Jose Mario Barichello

Global Gene Expression Profiling in Cultured Cells Is Strongly Influenced by Treatment with siRNA–Cationic Liposome Complexes

PurposeThe purpose of this study is to determine if the treatment with siRNA-lipoplexes significantly influences on global gene expression in the treated cells.MethodsWe investigated global gene expression in a HT1080 cell line by a cDNA microarray. We also evaluated the effect of lipofection on global gene expression by determining the change of the expression of an exogenous gene, green fluorescence protein (GFP), and also determined treatment-related cytotoxicity.ResultsTreatment of the cells with either siRNA-lipoplexes or cationic liposomes altered the expression of approximately 2,500 genes. When lipoplexes containing non-specific siRNAs were used, GFP expression was enhanced. In this case the effect was independent on the dose and type of siRNA in the formulation. By contrast, when lipoplexes containing a specific siRNA against GFP was used, GFP expression was markedly diminished. These results clearly indicate that an efficient reduction of a targeted gene expression by a specific siRNA is accompanied by a significant alteration of the expression of numerous non-targeted genes. In addition, treatment-related cytotoxicity increased with siRNA- and cationic lipid-doses, but was not dependent on siRNA type.ConclusionNon-specific effects of siRNA-lipoplexes may either enhance, attenuate or even fully mask the desired outcomes of siRNA-based biochemical studies and therapies.

Complexation of siRNA and pDNA with Cationic Liposomes: The Important Aspects in Lipoplex Preparation

In the last two decades, cationic liposomes have been investigated as vehicles for nucleic acids [plasmid DNA (pDNA) and small interfering RNA (siRNA)] delivery in vitro and in vivo. The formation of cationic liposomes-nucleic acids complexes, termed lipoplexes, depends on a number of experimental variables. The quality of the nucleic acid and the cationic liposome as well as the selection of diluents for diluting the concentrated stocks strongly affect the resulting lipoplexes and their efficiency of gene-expression or gene-silencing effect following transfection. In addition, the molar ratio of cationic lipid nitrogen (N) to siRNA or pDNA phosphate (P) (N/P ratio) influences the final characteristics of the lipoplexes, such as size, surface zeta potential, and reproducibility, thereby reflecting their efficiency following transfection. The methods presented in this chapter could be helpful to obtain reliable and reproducible lipoplexes and experimental results.

Agitation during lipoplex formation improves the gene knockdown effect of siRNA

The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.

Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes.

We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA - responsible for the characteristic bands of the A-form helix - and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.

Combined effect of liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate in rat skin

In this report, we investigated the combined effect of drug liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate (ISN) in rat abdominal skin in vitro. Occlusive application of both liposomal and aqueous ISN solution, with and without addition of 5% glycerol, showed that drug liposomalization and addition of glycerol has far-reaching implications for ISN permeation and accumulation in 4 and 8 weeks old rat abdominal skin. Using 8 weeks old rat abdominal skin, the optimal concentration of glycerol to be added to liposomal ISN was found to be 5%. The ISN mean values permeated through and accumulated in stripped 8 weeks old rat abdominal skin from those formulations described above were not significant different, which might indicate the combined effect of glycerol and liposomal ISN resides solely in the stratum corneum (SC). Based on previous reports, the enhancement effect of glycerol might be due to an increase in the SC hydration, and perhaps due to subtle changes in the lipid organization caused by penetration of liposomal lipids within the SC intercellular spaces. These data might provide evidence that glycerol action on SC is useful to facilitate skin permeation and accumulation of drugs formulated in liposome.

Argonaute2 is a potential target for siRNA-based cancer therapy for HT1080 human fibrosarcoma

Small interfering RNAs (siRNAs) are small RNA molecules that have a potent, sequence-specific gene silencing effect and therefore show promise for therapeutic use as molecular-targeted drugs for the treatment of various genetic diseases, including cancer. The aim of the present study was to evaluate whether Argonaute2 (Ago2) is a therapeutically effective target for siRNA-based cancer therapy. Ago2 is the key protein in mammalian RNAi and is also known as the only member of the Ago family that mediates the microRNA (miRNA)-dependent cleavage of targeted mRNAs. It is assumed that these unique properties of the Ago2 protein can play a central role in the regulation of the miRNA pathway and subsequent translational inhibition of miRNA-targeted mRNAs, including cell survival and cancer progression. To assess its therapeutic effect, siRNA against Ago2 (Ago2-siRNA) was transfected into HT1080 human fibrosarcoma cells, which are malignant cancer cells. Ago2 gene silencing resulted in the inhibition of cell growth and the induction of apoptosis and G0/G1 arrest in the cell cycle. In addition, Ago2 knockdown induced morphological changes and actin stress fiber formation in the cells. The results of a microarray study showed that Ago2 suppression stimulated several crucial genes related to apoptosis, the cell cycle, immune response, cell adhesion, metabolism, etc. Repeated intratumoral injection of Ago2-siRNA/cationic liposome complex induced tumor growth suppression in an HT1080 xenograft model. These results suggest that the suppression of the Ago2 gene may be useful for the inhibition of cancer progression and that Ago2 may be a desirable target for siRNA-based cancer therapy.