Macrophages are one of the most important candidates of innate immune response with pronounced phagocytic activity. As a part of the defense mechanism of our system, an increased level of particular chemokines initiate the activation and further differentiation of the macrophages into two major phenotypes, M1 (classically activated) and M2 (alternatively activated). M1 macrophages promote ‘pro-inflammatory’ activities, whereas, M2 macrophages show ‘anti-inflammatory’ activities. Now, normally a certain ratio of M1/M2 macrophages is maintained in healthy individuals. However, at certain disease conditions, a shift in the M1 to M2 or M2 to M1 macrophage population is observed. An assessment of the M1/M2 ratio would readily predict the health condition of the individual. Here, we propose a novel approach to identify M1 and M2 populations using simple flow cytometry and Gold nanorods (GNRs). According to reports, macrophages can readily internalize GNRs by phagocytosis. Now, this internalization of highly scattering GNRs will increase the cellular scattering of macrophages and thus can be identified by the Flow Cytometric technique. For the first time, we are reporting about the differential uptake of polyallylamine hydrochloride (PAH) coated GNRs by M1 and M2 macrophages (differentiated from THP1 cells). A 24 h incubation with the 100μg/ml PAH-GNRs results in a greater intake of PAH-GNRs by M2 cells compared to M1, which leads to an increased side scatter for M2 cells in Flow cytometry. Overall, this study opens a new avenue for simple identification of M1 and M2 cell types.
Currently, gold nanorods (GNRs), with versatile optical behaviors, are in the immense study to achieve a variety of applications in the fields of diagnostics and therapy. Macrophages, the proficient phagocytic immune cells, mainly differentiated from monocytes, play a major role in our innate immunity. Depending on the state of a disease, an enhancement of local macrophage population occurs as a normal protective measure of our body. We want to monitor this increased level and use it as a prediction index of a disease. The advantage of working with macrophages is that they willingly internalize GNRs by phagocytosis. Now, the macrophages with GNRs can be identified by Flow Cytometric technique through the high scattering of GNRs. Moreover, the surface charge of GNRs can significantly change the uptake and cytotoxicity pattern of GNRs. Here we compared the uptake of GNRs, with different zeta potentials, among three different types of macrophages –macrophages isolated from the blood of a healthy donor; macrophages differentiated from THP1 human monocyte cell lines and RAW 264.7 murine macrophage cell line. GNRs with positive zeta i.e. cetyl trimethylammonium bromide (CTAB), poly allylamine hydrochloride (PAH) higher uptake than the GNRs with null zeta i.e. polyethylene glycol (PEG) or negatively charged surface i.e., poly sodium 4-styrenesulfonate (PSS) and citrate. Overall, this study showed an application of the scattering property of GNRs as a tool to identify macrophages and how modifications of the GNR surface monitor their uptake by macrophages.
Atherosclerosis, the leading cause of morbidity and mortality of cardiovascular disease, occur due to hardening and narrowing of arteries for development of vulnerable plaques made of cholesterols, tissue macrophages, foam cells and smooth muscle cells. Early detection of atherosclerosis is essential for proper treatment. Our group has already reported about the potential application of the non-invasive diffusion reflection (DR) technique in the detection of atherosclerosis using gold nanorods (GNRs) as contrast agent in carotid artery injured mice model. The basics of the study lie on the uptake GNRs by macrophages that located at the vulnerable plaques, which act as a good absorption contrast for DR measurement. Accumulations of GNRs cause changes in the optical property of the tissues and in turn cause changes in DR profile. In this study, we report the potential application of DR measurement in the detection of atherosclerosis in high-fat diet mice. Here, we have used PEG-coated GNRs with absorption maxima around 660nm. The time kinetics showed that after 24h of GNR injection the DR can find the atherosclerotic plaques and with time (up to 72h) the GNR accumulation in plaques were faded out, but still can be detectable by DR. Our result strongly suggests that in future DR can be used for early detection of atherosclerosis.