Cell Internalizable and Intracellularly Degradable Cationic

Jun 30, 2014 - Star-shaped amphiphilic block polyurethane with pentaerythritol core for a hydrophobic drug delivery carrier. Shihai Wang , Yu Zhou , B...
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Cell Internalizable and Intracellularly Degradable Cationic Polyurethane Micelles as a Potential Platform for Efficient Imaging and Drug Delivery Mingming Ding,† Xin Zeng,† Xueling He,‡ Jiehua Li,† Hong Tan,*,† and Qiang Fu† †

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China ‡ Laboratory Animal Center, Sichuan University, Chengdu 610041, China S Supporting Information *

ABSTRACT: A cell internalizable and intracellularly degradable micellar system, assembled from multiblock polyurethanes bearing cell-penetrating gemini quaternary ammonium pendent groups in the side chain and redox-responsive disulfide linkages throughout the backbone, was developed for potential magnetic resonance imaging (MRI) and drug delivery. The nanocarrier is featured as a typical “cleavable core−internalizable shell−protective corona” architecture, which exhibits small size, positive surface charge, high loading capacity, and reduction-triggered destabilization. Furthermore, it can rapidly enter tumor cells and release its cargo in response to an intracellular level of glutathione, resulting in enhanced drug efficacy in vitro. The magnetic micelles loaded with superparamagnetic iron oxide (SPIO) nanoparticles demonstrate excellent MRI contrast enhancement, with T2 relaxivity found to be affected by the morphology of SPIO-clustering inside the micelle core. The multifunctional carrier with good cytocompatibility and nontoxic degradation products can serve as a promising theranostic candidate for efficient intracellular delivery of anticancer drugs and real-time monitoring of therapeutic effect.

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to intracellular stimuli, including pH,7 temperature,8 redox,9,10 enzymes,11 etc., constitutes a topical area of research.12 In particular, reduction-responsive nanovehicles containing disulfide bonds have received a tremendous amount of interest because of the great difference in the reducing environments of intracellular and extracellular milieu.9 Disulfide bonds can be cleaved in the presence of the reducing agent glutathione (GSH), enriched within tumor cells (∼10 mM), thus allowing a triggered release of encapsulated drugs. Nonetheless, most studies on redox-sensitive vehicles have focused on diblock or triblock copolymers containing a single disulfide linkage between hydrophobic and hydrophilic blocks,13 which generally lack control of responsiveness and micellization properties. Recently, multiblock copolymers have attracted considerable attention in the field of drug delivery due to their increased structural diversity and additional functionality in comparison with traditional linear block copolymers.14 As a typical multiblock copolymer, polyurethane has been successfully used in various biomedical applications.15 Its good biocompatibility, facile preparation, and highly tunable character enable the incorporation of different hydropobic and hydrophilic segments,16,17 cell penetrating molecules,6 targeting ligands,18 and

INTRODUCTION Over the past decades, nanoparticle-based drug delivery systems have shown great potential for cancer therapy.1 They can improve the pharmacokinetics and biodistribution of therapeutic agents and minimize unwanted side effects through preferential accumulation at target sites via an enhanced permeability and retention (EPR) effect. However, even if a high level of drug formulations can reach the tumor tissues, their poor cell internalization and inadequate release of payloads in tumor cells generally result in low drug availability and insufficient therapeutic response to kill the cancer cells, which may also induce multiple drug resistance (MDR).2 To promote the cellular uptake of nanocarriers, a promising approach may be the use of cell internalizable ligands,3 such as cell-penetrating peptides (CPPs), a class of nonviral vectors that allow the delivery of conjugated biomolecules and nanoparticles to overcome biological barriers.4 Alternatively, several synthetic peptide mimetics and other compounds such as quaternary ammonium (QA) groups have also shown excellent penetrating activity, which can be utilized to increase the permeability of nanovehicles across the cell membrane for enhanced cell internalization and delivery efficiency.5,6 To further increase the dosages and bioavailability of therapeutics in tumor cells, drugs must be released specifically at the target site at a high enough level to maximize the therapeutic efficacy. Therefore, the design of switchable nanosystems capable of unloading their payloads in response © XXXX American Chemical Society

Received: April 7, 2014 Revised: June 28, 2014

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Figure 1. (A) A typical molecular structure of cell internalizable and redox-degradable cationic polyurethanes. Color code: blue, PCL; yellow, Llysine ethyl ester diisocyanate (LDI); purple, bis(2-hydroxyethyl) disulfide (DHDS); red, gemini quaternary ammonium (GQA); green, methoxylpoly(ethylene glycol) (MPEG). The wavy line represents the PCL segment. (B−D) Schematic illustration of cationic multiblock polyurethanes (B) and their micelles loading SPIO nanoparticles (C) and antitumor drugs (D).

stimuli-sensitive linkages19,20 into the polymer chains to fabricate multifunctional drug delivery systems, as demonstrated previously by others and us. On the other hand, to offer more control over intracellular location and the possibility of real-time detection of tumors, nanocarriers combined with magnetic resonance imaging (MRI), one of the most powerful diagnostic tools, have emerged as a versatile platform to monitor the delivery efficiency.21,22 Superparamagnetic iron oxide (SPIO) nanoparticles are the most extensively researched contrast enhancement agents used to improve the imaging resolution of MRI, because of their intrinsic nontoxicity, large surface area, and excellent superparamagnetic properties.23 For example, Gao and co-workers reported a series of diblock copolymer micelles based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) or polylactide (PLA), which contain a cluster of SPIO particles for ultrasensitive MRI and effective delivery of doxorubicin (DOX).24,25 Furthermore, various micelle and vesicle systems constructed from other diblock, triblock, graft, and hyperbranched copolymers have also been employed as building blocks for the development of iron oxide-based imaging systems.26 However, to our knowledge, there is no report so far on SPIO-loaded polyurethane nanovehicles for multifunctional drug delivery and MRI applications.

In this work, we report a novel cell internalizable and intracellularly degradable micellar system loading SPIO nanoparticles for MR imaging and drug delivery (Figure 1). The micelles, self-assembled from redox-responsive cationic multiblock polyurethanes, were composed of a biodegradable PCL core, cell-penetrating gemini QA shell, protective PEG corona, and reduction-cleavable disulfide linkages throughout the polymeric backbone. Oleic acid modified Fe3O4 nanoparticles as a contrast agent were synthesized and encapsulated into the core of micelles. The bulk properties, self-assembly behavior, loading capacity, stimuli-responsiveness, and magnetic properties of polyurethane micelles were fully investigated. To study the cell internalization and intracellular delivery of nanoformulations into tumor cells, flow cytometry and confocal laser scanning microscopy (CLSM) were carried out. In addition, in vitro cytotoxicity and MR relaxivity were determined to assess the potential of polyurethane micelles for MR imaging and drug delivery.

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EXPERIMENTAL SECTION

Materials. GQA diamine and LDI were synthesized in our laboratory according to ref 16. PCL (MW 2000, Dow Chemical) was dehydrated under reduced pressure at 100 °C for 1 h before use. MPEG (MW 550, Acros Organics) was dried under vacuum. 1,4-Butanediol (BDO, Flaka chemika, Switzerland) was distilled under vacuum. DHDS (Alfa Aesar) was used as received. N,N-Dimethylacetamide (DMAc) was distilled B

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under vacuum. Triptolide (TP, ≥99.5%) was purchased from Nanjing Zelang Medicine Technology Co., China. Iron(III) chloride hexahydrate (FeCl3·6H2O), oleic acid (OA), ammonium hydroxide solution (NH3· H2O), ethanol, hexane, and tetrahydrofuran (THF) were purchased from Chengdu Kelong Chemical Reagent Company, China. Iron(II) chloride tetrahydrate (FeCl2·4H2O) was supplied by Tianjin Damao Chemical Reagent Factory, China. Fluorescein isothiocyanate isomer I (FITC, 90%) was from Acros Organics (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) was obtained from Sigma-Aldrich (USA). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Roche Diagnostics (Germany). Characterization. Gel permeation chromatography (GPC) was performed on a PL-GPC 220 (Polymer Laboratory Ltd., England) using N,N-dimethyl-formamide (DMF)/LiBr as eluent and poly(methyl methacrylate) (PMMA) as a reference. The sample concentration was 1.000 mg mL−1, and the flow rate was 1.000 mL min−1. Proton nuclear magnetic resonance (1H NMR, 400 MHz) was recorded on a Bruker AV II-400 MHz spectrometer using tetramethylsilane (TMS) as an internal standard in DMSO-d6 and D2O. Fourier transform infrared (FTIR) spectra were obtained on a Nicolet-560 spectrophotometer between 4000 and 600 cm−1 with the resolution of 4 cm−1. The analysis to fit to a Gaussian curve was performed using Origin Pro 8 SR4 software. Differential scanning calorimetry (DSC) was measured on a TA Q-20 instrument (USA) at a heating rate of 10 °C min−1 in the range of −100 to 100 °C under a steady flow of nitrogen. Transmission electron microscopy (TEM, Hitachi H-600-4, Japan) and high-resolution TEM (HRTEM, Tecnai G2 F20 S-TWIN, FEI, USA) were recorded with accelerating voltages of 75 and 200 kV, respectively. A drop of sample solution was placed on a copper grid with Formvar film, and then the liquid was blotted off and air-dried before measurement. For the observation of SPIO-free micelles, samples were stained with 1% (w/v) phosphotungstic acid. Sizes and ζ potentials of polyurethane micelles were measured with a Zetasizer Nano ZS dynamic light-scattering (DLS) instrument (Malvern, UK) at 25 °C at an angle of 90°. Fluorescence measurements were performed using pyrene as a probe. Pyrene dissolved in acetone was added to a series of vials, and acetone was subsequently evaporated. Then to each vial was added a certain amount of micellar solution to make a final pyrene concentration of 5.0 × 10−7 M. All the samples were equilibrated upon shaking for 2 h at 40−50 °C and incubated overnight at room temperature. Steady-state fluorescence spectra were recorded using an F-7000 FL spectrophotometer with bandwidths of 10.0 nm for excitation and 2.5 nm for emission. The excitation wavelength was 334 nm for emission spectra and 373 nm for excitation spectra. Crystallographic analysis was performed on an X-ray diffractometer (XRD, Phlips X’Pert PRO, XL-30). The diffraction patterns were taken from 20° to 90° (2θ) using Cu Kα radiation. The phase was determined by using standard powder diffraction files from the Joint Committee for Powder Diffraction Studies (JCPDS). The crystallite size is calculated from the full width at half-maximum (fwhm) of the diffraction peaks of the samples using the Scherrer equation, D = Kλ/ (b cos θ), where D is the crystallite size, K is a shape factor (about 0.89 for magnetite), λ is the wavelength of the X-rays (1.5406 Å), b is the full-width of the XRD peak at half-maximum, and θ is the Bragg angle. Thermogravimetric analysis (TGA) was carried out on a thermogravimetric analyzer (TGA Q500, TA Instruments, USA) under flowing nitrogen atmosphere. The dried powder samples were heated from room temperature to 700 °C at a rate of 10 °C min−1. Magnetic properties were measured using a vibrating sample magnetometer (VSM, Lakeshore 7410, USA) at 298 K under an applied magnetic field varying between −15 and 15 kOe. Synthesis of Redox-Responsive Cationic Multiblock Polyurethanes. Biodegradable redox-responsive cationic multiblock polyurethanes were prepared from LDI, PCL, GQA, BDO, DHDS, and MPEG via a facile three step polymerization. Briefly, LDI (1.19 g) and PCL (4 g) were first dissolved in 28 mL of DMAc and prepolymerized for 1 h at 60 °C under a dry nitrogen atmosphere. Afterward, BDO (0.11 g) or DHDS (0.19 g) as a chain extender and 1‰ stannous

octoate as a catalyst were added to react at 65 °C for 1 h; then GQA (1.06 g) was added, and the reaction was kept for 1 h at room temperature, followed by another 2 h at 60 °C. Finally, MPEG (0.55 g) was added, and the reaction was continued at 60 and 85 °C for 1 and 5 h, respectively. The resultant solutions are precipitated in a mixture of methanol and diethyl ether and dried under vacuum at 60 °C for 72 h (yield ∼70%). FTIR (cm−1): 3360−3300 (w, broad, ν N−H), 1800−1600 (w, ν CO), 2945, 2866 (s, ν CH2, CH3), 1246, 1047 cm−1 (w, ν C−O− C). 1H NMR (400 MHz, DMSO-d6, TMS, δ in ppm): 4.07 (m, −CH2OCO−), 3.98 (t, −CH2O), 3.62 (t, −S−CH2−CH2−), 3.51 (t, −CH2− in PEG), 3.07 (m, −CH2−N+(CH3)2−), 2.80 (t, −S−CH2−), 2.27 (t, −CH2COO−), 1.66 (−CH2CH2−N+), 1.53 (t, −CH2− in PCL), 1.29 (m, −CH2− in PCL and GQA), 1.17 (t, −CH3 in LDI), 0.90 (t, −CH3 in GQA). Preparation and Characterization of Polyurethane Micelles. Redox-responsive cationic multiblock polyurethane micelles were prepared by a dialysis method. Ten milliliters of polymer solution in DMAc was added dropwise to 50 mL of deionized water. The solution was then transferred to a dialysis tube (MWCO 3500) and dialyzed against deionized water for about 3 days to remove the organic solvent at room temperature. The micelle solution was centrifuged at 3000 rpm for 10 min and passed through a 0.45 μm pore-sized syringe filter (Millipore, Carrigtwohill, Co. Cork, Ireland). GSH (2 μM or 10 mM) was added into the micellar solutions to simulate the extracellular or intracellular reduction environment of tumor cells, respectively. The changes of size and size distributions of micelles in response to GSH was measured at different time intervals. The degradation products obtained were lyophilized and analyzed by a Waters-1515 (USA) GPC using DMF/LiBr as eluent and PMMA as a standard. TP as a model drug was loaded into polyurethane micelles by a micelle extraction technique. A measured amount of drug stock solution in acetone was added to the empty vial, and the solvent was evaporated under nitrogen. Then 10 mL of micelle solution was transferred into the vial and ultrasonicated for 2 h. The solution was centrifuged at 3000 rpm for 10 min and passed through a 0.45 μm pore-sized syringe filter to remove any insoluble drug. The amount of drugs loaded inside micelles was determined by a UV−vis spectrometer (UV-1800PC, Shanghai Mapada Instruments Co., Ltd., China). The release of TP was evaluated with a dialysis method in phosphate buffer solution (PBS, 10 mM, pH 7.4) containing 10 mM GSH at 37 °C with shaking. At desired time intervals, 3 mL of release media was sampled and replenished with an equal volume of fresh media. The release experiments were conducted in triplicate. The amount of TP released was determined by UV−vis spectrometer. Preparation of Oleic Acid Modified Fe3O4 Nanoparticles. The SPIO nanoparticles were prepared by a modified chemical coprecipitation method.26e In brief, FeCl3·6H2O (6.75 g) and FeCl2· 4H2O (3 g) were dissolved in deionized water (75 mL) purged with nitrogen gas. Then NH3·H2O (20 mL) was added, and the reaction was performed with vigorous stirring at 80 °C for 1 h. Afterward, oleic acid (3 g) was added slowly, and the reaction was continued for another 1 h. After the mixture was cooled to room temperature, the magnetic nanoparticles were harvested magnetically and washed with water and ethanol several times. The resultant black slurry was further dispersed in hexane for storage. Preparation of Encapsulated-SPIO Polyurethane Micelles. The hydrophobic SPIO nanoparticles were encapsulated into the core of polyurethane micelles during self-assembly. Redox-responsive polyurethanes (25 mg) and SPIO nanoparticles (18 mg) were dissolved in 3 mL of a mixed solvent consisting of THF and DMAc (1:9 v/v). The above solution was added dropwise into 10 mL of deionized water under sonication and then dialyzed against deionized water for 3 days to remove organic solvents. The micelle solution was centrifuged at 3000 rpm for 10 min and passed through a 0.45 μm pore-sized syringe filter. Cell Culture. Lewis lung carcinoma cells (LLCs) were cultivated in RPMI 1640 complete growth media (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 2 mM L-glutamine, and 1% antibiotics mixture (10000 U of C

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penicillin and 10 mg of streptomycin) (Gibco) at 37 °C in a humidified atmosphere of 5% CO2 (Sanyo Incubator, MCO-18AIC, Japan). Cellular Uptake. Physical entrapment of hydrophobic fluorescent probe FITC was used to prepare fluorescent-labeled polymeric micelles. In brief, a measured amount of FITC solution in acetone was added to an empty vial, and the solvent was evaporated under nitrogen in the dark. Then 2 mL of micelle solution was transferred into the vial and ultrasonicated for 2 h. The solution was centrifuged at 3000 rpm for 10 min and passed through a 0.2 μm pore-sized syringe filter to remove any insoluble dye. LLCs were seeded into 6-well culture plates and grown for 24 h. The cells were then treated with FITC-encapsulated polyurethane micelles at a concentration of 0.1 mg mL−1 at 37 °C for 30 and 60 min. Thereafter, culture medium was removed, and cells were washed with PBS three times and treated with trypsin. After removal of the supernatant, the cells were resuspended in 0.5 mL of PBS and analyzed using a FACS Aria flow cytometer (BD biosciences) by counting 10000 events. LLCs were seeded at a density of 1 × 105 cells/well in two 6-well chamber slides for 24 h. The cells were incubated with FITC-loaded micelles for 30 min at 37 °C. After removal of the medium, the cells were washed three times with cold PBS, fixed with 1 mL of 4% paraformaldehyde in PBS for 30 min at 4 °C, and stained with DAPI for 10 min. Finally, the slides were mounted with 10% glycerol solution and observed by a Zeiss 510 LSM microscope. Cytotoxicity Assay. LLCs were seeded in 96-well plates at 1 × 103 cells/well and incubated for 24 h. The culture medium was removed and replaced with 100 μL of medium containing various concentrations of micelle and drug-loaded micelle solutions for another 24 h of incubation. Then 20 μL of MTT solution (5 mg mL−1, Sigma) was added to each well. After the cells were incubated for 4 h, the MTT solution was removed, and the insoluble formazan crystals were dissolved in 110 μL dimethyl sulfoxide (DMSO). The absorbance was measured at a wavelength of 490 nm. The cell viability was normalized to that of cells cultured in the full culture media. The cell morphology was observed with phase-contrast microscopy images from an Optimas 5.2 image analysis system (Optimas Ltd., USA) attached to an IX2-SL inverted phase contrast microscope (Olympus Ltd., Japan) via a Nikon digital camera. The cytotoxicity of polyurethane degradation products was evaluated by completely degrading the polymers under accelerated conditions and exposing them to the cultured cells. Briefly, the polymer discs (200 mg) were placed in 1 mL of NaOH solution (1 M) and incubated at 70 °C for 4 days to degrade. The solution was then treated with HCl solution (1 M) to pH ∼1, and the degradation was continued for 2 days. Afterward, the pH of media was adjusted to 7.4, and PBS (10 mM, pH 7.4) was added to make a final volume of 4 mL. The resultant solution was filtered through a 0.2 μm membrane filter for sterilization and then diluted by 5, 10, and 100 times with culture media. Sterile PBS was similarly diluted as a control. The solutions were added to the cells cultured in the 96 well plates (100 μL/well), which were then incubated at 37 °C, 95% relative humidity, and 5% CO2 for 24 h. Cell viability was evaluated employing the MTT assay as described above and was normalized to that of fibroblasts grown in culture media with negative control. In Vitro MR Imaging. SPIO nanoparticles and magnetomicelles with different atomic Fe concentrations (0−0.5 mM) suspended in an agarose gel were transferred to tubes and tested using a 1.5 T clinical MRI instrument (Siemens, Germany). A multiecho fast spin−echo sequence was set as follows: repetition time (TR) = 5000 ms; echo time (TE) = 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 130, 150, 170, and 200 ms; field of view (FOV) = 103 mm × 206 mm; slice thickness = 1.5 mm. The T2 relaxation rates calculated from signal intensity (SI) of regions of interest (ROI) were plotted versus iron concentration, and the relaxivity was computed by linear regression analysis using OriginPro 8 software (OriginLab Corporation). Statistical Analysis. Data are expressed as means ± standard deviations (SD). The significance of difference in this study was analyzed by one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS, version 17.0) software.

Article

RESULTS AND DISCUSSION

Synthesis of Redox-Responsive Cationic Multiblock Polyurethanes. A series of biodegradable multiblock polyurethanes bearing disulfide linkages throughout the backbones, GQA cationic groups in the side chains, and MPEG oligomers attached to the chain end were readily prepared via a facile three-step copolymerization. The illustrated structure of polyurethane is shown in Figure 1A. The resultant polymers exhibit moderate molecular weights (Mw 16700−20600) with quite narrow molecular weight distributions (Figure S1 and Table S1, Supporting Information). The amount of gemini cationic QA groups, disulfide bonds, and PEG chains in the polymer structure can be easily adjusted by changing the feed ratios. Taking polyurethane with the highest content of disulfide bonds (100% in chain extender, GSSPU) and that without reduction-sensitive linkage (GPU) as examples, 1H NMR spectra of polymers are presented in Figure 2A. The peaks at 3.98

Figure 2. 1H NMR spectra of cell internalizable and redox-degradable cationic polyurethanes in DMSO-d6 (A) and their micelles in D2O (B). The inset shows a cross-sectional view of the core−shell−corona type micellar structure of cationic polyurethanes from DPD simulations. Color code: blue, PCL; red, GQA; yellow, LDI; green, PEG; magenta, DHDS.

(−CH2O−), 2.27 (−CH2COO−), 1.53 (−CH2CH2CH2−), and 1.29 (−CH2CH2CH2−) ppm are assigned to the methylene protons of PCL units. The chemical shifts of methylene and methyl protons in the ethoxyl group of LDI are found at 4.07 (−CH2OCO−) and 1.17 ppm (−CH3), respectively. Signal at 3.51 ppm is ascribed to the methylene groups of MPEG (−CH2CH2O−). Peak at 3.07 ppm (−CH2−N+(CH3)2−) is ascribed to the methylene and methyl protons of GQA polar D

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groups, while those at 1.66 (−CH2−CH2−N+) and 0.90 (−CH3) ppm belong to the methylene and terminal methyl of octyl group, respectively. In addition, two triplets observed at 2.80 and 3.62 ppm in the spectra of GSSPU are attributed to the hydrogen atoms in α-methylene (−S−CH2−) and β-methylene (−S−CH2− CH2−) next to the disulfide bond, respectively. These results demonstrate that all the desired functional moieties have been successfully incorporated into the multiblock polyurethanes. The FTIR spectra of multiblock polyurethanes are depicted in Figure 3 and Figure S2, Supporting Information. A broad

determined and given in Table S1, Supporting Information. It was found that the fraction of hydrogen-bonded carbonyl in urethane and urea decreases from 91.9% and 39.6%, respectively, to 51.6% and 18.1%, as disulfide linkages were introduced. The result agrees well with N−H shift and suggests that the hydrogen bonding within the hard segments is weakened and the phase mixing is thus increased with incorporation of disulfide linkages into the hard segments of polyurethanes. To further investigate the bulk behavior, thermal analysis of polyurethanes was carried out using DSC, and the results are depicted in Figure 3C and Table S3, Supporting Information. One can observe that the glass transition temperatures (Tg) of GSSPU (−58.0 °C) is relatively higher than that of GPU (−61.7 °C), indicating that the incorporation of disulfide decreases the degree of microphase separation of polyurethane. It is noteworthy that GPU shows no melting or crystallizing peak during the heating and cooling process, while GSSPU exhibits a sharp melting endotherm on the first heating curve (Figure 3D). This is possibly because the crystallization of PCL segments is improved by the disulfide-containing hard segments. However, further study is needed to better understand the interesting phenomenon. Construction of Cell Internalizable and ReductionDegradable Polyurethane Micelles. Owing to the unique surfactant nature of GQA groups and attractive amphiphilicity of polymers, the prepared multiblock polyurethanes can easily self-assemble in an aqueous solution to form a micellar structure, where PCL segments form the hydrophobic core to encapsulate water-insoluble drugs, GQA groups confer a cationic shell to improve the cell penetration ability, and PEG chains act as an outer corona that stabilizes the micelles in blood circulation and reduces uptake at reticuloendothelial sites (Figure 1). The formation of micelles was first studied using a pyrene fluorescence probe technique. The (0,0) absorption band in the excitation spectra shifts from 334.0 to 337.2 nm with the increase of polyurethane concentration in an aqueous solution of pyrene (Figure S7A, Supporting Information), suggesting that pyrene molecules are transferred from water solution to the microenvironment within micellar core. The core−shell− corona structure of polyurethane micelles was then characterized with 1H NMR. As seen from Figure 2B, all the characteristic peaks attributed to PCL, LDI, MPEG, and GQA units are clearly detected in DMSO-d6. However, the 1H NMR spectrum in D2O displays a significantly weakened resonance of PCL and amplified signals of PEG and GQA, strongly indicating the formation of multiblock polyurethane micelles. To visually demonstrate the fascinating architecture of polyurethane micelles, computational simulation was performed using a dissipative particle dynamics (DPD) method (Supporting Information). As seen from the cross-section view and density profiles of nanocarriers (Figure 2 and Figure S8, Supporting Information), a multilayered micellar structure comprising PCL core, cationic GQA shell, and PEG outer corona is well-defined, which is in good agreement with 1H NMR results. More powerful direct evidence of micelle formation comes from TEM images, where well dispersed individual particles with regular spherical shape are seen (Figure 4). The diameters of micelles range from 34 to 52 nm, and the surface charges are in the range of 36.4−38.7 mV, as determined by DLS and ζ potential measurements (Figure 4). Such small size and positively charged surface may be helpful to the cell internalization of polyurethane micelles, as preliminarily demonstrated in our

Figure 3. FTIR spectra in the (A) NH and (B) curve fitted CO stretching regions and DSC thermograms in the (C) glass transition regions and (D) melting and crystallizing regions of cell internalizable and redox-degradable cationic polyurethanes.

stretching band observed at 3300−3360 cm−1 is mainly attributed to N−H stretching vibration. The stretching band in the 1600−1800 cm−1 region is due to the absorption of ester carbonyl groups of PCL and free and hydrogen-bonded carbonyl of urethane groups, where a shoulder observed at 1650 cm−1 is ascribed to the hydrogen-bonded carbonyl of urea groups, further confirming that GQA groups are successfully copolymerized into the multiblock polyurethanes. Interestingly, with disulfide incorporation, a blue shift of the N−H stretching band from 3338 to 3367 cm−1 occurs (Figure 3A), and the shape of the CO stretching band changes accordingly (Figure 3B). To better understand this phenomenon, deconvolution of the carbonyl stretching region between 1600 and 1800 cm−1 was performed using a curve-fitting program, and the concentrations of free and hydrogen-bonded carbonyls of urethane and urea are E

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Figure 4. (A, B) Size and ζ potentials and (C, D) TEM micrographs of polyurethane micelles without (A, C) and with (B, D) redox-responsive disulfide linkages in the polymer backbone. The bars are 100 nm.

Figure 5. (A) Loading content and encapsulation efficiency of TP in polyurethane micelles. Changes of size (B) and size distributions (C) of cell internalizable and redox-degradable cationic polyurethane micelles in response to 10 mM or 2 μM GSH. (D) Release of TP from polyurethane micelles in PBS buffer solutions (pH 7.4, 10 mM) with or without 10 mM GSH.

previous work.6 The CMC values determined by fluorescence method are around 2.5 × 10−3 mg mL−1 (Figure S7B, Supporting Information), which are much lower than those reported for

conventional diblock, triblock, and branched PCL and PEG copolymers27 as well as biodegradable poly(ester urethane)s,28 implying that the polyurethane nanovehicles are highly stable. F

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assay. Evidently, a much smaller number of viable cells were observed for GSSPU micelles loaded with TP compared with drug-loaded GPU micelles, revealing a high drug efficacy for

To investigate the loading capacity of cationic polyurethane micelles as a carrier, TP, a diterpene triepoxide derived from the Chinese herb Tripterygium wilfordii Hook F with strong antitumor effects on various cancer cells,29 was chosen as a model hydrophobic drug. The loading of TP into polyurethane micelles was performed via an extraction method and determined by varying the feed weight ratio of drugs to the polymers. It was found that the nanocarriers can encapsulate TP efficiently, with maximum drug loading content (LC) and encapsulation efficiency (EE) of nearly 26% and 87%, respectively (Figure 5A). The drug loaded micelles show a unimodal size distribution with no appreciable change of particle size and ζ potential compared with blank micelles (Figure S9, Supporting Information), further suggesting a good physical stability of these formulations. It is known that the disulfide bonds can be reductively degraded in the presence of GSH predominantly found inside cells. Herein, to study the potential intracellular degradation of polyurethane nancarriers, the change of micellar size and size distributions in response to an intracellular level of GSH was monitored with DLS. Evidently, the average diameter of GSSPU micelles increases significantly, and the size distribution is greatly broadened after incubation with 10 mM GSH (Figure 5B,C), while negligible change of size and size distributions were observed for GSSPU micelles incubated with an extracellular concentration of GSH (∼2 μM) and for reduction insensitive GPU micelles treated with 10 mM GSH (Figure 5B and Figures S3−5, Supporting Information). This is probaly because the disulfide bonds embedded in the backbone are reductively degraded, resulting in the destabilization or reaggregation of polyurethane micelles. The degradation of polyurethanes was further confirmed with GPC results, where the molecular weight of GSSPU decreased to be undetectable in response to 10 mM GSH, while that of GPU remain unchanged under the same conditions (Figure S6, Supporting Information). Owing to the reduction-degradable character, GSSPU micelles released TP rapidly in a reductive environment (10 mM GSH, Figure 5D). In contrast, much slower drug release rates were observed for reduction insensitive GPU micelles under the same conditions, as well as for GSSPU micelles in the absence of GSH (Figure 5D). Considering that the reducing agent GSH presents in millimolar concentrations (∼10 mM) in the cytosol of tumor cells,9 a triggered intracellular release and subsequently an increased cytotoxicity can be anticipated for GSSPU formulations. To verify this potential, the cellular uptake of micelles and intracellular release of payloads were investigated by flow cytometry and confocal laser scanning microscopy (CLSM) using fluorescein isothiocyanate isomer I (FITC) as a hydrophobic fluorescent probe. It was found that both GPU and GSSPU can rapidly enter LLCs within 30 min (Figures 6 and 7), owning to the incorporated GQA group, which has shown excellent cell penetrating activity to promote the cell internalization of nanocarriers.6 Moreover, as expected, the mean fluorescence intensity of GSSPU micelles in tumor cells is moderately higher than that for insensitive GPU micelles at all tested time intervals (Figure 7), which should be due to the increased cellular uptake and triggered release of FITC following the cleavage of disulfidelinkages of GSSPU within tumor cells. The enhanced cell entry and intracellular drug release can rapidly increase the drug concentration in tumor cells and, therefore, effectively kill the cancer cells. The cytotoxicity of TP-loaded micelles against LLCs was assessed using MTT

Figure 6. Flow cytometry histogram profiles of Lewis lung carcinoma cells incubated with cell internalizable and redox-responsive polyurethane micelles prepared from GPU (A) and GSSPU (B) for 30 and 60 min. Polyurethane micelles were labeled with FITC.

reduction-responsive formulations (Figure 8A, p < 0.05). This result is in agreement with that reported by Zhong et al., where dextran-SS-poly(ε-caprolactone) micelles exhibited more efficient delivery and release of DOX into cytoplasm and nucleus in comparison with their nonsensitive counterparts, leading to a markedly enhanced drug efficacy toward tumor cells.13d Moreover, the blank micelles and their degradation products do not show any cytotoxicity at high concentrations up to 0.1 and 10 mg mL−1, respectively (Figure 8B), suggesting that the cationic polyurethane itself is not responsible for the cytotoxicity, and these cell internalizable and intracellulardegradable polyurethane micelles are potentially safe to be used as pharmaceutical nanovehicles. Preparation and Characterization of Magnetic Polyurethane Micelles. Fe3O4 nanoparticles were synthesized by chemical coprecipitation followed by coating with oleic acid. The synthesized SPIO particles can be well dispersed in hexane due to their hydrophobic nature after organic modification (Figure 9A). In addition, they exhibit spherical morphology with an average diameter of 15 nm, as determined by DLS measurement and TEM observation (Figure 9A,D). HRTEM micrograph shows a single iron oxide nanocrystal (Figure 9D), in which the individual lattice planes are clearly visible. The measured d-spacing of 2.53 Å matches with the (311) plane of magnetite. The XRD pattern of the sample is presented in Figure 10A. The diffraction peaks are consistent with those of standard magnetite pattern (JCPDS Card No. 19-0629), corresponding to face-centered cubic (fcc) Fe3O4 phases. The G

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Figure 7. CLSM images of Lewis lung carcinoma cells incubated with cell internalizable and redox-responsive polyurethane micelles prepared from GPU and GSSPU for 30 and 60 min. Polyurethane micelles were labeled with FITC (green); nuclei of cells were stained with DAPI (blue).

crystallite size calculated using the Scherrer equation is about 13.8 nm, which agrees well with those attained from DLS and TEM measurement, indicating that the nanoparticles are single crystals. The hydrophobic SPIONs were physically encapsulated into the core of polyurethane micelles. As a result, they can be transferred into an aqueous phase with high stability (Figure 9C). After SPIO loading, the polyurethane micelles maintain their positive surface charges (31−34 mV), while the size increases from 34−52 nm to over 100 nm in comparison with blank micelles (Figure 9B,C). The SPIO nanoparticles were found to be uniformly packed and form clusters in the cores of micelles. Interestingly, GPU magnetomicelles are spherical in shape (Figure 9E), while GSSPU−SPIO micelles display a short rodlike morphology (Figure 9F). This is possible because SPIO at the core of the micelle might affect the interactions between hydrotropic blocks and disturb the force balance governing the aggregation structures.30,31 The observation is particularly advantageous since rod-like micelles have manifested improved pharmacological effects compared with their spherical counterparts,32 such as a larger core volume for the encapsulation of therapeutics and imaging agents.33 To test this potential, the content of iron oxide in the magnetomicelles was determined with TGA. As shown in Figure 10B, the weight loss in the range of 200−480 °C corresponds to the decomposition of oleic acid and polyurethanes on the SPIO nanoparticles. The Fe3O4 content in the magnetomicelles can be calculated from the residual weight percentages. The result verifies a much higher Fe3O4 loading content (30.3%) of GSSPU micelles than that of GPU micelles (11.8%) at the same feed ratio (Figure 10B), which is also higher those reported for SPIO nanoparticles loaded in other micellar systems.31,34,35 The SPIO-loaded micelles possess a strong magnetism due to the Fe3O4 encapsulated. They appear as homogeneous

Figure 8. Viability of Lewis lung carcinoma cells measured by MTT assay after 24 h of incubation with various concentrations of blank and drug loaded micelles (A) and degradation products of polyurethanes (B). Error bars represent means ± standard deviation for n = 4. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.005.

dispersions in water and display a sensitive response to an external magnetic field (Figure 10C). The magnetization loops of SPIO nanoparticles and magnetomicelles determined by a field-dependent magnetization measurement are presented in Figure 10D. All the samples display typical superparamagnetic behavior, with negligible coercivity and remanence. The saturation magnetizations (Ms) of SPIO nanoparticles, GPU− SPIO, and GSSPU−SPIO at room temperature were determined to be 6.9, 20.4, and 79.4 emu g−1, respectively. The decreased saturation magnetization of magnetomicelles can be attributed to the presence of amphiphilic polyurethanes. These results suggest that the polyurethane micelles are promising for use as an MRIvisible nanosystem for imaging and drug delivery applications. In Vitro MRI Contrast Effect. SPIO nanoparticles are known to shorten the transverse (spin−spin) relaxation time (T2) of water protons and exert a dark or negative contrast in MRI.22,23 To assess the MRI contrast effect and T2 relaxation properties of nanocarriers, the micellar solutions with different iron concentrations were evaluated by T2-weighted MRI and compared with SPIO nanoparticles dispersed in agarose gel. As shown in Figure 11A, the signal intensity of MRI decreases with the increase of Fe concentration, suggesting an effective negative contrast enhancement in MRI. The specific relaxivity rate (r2), which reflects the effectiveness of magnetite nanoparticles to shorten the T2, was obtained from a plot of relaxation rate (T2−1) versus Fe concentration (Figure 11B). The T2 relaxivity coefficient of SPIO itself was calculated to be H

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Figure 9. (A−C) Size distributions and (D−F) TEM images of oleic acid modified Fe3O4 nanoparticles in hexane (A, D) and SPIO-loaded GPU (B, E) and GSSPU (C, F) magnetomicelles in water. Insets in panels A and C show oleic acid modified Fe3O4 nanoparticles and SPIO-loaded GSSPU micelles dispersed in hexane and water, respectively. Inset in panel D presents a HRTEM image of single crystallite showing the (311) lattice fringe at 2.53 Å. Insets in panels E and F display enlarged TEM images of SPIO-loaded GPU and GSSPU micelles, respectively. Unless otherwise noted, all the bars are 100 nm.

Figure 10. XRD patterns (A) and TGA curves (B) of oleic acid modified Fe3O4 nanoparticles and SPIO-loaded polyurethane micelles, (C) SPIO-loaded polyurethane micelles in response to an external magnetic field for 0 h (left) and 24 h (right), and (D) magnetic hysteresis loops of oleic acid modified Fe3O4 nanoparticles and SPIO-loaded polyurethane micelles.

55 mM−1 s−1, which was greatly increased after loading into polyurethane micelles due to the clustering of SPIO within the micellar core.24 Interestingly, SPIO-loaded GSSPU micelles show a considerably larger T2 relaxivity (r2 = 143 mM−1 s−1) than GPU−SPIO micelles (r2 = 90.6 mM−1 s−1), as indicated

by the steeper slope in Figure 11B. It was reported that the T2 relaxivity of SPIO-loaded nanovehicles was affected by the surrounding magnetic field, particle size, compositions of SPIO nanocrystals, and degree of SPIO clustering.26g Herein, the rod-like shape of magnetic GSSPU micelles with higher loading I

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results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (Grants 51203101, 51273126, and 51073104), China Postdoctoral Science Foundation (Grants 2011M500147 and 2012T50776), and Changjiang Scholars and Innovative Research Team in University (Grant IRT1163) are acknowledged.

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(1) (a) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotechnol. 2007, 2, 751−760. (b) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Science 2012, 338, 903−910. (2) (a) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002, 2, 48−58. (b) Dai, J.; Lin, S.; Cheng, D.; Zou, S.; Shuai, X. Angew. Chem., Int. Ed. 2011, 50, 9404−9408. (3) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Nat. Mater. 2009, 8, 543−557. (4) (a) Zorko, M.; Langel, Ü . Adv. Drug Delivery Rev. 2005, 57, 529− 545. (b) Grunwald, J.; Rejtar, T.; Sawant, R.; Wang, Z.; Torchilin, V. P. Bioconjugate Chem. 2009, 20, 1531−1537. (c) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Science 2010, 328, 1031−1035. (d) MacEwan, S. R.; Chilkoti, A. Nano Lett. 2012, 12, 3322−3328. (e) Jin, E.; Zhang, B.; Sun, X.; Zhou, Z.; Ma, X.; Sun, Q.; Tang, J.; Shen, Y.; Van Kirk, E.; Murdoch, W. J.; Radosz, M. J. Am. Chem. Soc. 2012, 135, 933−940. (5) (a) Verma, A.; Uzun, O.; Hu, Y.; Hu, Y.; Han, H.; Watson, N.; Chen, S.; Irvine, D. J.; Stellacci, F. Nat. Mater. 2008, 7, 588−595. (b) Kolonko, E. M.; Kiessling, L. L. J. Am. Chem. Soc. 2008, 130, 5626−5627. (c) Ding, J.; Xu, W.; Zhang, Y.; Sun, D.; Xiao, C.; Liu, D.; Zhu, X.; Chen, X. J. Controlled Release 2013, 172, 444−455. (d) Li, M.; Tang, Z.; Sun, H.; Ding, J.; Song, W.; Chen, X. Polym. Chem. 2013, 4, 1199−1207. (6) Ding, M.; He, X.; Wang, Z.; Li, J.; Tan, H.; Deng, H.; Fu, Q.; Gu, Q. Biomaterials 2011, 32, 9515−9524. (7) (a) MacKay, J. A.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chilkoti, A. Nat. Mater. 2009, 8, 993−999. (b) Alani, A. W. G.; Bae, Y.; Rao, D. A.; Kwon, G. S. Biomaterials 2010, 31, 1765−1772. (c) Jin, Y.; Song, L.; Su, Y.; Zhu, L.; Pang, Y.; Qiu, F.; Tong, G.; Yan, D.; Zhu, B.; Zhu, X. Biomacromolecules 2011, 12, 3460−3468. (d) Du, J.; Du, X.; Mao, C.; Wang, J. J. Am. Chem. Soc. 2011, 133, 17560−17563. (8) Wei, H.; Cheng, S.; Zhang, X.; Zhuo, R. Prog. Polym. Sci. 2009, 34, 893−910. (9) Meng, F.; Hennink, W. E.; Zhong, Z. Biomaterials 2009, 30, 2180−2198. (10) (a) Sun, Y.; Yan, X.; Yuan, T.; Liang, J.; Fan, Y.; Gu, Z.; Zhang, X. Biomaterials 2010, 31, 7124−7131. (b) Ding, J.; Shi, F.; Xiao, C.; Lin, L.; Chen, L.; He, C.; Zhuang, X.; Chen, X. Polym. Chem. 2011, 2, 2857−2864. (c) Li, J.; Huo, M.; Wang, J.; Zhou, J.; Mohammad, J. M.; Zhang, Y.; Zhu, Q.; Waddad, A. Y.; Zhang, Q. Biomaterials 2012, 33, 2310−2320. (d) Wang, K.; Liu, Y.; Yi, W.; Li, C.; Li, Y.; Zhuo, R.; Zhang, X. Soft Matter 2013, 9, 692−699. (11) Wang, C.; Chen, Q.; Wang, Z.; Zhang, X. Angew. Chem., Int. Ed. 2010, 49, 8612−8615. (12) (a) Wei, H.; Zhuo, R.; Zhang, X. Prog. Polym. Sci. 2013, 38, 503−535. (b) Li, Y.; Gao, G. H.; Lee, D. S. Adv. Healthcare Mater. 2013, 2, 388−417.

Figure 11. (A) T2-weighted MR images of oleic acid modified Fe3O4 nanoparticles and SPIO-loaded polyurethane micelles dispersed in 1.5% agarose gel at different Fe concentrations (mM). (B) T2 relaxation rates as a function of Fe concentrations (mM).

content and more concentrated clustering of SPIO nanoparticles may be response for the enhanced contrast efficiency.24,36 More work is warranted to better understand this phenomenon.

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CONCLUSION A novel cell internalizable and intracellularly degradable polyurethane micelle was developed for potential MR imaging and drug delivery applications. The nanovehicle displays a core−shell−corona nanostructure in aqueous solution, as verified by fluorescence measurement, 1H NMR, DLS, TEM, and DPD simulations. The nanocarriers exhibit high loading capacity and triggered release of therapeutics in response to an intracellular level of GSH. Furthermore, they can rapidly enter tumor cells and show an enhanced release of payloads within tumor cells, leading to an improved drug efficacy in vitro. In addition, oleic acid modified Fe3O4 nanoparticles were synthesized and encapsulated into the micellar core to form nanoclusters with different morphologies. The SPIO-loaded vehicles show good magnetic responsiveness and superparamagnetic behavior, as well as excellent MRI contrast effects and T2 relaxation properties. These nanosystems hold great promise to maximize the delivery efficiency and MRI relaxivity for cancer chemotherapy and diagnosis. Further work will focus on the combination of SPIO and anticancer drugs into a single multiblock polyurethane system to construct theranostic nanomedicines.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

Details of computational simulation, deconvolution analysis of FTIR spectra, GPC diagrams, fluorescence excitation spectra, additional FITR spectra, TEM images, and DLS and ζ potential J

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K

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