Senescence recovering by dual drug-encapsulated liposomal nanoparticles for large-scale human cell expansion.

Although regenerative therapy and bioartificial tissues and organs require a sufficient number of human cells, current cell expansion processes are accompanied by accumulation of senescent cells that are related to deterioration of cellular functions and induction of senescence-associated secretory phenotype (SASP). Therefore, suppression of replicative senescence during expansion is one of the crucial issues for dissemination of regenerative medicine. We herein developed dual drug-encapsulated liposomal nanoparticles (LNPs) to suppress cellular senescence in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and natural killer (NK) cells by removal of dysfunctional mitochondria from the senescent cells. We found that LNP treatment reduced senescent makers; downregulation of p21 expression and reduction of SA-ß-Gal activity in both cells provably due to mitophagy reactivation in the cells. Moreover, SASP secretion in hAT-MSCs and tumor cytotoxicity in NK cells were also improved upon LNP treatments. These findings may contribute to the production of highly effective expanded cells for regenerative medicine and bioartificial tissues and organs.

Gas Adsorption and Diffusion Behaviors in Interfacial Systems Composed of a Polymer of Intrinsic Microporosity and Amorphous Silica: A Molecular Simulation Study.

We investigate the adsorption and diffusion behaviors of CO2, CH4, and N2 in interfacial systems composed of a polymer of intrinsic microporosity (PIM-1) and amorphous silica using grand canonical Monte Carlo (GCMC) and molecular dynamics (MD) simulations. We build model systems of mixed matrix membranes (MMMs) with PIM-1 chains sandwiched between silica surfaces. Gas adsorption analysis using GCMC simulations shows that gas molecules are preferentially adsorbed in microcavities distributed near silica surfaces, resulting in an increase in the solubility coefficients of CO2, CH4, and N2 compared to bulk PIM-1. In contrast, diffusion coefficients obtained from MD simulations and then calibrated using the dual-mode sorption model show different tendencies depending on gas species: CO2 diffusivity decreases in MMMs compared to PIM-1, whereas CH4 and N2 diffusivities increase. These differences are attributed to competing effects of silica surfaces: the emergence of larger pores as a result of chain packing disruption, which enhances gas diffusion, and a quadrupole-dipole interaction between gas molecules and silica surface hydroxyl groups, which retards gas diffusion. The former has a greater impact on CH4 and N2 diffusivities, whereas the latter has a greater impact on CO2 diffusivity due to the strong quadrupole-dipole interaction between CO2 and surface hydroxyls. These findings add to our understanding of gas adsorption and diffusion behaviors in the vicinity of PIM-1/silica interfaces, which are unobtainable in experimental studies.

Byproduct-Free Intact Modification of Insulin by Cholesterol End-Modified Poly(ethylene glycol) for in Vivo Protein Delivery.

Insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. To maximize the effect of the treatment of these diseases, the addition of poly(ethylene glycol) (PEGylation) methods for the insulin are widely developed. Here, to make these PEGylation methods the simplest, we report the byproduct-free intact modification of insulin by cholesterol end-modified poly(ethylene glycol) with urethane, propyl, and methoxy groups (that is, Chol-U-Pr-mPEG). The noncovalent PEGylation by the Chol-U-Pr-mPEG has been achieved by the simple mixing of insulin with the Chol-U-Pr-mPEG in aqueous solution, followed by freeze-drying. The formation of the Chol-U-Pr-mPEG/insulin complex has proceeded without byproducts, such as N-hydroxysuccinimide, formed by the conventional covalent PEGylation using an active ester group. The byproduct-free PEGylation has preserved insulin conformation as well as primary structure. The intact PEGylation has protected insulin from hydrolysis by protease. The resulting insulin modified by the Chol-U-Pr-mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice. Consequently, the Chol-U-Pr-mPEG/insulin complex formation offers the byproduct-free intact PEGylation of insulin for in vivo protein delivery.

Plasmid DNA mono-ion complex stabilized by hydrogen bond for in vivo diffusive gene delivery.

Our original concept of the mono-ion complex (MIC) between plasmid DNA (pDNA) and a monocationic biocompatible polymer has been stabilized by hydrogen bond formation. To form the hydrogen bond with pDNA, ω-amide-pentylimidazolium end-modified poly(ethylene glycol), that is, APe-Im-PEG, has been synthesized. Agarose gel retardation assay and circular dichroism measurement have revealed that the MIC between pDNA and APe-Im-PEG has been stabilized by the hydrogen bond between pDNA and the ω-amide group and that the stable MIC has surprisingly further migrated into gel, as compared with naked pDNA. The rise of melting temperature suggests that the specific hydrogen bond forms between an adenine-thymine base pair and the ω-amide group. The resulting pDNA MIC with APe-Im-PEG has enhanced gene expression by intramuscular administration in mice, as compared with a poly(ethylenimine) polyion complex (PIC). These results suggest that the pDNA MIC is diffusive in vivo administration site, as compared with pDNA PICs. Our methodology for MIC stabilization by a ω-amide group is expected to offer superior supramolecular systems to those by ubiquitous PICs for in vivo diffusive gene delivery.

Alkylimidazolium end-modified poly(ethylene glycol) to form the mono-ion complex with plasmid DNA for in vivo gene delivery.

In this study, we consider that the decrease in the transfection activity of polycations in vivo, compared with that in vitro, results from their polyion complex formation. Namely, owing to cross-linking between polycations and plasmid DNAs (pDNAs), the disadvantage of in vivo gene delivery mainly stems from the difficulty in controlling the properties of the resulting polyion complex at the nanoscale size. To avoid the cross-linking by polycations, we have establish the concept of "mono-ion complex" formation between pDNA and a monocationic biocompatible polymer. Here we have synthesized alkylimidazolium end-modified poly(ethylene glycol), that is, R-Im-PEG, and have tuned the electrostatic interaction between the resulting alkylimidazolium group and the phosphate group of pDNA by the length of the alkyl chain to achieve "mono-ion complex" formation with pDNA for in vivo gene delivery. Instead of a polyion complex, our original concept of the "mono-ion complex" consisting of the Bu-Im-PEG and pDNA is expected to offer unique tools to break through the barriers of in vivo gene delivery. As well as the field of gene delivery, this study is considered to have exploded the common sense that it is impossible to form not a polyion complex but a "mono-ion complex" under aqueous conditions for all fields of the modification of biomacromolecules.

Possibility for the development of cosmetics with PLGA nanospheres.

The optimized preparation of Poly-(lactide-co-glycolic acid) (PLGA) nanospheres containing ubiquinone (UQ) for cosmetic products was pursued. By investigating various conditions for the preparation of UQ/PLGA nanospheres such as the molecular weight of PLGA, PLGA concentration, and UQ concentration, UQ/PLGA nanospheres with increased stability and slower drug release at a higher drug loading efficiency were prepared. Permeation tests on the prepared nanospheres using iontophoresis via electric dermal administration on membrane filters (200 nm pore size) and hairless mouse skin samples were also carried out. After iontophoresis, the nanospheres choked the membrane filter and remained on the horny layer of the hairless mouse skin, even after washing. Therefore, the prepared UQ/PLGA nanospheres and the established iontophoresis technique with the PLGA nanospheres in the present study can be applied to the future development of cosmetics.

Synthesis and characterization of methylated poly(L-histidine) to control the stability of its siRNA polyion complexes for RNAi.

Poly(L-histidine) (PLH) with dimethylimidazole groups has been synthesized as a pH-sensitive polypeptide to control the stability of its small interfering RNA (siRNA) polyion complexes for RNA interference (RNAi). The resulting methylated PLH (PLH-Me) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurement. Agarose gel retardation assay proved that the quaternary dimethylimidazole groups worked as cationic groups to retain siRNA. The stability of the PLH-Me/siRNA complexes has depended on the content of hydrophobic groups, that is, τ/π-methylimidazole groups as well as deprotonated imidazole groups. PLH-Me exhibited no significant cytotoxicity despite the existence of cationic dimethylimidazole groups. By use of PLH-Me as a pH-sensitive siRNA carrier, the PLH-Me/siRNA complexes mediated efficient siRNA delivery attributed to the dimethylimidazole groups, and the gene silencing depended on the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups. These results suggest that PLH-Me controls the stability of siRNA polyion complexes by enhancing noncytotoxic siRNA delivery by optimizing the content balance of dimethyl, τ/π-methyl, and unmodified imidazole groups.

Up-regulation of gene expression by transfection to hepatocyte spheroids.

The gene expression is up-regulated by transfection to spheroids rather than monolayered cells. Monolayered hepatocytes form spheroids on rubbed polyimide membrane after 3 day incubation. The transfection of a rhodamine-labeled plasmid DNA or a green fluorescent protein (GFP) reporter gene to monolayered hepatocytes makes the spheroids exhibiting the fluorescence from the rhodamine or GFP inside the resulting spheroids. On the other hand, the transfection to hepatocyte spheroids makes the spheroids exhibit the fluorescence only outside the resulting spheroids. However, the whole gene expression of the luciferase reporter plasmid DNA from the lysate of the transfected spheroids is up-regulated in early incubation time, as compared to that of the transfected monolayered hepatocytes. Furthermore, the secretion of vascular endothelial growth factor (VEGF) from the VEGF-transfected spheroids is higher than that from the transfected monolayered hepatocytes. These results suggest that the up-regulation of exogenous gene expression is achieved by the control of differentiation and proliferation of hepatocytes via spheroid formation.

Pharmaceutical effect of manganese porphyrins on manganese superoxide dismutase deficient mice.

Mice lacking manganese-superoxide dismutase (Mn-SOD) activity exhibit typical pathology of dilated cardiomyopathy (DCM). In the present study, the structure-activity relationship between the water-soluble manganese (Mn) porphyrin with SOD activity and the in vivo pharmaceutical effect on DCM is reported. The Mn-SOD-deficient mice were treated with Mn-porphyrins for 3 weeks. The treatment of a Mn-porphyrin, MnM2Py(2)P, suppressed the progression of cardiac dilation. These results suggest that the Mn-porphyrin MnM2Py(2)P treatment is proposed as a potential therapy for DCM.

Zinc-chelated poly(1-vinylimidazole) and a carbohydrate ligand polycation form DNA ternary complexes for gene delivery.

Zinc-chelated poly(1-vinylimidazole) (PVIm-Zn) and a carbohydrate ligand polycation, a poly(l-lysine) conjugated with lactose molecules (PLL-Lac), have formed DNA ternary complexes for gene delivery. The particle size of the PVIm-Zn/DNA complexes with negative zeta potential was decreased by the addition of the PLL-Lac. The resulting PLL-Lac/PVIm-Zn/DNA ternary complexes, which exhibited the pH-dependent dissociation of the PLL-Lac, mediated more gene expression than the PVIm/DNA binary complexes. The PLL-Lac/PVIm-Zn/DNA complexes with the specific recognition of cell surface receptors mediated the highest gene expression without cytotoxicity at a relatively lower charge ratio (positive/negative = 2.5). These results suggest that the pH-dependent dissociation of the carbohydrate ligands after the recognition of cell surface receptors, including the physicochemical and biochemical function of PVIm-Zn, played an important role in gene expression.

Aligned electrospun nanofiber composite membranes for fuel cell electrolytes.

We have synthesized the novel composite membranes composed of sulfonated polyimide nanofibers and sulfonated polyimide for proton exchange membrane fuel cell. It was clear that the polyimides within nanofiber were significantly oriented or aggregated when electrospun; as the result, the membrane stability, such as oxidative and hydrolytic stabilities, of the composite membrane was significantly improved with an increase in nanofiber, and oxygen permeability of the composite membrane also decreased when compared to that determined in the membrane without nanofibers. In addition, the proton conductivity of the membrane in the parallel direction indicated a significantly higher value when compared to that determined for the membrane in the perpendicular direction or for the membrane without nanofibers prepared with conventional solvent-casting method. Consequently, nanofibers proved to be promising materials as a proton exchange membrane and the composite membrane containing nanofibers may have potential application for use in fuel cells.

Fabrication and Electrolyte Characterizations of Nanofiber Framework-Based Polymer Composite Membranes with Continuous Proton Conductive Pathways.

For future fuel cell operations under high temperature and low- or non-humidified conditions, high-performance polymer electrolyte membranes possessing high proton conductivity at low relative humidity as well as suitable gas barrier property and sufficient membrane stability are strongly desired. In this study, novel nanofiber framework (NfF)-based composite membranes composed of phytic acid (Phy)-doped polybenzimidazole nanofibers (PBINf) and Nafion matrix electrolyte were fabricated through the compression process of the nanofibers. The NfF composite membrane prepared from the pressed Phy-PBINf showed higher proton conductivity and lower activation energy than the conventional NfF composite and recast-Nafion membranes, especially at low relative humidity. It is considered that the compression process increased the nanofiber contents in the composite membrane, resulting in the construction of the continuously formed effective proton conductive pathway consisting of the densely accumulated phosphoric acid and sulfonic acid groups at the interface of the nanofibers and the Nafion matrix. Since the NfF also improved the mechanical strength and gas barrier property through the compression process, the NfF composite polymer electrolyte membranes have the potential to be applied to future fuel cells operated under low- or non-humidified conditions.

Synthesis and characterization of alkylated poly(1-vinylimidazole) to control the stability of its DNA polyion complexes for gene delivery.

Poly(1-vinylimidazole) (PVIm) with alkylated imidazole groups has been synthesized as a pH-sensitive polycation to control the stability of its DNA polyion complexes for gene delivery. The resulting alkylated PVIm (PVIm-R) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurements. Agarose gel retardation assay proved that the alkylated imidazole groups worked as anchor groups to retain DNA. Pyrene fluorescence measurement showed that the hydrophobic domain of the DNA complex with butylated PVIm (PVIm-Bu) increased after the protonation of imidazole groups of the PVIm-Bu to enhance the membrane disruptive activity. The PVIm-Bu exhibited no significant cytotoxicity in spite of the existence of cationic groups. The resulting PVIm-Bu/DNA complexes easily released DNA, as compared with the octylated PVIm, which was examined by competitive exchange with dextran sulfate. As a result, the PVIm-R/DNA complexes mediated efficient gene delivery, and the gene expression depended on the length and density of the alkyl chains. These results suggest that pH-sensitive PVIm-R's control of the stability of DNA polyion complexes enhanced noncytotoxic gene delivery by the optimized alkylated imidazole groups.

Effects of a calcium-channel blocker (CV159) on hepatic ischemia/reperfusion injury in rats: evaluation with selective NO/pO2 electrodes and an electron paramagnetic resonance spin-trapping method.

Nitric oxide (NO) and the partial pressure of oxygen (pO(2)) in the liver were simultaneously quantified in rats with partial hepatic ischemia/reperfusion injury (PHIRI). Real-time NO/pO(2) monitoring and immunohistochemical analysis for superoxide dismutase and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) were performed to evaluate the protective effects of a dihydropyridine-type calcium-channel blocker--CV159--on PHIRI. Serum high-mobility-group box-1 (HMGB-1) was measured to assess cellular necrosis. Moreover, we used in vitro/ex vivo electron paramagnetic resonance spin trapping to assess the hydroxyl radical (*OH)-scavenging activity (OHSA) of CV159 and the liver tissue. The NO levels were significantly higher in CV159-treated rats than in control rats throughout the ischemic phase. Immediately after reperfusion, the levels temporarily increased in waves and then gradually decreased in the treated rats but remained constant in the control rats. pO(2) was continually higher in the treated rats. In these rats, hepatic eNOS expression increased, whereas iNOS expression decreased. The treated rats exhibited significantly higher cytosolic and mitochondrial concentrations NOx (NO(2)+NO(3)). The serum HMGB-1 levels significantly decreased in the treated rats. Moreover, CV159 directly scavenged *OH and both mitochondrial and cytosolic OHSA were preserved in the treated rats. Thus, CV159-mediated inhibition of intracellular Ca(2+) overloading may effectively minimize organ damage and also have *OH-scavenging activity and the cytoprotective effects of eNOS-derived NO.

Control of drug loading efficiency and drug release behavior in preparation of hydrophilic-drug-containing monodisperse PLGA microspheres.

We prepared monodisperse poly(lactide-co-glycolide) (PLGA) microspheres containing blue dextran (BLD)--a hydrophilic drug--by membrane emulsification technique. The effects of electrolyte addition to the w(2) phase and significance of the droplet size ratio between primary (w(1)/o) and secondary (w(1)/o/w(2)) emulsions during the preparation of these microspheres was examined. The droplet size ratio was evaluated from the effect of stirring rate of the homogenizer when preparing the primary emulsion. The drug loading efficiency of BLD in these microspheres increased with stirring rate. It increased to approximately 90% when 2.0% NaCl was added to the w(2) phase. Drug release from these microspheres was slower than that when they were prepared without electrolyte addition. Despite the very high efficiency drug release was gradual because BLD was distributed at the microspheres core. Relatively monodisperse hydrophilic-drug-containing PLGA microspheres with controlled drug loading efficiency and drug release behavior were prepared.

The potential of lipid-polymer nanoparticles as epigenetic and ROS control approaches for COPD.

Chronic obstructive pulmonary disease (COPD) is a lung disease caused by an inflammatory response to various inhaled toxins, especially cigarette smoke. Reactive oxygen species (ROS) and epigenetic abnormality are intimately related to the pathology of COPD, and the overproduction of ROS results in a decrease of histone deacetylase 2 (HDAC2), leading to glucocorticoid resistance. Therefore, a novel treatment that simultaneously reduces ROS level and glucocorticoid resistance is urgently needed. In this study, we developed a codelivery system using core-shell type lipid-polymer nanoparticles (LPNs) composed of a poly(lactic acid) (PLA) core encapsulating a potent antioxidant Mn-porphyrin dimer (MnPD) and a cationic lipid (DOTAP) shell that binds HDAC2-encoding plasmid DNA (pHDAC2), as a new therapeutic approach toward COPD. The transfection of pHDAC2 combined with the elimination of ROS by MnPD exhibited a significant enhancement of intracellular HDAC2 expression levels, suggesting that the multi-antioxidative activity of MnPD plays a crucial role in the expression of HDAC2. Moreover, treatment with LPNs efficiently ameliorated the steroid resistance in COPD models in vitro as evidenced by the lowered expression levels of IL-8. Recovery from mitochondrial dysfunction may be the mechanism underlying the action of LPNs. The PLA-MnPD/DOTAP/pHDAC2 system proposed offers a new therapeutic approach for COPD based on the synergism of ROS elimination and HDAC2 expression.

Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

To cleave biocompatible poly(ethylene glycol) (PEG) from the mono-ion complex (MIC) for sustainable cellular uptake in vivo, ω-amide-pentylimidazolium end-modified PEG with an ester bond, that is, APe-Im-E-PEG, has been synthesized. The hydrolysis of the resulting APe-Im-E-PEG proceeded during the incubation for 2 weeks under physiological conditions, which was confirmed by gel filtration chromatography. APe-Im-E-PEG formed the MIC with plasmid DNA (pDNA), assessed by agarose gel retardation assay. Furthermore, dynamic light scattering measurement and transmission electron microscopy observations have estimated that the particle size of the resulting MIC was approximately 30 nm, with a rather flexible structure. The APe-Im-E-PEG/pDNA MIC incubated for 2 weeks exhibited hemolytic activity at endosomal pH, presumably because the pH-sensitive carboxyl groups revealed after the hydrolysis of an ester bond of APe-Im-E-PEG. The APe-Im-E-PEG/pDNA MIC enhanced the gene expression 2 weeks after transfection in vivo by intramuscular administration in mice. Consequently, in vivo sustainable gene expression has been achieved by the molecular design of APe-Im-E-PEG for cellular uptake and endosomal escape proceeded by temporal hydrolysis of the ester bond.

Preparation and properties of PLGA microspheres containing hydrophilic drugs by the SPG (shirasu porous glass) membrane emulsification technique.

In the present paper, monodisperse poly (lactide-co-glycolide) (PLGA) microspheres containing the hydrophilic model drug, blue dextran (BLD), were manufactured by the solvent evaporation method and the shirasu porous glass (SPG) membrane emulsification technique. In order to prepare PLGA microspheres with a higher drug loading efficiency by the membrane emulsification technique, the test of stability and productivity of the primary emulsion (w(1)/o emulsion) was preliminary examined by change species or concentration of the oil-soluble surfactant and the ratio of water and organic solvent. The primary emulsion (w(1)/o) composed of the BLD aqueous solution and dichloromethane (DCM) dissolved PLGA was prepared with the micro homogenizer. The secondary emulsion (w(1)/o/w(2)) was prepared by the SPG membrane emulsification technique. BLD/PLGA microspheres of various micro level sizes of 2.0-10 microm prepared by variation of pore size of the using SPG membrane. The highly monodisperse BLD/PLGA microspheres were also manufactured by added polyethylene glycol (PEG) into the water phase, as reported in a previous paper. The initial release rate of the drug from such microspheres controlled than the sample manufactured without an additive.

Effect of polyethylene glycol on preparation of rifampicin-loaded PLGA microspheres with membrane emulsification technique.

Monodisperse poly(lactide-co-glycolide) (PLGA) microspheres containing rifampicin (RFP), anti-tubercle drug, as hydrophobic model drug were prepared by solvent evaporation method with a membrane emulsification technique using Shirasu Porous Glass (SPG) membranes. Five kinds of rifampicin-loaded PLGA (RFP/PLGA) microspheres with different sizes were prepared by changing pore size of the membranes. Effect of polyethylene glycol (PEG) added to polyvinyl alcohol (PVA) solution (continuous phase) upon the monodispersity of microspheres was studied. PEG was used as a stabilizer for microspheres dispersing in PVA solution. The most suitable molecular weight of PEG as a stabilizer was 20,000. RFP/PLGA microspheres prepared with PEG20000 were apparently more uniform than those prepared without PEG. The yield of RFP/PLGA microspheres was 100%. The initial burst observed in the release of RFP from RFP/PLGA microspheres was suppressed by the addition of PEG.

Preparation of pH-sensitive liposomes retaining SOD mimic and their anticancer effect.

We prepared an anticancer drug based on a pH-sensitive liposome retaining Fe-porphyrin as an SOD mimic. The liposomes contained cationic/anionic lipid combinations and were composed of Fe-porphyrin, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine, dimethylditetradecylammonium bromide, sodium oleate, and Tween-80. The Fe-porphyrin was released from the liposome at low pH, and the cytotoxicity for cancer cells by the liposome depended on the acidic environments of the endosomes in the cells. Furthermore, although the liposome exhibited an excellent anticancer effect on a gastric cancer cell line, the SOD activity of Fe-porphyrin was shown to have a significant influence on the cytotoxicity toward cancer cells. These findings suggest that the pH-sensitive liposome retaining the Fe-porphyrin as an SOD mimic promises to be a novel anticancer drug for endosomal escape.