Preparation method and application of nanochloroplasts with targeted mitochondrial repair function

Abstract

This invention discloses a method for preparing and applying nanochloroplasts with targeted mitochondrial repair function, belonging to the field of biomedical technology. The preparation method includes the following steps: mixing energy module nanoparticles and catalytic module nanoparticles with a cell membrane solution, and co-encapsulating them in a compartmentalized manner using membrane extrusion technology to form a nanochloroplast core; then, co-modifying the surface of the nanochloroplast core with upconversion nanoparticles through asymmetric modification. The nanochloroplasts of this invention can simulate the partitioned structure of natural chloroplasts, achieving phototaxis, energy supply, and carbon fixation functions, and can target mitochondria for restoring mitochondrial metabolism. This system can reconstruct the mitochondrial oxygen-carbon metabolism network in an ischemic model and is expected to develop into a novel nanomaterial for more effective treatment of ischemic diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying nanochloroplasts with targeted mitochondrial repair function. Background Technology

[0002] Mitochondrial dysfunction is closely related to the development of various diseases, especially cardiovascular and cerebrovascular diseases. The fundamental pathology lies in the collapse of the mitochondrial metabolic network caused by a pathological environment, leading to irreversible functional cellular damage. In the acute phase of the disease, the lack of nutrient substrates caused by vascular occlusion disrupts mitochondrial oxidative phosphorylation (OXPHOS, oxygen metabolism) and the tricarboxylic acid cycle (TCA, carbon cycle), forcing cellular metabolism to shift towards compensatory glycolysis, thereby accumulating large amounts of cytotoxic lactic acid and CO2. Although blood flow is restored during reperfusion, the subsequent surge in reactive oxygen species (ROS) and metabolic waste further exacerbates mitochondrial dysfunction, forming a vicious cycle of energy shortage and cell death.

[0003] In nature, mitochondria and chloroplasts have formed a natural symbiotic relationship during the evolution of life. Specifically, chloroplasts produce oxygen and energy (O2 / ATP / NADPH) through photophosphorylation on the thylakoid membrane (as a light reaction), and then initiate the Calvin cycle in the stroma (as a dark reaction) to fix CO2. These products (O2 / ATP / NADPH) produced in chloroplasts can precisely supply the substrates needed to repair ischemic mitochondrial metabolism, while simultaneously recycling the metabolic waste CO2 produced by mitochondria through the dark reaction of chloroplasts, thus forming a typical closed loop of oxygen and carbon metabolism in nature. This metabolic complementarity originating from the origin of life suggests that a systematic and targeted intervention system based on the engineered reconstruction of photosynthetic reactions may provide a reliable solution for reshaping mitochondrial oxygen and carbon metabolism. In recent years, the biomedical development of photosynthetic functional components based on nanotechnology has shown breakthrough potential. In-situ oxygen and energy supply to lesion areas can be achieved by biomimetic assembly of light reaction units (such as nanothylakoids), or artificial carbon fixation modules can be constructed to improve CO2 conversion efficiency. However, existing single-module systems can only simulate a single link in the light reaction (energy and oxygen supply) or the dark reaction (carbon fixation). They cannot replicate the full-function biomimicry of natural chloroplasts' "phototaxis-energy supply-carbon fixation", nor are they deeply integrated with mitochondrial oxygen and carbon metabolism.

[0004] The prerequisite for efficiently replicating the natural photosynthetic system with nanochloroplasts is the biomimetic compartmentalized structural design of natural chloroplasts. Chloroplasts construct a physical barrier through the thylakoid's double-membrane system, precisely compartmentalizing the light reaction (photophosphorylation) and the dark reaction (Calvin's carbon cycle). This prevents electron transport damage to key enzymes in the dark reaction caused by the light reaction, ensuring that the light and dark reactions occur simultaneously without interference. A simple, modular assembly strategy would not only disrupt the energy conduction integrity of the photosynthetic electron transport chain within the nanothylakoids but could also severely restrict light capture efficiency and CO2 conversion capacity due to the shielding of active sites on the surface of the nanocatalysts, ultimately leading to a decline in the overall efficiency of the light-dark dual-reaction system. Therefore, constructing a nanochloroplast system with synergistic metabolic functions by mimicking the spatial partitioning characteristics of natural chloroplasts through multi-scale biomimetic design presents a significant scientific challenge.

[0005] Chinese patent application CN118028210A discloses a targeted nanothylakoid membrane, its preparation method, and its application. This patent obtains highly efficient and stable nanothylakoids by modifying the surface of nanovesicles with PEG and folic acid with targeting function. This overcomes the technical problems of existing in vitro thylakoid membranes, such as rapid decline in in vitro activity, short lifespan, poor targeting, short metabolic time, and unsatisfactory effects. However, this patent only modifies thylakoids and does not form fully functional photosynthetic nanochloroplasts, so further research and improvement are needed. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide a nanochloroplast that structurally mimics the compartmentalization characteristics of natural cells for repairing mitochondrial metabolic function.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] This invention proposes a nanochloroplast with targeted mitochondrial repair function, comprising: (1) A light-driven nanomotor module comprising a cell membrane and upconversion nanoparticles; wherein the upconversion nanoparticles are asymmetrically modified on the surface of the cell membrane; (2) Energy module nanoparticles, encapsulated inside the light-driven nanomotor module, are used to perform the light reaction of photosynthesis; (3) Catalytic module nanoparticles, which are encapsulated together with the energy module nanoparticles inside the light-driven nanomotor module and are spatially isolated from the energy module nanoparticles, are used to perform the dark reaction of photosynthesis; The energy module nanoparticles and the catalytic module nanoparticles are encapsulated in a compartmentalized manner using membrane self-assembly technology.

[0009] The nanochloroplasts of the present invention are structurally replicated functional photosynthetic units of natural chloroplasts, namely phototactic modules, light reaction modules, and dark reaction modules, which are respectively named light-driven nanomotor modules, energy module nanoparticles, and catalytic module nanoparticles in the nanochloroplast system.

[0010] Preferably, the upconversion nanoparticles in the light-driven nanomotor module serve as a nanoscale light source to convert near-infrared light (740 nm - 1700 nm) into visible light (380 nm - 780 nm). They are produced using a classic hydrothermal synthesis technique with NaYF4 as the matrix and nanoparticles doped with different rare earth ions, with an average particle size of approximately 5-30 nm.

[0011] More preferably, the different rare earth ions include, but are not limited to, any two or more of yttrium ions, ytterbium ions, erbium ions, and gadolinium ions.

[0012] Preferably, the cell membrane (with targeting inflammation) in the light-driven nanomotor module is selected from any one of platelet membrane, macrophage membrane, erythrocyte membrane, neutrophil membrane or stem cell membrane, and is prepared by repeated freeze-thaw method, with a particle size range of 80-180 nm.

[0013] Preferably, the energy module nanoparticles are selected from any one of spinach, lettuce, cabbage, kale, pea sprouts or Arabidopsis thaliana, and are prepared by differential centrifugation and membrane extrusion, with a particle size range of 50-150 nm.

[0014] Preferably, the catalytic module nanoparticles are CO2 nanocatalysts prepared with different metal substrates, and the particle size range is 5-30 nm.

[0015] Preferably, the asymmetric modification is selected from any one of click chemistry reaction, template-assisted modification, and microfluidic preparation method.

[0016] This invention also proposes a method for preparing the above-mentioned nanochloroplasts, comprising the following steps: Energy module nanoparticles and catalytic module nanoparticles are mixed with cell membrane solution and co-encapsulated in a compartmentalized manner using membrane extrusion technology to form a nanochloroplast core; upconversion nanoparticles are then co-modified on the surface of the nanochloroplast core through asymmetric modification.

[0017] (Light-driven nanomotor module: as the outer structure of nanochloroplasts, specifically, it is composed of upconversion nanoparticles asymmetrically modified into cell membranes with targeted inflammation) The specific steps include: Energy module nanoparticles and catalytic module nanoparticles with a certain mass ratio and moderate surface charge repulsion are dispersed in a suitable solvent. After a period of time, a certain amount of light-driven nanomotor is added and mixed for a period of time. Then, the mixture is processed by membrane extrusion using polycarbonate templates of different sizes. Finally, the supernatant is removed by centrifugation to obtain nanochloroplasts.

[0018] Preferably, the mass ratio of the energy module nanoparticles to the catalytic module nanoparticles is in the range of 1:10 to 10:1.

[0019] Preferably, the surface charge ratio of the energy module nanoparticles and the catalytic module nanoparticles with moderate surface charge repulsion is in the range of 2:1 to 7:1.

[0020] Preferably, the suitable solvent is any one of phosphate (PBS), tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), or 3-morpholinopropanesulfonic acid (MOPS) buffer.

[0021] Preferably, the time range for the two modules to be mixed is 5-30 minutes.

[0022] Preferably, the mass range of the aforementioned amount of light-driven nanomotor is 2-10 mg.

[0023] Preferably, the blending time is 10-50 minutes.

[0024] Preferably, the size range of the polycarbonate templates of different sizes is 50-200 nm.

[0025] The present invention also proposes the application of the above-mentioned nanochloroplasts in the preparation of drugs for targeted repair of mitochondrial metabolic dysfunction.

[0026] Preferably, the nanochloroplasts are used to penetrate physiological barriers (including thrombi and the blood-brain barrier) under near-infrared light driving.

[0027] This invention also proposes the application of the above-mentioned nanochloroplasts in targeted mitochondrial repair, including the following steps: cellular-level targeted mitochondrial effects (observing the co-localization of Cy5-labeled UT-PNS with mitochondria using confocal microscopy, and characterizing functional repair through experiments on mitochondrial morphology (observing mitochondrial morphological changes using transmission electron microscopy), glycolysis (determining glycolysis, glycolytic capacity, and non-glycolytic acidification using extracellular acidification rate (ECAR), and detecting lactate (LA) and pyruvate (PA) using a kit), TCA cycle (detecting oxaloacetate (OAA), succinate (SUCN), and citrate (CA) using a kit), and oxidative phosphorylation (detecting basal respiration, ATP production, and maximal respiration capacity using oxygen consumption rate (OCR), and detecting the activity of mitochondrial complex III using a kit).

[0028] The present invention has the following beneficial effects: 1. This invention utilizes a photochemotactic module with autonomous movement as a carrier. Through membrane self-assembly technology, an energy module (nanothylakoids) and a catalytic module (nanocatalyst) are encapsulated within the cell membrane in a compartmentalized manner. Furthermore, upconversion and mitochondrial targeting molecules are modified onto the surface of these modules to construct biomimetic nanochloroplasts. These nanochloroplasts possess dual targeting capabilities (tissue and organelle) and, under near-infrared light irradiation, repair damaged mitochondria by simulating photosynthesis.

[0029] 2. The nano-chloroplasts of this invention can mimic the partitioned structure of natural chloroplasts, achieving phototaxis, energy supply, and carbon fixation functions. They can also target mitochondria to restore mitochondrial metabolism. This system can reconstruct the mitochondrial oxygen-carbon metabolism network in ischemic models and holds promise for developing into a novel nanomaterial for more effective treatment of ischemic diseases. Attached Figure Description

[0030] Figure 1 Representative transmission electron microscope (TEM) images of the light-driven upconversion nanomotor, nanothylakoids, and nanocatalysts prepared in Example 1 are shown. Figure 2 Representative transmission electron microscopy (TEM) images of the three structures of the intermediate product (PNS) prepared in Example 1 are shown. Figure 3 The intermediate products prepared in Example 1, as well as the average particle size and Zeta potential characterization results of the nanochloroplasts, are shown. Figure 4 Representative transmission electron microscopy (TEM) images of the nanochloroplasts prepared in Example 1 are shown; Figure 5The effects of the three structures of the intermediate product PNS prepared in Example 1 on the following indicators are shown: a) oxygen generation rate, b) supernatant pH, and c) relative ATP content. Figure 6 The symmetrically modified product (UTPNS) prepared for Comparative Example 2 and the movement trajectory and mean square displacement of nanochloroplasts under near-infrared irradiation; Figure 7 The diagram shows the mitochondrial targeting effect of the nanochloroplasts prepared in Example 1; Figure 8 Representative transmission electron microscopy (TEM) images of mitochondrial morphology in cells treated with nanochloroplasts prepared in Example 1 are shown. Figure 9 The restoration of mitochondrial function in cells after treatment with the nanochloroplasts prepared in Example 1 is shown. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0032] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0033] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0034] Example 1: S1. Fabrication of a light-driven nanomotor module (1) Preparation of upconversion: Preparation of upconversion cores (NaYF4:Yb, Er): YCl3·6H2O, YbCl3·6H2O, and ErCl3·6H2O were added to a mixed solvent of oleic acid and octadecene, dissolved, and then heated to react (60℃, 1h) to obtain a clear solution. After cooling, a methanol solution containing sodium hydroxide and ammonium fluoride was added and reacted for 1h. Subsequently, the temperature was raised under inert gas protection and reacted for a period of time (80℃, 1h). Finally, the core nanoparticles were obtained by centrifugation and washing.

[0035] Preparation of core-shell structures (NaYF4: Yb, Er@NaGdF4): GdCl3·6H2O was heated in an oleic acid and octadecene solvent (80℃, 1h). After cooling, a cyclohexane solution containing the core nanoparticles was added. After removing the solvent, a methanol solution of sodium hydroxide and ammonium fluoride was added again to initiate the reaction. The temperature was then raised and the reaction continued for a period of time (80℃, 1h). After the reaction was completed, centrifugation was performed to obtain core-shell nanoparticles, which were then dispersed in an organic solvent.

[0036] Surface functionalization (UCNPs-Mal): Core-shell nanoparticles were surface-modified using an ultrasonic thin-film hydration method. Nanoparticles were mixed with the amphiphilic polymer DSPE-PEG-Mal (1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)]) in chloroform, and a uniform thin film was formed by rotary evaporation. The film was then ultrasonically hydrated with ultrapure water and finally purified by centrifugation to obtain surface-functionalized upconversion nanoparticles.

[0037] (2) Preparation of platelet membrane: Platelets were suspended in H2O and subjected to repeated freeze-thaw cycles at -80°C and 25°C. The solution was then centrifuged and washed with PBS buffer to obtain platelet membranes.

[0038] S2. Preparation of nano-thylakoids: Fresh spinach leaves were pre-cooled at 4°C, then stirred and filtered in a buffer solution (pH 7.8) containing 20 mM Tricine, 0.4 mM sucrose, 2 mM ascorbic acid, 2 mM MgCl2, 40 mM NaCl, and 0.2% bovine serum albumin. The filtrate was centrifuged to collect intact chloroplast precipitates, which were then resuspended in hypotonic lysis buffer (10 mM HEPES, pH 8.0) for lysis. After centrifugation, crude thylakoid extract was obtained and washed repeatedly. The obtained thylakoids were resuspended in PBS buffer containing 0.4 mM sucrose, 20 mM HEPES, 5 mM MgCl2, and 15 mM NaCl and washed by centrifugation. Finally, the extract was sonicated and extruded through a polycarbonate membrane multiple times to obtain nano-thylakoids.

[0039] S3. Preparation of catalyst nanoparticles: Catalyst nanoparticles were prepared using a hydrothermal method. Under continuous magnetic stirring, 0.6 mmol sodium dodecylbenzenesulfonate (SDBS), 0.25 mmol SnCl4·5H2O, and 2 mmol L-cysteine ​​were dissolved sequentially in a mixed solvent of 15 mL water and 15 mL ethylene glycol. After reacting for 10 minutes, the mixture was transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 160°C for 10 hours, followed by natural cooling to room temperature. The resulting product was washed repeatedly with deionized water and ethanol, and then freeze-dried overnight in a vacuum freeze-drying apparatus for subsequent use.

[0040] S4. Preparation of nanochloroplasts: Nanochloroplasts were prepared using a two-step method. First, the nanochloroplast core was prepared via membrane coating: 4 mL of platelet membrane solution (20 mg / mL) was coated with the membrane. -1 ), 4 mL of nano-thylakoid solution (20 mg / mL) -1 The mixture of 8 mg of nanocatalyst and 8 mg of nanocatalyst was uniformly mixed, and then extruded repeatedly 50 times through a 120 nm polycarbonate porous membrane to prepare compartmentalized PNS.

[0041] Subsequently, upconversion nanoparticles and TPP-NH2 were co-modified onto the PNS surface. Specifically: 400 μL of PNS solution was carefully added to a 12-well plate and allowed to stand overnight. After washing with PBS to remove excess PNS, 400 μL of TPP-NH2 (1 mg·mL⁻¹) was added. - ¹) with maleimide-modified upconversion nanoparticles (1 mg·mL⁻¹) - ¹) Incubate in the solution for 1 hour. Finally, remove the supernatant and collect the precipitate with PBS to obtain nanochloroplasts.

[0042] like Figure 1 As shown, the light-driven nanomotor, energy module nanoparticles, and catalytic module nanoparticles in the nanochloroplasts are all uniform spherical structures.

[0043] like Figure 2 As shown, TEM confirmed the three module configurations of PNS (hybrid, embedded, and compartmentalized). The hybrid form includes membrane fusion of NTU and PM, followed by SnS2 loading; the embedded form includes NTU loaded with SnS2, followed by encapsulation by PM; and the compartmentalized form in which NTU and SnS2 do not mix with each other but are both within PM.

[0044] like Figure 3As shown, the intermediate products of nanochloroplasts, PM, has an average particle size of about 120 nm, NTU about 80 nm, and SnS2 about 8 nm. PM and nanochloroplasts have similar particle sizes, with an average particle size of about 120 nm. PM, NTU, and SnS2 are all negatively charged. After modification with positively charged TPP-NH2, the potential of UT-PNS increases, but UT-PNS remains negatively charged.

[0045] like Figure 4 As shown, the compartmentalized structure of UT-PNS can be clearly seen by TEM, which further demonstrates the successful preparation of compartmentalized nanochloroplasts (UT-PNS).

[0046] like Figure 5 As shown, the photosynthetic performance of the three PNS module configurations (mixed, embedded, and compartmentalized) was evaluated from the perspective of maximizing the light / dark reactions. O2 production and ATP synthesis were measured as indicators of the light reaction, while solution pH was measured as an indicator of the dark reaction. Compared to the other two configurations, the compartmentalized PNS configuration significantly improved O2 production and ATP synthesis while decreasing the solution pH.

[0047] like Figure 6 As shown, the trajectories and mean square displacements of the symmetric nanomaterials UTPNS and nanochloroplasts prepared in Comparative Example 3 under near-infrared irradiation were observed. The trajectories of UT-PNS and UTPNS with fluorescent labeling (Cy5) were observed using fluorescence microscopy, revealing that UT-PNS exhibited a significant movement distance. Furthermore, the parabolic relationship between the mean square displacement (MSD) and the time interval (Δt) indicates that UT-PNS possesses autonomous movement capabilities, while the symmetric nanomaterial UTPNS does not.

[0048] Example 2: It is basically the same as Example 1, except that the mass ratio of nano-thylakoids to platelet membranes and nano-catalysts is 1:1:1.

[0049] Example 3: The process is basically the same as in Example 1, except that: after the nano-thylakoids, platelet membrane and nano-catalyst are mixed evenly, they are repeatedly extruded through a 120 nm polycarbonate porous membrane 25 times using an extruder.

[0050] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that: 4 mL of platelet membrane solution (20 mg / mL) was used. -1 ) and 4 mL of nano-thylakoid solution (20 mg / mL) -1After extruding through a 120 nm polycarbonate porous membrane 50 times, 8 mg of SnS2 nanocatalyst was added and ultrasonically mixed until homogeneous. This mixture was then extruded again through the 120 nm polycarbonate porous membrane 50 times to prepare a hybrid PNS, as shown below. Figure 2 As shown. The poor performance of the hybrid spatial configuration is mainly due to its inability to effectively isolate the light-reaction and dark-reaction modules. In the hybrid structure, the nanothylakoids (NTUs) are in direct contact with the SnS2 nanocatalyst. The active electrons generated by the NTUs under photoexcitation are uncontrollably transferred to SnS2, severely disrupting the integrity of the NTU's own photosynthetic electron transport chain, leading to a sharp drop in the O2 / ATP / NADPH generation efficiency. Simultaneously, the surface of SnS2 is largely covered by NTUs, obscuring its catalytic active sites and limiting its CO2 fixation capacity, such as... Figure 5 As shown.

[0051] By altering the assembly sequence of thylakoid nanoparticles (NTU), SnS2 nanocatalysts, and platelet membranes (PM), three spatial configurations—mixed, embedded, and compartmentalized—were prepared. Characterization and performance evaluation confirmed that only the compartmentalized structure optimally mimics the compartmentalized design of natural chloroplasts, precisely separating the light-dependent reactions (NTU) and dark-dependent reactions (SnS2) at different spatial sites within the membrane structure. This physical isolation protects the integrity of the photosynthetic electron transport chain within the thylakoid nanoparticles while preventing them from obscuring the light absorption efficiency of the SnS2 catalyst, thus ensuring that the light and dark reactions can proceed simultaneously and efficiently. The mixed and embedded spatial configurations exhibited poor performance, primarily due to their inability to effectively isolate the light-dependent and dark-dependent reaction modules. In the mixed structure, the thylakoid nanoparticles (NTU) are in direct contact with the SnS2 nanocatalyst, and the active electrons generated by the NTU under photoexcitation are uncontrollably transferred to SnS2, severely disrupting the integrity of the NTU's own photosynthetic electron transport chain and leading to a sharp drop in the generation efficiency of O2 / ATP / NADPH. Meanwhile, the surface of SnS2 is largely covered by the NTU, obscuring its catalytic active sites and thus limiting its CO2 fixation capacity. Although the embedded structure encapsulates SnS2 within the NTU, the close proximity of the two still cannot avoid the aforementioned electronic interference problems, further limiting the mass transfer efficiency between the substrate CO2 and the product CO.

[0052] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that: 4 mL of nano-thylakoid solution (20 mg / mL) was used. -1 The mixture was ultrasonically mixed with 8 mg of SnS2 nanocatalyst until homogeneous. The mixture was then extruded repeatedly through a 120 nm polycarbonate porous membrane 50 times. Finally, 4 mL of platelet membrane solution (20 mg / mL) was added. -1The embedded PNS was prepared by repeatedly extruding the material through a 120 nm polycarbonate porous membrane 50 times using an extruder. Figure 2 As shown. Although the embedded structure encapsulates SnS2 within the NTU, the close proximity of the two components still leads to unavoidable electronic interference, further limiting the mass transfer efficiency between the substrate CO2 and the product CO, such as... Figure 5 As shown.

[0053] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that the upconversion nanoparticles were uniformly and symmetrically modified on PNS, without asymmetric modification. (When upconversion nanoparticles (UCNPs) are uniformly and symmetrically modified on the PNS surface, the oxygen (O2) generated under near-infrared light excitation will be uniformly released in all directions of the nanoparticles. This symmetrical gas release cannot form a directional concentration gradient or net thrust around the particles; the forces in each direction cancel each other out, therefore chemical energy cannot be converted into kinetic energy for directional motion, resulting in a lack of autonomous motion capability, such as...) Figure 6 As shown.

[0054] Nanochloroplasts achieve precise mitochondrial targeting primarily through the lesion-specific recognition and efficient transmembrane capacity conferred by the platelet membrane, enabling their accumulation and internalization in ischemic areas. Subsequently, their photo-driven nanomotor properties provide autonomous movement within the cytoplasm, overcoming intracellular diffusion resistance. Finally, through the surface-modified mitochondrial targeting molecule TPP-NH2, an active targeting mechanism driven by mitochondrial membrane potential ensures the efficient targeting of nanochloroplasts to mitochondria. Figure 7 As shown, the red fluorescence of UT-PNS and the green fluorescence of mitochondria colocalize. Quantitative analysis yielded a Pearson correlation coefficient (Pearson R value) of 0.86 and a mean relative fluorescence intensity (MFI) of 2.73 ± 0.08. These values ​​indicate a significant degree of colocalization, confirming the mitochondrial targeting of UT-PNS.

[0055] We conducted a comprehensive analysis of the repair effects of UT-PNS on damaged mitochondrial function using morphological and metabolic function assays. For example... Figure 8 As shown, transmission electron microscopy revealed abnormal swelling and cristae breakage in mitochondria in the model (indicated by white arrows). However, UT-PNS treatment effectively reversed the abnormal swelling and cristae disruption of mitochondria in the model group cells, restoring their normal structure. At the metabolic level, such as... Figure 9As shown, UT-PNS exhibits a comprehensive ability to remodel the mitochondrial energy network. Regarding glycolytic flux, UT-PNS treatment significantly reduced the OGD / R-induced increase in extracellular acidification rate (ECAR), decreased lactate (LA) accumulation, and restored pyruvate (PA) levels, indicating a return of cellular energy metabolism from over-reliance on anaerobic glycolysis to efficient oxidative metabolism. Analysis of key intermediates in the tricarboxylic acid cycle (TCA cycle) revealed significantly higher levels of oxaloacetate (OAA), succinate (SUCN), and citrate (CA) in the UT-PNS-treated group compared to the model group, confirming effective restoration of the TCA cycle. Further functional assessment of oxidative phosphorylation (OXPHOS) showed that UT-PNS treatment not only enhanced basal respiration, ATP production, and maximal respiration capacity (measured by OCR), but also increased the activity of mitochondrial complex III, indicating a synergistic improvement in electron transport chain function and ATP synthesis efficiency.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanochloroplast with targeted mitochondrial repair function, characterized in that, include: (1) A light-driven nanomotor module comprising a cell membrane and upconversion nanoparticles; wherein the upconversion nanoparticles are asymmetrically modified on the surface of the cell membrane; (2) Energy module nanoparticles, encapsulated inside the light-driven nanomotor module, are used to perform the light reaction of photosynthesis; (3) Catalytic module nanoparticles, which are encapsulated together with the energy module nanoparticles inside the light-driven nanomotor module and are spatially isolated from the energy module nanoparticles, are used to perform the dark reaction of photosynthesis; The energy module nanoparticles and the catalytic module nanoparticles are encapsulated in a compartmentalized manner using membrane self-assembly technology.

2. The nanochloroplast according to claim 1, characterized in that, The upconversion nanoparticles in the light-driven nanomotor module serve as a nanoscale light source to convert near-infrared light into visible light. They are produced using a classic hydrothermal synthesis technique with NaYF4 as the matrix and nanoparticles doped with different rare earth ions, with an average particle size of approximately 5-30 nm.

3. The nanochloroplast according to claim 1, characterized in that, The cell membrane in the light-driven nanomotor module is selected from any one of platelet membrane, macrophage membrane, erythrocyte membrane, neutrophil membrane or stem cell membrane, and is prepared by repeated freeze-thaw method, with a particle size range of 80-180 nm.

4. The nanochloroplast according to claim 1, characterized in that, The energy module nanoparticles are selected from any one of spinach, lettuce, cabbage, kale, pea sprouts or Arabidopsis thaliana, and are prepared by differential centrifugation and membrane extrusion, with a particle size range of 50-150 nm.

5. The nanochloroplast according to claim 1, characterized in that, The catalytic module nanoparticles are CO2 nanocatalysts prepared with different metal substrates, and the particle size range is 5-30 nm.

6. The nanochloroplast according to claim 1, characterized in that, The asymmetric modification method is selected from any one of click chemistry reaction, template-assisted modification, and microfluidic preparation method.

7. The method for preparing nanochloroplasts according to any one of claims 1-6, characterized in that, Includes the following steps: Energy module nanoparticles and catalytic module nanoparticles are mixed with cell membrane solution and co-encapsulated in a compartmentalized manner using membrane extrusion technology to form a nanochloroplast core; upconversion nanoparticles are then co-modified on the surface of the nanochloroplast core through asymmetric modification.

8. The use of the nanochloroplasts according to any one of claims 1-6 in the preparation of a medicament for targeted repair of mitochondrial metabolic dysfunction.

9. Use of the nanochloroplasts according to any one of claims 1-6 in the preparation of reagents for penetrating physiological barriers.

10. The use according to claim 9, characterized in that, The physiological barriers include thrombosis and the blood-brain barrier.

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