pH-responsive micelles based on (PCL)2(PDEA-b-PPEGMA)2 miktoarm polymer: controlled synthesis, characterization, and application as anticancer drug carrier
© Lin et al.; licensee Springer. 2014
Received: 26 February 2014
Accepted: 4 May 2014
Published: 18 May 2014
Amphiphilic A2(BC)2 miktoarm star polymers [poly(ϵ-caprolactone)]2-[poly(2-(diethylamino)ethyl methacrylate)-b- poly(poly(ethylene glycol) methyl ether methacrylate)]2 [(PCL)2(PDEA-b-PPEGMA)2] were developed by a combination of ring opening polymerization (ROP) and continuous activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). The critical micelle concentration (CMC) values were extremely low (0.0024 to 0.0043 mg/mL), depending on the architecture of the polymers. The self-assembled empty and doxorubicin (DOX)-loaded micelles were spherical in morphologies, and the average sizes were about 63 and 110 nm. The release of DOX at pH 5.0 was much faster than that at pH 6.5 and pH 7.4. Moreover, DOX-loaded micelles could effectively inhibit the growth of cancer cells HepG2 with IC50 of 2.0 μg/mL. Intracellular uptake demonstrated that DOX was delivered into the cells effectively after the cells were incubated with DOX-loaded micelles. Therefore, the pH-sensitive (PCL)2(PDEA-b-PPEGMA)2 micelles could be a prospective candidate as anticancer drug carrier for hydrophobic drugs with sustained release behavior.
Over the past several decades, great efforts have been made to improve the available anticancer therapies. Unfortunately, the majority of chemotherapy, which has a substantial hydrophobic component, is usually hampered by problems such as lack of tumor selectivity, poor water solubility, uncontrollable pharmacokinetic processes, and the possible incurrence of severe side effects [1–3]. To improve therapeutic efficacy as well as minimize side effects, tremendous drug delivery vehicles based on polymer micelles have been exploited. Polymeric micelles, with nanoscopic core-shell structures self-assembled by amphiphilic copolymers, have attracted the attention of researchers as hydrophobic drug carriers owing to their unique properties, including higher loading capacity, improved water solubility, passive and active targeting capabilities, prolonged in vivo circulation duration, enhanced therapeutic efficacy, and negligible side effects [4–8].
In recent years, stimulus-responsive polymer materials, which can accept appropriate changes in response to specific environmental fluctuations or imposed variations of control parameters, are recognized as one of the most promising modalities in drug delivery systems due to their unique behaviors and intelligent properties [9, 10]. Although many types of stimuli have been extensively studied as drug carriers, including their responsive abilities to pH, temperature, redox, light, ionic strength, enzyme and so forth, a variety of the researches have focused on utilizing pH-responsive polymeric micelles [11–15]. The vital reason for the promising use of pH-responsive polymeric micelles aiming at tumor-targeting is attributed to the different conditions in normal tissues and tumor tissues. Since the intracellular pH values of endosomal and lysosomal environment are typically acidic (pH 5.0 to 6.0 and 4.5 to 5.0, respectively) and the extracellular pH values in tumor tissues are around 6.5 to 7.0, when compared with the neutral pH 7.4 of the normal physiological environment. An ideal anticancer drug pH-responsive polymeric micelles can escape releasing of drug in normal tissues (pH 7.4) and destabilize at an early endosomal pH 6.0 [16–18]. Poly(2-(diethylamino)ethyl methacrylate) (PDEA), a kind of cationic polyelectrolyte with a pKb of 6.9, can be soluble in water under pH 6.9 but become hydrophobic and insoluble at normal physiological conditions. The responsiveness to the weakly acidic condition indicates that PDEA copolymers can be latent pH-sensitive polymeric micelles for tumor-targeting drug delivery [16, 19].
Star-shaped polymers, one kind of dendritic polymers with well-defined architecture and multiple polymer chains emanating from the central core, have similar topological structures to polymeric micelles and can form more stable nanoscale assemblies in selective solvents, as compared with the corresponding linear block analogues. Hence, star polymers have been actively investigated currently for potential utility as nanoreactors, catalysts, sensors, polymer and electrolytes and in biomedical and therapeutic applications [20–23]. Amphiphilic star polymer can be divided into amphiphilic homo-arm star block polymer (AB)n and amphiphilic miktoarm star polymers (AmBn). With same polymer chains emanating from the central core, amphiphilic homo-arm star block polymers have been prepared and used particularly in drug and gene delivery [24, 25]. For example, He and coworkers synthesized well-defined four-arm PEO-b-PDEAEMA, which could form pH-responsive micelles. And the four-arm PEO-b-PDEAEMA micelles were suggested high gene transfection efficiency for the delivery of DNA [26, 27]. Knop's group developed amphiphilic star-shaped block copolymers (PCLa-b-POEGMAb)4 for loading the novel fungicide to provoke an inhibition of the growth of different fungal strains . A series of amphiphilic four- and six-armed star triblock copolymers 4/6AS-PCL-b-PDEAEMA-b-PPEGMA were also developed recently by our group for the intracellular delivery of the anticancer drug doxorubicin (DOX) .
Amphiphilic miktoarm star polymers with at least two different polymer chains emanating from the central core such as A2B2, A3B3, A2B, A3B, ABC, AB2C2, ABCD, etc., especially for A2B2 and A3B3, have been used in self-assembly and responsive behavior. Gou's group synthesized a series of A2B2 miktoarm star copolymer C4S(PCL)2-(PEG)2, which could self-assemble into various morphologies in aqueous solution controlled by both the macromolecular architectures and the compositions of the copolymer . Well-defined (PNIPAAM)2-(PNVP-b-PAA)2 and (PNIPAAM-b-PAA)2-(PNVP)2 were developed by Zhang's group, and by tuning pH values and temperatures of aqueous solution of these two copolymers, three types of micellar aggregates and the unimer state could undergo reversible switch on and off in size and morphology . However, limited work of A2B2 and A3B3 type miktoarm polymers was reported on drug and gene delivery.
Pentaerythritol was dried under reduced pressure overnight prior to use. ϵ-Caprolactone (ϵ-CL, 99%, Aldrich, St. Louis, MO, USA) was dried over calcium hydride and distilled under reduced pressure before use. 2-(Diethylamino)ethyl methacrylate (DEA, TCI-EP) was distilled from calcium hydride and stored under argon at −20°C. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 475 Da, 99%, Aldrich) was purified by passing through a column filled with neutral alumina to remove inhibitor. Tetrahydrofuran (THF) was dried over sodium using benzophenone as a dryness indicator and distilled under nitrogen prior to use. Toluene was distilled from calcium hydride. Doxorubicin hydrochloride (DOX∙HCl) was purchased from Beijing Huafeng United Technology Co., Ltd., Beijing, China. Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were all purchased from Invitrogen, Carlsbad, CA, USA. HepG2 cells were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA, and cultured under the recommended conditions according to the supplier. 3-(4,5-Dimethyltlliazol-2-yl)-2,5-diphenyltetrazoxium bromide (MTT) and Hoechst 33324 were purchased from Sigma Chemical Co. Pyrene (99%, Aldrich), 2-bromoisobutyryl bromide (98%, Alfa Aesar, Ward Hill, MA, USA), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 99%, Aldrich), paraformaldehyde (99%, Aldrich), CuBr2, methanol, stannous octoate (Sn(Oct)2), triethylamine (TEA), dimethyl sulfoxide (DMSO), acetone, and all other reagents were used as received.
Synthesis of difunctional initiator pentaerythritol bis(2-bromoisobutyrate) [(OH)2-Br2]
(OH)2-Br2 was synthesized as follows: to a flame-dried 250 mL Schlenk flask with a magnetic stirring bar, which was evacuated and flushed with argon thrice, pentaerythritol (6.80 g, 0.05 mmol), anhydrous THF (150 mL), and TEA (13.89 mL, 0.10 mmol) were added in turn at 0°C. Then, 2-bromoisobutyryl bromide (12.36 mL, 0.10 mmol) was injected dropwise for a period of 2 h with vigorous stirring. The reaction was continued at 0°C for 5 h and then at room temperature for another 24 h. The reaction mixture was cooled, extracted with 300 mL diethyl ether thrice, and then the diethyl ether layer was washed successively with water, saturated NaHCO3, and water and dried over MgSO4 overnight followed by rotary evaporation to remove the solvent. The colorless liquid product (OH)2-Br2 was collected by distillation under reduced pressure. 1H NMR (d6-DMSO as solvent, in Additional file 1: Figure S1): −O-CH2- δ = 3.65 ppm (4H), −COO-CH2- δ = 4.31 ppm (4H), −C(CH3)2-Br δ = 1.96 ppm (12H); Element Analysis, calculated (%): C 35.94, H 5.37; found (%): C 35.83, H 4.85.
Synthesis of bromide-terminated two-arm poly(ϵ-caprolactone) macroinitiator [(PCL)2-Br2]
(PCL)2-Br2 was synthesized by ROP of ϵ-CL using (OH)2-Br2 as initiator [32, 33]. Typically, a flame-dried 100 mL Schlenk flask equipped with a magnetic stirring bar was charged with difunctional initiator [(OH)2-Br2] (0.434 g, 1 mmol), and the flask was evacuated and flushed with argon three times. Subsequently, the freshly distilled ϵ-CL (6 g) and a required amount of Sn(Oct)2 (0.1 wt.% of ϵ-CL, 0.006 g) solution were injected into the flask by syringe and three ‘freeze-pump-thaw’ cycles were performed to remove any oxygen from the solution. The flask was immersed into a thermostated oil bath at 130°C for 24 h. The crude polymer was dissolved in approximately 50 mL THF followed by adding dropwise to 500 mL water/methanol (1:1, v/v) mixture to precipitate the product, which was collected and dried under vacuum for 24 h, resulting in powdery (PCL)2-Br2.
Synthesis of A2(BC)2 miktoarm star polymers (PCL)2(PDEA-b-PPEGMA)2
The continuous ARGET ATRP of DEA and PEGMA was in situ monitored by ReactIR iC10 (Metter-Toledo AutoChem, Columbia, MD, USA) equipped with a light conduit and DiComp (diamond composite) insertion probe [34, 35]. The FTIR spectra were collected every minute, and the change of absorbance at 938 cm−1 (=CH2 wags of the DEA and PEGMA) was used to provide the conversion of monomers during the polymerization calculated by ReactIR 4.1 software. In a typical synthesis procedure, a previously dried 100 mL Schlenk flask equipped with a magnetic stirring bar was charged with (PCL)2-Br2 (4.0 g, 0.8 mmol) and CuBr2 (0.0143 g, 0.064 mmol). The real-time FTIR probe was introduced into the flask, and the flask was then evacuated and flushed with argon thrice. Anhydrous toluene (18 mL), DEA (4.8 g), and ligand HMTETA (0.164 mL, 0.64 mmol) were injected into the flask using degassed syringes in order. The mixture was stirred for 10 min, and a required amount of Sn(Oct)2 (0.259 g, 0.64 mmol) solution in toluene (2 mL) was added into the flask by syringe. The flask was placed in a preheated oil bath maintained at 70°C, and the FTIR spectra were collected at the time. After 5 h, the absorbance of 938 cm−1 was kept almost constant and the second monomer PEGMA (Mn = 475, 6.4 g) was then introduced by syringe to continue the polymerization for another 20 h. Then, the flask was removed from the oil bath and cooled to room temperature. THF (50 mL) was added into the flask, and the mixture was then passed through a neutral alumina column to remove the catalyst. After removing the catalyst, the product was recovered by being precipitated into tenfold excess of n-hexane, filtered, and finally dried under vacuum for 24 h.
The critical micelle concentration (CMC) values of (PCL)2(PDEA-b-PPEGMA)2 were determined by the fluorescence probe technique using pyrene as a fluorescence probe. Pyrene dissolved in acetone was added into deionized water (pH 7.4) to make a concentration of 12 × 10−7 M following by removed acetone 2 h through evaporation. The final concentration of pyrene was adjusted to 6 × 10−7 M. The (PCL)2-(PDEA-b-PPEGMA)2 (5 mg) was first dissolved into 50 mL deionized water and then diluted to a series of concentrations from 0.0001 to 0.1 mg/mL with deionized water. Then, 10 mL of polymer solutions at different concentrations were added to the pyrene-filmed vials, respectively, and the combined solutions were equilibrated at room temperature in the dark for 24 h before measurement. The fluorescence excitation spectra of polymer/pyrene solutions were measured and used for determining the CMC values.
Preparation of empty and DOX-loaded micelles
The empty and DOX-loaded (PCL)2(PDEA-b-PPEGMA)2 self-assembled micelles were prepared according to the diafiltration method. Typically, (PCL)2(PDEA-b-PPEGMA)2 (40 mg) was dissolved in 20 mL of DMSO (40 mL for empty micelles) at room temperature 25°C, followed by adding a predetermined amount of DOX∙HCl (10 mg) and double molar amount of TEA in another 20 mL of DMSO and then stirring for 4 h. Then, the mixture solution was transferred to dialysis bag (MWCO = 3.5 kDa) and dialyzed against deionized water for 24 h to remove the organic solvents and free DOX. The deionized water was changed every 4 h for the first 8 h and then replaced every 6 h. After dialysis, the micelles were filtered by a membrane filter (0.45-μm pore) to remove aggregated particles. Then, half of the empty and DOX-loaded micelles were used to study the pH-responsive behavior by the addition of NaOH or HCl (0.01 M) solution. And the remaining empty and DOX-loaded micelles were collected by freeze-drying to obtain dried product. The received white powder was stored at −20°C until further experiments. The values of Dhs and morphologies of the empty and DOX-loaded micelles were monitored by DLS and TEM. DOX-loaded micelles were dissolved in 10 mL of DMSO under vigorous vortexing and analyzed by UV-vis spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) at 480 nm to obtain DOX loading content (LC), wherein a calibration curve was obtained with DOX-DMSO solutions with different DOX concentrations. The LC values were around 10% in the current work.
In vitro DOX release
The release profiles of DOX from the DOX-loaded micelles at a concentration of 1 mg/mL were studied in different media (pH 5.0, pH 6.5, and pH 7.4). Briefly, 5 mg of DOX-loaded micelles were immersed in 5 mL of PBS buffer (pH 7.4 or pH 6.5) or acetate buffer (pH 5.0) and then placed in a pre-swollen cellulose membrane bag (MWCO = 3.5 kDa). The whole bag was placed into 40 mL of PBS or acetate buffer with constant shaking (100 rpm) at 37°C (Dissolution Tester RCZ-8B, TDTF, Tianjin, China). At predetermined time intervals, a 4-mL buffer solution outside the dialysis bag was extracted and it was replaced by an equal volume of fresh media to keep the sink condition. The amounts of released DOX in different buffers were monitored by UV-vis spectrophotometer at 480 nm. Each experiment was done in triplicate, and the results were the average data.
Cell culture and cytotoxicity assay
The in vitro cytotoxicity tests of the free DOX, empty, and DOX-loaded micelles were evaluated by the standard MTT assay against HepG2 cells. The HepG2 cells were first seeded on a 96-well plate at an initial density of 1 × 104 cells/well in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C in a CO2 (5%) incubator for 3 days to reach 60% to 70% confluence. Then, the empty micelles with the final concentration from 1 to 400 μg/mL were added. After 48 h, 20 μL of MTT solution (5 mg/mL in PBS buffer) was added into each well and incubated for another 4 h. Afterwards, the medium in each well was then removed and 200 μL of DMSO was added to dissolve the internalized purple formazan crystals. The absorbance was measured at a wavelength of 490 nm by a microplate reader (Multiskan Spectrum, Thermo Scientific, Vantaa, Finland). Data were expressed as average ± SD (n = 3).
HepG2 cells were incubated with free DOX and DOX-loaded micelles with DOX final concentration from 0.1 to 20 μg/mL in culture medium. After 24 and 48 h, 20 μL of MTT solution (5 mg/mL in PBS buffer) was added into each well and incubated for another 4 h. Afterwards, the culture medium was removed, the obtained crystals were dissolved in 200 μL of DMSO, and the absorbance was measured at a wavelength of 490 nm by a microplate reader. Data were expressed as average ± SD (n = 3).
Confocal laser scanning microscopy (CLSM, Zeiss, LSM 510, Oberkochen, Germany) was employed to examine the intracellular distribution of DOX. HepG2 cells were seeded on slides on a 6-well plate at a density of 4 × 105 cells/well in 2 mL of DMEM and were cultured for 24 h at 37°C in 5% CO2 atmosphere. The cells were then incubated with free DOX and DOX-loaded micelles at a final DOX concentration of 50 μg/mL in DMEM for 4 or 24 h at 37°C. At each predetermined time, the culture media were removed and the cells were washed with PBS (1 min × 3) to remove the DOX-loaded micelles that were not ingested by the cells. Subsequently, the cells were fixed with 4% (w/v) paraformaldehyde aqueous solution for 30 min at room temperature. The slides were then rinsed with PBS (2 min × 3). Finally, the cells were stained with Hoechst 33324 (5 mg/mL in PBS) at 37°C for 15 min, and the slides were rinsed with PBS (2 min × 3). The prepared slides were obtained by CLSM.
1H NMR spectra measurements were examined in d6-DMSO and CDCl3 at 25°C using Bruker AVANCE ΙΙΙ 400 (Madison, WI, USA) operating at 400 MHz. The number average molecular weight (Mn) and polydispersity index (Mw/Mn) were determined by gel permeation chromatography (GPC) adopting an Agilent 1200 series GPC system (Santa Clara, CA, USA) equipped with a LC quant pump, PL gel 5 mm 500, 104, and 105 Å columns in series, and RI detector. The column system was calibrated with a set of monodisperse polystyrene standards using HPLC grade THF as mobile phase with a flow rate of 1.0 mL/min at 30°C. Fluorescence spectra were recorded using a fluorescence spectrophotometer (F-4500, Hitachi, Chiyoda-ku, Japan). The hydrodynamic diameter (Dh) and distribution (PDI) of micelles were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano S, Malvern, WR, UK). Morphologies of micelles were investigated by transmission electron microscopy (TEM, Hitachi H-7650) operating at 80 kV.
Results and discussion
Synthesis and characterization of (PCL)2(PDEA-b-PPEGMA)2
GPC and 1 H NMR data of (PCL) 2 (PDEA- b -PPEGMA) 2 polymers
M n, GPC b
M n, NMR c
M n, RealIR d
The molecular weights of the serial (PCL)2(PDEA-b-PPEGMA)2 were determined by GPC and summarized in Table 1. It can be seen that the GPC curves presented in Additional file 1: Figure S2 appeared monomodal symmetric distribution and the values of Mw/Mn were below 1.50, which are acceptable for further application of delivering drugs. It was also found that GPC analysis for (PCL)2(PDEA-b-PPEGMA)2 tended to underestimate the molecular weight (which was typically smaller) as compared to their linear counterpart due to the reduced hydrodynamic volumes. The characterization of the molar masses of star polymers by GPC is not straightforward. Since standard samples with exactly the same topology and with known molar masses do not exist, the calibration with narrow standards cannot be applied [38, 39].
Characterization of the empty and DOX-loaded micelles
The variations of the Dhs and zeta potentials of the empty micelles and DOX-loaded micelles were investigated from the facile pH adjusting. As shown in Figure 6, when decreasing pH from 10 to 2, the Dhs and zeta potentials increased gradually followed by abrupt descend because the micelles underwent shrinking-swelling-dissociating conformational transition. The Dhs of the micelles showed slightly increase owing to incorporation of DOX molecules in the core of micelles compared to the empty micelles. At higher pH above 8, both micelles were in a compact, collapsed form with the Dhs remained almost constant because the PDEA segments were deprotonated. And the zeta potentials at higher pH (like pH 10) were negative with increasing OH− in the solution. As the pH values were ranging from 8 to 4, both micelles exhibited the gradually stretched conformation with significant increase of Dhs and zeta potentials due to gradual protonation of DEA block and the increasing hydrophilicity of PDEA. At pH < 4, the Dhs and zeta potentials of both micelle solutions showed sharp decrease, indicating that the PDEA segments were fully protonated with imparting a hydrophilic characteristic and the extremely strong electrostatic repulsion between polymer chains, which might cause the decrease of the aggregation number of the polymers or even slight dissociation of the micelle structures .
In vitro drug release profiles and cell experiments
Where Mt and M∞ are the absolute cumulative amount of drug released at time t and infinite time respectively, n is the release exponent indicating the drug release mechanism and k is a constant incorporating structural and geometric characteristic of the device. For spherical particles, the value of n is equal to 0.43 for Fickian diffusion and 0.85 for non-Fickian mechanism, n < 0.43 is due to the combination of diffusion and erosion control, and 0.43 < n < 0.85 corresponds to anomalous transport mechanism .
The fitting parameters, including the release exponent n, rate constant k, and the correlation coefficient R2, were shown in Additional file 1: Table S1. The release of DOX at different pH conditions were divided into two stages with good linearity, one is from 0 to 12 h, and the other is from 12 to 96 h. The results showed that the pH values have major influence on DOX release process. In the first 12 h, the n values of pH 7.4, 6.5, and 5.0 were 0.28, 0.49, and 0.63, respectively. The drug release rates were significantly accelerated and the mechanism of DOX transformed from the combination of diffusion and erosion control to anomalous transport mechanism action when changing pH from 7.4 to 5.0. After 12 h, drug release was controlled by anomalous transport mechanism action with the n values of pH 7.4, 6.5, and 5.0 were 0.48, 0.49, and 0.50, respectively.
Serial amphiphilic miktoarm star polymers (PCL)2(PDEAEMA-b-PPEGMA)2 were successfully prepared by a combination of ROP and continuous ARGET ATRP. A good first-order kinetic characteristic was observed for the continuous ARGET ATRP of DEA and PEGMA. The CMC values of (PCL)2(PDEA-b-PPEGMA)2 were extremely low (0.0024 to 0.0043 mg/mL). The self-assembled empty and DOX-loaded micelles were spherical in morphologies with average sizes of 63 and 110 nm depending on the architecture of the copolymers. The pH responsiveness and in vitro release properties from the micelles exhibited desired pH dependence owing to the protonation of tertiary amine groups of DEA. The in vitro release study showed that the release of DOX at pH 5.0 was much faster than that at pH 7.4 and pH 6.5. Moreover, in vitro cytotoxicity of DOX-loaded micelles suggested that they could effectively inhibit the growth of cancer cells HepG2 with IC50 of 2.0 μg/mL, indicating that the DOX-loaded (PCL)2(PDEA-b-PPEGMA)2 micelles could exhibit similar antitumor activities to free DOX. Intracellular uptake demonstrated that DOX was delivered into the cells effectively after the cells were incubated with DOX-loaded micelles. The characteristics demonstrated that these pH-sensitive (PCL)2(PDEA-b-PPEGMA)2 micelles would be efficient and hopeful platforms for cancer therapy.
WJL and DX are doctoral candidates, SYN is a master student. JFW is a professor in the School of Bioscience & Bioengineering, South China University of Technology, Guangzhou, People’s Republic of China. XDG is an assistant professor, and LJZ is a professor in the School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, People's Republic of China.
This work was financially supported by the National Natural Science Foundation of China (No. 21176090), Team Project of Natural Science Foundation of Guangdong Province, China (No. S2011030001366), Science and Technology Foundation of Guangdong Province, China (No. 2012B050600010, 2011B050400016), and Fundamental Research Funds for the Central Universities, China (No. 2013ZP0010, 2014ZP0020).
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