A visible light-activated azo-fluorescent switch for imaging-guided and light-controlled release of antimycotics

The study addresses the need for visible light-activated molecular switches that can both modulate biological processes and provide a fluorescence readout to monitor these events in real time. Azo compounds are established photoswitches that isomerize between E and Z forms under light and have been adapted for biological applications due to the non-invasive, biocompatible nature of visible light. However, conventional azo-fluorescent switch designs that directly append fluorophores to azo units often suffer from fluorescence quenching via FRET, reduced photoisomerization efficiency, and potential cytotoxicity, in addition to synthetic complexity. The authors hypothesize that building a donor–acceptor (D–A) fluorogenic system using the azo N=N as a conjugated bridge between an electron donor and acceptor could yield a visible-light-activated azo-fluorescent switch with improved fluorescence, robust isomerization, and imaging capability. They further posit that inhibiting the twisted intramolecular charge transfer (TICT) process—by replacing a flexible N,N-dimethylamino donor with a rigid julolidine donor—will enhance fluorescence while retaining efficient photoisomerization. Finally, they propose that the larger steric volume associated with the julolidine donor will produce measurable nanocavity changes in liposome-encapsulated nanoparticles upon isomerization, enabling light-controlled, fluorescence-monitored drug release.

Visible light-activated azo switches have been widely explored for material and biological systems, enabling optical control of biomolecules, cellular processes, and supramolecular systems. Traditional strategies attach fluorophores directly to the azo unit, but these often lead to fluorescence quenching via FRET, compromised photoisomerization, and potential biocompatibility issues. Azo-heteroarenes with D–A backbones (e.g., arylazopyrazoles) exhibit efficient visible-light E–Z isomerization and are attractive platforms for switches. However, many such systems show weak fluorescence due to nonradiative internal conversion associated with E–Z isomerization and TICT. Prior works have improved visible/NIR activation ranges and photostationary state control but less focus has been placed on concurrent strong fluorescence readouts. The authors build on these insights by designing a D–A azo-fluorescent switch (AzoPJ) that restricts TICT via a rigid julolidine donor while maintaining a pyrazole acceptor, aiming to circumvent quenching and synthetic complexity, and to integrate optical switching with fluorescence imaging and nanocarrier-controlled release.

Design and synthesis: The D–A azo-fluorescent switch AzoPJ was designed by replacing the N,N-dimethylamino donor in AzoPNMe2 with a rigid julolidine donor to inhibit TICT while retaining the pyrazole acceptor and the azo N=N conjugated bridge. AzoPJ was synthesized via diazo coupling between the diazonium salt of 4-amino-1,3,5-trimethylpyrazole and julolidine, affording AzoPJ in 65% yield. Structure confirmation used 1H/13C NMR and HRMS; single crystals grown by slow diffusion (hexane into dichloromethane) were analyzed by X-ray crystallography (CCDC 2337626). Computational analysis: TD-DFT (B3LYP/6-31G*) was used to compute electronic transitions and orbital distributions for E- and Z-AzoPJ; molecular volumes were calculated at RHF/6-31G**. Calculations indicated strong ICT character and predicted key absorption bands for E and Z isomers. Spectroscopy and photoisomerization: UV–Vis and fluorescence spectra were recorded in DMSO. Photoisomerization was initiated with 440 nm irradiation (50 mW cm−2 or 20 W cm−2) and reversed with 535 nm (50 mW cm−2 or 15 W cm−2). Natural light was simulated using an artificial climate box (MGC-450HP-2L). Fatigue resistance was evaluated over 10 alternating 440/535 nm cycles. Fluorescence measurements for AzoPJ were performed at λex = 350 nm; single-crystal fluorescence used λex = 430 nm. AzoPNMe2 fluorescence was measured at λex = 328 nm. Nanoparticle preparation and characterization: Liposomal nanoparticles (NPs) were prepared by matrix encapsulation using DSPE-mPEG5000. AzoPJ NPs: DSPE-mPEG5000 (9 mg) in water (9 mL) was mixed with AzoPJ (1 mg) in THF, sonicated 30 min, THF removed under N2, and concentrated via 50 kDa centrifugal filtration; stored at 4 °C. Drug-loaded NPs (AzoPJ-PEPA NPs) were similarly prepared by co-encapsulation of AzoPJ (1 mg) and flubeneteram (PEPA, 0.2 mg). Control PEPA NPs lacking AzoPJ were also prepared. DLS (Zetasizer ZEN3600) measured hydrodynamic diameter and zeta potential before and after 440 nm irradiation (20 W cm−2). TEM (Tecnai G2 F20, 200 kV) imaged morphology. Stability was assessed over time, and across pH, by UV–Vis, DLS, and zeta potential. Drug encapsulation and release: Encapsulation efficiency for PEPA in AzoPJ-PEPA NPs was determined by centrifugation (1500 × g, 20 min) and UV–Vis quantification of unencapsulated PEPA. In vitro release used dialysis (MWCO: 14 kDa) at 37 °C in PBS with irradiation at 440 nm (20 W cm−2), natural light, or dark; PEPA release quantified from absorbance at ~260 nm and standard curves. NP stability during release was tracked by DLS and zeta potential time courses (0–24 h). Biocompatibility assays: B16 cells were incubated with AzoPJ NPs or AzoPJ-PEPA NPs (0–40 μg mL−1) for 24 h; viability assessed by MTT assay (absorbance at 490 nm). Fluorescence imaging in microbes: Rhizoctonia solani was cultured on PDA at 28 °C. For concentration dependence, hyphae were incubated with AzoPJ NPs (0–1 mg mL−1) for 24 h, washed (PBS ×3), and imaged by CLSM (Leica TCS SP8 X; λex 405 nm; λem 460–580 nm). Time dependence used 1 mg mL−1 AzoPJ NPs for 0–48 h. Photoswitch imaging: after 48 h incubation with AzoPJ NPs (1 mg mL−1), hyphae were irradiated with 440 nm (20 W cm−2, 1 h) and re-imaged. Staphylococcus aureus was treated analogously for imaging and photoswitching studies. Antifungal efficacy in vitro: AGAR dilution assays evaluated concentration- and time-dependent inhibition of R. solani with AzoPJ-PEPA NPs under 440 nm or natural light irradiation. Growth diameters were measured daily; inhibition ratios calculated. Persistent effect assays compared seven groups: (I) Blank, (II) Natural light, (III) AzoPJ-PEPA NPs (PEPA 5 mg L−1), (IV) PEPA 5 mg L−1 + natural light, (V) PEPA 7.5 mg L−1 + natural light, (VI) PEPA 10 mg L−1 + natural light, (VII) AzoPJ-PEPA NPs (PEPA 5 mg L−1) + natural light, with natural light exposure 4 h/day; growth followed until group IV reached its limit. PI staining (λem 590–700 nm) assessed membrane damage post-treatments. Rice leaf assays: Detached rice leaves (tillering stage) were inoculated with R. solani mycelial plugs (~6 mm), treated with the seven groups above, and incubated at 28 °C, 80% RH, 12 h light/dark; natural light from climate box. Lesion development was recorded daily over 7 days; contact angle measurements evaluated leaf wettability of AzoPJ-PEPA NPs vs PEPA and water. Plant biosafety was assessed by germinating rice seeds for 48 h in 0–1 mg mL−1 AzoPJ-PEPA NPs and measuring germination rate, shoot/root length.

Molecular design and photophysics: Substituting a rigid julolidine donor into a D–A azo-heteroarene (AzoPJ) inhibits TICT and enhances fluorescence versus AzoPNMe2. AzoPJ single crystals show bright green emission with peaks at 535 and 569 nm (λex 430 nm). In DMSO solution, AzoPJ fluorescence intensity is ~4-fold higher than AzoPNMe2 and exhibits emission centered near 507 nm (λex 350 nm). TD-DFT indicates strong ICT character: for E-AzoPJ, major transitions include S0–S2 at ~418.6 nm (oscillator strength ~1.00, 71% HOMO→LUMO) and S0–S5 at ~283.4 nm (0.246, 55% HOMO−2→LUMO). For Z-AzoPJ: S0–S1 at ~496.7 nm (0.218, 61% HOMO→LUMO) and S0–S2 at ~371.7 nm (0.315, 58% HOMO−1→LUMO). Visible-light photoisomerization and fluorescence switching: AzoPJ displays a broad absorption (315–600 nm, max ~440 nm) consistent with E-form in DMSO. 440 nm irradiation produces a photostationary E/Z mixture with peaks at 376 and 453 nm and an isosbestic point at 478 nm; 535 nm irradiation recovers the E-form spectrum. Reversible switching is maintained over at least 10 cycles. Natural light also drives isomerization. Fluorescence responds to isomerization: upon 440 nm irradiation, the 507 nm emission decreases while a 420 nm band increases; 535 nm irradiation reverses these changes, enabling ratiometric-like fluorescence monitoring of the switching process. Nanoparticle light-responsiveness: AzoPNMe2 NPs showed minor changes upon irradiation (size ~179.8→193.1 nm; zeta −49.85→−40.08 mV) with little morphology change. In contrast, AzoPJ NPs undergo substantial, reversible size and zeta potential changes under 440 nm: DLS size ~117.5→165.4 nm; zeta −53.9→29.88 mV; TEM confirms enlargement post-irradiation. UV–Vis of NPs reflects the AzoPJ isomerization trend. NPs are stable over time and across pH; B16 cell viability remains high at 0–40 μg mL−1. Fungal and bacterial imaging and in-cell photoswitching: AzoPJ NPs label R. solani hyphae with green fluorescence in a concentration- and time-dependent manner, resolving septa at 1 mg mL−1 and 48 h. 440 nm irradiation of stained hyphae markedly diminishes green fluorescence, evidencing intracellular photoisomerization; similar behavior is observed in S. aureus. Drug-loaded NPs and light-controlled release: AzoPJ-PEPA NPs show light-induced size and zeta shifts (DLS 136.1→230.7 nm; zeta −30.97→−1.39 mV) and TEM-confirmed enlargement upon 440 nm irradiation; control PEPA NPs lacking AzoPJ show minimal change. Encapsulation efficiency for PEPA is ~80%. In vitro release reaches ~40% cumulative under 440 nm and a similar level under natural light; release in the dark is ~3% over the same period. During light exposure, NP size increases and zeta potential becomes less negative over 0–24 h; both remain stable in the dark. AzoPJ-PEPA NPs exhibit low cytotoxicity toward B16 cells (0–40 μg mL−1). Imaging-guided release and antifungal effects: In R. solani, AzoPJ-PEPA NPs yield strong green fluorescence that weakens under 440 nm irradiation correlating with azo isomerization and drug release. Post-irradiation, hyphae display deformation and rupture; PI staining shows strong red fluorescence for PEPA alone and for AzoPJ-PEPA NPs with 440 nm, but negligible for AzoPJ-PEPA NPs without light, confirming light-triggered fungicidal action. Antifungal performance in vitro: In agar dilution assays, AzoPJ-PEPA NPs achieve concentration- and irradiation time-dependent inhibition. At PEPA 10 mg L−1 within the NPs, inhibition of R. solani reaches 86.3% (440 nm) and 89.2% (natural light); time-dependent inhibition under 440 nm reaches ~80.4% at 24 h. In persistent-effect comparisons, AzoPJ-PEPA NPs with PEPA 5 mg L−1 under natural light match the efficacy of PEPA 10 mg L−1 alone while halving the active dose. At equal 5 mg L−1 PEPA, the nano formulation doubles the holding period: small-molecule PEPA effect lasts ~7 days vs ~14 days for AzoPJ-PEPA NPs under natural light. Rice sheath blight control and plant compatibility: On rice leaves inoculated with R. solani, AzoPJ-PEPA NPs plus natural light prevent lesion development over 7 days (only slight yellowing), whereas controls and lower-dose PEPA alone show earlier and more extensive lesions. Contact angle measurements indicate improved leaf wettability of AzoPJ-PEPA NPs compared to PEPA, suggesting enhanced bioavailability. AzoPJ-PEPA NPs (0–1 mg mL−1) have negligible effects on rice seed germination and seedling growth, supporting plant biocompatibility.

The work validates a design strategy for azo-fluorescent switches that integrates visible-light photoswitching with enhanced fluorescence readout by constructing a donor–acceptor system and suppressing TICT via a rigid julolidine donor. The resulting switch, AzoPJ, undergoes efficient E/Z photoisomerization under 440/535 nm and even natural light, while concomitant fluorescence changes enable real-time optical tracking. When formulated in PEGylated liposomal nanoparticles, the switch’s isomerization induces significant and reversible nanocavity changes, which can be harnessed to modulate encapsulation and release of small molecules. This underpins a controllable nanoplatform that couples fluorescence monitoring with light-triggered delivery of the antimycotic PEPA. The platform demonstrates strong antifungal activity against Rhizoctonia solani: imaging confirms intracellular photoswitching; in vitro release reaches substantial levels under visible or natural light but remains minimal in the dark; and antifungal assays show dose-sparing (half the active ingredient for comparable efficacy) and a prolonged holding period (approximately doubled) relative to the small molecule. On rice leaves, the nanoformulation under natural light suppresses sheath blight lesion development over a week, indicating translational potential for agricultural disease management. Together, these findings directly address the research aim of coupling visible-light control with fluorescence-guided monitoring for drug release, offering a generalizable approach for precision delivery in biology and agriculture.

This study introduces AzoPJ, a visible light-activated azo-heteroarene fluorescent switch that combines a D–A architecture with TICT suppression to achieve strong fluorescence and robust, reversible photoisomerization. Incorporation into PEGylated liposomal nanoparticles yields a light-responsive nanocavity that enables fluorescence-imaged, light-controlled release of an antimycotic cargo. The system effectively kills Rhizoctonia solani in vitro and on rice leaves under natural light, while reducing required active dose and extending the effective period compared to the free drug. The approach provides a blueprint for designing imaging-guided, light-controlled, releasable nanomedicines and suggests broader applicability to diverse therapeutics and agricultural agents. Future work could optimize release efficiency and kinetics, extend the spectral responsiveness further into the red/NIR for deeper penetration, evaluate long-term environmental and ecological safety, and conduct field-scale trials to validate agricultural performance.

The release plateau reached approximately 40% under visible or natural light, indicating incomplete cargo liberation under the tested conditions. Irradiation intensities and durations used for in vitro switching and release (e.g., 20 W cm−2 for up to several hours) may be higher than typical environmental light exposure, potentially limiting real-world applicability without optimization. Antifungal tests primarily targeted Rhizoctonia solani; broader-spectrum efficacy against other pathogens was not evaluated. In vivo assessments were limited to detached leaf assays in controlled climate conditions; field-scale performance, stability, and environmental impact remain to be tested. While cytotoxicity toward B16 cells and plant seedlings appeared low, comprehensive biosafety, phototoxicity, and long-term ecological studies are needed. Zeta potential changes, including sign inversion in some NP formulations, were observed and warrant mechanistic investigation regarding colloidal stability and interactions in complex biological matrices.