Polyplexes assembled from poly(aspartamide) derivatives bearing 1,2-diaminoethane side chains, [PAsp(DET)] display amplified in vitro and in vivo transfection activity with minimal cytotoxicity. To elucidate the molecular mechanisms involved in this unique function of PAsp(DET) polyplexes, the physicochemical and biological properties of PAsp(DET) were thoroughly evaluated with a control bearing 1,3-diaminopropane side chains, PAsp(DPT). Between PAsp(DET) and PAsp(DPT) polyplexes, we observed negligible physicochemical differences in particle size and zeta-potential. However, the one methylene variation between 1,2-diaminoethane and 1,3-diaminopropane drastically altered the transfection profiles. In sharp contrast to the constantly high transfection efficacy of PAsp(DET) polyplexes, even in regions of excess polycation to plasmid DNA (pDNA) (high N/P ratio), PAsp(DPT) polyplexes showed a significant drop in the transfection efficacy at high N/P ratios due to the progressively increased cytotoxicity with N/P ratio. The high cytotoxicity of PAsp(DPT) was closely correlated to its strong destabilization effect on cellular membrane estimated by hemolysis, leakage assay of cytoplasmic enzyme (LDH assay), and confocal laser scanning microscopic observation. Interestingly, PAsp(DET) revealed minimal membrane destabilization at physiological pH, yet there was significant enhancement in the membrane destabilization at the acidic pH mimicking the late endosomal compartment (pH approximately 5). Apparently, the pH-selective membrane destabilization profile of PAsp(DET) corresponded to a protonation change in the flanking diamine unit, i.e., the monoprotonated gauche form at physiological pH and diprotonated anti form at acidic pH. These significant results suggest that the protonated charge state of 1,2-diaminoethane may play a substantial role in the endosomal disruption. Moreover, this novel approach for endosomal disruption neither perturbs the membranes of cytoplasmic vesicles nor organelles at physiological pH. Thus, PAsp(DET) polyplexes, residing in late endosomal or lysosomal states, smoothly exit into the cytoplasm for successful transfection without compromising cell viability.

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.

A block catiomer polyplex, showing a high stability in the extracellular medium and an efficient release of plasmid DNA (pDNA) in the intracellular compartment, was developed by controlling both the cationic charge and disulfide cross-linking densities of the backbone polycations. Poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) was thiolated using either of two thiolation reagents, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (Traut's reagent), to investigate the effects of both the charge and disulfide cross-linking densities on the properties of the polyplexes. The introduction of thiol groups by SPDP proceeded through the formation of amide linkages to concomitantly decrease the cationic charge density of PLL segment, whereas Traut's reagent promoted the thiolation with the introduction of cationic imino groups to keep the charge density constant. These thiolated PEG-PLLs were complexed with pDNA to form the disulfide cross-linked block catiomer polyplexes, which had the size of approximately 100 nm. Both thiolation methods were similarly effective in introducing disulfide cross-links to prevent the polyplex from the dissociation through a counter polyanion exchange in the extracellular oxidative condition. On the other hand, the efficient release of pDNA responding to the reductive condition mimicking the intracellular environment was only achieved for the polyplex thiolated with SPDP, a system compensating for the decrease in the charge density with the disulfide cross-linking. This distinctive sensitivity toward oxidative and reductive environments was nicely correlated with the remarkable difference in the transfection efficiency between these two types of thiolated polyplexes (SPDP and Traut's reagent types): the former revealed approximately 50 times higher transfection efficiency toward 293T cells than the latter. Obviously, the balance between the densities of the cationic charge and disulfide cross-linking in the thiolated polyplex played a crucial role in the delivery and controlled release of entrapped pDNA into the microenvironment of intracellular compartment to achieve the high transfection efficiency.

Fabrication of monodispersed, submicrometer-sized vesicles (nanosomes) that form through self-assembly possessing a thin and permeable membrane remains a significant challenge. Conventional fabrication of nanosomes through self-assembly of amphiphilic molecules often requires cumbersome processes using organic solvents combined with physical procedures (e.g., sonication, thermal treatment, and membrane filtration) to obtain unilamellar structures with a controlled size distribution. Herein, we report the first example of spontaneously formed submicrometer-sized unilamellar polyion complex vesicles (Nano-PICsomes) via self-assembly of a pair of oppositely charged PEG block aniomer and homocatiomer in an aqueous medium. Detailed dynamic light scattering and transmission electron microscopic analysis revealed that vesicle sizes can be controlled in the range of 100-400 nm with a narrow size distribution, simply by changing the total polymer concentration. Also, each Nano-PICsome was composed of a uniform single PIC membrane, the thickness of which is around 10-15 nm, regardless of its size. Fluorescence correlation spectroscopy measurement verified that Nano-PICsomes were able to encapsulate water-soluble fluorescent macromolecules in the inner water phase and release them slowly into the exterior. Moreover, cross-linking of the vesicle membrane allows tuning of permeability, enhancement in stability under physiological conditions, and preservation of size and structure even after freeze-drying and centrifugation treatment. Finally, Nano-PICsomes showed a long circulation time in the bloodstream of mice. Precise control of the particle size and structure of hollow capsules through simple aqueous self-assembly and easy modification of their properties by cross-linking is quite novel and fascinating in terms of ecological, low-cost, and low-energy fabrication processes as well as the potential utility in the biomedical arena.

Ferroelectric transition has been detected in a ferrimagnetic spinel oxide of CoCr2O4 upon the transition to the conical spin order below 25 K. The direction [110] of the spontaneous polarization is normal to both the magnetization easy axis [001] and to the propagation axis [110] of the transverse spiral component, in accord with the prediction based on the spin-current model. The reversal of the spontaneous magnetization by a small magnetic field (approximately 0.1 T) induces the reversal of the spontaneous polarization, indicating the clamping of the ferromagnetic and ferroelectric domain walls.