Nano-bioconjugates Used In Gene Silencing And Therapy For Cancer Cells

Nowadays malignant tumors (cancers) have became one of the most seriousdiseases affecting human health. Increasing social stress, worse physique, worseenvironment, all of these lead to rising cancer cases. Radiotherapy, chemotherapy andsurgery are the most common methods used in cancer therapy. Chemotherapy is themost used therapy and suitable for kinds of cancers. However chemotherapy usuallyresults in severe side effect and the therapeutic effect will decrease along with thedevelopment of cancers.Nanocarriers have a lot of advantages, such as goog biocompatibility andexcellent modificability. Thereby, in the past decades nanocarriers had been developedrapidly to be used in cancer therapy and detection. Gene silence has been discovered formany years and lots of efforts have been given to utilize it in cancer therapy. Because ofthe difference in gene expression between healthy cells and cancer cells usually revealsthe reason leads to canceration, gene therapies, especially gene silencing therapies,targeting the abnormal expressed gene show good therapeutic effect. Nanocarriers basedon graphene attract many attention in recent years due to its native features includinghuge specific surface, good optical properties, easy modificability through π-πinteraction and well biocompatibility. But the therapy using nanocarriers oftendissatisfactory because of:1. Bad targeting effect of the nanocarriers results in toxic andside effects for healthy cells;2. Single mode therapy limits the therapeutic effect. Hence,it is significant to develop nanocarriers capable of killing cancer cells effectively andspecifically as well as friendly to healthy cells.In this dissertation we designed a series of nanocarriers for cancer cells imagingand therapy using gold nanoparticles (GNPs), molecular beacon (MB), siRNA, nuclear localization signal (NLS) peptides, carboxyl graphene and cell membrane receptorantibody. All of these nanocarriers were based on the abnormal expressed mRNA orDNA and cell membrane receptors. It mainly includes the following sections:1. Nanocarrier using GNPs as core. Photosensitizer Ce6and sulfydryl modified MBswere assembled on the surface of GNPs through Au-S bond, then Doxorubicin(Dox) can intercalate into the doublestranded GC or CG sequences of the stemregion of MBs in which the fluorescence can be quenched and the cytotoxicity ofDox was decreased. The GNPs have a strong surface plasmon absorption band inthe visible region and can effectively quench the fluorescence of the photosensitizerand Dox. In the presence of targeting mRNA, more stable duplexes are formed bythe hybridization of loop sequences of MBs and targeting mRNA sequences,causing the opening of MB and the release of Dox and Ce6molecules. Thefluorescence and cytotoxicity of Dox were recovered. At the same time, the Ce6was activated for PDT and singlet oxygen would generate upon655nm laserirradiation. Dox and singlet oxygen formed multimodal imaging and therapy forcancer cells. This nanocarrier can respond to the targeting mRNA rapidly and hasgood biocompatibility. Compared with single modal therapy, multimodal therapyhas a better therapeutic effect.2. We designed a nanocarrier based on gold nanoparticles (GNPs). Sulfydryl modifiedsiRNA and NLS were assemble on the surface of GNPs through Au-S bond by atwo step synthesis. The NLS would lead nanocarriers to nuclear after nanocarrierswere internalized by cancer cells. Then the siRNA would direct methylation andtranscriptional silencing of homologous promoters. Compared withpost-transcriptional gene silencing, DNA methylation had better gene silence effectand leaded to less tumorigenesis. We also found that internalization of thenanocarriers had little difference between different cancaer cells.3. We designed a nanocarrier, based on carboxyl graphene, called “DR4Capturer”.DR4antibody was linked to the surface of carboxyl graphene through covalent bond and could combine with DR4. Taking anvantage of the rigidity of grapheneand fluidity of cell membrance, DR4Capturer would achieve capture of DR4andthereby induce the clustering of death receptors to promote apoptosis signallingpathways. Clustering of DR4forms the death-inducing signalling complex (DISC)containing the Fas-associated death domain (FADD) and procaspase-8. In the DISC,procaspase-8is cleaved to active caspase-8, which leads to further activation ofcaspase-3. PI3K signalling pathway was over active in cancer cells and AKT,which was very important in PI3K pathway, could inhibit the activity of caspase-3.Given to this, we load AKT siRNA on the surface of GO to decrease the expressionof AKT. We found that DR4Capturer could kill cancer cells effectively with highspecificity and avoid the damage of healthy cells. The therapeutic effect wasenhanced after the addition of AKT siRNA.