Alexa Fluor 680-labeled transferrin-cationic (NBD-labeled DOPE-DOTAP) liposome-encapsulated gadopentetate dimeglumine complex (TfNIR-LipNBD-CA complex) is a dual (multimodality) molecular imaging probe with fluorescent and magnetic properties that can be used for imaging tumors with overexpressed transferrin (Tf) receptors (1). Alexa Fluor 680 is a near-infrared (NIR) fluorescence dye with an absorption maximum of 679 nm, an emission maximum of 720 nm, and an extinction coefficient of 180,000 cm−1M−1 (2). Gadopentetate dimeglumine (Gd-DTPA) is a water-soluble paramagnetic contrast agent approved by the United States Food and Drug Administration for contrast enhancement in magnetic resonance imaging (MRI) (3).
Tf is part of a family of proteins that includes serum Tf, ovotransferrin, and lactoferrin (4). Serum Tf is a monomeric glycoprotein (molecular mass, 80 kDa) that binds Fe3+ for delivery to vertebrate cells through receptor-mediated endocytosis (1). The Tf receptor (TfR) mediates the internalization of iron-loaded Tf into cells (4, 5). The TfR (CD71) is a type II transmembrane glycoprotein and is found primarily as a homodimer (molecular mass, 180 kDa). It also contains other growth regulatory properties in certain normal and malignant cells. The elevated levels of TfR in some malignancies (e.g., 74% breast carcinomas, 76% lung adenocarcinomas, and 93% lung squamous cell carcinomas) and the extracellular accessibility of this molecule make TfR a potential molecular target for cancer imaging or therapy.
Liposomes (Lips) are small nontoxic vesicles composed of lipid bilayers enclosing aqueous volume, and they are versatile carriers of both therapeutic drugs and imaging agents (5-7). Although Lips are naturally taken up by the reticuloendothelial system, the size, charge, and surface of Lips can be modified for targeting purposes. Because of the intrinsically low sensitivity of MRI and the low penetration of light, optical and MRI multifunctional probes have been developed as one of the possible approaches to enhance the clinical and research applications of both imaging modalities (1, 8, 9). Shan et al. (1) reported the preparation of a dual probe with fluorescent and magnetic properties based on Lips targeting to TfR-overexpressed tumors. In this TfNIR-LipNBD-CA complex, Alexa Fluor 680-labeled Tf was linked on the surface of cationic Lips with Gd-DTPA encapsulated inside the vesicles.
The TfNIR-LipNBD-CA complex was synthesized with the Alexa Fluor 680 conjugate of human Tf (TfNIR), cationic Lips (LipNBD) and commercially available Gd-DTPA (CA) (1). The cationic LipNIB used the green fluorescent formula of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a ratio of 1:1 (w/w) plus 0.1% DOPE-N-(7-nitro-2-1,3-benzoxadiazole-4-yl) (NBD-DOPE). Briefly, the lipids were mixed in chloroform and then dried under a nitrogen stream. The LIPNBD-CA was formed by reconstituting the dried lipid mixture with 50 μl water containing 12 μl of Gd-DTPA (commercial formulation of 469.01 μg/μl). The LipNBD-CA was homogenized and incubated for 10 min. The Lip volume was adjusted to 175 μl with water and then sequentially downsized by sonication (80–90 W, 10 min) and filtration with decreasing pore diameter from 0.2 to 0.1 μm. Finally, TfNIR (5 mg/ml) was added and incubated for at least 10 min. Unencapsulated CA and free TfNIR were removed by gel filtration chromatography. The final Lip:Tf:Gd-DTPA ratio was 10:12.50:0.56 (nmol:μg:mg).
In Vitro Studies: Testing in Cells and Tissues
Shan et al. (1) conducted cellular uptake tests of Tf-LipNBD-NIR dye with TfR-overexpressed MDA-MB-231-luc human breast cancer cells. In this study, Tf was not labeled with Alexa Fluor 680. Instead, Alexa Fluor 680 (red dye) was encapsulated by LipNBD (green fluorescent) so that the encapsulated reagent (the red dye in place of CA) and the Lip could be observed by confocal microscopy. After incubation with the cells, both the green LipNBD and the encapsulated red dye were observed in the cell cytoplasm as early as 5 min. The fluorescent intensity (FI) within the cytoplasm increased gradually and reached a maximum at ~1 h. The LipNBD and red dye then accumulated again to form multiple endosomes at the peripheral area of the cytoplasm. The authors suggested that this might represent the release or degradation of the probe through the action of lysosomal enzymes. In comparison, when the red dye alone was incubated with the cells, no cellular uptake was observed. The efficiency of the uptake was quantified on cell pellets after 1 h of incubation. The red dye FI values (p/s/cm2/steradian (sr) × 109; n = 3) were 6.88 ± 0.59, 4.99 ± 0.51, and 0.23 ± 0.006 for Tf-LipNBD dye, LipNBD -dye, and dye alone, respectively. The green dye FI values (p/s/cm2/sr × 107; n = 3) were 2.03 ± 0.14, 1.64 ± 0.09, and 1.10 ± 0.13 for Tf-LipNBD-dye, LipNBD-dye, and dye alone, respectively. In the blocking study in which the cells were pretreated with unlabeled Tf (three-fold higher amount) before incubation with Tf-LipNBD dye, the red dye FI value decreased from 3.42 × 109 p/s/cm2/sr to 2.45 × 109 p/s/cm2/sr with a 65.6% blockage of dye uptake. The green dye FI value for LipNBD dye decreased from 3.45 × 107 p/s/cm2/sr to 2.57 × 107 p/s/cm2/sr with a 71.0% blockage of LipNBD uptake.
In another study, where TfNIR-LipNBD-CA was incubated with the cells, optical imaging and confocal microscopy confirmed that both TfNIR and LipNBD were colocalized within cell cytoplasm after 5 min of incubation (1). To measure the presence of CA within the cells, MRI (400-MHz NMR spectrometer) was also performed on cell pellets obtained from cells incubated with TfNIR-LipNBD-CA. The T1 relaxation times (n = 3) were 408.1 ± 13.8 ms, 374 ± 17.3 ms, and 366 ± 17.1 ms for cells incubated with CA, LipNBD-CA, and TfNIR-LipNBD-CA, respectively.
Shan et al. (1) evaluated the tumor signal enhancement in nude mice (n = 10) bearing the subcutaneous MDA-MB-231-luc human breast cancer (0.4–1.2 cm diameter). Each mouse received 200 μl of TfNIR-LipNBD-CA (containing 12 μl Gd-DTPA) by i.v. injection. MRI imaging using a 400-MHz NMR spectrometer and a T1-weighted spin-echo sequence (repetition time = 800 ms, echo time = 11.4 ms) showed significant tumor contrast enhancement as early as 10 min and reached a maximum at 90–120 min. The enhancement appeared to be more heterogeneous in larger tumors but more uniform in smaller tumors. Pathology results showed that the highly enhanced regions represented the more actively proliferating tumor cells. The weakly enhanced areas contained dying cells and the necrotized regions. Giving the CA alone enhanced the tumor contrast only slightly. The CA enhancement started from the peripheral area to the center and reached the maximum in 30–60 min. CA containing Lip without linkage to Tf showed an even weaker signal enhancement. No blocking study was performed.
Optical imaging of TfNIR-LipNBD-CA based on TfNIR showed clear tumor signal as early as 10 min and reached a maximum at 90–120 min (1). The FI was related to the tumor sizes and showed detectable FI in larger tumors (>0.8 cm diameter) after 2 days. The FI of LipNBD was too weak to be detected. The probe was rapidly distributed throughout the body. It was taken up by well-perfused organs and then rapidly washed out. The probe activity remained in the tumor and was not washed out. The tumor/contralateral muscle ratios varied from 1.3 to 3.4 within 10 min to 48 h. This ratio appeared to be dependent on tumor sizes. Small tumors (<3 mm diameter) showed less FI than the bigger tumors. In comparison, administration of the red dye containing Lip without linkage to Tf showed no tumor signal enhancement.
Other Non-Primate Mammals
No publication is currently available.
No publication is currently available.
No publication is currently available.
NCRR 2G12RR003048, NIH 5U54CA091431.
- Shan L., Wang S., Sridhar R., Bhujwalla Z.M., Wang P.C. Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts. Mol Imaging. 2007;6(2):85–95. [PubMed: 17445503]
- Berlier J.E., Rothe A., Buller G., Bradford J., Gray D.R., Filanoski B.J., Telford W.G., Yue S., Liu J., Cheung C.Y., Chang W., Hirsch J.D., Beechem J.M., Haugland R.P., Haugland R.P. Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem. 2003;51(12):1699–712. [PubMed: 14623938]
- Runge V.M. Safety of approved MR contrast media for intravenous injection. J Magn Reson Imaging. 2000;12(2):205–13. [PubMed: 10931582]
- Daniels T.R., Delgado T., Rodriguez J.A., Helguera G., Penichet M.L. The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin Immunol. 2006;121(2):144–58. [PubMed: 16904380]
- Daniels T.R., Delgado T., Helguera G., Penichet M.L. The transferrin receptor part II: targeted delivery of therapeutic agents into cancer cells. Clin Immunol. 2006;121(2):159–76. [PubMed: 16920030]
- Cheng K.T., Seltzer S.E., Adams D.F., Blau M. The production and evaluation of contrast-carrying liposomes made with an automatic high-pressure system. Invest Radiol. 1987;22(1):47–55. [PubMed: 3818235]
- Hatakeyama H., Akita H., Maruyama K., Suhara T., Harashima H. Factors governing the in vivo tissue uptake of transferrin-coupled polyethylene glycol liposomes in vivo. Int J Pharm. 2004;281(1-2):25–33. [PubMed: 15288340]
- Veiseh O., Sun C., Gunn J., Kohler N., Gabikian P., Lee D., Bhattarai N., Ellenbogen R., Sze R., Hallahan A., Olson J., Zhang M. Optical and MRI multifunctional nanoprobe for targeting gliomas. Nano Lett. 2005;5(6):1003–8. [PubMed: 15943433]
- Kircher M.F., Mahmood U., King R.S., Weissleder R., Josephson L. A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res. 2003;63(23):8122–5. [PubMed: 14678964]
This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.
Created: October 18, 2007; Last Update: December 17, 2007.
National Center for Biotechnology Information (US), Bethesda (MD)
Cheng KT, Wang PC, Shan L. Alexa Fluor 680-labeled transferrin-cationic (NBD-labeled DOPE-DOTAP) liposome-encapsulated gadopentetate dimeglumine complex. 2007 Oct 18 [Updated 2007 Dec 17]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.