Photoredox molecular swimmer

Abstract

A photoredox molecular swimmer, structurally comprising a photoredox center, a redox shuttle, and a flexible PEG chain covalently linking the photoredox center and the redox shuttle. The photoredox molecular swimmer of the present invention enhances cell membrane permeability, promotes entry of extracellular chemicals into living cells, and induces on-demand apoptosis.

Description

A photo-oxidation-reduction molecular swimming machine Technical Field

[0001] This invention relates to the field of artificial molecular machines, and more specifically to a photo-oxidation-reduction molecular swimming machine. Background Technology

[0002] Micro and nanoswimming machines are a class of small-scale machines that convert the free energy of their surroundings into kinetic energy, overcoming thermal fluctuations and exhibiting autonomous propulsion. This property makes them widely applicable in fields such as healthcare and environmental remediation (Acc. Chem. Res. 50, 2-11 (2017)). To propel such small-scale swimming machines, sufficient driving force is needed to overcome the effects of thermal motion and fluidity. For example, bacteria and algae can propel themselves by exerting a backward force on the surrounding liquid through their flagella (J. Fluid Mech. 705, 58-76 (2012)). However, replicating the structure and function of natural counterparts in artificial micro / nanoswimming machines is very difficult. At the microscale, researchers often employ simple asymmetric structures, such as Janus particles or triangular structures, to induce autonomous propulsion in micromachines. In addition, translational propulsion can be generated by introducing external fields with asymmetry, such as magnetic fields, electric fields, acoustic fields, or optical fields (Nat. Rev. Chem. 3, 536-551 (2019)).

[0003] As machine size shrinks to the nanoscale, the propulsion of artificial machines becomes increasingly complex due to fluctuations in position and orientation introduced by Brownian motion (Nat. Phys. 7, 576–580 (2011)). Recently, researchers have discovered several natural molecular machines, such as diverse and complex protein motors including actin and dynein, which can move linearly along tubulin and play indispensable roles in muscle contraction, cellular cargo transport, DNA unfolding, and repair (Nature 422, 759–765 (2003)). Meanwhile, researchers have been working to create artificial molecular machines with similar functions, employing frameworks such as alkenes, alkanes, and rotaxanes. In 2016, Jean-Pierre Sauvage, Sir J. Fraser Stoddart, and Bernard L. Feringa were awarded the Nobel Prize in Chemistry for their outstanding contributions to the field, an honor that highlights the importance and value of molecular motors in scientific research and technological development (Angew. Chem. Int. Ed. Engl. 56, 11060-11078 (2017)).

[0004] Molecular swimming machines have long been envisioned as versatile tools for manipulating biomolecules and cellular activity by utilizing their mechanical effects. For example, molecular hand drills have recently been developed to enhance cell membrane permeability and induce apoptosis. However, because previous molecular machines could not swim, their molecular motion could not be directly imaged, making their exact functional mechanisms elusive.

[0005] Furthermore, unresolved limitations in current research on artificial molecular machines include: First, the complex structure and function of molecular motor systems make their construction and operation difficult. At the molecular scale, Brownian motion dominates, making it extremely difficult to control the motion of molecular motors. Therefore, to drive molecular motors to perform specific motions rather than random vibrations, thermodynamic equilibrium must be broken. Additionally, at the microscopic or nanoscale, the inertial forces necessary for macroscopic motion are lacking, with viscosity playing a dominant role. The Reynolds number (Re) represents the ratio of inertial to viscous forces; at the nanoscale and molecular level, the extremely small size of objects results in extremely low Reynolds numbers, requiring molecular motors to break time-reversal symmetry to produce net displacement.

[0006] Secondly, real-time observation of molecular motor motion faces obstacles: researchers have used nuclear magnetic resonance (NMR) technology to discover that the diffusion coefficient (D) of enzyme molecules or molecular reactants changes with chemical reactions. Inconsistencies exist regarding whether diffusion is enhanced or weakened due to factors such as data fitting, interpretation, and reference selection. With the development of single-molecule tracking (SMT) technology, researchers can directly observe molecular motion and interactions at the nanoscale. However, observing the motion of a single molecule is not easy; it depends on the designed and synthesized molecular motor structure and its motion mechanism. Summary of the Invention

[0007] To address the aforementioned problems with current artificial molecular machines, this invention provides the following solutions:

[0008] According to a first aspect of the present invention, the present invention provides a photoredox molecular swimming machine, the structure of which includes a photoredox center, a redox shuttle, and a flexible PEG chain covalently connecting the photoredox center and the redox shuttle.

[0009] Preferably, the structure of the photo-oxidation-reduction molecular swimming machine is as shown in compound 5:

[0010] Where n is an integer between 0 and 9.

[0011] Preferably, n in compound 5 is 0, 3, or 9.

[0012] According to a second aspect of the present invention, the present invention provides a method for preparing the photo-oxidation-reduction molecular swimming machine described in the first aspect above, wherein the synthetic route of the preparation method is as follows:

[0013] Where n is an integer between 0 and 9.

[0014] Preferably, n in compound 5 is 0, 3, or 9.

[0015] Preferably, all reactions are carried out in a dry solvent under an inert argon atmosphere using the standard Schlenk technique.

[0016] According to a third aspect of the invention, the invention provides uses of the photo-oxidation-reduction molecular swimming machine, the uses including at least one of the following:

[0017] (1) Regulates lipid droplet movement;

[0018] (2) Regulates cell membrane permeability;

[0019] (3) Used to prepare pharmacodynamic enhancers that can promote drug entry into cells; or

[0020] (4) Drug delivery.

[0021] Preferably, the drug is a cancer cell killer, specifically a cancer cell killer that acts within cells.

[0022] In this invention, the efficacy enhancer is a substance that is not used alone but in combination with a drug to enhance its efficacy. The photo-oxidation-reduction molecular swimming machine capable of regulating cell membrane permeability provided by this invention can promote the transfer of drugs with efficacy from outside the cell to inside the cell, thereby improving the drug's efficacy.

[0023] In this invention, the drug delivered is a drug that needs to be delivered from outside the cell to inside the cell to exert its effect inside the cell.

[0024] By employing the aforementioned technical solutions, this invention addresses the difficulty in constructing and operating complex molecular motor systems in existing technologies. It is the first to construct a self-propelled molecular swimming machine, thus solving this problem. Furthermore, addressing the difficulty in real-time observation of molecular motor motion, the molecular swimming machine designed and provided by this invention solves the problem of diffusion measurement. Utilizing its unique performance, it can be monitored in real-time under SMT (Surface Mount Technology) conditions, thereby revealing its motion patterns and enabling wider applications.

[0025] The advantages of this invention are also reflected in the following aspects: First, this invention is the first to design and prepare a molecular swimming machine and achieve translational diffusion swimming behavior—this invention designs and synthesizes a photoredox molecular swimming machine, covalently connecting the photoredox center and the redox shuttle through flexible PEG chains. Although the photooxidation reaction occurs inside the molecular swimming machine, autonomous propulsion is still observed, indicating that the interaction between the photoredox center and the redox shuttle is inherently asymmetric, and surrounding solvent molecules must also participate in this chemomechanical coupling, which can overcome the influence of Brownian motion in the fluid and achieve swimming behavior.

[0026] Secondly, the molecular swimming machine of this invention has high biocompatibility and can regulate cell membrane permeability through light, promoting the entry of extracellular substances into the cell to exert corresponding effects on the cell, such as killing cancer cells. The photo-oxidation-reduction molecular swimming machine provided by this invention has lipophilic properties, can be inserted into the phospholipid bilayer, and can be observed in real time using a TIRF microscope.

[0027] By utilizing the swimming ability and excellent biocompatibility of the photo-oxidation-reduction molecular swimming machine of the present invention, cell membrane permeability can be enhanced, and extracellular chemicals can be introduced into living cells, inducing apoptosis on demand. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 shows the NMR spectra of compounds NRT0, NRT3, and NRT9. a, 1H NMR spectrum of NRT0. b, 1C NMR spectrum of NRT0. c, 1H NMR spectrum of NRT3. d, 1C NMR spectrum of NRT3. e, 1H NMR spectrum of NRT9. f, 1C NMR spectrum of NRT9.

[0030] Figure 2 illustrates the photoredox reaction between NR and TEMPO, leading to enhanced NR diffusion. a) Schematic diagram of the reaction between NR molecules (red cuboids) and TEMPO (blue spheres) in a lipid bilayer under photoexcitation, resulting in mechanical propulsion and NR swimming. b) Representative trajectories of NR molecules show that propulsion increases with increasing TEMPO concentration and light intensity. Scale bar is 2 μm. c) Time-dependent MSD of NR molecules at different light intensities at a 5 mM TEMPO concentration. d) Corresponding diffusion coefficients (D) at different light intensities. e) At a fixed light intensity of 585 mW / mm².-2 Below, the time-dependent MSD of NR molecules at different TEMPO concentrations is shown. f represents the corresponding diffusion coefficient (D) at different TEMPO concentrations, with darker colors indicating higher light intensity or concentration.

[0031] Figure 3 shows the NR molecular trajectory illustrating the Lévy flight behavior. a, NR in Δt (from 12 to 48 ms) as light intensity increases from 130 mW / mm -2 Increased to 585mW mm -2 b, The step size PDF. The TEMPO concentration was fixed at 5 mM. b, The dependence of NR diffusion MSD on time interval shows an exponential factor α that increases at higher light intensities. c, At 585 mW mm -2 The typical trajectory of NR is shown below, marked with red circles indicating the inflection point (10°). The PDF of the waiting time Δt between the inflection points (5°, 10°, and 20°) shows power-law decay, where the exponential factor μ decreases with increasing light intensity, from 2.9 (green, 130mW / mm²). -2 ), 2.8 (blue, 390mW mm) -2 ) to 2.3 (red, 585mWmm) -2 Square, circular, and triangular patterns represent 5°, 10°, and 20° turns, respectively. The results were obtained by averaging ten sets of data.

[0032] Figure 4 illustrates the swimming behavior of a molecular swimming machine driven by intramolecular redox reactions. a, A sketch of a molecular swimming machine composed of NR (pink cubes) and TEMPO (blue spheres), where the non-interaction of the two components leads to overall mechanical propulsion. b, Molecular structures of molecular swimming machines with different PEG chain lengths (i.e., NRT0, NRT3, and NRT9). c, The diffusion coefficients D of NRT0 (red), NRT3 (yellow), and NRT9 (blue) under illumination are independent of molecular density, indicating that the reaction is intramolecular. This figure shows molecular photographs at different molecular densities. Scale bar 2 μm. d, The diffusion coefficients D of NRT0, NRT3, and NRT9 increase with increasing light intensity, while NRT0 shows the highest mobility. e, DFT calculations of the electrostatic potentials of the molecular swimming machine in the ground (red) and excited states (blue), interconverted via charge transfer during the excitation and regeneration steps. f. Typical MD simulation trajectories of the molecular swimming machine under no-charge-discharge (red), fixed-charge but no-discharge (green), and charge-discharge cycles (blue) show enhanced molecular diffusion under redox cycles. g. The MSD of the MD simulation trajectories as a function of the time delay is shown, with slopes of 0.90, 0.90, and 1.07 nm, respectively. 2 ns -1 The figure shows the corresponding PDF for the step size.

[0033] Figure 5 illustrates the molecular swimming machinery modulating cell membrane permeability. a, Schematic diagram of the molecular swimming machinery manipulating lipid droplets (LDs) and modulating membrane permeability. Yellow spheres represent LDs, propelled by the molecular swimming machinery (pink blocks and blue spheres). b, c, at 8mW mm -2 Trajectories of intracellular LDs treated with NR molecules (b) and NRT9 swimming machines (c) under light intensity. Scale bar 8 μm. d, Comparison of MSD of LDs treated with NR and NRT9 under light illumination, showing the aggregation effect of molecular swimming machines enhancing diffusion. The figure shows the corresponding diffusion coefficients of LDs. e, Schematic diagram of molecular swimming machines opening the cell membrane (pink blocks with blue spheres), facilitating the penetration of DAPI molecules (green spheres). f, Using NRT9 or NR (10 min), no (-L) or with (+L) light (20 mW mm) -2 Representative fluorescent DAPI images of HeLa cells treated with light, where only illuminated NRT cells show significant DAPI penetration. Scale bar 20 μm. g, (f) shows the changes in intranuclear DAPI fluorescence intensity for the corresponding treatments. h, NRT9 swimming machine at 0 to 20 mW / mm². -2 The time dependence of DAPI intensity under different light intensities (light blue to dark blue) is shown in the figure. This figure depicts the relationship between extracted cell membrane permeability and light intensity, expressed as K / K0 ~ I. 0.5 Fitting. i, Fluorescence images of Hoechst and SYTOX signals under three conditions: PTX with light, PTX without light, and light without PTX. Scale bar 30 μm. j, Comparison of intracellular SYTOX signal evolution at different doses of paclitaxel (PTX), shown with light (red) and without light (green), indicating enhanced efficacy due to increased membrane permeability caused by molecular swimming machinery. The color gradient from bright to dark represents PTX concentration increasing from 0 to 6.6 μM.

[0034] Figure 6 shows the phototoxicity assessment of the molecular swimming machinery in HeLa cells. a) shows the CCK-8 assay results, demonstrating NRT9 cell viability after 30 minutes of incubation without light at a specific threshold concentration. b) NRT9 cell viability was assessed under light conditions (light intensity 15 mW cm⁻¹). -2 (20 minutes). Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1: Detection of the properties of photoredox molecules in photoredox reactions

[0037] In this embodiment, the behavior of Nile red (NR) as the photoredox center and 2,2,6,6-tetramethylpiperidoxyl (TEMPO) as the redox shuttle in the photoredox molecular swimming machine was investigated in the photoredox reaction without PEG linkage. The results showed that NR molecules can diffuse in the presence of TEMPO.

[0038] (1) Preparation of lipid bilayer

[0039] Lipid bilayers were fabricated using a microfluidic chip. The master mold of the microfluidic chip was fabricated using a SMARTPRINT UV maskless lithography system (Microlight3D) on a silicon wafer via photolithography on an SU-82050. The microfluidic channel width was 100 μm. The polydimethylsiloxane (PDMS) microfluidic chip was replicated by pouring a PDMS mixture onto a prepared silicon wafer and then baking it at 65 °C for 10 hours. Subsequently, the PDMS and glass slides were cleaned and exposed to reactive oxygen plasma, where they bonded together to form the complete device. This device was designed to fabricate lipid bilayers and facilitate the exchange of external solutions. The lipid solution was prepared using L-α-phosphatidylcholine in chloroform to obtain a lipid stock solution with a concentration of 15 mg / mL, which was then dried to form a lipid membrane. Subsequently, appropriate amounts of isopropanol and a fluorescent dye were used to dissolve the dried lipid membrane. To prepare the bilayer, isopropanol was injected through the inlet of the device at a flow rate of 1000 μL / h for 10 minutes. Subsequently, the prepared lipid solution containing fluorescent dye was injected to ensure complete exchange, without dilution, at a flow rate of 10 μL / h for 30 minutes. Water was then introduced into the microfluidic chamber at a flow rate of 500 μL / h for 15 minutes to complete the lipid bilayer preparation procedure.

[0040] (2) The diffusion of NR molecules is enhanced in the photo-redox reaction.

[0041] The diffusion properties of NR molecules in photoredox reactions were investigated using single-molecule tracking (SMT). Specifically, total internal reflection fluorescence microscopy (TIRF) with a TIRF 100× objective was employed. Time-series images were captured using an ORCA-Fusion BT camera (C15440) at light intensities of 455, 520, 585, and 650 mW mm. -2 Below, the image sequence was recorded at a frame rate of 130fps. For values ​​below 455mWmm... -2The light intensity was measured, and the image sequence was recorded at a frame rate of 80 fps. The molecular trajectories were analyzed using TrackMate in ImageJ.

[0042] The dynamic behavior of NR molecules on a lipid bilayer was investigated using SMT technology under a TIRF microscope. As shown in Figure 2a, NR molecules are inserted into a lipid bilayer immersed in an aqueous TEMPO solution. Under 561 nm laser excitation, photoinduced electron transfer (PET) between NR and TEMPO molecules was induced, with the reaction rate modulated by light intensity and TEMPO concentration. The trajectory of NR molecules within the liquid lipid layer was recorded simultaneously (Figure 2b). The TEMPO concentration increased from 0 mM to 10 mM, and the light intensity increased from 130 mW to 585 mmW. -2 In the presence of TEMPO, the trajectory of NR molecules significantly increases. On the other hand, under constant light intensity, higher TEMPO concentrations induce higher PET rates, which also promotes NR migration, suggesting a correlation between molecular swimming behavior and the PET process.

[0043] The enhanced mobility and self-driven behavior of photoredox molecules can be further quantified by the time-dependent mean square shift (MSD) of NR molecules. The MSDs of NR at different light intensities and at different TEMPO concentrations are plotted in Figures 2c and 2e, and conform to the power-law relationship MSD = 4DΔt. α Where D is the diffusion coefficient, Δt is the hysteresis time, and α represents the scale exponent of MSD. As shown in Figures 2d and f, the MSD increases from 130 to 650 mW / mm². -2 The light intensity significantly increased D by tenfold, from 1.2±0.1 to 9.8±0.4 μm. 2 s -1 Similarly, as the TEMPO concentration increased from 0 to 10 mM, D also increased accordingly, from 0.29 ± 0.02 to 9.2 ± 0.5 μm. 2 s -1 .

[0044] (3) NR diffusion exhibits the motion pattern of Levi's flight.

[0045] The motion pattern of NR diffusion was observed using SMT technology. The results showed that the motion pattern of NR molecules is Lévy flight: the molecular motion deviates significantly from Brownian motion due to the mechanical propulsion generated by the photo-redox reaction. This is mainly because the photo-redox reaction contributes primarily to the translational degrees of freedom rather than the rotational degrees of freedom. Figure 3a illustrates the motion at light intensities of 130, 390, and 585 mW / mm². -2The probability density function of molecular displacements is shown below, with a fixed time interval ranging from 12 to 48 milliseconds. As the time interval increases, the distribution of molecular displacements exhibits a more pronounced long tail with higher light intensity. Furthermore, Figure 3b presents the power-law dependence of the mean square displacement of NR molecules as a function of time intervals under different light intensities. As the light intensity increases from 130 mW / mm... -2 Increased to 585mW mm -2 Besides the obvious enhanced diffusion, the scale exponent α of the MSD also appears to increase slightly from 1.05 to 1.15, indicating that molecular motion seems to become increasingly diffuse under stronger illumination, which can be attributed to enhanced self-propulsion. The long-tailed distribution and enhanced diffusion behavior observed under stronger illumination suggest that NR molecules exhibit a motion pattern similar to Lévy flight under stronger illumination, with displacement jumps larger than Brownian motion. Furthermore, the significant non-uniform propulsion velocity of NR molecules implies that NRs are performing Lévy flight rather than Lévy walking. We can further analyze molecular motion by defining "turning points" in the NR molecular trajectory where the rotation angle exceeds a certain threshold. Figure 3c shows a typical trajectory of NR molecules with turning points (marked with red circles), where turning points are defined as rotation angles > 10°, showing that NR molecules mainly undergo local fluctuations near fixed points, occasionally with long-distance jumps: a characteristic of Lévy flight. In addition, the distance between consecutive turning points in a given trajectory can be regarded as a migration segment ΔL, which is usually proportional to its duration Δt. -μ Figure 3d illustrates the segment length density as a function of Δt under different light intensities, showing that the tail of the segment length density decays exponentially as μ decreases from 2.9 to 2.3, and the light intensity increases from 130 mW / mm². -2 Increased to 585mW mm -2 According to Lévy flight theory, the migration type can be considered as Lévy flight with 1 < μ < 3. These results confirm that under photoredox reactions, molecules not only self-propel themselves beyond rotational fluctuations via translational motion, i.e., diffusion enhancement, but also that molecular propulsion is intermittent. Therefore, the observed molecular swimming machine migration step distribution is Lévy, rather than a Gaussian distribution like that of Brownian particles, thus realizing a macroscopic phase transition in a non-equilibrium thermodynamic state.

[0046] Example 2: Synthesis and Motion Performance Study of a Photoredox Molecular Swimming Machine

[0047] In this embodiment, the non-reciprocal nature of redox interactions at the single-molecule level is demonstrated by showing that the molecular swimming machine can drive itself through an intramolecular photoredox reaction. Furthermore, by constructing a self-contained, intramolecularly reaction-driven molecular swimming machine, the adaptability and applicability of the molecular swimming machine are significantly enhanced because operation does not require additional chemical fuels. In this embodiment, we covalently link the photoredox center (NR) to the redox shuttle (TEMPO) using flexible PEG chains to form a photoredox molecular swimming machine (NR-TEMPO, abbreviated as NRT), where the length of the PEG chain can be adjusted to tune the redox reaction rate and molecular stiffness (Figure 4a). Figure 4b shows the structures of three molecular swimming machines with different PEG chain lengths.

[0048] (1) Synthesis of photo-oxidation-reduction molecular swimming machinery

[0049] In this embodiment, all reactions were carried out in a dry solvent under an inert argon atmosphere using the standard Schlenk technique. 1 H and 13 C was recorded on a Bruker–400MHz NMR ARX400. 1 H and 13 The chemical shifts of the C10 NMR signals were referenced as internal standards for tetramethylsilane (δ = 0.00 ppm) and CDCl3 (δ = 77.00 ppm). Mass spectra were recorded on a Bruker APEX IV mass spectrometer. The synthetic routes used to obtain compounds 5 (n = 0, 3, and 9) are shown below.

[0050] Synthesis of Compound 3: 3-Diethylaminophenol (1 mM, Compound 1) was dissolved in a NaNO2 HCl / H2O mixed solution (0 °C, 2.5 h) to obtain nitrosophenol (Compound 2), which was then refluxed with 1,3-dihydroxynaphthalene in dimethylformamide (DMF) (110 °C, 45 min) to obtain Compound 3.

[0051] Synthesis of Compound 4: Compound 3 (1 mM), the PEG chain derivative (3 mM), and K₂CO₃ (5 mM) were dissolved in acetone (50 mL). The mixture was stirred overnight at 60 °C. The solution was cooled to room temperature and poured into water. The crude product was extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The desired product was separated by silica gel column chromatography.

[0052] Synthesis of Compound 5: Compound 4 (1 mM), 4-hydroxyTEMPO (5 mM), and NaH (8 mM) were dissolved in dimethylformamide (DMF). The solution was then cooled to room temperature and the DMF was removed under reduced pressure. To further purify the crude product, it was dissolved in ethyl acetate and then adsorbed onto silica gel. Finally, the desired products of Compound 5 (designated NRT0, NRT3, and NRT9, with n values ​​of 0, 3, and 9, respectively) were obtained by silica gel column chromatography. 1 H NMR, 13 The structures of NRT0, NRT3, and NRT9 were confirmed by C NMR and mass spectrometry, respectively, and the results are as follows.

[0053] (2) Evaluate the propulsion efficiency and diffusion properties of the photo-redox molecular swimming machine.

[0054] To evaluate the propulsion efficiency of the molecular swimming machines, the synthesized NRT0, NRT3, and NRT9 molecular swimming machines (Figures 4a-b) were inserted into a lipid bilayer prepared according to the method of Example 1. Their trajectories were acquired under different light intensities, and the mean square displacement (MSD) and corresponding diffusion coefficient D of the molecules could be extracted. It was assumed that the interaction between NR and TEMPO was mutual, and the forces between the two reactive sites within the molecule would cancel each other out, leading to the elimination of molecular swimming motion. However, surprisingly, all NRT molecules exhibited similar light intensity-dependent swimming behavior. By measuring different molecular densities ranging from 0.4 to 1.2 molecule μm... -2 The diffusion of NRT was evaluated, and it can be seen that under a given illumination intensity, D is independent of molecular density, and is approximately 3.1 ± 0.04, 2.1 ± 0.1, and 1.6 ± 0.05 μm for NRT0, NRT3, and NRT9, respectively. 2 s -1 (Figure 4c). This stable migration confirms that the redox reactions associated with molecular swimming are intramolecular rather than intermolecular, indicating that the photoredox interaction between the photoredox center (NR) and the redox shuttle (TEMPO) is non-reciprocal and responsible for the propulsion of the molecular swimming machine. Subsequently, the diffusion coefficients D of NRT0, NRT3, and NRT9 were extracted based on their MSDs, using the same method previously used for NR. As shown in Figure 4d, all three molecular swimming machines exhibited enhanced diffusion with increasing light intensity, with NRT0 showing a higher D than NRT3 and NRT9. This result suggests that the shorter chain length between NR and TEMPO leads to a higher redox reaction rate, enhancing propulsion efficiency.

[0055] Figure 4e illustrates the charge distribution of NRT3 undergoing a charge / discharge cycle consisting of TEMPO charge transfer and regeneration. Under illumination, electron transfer from TEMPO to NR increases the potential difference at the molecular surface, while the subsequent regeneration step transfers electrons back to TEMPO, restoring its initial surface potential. Molecular dynamics (MD) simulations were used to study the dynamic behavior of the molecule under this cyclic surface potential, and the results were compared with two control groups with zero or stationary charge. Typical trajectories of the NRT3 molecule under these three settings are shown in Figure 4f, demonstrating that NRT3 diffuses faster through the cyclic surface potential compared to the two control groups with zero or stationary charge. Statistical analysis data show that the mean square displacement of the step size and the probability density function indicate enhanced diffusion of the NRT molecule with charge / discharge cycling (blue), while the molecule with zero or stationary charge shows relatively slow diffusion (Figure 4g).

[0056] Example 3: In vitro study of a photo-oxidation-reduction molecular swimming machine

[0057] Cellular behavior of HeLa cells was investigated using a Leica DM18 confocal laser scanning microscope. Cells were cultured at 5% CO2 and 37°C in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL of [unspecified ingredient]. -1 Penicillin and 100 μg mL -1 Streptomycin (all from Invitrogen).

[0058] (1) Research on the regulation of lipid droplet motion by photo-oxidation-reduction molecular swimming machinery

[0059] To investigate lipid droplet (LD) dynamics, HeLa cells were collected and incubated for 10 min with 5 μM NR (as a control) or NRT9. Subsequently, the cells were washed three times with phosphate-buffered saline (PBS, Invitrogen) and then incubated with 1 × 10⁻⁶ cells. 5 Cells mL -1 Cells were seeded at a density in confocal culture dishes (ibidi, LOT052923A001). After attaching to the culture dishes, the cells were washed three times with PBS and fresh cell culture medium was added. The confocal culture dishes were maintained at a constant 37°C and 5% CO2 environment to ensure cell viability and were exposed to a 552nm laser. Video was recorded at 0.2fps, and data analysis was performed using TrackMate in ImageJ.

[0060] The results showed that the study was conducted in HeLa cells containing molecular swimming machinery and NR, where NR and NRT selectively concentrated on lipid droplets and could be tracked individually (Fig. 5a). As shown in Fig. 5b-d, lipid droplets containing molecular swimming machinery exhibited significantly longer trajectories and a corresponding diffusion coefficient D that was approximately 3-fold enhanced compared to lipid droplets containing NR. This observation indicates that the intramolecular photoreduction reaction of NRT not only propels the lipid droplets at the molecular scale but also enhances their diffusion. Subsequently, we calculated the MSD of the lipid droplets, showing a superdiffusion property with an α of approximately 1.25 for lipid droplets containing NRT9. Furthermore, an attenuation parameter μ = 1.7 ± 0.2 was observed in lipid droplets loaded with NRT, while μ = 3.4 ± 0.3 was observed for lipid droplets without active agitation. Since 1 < μ < 3 was observed for LDs-NRT, it can be seen that the migration of lipid droplets under the propulsion of molecular swimming machinery is also a Lévy flight.

[0061] (2) Research on the regulation of cell membrane permeability by photo-oxidation-reduction molecular swimming machinery

[0062] Because molecular swimming machines migrate much faster than surrounding lipid molecules, membrane dynamics cannot match them. Therefore, molecular swimming machines can temporarily open the membrane, enhancing membrane permeability and cellular uptake rates. To assess this, the membrane-impermeable DNA staining agent 4′,6-diamino-2-phenylindole (DAPI) was used to test the regulation of cell membrane permeability by molecular swimming machine movement (Figure 5e). Specifically, HeLa cells were treated with 5 μM NR, NRT9, or TEMPO, respectively, following the previously mentioned procedures. Cell culture medium containing 1 μM DAPI was added to the cells. Subsequently, the cells were immediately exposed to a light beam at a wavelength of 552 nm for 10 minutes. Furthermore, the DAPI intensity was detected using 405 nm light. To ensure cell viability, confocal culture dishes were maintained at a constant 37°C and 5% CO2. Video was recorded at a frame rate of 1 fps, and data analysis was performed using ImageJ.

[0063] Regarding the results of the photo-redox molecular swimming machinery regulating cell membrane permeability, Figure 5f shows sequential fluorescence images of adherent HeLa cell clusters treated with the NRT9 swimming machinery and DAPI under light stimulation. It reveals that 10 minutes after treatment (Figure 5g), DAPI entry into the cell nucleus was significantly increased under the action of light and molecular swimming machinery, while cells treated with NR and TEMPO under light or no light showed negligible DAPI penetration, confirming that the enhanced cell membrane permeability should be attributed to molecular swimming motion.

[0064] Since the swimming speed of molecules is proportional to light intensity, cell membrane permeability should also be positively correlated with light intensity. This conclusion was confirmed by monitoring the evolution of DAPI fluorescence intensity in the cell nucleus with light intensity (Figure 5h). To quantitatively understand the relationship between cell membrane permeability and light intensity, a theoretical model was used to estimate intracellular transmembrane processes, thereby extracting membrane permeability under different light intensities. In this model, DAPI molecules crossing the cell and nuclear membranes are considered as a diffusion process described by Fick's law. As shown in Figure 5h, the normalized cell membrane permeability coefficient, denoted as K / K0, is given by K / K0 ~ I^ 0.5 Scaling, where K0 represents the membrane permeability in the absence of illumination.

[0065] (3) Research on photo-oxidation-reduction molecular swimming machinery to promote drug killing of cancer cells

[0066] Given the ability of molecular swimming machines to enhance cell membrane permeability, we demonstrated their enhancing effect on paclitaxel (PTX)-induced cancer cell necrosis. We evaluated the necrosis effect of NRT9-treated cancer cells under different PTX concentrations and illumination conditions using standard SYTOX and Hoechst staining. Specifically, to investigate cell viability, the nucleic acid dye SYTOX Green (Thermo Fisher, S7020) was used as an indicator of dead cells. Specific amounts of paclitaxel (PTX) and 1 μM SYTOX Green were added to the cell culture medium. The treated molecular swimming machine cells were immediately exposed to a 552 nm light beam for 1 hour. Furthermore, 488 nm light was used to detect dead cells, while 405 nm light was used to locate the nuclei and viable cells. To ensure cell viability, confocal culture dishes were maintained at a constant 37°C and 5% CO2. Video was captured at a frame rate of 0.25 fpm, and data analysis was performed using ImageJ.

[0067] The results, shown in Figure 5i, depict typical fluorescence images of cancer cells under three conditions: 3.3 μM PTX under illumination, 3.3 μM PTX without illumination, and PTX without illumination. Only PTX under illumination induced significant apoptosis labeled SYTOX, indicating that molecular swimming enhances intracellular penetration and uptake of the drug. Quantitatively, the enhanced PTX potency under illumination and without illumination was monitored using SYTOX staining. As shown in Figure 5j, under illumination, extremely low doses of PTX were required to induce apoptosis, while the time required for SYTOX signal saturation decreased with higher PTX doses. Notably, at a drug concentration of 6.6 μM, apoptosis occurred within just 24 minutes due to rapid drug uptake. In contrast, no apoptosis was observed under no illumination, further confirming that the enhanced drug potency was induced by molecular swimming mechanisms.

[0068] Example 4: Study on phototoxicity of photoredox molecular swimming machinery

[0069] This example evaluated the phototoxicity of NRT9 in HeLa cells. In this example, a total of 5000 cells were seeded into 96-well plates. Subsequently, the cells were treated with a series of concentrations of NRT9 for 30 minutes. After treatment with a molecular swimming apparatus, the cells were exposed to light at an intensity of 15 mW cm⁻¹. -2 Cells were exposed to green light for 20 minutes. Subsequently, they were washed three times and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was then carefully removed, and a CCK-8 solution was prepared by diluting the CCK-8 solution with fresh culture medium at a ratio of 1:10. The prepared solution was added to the cells, and the absorbance of the cells was measured at 450 nm using a microwell reader (SpectraMax iD5, Molecular Devices). Cell viability was calculated according to the CCK-8 protocol (C0046, Beyotime).

[0070] The results showed almost no cytotoxicity in cells without light, while at a light intensity of 15 mW / cm², the cytotoxicity was significantly higher. -2 At times, exceeding 116 μg / mL -1 An overdose of NRT9 resulted in mild cell damage (Figure 6). These findings lay the foundation for the future application of molecular swimming machines in cancer treatment and drug delivery, paving the way for more effective therapeutic interventions.

[0071] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A photoredox molecular swimming machine, the structure of which includes a photoredox center, a redox shuttle, and a flexible PEG chain covalently connecting the photoredox center and the redox shuttle.

2. The photo-oxidation-reduction molecular swimming machine according to claim 1, wherein the structure of the photo-oxidation-reduction molecular swimming machine is as shown in compound 5: Where n is an integer between 0 and 9.

3. The photo-oxidation-reduction molecular swimming machine according to claim 2, wherein n is 0, 3, or 9.

4. A method for preparing the photo-oxidation-reduction molecular swimming machine according to claim 2 or 3, wherein the synthetic route of the preparation method is as follows: Where n is an integer between 0 and 9.

5. The preparation method according to claim 4, wherein n is 0, 3, or 9.

6. The preparation method according to claim 4, wherein all reactions are carried out in a dry solvent under an inert argon atmosphere using standard Schlenk techniques.

7. Use of the photo-oxidation-reduction molecular swimming machine according to claim 1, wherein the use includes at least one of the following: (1) Regulates lipid droplet movement; (2) Regulates cell membrane permeability; (3) Used to prepare pharmacodynamic enhancers that can promote drug entry into cells; or (4) Drug delivery.

8. The use according to claim 7, wherein the drug is a cancer cell killer.

9. The use according to claim 7, wherein the drug in the drug delivery is a drug that needs to be delivered from outside the cell to inside the cell and exert its pharmacological effect inside the cell.

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