Gene therapy has emerged as an influential tool for treating the genetic and specific acquired disorders. Among all kinds of gene delivery systems, the cationic polymer polyethyleneimine (PEI) is considered as a promising non-viral gene delivery vector, although there are still concerns about its magnitude of cytotoxicity. While any cell insult leads to unfolded/misfolded protein accumulation and its consequent unfold protein response, evaluating the expression profile of ER-stress genes would be a sensitive indicator of cell stress. Beside cytotoxicity assays, real-time RT-PCR was used to investigate the effects of PEI nanoparticles on the endoplasmic reticulum. Treating Neuro2A cells revealed that PEI can induce cell toxicity in a concentration-dependent manner. Also, It increased the transcript levels of Grp78 (Bip), Atf4 and Chop, and splicing of Xbp1. To further optimize the transfection properties in Neuro2A cells, PEI was used to deliver a plasmid DNA containing GFP reporter. While different PEI/plasmid ratios revealed similar transfection efficiency, increasing the PEI/plasmid ratio led to induction of ER-stress markers. These results underscored that beside the effectiveness of PEI, using the lowest possible ratio of PEI/plasmid would minimize the detrimental effects of PEI on cells and confer it a beneficial therapeutic importance in nucleic acid delivery.
Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.
Ischemia/reperfusion (I/R) is one of the main causes of acute renal failure, leading to generation of reactive oxygen species (ROS) and ensuing oxidative stress, which results in unfolded/misfolded protein accumulation and its dependent response pathways, generally known as unfolded protein response (UPR), in the endoplasmic reticulum (ER). We studied the effects of renal I/R on the expression of ER-stress genes, as well as concomitant effects of oral pretreatment with pomegranate (Punica granatum L.) pith and carpellary membrane (PPCM). An in vivo model of rat kidney I/R followed by conventional and real-time RT-PCR was used to evaluate the modulation of GRP78 and XBP1 as central UPR and ER-stress markers. Ischemia followed by reperfusion (60 and 150 min, respectively) resulted in decreased antioxidant properties of plasma (lowered ferric reducing ability of plasma [FRAP] value) and GRP78 transcript levels. Oral administration of PPCM aqueous extract for 10 days with or without ischemia (PPCM and PPCM/Isc groups, respectively) was able to further decrease the GRP78 expression, while it increased the FRAP value. PPCM pretreatment also reduced the XBP1 splicing in the PPCM/Isc group compared to the Isc group. These results suggest that UPR is activated during oxidative insults induced by I/R, while PPCM can reduce I/R-induced ER stress in rat kidneys via antioxidant defense mechanisms.
Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated.In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30nm and suitable homogeneity with minimum 12-month stability at 4◦C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 hours in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.
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