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Liposomes composed of egg phosphatidylcholine and cholesterol were modified with the temperature-responsive polymer poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)), and exhibited reversible surface properties with temperature. Completely reversible liposome aggregation due to P(NIPAAm-co-DMAAm) hydration/dehydration was demonstrated over four successive cycles of heating and cooling. The P(NIPAAm-co-DMAAm) polymer was hydrated during cooling, which dispersed the liposomes. The rigidity of the liposomal membrane was one of the factors in the reversible aggregation, as was the modification density of the polymer on the liposomes. A low density on relatively rigid liposomes could maintain the polymer property of reversible hydrated layers below critical solution temperature (LCST) boundary. Above the LCST, temperature-responsive polymers could also transport negatively charged liposomes into cells. The reversible behavior of the temperature-responsive polymer-modified liposomes has not been reported previously and could enable new applications for switching deposit forms as alternative drug carriers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Small interfering RNAs (siRNAs) have been attracting significant attention owing to their gene silencing properties, which can be utilized to treat intractable diseases. In this study, two temperature-responsive liposomal siRNA carriers were prepared by modifying liposomes with different polymers-poly( N -isopropylacrylamide- co - N , N -dimethylaminopropyl acrylamide) (P(NIPAAm- co -DMAPAAm)) and poly( N -isopropylacrylamide- co - N , N -dimethylacrylamide) P(NIPAAm- co -DMAAm). The phase transition of P(NIPAAm- co -DMAPAAm) was sharper than that of P(NIPAAm- co -DMAAm), which is attributed to the lower co-monomer content. The temperature dependent fixed aqueous layer thickness (FALT) of the prepared liposomes indicated that modifying liposomes with P(NIPAAm- co -DMAPAAm) led to a significant change in the thickness of the fixed aqueous monolayer between 37 °C and 42 °C; while P(NIPAAm- co -DMAAm) modification led to FALT changes over a broader temperature range. The temperature-responsive liposomes exhibited cellular uptake at 42 °C, but were not taken up by cells at 37 °C. This is likely because the thermoresponsive hydrophilic/hydrophobic changes at the liposome surface induced temperature-responsive cellular uptake. Additionally, siRNA transfection of cells for the prevention of luciferase and vascular endothelial growth factor (VEGF) expression was modulated by external temperature changes. P(NIPAAm- co -DMAPAAm) modified liposomes in particular exhibited effective siRNA transfection properties with low cytotoxicity compared with P(NIPAAm- co -DMAAm) modified analogues. These results indicated that the prepared temperature-responsive liposomes could be used as effective siRNA carriers whose transfection properties can be modulated by temperature., Competing Interests: The authors declare no conflict of interest.

The development of antiviral agents that have novel mechanisms of action is urgently required in the topical therapy of herpes simplex virus type 2 (HSV-2) infections. We reported previously that topical application of branched 3610-Da polyethylenimine (PEI) exhibited preventative antiviral activity. In this study, to develop therapeutic anti-HSV-2 agents, the most potent PEI combined with ~200 nm-sized liposomes with or without oleic acid (liposomes/PEI) was selected in vitro and further evaluated using in vivo studies. The mechanism of action in vivo was elucidated using PEIs with decreased numbers of primary amine residues, resulting from ethylene carbonate treatment, and polyallylamine, a linear polyamine consisting of primary amines. Cytotoxicity and antiviral activity in vitro, and the appearance of acute herpetic disease and virus yields in mice intravaginally administered with liposomes/PEI were evaluated in cell culture assays and a mouse genital herpes model, respectively. In addition, the cellular association of liposome/PEI was examined by flow cytometry and confocal microscopy. PEI showed higher antiviral activity postinfection than preinfection in vivo. Liposome/PEI and PEI with decreased numbers of primary amine residues at a dose of 0.2 mg PEI/mouse exhibited more potent therapeutic antiviral activity than acyclovir and PEI alone without acute lesion appearance or toxicity pre- or postinfection, but polyallylamine was moderately effective only preinfection. Liposome concentrations were important for the effectiveness of liposome/PEI. This finding suggests that PEI combined with liposomes and with slightly decreased numbers of primary amines may be an effective vaginally administrated antiviral drug, and secondary and tertiary amine residues of PEI may contribute to the inhibitory efficiency against viral infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Injections of poly(ethylene glycol)-modified liposomes (PEG-liposomes) cause rapid clearance of the second dose of PEG-liposomes. This phenomenon is known as the accelerated blood clearance (ABC) phenomenon. Previous studies have suggested that PEG-specific IgM (anti-PEG IgM) can play a major role in the ABC phenomenon. In our previous study, however, a PEG-shell-possessing polymeric micelle with hydrophilic inner core (PEG-P(Lys-DOTA-Gd) micelle) did not induce the ABC phenomenon nor the IgM responses, and exhibited no change in its plasma concentration in PEG-liposome-injected mice. In the present paper, we studied the ABC-phenomenon in more detail by comparing the behaviors between PEG-liposomes, PEG-P(Lys-DOTA-Gd) micelle, and hydrophobic-core-possessing PEG-PBLA micelles. We demonstrated that the PEG-PBLA micelle induced similar IgM responses as observed in PEG-liposome; however, the second dose of PEG-PBLA micelle exhibited no decreases in their plasma concentration, while the second dose of PEG-liposome did exhibit rapid clearances. Furthermore, we did not observe any PEG main chain specific IgM in PEG-liposome injected mice by sandwich ELISA which can measure more specific IgM to the PEG main chain theoretically. These results suggested that the induced IgM recognizes an interface between PEG chain and hydrophobic chain, rather than PEG main chain, and the anti-PEG IgM hypothesis should be re-evaluated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mercaptoundecahydrododecaborate (BSH)-encapsulating 10% distearoyl boron lipid (DSBL) liposomes were developed as a boron delivery vehicle for neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in addition to its encapsulated agents. BSH-encapsulating 10% DSBL liposomes have high boron content (B/P ratio: 2.6) that enables us to prepare liposome solution with 5000 ppm boron concentration. BSH-encapsulating 10% DSBL liposomes displayed excellent boron delivery efficacy to tumor: boron concentrations reached 174, 93, and 32 ppm at doses of 50, 30, and 15 mg B/kg, respectively. Magnescope was also encapsulated in the 10% DSBL liposomes and the real-time biodistribution of the Magnescope-encapsulating DSBL liposomes was measured in a living body using MRI. Significant antitumor effect was observed in mice injected with BSH-encapsulating 10% DSBL liposomes even at the dose of 15 mg B/kg; the tumor completely disappeared three weeks after thermal neutron irradiation ((1.5-1.8) × 10(12) neutrons/cm(2)). The current results enabled us to reduce the total dose of liposomes to less than one-fifth compared with that of the BSH-encapsulating liposomes without reducing the efficacy of boron neutron capture therapy (BNCT).

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.

For the optimization of plasmid DNA (pDNA)-cationic lipid complexes and lipoplex delivery, proper indexes of the physicochemical properties of lipoplexes are required. In general, the characteristics of lipoplexes are defined by particle size and zeta-potential at various mixing ratios of cationic liposomes and pDNA. In this study, we characterized the hydration level of surfactant-modified and PEGylated cationic cholesterol-based (OH-Chol) liposomes and their lipoplexes by monitoring both the fluorescent probe laurdan and the dielectric relaxation time. Fluorescence measurement using laurdan detected hydration of the headgroup of lipids in surfactant-modified liposomes and PEGylated DOTAP-liposomes, but hardly any fluorescence was detected in PEGylated OH-Chol-liposomes because the PEG layers may extend and cover the fluorescent maker. On the other hand, the measurement of dielectric relaxation time of water molecules revealed total hydration, including hydration of the PEG layer and the headgroup of cationic lipids. Furthermore, we found an inverse correlation between hydration level and cellular uptake of PEGylated lipoplexes (R=0.946). This finding indicated that the dielectric relaxation time of water molecules provides an important indicator of hydration of liposome and lipoplexes along with the fluorescence intensity of laurdan., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Folate-polymer-coated liposomes were developed for targeted chemotherapy using doxorubicin (DXR) as a model drug. Folate-poly(L-lysine) (F-PLL) conjugates with a folate modification degree of 16.7 mol% on epsilon amino groups of PLL were synthesized. DXR-loaded anionic liposomes were coated with F-PLL, and the cellular association of F-PLL-coated liposomes was evaluated by flow cytometry, and confocal microscopy in human nasopharyngeal carcinoma KB cells overexpressing folate receptors (FRs), and human lung adenocarcinoma A549 cells [FR (-)]. The existence of a polymer layer on the surface of F-PLL-coated liposomes was confirmed by zeta potential analysis. The KB cellular association of F-PLL-coated liposomal DXR was increased compared with that of PLL-coated liposomes and was inhibited in the presence of free folic acid. Twofold higher cytotoxicity of F-PLL-coated liposomal DXR was observed compared with that of the PLL-coated liposomal DXR in KB cells, but not in A549 cells, suggesting the presence of FR-mediated endocytosis. These results indicated that folate-targeted liposomes were prepared successfully by coating the folate-polymer conjugate F-PLL. This novel preparation method of folate-targeted liposomes is expected to provide a powerful tool for the development of a folate-targeting drug nanodevice as coating with ligand-polymer conjugates can be applicable to many kinds of particles, as well as to lipid-based particles.

Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strategy for various cancers. In this study, we hypothesized that a liposomal formulation of HDACI might efficiently deliver HDACI into tumors. To incorporate HDACI efficiently into the liposomal membrane, we synthesized six HDACI-lipid conjugates, in which polyethylene glycol(2000) (PEG(2000))-lipid or cholesterol (Chol) was linked with a potent hydroxamic acid, HDACI, SAHA or K-182, by cleavable linkers, such as ester, carbamide and disulfide bonds. Liposomal HDACI-lipid conjugates were prepared with distearoylphosphatidylcholine (DSPC) and HDACI-Chol conjugate or with DSPC, Chol and HDACI-PEG-lipid conjugates, and their cytotoxicities were evaluated for human cervix tumor HeLa and mouse colon tumor Colon 26 cells. Among the liposomes, liposomal oleyl-PEG(2000)-SAHA conjugated with SAHA and oleyl-PEG(2000) via a carbamate linker showed higher cytotoxicity via hyperacetylation of histone H3 and induction of caspase 3/7 activity. These results suggested that liposomal HDACI-lipid conjugates may be a potential tool for cancer therapy.

Cationic liposomes composed of 3beta-[N-(N'N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1:1 or 3:2) prepared by the dry-film method have been often used as non-viral gene delivery vectors. We have shown that a more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1:2) than those (3:2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method. The enhanced transfection is related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in opti-MEM I reduced-serum medium (optiMEM), not to an increased cellular association with the lipoplex. Cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery.

As reported previously, cationic liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) and N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol (MHAPC-liposomes) achieved efficient gene transfection in the mouse lung following intratracheal injection. We have studied here the role of surfactants, mannosylerythritol lipid-A (MEL-A) and polysorbate 80 (Tween 80), in affecting gene transfection of MHAPC-lipoplexes (complex with pCMV-luc DNA) in A549 cells and in the mouse lung. MEL-A increased gene transfection of MHAPC-lipoplexes significantly in vitro and slightly in the mouse lung, while Tween 80 decreased it both in vitro and in vivo. As assessed by confocal laser scanning microscopy and fluorescence imaging, MEL-A might faciliate gene dissociation from MHAPC-lipoplexes with fluorescein-labeled oligodeoxynucleotide (FITC-ODN) after internalization into the cells and retained the lipoplexes in the mouse lung for prolonged time, while Tween 80 was inefficient to deliver foreign gene into target cells and in the lung. These results demonstrated that MEL-A is advantageous to Tween 80 in the modification of cationic liposomes as gene delivery vectors in the lung.

The surface properties of cationic liposomes and lipoplexes largely determine the cellular association and gene transfection efficiency. In this study, we measured the surface properties, such as zeta potentials, surface pH and hydration levels of MHAPC- and OH-Chol-lipoplexes and their cellular association, without and with the modification of biosurfactant mannosylerythritol lipid-A (MEL-A) or Tween 80 (MHAPC=N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol; OH-Chol=cholesteryl-3beta-carboxyamindoethylene-N-hydroxyethylamine). Compared to OH-Chol-lipoplexes, the higher cellular association of MHAPC-lipoplexes correlated with the significantly higher zeta potentials, lower surface pH levels and "drier" surface, as evaluated by the generalized polarization of laurdan. Both MEL-A and Tween 80 modification of MHAPC-lipoplexes did not significantly change zeta potentials and surface pH levels, while MEL-A modification of OH-Chol-lipoplexes seriously decreased them. MEL-A hydrated the liposomal surface of MHAPC-lipoplexes but dehydrated that of OH-Chol-lipoplexes, while Tween 80 hydrated those of MHAPC- and OH-Chol-lipoplexes. In all, cationic liposomes composed of lipids with secondary and tertiary amine exhibited different surface properties and cellular associations of lipoplexes, and modification with surfactants further enlarged their difference. The strong hydration ability of Tween 80 may relate to the low cellular association of lipoplexes, while the dehydration of MEL-A-modified OH-Chol-lipoplexes seemed to compensate the negative zeta potential for the cellular association of lipoplexes.

The efficacy of folate-targeted liposomal drug delivery has not been fully achieved in part because of the slow release of the encapsulated drugs following uptake of the liposomes by target cells. Since liposomal mitoxantrone (MXN) composed of lipids with high fluidity was reported to achieve strong anticancer effects in vivo, we hypothesized that folate-targeted liposomal MXN uptake via folate receptor (FR)-mediated endocytosis could effectively release drugs into the endosomal compartment. Folate-targeted liposomal MXN was prepared using two lipids with different fluidities. MXN was released slowly from all types of liposome into PBS, indicating that the cellular uptake of MXN was considered to be in the liposomal form. Folate-targeted liposomes with high fluidity exhibited lower cellular uptake of loaded FITC-labeled dextran into FR (+) KB cells, but, when MXN was loaded, higher cytotoxicity than liposomes with lower fluidity. On the other hand, the cellular uptake of non-folate liposomes was not affected by the membrane fluidity, but higher cytotoxicity was observed in liposomal MXN with high fluidity, which suggested a higher rate of release of the drug from the liposomes. High levels of cytotoxic activity were achieved with folate-targeted liposomal MXN though the cellular uptake rate was restricted by selecting liposomes with higher lipid membrane fluidity. This finding provides a new insight into folate-targeted carrier drug delivery.

Camptothecin (CPT) is a strong antitumor agent, but its use limited by its low solubility and the instability of the active lactone form. To overcome these difficulties, liposomes incorporating CPT (CPT liposomes) were designed and tested. CPT liposomes were formulated by the addition of 3,5-bis(dodecyloxy)benzoic acid (DB) to polyethylene glycol-containing liposomes, and by coating the surface of the liposomes with human serum albumin (HSA, HSA-DB-L). HSA-DB-L successfully entrapped CPT with about 80% efficiency and with a particle size of about 150 nm. HSA-DB-L showed attenuated drug release and storage stability. Pharmacokinetics studies in mice showed that i.v. injection of HSA-DB-L (2.5 mg/kg) led to prolonged circulation in the plasma; the area under the curve was 22-fold higher than that of CPT solution. The tumor growth in mice with subcutaneous transplantation of colon 26 tumor cells was significantly inhibited after a single i.v. injection of HSA-DB-L at a dose of 15 mg/kg without any significant body weight loss. HSA-DB-L increased the accumulation of CPT in tumor tissue significantly (9.6-fold) more efficiently than CPT solution 24 h after i.v. injection. These findings suggest that HSA-DB-L could increase the stability and the antitumor effect of CPT. CPT delivery by novel liposome formulations is a potential approach for effective treatment of cancer.

Camptothecin (CPT) has anticancer activity. While only the lactone form of CPT is biologically active, this form exhibits poor aqueous solubility. Pharmaceutical formulation of CPT incorporated in liposomes is of significant importance to develop the therapeutic utilization of CPT. The aim of this study was to increase incorporation efficiency and stability of CPT in liposomes composed of hydrogenated soybean phosphatidylcholine, cholesterol, and oleic acid (7 : 3 : 1, molar ratio), by incorporating three kinds of artificial lipids (DBs) (DB-liposome); 4-n-(M12B), 3,5-bis(B12B) and 3,4,5-tris(dodecyloxy)benzoic acid (T12B). The interaction of CPT with DB in the state of liposomes, was examined. In DB-liposomes presenting mean diameters of 150 nm, incorporation efficiency of CPT up to 55% and final drug to lipid molar ratio up to 0.07 were obtained when the liposomes were prepared at a feeding ratio of 1/30 (w/w) CPT/total lipid. However, in the optimal formulations, incorporated DB mol% was different; T12B and D12B were incorporated about one third and half mol% of M12B, respectively. Moreover, we demonstrated that T12B stabilized CPT in liposomes significantly compared with other DBs as measured by CPT release, and by steady state fluorescence polarization degree of CPT using intrinsic fluorescence of CPT. These findings suggested that in addition of contribution of phenyl group of DB, dodecyloxy group may interact strongly with lactone ring of CPT. The capacity to contain CPT interacted with DBs may be limited in liposomes. T12B may be incorporated in the interior of the bilayers, resulting in increase of incorporation stability of CPT. This finding demonstrates a potential application of the novel liposome formulation of CPT in drug delivery.

Cationic liposomes composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1:1 or 3:2) prepared by the dry-film method have been often used as non-viral gene delivery vectors. The formulation and preparation of DC-Chol/DOPE liposomes, as well as the formation of their lipoplexes were investigated in an attempt to improve transfection efficiency in vitro. A more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1:2) than those (3:2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method. The enhanced transfection might be related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in optiMEM, not to an increased cellular association with the lipoplex. The use of a modified ethanol injection method might enhance the role of DOPE that is aid in destabilization of the plasma membrane and/or endosome. These findings suggested that cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery.

In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.

The intracellular delivery of proteins using cell-penetrating peptides (CPPs) including oligoarginine (oligo-Arg) carriers raises the possibility of establishing novel therapeutic methods. We compared the effect of the length of oligo-Arg in modified liposomes ((Arg)(n)-L; n = 4, 6, 8, 10) on the delivery of proteins by flow cytometry, fluorescence microscopy, and spectrofluorimetry. As a free liposome, Arg4-modified liposome Arg4-L was most efficiently internalized in cells. The efficiency decreased depending on the length of oligo-Arg. For the intracellular delivery of proteins, (Arg)(n)-L was physically associated with proteins. Concerning the effect of oligo-Arg length, liposome/protein complexes showed a different behavior. Arg4-L carried bovine serum albumin (BSA, 66 kDa) and beta-galactosidase (beta-Gal, 120 kDa) 6-fold higher than free BSA and free beta-Gal. Arg10-L showed similar performance for these two proteins to Arg4-L. The enzymatic activity of beta-Gal in the cells showed that proteins were transported as a biologically active form. Arg10-L carried 100-fold more immunoglobulin G (IgG, 150 kDa) than free IgG, and 3-fold more than Arg4-L into cells. Shorter oligo-Arg chain on liposomes may be enough for liposomes alone to be taken up in cells, but more Arg residues may be needed to form a complex with high molecular weight proteins and deliver them into cells. This information will aid in the design of (Arg)(n)-L as a carrier for delivering proteins into cells.

The design, synthesis, and evaluation of in vitro gene delivery efficacy of a novel series of oligo-Arg-lipid conjugates are described. 3,5-Bis(dodecyloxy)benzamide (BDB) was employed as the lipid component, and a poly(ethylene glycol) (PEG) spacer was introduced between the C-terminal of oligo-Arg and the amide group of BDB. Four derivatives with various oligo-Arg lengths (ArgN-PEG-BDB; N = 4, 6, 8, 10: the number of arginine residues) were prepared, and the effect of oligo-Arg length on the gene transfection was investigated in HeLa cells. Transfection efficiency increased as the number of arginine residues increased. Arg10-PEG-BDB showed the highest transfection efficiency, without severe toxicity to cells. These findings well corresponded to the cellular association of the Arg-PEG-BDB/DNA complex determined by flow cytometry. Even in the presence of serum, Arg10-PEG-BDB achieved appreciable cellular association and attained high gene expression. Thus, Arg10-PEG-BDB is potentially a simple and useful gene delivery tool, because one need only to mix it with plasmid DNA and apply the complexes to the cells even in a serum-containing medium.

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG2 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at approximately 200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG2 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatocytes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL-mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting.

Formulations of ultra-deformable liposomes containing bleomycin (Bleosome) have previously been described and proposed for topical treatment of skin cancer [Lau, K.G., Chopra, S., Maitani, Y., 2003. Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13, 237-239]. In this study, the stability of various Bleosome formulations was characterised and a purification process was established to isolate Bleosome for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 degrees C or at room temperature. Serially-diluted samples of this purified, high-strength product, 'high dose' were applied onto keratinocyte cell cultures to elucidate Bleosome LD50 profiles. In vitro data revealed that the LD50 of bleomycin encapsulated in Bleosome was approximately three-fold higher than free bleomycin solution for SCC cells, and nearly 30 times higher for NEB-1 cells. However, Bleosome containing 30 microg/ml of active bleomycin killed more than twice as many SCC cells than NEB-1 cells. At that concentration, the potency of liposomal bleomycin on causing cell death of SCC cells was found to be similar to that of free bleomycin solution. This effect was not seen on NEB-1 cells. It seems that SCC cells were particularly susceptible to Bleosome containing high levels of bleomycin. Results from these experiments promote the development of a novel product for the topical treatment of skin cancer.

In the present study, cholesteryl hemisuccinate (CHEMS) was evaluated for use as a membrane stabilizer in dipalmitoylphosphatidylcholine (DPPC) liposomes. Differential scanning calorimetry (DSC) and a calcein release study showed that CHEMS was more effective than cholesterol (CHOL) in increasing DPPC membrane stability. The findings of Fourier transform infrared spectroscopy (FT-IR) also suggested that CHEMS interacts with DPPC via both hydrogen bonding and electrostatic interaction. More importantly, CHEMS did not interact with saikosaponin-d (SSD), a triterpene saponin from Bupleurum species, unlike CHOL. SSD-containing liposomes with DPPC, CHEMS and DSPE-PEG could greatly decrease the hemolytic activity of SSD. This study demonstrated that CHEMS has more stabilization ability than CHOL since CHEMS may exhibit both hydrogen bond interaction and electrostatic interaction with DPPC membrane while CHOL only has hydrogen bond interaction, resulting in stable and low-hemolytic SSD-liposomes.

Recent advances in biotechnology have promoted biomolecular targeting of drugs, peptides and genes in the treatment and management of major diseases and infections. Therapeutic development of drugs and delivery systems may have various objectives: Systemic drugs require optimal delivery and uptake at target sites; peptide drugs require alternative routes of administration, such as nasal or intestinal absorption; gene medicines need to be delivered efficiently, safely and selectively to diseased areas. The propensity of ligand-modified liposomes to carry drugs and genes to desirable sites has been extensively examined and current reports show considerable progress in this field. Sterylglucoside (SG) is a novel absorption-enhancer of peptide drugs across nasal and intestinal mucosae. Physico-chemical properties and biodistribution of liposomes incorporating SG were studied and compared against the profiles of aglycon and sitosterol derivatives of SG. It was shown that SG particles aided colon drug delivery and increased bioavailability of peptide drugs after nasal and intestinal administration. In addition, they were able to enhance anticancer effects in liver cancer chemotherapy. Biological fate and interaction of SG with hepatocytes support the novel proposition of liver-targeting SG-liposomes.

The aim of this study was to examine the interaction of soybean-derived sterylglucoside (SG) with the human hepatoblastoma cell line HepG2 with regard to the penetration-enhancing effect of beta-sitosterol glucoside (Sit-G) to clarify the accumulation of SG-containing liposomes (SG-liposomes) to the liver in vivo. The approach was based on measurement of the association of SG-liposomes labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) in terms of asialoglycoprotein receptor (ASGP-R)-mediated endocytosis, affinity of Sit-G using lAsys and the association of FITC-dextran 4400 (FD-4) increased by Sit-G with the cells. The association of SG-liposomes was decreased by addition of asialofetuin, suggesting that SG-liposomes might be taken up via ASGP-R. Sit-G showed higher affinity with HepG2 cells than HeLa cells, and enhanced the association of FD-4 depending on the incubation time and Sit-G concentrations. Significant positive correlations were found between Sit-G and FD-4 association with the cells, indicating that Sit-G enhanced the drug penetration by distribution in cell membranes. The high degree of liver association of SG-liposomes in vivo might be related to recognition of glucose residues of SG by ASGP-R and to the high affinity and penetration-enhancing effect of Sit-G with hepatocytes.

We investigated the interaction of liposomes surface-modified with soybean-derived sterylglucoside (SG) (SG-liposomes) with HepG2 cells in the point of involvement of asialoglycoprotein receptor (ASGP-R) mediated endocytosis and examined the efficiency of SG-liposomes as drug carriers using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as a maker of liposome, carboxylated polystyrene microspheres (Fluoresbrite) as a model drug not taken up in cells and doxorubicin (DXR). SG-liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Ch) and SG (DPPC/Ch/SG=6:3:1, molar ratio) and DiI, Fluoresbrite and DXR were entrapped in SG-liposomes, respectively. Each SG-liposome was incubated with HepG2 cells at 4 or 37 degrees C, and co-incubated with asialofetuin (AF) as a competitor of ASGP-R. The association of DiI, Fluoresbrite or DXR entrapped in SG-liposomes with HepG2 cells at 37 degrees C was significantly higher than that in liposomes containing no SG. That of DiI and Fluoresbrite was reduced significantly by the incubation with AF, but that of DXR was not affected. These findings suggest that Fluoresbrite behaves like the lipid component of SG-liposomes, but DXR in SG-liposomes does not behave similar to the lipid component of SG-liposomes, thus, its drug behavior released from liposomes may be due to its physicochemical properties. SG-liposomes are potentially useful drug carriers to the liver, because the glucose residue may work as a kind of ligand for ASGP-R.

The effects of soybean-derived sterylglucoside (SG) on the fluidity of liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) were investigated compared with those of soybean-derived sterol (SS) and cholesterol (Ch) using an electron spin resonance spectrometer. Three kinds of liposomes were prepared in the molar ratio of DPPC/X=7/4, where X is SS, Ch or SG. The fluidity close to the polar head groups increased with an increase of temperature in the DPPC membrane containing SS, Ch and SG in the range 35 to 45 degrees C. Those near the hydrophobic end changed with an increase in temperature in liposomes containing SS, Ch and SG, which had a fluidizing effect on the DPPC membrane below the transition temperature (Tm, 41.9 degrees C) and a condensing effect over the Tm. The fluidizing effects of these compounds around 37 degrees C near the polar head group and the hydrophobic end increased in the following order: Ch < SG < or = SS and SS < Ch < SG, respectively. SG increased the fluidity of liposomal membrane dramatically above the Tm (35.4 degrees C). These results suggest that the high fluidity close to the hydrophobic end of the liposomal membranes around 37 degrees C, the decrease of Tm, and the sigmoidal nature of fluidity vs. temperature are important factors in the effectiveness of liposomes containing SG as a carrier of drugs.

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.

The blood clearance, tissue uptake and antitumor efficacy against liver metastasis of M5076 reticulosarcoma in mice and against primary liver cancer in rats of doxorubicin (DOX) encapsulated in two types of liposomes, with and without a soYbean-derived sterylglucoside mixture (SG), were examined. Liposomes entrapping DOX were composed of dipalmitoylphosphatidylcholine (DPPC), SG and cholesterol (Ch) at a molar ratio of 6:1:3, (SG-liposomes) and 6:0:4 (non-SG-liposomes). Pharmacokinetic analysis of drug disposition was based on the area under the curve (AUC) for liposomes up to 24 h following i.v. injection. SG-liposomes showed lower DOX concentrations in blood and higher concentrations in liver compared with non-SG-liposomes. The highest AUC of SG-liposomes in tissue was in liver, 2.4 times higher than that of the free drug. The antitumor efficacy of SG-liposomes was compared with that of free DOX and non-SG-liposomes at a dose of 5 mg DOX/kg. SG-liposomes displayed stronger antitumor activity than the free drug and non-SG-liposomes in murine reticulosarcoma M5076 tumor models and primary liver cancer models reflecting accumulation in hepatocytes. The antitumor activity of SG-liposomes in rats with primary liver cancer was significantly higher compared with free DOX and non-SG-liposomes (ILS: 92.7%).

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.

In this study, we investigated a liposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) for targeting the liver in mice. We found two distribution profiles of liposomes (reverse-phase evaporation vesicles, REV) in the liver depending on the concentration of Ch and SG in vivo; Ch tends to enhance liposomes containing 10 mol% SG accumulation in the liver, but to decrease those containing 20 mol% SG, and it prolongs the circulation in blood. Dicetylphosphate did not enhance liver targeting by liposomes with SG and Ch. More DPPC/SG/Ch-liposomes (6:1:3) were significantly taken up by cultivated hepatocytes than DPPC/SG/Ch-liposomes (6:0:4), suggesting a glucose residue. The optimal composition for the maximal liver targeting was a mixture of 0.2 micron DPPC/SG/Ch-liposomes (REV) at a molar ratio of 6:1:3. This composition of liposomes was distributed 3 times greater in hepatocytes than non-parenchymal cells 1 h after intravenous injection.

With a rapid demand to decrease the side effect of drug, a variety of drug delivery systems have been developed. This review will focus on the development of liposomes with soybean-derived sterols and their glucosides for drug carriers. Current status and further perspectives in this research field are reviewed mainly based on the results obtained in our laboratory. First we studied the different physicochemical properties of dipalmitoylphos-phatidylcholine (DPPC) liposomes with soybean-derived sterols (SS) and their glucosides (SG). SS rigidifies the liposomal membrane but SG fluidizes it. SS stabilizes liposomes more than cholesterol that is conventionally used as stabilizing agents in liposomes. On the basis of this information, we developed liposomes with SS and SG for a drug carrier. Secondly we studied the stability of liposomes in the blood and biodistribution and found that liposomes with SS were stable as expected in vitro results. In particular, DPPC: SS (7:4, molar ratio) size 0.2 micron showed long circulation. Thirdly successful targeting of the drugs to the liver was achieved by liposomes with SG. Finally, we succeeded in developing liposomal erythropoietin and doxorubicin using liposomes with SS for sustained release of drugs. Liposomal drugs increased the pharmacological effect compared with free drugs, suggesting a decrease of side effect and long circulation. The attempt for oral administration using liposomes of peptide drugs was carried out successfully. We have established that the study of physicochemical properties of liposomes is needed rationally as the distribution of drugs in liposomes and the rigidity of liposomal membrane, prior to the development of the drug carrier of liposomes. SS is useful to stabilize liposomes and SG to targeting to the liver parenchymal cells. This information can be useful and practical for the development of liposomes for drug carriers.

Intestinal absorption of recombinant human erythropoietin (Epo) encapsulated in liposomes (Epo/liposomes) was examined by measuring the pharmacological effects of Epo after oral administration in rats. Circulating reticulocyte counts after oral administration of Epo/liposomes showed a profile different from that after intravenous administration. Epo/liposomes 0.1 micron in diameter were absorbed more effectively than those 0.2 micron in diameter. In the 0.1 micron Epo/liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterols (SS), cholesterol (Ch), or soybean-derived sterylglucosides (SG), DPPC/SS (in molar ratio 7/2) and DPPC/Ch (7/2) showed higher efficiency in intestinal absorption than DPPC/Ch (7/4) and DPPC/SG (7/2) at a low dose by the sysmex method. Pharmacological availabilities for oral administration of Epo/liposomes were 0.74-31% and 3.3-30% as evaluated by circulating reticulocyte counts and percentage circulating reticulocytes of erythrocytes, respectively, in comparison to those for intravenous administration of the same dose.

We investigated the transfection efficiency of β-sitosterol βD-glucoside (Sit-G)-containing liposome/DNA complex (SitG-liposome/DNA complex) for liver targeting. The Sit-Gliposome/DNA complex was composed of Tfx-20 reagent (Tfx), ie synthetic cationic lipid [N,N,N′,N′-tetramethyI-N,N′bis (2-hydroxyethyl) -2, 3-di (oleo yloxy)-1, 4-butanediammonium iodide] with L-dioleoylphosphatidylethanolamine (DOPE), 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and Sit-G with plasmid DNA. The in vitro studies were performed in HepG2 cells in serum-containing medium and the in vivo studies were carried out in the mice following intravenous injection. The Sit-G-liposome produced a Sit-Gliposome/DNA complex of relatively small size (100-250 nm). Transfection efficiency of the luciferase marker gene by Sit-G-liposome/DNA complex was increased in the presence of 10% serum in vitro, and was selectively high in the mouse liver reaching expression values up to an average of 14.9 pg luciferase/mg tissue protein, compared with Tfx/DNA complex, which showed approximately three-fold higher gene expression than Sit-G-liposome/DNA complex in vitro. High in vitro transfection efficiency by Sit-G-liposome/DNA complex seemed to be possible even with large lipid precipitates, whereas high in vivo activity seemed to be related to small and dispersed complexes. The interaction of liposome/DNA complexes with serum may be a key point to predict the in vivo efficiency of a liposome vector. [ABSTRACT FROM AUTHOR]