Uptake of the carborane derivative of cholesteryl ester by glioma cancer cells is mediated through LDL receptors.

PURPOSE: This study was to elucidate the mechanism of cellular uptake of cholesteryl 1,12-dicarba-closo-dodecaboranel-carboxylate (BCH), a new anti-cancer carborane derivative of cholesteryl ester, by glioma cancer cells. METHODS: BCH (solubilized with liposomal formulation) was incubated with SF-763 and SF-767 glioma cell lines in the presence of different amounts of monoclonal anti-LDL receptor antibody for cellular uptake studies. Various amounts of lipoprotein deficient serum (LPDS) were also used during the uptake. The effect of calcium ion and low temperature on BCH uptake were investigated. In addition, the transfer of BCH from liposomes to low-density lipoprotein (LDL) particles was determined through gradient ultracentrifugation. RESULTS: BCH uptake by these glioma cells was significantly inhibited by the monoclonal antibody. The uptake by both cell lines was reversely correlated with the amount of LPDS. The presence of calcium ion promoted the BCH uptake, whereas the low temperature decreased the BCH uptake. After 16 h incubation, about 46% of BCH was transferred from liposomes to LDL particles. CONCLUSIONS: These results strongly suggested that the cellular uptake of BCH (in liposomal formulation) by SF-763 and SF-767 glioma cell lines is mediated through LDL receptors.

In vitro gene transfection in human glioma cells using a novel and less cytotoxic artificial lipoprotein delivery system.

PURPOSE: To develop and evaluate a novel artificial lipoprotein delivery system for in vitro gene transfection in human glioma cells. METHOD: Nanoemulsion was formulated with similar lipid compositions present in natural lipoproteins. The oil phase of nanoemulsion was composed of triolein (70%), egg phosphatidylcholine (22.7%), lysophosphatidylcholine (2.3%), cholesterol oleate (3.0%), and cholesterol (2.0%). To replace the surface protein as in natural lipoprotein, poly-L-lysine was modified to add palmitoyl chains at a basic condition and was incorporated onto the nanoemulsion particles through hydrophobic interaction. A model plasmid DNA, pSV-beta-Gal containing a reporter gene for beta-galactosidase was carried by the nanoemulsion/poly-L-lysine particles. The charge variation of soformed complex was examined by agarose gel electrophoresis and zeta potential measurement. In vitro transfection was conducted on human SF-767 glioma cell line using this new system. After standard X-Gal staining, transfected cells were observed under light microscope. The effect of chloroquine on the transfection was examined and, finally, the cytotoxicity of this new system was evaluated in comparison with commercial Lipofectamine gene transfection system. RESULTS: The plasmid DNA was effectively carried by this artificial lipoprotein delivery system and the reporter gene was expressed in the glioma cells. Transfection efficiency was significantly increased by the treatment of chloroquine, indicating that endocytosis possibly was the major cellular uptake pathway. Compared to Lipofectamine system, this new delivery system demonstrated similar transfection efficiency but a much lower cytotoxicity. In the experiment, the cell viability showed up to 75% using this system compared to only 24% using Lipofectamine system. CONCLUSION: A new artificial lipoprotein delivery system was developed for in vitro gene transfection in tumor cells. The new system showed similar transfection efficiency but a much lower cytotoxicity compared with commercial Lipofectamine system.

Synthesis, preformulation and liposomal formulation of cholesteryl carborane esters with various fatty chains.

The elevated expression of LDL receptor on tumor cells provides one attractive approach for targeted drug delivery to tumor cells. Suitable antitumor compounds, however, need to be synthesized and developed which mimic the native cholesteryl esters (as major constituent of LDL) in chemical structure for targeted delivery to tumor cells through the over-expressed LDL receptors. In the present study, new antitumor compounds were designed containing cholesterol, fatty chain and carborane which is used as the antitumor unit. Three new compounds were synthesized with a three-step reaction scheme. Similar to the native cholesteryl esters, these compounds are extremely hydrophobic and, before any further biological studies, suitable liposomal formulations for these new compounds are required. Various liposomal formulations as well as the preformulation characterization of these new compounds were thus examined. The incorporation efficiency of the compounds in liposomes was found to vary significantly depending on the type of fatty chain attached and the ratio of cholesterol:phospholipid used as the excipients of liposomal formulation.

Sequential first-pass metabolism of nortilidine: the active metabolite of the synthetic opioid drug tilidine.

The disposition of nortildine, the active metabolite of the synthetic opioid drug tilidine, was investigated in healthy volunteers in a randomized, single-dose, three-way crossover design. Three different treatments were administered: tilidine 50 mg intravenously, tilidine 50 mg orally, and nortilidine 10 mg intravenously. The plasma concentrations of tilidine, nortilidine, and bisnortilidine were determined and subjected to pharmacokinetic analysis using noncompartmental methods. The systemic bioavailability of tilidine was low (7.6% +/- 5.3%) due to a pronounced first-pass metabolism. The areas under the plasma concentration versus time curves (A UC) of nortilidine were similar following either oral or intravenous administration of tilidine 50 mg (375 +/- 184 vs. 364 +/- 124 ng.h.ml(-1)). AUC of nortilidine was 229 +/- 42 ng.h.ml(-1) after IV infusion of nortilidine 10 mg and thus much greater than after IV tilidine corrected for differences in dose. Nortilidine had a much lower volume of distribution (275 +/- 79 vs. 1326 +/- 477 L) and a somewhat lower clearance (749 +/- 119 vs. 1198 +/- 228 ml/min) than tilidine. About two-thirds of the dose of tilidine was metabolized to nortilidine, although only half of the latter fraction was available in the peripheral circulation. Nortilidine was subsequently metabolized to bisnortilidine. The mean ratio of the AUC of bisnortilidine to nortilidine was 0.65 +/- 0.14 following IV administration of nortilidine but 1.69 +/- 0.38 and 1.40 +/- 0.27 following oral and intravenous administration of tilidine, respectively. The shapes of the plasma concentration-time curves of the metabolites and parent drug declined in parallel, indicating that the disposition of the metabolites is formation rate limited. Thus, although two-thirds of the dose of tilidine is metabolized to nortilidine, only one-third of the dose is available systemically as nortilidine for interaction with the opiate receptors after both intravenous and oral dosing of tilidine. The remaining part of nortilidine is retained in the liver and is subsequently metabolized to bisnortilidine and yet unknown compounds.

VLDL-resembling phospholipid-submicron emulsion for cholesterol-based drug targeting.

The objective of the current study was to develop and evaluate VLDL-resembling phospholipid-submicron emulsion (PSME) as a carrier system for new cholesterol-based compounds for targeted delivery to cancer cells. BCH, a boronated cholesterol compound, was originally developed in our laboratory to mimic the cholesterol esters present in the LDL and to follow a similar pathway of cholesterol transport into the rapidly dividing cancer cells. The VLDL-resembling system was then designed to solubilize BCH, facilitate the interaction with LDL, and thus assist the BCH delivery to cancer cells. BCH-containing PSME was prepared by sonication. Chemical compositions and particle sizes of different PSME fractions were determined. The lipid structure of PSME and location of BCH in the formulation were assessed based on experimental results. Density gradient ultracentrifugation fractionated the emulsion into three particle-size populations with structures and compositions resembling native VLDL. In vitro interaction between PSME and LDL was evident by agarose electrophoresis, as both formed a single band with an intermediate mobility. The transfer of BCH from PSME to LDL was also observed in the presence of other serum components including serum proteins. Cell culture data showed sufficient uptake of BCH in rat 9L glioma cells (> 50 microg boron/g cells). In conclusion, this system has the capability to incorporate the cholesterol-based compound, interact with native LDL, and assist the delivery of this compound into cancer cells in vitro.