Azo compounds, known for their reversible *E*/ *Z* isomerization upon light exposure, are valuable components in light-responsive systems. Their non-invasive and biocompatible nature makes them attractive for biological applications, particularly in controlling biomolecule functions via light-induced conformational changes and manipulating cellular processes. However, challenges remain in designing azo switches that efficiently coordinate light control with fluorescence imaging for real-time monitoring. Traditional strategies involving direct fluorophore modification often suffer from fluorescence quenching (FRET), reduced photoisomerization efficiency, and cytotoxicity. This work explores an alternative approach using azoarenes as conjugating bridges between electron donors and acceptors to create a Donor-Acceptor (D-A) system, aiming to overcome these limitations and develop a visible light-activated azo-fluorescent molecular switch suitable for imaging-guided, light-controlled drug delivery.

Existing research extensively documents the use of azo compounds as molecular switches in material science and their growing application in biological systems. Visible light-activated azo optical switches have shown promise in engineering biomolecules (proteins and nucleic acids) for light-controlled function. Recent studies highlight the use of azo switch-mediated optical control to explore and manipulate cellular processes. However, most azo switches lack integrated fluorescence monitoring, hindering real-time assessment of light-regulated events. Existing azo-fluorescent switches, often created by direct fluorophore modification, suffer from issues such as fluorescence resonance energy transfer (FRET), reduced photoisomerization efficiency, cytotoxicity, and complex synthesis. Therefore, there is a clear need for a novel design that addresses these limitations.

The researchers designed a visible light-activated azo-fluorescent molecular switch (AzoPJ) with a D-A structure, using an azoarene as a conjugating bridge between a julolidine electron donor and a pyrazole electron acceptor. AzoPJ was synthesized via a diazo coupling reaction. Its structure was characterized using NMR, mass spectrometry, and X-ray crystallography. Photoisomerization properties were assessed using UV-Vis absorption spectroscopy, showing reversible *E*/ *Z* isomerization under 440 nm (E to Z) and 535 nm (Z to E) light irradiation, and also with natural light. Fluorescence properties were evaluated using fluorescence spectroscopy, demonstrating a significant enhancement compared to a related compound (AzoPNMe2) due to the inhibition of the TICT (twisted intramolecular charge transfer) process by the rigid julolidine group. The volume of AzoPJ was computationally determined to be larger than AzoPNMe2, suggesting potential for cavity size changes in nanoparticles upon isomerization. AzoPJ and AzoPNMe2 nanoparticles (NPs) were prepared using a matrix encapsulation method with DSPE-mPEG5000. Their size and zeta potential were measured by DLS and TEM before and after light irradiation, revealing significant size changes in AzoPJ NPs after 440 nm light irradiation. The biocompatibility and stability of AzoPJ NPs were evaluated using MTT assays on B16 cells and UV-Vis spectroscopy under varying pH conditions and time. For fungal imaging, *Rhizoctonia solani* was incubated with AzoPJ NPs, and fluorescence imaging was performed using confocal laser scanning microscopy (CLSM). The light-controlled release of antimycotics was investigated using AzoPJ-PEPA NPs (PEPA: flubeneteram). The size and zeta potential of AzoPJ-PEPA NPs were monitored by DLS and TEM, demonstrating light-induced size increase and potential change. *In vitro* drug release was measured using a dialysis method under 440 nm light, natural light and dark conditions. Antifungal activity of AzoPJ-PEPA NPs against *Rhizoctonia solani* was evaluated using the agar dilution method and mycelial growth rate method. *In vivo* experiments were conducted by spraying rice leaves inoculated with *Rhizoctonia solani* with different solutions, observing lesion development under natural light. The interface affinity of AzoPJ-PEPA NPs to rice leaves was determined using contact angle measurement. The effect of AzoPJ-PEPA NPs on rice seedlings was evaluated.

The study successfully synthesized and characterized AzoPJ, a visible light-activated azo-fluorescent switch with enhanced fluorescence compared to AzoPNMe2 due to TICT inhibition. AzoPJ exhibited reversible *E*/ *Z* photoisomerization under visible light, including natural light, which was confirmed by UV-Vis spectroscopy. Fluorescence spectroscopy showed distinct fluorescence changes upon isomerization, enabling fluorescence imaging. The incorporation of the julolidine group in AzoPJ resulted in significant nanocavity size changes in its liposome-encapsulated nanoparticles upon light irradiation, forming a nanoscale light-controlled switch. This property was harnessed to create a light-controlled drug delivery system (AzoPJ-PEPA NPs). *In vitro* studies demonstrated light-triggered release of the antimycotic flubeneteram (PEPA), confirmed by UV-Vis and DLS analysis. Fluorescence imaging of *Rhizoctonia solani* showed that AzoPJ NPs accumulated in fungal cells and displayed fluorescence changes upon light irradiation indicating *in vivo* isomerization and drug release. Antifungal assays showed significant growth inhibition of *Rhizoctonia solani* under light irradiation with AzoPJ-PEPA NPs, outperforming small molecule antimycotics in terms of effectiveness and duration. *In vivo* experiments on rice leaves demonstrated the effectiveness of AzoPJ-PEPA NPs against rice sheath blight disease caused by *Rhizoctonia solani*. The AzoPJ-PEPA NPs showed enhanced efficacy and prolonged antifungal activity compared to small-molecule PEPA alone, at a lower dose.

This research successfully addressed the challenges in designing azo-fluorescent switches for imaging-guided and light-controlled drug delivery by introducing a novel D-A structure that effectively overcomes FRET and TICT issues. The results demonstrated the potential of this system for precise and efficient drug release in a controllable manner using visible light, including natural light. The enhanced antifungal activity and prolonged duration of action of AzoPJ-PEPA NPs compared to free PEPA highlight the significant benefits of this nano-delivery system. The *in vivo* study on rice sheath blight disease showcased the translational potential of this approach for agricultural applications.

This study presents a novel visible light-activated azo-fluorescent switch (AzoPJ) with enhanced fluorescence and reversible photoisomerization properties. The switch enables the construction of a light-controlled nanoplatform for targeted drug delivery, demonstrated by the effective release of an antimycotic agent against *Rhizoctonia solani*. The system shows improved antifungal activity and prolonged efficacy compared to the small molecule drug alone. This strategy holds significant promise for agricultural applications in controlling plant diseases and opens new avenues for developing imaging-guided light-controlled nanomedicines. Future work could explore the applicability of this system to other drugs and diseases, along with optimizing the nanoparticle design for improved biodistribution and targeting.

The study primarily focused on *Rhizoctonia solani*, and further investigation is needed to determine the efficacy against a broader range of fungal pathogens. While the *in vivo* study on rice leaves showed promising results, large-scale field trials are required to validate the long-term efficacy and safety. The mechanism of antifungal action warrants deeper exploration. Further research could focus on optimizing the nanoparticle formulation for improved stability and biocompatibility.