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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

OLYMPUS OM-4 A decade ago, Olympus introduced the OM-1, first of a generation of extremely compact, full-featured 35mm SLRs. Three years later, it brought forth the equally compact OM-2, the [...]

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]

The effect and mechanism of action of β-sitosterol β-d-glucoside (Sit-G) on the in vitro and in vivo nasal absorption of FITC-dextran (molecular weight, 4400; FD-4) in rabbits were studied in comparison with β-sitosterol (Sit). The FD-4 permeation in the powder dosage form was increased by Sit-G and Sit and related to the uptake of Sit-G and Sit with no changes in the amount of cholesterol in the excised nasal mucosa. The application of Sit and Sit-G increased FD-4 permeation with and without a decrease in transepithelial resistance (TEER), respectively. These results suggested that the mechanism of the enhancement by Sit-G was different from those of Sit and sodium caprate; Sit-G may exert its effects mainly via the transcellular pathway due to perturbation of the mucosal membrane. [Copyright &y& Elsevier]

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.

Short interfering RNA (siRNA) delivery systems using nanoparticle carriers have been limited by inefficient intracellular delivery. One drawback is the poor cellular uptake of siRNA/particle complexes through the plasma membrane and release of the nucleic acids into the cytosol. In this study, to develop the temperature-responsive liposome as a novel carrier for siRNA delivery, we prepared lipoplexes and assessed cellular uptake of siRNA and gene silencing activity of target genes, compared with those of a commercial transfection reagent, Lipofectamine RNAiMAX, and non-modified or PEGylated liposomes. The temperature-responsive polymer, N-isopropylacrylamide-co-N,N'-dimethylaminopropylacrylamide [P(NIPAAm-co-DMAPAAm)]-modified liposome induced faster intracellular delivery because P(NIPAAm-co-DMAPAAm) exhibits a lower critical solution temperature (LCST) changing its nature from hydrophilic to hydrophobic above the LCST. The temperature-responsive liposomes showed significantly higher gene silencing activity than other carriers with less cytotoxicity. Furthermore, we showed that the temperature-responsive lipoplexes were internalized mainly via microtubule-dependent transport and also by the clathrin-mediated endocytosis pathway. This is the first report that temperature-responsive polymer-modified liposomes thermally enhanced silencing activity of siRNA. The dehydrated polymer on the liposomes, and its aggregation caused around the LCST, can probably be attributed to effective cellular uptake of the lipoplexes for gene silencing activity by interaction with the cell membrane., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Surface PEGylation on nanoparticles has greatly helped prolong their blood circulation half-lives. However, The injection of PEGylated nanoparticles into mice induced poly(ethylene glycol) (PEG)-specific IgM antibodies (anti-PEG IgMs), significantly changing PEG-liposomes' pharmacokinetics. In this study, we used various PEG-conjugates to conduct a mechanistic study of anti-PEG IgMs' binding behavior. The conventional belief has been that anti-PEG IgMs bind to PEG main chains; however, our findings reveal that anti-PEG IgMs did not bind to PEG main chains, whereas anti-PEG IgMs did bind to PEG-hydrophobic polymer blocks. The insertion of a hydrophilic polymer between each PEG chain and each hydrophobic polymer block suppressed anti-PEG IgMs' binding. We prove here that hydrophobic blocks are essential to anti-PEG IgMs' binding, and also that anti-PEG IgMs do not bind to intact PEGs without hydrophobic moiety. These results support our conclusion that anti-PEG IgMs exhibit specificity to PEG; however, the presence of a hydrophobic block at a proximity position from each PEG chain is essential for the binding. Also in the present study, we elucidate relations between anti-PEG IgMs and PEGylated nanoparticles. In one of our previous studies, anti-PEG IgMs scarcely affected the pharmacokinetics of PEG-b-poly(β-benzyl l-aspartate) block copolymer (PEG-PBLA) micelles, whereas anti-PEG IgMs significantly decreased PEG-liposomes' blood circulation half-life. Finally, we found that the ratio of anti-PEG IgM molecules to PEG-liposome particles is critical to these pharmacokinetic changes, and that a 10-fold increase in the number of anti-PEG IgM molecules permitted them to capture the PEG-liposome particles, thus leading to the aforementioned changes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We evaluated structural factors characterizing PEG-b-P(Asp-Bzl) micelles including core size, aggregation number (Nagg), and core surface PEG density by means of small-angle X-ray scattering (SAXS), field flow fractionation with multi-angle light scattering (FFF-MALS) analysis, and DLS. Furthermore, we evaluated the stability of PEG-b-P(Asp-Bzl) micelles by means of GPC. This paper reports the correlation between the evaluated micelles' structural factors and the micelles' behaviors including the micelles' in vivo pharmacokinetic behaviors. One micelle PEG(12)-b-P(Asp-Bzl) (PEG=12,000) exhibited a high core surface density (~0.99 chain/nm(2)). In these circumstances, PEG(12)-b-P(Asp-Bzl) micelles exhibited a highly stretched PEG brush form. However, the evaluated core surface PEG densities could not fully explain the micelles' in vivo pharmacokinetic behaviors. In contrast, GPC will become a strong tool for predicting PEG(12)-b-P(Asp-Bzl) micelles' in vivo behaviors, as well as the micelles' in vitro behaviors. The stability results correlated strongly with the area-under-the-curve (AUC) values of PEG-b-P(Asp-Bzl) micelles' in vivo pharmacokinetics. Finally, we evaluated PEG(12)-b-P(Asp-Bzl) micelles' most effective structural factor for determining the micelles' behaviors, and the micelles' outermost shell surface's PEG density (DOS, PEG) correlated with the micelles' behaviors. We revealed that the evaluated DOS, PEG is the most important factor for understanding PEG(12)-b-P(Asp-Bzl) micelles' behaviors., (Copyright © 2015. Published by Elsevier B.V.)

Context: Cationic liposomes can efficiently deliver siRNA to the lung by intravenous injection of cationic liposome/siRNA complexes (lipoplexes)., Objective: The aim of this study was to examine a formulation of cationic liposomes for siRNA delivery to lung metastasis of breast tumor., Materials and Methods: For the preparation of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDAB) as a cationic lipid and cholesterol (Chol) or 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) as a neutral lipid were used. In vitro and in vivo gene silencing effects by cationic lipoplexes were evaluated after transfection into stably luciferase-expressing human breast tumor MCF-7-Luc cells and after intravenous injection into mice with lung MCF-7-Luc metastasis, respectively. Intracellular localization of siRNA after transfection into MCF-7 cells by cationic lipoplexes and biodistribution of siRNA after intravenous injection of cationic lipoplexes into the mice with lung metastasis were examined by confocal and fluorescent microscopy analyses, respectively., Results: In in vitro transfection, DOTAP/DOPE and DDAB/DOPE lipoplexes of luciferase siRNA strongly suppressed luciferase activity in MCF-7-Luc cells, but DOTAP/Chol and DDAB/Chol lipoplexes did not, although DOTAP/Chol and DDAB/Chol lipoplexes exhibited higher cellular uptake than DOTAP/DOPE and DDAB/DOPE lipoplexes. When their cationic lipoplexes were intravenously injected into mice with lung MCF-7-Luc metastasis, siRNAs were mainly accumulated in the lungs; however, the reduced luciferase activities in the lung-metastasized tumors were observed only by injections of DOTAP/Chol and DOTAP/DOPE lipoplexes, but not by DDAB/Chol and DDAB/DOPE lipoplexes., Conclusions: DOTAP-based liposomes might be useful as an in vivo siRNA delivery carrier that can induce gene silencing in lung-metastasized tumors.

Purpose: NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets., Methods: The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy., Results: The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry., Conclusions: These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets.

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) cause genital herpes, which can enhance the acquisition of human immunodeficiency virus. The development of anti-HSV agents with novel mechanisms of action is urgently required in the topical therapy of genital herpes. In this study, the in vitro and in vivo anti-HSV effects of Epomin SP-012(®), a highly cationic polyethylenimine, were evaluated. When the in vitro antiviral effects of SP-012 were assessed, this compound showed potent activity against HSV-1 and HSV-2. It inhibited the attachment of HSV-2 to host cells and cell-to-cell spread of infection in a concentration-dependent manner and exerted a virucidal effect. No SP-012-resistant HSV-2 was found when the virus was successively passaged in the presence of SP-012. In a mouse genital herpes model, topically administered SP-012 inhibited the progression of the disease caused by HSV infection. These data illustrate that SP-012 may be a novel class of HSV inhibitor that would be acceptable for long-term topical application.