Synthesis of Hyperbranched Polypeptide and PEO Block Copolymer

Aug 19, 2013 - Byrne , M.; Thornton , P. D.; Cryanb , S. A.; Heise , A. Polym. Chem. 2012, 3, 2825– 2831. [Crossref], [CAS]. 54. Star polypeptides b...
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Synthesis of Hyperbranched Polypeptide and PEO Block Copolymer by Consecutive Thiol-Yne Chemistry Xiao Chang and Chang-Ming Dong* Department of Polymer Science & Engineering, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China S Supporting Information *

ABSTRACT: Hyperbranched poly(ε-benzyloxycarbonyl-L-lysine) (HPlys) with multiple alkyne peripheries was synthesized through the click polycondensation of an AB2 type Plys macromonomer with α-thiol and ω-alkyne terminal groups (thiol is the A unit, and each π bond in alkyne is the B unit), and the resulting HPlys was further conjugated with thioltermined poly(ethylene oxide) (PEO) to generate HPlys-bPEO block copolymer by consecutive thiol-yne chemistry. Their molecular structures and physical properties were characterized in detail by FT-IR, 1H NMR, gel permeation chromatography, differential scanning calorimetry, wide-angle X-ray diffraction, and polarized optical microscopy. HPlys and HPlys-b-PEO mainly assumed an α-helix conformation similar to the linear precursors, while the liquid crystalline phase transition of Plys segment disappeared within HPlys and HPlys-b-PEO. HPlys-b-PEO self-assembled into nearly spherical micelles in aqueous solution, while it gave a 5-fold lower critical aggregation concentration (8.9 × 10−3 mg/mL) than a linear counterpart (4.5 × 10−2 mg/mL), demonstrating a dendritic topology effect. Compared with a linear counterpart, HPlys-b-PEO gave a higher drug-loading capacity and efficiency for the anticancer drug doxorubicin (DOX) and a slower drug-release rate with an improved burst-release profile, enabling them useful for drug delivery systems. Importantly, this work provides a versatile strategy for the synthesis of hyperbranched polypeptides and related block copolymers by utilizing thiol-yne chemistry.

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INTRODUCTION Hyperbranched polymers, as alternatives to dendrimers, have been increasingly investigated for molecular diagnosis and drug/gene delivery vesicles, because they have available cavities for the encapsulation of probes/drugs and demonstrate unique self-assembly properties and multivalent characteristics.1−13 Reminiscently, naturally branched peptide-containing biomacromolecules (e.g., collagen and glycoprotein) not only are important components existing in the connective tissues and the extracellular matrices of mammalians but also play unique recognition and communication functions.14−16 Therefore, developing synthetic polypeptides with tunable dendritic topologies provides an important platform for understanding structure−property relationship as a biomimetic model and for biomedical applications.17−28 In the aspect of dendritic polypeptides, Klok et al. reported the facile synthesis of dendritic-graft poly(ε-benzyloxycarbonylL-lysine) and highly branched polylysines by repetitive ringopening polymerization of N-carboxyanhydride monomers in multigram quantities.29,30 As hyperbranched polymers can be easily synthesized from the polycondensations of ABx (x ≥2) and/or A2 + Bx (x ≥ 2) monomers in one pot,13 and the thermal polymerizations of inherent AB2 amino acids (e.g., Llysine and L-glutamic acid) have been intensively investigated.31−33 Compared with that of the purely statistical AB2 monomer, the thermal polymerization of L-lysine produced the © XXXX American Chemical Society

hyperbranched polylysines with a relatively lower degree of branching (DB, 0.35−0.45) due to the unequal reactivity of two amine groups.32,33 Notably, it is still challenging to synthesize dendritic polypeptides especially with a high DB, which hampers their wide applications.23 Owing to high efficiency and easiness to use light irradiation, the click reactions such as thiol-yne and thiol-ene additions proved to be powerful tools for the synthesis and functionalization of various complex architectures (e.g., dendritic polymers) and biologically relevant systems.34−42 For example, hyperbranched polystyrene and the block copolymers have been synthesized through the click polycondensation of an AB2 macromonomer by utilizing thiol-yne chemistry.43−46 Savin et al. reported the modular synthesis of polypeptide-based 3-arm star polymers by using thiol-yne chemistry, which self-assembled into spherical vesicles independent of block composition.47 We found that hyperbranched biodegradable polyester poly(ε-caprolactone) (PCL) could be click polymerized using an AB2 PCL macromonomer.48 However the cross-linking side reactions existed during the click polymerization of PCL, which could be prohibited by enhancing the rigidity of the macromonomer Received: July 1, 2013 Revised: August 14, 2013

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Scheme 1. A) Synthesis of an AB2 Type Poly(ε-benzyloxycarbonyl-L-lysine) with α-Thiol and ω-Alkyne Terminal Groups (PAPLys-SH) and B) Hyperbranched Poly(ε-benzyloxycarbonyl-L-lysine) (HPlys) and the Related HPlys-b-PEO Block Copolymer Synthesized via Thiol-Yne Chemistry

1,6-Diphenyl-1,3,5-hexatriene (DPH), 1,4-dithiothreitol (DTT, ≥99%), and poly(ethylene glycol) methyl ether (PEO, Mn = 2000) were purchased from Aldrich and used as received. ε-Benzyloxycarbonyl-L-lysine N-carboxyanhydride (lys-NCA) monomer was synthesized from ε-benzyloxycarbonyl-L-lysine with triphosgene according to the literature.49 The thiol-terminated PEO (PEO-SH, Mn = 2000) was synthesized according to the previous publication.40 The amine-terminated PEO (PEO-NH2, Mn = 2000, Adamas-beta) was used for the synthesis of linear block copolymer LPlys-b-PEO. Methods. FT-IR spectra were recorded on a Perkin-Elmer Paragon 1000 spectrometer at frequencies ranging from 400 to 4000 cm−1. Samples were thoroughly mixed with KBr and pressed into pellet form. 1 H NMR (400 MHz) spectrum was collected at room temperature on a Varian Mercury-400 spectrometer. All samples were dissolved in mixed solvents of CF3COOD and CDCl3 (CF3COOD: CDCl3 1:4, v/ v). Tetramethylsilane was used as an internal standard. Molecular weights and polydispersities (Mw: weight-average molecular weight, Mn: number-average molecular weight; Mw/Mn) of polymers were determined on a gel permeation chromatograph (GPC, HLC-8320, Tosoh Corporation, Japan) equipped with two HLC-8320 columns (TSKgel Super AWM-H, pore size: 9 μm; 6 × 150 mm, Tosoh Corporation) and a double-path, double-flow refractive index (RI) detector (Bryce) at 30 °C. The injection volume and concentration of polymers was 20 μL and 2 mg/mL, respectively. The elution phase was DMF-LiBr (0.01 mol·L−1) with an elution rate of 0.6 mL·min−1, and a series of polymethyl methacrylate was used as the calibration standard. The differential scanning calorimetry (DSC) analysis was carried out using a Perkin-Elmer Pyris 1 instrument under nitrogen flow (10 mL/min). All samples were first heated from 20 to 220 °C at 10 °C/min and held for 2 min to erase the thermal history, then

chain (e.g., forming polypseudorotaxanes of PCL macromonomer). As biodegradable polypeptide poly(ε-benzyloxycarbonyl-L-lysine) (Plys) exhibits a rigid α-helical conformation, it is reasoned that the rod AB2 type Plys can be used to prepare hyperbranched HPlys with a high DB by utilizing the thiol-yne chemistry (Scheme 1). The as-synthesized HPlys had a clickable periphery terminated by multiple alkyne groups, which was further conjugated with thiol-terminated poly(ethylene oxide) (PEO) to generate the hyperbranched block copolymer HPlys-b-PEO by consecutive thiol-yne chemistry. To the best of our knowledge, these have not been studied. The molecular structures and physical properties of HPlys and HPlys-b-PEO were fully investigated and compared with their linear analogues. Importantly, the self-assembled micelles of HPlys-b-PEO presented a higher thermodynamic stability, a better drug-loaded property, and an improved burst-release profile than the linear counterpart.

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EXPERIMENTAL SECTION

Materials. Dimethylformamide (DMF, ≥99.5%) and tetrahydrofuran (THF, ≥99%) were distilled from calcium hydride under reduced pressure and stored over molecular sieves, respectively. εBenzyloxycarbonyl-L-lysine (98%), cystamine dihydrochlodride (98%), dicyclohexylcarbodiimide (DCC, 99.2%), 4-dimethylaminopyridine (DMAP, 99.1%), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), 3,3′-dithiobis(propionic acid) (DTPA, 99%), N-hydroxybenzotrizole (HOBT, 99%), 4-pentynoic acid (PA, 99%), and triphosgene (99%) were purchased from local corporations and used as received. B

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cooled to 20 °C at 10 °C/min, and finally heated to 220 °C at 10 °C/ min. The melting temperature (Tm) of PEO and the liquid crystalline phase transition temperature (TLC) of all samples were taken as the maximum temperature of endothermic peak. The indium standard was used for temperature and enthalpy calibrations. The liquid crystal (LC) phase of polymer was observed using a Leica DMLP polarized optical microscope (Leica Microsystems GmbH, Germany). The powder sample was sandwiched between two glass plates, heated to 150−180 °C by 10 °C/min and held for 5 min (appearance of LC phase), and continued heating to 220 °C for 3 min, and finally quenched by 20 °C/min to the room temperature. 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 CuKα radiation source (wavelength = 1.54 Å). The supplied voltage and current were set to 40 kV and 30 mA, respectively. Samples were exposed at a scan rate of 2θ = 4° min−1 between 2θ = 2o and 40°. UV−vis spectra of samples were recorded at room temperature using a Spectrumlab54 UV−vis spectrophotometer. The mean size of nanoparticles was determined by dynamic light scattering (DLS) using a Malvern Nano_S instrument (Malvern, UK), and the solution of nanoparticles was performed at a scattering angle of 90° and at room temperature of 25 °C. All 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. Preparation of Thiol-Terminated Poly(ε-benzyloxycarbonylL-lysine). The thiol-terminated poly(ε-benzyloxycarbonyl-L-lysine) (Plys-SH) was synthesized from the ROP of lys-NCA in DMF solution at room temperature followed by a DTT reduction reaction. In a representative procedure, lys-NCA (765 mg, 2.5 mmol) was dissolved in 7 mL of DMF under nitrogen atmosphere, and then a degassed solution of cystamine (Cys) in 380 μL of DMF (20 mg/mL) was added via a syringe. The resulting solution was stirred vigorously at room temperature for 48 h and then precipitated dropwise into a large excess of diethyl ether (50 mL). The white precipitate was filtered and dried in vacuo at 40 °C to give 598 mg of Cys-Plys20 (91.3%, yield). 1H NMR of Cys-Plys20 (CDCl3/CF3COOD, v:v = 4:1): δ (ppm) = 1.21−1.44 (m, CH2CH2CH2), 1.55−1.75 (m, CH2CH2CH), 2.65−2.75 (t, SCH2CH2), 3.00−3.20(q, CH2CH2NH), 3.44−3.68 (t, SCH2CH2), 4.44 (t, CH2CH2CH), 5.11 (s, CH2Ph), 7.27−7.38 (m, CH2Ph). FT-IR (KBr, cm−1): 3300 (νN−H), 2937 (νC−H), 1734 (νCO), 1651 (amide I), 1550 (amide II), 1130 (νC− O−C). Mw,GPC = 14680, Mw/Mn = 1.46. Cys-Plys20 (531.6 mg, 0.05 mmol) and DTT (30.8 mg, 0.2 mmol) were dissolved into 5 mL of DMF under N2 atmosphere. The reaction mixture was stirred vigorously at room temperature for 24 h. The solution was concentrated under reduce pressure and then precipitated into a large excess of diethyl ether (50 mL). The precipitate was washed 3 times by using ether (20 mL × 3) and then dried in vacuo to give the product Plys20-SH (482.5 mg, yield 91.6%). 1H NMR of NH2Plys20-SH (CDCl3/CF3COOD, v:v = 4:1): δ (ppm) = 1.21−1.44 (m, CH2CH2CH2), 1.55−1.75 (m, CH2CH2CH), 2.60 (t, SCH2CH2), 3.00−3.20 (q, CH 2 CH 2 NH), 3.42 (t, SCH 2 CH 2 ), 4.44 (t, CH2CH2CH), 5.11 (s, CH2Ph), 7.27−7.38 (m, CH2Ph). FT-IR (KBr, cm‑1): 3300 (νN−H), 2937 (νC−H), 1734 (νCO), 1651 (amide I), 1550 (amide II), 1130 (νC−O−C). Preparation of AB2 Type Poly(ε-benzyloxycarbonyl-L-lysine) with α-Thiol and ω-Alkyne Terminal Groups (PA-PLys-SH). PA (11.8 mg, 0.12 mmol), DCC (37.1 mg, 0.18 mmol), and HOBT (24.3 mg, 0.18 mmol) were dissolved into 1 mL of DMF under N2 atmosphere. After having been stirred vigorously at room temperature for 8 h, Plys20-SH (425.3 mg, 0.08 mmol) in 4 mL of DMF was added dropwise into the PA solution. The mixture was stirred vigorously at room temperature for 36 h. After adding several drops of acetone, the mixture was filtered to remove dicyclohexylurea (DCU). The solution was concentrated under reduced pressure and then precipitated into a

large excess of ether (50 mL). The precipitate was washed 3 times with cold methanol (20 mL × 3) and then dried in vacuo to give the product PA-PLys20-SH (383.6 mg, yield 89%). 1H NMR of PA-PLysSH (CDCl3/CF3COOD, v:v = 4:1): δ (ppm) = 1.21−1.44 (m, CH2CH2CH2), 1.55−1.75 (m, CH2CH2CH), 2.0 (t, HCCCH2CH2), 2.42−2.5 (m, HCCCH2CH2), 2.60 (t, SCH2CH2), 3.00−3.20(q, CH2CH2NH), 3.42 (t, SCH2CH2), 4.44 (t, CH2CH2CH), 5.11 (s, CH2Ph), 7.27−7.38 (m, CH2Ph). FT-IR (KBr, cm−1): 3300 (νN−H), 2937 (νC−H), 1734 (νCO), 1651 (amide I), 1550 (amide II), 1130 (νC−O−C). Mw,GPC = 11490, Mw/Mn = 1.29. Synthesis of Hyperbranched Poly(ε-benzyloxycarbonyl-Llysine) (HPLys) via Thiol-Yne Chemistry. A typical click polycondensation example follows: both PA-PLys20-SH (50 mg) and DMPA photoinitiator (2.5 mg, 5 wt %) were dissolved into 0.5 mL of DMF in a 5 mL tube, where the exhausting-refilling process was carried out three times using a Schlenk line. The solution was then irradiated under a 365 nm high-pressure mercury lamp (150 W) for a predetermined time (i.e., 2, 5, 10, 30 min), where the distance between the lamp and the tube was 15 cm. The resulting solution was precipitated into 10 mL of ether and then dried in vacuo to give the product HPlys. 1H NMR of HPLys-10 (CDCl3/CF3COOD, v:v = 4:1): δ (ppm) = 1.21−1.44 (m, CH2CH2CH2), 1.55−1.75 (m, CH2CH2CH, SCHCH2CH2), 2.0 (t, HCCCH2CH2), 2.25−2.42 (m, SCHCH2CH2), 2.42−2.5 (m, HCCCH2CH2), 2.57−2.64 (m, NHCH 2 CH 2 S), 2.66−2.74 (m, SCH 2 CHS), 2.76−2.84 (m, SCH2CHS), 3.00−3.20(q, CH2CH2NH), 3.42 (t, NHCH2CH2S), 4.44 (t, CH2CH2CH), 5.11 (s, CH2Ph), 7.27−7.38 (m, CH2Ph). FTIR (KBr, cm−1): 3300 (νN−H), 2937 (νC−H), 1734 (νCO), 1651 (amide I), 1550 (amide II), 1130 (νC−O−C). Mw,GPC = 21380, Mw/Mn = 2.52. Synthesis of Hyperbranched Block Copolymer HPLys-b-PEO via Consecutive Thiol-Yne Chemistry. HPlys-10 (108.3 mg, alkyne group =0.01 mmol), PEO-SH (50.1 mg 0.024 mmol, 1.2 eqv), and DMPA photoinitiator (1.0 mg, 2 wt %) were dissolved into 1.5 mL of DMF in a 5 mL tube, where the exhausting-refilling process was carried out three times using a Schlenk line. The solution was then irradiated under a 365 nm high-pressure mercury lamp (150 W) for 30 min, where the distance between the lamp and the tube was 15 cm. The resulting solution was precipitated into 10 mL of ether to give the precipitate, which was then washed 3 times with ether/methanol solvents (v:v = 3/1, 20 mL × 3) to give the final product HPlys-b-PEO (127.3 mg, 80% yield). 1H NMR of HPlys-b-PEO (CDCl3/ CF3COOD, v:v = 4:1): δ (ppm) = 1.21−1.44 (m, CH2CH2CH2), 1.55−1.75 (m, CH2CH2CH), 3.00−3.20(q, CH2CH2NH), 3.49 (t, CH3O), 3.62−3.85 (s, OCH2CH2O), 4.01 (t, CH2CH2O), 4.44 (t, CH2CH2CH), 5.11 (s, CH2Ph), 7.27−7.38 (m, CH2Ph). FT-IR (KBr, cm−1): 3300 (νN−H), 2888(νC−H for PEO), 2937 (νC−H), 1734 (νCO), 1651 (amide I), 1550 (amide II), 1113 (νC−O−C for PEO). Mw,GPC = 31220, Mw/Mn = 1.91. Preparation of Doxorubicin-Loaded HPlys-b-PEO Micelles. The doxorubicin-loaded micelles were fabricated in aqueous solution according to our previous publications.49,50 HPlys-b-PEO copolymers (10 mg) and doxorubicin hydrochloride (5 mg) were dissolved in 10 mL of DMF, in which 1.5-fold of Et3N (12.9 μmoL) was added to neutralize HCl in solution. Distilled water (2 mL) was then added gradually at a speed of 20 μL/min using a microsyringe until the formation of nanoparticles. The resulting DOX-loaded micelles solution was then put into a dialysis bag and subjected to dialysis against 8 × 1 L of distilled water for 2 days. The drug loading capacity of micelles can be determined at 500 nm by UV−vis spectroscopy. In Vitro Doxorubicin Release from Drug-Loaded Micelles. The drug-loaded micelles solution (5 mL) was put into a dialysis bag, which was then put in a 15 mL buffer solution (pH = 7.4 or 5.7) in a tube at 37 °C. The drug-release solution was changed periodically (2, 4, 6, 8, 10, 12, 24 h, etc.), and the amount of doxorubicin released from micelles was measured by UV−vis at 500 nm. All release experiments were carried out in duplicate, and all data were averages of six determinations used for drawing figures. C

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Moreover, the integral ratio of α-thiol-connected methylene units or ω-alkyne of PA-Plys20-SH to the methine unit of Plys backbone was very close to the theoretical value (Hj+k/Hl/Hh/ Hg = 4//1/2/20). The number-average molecular weight calculated by 1H NMR is 5400. In addition, PA-Plys20-SH has a weight-average molecular weight (M w ) of 11490 and polydispersity (Mw/Mn) of 1.29. Collectively, these results confirmed that an AB2 type polypeptide macromonomer PAPlys-SH with α-thiol and ω-alkyne terminal groups was successfully synthesized by the combination of ROP and DCC/HOBT conjugation reaction. Synthesis of Hyperbranched Polypeptides HPlys via Thiol-Yne Chemistry. The AB2 type polypeptide macromonomer PA-Plys20-SH was photopolymerized by 2,2dimethoxy-2-phenylacetophenone (DMPA) under UV irradiation at 365 nm to produce HPlys (Scheme 1B), and the detailed results were summarized in Table 1. It is observed that

RESULTS AND DISCUSSION Synthesis of AB2 Polypeptide Macromonomer Poly(εbenzyloxycarbonyl-L-lysine) with α-Thiol and ω-Alkyne Terminal Groups (PA-Plys-SH). The AB2 polypeptide macromonomer PA-Plys-SH was synthesized by the following two steps. First, the polypeptide poly(ε-benzyloxycarbonyl-Llysine) with α-thiol and ω-amine terminal groups (Plys20-SH, the subscript denotes the degree of polymerization) was synthesized by cystamine initiated ring-opening polymerization (ROP) of lys-NCA in DMF at room temperature, followed by the reduction of a disulfide bond (-S-S-) into thiol (-SH) using 1,4-dithiothreitol in DMF at room temperature (Scheme 1 A). Besides the typical proton signals of Plys backbone (Figure 1A), the 1H NMR spectrum of Plys20-SH clearly shows that the

Table 1. Synthesis of Hyperbranched HPlys and HPLys-bPEO by Using Thiol-Yne Chemistry sample

a

PA-PlysSH HPlys-2 HPlys-5 HPlys-10 HPlys-30 HPlys-bPEO LPlys-bPEOe PEO-SH

click efficiencyc (%)

yieldd (%)

irradiation time (min)

Mw,GPC

0

11490

1.29

0

92.7

2 5 10 30 30

15570 21440 21380 21310 31220

2.12 2.36 2.52 2.36 1.91

65.4 88.4 93.2 99.0 100

86.7 78.3 74.6 67.9 80.6



14035

1.41



92.3



2250

1.23



78.1

b

Mw/Mn

b

a

The click polymerization was irradiated under 365 nm using 2 wt % or 5 wt % DMPA photoinitiator. bBoth the weight-averaged molecular weights (Mw,GPC) and the polydispersity (Mw/Mn) of polymers were determined by GPC. cClick efficiency was calculated based on the alkyne conversion determined by 1H NMR. dThe yield polymers were determined gravimetrically. eLPlys-b-PEO represents linear block copolymer used as a control.

the Mw of HPlys gradually increased with the light irradiation time and then leveled off in the range of 5 to 30 min. Meanwhile, the polydispersity progressively became broadening from 1.3 to about 2.4 and then kept stable. As shown in Figure 2, the GPC traces showed that the elution peak of HPlys gradually shifted toward the higher molecular weight region with a broad polydispersity compared with that of the linear precursor PA-Plys20-SH. The shoulders in GPC traces are probably induced by the stronger interactions between hyperbranched polypeptides and GPC columns.15,16 Besides the typical proton signals of Plys backbone, the 1H NMR spectrum of HPlys clearly shows new signals at 2.8 (m) and 2.7 ppm (n) assignable to methylene (S-CH2) and methine (SCH) (Figure 3). Moreover, the integral of the alkyne terminals progressively decreased over the click polymerization and the click efficiency increased from 65% to 99% over the irradiation time ranging from 2 to 30 min. As each alkyne reacted with two thiols, 50% of the alkyne terminals retained in the periphery of HPlys, which was in good agreement with the theoretical value. In addition, no discernible peak for the possible alkene intermediate (-S-CHCH-) is observed at 5.4−6.5 ppm, indicating that no alkene would be produced by one thiol

Figure 1. 1H NMR spectra of PLys20-SH (A) and PA-PLys20-SH (B).

two methylene (HS-CH2-CH2-) groups adjacent to thiol terminal appeared at 3.4 (h) and 2.6 ppm (i). Moreover, the integral ratio of the methylene units (h or i) of NH2-Plys20-SH to the methine (-CH-, g) of the Plys backbone was equal to the theoretical value (Hh/Hi/Hg = 2/2/20). In the second step, using the DCC/HOBT catalyzed conjugation reaction, the intermediate Plys-SH was connected with 4-pentynoic acid (PA) to produce PA-Plys20-SH. Compared with Plys20-SH, PAPlys20-SH showed new proton signals at 2.5 ppm and 2.0 ppm assignable to the methylene groups (-NHCO-CH2CH2-C≡CH, j+k) and the alkyne terminal (-C≡CH, l) (Figure 1B). D

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Figure 2. GPC traces of HPlys, HPlys-b-PEO, PA-Plys-SH, and PEO-SH.

segments, 1H NMR spectrum of HPlys-b-PEO showed that the proton signal at 2.0 ppm assignable to the original alkyne within HPlys disappeared completely, suggesting that the alkyne periphery of HPlys was fully reacted by PEO-SH (Supporting Information, Figure S1). Collectively, these results demonstrate that hyperbranched HPlys-b-PEO block copolymer can be click conjugated between HPlys and PEO-SH by consecutive thiolyne chemistry (Scheme 1B). FT-IR, WAXD, and DSC Analyses. FT-IR spectra of hyperbranched HPlys and HPlys-b-PEO showed the typical amide I and II bands at 1651 cm−1 and 1550 cm−1 similar to linear precursor PA-Plys-SH (Figure S2). This indicated that they mainly assumed an α-helix conformation in solid state and at room temperature.15,16,49 WAXD was further used to demonstrate the secondary conformation of HPlys and HPlys-b-PEO in solid state, as shown in Figure 4. Compared with their linear counterparts, HPlys and HPlys-b-PEO similarly presented a set of three Bragg peaks at about 0.43 Å−1, 0.75 Å−1, and 0.87 Å−1 with a ratio of 1:31/2:2. This

Figure 3. 1H NMR spectrum of a representative sample HPLys-30.

addition to alkyne for HPlys. This result implies that the degree of branching (DB) of HPlys is close to 1 within the error of 1H NMR measurement.43−45,48 Taken together these results demonstrate that the click polycondensation of PA-Plys-SH occurred in a fast manner under 365 nm UV irradiation and produced HPlys with a high DB. To the best of our knowledge, this is the first example that describes the synthesis of biodegradable and hyperbranched polypeptides HPlys with clickable alkyne periphery, which can be further conjugated by consecutive thiol-yne chemistry.34−48 The click conjugation reaction between as-synthesized HPlys and thiol-terminated PEO (PEO-SH) in DMF was conducted using DMPA initiator (2 wt %) and 365 nm UV irradiation for 30 min. Compared with both HPlys-10 (Mw,GPC = 21380) and PEO-SH (Mw,GPC = 2250) precursors, the resulting HPlys-bPEO block copolymer apparently moved toward a higher molecular weight region (Mw,GPC = 31220) in GPC traces (Figure 2 and Table 1), convincingly confirming the successful synthesis. Besides the characteristic signals of PEO and Plys

Figure 4. WAXD patterns of HPlys, HPlys-b-PEO, LPlys-b-PEO, PAPlys-SH, and PEO-SH. E

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Figure 5. DSC curves of HPlys, HPlys-b-PEO, LPlys-b-PEO, PA-Plys-SH, and PEO-SH in the first heating run.

confirmed that they mainly assumed an α-helix conformation, which is consistent with the above FT-IR analysis.49−52 Note that the broad diffraction peak at 1.4 Å−1 corresponds to a helical pitch of ∼5 Å. In addition, both HPlys-b-PEO and LPlys-b-PEO showed very weak Bragg diffraction peaks at about 1.34 Å−1 and 1.63 Å−1 compared with the PEO homopolymer.15,49,50 This result demonstrates that the crystallinity of PEO block within copolymers was greatly prohibited, which is clarified by the following DSC analysis. DSC can characterize the liquid crystalline (LC) structure of Plys and crystallinity of PEO in solid state, as shown in Figure 5 and Table S1. Linear precursor PA-Plys-SH presented a clear LC phase transition temperature (TLC) at 192.2 °C in the first heating run.49−52 However, the LC phase transition gradually disappeared within hyperbranched polypeptides such as HPlys2 and HPlys-10, indicating that the ill-defined hyperbranched topology prohibited the formation of LC phase. Similarly, the LC phase transition appeared in linear block copolymer LPlysb-PEO, while it disappeared in hyperbranched block copolymer HPlys-b-PEO. Note that the LC phase transition disappeared in the second heating run during DSC measurement, suggesting that the LC phase transition was irreversible (Figure S3).49−53 The LC phase was further monitored by polarized optical microscopy (POM). The LC phase appeared at about 5 min and grew slowly after linear PA-Plys-SH and/or LPlys-b-PEO were annealed at temperature of 150−180 °C (Figure S4). Meanwhile, the LC phase retained even if the temperature increased to 220 °C and then decreased to 20 °C at a cooling rate of 20 °C/min. This thermotropic LC phase was possibly induced by the nematic orientation of the rodlike Plys.50,51 In addition, HPlys-b-PEO gave a lower degree of crystallinity (Xc = 15.2%) compared with PEO having a high Xc of 89.5%. This result demonstrated that the crystallinity of PEO was greatly attenuated within HPlys-b-PEO, which was consistent with the above WAXD analysis. In addition, the biodegradation of synthetic polypeptides is usually through a proteolytic mechanism.20,54 In our case, the hyperbranched topology prohibited the formation of liquid crystalline mesophase compared with linear counterpart, which might be beneficial for the biodegradation. On the other hand, the steric hindrance induced by dendritic topology will to some

extent inhibit the enzymatic activity.54 So, how the hyperbranched topology affects on the biodegradation of polypeptides needs further investigation. Self-Assembled Micelles and Drug-Release Properties of HPlys-b-PEO. Because of the amphiphilic nature of the HPlys-b-PEO block copolymer, the critical aggregation concentration (cac) that represents the thermodynamic stability was measured by UV−vis spectroscopy using 1,6-diphenyl1,3,5-hexatriene (DPH) dye.49,50 Figure 6 shows that the

Figure 6. The relationship of the absorbance intensity of DPH as a function of HPlys-b-PEO or LPlys-b-PEO concentration in aqueous solution.

absorbance intensity of DPH remained constant and then increased sharply, reflecting the spontaneous self-assembly.49,50 HPlys-b-PEO gave a cac of 8.9 × 10−3 mg/mL, which is 5-fold lower than that of the linear counterpart LPlys-b-PEO (4.5 × 10−2 mg/mL). This result demonstrates that the hyperbranched topology might greatly increase the thermodynamic stability of self-assembled aggregates in aqueous solution.6,55 As shown in Figures 7 and 8, both the average size and the morphology of the self-assembled aggregates were characterized F

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Figure 7. Nanoparticle size distribution of copolymer micelles (A, C) and drug-loaded micelles (B, D) measured by DLS.

Figure 8. TEM photographs of copolymer micelles (A, C) and drug-loaded micelles (B, D): A, HPlys-b-PEO; B, HPlys-b-PEO/DOX; C, LPlys-bPEO; D, LPlys-b-PEO/DOX.

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by means of DLS and TEM. HPlys-b-PEO self-assembled into nearly spherical aggregates with a smaller diameter (166.2 ± 12.6 nm) than LPlys-b-PEO ones with a size of 253.6 ± 10.3 nm, which was partially due to the lower cac of hyperbranched copolymer.6,55 Note that the DLS-determined size is largely bigger than that determined by TEM, which is induced by two following facts. One is the loose PEO corona of the selfassembled aggregates cannot be discerned as the aggregates were not negatively stained during TEM measurement, and the other is the aggregates dehydration under the high vacuum condition.15,55,56 As a first-line anticancer drug in clinic,49,50,56 doxorubicin (DOX) was used to study the drug-loading and in vitro drugrelease properties of the nanoparticles, and the drug-loading parameters were summarized in Table 2. Besides a higher yield Table 2. Drug-Loading Parameters for the DOX-Loaded Micelles of HPlys-b-PEO and LPlys-b-PEO Block Copolymers sample

HPlys-b-PEO/DOX

LPlys-b-PEO/DOX

DLCa DLEb yieldc

15.2% 38.2% 85.5%

12.5% 13.9% 37.3%

Figure 9. In vitro drug-release profiles of drug-loaded micelles at 37 °C and at aqueous pH 7.4 or 5.7 (K denotes the initial drug-release rate).

a

DLC denotes the drug-loading capacity of micelles, which is calculated as the weight ratio of actual drug to drug-loaded nanoparticles. bDLE denotes the drug loading efficiency of micelles, which is calculated as the weight ratio of actual and added drug content. cYield denotes the weight ratio of the forming DOX-loaded micelles to the mixture of copolymer and drug added.

7.4. It is known that the doxorubicin release from nanoparticles was mainly controlled by a drug diffusion mechanism, which was related to the water uptake by hydrophilic PEO.20,49 That is to say, both each block length and copolymer composition are the most important parameters compared with the whole molecular weight of copolymer. As hydrophobic Plys and hydrophilic PEO were designed the same in hyperbranched and linear copolymers, the hyperbranched topology apparently affected the drug release properties. At pH 5.7 similar to the weakly acidic environment of endosome or lysosome inside a cell, all the DOX-loaded nanoparticles gave a relatively faster drug-release profile than those at pH 7.4, but the pH effect was less than the copolymer topology. Collectively, compared with those of a linear analogue, the self-assembled aggregates of hyperbranched block copolymer HPlys-b-PEO showed a 5-fold lower cac and a higher thermodynamic stability, a better drug-loading property (i.e., higher yield of forming drug-loaded nanoparticles, higher DLE and DLC), and a slower apparent drug-release rate with a smoothed burst-release profile. These properties might enhance hyperbranched polypeptide copolymer HPlys-b-PEO useful for drug delivery systems.6,55,56

of forming drug-loaded nanoparticles, HPlys-b-PEO gave a higher drug-loading efficiency (DLE) of 38.2% and a higher drug-loading capacity (DLC) of 15.2% than LPlys-b-PEO. These results suggest that hyperbranched polypeptide copolymers might provide larger cavities for encapsulating drugs than linear counterpart.6 The DOX-loaded nanoparticles of HPlys-bPEO showed a spherical morphology with an inner dark core (see red arrow, Figure 8 and Figure S5) and a diameter of 205.5 ± 31.0 nm, which was due to the drug enhanced contrast in the core of nanoparticles. Note that the drug-loaded nanoparticles had a higher variation in size than the blank ones, which was due to the fact that the hydrophobic doxorubicin drug changed the hydrophobic−hydrophilic ratio of hyperbranched polypeptide copolymers.49,50 Moreover, the fluorescence intensity of DOX-loaded nanoparticles (e.g., 0.05 mg/mL) greatly quenched and was about 17-fold lower than that of free DOX (Figure S6). This result also verified that DOX was indeed entrapped into the micellar core. It is known that the drug-loaded polymeric micelles or vesicles often present a premature burst-release behavior.49,50,56 Does the hyperbranched topology of copolymer affect the drugrelease profile? In vitro drug-release property of these DOXloaded nanoparticles was studied in PBS and at pH 7.4 or 5.7 (Figure 9), and all DOX-loaded nanoparticles mainly showed a biphasic drug-release profile.49,50 During initial drug-release stage of 48 h, the DOX-loaded nanoparticles of HPlys-b-PEO released 24.7% DOX at pH 7.4, while those of LPlys-b-PEO released 44.3% DOX. These results demonstrate that the hyperbranched topology improved the burst-release behavior of DOX-loaded nanoparticles. Meanwhile, the apparent drugrelease rate of HPlys-b-PEO was about 1.7-fold slower than that of LPlys-PEO, and 42.8% DOX was released over 324 h at pH

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CONCLUSIONS Hyperbranched polypeptide HPlys with clickable alkyne periphery was synthesized by the polycondensation of an AB2 Plys macromonomer with α-thiol and ω-alkyne terminal groups and then conjugated with PEO-SH to generate HPlys-b-PEO block copolymer using consecutive thiol-yne chemistry. Their molecular structures and physical properties were thoroughly investigated. HPlys and HPlys-b-PEO mainly presented an αhelix conformation similar to the linear analogues, while the liquid crystalline phase transition disappeared within HPlys and HPlys-b-PEO. Similar to a linear counterpart, HPlys-b-PEO self-assembled into nearly spherical aggregates in aqueous solution, while it gave a 5-fold lower cac, a better drug-loading property, and a slower apparent drug-release rate with a smoothed burst-release profile. This work establishes a platform for the synthesis of hyperbranched polypeptides and related H

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block copolymers, and the drug-loaded nanoparticles exhibited better properties useful for drug delivery systems.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1, 1H NMR, FT-IR, DSC, POM, and fluorescence spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-21-54748916. Fax: 86-21-54741297. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support of National Natural Science Foundation of China (21274086 and 21074068) and Shanghai Leading Academic Discipline Project (B202). The assistance of Instrumental Analysis Center of SJTU is also appreciated.

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