摘要
鼻咽癌是头颈部较常见恶性肿瘤,是我国南方、东南亚及北非等地区最常见的恶性肿瘤之一,其发病率男性高达27.49／万,女性达10.51／万,居所有恶性肿瘤发病率的第三位,死亡率高居第四位。近年来,用顺铂(Cisplatin+氟尿嘧啶(Fluorouracil)方案的诱导化疗及在放疗的同时使用顺铂(Cisplatin)同步化疗已成为局部区域晚期鼻咽癌的标准治疗模式。但尽管采用同期放化疗,目前总的放疗剂量都没有降低,势必引起较大的放疗并发症,包括严重的放射性粘膜炎、局部软组织损伤等,导致患者生活质量较差,对于晚期病例以及放射线不敏感的鼻咽癌病例治疗效果不理想。如何降低化疗药物顺铂的毒性,从而提高晚期鼻咽癌治疗后的生存率和生活质量一直是困扰临床医生的难题。
由于肿瘤具有快速消耗和增殖的病理特点,一般肿瘤组织的微环境是呈酸性,pH大约为6.5,当纳米粒子进入细胞内部后,粒子会遇到pH值更低的溶酶体和内涵体(pH5.0～5.5),而正常组织的pH为7.4,利用正常组织与病理组织的pH值差异可设计出靶向于肿瘤组织或其他特定器官进行药物传递的pH敏感药物载体,增加药物在病灶部位的浓度,提高生物利用度,这种pH敏感药物载体可用于药物、基因、蛋白质等的控制释放。叶酸受体在肿瘤细胞膜表面高度表达,而在绝大多数正常组织中几乎不表达。利用叶酸和叶酸受体的高度亲和性,在叶酸受体的介导下将与叶酸偶联的药物转运入肿瘤细胞内,在溶酶体和内涵体低pH值酸性环境下药物释放,达到细胞内靶向化疗的目的。我们课题组前期研究发现鼻咽癌组织及鼻咽癌细胞株HNE-1和喉癌细胞株Hep-2高度表达叶酸受体,并已成功制备了叶酸靶向载顺铂和载基因磁性纳米药物,在鼻咽癌、喉癌基质金属蛋白酶2基因沉默的治疗研究中取得良好效果。但发现药物的靶向性和稳定性尚不满意,本课题拟在原研究基础上,根据肿瘤微环境的酸性特征,引入pH敏感细胞内释放药物等新的研究成果,设计、筛选、构建出一个新型的具有分子靶向、磁靶向、通过肿瘤酸性微环境完成PH敏感定向释药、在外置交变磁场的作用下同时具有热疗和放疗增敏作用的智能靶向给药系统。采用透射电镜、振动样品磁强计(Vibrating sample magnetometer, VSM)、激光动态粒径仪、紫外分光光度计等对其理化性状等进行表征,同时以叶酸受体表达阳性鼻咽癌细胞株HNE-1作为实验模型,通过药物摄取和体外抑制及凋亡实验评价这种新型的叶酸修饰的载顺铂磁性纳米pH敏感靶向药物的靶向性和治疗效果,为开发新的智能抗癌药物奠定基础。共分三章：
第一章,采用免疫组织化学法检测30例鼻咽癌组织标本中叶酸受体(FR α)的表达,同时采用Western-Blot和RT-PCR的方法进一步检测鼻咽癌组织中FR α在蛋白质和mRNA水平的表达,筛选肿瘤靶分子。免疫组化结果显示鼻咽癌组织FR α表达阳性率达为86.7%,临床分期晚的病例阳性率较临床分期早的病人更高。Western-Blot和RT-PCR结果也表明鼻咽癌组织FR α高度表达。这些结果提示将FR a作为治疗鼻咽癌的靶点具有高度的可行性。
第二章,叶酸修饰的载顺铂磁性纳米pH敏感靶向药物的制备和表征。
采用化学共沉淀法制备醛基化海藻酸钠改性的四氧化三铁纳米粒(MNPs),海藻酸钠表面的羧基通过配位络合作用与顺铂连接,制备偶联叶酸的双肼基PEG,其肼基端与海藻酸钠的醛基通过腙键连接包裹载药纳米粒,经纯化获得叶酸修饰的载顺铂磁性纳米pH敏感靶向抗癌药物(FA-PEG-NH-N=MNPs-CDDP).表征结果显示：透射电镜下该磁性纳米pH敏感靶向药物的磁核平均粒径约为10.2±1.5nm,激光粒度仪测得平均水动力学直径176.6±1.1nm, zeta电位为-20.91±1.76mv,顺铂含量为0.773mg/ml,铁含量约为1.908mg/ml,最大饱和磁化强度为16.3±0.2emu/g,具有良好的稳定性和磁响应性,在pH值低于6.5的酸性条件下释放药物,具有pH敏感性。
第三章,叶酸修饰的载顺铂磁性纳米pH敏感靶向药物对鼻咽癌细胞的靶向性实验研究。
以叶酸受体阳性HNE-1细胞作研究对象,叶酸受体阴性鼻咽癌细胞CNE-2作对照,通过细胞摄取铁染色、透射电镜观察叶酸修饰的载顺铂磁性纳米pH敏感靶向药物(FA-PEG-NH-N=MNPs-CDDP)的靶向性。采用MTS法、流式细胞分析、TUNNEL实验和透射电镜方法检钡(?)FA-PEG-NH-N=MNPs-CDDP对鼻咽癌细胞HNE-1的体外抑制效应和细胞凋亡。结果显示：FA-PEG-NH-N=MNPs-CDDP易被叶酸受体阳性鼻咽癌细胞HNE-I摄取,而不易被叶酸受体阴性的CHE-2摄取,纳米药物被细胞摄取后存在于细胞浆中,提示该纳米药物具有良好的分子靶向性。MTS结果显示：FA-PEG-NH-N=MNPs-CDDP (7.4) FA-PEG-NH-N=MNPs-CDDP(6.5)和CDDP三者对HNE-1的抑制率均存在明显的剂量依赖性和时间依赖性,而MNPs对HNE-1的生长无影响。FA-PEG-NH-N=MNPs-CDDP(6.5)对细胞的抑制大于PH=7.4组,提示药物在低pH值6.5时有释放。流式细胞、Tunel法检测结果与MTT一致,FA-PEG-NH-N=MNPs-CDDP实验组(pH6.5)、实验组(pH7.4)和CDDP分别与HNE-1细胞共培养48小时后,细胞均发生明显凋亡,且凋亡率随药物浓度增加而升高。低浓度时均导致细胞G2/M期阻滞,高浓度时引起S期阻滞。透射电镜结果表明摄取FA-PEG-NH-N=MNPs-CDDP的细胞出现明显凋亡形态改变,而摄取MNPs的细胞形态无改变。
以上结果显示叶酸受体在鼻咽癌组织不同水平均高度表达,适合作为药物分子靶点,叶酸修饰的载顺铂磁性纳米pH敏感靶向药物能被HNE-1细胞摄取,对其生长的体外抑制效应与单纯顺铂相同,FA-PEG-NH-N=MNPs-CDDP实验组(pH6.5)、实验组(pH7.4)具有缓释作用,而实验组(pH6.5)则具有pH敏感释放药物特性,并通过阻滞细胞周期诱导细胞凋亡,而载体本身无细胞毒性。
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.
引文
[1]全国肿瘤防治研究办公室,等.2010中国肿瘤登记年报-中国肿瘤登记地区2007年肿瘤发病死亡[M].北京：军事医学科学出版社,009.
[2]马骏.鼻咽癌治疗的研究进展.中山大学学报(医学科学版).2010(3)
[3]Kuang Y, Liu J, Liu Z, et al.Cholesterol-based anionic long-circulating cisplatin liposomes with reduced renal toxicity. Biomaterials.2011 Nov 11.
[4]Sastry J, Kellie SJ. Severe neurotoxicity, ototoxicity and nepHrotoxicityfollowing high-dose cisplatin and amifostine [J]. Pediatr Hematol Oncol,2005,22:441-5.
[5]Ying Kuang, Jia Liu, Zhilan Liu,et al.Cholesterol-based anionic long-circμlating cisplatin liposomes with reduced renal toxicity. Biomaterials. 2011Nov11
[6]XIE M, CHEN S, XU X, et a l.Preparation of two kinds of superpara magnetic carriers supported cisplatinum comp lexes and the comparison of their characteristics[J]. Chin Sci Bull(科学通报),2006,51(2):151-157
[7]Okeyoshi K, Suzuki D, Yoshida R. Effect of nanointegration on pHotoinduced hydrogen-generating nanogel systems. Langmuir.2012 Jan 17;28(2):1539-44.
[8]J. Kim, H.J. Yoon, S. Kim, K. Wang, T. Ishii and Y.R. Kim, et al. Polymer-metal complex micelles for the combination of sustained drμg releasing and pHotodynamic therapy. J Mater Chem,19 (2009), pp. 4627-4631
[9]E. Monti, M.B. Gariboldi, R. Ravizza, R. Molteni, K. Sparnacci and M. Laus, et al. Poly (methylmetacrylate) (PMMA) core-shell nanospHeres act as efficient pHarmacopHores for the antiproliferative [PtCl3(NH3)] complex by forming ionic couples. Inorganica Chim Acta,362 (2009), pp.4099-4109
[10]Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, Bergemann C, Huenges E, Nawroth T, Arnold W, Parak FG.. Targeting cancer cells: magnetic nanoparticles as drμg carriers. European BiopHysics Journal, 2006;35(5):446-450.
[11]Alexiou C, Jurgons R, Seliger C, Brunke O, Iro H, Odenbach S. Delivery of superparamagnetic nanoparticles for local chemotherapy after intraarterial infusion and magnetic drμg targeting. Anticancer Res, 2007;27(4A):2019-2022.
[12]刘洁,谢民强,张涛,张宏征,许熠铭.叶酸分子靶向载顺铂磁性纳米药物制备及表征[J].中国组织工程研究与临床康复,2011,15(25)：4631-4637
[13]黄水仙；谢民强；李勇,袁晓阳,陈帅君.载顺铂磁性纳米药物在鼻咽癌裸鼠移植瘤组织内的分布及靶向性研究.医学研究杂志,2011,(2)
[14]Sebolt-Leopold JS, English JM. Mechanisms of drμg inhibition of signalling molecμles. Nature.2006 May 25; 441(7092):457-462.
[15]Chen YC, Chang WH, Wang SJ, et al. Fluorescent Magnetic Nanoparticles with Specific Targeting Functions for Combinded Targeting, Optical Imaging and Magnetic Resonance Imaging.J Biomater Sci Polym Ed.2011 Oct 17.
[16]Zhang Y, Xiang J, Liu Y, et al. Constructing transferrin receptor targeted drμg delivery system by using doxorubicin hydrochloride and vanadocene dichloride.Bioorg Med Chem Lett.2011 Oct 1;21(19):5982-6.
[17]Kolhatkar R, Lote A, Khambati H. Active tumor targeting of nanomaterials using folic acid, transferrin and integrin receptors.Curr Drμg Discov Technol. 2011Sep1;8(3):197-206.
[18]Salazar M D, Ratnam M. The folate receptor:what does it promise in tissue-targeted therapeutics?. Cancer Metastasis Rev,2007,26(1):141-152.
[19]Ross JF, Chaudhuri PK,Ratnam M. Differential regμlation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. PHysiologic and clinical implications. Cancer,1994,73:2432-43.
[20]Low PS, Kμlaratne SA. Folate-targeted therapeutic and imaging agents for cancer. Curr Opin Chem Biol.2009 Jun;13(3):256-62.
[21]张宏征,谢民强,李仲汉,郭灵.叶酸受体在鼻咽癌组织及细胞中的表达与意义.中国现代医学杂志,2009,19(6)：833-6.
[22]张宏征,谢民强,康庄,沈辉,王蕾.顺铂磁性纳米药物体内靶向性研究[J].南 方医科大学学报,2008,(10)
[23]Zhao H, Yung L Y. Selectivity of folate conjμgated polymer micelles against different tumor cells. Int J PHarm,2008;349(1-2):256-268.
[24]Huang S, Lin B, Qin J.Microfluidic synthesis of tunable poly-(N-isopropylacrylamide) microparticles via PEG adjustment.ElectropHoresis. 2011 Nov 11.
[25]Nagase K, Mizutani Akimoto A, Kobayashi J,et al.Effect of reaction solvent on the preparation of thermo-responsive stationary pHase throμgh a surface initiated atom transfer radical polymerization.J Chromatogr A.2011 Dec 2;1218(48):8617-28.
[26]Muthiah P, Hoppe SM, Boyle TJ, et al.Thermally Tunable Surface Wettability of Electrospun Fiber Mats:Polystyrene/Poly (N-isopropy lacrylamide) Blended versus Crosslinked Poly [(N-isopropy lacrylamide)-co-(methacrylic acid)].Macromol Rapid Commun.2011 Nov 1;32(21):1716-21.
[27]Yu S, Wu G, Gu X, Wang J, Wang Y, Gao H, Ma J. Magnetic and pH-sensitive nanoparticles for antitumor drμg delivery. Colloids Surf B Biointerfaces. 2012.Nov 1;103C:15-22.
[28]Wuang SC, Neoh KG, Kang ET, Leckband DE, Pack DW. Acid-Sensitive Magnetic Nanoparticles as Potential Drμg Depots. AIChE J.2011 Jun;57(6):1638-1645.
[29]She W, Luo K, Zhang C, Wang G, Geng Y, Li L, He B, Gu Z. The potential of self-assembled, pH-responsive nanoparticles of mPEGylated peptide dendron-doxorubicin conjμgates for cancer therapy. Biomaterials. 2013Feb;34(5):1613-23.
[30]Chen B, van der Poll DG, Jerger K, Floyd WC, Frechet JM, Szoka FC. Synthesis and properties of star-comb polymers and their doxorubicin conjμgates. Bioconjμg Chem.2011 Apr 20;22(4):617-24.
[31]Lu PL, Chen YC, Ou TW, Chen HH, Tsai HC, Wen CJ, Lo CL, Wey SP, Lin KJ, YenTC, Hsiue GH. Mμltifunctional hollow nanoparticles based on graft-diblock copolymers for doxorubicin delivery. Biomaterials.2011 Mar;32(8):2213-21.
[32]Fang C, Kievit FM, Veiseh O, StepHen ZR, Wang T, Lee D, Ellenbogen RG, Zhang M. Fabrication of magnetic nanoparticles with controllable drμg loading and release throμgh a simple assembly approach. J Control Release. 2012 Aμ20;162(1):233-41.
[33]Kobayashi T. Cancer hyperthermia using magnetic nanoparticles. Biotechnol J.2011 Nov;6(11):1342-7.
[34]Sneed PK, Stauffer PR, McDermott MW, et al. Survival benefit of hyperthermia in a prospective randomized trial of brachytherapy boost +/-hyperthermia for glioblastoma mμ1tiforme. Int J Radiat Oncol Biol PHys.1998;40:287-295
[35]Stahl H, Wust P, Maier-Hauff K, Seebass M, et al. The use of an early postoperative interstitial-hyperthermia combination therapy in malignant gliomas. Strahlenther Onkol.1995;171:510-524
[36]Silva AC, Oliveira TR, Mamani JB, Malheiros SM, Malavolta L, Pavon LF, Sibov TT, Amaro E Jr, Tannus A, Vidoto EL, Martins MJ, Santos RS, Gamarra LF.Application of hyperthermia induced by superparamagnetic iron oxide nanoparticles in glioma treatment. Int J Nanomedicine.2011;6:591-603.
[37]Itoh Y, Yamada Y, Kazaoka Y, Ishiguchi T, Honda N. Combination of chemotherapy and mild hyperthermia enhances the antitumor effects of cisplatin and adriamycin in human bladder cancer T24 cells in vitro. Exp Ther Med.2010;1:319-23.
[38]Conti M, De Giorgi U, Tazzari V,et al. Clinical pHarmacology of intraperitoneal cisplatin-based chemotherapy.J Chemother.2004 Nov;16 Suppl 5:23-5.
[39]Hergt R, Dutz S, Mμller R, Zeisberger M. Magnetic particle hyperthermia: nanoparticles magnetism and materials development for cancer therapy. J PHys Condens Matter.2006; 18:2919-34.
[40]Jin, H., Kang, K.A. Application of novel metal nanoparticles as optical/thermal agents in optical mammograpHy and hyperthermic treatment for breast cancer.Adv. Exp. Med. Biol.2007.599,45-52.
[41]Johannsen, M., Gneveckoow, U., Thiesen, B.et al. Thermotherapy ofprostate cancer using magnetic nanoparticles:feasibility, imaging, and threedimensional temperature distribution. Eur. Urol.2007.52,1653-1662.
[42]Wust, P., Gneveckow, U., Johannsen, et al. Magnetic nanoparticles for interstitial thermotherapy-feasibility, tolerance and achieved temperatures. Int. J. Hyperperthermia 2006.22,673-685
[43]Tschoep-Lechner KE, Milani V, Berger F, Dieterle N, Abdel-Rahman S, Salat C,Issels RD. Gemcitabine and cisplatin combined with regional hyperthermia as second-line treatment in patients with gemcitabine-refractory advanced pancreatic cancer. Int J Hyperthermia.2013;29(1):8-16.
[44]Kato S, Kimura M, Kageyama K, Tanaka H, Miwa N. Enhanced radiosensitization by liposome-encapsμlated pimonidazole for anticancer effects on human melanoma cells. J Nanosci Nanotechnol.2012 Jun;12(6):4472-7.
[45]王旭飞.高Z重金介质的肿瘤放疗增敏效应研究现状.中国医学物理学杂志.2012.29(3)：3337-3345.
[46]Hainfeld JF, Smilowitz HM, O'Connor MJ, Dilmanian FA, Slatkin DN. Gold nanoparticle imaging and radiotherapy of brain rumors in mice. Nanomedicine (Lond).2012 Dec 24.
[47]Roa W, Zhang X, Guo L, Shaw A, Hu X, Xiong Y, Gμlavita S, Patel S, Sun X, Chen J, Moore R, Xing JZ. Gold nanoparticle sensitize radiotherapy of prostate cancer cells by regμlation of the cell cycle. Nanotechnology.2009 Sep 16;20(37):375101.
[48]谢民强,陈帅君,徐雪青,李仲汉,沈辉,许家瑞.两种顺铂磁性纳米颗粒制备及其特性的比较.《科学通报》.2005.(19)
[49]李仲汉,谢民强,陈帅君,王蕾.改性载顺铂磁性纳米药物治疗鼻咽癌的体外实验。中国组织工程研究与临床康复,2009；13(29)：5637-5640
[1]Chen Y, Sun Y, Liang SB, Zong JF, Li WF, Chen M, Chen L, Mao YP, Tang LL, Guo Y, Lin AH, Liu MZ, Ma J. Progress report of a randomized trial comparing long-term survival and late toxicity of concurrent chemoradiotherapy with adjuvant chemotherapy versus radiotherapy alone in patients with stage III to IVB nasopharyngeal carcinoma from endemic regions of China. Cancer.2013 Apr 10.
[2]Niu X, Hu C, Kong L. Experience with combination of cetuximab plusintensity-modulated radiotherapy with or without chemotherapy for locoregionally advanced nasopharyngeal carcinoma. J Cancer Res Clin Oncol.2013 Mar 24.
[3]Salvati A, Pitek AS, Monopoli MP, Prapainop K, Bombelli FB, Hristov DR, Kelly PM, Aberg C, Mahon E, Dawson KA. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.Nat Nanotechnol.2013 Feb;8(2):137-43.
[4]Mehra NK, Mishra V, Jain NK. Receptor-based targeting of therapeutics. Ther Deliv.2013 Mar;4(3):369-94.
[5]陈燕,陈鸿雁.叶酸受体在鼻咽癌细胞系及鼻咽癌组织中的表达[J].肿瘤学杂志,2009,07：627-628.
[6]6.陈素钻,刘璇芝,郭光华,et a1.叶酸受体a在食管癌的表达及与食管癌临床病理关系的研究.实用肿瘤杂志[J],2008,23(3)：200-205
[7]Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem.2005 Mar 15;338(2):284-93.
[8]张宏征,谢民强,李仲汉,郭灵.叶酸受体在鼻咽癌组织及细胞中的表达与意义.中国现代医学杂志,2009,19(6)：833-6.
[9]Elnakat H, Ratnam M. Distribution, functionality and gene regulation of folate receptor isoforms:implications in targeted therapy. Adv Drug Deliv Rev.2004;56(8):1067-1084.
[10]Teng L, Xie J, Teng L, Lee RJ. Clinical translation of folate receptor-targeted therapeutics. Expert Opin Drug Deliv.2012 Aug;9(8):901-8.
[11]O'Shannessy DJ, Somers EB, Smale R, Fu YS. Expression of Folate Receptor-a(FRA) in Gynecologic Malignancies and its Relationship to the Tumor Type. Int JGynecol Pathol.2013 May;32(3):258-68.
[12]Cagle PT, Zhai QJ, MurpHy L, Low PS. Folate receptor in adenocarcinoma and squamous cell carcinoma of the lung:potential target for folate-linked therapeutic agents. Arch Pathol Lab Med.2013 Feb;137(2):241-4.
[13]Iwakiri S, Sonobe M, Nagai S, Hirata T, Wada H, Miyahara R. Expression status of folate receptor alpHa is significantly correlated with prognosis in non-small-cell lung cancers. Ann Surg Oncol.2008 Mar;15(3):889-99.
[14]Despierre E, Lambrechts S, Leunen K,et al. Folate receptor alpHa (FRa) expression remains unchanged in epithelial ovarian and endometrial cancer after chemotherapy. Gynecol Oncol.2013 Apr 2. doi:pii:S0090-8258
[l]马骏.鼻咽癌治疗的研究进展.中山大学学报(医学科学版).2010(3)
[2]Kuang Y, Liu J, Liu Z, et al.Cholesterol-based anionic long-circμlating cisplatin liposomes with reduced renal toxicity. Biomaterials.2011 Nov 11
[3]Kolhatkar R, Lote A, Khambati H. Active tumor targeting of nanomaterials using folic acid, transferrin and integrin receptors.Curr Drμg Discov Technol. 2011 Sep 1;8(3):197-206.
[4]Yu S, Wu G, Gu X, Wang J, Wang Y, Gao H, Ma J. Magnetic and pH-sensitive nanoparticles for antitumor drμg delivery. Colloids Surf B Biointerfaces.2012.Nov 1;103C:15-22.
[5]Hu X, Liu S, Huang Y, Chen X, Jing X. Biodegradable block copolymer-doxorubicin conjugates via different linkages:preparation, characterization, and in vitro evaluation. Biomacromolecules.2010, 11(8):2094-102.
[6]Ohguchi Y Kawano K Hattori Y et al. Selective delivery of folate-PEG-lirlked, nanoemulsion-loaded aclacinomycin A to KB nasopharyngeal cells and xenografl:effect ofchain length and amount of folate-PEG linker[J]. J Drug Target,2008,16(9):660-7.
[7]Lanrienzoa P, Malinconicoa M, Mottab A et al. Synthesis and characterization of a novel alginate-poly(ethylene glycol) graft copolymer[J]. Carbohydrate Polymers.2005,62(3):274-282.
[8]刘洁,谢民强,张涛,张宏征,许熠铭.叶酸分子靶向载顺铂磁性纳米药物制备及表征[J].中国组织工程研究与临床康复,2011,15(25)：4631-4637
[9]XIE M, CHEN S, XU X, et a 1.Preparation of two kinds of superpara magnetic carriers supported cisplatinum comp lexes and the comparison of their characteristics[J]. Chin Sci Bu11(科学通报),2006,51(2)：151-157
[10]Nguyen R,Huc I.Optimizing the reversibility of hydranzone formation for dynamic combinatorial chemistry.chem commun.2003.9(8):942-943
[11]邱小香.分光光度法测定水中全铁的含量[J].西南民族大学学报(自然科学版),2011,01：111-113.
[12]Dody Avichezer, Bilha Schechter, Ruth Arnon. Functional polymers in drug delivery:carrier-supported CDDP (cis-platin) complexes of polycarboxylates-effect on human ovarian carcinoma. Reactive & Functional Polymers,1998,36:59-69
[13]Boonthcekul T, Kong HJ, Mooney DJ. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution[J]. Biomaterials,2005,26(15):2455-65.
[14]akila E, Bimbo LM, Kaasalainen M, Herranz B, Airaksinen AJ, Heinonen M, Kukk E, Hirvonen J, Santos HA, Salonen J. Amine modification of thermally carbonized porous silicon with silane coupling chemistry. Langmuir. 2012;28(39):14045-54.
[15]Weissleder R,Stark DD,Engelstad BL,etal.Superparamagnetic iron oxides:pHarmacokinerics and toxicity[J].AM J Roent.1989,152(1):167-173
[16]陈帅君,谢民强,王蕾,龙镇,李仲汉,张宏征.顺铂磁性纳米药物的安全性评价[J].中国药房,2008,25：1941-1943.
[17]王琴梅,廖燕红,滕伟,et a1.盐酸羟胺2电位滴定法测定氧化海藻酸钠上的醛基浓度.分析试验室.2008,27(Suppl)：83-86
[18]Parveen S, Sahoo SK. Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery. Eur J PHarmacol.2011 Nov 30;670(2-3):372-83.
[19]Azzam T,Eisenberg A.Control of vesicular morpHologies through hydropHobic block length.Angew Chem Int Ed.2006,45(44),7443-7447
[20]江明,A.艾森伯格,刘国军等,大分子自组装.北京,科技出版社,2006
[1]Xiao H, Yan L, Zhang Y, Qi R, Li W, Wang R, Liu S, Huang Y, Li Y, Jing X. Adual-targeting hybrid platinum(IV) prodrug for enhancing efficacy. Chem Commun (Camb).2012 Nov 11;48(87):10730-2.
[2]Wahajuddin, Arora S. Superparamagnetic iron oxide nanoparticles:magnetic nanoplatforms as drug carriers. Int J Nanomedicine.2012;7:3445-71.
[3]Okamatsu A, Motoyama K, Onodera R, Higashi T, Koshigoe T, Shimada Y, Hattori K, Takeuchi T, Arima H. Folate-Appended β-Cyclodextrin as a Promising Tumor Targeting Carrier for Antitumor Drugs in Vitro and in Vivo. Bioconjug Chem.2013 Apr 17;24(4):724-33.
[4]刘洁,谢民强,张涛,张宏征,许熠铭.叶酸分子靶向载顺铂磁性纳米药物制备及表征[J].中国组织工程研究与临床康复,2011,25：4631-4637.
[5]Du C, Deng D, Shan L, Wan S, Cao J, Tian J, Achilefu S, Gu Y. A pH-sensitive doxorubicin prodrug based on folate-conjugated BSA for tumor-targeted drug delivery. Biomaterials.2013 Apr;34(12):3087-97.
[6]王玉,王东凯,孙念.pH-敏感型纳米制剂概述[J].中国药剂学杂志(网络版),2009,(02)：72-76.
[7]李苹,蔡绍皙,吴泽志,邓传斌.MTS法在磁控加载环境中细胞增殖测定中的应用[J].西南农业大学学报(自然科学版),2006,04：566-568.
[8]Martens-de Kemp SR, Dalm SU, Wijnolts FM, Brink A, Honeywell RJ, Peters GJ,Braakhuis BJ, Brakenhoff RH. DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells. PLoS One.2013 Apr 17;8(4):e61555.
[9]谢民强,陈帅君,徐雪青,李仲汉,沈辉,许家瑞.两种顺铂磁性纳米颗粒制备及其特性的比较[J].科学通报,2005,19：33-38.
[10]陈帅君,谢民强,王蕾,龙镇,李仲汉,张宏征.顺铂磁性纳米药物的安全性评价[J].中国药房,2008,(25)：1941-1943.
[11]李仲汉,谢民强,陈帅君,王蕾.改性载顺铂磁性纳米药物治疗鼻咽癌的体外实验(英文)[J].中国组织工程研究与临床康复,2009,(29)：5637-5640.
[12]]谢民强,王蕾,陈帅君,徐雪青,沈辉.载顺铂磁性纳米药物在小鼠体内的组织分布[J].中国药学杂志,2010,(21)：1644-1647.
[13]张宏征,谢民强,张涛,陈帅君.叶酸-顺铂纳米药物的制备及其对人鼻咽癌细胞的靶向治疗作用[J].实用医学杂志,2011,(10)：1729-1732.
[14]王琳,贾琴,范能全.评价细胞毒性方法的探讨[J].泸州医学院学报,2005,(02)：132-134.
[15]王双苗,杨江勇,杜丽娜,金义光,周兰兰.载氟尿嘧啶pH敏感树枝状聚合物纳米载体的体外释药及对人肝癌细胞药效学评价[J].国际药学研究杂志,2011,(02)：148-152.
[16]Reddy AM, Kwak BK, Shim HJ, et al. In vivo tracking of mesenchymal stem cells labeled with a novel chitosan-coated superparamagnetic iron oxide nanoparticles using 3. OT MRI. J Korean Med Sci.2010;25(2):211-219
[17]Wong TH, Tekant Y, Ngoi SS, et al, The suirgical treatment of squamous cell carcinoma of the oesophagus in Singapore[J]. Ann Acad Med Singapore,1990,19 (6):807-810
[18]庞荣清,刘春生,潘兴华,张步振,王桂华,吴秀琴,陈志龙.顺铂对人肺腺癌细胞SLC-89作用的机理探讨[J].中国肺癌杂志,2003,(06)：469-472
[19]黄伟,张瑶珍,周剑锋,刘文励.Chk1/2反义寡核苷酸对DDP诱导白血病原代细胞凋亡影响的研究[J].临床血液学杂志,2006,(06)：362-364
[20]Wlodkowic D, Skommer J, Darzynkiewicz Z. Flow cytometry-based apoptosis detection. Methods Mol Biol.2009:559:19-32.
[21]Loo DT. In situ detection of apoptosis by the TUNEL assay:an overview of techniques. Methods Mol Biol.2011;682:3-13.