Exportin-inspired artificial cell nuclear-exporting nanosystems.

Inspired by the structural and chemical features of naturally occurring importin/exportin that allows them to pass through the nuclear pore complexes, we successfully developed an artificial nuclear-exporting nanosystem capable of eliminating compounds accumulated abnormally in the nucleus.

Angiogenesis Promotion by Combined Administration of DFO and Vein Endothelial Cells Using Injectable, Biodegradable, Nanocomposite Hydrogel Scaffolds.

Desferrioxamine (DFO) upregulates HIF-1α and stimulates expression of vascular endothelial growth factor (VEGF), thereby accelerating neovascularization. As DFO acts primarily upon surrounding vein endothelial cells to stimulate angiogenesis, the angiogenic efficacy of DFO could be reduced in severely injured tissues lacking a sufficient number of vein endothelial cells. We hypothesized that combined administration of DFO and vein endothelial cells is a promising tissue engineering approach for promoting neovascularization. In this study, we evaluated the applicability of this approach using injectable, biocompatible, biodegradable nanocomposite gels consisting of poly(dl-lactide-co-glycolide)-b-polyethylene glycol-b-poly(dl-lactide-co-glycolide) (PLGA-PEG-PLGA) copolymers and clay nanoparticle LAPONITE. The nanocomposites exhibited irreversible thermo-gelation in the presence of DFO, and the mechanical strength was strongly affected by the amount of DFO. The storage moduli of the gels increased with increasing amount of DFO. These results indicate that the interaction between DFO and LAPONITE works as physical cross-linking points and facilitates the formation of the gel network. The nanocomposite gels achieved sustained slow release of DFO due to interactions between DFO and LAPONITE. Human umbilical vein endothelial cells (HUVECs) cultured on DFO-loaded nanocomposite gels exhibited a higher degree of vascular tube formation than cells cultured on nanocomposite gels without DFO. Moreover, the number of branching points and the diameter of the blood vessels regenerated in the gels significantly increased with increasing DFO amount, indicating that DFO released from the gels facilitates vascular tube-forming capacity. As a proof of concept, we demonstrate that the combined administration of DFO and vein endothelial cells using nanocomposite gels promotes greater angiogenesis than DFO administration alone using the same gels by in vivo experiments, confirming the validity of our hypothesis. Considering the multiple advantages of nanocomposite gels with regard to potential vascularization capacity, certain biocompatibility, biodegradability, and injectable cell- and drug-delivery capacity, we concluded that the nanocomposite gels have potential utility as scaffolding biomaterials for vascularization in tissue engineering applications.

Injectable Biocatalytic Nanocomposite Hydrogel Factories for Focal Enzyme-Prodrug Cancer Therapy.

Systemic enzyme-prodrug therapy (EPT) using nanofactories, nanoparticles encapsulating prodrug-activating enzymes, is a promising concept for anticancer therapy. However, systemic delivery systems can be problematic. As nanofactories are typically carried by the blood circulation to tissues throughout the body, conversion of anticancer drugs in normal tissues can cause severe side effects. To overcome this problem, we developed a novel focal EPT approach utilizing nanocomposite hydrogels composed of a poly(dl-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(dl-lactide-co-glycolide) (PLGA-PEG-PLGA) copolymer, LAPONITE, and ß-galactosidase (ß-gal). The nanocomposite gels can be easily injected locally, and the inherent enzyme activity of ß-gal can be preserved long-term. Prodrug 5-FU-ß-gal readily permeated into the interior space of gels and was converted into the active anticancer drug 5-FU. Importantly, a single local injection of nanocomposite gels and prodrug 5-FU-ß-gal provided long-lasting antitumor activity in vivo without observable side effects, demonstrating the potential utility of injectable biocatalytic hydrogel factories for novel focal EPT systems.

Biological Tissue-Inspired Living Self-Healing Hydrogels Based on Cadherin-Mediated Specific Cell-Cell Adhesion.

Regarding synthetic self-healing materials, as healing reactions occur at the molecular level, bond formation occurs when healing chemicals are nanometer distances apart. However, motility of healing chemicals in materials is quite limited, permitting only passive diffusion, which reduces the chance of bond formation. By contrast, biological-tissues exhibit significant high-performance self-healing, and cadherin-mediated cell-cell adhesion is a key mechanism in the healing process. This is because cells are capable of a certain level of motility and actively migrate to damage sites, thereby achieving cell-cell adhesion with high efficacy. Here, we report biological-tissue-inspired, self-healing hydrogels in which azide-modified living cells are covalently cross-linked with alkyne-modified alginate polymers via bioorthogonal reactions. As a proof-of-concept, we demonstrate their unique self-healing capabilities originating from cadherin-mediated adhesion between cells incorporated into the gels as mobile healing mechanism. This study provides an example of self-healing material incorporating living components into a synthetic material to promote self-healing.

Bioinspired Cell Nuclear Nanotransporters Generated by Self-Assembly of Amphiphilic Polysaccharide-Amino Acid Derivatives Conjugates.

Development of nanomaterials that surely transport functional biomacromolecules and bioactive synthetic compounds into the cell nucleus must be promising for the generation of nuclear-targeting new technologies. However, the development of nuclear transporting nanomaterials thus still remains a significant challenge, because molecular transport between the cytoplasm and the nucleus of a eukaryotic cell is strictly regulated by the sole gateway through the nuclear envelope, the nuclear pore complexes (NPCs). Here, the rational design of novel artificial nuclear nanotransporters (NucPorters), inspired by importin, naturally occurring nuclear transporters is shown. The NucPorter is generated by simple molecular design: self-assembly of amphiphilic polymers, where a few numbers of hydrophobic amino-acid derivatives with phenyl groups are conjugated to negatively charged hydrophilic heparin. The NucPorter can mimic essential structural and chemical features of importin machinery to pass through the NPCs. Importantly, the NucPorter demonstrates remarkable rapid and high efficient nuclear transport in cultured cells, tissue/organ, and living mice. Moreover, the NucPorter successfully imports both enzymes and synthetic anticancer drugs into the nucleus while maintaining their bioactivity. Thus, the NucPorter provides a promising new route to generate innovative nuclear-targeting medicines, diagnostics, cell imaging and engineering techniques, and drug delivery systems.

A thin hydrogel barrier linked onto cell surface sialic acids through covalent bonds induces cancer cell death in vivo.

Hypersialylation is the aberrant expression of sialic acid in cell surface glycans and is pervasive in cancer cells. Recent studies have shown that hypersialylation provides a microenvironment conducive to cancer progression, mediated by the interaction between sialic acid and sialic acid-binding receptors. Therefore, a technique to block the interaction between the overexpressed sialic acid on cancer cell surfaces and its receptors is a promising approach to develop new cancer therapies. We focused on hydrogels as an artificial barrier to block this interaction and present here the development of a novel technique for selectively covalently binding a thin hydrogel barrier on sialic acid residues on cancer cell surfaces. This technique effectively inhibited cancer cell adhesion, motility and growth, caused cancer cell death in vitro, and completely suppressed tumor growth in vivo, thereby clearly demonstrating a potent antitumor effect.

Nanocomposite injectable gels capable of self-replenishing regenerative extracellular microenvironments for in vivo tissue engineering.

Injectable hydrogels are biomaterials that have the potential to provide scaffolds to cells for in situ tissue regeneration with a minimally invasive implantation procedure. The success of in vivo tissue engineering utilizing injectable gels depends on providing cells with appropriate scaffolds that present an instructive extracellular microenvironment, which strongly influences the survival, proliferation, organization, and function of cells encapsulated within gels. One of the most important abilities of injectable gels to achieve this function is to adsorb and retain a wide variety of requisite bioactive molecules including nutrients, extracellular matrices, and growth/differentiation factors within gels. Previously, we developed nanocomposite injectable gels fabricated by simple combination of common biodegradable copolymers, poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), and synthetic clay nanoparticles (LAPONITE®). We revealed that the nanocomposite injectable gels strongly adsorb ECM molecules including collagen and heparin within gels and retain them due to the ability of LAPONITE® in synchronization with the degradation of PLGA-PEG-PLGA and subsequent release of the degradation products. Human dermal fibroblast cells cultured on the nanocomposite gels showed enough high cell viability and proliferation for at least a week. Moreover, various kinds of human cells encapsulated within the nanocomposite gels exhibited significantly higher survival, proliferation, and three-dimensional organization in comparison with the PLGA-PEG-PLGA gel, LAPONITE® gel, and Matrigel. Furthermore, transplantation of mouse myoblast cells with the nanocomposite gels in model mice of skeletal muscle injury dramatically enhanced tissue regeneration and functional recovery, whereas cell transplantation with the PLGA-PEG-PLGA gel did not. Thus, the nanocomposite injectable gels possess unique abilities to self-replenish the regenerative extracellular microenvironment within the gels in the body, demonstrating the potential utility of the nanocomposite injectable gels for in vivo tissue engineering.