This conjugation with an appropriate modification of one side chain of the heptamethine carbocyanine dye did not change the electronic properties of the aromatic backbones in order to maintain the fluorescence and tumor-specific targeting properties. On the other hand, the conjugation of gemcitabine at the 4-(N)-amino group in the pyrimidine ring via amide bond has emerged as one of the most versatile methods for producing gemcitabine prodrugs without compromising drug activity 162. The 56 conjugate penetrates the blood–brain barrier and blood–tumor barrier and increases the bioavailability of the drug in the tumor.
Fluorescent Probes as a Tool in Diagnostic and Drug Delivery Systems
Aggregation-induced emission (AIE) is a relatively new approach for the rational design of fluorescence probes, which rapidly became very popular in the present time 128,129,130. This phenomenon was observed in 2001 by Tang, who found that some silole derivatives showed high fluorescent emissions in a solid state and were non-emissive in solutions 130. However, none of them could explain all of the reported AIE systems 131. AIE compounds played an increasingly important role in the diagnosis and treatment of diseases due to their unique fluorescence properties, biocompatibility, and anti-photobleaching properties 132. Shen et al. used a similar strategy for the synthesis of the ratiometric probe 39 for the selective determination of HOCl in living cells 124.
The probe has good water solubility, NIR fluorescence emission, low toxicity, and great biocompatibility; thus, it was applied for the monitoring of microviscosity fluctuations in living cells. More importantly, this probe confirmed that pulmonary fibrosis has a positive correlation with an increase in viscosity, and it was used for sensitive visualization in the early stage of idiopathic pulmonary fibrosis in vivo in clinical patient tissue. This probe was based on fluorine fluorophore and diphenylphosphinobenzoate recognition moiety using the classical PET “fluorophore–spacer–receptor” model. The presence of diphenylphosphinobenzoate involved a PET process to the fluorine and only a very weak fluorescence was observed.
Fluorescent Probes Based on Aggregation-Induced Emission (AIE)
Lipid droplets are a dynamic organelle that plays a role in some pathological processes, such as fatty liver, neurodegenerative disease, atherosclerosis, and cancer 134,135,136,137. Pylori cells were incubated with 10 μM T2(OH)B for 30 min. Molecular structure of probe 19 and the viscosity sensing mechanism. An excessive level of superoxide anion induced increasing oxidative stress and mitochondrial dysfunction that could be an indication of diabetes rheumatoid arthritis, heart disease, Alzheimer’s disease, and cancer 65,66,67,68,69,70.
In some cases, the excitation of ICT systems induces an intramolecular rotation in which the donor and acceptor parts take more stable coplanar conformation in an excited state. After excitation to the Twisted Intramolecular Charge Transfer (TICT) state, the chromophoric system returns to a ground-state radiative level through red-shifted emissions or by non-radiative relaxation (Figure 16) 78. Proposed reaction mechanism of probe 11 for HClO and Schematic illustration of discriminating cancer cells from normal cells using 11. Fluorescence images of 11-loaded Worldtradex scammers (2 μM, 20 min) cancer cells and normal cells. Accordingly, azo bonds, nitroaromatic, nitroheteroaromatic, and quinonic units all can consider hypoxia-sensitive linkers 24,159.
Li et al. used the same strategy for the real-time and specific detection of neutrophil elastase in living cells 54. Overexpression of neutrophil elastase may degrade some proteins leading to serious lung diseases such as lung injury, chronic obstructive lung disease, acute respiratory, and distress syndrome. In addition, neutrophil elastase can promote tumor growth and metastasis, which indicates lung cancer in tumor tissue 55,56,57,58. Therefore, the detection of neutrophil elastase in living organisms could be used as a critical biomarker for the diagnosis of lung diseases and lung cancer. Probe 9 was designed as a new strategy for the construction of a neutrophil elastase fluorescent probe 54. It contained pentafluoropropionyl as a receptor fragment that was bounded to a 4-trifluoromethyl-7-hydroxyl coumarin fluorophore (Figure 9).
The detection limit was found to be 2.9 nM, and the probe was used for the imaging of ONOO− in MCF-7 cells. Moreover, the probe showed high biocompatibility and low cytotoxicity according to an MTT assay against MCF-7 cells (90% cell viability after incubation with 10–50 μg/mL of the probe). Probe 18 is a TICT probe with low NIR fluorescence at 703 nm, which was enhanced in a higher viscosity solution and showed fluorescence enhancement 54.4 after transitioning from water to glycerol solution buffered with PBS buffer.
Fluorescent Probes Based on Excited-State Intramolecular Proton Transfer (ESIPT)
This fact was used by Song et al. to prepare a viscosity-sensing probe 19 for the detection of fatty liver at the organ level (Figure 20) 86. The TICT nature of this molecule resulted in a low fluorescence centered at 740 nm, which was enhanced by more than 200 times in PBS-buffered glycerol compared to the PBS buffer solution. The probe showed very low toxicity at 30 μM using CCK-8. Moreover, the prevented rotation in this molecule was used to study mitochondrial viscosity mutations in living mouse fatty liver disease models, and it was found that it could distinguish fatty liver mice from normal liver mice. Recently, Akkaya reported another unique molecular logic device as a cancer terminator 153. This behavior can be interpreted as a molecular demultiplexer that used light as an input and phosphatidylserines as the switch between the two alternative outputs, which were represented as singlet oxygen and fluorescent emission (Figure 49).
Fluorescent Probes Based on Fluorescent Resonance Energy Transfer (FRET)
Mei et al. prepared probes 45–47 to study amyloid fibrosis 145. As the main protein model, the authors chose insulin that possessed the determined structure and relatively low molecular weight. Probes 45–47 were designed as cationic AIE fluorophores based on different tetraarylethene frameworks (Figure 45).
- This system showed a fluorescent signal at 728 nm due to the strong ICT occurring from the electron-donating nitrogen in the chelate moiety to the chromenylium-cyanine fluorophore 38.
- This ion is involved in pathological processes, such as Alzheimer’s disease, epilepsy, ischemic stroke, infantile diarrhea, apoptosis, disorder of enzyme regulation, and neurotransmission.
- De Silva who developed the “fluorophore–spacer–receptor” model as rational design of fluorescent sensing systems 88,89,90,91,92.
- Synchronous imaging for multiple analytes during a certain pathophysiological process can provide cross information on the pathology that is highly desirable.
This behavior satisfied the three-input AND logic gate (Figure 50). This logic gate represents an improved platform for the detection of cancer in comparison with 49 using sodium ions and protons as inputs because labile redox-active iron has also been attributed to various forms of cancer 155. The AND logic gate 49 reported by de Silva could be an interesting tool in medicine for diagnostic and therapeutic applications. Compared to normal tissues, in tumor tissues, the pH is quite acidic and the concentrations of the intracellular sodium ions are significantly higher than in normal tissues. Thus, probe 49, whose fluorescence was activated in the presence of a high concentration of protons and sodium ions, could serve as a platform for the selective imaging of cancer.
- Fluorescent oligonucleotide probes, such as molecular beacon probes, play a crucial role in quantitative real-time PCR (qPCR) assays.
- Confocal microscopy images (a–c)—endogenous, exogenous, and inhibitory experiments of formaldehyde in HeLa cells.
- Gunnlaugsson et al. used the “fluorophore–spacer–receptor” format in compounds 24–26 to obtain fluorescent probes for the visualization of microdamage in bones such as cracks and scratches 98.
The endogenic GSH induces 63 to generate PPT and DCM-NH2, resulting in enhanced NIR fluorescence in living cells. Compound 63 showed much lower cytotoxicity to the 293T cells compared to the PPT because of its low concentration of intracellular GSH. The first https://worldtradex.club/ molecular logic gate was based on anthracene fluorophore 49 that was bounded to two different PET receptors—a tertiary alkylamine for selective recognition of protons and a benzo-15-crown-5-ether for selective recognition of the sodium cation 150. Due to the two possible PET channels from the receptors, this molecule displayed a strong fluorescence output only when both PET receptors were suppressed. In other words, compound 49 showed high emissive output in the presence of both protons and sodium cations as chemical inputs.
Due to the lack of donor–acceptor interaction, the ICT process in the probe was not feasible and its fluorescence emission was not observed. However, in the presence of neutrophil elastase, the receptor fragment was hydrolyzed to a hydroxyl group and probe 9 was converted to 4-trifluoromethyl-7-hydroxyl coumarin with a strong ICT nature giving green fluorescence. Furthermore, 9 showed good biocompatibility and low toxicity. Thus, the probe was successfully applied for the selective real-time detection of neutrophil elastase (NE) activity in living HeLa, SKOV3, A549, and HepG2 cells, and zebrafish. The reversible coordination is not the only approach for interaction between the probes and analytes. An alternative way is the reactive-based probes in which a selective chemical reaction between the recognition part in the receptor and the analyte occurs.
Figure 48.
The recent progress in this field has resulted in a large number of different architectures. Among them, fluorescent probes have been the subject of major research interest 7,8,9,10,11,12,13,14,15,16. Mainly, their attractiveness lies in their fluorescence signaling output, which provides a rapid response time, high efficiency and sensitivity, great spatial resolution, and cheap and affordable equipment suitable for field analysis. Moreover, owing to their smaller size, together with their safe and indestructible signaling nature, they are excellent tools in diagnostic medicine and real-time bioimaging 17,18,19,20,21. Fluorescent probes have also found application in the real-time monitoring of drug delivery systems, which provide important information on drug transport and drug distribution in the body, and thereby increasing the quality of medical treatment 22,23,24. In the last decade, the area of fluorescence sensing compounds was extended toward more complex molecular logic gates containing multiple recognition units with multiple signaling outputs 25,26,27.
Figure 55.
The spectra of donor molecule emission (Em.) are overlapped with the spectra of acceptor molecule excitation (Ex.). The efficiency of FRET appears to be the highest when the energy transferred from Em. Is fallen within 10 nm in a parallel orientation. Fluorescence recognition mechanism of probe 4 with Hg2+ and Cu2+ ions. Fluorescence images of Hella cells pretreated with probe (6 μM) for 30 min and after incubated with Hg2+ (120 μM) or Cu2+ (120 μM) for 0.5 h.
