Effects of lipoprotein nanoparticles' composition and size on their internalization in plant and mammalian cells.

The internalization of engineered high-density lipoprotein nanoparticles (engineered lipoproteins [eLPs]) with different lipid and protein compositions, zeta potentials, and/or sizes were analyzed in representative plant and mammalian cells. The impact of the addition of a cell-penetrating peptide to eLPs on the internalization was very small in Bright Yellow (BY)-2 protoplasts compared with HeLa cells. When eLPs were prepared with one of the abundant lipids in BY-2 cells, digalactosyldiacylglycerol (DGDG) (eLP4), its internalization was dramatically increased only in HeLa cells. Such an increase in HeLa cells was also obtained for liposomes containing DGDG in a DGDG content-dependent manner. Increasing the size and zeta potential of eLPs improved their internalization in both HeLa cells and in BY-2 protoplasts but to quite varying degrees. Although eLPs tended to stay at the plasma membrane (PM) in BY-2 protoplasts with much less internalization, the PM-bound eLPs somehow promoted the internalization of coexisting nanobeads in cell culture media. These results provide fundamental insight into the future design of lipid nanoparticles for drug delivery in mammalian and plant cells.

Synthesis of Butterfly-Like Shaped Gold Nanomaterial: For the Regulation of Liquid-Liquid Phase-Separated Biomacromolecule Droplets.

Nanotechnology is a critical tool to manipulate the sophisticated behavior of biological structures and has provided new research fields. Liquid-liquid phase-separated (LLPS) droplets gather attention as basic reaction fields in a living cell. Droplets play critical roles in regulating protein behavior, including enzyme compartmentalization, stress response, and disease pathogenesis. The dynamic manipulation of LLPS droplet formation/deformation has become a crucial target in nanobiotechnology. However, the development of nanodevices specifically designed for this purpose remains a challenge. Therefore, this study presents butterfly-shaped gold nanobutterflies (GNBs) as novel nanodevices for manipulating LLPS droplet dynamics. The growth process of the GNBs is analyzed via time-lapse electroscopic imaging, time-lapse spectroscopy, and additives assays. Interestingly, GNBs demonstrate the ability to induce LLPS droplet formation in systems such as adenosine triphosphate/poly-l-lysine and human immunoglobulin G, whereas spherical and rod-shaped gold nanoparticles exhibit no such capability. This indicates that the GNB concave surface interacts with the droplet precursors facilitating the LLPS droplet formation. Near-infrared-laser irradiation applied to GNBs enables on-demand deformation of the droplets through localized heat effects. GNB regulates the enzymatic reaction of lysozymes. The innovative design of GNBs presents a promising strategy for manipulating LLPS dynamics and offers exciting prospects for future research.

Phase-Diagram Observation of Liquid-Liquid Phase Separation in the Poly(l-lysine)/ATP System and a Proposal for Diagram-Based Application Strategy.

Liquid-liquid phase separation (LLPS) is essential to understanding the biomacromolecule compartmentalization in living cells and to developing soft-matter structures for chemical reactions and drug delivery systems. However, the importance of detailed experimental phase diagrams of modern LLPS systems tends to be overlooked in recent times. Even for the poly(l-lysine) (PLL)/ATP system, which is one of the most widely used LLPS models, any detailed phase diagram of LLPS has not been reported. Herein, we report the first phase diagram of the PLL/ATP system and demonstrate the feasibility of phase-diagram-based research design for understanding the physical properties of LLPS systems and realizing biophysical and medical applications. We established an experimentally handy model for the droplet formation-disappearance process by generating a concentration gradient in a chamber for extracting a suitable condition on the phase diagram, including the two-phase droplet region. As a proof of concept of pharmaceutical application, we added a human immunoglobulin G (IgG) solution to the PLL/ATP system. Using the knowledge from the phase diagram, we realized the formation of IgG/PLL droplets in a pharmaceutically required IgG concentration of ca. 10 mg/mL. Thus, this study provides guidance for using the phase diagram to analyze and utilize LLPS.

Control of Lipid Bilayer Phases of Cell-Sized Liposomes by Surface-Engineered Plasmonic Nanoparticles.

Liquid-ordered (Lo)-phase domains, a cholesterol-rich area on lipid bilayers, have attracted significant attention recently because of their relevance to lipid rafts, the formation/collapse of which is associated with various kinds of information exchange through the plasma membrane. Here, we demonstrate that the formation/collapse of Lo-phase domains in cell-sized liposomes, that is, giant unilamellar vesicles (GUVs), can be controlled with bioactive plasmonic nanoparticles and light. The nanoparticles were prepared by surface modification of gold nanorods (AuNRs) using a cationized mutant of high-density lipoprotein (HDL), which is a natural cholesterol transporter. Upon the addition of surface-engineered AuNRs to GUVs with the mixed domains of Lo and liquid-disorder (Ld) phases, the Lo domains collapsed and solid-ordered (So)-phase domains were formed. The reverse phase transition was achieved photothermally, with the AuNRs loaded with cholesterol. During these transitions, the AuNRs appeared to be selectively localized on the less fluidic domain (Lo or So) in the phase-mixed GUVs. These results indicate that the phase transitions occur through the membrane binding of the AuNRs followed by spontaneous/photothermal transfer of cholesterol between the AuNRs and GUVs. Our strategy to develop bioactive AuNRs potentially enables spatiotemporal control of the formation/collapse of lipid rafts in living cells.

Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression.

Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs' properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations.

Interfacial and intrinsic molecular effects on the phase separation/transition of heteroprotein condensates.

Liquid-liquid phase separation (LLPS) and droplet formation by LLPS are key concepts used to explain compartmentalization in living cells. Protein contact to a membrane surface is considered an important process for protein organization in a liquid phase or during transition to a solid or liquid dispersion state. The direct experimental comprehensive investigation is; however, not performed on the surface-droplet interaction and phase transition. In the present study, we constructed simple and reproducible experiments to analyze the structural transition of aggregates and droplets in an ovalbumin (OVA) and lysozyme (LYZ) complex on glass slides with various coatings. The difference in droplet-surface interaction may only be important in the boundary region between aggregates and droplets of a protein mixture, as shown in the phase diagram. Co-aggregates of OVA-LYZ changed to droplet-like circular forms during incubation. In contrast, free l-lysine resulted in the uniform droplet-to-solid phase separation at lower concentrations and dissolved any structures at higher concentrations. These results represent the first phase-diagram-based analysis of the phase transition of droplets in a protein mixture and a comparison of surface-surface and small molecular-droplet structure interactions.

Reentrant condensation of a multicomponent cola/milk system induced by polyphosphate.

Reentrant condensation (RC) is a protein behavior in which the protein solution shifts between the one- and two-phase state more than twice by increasing a single parameter. Although RC would be a candidate mechanism for the physicochemical design of food additives, no realistic model has been established under diverse contaminants like food materials. Here, we found that a mixture of cola and milk yielded RC. At pH 3.2-3.6, cola induced milk condensation at 30-40%, while lower or higher concentrations of cola did not. Furthermore, we reduced this cola/milk system to two pure components, casein in milk and polyphosphate (polyP) in cola, and investigated the characteristics of casein concentration and zeta potential. This was the first experimental demonstration of RC occurrence in a multicomponent system. The well-characterized cola/milk system would explore both the universal nature of proteins and the industrial application of RC.

Membrane fusogenic high-density lipoprotein nanoparticles.

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.