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

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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Survivin specified small interfering RNA-CLIO-Cy5.5

siSurvivin-CLIO-Cy5.5
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: May 12, 2008.

Chemical name:Survivin specified small interfering RNA-CLIO-Cy5.5
Abbreviated name:siSurvivin-CLIO-Cy5.5
Synonym:MN-NIFR-siSurvivin
Agent category:Nucleic acid, small molecule (nanoparticle)
Target:RNAse III
Target category:Enzyme
Method of detection:Magnetic resonance imaging (MRI), near-infrared (NIFR) optical imaging
Source of signal/contrast:Iron oxides, Cy5.5
Activation:No
Studies:
  • Checkbox Rodents
No structure is Currently available in PubChem.

Background

[PubMed]

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).

Synthesis

[PubMed]

The synthesis of siSurvivin-CLIO-Cy5.5 was conducted in multiple steps (6). Initially, monocrystalline iron oxide (MION) was synthesized by neutralization of ferrous salts, ferric salts, and dextran with ammonium hydroxide, followed by ultrafiltration (16). The obtained MION was cross-linked in strong base with epichlorohydrin and then reacted with ammonia to produce CLIO-NH2. Next, N-hydroxysuccinimide ester of Cy5.5 was reacted with the CLIO-NH2 (17). The produced CLIO-Cy5.5 was conjugated with a heterobifunctional cross-linker, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), via N-hydroxysuccinimide ester. Then, Myr-Ala-(ARg)7-Cys-CONH2 MPAPs were attached to this linker via a sulfhydryl reactive pyridyl disulfide residue (pH 7). The produced CLIO(MPAP)-Cy5.5 was coupled with an m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) cross-linker (pH 8.5). At the same time, a commercial siRNA duplex to human survivin was modified with a thiol moiety via a hexyl spacer (5’-S-S-(CH2)6-) for bioconjugation. Finally, the free thiol single-stranded RNA was reacted with the CLIO(MPAP)-Cy5.5 via the MBS cross-linker to produce siSurvivin-CLIO-Cy5.5. The analysis results demonstrated that on average there were three Cy5.5, four MPAP, and five siSurvivin molecules per paramagnetic nanoparticle.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

No publication is currently available.

Animal Studies

Rodents

[PubMed]

Medarova et al. studied the in vivo effect of siSurvivin-CLIO-Cy5.5 in tumors (6). Nude mice (n = 5) were implanted with human colon adenocarcinoma LS174T cells on the right flank. The delivery of siSurvivin-CLIO-Cy5.5 was examined with MRI at 9.4 T; T2-weighted images were collected at 10–14 days after the inoculation, when tumors grew to ~0.5 cm in diameter. siSurvivin-CLIO-Cy5.5 was injected intravenously at a dose of 10 mg Fe/kg (440 nmol siSurvivin/kg) twice per week for 2 weeks. There was a significant drop in T2 relaxation times in tumors after injection of siSurvivin-CLIO-Cy5.5, which was further confirmed by near-infrared optical imaging at Cy5.5 channel. The injection of siSurvivin-CLIO-Cy5.5 did not appear to induce serum interferon-α activation. The specific silencing efficiency of siSurvivin-CLIO-Cy5.5 was examined with in vitro reverse-transcription polymerase chain reaction. The transcript levels of Survivin in the tumors treated with siSurvivin-CLIO-Cy5.5 were 97 ± 2% lower than the levels observed in the controls, which were treated with the parental magnetic nanoparticles; the levels were 83 ± 2% lower than the levels seen in mice treated with the mismatch control siRNA. Frozen sections of 7-mm-thick were used to assess the apoptosis and necrosis in tumors. The level of apoptosis was evaluated by a terminal deoxynucldotidyl transferase dUTP nick end labeling (TUNEL) assay and the level of necrosis was evaluated by hematoxylin and eosin (H&E) staining. Both apoptosis and necrosis were also much higher in the tumors than in the controls.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

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Chiu Y.L. , Ali A. , Chu C.Y. , Cao H. , Rana T.M. Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem Biol. 2004;11(8):1165–75. [PubMed: 15324818]
2.
Dykxhoorn D.M. , Novina C.D. , Sharp P.A. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 2003;4(6):457–67. [PubMed: 12778125]
3.
Fuchs U. , Borkhardt A. The application of siRNA technology to cancer biology discovery. Adv Cancer Res. 2007;96:75–102. [PubMed: 17161677]
4.
Tiscornia G. , Singer O. , Ikawa M. , Verma I.M. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci U S A. 2003;100(4):1844–8. [PMC free article: PMC149921] [PubMed: 12552109]
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Mahmood Ur R. , Ali I. , Husnain T. , Riazuddin S. RNA interference: The story of gene silencing in plants and humans. Biotechnol Adv. 2008;26(3):202–9. [PubMed: 18221853]
6.
Medarova Z. , Pham W. , Farrar C. , Petkova V. , Moore A. In vivo imaging of siRNA delivery and silencing in tumors. Nat Med. 2007;13(3):372–7. [PubMed: 17322898]
7.
Pennati M. , Folini M. , Zaffaroni N. Targeting survivin in cancer therapy. Expert Opin Ther Targets. 2008;12(4):463–76. [PubMed: 18348682]
8.
Zaffaroni N. , Pennati M. , Daidone M.G. Survivin as a target for new anticancer interventions. J Cell Mol Med. 2005;9(2):360–72. [PubMed: 15963255]
9.
Altieri D.C. Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer. 2008;8(1):61–70. [PubMed: 18075512]
10.
Ernst L.A. , Gupta R.K. , Mujumdar R.B. , Waggoner A.S. Cyanine dye labeling reagents for sulfhydryl groups. Cytometry. 1989;10(1):3–10. [PubMed: 2917472]
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Lin Y. , Weissleder R. , Tung C.H. Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjug Chem. 2002;13(3):605–10. [PubMed: 12009952]
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Ballou B. , Fisher G.W. , Hakala T.R. , Farkas D.L. Tumor detection and visualization using cyanine fluorochrome-labeled antibodies. Biotechnol Prog. 1997;13(5):649–58. [PubMed: 9336985]
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Pham W. , Zhao B.Q. , Lo E.H. , Medarova Z. , Rosen B. , Moore A. Crossing the blood-brain barrier: a potential application of myristoylated polyarginine for in vivo neuroimaging. Neuroimage. 2005;28(1):287–92. [PubMed: 16040255]
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Josephson L. , Perez J.M. , Weissleder R. Magnetic nanosensors for the detection of oligonucleotide sequences. Angew. Chem. Int. Ed. Engl. 2001;40:3204–06.
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Bulte J.W. , Brooks R.A. , Moskowitz B.M. , Bryant L.H. Jr, Frank J.A. T1 and T2 relaxometry of monocrystalline iron oxide nanoparticles (MION-46L): theory and experiment. Suppl 1Acad Radiol. 1998;5:S137–40. [PubMed: 9561064]
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Wunderbaldinger P. , Josephson L. , Weissleder R. Crosslinked iron oxides (CLIO): a new platform for the development of targeted MR contrast agents. Suppl 2Acad Radiol. 2002;9:S304–6. [PubMed: 12188255]
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Montet X. , Montet-Abou K. , Reynolds F. , Weissleder R. , Josephson L. Nanoparticle imaging of integrins on tumor cells. Neoplasia. 2006;8(3):214–22. [PMC free article: PMC1578521] [PubMed: 16611415]

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