Article pubs.acs.org/Biomac
Disulfide-Linked Amphiphilic Polymer-Docetaxel Conjugates Assembled Redox-Sensitive Micelles for Efficient Antitumor Drug Delivery Pei Zhang,† Huiyuan Zhang,† Wenxiu He,† Dujuan Zhao,† Aixin Song,‡ and Yuxia Luan*,† †
School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road, Jinan, Shandong Province 250012, People’s Republic of China ‡ Key Lab of Colloid and Interface Chemistry, Ministry of Education, Shandong University, Jinan, Shandong Province 250100, People’s Republic of China
ABSTRACT: Here, we prepared novel redox-sensitive drug delivery system based on copolymer-drug conjugates methoxy poly(ethylene glycol)-poly(γ-benzyl L-glutamate)-disulfide-docetaxel (mPEG-PBLG-SS-DTX) to realize the desirable cancer therapy. First, copolymers of methoxy poly(ethylene glycol)-poly(γ-benzyl L-glutamate) (mPEG-PBLGs) with different molecular weight (mPEG2000-PBLG1750 and mPEG5000-PBLG1750) were synthesized via the ring open polymerization (ROP) of 5-benzyl-L-glutamate-N-carboxyanhydride (γ-Bzl-L-Glu-NCA) initiated by monoamino-terminated mPEG (mPEG-NH2). Then, the docetaxel (DTX) was conjugated to the block polymers through a linkage containing disulfide bond to obtain mPEG-PBLGSS-DTXs, including mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX. The obtained copolymer-drug conjugates mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX could self-assemble into nanosized micelles in aqueous environment via dialysis method with a low critical micelle concentration (CMC, 3.98 and 6.94 μg/mL, respectively). The size of the micelles was approximately 101.3 and 148.9 nm, respectively, with a narrow size distribution. They released approximately 40% DTX in a sustained way in the presence of 50 mM DTT after 120 h in comparison with only approximately 10% DTX released from micelles in the absence of DTT. The high cytotoxicity was identified for mPEG-PBLG-SS-DTXs micelles against MCF-7/ADR and A549 cells, and the IC50 of mPEG-PBLG-SS-DTXs micelles against MCF-7/ADR for 24 h was roughly a 15th of the value of free DTX. Moreover, the mPEG-PBLG-SS-DTXs micelles could be efficiently uptaken by MCF-7/ADR and A549 cells. Thus, the present constructed mPEG-PBLG-SS-DTXs micelles were very promising for effective cancer therapy.
1. INTRODUCTION Over the past decades, nanocarriers have attracted abundant attention as practical strategies for target drug delivery and controlled drug delivery system for anticancer therapy. To date, many nanoscale formulations including nanoparticles, liposomes, microcapsules, nanogels, polymeric micelles, and so forth have been prepared.1−5 Among these carriers, polymeric micelles have attracted great interest because of several merits such as the enhanced water solubility, decreased side effects, improved biocompatibility, passive accumulation of the drugs in the tumor tissues and prolonged circulating time.6,7 It should be noted that polymeric micelles with loaded doxorubicin (NK911) and paclitaxel (Genexol-PM) have already been advanced to clinical trials in several countries.6,7 © XXXX American Chemical Society
As one of the most well-known hydrophilic block, poly(ethylene glycol) (PEG) is a nonimmunogenic and nontoxic polymer forming the hydrated outer shell.8 It is noteworthy that micelles with PEG shell possess a dramatic prolonged circulation time and reduced uptake by the macrophages of the reticuloendothelial system.9 The hydrophobic moiety, poly(γ-benzyl L-glutamate) (PBLG), is biodegradable and forms the inner core of micelles and acts as a drug reservoir, especially for the hydrophobic drugs.10,11 Given the predominant characteristics including solubilization of hydrophobic Received: December 29, 2015 Revised: February 26, 2016
A
DOI: 10.1021/acs.biomac.5b01758 Biomacromolecules XXXX, XXX, XXX−XXX
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Scheme 1. Schematic Diagram of mPEG-PBLG-SS-DTXs Micelles Self-Assembly, Endocytosis by Tumor Cells, as Well as the Redox-Triggered Drug Release
example, Chen et al.28 developed doxorubicin (DOX)-loaded micelles based on poly(ethylene glycol)-SS-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) (PEG-SSPTMBPEC) copolymer, which could respond to endosomal pH as well as cytoplasmic GSH. Wu et al.29 prepared camptothecin (CPT) loaded micelles based on mPEG-SSNH-g-PHAsp, which released CPT and revealed the enhanced cellular uptake as a result of the breakaway of PEG shells in response to DTT. Thambi et al. as well as Guo et al.30−32 developed DOX-, CPT-, and 7-ethyl-10-hydroxy-camptothecin (SN-38)-loaded micelles based on amphiphilic diblock copolymer bearing the redox-sensitive linker, composed of mPEG-SS-PBLG. However, the drugs were encapsulated physically into the micelles and it is hard to achieve the stability in blood circulation. In order to construct a more stable drug delivery system, we conjugated the anticancer drug DTX to our synthesized block polymers mPEG-PBLG via disulfide bond to obtain mPEGPBLG-SS-DTXs that was used to prepare the redox-responsive drug delivery system for cancer therapy. Two series copolymerdrug conjugates, mPEG2000-PBLG1750-SS-DTX and mPEG5000PBLG1750-SS-DTX, were synthesized and both of them could form unique core−shell micelles with redox-responsive properties. As shown in Scheme 1, the mPEG-PBLG-SS-DTXs copolymers self-assemble into micelles in aqueous environment and are expected to be uptaken into tumor cells by endocytosis. The linkage containing disulfide bond between DTX and amphiphilic copolymers mPEG-PBLG is supposed to release the DTX in response to the reducing environment in malignance. The copolymers mPEG-PBLG with different molecular weight are synthesized via the ROP of γ-Bzl-L-GluNCA initiated by mPEG-NH2, followed by conjugating DTX to it through a linkage containing disulfide bond. Subsequently, the characteristics, in vitro reduction-sensitive drug release behavior, hemolysis, cytotoxicity and cellular uptake, are investigated.
drugs, sustained and selective release of drugs, as well as prolonged circulation time, micelles based on PEG-PBLG have drawn more and more attention.8,11,12 However, in view of reports in the literature there are a few of challenges that need to be managed in order to accomplish a wide clinical application of polymeric micelles. It should be noted that a considerable amount of drug may release from the micelles before reaching the pathological tissues and/or the neoplastic cells because of the lacking stability upon intravenous injection against attenuation and interactions with substances in plasma.13,14 This pragmatic limit is a critical account for poor targeted delivery and low therapy efficiency of many polymeric micelles. Aliphatic polyesters such as poly(εcaprolactone) (PCL), poly(lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) (PLGA) generally show continuing degradation kinetics inside the body, which may last days, weeks, or even months.6,15 Furthermore, rapid drug release of polymeric micelles upon reaching tumor tissues is necessary for improving antitumor efficacy as well as circumventing drug resistance in pathological cells. In this regard, smart stimuli-sensitive polymeric micelles have been explored for achieving enhanced drug release at particular site and/or time in response to appropriate environment factors, for example, pH, ultrasound, magnetic, light, temperature, reductive environment, or their combinations.16−23 These interesting characters capacitate polymeric micelles to be promising candidates for controlled drug carriers with prolonged circulation time and optimal efficiency of drug loading and releasing. Among the various applied stimulisensitive antitumor drug carriers, reduction-sensitive disulfide linkage-employing polymeric micelles have attracted more and more attention given the remarkable redox potential difference between the poorly reducing extracellular environments and the relatively more reducing intracellular spaces. The bioreducible linker containing disulfide bond can cleave when the micelles in which it is comprised encounter reductive substances, for instance, glutathione (GSH). GSH is a tripeptide involved glutamate, cysteine, and glycine that can cleave the disulfide bond through thiol−disulfide exchange reaction. The disulfide bond is relatively stable in extracellular compartment in which the GSH concentration is 2−20 μM.24 On the contrary, it can be reversibly cleaved in the intracellular fluid where it contains 2−10 mM GSH.24−27 Furthermore, tumor cells typically exhibit elevated GSH concentrations, which is typically at least four times higher than the values of normal cells. It has been a hot topic to construct the redox-sensitive drug delivery system, taking advantage of the difference of redox potential for controlling the shedding of micelle hydrophilic shell. For
2. MATERIALS AND METHODS 2.1. Materials. Methoxy-poly(ethylene glycol) (mPEG) with Mw of 2000 and 5000 g/mol were purchased from SigmaAldrich (St. Louis, MO) and used without further purification. Dichloromethane (DMC) and dimethylformamide (DMF) were dried by calcium hydride (CaH2) distillation. All other reagents were used as received without further purification. γBzl-L-Glu-NCA was purchased from J&K Scientific Ltd. DLdithiothreitol (DTT), 3,3′-dithiodipropionic acid (DTDP), acetyl chloride, 4-dimethylaminopyridine (DMAP), triethylB
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Biomacromolecules amine (TEA), N,N′-dicyclohexylcarbodiimide (DCC), and Nhydroxysuccinimide (NHS) were purchased from Aladdin Industrial Inc. and DTX was supplied by Melone Pharmaceutical Co. Ltd. The acetonitrile and tetrahydrofuran (THF) were for HPLC, and all other reagents were of analytical grade. 2.2. Synthesis of mPEG-PBLG. 2.2.1. Synthesis of TosylTerminated mPEG (mPEG-OTs). The mPEG-OTs was prepared taking advantage of toluene sulfonate esterification with mPEG. Briefly, mPEG (2 mmol) was dissolved into anhydrous DMC. Then excessive amount of 4-toluene sulfonyl chloride (TsCl, 13 mmol), which was in a solution of pyridine (25 mL), was added dropwise and the mixture was stirred in dark at 30 °C for 24 h. The reaction mixture was washed with hydrochloric acid solution (3 mol/L) to remove superfluous pyridine, and whereafter, a good deal of sodium bicarbonate was added to the organic phase in order to neutralize the residual hydrochloric acid. The organic phase was concentrated and the precipitates were obtained by the addition of ice-cold diethyl ether. The product was further desiccated under vacuum, herein denoted mPEG-OTs. 2.2.2. Synthesis of mPEG-NH2. A sample containing 0.5 mmol mPEG-OTs and 20 mL of ammonia water was added to a high-pressure reactor and reacted under 140 °C for 6 h. The reaction mixture was extracted by DMC and sodium hydroxide solution (1 mol/L) was added into the organic phase to stir for 4 h, which was followed by abstersion with saturated salt water and a concentration of the organic phase. The resultant product was desiccated under vacuum to obtain mPEG-NH2. 2.2.3. Synthesis of mPEG-PBLG. The mPEG-PBLG block copolymers were synthesized via the ROP reaction of γ-Bzl-LGlu-NCA initiated by mPEG-NH2 in DCM.11,12,33 After reaction at 30 °C for 72 h, the mixture was concentrated and a large excess of diethyl ether was poured into it to precipitate the mPEG-PBLG copolymers, which was dried under vacuum afterward. 2.3. Synthesis of DTX and mPEG-PBLG Conjugation Linked by Disulfide Bond (mPEG-PBLG-SS-DTX). 2.3.1. Synthesis of Dithiodipropionic Anhydride (DTDPA). The DTDPA was prepared through dehydration reaction as previously reported.7,21,34 In brief, 3,3′-thiodipropionic acid (DTDP, 1.0 g) was refluxed in acetyl chloride (3 mL) at 65 °C for 2 h. After solvent removal, the residue was precipitated via pouring excess ethyl ether in order to afford DTDPA and vacuum-dried. 2.3.2. Synthesis of Disulfide-Functionalized mPEG-PBLG (mPEG-PBLG-SS-COOH). mPEG-PBLG-SS-COOH was synthesized via esterification of mPEG-PBLG with cyclic anhydride DTDPA. Briefly, the block copolymers PEG-PBLG (0.1 mmol) dissolved in 5 mL of anhydrous DMF were added with DTDPA (0.2 mmol) and DMAP (0.1 mmol). After all of the substances were completely dissolved, triethylamine (TEA, 0.2 mmol) was added. The reaction mixture was maintained at 35 °C under nitrogen atmosphere for 36 h. Subsequently, the reaction solution was precipitated into excess ice cold ethyl ether and vacuum-dried to give mPEG-PBLG-SS-COOH. 2.3.3. Synthesis of mPEG-PBLG-SS-DTX. mPEG-PBLG-SSCOOH (0.1 mmol), NHS (0.2 mmol) were codissolved in 4 mL of anhydrous DCM. Subsequently DCC (0.2 mmol), which was dissolved in anhydrous DCM (1 mL), was added dropwise at 0 °C. The mixed solution was stirred at 0 °C under nitrogen atmosphere for 2 h as well as at room temperature for another 22 h and then filtered to remove the byproduct, N,N′dicyclohexylurea (DCU). The filtrate was added dropwise to a
solution of DTX (0.2 mmol) in anhydrous DCM and DMAP (0.2 mmol) was added after 5−10 min. The reaction proceeded at room temperature under nitrogen atmosphere for 48 h. Then, the product was purified by precipitating via pouring excess ice cold ethyl ether and dried in vacuum. 2.4. Preparation of mPEG-PBLG-SS-DTX Micelles and Their CMC Measurements. The mPEG-PBLG-SS-DTXs micelles were prepared by dialysis. Detailed steps were described as follows: 10 mg of mPEG-PBLG-SS-DTXs was dissolved in a mixed solvent of THF/DMF [30/70, v/v]. Subsequently, the mixed solution was dialyzed against distilled water utilizing cellulose dialysis tubing (MWCO = 3500 Da) to form micelles and remove the THF and DMF at room temperature for approximately 24 h. The distilled water was exchanged at intervals of 4 h. The CMC of mPEG-PBLG-SS-DTXs micelles was investigated via pyrene fluorescent probe method. A series of mPEGPBLG-SS-DTXs micelles solutions with different concentrations from 1.0 × 10−5 mg/mL to 0.5 mg/mL were prepared and the concentration of pyrene was set to be 6.0 × 10−6 M. The fluorescence spectra of all solutions were recorded by fluorescence spectrophotometer (Hitachi F-7000, Japan). An emission spectrum of each sample was recorded from 300 to 450 nm with excitation set to 334 nm. CMC was estimated by plotting lg (concentration) versus the ratio of the fluorescence intensities at 373 nm (I1) and 383 nm (I3) nm. 2.5. Characterization. The 1H nuclear magnetic resonance 1 ( H NMR) spectra were recorded on a Bruker Avance 400 spectrometer (Germany) operating at 400 MHz at room temperature in CDCl3 (+0.03% TMS) or DMSO-d6 (+0.03% TMS) to verify the structure of the polymers. Fourier transform infrared (FTIR) spectra was performed on Nicolet 6700 Fourier transform infrared spectrometer (Thermo Fisher Scientific, U.S.A.) to provide additional evidence for the successful synthesis of polymers. The transmission electron microscopy (TEM, JEM-200CX) was employed to record the morphology of micelles. Samples were prepared by dropcasting mPEG-PBLG-SS-DTXs micelles solutions on 300-mesh copper grids without staining and then air drying at room temperature before measurement. Dynamic light scattering (DLS, BIC·Brook-Haven) was employed to detect their average size and size distribution, which was implemented with a scattering angle of 90° at 25 °C. Considering that DTX as well as mPEG-PBLG-SS-DTXs micelles have maximum absorbance wavelength at 230 nm, the UV−visible spectrum was used to further demonstrate the drug loaded content (DL) of DTX in mPEG-PBLG-SS-DTXs micelles, which was calculated according to the following formulas DL(%) =
Weight of drug in micelles × 100 Total weight of micelles
2.6. In Vitro Drug Release Study. DTT was chosen as a substitution of GSH in the present study.35−38 The solution of free DTX and mPEG-PBLG-SS-DTXs micelles (containing 0.2 mg of DTX) was put in cellulose dialysis tubing (MWCO = 3500 Da) and exchanged against phosphate buffer saline (PBS, pH = 7.4) containing 0.5% (w/w) Tween-80 with or without DTT (50 mM) in a shaking table at 37 °C. At predetermined time intervals, an aliquot of the external buffer phase was withdrawn and replaced with fresh PBS buffer or PBS buffer with 50 mM DTT for keeping the sinking condition and the C
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Figure 1. (A) The synthetic routes, reagents, and conditions. (B) The mechanism of synthesizing copolymer PEG−PBLG via ROP reaction.
(1.1 mL) were added to mPEG-PBLG-SS-DTXs micelles (0.15 mL) at systematically varied concentrations, which was followed by incubation at 37 °C in a thermostatic water bath for 2 h. Normal saline and distilled water were utilized as negative and positive controls, respectively. Subsequently, the RBCs were centrifuged for 15 min at 1500 rpm. The supernate was measured via ultraviolet spectrophotometer and the absorbance at 540 nm was serviceable. All the tests were performed in triplicate. The hemolysis ratio (HR) of RBCs was calculated using the following formula
amount of released DTX was determined by high-performance liquid chromatography (HPLC) at 230 nm with acetonitrile/ water (65:35, v/v) as mobile phase. Each experiment was carried out in triplicate. Standard DTX solutions (1−10 μg/ mL) were prepared via dilution from a stock solution. 2.7. Hemolysis Assay. Hemolytic activity of mPEG-PBLGSS-DTXs micelles was evaluated as previously reported with minor alteration.39,40 In brief, fresh rabbit blood that had been diluted by physiological saline was centrifuged to isolate red blood cells (RBCs). After carefully washing and diluting, the RBC suspension (1.25 mL, 2%, v/v) as well as normal saline D
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Biomacromolecules HR (%) =
A sample − A negative control A positive control − A negative control
3.1. Synthesis and Characterization of mPEG-PBLG. The biocompatible mPEG-PBLGs copolymers were synthesized in three steps, as outlined in Figure 1A. mPEG-OTs were first prepared via a toluene sulfonate esterification reaction. The reaction was performed in a DCM/pyridine system with the simultaneous dropping of TsCl into the mPEG solution. Pyridine could neutralize the hydrogen chloride that was constantly produced by the hydrolysis of excessive TsCl. The reaction time was prolonged to 24 h considering the steric hindrance of mPEG as well as the rather low reaction activity of TsCl. The product yield of mPEG2000-OTs and mPEG5000-OTs were 78.8% and 93.5% respectively. 1H NMR (CDCl3-0.03% TMS, ppm, Figure 2A,B) indicated the structure of mPEG and mPEG-OTs. Figure 2A: δ = 3.38 (s, 3H, PEG-OCH3); 3.60−
× 100
2.8. Cell Culture. The multidrug resistant variant breast cancer (MCF-7/ADR) cells and human lung carcinoma (A549) cells were cultured at 37 °C in a 5% CO2 atmosphere in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS). 2.9. Cytotoxicity Assay. The cytotoxicity of mPEG-PBLGSS-DTXs micelles were evaluated employing MCF-7/ADR and A549 cells with in vitro proliferation via the MTT method. Briefly, the MCF-7/ADR as well as A549 cells were seeded at 5000 cells/well in a 96-well plate and were allowed to incubate for 24 h. Subsequently, the culture medium was replaced with 200 μL of fresh medium containing free DTX, mPEG2000PBLG1750-SS-DTX, and mPEG5000-PBLG1750-SS-DTX micelles with systematically varied concentrations, and the cells were further cultured for 24 and 48 h, respectively. Afterward, 10 μL of MTT solution (5 mg/mL in PBS) was added and the cells were incubated at 37 °C for another 4 h. The medium was replaced by 150 μL of DMSO for the dissolution of MTT formazan crystals. The cell inhibition rate was calculated by measuring the absorbance at 490 nm by ELIASA of PerkinElmer. The culture medium with or without cells served as control and blank, respectively. All experiments were performed in triplicate. The following formula was employed to calculate the cell inhibition rate Cell inhibition rate (%) =
Acontrol − A sample Acontrol − A blank
× 100
2.10. Cellular Uptake of Micelles. The cellular uptake of micelles was estimated by fluorescent inverted microscope (Olympus, Tokyo, Japan) utilizing coumarin-6 (C-6) as a fluorescence probe. Briefly, MCF-7/ADR and A549 cells were seeded at a density of 2 × 105 cells/well into 6-well plates and incubated for 24 h at 37 °C. Then the original medium was replaced with fresh medium containing free C-6, mPEG2000PBLG1750-SS-DTX/C-6, or mPEG5000-PBLG1750-SS-DTX/C-6 micelles. The cells were incubated for 2 or 4 h at 37 °C and were observed under fluorescent inverted microscope. 2.11. Statistical Analysis. Data were representative of at least three individual experiments and expressed as means ± standard deviation. Statistical significances were performed via a paired t-test (Student’s t-test). p < 0.05 was considered to be statistically significant, and p < 0.01 was considered to be highly significant.
3. RESULTS AND DISCUSSION mPEG-PBLGs copolymers, including mPEG2000-PBLG1750 and mPEG5000-PBLG1750, were synthesized via the ROP of γ-Bzl-LGlu-NCA initiated by mPEG-NH2.11,12,21 Subsequently, the antitumor drug DTX was conjugated to the mPEG-PBLGs copolymers via a disulfide linkage (Figure 1A). The 1H NMR and FTIR spectra of mPEG2000-PBLG1750-SS-DTX and its intermediate products were listed as examples to illustrate their structure because the only difference between mPEG2000PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX as well as their intermediate products was the polymerization degree of mPEG. The micelles based on these conjugates were of particular interest because they exhibited excellent reduction sensitivity and prominent antitumor activity.
Figure 2. 1H NMR spectra of mPEG (A); mPEG-OTs (B); mPEGNH2 (C); mPEG-PBLG (D). E
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Biomacromolecules 3.72 (m, 156H, −OCH2CH2−). Figure 2B: δ = 2.45 (s, 3H, −C6H4CH3); 3.38 (s, 3H, PEG-OCH3); 3.60−3.72 (m, 156H, −OCH2CH2−); 7.32−7.80 (dd, 4H, CH3−C6H4−). Their 1H NMR spectrum indicated the successful toluene sulfonate esterification of mPEG in which new peaks belonged to protons of benzene ring and methyl in OTs residue at 7.32−7.80 and 2.45 ppm, respectively, were observed. Subsequently, the obtained mPEG-OTs, which could attack ammonia as alkylating agents, reacted with ammonia water via the electrophilic substitutive reaction in high-pressure reactor, resulting in the formation of mPEG-NH2. Excess amount of ammonia water was employed to ensure complete reaction. During this progress, the leaving of OTs residue was the most important part and it is worth noting that the OTs residue was an excellent leaving group with approximate 100% probability. The product yield of mPEG2000-NH2 and mPEG5000-NH2 were 64.4% and 40.2%, respectively. The structure of product was confirmed by 1H NMR spectra (CDCl3-0.03% TMS, ppm, Figure 2C). δ = 2.96 (t, 2H, −NH2); 3.38 (s, 3H, PEG-OCH3); 3.60−3.72 (m, 156H, −OCH2CH2−). Compared with that of mPEG-OTs, evaporated peaks at 7.32−7.80 and 2.45 ppm assigned to protons of benzene ring and methyl in OTs residue as well as additional peaks at 2.96 ppm belonged to protons of amino declared that the mPEG-NH 2 was synthesized successfully. Next, mPEG-PBLG was obtained by the ROP reaction of γBzl-L-Glu-NCA initiated by mPEG-NH2 in DMC at 30 °C for 72 h.11,12,33 The reaction mechanism was presented in Figure 1B. The mPEG-NH2 attacked the C-5 of γ-Bzl-L-Glu-NCA monomer to give intermediate 2, which would eliminate CO2 and produce structure 3. And it could abidingly react with γBzl-L-Glu-NCA monomer to give copolymer mPEG-PBLG. The product yield of mPEG2000-PBLG1750 and mPEG5000PBLG1750 were 94.6% and 95.1% respectively. The 1H NMR and FTIR spectra could indicate the structure of the copolymer mPEG-PBLG. 1H NMR (DMSO-d6 + 0.03% TMS, ppm, Figure 2D): δ = 1.72−2.46 (m, 32H, −CH2CH2− of PBLG); 3.24 (s, 3H, PEG-OCH 3 ); 3.45−3.64 (m, 156H, −OCH2CH2−); 4.90−5.14 (m, 16H, C6H5−CH2-); 7.15− 7.41 (m, 40H, C6H5−). The resonances appeared at 4.90−5.14 and 7.15−7.41 ppm could testify that the block copolymers mPEG-PBLG were synthesized successfully as well as the degree of polymerization and molecular weight of PBLG was approximately 8 and 1750, respectively. The FTIR spectra of mPEG and mPEG-PBLG were shown in Figure 4A,B. Compared with the FTIR spectra of mPEG, the appearance of primary amine (doublet) at 3446 cm−1 as well as peptide bond at 1652 and 1547 cm−1 also indicated the successful synthesis of the copolymer mPEG-PBLG. 3.2. Synthesis and Characterization of mPEG-PBLGSS-DTX. The antitumor drug DTX was conjugated to the block copolymer mPEG-PBLG via a linkage containing disulfide bond through three steps as shown in Figure 1A. Considering costeffectiveness of DTDP and high reactivity of DTDPA, they were selected as a disulfide donor in the present study.7,21,34 DTDP was first refluxed in acetyl chloride that acted as a dehydration reagent to give DTDPA as a white solid in ∼50% yield. The structure of DTDP (DMSO-d6 + 0.03% TMS, ppm, Figure 3A) and DTDPA (DMSO-d6 + 0.03% TMS, ppm, Figure 3B) was confirmed by 1H NMR spectroscopy. Figure 3A: δ = 2.59−2.64 (t, 4H, −CH2−CH2−SS-CH2−CH2−); 2.85−2.91 (t, 4H, −CH2−CH2−SS−CH2−CH2−); 12.35 (s, 2H, −COOH). Figure 3B: δ = 2.60−2.66 (t, 4H, −CH2−
Figure 3. 1H NMR spectra of DTDP (A); DTDPA (B); mPEGPBLG-SS-COOH (C); DTX (D); mPEG-PBLG-SS-DTX (E).
CH2−SS-CH2−CH2−); 2.87−2.93 (t, 4H, −CH2−CH2−SS− CH2−CH2−). The peaks at 12.35 ppm belonged to protons of carboxyl terminal disappeared, which could demonstrated that successful synthesized of anhydride. In addition, the melting point of DTDP (153−155 °C) and DTDPA (65−70 °C) could also certify the accomplish of the progress. Then, the mPEG-PBLG-SS-COOH was synthesized from esterification of mPEG-PBLG with DTDPA. As shown in Figure 1A, the terminal amino group of mPEG-PBLG F
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Biomacromolecules
The resonance of this proton was likely shifted to the lower field and mixed with the peaks at 4.90−5.14 ppm belonged to the protons of PBLG. According to the 1H NMR spectrum, the molecular weights of mPEG2000-PBLG1750 -SS-DTX and mPEG5000-PBLG1750-SS-DTX were 4702 and 7702 g·mol−1, respectively. FTIR spectrum was also chosen to provide additional evidence for the successful synthesis of the copolymers. The FTIR spectra of DTX and mPEG-PBLG-SSDTX was presented in Figure 4D,E, resonance near 3301 cm−1 (−OH) and 1168 cm−1(C−O) of hydroxide radical, which appeared at 3377 and 1168 cm−1, respectively, in the spectrum of DTX, might also indicated the successful conjugation of DTX onto mPEG-PBLG-SS-COOH. 3.3. Characteristics of mPEG-PBLG-SS-DTX. Because of its amphiphilic nature, the synthesized mPEG-PBLG-SS-DTXs could easily self-assemble into micelles in aqueous solution via dialysis method. The drug loading contents were about 13.9% and 9.2% for mPEG2000-PBLG1750-SS-DTX and mPEG5000PBLG1750-SS-DTX micelles, respectively and the micelles can be stable for more than 30 days. Some characteristics of mPEGPBLG-SS-DTXs were listed in Table 1. The zeta potentials of mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750-SSDTX micelles were −20.1 ± 0.1 and −14.5 ± 0.1 mV, respectively, which indicated that there was strong repulsion among micelles to prevent their fusion. The micellization behaviors of mPEG-PBLG-SS-DTXs could be confirmed through measuring the CMC value utilizing a pyrene fluorescence probe.47 The fluorescence spectra of pyrene enhanced as the polymer concentration increased as shown in Figure 5A,B. The CMC was determined by plotting emission intensity ratio of I1/I3 against the lg of the polymer concentrations and the onset of the sharp change in the slope of the line was taken as the CMC.48 As the sigmoid curve presented in Figure 5C, the intensity ratio of I1/I3 kept up almost constant at relatively low polymer concentration, revealing the characteristics of pyrene located in water environment. While the ratio value declined dramatically when the polymer concentration increased to CMC, intimating the hydrophobic environment that pyrene was existing and the formation of polymeric micelles. Compared with mPEG5000PBLG1750-SS-DTX, the CMC value for mPEG2000-PBLG1750SS-DTX in water was of a relatively low level, indicating it possessed comparatively excellent self-assembly ability in aqueous environments, which was coincident with its shorter hydrophilic block. TEM micrographs and size distributions of mPEG-PBLG-SS-DTXs micelles were presented in Figure 6A,B. TEM images demonstrated that the spherical micelles were formed in the two series of mPEG-PBLG-SS-DTXs aqueous solutions with the size of 101.3 ± 1.4 and 148.9 ± 1.4 nm for mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750SS-DTX micelles, respectively. The larger size of mPEG5000PBLG1750-SS-DTX micelles than that of mPEG2000-PBLG1750SS-DTX micelles is probably due to the longer hydrophilic block of the conjugate.49,50 3.4. In Vitro Reduction-Sensitive Release Behavior of DTX from Micelles. The release behavior of DTX was
copolymer was coupled with DTDPA by amidation reaction using DMAP as a catalyzer to produce the intermediate mPEGPBLG-SS-COOH.41−46 The product yield of mPEG2000PBLG1750-SS-COOH and mPEG5000-PBLG1750-SS-COOH were 66.2% and 82.8%, respectively. The reaction of mPEGPBLG with DTDPA and the structure of the intermediate mPEG-PBLG-SS-COOH were studied by 1H NMR and FTIR spectra. 1H NMR (DMSO-d6 + 0.03% TMS, ppm, Figure 3C): δ = 1.73−2.44 (m, 32H, −CH2CH2− of PBLG); 2.58−2.65 (t, 3H, −CH2−CH2 −SS−CH 2−CH2−); 2.85−2.92 (t, 3H, −CH2−CH2−SS−CH2−CH2−); 3.24 (s, 3H, PEG-OCH3); 3.45−3.66 (m, 156H, −OCH2CH2−); 4.90−5.14 (m, 16H, C6H5−CH2−); 7.15−7.41 (m, 40H, C6H5−). Compared with that of mPEG-PBLG, new triplet peaks at 2.58−2.65 and 2.85− 2.92 ppm pertaining to DTDPA were observed, which could certify the definite acquisition of mPEG-PBLG-SS-COOH. The FTIR spectra of mPEG-PBLG-SS-COOH were shown in Figure 4C. The substitution of primary amine (doublet) at 3446 cm−1 with secondary amine (singlet) at 3445 cm−1 might make a contribution to the certifying of mPEG-PBLG-SSCOOH.
Figure 4. FTIR spectra of mPEG (A); mPEG-PBLG (B); mPEGPBLG-SS-COOH (C); DTX (D); mPEG-PBLG-SS-DTX (E).
Finally, mPEG-PBLG-SS-DTX was synthesized by coupling the terminal hydroxyl group of antitumor drug DTX with DCC-activated carboxyl groups of mPEG-PBLG-SS-COOH. The product yield of mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX were 79.5% and 85.4%, respectively. The 1H NMR spectra of DTX (DMSO-d6 + 0.03% TMS, ppm) and mPEG-PBLG-SS-DTX (DMSO-d6 + 0.03% TMS, ppm) were shown in Figure 3D,E. The effective conjugation between mPEG-PBLG-SS-COOH and DTX could be verified by the following two facts. One was that the characteristic resonances for DTX were observed in the 1H NMR spectrum for mPEG-PBLG-SS-DTX. Another was the resonance at 4.49 ppm (br, s, 1H) appeared in the spectrum of DTX and belonged to the proton of the 2′ carbon in DTX greatly weaken in the spectrum of the mPEG-PBLG-SS-DTX, which declared that mPEG-PBLG-SS-COOH was conjugated at this position.
Table 1. Characteristics of mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX Micelles polymer
size (nm)
PDI
CMC (μg/mL)
DL (%)
zeta potential (mV)
mPEG2000-PBLG1750-SS-DTX mPEG5000-PBLG1750-SS-DTX
101.3 ± 1.4 148.9 ± 1.4
0.14 ± 0.03 0.18 ± 0.02
3.98 6.94
13.9 ± 0.8 9.2 ± 0.4
−20.1 ± 0.1 −14.5 ± 0.1
G
DOI: 10.1021/acs.biomac.5b01758 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Without DTT treatment, the accumulative release of DTX from mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750SS-DTX micelles reached 6.6% and 11.4%, respectively, within 12 h. The subsequent release rate was rather slower and only 12.0% and 17.2% of DTX released from the mPEG2000PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX micelles, respectively, after 120 h. In contrast, the mPEG-PBLG-SSDTXs micelles showed much different release profiles in the presence of 50 mM DTT and the accumulative release of DTX from the mPEG 2000 -PBLG 1750 -SS-DTX and mPEG 5000 PBLG1750-SS-DTX micelles both dramatically increased to approximately 40% after 120 h. The drug release rate of mPEGPBLG-SS-DTXs micelles with 50 mM DTT was significantly faster compared with that without DTT. These results suggested that the mPEG-PBLG-SS-DTXs micelles, as potential drug delivery systems, were fairly stable in a nonreducing environment and could achieve rapid drug release in a reducing compartment. According to the literatures,51−53 less than 50% drug released in 120 h is acceptable in drug delivery systems for inhibiting tumor growth efficiently in vivo and the mPEG−PBLG copolymer is efficient for escaping the capture of mononuclear phagocyte system. Therefore, our developed micelles are desirable to be a long-circulating drug carrier system and they can survive such long time in vivo. Moreover, the reported works8,11 used the slow in vitro release rate to direct in vivo study and they found that polymeric micelles with slow in vitro release property exhibited favorable pharmacokinetic characteristics. Therefore, our in vitro release test could be useful for guiding the in vivo study based on the in vivo−in vitro correlation and in vivo studies will be performed in the follow-on work. 3.5. Hemolysis. Blood compatibility is necessary for drug carriers because they are finally injected into blood via intravenous injection. In this study, a hemolysis assay was performed according to the previous report employing rabbit red blood cells.39,40 During the experiments, the concentrations of DTX in the two micelles were kept the same and therefore the concentrations of mPEG2000-PBLG1750-SS-DTX and mPEG5000-PBLG1750-SS-DTX were different. As shown in Figure 8, mPEG-PBLG-SS-DTXs micelles showed negligible hemolysis toxicity (
