J Mater Sci: Mater Med (2014) 25:691–701 DOI 10.1007/s10856-013-5109-9
Preparation and characterization of galactosylated glycol chitosan micelles and its potential use for hepatoma-targeting delivery of doxorubicin Jing-Mou Yu • Wei-Dong Li • Lu Lu • Xue-Yun Zhou • Dian-Yuan Wang • Hui-Min Li • Xiao-Yuan Xu • Jian Chen
Received: 29 April 2013 / Accepted: 29 November 2013 / Published online: 14 December 2013 Ó Springer Science+Business Media New York 2013
Abstract This study aimed to develop novel galactosylated cholesterol modified-glycol chitosan (Gal-CHGC) micelles for targeting delivery of doxorubicin (DOX) in live cancer cells. Three kinds of Gal-CHGC conjugates were synthesized and characterized. The mean particle size and critical aggregation concentration of these polymeric micelles increased with the increase of galactose substitution degree. The DOX-loaded micelles were prepared by an o/w method. The mean diameters of DOX-loaded galactosylated micelles were in the range of 387–497 nm. DOX released from drug-loaded micelles displayed a biphasic way. Cellular uptake studies demonstrated that DOX-loaded galactosylated micelles could enhance the uptake of DOX into HepG2 cells. Moreover, the cytotoxicity of DOX-loaded galactosylated micelles against HepG2 cells significantly improved in contrast with free DOX and DOX-loaded micelles without galactosylation. These results suggested that Gal-CHGC micelles could be a potential carrier for hepatoma-targeting drug delivery.
J.-M. Yu (&) L. Lu H.-M. Li J. Chen Department of Pharmacy, College of Basic Medical Science, Jiujiang University, 17 Lufeng Road, Jiujiang 332000, People’s Republic of China e-mail: [email protected] W.-D. Li (&) X.-Y. Xu Key Laboratory of Systems Biology Medicine of Jiangxi Province, College of Basic Medical Science, Jiujiang University, Jiujiang 332000, People’s Republic of China e-mail: [email protected] X.-Y. Zhou D.-Y. Wang College of Science, Jiujiang University, Jiujiang 332005, People’s Republic of China
1 Introduction Polymeric micelles, especially used in drug delivery system, have played an important role on enhancing drug water solubility, targeting drug delivery to the desirable action site, decreasing side effects, and improving drug bioavailability, etc. [1–5]. The micelles are composed of hydrophobic segments as the internal core and hydrophilic segments as the outer shell in an aqueous media. Glycol chitosan is a chitosan derivative and widely chosen as hydrophilic group in amphiphilic copolymer because of its solubility in water at all pH values and biocompatibility. Amphiphilic glycol chitosan derivatives such as glycol chitosan bearing 5b-cholanic acid [6], glycol chitosan bearing adriamycin [7] and quaternary ammonium palmitoyl glycol chitosan [8] have been reported as efficient drug carriers. In previous studies, Yu et al. have synthesized cholesterol-modified glycol chitosan (CHGC) conjugate which possessed high molecular weight glycol chitosan, and the self-aggregated CHGC nanoparticles were prepared in aqueous medium [9]. Moreover, doxorubicin (DOX)loaded CHGC nanoparticles demonstrated the prolonged circulation time in rat plasma, and exhibited higher antitumor efficacy in vivo than free DOX [10]. In recent years, increasing attention has been paid to the fabrication of targeting polymeric micelles with excellent cell internalization and intracellular drug release performance. In order to endow specific recognition of the micelles by a specific site, targeting moieties or ligands have been adopted and introduced to the outer surface of polymeric micelles [11–13]. It has been documented that asialoglycoprotein receptor (ASGPR) is an integral membrane protein and overexpressed on the surface of mammalian hepatocytes [14]. The ASGPR can specifically recognize b-D-galactose, N-acetylgalactosamine or lactose
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ligands, and bring it into the hepatocytes through endocytic process as a ligand-receptor complex. This provided a unique way for the development of liver-targeting drug delivery systems. Galactose-containing polymeric micelles as targeting agents for ASGPR have been a useful tool for specific therapy of liver diseases [15–17]. This type of polymer-galactose conjugates has amphiphilic structures which can entrap hydrophobic drug in the hydrophobic inner core and exhibit a hydrophilic outer shell with pending galactose moieties available for ASGPR recognition. For example, Wu et al. [18] reported that galactosylated and fluorescein isothiocyanate (FITC) labeled polycaprolactone-g-dextran (Gal-PCL-g-Dex-FITC) micelles exhibited apparent targetability to HepG2 cells and liver tissue, indicating that the galactosylated micelles have great potential as a liver targeting drug carrier. Herein, the primary objective of this study is to construct galactosylated cholesterol modified-glycol chitosan (Gal-CHGC) micelles for hepatoma-targeting delivery of DOX. Gal-CHGC conjugates with different degree of galactosylated modification were synthesized and characterized. DOX was physically entrapped in the micelles by an o/w method. We also investigated the physicochemical properties of DOX-loaded micelles, drug release behavior in vitro. Moreover, the cellular uptake and cytotoxicity of drug-loaded micelles were carried out in ASGPR-overexpressed HepG2 cells.
2 Materials and methods 2.1 Materials Glycol chitosan (Mw = 83.0 kDa) was obtained by enzymatic degradation of 75.2 % deacetylated glycol chitosan [19], which was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cholesterol, succinic anhydride, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) were obtained from SigmaAldrich (St. Louis, MO). Lactobionic acid and potassium poly(vinyl sulfate) were supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Pyrene was purchased from Acros Organics (New Jersey, USA). DOX hydrochloride was supplied by Beijing Huafeng United Technology Co., Ltd. (Beijing, China). RPMI 1640 medium and trypsin– EDTA were purchased from Jinuo Biotechnology Company (Hangzhou, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biologic Co. Ltd. (Zhejiang, China). 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). Unless stated otherwise, all other chemicals were of analytical grade and used as received.
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2.2 Synthesis of CHGC conjugate CHGC conjugate was synthesized by using our previous method with minor modification [19]. Briefly, cholesterol (4.0 g, 10.4 mmol) was dissolved in pyridine, and succinic anhydride (2.88 g, 28.0 mmol) was added. The reaction proceeded for 24 h at room temperature. Then, the mixture was precipitated in water. The produced precipitate was filtered and dissolved in ether. After filtration, the clear solution was obtained and dried under reduced pressure. The cholesterol hemisuccinate (CHS) product was obtained by recrystallization in acetone. Glycol chitosan (500 mg) was dissolved in distilled water overnight and diluted with ethanol. EDC (100.5 mg, 0.524 mmol) and CHS (127.7 mg, 0.262 mmol) were slowly added. After the reaction mixture was magnetically stirred for 72 h at room temperature, it was sequentially dialyzed (MWCO: 14 kDa) against the excess amount of ethanol/distilled water solution (88:12, v/v) and distilled water. The dialyzed solution was lyophilized to obtain CHGC conjugate. 2.3 Synthesis of Gal-CHGC conjugates Gal-CHGC conjugates were synthesized by carbodiimide chemistry. CHGC (120 mg) was dissolved in 40 ml of distilled water overnight, and followed by sonication using a probe type sonifier (Ningbo Scientz Biotechnology Co. Ltd, China) at 200 W for 40 times in an ice bath. Lactobionic acid (40 mg, 0.112 mmol) and EDC (85.8 mg, 0.448 mmol) were added, and the solution was stirred for 10 h at 70 °C. The resultant solution was dialyzed (MWCO: 14 kDa) in distilled water for 48 h and followed by freeze-drying. The copolymer obtained was denoted as Gal-CHGC-1. The other Gal-CHGC conjugates were prepared analogously using different feed mole ratios. For Gal-CHGC-2, 120 mg CHGC, 60 mg lactobionic acid (0.167 mmol) and 128.7 mg EDC (0.671 mmol) was used, while for GalCHGC-3, 80 mg lactobionic acid (0.223 mmol) and 171.6 mg EDC (0.895 mmol) was employed. 2.4 Characterization of polymeric micelles 1
H-Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DMX500 spectrometer using D2O as the solvent. The degree of substitution (DS), defined as the number of cholesterol groups per 100 sugar residues of glycol chitosan was determined by a colloidal titration method [6, 19], which is based on the reaction between positively charged glycol chitosan and negatively charged potassium poly(vinyl sulfate).
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The hydrodynamic diameters of the micelles were measured by dynamic light scattering (90Plus, Brookhaven Instruments Corp., Holtsville, NY, USA). The micelles were dispersed in deionized water at the concentration of 1 mg/ml. The particle size analysis was carried out at 25 °C. And the morphology was observed through a TEM (JEM-1230, Jeol, Japan) operated at 80 kV. For TEM sample preparation, a drop of sample solution was placed onto a 300-mesh copper grid coated with carbon and the extra solution was blotted with filter paper, followed by air drying. 2.5 Determination of critical aggregation concentration (CAC) The CAC of Gal-CHGC conjugates were determined using pyrene as a fluorescence probe. The Gal-CHGC conjugate suspension was prepared by sonication and adjusted to various conjugate concentrations. A known amount of pyrene in acetone was added into each of 10 ml vials and the final concentration of pyrene was 6.0 9 10-7 M. After the acetone was evaporated, a total of 10 ml of various concentrations of copolymer solution were introduced to each vial and then heated at 50 °C for 10 h to equilibrate the pyrene and the micelles, and left to cool overnight at room temperature. The fluorescence spectra were recorded using a fluorescence spectrophotometer (Hitachi F-4500, Japan) with a slit width of 2.5 nm. The excitation and emission wavelength was set at 339 and 390 nm, respectively. 2.6 Preparation and characterization of DOX-loaded micelles The DOX-loaded micelles were prepared by the o/w method [20, 21]. Firstly, copolymer samples (30 mg) were dissolved in distilled water (60 ml), followed by probe sonication as stated above. Secondly, DOX hydrochloride (15 mg) was dissolved in 10 ml of CHCl3 in the presence of triethylamine (3 times molar quantity of DOX) to obtain a free DOX containing solution. Then 3 ml of DOX organic phase solution were slowly added to the above aqueous phase under vigorously stirring to form the o/w emulsion. CHCl3 in the emulsion was removed by stirring under reduced pressure. The micelle solution was filtered through a 0.8-lm membrane and eluted through a Sephadex G25 fine column to remove non-encapsulated DOX. The resultant DOX-loaded micelles were obtained by freeze drying. For drug loading quantification, freeze-dried micelles were dispersed in aqueous solution and disrupted by the addition of DMSO. This solution was stirred and followed by sonication in an ice bath. The absorbance of the solution
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was measured using UV–vis spectrophotometer at k = 481 nm and calculated using following equations: LC (loading content) =
weight of DOX loaded weight of DOX - loaded micelles 100% ð1Þ
EE (encapsulation efficiency) =
weight of DOX loaded weight of DOX in feed 100% ð2Þ
The hydrodynamic diameters of the micelles were also investigated by dynamic light scattering (90Plus, Brookhaven Instruments Corp., Holtsville, NY, USA). The analysis conditions were the same as described above. The morphological characteristics of DOX-loaded micelles were examined through a TEM (JEM-1230, Jeol, Japan) as mentioned above. 2.7 In vitro DOX release study DOX release behavior was explored by using a dynamic dialysis method. The release experiments were conducted in PBS (pH 7.4) at 37 °C. Typically, 1 ml of DOX-loaded micelles was placed in a dialysis bag (MWCO: 14 kDa) and dialyzed against 20 ml PBS in an air-bath shaker at 100 rpm. At predetermined time points, the entire medium was taken out with replenishing equal volume of fresh medium. The amount of DOX released was determined by fluorescence spectrophotometer (Hitachi F-4500, Japan). Measurements were made at an excitation wavelength of 470 nm and an emission wavelength of 585 nm. The accumulated drug was calculated, and each study was conducted in triplicate. 2.8 Cell culture Human hepatocellular carcinoma cell line (HepG2) was obtained from Shanghai Institutes of Biology Sciences, Chinese Academy of Sciences (Shanghai, China). HepG2 cells were routinely cultured in RPMI-1640 medium containing 10 % FBS and 1 % penicillin–streptomycin at 37 °C and 5 % CO2 humidified atmosphere. 2.9 Cellular uptake study In order to visualize cellular uptake of DOX by HepG2 cells, the cells were examined by confocal laser scanning microscopy (CLSM) (Carl Zeiss LSM-510, Germany). The cells were seeded into 6-well plates (Costar, Corning, NY, USA) at a density of 2 9 105 cells/ml and cultured overnight at 37 °C in 5 % CO2. After removed the cultured
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media, the cells were incubated with free DOX, DOXloaded CHGC (DOX-CHGC), or DOX-Gal-CHGC-1 micelles (equivalent DOX concentration: 10 lg/ml) in RPMI 1640 medium supplemented with 10 % FBS. After 4 or 6 h incubation, the cells were washed three times with PBS (pH 7.4) and fixed in 4 % paraformaldehyde solution at 4 °C for 1 h. The intracellular localization of DOX was observed by confocal microscope at 488 nm for excitation and an LP filter of 560 nm for DOX detection. Flow cytometry was also used to investigate the uptake of DOX into HepG2 cells. In the same way described above, HepG2 cells were incubated with different DOX formulations in 6-well plates. After 4 or 6 h treatment, the cells were harvested with 0.2 % (w/v) trypsin-0.1 % (w/v) EDTA solution. The cells were centrifuged and washed with icecold PBS for 3 times. Then the cells were re-suspended with PBS before the fluorescence-activated cell sorting (FACS) analysis using a flow cytometer (Becton–Dickinson FACSCalibur, USA) equipped with a 488-nm argon ion laser. 2.10 In vitro cytotoxicity study The cytotoxicity of free DOX, DOX-CHGC and DOX-GalCHGC-1 micelles against HepG2 cells was measured by an MTT-based assay. The cells were seeded at a density of 1 9 104 cells/well in 96-well plates (Costar, Corning, NY, USA) and incubated for 24 h at 37 °C and 5 % CO2. After removing the medium, 200 ll of RPMI 1640 medium containing various concentrations of free DOX, DOXCHGC, DOX-Gal-CHGC-1, CHGC or Gal-CHGC-1 micelles were added, and further incubated for 48 h. Then, 20 ll of MTT solution (5 mg/ml in PBS) were added to each well. After an additional 4 h incubation, the culture medium was discarded, and the formazan crystals in cells were solubilized with 200 ll of DMSO for 15 min. The absorbency values were measured by microplate reader (Thermo Scientific Multiskan MK3, USA) at wavelength 570 nm (OD570), and cell viability was calculated by dividing OD570 (sample) with OD570 (blank).
3 Results and discussion
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amino group of glycol chitosan (Scheme 1). As presented in Fig. 1, 1H NMR spectra were used to confirm the CHGC conjugate. Compared with glycol chitosan, the new proton peaks of the methene hydrogen signals of CHS (chemical shift 1.25–1.27 ppm) were observed in spectrum of CHGC [19]. These results demonstrated that the CHS was successfully grafted onto the chains of glycol chitosan. To determine the number of cholesterol groups conjugated on the glycol chitosan backbone, a colloidal titration method was used. The DS was 8.2 cholesterol groups per 100 sugar residues of glycol chitosan. The mean diameter of CHGC determined by dynamic light scattering (DLS) was 264 nm. The principle of the chemical reaction in synthesis of Gal-CHGC is also the formation of amide bonds. The carboxyl groups of lactobionic acid were conjugated with free amino groups of CHGC via the coupling reagent EDC. The structure changes of Gal-CHGCs were confirmed by the 1H NMR technique. The number of hydrogens in GalCHGC at d 3.0–4.2 was higher than that in CHGC, implying that galactose was introduced to CHGC (Fig. 1c– e). And the intensity increased as the feed amount of lactobionic acid in the synthesis increased. All these results demonstrated three Gal-CHGC conjugates with different levels of galactose substitution were successful obtained. The substitution degree of galactose (DSGal) was the number of galactose groups per 100 sugar residues of backbone glycol chitosan in Gal-CHGC, and it was estimated by 1H NMR via the following equation [22]: DSGal ¼
HGalCHGCðd3:04:2Þ HCHGCðd3:04:2Þ 12
ð3Þ
HGal-CHGC (d 3.0–4.2) was the number of hydrogens of Gal-CHGC at d 3.0–4.2. HCHGC (d3.04.2) was the number of hydrogens of CHGC at d 3.0–4.2. The DSGal of GalCHGC-1, Gal-CHGC-2 and Gal-CHGC-3 was 7.9, 11.5 and 18.4, respectively. However, the typical resonance peaks of cholesterol group were hardly observed in GalCHGC conjugates in D2O (Fig. 1), which was due to restricted motions of these protons in the micelle core. This characteristic of the 1H NMR spectra further suggested the micelle formation by Gal-CHGC cojugates in an aqueous solution. Similar results were reported by other researchers [22, 23].
3.1 Synthesis and characterization of Gal-CHGC conjugate 3.2 Physicochemical properties of Gal-CHGC micelles As shown in Scheme 1, the synthesis of Gal-CHGC conjugates involved three steps: (1) carboxylation of cholesterol; (2) synthesis of CHGC; (3) galactosylation of CHGC. Firstly, cholesterol was synthesized with succinic anhydride, and then CHS was produced. In the presence of EDC, CHGC was obtained by the formation of amide groups between carboxyl group of CHS and the primary
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The self-aggregation behavior of Gal-CHGC micelles in aqueous media was investigated by fluorescence spectroscopy in the presence of pyrene [24]. Plots of the intensity ratio I338/I333 from excitation spectra were shown in Fig. 2 as a function of the copolymer concentration. At the low copolymer concentration, the values of I338/I333 ratio were
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Scheme 1 Synthesis of GalCHGC conjugate
low and the plots were almost flat. As the ratio values significantly increased, it implied that pyrene molecule was transferred from a polar microdomain to a more hydrophobic one. The CAC value was determined from the concentration dependence of the I338/I333 ratios. CAC was designated as the intersection of two straight line segments, which was drawn from the points at the lowest polymer concentrations and the ones on the rapidly rising part of the plot. The CAC values of Gal-CHGC-1, Gal-CHGC-2 and Gal-CHGC-3 were respectively 0.0161, 0.0243 and 0.0453 mg/ml, which increased as the degree of galactose substitution increased. These results were attributed to the changes of hydrophilic segment in the Gal-CHGC.
Moreover, the low CAC means that the Gal-CHGC conjugates can form nano-scaled micelles under highly diluted condition. The mean diameters of Gal-CHGC-1, Gal-CHGC-2 and Gal-CHGC-3 determined by DLS were 359, 436 and 471 nm, respectively. The size of Gal-CHGCs micelles was larger than that of blank CHGC micelles described above. It was clearly seen that the lower amount of galactose substitution in the copolymer resulted in the formation of smaller particles. This is because the polymer chains were not likely to form more compact aggregates after hydrophilic galactose conjugation. As shown in Fig. 3a, TEM depicted that the shape of Gal-CHGC-1
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Fig. 1 1H NMR spectra of a glycol chitosan, b CHGC, c Gal-CHGC-1, d Gal-CHGC-2 and e Gal-CHGC-3
self-aggregates was roughly spherical. And the particle size was similar with the average diameter of Gal-CHGC-1 self-aggregates, which was determined by DLS analysis. 3.3 Preparation and characterization of DOX-loaded micelles DOX-loading of polymeric micelles was adopted by an oilin-water emulsion method as previously reported [25]. The similar procedures were used in the present study. DOX hydrochloride was deprotonated by triethylamine in the
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chloroform organic phase. The molar ratio of triethylamine/DOX was set to 3.0. Table 1 demonstrated the physicochemical properties of drug-loaded micelles. The particle size of the polymeric micelles assembled in aqueous solution was determined by DLS. The mean diameters of three kinds of DOX-Gal-CHGC micelles were in the range of 387–497 nm. It was found that the particle size of DOX-CHGC and DOX-Gal-CHGC micelles was larger than that of their blank polymeric micelles. We inferred that DOX molecules were entrapped into the micelles and then the size of these micelles increased.
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3.4 In vitro drug release study To study the release behavior of DOX in vitro, DOXCHGC or DOX-Gal-CHGCs micelles were incubated in a dialysis bag under shaking at 37 °C, and the released DOX was quantified using fluorescence spectrophotometer. As shown in Fig. 4, the total amount of DOX released from DOX-CHGC micelles in 96 h was 49.8 %, and was higher than that of DOX-Gal-CHGCs micelles. This result may be due to DOX-CHGC micelles with the smaller particle size and non-galactose in the outer shell. In addition, DOX-GalCHGC-1 micelles showed faster drug release in 96 h than DOX-Gal-CHGC-2 and DOX-Gal-CHGC-3 micelles. The results revealed that the release rate may be different when the degree of galactose substitution was changed. In addition, DOX appeared to be released from DOX-loaded micelles in a biphasic way. The first phase of release was completed within 6 h, considering as an initial burst. After this initial effect, DOX was released a second phase showing slow sustained release for upto 96 h. As previously described, the release kinetic is associated with both dissolution and diffusion based on the Noyes-Whitney law of dissolution and Fick’s first law of diffusion [26]. For consideration of both the particle size and drug-loading rate, the DOX-Gal-CHGC-1 micelles were selected for further research. 3.5 Cellular uptake of study
Fig. 2 Polts of the intensity ratio I338/I333 vs. log C for Gal-CHGC conjugates with various compositions: a Gal-CHGC-1, b Gal-CHGC2 and c Gal-CHGC-3
Moreover, as the degree of galactose substitution in the Gal-CHGC conjugates increased, the reduction of drug loading content and encapsulation efficiency in DOX-GalCHGC micelles were observed. This was mainly due to the fact that Gal-CHGC micelles containing the more hydrophilic groups displayed poorer capacity to load drug. As shown in Fig. 3b, TEM observation demonstrated that the shape of DOX-Gal-CHGC-1 self-aggregates was nearly spherical. The average diameter of DOX-Gal-CHGC-1 micelles was similar with the size determined by DLS.
CLSM was used to directly monitor the internalization of DOX-loaded micelles and free DOX into HepG2 cells (Fig. 5). When HepG2 cells were incubated with free DOX for 4 h, only weak red fluorescence were observed in the nucleus. For DOX-CHGC and DOX-Gal-CHGC-1 aggregate systems, however, the DOX fluorescence is observed in both cytoplasm and nucleus. After 6 h of cell incubation with the free DOX, stronger fluorescence was observed in cell nuclei in addition to the weak fluorescence in cytoplasm. And the cells incubated with the DOX-CHGC and DOX-Gal-CHGC-1 micelles also emitted significantly increased fluorescence. As comparatively estimated by CLSM, cellular uptake extent of DOX-Gal-CHGC-1 micelles was higher than that of DOX-CHGC micelles without galactosylation and free DOX after exposure of 4 and 6 h under the same condition. Moreover, HepG2 cells incubated with free DOX showed lower red fluorescence than the cells treated with DOX-CHGC micelles. It implied that the internalization mechanism of the DOX-loaded micelles would be different from that of free DOX. The free DOX molecules were transported into the cytoplasm of the cells and reached into the nucleus in a passive diffusion manner. The micellar systems were internalized through an endocytosis pathway, and then be localized in
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Fig. 3 TEM images of a GalCHGC-1 and b DOX-GalCHGC-1 micelles
Table 1 Physicochemical properties of drug-loaded copolymer micelles Sample
DOX/carriera
Size (nm)b
PIc
LC (%)d
EE (%)e
DOX-CHGC
1.5/10
298 ± 17.1
0.246 ± 0.038
10.7 ± 0.51
80.0 ± 4.25
DOX-Gal-CHGC-1
1.5/10
387 ± 29.1
0.252 ± 0.031
9.66 ± 0.43
71.6 ± 3.56
DOX-Gal-CHGC-2
1.5/10
451 ± 25.7
0.223 ± 0.063
8.79 ± 0.24
64.5 ± 1.89
DOX-Gal-CHGC-3
1.5/10
497 ± 24.6
0.242 ± 0.072
8.09 ± 0.31
58.9 ± 2.44
The results represent the mean ± SD (n = 3) a
The ratio of DOX to carrier, based on feed amount (mg/mg)
b
Measured by dynamic light scattering
c
Polydispersity index
d
Loading content
e
Encapsulation efficient
Fig. 4 Release profiles of DOX from DOX-CHGC, DOX-GalCHGC-1, DOX-Gal-CHGC-2 and DOX-Gal-CHGC-3 micelles at 37 °C in PBS at pH 7.4
acidic endocytic compartments [27]. As mentioned above, DOX-loaded micelles displayed sustained release behavior in vitro, whereas free DOX could freely diffuse in the
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media. Additionally, it is well-known that the human hepatocellular carcinoma cells (HepG2) express the ASGP receptors on their surfaces. The improved cellular uptake of DOX-Gal-CHGC-1 micelles might be attributed to the effective targeting of HepG2 cells by the galactoseexpressing nanoparticles. Therefore, it could be concluded that Gal-CHGC-1 micelles could enhance cellular uptake of DOX in HepG2 cells. To further analyze the cellular uptake of free DOX, DOX-CHGC and DOX-Gal-CHGC-1 micelles by HepG2 cells, flow cytometry was performed. As seen in Fig. 6a, the cells incubated with DOX-Gal-CHGC-1 micelles for 4 h showed higher fluorescent intensity than the cells treated with free DOX and DOX-CHGC micelles. As the incubation time increased to 6 h, the amounts of DOX fluorescence emitted from these DOX formulations simultaneously increased (Fig. 6b). It was clearly seen that the orders of fluorescent intensity emitted from DOX formulations were DOX-Gal-CHGC-1 [ DOX-CHGC [ free DOX in 4 and 6 h. This result was consistent with confocal microscopic observation. We inferred that DOX-loaded micelles could improve the cytotoxicity of DOX.
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Fig. 5 CLSM images of HepG2 cells after incubation with free DOX, DOX-CHGC and DOX-Gal-CHGC-1 micelles for 4 or 6 h
Fig. 6 Flow cytometric analysis of HepG2 cells incubated with free DOX, DOX-CHGC and DOX-GalCHGC-1 micelles for a 4 h or b6h
3.6 In vitro cytotoxicity study The cytotoxicities of free DOX, DOX-CHGC and DOXGal-CHGC-1 micelles were investigated in HepG2 cell after 48 h incubation. As shown in Fig. 7a, DOX-GalCHGC-1 and DOX-CHGC micelles showed higher cytotoxicity in contrast with free DOX. The half inhibitory concentration (IC50) of free DOX was 0.985 lg/ml, while that of DOX-Gal-CHGC-1 and DOX-CHGC micelles was 0.236 and 0.586 lg/ml, respectively. The IC50 value of DOX-Gal-CHGC-1 micelles against HepG2 cells was nearly 4.17 times lower than that of free DOX, indicating that Gal-CHGC-1 micelles could be efficient targeting delivery of DOX in hepatocellular carcinoma cells. As mentioned above, DOX released from DOX-Gal-CHGC-1
and DOX-CHGC micelles in 48 h were 44.3 and 48.2 %, respectively. The cytotoxicity improved is mainly originated from the enhanced cellular uptake of DOX-loaded micelles. These results agreed with flow cytometry and confocal microscopic analysis. Therefore, we confirmed that Gal-CHGC polymeric micelles could improved the cytotoxicity of DOX in ASGPR-overexpressed HepG2 cells. As shown in Fig. 7b, blank CHGC and Gal-CHGC1 micelles did not exhibit notable cytotoxicity against HepG2 cells. IC50 of CHGC and Gal-CHGC-1 micelles was 451.8 and 407.7 lg/ml, respectively. These results demonstrated that blank CHGC and Gal-CHGC-1 micelles showed low cytotoxicity. Taken together, the Gal-CHGC micelles have great potential in liver tumortargeting chemotherapy.
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References
Fig. 7 In vitro cytotoxicity of a free DOX, DOX-CHGC, DOX-GalCHGC-1 micelles, and b CHGC, Gal-CHGC-1 micelles against HepG2 cells after 48 h incubation
4 Conclusion In summary, three kinds of Gal-CHGC copolymers with different levels of galactose substitution were successfully synthesized and characterized. DOX was physically loaded into the Gal-CHGC micelles. DOX release from DOX-Gal-CHGC micelles in vitro showed an initial burst effect and followed by a slow drug release. In cellular uptake study, DOX-Gal-CHGC micelles showed galactose-mediated cancer cell targeting and enhanced cellular uptake in HepG2 cells. Moreover, the antitumor efficiency of DOX-Gal-CHGC micelles was higher than that of DOX-CHGC micelles and free DOX in HepG2 cells. Therefore, these results implied that Gal-CHGC micelles could be promising drug carriers for specific delivery to liver cancer cells. Acknowledgments We are grateful for partially financial support of National Nature Science Foundation of China (No. 81001418), Natural Science Foundation of Jiangxi Province (No. 20114BAB215017), and Scientific Research Fund of Jiangxi Education Department (No. GJJ11241).
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Preparation and characterization of galactosylated glycol chitosan micelles and its potential use for hepatoma-targeting delivery of doxorubicin Jing-Mou Yu • Wei-Dong Li • Lu Lu • Xue-Yun Zhou • Dian-Yuan Wang • Hui-Min Li • Xiao-Yuan Xu • Jian Chen
Received: 29 April 2013 / Accepted: 29 November 2013 / Published online: 14 December 2013 Ó Springer Science+Business Media New York 2013
Abstract This study aimed to develop novel galactosylated cholesterol modified-glycol chitosan (Gal-CHGC) micelles for targeting delivery of doxorubicin (DOX) in live cancer cells. Three kinds of Gal-CHGC conjugates were synthesized and characterized. The mean particle size and critical aggregation concentration of these polymeric micelles increased with the increase of galactose substitution degree. The DOX-loaded micelles were prepared by an o/w method. The mean diameters of DOX-loaded galactosylated micelles were in the range of 387–497 nm. DOX released from drug-loaded micelles displayed a biphasic way. Cellular uptake studies demonstrated that DOX-loaded galactosylated micelles could enhance the uptake of DOX into HepG2 cells. Moreover, the cytotoxicity of DOX-loaded galactosylated micelles against HepG2 cells significantly improved in contrast with free DOX and DOX-loaded micelles without galactosylation. These results suggested that Gal-CHGC micelles could be a potential carrier for hepatoma-targeting drug delivery.
J.-M. Yu (&) L. Lu H.-M. Li J. Chen Department of Pharmacy, College of Basic Medical Science, Jiujiang University, 17 Lufeng Road, Jiujiang 332000, People’s Republic of China e-mail: [email protected] W.-D. Li (&) X.-Y. Xu Key Laboratory of Systems Biology Medicine of Jiangxi Province, College of Basic Medical Science, Jiujiang University, Jiujiang 332000, People’s Republic of China e-mail: [email protected] X.-Y. Zhou D.-Y. Wang College of Science, Jiujiang University, Jiujiang 332005, People’s Republic of China
1 Introduction Polymeric micelles, especially used in drug delivery system, have played an important role on enhancing drug water solubility, targeting drug delivery to the desirable action site, decreasing side effects, and improving drug bioavailability, etc. [1–5]. The micelles are composed of hydrophobic segments as the internal core and hydrophilic segments as the outer shell in an aqueous media. Glycol chitosan is a chitosan derivative and widely chosen as hydrophilic group in amphiphilic copolymer because of its solubility in water at all pH values and biocompatibility. Amphiphilic glycol chitosan derivatives such as glycol chitosan bearing 5b-cholanic acid [6], glycol chitosan bearing adriamycin [7] and quaternary ammonium palmitoyl glycol chitosan [8] have been reported as efficient drug carriers. In previous studies, Yu et al. have synthesized cholesterol-modified glycol chitosan (CHGC) conjugate which possessed high molecular weight glycol chitosan, and the self-aggregated CHGC nanoparticles were prepared in aqueous medium [9]. Moreover, doxorubicin (DOX)loaded CHGC nanoparticles demonstrated the prolonged circulation time in rat plasma, and exhibited higher antitumor efficacy in vivo than free DOX [10]. In recent years, increasing attention has been paid to the fabrication of targeting polymeric micelles with excellent cell internalization and intracellular drug release performance. In order to endow specific recognition of the micelles by a specific site, targeting moieties or ligands have been adopted and introduced to the outer surface of polymeric micelles [11–13]. It has been documented that asialoglycoprotein receptor (ASGPR) is an integral membrane protein and overexpressed on the surface of mammalian hepatocytes [14]. The ASGPR can specifically recognize b-D-galactose, N-acetylgalactosamine or lactose
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ligands, and bring it into the hepatocytes through endocytic process as a ligand-receptor complex. This provided a unique way for the development of liver-targeting drug delivery systems. Galactose-containing polymeric micelles as targeting agents for ASGPR have been a useful tool for specific therapy of liver diseases [15–17]. This type of polymer-galactose conjugates has amphiphilic structures which can entrap hydrophobic drug in the hydrophobic inner core and exhibit a hydrophilic outer shell with pending galactose moieties available for ASGPR recognition. For example, Wu et al. [18] reported that galactosylated and fluorescein isothiocyanate (FITC) labeled polycaprolactone-g-dextran (Gal-PCL-g-Dex-FITC) micelles exhibited apparent targetability to HepG2 cells and liver tissue, indicating that the galactosylated micelles have great potential as a liver targeting drug carrier. Herein, the primary objective of this study is to construct galactosylated cholesterol modified-glycol chitosan (Gal-CHGC) micelles for hepatoma-targeting delivery of DOX. Gal-CHGC conjugates with different degree of galactosylated modification were synthesized and characterized. DOX was physically entrapped in the micelles by an o/w method. We also investigated the physicochemical properties of DOX-loaded micelles, drug release behavior in vitro. Moreover, the cellular uptake and cytotoxicity of drug-loaded micelles were carried out in ASGPR-overexpressed HepG2 cells.
2 Materials and methods 2.1 Materials Glycol chitosan (Mw = 83.0 kDa) was obtained by enzymatic degradation of 75.2 % deacetylated glycol chitosan [19], which was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cholesterol, succinic anhydride, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) were obtained from SigmaAldrich (St. Louis, MO). Lactobionic acid and potassium poly(vinyl sulfate) were supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Pyrene was purchased from Acros Organics (New Jersey, USA). DOX hydrochloride was supplied by Beijing Huafeng United Technology Co., Ltd. (Beijing, China). RPMI 1640 medium and trypsin– EDTA were purchased from Jinuo Biotechnology Company (Hangzhou, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biologic Co. Ltd. (Zhejiang, China). 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). Unless stated otherwise, all other chemicals were of analytical grade and used as received.
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2.2 Synthesis of CHGC conjugate CHGC conjugate was synthesized by using our previous method with minor modification [19]. Briefly, cholesterol (4.0 g, 10.4 mmol) was dissolved in pyridine, and succinic anhydride (2.88 g, 28.0 mmol) was added. The reaction proceeded for 24 h at room temperature. Then, the mixture was precipitated in water. The produced precipitate was filtered and dissolved in ether. After filtration, the clear solution was obtained and dried under reduced pressure. The cholesterol hemisuccinate (CHS) product was obtained by recrystallization in acetone. Glycol chitosan (500 mg) was dissolved in distilled water overnight and diluted with ethanol. EDC (100.5 mg, 0.524 mmol) and CHS (127.7 mg, 0.262 mmol) were slowly added. After the reaction mixture was magnetically stirred for 72 h at room temperature, it was sequentially dialyzed (MWCO: 14 kDa) against the excess amount of ethanol/distilled water solution (88:12, v/v) and distilled water. The dialyzed solution was lyophilized to obtain CHGC conjugate. 2.3 Synthesis of Gal-CHGC conjugates Gal-CHGC conjugates were synthesized by carbodiimide chemistry. CHGC (120 mg) was dissolved in 40 ml of distilled water overnight, and followed by sonication using a probe type sonifier (Ningbo Scientz Biotechnology Co. Ltd, China) at 200 W for 40 times in an ice bath. Lactobionic acid (40 mg, 0.112 mmol) and EDC (85.8 mg, 0.448 mmol) were added, and the solution was stirred for 10 h at 70 °C. The resultant solution was dialyzed (MWCO: 14 kDa) in distilled water for 48 h and followed by freeze-drying. The copolymer obtained was denoted as Gal-CHGC-1. The other Gal-CHGC conjugates were prepared analogously using different feed mole ratios. For Gal-CHGC-2, 120 mg CHGC, 60 mg lactobionic acid (0.167 mmol) and 128.7 mg EDC (0.671 mmol) was used, while for GalCHGC-3, 80 mg lactobionic acid (0.223 mmol) and 171.6 mg EDC (0.895 mmol) was employed. 2.4 Characterization of polymeric micelles 1
H-Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DMX500 spectrometer using D2O as the solvent. The degree of substitution (DS), defined as the number of cholesterol groups per 100 sugar residues of glycol chitosan was determined by a colloidal titration method [6, 19], which is based on the reaction between positively charged glycol chitosan and negatively charged potassium poly(vinyl sulfate).
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The hydrodynamic diameters of the micelles were measured by dynamic light scattering (90Plus, Brookhaven Instruments Corp., Holtsville, NY, USA). The micelles were dispersed in deionized water at the concentration of 1 mg/ml. The particle size analysis was carried out at 25 °C. And the morphology was observed through a TEM (JEM-1230, Jeol, Japan) operated at 80 kV. For TEM sample preparation, a drop of sample solution was placed onto a 300-mesh copper grid coated with carbon and the extra solution was blotted with filter paper, followed by air drying. 2.5 Determination of critical aggregation concentration (CAC) The CAC of Gal-CHGC conjugates were determined using pyrene as a fluorescence probe. The Gal-CHGC conjugate suspension was prepared by sonication and adjusted to various conjugate concentrations. A known amount of pyrene in acetone was added into each of 10 ml vials and the final concentration of pyrene was 6.0 9 10-7 M. After the acetone was evaporated, a total of 10 ml of various concentrations of copolymer solution were introduced to each vial and then heated at 50 °C for 10 h to equilibrate the pyrene and the micelles, and left to cool overnight at room temperature. The fluorescence spectra were recorded using a fluorescence spectrophotometer (Hitachi F-4500, Japan) with a slit width of 2.5 nm. The excitation and emission wavelength was set at 339 and 390 nm, respectively. 2.6 Preparation and characterization of DOX-loaded micelles The DOX-loaded micelles were prepared by the o/w method [20, 21]. Firstly, copolymer samples (30 mg) were dissolved in distilled water (60 ml), followed by probe sonication as stated above. Secondly, DOX hydrochloride (15 mg) was dissolved in 10 ml of CHCl3 in the presence of triethylamine (3 times molar quantity of DOX) to obtain a free DOX containing solution. Then 3 ml of DOX organic phase solution were slowly added to the above aqueous phase under vigorously stirring to form the o/w emulsion. CHCl3 in the emulsion was removed by stirring under reduced pressure. The micelle solution was filtered through a 0.8-lm membrane and eluted through a Sephadex G25 fine column to remove non-encapsulated DOX. The resultant DOX-loaded micelles were obtained by freeze drying. For drug loading quantification, freeze-dried micelles were dispersed in aqueous solution and disrupted by the addition of DMSO. This solution was stirred and followed by sonication in an ice bath. The absorbance of the solution
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was measured using UV–vis spectrophotometer at k = 481 nm and calculated using following equations: LC (loading content) =
weight of DOX loaded weight of DOX - loaded micelles 100% ð1Þ
EE (encapsulation efficiency) =
weight of DOX loaded weight of DOX in feed 100% ð2Þ
The hydrodynamic diameters of the micelles were also investigated by dynamic light scattering (90Plus, Brookhaven Instruments Corp., Holtsville, NY, USA). The analysis conditions were the same as described above. The morphological characteristics of DOX-loaded micelles were examined through a TEM (JEM-1230, Jeol, Japan) as mentioned above. 2.7 In vitro DOX release study DOX release behavior was explored by using a dynamic dialysis method. The release experiments were conducted in PBS (pH 7.4) at 37 °C. Typically, 1 ml of DOX-loaded micelles was placed in a dialysis bag (MWCO: 14 kDa) and dialyzed against 20 ml PBS in an air-bath shaker at 100 rpm. At predetermined time points, the entire medium was taken out with replenishing equal volume of fresh medium. The amount of DOX released was determined by fluorescence spectrophotometer (Hitachi F-4500, Japan). Measurements were made at an excitation wavelength of 470 nm and an emission wavelength of 585 nm. The accumulated drug was calculated, and each study was conducted in triplicate. 2.8 Cell culture Human hepatocellular carcinoma cell line (HepG2) was obtained from Shanghai Institutes of Biology Sciences, Chinese Academy of Sciences (Shanghai, China). HepG2 cells were routinely cultured in RPMI-1640 medium containing 10 % FBS and 1 % penicillin–streptomycin at 37 °C and 5 % CO2 humidified atmosphere. 2.9 Cellular uptake study In order to visualize cellular uptake of DOX by HepG2 cells, the cells were examined by confocal laser scanning microscopy (CLSM) (Carl Zeiss LSM-510, Germany). The cells were seeded into 6-well plates (Costar, Corning, NY, USA) at a density of 2 9 105 cells/ml and cultured overnight at 37 °C in 5 % CO2. After removed the cultured
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media, the cells were incubated with free DOX, DOXloaded CHGC (DOX-CHGC), or DOX-Gal-CHGC-1 micelles (equivalent DOX concentration: 10 lg/ml) in RPMI 1640 medium supplemented with 10 % FBS. After 4 or 6 h incubation, the cells were washed three times with PBS (pH 7.4) and fixed in 4 % paraformaldehyde solution at 4 °C for 1 h. The intracellular localization of DOX was observed by confocal microscope at 488 nm for excitation and an LP filter of 560 nm for DOX detection. Flow cytometry was also used to investigate the uptake of DOX into HepG2 cells. In the same way described above, HepG2 cells were incubated with different DOX formulations in 6-well plates. After 4 or 6 h treatment, the cells were harvested with 0.2 % (w/v) trypsin-0.1 % (w/v) EDTA solution. The cells were centrifuged and washed with icecold PBS for 3 times. Then the cells were re-suspended with PBS before the fluorescence-activated cell sorting (FACS) analysis using a flow cytometer (Becton–Dickinson FACSCalibur, USA) equipped with a 488-nm argon ion laser. 2.10 In vitro cytotoxicity study The cytotoxicity of free DOX, DOX-CHGC and DOX-GalCHGC-1 micelles against HepG2 cells was measured by an MTT-based assay. The cells were seeded at a density of 1 9 104 cells/well in 96-well plates (Costar, Corning, NY, USA) and incubated for 24 h at 37 °C and 5 % CO2. After removing the medium, 200 ll of RPMI 1640 medium containing various concentrations of free DOX, DOXCHGC, DOX-Gal-CHGC-1, CHGC or Gal-CHGC-1 micelles were added, and further incubated for 48 h. Then, 20 ll of MTT solution (5 mg/ml in PBS) were added to each well. After an additional 4 h incubation, the culture medium was discarded, and the formazan crystals in cells were solubilized with 200 ll of DMSO for 15 min. The absorbency values were measured by microplate reader (Thermo Scientific Multiskan MK3, USA) at wavelength 570 nm (OD570), and cell viability was calculated by dividing OD570 (sample) with OD570 (blank).
3 Results and discussion
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amino group of glycol chitosan (Scheme 1). As presented in Fig. 1, 1H NMR spectra were used to confirm the CHGC conjugate. Compared with glycol chitosan, the new proton peaks of the methene hydrogen signals of CHS (chemical shift 1.25–1.27 ppm) were observed in spectrum of CHGC [19]. These results demonstrated that the CHS was successfully grafted onto the chains of glycol chitosan. To determine the number of cholesterol groups conjugated on the glycol chitosan backbone, a colloidal titration method was used. The DS was 8.2 cholesterol groups per 100 sugar residues of glycol chitosan. The mean diameter of CHGC determined by dynamic light scattering (DLS) was 264 nm. The principle of the chemical reaction in synthesis of Gal-CHGC is also the formation of amide bonds. The carboxyl groups of lactobionic acid were conjugated with free amino groups of CHGC via the coupling reagent EDC. The structure changes of Gal-CHGCs were confirmed by the 1H NMR technique. The number of hydrogens in GalCHGC at d 3.0–4.2 was higher than that in CHGC, implying that galactose was introduced to CHGC (Fig. 1c– e). And the intensity increased as the feed amount of lactobionic acid in the synthesis increased. All these results demonstrated three Gal-CHGC conjugates with different levels of galactose substitution were successful obtained. The substitution degree of galactose (DSGal) was the number of galactose groups per 100 sugar residues of backbone glycol chitosan in Gal-CHGC, and it was estimated by 1H NMR via the following equation [22]: DSGal ¼
HGalCHGCðd3:04:2Þ HCHGCðd3:04:2Þ 12
ð3Þ
HGal-CHGC (d 3.0–4.2) was the number of hydrogens of Gal-CHGC at d 3.0–4.2. HCHGC (d3.04.2) was the number of hydrogens of CHGC at d 3.0–4.2. The DSGal of GalCHGC-1, Gal-CHGC-2 and Gal-CHGC-3 was 7.9, 11.5 and 18.4, respectively. However, the typical resonance peaks of cholesterol group were hardly observed in GalCHGC conjugates in D2O (Fig. 1), which was due to restricted motions of these protons in the micelle core. This characteristic of the 1H NMR spectra further suggested the micelle formation by Gal-CHGC cojugates in an aqueous solution. Similar results were reported by other researchers [22, 23].
3.1 Synthesis and characterization of Gal-CHGC conjugate 3.2 Physicochemical properties of Gal-CHGC micelles As shown in Scheme 1, the synthesis of Gal-CHGC conjugates involved three steps: (1) carboxylation of cholesterol; (2) synthesis of CHGC; (3) galactosylation of CHGC. Firstly, cholesterol was synthesized with succinic anhydride, and then CHS was produced. In the presence of EDC, CHGC was obtained by the formation of amide groups between carboxyl group of CHS and the primary
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The self-aggregation behavior of Gal-CHGC micelles in aqueous media was investigated by fluorescence spectroscopy in the presence of pyrene [24]. Plots of the intensity ratio I338/I333 from excitation spectra were shown in Fig. 2 as a function of the copolymer concentration. At the low copolymer concentration, the values of I338/I333 ratio were
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Scheme 1 Synthesis of GalCHGC conjugate
low and the plots were almost flat. As the ratio values significantly increased, it implied that pyrene molecule was transferred from a polar microdomain to a more hydrophobic one. The CAC value was determined from the concentration dependence of the I338/I333 ratios. CAC was designated as the intersection of two straight line segments, which was drawn from the points at the lowest polymer concentrations and the ones on the rapidly rising part of the plot. The CAC values of Gal-CHGC-1, Gal-CHGC-2 and Gal-CHGC-3 were respectively 0.0161, 0.0243 and 0.0453 mg/ml, which increased as the degree of galactose substitution increased. These results were attributed to the changes of hydrophilic segment in the Gal-CHGC.
Moreover, the low CAC means that the Gal-CHGC conjugates can form nano-scaled micelles under highly diluted condition. The mean diameters of Gal-CHGC-1, Gal-CHGC-2 and Gal-CHGC-3 determined by DLS were 359, 436 and 471 nm, respectively. The size of Gal-CHGCs micelles was larger than that of blank CHGC micelles described above. It was clearly seen that the lower amount of galactose substitution in the copolymer resulted in the formation of smaller particles. This is because the polymer chains were not likely to form more compact aggregates after hydrophilic galactose conjugation. As shown in Fig. 3a, TEM depicted that the shape of Gal-CHGC-1
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Fig. 1 1H NMR spectra of a glycol chitosan, b CHGC, c Gal-CHGC-1, d Gal-CHGC-2 and e Gal-CHGC-3
self-aggregates was roughly spherical. And the particle size was similar with the average diameter of Gal-CHGC-1 self-aggregates, which was determined by DLS analysis. 3.3 Preparation and characterization of DOX-loaded micelles DOX-loading of polymeric micelles was adopted by an oilin-water emulsion method as previously reported [25]. The similar procedures were used in the present study. DOX hydrochloride was deprotonated by triethylamine in the
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chloroform organic phase. The molar ratio of triethylamine/DOX was set to 3.0. Table 1 demonstrated the physicochemical properties of drug-loaded micelles. The particle size of the polymeric micelles assembled in aqueous solution was determined by DLS. The mean diameters of three kinds of DOX-Gal-CHGC micelles were in the range of 387–497 nm. It was found that the particle size of DOX-CHGC and DOX-Gal-CHGC micelles was larger than that of their blank polymeric micelles. We inferred that DOX molecules were entrapped into the micelles and then the size of these micelles increased.
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3.4 In vitro drug release study To study the release behavior of DOX in vitro, DOXCHGC or DOX-Gal-CHGCs micelles were incubated in a dialysis bag under shaking at 37 °C, and the released DOX was quantified using fluorescence spectrophotometer. As shown in Fig. 4, the total amount of DOX released from DOX-CHGC micelles in 96 h was 49.8 %, and was higher than that of DOX-Gal-CHGCs micelles. This result may be due to DOX-CHGC micelles with the smaller particle size and non-galactose in the outer shell. In addition, DOX-GalCHGC-1 micelles showed faster drug release in 96 h than DOX-Gal-CHGC-2 and DOX-Gal-CHGC-3 micelles. The results revealed that the release rate may be different when the degree of galactose substitution was changed. In addition, DOX appeared to be released from DOX-loaded micelles in a biphasic way. The first phase of release was completed within 6 h, considering as an initial burst. After this initial effect, DOX was released a second phase showing slow sustained release for upto 96 h. As previously described, the release kinetic is associated with both dissolution and diffusion based on the Noyes-Whitney law of dissolution and Fick’s first law of diffusion [26]. For consideration of both the particle size and drug-loading rate, the DOX-Gal-CHGC-1 micelles were selected for further research. 3.5 Cellular uptake of study
Fig. 2 Polts of the intensity ratio I338/I333 vs. log C for Gal-CHGC conjugates with various compositions: a Gal-CHGC-1, b Gal-CHGC2 and c Gal-CHGC-3
Moreover, as the degree of galactose substitution in the Gal-CHGC conjugates increased, the reduction of drug loading content and encapsulation efficiency in DOX-GalCHGC micelles were observed. This was mainly due to the fact that Gal-CHGC micelles containing the more hydrophilic groups displayed poorer capacity to load drug. As shown in Fig. 3b, TEM observation demonstrated that the shape of DOX-Gal-CHGC-1 self-aggregates was nearly spherical. The average diameter of DOX-Gal-CHGC-1 micelles was similar with the size determined by DLS.
CLSM was used to directly monitor the internalization of DOX-loaded micelles and free DOX into HepG2 cells (Fig. 5). When HepG2 cells were incubated with free DOX for 4 h, only weak red fluorescence were observed in the nucleus. For DOX-CHGC and DOX-Gal-CHGC-1 aggregate systems, however, the DOX fluorescence is observed in both cytoplasm and nucleus. After 6 h of cell incubation with the free DOX, stronger fluorescence was observed in cell nuclei in addition to the weak fluorescence in cytoplasm. And the cells incubated with the DOX-CHGC and DOX-Gal-CHGC-1 micelles also emitted significantly increased fluorescence. As comparatively estimated by CLSM, cellular uptake extent of DOX-Gal-CHGC-1 micelles was higher than that of DOX-CHGC micelles without galactosylation and free DOX after exposure of 4 and 6 h under the same condition. Moreover, HepG2 cells incubated with free DOX showed lower red fluorescence than the cells treated with DOX-CHGC micelles. It implied that the internalization mechanism of the DOX-loaded micelles would be different from that of free DOX. The free DOX molecules were transported into the cytoplasm of the cells and reached into the nucleus in a passive diffusion manner. The micellar systems were internalized through an endocytosis pathway, and then be localized in
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Fig. 3 TEM images of a GalCHGC-1 and b DOX-GalCHGC-1 micelles
Table 1 Physicochemical properties of drug-loaded copolymer micelles Sample
DOX/carriera
Size (nm)b
PIc
LC (%)d
EE (%)e
DOX-CHGC
1.5/10
298 ± 17.1
0.246 ± 0.038
10.7 ± 0.51
80.0 ± 4.25
DOX-Gal-CHGC-1
1.5/10
387 ± 29.1
0.252 ± 0.031
9.66 ± 0.43
71.6 ± 3.56
DOX-Gal-CHGC-2
1.5/10
451 ± 25.7
0.223 ± 0.063
8.79 ± 0.24
64.5 ± 1.89
DOX-Gal-CHGC-3
1.5/10
497 ± 24.6
0.242 ± 0.072
8.09 ± 0.31
58.9 ± 2.44
The results represent the mean ± SD (n = 3) a
The ratio of DOX to carrier, based on feed amount (mg/mg)
b
Measured by dynamic light scattering
c
Polydispersity index
d
Loading content
e
Encapsulation efficient
Fig. 4 Release profiles of DOX from DOX-CHGC, DOX-GalCHGC-1, DOX-Gal-CHGC-2 and DOX-Gal-CHGC-3 micelles at 37 °C in PBS at pH 7.4
acidic endocytic compartments [27]. As mentioned above, DOX-loaded micelles displayed sustained release behavior in vitro, whereas free DOX could freely diffuse in the
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media. Additionally, it is well-known that the human hepatocellular carcinoma cells (HepG2) express the ASGP receptors on their surfaces. The improved cellular uptake of DOX-Gal-CHGC-1 micelles might be attributed to the effective targeting of HepG2 cells by the galactoseexpressing nanoparticles. Therefore, it could be concluded that Gal-CHGC-1 micelles could enhance cellular uptake of DOX in HepG2 cells. To further analyze the cellular uptake of free DOX, DOX-CHGC and DOX-Gal-CHGC-1 micelles by HepG2 cells, flow cytometry was performed. As seen in Fig. 6a, the cells incubated with DOX-Gal-CHGC-1 micelles for 4 h showed higher fluorescent intensity than the cells treated with free DOX and DOX-CHGC micelles. As the incubation time increased to 6 h, the amounts of DOX fluorescence emitted from these DOX formulations simultaneously increased (Fig. 6b). It was clearly seen that the orders of fluorescent intensity emitted from DOX formulations were DOX-Gal-CHGC-1 [ DOX-CHGC [ free DOX in 4 and 6 h. This result was consistent with confocal microscopic observation. We inferred that DOX-loaded micelles could improve the cytotoxicity of DOX.
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Fig. 5 CLSM images of HepG2 cells after incubation with free DOX, DOX-CHGC and DOX-Gal-CHGC-1 micelles for 4 or 6 h
Fig. 6 Flow cytometric analysis of HepG2 cells incubated with free DOX, DOX-CHGC and DOX-GalCHGC-1 micelles for a 4 h or b6h
3.6 In vitro cytotoxicity study The cytotoxicities of free DOX, DOX-CHGC and DOXGal-CHGC-1 micelles were investigated in HepG2 cell after 48 h incubation. As shown in Fig. 7a, DOX-GalCHGC-1 and DOX-CHGC micelles showed higher cytotoxicity in contrast with free DOX. The half inhibitory concentration (IC50) of free DOX was 0.985 lg/ml, while that of DOX-Gal-CHGC-1 and DOX-CHGC micelles was 0.236 and 0.586 lg/ml, respectively. The IC50 value of DOX-Gal-CHGC-1 micelles against HepG2 cells was nearly 4.17 times lower than that of free DOX, indicating that Gal-CHGC-1 micelles could be efficient targeting delivery of DOX in hepatocellular carcinoma cells. As mentioned above, DOX released from DOX-Gal-CHGC-1
and DOX-CHGC micelles in 48 h were 44.3 and 48.2 %, respectively. The cytotoxicity improved is mainly originated from the enhanced cellular uptake of DOX-loaded micelles. These results agreed with flow cytometry and confocal microscopic analysis. Therefore, we confirmed that Gal-CHGC polymeric micelles could improved the cytotoxicity of DOX in ASGPR-overexpressed HepG2 cells. As shown in Fig. 7b, blank CHGC and Gal-CHGC1 micelles did not exhibit notable cytotoxicity against HepG2 cells. IC50 of CHGC and Gal-CHGC-1 micelles was 451.8 and 407.7 lg/ml, respectively. These results demonstrated that blank CHGC and Gal-CHGC-1 micelles showed low cytotoxicity. Taken together, the Gal-CHGC micelles have great potential in liver tumortargeting chemotherapy.
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References
Fig. 7 In vitro cytotoxicity of a free DOX, DOX-CHGC, DOX-GalCHGC-1 micelles, and b CHGC, Gal-CHGC-1 micelles against HepG2 cells after 48 h incubation
4 Conclusion In summary, three kinds of Gal-CHGC copolymers with different levels of galactose substitution were successfully synthesized and characterized. DOX was physically loaded into the Gal-CHGC micelles. DOX release from DOX-Gal-CHGC micelles in vitro showed an initial burst effect and followed by a slow drug release. In cellular uptake study, DOX-Gal-CHGC micelles showed galactose-mediated cancer cell targeting and enhanced cellular uptake in HepG2 cells. Moreover, the antitumor efficiency of DOX-Gal-CHGC micelles was higher than that of DOX-CHGC micelles and free DOX in HepG2 cells. Therefore, these results implied that Gal-CHGC micelles could be promising drug carriers for specific delivery to liver cancer cells. Acknowledgments We are grateful for partially financial support of National Nature Science Foundation of China (No. 81001418), Natural Science Foundation of Jiangxi Province (No. 20114BAB215017), and Scientific Research Fund of Jiangxi Education Department (No. GJJ11241).
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