Stable and sensitive probes for lysosomes: Cell‐penetrating fluorescent calix[4]arenes accumulate in acidic vesicles

Abstract

The uptake of a fluorescently labeled cationic calix [4] (NBDCalAm) in live, nonfixed cells has been investigated. The compound is taken into the cells rapidly and shows distinct endosomal distribution after 2 hours. This distribution pattern shows colocalization with lysosomal staining. The uptake is not altered by inhibition of clathrin or caveolae dependent pathways nor by depletion of the cellular ATP-pool. Immediately after uptake the probe is localized in the Golgi and brefeldin A treatment prevents transport to lysosomes. Pulse chase experiments with bafilomycin A1, monensin, and sodium azide showed that accumulation and retention of the probe in lysosomes is primarily driven by the activity of vacuolar ATPases. The NBD labeled calix[4]arene provides a very stable and sensitive marker for lysosomes, and has a considerable advantage over some commercially available lysosomal markers in so far that the fluorescent signal is stable even when the cells are incubated in dye-free medium after staining. ' 2010 International Society for Advancement of Cytometry Key termslysosome; calixarene; cell-penetration; immunofluorescence CALIXARENES (1) are a family of readily synthesized and functionalized macrocycles which have found applications in a number of research areas. In particular the ability of derivatives to complex anions (2,3) and cations (4,5) has been exploited for the development of sensors. Recently, interest has focused on the use of calixarene derivatives in biological systems be it as drugs, drug delivery systems, or imaging agents. In the design of drug molecules, particular attention has been paid to calixarenes as antimicrobials (6-8), as vaccines (9) and as anticancer agents (10). In the field of drug delivery, agents for nucleic acids based on single calixarenes (11-13) have shown promise and we have recently shown that larger arrays of cationic calix[4]arene, so-called multicalixarenes, are able to transfect DNA effectively (14). In addition a number of studies have combined the ability of calixarene derivatives to complex cations with their low toxicity in the development of noncovalently labeled protein MRI imaging agents (15,16).While the nontoxic (17) and non-immunogenicity (18,19) status of calixarenes has been established, less information is available on how calixarenes are processed within cells and their cellular fate, both of which are important features which must be characterized before such derivatives are taken further for clinical development. We recently demonstrated that fluorescently labeled calix [4]arene have the potential to track progress of the macrocycle in cells, as an NBD labeled cationic derivative was readily and speedily taken up into cells and provided a very stable, nontoxic read-out system to investigate cellular localization (20). The absorbance and emission spectra for the NBDCalAm showed that it can easily be visualized using a GFP filter (20).

Indicator Displacement Assays Inside Live Cells

“…These fluorescence enhancements demonstrate that the analytes were taken up within 10–15 min into the cells, where they displaced LCG from the macrocyclic complex to result in enhanced cellular fluorescence. Incubation times longer than 20 min led to relatively smaller fluorescence recoveries, and no significant increase was observed after 5 h of incubation, thus suggesting that the reporter pair proceeds from the cytoplasm into different cellular compartments 18b. The staining pattern of the released LCG is punctate, in agreement with previous cellular imaging studies of the dye alone 18d.…”

Calix[4]arene C-99 inhibits myosin ATPase activity and changes the organization of contractile filaments of myometrium

“…These differences maybearesultofcalix [4]arenepenetrationintoa cell and its interaction with contractile proteins. The abilityofcalix [4]arenestopenetrateinsidethecell wasshownbyotherauthorsusingfluorescentderivativesofsomecalix [4]arenes [11,12]. Similarresultsconcerningtheeffectofsome compoundsontheintegrityofactomyosincomplex havebeendescribedinpublisheddata.Inparticular, theabilityoftapsigargin [13]andphorbolester [14] to affect actin and myosin association was shown by the method of confocal microscopy on the culture of А7r5SMcells;thatisaccompaniedbytransformationofactinfilamentsintodiscreteperipheralcorpusclesandbydiffuselocalizationofmyosin.Such effect is a result of penetration of these compounds to A7r5 cell cytoplasm.…”

Design and synthesis of a multivalent fluorescent folate–calix[4]arene conjugate: cancer cell penetration and intracellular localization

“…25 NBD was selected as a fluorochrome because of its known biocompatibility, easy conjugability, and stability inside the cell. 21,26,27 The CuCAA click reaction was chosen because it can be carried out under mild reaction conditions and generate a 1,2,3-triazole linker biocompatible and stable in biological environments. 28 The presence of resonances relative to the NBD fluorophore (6.34 and 8.46 ppm for the ArH protons and 7.21 for the NH proton) and the triazole ring (7.01 ppm and 124.4 ppm for the CH group) in the 1 H-and 13 C-NMR spectra of 6 evidenced the success of the click reaction.…”