Efficacy of high-affinity liposomal astaxanthin on up-regulation of age-related markers induced by oxidative stress in human corneal epithelial cells

Abstract

Decreases in tear volume, unstable tear films and excessive tear evaporation are known to cause desiccation and hyperosmolar stress. These, in turn, induce oxidative stress that is thought to cause dry eye, which is also considered to be age-related disease. We hypothesized that oxidative stress induces up-regulation of age-related markers, and that the antioxidant astaxanthin prepared as a liposomal formulation may be a candidate for the treatment of dry eye. Herein, we examined age-related markers in an in vitro dry eye model, and evaluated the efficacy of high-affinity liposomes containing astaxanthin. The in vitro dry eye model showed desiccation time-dependent increases in reactive oxygen species. We confirmed the up-regulation of p53, p21 and p16 as a function of desiccation time. Pretreatment with both neutral and slightly-positively-charged astaxanthin liposomal formulations showed significant suppression of up-regulation of all markers, with the positively-charged liposomes exhibiting the greatest efficacy. Furthermore, positively-charged liposomes labeled with fluorescent dyes demonstrated much higher affinity to normal human corneal epithelial cells (HCECs) than neutral liposomes. Taken together, we confirmed the up-regulation of age-related markers, especially p16, in an in vitro dry eye model, and demonstrated the potential of high-affinity liposomal astaxanthin for the treatment of dry eye.

Protective effect of high-affinity liposomes encapsulating astaxanthin against corneal disorder in the <i>in vivo</i> rat dry eye disease model

“…Liposomal formulations comprising only EPC had a mostly neutral charge, and the zeta potential for liposomal formulations composed of EPC and DOTAP showed a slight positive charge (<10 mV), which is consistent with our previous report (Table 2). (23) The actual concentration of Asx encapsulated in E/Asx-lipo and E/D/Asx-lipo was nearly equal to theoretical values at 98.6% and 101.9% for E/Asx-lipo and E/D/ Asx-lipo, respectively ( Table 2).…”

“…(22) In our previous report, the liposomal formulation of Asx could suppress these phenomena in the in vitro DED model using HCECs. (23) We also demonstrated that liposomes comprising both neutral and cationic lipids that bore a slight positive charge exerted superior antioxidative effects compared to liposomes consisting of only neutral lipids. The positive surface charge is predicted to increase affinity of the liposomes for cell membranes, and thus high affinity, positively-charged liposomes should be advantageous for corneal treatments involving eye drops that typically exhibit low bioavailability due to the drainage system associated with tear flow.…”

“…(1) Previously, we reported that desiccative stress indeed results in intracellular ROS generation that is associated with up-regulated expression of age-related markers in the in vitro DED model, and demonstrated that Asx encapsulated in high-affinity liposomes could be a potential candidate therapeutic agent for DED. (23) In this study, we investigated the correlation between SPK as an indicator of corneal damage and expression of the age-related markers p53, p21 and p16 using the in vivo rat DED model. We demonstrated decreases in tear volume and up-regulated expression of p53, p21 and p16 as age-related markers, as well as increased fluorescein score as an indicator of SPK in the in vivo rat DED model (Fig.…”

“…EPC or EPC/ DOTAP liposomes encapsulating Asx (E/Asx-lipo or E/D/Asx-lipo, respectively) were prepared according to our previous report. (23) Briefly, a chloroform solution containing 20 mM EPC or 18 mM EPC and 2 mM DOTAP with 200 mM of Asx was dried to a thin film in a test tube using nitrogen gas. Dried lipid films were hydrated with 10% sucrose solution at room temperature and sonicated in a bath type sonicator (Bransonic, Branson Ultrasonics Corporation, Danbury, CT) to obtain a liposomal suspension of small vesicles.…”

“…The positive surface charge is predicted to increase affinity of the liposomes for cell membranes, and thus high affinity, positively-charged liposomes should be advantageous for corneal treatments involving eye drops that typically exhibit low bioavailability due to the drainage system associated with tear flow. (23) In this study we expanded on our earlier findings by using the in vivo rat DED model to clarify the relationship between superficial punctate keratopathy (SPK), which is a major symptom of DED, (24)(25)(26) and the behavior of age-related markers. We also verified the correlation of findings for the in vivo rat DED model with those reported previously for the in vitro DED model.…”