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Yoshie Maitani,1 Yuri Nakamura,1 Masao Kon,1 Emi Sanada,1 Kae Sumiyoshi,1 Natsuki Fujine,1 Masumi Asakawa,2 Masaki Kogiso,2 Toshimi Shimizu21Institute of Medicinal Chemistry, Hoshi University, Tokyo, Japan; 2Nanotube Research Center (NTRC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, JapanAbstract: The size and shape of intravenously injected particles can affect their biodistribution and is of importance for the development of particulated drug carrier systems. In this study, organic nanotubes (ONTs) with a carboxyl group at the surface, a length of approximately 2 µm and outer diameter of 70–90 nm, were injected intravenously into tumor-bearing mice. To use ONTs as drug carriers, the biodistribution in selected organs of ONTs postinjection was examined using irinotecan, as an entrapped water-soluble marker inside ONTs, and gadolinium-chelated ONT, as an ONT marker, and compared with that of a 3 µm fluorescently labeled spherical microparticle which was similar size to the length of ONTs. It was found that for irinotecan, its active metabolite and gadolinium-chelated ONTs were highly accumulated in the lung, but to a lower level in the liver and spleen. On the other hand, microparticles deposited less in the lung and more highly in the liver. Moreover, histologic examination showed ONTs distributed more in lung tissues in part, whereas microparticles were present in blood vessels postinjection. These preliminary results support the notion of using negatively charged ONTs as intravascular carriers to maximize accumulation in the lung whilst reducing sequestration by the liver and spleen. This finding suggested that ONTs are potential carriers for lung-targeting drug delivery.Keywords: organic nanotube, lung, biodistribution, microparticle, particle shape

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

Background: To enhance the success rate of nanocarrier-mediated chemotherapy combined with an anti-angiogenic agent, it is crucial to identify parameters for tumour vasculature that can predict a response to the treatment of the anti-angiogenic agent.Methods: To apply transforming growth factor (TGF)-beta type I receptor (TbetaR-I) inhibitor, A-83-01, to combined therapy, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was carried out in mice bearing colon 26 cells using gadolinium (Gd)-DTPA and for its liposomal formulation to evaluate changes in tumour microvasculature following A-83-01. Tumour vascular parameters from DCE-MRI were compared with histological assessment and apparent diffusion coefficient of water in tumour generated by diffusion-weighted MRI.Results: Contrary to evaluations reported for anti-angiogenic agents, A-83-01 treatment increased the initial area under the Gd concentration-time curve (IAUGC60), volume transfer constant (K(trans)) and fractional plasma volume (v(p)) significantly within 24 h, that was positively related to alpha-smooth muscle actin-positive pericyte coverage and tumour cell proliferation, and was correlated inversely with the apparent diffusion coefficient. The vascular function of the tumour improved by A-83-01 treatment was well assessed on post-liposomal Gd-DTPA-enhanced MR images, which predicted delivery of a liposomal drug to the tumour.Conclusion: These findings suggest that DCE-MRI and, in particular, K(trans) and v(p) quantitation, provide important additional information about tumour vasculature by A-83-01 treatment. [ABSTRACT FROM AUTHOR]

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

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

When flying in a group, echolocating bats have to separate their own echoes from pulses and echoes belonging to other individuals to extract only the information necessary for their own navigation. Previous studies have demonstrated that frequency-modulated (FM) bats change the terminal frequencies (TFs) of downward FM pulses under acoustic interference. However, it is not yet clear which acoustic characteristics of the jamming signals induce the TF shift according to the degree of acoustic interference. In this study, we examined changes in the acoustic characteristics of pulses emitted by Miniopterus fuliginosus while presenting jamming stimuli with different FM patterns to the bat flying alone. Bats significantly altered their TFs when responding to downward (dExp) and upward (uExp) exponential FM sounds as well as to a constant-frequency (CF) stimulus, by approximately 1-2 kHz (dExp: 2.1±0.9 kHz; uExp: 1.7±0.3 kHz; CF: 1.3±0.4 kHz) but not for linear FM sounds. The feature common to the spectra of these three jamming stimuli is a spectrum peak near the TF frequency, demonstrating that the bats shift the TF to avoid masking of jamming sounds on the TF frequency range. These results suggest that direct frequency masking near the TF frequency range induces the TF shift, which simultaneously decreases the similarity between their own echolocation sounds and jamming signals., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Echolocating bats need to solve the problem of signal jamming by conspecifics when they are in a group. However, while several mechanisms have been suggested, it remains unclear how bats avoid confusion between their own echoes and interfering sounds in a complex acoustic environment. Here, we fixed on-board microphones onto individual frequency-modulating bats flying in groups. We found that group members broaden the inter-individual differences in the terminal frequencies of pulses, thereby decreasing the similarity of pulses among individuals. To understand what features most affect similarity between pulses, we calculated the similarity of signals mimicking pulses. We found that the similarity between those artificial signals was decreased most by manipulation of terminal frequency. These results demonstrate that the signal jamming problem is solved by this simple strategy, which may be universally used by animals that use active sensing, such as echolocating bats and electric fish, thereby transcending species and sensory modalities., Competing Interests: The authors declare no competing interests.

Drug delivery by nanoparticle carriers has been limited by inefficient intracellular drug delivery. Temperature-responsive poly( N -isopropylacrylamide) (PNIPAAm)-modified liposomes can release their content following heating. In this study, we synthesized the temperature-responsive polymer poly( N -isopropylacrylamide)- co - N , N '-dimethylaminopropylacrylamide (P(NIPAAm- co -DMAPAAm)) and investigated the properties of liposomes modified with P(NIPAAm- co -DMAPAAm) for intracellular drug carriers. The copolymer displayed a thermosensitive transition at a lower critical solution temperature (LCST) that is higher than body temperature. Above the LCST, the temperature-responsive liposomes started to aggregate and release. The liposomes showed a fixed aqueous layer thickness (FALT) at the surface below the LCST, and the FALT decreased with increasing temperature. Above 37 °C, cytosolic release from the temperature-responsive liposomes was higher than that from the PEGylated liposomes, indicating intracellular uptake. Here, we showed that the tunable surface properties of the temperature-responsive polymer-modified liposomes possibly enabled their dehydration by heating, which likely induced a faster cellular uptake and release. Therefore, the liposomes could be highly applicable for improving intracellular drug-delivery carriers., Competing Interests: The authors declare no competing financial interest.

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.

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.

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.)

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.

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.

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.

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.)

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.

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.

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.

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.

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

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

Purpose: Tumor cell targeting is a promising strategy for enhancing the therapeutic potential of chemotherapy agents. Polyethylene glycol (PEG)-coated (sterically stabilized) liposomes show enhanced accumulation on the surface of tumors, but steric hindrance by PEGylation reduces the association of the liposome-bound ligand with its receptor. To increase folate receptor (FR) targeting, we optimized the concentration and PEG spacer length of folate-PEG-lipid in liposomes., Experimental Design: Three types of folate-linked liposomal doxorubicin were designed and prepared by optimizing the concentration and PEG spacer length of folate-PEG-lipid in PEGylated or non-PEGylated liposomes and by masking folate-linked liposomes where the folate ligand is "masked" by adjacent PEG spacers. The liposome targeting efficacy was evaluated in vitro and in vivo., Results: In human oral carcinoma KB cells, which overexpress FR, modification with sufficiently long PEG spacer and a high concentration of folate ligand to non-PEGylated liposomes increased the FR-mediated association and cytotoxicity more than with PEGylated and masked folate-linked liposomes. On the contrary, in mice bearing murine lung carcinoma M109, modification with the folate ligand in PEGylated and masked folate-linked liposomes showed significantly higher antitumor effect than with non-PEGylated liposomes irrespective of the length of time in the circulation after intravenous injection., Conclusions: The results of this study will be beneficial for the design and preparation of ligand-targeting carriers for cancer treatment.

The RNA interference (RNAi) effect is an alternative technology to antisense DNA as an experimental method of down-regulating a specific target protein. Optimal gene therapy for tumors must deliver synthetic small interfering RNA (siRNA) to tumor cells with high efficiency and minimal toxicity. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA with high transfection efficiency. In this study, to apply NP-OH for siRNA transfection, we optimized the charge ratio (+/-) of NP-OH/siRNA and nanoplex-forming solution, and evaluated the transfection efficiency into PC-3 cells. Positively charged nanoplex prepared in the presence of sodium chloride exhibited efficient siRNA transfer. The distribution of siRNA after transfection was strongly detected both in the cytoplasm and nucleus. Furthermore, we demonstrated that NP-OH nanoplex of bcl-2 siRNA significantly inhibited tumor growth compared with control siRNA, and the efficacy was comparable to commercial products. The results of the experiments showed that NP-OH nanoparticles have potential as a viable vector candidate for synthetic siRNA delivery.

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

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.

Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin alpha2beta1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArray) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.

A two-step transcriptional amplification system (TSTA) was used to enhance the efficacy of suicide gene therapy for treatment of prostate cancer. We designed a TSTA system and constructed two types of plasmid: one containing GAL4-VP16 fusion protein under the control of a tumor-specific promoter, the other containing luciferase or herpes simplex virus thymidine kinase (HSV-tk) under the control of a synthetic promoter. The TSTA systems using nanoparticles based on lipids were evaluated by measuring the amount of induced luciferase activity as a function of prostate-specific membrane antigen (PSMA) and midkine (Mk) promoters, specific for LNCaP and PC-3 prostate cancer cells, respectively. In LNCaP cells that were PSMA-positive, the TSTA system featuring the PSMA enhancer and promoter exhibited activity that was 640-fold greater than a system consisting of one-step transcription with the PSMA promoter. In contrast, this difference in activity did not occur in PSMA-negative PC-3 cells. In Mk-positive PC-3 cells, the TSTA system with the Mk promoter exhibited a five-fold increase in activity over one-step transcription, but such activity was not induced in Mk-negative LNCaP cells. When using HSV-tk for suicide gene therapy, TSTA systems featuring the PSMA or Mk promoter inhibited in vitro cell growth in the presence of ganciclovir. Furthermore, the TSTA system featuring the Mk promoter suppressed in vivo growth of PC-3 tumor xenografts to a greater extent than one-step transcription. These findings show that TSTA systems can enhance PSMA and Mk promoter activities and selectively inhibit PC-3 cell growth in tumors. This suggests that TSTA systems featuring tumor-specific promoters are suitable for cancer treatment by gene therapy.

Activated macrophages are the key effector cells in rheumatoid arthritis (RA) and secrete multiple mediators of inflammation including proinflammatory cytokines. We investigated delivery of a nuclear factor kappa B (NFkappaB) decoy by folate-linked lipid-based nanoparticles (NP-F) into murine macrophages. The expression of folate receptor (FR) in RAW264.7 cells activated by lipopolysaccaride was confirmed by strong expression of FR mRNA, and association of FITC-labeled folate-BSA conjugate. When transfected via NP-F, the NFkappaB decoy was strongly detected in the cytoplasm, and an inhibitory effect on the translocation of NFkappaB into the nucleus was observed at 0.03 microM of the decoy, suggesting that NP-F effectively delivered the NFkappaB decoy into the cytoplasm. This information is of value for the design of NFkappaB decoy carrier systems targeting FR in activated macrophages in gene therapy for autoimmune diseases such as RA.

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.

Purpose: To establish a novel formulation tumor-targeted drug carrier of lipophilic antitumor antibiotics, aclacinomycin A (ACM), folate-linked microemulsions were prepared and investigated both in vitro and in vivo., Experimental Design: Three kinds of folate-linked microemulsions with different polyethylene glycol (PEG) chain lengths loading ACM were formulated with 0.24 mol% folate-PEG(2000)-distearoylphosphatidylethanolamine (DSPE), folate-PEG(5000)-DSPE, and folate-lipid (without PEG linker) in microemulsions. In vitro studies were done in a human nasopharyngeal cell line, KB, which overexpresses the folate receptor (FR), and a human hepatoblastoma cell line, [FR(-)] HepG2. In vivo experiments were done in a KB xenograft by systemic administration of folate-linked microemulsions loading ACM., Results: The association of folate-linked microemulsions to KB cells could be blocked by 2 mmol/L free folic acid. Selective FR-mediated cytotoxicity of folate-linked microemulsions loading ACM was obtained in KB but not in HepG2 cells. The association of the folate-PEG(5000)-linked microemulsion and folate-PEG(2000)-linked microemulsion with the cells was 200- and 4-fold higher, whereas their cytotoxicity was 90- and 3.5-fold higher than those of nonfolate microemulsion, respectively. The folate-PEG(5000)-linked microemulsions showed 2.6-fold higher accumulation in solid tumors 24 hours after i.v. injection and greater tumor growth inhibition than free ACM., Conclusion: These findings suggest that a folate-linked microemulsion is feasible for tumor-targeted ACM delivery. This study shows that folate modification with a sufficiently long PEG chain on emulsions is an effective way of targeting emulsion to tumor cells.

Retinoic acid (RA), a potent inducer of cell differentiation, and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), a potent inducer of apoptosis, are well known as anticancer agents that are administered orally to patients for leukemia, breast and prostate cancer, respectively. However, it has not been studied whether both retinoids are effective on metastatic cancer. In mice implanted with M5076 cells, murine reticulum cell sarcoma survival times were prolonged by i.v. treatment of RA and 4-HPR entrapped in liposomes containing soybean-derived sterylglucoside mixture (SG), which accumulates in liver. In contrast, free RA and 4-HPR were inactive. These results indicate that RA and 4-HPR in SG-liposomes exhibit anticancer efficacy on metastatic cancers, and may have great potential for clinical use in the treatment of various cancers.

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

Falconensones A and B are new type of yellow pigment isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis. To date, these falconensones and their derivatives, falconensone A p-bromophenylhydrazone and falconensone A dioxime are known to exhibit biological activities, which include growth inhibition and both induction of differentiation and apoptosis of HL60 human leukemia cells. The synthetic derivatives have been shown to be more potent than natural falconensone A and B in eliciting these activities. Herein, we investigate whether falconensones inhibit growth of other cancer cell lines in vitro, and we evaluate their ability to modify survival in C57 BL/6J mice using M5076 murine reticulosarcoma in vivo, which is established as the metastasis model. Falconensone A, falconensone A p-bromophenylhydrazone, and falconensone A dioxime inhibit growth of human myeloid leukemia cell lines, HL60 and HL60R, human hepatoma cell line HepG2, human prostate cancer cell line DU-145, and human breast cancer cell line MCF-7/Adr(R), whereas falconensone B, the 4'-nor-methyl derivative of falconensone A, shows extremely low or no activity. In contrast, all of the falconensones are active in growth inhibition of human breast cancer cell line MCF-7. Survival time of M5076-implanted mice was prolonged by treatment with falconensones, particularly falconensone A dioxime. These results indicate that falconensone A and its derivatives exhibit anticancer efficacy in a broad spectrum of cancer cell lines. These agents may have great potential for clinical use in the treatment of various cancers.

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

Remote loading of insulin and bovine serum albumin (BSA) into neutral or positively-charged liposomes by incubation under a transmembrane pH gradient or non-pH gradient was investigated. Trapping efficiencies in several incubation conditions were compared with those of the conventional reverse-phase evaporation vesicle method (c-REV method). For neutral liposomes, insulin could not be effectively loaded into the liposomes by incubation, regardless of the incubation conditions. The trapping efficiency of insulin into positively-charged liposomes was higher than that of neutral liposomes, especially by the pH-gradient method. Insulin could be loaded into positively-charged liposomes about twofold more efficiently than by the pH-gradient method, compared with the c-REV method. Insulin distributed more on the surface of liposomes by the pH-gradient method than by the c-REV method. BSA showed significantly higher affinity to positively-charged liposomes than to neutral ones by various methods. However, the transmembrane pH-gradient method did not increase BSA loading into liposomes, compared with the c-REV and the non-pH-gradient methods. Our results suggest that the pH-gradient method, combining electrostatic interactions, may be useful for preparation of liposomal insulin and that the high hydrophobicity of BSA may not increase the remote loading of BSA into liposomes by the pH-gradient method.

The effect of shikonin (SK) on granulation tissue formation was biochemically evaluated and the biological effect of SK on cotton pellet induced granulation tissue formation and the induction of hind paw edema was compared with that of lambda-carrageenan (carrageenan) in rats. The dry weight of granulation tissue formed was increased by SK. The amounts of hemoglobin and hydroxyproline in granulation tissue were also increased by SK. These results suggest that SK has an enhancing effect on the formation of granulation tissue, accompanied by the proliferation of capillaries and the increased production of collagen in rats. SK showed mild stimulation toward swelling when injected into the hind paws of rats, as did carrageenan, while it showed a marked enhancing effect on granulation tissue formation compared with carrageenan. These results suggest that the mechanism underlying the biological effect of SK on granulation tissue formation is different from that of carrageenan, though the mild stimulation by SK might still contribute in part to the enhancing effect on granulation tissue formation.

The effect of a soybean-derived sterol mixture (SS) and a steryl glucoside mixture (SG) as enhancers of the nasal absorption of insulin in rabbits was investigated. SS consists of beta-sitosterol (Sit), campesterol (Camp), stigmasterol (Stig) and brassicasterol (Bras), and SG is a mixture of their monoglucosides. For each component of SS tested for efficacy in promoting the systemic absorption of nasally administered insulin, the following order was observed: Sit> or =Camp>Stig. This finding was in agreement with the order of the enhancers' lipophilicity. In the case of SG, the effect of beta-sitosterol beta-D-glucoside (Sit-G) was significantly greater than that of SG. The pharmacological bioavailability was 6.7% for SG and 11.3% for Sit-G in the suspension dosage forms. SG showed a greater degree of enhancement of insulin permeation through the nasal mucosa than SS. To elucidate the contribution of SG to the enhanced absorption, insulin permeation through an artificial membrane and the nasal mucosa was investigated in vitro, and the results were compared with those for SS. The findings suggest that SG and SS have some effect on nasal mucosa lipids.

A soybean-derived sterylglucoside mixture (SG) is a potential enhancer of the nasal absorption of insulin. The aim of the study was to examine the absorption of insulin given as the powder dosage form with SG and excipient and to determine the subchronic effects of SG on the morphology of rabbit nasal epithelium. The insulin powder dosage form with SG was administered to the rabbit nasal cavity for five successive days. The average bioavailability and the average pharmacological bioavailability of insulin were about 25.0 and 61.6%, respectively. The nasal mucosa was taken from the nasal cavity and side-effects were investigated using an optiphoto light microscope. The insulin powder dosage form with SG produced no signs of inflammation, erosion or squamous metaplasia. These findings indicate that SG can be considered as a safe and effective enhancer and excipient in the nasal insulin powder dosage form.