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Development of an efficient small interfering RNA (siRNA) delivery method using non-viral carriers is necessary to determine potent therapeutic effects of RNA interference. Inflammatory responses induced by siRNA interaction with Toll-like receptors and retinoic-acid-inducible gene I protein/melanoma differentiation-associated gene 5 (RIG-I/MDA-5) are obstacles to the application of siRNAs in clinically. Here, we evaluated the effects on inflammatory responses by our siRNA delivery method using bubble lipoplexes with ultrasound (US) exposure in cultured macrophages. The effective gene suppression effects were obtained under low-toxic conditions in this siRNA transfer method. The interferon (IFN)-α after siRNA transfer using lipoplexes/bubble lipoplexes with US exposure was not detected. However, low levels of type I IFN mRNA production were induced through interaction of siRNA and cytoplasmic RIG-I/MDA-5, but not Toll-like receptors. Our findings indicate that it is possible to develop a safe and efficient siRNA delivery technique using mannosylated bubble lipoplexes and US exposure.

Tumour-associated macrophages (TAM) exhibit an M2 phenotype that promotes tumour progression, and conversion of M2 TAM toward a tumouricidal M1 phenotype is a promising anti-cancer therapy. As NF-κB is a key regulator of macrophage polarization, we developed an in vivo TAM-targeting delivery system that combines mannose-modified bubble liposomes/NF-κB decoy complexes (Man-PEG bubble lipoplexes) and ultrasound (US) exposure. We investigated the effects of NF-κB decoy transfection on TAM phenotype in solid tumour-bearing mice. Post-transfection tumour growth and survival rates were also recorded. Th2 cytokine (IL-10) level in TAM was significantly lower by NF-κB decoy transfection using Man-PEG bubble lipoplexes and US exposure, while Th1 cytokine levels (IL-1β, TNF-α and IL-6) were significantly higher when compared with controls. In addition, mRNA levels of vascular endothelial growth factor, matrix metalloproteinase-9 and arginase were significantly lower in TAM post-NF-κB decoy transfection. Importantly, TAM-targeted NF-κB decoy transfection inhibited tumour growth and prolonged survival rates in mice. Therefore, TAM-targeted NF-κB decoy transfection using Man-PEG bubble lipoplexes and US exposure may be an effective approach for anti-cancer therapy based on TAM phenotypic conversion from M2 toward M1.

Recently, our group developed ultrasound (US)-responsive and mannose-modified gene carriers (Man-PEG(2000) bubble lipoplexes), and successfully obtained a high level of gene expression in mannose receptor-expressing cells following gene transfection using Man-PEG(2000) bubble lipoplexes and US exposure. We also reported that large amounts of plasmid DNA (pDNA) were transferred into the cytoplasm of the targeted cells in the gene transfection using this method. In the present study, we investigated the involvement of transcriptional processes on enhanced gene expression obtained by unmodified and Man-PEG(2000) bubble lipoplexes with US exposure. The transcriptional process related to activator protein-1 (AP-1) and nuclear factor-κB (NFκB) was activated by US exposure, and was founded to be involved in enhanced gene expression obtained by gene transfection using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure. On the other hand, activation of AP-1 and NFκB pathways followed by US exposure was hardly involved in the inflammatory responses in the gene transfection using this method. These findings suggest that activation of AP-1 and NFκB followed by US exposure is involved in the enhanced gene expression using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure, and the selection of pDNAs activated by US exposure is important in this gene transfection method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG(2000) bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG(2000) bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG(2000) bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

DNA vaccination has attracted much attention as a promising therapy for the prevention of metastasis and relapse of malignant tumors, especially highly metastatic tumors such as melanoma. However, it is difficult to achieve a potent cancer vaccine effect by DNA vaccination, since the number of dendritic cells, which are the major targeted cells of DNA vaccination, is very few. Here, we developed a DNA vaccination for metastatic and relapsed melanoma by ultrasound (US)-responsive and antigen presenting cell (APC)-selective gene carriers reported previously, named Man-PEG₂₀₀₀ bubble lipoplexes. Following immunization using US exposure and Man-PEG(2000) bubble lipoplexes constructed with pUb-M, which expresses ubiquitylated melanoma-specific antigens (gp100 and TRP-2), the secretion of Th1 cytokines (IFN-γ and TNF-α) and the activities of cytotoxic T lymphocytes (CTLs) were specifically enhanced in the presence of B16BL6 melanoma antigens. Moreover, we succeeded in obtaining potent and sustained DNA vaccine effects against solid and metastatic tumor derived from B16BL6 melanoma specifically. The findings obtained from this study suggest that the gene transfection method using Man-PEG₂₀₀₀ bubble lipoplexes and US exposure could be suitable for DNA vaccination aimed at the prevention of metastatic and relapsed cancer.

Purpose: To explore the effect of NFκB activation in macrophages on osteoclastogenesis of bone marrow cells for potential application as a new type of therapy for preventing bone loss., Methods: Primary cultured macrophages and bone marrow cells were prepared from mice. As macrophage-targeted carriers, Mannosylated cationic liposomes (Man-liposomes) were prepared and were allowed to form complexes with NFκB decoy (a double-stranded oligonucleotide). Cellular uptake, inhibition of NFκB activation, and cytokine production were evaluated using macrophages. Osteoclastogenesis was investigated using bone marrow cells, which were cultured in the conditioned medium prepared from macrophages with or without Man-liposome/NFκB decoy complexes treatment., Results: Cellular accumulation of NFκB decoy was enhanced by Man-liposome. NFκB activation in macrophages and TNF-α production were suppressed in macrophages by Man-liposome/NFκB decoy complexes but not by the naked NFκB decoy, Gal-liposome/NFκB decoy complexes, or Man-liposome/random decoy complexes. Osteoclastogenesis of bone marrow cells was induced in the conditioned medium prepared from activated macrophages but not by activated macrophages treated with Man-liposome/NFκB decoy complexes., Conclusion: Osteoclastogenesis induced by activated macrophages could be suppressed by the treatment macrophages with Man-liposome/NFκB decoy complexes. Macrophage-targeted delivery of NFκB decoys using Man-liposomes may be promising in its use for the remediation of bone loss.

The nuclear factor kappa B (NFκB) signaling pathway is a key mechanism in the pathophysiology of lung inflammation. NFκB is critically responsible for the expression of pro-inflammatory mediators following activation. The specific inhibition of NFκB by a NFκB decoy via inhalation appears to improve therapeutic effects. However, administration of naked NFκB decoy limits the efficacy of the decoy strategy due to low targeting ability to immune cells such as alveolar macrophages. In this study, we have assessed the effect of alveolar macrophage-targeted NFκB decoy by mannosylated (Man) cationic liposomes in a LPS-induced lung inflammation model after intratracheal administration. The complex of Man-cationic liposome/NFκB decoy was physically stable during spraying. Man-cationic liposome/NFκB decoy complex was selectively delivered to alveolar macrophages for subsequent localization of NFκB decoy in the cytoplasm and to a lesser extent in the nucleus. In the LPS-induced lung inflammation model, pre-treatment with Man-cationic liposome/50μg NFκB decoy complex significantly inhibited the release of TNF-α, IL-1β and CINC-1, neutrophil infiltration and NFκB activation compared with naked NFκB decoy, cationic liposome/NFκB decoy complex and Man-cationic liposome/scrambled decoy complex treatments. This study demonstrates the sufficient targeting of NFκB decoy using Man-cationic liposomes in a novel effective anti-inflammatory therapy for lung inflammation., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG(2000) bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG(2000) bubble lipoplexes and US exposure enabled approximately 500-800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers., (Copyright 2010 Elsevier Ltd. All rights reserved.)

To achieve effective gene therapy, it is necessary to selectively and efficiently transfect therapeutic gene into targeted cells. In this study, we developed a combination method using mannosylated lipoplexes, which show selectivity to antigen-presenting cells such as macrophages and dendritic cells, and bubble liposomes (BLs), which are known to enhance transfection efficiency on exposure to ultrasound (US). In cultured mouse macrophages, known for the expression of mannose receptors, the transfection efficiency of this combination method using mannosylated lipoplexes and BLs with US was higher than that of naked pDNA or unmodified lipoplexes and BLs. In the liver and spleen, the in vivo transfection efficiency of this combination method was significantly higher than that of naked pDNA or unmodified lipoplexes and BLs with US. Transfection in hepatic nonparenchymal cells using this combination method was about 12 times higher than that in hepatic parenchymal cells. As far as splenic transfection activities were concerned, the transfection efficiency of this combination method in CD11c(+) cells was significantly higher than that in CD11c(-) cells. In conclusion, we demonstrated that the gene transfection efficiency in macrophages and dendritic cells was significantly increased by this combination method using mannosylated lipoplexes and BLs with US exposure.

The success of targeting systems to alveolar macrophages critically depends on internalization into these cells for pharmacological intervention. Direct respiratory delivery via inhalation of mannose modified liposomal carriers to alveolar macrophages is of great interest. To evaluate the targeting efficiency to alveolar macrophages by intratracheal administration of mannosylated liposomes (Man-liposomes), Man-liposomes with various ratio of mannosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) as mannose receptor ligand were investigated with regard to their in vitro uptake in primary cultured alveolar macrophages and in vivo intratracheal administration in rats. The in vitro uptake of Man-liposomes took place in a concentration-dependent manner. The internalization of Man-liposomes with 7.5% (Man-7.5-liposomes) and 5.0% (Man-5.0-liposomes) Man-C4-Chol was considerably higher than that of Man-liposomes with 2.5% of Man-C4-Chol (Man-2.5-liposomes) and Bare-liposomes and significantly inhibited by an excess of mannan, suggesting mannose receptor-mediated endocytosis. After intratracheal administration of Man-7.5 and Man-5.0-liposomes in rats, a significantly high internalization and selective targeting to alveolar macrophages was observed. The enhanced cellular uptake in alveolar macrophages related to the mannose density of Man-liposomes was also confirmed both in vitro and in vivo confocal microscopy studies. These results demonstrate the efficient targeting to alveolar macrophages by the intratracheally administered Man-liposomes via mannose receptor-mediated endocytosis.

Carbohydrate grafted emulsions are one of the most promising cell-specific targeting systems for lipophilic drugs. We have previously reported that mannosylated (Man-) emulsions composed of soybean oil, EggPC and cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) with a ratio of 70:25:5 were significantly delivered to liver non-parenchymal cells (NPC) via mannose receptor-mediated mechanism after intravenous administration in mice. Since the efficient targeting through a receptor-mediated mechanism is largely controlled by ligand-receptor interaction, the effect of mannose density on Man-emulsions was studied with regard to both the disposition in vivo in mice and the uptake in vitro, using elicited macrophages which express a number of mannose receptors. After intravenous injection, Man-emulsions with 5.0% (Man-5.0-emulsions) and 7.5% (Man-7.5-emulsions) of Man-C4-Chol were rapidly eliminated from the blood circulation and preferentially accumulated in the liver-NPC compared with Man-emulsions with 2.5% of Man-C4-Chol (Man-2.5-emulsions) and bare emulsions (Bare-emulsions). The in vitro study showed increased internalization of Man-5.0- and Man-7.5-emulsions and significant inhibition of uptake in the presence of mannan. The enhanced uptake of Man-emulsions was related to the increasing of Man-C4-Chol content that corresponded to confocal microscopy study. These results suggest that the mannose density of Man-emulsions plays an important role in both cellular recognition and internalization via a mannose receptor-mediated mechanism.

The purpose of this study was to inhibit endotoxin induced cytokines production and liver injury by liver non-parenchymal cell (NPC) selective delivery of nuclear factor kappaB (NFkappaB) decoy using mannosylated cationic liposomes (Man-liposomes). In this study, we examined the distribution, inhibitory effect on cytokines production and ALT/AST of intravenously injected Man-liposome/NFkappaB decoy complex. Man-liposome/[(32)P] NFkappaB decoy complexes mostly accumulated in the liver, preferentially in NPC. In a murine lipopolysaccharide-induced liver failure model, the production of tumor necrosis factor-alpha (TNFalpha), IFNgamma, IL1-beta, ALT and AST were effectively reduced by Man-liposome complexes. However, cationic or galactosylated cationic liposome complexes could not inhibit TNFalpha production.

The kinetics and specificity of the molecular interaction between proteins modified with varying numbers of mannose residues and isolated rabbit mannan-binding lectin (MBL) were characterized by using surface plasmon resonance spectroscopy (SPR). Mannosylated bovine serum albumin (Man-BSA) with different numbers of mannoses and other mannosylated derivatives of lysozyme (LZM), soybean trypsin inhibitor (STI), superoxide dismutase (SOD) and bovine gamma-immunoglobulin (IgG) were synthesized. Rabbit MBL was isolated by affinity column chromatography and immobilized on the SPR sensor chip via avidin-biotin binding. Binding of Man-BSAs to immobilized rabbit MBL increased with an increase in the number of mannose residues, primarily due to the reduction in dissociation rate. On the other hand, the association rate constant was similar for five mannosylated proteins investigated, whereas the dissociation rate constant differed markedly in spite of the same degree of mannosylation. Specific binding of mannosylated proteins to MBL may depend on the number of mannose residues and their steric configurations.

In our previous studies, serum mannan-binding protein (MBP) accelerated the uptake by cultured macrophages. The present study was initiated to investigate the kinetics of molecular interaction between mannosylated liposomes and MBP in more details and the effects of lipid composition on the interaction. The analysis was carried out by surface plasmon spectroscopy (SPR) methods, using rabbit serum MBP isolated by affinity chromatography. In SPR studies, neither conventional liposomes nor galactosylated liposomes indicated any interaction, but each mannosylated liposomes had a high response signal corresponding to molecular interaction with immobilized MBP. Association of mannosylated liposomes to serum MBP was not dependent on the lipid composition, suggesting a diffusion-controlled association. Dissociation of the mannosylated liposomes from serum MBP was extremely slow. DSPC/Chol/Man-C4-Chol (90 : 5 : 5, molar ratio) exhibited a slower dissociation rate than DSPC/Chol/Man-C4-Chol (60 : 35 : 5). Clustering of mannose residues on liposomal surfaces might be important in determining the binding affinity of mannosylated liposomes with MBP.

We have previously reported that mannosylated cationic liposome consisting with the mannosylated cationic cholesterol derivative Man-C4-Chol (Man) and dioleoylphosphatidylethanolamine (DOPE) (Man/DOPE) could deliver DNA to the liver by intravenous administration via mannose receptor-mediated endocytosis, however, rapid degradation in lysosomes might be a rate-limiting step in its gene transfection. In this study, we tried to evaluate the role of DOPE in in vivo gene transfer by comparing its transfection efficacy with mannosylated liposomes composed of Man and dioleoylphosphatidylcholine (DOPC) (Man/DOPC). In vitro studies showed that the cellular association of both liposome/pCMV-Luc complexes was almost the same, although Man/DOPE complex showed about 10-fold higher transfection activity than Man/DOPC complex. After intraportal administration into mice, Man/DOPE complex showed higher gene expression than Man/DOPC complex, suggesting that DOPE improves intracellular trafficking in target cells under in vivo conditions. An intravenous administration study demonstrated that Man/DOPE complex was accumulated in the liver more efficiently and achieved a higher gene expression in the liver than Man/DOPC complex. Thus, we conclude that the property of DOPE in mannosylated liposomes contributes to the efficient gene expression in the target site through enhanced distribution to the target site and intracellular sorting in the target cells under in vivo conditions.

Cell-specific drug delivery is one of the most promising strategies for improving therapeutic efficiency and minimizing systemic toxicity. Carrier systems devoted to receptor-mediated targeting need to be developed. In the case of liver-non-parenchymal cell-specific targeting systems, glycosylated emulsions have been developed as carriers for lipophilic drugs and/or peptides. This present study demonstrates the in vivo disposition behaviour and pharmacokinetic characteristics of mannosylated (Man-) and fucosylated (Fuc-) emulsions incorporated with cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and its fucosylated derivatives (Fuc-C4-Chol), respectively. Man- (or Fuc-) emulsions are composed of soybean oil, EggPC and Man-C4-Chol (or Fuc-C4-Chol) in a weight ratio of 70:25:5. After intravenous administration to mice, these two types of [(3)H]cholesteryl hexadecyl ether (CHE)-labelled glycosylated emulsions were rapidly eliminated from the blood circulation and preferentially recovered in the liver. In contrast, bare (Bare-) emulsions composed of soybean oil:EggPC:cholesterol (Chol) in a weight ratio of 70:25:5 were more retained in the blood circulation. The hepatic uptake clearances of Man- and Fuc-emulsions were 3.3- and 4.0-times greater than that of Bare-emulsions. Interestingly, the hepatic uptake clearance of Fuc-emulsions was significantly higher that that of Man-emulsions. The uptake ratios by non-parenchymal cells (NPC) and parenchymal cells (PC) (NPC/PC ratio) for Bare-, Man- and Fuc-emulsions were found to be 0.4, 2.0 and 2.9, respectively. The hepatic uptakes of [(3)H]CHE-labelled Man- and Fuc-emulsions were reduced by pre-dosing with glycosylated proteins and liposomes. These results clearly support the conclusion that Man- and Fuc-emulsions are promising carrier systems for liver NPC-specific targeting via receptor-mediated mechanism.

The purpose of this study is to delineate uptake characteristics of mannosylated and fucosylated proteins in primary cultured sinusoidal endothelial cells and Kupffer cells. In cultured sinusoidal endothelial cells, uptake of mannosylated and fucosylated bovine serum albumin (BSA) was significantly inhibited by excess mannosylated and fucosylated BSAs but not by galactosylated BSA, suggesting that both glycosylated proteins might be primarily taken up via mannose receptors. In cultured Kupffer cells, uptake of fucosylated BSA was significantly inhibited by excess galactosylated BSA as well as mannosylated and fucosylated BSAs, although that of mannosylated BSA was inhibited only by mannosylated and fucosylated BSAs. This suggests that uptake of fucosylated BSA by Kupffer cells might be mediated by both Kupffer cell lectin (fucose receptor) and mannose receptor. On the other hand, in vivo hepatic uptake of fucosylated BSA was inhibited to a greater extent by GdCl3 pretreatment than that of mannosylated BSA. Based on in vitro and in vivo experiments, it was concluded that fucosylated BSA is more Kupffer cell-selective because it exhibited a lower sinusoidal endothelial cell uptake than mannosylated BSA.

We have previously reported that, unlike a lipoplex and mannosylated (Man) lipoplex underwent gene transfer to liver nonparenchymal cells (NPC) that possess mannose receptors after intravenous administration in mice. In this study, the tissue, intrahepatic distribution, and subcellular localization of the lipoplex after intravenous administration were investigated. DC-Chol liposome was selected as a cationic liposomes. After administration of lipoplex and Man lipoplex, the high gene expression was observed in the lung and liver, respectively. After administration of [32P]Man lipoplex, about 80% of [32P]plasmid DNA (pDNA) was accumulated in the liver. As for the intrahepatic distribution, the NPC/parenchymal cells (PC) ratio of [32P]Man lipoplex was 9.64, whereas the NPC/PC ratio of [32P]lipoplex was 1.93. The radioactivity in the cytosolic fraction of liver homogenate of [111In]Man lipoplex was two-fold higher than that of [111In]lipoplex, indicating that Man liposomes facilitate the release of pDNA into the cytosolic space. However, a rapid sorting of the radioactivity from endosomes to lysosomes was observed with the [111In]Man lipoplex. Also, amplification of pDNA by PCR suggested that the Man lipoplex is more rapidly degraded within the intracellular vesicles than the lipoplex. These results suggested that modulation of its intracellular sorting could improve the transfection efficiency of Man lipoplex.

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.

Mannosylated liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar liposomes consisting of cholesterol and distearoyl phosphatidylcholine (DSPC). The biodistribution of liposomes labeled with [3H]cholesteryl hexadecyl ether was examined in mice. The rate and extent of the hepatic uptake of those [3H]liposomes increased proportionally on increasing the mixing ratio of Man-C4-Chol. Their hepatic uptake was reduced by increasing the administered dose due to the limited number of mannose receptors. The liver uptake of [3H]Man-liposomes was preferentially mediated by liver non-parenchymal cells (NPC) and significantly inhibited by co-injection with an excess of Man-BSA, indicating the involvement of a mannose receptor-mediated mechanism in the hepatic uptake of Man-liposomes. Muramyl dipeptide (MDP), an immunomodulator, was also incorporated into the liposomes and its inhibitory effect in an experimental liver metastasis model was examined. In contrast to free MDP treatment, which showed little effect on the inhibition of metastasis, liposomal MDP significantly reduced the number of metastatic colonies in the liver. Active targeting of MDP to liver NPC by Man-liposomes resulted in more effective inhibition than delivery of MDP by liposomes without mannose. Treatment with MDP/Man-liposomes further increased the survival of the tumor-bearing mice. These results suggest that Man-liposomes are effective carriers for targeted delivery of bioactive compounds to liver NPC.

Presents information on a study which investigated in vivo recognition of mannosylated proteins by hepatic mannose receptors and serum mannan-binding protein. Materials and methods; Results of the study; Discussion.

A method involving the use of doxorubicin-loaded polyethylene-glycol-modified liposomes and transfection using mannose-modified bubble lipoplexes in combination with ultrasound irradiation may be a promising approach to cancer treatment; it could not only suppress early-stage tumor growth but also enhance transfection efficacy in antigen-presenting cells, thus enhancing the therapeutic potential of a DNA vaccine. However, to date only limited research has been carried out regarding this combination DNA vaccination method for use in cancer therapy. In this study, we examined the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier combined with doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Doxorubicin-encapsulated PEGylated liposomes activated transcriptional factors, such as nuclear factor-κB and AP-1 in the spleen; subsequently pUb-M, ubiquitylated melanoma-related antigen encoding plasmid DNA expression in splenic cells was significantly enhanced. Moreover, effective cytotoxic T-lymphocyte activities were stimulated by DNA vaccination combined with the administration of doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Furthermore, potent DNA vaccine effects against established solid tumor and metastatic tumor derived from B16BL6 melanoma were observed. These results suggest that the combined use of DNA vaccination with doxorubicin-encapsulated polyethylene-glycol-modified liposomes could be an effective method for the treatment of melanoma using immunotherapy. [ABSTRACT FROM AUTHOR]