Fluorescent Method For Monitoring Gene/Drug Delivery And Fluoride Ion Detection

In this dissertation, we conbine the technology of controlled/living radical polymerizations and click chemistry as well as Michael addition chemistry, a series of well-defined DHBCs were synthesized for the visualization of the drug delivery process, and a novel gene delivery system was also developed for the visualization of the gene delivery process. Thermo-responsive double hydrophilic diblock copolymers functionalized by fluorescent turn-on probes for the detection of fluoride ions were discussed at last. The dissertation includes the following four parts:1. Supramolecular aggregates of stimuli-responsive block copolymers are increasingly utilized as drug nanocarriers. Although in situ tracking their triggered disintegration and drug release processes at the cellular level is highly desirable, it remains a considerable challenge. We report the fabrication of double hydrophilic block copolymers covalently conjugated with α,β-unsaturated ketone-caged coumarin functionalities in the thermoresponsive block; upon thermo-induced micellization and cellular uptake, Michael addition reaction of unsaturated ketone moieties with thiol compounds (GSH) in the reductive subcellular compartments leads to micelle-to-unimer transition; this is accompanied with concomitant fluorescence emission turn-on and triggered drug release, allowing for the process visualization.2. Gene therapy has emerged as a promising tool in the treatment of severe genetic diseases. Numerous gene carriers have been developed to increase gene transfection efficiency and decrease cytotoxicity. However, intracellular visible gene delivery process remains a considerable challenge. Herein, a cationic amphiphile consisting of poly(2-dimethylaminoethyl methacrylate)(PDMAEMA) and thiol-reactive moiety (i.e., PyCMA) in the distal end derived from coumarin chromophore bearing α,β-unsaturated ketone residue was applied for gene delivery. The resultant cationic PyCMA-PDMAEMA amphiphiles self-assemble into micellar nanoparticles and complex with oppositely charged DNA with the formation of polyplexes, which are internalized into cells via endocytosis, followed by endosomal escape into cytosol and subjected to GSH-involved Michael addition reaction of PyCMA moieties at neutral pH, resulting in turn-on blue fluorescence, disintegration of polyplexs, and release of DNA payloads. This work represents a novel strategy to construct gene carriers, where intracellular endogenous stimuli are applied to regulate the self-assembled polyplexes, thereby visualizing the release of DNA payloads in specific sites through turn-on fluorescent signals and exhibiting optimized gene transfection and decreased cytotoxicity.3. We report on the fabrication of a novel type of responsive double hydrophilic block copolymer (DHBC)-based highly selective and sensitive fluorescence "turn-on" reactive probes for fluoride ions (F") working in purely aqueous media by exploiting F"-induced cyclization reaction of nonfluorescent moieties to induce the formation of fluorescent coumarin moieties within the thermoresponsive block. Diblock copolymers bearing F--reactive moieties (SiCouMA) in the thermoresponsive block, PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA), were synthesized at first via reversible addition-fragmentation chain transfer (RAFT) technique followed by post-modification, where PEO, MEO2MA, and OEGMA are poly(ethylene glycol), di(ethylene glycol) monomethyl ether methacrylate, and oligo(ethylene glycol) monomethyl ether methacrylate, respectively. As-synthesized diblock copolymers molecularly dissolve in water at room temperature and self-assemble into micellar nanoparticles above the critical micellization temperature (33℃). In the presence of F" ions, deprotection of nonfluorescent SiCouMA moieties followed by spontaneous cyclization reaction leads to the formation of highly fluorescent coumarin residues (CouMA). Thus, PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) diblock copolymers can serve as highly efficient and selective fluorescence "turn-on" reaction probes for F" ions in aqueous media. In the range of0-1600equiv. F-ions, diblock unimers and micellar solutions at20℃and40℃exhibit～88-fold and～30-fold increase in fluorescence emission intensity (20min incubation time), respectively. The detection limits were determined to be0.065ppm and0.05ppm for diblock unimers and micelles, respectively. Most importantly, in the low F" concentration range, excellent linear correlation between F-concentration and emission intensity was observed (0～15ppm for unimers at20℃and0-8ppm for micelles at40℃). Interestingly, upon complete transformation of nonfluorescent SiCouMA moieties into fluorescent CouMA, the emission intensity of diblock copolymer solution decreases linearly with temperatures in the range of20-60℃, suggesting its further application as fluorometric temperature sensors. 4. We also report on a fluorometric chemosensor for fluoride ions working in both purely aqueous media and THF solution based on4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBDAE) and t-butyldimethylsilyl caged F"-reactive moieties (SiCouMA) labeled diblock copolymers. P(St-co-SiCouMA)-b-P(NIPAM-co-NBDAE) diblock copolymers were synthesized at first via reversible addition-fragmentation chain transfer (RAFT) technique followed by postmodification, where St and NIPAM are styrene and N-Isopropyl acrylamide respectively. While synthesized diblock copolymers molecularly dissolve in THF solution (4v/v%, water), upon gradual addition of TBAF, the deprotection of nonfluorescent SiCouMA moieties followed by spontaneous cyclization reaction leads to the formation of highly fluorescent coumarin moieties and the blue fluorescence emission of coumarin moieties gradually turn on, the green fluorescence emission of NBDAE moieties can be dramatically quenched due to the formation of hydrogen-bonding (H-bonds) interactions, accompanied with the colorimetric transition from green to pale yellow. While the diblock copolymer self-assembled into micelles in water, upon treating with NaF, the blue fluorescence emission of coumarin moieties gradually turn on for the deprotection of SiCouMA moieties, while the green fluorescence emission of NBDAE moieties remain almost constantly because of the influence on H-bonds interaction of NBDAE moieties and fluoride ions by water. The detection limit of F" ions can be down to0.009ppm in THF and0.11ppm in water respectively. Importantly, in the low F-concentration range, excellent linear correlation between F" concentration and emission intensity ratio (F408/F530) was observed (0-30ppm in THF and0-10ppm in water respectively). This work presents a new example of F" probes for both tetrabutylammonium fluoride salts and water soluble sodium fluoride salts. Furthermore, upon complete transformation of nonfluorescent SiCouMA moieties into fluorescent coumarin in water, the emission intensity ratio (F408/F530) of diblock copolymer solution increase almost linearly with temperatures in the range of20-45℃, suggesting its further application as ratiometric temperature sensors.