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Purpose: NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets., Methods: The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy., Results: The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry., Conclusions: These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets.

Purpose: To evaluate effect of a vascular disrupting agent, a combretastatin derivative (Cderiv), on tumor targeting for polymeric micelle carrier systems, containing either a diagnostic MRI contrast agent or a therapeutic anticancer drug., Methods: Cderiv was pre-administered 72 h before polymeric micelle MRI contrast agent injection. Accumulation of the MRI contrast agent in colon 26 murine tumor was evaluated with or without pretreatment of Cderiv by ICP and MRI., Results: Significantly higher accumulation of the MRI contrast agent was found in tumor tissues when Cderiv was administered at 72 h before MRI contrast agent injection. T(1)-weighted images of the tumor exhibited substantial signal enhancement in tumor area at 24 h after the contrast agent injection. In T(1)-weighted images, remarkable T(1)-signal enhancements were observed in part of tumor, not in whole tumor. These results indicate that Cderiv pretreatment considerably enhanced the permeability of the tumor blood vessels. Antitumor activity of adriamycin encapsulated polymeric micelles with the Cderiv pretreatment suppressed tumor growth in 44As3 human gastric scirrhous carcinoma-bearing nude mice., Conclusions: Pretreatment of Cderiv enhanced tumor permeability, resulting in higher accumulation of polymeric micelle carrier systems in solid tumors.

Purpose: Accelerated blood clearance (ABC) is induced by repeated injections of PEGylated liposomes. In this study, the ABC was investigated for a gadolinium-containing PEG-poly(L-lysine)-based polymeric micelle (Gd-micelle) and PEGylated liposome (Gd-liposome) in mice., Materials and Methods: Effects of the first injection of Gd-micelle on the tissue distribution of the second dose of Gd-micelle were studied. Additionally, effects of the first injection of Gd-micelle, Gd-liposome, empty liposome, polyethyleneglycol (PEG(500,000)), and PEG-lipid on the distribution of the second dose of the Gd-liposome were evaluated., Results: Results indicated that the tissue distribution of the second injection of the Gd-micelle at a dose of 33, 5, or 2 micromol Gd/kg was not affected by the first injection of the Gd-micelle at different doses and time intervals or of the empty PEGylated liposome 7 days before. ABC of Gd-liposome at a dose of 2.3 micromol Gd/kg (corresponding to 10 micromol lipids/kg) was observed when the empty PEGylated liposome or Gd-liposome, but not the Gd-micelle, PEG(500,000) or PEG-lipid, was pre-administered., Conclusions: The hydrophobic core of the micelle or lipid bilayer of PEGylated liposome has a major effect on this phenomenon. These studies have significant implications for the evaluation of PEG-poly(L-lysine)-based micellar formulation of Gd-based contrast agents.

Purpose: Polymeric micelles were designed for targeting of a water-insoluble anticancer agent, camptothecin (CPT). Chemical structures of inner core segment were optimized to achieve high incorporation efficiency and stable CPT-loaded micelles., Methods: Poly(ethylene glycol)-poly(beta-benzyl L-aspartate) block copolymer (PEG-PBLA) was synthesized. The PBLA chain was modified by alkaline hydrolysis of its benzyl group followed by esterification with benzyl, n-butyl, and lauryl groups. Incorporation of CPT into micelles was carried out by an evaporation method. The stability of drug-loaded micelles was studied by gel-permeation chromatography (GPC), and their in vitro release behaviors were analyzed., Results: CPT was incorporated into polymeric micelles constructed by various block copolymers. Among the esterified groups, block copolymers with high benzyl ester contents showed high CPT loading efficiency and stable CPT-loaded micelles. In chain lengths, 5-27 Bz-69 showed the highest incorporation efficiency. In contrast, 5-52 Bz-67, which had a longer hydrophobic chain, showed low incorporation efficiency. Release of CPT from the micelles was dependent on the benzyl contents and chain lengths. Sustained release was obtained when the benzyl content was high., Conclusions: CPT was successfully incorporated into polymeric micelles with high efficiency and stability by optimizing chemical structures of the inner core segment.

Purpose: To develop paclitaxel carried by injectable PEGylated emulsions, an artificial neural network (ANN) was used to optimize the formulation--which has a small particle size, high entrapment efficiency, and good stability--and to investigate the role of each ingredient in the emulsion., Methods: Paclitaxel emulsions were prepared by a modified ethanol injection method. A computer optimization technique based on a spherical experimental design for three-level, three factors [soybean oil (X1), PEG-DSPE (X2) and polysorbate 80 (X3)] were used to optimize the formulation. The entrapment efficiency of paclitaxel (Y1) was quantified by HPLC; the particle size of the emulsions (Y2) was measured by dynamic laser light scattering and the stability of paclitaxel emulsions was monitored by the changes in drug concentration (Y3) and particle size (Y4) after storage at 4 degrees C., Results: The entrapment efficiency, particle size and stability of paclitaxel emulsions were influenced by PEG-DSPE, polysorbate 80, and soybean oil. Paclitaxel emulsions of small size (262 nm), high entrapment efficiency (96.7%), and good stability were obtained by the optimization., Conclusions: A novel formulation for paclitaxel emulsions was optimized with ANN and prepared. The contribution indices of each component suggested that PEG-DSPE mainly contributes to the entrapment efficiency and particle size of paclitaxel emulsions, while polysorbate 80 contributes to stability.

Purpose: The aim of this study was to prepare a novel vaccine carrier particulate system (nanoparticles and emulsions) with chitosan and to evaluate the effect of this system on the immune response for intranasal delivery., Methods: Chitosan nanoparticles (NP) and chitosan-coated emulsions (CC-Emul) were prepared by improvement of the method previously reported and by modified ethanol injection methods, respectively. The rats were immunized with the particles adsorbed with ovalbumin (OVA) and cholera toxin (CT) by intranasal (i.n.) and intraperitoneal (i.p.) administration., Results: NP and CC-Emul could be prepared with particle diameter from about 0.4 microm to 3 microm. IgG induced by i.n. of NP was comparable with that by i.p., and IgA induced by i.n. of 0.4-microm- and 1-microm-size NP was significantly higher than control (OVA and CT). IgG and IgA induced by i.n. of 2-microm-size CC-Emul were significantly higher than those with control., Conclusions: The novel chitosan particles used simple preparation methods showed high OVA adsorption. When administered intranasally, NP and CC-Emul induced systemic immune response in rats. These findings suggested that CC-Emul and the smaller-size (0.4 microm) NP are effective for targeting to nasal-associated lymphoid tissues (NALTs) in nasal vaccine delivery.

Purpose: The aim of the study was to prepare stealth and remote loading proliposome (SRP-L) to carry doxorubicin (DXR) and evaluate the pharmacokinetics, acute toxicity, and anticancer effect of DXR carried with SRP-L., Methods: SRP-L was transparent solution. When SRP-L was injected into 0.9%, NaCl aqueous solution containing DXR. liposomes formed and automatically loaded DXR (SRP-L-DXR). The long circulation of SRP-L-DXR was evaluated using the pharmacokinetics of SRP-L-DXR. cardiolipin liposomal DXR (CL-DXR) and free DXR (F-DXR). The acute toxicity and anticancer effect of SRP-L-DXR were evaluated in C57BL/6 mice and murine hystocytoma M5076 tumor model., Results: The average diameter of SRP-L-DXR in pure water was 112.9 +/- 8.6 (nm) and the encapsulation efficiency of SRP-L-DXR was 96.5 +/- 0.2% in pure water, 95.5 +/- 0.1% in 5% glucose and 98.01 +/- 0.6% in 0.9% NaCl. The plasma concentration of SRP-L-DXR was much higher than those of F-DXR and CL-DXR. Compared with that of F-DXR, the SRP-L-DXR had lower acute toxicity and its anticancer effects depended upon the therapeutic treatment., Conclusions: A novel proliposome (SRP-L) was developed, which could automatically load DXR and form SRP-L-DXR with excellent characteristics. SRP-L-DXR had lower acute toxicity but was not always more effective for the treatment of the ascitic M5076 than F-DXR.

Purpose: Recombinant human erythropoietin (Epo) is used frequently through intravenous (i.v.) and subcutaneous (s.c.) administration for the clinical treatment of the last stage of renal anemia. We encapsulated Epo in liposomes to develop an alternative administration route. The purpose of our study was to evaluate the pharmacokinetics and the pharmacological effects of liposomal Epo in comparison with the Epo after i.v. and s.c. administration to rats., Methods: Epo was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) prepared by the reversed-phase evaporation vesicle method. After filtration through a 0.1 micron polycarbonate membrane, liposomes were gel filtered (Epo/liposomes)., Results: Epo/liposomes showed higher pharmacological activity than Epo/liposomes before gel filtration after i.v. administration to rats. Non-encapsulated Epo lost its activity, whereas encapsulated Epo in liposomes retained it. The pharmacological effects of Epo/liposomes were greater than those of Epo after i.v. administration. Epo/liposomes afforded 3-9 times higher AUC, lower clearance and lower steady-state volume of distribution than Epo after both i.v. and s.c. administrations. Epo/liposomes had an improved pharmacokinetics profile compared with Epo. S.c. administration of Epo/liposomes at 7 h may penetrate primarily (40% of dose) through the blood as a liposome and partly (7% of dose) in lymph. CONCLUSIONS. Epo/liposomes may reduce the frequency of injections required for a certain reticulocyte effect in comparison to Epo. The lower clearance of Epo/liposomes may increase the plasma concentrations of Epo, which increases the efficacy.

The systemic degradation and the reticuloendothelial system (RES) uptake of calcein entrapped in dipalmitoylphosphatidylcholine (DPPC) liposomes with soybean-derived sterols (SS) were examined after intravenous administration to mice by measuring the free and liposomal calcein levels in the blood. The results indicate that the rates of systemic degradation and the RES uptake of liposomes decrease with the addition of SS in DPPC liposomes since the SS has the ability to stabilize the liposomes. The rate of uptake by RES is larger than the rate of systemic degradation. The rate of leakage of calcein from liposomes by incubation in plasma in vitro is almost the same as that of systemic degradation in vivo.