Study On Synthesis And Antitumor Activity Of FA/PEG-functionalized DGL As Gene Nanovector

In recent years,the gene therapy represented by antisense technique is regarded as one of the most promising approaches for cancer treatment.With the development of genomics research,it has been found that some genes can be used as therapeutic targets because they play very important roles in the process of tumor transformation and development.Researchers have found that hypoxia-inducible factor-1?(HIF-1?)is overexpressed in various malignancies such as liver cancer,and is associated with biological characteristics such as tumor invasion,metastasis and prognosis.Therefore,HIF-1? can be used as a potential molecular target.It is a promising approach of cancer treatment by competitively binding HIF-1? antisense oligonucleotide(ASODN)fragments to HIF-1? mRNA to degrade the base-complementary sequence of HIF-1? mRNA,or to silence HIF-1? protein expression.The bare HIF-1? antisense oligodeoxynucleotide fragment,however,can be degraded by nuclease from both tissues and cells,and has poor intracellular delivery.Therefore,this paper starts with the development of a nanovector to effectively deliver HIF-1? antisense oligonucleotide fragments.Firstly,dendrigraft poly-L-lysine(DGL)with high biocompatibility and biodegradability was chosen as the core of carrier.Polyethylene glycol(PEG)and folic acid(FA)molecules were used as ligands and covalently bound to the surface of DGL.The introduction of PEG can not only reduce the cation toxicity of DGL but also avoid DGL from being intercepted by reticulo-endothelial system(RES),which prolonged blood circulation time and increased the distribution in the tumor sites through the enhanced permability and retention(EPR)effect.Furthermore,the functionalization of FA allowed the carrier to actively target tumor cells,and then promoted targeted endocytosis by folate receptor-mediated endocytosis.Thus,a safe,efficient and target-deliverable gene vector was synthesized.Secondly,the optimal ratio of the vector to HIF-1? antisense oligonucleotide fragments was further found out.The HIF-1? antisense oligonucleotide fragments were uploaded to the DGL core by electrostatic attraction,so as to synthesize the gene nanovector system.The morphology,corresponding size distributions and composition of the gene nanovector were characterized by means of transmission electron microscope,dynamic light scattering and infrared spectroscopy.The gene nanovector was spherical nanoparticle with particle size of 30-34 nm.Agarose gel retardation assay showed that the combination of vector with the gene was stable,and vector can effectively load the gene.According to the cellular uptake measurement and cell imaging observation,they conformably indicated that the gene nanovector system can enhance the uptake of ASODN by tumor cells and obviously achieve targeting endocytosis of ASODN.The results of MTT assay showed that the cytotoxicity of the PEG-functionalized empty vector was significantly lower than that of DGL.And FA-modified drug-loaded gene-nanovector showed high antitumor activity.It was further proved that gene-nanocarrier system could achieve cell internalization by qRT-PCR and ELISA experiments,so that gene drugs could be effectively deliver to target genes and reduced the expression of target genes at the mRNA and protein levels.The in vivo targeting and antitumor efficacy studies on tumor-bearing mice model further demonstrated that the modification of PEG and FA significantly increased the blood circulation time and improved the specificity of the vector,so that the vector could deliver more gene drugs to the tumor sites,achieving significant tumor inhibition with a tumor inhibition rate of 68.23%.To sum up,the resultant gene nanovector system in this paper can achieve safe,efficient and targeting delivery of HIF-1? antisense oligonucleotide fragments,which will provide certain reference for gene therapy of tumors.