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Nanoparticles of zeolitic imidazole framework-8 (ZIF-8 NPs), which are the subclass of metal-organic frameworks consisting of Zn ion and 2-methylimidazole, have been identified as promising drug carriers since their large microporous structure is suited for encapsulating hydrophobic drug molecules. However, one of the limitations of ZIF-8 NPs is their low stability in physiological solutions, especially in the presence of water and phosphate anions. These molecules can interact with the coordinatively unsaturated Zn sites at the external surface to induce the degradation of ZIF-8 NPs. In this study, herein a facile approach is reported to enhance the chemical stability of ZIF-8 NPs by surface coating with polyacrylic acid (PAA). The PAA-coated ZIF-8 (PAA-ZIF-8) NPs are prepared by mixing ZIF-8 NPs and PAA in water. PAA coating inhibits the degradation of ZIF-8 NPs in water as well as phosphate-buffered saline over 6 days, which seems to be due to the coordination of carboxyl groups of PAA to the reactive Zn sites. Furthermore, the PAA-ZIF-8 NPs loaded with the anticancer drug doxorubicin (Dox) show cytotoxicity in human colon cancer cells. These results clearly show the feasibility of the PAA coating approach to improve the chemical stability of ZIF-8 NPs without impairing their drug delivery capability., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Nanoparticle-based drug carriers that can respond to oxidative stress in tumor tissue have attracted attention for site-specific drug release. Taking advantage of the characteristic microenvironment in tumors, one of the attractive directions in drug delivery research is to design drug carriers that release drugs upon oxidation. A strategy to incorporate oxidation-sensitive thioether motifs such as thiomorpholine acrylamide (TMAM) to drug carriers has been often used to achieve oxidation-induced dissociation, thereby targeted drug release. However, those delivery systems often suffer from a slow dissociation rate due to the intrinsic hydrophobicity of the thioether structures. In this study, we aimed to enhance the dissociation rate of TMAM-based micelles upon oxidation. The random copolymers of N -isopropylacrylamide and TMAM (P(NIPAM/TMAM)) were designed as an oxidation-sensitive segment that showed a fast response to oxidative stress. We first synthesized P(NIPAM/TMAM) copolymers with different NIPAM:TMAM molar ratios. Those copolymers exhibited low critical solution temperatures (LCSTs) below 32 °C, which shifted to higher temperatures after oxidation. The changes in LCSTs depend on the NIPAM:TMAM molar ratios. At the NIPAM:TMAM molar ratio of 82:18, the LCSTs before and after oxidation were 17 and 54 °C, respectively. We then prepared micelles from the diblock copolymers of poly( N -acryloyl morpholine) (PAM) and P(NIPAM/TMAM). The micelles showed an accelerated dissociation rate upon oxidation compared to the micelles without NIPAM units. Furthermore, the doxorubicin (Dox)-loaded micelles showed enhanced relative toxicity in human colorectal cancer (HT29) cells over human umbilical vein endothelial cells (HUVECs). Our novel strategy to design an oxidation-sensitive micellar core comprising a P(NIPAM/TMAM) segment can be used as a chemotherapeutic delivery system that responds to an oxidative tumor microenvironment in an appropriate time scale.

Per/polysulfide species that are generated from endogenously produced hydrogen sulfide have critical regulatory roles in a wide range of cellular processes. However, the lack of delivery systems that enable controlled and sustained release of these unstable species in biological systems hinders the advancement of sulfide biology research, as well as the translation of knowledge to therapeutic applications. Here, a novel approach is developed to generate per/polysulfide species in cells by combining an H 2 S donor and manganese porphyrin-containing polymeric micelles (MnPMCs) that catalyze oxidization of H 2 S to per/polysulfide species. MnPMCs serve as a catalyst for H 2 S oxidation in aerobic phosphate buffer. HPLC-MS/MS analysis reveals that H 2 S oxidation by MnPMCs in the presence of glutathione results in the formation of glutathione-SnH (n = 2 and 3). Furthermore, co-treatment of human umbilical vein endothelial cells with the H 2 S donor anethole dithiolethione and MnPMCs increases intracellular per/polysulfide levels and induces a proangiogenic response. Co-delivery of MnPMCs and an H 2 S donor is a promising approach for controlled delivery of polysulfides for therapeutic applications., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Gaseous signaling molecules such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S) have recently been recognized as essential signal mediators that regulate diverse physiological and pathological processes in the human body. With the evolution of gaseous signaling molecule biology, their therapeutic applications have attracted growing attention. One of the challenges in translational research of gaseous signaling molecules is the lack of efficient and safe delivery systems. To tackle this issue, researchers developed a library of gas donors, which are low molecular weight compounds that can release gaseous signaling molecules upon decomposition under physiological conditions. Despite the significant efforts to control gaseous signaling molecule release from gas donors, the therapeutic potential of gaseous signaling molecules cannot be fully explored due to their unfavorable pharmacokinetics and toxic side effects. Recently, the use of nanoparticle-based gas donors, especially self-assembled polymeric gas donors, have emerged as a promising approach. In this review, we describe the development of conventional small gas donors and the challenges in their therapeutic applications. We then illustrate the concepts and critical aspects for designing self-assembled polymeric gas donors and discuss the advantages of this approach in gasotransmistter delivery. We also highlight recent efforts to develop the delivery systems for those molecules based on self-assembled polymeric nanostructures. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies., (© 2023 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.)

Polymeric nanoparticles with reactive functional groups are an attractive platform for drug carriers that can be conjugated with drugs through a cleavable covalent linkage. Since the required functional groups vary depending on the drug molecule, there is a need for development of a novel post-modification method to introduce different functional groups to polymeric nanoparticles. We recently reported phenylboronic acid (PBA)-containing nanoparticles (BNP) with a unique framboidal morphology created via one-step aqueous dispersion polymerization. Since BNPs have high surface area due to their framboidal morphology and contain a high density of PBA groups, these particles can be used as nanocarriers for drugs that can bind to PBA groups such as curcumin and a catechol-bearing carbon monoxide donor. To further explore the potential of BNPs, in this article we report a novel strategy to introduce different functional groups to BNPs via the palladium-catalyzed Suzuki-Miyaura cross-coupling reaction between the PBA groups and iodo- and bromo-coupling partners. We developed a new catalytic system that efficiently catalyzes Suzuki-Miyaura reactions in water without the need for an organic solvent, as confirmed by NMR. Using this catalyst system, we show that BNPs can be functionalized with carboxylic acids, aldehyde, and hydrazide groups while keeping their original framboidal morphology as confirmed via IR, alizarin red assay, and TEM. Furthermore, the potential of the functionalized BNP in drug delivery applications was demonstrated by conjugating the hydrogen sulfide (H 2 S)-releasing compound anethole dithiolone to carboxylic acid-functionalized BNPs and show their H 2 S-releasing capability in cell lysate.

Hydrogen sulfide (H 2 S) is a gaseous signaling molecule in the human body and has attracted attention in cancer therapy due to its regulatory roles in cancer cell proliferation and migration. Accumulating evidence suggests that continuous delivery of H 2 S to cancer cells for extended periods of time suppresses cancer progression. However, one major challenge in therapeutic applications of H 2 S is its controlled delivery. To solve this problem, polymeric micelles are developed containing H 2 S donating-anethole dithiolethione (ADT) groups, with H 2 S release profiles optimal for suppressing cancer cell proliferation. The micelles release H 2 S upon oxidation by reactive oxygens species (ROS) that are present inside the cells. The H 2 S release profiles can be controlled by changing the polymer design. Furthermore, the micelles that show a moderate H 2 S release rate exert the strongest anti-proliferative effect in human colon cancer cells in in vitro assays as well as the chick chorioallantoic membrane cancer model, while the micelles do not affect proliferation of human umbilical vein endothelial cells. This study shows the importance of fine-tuning H 2 S release profiles using a micelle approach for realizing the full therapeutic potential of H 2 S in cancer treatment., (© 2023 Wiley-VCH GmbH.)

Angiogenesis is regulated in coordinated fashion by chemical and mechanical cues acting on endothelial cells (ECs). However, the mechanobiological mechanisms of angiogenesis remain unknown. Herein, we demonstrate a crucial role of blood flow-driven intraluminal pressure (IP) in regulating wound angiogenesis. During wound angiogenesis, blood flow-driven IP loading inhibits elongation of injured blood vessels located at sites upstream from blood flow, while downstream injured vessels actively elongate. In downstream injured vessels, F-BAR proteins, TOCA1 and CIP4, localize at leading edge of ECs to promote N-WASP-dependent Arp2/3 complex-mediated actin polymerization and front-rear polarization for vessel elongation. In contrast, IP loading expands upstream injured vessels and stretches ECs, preventing leading edge localization of TOCA1 and CIP4 to inhibit directed EC migration and vessel elongation. These data indicate that the TOCA family of F-BAR proteins are key actin regulatory proteins required for directed EC migration and sense mechanical cell stretching to regulate wound angiogenesis., (© 2022. The Author(s).)

Oxidation-sensitive drug delivery systems (DDSs) have attracted attention due to the potential to improve efficacy and safety of chemotherapeutics. These systems are designed to release the payload in response to oxidative stress conditions, which are associated with many types of cancer. Despite extensive research on the development of oxidation-sensitive DDS, the lack of selectivity toward cancer cells over healthy cells remains a challenge. Here, we report the design and characterization of polymeric micelles containing thioether groups with varying oxidation sensitivities within the micellar core, which become hydrophilic upon thioether oxidation, leading to destabilization of the micellar structure. We first used the thioether model compounds, 3-methylthiopropylamide (TPAM), thiomorpholine amide (TMAM), and 4-(methylthio)benzylamide (TPhAM) to investigate the effect of the chemical structures of the thioethers on the oxidation by hydrogen peroxide (H 2 O 2 ). TPAM shows the fastest oxidation, followed by TMAM and TPhAM, showing that the oxidation reaction of thioethers can be modulated by changing the substituent groups bound to the sulfur atom. We next prepared micelles containing these different thioether groups within the core (TP, TM, and TPh micelles). The micelles containing the thioether groups with a higher oxidation sensitivity were destabilized by H 2 O 2 at a lower concentration. Micelle destabilization was also tested in human liver cancer (HepG2) cells and human umbilical vein endothelial cells (HUVECs). The TP micelles having the highest oxidation sensitivity were destabilized in both HepG2 cells and HUVECs, while the TPh micelles, which showed the lowest reactivity toward H 2 O 2 , were stable in these cell lines. The TM micelles possessing a moderate oxidation sensitivity were destabilized in HepG2 cells but were stable in HUVECs. Furthermore, the micelles were loaded with doxorubicin (Dox) to evaluate their potential in drug delivery applications. Among the micelles, the TM micelles loaded with Dox showed the enhanced relative toxicity in HepG2 cells over HUVECs. Therefore, our approach to fine-tune the oxidation sensitivity of the micelles has potential for improving therapeutic efficacy and safety of drugs in cancer treatment.

Hydrogen sulfide (H 2 S), an important gaseous signalling molecule in the human body, has been shown to be involved in many physiological processes such as angiogenesis. Since the biological activities of H 2 S are known to be significantly affected by the dose and exposure duration, the development of H 2 S delivery systems that enable control of H 2 S release is critical for exploring its therapeutic potential. Here, we prepared polymeric micelles with different H 2 S release profiles, which were prepared from amphiphilic block copolymers consisting of a hydrophilic poly( N -acryloyl morpholine) segment and a hydrophobic segment containing H 2 S-releasing anethole dithiolethione (ADT) groups. The thermodynamic stability of the micelles was modulated by altering the ADT content of the polymers. The micelles with higher thermodynamic stability showed significantly slower H 2 S release. Furthermore, the sustained H 2 S release from the micelles enhanced migration and tube formation in human umbilical vein cells (HUVECs) and induced vascularlization in the in ovo chick chorioallantoic membrane (CAM) assay., Competing Interests: Conflicts of interest There are no conflicts to declare.

Curcumin (Cur) has a wide range of bioactivities that show potential for the treatment of cancer as well as chronic diseases associated with inflammation and aging. However, the therapeutic efficacy of Cur has been hampered by its rapid degradation under physiological conditions and low aqueous solubility. To address these problems, we prepared Cur-loaded polymeric nanoparticles (CNPs), in which Cur was complexed with phenylboronic acid-containing framboidal nanoparticles (NPs), by simple mixing of Cur and NPs in an aqueous solution. CNPs showed improved chemical stability of Cur and released it in a sustained manner under physiological conditions. Furthermore, CNPs significantly enhanced the antiangiogenic and anticancer activities of Cur in chicken chorioallantoic membrane models.

Two polyamine derivatives of protoporphyrin IX (PPIX) were tested as photodynamic therapy (PDT) agents in HT29 colorectal cancer and HEP3B liver cancer cell lines. These compounds exhibit excellent singlet oxygen quantum yields and show strong in vitro PDT efficacy after 660 nm laser irradiation, whereas exogenous PPIX itself exhibits much weaker PDT effects. Confocal microscopy imaging studies reveal that a protoporphyrin derivative with eight amine moieties has excellent water solubility, and localizes mainly in the mitochondria of both HT29 and HEP3B cells, whereas the cellular distribution of a protoporphyrin derivative with four amine moieties is not as specific. This work demonstrates that polyamine moieties on macrocycles can enhance PDT efficacy by targeting mitochondria., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Furoxans, or 1,2,5-oxadiazole-N-oxides, are a class of nitric oxide (NO)-donating compounds that release NO in response to thiol-containing molecules. In this study, polymeric micelles bearing furoxan moieties are prepared from an amphiphilic block copolymer consisting of a hydrophobic furoxan-bearing block and a hydrophilic poly(N-acryloylmorpholine) block. The block copolymer is prepared using a combination of the reversible addition-fragmentation chain transfer polymerization and the copper-catalyzed Huisgen cycloaddition techniques. The block copolymers form spherical micelles with a diameter of 50 nm by self-assembly in water. The micelles release NO in response to cysteine and show improved stability against hydrolytic decomposition. Furthermore, the micelles show a synergistic anti-proliferative effect with ibuprofen in human colon cancer cells., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbon monoxide (CO) is an essential gaseous signaling molecule in the human body. Toward the controlled delivery of CO to the target tissues or cells, nanomaterial-based CO donors have attracted growing attention. Here, we present CO-releasing polymeric nanoparticles (CONPs) prepared by simple mixing of phenylboronic acid-containing framboidal nanoparticles with the catechol-bearing CO-donor Ru(CO)3Cl(L-DOPA) via phenylboronic acid-catechol complexation. The CONPs release CO in response to cysteine and suppress the production of the pro-inflammatory mediators interleukin 6 (IL-6) and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated murine macrophages. This CONP platform may show promise in therapeutic applications of CO.

This data article provides supplementary figures to the research article entitled, "Phase separation approach to a reactive polycarbonate monolith for "click" modifications" (Xin et al., Polymer, 2015, doi:10.1016/j.polymer.2015.04.008). Here, the nitrogen adsorption/desorption isotherms of the prepared porous polycarbonate monolith are shown to classify its inner structure and calculate the specific surface area. The monoliths were modified by using the thiol-ene click chemistry and the olefin metathesis, which was examined by contact angle measurements, FT-IR, solid state 13 C NMR spectroscopy as well as thermogravimetric analysis.

Functional polymeric nanoparticles have been used for various applications in the biomaterials field. Recently, we reported phenylboronic acid-containing nanoparticles (PBA NPs) having an unique framboidal morphology, prepared in a single-step by the aqueous dispersion polymerization of N-acryloyl-3-aminophenylboronic acid (PBAAM) in the presence of poly(ethylene glycol) acrylamide (PEGAM) as a polymerizable dispersant and N,N'-methylenebisacrylamide (MBAM) as a crosslinker. In this study, we prepared mannosylated and fluorescent PBA NPs that could be used for different applications such as drug delivery and bioimaging. Fluorescent PBA NPs were synthesized by including the fluorescent Nile Blue acrylamide monomer in the reaction mixture during the dispersion polymerization of PBAAM. By using a carboxyl group-bearing PEGAM dispersant, carboxyl group-bearing PBA NPs were prepared that were modified with mannosamine to yield mannosylated PBA NPs. Cellular uptake studies showed that the mannosylated PBA NPs were selectively taken up by murine RAW264.7 macrophages. These results show that PBA NPs allow for flexible modification with various functionalities and could therefore be a potential platform for targeted delivery of drugs to macrophages., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Back Cover: The micellar prodrugs of desmethyl anethole dithiolethione (ADT-OH) with different hydrolysis rates prepared from block copolymers having ADT-OH linked via an ester bond using glycine and isoleucine linkers are presented. Micelles having a glycine linker inhibit proliferation of cancer cells. Further details can be found in the article by U. Hasegawa, N. Tateishi, H. Uyama, A. J. van der Vlies on page 1512., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prodrug micelles carrying 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), a compound possessing chemopreventive properties, are prepared from amphiphilic block copolymers linking ADT-OH via an ester bond using glycine (PAM-PGlyADT) and isoleucine linkers (PAM-PIleADT). The release of ADT-OH from the PAM-PIleADT micelles is much slower than the PAM-PGlyADT micelles. The PAM-PGlyADT micelles show comparable toxicity with ADT-OH in different cancer cell lines, whereas the PAM-PIleADT micelles are not toxic up to 400 µM. This ADT-ester prodrug micelle approach enables to modulate the release rate of ADT-OH and thus might find application in cancer therapy and prevention., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Here, we provide the NMR spectra and AFM data for antioxidant micelles prepared from amphiphilic PAM-PDA block copolymers composed of a poly(N-acryloyl morpholine) and a redox-active catechol-bearing block with different catechol content. We also provide details of the electrochemical analysis that showed micelles higher catechol content had a similar redox potential with the small catechol compound dopamine, but slowed down the redox reaction (Hasegawa et al., Polymer (in press)).

Antioxidant micelles capable of scavenging reactive oxygen species (ROS) are prepared from poly(ethylene glycol)-b-poly(dopamine) block copolymers. The micelles inhibit tube formation of human umbilical vein endothelial cells (HUVECs) by scavenging endogenous ROS. Furthermore, the micelles inhibit angiogenesis in the chicken ex ovo chorioallantoic membrane assay. The results show that antioxidant micelles containing catechol moieties may be useful in anti-angiogenic therapy to treat various diseases such as cancer., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrogen sulfide (H2S) is a gaseous signaling molecule that has several important biological functions in the human body. Because of the difficulties of handling H2S gas, small organic compounds that release H2S under physiological conditions have been developed. The observed bioactivities of these H2S donors have generally been directly correlated with their H2S release properties. However, apart from H2S release, these H2S donors also exert biological effects by direct interaction with intracellular components within the cytoplasm after passive diffusion across cellular membranes. Here we report polymeric H2S donors based on ADT-OH which would alter cellular trafficking of ADT-OH to minimize the unfavorable interactions with intracellular components. We designed and synthesized a poly(ethylene glycol)-ADT (PEG-ADT) conjugate having ADT linked via an ether bond. Whereas ADT-OH significantly reduced cell viability in murine macrophages, the PEG-ADT conjugate did not show obvious cytotoxicity. The PEG-ADT conjugate released H2S in murine macrophages but not in the presence of serum proteins. The PEG-ADT conjugate was taken up by the cell through the endocytic pathway and stayed inside endolysosomes, which is different from the small amphiphilic donor ADT-OH that can directly enter the cytoplasm. Furthermore, PEG-ADT was capable of potentiating LPS-induced inflammation. This polymeric H2S donor approach may help to better understand the H2S bioactivities of the H2S donor ADT-OH.

A hybrid monolith of poly(γ-glutamic acid) and hydroxyapatite (PGA/HAp monolith) was prepared via biomineralization and used as a macroporous cell scaffold in bone tissue engineering. The PGA monolith having a bimodal pore size distribution was used as a substrate to induce biomineralization. The PGA/HAp monolith was obtained by immersing the PGA monolith in simulated body fluid. Pretreatment with CaCl2 enhanced the apatite-forming ability of the PGA monolith. Murine osteoblastic MC3T3-E1 cells efficiently attached and proliferated on the PGA/HAp monolith. MTT assay showed that both the PGA and PGA/HAp monolith did not have apparent cytotoxicity. Moreover, the PGA and PGA/HAp monoliths adsorbed bone morphogenetic protein-2 (BMP-2) by electrostatic interaction which was slowly released in the medium during cell culture. The PGA/HAp monolith enhanced BMP-2 induced alkaline phosphatase activity compared to the PGA monolith and a polystyrene culture plate. Thus, these PGA/HAp monoliths may have potential in bone tissue engineering.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat acute pain, fever, and inflammation and are being explored in a new indication in cancer. Side effects associated with long-term use of NSAIDs such as gastrointestinal damage and elevated risk of stroke, however, can limit their use and exploration in new indications. Here we report a facile method to prepare well-defined amphiphilic diblock copolymer NSAID prodrugs by direct reversible addition-fragmentation transfer (RAFT) polymerization of the acrylamide derivative of ibuprofen (IBU), a widely used NSAID. The synthesis and self-assembling behavior of amphiphilic diblock copolymers (PEG-PIBU) having a hydrophilic poly(ethylene glycol) block and a hydrophobic IBU-bearing prodrug block were investigated. Release profiles of IBU from the micelles by hydrolysis were evaluated. Furthermore, the antiproliferative action of the IBU-containing micelles in human cervical carcinoma (HeLa) and murine melanoma (B16-F10) cells was assessed.

Colloidal drug and prodrug conjugates have unique targeting characteristics for tumor vasculature from the blood and for the lymphatics draining a tissue injection site. Tioguanine and tioguanine-generating prodrugs have been investigated as anticancer and immunosuppressive agents, including use in cancer immunotherapy. Recently we developed block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide) that self-assemble in aqueous solutions to form micellar structures. Since the polymers carry a free terminal thiol group resulting from the ring-opening polymerization of the propylene sulfide monomer, we sought to prepare prodrug block copolymers with tioguanine linked by a reduction-sensitive disulfide bond. The synthesis involved a disulfide exchange between the oxidized form of tioguanine and the polymer. Spectroscopic data is presented to support the proposed reaction. The polymers self-assembled when dispersed in water to form tioguanine prodrug micelles with a size range between 18 and 40 nm that released tioguanine in response to cysteine and serum as shown spectroscopically. In comparison with a poly(ethylene glycol) prodrug polymer, we show that the rate of tioguanine release can be controlled by changing the poly(propylene sulfide) block length and that the tioguanine remains bioactive with cultured cells.

Analytical ultracentrifugation (AUC) primarily serves to investigate hydrodynamic and thermodynamic properties of natural and synthetic macromolecules and colloids in solution, dispersion or suspension. Beside such more conventional use, AUC can support materials development particularly by combining different optical systems, if the AUC is equipped with such, or using complementary data evaluation approaches. In this context, an Optima XL-I equipped with absorbance (AO) and interference optics (IO) was used alone or complementary to study the success of conjugation of biopolymers, to evaluate the completeness of the incorporation of macromolecules into micelles and vesicles, and to analyze the composition and homogeneity of macromolecular assemblies. The combination of AO and IO proved covalent binding of concanavalin A to dextran without macromolecular degradation as well as the formation of mixed micelles composed of two types of block copolymers. Further, AUC contributed to analyze the homogeneity, purity, size and size distribution of carbon monoxide-releasing macromolecular assemblies. These case studies revealed that the application possibilities of AUC are by far not completely discovered but can still be extended., (Copyright © 2010 Elsevier Inc. All rights reserved.)

With the discovery of important biological roles of carbon monoxide (CO), the use of this gas as a therapeutic agent has attracted attention. However, the medical application of this gas has been hampered by the complexity of the administration method. To overcome this problem, several transition-metal carbonyl complexes, such as Ru(CO)(3)Cl(glycinate), [Ru(CO)(3)Cl(2)](2), and Fe(η(4)-2-pyrone)(CO)(3), have been used as CO-releasing molecules both in vitro and in vivo. We sought to develop micellar forms of metal carbonyl complexes that would display slowed diffusion in tissues and thus better ability to target distal tissue drainage sites. Specifically, we aimed to develop a new CO-delivery system using a polymeric micelle having a Ru(CO)(3)Cl(amino acidate) structure as a CO-releasing segment. The CO-releasing micelles were prepared from triblock copolymers composed of a hydrophilic poly(ethylene glycol) block, a poly(ornithine acrylamide) block bearing Ru(CO)(3)Cl(ornithinate) moieties, and a hydrophobic poly(n-butylacrylamide) block. The polymers formed spherical micelles in the range of 30-40 nm in hydrodynamic diameter. Further characterization revealed the high CO-loading capacity of the micelles. CO-release studies showed that the micelles were stable in physiological buffer and serum and released CO in response to thiol-containing compounds such as cysteine. The CO release of the micelles was slower than that of Ru(CO)(3)Cl(glycinate). In addition, the CO-releasing micelles efficiently attenuated the lipopolysaccharide-induced NF-κB activation of human monocytes, while Ru(CO)(3)Cl(glycinate) did not show any beneficial effects. Moreover, cell viability assays revealed that the micelles significantly reduced the cytotoxicity of the Ru(CO)(3)Cl(amino acidate) moiety. This novel CO-delivery system based on CO-releasing micelles may be useful for therapeutic applications of CO.

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen-Pluronic conjugate to the polymerization mixture ( Reddy , S. T. , et al. ( 2007 ) Nat. Biotechnol. 25, 1159 ). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteamine pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented.

Raspberry-like assembly of nanogels (A-CHPNG) with a high potential as a carrier for protein delivery was prepared. Cross-linking of acrylate group-modified cholesterol-bearing pullulan nanogel (CHPANG) with thiol group-modified poly (ethylene glycol) (PEGSH) by Michael addition yielded A-CHPNG with narrow size distribution. The size of A-CHPNGs was controlled in the range of 40-120 nm by changing the concentration of CHPANG and PEGSH. A-CHPNG gradually degraded by hydrolysis under physiological condition and seemed to dissociate back to original nanogel. A-CHPNG encapsulated interleukin-12 (IL-12) efficiently (96%) and stably kept it in the presence of BSA (50 mg/ml). In addition, A-CHPNG had a high potential to maintain a high IL-12 level in plasma after subcutaneous injection in mice. Therefore, A-CHPNG is a promising carrier for long-term medications.

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.

Ectopic bone formation after joint replacement or brain injury in humans is a serious complication that causes immobility of joints and severe pain. However, mechanisms underlying such ectopic bone formation are not fully understood. Bone morphogenetic protein (BMPs) are defined as inducers of ectopic bone formation, and they are regulated by several types of inhibitors. ANA is an antiproliferative molecule that belongs to Tob/BTG family, but its activity in bone metabolism has not been known. Here, we examined the role of ANA on ectopic bone formation activity of BMP. In ANA-deficient and wild-type mice, BMP2 was implanted to induce ectopic bone formation in muscle. ANA deficiency increased mass of newly formed bone in vivo compared with wild-type based on 3D-muCT analyses. ANA mRNA was expressed in bone in vivo as well as in osteoblastic cells in vitro. Such ANA mRNA levels were increased by BMP2 treatment in MC3T3-E1 osteoblastic cells. Overexpression of ANA suppressed BMP-induced expression of luciferase reporter gene linked to BMP response elements in these cells. Conversely, ANA mRNA knockdown by small interference RNA enhanced the BMP-dependent BMP response element reporter expression. It also enhanced BMP-induced osteoblastic differentiation in muscle-derived C2C12 cells. Immunoprecipitation assay indicated that ANA interacts with Smad8. Thus, ANA is a suppressor of ectopic bone formation induced by BMP, and this inhibitory ANA activity is a part of the negative feedback regulation of BMP function.

Quantum dots (QDs) have attracted attention for their potential as a cell imaging regent. However, the development of effective intracellular delivery system for QDs is needed to apply various cell lines without affecting cellular function. We reported here new QDs delivery system by using cationic nanogel consisting of cholesterol-bearing pullulan modified with an amino group (CHPNH2). The uptake of hybrid nanoparticles into HeLa cells was followed by flow cytometry, and confocal laser scanning fluorescence microscopy. Protein-conjugated QDs were effectively internalized into cells by the nanogel compared with a cationic liposome system. The hybrid nanoparticle was used to stain rabbit mesenchymal stem cells (MSCs) so as to evaluate their effect on cell function. CHPNH2-QD hybrid nanoparticles remained detectable inside MSCs for at least 2 weeks of culture and had little effect on the in vitro chondrogenic ability of MSCs. The hybrid nanoparticles are a promising candidate as a cell tracer in tissue engineering.

For a valid cytokine immunotherapy of malignancies, a suitable delivery system that ensures slow-release of cytokines is required, because short half-life in vivo of the molecules ruins therapeutic efficacy while causing severe systemic toxic effects. We previously showed that the cholesterol-bearing pullulan (CHP)-based hydrogel nanoparticles, or nanogel, encapsulates, stabilizes and releases various molecules. Here we applied this nanogel to administration in vivo of interleukin-12 (IL-12). Recombinant murine IL-12 (rmIL-12) was successfully incorporated into CHP nanogel simply by incubated with CHP at room temperature. After subcutaneously injected into mice, the CHP/rmIL-12 complex led to a prolonged elevation in IL-12 concentration in the sera. Repetitive administrations of the CHP/rmIL-12, but not rmIL-12 alone, induced drastic growth retardation of preestablished subcutaneous fibrosarcoma without causing any serious toxic event. The present study proposes a novel therapeutic intervention technology, taking advantage of slow and sustained release of bioactive cytokines from the self-assembling biocompatible nanoparticles.

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.)

Nanogels of cholesterol-bearing pullulan modified with amino-groups (CHPNH(2)) were utilized as a career to introduce quantum dots (QDs) into periodontal ligament (PDL) cells. Nanogel-QD effectively labeled PDL cells over 3 weeks. These results indicate that CHPNH(2)nanogels have a potential as a research tool in the studies of intracellular delivery system.

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2). The CHPNH2-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.