Supramolecular Assembly-Activated Single-Molecule Phosphorescence Resonance Energy Transfer for Near-Infrared Targeted Cell Imaging

In recent years, pure organic phosphorescence resonance energy transfer (PRET) research has become a hot topic. In this paper, the authors constructed a single-molecule PRET system with a large Stokes shift (367 nm) and near-infrared (NIR) emission using the host molecule alkyl-bridged methoxy-tetraphenylethylene-based phenylpyridine derivatives (TPE-dpy), cucurbit[n]urils (CB[n], n = 7, 8) with different parameters, and hyaluronic acid-modified β-cyclodextrin (HAcd). The authors successfully applied this system for targeted imaging of mitochondria in cancer cells.

Research Background

Supramolecular assembly has long been a focus of attention due to its important applications in molecular recognition, catalysis, luminescent materials, medicine, and sensing. In particular, supramolecular assembly based on macrocyclic compounds has attracted significant research interest due to its ability to suppress vibrational deactivation of singlet or triplet excitons, thereby inducing and improving the photophysical properties of guest molecules, especially room-temperature phosphorescence (RTP). Although rapid progress has been made in RTP systems, achieving tunable phosphorescence emission, particularly in the NIR region, still faces significant challenges. This limitation is mainly due to the constraints of the energy gap law.

Paper Source

This paper was written by researchers Xiaolu Zhou, Xue Bai, Fangjian Shang, Heng-yi Zhang, Li-hua Wang, Xiufang Xu, and Yu Liu from the State Key Laboratory of Elemento-Organic Chemistry, Department of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University. The research was accepted on May 28, 2024, and can be found in Volume 15, Issue 4787 of Nature Communications.

Research Workflow

Research Subjects

The research mainly focused on how to construct a single-molecule PRET system for targeted mitochondrial imaging using cucurbit[n]urils and hyaluronic acid-modified cyclodextrin. In the study, the authors explored the topological conformations and binding behaviors of supramolecular assemblies, as well as the activation of single-molecule PRET processes through secondary assembly.

Experimental Steps

1. Synthesis and Characterization of Guest Molecules:

   • Methoxy-tetraphenylethylene was synthesized with different amounts of alkyl-bridged phenylpyridines via Mizoroki-Heck reaction and alkyl substitution.
   • Detailed characterization was performed using ^1H NMR, ^13C NMR, 2D COSY, and high-resolution mass spectrometry (HRMS).

2. Exploration of Cucurbituril Binding Behavior and Topological Conformations:

   • The binding behavior of TPE-dpy with CB[7] and CB[8] was investigated using ^1H NMR, UV-Vis titration, and 2D ROESY/DOSY spectra.
   • Changes in the conformations of different assemblies were observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

3. Secondary Supramolecular Assembly and Single-Molecule PRET Activation:

   • HAcd was introduced into the TPE-dpy/CB[7]/CB[8] system to construct secondary assemblies, and the changes in topological conformations were observed.
   • The secondary assemblies were further characterized using dynamic light scattering (DLS), TEM, and zeta potential experiments, and the optical properties of the PRET process were studied.

4. Photophysical Properties and Mechanistic Studies:

   • The photophysical properties of different assemblies and the underlying mechanisms, including RTP and PRET behavior, were explored.
   • Density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations were used to support the experimental results.

5. Cancer Cell Targeted Imaging:

   • Targeted imaging experiments were performed using human cervical cancer cells (HeLa) and human embryonic kidney cells (293T), and the NIR luminescence signal within the cells was observed using confocal laser scanning microscopy (CLSM).

Key Experimental Details

Synthesis of TPE-dpy

Under nitrogen protection, 4,4’-[[2,2-bis(4-methoxyphenyl)ethylidene]bis(4,1-phenylenevinylene)]pyridine (54 mg, 0.09 mmol) and py-1 (94.5 mg, 0.22 mmol) were dissolved in 5 mL of acetonitrile. The mixture was heated at 85°C for 36 hours. After the reaction, the acetonitrile solvent was removed by ultrasonication, and the residue was washed with acetone. Finally, the product was obtained by heat filtration and recrystallization.

Investigation of TPE-dpy Binding with Cucurbiturils

1. ^1H NMR and UV-Vis spectra of TPE-dpy with CB[8] and CB[7] systems were collected, and the binding constants were calculated using the Job’s plot method.
2. The binding modes were explored using 2D ROESY and 2D DOSY spectra, ultimately confirming a 1:1 binding stoichiometry between TPE-dpy and CB[8], and a 1:4 binding stoichiometry with CB[7].

Photophysical Properties Studies

When the binary assemblies of TPE-dpy with CB7 or CB[8] changed, the absorption peaks showed different degrees of red shifts. In the systems of TPE-dpy with 2CB[7], 4CB[7], CB[8], and CB[7]/CB[8], the PL spectra displayed substantial emission peaks around 530 nm, indicating that the system exhibited long-lived characteristics.

Secondary Assembly and PRET Activation: 1. The structural changes of TPE-dpy with CB[7]/CB[8] upon the addition of HAcd were determined, and the assembly behavior was explored using DLS, TEM, and zeta potential experiments. 2. The PRET reaction was demonstrated under 330 nm excitation, with the observation of NIR delayed fluorescence at 700 nm.

Cancer Cell Imaging Application: 1. The imaging performance of TPE-dpy/CB[7]/CB[8]@HAcd in HeLa and 293T cells was compared. 2. The colocalization analysis confirmed that the NIR signal was focused on mitochondria.

Research Conclusions and Significance

This paper successfully activated a single-molecule PRET system based on macrocyclic constraints and achieved NIR delayed fluorescence emission in cancer cells. With the aid of different cavity sizes of CB[7] and CB[8], the primary assembly of TPE-dpy exhibited tunable topological conformations. The secondary assembly of HAcd further activated the single-molecule PRET, generating NIR delayed fluorescence emission at 700 nm. This system exhibited a large Stokes shift (367 nm) and demonstrated successful application in targeted imaging of cancer cell mitochondria, providing a feasible pathway for the construction and application of single-molecule PRET.

Research Highlights

1. Large Stokes Shift (367 nm): The significant large Stokes shift of this system imparts distinct optical characteristics.
2. Supramolecular Topological Conformations Constructed by Cucurbiturils: At different binding stoichiometries, cucurbiturils participate in the system assembly, exhibiting an evolution of conformations from nanospheres to nanorods and multilayered nanoplates.
3. Single-Molecule PRET and NIR Emission Imaging: Successful NIR (700 nm) delayed fluorescence emission was achieved in cancer cell imaging, demonstrating its potential in medical imaging applications.

This research not only provides a novel approach to activate single-molecule PRET systems but also offers new possibilities for NIR targeted imaging applications. Future research directions may include further optimization of the PRET system to improve luminescence efficiency and exploration of its applications in other biomedical fields.