Controlled Dissociation of Polymeric Micelles in Response to Oxidative Stress.

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.

Manganese Porphyrin-Containing Polymeric Micelles: A Novel Approach for Intracellular Catalytic Formation of Per/Polysulfide Species from a Hydrogen Sulfide Donor.

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 H2 S donor and manganese porphyrin-containing polymeric micelles (MnPMCs) that catalyze oxidization of H2 S to per/polysulfide species. MnPMCs serve as a catalyst for H2 S oxidation in aerobic phosphate buffer. HPLC-MS/MS analysis reveals that H2 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 H2 S donor anethole dithiolethione and MnPMCs increases intracellular per/polysulfide levels and induces a proangiogenic response. Co-delivery of MnPMCs and an H2 S donor is a promising approach for controlled delivery of polysulfides for therapeutic applications.

Recent advance in self-assembled polymeric nanomedicines for gaseous signaling molecule delivery.

Gaseous signaling molecules such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2 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.

Functionalization of Framboidal Phenylboronic Acid-Containing Nanoparticles via Aqueous Suzuki-Miyaura Coupling Reactions.

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 (H2S)-releasing compound anethole dithiolone to carboxylic acid-functionalized BNPs and show their H2S-releasing capability in cell lysate.

Reactive Oxygen Species-Triggered Hydrogen Sulfide Release and Cancer-Selective Antiproliferative Effect of Anethole Dithiolethione-Containing Polymeric Micelles.

Hydrogen sulfide (H2 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 H2 S to cancer cells for extended periods of time suppresses cancer progression. However, one major challenge in therapeutic applications of H2 S is its controlled delivery. To solve this problem, polymeric micelles are developed containing H2 S donating-anethole dithiolethione (ADT) groups, with H2 S release profiles optimal for suppressing cancer cell proliferation. The micelles release H2 S upon oxidation by reactive oxygens species (ROS) that are present inside the cells. The H2 S release profiles can be controlled by changing the polymer design. Furthermore, the micelles that show a moderate H2 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 H2 S release profiles using a micelle approach for realizing the full therapeutic potential of H2 S in cancer treatment.

Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins.

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.

Thioether-Based Polymeric Micelles with Fine-Tuned Oxidation Sensitivities for Chemotherapeutic Drug Delivery.

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 (H2O2). 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 H2O2 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 H2O2, 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.

Framboidal Nanoparticles Containing a Curcumin-Phenylboronic Acid Complex with Antiangiogenic and Anticancer Activities.

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.

Mitochondria-Targeting Polyamine-Protoporphyrin Conjugates for Photodynamic Therapy.

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.

Data in support of preparation and functionalization of a clickable polycarbonate monolith.

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 13C NMR spectroscopy as well as thermogravimetric analysis.

Polymeric Framboidal Nanoparticles Loaded with a Carbon Monoxide Donor via Phenylboronic Acid-Catechol Complexation.

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.

Furoxan-Bearing Micelles for Nitric Oxide Delivery.

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.

Mannose-displaying fluorescent framboidal nanoparticles containing phenylboronic acid groups as a potential drug carrier for macrophage targeting.

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.

Back Cover: Macromol. Biosci. 11/2015.

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.

NMR spectra and electrochemical behavior of catechol-bearing block copolymer micelles.

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)).

Hydrolysis-Sensitive Dithiolethione Prodrug Micelles.

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.

Inhibition of angiogenesis by antioxidant micelles.

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.

Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis.

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.

Preparation of poly(γ-glutamic acid)/hydroxyapatite monolith via biomineralization for bone tissue engineering.

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.

Design and synthesis of polymeric hydrogen sulfide donors.

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.