Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells

Abstract

Jurkat, an immortalized cell line derived from human leukemic T lymphocytes, has been employed as an excellent surrogate model of human primary T-cells for the advancement of T-cell biology and their applications in medicine. However, presumably due to its T-cell origin, Jurkat cells are very difficult to transfect. Thus, for the genetic modification of Jurkat cells, expensive and time-consuming viral vectors are normally required. Despite many previous efforts, non-viral vectors have not yet overcome the hurdles of low transfection efficiency and/or high toxicity in transfection of Jurkat cells. Here, we report that a simple addition of calcium ions (Ca 2+ ) into culture media at optimal concentrations can enhance the efficiency of the polyplex-mediated transfection using poly(ethylene imine) (PEI) by up to 12-fold when compared to the polyplex-only control. We show that calcium enhances the association between polyplex and Jurkat, which is at least partially responsible for the increase in transmembrane delivery of polyplex and consequential enhancement in expression of transgene. Other cations, Mg 2+ or Na + did not show similar enhancement. Interestingly, addition of Ca 2+ was rather detrimental for the transfection of lipoplex on Jurkat cells. Observation of significant enhancement in the transfection of non-viral vectors with a simple and physiologically relevant reagent like Ca 2+ in the engineering of hard-to-transfect cells such as Jurkat warrants further investigation on similar strategies.

A Polyplex in a Shell: The Effect of Poly(aspartic acid)-Mediated Calcium Carbonate Mineralization on Polyplexes Properties and Transfection Efficiency

“…P2 was probably more susceptible to dissociation due to the higher pAsp amount on its composition, which increases the polyanionic content of the polyplex and offers less free Ca 2+ to sacrifice for binding with heparin. Interestingly, it was reported by others that incubation of cationic polyplexes with calcium salts shows the opposite effect: Ca 2+ in such cases decreases pDNA binding of the polyplexes by competition with the polycation used. , This could explain why a decrease in pDNA binding was found recently when submitting cationic polyplexes to mineralization . Since the polyplexes were cationic, the authors probably failed to mineralize their polyplexes, precipitating particles by homogeneous nucleation, and the excess Ca 2+ from the mineralization reaction caused decrease in pDNA binding.…”

“…69 It is also proposed that specific pDNA and polyplex interactions with Ca 2+ resulting in smaller polyplex size might be one reason for improved transfection efficiency. 57 We believe that mineralization of the polyplexes could enable new applications based on the robustness of the mineral shell and specific mineral biointeractions. The effects observed with mineralized viruses in vivo are perhaps what could be expected to be achieved with the synthetic vector, considering that these properties are believed to be material-specific.…”

“…Indeed, there are studies that suggest that an increase in cytosolic Ca 2+ can increase endocytosis rates. , The effect of calcium has been recently highlighted in bone-marrow-derived stem cells transfected with PEI/pDNA polyplexes . It is also proposed that specific pDNA and polyplex interactions with Ca 2+ resulting in smaller polyplex size might be one reason for improved transfection efficiency …”

MicroRNA miR-27b-3p regulate microglial inflammation response and cell apoptosis by inhibiting A20 (TNF-α-induced protein 3)

“…Following 24 h treatment of the microglial cells with 100 ng/mL LPS, we conducted vector construction and transfection in mouse microglial cells [ 32 ]. The miR-27b-3p mimics, lentiviral recombinant vector LV-A20 overexpression, LV-siRNA-A20, miR-27b-3p inhibitor, as well as relevant negative controls (NC) were all sourced (GenePharma, China).…”

Nanotechnology-enabled immunoengineering approaches to advance therapeutic applications

“…In another study, Richter et al developed a block copolymer micelle incorporating lipoic acid into poly{[2-(dimethylamino)ethyl methacrylate] 101 - b -[ n -(butyl methacrylate) 124 - co -(lipoic acid methacrylate) 22 ]} (p(DMAEMA 101 - b -[ n BMA 124 - co -LAMA 22 ])) and showed ~ 29% transfection efficiency in K-562 cells while cells transfected with pDMAEMA showed negligible expression of the reporter [ 102 ]. Ayyadevara et al had found that the mere addition of Ca 2+ could increase the transfection of polyplex into Jurkat cells compared to polyplex-only control, while other cations such as Na + or Mg 2+ did not lead to such effect [ 103 ]. The increase in transfection was partially attributed to the increased association of polyplex with the cell membrane.…”