classic both BBB targeting and tumor targeting ligand,34 transferrin, was conjugated to the surfaces of polymersomes to prepare “Trojan horse” (Tf-PO
DOX) for glioma drug delivery. A single targeting ligand that could mediate both transport across the BBB and internalization into glioma cells represented the simplest approach for secondary targeting delivery. Enhanced intracellular delivery and glioma drug delivery were investigated in glioma bearing rats and then verified by pharmacodynamics tests including tumor volume, survival monitoring, and tumor cell apoptosis.

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’ MATERIALS AND METHODS Materials and Animals. The MPEG3k-PCL15.8k or Maleimide-PEG3.4k-PCL16k diblock copolymers were synthesized by ring-opening polymerization of ε-CL using MPEG (Mw 3000 Da, NOF Corporation, Japan) or Maleimide-PEG (Mw 3400 Da, NEKTAR, USA) as described elsewhere.35 Doxorubicin (DOX) and daunorubicin was purchased from Beijing Huafeng United Technology (China) and National Institute for the Control of Pharmaceutical and Biological Products (China), respectively. Holo-transferrin, 2-iminothiolane (Traut’s reagent), and phenylmethyl sulfonyl fluoride (PMSF) were ordered from Sigma and 5,5-dithiobis (2-nitrobenzoicacid) (Ellmann’s reagent) was from Acros (Belgium). Human transferrin ELISA quantitation kit was obtained from Bethyl (Montgomery, TX, USA). NeuroTACS II In Situ Apoptosis Detection Kit was purchased from R&D systems (Minneapolis, MN). C6 glioma cell line from rat was obtained from the American Type Culture Collection. Fetal bovine serum (FBS) and related cell culture medium were purchased from Invitrogen (Gibco, Carlsbad, CA). Plastic cell culture dishes, plates, and flasks were ordered from Corning Incorporation (Lowell, MA). Double distilled water was purified using a Millipore Simplicity System (Millipore, Bedford, MA, USA). All the other chemicals were analytical reagent grades and used without further purification. Wistar strain rats (weighing 200
230 g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The animals involved in this study were treated according to protocols evaluated and approved by the ethical committee of Fudan University. Preparation of Transferrin Conjugated Biodegradable Polymersomes. Biodegradable polymersomes were prepared with a blend of MPEG-PCL and Maleimide-PEG-PCL by the nanoprecipitation method.12 Briefly, 20 mg of MPEG-PCL and 1 mg of Maleimide-PEG-PCL were dissolved in 1.0 mL of tetrahydrofuran (THF). The polymer solution was then injected slowly into 10 mL of 0.2 M citrate buffer (pH 4.0). Mild stirring was performed to induce self-assembly for 30 min. To remove THF and exchange the external buffer, the solution was dialyzed against 0.01 M PBS buffer (pH 7.4) for 12 h using dialysis membranes (MW cutoff, 14 000 Da) and the buffer solution is replaced by fresh PBS every 4 h. For DOX loading, 2 mg of DOX was added and the mixed solution was incubated in the dark at 40 °C for 24 h. By means of ultrafiltration, unencapsulated DOX was eluted and the vesicle dispersion was concentrated to yield DOX-loaded polymersome (PO
DOX). Transferrin conjugation with DOX-loaded polymersomes was made as described previously.35 In brief, transferrin was thiolated using 2-iminothiolane (Traut’s reagent), then incubated with polymersomes for 6 h with a 1:3 molar ratio of thiolated transferrin to maleimide. By means of ultrafiltration again, unconjugated protein was removed and Tf-conjugated DOX-loaded polymersome (TfPO
DOX) solution was concentrated. All operations were conducted away from light. Characterization of Tf-Conjugated DOX-Loaded Polymersome. The formation of blank polymersomes was visualized by cryo-transmission electron micrograph (Cryo-TEM, JEOL 2010, Japan).35 The shape and size of unloaded polymersomes were observed by transmission electron microscope (TEM, JEM1230, JEOL, Japan) following negative staining with 1% uranyl acetate solution. The mean diameters and zeta potential of TfPO
DOX were determined by dynamic light scattering (DLS) using a Zeta Potential/Particle Sizer NICOMP 380 ZLS (PSS. 1172

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Bioconjugate Chemistry NICOMP Particle Size System, Santa Barbara, CA, USA). Surface transferrin densities were determined by ELISA method combined with turbidimetry method as previously described.35 Drug loading efficiency and in vitro release of DOX from TfPO
DOX was determined using fluorescence spectrometry (Ex: 480 nm and Em: 580 nm). In the latter, drug loaded polymersomes were suspended in a dialysis system which was monitored by periodic withdrawals of drug released into 0.01 M PBS (pH 4 and 7.4) or 10% rat plasma in PBS.36 Cell Uptake and Intracellular Drug Distribution of DOXLoaded Polymersomes. A 24-well plate was seeded with C6 cells at a density of 105 per well, and the cells were allowed to attach for 24 h. Dispersions of PO
DOX and Tf-PO
DOX as well as free DOX solution were each added into the medium in triplicate to make a final DOX concentration at 4 μg/mL in each well. After 4 h incubation at 37 °C, the medium was removed and the cells were stained with 1 μg/mL of 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 10 min at room temperature. After PBS washing, the plate was subjected to observation under the fluorescence microscope (Olymus IX71, Japan). Quantitative Determination of Cell Uptake of DOXLoaded Polymersomes. Similarly to the above, C6 cells were seeded onto 24-well plates at a density of 105 per well, and the cells were allowed to attach for 24 h. Three groups of DOX preparations were respectively added into the medium to make a final DOX concentration at 4 μg/mL in each well (triplicate for each formulation). The plates were incubated for 1, 2, 4, 8, or 12 h at 37 °C. For each time point, cells in each well of the corresponding plate were washed three times with ice-cold PBS to remove surface-bound polymersomes and further incubated with 0.4 mL of 1% Triton X-100 at 4 °C overnight. Afterward, 25 μL of the cell lysate from each well was sampled to determine the total cell protein content using the BCA protein assay kit. The rest of the cell lysate was used for extraction and HPLC determination of DOX (Agilent 1200) with fluorescence detector at an excitation wavelength of 480 nm and an emission wavelength of 550 nm.37 The uptake index (UI) was expressed as DOX (μg)/ cellular protein (mg). Intracellular Drug Distribution. Intracellular drug distribution studies were carried out as previously described with a little modification.38 Briefly, C6 cells were seeded onto 6-well plates at 106 cells per well and allowed to attach for 12 h. Three groups of DOX preparations were respectively added into the medium to make a final DOX concentration at 4 μg/mL in each well (triplicate for each formulation). The plates were incubated for 1, 2, 4, 8, or 12 h at 37 °C. For each time point, cells in each well of the corresponding plate were washed three times with ice-cold PBS to remove surface-bound polymersomes and further collected by centrifugation at 3000 rpm for 5 min after cell dissociation. The resulting cell pellets were washed thrice with 3 mL ice-cold PBS, resuspended in 300 μL TM-2 buffer (0.01 M Tris
HCl, pH 7.4, 0.002 M MgCl2, 0.0005 M PMSF), and incubated at room temperature for 1 min followed by ice water for 5 min. Triton X-100 was added to the suspension to a final concentration of 1% (v/v) and the suspension was incubated in ice water for an additional 5 min. After centrifugation at 1000 rpm at 4 °C for 10 min, the supernatant was transferred to determine the drug amount in cytoplasm and the isolated pellet was resuspended in 0.5 mL of TM-2 buffer for determination of the drug levels in nuclei. The drug levels of both cytoplasm and nuclei were determined by HPLC method as described above. The corresponding protein concentrations

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were measured using BCA protein assay kit. The uptake index (UI) was expressed as DOX (μg)/cellular protein (mg). In Vitro Cytotoxicity of DOX-Loaded Polymersomes against C6 Cells. C6 cells were seeded onto a 96-well plate at a density of 104 per well and incubated for 24 h before exposure to different DOX formulations with a series of concentrations for a further 24 h at 37 °C. Eight wells for untreated cells were prepared as controls. After exposure, the cytotoxicity of these formulations was assayed by the MTT method. The experiments were performed in quadruplicate. Tumor Implantation. Male rats were maintained at 22 ( 2 °C on a 12 h light
dark cycle with access to food and water ad libitum. For tumor implantation, animals were anesthetized with chloral hydrate (0.4 g/kg) intraperitoneally and then subjected to stereotaxic injection of approximately 3  105 of C6 cells (in 5 μL of RPMI-1640 medium) into the caudate nucleus region.39 The injection location was 1 mm posterior and 2 mm right to the bregma, and 4.5 mm beneath the exposed dura mater. The burr hole in the skull was sealed with bone wax and the scalp incision was sutured. Pharmacokinetics and Tissue Distribution of Tf-PO
DOX. Animal experiments were performed as previously described.32 In brief, 12 days after tumor implantation, male rats were randomly divided into 3 groups for pharmacokinetics or tissue distribution studies and administrated intravenously with a dose of 5 mg/kg DOX in solution, PO and Tf-PO. For pharmacokinetics study, blood samples were collected from the retro-orbital plexus at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 h following i.v. injection. At 24 h, the animals were decapitated and the brain tissues including cerebral cortex, left striatum, and right striatum (tumor tissue) were excised followed by quick washing with cold saline to remove surface blood. Blood sample and major organs including liver, spleen, kidney, lung, and heart were also collected. For tissue distribution at 4 h, the animals were decapitated and the brain tissues, blood sample, and other major organs were collected as described above. Fluorescent imaging of brain and other major organs was investigated by a CRi in vivo fluorescent imaging system (CRI, Woburn, MA, USA) consisting of a highly sensitive Peltier cooled backlit CCD camera (2184  1472 pixels). In brief, the organs were placed in the light-tight chamber, the filter for emission was set at 550 nm, respectively, and the photon emission was integrated over a period of 5 s. After that, a grayscale reference image was collected under white light. For localization of the photon emission, grayscale and pseudocolor images were merged using WinLight 32 software. HPLC analysis was performed for determining levels of DOX in biological specimen.37 In brief, the tissue samples were homogenized on ice with 3-fold volumes of deionized water followed by addition of 10 μL of daunorubicin (internal standard, 10 μg/mL). The samples were extracted with 2 mL of chloroform/methanol (4:1, v/v) following intense vortexing for 2 min. After centrifugation at 4000 g for 10 min, the subnatant was transferred and evaporated under a gentle stream of nitrogen at 40 °C. The residue was dissolved in 200 μL of methanol, and 20 μL was injected into the HPLC system. The blood plasma samples were extracted without the addition of water and reconstituted in the same manner. The chromatographic analysis was performed on the HPLC system (Shimadzu Scientific Instrument Inc., Japan) including a pump (model LC-10AT) and a fluorescence detector (model RF-10AXL; λex = 480 nm, λem = 550 nm). Using potassium dihydrogen phosphate/ 1173

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Figure 1. (A) Cryo-TEM images of polymersomes in water (bar 100 nm). The hydrophobic cores of polycaprolactone are the darker areas. (B) TEM images of polymersomes negatively stained with 1% uranyl acetate solution (bar 200 nm).

acetonitrile/glacial acetic acid (70:30:0.3, v/v) with pH 3.0 as the mobile phase at a flow rate of 1.2 mL/min, chromatographic separations were achieved with a C18 analytical column (5 μm, 4.6  200 mm, Diamonsil, Dikma) equipped with a guard column (Nova-Pak, Waters) at 35 °C. The retention time of DOX and internal standard (daunomycin) was about 3.7 and 8.2 min, respectively. For data analysis, the plasma data were fitted to a biexponential equation: A(t) = A1e
k1t þ A1e
k2t, where A(t) was %ID per mL of plasma (%ID, percentage of injected dose).40 Pharmacokinetic parameters, such as plasma clearance (Cl) and steady-state volume of distribution (Vss), were calculated from A1, A2, k1, and k2. Area under the blood concentration curve (AUC) was calculated by noncompartmental data analysis of blood concentrations. The brain permeability surface area (PS) product and brain uptake (expressed as %ID per g of tissue) were calculated as described elsewhere.41 Antitumor Efficacy in Rats Bearing Glioma. Tumor-bearing animals were equally and randomly divided into 4 groups, which received saline, DOX solution, PO
DOX, and Tf-PO
DOX, respectively. These preparations were i.v. injected at 2, 5, 8, and 11 days after glioma implantation, dosing at 1.5 mg/kg DOX.32,42 On the 7th and 14th days, 6 rats per group were randomly sacrificed. Heart perfusion with 10% neutral buffered formalin was conducted after sacrifice, and the brain tissue sample was collected and fixed with 10% neutral buffered formalin for at least 48 h and then embedded in paraffin followed by routine hematoxylin and eosin (H&E) staining on 5-μm-thick sections using routine protocols.43 The maximum diameter (a) and minimum diameter (b) of glioma tissue was measured under stereomicroscope, and the tumor volume (V) in each animal was calculated as V = 0.5ab2. Paraffin-embedded brain tumor tissue sections (5 μm) were also prepared for apoptosis detection by Tdt-mediated dUTP nick-end labeling (TUNEL) according to the protocol of NeuroTACS II In Situ Apoptosis Detection Kit. Samples were counterstained with hematoxylin while positive signals were developed by diaminobenzidine (DAB). Twelve tumor-bearing rats of each group were followed by survival monitoring. Survival data were analyzed with log-rank test in a Kaplan
Meier nonparametric analysis and summarized descriptively by means of median survival times with their respective 95% confidence intervals. Statistical Analysis. Statistical differences in uptake index, pharmacokinetic parameters, and tumor volume were determined by one-way analysis of variance (ANOVA), followed

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Figure 2. Cumulative releases (%) of doxorubicin from Tf-PO
DOX in different media.

by post hoc analysis of Bonferroni for multigroup comparison. P < 0.05 was considered significant.

’ RESULTS Characterization of DOX-Loaded Polymersomes. CryoTEM results revealed that a blend of MPEG3k-PCL15.8k and Maleimide-PEG3.4k-PCL16k spontaneously assembled into polymersomes which were generally round with a diameter of approximately 100 nm. Moreover, the hydrophobic core of the membrane provided the contrast, and the membrane thickness was homogeneous with a measured width d of about 16 nm (Figure 1A), which is considerably greater than any previously studied lipid system. The self-assembled bilayers was also thicker than that we previously reported (10 nm)35 due to the higher molecular weight of block polymer PCL, as this is clear and already has been demonstrated.44 The TEM examination demonstrated that polymersomes were of vesicle-like shape with averaged diameters around 100 nm (Figure 1B), in good accordance with DLS results. A slight diameter increase was observed following DOX loading and Tf conjugation. The final average diameters of PO
DOX and Tf-PO
DOX were 101.8 ( 9.2 nm and 107.7 ( 11.3 nm, respectively. The zeta potential values of PO
DOX and Tf-PO
DOX in 0.001 M NaCl solution were
9.1 ( 0.6 mV and
9.3 ( 0.5 mV, respectively, indicating Tf conjugation had no influence on the zeta potential value of polymersomes. Drug loading capacity of DOX in the polymersomes was about 4.4% with entrapment efficiency above 96%. The mean Tf molecule numbers per polymersome on the surface of Tf-PO
DOX was 35.1 ( 6.2, which was a reasonable density for brain targeting justified in other studies.41,45 As shown in Figure 2, under three releasing conditions, the release profile of DOX from Tf-PO
DOX displayed a biphasic pattern that was characterized by a first rapid release (∼17% of DOX was released within 4 h) followed by a slower and sustained release. After a 48 h incubation period, about 34.3%, 24.1%, and 38.4% of DOX were released from Tf-PO
DOX in pH 4.0, 7.4 PBS, and rat plasma, respectively. DOX was released much more rapidly in pH 4.0 than in pH 7.4 PBS, which might be attributed to acid-catalyzed hydrolysis of the PCL membrane46 and increased hydrophilicity of DOX in acid condition.47 The cumulative release of DOX from Tf-PO
DOX at 8 h in rat plasma was below 25% indicating that DOX entrapped in Tf-PO might be mostly preserved during the brain uptake process of TfPO
DOX. 1174

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Figure 3. Cell uptake and intracellular distribution of free DOX, PO
DOX, and Tf-PO
DOX (4 μg/mL DOX) in C6 cell line from 1 to 12 h. Relative uptake in cell (A), cytoplasm (B), and nuclei (C). (D) C6 viability inhibition-logarithmic doxorubicin concentration curve of DOX loaded polymersomes.

Figure 4. Fluorescence photographs of C6 cells after incubation for 4 h with free DOX, PO
DOX, and Tf-PO
DOX (4 μg/mL DOX).

Cell Uptake and Intracellular Distribution. Uptake of DOXloaded polymersomes by C6 cells increased with time (Figure 3A).

Tf-PO
DOX accumulated within the cells at a faster rate and to a larger extent than PO
DOX. After 12 h incubation, the UI for 1175

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Figure 5. Pharmacokinetics and brain delivery of doxorubicin-loaded polymersomes. (A) %ID/mL of plasma of doxorubicin is plotted versus various times after i.v. injection of 5 mg/kg of doxorubicin solution, doxorubicin loaded Tf-PO, and PO in C6 glioma transplanted rats. (B) Permeability surface area (PS) product and (C) brain uptake of doxorubicin at 4 h after intravenous injection in C6 glioma transplanted rats. (D) Fluorescent imaging of main organs at 4 h after i.v. injection of 5 mg/kg of doxorubicin solution, PO
DOX, and Tf-PO
DOX in C6 glioma transplanted rats. Data represent means ( SD (n = 4). *P < 0.05, **P < 0.01 vs Tf-PO
DOX group. Data are means ( SD of n = 4 rats/point.

Tf-PO
DOX and PO
DOX were 4.8 and 3.3 μg/mg, respectively. The uptake of polymersomes in cytoplasm presented an initial rapid increase before 4 h (Figure 3B). After that, the uptake curve for polymersomes showed a slow decrease and tended to reach a plateau as the incubation time prolonged. A low cytoplasmic uptake of free DOX was obviously observed during the incubation period. In nuclei, the uptake for DOX-loaded polymersomes increased with time, while the uptake for free DOX rapidly reached the highest value at 4 h (Figure 3C). By 12 h, the uptake for Tf-PO
DOX into nuclei was significantly more than that for PO
DOX (P < 0.01), which might be attributed to higher cytoplasmic uptake for Tf-PO
DOX. MTT assay on C6 cells viability showed that the cytotoxicity of Tf-PO
DOX and PO
DOX depended on their concentration. Viability
concentration curves (Figure 3D) demonstrated that the IC50 (DOX concentration of 50% inhibition) of these two formulations was 3.6 and 13.5 μg/mL, respectively, manifesting a 3.75 intensification of cell inhibition of Tf-PO
DOX compared to PO
DOX. The results indicated a significantly improved cytotoxicity mediated by Tf, which was in agreement with the higher intracellular delivery and higher nuclei uptake of Tf-PO
DOX than those of PO
DOX as presented above. Intracellular distribution of polymersomes into C6 cells was also demonstrated by fluorescence microscopy photographs in which red fluorescence represented DOX and blue fluorescence referred to nuclei stained with DAPI (Figure 4). For free DOX, the red fluorescence nearly completely overlaid with the blue fluorescence, indicating that free DOX diffused into the cells and mainly concentrated at the nucleus, which agreed well with the

results of quantitative determination. With Rregard to polymersomes, the same as the quantitative results, the red DOX fluorescence distributed mainly in the cytoplasm of cells except for partly in the nucleus, indicating that cell uptake of DOX-loaded polymersomes was not through the same diffusion mechanism as free DOX, but in a different way, possibly by endocytosis. Pharmacokinetics and Tissue Distribution of Tf-PO
DOX. As shown in Figure 5 A, the blood clearance of DOX for free DOX, PO
DOX, and Tf-PO
DOX in rats occurred in a biexponential manner. In the group of free DOX, disappearance of DOX from the blood circulation was very rapid with the plasma concentration of only 0.19%ID/mL at 15 min. On the contrary, the plasma DOX concentration for PO
DOX and Tf-PO
DOX remained high for a long time. Even at 24 h after administration, the plasma DOX concentrations for PO
DOX and TfPO
DOX were 0.06%ID/mL and 0.03%ID/mL, which were 11.9-fold and 6.3-fold that for free DOX, respectively, exhibiting a remarkable prolonged plasma clearance. Pharmacokinetic parameters calculated from Figure 5 revealed that Tf-PO
DOX and PO
DOX significantly decreased the plasma clearance (Cl) of DOX from 1038 mL/h/kg to 101 and 76 mL/h/kg, respectively (P < 0.01). Meanwhile, AUC increased by 19.1- and 24.8-fold as compared to free DOX. However, there was no significant difference in Cl and AUC between Tf-PO
DOX and PO
DOX (P > 0.05), indicating that surface modification of Tf on polymersomes did not significantly change the blood clearance of polymersomes. In order to investigate the brain delivery property of TfPO
DOX, brain uptake and PS product of brain tissue including 1176

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Figure 6. Antitumor effects of DOX-loaded polymersomes in gliomabearing rats. (A) Glioma volume of different therapeutic groups at the 7th and 14th days (n = 4). ** P < 0.01 vs Tf-PO
DOX group. (B) Percentage of survival (Kaplan
Meier plot) of C6 glioma rat models.

cortex, left striatum, and glioma were quantitatively analyzed (Figure 5B,C). Although free DOX has the highest PS value of 0.040 mL/h/g (Figure 5B), the average brain uptake for free DOX was only around 0.007%ID/g (Figure 5C), which might be attributed to the rapid plasma clearance of free DOX. For free DOX, there was no significant difference of PS value and tissue uptake between cortex, left striatum, and glioma. For PO
DOX, despite the marked increase in plasma AUC, the BBB PS value was reduced to almost zero, resulting in poor uptake in brain cortex and left striatum. The glioma uptake of PO
DOX was as 2.4-fold that of free DOX, which might be due to the EPR effect of tumor tissue. Conversely, the use of Tf conjugated polymersomes significantly increased the PS value and brain uptake (P < 0.01). As compared to PO
DOX, the PS values of TfPO
DOX for cortex, left striatum, and glioma at 4 h were 3.7fold, 3.8-fold, and 3.2-fold those of PO
DOX, respectively. The tissue uptake of Tf-PO
DOX for cortex, left striatum, and glioma at 4 h were 3.6-fold, 3.6-fold, and 3.0-fold those of PO
DOX, respectively. Although the PS value was only 37% that of free DOX, the average %ID/g glioma tissue for Tf-PO
DOX was 0.053%, which increased by 7.1-fold compared with free DOX, suggesting that Tf-PO might be a promising carrier for brain drug delivery, especially for glioma drug delivery. Brain delivery and tissue distribution of DOX-loaded polymersomes were also determined by fluorescent imaging of main organs at 4 h after i.v. injection of 5 mg/kg of doxorubicin solution, PO
DOX, and Tf-PO
DOX in C6 glioma transplanted rats. As shown in Figure 5D, although PO
DOX showed intensified fluorescence intensity at the glioma site compared with free

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DOX, Tf-PO
DOX group demonstrated the highest brain fluorescence intensity especially at the glioma site among three groups, which agreed well with quantitative results presented above. DOX distribution in other tissues showed that polymersomes accumulated most in the liver but little in the heart, suggesting that polymersome DOX might have similar potential as Doxil (liposomal doxorubicin) to decrease heart toxicity of DOX. Pharmacodynamics Experiments in Glioma Model Rats. The antitumor effects of DOX-loaded polymersomes in gliomabearing rats were shown in Figure 6. The Tf-PO
DOX group presented the smallest glioma volume for rats sacrificed on the same day (Figure 6A), especially on the 14th day. On day 14, the tumor volume of the Tf-PO
DOX group was significantly lower than that of any other group (P < 0.01), indicating that TfPO
DOX treatment effectively inhibited the rapid growth of glioma. Life-span extension treated with a multidose of 1.5 mg/ kg DOX on days 2, 5, 8, and 11 after glioma implantation was shown in Figure 6B. By log-rank test (Table 1), the median survival times of two polymersome groups were all significantly prolonged compared with that of saline control. However, the increase in survival times (IST) of the Tf-PO
DOX group was more considerable when compared with any other group (P < 0.05), which reached nearly 100% and 70% life-span extension compared with the saline control and free DOX group, respectively, indicating that Tf-PO
DOX possessed the most powerful antitumor activity. The TUNEL assay further detected DNA fragmentation, a marker of late apoptosis, in nuclei of tumor cells on days 7 and 14 following chemotherapy with 4 different formulations (Figure 7). As shown in Figure 7D, for rats treated with Tf-PO
DOX, significant apoptotic responses of tumor cells in the periphery of tumor were obviously observed on day 7. However, little apoptosis was detected in any tumor tissues treated by free DOX and PO
DOX when compared with saline control (Figure 7B,C). Furthermore, much more apoptotic tumor cells in Tf-PO
DOX treated tumor tissues after 14 days (Figure 7H) were detected compared with any other treatment (Figure 7), although many were presented in PO
DOX treatment.

’ DISCUSSION The thin film hydration method was commonly used to prepare biodegradable polymersomes; however, the hydration process needed a high temperature for a long time,46 which might cause hydrolysis of polyesters block and unfavorable formation of polymersomes. The relative broad size distribution and large vesicle size32,46 were also unfavorable defects for in vivo drug delivey.48 Thus, in this study, the nanoprecipitation method was applied to prepare polymersomes, and small-sized polymersomes (107 nm) with a narrow size distribution (polydispersity index, PDI < 0.16 as determined from DLS) were formed, which agreed well with previous reports.19 Compared with our previously reported large polymersomes (220 nm), small-sized polymersomes with the same zeta potential showed a higher cell uptake by C6 cells than that (1.5 μg/mg) for large polymersomes,32 indicating that C6 cells favored the uptake of smaller polymersomes. Moreover, blood clearance of small-sized polymersomes was slower than that (156 mL/h/kg, unpublished data) for large polymersomes. These results agreed with previous research that particle size played a key role in cell uptake of stealth particles. For macrophage, large particle sizes were phagocytized more 1177

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Table 1. Median Survival Time for Glioma Implanted Rats of Different Therapeutic Groups (n = 12) groupa

median

standard error

95% confidence interval

Log-rank test

ISTc

ISTs

Control

17.0

1.3

14.5
19.5

-

-

-

Free DOX

20.0

3.0

14.1
25.9

PO
DOX

24.0

2.5

19.0
29.0

b

41.2

Tf-PO
DOX

34.0

3.0

c,d,e

28.1
39.9

100.0

70.0

Dosage of DOX is 4  1.5 mg/kg. P < 0.05, P < 0.01 vs control. P < 0.01 vs DOX solution. P < 0.05 vs PO
DOX. The increases in survival times (%) are compared to control (ISTC) or to DOX solution (ISTS). a

b

c

d

e

Figure 7. TUNEL staining of tumor tissue sections obtained from rat models 7 days (upper row) and 14 days (lower row) post C6 implantation, following i.v. injection of 1.5 mg/kg DOX on days 2, 5, 8, and 11. Saline control group, (A) and (E); Free DOX treatment, (B) and (F); PO
DOX treatment, (C) and (G); Tf-PO
DOX treatment, (D) and (H). Positive signals were developed by DAB. Cell nuclei were counterstained with hematoxylin.

efficiently while nonphagocytic uptake favored smaller particles.49 Thus, small-sized polymersomes might have the advantages of both decreasing blood clearance and increasing intracellular delivery to glioma cells. Considering that chemical and physical properties of the nanoparticles, including size, surface charge, and surface chemistry, are important factors that determine their pharmacokinetics (PK) and biodistribution, it was of significance to control vesicle size of polymersomes to around 100 nm, which is most optimal for improving the PK of polymersomes48 and advantageous for endocytosis by brain capillary endothelial cells.41,50 The zeta potential of drug-loaded polymersomes ranged from
10 to
9 mV, which agreed well with the optimal zeta potential within 10 mV, and might help to decrease MPS uptake and prolong blood circulation compared to the charged ones.31,49 Moreover, the fully hydrophilic PEG (100%) content on the surface of polymersomes could confer good resistance to opsonization and extend vesicle circulation times.13 Therefore, in this study, Tf-PO
DOX and PO
DOX greatly decreased the plasma clearance of DOX and increased AUC, which might be attributed to the optional particle size, surface charge, and surface chemistry of biodegradable polymersomes. Inclusion of active targeting ligands in nanosized drug delivery systems was usually used to increase the rate of intracellular delivery and bioavailability of the drug.31 In this study, Tf molecules with moderate density were conjugated to the polymersome surface to increase both the BBB permeability and intracellular delivery to glioma cells. In fact, despite the fact that PO
DOX could intensively increase the plasma AUC, the brain uptake was very low because of its poor BBB permeability. On the contrary, as compared to PO
DOX, Tf conjugated polymersomes significantly increased the BBB permeability and brain

uptake by 3.7-fold and 3.6-fold, respectively, indicating that Tf modification with moderate density greatly facilitated the delivery of polymersomes across BBB. To create pharmacological efficacy, a major challenge was that the encapsulated drug must be delivered and released to the target cells. Although in vitro release of DOX from polymersomes was relatively slow, cell uptake and intracellular distribution results revealed that surface Tf conjugation to polymersomes significantly improved cell uptake and nuclei uptake of DOX, manifesting a significantly enhanced cytotoxicity against C6 cells. Moreover, the enhanced cytotoxicity was consistent with the in vivo efficacy of the Tf-PO
DOX in glioma rats. Compared with PO
DOX and free DOX, TfPO
DOX exhibited the most powerful antitumor activity by reducing the tumor volume, increasing median survival time of glioma rats, and extensively producing tumor cell apoptosis. The effect might be attributed to three factors as follows. First, optional chemical and physical properties of the polymersomes decreased the plasma clearance of DOX in the circulation and increased the possibility to cross BBB. Second, the Tf conjugation facilitated the BBB delivery of polymersomes and increased intracellular delivery of DOX in glioma cells, especially in nuclei, where its cytotoxicity was exerted. Finally, EPR effects at advanced stages of solid brain tumor also contributed much to accumulation of polymersomes in glioma.51

’ AUTHOR INFORMATION Corresponding Author

*Tel.: þ86-21-5198-0067; Fax: þ86-21-5198-0069. E-mail address: [email protected]. Postal address: Department of Pharmaceutics, School of Pharmacy, Fudan University (Zhangjiang Campus). 826 Zhangheng Rd, Shanghai 201203, P.R. China. 1178

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’ ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 Program) 2007CB935800, National Science and Technology Major Project 2009ZX09310-006, National Natural Science Foundation of China (30762544), and the young teacher’s initiative foundation of Fudan University. The authors acknowledge Dr. Kunpeng Li, School of Life Sciences, Zhongshan University, China, for his precious help on Cryo-TEM of polymersomes. ’ REFERENCES (1) Orive, G., Ali, O. A., Anitua, E., Pedraz, J. L., and Emerich, D. F. (2010) Biomaterial-based technologies for brain anti-cancer therapeutics and imaging. Biochim. Biophys. Acta 1806, 96–107. (2) Bidros, D. S., and Vogelbaum, M. A. (2009) Novel drug delivery strategies in neuro-oncology. Neurotherapeutics 6, 539–46. (3) Juillerat-Jeanneret, L. (2008) The targeted delivery of cancer drugs across the blood-brain barrier: chemical modifications of drugs or drug-nanoparticles? Drug Discovery Today 13, 1099–106 (4) Bredel, M., and Zentner, J. (2002) Brain-tumour drug resistance: the bare essentials. Lancet Oncol. 3, 397–406. (5) Hynynen, K. (2008) Ultrasound for drug and gene delivery to the brain. Adv. Drug Delivery Rev. 60, 1209–17. (6) Liu, H. L., Hua, M. Y., Chen, P. Y., Chu, P. C., Pan, C. H., Yang, H. W., Huang, C. Y., Wang, J. J., Yen, T. C., and Wei, K. C. (2010) Bloodbrain barrier disruption with focused ultrasound enhances delivery of chemotherapeutic drugs for glioblastoma treatment. Radiology 255, 415–25. (7) Deeken, J. F., and Loscher, W. (2007) The blood-brain barrier and cancer: transporters, treatment, and Trojan horses. Clin. Cancer Res. 13, 1663–74. (8) Caruso, G., Raudino, G., Caffo, M., Alafaci, C., Granata, F., Lucerna, S., Salpietro, F. M., and Tomasello, F. (2010) Nanotechnology platforms in diagnosis and treatment of primary brain tumors. Recent Pat. Nanotechnol. 4, 119–24. (9) Ding, H., Inoue, S., Ljubimov, A. V., Patil, R., Portilla-Arias, J., Hu, J., Konda, B., Wawrowsky, K. A., Fujita, M., Karabalin, N., Sasaki, T., Black, K. L., Holler, E., and Ljubimova, J. Y. (2010) Inhibition of brain tumor growth by intravenous poly (beta-L-malic acid) nanobioconjugate with pH-dependent drug release [corrected]. Proc. Natl. Acad. Sci. U.S.A. 107, 18143–8. (10) Chen, Q., Schonherr, H., and Vancso, G. J. (2009) Blockcopolymer vesicles as nanoreactors for enzymatic reactions. Small 5, 1436–45. (11) Christian, D. A., Cai, S., Bowen, D. M., Kim, Y., Pajerowski, J. D., and Discher, D. E. (2009) Polymersome carriers: from selfassembly to siRNA and protein therapeutics. Eur. J. Pharm. Biopharm. 71, 463–74. (12) Meng, F., Engbers, G. H., and Feijen, J. (2005) Biodegradable polymersomes as a basis for artificial cells: encapsulation, release and targeting. J. Controlled Release 101, 187–98. (13) Discher, D. E., Ortiz, V., Srinivas, G., Klein, M. L., Kim, Y., Christian, D., Cai, S., Photos, P., and Ahmed, F. (2007) Emerging applications of polymersomes in delivery: from molecular dynamics to shrinkage of tumors. Prog. Polym. Sci. 32, 838–857. (14) Discher, D. E., and Eisenberg, A. (2002) polymer vesicles. Science 297, 967–973. (15) Du, J., and O’Reilly, R. K. (2009) Advances and challenges in smart and functional polymer vesicles. Soft Matter 5, 3544–3561. (16) Photos, P. J., Bacakova, L., Discher, B., Bates, F. S., and Discher, D. E. (2003) Polymer vesicles in vivo: correlations with PEG molecular weight. J. Controlled Release 90, 323–34. (17) Upadhyay, K. K., Agrawal, H. G., Upadhyay, C., Schatz, C., Le Meins, J. F., Misra, A., and Lecommandoux, S. (2009) Role of block copolymer nanoconstructs in cancer therapy. Crit. Rev. Ther. Drug 26 (2), 157–205.

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