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A natural polysaccharide scaffold, referred to as "freeze-dry nanogel-crosslinked-porous" (FD-NanoCliP) gel, was tested in comparison with an atelocollagen scaffold with respect to osteogenesis versus the mouse mesenchymal progenitor cell line KUSA-A1. The amphiphilic polysaccharide network, engineered in its structure to fit chemically crosslinked nanogels as building blocks into a physically crosslinked porous gel, revealed a superior osteointegrative performance as compared to the soluble atelocollagen network and a peculiar c-plane orientation growth of apatite crystallites, which resembled the structure of natural enamel. Besides evaluating osteogenesis in the FD-NanoCliP gel scaffold, an additional purpose of this study was to assess its chemical composition at the nanoscale and, through its knowledge, to interpret the osteogenic response of mesenchymal cells. In addition to conventional (optical and electron) microscopy and biological evaluation kits, the peculiar chemistry of the FD-NanoCliP gel scaffold and the formation of apatite on it were characterized by means of several independent analytical probes at the molecular scale, which included Raman, cathodoluminescence, energy dispersive X-ray, and X-ray fluorescence spectroscopies. This body of information consistently provided evidence for a peculiar chemistry developed in osteogenesis at the polysaccharide scaffold surface. Such chemistry is not available in soluble atelocollagen and it is key in the superior bioactivity found in the polysaccharide network., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Amphiphilic polysaccharide self-assembled (SA) nanogels are promising protein carriers owing to their chaperone-like activity that allows them to nanoencapsulate proteins within their polymer networks. The chaperoning function is an important concept that has led to breakthroughs in the development of effective protein drug delivery systems by stabilizing formulations and controlling the quality of unstable proteins. Recently, nanogel-tectonic materials that integrate SA nanogels as building blocks have been designed as new hydrogel biomaterials. This article describes recent progress and applications of SA nanogel tectonic materials as protein delivery systems for tissue engineering., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transplantation of engineered three-dimensional (3D) bone tissue may provide therapeutic benefits to patients with various bone diseases. To achieve this goal, appropriate 3D scaffolds and cells are required. In the present study, we devised a novel nanogel tectonic material for artificial 3D scaffold, namely the nanogel-cross-linked porous (NanoCliP)-freeze-dried (FD) gel, and estimated its potential as a 3D scaffold for bone tissue engineering. As the osteoblasts, directly converted osteoblasts (dOBs) were used, because a large number of highly functional osteoblasts could be induced from fibroblasts that can be collected from patients with a minimally invasive procedure. The NanoCliP-FD gel was highly porous, and fibronectin coating of the gel allowed efficient adhesion of the dOBs, so that the cells occupied the almost entire surface of the walls of the pores after culturing for 7 days. The dOBs massively produced calcified bone matrix, and the culture could be continued for at least 28 days. The NanoCliP-FD gel with dOBs remarkably promoted bone regeneration in vivo after having been grafted to bone defect lesions that were artificially created in mice. The present findings suggest that the combination of the NanoCliP-FD gel and dOBs may provide a feasible therapeutic modality for bone diseases.

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.

Boron neutron capture therapy, based on the release of thermal neutron irradiation from boron, is a targeted radiation therapy for cancer. Targeted and sufficient accumulation of boron in tumor cells to achieve cytotoxic efficacy and reduce off-target effects remains a challenge. Carborane has been investigated for use as a delivery agent in boron neutron capture therapy because of its high boron content and chemical stability; however, it is cytotoxic, making safe delivery difficult. The aim of this study was to investigate the potential of carborane-bearing pullulan nanogels to safely and effectively deliver boron to tumor cells in vitro and in vivo and, consequently, assess their potential as a boron neutron capture therapeutic. Murine fibrosarcoma cells (CMS5a) were used for in vitro investigations of nanogel cytotoxicity, cell uptake. A mouse fibrosarcoma xenograft model was used to investigate the bio-distribution of nanogels after intravenous administration. The nanogels produced no apparent cytotoxicity and underwent cell uptake in CMS5a cells after a 24 h incubation at up to 2000 μg/mL and 400 μg/mL, respectively. The internalized nanogels were localized around the nuclear membrane. The nanogels were administered intravenously to mice bearing fibrosarcoma xenografts. Nanogel tumor localization likely occurred through the enhanced permeation and retention effect. The nanogels successfully reduced the cytotoxicity of carborane, were internalized into tumor cells, acted as a dual-delivery therapeutic and accumulated in tumors in vivo. Consequently, they demonstrate significant potential as a boron neutron capture therapeutic., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Protein pharmaceuticals show great therapeutic promise, but effective intracellular delivery remains challenging. To address the need for efficient protein transduction systems, we used a magnetic nanogel chaperone (MC): a hybrid of a polysaccharide nanogel, a protein carrier with molecular chaperone-like properties, and iron oxide nanoparticles, enabling magnetically guided delivery. The MC complexed with model proteins, such as BSA and insulin, and was not cytotoxic. Cargo proteins were delivered to the target HeLa cell cytosol using a magnetic field to promote movement of the protein complex toward the cells. Delivery was confirmed by fluorescence microscopy and flow cytometry. Delivered β-galactosidase, inactive within the MC complex, became enzymatically active within cells to convert a prodrug. Thus, cargo proteins were released from MC complexes through exchange interactions with cytosolic proteins. The MC is a promising tool for realizing the therapeutic potential of proteins., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We investigated the biological activity of W9, a bone resorption inhibitor peptide, using NanoClik nanoparticles as an injectable carrier, where acryloyl group-modified cholesterol-bearing pullulan (CHPOA) nanogels were crosslinked by pentaerythritol tetra (mercaptoethyl) polyoxyethylene. Thirty 5-week-old male C57BL/6J mice were fed a low calcium diet and received once-daily subcutaneous injections of the carrier alone, W9 24 mg/kg/day alone, W9 24 mg/kg/day incorporated in cholesterol bearing pullulan (CHP) nanogels, or W9 (8 and 24 mg/kg/day) incorporated in NanoClik nanoparticles for 4 days (n=5). Mice that received a normal calcium diet with NanoClik nanoparticle injections without W9 were used as a control group. Radiological analyses showed that administration of W9 24 mg/kg/day significantly prevented low calcium-induced reduction of bone mineral density in the long bones and lumbar vertebrae, but only when the NanoClik nanoparticles were used as a carrier. Histomorphometric analyses of the proximal tibiae revealed that W9 24 mg/kg/day incorporated in NanoClik nanoparticles prevented the increase in bone resorption indices induced by a low calcium diet, which was confirmed by measurement of serum bone resorption markers. These data suggest that NanoClik nanoparticles could be a useful carrier for peptide therapeutics, and also demonstrate that daily subcutaneous injections of the W9 peptide with the nanoparticles were able to inhibit bone loss in vivo. An osteoclastogenesis inhibition assay performed in vitro confirmed a slower release profile of W9 from NanoClik nanoparticles compared with conventional CHP nanogels.

Unmethylated CpG oligodeoxynucleotides induce inflammatory immune responses through cytokine production and have attracted increasing attention as an immunostimulator. However, there remains a challenging issue of the use of 'native CpG DNA'. In the present study, we prepared cationic nanometer-sized gels (nanogels) consisting of cycloamylose modified with cholesterol and diethylaminoethane to form hydrophobic cross-linking points and to add positively charged groups, respectively. The cationic nanogels and native CpG DNA formed nanometer-sized complexes. Complexes of native CpG DNA with cationic nanogels delivered native CpG DNA to macrophage-like cells and induced cytokine production. In addition, complexes of negative control oligonucleotides with cationic nanogels did not induce cytokine production, and the induction of cytokines using complexes of phosphorothioate-modified CpG with cationic nanogels was lower than that of native CpG DNA. These results suggest that the complex of native CpG DNA with cationic nanogels is a promising strategy for nucleic acid adjuvants.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

To establish a safer and more effective vaccine against pneumococcal respiratory infections, current knowledge regarding the antigens common among pneumococcal strains and improvements to the system for delivering these antigens across the mucosal barrier must be integrated. We developed a pneumococcal vaccine that combines the advantages of pneumococcal surface protein A (PspA) with a nontoxic intranasal vaccine delivery system based on a nanometer-sized hydrogel (nanogel) consisting of a cationic cholesteryl group-bearing pullulan (cCHP). The efficacy of the nanogel-based PspA nasal vaccine (cCHP-PspA) was tested in murine pneumococcal airway infection models. Intranasal vaccination with cCHP-PspA provided protective immunity against lethal challenge with Streptococcus pneumoniae Xen10, reduced colonization and invasion by bacteria in the upper and lower respiratory tracts, and induced systemic and nasal mucosal Th17 responses, high levels of PspA-specific serum immunoglobulin G (IgG), and nasal and bronchial IgA antibody responses. Moreover, there was no sign of PspA delivery by nanogel to either the olfactory bulbs or the central nervous system after intranasal administration. These results demonstrate the effectiveness and safety of the nanogel-based PspA nasal vaccine system as a universal mucosal vaccine against pneumococcal respiratory infection.

A self-assembled nanogel, derived from an acid-labile cholesteryl-modified pullulan (acL-CHP), was prepared by grafting vinyl ether-cholesterol substituents onto a 100 kD pullulan main chain polymer backbone. Stable nanogels are formed by acL-CHP self-assemblies at neutral pH. The hydrodynamic radius of the nanogels, observed to be 26.5 ± 5.1 nm at pH 7.0, increased by ~135% upon acidification of the solution to pH 4.0. SEC analysis of the acL-CHP nanogel at pH 4.0 showed that the grafts were nearly 80% degraded after 24 h, whereas little or no degradation was observed over the same time period for a pH stable analog (acS-CHP) at pH 4.0 or the acL-CHP at pH 7.0. Complexation of BSA with the acL-CHP nanogel was observed at pH 7.0 with subsequent release of the protein upon acidification. These findings suggest that stimuli-responsive, self-assembled nanogels can release protein cargo in a manner that is controlled by the degradation rate of the cholesterol-pullulan grafting moiety.

Nasal vaccination is considered a potent and practical immunization route for the induction of effective immunity to infectious diseases. Successful nasal vaccines require efficient delivery to, and retention of antigens within, nasal mucosa, including both the inductive (e.g., nasopharynx-associated lymphoid tissues) and effector (e.g., turbinate covered with single-layer epithelium) tissues, where antigen-specific immune responses are initiated and executed, respectively. We developed an approach towards successful nasal vaccination by using self-assembled nano-sized hydrogel particles, known as nanogels, which are composed of a cationic type of cholesteryl group-bearing pullulan. Here, we review the merging of nanotechnological and immunological concepts leading to the development of next-generation nasal vaccines, and demonstrate the applicability of novel nanogel-based vaccine for the prevention of infectious diseases.

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cholesterol-bearing pullulan (CHP) nanogel is a synthetic degradable biomaterial for drug delivery with high biocompatibility. Guided bone regeneration (GBR) is a bone augmentation technique in which a membrane is used to create and keep a secluded regenerative space. The purpose of the present study was to evaluate the effects of the novel CHP nanogel membrane in GBR. Thirty-six adult Wistar rats were used and bilaterally symmetrical full-thickness parietal bone defects of 5 mm diameter were created with a bone trephine burr. Each defect was covered with the collagen membrane or the CHP nanogel membrane or untreated without any membrane. The animals were sacrificed at 2, 4 and 8 weeks and analysed radiologically and histologically. Furthermore, after incubating human serum with CHP nanogel or collagen, the amount of PDGF in the serum was measured using ELISA. New bone formation in terms of bone volume was higher in the nanogel group than in the control or collagen groups at 2 and 4 weeks. At 8 weeks, both membrane groups showed higher bone volumes than the control group. Notably, the newly-formed bone in the bone defect in the nanogel group was uniform and histologically indistinguishable from the original bone, whereas in the collagen group the new bone showed an irregular structure that was completely different from the original bone. After incubating with CHP nanogel, the amount of PDGF in the serum decreased significantly. CHP nanogel GBR membrane favourably stimulated bone regeneration, in which a unique characteristic of CHP nanogel, the storage of endogenous growth factors, was likely implicated., (Copyright © 2011 John Wiley & Sons, Ltd.)

We developed a novel gene delivery system capable of endosome disruption using a polysaccharide-based cationic nanogel composed of a hexadecyl group-bearing cationic cycloamylose nanogel (C16-catCA nanogel) and phospholipaseA(2) (PLA(2)) to hydrolyze membrane phospholipids. C16-catCA nanogel formed nanoparticles with PLA(2) and pDNA by hydrophobic and electrostatic interactions. Both pDNA and PLA(2) were effectively internalized into cells by the C16-catCA nanogel. In addition, the pDNA expression level was enhanced when complexed with specific concentrations of PLA(2). PLA(2) complexed with C16-catCA nanogel also showed a similar hemolytic activity against red blood cells to that observed using native PLA(2). These results suggest that the C16-catCA nanogel/PLA(2) complex possesses membrane disruption ability when delivered into cells and triggers the subsequent release of pDNA from the endosome to the cytoplasm. This is the first report of co-delivery of pDNA and PLA(2) using the same carrier to achieve effective gene delivery., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A cell specific peptide (Arg-Gly-Asp; RGD)-modified nanogel was prepared and evaluated for its potential to act as a protein delivery carrier. A bovine serum albumin (BSA)/RGD-modified nanogel complex was efficiently internalized into cells through integrin-mediated endocytosis. Endosomal escape of the RGD-modified nanogel was observed after 24 h incubation. The nanogel proved useful for targeted protein delivery., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Regeneration of bone requires the combination of appropriate drugs and an appropriate delivery system to control cell behavior. However, the delivery of multiple drugs to heal bone is complicated by the availability of carriers. The aim of this study was to explore a new system for delivery of a selective EP4 receptor agonist (EP4A) in combination with low-dose bone morphogenetic protein 2 (BMP-2)., Methods: Combined delivery of EP4A and BMP-2 was carried out with a nanogel-based scaffold in the shape of a disc, to repair critical-size circle-shaped bone defects in calvariae that otherwise did not heal spontaneously., Results: Combination treatment with EP4A and low-dose BMP-2 in nanogel efficiently activated bone cells to regenerate calvarial bone by forming both outer and inner cortical plates as well as bone marrow tissue to regenerate a structure similar to that of intact calvaria. EP4A enhanced low-dose BMP-2-induced cell differentiation and activation of transcription events in osteoblasts., Conclusion: These data indicate that combined delivery of EP4A and low-dose BMP-2 via nanogel-based hydrogel provides a new system for bone repair., (Copyright © 2011 by the American College of Rheumatology.)

Nanosize hydrogels (nanogels) are polymer nanoparticles with three-dimensional networks, formed by chemical and/or physical cross-linking of polymer chains. Recently, various nanogels have been designed, with a particular focus on biomedical applications. In this review, we describe recent progress in the synthesis of nanogels and nanogel-integrated hydrogels (nanogel cross-linked gels) for drug-delivery systems (DDS), regenerative medicine, and bioimaging. We also discuss chaperone-like functions of physical cross-linking nanogel (chaperoning engineering) and organic-inorganic hybrid nanogels., (Copyright © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.)

Bone regeneration for the defects in revision surgery of joint replacement is an increasingly important issue. To repair bone defects, bone cell activation by growth factors using synthetic resorbable scaffold is a useful and safe option. We examine the efficiency of nanogel-crosslinking hydrogel as a novel synthetic scaffold for BMP to stimulate osteoblasts and to induce bone formation. Cholesterol-bearing pullulan nanogel-crosslinking hydrogel (CHPA/Hydrogel) was used to deliver BMP. The CHPA hydrogel pellets were implanted in vivo. Single implantation of CHPA/hydrogel containing low amounts of BMP induced osteoblastic activation and new bone formation in vivo. Furthermore, nanogel in a disc shape established recruitment of osteoblastic cells that vigorously formed bone to heal the calvarial defects, which did not heal spontaneously without it. In conclusion, CHPA/hydrogel serves as an efficient and versatile scaffold for the stimulation of osteoblasts to form bone and to repair defects via delivery of BMP.

An effective intracellular protein delivery system with self-assembled cationic nanogels is reported. Interaction of proteins with self-assembled nanogels of cationic cholesteryl group-bearing pullulans (CHPNH 2) was investigated by dynamic light scattering (DLS), transmission electron micrograph (TEM), fluorescence resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS). The cationic nanogels strongly interacted with bovine serum albumin (BSA) and formed monodispersed nanoparticels (<50 nm). The complex more effectively internalized into HeLa cells than cationic liposomes and a protein transduction domain (PTD) based carrier even in the presence of serum. The higher efficiency of the nanogel carrier is probably due to the formation of colloidally stable nanoparticles with the protein. The enzymatic activity of beta-galactosidase (beta-Gal) was retained after internalization into cells. The nanogel carrier promoted nuclear delivery of a GFP-conjugated nuclear localization signal and Tat as a PTD (Tat-NLS-GFP). A blocking experiment with chemical inhibitors revealed the possible involvement of macropinocytosis in the uptake of the nanogel complex. After cellular uptake, the complex of the nanogel-protein was dissociated and the protein was released inside the cell. Such a self-assembled cationic nanogel system should create opportunities for novel applications of protein delivery.

Recovery of bone loss is one of the active research issues in bone medicine due to the need for efficient measures for bone gain. We examined here a novel drug delivery system using a nanogel of cholesterol-bearing pullulan (CHP) in combination with prostaglandin E2 (PGE2). PGE2 or PGE2/CHP, vehicle (saline containing 0.06% ethanol and 0.02% Tween 80) or CHP were injected on to the calvariae of mice once every day for 5 days per week for 4 weeks. Low dosage of PGE2 (0.6 microg) alone or CHP alone did not induce new bone formation in this system. In contrast, PGE2 (0.6 microg)/CHP induced new bone formation. Bone formation activities of PGE2 was enhanced by CHP nanogels only at the site of injection (calvaria) but not in the distant sites of the skeleton, showing that PGE2/CHP could avoid systemic effects. In spite of the fact that previously reported animal models of bone formation by PGE2 were associated with loss of body weight, bone formation based on PGE2/CHP did not associate with loss of body weight. Furthermore, only a single application of PGE2 in combination with nanogel cross-linking hydrogel sphere (PGE2/CHP-PEO) induced new bone formation. Thus, nanogel-based delivery system is an efficient delivery system of bone anabolic agent, PGE2., ((c) 2007 Wiley-Liss, Inc.)

The formation of fibrils by amyloid beta-protein (Abeta) is considered as a key step in the pathology of Alzheimer's disease (AD). Inhibiting the aggregation of Abeta is a promising approach for AD therapy. In this study, we used biocompatible nanogels composed of a polysaccharide pullulan backbone with hydrophobic cholesterol moieties (cholesterol-bearing pullulan, CHP) as artificial chaperones to inhibit the formation of Abeta-(1-42) fibrils with marked amyloidgenic activity and cytotoxicity. The CHP-nanogels incorporated up to 6-8 Abeta-(1-42) molecules per particle and induced a change in the conformation of Abeta from a random coil to alpha-helix- or beta-sheet-rich structure. This structure was stable even after a 24-h incubation at 37 degrees C and the aggregation of Abeta-(1-42) was suppressed. Furthermore, the dissociation of the nanogels caused by the addition of methyl-beta-cyclodextrin released monomeric Abeta molecules. Nanogels composed of amino-group-modified CHP (CHPNH(2)) with positive charges under physiological conditions had a greater inhibitory effect than CHP-nanogels, suggesting the importance of electrostatic interactions between CHPNH(2) and Abeta for inhibiting the formation of fibrils. In addition, CHPNH(2) nanogels protected PC12 cells from Abeta toxicity.

Chaperone-like activity (to trap proteins in biomaterials without their aggregations and to control release of proteins in a native form) is important to design protein delivery systems as well as protein engineering. In general, irreversible adsorption of proteins is unavoidable in trapping them in hydrogel biomaterials because it is difficult to control the mesh size of the hydrogel matrix. We suggested that physically cross-linked nanogels with a size comparable to that of proteins are useful for these purposes. Tailor-made functional nanogels and hydrogels were designed by self-assembly of functional associating polysaccharides such as cholesterol-bearing pullulans. The nanogels can trap hydrophobic molecules, proteins and nucleic acids. They are useful as artificial molecular chaperones and also polymeric nanocarriers in DDS.

Dynamic CHP-CD nanogels, which consisted of a self-assembly of cholesteryl-group-bearing pullulan (CHP) and beta-cyclodextrin (CD), were characterized by SEC and SEC-MALS methods. The nanogels prevented the thermal aggregation of carbonic anhydrase B (CAB) by selective trapping of the heat-denatured protein. After the complex between the CHP-CD nanogels and CAB was cooled, the enzyme activity of CAB spontaneously recovered upon release from the complex. The dynamic nanogels self-regulated an association of heat denatured protein and dissociation of native protein depending on the concentration of CD. The thermal stability of CAB was improved by thermoresponsive controlled association between the proteins and the artificial molecular chaperone.

Novel photoresponsive nanogels were prepared by the self-assembly of spiropyrane-bearing pullulan (SpP). The solution properties of the nanogels could be controlled by photostimulation via isomerization between hydrophobic spiropyrane and hydrophilic merocyanine. The molecular chaperone-like activity of the nanogels in protein refolding was investigated. The activity of citrate synthase significantly increased when the amphiphilicity of SpP nanogels was switched by photostimulation.