Pharmaceutical NanotechnologySynthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme
Introduction
Glioblastoma multiforme (GBM) is a highly aggressive malignancy that accounts for approximately 85% of primary brain tumors in adults. Current treatment consisting of surgery, radiation and chemotherapy has had limited success and median survival time is approximately 1 year from diagnosis. A study on seven GBM cell lines showed that these cells have upregulated low density lipoprotein receptors (LDLR)s and that receptor numbers per cell varied between 125,000 and 950,000 receptors (Maletinska et al., 2000). Studies on the distribution of LDLR in normal rat and monkey brain tissue suggest that normal brain tissue, particularly neurons, has relatively low LDLR (Pitas et al., 1987). Thus is appears that the LDLR is a potential molecular target for the selective delivery of anti-tumor compounds to GBM.
Low density lipoprotein (LDL) is the major ligand for the LDLR and it is also the major transporter of cholesterol in the plasma. The cholesterol transported by LDL is used for cell growth and membrane repair. LDL is a 22–27 nm particle composed of a core of hydrophobic lipids, primarily cholesteryl esters with a small amount of triglyceride, and has a surface coat of phospholipids, unesterified cholesterol, and a single molecule of apolipoprotein B-100 (apoB) (Grundy, 1990). ApoB-100 is a 550,000 Da glycoprotein with nine amino acids (3359–3367) serving as the binding domain for the LDL receptor (Segrest et al., 2001). Upon binding to LDLR in clathrin coated pits, LDL is internalized via endocytosis and moves into the endosome where a drop in pH causes the receptor to dissociate from the LDL. The receptor is recycled back to the surface of the cell while the LDL is moved into the lysosome where the particle is degraded (Goldstein et al., 1985). This pathway could be useful for the delivery and concentration of drugs into tumors expressing LDLR.
Tumor cells generally have high cholesterol requirements as they are rapidly dividing cells. Increased LDL requirement and receptor activity has been observed in colon cancer (Niendorf et al., 1995), prostate tumors (Chen and Hughes-Fulford, 2001), adrenal tumors (Nakagawa et al., 1995), hormone unresponsive breast tumors (Stranzl et al., 1997), cancers of gynecological origin (Gal et al., 1981), lung tumor tissues (Vitols et al., 1992), leukemia (Tatidis et al., 2002, Vitols et al., 1994, Vitols et al., 1984, Ho et al., 1978), and malignant brain tumors (Rudling et al., 1990). It was previously suggested that plasma-derived LDL could be used as a drug delivery system for tumors expressing LDLR since its hydrophobic core has the possibility of incorporating lipophilic drugs (Firestone, 1994, Rensen et al., 2001). Drugs have been either directly loaded onto plasma LDL or the core lipids of LDL were replaced with drugs (Callahan et al., 1999, Ji et al., 2002, Chu et al., 2001, Vitols et al., 1985, Firestone et al., 1984, Dubowchik and Firestone, 1995, Vitols et al., 1990). Plasma LDL, however, is less than ideal as a targeting agent since it is difficult to isolate in large quantities and is variable in composition and size. Another approach used reconstituted LDL consisting of a lipid emulsion containing drugs stabilized by purified apoB-100 (Lundberg, 1987, Lundberg, 1994, Lundberg and Suominen, 1984, Masquelier et al., 2006). The apoB-100 protein is difficult to isolate due to its large size and propensity to aggregate and is therefore not useful for generation of large batches of reconstituted LDL.
Recent studies have shown the feasibility of creating a synthetic LDL particle as a replacement for serum in cell culture media using a lipid emulsion and a peptide composed of the LDLR binding domain of apoB (Baillie et al., 2002, Hayavi and Halbert, 2005). This synthetic particle was able to support cell growth by delivering cholesterol to cells via the LDL receptor. We hypothesized that a synthetic nano-LDL (nLDL) particle that mimics the binding and uptake properties of plasma-derived LDL could be useful for targeting GBM cells. In the present report, we describe a synthetic nano-LDL particle composed of a lipid emulsion and a synthetic bifunctional peptide which contains a lipid binding domain and the nine amino acid LDLR binding domain. We demonstrate that these novel particles mimic the behavior of plasma-derived LDL and are capable of targeting the LDLR on GBM cells.
Section snippets
Materials
Triolein (TO), cholesteryl oleate (CO), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), suramin, butylated hydroxytoluene (BHT), and 1% fatty acid free bovine serum albumin (BSA) were obtained from Sigma. Lysotracker Blue DND 22 and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) were obtained from Invitrogen (Molecular Probes). Egg yolk phosphatidyl choline (PC) was obtained from Avanti Polar Lipids. Plasma-derived LDL and high density lipoprotein (HDL) were
Characterization of nLDL
The 29 amino acid bifunctional peptide was observed to be highly water soluble and readily bound to lipid emulsions. Unbound peptide was removed by dialysis; the amount of original peptide remaining bound to nLDL was 82 ± 9% (n = 11). FPLC analysis of the nLDL (Fig. 1) indicated particles were primarily intermediate between LDL and HDL in size. In addition, lipid-poor particles smaller than HDL (fractions 17–19) were also observed.
In order to remove lipid-poor peptide complexes from the lipid-rich
Discussion
High-grade gliomas are difficult to treat since they grow aggressively and islets of cells often remain after surgical excision of the tumor. These residual cells lead to recurrence of the tumor. In recent years the emphasis for treatment of gliomas, as well as other types of tumors, has been the identification of specific molecular targets for the delivery of drugs. Such molecular targets would minimize toxic effects on surrounding normal tissue. The epidermal growth factor receptor (Barth et
Acknowledgements
The authors thank Terren Trott, Chris Rosen, and Peter Wang for their technical assistance with the project and Dr. Robert Ryan for helpful discussions. This work was supported in part by CHORI Institutional Funds and the United States Department of Energy under contracts DE-ACO3-76SF00098 and DE-ACO2-05CH11231.
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