Redox Polyion Complex Micelle-Based Injectable Hydrogel as Local

Mar 20, 2019 - Since PIC-based hydrogel possesses a nitroxide radical as a catalytic ROS scavenger covalently conjugated in the polymer backbone, ...
0 downloads 0 Views 1MB Size
Download PDF
Downloaded via BETHEL UNIV on March 31, 2019 at 15:41:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Chapter 12

Redox Polyion Complex Micelle-Based Injectable Hydrogel as Local Reactive Oxygen Species Scavenging Therapeutics Long Binh Vong1,2 and Yukio Nagasaki*,2,3,4 1Department of Biochemistry, Faculty of Biology and Biotechnology, University of Science, Vietnam National University Ho Chi Minh City, Ho Chi Minh City, 702500, Vietnam 2Department of Material Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8573, Japan 3Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan 4Center for Research in Isotopes and Environmental Dynamics, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan *E-mail: [email protected].

The overproduction of reactive oxygen species (ROS) induces oxidative stress on functional biomolecules, leading to numerous human diseases, including inflammation, aging, diabetes, neurodegenerative disease, myocardial infarction, and cancer. The regulation of local ROS level and redox equilibrium plays a critical role in preventing and curing biological disorders. Various endogenous redox enzymes are balanced to maintain physiological hemostasis in vivo, but this balance is lost when ROS is excessively produced, leading to strong oxidative stress and diseases. Not only natural vitamins C and E but also many antioxidants are synthesized to eliminate excessively produced ROS. However, most antioxidants have been largely ineffective in clinical settings, due to their low stability and nonspecific and short activity in physiological conditions. One of the other important problems of these low-molecular-weight antioxidants is that

© 2019 American Chemical Society Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

they cause the dysfunction of normal cells by destroying normal redox reactions such as an electron transport chain after their nonspecific cellular internalization. In order to suppress these types of adverse side effects, we have been developing polymer antioxidants, which prevent their internalization in healthy cells to protect important normal redox reactions. Antioxidant polymers composed of an amphiphilic block copolymer self-assemble in water to become redox nanoparticles (RNPs). To date, we have proven that RNPs exert a high therapeutic efficacy on diseases strongly dependent on various oxidative stresses, including ulcerative colitis, Alzheimer;s disease, ischemia reperfusion injuries such as brain, heart, kidney, and intestinal ischemia reperfusion, and cancer. More recently, we started to develop new antioxidant nanomedicines for local diseases related to oxidative stress. For these objectives, we designed novel redox polyion complex micelles (PIC) that form irreversible temperature-responsive hydrogels at body temperature for local therapies. Since PIC-based hydrogel possesses a nitroxide radical as a catalytic ROS scavenger covalently conjugated in the polymer backbone, it exhibits therapeutic function by regulating the local redox equilibrium. After conversion to a hydrogel, it continuously eliminates ROS locally and suppresses rapid diffusion at injected sites. In this chapter, we describe the design, preparation, and characterization of developed redox injectable hydrogel and its medical applications for arthritis, periodontal disease, local anesthesia, as well as its application as an anti-adhesive gel and its sustained release of bioactive molecules in other medical therapies.

Injectable Hydrogel and Biomedical Applications Hydrogels have recently received great attention in polymer and material sciences for biomedical applications. Basically, hydrogels are materials composed of synthetic and/or natural polymers to form cross-linked networks with three-dimensional structures. Hydrogels are also known as soft materials that contain a large amount of water in their matrix, exhibiting excellent biocompatibility due to their physical similarity to human tissue (1, 2). In the 1980s, one of the initial and important studies developed microencapsulated islets as bioartificial pancreas using a calcium alginate hydrogel to improve streptozotocin-induced diabetes in rats (3). For many years, the versatile applications of hydrogels in biomedical fields have emerged, including their use as biosensors, in tissue engineering, and in drug and cell delivery (4, 5). Basically the hydrogels are not homogenous and can be divided into chemical hydrogels and physical hydrogels, as shown in Figure 1. The former hydrogels are typical hydrogels formed by the chemical cross-linking of water-soluble polymers using 288 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

various cross-linkers, which can be incorporated into a proteolytically degradable sequence to improve their degradability. Several approaches have been used for chemical hydrogel preparation, including enzymatic cross-linking, Schiff base cross-linking, Michael addition, click chemistry, and photo-induced cross-linking methods (6). Since these chemical cross-linking agents are usually toxic to cellular organisms and induce immune responses, they must be removed completely from the prepared hydrogels. Alternatively, physical hydrogels are generated without covalent cross-linking, but physical cross-linking such as crystallization, hydrogen and van der Waals bonds and hydrophobic and electrostatic interactions can also be utilized to stabilize the hydrogel matrix (4), as shown in Figure 1.

Figure 1. Schematic of versatile fabrications of chemical hydrogels (a) and physical hydrogels (b).

The hydrogels are particularly composed of polymers having high water solubility and/or water affinity. Many natural polymers or biopolymers have been reported for hydrogel fabrication. For example, polysaccharides such as chitosan, chondroitin sulfate, hyaluronic acid, alginate, agarose, and dextran have been widely used in biomaterials for tissue engineering and drug delivery systems (7). In addition, the fibrous proteins such as collagens and their extract (gelatin) are also used to prepare hydrogels for tissue engineering because of their adhesive properties with cells (8, 9). Although these natural polymers exhibit high biocompatibility as well as biodegradability, further improvements are necessary for optimization. In order to promote their use in biological systems, improvement of their mechanical strength is required. Chemical cross-linking of these polymers is one important way to solve these problems. Recently, 289 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

“stimuli-responsive hydrogel” has attracted attention as another important characteristic of the gel matrix to develop in biomedical fields. In 1978, Toyoichi Tanaka reported on the concept of the “volume-phase transition” of polymeric gels composed of stimuli-responsive polymers, which have a coil-globule phase transition in response to the environmental stimuli such as pH, temperature, ion strength, light, and specific biomolecules (10). Since then, versatile applications have been reported by these stimuli-responsive hydrogels. Among them, temperature is one of the most useful stimuli, especially in vivo. Many types of temperature-responsive polymers are designed and used as the main component of the hydrogels. For example, poly(N-isopropylacrylamide) (PNIPAAM) possesses a lower critical solution temperature (viz., it is soluble under the low temperature and precipitates at the elevated temperature). Several other polymers such as poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) and poly(N,N-diethylacrylamide) also exhibiting a lower critical solution temperature have been widely used as injectable biodegradable hydrogels since the transition point of these polymers is around body temperature (37 °C); thus, they are useful for temperature-responsive drug carriers and injectable hydrogels (11). Biocompatibility is one of the most important issues of chemical hydrogel materials to the cell and human body. Unlike natural polymer-forming hydrogels exhibiting high biocompatibility, it has been reported that synthetic polymers may induce an inflammatory response after injection. PNIPAAM-based hydrogels caused a chronic inflammatory response in the muscle injury by the induction of CD8+ T cell infiltration and increased class 1 major histocompatibility complex-positive regenerating myofibers (12). PLGA is a popular biodegradable polymer approved by the United States Food and Drug Administration; however, the release of lactic acid and glycolic acid from PLGA may lead to lower pH levels and local inflammation (13). Improvement of the biocompatibility of chemical hydrogel-forming polymers is required to minimize immune response and toxicity after injection. Injectable hydrogel, where a key biomaterial can be injected as a solution and form a solid gel in the tissue, has been widely used in biomedical applications, particularly cell-based tissue engineering, regenerative medicine, and drug/protein delivery systems (8, 14, 15). Conventional stem cell transplantation is a promising approach for tissue healing and regeneration to improve tissue damage and organ functions; however, most transplanted cells lose their stemness property and exhibit low viability and low retention at the targeted sites after injection to the body due to the mechanical disruption of cells during injection and harsh microenvironments at injected sites (16). Hydrogels possess polymeric networks that closely mimic the native extracellular matrix to maintain cellular properties and prolong the cellular viability and retention at targeted tissues. For example, hyaluronan and methylcellulose-based hydrogels were reported to significantly improve the survival and distribution of neural stem cells in the adult brain after transplantation (17). In addition, it has been reported that tunable three-dimensional microenvironments and the mechanical properties of hydrogels modulate the cellular condensation and stem cell differentiation as well as cellular morphology (18, 19). Injectable gels can be also utilized for drug encapsulation and controlled release from an injected local site. Conventional 290 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

drug treatments require high doses and repeated administration to achieve the desired therapeutic efficacy; this leads to an inconvenience and complications for patients and possible adverse effects due to the nonspecific drug distribution through the entire body. Injectable hydrogels are capable of incorporating large amounts of hydrophilic drugs and other water-soluble bioactive molecules, which can be controllably and sustainably released at diseased tissues specifically (20–22). For instance, the pH-responsive hydrogel composed of N-carboxyethyl chitosan and dibenzaldehyde-terminated PEG was developed for the delivery and controlled release of doxorubicin for cancer therapy (23). Shibata et al. developed glucose-responsive fluorescence that immobilized in poly(acrylamide)-based injectable hydrogel beads for continuous blood glucose monitoring (24). In this design, the glucose-responsive fluorescent monomer was composed of a glucose-recognition site, fluorescent probe, spacer, and polymerizable group. In another study, Sorrano et al. developed a local sustained delivery system of interleukin-10 (IL-10) using a hyaluronic acid-based injectable hydrogel, effectively alleviating the local and systemic inflammation induced in acute kidney injury (25). Wang et al. described the microRNA-based therapy using a hyaluronic acid injectable hydrogel for local and sustained miRNA-302 delivery to improve the outcome in cardiomyocyte proliferation (26). Purcell et al. developed a polysaccharide-based hydrogel system for the delivery and release of a recombinant tissue inhibitor of matrix metalloproteinase in response to enzyme activity to attenuate the adverse left ventricular remodeling in a cardiovascular disease porcine model (27).

Introduction of Reactive Oxygen Species and Oxidative Injuries It has been reported that reactive oxygen species (ROS) are important intracellular signaling molecules in homeostasis (28). ROS, highly reactive chemical molecules containing oxygen such as singlet oxygen, hydrogen peroxide, superoxide, and hydroxyl radicals, are physiologically generated through reactions in the electron transport chain on the mitochondria membrane to produce adenosine triphosphate, essential energy-carrying molecules. In addition, these oxidants have been reported to simulate the secondary protective response in the host organism against oxidative damages (29). Therefore, maintaining these important ROS is critical to maintain the redox hemostasis and normal physiological conditions. However, the overproduction of ROS causes an increase in oxidative stress and damages to biomacromolecules, including proteins, lipids, and nucleic acids. Thus, the accelerating accumulation of ROS production is responsible for numerous human diseases such as inflammation (30), aging (31), infection (32), cardiovascular disease (33), neurodegeneration (34), and cancer (35). Antioxidants have been used to counteract the toxic overproduction of ROS to prevent and treat these oxidative stress-related diseases. Several antioxidant drugs were approved for clinical use in various diseases such as edaravone for ischemic stroke, N-acetylcysteine for acetaminophen overdose-induced liver injury, and α-lipoic acid for diabetic neuropathy (36). However, many antioxidants or ROS scavengers such as vitamin A, vitamin C, N-acetylcysteine, or glutathione 291 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

have been used in numerous preclinical studies presenting negative efficacy in vivo, although their effectiveness is clearly shown in in vitro conditions. It can also be explained that when these low-molecular-weight (LMW) antioxidants are administered, they spread nonspecifically to the entire body and are rapidly metabolized, leading to low bioavailability and low activity at the target sites. In addition, LMW antioxidants facilely internalize into the healthy cells and may interrupt the important electron transfer reactions in mitochondria, leading to unwanted adverse effects (37). Consequently, most antioxidants have failed in clinical practices. Therefore, improvements in the bioavailability, stability, and diseased specificity of antioxidants need to be addressed to achieve higher antioxidant therapeutic efficacies.

The Design of Redox Injectable Hydrogel for Local ROS Scavenging Stable nitroxide radicals such as 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) have recently attracted much attention because their molecular structures are capable of counteracting with free radicals as strong antioxidants. In addition, nitroxide radicals possess an impair electron, which can be monitored by electron spin resonance (ESR) as a biophysical tool. For biomedical applications, these nitroxide radicals have been studied and showed great potential in radioprotection, functional imaging, antioxidants, and anticancer treatments (38, 39). Under physiological environments, however, treatment of these LMW nitroxide radicals exhibits a low therapeutic window because of many issues, including preferential renal clearance, rapid metabolism to the low activity hydroxylamine form, and nonspecific distribution to normal tissues, leading to adverse effects, as described above. To overcome the issue of LMW compounds, we previously developed redox nanoparticles (RNPs), prepared by an amphiphilic block copolymer (A–B type) containing stable nitroxide radicals in the hydrophobic segment as a side chain. Nitroxide radicals can be covalently conjugated to a hydrophobic chain via an amine linkage to form a nanoparticle with a pH-sensitive character (RNPN) or via ether linkage to form a nanoparticle with a pH-insensitive character (RNPO), as shown in Figure 2. By simply self-assembling in the physiological environment, the amphiphilic copolymer forms the core-shell–type micelle structure with a diameter of approximately several tens of nanometers, in which nitroxide radicals are confined in the core (40). For several years, we have investigated the antioxidant therapies of RNPs in oxidative stress injury models in mice such as intestinal inflammation (41, 42), renal failure (43), liver injury (44, 45), neurodegenerative diseases (46, 47), and cancer (48). Our results showed that RNPs presented significantly higher therapeutic efficacy in these diseased mice models when compared to LMW nitroxide radicals. It should be noted that RNP does not cause serious damage to normal cells, tissues, and organs, as it is not easily internalized into normal cells, unlike LMW antioxidants. It is interesting that the combination of RNPs with conventional chemotherapies such as doxorubicin, irinotecan, and pioglitazone showed the synergistic efficacy in several cancer model mice, while 292 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

simultaneously minimizing the adverse effects of these chemotherapies (49–51). RNPs not only could improve the bioavailability of conventional antioxidants but also could suppress the oxidative degradation of antioxidants such as curcumin (52). However, RNPs may not be suitable for local antioxidant therapies because of their high colloidal dispersion characteristics and because they are hard to retain in local sites. Therefore, a novel system must be developed for the effective treatment of local antioxidant therapies.

Figure 2. Redox nanoparticles as ROS-scavenging nanomedicine. Reproduced with permission from reference (50). Copyright 2016 American Chemical Society. In order to overcome the issue of RNPs, we recently designed and developed a novel redox injectable hydrogel (RIG) to locally scavenge ROS at diseased tissues. The RIG was prepared from polyion complex (PIC) micelles, which consist of nitroxide radicals containing the A–B–A type triblock copolymer poly[4(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene]-b-poly(ethylene glycol)-b-poly[ 4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PMNT-b-PEG-b-PMNT) and polyanionic polymers such as poly(acrylic acid) (PAAc) and chondroitin sulfate (CS) (Figure 3). Polycationic PMNT-b-PEG-bPMNT possesses nitroxide radical TEMPO moieties as ROS scavengers, while polyanionic PAAc and CS are well-known biodegradable polymers. By simply mixing the polycation and polyanion, the flower-like PIC micelles were formed with a diameter of approximately several tens to a hundred nanometers in a solution at room temperature (Figure 3). When the ionic strength and temperature increased, the viscosity of the PIC solution dramatically increased by four orders of magnitude, and the sol-gel transition temperature (where storage modulus [G’] and loss modulus [G”] became the same, G’ = G”) occurred at around 30 °C due to the destabilization of PIC flower micelles via increased thermal motion energy, and both moduli (G’ and G”) did not change when the temperature decreased, indicating irreversible gel formation (Figure 3). On the contrary, the conventional A–B–A-type triblock copolymers, possessing the hydrophobic A-segments and the hydrophilic B-segment, formed a temperature-responsive reversible gel due 293 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

to the hydrophobicity-induced aggregation (53). In the RIG system, a partial disintegration of the PIC core due to the elevated ionic strength might contribute to the irreversible cross-linking between the PIC micelles via both electrostatic cross-linking and hydrophobic interaction.

Figure 3. Schematic illustration of RIG for scavenging local ROS. Reproduced with permission from references (54) and (55). Copyright 2013 and 2018 Elsevier B.V.

PMNT-b-PEG-b-PMNT was simply synthesized on a large scale using a two-step protocol. In the first step, the poly(chloromethylstyrene)b-PEG-b-poly(chloromethylstyrene) (PCMS-b-PEG-b-PCMS) triblock copolymer was synthesized by reversible addition-fragmentation chain-transfer polymerization. In the second step, the nitroxide radical amino-TEMPO could be easily covalently conjugated to the PCMS hydrophobic segment to obtain PMNT-b-PEG-b-PMNT triblock polymers. By possessing an amino group, 294 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

pH-responsive PMNT-b-PEG-b-PMNT could be partially protonated under a neutral to acidic pH to produce positive charge complexing with anionic polymers forming a redox flower micelle via electrostatic interaction. In addition, the hydrophobicity of PMNT segments can also stabilize the forming hydrogel via hydrophobic interaction. For the design, our redox PIC flower micelle was composed of: (1) hydrophilic PEG loops layered as an outer shell to prevent aggregation among micelles for enhancing the injectability as a free-flowing solution even at high concentrations, (2) a polyion core as a nanoreservoir for charged drugs/proteins, and (3) nitroxide radicals with ROS-scavenging capacity covalently conjugated into the core of PIC. Under physiological conditions, the flower micelles are expected to collapse, rearrange, and form a physically cross-linking network among micelles, which contributes to in situ gelation and thus leads to long-term retention of the drug after the subcutaneous injection at a local site. In this chapter, we describe the local therapies using RIG for several oxidative injury model mice.

Medical Applications of Redox Injectable Hydrogel Inflammation and Periodontal Disease (54, 56) Local inflammation is characterized by a high infiltration of immune cells and an overproduction of ROS, which lead to systemic diseases. As described above, the retention of antioxidants at the injury site is one of the most critical factors to achieve the effective ROS scavenging at the focused local site. Nitroxide radical TEMPO not only has the antioxidant potential but can also be measured by ESR quantitatively. Furthermore, the local retention time of RIG, where TEMPO is covalently conjugated into the polymer network, can be observed noninvasively using in vivo imaging by L-band ESR, which allows real-time monitoring in clinical use. It should be noted that LMW TEMPO shows high signal intensity at the early stage after injection in the mice’s hind paws, and it rapidly decreases just 30 min after injection. In contrast, the signal intensity of RIG was initially weak (probably due to the quench in the gel matrix) but gradually increased and remained 40% even three days after injection, indicating the enhanced local retention time of nitroxide radicals by RIG treatment. We next investigated the local inflammatory response of the injectable hydrogel at injected sites. The injection of nRIG (non-redox injectable hydrogel without ROS-scavenging activity) itself induced an immune response and severe inflammation at the local injected site, while RIG treatment did not exhibit significant difference when compared to untreated mice. As mentioned above, the biocompatibility is one of the most important properties of hydrogel for biomedical treatment, and this is characterized by physical and chemical properties, biodegradability, and stability (11, 57). Although nRIGs are mainly composed of highly biocompatible PEG and polystyrene, the elevation of immune response was observed in nRIG treatment, while RIG treatment did not significantly stimulate the inflammatory response. This data indicates that the ROS-scavenging capacity of RIG plays an important role in suppressing inflammatory responses, improving its biocompatibility. When a mouse model of carrageenan-induced arthritis disease was used to 295 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

investigate the effect of RIG in suppressing inflammation, the carrageenan was injected into the right hind paw to cause local inflammation where the production of pro-inflammatory cytokines such as myeloperoxidase (MPO), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α) were elevated. We found that the treatment of RIG significantly suppressed the production of these pro-inflammatory cytokines in carrageenan-induced arthritic mice, while the nRIG treatment induced the severe inflammation not only in local injected sites but also in the entire body. It was also confirmed that LMW amino-TEMPO and RNPN exhibited low therapeutic treatment due to their rapid diffusion from the local injected site. The data indicates the effectiveness of RIG treatment in reducing the local inflammation that related to oxidative stress. In another study, RIG was locally injected into a periodontal pocket to treat periodontitis in model rats. Interestingly, by simply scavenging ROS in the periodontal pocket, RIG could recover the gingival blood flow, inhibit the P. gingivalis-induced bone loss, and suppress the differentiation of osteoclast in vitro and in vivo in rat models. RIG presents a promising approach for local inflammation treatment.

Figure 4. Several biomedical applications of RIG have been investigated so far.

RIG for Anti-Adhesion (58) Tissue adhesion is a serious issue for patients after abdominal surgeries, and it requires readmission and reoperation, leading to a low quality of life (59). Postoperative peritoneal adhesion is strongly related to oxidative stress caused during surgical procedures, and the consequences of tissue adhesion include chronic intestinal inflammation, pelvic pain, and even infertility (60–62). 296 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

To prevent postsurgical adhesions, several materials have been developed as physical barriers to separate traumatized tissue from other surrounding tissues. For example, a treatment of pure alginate gel showed the efficacy to suppress the incidence of postoperative adhesion in a rat model (63). Several other injectable hydrogels prepared from carboxymethyl chitosan with aldehyde-installed hyaluronic acid, or methyl cellulose, have been studied to prevent the postsurgical peritoneal adhesion (64–66). However, these materials are simply physical barriers, and they are not able to effectively prevent inflammation, which may cause adhesion relapse. To develop an innovative anti-adhesion agent, an injectable hydrogel with antioxidant potential would be a great material for postsurgical adhesion. Therefore, in this study, we investigated the anti-adhesion effect of the RIG system, which functions not only as a physical barrier hydrogel but also as a suppressor of the inflammation by ROS scavenging on talc-induced adhesion model mice. It is well known that the intraperitoneal injection of talc causes severe inflammation, resulting in adhesion in the peritoneum (67, 68). Seprafilm from Kaken Pharmaceutical Company, Ltd. (Tokyo, Japan) was used as an available anti-adhesion material. Our results showed that the effect of an LMW TEMPO treatment was insufficient to suppress the inflammation due to its rapid diffusion from local treatment sites. nRIG can be used as a spray for the treatment area and showed almost the same level of commercially available Seprafilm, indicating that they are at the same level as physical barriers of organs, which prevent adhesion to each other. However, some mice still suffered from peritoneal adhesion after the treatment. On the contrary, the adhesion from mice treated with RIG significantly decreased. It was confirmed that only the local administration of RIG effectively suppresses superoxide levels and the inflammatory mediators such as TNF-α, interleukin-6 (IL-6), and MPO in talc-treated mice when compared to other treatments in addition of the physical separation of organs from each other. Furthermore, the fibrin and collagen formation in the peritoneal membrane were significantly decreased in RIG treatment, while a massive amount of these extracellular matrices was observed in Seprafilm and other treatments. This result indicates that the RIG system exhibits a combination of the physical separation of gel property and ROS-scavenging capacity to suppress inflammation; thus, RIG presents a great innovation as a high-performance anti-adhesion biomaterial. Bioactive Molecule Entrapment for Local Anesthetizers and Cancer Immunotherapy (69–71) Because our RIG possesses a hydrophobic core, which is composed of an opposite-charge polymeric complex, we have confirmed the encapsulation of LMW drugs, peptides, and proteins for high-performance local delivery systems. Several examples are described in this section. To relieve the pain in surgery and minimize injury, versatile anesthetic drugs have been developed (72, 73). Because these anesthetics generally display a short duration of action, continuous administration of the drug is required, which decreases quality of life. Several drug delivery nanocarriers and injectable responsive hydrogels have been investigated for sustained drug release to achieve prolonged anesthesia (74). However, most of them did not show a remarkable effect. Because most 297 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

local anesthetic drugs possess an amino group and tend to protonate in slightly acidic injured tissue, which decreases the internalization of the drug into the cell, they have a limited anesthetic efficiency (75). Since RIG eliminates ROS and suppresses local inflammation, we anticipate it can reduce the protonation of the anesthetic drugs by preventing a decrease in pH. An anesthetic drug, lidocaine-incorporated RIG (Lido@RIG), was applied to the carrageenan-induced inflammation in mice’s hind paws; we evaluated the efficacy using the paw withdrawal threshold (PWT) via a von Frey test. In the mice treated with carrageenan, PWT decreased significantly due to the inflammation-induced pain. The PWT in the treatment of lidocaine-loaded nRIG (control injectable hydrogel prepared from conventional polyamine-PEG-polyamine without ROS-scavenging nitroxide radicals) also decreased significantly and did not recover in three days. In contrast, a decrease in PWT from mice treated with lidocaine-loaded RIG was not observed, indicating that the suppression of inflammation by RIG could prolong the therapeutic effect of lidocaine in reducing the patient’s pain. For cancer immunotherapy, molecular-targeting bioactive proteins have been attractive to stimulate the patient’s own immune system against cancer cells. However, these biomolecules are not stable under physiological environments such as degradation by enzyme and acidic pH, leading to the reduction of their bioactivity before reaching targeted cells. Furthermore, the bioactive proteins in cancer immunotherapy have been reported to induce severe adverse effects. For examples, the administration of interleukin-12 (IL-12) resulted in an increased concentration of interferon gamma (IFN-γ) to cause systemic toxicities, including death in a clinical trial (76–78). Since the electrostatic interaction between PMNT-b-PEG-b-PMNT polycation and PAAc polyanion is the main driving force to form PIC micelles, RIG presents a high potential to incorporate charged biomolecules such as protein and peptide for a sustained release. In fact, we have confirmed the encapsulation capacity of RIG using various proteins with different molecular weights and charges. The results of increased size, shielding in surface charge, and confirmed fluorescent resonance energy transfer clearly indicate the successful incorporation of different types of proteins into the core of PIC micelles. It was confirmed that protein-loaded RIG (bovine serum albumin was used as a protein model) prolonged the retention of protein for two weeks after subcutaneous injection, while the protein was rapidly diffused and completely disappeared 24 hours after the injection of the naked protein. IL-12–loaded RIG was also prepared stably, and its local administration was employed for cancer immunotherapy using the tumor-bearing mice model. The results showed that IL-12–loaded RIG significantly inhibited the tumor growth when compared to IL-12 alone, although the dose of IL-12 was twice less. In addition, the IL-12–loaded RIG treatment did not show any noticeable toxicity while systemic inflammation and liver injury were observed in mice treated with IL-12 alone. This data suggests that the improved retention and sustained release of IL-12 by RIG with ROS-scavenging capacity significantly suppressed the tumor progression and adverse effects of IL-12. Based on these data, RIG presents a promising approach as a drug and biomolecule carrier for medical applications.

298 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

RIG Improves Nitric Oxide Generation for Myocardial Infarction Therapy (55) Cardiovascular disease is the leading cause of death worldwide; however, the effectiveness of current therapies is limited. To improve the angiogenesis and cardiac repair, the use of injectable hydrogel to deliver cells and biomolecules has recently garnered attention as a promising approach (79). Stem cell-encapsulated dextran-g-poly(ε-caprolactone)-2-hydroxylethyl methacrylate/PNIPAAm-based or oligoPEG fumarate-based hydrogels significantly increased stem cell retention and viability in myocardium to improve heart function and revascularization after infarction (80, 81). The local treatment of methacrylated gelatin hydrogel composed of poly(ethylenimine) functionized graphene oxide complexing with the vascular endothelial growth factor proangiogenic gene significantly increased myocardial capillary density and reduced scar area in the infarcted heart (82). Our strategy is to develop the nitric oxide (NO) for angiogenesis therapy in the treatment of myocardial infarction (MI). NO, a soluble gas continuously synthesized by endothelium, plays an important role in the modulation of angiogenesis and in the protection against the onset and progression of cardiovascular disease (83). Basically, NO is generated from L-arginine by a family of NO synthases (NOS), including endothelial NOS (eNOS) from endothelial cells, neuronal NOS (nNOS) from neuronal cells, and inducible NOS (iNOS) mainly from macrophages. NO and NOS systems act as key regulators of cardiac function and angiogenesis after infarction (84). Due to an extremely short half-life and low bioavailability, the therapeutic use of NO in cardiovascular disease is limited. In addition, overproduced ROS (in particular, superoxide anions) at diseased tissues rapidly interact with NO to form other nitrogen reactive species such as peroxynitrite, which further induces myocardial injury and reduces NO level (85, 86). It has been reported that antioxidant/ROS-scavenger and L-arginine treatments increase NO production to improve vascularization (87, 88). Recently, we developed a nanoparticle-assisted L-arginine delivery system using PEG-b-poly(L-arginine) for NO-based cancer therapy, and the results clearly showed the tumor progression and antitumor activities are investigated depending on PEG-b-poly(L-arginine) doses (89). Tumor progression was observed with the low-dose treatment of PEG-b-poly(L-arginine), while the high-dose treatment induced tumor apoptosis and suppression, which was strongly related to the NO generation level (90, 91). At the inflammation and tumor tissues, there was an elevated infiltration of activated macrophages (92), expressing a high activity of iNOS to catalyze the L-arginine monomer to NO. However, LMW L-arginine is rapidly metabolized and easily spread through the entire body after administration, resulting in low bioavailability and low therapeutic efficacy. For the local sustained release of NO for myocardial infarction therapy, we designed a triblock copolymer poly(L-arginine)-b-PEG-b-poly(L-arginine) (PArgb-PEG-b-PArg)-based injectable hydrogel by complexing with a PAAc polyanion in combination with the ROS-scavenging PMNT-b-PEG-b-PMNT polymer. Our hypothesis is that PArg-b-PEG-b-PArg can be hydrolyzed by macrophage protease to sustainably produce the L-arginine monomer as the substrate of iNOS for continuous NO generation, while the PMNT-b-PEG-b-PMNT-possessing 299 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

nitroxide radical can scavenge overproduced ROS at the injected site to suppress the inflammation and maintain the NO bioavailability. This injectable hydrogel presented two biofunctional properties, including NO-releasing activity and ROS-scavenging activity, so-called NO-RIG. When compared to free polymers, whose retention was only a few hours, NO-RIG prolonged the retention of polymers in myocardium for 10 days after intramyocardial injection. Interestingly, the treatment of NO-RIG significantly induced the NO generation and neovascularization as compared to other controls such as single NO-releasing or single ROS-scavenging injectable hydrogels. As a result, NO-RIG effectively improved cardiac functions and reduced the infarcted size in the ligation of left anterior descending artery models in mice, while other control treatments did not show any effectiveness at all. This result suggests that NO-RIG will be a promising controllable NO-releasing hydrogel for the treatment of heart failure by angiogenesis therapeutics.

Conclusions and Future Perspectives In this chapter, we described an overview of injectable hydrogel use in biomedical applications. Versatile injectable hydrogel systems were introduced to deliver and sustainably release drugs/biomolecules as drug delivery systems, or to maintain normal cellular properties and cell viability as cell delivery in tissue engineering and regenerative medicine. We also described our design and development of RIG, which is an ROS-scavenging hydrogel, as polymeric therapeutics in the local treatment of inflammation, anti-adhesion, cardiovascular disease, etc. (Figure 4). It should be emphasized that our developed RIG itself presented the therapeutic activity without the entrapment of conventional drugs and biomolecules due to the ROS-scavenging capacity of hydrogel-forming polymers, including PMNT-b-PEG-b-PMNT and PArg-b-PEG-b-PArg. Therefore, RIG is a promising injectable hydrogel for the delivery of drugs and biomolecules; furthermore, through ROS-scavenging activity, RIG can effectively maintain the cellular properties, in particular maintaining the stemness and viability of stem cells for regenerative medicine.

Acknowledgments A part of this work was supported by Grant-in-Aid for Scientific Research A (21240050) and Grant-in-Aid for Research Activity Start-up (22800004) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. This work was also partly supported by Young Scientist B (16K16397) from MEXT of Japan, and Vietnam National Foundation for Science and Technology Development (NAFOSTED, under Grant number 108.05-2017.327) to L. B. Vong.

300 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

References 1. 2.

3. 4. 5.

6.

7. 8. 9.

10. 11. 12.

13.

14. 15. 16.

17.

Seliktar, D. Designing Cell-Compatible Hydrogels for Biomedical Applications. Science 2012, 336, 1124–1128. Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165–13307. Lim, F.; Sun, A. M. Microencapsulated Islets as Bioartificial Endocrine Pancreas. Science 1980, 210, 08–910. Hoffman, A. S. Hydrogels for Biomedical Applications. Adv. Drug Delivery Rev. 2002, 54, 3–12. Caló, E.; Khutoryanskiy, V. V. Biomedical Applications of Hydrogels: A Review of Patents and Commercial Products. Eur. Polym. J. 2015, 65, 252–267. Liu, M.; Zeng, X.; Ma, C.; Yi, H.; Ali, Z.; Mou, X.; Li, S.; Deng, Y.; He, N. Injectable Hydrogels for Cartilage and Bone Tissue Engineering. Bone Res. 2017, 5, 17014. Vermonden, T.; Censi, R.; Hennink, W. E. Hydrogels for Protein Delivery. Chem. Rev. 2012, 112, 2853–2888. Lee, C. H.; Singla, A.; Lee, Y. Biomedical Applications of Collagen. Int. J. Pharm. 2001, 221, 1–22. Yue, K.; Trujillo-de Santiago, G.; Alvarez, M. M.; Tamayol, A.; Annabi, N.; Khademhosseini, A. Synthesis, Properties, and Biomedical Applications of Gelatin Methacryloyl (GelMA) Hydrogels. Biomaterials 2015, 73, 254–271. Tanaka, T. Collapse of Gels and the Critical Endpoint. Phys. Rev. Lett. 1978, 40, 820–823. Klouda, L.; Mikos, A. G. Thermoresponsive Hydrogels in Biomedical Applications. Eur. J. Pharm. Biopharm. 2008, 68, 34–45. Zhu, R.; Wu, G.; Liu, X.; Shi, D.; Cao, B.; Gu, R.; Xiao, J.; Liao, H. PNIPAM Hydrogel Induces Skeletal Muscle Inflammation Response. RSC Adv. 2015, 5, 28023–28029. Athanasiou, K. A.; Niederauer, G. G.; Agrawal, C. M. Sterilization, Toxicity, Biocompatibility and Clinical Applications of Polylactic Acid/Polyglycolic Acid Copolymers. Biomaterials 1996, 17, 93–102. Balakrishnan, B.; Banerjee, R. Biopolymer-Based Hydrogels for Cartilage Tissue Engineering. Chem. Rev. 2011, 111, 4453–4474. Hoare, T. R.; Kohane, D. S. Hydrogels in Drug Delivery: Progress and Challenges. Polymer 2008, 49, 1993–2007. Aguado, B. A.; Mulyasasmita, W.; Su, J.; Lampe, K. J.; Heilshorn, S. C. Improving Viability of Stem Cells during Syringe Needle Flow through the Design of Hydrogel Cell Carriers. Tissue Eng., Part A 2012, 18, 806–815. Ballios, B. G.; Cooke, M. J.; Donaldson, L.; Coles, B. L.; Morshead, C. M.; van der Kooy, D.; Shoichet, M. S. A Hyaluronan-Based Injectable Hydrogel Improves the Survival and Integration of Stem Cell Progeny Following Transplantation. Stem Cell Reports 2015, 4, 1031–1045.

301 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

18. Toh, W. S.; Lim, T. C.; Kurisawa, M.; Spector, M. Modulation of Mesenchymal Stem Cell Chondrogenesis in a Tunable Hyaluronic Acid Hydrogel Microenvironment. Biomaterials 2012, 33, 3835–3845. 19. Seidlits, S. K.; Khaing, Z. Z.; Petersen, R. R.; Nickels, J. D.; Vanscoy, J. E.; Shear, J. B.; Schmidt, C. E. The Effects of Hyaluronic Acid Hydrogels with Tunable Mechanical Properties on Neural Progenitor Cell Differentiation. Biomaterials 2010, 31, 3930–3940. 20. Li, J.; Mooney, D. J. Designing Hydrogels for Controlled Drug Delivery. Nat. Rev. Mater. 2016, 1, 16071. 21. Phan, V. H. G.; Thambi, T.; Duong, H. T. T.; Lee, D. S. Poly(amino carbonate urethane)-Based Biodegradable, Temperature and pH-Sensitive Injectable Hydrogels for Sustained Human Growth Hormone Delivery. Sci. Rep. 2016, 6, 29978. 22. Lee, F.; Chung, J. E.; Kurisawa, M. An Injectable Hyaluronic Acid-Tyramine Hydrogel System for Protein Delivery. J. Controlled Release 2009, 134, 186–193. 23. Qu, J.; Zhao, X.; Ma, P. X.; Guo, B. pH-Responsive Self-Healing Injectable Hydrogel Based on N-carboxyethyl Chitosan for Hepatocellular Carcinoma Therapy. Acta Biomater. 2017, 58, 168–180. 24. Shibata, H.; Heo, Y. J.; Okitsu, T.; Matsunaga, Y.; Kawanishi, T.; Takeuchi, S. Injectable Hydrogel Microbeads for Fluorescence-Based In Vivo Continuous Glucose Monitoring. Proc. Natl. Acad. Sci., U.S.A. 2010, 107, 17894–17898. 25. Soranno, D. E.; Rodell, C. B.; Altmann, C.; Duplantis, J.; AndresHernando, A.; Burdick, J. A.; Faubel, S. Delivery of Interleukin-10 via Injectable Hydrogels Improves Renal Outcomes and Reduces Systemic Inflammation following Ischemic Acute Kidney Injury in Mice. Am. J. Physiol. Renal. Physiol. 2016, 311, 362–372. 26. Wang, L. L.; Liu, Y.; Chung, J. J.; Wang, T.; Gaffey, A. C.; Lu, M.; Cavanaugh, C. A.; Zhou, S.; Kanade, R.; Atluri, P.; Morrisey, E. E.; Burdick, J. A. Local and Sustained miRNA Delivery from an Injectable Hydrogel Promotes Cardiomyocyte Proliferation and Functional Regeneration after Ischemic Injury. Nat. Biomed. Eng. 2017, 1, 983–992. 27. Purcell, B. P.; Lobb, D.; Charati, M. B.; Dorsey, S. M.; Wade, R. J.; Zellars, K. N.; Doviak, H.; Pettaway, S.; Logdon, C. B.; Shuman, J. A.; Freels, P. D.; Gorman, J. H., 3rd; Gorman, R. C.; Spinale, F. G.; Burdick, J. A. Injectable and Bioresponsive Hydrogels for On-Demand Matrix Metalloproteinase Inhibition. Nat. Mater. 2014, 13, 653–661. 28. Ray, P. D.; Huang, B. W.; Tsuji, Y. Reactive Oxygen Species (ROS) Homeostasis and Redox Regulation in Cellular Signaling. Cell Signal. 2012, 24, 981–990. 29. Ma, Q. Role of nrf2 in Oxidative Stress and Toxicity. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 401–426. 30. Mittal, M.; Siddiqui, M. R.; Tran, K.; Reddy, S. P.; Malik, A. B. Reactive Oxygen Species in Inflammation and Tissue Injury. Antioxid. Redox Signaling 2014, 20, 1126–1167. 302 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

31. Liochev, S. I. Reactive Oxygen Species and the Free Radical Theory of Aging. Free Radical Biol. Med. 2013, 60, 1–4. 32. Paiva, C. N.; Bozza, M. T. Are Reactive Oxygen Species Always Detrimental to Pathogens? Antioxid. Redox Signaling 2014, 20, 1000–1037. 33. Sugamura, K.; Keaney, J. F., Jr. Reactive Oxygen Species in Cardiovascular Disease. Free Radical Biol. Med. 2011, 51, 978–992. 34. Kim, G. H.; Kim, J. E.; Rhie, S. J.; Yoon, S. The Role of Oxidative Stress in Neurodegenerative Diseases. Exp. Neurobiol. 2015, 24, 325–340. 35. Waris, G.; Ahsan, H. Reactive Oxygen Species: Role in the Development of Cancer and Various Chronic Conditions. J. Carcinog. 2006, 5, 14. 36. Firuzi, O.; Miri, R.; Tavakkoli, M.; Saso, L. Antioxidant Therapy: Current Status and Future Prospects. Curr. Med. Chem. 2011, 18, 3871–3888. 37. Vong, L. B.; Kobayashi, M.; Nagasaki, Y. Evaluation of the Toxicity and Antioxidant Activity of Redox Nanoparticles in Zebrafish (Danio rerio) Embryos. Mol. Pharm. 2016, 13, 3091–3097. 38. Soule, B. P.; Hyodo, F.; Matsumoto, K.; Simone, N. L.; Cook, J. A.; Krishna, M. C.; Mitchell, J. B. The Chemistry and Biology of Nitroxide Compounds. Free Radical Biol. Med. 2007, 42, 1632–1650. 39. Nagasaki, Y. Nitroxide Radicals and Nanoparticles: A Partnership for Nanomedicine Radical Delivery. Ther. Delivery 2012, 3, 165–179. 40. Yoshitomi, T.; Miyamoto, D.; Nagasaki, Y. Design of Core-Shell–Type Nanoparticles Carrying Stable Radicals in the Core. Biomacromolecules 2009, 10, 596–601. 41. Vong, L. B.; Tomita, T.; Yoshitomi, T.; Matsui, H.; Nagasaki, Y. An Orally Administered Redox Nanoparticle that Accumulates in the Colonic Mucosa and Reduces Colitis in Mice. Gastroenterology 2012, 143, 1027–1036. 42. Sha, S.; Vong, L. B.; Chonpathompikunlert, P.; Yoshitomi, T.; Matsui, H.; Nagasaki, Y. Suppression of NSAID-Induced Small Intestinal Inflammation by Orally Administered Redox Nanoparticles. Biomaterials 2013, 34, 8393–8400. 43. Yoshitomi, T.; Hirayama, A.; Nagasaki, Y. The ROS Scavenging and Renal Protective Effects of pH-Responsive Nitroxide Radical-Containing Nanoparticles. Biomaterials 2011, 32, 8021–8028. 44. Boonruamkaew, P.; Chonpathompikunlert, P.; Nagasaki, Y. Redox Nanoparticle Therapeutics for Acetaminophen-Induced Hepatotoxicity in Mice. Oxid. Med. Cell. Longevity 2016, 2016, 4984597. 45. Eguchi, A.; Yoshitomi, T.; Lazic, M.; Johnson, C. D.; Vong, L. B.; Wree, A.; Povero, D.; Papouchado, D.; Nagasaki, Y.; Feldstein, A. E. Redox Nanoparticles as a Novel Treatment Approach for Inflammation and Fibrosis Associated with Nonalcoholic Steatohepatitis. Nanomedicine (London) 2015, 10, 2697–2708. 46. Boonruamkaew, P.; Chonpathompikunlert, P.; Vong, L. B.; Sakaue, S.; Tomidokoro, Y.; Ishii, K.; Tamaoka, A.; Nagasaki, Y. Chronic Treatment with a Smart Antioxidative Nanoparticle for Inhibition of Amyloid Plaque Propagation in Tg2576 Mouse Model of Alzheimer’s Disease. Sci. Rep. 2017, 7, 3785. 303 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

47. Chonpathompikunlert, P.; Fan, C. H.; Ozaki, Y.; Yoshitomi, T.; Yeh, C. K.; Nagasaki, Y. Redox Nanoparticle Treatment Protects against Neurological Deficit in Focused Ultrasound-Induced Intracerebral Hemorrhage. Nanomedicine (London) 2012, 7, 1029–1043. 48. Vong, L. B.; Yoshitomi, T.; Matsui, H.; Nagasaki, Y. Development of an Oral Nanotherapeutics using Redox Nanoparticles for Treatment of ColitisAssociated Colon Cancer. Biomaterials 2015, 55, 54–63. 49. Thangavel, S.; Yoshitomi, T.; Sakharkar, M. K.; Nagasaki, Y. Chronic Treatment with a Smart Antioxidative Nanoparticle for Inhibition of Amyloid Plaque Propagation in Tg2576 Mouse Model of Alzheimer’s Disease. Biomaterials 2016, 99, 109–123. 50. Vong, L. B.; Nagasaki, Y. Combination Treatment of Murine Colon Cancer with Doxorubicin and Redox Nanoparticles. Mol. Pharm. 2016, 13, 449–455. 51. Yoshitomi, T.; Ozaki, Y.; Thangavel, S.; Nagasaki, Y. Redox Nanoparticle Therapeutics to Cancer—Increase in Therapeutic Effect of Doxorubicin, Suppressing Its Adverse Effect. J. Controlled Release 2013, 172, 137–143. 52. Thangavel, S.; Yoshitomi, T.; Sakharkar, M. K.; Nagasaki, Y. Redox Nanoparticles Inhibit Curcumin Oxidative Degradation and Enhance Its Therapeutic Effect on Prostate Cancer. J. Controlled Release 2015, 209, 110–119. 53. Shim, M. S.; Lee, H. T.; Shim, W. S.; Park, I.; Lee, H.; Chang, T.; Kim, S. W.; Lee, D. S. Poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly (D,L-lactic acid-co-glycolic acid) Triblock Copolymer and Thermoreversible Phase Transition in Water. J. Biomed. Mater. Res. 2002, 61, 188–196. 54. Pua, M. L.; Yoshitomi, T.; Chonpathompikunlert, P.; Hirayama, A.; Nagasaki, Y. Redox-Active Injectable Gel Using Thermo-Responsive Nanoscale Polyion Complex Flower Micelle for Noninvasive Treatment of Local Inflammation. J. Controlled Release 2013, 172, 914–920. 55. Vong, L. B.; Bui, T. Q.; Tomita, T.; Sakamoto, H.; Hiramatsu, T.; Nagasaki, Y. Novel Angiogenesis Therapeutics by Redox Injectable Hydrogel—Regulation of Local Nitric Oxide Generation for Effective Cardiovascular Therapy. Biomaterials 2018, 167, 143–152. 56. Saita, M.; Kaneko, J.; Sato, T.; Takahashi, S. S.; Wada-Takahashi, S.; Kawamata, R.; Sakurai, T.; Lee, M. C.; Hamada, N.; Kimoto, K.; Nagasaki, Y. Novel Antioxidative Nanotherapeutics in a Rat Periodontitis Model: Reactive Oxygen Species Scavenging by Redox Injectable Gel Suppresses Alveolar Bone Resorption. Biomaterials 2016, 76, 292–301. 57. Lutolf, M. P.; Weber, F. E.; Schmoekel, H. G.; Schense, J. C.; Kohler, T.; Müller, R.; Hubbell, J. A. Repair of Bone Defects Using Synthetic Mimetics of Collagenous Extracellular Matrices. Nat. Biotechnol. 2003, 21, 513–518. 58. Nakagawa, H.; Matsumoto, Y.; Matsumoto, Y.; Miwa, Y.; Nagasaki, Y. Design of High-Performance Anti-Adhesion Agent Using Injectable Gel with an Anti-Oxidative Stress Function. Biomaterials 2015, 69, 165–173. 59. Ellis, H. The Clinical Significance of Adhesions: Focus on Intestinal Obstruction. Eur. J. Surg. Suppl. 1997, 577, 5–9. 304 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

60. Schenker, J. G.; Margalioth, E. J. Intrauterine Adhesions: An Updated Appraisal. Fertil. Steril. 1982, 37, 593–610. 61. DiZerega, G. S. Use of Adhesion Prevention Barriers in Pelvic Reconstructive and Gynecologic Surgery. Pelvic Surg. 1997, 19, 188–209. 62. Bosteels, J.; Weyers, S.; Mol, B. W.; D’Hooghe, T. Anti-Adhesion Barrier Gels Following Operative Hysteroscopy for Treating Female Infertility: A Systematic Review and Meta-Analysis. Gynecol. Surg. 2014, 11, 113–127. 63. Chaturvedi, A. A.; Lomme, R. M.; Hendriks, T.; van Goor, H. Prevention of Postsurgical Adhesions Using an Ultrapure Alginate-Based gel. Br. J. Surg. 2013, 100, 904–910. 64. Song, L.; Li, L.; He, T.; Wang, N.; Yang, S.; Yang, X.; Zeng, Y.; Zhang, W.; Yang, L.; Wu, Q.; Gong, C. Peritoneal Adhesion Prevention with a Biodegradable and Injectable N,O-carboxymethyl Chitosan-Aldehyde Hyaluronic Acid Hydrogel in a Rat Repeated-Injury Model. Sci. Rep. 2016, 6, 37600. 65. Becker, J. M.; Dayton, M. T.; Fazio, V. W.; Beck, D. E.; Stryker, S. J.; Wexner, S. D.; Wolff, B. G.; Roberts, P. L.; Smith, L. E.; Sweeney, S. A.; Moore, M. Prevention of Postoperative Abdominal Adhesions by a Sodium Hyaluronate-Based Bioresorbable Membrane: A Prospective, Randomized, Double-Blind Multicenter Study. J. Am. Coll. Surg. 1996, 183, 297–306. 66. Zhang, Y.; Gao, C.; Li, X.; Xu, C.; Zhang, Y.; Sun, Z.; Liu, Y.; Gao, J. Thermosensitive Methyl Cellulose-Based Injectable Hydrogels for Post-Operation Anti-Adhesion. Carbohydr. Polym. 2014, 101, 171–178. 67. Frazier-Jessen, M. R.; Mott, F. J.; Witte, P. L; Kovacs, E. J. Estrogen Suppression of Connective Tissue Deposition in a Murine Model of Peritoneal Adhesion Formation. J. Immunol. 1996, 156, 3036–3042. 68. Heuvel, M. M. V. D.; Smit, H. J.; Barbierato, S. B; Havenith, C. E.; Beelen, R. H.; Postmus, P. E. Talc-Induced Inflammation in the Pleural Cavity. Eur. Respir. J. 1998, 12, 1419–1423. 69. Nagasaki, Y.; Mizukoshi, Y.; Gao, Z.; Feliciano, C. P.; Chang, K.; Sekiyama, H.; Kimura, H. Development of a Local Anesthetic LidocaineLoaded Redox-Active Injectable Gel for Postoperative Pain Management. Acta Biomater. 2017, 57, 127–135. 70. Ishii, S.; Kaneko, J.; Nagasaki, Y. Dual Stimuli-Responsive Redox-Active Injectable Gel by Polyion Complex Based Flower Micelles for Biomedical Applications. Macromolecules 2015, 48, 3088–3094. 71. Ishii, S.; Kaneko, J.; Nagasaki, Y. Development of a Long-Acting, ProteinLoaded, Redox-Active, Injectable Gel Formed by a Polyion Complex for Local Protein Therapeutics. Biomaterials 2016, 84, 210–218. 72. Challapalli, V.; Tremont-Lukats, I. W.; McNicol, E. D.; Lau, J.; Carr, D. B. Systemic Administration of Local Anesthetic Agents to Relieve Neuropathic Pain. Cochrane Database Syst. Rev. 2005, 4, CD003345. 73. Rowbotham, M. C.; Reisner-Keller, L. A.; Fields, H. L. Both Intravenous Lidocaine and Morphine Reduce the Pain of Postherpetic Neuralgia. Neurology 1991, 41, 1024–1028.

305 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

74. Bagshaw, K. R.; Hanenbaum, C. L.; Carbone, E. J.; Lo, K. W.; Laurencin, C. T.; Walker, J.; Nair, L. S. Pain Management via Local Anesthetics and Responsive Hydrogels. Ther. Delivery 2015, 6, 165–176. 75. Al-Saffar, M. T.; AL-Sandook, T. A.; Y-Taha, M. A Possible New Concept in the Mechanism of Action of Local Anesthesia. Am. J. Med. Biol. Res. 2013, 1, 134–137. 76. Cohen, J. IL-12 Deaths: Explanation and a Puzzle. Science 1995, 270, 908. 77. Leonard, J. P.; Sherman, M. L.; Fisher, G. L.; Buchanan, L. J.; Larsen, G.; Atkins, M. B.; Sosman, J. A.; Dutcher, J. P.; Vogelzang, N. J.; Ryan, J. L. Effects of Single-Dose Interleukin-12 Exposure on Interleukin-12-Associated Toxicity and Interferon-Gamma Production. Blood 1997, 90, 2541–2548. 78. Car, B. D.; Eng, V. M.; Lipman, J. M.; Anderson, T. D. The Toxicology of Interleukin-12: A Review. Toxicol. Pathol. 1999, 27, 58–63. 79. Hasan, A.; Khattab, A.; Islam, M. A.; Hweij, K. A.; Zeitouny, J.; Waters, R.; Sayegh, M.; Hossain, M. M.; Paul, A. Injectable Hydrogels for Cardiac Tissue Repair after Myocardial Infarction. Adv. Sci. (Weinheim) 2015, 2, 1500122. 80. Li, X. Y.; Wang, T.; Jiang, X. J.; Lin, T.; Wu, D. Q.; Zhang, X. Z.; Okello, E.; Xu, H. X.; Yuan, M. J. Injectable Hydrogel Helps Bone Marrow-Derived Mononuclear Cells Restore Infarcted Myocardium. Cardiology 2010, 115, 194–199. 81. Wang, H.; Liu, Z.; Li, D.; Guo, X.; Kasper, F. K.; Duan, C.; Zhou, J.; Mikos, A. G.; Wang, C. Injectable Biodegradable Hydrogels for Embryonic Stem Cell Transplantation: Improved Cardiac Remodelling and Function of Myocardial Infarction. J. Cell. Mol. Med. 2012, 16, 1310–1320. 82. Paul, A.; Hasan, A.; Kindi, H. A.; Gaharwar, A. K.; Rao, V. T.; Nikkhah, M.; Shin, S. R.; Krafft, D.; Dokmeci, M. R.; Shum-Tim, D.; Khademhosseini, A. Injectable Graphene Oxide/Hydrogel-Based Angiogenic Gene Delivery System for Vasculogenesis and Cardiac Repair. ACS Nano 2014, 8, 8050–8062. 83. Naseem, K. M. The Role of Nitric Oxide in Cardiovascular Diseases. Mol. Aspects Med. 2005, 26, 33–65. 84. Massion, P. B.; Feron, O.; Dessy, C.; Balligand, J. L. Nitric Oxide and Cardiac Function: Ten Years After, and Continuing. Circ. Res. 2003, 93, 388–398. 85. Ferdinandy, P.; Danial, H.; Ambrus, I.; Rothery, R. A.; Schulz, R. Peroxynitrite is a Major Contributor to Cytokine-Induced Myocardial Contractile Failure. Circ. Res. 2000, 87, 241–247. 86. Lundberg, J. O.; Gladwin, M. T.; Weitzberg, E. Strategies to Increase Nitric Oxide Signalling in Cardiovascular Disease. Nat. Rev. Drug Discovery 2015, 14, 623–641. 87. de Lorgeril, M. Dietary Arginine and the Prevention of Cardiovascular Diseases. Cardiovasc. Res. 1998, 37, 560–563. 88. Benito, S.; Lopez, D.; Sáiz, M. P.; Buxaderas, S.; Sánchez, J.; PuigParellada, P.; Mitjavila, M. T. A Flavonoid-Rich Diet Increases Nitric Oxide Production in Rat Aorta. Br. J. Pharmacol. 2002, 135, 910–916. 306 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

89. Kudo, S.; Nagasaki, Y. Novel Nitric Oxide-Based Anticancer Therapeutics by Macrophage-Targeted poly(L-arginine)-Based Nanoparticles. J. Controlled Release 2015, 217, 256–262. 90. Thomas, D. D.; Espey, M. G.; Ridnour, L. A.; Hofseth, L. J.; Mancardi, D.; Harris, C. C.; Wink, D. A. Hypoxic Inducible Factor 1α, Extracellular Signal-Regulated Kinase, and p53 Are Regulated by Distinct Threshold Concentrations of Nitric Oxide. Proc. Natl. Acad. Soc., U.S.A. 2004, 101, 8894–8899. 91. Jaffrey, S. R.; Erdjument-Bromage, H.; Ferris, C. D.; Tempst, P.; Snyder, S. H. Protein S-nitrosylation: A Physiological Signal for Neuronal Nitric Oxide. Nat. Cell Biol. 2001, 3, 193–197. 92. Balkwill, F.; Mantovani, A. Inflammation and Cancer: Back to Virchow? Lancet 2001, 357, 539–545.

307 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.