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Context: Cationic liposomes can efficiently deliver siRNA to the lung by intravenous injection of cationic liposome/siRNA complexes (lipoplexes)., Objective: The aim of this study was to examine a formulation of cationic liposomes for siRNA delivery to lung metastasis of breast tumor., Materials and Methods: For the preparation of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDAB) as a cationic lipid and cholesterol (Chol) or 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) as a neutral lipid were used. In vitro and in vivo gene silencing effects by cationic lipoplexes were evaluated after transfection into stably luciferase-expressing human breast tumor MCF-7-Luc cells and after intravenous injection into mice with lung MCF-7-Luc metastasis, respectively. Intracellular localization of siRNA after transfection into MCF-7 cells by cationic lipoplexes and biodistribution of siRNA after intravenous injection of cationic lipoplexes into the mice with lung metastasis were examined by confocal and fluorescent microscopy analyses, respectively., Results: In in vitro transfection, DOTAP/DOPE and DDAB/DOPE lipoplexes of luciferase siRNA strongly suppressed luciferase activity in MCF-7-Luc cells, but DOTAP/Chol and DDAB/Chol lipoplexes did not, although DOTAP/Chol and DDAB/Chol lipoplexes exhibited higher cellular uptake than DOTAP/DOPE and DDAB/DOPE lipoplexes. When their cationic lipoplexes were intravenously injected into mice with lung MCF-7-Luc metastasis, siRNAs were mainly accumulated in the lungs; however, the reduced luciferase activities in the lung-metastasized tumors were observed only by injections of DOTAP/Chol and DOTAP/DOPE lipoplexes, but not by DDAB/Chol and DDAB/DOPE lipoplexes., Conclusions: DOTAP-based liposomes might be useful as an in vivo siRNA delivery carrier that can induce gene silencing in lung-metastasized tumors.

In this study, we developed an anionic lipoplex by coating cationic lipoplex with anionic polymers such as hyaluronan (HA), chondroitin sulfate C (CS) and poly-l-glutamic acid (PLE) to deliver the plasmid DNA efficiently into the tumor by avoiding interaction with erythrocytes. The sizes of HA-, CS- and PLE-coated lipoplexes were ∼200 nm and the ζ-potentials were negative. CS- and PLE-coated lipoplexes did not induce agglutination after mixing with erythrocytes, but cationic and HA-coated lipoplexes exhibited agglutination. In terms of biodistribution and gene expression after intravenous administration, cationic and HA-coated lipoplexes largely accumulated and induced gene expression in the lung. In contrast, CS- and PLE-coated lipoplexes did not exhibit high gene expression in the lung and mainly accumulated in the liver. However, in tumor, differences in lipoplex accumulation and gene expression were not observed among the lipoplexes. In terms of toxicity after intravenous injection, CS- and PLE-coated lipoplexes did not increase tumor necrosis factor-α, aspartate aminotransferase and alanine aminotransferase concentrations in blood. From these findings, CS and PLE coatings for cationic lipoplex might produce safe systemic vectors, although they did not increase gene expression in tumor.

Pheochromocytomas are highly angiogenic neuroendocrine tumors. The side effects of treatment with cytotoxic agents frequently outweigh the benefits. Neuroendocrine tumors are highly angiogenic, dependent on vascular endothelial growth factor and receptor (VEGFR) activation. Sunitinib has antitumor and antiangiogenic activities that target VEGFRs. We investigated the antitumor activity of liposomal sunitinib and irinotecan alone and in combination. Liposomal sunitinib and irinotecan, and liposomes co-loaded with both drugs were prepared, and antitumor activity and biodistribution were examined in nude mice bearing PC12 tumors. Liposomal sunitinib increased in life span (ILS, 14.3%) compared with free sunitinib (-17.1% ILS) with moderate tumor growth suppression, whereas liposomal irinotecan suppressed tumor growth significantly without a survival benefit compared with free irinotecan (-21.7 and -13.3% ILSs, respectively). The combination of liposomal sunitinib plus liposomal irinotecan, and liposomes co-loaded with both drugs, induced significant inhibition of tumor growth and increased life-span more than the combination of free drugs. Accumulation of irinotecan in tumors by the combination of the two liposomal drugs and liposomes co-loaded with both drugs was significantly increased compared with the combination of free drugs. This study provides novel formulations of sunitinib and irinotecan in combination for the treatment of pheochromocytoma.

Cell-penetrating peptides (CPPs) are small peptides that can facilitate the uptake of macromolecular drugs, such as proteins or nucleic acids, into mammalian cells. Cytosolic delivery of CPPs could be beneficial to bypass conventional endocytosis in order to avoid degradation in the lysosomes. Oligoarginine conjugates have characteristics similar to CPPs in terms of cell translocation and are used in the intracellular delivery of plasmid DNA. In these cases, oligoarginine length and/or charge are important factors in the cellular uptake of oligoarginine alone. The arginine moiety of oligoarginine-modified particles may also be a decisive factor for vectors to deliver plasmid DNA. Oligoarginine-PEG-lipids can form self-assembled particles and modify the surface of lipid- and polymer-based particles. This review focuses on the influence of: i) oligoarginine-modified particles such as micelles, liposomes and polymer-based particles; ii) the morphology of oligoarginine-PEG-lipid complexed with plasmid DNA by decreasing the charge ratio; and iii) the oligoarginine length in the complex on its cellular uptake, transfection efficiency and uptake mechanism. The oligoarginine length of oligoarginine-modified particle complexed with plasmid DNA governs the cellular uptake pathway that determines the destiny of intracellular trafficking and finally transfection efficiency. The new aspects of surface-functionalized particle vectors with oligoarginine are discussed.

To investigate the use of folate-targeted nanoemulsion-loaded aclacinomycin A (ACM) to folate receptor (FR)-positive cells, we attempted to optimize the targeting ability of nanoemulsions by modifying the chain length and amount of the folate-PEG linker. Folate-linked, nanoemulsion-loaded ACM were formulated with 0.24 mol% of folate-poly (ethylene glycol)(3400)- (folate-PEG(3400)-) and folate-PEG(5000)-distearoylphosphatidylethanolamine (DSPE), and 0.03 mol% of folate-PEG(5000)-DSPE in nanoemulsions. Selective FR-mediated uptake was achieved in a human nasopharyngeal tumor cell line, KB, which overexpresses FR, but not in a human hepatoblastoma cell line, (FR(-)) HepG2. At the same amount of folate modification, the association with KB cells was increased with increasing the PEG-chain length. The association of 0.03 and 0.24 mol% folate-PEG(5000)-linked nanoemulsions with cells was 5- and 3.3-fold higher than that of non-folate nanoemulsion, respectively, while their cytotoxicity was similar. Both 0.03 and 0.24 mol% folate-PEG(5000)-linked nanoemulsions and non-folate nanoemulsion following intravenous injection inhibited tumor growth more significantly than ACM solution on day 24 following tumor inoculation (p < 0.01). This study demonstrates that a folate-linked nanoemulsion is feasible for tumor-targeted ACM delivery, and that folate modification with a sufficiently long PEG-chain and a small amount of nanoemulsion is an effective way of targeting nanoemulsion to tumor cells.

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as beta-sitosterol beta-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.

A water-insoluble anti-cancer agent, camptothecin (CPT) was incorporated to a polymeric micelle carrier system forming from poly(ethylene glycol)-poly(aspartate) block copolymers. Incorporation efficiency and stability were analyzed in correlation with chemical structures of the inner core-forming hydrophobic blocks as well as with incorporation methods. Among three incorporation methods (dialysis, emulsion and evaporation methods), an evaporation method brought about much higher CPT yields with less aggregation than the other two methods. By the evaporation method, CPT was incorporated to polymeric micelles in considerably high yields and with high stability using block copolymers possessing high contents of benzyl and methylnaphtyl ester groups as hydrophobic moieties. This indicates importance of molecular design of the hydrophobic block chain to obtain targeting using polymeric micelle carriers as well as importance of the drug incorporation method.

The mechanism of drug release from progesterone suppositories that consist of two types of hard fat (Witepsol W35 and Witepsol E85) was investigated. The strength, the thermodynamic characteristics, the surface structures, the drug release property, methylene blue penetration into suppositories, and change of surface structure after the dissolution test were employed for detecting characteristics of progesterone suppositories. The formulation with a mixing ratio of Witepsol W35 and Witepsol E85 at a 1:1 ratio showed the maximum strength value. The peak temperature of the suppositories showed a tendency to increase with increases in the ratio of Witepsol E85. The maximum height of the profiles measured with laser microscopy, from 20.8 microm to 29.2 microm, reached a maximum after 3 h of the dissolution test. When the suppositories were immersed in pH 7.4 phosphate buffer containing 0.5% methylene blue at 37 degrees C, the penetrating area increased with time. The weight of the suppositories also increased with time. According to these findings, it was suggested that the release of drug from a mixed type of suppository containing progesterone was via the matrix and pores.

A modified ethanol injection method for liposomes containing soybean phosphatidylcholine (SPC), cholesterol (Ch), beta-sitosterol beta-D-glucoside (Sit-G) and oleic acid (OA) was developed, that can produce homogeneous unilamellar liposomes without the use of sonication and dialysis. In this method, water is poured into a concentrated lipid-ethanol solution and then ethanol is removed in an evaporator. Dilution with water causes spontaneous formation of small and homogenous unilamellar vesicles from micellar aggregate. The size of liposomes can be controlled by the ratio of ethanol to water. OA and Sit-G were distributed at the surface of liposomes and were recognized by Concanavalin A, respectively. This easy and quick method for preparation of liposomes may be applicable in many areas.

The relationship between the rigidity of the liposomal membrane and the absorption of insulin after nasal administration of liposomes modified with an enhancer containing insulin was investigated for the nasal delivery of peptide drugs in rabbits. The rigid liposomal membrane makes liposomes stable, protecting insulin from enzymatic degradation. Soybean-derived sterol (SS) or its sterylglucoside (SG) was used as an enhancer. Dipalmitoylphosphatidylcholine (DPPC) liposomes modified with SG had increased fluidity of the hydrophobic group of the liposome bilayer compared with the liposomes modified with cholesterol (Ch) or SS, as shown by measurements of the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5,-hexatriene (DPH); however, the fluidity of the polar group of the liposome bilayer was decreased according to measurements of steady-state fluorescence anisotropy of dansylhexadecylamine (DSHA) at 37 degrees C. These findings suggest that the fluidity of the hydrophobic group of the liposome bilayer is responsible for the increase of liposomal leakage and instability of the liposomes. When insulin was administered nasally to rabbits as a solution, no hypoglycemic effect was observed. The administration of insulin contained in DPPC/SG (7/4, mole) liposomes with high fluidity caused a high glucose reduction of long duration (8 hr). DPPC/SS and DPPC/Ch (7/4) liposomes with low fluidity caused low glucose reductions. These results demonstrated that liposomes modified with SG can be useful as carriers of insulin administered nasally.

We investigated multilamellar vesicle (MLV) liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and a soybean-derived sterylglucoside mixture (SG) (DPPC/SG-liposomes) for targeting liver parenchymal cells after administration via the tail vein in mice, using liposome-entrapping calcein. The accumulation of DPPC/SG(7:2, mole ratio, DPPC:SG = 7:2)-liposomes in the liver was the highest among DPPC/SG-liposomes. About 80% and 40% of the dose of DPPC/SG(7:2)-liposomes accumulated in the liver at 15 min and 2 h, respectively, but about 20% of DPPC/SG(7:3.5, 7:7)-liposomes accumulated at 2 h after an intravenous administration. However, the uptake of DPPC/SG(7:2, 7:3.5, 7:7)-liposomes by liver parenchymal cells was about 7 times greater than that in non-parenchymal cells irrespective of the SG concentration in liposomes. The uptake of DPPC/SG(7:2)-liposomes in liver was almost the same level as that by liposomes containing lactocylceramide (LC) (LC-liposomes) that were already known to be taken up in liver parenchymal cells by the asialoglycoprotein receptor. DPPC/SG(7:2)-liposomes effectively targeted liver, having optimal stability and SG.