ARTICLE pubs.acs.org/Biomac
Bioreducible Micelles Self-Assembled from Amphiphilic Hyperbranched Multiarm Copolymer for Glutathione-Mediated Intracellular Drug Delivery Jinyao Liu,† Yan Pang,† Wei Huang,* Xiaohua Huang, Lili Meng, Xinyuan Zhu, Yongfeng Zhou, and Deyue Yan* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic of China
bS Supporting Information ABSTRACT: A new type of biodegradable micelles for glutathione-mediated intracellular drug delivery was developed on the basis of an amphiphilic hyperbranched multiarm copolymer (H40-star-PLA-SS-PEP) with disulfide linkages between the hydrophobic polyester core and hydrophilic polyphosphate arms. The resulting copolymers were characterized by nuclear magnetic resonance (NMR), Fourier transformed infrared (FTIR), gel permeation chromatography (GPC), and differential scanning calorimeter (DSC) techniques. Benefiting from amphiphilic structure, H40-star-PLA-SS-PEP was able to selfassemble into micelles in aqueous solution with an average diameter of 70 nm. Moreover, the hydrophilic polyphosphate shell of these micelles could be detached under reductionstimulus by in vitro evaluation, which resulted in a rapid drug release due to the destruction of micelle structure. The glutathionemediated intracellular drug delivery was investigated against a Hela human cervical carcinoma cell line. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that H40-star-PLA-SS-PEP micelles exhibited a faster drug release in glutathione monoester (GSH-OEt) pretreated Hela cells than that in the nonpretreated cells. Cytotoxicity assay of the doxorubicin-loaded (DOX-loaded) micelles indicated the higher cellular proliferation inhibition against 10 mM of GSH-OEt pretreated Hela cells than that of the nonpretreated ones. As expected, the DOX-loaded micelles showed lower inhibition against 0.1 mM of buthionine sulfoximine (BSO) pretreated Hela cells. These reduction-responsive and biodegradable micelles show a potential to improve the antitumor efficacy of hydrophobic chemotherapeutic drugs.
’ INTRODUCTION Despite the discovery of various antitumor drugs for cancer therapy, clinical outcomes have been disappointing because most of them exhibit severe side effects. As one of the most promising nanocarrier systems, self-assembled polymeric micelles have been employed to overcome the problems.14 The polymeric micelle carriers possess several unique features, such as enhancing the aqueous solubility, prolonging the circulation time, improving the preferential accumulation at the tumor site by the enhanced permeability and retention (EPR) effect, and reducing systemic side effects.5,6 However, the effective concentration of the active anticancer drug is often insufficient within cancer cells due to the poor drug release from the polymeric micelles into the cytoplasm. The rapidly intracellular drug release is desirable after the micelles arrive at the cytoplasm, which may enhance the anticancer efficacy as well as reduce drug resistance in cancer cells. Driven by a need to rapidly drug release in response to an appropriate stimulus, various stimuli-responsive micelles have r 2011 American Chemical Society
been developed as smart drug carriers and investigated extensively in the past few years, including temperature-, pH-, ultrasound-, redox-, and enzyme-responsive ones.716 Upon reaching the target tumor cells, these drug-loaded micelles can be localized intracellularly and subsequently provoked by the stimulus to release the drugs rapidly, which results in aggressive activity within tumor cells and enhances the therapeutic efficacy with relatively low side effects.16 Among these smart drug carriers, reduction-sensitive micelles have received great attention for intracellular drug delivery due to the existence of a large difference in the redox potential between the mildly oxidizing extracellular milieu and the reducing intracellular fluids.17 The micelles containing disulfide bonds have been explored for this purpose, which can be cleaved in the presence of reducing agents such as glutathione (GSH). As a thiol-containing tripeptide, Received: December 15, 2010 Revised: March 23, 2011 Published: April 04, 2011 1567
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Biomacromolecules GSH is found in the blood plasma of humans with micromolar concentrations, whereas it is around 10 mM in the cytosol.18 Especially, the concentration level of cytosolic GSH in some tumor cells has been found to be several times higher than that in normal cells.19 Therefore, disulfide-containing polymeric micelles can facilitate intracellular release of the encapsulated drugs by the cleavage of this bond.2024 Hyperbranched polymers are highly branched macromolecules with dendritic units, linear units, terminal units, and threedimensional torispherical architecture, which have attracted increasing attention in biomedicine, especially for drug delivery applications.2531 Comparing to dendrimer with perfectly branched and monodisperse structure,32 hyperbranched polymer is rather irregular due to the random distribution of these structure units in the backbone and can be prepared by facile onepot synthesis.29,30 During the past decade, micelles from amphiphilic hyperbranched multiarm copolymers have displayed great potential to construct promising drug delivery systems with improved micelle stability and drug loading ability.3338 However, to the best of our knowledge, self-assembled micelles based on amphiphilic hyperbranched multiarm copolymers containing unique disulfide bonds for glutathione-mediated intracellular drug delivery has not yet been reported. It is well-known that the biodegradability of polymeric micelles is an essential parameter for drug delivery applications. In other words, nonbiodegradable drug carriers may lead to an accumulation in different organs and cause toxic effects or inflammation.39,40 Our previous research demonstrates that the micelles self-assemble from amphiphilic hyperbranched multiarm copolymers containing biodegradable polyphosphate moiety are promising drug carriers.4143 In this work, a new type of biodegradable amphiphilic hyperbranched multiarm copolymer with disulfide-linkages between hydrophobic and hydrophilic moieties was synthesized and used to construct a drug delivery system for intracellular drug release mediated by GSH in tumor cells. This copolymer was able to self-assemble into micelles in aqueous solution, and the hydrophilic shell of micelles could be detached under reduction-stimulus and resulted in a rapid drug release with the destruction of micelles. The glutathionemediated intracellular drug delivery was also investigated against a Hela human cervical carcinoma cell line by pretreating these cells with glutathione monoester (GSH-OEt) or buthionine sulfoximine (BSO), which could increase or decrease the concentration of GSH in the cytoplasm, respectively.
’ EXPERIMENTAL SECTION Materials. 2-Ethoxy-2-oxo-1,3,2-dioxaphospholane (EP) was synthesized by a method described previously and distilled under reduced pressure just before use.41 Propargyl alcohol and N,N-dimethylformamide (DMF) were dried over calcium hydride and then purified by vacuum distillation. Tetrahydrofuran (THF) was dried by refluxing with the fresh sodium-benzophenone complex under N2 and distilled just before use. L-Lactide (LA) was purchased from Sigma and recrystallized from ethyl acetate before use. Boltorn H40 (H40) was obtained from Perstorp Polyols AB, Sweden. Tin(II) octoate (Sn(Oct)2), 3,30 -dithiodipropionic acid, DL-1,4-dithiothreitol (DTT), glutathione monoester (GSH-OEt), buthionine sulfoximine (BSO), N,N-dicyclohexylcarbodiimide (DCC), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and 4-dimethylamino-pyridine (DMAP) were purchased from Sigma and used as received. Doxorubicin hydrochloride (DOX 3 HCl) was purchased from Beijing Huafeng United Technology
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Corporation and used as received. Clear polystyrene tissue culture treated 12- and 96-well plates were obtained from Corning Costar. All other reagents and solvents were purchased from the domestic suppliers and used as received. Measurements. Nuclear magnetic resonance (NMR) analyses were recorded on a Varian Mercury Plus 400 MHz spectrometer with deuterated chloroform (CDCl3) as the solvent. The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) were measured by gel permeation chromatography (GPC). GPC was performed on a Perkin-Elmer series 200 system (10 μm PL gel 300 7.5 mm mixed-B and mixed-C column, linear polystyrene calibration) equipped with a refractive index (RI) detector. DMF containing 0.01 mol/L lithium bromide was used as the mobile phase at a flow rate of 1 mL/min at 70 °C. Fourier transform infrared spectrometer (FTIR) spectra were recorded on a Paragon 1000 instrument by KBr sample holder method. Differential scanning calorimeter (DSC) was performed on a Perkin-Elmer Pyris 1 in nitrogen atmosphere. Both In and Zn standards were used for temperature and enthalpy calibrations. First, all the samples (about 5.0 mg in weight) were heated from room temperature to 150 °C, held at this temperature for 3 min to remove the thermal history and quenched to 80 °C. Then all samples were heated again from 80 to 150 °C at 10 °C/min to determine the glass transition temperature (Tg). Dynamic light scattering (DLS) measurements were performed in aqueous solution using a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples of 1 mg/mL were measured at 20 °C and at a scattering angle of 173°. Transmission electron microscopy (TEM) studies were performed with a JEOL 2010 instrument operated at 200 kV. The samples were prepared by directly dropping the solution of micelles onto carbon-coated copper grids and dried at room temperature overnight without staining before measurement. Purification of H40. A total of 2.50 g commercially available H40 was dissolved in 25 mL of acetone. A total of 25 mL of ethyl ether was slowly added into the polymer solution under vigorous stirring. After 12 h, the precipitates were filtered, washed twice with acetone/ether mixture (v/v, 1:1), and dried under vacuum, and finally, 1.06 g of purified H40 was obtained. Synthesis of H40-star-poly(L-lactide) (H40-star-PLA). H40star-PLA was prepared by the ring-opening polymerization (ROP) of LA using the purified H40 as a macroinitiator and Sn(Oct)2 as a catalyst. In a typical polymerization procedure: H40 (250 mg, 5.65 mmol hydroxyl groups) was added into a 50 mL flask and dried under vacuum at 100 °C for 1 h. Then LA (5.70 g, 39.6 mmol) and Sn(Oct)2 (16.2 mg, 0.040 mmol) were also added into the flask in a glovebox with the water content less than 0.1 ppm. The flask was immerged in an oil bath at 125 °C and the reaction mixture was stirred for 20 h. The crude H40star-PLA was diluted with THF and precipitated in cold diethyl ether. Then it was purified by dissolving in THF, passing through a neutral alumina column, concentrating, precipitating in diethyl ether, and finally drying under vacuum; 83.2% yield, Mn,GPC = 7.5 104 g/mol, Mw/Mn = 1.3, Mn,NMR = 10.9 104 g/mol. 1H NMR (CDCl3, ppm): 5.15 (CCH(CH3)O), 4.34 (CCH(CH3)OH), 4.24 (C(CH3)CH2O), 1.56 (CCH(CH3)O), 1.24 (C(CH3)CH2O).
Synthesis of Poly(2-ethoxy-2-oxo-1,3,2-dioxaphospholane) (PEP). PEP was prepared by the ROP of EP using propargyl alcohol as an initiator and Sn(Oct)2 as a catalyst. In a typical polymerization procedure, propargyl alcohol (61.4 mg, 1.10 mmol), EP (5.00 g, 32.9 mmol), and 20 mL of THF were added to a 25 mL flask in a glovebox with the water content less than 0.1 ppm. Then, Sn(Oct)2 (222 mg, 0.548 mmol) was also added into the flask. The reaction mixture was placed in an oil bath at 35 °C for 3 h. The solution was precipitated into cold diethyl ether containing 10% methanol (v/v) twice. Finally, the purified PEP was obtained by vacuum-drying overnight; 78.2% yield, Mn, 4 4 1 GPC = 0.29 10 g/mol, Mw/Mn = 1.3, Mn,NMR = 0.28 10 g/mol. H 1568
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Biomacromolecules NMR (CDCl3, ppm): 4.20 (OCH2CH2O), 4.14 (OCH2CH3), 3.74 (OCH2CH2OH), and 1.32 (OCH2CH3).
Synthesis of Disulfide-Linked Carboxyl-Terminal PEP (PEP-SS-COOH). PEP-SS-COOH was prepared by coupling 3,30 -
dithiodipropionic acid with PEP. In a typical procedure, 3,30 -dithiodipropionic acid (0.420 g, 2.00 mmol) was dissolved in 15 mL of anhydrous DMF under N2 for 15 min. DCC (0.412 g, 2.00 mmol) in 5 mL of DMF was added and stirred for an additional 30 min. After the addition of a catalytic quantity of DMAP (0.123 g, 1.00 mmol) and the corresponding PEP (2.90 g, 1.00 mmol), the reaction mixture was stirred for 8 h at room temperature. Then the reaction mixture was filtered to remove dicyclohexylurea and the filtrate was concentrated at 30 °C under vacuum. The residue was precipitated into cold diethyl ether. After filtration, the crude product was purified by dissolving in CH2Cl2, passing through a neutral alumina column, concentrating, precipitating in cold diethyl ether, and finally drying under vacuum; 54.2% yield, Mn, 4 4 1 GPC = 0.31 10 g/mol, Mw/Mn = 1.4, Mn,NMR = 0.30 10 g/mol. H NMR (CDCl3, ppm): 4.20 (OCH2CH2O), 4.14 (OCH2CH3), 2.91 (CH2CH2SSCH2CH2), 2.73 (CH2CH2SSCH2CH2), and 1.32 (OCH2CH3). Synthesis of H40-star-PLA-SS-PEP. H40-star-PLA-SS-PEP was prepared by coupling PEP-SS-COOH with H40-star-PLA. In a typical procedure, H40-star-PLA (518 mg, 0.304 mmol of hydroxyl groups), PEP-SS-COOH (1.368 g, 0.456 mmol), DCC (93.9 mg, 0.406 mmol), and DMAP (37.1 mg, 0.304 mmol) were dissolved in 30 mL of dry DMF and kept under vigorous stirring at room temperature for 16 h. Then the reaction mixture was filtered to remove dicyclohexylurea, and the filtrate was concentrated under vacuum. The residue was precipitated in diethyl ether. After filtration, the crude product was purified by dissolving in dimethyl sulfoxide (DMSO), dialyzing (MWCO = 50000 g/mol) against DMSO for 3 h, then dialyzing against deionized water for 48 h (Note: Exchanged deionized water at appropriate intervals), and finally freeze-drying to obtain the pure H40-star-PLA-SS-PEP; 85.5% yield, Mn,GPC = 10.4 104 g/mol, Mw/Mn = 1.3, Mn,NMR = 20.5 104 g/mol. 1H NMR (CDCl3, ppm): 5.15 (CCH(CH3)O), 4.68 (CHCCH2O), 4.25 (OCH2CH2O), 4.16 (OCH2CH3), 2.92 (CH2CH2SSCH2CH2), 2.79 (CH2CH2SSCH2CH2), 2.64 (CHCCH2O), 1.56 (CCH(CH3)O), and 1.35 (OCH2CH3). Preparation of Self-Assembled Micelles. Polymeric micelles were prepared by a dialysis method. A total of 20 mg H40-star-PLA-SSPEP was dissolved in 2 mL of DMF and stirred at room temperature for 2 h. Then the polymer solution was slowly added into 5 mL of deionized water under vigorous stirring. After 2 h, the solution was transferred into dialysis membrane tubing (MWCO = 15000 g/mol) and dialyzed against deionized water for 24 h to remove the DMF. During the dialyzing process, the water was exchanged at appropriate intervals.
Reduction-Triggered Destabilization of H40-star-PLA-SSPEP Micelles. The size change of micelles in response to 10 mM of DTT in phosphate buffered saline (PBS; 50 mM, pH 7.4) was determined by DLS measurement. Briefly, 10 mM of DTT was added into a 10 mL solution of H40-star-PLA-SS-PEP micelles (1 mg/mL) in PBS (50 mM, pH 7.4), which was degassed in advance with nitrogen for 30 min. Then the solution was mildly stirred at 37 °C. At different time intervals, the size was measured by DLS. Preparation of DOX-Loaded Micelles. DOX-loaded micelles were prepared as follows: 20 mg H40-star-PLA-SS-PEP was dissolved in 2 mL of DMF, followed by adding a predetermined amount of DOX 3 HCl and 2 molar equiv of triethylamine and stirred at room temperature for 2 h. Then the mixture was slowly added into 5 mL of deionized water and stirred for another 2 h. Subsequently, the solution was dialyzed against deionized water for 24 h (MWCO = 15000 g/mol) and the deionized water was exchanged every 4 h. To determine the total loading of the drug, the DOX-loaded micelle solution was lyophilized and then
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dissolved in DMF again. The UV absorbance of the solution at 500 nm was measured to determine the total loading of DOX. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula: DLCðwt%Þ ¼ ðweight of loaded drug=weight of polymerÞ 100% DLEð%Þ ¼ ðweight of loaded drug=weight in feedÞ 100%
Reduction-Triggered Drug Release. The DOX-loaded micelles of H40-star-PLA-SS-PEP were diluted to 1 mg/mL and then 0.7 mL of this solution was transferred into a membrane tubing (MWCO = 15000). It was immersed into a glass bottle containing 20 mL of PBS (50 mM, pH 7.4) or PBS with 0.1 mM of DTT in a shaking water bath at 37 °C to acquire sink conditions. At predetermined time intervals, 6 mL of external buffer solution was withdrawn and replaced with 6 mL of fresh PBS or PBS with 0.1 mM of DTT. The amount of DOX released was determined by using fluorescence measurements (LS-50B luminescence spectrometer, excitation at 480 nm). All DOX-release experiments were conducted in triplicate and the results are expressed as the average data with standard deviations. Cell Culture. Hela cells (a human cervical carcinoma cell line) were cultivated in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum) and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Intracellular Drug Release. The experiments of intracellular drug release were performed on flow cytometry and confocal laser scanning microscopy (CLSM). Flow Cytometry. Hela cells were seeded in six-well plates at 5 105 cells per well in 1 mL of complete DMEM and cultured for 24 h, and then treated with GSH-OEt for 2 h. Cells were washed by PBS and incubated at 37 °C for an additional 1 h with DOX-loaded H40-starPLA-SS-PEP micelles at a final DOX concentration of 0.90 μg/mL in complete DMEM. Cells without GSH-OEt treatment were used as the control. Thereafter, culture medium was removed and cells were washed with PBS three times and treated with trypsin. Then, 2 mL of PBS was added to each culture well and the solutions were centrifugated for 5 min at 1000 rpm. After the removal of supernatants, the cells were resuspended in 0.5 mL of PBS. Data for 1 104 gated events were collected and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. CLSM. Hela cells were seeded in six-well plates at 2 105 cells per well in 1 mL of complete DMEM, cultured for 24 h, and then treated with GSH-OEt for 2 h. Cells were washed by PBS and incubated at 37 °C for an additional 1 h with DOX-loaded H40-star-PLA-SS-PEP micelles at a final DOX concentration of 0.90 μg/mL in complete DMEM. Cells without GSH-OEt treatment were used as the control. Then the culture medium was removed and cells were washed with PBS three times. Thereafter, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS for three times. Finally, the cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min, and the slides were rinsed with PBS three times. The slides were mounted and observed by a LSM 510META. Activity Analyses. The cytotoxicity of DOX-loaded H40-starPLA-SS-PEP micelles against Hela cells was evaluated in vitro by MTT assay. Hela cells were seeded into 96-well plates at 1 104 cells per well in 200 μL of medium and further incubated for 24 h. The cells were then treated with GSH-OEt for 2 h or BSO for 12 h. Cells without pretreatment were used as the control. After washing off the GSH-OEt or BSO with PBS, DOX-loaded H40-star-PLA-SS-PEP micelles diluted in complete DMEM (200 μL) with the final DOX concentration from 0.018 to 0.90 μg/mL were added to cells and the cells were further 1569
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Biomacromolecules
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Scheme 1. Detailed Synthetic Route of H40-star-PLA-SS-PEP
incubated for 48 h. After the incubation, the culture medium was removed and washed with PBS twice. Then 200 μL of DMEM and 20 μL of 5 mg/mL MTT assays stock solution in PBS were added. After incubating the cells for 4 h, the medium containing unreacted MTT was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL/well DMSO, and the absorbance was measured in a PerkinElmer 1420 Multilabel counter at a wavelength of 490 nm. The resulting data are expressed as average ( standard deviations (n = 6).
’ RESULTS AND DISCUSSION Synthesis and Characterization of H40-star-PLA-SS-PEP. We designed and synthesized an amphiphilic hyperbranched multiarm copolymer (H40-star-PLA-SS-PEP) on the basis of H40, PLA, and PEP with disulfide-linkage between the hydrophobic and the hydrophilic moieties. As a kind of biodegradable hyperbranched aliphatic polyester, H40 has received much attention in the field of drug delivery systems.44 In recent years, some amphiphilic hyperbranched multiarm copolymers based on H40 have been synthesized and self-assembled into micelles with high stability and drug loading ability for targeted drug delivery.4548 PLA is another kind of frequently used biodegradable polymer used in drug delivery systems with high mechanical strength and excellent shaping and molding properties.49 Thus, H40 and PLA were separately used as the hydrophobic core and inner shell of the amphiphilic multiarm copolymer. On the other hand, polyphosphates are also an important class of biomaterials with good biocompatibility, biodegradability, and the structural similarity to nucleic and teichoic acids.5053 Not only can they be degraded naturally into harmless low molecular weight products through hydrolysis or enzymatic digestion under the physiological conditions, but they also still exhibit good flexibility in adjusting their pendant groups and physicochemical properties by the convenient
functionalization of pentavalent phosphorus.5456 Here polyphosphates were selected as the outer hydrophilic shell of the amphiphilic multiarm copolymer. The detailed synthetic route of H40-star-PLASS-PEP is illustrated in Scheme 1. The detailed characterization of the purified H40 and H40-star-PLA can refer to our previous report.41 Here the degree of branching (DB), degree of polymerization (DP), and Mn of the purified H40 were 0.375, 61, and 0.73 104 g/mol, respectively, on the basis of its 13C NMR spectrum (Figure S1A in the Supporting Information). The purified H40 was used as macroinitiator to initiate the ROP of LA to produce the intermediate H40-star-PLA. Meanwhile, propargyl alcohol and Sn(Oct)2 were used as the initiator and the catalyst respectively to prepare PEP by the ROP of EP in THF. Then the terminal hydroxyl group of PEP was coupled with one of the carboxyl group in 3,30 dithiodipropionic acid by esterification in the presence of DCC and DMAP to produce another intermediate, PEP-SS-COOH. Finally, H40-star-PLA-SS-PEP was obtained by the condensation reaction of the carboxyl group in PEP-SS-COOH and the terminal hydroxyl groups in H40-star-PLA. At the beginning, we tried to synthesize an amphiphilic hyperbranched multiarm copolymer by directly grafting PEP-SS-COOH to H40 core with DCC and DMAP as catalysts. Unfortunately, the grafting ratio of PEP-SS-COOH was very low. This might be caused by the steric hindrance and the low reactivity of the terminal hydroxyl groups in the H40 core. According to the previous report, these hydroxyl groups can efficiently initiate the ROP of LA in bulk and the newly formed hydroxyl end-groups in the resulting polymer display relatively high reactivity and couple with the terminal carboxyl groups of other polymers.46 Therefore, the PLA segments, which were introduced into the hyperbranched multiarm copolymer, not only adjust the content of the hydrophobic moiety in H40-star-PLA-SSPEP, but also facilitate the grafting of the hydrophilic polyphosphate shell. 1570
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Figure 1. 1H NMR spectra of PEP and PEP-SS-COOH in CDCl3.
Figure 3. FTIR spectra (A) and GPC profiles (B) of the intermediates and the resulting copolymers.
Figure 2. 1H NMR spectra of H40-star-PLA and H40-star-PLA-SS-PEP in CDCl3.
The chemical structure of the resulting polymers was first characterized by a 1H NMR spectrum. As shown in Figure 1, three strong peaks are observed at 4.20, 4.14, and 1.32 ppm in the 1 H NMR spectrum of PEP and assigned to the protons of OCH2CH2O, OCH2CH3, and OCH2CH3 in the PEP backbone, respectively. The weak peak at 3.74 ppm is attributed to the protons of OCH2CH2OH in the one end of PEP chains. The peaks at 4.64 and 2.61 ppm belong to the protons of CHCCH2 and CHCCH2 in the another end of PEP chains, which means that propargyl alcohol initiates the ROP of EP successfully. The Mn and DP of PEP were about 0.28 104 g/ mol and 18, respectively, by calculating the relative intensity of the signal at 3.74 ppm (OCH2CH2OH in PEP) and the signal at 4.20 ppm (OCH2CH2O in PEP). Comparing with the
spectrum of PEP, the new signals at 2.91 and 2.73 ppm in the 1H NMR spectrum of PEP-SS-COOH are ascribed to the protons of CH2CH2SSCH2CH2 and CH2CH2SSCH2CH2, respectively. The signal at 3.74 ppm (OCH2CH2OH in PEP) decreased obviously in the 1H NMR spectrum of PEP-SSCOOH, which means the high esterification efficiency in the coupling reaction of PEP and 3,30 -dithiodipropionic acid. The coupling efficiency is about 98% according to the integrals of signal at 3.74 ppm (f) in the 1H NMR spectrum of PEP-SSCOOH. The 1H NMR spectra of H40-star-PLA and H40-starPLA-SS-PEP are given in Figure 2. The Mn and DP of the PLA blocks were about 0.16 104 g/mol and 11, respectively, by calculating the relative intensity of the peak at 4.34 ppm (the methine groups adjacent to the terminal hydroxyl groups in PLA blocks) and the peak at 5.15 ppm (the rest of methine groups in PLA blocks). On the basis of the quaternary 13C NMR spectrum of H40-star-PLA (Figure S1B in the Supporting Information), all the terminal hydroxyl groups in H40 initiated the polymerization of LA and the Mn of H40-star-PLA is about 10.9 104 g/mol. Furthermore, the grafting ratio of PEP-SS-COOH onto H40star-PLA can be calculated from the relative integral values of the methyl protons (h) in PEP and methine protons (a) in PLA segments in the 1H NMR spectrum and the result is about 50%. The FTIR spectra of the obtained polymers are displayed in Figure 3A. As exhibited in the FTIR spectrum of PEP, the absorptions at 1268 and 1178 cm1 can be attributed to the asymmetrical and symmetrical PdO stretching, respectively.50 The POC stretching is also found at 986 cm1. Contrast to the FTIR spectra of PEP, the peaks at 1734 and 1645 cm1 are the characteristic absorption bands of ester and acid carbonyl 1571
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Biomacromolecules
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Table 1. Characterization Data of the Intermediates and the Resulting Copolymers Mwa (g/mol; 104)
Mna (g/mol; 104)
Mw/Mna
Mnb (g/mol; 104)
H40
0.5c
0.3c
1.8c
0.73
H40-star-PLA
9.8
7.5
1.3
10.9
37.1
PEP
0.38
0.29
1.3
0.28
54.3
PEP-SS-COOH
0.43
0.31
1.4
0.30
53.9
H40-star-PLA-SS-PEP
13.7
10.4
1.3
20.5
55.4/34.2
sample
a
Tgd (°C) 26.8
Determined by GPC. b Calculated on the basis of NMR results. c From Perstorp data sheet. d Measured by DSC.
groups in PEP-SS-COOH, respectively. As expected, most of the above-mentioned characteristic peaks are observed clearly in the FTIR spectrum of H40-star-PLA-SS-PEP. This further confirms the successful synthesis of the amphiphilic hyperbranched multiarm copolymer. Moreover, the molecular weights of all intermediates and H40-star-PLA-SS-PEP were measured by GPC and calculated by 1H NMR spectrum, respectively, and summarized in Table 1. The representative GPC profiles of H40, H40-starPLA, and H40-star-PLA-SS-PEP are shown in Figure 3B and looks relatively monomodal and symmetric. A clear shift toward higher molecular weights was observed by comparing the GPC curve of H40-star-PLA-SS-PEP with those of the intermediate H40-star-PLA and the precursor H40. This indicates the corresponding grafting and coupling reactions are successful. The number-averaged molecular weight of H40-star-PLA-SS-PEP is 10.4 104 g/mol with a PDI value of 1.3 by GPC using linear polystyrene as calibration. This result may be smaller than the real molecular weights of H40-star-PLA-SS-PEP because dendritic polymers generally have smaller sizes than linear polymers with the same molecular weight and can hardly be expanded in solution.57 Thus, we also calculated the number-averaged molecular weight of H40-star-PLA-SS-PEP according to the 1 H NMR spectrum and the result is 20.5 104 g/mol, which is much higher than that of GPC. Furthermore, the thermal property of the intermediates and the resulting copolymers was characterized by DSC measurement and all resulting DSC curves were displayed in Figure S2 (see the Supporting Information). The glass transition of H40 occurs at 26.8 °C, while the glass transition temperature (Tg) of PLA segments is 37.1 °C. For H40-star-PLA-SS-PEP, two transitions at 55.4 and 34.2 °C can be observed, which correspond to the glass transitions of PEP and PLA segments, respectively. The glass transition of H40 core can not be found in both DSC curves of H40-star-PLA and H40star-PLA-SS-PEP because of the low percentage of H40. The detailed data of Tgs are also summarized in Table 1. Both GPC and DSC results further confirm the successful preparation of H40-star-PLA-SS-PEP. Fabrication and Characterization of Micelles. The amphiphilic nature of H40-star-PLA-SS-PEP provides an opportunity for itself to self-assemble into nanometer aggregates in aqueous solution. After dissolved in DMF and dialyzed against water, H40-star-PLA-SS-PEP was able to spontaneously self-assemble into micelles driven by the amphiphilicity of hydrophobic H40 core as well as PLA inner segments and hydrophilic polyphosphate outer shell. Furthermore, the hydrophilic polyphosphate outer shell maintains a hydration barrier to provide the micelle stability.38,41 The formation of H40-star-PLA-SS-PEP micelles was first confirmed by fluorescence technique using pyrene as a probe. The fluorescence spectra of pyrene in copolymer solutions with the different concentration of H40-star-PLA-SS-PEP
Figure 4. DLS plots (A) and TEM photos (B) of H40-star-PLA-SSPEP micelle.
are shown in Figure S3 (see the Supporting Information). A red shift from 334 to 337 nm was observed when the concentration of H40-star-PLA-SS-PEP increased from 0.5 103 to 0.5 mg/ mL. This red shift suggested the pyrene molecules were transferred from the water into the hydrophobic core and suggested the formation of micelles.58 The size of micelles is a very important parameter for intracellular drug delivery because the small size (
