Ratiometric afterglow luminescent nanoplatform enables reliable quantification and molecular imaging

The study addresses key limitations of afterglow luminescence for molecular imaging in vivo: paucity of reactive sites for activatable probe design and unreliable quantification due to time-dependent signal attenuation and sensitivity to laser/measurement parameters. Building on the concept of energy transfer strategies like FRET, the authors hypothesize that an excitation-free afterglow donor coupled to a responsive acceptor could enable ratiometric self-calibration. They propose an afterglow resonance energy transfer (ARET) strategy to create a universal ratiometric afterglow nanoplatform (RAN) capable of specific, quantitative detection of analytes (e.g., NO, ONOO−, pH) with high reliability and low background, aimed at improving in vivo imaging accuracy and enabling real-time evaluation of biological processes such as macrophage-mediated tumor immunotherapy.

Prior work highlights fluorescence imaging drawbacks (photobleaching, autofluorescence) and the advantages of afterglow luminescence for high SBR, deep-tissue imaging, and various applications (cell tracking, cancer imaging, lymph node mapping, vascularization, temperature monitoring). However, existing afterglow materials are structurally inert, limiting activatable probe design, and their signals attenuate over time, complicating quantification. FRET is a well-established ratiometric strategy using donor-acceptor pairs with distinct reactive sites, enabling flexible probe construction. The authors leverage these insights to develop an ARET approach, replacing the photoexcited donor with an afterglow substrate to achieve excitation-free, ratiometric afterglow sensing that overcomes attenuation and parameter dependencies.

Design and mechanism: The authors introduce afterglow resonance energy transfer (ARET), where an afterglow substrate (MEHPPV) serves as the excitation-free donor. After pre-irradiation, afterglow initiators (BDP or TPP) generate singlet oxygen to form PPV-dioxetane intermediates in MEHPPV, storing energy that is released as afterglow (AF1). Via spectral overlap and proximity, energy is transferred to an acceptor responsive molecule to yield a longer-wavelength afterglow (AF2). The ratiometric AF2/AF1 serves as the quantitative readout, designed to be independent of laser and acquisition parameters and robust to attenuation. Nanoplatform construction: RANs were assembled by nanoprecipitation using amphiphilic polymers/surfactants. Components: MEHPPV (afterglow substrate), BDP or TPP (afterglow initiators), responsive molecules (NRM for NO, ORM for ONOO−, PRM for pH), and surfactants (F127, F127-PSMA, or PSMA-PEG). THF solutions containing components were rapidly injected into water under sonication, followed by THF removal and ultrafiltration. Doping levels of acceptors and initiator types were optimized. Particle size and morphology were evaluated by DLS and TEM; colloidal stability was monitored in DPBS/DMEM. Responsive probes: - RAN1 (NO-responsive): NRM contains benzo[c][1,2,5]thiadiazole-5,6-diamine (weak acceptor); reaction with NO yields a stronger triazolobenzothiadiazole acceptor (NRM-NO) via ICT enhancement, red-shifting absorption/emission. ARET from MEHPPV AF1 (~600 nm) to NRM-NO produces AF2 (~830 nm). - RAN2 (ONOO−-responsive): ORM derived from IR780 with ONOO−-sensitive functionality; ARET yields ratiometric changes (AF1 ~820 nm; AF2 ~600 nm). - RAN3 (pH-responsive): PRM constructed from CS dye with pH-dependent spectral changes, enabling ratiometric afterglow (AF1 ~600 nm; AF2 ~750 nm). Spectroscopy and imaging in solution: Absorption and fluorescence spectra were recorded; afterglow imaging performed after 660 nm or white light pre-irradiation using IVIS with appropriate emission filters (MEHPPV: 550–650 nm; acceptors: 800–875 nm). Kinetics, linearity ranges, detection limits, and specificity were assessed. Attenuation studies collected afterglow at intervals after irradiation. Dependence on laser power, irradiation time, exposure time, and decay time was evaluated. Tissue penetration: After pre-irradiation, RAN1 solutions (±NO) were imaged through chicken tissues of varying thickness (0–0.6 cm) under fluorescence and afterglow modes; SBR and AF2/AF1 vs depth were quantified. In vitro cell studies: Cytotoxicity (CCK-8) in 4T1 and RAW264.7 cells; subcellular localization (lysosomes) via colocalization. Macrophage polarization in RAW264.7 cells using IFN-γ, BLZ945, pexidartinib, and chloroquine; imaging with RAN1; NO scavenging control (Carboxy-PTIO). Flow cytometry quantified CD80 and CD86 expression; iNOS immunofluorescence staining validated polarization. In vivo models and imaging: - Inflammation model: LPS injected intradermally into mouse rear paws; RAN1 injected i.d.; fluorescence and afterglow imaging over time, ratiometric analysis. - LPS-induced liver injury in Nos2−/− vs WT mice: LPS or PBS i.p., RAN1 i.v.; in vivo afterglow and fluorescence imaging; ex vivo iNOS staining and flow cytometry of liver cells. - Tumor model: 4T1 xenografts in BALB/c mice; RAN1 i.v.; longitudinal fluorescence and afterglow imaging to assess tumor accumulation (EPR) and endogenous NO dynamics; analysis of attenuation behavior in vivo. - Macrophage-modulated immunotherapy: Intratumoral administration of modulators (IFN-γ, BLZ945, pexidartinib, chloroquine) followed by RAN1 i.v.; longitudinal ratiometric imaging; ex vivo flow cytometry of TAMs (CD11b, F4/80) and M1 marker (CD80); tumor growth monitoring; histology (H&E), apoptosis (TUNEL); safety (body weight, organ histology). Synthesis and characterization: Detailed synthetic procedures and characterization (1H/13C NMR, MS) for BDP, NRM, IR780, ORM, CS, PRM are provided. Imaging parameters (laser power/time, acquisition time) and data analysis methods (ROIs, Living Image, GraphPad) are specified. Ethics approvals and animal handling protocols are included.

- Universal ARET-based ratiometric afterglow nanoplatform (RAN) enables customizable activatable probes for specific analytes (NO, ONOO−, pH) with reliable quantification via AF2/AF1. RAN1 (NO): - Spectral response: Upon increasing NO, absorption at 660 nm increases; fluorescence at 830 nm increases while emission at 600 nm decreases; afterglow AF2 (830 nm) increases and AF1 (600 nm) decreases. - Quantification: AF2/AF1 ratio linearly correlates with NO from 0–20 μM; detection limit 0.21 μM (3σ/slope). Reaction reaches plateau in ~30 min. High specificity for NO over other ROS/RNS. - Reliability: AF2/AF1 ratio is independent of laser power, irradiation time, exposure time, and attenuation time, while individual AF1/AF2 intensities vary with these parameters. Afterglow attenuation cannot be prevented by increasing NRM doping, but AF2/AF1 remains constant over decay and scales with NRM content. - Tissue penetration: Through 0–0.6 cm chicken tissue, afterglow SBR vastly exceeds fluorescence (∼1200-fold improvement for AF1). AF2/AF1 ratio remains constant with depth, whereas FL2/FL1 decreases due to tissue background. - In vivo inflammation: In LPS-induced paw inflammation, both fluorescence and afterglow ratios increase more rapidly in LPS vs PBS regions (1–3 h), demonstrating endogenous NO detection; afterglow shows higher SBR. - LPS liver injury, genetic validation: In WT mice, LPS significantly increases AF2/AF1 in liver; Nos2−/− mice show no increase, consistent with lack of NO production. iNOS staining/flow cytometry corroborate higher iNOS in LPS-treated WT vs Nos2−/−. - Tumor imaging: RAN1 accumulates in tumors (EPR); normalized afterglow and fluorescence ratios increase from 1 to 18 h, indicating endogenous NO detection in TME; afterglow SBR exceeds fluorescence. In vivo AF2/AF1 remains stable over attenuation time after light cessation. - Macrophage polarization in vitro: RAW264.7 cells treated with IFN-γ, BLZ945, pexidartinib, or chloroquine show increased AF2/AF1 and FL2/FL1; NO scavenger reduces ratios. AF2/AF1 responds more strongly than FL2/FL1 due to lower background. Increased CD80/CD86 and iNOS expression validate M1 polarization, correlating with ratiometric signals. - Macrophage-modulated immunotherapy in vivo: After intratumoral modulators, afterglow ratios at 36 h (AF2/AF1) increase markedly: control 1.86; IFN-γ 3.98; BLZ945 2.87; pexidartinib 3.39; chloroquine 3.27, outperforming fluorescence ratios (FL2/FL1: control 1.41; IFN-γ 1.64; BLZ945 1.54; pexidartinib 1.57; chloroquine 1.56). Flow cytometry shows increased TAMs and M1 markers (CD80+F4/80+), highest in IFN-γ group, aligning with AF2/AF1. Tumor growth is significantly inhibited in modulator groups vs control; H&E and TUNEL indicate greater necrosis/apoptosis; no significant body weight changes or organ pathology observed. RAN2 (ONOO−): - Ratiometric response with decreasing AF1 (820 nm) and constant AF2 (600 nm) upon ONOO−; rapid kinetics; linear quantification with detection limit 41.2 nM. RAN3 (pH): - Robust ratiometric afterglow/fluorescence response across pH with pKa = 4.73; effective acidic pH sensing. Overall: ARET-based ratiometric afterglow probes deliver high SBR, deep-tissue reliability, and parameter-independent quantification, enabling accurate in vitro/in vivo analyte measurement and real-time evaluation of macrophage-mediated immunotherapy.

The ARET strategy merges the self-calibration advantages of FRET with excitation-free afterglow donors to address limitations in afterglow imaging: persistent attenuation, insufficient reactive sites, and sensitivity to laser/measurement parameters. By transferring stored afterglow energy from MEHPPV to responsive acceptors, RAN achieves ratiometric outputs (AF2/AF1) that remain stable across laser power, irradiation/exposure durations, decay times, and tissue depths, thus substantially improving quantification reliability and imaging robustness. High SBR from afterglow eliminates background effects that compromise fluorescence, producing accurate readouts even in deeper tissues. The platform’s modularity is demonstrated by NO-, ONOO−-, and pH-responsive probes with low detection limits, confirming universality through spectral matching of acceptors with the afterglow donor. Biologically, real-time imaging of NO—a hallmark of M1 macrophage polarization—enabled noninvasive assessment of inflammation, genetic validation in Nos2−/− mice, and evaluation of macrophage-modulated tumor immunotherapy. Correlations between AF2/AF1, iNOS/CD80/CD86 expression, and therapeutic outcomes substantiate the platform’s utility for monitoring immune responses and predicting treatment efficacy.

This work introduces a universal ratiometric afterglow nanoplatform (RAN) based on ARET that enables sensitive, reliable, and quantitative imaging of specific analytes in complex biological environments. The platform overcomes afterglow attenuation and parameter dependencies by leveraging an internal ratiometric readout (AF2/AF1) with markedly enhanced SBR and deep-tissue reliability. Demonstrations with RAN1 (NO), RAN2 (ONOO−), and RAN3 (pH) validate universality, with low detection limits and robust in vitro and in vivo performance. As a proof-of-concept, RAN1 enables noninvasive monitoring of M1 macrophage-derived NO to evaluate macrophage-modulated immunotherapy and correlate immune polarization with therapeutic efficacy. Future directions include expanding the responsive library to additional biomarkers (e.g., enzymes, metabolites, ions), optimizing spectral matching for multiplexed ratiometric afterglow imaging, enhancing targeting strategies beyond EPR, and translating the technology to larger animal models and clinical-relevant settings.

- Afterglow intensity attenuation persists over time even with altered doping; while AF2/AF1 remains stable, absolute signal decay may limit very long imaging windows. - Depth reliability was demonstrated ex vivo up to 0.6 cm in chicken tissue; performance at greater depths or in larger animals remains to be established. - In vivo validation focused on select analytes (NO, ONOO−, pH) and mouse models (inflammation, liver injury, 4T1 tumors); broader generalizability across diseases and species requires further study. - The approach requires pre-irradiation and specialized imaging systems (e.g., IVIS with specific filters), which may impact translational practicality. - Probe design depends on spectral matching between MEHPPV afterglow and acceptor dyes; not all targets may have readily available compatible responsive fluorophores.