Materials Science and Engineering C 50 (2015) 341–347
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Stimuli-responsive PEGylated prodrugs for targeted doxorubicin delivery Minghui Xu, Junmin Qian ⁎, Xuefeng Liu, Ting Liu, Hongjie Wang State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 710049, China
a r t i c l e
i n f o
Article history: Received 7 November 2014 Received in revised form 22 January 2015 Accepted 30 January 2015 Available online 31 January 2015 Keywords: Targeted prodrug Reduction/pH dual-sensitivity Poly(ethylene glycol) Doxorubicin Liver cancer
a b s t r a c t In recent years, stimuli-sensitive prodrugs have been extensively studied for the rapid “burst” release of antitumor drugs to enhance chemotherapeutic efficiency. In this study, a novel stimuli-sensitive prodrug containing galactosamine as a targeting moiety, poly(ethylene glycol)–doxorubicin (PEG–DOX) conjugate, was developed for targeting HepG2 human liver cancer cells. To obtain the PEG–DOX conjugate, both galactosamine-decorated poly(ethylene glycol) aldehyde (Gal-PEG-CHO) and methoxy poly(ethylene glycol) aldehyde (mPEG-CHO) were firstly synthesized and functionalized with dithiodipropionate dihydrazide (TPH) through direct reductive amination via Schiff's base formation, and then DOX molecules were chemically conjugated to the hydrazide end groups of TPH-functionalized Gal-/m-PEG chains via pH-sensitive hydrazone linkages. The chemical structures of TPH-functionalized PEG and PEG–DOX prodrug were confirmed by 1H NMR analysis. The PEG–DOX conjugate could self-assemble into spherical nanomicelles with a mean diameter of 140 nm, as indicated by transmission electron microscopy and dynamic light scattering. The drug loading content and loading efficiency in the prodrug nanomicelles were as high as 20 wt.% and 75 wt.%, respectively. In vitro drug release studies showed that DOX was released rapidly from the prodrug nanomicelles at the intracellular levels of pH and reducing agent. Cellular uptake and MTT experiments demonstrated that the galactosamine-decorated prodrug nanomicelles were more efficiently internalized into HepG2 cells via a receptor-mediated endocytosis process and exhibited a higher toxicity, compared with pristine prodrug nanomicelles. These results suggest that the novel Gal-PEG–DOX prodrug nanomicelles have tremendous potential for targeted liver cancer therapy. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy is one of the most common treatments in the fight against cancer. However, the clinical efficacy of conventional therapeutic drugs is always limited by their non-specificity and high systemic toxicity [1]. Doxorubicin (DOX), an anthracyclinic antibiotic, is commonly used as an anticancer drug in the treatment of various cancers, such as ovarian, breast, prostate, cervix and liver cancers [2]. However, the clinical application of DOX is severely hampered by its short halflife and severe toxicity to normal tissues, e.g. cardiotoxicity and myelosuppression [3]. In the past decades, targeted drug delivery strategies for cancer therapy have emerged as a most promising technology platform to overcome these problems [4,5]. Up until now, a large number of drug delivery systems, such as synthetic polymer–drug conjugates [6], liposomes [7], nanomicelles [8], nanogels [9], quantum dots [10] and nanoparticles [11], have been extensively investigated for targeted intracellular delivery of chemotherapeutics. These delivery systems offer several advantages, such as (i) increasing the water solubility of hydrophobic drugs, (ii) protecting drugs from aggregation, degradation and/or deactivation, (iii) enhancing drug bioavailability, (iv) ⁎ Corresponding author. E-mail address: [email protected] (J. Qian).
http://dx.doi.org/10.1016/j.msec.2015.01.098 0928-4931/© 2015 Elsevier B.V. All rights reserved.
prolonging blood circulation time and (v) enabling passive or active targeting of drugs to the tumor tissues and cells [12]. Amongst the various delivery systems, polymer–drug conjugates, in which drugs are chemically coupled on functional end groups or side groups of polymer molecules, have received tremendous interest because of their high loading, controlled drug release and long blood circulation half-life [13,14]. Numerous polymeric prodrugs are investigated in various phases of clinical trials [15]. As a well-known water soluble, non-toxic and non-immunogenic biomedical polymer, poly(ethylene glycol) (PEG) has been extensively used in the preparation of drug delivery systems in order to increase water solubility, reduce immunogenicity, prolong plasma circulation time and improve tumor targeting ability [16]. Many PEG-based prodrugs containing doxorubicin [17], paclitaxel [18] or camptothecin [19] have been developed, and several of them have received market approval. Generally, polymer–drug prodrugs can arrive at tumor cells through passive targeting via the enhanced permeability and retention (EPR) effect. Moreover, targeting ligands such as folate [20], biotin [21] and heparin [22] attached to prodrugs allow active targeting by binding to the receptors overexpressed in cancer cells. As an active targeting moiety for liver cancer, galactosamine (Gal) is able to recognize and bind to the surface asialoglycoprotein (ASGP) receptors overexpressed in human hepatocellular carcinoma cells [23]. Gal-conjugated nanocarriers have been
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successfully developed as targeted drug delivery systems for liver cancer therapy [24,25]. Recent studies indicate that the enhanced therapeutic efficacy is able to be achieved if nanocarriers can accomplish rapid drug release once they arrive at the tumor site [26]. Therefore, new drug delivery systems with smart functions such as active targeting to tumor site and stimulitriggered burst drug release only at targeting site are of high demand in cancer treatment. However, the traditional drug carriers based on biodegradable polymers such as polyesters, polyamino acids and polyurethanes usually undergo gradual degradation kinetics in vivo, which display an extended release of drugs over a period of days to weeks and decrease drug efficiency [27–29]. In the past decade, the tumor microenvironment-activated prodrugs have been extensively studied for the rapid “burst” release of antitumor drugs [30]. A reducing acidic environment in tumor is the premise of designing stimuli-sensitive nanomicelles available for tumor-specific drug delivery. It is reported that the DOX-conjugated prodrugs via hydrazone linkages are stable under physiological conditions (pH 7.4) and can selectively release payloads rapidly under intracellular conditions [31]. Recently, some reduction-sensitive nanocarriers which contain bioreducible disulfide linkages have been designed to trigger burst release of drugs [32]. The disulfide bonds are perceived stable in the slightly oxidizing extracellular surrounding, but may be rapidly cleaved via thiol–disulfide exchange reactions with intracellular reducing molecules, especially with glutathione (GSH), a most abundant thiol species in the cytoplasm [33]. Up to now, stimuli-responsive prodrugs for targeted cancer therapy have attracted special attentions [34,35]. The aim of this study was to develop a novel stimuli-sensitive poly(ethylene glycol)-based prodrug for targeted intracellular delivery of DOX in liver cancer treatment. In the prodrug, DOX was conjugated to poly(ethylene glycol) via a functional linkage containing both bioreducible disulfide and acid-labile hydrazone bonds. The prodrug was expected to be stable under physiological conditions, be selectively taken up by hepatoma carcinoma cells via ASGP receptor-mediated endocytosis, and achieve rapid disassembly at the intracellular levels of pH and glutathiose after internalization into tumor cells, thereby improving the antitumor efficacy of DOX. The chemical structures and drugloading capacity of the prodrug as well as the morphology and physicochemical characteristics of the self-assembled nanomicelles from the prodrugs were investigated in detail. The in vitro evaluation of reduction/pH dual-sensitive drug release behavior was carried out in different reducing acidic environments. The cellular uptake and cytotoxicity of the prodrugs were studied using human liver carcinoma cell line HepG2. The preliminary in vitro results indicated the great potential of the prodrug in improving the efficiency of DOX for liver cancer treatment. 2. Materials and methods 2.1. Materials Galactosamine hydrochloride (Gal·HCl), DL-dithiothreitol (DTT), sodium cyanoborohydride (NaBH3CN), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT), dihydroxy-terminal poly(ethylene glycol) (HO-PEG-OH) and methoxy poly(ethylene glycol) (mPEG-OH) (molecular weight: 2 kDa) were purchased from Sigma-Aldrich. Hydrazine hydrate and 3,3′-dithiodipropionic acid were purchased from Aladdin Reagent Inc. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen. All other chemicals were of analytical grade and were used without further purification. All aqueous solutions were prepared using ultrapure water with a resistance of 18.25 MΩ.
2.2. Synthesis of PEG–DOX prodrug 2.2.1. Synthesis of dithiodipropionate dihydrazide functionalized PEG (PEGTPH) Dithiodipropionate dihydrazide (TPH) was synthesized according to the reported procedure [36,37]. Briefly, 3,3′-dithiodipropionic acid (10 g, 47.6 mmol) and ethanol (22.1 g, 480 mmol) were dissolved in 30 mL of toluene containing p-toluenesulfonic acid (0.4 g, 2.33 mmol) catalyst, and the reaction mixture was heated under reflux for 5 h using a Dean–Stark trap. After the solvent had been removed by vacuum evaporation, the intermediate product dithiodipropionate diethyl was washed with 5% aqueous sodium bicarbonate solution and distilled water until the filtrate became neutral. After drying in vacuo at 40 °C, dithiodipropionate diethyl was obtained in 94% yield. After that, dithiodipropionate diethyl (10 g, 37.5 mmol) and hydrazine hydrate (12 g, 0.24 mol) were dissolved in 20 mL of ethanol, and the reaction mixture was heated under reflux for 4 h and then left at 4 °C overnight. The precipitated solid was collected, recrystallized from a 1:3 water/ ethanol mixture and dried at 40 °C for 48 h in vacuo. Dithiodipropionate dihydrazide was obtained in 68% yield. m.p.: 131–132 °C. 1H NMR (DMSO-d6, Bruker 400 MHz, δppm): 2.53 (t, 2H,\NH\CH2\), 2.85 (t, 2H,\S\CH2\), 4.2 (s, 2H,\NH2), 9.1 (s, H,\NH\). Poly(ethylene glycol)-dialdehyde (OHC-PEG-CHO) and methoxy poly(ethylene glycol)-aldehyde (mPEG-CHO) were synthesized by the oxidation of HO-PEG-OH and mPEG-OH with dimethylsulfoxide/acetic anhydride according to the reported procedure [38,39], respectively. Gal-conjugated PEG-aldehyde (Gal-PEG-CHO) was synthesized via the selective reductive amination of Schiff's base. Briefly, Gal·HCl (172.4 mg, 0.8 mmol) was dissolved in 2 mL of water and neutralized with a 0.1 mol/L sodium hydrate solution. The obtained solution was added dropwise under vigorous stirring to 10 mL of aqueous solution of OHC-PEG-CHO (2 g, 1 mmol), and then an aqueous solution of NaBH3CN (100 mg, 1.6 mmol, in 4 mL water) was added. The solution was stirred for 24 h at room temperature, and the product was extracted with dichloromethane for three times. The collected organic phase was concentrated and precipitated in excess of diethyl ether. After drying under vacuum, Gal-PEG-CHO was obtained as a pale yellow powder. IR data (IR Prestige-21 FTIR spectrometer, Shimadzu, Japan): 1736 cm− 1 for C_O stretching vibrations in OHC-PEG-CHO; 1728 cm− 1 for C_O stretching vibrations in Gal-PEG-CHO, 1060 cm−1 for its C\N stretching vibrations [40]. Dithiodipropionate dihydrazide functionalized PEG (PEG-TPH) was synthesized through reductive amination via Schiff's base formation. A mixture of mPEG-CHO (950 mg, 0.475 mmol) and Gal-PEG-CHO (50 mg, 0.025 mmol) was dissolved in water (10 mL), and then TPH (1.785 g, 7.5 mmol, 15 eq) was added. The pH value of solution was adjusted to 6.5 using 0.1 mol/L hydrochloric acid. The mixture solution was stirred for 24 h, and then NaBH3CN (125.7 mg, 5 mmol, 10 eq, with respect to aldehyde groups) was added. The solution was stirred for another 24 h at room temperature. The final solution was dialyzed with dialysis membrane (MWCO 1000) against distilled water for 2 days to remove unreacted TPH. Lyophilized PEG-TPH was obtained as a white powder. 2.2.2. Chemical conjugation of DOX to PEG-TPH DOX was chemically conjugated to PEG-TPH via a hydrazone linkage formed between the ketone group of DOX and the hydrazide end group of PEG-TPH. In a typical synthesis, PEG-TPH (30 mg) dissolved in 10 mL of dimethyl sulfoxide (DMSO) was reacted with DOX·HCl (8 mg) in the presence of triethylamine (1.92 μL) for 24 h at room temperature. The solution was dialyzed against 2 mM phosphate buffered saline (PBS) buffer (pH 7.4) until no further color change was observed. The solution was protected from light all the time. The amount of free DOX in PBS buffer was determined by UV/Vis spectrophotometry at 485 nm using a standard calibration curve (1–100 μg/mL). Drug loading content (DLC) and drug loading
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Scheme 1. Synthetic scheme of reduction/pH dual-sensitive PEG–DOX prodrug and illustration of PEG–DOX prodrug nanomicelle.
efficiency (DLE) were calculated according to Eqs. (1) and (2), respectively. DLCðwt:%Þ ¼
weight of loaded drug 100 weight of polymer
ð1Þ
DLEðwt:%Þ ¼
weight of loaded drug 100 weight of drug in feed
ð2Þ
2.3. Preparation and characterization of PEG–DOX prodrug nanomicelles To obtain nanomicelles, PEG–DOX prodrug (3 mg) was added to 3 mL of H2O, and the system was sonicated for 10 min using a probetype ultrasonicator (JY 92-2D; Ningbo Scientz Biotechnology Co., Ltd, Nanjing, China) at 100 W in an ice bath. The diameter of the nanomicelles self-assembled from the PEG–DOX prodrug was measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 instrument (Malvern instruments, UK). The DLS measurements were performed at a fixed scattering angle of 90°. The morphology and size of the nanomicelles were observed by a transmission electron microscope (JEM-2000CX, JEOL, Japan).
desired time intervals, 100 μL aliquots from the bags were analyzed by UV/Vis spectrophotometry, and the DOX concentration was calculated based on the absorbance intensity at 485 nm. Meanwhile, the diameters of the nanomicelles were measured by DLS at predefined time points. The release experiments were conducted in triplicate, and the presented results were average values. 2.5. Cellular uptake study HepG2 cells were seeded in a 6-well plate at a density of 1 × 105 cells/well and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS for 24 h in a humidified atmosphere with 5% CO2 at 37 °C. The media were replaced with 2 mL of fresh media containing 2 mg of PEG–DOX or Gal-decorated PEG–DOX prodrug nanomicelles. After incubation at 37 °C for 1, 4 and 24 h, the culture media were discarded, and the cells were rinsed three times with PBS to remove the prodrug nanomicelles that were not taken up by cells. After that, the cells were fixed with 4% paraformaldehyde, and 200 μL of DAPI solution was added into each well to stain cell nuclei. The cellular uptake of PEG–DOX prodrugs was observed and imaged using fluorescence microscopy. 2.6. Cytotoxicity assay
2.4. In vitro release of DOX from nanomicelles triggered by DTT and pH The in vitro release profiles of DOX from the prodrug nanomicelles were studied using a dialysis method in six different media: PBS buffer (pH 7.4, 6.0 and 5.0) with and without 10 mM DTT. Briefly, a suspension of 5 mg of PEG–DOX prodrug nanomicelles in 5 mL of water was placed in a dialysis tube (MWCO 1000), and the tube was immersed in PBS buffer and shaken at a speed of 150 rpm at room temperature. At
The cytotoxicity of Gal-PEG–DOX, mPEG–DOX and PEG-TPH to HepG2 cells was studied using MTT assay. In brief, HepG2 cells (3 × 103) were seeded in 96-well plates and incubated in DMEM containing 10% FBS overnight. The media were replaced with fresh media containing PEG–DOX prodrugs at various DOX concentrations of 5–80 μg/mL or PEG-TPH at concentrations ranging from 0.001 to 8 mg/mL (up to 40 × polymer content in the PEG–DOX prodrug at a
Fig. 1. 1H NMR spectra (in DMSO-d6) of (A) mPEG-TPH and (B) mPEG–DOX prodrug.
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Fig. 2. (A) Size distribution and (B) morphology of PEG–DOX prodrug nanomicelles as measured by DLS and TEM, respectively.
DOX concentration of 40 μg/mL) for 48 h at 37 °C in a humidified 5% CO2-containing atmosphere, using the cells grown in a pristine medium as a control. Afterwards, a stock solution (20 μL) containing 0.1 mg of MTT in PBS was added into each well. The plates were then incubated at 37 °C for 4 h to allow MTT metabolism. After the media had been discarded, 150 μL of DMSO was added to each well and the plates were incubated for 10 min at room temperature in the dark. The absorbance at a wavelength of 490 nm of each well was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 490 nm with control wells containing only a cell culture medium. Data were presented as mean ± standard deviation (SD) (n = 3). 3. Results and discussion 3.1. Synthesis of PEG–DOX prodrug The aim of the present study was to synthesize a novel reduction/pH dual-sensitive PEG-based prodrug system, which could self-assemble in aqueous media into the nanomicelles. The nanomicelles were expected to be stable under physiological conditions, be selectively taken up by liver cancer cells via ASGP receptor-mediated endocytosis, and achieve rapid disassembly at the intracellular levels of pH and reducing agent. The synthetic route of PEG–DOX prodrug is shown in Scheme 1. A mixture of Gal-PEG-CHO and mPEG-CHO was firstly functionalized with TPH by the reduction amination of the Schiff's base formed between the aldehyde groups of Gal-/m-PEG-CHO and the amino groups of TPH molecules. DOX was conjugated onto the TPH-functionalized PEG through a hydrazone linkage formed between the ketone group of DOX and the hydrazide end group of PEG-TPH. The obtained PEG–DOX prodrug was amphiphilic and could self-assemble into nanomicelle in an aqueous solution with DOX as the core and PEG as the shell. The chemical structures of mPEG-TPH and mPEG–DOX prodrug were characterized by 1H NMR spectra, as shown in Fig. 1. The proton signals of TPH at 2.91 and 3.43 ppm appeared in Fig. 1A, suggesting the successful synthesis of PEG-TPH. As revealed in Fig. 1B, the chemical shifts at 1.22, 2.02, 4.59, 5.48, and 7.69–7.95 ppm originated from DOX demonstrated that the DOX molecules were successfully conjugated onto the PEG-TPH molecules [41,42].
with an average size of ~100 nm (Fig. 2B). Notably, the average diameter determined by TEM was smaller than that measured by DLS. It is most likely because DLS data reflected the hydrodynamic size of the nanomicelles while TEM image showed the size of dry nanomicelles. The nanomicelles would shrink during the drying process [44]. It has been reported that the nanomicelles with the size of b200 nm will accumulate in tumor sites due to the EPR effect [45,46]. Therefore, the prodrug synthesized in this study should be a promising drug delivery system. 3.3. DOX loading and in vitro DOX release In order to conjugate DOX onto PEG-TPH, the reaction between PEGTPH and DOX·HCl was performed in DMSO for 24 h and then the solution was dialyzed against 2 mM of PBS buffer (pH 7.4) to remove unconjugated DOX molecules. The DLE of PEG–DOX was 75%, and the DLC was as high as 20%. The high DLC should be the result of the loading of DOX in the form of PEG-based prodrug. Recent studies reveal that prodrugs with a stimuli-responsive character are more ideal for drug delivery [30]. The prodrug designed in the present study was expected to display a pH and reduction dualresponsive fashion. The in vitro release of DOX from the nanomicelles was investigated using a dialysis method in PBS buffer (pH 7.4, 6.0 and 5.0) in the presence or absence of 10 mM DTT. The pH and DTT levels were chosen to simulate those in the endosomal and lysosomal vesicles inside the cancer cells, whose pH values and GSH concentrations are 4–6 and 2–10 mM, respectively. As shown in Fig. 3, the release of DOX exhibited a biphasic pattern, in which a rapid release in the initial stage of 12 h was followed by a sustained release over a prolonged time period of up to 120 h. Similar phenomenon was observed in
3.2. Characterization of PEG–DOX prodrug nanomicelles The size distribution of nanocarriers plays an important role in intravenous administration [43]. To obtain a well-distributed nanomicelle solution, PEG–DOX prodrug nanomicelles were prepared by the ultrasonic method. The DLS results showed that the nanomicelles formed from the PEG–DOX prodrug had an average diameter of 140 ± 5.4 nm (Fig. 2A). Transmission electron microscopy (TEM) micrograph revealed that the PEG–DOX prodrug nanomicelles showed a spherical morphology
Fig. 3. In vitro reduction/pH-triggered release profiles of DOX from PEG–DOX prodrug nanomicelles in PBS buffer (pH 7.4, 6.0, and 5.0) with and without 10 mM DTT.
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Fig. 4. Change in the size of PEG–DOX prodrug nanomicelles in PBS buffer (pH 7.4 with and without DTT, pH 5.0 with 10 mM DTT) with increasing release time.
other DOX-based prodrug systems [47]. It was found that a minimal amount of drug (b 15%) was released prior to the appearance of the plateau at pH 7.4 in the absence of DTT, which revealed that hydrazone linkages were relatively stable under neutral pH environment. The release rate of DOX was markedly accelerated when decreasing pH from 7.4 to 5.0 regardless of DTT. The faster release of DOX was attributed to the acidic catalytic cleavage of hydrazone linkage between PEG and DOX, thus releasing the conjugated DOX [48]. In addition, because of the basic nature of DOX (pKa = 8.3), the released DOX would become water-soluble in the acidic environments [49]. Compared with the release profiles in the absence of DTT, the release rate of DOX from the prodrugs in the presence of DTT (10 mM) was much higher regardless of pH values. For example, more than 50% of DOX was released under acidic reducing conditions (pH 5.0, 10 mM DTT) within the first 12 h, and over 90% of DOX was released after 120 h. The accelerated release was due to the dissociation of prodrug caused by the reductiontriggered cleavage of disulfide linkage between PEG and DOX via
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thiol–disulfide exchange reactions [50]. The results meant that the prodrug was stable under physiological conditions, and its pH/ reduction-dependent DOX release property might ensure the intracellular rapid release of DOX in the reductive acidic microenvironment of cancer cells. Therefore, the PEG–DOX prodrug is an ideal vehicle for targeted intracellular delivery. Some studies suggest that the detachment of hydrophobic drug from the micelles can trigger the destabilization of the hydrophobic core and aggregate the micelles together [51,52]. In this study, the change in nanomicelle size during the release of DOX was monitored by DLS measurement, and the results are shown in Fig. 4. Compared with the average diameter of the nanomicelles in PBS (pH 7.4 with 10 mM DTT) which increased from 140 to 413 nm in the first 12 h and increased to 762 nm at 60 h, it is found that the average diameter of the nanomicelles in PBS (pH 5.0 with 10 mM DTT) increased faster from 140 to 890 nm in the first 12 h and increased to 1660 nm at 60 h time point. In contrast, the diameter of the nanomicelles in PBS (pH 7.4) slightly increased from 140 to 210 nm in the initial stage of 12 h and kept stable in the remaining time. The trend was according with that in the drug release studies. The faster increase in the micelle size in the reducing acidic microenvironment (pH 5.0, 10 mM DTT) than under the physiological conditions (pH 7.4) was attributed to the combination of pH-sensitive cleavage of hydrazone bond and reduction-triggered breakage of disulfide bond between the PEG and DOX, which resulted in the rapid release of DOX. The detachment of hydrophobic drug from the micelles aggregated the micelles together [53,54]. 3.4. Cellular uptake study It has been proved that the ASGP receptor is abundantly expressed in various human hepatocellular carcinoma cell lines, including HepG2 [55]. To visually investigate the cellular uptake of PEG–DOX prodrug nanomicelles, the cell nuclei were fluorescently stained blue with DAPI. To assess the internalization capacity of the nanomicelles in HepG2 cells, HepG2 cells were incubated with Gal-PEG–DOX prodrug nanomicelles for 1, 4 and 24 h, using the HepG2 cells treated with Gal-unconjugated prodrug nanomicelles as a control group. Their
Fig. 5. Fluorescent images of HepG2 cells incubated with (left two columns) mPEG–DOX and (right two columns) Gal-PEG–DOX prodrug nanomicelles for (A, B) 1, (C, D) 4 and (E, F) 24 h. In each panel, the two fluorescent images from left to right show the fluorescent image of DOX and the overlaid fluorescent image of DOX and DAPI, respectively. Scale bars correspond to 40 μm.
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Fig. 6. Cytotoxicity of PEG-TPH to HepG2 cells at a concentration range of 0.001 to 8 mg/mL after incubation for 48 h. The cell viability was determined by MTT assay (n = 3).
fluorescent images are shown in Fig. 5. As revealed in Fig. 5, in the experimental group, the fluorescence intensity of DOX in the cytoplasm significantly increased with the prolongation of incubation time. In contrast, the fluorescence intensity of DOX in HepG2 cells in the control group was much weaker than that in the experimental group cells at the same culture time. Such results indicated that the Gal-PEG–DOX prodrug nanomicelles could be taken up more effectively by HepG2 cells than the mPEG–DOX prodrug nanomicelles. The most possible reason is that HepG2 cells overexpress ASGP receptors, and the ligand Galdecorated prodrug nanomicelles can recognize and bind to the ASGP receptors and be taken up rapidly and specifically by HepG2 cells via ASGP receptor-mediated endocytosis [56]. These results demonstrated that the Gal-PEG–DOX prodrug would be a great promising approach for the targeted delivery of DOX to hepatocellular carcinoma. 3.5. In vitro cytotoxicity studies The cytotoxicity of PEG-TPH and PEG–DOX prodrug against HepG2 cells was investigated by MTT assay. HepG2 cells were incubated with PEG-TPH or PEG–DOX prodrug for 48 h. The results (Fig. 6) revealed that PEG-TPH was non-toxic to HepG2 cells at concentrations of up to 0.5 mg/mL, which approximated the content of polymer in the PEG– DOX prodrug at a DOX concentration of 100 μg/mL. PEG-TPH still did not show any evident toxicity even when its concentration was up to 8 mg/mL, indicating that PEG-TPH possessed excellent biocompatibility similar to PEG [57]. However, under otherwise the same conditions,
PEG–DOX prodrug showed pronounced cytotoxic effects (Fig. 7). For example, Gal-PEG–DOX and mPEG–DOX prodrugs significantly decreased the cell viability of HepG2 cells by 40.8% and 58% at a DOX concentration of 40 μg/mL, respectively. At a higher DOX concentration of 80 μg/mL, the percentages of viable HepG2 cells further decreased to 34.1% and 42.6%, respectively. The IC50 (i.e., inhibitory concentration to produce 50% cell death) of Gal-PEG–DOX prodrug was obviously different from that of mPEG–DOX prodrug. Their IC50 values for HepG2 cells were approximately 6.54 and 60.8 μg/mL (equivalent DOX concentrations), respectively. The results indicated that Gal-PEG–DOX prodrug could be taken up more effectively by HepG2 cells in comparison with mPEG– DOX prodrug, because Gal-decorated prodrug nanomicelles could recognize the ASGP receptors expressed on the surface of HepG2 cells [58]. Therefore, the more DOX had entered HepG2 cells, the more toxicity. Moreover, the prodrugs have to be activated in endosomal compartments to exert the cytostatic effects. All the results showed that the cellular uptake and antitumor activity of PEG–DOX prodrug could be enhanced through the installment of a specific targeting ligand, which made this system particularly attractive for tumor-targeted drug delivery applications. 4. Conclusions In this study, a novel stimuli-sensitive PEG–DOX prodrug has been successfully synthesized. DOX was conjugated onto poly(ethylene glycol) via a functional linkage containing disulfide and hydrazone bonds. The PEG–DOX prodrug could self-assemble in an aqueous solution into spherical nanomicelles with an average diameter of 140 nm. The release of DOX from the nanomicelles at neutral pH exhibited a slow and sustained behavior, while more than 50% of total loaded DOX amount could be released within the first 12 h under acidic reducing microenvironment mimicking the intercellular conditions in tumor. The cellar uptake and cytotoxicity studies confirmed that the Gal-decorated PEG– DOX prodrug nanomicelles could specifically interact with HepG2 cells and be internalized into HepG2 cells via receptor-mediated endocytosis, showing enhanced cytotoxicity. Therefore, the novel PEG–DOX prodrug nanomicelles have great potential for targeted intracellular delivery of DOX and for improving the anticancer efficiency of DOX. Acknowledgments The authors gratefully acknowledge the National Natural Science Foundation of China (50773062, 50603020), the Natural Science Basic Research Plan in Shaanxi Province of China (2013K09-27, 2011K1301-09) and the Fundamental Research Funds for the Central Universities (XJJ2014124, XJJ2013130, XJJ2012146) for their financial support of this research. References
Fig. 7. Cytotoxicity of mPEG–DOX and Gal-PEG–DOX prodrugs to HepG2 cells at various DOX concentrations of 5–80 μg/mL after incubation for 48 h. The cell viability was determined by MTT assay (n = 3).
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Stimuli-responsive PEGylated prodrugs for targeted doxorubicin delivery Minghui Xu, Junmin Qian ⁎, Xuefeng Liu, Ting Liu, Hongjie Wang State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 710049, China
a r t i c l e
i n f o
Article history: Received 7 November 2014 Received in revised form 22 January 2015 Accepted 30 January 2015 Available online 31 January 2015 Keywords: Targeted prodrug Reduction/pH dual-sensitivity Poly(ethylene glycol) Doxorubicin Liver cancer
a b s t r a c t In recent years, stimuli-sensitive prodrugs have been extensively studied for the rapid “burst” release of antitumor drugs to enhance chemotherapeutic efficiency. In this study, a novel stimuli-sensitive prodrug containing galactosamine as a targeting moiety, poly(ethylene glycol)–doxorubicin (PEG–DOX) conjugate, was developed for targeting HepG2 human liver cancer cells. To obtain the PEG–DOX conjugate, both galactosamine-decorated poly(ethylene glycol) aldehyde (Gal-PEG-CHO) and methoxy poly(ethylene glycol) aldehyde (mPEG-CHO) were firstly synthesized and functionalized with dithiodipropionate dihydrazide (TPH) through direct reductive amination via Schiff's base formation, and then DOX molecules were chemically conjugated to the hydrazide end groups of TPH-functionalized Gal-/m-PEG chains via pH-sensitive hydrazone linkages. The chemical structures of TPH-functionalized PEG and PEG–DOX prodrug were confirmed by 1H NMR analysis. The PEG–DOX conjugate could self-assemble into spherical nanomicelles with a mean diameter of 140 nm, as indicated by transmission electron microscopy and dynamic light scattering. The drug loading content and loading efficiency in the prodrug nanomicelles were as high as 20 wt.% and 75 wt.%, respectively. In vitro drug release studies showed that DOX was released rapidly from the prodrug nanomicelles at the intracellular levels of pH and reducing agent. Cellular uptake and MTT experiments demonstrated that the galactosamine-decorated prodrug nanomicelles were more efficiently internalized into HepG2 cells via a receptor-mediated endocytosis process and exhibited a higher toxicity, compared with pristine prodrug nanomicelles. These results suggest that the novel Gal-PEG–DOX prodrug nanomicelles have tremendous potential for targeted liver cancer therapy. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy is one of the most common treatments in the fight against cancer. However, the clinical efficacy of conventional therapeutic drugs is always limited by their non-specificity and high systemic toxicity [1]. Doxorubicin (DOX), an anthracyclinic antibiotic, is commonly used as an anticancer drug in the treatment of various cancers, such as ovarian, breast, prostate, cervix and liver cancers [2]. However, the clinical application of DOX is severely hampered by its short halflife and severe toxicity to normal tissues, e.g. cardiotoxicity and myelosuppression [3]. In the past decades, targeted drug delivery strategies for cancer therapy have emerged as a most promising technology platform to overcome these problems [4,5]. Up until now, a large number of drug delivery systems, such as synthetic polymer–drug conjugates [6], liposomes [7], nanomicelles [8], nanogels [9], quantum dots [10] and nanoparticles [11], have been extensively investigated for targeted intracellular delivery of chemotherapeutics. These delivery systems offer several advantages, such as (i) increasing the water solubility of hydrophobic drugs, (ii) protecting drugs from aggregation, degradation and/or deactivation, (iii) enhancing drug bioavailability, (iv) ⁎ Corresponding author. E-mail address: [email protected] (J. Qian).
http://dx.doi.org/10.1016/j.msec.2015.01.098 0928-4931/© 2015 Elsevier B.V. All rights reserved.
prolonging blood circulation time and (v) enabling passive or active targeting of drugs to the tumor tissues and cells [12]. Amongst the various delivery systems, polymer–drug conjugates, in which drugs are chemically coupled on functional end groups or side groups of polymer molecules, have received tremendous interest because of their high loading, controlled drug release and long blood circulation half-life [13,14]. Numerous polymeric prodrugs are investigated in various phases of clinical trials [15]. As a well-known water soluble, non-toxic and non-immunogenic biomedical polymer, poly(ethylene glycol) (PEG) has been extensively used in the preparation of drug delivery systems in order to increase water solubility, reduce immunogenicity, prolong plasma circulation time and improve tumor targeting ability [16]. Many PEG-based prodrugs containing doxorubicin [17], paclitaxel [18] or camptothecin [19] have been developed, and several of them have received market approval. Generally, polymer–drug prodrugs can arrive at tumor cells through passive targeting via the enhanced permeability and retention (EPR) effect. Moreover, targeting ligands such as folate [20], biotin [21] and heparin [22] attached to prodrugs allow active targeting by binding to the receptors overexpressed in cancer cells. As an active targeting moiety for liver cancer, galactosamine (Gal) is able to recognize and bind to the surface asialoglycoprotein (ASGP) receptors overexpressed in human hepatocellular carcinoma cells [23]. Gal-conjugated nanocarriers have been
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successfully developed as targeted drug delivery systems for liver cancer therapy [24,25]. Recent studies indicate that the enhanced therapeutic efficacy is able to be achieved if nanocarriers can accomplish rapid drug release once they arrive at the tumor site [26]. Therefore, new drug delivery systems with smart functions such as active targeting to tumor site and stimulitriggered burst drug release only at targeting site are of high demand in cancer treatment. However, the traditional drug carriers based on biodegradable polymers such as polyesters, polyamino acids and polyurethanes usually undergo gradual degradation kinetics in vivo, which display an extended release of drugs over a period of days to weeks and decrease drug efficiency [27–29]. In the past decade, the tumor microenvironment-activated prodrugs have been extensively studied for the rapid “burst” release of antitumor drugs [30]. A reducing acidic environment in tumor is the premise of designing stimuli-sensitive nanomicelles available for tumor-specific drug delivery. It is reported that the DOX-conjugated prodrugs via hydrazone linkages are stable under physiological conditions (pH 7.4) and can selectively release payloads rapidly under intracellular conditions [31]. Recently, some reduction-sensitive nanocarriers which contain bioreducible disulfide linkages have been designed to trigger burst release of drugs [32]. The disulfide bonds are perceived stable in the slightly oxidizing extracellular surrounding, but may be rapidly cleaved via thiol–disulfide exchange reactions with intracellular reducing molecules, especially with glutathione (GSH), a most abundant thiol species in the cytoplasm [33]. Up to now, stimuli-responsive prodrugs for targeted cancer therapy have attracted special attentions [34,35]. The aim of this study was to develop a novel stimuli-sensitive poly(ethylene glycol)-based prodrug for targeted intracellular delivery of DOX in liver cancer treatment. In the prodrug, DOX was conjugated to poly(ethylene glycol) via a functional linkage containing both bioreducible disulfide and acid-labile hydrazone bonds. The prodrug was expected to be stable under physiological conditions, be selectively taken up by hepatoma carcinoma cells via ASGP receptor-mediated endocytosis, and achieve rapid disassembly at the intracellular levels of pH and glutathiose after internalization into tumor cells, thereby improving the antitumor efficacy of DOX. The chemical structures and drugloading capacity of the prodrug as well as the morphology and physicochemical characteristics of the self-assembled nanomicelles from the prodrugs were investigated in detail. The in vitro evaluation of reduction/pH dual-sensitive drug release behavior was carried out in different reducing acidic environments. The cellular uptake and cytotoxicity of the prodrugs were studied using human liver carcinoma cell line HepG2. The preliminary in vitro results indicated the great potential of the prodrug in improving the efficiency of DOX for liver cancer treatment. 2. Materials and methods 2.1. Materials Galactosamine hydrochloride (Gal·HCl), DL-dithiothreitol (DTT), sodium cyanoborohydride (NaBH3CN), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT), dihydroxy-terminal poly(ethylene glycol) (HO-PEG-OH) and methoxy poly(ethylene glycol) (mPEG-OH) (molecular weight: 2 kDa) were purchased from Sigma-Aldrich. Hydrazine hydrate and 3,3′-dithiodipropionic acid were purchased from Aladdin Reagent Inc. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen. All other chemicals were of analytical grade and were used without further purification. All aqueous solutions were prepared using ultrapure water with a resistance of 18.25 MΩ.
2.2. Synthesis of PEG–DOX prodrug 2.2.1. Synthesis of dithiodipropionate dihydrazide functionalized PEG (PEGTPH) Dithiodipropionate dihydrazide (TPH) was synthesized according to the reported procedure [36,37]. Briefly, 3,3′-dithiodipropionic acid (10 g, 47.6 mmol) and ethanol (22.1 g, 480 mmol) were dissolved in 30 mL of toluene containing p-toluenesulfonic acid (0.4 g, 2.33 mmol) catalyst, and the reaction mixture was heated under reflux for 5 h using a Dean–Stark trap. After the solvent had been removed by vacuum evaporation, the intermediate product dithiodipropionate diethyl was washed with 5% aqueous sodium bicarbonate solution and distilled water until the filtrate became neutral. After drying in vacuo at 40 °C, dithiodipropionate diethyl was obtained in 94% yield. After that, dithiodipropionate diethyl (10 g, 37.5 mmol) and hydrazine hydrate (12 g, 0.24 mol) were dissolved in 20 mL of ethanol, and the reaction mixture was heated under reflux for 4 h and then left at 4 °C overnight. The precipitated solid was collected, recrystallized from a 1:3 water/ ethanol mixture and dried at 40 °C for 48 h in vacuo. Dithiodipropionate dihydrazide was obtained in 68% yield. m.p.: 131–132 °C. 1H NMR (DMSO-d6, Bruker 400 MHz, δppm): 2.53 (t, 2H,\NH\CH2\), 2.85 (t, 2H,\S\CH2\), 4.2 (s, 2H,\NH2), 9.1 (s, H,\NH\). Poly(ethylene glycol)-dialdehyde (OHC-PEG-CHO) and methoxy poly(ethylene glycol)-aldehyde (mPEG-CHO) were synthesized by the oxidation of HO-PEG-OH and mPEG-OH with dimethylsulfoxide/acetic anhydride according to the reported procedure [38,39], respectively. Gal-conjugated PEG-aldehyde (Gal-PEG-CHO) was synthesized via the selective reductive amination of Schiff's base. Briefly, Gal·HCl (172.4 mg, 0.8 mmol) was dissolved in 2 mL of water and neutralized with a 0.1 mol/L sodium hydrate solution. The obtained solution was added dropwise under vigorous stirring to 10 mL of aqueous solution of OHC-PEG-CHO (2 g, 1 mmol), and then an aqueous solution of NaBH3CN (100 mg, 1.6 mmol, in 4 mL water) was added. The solution was stirred for 24 h at room temperature, and the product was extracted with dichloromethane for three times. The collected organic phase was concentrated and precipitated in excess of diethyl ether. After drying under vacuum, Gal-PEG-CHO was obtained as a pale yellow powder. IR data (IR Prestige-21 FTIR spectrometer, Shimadzu, Japan): 1736 cm− 1 for C_O stretching vibrations in OHC-PEG-CHO; 1728 cm− 1 for C_O stretching vibrations in Gal-PEG-CHO, 1060 cm−1 for its C\N stretching vibrations [40]. Dithiodipropionate dihydrazide functionalized PEG (PEG-TPH) was synthesized through reductive amination via Schiff's base formation. A mixture of mPEG-CHO (950 mg, 0.475 mmol) and Gal-PEG-CHO (50 mg, 0.025 mmol) was dissolved in water (10 mL), and then TPH (1.785 g, 7.5 mmol, 15 eq) was added. The pH value of solution was adjusted to 6.5 using 0.1 mol/L hydrochloric acid. The mixture solution was stirred for 24 h, and then NaBH3CN (125.7 mg, 5 mmol, 10 eq, with respect to aldehyde groups) was added. The solution was stirred for another 24 h at room temperature. The final solution was dialyzed with dialysis membrane (MWCO 1000) against distilled water for 2 days to remove unreacted TPH. Lyophilized PEG-TPH was obtained as a white powder. 2.2.2. Chemical conjugation of DOX to PEG-TPH DOX was chemically conjugated to PEG-TPH via a hydrazone linkage formed between the ketone group of DOX and the hydrazide end group of PEG-TPH. In a typical synthesis, PEG-TPH (30 mg) dissolved in 10 mL of dimethyl sulfoxide (DMSO) was reacted with DOX·HCl (8 mg) in the presence of triethylamine (1.92 μL) for 24 h at room temperature. The solution was dialyzed against 2 mM phosphate buffered saline (PBS) buffer (pH 7.4) until no further color change was observed. The solution was protected from light all the time. The amount of free DOX in PBS buffer was determined by UV/Vis spectrophotometry at 485 nm using a standard calibration curve (1–100 μg/mL). Drug loading content (DLC) and drug loading
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Scheme 1. Synthetic scheme of reduction/pH dual-sensitive PEG–DOX prodrug and illustration of PEG–DOX prodrug nanomicelle.
efficiency (DLE) were calculated according to Eqs. (1) and (2), respectively. DLCðwt:%Þ ¼
weight of loaded drug 100 weight of polymer
ð1Þ
DLEðwt:%Þ ¼
weight of loaded drug 100 weight of drug in feed
ð2Þ
2.3. Preparation and characterization of PEG–DOX prodrug nanomicelles To obtain nanomicelles, PEG–DOX prodrug (3 mg) was added to 3 mL of H2O, and the system was sonicated for 10 min using a probetype ultrasonicator (JY 92-2D; Ningbo Scientz Biotechnology Co., Ltd, Nanjing, China) at 100 W in an ice bath. The diameter of the nanomicelles self-assembled from the PEG–DOX prodrug was measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 instrument (Malvern instruments, UK). The DLS measurements were performed at a fixed scattering angle of 90°. The morphology and size of the nanomicelles were observed by a transmission electron microscope (JEM-2000CX, JEOL, Japan).
desired time intervals, 100 μL aliquots from the bags were analyzed by UV/Vis spectrophotometry, and the DOX concentration was calculated based on the absorbance intensity at 485 nm. Meanwhile, the diameters of the nanomicelles were measured by DLS at predefined time points. The release experiments were conducted in triplicate, and the presented results were average values. 2.5. Cellular uptake study HepG2 cells were seeded in a 6-well plate at a density of 1 × 105 cells/well and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS for 24 h in a humidified atmosphere with 5% CO2 at 37 °C. The media were replaced with 2 mL of fresh media containing 2 mg of PEG–DOX or Gal-decorated PEG–DOX prodrug nanomicelles. After incubation at 37 °C for 1, 4 and 24 h, the culture media were discarded, and the cells were rinsed three times with PBS to remove the prodrug nanomicelles that were not taken up by cells. After that, the cells were fixed with 4% paraformaldehyde, and 200 μL of DAPI solution was added into each well to stain cell nuclei. The cellular uptake of PEG–DOX prodrugs was observed and imaged using fluorescence microscopy. 2.6. Cytotoxicity assay
2.4. In vitro release of DOX from nanomicelles triggered by DTT and pH The in vitro release profiles of DOX from the prodrug nanomicelles were studied using a dialysis method in six different media: PBS buffer (pH 7.4, 6.0 and 5.0) with and without 10 mM DTT. Briefly, a suspension of 5 mg of PEG–DOX prodrug nanomicelles in 5 mL of water was placed in a dialysis tube (MWCO 1000), and the tube was immersed in PBS buffer and shaken at a speed of 150 rpm at room temperature. At
The cytotoxicity of Gal-PEG–DOX, mPEG–DOX and PEG-TPH to HepG2 cells was studied using MTT assay. In brief, HepG2 cells (3 × 103) were seeded in 96-well plates and incubated in DMEM containing 10% FBS overnight. The media were replaced with fresh media containing PEG–DOX prodrugs at various DOX concentrations of 5–80 μg/mL or PEG-TPH at concentrations ranging from 0.001 to 8 mg/mL (up to 40 × polymer content in the PEG–DOX prodrug at a
Fig. 1. 1H NMR spectra (in DMSO-d6) of (A) mPEG-TPH and (B) mPEG–DOX prodrug.
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Fig. 2. (A) Size distribution and (B) morphology of PEG–DOX prodrug nanomicelles as measured by DLS and TEM, respectively.
DOX concentration of 40 μg/mL) for 48 h at 37 °C in a humidified 5% CO2-containing atmosphere, using the cells grown in a pristine medium as a control. Afterwards, a stock solution (20 μL) containing 0.1 mg of MTT in PBS was added into each well. The plates were then incubated at 37 °C for 4 h to allow MTT metabolism. After the media had been discarded, 150 μL of DMSO was added to each well and the plates were incubated for 10 min at room temperature in the dark. The absorbance at a wavelength of 490 nm of each well was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 490 nm with control wells containing only a cell culture medium. Data were presented as mean ± standard deviation (SD) (n = 3). 3. Results and discussion 3.1. Synthesis of PEG–DOX prodrug The aim of the present study was to synthesize a novel reduction/pH dual-sensitive PEG-based prodrug system, which could self-assemble in aqueous media into the nanomicelles. The nanomicelles were expected to be stable under physiological conditions, be selectively taken up by liver cancer cells via ASGP receptor-mediated endocytosis, and achieve rapid disassembly at the intracellular levels of pH and reducing agent. The synthetic route of PEG–DOX prodrug is shown in Scheme 1. A mixture of Gal-PEG-CHO and mPEG-CHO was firstly functionalized with TPH by the reduction amination of the Schiff's base formed between the aldehyde groups of Gal-/m-PEG-CHO and the amino groups of TPH molecules. DOX was conjugated onto the TPH-functionalized PEG through a hydrazone linkage formed between the ketone group of DOX and the hydrazide end group of PEG-TPH. The obtained PEG–DOX prodrug was amphiphilic and could self-assemble into nanomicelle in an aqueous solution with DOX as the core and PEG as the shell. The chemical structures of mPEG-TPH and mPEG–DOX prodrug were characterized by 1H NMR spectra, as shown in Fig. 1. The proton signals of TPH at 2.91 and 3.43 ppm appeared in Fig. 1A, suggesting the successful synthesis of PEG-TPH. As revealed in Fig. 1B, the chemical shifts at 1.22, 2.02, 4.59, 5.48, and 7.69–7.95 ppm originated from DOX demonstrated that the DOX molecules were successfully conjugated onto the PEG-TPH molecules [41,42].
with an average size of ~100 nm (Fig. 2B). Notably, the average diameter determined by TEM was smaller than that measured by DLS. It is most likely because DLS data reflected the hydrodynamic size of the nanomicelles while TEM image showed the size of dry nanomicelles. The nanomicelles would shrink during the drying process [44]. It has been reported that the nanomicelles with the size of b200 nm will accumulate in tumor sites due to the EPR effect [45,46]. Therefore, the prodrug synthesized in this study should be a promising drug delivery system. 3.3. DOX loading and in vitro DOX release In order to conjugate DOX onto PEG-TPH, the reaction between PEGTPH and DOX·HCl was performed in DMSO for 24 h and then the solution was dialyzed against 2 mM of PBS buffer (pH 7.4) to remove unconjugated DOX molecules. The DLE of PEG–DOX was 75%, and the DLC was as high as 20%. The high DLC should be the result of the loading of DOX in the form of PEG-based prodrug. Recent studies reveal that prodrugs with a stimuli-responsive character are more ideal for drug delivery [30]. The prodrug designed in the present study was expected to display a pH and reduction dualresponsive fashion. The in vitro release of DOX from the nanomicelles was investigated using a dialysis method in PBS buffer (pH 7.4, 6.0 and 5.0) in the presence or absence of 10 mM DTT. The pH and DTT levels were chosen to simulate those in the endosomal and lysosomal vesicles inside the cancer cells, whose pH values and GSH concentrations are 4–6 and 2–10 mM, respectively. As shown in Fig. 3, the release of DOX exhibited a biphasic pattern, in which a rapid release in the initial stage of 12 h was followed by a sustained release over a prolonged time period of up to 120 h. Similar phenomenon was observed in
3.2. Characterization of PEG–DOX prodrug nanomicelles The size distribution of nanocarriers plays an important role in intravenous administration [43]. To obtain a well-distributed nanomicelle solution, PEG–DOX prodrug nanomicelles were prepared by the ultrasonic method. The DLS results showed that the nanomicelles formed from the PEG–DOX prodrug had an average diameter of 140 ± 5.4 nm (Fig. 2A). Transmission electron microscopy (TEM) micrograph revealed that the PEG–DOX prodrug nanomicelles showed a spherical morphology
Fig. 3. In vitro reduction/pH-triggered release profiles of DOX from PEG–DOX prodrug nanomicelles in PBS buffer (pH 7.4, 6.0, and 5.0) with and without 10 mM DTT.
M. Xu et al. / Materials Science and Engineering C 50 (2015) 341–347
Fig. 4. Change in the size of PEG–DOX prodrug nanomicelles in PBS buffer (pH 7.4 with and without DTT, pH 5.0 with 10 mM DTT) with increasing release time.
other DOX-based prodrug systems [47]. It was found that a minimal amount of drug (b 15%) was released prior to the appearance of the plateau at pH 7.4 in the absence of DTT, which revealed that hydrazone linkages were relatively stable under neutral pH environment. The release rate of DOX was markedly accelerated when decreasing pH from 7.4 to 5.0 regardless of DTT. The faster release of DOX was attributed to the acidic catalytic cleavage of hydrazone linkage between PEG and DOX, thus releasing the conjugated DOX [48]. In addition, because of the basic nature of DOX (pKa = 8.3), the released DOX would become water-soluble in the acidic environments [49]. Compared with the release profiles in the absence of DTT, the release rate of DOX from the prodrugs in the presence of DTT (10 mM) was much higher regardless of pH values. For example, more than 50% of DOX was released under acidic reducing conditions (pH 5.0, 10 mM DTT) within the first 12 h, and over 90% of DOX was released after 120 h. The accelerated release was due to the dissociation of prodrug caused by the reductiontriggered cleavage of disulfide linkage between PEG and DOX via
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thiol–disulfide exchange reactions [50]. The results meant that the prodrug was stable under physiological conditions, and its pH/ reduction-dependent DOX release property might ensure the intracellular rapid release of DOX in the reductive acidic microenvironment of cancer cells. Therefore, the PEG–DOX prodrug is an ideal vehicle for targeted intracellular delivery. Some studies suggest that the detachment of hydrophobic drug from the micelles can trigger the destabilization of the hydrophobic core and aggregate the micelles together [51,52]. In this study, the change in nanomicelle size during the release of DOX was monitored by DLS measurement, and the results are shown in Fig. 4. Compared with the average diameter of the nanomicelles in PBS (pH 7.4 with 10 mM DTT) which increased from 140 to 413 nm in the first 12 h and increased to 762 nm at 60 h, it is found that the average diameter of the nanomicelles in PBS (pH 5.0 with 10 mM DTT) increased faster from 140 to 890 nm in the first 12 h and increased to 1660 nm at 60 h time point. In contrast, the diameter of the nanomicelles in PBS (pH 7.4) slightly increased from 140 to 210 nm in the initial stage of 12 h and kept stable in the remaining time. The trend was according with that in the drug release studies. The faster increase in the micelle size in the reducing acidic microenvironment (pH 5.0, 10 mM DTT) than under the physiological conditions (pH 7.4) was attributed to the combination of pH-sensitive cleavage of hydrazone bond and reduction-triggered breakage of disulfide bond between the PEG and DOX, which resulted in the rapid release of DOX. The detachment of hydrophobic drug from the micelles aggregated the micelles together [53,54]. 3.4. Cellular uptake study It has been proved that the ASGP receptor is abundantly expressed in various human hepatocellular carcinoma cell lines, including HepG2 [55]. To visually investigate the cellular uptake of PEG–DOX prodrug nanomicelles, the cell nuclei were fluorescently stained blue with DAPI. To assess the internalization capacity of the nanomicelles in HepG2 cells, HepG2 cells were incubated with Gal-PEG–DOX prodrug nanomicelles for 1, 4 and 24 h, using the HepG2 cells treated with Gal-unconjugated prodrug nanomicelles as a control group. Their
Fig. 5. Fluorescent images of HepG2 cells incubated with (left two columns) mPEG–DOX and (right two columns) Gal-PEG–DOX prodrug nanomicelles for (A, B) 1, (C, D) 4 and (E, F) 24 h. In each panel, the two fluorescent images from left to right show the fluorescent image of DOX and the overlaid fluorescent image of DOX and DAPI, respectively. Scale bars correspond to 40 μm.
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Fig. 6. Cytotoxicity of PEG-TPH to HepG2 cells at a concentration range of 0.001 to 8 mg/mL after incubation for 48 h. The cell viability was determined by MTT assay (n = 3).
fluorescent images are shown in Fig. 5. As revealed in Fig. 5, in the experimental group, the fluorescence intensity of DOX in the cytoplasm significantly increased with the prolongation of incubation time. In contrast, the fluorescence intensity of DOX in HepG2 cells in the control group was much weaker than that in the experimental group cells at the same culture time. Such results indicated that the Gal-PEG–DOX prodrug nanomicelles could be taken up more effectively by HepG2 cells than the mPEG–DOX prodrug nanomicelles. The most possible reason is that HepG2 cells overexpress ASGP receptors, and the ligand Galdecorated prodrug nanomicelles can recognize and bind to the ASGP receptors and be taken up rapidly and specifically by HepG2 cells via ASGP receptor-mediated endocytosis [56]. These results demonstrated that the Gal-PEG–DOX prodrug would be a great promising approach for the targeted delivery of DOX to hepatocellular carcinoma. 3.5. In vitro cytotoxicity studies The cytotoxicity of PEG-TPH and PEG–DOX prodrug against HepG2 cells was investigated by MTT assay. HepG2 cells were incubated with PEG-TPH or PEG–DOX prodrug for 48 h. The results (Fig. 6) revealed that PEG-TPH was non-toxic to HepG2 cells at concentrations of up to 0.5 mg/mL, which approximated the content of polymer in the PEG– DOX prodrug at a DOX concentration of 100 μg/mL. PEG-TPH still did not show any evident toxicity even when its concentration was up to 8 mg/mL, indicating that PEG-TPH possessed excellent biocompatibility similar to PEG [57]. However, under otherwise the same conditions,
PEG–DOX prodrug showed pronounced cytotoxic effects (Fig. 7). For example, Gal-PEG–DOX and mPEG–DOX prodrugs significantly decreased the cell viability of HepG2 cells by 40.8% and 58% at a DOX concentration of 40 μg/mL, respectively. At a higher DOX concentration of 80 μg/mL, the percentages of viable HepG2 cells further decreased to 34.1% and 42.6%, respectively. The IC50 (i.e., inhibitory concentration to produce 50% cell death) of Gal-PEG–DOX prodrug was obviously different from that of mPEG–DOX prodrug. Their IC50 values for HepG2 cells were approximately 6.54 and 60.8 μg/mL (equivalent DOX concentrations), respectively. The results indicated that Gal-PEG–DOX prodrug could be taken up more effectively by HepG2 cells in comparison with mPEG– DOX prodrug, because Gal-decorated prodrug nanomicelles could recognize the ASGP receptors expressed on the surface of HepG2 cells [58]. Therefore, the more DOX had entered HepG2 cells, the more toxicity. Moreover, the prodrugs have to be activated in endosomal compartments to exert the cytostatic effects. All the results showed that the cellular uptake and antitumor activity of PEG–DOX prodrug could be enhanced through the installment of a specific targeting ligand, which made this system particularly attractive for tumor-targeted drug delivery applications. 4. Conclusions In this study, a novel stimuli-sensitive PEG–DOX prodrug has been successfully synthesized. DOX was conjugated onto poly(ethylene glycol) via a functional linkage containing disulfide and hydrazone bonds. The PEG–DOX prodrug could self-assemble in an aqueous solution into spherical nanomicelles with an average diameter of 140 nm. The release of DOX from the nanomicelles at neutral pH exhibited a slow and sustained behavior, while more than 50% of total loaded DOX amount could be released within the first 12 h under acidic reducing microenvironment mimicking the intercellular conditions in tumor. The cellar uptake and cytotoxicity studies confirmed that the Gal-decorated PEG– DOX prodrug nanomicelles could specifically interact with HepG2 cells and be internalized into HepG2 cells via receptor-mediated endocytosis, showing enhanced cytotoxicity. Therefore, the novel PEG–DOX prodrug nanomicelles have great potential for targeted intracellular delivery of DOX and for improving the anticancer efficiency of DOX. Acknowledgments The authors gratefully acknowledge the National Natural Science Foundation of China (50773062, 50603020), the Natural Science Basic Research Plan in Shaanxi Province of China (2013K09-27, 2011K1301-09) and the Fundamental Research Funds for the Central Universities (XJJ2014124, XJJ2013130, XJJ2012146) for their financial support of this research. References
Fig. 7. Cytotoxicity of mPEG–DOX and Gal-PEG–DOX prodrugs to HepG2 cells at various DOX concentrations of 5–80 μg/mL after incubation for 48 h. The cell viability was determined by MTT assay (n = 3).
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