Effects Of Nanoparticle Size And Transfection Methods On Gene Transfection

Gene therapy is a therapeutic modality that relies on the delivery of the nucleic acid drugs to the target cells,by which the desired gene products(nucleic acids or proteins)are expressed to replace or correct the defective genes in patients.Because of its high efficacy and low toxicity,it has become a promising treatment for cancers and other genetic diseases.The methods of gene delivery mainly include viral vector delivery,non-viral vector delivery,and physical delivery.Compared with viral vectors,non-viral vectors have the advantages of low immunogenicity,ready scale-up manufacture,and convenience in modification of the vectors.However,insufficient delivery efficiency is the primary barrier against the clinical translation of gene therapy using non-viral vectors.There still remains too much unknown in the gene delivery mechanisms,even for the most investigated polymeric carriers(i.e.,polyethyleneimine,PEI).The conflicting results have been often seen in literature due to the large variability in the experimental conditions and operation.Therefore,some key parameters should be identified and thus strictly controlled in the formulation process.The effect of the formulation processing parameters(e.g.,reaction concentration or volume)and the resulting nano-structure properties on gene transfection have been rarely investigated.In this work,the effect of reaction concentration(or volume)on the formation of PEI/DNA complex and the nano properties was investigated.It was found that the highconcentration process(i.e.,small reaction-volume)for mixture resulted in the largesized PEI/DNA NPs that had a higher efficiency of gene transfection,compared to the small-sized counterpart that was prepared at a low concentration.The microstructural experiments showed that the prepared small-sized NPs were firmly condensed,whereas the large-sized NPs were bulky and botryoid-shaped.The transfection mechanisms were investigated through various microscopic methods.It was revealed that the large NPs entered the tumor cells via the macropinocytosis pathway,and then efficiently dissociated in the cytoplasm and released DNA,thus promoting the intranuclear delivery.The enhanced therapeutic efficacy of the large NPs was demonstrated in the cervical subcutaneous xenograft and peritoneal metastasis mouse models.This work provides a better understanding of the effect of formulation process on nano-structural properties and gene transfection,laying a theoretic basis for the rational design of the experimental process.How to achieve the effective transfection of the primary cells(e.g.,dendritic cells,DCs)is pressing need to solve in gene delivery.For instance,DC is the target of DNA vaccination,but its transfection efficiency is very low.Various methods for DC transfection were investigated and compared in this work.The transfection study was caried out in the murine bone marrow derived DCs using the following three transfection methods: transfection of PEI/DNA nanoparticles(PEI/DNA),electrotransfection of naked DNA(electro-nDNA),and electrotransfection of PEI/DNA NPs(electro-NPs).The results showed that only electro-nDNA led to relative good transfection efficiency in BMDCs.The viability,apoptosis,proliferation,and cell cycle of BMDCs after transfection were detected.And the results showed that the low proliferation of BMDCs was closely associated with their poor transfection efficiency.For the PEI/DNA group,the low transfection efficiency was mainly due to the relatively low cellular uptake and S-phase cell cycle arrest.For the electro-NPs group,the strong cytotoxicity and S-phase cell cycle arrest seriously suppress the gene transfection.