Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Polymeric micelles with small lipophilic moieties for drug delivery Dong Li, Yan Liang, Yusi Lai, Gang Wang, Bin He ∗ , Zhongwei Gu ∗ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
a r t i c l e
i n f o
Article history: Received 17 April 2013 Received in revised form 15 September 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: 9-nitro-20(s)-camptothecin Micelle Drug delivery Cytotoxicity Cellular uptake
a b s t r a c t The aim of this study was to develop a new polymeric micelle delivery system for antitumor drugs. An amphiphile of methoxypoly(ethylene glycol)-histidine-di(cinnamic acid) (PEGHC) with a small lipophilic moiety instead of a hydrophobic biodegradable polymer chain was synthesized and characterized. The PEGHC self-assembled into micelles. The critical micelle concentration (CMC) was tested. 9-Nitro-20(s)camptothecin (9-NC) was used as a model drug for encapsulation. The size and morphology of both blank and 9-NC loaded micelles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release profile of 9-NC loaded micelles was studied. HepG2 liver cancer cells were incubated with the drug-loaded micelles to investigate the in vitro anticancer efficiency. The results showed that the 9-NC loaded micelles exhibited high accumulated release rate (>85%) and efficient in vitro anticancer activity. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Drug delivery systems have been widely used for cancer chemotherapy [1]. Polymeric amphiphiles could self-assemble into nanoparticles (NPs) such as micelles [2–4] and vesicles [5], which were used as carriers for anticancer drug delivery. The core shell structure of micelles [6,7] was convenient to load hydrophobic anticancer drugs, including paclitaxel (PTX) and doxorubicin (DOX). Nanovesicles were efficient carriers for hydrophilic drugs such as doxorubicin hydrochloride (DOX HCl) [8]. The encapsulation of polymeric nanoparticles not only reduce the side effects [9] but also improve the bioavailability [10–12] of anticancer drugs. PEGylation of NPs was considered as an ideal strategy to design anticancer drug delivery systems as it could avoid the uptake of reticuloendothelial system (RES) and prolong circulation time in blood vessel transportation. Drug loaded NPs with a diameter lower than 200 nm could passively target tumor tissues via the enhanced permeability and retention (EPR) effect [13–16]. In most cases, block copolymers with poly(ethylene glycol) (PEG) as the hydrophilic segments and biodegradable chains of poly(lactic acid), poly(ε-caprolactone), and poly(amino acid) [17] as the lipophilic segments have been fabricated as drug carriers [18,19]. It has been reported that
∗ Corresponding authors at: National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China. Tel.: +86 28 85412923; fax: +86 28 85410653. E-mail addresses: [email protected] (B. He), [email protected] (Z. Gu).
different ratio of the hydrophilic and hydrophobic segments could affect the pattern of NPs [20] and drug release behaviors. Although, polymeric micelles self-assembled from block copolymer amphiphiles exhibited long sustaining release; however, the final accumulated release rate was low. Thus, seeking polymeric micelles with high-accumulated release rate of anticancer drug is an attractive topic in anticancer drug delivery. Camptothecin (CPT) was first extracted from the Chinese tree Camptotheca acuminata in the early 1960s [21]. CPT and its derivatives such as 9-nitro-20(s)-camptothecin (9-NC) have shown significant activity against a broad range of tumors [22]. CPT has a lactone ring, which is selectively targeted to topoisomerase I [23]. In physiological conditions, the lactone ring is easily opened and the antitumor activity of CPT will be lost [24,25]. The major limitations of CPTs as anticancer drugs are poor solubility and the instability of its lactone ring under physiological conditions [26,27]. Many strategies have been adopted to improve the solubility and instability of 9-NC. Encapsulation of 9-NC into polymeric micelles produced promising results, over 90% of 9-NC retained the lactone ring after incubated in phosphate-buffered saline (PBS) for 100 min [28], which was better than the conjugation of CPTs to carriers to protect the lactone ring of CPTs [29–31]. In this study, polymeric micelles with small lipophilic moieties instead of hydrophobic chains were synthesized. The small lipophilic moieties were expected to enhance the accumulated release rate of 9-NC. An l-histidine (His) moiety conjugated with two cinnamic acid (Cin) molecules was synthesized as the lipophilic segment, whereas mPEG was used as the hydrophilic segment. 9-NC was encapsulated in the methoxypoly(ethylene
0927-7765/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.10.032
628
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
glycol)-histidine-di(cinnamic acid) (PEGHC) micelles. The size, morphology, and critical micelle concentration (CMC) of PEGHC micelles were measured. The release profile and in vitro anticancer activity of 9-NC-loaded micelles were investigated. The PEGHC micelles exhibited a high accumulated release rate of 9-NC (>85%) and an efficient inhibition effect on the proliferation of HepG2 liver cancer cells. 2. Materials and methods 2.1. Materials Methoxy poly(ethylene glycol) (mPEG, Mn = 2000) was purchased from Sigma–Aldrich Chemicals. 9-NC was purchased from Shanghai Future Reagent Co. Ltd. (Shanghai, China). N␣Boc-l-histidine (Boc-His-OH), methyl l-lysinate dihydrochloride (H-Lys-OMe 2HCl), N,N-diisopropylethylamine (DIPEA), dicyclohexylcarbodiimide (DCC), trifluoroacetic acid (CF3 COOH), HBTU and N(o-benzotriazol-1-yl-N,N,N ,N -tetramethyluronium), hydroxybenzotrizole (HOBt) were purchased from GL Biochem Ltd. (Shanghai, China). Cinnamic acid was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methanesulfonyl chloride (MeSO2 Cl), ammonia water (25 wt.%), and triethylamine (Et3 N) were purchased from Aladdin Reagents (Shanghai, China). Dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dichloromethane (CH2 Cl2 ), chloroform (CHCl3 ), methanol (MeOH), and diethyl ether were purchased from Kelong Chemical Co. (Chengdu, China). DMF was distilled under reduced pressure. CH2 Cl2 and CHCl3 were dried over CaH2 , and distilled before use. 2.2. Synthesis of methoxypoly(ethylene glycol)-histidine-di(cinnamic acid) (PEGHC) 2.2.1. Synthesis of methoxy-amino-poly(ethylene glycol) (mPEG-NH2 ) mPEG (5.0 g, 2.5 mmol) and methanesulfonyl chloride (MeSO2 Cl; 0.97 mL, 1.25 mmol) were added to a flask under nitrogen atmosphere and dissolved in dry CH2 Cl2 (50 mL) with stirring. Et3 N (1.75 mL, 1.25 mmol) was added dropwise to the mixture in an ice bath. After the mixture was reacted at room temperature for 24 h [32], ammonia water (25 wt.%) was added to the solution and stirred at room temperature for 3 days. mPEG-NH2 was obtained as a white powder after being extracted with CH2 Cl2 and precipitated in diethyl ether. The product was vacuum-dried (4.0 g; yield: 80%). 2.2.2. Synthesis of methoxypoly(ethylene glycol)-histidine-NH2 (mPEG-His-NH2 ) Boc-His-OH (0.5106 g, 2 mmol) and HOBt (0.2702 g, 2 mmol) were added to a flask under nitrogen atmosphere and dissolved in 25 mL of dry CHCl3 with stirring. DCC (0.816 g, 4 mmol) in 10 mL of CHCl3 was added dropwise. After reacted for 4 h, mPEG-NH2 (2.023 g, 1 mmol) and DIPEA (160 L, 1 mmol) in 10 mL of CHCl3 were added dropwise to the solution. The solution was stirred at 30 ◦ C for 48 h. The mixture was precipitated in dry diethyl ether. The product was further reacted with CF3 COOH (0.77 mL, 10 mmol) in 1 mL of CH2 Cl2 to remove the Boc groups. mPEG-His-NH2 was obtained as light yellow powder after being precipitated in diethyl ether. The product was vacuum-dried (2.047 g; yield: 87%). 2.2.3. Synthesis of HOOC-Lysine-di(cinnamic acid) (HOOC-Lys-DCin) H-Lys-OMe 2HCl (1.014 g, 4.18 mmol), cinnamic acid (1.227 g, 8.36 mmol), HOBt (1.120 g, 8.36 mmol), and HBTU (3.143 g,
8.36 mmol) were added into a flask under nitrogen atmosphere and dissolved in 30 mL of dry DMF with stirring. DIPEA (34.5 L, 2.08 mmol) in 10 mL of DMF was added dropwise. The reaction was continued at room temperature for 48 h; the mixture was washed with saturated sodium chloride solution (30 mL), sodium hydrogen sulfate (1 M, 30 mL), and sodium carbonate (1 M, 30 mL), respectively. The product was further reacted with sodium hydroxide in ethanol (1 M, 30 mL) to remove methyl ester groups from lysine. HOOC-Lys-DCin was obtained as light yellow powder after being extracted with CHCl3 . The product was vacuum-dried (1.826 g; yield: 96%). 2.2.4. Synthesis of methoxypoly(ethylene glycol)-histidine-di(cinnamic acid) (PEGHC) HOOC-Lys-DCin (0.114 g, 0.312 mmol) and HOBt (0.056 g, 0.416 mmol) were added to a flask under nitrogen atmosphere and dissolved in 10 mL of dry CHCl3 . DCC (0.340 g, 1.66 mmol) in 10 mL of CHCl3 was added dropwise. After being stirred for 4 h, mPEGHis-NH2 (0.506 g, 0.208 mmol) and DIPEA (34.5 L, 0.208 mmol) in 10 mL of CHCl3 were added. The reaction was kept at 30 ◦ C for 48 h. The mixture was filtered to remove N,N -dicyclohexylure (DCU). The filtrate was concentrated, purified by precipitation twice in dry diethyl ether, and dialyzed (MWCO 1000 Da; Spectrum Laboratories, Inc) against deionized water. PEGHC was light yellow powder after freeze-drying (0.410 g; yield: 71%). 2.2.5. Characterization The products were confirmed by Bruker Avance II NMR spectrometer at 400 MHz (1 H NMR) with tetramethylsilane (TMS) as the internal standard. The molecular weights were measured by Autoflex MALDI-TOF-MS (Bruker, USA) with 2,5-dihydroxybenzoic acid (DHB) as the matrix. 2.3. Preparation and characterization of PEGHC micelles 2.3.1. Preparation of blank PEGHC micelles PEGHC (15 mg) was dissolved in DMSO (2.0 mL) and dropped slowly into 10 mL of deionized water with stirring at room temperature. The solution was dialyzed for 24 h against deionized water to remove DMSO using dialysis tubing (MWCO 1000 Da). After the dialysis, the solution in the dialysis tubing was filtered through a 0.45-m filter (Millipore). The filtrate was lyophilized to receive blank PEGHC micelles. The CMC was determined by fluorescence technique with pyrene as a probe [33]. The pyrene aqueous solution (6.0 × 10−7 M) was prepared. The solutions of PEGHC with concentrations from 0.10 × 10−4 g/L to 0.25 g/L were prepared. The emission wavelength was 395 nm. The fluorescent intensity at 334 and 338 nm were recorded. The value (I338 /I334 ) was calculated and plotted against the logarithm of the PEGHC concentration. 2.3.2. Preparation of drug-loaded micelles 9-NC was used as the model drug. PEGHC (20 mg) and 9-NC (2 mg) were dissolved in DMSO (3 mL). The solution was dropped slowly into 10 mL of deionized water with stirring at room temperature. The solution was dialyzed (MWCO 1000 Da) for 12 h against deionized water at 4 ◦ C to remove free drug. The deionized water was replaced every 3 h. The non-encapsulated drug was further removed by centrifugation (3000 rpm for 5 min at 4 ◦ C). The 9-NC-loaded PEGHC (9-NC/PEGHC) micelles were obtained after freeze-dry. The drug-loading content (DLC) and encapsulation efficiency (EE) were determined as follows: 1 mg of 9-NC-loaded micelles was dissolved in a mixture of methanol-acetate buffer solution (7:3 v/v, pH 4.5). The amount of 9-NC in the micelles was determined by
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
629
UV–vis absorbance at 376 nm using a standard calibration curve. The DLC and EE were calculated from Eqs. (1) and (2), respectively: DLC (wt.%) = EE (wt%) =
mass of drug in micelles mass of drug − loaded micelles
mass of drug in micelles mass of drug fed initially
× 100%
× 100%
(1) (2)
2.3.3. Characterization of PEGHC micelles The morphology of the PEGHC micelles was characterized by transmission electron microscopy (TEM, JEOL, Japan). The sample was prepared by dropping the PEGHC micelle solution (0.5 mg/mL) onto a copper grid coated with carbon film. The samples were negatively stained with phosphotungstic acid aqueous solution. The hydrodynamic diameter and the particle size distribution of the PEGHC micelles were determined by dynamic light scattering (DLS; Malvern Zetasizer Nano ZS, UK). Before the measurement, the samples were dispersed in deionized water (0.5 mg/mL). The measurements were performed at an angle of 173◦ to the incident light at 25 ◦ C. 2.4. In vitro drug release The in vitro release experiments were carried out using a dialysis method. Freeze-dried 9-NC/PEGHC micelles (2.3 mg; DLC 4.5%) were dispersed into 2 mL of PBS buffer (pH 7.4, 0.01 M); the solution was put into a dialysis membrane (MWCO 1000 Da). As a control, 0.1 mg of 9-NC in 2 mL of methanol and PBS solution (1:9, v/v) was passed through a dialysis membrane in the similar manner. The dialysis membranes were immersed into vials containing 25 mL of PBS in a shaking bed at 37 ◦ C. Tween 80 (0.1% v/v) was added into the release medium [34] to improve the solubility of 9-NC in PBS solution (pH 7.4, 0.01 M). The amount of released 9-NC was detected by UV absorbance at 376 nm via a standard calibration curve.
Scheme 1. Schematic illustration of synthesis routes of PEGHC.
2 h incubation at 37 ◦ C, the medium was removed from each dish, and the cells were washed with PBS (pH 7.4, 0.01 M) for three times. The cells were imaged by a confocal laser scanning microscope (Leica TCP SP5) using 405 nm (blue channel) lasers. 3. Results and discussion
2.5. Cell viability assay
3.1. Synthesis and characterization of PEGHC
2.5.1. Cytotoxicity assay The biocompatibility of PEGHC micelles was evaluated against NIH3T3 fibroblasts and HepG2 liver cancer cells. Cells (5 × 103 ) were seeded in 96-well plates for 24 h. The culture medium was removed and replaced with fresh medium containing the appropriate amount of blank PEGHC micelles at the concentrations ranged from 1 to 500 g/mL. After 48 h of incubation at 37 ◦ C, the culture medium was removed. 10 L of Cell Counting Kit-8 (CCK-8) was added to each well. After additional incubation for 2 h, the absorbance at 450 nm was measured by an enzyme-labeled instrument.
PEGHC was synthesized as the route shown in Scheme 1. The structure of mPEG-His(Boc) and PEGHC were characterized by 1 H NMR spectroscopy. Fig. 1a shows the 1 H NMR spectrum of mPEGHis(Boc). The chemical shift at ı = 1.42 ppm (g) is attributed to the methyl protons (–CCH3 ) in the His moieties. The chemical shift at ı = 3.64 ppm (b) is assigned to methylene protons (–O–CH2 –) in mPEG. Fig. 1b shows the 1 H NMR spectrum of PEGHC. The chemical shifts at ı = 1.47−1.86 ppm (i4, i5, i6) are attributed to the methylene protons (–CH2 –) in the lysine moieties, and the broad peak at ı = 7.47 ppm (p2, p3) is associated with protons in Cin. According to the 1 H NMR measurements, it indicates the successful synthesis of PEGHC. The PEGHC was further confirmed by mass spectroscopy (MS; see Supplementary data, Fig. S1). Fig. S1a shows the MS spectrum of mPEG-His(Boc) with a molecular weight of 2447 Da (Na+ ). Fig. S1b shows the MS spectrum of PEGHC with a molecular weight of 2735 Da (Na+ ). The result of MS spectra demonstrates that PEGHC was synthesized.
2.5.2. In vitro antitumor activity HepG2 cells were seeded in 96-well plates at the density of 5 × 103 per well for 24 h. The culture medium was removed and replaced with fresh medium containing the appropriate amount of 9-NC/PEGHC micelles with the concentrations ranging from 0.0001 to 20 g/mL. The medium was removed after 48 h incubation at 37 ◦ C. 10 L of CCK-8 was added to each well. After an additional incubation for 2 h, the absorbance at 450 nm was measured by an enzyme-labeled instrument. The cell morphology and survival rate were observed using an inverted fluorescence microscope. 2.6. Confocal microscopic study HepG2 cells were seeded on a 35 mm glass-bottom dish at a density of 1 × 104 cells per well for 24 h. The culture medium (200 L) was removed and replaced with fresh medium containing free 9NC, and 9-NC/PEGHC micelles at a concentration of 20 g/mL. After
3.2. CMC determination Amphiphiles of PEGHC could self-assemble into micelles in aqueous solution. The micelle formation was investigated via the fluorescence technique using pyrene as a probe (see Supplementary data, Fig. S2). The excitation spectrum of the fluorescence probe has different fluorescence properties in different polar microenvironments. At low concentrations, the ratio (I338 /I334 ) closely matched pyrene in water; whereas at high concentrations, the ratio matched pyrene in hydrophobic environment [35]. Fig. S2 shows that an
630
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
Fig. 1.
1
H NMR spectra of mPEG-His(Boc) (a) and PEGHC (b).
Fig. 2. Transmission electron microscopic images of micelles prepared from blank PEGHC micelles (a) and 9-NC/PEGHC micelles (b).
3.3. Preparation and characterization of 9-NC/PEGHC micelles 3.3.1. Drug-loading content (DLC) determination 9-NC was used as a model drug for the encapsulation in PEGHC micelles. 9-NC loaded micelles were successfully prepared with DLC of 4.5% and EE of 49.5% via a dialysis method.
3.3.2. Morphology of the PEGHC micelles PEGHC self-assembled into micelles in water through hydrophilic and hydrophobic interactions. The particle size and distributions were studied by DLS. As shown in Fig. S3, the average hydrodynamic diameters of blank PEGHC micelles (a) and 9-NC/PEGHC micelles (b) are 64 ± 23 nm and 71 ± 30 nm, respectively. The results show that the mean particle size of 9-NC/PEGHC micelles is bigger than that of blank PEGHC micelles, and both sizes are less than 100 nm. Fig. 2a and Fig. 2b show the TEM images of blank PEGHC micelles and 9-NC/PEGHC micelles at 25 ◦ C, which indicate that the PEGHC micelles are spherical nanoparticles. The average diameters of blank PEGHC micelles and 9-NC/PEGHC micelles are approximately 60 nm and 80 nm, respectively.
3.4. In vitro drug release The release behavior of free 9-NC and 9-NC/PEGHC micelles (DLC = 4.5%) was evaluated by a dialysis technique. Tween 80 (0.1% v/v) was added into the release media (pH 7.4, 0.01 M) to improve the solubility of 9-NC [36]. The results are shown in Fig. 3. Free drug shows a rapid release where more than 90% of 9-NC is released within 2 h [28]. However, 9-NC/PEGHC micelles show the relatively slow release behavior. The release profile of 9-NC/PEGHC micelles shows a burst release of 20% in the first 2 h, and then the drug
Accumulated drug Release (%)
abrupt increase is detected from the plot of I338 /I334 versus the logarithm of the concentration with increasing PEGHC concentrations. The measured CMC value of PEGHC is 34.5 mg/L at room temperature, indicating that PEGHC micelles are stable at low concentrations.
100 80 60 40 9-NC 9-NC/PEGHC micelles
20 0 0
10
20 30 Time (h)
40
50
Fig. 3. Release profiles of free drug (black) and 9-NC/PEGHC micelles (red) in PBS (pH 7.4, 0.01 M, 0.1% v/v Tween 80) at 37 ◦ C. Means ± SD (n = 3).
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
631
release is much slower. After the first burst release, 40% of drug in 9-NC/PEGHC micelles is released in 8 h. The accumulated release rate is as high as 85% in 48 h. The release behavior of PEGHC micelles was different from micelles consisting of long hydrophobic chains. However, the release behavior of 9-NC/PEGHC micelles was similar to 9-NCloaded liposomes [37]. A similar carrier with two Cin but without His as the lipophilic moiety has been used for 9-NC release in our previous work [38]; however, the accumulated release of 9-NC was less than 40% even with a release time of 72 h, most of the 9-NC could not be released from mPEG-DCIN micelles. Interestingly, when a His molecule was introduced as a linker between the hydrophilic mPEG segment and the liphophilic Lys-DCin lipophilic moiety, the accumulated release rate was higher than 85% within 48 h, demonstrating a significantly improved accumulated release rate. These results indicated that His could be used as an accelerator to enhance the release of 9-NC.
3.5.1. Material cytotoxicity The cytotoxicity of blank PEGHC micelles was evaluated with HepG2 cells (see Fig. S4) and NIH3T3 fibroblasts (see Fig. 4). As shown in Fig. S4 and Fig. 4, blank PEGHCs are non-toxic to either HepG2 cells or NIH3T3 fibroblasts even at a high concentration (0.5 mg/mL). At all concentrations, the cell viabilities of NIH3T3 fibroblasts and HepG2 cells are higher than 90%. These results demonstrate that PEGHC micelles could be potentially used as drug carriers. 3.5.2. In vitro antitumor activity The antitumor activity of 9-NC/PEGHC micelles was evaluated in vitro. As shown in Fig. 5, the results show that 9-NC/PEGHC micelles could kill HepG2 cells in a dose-dependent manner. The value of the half maximal (50%) inhibitory concentration (IC50 ) of 9NC/PEGHC NPs is 0.03 mg/L (48 h), whereas the IC50 values of 9-NC reported by Zhang et al. were 2.46 mg/L (48 h) and 0.06 mg/L (72 h) [39]. This result indicates that the HepG2 cells are more sensitive to 9-NC/PEGHC micelles. It demonstrates that the anticancer activity of 9-NC is enhanced via the encapsulation of PEGHC micelles.
Fig. 4. Cytotoxicity of blank PEGHC micelles cultured with NIH3T3 for 48 h. Means ± SD (n = 3).
100
Cell viability (%)
3.5. Cell viability assay
80 60 40 20 0 1E-3
0.01
0.1
1
10
Concentration (µg/mL) Fig. 5. In vitro anticancer activities of 9-NC/PEGHC micelles against HepG2 cells with a 48-h incubation time. Means ± SD (n = 3).
Fig. 6. Confocal laser scanning microscopic images of HepG2 cells incubated with free 9-NC (a, b), 9-NC/PEGHC NPs (c, d), and the control group (e, f).
632
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
The morphology of HepG2 cells incubated with blank PEGHC micelles and 9-NC/PEGHC micelles was observed by inverted microscope (see Supplementary data, Fig. S5). The cells incubated with 9-NC/PEGHC micelles are shrunk and aggregated, which indicates that almost all the HepG2 cells were killed by 9-NC (Fig. S5b). 3.6. Cellular uptake study The cellular uptake behavior of 9-NC and 9-NC/PEGHC micelles against HepG2 cell for 2 h was studied by confocal laser scanning microscopy (CLSM). The fluorescence was detected by confocal microscope with a laser wavelength of 405 nm [40,41]. As shown in Fig. 6, the control group (f) does not show fluorescence. However, significant 9-NC fluorescence is observed in cells incubated with 9-NC/PEGHC micelles (d), which indicates the successful delivery of 9-NC into the HepG2 cells. As PEGHC micelles improved the solubility of 9-NC greatly, the fluorescence intensity in HepG2 cells incubated with 9-NC/PEGHC micelles (Fig. 6d) is stronger than that of free 9-NC (Fig. 6b). 9-NC (20 g/mL) formed aggregates and deposited at the bottom of the dish as bright fluorescent spots (Fig. 6b and Fig. 6d) due to the poor solubility of 9-NC in water. These results demonstrate the rapid cellular uptake and dispersion of 9-NC/PEGHC micelles in the cytosol of cells. 4. Conclusion PEGHC micelles, with small molecules as lipophilic moieties, were successfully designed and synthesized. The anticancer drug 9-NC was used to investigate the release properties of PEGHC micelles. The DLC of PEGHC micelles was 4.5% with the particle size less than 100 nm. The release of 9-NC from PEGHC micelles could be sustained, and the accumulated release rate was higher than 85%, which was considerably higher than that of other polymeric micelles. Histidine acted as an accelerator to enhance the release of 9-NC from PEGHC micelles. The IC50 of 9-NC-loaded PEGHC micelles was as low as 0.030 mg/L. The drug-loaded PEGHC micelles exhibited efficient cellular endocytosis and inhibited the proliferation of HepG2 liver cancer cells efficiently. PEGHC micelles are potential promising carriers for 9-NC delivery. Acknowledgments This research work was supported by the National Basic Research Program of China (National 973 program, No. 2011CB606206), National Science Foundation of China (NSFC, No. 31170921, 50830105, 51133004), National Science Foundation for Excellent Young Scholars (No. 51222304), and Program for Changjiang Scholars and Innovative Research Team in University (IRT1163). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.10.032.
References [1] V. Torchilin, Pharmaceut. Res. 24 (2007) 1–16. [2] L. Fan, F. Li, H. Zhang, Y. Wang, C. Cheng, X. Li, C. Gu, Q. Yang, H. Wu, S. Zhang, Biomaterials 31 (2010) 5634–5642. [3] A.L.Z. Lee, Y. Wang, H.Y. Cheng, S. Pervaiz, Y.Y. Yang, Biomaterials 30 (2009) 919–927. [4] H. Wang, P. Zhao, W. Su, S. Wang, Z. Liao, R. Niu, J. Chang, Biomaterials 31 (2010) 8741–8748. [5] M. Saad, O.B. Garbuzenko, T. Minko, Nanomedicine 3 (2008) 761–776. [6] S.A. Jenekhe, X.L. Chen, Science 279 (1998) 1903–1907. [7] C.L. Lo, K.M. Lin, G.H. Hsiue, J. Controlled Release 104 (2005) 477–488. [8] G. Gaucher, M.H. Dufresne, V.P. Sant, N. Kang, D. Maysinger, J.C. Leroux, J. Controlled Release 109 (2005) 169–188. [9] P. Couvreur, B. Kante, L. Grislain, M. Roland, P. Speiser, J. Pharm. Sci. 71 (1982) 790–792. [10] Y. Gao, L. Chen, W. Gu, Y. Xi, L. Lin, Y. Li, Mol. Pharm. 5 (2008) 1044–1054. [11] G.S. Kwon, Crit. Rev. Ther. Drug. Carrier Syst. 20 (2003) 357. [12] N. Tang, G. Du, N. Wang, C. Liu, H. Hang, W. Liang, J. Natl. Cancer I 99 (2007) 1004–1015. [13] H. Maeda, Adv. Drug Delivery Rev. 46 (2001) 169–185. [14] H. Maeda, L.W. Seymour, Y. Miyamoto, Bioconjugate Chem. 3 (1992) 351–362. [15] F.M. Muggia, Clin. Cancer Res. 5 (1999) 7–8. [16] P.A. Vasey, S.B. Kaye, R. Morrison, C. Twelves, P. Wilson, R. Duncan, A.H. Thomson, L.S. Murray, T.E. Hilditch, T. Murray, Clin. Cancer Res. 5 (1999) 83–94. [17] R. Liu, B. He, D. Li, Y. Lai, J. Chang, J.Z. Tang, Z. Gu, Int. J. Nanomed. 7 (2012) 4433. [18] M.C. Jones, J.C. Leroux, Eur. J. Pharm. Biopharm. 48 (1999) 101–111. [19] A. Rösler, G.W.M. Vandermeulen and H.A. Klok, Adv Drug Delivery Rev, 64, Supplement (2012) 270-279. [20] L. Zhang, A. Eisenberg, Science 268 (1995) 23. [21] H. Ulukan, P.W. Swaan, Drugs 62 (2002) 2039–2057. [22] R. Garcia-Carbonero, J.G. Supko, Clin. Cancer Res. 8 (2002) 641–661. [23] J. Fassberg, V.J. Stella, J. Pharm. Sci. 81 (1992) 676–684. [24] M.E. Wall, M.C. Wani, A.W. Nicholas, G. Manikumar, C. Tele, L. Moore, A. Truesdale, P. Leitner, J.M. Besterman, J. Med. Chem. 36 (1993) 2689–2700. [25] M.C. Wani, A.W. Nicholas, M.E. Wall, J. Med. Chem. 30 (1987) 2317–2319. [26] W.D. Kingsbury, J.C. Boehm, D.R. Jakas, K.G. Holden, S.M. Hecht, G. Gallagher, M.J. Caranfa, F.L. McCabe, L.F. Faucette, R.K. Johnson, R.P. Hertzberg, J. Med. Chem. 34 (1991) 98–107. [27] U. Schaeppi, R. Fleischman, D. Cooney, Cancer Chemother. Rep. 5 (1974) 25. [28] J. Gao, J. Ming, B. He, Y. Fan, Z. Gu, X. Zhang, Eur. J. Pharm. Sci. 34 (2008) 85–93. [29] F.M.H. de Groot, G.F. Busscher, R.W.M. Aben, H.W. Scheeren, Bioorg. Med. Chem. Lett. 12 (2002) 2371–2376. [30] H.G. Lerchen, J. Baumgarten, K. von dem Bruch, T.E. Lehmann, M. Sperzel, G. Kempka, H.H. Fiebig, J. Med. Chem. 44 (2001) 4186–4195. [31] N. Pessah, M. Reznik, M. Shamis, F. Yantiri, H. Xin, K. Bowdish, N. Shomron, G. Ast, D. Shabat, Bioorgan Med. Chem. 12 (2004) 1859–1866. [32] R. Liu, D. Li, B. He, X. Xu, M. Sheng, Y. Lai, G. Wang, Z. Gu, J. Controlled Release 152 (2011) 49–56. [33] F.M. Winnik, S.T. Regismond, Colloids Surf., A 118 (1996) 1–39. [34] J.L. Lu, J.C. Wang, S.X. Zhao, X.Y. Liu, H. Zhao, X. Zhang, S.F. Zhou, Q. Zhang, Eur. J. Pharm. Biopharm. 69 (2008) 899–907. [35] M. Wilhelm, C.L. Zhao, Y. Wang, R. Xu, M.A. Winnik, J.L. Mura, G. Riess, M.D. Croucher, Macromolecules 24 (1991) 1033–1040. [36] A. Kumari, S.K. Yadav, S.C. Yadav, Colloids Surf., B 75 (2010) 1–18. [37] L.J. Zhang, B. Xing, J. Wu, B. Xu, X.L. Fang, Pulm Pharmacol. Ther. 21 (2008) 239–246. [38] Y. Liang, Y. Lai, D. Li, B. He, Z. Gu, Mater. Lett. 97 (2013) 4–7. [39] S. Zheng, S. Chang, J. Lu, Z. Chen, L. Xie, Y. Nie, B. He, S. Zou, Z. Gu, PloS one 6 (2011) e21064. [40] H. Dong, C. Dong, Y. Feng, T. Ren, Z. Zhang, L. Li, Y. Li, Med. Chem. Commun. 3 (2012) 1555–1561. [41] P. Pramod, K. Takamura, S. Chaphekar, N. Balasubramanian, M. Jayakannan, Biomacromolecules 13 (2012) 3627–3640.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Polymeric micelles with small lipophilic moieties for drug delivery Dong Li, Yan Liang, Yusi Lai, Gang Wang, Bin He ∗ , Zhongwei Gu ∗ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
a r t i c l e
i n f o
Article history: Received 17 April 2013 Received in revised form 15 September 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: 9-nitro-20(s)-camptothecin Micelle Drug delivery Cytotoxicity Cellular uptake
a b s t r a c t The aim of this study was to develop a new polymeric micelle delivery system for antitumor drugs. An amphiphile of methoxypoly(ethylene glycol)-histidine-di(cinnamic acid) (PEGHC) with a small lipophilic moiety instead of a hydrophobic biodegradable polymer chain was synthesized and characterized. The PEGHC self-assembled into micelles. The critical micelle concentration (CMC) was tested. 9-Nitro-20(s)camptothecin (9-NC) was used as a model drug for encapsulation. The size and morphology of both blank and 9-NC loaded micelles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release profile of 9-NC loaded micelles was studied. HepG2 liver cancer cells were incubated with the drug-loaded micelles to investigate the in vitro anticancer efficiency. The results showed that the 9-NC loaded micelles exhibited high accumulated release rate (>85%) and efficient in vitro anticancer activity. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Drug delivery systems have been widely used for cancer chemotherapy [1]. Polymeric amphiphiles could self-assemble into nanoparticles (NPs) such as micelles [2–4] and vesicles [5], which were used as carriers for anticancer drug delivery. The core shell structure of micelles [6,7] was convenient to load hydrophobic anticancer drugs, including paclitaxel (PTX) and doxorubicin (DOX). Nanovesicles were efficient carriers for hydrophilic drugs such as doxorubicin hydrochloride (DOX HCl) [8]. The encapsulation of polymeric nanoparticles not only reduce the side effects [9] but also improve the bioavailability [10–12] of anticancer drugs. PEGylation of NPs was considered as an ideal strategy to design anticancer drug delivery systems as it could avoid the uptake of reticuloendothelial system (RES) and prolong circulation time in blood vessel transportation. Drug loaded NPs with a diameter lower than 200 nm could passively target tumor tissues via the enhanced permeability and retention (EPR) effect [13–16]. In most cases, block copolymers with poly(ethylene glycol) (PEG) as the hydrophilic segments and biodegradable chains of poly(lactic acid), poly(ε-caprolactone), and poly(amino acid) [17] as the lipophilic segments have been fabricated as drug carriers [18,19]. It has been reported that
∗ Corresponding authors at: National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China. Tel.: +86 28 85412923; fax: +86 28 85410653. E-mail addresses: [email protected] (B. He), [email protected] (Z. Gu).
different ratio of the hydrophilic and hydrophobic segments could affect the pattern of NPs [20] and drug release behaviors. Although, polymeric micelles self-assembled from block copolymer amphiphiles exhibited long sustaining release; however, the final accumulated release rate was low. Thus, seeking polymeric micelles with high-accumulated release rate of anticancer drug is an attractive topic in anticancer drug delivery. Camptothecin (CPT) was first extracted from the Chinese tree Camptotheca acuminata in the early 1960s [21]. CPT and its derivatives such as 9-nitro-20(s)-camptothecin (9-NC) have shown significant activity against a broad range of tumors [22]. CPT has a lactone ring, which is selectively targeted to topoisomerase I [23]. In physiological conditions, the lactone ring is easily opened and the antitumor activity of CPT will be lost [24,25]. The major limitations of CPTs as anticancer drugs are poor solubility and the instability of its lactone ring under physiological conditions [26,27]. Many strategies have been adopted to improve the solubility and instability of 9-NC. Encapsulation of 9-NC into polymeric micelles produced promising results, over 90% of 9-NC retained the lactone ring after incubated in phosphate-buffered saline (PBS) for 100 min [28], which was better than the conjugation of CPTs to carriers to protect the lactone ring of CPTs [29–31]. In this study, polymeric micelles with small lipophilic moieties instead of hydrophobic chains were synthesized. The small lipophilic moieties were expected to enhance the accumulated release rate of 9-NC. An l-histidine (His) moiety conjugated with two cinnamic acid (Cin) molecules was synthesized as the lipophilic segment, whereas mPEG was used as the hydrophilic segment. 9-NC was encapsulated in the methoxypoly(ethylene
0927-7765/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.10.032
628
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
glycol)-histidine-di(cinnamic acid) (PEGHC) micelles. The size, morphology, and critical micelle concentration (CMC) of PEGHC micelles were measured. The release profile and in vitro anticancer activity of 9-NC-loaded micelles were investigated. The PEGHC micelles exhibited a high accumulated release rate of 9-NC (>85%) and an efficient inhibition effect on the proliferation of HepG2 liver cancer cells. 2. Materials and methods 2.1. Materials Methoxy poly(ethylene glycol) (mPEG, Mn = 2000) was purchased from Sigma–Aldrich Chemicals. 9-NC was purchased from Shanghai Future Reagent Co. Ltd. (Shanghai, China). N␣Boc-l-histidine (Boc-His-OH), methyl l-lysinate dihydrochloride (H-Lys-OMe 2HCl), N,N-diisopropylethylamine (DIPEA), dicyclohexylcarbodiimide (DCC), trifluoroacetic acid (CF3 COOH), HBTU and N(o-benzotriazol-1-yl-N,N,N ,N -tetramethyluronium), hydroxybenzotrizole (HOBt) were purchased from GL Biochem Ltd. (Shanghai, China). Cinnamic acid was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methanesulfonyl chloride (MeSO2 Cl), ammonia water (25 wt.%), and triethylamine (Et3 N) were purchased from Aladdin Reagents (Shanghai, China). Dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dichloromethane (CH2 Cl2 ), chloroform (CHCl3 ), methanol (MeOH), and diethyl ether were purchased from Kelong Chemical Co. (Chengdu, China). DMF was distilled under reduced pressure. CH2 Cl2 and CHCl3 were dried over CaH2 , and distilled before use. 2.2. Synthesis of methoxypoly(ethylene glycol)-histidine-di(cinnamic acid) (PEGHC) 2.2.1. Synthesis of methoxy-amino-poly(ethylene glycol) (mPEG-NH2 ) mPEG (5.0 g, 2.5 mmol) and methanesulfonyl chloride (MeSO2 Cl; 0.97 mL, 1.25 mmol) were added to a flask under nitrogen atmosphere and dissolved in dry CH2 Cl2 (50 mL) with stirring. Et3 N (1.75 mL, 1.25 mmol) was added dropwise to the mixture in an ice bath. After the mixture was reacted at room temperature for 24 h [32], ammonia water (25 wt.%) was added to the solution and stirred at room temperature for 3 days. mPEG-NH2 was obtained as a white powder after being extracted with CH2 Cl2 and precipitated in diethyl ether. The product was vacuum-dried (4.0 g; yield: 80%). 2.2.2. Synthesis of methoxypoly(ethylene glycol)-histidine-NH2 (mPEG-His-NH2 ) Boc-His-OH (0.5106 g, 2 mmol) and HOBt (0.2702 g, 2 mmol) were added to a flask under nitrogen atmosphere and dissolved in 25 mL of dry CHCl3 with stirring. DCC (0.816 g, 4 mmol) in 10 mL of CHCl3 was added dropwise. After reacted for 4 h, mPEG-NH2 (2.023 g, 1 mmol) and DIPEA (160 L, 1 mmol) in 10 mL of CHCl3 were added dropwise to the solution. The solution was stirred at 30 ◦ C for 48 h. The mixture was precipitated in dry diethyl ether. The product was further reacted with CF3 COOH (0.77 mL, 10 mmol) in 1 mL of CH2 Cl2 to remove the Boc groups. mPEG-His-NH2 was obtained as light yellow powder after being precipitated in diethyl ether. The product was vacuum-dried (2.047 g; yield: 87%). 2.2.3. Synthesis of HOOC-Lysine-di(cinnamic acid) (HOOC-Lys-DCin) H-Lys-OMe 2HCl (1.014 g, 4.18 mmol), cinnamic acid (1.227 g, 8.36 mmol), HOBt (1.120 g, 8.36 mmol), and HBTU (3.143 g,
8.36 mmol) were added into a flask under nitrogen atmosphere and dissolved in 30 mL of dry DMF with stirring. DIPEA (34.5 L, 2.08 mmol) in 10 mL of DMF was added dropwise. The reaction was continued at room temperature for 48 h; the mixture was washed with saturated sodium chloride solution (30 mL), sodium hydrogen sulfate (1 M, 30 mL), and sodium carbonate (1 M, 30 mL), respectively. The product was further reacted with sodium hydroxide in ethanol (1 M, 30 mL) to remove methyl ester groups from lysine. HOOC-Lys-DCin was obtained as light yellow powder after being extracted with CHCl3 . The product was vacuum-dried (1.826 g; yield: 96%). 2.2.4. Synthesis of methoxypoly(ethylene glycol)-histidine-di(cinnamic acid) (PEGHC) HOOC-Lys-DCin (0.114 g, 0.312 mmol) and HOBt (0.056 g, 0.416 mmol) were added to a flask under nitrogen atmosphere and dissolved in 10 mL of dry CHCl3 . DCC (0.340 g, 1.66 mmol) in 10 mL of CHCl3 was added dropwise. After being stirred for 4 h, mPEGHis-NH2 (0.506 g, 0.208 mmol) and DIPEA (34.5 L, 0.208 mmol) in 10 mL of CHCl3 were added. The reaction was kept at 30 ◦ C for 48 h. The mixture was filtered to remove N,N -dicyclohexylure (DCU). The filtrate was concentrated, purified by precipitation twice in dry diethyl ether, and dialyzed (MWCO 1000 Da; Spectrum Laboratories, Inc) against deionized water. PEGHC was light yellow powder after freeze-drying (0.410 g; yield: 71%). 2.2.5. Characterization The products were confirmed by Bruker Avance II NMR spectrometer at 400 MHz (1 H NMR) with tetramethylsilane (TMS) as the internal standard. The molecular weights were measured by Autoflex MALDI-TOF-MS (Bruker, USA) with 2,5-dihydroxybenzoic acid (DHB) as the matrix. 2.3. Preparation and characterization of PEGHC micelles 2.3.1. Preparation of blank PEGHC micelles PEGHC (15 mg) was dissolved in DMSO (2.0 mL) and dropped slowly into 10 mL of deionized water with stirring at room temperature. The solution was dialyzed for 24 h against deionized water to remove DMSO using dialysis tubing (MWCO 1000 Da). After the dialysis, the solution in the dialysis tubing was filtered through a 0.45-m filter (Millipore). The filtrate was lyophilized to receive blank PEGHC micelles. The CMC was determined by fluorescence technique with pyrene as a probe [33]. The pyrene aqueous solution (6.0 × 10−7 M) was prepared. The solutions of PEGHC with concentrations from 0.10 × 10−4 g/L to 0.25 g/L were prepared. The emission wavelength was 395 nm. The fluorescent intensity at 334 and 338 nm were recorded. The value (I338 /I334 ) was calculated and plotted against the logarithm of the PEGHC concentration. 2.3.2. Preparation of drug-loaded micelles 9-NC was used as the model drug. PEGHC (20 mg) and 9-NC (2 mg) were dissolved in DMSO (3 mL). The solution was dropped slowly into 10 mL of deionized water with stirring at room temperature. The solution was dialyzed (MWCO 1000 Da) for 12 h against deionized water at 4 ◦ C to remove free drug. The deionized water was replaced every 3 h. The non-encapsulated drug was further removed by centrifugation (3000 rpm for 5 min at 4 ◦ C). The 9-NC-loaded PEGHC (9-NC/PEGHC) micelles were obtained after freeze-dry. The drug-loading content (DLC) and encapsulation efficiency (EE) were determined as follows: 1 mg of 9-NC-loaded micelles was dissolved in a mixture of methanol-acetate buffer solution (7:3 v/v, pH 4.5). The amount of 9-NC in the micelles was determined by
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
629
UV–vis absorbance at 376 nm using a standard calibration curve. The DLC and EE were calculated from Eqs. (1) and (2), respectively: DLC (wt.%) = EE (wt%) =
mass of drug in micelles mass of drug − loaded micelles
mass of drug in micelles mass of drug fed initially
× 100%
× 100%
(1) (2)
2.3.3. Characterization of PEGHC micelles The morphology of the PEGHC micelles was characterized by transmission electron microscopy (TEM, JEOL, Japan). The sample was prepared by dropping the PEGHC micelle solution (0.5 mg/mL) onto a copper grid coated with carbon film. The samples were negatively stained with phosphotungstic acid aqueous solution. The hydrodynamic diameter and the particle size distribution of the PEGHC micelles were determined by dynamic light scattering (DLS; Malvern Zetasizer Nano ZS, UK). Before the measurement, the samples were dispersed in deionized water (0.5 mg/mL). The measurements were performed at an angle of 173◦ to the incident light at 25 ◦ C. 2.4. In vitro drug release The in vitro release experiments were carried out using a dialysis method. Freeze-dried 9-NC/PEGHC micelles (2.3 mg; DLC 4.5%) were dispersed into 2 mL of PBS buffer (pH 7.4, 0.01 M); the solution was put into a dialysis membrane (MWCO 1000 Da). As a control, 0.1 mg of 9-NC in 2 mL of methanol and PBS solution (1:9, v/v) was passed through a dialysis membrane in the similar manner. The dialysis membranes were immersed into vials containing 25 mL of PBS in a shaking bed at 37 ◦ C. Tween 80 (0.1% v/v) was added into the release medium [34] to improve the solubility of 9-NC in PBS solution (pH 7.4, 0.01 M). The amount of released 9-NC was detected by UV absorbance at 376 nm via a standard calibration curve.
Scheme 1. Schematic illustration of synthesis routes of PEGHC.
2 h incubation at 37 ◦ C, the medium was removed from each dish, and the cells were washed with PBS (pH 7.4, 0.01 M) for three times. The cells were imaged by a confocal laser scanning microscope (Leica TCP SP5) using 405 nm (blue channel) lasers. 3. Results and discussion
2.5. Cell viability assay
3.1. Synthesis and characterization of PEGHC
2.5.1. Cytotoxicity assay The biocompatibility of PEGHC micelles was evaluated against NIH3T3 fibroblasts and HepG2 liver cancer cells. Cells (5 × 103 ) were seeded in 96-well plates for 24 h. The culture medium was removed and replaced with fresh medium containing the appropriate amount of blank PEGHC micelles at the concentrations ranged from 1 to 500 g/mL. After 48 h of incubation at 37 ◦ C, the culture medium was removed. 10 L of Cell Counting Kit-8 (CCK-8) was added to each well. After additional incubation for 2 h, the absorbance at 450 nm was measured by an enzyme-labeled instrument.
PEGHC was synthesized as the route shown in Scheme 1. The structure of mPEG-His(Boc) and PEGHC were characterized by 1 H NMR spectroscopy. Fig. 1a shows the 1 H NMR spectrum of mPEGHis(Boc). The chemical shift at ı = 1.42 ppm (g) is attributed to the methyl protons (–CCH3 ) in the His moieties. The chemical shift at ı = 3.64 ppm (b) is assigned to methylene protons (–O–CH2 –) in mPEG. Fig. 1b shows the 1 H NMR spectrum of PEGHC. The chemical shifts at ı = 1.47−1.86 ppm (i4, i5, i6) are attributed to the methylene protons (–CH2 –) in the lysine moieties, and the broad peak at ı = 7.47 ppm (p2, p3) is associated with protons in Cin. According to the 1 H NMR measurements, it indicates the successful synthesis of PEGHC. The PEGHC was further confirmed by mass spectroscopy (MS; see Supplementary data, Fig. S1). Fig. S1a shows the MS spectrum of mPEG-His(Boc) with a molecular weight of 2447 Da (Na+ ). Fig. S1b shows the MS spectrum of PEGHC with a molecular weight of 2735 Da (Na+ ). The result of MS spectra demonstrates that PEGHC was synthesized.
2.5.2. In vitro antitumor activity HepG2 cells were seeded in 96-well plates at the density of 5 × 103 per well for 24 h. The culture medium was removed and replaced with fresh medium containing the appropriate amount of 9-NC/PEGHC micelles with the concentrations ranging from 0.0001 to 20 g/mL. The medium was removed after 48 h incubation at 37 ◦ C. 10 L of CCK-8 was added to each well. After an additional incubation for 2 h, the absorbance at 450 nm was measured by an enzyme-labeled instrument. The cell morphology and survival rate were observed using an inverted fluorescence microscope. 2.6. Confocal microscopic study HepG2 cells were seeded on a 35 mm glass-bottom dish at a density of 1 × 104 cells per well for 24 h. The culture medium (200 L) was removed and replaced with fresh medium containing free 9NC, and 9-NC/PEGHC micelles at a concentration of 20 g/mL. After
3.2. CMC determination Amphiphiles of PEGHC could self-assemble into micelles in aqueous solution. The micelle formation was investigated via the fluorescence technique using pyrene as a probe (see Supplementary data, Fig. S2). The excitation spectrum of the fluorescence probe has different fluorescence properties in different polar microenvironments. At low concentrations, the ratio (I338 /I334 ) closely matched pyrene in water; whereas at high concentrations, the ratio matched pyrene in hydrophobic environment [35]. Fig. S2 shows that an
630
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
Fig. 1.
1
H NMR spectra of mPEG-His(Boc) (a) and PEGHC (b).
Fig. 2. Transmission electron microscopic images of micelles prepared from blank PEGHC micelles (a) and 9-NC/PEGHC micelles (b).
3.3. Preparation and characterization of 9-NC/PEGHC micelles 3.3.1. Drug-loading content (DLC) determination 9-NC was used as a model drug for the encapsulation in PEGHC micelles. 9-NC loaded micelles were successfully prepared with DLC of 4.5% and EE of 49.5% via a dialysis method.
3.3.2. Morphology of the PEGHC micelles PEGHC self-assembled into micelles in water through hydrophilic and hydrophobic interactions. The particle size and distributions were studied by DLS. As shown in Fig. S3, the average hydrodynamic diameters of blank PEGHC micelles (a) and 9-NC/PEGHC micelles (b) are 64 ± 23 nm and 71 ± 30 nm, respectively. The results show that the mean particle size of 9-NC/PEGHC micelles is bigger than that of blank PEGHC micelles, and both sizes are less than 100 nm. Fig. 2a and Fig. 2b show the TEM images of blank PEGHC micelles and 9-NC/PEGHC micelles at 25 ◦ C, which indicate that the PEGHC micelles are spherical nanoparticles. The average diameters of blank PEGHC micelles and 9-NC/PEGHC micelles are approximately 60 nm and 80 nm, respectively.
3.4. In vitro drug release The release behavior of free 9-NC and 9-NC/PEGHC micelles (DLC = 4.5%) was evaluated by a dialysis technique. Tween 80 (0.1% v/v) was added into the release media (pH 7.4, 0.01 M) to improve the solubility of 9-NC [36]. The results are shown in Fig. 3. Free drug shows a rapid release where more than 90% of 9-NC is released within 2 h [28]. However, 9-NC/PEGHC micelles show the relatively slow release behavior. The release profile of 9-NC/PEGHC micelles shows a burst release of 20% in the first 2 h, and then the drug
Accumulated drug Release (%)
abrupt increase is detected from the plot of I338 /I334 versus the logarithm of the concentration with increasing PEGHC concentrations. The measured CMC value of PEGHC is 34.5 mg/L at room temperature, indicating that PEGHC micelles are stable at low concentrations.
100 80 60 40 9-NC 9-NC/PEGHC micelles
20 0 0
10
20 30 Time (h)
40
50
Fig. 3. Release profiles of free drug (black) and 9-NC/PEGHC micelles (red) in PBS (pH 7.4, 0.01 M, 0.1% v/v Tween 80) at 37 ◦ C. Means ± SD (n = 3).
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
631
release is much slower. After the first burst release, 40% of drug in 9-NC/PEGHC micelles is released in 8 h. The accumulated release rate is as high as 85% in 48 h. The release behavior of PEGHC micelles was different from micelles consisting of long hydrophobic chains. However, the release behavior of 9-NC/PEGHC micelles was similar to 9-NCloaded liposomes [37]. A similar carrier with two Cin but without His as the lipophilic moiety has been used for 9-NC release in our previous work [38]; however, the accumulated release of 9-NC was less than 40% even with a release time of 72 h, most of the 9-NC could not be released from mPEG-DCIN micelles. Interestingly, when a His molecule was introduced as a linker between the hydrophilic mPEG segment and the liphophilic Lys-DCin lipophilic moiety, the accumulated release rate was higher than 85% within 48 h, demonstrating a significantly improved accumulated release rate. These results indicated that His could be used as an accelerator to enhance the release of 9-NC.
3.5.1. Material cytotoxicity The cytotoxicity of blank PEGHC micelles was evaluated with HepG2 cells (see Fig. S4) and NIH3T3 fibroblasts (see Fig. 4). As shown in Fig. S4 and Fig. 4, blank PEGHCs are non-toxic to either HepG2 cells or NIH3T3 fibroblasts even at a high concentration (0.5 mg/mL). At all concentrations, the cell viabilities of NIH3T3 fibroblasts and HepG2 cells are higher than 90%. These results demonstrate that PEGHC micelles could be potentially used as drug carriers. 3.5.2. In vitro antitumor activity The antitumor activity of 9-NC/PEGHC micelles was evaluated in vitro. As shown in Fig. 5, the results show that 9-NC/PEGHC micelles could kill HepG2 cells in a dose-dependent manner. The value of the half maximal (50%) inhibitory concentration (IC50 ) of 9NC/PEGHC NPs is 0.03 mg/L (48 h), whereas the IC50 values of 9-NC reported by Zhang et al. were 2.46 mg/L (48 h) and 0.06 mg/L (72 h) [39]. This result indicates that the HepG2 cells are more sensitive to 9-NC/PEGHC micelles. It demonstrates that the anticancer activity of 9-NC is enhanced via the encapsulation of PEGHC micelles.
Fig. 4. Cytotoxicity of blank PEGHC micelles cultured with NIH3T3 for 48 h. Means ± SD (n = 3).
100
Cell viability (%)
3.5. Cell viability assay
80 60 40 20 0 1E-3
0.01
0.1
1
10
Concentration (µg/mL) Fig. 5. In vitro anticancer activities of 9-NC/PEGHC micelles against HepG2 cells with a 48-h incubation time. Means ± SD (n = 3).
Fig. 6. Confocal laser scanning microscopic images of HepG2 cells incubated with free 9-NC (a, b), 9-NC/PEGHC NPs (c, d), and the control group (e, f).
632
D. Li et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 627–632
The morphology of HepG2 cells incubated with blank PEGHC micelles and 9-NC/PEGHC micelles was observed by inverted microscope (see Supplementary data, Fig. S5). The cells incubated with 9-NC/PEGHC micelles are shrunk and aggregated, which indicates that almost all the HepG2 cells were killed by 9-NC (Fig. S5b). 3.6. Cellular uptake study The cellular uptake behavior of 9-NC and 9-NC/PEGHC micelles against HepG2 cell for 2 h was studied by confocal laser scanning microscopy (CLSM). The fluorescence was detected by confocal microscope with a laser wavelength of 405 nm [40,41]. As shown in Fig. 6, the control group (f) does not show fluorescence. However, significant 9-NC fluorescence is observed in cells incubated with 9-NC/PEGHC micelles (d), which indicates the successful delivery of 9-NC into the HepG2 cells. As PEGHC micelles improved the solubility of 9-NC greatly, the fluorescence intensity in HepG2 cells incubated with 9-NC/PEGHC micelles (Fig. 6d) is stronger than that of free 9-NC (Fig. 6b). 9-NC (20 g/mL) formed aggregates and deposited at the bottom of the dish as bright fluorescent spots (Fig. 6b and Fig. 6d) due to the poor solubility of 9-NC in water. These results demonstrate the rapid cellular uptake and dispersion of 9-NC/PEGHC micelles in the cytosol of cells. 4. Conclusion PEGHC micelles, with small molecules as lipophilic moieties, were successfully designed and synthesized. The anticancer drug 9-NC was used to investigate the release properties of PEGHC micelles. The DLC of PEGHC micelles was 4.5% with the particle size less than 100 nm. The release of 9-NC from PEGHC micelles could be sustained, and the accumulated release rate was higher than 85%, which was considerably higher than that of other polymeric micelles. Histidine acted as an accelerator to enhance the release of 9-NC from PEGHC micelles. The IC50 of 9-NC-loaded PEGHC micelles was as low as 0.030 mg/L. The drug-loaded PEGHC micelles exhibited efficient cellular endocytosis and inhibited the proliferation of HepG2 liver cancer cells efficiently. PEGHC micelles are potential promising carriers for 9-NC delivery. Acknowledgments This research work was supported by the National Basic Research Program of China (National 973 program, No. 2011CB606206), National Science Foundation of China (NSFC, No. 31170921, 50830105, 51133004), National Science Foundation for Excellent Young Scholars (No. 51222304), and Program for Changjiang Scholars and Innovative Research Team in University (IRT1163). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.10.032.
References [1] V. Torchilin, Pharmaceut. Res. 24 (2007) 1–16. [2] L. Fan, F. Li, H. Zhang, Y. Wang, C. Cheng, X. Li, C. Gu, Q. Yang, H. Wu, S. Zhang, Biomaterials 31 (2010) 5634–5642. [3] A.L.Z. Lee, Y. Wang, H.Y. Cheng, S. Pervaiz, Y.Y. Yang, Biomaterials 30 (2009) 919–927. [4] H. Wang, P. Zhao, W. Su, S. Wang, Z. Liao, R. Niu, J. Chang, Biomaterials 31 (2010) 8741–8748. [5] M. Saad, O.B. Garbuzenko, T. Minko, Nanomedicine 3 (2008) 761–776. [6] S.A. Jenekhe, X.L. Chen, Science 279 (1998) 1903–1907. [7] C.L. Lo, K.M. Lin, G.H. Hsiue, J. Controlled Release 104 (2005) 477–488. [8] G. Gaucher, M.H. Dufresne, V.P. Sant, N. Kang, D. Maysinger, J.C. Leroux, J. Controlled Release 109 (2005) 169–188. [9] P. Couvreur, B. Kante, L. Grislain, M. Roland, P. Speiser, J. Pharm. Sci. 71 (1982) 790–792. [10] Y. Gao, L. Chen, W. Gu, Y. Xi, L. Lin, Y. Li, Mol. Pharm. 5 (2008) 1044–1054. [11] G.S. Kwon, Crit. Rev. Ther. Drug. Carrier Syst. 20 (2003) 357. [12] N. Tang, G. Du, N. Wang, C. Liu, H. Hang, W. Liang, J. Natl. Cancer I 99 (2007) 1004–1015. [13] H. Maeda, Adv. Drug Delivery Rev. 46 (2001) 169–185. [14] H. Maeda, L.W. Seymour, Y. Miyamoto, Bioconjugate Chem. 3 (1992) 351–362. [15] F.M. Muggia, Clin. Cancer Res. 5 (1999) 7–8. [16] P.A. Vasey, S.B. Kaye, R. Morrison, C. Twelves, P. Wilson, R. Duncan, A.H. Thomson, L.S. Murray, T.E. Hilditch, T. Murray, Clin. Cancer Res. 5 (1999) 83–94. [17] R. Liu, B. He, D. Li, Y. Lai, J. Chang, J.Z. Tang, Z. Gu, Int. J. Nanomed. 7 (2012) 4433. [18] M.C. Jones, J.C. Leroux, Eur. J. Pharm. Biopharm. 48 (1999) 101–111. [19] A. Rösler, G.W.M. Vandermeulen and H.A. Klok, Adv Drug Delivery Rev, 64, Supplement (2012) 270-279. [20] L. Zhang, A. Eisenberg, Science 268 (1995) 23. [21] H. Ulukan, P.W. Swaan, Drugs 62 (2002) 2039–2057. [22] R. Garcia-Carbonero, J.G. Supko, Clin. Cancer Res. 8 (2002) 641–661. [23] J. Fassberg, V.J. Stella, J. Pharm. Sci. 81 (1992) 676–684. [24] M.E. Wall, M.C. Wani, A.W. Nicholas, G. Manikumar, C. Tele, L. Moore, A. Truesdale, P. Leitner, J.M. Besterman, J. Med. Chem. 36 (1993) 2689–2700. [25] M.C. Wani, A.W. Nicholas, M.E. Wall, J. Med. Chem. 30 (1987) 2317–2319. [26] W.D. Kingsbury, J.C. Boehm, D.R. Jakas, K.G. Holden, S.M. Hecht, G. Gallagher, M.J. Caranfa, F.L. McCabe, L.F. Faucette, R.K. Johnson, R.P. Hertzberg, J. Med. Chem. 34 (1991) 98–107. [27] U. Schaeppi, R. Fleischman, D. Cooney, Cancer Chemother. Rep. 5 (1974) 25. [28] J. Gao, J. Ming, B. He, Y. Fan, Z. Gu, X. Zhang, Eur. J. Pharm. Sci. 34 (2008) 85–93. [29] F.M.H. de Groot, G.F. Busscher, R.W.M. Aben, H.W. Scheeren, Bioorg. Med. Chem. Lett. 12 (2002) 2371–2376. [30] H.G. Lerchen, J. Baumgarten, K. von dem Bruch, T.E. Lehmann, M. Sperzel, G. Kempka, H.H. Fiebig, J. Med. Chem. 44 (2001) 4186–4195. [31] N. Pessah, M. Reznik, M. Shamis, F. Yantiri, H. Xin, K. Bowdish, N. Shomron, G. Ast, D. Shabat, Bioorgan Med. Chem. 12 (2004) 1859–1866. [32] R. Liu, D. Li, B. He, X. Xu, M. Sheng, Y. Lai, G. Wang, Z. Gu, J. Controlled Release 152 (2011) 49–56. [33] F.M. Winnik, S.T. Regismond, Colloids Surf., A 118 (1996) 1–39. [34] J.L. Lu, J.C. Wang, S.X. Zhao, X.Y. Liu, H. Zhao, X. Zhang, S.F. Zhou, Q. Zhang, Eur. J. Pharm. Biopharm. 69 (2008) 899–907. [35] M. Wilhelm, C.L. Zhao, Y. Wang, R. Xu, M.A. Winnik, J.L. Mura, G. Riess, M.D. Croucher, Macromolecules 24 (1991) 1033–1040. [36] A. Kumari, S.K. Yadav, S.C. Yadav, Colloids Surf., B 75 (2010) 1–18. [37] L.J. Zhang, B. Xing, J. Wu, B. Xu, X.L. Fang, Pulm Pharmacol. Ther. 21 (2008) 239–246. [38] Y. Liang, Y. Lai, D. Li, B. He, Z. Gu, Mater. Lett. 97 (2013) 4–7. [39] S. Zheng, S. Chang, J. Lu, Z. Chen, L. Xie, Y. Nie, B. He, S. Zou, Z. Gu, PloS one 6 (2011) e21064. [40] H. Dong, C. Dong, Y. Feng, T. Ren, Z. Zhang, L. Li, Y. Li, Med. Chem. Commun. 3 (2012) 1555–1561. [41] P. Pramod, K. Takamura, S. Chaphekar, N. Balasubramanian, M. Jayakannan, Biomacromolecules 13 (2012) 3627–3640.
