Reduction in the size of layered double hydroxide nanoparticles enhances the efficiency of siRNA delivery

Abstract

Small interfering RNAs (siRNA) are a potentially powerful new class of pharmaceutical drugs for many diseases, while the delivery of unprotected siRNAs is ineffective due to their susceptibility to degradation by ubiquitous nucleases under physiological conditions. Layered double hydroxide nanoparticles (LDHs) have been found to be efficient carriers of anionic drugs and nucleic acids. Our previous research has shown that LDHs (with the Z-average particle size of approximately 110 nm) can mediate siRNA delivery in mammalian cells, resulting in gene silencing. However, short double-stranded nucleic acids are mostly adsorbed onto the external surface and not well protected by LDHs. In order to enhance the intercalation of siRNA into the LDH interlayer and the efficiency of subsequent siRNA delivery, we prepared smaller LDHs (with the Z-average particle size of approximately 45 nm) with an engineered nonaqueous method. We demonstrate here that dsDNA/siRNA is more effectively intercalated into these small LDH nanoparticles, more dsDNA/siRNA is transfected into HEK 293T cells, and more efficient silencing of the target gene is achieved using smaller LDHs. Thus smaller LDH particles have greater potential as a delivery system for the application of RNA interference.

Particle size- and number-dependent delivery to cells by layered double hydroxide nanoparticles

“…The Z-average size here is the intensity weighted mean hydrodynamic diameter of particles (more details can be referred to Malvern Instruments Frequently Asked Question ''What is the Z-average''). Both sLDH and L-LDH NPs have a zeta potential of $40 mV, which is in agreement with earlier reports for LDH [48,54,55].…”

“…The mean fluorescence intensity of cells treated with sLDH-5%FITC at these concentrations did not change (P5.0 lg/mL), indicating that cellular uptake was saturated under these conditions. Our previous confocal images have confirmed that hexagonal LDH NPs are taken up by various cell types and are mostly located in the cytoplasm [48,54,58,59].…”

“…Small LDH (sLDH) NPs were prepared by a non-aqueous precipitation method [48,49]. Briefly, 10 mL methanol (Fluka, P99.0%) solution containing 6 mmol of Mg(NO 3 ) 2 Á6H 2 O and 2 mmol of Al(NO 3 ) 3 Á9H 2 O (Fluka, >99.0%) was added drop-wise to a 40 mL solution of methanol containing 16 mmol NaOH (Fluka, >97.0%, pellets) under vigorous stirring.…”

“…Choi and Choy's group studied the cellular uptake behaviours and mechanisms of LDH NPs (with sheet-like shape), finding 50 nm LDH NPs showed higher cellular uptake than 100-200 nm and 350 nm ones [46,47]. Recently we found 40 nm LDH NPs can carry 1-4 times more dsDNA than 100 nm ones at LDH:DNA mass ratio of 1:1 and 5:1, contributing to enhanced gene delivery [48,49].…”

“…Some have used the mass concentration while others used the particle number concentration. The third one is the delivery efficiency, often expressed as the NP number [6,16,43,51], the mass amount (pg/cell) [16,41], or the relative amount of labelled fluorescence dye [48,52] taken up by each cell. In addition, the difference in the shape and surface property from a variety of materials and surface modified functional groups may also cause the inconsistency.…”