Amido-functional Fe₃O₄ Nanoparticles-mediated Wt-p53 Gene Therapy For Hepatocellular Carcinoma In Vitro And In Vivo

Recently,the methods of gene delivery are gradually developing and have made great progresses.So the gene therapy of HCC has been as a hot spot on the biology and clinical medicine field.The gene delivery vectors and vectors associated immune response, cell toxicity and safety are the bottleneck of research and clinic application of gene therapy.The traditional gene delivery vectors including viral vectors and non-viral vectors have some disadvantages that are hard to overcome.Nanoparticle(NP) as a new non-viral gene delivery vector is recently developed.Gene therapeutical molecules including DNA,RNA,etc,which are encapsulated into or loaded on the surface of nanoparticles.While specific monoclonal antibodies of the target cell are linked on the surface of nanoparticle.The nanoparticles/DNA complexes can be taken into target cells by receptor-mediated endocytosis resulting in an effective target gene therapy.Selecting proper materials is the key point for nanoparticle-mediated gene delivery and gene therapy.The nanoparticle materials must be very good to biodegradability or easy to be discharged,and to have non-immunogenic.The nanoparticles made by biodegradable polymer have some advantages,such as stability,non-toxicity,good biocompatibility and the loaded DNA delivery can be controlled and protected from degradation.It is proved that the binding between nanoparticles and DNA was accomplished by electrostatic attraction.The negatively charge of phosphate backbone of DNA could be binded to the positively charged of vector.Therefore,modification of the nanoparticles surface properties with biomolecules makes the nanoparticles surface carring possitve charge which can protect the condensotion of NPs,it is avaliable to the carried gene delivery.In this study,we used functional Fe₃O₄ nanoparticles modified by APTES as a gene delivery vector,and found that functional Fe₃O₄ nanoparticles -DNA complexes could protect the DNA from degradation of DNaseI and clip of ultrasound.While we found that Fe₃O₄ nanoparticles had no obvious cell toxicity to HepG2 at lower concentration,but showed cell toxicity at high concentration.The pEGEP-C1 expression plasmid could be transfected effectively into HepG2 cells by APTES-modified Fe₃O₄ nanoparticles in vitro.The transfection rate was 39%,which was slightly higher than that of liposome(P＞0.05),but the transfecation rate could be increased by addition of extra-magnetic field.Using functional Fe₃O₄ NPs as gene delivery vector and eucryotic expression plasmid pEGEP-C1 as therapeutical gene were performed in this study,and the expression of wt-p53 transfected in HepG2 cells was measured by RT-PCR and Western blot.The results showed that transfection of exogenous wild-type p53 gene to HepG2 cells was successful and could be expression of p53 protein.In this experiment,the nude rats were randomly diveded into 4 groups:control group,local thermotherapy group,amino-functional of Fe₃O₄ nanoparticles for transduction pcDNA3.1(+)-p53 gene(functional gene) therapy group,and combination of thermotherapy and group functional gene therapy.The results showed that thermotherapy and functional gene therapy can inhibit the growth of metastatic tumors in nude mice.However the effect of combined therapy was better than that of the thermotherapy or functional gene therapy alone.The inhibiting rate of the tumor growth in combined therapy group,functional gene therapy group and thermotherapy group was 84.07%,59.43%and 47.19%, respectively.