| Nasopharyngeal carcinoma(NPC)is one of the most common malignancies in all the Head and neck neoplasms in south China, Southeast America and North Africa especially in Guangdong provinces. The nasopharyngeal cancer incidence rates is up to27.49per10,000in men and10.51per10,000in women. The incidence rates of nasopharyngeal cancer were ranked fourth in the world, while the mortality rate was ranked fourth in the world.In recent years, the program of using Cisplatin+Fluorouracil to carry out inducing chemotherapy and the program of using Cisplatin+Fluorouracil to carry out concurrent radiochemotherapy have became standard treatment mode for local regional later-period nasopHarynx cancer.But although concurrent radiochemotherapy is adopted, current general radiotherapy dose is not reduced, so it firmly will cause relevant large radiation therapy complications, including serious radiation mucositis, local soft tissue injury, etc. This will cause very poor living quality of patient; therefore it is not idea for later-period case and nasopharyngeal carcinoma case without sensitive to radiation. How to reduce the toxicity of chemotherapeutics Cisplatin and improve survival rate and living quality after later-period nasopharynx cancer treatment,this is always a difficult problem for doctors.Because tumor has pathological characters such as rapid consumption and proliferation,generally the pH environment of tumor tissue is acidic,the value is about6.5,when nano particle entering cell,the particle will meet lysosomes and endosome (pH5.0～5.5)with lower pH value,but the pH of normal tissue is7.4, by using the difference of pHysiological pH value of normal tissue and pathological tissue, it can design numerous pH sensitive drug carriers targeting tumor tissue or other specific organ carrying out drug delivery increasing the concentration of drug at lesion and improving bioavailability,and it is widely used in control releasing of drug,gene,protein etc.The pH sensitive Nano preparation will be a promising targeting drug-delivery system.As a molecular target for molecular targeted tumor therapy, folate receptor is highly expressed on the surface of tumor cell,but it is almost not expressed in a majority of normal tissues.By using the high affinity of folate and folate receptor to supplement the insufficiency of magnetic targeting, to targeting the drug coupling folate receptor to tumor in acid environment, reaching pH sensitive drug releasing in acid environment, after drug entering cell under the guidance of folate receptor, further releasing drug when meeting lysosomes and endosome with lower pH value, achieving the effect of chemotherapy in the cell.It was discovered by our research group in previous period that nasopharyngeal carcinoma tissue, CNE-2and Laryngeal cancer cell line Hep-2highly express folate receptor,and our group has successfully prepared folate targeted carried Cisplatin and gene-carried magnetic nano drug, achieving excellent effects in the gene silence treatment research of nasopharynx-cancer based and laryngocarcinoma based metalloprotease2.But it is found that the targeting and stability of drug is still not completely satisfied,this theme is proposed to introduce some new innovative highlights such as releasing drug within PH sensitive cell,in the purpose of design,screen and construct a new type intelligent targeting drug delivery system featuring in having molecular targeting and magnetic targeting, complete PH sensitive orientation drug releasing through tumor’s acidic microenvironment, simultaneously having thermal theropy and radiotherapy sensitization function under the function of external alternating magnetic field.By using TEM,VSM, ultraviolet spectroscopy and other technologies to carry out characterization on its physical and chemical properties, and conducting research on the characteristics of pH sensitive drug releasing,meanwhile taking folate receptor expressing positive nasopharyngeal carcinoma cell line HNE-1as experiment model,to evaluate the targeting and treatment effects of PH sensitive carried Cisplatin magnetic nano intelligent targeting drug of such new type of folate decoration through drug undertaking, external inhibition and apoptosis experiment, so as to make a foundation for development of intelligent anti-cancer drug.It is totally divided into three chapters.Chapter I. Respectively using immunohistochemical method to detect the expression of folate receptor a in30nasopharynx cancer tissues,meanwhile using Western-Blot and RT-PCR to detect the expression of folate receptor on protein and mRNA level,so as to screen target molecule of tumor. The immunohistochemical results show that positive rate in folate receptor expression in nasopharynx cancer tissue are86.7%, the positive rate of case having a later clinical stage is higher that those patients having a early clinical stage, which is accord with the Western-Blot and RT-PCR. These reusults show that as the target of treatment of nasopharynx cancer, folate receptor has relevant high practicability.Chapter Ⅱ.Preparation and characterization of PH sensitive carried Cisplatin magnetic nano intelligent targeting drug decorated by folate.Adopting chemical coprecipitation method to prepare Ferroferric oxide nanoparticles (MNP)modified by aldehyde sodium alginate, coordination complexation of sodium alga acid and Cisplatin on the surface of nano particles is carried on chemotherapeutic drug Cisplatin, meanwhile it prepares out double hydrazino-poly PEG, the carboxyl group of hydrazino-folate on one side of double hydrazino-poly PEG, obtaining folate-hydrazino poly PEG-diazanyl, then folate-hydrazino poly PEG-diazanyl connects the aldehyde group of sodium alga acid on MNP to wrap up drug, making Cisplatin to be hidden within carrier, obtaining PH sensitive carried Cisplatin magnetic nano intelligent targeting drug connecting folate decoration. Finally obtain Cisplatin carried magnetic nano drug targeted by folate receptor (FA-PEG-NH-N=MNPs-CDDP). Characterization results show:the average magnetic nuclei particle size of PH sensitive Cisplatin-carried magnetic nano intelligent targeting drug connecting folate decoration is about10.2±.5nm, the average hydrodynamic diameter detected by laser particle size instrument is176.6±.1nm, Zeta electric potential is-20.91±1.76mv, the content of Cisplatin is0.773mg/ml, content of iron is about1.908mg/ml, maximum saturated magnetization intensity is16.3±0.2emu/g, having excellent stability and mangnetic responsiveness, and releasing drug under low pH value while having PH sensitiveness.Chapter III.The targeting experiment research carried out on nasopHarynx cancer cell by taking folate decorated pH sensitive Cisplatin-carried magnetic nano intelligent targeting drug takes the positive HNE-1cell of folate receptor as research object, takes negative nasopharynx cancer cell CNE-2of folate receptor as comparison, by using folate receptor undertaking iron stain and TEM to observ the targeting of pH sensitive Cisplatin-carried magnetic nano intelligent targeting drug decorated by folate (FA-PEG-NH-N=MNPs-CDDP). External inhibition effects adopt MTS method,flow cytometry analysis,TUNNEL experiment and TEM method to detect the external inhabition effect and cell toxicity of FA-PEG-NH-N=MNPs-CDDP and MNPs on nasopharynx cancer cell HNE-1. The targeting results show:FA-PEG-NH-N=MNPs-CDDP is easy to be undertaken by nasopHarynx cancer cell HNE-1with positive expression of folate receptor,but it is not easy to be understaken by nasopharynx cancer cell CNE-2with negative expression of folate receptor.The nano drug exists in cytoplasm after being undertaken by cell,the result indicates its excellent molecular targeting. MTS result show:FA-PEG-NH-N=MNPs-CDDP (7.4),FA-PEG-NH-N=MNPs-CDDP(6.5) and CDDP all have obvious dosage dependency and time dependency,however MNPs has no impact on the growth of HNE-1. The detection result of FCM is consistent with MTT,after jointly culturing FA-PEG-NH-N=MNPs-CDDP,CDDP and HNE-1cell for48hours,the cells all appear obvious apoptosis, and the apoptosis rate shall be increased along with the increasing of drug concentration,but the difference between both is no obvious (P>0.05). When it is in low concentration, it will cause retarding in S stage of cell, it will not influence cell cycle (P>0.05)when it is in high concentration,but FA-PEG-NH-N=MNPs-CDDP in same concentration does not influence HNE-1apoptosis, however it can make cell cycle transferring to S stage. TEM results indicate that the cell of FA-PEG-NH-N=MNPs-CDDP appears obvious apoptosis shape changing,but the cell shape of undertaking MNPs has no change.Above results show that folate decorated pH sensitive Cisplatin-carried magnetic nano intelligent targeting drug prepared by us still has same effect of external inhibiting HNE-1cell growth of pure Cisplatin.But the carrier itself has no cell toxicity and it can be undertaken by HNE-1cell, it may affect cell cycle when it is in high concentration. |
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