A method to determine the incorporation capacity of camptothecin in liposomes

Abstract

The purpose of this study was to establish a new experimental approach to determine the maximum amount of camptothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultracentrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubility of CPT in the buffer was improved by approximately a factor of 10 (from ~2.7 to 15-50 µg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ~100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ~20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrinsic water solubility of the drug.

Targeted drug delivery using genetically engineered diatom biosilica

“…Camptothecin (CPT; partition coefficient, logP ¼ 1.7) and its more potent derivative 7-ethyl-10-hydroxy-CPT (SN38; logP ¼ 2.7) were selected as model compounds for poorly water-soluble chemotherapy drugs. CPT-loaded liposomes were prepared with the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) 29 and SN38 was encapsulated into cetyltrimethylammonium bromide (CTAB) micelles 30 .…”

Liposomal formulations of poorly soluble camptothecin: drug retention and biodistribution

“…In our earlier studies we investigated the CPT incorporation capacity for different liposome formulations (Saetern et al, 2004b). These studies revealed that the cationic EPC/DOTAP containing liposomes had a superior CPT incorporation capacity compared with the other formulations, whereas the formulations that contained DMPC or cholesterol resulted in stiffening of the bilayer and showed the lowest incorporation (Saetern et al, 2004b).…”

“…Several patents (Burke, 1996, Perez-Soler et al, 1998 are available and numerous studies (Sugarman et al, 1996, Proulx et al, 2001, Burke et al, 1992, Saetern et al, 2004b, Watanabe et al, 2008, Eichhorn et al, 2007, Clements et al, 1996, Daoud et al, 1995 reported on liposomal formulations of camptothecins, whereof the majority of studies is on liposomal CPT-formulations investigated water soluble CPT-derivatives such as topotecan (Yang et al, 2012, Tardi et al, 2000, Liu et al, 2002, Subramanian et al, 1995, Zucker et al, 2012, Drummond et al, 2010, Dadashzadeh et al, 2008, irinotecan (Chou et al, 2003, Sadzuka, 2000, Sadzuka et al, 1998, Sadzuka et al, 1999, Sadzuka et al, 1997, Drummond et al, 2006, Zhang et al, 2012, Hattori et al, 2009), lurtotecan (MacKenzie et al, 2004, Loos et al, 2000, Desjardins et al, 2001, Colbern et al, 1998, SN-38 {Zhang, 2004Atyabi, 2009Sadzuka, 2005Lei, 2004#1330}, 9-nitro-CPT (Chen et al, 2008, Chen et al, 2006, Gilbert et al, 2002, Koshkina et al, 1999 and DB-67 (Bom et al, 2001. The focus on the present study is in contrast on poorly water soluble and lipophilic parent CPT-compound.…”

“…Previously, several studies have been performed on incorporation of CPT in liposomes, however the experimental designs used were different and none besides the one from Saetern and colleagues has reported a thorough study of many different liposome compositions. Despite of this the results from the mentioned studies could be summarized to relieve the following effect of different types of lipids: Inclusion of cationic lipids has shown to increase the incorporation of CPT (Eichhorn et al, 2007, Saetern et al, 2004b, Sugarman et al, 1996, the same has been shown with anionic lipids even though not to the same extent (Burke et al, 1993, Saetern et al, 2004b, Sugarman et al, 1996. Inclusion of fatty acids with increased degree of saturation as well as cholesterol has in most studies shown to decrease incorporation (Daoud et al, 1995, Saetern et al, 2004b, Sugarman et al, 1996 but in some cases the opposite effect has been observed when fatty acids with increased degree of saturation has been included in liposome formulations (Burke et al, 1993).…”

“…In an earlier study we performed in vitro characterization of CPT-containing liposomes (Saetern et al, 2004b). In this study certain DOTAP-based liposome formulations were found to have a superior and sufficient incorporation capacity for CPT to reach therapeutically relevant concentrations (approximately 50 mg/ml) using physiological acceptable formulation characteristics.…”