Tatsuaki Tagami

Effect of siRNA in PEG-coated siRNA-lipoplex on anti-PEG IgM production

For efficient delivery of small interfering RNA (siRNA) in vivo, it is important to control the blood circulation of the delivery vehicle. Surface modification of the siRNA/cationic liposome complex (siRNA-lipoplex) with polyethylene glycol (PEG) is expected to enhance circulation time in blood. However, we have recently reported that anti-PEG IgM production after the first injection of PEG-coated liposome is responsible for a reduction in the blood circulation of the second dose of the liposome, which is known as the accelerated blood clearance (ABC) phenomenon. It is unknown whether a PEG-coated siRNA-lipoplex (PSCL) can cause the ABC phenomenon and anti-PEG IgM production. In this study, an anti-PEG IgM response to PSCL was detected and was inversely related to the PSCL dose. Interestingly, the anti-PEG IgM response was significantly lower for PSCL than it was for PEG-coated naked cationic liposomes (PCL). The studies with splenectomized mice and nude mice indicated that anti-PEG IgM production was closely related to an interaction of PSCL and PCL with the spleen, which is associated with a T cell-independent mechanism. In addition, PSCL induced apoptosis on IgM-expressing splenic cells more strongly than PCL did, which suggests that accumulation in the spleen and the apoptotic effect of PEG-coated substances on splenic B cells could affect the potency of anti-PEG IgM production.

The effect of simvastatin-loaded polymeric microspheres in a critical size bone defect in the rabbit calvaria

The present study describes the development of a microsphere capsule based on polylactide-co-glycolide (PLGA) loaded with simvastatin that was subsequently incorporated into synthetic bone cement. The osteogenic effect of simvastatin-loaded bone cement was in a critical sized defect in vivo to test the hypothesis the biologic response would be different depending on the dosage of simvastatin applied to bone cement. Our results showed that simvastatin loaded PLGA microspheres can be successfully obtained through O/W emulsion/solvent evaporation method with appropriate morphologic characteristics and high encapsulation efficiency for incorporation in bone cements. The biodegradable characteristic of the microspheres successfully presented a slow release and the duration of the release lasted for more than 1 month. The in vivo experiment revealed that the microspheres containing simvastatin significantly enhanced bone formation in the rabbit calvaria critical size defect.

Improvement of survival in C6 rat glioma model by a sustained drug release from localized PLGA microspheres in a thermoreversible hydrogel.

A local drug delivery system based on sustained drug release is an attractive approach to treat brain tumors. We have developed a novel device using drug-incorporated poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in thermoreversible gelation polymer (TGP) formulation (drug/PLGA/TGP formulation). TGP forms a gel at body temperature but sol at room temperature. Therefore, when this formulation is injected into the brain tumor, the PLGA microspheres in TGP gel are localized at the injection site and do not diffuse throughout the brain tissue; eventually, sustained drug release from PLGA microspheres is achieved at the target site. In this study, two chemotherapeutic drugs (camptothecin (CPT) or vincristine (VCR)) were incorporated into PLGA microspheres to prepare drug/PLGA/TGP formulations. VCR/PLGA microspheres exhibited the higher encapsulation efficiency than CPT/PLGA microspheres (70.1% versus 30.1%). In addition, VCR/PLGA microspheres showed a higher sustained release profile than CPT/PLGA microspheres (54.5% versus 72.5% release, at 28 days). Therapeutic effect (mean survival) was evaluated in the C6 rat glioma model (control group, 18 days; CPT/PLGA/TGP treatment group, 24 days; VCR/PLGA/TGP treatment group, 33 days). In particular, the VCR/PLGA/TGP formulation produced long-term survivors (>60 days). Therefore, this formulation can be therapeutically effective formulation for the glioma therapy.

Global Gene Expression Profiling in Cultured Cells Is Strongly Influenced by Treatment with siRNA–Cationic Liposome Complexes

PurposeThe purpose of this study is to determine if the treatment with siRNA-lipoplexes significantly influences on global gene expression in the treated cells.MethodsWe investigated global gene expression in a HT1080 cell line by a cDNA microarray. We also evaluated the effect of lipofection on global gene expression by determining the change of the expression of an exogenous gene, green fluorescence protein (GFP), and also determined treatment-related cytotoxicity.ResultsTreatment of the cells with either siRNA-lipoplexes or cationic liposomes altered the expression of approximately 2,500 genes. When lipoplexes containing non-specific siRNAs were used, GFP expression was enhanced. In this case the effect was independent on the dose and type of siRNA in the formulation. By contrast, when lipoplexes containing a specific siRNA against GFP was used, GFP expression was markedly diminished. These results clearly indicate that an efficient reduction of a targeted gene expression by a specific siRNA is accompanied by a significant alteration of the expression of numerous non-targeted genes. In addition, treatment-related cytotoxicity increased with siRNA- and cationic lipid-doses, but was not dependent on siRNA type.ConclusionNon-specific effects of siRNA-lipoplexes may either enhance, attenuate or even fully mask the desired outcomes of siRNA-based biochemical studies and therapies.

Novel pharmaceutical cocrystal consisting of paracetamol and trimethylglycine, a new promising cocrystal former

Paracetamol (APAP), a frequently used antipyretic drug, has poor compression moldability. In this study, we identified a novel cocrystal consisting of APAP and trimethylglycine (TMG) that exhibits improved tabletability. TMG was used instead of oxalic acid (OXA), which is a coformer reported previously. The cocrystal (APAP-TMG at a molar ratio of 1:1) was characterized by X-ray analysis, infrared spectroscopy, and thermal analysis. The crystal structure of APAP-TMG revealed that it was a cocrystal, since no proton was transferred between the APAP and TMG molecules. The compression and dissolution properties of APAP-TMG were similar to that of the APAP-OXA cocrystal. In addition, taste sensing measurements suggested that TMG has a sweet and umami taste, indicating that TMG should suppress the bitterness of APAP. From these results, TMG could be a safe and promising cocrystal former that could replace OXA, which can irritate tissues.

Simple and effective preparation of nano-pulverized curcumin by femtosecond laser ablation and the cytotoxic effect on C6 rat glioma cells in vitro

The pulverization of poorly water-soluble drugs and drug candidates into nanoscale particles is a simple and effective means of increasing their pharmacological effect. Consequently, efficient methods for pulverizing compounds are being developed. Femtosecond lasers, which emit ultrashort laser pulses, can be used to generate nanoscale particles without heating and are finding in various fields, including pharmaceutical science. Laser ablation holds promise as a novel top-down pulverization method for obtaining drug nanoparticles. We used a poorly water-soluble compound, curcumin (diferuloyl methane), to understand the characteristics of femtosecond laser pulverization. Various factors such as laser strength, laser scan speed, and the buffer solution affected the size of the curcumin particles. The minimum curcumin particle size was approximately 500 nm; the particle size was stable after 30 days. In vitro studies suggested that curcumin nanoparticles exhibited a cytotoxic effect on C6 rat glioma cells, and remarkable intracellular uptake of the curcumin nanoparticles was observed. The results suggest that femtosecond laser ablation is a useful approach for preparing curcumin nanoparticles that exhibit remarkable therapeutic effects.

3D Printing Factors Important for the Fabrication of Polyvinylalcohol Filament-Based Tablets

Three-dimensional (3D) printers have been applied in many fields, including engineering and the medical sciences. In the pharmaceutical field, approval of the first 3D-printed tablet by the U.S. Food and Drug Administration in 2015 has attracted interest in the manufacture of tablets and drugs by 3D printing techniques as a means of delivering tailor-made drugs in the future. In current study, polyvinylalcohol (PVA)-based tablets were prepared using a fused-deposition-modeling-type 3D printer and the effect of 3D printing conditions on tablet production was investigated. Curcumin, a model drug/fluorescent marker, was loaded into PVA-filament. We found that several printing parameters, such as the rate of extruding PVA (flow rate), can affect the formability of the resulting PVA-tablets. The 3D-printing temperature is controlled by heating the print nozzle and was shown to affect the color of the tablets and their curcumin content. PVA-based infilled tablets with different densities were prepared by changing the fill density as a printing parameter. Tablets with lower fill density floated in an aqueous solution and their curcumin content tended to dissolve faster. These findings will be useful in developing drug-loaded PVA-based 3D objects and other polymer-based articles prepared using fused-deposition-modeling-type 3D printers.

Active Drug Targeting of a Folate-Based Cyclodextrin–Doxorubicin Conjugate and the Cytotoxic Effect on Drug-Resistant Mammary Tumor Cells In Vitro

Active drug targeting is an effective therapeutic approach for the treatment of malignant cancers and novel types of drug carriers have been developed. In this study, we developed a cyclodextrin (CD)-based novel carrier-drug conjugate, called per-FOL-β-CD-ss-DOX, which has folic acid (FA) molecules at the end of primary hydroxyl groups of β-CD and a pH-cleavable spacer with an anticancer drug, doxorubicin (DOX), at the end of secondary hydroxyl groups. This per-FOL-β-CD-ss-DOX exhibited a significant cellular uptake as compared with the free DOX solution by EMT6/P cells, which activate the expression of folate receptor (FR). Cellular uptake of per-FOL-β-CD-ss-DOX was significantly inhibited in the presence of FA and was also inhibited at 4°C. The conjugate exhibited remarkable cytotoxic effects in EMT6/AR1 cells, which are resistant to DOX, whereas free DOX solution did not exhibit this effect. These results suggest that per-FOL-β-CD-ss-DOX can be taken up into cells via FR-related endocytosis and the cleaved DOX derived from it in endosomes could escape the efflux caused by P-glycoprotein, resulting in the cytotoxic effect. Therefore, the drug delivery by per-FOL-β-CD-ss-DOX may be a useful approach for drug delivery to FR-expressing cells such as drug-resistant malignant cancers.

Agitation during lipoplex formation improves the gene knockdown effect of siRNA

The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.

Effective Remote Loading of Doxorubicin into DPPC/Poloxamer 188 Hybrid Liposome to Retain Thermosensitive Property and the Assessment of Carrier-Based Acute Cytotoxicity for Pulmonary Administration

Functional liposome administration via the pulmonary route is of interest to treat pulmonary diseases, including cancer. Here, a block copolymer used as a medical additive, Poloxamer 188 (P188), was incorporated into liposome membranes, and the thermosensitive characteristics of the DPPC/P188 hybrid liposomes were assessed. An increase in P188 incorporation in DPPC liposomes enhanced the release of calcein, a fluorescence marker, from liposomes at 42°C in vitro; calcein release was significantly slower at 37°C. The lipid composition was optimal at a DPPC/P188 ratio of 3:0.4 (molar ratio). DPPC/P188 liposomes did not exhibit in vitro cytotoxicity against A549 cells and Raw 264.7 cells used as models of pulmonary cells or trigger in vivo acute inflammation as determined by the secretion of tumor necrosis factor alpha. Next, an anticancer drug, doxorubicin (DOX), was loaded with approximately 90% efficiency into DPPC/P188 liposomes using a remote-loading method and a DOX-phospholipid ratio of 1:20 (w/w). The interior buffer of liposome has remarkably changed DOX release at 42°C. DOX released from DPPC/P188 liposomes at 42°C exhibited cytotoxic effects toward A549 cells comparable with free DOX solution. These results suggest that a DOX-loaded DPPC/P188 liposome formulation administered via the pulmonary route may be useful for treating lung cancer.