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.

Enhanced Immunostimulating Activity of Lactobacilli-Mimicking Materials by Controlling Size.

The design and synthesis of materials capable of activating the immune system in a safe manner is of great interest in immunology and related fields. Lactobacilli activate the innate immune system of a host when acting as probiotics. Here, we constructed lactobacilli-mimicking materials in which polysaccharide-peptidoglycan complexes (PS-PGs) derived from lactobacilli were covalently conjugated to the surfaces of polymeric microparticles with a wide variety of sizes, ranging from 200 nm to 3 µm. The artificial lactobacilli successfully stimulated macrophages without cytotoxicity. Importantly, we found that the size of artificial lactobacilli strongly influenced their immunostimulating activities, and that artificial lactobacilli of 1 µm exhibited 10-fold higher activity than natural lactobacilli. One major advantage of the artificial lactobacilli is facile control of size, which cannot be changed in natural lactobacilli. These findings provide new insights into the design of materials for immunology as well as the molecular biology of lactobacillus.

Synthesis and immunestimulating activity of lactobacilli-originated polysaccharide-polymeric microparticle conjugates.

The design and synthesis of biomaterials capable of activating the immune system are of interest in immunology-related fields because of their ability to tune up the immune defenses of the host. Lactobacilli are a major constituent of normal human indigenous flora, and some specific strains are known to activate the immune system of the host as probiotics. In this study, we first fabricated novel biohybrid materials in which lactobacilli (L. casei strain Shirota, LcS)-originated polysaccharide-peptidoglycan complexes (PS-PGs) are conjugated with polymeric microparticles (MPs). PS-PGs conjugated onto polymeric MPs surfaces bound its specific antibody, suggesting that PS-PGs kept their original molecular recognition ability. The PS-PGs-based hybrid MPs with an appropriate density of conjugated PS-PGs effectively induced high levels of IL-12 production from macrophages without cytotoxicity. These results suggest that LcS-originated PS-PGs could be available bio-originated materials for developing novel biomaterials capable of activating the immune system in a safe manner.

Self-assembling polymer micelle/clay nanodisk/doxorubicin hybrid injectable gels for safe and efficient focal treatment of cancer.

The purpose of this study was to fabricate a safe and effective doxorubicin (DOX)-delivery system for focal cancer chemotherapy. A novel biodegradable injectable gel was developed through self-assembly of poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA) copolymer micelles, clay nanodisks (CNDs), and DOX. We discovered that DOX loaded in the hybrid gels acts as an anticancer drug and as a building block to organize new gel networks. Accordingly, long-term sustained release of DOX from hybrid injectable gels without initial burst release was achieved. Moreover, it was revealed that the DOX incorporated into gel networks controls its own release profile. This hybrid injectable gel is a self-controlled drug release system, which is a novel concept in controlled drug release. Importantly, a single injection of PLGA-PEG-PLGA/CND/DOX hybrid gel provides long-term sustained antitumor activity in vivo against human xenograft tumors in mice, suggesting the potential of hybrid gels as a valuable local DOX-delivery platform for cancer focal therapy.

Anticancer drug-based multifunctional nanogels through self-assembly of dextran-curcumin conjugates toward cancer theranostics.

Curcumin (CCM) has been received much attention in cancer theranostics because CCM exhibits both anticancer activity and strong fluorescence available for bio-imaging. However, CCM has never been utilized in clinical mainly due to its extremely low water solubility and its low cellular uptake into cancer cells. We fabricated novel CCM-based biodegradable nanoparticles through self-assembly of amphiphilic dextran-CCM conjugates. Significantly high CCM loading contents in the nanoparticles and the high water solubility were achieved. Importantly, the dextran-CCMs nanoparticles were effectively delivered into HeLa cells and exhibited strong fluorescence available for live-cell imaging, although the nanoparticles were not delivered into normal cells. Thus, the dextran-CCMs nanoparticles could be a promising for creation of novel CCM-based cancer theranostics with high efficacy.

Thixotropic ß-Chitin Nanofiber Hydrogel with a Living Body-Responsive Self-Hardening Property and Its Application as a Sprayable Antiadhesion Barrier.

Considering recent advances in surgical techniques, sprayable antiadhesion barriers that are compatible with minimally invasive procedures are needed. However, the relatively low mechanical stiffness of the current thixotropic reversible sol-to-gel transition hydrogels has hindered their medical application. Herein, we show a thixotropic sprayable ß-chitin nanofiber hydrogel that spontaneously lost the thixotropic property in response to the environments within the living body. Furthermore, interactions between hydrogels and the biological environment result in a significant increase in mechanical stiffness. Due to these advantageous properties, ß-chitin nanofiber hydrogels administered by spray prevent postoperative abdominal adhesions and are thus promising sprayable antiadhesion barriers.

Two-Dimensional Metal-Organic Framework-Based Cellular Scaffolds with High Protein Adsorption, Retention, and Replenishment Capabilities.

Metal-organic frameworks (MOFs) are porous materials with adsorption, storage, and separation capabilities due to their high specific surface areas and large pore volumes. MOFs are thus used in biomedical applications, and MOF nanoparticles have been widely studied as nanocarriers for drug delivery systems. Several research groups recently reported that specific MOF nanoparticles can adsorb and retain proteins, suggesting to us that MOF nanoparticles may have advantages as novel cell culture scaffolds. However, MOF nanoparticles cannot be used as two-dimensional scaffolds for cells. We therefore established a bottom-up technique to construct two-dimensional MOFs [MIL-53 (Al)] on polymer films. The developed two-dimensional MIL-53 (Al) film [fMIL-53 (Al)] exhibited high serum protein adsorption, retention, and replenishment capabilities as compared to conventional cell culture scaffolds. ß-Galactosidase, used as a model protein, adsorbed on fMIL-53 (Al) exhibited original enzymatic activity, indicating that proteins are not denatured during the adsorption process. The viability of mouse myoblast cells (C2C12) cultured on fMIL-53 (Al) was 100%, indicating the cell compatibility of fMIL-53 (Al). Importantly, C2C12 cells cultured on serum protein-preadsorbed fMIL-53 (Al) exhibited excellent long-term adhesion, morphology, and proliferation even in a medium lacking serum proteins, demonstrating an important advantage of fMIL-53 (Al) as a cell culture scaffold, given that conventional cell culture scaffolds typically require a serum-containing medium to support stable cell adhesion and proliferation. To our knowledge, this is the first report regarding the application of MOFs as cell culture scaffolds and will serve as a starting point for studying two- and three-dimensional MOF-based cellular scaffolds for cell culture systems and for in vitro and in vivo tissue engineering.

Covalent Stem Cell-Combining Injectable Materials with Enhanced Stemness and Controlled Differentiation In Vivo.

Biohybrid materials, which are defined as engineered functional materials combining living components with nonliving synthetic materials, are considered promising bioactive materials for applications in in vivo tissue engineering. However, the rational design of biohybrid materials applicable to in vivo tissue engineering faces major challenges associated with techniques for combining living cells with nonliving synthetic materials and cell sources. Here, we report injectable covalent stem cell-combing biohybrid materials prepared via a bio-orthogonal click cross-linking reaction of azide-modified adipose-derived stem cells (N3[+]ADSCs), one of the most promising cell sources utilized clinically, with alkyne-modified biocompatible alginate polymers. The mechanical properties of the covalent stem cell-combining biohybrid materials can be adapted to the mechanical properties of the surrounding environment in which they are transplanted by alternating the number of N3[+]ADSCs, the concentration of alkyne-modified alginate, and the number of alkyne groups. Importantly, ADSCs in the covalent biohybrid materials expressed a high level of CD-105, a marker for undifferentiated mesenchymal stem cells, in the body in the absence of differentiation signals, whereas very little CD-105 was expressed in the control physical cell-loading materials, demonstrating that this covalent stem cell-combining approach results in enhanced retention of the material's "stemness" and controlled differentiation in the body. We assessed the potential utility of the covalent stem cell-combining biohybrid materials for in vivo tissue engineering using a murine severe skeletal muscle defect-healing model. Importantly, all of the tissues regenerated by the covalent biohybrid material treatment expressed MYH3, a myogenic marker protein, whereas no expression of MYH3 was detected in the tissues reconstructed by treatment with control physical stem cell-loading materials and Matrigel, indicating that this covalent stem cell-combining approach results in controlled differentiation in the body. Our data demonstrate the potential utility of covalent stem cell-combining biohybrid materials with host tissue-integrative and controlled differentiation capabilities available for in vivo tissue engineering.

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.

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.

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.

Application of the Southwell Plot method to determine equilibrium time in phosphate adsorption.

Among the different factors that influence the liquid-solid adsorption technique, equilibrium time is one of the most relevant and requires a large number of experiments over a long period of time for its determination. This work evaluates the Southwell Plot as a further tool that can contribute to determining the equilibrium time in adsorption processes. It can also optimize the operating conditions in a batch system for the removal of phosphate in adsorbents produced from domestic sewage sludge and clam shell residue. Sewage sludge and clam shell residues were ground, sieved and sintered at 700 °C for 1 h. The material was characterized by thermal analyses (TG/DTG), chemical analysis (EDX), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and adsorption studies. The kinetic studies were investigated by varying the initial concentration of the phosphate solution and mass of the adsorbent. The equilibrium time was determined by applying the Southwell Plot method to the kinetic data and the results showed some fluctuations as a function of the adsorbent mass. At 0.30 g of the adsorbent in 30 mL of the phosphate solution, regardless of the initial phosphate concentration, the equilibrium time determined by the Southwell Plot was 4 h. The maximum phosphate adsorption capacity in this condition, determined by the Langmuir equation, was 49.45 mg g-1.

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.

Biodegradable shape-memory polymers exhibiting sharp thermal transitions and controlled drug release.

Biodegradable shape-memory polymer networks prepared by cross-linking star shape branched oligo(ε-caprolactone) (bOCL) with hexamethylene diisocyanate are introduced. The thermal and mechanical properties of these networks were investigated using differential scanning calorimetry and tensile testing, respectively, and the morphology of the phase structure was characterized by polarized optical microscopy. The shape-memory properties of the networks were quantified using thermomechanical tensile experiments and showed strain fixity rates R(f) higher than 97% and strain recovery rates R(r) as high as 100%. Of note, networks of OCL segments with a lower degree of polymerization (DP; 10) exhibited significantly improved temperature-sensitive shape recovery: 90% of the permanent shape was recovered upon heating to within a 2 °C range (37-39 °C). The networks exhibited complete shape recovery to the permanent shape within 10 s at 42 °C. Theophylline-loaded (10 and 20 wt %) shape-memory materials, prepared by cross-linking bOCL with hexamethylene diisocyanate in the presence of theophylline, are also described as a model for a controlled drug release device. The 10 wt % loaded material was sufficiently soft and flexible for complex shape transformation and also showed high R(f) (98%) and R(r) (99%). Sustained release of loaded theophylline was achieved over 1 month without initial burst-release in a phosphate buffer solution (PBS; pH 7.4) at 37 °C.

Polyrotaxane composed of poly-L-lactide and alpha-cyclodextrin exhibiting protease-triggered hydrolysis.

Biodegradable polyrotaxanes (PRXs) were synthesized from bis-amino-terminated poly(L-lactide) (PLLA) and alpha-cyclodextrin (alpha-CD), combined with capping by bulky end groups at the amino-termini of PLLA through enzymatically cleavable peptide linkages. The crystalline structure of the PRXs in the solid state was investigated by wide-angle X-ray diffraction (WAXRD), and the results suggested that PRX forms a column-type crystalline structure. Hydrolysis of ester bonds of the PLLA in PRX was prevented due to the supramolecular structure. However, in the presence of a protease (papain), the hydrolysis of PLLA in PRX was induced. The removal of the bulky end groups by the protease acted as a trigger for the release of alpha-CD and allowed hydrolysis of the PLLA ester bonds. Such unique hydrolysis behavior, that is, proteinase-triggered degradation of a polyester, was achieved by the combination of the supramolecular architecture, biodegradable PLLA, and enzymatically cleavable end groups.

Nuclease resistant methylphosphonate-DNA/LNA chimeric oligonucleotides.

Synthesis of chimeric 9-mer oligonucleotides containing methylphosphonate-linkages and locked nucleic acid (LNA) monomers, their binding affinity towards complementary DNA and RNA, and their 3'-exonucleolytic stability are described. The obtained methylphosphonate-DNA/LNA chimeric oligonucleotides display similarly high RNA affinity and RNA selectivity as a corresponding 9-mer DNA/LNA chimeric oligonucleotide, but much higher resistance towards 3'-exonucleolytic degradation.

Living functional hydrogels generated by bioorthogonal cross-linking reactions of azide-modified cells with alkyne-modified polymers.

To date, many scientists have thoroughly investigated both cells and cellular functions, resulting in the identification of numerous molecular mechanisms underlying the cellular functions. Based on these findings, medical scientists and pharmacologists have developed many technological applications for cells and cellular functions in medicine. How can material scientists utilize cells and cellular functions? Here, we show a concept for utilizing cells and their functions from the viewpoint of materials science. In particular, we develop cell cross-linked living bulk hydrogels by bioorthogonal click cross-linking reactions of azide-modified mammalian cells with alkyne-modified biocompatible polymers. Importantly, we demonstrate the unique functionalities of the living hydrogels, originating from the basic functions of the cells incorporated in the living hydrogels as active cross-linking points. The findings of this study provide a promising route to generating living cell-based next-generation innovative materials, technologies, and medicines.

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.

Exhibition of soft and tenacious characteristics based on liquid crystal formation by introduction of cholesterol groups on biodegradable lactide copolymer.

Cholesterol side-functionalized poly(depsipeptide- co- dl-lactide) (PGD- dl-LA-(cholesterol) n ) and poly(depsipeptide) (PGD-(cholesterol) n) were prepared as novel biodegradable liquid crystalline (LC) polymers. These polymer films exhibited different LC phases depending on the cholesterol unit content in the polymers. The thermodynamic stability of these LC phases was quite high, and PGD-(cholesterol) n film exhibited continuous LC phases up to 202 degrees C. The resulting cholesterol LC phases were indicated to act as physical cross-linking points to form noncovalent network structures among the polymer chains. Therefore, PGD- dl-LA-(cholesterol) n film exhibited a rubbery and stretchy nature at 37 degrees C due to physical cross-linking points based on cholesterol LC phase well-dispersed in the film. The cholesterol side-group effects leading to rubbery character and hydrolytic resistance reported herein are rather unique. The biodegradable LC material exhibiting a soft and tenacious nature is a promising candidate for a new class of implant biomaterials used with dynamic organs of the body such as the heart and blood vessels.