NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

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

Cover of Molecular Imaging and Contrast Agent Database (MICAD)

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

Show details

Quinolinium, 1,1'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3H)-benzothiazolylidene)-1-propen-1-yl]-,iodide (1:4)

, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: May 2, 2012.

Chemical name:Quinolinium, 1,1'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3H)-benzothiazolylidene)-1-propen-1-yl]-,iodide (1:4)Image TOTO3.jpg
Abbreviated name:TOTO-3
Agent Category:Compounds
Target:Nucleic acids (DNA and RNA)
Target Category:Nucleic acids
Method of detection:Optical imaging
Source of signal / contrast:TOTO-3
  • Checkbox In vitro
  • Checkbox Rodents
Structures of TO-PRO-3 and TOTO-3 (1).



Quinolinium, 1,1'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3H)-benzothiazolylidene)-1-propen-1-yl]-,iodide (1:4), abbreviated as TOTO-3, is a cell-impermeable cyanine acid dye that has a high affinity for nucleic acids (1, 2). TOTO-3 shows weak fluorescence in solution but generates significantly increased quantum yield (100- to 1,000-fold increase) when binding to DNA or RNA (1, 3, 4). Furthermore, TOTO-3 has a relatively long wavelength (maximum excitation/emission, 640/660 nm). These optical properties make it suitable to assess plasma membrane integrity and to detect DNA with optical imaging (2, 5). TOTO-3 is the dimeric form of quinolinium, 4-[3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-1-[3-(trimethylammonio)propyl]-,diiodide (TO-PRO-3).

To date, chemotherapy remains the treatment of choice for advanced cancer. However, anticancer agents are often much less efficient against tumor cells in vivo compared with their effect in vitro. Several characteristics of tumors cause this inefficiency, such as high interstitial pressure, increased collagen fibers in the extracellular matrix, acidic microenvironment, and cell membrane (2). Ultrasound has been shown to facilitate the penetration of drugs into tumors by several mechanisms, such as ultrasound-induced hyperthermia, which can enhance drug accumulation in tumor tissue and trigger the release of the drug payload from temperature-sensitive carriers, and ultrasound-induced pores in cell membrane, which can improve cell internalization by increasing the membrane permeability (6, 7).

Molecular imaging has been investigated to monitor and quantify the processes of ultrasound-mediated intracellular drug delivery (2). For this purpose, contrast agents have usually been coupled to or loaded into drug carriers. However, coupling contrast agents to small molecules, as with chemotherapeutic drugs, may change their pharmacokinetics and pharmacodynamics (2). An alternative strategy is co-delivery of drug-mimicking contrast agents and small molecule drugs. Deckers et al. tested the feasibility of using a fluorescent nuclear acid staining dye (TOTO-3) as a model drug to monitor ultrasound-mediated delivery in real time (2). In vivo fluorescence imaging demonstrated that an optical contrast agent with characteristics similar to an anti-cancer drug may be used for in vivo monitoring of the drug delivery process in real time (2). This chapter summarizes the data obtained with TOTO-3.



TOTO-3 is commercially available.

In Vitro Studies: Testing in Cells and Tissues


Deckers et al. analyzed the cell internalization of TOTO-3 after incubation with mouse rectal carcinoma line CMT-93 cells without or with preliminary permeabilization with 0.2% Tween (2). When cells were permeabilized before incubation with TOTO-3, strong fluorescent staining of the cytoplasmic RNA and nucleoli was observed. On the contrary, the TOTO-3 fluorescence was not detectable in cells without permeabilization, confirming that TOTO-3 is a cell-impermeable dye.

Animal Studies



Deckers et al. tested the feasibility of TOTO-3 as a smart probe for in vivo monitoring of ultrasound-mediated intracellular drug delivery (2). RAG 2/g_C mice bearing a CMT-93 tumor on both flanks (n = 8 mice) were first co-injected with TOTO-3 and microbubbles directly into both tumors, and ultrasound (1.5 MHz for 1 min) was then applied to one tumor with the second tumor serving as a control. The time course of ultrasound-mediated intracellular delivery of TOTO-3 was measured with fluorescence imaging. A distinct difference in the fluorescence intensity was observed between the ultrasound-treated and control tumors at 2 h and 4 h after ultrasound application. At these time points, two-fold higher mean signal intensity and an even larger difference in the total and maximum intensities were observed in the ultrasound-treated tumor.

Epifluorescence microscopy was performed on the excised tumor sections to demonstrate the internalization of TOTO-3 (2). Relatively large areas with TOTO-3 signal were observed in the ultrasound-exposed tumor sections. In contrast, control tumor sections typically showed no fluorescence or only single cells with TOTO-3 uptake. The overall signal intensity was quite weak in both cases.

The investigators hypothesized that phagocytic cells, such as macrophages, caused ultrasound-independent uptake of TOTO-3, which may explain the observed fluorescence in the control tumors (2). Anti-CD68 staining revealed a significant presence of macrophages in both control and ultrasound-treated tumors. However, TOTO-3 signal was strongly associated with macrophages only in the control tumors. Ultrasound-independent uptake was further confirmed with isolated intraperitoneal macrophages from female Rag-gamma mice after incubation of TOTO-3 with the macrophages (2). Flow cytometry and confocal microscopy confirmed that macrophages could take up TOTO-3 in an ultrasound-independent manner. The peak fluorescent intensity was in the range of 1 × 103 to 1 × 104, comparable to the intensity observed for the positive control macrophages (permeabilized with 0.2% Tween before incubation with TOTO-3). The negative control sample (incubation without TOTO-3 and Tween) showed negligible fluorescence signal.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


Milanovich N. et al. Binding of TO-PRO-3 and TOTO-3 to DNA: fluorescence and hole-burning studies. Journal of Physical Chemistry. 1996;100:9181–6.
Deckers R. et al. A fluorescent chromophore TOTO-3 as a 'smart probe' for the assessment of ultrasound-mediated local drug delivery in vivo. Contrast Media Mol Imaging. 2011;6(4):267–74. [PubMed: 21861287]
Martin R.M., Leonhardt H., Cardoso M.C. DNA labeling in living cells. Cytometry A. 2005;67(1):45–52. [PubMed: 16082711]
Suzuki T. et al. DNA staining for fluorescence and laser confocal microscopy. J Histochem Cytochem. 1997;45(1):49–53. [PubMed: 9010468]
Zuliani T. et al. Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: interest for cell death investigations. Cytometry A. 2003;54(2):100–8. [PubMed: 12879456]
Frenkel V. Ultrasound mediated delivery of drugs and genes to solid tumors. Adv Drug Deliv Rev. 2008;60(10):1193–208. [PMC free article: PMC2491332] [PubMed: 18474406]
Khaibullina A. et al. Pulsed high-intensity focused ultrasound enhances uptake of radiolabeled monoclonal antibody to human epidermoid tumor in nude mice. J Nucl Med. 2008;49(2):295–302. [PubMed: 18199622]
PubReader format: click here to try


  • PubReader
  • Print View
  • Cite this Page
  • PDF version of this page (593K)
  • MICAD Summary (CSV file)

Search MICAD

Limit my Search:

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to pubmed

Related citations in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...