Background

[PubMed]

The saposin C (SapC)-dioleylphosphatidylserine (DOPS) nanovesicle coupled with iron oxide, abbreviated as SapC-DOPS-IO, is a contrast agent developed by Kaimal et al. for extracellular phospholipid (PS)-targeted magnetic resonance imaging (MRI) (1).

Saposins are a group of water-soluble lysosomal glycoproteins (SapA, SapB, SapC, and SapD) with molecular weights of 8–11 kDa (1-3). They are generated by the proteolytic processing of the common precursor prosaposin. Saposins localize primarily in the lysosomes and are required for the catabolism of glycosphingolipids (4, 5). The four saposins have a high degree of structural similarity and share lipid-binding and membrane-perturbing properties; however, they behave differently and exhibit different specificities (2, 3). SapC is the second saposin to be discovered, and it binds to membranes in a pH-controlled manner (6). SapC can directly activate enzymes and stimulate the hydrolysis of glycocerebroside and galactocerebroside (7). Deficiency of SapC leads to an abnormal juvenile form of Gaucher disease with accumulation of glucosylceramide in various organs, including the brain (5).

SapC preferentially interacts with unsaturated, negatively charged PS such as DOPS at acidic pH (6, 8). PS is present in all cells and constitutes ~2%–10% of total cellular lipids. In normal tissues, PS is localized in the cell membrane leaflets that face the cytosol. However, in pathological conditions such as tumors, PS translocates to the outer leaflet of the plasma membrane, where it activates and participates in various cellular processes, including apoptosis and necrosis (9). Phagocytes in healthy tissues rapidly and efficiently remove the PS-expressing cells and cell remnants, but these PS-expressing cells and cell remnants accumulate in diseased tissues as a result of the activation of the cell death process and the insufficient clearance of cells with externalized PS (9).

Because tumors express abundant PS on the cell surface and have a lower extracellular pH (pH ~6) than normal tissues (pH ~7), the SapC-PS interaction provides a valuable system for targeted tumor imaging and therapy (4, 9, 10). Qi et al. developed a SapC-DOPS nanovesicle system that induced apoptosis of tumor cells in vitro and inhibited growth of neuroblastomas and malignant peripheral nerve sheath tumors in animal models (2). Fluorescently labeled SapC-DOPS (CVM-SapC-DOPS) has been shown to preferentially accumulate in tumor xenografts (1, 2). Kaimal et al. synthesized SapC-DOPS-IO by encapsulating IO particles into the SapC-DOPS nanovesicles, and they showed that SapC-DOPS-IO was effectively detected in tumors with the use of MRI (1). These studies indicate that SapC-DOPS nanovesicles are promising as a new and robust theranostic agent for cancer-selective detection, visualization, and therapy (1, 2). This chapter summarizes the data obtained with SapC-DOPS-IO. The data obtained with CVM-SapC-DOPS are summarized in another chapter in MICAD.

Synthesis

[PubMed]

Qi et al. described the synthesis of SapC-DOPS nanovesicles in detail (2). Briefly, recombinant human SapC was expressed in Escherichia coli cells. Pure SapC was then mixed with lipids in acid buffer (pH 5). The protein–lipid mixture was gently sonicated and ultracentrifuged to produce SapC-DOPS nanovesicles in pellet form. No SapC was detected in the supernatant fraction, indicating a very high loading/coupling efficiency. The sonicated SapC-DOPS complexes contained monodispersed, unilamellar vesicles with a mean diameter of ~190 nm.

To encapsulate IO particles (ferumoxtran-10; ~20 nm in diameter) in nanovesicles, Kaimal et al. first oxidized the dextran coating on the IO particles to generate aldehyde groups (1). The oxidized IO particles were then mixed with SapC and lipids to generate SapC-DOPS-IO nanovesicles. The surface aldehydes of the IO particles could form covalent Schiff bonds at high pH with amines of DOPS. The vesicles were sized by passing through a 200-nm polycarbonate membrane. To remove the IO particles attached to the outer surface of the vesicles, the solution was dialyzed against a low pH solution (pH 4.5) of sodium chloride and sodium citrate. The unencapsulated IO particles were removed by passing the solution through a Con-A Sepharose 4B column. The SapC-DOPS-IO nanovesicles were stabilized in 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffered to pH 8 (20 mM final concentration).

The SapC-DOPS-IO nanovesicles were bilayer-spherical in shape and had a mean diameter of ~200 ± 27 nm. The molar ratio of SapC/DOPS and the mean iron concentration in the SapC-DOPS-IO were determined to be 1:10 and 94.8 ± 12.8 μg/ml, respectively. The relaxivity r₂ value was measured to be 101.21 ± 4.9 mM^-1s^-1.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

MRI and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) were performed after incubation of human neuroblastoma cells (CHLA-20) with SapC-DOPS-IO nanovesicles for various times (1). MRI scans of the cells fixed in agarose showed an increasing number of signal voids in cells incubated with SapC-DOPS-IO for longer periods. The R2 and R2* relaxation rates of cells in agarose were 14.64 s^-1 and 26.74 s^-1, respectively, for cells exposed to SapC-DOPS-IO, and 7.84 s^-1 and 11.04 s^-1, respectively, for control cells exposed to free IO particles for 24 h. The average cellular iron content was 1.48, 2.12, and 3 pg/cell for cells incubated with SapC-DOPS-IO for 12, 18, and 24 h, respectively. When the cells were exposed to various concentrations of SapC-DOPS-IO for 12 h, the cell uptake was proportional to the initial concentration in the growth medium.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

References

1.

Kaimal, V., Z. Chu, Y.Y. Mahller, B. Papahadjopoulos-Sternberg, T.P. Cripe, S.K. Holland, and X. Qi, Saposin C Coupled Lipid Nanovesicles Enable Cancer-Selective Optical and Magnetic Resonance Imaging. Mol Imaging Biol, 2010. [PMC free article: PMC4627685] [PubMed: 20838909]

2.

Qi X., Chu Z., Mahller Y.Y., Stringer K.F., Witte D.P., Cripe T.P. Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein. Clin Cancer Res. 2009;15(18):5840–51. [PubMed: 19737950]

3.

Rossmann M., Schultz-Heienbrok R., Behlke J., Remmel N., Alings C., Sandhoff K., Saenger W., Maier T. Crystal structures of human saposins C andD: implications for lipid recognition and membrane interactions. Structure. 2008;16(5):809–17. [PubMed: 18462685]

4.

Wang Y., Grabowski G.A., Qi X. Phospholipid vesicle fusion induced by saposin C. Arch Biochem Biophys. 2003;415(1):43–53. [PubMed: 12801511]

5.

Atrian S., Lopez-Vinas E., Gomez-Puertas P., Chabas A., Vilageliu L., Grinberg D. An evolutionary and structure-based docking model for glucocerebrosidase-saposin C and glucocerebrosidase-substrate interactions - relevance for Gaucher disease. Proteins. 2008;70(3):882–91. [PubMed: 17803231]

6.

Abu-Baker S., Qi X., Newstadt J., Lorigan G.A. Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing 31P and 2H solid-state NMR spectroscopy. Biochim Biophys Acta. 2005;1717(1):58–66. [PubMed: 16289479]

7.

Nieh M.P., Pencer J., Katsaras J., Qi X. Spontaneously forming ellipsoidal phospholipid unilamellar vesicles and their interactions with helical domains of saposin C. Langmuir. 2006;22(26):11028–33. [PubMed: 17154581]

8.

Abu-Baker S., Qi X., Lorigan G.A. Investigating the interaction of saposin C with POPS and POPC phospholipids: a solid-state NMR spectroscopic study. Biophys J. 2007;93(10):3480–90. [PMC free article: PMC2072076] [PubMed: 17704143]

9.

Schutters K., Reutelingsperger C. Phosphatidylserine targeting for diagnosis and treatment of human diseases. Apoptosis. 2010;15(9):1072–82. [PMC free article: PMC2929432] [PubMed: 20440562]

10.

Zhang X., Lin Y., Gillies R.J. Tumor pH and its measurement. J Nucl Med. 2010;51(8):1167–70. [PMC free article: PMC4351768] [PubMed: 20660380]