Construction of Targeting-Clickable and Tumor-Cleavable

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Construction of Targeting-Clickable and Tumor-Cleavable Polyurethane Nanomicelles for Multifunctional Intracellular Drug Delivery Nijia Song,† Mingming Ding,† Zhicheng Pan, Jiehua Li, Lijuan Zhou, Hong Tan,* and Qiang Fu College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China ABSTRACT: New strategies for the construction of versatile nanovehicles to overcome the multiple challenges of targeted delivery are urgently needed for cancer therapy. To address these needs, we developed a novel targeting-clickable and tumor-cleavable polyurethane nanomicelle for multifunctional delivery of antitumor drugs. The polyurethane was synthesized from biodegradable poly(ε-caprolactone) (PCL) and L-lysine ethyl ester diisocyanate (LDI), further extended by a new designed L-cystine-derivatized chain extender bearing a redoxresponsive disulfide bond and clickable alkynyl groups (Cys-PA), and finally terminated by a detachable methoxyl-poly(ethylene glycol) with a highly pH-sensitive benzoic-imine linkage (BPEG). The obtained polymers show attractive self-assembly characteristics and stimuliresponsiveness, good cytocompatibility, and high loading capacity for doxorubicin (DOX). Furthermore, folic acid (FA) as a model targeting ligand was conjugated to the polyurethane micelles via an efficient click reaction. The decoration of FA results in an enhanced cellular uptake and improved drug efficacy toward FA-receptor positive HeLa cancer cells in vitro. As a proof-of-concept, this work provides a facile approach to the design of extracellularly activatable nanocarriers for tumor-targeted and programmed intracellular drug delivery.

targeting specificity and inhibiting effective cellular uptake.6 To address this dilemma, detachable PEG corona could be employed to “maximize the stealth” in circulation and then detach in the tumor area to “maximize the targeting” of nanocarriers.5,7 Furthermore, drugs must be rapidly released once inside the target cell for realizing a sufficiently high intracellular level of therapeutics to enhance the drug bioavailability and overcome MDR. Hence, it is highly desirable to construct smart nanomicelles capable of responding to the intracellular environment and releasing their payloads in a triggered/controlled manner.8−10 To address the multiple challenges of targeted delivery, a “multifunctional” polymeric carrier system that integrates various desired functions has emerged as the next generation of nanomedicines to improve the therapeutic efficacy and ultimate clinical outcome.11 However, the fabrication of micellar systems with multiple functionalities still remains a great challenge, as conventional diblock and triblock copolymers always lack reactive sites for functionalization. Therefore, much research has recently been focused on the studies of multiblock copolymers and multicomponent micelles as drug carriers in the hunt for improved cancer therapy.12−14 Lately, we proposed a facile “molecular engineering” strategy for the design of multifunctional nanocarriers using multiblock

1. INTRODUCTION Over the past decades, studies on targeted polymeric nanocarriers to enhance the therapeutic efficiency of anticancer drugs have been rapidly developed in pharmaceutical research and clinical trials. Among such carriers, polymeric micelles, a kind of core−shell-type supramolecular nanostructure assembled from amphiphilic block copolymers, have attracted significant attention in the field of drug delivery. The hydrophobic core of the micelles is a loading space that accommodates waterinsoluble agents, and the hydrophilic shell is a protective corona that stabilizes the micelles in aqueous solution.1 The appropriate size (10−100 nm) of micelles allows an increased circulation time in the bloodstream and a passive accumulation in vascularized solid tumors through the enhanced permeability and retention (EPR) effect.2 However, the lack of cell-specific interactions and insufficient uptake at tumor sites may decrease the therapeutic efficacy and even induce multiple drug resistance (MDR).3 A facile approach to achieve active tumor targeting is to introduce targeting ligands, such as folic acid (FA) and antibodies, to the micellar surface through various conjugation chemistries.4 Nonetheless, the targeting molecules can expose nanocarriers to the reticuloendothelial system (RES) and result in opsonization-mediated clearance of nanocarriers.5 Coating the micellar surface with polyethylene glycol (PEG; i.e., PEGylation) can increase circulation time by reducing the interactions with serum proteins and alleviate uptake by phagocytic cells, whereas it also prevents the interactions between nanocarriers and cell surface, thus, compromising the © XXXX American Chemical Society

Received: September 7, 2013 Revised: November 7, 2013

A

dx.doi.org/10.1021/bm401342t | Biomacromolecules XXXX, XXX, XXX−XXX

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to ensure the drug loading capacity and biocompatibility/ biodegradability for safe drug administration, which also includes redox-cleavable linkages for triggered release of drugs under reduction intracellular environment.25 The side chain bears a variety of alkyne groups serving as active sites for “postfunctionalization” of polyurethane micelles with different targeting ligands and other functional elements via a facile click reaction. “Click” chemistry is a library of efficient and reliable reactions used to engineer the architecture and function of materials.26 The best-documented example is the copper catalyzed alkyne−azide cycloaddition (CuAAC), which can proceed rapidly in mild aqueous conditions, with negligible side reactions and minimal byproducts.27 Furthermore, to achieve “maximal stealth and maximal targeting” of nanocarriers, a protective poly(ethylene glycol) (PEG) coating was covalently incorporated through a highly acid-sensitive benzoic-imine linker. The linkage is stable at neutral and basic environment but cleaves in slight acidic conditions (e.g., extracellular environment of solid tumor, pH ∼6.5−7.2),28 which appears more promising for the construction of extracellularly activated “temporarily stealth” nanovehicles, compared with hydrazone,29 acetal,30 and orthoester31 that dissociate primarily in endosomal condition within tumor cells (pH ∼ 5). To obtain these polyurethanes, a novel multifunctional L-cystine-derivatized diamine chain extender containing redoxresponsive disulfide bond and clickable alkynyl groups (CysPA) and a pH-sensitive methoxyl-poly(ethylene glycol) with a benzoic-imine linkage (BPEG) was first designed and synthesized. Then a series of clickable and cleavable polyurethanes were synthesized from PCL, LDI, Cys-PA and BPEG. The bulk, micellization, stimuli-sensitivity, and drug loading properties of the resultant polyurethanes were fully characterized. Additionally, folate chosen as a model targeting molecule was modified with azide and attached onto the polymeric micelles through click chemistry. Subsequently, flow cytometry, confocal laser scanning microscopy (CLSM), and methyl tetrazolium (MTT) assay were carried out to investigate the in vitro cellular uptake, antitumor activity, and cytocompatibility of multifunctional polyurethane micelles.

Scheme 1. Design and Construction of Targeting-Clickable and Tumor-Cleavable Polyurethane Nanomicelles: (A) Schematic molecular structure of multiblock polyurethanes; (B) Self-assembled clickable polyurethane nanomicelles; (C) Conjugation of folate ligand via click chemistry; (D) Extracellular pH-activated detachment of PEG shell through the cleavage of benzoic-imine linkage; (E) Intracellular drug release triggered by the cleavage of disulfide bond in response to GSH

polyurethanes (MPU).15,16 The highly variable chemistry of polyurethanes17 enables the incorporation of different hydrophobic/hydrophilic segments and specific functional components into the polymeric chains to generate MPUs that exhibit rapid and adjustable degradation rates,18 controllable size and shape,19 as well as efficient cellular uptake of nanoassemblies.20 Moreover, pH-sensitive hydrazone16,21 and redox-responsive disulfide linkages15 were utilized to fabricate stimuli-responsive polyurethanes with stepwise bulk degradation and triggered drug release behaviors. In addition, folate as an end-capping reagent was introduced into the polyurethane structure for active targeting.22 Folate has been widely used as a ligand to target folate receptors (FRs) overexpressed in various types of human epithelial cancer cells and restricted in normal tissues.23 However, precoupling of folate to polymeric chain end prior to micellization does not facilitate the control of ligand density and type. Moreover, it is advantageous for conjugation methodologies to be applicable to a wide range of ligands and delivery systems with minimal chance for undesired side reactions with other active moieties in polymer structure.24 Herein, we report a novel targeting-clickable and tumorcleavable polyurethane micellar system for multifunctional intracellular drug delivery (Scheme 1). The backbone of the polyurethane comprises hydrophobic poly(ε-caprolactone) (PCL) and nontoxic L-lysine ethyl ester diisocyanate (LDI)

2. MATERIALS AND METHODS 2.1. Materials. N,N-Dimethylacetamide (DMAc) was dried over CaH2, vacuum distilled, and stored in the presence of 4 Å molecular sieves. PEG (Mn = 1900, Alfa Aesar, U.K.) and PCL (Mn = 2000, Dow Chemical, U.S.A.) were dehydrated under reduced pressure at 90 °C for 2 h before use. LDI was synthesized and purified according to a previous report.18a 4-Formylbenzoic acid (CBA, 98.0%) and propargylamine (PA, 98.0%) were purchased from Energy-chemical, China. N,N′-Dicyclohexylcarbodiimide (DCC, ≥98.0%) and L-cysteine (≥99.0%) were obtained from Chengdu Kelong Chemical Co., Ltd., China. Di-tert-butyl dicarbonate (Boc2O, ≥98.0%) and 4-dimethylaminopyridine (DMAP, ≥99.0%) were bought from Asta Tech Pharmaceutical Co., Ltd., China. 2-Bromoethylamine hydrobromide (98.0%) and sodium ascorbate (99.0%) were purchased from Aladdin Reagent Database Inc., China. 1-Hydroxybenzotrizole (HoBt; Shanghai Yuanju Biological Technology Co., Ltd., China, 99.0%), ethanolamine (Tianjin Bodi Chemical Co., Ltd., China, ≥99.0%), sodium azide (Sanland-chem, International Inc., China, ≥99.5%), 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC; J&K Chemical Ltd., China, 98.0%), N-Hydroxysuccinimide (NHS; Alfa Aesar, U.K., ≥98.0%), folic acid (Sinopharm Chemical Reagent Co., Ltd., China, ≥97.0%), and doxorubicin hydrochloride (DOX·HCl, Tecoland Corporation, U.S.A.) were used as received. 2.2. Characterization. Proton nuclear magnetic resonance spectroscopy (1H NMR) was obtained on a Varianunity Inova-400 spectrometer B


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2.4. Synthesis of Cys-PA. DCC (4.33 g) and HoBt (4.05 g) were added into a solution of Boc-Cys (4.40 g) in CH2Cl2 kept in an ice salt bath, and then propargylamine (1.38 g) was added. The mixture was stirred for 24 h at room temperature. After reaction, ethyl acetate with dilute hydrochloric acid was added to extraction product. The ethyl acetate containing Boc-Cys-PA was washed with saturated NaHCO3 solution, saturated solution of sodium chloride and distilled water, and dried over anhydrous MgSO4. At last, the solvent ethyl acetate was removed by evaporation. 1 H NMR (400 MHz, DMSO, TMS, δ in ppm): 1.38 (s, 18H, -CH3), 2.80 (t, 2H, -CCH), 3.07 (m, 4H, -S−CH2-), 3.84 (s, 4H, -NH-CH2-CC-), 4.19 (s, 2H, -CH-), 7.06 (d, 2H, Boc-NH-), 8.41 (d, 2H, -HC−CO-NH-CH2-). FTIR (cm−1): 3335.50, 3059.35 (s, ν N−H), 2977.23 (s, ν CH3), 2932.40 (s, ν CH2), 2860.00 (m, ν CH2−S), 1688.79 (s, ν CO, free), 1659.97 (s, ν CO, H-boned), 1520.33 (s, δ C−N−H), 1446.31 (m, δ CH2), 1416.12 (m, δ CH2−S), 1366.41, 1389.65 (s, δ CH3), 641.00 (m, broad, δ ≡CH). The Boc-protected amine groups of Boc-Cys-PA were converted to primary amine groups (Cys-PA) with 20 mL of hydrogen chloride saturated ethyl acetate. Then the solvent was removed under reduced pressure. The product was redissolved in methanol and the pH was adjusted around 8.5 with NaHCO3. Impurity was removal by suction filtration, and the filtrate was concentrated by evaporation (yield: 50−60%). 1 H NMR (400 MHz, DMSO, TMS, δ in ppm): 2.75 (t, 2H, -C CH), 3.03 (m, 4H, -S−CH2-), 3.51 (m, 2H, H2N-CH-), 3.86 (s, 4H, -NH-CH2-CC-), 8.48 (d, 2H). FTIR (cm−1): 3390.00, 3300.00 (s, ν N−H), 2932.40 (s, ν CH2), 2860.00 (m, ν CH2−S), 1723.03 (s, ν CO, free), 1660.49 (s, ν CO, H-boned), 1565.58 (s, δ C−N−H), 1453.09 (m, δ CH2), 1416.12 (m, δ CH2−S), 626.25 (m, broad, δ ≡CH). MS (APCI, positive) m/z: Calcd, 314.00 g mol−1; found, 314.99 g mol−1. 2.5. Synthesis of MPEG-CBA. CBA (15.20 g) was added to a solution of MPEG (11.93 g) in dichloromethane. Subsequently, DCC (16.50 g) and DMAP (2.44 g) were added, and the mixture was stirred mildly for 60 h at room temperature. After filtration to remove the precipitated dicyclohexylurea (DCU), the solvent was evaporated under vacuum. The product was purified by recrystallization three times in isopropanol, and dried under vacuum at 45 °C for 3 days (yield: 90%). 1 H NMR (400 MHz, CDCl3, TMS, δ in ppm): 3.38 (s, 3H, -OCH3), 3.64 (m, -CH2- of PEG), 3.86 (t, 2H, -O−CH2-), 4.52 (t, 2H, -CO-CH2-), 7.95 (d, 2H, -CH- of benzene ring), 8.23 (d, 2H, -CH- of benzene ring), 10.12 (s, 1H, -CHO). FTIR (cm−1): 2883.00 (s, ν CH2), 2850.00 (s, ν CH3), 2820.28, 2740.07 (w, ν C−H of −CHO), 1720.93 (s, ν CO), 1702.51 (s, ν CO, −CHO), 1598.49, 1575.44, 1498.58 (w, ν CC of benzene), 1466.67 (m-s, δ CH2), 1343.57 (m, δ CH3), 1147.46 (s, ν C−O−C, COOC), 1114.97 (s-m, ν C−O−C, PEG), 760.16 (s, δ C−H of benzene). 2.6. Synthesis of BPEG. Ethanolamine (3.66 g) was added to a solution of MPEG-CBA (13.00 g) in tetrahydrofuran (THF, 280 mL) and the mixture was stirred at 40 °C for 12 h. The solvent was evaporated under reduced pressure. The product was dissolved in methanol, precipitated in anhydrous ethyl ether to remove impurities,

(400 MHz, U.S.A.) using tetramethylsilane (TMS) as an internal standard and CDCl3 or DMSO-d6 as solvents. Fourier transform infrared spectroscopy (FTIR) was recorded on a Nicolet 6700 spectrometer (Thermo Electron Corporation, U.S.A.) between 4000 and 600 cm−1, with a resolution of 4 cm−1. Gel permeation chromatography (GPC) was performed with Waters-1515 (U.S.A.) using N,N-dimethylformamide (DMF)/LiBr as eluent. The molecular weights are relative to polymethyl methacrylate (PMMA) standards. The flow rate was 1.0 mL min−1 at 40 °C. Differential scanning calorimetry (DSC) was carried out on a TA Q20 instrument (U.S.A.) at a heating rate of 10 °C min−1 from −100 to 100 °C under a steady flow of nitrogen. Transmission electron microscopy (TEM) was performed on a Hitachi model H-600-4 transmission electron microscope (Japan) with an accelerating voltage of 75 KV. A drop of micellar solution stained by 1% (w/v) phosphotungstic acid was placed on a copper grid with Formvar film, and then the liquid was blotted off and air-dried before measurement. Sizes and zeta potentials of micelles were measured with a Zetasizer Nano ZS dynamic light-scattering (DLS) instrument (Malvern, U.K.) at 25 °C at an angle of 90°. Turbidity measurements were performed using a SGZ-500IT nephelometer (Shanghai Yuefeng Instruments and Meters Co. Ltd., China), and the results were reported in nephelometric turbidity units (NTU). Fluorescence measurements were carried out on a 970 CRT fluorescence spectrophotometer (Shanghai Precision and Scientific Instrument Co., Ltd., China). As a hydrophobic probe, certain amounts of pyrene in acetone were added into a series of vials. After acetone was evaporated, to each vial was added micellar solution with different concentrations. The final concentration of pyrene was 5.0 × 10−7 M. All samples were immersed in a sonicator for 4 h at room temperature. Steady-state fluorescence spectra were recorded with bandwidths of 5.0 nm for excitation and 2.0 nm for emission, respectively. The excitation spectra (λem = 372.0 nm) were collected, and the intensity ratio of the peak at 336.5 nm to that at 333.8 nm from excitation spectra is plotted against the log of the micelle concentration. Critical micelle concentrations (CMCs) were calculated according to the plot. 2.3. Synthesis of Boc-Cys. Triethylamine (15.20 g) was added to an aqueous solution of cysteine (12.00 g) cooled in an ice water bath with stirring. Then Boc2O (32.70 g) was added. The reaction was kept for 2 h, and the solvent was evaporated under reduced pressure. The product was dissolved in ethyl acetate and washed with 10% ice hydrochloric acid, saturated solution of sodium chloride and distilled water, and dried over anhydrous MgSO4. Then, after filtration of the solution, the solvent was removed (yield: 86%). 1 H NMR (400 MHz, DMSO, TMS, δ): 1.37 (s, 18H, −CH3), 2.85 (m, 4H, -S−CH2-), 4.25 (s, 2H, -CH-), 6.36 (d, 2H, -NH-). FTIR (cm−1): 3370.25, 3167.84 (s, ν N−H), 2983.21 (s, ν CH3), 2934.29 (s, ν CH2), 2860.00 (m, ν CH2−S), 1710.23 (s, ν CO), 1688.92 (s, ν CO), 1518.33 (s, δ C−N−H), 1445.00 (m, δ CH2), 1416.12 (m, δ CH2−S), 1368.33, 1392.52 (s, δ CH3), 929.25 (m, ρ O−H, two association).

Table 1. Theoretical Composition, Molecular Weight, and Thermal Properties of Clickable and Cleavable Multiblock Polyurethanes molecular weightsb (g mol−1)

feed ratio (mol) samples

a

C100B0 C100B25 C100B50 C100B75 C100B100

Tgc (°C)

LDI

PCL

MPEG

BPEG

Cys-PA

Mn

Mw

Mw/Mn

2 2 2 2 2

0.8 0.8 0.8 0.8 0.8

0.4 0.3 0.2 0.1 0

0 0.1 0.2 0.3 0.4

1 1 1 1 1

14200 14600 12300 14200 15500

18800 18700 17200 18400 18700

1.33 1.28 1.40 1.30 1.21

−60.4 −60.2 −60.9 −61.4 −60.6

a Multiblock polyurethanes are denoted as CXBY, where C is for Cys-PA, B is for BPEG, X and Y are for the molar fraction of Cys-PA in chain extender and BPEG in soft segment, respectively. bMolecular weights and molecular weight distributions were determined by GPC. cTg is measured by DSC.

C


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Scheme 2. Synthesis of Multifunctional Chain Extender Cys-PA

Scheme 3. Synthesis of pH-Sensitive BPEG

Figure 1. 400 MHz 1H NMR spectra of clickable and cleavable polyurethanes in DMSO-d6: (A) C100B100, (B) C100B0, and (C) C0B50. (520.00 mg) and EDC (880.00 mg) was added and stirred for 1 h. Then a solution of N3EA (473.00 mg) in DMF was mixed into the system. The reaction was kept at room temperature for 36 h. Subsequently, the product was precipitated in water, washed with acetone and dried under vacuum (yield: 70%). 1 H NMR (400 MHz, DMSO, TMS, δ in ppm): 1.87 (m, 1H, -CHCH2-CH2-), 2.03 (m, 1H, -CH-CH2-CH2-), 2.19 (t, 2H, -CH2-CH2CO-), 2.31 (t, 2H, -CH2-N3), 3.23 (m, 2H, -CH2-CH2-N3), 4.32 (m, 1H, -CH-CH2-), 4.48 (d, 2H, -CH2-NH-Ph), 6.64 (d, 2H, -CH- of benzene ring), 6.93 (t, 2H, -NH2), 7.66 (m, 2H, -CH- of benzene ring), 8.10 (m, 2H, s, -CO-NH-CH-), 8.64 (s, 1H, -CH of pteridine group). FTIR (cm−1): 3357.00 (s, ν NH2), 2102.35 (s, ν −N+N). MS (APCI, positive) m/z: Calcd, 509.40 g mol−1; found, 508.11 g mol−1. 2.9. Synthesis of Clickable and Cleavable Polyurethanes. A series of biodegradable multiblock polyurethanes bearing reductionresponsive disulfide bonds, pH-sensitive benzoic-imine linkages, and clickable alkynyl groups were synthesized from LDI, PCL, MPEG/ BPEG, and Cys-PA using a multistep solution polymerization in DMAc. The feed ratios are shown in Table 1. PCL was first copolymerized with LDI at 60 °C under a dry nitrogen atmosphere in the presence of stannous octoate catalyst for 1 h. After cooling to room temperature, chain extender Cys-PA was added and allowed to react with prepolymers for 1 h at room temperature, followed by another 2 h at 60 °C. Finally, MPEG and BPEG with different molar ratios (Table 1) were added to react for 6 h. The polymer obtained was precipitated in anhydrous ethyl ether and dried under vacuum at 60 °C for 3 days. 2.10. Preparation of Multiblock Polyurethane Micelles. To prepare clickable and cleavable polyurethane micelles, 5 mL of polymer solution in DMAc (10 mg mL−1) was dropped (1 drop every 30 s) into 20 mL of phosphate buffered saline (PBS, pH 7.4). The solution was then transferred to a dialysis tube (MWCO 3500) and dialyzed against PBS for about 3 days to remove the organic solvent at room

and further recrystallized in isopropanol. The pure BPEG was dried under vacuum at 40 °C for 3 days. 1 H NMR (400 MHz, CDCl3, TMS, δ in ppm): 3.38 (s, 3H, -O− CH3), 3.64 (m, -CH2- of PEG), 3.77 (m, 2H, -CN-CH2-CH2-), 3.83 (m, 2H, -O−CH2-), 3.94 (m, 2H, -CN-CH2-CH2-), 4.51 (t, 2H, -CH2O−CO-), 7.81 (d, 2H, -CH- of benzene ring), 8.11 (d, 2H, -CH- of benzene ring), 8.41 (s, 1H, -CHN-). FTIR (cm−1): 3300.00 (w, broad, ν O−H, H-boned), 2883.89 (s, ν CH2), 2850−2815 (s, ν CH3), 1721.41 (s, ν CO), 1608.91, 1557.35, 1503.28 (w, ν CC of benzene), 1467.34 (s, δ CH2), 1450 (s, δ CH3), 1148.64 (s, ν C−O− C, COOC), 1116.21 (s-m, ν C−O−C, PEG), 1060 (s, ν C−O of −CH2−OH), 760.58 (s, δ −CH of benzene). 2.7. Synthesis of N3EA. 2-Bromoethylamine hydrobromide (10.25 g) was dissolved in distilled water with mild stirring. Sodium azide (9.75 g) was added carefully and the mixture was kept at 70 °C with refluxing for 12 h. Thereafter, the system was cooled to 0 °C and NaOH (7.00 g) was added. The product was extracted with anhydrous ethyl ether and dried over MgSO4. After filtration of the solution, the solvent was allowed to evaporate under atmospheric conditions (yield: 50−60%). 1 H NMR (400 MHz, CDCl3, TMS, δ in ppm): 1.54 (s, 2H, -NH2), 2.89 (t, 2H, -CH2-NH2), 3.38 (t, 2H, N3-CH2-). FTIR (cm−1): 3357.00 (s, ν N−H), 2931.63 (s, ν CH2), 2102.35 (s, ν -N+N), 1584.00 (s, δ NH2), 1473.81 (s, δ CH2). 2.8. Synthesis of N3FA. After folic acid (2.00 g, 4.4 mmol) was completely dissolved in DMF precooled in an ice water bath, NHS D


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Figure 2. FTIR spectra (A) and DSC thermograms (B, C) of clickable and cleavable polyurethanes: (a) C100B0, (b) C100B25, (c) C100B50, (d) C100B75, (e) C100B100, and (f) C0B50.

Figure 3. (A) Typical fluorescence excitation spectra (λem = 372 nm) of clickable and cleavable polyurethane nanomicelles. (B) I336.5/I333.8 ratios in the excitation spectra as a function of micellar concentrations (log C). The CMCs are obtained from the intersection of the two tangent lines shown by the arrows, and summarized in Table 2.

temperature. The micelle solution was centrifugalized at 3000 r min−1 for 20 min and passed through a 0.45 μm pore-sized syringe filter (Milipore, Carrigtwohill, Co. Cork, Ireland). 2.11. Stability and Stimuli-Responsiveness. To study the stability of polyurethane micelles, the samples were incubated at 37 °C in a shaker, and the size was determined at the scheduled time. Moreover, the micelles were further diluted in PBS (1:30) and equilibrated for 1 h before turbidity measurement. To investigate the stimuli-responsive properties of micelles, HCl or GSH were added into the micellar solutions to simulate the excellular acidic (pH ∼6.5) or intracellular reduction environments (10 mM GSH) in the tumor. All the samples were incubated at 37 °C with shaking (110 r min−1), and monitored by size measurement and TEM observation. 2.12. Drug Loading. DOX as a model drug was loaded into C100BPU micelles using a dialysis method. First, DOX·HCl and polyurethanes with different weight ratios were codissolved in DMAc solution, and treated with triethylamine for 2 h with stirring to remove hydrochloride. Afterward, the solution was dropped into PBS to induce micellization. The micelle solutions was dialyzed, centrifugalized and filtered as described in the previous section. The amount of DOX loaded inside micelles was determined using a Hitachi F-7000 FL spectrophotometer (excitation at 480 nm). Drug loading content and encapsulation efficiency were calculated according to the following equations:

Table 2. Size, Size Distribution (PdI), Zeta Potential, and Critical Micelle Concentration (CMC) of Clickable and Cleavable Multiblock Polyurethanes sample

size (nm)

PdI

zeta potential (mV)

CMC (10−3 mg mL−1)

C100B0 C100B25 C100B50 C100B75 C100B100 C100B100-FA

105.0 122.0 67.3 153.0 99.8 103.0

0.306 0.217 0.424 0.209 0.239 0.459

−41.7 −30.6 −23.6 −21.1 −15.0 −14.6

3.4 2.0 2.6 4.0 4.2 2.9

2.13. Conjugation of Folate via Click Reaction. Folate was chemically attached onto the polyurethane nanomicelles through a CuAAC click reaction. Briefly, drug-loaded or drug-free micelle solutions (50 mL) were mixed with N3FA (10.00 mg) in the presence of sodium ascorbate (1.00 mg) and CuSO4·5H2O (0.50 mg) to react at room temperature under moderate stirring for 24 h. Afterward, the solution was transferred to a dialysis tube (MWCO 3500) and dialyzed against PBS for about 5 days to remove the unreacted N3FA and traces of the catalyst. Then the micellar solution was centrifugalized at 3000 r min−1 for 20 min and passed through a 0.45 μm pore-sized syringe filter. To determine the amount of folate conjugated, ultraviolet−visible (UV−vis) spectroscopy was performed on a UV-1800PC spectrophotometer (Mapada Instruments, China), using calibration curve obtained from PBS solutions with different N3FA concentrations.

loading content (%) = weight of loaded drugs/weight of drug-loaded micelles × 100%

encapsulation efficiency (%) = weight of loaded drugs/weight of feeding drugs × 100% E


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Figure 4. (A) Time-dependent size and turbidity of polyurethane micelles under physiological conditions. (B) The change of micellar size in response to acidic (pH 6.5) and redox (10 mM GSH) environments. Schematic structures (C) and TEM images (D−F) of polyurethane micelles before (E) and after incubation at pH 6.5 (D) and 10 mM GSH (F) for 24 h. The bars are 100 nm. (DAPI, 10 μg mL−1, Sigma-Aldrich, U.S.A.) for 10 min. The slides were mounted with 10% glycerol solution and examined using a Leica TCS SP5 (Leica Microscopy Systems Ltd., Germany). 2.16. Cytotoxicity Assay. MTT assay was carried out to evaluate the cytotoxicity of blank polyurethane nanomicelles and antitumor activity of DOX-loaded micelles in vitro. L929 mouse fibroblasts and HeLa cells were harvested in a logarithmic growth phase with 0.25% (w/v) trypsin in D-Hanks solution (Gibco), and seeded in 96-well plates (Corning, U.S.A.) at a density of 5 × 103 cells/well for 24 h of incubation. The cells were treated with 100 μL medium containing various concentrations of drug-free and DOX-loaded micelles. Sterile PBS and free DOX solutions diluted to the same concentrations with culture media were set as negative control and positive control, respectively. At the designated time intervals (24 and 72 h), 20 μL of MTT solution in PBS (5 mg mL−1, Sigma-Aldrich, U.S.A.) was added to each well. After incubation for 4 h at 37 °C, the MTT solution was removed and the insoluble formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO). The plates were shaken for 10 min, and the absorbance of formazan product was measured at 490 nm on a microplate reader (DNM-9602, Nanjing Perlove Medical Equipment Co., Ltd., China). The cell viability was normalized to that of untreated cells. The dose−effect curves were plotted and the median inhibitory concentration (IC50) was determined using the software GraphPad Prism 5 for Windows (GraphPad Software, Inc., San Diego, CA).

2.14. Cell Culture. L929 mouse fibroblasts were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and HeLa cells were supplied by West China Medical Center of Sichuan University. All the cells were maintained in a RPMI 1640 medium (HyClone, U.S.A.) containing 10% fetal bovine serum (FBS, Gibco Life, U.S.A.) and 5% penicillin-streptomycin (Gibco Life, U.S.A.) in a humidified atmosphere of 5% CO2 at 37 °C. 2.15. Cellular Uptake. For flow cytometry, HeLa cells were seeded in cell culture flasks (25 × 25 mm, Corning, U.S.A.) as usual. When the cells reached 70−80% confluence, the medium was removed, and the cells were washed twice with PBS (pH 7.4) and treated with DOX-loaded micelles in a humidified incubator with 5% CO2 atmosphere at 37 °C. After 2 and 4 h of incubation, the medium was discarded and the cells were washed twice with PBS. The cells were then detached by 0.25% (w/v) trypsine, dispersed in 5 mL of medium, and centrifuged three times. At last, the cells were resuspended in 0.5 mL of PBS, and analyzed based on the DOX fluorescence using a FACS Aria flow cytometer (BD Biosciences). For confocal laser scanning microscope (CLSM) studies, HeLa cells were seeded in six-well plates (Corning, U.S.A.; a clean coverslip was put in each well) at a density of 1× 105 cells/well and grown for 24 h. The cells were incubated with DOX-loaded micelles for 2 and 4 h at 37 °C. After removal of the supernatant, the cells were washed three times with cold PBS, fixed with 1 mL of 4% paraformaldehyde for 30 min at 4 °C, and stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride F


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Figure 5. (A) Schematic illustration of folate-conjugation via click chemistry. (B−E) TEM images (B), size distributions (C), 1H NMR spectra (D), and UV−vis spectra (E) of clickable and cleavable polyurethane micelles before (a) and after (b) conjugation with folate. Scale bars in (B) are 100 nm, and insets in (D) show enlarged 1H NMR spectra and illustrated molecular structure of FA residue.

using 1H NMR and FTIR, as clarified in the Materials and Methods. Benzoic-imine bond is a highly pH-sensitive bond that hydrolyzes under quite weak acidic conditions (pH ∼ 6.5− 6.8), whereas stable at neutral and basic environment because of the proper π−π conjugation extent.28 It is well-known that most extracellular pH at a solid tumor site is 6.5−7.2, compared with that at normal tissue (7.4).32 Therefore, the incorporation of BPEG containing benzoic-imine linkage is expected to provide good protection for carriers and ligands in circulation and to be detached under extracellular environment of tumor tissues for maximizing the targeting effect and intracellular delivery of antitumor drugs. 3.3. Synthesis of Clickable and Cleavable Polyurethanes. A series of biodegradable multiblock polyurethanes bearing reduction-responsive disulfide bonds, pH-sensitive benzoicimine linkages, and clickable alkynyl groups were synthesized from PCL, LDI, Cys-PA, and BPEG using a solution polymerization. The structures of the multiblock polyurethanes are illustrated in Scheme 1. By changing the feed ratios of monomers, the amount of alkynyl sites as well as the degree of stimuli sensitivity can be controlled, as listed in Table 1. The obtained multiblock polyurethanes are denoted as CXBY, where C is for Cys-PA, B is for BPEG, X and Y are for the fed molar fractions of Cys-PA in chain extender and BPEG in soft segment, respectively. All the polymers present moderate molecular weights (Mn is about 12000−15000), with monodisperse and quite narrow molecular weight distributions (Table 1).

3. RESULTS AND DISCUSSION 3.1. Synthesis of Multifunctional Chain Extender. To prepare novel clickable and cleavable polyurethanes, a multifunctional L-cystine-derivatized diamine chain extender containing a redox-responsive disulfide bond in the backbone and two clickable alkynyl groups in the side chains (Cys-PA) was first designed and synthesized. The synthesis route is illustrated in Scheme 2. The structures of Cys-PA and its intermediates were identified with 1H NMR, FTIR and MS, as described in the Materials and Methods. The disulfide linkage incorporated is in favor of triggered release of payloads in response to intracellular levels of glutathione (GSH), while the alkynyl groups can provide active sites for further conjugation of targeting ligands through click chemistry. In addition, cystine and its reduced product cysteine are both amino acids used as protein building blocks throughout the body. Thus, the inclusion of Cys-PA may ensure the biocompatibility of polyurethanes as well as their degradation products. 3.2. Synthesis of pH-Detachable End-Capping Agent. To achieve the “temporarily stealth” property of polyurethane nanocarriers, MPEG was first functionalized by reacting with CBA in the presence of DCC and DMAP. Then the aldehydeended PEG was further reacted with ethanolamine to yield a pH-sensitive end-capping monomer with a benzoic-imine linkage. The synthetic procedure is shown in Scheme 3. The chemical structures of MPEG-CBA and BPEG were examined G


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Scheme 4. Synthesis of (A) N3EA and (B) N3FA

The 1H NMR spectra of polyurethanes are depicted in Figure 1, where all characteristic peaks of PCL, PEG, and LDI can be found. The peaks at 3.97 (-COOCH2-), 2.26 (-CH2COO-), 1.52 (-CH2CH2CH2-), and 1.30 ppm (-CH2CH2CH2-) are assigned to the methylene protons of PCL unit. The sharp peak at 3.50 ppm is attributed to the methylene protons of PEG block (-CH2CH2O-). The chemical shifts of methylene (-CH2-OCO-) and methyl protons (-CH3) in the ethoxyl group of LDI are at 4.07 and 1.15 ppm, respectively. The characteristic peak of the imine proton (-HCN-) is at 8.20 ppm, and the signals at 8.02 and 7.89 ppm are originated from benzene ring of BPEG. These peaks can not be found in the spectrum of C100B0 without BPEG, suggesting that corresponding polyurethane (C100B100) is terminated by BPEG. In addition, peaks at 2.73 and 3.07−3.16 ppm are ascribed to the alkynyl proton (-CCH) and methylene protons next to the disulfide bond (-S−S−CH2-) in Cys-PA, respectively, demonstrating that the multifunctional chain extender has been successfully introduced into the chains of polyurethanes. The FTIR spectra of polyurethanes are shown in Figure 2A. The stretching band in the 1600−1800 cm−1 region is overlapped by the absorption of ester carbonyl groups of PCL, free and hydrogen-bonded carbonyl of urethane groups, where a shoulder observed at 1654 cm−1 is ascribed to the hydrogenbonded carbonyl of urea groups. A broad stretching band around 3340 cm−1 is mainly attributed to the hydrogen-bonded N−H stretching vibration.33,34 These results suggest an existence of microphase separation in the multiblock polyurethanes.34,35 To better understand the bulk property, DSC measurement was carried out to investigate the thermal behavior of polyurethanes. The glass transition temperatures (Tg) of soft segment, frequently used as an indicator of the degree of phase separation,34,36 was shown in Figure 2B and listed in Table 1. Evidently, the Tgs of polyurethanes are in the range of −62 ∼ −60 °C, which are close to those of pure PEG (−53 to −64 °C) and PCL (−58 °C),37 and much lower than those for other PCL- and PEG-based polyurethane systems reported in our previous work.18a,38 This result agrees with

Figure 6. (A) Drug loading content and encapsulation efficiency for DOX loaded in clickable and cleavable polyurethane micelles (C100B100) at different drug feed ratios. (B) Drug loading results for C100B100 and C100B100-FA micelles.

Figure 7. Flow cytometry analysis of HeLa cells treated with DOXloaded polyurethane micelles with or without FA conjugation for different times.

FTIR analysis and further demonstrates a high degree of phase separation between hard and soft segments in these polyurethanes.34 In addition, no thermal transition related to hard segment is observed in all DSC curves (Figure 2C). This is possibly due to the fact that structurally asymmetric LDI bearing ethyl ester side chains can hinder the chain packing of hard segment.39 Interestingly, no melting peak was recorded for all the samples except C0B50. This might be attributed to the presence of Cys-PA side chains that increases the steric H


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Figure 8. CLSM images of HeLa cells incubated with DOX-loaded polyurethane micelles with (B, D) or without (A, C) FA conjugation for 2 h (A, B) and 4 h (C, D): Nuclei of cells were stained with DAPI.

hindrance for crystallization of soft segment.40 Furthermore, the mixing of PEG and PCL phase in the soft segment may also account for the inhibited crystallization ability.41 Further work is needed to better understand this phenomenon. 3.4. Preparation and Characterization of Polyurethane Micelles. The prepared multiblock polyurethanes can selfassemble into nanomicelles in an aqueous solution, as verified by fluorescence measurement using pyrene as a probe. A typical fluorescence excitation spectrum of polyurethane micelles is given in Figure 3A. It was found that the (0, 0) absorption band shifts from 333.8 to 336.5 nm with an increase of polyurethane concentration in aqueous solution of pyrene, suggesting that pyrene molecules are transferred from an aqueous environment to a hydrophobic microenvironment within the micellar core.42 The diameters of the micelles determined by DLS are in the range of 67−153 nm, and the polydispersity indices (PdI) are between 0.2 and 0.5, with bimodal size distributions (Table 2). The nanocarriers exhibit well-dispersed individual particles with a regularly spherical shape, as evidenced by TEM observation (Figures 4E and 5B). The zeta potentials of nanoparticles in PBS solution are listed in Table 2. All the micelles display negative surface charges ranging from −41.7 to −14.6 mV, which may be attributed to the presence of ionized carboxyl groups of PCL segments and the polarization of water molecules under the effect of PEG.43

Interestingly, it was noticed that the absolute zeta potential values of the micelles decreased with the increasing content of BPEG (Table 2). This is probably due to that the increased amount of benzoic-imine linkages between micellar core and PEG corona may affect the flexibility and spatial organization of PEG chains on micelle surface.43a To determine the CMC of polyurethane micelles, the intensity ratios of I336.5/I333.8 from the excitation spectra of pyrene were plotted against the log of polyurethane concentrations (Figure 3B). The CMCs are obtained from the intersection of the two tangent lines and summarized in Table 2. These values (2.0−4.2 × 10−3 mg mL−1) are much lower than those reported for traditional diblock,44 triblock,45 branched,46 and star-shaped47 copolymer micelles based on PCL and PEG, implying that the polyurethane micelles are thermodynamically stable. The stability of polyurethane micelles was further verified by monitoring the size and turbidity under a simulative physiological condition. It was found that the micelles are rather stable in aqueous solution, with size and turbidity almost unchanged during the incubation times (Figure 4A). However, as the solution pH changes from 7.4 to 6.5, the micellar diameter increases moderately and the surface of particles appears uneven (Figure 4B,D). This is probably due to the cleavage of benzoic-imine bond under acidic conditions leading to partial detachment of PEG corona and further aggregation of micelles. I


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Moreover, due to the cleavage of disulfide linkages in the polymer backbone, the micelles disintegrate in the presence of 10 mM GSH, with size dramatically increased and visible precipitates observed in the micelle solution (Figure 4B,F). These results suggest that the multiblock polyurethane micelles can respond to acidic and redox environments in tumors, which are potentially useful for intracellular delivery of therapeutics. More work is needed and ongoing to investigate the kinetics of shelldetachment and core-breakage of polyurethane micelles, as well as their effects on the drug release profiles and cell internalization ability. 3.5. Conjugation of FA via Click Chemistry. To improve the efficiency and specificity of nanocarriers, folate was chosen as a model targeting ligand for further functionalization of polyurethane nanomicelles. Toward this goal, the γ-carboxylic group of folate was first reacted with N3EA to give the azide folate (N3FA; Scheme 4). The chemical structures of N3FA and its intermediates were confirmed using 1H NMR, FTIR, and MS as described in the Materials and Methods. Taking C100B100 as an example, azide-modified folate was covalently attached onto the polyurethane micelles via a facile click reaction (Figure 5A). After conjugation, the targeted micelles present some irregular morphology (Figure 5B), with mean diameter and zeta potential nearly unchanged, and size distribution increased slightly (Figure 5C and Table 2). The success of folate conjugation was verified using 1H NMR and UV−vis spectroscopy. As seen from the 1H NMR spectra (Figure 5D), the signal of alkynyl proton (-CCH) at 2.73 ppm in the 1H NMR spectrum of C100B100 disappears, while a new peak could be observed at 7.74 ppm in the spectrum of C100B100-FA, which was attributed to the 1,2,3triazole proton resulted from CuAAC click reaction. Moreover, characteristic peaks of folate can be found at 8.63 (-CHN), 7.67 (Ph-C2H and Ph-C6H), 6.94 (-NH2), and 6.63 ppm (PhC3H and Ph-C5H). All these signals can not be observed in the spectrum of C100B100, indicating that folate ligand has been chemically conjugated to the polyurethane micelles. In addition, the UV−vis spectra of C100B100 and C100B100-FA micelles are depicted in Figure 5E. The appearance of maximum absorption peak of C100B100-FA at 280 nm corresponding to the aromatic chromophore of folate informs that the multiblock polyurethane micelles have been click-functionalized with folate. According to the calibration curve generated from the N3FA standard solutions at known concentrations, the content of folate conjugated was calculated to be about 3.7 w/w %, which is deemed sufficient to provide active targeting for polyurethane nanovehicles.48 Furthermore, the folate concentration can be easily controlled trough the amount of clickable alkynyl sites and the feed ratios of N3FA, as verified in our ongoing work. 3.6. Drug Loading Study. To evaluate the loading capacity of polyurethane nanomicelle as a vehicle for chemotherapeutic agents, doxorubicin (DOX) was chosen as a model hydrophobic drug. DOX is one of the most potent drugs frequently used to address a broad number of different kinds of cancers.49 However, like most anticancer drugs, DOX suffers from its poor solubility and acute toxicity to normal tissue. To address these issues, DOX was loaded into multiblock polyurethane micelles using a dialysis method. As illustrated in Figure 6A, the polyurethane nanovehicles can encapsulate DOX efficiently, with maximum drug loading content (LC) over 23%, which is much higher than those reported for conventional amphiphilic block copolymer micelles.14,50,51 Interestingly, the FA-conjugated micelles

Figure 9. Cytotoxicity of DOX-loaded polyurethane micelles against HeLa cells for 24 h (A) and 72 h (B) of incubation. Free DOX was used as a positive control. (C) IC50 values of various DOX formulations toward HeLa cells for different incubation times.

exhibit relatively lower drug loading capacity compared with nonconjugated nanomicelles (Figure 6B). A possible explanation could be that the “post-conjugation” of folate molecules after drug loading may lead to premature leakage and release of drugs from the micelles during click reaction and dialysis process. 3.7. In Vitro Cellular Uptake. Flow cytometry analysis was performed to investigate the uptake and internalization of multiblock polyurethane micelles with or without “clicked” FA moiety against folate receptor (FR) positive HeLa cells. Since DOX itself is fluorescent, it can be used directly to measure cellular uptake without additional fluorescent markers. J


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Figure 10. Cell viability of HeLa cells (A, B) and L929 mouse fibroblasts (C, D) after 24 h (A, C) and 72 h (B, D) of incubation with various concentrations of clickable and cleavable polyurethane micelles.

Figure 7 shows the flow cytometry data for DOX-loaded C100B100 and C100B100-FA micelle after incubation with HeLa cells for different times. Surprisingly, it appears that targeted nanoparticle does not enter tumor cells as fast as expected, with internalization efficiency even slightly lower than that for nontargeted nanomicelles at the first 2 h of incubation. However, with longer incubation time up to 4 h, C100B100-FA micelles showed an enhanced cellular uptake compared with C100B100. To better understand this phenomenon, CLSM was employed to visualize the cellular uptake of micellar formulations. As shown in Figure 8, C100B100 and C100B100-FA display similar fluorescence intensities at 2 h and the fluorescence increases with time. Particularly, FA-targeted nanomicelles exhibit significantly higher cellular uptake than nontargeted micelles after 4 h of incubation, and the DOX fluorescence was observed mainly in the cytoplasm of the cells. The CLSM result is in good agreement with flow cytometry analysis, indicating that the cellular uptake of C100B100-FA micelles is probably based on an FR-mediated endocytosis mechanism leading to a larger amount of micelles internalized into tumor cells.52 It is noteworthy that the targeting efficiency observed herein is somewhat lower than those reported for other nano delivery systems decorated with folate.53 A possible reason is that FA was conjugated to the polyurethane micelles with a quite short spacer and further shielded by the long chain PEG corona, which shows a decreased mobility and interaction with receptor-bearing cells.54 In fact, we have found that both

lengthened spacer and pH-triggered cleavage of PEG corona are helpful to cellular targeting and subsequent cell internalization of nanocarriers. This work is ongoing in our laboratory and will be published elsewhere. 3.8. In Vitro Antitumor Activity and Cytocompatibility. To demonstrate the potential antitumor utility of polyurethane micelles, HeLa cell line was employed to investigate the cytotoxicity of DOX-loaded C100B100 and C100B100-FA micelles, using free DOX as a positive control. Figure 9 shows the cell viability of HeLa cells cultured with various drug formulations at different DOX concentrations for 24 and 72 h. It was found that cytotoxicity of the tested samples is dose-dependent. The IC50 was calculated and illustrated in Figure 9C. Evidently, free DOX appears more toxic than DOXloaded polyurethane micelles, with IC50 values much lower than those for micellar formulations. This is because free DOX was internalized and transported to the nucleus faster than micelles.55 In addition, FA-targeted micelles demonstrate a higher cytotoxicity compared with nontargeted formulations, which is consistent with its higher cellular uptake observed with flow cytometry and CLSM. The result again indicates that folate conjugation can improve the targeting and intracellular delivery of DOX, thus, resulting in an increased drug efficacy. To exclude the possibility that polyurethane micelles themselves can inhibit tumor cells, the viability of HeLa cells incubated with DOX-free micelles was measured by an MTT assay. DOX-loaded C100B100 was set as a positive control. K


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(3) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002, 2, 48−58. (4) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotechnol. 2007, 2, 751−760. (5) Gullotti, E.; Yeo, Y. Mol. Pharmaceutics 2009, 6, 1041−1051. (6) Ashley, C. E.; Carnes, E. C.; Phillips, G. K.; Padilla, D.; Durfee, P. N.; Brown, P. A.; Hanna, T. N.; Liu, J.; Phillips, B.; Carter, M. B.; Carroll, N. J.; Jiang, X.; Dunphy, D. R.; Willman, C. L.; Petsev, D. N.; Evans, D. G.; Parikh, A. N.; Chackerian, B.; Wharton, W.; Peabody, D. S.; Brinker, C. J. Nat. Mater. 2011, 10, 389−397. (7) Tian, L.; Bae, Y. H. Colloids Surf., B 2012, 99, 116−126. (8) (a) Rapoport, N. Prog. Polym. Sci. 2007, 32, 962−990. (b) Wei, H.; Zhuo, R.; Zhang, X. Prog. Polym. Sci. 2013, 38, 503−535. (9) Yu, S.; He, C.; Ding, J.; Cheng, Y.; Song, W.; Zhuang, X.; Chen, X. Soft Matter 2013, 9, 2637−2645. (10) Ding, J.; Chen, J.; Li, D.; Xiao, C.; Zhang, J.; He, C.; Zhuang, X.; Chen, X. J. Mater. Chem. B 2013, 1, 69−81. (11) (a) Torchilin, V. P. Adv. Drug Delivery Rev. 2012, 64, 302−315. (b) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Science 2012, 338, 903−910. (12) (a) Yang, X.; Grailer, J. J.; Rowland, I. J.; Javadi, A.; Hurley, S. A.; Matson, V. Z.; Steeber, D. A.; Gong, S. ACS Nano 2010, 4, 6805− 6817. (b) Wang, W.; Cheng, D.; Gong, F.; Miao, X.; Shuai, X. Adv. Mater. 2012, 24, 115−120. (c) Li, Y.; Xiao, W.; Xiao, K.; Berti, L.; Luo, J.; Tseng, H. P.; Fung, G.; Lam, K. S. Angew. Chem., Int. Ed. 2012, 51, 2864−2869. (13) Abeylath, S. C.; Ganta, S.; Iyer, A. K.; Amiji, M. Acc. Chem. Res. 2011, 44, 1009−1017. (14) Xiong, X.; Lavasanifar, A. ACS Nano 2011, 5, 5202−5213. (15) Ding, M.; Li, J.; He, X.; Song, N.; Tan, H.; Zhang, Y.; Zhou, L.; Gu, Q.; Deng, H.; Fu, Q. Adv. Mater. 2012, 24, 3639−3645. (16) Ding, M.; Song, N.; He, X.; Li, J.; Zhou, L.; Tan, H.; Fu, Q.; Gu, Q. ACS Nano 2013, 7, 1918−1928. (17) Ding, M.; Li, J.; Tan, H.; Fu, Q. Soft Matter 2012, 8, 5414− 5428. (18) (a) Ding, M.; Li, J.; Fu, X.; Zhou, J.; Tan, H.; Gu, Q.; Fu, Q. Biomacromolecules 2009, 10, 2857−2865. (b) Wang, Z.; Yu, L.; Ding, M.; Tan, H.; Li, J.; Fu, Q. Polym. Chem. 2011, 2, 601−607. (c) Ding, M.; Qian, Z.; Wang, J.; Li, J.; Tan, H.; Gu, Q.; Fu, Q. Polym. Chem. 2011, 2, 885−891. (19) (a) Ding, M.; He, X.; Zhou, L.; Li, J.; Tan, H.; Fu, X.; Fu, Q. J. Controlled Release 2011, 152, e87−e89. (b) Ding, M.; Zhou, L.; Fu, X.; Tan, H.; Li, J.; Fu, Q. Soft Matter 2010, 6, 2087−2092. (c) Tan, H.; Wang, Z.; Li, J.; Pan, Z.; Ding, M.; Fu, Q. ACS Macro Lett. 2013, 2, 146−151. (20) Ding, M.; He, X.; Wang, Z.; Li, J.; Tan, H.; Deng, H.; Fu, Q.; Gu, Q. Biomaterials 2011, 32, 9515−9524. (21) (a) Zhou, L.; Liang, D.; He, X.; Li, J.; Tan, H.; Li, J.; Fu, Q.; Gu, Q. Biomaterials 2012, 33, 2734−2745. (b) Zhou, L.; Yu, L.; Ding, M.; Li, J.; Tan, H.; Wang, Z.; Fu, Q. Macromolecules 2011, 44, 857−864. (22) Yu, L.; Zhou, L.; Ding, M.; Li, J.; Tan, H.; Fu, Q.; He, X. J. Colloid Interface Sci. 2011, 358, 376−383. (23) (a) Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Acc. Chem. Res. 2008, 41, 120−129. (b) Sudimack, J.; Lee, R. J. Adv. Drug Delivery Rev. 2000, 41, 147−162. (c) Chen, C.; Ke, J.; Zhou, X. E.; Yi, W.; Brunzelle, J. S.; Li, J.; Yong, E.; Xu, H. E.; Melcher, K. Nature 2013, 500, 486−489. (24) (a) De, P.; Gondi, S. R.; Sumerlin, B. S. Biomacromolecules 2008, 9, 1064−1070. (b) Kamphuis, M. M. J.; Johnston, A. P. R.; Such, G. K.; Dam, H. H.; Evans, R. A.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. J. Am. Chem. Soc. 2010, 132, 15881−15883. (25) Meng, F.; Hennink, W. E.; Zhong, Z. Biomaterials 2009, 30, 2180−2198. (26) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. (b) Sumerlin, B. S.; Vogt, A. P. Macromolecules 2010, 43, 1−13. (27) Such, G. K.; Johnston, A. P. R.; Liang, K.; Caruso, F. Prog. Polym. Sci. 2012, 37, 985−1003.

The results are shown in Figure 10 A and B. It was found that the cell viability is higher than 90% after 24 and 72 h of incubation, while the DOX-loaded micelles show evident unfriendly impact on tumor cells. To evaluate the biocompatibility of multifunctional polyurethane nanovehicles, the cytotoxicity of drug-free micelles toward L929 fibroblast cells was also accessed. As seen from Figure 10C,D, all the blank polyurethane micelles do not show significant toxic effect toward L929 cells after 24 and 72 h of incubation at a high concentration up to 0.1 mg mL−1. It is worth mentioning that both L929 and HeLa cells exhibit good cell growth state when incubated with C100B100FA micelles, revealing that the amounts of copper added in the click reaction have been reduced to a safe level. The result demonstrates that these clickable and cleavable nanocarriers are potentially safe for application in drug delivery.

4. CONCLUSIONS In summary, a series of novel targeting-clickable and tumorcleavable polyurethanes have been successfully synthesized using PCL, LDI, a new designed multifunctional chain extender bearing redox-responsive disulfide bond and clickable alkynyl groups (Cys-PA), and a highly pH-sensitive end-capping monomer containing benzoic-imine linkages (BPEG). The obtained multiblock polyurethanes exhibit attractive micellization properties, multiple stimuli-responsiveness, and high loading capacity for DOX. Folate as a targeting ligand was “post-conjugated” to the micelles via an efficient CuAAC click reaction. In vitro experiments suggest that the FA-decorated polyurethane micelles can target FR positive HeLa cells, resulting in an enhanced cellular uptake and increased drug efficacy compared with nontargeted micelles, while the blank micelles do not show significant toxic effect toward L929 cells. Therefore, these multifunctional nanomicelles are favorable candidates as biodegradable carriers for targeted intracellular drug delivery applications. Further studies will focus on the effects of FA content, and the cleavage of PEG corona and disulfide linkage on the cellular targeting and delivery specificity of polyurethane micelles.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work (N.S. and M.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Shaobing Zhou (Southwest Jiaotong University, China) for his help in size and zeta potential measurements. This work was supported by the National Natural Science Foundation of China (51203101, 51073104, 51273126), China Postdoctoral Science Foundation (2011M500147, 2012T50776), and Changjiang Scholars and Innovative Research Team in University (IRT1163).

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REFERENCES

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Biomacromolecules

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