ACS Nano: reversible self-assembled nanoprobe for dynamic detection of intracellular pH changes

Self-assembly is a powerful tool to make basic molecular units into functional nanostructures, which plays an important role in optical sensing and drug delivery control Molecular self-assembly is mainly driven by a variety of weak and reversible non covalent interactions, including electrostatic interactions, π - π stacking, van der Waals interactions and hydrophobic effects However, the stability of self-assembled nanostructures is often destroyed by the complex and changeable intracellular environment Therefore, how to build reversible self-assembly system in vivo is still a huge challenge Recently, a reversible self-assembly system with hydrophobic gold nanoparticles (AuNP) as the core and pH responsive dye-doped copolymers as the shell was designed and synthesized by Liu dingbin's research group, School of chemistry, Nankai University The reversible assembly disassembly process can be precisely controlled by adjusting the intermolecular force between the copolymers and AuNP Relevant achievements were published in acsnano (DOI: 10.1021 / acsnano 8b07054) under the title of "reversible self assembly of nanoprobes in live cells for dynamic internal pH imaging" Firstly, a series of pH responsive block copolymers were synthesized by atom transfer radical polymerization (ATRP) These copolymers are composed of hydrophilic polyethylene glycol (PEG) chains and hydrophobic blocks with tertiary amines (DPA, Dipa and HA), and are conjugated with pH insensitive Cy5 dyes as imaging beacons Subsequently, the author used dodecyl mercaptan to cover the AuNP, making the NP core hydrophobic, thus initiating the self-assembly of the copolymer onto the AuNP (Fig 1a) The results of UV-Vis spectrum analysis show that the characteristic absorption band of AuNP is red shifted from 525 nm to 535 nm, and the absorption band of Cy5 appears at about 600 nm AuNP has high fluorescence quenching efficiency When Cy5 is very close to the surface of AuNP, its surface fluorescence is effectively quenched (Fig 1b) The transmission electron microscope (TEM) image clearly shows the core-shell structure of the nanomodule, in which the thickness of the organic layer around the AuNP core is about 3 nm (Fig 1c) Then, the disassembly behavior of the nano module was investigated When the solution was adjusted to acidity, it was found that the fluorescence intensity of Cy5 increased significantly, which indicated that the copolymer had been released from AuNP The main reason for this phenomenon is that under acidic condition, Dipa group is positively charged, which weakens its hydrophobic interaction with the core of AuNP covered by dodecyl mercaptan Furthermore, DPA and HA were conjugated with tetramethylrhodamine (TMR) and cy7.5 dyes respectively, which also proved the assembly and disassembly process of pH dependent nanomodules Surprisingly, the three nanomodules showed significant fluorescence transitions at different pH values (Fig 1E) In order to quantitatively evaluate the pH response properties, the fluorescence intensity was normalized by the ratio (f-f min) / (f max-f min), where f was the fluorescence intensity at the desired pH value, and f Max and f min were the fluorescence intensity of completely restored fluorescence and fluorescence quenched state The extremely low Δ pH value of 10-90% (the pH range of the normalized fluorescence intensity varies from 10% to 90%) fully demonstrates the high sensitivity of the nanomodule to the change of pH (Fig 1F) (source: acsnano) then, the molecular dynamics simulation of the assembly disassembly process of nano module was carried out In order to reduce the calculation cost, the molecular structure of the copolymer is simplified into three repeat units of Dipa conjugated peg The simulation results show that when the pH is more than 6.2, the copolymers are adsorbed on the surface of AuNP, and the Dipa chain segment is anchored in the dodecyl mercaptan; when the pH is 6.2, it is easier to bind on the surface of AuNP than the protonated Dipa (pH < 6.2), which shows that there is a stronger interaction between the non protonated Dipa and AuNP The free energy curve of the interaction between the two DIPAS shows that there is an obvious repulsion between the two protonated DIPAS (Fig 2D), which is significantly stronger than the attraction between Dipa and AuNP (Fig 2C) Therefore, the pH induced disassembly is attributed to the weak interaction between the protonated Dipa chain segment and the hydrophobic AuNP surface, as well as the strong repulsion between protonated Dipa under acidic conditions Furthermore, the electrostatic potential (ESP) of Dipa in different states is calculated by Gaussin 09 program, and the result is highly consistent with that of free energy (Fig 2e) (source: acsnano) is based on the reversible ionization property of tertiary amine (TA) group The author controls the assembly disassembly process reversibly by changing the pH value of the solution Under the condition of pH 6.0-7.4, the fluorescence of the nanomodule was restored and quenched three times (Fig 3a and 3b), respectively, which showed the reversibility of the assembly disassembly process Then, the author explored whether the protein affects its recombination process The results show that protein has a great influence on the process of copolymer reconstitution (Fig 3C), but does not affect the recombination of copolymer based on AuNP (Fig 3D) It is gratifying that the calculation results of the binding free energy of the copolymer with AuNP and BSA are also consistent with the experimental results (Fig 3E and Fig 3f) The above results show that the copolymers have higher affinity with AuNP (source: acsnano) based on the good results of in vitro experiments, the authors used nano modules to monitor the pH changes of different organelles Firstly, the Au NP modified by peg-b - (pdipa-r-cy5) and the Au NP modified by peg-b - (pdpa-r-tmr) were assembled with the copolymer formed by RGD with the target function to form the probe pH t 6.2 and pH t 4.4, respectively The two probes were incubated with HepG2 cells and the imaging results were recorded by confocal microscopy After the probe entered the cell, it was activated Within 4 hours, the fluorescence signals of Cy5 and TMR channels gradually recovered, while after incubation for 5-7 hours, the fluorescence signals gradually weakened (Fig 4) The authors speculate that reversible pH responsive fluorescence activation quenching may be caused by the reversible assembly of polymer to the core of AuNP, which can be verified by TEM imaging of cell sections It was further found that the nanoprobe mainly undergoes a cycle of endocytosis and exocytosis In cells, the nanoprobe first encounters the acidic primary endosome (pH = 6.0-6.5), then transports to the secondary endosome / lysosome (pH = 4.0-5.5), and finally transports to organelles such as Golgi body (pH = 7.4) (source: acsnano) finally, the transport of probes from primary endosome to secondary endosome / lysosome in cells was studied HepG2 cells were transfected with green fluorescent protein (GFP) fused RAB5A and lamp1 biomarkers The corresponding biomarkers can specifically recognize primary and secondary endosomes / lysosomes, respectively Then, the GFP stained cells were incubated with the probe, and the probe and specific organelles were Co located by confocal imaging The results showed that probe pH t 6.2 remained in the primary endosome within 4 hours, while other probes gradually activated in the secondary endosome / lysosome (Fig 5a) After 6 hours, probe pH t 6.2 completely transferred from primary endosome to secondary endosome / lysosome, while other probes have transferred to organelles such as Golgi body In contrast, probe pH t 4.4 remained "silent" in both primary and secondary endosomes / lysosomes within 2 hours After incubation for 4 hours, the fluorescence of probe pH t4.4 gradually recovered These results show that although the two pH probes can enter the same compartment, their fluorescence activation behavior is different Based on the above results, the dynamic disassembly recombination process of two probes in different organelles was proposed After entering the cell, the pH responsive polymer in probe pH t 6.2 can be rapidly released into the primary endosome When the probe enters the secondary endosome / lysosome, the decomposition process reaches saturation On the contrary, the decomposition of probe pH t 4.4 occurred only in the secondary endosome / lysosome When the probe was transferred to organelles such as Golgi apparatus, the released polymer was reassembled on the surface of AuNP and fluorescence quenching occurred again (Fig 5C) (source: acsnano) in a word, the author designed and synthesized a series of pH responsive copolymers, which can be reversibly assembled on the core of AuNP in aqueous solution to build nano components The assembly and disassembly process is initiated by reversible protonation of Ta Group, and realized by adjusting the interaction between the copolymer and AuNP nucleus The nanomodule has the following advantages: 1 It can be assembled and disassembled reversibly under physiological conditions; 2 It is a good fluorescence quenching agent while being used as a self-assembly template; 3 A few copolymers on the nanomodule can still be used for biological imaging; 4 The nanomodule is highly sensitive to pH; 5 The pH t value of the nanomodule can be adjusted The discovery of these pH responsive nanocomposites will help to track the transport of nanoparticles in living cells, and can be applied in biological and biomedical fields.