Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs

COREL-07612; No of Pages 13 Journal of Controlled Release xxx (2015) xxx–xxx

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Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs Chuan Yang a,1, Shao Qiong Liu a,1, Shrinivas Venkataraman a, Shu Jun Gao a, Xiyu Ke a, Xin Tian Chia a, James L. Hedrick b, Yi Yan Yang a,⁎ a b

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA

a r t i c l e

i n f o

Article history: Received 26 November 2014 Received in revised form 20 March 2015 Accepted 21 March 2015 Available online xxxx Keywords: Star-like polycarbonate Vesicles Doxorubicin Biodistribution Anti-tumor activity

a b s t r a c t Amphiphilic polycarbonate/PEG copolymer with a star-like architecture was designed to facilitate a unique supramolecular transformation of micelles to vesicles in aqueous solution for the efficient delivery of anticancer drugs. The star-shaped amphipilic block copolymer was synthesized by initiating the ring-opening polymerization of trimethylene carbonate (TMC) from methyl cholate through a combination of metal-free organocatalytic living ring-opening polymerization and post-polymerization chain-end derivatization strategies. Subsequently, the self-assembly of the star-like polymer in aqueous solution into nanosized vesicles for anti-cancer drug delivery was studied. DOX was physically encapsulated into vesicles by dialysis and drug loading level was significant (22.5% in weight) for DOX. Importantly, DOX-loaded nanoparticles self-assembled from the star-like copolymer exhibited greater kinetic stability and higher DOX loading capacity than micelles prepared from cholesterol-initiated diblock analogue. The advantageous disparity is believed to be due to the transformation of micelles (diblock copolymer) to vesicles (star-like block copolymer) that possess greater core space for drug loading as well as the ability of such supramolecular structures to encapsulate DOX. DOX-loaded vesicles effectively inhibited the proliferation of 4T1, MDA-MB-231 and BT-474 cells, with IC50 values of 10, 1.5 and 1.0 mg/L, respectively. DOX-loaded vesicles injected into 4T1 tumor-bearing mice exhibited enhanced accumulation in tumor tissue due to the enhanced permeation and retention (EPR) effect. Importantly, DOX-loaded vesicles demonstrated greater tumor growth inhibition than free DOX without causing significant body weight loss or cardiotoxicity. The unique ability of the star-like copolymer emanating from the methyl cholate core provided the requisite modification in the block copolymer interfacial curvature to generate vesicles of high loading capacity for DOX with significant kinetic stability that have potential for use as an anti-cancer drug delivery carrier for cancer therapy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Doxorubicin (DOX) is commonly used in the standard treatment for a wide range of cancers including osteosarcomas, Hodgkin's lymphoma, acute myeloid leukemia, soft tissue sarcomas and cancers of the breast, bile duct, liver and bladder [1]. However, DOX chemotherapy is deemed to be unsatisfactory due to undesirable side effects, such as cardiac, kidney, hematological and nephro-toxicities [2–4]. In particular, DOXinduced cardiotoxicity is dosage-limiting and irreversible, which contributed significantly to morbidity and mortality [1,4]. Moreover, the inherent hydrophilicity of the compound makes it difficult to encapsulate and transport to the targeted region. The use of nanocarriers for targeted and sustained drug delivery in cancer therapy has demonstrated immense promise [5,6]. By exploiting ⁎ Corresponding author. E-mail address: [email protected] (Y.Y. Yang). 1 These authors contributed to the study equally.

the enhanced permeability and retention (EPR) effect in cancerous tissues, nanocarriers are able to achieve a specific biodistribution profile which concentrates chemotherapeutic agents in the tumor sites, while exhibiting selective cytotoxicity [5]. Extensive research has been conducted on a variety of nanostructures, including liposomes, self-assembled micellar nanoparticles and dendrimers, to realize the therapeutic potential of anticancer drugs [7–17]. Among these, selfassembled polymeric nanoparticles have been reported as promising drug carriers in cancer therapy as these nanoparticles offer an attractive approach for drug delivery by the virtue of the well-defined core–shell structure. The hydrophobic core serves as a drug reservoir, while the hydrophilic exterior increases water solubility of hydrophobic therapeutics, protects the cargos from enzymatic degradation and stabilizes the nanoparticles [16,18,19]. Likewise, polymeric nanoparticles confer prolonged blood circulation and allow for chemical modification to install targeting ligands and functional groups through advanced chemistry [19,20]. Despite the momentous advantages of polymeric micellar drug delivery systems, there has been limited success in clinic, and only a few

http://dx.doi.org/10.1016/j.jconrel.2015.03.027 0168-3659/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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C. Yang et al. / Journal of Controlled Release xxx (2015) xxx–xxx

polymeric micelle systems have entered phase II clinical trials [21–24]. Therefore, there remains a need to overcome specific drawbacks that limit the therapeutic outcomes of the nanocarriers. One major challenge of polymeric micelles is to maintain in vivo stability [12]. Upon intravenous injection, the nanoparticles are exposed to numerous environmental changes comprising of dilution, pH and salt changes, and interactions with blood proteins. This may result in unwarranted disassociation of polymeric micellar nanoparticles and premature drug release, leading to undesirable toxicity to healthy tissues [25]. Studies have indicated that the thermodynamic stability of micelles is associated with the critical micelle concentration (CMC), where lower CMCs reflect higher thermodynamic stability in the bloodstream [26]. To circumvent instability, there has been increasing interest in utilizing chemical crosslinking of the core or shell of micelles [27,28], or non-covalent interactions such as ionic interaction [12] or stereocomplexation [29]. Another prevailing issue of polymeric micellar drug delivery systems is inadequate drug loading capacity, which is determined by the interactions between the loaded drugs and the micellar core [18]. Recent studies have proposed the use of non-covalent interactions such as hydrogen bond, hydrophobic and ionic interactions to increase drug loading capacity. We have recently reported that the addition of acid functional groups to the core-forming moiety of the polymeric micelles provided sites for ionic interactions with the basic amine group on DOX, significantly increasing drug loading level [7,12]. However, such strategies invariably hindered DOX release due to the strong ionic interaction. The macromolecular design with appropriate functionalities can not only improve drug-loading content and stability, but it can also be utilized to engineer aqueous self-assembly to result in nanostructures of different shapes. Precise control of nanostructural shapes in drug-delivery applications has been recognized as an important handle to tailor in vivo outcome [30]. Among different readily accessible nanostructural shapes, polymeric vesicles constitute an important class with unique ability to encapsulate both hydrophobic and hydrophilic cargoes. Apart from the versatility in the choice of cargoes, polymer vesicles also offer numerous possibilities to engineer their chemical, physical and biological properties through effective macromolecular design. Recently, amphiphilic branched star and star-like polymers with three-dimensional structures have demonstrated to have great potential to form vesicles with excellent storage stability, particularly for the nanosized vesicles [31]. Enhancement in the physico-chemical properties along with flexibility in both the design and applications renders amphiphilic star-like polymers an attractive starting point for the development of novel drug delivery vehicles. With the advances in the synthesis of dendrimers and other branched polymers, innumerable amphiphilic branched star and star-like polymers can be in principle designed with their properties, specifically tailored to meet the requirements for drug delivery applications [32]. Though the use of a dendrimer as a branched core would enable precise control over a multitude of properties, the iterative synthesis of these functional precursors would be a concern due to increased production cost. Cholic acid, a natural bile acid, has been recognized as a cheap and readily available platform to initiate branched degradable polymers. The three hydroxyl groups of cholic acid and its derivatives have been used as initiating sites to access well-defined star-like polymers through the polymerization of monomers such as lactide [33], TMC [34], and allyl glycidyl ether [35], to name a few. Though in these preceding works, star-like polymers were readily synthesized, incorporating hydrophilic components to impart amphiphilicity was not straightforward [36]. For instance in an elegant study reported by Zhu and colleagues [36], sequential anionic polymerization of allyl glycidyl ether and ethylene glycol initiated from cholic acid derivative yielded well-defined amphiphilic star-like polymers. Stringent reaction conditions imposed by the use of anionic polymerization can be a concern for the wide applicability of this approach. Clearly there is a need to have simple synthetic routes to access polymeric amphiphiles with ability to exert control over the

ratio of hydrophobic and hydrophilic components. In this study, we used the three hydroxyl groups of methyl cholate to initiate ROP of trimethylene carbonate (TMC) to result in a 3-arm star polymer with hydroxyl chain ends, which were in turn conjugated with an unsymmetrically functionalized polyethylene glycol (vinyl sulfone-PEGcarboxylic acid, i.e. VS-PEG-COOH). Vinyl sulfone group may be used to install a biological ligand for specific tumor targeting. In order to understand the role of molecular structure on aqueous self-assembly, drug loading capacity and stability, an amphiphilic diblock copolymer analogue initiated from the single hydroxyl group of cholesterol, was synthesized in the same fashion as star-like polymer to facilitate comparison. DOX was physically encapsulated into nanoparticles using the star-like block copolymer and diblock copolymer, and characterized for particle size, zeta potential, drug loading, critical micelle concentration (CMC), stability in serum- and SDS-containing media and in vitro drug release. The uptake of free DOX and DOXloaded nanoparticles by BT-474 and MDA-MB-231 human breast cancer cell lines was studied by confocal microscopy and flow cytometry. The cytotoxicity of free DOX and DOX-loaded nanoparticles was evaluated against BT-474, MDA-MB-231 and 4T1 (mouse breast cancer) cell lines. The in vivo biodistribution of the nanoparticles was studied by non-invasive fluorescence imaging. The in vivo anti-cancer efficacy of DOX-loaded nanoparticles was investigated in a 4T1 mouse breast cancer model in comparison with free DOX. 2. Materials and methods 2.1. Materials All chemicals were purchased from Sigma-Aldrich and utilized as received unless otherwise indicated. All solvents were of analytical grade, purchased from Fisher Scientific and used as received. Trimethylene carbonate (TMC) was purchased from Boehringer Ingelheim (Ingelheim, Germany), and dried extensively by freezedrying under high vacuum. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) was distilled from CaH2 under dry N2 and transferred to a glove box. N-(3,5-Trifuluoromethyl)phenyl-N′-cyclohexylthiourea (TU) catalyst was prepared as described previously [37]. Before transferring into the glove box, monomers and other reagents (like VS-PEG-COOH) were dried extensively by freeze-drying under high vacuum. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma, U.S.A., and dissolved in phosphate-buffered saline (PBS, pH 7.4) with a concentration of 5 mg/mL, and the solution was filtered with a 0.22 μm filter to remove blue formazan crystals prior to use. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) was purchased from Caliper Life Sciences, U.S.A. MDA-MB-231, BT-474 human breast cancer cell lines and 4T1 mouse mammary carcinoma cell line were obtained from ATCC, U.S.A., and cultured according to ATCC's recommendation. MDA-MB-231 cells were cultured in Dulbecco's modified eagle medium (DMEM), while BT-474 and 4T1 were cultured in Roswell Park Memorial Institute (RPMI 1640) medium. All media were supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, U.S.A.). 2.2. Polymer synthesis 2.2.1. Synthesis of methyl cholate-initiated star-like PTMC (Scheme 1) In a glove box, methyl cholate (MC, 0.061 g, 0.144 mmol) and TMC (0.221 g, 2.16 mmol) were dissolved in 1.5 mL of dichloromethane (DCM), followed by adding DBU (0.13 mL, 0.864 mmol) to initiate the polymerization. The reaction solution was stirred overnight before benzoic acid (127 mg, 1.04 mmol) was added to quench the reaction. Then, the solution was precipitated in cold methanol (MeOH), centrifuged and washed 3 times with cold MeOH before dried in vacuum, giving MC-initiated PTMC as sticky white solid (0.26 g, 92%). PDI: 1.20. 1H NMR (400 MHz, CDCl3, 22 °C): δ 4.23 (t, 104H, H-a of PTMC), 2.04 (m,

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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Scheme 1. Synthesis procedures and chemical structures of MC-initiated star-like polycarbonate/PEG block copolymer 1 and CHOL-initiated PTMC/PEG diblock copolymer 2.

52H, H-b of PTMC), 0.65–1.00 (2 s and d, 9H, H-c, H-d and H-e of methyl cholate). Cholesterol (CHOL)-initiated PTMC with a similar DP as that of MCinitiated star-like PTMC was also prepared as a control polymer. Yield, 88%; PDI, 1.23. 1H NMR (400 MHz, CDCl3, 22 °C): δ 4.23 (t, 120H, H-a of PTMC), 2.05 (m, 60H, H-b of PTMC), 0.65–1.05 (2 s and 2 d, 15H, methyl group of cholesterol). 2.2.2. Synthesis of VS-PEG-COOH (Scheme 1) In a nitrogen gas atmosphere, triethylamine (0.107 mL, 0.743 mmol) was added to a solution of HS-PEG-COOH (0.488 g, 0.162 mmol) in dry MeOH (5 mL). The resulting solution was added dropwise to 5 mL of MeOH containing divinyl sulfone (0.46 mL, 4.46 mmol) under stirring. The reaction mixture was heated to 40 °C and reacted for 6 h before being cooled down to room temperature, and concentrated to dryness. The residue was dissolved in 10 mL of MeOH, and 0.23 mL of formic acid was added to the solution. After stirring for 1 h, the solution was concentrated to dryness, and the residue was purified by column chromatography on a Sephadex LH-20 column with THF as eluent, giving VS-PEG-COOH as white powder (0.48 g, 95%). 1H NMR (400 MHz, CDCl3, 22 °C): δ 6.69 (q, 1H, H of vinyl group), 6.48 (d, 1H, H of vinyl group), 6.21 (d, 1H, H of vinyl group), 3.64 (s, 273 H, H of PEG). 2.2.3. Synthesis of MC-initiated star-like polycarbonate/PEG copolymers In a nitrogen gas atmosphere, MC-initiated 3-arm star-like PTMC (0.096 g, 0.03 mmol), VS-PEG-COOH (0.444 g, 0.145 mmol) and 4dimethylaminopyridine (DMAP, 3.6 mg, 0.0294 mmol) were dissolved in 1 mL of DCM, followed by adding a solution of N, N′dicyclohexylcarbodiimide (DCC, 0.488 g, 0.162 mmol) in 1 mL of DCM. The reaction solution was stirred for 48 h before removing the byproduct DCU by filtration, and concentrated to dryness. The star-like copolymer was re-dissolved in DCM and MeOH, and further purified

by fractionation (0.2 g, 68%). PDI: 1.17. 1H NMR (400 MHz, CDCl3, 22 °C): δ 6.69 (q, 2H, H of vinyl group), 6.48 (d, 2H, H of vinyl group), 6.20 (d, 2H, H of vinyl group), 4.24 (t, 104H, H-a of PTMC), 3.63 (s, 546 H, H of PEG), 2.03 (m, 52H, H-b of PTMC), 0.65–1.00 (2 s and d, 9H, H-c, H-d and H-e of methyl cholate). Similarly, CHOL-initiated PTMC-PEG diblock copolymer was made. Yield, 62%; PDI, 1.19. 1H NMR (400 MHz, CDCl3, 22 °C): δ 6.69 (q, 1H, H of vinyl group), 6.46 (d, 1H, H of vinyl group), 6.21 (d, 1H, H of vinyl group), 4.22 (t, 120H, H-a of PTMC), 3.63 (s, 273 H, H of PEG), 2.04 (m, 60H, H-b of PTMC), 0.65–1.05 (2 s and 2 d, 15H, methyl group of cholesterol). 2.3. Gel permeation chromatography (GPC) GPC analysis for both star-like and diblock copolymers was carried out on a GPC system (Waters 2690, MA, U.S.A.) with an Optilab rEX differential refractometer detector (Wyatt Technology Corporation, U.S.A.) and Waters HR-4E column. The mobile phase used was THF with a flow rate of 1 mL/min. Weight average molecular weights and polydispersity indices were calculated from a calibration curve obtained using a series of polystyrene standards (Polymer Laboratories Inc., MA, U.S.A., with molecular weight ranging from 1350 to 151,700). 2.4. Nuclear magnetic resonance (NMR) 1

H NMR analyses of the star-like and diblock copolymers as well as their precursor polymers were performed on a Bruker Advance 400 NMR spectrometer at 400 MHz at room temperature. The 1H NMR measurement parameters: acquisition time of 3.2 s, pulse repetition time of 2.0 s, 30 °C pulse width, 5208-Hz spectral width, and 32 K data points. Chemical shifts were referred to the solvent peak (δ = 7.26 for CDCl3).

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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2.5. Fluorescence measurements

2.8. Kinetic stability study of DOX-loaded nanoparticles

The critical micelle concentration (CMC) of the block copolymers in DI water was estimated by fluorescence spectroscopy using pyrene as a probe. Fluorescence spectra were recorded by a Fluoromax-4 spectrofluorometer at room temperature. Aliquots of pyrene solutions in acetone (6.16 × 10− 6 M, 100 μL) were added to 1 mL volumetric flasks, and the acetone was allowed to evaporate. Then, 1 mL of polymer solutions with different concentrations ranging from 0.01 ppm to 1000.0 ppm was added to the vials. The final pyrene concentration is 6.16 × 10−7 M. The solutions were equilibrated for 24 h at 20 °C. The excitation spectra were recorded from 300 to 360 nm with an emission wavelength of 395 nm. Both excitation and emission bandwidths were set at 2.5 nm. The intensity ratios of I338 to I335 were plotted as a function of logarithm of polymer concentration. The CAC value was taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations.

Kinetic stability of DOX-loaded nanoparticles in DI water was examined using sodium dodecyl sulfate (SDS) as a destabilizing agent [38]. Briefly, SDS aqueous solution was added to the nanoparticle suspension at a final concentration of 2.23 mg/mL and mixed by vortexing. The change in scattered light intensity was recorded by DLS using the Zetasizer 3000 HAS.

2.6. Nanoparticle preparation Blank and DOX-loaded nanoparticles were prepared by a membrane dialysis method. For blank nanoparticles, the polymer (10.0 mg) was dissolved in 2 mL of N, N-dimethylacetamide (DMAc). The solution was then dialyzed against de-ionized (DI) water at 20 °C for 24 h using a membrane dialysis bag with a molecular weight cut-off of 1000 (Spectra/Por 7, Spectrum Laboratories Inc.). The water was replaced at 3, 6 and 24 h. For DOX-loaded nanoparticles, the polymer (10.0 mg) was dissolved in 1 mL of DMAc. DOX (5.0 mg) was neutralized with three moles excess triethylamine in 1 mL of DMAc. The DOX solution was added into the polymer solution and mixed by vortexing for 2 min. The mixture was dialyzed against DI water at 20 °C for 24 h using a membrane dialysis bag with a molecular weight cut-off of 1000. After dialysis, the solution in the dialysis bag was collected. To determine DOX loading level, a known amount of DOX-loaded nanostructures was dissolved in 1 mL of DMAc. The DOX concentration was estimated by using a UV–VIS spectrophotometer (UV 2501 PC Shimadzu, Japan) at 480 nm. The drug loading was calculated based on the standard curve obtained from DOX in DMAc. The yield of nanoparticles was calculated as the weight ratio of nanoparticles recovered to initial polymer and drug.

2.7. Particle size and zeta potential analyses The particle size and zeta potential of the nanoparticles were measured by a Zetasizer 3000 HAS (Malvern Instrument Ltd., Malvern, UK) equipped with a He–Ne laser beam at 658 nm (scattering angle: 90°). The concentration of nanoparticles is 1.0 mg/mL. Each measurement was repeated 5 times. An average value was obtained from three measurements. Particle size of DOX-loaded nanoparticles in PBS containing 10% FBS was recorded as a function of time. The size measurements were performed by multimodel analysis.

2.9. Transmission electron microscopy (TEM) The morphologies of blank and DOX-loaded nanoparticles were analyzed by TEM (FEI Tecnai G2 F20 electron microscope) using an acceleration voltage of 200 keV. The polymers were dissolved in dimethyl formamide (DMF, 5 mg/mL), and the solution was then dialyzed against DI water at 20 °C for 24 h using a dialysis membrane with a molecular weight cut-off of 1000 (Spectra/Por 7, Spectrum Laboratories Inc.). TEM samples were prepared by first placing a drop of selfassembled polymer solution (4.0 μL) onto a formvar coated 200 mesh copper grid (Ted Pella Inc., U.S.A.). After 1 min, the excess solution was wicked off by using filter paper. The staining agent phosphotungstic acid (1% w/v; 4.0 μL) was placed on the grid. After 1 min, the excess solution was wicked off and the grid was left to dry under ambient conditions. 2.10. In vitro release study of DOX-loaded nanoparticles DOX release from the DOX-loaded nanoparticles was investigated using the dialysis method. The micelles (2 mL) were transferred to a membrane dialysis bag with a molecular weight cut-off of 1000 (Spectra/Por 7, Spectrum Laboratories Inc.). The bag was immersed in a bottle containing 40 mL of PBS with pH 7.4 or pH 5. 0. This was kept shaking on an orbital shaker at 100 rpm at 37 °C. The solution from the release medium (1 mL) was withdrawn at specific time points and replaced with 1 mL of fresh PBS buffer. The DOX content in the samples was analyzed using the UV–VIS spectrophotometer at 480 nm. 2.11. In vitro cytotoxicity study MDA-MB-231 cells were cultured in DMEM, and BT-474 as well as 4T1 cells in RPMI1640 supplemented with 10% FBS, 5% penicillin, 2 mM L-glutamine (Sigma) and incubated at 37 °C, 5% CO2. The cells were seeded onto 96-well plates at 10, 000 cells per well and incubated for one day. Free DOX and DOX-loaded nanoparticles in DMEM or RPMI1640 were diluted with the growth medium to give final DOX concentrations of 0.1, 0.5, 1.0, 5.0, 10.0, 25.0 and 50.0 mg/L. The blank nanoparticles in DMEM or RPMI1640 were diluted to 10, 25, 50.0, 100.0, 250.0 and 500.0 mg/L. The media in the wells were replaced with 100 μL of the pre-prepared samples. The plates were then returned to the incubator and maintained in 5% CO2 at 37 °C for 48 h. Fresh growth media (90 μL) and 10 μL of MTT solution were used to replace the mixture in each well after the designated period of exposure. The plates were then returned to the incubator and maintained

Table 1 Physical properties of polymers, blank and DOX-loaded nanoparticles. Polymers

Polymer properties Mna (g/mol)

1 2 a b c d

9,310 6,564

PDI

1.17 1.19

Nanoparticle properties CMCb (mg/L)

5.2 3.0

wPtmcc (%)

35.6 54.3

Dhd (nm)

PDI

Blank

DOX-loaded

Blank

DOX-loaded

109 ± 1 33 ± 1

196 ± 1 92 ± 1

0.26 ± 0.01 0.20 ± 0.01

0.12 ± 0.01 0.23 ± 0.01

Zeta potential (mV) of DOX-loaded

Drug loading (wt.%)

–5.6 ± 0.3 –6.8 ± 0.1

22.5 ± 0.1 7.3 ± 0.1

Molecular weight was determined by 1H NMR spectroscopy. Critical micelle concentration obtained by fluorescence spectroscopy. Weight fraction of Ptmc as determined by 1H NMR spectroscopy. Hydrodynamic diameter and polydispersity index were measured by dynamic light scattering.

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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Fig. 1. 1H NMR spectrum of MC-initiated star-like PTMC/PEG copolymer 1 in CDCl3.

in 5% CO2, at 37 °C, for a further 3 h. The growth medium and excess MTT in each well were then removed before 150 μL of DMSO was added to each well to dissolve the internalized purple formazan crystals. An aliquot of 100 μL was taken from each well and transferred to a fresh 96-well plate. Each sample was tested in eight replicates per plate. The plates were then assayed at 550 nm and 690 nm. The absorbance readings of the formazan crystals were taken to be that at 550 nm subtracted

by that at 690 nm. The results were expressed as a percentage of the absorbance of the blank control. 2.12. Cellular distribution and uptake of DOX-loaded nanoparticles BT-474 cells were seeded at a density of 5 × 104 cells/well onto a 4well borosilicate coverglass chamber (NUNC). After overnight culture,

Fig. 2. TEM images of blank vesicles (a) and DOX-loaded vesicles (b) self-assembled from star-like copolymer 1 (Scale bar: 500 nm for a and 200 nm for b); blank micelles (c) and DOXloaded micelles (d) self-assembled from diblock copolymer 2 (Scale bar: 50 nm for c and 100 nm for d).

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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the medium was replaced with fresh medium containing free DOX (2.5 mg/L) and DOX-loaded nanoparticles (DOX concentration: 2.5 mg/L) for various periods of time. The medium was removed and washed three times with PBS and then stained with Lysotracker™ Green (Life Technology, 75 nM) and Hoechest 33258 (5 μg/mL) for 20 min at 37 °C. The cells were washed again twice with PBS, fixed in 10% formalin solution for 15 min at room temperature, followed by washing twice with PBS and visualized by a confocal laser scanning microscope (CLSM, Carl Zeiss LSM 510 META inverted confocal microscope, Germany). DOX was excited at 532 nm with emission at 595 nm, Lysotracker™ Green was excited at 488 nm with emission at 532 nm, whereas Hoechest dye was excited at 350 nm with emission at 480 nm. All observations were conducted under the same conditions. The cellular uptake of DOX was further evaluated quantitatively via flow cytometry (FACS Calibur BD Bioscience). BT-474 cells were seeded at a density of 5 × 104 cells/well onto 24 well pates (NUNC). After overnight culture, the medium was replaced with RPMI 1640 medium containing free DOX (1.0 mg/L) and DOX-loaded nanoparticles (DOX concentration: 1.0 mg/L) for 4 h. The medium was removed and the cells were washed three times with PBS, trypsinized, and harvested by centrifugation. The cells were then suspended in PBS for analysis. 2.13. In vivo biodistribution of nanoparticles To evaluate the in vivo biodistribution of nanoparticles, a nearinfrared fluorophore DiR was loaded into the nanoparticles. The polymer (10 mg) and 0.5 mg of DiR were dissolved in 1 mL of DMF, and dialyzed against DI water at 20 °C for 24 h using a dialysis membrane bag with a molecular weight cut-off of 1000. To determine the loading level of DiR, the dye-loaded nanoparticles were dissolved in DMSO and absorbance was measured against a standard curve of free dye dissolved in DMSO at 759 nm wavelength. Animal studies were carried out according to the protocols approved by the Singapore Biological Research Center (BRC)'s Institutional Animal Care and Use Committee. Briefly, female balb/c mice (body weight 19–25 g) were subcutaneously implanted with an ATCC 4T1 mice breast cancer epidermal carcinoma xenograft cell line (0.75 × 106 cells per mouse). Tumors were allowed to grow for 10 days. The mice were then administrated with 200 μL of DiR-loaded nanoparticles (DiR concentration: 30 mg/L) via tail vein injection. Non-invasive fluorescence imaging at various time points up to 5 days post-injection was performed using the IVIS 100 (Caliper Life Sciences, U.S.A.). Anesthetized mice were placed on an animal plate heated to 37 °C. The near-infrared fluorescence was imaged using the ICG filter pairs and exposure time was set to 3 s. Scans were performed at 5 min, 5 h, 24 h, 48 h and 5 days post-administration. The mice were then sacrificed at day 5 to evaluate the biodistribution of nanoparticles in various tissues (tumor, heart, liver, spleen, lungs and kidneys). Free DiR (30 mg/L) and PBS were used as controls.

Fig. 3. Nanoparticle stability. (a) Size of DOX-loaded nanoparticles made from the star-like (1) and diblock (2) copolymers in PBS containing 10% fetal bovine serum changes as a function of time at 37 °C. (b) Relative scattered light intensity of DOX-loaded nanoparticles changes as a function of time after being challenged with SDS. Relative intensity (%) means the percentage of the scattered light intensity at time x in relative to that at time 0.

tumor sections were quantified by counting the number of TUNELpositive nuclei in 5 representative fields of magnification of 20 and obtaining the mean value for each sample. Images were acquired and analyzed using a light microscope (Olympus, Japan). 2.15. Statistics The data are expressed as mean ± standard deviation. Standard deviation is indicated by the error bars. Student's t-test was used to 80.0

The 4T1 mouse breast cancer model was established as described above. The mice were randomly divided into 3 groups of 6 mice each. Free DOX and nanoparticles containing an equivalent amount of DOX (5 mg/kg) were administrated through tail vein at the predetermined time points (Days 0, 4, 7 and 11). PBS was used as control. Tumor volume and body weight were monitored to assess tumor inhibition activity and overall toxicity of each formulation. The tumor dimensions were measured with a Vernier caliper, and the tumor volume was calculated using the formula: Tumor volume = (width)2 × length × ½. At the end of the in vivo study, the mice were sacrificed and the tumors were excised. For histological examination, the samples were fixed in 10% formalin solution followed by paraffin embedding and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining using standard techniques. The slides were counterstained with hematoxylin to visualize nuclei. Apoptotic cells in the

Cumulative release (%)

2.14. In vivo therapeutic efficacy

60.0 pH 7.4 pH 5.0

40.0

20.0

0.0 0

5

10

15

20

25

30

Time (hours) Fig. 4. In vitro release profiles of DOX-loaded vesicles in PBS buffers (pH 7.4 and pH 5.0 respectively) at 37 °C.

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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Fig. 5. Cellular uptake and subcellular distribution of DOX fluorescence (red) in BT-474 cells after incubation with DOX-loaded vesicles at 37 °C for various periods of time. Cell nuclei and acidic organelles are stained with Hochest (blue) and Lysotracker Green (green), respectively. The concentration of DOX equivalent was 2.5 mg/L. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

determine significance among groups. A value of p b 0.05 was considered to be significant.

3. Results and discussion 3.1. Polymer synthesis and characterization The star-like copolymer was synthesized by utilizing three hydroxyl groups of methyl cholate as the core. Methyl cholate was used to initiate ROP of TMC in the presence of DBU as a catalyst (Scheme 1). To achieve a full conversion of TMC, a relatively higher amount of DBU was required (molar ratio of DBU to TMC was 1:4). After the reaction was competed, excess benzoic acid was added to quench the polymerization. DBU, benzoic acid and unreacted TMC were easily removed after precipitation in cold MeOH. An unsymmetrically functionalized PEG with reactive chain ends, VS-PEG-COOH was obtained by reacting HS-PEGCOOH with excess divinyl sulfone (DVS) in the presence of triethylamine. The carboxylic acid group of VS-PEG-COOH was recovered by the addition of formic acid after the addition reaction was completed. The star-like amphiphilic PTMC/PEG block copolymer was synthesized by the condensation of MC-initiated 3-arm star polymer PTMC-triol and VS-PEG-COOH in the presence of DCC/DMAP. The feed ratio of VS-PEG-COOH to MC-PTMC-triol was 1.5:1. The star-like copolymer was purified from the reaction mixture by repeated precipitation and fractionation. From the GPC chromatographs of the polymer before and after purification, the trace for the crude product shows bimodal distribution and the shoulder peak is attributed to unreacted VS-PEGCOOH. After purification, this component was completely removed and its corresponding peak disappeared, giving rise to a single unimodal peak that corresponds to the polymer. Both star-like and diblock copolymers were obtained with narrow molecular weight distributions (PDI: 1.17 and 1.19 respectively) (Table 1).

In addition to molecular weight analysis, the chemical structures of the polymers were characterized by 1H NMR analysis. For example, Fig. 1 shows the proton spectrum of methyl cholate-initiated star-like copolymer, in which all peaks attributed to PEG, PTMC, methyl cholate and vinyl sulfone group were clearly observed. Quantitative comparisons between the integral intensities of the peaks of methyl groups in methyl cholate at 0.65–1.00 ppm, methylene group of PTMC at 2.03 ppm, ethylene groups of PEG at 3.63 ppm, and vinyl hydrogen in the vinyl sulfone group at 6.20–6.70 ppm gave the composition of the polymer. Interestingly, the ratio of PEG block to PTMC block is 2 to 1 instead of 3 to 1, indicating that there are two PEG blocks in the polymer.

Fig. 6. Fluorescent intensity of BT-474 cells incubated with free DOX and DOX-loaded vesicles for 4 h at a DOX concentration of 1.0 mg/L, where cells without treatment were used as control (* means p b 0.05).

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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This is most probably because the condensation reaction only happens between the hydroxyl chain ends of 3-arm MC-PTMC and the carboxylic acid end of VS-PEG-COOH, and the DCC/DMAP reaction merely enabled 2 out of 3 alcohol ends of MC-PTMC to be grafted by VS-PEG-COOH even though excess VS-PEG-COOH was used (the molar ratio of carbolic acid groups to hydroxyl groups is 1.5 to1). The disparity is likely due to partial micelle formation precluding quantitative conversion. Thus, for the methyl cholate-initiated star-like polymer, the two hydrophobic PTMC blocks were flanked by two hydrophilic PEG blocks. The cholesterol-

initiated PTMC/PEG diblock copolymer with the same DP of PTMC as the star-like polymer was synthesized as a comparison. Vinyl sulfone groups at the distal end of PEG block can be used to attach targeting ligands. 3.2. Self-assembly of star-like and diblock copolymers Aqueous self-assembly of biodegradable polymeric amphiphiles has paved access to discrete functional nanocarriers suitable for drug delivery. Under aqueous conditions, both star-like and diblock copolymers were found to self-assemble to form nanostructures. Critical micelle concentration (CMC) or the concentration above which the polymers exist as aggregates, was determined in DI water by using the well-established pyrene probe method. The star-like and diblock polymers have CMC values of 5.2 and 3.0 mg/L, respectively (Table 1 and Fig. S2). The relatively low CMC values for the polymers suggest that the nanostructures can be formed at low concentrations, allowing their use in body fluids where the formulation would undergo an extensive dilution after administration. In principle, the star-like polymer could exist as a unimolecular micelle; however the manifestation of a CMC clearly shows that it tends to aggregate in solution. Dynamic light scattering (DLS) technique was used to measure the hydrodynamic size of the self-assembled nanostructures. Hydrodynamic diameters of the self-assembled nanostructures from star-like and diblock copolymers were found to be 109 and 33 nm, respectively (Table 1). As observed by transmission electron microscopy (TEM) the star-like polymer formed vesicles (Fig. 2a), while diblock copolymer formed micelles (Fig. 2c). It is interesting to note that the star-like copolymer with a high hydrophilic content (~64 wt.%) formed vesicles, while the control diblock polymer with a lower hydrophilic content (~46 wt.%) formed micelles. This hydrophilic content for the star-like copolymer is significantly higher than typical hydrophilic contents (~20–42 wt.%) reported for the formation of polymeric vesicles [39], suggesting that the placement of methyl cholate at the center along with the presence of PTMC segments affects the interfacial curvature to generate the targeted vesicles. The formation of vesicles from star-like copolymers is consistent with other reports, having amphiphilic branched multi-arm polymers [31]. Apart from the overall amphiphilicity, the exact physicochemical nature of the individual segments has been previously shown to influence morphological outcome [40–42]. Doxorubicin (DOX) was physically loaded into nanoparticles via a simple membrane dialysis method. The particle sizes of DOX-loaded nanoparticles formed from the star-like and diblock copolymers were 196 and 92 nm, respectively, which were larger than the blank nanoparticles due to drug loading. The vesicle and micelle structures were retained even after DOX encapsulation as observed by TEM for the star-like copolymer (Fig. 2b) and diblock copolymer (Fig. 2d), respectively. 3.3. DOX loading The drug loading capacity of polymeric micelles is affected by various parameters including polymer architecture, core-forming component of the polymer, compatibility and interaction between the polymer and drug. Importantly, the nanoparticles prepared from the starlike copolymer had significantly higher drug loading capacity (22.5%) as compared to the micelles made from the diblock copolymer (7.3%) (Table 1). As shown in Fig. 2, polymer 1 formed vesicles with greater core space for drug loading, while polymer 2 formed micelles with a solid core, leaving less space available for housing drug molecules. 3.4. Stability of DOX-loaded nanoparticles

Fig. 7. Viability of (a) 4T1, (b) MDA-MB-231 and (c) BT-474 cells after incubation with free DOX and DOX-loaded vesicles for 48 h at 37 °C.

It is essential for the nanoparticles to be stable for in vivo application when exposure to the complex bloodstream environment. It has been reported that proteins bind to the surface of nanoparticles to form protein corona, leading to aggregation and clearance by the mononuclear

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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phagocyte system (MPS) [12]. The DOX-loaded nanoparticles were incubated with FBS-containing PBS at 37 °C, and the particle sizes were monitored as a function of time. The sizes of DOX-loaded nanoparticles self-assembled from both the star-like and diblock copolymers experienced ~30 nm drop within the first 2 h, which was probably attributed to the contribution of particle size of FBS (~15 nm, Fig. S3). The particle sizes did not change significantly over the following 2 days of incubation (Fig. 3a), implying good stability. This can be explained that PEG shell covered the nanoparticles, and prevented protein adsorption and nanoparticle aggregation. The kinetic stability of the DOX-loaded

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nanoparticles was further investigated by challenging the nanoparticle suspension with sodium dodecyl sulfate (SDS). As shown in Fig. 3b, DOX-loaded nanoparticles formed from the diblock copolymer experienced ~ 15% reduction in the relative scattering light intensity within 2 h after SDS treatment, indicating that the nanoparticles were partially dissociated by SDS as a result of hydrophobic competition [43]. Notably, the DOX-loaded nanoparticles formed from the star-like copolymer were more stable with less than 10% decrease in the scattered light intensity in the first 2 h, and remained stable after 48 h of SDS treatment. This indicates that the vesicle structure of DOX-loaded nanoparticles 1

Fig. 8. In vivo biodistribution of nanoparticles. NIRF images of 4T1 tumor-bearing mice following intravenous administration of DiR-loaded vesicles (a) and DiR-loaded micelles (c); NIRF images of various organs at 5 days post-administration of DiR-loaded vesicles (b) and DiR-loaded micelles (d).

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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was more stable. In addition, there might be more pi–pi stacking of DOX molecules in the core of vesicles due to higher DOX loading level. Overall, DOX-loaded vesicles achieved high drug loading capacity and excellent kinetic stability, and were therefore selected for further studies.

3.5. In vitro drug release To evaluate the applicability of the nanoparticles as drug delivery carrier, DOX release kinetics and in vitro cytotoxicity against cancer cells were investigated. As illustrated in Fig. 4, the release profile of DOX-loaded vesicles was characterized by a relatively faster release during the first 8 h (~ 50%), followed by a slow and sustained release up to 24 h. The cumulative DOX release from the vesicles was ~65% at pH 7.4 after 24 h of incubation. After that, the release was too slow to be detected under the in vitro release conditions. DOX molecules that formed pi–pi stacking and/or trapped in the shell might contribute to the slow release phase. Drug release from DOX-loaded vesicles was further investigated at pH 5.0 to simulate the endosomal and/or liposomal environment [44]. The cumulative release of DOX at pH 5.0 was higher as compared to that at pH 7.4. This phenomenon was also observed in other polymeric micellar systems. This was attributed to the fact that more DOX molecules were protonated at lower pH as the pKa of DOX was estimated by titration to be pH 8.2, leading to increased water solubility [7].

3.6. In vitro cellular uptake of DOX To explore the internalization and intracellular distribution of DOXloaded nanoparticles, BT-474 cells were cultured with either free DOX or DOX-loaded vesicles for various periods of time, and then observed under CLSM. As shown in Fig. 5, red fluorescence of DOX molecules was observed in the Lysotracker Green-labeled acidic organelles after 1 h of incubation with DOX-loaded vesicles, indicating that they were mainly internalized via endocytosis and localized in the acidic organelles (endosomes and lysosomes). In contrast, red fluorescence accumulated in the cell nucleus after 1 h of incubation with free DOX (image not shown), suggesting that free DOX was rapidly localized in the nucleus by diffusion pathway [11]. After 4 h of incubation, the red fluorescence from DOX-loaded vesicles was seen in both the acidic organelles and nucleus of the cells. At 24 h post-treatment, the red fluorescence from DOX-loaded vesicles was primarily seen in the nucleus due to intracellular DOX release from the vesicles and subsequent accumulation in the nucleus (Fig. 5). The intracellular uptake of DOX molecules in BT-474 cells was further analyzed quantitatively using flow cytometry. Fig. 6 shows that DOX-loaded vesicles yielded higher fluorescence intensity than that of free DOX after 4 h of incubation, suggesting that endocytosis led to greater cellular uptake of DOX by BT-474 cells when compared to the free diffusion pathway. 3.7. In vitro cytotoxicity The in vitro cytotoxicity of DOX was evaluated against 4T1, MDAMB-231 and BT-474 cells using DOX-loaded vesicles in comparison with free DOX (Fig. 7). DOX inhibited the proliferation of 4T1 cells more effectively when treated by free DOX than DOX-loaded vesicles, and the IC50 values for free DOX and DOX-loaded micelles were estimated to be 5.0 and 10.0 mg/L, respectively (Fig. 7a). A lower in vitro anticancer effect of encapsulated DOX against 4T1 cells was also reported in other nanoparticulate formulations due to slow intracellular release of DOX [45]. However, in MDA-MB-231 cells, the cytotoxicity of DOX (IC50: 1.5 mg/L) was similar between free DOX and DOX-loaded vesicles. The comparable IC50 values for free DOX and DOX-loaded vesicles implied a more rapid and complete intracellular release of DOX from the vesicles due to low pH in the endosomes and lysosomes, which was evidenced by its pH-dependent release characteristics, and enzymatic degradation of the polymers in the presence of cells [12]. Notably, in BT-474 cells, DOX-loaded vesicles showed a higher inhibitory effect than free DOX (IC50: 3.0 and 1.0 mg/L for free DOX and DOX-loaded micelles, respectively). The greater inhibitory effect of DOX-loaded vesicles might be due to greater cellular uptake of DOX (Fig. 6). The released DOX intercalates with the chromosomal DNA and induced cell death. These results suggest that the cellular uptake and intracellular DOX release depended on cell type. It is worth noting that the star-like copolymer did not induce significant cytotoxicity against all the cells studied even at concentrations up to 500 mg/L (Fig. S4). 3.8. In vivo biodistribution of nanoparticles in tumor-bearing mice

Fig. 9. In vivo anti-tumor efficacy and overall toxicity of free DOX and DOX-loaded vesicles in a 4T1 mouse breast cancer model. (a) Tumor volume changes as a function of time; (b) relative mouse weight over time during the treatment. Percentage of tumor volume or relative mouse weight was calculated by dividing the tumor volume or relative mouse weight at a given time point over the respective values at day 0 and being multiplied by 100% (a, * means p b 0.05, free DOX or DOX-loaded vesicles vs. control; + means p b 0.05, DOX-loaded vesicles vs. free DOX. b, * means p b 0.05, free DOX vs. control or DOX-loaded vesicles).

To investigate the in vivo real-time biodistribution and tumor targeting properties of nanoparticles, the vesicles formed from starlike copolymer were incorporated with a near infra-red fluorescent (NIRF) dye, DiR, and injected via the tail vein into BALB/c mice bearing subcutaneous 4T1 tumor [46]. The biodistribution and tumor targeting properties of vesicles were then monitored through noninvasive NIRF imaging over 5 days. As illustrated in Fig. 8a, within 5 min postadministration, DiR fluorescence signals were detected in the whole bodies of mice as a result of extensive circulation of the vesicles in the bloodstream. The contrast between the subcutaneous tumor and the normal tissue became apparent after 24 h post-injection. DiR intensity gradually increased and the strongest fluorescence signals were seen in the tumor tissue at 48 h post-administration, indicating

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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enhanced accumulation of DiR-loaded vesicles in the tumor tissue. The biodistribution and tumor targeting properties of micelles formed from diblock copolymer were conducted as a comparison. It is noted that the contrast between the subcutaneous tumor and the normal tissues was seen after 48 h post-injection of the micelles (Fig. 8c). Free DiR dye was used as control and injected at an equivalent dose as that in the vesicle formulation. From Fig. S5a, NIRF signals in the whole body of mice were much lower than that of nanoparticle formulation over the whole period of time observed, indicating that DiR was rapidly eliminated from the mice's blood after injection. Moreover, the mice with free DiR always showed much lower NIRF intensity at the tumor tissue as compared to those with nanoparticle formulations (Fig. S4). The biodistribution of nanoparticles in the tumor-bearing mice was further assessed from the images of the major organs at day 5 postinjection. As shown in Fig. 8b, NIRF signals were detected in the liver and spleen, suggesting the retention of DiR-loaded vesicles in these organs. This finding agrees with previous reports, showing that nanoparticles were preferentially taken up by macrophages in reticuloendothelial systems (RES) [47,48]. Importantly, the highest NIRF signals were observed in the tumor tissue, which is consistent with those obtained from real-time images of the mice (Fig. 8a). DiR-loaded micelles showed a similar biodistribution pattern as that of vehicles (Fig. 8d), but the intensity of DiR signal was stronger in the lungs as compared to the vesicles. This high tumor targeting property of vesicles and micelles was attributed to the stability and prolonged circulation of the vesicles and micelles in the blood and the EPR effect in the tumor tissue.

Control

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3.9. In vivo antitumor activity In vivo antitumor activity of DOX-loaded vesicles was investigated using 4T1-tumor bearing BALB/c mice via tail vein injection (Fig. 9a). The mice were randomly grouped and treated with three solutions: PBS (control), free DOX and DOX-loaded vesicles, where DOX dosage was 5.0 mg/kg. It should be mentioned that lyophilized DOX-loaded micelles were unable to re-disperse in water to get 5 mg/kg DOX dose due to lower drug loading capacity. Therefore, in vivo therapeutic efficacy of micelles was not included as a comparison. The tumor growth rate was the highest in the control group of mice treated with PBS, where tumor size rapidly increased by 13.9 ± 1.3-fold as compared to the original size. The tumor growth in mice injected with free DOX showed a marked reduction as compared to the control, increasing by 8.4 ± 0.9-fold at day 18 (p b 0.05). Importantly, DOX-loaded vesicles demonstrated a significantly higher antitumor effect when compared to the PBS- and DOX-treated groups over 18 days of treatment (5.1 ± 0.6 folds vs. PBS, p b 0.01; vs. DOX, p b 0.05), which was attributed to the EPR effect of the vesicles in the tumor tissue (Fig. 8). To evaluate the general toxicity of free DOX and DOX-loaded vesicle formulations, the body weight of mice in each group was monitored over the treatment period (Fig. 9b). Mice treated with free DOX showed ~ 20% loss in body weight at day 18, implying DOX toxicity. The body weight of the untreated mice decreased by 9% at day 18, where the body weight loss was compromised by tumor growth. There was no significant difference in the body weight between the group treated with

DOX

DOX-loaded vesicles

a

b

c

d

e

f

Tumor

Heart

g

h

Fig. 10. TUNEL staining of tumor and heart tissues at 18 days post-injection of free DOX and DOX-loaded vesicles. (a, d) Tumor and heart sections from the mice treated with control (PBS); (b, e) tumor and heart sections from mice treated with free DOX; (c, f) tumor and heart sections from mice treated with DOX-loaded vesicles; quantitation of mean apoptotic bodies per field (objective ×20) in tumor (g) and heart (h) tissues (* means p b 0.05, DOX-loaded vesicles vs. free DOX; + means p N 0.1, DOX-loaded vesicles vs. control). Scale bar: 50 μm.

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

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DOX-loaded vesicles and the control group (p N 0.10). These results revealed that vesicles with greater stability delivered DOX to the tumor more effectively than free DOX and attenuated the toxicity associated with free DOX. In the future, in addition to passive tumor targeting, active targeting will be performed by conjugation of thiolcontaining targeting peptides to the vinyl sulfone-functionalized polymeric vesicles to further enhance in vivo antitumor efficacy. Histological examination of tumor and heart tissues excised from the mice was conducted. The presence of apoptotic cells in the tissues can be observed by the brown regions resulting from apoptosis using TUNEL assay. As shown in Fig. 10a–c and f, the treatment with DOXloaded vesicles led to a higher number of apoptotic cells in the tumor tissue as compared to free DOX treatment. The antitumor effect of DOX-loaded vesicles was further evaluated by quantifying the number of apoptotic bodies in the tumor tissue. A higher number of apoptotic bodies were observed in the tumor tissue treated by DOX-loaded vesicles (p b 0.05) (Fig. 10g). These results further proved that the antitumor efficacy of the DOX-loaded vesicles was higher than free DOX. Notably, there was no significant apoptosis in the heart tissue treated with the DOX-loaded vesicles as compared to the control without any treatment (Fig. 10d vs. f, Fig. 10h, p N 0.1), while the free DOX treatment induced a significantly higher number of apoptotic cells in the heart tissue (Fig. 10d vs. e, Fig. 10h, p b 0.05). This is because DOX-loaded vesicles mainly accumulated in the tumor tissue, eliminating cardiotoxicity. 4. Conclusions A star-like PTMC/PEG amphiphilic block copolymer, synthesized through metal-free organo-catalytic living ring-opening polymerization, was shown to self-assemble into vesicles for anticancer drug delivery. DOX was physically encapsulated into vesicles, and the resulting DOX-loaded vesicles exhibited nanosize, greater kinetic stability and higher DOX loading capacity as compared to micelles formed from a diblock copolymer analogue. When injected into 4T1 tumor-bearing mice, the DOX-loaded vesicles exhibited enhanced accumulation in the tumor tissue and greater anti-tumor efficacy than free DOX without causing significant body weight loss or cardiotoxicity. The star-like amphiphilic block copolymer capable of directing the supramolecular assembly holds potential as an anticancer drug delivery carrier. Acknowledgments The study was funded by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2015.03.027. References [1] P.K. Singal, N. Iliskovic, Doxorubicin-induced cardiomyopathy, N. Engl. J. Med. 339 (1998) 900–905. [2] C.L. Du, D.W. Deng, L.L. Shan, S.N. Wan, J. Cao, J.M. Tian, S. Achilefu, Y.Q. Gu, A pHsensitive doxorubicin prodrug based on folate-conjugated BSA for tumor-targeted drug delivery, Biomaterials 34 (2013) 3087–3097. [3] D. Morelli, S. Menard, M.I. Colnaghi, A. Balsari, Oral administration of antidoxorubicin monoclonal antibody prevents chemotherapy-induced gastrointestinal toxicity in mice, Cancer Res. 56 (1996) 2082–2085. [4] D.D. Vonhoff, M.W. Layard, P. Basa, H.L. Davis, A.L. Vonhoff, M. Rozencweig, F.M. Muggia, Risk-factors for doxorubicin-induced congestive heart-failure, Ann. Intern. Med. 91 (1979) 710–717. [5] D. Peer, J.M. Karp, S. Hong, O.C. FaroKhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751–760. [6] A.Z. Wang, R. Langer, O.C. Farokhzad, Nanoparticle delivery of cancer drugs, Annu. Rev. Med. 63 (2012) 185–198.

[7] A.B.E. Attia, C. Yang, J.P.K. Tan, S.J. Gao, D.F. Williams, J.L. Hedrick, Y.Y. Yang, The effect of kinetic stability on biodistribution and anti-tumor efficacy of drug-loaded biodegradable polymeric micelles, Biomaterials 34 (2013) 3132–3140. [8] J.E. Chung, S. Tan, S.J. Gao, N. Yongvongsoontorn, S.H. Kim, J.H. Lee, H.S. Choi, Selfassembled micellar nanocomplexes comprising green tea catechin derivatives and protein drugs for cancer therapy, Nat. Nanotechnol. 9 (2014) 907–912. [9] K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushima, K. Okamoto, G.S. Kwon, Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyll-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance, J. Control. Release 64 (2000) 143–153. [10] S.E. Krown, D.W. Northfelt, D. Osoba, J.S. Stewart, Use of liposomal anthracyclines in Kaposi's sarcoma, Semin. Oncol. 31 (2004) 36–52. [11] J.A. MacKay, M. Chen, J.R. McDaniel, W. Liu, A.J. Simnick, A. Chilkoti, Self-assembling chimeric polypeptide–doxorubicin conjugate nanoparticles that abolish tumours after a single injection, Nat. Mater. 8 (2009) 993–999. [12] C. Yang, A.B.E. Attia, J.P.K. Tan, X.Y. Ke, S.J. Gao, J.L. Hedrick, Y.Y. Yang, The role of non-covalent interactions in anticancer drug loading and kinetic stability of polymeric micelles, Biomaterials 33 (2012) 2971–2979. [13] X.Z. Yang, X.J. Du, Y. Liu, Y.H. Zhu, Y.Z. Liu, Y.P. Li, J. Wang, Rational design of polyion complex nanoparticles to overcome cisplatin resistance in cancer therapy, Adv. Mater. 26 (2014) 931–936. [14] A.L.Z. Lee, Y. Wang, H.Y. Cheng, S. Pervaiz, Y.Y. Yang, The co-delivery of paclitaxel and Herceptin using cationic micellar nanoparticles, Biomaterials 30 (2009) 919–927. [15] C.C. Lee, J.A. MacKay, J.M.J. Frechet, F.C. Szoka, Designing dendrimers for biological applications, Nat. Biotechnol. 23 (2005) 1517–1526. [16] Y. Wang, S.J. Gao, W.H. Ye, H.S. Yoon, Y.Y. Yang, Co-delivery of drugs and DNA from cationic core–shell nanoparticles self-assembled from a biodegradable copolymer, Nat. Mater. 5 (2006) 791–796. [17] H. Fan, Q.D. Hu, F.J. Xu, W.Q. Liang, G.P. Tang, W.T. Yang, In vivo treatment of tumors using host–guest conjugated nanoparticles functionalized with doxorubicin and therapeutic gene pTRAIL, Biomaterials 33 (2012) 1428–1436. [18] M.C. Jones, J.C. Leroux, Polymeric micelles — a new generation of colloidal drug carriers, Eur. J. Pharm. Biopharm. 48 (1999) 101–111. [19] N. Wiradharma, Y. Zhang, S. Venkataraman, J.L. Hedrick, Y.Y. Yang, Self-assembled polymer nanostructures for delivery of anticancer therapeutics, Nano Today 4 (2009) 302–317. [20] N.E. Kamber, W. Jeong, R.M. Waymouth, R.C. Pratt, B.G.G. Lohmeijer, J.L. Hedrick, Organocatalytic ring-opening polymerization, Chem. Rev. 107 (2007) 5813–5840. [21] N. Nishiyama, K. Kataoka, Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery, Pharmacol. Ther. 112 (2006) 630–648. [22] S.C. Owen, D.P.Y. Chan, M.S. Shoichet, Polymeric micelle stability, Nano Today 7 (2012) 53–65. [23] J.W. Valle, A. Armstrong, C. Newman, V. Alakhov, G. Pietrzynski, J. Brewer, S. Campbell, P. Corrie, E.K. Rowinsky, M. Ranson, A phase 2 study of SP1049C, doxorubicin in P-glycoprotein-targeting pluronics, in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction, Invest. New Drugs 29 (2011) 1029–1037. [24] H. Cabral, K. Kataoka, Progress of drug-loaded polymeric micelles into clinical studies, J. Control. Release 190 (2014) 465–476. [25] H.S. Han, J. Lee, H.R. Kim, S.Y. Chae, M. Kim, G. Saravanakumar, H.Y. Yoon, D.G. You, H. Ko, K. Kim, I.C. Kwon, J.C. Park, J.H. Park, Robust PEGylated hyaluronic acid nanoparticles as the carrier of doxorubicin: mineralization and its effect on tumor targetability in vivo, J. Control. Release 168 (2013) 105–114. [26] J.B. Liu, F.Q. Zeng, C. Allen, In vivo fate of unimers and micelles of a poly(ethylene glycol)-block-poly(caprolactone) copolymer in mice following intravenous administration, Eur. J. Pharm. Biopharm. 65 (2007) 309–319. [27] E.S. Read, S.P. Armes, Recent advances in shell cross-linked micelles, Chem. Commun. (2007) 3021–3035. [28] Y.L. Wu, W. Chen, F.H. Meng, Z.J. Wang, R. Cheng, C. Deng, H.Y. Liu, Z.Y. Zhong, Corecrosslinked pH-sensitive degradable micelles: a promising approach to resolve the extracellular stability versus intracellular drug release dilemma, J. Control. Release 164 (2012) 338–345. [29] K. Fukushima, R.C. Pratt, F. Nederberg, J.P.K. Tan, Y.Y. Yang, R.M. Waymouth, J.L. Hedrick, Organocatalytic approach to amphiphilic comb-block copolymers capable of stereocomplexation and self-assembly, Biomacromolecules 9 (2008) 3051–3056. [30] S. Venkataraman, J.L. Hedrick, Z.Y. Ong, C. Yang, P.L. Ee, P.T. Hammond, Y.Y. Yang, The effects of polymeric nanostructure shape on drug delivery, Adv. Drug Deliv. Rev. 63 (2011) 1228–1246. [31] W. Jiang, Y. Zhou, D. Yan, Hyperbranched polymer vesicles: from self-assembly, characterization, mechanisms, and properties to applications, Chem. Soc. Rev. (2015) Advance Article, http://dx.doi.org/10.1039/C4CS00274A. [32] E.R. Gillies, J.M. Frechet, Dendrimers and dendritic polymers in drug delivery, Drug Discov. Today 10 (2005) 35–43. [33] H.L. Fu, T. Zou, S.X. Cheng, X.Z. Zhang, R.X. Zhuo, Cholic acid functionalized star poly(DL-lactide) for promoting cell adhesion and proliferation, J. Tissue Eng. Regen. Med. 1 (2007) 368–376. [34] T. Zou, S.L. Li, Z.Y. Hu, S.X. Cheng, R.X. Zhuo, Synthesis and properties of star oligo/poly(trimethylene carbonate)s with cholic acid moieties as cores, J. Biomater. Sci. Polym. Ed. 18 (2007) 519–530. [35] G. Giguere, X.X. Zhu, Functional star polymers with a cholic acid core and their thermosensitive properties, Biomacromolecules 11 (2010) 201–206. [36] C. Li, C. Lavigueur, X.X. Zhu, Aggregation and thermoresponsive properties of new star block copolymers with a cholic acid core, Langmuir 27 (2011) 11174–11179.

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027

C. Yang et al. / Journal of Controlled Release xxx (2015) xxx–xxx [37] R.C. Pratt, F. Nederberg, R.M. Waymouth, J.L. Hedrick, Tagging alcohols with cyclic carbonate: a versatile equivalent of (meth)acrylate for ring-opening polymerization, Chem. Commun. (Camb.) (2008) 114–116. [38] V.M. Gaspar, C. Goncalves, D. de Melo-Diogo, E.C. Costa, J.A. Queiroz, C. Pichon, F. Sousa, I.J. Correia, Poly(2-ethyl-2-oxazoline)-PLA-g-PEI amphiphilic triblock micelles for co-delivery of minicircle DNA and chemotherapeutics, J. Control. Release 189 (2014) 90–104. [39] F. Ahmed, D.E. Discher, Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis-triggered controlled release vesicles, J. Control. Release 96 (2004) 37–53. [40] S.H. Kim, F. Nederberg, R. Jakobs, J.P. Tan, K. Fukushima, A. Nelson, E.W. Meijer, Y.Y. Yang, J.L. Hedrick, A supramolecularly assisted transformation of block-copolymer micelles into nanotubes, Angew. Chem. Int. Ed. Engl. 48 (2009) 4508–4512. [41] J.P. Tan, S.H. Kim, F. Nederberg, K. Fukushima, D.J. Coady, A. Nelson, Y.Y. Yang, J.L. Hedrick, Delivery of anticancer drugs using polymeric micelles stabilized by hydrogen-bonding urea groups, Macromol. Rapid Commun. 31 (2010) 1187–1192. [42] S. Venkataraman, Z.A. Chowdhury, A.L. Lee, Y.W. Tong, I. Akiba, Y.Y. Yang, Access to different nanostructures via self-assembly of thiourea-containing PEGylated amphiphiles, Macromol. Rapid Commun. 34 (2013) 652–658.

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[43] N. Kang, M.E. Perron, R.E. Prud'homme, Y. Zhang, G. Gaucher, J.C. Leroux, Stereocomplex block copolymer micelles: core–shell nanostructures with enhanced stability, Nano Lett. 5 (2005) 315–319. [44] Y. Bae, S. Fukushima, A. Harada, K. Kataoka, Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change, Angew. Chem. Int. Ed. Engl. 42 (2003) 4640–4643. [45] S.Q. Liu, Y.W. Tong, Y.Y. Yang, Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N, Ndimethylacrylamide)-b-poly(D, L-lactide-co-glycolide) with varying compositions, Biomaterials 26 (2005) 5064–5074. [46] V. Ntziachristos, C. Bremer, R. Weissleder, Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging, Eur. Radiol. 13 (2003) 195–208. [47] K.Y. Choi, K.H. Min, H.Y. Yoon, K. Kim, J.H. Park, I.C. Kwon, K. Choi, S.Y. Jeong, PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo, Biomaterials 32 (2011) 1880–1889. [48] C.Y. Sun, S. Dou, J.Z. Du, X.Z. Yang, Y.P. Li, J. Wang, Doxorubicin conjugate of poly(ethylene glycol)-block-polyphosphoester for cancer therapy, Adv. Healthc. Mater. 3 (2014) 261–272.

Please cite this article as: C. Yang, et al., Structure-directing star-shaped block copolymers: Supramolecular vesicles for the delivery of anticancer drugs, J. Control. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.03.027