Intracellular temperature plays a prominent role in cellular functions and biochemical activities inside living cells, but effective intracellular temperature sensing and imaging is still in its infancy. Herein, thermoresponsive double hydrophilic block copolymers (DHBCs)-based fluorescent thermometers were fabricated to investigate their application in intracellular temperature imaging. Blue-emitting coumarin monomer, CMA, green-emitting 7-nitro-2,1,3-benzoxadiazole (NBD) monomer, NBDAE, and red-emitting rhodamine B monomer, RhBEA, were copolymerized separately with N-isopropylacrylamide (NIPAM) to afford dye-labeled PEG-b-P(NIPAM-co-CMA), PEG-b-P(NIPAM-co-NBDAE), and PEG-b-P(NIPAM-co-RhBEA). Because of the favorable fluorescence resonance energy transfer (FRET) potentials between CMA and NBDAE, NBDAE and RhBEA, as well as the slight tendency between CMA and RhBEA fluorophore pairs, three polymeric thermometers based on traditional one-step FRET were fabricated by facile mixing two of these three fluorescent DHBCs, whereas exhibiting limited advantages. Thus, two-step cascade FRET among three polymeric fluorophores was further interrogated, in which NBD acted as a bridging dye by transferring energy from CMA to RhBEA. Through the delicate optimization of the molar contents of three polymeric components, a ∼8.4-fold ratio change occurred in the temperature range of 20–44 °C, and the detection sensitivity improved significantly, reached as low as ∼0.4 °C, which definitely outperformed other one-step FRET thermometers in the intracellular temperature imaging of living cells. To our knowledge, this work represents the first example of polymeric ratiometric thermometer employing thermoresponsive polymer-based cascade FRET mechanism.Keywords: cascade FRET; intracellular temperature imaging; PNIPAM; polymeric ratiometric thermometers; thermoresponsive DHBCs;

Supramolecular vesicles, also referred to as polymersomes, self-assembled from amphiphilic polymers capable of synchronically loading with both hydrophilic and hydrophobic payloads have shown promising potential in drug delivery application. Herein, we report the fabrication of pH-responsive polymersomes via supramolecular self-assembly of amphiphilic diblock copolymers, poly(ethylene oxide)-b-poly(2-((((5-methyl-2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl)methoxy)carbonyl)amino)ethyl methacrylate) (PEO-b-PTTAMA), which were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization of a pH-responsive monomer (i.e., TTAMA) using a PEO-based macroRAFT agent. The resultant amphiphilic diblock copolymer then self-assembled into vesicles consisting of hydrophilic PEO coronas and pH-responsive hydrophobic bilayers, as confirmed by TEM and DLS measurements. The polymersomes containing cyclic benzylidene acetals in the hydrophobic bilayers were relatively stable under neutral pH, whereas they underwent hydrolysis with the liberation of hydrophobic 2,4,6-trimethoxybenzaldehyde and the simultaneous generation of hydrophilic diol moieties upon exposure to acidic pH milieu, which could be monitored by UV/vis spectroscopy, SEM, and TEM observations. By loading hydrophobic model drug (Nile red) as well as hydrophilic chemotherapeutic drug (doxorubicin hydrochloride, DOX·HCl) into the bilayer and aqueous interior of the polymersomes, the subsequent release of Nile red and DOX·HCl payloads was remarkably regulated by the solution pH values, and a lower pH value led to a faster drug release profile. In vitro experiment, observed by a confocal laser scanning microscope (CLSM), revealed that the pH-responsive polymersomes were easily taken up by HeLa cells and were primarily located in the acidic organelles after internalization, where the pH-responsive cyclic acetal moieties were hydrolyzed and the embedded payloads were therefore released, allowing for on-demand release of the encapsulants mediated by intracellular pH. In addition to small molecule chemotherapeutic drugs, biomacromolecules (alkaline phosphatase, ALP) can also be encapsulated into the aqueous lumen of the polymersomes. Significantly, the pH-triggered degradation of polymersomes could also regulate the release of encapsulated ALP, as confirmed by ALP-activated fluorogenic reaction.

Adequate retention in blood circulation is a prerequisite for construction of gene delivery carriers for systemic applications. The stability of gene carriers in the bloodstream requires them to effectively resist protein adsorption and maintain small size in the bloodstream avoiding dissociation, aggregation, and nuclease digestion under salty and proteinous medium. Herein, a mixture of two block catiomers consisting of the same cationic block, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), but varying shell-forming blocks, poly[2-(2-methoxyethoxy) ethyl methacrylate] (PMEO2MA), and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), was used to complex with plasmid DNA (pDNA) to fabricate polyplex micelles with mixed shells (MPMs) at 20 °C. The thermoresponsive property of PMEO2MA allows distinct phase transition from hydrophilic to hydrophobic by increasing incubation temperature from 20 to 37 °C, which results in a distinct heterogeneous corona containing hydrophilic and hydrophobic regions at the surface of the MPMs. Subsequent study verified that this transition promoted further condensation of pDNA, thereby giving rise to improved complex and colloidal stability. The proposed system has shown remarkable stability in salty and proteinous solution and superior tolerance to nuclease degradation. As compared with polyplex micelles formed from single POEGMA-b-PAsp(DET) block copolymer, in vivo circulation experiments in the bloodstream further confirmed that the retention time of MPMs was prolonged significantly. Moreover, the proposed system exhibited remarkably high cell transfection activity especially at low N/P ratios and negligible cytotoxicity and thus portends promising utility for systemic gene therapy applications.

We report on the fabrication of water-dispersible composite silica nanospheres covalently anchored with gold nanoparticles (AuNPs) possessing thermo-tunable spatial distributions at the outer periphery of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) brushes. Starting from initiator-functionalized silica nanoparticles, surface-initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAM) afforded hybrid silica nanoparticles coated with PNIPAM brushes. The substitution reaction of halogen terminal groups of grafted PNIPAM chains with sodium azide and subsequent click reaction with 1,2-dithiolane-3-pentanoic acid-N-propargylamide afforded hybrid silica nanoparticles coated with 1,2-dithiolane end-capped PNIPAM brushes. AuNPs were then covalently anchored to the outer periphery of hybrid silica nanoparticles by utilizing strong chemisorption of surface-attached dithiolane moieties to AuNPs. Dynamic laser light scattering (LLS) measurements revealed that thermosensitive PNIPAM brushes at the surface of hybrid silica nanoparticles exhibit reversible thermo-induced collapse/swelling transitions, leading to the facile thermo-modulation of spatial distributions of AuNPs covalently attached at the periphery of composite silica nanospheres and thermo-reversible surface plasmon absorption band shift. The reported strategy of covalent assembly of AuNPs into well-defined composite nanospheres possessing thermo-tunable characteristics might be further exploited for colorimetric temperature sensing and responsive SERS detection purposes.

In this work, we integrated the concept of aggregation-induced emission (AIE) with the specific supramolecular recognition between K+ ions and crown ether moieties to develop more effective fluorometric K+ probes. We synthesized a novel crown ether-functionalized tetraphenylethene (TPE) derivative, TPE-(B15C5)4, via the thiol-ene click reaction of thiol-derivatized TPE, TPE-(SH)4, with maleimide-functionalized benzo-15-crown-5 (B15C5). In TPE-(B15C5)4, the TPE core and four outer B15C5 moieties serve as the AIE-active motif and supramolecular K+-recognizing functionalities, respectively. TPE- (B15C5)4 molecularly dissolves in THF with negligible fluorescence emission. As we have envisaged, upon K+ addition, TPE-(B15C5)4 can be effectively induced to aggregate due to K+-mediated cross-linking via the formation of K+/B15C5 (1/2 molar ratio) molecular recognition complex in a sandwiched manner. This process is concomitantly accompanied with the turn-on of fluorescence emission via the AIE mechanism. Thus, TPE-(B15C5)4 can serve as highly sensitive and selective fluorometric off–on K+ probes.

We report on the fabrication of highly sensitive ratiometric fluorescent pH and temperature probes based on thermoresponsive double hydrophilic block copolymers (DHBCs) with the two blocks labeled with two types of dyes possessing different pH-switchable emission characteristics. P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs were synthesized via consecutive reversible addition–fragmentation chain transfer (RAFT) polymerizations in combination with post-modifications, where NIPAM, OEGMA, FITC, and RhBAM are N-isopropylacrylamide, oligo(ethylene glycol) monomethyl ether methacrylate, fluorescein isothiocyanate, and rhodamine B-based derivatives, respectively. Due to that FITC and RhBAM moieties exhibit prominent decrease and increase in emission intensities with decreasing solution pH, respectively, intensity ratios of characteristic RhBAM and FITC emission bands, I582/I522, of P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) unimers at 25 °C exhibit ∼39-fold changes in the range of pH 2–10. At elevated temperatures, thermo-induced formation of PNIPAM-core micelles enables effective fluorescence resonance energy transfer (FRET) between FITC and RhBAM moieties respectively located within micellar cores and coronas, and I582/I522 exhibits ∼52.5-fold changes in the same pH range. The reported dually modulated multicolor-emitting P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs are capable of ultrasensitive fluorometric detection of solution pH and temperature in a ratiometric manner, which augurs well for their practical applications in sensing, imaging, and the fabrication of new generation of theranostic systems.

We report on the fabrication of water-dispersible composite silica nanospheres covalently anchored with gold nanoparticles (AuNPs) possessing thermo-tunable spatial distributions at the outer periphery of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) brushes. Starting from initiator-functionalized silica nanoparticles, surface-initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAM) afforded hybrid silica nanoparticles coated with PNIPAM brushes. The substitution reaction of halogen terminal groups of grafted PNIPAM chains with sodium azide and subsequent click reaction with 1,2-dithiolane-3-pentanoic acid-N-propargylamide afforded hybrid silica nanoparticles coated with 1,2-dithiolane end-capped PNIPAM brushes. AuNPs were then covalently anchored to the outer periphery of hybrid silica nanoparticles by utilizing strong chemisorption of surface-attached dithiolane moieties to AuNPs. Dynamic laser light scattering (LLS) measurements revealed that thermosensitive PNIPAM brushes at the surface of hybrid silica nanoparticles exhibit reversible thermo-induced collapse/swelling transitions, leading to the facile thermo-modulation of spatial distributions of AuNPs covalently attached at the periphery of composite silica nanospheres and thermo-reversible surface plasmon absorption band shift. The reported strategy of covalent assembly of AuNPs into well-defined composite nanospheres possessing thermo-tunable characteristics might be further exploited for colorimetric temperature sensing and responsive SERS detection purposes.

We report on the fabrication of highly sensitive ratiometric fluorescent pH and temperature probes based on thermoresponsive double hydrophilic block copolymers (DHBCs) with the two blocks labeled with two types of dyes possessing different pH-switchable emission characteristics. P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs were synthesized via consecutive reversible addition–fragmentation chain transfer (RAFT) polymerizations in combination with post-modifications, where NIPAM, OEGMA, FITC, and RhBAM are N-isopropylacrylamide, oligo(ethylene glycol) monomethyl ether methacrylate, fluorescein isothiocyanate, and rhodamine B-based derivatives, respectively. Due to that FITC and RhBAM moieties exhibit prominent decrease and increase in emission intensities with decreasing solution pH, respectively, intensity ratios of characteristic RhBAM and FITC emission bands, I582/I522, of P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) unimers at 25 °C exhibit ∼39-fold changes in the range of pH 2–10. At elevated temperatures, thermo-induced formation of PNIPAM-core micelles enables effective fluorescence resonance energy transfer (FRET) between FITC and RhBAM moieties respectively located within micellar cores and coronas, and I582/I522 exhibits ∼52.5-fold changes in the same pH range. The reported dually modulated multicolor-emitting P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs are capable of ultrasensitive fluorometric detection of solution pH and temperature in a ratiometric manner, which augurs well for their practical applications in sensing, imaging, and the fabrication of new generation of theranostic systems.