【Angew.Chem.】突破能量间隙律！126.1 ms超长寿命深红/近红外有机余辉纳米颗粒助力高对比度活体成像
文章标题：White‐Light‐Excitable Deep‐Red/NIR Organic Afterglow Nanoparticles for High‐Contrast In Vivo Imaging
通讯作者：Bin Liu
文章概要
引言
近红外有机余辉探针因其长寿命发光和能消除背景荧光干扰的特性，在深层组织高对比度生物成像中展现出巨大潜力。然而，受限于能量间隙律，如何在单一有机发光体中同时实现白光激发、长寿命近红外磷光以及均匀纳米颗粒的制备，一直是该领域面临的重大挑战。传统的“自上而下”晶体粉碎法往往会导致颗粒尺寸不均和材料损失，限制了其临床转化与实际应用。
Rational design and photophysical properties of white-light-excited NIR organic RTP materials. (a) Chemical structures of host and guest molecules. (b) Schematic illustration of the lattice-matched host–guest doping strategy. (c) Absorption, fluorescence, and phosphorescence spectra of PyC and BPC in toluene solution (1 × 10−5 M). (d) Normalized steady-state and delayed (10 ms) emission spectra of the doped systems (inset: photographs of luminescence from the doped systems under UV irradiation [on] and immediately after UV removal [off]). (e) Decay curves monitored at 637 nm for BMC&PyC and BMC&BPC. (f) Phosphorescence excitation spectra (with 1 ms delay) recorded at 637 nm emission for BMC&PyC and BMC&BPC. (g, h) Excitation–delayed emission mapping (with 1 ms delay) for BMC&PyC (g) and BMC&BPC (h).
主要实验及结论
为了攻克这一难题，研究团队创新性地提出了一种“自下而上”的策略，通过将具有外延共轭结构的苯基咔唑客体分子（PyC或BPC）引入到刚性的BMC宿主晶格中，成功构建了晶格匹配的宿主-客体纳米晶体。实验结果表明，该掺杂体系展现出客体主导的深红/近红外余辉发射，最长振动带达到762 nm，并可被高达475 nm的可见光有效激活，实现了高达126.1 ms的超长寿命和2.7%的磷光量子产率。单晶衍射与理论模拟进一步证实，宿主与客体之间极佳的几何相容性提供了稳固的刚性微环境，大幅抑制了非辐射衰减。
Theoretical simulation studies of BMC&PyC and BMC&BPC. (a) Optimized geometries and excited-state properties calculated for BMC&PyC and BMC&BPC using quantum mechanics/semi-empirical extended tight-binding (QM/xTB) methods. (b) Hirshfeld surface analyses for the BMC cluster, BMC&PyC, and BMC&BPC. (c) Distribution of C–H distances (_d_i + _d_e) within 2.70 Å and corresponding contact areas between central guest and surrounding host molecules in the BMC cluster, BMC&PyC, and BMC&BPC systems. (d) Calculated energy levels and spin–orbit coupling (SOC) constants for the studied systems. (e) Root mean square displacement/deviation (RMSD) between S0 and T1 geometries for isolated PyC, PyC in BMC&PyC, isolated BPC, and BPC in BMC&BPC.
Energy transfer mechanism investigation of BMC&PyC and BMC&BPC. (a) Photoluminescence (PL) spectra of pure BMC and BMC&PyC under 310 nm excitation. (b) PL spectra of BMC&PyC under 310 nm excitation at various doping concentrations. (c) Delayed-emission excitation spectra (1 ms delay) monitored at 637 nm for BMC&PyC at varying doping concentrations. (d) Enhancement factors of the 1 ms delayed-emission intensity at 637 nm (relative to the intensity at 0.05% doping) for BMC&PyC at various doping concentrations excited at 310 and 390 nm (mean ± SD, n = 5). (e) Fluorescence excitation spectra (monitored at 479 or 525 nm) and phosphorescence excitation spectra (monitored at 637 nm) for BMC&PyC and BMC&BPC. (f) Jablonski diagram illustrating the proposed energy transfer routes in the lattice-matched host–guest systems (ET, energy transfer; ISC, intersystem crossing).
在此基础上，团队利用简单的高分子辅助重沉淀法，直接制备出单分散的发光纳米颗粒，有效避免了传统制备工艺中的尺寸异质性。在生物应用中，这些纳米颗粒展现出极低的细胞毒性和优异的细胞内吞能力。体内成像实验表明，在白光预照射后，纳米颗粒在小鼠体内的深红/近红外余辉可持续超过720秒，且能穿透厚度超过8 mm的肌肉组织。更重要的是，利用白光的原位循环激活特性，研究人员成功实现了对小鼠淋巴肿瘤转移与动态输运过程的实时、高对比度追踪。
Bottom-up fabrication and characterization of NPs. (a) Schematic of polymer-assisted nanoprecipitation used to formulate BMC&PyC and BMC&BPC nanoparticles (NPs) (inset: fluorescence and phosphorescence images). (b) Steady-state and delayed emission spectra of BMC&PyC and BMC&BPC NPs. (c, d) Particle-size distributions of BMC&PyC and BMC&BPC NPs (insets: Transmission electron microscopy (TEM) micrographs; scale bar: 100 nm). (e, f) Cell viability of BMC&PyC, BMC&BPC, and their NPs in 4T1 cells (mean ± SD, n = 3). (g, h) Time-gated phosphorescence cellular imaging of BMC&PyC and BMC&BPC NPs in 4T1 cells at increasing incubation times (scale bar: 20 µm).
Bioimaging performance of BMC&PyC and BMC&BPC NPs. (a) Time-dependent phosphorescence images of BMC&PyC and BMC&BPC NPs acquired on an IVIS imaging system (bioluminescence mode) after 60 s of white-light pre-irradiation. (b) Through-tissue phosphorescence images of NPs covered by chicken breast tissue of increasing thickness after 60 s of white-light pre-irradiation. (c) Quantification of integrated phosphorescence signal versus tissue thickness for (b) (mean ± SD, n = 3). (d) In vivo phosphorescence images of mice with subcutaneous injections of BMC&BPC NPs acquired under two imaging modes (pre-excitation mode and post-excitation mode; white-light irradiation for 60 s). (e) Quantitative analysis of phosphorescence signals in (d) (mean ± SD, n = 3). (f) In vivo phosphorescence images of mouse hind paw and lymph node tumor injected with BMC&BPC NPs (100 µL, 2 mg/mL) following 60 s of white-light pre-irradiation. (g) Quantification of signals corresponding to (f) (mean ± SD, n = 3).
总结及展望
本研究成功搭建了一个“自下而上”的白光激发深红/近红外有机余辉纳米晶体平台，为安全、高对比度的临床生物成像提供了全新方案。这种将晶格匹配的宿主-客体光物理学与底部分散制备相结合的设计思路，有效突破了传统余辉材料的性能瓶颈。展望未来，研究团队将致力于开发具有更长余辉寿命的分子体系，并探索基于双光子或上转换激发的近红外激活技术，以进一步提升活体深层组织的原位激发深度，推动该技术在术中内窥镜导航及长效成像追踪中的实际落地。