Mitochondria with in-situ modification of mitochondrial surface by polymers, and preparation method and application thereof

Abstract

The application provides mitochondria with in-situ modification of a mitochondrial surface polymer, a preparation method and application thereof, and belongs to the field of biomedical engineering. The mitochondria with in-situ modification of a mitochondrial surface polymer are prepared by in-situ "click" polymerization in cells to precisely and mildly modify the mitochondrial surface. The "modification first and extraction later" mode proposed by the application solves the three inherent defects of the traditional "extraction first and modification later" mode. The polymer modification layer can form a protective barrier, reduce mechanical damage, and maintain the structural and functional integrity. The charge repulsion with the target cell membrane is reduced, and the internalization efficiency of mitochondria to the target cell is significantly improved. The mitochondria with in-situ modification of a polymer provided by the application have wide application prospects in the fields of biomedical technologies such as neurodegenerative diseases and sub-health intervention.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a mitochondrial surface polymer-modified mitochondria, its preparation method and application. Background Technology

[0002] Mitochondria, as the energy suppliers of cells, directly affect normal cellular physiological activities due to their structural stability and functional integrity. Mitochondrial transplantation, as a novel cell therapy, has shown broad application prospects in the field of medical technology.

[0003] To enhance the stability of mitochondrial structures and regulate their surface properties to suit transplantation requirements, mitochondrial surface modification is typically required before transplantation. Current modification methods employ an "extraction-then-modification" approach, where mitochondria are first isolated from host cells and then their surface is modified extracellularly. Modification methods include polymer modification, peptide modification, phospholipid modification, and small molecule modification. Polymer modification is the most important, typically involving the non-covalent adsorption of pre-prepared polymers onto the mitochondrial surface, and is currently the core modification method for achieving mitochondrial structural stability and functional optimization. However, existing polymer modification strategies face two major challenges, making it difficult to meet the actual needs of clinical transplantation: First, during the extracellular modification of polymers, mitochondria are separated from their original intracellular microenvironment and are affected by external mechanical shearing, environmental changes, and other factors, which will lead to structural damage and functional decline. At the same time, the extracellular modification steps are complicated, which will reduce the extraction efficiency and activity retention rate of mitochondria.

[0004] Second, the fixed chain length and spatial conformation of the pre-prepared polymer lead to poor compatibility between the polymer and the mitochondrial surface. At the same time, this modification method cannot precisely control the modification density and thickness of the polymer on the mitochondrial surface, resulting in low mitochondrial delivery efficiency.

[0005] In summary, the "extract first, modify later" model is the inherent root cause of the two challenges mentioned above. Therefore, only by changing the current "extract first, modify later" model can this core challenge be overcome. Summary of the Invention

[0006] To address the aforementioned issues, this invention proposes a polymer-modified mitochondria on its surface, its preparation method, and its applications. The method employs a "modification-then-extraction" approach, where mitochondria are first modified in situ within the host cell, followed by the separation and extraction of the modified mitochondria. This intracellular in-situ modification method ensures that mitochondria remain within their own physiological microenvironment, effectively avoiding structural damage and functional decline caused by separation from the microenvironment during extracellular modification. Furthermore, this method allows for precise control of the polymer modification density and spatial conformation, achieving a perfect fit between the polymer and the mitochondrial surface. This solves the stability problem during extraction and effectively shields the negative charge on the mitochondrial surface, enhancing interaction with target cells and overcoming the challenge of low delivery efficiency.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a mitochondria with in situ polymer-modified mitochondria, wherein the mitochondria with in situ polymer-modified mitochondria are formed by in situ "click" polymerization of mitochondria in the host cell, so that the polymer is polymerized and modified in situ on the mitochondrial surface.

[0008] This invention also provides a method for preparing mitochondria with in-situ modified mitochondrial surface polymers as described above, comprising the following steps: 1) Preparation of "click" polymerization initiators and monomers; 2) After incubating the "click" polymerization initiator with the host cells for 1-5 hours, wash 1-5 times; 3) Add monomers, copper sulfate and sodium ascorbate to the cells and continue incubation for 1-4 hours. Wash 1-5 times to complete the in-situ polymerization modification of the mitochondrial surface in the cells. 4) Mitochondria modified with polymer on the mitochondrial surface were extracted by centrifugation.

[0009] Preferably, the molar ratio of the monomer to the "click" polymerization initiator is 50:1 to 1000:1; the molar ratio of the "click" polymerization initiator to copper sulfate is 1:1 to 1:5; and the concentration of sodium ascorbate is 1 to 10 mM.

[0010] Preferably, the chemical structure of the "click" polymerization initiator includes: a tri(triazole methyl)amine group, an R group, and an initiation site.

[0011] Preferably, the R group comprises a triphenylphosphine salt group or a mitochondrial-targeting peptide; the initiation site comprises an azide group or an alkynyl group.

[0012] Preferably, the monomer has a structure comprising an azide group and an alkynyl group, and the two groups are connected by two types of functional groups; the functional groups include hydrophilic spacer groups and functional modification units.

[0013] Preferably, the hydrophilic spacer group comprises an ethylene glycol repeating unit; the functional modification unit comprises one or more of a charge-regulating unit, a targeting unit, or a fluorescent group; the charge-regulating unit comprises a guanidine group, a carboxyl group, or an amino group; the targeting unit comprises biotin, RGD peptide, or folic acid; and the fluorescent group is selected from FITC, rhodamine, or CY5.

[0014] Preferably, the centrifugation includes the following steps: lysing the cells treated with in situ polymerization, centrifuging at 800×g and 4℃ for 10 min to remove the cell nucleus and cell debris, collecting the supernatant and centrifuging at 12000×g and 4℃ for 20 min to obtain mitochondrial precipitate, washing with mitochondrial preservation solution 1 to 3 times, and resuspending to obtain purified polymer-modified mitochondria.

[0015] The present invention also provides the application of mitochondria modified in situ with mitochondrial surface polymers as described in the above-described scheme, and mitochondria modified in situ with mitochondrial surface polymers prepared by the preparation method described in the above-described scheme, in mitochondrial transplantation.

[0016] Preferably, the mitochondria modified in situ with the mitochondrial surface polymer can repair energy metabolism disorders in dopaminergic neurons.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: 1. Breaking through the bottleneck of the traditional "extraction first, modification later": This invention uses in-situ intracellular click polymerization to directly modify the polymer surface of mitochondria in the host cell, avoiding structural damage and functional decline caused by mitochondria leaving the cell microenvironment, and significantly improving the extraction efficiency and activity retention rate of mitochondria; 2. Enhance mitochondrial structural stability: The polymer modification layer can form a protective barrier, reducing mechanical damage to mitochondria during extraction, preservation and transplantation, while regulating the surface charge of mitochondria to prevent mitochondrial aggregation and maintain their structural and functional integrity. 3. Improve mitochondrial delivery efficiency: The polymer-modified layer can shield the negative charge on the mitochondrial surface, reduce the charge repulsion with the target cell membrane, and enhance the specific recognition of the target cell through the targeting motif in the monomer, thus significantly improving the internalization efficiency of mitochondria into the target cell; 4. Good biocompatibility and strong controllability: The structural design of the initiator and monomer meets the requirements of the intracellular physiological environment, the polymerization reaction conditions are mild (37℃, physiological concentration), and there is no obvious cytotoxicity; by adjusting parameters such as the ratio of monomer to initiator and polymerization time, the molecular weight and modification density of the polymer can be precisely controlled. 5. Broad application prospects: Modified mitochondria can be widely used in the treatment of various mitochondrial dysfunction-related diseases, especially in neurodegenerative diseases such as Parkinson's disease. They can effectively repair the energy metabolism of diseased cells and provide a new technical path for disease treatment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0019] Figure 1 The above is the 1H NMR spectrum of the "click" polymerization initiator in Example 1; Figure 2 This is the mass spectrum of the "click" polymerization initiator in Example 1; Figure 3 The hydrogen nuclear magnetic resonance spectrum of the glycol monomer in Example 1; Figure 4 This is the mass spectrum of the glycol monomer in Example 1; Figure 5 The above is the 1H NMR spectrum of the guanidine-containing monomer in Example 1; Figure 6 GPC curves of mitochondrial surface polymers; Figure 7 Super-resolution fluorescence microscopy image of polymers on the mitochondrial surface; Figure 8 These are transmission electron microscopy (TEM) images of primitive mitochondria and mitochondria modified with surface polymers. Image A is a TEM image of primitive mitochondria, and image B is a TEM image of mitochondria modified with surface polymers. Figure 9 A comparison chart of uptake rates in different mitochondrial cells; Figure 10 A comparison of ATP production in cells after different mitochondrial transplantations; Figure 11 This is a comparison of cellular oxygen consumption rates after different mitochondrial transplantations. Detailed Implementation

[0020] The present invention provides a mitochondria with in situ polymer-modified mitochondria, wherein the mitochondria with in situ polymer-modified mitochondria are formed by in situ "click" polymerization of mitochondria in the host cell, so that the polymer is polymerized and modified in situ on the mitochondrial surface.

[0021] This invention also provides a method for preparing mitochondria with in-situ modified mitochondrial surface polymers as described above, comprising the following steps: 1) Preparation of "click" polymerization initiators and monomers; 2) After incubating the "click" polymerization initiator with the host cells for 1-5 hours, wash 1-5 times; 3) Add monomers, copper sulfate and sodium ascorbate to the cells and continue incubation for 1-4 hours. Wash 1-5 times to complete the in-situ polymerization modification of the mitochondrial surface in the cells. 4) Mitochondria modified with polymer on the mitochondrial surface were extracted by centrifugation.

[0022] In this invention, the chemical structure of the initiator preferably includes: a tri(triazole methyl)amine group, an R group, and an initiation site; wherein, the triamine structure of the tri(triazole methyl)amine group is a cuprous ion complexation site, which can efficiently complex cuprous ions to form a catalytic core and initiate a click polymerization reaction; the R group consists of two mitochondrial targeting sites, including a triphenylphosphine salt group or a mitochondrial targeting peptide, preferably triphenylphosphine (TPP) or CGKRK polypeptide, which can specifically target mitochondria through mitochondrial membrane potential, ensuring that the initiator is anchored to the mitochondrial surface; the initiation site is an azide group (-N3) or an alkyne group (-C≡CH), which can undergo a click reaction with the corresponding functional group of the monomer to initiate the growth of the polymer chain. The spacer group between each functional site in the initiator is an ethylene glycol repeating unit, and its repetition number n = 1~5.

[0023] In this invention, the monomer comprises an azide group and an alkynyl group, connected by two types of functional groups. The functional groups include hydrophilic spacer groups or functional modification motifs; wherein the hydrophilic spacer group is an ethylene glycol repeating unit with a repetition frequency n=1~6, used to regulate the water solubility of the monomer and the flexibility of the polymer, avoiding polymer aggregation that could damage the mitochondrial structure; the functional modification motifs are preferably one or more of charge-regulating motifs, targeting motifs, or fluorescent motifs; wherein the charge-regulating motifs preferably include guanidine, carboxyl, or amino groups, used to regulate the mitochondrial surface charge and reduce charge repulsion during delivery; the targeting motifs include biotin, RGD peptide, and folic acid, used to enhance the specific recognition of target cells by modified mitochondria; the fluorescent motifs include FITC, rhodamine, and CY5, used to track the modification process, extraction efficiency, and delivery pathway of mitochondria.

[0024] In this invention, copolymerization reactions can be carried out using various combinations of monomers containing two types of functional groups during the polymerization process, including but not limited to: 1. A combination of monomers with two different functional modification motifs, such as a combination of a monomer containing a charge regulation motif and a monomer containing a targeting motif, to simultaneously achieve mitochondrial surface charge regulation and targeted recognition; 2. Combination of functional groups containing hydrophilic spacer groups and functional groups containing functional modification units, taking into account both the water solubility and functionalization requirements of the polymer; 3. A combination of functional groups from two different fluorescent groups for multi-channel tracking of mitochondrial modification and delivery processes.

[0025] The chemical structure of the initiator for the "click" polymerization is shown below: .

[0026] In the formula, the spacer group between each functional site in the initiator is an ethylene glycol repeating unit, and n is the number of repetitions of the ethylene glycol repeating unit, which is n=1~5.

[0027] The chemical structure of the monomer with added hydrophilic spacer groups is shown below: .

[0028] In the formula, the hydrophilic spacer group is an ethylene glycol repeating unit, and n is the number of repetitions of the ethylene glycol repeating unit, which is n=1~6.

[0029] The chemical structure of the monomer with added functional modifying units is shown below: .

[0030] In this invention, the host cells preferably include NIH-3T3, RAW264.7, HEK293T, mMSCs, HepG2, and more preferably NIH-3T3 fibroblast cell line cells.

[0031] In this invention, copper sulfate is a catalyst precursor; sodium ascorbate is a reducing agent, which reduces Cu... 2+ Reduced to Cu + To initiate a click-aggregate reaction.

[0032] In this invention, the molar ratio of the monomer to the initiator is preferably 50:1 to 1000:1, which can be adjusted according to the molecular weight and modification density of the target polymer; the molar ratio of the initiator to copper sulfate in the "click" polymerization is preferably 1:1 to 1:5, which ensures sufficient Cu. + Initiate click polymerization while avoiding Cu + Excessive use leads to cytotoxicity; the sodium ascorbate concentration is 1-10 mM, which can efficiently remove Cu. 2+ Reduced to Cu + It does not cause damage to cells and mitochondria.

[0033] In this invention, the incubation time between the initiator and the host cell is preferably 1-5 hours, more preferably 3 hours; the number of washes is preferably 1-5 times, more preferably 3 times; the incubation conditions between the initiator and the host cell are: temperature 35℃-40℃, preferably 37℃; CO2 concentration 3%-10%, preferably 5%; under these conditions, the initiator can specifically target and anchor to the mitochondrial surface within the cell; the washing solution is PBS buffer, which can remove unbound free initiator. The incubation process after adding monomer, copper sulfate, and sodium ascorbate must maintain the incubation conditions between the initiator and the host cell. Under these conditions, the monomer can polymerize under the action of the initiator to form a polymer, achieving modification of the mitochondrial surface; after the reaction, the washing solution is PBS buffer, which removes unreacted monomer, copper sulfate, and sodium ascorbate. In this invention, the total polymerization time is 12-48 hours.

[0034] In this invention, the centrifugation includes the following steps: lysing the host cells treated with in situ polymerization, centrifuging at 800×g and 4℃ for 10 min to remove the cell nucleus and cell debris, collecting the supernatant and centrifuging at 12000×g and 4℃ for 20 min to obtain mitochondrial precipitate, washing with mitochondrial preservation solution 1 to 3 times, and resuspending the washed mitochondrial precipitate to obtain purified mitochondrial surface polymer-modified mitochondria.

[0035] In this invention, the lysis is performed using pre-cooled cell lysis buffer; the lysis conditions are: sonication in an ice bath at 4°C for 3-5 minutes, which ensures complete cell lysis without damaging the mitochondrial structure. The mitochondrial preservation solution must be pre-cooled; the purpose of washing with the mitochondrial preservation solution is to remove residual cell lysis buffer and unbound polymers.

[0036] The present invention also provides the application of mitochondria modified in situ with mitochondrial surface polymers as described above, and mitochondria modified in situ with mitochondrial surface polymers prepared by the above preparation method in mitochondrial transplantation.

[0037] In this invention, modifying mitochondria with the extracted polymers for mitochondrial transplantation at the cellular or animal level can significantly improve the delivery efficiency of mitochondria to target cells, while enhancing the structural stability and functional integrity of mitochondria during transplantation, thereby achieving energy metabolism repair and functional improvement of target cells.

[0038] In this invention, polymer-modified mitochondria are transplanted into the diseased brain tissue of Parkinson's disease model animals or patients. By supplementing the functional mitochondria, the energy metabolism disorder of dopaminergic neurons is repaired, and the symptoms of Parkinson's disease such as motor disorders and muscle stiffness are relieved, thereby achieving the treatment of Parkinson's disease.

[0039] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a mitochondrial surface polymer-modified mitochondria, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1: Synthesis of initiators and monomers The synthetic route for the "click" polymerization initiator is shown below: .

[0041] Synthesis of compound 1a: N-Boc-3-aminopropyl bromide (10.0 g, 1.0 eq) was dissolved in an acetone / water mixture, followed by the addition of potassium azide (5.1 g, 1.5 eq), and the reaction was carried out overnight at 60 °C. After the reaction was completed, the mixture was cooled to room temperature, the reaction was quenched with water, and the mixture was extracted three times with 100 mL of dichloromethane (DCM). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a colorless oily compound 1a.

[0042] Synthesis of compound 1b: Triacetylpropylamine (935.9 mg, 1.0 eq) and compound 1a (3.0 g, 2.1 eq) were dissolved in tetrahydrofuran (THF). Sodium ascorbate (0.3 g, 0.2 eq) and anhydrous copper sulfate (113.9 mg, 0.1 eq) were dissolved separately in water. Sodium ascorbate was added to the reaction solution first, and after thorough mixing, copper sulfate solution was added dropwise. The reaction was carried out at room temperature under N2 protection for 48 h, with TLC monitoring the reaction progress (developing solvent: DCM:EA = 1:1). After the reaction was complete, water was added to quench the reaction, and the mixture was extracted three times with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a white solid, 1b.

[0043] Synthesis of compound 1c: Compound 1b (1.0 g, 1.0 eq) and azidopolyethylene glycol (395.4 mg, 1.2 eq) were dissolved in THF. Sodium ascorbate (74.5 mg, 0.2 eq) and anhydrous copper sulfate (30 mg, 0.1 eq) were dissolved separately in water. Sodium ascorbate was added to the reaction mixture, and after thorough mixing, copper sulfate solution was added dropwise. The reaction was carried out at room temperature under N2 protection for 48 h, with TLC monitoring the reaction progress (evolving solvent: MeOH:DCM = 1:10). After the reaction was complete, water was added to quench the reaction, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a pale yellow oil, 1c.

[0044] Synthesis of compound 1d: Compound 1c (1.0 g, 1.0 eq) was dissolved in DCM and stirred for 10 min in an ice-water bath. Then, p-toluenesulfonyl chloride (404.6 mg, 1.5 eq) was added to the reaction solution, and after thorough mixing, triethylamine (TEA) and 4-dimethylaminopyridine (DMAP) were added. The reaction was carried out overnight at room temperature under N2 protection, with TLC monitoring the reaction progress (evolving solvent: MeOH:DCM = 1:10). After the reaction was completed, the reaction was quenched with water, extracted three times with dichloromethane, and the organic phases were combined, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a pale yellow oil, 1d.

[0045] Synthesis of compound 1e: Compound 1d (500.0 mg, 1.0 eq) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and KN3 (192.0 mg, 4.0 eq) was added. The mixture was heated to 55 °C for 24 h under N2 protection, and the reaction progress was monitored by TLC (evolving solvent: MeOH:DCM = 1:10). After the reaction was completed, the reaction was quenched with water, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a pale yellow oil, 1e.

[0046] Synthesis of compound 1f: 500.0 mg of compound 1e was dissolved in 6.0 mL of DCM, and then 2 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated to dryness, and then acetonitrile was added and evaporated to dryness three times at 40 °C to remove as much TFA as possible, finally yielding a pale yellow oily compound 1f.

[0047] Synthesis of compound 1g: (3-propanoyl)triphenylphosphine bromide (710.6 mg, 4.4 eq) was dissolved in DCM, and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (171.7 mg, 1.2 eq) was added for activation for 30 min. Then, compound 1f (200.0 mg, 1.0 eq) was added to the reaction solution, along with 0.5 mL of N,N-diisopropylethylamine. The reaction was carried out at room temperature for 48 h. The reaction progress was monitored by TLC (developing solvent: MeOH:DCM = 1:10). After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 1g of pale yellow solid.

[0048] The synthetic route for monomeric glycol is shown below: .

[0049] Synthesis of compound 2a: First, 11.64 g (60 mmol) of tetraethylene glycol and 1.23 g (22 mmol) of KOH were weighed and added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred vigorously. After stirring at 40 °C for 30 min, 2.1 g (20 mmol) of 3-bromopropyne was added. The mixture was then heated to 60 °C and stirred for 3 h. The reaction progress was monitored by TCL. After the reaction was complete, the mixture was diluted with water (50 mL) and acidified to pH 1 with 1 M hydrochloric acid. The aqueous layer was extracted with ethyl acetate (3 × 150 mL), and the organic phases were combined. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (200-300 mesh) using ethyl acetate / n-hexane (v / v = 1:2) as the eluent to obtain compound 2a as a colorless oil (4.3 g, yield: 92%).

[0050] Synthesis of compound 2b: 2a (1.35 g, 5.82 mmol) was dissolved in dichloromethane (10 mL) and transferred to a 100 mL round-bottom flask equipped with a magnetic stirrer. p-Toluenesulfonyl chloride (1.2 g, 6.4 mmol) was added, and the reaction solution was cooled to 0 °C in an ice-water bath. KOH (1.3 g, 23.28 mmol) was slowly added, and the mixture was stirred vigorously for 2 h. The mixture was then poured into ice water, and the crude product was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine, and dried over an appropriate amount of anhydrous magnesium sulfate. The mixture was then concentrated by vacuum evaporation without further treatment to obtain colorless oil 2b (2.2 g, yield: 98%).

[0051] Synthesis of compound 2c: 2b (1.83 g, 4.7 mmol) was weighed and dissolved in N,N-dimethylformamide (10 mL), then transferred to a 25 mL round-bottom flask equipped with a magnetic stirrer. Potassium azide (1.14 g, 14.1 mmol) was added to the reaction system, followed by triethylamine (4.3 g, 42.3 mmol). The mixture was stirred for 24 hours, and the reaction progress was monitored using a TCL (Total Discharge Chromatography-Concentration System). N,N-dimethylformamide was first evaporated to dryness using a rotary evaporator, washed with saturated sodium bicarbonate solution (50 mL), and the aqueous layer was extracted with ethyl acetate (3 × 150 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated crude product was purified by silica gel column chromatography (200–300 mesh) using ethyl acetate / n-hexane (v / v = 1 / 3) as the eluent to obtain pale yellow oil 2c (918 mg, yield: 76%).

[0052] The synthetic route for guanidine-containing monomers is shown below: .

[0053] Synthesis of compound 3a: 2.14 g (10 mmol) of tert-butyl (2-acrylamidoethyl)carbamate and propylthiolamidine (1.21 g, 22 mmol) were dissolved in 25 mL of methanol and transferred to a 100 mL round-bottom flask equipped with a magnetic stirrer. The mixture was refluxed and stirred at 50 °C for 48 hours. The reaction progress was monitored using a thin-layer chromatography plate (TCL). After the reaction was complete, the methanol was removed by vacuum distillation, and the residue was dissolved in dichloromethane (200 mL). The mixture was extracted and washed three times with saturated brine (3 × 150 mL), and the combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated crude product was purified by silica gel column chromatography (200–300 mesh) using a DCM:methanol ratio of 20:1 to obtain the yellow oil product 3a (1.23 g, yield: 46%).

[0054] Synthesis of compound 3b: Product 3a (0.5 g, 1.86 mmol) was completely dissolved in 20 mL of anhydrous dichloromethane (DCM) and transferred to a 100 mL round-bottom flask equipped with a magnetic stir bar. Azideacetic acid (0.28 g, 2.79 mmol) was then added and stirred until fully dissolved. Subsequently, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 0.11 g, 2.27 mmol) and 4-dimethylaminopyridine (DMAP, 0.02 g, 0.18 mmol) were added sequentially, and the reaction was stirred at 25 °C for 24 h. The reaction progress was monitored by thin-layer chromatography (TLC), and the reaction was considered complete when the starting material spot completely disappeared. After the reaction, the system was diluted with 200 mL of DCM, washed three times with saturated brine (3 × 100 mL), the aqueous phase was discarded, and the organic phase was dried over anhydrous Na₂SO₄ and concentrated under vacuum to remove the solvent. The crude product was purified by column chromatography using DCM:methanol = 60:1 as the eluent. The concentrated crude product was purified by silica gel column chromatography with a mesh size of 200-300 to obtain a yellow-brown oily solid product 3b (543 mg, 81%).

[0055] Synthesis of compound 3c: Purified compound 3b (500 mg, 1.42 mmol) was dissolved in ultradry dichloromethane (10 mL) and transferred to a 50 mL round-bottom flask equipped with a magnetic stir bar. Trifluoroacetic acid (10 mL) was added to the mixture. After stirring at room temperature for 12 h, the trifluoroacetic acid was evaporated under reduced pressure. After the trifluoroacetic acid was completely removed, a large amount of brown oily product 3c (358 mg, yield 83%) was observed to be generated.

[0056] Synthesis of compound 3d: (2-Acrylamidoethyl)carbamic acid (0.4 g, 1.59 mmol) was added to 10 mL of anhydrous dichloromethane and stirred until completely dissolved. The solution was then transferred to a 100 mL round-bottom flask equipped with a magnetic stir bar. Compound 3c (0.49 g, 1.59 mmol) and triethylamine (0.59 g, 5.85 mmol) were then added, and the mixture was stirred at 25 °C for 24 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, the system was diluted with 200 mL of DCM and washed successively with saturated brine (3 × 100 mL) to remove unreacted reagents and byproducts. The organic phase was dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated under vacuum to remove the solvent. The crude product was purified by column chromatography: first, unreacted raw material impurities were eluted with pure DCM, and then the elution system was switched to DCM:methanol = 60:1 to obtain the target product 3d (0.41g, yield 52%).

[0057] Synthesis of compound 3e: Purified compound 3d (0.4 g, 0.8 mmol) was dissolved in ultradry dichloromethane (10 mL) and transferred to a 50 mL round-bottom flask equipped with a magnetic stir bar. Trifluoroacetic acid (10 mL) was added to the mixture. After stirring at room temperature for 12 h, the trifluoroacetic acid was evaporated under reduced pressure. After the trifluoroacetic acid was completely removed, a large amount of yellow oily product 3e (0.21 g, yield 88%) was observed to be generated.

[0058] Example 2: Acquisition of 1H NMR and Mass Spectra The "click" polymerization initiator, glycol monomer, and guanidine-containing monomer prepared in Example 1 were sampled respectively. The "click" polymerization initiator and guanidine-containing monomer were placed in a high-field nuclear magnetic resonance (NMR) spectrometer to collect 1H NMR spectra, and the glycol monomer was placed in a liquid superconducting NMR spectrometer to collect 1H NMR spectra. The "click" polymerization initiator and glycol monomer were placed in a single quadrupole liquid chromatography-mass spectrometry system to collect mass spectra.

[0059] Among them, the 1H NMR spectrum of the "click" polymerization initiator is as follows: Figure 1 As shown, the mass spectrum is as follows Figure 2 As shown; the 1H NMR spectrum of the glycol monomer is as follows Figure 3 As shown, the mass spectrum is as follows Figure 4 As shown; the 1H NMR spectrum of the guanidine-containing monomer is as follows Figure 5 As shown.

[0060] Example 3: GPC curves of mitochondrial surface polymers HepG2 cells were first seeded in 60 mm cell culture dishes. Then, using 10 mL of LDMMEM as the solvent, 1 g of 2 mM initiator (dissolved in dimethyl sulfoxide, DMSO) was added, and the cells were incubated at 37°C under 5% CO2 humidification for 3 hours. After 3 hours of incubation, the culture medium was discarded, and 5 mL of fresh phosphate-buffered saline (PBS) was added. The cells were washed three times with PBS (5 mL each time). The culture medium was then replaced with 10 mL of fresh medium containing 20 μL of 20 mM monomer 2c (dissolved in PBS), 10 μL of 10 mM copper sulfate (CuSO4, dissolved in PBS), and 40 μL of 100 mM ascorbic acid (AA, dissolved in PBS), and incubated at 37°C under 5% CO2 humidification for 3 hours. After incubation, 5 mL of PBS was added, and the cells were washed three times with PBS (5 mL each time). Discard the PBS, add 1 mL of trypsin solution and digest for 3 minutes. Then discard the PBS, add 4 mL of fresh culture medium to stop the digestion, and carefully pipette the cells from the bottom of the culture dish. Centrifuge the cell suspension at 1300×g for 5 minutes at room temperature, discard the supernatant and collect the cell pellet. Add 1.5 mL of mitochondrial isolation reagent to the pellet, gently resuspend the cells, and place on ice for 15 minutes. Then transfer the cell suspension to a suitable glass homogenizer and homogenize approximately 30 times. Centrifuge the homogenate at 600×g for 10 minutes at 4°C, carefully transfer the supernatant to another centrifuge tube, centrifuge at 11000×g for 10 minutes at 4°C, discard the supernatant, and the resulting pellet is the isolated mitochondria. The isolated mitochondrial samples were lysed with 200 μL of mitochondrial lysis buffer containing PMSF added before use. The mitochondria were extracted with 50 mL of dichloromethane. The organic phase was concentrated under reduced pressure to remove the solvent, and the resulting polymer was analyzed by gel permeation chromatography (GPC). The eluent was N,N-dimethylformamide. GPC tests were then performed using a Wates 2414 pump and a Wates 1515 differential detector.

[0061] The results are as follows Figure 6 As shown in the figure. The results indicate that there is significant polymer enrichment inside the mitochondria, with a number-average molecular weight (M... n The value is 15.4 kDa, and the dispersion is ( The value was 1.43 (using polyethylene oxide as a standard), confirming that the above in-situ polymerization process has precise subcellular localization characteristics.

[0062] Example 4: Super-resolution fluorescence microscopy images of mitochondrial surface polymers HepG2 cells were first seeded in 60 mm cell culture dishes. Then, 10 μL of 1 g of 2 mM initiator (dissolved in dimethyl sulfoxide, DMSO) was added to 10 mL of DMEM, and the cells were incubated at 37°C under 5% CO2 humidification for 3 hours. After 3 hours of incubation, the culture medium was discarded, and 5 mL of fresh phosphate-buffered saline (PBS) was added. The cells were washed three times with 5 mL of PBS each time. The culture medium was then replaced with 10 mL of fresh medium containing 20 mM monomer 2c (dissolved in PBS), 2 μL of 20 mM CY5 monomer (dissolved in DMSO), 10 μL of 10 mM copper sulfate (CuSO4, dissolved in PBS), and 40 μL of 100 mM ascorbic acid (AA, dissolved in PBS), and incubated at 37°C under 5% CO2 humidification for 3 hours. Cells were directly imaged using a high-sensitivity structured light illumination microscope (HIS-SIM).

[0063] The results are as follows Figure 7 As shown, the results, including cross-sectional analysis and 3D reconstruction, confirm that the polymer is mainly located on the outer and inner mitochondrial membranes (including the cristae). Given the low permeability of the mitochondrial membrane to macromolecules, the accumulation of polymers on the cristae is particularly significant. This phenomenon can be attributed to the following mechanism: a typical "click" polymerization initiator is first transported to the inner mitochondrial membrane via triphenylphosphine (TPP)-mediated targeting; subsequently, small monomers permeate into the membrane structure; and then the polymerization reaction occurs in situ on the mitochondrial membrane. The resulting high-molecular-weight polymer cannot diffuse out of the mitochondria due to size exclusion effects, thus being effectively captured within the organelle.

[0064] Transmission electron microscopy image of polymer-modified mitochondria in Example 5 To further characterize the polymer-modified mitochondria, we isolated mitochondria from host cells after the polymerization reaction, and then purified them by density gradient centrifugation to obtain the polymer-modified mitochondria. The specific preparation method is described in Example 3.

[0065] Cryo-transmission electron microscopy (Cryo-TEM) revealed the characteristics of surface modifications at the structural level (such as... Figure 8 (As shown). Unmodified mitochondria have a clear outer membrane and well-structured mitochondrial cristae; while polymer-modified mitochondria show a distinct polymer layer on their outer membrane and significant polymer deposition on their mitochondrial cristae.

[0066] Example 6: Efficiency of mitochondrial delivery into cells modified with surface polymers To evaluate the efficacy of mitochondrial transplantation, this embodiment designed three experimental groups: unmodified mitochondria, mitochondria modified with 1g of initiator (non-polymerized), and engineered mitochondria based on monomer 2c polymerization mediated by 1g of initiator. After isolating these mitochondria from HepG2 cells, they were directly co-cultured with target HepG2 cells at a dose of approximately 50,000 mitochondria without additional treatment. After 2 hours of culture, the internalization of mitochondria was assessed by flow cytometry.

[0067] This indicator can accurately reflect the level of internalization (results are as follows). Figure 9 (As shown in the image). The results showed that both unmodified and initiator-modified mitochondria had low cellular uptake efficiency, with over 70% of mitochondria remaining extracellularly. This limitation may be related to their negative surface charge. In contrast, polymer-coated mitochondria exhibited significantly enhanced cellular uptake capacity, reaching approximately 80%. This phenomenon is attributed to the fact that surface polymer modification can effectively reduce surface electronegativity, thereby effectively promoting receptor-mediated endocytosis.

[0068] Example 7: Cellular ATP production after mitochondrial transplantation To evaluate the effects of different modification strategies on cellular energy metabolism after mitochondrial transplantation, this study used intracellular ATP production as an indicator to characterize the functional effects of mitochondrial transplantation. Three control groups were set up: an unmodified mitochondrial transplantation group, a mitochondrial transplantation group modified with 1g of initiator (non-polymerized), and engineered mitochondria based on monomer 2c polymerization mediated by 1g of initiator. After isolating mitochondria from HepG2 cells, each group was co-cultured with target HepG2 cells at the same dosage. After 24 hours of culture, the ATP levels of each group were measured using a multi-mode microplate reader.

[0069] The results show (e.g.) Figure 10 While ATP production in unmodified and initiator-modified mitochondria showed slight increases after transplantation, the levels in both groups were similar and remained low. In contrast, ATP production in polymer-modified mitochondria was significantly higher, the highest among the three groups, indicating that polymer-modified mitochondria more effectively enhance the energy metabolism of target cells. This result is consistent with the significant improvement in mitochondrial internalization efficiency caused by polymer modification, suggesting that polymer modification not only promotes cellular uptake of mitochondria but also further enhances their functional activity within cells, thereby more effectively restoring the cell's ATP synthesis capacity.

[0070] Example 8: Cellular Oxygen Consumption After Mitochondrial Transplantation To evaluate the impact of different modification strategies on the recovery of cellular respiratory function after mitochondrial transplantation, this study characterized the functional effect of mitochondrial transplantation by measuring cellular oxygen consumption rate (OCR). Four control groups were set up: pre-transplantation (control group), unmodified mitochondrial transplantation group, mitochondrial transplantation group modified with 1g initiator (non-polymerized), and engineered mitochondrial transplantation group based on monomer 2c polymerization mediated by 1g initiator. Mitochondria from each group were isolated from HepG2 cells and co-cultured with target HepG2 cells at the same dosage. The basal oxygen consumption rate of each group was then measured using a fluorescence spectrophotometer.

[0071] The results show (e.g.) Figure 11 Before transplantation, the oxygen consumption rate of cells was at a low level. While both unmodified and initiator-modified mitochondria showed some increase in oxygen consumption rate after transplantation, the increase was limited, and there was no significant difference between the two groups. In contrast, the polymer-modified mitochondrial transplantation group showed a significantly higher oxygen consumption rate, the highest among the four groups, suggesting that polymer-modified mitochondria can more effectively enhance the respiratory function of target cells after transplantation. This result is consistent with the conclusion that polymer modification significantly improves mitochondrial internalization efficiency and ATP production after mitochondrial transplantation, indicating that polymer modification not only promotes cellular uptake of mitochondria but also further enhances their functional activity within cells, thereby more effectively improving cell viability.

[0072] Based on the experimental results of Examples 6-8, it is shown that the polymer-modified layer can shield the negative charge on the surface of mitochondria, reduce the charge repulsion with the target cell membrane, and enhance the specific recognition of target cells through the targeting motif in the monomer, significantly improving the internalization efficiency of mitochondria into target cells. After transplantation, it can more effectively improve the energy metabolism level of target cells, enhance their functional activity in the cell, thereby more effectively restoring the cell's ATP synthesis capacity and improving cell activity.

[0073] Example 9: Animal experiment on polymer-modified mitochondrial transplantation for the treatment of Parkinson's disease The experimental materials were 6-8 week old C57BL / 6J mice. The reagents and instruments included MPTP, mitochondrial extraction kit, and brain stereotaxic instrument. Mitochondria were extracted from NIH-3T3 cells and free mitochondrial suspension and mitochondrial capsule suspension were prepared for later use.

[0074] Six- to eight-week-old C57BL / 6J mice were selected and injected intravenously five times a week with 2.5 mg / kg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). After successfully establishing a Parkinson's disease model, the mice were randomly divided into two groups: one group received intravenous injections containing 1×10⁻⁶ MPTP twice a week. 8One group of mice received a solution of free mitochondria or polymer-modified mitochondria (100 μL) for one month of continuous intervention; another group of mice received an equal volume of PBS solution intravenously as a control. Simultaneously, untreated C57BL / 6J mice served as wild-type controls. The mice's motor learning ability, coordination, and balance were assessed using open field tests, pole climbing tests, and rotarod tests. Oxygen consumption, carbon dioxide production, and activity data were collected using metabolic cage assays.

[0075] Behavioral testing results showed that, compared with the model control group, the total movement distance, freezing time, pole climbing time, and rotarod latency of mice in the polymer-modified mitochondria treatment group increased, and were close to those of the wild-type control group. The mitochondrial capsule group showed even better results. Metabolic index testing showed that the O2 consumption, CO2 production, and energy consumption of mice in the treatment group increased, the respiratory quotient tended to normalize, and food intake returned to normal levels. This confirmed that mitochondrial transplantation can effectively improve the symptoms of Parkinson's disease mice and has good therapeutic effects and universality.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A mitochondrial surface polymer-modified mitochondrial, characterized in that, The mitochondria modified with polymer on the mitochondrial surface are formed by in-situ "click" polymerization of polymers on the mitochondrial surface within the host cell.

2. The method for preparing mitochondria with in-situ modified mitochondrial surface polymers as described in claim 1, characterized in that, Includes the following steps: 1) Preparation of "click" polymerization initiators and monomers; 2) After incubating the "click" polymerization initiator with the host cells for 1-5 hours, wash 1-5 times; 3) Add monomers, copper sulfate and sodium ascorbate to the cells and continue incubation for 1-4 hours. Wash 1-5 times to complete the in-situ polymerization modification of the mitochondrial surface in the cells. 4) Mitochondria modified with polymer on the mitochondrial surface were extracted by centrifugation.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the monomer to the "click" polymerization initiator is 50:1 to 1000:1; the molar ratio of the "click" polymerization initiator to copper sulfate is 1:1 to 1:5; and the concentration of sodium ascorbate is 1 to 10 mM.

4. The preparation method according to claim 2, characterized in that, The chemical structure of the "click" polymerization initiator includes: a tri(triazole methyl)amine group, an R group, and an initiation site.

5. The preparation method according to claim 4, characterized in that, The R group includes a triphenylphosphine salt group or a mitochondrial-targeting peptide; the initiation site includes an azide group or an alkynyl group.

6. The preparation method according to claim 2, characterized in that, The monomer has a structure comprising an azide group and an alkynyl group, which are connected by two types of functional groups; the functional groups include hydrophilic spacer groups and functional modification units.

7. The preparation method according to claim 6, characterized in that, The hydrophilic spacer group includes an ethylene glycol repeating unit; the functional modification unit includes one or more of a charge-regulating unit, a targeting unit, or a fluorescent group; the charge-regulating unit includes a guanidine group, a carboxyl group, or an amino group; the targeting unit includes biotin, RGD peptide, or folic acid; the fluorescent group is selected from FITC, rhodamine, and CY5.

8. The preparation method according to claim 2, characterized in that, The centrifugation includes the following steps: lysing cells treated with in situ polymerization, centrifuging at 800×g and 4℃ for 10 min to remove cell nuclei and cell debris, collecting the supernatant, centrifuging at 12000×g and 4℃ for 20 min to obtain mitochondrial precipitate, washing with mitochondrial preservation solution 1-3 times, and resuspending to obtain purified polymer-modified mitochondria.

9. The application of mitochondria modified in situ with mitochondrial surface polymer as described in claim 1, and mitochondria modified in situ with mitochondrial surface polymer prepared by the preparation method according to any one of claims 2 to 8, in mitochondrial transplantation.

10. The application according to claim 9, characterized in that, Mitochondria modified in situ with the polymer on the mitochondrial surface can repair energy metabolism disorders in dopaminergic neurons.

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