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In this study, we developed anionic polymer-coated liposome/siRNA complexes (lipoplexes) with chondroitin sulfate C (CS), poly-l-glutamic acid (PGA) and poly-aspartic acid (PAA) for siRNA delivery by intravenous injection, and evaluated the biodistribution and gene silencing effect in mice. The sizes of CS-, PGA- and PAA-coated lipoplexes were about 200?nm and their ?-potentials were negative. CS-, PGA- and PAA-coated lipoplexes did not induce agglutination after mixing with erythrocytes. In terms of biodistribution, siRNAs after intravenous administration of cationic lipoplexes were largely observed in the lungs, but those of CS-, PGA- and PAA-coated lipoplexes were in both the liver and the kidneys, indicating that siRNA might be partially released from the anionic polymer-coated lipoplexes in the blood circulation and accumulate in the kidney, although the lipoplexes can prevent the agglutination with blood components. To increase the association between siRNA and cationic liposome, we used cholesterol-modified siRNA (siRNA-Chol) for preparation of the lipoplexes. When CS-, PGA- and PAA-coated lipoplexes of siRNA-Chol were injected into mice, siRNA-Chol was mainly observed in the liver, not in the kidneys. In terms of the suppression of gene expression in vivo, apolipoprotein B (ApoB) mRNA in the liver was significantly reduced 48?h after single intravenous injection of PGA-coated lipoplex of ApoB siRNA-Chol (2.5?mg?siRNA/kg), but not cationic, CS- and PAA-coated lipoplexes. In terms of toxicity after intravenous injection, CS-, PGA- and PAA-coated lipoplexes did not increase GOT and GPT concentrations in blood. From these findings, PGA coatings for cationic lipoplex of siRNA-Chol might produce a systemic vector of siRNA to the liver.

To enhance tumor magnetic resonance imaging (MRI) signals via the selective accumulation of contrast agents, we prepared folate-modified gadolinium-lipid-based nanoparticles as MRI contrast agents. Folate-modified nanoparticles were comprised of polyethylene glycol (PEG)-lipid, gadolinium diethylenetriamine pentaacetic acid lipid, cationic cholesterol derivatives, folate-conjugated PEG-lipid, and Cy7-PEG-lipid. Folate receptor-mediated cellular nanoparticle association was examined in KB cells, which overexpress the folate receptor. The biodistribution of nanoparticles after their intravenous injection into KB tumor-bearing mice was measured. Mice were imaged through in vivo fluorescence imaging and MRI 24 h after nanoparticle injection, and the intensity enhancement of the tumor MRI signal was evaluated. Increased cellular association of folate-modified nanoparticles was inhibited by excess free folic acid, indicating that nanoparticle association was folate receptor-mediated. Irrespective of folate modification, the amount of nanoparticles in blood 24 h after injection was ca. 10% of the injected dose. Compared with non-modified nanoparticles, folate-modified nanoparticles exhibited significant accumulation in tumor tissues without altering other biodistribution, as well as enhanced tumor fluorescence and MRI signal intensity. The results support the feasibility of MRI- and in vivo fluorescence imaging-based tumor visualization using folate-modified nanoparticles and provide opportunities to develop folate targeting-based imaging applications.

Purpose: To evaluate the accuracy of an equilibrium magnetization (M0 ) map obtained using a two-dimensional (2D) spoiled gradient-recalled echo (SPGR) pulse sequence with variable flip angle (VFA)., Materials and Methods: Single-slice 2D SPGR images of 4% agar gel phantoms with different gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) concentrations (0-1 mM) were obtained with a VFA (2-30°). The 2D SPGR-VFA data were acquired with different repetition times (TRs; 7.8-117.2 ms), Gaussian and sinc RF pulses, and different field strengths (4.7, 7, and 9.4 Tesla). M0 and T1 maps were calculated from the 2D SPGR-VFA data. M0 and T1 values were compared with those calculated from free-relaxed 2D gradient-recalled echo (GRE) images and inversion recovery-prepared 2D SPGR images. The M0 and T1 slice profiles were also investigated., Results: Consistent M0 values were obtained, regardless of the different Gd concentrations, TRs, and pulse sequences. The M0 slice profiles calculated from the sliced SPGR-VFA data quantitatively reproduced those calculated from the free-relaxed sliced GRE. In contrast, the T1 values calculated from the 2D SPGR-VFA data were underestimated at a high Gd concentration, short TR, and Gaussian RF pulse., Conclusion: M0 values calculated from 2D SPGR-VFA images are highly quantitative., (Copyright © 2013 Wiley Periodicals, Inc.)

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

Highly relevant mouse models of human neuroblastoma (NB) are needed to evaluate new therapeutic strategies against NB. In this study, we characterized transgenic mice with bilateral adrenal tumors. On the basis of information from the tumoral gene expression profiles, we examined the antitumor effects of unencapsulated and liposomal doxorubicin (DXR), alone and in combination with gefitinib, on adrenal NB. We showed that intravenous injection of unencapsulated or liposomal DXR alone inhibited tumor growth in a dose-dependent manner, as assessed by magnetic resonance imaging (MRI). However, liposomal DXR did not exhibit greater antitumor effect than unencapsulated DXR. Immunohistochemical analysis revealed that the adrenal tumor vasculature with abundant pericyte coverage was a less leaky structure for liposomes. Combination therapy with unencapsulated or liposomal DXR plus gefitinib strongly suppressed tumor growth and delayed tumor regrowth than treatment with unencapsulated or liposomal DXR alone, even at a lower dose of DXR. Dynamic contrast-enhanced MRI analysis revealed that gefitinib treatment increased blood flow in the tumor, indicating that gefitinib treatment changes the tumor vascular environment in a manner that may increase the antitumor effect of DXR. In conclusion, the combination of gefitinib and DXR induces growth inhibition of adrenal NBs in transgenic mice. These findings will provide helpful insights into new treatments for NB.

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.)

Previously, we reported that cationic nanoparticles (NP) composed of cholesteryl diamine (OH-Chol, (3S)-N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide) and Tween 80 could deliver plasmid DNA (pDNA) and small interfering RNA (siRNA) with high transfection efficiency into various tumor cells. In this study, to facilitate the endosomal escape of siRNA transfected by lipid-based nanoparticles, we synthesized new cationic cholesteryl triamine (OH-N-Chol, (3S)-N-(2-(2-(2-hydroxyethylamino)ethylamino)ethyl)cholesteryl-3-carboxamide) with an ethylenimine extension and prepared cationic nanoparticles (NP-N) composed of cholesteryl triamine and Tween 80. Although NP-N/siRNA complex (NP-N nanoplex) after mixing NP-N with siRNA was >350 nm in size, the vortex-mixing during the nanoplex formation decreased it to about 200 nm, which was an injectable size. NP-N nanoplex was mainly internalized by macropinocytosis-mediated endocytosis, as was NP nanoplex, and showed higher gene knockdown efficiency than NP nanoplex in human cervical carcinoma SiHa cells. From these results, cationic nanoparticles composed of OH-N-Chol and Tween 80 may have potential as a gene vector for siRNA transfection to tumor cells., (Copyright © 2012 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).

Previously, we prepared cationic nanoparticles (NP and NP-N) composed of cholesteryl diamine (OH-Chol, (3S)-N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide) and cholesteryl triamine (OH-N-Chol, (3S)-N-(2-(2-(2-hydroxyethylamino)ethylamino)ethyl)cholesteryl-3-carboxamide), respectively, with Tween 80 for small interfering RNA (siRNA) delivery into tumor cells. In this study, we prepared NP-0.25 N composed of OH-Chol and OH-N-Chol at a molar ratio of 3/1 with Tween 80, and evaluated the transfection efficiency of plasmid DNA (pDNA) into tumor cells. NP-N exhibited lower transfection activity than NP; however, NP-0.25 N showed higher transfection activity than both NP and NP-N in various tumor cells. NP-0.25 N increased the amount of internalized pDNA by increased cellular association, and improved the escape from endosomes after clathrin-mediated endocytosis. The results of the experiments suggested that cholesteryl triamine may have potential as a helper lipid to increase the transfection for pDNA delivery by cationic cholesterol-based nanoparticles.

We developed elastic cationic liposomal vectors for transdermal siRNA delivery. These liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid and sodium cholate (NaChol) or Tween 80 as an edge activator. When NaChol or Tween 80 was included at 5, 10, and 15% (w/w) into DOTAP liposomal formulations (C5-, C10-, and C15-liposomes and T5-, T10-, and T15-liposomes), C15- and T10-liposomes showed 2.4- and 2.7-fold-higher elasticities than DOTAP liposome, respectively. Although the sizes of all elastic liposomes prepared in this study were about 80-90 nm, the sizes of C5-, C10- and C15-liposome/siRNA complexes (lipoplexes) were about 1,700-1,800 nm, and those of T5-, T10-, and T15-lipoplexes were about 550-780 nm. Their elastic lipoplexes showed strong gene suppression by siRNA without cytotoxicity when transfected into human cervical carcinoma SiHa cells. Following skin application of the fluorescence-labeled lipoplexes in mice, among the elastic lipoplexes, C15- and T5-lipoplexes showed effective penetration of siRNA into skin, compared with DOTAP lipoplex and free siRNA solution. These data suggest that elastic cationic liposomes containing an appropriate amount of NaChol or Tween 80 as an edge activator could deliver siRNA transdermally.

The α3β1 integrin is an adhesion receptor for extracellular matrix proteins, and plays crucial roles in cell motility, proliferation, and differentiation. The aberrant expression of this adhesion molecule on tumor cells is frequently associated with their malignant behaviors. We previously reported that the Ets transcription factor-binding consensus sequence 133 bp upstream of the mouse α3 integrin gene is an important element for its expression in various tumor cell lines. In the present study, we attempted to identify a transcription factor bound to the Ets-consensus sequence, and found that Ets-1 bound to this sequence in an electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and pull-down assay with a tandem repeat of the sequence as adsorbent. We next examined the role of Ets-1 in α3 integrin gene expression by use of a luciferase assay with a reporter plasmid containing the 5'-flanking region of the α3 integrin gene. Cotransfection of HEK293T cells with an Ets-1 expression construct and the reporter plasmid increased luciferase activity. By contrast, transfection of HT1080 cells (high α3 integrin expresser) with a dominant-negative mutant of Ets-1 decreased luciferase activity. Overexpression of Ets-1 in HepG2 hepatocellular carcinoma cells (low α3 integrin expresser) upregulated α3 integrin expression as assessed by immunoprecipitation. Finally, the induction of α3 integrin gene expression in HepG2 cells after transforming growth factor-β1 treatment was abrogated by the dominant-negative mutant of Ets-1. These results suggest that Ets-1 is involved in transcriptional activation of the α3 integrin gene through its binding to the Ets-consensus sequence at -133 bp., (© 2012 The Authors Journal compilation © 2012 FEBS.)

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.

Neuroblastoma is the second most common type of solid tumor in children and is commonly found in the adrenal medulla. Recently, we developed transgenic mice exhibiting tumors bilaterally in the adrenal medulla through the expression of SV40 T-antigen. Since these transgenic mice facilitate the development of new therapeutic approaches for neuroblastoma, non-invasive monitoring methods are required for serial measurement of tumor progression. In this study, we monitored the serial progression of adrenal tumors in transgenic mice by magnetic resonance imaging (MRI) of 9.4 T vertical type, and calculated the tumor volume. The accuracy of the tumor volume determination by MRI was verified by standard volume measurements at autopsy. Adrenal tumors as small as 1.5 mm in diameter were detected and quantitatively measured in the transgenic mice by in vivo MRI without using exogenous contrast agents on T(2)-weighted spin echo images. The tumor sizes by MRI correlated better with tumor weight than the volume by calculation with a caliper. Furthermore, we monitored the change of tumor volume following administration of doxorubicin at weekly intervals. The tumor progression and regression following doxorubicin treatment in the individual mice could be observed by serial MRI. From these findings, non-invasive MRI is likely to be useful for monitoring the response of spontaneous tumors to therapeutic drugs.

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.)

Background: Decaarginine-polyethylene glycol-conjugated 3,5-bis(dodecyloxy)benzamide/plasmid DNA [Arg10-polyethylene glycol (PEG)-lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated., Methods: R10B/DNA complexes prepared at a N/P ratio of 8.5/1 or 42.5/1, corresponding to 5 µm or 25 µm R10B, respectively, were added to HeLa cells, and their uptake and subsequent intracellular fate were examined by cell imaging using electron microscopy (EM) and correlative light-electron microscopy (CLEM)., Results: EM and CLEM analyses revealed that R10B/DNA complexes formed at the lower N/P ratio were mainly taken up by the cells through macropinocytosis, whereas R10B/DNA complexes formed at the higher N/P ratio bound to protruding membrane structures or permeated into the cells by a different pathway. In cells expressing the transgene, R10B/DNA complexes were observed both in macropinosomes and in the cytoplasm. In addition, these cells had macropinosomes with disrupted membranes. These results suggest that cellular uptake through macropinocytosis and subsequent disruption of the macropinosome membrane may be a critical step for R10B-mediated gene delivery., Conclusions: We have shown that the existence of R10B/DNA complexes in macropinosomes at the early stages of gene delivery correlates with high efficiency R10B-mediated gene delivery. This finding will provide valuable insights for the engineering of more efficient gene delivery systems based on oligoarginine-mediated carriers., (Copyright © 2012 John Wiley & Sons, Ltd.)

Elevated interstitial fluid pressure (IFP) in a tumor is a barrier to tumor accumulation of systemic delivery of nanocarriers. In this study, we investigated whether intravenous injection of type I collagenase (collagenase-1) reduced IFP in tumors and increased the accumulation and gene expression of cationic liposome/plasmid DNA complex (lipoplex) in tumors after intravenous injection into mice bearing mouse lung carcinoma LLC tumors. Collagenase-1 reduced the amount of type I collagen in the tumor, and significantly decreased IFP by 65% at 1h after injection. Therefore, collagenase-1 induced 1.5-fold higher accumulation and 2-fold higher gene expression of lipoplex in tumors after intravenous injection. These findings indicated that intravenous injection of collagenase-1 improved the accumulation of lipoplex by decreasing IFP in tumors. These results support the potential use of collagen digestion as a strategy to improve systemic gene delivery into tumors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Medullary thyroid carcinoma is a rare endocrine tumor, which shows overexpression of somatostatin receptor subtype 2. There is no systemic therapy for medullary thyroid carcinoma. Previously we reported that octreotide-PEG liposomes loaded with irinotecan, which target somatostatin receptor subtype 2, showed high therapeutic efficacy for medullary thyroid carcinoma xenografts compared with free irinotecan or non-targeted non-PEGylated liposomal irinotecan. In this study, we evaluated octreotide-PEG liposomes loaded with irinotecan in terms of the biodistribution of irinotecan and its active metabolite, and its therapeutic efficacy, compared with PEGylated liposomes. Furthermore, to elucidate the effect of octreotide ligand after cellular association, we assessed the cytotoxicity in tumor cells and the inhibition of protein phosphorylation in the tumor cells and xenografts using empty octreotide-PEG liposomes, which were loaded with no drug. In a therapeutic study, octreotide-PEG liposomes loaded with irinotecan significantly improved median survival compared with PEGylated liposomes. In tumor tissue at 6 h after injection, octreotide-PEG liposome-treated mice showed significantly higher concentrations of irinotecan and 7-ethyl-10-hydrocamptothecin compared with PEGylated liposome-treated mice, indicating that octreotide-PEG liposomes accumulated rapidly and to a high level in the tumor. Furthermore, empty octreotide-PEG liposome inhibited the phosphorylation of p70S6K in vitro and in vivo. These findings indicated that octreotide-PEG liposomal irinotecan has dual functions with targeted tumor delivery and assistance of cellular cytotoxicity, which led to higher therapeutic efficacy than PEGylated liposomes for medullary thyroid carcinoma xenografts., (© 2011 Japanese Cancer Association.)

Objectives: In 2007, measles prevailed among the youth and young adult population in Japan, creating in a serious social problem. Among the developed countries, Japan has a relatively high incidence of measles. The objective of this study was to assess the effect of improvements in the vaccination policy against measles through simulations., Methods: We developed an age-time two-dimensional model for the transmission of measles to reflect an age structure, enabling contact rate to be selected by age. Introduction of the maternal immunity class into the model allowed natural infection and vaccination to be discriminated along the course of an infant's mother acquiring the immunity, thereby resulting in an improved accuracy of the simulations in infants. Several vaccination scenarios were attempted in order to assess the influence of various vaccination policies on the prevention of a measles epidemic., Results: The results of this quantitative study indicated that suppression of a measles outbreak requires the maintenance of high vaccine coverage and that a decline in vaccine coverage may result in a measles epidemic., Conclusions: The present standard immunization program for measles will maintain an acceptable level of immunity and is therefore associated with a low risk of an epidemic after discontinuation of the third and fourth stages as scheduled--as long as at least 90% vaccine coverage of the first and second is maintained. The simulation results show that discontinuation of the third and fourth stages of vaccination as scheduled should be accompanied by endeavors to maintain appropriate high vaccine coverage of the first and second stages.

It was believed for a long time that mRNA is very unstable, and can not be used for therapeutic purposes. In the last decade, however, many research groups proved its transfection feasibility along with advantages and applications. Our investigation is aimed at establishing a potent and efficient mRNA delivery system. We previously reported that an inorganic-organic hybrid carrier by exploiting the advantages of inorganic nano apatite particles onto organic carrier DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} and showed potential effect of carbonate apatite particles on each of the mRNA delivery steps in dividing and non-dividing cell. Here, we report on the development of a more efficient mRNA carrier by complexing ECM protein, fibronectin with the DOTAP-apatite carrier. The carrier showed enhanced uptake of luciferase mRNA both qualitatively and quantitatively. Accelerated cellular endocytosis rate was evaluated using labeled endosome. Finally expression of lucifearse mRNA was higher for fibronectin complexed carrier in compared to the uncoated one.

In this study, we investigated the effect of manufacturing factors such as particle size, water content and manufacturing method on the physical stability and solubility of solid dispersion formulations of a low-glass-transition-temperature (T(g)) drug. Solid dispersions were prepared from polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) by hot melt extrusion or spray drying. Water content of solid dispersions prepared by hot melt extrusion determined by dynamic moisture sorption measurement was increased drastically with relative humidity below a certain level of particle size. The blends with a lower water content (0.8%) prepared by hot melt extrusion during storage were more stable than those with a higher water content (3.5%) prepared by spray drying, which caused rapid recrystallization. Physical stability in the hot melt blends may be attributed to reduced molecular mobility due to a higher T(g). Dissolution study revealed that solid dispersions prepared by hot melt extrusion with the smallest particle size showed decreased solubility, attributed to reduced wetting properties (surface energy), which is not predictable by the Noyes-Whitney equation. Taken together, these results indicate that the control of particle size concerned in water content or wetting properties is critical to ensuring the physical stability or enhancing solubility of low-T(g) drugs. Further, hot melt extrusion, which can reduce water content, is a suitable manufacturing method for solid dispersions of low-T(g) drugs.

The purpose of this study was establishing a solid dispersion formulation containing a low glass transition temperature (T(g)) and poorly water-soluble drug. Drug/polymer blends with differing physicochemical stabilities and oral absorption were prepared from copolyvidone (PVP-VA), polyvinylpyrrolidone (PVP) or hydroxypropylmethylcellulose (HPMC) by a hot melt extrusion. HPMC drastically increased the drug oral absorption property, while PVP-VA or PVP stabilized solid dispersions during storage by increasing the T(g) in proportion to polymer concentration. Experimental T(g) values corresponded closely with theoretical T(g) values; indeed, the T(g) values of solid dispersion with HPMC did not increase significantly compared to the T(g) value for the drug alone. A solid dispersion formulation incorporating two different polymers-HPMC and either PVP-VA or PVP-maintained increased T(g), physicochemical stability, solubility, and bioavailability of the solid dispresions owing to each polymer. These findings suggested that both oral absorption and physicochemical stability of low-T(g) drug will be improved using less amount of solid dispersion of combined two polymers than polymer alone.

Everolimus has demonstrated antitumor efficacy for various cancers as a result of its inhibition of the mammalian target of rapamycin (mTOR) signaling cascade, which activates cell growth and cell proliferation. However, the low water solubility and low bioavailability of everolimus have prevented its clinical development as an anticancer drug. Therefore, to address the unsuitable characteristic of everolimus, we attempted to prepare liposomal everolimus as a viable drug delivery system, and then evaluated the anticancer efficacy of this system against a medullary thyroid carcinoma cell line (TT cells), a breast cancer cell line (MCF-7 cells) and a small lung carcinoma cell line (NCI-H446 cells). The particle size and entrapment efficacy of liposomal everolimus was ca. 80 nm and more than 90%, respectively. Liposomal everolimus showed higher cytotoxicity against NCI-H446 cells compared with TT cells. Against NCI-H446 tumors, significant suppression of the tumor volume was observed in liposomal everolimus-treated mice by intravenous injection, compared with free everolimus-treated mice by intraperitoneal injection, at a dose of 5 mg/kg without body weight loss. This study showed that liposomal everolimus could be a powerful formulation with anticancer efficacy for some cancers.

Menthosomes, novel deformable carriers for the enhancement of transdermal delivery are introduced in this study. Meloxicam (MX)-loaded menthosomes were formulated, and their physicochemical characteristics and skin permeability were evaluated. A two-factor spherical and second-order composite experimental design was used to prepare the formulation of the menthosomes. Ten formulations of menthosomes composed of a phospholipid as the lipid bilayer carrier, cholesterol (Chol) as a stabilizer and cetylpyridinium chloride (CPC) and L-menthol as penetration enhancers were prepared. The amounts of Chol and CPC were selected as causal factors. Physicochemical characteristics (particle size, size distribution, zeta potential, elasticity and drug content) and an in vitro skin-permeation study of meloxicam-loaded menthosomes were evaluated. The concentrations of MX that permeated the skin at 2-12 h and the flux were selected as response variables. The optimal formulation was estimated using a nonlinear response-surface method incorporating thin-plate spline interpolation. The experimental values were very close to the values predicted by the computer programs in this study. A Bayesian network analysis was applied to gain a mechanistic understanding of the relationships between causal factors and response variables.

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

Organic nanotubes made of synthetic amphiphilic molecules are novel materials that form by self-assembly. In this study, organic nanotubes with a carboxyl group (ONTs) at the surface were used as a carrier for the anticancer drug doxorubicin, which has a weak amine group. The IC(50) values of ONT for cells were higher than that of conventional liposomes, suggesting that ONTs are safe. The results showed that the drug loading of ONTs was susceptible to the effect of ionic strength and H(+) concentration in the medium, and drug release from ONTs was promoted at lower pH, which is favorable for the release of drugs in the endosome after cellular uptake. ONTs loaded with the drug were internalized, and the drug was released quickly in the cells, as demonstrated on transmission electron microscopy images of ONTs and the detection of a 0.05% dose of ONT chelating gadolinium in the cells. Moreover, ONT could be modified chemically with folate by simply mixing with a folate-conjugate lipid. Therefore, these novel, biodegradable organic nanotubes have the potential to be used as drug carriers for controlled and targeting drug delivery., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, but treatment with irinotecan (CPT-11) is limited because of side effects. MTC is known to overexpress the somatostatin receptor subtype 2 (SSTR2). Octreotide (Oct) is a somatostatin analogue that has a high binding affinity for SSTR and can be used as a tumor-targeting ligand. We prepared Oct-targeted liposomes loaded with CPT-11 using Oct-poly (ethylene glycol) (PEG)-lipid and evaluated Oct-mediated association and cytotoxicity of the liposomes with an MTC cell line TT. The association of higher concentrations of modified Oct-targeted liposomes with TT cells was significantly higher than PEGylated liposomes and was significantly inhibited by empty Oct-targeted liposomes but not by free Oct. With exposure for 96 h, the cytotoxicity of Oct-targeted liposomal CPT-11 (IC50: 1.05 ± 0.47 μM) was higher than free CPT-11 (IC50: 3.76 ± 0.61 μM) or PEGylated liposomal CPT-11 (IC50: 3.05 ± 0.28 μM). In addition, empty Oct-targeted liposomes showed significantly higher cytotoxicity than empty nontargeted liposomes at a concentration where free Oct did not show cytotoxicity, suggesting that Oct as a ligand showed cytotoxicity. Moreover, Oct-targeted liposomal CPT-11 led to significantly higher antitumor activity and prolonged the survival time compared with nontargeted liposomal and free CPT-11 at a one-third dose and lower administration times with free CPT-11. These findings indicated that Oct-targeted liposomes loaded with CPT-11 may offer considerable potential for MTC chemotherapy because cytotoxicity of both CPT-11 and Oct was enhanced by effective cellular uptake via SSTR2.

Previously, we have reported that decaarginine-conjugated PEG-lipids (R10B) efficiently delivered plasmid DNA (pDNA) into human cervical carcinoma HeLa cells via macropinocytosis; however, the mechanism of cellular uptake by R10B was not evaluated in other cell lines. In this study, we investigated the internalization mechanism by R10B/pDNA complex (R10B-lipoplex) in human prostate tumor PC-3 and human nasopharyngeal tumor KB cells, and compared with that in HeLa cells. Although it was necessary for R10B-lipoplex to associate with heparan sulfate (HS) on the cell surface in all cell lines, the R10B-lipoplex was internalized primarily through clathrin-mediated endocytosis in PC-3 and KB cells, and macropinosytosis in HeLa cells. In HeLa cells, treatment with the R10B-lipoplex induced the formation of lamellipodia for macropinocytosis, but did not in KB and PC-3 cells. Furthermore, the highest transfection efficiency by R10B-lipoplex was observed in HeLa cells. These findings indicated that the R10B-lipoplex induced the formation of lamellipodia in HeLa cells after binding to HS on the cells and was then internalized by macropinocytosis, which could induce high gene expression because of escaping degradation in lysosomes. Cell physiology might be a critical factor in cellular internalization and efficient transfection by cell penetration peptide., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We incorporated an anticancer agent, camptothecin (CPT), into polymeric micelle carriers by using two different solvents (TFE and chloroform) in the solvent-evaporation drug incorporation process. We observed significant differences in the drug-incorporation behaviors, in the morphologies of the incorporated drug and the polymeric micelles, and in the pharmacokinetic behaviors between the two solvents' cases. In particular, the CPT-incorporated polymeric micelles prepared with TFE as the incorporation solvent exhibited more stable circulation in blood than those prepared with chloroform. This contrast indicates a novel technological perspective regarding the drug incorporation into polymeric micelle carriers. Morphological analyses of the inner core have revealed the presence of the directed alignment of the CPT molecules and CPT crystals in the micelle inner core. This is the first report of the morphologies of the drug incorporated into the polymeric micelle inner cores. We believe these analyses are very important for further pharmaceutical developments of polymeric micelle drug-carrier systems., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In this study, we investigated whether the therapeutic efficacy of liposomal doxorubicin (DXR-SL) could be enhanced by angiotensin II (AT)-induced hypertension. AT-induced hypertension increased the volume of tumor blood flow in mice bearing a poorly vascularized Lewis lung carcinoma (LLC) tumor, but only slightly in mice bearing a well-vascularized colon carcinoma Colon 26 (C26) tumor. In therapeutic efficacy, AT-induced hypertension enhanced the antitumor activity of DXR-SL in mice bearing LLC and C26 tumors. Localization of DXR-SL after injection by AT-induced hypertension was observed outside tumor blood vessels in LLC and C26 tumors, but within them under the normotension. From these findings, AT-induced hypertension had potential to improve the delivery of DXR-SL to both well- and poorly vascularized solid tumors., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

The folate receptor is an attractive target for selective tumor delivery of liposomal doxorubicin (DXR) because it is abundantly expressed in a large percentage of tumors. This study examined the effect of polyethylene glycol (PEG) spacer length and folate ligand density on the targeting ability of folate-modified liposomes. Liposomes were modified with folate-derivatized PEG-distearoylphosphatidylethanolamine with PEG molecular weights of 2000, 3400, or 5000. The association of DXR-loaded liposomes with KB cells, which overexpress the folate receptor, was evaluated by flow cytometry at various ratios of folate modification. A low ratio of folate modification with a sufficiently long PEG chain showed the highest folate receptor-mediated association with the cells, but did not show the highest in vitro cytotoxicity. DXR release from folate-modified liposomes in endosomes might be different. These findings will be useful for designing folate receptor-targeting carriers.

The structure of tumor vasculature is crucial for the nanocarrier-mediated chemotherapy. Recently, transforming growth factor-β (TGF-β) inhibitor was reported to increase the tumor accumulation of nanocarriers by changing the structure of tumor vasculature. To identify the parameters of tumor vasculature function following TGF-β inhibitor (A-83-01) treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed using Gd-DTPA and its liposomal formulation (Gd-L) as contrast agents. Observation of tumor MR image before, during, and after injection of contrast agent could calculate the parameters of vascular function, such as volume transfer constant between blood plasma and extracellular space (K(trans)) and fractional plasma volume (v(p)). A-83-01 treatment significantly increased these parameters within 24 h that was positively related to pericyte coverage and tumor cell proliferation. Furthermore, apparent diffusion coefficient (ADC) determined by diffusion-weighed imaging was decreased by A-83-01 treatment, suggesting the decrease of tumor interstitial fluid pressure. Vascular function of the tumor improved by A-83-01 treatment well assessed on post-Gd-L-enhanced MR images, which predicted delivery of liposomal drug to the tumor. These findings suggest that DCE-MRI and, in particular, K(trans) and v(p) quantitation, provide important additional information about tumor vasculature by A-83-01 treatment.

The current study synthesized macromolecular magnetic resonance imaging (MRI) contrast agents constituted of the poly(ethylene glycol)-b-poly(L-lysine) block copolymer (PEG-P(Lys)). A chelate group, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), was attached to the primary amino group of the block copolymer in desired contents. Gd-DOTA-based macromolecular contrast agents were prepared from PEG-P(Lys) having DOTA (PEG-P(Lys-DOTA) and Gd(III) ions. All of the PEG-P(Lys) block copolymers having gadolinium ions (PEG-P(Lys-DOTA-Gd)) showed higher T(1) relaxivity (per gadolinium), r(1)=5.6-7.3mM(-1)s(-1), than that of a low-molecular-weight gadolinium-chelate, diethylenetriaminepentaacetic acid-gadolinium(III) (Gd-DTPA) at 9.4T. The study prepared the polyion complex (PIC) micelles from the amino groups of the lysine units and an oppositely charged polyanion, poly(methacrylic acid) or dextran sulfate, in an aqueous medium. In contrast, the fully DOTA-attached PEG-P(Lys-DOTA-Gd) formed a PIC with a polycation. Compared with partially DOTA-attached cationic PEG-P(Lys-DOTA-Gd), this PIC micelle yielded a forty percent decrease of r(1). This r(1) decrease was considered to result from a change in the accessibility of water molecules to gadolinium ions in the micelles' inner core. The r(1) was decreased upon formation of the PIC micelle, and this change proved that our concept worked in vitro. Blood-circulation characteristics of PIC micelles were controlled by means of changing the molecular weight of the counter anion. The PIC micelles accumulated in tumor tissues, and MRI study showed T1W image of axial slice of tumor area was significantly enhanced at 24h after the injection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Tumor cell targeting of drug carriers is a promising strategy and uses the attachment of various ligands to enhance the therapeutic potential of chemotherapy agents. Folic acid is a high-affinity ligand for folate receptor, which is a functional tumor-specific receptor. The transforming growth factor (TGF)-β type I receptor (TβR-I) inhibitor A-83-01 was expected to enhance the accumulation of nanocarriers in tumors by changing the microvascular environment. To enhance the therapeutic effect of folate-linked liposomal doxorubicin (F-SL), we co-administrated F-SL with A-83-01. Intraperitoneally injected A-83-01-induced alterations in the cancer-associated neovasculature were examined by magnetic resonance imaging (MRI) and histological analysis. The targeting efficacy of single intravenous injections of F-SL combined with A-83-01 was evaluated by measurement of the biodistribution and the antitumor effect in mice bearing murine lung carcinoma M109. A-83-01 temporarily changed the tumor vasculature around 3 h post injection. A-83-01 induced 1.7-fold higher drug accumulation of F-SL in the tumor than liposome alone at 24 h post injection. Moreover F-SL co-administrated with A-83-01 showed significantly greater antitumor activity than F-SL alone. This study shows that co-administration of TβR-I inhibitor will open a new strategy for the use of FR-targeting nanocarriers for cancer treatment., (© 2010 Japanese Cancer Association.)

Adrenal neuroblastoma and pheochromocytoma have the same embryonic origin from neural crest cells and mainly arise from the adrenal medulla. Recently, transgenic mice exhibiting tumors in the bilateral adrenal medulla by the expression of SV40 T-antigen were developed. In this study, we investigated mRNA expression in adrenal tumors of transgenic mice and compared them with human pheochromocytoma by DNA microarray analysis. To compare mouse adrenal tumors and human pheochromacytoma, we found that the expressions of noradrenergic neuron-related genes, including dopa decarboxylase, phenylethanolamine-N-methyltransferase and chromogranin B, were up-regulated in humans but not in mice; however, the expression of neuroblastoma-related genes, including Mycn, paired-like homeobox 2b, gamma-aminobutyric acid A receptor beta3 subunit, islet 1 and kinesin family member 1A, was up-regulated in both species. From the gene expression profiles, the characterization of mouse adrenal tumor, may be similar to that of human adrenal neuroblastoma rather than pheochromacytomas. This mouse model would be a useful tool for the development of anti-cancer drugs and for understanding the etiology of adrenal neuroblastoma.

RhoA protein is involved in the Ca(2+) sensitization of bronchial smooth muscle (BSM) contraction, and an upregulation of RhoA in BSMs has been suggested in allergic bronchial asthma. However, the mechanism of upregulation of RhoA remains poorly understood. In the present study, the transcriptional regulation of human RhoA gene was investigated in cultured human BSM cells stimulated with IL-13 and TNF-alpha, both of which have an ability to upregulate RhoA protein. Luciferase-based assay showed that the RhoA promoter activity was augmented by both IL-13 and TNF-alpha. The deletion studies revealed a significant level of promoter activity between the 112 bp upstream and the transcription start site, which contains the STAT6 (78-70 bp upstream) and NF-kappaB (84-74 bp upstream) binding regions. The promoter activity was also decreased significantly by the mutations of these regions. Thus, the current study for the first time characterized the transcriptional regulation of the human RhoA gene. The findings also suggest that STAT6 and NF-kappaB are important for the upregulation of RhoA in human BSM induced by IL-13 and TNF-alpha, both of which are major cytokines in the pathogenesis of allergic bronchial asthma., ((c) 2009 Elsevier Ltd. All rights reserved.)

Therapeutic agents targeting HER-2/neu have been intensively addressed over the past decades. Previously, we reported that HER-2 synthetic small interfering RNA (HER-2 siRNA) could suppress the growth of human nasopharyngeal KB tumor xenografts by intratumoral injection with lipid-based nanoparticles; however, complete regression of the tumor was not achieved. In this study, we investigated antitumor activity by RNA interference in combination with paclitaxel (PTX) for KB cells using HER-2 siRNA and HER-2 short hairpin RNA-expressing plasmid DNA (HER-2 shRNA pDNA). Suppression of HER-2 expression by siRNA or shRNA pDNA caused significant reduction of proliferation by inducing apoptosis and enhancing the sensitivity for PTX in HER-2 positive KB cells. Interestingly, an HER-2 antibody trastuzumab could not increase the antitumor effect by PTX in KB xenografts. Combination therapy by intratumoral injection of HER-2 siRNA or HER-2 shRNA pDNA with PTX significantly inhibited the tumor growth of xenografts compared with each therapy used individually. In particular, HER-2 shRNA pDNA plus PTX largely extended the mean survival days compared with HER-2 siRNA plus PTX. Collectively, these findings suggest that HER-2 shRNA-based combined therapy with PTX could be a novel strategy to inhibit the progression of HER-2-positive cancer.

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, and treatment with irinotecan (CPT-11) is limited because of side effects. Mutations in the Rearranged during transfection (RET) proto-oncogene are considered the causative event of MTC. The objective of this study was to examine whether small interfering RNA (siRNA) and its combined treatment with CPT-11 could inhibit MTC cell growth in vitro and in vivo. The transfection of RET siRNA suppressed RET expression, reduced proliferation, and increased caspase-3/7 activity via the down-regulation of Bcl-2 expression. Combined treatments with CPT-11 or SN-38 significantly increased caspase 3/7 activity compared with RET siRNA, CPT-11 or SN-38 treatment alone. Importantly, intratumoral injection of RET siRNA along with intravenous injection of CPT-11 significantly inhibited the tumor growth of MTC xenografts via an increased apoptotic effect. These findings that RET siRNA enhanced sensitivity for CPT-11 will provide a novel strategy for the treatment of MTC with RET mutation.

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

Background: Dynorphin A in the spinal cord is considered to contribute to nociceptive stimuli. However, it has not yet been determined whether activation of the spinal dynorphinergic system under nociceptive stimuli plays a role in direct acceleration of the ascending nociceptive pathway. In this study, the authors investigated the role of spinal dynorphinergic transmission in ongoing brain activation under noxious stimuli in mice., Methods: The changes in prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity in the mouse spinal cord were determined after the intraplantar injection of complete Freund's adjuvant in mice. The signal intensity in different brain regions after the intraplantar injection of complete Freund's adjuvant or intrathecal injection of dynorphin A (1-17) was measured by a pharmacological functional magnetic resonance imaging analysis., Results: Complete Freund's adjuvant injection produced pain-associated behaviors and induced a dramatic increase in signal intensity in the mouse cingulate cortex, somatosensory cortex, insular cortex, and thalamic nuclei. These effects were not seen in prodynorphin knockout mice. Prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity on the ipsilateral side of the spinal cord were markedly increased in complete Freund's adjuvant-injected mice. Furthermore, intrathecal injection of dynorphin A (1-17) at relatively high doses caused pain-associated behaviors and a remarkable increase in the activities of the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei in mice., Conclusions: These findings indicate that spinally released dynorphin A (1-17) by noxious stimuli leads to the direct activation of ascending pain transmission.

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.

In this study, to establish the conditions of a drug release assay for PEGylated liposome formulations that relates with the drug stability profile in serum in vivo, the influences of incubation temperature and serum protein in the release buffer were examined using liposomal doxorubicin (DXR). In in vitro drug release assays, a PEGylated liposomal DXR in phosphate buffered saline (PBS) at 37 degrees C showed higher drug release rate than non-PEGylated formulation although PEGylated liposomal DXR had higher stability than an equivalent non-PEGylated formulation following intravenous injection. When bovine serum albumin (BSA) and increased temperature, 50 degrees C, were used to accelerate drug release from the liposomes and to mimic in vivo result, non-PEGylated liposomal DXR showed conversely higher release than a PEGylated formulation. Since high temperature increased BSA adsorption onto liposomes, BSA may cause non-PEGylated liposomes instability more than PEGylated ones, resulting in the reverse of the drug release rate of both liposomes. This finding suggested that the conditions in the drug release assay with PEGylated liposomal DXR may be able to be set by a combination of BSA and providing additional thermal energy.

We developed histone deacetylase inhibitor (HDACI) prodrugs to enhance the expression of the external genes transfected into human cells with cationic nanoparticles (NPs). We synthesized five kinds of lipid-linked HDACI prodrugs in which n-dodecanoic acid or cholesterol is linked with a potent HDACI, K-182, by an ester bond or a disulfide carbonate linker. The prodrugs were able to admix as a component of NPs, although the intact K-182 was not incorporated into NPs. Namely, NPs composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 with the 10 mol% K-182 prodrug were prepared as a DNA vector to transfect plasmid DNAs into human prostate cancer cells, PC-3, or human breast cancer cells, Sk-Br-3. The NPs containing K-182 prodrugs with n-dodecanoic acid exhibited two to four times higher the gene expression than the original NPs. The enhancement of the gene expression will be due to the hyperacetylation of core histones caused by intact K-182 degraded from the prodrug in the vector incorporated into the cells.

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

A polymeric micelle drug carrier system was applied to the targeting of an MRI (magnetic resonance imaging) contrast agent. A block copolymer, PEG-b-poly(L-lysine), was used for conjugation of gadolinium ions through chelating moieties, DOTA. The DOTA moieties were successfully conjugated to all primary amine groups of the lysine residues. The obtained block copolymer, PEG-b-poly(L-lysine-DOTA), formed a polymeric micelle. The polymeric micelle structure was maintained even after partial gadolinium chelation ( approximately 40%) to the DOTA moieties. The prepared polymeric micelle MRI contrast agent was injected into a mouse tail vein at a dose of 0.05 mmol Gd/kg. The polymeric micelle-based MRI contrast agent exhibited stable blood circulation. A considerable amount (6.1+/-0.3% of ID/g of the polymeric micelle) was found to accumulate at solid tumors 24 h after intravenous injection by means of the EPR effect. An MRI analysis revealed that the signal intensity of the tumor was enhanced 2.0-fold by the use of this contrast agent.

Phytic acid (IP-6) is a polyphosphorylated carbohydrate with antitumor activity for many kinds of tumors. In this study, we developed a novel method of loading irinotecan (CPT-11) into liposomes using IP-6, and evaluated its antitumor effect on colon tumors in vivo. Liposomal CPT-11 was prepared by loading CPT-11 to distearoylphosphatidylcholine/cholesterol/methoxy-poly(ethyleneglycol)-distearylphosphatidylethanolamine liposomes prepared in IP-6 solution, CuSO(4) solution and citrate buffer, respectively (IP6-, Cu- and Cit-L). CPT-11 loading efficiency for IP6-L (90-100%) was higher than that for Cit-L (less than 40%), and similar to Cu-L when CPT-11 to total lipid weight ratio was increased from 0.2 to 0.6. Plasma elimination and biodistribution of liposomal CPT-11 and its metabolite SN-38 were measured after intravenous administration. IP6-L following i.v. injection showed 1.3- and 1.7-fold higher plasma area under the curves of CPT-11 and SN-38, respectively, than Cu-L. Finally, therapeutic activity was determined in mouse Colon 26 and human COLO 320DM tumor xenografts in mice. IP6-L significantly exhibited superior anticancer activity to Cu-L and free CPT-11 in Colon 26 tumor. Using IP-6 as a drug-trapping agent in liposome, IP6-L improved CPT-11 pharmacokinetics and increased antitumor activity in colon tumors.

Recent microarray analyses revealed that a disintegrin and metalloproteinase (ADAM) 8 (ADAM8; also called CD156) is one of the asthma candidate genes. However, the function of ADAM8 and its localization in the airways are still poorly understood. In the present study, the changes in the expression and localization of ADAM8 in the airways of a mouse model of allergic bronchial asthma were investigated. Male BALB/c mice were sensitized and repeatedly challenged with ovalbumin antigen to induce asthmatic response. After the final antigen challenge, mRNA and protein expressions of ADAM8 were elucidated by quantitative RT-PCR and immunohistochemistry. The mRNA expression of ADAM8 in the airways was significantly increased in this animal model of asthma compared with naive animals. Immunohistochemical examinations revealed that ADAM8 was located in airway epithelia, airway smooth muscles, and infiltrated cells (mainly macrophages) into lung parenchyma. A distinctly stronger immunostaining of ADAM8 was observed in these airway cells of the repeatedly antigen-challenged mice compared with those of the sensitized control animals. An upregulation of ADAM8 in the airways might be involved in the pathogenesis of airway inflammation and/or hyperresponsiveness, characteristic features of allergic bronchial asthma.

Oligoarginine-conjugated lipids ((Arg)n-PEG-lipid) (n=4, 6, 8, and 10: number of arginine residues) are novel gene delivery vectors. We prepared two oligoarginine-modified liposomes using (Arg)n-lipid without and with poly(ethylene glycol) (PEG) spacer ((Arg)n-L and (Arg)n-PEG-L), and investigated the effect of PEG spacer and oligoarginine length of liposomes on cellular uptake, gene transfection, and its mechanism in HeLa cells, using complexes with plasmid DNA (DNA) or oligodeoxynucleotide. Transfection efficiency increased as the number of arginine residues increased and Arg10-PEG-L/DNA complexes (lipoplexes) showed the highest gene transfection efficiency among (Arg)n- and (Arg)n-PEG-lipoplexes. Arg4- and Arg4-PEG-lipoplexes were taken up greatly into cells, but showed lower transfection efficiency than Arg10- and Arg10-PEG-lipoplexes, respectively. The different gene expression by Arg4-L to Arg10-L with or without PEG spacer may be explained by the different intracellular uptake mechanism. The main cellular uptake mechanism of Arg10-L and Arg10-PEG-L was the macropinocytosis pathway, whereas that of Arg4-L and Arg4-PEG-L was not. PEG spacer was more effective for intracellular trafficking than Arg length and surface charge of lipoplex which depends on Arg length at the almost same size of lipoplexes. The findings suggested that Arg10-PEG-L was a superior vector since Arg10 induced the macropinocytosis uptake pathway.

The hydrodynamic method by rapid intravenous injection of a large volume of plasmid DNA is known to be an efficient and liver-specific method of in vivo gene delivery and achieves high levels of foreign gene expression, particularly in hepatocytes. Low transgene activities have also been observed in other organs such as the spleen and lung; however, the expression profiles of mRNA and protein are still unknown. Therefore, we investigated the localization of luciferase mRNA by in situ hybridization and luciferase activity in mice after transfection of pCMV-luc encoding the luciferase gene under the control of cytomegalo virus (CMV) promoter. We found that hydrodynamic injection effectively induced mRNA expression of the transgene only in the liver although transgene activities were observed in other organs. The transgene activity observed in other organs may be due to leakage from hepatocyte gene expression by transient increase in the permeability of the hepatocyte cellular membrane caused by increased pressure by hydrodynamic injection.