Targeting macrophages with Astaxanthin-loaded microparticles: a strategy to attenuate radiation-induced fibrosis

Radiation-induced fibrosis (RIF) is one of the common adverse effects of radiotherapy (overall incidence between 10-20%) and it results in a multitude of symptoms that can significantly impact the quality of life of cancer patients. RIF is a complex multi-factorial process that ultimately results in an excess accumulation of collagen and other extracellular matrix components in many organs, especially in the lung, skin, small bowel, breast, liver, and kidney, that in turn impairs their own functions. RIF is initiated by direct radiation-induced cell damage and by indirect mechanisms which, in turn, involve the generation of reactive oxygen and nitrogen species (ROS and RNS, respectively) through water radiolysis and activation of nitric oxide synthase. Central to RIF is the role of tissue-resident leukocytes, such as macrophages, which, in response to ROS accumulation and tissue damage, activate the expression of pro-inflammatory cytokines, such as transforming growth factor beta (TGF-β). TGF-β may, in turn, increase ROS production through suppression of antioxidant enzymes, thus leading to a positive feedback that sustains oxidative stress, inflammation and tissue damage. f-β, finally, promotes fibroblasts recruitment and local deposition of extracellular matrix components. Our understanding of the processes involved in the pathology of RIF have improved. However, to date no successful therapeutic approaches are available in the clinical setting. Importantly, it has been reported that post- radiation antioxidant therapy significantly reduces RIF in animal models. This shows that it is indeed possible to halt the positive feedback between ROS accumulation and inflammation at the basis of the disease. Astaxanthin (3,3′-dihydroxy-β-β′-carotene-4,4′-dione; ASX) is a xantophyll ketocarotenoid present in several marine and freshwater organisms, including microorganisms, crustaceans and fishes. ASX is a potent antioxidant, with rate constants for radical scavenging approximately 10 times higher than other carotenoids, such as lutein and β-carotene, and 100 times more than that of α- tocopherol. ASX might, therefore, be used to reduce or prevent RIF. ASX, however, is not soluble in water-based biological fluids and is sensitive to temperature, light and oxygen, all factors that limit its stability and bioavailability and that can be controlled by ASX encapsulation into appropriate chemical matrices. Encapsulation of ASX into micrometer-sized particles might also represent an excellent strategy to deliver ASX specifically to macrophages and other innate immune cells that are characterized by their unique ability to engulf, -5- uptake and degrade particles of this size through phagocytosis. In contrast, tumor cells do not phagocytose and hence will not be provided direct protection against antioxidants conferred by ASX. This project aims at demonstrating the feasibility of this possible therapeutic approach against RIF, albeit in a cell-based in vitro setting. The results clearly indicate that ASX microparticles interfere with the positive feedback between ROS and TGF-β in phagocytic cells, and that pentoxifylline, a drug currently investigated for its ability to reduce RIF, amplifies the effects of astaxanthin microparticles on macrophages. Treatment strategies involving ASX microparticles, eventually in combination with other drugs such as pentoxifylline, should be further studied to examine their potential to reduce inflammation and inhibit radiation-induced fibrosis.