A novel nitric oxide-based anticancer therapeutics by macrophage-targeted poly(l-arginine)-based nanoparticles

Abstract

In the immune system, macrophages in tumor tissue generate nitric oxide (NO), producing versatile effects including apoptosis of tumor cells, because inducible NO synthase (iNOS) in the cytoplasm of a macrophage produces NO using l-arginine as a substrate. Here, we propose novel NO-triggered immune therapeutics based on our newly designed nanoparticle system. We designed a poly(ethylene glycol)-block-poly(l-arginine) (i.e., PEG-b-P(l-Arg)) block copolymer and prepared polyion complex micelles (PEG-b-P(l-Arg)/m) composed of PEG-b-P(l-Arg) and chondroitin sulfate for systemic anticancer immunotherapy. iNOS treatment of PEG-b-P(l-Arg) did not generate NO, but NO molecules were detected after trypsin pretreatment, indicating that hydrolysis of P(l-Arg) to monomeric arginine was taking place in vitro. RAW264.7 macrophages abundantly generated NO from the PEG-b-P(l-Arg)/m in comparison with control micelles; this finding is indicative of robustness of the proposed method. It is interesting to note that systemic administration of PEG-b-P(l-Arg)/m had no noticeable adverse effects and suppressed the tumor growth rate in C26 tumor-bearing mice in a dose-dependent manner. Our newly designed nanoparticle-assisted arginine delivery system seems to hold promise as an NO-mediated anticancer immunotherapy.

Introduction

Anticancer immunotherapy has been attracting increasing attention, as a possible replacement of conventional cancer treatments such as surgical interventions, chemotherapy, and radiation therapy [1], [2], [3], [4]. During anticancer immunotherapy, endogenous immune cells are activated and attack tumor tissues. Initially, T-lymphocytes and natural killer cells were mainly targeted to improve the immune system. For example, TGF-β inhibitor and/or the immunostimulatory cytokine IL-2, were employed for activation or expansion of tumor-specific cytotoxic T lymphocytes. Most recently, macrophages were targeted to increase their antitumor activities [5], [6]. Macrophages in tumor tissues are classified into the M1 phenotype and M2 phenotype [7], [8], [9], [10], [11]. At an early stage of cancer, M1 macrophages overexpress inducible nitric oxide synthase (iNOS) intracellularly, which generates the cytotoxic substance nitric oxide (NO) from l-arginine as a substrate; this is a major mechanism of tumor-cytotoxic activities of macrophages.

In this study, we focused on these M1 macrophages, which have a much longer lifespan than other immune cells. It was reported that M1 macrophages are a major component of the leukocytic infiltrates into tumor tissues at an early stage of cancer [12]. Although M1 macrophages have not been studied much as mediators of anticancer immunotherapy, some recent research showed that activated macrophages can indeed enhance the efficacy of anticancer immunotherapy. For instance, at the malignant stage of a tumor, the M1 phenotype converts to the M2 phenotype, which accelerates activities of various types of tumors in terms of initiation, proliferation, metastasis, and angiogenesis. Zhang's group reported that cationic polymers such as cationic dextran specifically induce M2 macrophages (via Toll-like receptor 4 signaling) to produce and secrete IL-12, which converts M2 macrophages back to the tumor-cytotoxic M1 phenotype, resulting in anticancer activity [5], [6]. As stated above, activated M1 macrophages overexpress iNOS to produce NO. This gaseous and lipophilic molecule rapidly diffuses throughout tumor tissues from the macrophages that infiltrate these tissues [13]. The liberated NO is reported to cause apoptosis via several mechanisms such as condensation with amine or thiol groups of certain proteins and DNA damage related to p53 signaling [14], [15], [16]. The NO molecule also reacts with superoxide to produce peroxynitrite that directly induces necrosis of cells. Although the intracellular arginine concentration in macrophages surrounding tumor tissue is at the saturation level, one research group reported that the addition of extracellular arginine further upregulates NO: the so-called arginine paradox [17]. On the basis of these mechanisms, we assumed that accumulation of arginine at a tumor site prevents tumor progression via site-specific NO production. Accordingly, in this study, we examined the activation of macrophages through arginine delivery into tumor tissues. Although there are several reports on direct administration of arginine, this method has almost no antitumor effect because of rapid diffusion of l-arginine throughout the entire body and easy metabolism and/or excretion. Here, we utilized a nanoparticle-assisted arginine delivery system. Our newly designed material is polyion complex (PIC) micelles composed of poly(ethylene glycol)-b-poly(l-arginine) (i.e., PEG-b-P(l-Arg)) block copolymers coupled with polyanions such as chondroitin sulfate (CS). Our strategy involves delivery of poly(l-arginine) to a tumor site by passive targeting of highly colloidally stable nanoparticles via the enhanced permeability and retention (EPR) effect, [18] followed by capture by strong phagocytic macrophages and enzymatic hydration of the internalized poly(l-arginine) resulting in monomeric arginine in the macrophages. Finally, iNOS in the macrophages generates NO, which suppresses tumor progression. It should be noted that a low concentration of NO improves angiogenesis, which enhances tumor growth, whereas a high concentration of NO causes apoptosis of tumor cells. Our results revealed that a high NO concentration has an antitumor effect.