Lysosomal Probe Research Articles

Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane.

Classical chemical probes are prone to dissipation from stressed organelles, as evidenced by the incapability of mitochondrial dyes to image mitophagy linked to multiple diseases. We herein reported mitophagy imaging via covalent anchoring of a lysosomal probe to the mitochondrial inner membrane (CALM). Utilizing DBCORC-TPP, an azide-conjugatable probe with acidity-triggered fluorescence, CALM is operated via ΔΨm-promoted probe accumulation in mitochondria and thereby bioorthogonal ligation of the trapped probe with azido-choline (Azcholine) metabolically installed on the mitochondrial membrane. Overcoming the limitation of synthetic probes to dissipate from stressed organelles, CALM enables signal-on fluorescence imaging of mitophagy induced by starvation and is further employed to reveal mitophagy in ferroptosis. These results suggest the potential of CALM as a new tool to study mitophagy.

A bifunctional sensitive fluorescence probe based on pyrene for the detection of pH and viscosity in lysosome

Lysosome is one of the important organelles in intracellular transport. It plays a significant role in the physiological process. The lysosomal microenvironment affects the functions of lysosome. When the original acidic environment of lysozyme is destroyed or the fluid viscosity increases gradually, various diseases are easily induced. However, most fluorescent probes can only locate in cells. The fewer probes of subcellular organelles were found and their functions are often single. So, it is of great importance to design multifunctional fluorescent probes with the capable of localizing in lysosome. In this study, a novel lysosome probe, 4-(4-Pyren-1-yl-but-3-enyl)-morpholine (PIM), was synthesized using pyrene as a fluorescent group and morpholine as a target group. The introduction of morpholine group made PIM localize in lysosome with high selectivity. The fluorescence will be enhanced with the increased viscosity because of restricting the rotation of CC bond and CN in PIM, and the detecting linear range is from 4.05 cP to 393.48 cP, which qualified the requirement of the viscosity monitoring in body. Meanwhile, the fluorescence intensity of PIM declines with the decrease of pH because the Schiff base of PIM is hydrolyzed, which was affirmed by 1H NMR, LC-MS and fluorescence spectra. Moreover, cell imaging and MTT experiments confirmed that PIM as a novel bifunctional probe can be used to detect pH and endogenous viscosity in lysosome.

One-pot synthesis of conjugated polymer dots with ultrasmall size below 10 nm through Schiff-base chemistry and their bioapplications in monitoring lysosomal pH

Abstract Developing new strategies for facile synthesis of conjugated polymer dots (CPdots) derived from novel monomer species is highly desirable to improve their application. This paper describes a super-simple method, based on Schiff base condensation, for facilely fabricating ultrasmall CPdots under room temperature. This novel CPdots, prepared by reacting the o-benzoquinone with o-phenylenediamine catalyzed by hydrochloric acid, defined as OOPdots, show a variety of superior properties, such as uniform small size, red emission and high quantum yield. With abundant imine and amino groups on the surface, the OOPdots exhibit an excellent pH responsiveness via modulation of the self-assemble or dissociation. Further experiments indicate that the resulting OOPdots are high-performance probes for labeling lysosomes in living cells and zebrafish. This work not only expands the application scope of OOPdots in the field of biological imaging, but also provides guidance for their rapid and large-scale preparation.

An AIEgen-based photosensitizer for lysosome imaging and photodynamic therapy in tumor

Lysosome is an indispensable organelle and its dysfunction often leads to some neurodegenerative diseases. Traditional fluorescent lysosome probes usually have conjugate structures and the π-π stacking can lead to aggregation-caused quenching (ACQ), which limits its application in bioimaging. In this work, we have designed an AIEgen-based photosensitizer MP-TPEDCH by introducing a lysosome-targeting moiety of 4-(2-chloroethyl)morpholine (MP) to a fluorescent AIE skeleton TPEDCH. The optimized structure calculated by density functional theory (DFT) reveals it has a propeller-shaped conformation, which severely inhibits the intermolecular π-π stacking interaction and therefore results in fluorescence quenching in the isolate state. Moreover, it has a broad emission wavelength up to 850 nm and exhibits typical AIE features. The electronic clouds on highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are effectively separated, suggesting MP-TPEDCH had a small singlet-triplet energy gap. Encouragingly, it has been demonstrated that MP-TPEDCH showed high photostability, excellent lysosome targeting specificity, well ROS generation ability and dramatically enhanced antitumor effect under NIR irradiation. The proposed probe not only expands the family of lysosome imaging agents but also provides a new strategy for preparing effective photosensitizers in cancer treatment.

Robust Packing of a Self‐Assembling Iridium Complex via Endocytic Trafficking for Long‐Term Lysosome Tracking

AbstractLive cell imaging of lysosome positioning and motility is critical to studying lysosome status and function for pharmacological interventions. To create a super stable lysosomal probe for long‐term live cell imaging, we have designed and synthesized an aromatic‐peptide‐conjugated cyclometalated iridium(III) complex that emits light via π–π stacking oriented self‐assembly in water at extremely low concentration. Through endocytic trafficking, self‐assemblies are transformed from nanoparticles into sturdily packed networks that are stabilized in lysosomal acidic environment. Upon short time/low dose treatment of the iridium complex at passage 0, live cell lysosomal tracking is applicable beyond the 14th passage of cells with high labelling rate and a mild decline in luminescence intensity. The illuminated lysosomes are trackable using super‐resolution imaging to study their response to cellular processes.

Robust Packing of a Self-Assembling Iridium Complex via Endocytic Trafficking for Long-Term Lysosome Tracking.

Live cell imaging of lysosome positioning and motility is critical to studying lysosome status and function for pharmacological interventions. To create a super stable lysosomal probe for long-term live cell imaging, we have designed and synthesized an aromatic-peptide-conjugated cyclometalated iridium(III) complex that emits light via π-π stacking oriented self-assembly in water at extremely low concentration. Through endocytic trafficking, self-assemblies are transformed from nanoparticles into sturdily packed networks that are stabilized in lysosomal acidic environment. Upon short time/low dose treatment of the iridium complex at passage 0, live cell lysosomal tracking is applicable beyond the 14th passage of cells with high labelling rate and a mild decline in luminescence intensity. The illuminated lysosomes are trackable using super-resolution imaging to study their response to cellular processes.

Fast fluorescence lifetime microscopy imaging of any number of discrete irregular regions of interest

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in biomedical research due to its high specificity, high sensitivity and quantification ability in cell microenvironment sensing. The fluorescence lifetime detection method based on time-correlated single photon counting (TCSPC) is one of the most commonly used techniques at present. However, due to the limitation of imaging principles and conditions, this technique has the disadvantages of long data acquisition time and consequently low imaging speed. In this paper, a fast FLIM technique for any number of discrete and irregular regions of interest (ROIs) in biological samples is developed. The technology uses acousto-optic deflectors (AODs) to achieve fast and flexible addressing scanning, optimize the synchronization strategy between AOD and TCSPC, and reconstruct the lifetime image through simple online feature analysis of the ROI shapes. For the case of multiple discrete irregular ROIs in biological samples, it can greatly save the time of data acquisition, thus realizing the fast FLIM imaging of these ROIs, which is benificial to the study of the heterogeneity of biological events in biological system. In particular, the fast fluorescence imaging result for 87 discrete points in the field of view shows that this method can obtain a fluorescence lifetime image in a very short acquisition time (only 52.2 ms) and thus achieving a very fast imaging speed in such a situation. Dynamic FLIM imaging of lysosome probe LysoSensor Green DND-189 in living cells stimulated by ammonium chloride is carried out to monitor the real-time change of pH value in lysosome lumen. The acquisition time for a single fluorescence lifetime image of lysosomes in two ROIs is only 200 ms. The results show that the rapid FLIM technology can be used to dynamically monitor the changes of microenvironment in biological samples, and will play an important role in the microenvironment sensing in living cells.

Highly Photostable Fluorescent Tracker with pH-Insensitivity for Long-Term Imaging of Lysosomal Dynamics in Live Cells

Visualizing and tracking lysosomal dynamic changes is crucially important in the fields of physiology and pathology. Most currently used pH-dependent small-molecule lysotrackers and sensors usually fail to visualize and track the changes due to (1) their leakage from lysosomes when the lysosomal pH increases and (2) their low photostability. Therefore, it is of significant interest to develop lysosomal probes for visualizing and tracking lysosomal dynamics independent of pH fluctuations and with high photostability. Herein, we found that the popular dicyanomethylene-4H-pyran (DCM) derivative DCM-NH2 can selectively target and label lysosomes with bright red fluorescence regardless of pH changes. The fluorescence enhancement in lysosomes has probably resulted from their microenvironment of polarity and viscosity. Compared with the commonly used commercial lysosomal molecular probes (LysoTracker Deep Red (LTDR) and LysoTracker Red DND-99), DCM-NH2 was demonstrated to exhibit a much stronger tolerance in lysosomes against various treatments and microenvironmental changes, and lysosomal membrane permeability could not cause DCM-NH2 to lose imaging of their targets as well. Moreover, DCM-NH2 exhibited a superior anti-photobleaching ability and low (photo-) cytotoxicity, which, along with pH-insensitivity, ensured its capability of long-term visualizing and tracking lysosomal dynamics. Lysosomal dynamic events such as the kiss-and-run process, fusion-fission, and mitophagy were successfully recorded with DCM-NH2. Our study thus confirms that DCM-NH2 is highly competitive for lysosomal imaging by overcoming the limitations of the commercial LysoTrackers and highlights the unexplored application of DCM-NH2 in bioimaging.

Time-Resolved Luminescent High-Throughput Screening Platform for Lysosomotropic Compounds in Living Cells.

Lysosomes are membrane-bound organelles that regulate protein degradation and cellular organelle recycling. Homeostatic alteration by lysosomotropic compounds has been suggested as a potential approach for the treatment of cancer. However, because of the high false-negative rate resulting from strong fluorescent background noise, few luminescent high-throughput screening methods for lysosomotropic compounds have been developed for cancer therapy. Imidazole is a five-membered heterocycle that can act within the acidic interior of lysosomes. To develop an efficient lysosomotropic compound screening system, we introduced an imidazole group to iridium-based complexes and designed a long-lifetime lysosomal probe to monitor lysosomal activity in living cells. By integrating time-resolved emission spectroscopy (TRES) with the novel iridium-based lysosomal probe, a high-throughput screening platform capable of overcoming background fluorescent interference in living cells was developed for discovering lysosomotropic drugs. As a proof-of-concept, 400 FDA/EMA-approved drugs were screened using the TRES system, revealing five compounds as potential lysosomotropic agents. Significantly, the most promising potent lysosomotropic compound (mitoxantrone) identified in this work would have showed less activity if screened using a commercial lysosomal probe because of interference from the intrinsic fluorescence of mitoxantrone. We anticipate that this TRES-based high-throughput screening system could facilitate the development of more lysosomotropic drugs by avoiding false results arising from the intrinsic fluorescence of both bioactive compounds and/or the cell background.

Simultaneous visualization of lipid droplets and lysosomes using a single fluorescent probe

Lipid droplets (LDs) and lysosomes, as the important subcellular organelles, play vital roles in cell metabolism and physiology. The finding that LDs can be degraded by lysosomes through lipophagy demonstrated the communication between LDs and lysosomes. Thus, simultaneous visualization of LDs and lysosomes is extremely valuable for studying the LD-lysosome interplay. However, single fluorescent probe for simultaneous imaging LDs and lysosomes in live cells and tissues has been rarely reported. Herein, we developed a polarity-sensitive naphthalimide derivative LD-Lys as fluorescent probe with high photostability and low cytotoxicity. Due to its TICT feature, LD-Lys is highly sensitive to the nuance of polarity between LDs and lysosomes, and show strong and weak fluorescence, respectively. The simultaneous two-color imaging of LDs with bright yellow emission and lysosomes with weak red emission is also realized by using the Lambda mode of confocal laser scanning microscope. By monitoring the location changes, real-time tracking the movement of LDs and lysosomes in cells by LD-Lys could be visualized. Interestingly, the size, morphology and distribution of LDs and lysosomes in different tissues could be also visualized clearly. These excellent properties made LD-Lys a promising fluorescent agent for studying the LD-lysosome interplay and metabolism diseases.

Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure

To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA). Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco's Modified Eagle's medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope. After incubating protamine sulfate, negative potential was transformed into positive one ( (-1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h ( P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less. Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.

Isolation of intact Leishmania amazonensis large parasitophorous vacuoles from infected macrophages by density gradient fractionation

As the causative agent of hard-to-treat diffuse cutaneous leishmaniasis, Leishmania (L.) amazonensis persists in the host organism sheltered within large Parasitophorous Vacuoles (PVs) formed mainly in macrophages. In the present study, I present a simple and efficient method for L. amazonensis PV isolation. Isolated PVs are intact as demonstrated by the conservation of lysosomal probes loaded into PVs before the procedure. The method is useful for studies aiming at a complete and accurate molecular profile of these structures, to better understand the biogenesis of this pathogen-containing vacuole and its implication in parasite persistence and in leishmaniasis pathogenesis.

From nucleus to mitochondria to lysosome selectivity switching in a cyanine probe: The phenolic to methoxy substituent conversion affects probe's selectivity.

A cyanine dye with R 2=-OH group has been recently reported to exhibit simultaneous selectivity toward cellular nucleus and mitochondria. In order to investigate the role of the substituents towards the organelle selectivity, probe 2 (with R2=-OR group) was synthesized in good yields. When applied to cellular study, probe 2 exhibited excellent selectivity to stain mitochondria of live cells without observing nucleus staining. The study indicated that the R2 group was the key component in tuning the observed organelle selectivity switching of the probe. This was further verified by removing the hydroxyl group (e.g. R2=-H), which revealed no selectivity to any organelles. The impact of the hydroxyl and alkoxy to intracellular organelle selectivity was further examined in a structurally related system, by replacing a cyanine fragment in probe 2 by a benzothiazole moiety to give a cyanine-benzothiazole hybrid system. In the cyanine-benzothiazole hybrid system, probe 4 (with R2=OCH3) revealed high selectivity towards intracellular lysosomes, which was similarly observed from its hydroxyl analogue (probe 3, R2=OH). Therefore, the impact of the substituent (from -OH to -OMe) to the organelle selectivity was also dependent on the probe structure. In summary, during the study of the substituent effect via structural modification, probe 2 was discovered to exhibit excellent mitochondria selectivity, while Probe 4 was identified as an interesting lysosome probe without affecting lysosomal pH.

One-Step Fabrication of Functional Carbon Dots with 90% Fluorescence Quantum Yield for Long-Term Lysosome Imaging.

Compared with semiconductor quantum dots and organic chromophores, carbon dots (CDs)-based lysosome probe with strong emission, an intrinsic targeting ability, and easy synthesis procedure were urgently desired in visualization imaging studies. Herein, we showed that it was possible to produce CDs with the desired properties for lysosome imaging via a one-step hydrothermal treatment of commercial reagents, rose bengal (RB) and branched polyethylenimine (bPEI). The prepared bPEI-RB CDs (P-R CDs) had a high fluorescence quantum yield (FLQY) of 90.49%, a narrow emission band of 30 nm, negligible phototoxicity, and dark toxicity. Moreover, P-R CDs had an intrinsic lysosome targeting ability without any postmodification of theligands. Long-term cell imaging displayed P-R CDs can anchor lysosomes for up to 48 h without leakage. In addition, experimental results confirmed that dehalogenation cross-linking and structural reorganization of the reactants were the main causes of the ultrahigh photoluminescence efficiency, low cytotoxicity, and passivated surface of the P-R CDs. This origin was attributed to the restricted intersystem crossing and nonradiative transition, the reduced production of singlet oxygen, and suitable -NH2 functional groups. Due to outstanding characteristics, P-R CDs may be developed for a promising tool in lysosome images. The concept of facile preparation will also pave a new avenue for developing ultrabright functional nanomaterial markers.

An azacyclo-localizing fluorescent probe for the specific labeling of lysosome and autolysosome

Understanding lysosome-related physiology needs specific lysosome probes to track the biological processes of lysosome in living cells. Here, we report an azacyclo-modified fluorescent probe that has a large Stokes shift, good photostability and negligible cytotoxicity for highly specific labeling of lysosome and autolysosome in living cells. The probes with different kinds of azacyclo groups on parent dye dansyl are screened to show that dansyl-cycleanine (DNS-C) with four nitrogen atoms possesses the best lysosome-localized ability. And DNS-C as a universal tracker exhibits excellent ability for lysosome labeling in different cell lines with high overlap coefficients (≥0.90). Different from a commercially available LysoTracker, the Stokes shift of DNS-C up to 240 nm (λex/em = 330/570 nm), is much larger than that of LysoTracker ~20 nm (λex/em = 573/595 nm). More importantly, the fluorescence of DNS-C keeps still high brightness after a time-lapsed imaging for 40 min in living cells, implying its remarkable photostability for long-term tracking. In addition, DNS-C can also clearly image the autolysosome, a critical subcellular compartment, forming by the fusion of lysosome with autophagosome in autophagy. These results suggest the promising utility of our probe as a powerful tool to real-time trace physiological processes of lysosomes.

Dual-functioning IQ-LVs as lysosomal viscosity probes with red-shifted emission and inhibitors of autophagic flux

Current fluorescent probes for lysosomes have practical limits due to their pH sensitivity, which makes them unsuitable for long-term tracking of lysosomes in live cell imaging. In addition, viscosity probes are also responsive to polarity, which imposes considerable challenges in cellular imaging. Here, we report IQ-LVs, which can serve as viscosity sensors at the lysosomal pH. In contrast to the majority of current molecular rotors that exhibit blue and green emission, IQ-LVs showed red-shifted emission (∼95 nm) upon increasing the viscosity at pH 4.5. Among the synthesized viscosity sensors, IQ-LV57 and IQ-LV70 lacking an aromatic ring at R1 displayed 15-fold and 116-fold enhancement of red-shifted emission, respectively, upon changes in viscosity. They showed negligible polarity dependency, while the pH sensitivity was minimized by tuning R2 as we envisioned. Our 1D 1H-NMR titrations along with TCSPC analysis revealed that IQ-LVs exhibit two emissions in lysosomes, viscosity-insensitive green emission (+HA’-IQH+) and viscosity-sensitive red-shifted emission (A’-IQH+), which enabled live cell imaging for tracking lysosomes during the autophagy process. In fluorescence confocal imaging, the red emission was enhanced upon the increase in lysosomal viscosity in HeLa and MCF7 cells, and the observed emission from IQ-LV57 and IQ-LV70 had a longer duration than that of LysoTracker™ Deep Red in time-lapse images of live MCF7 cells. Furthermore, treatment of IQ-LV37 in MCF7 cells resulted in increased autophagosomes and autolysosomes during the autophagy process. Further western blot analysis revealed that IQ-LV37 and IQ-LV57 block the degradation of autophagosomes, serving as autophagy inhibitors.

A novel carbazolyl GFP chromophore analogue: synthesis strategy and acidic pH-activatable lysosomal probe for tracing endogenous viscosity changes

A novel, acidic pH-activatable carbazolyl GFP chromophore analogue was designed for tracing lysosomal viscosity changes.

Super-resolution observation of lysosomal dynamics with fluorescent gold nanoparticles.

Because lysosomes play critical roles in multiple cellular functions and are associated with many diseases, studying them at the subcellular level could elucidate their functionality and support the discovery of therapeutic drugs for treating those diseases. The commonly used dyes for super-resolution imaging of lysosomes are the commercial molecular LysoTrackers. But the tolerance to changes in the lysosomal microenvironment and to lysosomal membrane permeabilization (LMP) and the photostability of the LysoTrackers are worrisome. The purpose of our study was to evaluate the feasibility of performing a fluorescent gold nanoprobe for super-resolution observation of lysosomal dynamics in living cells and compare it to the commercial LysoTrackers.Methods: The nanoprobe Cy5@Au NP contained three parts: a bio-inert gold core, a biocompatible polyethylene glycol spacer, and a fluorophore cyanine 5. Structured illumination microscopy (SIM) was employed to capture the fluorescence of Cy5@Au NPs in cells. The tolerance assays to changes in the lysosomal microenvironment and to LMP, the photobleaching assay, and the long-term lysosomes labelling assay of Cy5@Au NPs were compared with commercial LysoTrackers. The super-resolution observation of lysosomal dynamics with Cy5@Au NPs was performed.Results: Cy5@Au NPs can light up lysosomes specifically under SIM. Compared with commercial lysosomal molecular probes, Cy5@Au NPs exhibited stronger tolerance in lysosomes during various treatments, and changes in the lysosomal microenvironment and LMP did not cause Cy5@Au NPs to lose track of their targets. Cy5@Au NPs demonstrated an excellent anti-photobleaching ability, and a long-term labelling assay revealed that they could label lysosomes more than 3 d. Biological events of lysosomes such as the kiss-and-run process, fusion, fission, and mitophagy were recorded with the fluorescent Cy5@Au NPs under SIM.Conclusions: The nanoprobe Cy5@Au NP was successfully used as a lysosomal probe for the super-resolution observation in living cells and found to overcome the limitations of commercial LysoTrackers. Our results thus confirm that nanoparticles can be useful tools for subcellular super-resolution imaging and highlight new avenues for using nanoparticles in biology.

Intrinsic lysosomal targeting fluorescent carbon dots with ultrastability for long-term lysosome imaging.

Lysosomes are crucial dynamic organelles which play key roles in different cellular processes such as autophagy, endocytosis and phagocytosis. Thus, long-term and real-time lysosomal imaging is desirable and essential to understand the dynamics and biological functions of lysosomes. Herein, ultra-stable carbon dots (CDs) which have shown many advantages such as pH-independence, high water solubility, good photostability and high biocompatibility for lysosome labeling and long-term tracking were synthesized using a one-pot pyrolysis method via microwave irradiation. Compared with the commercial lysosome probe (LysoTracker™ Deep Red), the fluorescent CDs show superior resistance to photobleaching and the HeLa cells were stably labeled by CDs for over 48 h. In addition, the CDs could stain lysosomes in different cell lines with high specificity and track lysosomal movements. Furthermore, the CDs could stain not only lysosomes in living cells, but also lysosomes in drug-induced apoptotic cells and fixed cells, suggesting that they are suitable for lysosomal tracking under both physiological and toxicological processes. All these excellent properties could be attributable to the ultrastability of CDs, which can be employed for constructing nanoplatforms for other applications such as drug carriers and signal guiders in biological processes. This study provides not only a strategy to synthesize green fluorescent CDs for tracking lysosomes, but also a promising candidate for drug loading and signal guidance of biological processes.

4-Amino-1,8-naphthalimide based fluorescent photoinduced electron transfer (PET) pH sensors as liposomal cellular imaging agents: The effect of substituent patterns on PET directional quenching

Four new fluorescent sensors (1–4) based on the 4-amino-1,8-naphthalimide fluorophores (Naps) have been synthesized based on the classical fluorophorespacer-receptor model. These four compounds all gave rise to emission bands centred at ca. 535 nm, which were found to be highly pH dependent, the emission being ‘switched on’ in acidic media, while being quenched due to PET from the amino moieties to the excited state of the Nap at more alkaline pH. The luminescent pH dependence for these probes was found to be highly dependent on the substitution on the imide site, as well as the polyamine chain attached to the position 4-amino moiety. In the case of sensor 2 the presence of the 4-amino-aniline dominated the pH dependent quenching. Nevertheless, at higher pH, PET quenching was also found to occur from the polyamine site. Hence, 2 is better described as a receptor1-spacer1-fluorophore-spacer2-receptor2 system, where the dominant PET process is due to (normally less favourable) ‘directional’ PET quenching from the 4-amino-aniline unit to the Nap site. Similar trends and pH fluorescence dependences were also seen for 3 and 4. These compounds were also tested for their imaging potential and toxicity against HeLa cells (using DRAQ5 as nuclear stain which does now show pH dependent changes in acidic and neutral pH) and the results demonstrated that these compounds have reduced cellular viability at moderately high concentrations (with IC50 values between ca. 8–30 µmol·L−1), but were found to be suitable for intracellular pH determination at 1 µmol ·L−1concentrations, where no real toxicity was observed. This allowed us to employ these as lysosomal probes at sub-toxic concentrations, where the Nap based emission was found to be pH depended, mirroring that seen in aqueous solution for 1–4, with the main fluorescence changes occurring within acidic to neutral pH.