Antitumor activity of sorafenib-incorporated nanoparticles of dextran/poly(dl-lactide-co-glycolide) block copolymer
- Do Hyung Kim†1, 2,
- Min-Dae Kim†3,
- Cheol-Woong Choi3,
- Chung-Wook Chung1,
- Seung Hee Ha1,
- Cy Hyun Kim1, 2,
- Yong-Ho Shim1,
- Young-Il Jeong1Email author and
- Dae Hwan Kang1, 2Email author
© Kim et al; licensee Springer. 2012
Received: 15 September 2011
Accepted: 27 January 2012
Published: 27 January 2012
Sorafenib-incoporated nanoparticles were prepared using a block copolymer that is composed of dextran and poly(DL-lactide-co-glycolide) [Dexb LG] for antitumor drug delivery. Sorafenib-incorporated nanoparticles were prepared by a nanoprecipitation-dialysis method. Sorafenib-incorporated Dexb LG nanoparticles were uniformly distributed in an aqueous solution regardless of the content of sorafenib. Transmission electron microscopy of the sorafenib-incorporated Dexb LG nanoparticles revealed a spherical shape with a diameter < 300 nm. Sorafenib-incorporated Dexb LG nanoparticles at a polymer/drug weight ratio of 40:5 showed a relatively uniform size and morphology. Higher initial drug feeding was associated with increased drug content in nanoparticles and in nanoparticle size. A drug release study revealed a decreased drug release rate with increasing drug content. In an in vitro anti-proliferation assay using human cholangiocarcinoma cells, sorafenib-incorporated Dexb LG nanoparticles showed a similar antitumor activity as sorafenib. Sorafenib-incorporated Dexb LG nanoparticles are promising candidates as vehicles for antitumor drug targeting.
Keywordssorafenib polymeric micelle dextran poly(DL-lactide-co-glycolide)
Nanoparticles have been extensively investigated as a means of specifically targeting drugs to a desirable site of action . Notably, nanoparticles having a hydrophobic inner core and hydrophilic outer shell have received great attention due to their superior properties in drug delivery [2–6]. They are regarded to be ideal vehicles for antitumor drug delivery because their hydrophobic inner core is an appropriate reservoir for hydrophobic anticancer drugs and because their hydrophilic outer shell facilitates avoidance of the reticuloendothelial system, long blood circulation, and the improvement of enhanced permeation and retention [EPR] effect in tumor tissue .
Cholangiocarcinoma [CC], a malignant tumor arising from the biliary tract, has a high mortality rate. Even though surgical resection is regarded as a curative method, most of patients diagnosed with a latent CC state are not considered for surgical resection . Furthermore, conventional radiation or chemotherapeutic treatment is known to have limited advantages . Therefore, novel treatment option is required to enhance therapeutic efficacy of CC.
Sorafenib inhibits tumor cell proliferation and vascularization by the activation of the receptor for tyrosine kinase signaling in the Ras/Raf/Mek/Erk cascade pathway . Sorafenib is an effective chemotherapeutic agent against various tumor types including CC  and inhibits proliferation, angiogenesis, and invasion of tumor cells [9, 10]. However, poor aqueous solubility and undesirable side effects limit the clinical application and local treatment of sorafenib. These side effects might be overcome by use of nanoparticles for tumor delivery and controlled release of sorafenib [11, 12].
In this study, we prepared sorafenib-incorporated Dexb LG nanoparticles as an antitumor drug delivery system. The properties of sorafenib-incorporated Dexb LG nanoparticles were studied in terms of core-shell structure, particle size, morphology, and drug release rate. Antitumor activity of sorafenib-incorporated Dexb LG nanoparticles was tested using human cholangiocarcinoma [HuCC-T1] cells.
Dextran from Leuconostoc spp. (average molecular weight [MW] approximately 6,000), hexamethylene diamine [HMDA], N,N-dicylohexylcarbodiimide [DCC], and N-hydroxysuccimide [NHS] were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib was purchased from LC Laboratories (Woburn, MA, USA). Spectra/Por™ dialysis membranes (MW cutoff [MWCO] = 2,000 g/mol and 8,000 g/mol) were purchased from Spectrum Labs (Rancho Dominguez, CA, USA). Poly(DL-lactic acid-co-glycolic acid) (PLGA-5005, MW = 5,000 g/mol) were purchased from Wako Pure Chemicals (Osaka, Japan).
Synthesis of Dexb LG copolymer
Dexb LG copolymer was synthesized as reported previously . Aminated dextran was prepared as follows. Dextran (180 mg) dissolved in dimethylsulfoxide [DMSO] was mixed with sodium cyanoborohydride and stirred for 24 h. After that, 10 equivalents of HMDA were added and stirred for 24 h at room temperature. The resulting aminated dextran was obtained by dialysis against deionized water and was lyophilized. N-hydroxysuccimide PLGA [PLGA-NHS] was prepared by reaction with DCC and NHS. Dexb LG copolymer was prepared by dissolving 120 mg of aminated dextran and 100 mg of PLGA-NHS in DMSO and undergoing reaction for 2 days. Reactants were dialyzed to remove unreacted dextran (MWCO of dialysis membrane = 8,000 g/mol), and the product was lyophilized. The resulting white powder was dissolved in chloroform to remove unreacted PLGA. Yield of the final product was about 89% (w/w).
Preparation of sorafenib-incorporated Dexb LG nanoparticles
Analysis of nanoparticles
The characterization of nanoparticles was performed in DMSO-d6 or D2O using 500 MHz1H nuclear magnetic resonance [NMR] spectroscopy (500 MHz superconducting FT-NMR spectrometer; Varian Unity-Inova 500; Agilent Technologies, Foster City, CA, USA). The morphology of nanoparticles was observed by transmission electron microscopy [TEM] using a JEM-2000 FX II microscope (JEOL, Tokyo, Japan). One drop of nanoparticle solution containing phosphotungstic acid (0.05% w/w) was placed onto a carbon film coated on a copper grid for TEM. Observation was done at an accelerating voltage of 80 kV. The particle size and zeta potential were measured by the Nano-ZS apparatus (Malvern Instruments, Malvern, UK). A sample solution prepared by dialysis was used to determine the particle size.
Drug release study in vitro
The release experiment was carried out in vitro. A sample solution prepared by dialysis was used directly. This solution was introduced into the dialysis membrane. Next, the dialysis membrane was placed in a 200-ml bottle with 100 ml of phosphate buffered saline [PBS] containing 1% (v/v) Tween 80 [PBST]. This bottle was placed in a shaking incubator with a stirring speed of 100 rpm at a temperature of 37°C. At specific times, the PBST was sampled for analysis of drug concentration. After each sampling, the entire volume of PBST was replaced with fresh PBST to prevent drug saturation. The concentration of the released sorafenib was determined by HPLC.
The Flexar HPLC system (PerkinElmer, Waltham, MA, USA) was equipped with a Solvent Manager 5-CH degasser, an autosampler, a quaternary LC pump, a column oven, and a UV-visible detector. Chromatography was performed on a guard column (SecurityGuard® Guard Cartridge Kit; Phenomenex, Torrance, CA, USA) and on a C18 column (Brownlee C18®, 5 μm, 150 × 4.6; PerkinElmer) at 37°C. Sorafenib was eluted isocratically with mobile phase (acetonitrile/methanol/1% acetic acid at a ratio of 35:38:27) at a flow rate of 1 ml/min and monitored at 254 nm. The chromatograms were recorded and integrated with the Chromera 2.1 system software (PerkinElmer).
Cell cytotoxicity test in vitro
HuCC-T1 cells maintained in RPMI 1640 (10% fetal bovine serum, 5% CO2 at 37°C) were used to evaluate the antitumor activity of sorafenib-incorporated nanoparticles. Viability of tumor cells was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT]-based cell proliferation assay. HuCC-T1 cells were seeded at a density of 2 × 103 cells/well in 96-well plates with 100 μl of medium before addition of polymeric micelles. Next, free sorafenib, sorafenib-incorporated polymeric micelles, or empty polymeric micelles were added to 96-well plates at 100 μl. Controls were treated with 0.1% (v/v) of DMSO. Cells were incubated for 48 h, and cell viability was then measured in triplicate using an established MTT assay protocol.
Results and discussion
Characterization of sorafenib-incorporated Dexb LG nanoparticles
Sorafenib (Figure 1a) is a relatively novel class of angiogenesis inhibitor, which was selected as an anticancer drug due to its poor aqueous solubility. Nanoparticles are regarded as an ideal candidate for these kinds of drugs because the abundant microvascular structure of the tumor tissue is a useful target for nanoparticulate drug delivery system via the EPR effect.
Characterization of sorafenib-incorporated nanoparticles
Drug contents (%, w/w)
46 ± 1.12
-36.4 ± 3.1
63 ± 0.58
-35.5 ± 2.2
133 ± 0.58
-36.0 ± 1.1
181 ± 1.15
-35.8 ± 0.9
In vitro cell cytotoxicity
In this study, we prepared sorafenib-incorporated nanoparticles by nanoprecipitation-dialysis method. Sorafenib-incorporated Dexb LG nanoparticles adopt a spherical shape with a size < 300 nm. The higher the initial drug feeding, the higher is the quality of incorporated sorafenib. The size of the nanoparticles was increased according to the amount of sorafenib. Increasing quantity of incorporated sorafenib decreases the release rate of the drug. Sorafenib-incorporated Dexb LG nanoparticles have a similar antitumor activity against tumor cells in vitro compared to sorafenib itself. The collective results indicate the promise of sorafenib-incorporated Dexb LG nanoparticles as vehicles for antitumor drug targeting.
This study was supported by a grant of the Korean Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (project number A091047).
- Allemann E, Gurny R, Doelker E: Drug loaded poly(lactic acid) nanoparticles produced by a reversible salting-out process: purification of an injected dosage form. Eur J Pharm Biopharm 1993, 39: 173–191.Google Scholar
- Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R: Biodegradable long-circulating polymeric nanospheres. Science 1994, 263: 1600–1603. 10.1126/science.8128245View ArticleGoogle Scholar
- La SB, Okano T, Kataoka K: Preparation and characterization of the micelle-forming polymeric drug indomethacin-incorporated poly(ethylene oxide)-poly(beta-benzyl L-aspartate) block copolymer micelles. J Pharm Sci 1996, 85: 85–90. 10.1021/js950204rView ArticleGoogle Scholar
- Kwon GS, Naito M, Yokoyama M, Okano T, Sakurai Y, Kataoka K: Physical entrapment of adriamycin in AB block copolymer micelles. Pharm Res 1995, 12: 192–195. 10.1023/A:1016266523505View ArticleGoogle Scholar
- Kwon GS, Naito M, Yokoyama M: Micelles based on AB block copolymers of poly(ethylene oxide) and poly(β-benzyl L-aspartate). Langmuir 1993, 9: 945–949. 10.1021/la00028a012View ArticleGoogle Scholar
- Kwon GS, Yokoyama M, Okano T, Sakurai Y, Kataoka K: Biodistribution of micelle-forming polymer-drug conjugates. Pharm Res 1993, 10: 970–974. 10.1023/A:1018998203127View ArticleGoogle Scholar
- Seehofer D, Kamphues C, Neuhaus P: Management of bile duct tumors. Expert OpinPharmacother 2008, 9: 2843–2856. 10.1517/146565188.8.131.5243View ArticleGoogle Scholar
- Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M: Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther 2008, 7: 3129–3140. 10.1158/1535-7163.MCT-08-0013View ArticleGoogle Scholar
- Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, Wilhelm S, Lynch M, Carter C: Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006, 66: 11851–11858. 10.1158/0008-5472.CAN-06-1377View ArticleGoogle Scholar
- Qun W, Tao Y: Effective treatment of advanced cholangiocarcinoma by hepatic arterial infusion chemotherapy combination with sorafenib: one case report from China. Hepatogastroenterology 2010, 57: 426–429.Google Scholar
- Wang XQ, Fan JM, Liu YO, Zhao B, Jia ZR, Zhang Q: Bioavailability and pharmacokinetics of sorafenib suspension, nanoparticles and nanomatrix for oral administration to rat. Int J Pharm 2011, 419: 339–346. 10.1016/j.ijpharm.2011.08.003View ArticleGoogle Scholar
- Zhang JY, He B, Qu W, Cui Z, Wang YB, Zhang H, Wang JC, Zhang Q: Preparation of the albumin nanoparticle system loaded with both paclitaxel and sorafenib and its evaluation in vitro and in vivo. J Microencapsul 2011, 28: 528–536. 10.3109/02652048.2011.590614View ArticleGoogle Scholar
- Jeong YI, Kim DH, Chung CW, Yoo JJ, Choi KH, Kim CH, Ha SH, Kang DH: Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer. Int J Nanomedicine 2011, 6: 1415–1427.View ArticleGoogle Scholar
- Jeong YI, Kim DG, Jang MK, Nah JW, Kim YB: All-trans retinoic acid release from surfactant-free nanoparticles of poly(DL-lactide-co-glycolide). Macromol Res 2008, 16: 717–724. 10.1007/BF03218586View ArticleGoogle Scholar
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