Density gradient separation of carborane-containing liposome from low density lipoprotein and detection by inductively coupled plasma spectrometry.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for analyzing the new cholesterol-based compounds (BCH, BCH-Da, BCH-Db and BCH-Dc) in liposomal formulations. Not only the boron compounds but also the phospholipid compositions of the liposome formulation were quantitatively analyzed. Reasonable limit of detection for boron (0.5 µg/ml) and phosphorous (0.09 µg/ml), respectively, was observed. ICP-MS was also utilized for analyzing BCH in a brain distribution study. The detection limit of boron analysis by ICP-MS is at least three orders of magnitude lower than of that of ICP-AES (1 ng B/ml). The method was linear in the range of 500-1 ng B/ml and the linearity correlation coefficient was 1. In addition, an ultracentrifugation method was developed to separate liposomes from low-density lipoprotein (LDL). Factors such as density gradient and size of liposomes were adjusted to optimize separation and it was observed that in conjunction to time, speed and density gradient, size of the liposome also had impact on the separation using centrifugation method. These findings show the importance of ICP-AES as an analytical method for the analysis of element-based compounds encapsulated in phospholipid vesicles.

A new bioimaging carrier for fluorescent quantum dots: phospholipid nanoemulsion mimicking natural lipoprotein core.

Fluorescent quantum dots (semiconductor nanocrystals) have the potential to revolutionize biological imaging, but their use has been limited by difficulties in obtaining quantum dots that are water soluble and biocompatible. The objectives of our research were to develop a methodology for encapsulation of cadnium-selenium (CdSe) quantum dots (QDs) in phospholipid nanoemulsion that mimics the natural lipoprotein core and to study their interactions with cultured non-small cell lung cancer cells (NSCLC). We found that CdSe QDs can be efficiently encapsulated in the phospholipid nanoemulsion. The QD nanoemulsion has a particle size approximately 80 nm and appears physically stable. The QD nanoemulsion interacts well with cells. The intensity of cellular fluorescence imaging increases with the cell incubation time, indicating more QDs were taken up by the cells, respectively. Two types of fluorescence microscopies confirm that QDs are primarily localized in the cytoplasm but not in the nucleus of the cells.

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.

Effective transfection of rabies DNA vaccine in cell culture using an artificial lipoprotein carrier system.

PURPOSE: To evaluate the transfection efficiency in cell culture of rabies plasmid DNA vaccine carried by a novel artificial lipoprotein system. METHODS: Phospholipid nanoemulsions resembling the lipid core of natural lipoproteins were prepared. The artificial lipoprotein carrier system for DNA was constructed by assembling of the nanoemulsion (NE)-palmitoyl-poly-L-lysine (p-PLL)-rabies DNA complex. Agarose gel electrophoresis, zeta potential, and mobility measurement were conducted to determine the surface charge balance in various complex compositions. Transfection and transfection efficiency were examined by fluorescence microscopy and flow cytometry, respectively. RESULTS: The artificial lipoprotein system was successfully constructed and the rabies DNA vaccine was effectively transfected in glioma cell line SF-767. The amount of p-PLL incorporated into the artificial lipoprotein formulations had a significant effect on transfection efficiency. The new system also proved to be more efficient in cellular transfection of rabies DNA vaccine than the commercial lipofectamine formulation. CONCLUSIONS: Effective transfection of rabies DNA vaccine in cell culture can be achieved using the novel artificial lipoprotein carrier system, and the charge balance of the NE-p-PLL-DNA complex appears an important factor.

An automated approach to salt selection for new unique trazodone salts.

PURPOSE: The purpose of this study was to establish an automated approach to salt selection and to search for unique trazodone salts for new applications. METHODS: Automated procedures were developed on a Biomek 2000 automation workstation with stacker and plate reader capabilities. Trazodone was dispensed into 96-well plates, and an automated method was set up to form 104 trazodone salts. Salts were observed under a polarized light microscope to determine crystallinity. After stepwise eliminations, the remaining salts were scaled-up and subjected to differential scanning calorimetry (DSC), powder x-ray diffraction (PXRD), hygroscopic, pH-solubility, density, surface area, and particle size analyses. RESULTS: Oils formed in several cases resulting in preliminary elimination of mesyl and esyl salts and four crystallizing solvents. Crystallinity was observed in 34 of 44 scaled-up trazodone salts. PXRD, DSC, and hygroscopic analyses indicated a number of new salts that were comparable in physicochemical parameters to the marketed HCl salt. Among them, the tosylate salt showed uniqueness for new applications. CONCLUSIONS: Automated procedures can be developed to increase the efficiency of pharmaceutical salt selection. The new tosylate salt gave a unique pH-solubility profile with low solubility over the entire pH range making it a potential candidate for a suspension or prolonged action formulation.

In vitro uptake of a new cholesteryl carborane ester compound by human glioma cell lines.

The cellular uptake and retention of a new cholesteryl carborane ester compound, cholesteryl 1,12-dicarba-closo-dodecaboranel-carboxylate (BCH), by two human glioma cell lines, glioblastoma multiforme SF-763 and SF-767, was evaluated. BCH, which is an extremely hydrophobic compound, was formulated into liposomes and incubated with two human glioma tumor cell lines and one human normal neuron cell line. The amount of BCH uptake by the cells was measured by high performance liquid chromatography. The effects of BCH concentration in the culture medium and the incubation time on the cellular uptake of BCH were studied. In addition, BCH uptake by tumor cells was examined in the presence and absence of lipoprotein in the culture medium. It was found that the amount of BCH taken by the glioma cell lines was much more (up to 14 times) than that by the normal neuron cell line. The cellular uptake of BCH was related to the amount of BCH in the medium as well as the incubation time. The cellular uptake of BCH by SF-763 and SF-767 cells after 16 h of incubation was 283.3 +/- 38.9 and 264.0 +/- 36.5 microg boron/g cells, respectively. The majority of BCH taken up in tumor cells was retained after the subsequent incubation. In the presence of lipoprotein, the cellular uptake of BCH by SF-767 tumor cells was about four times as much as that in the absence of lipoprotein. In conclusion, the cellular uptake of BCH by glioma cells was about 14 times higher than by normal neuron cells. The uptake in glioma cells was up to 10 times higher than that required for successful cancer treatment and BCH was well retained in the tumor cells. Lipoprotein seemed to have an important role in the BCH uptake by glioma cells.

Safety and utilization of blood components as therapeutic delivery systems.

In recent years, natural blood components have been extensively studied as the advanced therapeutic delivery systems. The blood components which can potentially be used as the therapeutic delivery systems include different types of cells, such as erythrocytes and lymphocytes, macromolecular complexes such as lipoproteins and antibody or albumin conjugates and other molecules. This review article covers the progress in this topic, specifically, including the safety issues and the utilization of these component. It can be seen through the literature that the blood components as the therapeutic delivery systems have a number of advantages over traditional pharmaceutical products. The efficacy and practice of the applications, however, require significant amount of development work in the near future.

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.

Synthesis of cholesterol-carborane conjugate for targeted drug delivery.

The cholesterol-carborane conjugate has been designed and synthesized to selectively deliver boron to tumor cells by means of reconstituted low-density lipoprotein. The chemical stability and cytotoxicity of the new compound have been examined. Several methods have been evaluated for incorporation of the compound into LDL.

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.