Photoactivated nanomotors via aggregation induced emission for enhanced phototherapy

The study addresses the challenge of translating synthetic nano/micromotors into biomedical applications by overcoming reliance on chemical fuels, non-biocompatible components, and environmental sensitivity. The authors propose creating motile nanosystems powered by non-chemical (radiative) energy that integrate propulsion and therapeutic function with spatial control. Aggregation-induced emission (AIE) is highlighted as a promising platform due to its stable fluorescence in the aggregated state, resistance to aggregation-caused quenching, and capability for photodynamic reactive oxygen species (ROS) generation, particularly with second-generation AIE luminogens that support two-photon near-infrared (TP-NIR) activation. To convert radiant energy into propulsion, plasmonic gold nanoshells, known for NIR-induced photothermal effects, are incorporated. The central hypothesis is that integrating AIEgenic polymersomes with an asymmetric Au nanoshell will yield synergistic behavior: AIE-mediated TP-NIR absorption and ROS generation combined with Au-driven thermophoresis for enhanced motility, enabling precise, site-specific phototherapy.

Prior research has developed various chemically and physically propelled nanomotors, yet biomedical translation is limited by requirements for high chemical fuel and biocompatibility issues. AIE luminogens have shown advantages over conventional photosensitizers by avoiding aggregation-induced quenching and photobleaching, and second-generation AIEgens improve TP-NIR absorption and ROS generation. TP-NIR provides deep tissue penetration and micron-scale spatial resolution for selective activation. AIEgens have been incorporated into diverse nanostructures, including polymersomes, but not previously to impart active motion. Plasmonic gold nanostructures exhibit strong NIR photothermal conversion for photothermal therapy and have been used to drive thermophoresis and promote cell membrane percolation with hemispherical coatings. The literature suggests that combining AIE-based phototherapeutics with plasmonic Au could enable dual phototactic/phototherapeutic nanosystems, but such synergistic, structure-inherent integration had not been reported.

- Polymer synthesis and AIE integration: Biodegradable amphiphilic PEG-PTMC copolymers were synthesized using pentafluorophenyl-activated trimethylene carbonate monomers enabling post-polymerization amine functionalization. A second-generation AIEgenic moiety containing tetraphenylethylene and dicyanovinyl (TPEDC) was synthesized and covalently incorporated into the PTMC block to yield PEG44-P(AIE)n (n=5,8,14,22; D≈1.1). Spectroscopic and structural characterization of TPEDC and copolymers are provided (Supplementary Figs. 1–24). - Self-assembly to polymersomes: Copolymers were assembled by solvent switch (THF→water to 50 vol% followed by dialysis). PEG44-P(AIE)5/8/14 formed vesicles of 300–500 nm with low PDI (≤0.1); membrane thickness ~8–14 nm by TEM/cryo-TEM. PEG44-P(AIE)22 formed ~70 nm micelles/nanoaggregates via fast precipitation. PEG44-P(AIE)14 was selected for subsequent work based on AIE density and size. AIE fluorescence upon aggregation was confirmed (λex=373 nm, λem=617 nm), with strong emission arising by 20% water addition. - Gold hemishell coating (Janus fabrication): AIE polymersome monolayers were formed on hydrophilic silica slides, dried, then sputter-coated with Au (Quorum K575X, 65 mV, 30 s) to create hemispherical nanoshells. Particles were re-dispersed by ultrasonication. Morphology and coating distribution were characterized by SEM, TEM, cryo-TEM, cryo-electron tomography, and EDX mapping, showing Au only on the exterior and intact vesicular membranes without aggregation; hydrodynamic size remained ~400 nm by DLS. - Cargo integrity tests: Hydrophilic (10 kDa dextran-TMR) and hydrophobic (Cy7) cargos were encapsulated in polymersomes prior to Au coating to confirm structural integrity during processing; leakage was assessed over time by fluorescence in supernatant (dextran-TMR) or bulk (Cy7). - Motility characterization by TP-CLSM: Motility was measured in PBS on a Leica TCS SP5X two-photon confocal microscope (760 nm TP-NIR). Particles were tracked via intrinsic AIE fluorescence. Trajectories, MSD, and velocities (Vxy) were extracted using ImageJ and Origin. Diffusion coefficients and rotational times were calculated: Dt=kBT/(6πηR), DR=kBT/(8πηR3), giving theoretical Dt≈1.07 μm² s−1, DR≈20 s−1, τR≈0.05 s for ~400 nm particles. Controls included symmetric AIE-polymersomes and commercial Au nanoshells (160 nm) under NIR. - Nanosight (NTA) motility and phototaxis: NanoSight NS300 recorded trajectories for AIE/Au, AIE polymersomes, and Au shells at ~5 μg mL−1. Illumination used 405 nm or 488 nm lasers for selective AIE excitation and an external 660 nm DPSS diode (0 or 1 W) for directional phototaxis. MSD was analyzed in 2D with fits to MSD=(4D)Δt+v2Δt2 to extract velocities. Directional illumination geometry enabled assessing negative phototaxis. - Cell studies (HeLa): Viability by MTT after 24 h exposure to varying concentrations of AIE polymersomes or AIE/Au nanomotors. TP-NIR irradiation controls (up to 200 s) without particles were performed. Membrane disruption: cells stained with WGA-Alexa488, Hoechst, PI; treated with AIE/Au (25 μg mL−1) and irradiated by TP-CLSM, imaging real-time PI influx. Intracellular ROS: CM-H2DCFDA-loaded cells treated with particles and irradiated; time-resolved fluorescence measured. Cell death: calcein-AM (live) and PI (dead) staining under TP-NIR activation. 3D multicellular spheroid experiments assessed penetration and crown toxicity upon activation. Imaging included 3D confocal z-stacks; lysotracker co-staining assessed intracellular trafficking. - Data analysis: MSD slopes distinguished Brownian vs propulsive motion; τR considerations used to interpret regimes (Δt<τR vs Δt>τR). Multiple repeats ensured reproducibility; error bars represent standard deviations from tracked particle/cell counts.

- Successful fabrication of AIE-rich PEG44-P(AIE)14 polymersomes (~300–500 nm, PDI≤0.1) exhibiting strong aggregation-induced fluorescence (λex=373 nm, λem=617 nm). Membrane thickness scaled with AIE block (~8–14 nm). - Hemispherical Au nanoshells were deposited asymmetrically on polymersomes without compromising vesicle integrity or causing aggregation; hydrodynamic diameters remained ~400 nm; Au increased absorbance across visible–NIR without quenching AIE emission. - Under TP-NIR (760 nm) activation, AIE/Au nanomotors exhibited autonomous, propulsive motion with velocities increasing with laser power; motion trajectories transitioned from diffusive to directional with increasing intensity. Theoretical Dt for 400 nm particles was ~1.07 μm² s−1; experimentally measured Dt without laser: 0.85 μm² s−1 (AIE/Au) and 0.93 μm² s−1 (Au-only controls). - MSD analyses showed parabolic profiles (indicative of propulsion) at Δt>τR (~0.05 s) for all applied powers. Symmetric AIE-polymersomes and symmetric Au nanoshells exhibited only enhanced Brownian diffusion under NIR, ruling out bulk heating as the cause of propulsion. - AIE/Au nanomotors achieved up to 45% higher velocities than Au-coated non-AIE control polymersomes, evidencing synergy wherein AIE transduces radiant energy to the Au shell to enhance thermophoresis. - Selective excitation experiments (NTA) showed propulsive motion at 405 nm (AIE-absorbing) but not at 488 nm for AIE/Au nanomotors; uncoated AIE polymersomes and non-AIE nanomotors remained Brownian at both wavelengths, confirming AIE→Au energy transfer drives enhanced motion. - Directional irradiation (660 nm) induced negative phototaxis (motion away from the light source), suggesting utility for penetration into tissues along intensity gradients. - Biocompatibility controls: AIE polymersomes and AIE/Au nanomotors showed relatively low dark toxicity (viability ~85% and ~70% at 200 μg mL−1, respectively). TP-NIR alone up to 200 s caused no detectable toxicity. - Phototherapy synergy in HeLa cells: AIE polymersomes plus TP-NIR induced low ROS and negligible toxicity up to 280 s. AIE/Au nanomotors under TP-NIR produced high intracellular ROS within ~48 s and rapid membrane disruption/percolation with PI uptake indicating apoptosis by ~80 s. Live/dead imaging showed loss of calcein-AM signal within 40–80 s in irradiated regions, with non-irradiated neighboring cells remaining viable, demonstrating high spatial selectivity. - Dose dependence: Lower nanomotor concentrations (25→2.5→0.5→0.25 μg mL−1) reduced cellular accumulation, PI sequestration, and loss of activity proportionally. - Controls lacking AIE (Au-coated polymersomes) did not induce ROS or TP-NIR-mediated cytotoxicity, underscoring the necessity of AIE functionality for PDT. - 3D spheroid model: TP-NIR-activated AIE/Au nanomotors promoted enhanced penetration and evident cytotoxicity at the spheroid crown, supporting potential for tissue-level application.

The integrated AIE/Au design addresses the need for non-chemical energy-driven nanomotors that combine propulsion with therapeutic action. AIE luminogens provide stable fluorescence, efficient TP-NIR absorption, and ROS generation, while the Au hemishell converts localized optical energy into thermal gradients to drive thermophoresis. The observed up to 45% velocity enhancement over non-AIE controls supports synergistic energy transduction from AIE to the Au layer, increasing propulsion efficiency. MSD analyses confirm autonomous propulsion under optical activation, and negative phototaxis suggests a mechanism for guided penetration into tissues along light intensity gradients. In cell studies, motility-assisted membrane percolation facilitates rapid intracellular access, amplifying AIE-mediated ROS production for potent, spatially confined phototoxicity under TP-NIR. The approach maintains low off-target effects: particles are relatively benign without activation, and TP-NIR alone is non-toxic, yielding precise on-demand cytotoxicity. Mechanistically, the authors hypothesize that highly localized, asymmetric thermal gradients around nanoscale motors reduce effective rotational randomization and enable ballistic-like motion beyond classical expectations; further theoretical and experimental work is warranted to fully elucidate propulsion at this scale.

The work introduces a synergistic phototactic/phototherapeutic nanomotor based on AIE-rich biodegradable polymersomes asymmetrically coated with a gold nanoshell. These hybrid nanomotors convert TP-NIR irradiation into enhanced thermophoretic propulsion and simultaneously generate ROS for photodynamic therapy, yielding rapid, spatially controlled cancer cell ablation in vitro, including in 3D spheroids. The study details synthesis, assembly, coating, and comprehensive physical and biological characterization, demonstrating superior motility relative to non-AIE controls and strong phototherapeutic efficacy. Future research should investigate the fundamental propulsion mechanisms at the nanoscale, optimize AIE structures and Au geometries for deeper tissue activation and improved efficiency, evaluate in vivo biodistribution and safety, and explore guided delivery strategies leveraging phototaxis and advanced optical focusing in complex tissues.

- Mechanistic understanding: The precise mechanism underlying the observed ballistic propulsion at the nanoscale remains speculative; non-classical effects from localized thermal gradients are proposed but require dedicated theoretical and experimental validation. - In vitro scope: Demonstrations are limited to in vitro cell cultures and 3D spheroids; in vivo performance, biodistribution, immune interactions, clearance, and long-term safety are unassessed. - Activation requirements: Effective operation relies on TP-NIR/optical setups (e.g., two-photon microscopes or focused NIR sources); translation will require clinically compatible light delivery systems and consideration of tissue scattering/absorption. - Processing artifacts: Gold coating involves drying and sputtering; while cryo-ET suggests structural recovery upon rehydration, subtle deformation effects cannot be entirely excluded. - Thermal effects control: Although controls suggest bulk heating is not responsible for propulsion, precise local temperature maps and thermometry during activation were not reported.