Research On The Methotrexate-biotinylated Chitosan Nanoparticles

Cancer still remains the first killer in human health, the treatment of which is still mainly relied on chemotherapy. However, in killing cancer cells, chemotherapeutic drugs are also of great killing effects to the peripheral normal cells. This causes agony to the cancer patients. In recent years, through the efforts of professionals, targeted preparations developed quickly. However, most of the present researches are limited to targeting cancerous tissues, unable to distinguish cancerous cells and normal cells. This limits the reduction of side effects of chemotherapy.Budavari discovered that the content of Vitamin H （Biotin） was noticeably higher in tumor cells than in normal tissues and the rapid proliferation of tumor cells needed more biotin. The present paper connected biotin and chitosan through the formation of amido bond. Nanoparticles were prepared using MTX as the model drug to achieve tumor cell targeting.Biotinylated chitosan （B-CS） was prepared in DMSO using chitosan and biotin as raw materials, under the catalysis of 4-Dimethylaminopyridine （DMAP） and Dicyclohexylcarbodimide （DCC）. Purification was made by dialysis. Influencing factors in the synthesis were studied and the product was characterized by UV、FT-IR、¹H-NMR and DSC. Results indicated that two materials were successfully linked together through the formation of amido bond.Methotrexate-biotinylated chitosan nanoparticles （MTX-B-CS-NP） were prepared by dialysis method. The technique was studied by single factor method and the optimum process was established. Nanoparticles was prepared using the optimum process. Particle size and zeta potential were measured using a particle size analyzer. An HPLC method was established to analyze the content of MTX and determine drug loading. The formulation stability at 40℃and the in-vitro release were studied. The particle size was 167.4±41.34 nm and the zeta potential was +29.3mV. Results of the stability tests indicated that the product was of good stability at 40℃for 6 months. Results of the in-vitro release tests showed that the carrier was able to sustain the release of MTX.The safety tests on MTX-B-CS-NP proved that the nanoparticle formulation had no hemolytic effect and it could be used in intraveneous injection. It had no irritability on administration site and blood vessel, no anaphylactic effect as caused by injection. It had varying degrees of proliferation inhibiting effects on human solid tumor cells, i.e. H₂₂、HeLa、MCF-7、A375-S2 and SGC-7901. When the sample concentration was less than 50μg/ml, tested sample and the injection used as a control had similar cell proliferation inhibiting effects. When the sample concentration was higher than 50μg/ml, the inhibition rate of sample was obvious. When the sample concentration was 100μg/ml, there was noticeable difference between sample and the injection used as a control, which was 50.44%(H₂₂)、49.45%（SGC-7901）、11.27%（A375-S2）、46.48% （MCF-7）�'�31.85% （HeLa） respectively. The sample showed certain dependence on dosage and the control injection had no great difference in inhibition rate among varying concentrations. Human solid tumor cell H22, which was of obvious difference in tumor inhibiting rate, was chosen for the in-vivo tumor inhibition. Tests indicated that the inhibition rate of nanoparticle formulation at a dosage of 2.5 mg/kg was similar to the injection used as a control at the dosage of 5.0 mg/kg.The Pharmacokinetics of MTX injection and drug containing nanoparticles in rat plasma, heart, solid tumor, spleen, liver and kidney was studied after intravenous injection. Results indicted that the in-vivo distribution process of MTX was obviously changed after it was made into a targeted formulation. The r_e and C_e value of solid tumor were the highest. Comparison of TeQ value indicated that in solid tumor the distribution of ordinary MTX formulation was only 0.2312, while 0.4438 of MTX in the nanoparticle formulation was rich in solid tumor.