Intraperitoneal administration of nanoparticles containing tocopheryl succinate prevents peritoneal dissemination.

Intraperitoneal administration of anticancer nanoparticles is a rational strategy for preventing peritoneal dissemination of colon cancer due to the prolonged retention of nanoparticles in the abdominal cavity. However, instability of nanoparticles in body fluids causes inefficient retention, reducing its anticancer effects. We have previously developed anticancer nanoparticles containing tocopheryl succinate, which showed high in vivo stability and multifunctional anticancer effects. In the present study, we have demonstrated that peritoneal dissemination derived from colon cancer was prevented by intraperitoneal administration of tocopheryl succinate nanoparticles. The biodistribution of tocopheryl succinate nanoparticles was evaluated using inductively coupled plasma mass spectroscopy and imaging analysis in mice administered quantum dot encapsulated tocopheryl succinate nanoparticles. Intraperitoneal administration of tocopheryl succinate nanoparticles showed longer retention in the abdominal cavity than by its intravenous (i.v.) administration. Moreover, due to effective biodistribution, tumor growth was prevented by intraperitoneal administration of tocopheryl succinate nanoparticles. Furthermore, the anticancer effect was attributed to the inhibition of cancer cell proliferation and improvement of the intraperitoneal microenvironment, such as decrease in the levels of vascular endothelial growth factor A, interleukin 10, and M2-like phenotype of tumor-associated macrophages. Collectively, intraperitoneal administration of tocopheryl succinate nanoparticles is expected to have multifaceted antitumor effects against colon cancer with peritoneal dissemination.

Tocopheryl Phosphate Inhibits Rheumatoid Arthritis-Related Gene Expression In Vitro and Ameliorates Arthritic Symptoms in Mice.

Anti-rheumatoid arthritis (RA) effects of α-tocopherol (α-T) have been shown in human patients in a double-blind trial. However, the effects of α-T and its derivatives on fibroblast-like synoviocytes (FLS) during the pathogenesis of RA remain unclear. In the present study, we compared the expression levels of genes related to RA progression in FLS treated with α-T, succinic ester of α-T (TS), and phosphate ester of α-T (TP), as determined via RT-PCR. The mRNA levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-3, and MMP-13 were reduced by treatment with TP without cytotoxicity, while α-T and TS did not show such effects. Furthermore, intraperitoneal injection of TP ameliorated the edema of the foot and joint and improved the arthritis score in laminarin-induced RA model mice. Therefore, TP exerted anti-RA effects through by inhibiting RA-related gene expression.

Rapid modification of antibodies on the surface of liposomes composed of high-affinity protein A-conjugated phospholipid for selective drug delivery.

Antibody-modified liposomes, immuno-liposomes, can selectively deliver encapsulated drug 'cargos' to cells via the interaction of cell surface proteins with antibodies. However, chemical modification of both the antibodies and phospholipids is required for the preparation of immuno-liposomes for each target protein using conventional methods, which is time-consuming. In the present study, we demonstrated that high-affinity protein A- (Protein A-R28: PAR28) displaying liposomes prepared by the post-insertion of PAR28-conjugated phospholipid through polyethylene glycol (PEG)-linkers (PAR28-PEG-lipo) can undergo rapid modification of antibodies on their surface, and the liposomes can be delivered to cells based on their modified antibodies. Anti-CD147 and anti-CD31 antibodies could be modified with PAR28-PEG-lipo within 1 h, and each liposome was specifically taken up by CD147- and CD31-positive cells, respectively. The cellular amounts of doxorubicin delivered by anti-CD147 antibody-modified PAR28-PEG-lipo were significantly higher than those of isotype control antibody-modified liposomes. PAR28-PEG-lipo can easily and rapidly undergo modification of various antibodies on their surface, which then makes them capable of selective drug delivery dependent on the antibodies.

α-Tocopheryl succinate stabilizes the structure of tumor vessels by inhibiting angiopoietin-2 expression.

α-Tocopheryl succinate (TS) is a tocopherol derivative and has multifaceted anti-cancer effects; TS not only causes cancer cell-specific apoptosis but also inhibits tumor angiogenesis. Although TS has the potential to be used as a well-tolerated anti-angiogenic drug, it is still unclear which step of the angiogenic process is inhibited by TS. Here, we show that TS inhibits the expression of angiopoietin (Ang)-2, which induces destabilization of vascular structure in the initial steps of the angiogenic process. In mouse melanoma cells, TS treatment decreased mRNA and extracellular protein levels of Ang-2; however, the mRNA level of Ang-1, which stabilizes the vascular structure, remained unchanged. Furthermore, aorta ring and Matrigel plug angiogenesis assays indicated that the conditioned medium from TS-treated cells (CM-TS) inhibited neovascularization and blood leakage from the existing blood vessels, respectively. Following immunohistochemical staining of the vessels treated with CM-TS, imaging studies showed that the vascular endothelial cells were highly packed with pericytes. In conclusion, we found that TS inhibits Ang-2 expression and, consequently, stabilizes the vascular structure during the initial step of tumor angiogenesis.

Low Electric Treatment activates Rho GTPase via Heat Shock Protein 90 and Protein Kinase C for Intracellular Delivery of siRNA.

Low electric treatment (LET) promotes intracellular delivery of naked siRNA by altering cellular physiology. However, which signaling molecules and cellular events contribute to LET-mediated siRNA uptake are unclear. Here, we used isobaric tags in relative and absolute quantification (iTRAQ) proteomic analysis to identify changes in the levels of phosphorylated proteins that occur during cellular uptake of siRNA promoted by LET. iTRAQ analysis revealed that heat shock protein 90 (Hsp90)α and myristoylated alanine-rich C-kinase substrate (Marcks) were highly phosphorylated following LET of NIH 3T3 cells, but not untreated cells. Furthermore, the levels of phosphorylated Hsp90α and protein kinase C (PKC)γ were increased by LET both with siRNA and liposomes having various physicochemical properties used as model macromolecules, suggesting that PKCγ activated partly by Ca2+ influx as well as Hsp90 chaperone function were involved in LET-mediated cellular siRNA uptake. Furthermore, LET with siRNA induced activation of Rho GTPase via Hsp90 and PKC, which could contribute to cellular siRNA uptake accompanied by actin cytoskeleton remodeling. Collectively, our results suggested that LET-induced Rho GTPase activation via Hsp90 and PKC would participate in actin-dependent cellular uptake of siRNA.

Carotenoid Stereochemistry Affects Antioxidative Activity of Liposomes Co-encapsulating Astaxanthin and Tocotrienol.

We previously found that antioxidative activity of liposomes co-encapsulating astaxanthin (Asx) and tocotrienols (T3s) was higher than the calculated additive activity, which results from intermolecular interactions between both antioxidants (J. Clin. Biochem. Nutr., 59, 2016, Kamezaki et al.). Herein, we conducted experiments to optimize Asx/α-T3 ratio for high antioxidative activity, and tried to elucidate details of intermolecular interaction of Asx with α-T3. Higher activity than calculated additive value was clearly observed at an Asx/α-T3 ratio of 2 : 1, despite two α-T3 would potentially interact with two terminal rings of one Asx. The synthetic Asx used in this study was a mixture of three stereoisomers, 3R,3'R-form (Asx-R), 3S,3'S-form (Asx-S) and 3R,3'S-meso form (Asx-meso). The calculated binding energy of the Asx-S/α-T3 complex was higher than those of Asx-R/α-T3 and Asx-meso/α-T3, suggesting that Asx-S and α-T3 is the most preferable combination for the intermolecular interaction. The optimal Asx-S/α-T3 ratio for antioxidation was shown to be 1 : 2. These results suggest that the Asx stereochemistry affects the intermolecular interaction of Asx/α-T3. Moreover, the absorption spectrum changes of Asx-S upon co-encapsulation with α-T3 in liposomes indicate that the electronic state of Asx-S is affected by intermolecular interactions with α-T3. Further, intermolecular interactions with α-T3 affected the electronic charges on the C9, C10 and C15 atoms in the polyene moiety of Asx-S. In conclusion, the intermolecular interaction of Asx/T3 depends on the Asx stereochemistry, and caused a change in the electronic state of the Asx polyene moiety by the presence of double bond in the T3 triene moiety.

Prevention of UV-Induced Melanin Production by Accumulation of Redox Nanoparticles in the Epidermal Layer via Iontophoresis.

UV rays induce melanin production in the skin, which, from a cosmetic point of view, is problematic. Reactive oxygen species (ROS) generated in the skin upon UV irradiation are thought to be responsible for melanin production. Thus, effective antioxidants are recognized as useful tools for prevention of UV-induced melanin production. Redox nanoparticles (RNPs) containing nitroxide radicals as free radical scavengers were previously developed, and shown to be effective ROS scavengers in the body. RNPs are therefore expected to be useful for effective protection against UV-induced melanin production. However, as the sizes of RNPs are typically larger than the intercellular spaces of the skin, transdermal penetration is difficult. We recently demonstrated effective transdermal delivery and accumulation of nanoparticles in the epidermal layer via faint electric treatment, i.e., iontophoresis, suggesting that iontophoresis of RNPs may be a useful strategy for prevention of UV-induced melanin production in the skin. Herein, we performed iontophoresis of RNPs on the dorsal skin of hairless mice that produce melanin in response to light exposure. RNPs accumulated in the epidermal layer upon application of iontophoresis. Further, the combination of RNPs with iontophoresis decreased UV-induced melanin spots and melanin content in the skin. Taken together, we successfully demonstrated that iontophoresis-mediated accumulation of RNPs in the epidermis prevented melanin production.

Tumor Microenvironment-Sensitive Liposomes Penetrate Tumor Tissue via Attenuated Interaction of the Extracellular Matrix and Tumor Cells and Accompanying Actin Depolymerization.

Delivery of anticancer drugs into tumor cores comprised of malignant cancer cells can result in potent therapeutic effects. However, conventional nanoparticle-based drug delivery systems used for cancer therapy often exhibit inefficient tumor-penetrating properties, thus, suggesting the need to improve the functional design of such systems. Herein, we focus on the interactions between cancer cells and the extracellular matrix (ECM) and demonstrate that liposomes modified with slightly acidic pH-sensitive peptide (SAPSp-lipo) can penetrate in vivo tumor tissue and in vitro spheroids comprised of cancer cells and ECM. We previously reported SAPSp-lipo, tumor microenvironment-sensitive liposomes, are effectively delivered to tumor tissue (Hama et al. J Control Release 2015, 206, 67-74). Compared with conventional liposomes, SAPSp-lipo could be delivered to deeper regions within both spheroids and tumor tissues. Furthermore, tumor penetration was found to be promoted at regions where actin depolymerization was induced by SAPSp-lipo and inhibited by the polymerization of actin. In addition, SAPSp-lipo attenuated the interaction between cancer cells and ECM, contributing to the penetration of SAPSp-lipo. These results suggest that SAPSp-lipo penetrates tumors via the interspace route and is accompanied by actin depolymerization. Taken together, SAPSp-lipo demonstrates potential as a novel tumor-penetrable drug carrier for induction of therapeutic effects against malignant cells that comprise tumor cores.

Development of flexible nanocarriers for siRNA delivery into tumor tissue.

Various non-viral delivery systems for small interfering RNAs (siRNA) have been developed. Such delivery systems generally exhibit tightly formed spherical structures. While such carriers have demonstrated good transfection activity in mono-layered cell systems, effects against solid tumors are often less apparent and difficult to demonstrate, likely due to the rigid structures of the carriers, which may prevent penetration to deeper regions within tumor tissue. Herein, we developed a flexible nanocarrier (FNC) system that is able to penetrate to deeper regions within tumor tissue. Specifically, we employed previously found flexible polyplexes comprised of siRNA and poly-l-lysine as wick structures for the preparation of FNCs. FNCs were constructed by coating the wick structures with lipids using a liposomal membrane fusion method. The diameters of the resulting FNCs were ca. 170nm, and the shapes were non-spherical. Lipid coating was confirmed using a nuclease resistance assay. Furthermore, FNCs showed significant RNA interference effects, comparable to Lipofectamine 2000, in a mono-layered cell system. To accelerate tumor penetration, the FNC surface was modified with polyethylene glycol (PEG) and the tight junction opener peptide AT1002. Surface-modified FNCs demonstrated effective penetrability into a cancer spheroid. Thus, we developed a novel and unique tumor-penetrable siRNA FNC system.

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment.

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

Synergistic antioxidative effect of astaxanthin and tocotrienol by co-encapsulated in liposomes.

Astaxanthin and vitamin E are both effective antioxidants that are frequently used in cosmetics, as food additives, and in to prevent oxidative damage. A combination of astaxanthin and vitamin E would be expected to show an additive anntioxidative effect. In this study, liposomes co-encapsulating astaxanthin and the vitamin E derivatives α-tocopherol (α-T) or tocotrienols (T3) were prepared, and the antioxidative activity of these liposomes toward singlet oxygen and hydroxyl radical was evaluated in vitro. Liposomes co-encapsulating astaxanthin and α-T showed no additive anntioxidative effect, while the actual scavenging activity of liposomes co-encapsulating astaxanthin and T3 was higher than the calculated additive activity. To clarify why this synergistic effect occurs, the most stable structure of astaxanthin in the presence of α-T or α-T3 was calculated. Only α-T3 was predicted to form hydrogen bonding with astaxanthin, and the astaxanthin polyene chain would partially interact with the α-T3 triene chain, which could explain why there was a synergistic effect between astaxanthin and T3 but not α-T. In conclusion, co-encapsulation of astaxanthin and T3 induces synergistic scavenging activity by intermolecular interactions between the two antioxidants.

Effective cytoplasmic release of siRNA from liposomal carriers by controlling the electrostatic interaction of siRNA with a charge-invertible peptide, in response to cytoplasmic pH.

Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is effectively released via electrostatic repulsion of siRNA with negatively charged SAPSP at cytoplasmic pH (7.4). The condensed complex of siRNA and positively-charged SAPSP at acidic pH (siRNA/SAPSP) was found to result in almost complete release of siRNA upon charge inversion of SAPSP at pH 7.4, with the resultant negatively-charged SAPSP having no undesirable interactions with endogenous mRNA. Moreover, liposomes encapsulating siRNA/SAPSP demonstrated knockdown efficiencies comparable to those of commercially available siRNA carriers. Taken together, SAPSP may be very useful as a siRNA condenser, as it facilitates effective cytoplasmic release of siRNA, and subsequent induction of specific RNAi effects.

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm.

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.

Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

Overcoming the polyethylene glycol dilemma via pathological environment-sensitive change of the surface property of nanoparticles for cellular entry.

Modification with polyethylene glycol (PEG) is currently considered an important strategy for anti-cancer drug delivery, because PEGylated-nanoparticles would be effectively delivered to tumor tissue by enhanced permeation and retention effects. However, PEGylation suppresses the cellular uptake of nanoparticles (NPs) to target cells (known as the PEG dilemma). Here, we propose a novel strategy, namely conferring a pathological environment-sensitive property of nanoparticles for overcoming the PEG dilemma. Specifically, although nanoparticles have an overall negative surface charge to avoid interactions with biogenic substances in blood circulation, inversion of surface charge (to positive) at the pH of the tumor microenvironment may allow the nanoparticles to be taken up by cancer cells. To prove this concept, charge-invertible nanoparticles modified with novel slightly acidic pH-sensitive peptide (SAPSP-NPs) were developed. The negatively-charged SAPSP-NPs were delivered to tumor tissue, and were successfully taken up by cancer cells upon inversion of the surface charge to positive at intratumoral pH. SAPSP-NPs may serve as an alternative carrier to the PEGylated NP for anti-cancer drug delivery.

Anti-cancer vaccination by transdermal delivery of antigen peptide-loaded nanogels via iontophoresis.

Transdermal vaccination with cancer antigens is expected to become a useful anti-cancer therapy. However, it is difficult to accumulate enough antigen in the epidermis for effective exposure to Langerhans cells because of diffusion into the skin and muscle. Carriers, such as liposomes and nanoparticles, may be useful for the prevention of antigen diffusion. Iontophoresis, via application of a small electric current, is a noninvasive and efficient technology for transdermal drug delivery. Previously, we succeeded in the iontophoretic transdermal delivery of liposomes encapsulating insulin, and accumulation of polymer-based nanoparticle nanogels in the stratum corneum of the skin. Therefore, in the present study, we examined the use of iontophoresis with cancer antigen gp-100 peptide KVPRNQDWL-loaded nanogels for anti-cancer vaccination. Iontophoresis resulted in the accumulation of gp-100 peptide and nanogels in the epidermis, and subsequent increase in the number of Langerhans cells in the epidermis. Moreover, tumor growth was significantly suppressed by iontophoresis of the antigen peptide-loaded nanogels. Thus, iontophoresis of the antigen peptide-loaded nanogels may serve as an effective transdermal delivery system for anti-cancer vaccination.

Effective capture of proteins inside living cells by antibodies indirectly linked to a novel cell-penetrating polymer-modified protein A derivative.

Antibodies against cytoplasmic proteins are useful tools that can control cellular function and clarify signaling mechanisms. However, it is difficult to capture proteins inside living cells, and thus appropriate methods for antibody delivery to the cytoplasm of living cells are required. Cell-penetrating materials, such as the TAT-peptide, have received attention for their ability to deliver various cargos into living cells. However, the direct modification of cargos with cell-penetrating materials is time-consuming and lacks versatility. Therefore, we conceived that protein A, which can bind to the fragment crystallizable region of an antibody, could indirectly link antibodies with cell-penetrating materials, creating an efficient and simple antibody delivery system. Here, we constructed a novel antibody delivery system using a cell-penetrating polymer-modified protein A derivative (CPP-pAd). Living cells treated with CPP-pAd/antibody complexes showed significantly higher antibody levels than those achieved with the commercially available reagent HVJ-E. Pre-treatment with sucrose prevented cellular uptake of the CPP-pAd/antibody complex, suggesting that the CPP-pAd/antibody internalization mechanism occurs through clathrin-dependent endocytosis. Interestingly, intracellularly delivered antibodies did not colocalize with endosome/lysosome markers, further suggesting that antibodies were delivered to the cytoplasm by escape from endosome/lysosome. Moreover, we observed that anti-nuclear pore complex antibodies, delivered to cells using CPP-pAd, localized to the nuclear membrane and inhibited nuclear factor κB dependent luciferase activity. Together, these results suggest that the antibodies delivered by CPP-pAd captured functional proteins, making CPP-pAd a promising strategy for effective capture of proteins inside living cells.

Lipocalin2 as a plasma marker for tumors with hypoxic regions.

Hypoxic tumors have been identified as appropriate indicators of tumor malignancy. However, no convenient plasma marker for hypoxic tumors has been described. Therefore, to identify a novel, convenient plasma marker for hypoxic tumors, we used microarray analysis to compare gene expression profiles of normoxic and hypoxic tumor tissues of mice bearing melanomas. Among the upregulated genes detected in hypoxic tumors, we chose to study the secretory protein lipocalin2 (LCN2) as a marker for hypoxic tumors. LCN2 protein levels in the plasma of mice bearing hypoxic tumors were significantly increased compared with those in mice bearing normoxic tumors. Interestingly, LCN2 mRNA levels were 17-fold higher in HIF-1α-positive hypoxic tumors than in HIF-1α-negative normoxic tumors. Furthermore, LCN2 mRNA levels were significantly higher in the B16-F1 cells and various human tumor cells cultured under hypoxic conditions than in cells cultured under normoxic conditions, while no changes in mRNA expression were observed in nontumor NIH-3T3 cells, even under hypoxic conditions. In cultured cells, the expression pattern of LCN2 was mostly consistent with that of HIF-1α, whereas that of a conventional hypoxic marker, carbonic anhydrase IX, was not. Collectively, our data suggested that LCN2 was a useful plasma marker for hypoxic tumors.

Development of nanoparticles incorporating a novel liposomal membrane destabilization peptide for efficient release of cargos into cancer cells.

In anti-cancer therapy mediated by a nanoparticle-based drug delivery system (DDS), overall efficacy depends on the release efficiency of cargos from the nanoparticles in the cancer cells as well as the specificity of delivery to tumor tissue. However, conventional liposome-based DDS have no mechanism for specifically releasing the encapsulated cargos inside the cancer cells. To overcome this barrier, we developed nanoparticles containing a novel liposomal membrane destabilization peptide (LMDP) that can destabilize membranes by cleavage with intramembranous proteases on/in cancer cells. Calcein encapsulated in liposomes modified with LMDP (LMDP-lipo) was effectively released in the presence of a membrane fraction containing an LMDP-cleavable protease. The release was inhibited by a protease inhibitor, suggesting that LMDP-lipo could effectively release its cargo into cells in response to a cancer-specific protease. Moreover, when LMDP-lipo contained fusogenic lipids, the release of cargo was accelerated, suggesting that the fusion of LMDP-lipo with cellular membranes was the initial step in the intracellular delivery. Time-lapse microscopic observations showed that the release of cargo from LMDP-lipo occurred immediately after association of LMDP-lipo with target cells. Consequently, LMDP-lipo could be a useful nanoparticle capable of effective release of cargos specifically into targeted cancer cells.

[Development of a novel nanocarrier focusing on the physicochemical properties of an anti-cancer therapy drug--development of anti-cancer nanoparticles containing vitamin E derivative with mulitifaceted anti-cancer effect].

Successful cancer gene therapy is dependent upon the development of nanocarriers that exhibit anti-cancer effects via delivery of nucleic acids to the target site in tumor tissue. The effectiveness of such systems has typically relied on the potency of the anti-cancer nucleic acids, as conventional carriers do not exhibit inherent anti-cancer activity, serving only as vehicles for delivery. Ideal nanocarriers for effective cancer gene therapy should not only serve as delivery systems for transporting anti-cancer nucleic acids to the target tumor tissue, but should also exhibit their own inherent anti-cancer activity. α-tocopheryl succinate (TS) has attracted attention as a unique anti-cancer agent for its ability to induce apoptosis in various cancer cells; moreover, TS readily forms nanovesicles (TS-NVs). Thus, vesicles comprised of TS represent prospective tools for use as drug delivery systems (DDS) for cancer therapy. Owing to the low vesicle stability in the presence of divalent cations or serum, however, TS-NVs are not suitable for encapsulating nucleic acids, and require passive targeting delivery to tumor tissue via an enhanced permeability and retention effect. To improve the stability of TS-NVs, we developed novel nanovesicles comprised of TS and egg phosphatidylcholine (EPC), which can form a stable lamellar structure (TS-EPC-NVs). In this review, we introduce the development of nanovesicles comprised of TS as a novel DDS carrier and demonstrate the anti-cancer activity of both the encapsulated nucleic acids and the carrier itself.