efficacy of methotrexate-impregnated hydroxyapatite composites on human mammary carcinoma cells, The

Journal of Orthopaedic Surgery, Apr 2007 by Vechasilp, J, Tangtrakulwanich, B, Oungbho, K, Yuenyongsawad, S

ABSTRACT

Purpose. To investigate the efficacy of local biodegradable composites of hydroxyapatite, plaster of Paris, and a binder of either alginate or chitosan impregnated with methotrexate on human mammary carcinoma cells.

Methods. An in vitro analysis of drug dissolution and a cytotoxicity test on human mammary carcinoma cells were performed over one month. Physicochemical properties of each composite were investigated using scanning electron microscopy, X-ray diffractometry, and Fourier transform infrared spectroscopy.

Results. Both composites with a binder of either alginate or chitosan could release methotrexate for over one month. The amount of methotrexate released depended on the amount of methotrexate loaded. The composite using alginate as a binder released a significantly greater amount of methotrexate than that using chitosan as a binder (p

Conclusion. Methotrexate-impregnated hydroxyapatite composites appear to be effective local skeletal methotrexate delivery systems against human mammary carcinoma cells in an in vitro model.

Key words: breast neoplasms; hydroxyapatite cement; methotrexate

INTRODUCTION

Chemotherapy is a standard treatment for most malignant bone tumours. To achieve optimal effect, a high systemic concentration of drug for a sufficiently long period is required to achieve both local and systemic control. However, parenteral administration of chemotherapy drugs in high dose is associated with adverse effects such as immune suppression, myelosuppression, hepatotoxicity, and cardiotoxicity.1,2 Methods have been proposed to reduce such effects associated with systemic delivery of anticancer agents. Development of local drug delivery systems has been one approach to reduce the risk of systemic complications.3 Various drug carriers such as polymers, hydroxyapatite, hydrogel, and polymethylmethacrylate have been studied with conflicting results.4-10 We have reported on the efficacy of a new composite of hydroxyapatite, plaster of Paris, and a binder of either chitosan or alginate for antibiotic and antifungal drugs.11-13 In the present study, we aimed to test the efficacy of these hydroxyapatite composites impregnated with methotrexate on human mammary carcinoma cells in an in vitro model.

MATERIALS AND METHODS

Hydroxyapatite (CA^sub 10^(PO^sub 4^)^sub 6^(OH)^sub 2^) from MTEC, Bangkok, Thailand; plaster of Paris from Merck, Darmstadt, Germany; and a binder of either alginate or chitosan (medium viscosity, 80% acetylation) from Fluka, Buchs, Switzerland were used to make the hydroxyapatite composite, which was impregnated with methotrexate from Zetate 50, Dabur, India. A geometric dilution technique was used: 4.25 g of hydroxyapatite was well mixed with plaster of Paris at a ratio of 85:15. Chitosan and alginate gel was prepared by dispersing in distilled water at 1% concentration. The plaster of Paris and hydroxyapatite were thoroughly mixed before the methotrexate solution was added and the mixture stirred until it became a homogeneous and smooth paste. It was then poured into a silastic mould (6.5 mm in diameter and 5.2 mm in thickness). Three different concentrations of methotrexate-0.5%, 1%, and 2% were impregnated in each of the 2 composites. One composite without methotrexate served as a control. After the cement tablets hardened, they were dried at 60°C for 2 hours and sterilised with ethylene oxide. Standard safety guidelines for using anticancer drugs were followed.

Five tablets of each preparation were tested for drug dissolution using a dissolution apparatus at 37°C with a stirring speed of 50 revolutions per minute. Each reactor contained an implant and 100 ml of phosphate buffer at pH7.4. Three reactors were used for each preparation. Two samples were taken at different time points and filtered with a millipore filter and then kept at -25°C. Dissolution was tested over one month. The amount of methotrexate in the sample was determined by ultraviolet absorbance at 304 nm with a spectrophotometer.

Human mammary carcinoma cells were grown as a monolayer in EMEM (Eagle Minimum Essential Medium, Biochrom KG Seromed, Berlin, Germany) with 10% newborn calf serum (Biowhittaker, Walkersville [MD], US) and penicillin/streptomycin/amphotericin B (100 µg/ml). The cells were typically grown to 70 to 80% confluence under 5% CO2 at 37°C in a CO2 incubator. Then the medium was changed and the cells used for the test procedure. The cells were removed from the culture flasks by washing with phosphate-buffered saline (Biochrom KG, Berlin, Germany) and trypsinised, and then incubated for 3 minutes at 37°C in a CO2 incubator and diluted with fresh medium. A total of 100 µl containing 3x104 cells was then added to a 96-well plate, which was then incubated for 24 hours in a CO2 incubator. Test samples were initially dissolved in dimethyl sulphoxide, then diluted into 10-fold serial dilutions with medium. Serial dilutions were performed and 100 µl was added to each well. After 72 hours of incubation, the cells were washed with fresh medium, cultured for an additional 72 hours, and then fixed with 100 µl of cold 40% aqueous trichloroacetic acid. The plates were kept at 4°C for one hour, washed with tap water 5 times and air-dried. The cells were stained using 0.4% sulphorhodamine B in 1% acetic acid (50 µl) for 30 minutes. Excess sulphorhodamine B solution was removed by washing 5 times with 1% aqueous acetic acid. Once dried, the bound dye was dissolved in 100 µl 10 mM Tris base pH10. The plate was shaken for 5 minutes, and the absorbance was measured at 492 nm using a microplate reader (Power Wave X, Bio-Tex Instruments, Winooski [VT], US). Finally, the results were reported in terms of median effective dose, calculated using toxicity percentage versus concentration. An extract was considered active when the median effective dose was