A Simple Colorimetric Assay of Bleomycin-Mediated DNA Cleavage Utilizing Double-Stranded DNA-Modified Gold Nanoparticles.

A colorimetric assay of DNA cleavage by bleomycin (BLM) derivatives was developed utilizing high colloidal stability on double-stranded (ds) DNA-modified gold nanoparticles (dsDNA-AuNPs) possessing a cleavage site. The assay was performed using dsDNA-AuNPs treated with inactive BLM or activated BLM (Fe(II)⋅BLM). A 10-min exposure in dsDNA-AuNPs with inactive BLM treatment resulted in a rapid color change from red to purple because of salt-induced non-crosslinking aggregation of dsDNA-AuNPs. In contrast, the addition of active Fe(II)⋅BLM retained the red color, probably because of the formation of protruding structures at the outermost phase of dsDNA-AuNPs caused by BLM-mediated DNA cleavage. Furthermore, the results of our model experiments indicate that oxidative base release and DNA-cleavage pathways could be visually distinguished with color change. The present methodology was also applicable to model screening assays using several drugs with different mechanisms related to antitumor activity. These results strongly suggest that this assay with a rapid color change could lead to simple and efficient screening of potent antitumor agents.

Fabrication of Hybrid Capsules via CaCO3 Crystallization on Degradable Coacervate Droplets.

Organic-inorganic CaCO3 capsules were prepared by crystallization of CaCO3 on Pickering emulsion prepared using coacervate droplets made from thermoresponsive and degradable poly(2-methylene-1,3-dioxepane- co-2-hydroxyethyl acrylate) (poly(MDO- co-HEA)) in sole aqueous medium. The diameters of CaCO3-based Pickering emulsion could be controlled by varying several parameters: diameter of CaCO3 powders, initial polymer concentration, and copolymer composition. The CaCO3 Pickering emulsion was able to load low-molecular-weight hydrophobic substances at temperatures above the lower critical solution temperature (LCST) due to formation of polymer-concentrated phases, i.e., coacervate droplets. The diameter of CaCO3 capsules prepared by crystallization also depended on the diameter of the CaCO3 Pickering emulsion. The CaCO3 shell was composed of calcite-type crystals, the most stable polymorph among known CaCO3 crystals. The facially prepared CaCO3 capsules are valuable for use in functional biomaterials, such as drug delivery carriers and cell culture scaffolds for noninvasive bone-regenerative medicine.

[A Case of a Retroperitoneal Liposarcoma with Long-Term Survival after Four Surgical Resections].

Retroperitoneal liposarcoma is a relatively rare tumor. The only established therapy is surgical resection and the tumor often recurs. This paper deals with a case of a retroperitoneal liposarcoma in which frequent surgical resections for recurrent tumors have provided relatively long-term survival for the patient. The patient was a 70-year-old woman who had undergone surgical resection for a right retroperitoneal tumor. The pathological diagnosis was dedifferentiated liposarcoma. Thereafter she experienced frequent recurrences which required 3 surgical resections. By means of positive margin for the last surgery, chemotherapy with eribulin was administered. There has been no recurrence 13 months after the last surgery.

Preparation of Degradable and Transformable Core-Corona-Type Particles that Control Cellular Uptake by Thermal Shape Change.

Particle-cell interactions, such as cellular uptake, vary depending on the particle size, shape, and surface properties. By dynamic control of the physical properties of particles, microparticle-cell interactions can intentionally be altered. Particle degradability is also necessary for their application in the body. In this study, we aimed to prepare degradable core-corona-type particles that are deformed near the body temperature and investigated particle shape-dependent cellular uptake. Degradable and transformable particles consisting of poly(2-methylene-1,3-dioxepane)-co-poly(ethylene glycol) with three-armed poly(ε-caprolactone) (PCL) were prepared. The particle melting point was controlled by the chain length of the three-armed PCL. Particle degradation occurred under both acidic and alkaline conditions via ester group hydrolysis in the polymer backbones. The rod-shaped microparticles prepared by uniaxial stretching at a temperature above the melting point of the core showed less uptake into macrophages than did the spherical microparticles. Therefore, the degradable transformable particles enable macrophage interaction control via stimuli-regulated particle shapes and are expected to be applied as drug delivery carriers that can be decomposed and excreted from the body.

Plasmid DNA Delivery into the Skin via Electroporation with a Depot-Type Electrode.

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (DEP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, DEP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. Methods: Green fluorescent protein (GFP)- and luciferase-encoding DNA were administrated, and GFP and luciferase were evaluated. Results: A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by DEP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by DEP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated DEP) at 20 V with 5 min intervals showed similar luciferase expression level to single DEP application with 100 V. Histological observations showed the skin became thicker after a single DEP at 100 V, whereas no apparent thickness changes were confirmed after repeated DEP at 20 V with 5 min intervals. Conclusions: This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective for DNA vaccine delivery into skin tissue.

Protein Removal from Hydrogels through Repetitive Surface Degradation.

Suppression of protein adsorption is a necessary property for materials used in the living body. In this study, thermoresponsive and degradable hydrogels were prepared by the radical polymerization of 2-methylene-1,3-dioxepane, 2-hydroxyethyl acrylate (HEA), and poly(ethylene glycol) monomethacrylate (PEGMA). The prepared hydrogels re-exposed PEG-grafted chains to the interface through surface degradation, which was confirmed by the maintenance of the chemical composition of the hydrogel surfaces after hydrolysis. Notably, adsorbed proteins can be removed from the hydrogel surfaces through hydrogel surface degradation at least thrice.

Facile preparation of multi-stimuli-responsive degradable hydrogels for protein loading and release.

Functional materials that can recognize the tumor microenvironment, characterized by acidic or reducing conditions, are needed for the designing of drug delivery carriers for cancer treatment. Hydrogels are potential protein drug carriers because they contain a large amount of water and stimuli-responsive functions can easily be introduced in them. However, it is difficult to introduce multi-stimuli-responsive functions and degradability at the same time. Here, we synthesized thermo- and pH-responsive hydrogels via a coupling reaction between poly(ethylene glycol) diglycidyl ether (PEGDE) and cystamine (CA). The prepared hydrogels showed lower critical solution temperature-type thermoresponsive behavior and pH-responsive swelling changes due to the protonation of secondary and/or tertiary amino groups arising from the crosslinking agent CA. Under reducing conditions, the hydrogels were degraded via the thiol exchange reaction in the presence of dithiothreitol or glutathione. The loading and release properties of FITC-labeled model proteins from the hydrogels were investigated. The loaded amount of the protein increased with decreasing molecular weight or hydrodynamic radius, which is based on the size of the network structure of the hydrogels. Notably, loaded proteins in the hydrogels were released only under reducing conditions, which mimic the tumor microenvironment. Thus, the prepared multi-responsive degradable hydrogels are expected to be used as functional drug delivery carriers for cancer treatment.

Preparation of thermoresponsive nanoparticles exhibiting biomolecule recognition ability via atom transfer radical dispersion polymerization.

Thermoresponsive core-corona type nanoparticles were prepared exhibiting biomolecule recognition ability on their surfaces. These thermoresponsive nanoparticles were prepared from a poly(N-isopropylacrylamide) (PNIPAAm) macro-initiator and styrene (St) in a polar solvent via atom transfer radical dispersion polymerization. The PNIPAAm macro-initiator contains an alkyl halide and/or phthalimide group on the terminated group of the polymer chain, and thus, the grafting of PNIPAAm on the PSt core resulted in terminating phthalimide end groups. These terminal groups were utilized to immobilize biomolecule recognition units, and the dispersion stabilities of the nanoparticles were found to change in aqueous solution at room temperature due to alteration of the terminating PNIPAAm groups by the presence of biomolecules at different concentrations.