The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R 2 > 0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

Understanding of principles governing selective and sensitive cancer targeting is critical for development of chemicals for cancer diagnostics and treatment. We determined the underlying mechanisms of how a novel fluorescent small organic molecule, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), selectively labels cancer cells but not normal cells. We show that BMVC is retained in the lysosomes of normal cells. In cancer cells, BMVC escapes lysosomal retention and localizes to the mitochondria or to the nucleus, where DNA-binding dramatically increases BMVC fluorescence intensity, allowing it to light up only cancer cells. Structure-function analyses of BMVC derivatives show that hydrogen-bonding capacity is a key determinant of lysosomal retention in normal cells, whereas lipophilicity directs these derivatives to the mitochondria or the nucleus in cancer cells. In addition, drug-resistant cancer cells preferentially retain BMVC in their lysosomes compared to drug-sensitive cancer cells, and BMVC can be released from drug-resistant lysosomes using lysosomotropic agents. Our results further our understanding of how properties of cellular organelles differ between normal and cancer cells, which can be exploited for diagnostic and/or therapeutic use. We also provide physiochemical design principles for selective targeting of small molecules to different organelles. Moreover, our results suggest that agents which can increase lysosomal membrane permeability may re-sensitize drug-resistant cancer cells to chemotherapeutic agents.

Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. We have introduced a G-quadruplex (G4) ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy. The GROs that form parallel G4 structures, such as PU22, T40214 and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form non-parallel G4 structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the fluorescence resonant energy transfer studies of fluorophore-labeled GROs show that the parallel G4 structures can be retained in CL1-0 cells, whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the non-parallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs.

We have applied a fluorescent molecule 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) for tumor targeting and treatment. In this study, we investigated the photo-induced antitumor effect of BMVC. In vitro cell line studies showed that BMVC significantly killed TC-1 tumor cells at light dose greater than 40 J/cm(2). The fluorescence of BMVC in the tumor peaked at 3 hours and then gradually decreased to reach the control level a. er 24 hours. In vivo tumor treatment studies showed BMVC plus light irradiation (iPDT) significantly inhibited the tumor growth. At day 24 a. er tumor implantation, tumor volume was measured to be 225 +/- 79 mm(3), 2542 +/- 181 mm(3), 1533 +/- 766 mm(3), and 1317 +/- 108 mm(3) in the iPDT, control, light-only, and BMVC-only groups, respectively. Immunohistochemistry studies showed the microvascular density was significantly lower in the iPDT group. Taken together, our results demonstrated that BMVC may be a potent tumor-specific photosensitizer (PS) for PDT.

ABSTRACT. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8  ns and that of interaction with the duplex structure of a calf thymus is ∼1.2  ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.