Photodynamic Therapy Using a Protease-Mediated Theranostic Agent Reduces Cathepsin-B Activity in Mouse Atheromata In Vivo

Abstract

M atrix-disorganizing proteases, such as cathepsins or matrix metalloproteinases from macrophages, can destabilize atheromata, followed by plaque rupture to cause thromboembolic stroke or myocardial infarction. [1][2][3][4] We previously showed that molecular imaging of cathepsin-B (CatB) or matrix metalloproteinase-2/9 protease activity reflected the inflammatory component of atherosclerotic pathology in mice 5 and human atheromata. 6 We also showed that the protease imaging could quantitatively demonstrate plaque-stabilizing effects of antiatherosclerotic drugs 5 and exercise training 7 in mice. The principle of photodynamic therapy (PDT) is to kill unwanted cells by using a combination of photosensitizers and light illumination to generate highly reactive oxygen species that locally destroys cells over short diffusion distances.8-10 A recent PDT study using macrophage-targeted photosensitizers preliminarily demonstrated the viability of near-infrared lightactivated therapeutic nanoagents in the treatment of atherosclerotic vascular disease by showing preferential destruction of macrophages in vitro and in mouse atheromata. Objective-To investigate whether an intravenously injected cathepsin-B activatable theranostic agent (L-SR15) would be cleaved in and release a fluorescent agent (chlorin-e6) in mouse atheromata, allowing both the diagnostic visualization and therapeutic application of these fluorophores as photosensitizers during photodynamic therapy to attenuate plaquedestabilizing cathepsin-B activity by selectively eliminating macrophages. Approach and Results-Thirty-week-old apolipoprotein E knock-out mice (n=15) received intravenous injection of L-SR15 theranostic agent, control agent D-SR16, or saline 3× (D0, D7, D14). Twenty-four hours after each injection, the bilateral carotid arteries were exposed, and Cy5.5 near-infrared fluorescent imaging was performed. Fluorescent signal progressively accumulated in the atheromata of the L-SR15 group animals only, indicating that photosensitizers had been released from the theranostic agent and were accumulating in the plaque. After each imaging session, photodynamic therapy was applied with a continuous-wave diode-laser. Additional near-infrared fluorescent imaging at a longer wavelength (Cy7) with a cathepsin-B-sensing activatable molecular imaging agent showed attenuation of cathepsin-B-related signal in the L-SR15 group. Histological studies demonstrated that L-SR15-based photodynamic therapy decreased macrophage infiltration by inducing apoptosis without significantly affecting plaque size or smooth muscle cell numbers. Toxicity studies (n=24) showed that marked erythematous skin lesion was generated in C57/BL6 mice at 24 hours after intravenous injection of free chlorin-e6 and ultraviolet light irradiation; however, L-SR15 or saline did not cause cutaneous phototoxicity beyond that expected of ultraviolet irradiation alone, neither did we observe systemic toxicity or neurobehavioral changes. Conclusions-This is the first study showing that macrophage-se...

Atherosclerosis Treatment with Stimuli‐Responsive Nanoagents: Recent Advances and Future Perspectives

“…Therefore, it can serve as both a stimulus and a target for drug delivery in atherosclerosis management. Shon et al developed protease‐mediated theranostic agents (L‐SR15 agents) for PDT that could suppress cathepsin B activity. L‐SR15 activation was mediated by cathepsin Bs that released Ce6 molecules leading to the reduction of foam cells via PDT .…”

“…To date, multi‐stimuli‐responsive AND‐based nanosystems have shown great promise in enhancing drug delivery efficiency to atherosclerotic lesions. Shon et al, for instance, have developed dual‐responsive theranostic nanoagents (L‐SR15 agents) that were active in response to light and enzyme (cathepsin B) stimuli. Without the presence of cathepsin Bs, the L‐SR15 nanoagents were still inactive.…”

Macrophages

“…Maiseyeu et al 156 showed that gadolinium nanoparticles conjugated with a neutralizing antibody against myeloid-related protein (Mrp)-8/14 (a secreted protein previously shown to promote leukocyte recruitment) limit Mrp-8/14-dependent inflammatory activation of bone marrow-derived macrophages in vitro and selectively image at CONS CALIFORNIA DIG LIB on November 19, 2014 http://atvb.ahajournals.org/ Downloaded from inflammatory cells in lesions of Apoe −/− mice, suggesting a potential theranostic approach for atherosclerosis. Others have developed photodynamic therapy to image and kill activated and detrimental macrophages within atherosclerotic lesions, 157 using a combination of photosensitizers and light illumination. In particular, the authors developed a cathepsin B-activatable theranostic agent, which becomes fluorescent and generates singlet oxygen on protease conversion followed by light therapy.…”

“…They showed selective imaging of macrophage-dependent cathepsin B activity in lesions of Apoe −/− mice, followed by significant reduction of macrophage content after application of light therapy, mostly because of macrophage apoptosis. 157 Future work should define the use and potential undesirable effects of this attractive see-and-treat approach. Induction of massive macrophage apoptosis in advanced lesions might promote necrotic core formation in the absence of effective efferocytosis pathways.…”

ROS-Responsive Activatable Photosensitizing Agent for Imaging and Photodynamic Therapy of Activated Macrophages

“…These activatable PDT agents were shown to be highly efficient in selectively killing target cancer cells while minimizing unwanted skin photosensitivity. Recently, we showed the first successful in vivo results demonstrating the potential utility of an enzyme-activatable PDT agent in near-infrared (NIR) fluorescence imaging and the subsequent PDT of atherosclerosis 31. The cathepsin B-activatable photodynamic theranostic agent reduced cathepsin-B activity in mouse atheromata and stabilized inflammatory plaques by selectively killing the activated macrophages in the diseased regions.…”