Gold nanoparticles enhanced electroporation for mammalian cell transfection

Abstract

Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials.

Figure 1

Schematic illustration on the mechanism of AuNPs enhancement on electroporation: (a) The pulse enhancement effect through minimizing the electric voltage consumed by the low conductive electroporation buffer during electroporation. By adding highly conductive AuNPs, more percentage of the overall electric voltage across the two electrodes is allocated on cells to have focused pulses when compared to the use of electroporation buffer alone; (b) localized electroporation when AuNPs are brought to the cell membrane through affinity binding with receptors there. The electric field is converged on the conductive AuNPs and these AuNPs could serve as virtual electrodes to polarize only limited area on the cell membrane when stay nearby.

Figure 2

Gold nanoparticles enhancement on electroporation of K562 cells with a commercial batch electroporation (labeled as “BTX”) system and a home-made semi-continuous microchannel system (labeled as “microchannel”). Panel (a) exhibits fluorescence and phase contrast microscopic images of pGFP plasmid transfection through BTX, BTX with AuNPs, Microchannel, and Microchannel with AuNPs. Panels (b) and (c) are quantitative results of the transfection efficiency (b) and the cell viability (c). The concentration of AuNPs used here is 5X or 0.05 wt% (0.5 mg/mL). n=6 and (***) represents p<0.005.

Figure 3

Simulation of the electric field focusing effect of AuNPs in electroporation. (a) The model and meshes setup for one gold nanoparticle embedded in the membrane of a K562 cell. (b) The calculated electric field lines around the AuNP. (c) The electric field around a transient pore on the cell membrane at the presence of one AuNP around.

Figure 4

Dependence of the pulse enhancement on the size and concentration of free AuNPs: panels (a-b) are the cell viability (a) and the transfection efficiency (b) with an overall pulse strength of 625 V/cm and panels (c-d) are the results with an overall pulse strength of 475 V/cm (panel c: the cell viability; panel d: the transfection efficiency). The blue and red dash lines refer to the cell viability and the transfection efficiency of electroporation with naked DNA at the optimal conditions (675 V/cm, single pulse of 10 ms), respectively. 1X AuNPs refers to 0.01 wt% or 0.1 mg/mL gold content.

Figure 4

Dependence of the pulse enhancement on the size and concentration of free AuNPs: panels (a-b) are the cell viability (a) and the transfection efficiency (b) with an overall pulse strength of 625 V/cm and panels (c-d) are the results with an overall pulse strength of 475 V/cm (panel c: the cell viability; panel d: the transfection efficiency). The blue and red dash lines refer to the cell viability and the transfection efficiency of electroporation with naked DNA at the optimal conditions (675 V/cm, single pulse of 10 ms), respectively. 1X AuNPs refers to 0.01 wt% or 0.1 mg/mL gold content.

Figure 5

Localized electroporation enhancement with Tf-AuNPs: (a) schematics of grafting Tf-AuNPs as virtual electrodes on the cell membrane, (b) the localized enhancement with Tf-AuNPs alone at various binding stages. The AuNPs used here are 20 nm with 1X concentration (0.1 mg/mL).

Figure 6

The combined enhancement of the pulse strength focusing and localized electroporation effects using a mixture of free AuNPs and Tf-AuNPs of various mixing ratios with a total AuNPs concentration of 1X (a) and 5X (b) with the pulse strength of 625 V/cm and 475 V/cm. AuNPs of 20nm were used here.

Figure 7

The confocal microscope images of the cellular uptake of AuNPs before and after electroporation: (a-b) for free FNPs and (c-d) for Tf-FNPs. Images in panels (a) and (c) were taken before electroporation and panels (b) and (d) were after electroporation. FNPs of 10X or 0.1 wt% (1.0 mg/mL) were used in all samples and Tf-FNPs were incubated with cells for 4 hr.