Gene Silencing via RNAi and siRNA Quantification in Tumor Tissue Using MEND, a Liposomal siRNA Delivery System

Abstract

Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5' RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.

The Polyethyleneglycol Dilemma: Advantage and Disadvantage of PEGylation of Liposomes for Systemic Genes and Nucleic Acids Delivery to Tumors

“…5 mol%) was sufficient for YSK05-MEND to circulate in the blood, unlike cationic doTAP-MENd. 124) This suggests that ySK05-MENd is a promising system for use in the in vivo delivery of nucleic acids to tumors. As another type of pH-sensitive lipid, we reported SS-cleavable protonactivated lipid-like material (ssPalm), which contains two tertiary amines that are positively charged at acidic pH and a disulfide bond that can be cleaved in a reducing environment.…”

RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal-siRNA system

“…YSK-MENDs were prepared as previously reported [10,11]. Briefly, 1,500 nmol of YSK05, 750 nmol of POPE, 750 nmol chol and 150 nmol PEG-DMG were dissolved in 400 μL of 90% (v/v) aqueous tertiary butanol (t-BuOH).…”

“…5' RACE-PCR for the detection of Cd31 mRNA cleaved by si-Cd31 was carried out as previously reported [11]. Briefly, GeneRacer Adaptor was ligated into cleaved Cd31 mRNA, and then reverse transcribed with Cd31 Gene Specific Primer by SuperScript III (Invitrogen).…”

“…We previously developed a liposomal siRNA system, a multi functional nano-device (MEND) [8,9]. In the past report, the use of a MEND composed of a pH-sensitive cationic lipid, YSK05 (YSK-MEND) caused a significant gene reduction in tumor tissue when intratumorally and intravenously injected into tumor-bearing mice [10,11]. A number of pH sensitive siRNA carriers, such as liposomes [12,13], polyplexes [14] and micelles [15], have been evaluated for use in tumor targeting.…”

“Programmed packaging” for gene delivery

“…We developed a tumor-targeting siRNA delivery system via the enhanced permeability and retention (EPR) effect, which involves the passive accumulation of a long-circulating macromolecule in tumor tissue [38]. We reported that the PEGylated YSK05-MEND retained encapsulated siRNA intact in the circulation and PEGylated YSK05-MEND suppressed a cancer-related gene via RNAi-mediated silencing using 5' rapid amplification and cDNA ends (5'-RACE)-PCR [39]. Likewise, the liver can be targeted by passive accumulation because neutral liposomes can accumulate in the liver through the fenestrae of liver endothelial cells and can be internalized into hepatocytes via a low density lipoprotein receptor through absorption of apolipoprotein E in the bloodstream [40].…”