Studies Of Enhanced PCR And Gene Delivery Application Using Pegylated Pei-entrapped Gold Nanoparticles

With the rapid development of nanotechnology,nanomaterials have become a research hotspot.Due to their unique physical and chemical properties,nanomaterials have been widely used in biomedical fields,and have achieved many valuable results.Polymerase chain reaction(PCR)is a molecular biology technique based on DNA amplification,which is also an important means of clinical medicine in the detection of disease.However,the presence of byproducts and low output are limitations generally associated with this technique.The introduction of the "nano PCR" provides a unique way for the improvement of PCR technique.In addition,gene therapy as a new method of treatment of disease has been considered a revolution in the field of medicine.Compared with the traditional treatment methods,gene therapy has been expected to fundamentally cure the disease and has become the focus of the current international academic research.However,carriers in gene delivery often have high toxicity or low transfection efficiency,non-viral vectors based on nanomaterials has received the greatest attention in recent research.In this dissertation,we synthesized a series of polyethylene glycol(PEG)-modified polyethylenimine(PEI)-entrapped gold nanoparticles(PEG-Au PENPs),having different gold loading contents.The role of the synthesized NPs in improving the PCR technique was then systematically evaluated and we also discussed a possible mechanism of PCR enhancement.At the same time,the PEG-Au PENPs as a gene vectors used in gene therapy research.We hope that through this dissertation,we can develope a kind of multi-functional nanomaterial which can optimize the PCR technology and can be used as an ideal gene transfer vector.In chapter 2,we used the low cost PEI as a substitute for the dendrimer,and polyethylene glycol(mPEG)modified PEI is used to improve the stability and biocompatibility of the nanomaterials.We used PEGylated PEI as a platform to synthesize Au NPs and the developed PEG-modified PEI NPs were used to prepare PEGylated PEI-entrapped Au NPs(PEG-Au PENPs)using different Au atom/PEI molar ratios via sodium borohydride reduction chemistry.In addition,the characterization data show that: The formed Au NPs presented relatively small size,showed to be quite uniform and good biological compatibility as well as high thermal conductivity.In chapter 3,according to the literatures,we established a screening platform for optimization of PCR,including two PCR amplification systems: the error-prone two-round PCR and GC-rich PCR(74% GC content)PCR.We used a series of optimization experiments and gel electrophoresis experiments to evaluate the optimization capability of synthesized PEG-Au PENPs.In addition,we have further explored the optimization mechanism of nanomaterials on PCR through abundant systematic experimental strategy.The results show that all the PEG-Au PENPs can improve the efficiency and specificity of PCR reaction.It is worth pointing out that we found that the nanomaterials may show different optimization mechanism in different PCR system.The improvement of the specificity and efficiency of the technique using the PEG-Au PENPs mainly depends on surface charge-mediated electrostatic interaction in the error-prone two-round PCR system,while in the GC-rich PCR system,thermal conduction may be the dominant factor.In chapter 4,to begin with,the ability that the PEG-Au PENPs compact pDNA was tested by agrose gel electrophoresis retardation assay,Then,through qualitative and quantitative analysis,the gene transfection efficiency was showed by using PEG-Au PENPs as a gene delivery vector to transfer both luciferase(Luc)reporter gene and enhanced green fluorescent protein(EGFP)gene to He La cells.Finally,the intracellular uptake capacity and intracellular trafficking of the carriers/pDNA compounds were investigated through flow cytometry and confocal images.Our results suggest the PEG-Au PENPs not only were able to transfer pDNA into cancer cells with dropped cytotoxicity than that of PEI alone,but also they possess a kind of ability with high transfection efficiency.