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Abstract: A novel liposomal formulation of paclitaxel targeting the folate receptor (FR) was synthesized and characterized. This formulation was designed to overcome vehicle toxicity associated with the traditional Cremophor EL-based formulation and to provide the added advantages of prolonged systemic circulation time and selective targeting of the FR, which is frequently overexpressed on epithelial cancer cells. The formulation had the composition of dipalmitoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol/monomethoxy-polyethylene glycol (PEG)2000-distearoyl phosphatidylethanolamine/folate-PEG3350-distearoyl phosphatidylethanolamine (DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE) at molar ratios of (85.5:9.5:4.5:0.5) and a drug-to-lipid molar ratio of 1:33. The liposomes were prepared by polycarbonate membrane extrusion. The mean particle size of the liposomes was 97.1nm and remained stable for at least 72h at 4°C. FR-targeted liposomes of the same lipid composition entrapping calcein were shown to be efficiently taken up by KB oral carcinoma cells, which are highly FR+. FR-targeted liposomes containing paclitaxel showed 3.8-fold greater cytotoxicity compared to non-targeted control liposomes in KB cells. Plasma clearance profiles of paclitaxel in the liposomal formulations were then compared to paclitaxel in Cremophor EL formulation. The liposomal formulations showed much longer terminal half-lives (12.33 and 14.23h for FR-targeted and non-targeted liposomes, respectively) than paclitaxel in Cremophor EL (1.78h). In conclusion, the paclitaxel formulation described in this study has substantial stability and favorable pharmacokinetic properties. The FR-targeted paclitaxel formulation is potentially useful for treatment of FR+ tumors and warrants further investigation. [Copyright &y& Elsevier]

Abstract: Folate polyethylene glycol-cholesterol hemisuccinate (folate-PEG-CHEMS) is a novel folate ligand firstly synthesized by our group and demonstrated good stability and potential targeting results on KB cells in vitro. The current study further explored endocytosis mechanisms of liposomes via folate receptor on L1210JF cells and assessed targeted therapeutic efficacy of folate-PEG-CHEMS anchored liposomes loading daunorubicin (F-L-DNR) in vivo. Folate-PEG-CHEMS was synthesized by a modified method. The liposome properties, cell cytotoxicity, intracellular and intratumoral localization, and therapeutic efficacy on a murine tumor model bearing L1210JF cells were evaluated. High encapsulation efficiency (95.1%±1.5%) and appropriate particle size (76.0±35.5nm) and zeta potential (−12.83±1.36mV) were achieved for F-L-DNR. IC50 of F-L-DNR on L1210JF cells was 2–3-folds lower than that of non-targeted liposomal daunorubicin (L-DNR). Anticancer efficacy on L1210JF tumor model indicated that mice survival time of F-L-DNR group at doses of 5mg/kg and 10mg/kg was significantly longer than that of L-DNR or free DNR. Confocal fluorescence photographs of F-L-DNR indicated enhanced endocytosis of liposomes via folate receptor on L1210JF cells, prolonged retaining time in tumors and improved drug release in the tumor site at 24h post intravenous injection of F-L-DNR. In conclusion, folate-PEG-CHEMS is an effective ligand for folate-targeted daunorubicin liposomes to achieve increased drug release in tumor and therapeutic efficacy. [Copyright &y& Elsevier]

Abstract: Our previous data demonstrated that folate receptor β (FR-β) targeted liposomal doxorubicin (FT-L-DOX) showed enhanced cytotoxicity relative to non-targeted liposomal doxorubicin (CON-L-DOX), and the effect was enhanced by selective FR-β upregulation by all-trans retinoic acid (ATRA) in AML blast cells. In this study, the enhanced cytotoxicity was investigated in the proliferating human AML clonogenic cells by combining FT-L-DOX with ATRA. Also, pharmacokinetic properties by pretreatment of ATRA were evaluated using FR-targeted liposomal calcein (FT-L-Calcein). Pharmacokinetic study showed that the area under the concentration curve (AUC) of FT-L-Calcein was decreased and total clearance was increased by pretreatment with ATRA. Meanwhile, the volume of distribution was significantly increased by pretreatment of ATRA. Moreover, calcein level in the liver, spleen and kidney was increased following intravenous administration of FT-L-Calcein by pretreatment of ATRA. In vitro cytotoxicity of FT-L-DOX was higher than that of CON-L-DOX and was increased by pretreatment with ATRA. Colony formation in AML cells was lower due to treatment with FT-L-DOX compared with CON-L-DOX and colony formation further decreased upon pretreatment with ATRA. Moreover, FT-L-DOX was more toxic to AML clonogenic cells than to AML blast cells. The results demonstrate that the efficiency of FR-mediated targeting of FT-L-DOX was preferentially enhanced by ATRA induced FR-β upregulation in AML clonogenic cells. [ABSTRACT FROM AUTHOR]

Abstract: Folate receptors (FRs) have been identified as cellular surface markers for cancer and leukemia. Liposomes containing lipophilic derivatives of folate have been shown to effectively target FR-expressing cells. Here, we report the synthesis of a novel lipophilic folate derivative, folate-polyethylene glycol-cholesterol hemisuccinate (F-PEG-CHEMS), and its evaluation as a targeting ligand for liposomal doxorubicin (L-DOX) in FR-expressing cells. Liposomes containing F-PEG-CHEMS, with a mean diameter of 120±20nm, were synthesized by polycarbonate membrane extrusion and were shown to have excellent colloidal stability. The liposomes were taken up selectively by KB cells, which overexpress FR-α. Compared to folate-PEG-cholesterol (F-PEG-Chol), which contains a carbamate linkage, F-PEG-CHEMS better retained its FR-targeting activity during prolonged storage. In addition, F-PEG-CHEMS containing liposomes loaded with DOX (F-L-DOX) showed greater cytotoxicity (IC50 =10.0μM) than non-targeted control L-DOX (IC50 =57.5μM) in KB cells. In ICR mice, both targeted and non-targeted liposomes exhibited long circulation properties, although F-L-DOX (t 1/2 =12.34h) showed more rapid plasma clearance than L-DOX (t 1/2 =17.10h). These results suggest that F-PEG-CHEMS is effective as a novel ligand for the synthesis of FR-targeted liposomes. [Copyright &y& Elsevier]

Phospholipids have been extensively evaluated as an anchor for both PEGylation and receptor-targeting in liposomal formulations. However, cholesterol, another important component in biomembranes, has not been fully investigated as an alternative anchor. In this study, the potential role of cholesterol for anchoring PEG and folate was investigated. Cholesterol derivatives were synthesized for PEGylation (mPEG-cholesterol) and folate receptor (FR) targeting (folate-PEG-cholesterol) and incorporated into the bilayer of FR-targeted liposomal doxorubicin. The colloidal stability of these cholesterol derivative-containing liposomes was superior to non-PEGylated liposomes, indicating that steric barrier provided by mPEG-cholesterol can efficiently inhibit aggregation of liposomes. FR-targeting activity of these liposomes was demonstrated by in vitro cell-binding studies on FR-overexpressing KB cells. In addition, in vivo circulation of cholesterol-anchored liposomes was prolonged compared to non-PEGylated liposomes. These studies suggest that cholesterol is a viable bilayer anchor for synthesis of PEGylated and FR-targeted liposomes. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2424–2435, 2007 [ABSTRACT FROM AUTHOR]