An assessment of the ocular safety of inactive excipients following sub-tenon injection in rabbits.

PURPOSE: This work characterized the safety and toleration of inactive excipients following sub-Tenon (ST) administration. METHODS: Rabbits were anesthetized and eyes received an ST injection of the following test excipients: carboxy methylcellulose (CMC; low [90 kDa], mid [250 kDa], and high [700 kDa] molecular weight [MW], 0.25%-1.0% w/v), polysorbate 80 (0.02 and 0.2% w/v), polyethylene glycol 3350 (PEG; 0.2 and 1.0% w/v), poloxamer 188 (0.01 and 0.25% w/v), poloxamer 182 (2% w/v), benzyl alcohol (BA; 4% w/v), benzalkonium chloride (BAC; 0.02%, 0.04%, and 0.05% w/v), and methylcellulose (MC; 0.25% w/v). After a 1-week observation period for clinical signs of ocular tolerability, the animals were euthanized and eyes were collected for histologic examination. RESULTS: The ocular tolerability of the tested excipients were ranked as follows from the innocuous to most deleterious: saline approximately PEG (1% w/v) approximately polysorbate 80 (0.2% w/v) > CMC (0.25% w/v, 90 kDa) > MC (0.25% w/v) approximately poloxomer 188 (0.25% w/v) approximately sodium citrate (pH 9) BAC (0.05% w/v) > CMC (0.5% w/v, 700 kDa) > poloxomer 182 (2% w/v) > BA (4% w/v). Clinical signs of ocular irritation were limited to redness and chemosis observed with most test excipients. The BA excipient also produced corneal opacity. Microscopic findings included histiocytic infiltration (BAC, BA, CMC, MC, and poloxamer 188), heterophilic inflammation (BA, CMC, and poloxamer 182), and edema (BAC, BA, CMC, and poloxamer 182) in episcleral tissue. The severity of the clinical and hisopathologic effects increased with the concentration of the test excipients administered. CONCLUSIONS: This research has evaluated the safety profile of inactive excipients that may be used to formulate new chemical entities for the treatment of ocular disease following a ST injection.

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.

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.