Surface engineering of gold nanoparticles for in vitro siRNA delivery

Abstract

Cellular uptake, endosomal/lysosomal escape, and the effective dissociation from the carrier are a series of hurdles for specific genes to be delivered both in vitro and in vivo. To construct siRNA delivery systems, poly(allylamine hydrochloride) (PAH) and siRNA were alternately assembled on the surface of 11.8 ± 0.9 nm Au nanoparticles (GNP), stabilized by denatured bovine serum albumin, by the ionic layer-by-layer (LbL) self-assembly method. By manipulating the outmost PAH layer, GNP-PAH vectors with different surface electric potentials were prepared. Then, the surface potential-dependent cytotoxicity of the resultant GNP-PAH particles was evaluated via sulforhodamine B (SRB) assay, while the surface potential-dependent cellular uptake efficiency was quantitatively analyzed by using the flow cytometry method based on carboxyfluorescein (FAM)-labeled siRNA. It was revealed that the GNP-PAH particles with surface potential of +25 mV exhibited the optimal cellular uptake efficiency and cytotoxicity for human breast cancer MCF-7 cells. Following these results, two more positively charged polyelectrolytes with different protonating abilities in comparison with PAH, i.e., polyethylenimine (PEI), and poly(diallyl dimethyl ammonium chloride) (PDDA), were chosen to fabricate similarly structured vectors. Confocal fluorescence microscopy studies indicated that siRNA delivered by GNP-PAH and GNP-PEI systems was better released than that delivered by the GNP-PDDA system. Further flow cytometric assays based on immunofluorescence staining of the epidermal growth factor receptor (EGFR) revealed that EGFR siRNA delivered by GNP-PAH and GNP-PEI exhibited similar down-regulation effects on EGFR expression in MCF-7 cells. The following dual fluorescence flow cytometry assays by co-staining phosphatidylserine and DNA suggested the EGFR siRNA delivered by GNP-PAH exhibited an improved silencing effect in comparison with that delivered by the commercial transfection reagent Lipofectamine 2000.

Gold nanostars coated with neutral and charged polyethylene glycols: A comparative study of in-vitro biocompatibility and of their interaction with SH-SY5Y neuroblastoma cells

“…These, in turn, may be loaded with anionic polymeric species, thanks to electrostatic interactions. In this connection, PAHcoated nanoparticles have been proposed for efficiently loading siRNA drugs in anticancer therapies [33,34]. Being involved in a long term research project for the use of siRNA against hepatocarcinoma, we also included GNS/PEGCOOH further coated with PAH in this study.…”

“…the PAH@GNS/PEGCOOH constructs, see Fig. 2, as PAH is a cationic polymer frequently used for enveloping nanoparticles and further loading the negatively charged NABDs (nucleic acid based drugs) [33,34]. In this perspective, we also checked the ability of the latter construct to be further loaded with a model anionic polymer, polystyrene sulfonate (PSS), see Fig.…”

Biomimetic gold nanocomplexes for gene knockdown: Will gold deliver dividends for small interfering RNA nanomedicines?

“…Alternatively, poly(allylamine hydrochloride) (PAH) has been used for different types of siRNA delivery constructs in order to overcome these barriers [35,125]. An LbL strategy has recently been reported in which bovine serum albumin (BSA) was first reduced by NaBH4, which resulted in denatured BSA (dBSA) [116]. This dBSA carried 35 thiol groups that were used for capping AuNPs [116].…”

“…An LbL strategy has recently been reported in which bovine serum albumin (BSA) was first reduced by NaBH4, which resulted in denatured BSA (dBSA) [116]. This dBSA carried 35 thiol groups that were used for capping AuNPs [116]. Due to the presence of numerous carboxyl groups on dBSA, dBSA-AuNPs were electrostatically coated with PAH to form a cationic layer (PAH-dBSA-AuNPs), which could efficiently bind siRNA (siRNA-PAHdBSA-AuNPs).…”

15 years on siRNA delivery: Beyond the State-of-the-Art on inorganic nanoparticles for RNAi therapeutics

“…When verifying protein knockdown, alternative techniques would need to be used such as western blots [143], flow cytometry [45] and In-Cell westerns [144]. A recent study by Zhao et al reported the development of AuNPs loaded with siRNA against the epidermal growth factor receptor (EGFR) gene, and observed a knockdown efficiency of between 16-38% in MCF- 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 REVIEW NANO TODAY 33 cells via flow cytometry (Figure 16) [145]. The use of the flow cytometer allowed for the distinction between populations of cells, with a better assessment of the siRNA knockdown; in contrast to western blots, which use a pooled protein sample from a cellular population.…”