In Vivo Cancer Targeting of Water-Soluble Taxol by Folic Acid Immobilization
Junichi Nakamura, Naoki Nakajima, Kazuaki Matsumura and Suong-Hyu Hyon*
Department of Medical Simulation Engineering, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
- *Corresponding Author:
- Suong-Hyu Hyon, Ph.D
Institute for Frontier Medical Sciences
53 Kawahara-cho, Shogoin
Sakyo-ku, Kyoto 606- 8507, Japan
Tel: 81 757514125
Fax: 81 757514141
E-mail: [email protected] kyoto-u.ac.jp
Received Date: December 05, 2010; Accepted Date: January 24, 2011; Published Date: January 24, 2011
Citation: Nakamura J, Nakajima N, Matsumura K, Hyon SH (2011) In Vivo Cancer Targeting of Water-Soluble Taxol by Folic Acid Immobilization. J Nanomedic Nanotechnol 2:106. doi:10.4172/2157-7439.1000106
Copyright: © 2011 Nakamura J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Paclitaxel is an anticancer drug used for lung, breast and ovarian tumors . Due to its poor water solubility, paclitaxel is generally administered as a mixture with poly (oxyethylene) castor oil (Cremophor EL) and dehydrated ethanol . Because Cremophor EL causes serious side-effects, such as irritation, in approximately 30% of patients , a steroid drug is required prior to its use to suppress these side effects . To exclude the need for Cremophor EL, paclitaxel conjugation with poly (L-glutamic acid)  and albumin , and drug delivery systems (DDS) such as liposomes, polymer micelles, and nanoparticles have been studied to enhance its water solubility and anticancer efficacy by systematic delivery [7, 8]. Folic acid (FA), a watersoluble vitamin that plays an important role in cell proliferation, has also been used as targeting molecule in micelle and liposome systems [9, 10]. Overexpression of the FA receptor in some cancer cells such as ovarian and brain carcinomas , and a human oral cancer cell line (KB) has been reported .
In our previous study, the water solubility of paclitaxel and the in vitro targeting activity were remarkably enhanced by the conjugation with dextran and FA, respectively . The anticancer effect was examined in vitro by using KB cells which overexpress FA receptors. FA was immobilized with dextran-conjugated paclitaxel (Dex-PXL) to provide cancer targeting to the FA receptor overexpressed on cancer cells. It was found that the water solubility of paclitaxel could be improved by dextran conjugation by as much as 2700 times, and that the cytotoxicity against KB cells could be enhanced 2-3 times by FA conjugation (Dex-PXL-FA) than that observed against a cell line without FA receptor over expression .
In vivo studies of DDS drugs such as micelles , liposomes , polymers , and nanoparticles  have already been reported. Recently, many studies have used antibodies  and peptides  as a ligand to target tumors. However, in spite of the specific antigen and antibody reaction for targeting, the presence of similar antigens in vivo would suppress the targeting efficacy of the drug . Maeda et al. compared the permeability of immature blood vessels of cancer tissue with that of normal tissue in 1986, and they found that compounds larger than about 100 nm in diameter accumulated to a higher extent in cancer tissues . Therefore, the drugs modified polymers to have a larger size show higher efficacy than those without the modification due to the accumulation of the larger drugs. Furthermore, these studies suggest that research should be focused on designing a drug with an enhanced permeability and retention (EPR) effect. Dextran was reported to have an EPR effect in DDS and to make a contribution to cancer-specific targeting . In particular, a molecular size of 50- 200nm in diameter was found to be crucially important for cancer tissue targeting. Even a modified agent is immediately excreted from the kidney when the size is smaller than 50nm . In contrast, if the size of the conjugated drug is larger than 200 nm, it becomes trapped in the liver and is degraded .
The present study further examined the in vivo antitumor efficacy of Dex-PXL-FA using a murine tumor xenograft model. In addition, the active (FA-targeting) and passive (EPR) effects of Dex-PXL-FA are also discussed in detail with regard to their molecular size evaluation.
Materials and Methods
The paclitaxel used for injection and for chemical modification were kindly donated by NIPPON KAYAKU Co., Ltd (Tokyo, Japan) and by Samyang Genex Corporation (Korea), respectively. Dextran (MW 70,000) was purchased from Meito Sangyo Co., Ltd. (Nagoya, Japan). Folic acid (FA), diethyl sulfoxide (DMSO), 1, 1 -carbonyldiimidazole (CDI), 1-ethyl-3 -(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and ethylenediamine were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and used without further purification.
KB cells (DS Pharma Biomedical Co., Ltd., Osaka, Japan) were cultured in RPMI1640 (folate-free, Invitrogen Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum and 100 g/ml of penicillinstreptomycin at 37°C under 5% CO2 in a humidified atmosphere. When the cells reached 80% confluence, they were detached by 0.25% (w/v) of trypsin containing 0.02% (w/v) of ethylenediamine tetracetic acid (EDTA) in phosphate buffered saline without calcium and magnesium (PBS(-)) and seeded on a new tissue cuture plate for subculture. The KB cells were used for each experiment within five passages.
Synthesis of amino-Dex, Dex-PXL, FA-adsorbed Dex-PXL and Dex-PXL-FA (covalent)
Dex-PXL, FA-adsorbed Dex-PXL, and Dex-PXL-FA (covalent) were synthesized according to the previous report . Briefly, synthesis method of their complex was shown as follows. Dextran (10 g) was dissolved in 70 ml DMSO and mixed with a solution of 2 g CDI in 5 ml DMSO, and the activation reaction was preceded at 50°C for 15 min. Subsequently, 5 ml of ethylenediamine was added and the mixture was stirred at 50°C for 18 h. After dialysis against running water for 24 h and distilled water (3 L, 1.5 h × 2) with a dialysis membrane (cut-off molecular weight of 14,000 Da), amino-Dex was recovered by air and vacuum drying. To activate the paclitaxel OH group, a solution of 0.7 g paclitaxel in 40 ml DMSO was added to a solution of 0.6 g CDI dissolved in 10 ml DMSO at 50°C for 15 min. Five grams of amino-Dex in 150 ml of DMSO was added to the solution, and the reaction proceeded at 50°C for 18 h. Dex-PXL was recovered by the same purification manner as described above. The yield of Dex-PXL was about 70. Recovered Dex- PXL and FA were dissolved in PBS at a concentration of 800 and 240 g/ ml, respectively. Here, a solution of 0.5 ml Dex-PXL was simply mixed with 0.5 ml of FA and stirred at 25°C for 18 h to prepare FA-adsorbed Dex-TXL. A solution of 0.1 g FA and 0.15 g NHS in 20 ml DMSO was mixed with 0.07 g of EDC in 10 ml DMSO, and the activation reaction of the FA COOH groups proceeded at 50°C for 5 min. Then, 0.5 g of Dex-PXL in 10 ml DMSO was added to the mixture and reacted at 50°C for 3 h. Dex-PXL-FA (covalent) was recovered after purification by dialysis against water and drying.
Molecular size evaluation by dynamic light scattering (DLS)
The hydrodynamic molecular size of the conjugates was measured at 25°C by DLS using a DLS-7000 instrument (Otsuka Electronics, Osaka, Japan) equipped with a He–Ne laser. The samples were dissolved in PBS and DMSO (for paclitaxel) at a concentration of 5000ppm, and 5ml of the solution was used for the evaluation.
Previously, folic acid receptor-targeted Dextran-Taxol-Folic acid (Dex-TXL-FA) has shown the in vitro superior and selective antitumor activity against human oral cancer cell line (KB) compared with the absent of folic acid, Dextran- Taxol. Present study is given for further investigation of in vivo antitumor efficacy of Dex-TXL-FA in the murine tumor xenograft model. To evaluate the antitumor effect of taxol, tumor bearing mice were prepared by s.c. inoculation of 1.0 × 10 6 KB cells in the back of nude mice. Seven days after inoculation, the administration of saline, paclitaxel for injection (PTX), Dex-TXL, FA-adsorbed Dex-TXL and Dex-TXL-FA (covalent) was started at a dose of 10mg/kg, by i.v. injection via the lateral tail vein three times ( on day 7, 9, and 11) and animal survival rate and tumor sizes were monitored.FA- adsorbed Dex-TXL and Dex-TXL-FA (covalent) showed approximately 3 times greater anticancer effect than that of taxol at the 30th day after tumor implantation. Furthermore, these FA immobilized TXL showed 2-3 month longer animal survival than that of taxol. These results suggest the conjugation with Dex and FA could provide an improvement in the anticancer therapy of taxol.