Synthesis, Self-Assembly, and In Vitro Doxorubicin Release Behavior

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Biomacromolecules 2009, 10, 2310–2318

Synthesis, Self-Assembly, and In Vitro Doxorubicin Release Behavior of Dendron-like/Linear/Dendron-like Poly(ε-caprolactone)-b-Poly(ethylene glycol)-b-Poly(ε-caprolactone) Triblock Copolymers Yang Yang, Chong Hua, and Chang-Ming Dong* Department of Polymer Science & Engineering, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China Received April 29, 2009; Revised Manuscript Received June 23, 2009

Dendron-like/linear/dendron-like poly(ε-caprolactone)-b-poly(ethylene glycol)-b-poly(ε-caprolactone) triblock copolymers with controlled molecular weights (Mn ) 9550-30 460) and low polydispersities were synthesized by a click conjugation between dendron-like poly(ε-caprolactone) and bifunctional azide-terminated poly(ethylene glycol) (copolymer yield ) 56-89%). Their molecular structures and physicochemical and self-assembly properties were thoroughly characterized by means of FT-IR, 1H NMR, multiangle laser light scattering coupled with gel permeation chromatography, differential scanning calorimetry, wide-angle X-ray diffraction, dynamic light scattering, and transmission electron microscopy. Using a nanoprecipitation method, these triblock copolymers self-assembled into spherical flower-like micelles with an average diameter of less than 50 nm in aqueous solution, and both the copolymer composition and the dendritic topology of the hydrophobic core had no apparent influence on the morphology of nanoparticles. The critical aggregation concentrations of these copolymers ranged from 0.034 to 0.048 mg/mL. However, the anticancer doxorubicin-loaded nanoparticles showed worm-like micelles similar to blank nanoparticles fabricated by a dialysis method, and the loaded doxorubicin drug hardly affected the final morphology of nanoparticles. Moreover, the doxorubicin-loaded nanoparticles fabricated from the dumbbell copolymer showed a higher drug loading efficiency of 18% and a longer drug-release time of 45 days than the linear counterpart. Consequently, this provides a versatile strategy not only for the synthesis of biodegradable and biocompatible dendron-like/linear/dendron-like triblock copolymers with dumbbell topology by using click chemistry but also for fabricating worm-like doxorubicin-loaded nanoparticles for anticancer drug release.

Introduction Since the pioneering works of Gitsov and Frećhet, lineardendritic macromolecular architectures (e.g., linear-dendron, dendron-linear-dendron, and dendronized polymers) received much attention in the past decades because they combine chainentangled linear polymers with densely chain-packed dendritic segments, which render them to possess unique solution, bulk, and especially self-assembly properties.1-15 Currently, lineardendritic amphiphiles have been shown to generate organogels, hydrogels, and other hierarchical micro/nanostructures useful for molecular diagnosis, drug/gene delivery vesicles, and tissue engineering scaffolds.16-23 From a synthetic viewpoint, the major strategies for the synthesis of linear-dendritic hybrids can be reduced into the following three methods: (a) coupling of dendron with a linear segment; (b) divergent growth of dendron along the terminal end of the linear segment; (c) controlled/“living” radical polymerizations using dendron macroinitiator.1 For example, linear-dendritic hybrids composed of hydrophobic poly(benzyl ether) dendrons and/or hydrophilic poly(amido amine) have been widely investigated.1,24,25 As an extension of these efforts, the hierarchical self-assembly of amphiphilic linear-dendritic hybrids has also been studied. Frećhet et al. reported the synthesis and fabrication of stimulus-responsive (e.g., pH and light) linear-dendritic nanostructures for drug delivery.26,27 * To whom correspondence should be addressed. Phone: 86-2154748916. Fax: 86-21-54741297. E-mail: [email protected].

Winnik and Manners et al. designed an organogelling dendronhelical polypeptide hybrid.28 Gitsov and Trollsås et al. studied dendrimer-like biodegradable poly(ε-caprolactone).29,30 However, studies on the biohybrids composed of dendritic biodegradable polyesters are quite rare, which might provide more insights for developing multifunctional and stimulus-responsive drug delivery vesicles for potential clinical applications.31-33 As a typical reaction of click chemistry, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been intensively investigated for the syntheses and modifications of complex modalities (e.g., polymers, films, tubes, and nanoparticles), biomacromolecules (e.g., proteins, enzymes, and DNA), and cells.34-44 Importantly, click chemistry, such as CuAAC and thiol-ene reactions, is proved to be a versatile method for the synthesis and functionalization of dendritic polymers (e.g., dendrimers, hyperbranched polymers, dendronized polymers, and linear-dendritic copolymers).45-53 The synthetic strategies for biodegradable polyester-based copolymers (e.g., poly(εcaprolactone), polylactides, and polyglycolide) have also been developed by utilizing click chemistry.47,54-59 On the other hand, as the U.S. Food and Drug Administration approved biomedical polymers, biodegradable poly(ε-caprolactone) (PCL), biocompatible poly(ethylene glycol) (PEG), and their copolymers have been increasingly investigated for various biomedical applications.60,61 Keeping these in mind, herein, a versatile strategy to design and prepare the dendron-like/linear/dendronlike poly(ε-caprolactone)-b-poly(ethylene glycol)-b-poly(ε-caprolactone) triblock copolymers (i.e., Dm-PCL-b-PEG-b-Dm-

10.1021/bm900497z CCC: $40.75  2009 American Chemical Society Published on Web 07/20/2009

Doxorubicin Release Behavior of Triblock Copolymers

Biomacromolecules, Vol. 10, No. 8, 2009

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Scheme 1. Synthesis of Dendron-like/Linear/Dendron-like Dm-PCL-b-PEG-Dm-PCL Triblock Copolymers by Using Click Chemistry

Scheme 2. Traditional Star-like Micelles Self-Assembled from Amphiphilic Diblock Copolymer (Left) and the Flower-like Micelles Self-Assembled from Our Dendron-like/Linear/Dendron-like Dm-PCL-b-PEG-Dm-PCl Triblock Copolymers (Right)

PCL; m ) 0, 1, 2, and 3) with dumbbell topology was successfully developed by using click chemistry (Scheme 1), and their molecular structures and physicochemical properties were thoroughly characterized. These amphiphilic triblock copolymers self-assembled into flower-like micelles with a biodegradable dendron-like PCL core surrounded by a looped PEG corona (Scheme 2), which should be capable of accommodating therapeutic drugs/probes and/or being easily functionalized with multiple stimulus-responsive components for intelligent drug delivery.31-33 Meanwhile, the dendron-like PCL cored micelles with a biocompatible PEG corona will have the stealthy and anti-absorbing protein properties and more dynamic stability compared with traditional star-like micelles. Moreover, the anticancer drug doxorubicin-loaded nanoparticles fabricated from the dumbbell copolymer showed a higher drug loading efficiency and a longer drug-release time than the linear counterpart, potentially making them useful for the anticancer drug delivery system.62

Experimental Section Materials. ε-Caprolactone (CL, Aldrich, 99%) and dimethylformamide (DMF, g99.5%) were distilled from calcium hydride under

reduced pressure and stored over molecular sieves, respectively. Dichloromethane (g99.5%) and toluene (g99.5%) were directly distilled from calcium hydride. Poly(ethylene glycol) (PEG, Mn ) 4000) was purchased from Shanghai Sinopharm Chemical Reagent Corporation and dried at 50 °C in vacuo overnight before use. Copper(I) bromide (99.999%, Aldrich), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, 99%, Aldrich), propargyl amine (99%, Acros), stannous octoate (SnOct2, Sigma, 95%), and toluene-4-sulfonyl chloride (98.5%, Aldrich) were used as received. 1,6-Diphenyl-1,3,5-hexatriene (DPH, 98%) was purchased from Aldrich and used as a probe molecule. Doxorubicin hydrocloride was purchased from Beijing Huafeng United Technology Corporation (Beijing, China) and used as received. Ethylenediamine (99%) and methyl acrylate (98%) were purchased from Shanghai Sinopharm Chemical Reagent Corporation and distilled before use. The propargyl focal point poly(amido amine) dendrons with 2m primary amine groups (i.e., Dm, m ) 0, 1, 2, and 3; Scheme 1) were synthesized using a protocol similar to that described by Lee et al.,63,64 as detailed in our previous publication.47 The clickable dendron-like poly(ε-caprolactone) with 2m PCL branches (i.e., Dm-PCL) was successfully synthesized from controlled ring-opening polymerization of CL monomer using Dm as initiator and SnOct2 as catalyst according to our previous publication,47 and their molecular weights were determined by the MALLS-GPC technique based on the dn/dc value of 0.042-0.073 mL/g. Methods. 1H NMR and 13C NMR spectroscopies were performed on a Varian Mercury-400 spectrometer. Tetramethylsilane was used as an internal standard. The actual molecular weights (Mn,LLS) and polydispersities (Mw/Mn) of the polymers were determined on a Waters 717 plus autosampler gel permeation chromatograph (GPC) equipped with Waters RH columns, a refractive index detector, and the DAWN EOS (Wyatt Technology) multiangle laser light scattering (MALLS) detector at 30 °C, THF as the eluent (1.0 mL/min). The dn/dc values of copolymers were detected by an Optilab DSP interferometric refractometer (Wyatt Technology) and directly obtained by its DNDC software, and they varied from 0.043 to 0.073 mL/g. The differential scanning calorimetry (DSC) analysis was carried out using a PerkinElmer Pyris 1 instrument under nitrogen flow (10 mL/min). All samples were first heated from 0 to 90 °C at 10 °C/min and held for 3 min to erase the thermal history, then cooled to 0 at 10 °C/min, and finally heated to 90 at 10 °C/min. Wide-angle X-ray diffraction (WAXD) patterns of powder samples were obtained at room temperature on a Shimadzu XRD-6000 X-ray diffractometer with a Cu KR radiation source (wavelength ) 1.54 Å). The supplied voltage and current were

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set to 40 kV and 30 mA, respectively. Samples were exposed at a scan rate of 2θ ) 4° min-1 between 2θ ) 5 and 40°. The mean size of nanoparticles was determined by dynamic light scattering (DLS) using a Malvern Nano_S instrument (Malvern, UK). The solution of nanoparticles was performed at a scattering angle of 90° and at 25 °C. All of the measurements were repeated three times, and the average values reported are the mean diameter ( standard deviation. Transmission electron microscopy (TEM) was performed using a JEM-2010/ INCA OXFORD TEM (JEOL/OXFORD) at a 200 kV accelerating voltage. Samples were deposited onto the surface of 300 mesh Formvar carbon film coated copper grids. Excess solution was quickly wicked away with a filter paper. The image contrast was enhanced by negative staining with phosphotungstic acid (0.5 wt %). Preparation of Bifunctional Azide-Terminated Poly(ethylene glycol) (N3-PEG-N3). Both PEG (1.00 g, 0.25 mmol) and toluene-4sulfonyl chloride (952.5 mg, 5 mmol) were completely dissolved in CH2Cl2 (10 mL) under N2 atmosphere. Triethylamine (1.04 mL, 7.5 mmol) was added dropwise to the above solution at ice-water bath, and then the resulting solution was stirred for 24 h at room temperature. The reaction solution was centrifuged and precipitated into 80 mL of diethyl ether, and then the powder was dried in vacuo at 25 °C to give the bifunctional tosylated PEG (Ts-PEG-Ts, 978.2 mg, 91% yield). 1H NMR of Ts-PEG-Ts (CDCl3): δ ) 2.42 (s, 6H), 3.62 (s, 360 H), 4.15 (t, 4H), 7.33 (d, 4H), 7.79 (d, 4H). Thus, sodium azide (546.0 mg, 8.4 mmol) was added to a solution of Ts-PEG-Ts (903.0 mg, 0.21 mmol) in dry DMF (10 mL) under N2 atmosphere, and the reaction mixture was stirred vigorously at room temperature for 24 h. DMF solvent was removed under reduced pressure, and then the product was dissolved in 80 mL of CH2Cl2. The mixture was extracted sequentially with NaCl (5 wt %) solution and distilled water, dried with anhydrous Na2SO4, and then precipitated in diethyl ether to yield 637.9 mg of bifunctional azide-terminated PEG (N3-PEG-N3, 76% yield). 1H NMR of N3-PEGN3 (CDCl3): δ ) 3.38 (t, 4H), 3.64 (s, 364H). Mn ) 4000, Mw/Mn ) 1.30. Synthesis of Dendron-like/Linear/Dendron-like Dm-PCL-b-PEGDm-PCL Triblock Copolymers via Click Chemistry. A typical procedure for the synthesis of Dm-PCL-b-PEG-Dm-PCL triblock copolymers was started with the feed ratio of reagents [N3-PEG-N3]/ [Dm-PCL]/[CuBr]/[PMDETA] ) 1/2.2/2.2/2.2. As a representative example, the click coupling reaction between N3-PEG-N3 (100.0 mg, 0.025 mmol) and D0-PCL24 (150.6 mg, 0.055 mmol alkyne unit) was conducted at 35 °C in a 25 mL Schlenk flask with 2 mL of DMF as solvent, and both CuBr (7.9 mg, 0.055 mmol) and PMDETA (243 µL, 0.055 mmol, diluted by 20-fold DMF) as catalyst. After 36 h, the copolymer solution was then precipitated in ethyl ether. The resulting copolymers were purified by solvent extraction using 8 mL of benzene and hexane (0.8: 1, v/v) to completely extract the excess D0-PCL24 precursor. The white powder was then dried in vacuo at 40 °C to give 199.2 mg of D0-PCL24-b-PEG-b-D0-PCL24 (89.2% yield). 1H NMR (CDCl3): δ (ppm) ) 1.38 (CH2, m, 96H), 1.63 (CH2, m, 192H), 2.30 (CH2, t, 96H), 3.65 (OCH2CH2O, s, 360H), 3.86 (CH2, s, 4H), 4.05 (CH2, t, 96H), 4.50 (CH2, s, 4H), 7.76 (CH-N, s, H). FT-IR (KBr, cm-1): 3440 (νN-H), 2940 (νC-H for PCL), 2888 (νC-H for PEO), 1725 (νCdO), 1600 (triazole). Mn ) 9550, Mw/Mn ) 1.22. Measurement of the Critical Aggregation Concentration of Dm-PCL-b-PEG-Dm-PCL Triblock Copolymers. The critical aggregation concentration (cac) of Dm-PCL-b-PEG-Dm-PCL triblock copolymers was determined employing the hydrophobic dye solubilization method using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe molecule.65 UV-vis spectra of samples were recorded in the range of 200-500 nm at room temperature. Fabrication of Dm-PCL-b-PEG-Dm-PCL Copolymers Nanoparticles in Aqueous Solution Using a Nanoprecipitation Method. Using a nanoprecipitation method,66-68 a typical procedure for the fabrication of copolymers nanoparticles in aqueous solution is as follows. Ten milligrams of Dm-PCL-b-PEG-Dm-PCL sample was dissolved completely in 2 mL of acetone at room temperature, and then the resulting

Yang et al. solution (5 mg/mL) was added dropwise to 10 mL of distilled water under vigorous stirring for about 15 min using a microsyringe. The solution was stirred vigorously for another 24 h at room temperature, and acetone was completely evaporated under reduced pressure. The obtained nanoparticle solution was stored at 4 °C before measurement, and both the mean size and morphology of nanoparticles were determined by DLS and TEM, respectively. Preparation of Doxorubicin-Loaded Nanoparticles in Aqueous Solution Using a Dialysis Method. Using a dialysis method,69,70 a typical procedure for the fabrication of doxorubicin-loaded nanoparticles in aqueous solution is as follows. Dm-PCL-b-PEG-Dm-PCL triblock copolymer (10 mg) and doxorubicin hydrochloride (5 mg, 8.6 µmoL) were dissolved in 7.5 mL of DMF, in which 1.5-fold of Et3N (12.9 µmoL) was added to neutralize HCL in solution. Distilled water (1.5 mL) was then added gradually at a speed of 30 µL/min using a microsyringe until the formation of nanoparticles. The nanoparticle solution was put into a dialysis bag and subjected to dialysis against 4 × 1 L of distilled water for 24 h. The drug-loaded nanoparticle solution was lyophilized and stored at 4 °C. The drug-loaded nanoparticles (1 mg) were dissolved in 5 mL of DMF and then analyzed by UV absorbance at 500 nm. The drug loading capacity of nanoparticles is calculated as the weight ratio of actual drug to drug-loaded nanoparticles, and the drug loading efficiency of nanoparticles is calculated as the weight ratio of actual and added drug content. Similarly, the blank nanoparticles were fabricated for the comparison study. In Vitro Doxorubicin Release from Anticancer Drug-Loaded Nanoparticles. The lyophilized drug-loaded nanoparticles (4 mg) were directly immersed into 1 mL of distilled water and then put into a dialysis bag. The dialysis bag was put in 15 mL of distilled water at 37 °C. The drug-released solution was changed periodically, and the amount of doxorubicin released from nanoparticles was measured by UV-vis at 500 nm at room temperature. All release experiments were carried out in duplicate, and all data were averages of six determinations used for drawing figures.

Results and Discussion Synthesis of Dendron-like/Linear/Dendron-like Dm-PCLb-PEG-b-Dm-PCL Triblock Copolymers via Click Chemistry. In this article, using biocompatible PEG and dendron-like poly(ε-caprolactone) having 2m PCL branches (Dm-PCL, m ) 0, 1, 2, and 3) as the precursors, a versatile strategy to prepare dendron-like/linear/dendron-like Dm-PCL-b-PEG-Dm-PCL triblock copolymers with dumbbell topology was successfully developed by using click chemistry. The dendron-like propargyl focal point Dm-PCL precursors with controlled molecular weights and low polydispersities were facilely synthesized by the controlled ring-opening polymerization of ε-caprolactone using the propargyl focal point dendrons Dm with 2m primary amine groups as initiators according to our previous publication.47 They can then be used for the click conjugation with bifunctional azide-terminated PEG (N3-PEG-N3) to produce these targeted triblock copolymers, as shown in Scheme 1. In order to prepare well-defined Dm-PCL-b-PEG-Dm-PCL triblock copolymers, 10% excess Dm-PCL precursor (based on N3 mol of N3-PEG-N3) was used for all the conjugation reactions, and the detailed results are compiled in Table 1. On the basis of the MALLS-GPC analysis (Figure 1), it is demonstrated that the excess Dm-PCL precursor can be completely removed from the resulting products by simple washing using mixed solvents of benzene and hexane (0.8: 1, v/v), and the purified triblock copolymer clearly showed the unimodal elution peak coupled with a narrow polydispersity. Note that the FT-IR analysis of washing solution also indicated that excess Dm-PCL precursor could be wholly extracted from the reactant product (Supporting Information S1). Moreover, the actual



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Table 1. Synthesis of Dendron-like/Linear/Dendron-like Dm-PCL-b-PEG-b-Dm-PCL Triblock Copolymers by Using Click Chemistry entrya

Mn,LLSb

Mw/Mnb

Mn,NMRc

PEG/PCL in copolymer (%)d

yield (%)

D0-PCL24 -b-PEG-b-D0-PCL241 D0-PCL681-b-PEG-b-D0-PCL681 D1-PCL222-b-PEG-b-D1-PCL222 D2-PCL74-b-PEG-b-D2-PCL74 D2-PCL194-b-PEG-b-D2-PCL194 D3-PCL138-b-PEG-b-D3-PCL138

9550 21200 13410 12730 25620 30460

1.22 1.19 1.34 1.07 1.02 1.33

9640 19680 14660 11920 22880 31100

41.5/58.5 20.3/79.7 27.3/72.7 33.6/66.4 17.5/82.5 12.9/87.1

89.2 84.2 85.0 56.5 76.5 70.3

1

a Both the subscript and the superscript numbers represent the degree of polymerization and the branch number of PCL block, respectively. b Both the actual molecular weight (Mn,LLS) and the polydispersity (Mw/Mn) of copolymer were determined by the MALLS-GPC technique. c Mn,NMR was determined by 1H NMR spectrum (e.g., Figure 5). d The weight percent of PEG or PCL in copolymer was calculated from the ratio of Mn,NMR of PEG or PCL to that of copolymer.

Figure 1. MALLS-GPC traces of D0-PCL241-b-PEG-b-D0-PCL241 triblock copolymer before and after simple washing using mixed solvents of benzene and hexane (0.8: 1, v/v).

Figure 3. 1H NMR spectra of N3-PEG-N3 (A) and D0-PCL241-b-PEGb-D0-PCL241 (B).

Figure 2. FT-IR spectra of PEG, N3-PEG-N3, and the Dm-PCL-bPEG-Dm-PCL triblock copolymers.

molecular weights of these triblock copolymers can be determined by both 1H NMR and MALLS-GPC, and Mn,NMR is basically consistent with Mn,LLS (Table 1). These results convincingly verified the successful synthesis of the purified triblock copolymers. As shown in Figure 2, FT-IR spectra also confirmed the click conjugation. Compared with N3-PEG-N3 precursor, the typical azide group at about 2097 cm-1 wholly disappeared within these triblock copolymers. In addition, these copolymers showed the distinct stretching bands at 2950 cm-1 (CH) and 1725 cm-1

(CdO) for PCL block, the intense stretching bands at 2883 and 840 cm-1 for PEG block, and the broad absorption at 3200-3600 cm-1 (NH) for the dendrons Dm. Besides the typical proton signals of both PCL and PEG blocks,71 the 1H NMR spectra of these copolymers clearly show that new signals at 7.76 ppm (singlet) typical of methine proton of the triazole ring and at 4.50 ppm of methylene protons (CH2) adjacent to triazole ring clearly appeared, and the original peak (CH2-N3, 3.38 ppm) within N3-PEG-N3 disappeared (Figure 3 and Supporting Information S2). Moreover, by taking into account the integration values of protons “b”, “f”, and “h”, the click conjugation efficiency ranged from 92 to 99% within the error of 1H NMR measurement. These results suggest that the propargyl focal point Dm-PCL precursor was quantitatively conjugated with N3PEG-N3 to produce the targeted triblock copolymers. Together these results indicate that the click conjugation between bifunctional N3-PEG-N3 and dendron-like propargyl focal point Dm-PCL precursors provides a versatile strategy for the synthesis of dendron-like/linear/dendron-like Dm-PCL-b-PEGb-Dm-PCL triblock copolymers with dumbbell topology, as shown in Scheme 1. To the best of our knowledge, this is the

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Yang et al.

and the central PEG block within the D3-PCL-b-PEG-D3-PCL sample exists in an amorphous state. In all, the above analyses indicate that the crystallization properties of these dumbbell triblock copolymers can be tuned from both the branch density or dendron generation of dendron-like Dm-PCL segments and the copolymer composition. These are very useful parameters for designing novel biodegradable polymeric biomaterials with controllable biodegradation rate and mechanical properties for biomedical applications.60,61

Figure 4. DSC curves of PEG and the Dm-PCL-b-PEG-Dm-PCL triblock copolymers in the cooling run (solid lines) and in the second heating run (dot lines), respectively.

first report that describes the synthesis of amphiphilic dumbbellshaped Dm-PCL-b-PEG-b-Dm-PCL triblock copolymers by utilizing click chemistry.34-59 DSC and WAXD Analyses. The melting and crystallization behaviors of these Dm-PCL-b-PEG-Dm-PCL copolymers were investigated by DSC, as shown in Figure 4 and Table 2. Compared with PEG and Dm-PCL precursors, most triblock copolymers with different compositions (PEG % ) 12.6-41.5 wt %) presented a superposed melting peak (Tm) and crystallization temperature (Tc) in the second heating run and in the cooling run, respectively, which was attributed to the fact that both PCL and PEG blocks had similar melting and crystallization temperature.47,72 Given the similar branch length of dendron-like Dm-PCL segments, the Tm of these copolymers had a decreased tendency over the branch density or dendron generation of Dm-PCL segments. This is attributable to the fact that both the intermolecular interactions (e.g., hydrogen bonding, van der Waals, and/or dipole-dipole interactions) among these copolymers and the constrained geometry of dendron-like DmPCL segments decreased the macromolecular mobility and rearrangement, inducing a decreased crystallization tendency. In addition, both Tm and Tc of copolymers apparently decreased with shortening PCL branch length (e.g., D2-PCL-b-PEG-D2PCL samples). Meanwhile, the degree of crystallinity (Xc) of both PCL and PEG blocks within copolymers similarly showed a decreased tendency over the branch density. As shown in Figure 5, WAXD was used to demonstrate the crystalline structure of these Dm-PCL-b-PEG-b-Dm-PCL triblock copolymers in solid state and at room temperature. The Dm-PCL and N3-PEG-N3 precursors presented prominent diffraction peaks at about 21.4 and 23.6° and about 19.1 and 23.2°, which are characteristic of the PCL and the PEG crystals, respectively.47,72 Except for the D3-PCL-b-PEG-D3-PCL sample, these triblock copolymers approximately presented the diffraction peaks of both PCL and PEG blocks, suggesting that these triblock copolymers formed microphase-separated crystalline materials (i.e., both crystalline PCL and crystalline PEG within copolymers) in solid state and at room temperature. Moreover, the relative peak intensity of PEG to PCL within these copolymers progressively decreased with increasing both the branch density and the weight percent of PCL segments. This suggests that the crystallization of the flank dendron-like PCL segments greatly restricted that of the central PEG segments,

Self-Assembly of Dm-PCL-b-PEG-b-Dm-PCL Triblock Copolymers. Using a nanoprecipitation method,66-68 these dumbbell triblock copolymers self-assembled into nanoparticles in aqueous solution at room temperature (Table 3). The critical aggregation concentration (cac) of amphiphilic copolymers was an important parameter for characterizing the thermodynamic stability of nanoparticles in aqueous solution,32,33 which was measured by the dye solubilization method.65 As shown in Figure 6, the absorbance intensity of 1,6-diphenyl-1,3,5hexatriene dye remained constant below a certain concentration, and then it increased substantially, reflecting the incorporation of dye in the hydrophobic region of nanoparticles. The cac of amphiphilic triblock copolymers ranged from 3.4 × 10-2 to 4.8 × 10-2 mg/mL, suggesting that the self-assembled micelles are thermodynamically stable in aqueous solution.32,33 Notably, the cac of these dumbbell copolymers has no apparent dependence on both the copolymer composition and the branch density or dendron generation of hydrophobic Dm-PCL segments. This is probably attributed to the fact that the flower-like micelles with a looped hydrophilic corona (often generated from triblock copolymers) are to some extent more stable than the conventional star-like micelles with a free corona (often generated from diblock copolymers) in aqueous solution (Scheme 2).73,74 Both the morphology and the average size of the selfassembled nanoparticles from these triblock copolymers were investigated by the techniques of TEM and DLS, and the detail results are shown in Figure 7 and Table 3. Except for the D3PCL138-b-PEG-D3-PCL138 sample, most triblock copolymers with different compositions (PEG % ) 12.6-41.5 wt %) selfassembled into spherical micelles with an average diameter of less than 50 nm, which makes them suitable for targeted drug delivery induced by the size-mediated accumulation.62 Notably, these micellar nanoparticles had a similar diameter with the conventional polymeric micelles usually with a diameter of 10-50 nm,33 suggesting that they had a dendron-like PCL core surrounded by a hydrophilic PEO corona. This result is different from both linear and dendritic diblock copolymers with a lower hydrophilic composition, which often self-assemble into the polymersomes and/or vesicles with a lower curvature.32 This is attributable to the fact that the looped PEG corona connected by a dendron-like core greatly decreased the repulsion among the corona chains, stabilizing the micelles with a relatively higher curvature. As for the highest generation D3-PCL138-b-PEG-D3-PCL138 copolymer with the lowest PEG composition (12.9%), a mixed morphology of spherical micelles and less amount of worm-like micelles was obtained, as shown in Figure 7D. In all, these results indicate that the dendron-like/linear/dendron-like triblock copolymers mainly self-assembled flower-like micelles with a diameter of less than 50 nm, and both the copolymer composition and the dendritic topology of the hydrophobic core had no apparent influence on the morphology of micelles. This also provides a convenient method to generate the flower-like micelles having a lower hydrophilic component in aqueous solution.



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Table 2. Melting and Crystallization Behaviors of the PEG, PCL Precursors, and the Dm-PCL-b-PEG-b-Dm-PCL Triblock Copolymers entry

Tc,PEGa (°C)

PEG D0-PCL241 D0-PCL241-b-PEG-b-D0-PCL241 D0-PCL681-b-PEG-b-D0-PCL681 D1-PCL222-b-PEG-b-D1-PCL222 D2-PCL74-b-PEG-b-D2-PCL74 D2-PCL194-b-PEG-b-D2-PCL194 D3-PCL138-b-PEG-b-D3-PCL138

39.7

Tc,PCLa (°C)

Tm,PEGb (°C)

Tm,PCLb (°C)

57.5, 62.0 32.4 21.9

27.4 38.4 30.3

16.1 6.6 26.1 20.9

52.0 48.1, 51.4

35.6 56.4 49.3 33.3 48.7 45.4

∆Hcc (J/g)

Xcd (%)

166.3 80.6 61.2 50.7 47.6 4.4 43.3 46.9

89.4 59.1 38.9 34.8 31.4 2.8 29.5 32.7

a Tc,PEG and Tc,PCL denote the crystallization temperature of PEG and PCL in the cooling run, respectively. b Tm,PEG and Tm,PCL denote the maximal melting temperature of PEG and PCL in the second heating run, respectively. c ∆Hc denotes the crystallization enthalpy of both PEG and PCL segments in the cooling run. d Xc denotes the degree of crystallinity of copolymers, where Xc ) ∆Hc/(WPEG∆H0c,PEG + WPCL∆H0c,PCL), ∆H0c,PEG ) 186.0 J/g, ∆H0c,PCL ) 136.4 J/g.

Figure 5. WAXD patterns of N3-PEG-N3, D0-PCL241, and the triblock copolymers.

Figure 6. Relationship of the absorbance intensity of DPH as a function of the triblock copolymer concentration in aqueous solution at room temperature.

Table 3. Self-Assembled Nanoparticles from the Triblock Copolymers in Aqueous Solution entry

average diameter (nm)a

PDIb

D0-PCL241-b-PEG-b-D0-PCL241 D0-PCL681-b-PEG-b-D0-PCL681 D1-PCL222-b-PEG-b-D1-PCL222 D2-PCL74-b-PEG-b-D2-PCL74 D2-PCL194-b-PEG-b-D2-PCL194 D3-PCL138-b-PEG-b-D3-PCL138

30.3 ( 2.6 41.5 ( 1.9 23.9 ( 1.1 14.1 ( 0.7 26.5 ( 3.4 160.9 ( 18.0

0.59 0.63 0.33 0.76 0.63 0.50

a The average diameter of nanoparticles was determined by DLS technique. b PDI denotes the polydispersities of nanoparticles in aqueous solution.

Doxorubicin-Loaded Nanoparticles and In Vitro Anticancer Drug-Release Behavior. Compared with the widely used but time-consuming dialysis method,69,70 the nanoprecipitation method proved to be an efficient and fast tool for the fabrication of polymeric nanoparticles in aqueous solution.66-68 However, as reported in our previous publication,75 the drugloaded polyester-based nanoparticles fabricated by the nanoprecipitation method gave a lower drug-loaded efficiency and a fast drug-release rate compared with those fabricated by the dialysis method. Therefore, in this article, we chose the dialysis method for the fabrication of drug-loaded nanoparticles in aqueous solution, and the blank nanoparticles were also prepared for the comparison study by using the same method. As a most common anticancer drug in clinical application,76,77 doxorubicin was successfully loaded in both the linear and dumbbell triblock copolymer nanoparticles. Both blank and drug-loaded nanoparticles were characterized by TEM. As shown in Figure 8, the

blank nanoparticles fabricated from the linear D0-PCL681-bPEG-D0-PCL681 copolymer showed the worm-like micelles, and the related drug-loaded nanoparticles gave a similar morphology in aqueous solution (Figure 8A,B). These results indicate that the loaded doxorubicin drug hardly affected on the final morphology of self-assembled nanoparticles. These worm-like micelles might be a better useful nanocontainer than spherical micelles for drug delivery, as recently reported by Discher et al.78 As a note, the D0-PCL681-b-PEG-D0-PCL681 nanoparticles fabricated by the nanoprecipitation method mainly showed spherical micelles compared with aforementioned worm-like micelles fabricated by the dialysis method (Figure 8C). This suggests that the preparation method to some extent affected the aggregation behavior of amphiphilic copolymers in aqueous solution partially because of using different organic solvents (e.g., DMF vs acetone), which potentially provides a facile method to fabricate different morphological nanoparticles with the same copolymer composition. In addition, both blank and drug-loaded D3-PCL138-b-PEG-D3-PCL138 copolymer nanoparticles fabricated by the dialysis method gave worm-like micelles and worm-like micelle networks, respectively (Figure 8D,E). Notably, the hydrophobic doxorubicin loaded within nanoparticles might serve as the physical cross-linking points for the interconnecting worm-like micelle networks. The doxorubicin loading efficiency of the dumbbell D3PCL138-b-PEG-D3-PCL138 copolymer was about 18%, higher than that (about 11%) for linear D0-PCL681-b-PEG-D0-PCL681 counterpart. Meanwhile, the doxorubicin loading capacity is

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Yang et al.

Figure 7. TEM photographs of the nanoparticles fabricated by a nanoprecipitation method: (A) D0-PCL241-b-PEG-b-D0-PCL241, (B) D2-PCL74b-PEG-b-D2-PCL74, (C) D2-PCL194-b-PEG-b-D2-PCL194, (D) D3-PCL138-b-PEG-b-D3-PCL138.

Figure 8. TEM photographs of the blank and doxorubicin-loaded nanoparticles fabricated by a dialysis method: (A) blank nanoparticles of D0-PCL681-b-PEG-b-D0-PCL681, (B) doxorubicin-loaded nanoparticles of D0-PCL681-b-PEG-b-D0-PCL681, (C) blank nanoparticles of D0-PCL681b-PEG-b-D0-PCL681 by a nanoprecipitation method, (D) blank nanoparticles of D3-PCL138-b-PEG-b-D3-PCL138, (E) doxorubicin-loaded nanoparticles of D3-PCL138-b-PEG-b-D3-PCL138.

about 11 and 9% for the dumbbell and linear copolymers, respectively. These suggest that the dendritic architecture of

copolymer was beneficial for the encapsulation of drug within nanoparticles, which is probably attributed to both the interior



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°C. As for the linear copolymer nanoparticles, the drug-release profile gave a similar triphasic pattern, that is, with a fast drugrelease rate (K1′ ) 2.60 h-1) within the initial 10 h (i.e., about 30% of drug was released), a moderate drug-release rate (K2 ) 0.46 h-1) within about 90 h (i.e., about 40% of drug was released), and then followed by a slow drug-release rate (K2′ ) 0.08 h-1) within the next 270 h (i.e., about 23% of drug was released). Moreover, the doxorubicin-loaded nanoparticles at pH 5.5 showed a triphasic drug-release profile with a much faster drug-release rate than those at pH 7. These results indicate that the doxorubicin release from nanoparticles is pH-dependent, which is due to both the faster biodegradation of copolymers and the increased aqueous solubility of doxorubicin at mildly acidic pH.83 All of the results demonstrate that the copolymer architecture has apparent effect on the drug-release rate of doxorubicin-loaded nanoparticles, and the dumbbell copolymer is more suitable for the fabrication of anticancer doxorubicin drug-release system with a relatively longer drug-release time than the linear counterpart.

Conclusions

Figure 9. In vitro drug-release profile of doxorubicin-loaded nanoparticles from both D0-PCL681-b-PEG-b-D0-PCL681 and D3-PCL138b-PEG-b-D3-PCL138 samples at 37 °C: pH )7 (A) and pH ) 5.5 (B).

cavity and the larger space within the dendron-like hydrophobic core.16,17,79 The drug-release behavior of the doxorubicin-loaded nanoparticles at aqueous pH 7 and 5.5 is shown in Figure 9. The drug-release profile of the dumbbell D3-PCL138-b-PEGD3-PCL138 copolymer nanoparticles at pH 7 shows a triphasic pattern,80 that is, with a fast drug-release rate (K1 ) 1.88 h-1) within initial 10 h (i.e., about 20% of drug was released), a moderate drug-release rate (K2 ) 0.33 h-1) within about 90 h (i.e., about 30% of drug was released), and then followed by a slow drug-release rate (K3 ) 0.05 h-1) within the next 1000 h (i.e., about 40% of drug was released). This can be attributed to the following reasons. One is that less amount of doxorubicin was probably encapsulated into the surface layer of the hydrophobic PCL core during the nanoparticle formation process, and the other is that the drug-loaded worm-like micelles had large surface areas because of their minimal size.81 These two factors resulted in the initially fast release of about 20% doxorubicin from the drug-loaded nanoparticles. On the basis of the degradation study of PCL-b-PEG-b-PCL copolymer nanoparticles,82 the hydrophobic and crystalline PCL core within nanoparticles should slightly degrade at pH 7 within our experimental period (about 45 days), suggesting that the degradation-induced drug release might be negligible. Thus, the following doxorubicin release was mainly controlled by the drug diffusion from nanoparticles, which was associated with enhanced water uptake by hydrophilic looped PEG corona at 37

A new class of dendron-like/linear/dendron-like Dm-PCL-bPEG-b-Dm-PCL triblock copolymers with dumbbell topology was successfully synthesized by using click chemistry. The click conjugation between dendron-like propargyl focal point DmPCL and bifunctional N3-PEG-N3 was confirmed by FT-IR, 1H NMR, and MALLS-GPC. The crystallization properties of these dumbbell triblock copolymers can be tuned by both the dendron generation of Dm-PCL segments and the copolymer composition. Using a nanoprecipitation method, these triblock copolymers mainly self-assembled into spherical flower-like micelles with an average diameter of less than 50 nm in aqueous solution, and both the copolymer composition and the dendron generation of PCL core had no apparent influence on the morphology of nanoparticles. However, the doxorubicin-loaded nanoparticles presented worm-like micelles similar to blank nanoparticles fabricated by a dialysis method, and the loaded doxorubicin drug hardly affected on the morphology of nanoparticles. The anticancer doxorubicin-loaded nanoparticles fabricated from the dumbbell copolymer showed a higher drug loading efficiency, demonstrating a triphasic release profile with a longer drugrelease time (about 45 days) at pH 7 than the linear analogue. This provides a promising platform not only for the synthesis of biodegradable and biocompatible triblock copolymers with dumbbell topology but also for fabricating worm-like doxorubicin-loaded nanoparticles for anticancer drug delivery system. Acknowledgment. The authors are grateful for the financial support of National Natural Science Foundation of China (20874058 and 20674050) and Shanghai Leading Academic Discipline Project (B202). The assistance of Instrumental Analysis Center of SJTU is also appreciated. Supporting Information Available. FT-IR for washing solution and 1H NMR for D2-PCL-b-PEG-D2-PCL. This material is available free of charge via the Internet at http:// pubs.acs.org.

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BM900497Z

Synthesis, Self-Assembly, and In Vitro Doxorubicin Release Behavior

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