A method for preparing a photosynthetic nanomotor powered by a biomolecular motor

Abstract

This invention discloses a photosynthetic nanomotor powered by a biomolecular motor and its preparation method, belonging to the field of colloidal motors. This invention aims to address the current limitations of research on the rotational dynamics of FoF1-ATP synthase molecular machines, which is limited to the motion of single and isolated objects. The photosynthetic nanomotor is prepared based on supramolecular self-assembly technology through the reconstruction of thylakoid vesicles and lecithin vesicles. The photophosphorylation system on the thylakoid membrane surface is reconstructed on the photosynthetic nanomotor. Utilizing the preserved biomolecular motor FoF1-ATPase as the power engine, under external visible light irradiation, protons generated by the photosystem accumulate in the photosynthetic liposome motor, driving the FoF1-ATPase to synthesize the energy currency ATP. Through metabolic reactions, light energy is converted into chemical energy ATP, ultimately generating self-propulsion. Due to its unique energy currency regeneration capability, the photosynthetic liposome motor has broad application prospects in the biomedical field.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of colloidal motors, and particularly relates to a preparation method of a photosynthetic nanomotor powered by a biomolecular motor. BACKGROUND

[0002] In vivo, biomolecular motors are responsible for energy conversion and movement from the molecular scale to the macro scale. The coordinated movement of multiple biomolecular motors can drive the movement of larger scale cell structures (larger scale organelles, cytoskeletons, and even multicellular tissues); inspired by this, driving mesoscopic systems by amplifying the action of molecular motors at the nanoscale can deepen the understanding of the basic principles of controlling the dynamic behavior of self-assembled subcellular structures, and can guide and design living artificial materials and machines far from equilibrium. In addition, the integration of biomolecular motors into mesoscopic materials to develop intelligent materials can obtain special materials such as movement, self-repair, and reproduction. The collection of biomolecular machines, such as myosin and kinesin, has been successfully integrated into micro-chemical systems, paving the way for the manufacture of new robots with cell-like functions. As a typical rotary molecular motor, FoF1-ATPase is a key enzyme for energy conversion in cells, powered by transmembrane proton potential, and synthesizes biological energy currency ATP with individual rotation. Current research on the rotary dynamics of FoF1-ATPase molecular machines only stays on the movement behavior of a single and isolated object. However, how the coordinated and synchronous rotation of multiple ATPase molecular motors controls energy dissipation at the microscopic scale and the influence of collective rotation on the movement behavior of colloidal scale materials has not been explored. SUMMARY

[0003] Based on the above technical deficiencies, the application develops and researches a photosynthetic nanomotor that can be driven by a rotary biomolecular motor, which can be propelled by the collective rotation of multiple FoF1-ATPases in the photosynthetic phosphorylation process. Based on the supramolecular self-assembly strategy of phospholipid phase separation principle, the application prepares a photosynthetic nanomotor with asymmetric distribution of biomolecular motor FoF1-ATPases. Light-induced proton accumulation in photosystem II leads to the accumulation of proton potential to drive rotary FoF1-ATPases to complete the process of photo-phosphorylation. The effective propulsion speed is positively correlated with the speed of photo-phosphorylation, indicating that the photosynthetic nanomotor is propelled by the coordinated rotation process of multiple FoF1-ATPases in the photo-phosphorylation process.

[0004] The application uses the reserved biomolecular motor FoF1-ATPase as a power engine, under the irradiation of an external visible light field, the protons generated by the photosystem accumulate in the photosynthetic liposome motor to drive the biomolecular motor FoF1-ATPase to synthesize energy currency ATP. Through metabolic reactions, light energy is converted into chemical energy ATP, and finally into self-propulsion. The photosynthetic liposome motor has broad application prospects in the biomedical field due to its unique energy currency regeneration ability.

[0005] The application provides a biomolecular motor energy supply photosynthetic nanomotor.

[0006] Step 1, homogenize spinach and separate the homogenate containing chloroplasts;

[0007] Step 2, then separate and purify chloroplasts;

[0008] Step 3, then lyse the chloroplasts and recover the thylakoids;

[0009] Step 4, then homogenize the thylakoids to obtain thylakoid membrane fragments:

[0010] Step 5, then extrude the thylakoid membrane fragments to obtain thylakoid vesicles;

[0011] Step 6, use egg yolk lecithin as a synthetic liposome raw material and cholesterol as a stabilizer to prepare lecithin liposome vesicles;

[0012] Step 7, reconstitute the thylakoid membrane and lecithin membrane by mechanical co-extrusion to obtain the photosynthetic nanomotor.

[0013] Further limitation, the preparation method of the thylakoid vesicles can only be prepared from spinach.

[0014] Further limitation, in step 1, homogenizing spinach and separating the homogenate containing chloroplasts, the specific steps are as follows:

[0015] ①, store the spinach in a 4℃ constant temperature refrigerator for 12h in the dark;

[0016] ②, wash 1kg of spinach leaves, separate the leaves and stems;

[0017] ③, tear the spinach leaves by hand, separate the meridians and recycle the leaf part, mix the leaf part with 50 ml chloroplast buffer (containing sucrose 68.56 g, KCl 0.3727 g, Na2HPO4 0.88995 g, KH2PO4 2.04135 g, MgCl2 0.2033 g, dissolved in water and made up to 500 mL) uniformly. Add 20 g of quartz sand to the mixture, grind uniformly with a mortar, centrifuge to separate the precipitate at 1000 rpm, and keep the supernatant. Adjust the speed to 4000 rpm and repeat the centrifugation to recover the precipitate.

[0018] Further limitation, in step 2, the chloroplast is separated and purified, specifically according to the following steps:

[0019] ①, resuspend the recovered precipitate in the chloroplast buffer to form a mixture.

[0020] ②, prepare 20% (w / w), 40% (w / w), 60% (w / w) sucrose solution, prepare gradient density centrifugation liquid, from top to bottom are 20% (w / w), 40% (w / w) and 60% (w / w) in turn, place the mixture of step ① in the sucrose gradient density centrifugation liquid, centrifuge at 4000 rpm for 5 min.

[0021] After the centrifuge stops, the chloroplast is in the 40% sucrose layer of the gradient density centrifugation liquid.

[0022] ③, recover the chloroplast dispersion layer obtained in step ② and resuspend it in the chloroplast buffer, centrifuge at a speed of 4000 rpm to recover the precipitate.

[0023] ④, repeat the washing and centrifugation step of step ③ to obtain purified chloroplasts.

[0024] Further limitation, in step 3, the chloroplasts are lysed and the thylakoids are recovered, specifically according to the following steps:

[0025] Disperse the purified chloroplasts in the chloroplast lysis solution (10 mL of 10 mM Hepes-NaOH at pH = 7.5, 0.50825 g of MgCl2 dissolved in water and made up to 500 mL), mix uniformly, stand for 10 min to 15 min, then centrifuge at a speed of 4000 rpm to recover the precipitate to obtain thylakoids. Place the thylakoids in the thylakoid lysis protection solution (2.5 mL of 10 mM Hepes-NaOH at pH = 7.5, 0.254125 g of MgCl2, 1.86375 g of KCl dissolved in water and made up to 500 mL) to obtain purified thylakoids.

[0026] Further limitation, in step 4, the thylakoids are homogenized to obtain thylakoid membrane fragments, specifically according to the following steps:

[0027] The thylakoid is dispersed in the thylakoid lysis protection solution, broken by a glass homogenizer for 5 min, fine quartz sand is added, and the thylakoid membrane fragments are obtained by grinding for 30 min, centrifugation at a centrifugal speed of 8000 rpm, removal of the quartz sand powder, and recovery of the supernatant.

[0028] Further limited, in step 5, the thylakoid membrane fragments are extruded to obtain thylakoid vesicles, which are specifically prepared according to the following steps:

[0029] The thylakoid membrane fragments are dispersed in the thylakoid lysis protection solution, and the thylakoid vesicles are obtained by extruding the polycarbonate membrane with a pore size of 3 μm, 1 μm, 800 nm, 400 nm, 200 nm, and 200 nm from large to small pore size by 21 times through a cell membrane extruder.

[0030] Further limited, in step 6, the lecithin liposome vesicles are prepared, which are specifically prepared according to the following steps:

[0031] The egg yolk lecithin is selected as the raw material for synthesizing the liposome, cholesterol is selected as the stabilizer, and the thylakoid lysis protection solution is selected as the buffer.

[0032] ①, 20 mg of lecithin and 5 mg of cholesterol are first weighed and dispersed in 3 ml of chloroform, and dissolved in a 1000 ml round-bottom flask.

[0033] ②, the chloroform organic solution obtained in step ① is evaporated by a rotary evaporator. After all the organic solvents are removed, nitrogen is blown for 1 h to ensure that the organic solvents are completely removed, and the round-bottom flask is vacuum dried for 12 h.

[0034] ③, 10 ml of lysis protection solution is added to the round-bottom flask obtained in step ②, and ultrasonic treatment is performed for 10 min to obtain lecithin vesicles.

[0035] ④, the lecithin vesicles are dispersed in the thylakoid lysis protection solution, and the lecithin vesicles with uniform size are obtained by extruding the polycarbonate membrane with a pore size of 3 μm, 1 μm, 800 nm, 400 nm, 200 nm, and 100 nm from large to small pore size by 21 times through a cell membrane extruder.

[0036] Further limited, in step 7, the photosynthetic nanomotor is prepared by co-extrusion, which is specifically prepared according to the following steps:

[0037] The lecithin vesicles and the photosynthetic nanovesicles are mixed uniformly at a volume ratio of 4:1, and the photosynthetic nanomotor with uniform size is obtained by extruding the polycarbonate membrane with a pore size of 200 nm by 21 times through a cell membrane extruder.

[0038] The application makes full use of the light response system of natural chloroplast structure, and studies the biomolecular motor FoF1-ATPase light reaction activity and ATP synthesis capacity of the photosynthetic nanomotor under visible light irradiation in view of the fact that the mixed film of the Janus photosynthetic nanomotor is derived from a thylakoid. 3- Under visible light irradiation, the ATP content in the environment is detected, and it is found that the ATP concentration gradually increases with the increase of the light irradiation time. The photosynthetic phosphorylation activity is further verified. Under the same condition, the motion behavior of the photosynthetic nanomotor is monitored, and it is found that the motion speed of the photosynthetic nanomotor is positively correlated with the photosynthetic phosphorylation speed.

[0039] The application studies the influence of light intensity on the motion behavior and photosynthesis of the photosynthetic nanomotor, and further counts the motion law and mechanism: it is found that there is a correlation between the ATP generation capacity and the motion of the photosynthetic nanomotor colloid motor under different light intensities. 2 Under the power of 30 mW / cm2, the photosynthetic nanomotor has the strongest ATP generation capacity and the best self-driving capacity. When the light intensity is lower or higher than the light intensity, the motion capacity is obviously decreased. When the light intensity is lower than the light saturation point, the photosynthesis intensity is low. Similar to the plant growth effect, when the light intensity is higher than the light intensity value, the light inhibition state is reached, and the ATP synthesis capacity is also decreased. Therefore, the photosynthetic nanomotor prepared in the application can control the photosynthesis, and then effectively control the motion of the photosynthetic nanomotor.

[0040] The photosynthetic nanomotor of the application can perform the photosynthetic phosphorylation process according to the change of the environmental light intensity, and the strength of the photosynthetic phosphorylation process affects the motion effect of the photosynthetic nanomotor.

[0041] The photosynthetic nanomotor of the application can respond to the environmental light, and has great development potential in drug carrying, in-vivo imaging and related disease treatment.

[0042] The beneficial effects of the application relative to the prior art are as follows:

[0043] The application can prepare the photosynthetic nanomotor powered by the biomolecular motor;

[0044] The application can regulate the capacity of the photosynthetic nanomotor (the motion control is completed by regulating the photosynthetic phosphorylation process through the light intensity);

[0045] The photosynthetic nanomotor is prepared by fusing natural thylakoid membrane and artificial lecithin membrane to form an asymmetric structure by using a supramolecular self-assembly strategy, so as to increase driving efficiency.

[0046] The photosynthetic nanomotor of the application retains natural thylakoid membrane related pigments and photosynthetic systems, and has good photochemical activity.

[0047] The photosynthetic nanomotor of the application has the ability of regenerating ATP in vitro, and has good biological catalytic reaction activity and self-driven movement ability.

[0048] In order to further understand the features and technical contents of the application, please refer to the following detailed description of the application and the accompanying drawings, however, the accompanying drawings are provided for reference and illustration only, and are not used to limit the application. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 It is a preparation method flowchart of the photosynthetic nanomotor of the application;

[0050] Figure 2 It is an atomic force microscope picture of the photosynthetic nanomotor;

[0051] Figure 3 It is a transmission electron microscope picture of the photosynthetic nanomotor;

[0052] Figure 4 It is the photosynthetic phosphorylation speed of the photosynthetic nanomotor under different light intensities;

[0053] Figure 5 It is a motion picture of the photosynthetic nanomotor under dark conditions and light intensity of 30mW / cm 2

[0054] Figure 6 It is a schematic diagram of self-driven movement of the photosynthetic nanomotor under visible light. DETAILED DESCRIPTION

[0055] The application will be further described below in conjunction with specific examples, which should be understood as only for illustrating the application but not for limiting the scope of the application.

[0056] The specific embodiments of the application are described in detail below, which are part of the specification but do not limit the application. The principles of the application are illustrated by the examples, and other aspects, features and advantages of the application will become apparent from the detailed description.

[0057] Example 1 combines Figure 1 ​Specifically, the biomolecule motor-powered photosynthetic nanomotor of the present embodiment is prepared by combining "top-down" and "bottom-up" chemical synthesis methods, as shown in the figure. First, spinach leaves are ground, and then the leaf cells are homogenized into a centrifuge tube, and then the chloroplasts are separated by differential centrifugation, and further purified by sucrose gradient density centrifugation. The precipitate containing chloroplasts, nuclei and other impurities is recovered. The chloroplasts are separated by differential centrifugation, and the chloroplasts are destroyed by low permeability lysis to obtain thylakoid membranes. The thylakoid membranes are homogenized for 10 minutes at low temperature using a glass homogenizer, and then homogenized with quartz sand for half an hour to obtain thylakoid membrane fragments. The thylakoid membrane fragments are then extruded to obtain thylakoid vesicles. After the thylakoid vesicles are mixed with blank egg phospholipid liposomes and incubated for one hour, they are extruded through a 200 nm polycarbonate membrane to obtain photosynthetic nanomotors with a particle size of about 230 nm.

[0058] The preparation of the photosynthetic nanomotor mainly includes seven parts:

[0059] (1) Homogenize spinach and separate the homogenate containing chloroplasts:

[0060] ①, store the spinach in a 4°C constant temperature refrigerator in the dark for 12 hours;

[0061] ②, wash 1 kg of spinach leaves, separate the leaves and stems;

[0062] ③, tear the spinach leaves by hand, separate the leaf meat part, and mix the leaf meat part with 50 ml of chloroplast buffer (containing sucrose 68.56 g, KCl 0.3727 g, Na2HPO4 0.88995 g, KH2PO4 2.04135 g, MgCl2 0.2033 g, dissolved in water and made up to 500 mL). Add 20 g of quartz sand to the mixture and grind it evenly with a mortar. Centrifuge at 1000 rpm to remove the precipitate, then adjust the speed to 4000 rpm and repeat the centrifugation to recover the precipitate.

[0063] (2) Isolation and purification of chloroplasts:

[0064] ①, resuspend the recovered precipitate in chloroplast buffer to form a mixture.

[0065] ②, prepare 20% (mass), 40% (mass), and 60% (mass) sucrose solutions, and prepare a gradient density centrifugation solution. From top to bottom, the gradient density centrifugation solution is 20% (mass), 40% (mass), and 60% (mass). Place the mixture of step ① in the sucrose gradient density centrifugation solution and centrifuge at 4000 rpm for 5 minutes.

[0066] After the centrifuge stops, the chloroplasts are in the 40% (mass) sucrose layer of the gradient density centrifugation solution.

[0067] ③, recover the chloroplast dispersion layer obtained in step 2, and resuspend in a chloroplast buffer, centrifuge at 4000 rpm, and recover the precipitate.

[0068] ④, repeat the washing and centrifugation step of step 3 to obtain purified chloroplasts.

[0069] (3) Lysis of chloroplasts and recovery of thylakoids:

[0070] Disperse the purified chloroplasts in a chloroplast lysis solution (10 mL of 10 mM Hepes-NaOH at pH 7.5, 0.50825 g of MgCl2 dissolved in water and made up to 500 mL), mix well, and centrifuge at 4000 rpm for 10 min to 15 min, and recover the precipitate to obtain thylakoids. Place the thylakoids in a thylakoid lysis protection solution (2.5 mL of 10 mM Hepes-NaOH at pH 7.5, 0.254125 g of MgCl2, 1.86375 g of KCl dissolved in water and made up to 500 mL) to obtain purified thylakoids.

[0071] (4) Homogenization of thylakoids to obtain thylakoid membrane fragments:

[0072] Disperse the thylakoids in the thylakoid lysis protection solution, break them up with a glass homogenizer for 5 min, add ultra-fine quartz sand, mix well, and grind for 30 min, centrifuge at 8000 rpm to remove the quartz sand powder, and recover the supernatant to obtain thylakoid membrane fragments.

[0073] (5) Extrusion of thylakoid membrane fragments to obtain thylakoid vesicles:

[0074] Disperse the thylakoid membrane fragments in the thylakoid lysis protection solution, and use a cell membrane extruder with pore sizes of 3 μm, 1 μm, 800 nm, 400 nm, and 200 nm, and extrude 21 times from large to small pore sizes using polycarbonate membranes to obtain thylakoid vesicles.

[0075] (6) Preparation of lecithin liposome vesicles:

[0076] Select egg yolk lecithin as the raw material for synthesizing liposomes, cholesterol as the stabilizer, and the thylakoid lysis protection solution as the buffer.

[0077] ⑤, first weigh 20 mg of lecithin and 5 mg of cholesterol, disperse them in 3 ml of chloroform, and dissolve them thoroughly in a 1000 mL round-bottom flask.

[0078] ⑥, rotary evaporate the chloroform organic solution obtained in step ① using a rotary evaporator. After all the organic solvents are removed, blow nitrogen for 1 h to ensure that the organic solvents are completely removed, and vacuum dry the round-bottom flask for 12 h.

[0079] ⑦ Add 10 ml of lysis protection solution to the round-bottom bottle obtained in step ②, and sonicate for 10 min to obtain lecithin vesicles.

[0080] ⑧. Disperse lecithin vesicles in a thylakoid lysis protection solution, and use a cell membrane extruder to extrude polycarbonate membranes with pore sizes of 3μm, 1μm, 800nm, 400nm, 200nm, and 100nm in stages from large pore size to small pore size 21 times to obtain lecithin vesicles with uniform size.

[0081] (7) Co-extrusion preparation of photosynthetic nanomotors:

[0082] After lecithin vesicles and photosynthetic nanovesicles were mixed evenly at a volume ratio of 4:1, they were extruded 21 times using a polycarbonate membrane with a pore size of 200 nm through a cell membrane extruder to obtain photosynthetic nanomotors with uniform size.

[0083] Example 2: Photosynthetic nanomotors were dispersed on mica sheets and freeze-dried. Their morphologies under an atomic microscope are shown below. Figure 2 As shown, the thickness of 14.7 nm corresponds exactly to the thickness of the phospholipid bilayer, indirectly confirming its vesicle structure.

[0084] The photosynthetic nanomotor obtained in Example 2 was stained with 0.1% phosphotungstic acid for four minutes and then observed under a transmission electron microscope. Figure 2 As shown, it has a single-layer vesicle structure with a particle size of 200 nm.

[0085] Example 3

[0086] When chloroplasts, thylakoids, and photosynthetic nanomotors are observed under an optical microscope, chloroplasts and thylakoids appear as flattened ellipsoids or spheres, while photosynthetic nanomotors are too small to be observed under an optical microscope. However, when excited by 488nm excitation light, the red fluorescence emitted by their chlorophyll can be observed, and they appear as red spots under the microscope.

[0087] Example 4

[0088] The hydration size of photosynthetic nanomotors was investigated using a Zetasizer particle size analyzer. Figure 4 The photosynthetic nanomotors were found to be uniform in size, with an average hydration diameter of 230 nm. This is consistent with the results obtained from transmission electron microscopy and atomic force microscopy.

[0089] Example 5

[0090] 10 mg of photosynthetic nanomotors and thylakoids were dispersed in 10 mL of deionized water, and their light absorption range of 400 nm-800 nm was measured. The results are as follows: Figure 5As shown, since part of the phospholipid membrane of the photosynthetic nanomotor comes from natural thylakoids, it has similar natural pigments to natural thylakoids, chlorophyll A, B and lutein carotenoids, so its absorption spectrum is similar to the absorption peaks at 480 nm and 680 nm. In addition, the fluorescence spectrum of the photosynthetic nanomotor and the thylakoid is also very similar, and the fluorescence emission peak is at 675 nm.

[0091] Example 6

[0092] Since the mixed membrane of the photosynthetic nanomotor comes from thylakoids, it has a photosynthetic phosphorylation mechanism. P680+ / P680 on the photosynthetic nanomotor is excited from the ground state to the excited state (P680+ / P680*). At the same time, the water is photolyzed by the photosystem II module to provide protons and photo-generated electrons. The transmembrane proton gradient is accumulated inside and drives the synthesis of ATP in the presence of ADP and PO4 3-

[0093]

[0094] Example 7

[0095] The ATP concentration was measured by high performance liquid chromatography (HPLC) as Figure 6 shown. 0.5 hours before measurement, DTT was added to the protein nanometer to make its final concentration 50 mM to reduce the enzyme. 3 mL of photosynthetic nanomotor was added to 1 mL of buffer solution (pH 8.0, 10 mM Tricine-NaOH, 20 μM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2, and 30 mM NaCl). Sample was taken every ten minutes to monitor the change of components. By adjusting the light intensity, it was found that when the light saturation point was reached, the light intensity was 30 mW / cm 2 , Under this light intensity, the ATP generation rate was the fastest, and the ATP concentration was 44 μmol / L after one hour of illumination.

[0096] Example 8

[0097] The movement of the photosynthetic nanomotor was observed and recorded using a fluorescence microscope. The photosynthetic nanomotor was labeled with Dii red blood cell membrane dye, and the green excitation light with an excitation wavelength of 534 nm was used. The movement was observed under 30 mW / cm 2 visible light intensity, and the CCD camera recorded the movement behavior of the photosynthetic nanomotor. The trackmate plug-in was used to track and calculate the movement of the photosynthetic nanomotor as Figure 5 ​As shown, the speed of the photosynthetic nanomotor under visible light is twice that under dark condition. At this time, the ATP synthesis speed of the photosynthetic nanomotor also reaches the maximum value, proving that the self-driven motion behavior of the photosynthetic nanomotor is closely related to the photosynthetic phosphorylation process in which the rotary molecular motor participates.

[0098] The raw materials listed in the present application, the upper and lower limits, interval values of each raw material of the present application, and the upper and lower limits, interval values of process parameters (such as temperature, time, etc.) can all achieve the present application, and examples are not listed one by one.

[0099] The above is only a preferred embodiment of the present application, of course, cannot limit the scope of the present application, should be noted that, for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and changes, these improvements and changes are also considered to be within the scope of the present application.

Claims

1. A biomolecule motor powered photosynthetic nanomotor, characterized in that, The photosynthetic nanomotor is a biological hybrid liposome colloidal motor, which is prepared by reconstituting lecithin vesicles and thylakoid vesicles through supramolecular self-assembly technology; and is prepared according to the following steps: first, thylakoid is homogenized to obtain thylakoid membrane fragments, and then the thylakoid membrane fragments are extruded 21 times through polycarbonate membranes with pore sizes of 3 μm, 1 μm, 800 nm, 400 nm and 200 nm from large pore size to small pore size to obtain thylakoid vesicles; lecithin liposome vesicles are prepared from egg yolk lecithin and cholesterol, and are extruded 21 times through polycarbonate membranes with pore sizes of 3 μm, 1 μm, 800 nm, 400 nm, 200 nm and 100 nm from large pore size to small pore size; then the thylakoid vesicles and the lecithin liposome vesicles are mixed in a volume ratio of 4:1, and are extruded 21 times through a polycarbonate membrane with a pore size of 200 nm to form a single-layer mixed vesicle structure.

2. The photosynthetic nanomotor according to claim 1, wherein, The thylakoid vesicle preparation method is prepared from spinach.

3. A method of preparing the photosynthetic nanomotor according to claim 1, characterized in that, The method comprises the following steps: Step 1, homogenize spinach and separate the homogenate containing chloroplasts; Step 2, then separate and purify the chloroplasts; Step 3, then lyse the chloroplasts and recover the thylakoids; Step 4, then homogenize the thylakoids to obtain thylakoid membrane fragments: Step 5, then extrude the thylakoid membrane fragments to obtain thylakoid vesicles; Step 6, prepare lecithin liposome vesicles using egg yolk lecithin as a synthetic liposome raw material and cholesterol as a stabilizer; Step 7, reconstitute the thylakoid membrane and the lecithin membrane by mechanical co-extrusion to obtain the photosynthetic nanomotor.

4. The preparation method according to claim 3, characterized in that, In step 1, first, the spinach is stored in a 4°C constant temperature refrigerator for 12 hours in the dark; then the spinach leaves are washed and torn by hand, the leaf meat part is separated and recovered, and the leaf meat part is mixed with 50 ml of chloroplast buffer; 20 g of quartz sand is added to the mixture and ground uniformly with a mortar, and after the precipitate is separated by centrifugation at 1000 rpm, the speed is adjusted to 4000 rpm, and the precipitate is repeatedly recovered by centrifugation; The chloroplast buffer is prepared by dissolving sucrose 68.56 g, KCl 0.3727 g, Na2HPO4 0.88995 g, KH2PO4 2.04135 g, MgCl2 0.2033 g in water and diluting to 500 mL.

5. The preparation method according to claim 4, characterized in that, In step 2, the chloroplasts are separated and purified: Step ①, resuspend the recovered precipitate in step 1 in the chloroplast buffer to form a mixture; Step ②, prepare 20% (w / w), 40% (w / w), and 60% (w / w) sucrose solutions to obtain gradient density centrifugation solutions, from top to bottom, 20% (w / w), 40% (w / w), and 60% (w / w), and place the mixture in step ① in the sucrose gradient density centrifugation solution, and centrifuge at a speed of 4000 rpm for 5 min; After the centrifuge stops, the chloroplasts are in the 40% sucrose layer of the gradient density centrifugation solution; Step ③, recover the dispersed layer of chloroplasts obtained in step ②, and resuspend in the chloroplast buffer, and centrifuge at a speed of 4000 r to recover the precipitate; Step ④, repeat the washing and centrifugation steps of step ③ to obtain purified chloroplasts.

6. The preparation method according to claim 3, characterized in that, In step 3, the purified chloroplasts are dispersed in a chloroplast lysis solution, mixed uniformly, and then centrifuged at a speed of 4000r for 10-15 min, and the precipitate is recovered to obtain thylakoids. The thylakoids are placed in a thylakoid lysis protection solution, and finally, purified thylakoids are obtained. The chloroplast lysis solution is prepared by dissolving 10 mL of 10 mM Hepes-NaOH with pH=7.5 and 0.50825 g of MgCl2 in water and diluting to 500 mL. The thylakoid lysis protection solution is prepared by dissolving 5 mL of 10 mM Hepes-NaOH with pH=7.5, 0.254125 g of MgCl2, and 1.86375 g of KCl in water and diluting to 500 mL.

7. The preparation method according to claim 3, characterized in that, In step 4, the thylakoids are dispersed in the thylakoid lysis protection solution, broken by a glass homogenizer for 5 min, and then ground with fine quartz sand for 30 min. The quartz sand powder is removed by centrifugation at a speed of 8000r, and the supernatant is recovered to obtain thylakoid membrane fragments.

8. The preparation method according to claim 3, characterized in that, In step 5, the thylakoid membrane fragments are dispersed in the thylakoid lysis protection solution, and the thylakoid vesicles are obtained by gradually extruding 21 times from a polycarbonate membrane with pore sizes of 3 μm, 1 μm, 800 nm, 400 nm, 200 nm, and 100 nm, respectively, using a cell membrane extruder.

9. The preparation method according to claim 3, characterized in that, In step 6, egg yolk lecithin is used as the raw material for synthesizing liposomes, cholesterol is used as a stabilizer, and the thylakoid lysis protection solution is used as a buffer to prepare lecithin liposome vesicles. The specific steps are as follows: ① 20 mg of lecithin and 5 mg of cholesterol are dispersed in 3 ml of chloroform and fully dissolved; ② The chloroform organic solution obtained in step ① is evaporated by a rotary evaporator. After all the organic solvents are removed, nitrogen is blown for 1 h to ensure that the organic solvents are completely removed. The round-bottom flask is vacuum dried for 12 h; ③ 10 ml of the lysis protection solution is added to the round-bottom flask obtained in step ②, and lecithin vesicles are obtained by ultrasonic treatment for 10 min; ④ The lecithin vesicles are dispersed in the thylakoid lysis protection solution, and the uniform-sized lecithin vesicles are obtained by gradually extruding 21 times from a polycarbonate membrane with pore sizes of 3 μm, 1 μm, 800 nm, 400 nm, 200 nm, and 100 nm, respectively, using a cell membrane extruder.

10. The preparation method according to claim 3, characterized in that, In step 7, the lecithin vesicles and the photosynthetic nanovesicles are mixed uniformly at a volume ratio of 4:1, and then the uniform-sized photosynthetic nanomotors are obtained by extruding 21 times using a polycarbonate membrane with a pore size of 200 nm using a cell membrane extruder.

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