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Survivin specified small interfering RNA-CLIO-Cy5.5.


Zhang H.


Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2008 Apr 17 [updated 2008 May 12].


Ribonucleic acid interference (RNAi) modulates intracellular activation via the use of small interfering RNA (siRNA) (1). RNAi suppresses gene expression through degradation of a specific, targeted mRNA, which leads to gene silencing. siRNA is a 21-23 nucleotide (nt) double-stranded RNA (dsRNA) with symmetric 2-3nt 3’ overhangs and 5’-phosphate and 3’-hydroxyl groups so that it is recognized by an RNAse III enzyme (2). In general, intracellular siRNA undergoes 5’-phosphorylation to unwind the RNA duplex, followed by association with RNA-induced silencing complex (RISC) (1). Then the activated RISC and the unwound anti-sense strand of the siRNA interact with the mRNA target to generate single site-specific cleavage at the mRNA target. The efficiency of gene silencing primarily relies on the optimal incorporation of siRNA into RISC and its stability in RISC, as well as a perfect complementary base pairing with the mRNA target (3). Mismatches can abolish the target degradation, the mRNA cleavage, and the RISC turnover. The high specificity in gene silencing makes siRNA a popular research tool for various gene-inactivation studies such as differentiation, apoptosis, and tumorigenesis (3). siRNA is also used in therapeutic applications to identify drug targets and to characterize gene functions in vivo without the use of gene knockout mice (4). Formulation of siRNAs with compounds to promote transit across cell membranes is being developed to address the major challenge of cellular delivery of siRNAs (5). Several imaging modalities have been used for localized in vivo delivery of siRNA (6). Survivin is a small protein (142 amino acids, 16.5 kDa) that belongs to the inhibitor of apoptosis protein (IAP) family (7). This type of protein can effectively suppress apoptosis induced by a variety of stimuli, including death receptor activation, growth factor withdrawal, ionizing radiation, viral infection, and genotoxic damage. Survivin comprises a single baculovirus IAP repeat (BIR) domain and an extended COOH-terminal α-helical coiled-coil domain but lacks the RING-finger domain found in the other IAPs. In addition to apoptosis, survivin is involved in many essential cellular functions, such as cell division, cell stress response, and surveillance checkpoints. Survivin is strongly expressed in the majority of human tumor types, including lung, breast, colon, gastric oesophageal, pancreatic, liver, bladder, uterine, and ovarian cancers, large-cell non-Hodgkins lymphomas, leukemias, neuroblastomas, brain tumors, pheochromocytomas, soft tissue sarcomas, melanomas, and other kinds of cancers (8). As a new target in cancer treatment, inhibition of survivin expression can be carried out through the use of anti-sense oligonucleotides, ribozymes, and siRNA (8). Survivin-specified siRNA (siSurvivin) is designed to target the anti-apoptotic gene Birc5 that encodes survivin (9). siSurvivin-crosslinked iron oxide nanoparticles (CLIO)-Cy5.5 (siSurvivin-CLIO-Cy5.5) is a magnetofluorescent nanoparticle used for multimodal imaging of the delivery and silencing of siSurvivin in tumors (6). This agent consists of four components: five siSurvivins to target survivin mRNA, three fluorescence probes (Cy5.5) for optical imaging, four myristoylated polyarginine peptides (MPAP) for mediating transportation to the cytoplasm, and an iron oxide nanoparticle core for enhancing magnetic resonance imaging (MRI) contrast and delivering siSurvivin to tumors. The siSurvivin is linked to the magnetic nanoparticles by a stable thioether bond without compromising silencing efficiency. Cy5.5 is a cyanine dye consisting of two quaternized heteroaromatic bases (A and A’) joined by a polymethine chain with five carbons (10), and it is directly bound to the nanoparticles. This dye possesses high quantum yield, good chemical stability, easy conjugation, and high sensitivity (mole extinction coefficient, ~250,000 mol/cm) (11, 12). As a membrane translocation module, MPAP has a hydrophobic 14-carbon moiety of myristic acid (Myr, -C(=O)-(CH2)12-CH3) linked to a polyarginine peptide to generate Myr-Ala-(ARg)7-Cys-CONH2 (13). MPAP can cross the cellular membrane of live cells efficiently and target the cytoplasm without registered toxicity (13). The nanoparticle contains an icosahedral core of superparamagnetic crystalline Fe3O4 (magnetite) that is caged by epichlorohydrin cross-linked dextran and functionalized with amine groups (CLIO-NH2) (14). They have a high magnetic susceptibility to induce a significant magnetization inside a magnetic field. This creates microscopic field gradients that diphase nearby protons and causes a shortening of T2 relaxation times (15). Enhanced permeability and retention effects in tumors and an increased fluid-phase endocytosis in tumor cells result in the accumulation of magnetic nanoparticles in the tumors (6). With the assistance of MPAP, sufficient siSurvivin can be delivered to the tumors. siSurvivin-CLIO-Cy5.5 allows for fine resolution (10–100 μm) and unlimited depth penetration of MRI with the high sensitivity (10-9–10-17 mol/L) and the short acquisition times of optical imaging (6).

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