[Show abstract][Hide abstract] ABSTRACT: Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of >90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins.
Full-text · Article · Dec 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: A self-assembled nanogel is constructed from light-sensitive cholesteryl pullulan (Ls-CHP) by using photo-labile ortho-nitrobenzyl (o-NB) units. The nanogel-based film is obtained by evaporation of an Ls-CHP nanogel solution. Exposure of the resulting nanogel-based film to light with a mask resulted in a patterned film that can encapsulate FITC-insulin.
No preview · Article · Nov 2015 · Chemical Communications
[Show abstract][Hide abstract] ABSTRACT: We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.
[Show abstract][Hide abstract] ABSTRACT: (This PDF file is proof version. Final published version is available from NPG journal page.)
Nanogels are nanometer-sized hydrogel nanoparticles with three-dimensional networks of crosslinked polymer chains. We have previously reported a method for constructing protein nanogels using vitamin B6 (pyridoxal)-bearing pullulan (PLPP) as a bio-crosslinker. To validate the potential for using this bio-crosslinker to prepare protein nanogels, we developed an improved method using metal ions to enhance the affinity between the protein and the bio-crosslinker. We investigated the binding of PLPP as a bio-crosslinker to a protein to produce protein nanogels using anionic bovine serum albumin (BSA) as a model protein. Ultraviolet-visible spectroscopic titration of PLPP with BSA showed that the binding of the PLPP to the BSA via the formation of Schiff bases was significantly enhanced in the presence of zinc ions because of the coordination of the Schiff base to metal ions, which shifted the equilibrium of the reaction to the Schiff base formation side. Dynamic light scattering measurements, high-performance liquid chromatography and transmission electron microscopy confirmed that the zinc ions enhanced the ability of PLPP to form nanogels by crosslinking with the anionic BSA despite the strong electrostatic repulsion between the two molecules.
[Show abstract][Hide abstract] ABSTRACT: A new organicinorganic hybrid was generated by self-assembly of nanosized polymer hydrogels (nanogels) and Fe3O4 nanoparticles (NPs) for potential biomedical applications. By using the tetrahydrofuran (THF) injection method, hydrophobized Fe3O4 NPs were complexed through hydrophobic interactions with amphiphilic polysaccharide nanogels. The obtained hybrid showed high colloidal stability and dispersibility in aqueous media. The number of Fe3O4 NP aggregates was controlled by changing the concentration of the Fe3O4 NPs in THF. The hybrid displayed greater enhancement than existing contrast media in T-2 magnetic resonance imaging because of the optimal agglomeration of Fe3O4 NPs following their complexation with nanogels. Furthermore, the hybrid generated heat in response to alternating magnetic-field irradiation. The increase in temperature was controlled by adjusting the concentration of the hybrid and the amplitude of the magnetic field, which indicates that the hybrid is also suitable for magnetic hyperthermia therapy.
[Show abstract][Hide abstract] ABSTRACT: We developed a new self-assembled amphiphilic nanogel-crosslinked porous (NanoCliP) gel that can trap proteins, liposomes, and cells. The NanoCliP gel was prepared by Michael addition of a self-assembled nanogel of acryloyl group-modified cholesterol-bearing pullulan to pentaerythritol tetra (mercaptoethyl) polyoxyethylene, followed by freezing-induced phase separation. Dynamic rheological analysis revealed that the storage modulus (G′) of the NanoCliP gel was approximately 10 times greater than that of a nonporous nanogel-crosslinked gel. Two-photon excitation deep imaging revealed that the NanoCliP gel comprises interconnected pores of several hundred micrometers in diameter. The NanoCliP gel trapped proteins and liposomes via hydrophobic interactions because its amphiphilic nanogels exhibit chaperone-like activity. Mouse embryonic fibroblasts penetrated the interconnected pores and adhered to the porous surface of fibronectin-complexed NanoCliP gel. In vivo, the NanoCliP gel enhanced cell infiltration, tissue ingrowth, and neovascularization without requiring exogenous growth factors, suggesting that the NanoCliP gel is a promising scaffold for tissue engineering.
[Show abstract][Hide abstract] ABSTRACT: Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder.
[Show abstract][Hide abstract] ABSTRACT: The effects of the supramolecular polyrotaxane (PRX) structure on cellular internalization are investigated by flow cytometry and confocal laser scanning microscopy. AF-545-labeled aminated PRXs (APRXs) containing different numbers of threaded α-cyclodextrins (CDs) and amino groups are synthesized; their cellular uptakes are analyzed using HeLa cells in serum. The APRX threaded CD number is discovered to be a more critical factor for enhancing cellular internalization than the APRX amine content. Additionally, APRXs are demonstrated to be more easily internalized than conventional linear cationic macromolecules. Because increased numbers of threaded CDs are related to increased PRX rigidity, the PRX rigid frame resulting from CD molecules threaded on a poly(ethylene glycol) (PEG) chain is suitable for intracellular tools in therapy and diagnosis.
No preview · Article · Mar 2014 · Macromolecular Bioscience
[Show abstract][Hide abstract] ABSTRACT: Successful gene therapy requires the development of an efficient intracellular nucleic acid delivery system. Cycloamylose (CA) is a large-ring cyclodextrin that has the potential to be used as a novel nanocarrier for intracellular nucleic acid delivery. A series of functionalized CAs were spontaneously complexed with nucleic acids, including double-stranded small interfering RNA or plasmid DNA, and they exhibited superior gene delivery performance over other delivery systems. Functional CAs and self-assembled nanogels were also used to develop a CA-based nucleic acid nanocarrier. The new gene delivery system was based on the ability of physically cross-linked cationic CA nanogels to encapsulate proteins. Using hydrophobic and electrostatic interactions, the cationic CA nanogel successfully and simultaneously delivered enzymes and plasmid DNA that were accommodated in individual carriers, with high gene transfer efficiency. The CA-based nucleic acid delivery system provides a useful foundation for developing multifunctional nucleic acid delivery systems.
[Show abstract][Hide abstract] ABSTRACT: Enzymatically synthesized glycogen (ESG) bearing hydrophobic groups were prepared to construct a new artificial molecular chaperone system. The radius of the amphiphilic ESG nanogel was approximately 15 nm. The nanogels prevented irreversible aggregation of carbonic anhydrase. Cyclodextrin induced the release of carbonic anhydrase in its active form via a one-step mechanism without dissociation of the nanogels.
[Show abstract][Hide abstract] ABSTRACT: The intracellular delivery of enzymes is an essential methodology to extend their therapeutic application. Herein, we have developed dissociable supermolecule-enzyme polyelectrolyte complexes based on reduction-cleavable cationic polyrotaxanes (PRXs) for the reactivation of delivered enzymes. These PRXs are characterized by their supramolecular frameworks of a polymeric chain threading into cyclic molecules, which can form polyelectrolyte complexes with anionic enzymes while retaining their three dimensional structure, although their enzymatic activity is reduced. Upon the addition of a reductant, the PRXs dissociate into their constituent molecules and release the enzymes, resulting in a complete recovery of enzymatic activity. Under the intracellular environment, the PRX-based enzyme complexes showed the highest intracellular enzymatic activity and efficient activation of anticancer prodrugs to induce cytotoxic effects in comparison with the non-dissociable complexes and the commercial cell-penetrating peptide-based reagents. Thus, the intracellularly dissociable supermolecules are an attractive system for delivering therapeutic enzymes into living cells.
[Show abstract][Hide abstract] ABSTRACT: Polysaccharide nanogels are one of the most attractive carriers for drug delivery systems. Nanogels encapsulate proteins in their hydrated polymer networks, and minimize the denaturation of proteins. In this study, we demonstrated the cross-linking of acryloyl group-modified polysaccharide nanogels via photopolymerization, which allowed the formation of novel hydrogel particles and macrogels. The mechanical properties of the resultant hydrogels depended on the concentrations of the nanogels and the cross-linkers. The most significant property of the nanogel-cross-linked hydrogel was the ability to encapsulate insulin via hydrophobic interactions. After incubation of the hydrogel containing insulin in water, the hydrogel was degraded by hydrolysis, and insulin was gradually released from the hydrogels over a period of 1 week. According to these results, this nanogel-cross-linked hydrogel prepared via photopolymerization has potential for innovative biomaterials.
No preview · Article · Jul 2013 · Reactive and Functional Polymers
[Show abstract][Hide abstract] ABSTRACT: Cationic lipoplexes, which consist of cationic lipids and DNA, are widely used in gene transfection. Recently, some reports suggest that the transfection efficiency of lipoplexes is affected by its formulation and physicochemical properties. To improve the lipoplex transfection efficiency, we examined gene transfection using lipoplexes treated with high hydrostatic pressure (HHP) that affects the phase behavior of the phospholipid bilayer. Lipoplexes were prepared with different cationic lipid to DNA ratios and then subjected to hydrostatic pressure at 5,000 and 10,000 atm. Non-pressurized lipoplex and HHP-treated lipoplex (after removing pressure) were evaluated. Dynamic light scattering measurement showed that the size of HHP-treated lipoplex increased compared with non-pressurized lipoplex, suggesting that formulation change and assembly of lipoplexes occurred under high hydrostatic pressure. Lipoplexes with and without HHP treatment were transferred into HeLa and COS-7 cells. The transfection efficiency of HHP-treated lipoplex increased compared with non-pressurized lipoplex, irrespective of the mixture ratio. Also, the transfection efficiency of HHP-treated lipoplex remained higher for several days. These results suggest that high hydrostatic pressure induces structural change in lipoplex, thereby affecting the transfection efficiency.
[Show abstract][Hide abstract] ABSTRACT: Cationic liposome (CL) is a promising vector for nucleic acid therapy. In the present study, we investigated the effect of high hydrostatic pressure (HHP) treatment to lipoplex on the lipoplex-based antisense oligodeoxynucleotides (AS-ODNs) delivery in order to improve the transfection efficacy of lipoplex. Cationic liposome consisting of DOTMA and DOPE was used. AS-ODNs were designed to inhibit the expression of firefly luciferase. The complexes of CL and AS-ODN were prepared at various C/A ratios and then pressurized hydrostatically at various atmospheres (∼10,000 atm) for 10 min (HHP treatment). After removal of pressure, the pressurized lipoplexes were used. The lipoplex with and without the HHP treatment was transferred into HeLa cells expressing firefly luciferase transiently. The luciferase activity using the HHP-treated lipoplex was decreased compared to that of the non-pressurized lipoplex. Also, for HEK293 cells expressing luciferase stably, the lipoplex with the HHP treatment could effectively suppress the luciferase expression. In order to elucidate relationship between the structure and the transfection efficiency of the HHP-treated lipoplex, the properties of the HHPtreated lipoplex were examined by various physicochemical analyses. The different physicochemical properties between the lipoplexes with and without HHP treatment were showed, suggesting that the nature of lipoplex was changed by the HHP treatment. We believe that this change of lipoplex properties by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and oligonucleotide delivery.
No preview · Article · Jan 2013 · MRS Online Proceeding Library
[Show abstract][Hide abstract] ABSTRACT: Nanosized hydroxyapatite (HAp) materials have received much attention in the context of their advanced biomedical applications, including tissue engineering and drug delivery systems. Hybridization of nanosized HAp with organic molecules is a promising approach to facilitate the preparation of HAp nanomaterials. Here, templated mineralization using self-assembled nanogels modified with tricarboxylate groups was performed to yield the hybrid HAp nanomaterial. In the pH gradient method, the nanogel acted as an excellent template for the formation of well-dispersed HAp particles. Transmission electron microscopy, selected area electron diffraction patterns and energy-dispersive X-ray spectroscopy of these particles revealed that amorphous nanoparticles of amorphous calcium phosphate formed first, followed by transformation to crystalline hydroxyapatite.
[Show abstract][Hide abstract] ABSTRACT: A nanosensory membrane device was constructed for detecting liposome fusion through changes in an enzymatic activity. Inspired by a biological signal transduction system, the device design involved functionalized liposomal membranes prepared by self-assembly of the following molecular components: a synthetic peptide lipid and a phospholipid as matrix membrane components, a Schiff's base of pyridoxal 5'-phosphate with phosphatidylethanolamine as a thermo-responsive artificial receptor, NADH-dependent L-lactate dehydrogenase as a signal amplifier, and Cu(2+) ion as a signal mediator between the receptor and enzyme. The enzymatic activity of the membrane device was adjustable by changing the matrix lipid composition, reflecting the thermotropic phase transition behavior of the lipid membranes, which in turn controlled receptor binding affinity toward the enzyme-inhibiting mediator species. When an effective fusogen anionic polymer was added to these cationic liposomes, membrane fusion occurred, and the functionalized liposomal membranes responded with changes in enzymatic activity, thus serving as an effective nanosensory device for liposome fusion detection.
[Show abstract][Hide abstract] ABSTRACT: New hybrid poly(ethylene glycol) (PEG) hydrogels crosslinked with both nanogels and nanogel-coated liposome complexes are obtained by Michael addition of the acryloyl group of a cholesterol-bearing pullulan (CHP) nanogel to the thiol group of pentaerythritol tetra(mercaptoethyl) polyoxyethylene. The nanogel-coated liposome complex is stably retained after gelation and the complexes are well dispersed in the hybrid gel. Microrheological measurements show that the strength and gelation time of the hybrid hydrogel can be controlled by changing the liposome:nanogel ratio. The hydrogel is gradually degraded by hydrolysis under physiological conditions. In this process, the nanogel is released first, followed by the nanogel-coated liposomes. Hybrid hydrogels that can incorporate various molecules into the nanogel and liposomes, and release them in a two-step controllable manner, represent a new functional scaffold capable of delivering multiple drugs, proteins or DNA.
[Show abstract][Hide abstract] ABSTRACT: IntroductionPreparation of Associating Polymer-Based NanogelsFunctions of Self-Assembled NanogelsApplication of Polysaccharide Nanogels to DDSIntegration of NanogelsConclusion and PerspectivesReferences