International Journal of Pharmaceutics 471 (2014) 28–36
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Polymeric micelles with citraconic amide as pH-sensitive bond in backbone for anticancer drug delivery Jun Cao a , Ting Su a , Longgui Zhang a , Rong Liu b , Gang Wang a, * , Bin He a , Zhongwei Gu a a b
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064 China College of Medical and Nursing,Chengdu University, Chengdu 610106 China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2014 Received in revised form 23 April 2014 Accepted 4 May 2014 Available online 13 May 2014
A novel pH-sensitive polymeric micelle was reported. Methoxy poly(ethylene glycol)-b-poly(e-caprolactone) copolymer with citraconic amide as pH-sensitive bond was synthesized (mPEG-pH-PCL). The copolymers self-assembled into micelles to encapsulate anticancer drug doxorubicin (DOX). The morphology, size and size distribution, drug release profile and in vitro anticancer activity of the DOX loaded mPEG-pH-PCL micelles were studied. The results showed that the mean size of the micelles was around 120 nm, the drug loading content and encapsulation efficiency of the mPEG-pH-PCL micelles were 6.8% and 54.3%, respectively. The mean diameter and size distribution of the mPEG-pH-PCL micelles increased significantly when soaking in medium with pH 5.5. The drug release of micelles in pH 5.5 was much faster than that in pH 7.4. The confocal laser microscopy and flow cytometry measurements indicated that the weak acidity of endosomes broke the citraconic amide bonds in the copolymer backbones and triggered the fast release of DOX. The in vitro IC50 of the drug loaded mPEG-pH-PCL micelles was lower than that of drug loaded polymeric micelles without pH-sensitivity to both HepG2 and 4T1 cancer cells. ã 2014 Published by Elsevier B.V.
Keywords: pH-sensitive Citraconic anhydride Polymeric micelle Drug delivery Doxorubicin
1. Introduction Biodegradable polymeric micelles are important carriers for anticancer drug delivery due to their excellent biocompatibility, biodegradability and long circulation in blood vessel transportation (Deng et al., 2012; Duncan, 2003; Kwon and Okano, 1996; Otsuka et al., 2012). A paclitaxel loaded poly(lactic acid)–poly (ethylene glycol) (PLA–PEG) copolymer micelle has been approved by FDA for cancer treatment, polyester-PEG micelles have attracted much interest to biomaterials scientists and pharmacists. Poly(e-caprolactone) (PCL) is a biodegradable polyester with low glass transition temperature (Tg) and melting point (Tm), the diffusion of hydrophobic anticancer drugs will be faster in the elastic PCL substrate, thus, PEG–PCL block copolymer micelles are extensively studied for anticancer drug delivery (Gu et al., 2013; Xin et al., 2012; Xue et al., 2012; Yang et al., 2014; Zhang et al., 2011). The PEG–PCL micelles are core-shell structured nanoparticles, hydrophobic anticancer drugs are loaded in the PCL cores. The drug release will last for several days, however, the low concentration of
* Corresponding author. Tel.: +86 28 85412923. E-mail address: [email protected] (G. Wang). http://dx.doi.org/10.1016/j.ijpharm.2014.05.010 0378-5173/ ã 2014 Published by Elsevier B.V.
released drug will not kill cancer cells efficiently, and it also has the risk to evoke multidrug resistance in cancer cells. Many pHsensitive polymeric micelles were designed and synthesized to resolve this problem (Bae et al., 2003, 2007; Cao et al., 2013), the drug release was triggered by the weak acidity of intracellular endosomes and/or lysomes and rapidly escaped from micelles. Two strategies were reported to fabricate pH-sensitive polymeric micelles. Introduction of imidazole groups in polymeric micelles was a practical approach, the imidazole groups were protonized in the medium with pH around 6.0, and the protonation transferred imidazole groups from hydrophobic to hydrophilic to swell the polymeric micelles to accelerate the release of drug. The molecules with imidazole groups such as L-histidine and 1-(3-aminopropyl) imidazole have been used to achieve the pH-sensitivity in polymeric micelles (Liu et al., 2011; Pu et al., 2014). Utilization of pH-cleavable hydrazone bond was another strategy to prepare pH-sensitive drug delivery systems (Pu et al., 2013). Anticancer drug was immobilized on nanocarriers via hydrazone bond to form drug-carrier conjugates, the hydrazone bonds were broken in endosomes and/or lysosomes to release the immobilized drug. Acetal bond was also a pH-sensitive bond introduced in polymeric micelles, the broken of acetal bond in acidic environment would change the internal structure of polymeric micelles to release the encapsulated drug (Gillies and Fréchet, 2005).
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Recently, a new pH-sensitive bond of citraconic amide was reported in drug/gene delivery system, which was called chargereversible delivery system (Prata et al., 2004; Xu et al., 2007; Yuan et al., 2012; Zhou et al., 2009). In this system, citraconic anhydride was reacted with amino group in nanoparticles to change the surface charge from positive to negative. The amide bond was broken in weak acidic condition in tumor tissues, the surface charge of nanoparticles was changed to positive and promoted the cellular uptake via the electrostatic interaction between positively charged nanoparticles and negatively charged cell membranes. As lack of functional groups for modification, PEG–PCL block copolymers were rarely reported to fabricate pH-sensitive drug carriers. Herein, we reported a novel pH-sensitive mPEG-PCL micelle. The hydroxyl terminal group of PEG was transferred into amino group, citraconic anhydride was used as a linker to connect PEG and PCL blocks, the pH-sensitive citraconic amide bond was formed between PEG and PCL blocks. Anticancer drug doxorubicin was encapsulated in the mPEG-pH-PCL micelles. Once the drug loaded micelles were internalized in cancer cells, the weak acidity in endosomes broke the citraconic amide bond to release the loaded drug (Scheme 1).
2. Materials and methods 2.1. Materials N,N-Dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), CDCl3, citraconic anhydride (CA), phenylmethanesulfonyl chloride (TsCl), e-caprolactone (CL) and methoxy-poly (ethylene glycol) (mPEG-OH, Mn = 2000 g/mol) were purchased from Sigma–Aldrich Co. (Steinheim, Germany) and used as received. Doxorubicin hydrochloride (DOXHCl, Shanghai Yingxuan Chempharm Co., Ltd., China) was dissolved in water, the pH value was adjusted to 9.6 to prepare doxorubicin. Benzyl alcohol, phosphate buffer saline (PBS) solution (pH 7.4, I = 0.01 M) containing disodium hydrogen phosphate (NaH2PO4), potassium dihydrogen phosphate (K2HPO4), sodium chloride (NaCl), potassium chloride (KCl) and sodium bicarbonate (NaHCO3) were purchased from Changzheng Chemical Co. (China). Sodium citrate buffer solution (CS) (pH 5.5) was composed of citric acid and sodium citrate. Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS) and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich Co. (Steinheim, Germany) and
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used for cells test. All the solvents were purchased from Kelong Chemical Co. (Chengdu, China) and used as received. 2.2. Measurements The solvent for 1H NMR measurement was CDCl3 containing 0.5% tetramethylsilane as the internal standard. The 1H NMR spectra were recorded on Bruker AVANCE-400 MHz NMR spectrometer, working at 400 MHz. The molecular weight (Mn and Mw) and molecular weight distribution (Mw/Mn) of the polymers were determined by gel permeation chromatography (GPC, Agilent 1100 Series) and analyzed with GPC–SEC data analysis software. The samples were analyzed at 25 C with CDCl3 as eluent at a flow rate of 1.0 mL/min. The MS spectra were performed on ESI-TOF MS (Waters Q-Tof Premier). 2.3. Synthesis of mPEG-NH2 Methoxy poly(ethylene glycol) (10 g, 5 mmol) was dissolved in anhydrous dichloromethane (150 mL) and cooled in an ice-water bath. Pyridine (1.58 g, 20 mmol) and TsCl (4.77 g, 25 mmol) in 50 mL of CH2Cl2 were added dropwise under stirring. The solution was stirred at 0 C for 8 h and further at room temperature for 4 days under nitrogen atmosphere. The mixture was filtrated, concentrated, and precipitated in excess cold diethyl ether. The product was recrystallized in ethanol, collected and dried in vacuum. The purified product was added to 100 mL of 25 wt% ammonia water solution. The mixture was vigorously stirred at room temperature for 3 days. The solution was extracted with dichloromethane for four times. The extract was dried with anhydrous magnesium sulfate and filtrated. The solvent was removed by rotary evaporator. The crude product was recrystallized in ethanol twice. The purified product was collected and dried in vacuum (yield = 60%). 2.4. Synthesis of pH-sensitive mPEG-pH-PCL mPEG-NH2 (5.0 g, 2.5 mmol), citraconic anhydride (1.12 g, 10 mmol) and pyridine (0.79 g, 10 mmol) were dissolved in 50 mL of anhydrous CH2Cl2, the reaction was carried out at room temperature for 24 h with vigorous stirring. After the solvent was evaporated using a rotary evaporator, the residue was dissolved in methylene chloride and precipitated in cold diethyl ether. The product was simplified mPEG-NH-CA-COOH. PCL was synthesized via ring opening polymerization (ROP) of e-caprolactone (e-CL) using Sn(Oct)2 (mass ratio 1/1000) as
Scheme 1. The illustration of drug release from pH-sensitive mPEG-pH-PCL micelles.
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catalyst and benzyl alcohol as initiator. e-CL (22.8 g, 0.2 mol) and benzyl alcohol (1.23 g, 11 mmol) were added into a dry polymerization tube, Sn(Oct)2 in anhydrous CH2Cl2 was added in the mixture. After purged with nitrogen for three times, the tube was sealed and put into an oil bath at 130 C for 48 h. The received PCL was dissolved in dichloromethane (CH2Cl2) and precipitated in cold diethyl ether.The white precipitated powder was filtered and vacuum-dried at 30 C (yield = 92.5%). mPEG-NH-CA-COOH (0.34 g, 0.17 mmol), DMAP (0.02 g, 0.18 mmol) and PCL (0.3 g, 0.68 mmol) were dissolved in 15 mL of anhydrous CH2Cl2 in an ice bath under nitrogen atmosphere. A solution of DCC (0.70 g, 3.39 mmol) in CH2Cl2 (10 mL) was added dropwise into the mixture. The mixture was stirred at room temperature for 48 h. The white solid dicyclohexylurea (DCU) precipitate was removed by filtration, the filtrate was concentrated and precipitated in cold diethyl ether. The product was dialyzed (Spectra/Por MWCO = 3500) for three days. The outer phase was replaced with fresh deionized water every 4 h. The solution in the tubing was freeze-dried to receive mPEG-pH-PCL (yield = 70%). 2.5. Critical micelle concentration (CMC) The CMC of the copolymers was determined by fluorescence spectra (F-7000, Hitachi Co., Japan) using pyrene as a probe. The solution concentrations of the micelles varied from 1.25 104 to 0.1 mg/mL1. The final pyrene concentration was 6 107 M. The fluorescence emission wavelength was fixed at 390 nm. The excitation spectrum from 300 to 360 nm was tested, the emission fluorescence at 334 and 336 nm was monitored. The CMC was estimated as the cross-point of extrapolating the intensity ratios I336/I334 at low and high concentration regions. 2.6. Preparation of drug loaded micelles The DOX loaded mPEG-pH-PCL micelles were prepared using dialysis approach (Cheon Lee et al., 2003; Kataoka et al., 2012). mPEG-pH-PCL (10 mg) and DOX (2.5 mg) were dissolved in 1 mL of DMSO. This solution was stirred at room temperature for 3 h before dropped into10 mL of deionized water with vigorous stirring. The solution was transferred to a dialysis tubing (Spectra/Por MWCO = 1000) and dialyzed against deionized water at 4 C for 24 h. The outer phase was replaced with fresh deionized water every 4 h. The solution in the tubing was lyophilized after centrifugation. The whole procedure was performed in the dark. The content of encapsulated DOX was determined by fluorescence measurement (excitation wavelength at 480 nm) in DMSO using calibration curve obtained from DOX/DMSO solutions with different DOX concentrations. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formulas: DLC ðwt%Þ ¼
mass of DOX in the micelles 100% mass of DOX loaded micelles
(1)
DLE ðwt%Þ ¼
the amount of DOX in micelles 100% the amount of DOX in feed
(2)
2.7. The morphology of micelles Atomic force microscopy (AFM, MFP-3D-BIO) was employed to observe the morphology of polymeric micelles. The AFM samples were prepared by casting a dilute micelle solution (0.05 mg/mL) on a mica substrate and dried. The AFM images were recorded with a Nanoscope III from Digital Instruments operated in the tapping mode in air using micro fabricated Si (type NCH) cantilevers with the spring constant between 27 and 53 N m1, the resonance frequency was in the range of 300–365 kHz.
2.8. Hydrodynamic size analysis The micelles were dispersed in pure water or PBS solution with different pH values, the particle size of the micelles were measured by Zetasizer Nan-ZS (Malvern Instrument Ltd., Malvern, UK) equipped with a He–Ne laser beam at 633 nm (scattering angle: 90 ). Each sample was filtered through a 450 nm syringe filter before analysis. The average size of the three measurements was recorded. 2.9. Drug release profile test The drug release of DOX loaded micelles was investigated by dialysis method. Briefly, the lyophilized DOX loaded micelles were dispersed in 1 mL of buffer solution (pH 7.4 and 5.5, ionic strength = 0.01 M). The solutions were put in dialysis tubings (Spectra/Por MWCO 1000). The dialysis tubings were immersed in vials containing 25 mL of buffer solution with different pH values (pH 7.4 and 5.5, ionic strength = 0.01 M). The vials were put in a shaking bed at 37 C with the shaking rate of 150 rpm. 1 mL of medium with released drug was taken out at predetermined time intervals and the same volumes of fresh media were added into the vials. The released DOX was detected by a fluorescence detector with an excitation wavelength at 480 nm and emission wavelength at 550 nm. 2.10. In vitro toxicity evaluation HepG2 cells were cultured in DMEM medium, 4T1 cells were cultured in RMPI 1640 medium. The media were supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 C in a humidified atmosphere with 5% CO2. The cells were harvested with 0.02% EDTA and 0.025% trypsin and rinsed. The cell suspension was used in the following experiments. The cytotoxicity of blank micelles was tested by Kit-8 assay (CCK8, Dojindo, Japan) against HepG2 cells. HepG2 cells were seeded in 96-well plates at 5 103 cells per well with 100 mL of DMEM. After 24 h incubation, the culture medium was removed and replaced with 100 mL of medium containing blank micelles. The cells were incubated for another 48 h. The culture medium was removed and the wells were rinsed with PBS (pH 7.4). 100 mL of CCK-8 (volume fraction 10%) solution in DMEM was added to each well. After incubated for 2 h, the absorbance was measured at a Thermo Scientific MK3 (Thermo Fisher, USA) at the wavelength of 450 nm. To study the cellular uptake, HepG2 and 4T1 cells at a logarithm phase were seeded on glass dishes (diameter = 35 mm) at acell density of 1 104 mL1. After incubated for 24 h, DOX loaded micelles were dissolved and diluted in PBS until the final DOX concentration was 5 mg/mL. The mixture was added into the glass dishes. After incubated at 37 C for 4 h, the medium was removed and the dishes were rinsed with PBS (pH 7.4). The cell nuclei were stained with DAPI and the culture medium was replaced with PBS. The cells were observed using confocal laser scanning microscopy (CLSM, Leica TCP SP5). DOX was excited at 480 nm with emission at 590 nm. For the flow cytometry tests, the HepG2 and 4T1 cells were seeded in 6-well plates at a density of 1 106 cells per well and incubated for 24 h, respectively. The cells were treated with DOX loaded micelles at the DOX concentration of 5 mg/mL for 3 h. The culture medium was eliminated, the cells were washed with PBS for three times and harvested by trypsinization. The cells were resuspended in PBS after centrifugation (1000 rpm, 5 min) and the fluorescence intensity was measured (excitation: 480 nm, emission: 590 nm) on a BD FACS Calibur flow cytometer (Beckton Dickinson). The in vitro anticancer activity was carried out in both HepG2 and 4T1 cells. The cells were harvested and seeded in 96-well
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Scheme 2. The synthetic routes of mPEG-pH-PCL (A) and mPEG-CA-PCL copolymers (B).
plates with 100 mL of medium and incubated for 24 h before the test. DOX loaded micelles in culture media were added to the medium-removed 96-well plates with different DOX concentrations (from 10 to 0.0001 mg/mL) and incubated for 48 h. The culture medium was removed and the wells were rinsed with PBS (pH 7.4). CCK8 in PBS (5 mg/mL, 10 mL) was added to each well. After the cells were incubated for additional 4 h, the medium was removed carefully, 100 mL of DMSO was added in each well. The absorbance was measured in a Thermo Scientific MK3 at the wavelength of 492 nm. 3. Results and discussion The synthesis of pH-sensitive mPEG-pH-PCL copolymer was presented in Scheme 2. In order to introduce pH-sensitive citraconic amide bond in the block copolymer, the terminal hydroxyl group in mPEG block was transferred to amino group. Citraconic anhydride was used as linker to couple mPEG and PCL blocks. The non-pH-sensitive mPEG-CA-PCL copolymer using citraconic anhydride to link mPEG and PCL blocks with both ester bonds was synthesized as control for the drug delivery study. The structure of the mPEG-pH-PCL copolymer was characterized by 1H NMR, the spectrum was presented in Fig. 1. In Fig. 1a, the amino group transition was clearly demonstrated as the CH2 neighboured to NH2 was upfield shifted from d = 3.6 to 2.9 ppm, the intensity calculation of peak c revealed that nearly all the
hydroxyl groups in mPEG blocks were tranformed into amino groups. The 1H NMR spectrum of citraconic anhydride modified mPEG was showed in Fig. 1b, the proton signals (e and f) in citraconic moiety were detected. The PCL block was synthesized via the ring opening polymerization with benzyl alcohol as initiator, the protons in the benzyl ring and CH2 were found in Fig. 1c. After the couple reaction, all the protons in both mPEG and PCL blocks were detected in the spectrum of Fig. 1d. This result implied that the two blocks were coupled via citraconic moiety. The 1H NMR spectrum and protons assignments of the non-pHsensitive mPEG-CA-PCL copolymer were in Fig. S1 in Supporting Information (SI). The couple reaction was further monitored by gel permeation chromotography (GPC) and mass spectroscopy (MS). In the GPC spectrum (Fig. S2 in SI), a main peak with high molecular weight of the diblock copolymer was detected, the weak peak with longer eluent time revealed that slight amount of mPEG and PCL blocks was not reacted. MS spectra clearly exhibited the increase of molecular weight, which revealed the success of the copolymer synthesis (Fig. S3 in SI). The molecular weights of the two mPEG-pH-PCL and mPEG-CAPCL copolymers calculated via GPC, 1H NMR and MS were summarized in Table 1. The GPC results were relative molecular weights to the standard samples of polystyenes (PS), they were the highest, the Mns of mPEG-pH-PCL and mPEG-CA-PCL copolymers were 4400 and 4600, respectively. Though the values of the three
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Fig. 1. The 1H NMR spectra of pH-sensitive mPEG-pH-PCLcopolymer.
molecular weights of the two copolymers were different, they were located in a narrow range around 4000, which was fitted to the theatrical designed molecular weights. The diblock copolymers self-assembled into micelles in aqueous medium, the critical micelle concentration (CMC), which indicated the stability of polymeric micelles, was tested. The CMCs of mPEG-pH-PCL and mPEG-CA-PCL micelles were 2.1 103 and 4.0 103 mg/mL, respectively. The fraction ratio of hydrophilic segment to hydrophobic segment was an important parameter to affect the CMC (Alexandridis et al., 1994; Ding et al., 2010). As the fraction of both hydrophobic and hydrophilic segments of the two copolymers was nearly the same, the CMCs of the two micelles were comparative. Anticancer drug doxorubicin was encapsulated in the two micelles, both drug loading content (DLC) and drug loading efficiency (DLE) were measured, the DLCs of mPEG-pH-PCL and
mPEG-CA-PCL micelles were 6.8% and 5.9%, and the corresponding DLEs were 54.3% and 48.6%. Both DLC and DLE of mPEG-pH-PCL micelles were a little higher than those of mPEG-CA-PCL micelles, which was probably related to the PDI of polymers as shown in Table 1. In order to evaluate the pH-sensitivity of the micelles, both blank mPEG-pH-PCL and mPEG-CA-PCL micelles were soaked in media with different pH values of 7.4 and 5.5. The mean size and size distribution of the micelles were tested by DLS. Both size and size distribution of mPEG-CA-PCL micelles varied little in the two
Table 1 The molecular weight of copolymers and parameters of micelles. Sample
mPEG-pHPCL mPEG-CAPCL a b c
Mnb
Molecular weighta
Mnc
CMC (mg/mL)
DLC (%)
DLE (%)
6.8
54.3
4600 8200 1.78 4040 4246 4.0 103 5.9
48.6
Mn
Mw
4400
7400 1.68 4060 4231 2.1 103
PDI
GPC results. Calculated from H NMR spectra. MS results.
Fig. 2. The mean size variations of mPEG-pH-PCL micelles in different pH values.
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Fig. 3. The AFM images of blank and drug loaded micelles, (a) blank mPEG-pH-PCL micelles; a’: DOX loaded mPEG-pH-PCL micelles; (b) blank mPEG-CA-PCL micelles; b’: DOX loaded mPEG-CA-PCL micelles.
media. When the mPEG-pH-PCL micelles were soaking in aqueous solution with pH 7.4, their mean sizes kept at around 120 nm even when the stored time was as long as 24 h. The size and size distribution of pH-sensitive mPEG-pH-PCL micelles in pH 5.5 were changed greatly (Fig. 2 and Fig. S4 in SI), it increased continuously from about 110–220 nm when the soaking time was 24 h. As the size tested by DLS was the hydrodynamic diameter of polymeric
micelles, it implied that the citraconic bonds in the backbone of mPEG-pH-PCL were broken and thus resulted in the dissociation of micelles to enlarge the hydrodynamic diameter. The PDI variation in Fig. S4 also supported the conclusion of micellar dissociation. Count rate is a parameter that represents the light scattering intensity of the sample which displays the number of photons detected per second (displayed in kcps-kilo counts per second)
Fig. 4. The release profiles of mPEG-pH-PCL and mPEG-CA-PCL micelles in different pH values.
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Fig. 5. The cytoxicity of blank mPEG-pH-PCL and mPEG-CA-PCL micelles incubated with HepG2 cells for 48 h.
(Xiong et al., 2012). The count rates of the mPEG-pH-PCL and mPEGCA-PCL micelles were measured in different soaking time. The count rates of mPEG-pH-PCL micelles decreased with increasing soaking time in both pH 7.4 and 5.5, and the count rate in pH 5.5 decreased very fast. The count rates of mPEG-CA-PCL micelles in pH 7.4 and 5.5 were nearly kept in a stable state (Fig. S5). These results indicated the pH-sensitivity of mPEG-pH-PCL micelles. The morphology of both blank and DOX loaded mPEG-pH-PCL and mPEG-CA-PCL micelles were observed by AFM (Fig. 3). The size
of blank mPEG-pH-PCL micelles was around 100 nm, which was smaller than the size tested by DLS. It was reasonable because the micelle in AFM was in dry state, which was smaller than the hydrodynamic diameter tested by DLS. After DOX was loaded in mPEG-pH-PCL micelles, the size was increased to about 150 nm. In the AFM image of mPEG-CA-PCL micelles, the size of micelles were not homogenous, aggregates with hundreds of nanometers were observed, however, the aggregates disappeared in the DOX loaded mPEG-CA-PCL micelles image. It was interesting that the dispersity of both blank micelles became better when the DOX was loaded in the micelles. It was probably attributed to the hydrophobic interaction between DOX and PCL blocks in micelles, as both PEG and PCL blocks with the molecular weight of 2000 were elastic polymers with low glass transition temperatures and crystallinity (Loh et al., 2008), the loaded hydrophobic DOX increased the hydrophobic interaction to compress the cores of micelles, thus, the aggregation of the micelles was prevented and the micelles were well dispersed. The release profiles of the two DOX loaded micelles were studied (Fig. 4). The pH values of the media were 7.4 and 5.5. Both drug loaded micelles exhibited burst release. To pH-sensitive mPEG-pH-PCL micelles, the drug release was much faster in pH 5.5 than that in pH 7.4. The encapsulated drug was released from mPEG-pH-PCL micelles and the cumulated release rate was as high as 80% within 90 h in pH 5.5. The cumulated release rate in pH 7.4 was only 40%. To mPEG-CA-PCL micelles, the drug release rate in pH 5.5 was also faster than that in pH 7.4, however, the cumulated release rate in pH 5.5 was much lower than that of mPEG-pH-PCL micelles in pH 5.5, it was less than 60%. The drug release rates of mPEG-pH-PCL and mPEG-CA-PCL micelles in pH 7.4 were
Fig. 6. Confocal microscopy images of HepG2 and 4T1 cells treated with DOX loaded micelles for 3 h, (A) mPEG-CA-PCL micelles; (B) mPEG-pH-PCL micelles; (C) DOXHCl. The DOX concentration of drug loaded micelles in A and B was 5 mg/mL, the concentration for DOXHCl in C was 10 mg/mL. The images from top to bottom were cells in bright field (1), DOX fluorescence in cells (2), cell nuclei stained by DAPI (3), and overlay images (4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. The flow cytometry results of HepG2 (a) and 4T1 (b) cells treated with drug loaded micelles for 3 h, (A) DOX loaded mPEG-CA-PCL micelles; (B) DOX loaded mPEG-pHPCL micelles; (C) DOXHCl. The DOX concentration of the micelle samples was 5 mg/mL, and the concentration of DOXHCl was10 mg/mL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
comparative. The fast release of mPEG-CA-PCL micelles in pH 5.5 was because of the protonation of doxorubicin in acid medium, which changed the DOX from hydrophobic to hydrophilic and the diffusion capacity of the protonated drug was largely strengthened. Comparing the two release profiles of drug loaded mPEG-pH-PCL and mPEG-CA-PCL micelles in pH 5.5, it could be concluded that
Fig. 8. The in vitro anticancer activity of drug loaded micelles, the calculated IC50s of DOX loaded mPEG-pH-PCL and mPEG-CA-PCL micelles were 0.695 mg/mL and 1.126 mg/mL to HepG2 cells (A), 3.922 mg/mL and 4.769 mg/mL to 4T1 cells (B).
the broken of citraconic amide bond in weak acidity accelerated the release of DOX. The two micelles were incubated with HepG2 cells to evaluate their cytotoxicity (Fig. 5). The concentrations of the micelles were ranged from 0.01 to 0.2 mg/mL. The cell viability of mPEG-pH-PCL micelles increased with the decrease of concentration, and that of mPEG-CA-PCL micelles was kept in nearly a same level. The cell viability for each sample was higher than 90%, which indicated that the two micelles were non-toxic to HepG2 cells. Cellular internalization is the key step for drug loaded micelles to fulfill their anticancer activity. In order to investigate the cellular uptake of DOX loaded micelles, the two drug loaded micelles were co-cultured with both HepG2 and 4T1 cells. Confocal laser microscopy and flow cytometry were used to study the cellular uptake of the two micelles. In confocal microscopy images (Fig. 6), both drug loaded mPEG-pH-PCL and mPEG-CA-PCL micelles could be internalized in both HepG2 and 4T1 cells. Strong red fluorescence of DOX was observed in cytoplasm, weak red fluorescence was observed in nuclei, it implied that most of the drug loaded micelles located in cytoplasm, the released DOX could diffuse into nuclei. The red fluorescence in cells treated with drug loaded mPEG-pH-PCL micelles was relatively stronger than that of mPEG-CA-PCL micelles. As a hydrophilic drug, DOXHCl diffused much faster in cells, thus, strong red fluorescence was observed in nuclei of cells treated with DOXHCl. The quantitative fluorescence intensity in the cells was measured by flow cytometry (Fig. 7). It was clear that the red fluorescence of cells treated with DOX loaded mPEG-pH-PCL micelles was stronger than that of cells treated with DOX loaded mPEG-CA-PCL micelles. It revealed that the pHsensitivity functionalized mPEG-pH-PCL micelles promoted the anticancer efficiency of DOX. The in vitro anticancer activity of the two drug loaded micelles was characterized by the half maximal inhibitory concentration (IC50) to HepG2 and 4T1 cells. The results were presented in Fig. 8. The calculated IC50s of DOX loaded pH-sensitive mPEG-pH-PCL micelles to HepG2 and 4T1 cells were 0.695 mg/mL and 3.922 mg/ mL, and those of DOX loaded mPEG-CA-PCL micelles were 1.126 mg/mL and 4.769 mg/mL. Both the IC50s of mPEG-pH-PCL micelles to HepG2 and 4T1 cells were lower than those of corresponding mPEG-CA-PCL micelles. The DOX encapsulated in mPEG-pH-PCL micelles exhibited better anticancer activity due to the pH-sensitivity. The IC50s of the two micelles to 4T1 cells were both higher than those to HepG2 cells, it implied that the anticancer efficiency of the drug loaded micelles was related to the types of cancer cells.
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4. Conclusion A novel pH-sensitive mPEG-pH-PCL copolymer micelle was synthesized, weak acid cleavable citraconic amide bond was introduced in the copolymer to receive pH-sensitivity. The characterizations of 1H NMR, GPC and MS demonstrated the successful synthesis of the diblock copolymers. The pH-sensitivity investigation showed that the citraconic amide bonds in the micelles were broken in pH 5.5 to result in size and size distribution enlargement of micelles. The drug loaded micelles exhibited excellent dispersion capability in aqueous medium with the mean size around 150 nm. The release of DOX loaded mPEGpH-PCL micelles was pH dependent, the release rate in pH 5.5 was much faster than that in pH 7.4. The confocal laser microscopy and flow cytometry measurements indicated that the weak acidity in endosomes could trigger the release of DOX efficiently. The in vitro IC50 tested revealed that the anticancer activity of DOX trapped in the pH-sensitive mPEG-pH-PCL micelles was enhanced. The introduction of citraconic amide in the copolymer backbone was a favorable strategy to fabricate pH-sensitive micellar drug delivery systems. Acknowledgements This research work was supported by National Science Foundation for Excellent Young Scholars (No. 51222304), Young teachers’ scientific research foundation of Sichuan University (2014SCU11015), National Science Foundation of China (NSFC, No. 31170921, 31370972, 51133004, 81361140343), National Basic Research Program of China (National 973 program, No. 2011CB606206), Program for Changjiang Scholars and Innovative Research Team in University (IRT1163). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.05.010. References Alexandridis, P., Holzwarth, J.F., Hatton, T.A., 1994. Micellization of poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymers in aqueous solutions: thermodynamics of copolymer association. Macromolecules 27, 2414–2425. Bae, Y., Fukushima, S., Harada, A., Kataoka, K., 2003. Design of environmentsensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change. Angew. Chem. Int. Ed. 42, 4640–4643. Bae, Y., Nishiyama, N., Kataoka, K., 2007. In vivo antitumor activity of the folateconjugated pH-sensitive polymeric micelle selectively releasing adriamycin in the intracellular acidic compartments. Bioconjugate Chem. 18, 1131–1139. Cao, J., Zhai, S., Li, C., He, B., Lai, Y., Chen, Y., Luo, X., Gu, Z., 2013. Novel pH-sensitive micelles generated by star-shape copolymers containing zwitterionic
sulfobetaine for efficientcellular internalization. J. Biomed. Nanotechnol. 9, 1847–1861. Cheon Lee, S., Kim, C., Chan Kwon, I., Chung, H., Young Jeong, S., 2003. Polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(e-caprolactone) copolymer as a carrier for paclitaxel. J. Control. Release 89, 437–446. Deng, C., Jiang, Y., Cheng, R., Meng, F., Zhong, Z., 2012. Biodegradable polymeric micelles for targeted and controlled anticancer drug delivery: promises, progress and prospects. Nano Today 7, 467–480. Ding, M., Zhou, L., Fu, X., Tan, H., Li, J., Fu, Q., 2010. Biodegradable gemini multiblock poly([varepsilon]-caprolactone urethane)s toward controllable micellization. Soft Matter 6, 2087–2092. Duncan, R., 2003. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2, 347–360. Gillies, E.R., Fréchet, J.M.J., 2005. pH-Responsive copolymer assemblies for controlled release of doxorubicin. Bioconjugate Chem. 16, 361–368. Gu, G., Xia, H., Hu, Q., Liu, Z., Jiang, M., Kang, T., Miao, D., Tu, Y., Pang, Z., Song, Q., Yao, L., Chen, H., Gao, X., Chen, J., 2013. PEG-co-PCL nanoparticles modified with MMP-2/9 activatable low molecular weight protamine for enhanced targeted glioblastoma therapy. Biomaterials 34, 196–208. Kataoka, K., Harada, A., Nagasaki, Y., 2012. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Deliv. Rev. 64, 37–48. Kwon, G.S., Okano, T., 1996. Polymeric micelles as new drug carriers. Adv. Drug Deliv. Rev. 21, 107–116. Liu, R., Li, D., He, B., Xu, X., Sheng, M., Lai, Y., Wang, G., Gu, Z., 2011. Anti-tumor drug delivery of pH-sensitive poly(ethylene glycol)–poly(L-histidine-)–poly(L-lactide) nanoparticles. J. Control. Release 152, 49–56. Loh, X.J., Colin Sng, K.B., Li, J., 2008. Synthesis and water-swelling of thermoresponsive poly(ester urethane)s containing poly(e-caprolactone), poly(ethylene glycol) and poly(propylene glycol). Biomaterials 29, 3185–3194. Otsuka, H., Nagasaki, Y., Kataoka, K., 2012. PEGylated nanoparticles for biological and pharmaceutical applications. Adv. Drug Deliv. Rev. 64, 246–255. Prata, C.A.H., Zhao, Y., Barthelemy, P., Li, Y., Luo, D., McIntosh, T.J., Lee, S.J., Grinstaff, M.W., 2004. Charge-reversal amphiphiles for gene delivery. J. Am. Chem. Soc. 126, 12196–12197. Pu, Y., Chang, S., Yuan, H., Wang, G., He, B., Gu, Z., 2013. The anti-tumor efficiency of poly(L-glutamic acid) dendrimers with polyhedral oligomeric silsesquioxane cores. Biomaterials 34, 3658–3666. Pu, Y., Zhang, L., Zheng, H., He, B., Gu, Z., 2014. Drug release of pH-sensitive poly (Laspartate)-b-poly(ethylene glycol) micelles with POSS cores. Polym. Chem. 5, 463–470. Xin, H., Sha, X., Jiang, X., Zhang, W., Chen, L., Fang, X., 2012. Anti-glioblastoma efficacy and safety of paclitaxel-loading Angiopep-conjugated dual targeting PEG–PCL nanoparticles. Biomaterials 33, 8167–8176. Xiong, M.-H., Bao, Y., Yang, X.-Z., Wang, Y.-C., Sun, B., Wang, J., 2012. Lipase-sensitive polymeric triple-layered nanogel for “on-demand” drug delivery. J. Am. Chem. Soc. 134, 4355–4362. Xu, P., VanKirk, E.A., Zhan, Y., Murdoch, W.J., Radosz, M., Shen, Y., 2007. Targeted charge-reversal nanoparticles for nuclear drug delivery. Angew. Chem. Int. Ed. 46, 4999–5002. Xue, B., Wang, Y., Tang, X., Xie, P., Wang, Y., Luo, F., Wu, C., Qian, Z., 2012. Biodegradable self-assembled MPEG-PCL micelles for hydrophobic oridonin delivery in vitro. J. Biomed. Nanotechnol. 8, 80–89. Yang, Q., Liao, J., Deng, X., Liang, J., Long, C., Xie, C., Chen, X., Zhang, L., Sun, J., Peng, J., Chu, B., Guo, G., Luo, F., Qian, Z., 2014. Anti-tumor activity and safety evaluation of fisetin-loaded methoxy poly(ethylene glycol)-b-poly(e-caprolactone) nanoparticles. J. Biomed. Nanotechnol. 10, 580–591. Yuan, Y.-Y., Mao, C.-Q., Du, X.-J., Du, J.-Z., Wang, F., Wang, J., 2012. Surface charge switchable nanoparticles based on zwitterionic polymer for enhanced drug delivery to tumor. Adv. Mater. 24, 5476–5480. Zhang, J., Men, K., Gu, Y., Wang, X., Gou, M., Guo, G., Luo, F., Qian, Z., 2011. Preparation of core cross-linked PCL-PEG-PCL micelles for doxorubicin delivery in vitro. J. Nanosci. Nanotechnol. 11, 5054–5061. Zhou, Z., Shen, Y., Tang, J., Fan, M., Van Kirk, E.A., Murdoch, W.J., Radosz, M., 2009. Charge-reversal drug conjugate for targeted cancer cell nuclear drug delivery. Adv. Funct. Mater. 19, 3580–3589.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Polymeric micelles with citraconic amide as pH-sensitive bond in backbone for anticancer drug delivery Jun Cao a , Ting Su a , Longgui Zhang a , Rong Liu b , Gang Wang a, * , Bin He a , Zhongwei Gu a a b
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064 China College of Medical and Nursing,Chengdu University, Chengdu 610106 China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2014 Received in revised form 23 April 2014 Accepted 4 May 2014 Available online 13 May 2014
A novel pH-sensitive polymeric micelle was reported. Methoxy poly(ethylene glycol)-b-poly(e-caprolactone) copolymer with citraconic amide as pH-sensitive bond was synthesized (mPEG-pH-PCL). The copolymers self-assembled into micelles to encapsulate anticancer drug doxorubicin (DOX). The morphology, size and size distribution, drug release profile and in vitro anticancer activity of the DOX loaded mPEG-pH-PCL micelles were studied. The results showed that the mean size of the micelles was around 120 nm, the drug loading content and encapsulation efficiency of the mPEG-pH-PCL micelles were 6.8% and 54.3%, respectively. The mean diameter and size distribution of the mPEG-pH-PCL micelles increased significantly when soaking in medium with pH 5.5. The drug release of micelles in pH 5.5 was much faster than that in pH 7.4. The confocal laser microscopy and flow cytometry measurements indicated that the weak acidity of endosomes broke the citraconic amide bonds in the copolymer backbones and triggered the fast release of DOX. The in vitro IC50 of the drug loaded mPEG-pH-PCL micelles was lower than that of drug loaded polymeric micelles without pH-sensitivity to both HepG2 and 4T1 cancer cells. ã 2014 Published by Elsevier B.V.
Keywords: pH-sensitive Citraconic anhydride Polymeric micelle Drug delivery Doxorubicin
1. Introduction Biodegradable polymeric micelles are important carriers for anticancer drug delivery due to their excellent biocompatibility, biodegradability and long circulation in blood vessel transportation (Deng et al., 2012; Duncan, 2003; Kwon and Okano, 1996; Otsuka et al., 2012). A paclitaxel loaded poly(lactic acid)–poly (ethylene glycol) (PLA–PEG) copolymer micelle has been approved by FDA for cancer treatment, polyester-PEG micelles have attracted much interest to biomaterials scientists and pharmacists. Poly(e-caprolactone) (PCL) is a biodegradable polyester with low glass transition temperature (Tg) and melting point (Tm), the diffusion of hydrophobic anticancer drugs will be faster in the elastic PCL substrate, thus, PEG–PCL block copolymer micelles are extensively studied for anticancer drug delivery (Gu et al., 2013; Xin et al., 2012; Xue et al., 2012; Yang et al., 2014; Zhang et al., 2011). The PEG–PCL micelles are core-shell structured nanoparticles, hydrophobic anticancer drugs are loaded in the PCL cores. The drug release will last for several days, however, the low concentration of
* Corresponding author. Tel.: +86 28 85412923. E-mail address: [email protected] (G. Wang). http://dx.doi.org/10.1016/j.ijpharm.2014.05.010 0378-5173/ ã 2014 Published by Elsevier B.V.
released drug will not kill cancer cells efficiently, and it also has the risk to evoke multidrug resistance in cancer cells. Many pHsensitive polymeric micelles were designed and synthesized to resolve this problem (Bae et al., 2003, 2007; Cao et al., 2013), the drug release was triggered by the weak acidity of intracellular endosomes and/or lysomes and rapidly escaped from micelles. Two strategies were reported to fabricate pH-sensitive polymeric micelles. Introduction of imidazole groups in polymeric micelles was a practical approach, the imidazole groups were protonized in the medium with pH around 6.0, and the protonation transferred imidazole groups from hydrophobic to hydrophilic to swell the polymeric micelles to accelerate the release of drug. The molecules with imidazole groups such as L-histidine and 1-(3-aminopropyl) imidazole have been used to achieve the pH-sensitivity in polymeric micelles (Liu et al., 2011; Pu et al., 2014). Utilization of pH-cleavable hydrazone bond was another strategy to prepare pH-sensitive drug delivery systems (Pu et al., 2013). Anticancer drug was immobilized on nanocarriers via hydrazone bond to form drug-carrier conjugates, the hydrazone bonds were broken in endosomes and/or lysosomes to release the immobilized drug. Acetal bond was also a pH-sensitive bond introduced in polymeric micelles, the broken of acetal bond in acidic environment would change the internal structure of polymeric micelles to release the encapsulated drug (Gillies and Fréchet, 2005).
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Recently, a new pH-sensitive bond of citraconic amide was reported in drug/gene delivery system, which was called chargereversible delivery system (Prata et al., 2004; Xu et al., 2007; Yuan et al., 2012; Zhou et al., 2009). In this system, citraconic anhydride was reacted with amino group in nanoparticles to change the surface charge from positive to negative. The amide bond was broken in weak acidic condition in tumor tissues, the surface charge of nanoparticles was changed to positive and promoted the cellular uptake via the electrostatic interaction between positively charged nanoparticles and negatively charged cell membranes. As lack of functional groups for modification, PEG–PCL block copolymers were rarely reported to fabricate pH-sensitive drug carriers. Herein, we reported a novel pH-sensitive mPEG-PCL micelle. The hydroxyl terminal group of PEG was transferred into amino group, citraconic anhydride was used as a linker to connect PEG and PCL blocks, the pH-sensitive citraconic amide bond was formed between PEG and PCL blocks. Anticancer drug doxorubicin was encapsulated in the mPEG-pH-PCL micelles. Once the drug loaded micelles were internalized in cancer cells, the weak acidity in endosomes broke the citraconic amide bond to release the loaded drug (Scheme 1).
2. Materials and methods 2.1. Materials N,N-Dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), CDCl3, citraconic anhydride (CA), phenylmethanesulfonyl chloride (TsCl), e-caprolactone (CL) and methoxy-poly (ethylene glycol) (mPEG-OH, Mn = 2000 g/mol) were purchased from Sigma–Aldrich Co. (Steinheim, Germany) and used as received. Doxorubicin hydrochloride (DOXHCl, Shanghai Yingxuan Chempharm Co., Ltd., China) was dissolved in water, the pH value was adjusted to 9.6 to prepare doxorubicin. Benzyl alcohol, phosphate buffer saline (PBS) solution (pH 7.4, I = 0.01 M) containing disodium hydrogen phosphate (NaH2PO4), potassium dihydrogen phosphate (K2HPO4), sodium chloride (NaCl), potassium chloride (KCl) and sodium bicarbonate (NaHCO3) were purchased from Changzheng Chemical Co. (China). Sodium citrate buffer solution (CS) (pH 5.5) was composed of citric acid and sodium citrate. Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS) and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich Co. (Steinheim, Germany) and
29
used for cells test. All the solvents were purchased from Kelong Chemical Co. (Chengdu, China) and used as received. 2.2. Measurements The solvent for 1H NMR measurement was CDCl3 containing 0.5% tetramethylsilane as the internal standard. The 1H NMR spectra were recorded on Bruker AVANCE-400 MHz NMR spectrometer, working at 400 MHz. The molecular weight (Mn and Mw) and molecular weight distribution (Mw/Mn) of the polymers were determined by gel permeation chromatography (GPC, Agilent 1100 Series) and analyzed with GPC–SEC data analysis software. The samples were analyzed at 25 C with CDCl3 as eluent at a flow rate of 1.0 mL/min. The MS spectra were performed on ESI-TOF MS (Waters Q-Tof Premier). 2.3. Synthesis of mPEG-NH2 Methoxy poly(ethylene glycol) (10 g, 5 mmol) was dissolved in anhydrous dichloromethane (150 mL) and cooled in an ice-water bath. Pyridine (1.58 g, 20 mmol) and TsCl (4.77 g, 25 mmol) in 50 mL of CH2Cl2 were added dropwise under stirring. The solution was stirred at 0 C for 8 h and further at room temperature for 4 days under nitrogen atmosphere. The mixture was filtrated, concentrated, and precipitated in excess cold diethyl ether. The product was recrystallized in ethanol, collected and dried in vacuum. The purified product was added to 100 mL of 25 wt% ammonia water solution. The mixture was vigorously stirred at room temperature for 3 days. The solution was extracted with dichloromethane for four times. The extract was dried with anhydrous magnesium sulfate and filtrated. The solvent was removed by rotary evaporator. The crude product was recrystallized in ethanol twice. The purified product was collected and dried in vacuum (yield = 60%). 2.4. Synthesis of pH-sensitive mPEG-pH-PCL mPEG-NH2 (5.0 g, 2.5 mmol), citraconic anhydride (1.12 g, 10 mmol) and pyridine (0.79 g, 10 mmol) were dissolved in 50 mL of anhydrous CH2Cl2, the reaction was carried out at room temperature for 24 h with vigorous stirring. After the solvent was evaporated using a rotary evaporator, the residue was dissolved in methylene chloride and precipitated in cold diethyl ether. The product was simplified mPEG-NH-CA-COOH. PCL was synthesized via ring opening polymerization (ROP) of e-caprolactone (e-CL) using Sn(Oct)2 (mass ratio 1/1000) as
Scheme 1. The illustration of drug release from pH-sensitive mPEG-pH-PCL micelles.
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catalyst and benzyl alcohol as initiator. e-CL (22.8 g, 0.2 mol) and benzyl alcohol (1.23 g, 11 mmol) were added into a dry polymerization tube, Sn(Oct)2 in anhydrous CH2Cl2 was added in the mixture. After purged with nitrogen for three times, the tube was sealed and put into an oil bath at 130 C for 48 h. The received PCL was dissolved in dichloromethane (CH2Cl2) and precipitated in cold diethyl ether.The white precipitated powder was filtered and vacuum-dried at 30 C (yield = 92.5%). mPEG-NH-CA-COOH (0.34 g, 0.17 mmol), DMAP (0.02 g, 0.18 mmol) and PCL (0.3 g, 0.68 mmol) were dissolved in 15 mL of anhydrous CH2Cl2 in an ice bath under nitrogen atmosphere. A solution of DCC (0.70 g, 3.39 mmol) in CH2Cl2 (10 mL) was added dropwise into the mixture. The mixture was stirred at room temperature for 48 h. The white solid dicyclohexylurea (DCU) precipitate was removed by filtration, the filtrate was concentrated and precipitated in cold diethyl ether. The product was dialyzed (Spectra/Por MWCO = 3500) for three days. The outer phase was replaced with fresh deionized water every 4 h. The solution in the tubing was freeze-dried to receive mPEG-pH-PCL (yield = 70%). 2.5. Critical micelle concentration (CMC) The CMC of the copolymers was determined by fluorescence spectra (F-7000, Hitachi Co., Japan) using pyrene as a probe. The solution concentrations of the micelles varied from 1.25 104 to 0.1 mg/mL1. The final pyrene concentration was 6 107 M. The fluorescence emission wavelength was fixed at 390 nm. The excitation spectrum from 300 to 360 nm was tested, the emission fluorescence at 334 and 336 nm was monitored. The CMC was estimated as the cross-point of extrapolating the intensity ratios I336/I334 at low and high concentration regions. 2.6. Preparation of drug loaded micelles The DOX loaded mPEG-pH-PCL micelles were prepared using dialysis approach (Cheon Lee et al., 2003; Kataoka et al., 2012). mPEG-pH-PCL (10 mg) and DOX (2.5 mg) were dissolved in 1 mL of DMSO. This solution was stirred at room temperature for 3 h before dropped into10 mL of deionized water with vigorous stirring. The solution was transferred to a dialysis tubing (Spectra/Por MWCO = 1000) and dialyzed against deionized water at 4 C for 24 h. The outer phase was replaced with fresh deionized water every 4 h. The solution in the tubing was lyophilized after centrifugation. The whole procedure was performed in the dark. The content of encapsulated DOX was determined by fluorescence measurement (excitation wavelength at 480 nm) in DMSO using calibration curve obtained from DOX/DMSO solutions with different DOX concentrations. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formulas: DLC ðwt%Þ ¼
mass of DOX in the micelles 100% mass of DOX loaded micelles
(1)
DLE ðwt%Þ ¼
the amount of DOX in micelles 100% the amount of DOX in feed
(2)
2.7. The morphology of micelles Atomic force microscopy (AFM, MFP-3D-BIO) was employed to observe the morphology of polymeric micelles. The AFM samples were prepared by casting a dilute micelle solution (0.05 mg/mL) on a mica substrate and dried. The AFM images were recorded with a Nanoscope III from Digital Instruments operated in the tapping mode in air using micro fabricated Si (type NCH) cantilevers with the spring constant between 27 and 53 N m1, the resonance frequency was in the range of 300–365 kHz.
2.8. Hydrodynamic size analysis The micelles were dispersed in pure water or PBS solution with different pH values, the particle size of the micelles were measured by Zetasizer Nan-ZS (Malvern Instrument Ltd., Malvern, UK) equipped with a He–Ne laser beam at 633 nm (scattering angle: 90 ). Each sample was filtered through a 450 nm syringe filter before analysis. The average size of the three measurements was recorded. 2.9. Drug release profile test The drug release of DOX loaded micelles was investigated by dialysis method. Briefly, the lyophilized DOX loaded micelles were dispersed in 1 mL of buffer solution (pH 7.4 and 5.5, ionic strength = 0.01 M). The solutions were put in dialysis tubings (Spectra/Por MWCO 1000). The dialysis tubings were immersed in vials containing 25 mL of buffer solution with different pH values (pH 7.4 and 5.5, ionic strength = 0.01 M). The vials were put in a shaking bed at 37 C with the shaking rate of 150 rpm. 1 mL of medium with released drug was taken out at predetermined time intervals and the same volumes of fresh media were added into the vials. The released DOX was detected by a fluorescence detector with an excitation wavelength at 480 nm and emission wavelength at 550 nm. 2.10. In vitro toxicity evaluation HepG2 cells were cultured in DMEM medium, 4T1 cells were cultured in RMPI 1640 medium. The media were supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 C in a humidified atmosphere with 5% CO2. The cells were harvested with 0.02% EDTA and 0.025% trypsin and rinsed. The cell suspension was used in the following experiments. The cytotoxicity of blank micelles was tested by Kit-8 assay (CCK8, Dojindo, Japan) against HepG2 cells. HepG2 cells were seeded in 96-well plates at 5 103 cells per well with 100 mL of DMEM. After 24 h incubation, the culture medium was removed and replaced with 100 mL of medium containing blank micelles. The cells were incubated for another 48 h. The culture medium was removed and the wells were rinsed with PBS (pH 7.4). 100 mL of CCK-8 (volume fraction 10%) solution in DMEM was added to each well. After incubated for 2 h, the absorbance was measured at a Thermo Scientific MK3 (Thermo Fisher, USA) at the wavelength of 450 nm. To study the cellular uptake, HepG2 and 4T1 cells at a logarithm phase were seeded on glass dishes (diameter = 35 mm) at acell density of 1 104 mL1. After incubated for 24 h, DOX loaded micelles were dissolved and diluted in PBS until the final DOX concentration was 5 mg/mL. The mixture was added into the glass dishes. After incubated at 37 C for 4 h, the medium was removed and the dishes were rinsed with PBS (pH 7.4). The cell nuclei were stained with DAPI and the culture medium was replaced with PBS. The cells were observed using confocal laser scanning microscopy (CLSM, Leica TCP SP5). DOX was excited at 480 nm with emission at 590 nm. For the flow cytometry tests, the HepG2 and 4T1 cells were seeded in 6-well plates at a density of 1 106 cells per well and incubated for 24 h, respectively. The cells were treated with DOX loaded micelles at the DOX concentration of 5 mg/mL for 3 h. The culture medium was eliminated, the cells were washed with PBS for three times and harvested by trypsinization. The cells were resuspended in PBS after centrifugation (1000 rpm, 5 min) and the fluorescence intensity was measured (excitation: 480 nm, emission: 590 nm) on a BD FACS Calibur flow cytometer (Beckton Dickinson). The in vitro anticancer activity was carried out in both HepG2 and 4T1 cells. The cells were harvested and seeded in 96-well
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Scheme 2. The synthetic routes of mPEG-pH-PCL (A) and mPEG-CA-PCL copolymers (B).
plates with 100 mL of medium and incubated for 24 h before the test. DOX loaded micelles in culture media were added to the medium-removed 96-well plates with different DOX concentrations (from 10 to 0.0001 mg/mL) and incubated for 48 h. The culture medium was removed and the wells were rinsed with PBS (pH 7.4). CCK8 in PBS (5 mg/mL, 10 mL) was added to each well. After the cells were incubated for additional 4 h, the medium was removed carefully, 100 mL of DMSO was added in each well. The absorbance was measured in a Thermo Scientific MK3 at the wavelength of 492 nm. 3. Results and discussion The synthesis of pH-sensitive mPEG-pH-PCL copolymer was presented in Scheme 2. In order to introduce pH-sensitive citraconic amide bond in the block copolymer, the terminal hydroxyl group in mPEG block was transferred to amino group. Citraconic anhydride was used as linker to couple mPEG and PCL blocks. The non-pH-sensitive mPEG-CA-PCL copolymer using citraconic anhydride to link mPEG and PCL blocks with both ester bonds was synthesized as control for the drug delivery study. The structure of the mPEG-pH-PCL copolymer was characterized by 1H NMR, the spectrum was presented in Fig. 1. In Fig. 1a, the amino group transition was clearly demonstrated as the CH2 neighboured to NH2 was upfield shifted from d = 3.6 to 2.9 ppm, the intensity calculation of peak c revealed that nearly all the
hydroxyl groups in mPEG blocks were tranformed into amino groups. The 1H NMR spectrum of citraconic anhydride modified mPEG was showed in Fig. 1b, the proton signals (e and f) in citraconic moiety were detected. The PCL block was synthesized via the ring opening polymerization with benzyl alcohol as initiator, the protons in the benzyl ring and CH2 were found in Fig. 1c. After the couple reaction, all the protons in both mPEG and PCL blocks were detected in the spectrum of Fig. 1d. This result implied that the two blocks were coupled via citraconic moiety. The 1H NMR spectrum and protons assignments of the non-pHsensitive mPEG-CA-PCL copolymer were in Fig. S1 in Supporting Information (SI). The couple reaction was further monitored by gel permeation chromotography (GPC) and mass spectroscopy (MS). In the GPC spectrum (Fig. S2 in SI), a main peak with high molecular weight of the diblock copolymer was detected, the weak peak with longer eluent time revealed that slight amount of mPEG and PCL blocks was not reacted. MS spectra clearly exhibited the increase of molecular weight, which revealed the success of the copolymer synthesis (Fig. S3 in SI). The molecular weights of the two mPEG-pH-PCL and mPEG-CAPCL copolymers calculated via GPC, 1H NMR and MS were summarized in Table 1. The GPC results were relative molecular weights to the standard samples of polystyenes (PS), they were the highest, the Mns of mPEG-pH-PCL and mPEG-CA-PCL copolymers were 4400 and 4600, respectively. Though the values of the three
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Fig. 1. The 1H NMR spectra of pH-sensitive mPEG-pH-PCLcopolymer.
molecular weights of the two copolymers were different, they were located in a narrow range around 4000, which was fitted to the theatrical designed molecular weights. The diblock copolymers self-assembled into micelles in aqueous medium, the critical micelle concentration (CMC), which indicated the stability of polymeric micelles, was tested. The CMCs of mPEG-pH-PCL and mPEG-CA-PCL micelles were 2.1 103 and 4.0 103 mg/mL, respectively. The fraction ratio of hydrophilic segment to hydrophobic segment was an important parameter to affect the CMC (Alexandridis et al., 1994; Ding et al., 2010). As the fraction of both hydrophobic and hydrophilic segments of the two copolymers was nearly the same, the CMCs of the two micelles were comparative. Anticancer drug doxorubicin was encapsulated in the two micelles, both drug loading content (DLC) and drug loading efficiency (DLE) were measured, the DLCs of mPEG-pH-PCL and
mPEG-CA-PCL micelles were 6.8% and 5.9%, and the corresponding DLEs were 54.3% and 48.6%. Both DLC and DLE of mPEG-pH-PCL micelles were a little higher than those of mPEG-CA-PCL micelles, which was probably related to the PDI of polymers as shown in Table 1. In order to evaluate the pH-sensitivity of the micelles, both blank mPEG-pH-PCL and mPEG-CA-PCL micelles were soaked in media with different pH values of 7.4 and 5.5. The mean size and size distribution of the micelles were tested by DLS. Both size and size distribution of mPEG-CA-PCL micelles varied little in the two
Table 1 The molecular weight of copolymers and parameters of micelles. Sample
mPEG-pHPCL mPEG-CAPCL a b c
Mnb
Molecular weighta
Mnc
CMC (mg/mL)
DLC (%)
DLE (%)
6.8
54.3
4600 8200 1.78 4040 4246 4.0 103 5.9
48.6
Mn
Mw
4400
7400 1.68 4060 4231 2.1 103
PDI
GPC results. Calculated from H NMR spectra. MS results.
Fig. 2. The mean size variations of mPEG-pH-PCL micelles in different pH values.
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Fig. 3. The AFM images of blank and drug loaded micelles, (a) blank mPEG-pH-PCL micelles; a’: DOX loaded mPEG-pH-PCL micelles; (b) blank mPEG-CA-PCL micelles; b’: DOX loaded mPEG-CA-PCL micelles.
media. When the mPEG-pH-PCL micelles were soaking in aqueous solution with pH 7.4, their mean sizes kept at around 120 nm even when the stored time was as long as 24 h. The size and size distribution of pH-sensitive mPEG-pH-PCL micelles in pH 5.5 were changed greatly (Fig. 2 and Fig. S4 in SI), it increased continuously from about 110–220 nm when the soaking time was 24 h. As the size tested by DLS was the hydrodynamic diameter of polymeric
micelles, it implied that the citraconic bonds in the backbone of mPEG-pH-PCL were broken and thus resulted in the dissociation of micelles to enlarge the hydrodynamic diameter. The PDI variation in Fig. S4 also supported the conclusion of micellar dissociation. Count rate is a parameter that represents the light scattering intensity of the sample which displays the number of photons detected per second (displayed in kcps-kilo counts per second)
Fig. 4. The release profiles of mPEG-pH-PCL and mPEG-CA-PCL micelles in different pH values.
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Fig. 5. The cytoxicity of blank mPEG-pH-PCL and mPEG-CA-PCL micelles incubated with HepG2 cells for 48 h.
(Xiong et al., 2012). The count rates of the mPEG-pH-PCL and mPEGCA-PCL micelles were measured in different soaking time. The count rates of mPEG-pH-PCL micelles decreased with increasing soaking time in both pH 7.4 and 5.5, and the count rate in pH 5.5 decreased very fast. The count rates of mPEG-CA-PCL micelles in pH 7.4 and 5.5 were nearly kept in a stable state (Fig. S5). These results indicated the pH-sensitivity of mPEG-pH-PCL micelles. The morphology of both blank and DOX loaded mPEG-pH-PCL and mPEG-CA-PCL micelles were observed by AFM (Fig. 3). The size
of blank mPEG-pH-PCL micelles was around 100 nm, which was smaller than the size tested by DLS. It was reasonable because the micelle in AFM was in dry state, which was smaller than the hydrodynamic diameter tested by DLS. After DOX was loaded in mPEG-pH-PCL micelles, the size was increased to about 150 nm. In the AFM image of mPEG-CA-PCL micelles, the size of micelles were not homogenous, aggregates with hundreds of nanometers were observed, however, the aggregates disappeared in the DOX loaded mPEG-CA-PCL micelles image. It was interesting that the dispersity of both blank micelles became better when the DOX was loaded in the micelles. It was probably attributed to the hydrophobic interaction between DOX and PCL blocks in micelles, as both PEG and PCL blocks with the molecular weight of 2000 were elastic polymers with low glass transition temperatures and crystallinity (Loh et al., 2008), the loaded hydrophobic DOX increased the hydrophobic interaction to compress the cores of micelles, thus, the aggregation of the micelles was prevented and the micelles were well dispersed. The release profiles of the two DOX loaded micelles were studied (Fig. 4). The pH values of the media were 7.4 and 5.5. Both drug loaded micelles exhibited burst release. To pH-sensitive mPEG-pH-PCL micelles, the drug release was much faster in pH 5.5 than that in pH 7.4. The encapsulated drug was released from mPEG-pH-PCL micelles and the cumulated release rate was as high as 80% within 90 h in pH 5.5. The cumulated release rate in pH 7.4 was only 40%. To mPEG-CA-PCL micelles, the drug release rate in pH 5.5 was also faster than that in pH 7.4, however, the cumulated release rate in pH 5.5 was much lower than that of mPEG-pH-PCL micelles in pH 5.5, it was less than 60%. The drug release rates of mPEG-pH-PCL and mPEG-CA-PCL micelles in pH 7.4 were
Fig. 6. Confocal microscopy images of HepG2 and 4T1 cells treated with DOX loaded micelles for 3 h, (A) mPEG-CA-PCL micelles; (B) mPEG-pH-PCL micelles; (C) DOXHCl. The DOX concentration of drug loaded micelles in A and B was 5 mg/mL, the concentration for DOXHCl in C was 10 mg/mL. The images from top to bottom were cells in bright field (1), DOX fluorescence in cells (2), cell nuclei stained by DAPI (3), and overlay images (4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. The flow cytometry results of HepG2 (a) and 4T1 (b) cells treated with drug loaded micelles for 3 h, (A) DOX loaded mPEG-CA-PCL micelles; (B) DOX loaded mPEG-pHPCL micelles; (C) DOXHCl. The DOX concentration of the micelle samples was 5 mg/mL, and the concentration of DOXHCl was10 mg/mL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
comparative. The fast release of mPEG-CA-PCL micelles in pH 5.5 was because of the protonation of doxorubicin in acid medium, which changed the DOX from hydrophobic to hydrophilic and the diffusion capacity of the protonated drug was largely strengthened. Comparing the two release profiles of drug loaded mPEG-pH-PCL and mPEG-CA-PCL micelles in pH 5.5, it could be concluded that
Fig. 8. The in vitro anticancer activity of drug loaded micelles, the calculated IC50s of DOX loaded mPEG-pH-PCL and mPEG-CA-PCL micelles were 0.695 mg/mL and 1.126 mg/mL to HepG2 cells (A), 3.922 mg/mL and 4.769 mg/mL to 4T1 cells (B).
the broken of citraconic amide bond in weak acidity accelerated the release of DOX. The two micelles were incubated with HepG2 cells to evaluate their cytotoxicity (Fig. 5). The concentrations of the micelles were ranged from 0.01 to 0.2 mg/mL. The cell viability of mPEG-pH-PCL micelles increased with the decrease of concentration, and that of mPEG-CA-PCL micelles was kept in nearly a same level. The cell viability for each sample was higher than 90%, which indicated that the two micelles were non-toxic to HepG2 cells. Cellular internalization is the key step for drug loaded micelles to fulfill their anticancer activity. In order to investigate the cellular uptake of DOX loaded micelles, the two drug loaded micelles were co-cultured with both HepG2 and 4T1 cells. Confocal laser microscopy and flow cytometry were used to study the cellular uptake of the two micelles. In confocal microscopy images (Fig. 6), both drug loaded mPEG-pH-PCL and mPEG-CA-PCL micelles could be internalized in both HepG2 and 4T1 cells. Strong red fluorescence of DOX was observed in cytoplasm, weak red fluorescence was observed in nuclei, it implied that most of the drug loaded micelles located in cytoplasm, the released DOX could diffuse into nuclei. The red fluorescence in cells treated with drug loaded mPEG-pH-PCL micelles was relatively stronger than that of mPEG-CA-PCL micelles. As a hydrophilic drug, DOXHCl diffused much faster in cells, thus, strong red fluorescence was observed in nuclei of cells treated with DOXHCl. The quantitative fluorescence intensity in the cells was measured by flow cytometry (Fig. 7). It was clear that the red fluorescence of cells treated with DOX loaded mPEG-pH-PCL micelles was stronger than that of cells treated with DOX loaded mPEG-CA-PCL micelles. It revealed that the pHsensitivity functionalized mPEG-pH-PCL micelles promoted the anticancer efficiency of DOX. The in vitro anticancer activity of the two drug loaded micelles was characterized by the half maximal inhibitory concentration (IC50) to HepG2 and 4T1 cells. The results were presented in Fig. 8. The calculated IC50s of DOX loaded pH-sensitive mPEG-pH-PCL micelles to HepG2 and 4T1 cells were 0.695 mg/mL and 3.922 mg/ mL, and those of DOX loaded mPEG-CA-PCL micelles were 1.126 mg/mL and 4.769 mg/mL. Both the IC50s of mPEG-pH-PCL micelles to HepG2 and 4T1 cells were lower than those of corresponding mPEG-CA-PCL micelles. The DOX encapsulated in mPEG-pH-PCL micelles exhibited better anticancer activity due to the pH-sensitivity. The IC50s of the two micelles to 4T1 cells were both higher than those to HepG2 cells, it implied that the anticancer efficiency of the drug loaded micelles was related to the types of cancer cells.
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4. Conclusion A novel pH-sensitive mPEG-pH-PCL copolymer micelle was synthesized, weak acid cleavable citraconic amide bond was introduced in the copolymer to receive pH-sensitivity. The characterizations of 1H NMR, GPC and MS demonstrated the successful synthesis of the diblock copolymers. The pH-sensitivity investigation showed that the citraconic amide bonds in the micelles were broken in pH 5.5 to result in size and size distribution enlargement of micelles. The drug loaded micelles exhibited excellent dispersion capability in aqueous medium with the mean size around 150 nm. The release of DOX loaded mPEGpH-PCL micelles was pH dependent, the release rate in pH 5.5 was much faster than that in pH 7.4. The confocal laser microscopy and flow cytometry measurements indicated that the weak acidity in endosomes could trigger the release of DOX efficiently. The in vitro IC50 tested revealed that the anticancer activity of DOX trapped in the pH-sensitive mPEG-pH-PCL micelles was enhanced. The introduction of citraconic amide in the copolymer backbone was a favorable strategy to fabricate pH-sensitive micellar drug delivery systems. Acknowledgements This research work was supported by National Science Foundation for Excellent Young Scholars (No. 51222304), Young teachers’ scientific research foundation of Sichuan University (2014SCU11015), National Science Foundation of China (NSFC, No. 31170921, 31370972, 51133004, 81361140343), National Basic Research Program of China (National 973 program, No. 2011CB606206), Program for Changjiang Scholars and Innovative Research Team in University (IRT1163). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.05.010. References Alexandridis, P., Holzwarth, J.F., Hatton, T.A., 1994. Micellization of poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymers in aqueous solutions: thermodynamics of copolymer association. Macromolecules 27, 2414–2425. Bae, Y., Fukushima, S., Harada, A., Kataoka, K., 2003. Design of environmentsensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change. Angew. Chem. Int. Ed. 42, 4640–4643. Bae, Y., Nishiyama, N., Kataoka, K., 2007. In vivo antitumor activity of the folateconjugated pH-sensitive polymeric micelle selectively releasing adriamycin in the intracellular acidic compartments. Bioconjugate Chem. 18, 1131–1139. Cao, J., Zhai, S., Li, C., He, B., Lai, Y., Chen, Y., Luo, X., Gu, Z., 2013. Novel pH-sensitive micelles generated by star-shape copolymers containing zwitterionic
sulfobetaine for efficientcellular internalization. J. Biomed. Nanotechnol. 9, 1847–1861. Cheon Lee, S., Kim, C., Chan Kwon, I., Chung, H., Young Jeong, S., 2003. Polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(e-caprolactone) copolymer as a carrier for paclitaxel. J. Control. Release 89, 437–446. Deng, C., Jiang, Y., Cheng, R., Meng, F., Zhong, Z., 2012. Biodegradable polymeric micelles for targeted and controlled anticancer drug delivery: promises, progress and prospects. Nano Today 7, 467–480. Ding, M., Zhou, L., Fu, X., Tan, H., Li, J., Fu, Q., 2010. Biodegradable gemini multiblock poly([varepsilon]-caprolactone urethane)s toward controllable micellization. Soft Matter 6, 2087–2092. Duncan, R., 2003. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2, 347–360. Gillies, E.R., Fréchet, J.M.J., 2005. pH-Responsive copolymer assemblies for controlled release of doxorubicin. Bioconjugate Chem. 16, 361–368. Gu, G., Xia, H., Hu, Q., Liu, Z., Jiang, M., Kang, T., Miao, D., Tu, Y., Pang, Z., Song, Q., Yao, L., Chen, H., Gao, X., Chen, J., 2013. PEG-co-PCL nanoparticles modified with MMP-2/9 activatable low molecular weight protamine for enhanced targeted glioblastoma therapy. Biomaterials 34, 196–208. Kataoka, K., Harada, A., Nagasaki, Y., 2012. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Deliv. Rev. 64, 37–48. Kwon, G.S., Okano, T., 1996. Polymeric micelles as new drug carriers. Adv. Drug Deliv. Rev. 21, 107–116. Liu, R., Li, D., He, B., Xu, X., Sheng, M., Lai, Y., Wang, G., Gu, Z., 2011. Anti-tumor drug delivery of pH-sensitive poly(ethylene glycol)–poly(L-histidine-)–poly(L-lactide) nanoparticles. J. Control. Release 152, 49–56. Loh, X.J., Colin Sng, K.B., Li, J., 2008. Synthesis and water-swelling of thermoresponsive poly(ester urethane)s containing poly(e-caprolactone), poly(ethylene glycol) and poly(propylene glycol). Biomaterials 29, 3185–3194. Otsuka, H., Nagasaki, Y., Kataoka, K., 2012. PEGylated nanoparticles for biological and pharmaceutical applications. Adv. Drug Deliv. Rev. 64, 246–255. Prata, C.A.H., Zhao, Y., Barthelemy, P., Li, Y., Luo, D., McIntosh, T.J., Lee, S.J., Grinstaff, M.W., 2004. Charge-reversal amphiphiles for gene delivery. J. Am. Chem. Soc. 126, 12196–12197. Pu, Y., Chang, S., Yuan, H., Wang, G., He, B., Gu, Z., 2013. The anti-tumor efficiency of poly(L-glutamic acid) dendrimers with polyhedral oligomeric silsesquioxane cores. Biomaterials 34, 3658–3666. Pu, Y., Zhang, L., Zheng, H., He, B., Gu, Z., 2014. Drug release of pH-sensitive poly (Laspartate)-b-poly(ethylene glycol) micelles with POSS cores. Polym. Chem. 5, 463–470. Xin, H., Sha, X., Jiang, X., Zhang, W., Chen, L., Fang, X., 2012. Anti-glioblastoma efficacy and safety of paclitaxel-loading Angiopep-conjugated dual targeting PEG–PCL nanoparticles. Biomaterials 33, 8167–8176. Xiong, M.-H., Bao, Y., Yang, X.-Z., Wang, Y.-C., Sun, B., Wang, J., 2012. Lipase-sensitive polymeric triple-layered nanogel for “on-demand” drug delivery. J. Am. Chem. Soc. 134, 4355–4362. Xu, P., VanKirk, E.A., Zhan, Y., Murdoch, W.J., Radosz, M., Shen, Y., 2007. Targeted charge-reversal nanoparticles for nuclear drug delivery. Angew. Chem. Int. Ed. 46, 4999–5002. Xue, B., Wang, Y., Tang, X., Xie, P., Wang, Y., Luo, F., Wu, C., Qian, Z., 2012. Biodegradable self-assembled MPEG-PCL micelles for hydrophobic oridonin delivery in vitro. J. Biomed. Nanotechnol. 8, 80–89. Yang, Q., Liao, J., Deng, X., Liang, J., Long, C., Xie, C., Chen, X., Zhang, L., Sun, J., Peng, J., Chu, B., Guo, G., Luo, F., Qian, Z., 2014. Anti-tumor activity and safety evaluation of fisetin-loaded methoxy poly(ethylene glycol)-b-poly(e-caprolactone) nanoparticles. J. Biomed. Nanotechnol. 10, 580–591. Yuan, Y.-Y., Mao, C.-Q., Du, X.-J., Du, J.-Z., Wang, F., Wang, J., 2012. Surface charge switchable nanoparticles based on zwitterionic polymer for enhanced drug delivery to tumor. Adv. Mater. 24, 5476–5480. Zhang, J., Men, K., Gu, Y., Wang, X., Gou, M., Guo, G., Luo, F., Qian, Z., 2011. Preparation of core cross-linked PCL-PEG-PCL micelles for doxorubicin delivery in vitro. J. Nanosci. Nanotechnol. 11, 5054–5061. Zhou, Z., Shen, Y., Tang, J., Fan, M., Van Kirk, E.A., Murdoch, W.J., Radosz, M., 2009. Charge-reversal drug conjugate for targeted cancer cell nuclear drug delivery. Adv. Funct. Mater. 19, 3580–3589.
