Generation of mucin gel particles with self-degradable and -releasable properties.

Herein, we focused on mucin, which is a large viscous glycoprotein in terms of materials science, and reported preparation of mucin gel particles and incorporation of enzymes to provide the particle with self-degradable and releasable properties. To expose the hydrophobic peptide cores, trimming of sugar moieties was carried out by ß-elimination reaction under alkaline conditions (tMucin). Nano-sized tMucin particles were prepared by the assembly of tMucin with the aid of a cationic surfactant. Then, cross-linking of tMucin particles was carried out via heat treatment (annealing) to induce thermal aggregation of the polypeptide chains. The hydrodynamic diameter of tMucin particles reversibly changed in response to calcium ions. Next, in an attempt to render the particle degradable, lysozyme was incorporated into the tMucin particles for the hydrolysis of oligosaccharide chains. These particles were gradually degraded upon enzymatic cleavage of the mucin molecules, facilitating the release of their incorporated substances. Also, the degradation of the mucin particles and the release of lysozyme were tunable by environmental conditions, such as temperature and calcium ions, in addition to the degree of cross-linking of the particles.

Preparation of free-standing hybrid colloidal membranes via assembly of liponanocapsules.

We intended to create a free-standing and organic-inorganic hybrid colloidal membrane. The colloidal membrane was assembled from polymeric capsules, which were prepared by coating of polymer layers over a liposome (liponanocapsules). In this study, two approaches were employed for mineralization over the membrane with calcium phosphate (CaP) to control its mechanical robustness and biodegradability (hybrid bioscaffold). One approach was based on CaP deposition over the liponanocapsules and their assembly into the hybrid membrane. CaP deposition was conducted via the counter-diffusion of phosphate ions and calcium ions across the capsule wall to obtain hybrid nanocapsules. Then, free-standing hybrid membrane was obtained by utilizing hybrid nanocapsules as building blocks for drying-mediated assembly. The obtained hybrid membrane was degraded into individual nanocapsules and degradation could be tuned by the crystal structure of CaP. In another approach, the free-standing membrane was assembled from DNA-coated liponanocapsules and then the counter-diffusion of ions was carried out across each assembled nanocapsule for CaP mineralization. The mechanical robustness of the membrane was significantly improved and its degradation was suppressed by CaP mineralization. This is probably because mineral cross-linkages were formed in the interspace between each nanocapsule. Fluorescent substances could be incorporated in each nanocapsule of the membrane and their release could be tuned by the control in crystal properties of CaP.

Preparation of protein nano-objects by assembly of polymer-grafted proteins.

We carried out surface-grafting from proteins and their assembling into objects with unique nanostructured materials (nano-objects). To immobilize polymer-initiating sites, amino groups of bovine serum albumin (BSA) were allowed to react with iniferter groups (BSA-i). Then, graft polymerization of N-isopropyl acrylamide (NIPAM) was performed by light-initiated living radical polymerization from immobilized iniferter moieties of BSA-i. The polymer-grafted BSA (BSA-g-PNIPAM) was assembled into nano-objects through the precipitation of PNIPAM graft chains and their sizes and morphologies were tuned by the chain length, the density and the chemical structure of graft polymers in addition to the environmental conditions such as temperature and pH. It was possible to retain the structures of nano-objects by thermal denaturation via heat treatment. Fluorescent substances were encapsulated in particulate nano-objects (nanoparticles) assembled from PNIPAM-g-BSA and their release could be regulated by tuning pH and temperature. Next, further graft polymerization from PNIPAM-grafted BSA was carried out by living radical polymerization of a cationic monomer, N,N-dimethylamino propyl acrylamide methyl chloride quaternary (DMAPAAQ). The grafted polymer was composed of a block copolymer of PNIPAM and a cationic polymer (PDMAPAAQ) and the gel-like nano-object was generated by increasing temperature. In contrast to PNIPAM-g-BSA, it became insoluble even when the temperature decreased, probably due to the electrostatic association between anionic regions of BSA and cationic regions of graft polymers. Coating of BSA-g-P(NIPAM-b-DMAPAAQ) enabled to form a uniform thin layer over a human hair. A free-standing membrane could be obtained by peeling from a water repellent substrate to create a porous membrane.

Fine-tuning in mineral cross-linking of biopolymer nanoparticle for incorporation and release of cargo.

We developed a mineral cross-linking strategy to prepare a biopolymer-based nanoparticle using calcium phosphate (CaP) as a cross-linker. Nanoparticles were first formed by mixing deoxyribonucleic acid (DNA) with cationic surfactants, and were cross-linked by CaP precipitation. After removal of the surfactants, we carried out the alternative dialysis of nanoparticles against CaCl2 aqueous solution and phosphate buffered solution for further mineral cross-linking. XRD and FT-IR studies revealed that the resultant nanoparticles were produced by mineral cross-linkages of hydroxyapatite (HAp) and the crystal amount and properties such as morphology and crystallinity could be well-controlled by the reaction conditions. Chemical dyes could be incorporated into nanoparticles via their affinities with crystal faces of HAp and DNA. Their release was tunable by crystal amount and properties of mineral cross-linkages. Also, the release could be triggered by mineral dissolution in response to pH. Such a mineral cross-linking will open up a potential way to provide a nanoparticle with versatile functions such as cleavable cross-linking, binding affinity for cargos, and pH-responsive release.

Preparation and assembly of poly(arginine)-coated liposomes to create a free-standing bioscaffold.

We created a free-standing membrane as a novel bioscaffold through the assembly of polymer-coated liposomes. Polyarginine (P(Arg)) possessing a cell-penetrating activity was used to form the polymer layer onto a negatively charged liposome (lipo-P(Arg)). The capsule wall of P(Arg) over liposomes made it possible to improve the mechanical property of capsules and to display deoxyribonucleic acid (DNA) over the vesicle surface through the electrostatic attraction (lipo-P(Arg)-DNA). The release rates of a fluorescent probe encapsulated in lipo-P(Arg) and lipo-P(Arg)-DNA were tunable by the number of polymeric layers of the capsule walls. To investigate the cell-membrane permeability of lipo-P(Arg)-DNA, polymer-coated liposomes were incubated with human umbilical vein endothelial cells (HUVECs) at 4 °C. It was found that lipo-P(Arg) underwent a significant cellular uptake, whereas bare liposomes and liposomes modified with chitosan were incapable of overcoming the plasma membrane barrier. To prepare a free-standing membrane composed of polymer-coated liposomes, a suspension of lipo-P(Arg)-DNA was cast over a mesh hole and dried up. SEM observation revealed that a free-standing membrane was obtained through drying-mediated assembly process without rupturing polymer-coated liposomes inside the membrane. On the other hand, it was not possible to obtain a complete membrane from a mixture of lipo-P(Arg) and DNA. In summary, lipo-P(Arg)-DNA capsules possess versatile functions as a drug carrier, and their assembly enables us to create a free-standing membrane applicable as a bioscaffold.

The preparation of sugar polymer-coated nanocapsules by the layer-by-layer deposition on the liposome.

We intended to combine the liposomal preparation and the layer-by-layer deposition to prepare a nanosized capsule. Chitosan (CHI) was deposited to form the cationic polymeric layer onto a negatively charged liposomal surface and further deposition was carried out using anionic polymers dextran sulfate (DXS) or deoxyribonucleic acid (DNA). zeta-Potentials of nanocapsules changed between positive and negative charges at each deposition. FE-TEM revealed that the liposome remained a spherical shape even after the layer-by-layer (LbL) deposition. The capsule wall showed a dramatic increase in stability against the surfactant Triton X-100 compared to a bare liposome, and the stability was controllable by the adsorption amount of the polymer. These suggest that the polymer multilayer was generated on the liposome surface by the layer-by-layer depositions of polysaccharides. The three kinds of chemical substances with different charges, 1-hydroxy pyrene-3,6,8-trisulfonic acid (HPTS), alendronate, and glucose, were encapsulated into nanocapsules and the release was suppressed by the polymeric capsule wall irrespective of charges. The release from DNA-deposited nanocapsules (liponano-CHI-DNA) was clearly increased by raising temperature from 25 to 60 degrees C. This indicates that the temperature-dependent release was achieved by applying DNA denaturation as a temperature-dependent "switch", which influenced the permeability of the capsule wall.