siRNAdelTM Gold Carrier Particles Optimized for
siRNA and Oligonucleotide Delivery
Supported by NIH Small Business Innovative Research grants, Seashell Technology, LLC has developed a novel family of gold carrier particles optimized for ballistic delivery of siRNA.
The particle surface has been modified for direct immobilization of nucleic acids including oligonucleotides and small interfering RNAs.
The functional delivery of inhibitory concentrations of siRNA has been demonstrated in several model systems, including
GFP expressing cell lines,
brain slices and
The siRNAdelTM gold carrier particles are supplied in three different sizes; 550 nm, 1000 nm and 1600 nm average diameter.
S550ri, S1000ri, S1600ri
Inhibition of Constitutive Green Fluorescent Protein Expression
The mouse C166-GFP endothelial cell line constitutively expresses green fluorescent protein (GFP). Seashell Technology siRNA binding particles were used to deliver both siRNA for GFP and a non-complementary to GFP negative control siRNA into C166-GFP cells. The cells were grown in culture and GFP fluorescence was monitored over a period of 6 days. The data in Figure 1
show that ballistic delivery of the negative control siRNA had no effect on GFP expression (Panel A), whereas delivery of siRNA for GFP resulted in rapid diminution of GFP expression after 24 hours. Consistent with published reports describing the recovery of expression following siRNA treatment, the cells treated with GFP siRNA began to accumulate GFP after 3 days, and greater than 90% of the original expression level was recovered after 6 days in culture. These data demonstrate that siRNA can be effectively delivered in a non-toxic method using Seashell Technology
siRNAdelTM gold carrier particles.
Figure 1: Delivery of siRNA into C166 mouse epithelial cells using Seashell Technology siRNAdelTM gold carrier particles. siRNAdelTM particles that contain either (A) siRNA non-complementary to GFP coding sequence, acting as a negative control, or (B) siRNA complementary to GFP coding sequence, were introduced into C166 cells constitutively expressing GFP using a ballistic device. The GFP fluorescence of the samples, at specified times after bombardment, was determined from digital images. Panels A and B are digital images that show the GFP expression level of monolayer cultures 24 hours following bombardment. A quantitative analysis of percent of "negative" control expression for cells at specified times following treatment with siRNA complementary to GFP coding sequence is shown in Panel C.
Inhibition of Reporter Plasmid Expression
Experiments performed by Denise Dunn in the laboratory of Dr. Don Lo at Duke University have demonstrated the functional activity of Seashell Technology's
siRNAdelTM particles to inhibit protein expression from reporter plasmid constructs co-delivered to rat brain slice explants. The particles are formulated both with siRNA complementary to yellow fluorescent protein (YFP) or non-complementary control RNA, and plasmids expressing YFP and red fluorescent protein from Discosoma (DsRed). Two days post-transfection, the expression levels are determined using fluorescence microscopy. The data of Figure 2 shows that delivery of siRNA complementary to YFP leads to a reduced level of YFP fluorescence. In contrast, non-complementary control siRNA has no detectable effect on the level of YFP fluorescence. These data demonstrate the
gold carrier particles are highly efficient for siRNA delivery and can be used for knockdown of proteins that are expressed at a high level from reporter construct plasmids in tissue explant models.
Figure 2: Delivery of siRNA into rat brain slice explants using siRNAdelTM gold carrier particles. Transfection of YFP siRNA significantly reduces YFP fluorescence (top right panel) on Day 2 following transfection compared to non-complementary control siRNA (top middle panel). The dsRED fluorescence (bottom panels) confirms specificity of silencing and is used as a marker for transfection efficiency and cell viability. Data provided with permission from Don Lo, Duke University.
Inhibition of Endogenous Gene Expression
Seashell Technology's siRNAdelTM gold carrier particles have also been used to knockdown expression of endogenous genes. Using a leech embryonic developmental model, the expression of neuronal guidance factor, netrin was eliminated in a subset of ventral longitudinal muscle cells in the whole embryo (Shefi et. al. Journal of Neuroscience (23):6619-6123).
This work was supported by NIH research grant 2R44EB001624-02A1.
This product is for research use only and is not intended for diagnostic use or to treat, cure or prevent any disease. The product is not available for resale. The safety of this material has not been determined and we recommend that this product and components are handled only by persons trained in laboratory techniques and are used in accordance with good laboratory practice. As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing. The BiolisticTM process is covered by patents owned by Dupont, Inc. and purchase of these products does not convey a license under these patents.