Acta Biomaterialia 10 (2014) 1259–1271
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Thermo- and pH-responsive copolymers based on PLGA-PEG-PLGA and poly(L-histidine): Synthesis and in vitro characterization of copolymer micelles Wei Hong a, Dawei Chen a,b, Li Jia a, Jianchun Gu a, Haiyang Hu a, Xiuli Zhao a, Mingxi Qiao a,⇑ a b
School of Pharmacy, PO Box 42, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, Liaoning Province 110016, People’s Republic of China School of Pharmacy, Medical College of Soochow University, Suzhou, Jiangsu Province 215123, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 6 July 2013 Received in revised form 2 November 2013 Accepted 16 December 2013 Available online 21 December 2013 Keywords: Thermoresponsive pH-responsive Copolymer micelles Tumor targeting Poly (L-histidine)
a b s t r a c t A series of novel thermo- and pH-responsive block copolymers of PHis-PLGA-PEG-PLGA-PHis composed of poly(ethylene glycol) (PEG), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-histidine) (PHis) were synthesized and used for the construction of stimuli-responsive copolymer micelles. The starting polymers of PLGA-PEG-PLGA and PHis were synthesized by ring-opening polymerization of DL-lactide and glycolide with PEG as an initiator and L-histidine N-carboxylanhydride with isopropylamine as an initiator, respectively. The final copolymer was obtained by the coupling reaction of PHis with PLGA-PEG-PLGA. The copolymer micelles were constructed to have an inner core consisting of two hydrophobic blocks (PLGA and deprotonated PHis) and an outer hydrophilic PEG shell. The temperature- and pH-induced structure changes of the micelles were characterized by an alteration in particle size, a decrease in pyrene florescence intensity, and a variation of 1H NMR spectra in D2O. It was speculated that the hydrophobic–hydrophilic transitions of PEG and PHis in response to temperature and pH variations accounted for the destabilization of micelles. In vitro release profiles, cell cytotoxicity and intracellular location studies further confirmed the temperature- and pH-responsive properties of the copolymer micelles. These results demonstrate the potential of the developed copolymers to be stimuli-responsive carriers for targeted delivery of anti-cancer drugs. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Polymeric micelles from amphiphilic block copolymers have attracted growing interest for anticancer drug delivery applications due to their various advantages over other types of nanoparticulates [1,2]. The principal advantages of such micelles are their high stability, high loading capacity for hydrophobic drugs, controlled drug release and potential for tumor targeting. Despite the efficient tumor targeting achieved by micelles, the relatively slow drug release kinetics from micelles at target sites has been considered a main problem compromising their therapeutic efficacy, especially for drug-resistant tumor cells [3,4]. It may also pose a risk to the development of multidrug resistance of initially drug-sensitive tumor cells. Micelles with drug release mechanisms triggered by a variety of physical or chemical stimuli, such as high temperature [5], pH [6] and ultrasound [7], have been developed to overcome the abovementioned problem. Among these stimuli, high temperature has been recognized as one of the best options because of its easy ⇑ Corresponding author. Tel.: +86 24 23986308; fax: +86 24 23986306. E-mail address: [email protected] (M. Qiao).
and safe medical application. The merits of high temperature are due to the substantial strides that have been made in applications of the hyperthermia therapy to solid tumors and the synergistic effect of hyperthermia and chemotherapy [8]. Studies have demonstrated that the use of local hyperthermia in combination with thermosensitive drug delivery resulted in significantly enhanced antitumor efficacy [9,10]. Besides temperature, pH has also proved another attractive stimulus because of the acidic prototype of tumor tissue [11]. Numerous thermoresponsive micelles have been reported over the past decade mainly based on the thermosensitive polymer poly(N-isopropylacrylamide) (PNIPAAm) [12]. PNIPAAm exhibits a lower critical solution temperature (LCST) of approximately 33 °C in aqueous solution, above which its solubility changes from hydrophilic to hydrophobic [13]. The change of solubility around the LCST destabilizes PNIPAAm-based micelles and triggers a burst-like release of the incorporated drug. However, the primary factors limiting the applications of PNIPAAm-based micelles were lack of biocompatibility and biodegradability as well as the poor micellar stability [4]. This makes biodegradable thermoresponsive copolymer of poly(DL-lactide-co-glycolide)–poly(ethyleneglycol)– poly(DL-lactide-co-glycolide) (PLGA-PEG-PLGA) more attractive
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.12.033
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than PNIPAAm. However, studies related to PLGA-PEG-PLGA copolymers have been mainly focused on the physical form of hydrogel rather than on micelles. A number of pH-responsive micelles based on poly(L-histidine) have been developed, such as poly(L-histidine) (polyHis, Mn 5 K)poly(ethylene glycol) (PEG, Mn 2 K) (PHis-PEG) diblock copolymer micelles [14], mixed micelles of PHis-PEG and poly(L-Lactide)-poly (ethylene glycol) (PLLA-PEG) [15] and the flower-like micelle constructed from poly(L-lactic acid) (PLA, Mn 3 K)-poly(ethylene glycol) (PEG, Mn 2 K)-poly(L-histidine) (polyHis, Mn 5 K) [16]. These micelles were found to undergo structural destabilization at slightly acidic pH due to the protonation of polyHis. However, it is very difficult to design pH-responsive micelles with a precise response to tumor acidic pH because of the subtle and complicated pH difference between the tumor and normal tissues. For example, the diblock copolymer of PHis-PEG had a higher triggering pH than extracellular tumor pH and began to dissociate below pH 7.4 [17]. More advanced polymers with both thermo- and pH-responsive properties were required to circumvent the above-mentioned problems. The dual-response properties of such copolymers would not only enable better on-demand drug release at the target site than solely thermo- or pH-responsive polymer but would also offer a promising synergistic effect of hyperthermia and chemotherapy. In this study, a new class of thermo- and pH-responsive pentablock copolymers of PHis-PLGA-PEG-PLGA-PHis was designed with the knowledge that a dual stimuli-responsive copolymer can be tailored that comprises both a thermoresponsive block and a pH-responsive block. The copolymer was expected to inherit its thermoresponsiveness from the PLGA-PEG-PLGA, and its pHresponsiveness from PHis. Compared to the previously reported PNIPAAm-based dual thermo- and pH-responsive polymers [18–20], the copolymers were superior in biodegradability and biocompatibility due to their composition. PLGA-PEG-PLGA copolymer end-capped with N-Boc-histidine was recently synthesized and applied as tumor extracellular pH-responsive micelles for drug delivery [21]. However, the synthesized copolymer has not previously exhibited thermoresponsiveness. The dual thermo- and pH-responsive properties of the PHis-PLGA-PEG-PLGA-PHis copolymers as well as their potential for the delivery of anticancer drugs have not been investigated to date. In addition, the copolymer featured a large proportion of hydrophobic blocks (PLGA and PHis) in its molecular structure, which could enable good drug-loading capacity and micellar stability and pH responsive property. The main objectives of this study were to synthesize the copolymer PHis-PLGA-PEG-PLGA-PHis, and to characterize the thermo- and pH-responsive properties of the copolymer-based micelles by particle size measurements, fluorescent probe technique, 1H nuclear magnetic resonance (NMR) study, in vitro drug release and cell cytotoxicity. In addition, the mechanisms of the temperature- and pH-responsiveness of the copolymer-based micelles were proposed.
2. Materials and methods 2.1. Materials DL-Lactide and glycolide were obtained from Beijing CONAN Polymer R&D Center (Beijing, PR China). Polyethylene glycol (PEG 2000), stannous 2-ethylhexanoate and 2-mercaptoethanol were purchased from Sigma–Aldrich (St Louis, MO, USA). Diethyl ether, methylene chloride, pyridine, succinic anhydride, thionyl chloride, isopropylamine, dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were purchased from Tianjin Bodi Chemical Co. Ltd. (Tianjin, PRChina). N,N0 -Carbonyldiimidazole (CDI) was purchased from Sigma (St Louis, MO, USA). Na-CBZ-Nim-DNP-L-histidine was pro-
vided by GL Biochem (Shanghai) Ltd. (Shanghai, PR China). Doxorubicin hydrochloride (Batch No. HF100113) was purchased from Beijing HuaFeng United Technology Co. Ltd. (Beijing, PR China). All the other chemicals were of reagent grade and used without further purification. 2.2. Synthesis and characterization of PHis-PLGA-PEG-PLGA-PHis To obtain the pentablock copolymer PHis-PLGA-PEG-PLGA-PHis, the triblock copolymer PLGA-PEG-PLGA and poly(Nim-DNP-L-histidine) were first synthesized, and then a coupling reaction between these products was conducted. Finally, a deprotection reaction was conducted to remove the protection group from poly(Nim-DNP-Lhistidine) to obtain the copolymer PHis-PLGA-PEG-PLGA-PHis. The overall scheme is presented in Fig. 1. 2.2.1. Synthesis of PLGA-PEG-PLGA copolymer The thermoresponsive PLGA-PEG-PLGA copolymer was synthesized by a ring-opening polymerization of DL-lactide and glycolide initiated by PEG (Mn: 2000 Da) with stannous 2-ethylhexanoate as a catalyst, as detailed in our previous publication [22]. The molar ratio of DL-lactide/glycolide was calculated by the integration area of the peaks from DL-lactide and glycolide in the 1H NMR spectrum. 2.2.2. Synthesis of CDI-activated PLGA-PEG-PLGA The triblock copolymer PLGA-PEG-PLGA and CDI were dissolved in 15 ml of dry acetonitrile. The CDI solution was added dropwise to the solution of PLGA-PEG-PLGA at room temperature over 2 h under nitrogen atmosphere. After addition, the mixture was kept stirring for an additional 24 h under a nitrogen atmosphere. The solution was concentrated in a rotary evaporator, and poured into an excess of ethyl ether. This process was repeated three times to remove unreacted CDI. The CDI-PLGA-PEG-PLGA-CDI was dried for 3 days under vacuum (yield 85%). 2.2.3. Synthesis of Nim-DNP-L-histidine carboxyanhydride hydrochloride Na-CBZ-Nim-DNP-L-histidine (1 g) was dried over phosphorus pentoxide in vacuum prior to use. It was dissolved in anhydrous THF (10 ml) followed by the addition of thionyl chloride (0.2 ml) under stirring. The reaction was carried out at room temperature for 1 h. An excess of anhydrous diethyl ether was added to obtain Nim-DNP-L-histidine carboxyanhydride hydrochloride (Nim-DNPL-histidine NCA HCl). The product was purified by dissolving it in nitromethane and crystallized by adding an excess amount of anhydrous diethyl ether. The precipitate was collected and dried under vacuum for 2 days at room temperature (yield 90%). 2.2.4. Synthesis of poly(Nim-DNP-L-histidine) Poly(Nim-DNP-L-histidine) was synthesized by a ring-opening polymerization of Nim-DNP-L-histidine NCA HCl initiated by a primary amine. Nim-DNP-L-histidine NCA HCl (1.0 g) was dissolved in 25 ml DMF and isopropylamine (5 ll) was added to the solution. The polymerization was conducted at room temperature for 3 days. After completion of the reaction, an excess of water was added to precipitate poly(Nim-DNP-L-histidine). The product was filtered out and dried under vacuum for 2 days at room temperature (yield 85%). 2.2.5. Synthesis of PHis-PLGA-PEG-PLGA-PHis The CDI-activated PLGA-PEG-PLGA was dissolved in 15 ml DMSO and added dropwise to the DMSO solution of poly(NimDNP-L-histidine) at room temperature over 2 h under a nitrogen atmosphere. The mixture was allowed to react for 3 days at room temperature. The product was purified by dialyzing (molecular weight cutoff size, 12000–14000) DMSO solution against deionized
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Fig. 1. Synthetic scheme of PHis-PLGA-PEG-PLGA-PHis copolymers.
water for 24 h. The purified product was further dried under vacuum for 2 days at room temperature (yield 80%). The product (2 g) was dissolved in DMSO (25 ml) and 2mercaptoethanol (13 ml) was added at room temperature for 1 day to cut off the dinitrophenyl (DNP) group from the poly(NimDNP-L-histidine). The product was purified by dialyzing (molecular weight cutoff size, 12000–14000) against deionized water for 3 days, followed by lyophilization (yield 80%). 2.2.6. Characterization of intermediates and PHis-PLGA-PEG-PLGAPHis The chemical structure of the intermediates and PHis-PLGAPEG-b-PLGA-PHis copolymer were characterized by a Bruker DRX-600 NMR instrument at 600 MHz. The molecular weight and molecular weight distribution of the PLGA-PEG-PLGA, PHis and PHis-PLGA-PEG-PLGA-PHis were also measured by a gel permeation chromatography (GPC) system equipped with a Waters 515 HPLC pump, a waters StyragelTM HT3 column (300 mm 7.8 mm) and a Waters 2410 refractive index detector. Tetrahydrofuran was used as eluent with a flow rate of 1 ml min1 at 40 °C. The molecular weight of the copolymer was determined relative to polystyrene standards. 2.2.7. Acid–base titration Acid–base titration was conducted on a ZDJ-4A Titrator (Shanghai REX Instrument Factory, PR China) connected to a PC controller where the software controls the experiment. Before performing the titration, the electrode was calibrated with standard pH solutions (pH 4.00, 6.86 and 9.18). 20 mg of two copolymer samples (PLGAPEG-PLGA and PHis9-PLGA-PEG-PLGA-PHis9) were dissolved in
20 ml deionized water in a vessel. A 0.1 N NaOH solution was added to adjust the pH of the solution to a suitable starting value. The titration was performed by the stepwise addition of 0.1 N HCl and the titration profile was recorded by the controller program. The pKa value was determined from the titration curve. 2.3. Critical micelle concentration determination The critical micelle concentration (CMC) of the copolymer was determined by the fluorescence probe technique with pyrene as a fluorescent probe. Aliquots of pyrene solution (2.4 105 M, 50 ll) in acetone were added to volumetric flasks and the acetone was removed by evaporation and mixed with 10 ml of the aqueous copolymer solutions with concentrations ranging from 1 104 to 1 101 g l1. The final pyrene concentration in the copolymer solution was fixed at 1.2 107 mol l1. All the solutions were stored in the dark place for 24 h to reach solubilization equilibrium prior to measurements. The fluorescence spectrum was recorded on a RF-5301 fluorescence spectrophotometer (Shimadzu, Japan). The intensity ratio (I385/I374) of the pyrene emission spectrum was plotted as a function of polymer concentration at the excitation wavelength of 350 nm. 2.4. Preparation of the copolymer micelles The copolymer micelles were prepared by a thin-film hydration method. Briefly, the copolymer (100 mg) was dissolved in acetonitrile (25 ml) in a round-bottomed flask. The solvent was evaporated under reduced pressure by rotary evaporation at 35 °C for 1 h to obtain a thin film. Residual acetonitrile in the film was
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removed under vacuum at room temperature for another 12 h. The resultant thin film was hydrated with 10 ml PBS (pH 8.0) at 35 °C for 30 min to obtain a micelle solution. The micellar solution was sonicated three times for 30 s each time with a KQ3200DB ultrasonicator at 400 W. 2.5. Incorporation of doxorubicin into copolymer micelles Doxorubicin (DOX) hydrochloride (20 mg) was stirred with triethylamine (molar ratio, 1/3) in 20 ml acetonitrile in a roundbottomed flask overnight to obtain a DOX base. The DOX base was then blended with 100 mg of copolymer in acetonitrile. The solvent was evaporated according to the operation in Sec. 2.4. The DOX-containing thin film was hydrated with 10 ml of phosphate buffer (pH 8.0) at 35 °C for 30 min. The micelle solution was sonicated three times for 30 s each time with a KQ3200DB ultrasonicator at 400 W. The resultant micellar solution was centrifuged (10000 rpm, 10 min) and filtrated through a 0.45 lm filter membrane to remove the unincorporated drug. To measure DOX loading content and efficiency, the micellar solution was diluted with acetonitrile prior to determination and the solution was assayed by UV–visible spectrophotometry (UV9100, Rayleigh, China) at 481 nm. A drug-free copolymer acetonitrile solution was used as blank. The drug loading content (DL%) and entrapment efficiency (EE%) were calculated by the following equation:
DL% ¼
Weight of the drug in micelles 100% Weight of the feeding polymer and drug
EE% ¼
Weight of the drug in micelles 100% Weight of the feeding drug
2.6. Transmission electron microscopy (TEM) observations The morphology of copolymer micelles was observed using a JEM-1230 microscope operating at an acceleration voltage of 80 kV without staining. TEM samples were prepared by dipping a copper grid into the micelle solution. A few minutes after the deposition, the extra solution was blotted away with a strip of filter paper and stained with phosphotungstic acid aqueous solution. The water was evaporated at room temperature for 2 h before TEM observation. 2.7. Dynamic light scattering Dynamic light scattering (DLS) was used to measure the mean hydrodynamic diameter and particle size distribution of the copolymer micelles. All the measurements were carried out on a Zetasizer Nano ZS90 (Malvern, UK) at 25 °C after equilibration for 5 min. The micellar solutions were filtered though a 0.45 lm disposable membrane filter prior to measurement. All the values were the average of at least three independent samples. 2.8. Characterization of the thermo- and pH-responsiveness of the copolymer micelles 2.8.1. Particle size and fluorescence determination The PHis9-PLGA-PEG-PLGA-PHis9 copolymer micelles prepared with pH 8.0 phosphate buffer were adjusted to different pH values (pH 7.4, 6.5, 6.0 and 5.0) by the addition of 0.1 N HCl. The copolymer micelle solution (pH 7.4, 6.0 and 5.0) was incubated at various temperatures (20–60 °C) for 15 min to reach equilibrium. The hydrodynamic diameters and particle size distributions of the samples were determined with a Zetasizer Nano ZS90 (Malvern, UK).
Each sample was filtered through a 0.45 lm disposable filter prior to measurements. Each measurement was repeated three times, and an average value was calculated. The copolymer micelles (0.1 g l1) contained pyrene (1.2 ( 107 mol l1) was prepared according to Section 2.3. The intensity ratio (I385/I374) of the pyrene emission spectrum was measured at different pHs (pH 7.4–5.0) and temperatures (30– 60 °C) with the excitation wavelength fixed at 350 nm. 2.8.2. 1H NMR measurements of the copolymer micelles in D2O The copolymer micelles were prepared in D2O by the thin-film hydration method as described in Sec. 2.4. The micelle solution was incubated at various temperatures (20–65 °C) for 15 min to reach equilibrium. The 1H NMR spectrum of the copolymer micelle was measured by a Bruker ARX-300 NMR instrument at 300 MHz at each designated temperature. 2.8.3. In vitro release of DOX from the copolymer micelles The DOX-loaded micelle solution (1.5 ml) was transferred into a dialysis bag (molecular weight cut-off size 5000) and immersed in a vessel. The drug-release tests were performed in 80 ml phosphate buffers of different pHs (pH 7.4, 6.8, 6.0 and 5.0) at 37 °C or in phosphate buffer (pH 7.4) at various temperatures (25, 37 and 41 °C) under sink conditions (0.5% Tween-80, V/V). At predetermined time intervals, 3 ml of the release medium was withdrawn and replaced with an equal volume of fresh medium. The DOX content was measured with a UV–visible spectrophotometer as described above. Each release experiment was performed in triplicate. 2.8.4. Cell cytotoxicity Human breast adenocarcinoma (MCF-7) cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yuanhengshengma Biological Reagent Institute, Beijing, PR China), penicillin (100 U ml1), streptomycin (100 lg ml1) and 0.2% NaHCO3 at 37 °C and 5% CO2. Cells in the exponential phase of growth were used in the experiments. The cells were seeded at 5 103 cells per well in 96-well plates (NUNC, Roskilde, Denmark) for 24 h. After removing growth medium, 100 ll RPMI-1640 medium containing free DOX (4 lg ml1) or equivalent DOX-loaded micelles were added to the plate. The pH of the culture medium was adjusted with 0.1 N HCl to pH 7.4, 7.0, 6.5 or 5.0. The cells were incubated for 48 h. The culture medium from each cell was removed and 20 ll MTT solution (5 mg ml1) was added. After 4 h further incubation, the culture medium was removed and 150 ll DMSO was added to solubilize the formazan crystals. The absorbance of each well was measured at 570 nm using a multifuctional microplate reader (Tecan, Austria). The cell viability expressed in this study is relative to those at each pH in the absence of DOX and micelles and the direct pH effect on the cell viability was not monitored with the MCF-7 cell line. 2.8.5. Confocal microscopy MCF-7 cells were seeded on coverslips placed in a 24-well plate (1 105 cells per well) and grown for 24 h. Free DOX, DOX-incorporated PLGA-PEG-PLGA micelles or PHis9-PLGA-PEG-PLGA-PHis9 micelles were then added and the cells were further incubated for 1, 4 and 8 h respectively. Thereafter, the cells were washed three times with cold PBS and 50 nM LysoTracker Green for 30 min to visualize endosome/lysosome, then fixed in 3.7% paraformaldehyde, and treated with 2 lM Hoechst 33258 for 10 min to dye nuclei. The coverslips were then mounted on microscope slides using a fluorescence-free buffered mounting medium. Images were acquired on a confocal laser scanning microscope (Olympus FV1000-IX81, Japan).
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Fig. 2. 1H NMR spectrum of NCA (A), poly (Nim-DNP-L-histidine) (B), PLGA-PEG-b-PLGA (C), CDI-PLGA-PEG-PLGA-CDI (D), poly(Nim-DNP-L-histidine)-PLGA-PEG-PLGApoly(Nim-DNP-L-histidine) (E), PHis-PLGA-PEG-PLGA-PHis (F).
Fig. 3. The typical GPC spectrum of poly(Nim-DNP-L-histidine) (A), PLGA-PEG-PLGA (B) and PHis9-PLGA-PEG-PLGA-PHis9 (C).
2.9. Statistical analysis
3. Results and discussion
All experiments were performed at least three times. Quantitative data are presented as the mean ± SD. Statistical comparisons were determined by the analysis of variance (ANOVA) among at least three groups or Student’s t-test between two groups. P-values
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Thermo- and pH-responsive copolymers based on PLGA-PEG-PLGA and poly(L-histidine): Synthesis and in vitro characterization of copolymer micelles Wei Hong a, Dawei Chen a,b, Li Jia a, Jianchun Gu a, Haiyang Hu a, Xiuli Zhao a, Mingxi Qiao a,⇑ a b
School of Pharmacy, PO Box 42, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, Liaoning Province 110016, People’s Republic of China School of Pharmacy, Medical College of Soochow University, Suzhou, Jiangsu Province 215123, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 6 July 2013 Received in revised form 2 November 2013 Accepted 16 December 2013 Available online 21 December 2013 Keywords: Thermoresponsive pH-responsive Copolymer micelles Tumor targeting Poly (L-histidine)
a b s t r a c t A series of novel thermo- and pH-responsive block copolymers of PHis-PLGA-PEG-PLGA-PHis composed of poly(ethylene glycol) (PEG), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-histidine) (PHis) were synthesized and used for the construction of stimuli-responsive copolymer micelles. The starting polymers of PLGA-PEG-PLGA and PHis were synthesized by ring-opening polymerization of DL-lactide and glycolide with PEG as an initiator and L-histidine N-carboxylanhydride with isopropylamine as an initiator, respectively. The final copolymer was obtained by the coupling reaction of PHis with PLGA-PEG-PLGA. The copolymer micelles were constructed to have an inner core consisting of two hydrophobic blocks (PLGA and deprotonated PHis) and an outer hydrophilic PEG shell. The temperature- and pH-induced structure changes of the micelles were characterized by an alteration in particle size, a decrease in pyrene florescence intensity, and a variation of 1H NMR spectra in D2O. It was speculated that the hydrophobic–hydrophilic transitions of PEG and PHis in response to temperature and pH variations accounted for the destabilization of micelles. In vitro release profiles, cell cytotoxicity and intracellular location studies further confirmed the temperature- and pH-responsive properties of the copolymer micelles. These results demonstrate the potential of the developed copolymers to be stimuli-responsive carriers for targeted delivery of anti-cancer drugs. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Polymeric micelles from amphiphilic block copolymers have attracted growing interest for anticancer drug delivery applications due to their various advantages over other types of nanoparticulates [1,2]. The principal advantages of such micelles are their high stability, high loading capacity for hydrophobic drugs, controlled drug release and potential for tumor targeting. Despite the efficient tumor targeting achieved by micelles, the relatively slow drug release kinetics from micelles at target sites has been considered a main problem compromising their therapeutic efficacy, especially for drug-resistant tumor cells [3,4]. It may also pose a risk to the development of multidrug resistance of initially drug-sensitive tumor cells. Micelles with drug release mechanisms triggered by a variety of physical or chemical stimuli, such as high temperature [5], pH [6] and ultrasound [7], have been developed to overcome the abovementioned problem. Among these stimuli, high temperature has been recognized as one of the best options because of its easy ⇑ Corresponding author. Tel.: +86 24 23986308; fax: +86 24 23986306. E-mail address: [email protected] (M. Qiao).
and safe medical application. The merits of high temperature are due to the substantial strides that have been made in applications of the hyperthermia therapy to solid tumors and the synergistic effect of hyperthermia and chemotherapy [8]. Studies have demonstrated that the use of local hyperthermia in combination with thermosensitive drug delivery resulted in significantly enhanced antitumor efficacy [9,10]. Besides temperature, pH has also proved another attractive stimulus because of the acidic prototype of tumor tissue [11]. Numerous thermoresponsive micelles have been reported over the past decade mainly based on the thermosensitive polymer poly(N-isopropylacrylamide) (PNIPAAm) [12]. PNIPAAm exhibits a lower critical solution temperature (LCST) of approximately 33 °C in aqueous solution, above which its solubility changes from hydrophilic to hydrophobic [13]. The change of solubility around the LCST destabilizes PNIPAAm-based micelles and triggers a burst-like release of the incorporated drug. However, the primary factors limiting the applications of PNIPAAm-based micelles were lack of biocompatibility and biodegradability as well as the poor micellar stability [4]. This makes biodegradable thermoresponsive copolymer of poly(DL-lactide-co-glycolide)–poly(ethyleneglycol)– poly(DL-lactide-co-glycolide) (PLGA-PEG-PLGA) more attractive
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than PNIPAAm. However, studies related to PLGA-PEG-PLGA copolymers have been mainly focused on the physical form of hydrogel rather than on micelles. A number of pH-responsive micelles based on poly(L-histidine) have been developed, such as poly(L-histidine) (polyHis, Mn 5 K)poly(ethylene glycol) (PEG, Mn 2 K) (PHis-PEG) diblock copolymer micelles [14], mixed micelles of PHis-PEG and poly(L-Lactide)-poly (ethylene glycol) (PLLA-PEG) [15] and the flower-like micelle constructed from poly(L-lactic acid) (PLA, Mn 3 K)-poly(ethylene glycol) (PEG, Mn 2 K)-poly(L-histidine) (polyHis, Mn 5 K) [16]. These micelles were found to undergo structural destabilization at slightly acidic pH due to the protonation of polyHis. However, it is very difficult to design pH-responsive micelles with a precise response to tumor acidic pH because of the subtle and complicated pH difference between the tumor and normal tissues. For example, the diblock copolymer of PHis-PEG had a higher triggering pH than extracellular tumor pH and began to dissociate below pH 7.4 [17]. More advanced polymers with both thermo- and pH-responsive properties were required to circumvent the above-mentioned problems. The dual-response properties of such copolymers would not only enable better on-demand drug release at the target site than solely thermo- or pH-responsive polymer but would also offer a promising synergistic effect of hyperthermia and chemotherapy. In this study, a new class of thermo- and pH-responsive pentablock copolymers of PHis-PLGA-PEG-PLGA-PHis was designed with the knowledge that a dual stimuli-responsive copolymer can be tailored that comprises both a thermoresponsive block and a pH-responsive block. The copolymer was expected to inherit its thermoresponsiveness from the PLGA-PEG-PLGA, and its pHresponsiveness from PHis. Compared to the previously reported PNIPAAm-based dual thermo- and pH-responsive polymers [18–20], the copolymers were superior in biodegradability and biocompatibility due to their composition. PLGA-PEG-PLGA copolymer end-capped with N-Boc-histidine was recently synthesized and applied as tumor extracellular pH-responsive micelles for drug delivery [21]. However, the synthesized copolymer has not previously exhibited thermoresponsiveness. The dual thermo- and pH-responsive properties of the PHis-PLGA-PEG-PLGA-PHis copolymers as well as their potential for the delivery of anticancer drugs have not been investigated to date. In addition, the copolymer featured a large proportion of hydrophobic blocks (PLGA and PHis) in its molecular structure, which could enable good drug-loading capacity and micellar stability and pH responsive property. The main objectives of this study were to synthesize the copolymer PHis-PLGA-PEG-PLGA-PHis, and to characterize the thermo- and pH-responsive properties of the copolymer-based micelles by particle size measurements, fluorescent probe technique, 1H nuclear magnetic resonance (NMR) study, in vitro drug release and cell cytotoxicity. In addition, the mechanisms of the temperature- and pH-responsiveness of the copolymer-based micelles were proposed.
2. Materials and methods 2.1. Materials DL-Lactide and glycolide were obtained from Beijing CONAN Polymer R&D Center (Beijing, PR China). Polyethylene glycol (PEG 2000), stannous 2-ethylhexanoate and 2-mercaptoethanol were purchased from Sigma–Aldrich (St Louis, MO, USA). Diethyl ether, methylene chloride, pyridine, succinic anhydride, thionyl chloride, isopropylamine, dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were purchased from Tianjin Bodi Chemical Co. Ltd. (Tianjin, PRChina). N,N0 -Carbonyldiimidazole (CDI) was purchased from Sigma (St Louis, MO, USA). Na-CBZ-Nim-DNP-L-histidine was pro-
vided by GL Biochem (Shanghai) Ltd. (Shanghai, PR China). Doxorubicin hydrochloride (Batch No. HF100113) was purchased from Beijing HuaFeng United Technology Co. Ltd. (Beijing, PR China). All the other chemicals were of reagent grade and used without further purification. 2.2. Synthesis and characterization of PHis-PLGA-PEG-PLGA-PHis To obtain the pentablock copolymer PHis-PLGA-PEG-PLGA-PHis, the triblock copolymer PLGA-PEG-PLGA and poly(Nim-DNP-L-histidine) were first synthesized, and then a coupling reaction between these products was conducted. Finally, a deprotection reaction was conducted to remove the protection group from poly(Nim-DNP-Lhistidine) to obtain the copolymer PHis-PLGA-PEG-PLGA-PHis. The overall scheme is presented in Fig. 1. 2.2.1. Synthesis of PLGA-PEG-PLGA copolymer The thermoresponsive PLGA-PEG-PLGA copolymer was synthesized by a ring-opening polymerization of DL-lactide and glycolide initiated by PEG (Mn: 2000 Da) with stannous 2-ethylhexanoate as a catalyst, as detailed in our previous publication [22]. The molar ratio of DL-lactide/glycolide was calculated by the integration area of the peaks from DL-lactide and glycolide in the 1H NMR spectrum. 2.2.2. Synthesis of CDI-activated PLGA-PEG-PLGA The triblock copolymer PLGA-PEG-PLGA and CDI were dissolved in 15 ml of dry acetonitrile. The CDI solution was added dropwise to the solution of PLGA-PEG-PLGA at room temperature over 2 h under nitrogen atmosphere. After addition, the mixture was kept stirring for an additional 24 h under a nitrogen atmosphere. The solution was concentrated in a rotary evaporator, and poured into an excess of ethyl ether. This process was repeated three times to remove unreacted CDI. The CDI-PLGA-PEG-PLGA-CDI was dried for 3 days under vacuum (yield 85%). 2.2.3. Synthesis of Nim-DNP-L-histidine carboxyanhydride hydrochloride Na-CBZ-Nim-DNP-L-histidine (1 g) was dried over phosphorus pentoxide in vacuum prior to use. It was dissolved in anhydrous THF (10 ml) followed by the addition of thionyl chloride (0.2 ml) under stirring. The reaction was carried out at room temperature for 1 h. An excess of anhydrous diethyl ether was added to obtain Nim-DNP-L-histidine carboxyanhydride hydrochloride (Nim-DNPL-histidine NCA HCl). The product was purified by dissolving it in nitromethane and crystallized by adding an excess amount of anhydrous diethyl ether. The precipitate was collected and dried under vacuum for 2 days at room temperature (yield 90%). 2.2.4. Synthesis of poly(Nim-DNP-L-histidine) Poly(Nim-DNP-L-histidine) was synthesized by a ring-opening polymerization of Nim-DNP-L-histidine NCA HCl initiated by a primary amine. Nim-DNP-L-histidine NCA HCl (1.0 g) was dissolved in 25 ml DMF and isopropylamine (5 ll) was added to the solution. The polymerization was conducted at room temperature for 3 days. After completion of the reaction, an excess of water was added to precipitate poly(Nim-DNP-L-histidine). The product was filtered out and dried under vacuum for 2 days at room temperature (yield 85%). 2.2.5. Synthesis of PHis-PLGA-PEG-PLGA-PHis The CDI-activated PLGA-PEG-PLGA was dissolved in 15 ml DMSO and added dropwise to the DMSO solution of poly(NimDNP-L-histidine) at room temperature over 2 h under a nitrogen atmosphere. The mixture was allowed to react for 3 days at room temperature. The product was purified by dialyzing (molecular weight cutoff size, 12000–14000) DMSO solution against deionized
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Fig. 1. Synthetic scheme of PHis-PLGA-PEG-PLGA-PHis copolymers.
water for 24 h. The purified product was further dried under vacuum for 2 days at room temperature (yield 80%). The product (2 g) was dissolved in DMSO (25 ml) and 2mercaptoethanol (13 ml) was added at room temperature for 1 day to cut off the dinitrophenyl (DNP) group from the poly(NimDNP-L-histidine). The product was purified by dialyzing (molecular weight cutoff size, 12000–14000) against deionized water for 3 days, followed by lyophilization (yield 80%). 2.2.6. Characterization of intermediates and PHis-PLGA-PEG-PLGAPHis The chemical structure of the intermediates and PHis-PLGAPEG-b-PLGA-PHis copolymer were characterized by a Bruker DRX-600 NMR instrument at 600 MHz. The molecular weight and molecular weight distribution of the PLGA-PEG-PLGA, PHis and PHis-PLGA-PEG-PLGA-PHis were also measured by a gel permeation chromatography (GPC) system equipped with a Waters 515 HPLC pump, a waters StyragelTM HT3 column (300 mm 7.8 mm) and a Waters 2410 refractive index detector. Tetrahydrofuran was used as eluent with a flow rate of 1 ml min1 at 40 °C. The molecular weight of the copolymer was determined relative to polystyrene standards. 2.2.7. Acid–base titration Acid–base titration was conducted on a ZDJ-4A Titrator (Shanghai REX Instrument Factory, PR China) connected to a PC controller where the software controls the experiment. Before performing the titration, the electrode was calibrated with standard pH solutions (pH 4.00, 6.86 and 9.18). 20 mg of two copolymer samples (PLGAPEG-PLGA and PHis9-PLGA-PEG-PLGA-PHis9) were dissolved in
20 ml deionized water in a vessel. A 0.1 N NaOH solution was added to adjust the pH of the solution to a suitable starting value. The titration was performed by the stepwise addition of 0.1 N HCl and the titration profile was recorded by the controller program. The pKa value was determined from the titration curve. 2.3. Critical micelle concentration determination The critical micelle concentration (CMC) of the copolymer was determined by the fluorescence probe technique with pyrene as a fluorescent probe. Aliquots of pyrene solution (2.4 105 M, 50 ll) in acetone were added to volumetric flasks and the acetone was removed by evaporation and mixed with 10 ml of the aqueous copolymer solutions with concentrations ranging from 1 104 to 1 101 g l1. The final pyrene concentration in the copolymer solution was fixed at 1.2 107 mol l1. All the solutions were stored in the dark place for 24 h to reach solubilization equilibrium prior to measurements. The fluorescence spectrum was recorded on a RF-5301 fluorescence spectrophotometer (Shimadzu, Japan). The intensity ratio (I385/I374) of the pyrene emission spectrum was plotted as a function of polymer concentration at the excitation wavelength of 350 nm. 2.4. Preparation of the copolymer micelles The copolymer micelles were prepared by a thin-film hydration method. Briefly, the copolymer (100 mg) was dissolved in acetonitrile (25 ml) in a round-bottomed flask. The solvent was evaporated under reduced pressure by rotary evaporation at 35 °C for 1 h to obtain a thin film. Residual acetonitrile in the film was
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removed under vacuum at room temperature for another 12 h. The resultant thin film was hydrated with 10 ml PBS (pH 8.0) at 35 °C for 30 min to obtain a micelle solution. The micellar solution was sonicated three times for 30 s each time with a KQ3200DB ultrasonicator at 400 W. 2.5. Incorporation of doxorubicin into copolymer micelles Doxorubicin (DOX) hydrochloride (20 mg) was stirred with triethylamine (molar ratio, 1/3) in 20 ml acetonitrile in a roundbottomed flask overnight to obtain a DOX base. The DOX base was then blended with 100 mg of copolymer in acetonitrile. The solvent was evaporated according to the operation in Sec. 2.4. The DOX-containing thin film was hydrated with 10 ml of phosphate buffer (pH 8.0) at 35 °C for 30 min. The micelle solution was sonicated three times for 30 s each time with a KQ3200DB ultrasonicator at 400 W. The resultant micellar solution was centrifuged (10000 rpm, 10 min) and filtrated through a 0.45 lm filter membrane to remove the unincorporated drug. To measure DOX loading content and efficiency, the micellar solution was diluted with acetonitrile prior to determination and the solution was assayed by UV–visible spectrophotometry (UV9100, Rayleigh, China) at 481 nm. A drug-free copolymer acetonitrile solution was used as blank. The drug loading content (DL%) and entrapment efficiency (EE%) were calculated by the following equation:
DL% ¼
Weight of the drug in micelles 100% Weight of the feeding polymer and drug
EE% ¼
Weight of the drug in micelles 100% Weight of the feeding drug
2.6. Transmission electron microscopy (TEM) observations The morphology of copolymer micelles was observed using a JEM-1230 microscope operating at an acceleration voltage of 80 kV without staining. TEM samples were prepared by dipping a copper grid into the micelle solution. A few minutes after the deposition, the extra solution was blotted away with a strip of filter paper and stained with phosphotungstic acid aqueous solution. The water was evaporated at room temperature for 2 h before TEM observation. 2.7. Dynamic light scattering Dynamic light scattering (DLS) was used to measure the mean hydrodynamic diameter and particle size distribution of the copolymer micelles. All the measurements were carried out on a Zetasizer Nano ZS90 (Malvern, UK) at 25 °C after equilibration for 5 min. The micellar solutions were filtered though a 0.45 lm disposable membrane filter prior to measurement. All the values were the average of at least three independent samples. 2.8. Characterization of the thermo- and pH-responsiveness of the copolymer micelles 2.8.1. Particle size and fluorescence determination The PHis9-PLGA-PEG-PLGA-PHis9 copolymer micelles prepared with pH 8.0 phosphate buffer were adjusted to different pH values (pH 7.4, 6.5, 6.0 and 5.0) by the addition of 0.1 N HCl. The copolymer micelle solution (pH 7.4, 6.0 and 5.0) was incubated at various temperatures (20–60 °C) for 15 min to reach equilibrium. The hydrodynamic diameters and particle size distributions of the samples were determined with a Zetasizer Nano ZS90 (Malvern, UK).
Each sample was filtered through a 0.45 lm disposable filter prior to measurements. Each measurement was repeated three times, and an average value was calculated. The copolymer micelles (0.1 g l1) contained pyrene (1.2 ( 107 mol l1) was prepared according to Section 2.3. The intensity ratio (I385/I374) of the pyrene emission spectrum was measured at different pHs (pH 7.4–5.0) and temperatures (30– 60 °C) with the excitation wavelength fixed at 350 nm. 2.8.2. 1H NMR measurements of the copolymer micelles in D2O The copolymer micelles were prepared in D2O by the thin-film hydration method as described in Sec. 2.4. The micelle solution was incubated at various temperatures (20–65 °C) for 15 min to reach equilibrium. The 1H NMR spectrum of the copolymer micelle was measured by a Bruker ARX-300 NMR instrument at 300 MHz at each designated temperature. 2.8.3. In vitro release of DOX from the copolymer micelles The DOX-loaded micelle solution (1.5 ml) was transferred into a dialysis bag (molecular weight cut-off size 5000) and immersed in a vessel. The drug-release tests were performed in 80 ml phosphate buffers of different pHs (pH 7.4, 6.8, 6.0 and 5.0) at 37 °C or in phosphate buffer (pH 7.4) at various temperatures (25, 37 and 41 °C) under sink conditions (0.5% Tween-80, V/V). At predetermined time intervals, 3 ml of the release medium was withdrawn and replaced with an equal volume of fresh medium. The DOX content was measured with a UV–visible spectrophotometer as described above. Each release experiment was performed in triplicate. 2.8.4. Cell cytotoxicity Human breast adenocarcinoma (MCF-7) cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yuanhengshengma Biological Reagent Institute, Beijing, PR China), penicillin (100 U ml1), streptomycin (100 lg ml1) and 0.2% NaHCO3 at 37 °C and 5% CO2. Cells in the exponential phase of growth were used in the experiments. The cells were seeded at 5 103 cells per well in 96-well plates (NUNC, Roskilde, Denmark) for 24 h. After removing growth medium, 100 ll RPMI-1640 medium containing free DOX (4 lg ml1) or equivalent DOX-loaded micelles were added to the plate. The pH of the culture medium was adjusted with 0.1 N HCl to pH 7.4, 7.0, 6.5 or 5.0. The cells were incubated for 48 h. The culture medium from each cell was removed and 20 ll MTT solution (5 mg ml1) was added. After 4 h further incubation, the culture medium was removed and 150 ll DMSO was added to solubilize the formazan crystals. The absorbance of each well was measured at 570 nm using a multifuctional microplate reader (Tecan, Austria). The cell viability expressed in this study is relative to those at each pH in the absence of DOX and micelles and the direct pH effect on the cell viability was not monitored with the MCF-7 cell line. 2.8.5. Confocal microscopy MCF-7 cells were seeded on coverslips placed in a 24-well plate (1 105 cells per well) and grown for 24 h. Free DOX, DOX-incorporated PLGA-PEG-PLGA micelles or PHis9-PLGA-PEG-PLGA-PHis9 micelles were then added and the cells were further incubated for 1, 4 and 8 h respectively. Thereafter, the cells were washed three times with cold PBS and 50 nM LysoTracker Green for 30 min to visualize endosome/lysosome, then fixed in 3.7% paraformaldehyde, and treated with 2 lM Hoechst 33258 for 10 min to dye nuclei. The coverslips were then mounted on microscope slides using a fluorescence-free buffered mounting medium. Images were acquired on a confocal laser scanning microscope (Olympus FV1000-IX81, Japan).
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Fig. 2. 1H NMR spectrum of NCA (A), poly (Nim-DNP-L-histidine) (B), PLGA-PEG-b-PLGA (C), CDI-PLGA-PEG-PLGA-CDI (D), poly(Nim-DNP-L-histidine)-PLGA-PEG-PLGApoly(Nim-DNP-L-histidine) (E), PHis-PLGA-PEG-PLGA-PHis (F).
Fig. 3. The typical GPC spectrum of poly(Nim-DNP-L-histidine) (A), PLGA-PEG-PLGA (B) and PHis9-PLGA-PEG-PLGA-PHis9 (C).
2.9. Statistical analysis
3. Results and discussion
All experiments were performed at least three times. Quantitative data are presented as the mean ± SD. Statistical comparisons were determined by the analysis of variance (ANOVA) among at least three groups or Student’s t-test between two groups. P-values
