Molecular motors perform unidirectional 360° rotation, comprising two photoisomerization steps and two THI steps. The mechanism of photoisomerization involves light-induced excitation from the ground state to an excited state, triggering rotation around the central C=C double bond and resulting in E/Z isomerization. In the subsequent THI step, the molecule undergoes a thermally activated helical inversion, driven by steric strain and thermal energy. During the rotation, four states can be observed: two stable states and two metastable states (Fig. 1a). The rotational speed of molecular motors can be tuned by molecular design. To achieve rapid responsiveness in LLPS regulation, amphiphiles were designed based on a second-generation molecular motor, exhibiting a half-life of seconds as monomers and extending to minutes within aqueous assemblies. Bis-urea groups relate to the molecule motors through C3 alkyl chains and are linked with hydrophilic oligoethylene glycol moieties through C6 alkyl chains, thus enabling urea groups in the hydrophobic pockets, which should offer hydrogen bonds for supramolecule assemblies. The oligoethylene glycol chains, comprising triethylene glycol (OEG3), tetraethylene glycol (OEG4), and hexaethylene glycol (OEG6), were incorporated to modulate the T. The synthesis of second-generation molecular motor amphiphiles 2MOEG6, 2MOEG4, and 2MOEG3 was described in the Supplementary Information, and all the molecules were characterized by H, C NMR, and high-resolution MS (Supplementary Figs. 1, 28-37 and 39-41).

The rotary process of molecular motors was investigated through the utilization of H NMR spectroscopy. Upon irradiation with 365 nm light for 70 min at -20 °C, proton signals of H (δ = 6.89 ppm) and H (δ = 2.62 ppm) shift downfield to 7.30 and 2.84 ppm, respectively (Fig. 2a), indicative of a conversion from the stable Z isomer (Z-2MOEG4) to the metastable E isomer (E-2MOEG4). The ratio of E-2MOEG4 to Z-2MOEG4 is 73:27 by integrating the H NMR signals at the photostationary state (PSS). Subsequently, upon warming the sample at 20 °C in the dark for 7 min, the proton signal of H exhibits a downfield shift, while the proton signal of H shifts upfield, which is in accordance with the transition of E-2MOEG4 to stable E isomer (E-2MOEG4) with full conversion. Irradiating the mixture with 365 nm light for 90 min at -20 °C results in an upfield shift of H and downfield shift of H, indicating the photoisomerization of E-2MOEG4 to metastable Z isomer (Z-2MOEG4) with a ratio of 76:24 at the PSS. After subsequent warming at 20 °C for 7 min, the proton signal of H shifts downfield while H shifts highfield, indicating the THI of Z-2MOEG4 to Z-2MOEG4. The final mixture after a rotation cycle contains Z-2MOEG4 and E-2MOEG4 with a ratio of 60:40. The rotation behavior was further monitored by time-dependent UV-Vis absorption spectroscopy at -3 °C. Upon irradiation, the absorption band at 300-385 nm of Z-2MOEG4 exhibits a decrease, accompanied by the emergence of a new band at 386-485 nm, indicating the formation of E-2MOEG4 (Fig. 2b). After light irradiation of 10 min, the system reaches the first photostationary state (PSS). Subsequently keeping the sample in the dark for 60 min resulted in the THI of E-2MOEG4 to E-2MOEG4 (THI) (Fig. 2c). A subsequent irradiation of E-2MOEG4 resulted in an increase in the absorption band at 386-485 nm until the second photostationary state (PSS), indicating the formation of Z-2MOEG4 (Fig. 2d). Finally, the band at 386-485 nm disappeared with an increase of absorption band at 300-385 nm when the sample was kept in the dark, indicating the THI of Z-2MOEG4 to Z-2MOEG4 (THI) (Fig. 2f). Isosbestic points at 386 nm were observed in all the processes, which indicates that the sole process occurred in each step. The results are consistent with those obtained from NMR analysis. 2MOEG6 showed the same rotary behavior as 2MOEG4 (Supplementary Fig. 2). In summary, H NMR and UV-Vis studies reveal that motors undergo unidirectional rotation, exhibiting two metastable and two stable states.

Cryogenic transmission electron microscopy (cryo-TEM) study revealed that molecular motor Z-2MOEG6 formed micelles with a diameter of 5 nm in water (Fig. 2f), while Z-2MOEG4 formed worm-like micelles with a length of approximately 20-50 nm and a comparable diameter to Z-2MOEG6 (Fig. 2g). Z-2MOEG3 was too hydrophobic to dissolve in water at room temperature (RT). The assembly morphology was found to be consistent with the packing parameters theory (P = V/al), where the smaller hydrophilic head groups (a) resulted in larger packing parameters P, and the increasing parameters resulted in the morphology transforming from micelles to worm-like micelles. Therefore, Z-2MOEG6 showed micelles (P < 1/3) while Z-2MOEG4 showed worm-like micelles (1/3 < P < 1/2) due to the shorter chain length of OEG that has a smaller area of hydrophilic head groups. Interestingly, Z-2MOEG4 were found to form fibers with a uniform diameter of 5 nm and lengths of several micrometers after one week of aging (Fig. 2h). Dynamic light scattering (DLS) measurements revealed that the micelles of Z-2MOEG6 showed a hydrodynamic diameter of around 6 nm. Z-2MOEG4 showed a hydrodynamic diameter of around 7 nm, whereas the aged sample of Z-2MOEG4 showed a hydrodynamic diameter of 150 nm. FTIR revealed a N-H stretching band at 3338 cm and a C=O stretching band at 1625 cm of aged Z-2MOEG4 (straight fibers), corresponding to approximately 20 cm shifts compared to the fresh assembly (Fig. 2j). These shifts suggest enhanced urea hydrogen bonding upon aging, supporting the transformation from worm-like micelles to the more ordered micrometer-scale straight fibers. Time-dependent DLS measurements revealed that Z-2MOEG4 transformed into the fibers in one day (Supplementary Fig. 7), while micelles of Z-2MOEG6 remained the same after one week (Supplementary Fig. 8). Z-2MOEG6 is more hydrophilic than Z-2MOEG4 due to its longer hydrophilic OEG chains, which may reduce the hydrophobic interactions necessary for forming more ordered assemblies upon aging.

The critical phase separation temperature (T) of stable isomers of 2MOEG4 and 2MOEG6 was investigated by temperature-dependent transmittance measurements (Supplementary Information for details, Supplementary Fig. 5). The aqueous solutions of E-2MOEG4, Z-2MOEG4, E-2MOEG6, and Z-2MOEG6 showed increased T (Fig. 3a). The higher T of 2MOEG6 compared to 2MOEG4 is due to the longer OEG chains with improved hydrophilicity of the molecules. E isomers of both molecular motors showed lower T than their Z isomers, possibly due to the greater hydrophobicity of the E isomer compared to the Z isomer. We further plotted the phase diagram of these two molecular motors with their isomers. The T of Z-2MOEG4 tended to decrease up to 0.1 mg/mL and then stabilized at around 29 °C as the concentration increased, whereas T of E-2MOEG4 stabilized at 18 °C (Fig. 3b). T of Z-2MOEG6 tended to decay until 0.1 mg/mL and then stabilized at 52 °C as concentration increased, whereas T of E-2MOEG6 stabilized at 41 °C (Fig. 3c). FTIR analysis showed N-H stretching at 3358 cm for both Z and E assemblies, indicating hydrogen-bonded urea moieties (Supplementary Fig. 11). A minor redshift in the C=O stretching band from 1641 cm (Z) to 1637 cm (E) suggests slightly stronger hydrogen bonding in E-2MOEG4. Nile red fluorescence assay was used to probe the internal hydrophobicity of assemblies. The critical aggregation concentration (CAC) of Z-2MOEG4 is 2.0 µM, higher than the 0.5 µM of E-2MOEG4, suggesting greater hydrophobicity of E isomer (Supplementary Fig. 12). This result is consistent with dipole moment calculations performed using density functional theory (DFT), which show that the Z-2MOEG4 exhibits a dipole moment of 9.65 D, while the E-2MOEG4 has a lower value of 6.49 D, indicating a lower polarity for the E isomer (Supplementary Information for details, Supplementary Fig. 26 and Supplementary Data 1). Notably, the T of the aged Z-2MOEG4 assembly solution decreased by 4 °C at the same concentration (Supplementary Fig. 6). The decrease of T may be attributed to the increased length of supramolecular polymers upon aging.

Confocal laser scanning microscopy (CLSM) was employed to investigate LLPS. The aqueous solution Z-2MOEG4 displayed homogeneity at 24 °C (Supplementary Fig. 13). At 45 °C (above T), the aqueous solution exhibited the formation of drops after phase separation (Fig. 3d). Frames from time-lapse movies of CLSM (Supplementary Movie 1) revealed that the droplets rapidly dissolved when the temperature decreased below the T (Fig. 3g). Upon reaching a temperature exceeding the T the generation of droplets was observed again (Supplementary Fig. 14, Supplementary Movie 2). The aqueous solution of E-2MOEG4 has been found to perform LLPS at RT (Fig. 3b). Droplets of E-2MOEG4 were observed in the bulk solution and glass substrates (Fig. 3e). The droplets in the bulk solution exhibited rapid motion (Supplementary Movie 3). To elucidate the internal dynamics of droplets, we performed fluorescence recovery after photobleaching (FRAP) measurements of droplets on the substrate. After photobleaching, E-2MOEG4 droplets exhibited a rapid recovery of fluorescence signals, with a half-life time (t) of 27.0 ± 5.3 s (Fig. 3f, h), indicating the liquid-like feature of droplets. DLS studies showed that the hydrodynamic diameter of the droplets of Z-2MOEG4 was around 1 µm after phase separation (Fig. 3i). The hydrodynamic diameter of the droplets formed by aged Z-2MOEG4 assemblies is around 400 nm upon phase separation (Supplementary Fig. 10). The hydrodynamic diameter of the Z-2MOEG6 droplets was approximately 360 nm (Fig. 3j), while the droplets of E-2MOEG6 exhibited a diameter of about 200 nm after phase separation (Fig. 3k). Notably, when the temperature decreases below T, all types of assemblies returned to their original sizes, confirming the reversibility of the LLPS process (Fig. 3i-k and Supplementary Fig. 10). DLS data revealed the size of droplets of molecular motors, and confirmed that motors with longer OEG chains have higher T, and Z isomers exhibit higher T than their corresponding E isomers.

The rotation of 2MOEG4 in assemblies was monitored by UV-Vis spectroscopy. In contrast to the rotation of 2MOEG4 as monomers, the rotation of 2MOEG4 in assemblies was accompanied by a reversible phase separation process at RT. Upon irradiation, the absorption band at 300-385 nm of Z-2MOEG4 exhibits a decrease, accompanied by the emergence of a new band at 386-485 nm, indicating the formation of E-2MOEG4 (Fig. 4a). An isosbestic point was observed at 386 nm was observed during the first 200 s of irradiation, which then disappeared with increasing full-spectrum absorption due to scattering from the phase separation of E-2MOEG4. The exponential curve of the absorption at 425 nm as the function of time until 200 s indicates photoisomerization of Z-2MOEG4 to E-2MOEG4 (Fig. 4b). After 200 s, the increase of the absorption at 500 nm is indicative of LLPS. The phase separation of E-2MOEG4 was further confirmed by DLS measurements. The hydrodynamic diameter of the assemblies increased from 7 nm to 490 nm after irradiation (Supplementary Fig. 9). The subsequent incubation of the sample in the dark for 50 min resulted in the THI of E-2MOEG4 to E-2MOEG4, accompanied by phase separation, as indicated by the reduction in absorption at 425 nm and an increase in absorption at 500 nm (Fig. 4c). The isomerization of motors during LLPS can be approximated by subtracting the absorbance at 425 nm from the absorbance at 500 nm (Abs@425 - 500 nm). Abs@425-500 nm demonstrates an exponential decline over time, which is indicative of the THI process (Fig. 4d). The enhanced scattering may be attributed to the increased size of the assembly during the THI process, as shown by DLS data, which indicate that the hydrodynamic diameter of the assemblies grew to 890 nm (Supplementary Fig. 9). A subsequent irradiation of E-2MOEG4 resulted in a slight increase in the absorption band at 425 nm, accompanied by a decrease in the full spectrum absorption band (Fig. 4e). The absorption at 500 nm showed a notable decline, whereas the Abs@425-500 nm increased exponentially (Fig. 4f). This implies that the transformation of E-2MOEG4 to Z-2MOEG4 is concomitant with the dissolution of droplets. Finally, the band at 386-485 nm disappeared with an increase of absorption band at 300-385 nm when the sample was kept in the dark, indicating the THI of Z-2MOEG4 to Z-2MOEG4 (Fig. 4g). An isosbestic point at 386 nm was observed during THI (Fig. 4g) and the absorption at 500 nm was maintained at zero in this process, thereby confirming the homogeneity of the solution (Fig. 4h). As a control, the rotation of E-2MOEG4 assemblies was also studied in water at lower temperatures. At a temperature below the T of four isomers, no phase separation behavior was observed upon the rotation of 2MOEG4 (Supplementary Fig. 3). The same behavior was also found for 2MOEG6 (Supplementary Fig. 4). These results indicate that the rotation-driven reversible LLPS is caused by the successively different T of the isomers. The T of Z isomers is higher than the operating temperature of the system, while the T of E isomers is lower than that, which results in the reversible LLPS during the rotation of the molecular motor.

To investigate the in situ generation of droplets, the rotation-driven phase separation was monitored using CLSM. The internal structure changes of assemblies were examined before and after phase separation using cryo-TEM. After photoinduced isomerization, the solution of Z-2MOEG4 supramolecular assemblies (Fig. 4j) underwent LLPS to form droplets (Fig. 4l). The worm-like micelles of Z-2MOEG4 (Fig. 4i) were transformed into tightly aggregated micelles of E-2MOEG4 as a consequence of phase separation (Fig. 4k). During the THI of E-2MOEG4 to E-2MOEG4, the assembled structure remains relatively unchanged (Fig. 4o), while the droplet size exhibits a slight increase (Fig. 4p). Upon subsequent irradiation, the aggregated micelles of E-2MOEG4 transformed into dispersed worm-like micelles of Z-2MOEG4 (Fig. 4m), and the droplets dissolved (Fig. 4n). Ultimately, keeping the sample in the dark led to the THI of Z-2MOEG4 to Z-2MOEG4, occurring in a homogeneous solution of supramolecular assemblies without droplets (Supplementary Fig. 15). The combined CLSM and cryo-TEM study indicate that the E-2MOEG4 and E-2MOEG4 isomers exhibit phase separation at RT, whereas the Z-2MOEG4 and Z-2MOEG4 isomers do not. Consequently, the reversible LLPS is achieved during the rotation of molecular motors, in accordance with the findings of UV-Vis spectroscopy.

To quantify the rotational speed of molecular motors in assemblies and droplets, we performed Eyring analysis on the THI processes from metastable to stable states and determined the energy landscape for motor rotation (Fig. 5, see Methods section for details). The Eyring analysis of the THI in water (assembly states) reveals an energy barrier (ΔG°) of 87.8 kJ mol from E-2MOEG6 to E-2MOEG6 (Fig. 5b), which is higher than that of 84.3 kJ mol in the monomeric state (Fig. 5a). ΔG° from Z-2MOEG6 to Z-2MOEG6 is 84.4 kJ mol for the monomer and 88.7 kJ mol in the assembly (Supplementary Figs. 16 and 17). Accordingly, the half-lifetime (t) of E-2MOEG6 is 2.0 min as a monomer and 8.3 min in assemblies, while the t of Z-2MOEG6 is 2.1 min as a monomer and 12.1 min in assemblies at 20 °C (Table S1). ΔG° from E-2MOEG4 to E-2MOEG4 and Z-2MOEG4 to Z-2MOEG4 are 87.9 and 89.0 kJ mol, respectively, which are also higher than the 84.0 and 84.2 kJ mol in the monomeric states (Supplementary Figs. 18-21). The t of E-2MOEG4 is 1.7 min as a monomer, while it is 8.7 min in assemblies, whereas the t of Z-2MOEG4 is 1.9 min as a monomer and 13.5 min in assemblies at 20 °C (Table S1). The slower speed of motors in assemblies can be attributed to the tight packing of neighboring molecules, which restrict the rotation. The energy barrier for two THI processes after phase separation is 87.5 and 88.1 kJ mol (Fig. 5c), and the half-lifetimes of E-2MOEG4 and Z-2MOEG4 are 7.4 min and 9.3 min at 20  °C, respectively, which are comparable to those in the assemblies before phase separation (Supplementary Figs. 22 and 23). The energy landscape of monomers and assemblies is shown in Fig. 5c. Overall, the rotational speeds of 2MOEG4 and 2MOEG6 monomers are nearly identical, but both are slower in supramolecular assemblies compared to their monomeric state. The rotational speed of 2MOEG4 remains largely unchanged after phase separation, indicating the good fluidity of the phase-separated droplets.

In summary, we have developed a molecular motor-driven LLPS of supramolecular assemblies, enabling orthogonal control by light and heat. Supramolecular assemblies of molecular motors exhibit different morphologies, deviating from spherical micelles to worm-like micelles and fibers, depending on the molecular structures and growth kinetics. The droplets formed through phase separation exhibit excellent fluidity, as demonstrated by the rapid recovery of fluorescence from the labeling dye and the uninhibited rotational speed of molecular motors. The critical phase separation temperatures are found to be closely correlated with the nature of hydrophilic ethylene glycol chain units and the configurations of molecular motors. The large geometrical differences between the isomers result in distinct critical phase separation temperatures, ranging from 18 to 52 °C. The reversible phase separation can be regulated by modifying the temperature, utilizing different critical phase separation temperatures. Benefiting from the multiple out-of-equilibrium states of molecular motors and their distinct critical phase separation temperatures, in situ reversible phase separation is realized during the unidirectional rotation of molecular motors. Achieving in situ control over the LLPS of supramolecular assemblies will facilitate the advancement of out-of-equilibrium biomedical materials with responsive capture and release functions.