Supramolecular assembly activated single-molecule phosphorescence resonance energy transfer for near-infrared targeted cell imaging

Pure organic phosphorescence resonance energy transfer is a research hotspot. Herein, a single-molecule phosphorescence resonance energy transfer system with a large Stokes shift of 367 nm and near-infrared emission is constructed by guest molecule alkyl-bridged methoxy-tetraphenylethylene-phenylpyridines derivative, cucurbit[n]uril (n = 7, 8) and β-cyclodextrin modified hyaluronic acid. The high binding affinity of cucurbituril to guest molecules in various stoichiometric ratios not only regulates the topological morphology of supramolecular assembly but also induces different phosphorescence emissions. Varying from the spherical nanoparticles and nanorods for binary assemblies, three-dimensional nanoplate is obtained by the ternary co-assembly of guest with cucurbit[7]uril/cucurbit[8]uril, accompanying enhanced phosphorescence at 540 nm. Uncommonly, the secondary assembly of β-cyclodextrin modified hyaluronic acid and ternary assembly activates a single intramolecular phosphorescence resonance energy transfer process derived from phenyl pyridines unit to methoxy-tetraphenylethylene function group, enabling a near-infrared delayed fluorescence at 700 nm, which ultimately applied to mitochondrial targeted imaging for cancer cells.

The authors present a supramolecular assembly activated single-molecule phosphorescence resonance energy transfer (PRET) system which is further useful in mitochondrial targeted imaging.Differing from previous PRET systems by dye-dope or noncovalently assembly of Donor and Acceptor, in this work a single molecule TPE-DPY (D and A covalently linked) is designed and its PRET feature is activated by the secondary supramolecular assembly.No double this interesting work provides a new strategy for the construction and application of PRET system based on singlemolecule.I thus recommend the publication of this work in Nat.Commun., after few points are addressed.1.Although the PRET system is based on the single molecule TPE-DPY, the PRET nature of this molecule does not display until it is activated by the secondary supramolecular assembly.The title "Single-Molecule Phosphorescence Resonance Energy Transfer for NIR Targeted Cell Imaging" overemphasizes "Single-Molecule" PRET and does not fit the work.More specific title, such as "supramolecular assembly activated single-molecule phosphorescence resonance …", is suggested.2. Although TPE-PY is selected as a reference compound for controlled experiments, its binding modes with CB7 or CB7/CB8 may not exactly fit the binding of TPE-DPY with CB7 or CB7/CB8, since the steric hindrance of CB7 should also be considered when assuming two CB7 binding on the two neighboring ethylene pyridine units.Since the following results (TPE-DPY/CB7/CB8 co-assembly) are based on the formation of TPE-PY-CB7 inclusion complex with binding ratio at 4:1, the authors are strongly suggested to provide more solid evidence such as ESI-MS to prove this 4:1 binding ratio.Similarly, how do the authors exclude the presence of TPE-DPY/CB[8] and TPE-DPY/CB [7] in the proposed co-assembly of TPE-DPY/CB[7]/CB[8]?The photophysical properties and morphology could be affected if the ternary co-assembly TPE-DPY/CB[7]/CB [8] is not exclusively formed.3. The authors mentioned that "the protons on methoxyphenyl (H1, H2) in TPE-1 shifted slightly to low-field, while the protons in styryl pyridiniums remained unchanged (Supplementary Fig. S38

Reviewer #3 (Remarks to the Author):
This manuscript by Zhou et al. reports the construction of a single-molecule phosphorescence resonance energy transfer (PRET) system with near-infrared (NIR) emission by using alkyl-bridged methoxy-tetraphenylethylene phenylpyridines derivative (TPE-DPY), cucurbit[n]uril (CB[n], n=7,8), and β-cyclodextrin modified hyaluronic acid (HACD).The authors have shown the RTP energy transfer in a single molecule containing both donor (phenyl pyridines unit) and acceptor (methoxytetraphenylethylene portion) through supramolecular confinement and generated an NIR delayed fluorescence emission at 700 nm which has been applied for mitochondrial-targeted imaging for cancer cells.I find that this work is a follow up/extension of their recently reported work (Adv.Mater. 2022, 34, 2203534), in which the authors have reported a supramolecular confinement RTPharvesting assembly G⊂CB[7]@HACD.This system shows efficient energy transfer to externally doped nile blue or tetrakis(4-sulfophenyl)porphyrin dye, accompanied by a long-lived NIR-emission (680 and 710 nm) which has also been applied for targeted NIR imaging of living tumor cells.On the overall examination and based on the points mentioned below, I don't see any novelty and clarity in this work and hence, I do not recommend the publication of this manuscript in the high impact Nature Communication journal.

Points of concern:
1.There are several literatures regarding the excimer formation in the presence of CB8.Generally, the excimers have the emission band in the longer wavelength region w.r.t the monomer emission as well as the lifetime increases to μs.TPE-DPY molecule has very low fluorescence yield.By forming self-assembly with CB8, the fluorescence intensity increases and the peak position at 540 nm matches well with the emission maxima of the aggregated TPE reported in the literature (J.Am.Chem.Soc. 2011, 133, 50, 20126).In the presence of CB8, TPE-DPY undergoes dimerization and shows fluorescence.The authors may have a look at the data from the above point as well.
2. The interpretation for phosphorescence spectrum of TPE-DPY in the presence of CB8 is mainly based on the qualitative data.The authors need to provide theoretical data to support their claim.
3. The authors state that "Due to the restriction of phenyl-pyridine unit motion by cucurbituril hydrophobic cavities through host-guest complexation, the binary assembly of CB [7] or CB [8] to TPE-DPY all induced a distinct intense phosphorescent emission around 530 nm" Mechanistically how does the restriction of phenyl-pyridine unit motion felicitates triplet population?If this is correct, restriction induced by any other means (say by rigid medium) also should do the same! 5. Why no role of TPE is discussed in the formation of assembly?Only place it was discussed is for the HACD interaction at the methoxy group.But what is the electronic mechanism (other than topological change) by which NIR delayed fluorescence is induced?6.It was stated that "…after the injection of Ar, the lifetime of TPE -DPY/CB[7]/CB[8] aqueous solution at 540 nm was significantly increased from 59.36 μs to 129.97 μs (Supplementary Fig .30) due to the avoidance of the triplet electron quenching caused by oxygen, further confirming the phosphorescence properties of emission peak at 540 nm" My observation on the trace of Fig. 30 says that the decay traces carry two lifetimes (one fast and other slow) and only change is in their relative amplitude contribution and the lifetime values may remain the same.Why a proper analysis is missed?7. The authors have qualitatively described and schematically shown the interactions and assembly formation without adequate quantitative date.How with the presence of such bulky macrocyclic groups the assembly formation is feasible?8. How do the authors justify the efficient energy transfer from one part of the complex to another part and also with HACD through space?It should have been adequately supported by the theoretical modelling justifying the placing of energy levels and their overlap function etc. 9.I agree that the provided NMR data indicate the interactions, but not the mechanism of triplet to singlet energy transfer.

Reply comments
Reply to reviewer 1. 1. On page 2, '… achieving tunable phosphorescence emission, especially in the nearinfrared (NIR) region still faces great challenges owing to the limitation of the energy gap law.28'The citation of reference 28 seems inappropriate, and the authors need to supplement and cite appropriate references.
), indicating the complexation of β-CD and methoxyphenyl unit".But in Fig S38, all proton signals at low field, including protons in styryl pyridinium showed downfield shifts as the same as protons in styryl pyridinium.The Binding mode and Ka should be reevaluated.4. Normally, the figures/schemes are numbered according to the text sequence.Based on that, Fig 3 and Fig 2 should be re-numbered reversely and removed to the corresponding word description.Fig S15 and Fig S23 should be removed to the compounds characterization part since they are COSY spectra of guest molecule rather than host-guest complexes.

Reviewer # 1 (
Remarks to the Author): This study involved the synthesis of a novel supramolecular assembly, consisting of alkyl-bridged methoxy-tetraphenylethylene-phenylpyridines derivative (TPE-DPY), cucurbit[n]uril, and β-CD grafted hyaluronic acid (HACD), leading to the formation of a single-molecule phosphorescence resonance energy transfer (PRET) which was successfully applied in tumor-targeted near-infrared imaging.Cucurbit[n]uril (CB[n]) is employed for inducing the phosphorescence of phenyl pyridines unit from TPE-DPY, while based on the different binding affinities, the primary assembly TPE-DPY/CB[n] presented controllable topological morphologies, realizing a transformation from nanosphere, rod-shaped pseudorotaxane to hierarchical self-assembled nanoplate.The introduction of HACD further changes the topological morphology from nanoplate to nanosphere and activates single intramolecular PRET from the phenyl pyridines unit to the methoxy-tetraphenylethylene portion, ultimately enabling near-infrared targeted imaging of cancer cells.Different from the dye-doped PRET system, this work releases a new concept that the macrocyclic confinement of CB[n] (n = 7, 8) and the cascade assembly of HACD could cooperatively activate the single molecule PRET to achieve a large Stokes shift.And, this work carries great significance in guiding the future development of supramolecular assembly and room-temperature phosphorescence.The present findings are highly valuable and, following some suggested revisions, so it is recommended for publication in Nature Communications.The specific comments for minor revision are as follows.Reply: We are grateful for the reviewer's positive comments.A point-by-point reply is attached below.