| Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors that attack human Head and neck, and the incidence is high in Southern China and Southeast Asia. Radiation therapy is the preferred treatment for NPC. Although radiotherapy equipment and technology updated, the average 5-year survival rate of advanced NPC radiotherapy is about 52%. Comprehensive treatment of radiotherapy combined with chemotherapy is the most common method in clinical applications. But because of the tumor metastasis,multi-drug resistance and adverse drug reaction, the overall 5-year survival rate is still far from optimistic.In recent years, chemotherapy is getting more and more attention in the field of nasopharyngeal carcinoma treatment research. Chemotherapy use chemical drugs to prevent cancer cell proliferation, invasion, metastasis, and finally kill cancer cells. Due to the weak selectivity of chemotherapy, chemical drugs is not only kill cancer cells but also damage the body’s normal cells inevitably. The anticancer drug cisplatin (CDDP) has been used generally in the treatment of solid tumors,such as melanoma, non-small cell lung carcinoma, hepatocellular carcinoma, cervical cancer, and some successes have been achieved. CDDP can be used in combination with 5-fluorouracil (5-FU) and other anti-cancer drugs, while it can produce a synergistic effect and no significant cross-resistance. However,the application of CDDP is compromised due to its poor aqueous solubility and serious adverse effects associated with nephrotoxicity myelosuppression and gastrointestinal adverse reactions. Furthermore, nonspecific systemic distribution of CDDP commonly gives rise to dose-dependent toxic effects and poor therapeutic outcomes. To absolve these problem,biodegradable magnetic nanoparticles functioned with polyethylene glycol(PEG) and poly(asparticacid)(PASP) on the surface have been successfully applied to enclose CDDP, achieving improved water solubility and bioavailability,less CDDP associated side effects as well as better therapeutic benefits.Cancer gene therapy is based on gene can alter human genetic material, and aslo have the effect of leading the tumor cells directly against tumor gene abnormalities which play a role in the treatment of tumors. Gene therapy has opened up new ways to treat cancer, while targeted gene therapy and combination chemotherapy in the treatment of NPC shows good prospects for clinical application.Tissue facor pathway inhibitor-2 (TFPI-2) can inhibit various members of the matrix metalloproteinase (MMP) family of proteolytic enzymes.TFPI-2 is also implicated in maintaining the integrity of extracellular matrix (ECM) structure, inhibiting the invasion and metastasis of tumor cells. TFPI-2 wildly distributed in the liver, kidney, heart, skeletal muscle and other normal tissues,while TFPI-2 reduced expression in pancreatic cancer,cervical cancer and nasopharyngeal carcinoma can consequently promote tumor invasion and metastasis.Further more,TFPI-2 can also significantly inhibit the invasion and metastasis of lung cancer, glioma, melanoma and ovarian cancer.Considering these data,we postulate that TFPI-2 gene therapy can inhibit the growth of some tumors. In gene therapy, gene carriers play a vital role. As a gene carrier, liposomes exhibit high toxicity, and adenovirus contains high risk of virus activation and lacks specific targeting.Nonviral vector is an attractive option although the current agents being used displayed disadvantages, such as low transfection efficiency and high toxicity.Over the recent years,molecular targeted therapy have been the major strategy in cancer research, because it can enhance the therapeutic effects,reduce side effects, prevent drug resistance,and so on. Folate receptor (FR) is a targeting ligand used for anti-cancer agents, since it’s often overexpressed in tumor cells,rarely found in normal tissue.FA which is a necessary vitamin of human cells has high affinity with folate receptor(FR). After coupling with other molecules,FA still maintain high affinity with FR,and can be absorb in cells through endocytosis pathway.The reported gene vectors which conjugated FA have FA-EPOPC-CHOL, FA-PEG-PEI etc,Compared with unconjugated carrier FA, the FR-positive HEK293T cells, C6 cells and KB cells can improve gene transfection efficiency and shown a stronger anti-tumor effect. FA-targeted chemotherapy drugs (paclitaxel, doxorubicin) can selectively identify and target into the KB cells, bel 7402 cells and xenografts in nude mice, increasing anti-tumor effects significantly. These characteristic of FA provided theoretical basis for targeted cancer therapy. This also provides the conditions for the enhancement of conventional chemotherapy and gene therapy specificity.Our previous study found that magnetic nanoparticle not only have general magnetic nanoparticle properties, such as small particle size, surface area, but also a magnetic targeting property. It has been found that the coupling FA iron oxide magnetic nanoparticles can specificially direct to the tumor target tissue,so it can use for diagnosis and treatment. After the nanoparticles and gene combined, nanoparticles can not only effectively protect gene but also improve gene transfection efficiency and delivery gene to targeted location. Our research group constructed magnetic nanocarrier which have folate-targeted effects. We used magnetic nanoparticles as a carrier to load cisplatin to study NPC targeted therapy effects,which showed good stability and therapeutic effect. Nanocarriers with a negative charge causesed it difficult to adsorb negatively charged genes. Polyethyleneimine (PEI) is a cationic organic polymer,forming a stable complex with DNA through electrostatic attraction, and it can be cellular uptake. Because cytotoxicity and poor solubility of PEI/DNA complexes, PEI be limited in its application as a gene carrier. Studies have shown that if the cationic copolymer of PEI nonionic link hydrophile, such as polyethylene glycol (PEG). They can not only solve the solubility problems,but cytotoxic of drug carriers to extend in vivo drug carrier cycle time and reduce non-specific adsorption.Experiments have been synthesized PEI-PEG copolymer wrapped Fe3O4 nanoparticles for gene carriers, but lack of effective molecular targeting. We prepared MPEG-PEI-FA copolymer by amidation,and we used it as a surface modifier to connect Fe3O4 nanoparticles modified by aldehyde sodium alginate(ASA-MNP). FA-MNP-CDDP/TFPI-2 connect ASA-MNP and MPEG-PEI-FA by electrostatic adsorption, containing CDDP and TFPI-2 plasmid. Folate receptor-positive HNE-1 cells and folate receptor-negative nasopharyngeal carcinoma CNE-2 cells was used to study the folate targeting effect and inhibiting effect.ObjectiveFA-MNP-CDDP/TFPI-2 was prepared to construct a novel multifunctional magnetic nanocarrier loading cisplatin (CDDP) and tissue factor pathway inhibitor-2 (TFPI-2), and evaluated comprehensive inhibition of this magnetic nanocomposites on nasopharyngeal carcinoma (NPC).Method1. Preparation of FA-MNP-CDDP/TFPI-2FA-MNP-CDDP/TFPI-2 was constructed with magnetic nanoparticles- cisplatin (MNP-CDDP) prepared by chemical co-precipitation and (FA-MPEG-PEI)TFPI-2 through electrostatic adsorption.2. Characterization of FA-MNP-CDDP/TFPI-2The morphology, particle size and zeta potential and CDDP concentration of nanocomposites were measured by Fourier transform infrared spectrum (FT-IR), transmission electron microscope (TEM) laser particle detector and OPDA (o-phenylenediamine) colourimetry, respectively.3. Experiment in vitroFlow cytometry was used to analyze the gene transfection efficiency of FA-MNP-CDDP/TFPI-2.The mRNA and protein expression levels of TFPI-2 were evaluated by RT-PCR and Western blot.Proliferation rate, cell apoptosis and invasion ability of HNE-1 cells cultured with FA-MNP-CDDP/TFPI-2 were measured by CCK-8, flow cytometry and matrigel invasion test.4. Experiment in vivoVivo imaging and nuclear magnetic resonance were used to observe the absorption of TFPI-2 plasmid and MNP in the same mice intravenously injected FA-MNP/CDDP/TFPI-2. Tunel assay observe the tumor tissue apoptotic and necrosis after intravenous injection of FA-MNP/CDDP/TFPI-2.ResultsThe results showed that FA-MNP/CDDP/TFPI-2 were successfully synthesized, with single magnetic core about 9nm,the average particle size 151 nm, Zeta potential +15.4 mV. OPDA showed that the CDDP concentration of nanocomposites was 150 μg/mL and the encapsulation efficiency of CDDP was 37.5%. The transfection efficiency of TFPI-2 in the HNE-1 was 37.4% higher than CNE-2 which was 27.2%. It indicated that FA-MNP/CDDP/TFPI-2 transfect gene with targeting function. Prussian iron staining results also show that HNE-1 cells uptake more magnetic nanocomposites than CNE-2 cells.The mRNA and protein expression levels of TFPI-2 in HNE-1 cells of FA-MNP-CDDP/TFPI-2 group were significantly increased compared with the control groups. The rate of growth inhibition and cell apoptosis were 37.2% and 44.6%, whereas that in MNP-CDDP group were 23.4% and 32.7%, in FA-TFP1-2 group were 17.3% and 13.9%, respectively (P<0.05). Matrigel invasion assay FA-MNP-CDDP/TFPI-2 group show HNE-1 cell invasion capability significantly lower than the control group. Flow cytometry show FA-MNP/CDDP/ TFPI-2 prevents cell proliferation mainly occured in the S and G2 phase. Vivo imaging display that FA-MNP/CDDP/TFPI-2 target to the FR-positive HNE-1 tumor, while no marked green fluorescent protein in the FR CNE-2-negative tumors. MRI showed FA-MNP/CDDP/TFPI-2 in HNE-1 tumor is significantly reduced tumor block signals than CNE-2.Tunnel experiments suggest that after intravenous injection of FA-MNP/CDDP/TFPI-2 tumor-bearing mice 23d,HNE-1 tumor cells death significantly higher than the CNE-2 tumor cells. FA-MNP-CDDP/TFPI-2 carrying TFPI-2 and cisplatin was successfully constructed and proved to be significantly inhibitory effect on the FR-positive HNE-l cells.ConclusionWe have successfully constructed a FA-targeted carring gene (TFPI-2) and cisplatin (CDDP) magnetic nanocomposites (FA-MNP/CDDP/TFPI-2), in vivo experiments showed that FR-positive NPC cells HNE-1 has good targeting and integrated inhibitory effect. |
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