Nanomicelles based on a boronate ester-linked diblock copolymer as the carrier of doxorubicin with enhanced cellular uptake

Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Nanomicelles based on a boronate ester-linked diblock copolymer as the carrier of doxorubicin with enhanced cellular uptake Yan Xu a , Yuanyuan Lu a , Lei Wang a , Wei Lu a , Jin Huang b , Ben Muir c , Jiahui Yu a,∗ a Shanghai Engineering Research Centre of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, PR China b College of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan, PR China c The Commonwealth Scientific and Industrial Research Organisation, Bayview Ave, Bag 10, Clayton South, Melbourne, Victoria 3169, Australia

a r t i c l e

i n f o

Article history: Received 26 November 2015 Received in revised form 21 January 2016 Accepted 25 January 2016 Available online 28 January 2016 Keywords: Acid-induced degradation Nanomicelle Boronate ester Enhanced endocytosis HepG2 cells

a b s t r a c t This study sought to develop a new type nanomicelle based on boronate ester-linked poly(ethylene glycol)-b-poly(benzyl glutamate) (PEG-BC-PBLG) diblock copolymer as the carrier of doxorubicin (Dox) to achieve acid-induced detachment of PEG shells and subsequent boronic acid-mediated enhanced endocytosis. In vitro studies revealed that the PEG-BC-PBLG copolymer was stable in neutral solutions but tend to hydrolysed under acidic conditions, which was attributed to the acid-sensitive properties of boronate ester bonds. The formation of PEG-BC@PBLG micelles was confirmed based on critical micelle concentration (CMC), particle size, and morphology observations. It was observed that these micelles were spherical with an average particle size of approximately 80 nm, as measured by dynamic laser scattering (DLS), suggesting their passive targeting to tumour tissue and endocytosis potential. Dox-loaded PEG-BC@PBLG micelles (PEG-BC@PBLG·Dox) showed sustained drug release profiles over 9 h, and their cumulative drug release was dependent on the pH value of the environment. Remarkably, cellular uptake ability of PEGBC@PBLG micelles was found to be higher than that of non-boronate ester-linked PEG@PBLG micelles due to boronic acid-mediated endocytosis, as revealed by confocal laser scanning microscopy (CLSM) imaging of fluorescein isothiocyanate (FITC) green-conjugated micelles, thereby providing higher cytotoxicity against HepG2 cells. The antitumour activity and toxicity of PEG-BC@PBLG·Dox micelles in vivo were evaluated in BLAB/c mice against HepG2 cell-derived tumours. Compared with Dox, PEG-BC@PBLG·Dox showed reduced toxicity, whereas its tumour growth inhibition rate was 17% higher than that of free Dox. These results indicate the great potential of PEG-BC@PBLG micelles as the carrier of various lipophilic anticancer drugs with improved anti-tumour efficacy. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Conventional chemotherapy has achieved great success in cancer therapy but remains limited by a lack of tumour-selectivity, drug resistance, and other side effects [1,2]. To overcome these limitations, substantial efforts has been devoted to the development of anticancer drug delivery carriers because of their prolonged circulation time and high passive targeting ability to tumour sites via the enhanced permeability and retention (EPR) effect [3,4]. For cancer therapy, it is desirable to achieve a sufficient drug concentration at tumour tissues, which may enhance the therapeutic efficacy and reduce the probability of drug resistance in cells [5,6]. Therefore, various stimuli-sensitive drug carriers have been designed in

∗ Corresponding author. Fax: +86 21 6223 7026. E-mail address: [email protected] (J. Yu). http://dx.doi.org/10.1016/j.colsurfb.2016.01.044 0927-7765/© 2016 Elsevier B.V. All rights reserved.

recent years based on the physiological properties of the tumour microenvironment or the expression of tumour-associated receptors [7–9]. The difference in the pH value between tumour and normal tissue is often used as a stimulus factor [10]. The pH value of normal plasma is 7.35–7.45, whereas the pH in tumour microenvironment decreases to 6.6–6.8 depending on the distance from blood vessels because anaerobic glycolysis is facilitated by the limited oxygen supply [11]. Previously designed pH-sensitive carriers are relatively stable in normal blood circulation but can degrade when they accumulate at target tumours, mainly owing to the breakage pH-sensitive chemical bonds [12,13]. Boronate ester has been demonstrated as a pH-sensitive chemical bond, which can be easily hydrolysed under acid conditions but relatively stable in neutral and alkaline solutions [14,15]. Levkin et al. [16] reported the formation of a boronate dextran polymer (B-Dex) that realised four-fold higher release of incorporated anticancer drug at pH 5.0 in comparison to pH 7.4. For delivery to solid

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

tumours, the surfaces of nanoparticles were modified with water soluble polymers, such as PEG, to afford a long circulation time in blood and passive targeting potential to the tumour mass [17,18]. However, PEGylation inhibits cellular uptake of nanoparticles [19]. Receptor-mediated cellular uptake of PEGylated nanoparticles is usually achieved by immobilization of antibodies or other ligands that recognize tumour-associated antigens or ligand receptors at the terminal end of PEG [20,21]. It has been reported that boronic acid shows high affinity with the polysaccharide called Sialyl Lewis X, which is abundant on the surface of HepG2 cells. Additionally, Zhong et al. revealed that the transfection of phenylboronic acidmodified PEIs was more efficient in HepG2 cells, which realised receptor-mediated endocytosis [22–24]. In this paper, a new strategy to prepare boronate esterlinked core-shell nanomicelles as the carrier of doxorubicin was developed to achieve acid-induced detachment of PEG shells and subsequent boronic acid-mediated enhanced endocytosis. Particularly, PEG-BC@PBLG nanomicelles were fabricated by self-assembly of boronate ester-linked PEG-BC-PBLG copolymer. Because boronate ester is stable in neutral environments, PEGBC@PBLG micelles could maintain their structure and integrate into blood circulation and could reach tumour tissues in a passive targeting manner through an EPR effect [25,26]. However, the pH at both primary and metastasised tumours (pH 6.5–6.8) is slightly lower than the pH of normal tissue, and intracellular components such as the endosomes (pH 6.0–6.5) or lysosomes (pH 5.0–5.5) are more acidic [27,28]. Therefore, the poly(ethylene glycol) (PEG) shells of PEG-BC@PBLG micelles would be detached in tumour tissues. The exposed boronic acid segment would then realise receptor-mediated endocytosis. Furthermore, the micelles would liberate the loaded drug and achieve improved cancer therapy (Scheme 1). The structure of PEG-BC-PBLG copolymer was characterised by 1 H NMR and gel permeation chromatography (GPC). The physicochemical properties of PEG-BC@PBLG micelles, such as the particle size, critical micelle concentration (CMC) and drug accumulative release, were studied. Subsequently, cellular uptake and cell toxicity were evaluated and compared with that of PEG@PBLG micelles that do not contain boronate ester bonds. The in vivo anti-tumour activity of PEG-BC@PBLG and Dox-loaded micelles in tumour-bearing nude mice were later evaluated in detail.

2. Experimentals 2.1. Materials N, N-diisopropylethylamine (DIPEA, 99%), 1-hydroxybenzotriazole (HOBT, 99%), 1-(3hydrochloride dimethylaminopropyl)-3-ethylcarbodiimide (EDC·HCl, 99%) and 3-aminobenzeneboronic acid (98%) were purchased from Sigma–Aldrich. Methoxypolyethylene glycol amine (CH3 O-PEG-NH2 , Mn = 2000, Fluka) was dried by azeotropic distillation from toluene. 3, 4-Dihydroxyphenylacetic acid (DA, 99%), fluorescein isothiocyanate (FITC, 97%) and 2, 4-dihydroxybenzoic acid (98%) were obtained from Aladdin. 5-Benzyl l-glutamate N-carboxyanhydride (BLG–NCA, 98%) was obtained from Alfa Aesar. Dialysis bags and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5diphenyltetrazolium bromide (MTT, 98%) were purchased from Vita Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure water was prepared using a Milli-Q system (Millipore, USA). Toluene and tetrahydrofuran (THF) were dried by refluxing over sodium wire and distilled before use. Triethylamine (TEA), dichloromethane (DCM), and dimethyl sulfoxide (DMSO) were dried by refluxing over CaH2 and distilled prior to use. Doxorubicin hydrochloride (>99%, Energy Chemical) and other reagents were used as received.

319

2.2. Cell line and culture Human hepatoma cell line HepG2 and human hepatocyte cell line HL-7702 was supplied by the Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. HepG2 cells were cultured in minimum essential medium (MEM), and HL-7702 cells were cultured in RPMI 1640 (Gibco BRL, Paris, France), both of which were supplemented with 10% foetal bovine serum (FBS, HyClone, Logan, UT), streptomycin at 100 ␮g/mL, and penicillin at 100 U/mL. All cells were incubated at 37 ◦ C in a humidified 5% CO2 atmosphere. The confluent cells were dissociated using a pre-warmed trypsin-EDTA solution at 37 ◦ C. 2.3. Synthesis of PEG-BC-PBLG DA was conjugated to the polymer mPEG-NH2 by an amidation reaction [29]. Briefly, mPEG-NH2 (1.000 g, 0.500 mmol) was first dissolved in 30 mL of anhydrous DCM; then, EDC·HCl (0.154 g, 0.800 mmol), HOBT (0.108 g, 0.800 mmol), DIPEA (0.260 mL, 1.500 mmol), and DA (0.126 g, 0.750 mmol) were added and protected by nitrogen. After stirring for 12 h at 25 ◦ C, the mixture was purified by column chromatography (dichloromethane:methanol = 30:1). The organic solvent was removed by low-pressure evaporation. The product (PEG-DA) was then precipitated into cold ethyl ether, isolated by filtration, and dried under high vacuum (yield: 78%). 3-Aminobenzeneboronic acid (0.137 g, 1.000 mmol) and PEGDA (0.430 g, 0.200 mmol) were dissolved in 100 mL of toluene and placed in a reactor fitted with a Dean-Stark apparatus for water removal [30–33]. The mixture was stirred and refluxed at 120 ◦ C for 8 h, and the reaction was then terminated by cooling to room temperature. The crude product was dissolved in anhydrous THF and dialysed for 48 h (molecular weight cut-off size: 1000). The resulting polymer was precipitated in cold ethyl ether, isolated by filtration, and dried under vacuum; this product was named PEG-BC (yield: 80%). PEG-BC-PBLG was synthesised by ring-opening polymerisation [34]. BLG-NCA (0.740 g, 2.800 mmol) and PEG-BC (0.897 g, 0.400 mmol) was dissolved in 30 mL of anhydrous THF. The reaction mixture was stirred for 24 h at 25 ◦ C and protected by N2 . The crude product was dissolved in anhydrous THF and dialysed for 48 h (molecular weight cut-off size: 3500). The resulting polymer was isolated by precipitation in cold ethyl ether, filtered, washed three times, and then dried under vacuum (yield: 95%). PEG-PBLG was synthesised by mPEG-NH2 and BLG-NCA in the same manner as described above. 2.4. Synthesis of PEG-BC-PBLG-FITC FITC (0.089 g, 0.230 mmol) and PEG-BC-PBLG (0.500 g, 0.150 mmol) were dissolved in10 mL of anhydrous THF and stirred for 12 h at 25 ◦ C [35]. The crude product was then evaporated and added to cold ethyl ether, washed several times to remove excess FITC, and dried under vacuum (yield: 91%). PEG-PBLG-FITC was synthesised in the same way. 2.5. Characterization of PEG-BC-PBLG diblock copolymer 1 H nuclear magnetic resonance (1 H NMR) spectra were recorded

using a Bruker Avarice TM 500 NMR spectrometer. The molecular weights of samples were measured using an Agilent 1200 gel permeation chromatography (GPC) system (Agilent Technologies Inc., Shanghai Branch). An Agilent 1200 refractive index detector and aqueous SEC start-up kit were used. Chromatography columns (PL aquagel-OH MIXED columns; Polymer Laboratories Ltd., Amherst, MA, USA) were calibrated using a poly (ethylene glycol) kit. The col-

320

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

Scheme 1. Illustration of PEG-BC@PBLG nanomicelles as the carrier of Dox for acid-induced detachment of PEG shells and boronic acid-mediated enhanced endocytosis. (A) Synthesis of the PEG-BC-PBLG diblock copolymer. (B) Self-assembly, endocytosis, and release of Dox from PEG-BC@PBLG nanomicelles.

umn temperature was maintained at 25 ◦ C. The mobile phase was a phosphate buffer solution (PBS, 0.01 M, pH 7.4), and the flow rate was 1.0 mL/min. 2.6. Fabrication and characterization of PEG-BC@PBLG micelles and Dox-loaded micelles PEG-BC-PBLG (2 mg) was dissolved in DCM (0.5 mL), and MilliQ water (10 mL) was then added under stirring in 500 r/min [36]. The solution was stirred until the dichloromethane volatilised

without low-pressure evaporation, subsequently dialysed against Milli-Q water (pH 7.4) for 24 h, during which the Milli-Q water was replaced every 6 h. The final solution was freeze-dried. The resultant nanoparticle was named PEG-BC@PBLG. Micelles of PEG-BC@PBLG·Dox were prepared under stirring with dropwise addition of Dox (2 mg) and PEG-BC-PBLG (10 mg) in DMSO (1 mL) to Milli-Q water (20 mL), followed by prolonged dialysis against MilliQ water for 48 h. The lyophilised product was stored under −20 ◦ C until use.

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

The critical micelle concentration (CMC) was determined using pyrene as a fluorescence probe. Pyrene was dissolved in acetone and evaporated completely to form a thin film at the bottom of the sample bottles. Various concentrations of micelle solutions (from 1 × 10−4 mg/mL to 0.5 mg/mL) were added to the bottles to maintain the concentration at 6.0 × 10−7 mol/L. The sample bottles were shaked using a thermostated oscillator at 30 ◦ C for 48 h prior to CMC measurement to sufficiently dissolve the pyrene. The fluorescence intensity ratio of I338 /I334 in the emission spectra of pyrene was analysed. The CMC was estimated as the cross-point when extrapolating the intensity ratio I338 /I334 in the low and high concentration ranges. The size distribution of micelles was determined using dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK) in Milli-Q water. For transmission electron microscopy (TEM), a 10 ␮L of sample solution (10−2 mg/mL) was carefully dropped onto clean copper grids and then dried at 25 ◦ C before imaging using a JEM-100C II microscope (LIBRA 120, Carl Zeiss, Germany). The drug loading content (DLC) and drug loading efficiency (DLE) of Dox in PEG-BC@PBLG micelles were obtained based on a standard curve of a Dox/DMSO solution using UV–vis spectroscopy (Varian Cary 100). Lyophilised PEG-BC@PBLG·Dox micelles were dissolved in DMSO, and the drug concentration was determined based on its UV absorbance at 480 nm. The DLC and DLE of PEG-BC@PBLG·Dox micelles were calculated according to the following formulas: DLC(%) = (weightofloadeddrug/weightofpolymer) × 100%

(1)

DLE(%) = (weightofloadeddrug/weightofdruginfeed) × 100%

(2)

2.7. Degradation studies of PEG-BC-PBLG copolymer and PEG-BC@PBLG micelles The degradation efficiency of PEG-BC-PBLG copolymer were calculated based on 1 H nuclear magnetic resonance (1 H NMR) spectroscopy. Ten milligrams of PEG-BC-PBLG was dissolved in 1 mL of DMSO-D6. Then, 100 ␮L of PB buffer (Na2 HPO4 /NaH2 PO4 buffer at pH 7.4, 6.5, 6.0 and 5.0; 0.1 M) prepared with D2 O was added to each vessel. Each sample was scanned once per hour, and the obtained 1 H NMR spectra were analysed. Size destabilization of PEG-BC@PBLG micelles in response to each pH buffer at different intervals was tracked using DLS measurements. One milligramme of lyophilised micelles was dissolved in 5 mL of PB buffer (pH 7.4, 6.5, 6.0, and 5.0), and the changes in size were quantified every 30 min. 2.8. Release of Dox from PEG-BC@PBLG·Dox micelles Release of Dox from PEG-BC@PBLG micelles was determined by the method described by Opanasopit et al. [37]. Five milligrams of lyophilised PEG-BC@PBLG·Dox was dissolved in 25 mL of PB buffer (pH 7.4 and 5.0). Each sample solution was transferred into dialysis bags (molecular weight cut-off size: 2000), which were then immersed in the corresponding pH buffer. At certain time intervals, a 1 mL aliquot of dialysis medium was removed, and the same volume of fresh buffer was added. The sample solution was analysed by UV–vis spectroscopy ( = 480 nm), and a calibration curve was established from known concentrations of free Dox. 2.9. Cell toxicity The cytotoxicity of PEG-BC@PBLG and PEG-BC@PBLG·Dox micelles was evaluated using an MTT assay. HepG2 cells were pre-incubated in a 96-well plate (9 × 103 cells/well) with 180 ␮L of culture medium per well in constant temperature incubator (Thermo, USA). After reaching 80% confluence at the time of treatment, the cell were further incubated with PEG-BC@PBLG,

321

PEG@PBLG·Dox, PEG-BC@PBLG·Dox, and free Dox at six concentrations (3.75, 7.5, 15, 30, 60, and 120 mg/L) for 24 h. The pH values of culture medium were 7.4, 6.5, and 5.0, respectively. Subsequently, the culture medium was amended with 10 ␮L of MTT solution (5 mg/mL). After further incubation for 4 h, 100 ␮L of DMSO was added to each well to replace the culture medium and dissolve insoluble formazan crystals. The absorbance of each well was measured at 570 nm using an automated BIO–TEK microplate reader (Powerwave XS, USA). Wells treated with 200 ␮L of PBS were used as a blank (ODblank ), and cells only treated with 200 ␮L of culture medium were used as a control (ODcontrol ). The cell viability was calculated as follows: Cellviability(%) = [(ODsample − ODblank )/(ODcontrol − ODblank )] × 100%

(3)

2.10. Cellular uptake The cellular uptake of PEG-BC@PBLG micelles was compared against that of HepG2 and HL-7702 cells using confocal laser scanning microscopy (CLSM, Leica TCS SP5). Pre-experiments were conducted to determine the concentration and incubation time of the micelles. Three mL of fresh growth medium with HepG2 and HL7702 cells was seeded into a 4-well glass plate (Greiner) (10 × 104 cells/well) and incubated for 24 h. The culture medium was then replaced by another 3 mL of fresh culture medium, which was prepared with PB buffer (pH value: 7.4, 6.5, 6.0 and 5.0) containing 0.4 mg/mL PEG-BC@PBLG-FITC micelles per well, and further cultured for 3 h. The cells were then rinsed with PBS three times and observed by CLSM (525 nm laser excitation). The fluorescence intensity of FITC in cells was analysed by flow cytometry (Guava easyCyte 6HT2L, USA). HepG2 and HL-7702 cells were cultured as described above. The anchorage-dependent cells were digested by pancreatic enzymes, collected by centrifugation (TG16-WS, China), and then washed three times with PBS. The collected cells were transferred to a 96-well plate, and the fluorescence intensity was measured by flow cytometry. The blank control (cells cultured with normal medium) and positive control (cells cultured with an equal amount of free FITC) were used as a baseline when we tested the fluorescence intensity. 2.11. Tumour induction and treatment Female BALB/c-nu mice (Slaccas Experimental Animal Co., Ltd., Shanghai) were used in vivo study. All mice (body weight: 18–20 g) were maintained in a laminar airflow cabinet (room temperature: 25.0 ± 0.5 ◦ C, relative humidity: 40–70%) with a 12-h light/dark cycle under specific pathogen-free conditions. Pre-experiments were conducted to determine the amount of subcutaneous cell injection and administration dosage. Subsequently, tumours were induced by subcutaneous injection of 5 × 105 HepG2 cells with a BD-Matrigel basement membrane matrix (50% of injection volume) into the left flanks of BALB/c-nu mice. When the tumour was palpable, animals were divided into four groups (n = 6) corresponding to treatments with saline solution (group A), PEG-BC@PBLG (group B), Dox (group C), and PEG-BC@PBLG·Dox (group D). The administration dosage of Dox was 5 mg/Kg of body mass, and each mouse was intravenously administered every 3 days up to a total number of 5 doses. Weights and deaths were carefully recorded throughout the period. The largest tumour diameter a and the second-largest diameter b perpendicular to a were measured using a digital calliper. The tumour volume (V, mm3 ) was calculated as follows: V = (a × b2 × )/6

(4)

322

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

Fig. 1. Size distribution determined by DLS (A) and morphology observed by TEM (B) of PEG-BC@PBLG nanomicelles.

2.12. Statistical data analysis Statistical data analysis was performed using Student’s t-test. 3. Results and discussion 3.1. Synthesis and characterization of PEG-BC-PBLG diblock copolymer PEG-BC-PBLG diblock copolymer was prepared using the synthetic approach as illustrated in Scheme 1 (A). In the first step, PEG-DA was synthesised by an amidation reaction, and the appearance of the characteristic peaks at 6.61–6.91 ppm assigned to DA and the new peak of Hd in the 1 H NMR spectrum of the purified product indicated the successful conjugation (Fig. S1). For the second step, PEG-BC was prepared through dehydration between PEG-DA and 3-aminobenzeneboronic acid. After removal of unreacted substrates by dialysis, the appearance of the peaks at 6.98–7.11 ppm assigned to 3-aminobenzeneboronic acid in Fig. S2 illustrated the correct conjugation. According to the 11 B NMR shown in Fig. S3, the obvious shift from ı 25 ppm (boronic acid) to ı 13 ppm (boronate ester) clearly demonstrated the formation of boronate ester in PEG-BC. Finally, PEG-BC-PBLG was synthesised by the ring-opening copolymerization of BLG-NCA using PEG-BC as an initiator. The disappearance of the peak at 4.20–4.29 ppm (t, CH) assigned to methine protons of BLG-NCA clearly indicated that the rings were thoroughly opened by the amino groups in PEG-BC and the 7.1-member rings of BLG-NCA were opened, as indicated by calculating the integral ratio of the peaks assigned to protons of DA (He–g ) and the peaks assigned to the new peak of methylene protons (Hq ) (Fig. S4). The molecular weight of the PEGBC-PBLG diblock copolymer measured by GPC was approximately Mn = 3538, Mw = 3774 and polydispersity index (PDI) = 1.06, which was in close agreement with the ring-opened number calculated by 1 H NMR. 3.2. Micellisation and characterisation Because the PBLG as hydrophobic moiety and the hydrophilic PEG chains were conjugated, it was expected that the selfassembly of PEG-BC-PBLG would produce nanomicelles in an aqueous medium, in which PBLG and PEG chains form the core and shell, respectively. To monitor the formation of PEG-BC@PBLG nanomicelles in an aqueous medium, fluorescence measurements were performed according to a previously reported method. The

CMC value of PEG-BC@PBLG nanomicelles was calculated to be 3.5 × 10−2 mg/mL based on the plot of the intensity ratio of I338 /I334 in the pyrene emission spectra (Fig. S5). In polymeric drug delivery systems, the size and zeta potential (␨) of nanomicelles are known to dramatically affect the pharmacokinetics. The particle size and size distribution of PEG-BC@PBLG nanomicelles were measured by DLS in an aqueous medium. As shown in Fig. 1 (A), the size of the PEG-BC@PBLG nanomicelles was 80 nm with a narrow size distribution, suggesting its efficient passive target potential towards tumour tissue. It was also noteworthy that nanomicelles with a positive surface charge are prone to precipitate due to interactions with serum protein in human blood. In the zeta potential measurement, PEG-BC@PBLG nanomicelles showed a neutral potential of 0.42 mV, suggesting its potential capacity for a prolonged circulation time in blood. To better characterize the shape of PEG-BC@PBLG nanomicelles, their morphologies were observed by TEM. Representative TEM images are shown in Fig. 1 (B). All of the assemblies were clearly spherical and had a good nanoscale dispersibility. The size of PEGBC@PBLG nanomicelles measured by TEM was ca. 50 nm, which was smaller than those measured by DLS (80 nm) due to the shrinking of polymer micelles during drying for the preparation of TEM specimens.

3.3. Degradation studies of PEG-BC-PBLG diblock copolymer and PEG-BC@PBLG micelles The degradation of PEG-BC-PBLG diblock copolymer was studied by 1 H NMR, which was based on its structural characteristics. The boronate ester bonds in PEG-BC-PBLG were prone to break into two parts under acid conditions, and the PEG shell could be dissolved in DMSO, whereas the hydrophobic PBLG component was insoluble in DMSO, which was synthesised individually to quantify its solubility. Therefore, the signal intensity of PBLG would be weakened with the increased extent of acidolysis and the amount of degradation and could be calculated from the integral ratio in 1 H NMR. As shown in Fig. 2, the degradation was studied in a simulated tumour microenvironment (pH 6.5, pH 6.0, and pH 5.0) and normal tissue (pH 7.4). The integral ratio of Hq in the PBLG component decreased in the more acidic environment, whereas the integral ratio of He–g in the PEG shell section remained unchanged. Notably, the boronate ester bond was more prone to break in a more acidic environment. Further, we used linear regression analysis of the degradation by calculating the integral ratio of He–g and Hq and determined the half-life time (t1/2 ) of PEG-BC-PBLG at each

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

323

Fig. 2. The degradation of PEG-BC-PBLG diblock copolymer monitored by 1 H NMR. The samples were dissolved in DMSO-D6, and PB buffer (Na2 HPO4 /NaH2 PO4 buffer of pH 8.5, 7.4, 6.5, 6.0 and 5.0; 0.1 M) prepared with D2 O was then added to each solution. Each sample was scanned once per hour, and the resulting 1 H NMR spectra were analysed.

Fig. 3. Changes in the size of PEG-BC@PBLG micelles in response to PB buffer with different pH values (pH 7.4, 6.5, 6.0, and 5.0; 0.1 M) determined based on DLS measurements after 24 h.

pH value: 75.48 h (pH 7.4), 12.45 h (pH 6.5), 10.07 h (pH 6.0) and 1.35 h (pH 5.0). These results indicated that PEG-BC-PBLG was relatively stable at pH 7.4 but degraded more quickly with decreasing pH. To further study the degradation process, the size destabilization of PEG-BC@PBLG micelles in response to different pH values at different intervals was determined using DLS measurements. The size of the assemblies in PB buffer at pH 6.5, pH 6.0, and pH 5.0 increased to 400, 950, and 2100 nm, respectively, whereas the size of the micelles at pH 7.4 remained at 100 nm (Fig. 3). The aggregation of the micelles might be attributed to the cleavage of boronate ester bonds, which resulted in the detachment of PEG shells and hence destroyed the hydrophilicity–hydrophobicity equilibrium

Fig. 4. Acid-induced release of Dox from PEG-BC@PBLG·Dox micelles. (A) PEGBC@PBLG·Dox (pH 7.4), (B) PEG-BC@PBLG·Dox (pH 6.5), (C) PEG-BC@PBLG·Dox (pH 6.0), (D) PEG-BC@PBLG·Dox (pH 5.0), (E) PEG@PBLG·Dox (pH 5.0) (mean ± SD, n = 4).

of the initial assemblies and their dispersion stability due to the absence of steric hindrance among PEG chains. 3.4. Release of Dox from PEG-BC@PBLG·Dox micelles The in vitro drug release from Dox-loaded micelles was investigated using dialysis tubing (MWCO: 2000 Da) at 37 ◦ C in buffers of varied pH values. As shown in Fig. 4, only approximately 10% of Dox was released within 9 h when the pH of the buffer was 7.4. However, the micelles released Dox more rapidly and completely with decreasing pH from 7.4 to 5.0, and the release of Dox was up to 85% when the pH was 5.0. In contrast, PEG@PBLG·Dox micelles

324

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

without boronate ester bonds maintained their architecture with only 5% release of Dox. It was thought that the rapid drug release from PEG-BC@PBLG·Dox micelles was triggered by the shedding of PEG shells due to the cleavage of boronate ester bonds in acid environments. 3.5. Cell toxicity The cytotoxicity of PEG-BC@PBLG micelles, PEG@PBLG·Dox micelles, PEG-BC@PBLG·Dox micelles and free Dox were evaluated with HepG2 cells in culture media having different pH values (pH 7.4 and pH 5.0) using an MTT assay. Representative concentrationgrowth inhibition bars are shown in Fig. 5. With the treatment of PEG-BC@PBLG micelles, cell viability remained at approximately 90%, suggesting low cytotoxicity. Although PEG-BC@PBLG·Dox and PEG@PBLG·Dox both showed low cytotoxicity when the pH of the culture medium was 7.4, the cytotoxicity of PEG@PBLG·Dox was obviously enhanced in a dose-dependent manner when the cell were treated with pH 5.0 medium; this effect might be attributed to the fact that boronate ester bonds in micelles can be rapidly split in response to an acidic tumour microenvironment, thereby causing boronic acid-mediated endocytosis. This manner was more obvious when the cell were treated with pH 5.0 medium because of more acidic condition. For non-boronate ester PEG@PBLG·Dox micelles, the encapsulated Dox presented a slow release even in pH 5.0 medium, and it can be assumed that PEG@PBLG·Dox maintained a high stability. However, PEG-BC@PBLG·Dox exhibited lower cytotoxicity than free Dox. It was likely that the loaded Dox was released after the micelles were endocytosed to the cells, which was a delayed process compared to the rapid diffusion of free Dox. These findings confirmed that the use of a boronate ester as a linker is beneficial for enhancing the anti-tumour activity of drug-loaded micelles. 3.6. Cellular uptake To better understand the cellular uptake ability of PEG-BC@PBLG micelles, the cellular uptake of micelles into HepG2 cells was also evaluated by CLSM and flow cytometry. The experiments were conducted using FITC as a fluorescent probe, which could be linked with PEG-BC-PBLG through a chemical bond, instead of Dox, which was only encapsulated into the hydrophobic core. As previously reported, boronic acid shows a high affinity with the polysaccharide called Sialyl Lewis X, which is abundant on the surface of HepG2 cells. To further quantify the affinity of PEG-BC@PBLG micelles towards HepG2 cells, we used HL-7702 cells as a control, which have much lower content of Sialyl Lewis X compared to HepG2 cells; non-boronate ester PEG@PBLG micelles were also used as a control. Pre-experiments were conducted to determine the drug concentration and incubation time of the micelles. Moreover, cells cultured with normal medium were used as a negative control, and free FITC was used as a positive control. As shown in Fig. 6, apparent intracellular FITC fluorescence was observed in HepG2 cells treated with PEG@PBLG-FITC and PEG-BC@PBLG-FITC micelles, and the fluorescence intensity of the PEG-BC@PBLG-FITC micelles was obviously enhanced as the pH decreasing from 7.4 to 5.0, indicating that endocytosis of PEG-BC@PBLG micelles was enhanced with greater exposure of the boronate acid segment. Moreover, the fluorescence intensity of PEG-BC@PBLG-FITC micelles incubated with HL-7702 cells showed little change, illustrating the lack of Sialyl Lewis X on HL-7702 cells and further confirming that the cellular uptake of PEG-BC@PBLG micelles was enhanced after degradation due to the high affinity of boronate acid towards HepG2 cells. The quantitative fluorescence intensities in each group were determined by flow cytometry. The results shown in Fig. 7 were consistent with the CLSM findings. The fluorescence intensity

Fig. 5. Cytotoxicity studies of PEG-BC@PBLG, PEG-BC@PBLG·Dox, PEG@PBLG micelles and free Dox against HepG2 cells after incubation in A) pH 7.4 medium, B) pH 5.0 medium, and C) pH 5.0 medium (mean ± SD, n = 6).

of PEG-BC@PBLG-FITC micelles in HepG2 cells increased with decreasing pH, and the strongest intensity was observed at pH 5.0; in contrast, the fluorescence intensity of micelles in HL-7702 cells remained low. A slight increase was observed for PEG@PBLGFITC micelles, probably because the acidic conditions enhanced

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

325

Fig. 6. Confocal laser scanning microscopy (CLSM) images of HepG2 cells and HL-7702 cells incubated with PEG@PBLG-FITC and PEG-BC@PBLG-FITC micelles in media with different pH values. (A) HepG2 cells treated with PEG@PBLG-FITC micelles, (B) HepG2 cells treated with PEG-BC@PBLG-FITC micelles, and (C) HL-7702 cells treated with PEG-BC@PBLG-FITC micelles.

cytosis by the exposed boronate acid segment of PEG-BC@PBLG micelles.

3.7. The in vivo anticancer activity of Dox-loaded PEG-BC@PBLG micelles

Fig. 7. Fluorescence intensity of HepG2 cells and HL-7702 cells incubated with PEG@PBLG-FITC and PEG-BC@PBLG-FITC micelles in PB buffer (pH 7.4, 6.5, 6.0, and pH 5.0, 0.1 M) (mean ± SD, n = 6).

the endocytosis. These results could be explained by the fact that boronate ester bonds in micelles are rapidly cleaved in response to the acidic tumour microenvironment, resulting in enhanced endo-

The antitumour efficacy of PEG-BC@PBLG·Dox micelles in vivo was evaluated by monitoring the tumour size after the administration of drugs. The administration dosage was set as 5 mg Dox/kg, which was determined in the pre-experiments. As shown in Fig. 8 (A), the volume of the tumour in the control group that was treated with normal saline increased significantly over time, indicating that normal saline had no inhibitory effect on tumour growth. By contrast, the tumour growth was efficiently inhibited in the mice treated with Dox or PEG-BC@PBLG·Dox micelles, suggesting a good anti-tumour activity. In addition, the average tumour inhibition rate of Dox and PEG-BC@PBLG·Dox micelles was 38.52% and 55.10%, respectively, indicating that PEG-BC@PBLG·Dox micelles had better antitumour activity than free Dox. It was thought that PEG-BC@PBLG·Dox accumulated in tumour tissue and realised boronic acid-mediated endocytosis into HepG2 cells, which enhanced the antitumour activity. Unexpectedly, the PEGBC@PBLG micelle drug carrier itself also exhibited toxicity, which lead to the inhibition of tumour growth.

326

Y. Xu et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 318–326

acid segment of micelles was exposed after boronate ester bonds were cleaved, which further induced receptor-mediated endocytosis without the use of ligands or antibodies. The results of in vitro studies demonstrated that PEG-BC@PBLG micelles had a markedly enhanced efficiency in killing cancer cells. In vivo studies also demonstrated that this smart drug delivery system enhances the antitumour efficacy of the loaded drug and reduces its systemic toxicity. This work provides a new strategy for the design of drug delivery systems and presents a promising therapeutic option for the treatment of cancers. Acknowledgements This study was supported by the National Natural Science Foundation of China (51573050) and the International Science and Technology Co-Operation Programme of China, Ministry of Science and Technology of China (2013DFG32340). 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.colsurfb.2016.01. 044. References [1] [2] [3] [4] [5] [6]

Fig. 8. In vivo anti-tumour efficacy. (A) Changes in the tumour volume caused by normal saline, Dox, PEG-BC@PBLG, and PEG-BC@PBLG·Dox. Each drug was injected i.v. five times at a dose of 5 mg Dox/kg. (B) Changes in the body weight of mice caused by normal saline, Dox, PEG-BC@PBLG, and PEG-BC@PBLG·Dox. The results represent the mean ± SD (n = 6). P < 0.05 in comparison with the control group and *P < 0.05 in comparison with PEG-BC@PBLG.

[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

A body-weight decrease can be a measure of drug side-effects. As shown in Fig. 8 (B), the body weight of the normal saline group showed little change, indicating that normal saline was nontoxic, and the small loss of body weight was probably caused by the addition of 0.1% DMSO as a control [38,39]. The free Dox group demonstrated a sharp decrease in the body weight of mice, with a loss of 18.8 ± 2.3% on the 15th day after treatment. Mice treated with PEG-BC@PBLG·Dox micelles showed a lower body weight loss. These results suggest that PEG-BC@PBLG micelles actually enhance the antitumour efficacy of Dox.

[23]

4. Conclusions

[33] [34] [35] [36] [37]

We prepared a pH-sensitive drug carrier using PEG-BC@PBLG micelles based on a boronate ester-linked PEG-BC-PBLG diblock copolymer. This drug delivery system responded to pH variations through its boronate ester bonds. Additionally, the boronate

[24] [25] [26] [27] [28] [29] [30] [31] [32]

[38] [39]

J.A. Hubbell, A. Chilkoti, Science 337 (2012) 303–305. Amit K. Rajora, Divyashree Ravishankar, Polymers 6 (2014) 2186–2220. Kazuo Maruyama, Adv. Drug Delivery Rev. 63 (2011) 161–169. A. Wicki, D. Witzigmann, V. Balasubramanian, J. Controlled Release 200 (2015) 138–157. Vladimir Torchilin, Eur. J. Pharm. Biopharm. 71 (2009) 431–444. Chien-Hsun Wu, Yi-Huei Kuo, Ruey-Long Hong, Sci. Trans. Med. 7 (290) (2015) 290ra91. Hirokazu Sakaguchi, Koji Uesugi, Invest. Ophthalmol. Vis. Sci. 55 (2014) 5272. Weipu Zhu, Ying Wang, Xia Cai, J. Mater. Chem. B 3 (2015) 3024–3031. Yake Xue, Xiaoxing Tang, Jin Huang, Colloids Surf. B 85 (2011) 280–288. Quang Nam Bui, Yi Li, Macromolecules 48 (2015) 4046–4054. R.K. Jain, Adv. Drug Delivery Rev. 46 (2001) 149–168. Jonathan D. Ashley, Jared F. Stefanick, J. Med. Chem. 57 (2014) 5282–5292. Juan Liu, Yuran Huang, Anil Kumar, Biotechnol. Adv. 32 (2014) 693–710. R. Bernardini, A. Oliva, A. Paganelli, E. Menta, Chem. Lett. 38 (2009) 750–751. Mingming Wang, Yu Wang, Ke Hu, Biomater. Sci. 3 (2015) 480–489. Linxian Li, Zewei Bai, Pavel A. Levkin, Biomaterials 34 (2013) 8504–8510. Jesse V. Jorkerst, Tatsiana Lobovkina, HHS Publ. Acess 6 (2011) 715–728. S.M. Moghimi, A.C. Hunter, J.C. Murray, Pharmacol. Rev. 53 (2001) 283–318. S. Takae, K. Miyata, M. Oba, J. Am. Chem. Soc. 130 (2008) 6001–6009. Yang Li, Martin Kroger, Biomaterials 35 (2014) 8467–8478. Ying Wang, Jiannan Xiang, Biomacromolecules 11 (2010) 3531–3538. Gregory A. Ellis, Michael J. Palte, Ronald T. Raines, J. Am. Chem. Soc. 134 (2012) 3631–3634. Wenqian Yang, Shouhai Gao, Xingming Gao, Bioorg. Med. Chem. Lett. 12 (2002) 2175–2177. Qi Peng, Renxi Zhuo, Zhenlin Zhong, Chem. Commun. 46 (2010) 5888–5890. Wei Scarano, Hien T.T. Duong, Hongxu Lu, Biomacromolecules 14 (2013) 962–975. Fang Jun, Hideaki Nakamura, Adv. Drug Delivery Rev. 63 (2011) 136–151. Suhong Wu, Ruogu Qi, Huihui Kuang, ChemPlusChem 00 (2012) 1–11. Y.Q. Yang, Bin Zhao, Wei Lin, Acta Biomater. 9 (2013) 7679–7690. Chen Wang, Haizhu Yu, Org. Biomol. Chem. 11 (2013) 2140–2146. Kamal Aziz Ketuly, A. Hamid, A. Hadi, Molecules 15 (2010) 2347–2356. Agnieszka Adamczyk-Wozniak, Krzysztof M. Borys, Karolina Czerwin ska, Spectrochim. Acta Part A 116 (2013) 616–621. Jing Su, Feng Chen, Vincent L. Cryns, J. Am. Chem. Soc. 133 (2011) 11850–11853. Jennifer N. Cambre, Brent S. Sumerlin, Polymer 52 (2011) 4631–4643. Dong Yang, Xiaohong Zhang, Prog. Nat. Sci. 19 (2009) 1305–1310. Min Huang, Zengshuan Ma, Pharm. Res. 19 (2002) 1488–1494. Zachary L. Tyrrell, Maciej Radosz, Prog. Polym. Sci. 35 (2010) 1128–1143. P. Opanasopit, M. Yokoyama, M. Watanabe, K. Kawano, Y. Maitani, T. Okanoa, J. Controlled Release 104 (2005) 313–321. Keizo Sato, Moritaka Suga, Am. J. Respir. Crit. Care Med. 157 (1998) 853–857. Afia Naaz, Srikanth Yellayi, Endocrinology 144 (2003) 3315–3320.