| Laryngeal carcinoma is one of the common malignant tumors that attack human head and neck, accounting for approximately 1% of overall malignant tumors. In China, the incidence rate of laryngeal carcinoma is about 1.5-3 per 10 million people. In the sites of ear, nose and throat, its incidence rate is just lower than that of nasopharyngeal carcinoma and or nasal cavity and sinus carcinoma with second in norther China and third in souther. As the early symptoms of laryngeal cancer is not obvious, its diagnosis is often delayed to advanced stage, especially in supraglottic cancer and hypopharyngeal cancer. Besides, in advanced laryngeal cancer, the rate of lymph node metastasis is high, while the surgical total resection rate is low, and the 5-year survival rate is less than 30%. Moreover, the patients who underwent total laryngectomy will lose speech function in all their life. The leading causes of death of patients with laryngeal carcinoma are local recurrence and cervical lymph node metastasis. The present treatments like surgery, radiotherapy or chemotherapy have little effctiveness on those patients with advanced laryngeal carcinoma. As a result, it has long been a problem for clinicians to improve the survival rate and life quality of patients after advanced laryngeal cancer treatments. Molecular targeted therapy is a new means of cancer treatment, specifically aiming at the target molecules of cancer cell which play a key role in tumorigenesis and development, or taking advantage of overexpression receptor of cancer cell surface, delivered targeting drug to the tumor cells through the coupling of special ligands to treat cancer and reduce systemic side-effects. The folate receptor as a molecular target for cancer therapy has already had its clinical application. Its ligand is a small molecular weight of vitamin folic acid, which is highly expressed in the tumor cell surface, whereas it has almost no expression in most normal tissues. Because of the high affinity between folic acid and the folate receptor, folic acid can be conjugated with drug and targeted to the tumor. Preliminary studies of our group have found the high expression of folate receptor in laryngeal cancer and successfully prepared folate modified magnetic nanoparticles loaded cisplatin and loaded targeting gene, which show satisfying results in the treatment of nasopharyngeal carcinoma and matrix metalloproteinase-2 gene silencing in laryngeal cancer cells. However, the stability of drug and capacity of drug loading are still not desirable. This study focuses on optimizing the drug preparation technology. With laryngeal cancer cells Hep-2 as the research object, it evaluates targeting property and therapeutic effect of this molecular targeting medicine optimized through drug intake and in vitro inhibit experiments. The study is divided into three parts as follows.The first part stresses on the optimization and characterization of the preparation process of folate modified magnetic nanoparticles loaded cisplatin.In order to achieve preparation process optimization, the ratio of glyco toluenesulfonyl chloride and PEG was increased from 4～5:1 to 6:1 during the preparation of amine-terminated poly-ethylene; as a result, the reaction was sped up significantly. Besides, during the folate carboxyl activation process, the proportion of each component was optimized, and the coupling of amino PEG was increased. Characterization results showed that the folate modified magnetic nanoparticles loaded cisplatin optimized had a good stability and magnetic magnetic responsiveness. Its average hydrodynamic diameter was 110.9+1.7 nm, zeta potential was-26.45±1.26 mV, cisplatin content was 1.3 mg/ml, iron content was about 1.39 mg/ml, and maximum saturation magnetization was 22.2 emu/g.The second part focuses on the study of the targeting property of folate modified magnetic nanoparticles loaded cisplatin for laryngeal cancer cells.In this study, the folic acid receptor-positive laryngeal cancer Hep-2 cells was used as the research subject, and folate receptor-positive nasopharyngeal carcinoma cells HNE-1 and folate receptor-negative nasopharyngeal carcinoma cell CNE-2 was used as control. The study investigated the targeting property of folate modified magnetic nanoparticles loaded cisplatin (FA-CDDP-MNPs) through the nanomedicine uptake of Hep-2 cells, Prussian iron staining and transmission electron microscopy. The results showed that folate modifide magnetic nanoparticles(FA-MNPs) and FA-CDDP-MNPs were susceptible uptaked by folate receptor-positive carcinoma cells, Hpe-2 and HNE-1, not susceptible uptaked by folate receptor expression negative CNE-2, and the magnetic nanoparticles were present in the cytoplasm after cellular uptake, which showed that FA-CDDP-MNPs has a good molecular targeting.The third part stresses on folate modified magnetic nanoparticles loaded cisplatin for laryngeal cancer cells in vitro inhibition effects:The study used MTT, flow cytometry and transmission electron microscopy to detect the in vitro inhibition effects and cytotoxicity of FA-CDDP-MNPs, CDDP, and FA-MNPs on Hep-2 laryngeal carcinoma cells. MTT Results showed that both the FA-CDDP-MNPs and CDDP were obvious dose dependent and time dependent for the inhibition rate to Hep-2. When the concentration of cisplatin is 8μg/ml and cultured for 48 h, the inhibition rate of FA-CDDP-MNPs is 82.2% and that of CDDP reached as high as 93.3%. There was no significant difference between them (P> 0.05), and the FA-MNPs had no effect on the growth of Hep-2.The flow cytometry results was consistent with that of MTT. Hep-2 cells were cultured for 48 hours with FA-CDDP-MNPs and CDDP respectively. Cells were obvious apoptosis, and apoptosis rate ware in positive proportion to the increase of drug concentration. However, there was no significant difference between the two kinds of drug (P>0.05). Low concentrations all resulted in cells G0/G1 phase blocking, and high concentration did not affect cell cycle, while the same concentration of FA-MNPs did not affect apoptosis of Hep-2, but made the cell cycle shift to S phase.TEM results showed that the morphology of the cells which uptaked FA-CDDP-MNPs were changed significantly, while the ones which uptaked FA-MNPs displayed no change in cell morphology. The above results suggested that in vitro FA-CDDP-MNPs had the same effects on inhibiting the growth of laryngeal cancer Hep-2 cells just as cisplatin did alone, while the carrier itself had no cytotoxicity, and could be uptaked by Hep-2, but high concentration might affect the cell cycle. |
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