Preparation method of high-activity mitochondrial targeting MAPS polypeptide preparation

Abstract

The invention discloses a preparation method of a high-activity mitochondrial targeting MAPS polypeptide preparation, and relates to the technical field of biologication.The preparation method comprises the steps of S1, polypeptide design and synthesis, S2, thiol oriented cross-linking, S3, cutting and purification, S4, liposome assembly and S5, freeze-drying preparation molding.The preparation method has the advantages that by designing an amphipathic alpha-spiral polypeptide structure, the high-activity mitochondrial targeting MAPS polypeptide preparation is obtained; a stable helical conformation is spontaneously formed in a physiological environment by utilizing the alternative arrangement characteristic of hydrophobic and hydrophilic residues; the hydrophobic surface of the conformation can effectively interact with the mitochondrial membrane lipid bilayer, so that the transmembrane resistance is reduced; meanwhile, the hydrophilic surface is combined with the high negative membrane potential of the mitochondrial inner membrane through charge interaction to form a directional driving effect, so that the active transfer efficiency of the polypeptide under the mitochondrial membrane potential is remarkably improved; in addition, the integrated mitochondrial positioning signal further specifically recognizes the mitochondrial outer membrane transporter, the polypeptide is assisted to be accurately anchored to the targeting site, and non-specific diffusion is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method of a highly active mitochondrion-targeted MAPS polypeptide preparation. Background Art

[0002] Mitochondrion-targeted polypeptide preparations are a novel therapeutic strategy for specifically delivering active ingredients to mitochondria and have important application potential in the fields of degenerative diseases, tumors, and metabolic disorders. Mitochondria, as the core organelles for cell energy metabolism and apoptosis regulation, are closely related to various pathological processes in terms of their dysfunction.

[0003] In the prior art, most mitochondrion-targeted systems rely on a single physicochemical driving mechanism (such as cation-dependent membrane adsorption) or simple sequence signals (such as mitochondrion localization peptides), lacking a coordinated design for the process of mitochondrion membrane penetration and being difficult to effectively coordinate the hydrophilic-hydrophobic balance of polypeptides, resulting in an increased resistance when crossing the double membrane of mitochondria. In view of the above problems, the following solutions are proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a highly active mitochondrion-targeted MAPS polypeptide preparation. By designing an amphiphilic α-helix polypeptide structure and utilizing the alternating arrangement characteristics of its hydrophobic and hydrophilic residues, it can effectively interact with the lipid bilayer of the mitochondrion membrane, reduce the transmembrane resistance, and solve the problem in the prior art that it is difficult to effectively coordinate the hydrophilic-hydrophobic balance of polypeptides, resulting in an increased resistance when crossing the double membrane of mitochondria.

[0005] To solve the above technical problems, the present invention is realized through the following technical solutions:

[0006] The present invention provides a preparation method of a highly active mitochondrion-targeted MAPS polypeptide preparation, including:

[0007] Step S1, polypeptide design and synthesis: Design a polypeptide containing an MLS targeting sequence, an amphiphilic α-helix structure, and a cysteine cross-linking site, and sequentially couple amino acids through solid-phase chemistry to construct a complete backbone;

[0008] Step S2, thiol-directed cross-linking: React the SMCC cross-linking agent with the reduced polypeptide thiol group to form a reversible covalent cross-linking structure;

[0009] Step S3, cleavage and purification: Cleave the resin and separate and purify the polypeptide by reverse-phase chromatography to ensure high purity and correct folding conformation;

[0010] Step S4, liposome assembly: Load the polypeptide into DOPE / DOTAP / cholesterol liposomes and form a nano-carrier with uniform particle size by ultrasonic extrusion method;

[0011] Step S5, Freeze-drying preparation forming: After adding the freeze-drying protectant, it is dried in stages to obtain a solid preparation that can be stored for a long time, and the targeting and transmembrane activities are retained after reconstitution.

[0012] Further, in the step S1, the polypeptide design and synthesis specifically include the following steps:

[0013] Step S11, Sequence design: Using KLAKLAK-NH2 as the mitochondrial targeting core, connecting the MLS sequence at the N-terminus, and inserting cysteine (Cys) at the C-terminus as the cross-linking site;

[0014] Alternately arranged hydrophobic (Leu, Ile) and hydrophilic (Lys, Arg) residues are inserted in the middle section to form an amphiphilic α-helix template (sequence such as: KLAKLAK-Lys-Leu-Arg-Ile-Lys-Ile-Arg-Leu-Cys);

[0015] Step S12, Solid-phase synthesis: Using the Fmoc chemical strategy, with Rink Amide MBHA resin as the carrier; HBTU / HOBt as the activator, the coupling time is 30 minutes, and the deprotection is carried out using a 20% piperidine / DMF solution, deprotecting 2×5 minutes each time, coupling amino acids successively from the C-terminus to the N-terminus, rinsing 3 times with DMF after each reaction step, and drying with nitrogen;

[0016] By combining computer simulation and empirical design, an amphiphilic α-helix polypeptide sequence is constructed, integrating the mitochondrial localization signal (MLS) and alternately hydrophobic / hydrophilic residues to form a transmembrane driving structure. Solid-phase synthesis uses the Fmoc chemical strategy to prepare a high-purity linear polypeptide with a resin as the carrier, ensuring precise control of the sequence, laying a molecular foundation for subsequent cross-linking and functionalization, and avoiding non-specific side reactions.

[0017] Further, in the step S11, the MLS sequence in the sequence design is specifically MLRVLLVLAAFSATAGSA;

[0018] In the step S12, in the solid-phase synthesis, the particle size of the Rink Amide MBHA resin is 100-200 mesh, and the substitution value is 0.5 mmol / g; the molar ratio of the activator HBTU to HOBt when mixed is 1:1; the preparation ratio of the 20% piperidine / DMF solution is a volume ratio.

[0019] Further, in the step S2, the thiol-directed cross-linking specifically includes the following steps:

[0020] Step S21, Thiol group activation: Immerse the synthesized polypeptide-resin complex in a 50 mM TCEP (tris(2-carboxyethyl)phosphine) solution with a pH of 6.0, shake at room temperature for 1 hour to reduce the thiol group on the Cys side chain;

[0021] Step S22: Bifunctional crosslinker coupling: Use SMCC (sulfosuccinimidyl-4-N-maleimidomethyl) cyclohexane-1-carboxylate) as a crosslinker, dissolve it in PBS buffer with a pH of 7.4 until the final concentration is 5 mmol / L; mix the polypeptide and SMCC at a molar ratio of 1:3, and react in the dark at 4°C for 12 hours to form a thioether bond crosslinked structure; add 10 volumes of pre-cooled ether for precipitation, centrifuge to collect the precipitate, with a centrifugation speed of 10000×g for 10 minutes;

[0022] By utilizing the thiol-maleimide click chemical reaction, a reversible crosslinking bond (thioether bond) is introduced at specific cysteine sites to enhance the stability of the polypeptide during intracellular transport. By controlling the concentration of the crosslinker (SMCC) and the reaction time, precise regulation of intramolecular or intermolecular crosslinking is achieved, balancing the structural rigidity and mitochondrial membrane potential responsiveness, and preventing premature degradation or aggregation of the polypeptide.

[0023] Furthermore, step S3, cleavage and purification specifically include the following steps:

[0024] Step S31: Resin cleavage: Prepare a cleavage solution of TFA (trifluoroacetic acid): TIS (triisopropylsilane): H2O = 95:2.5:2.5, and pre-cool it in an ice bath; immerse the crosslinked polypeptide-resin in the cleavage solution, shake at room temperature for 3 hours, filter to remove the resin, and concentrate it to 1 / 10 of the original volume by nitrogen purging;

[0025] Step S32: Reverse-phase chromatography purification: Use a C18 preparative column and perform gradient elution with mobile phase A and mobile phase B;

[0026] During the gradient elution process, the concentration of mobile phase B gradually increases from 5% to 60% over 30 minutes, with a flow rate of 10 mL / min. When detecting the polypeptide by ultraviolet, the wavelength used is 220 nm. Collect the main peak eluate with a retention time of 18 - 22 minutes, and lyophilize to obtain a white powder;

[0027] By using a TFA cleavage solution to release the polypeptide chain from the resin, simultaneously removing the protecting groups and retaining the active functional groups, reverse-phase chromatography purification separates impurities through hydrophobic interaction to ensure the purity of the final product, eliminates the interference of uncrosslinked fragments or truncated sequences, and guarantees the consistency of the subsequent liposome drug loading efficiency and biological function.

[0028] Furthermore, in step S31, the preparation ratio of the cleavage solution in resin cleavage is a volume ratio;

[0029] In step S32, for reverse-phase chromatography purification, the particle size of the C18 preparative column is 5 μm, and the pore size is Mobile phase A is an aqueous solution containing 0.1% TFA, mobile phase B is an acetonitrile solution containing 0.1% TFA, and the elution time range of the target polypeptide in the chromatographic column.

[0030] Further, in step S4, the liposome assembly specifically includes the following steps:

[0031] Step S41, lipid film preparation: Weigh DOPE (dioleoyl phosphatidylethanolamine): DOTAP (dioleoyl trimethylammonium propane): cholesterol with a molar ratio of 5:3:2, and dissolve it in a mixed solvent of chloroform and methanol; Rotate and evaporate to form a film, and purge with nitrogen for 30 minutes to remove residual solvents;

[0032] Step S42, polypeptide loading: Dissolve the purified polypeptide in HEPES buffer, and the final concentration of the dissolved polypeptide is 2 mg / mL; Then mix the lipid and the polypeptide at a mass ratio of 10:1, vortex for 2 minutes, perform ultrasonic treatment, and finally extrude through a 0.22 μm polycarbonate membrane 3 times to obtain homogeneous liposomes;

[0033] The polypeptide is encapsulated in cationic liposomes (DOPE / DOTAP / cholesterol) by the thin film hydration method, and the electrostatic interaction is used to enhance the binding efficiency between the polypeptide and the lipid membrane; Extrusion homogenization controls the liposome particle size to 100 ± 20 nm, optimizes cell uptake and mitochondrial targeting, reduces immunogenicity at the same time, and improves in vivo delivery stability.

[0034] Further, in step S41, the volume ratio of the mixed solvent of chloroform and methanol in lipid film preparation is 9:1; The temperature for rotary evaporation to form a film is 40 °C, and the vacuum degree is -0.09 MPa;

[0035] In step S42, the concentration of HEPES buffer in polypeptide loading is 10 mmol / L, pH is 7.4, and it contains 150 mmol / L sodium chloride; The frequency of ultrasonic treatment is 40 kHz, the power is 200 W, and the duration is 5 minutes; The particle size range of the liposomes is 100 ± 20 nm, and PDI < 0.2.

[0036] Further, in step S5, the freeze-dried preparation forming specifically includes the following steps:

[0037] Step S51, pre-freeze-drying treatment: Add a freeze-drying protectant to the liposome suspension and equilibrate in a 4 °C environment for 2 hours;

[0038] Step S52, freeze-drying: Place the liposome suspension in an environment of -80 °C and quick-freeze for 4 hours; Then transfer it to a freeze-dryer and perform primary drying for 24 hours under a vacuum condition of -45 °C and 0.1 mbar; Finally, raise the temperature to 25 °C and reduce the vacuum degree to 0.01 mbar, and continue secondary drying for 6 hours to finally obtain a porous and loose freeze-dried powder;

[0039] Trehalose and mannitol are added as lyoprotectants, and water is removed through pre-freezing and gradient drying to form porous freeze-dried powder. This process preserves the structural integrity of liposomes, extends the storage stability of the preparation (>12 months at 2-8°C), facilitates the rapid restoration of nanoparticle dispersibility after reconstitution, and ensures the convenience of clinical use.

[0040] Furthermore, in step S51, the concentration of the lyoprotectant in the pre-freeze-drying treatment is expressed as weight / volume percentage, containing 5% trehalose and 1% mannitol.

[0041] The present invention has the following beneficial effects:

[0042] 1. By designing an amphiphilic α-helical polypeptide structure, the present invention utilizes the alternating arrangement characteristics of its hydrophobic and hydrophilic residues to spontaneously form a stable helical conformation in a physiological environment. The hydrophobic surface of this conformation can effectively interact with the mitochondrial membrane lipid bilayer, reducing the transmembrane resistance. At the same time, the hydrophilic surface binds to the highly negative membrane potential of the inner mitochondrial membrane through charge interaction, forming an oriented driving effect, significantly enhancing the active transport efficiency of the polypeptide under the mitochondrial membrane potential. In addition, the integrated mitochondrial localization signal further assists the polypeptide to precisely anchor to the target site by specifically recognizing the outer mitochondrial membrane transport protein, reducing non-specific diffusion.

[0043] 2. By introducing reversible thiol cross-linking sites into the polypeptide sequence and using bifunctional cross-linking agents to form dynamic covalent bonds, an intramolecular or intermolecular cross-linking network is constructed. This cross-linked structure remains stable in the extracellular neutral environment, avoiding protease degradation or interference from serum components. After entering the mitochondria, the high concentration of glutathione in the mitochondrial matrix can trigger the reversible cleavage of the thioether bond, restoring the biological activity of the polypeptide.

[0044] 3. By integrating the mitochondrial localization signal with the amphiphilic α-helical structure, the targeting of the polypeptide no longer depends on a single physicochemical interaction, but rather achieves dual targeting through the specific recognition of the MLS and mitochondrial membrane receptors and the membrane penetration of the α-helix. This design can reduce non-specific adsorption to non-target tissues or organelles and reduce off-target toxicity. At the same time, encapsulating the polypeptide with a liposome carrier and simulating the natural membrane structure by adjusting the phospholipid composition can further improve the cell membrane fusion efficiency and biocompatibility of the preparation. The surface modification of the liposome can also avoid immune system recognition and extend the residence time of the drug in the circulatory system.

[0045] Of course, it is not necessary for any product implementing the present invention to simultaneously achieve all the above-mentioned advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0047] Figure 1 It is a schematic flow chart of a preparation method of a highly active mitochondrial-targeted MAPS polypeptide preparation of the present invention. Detailed implementation manners

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0049] Please refer to Figure 1 As shown, the present invention is a preparation method of a highly active mitochondrial-targeted MAPS polypeptide preparation, including:

[0050] Step S1, polypeptide design and synthesis:

[0051] Step S11, sequence design: Using KLAKLAK-NH2 as the mitochondrial targeting core, connecting the MLS sequence at the N-terminus, and inserting cysteine (Cys) at the C-terminus as a crosslinking site;

[0052] Insert alternating hydrophobic (Leu, Ile) and hydrophilic (Lys, Arg) residues in the middle section to form an amphiphilic α-helix template (sequence such as: KLAKLAK-Lys-Leu-Arg-Ile-Lys-Ile-Arg-Leu-Cys);

[0053] Step S12, solid-phase synthesis: Using the Fmoc chemical strategy, with Rink Amide MBHA resin as the carrier; HBTU / HOBt as the activator, the coupling time is 30 minutes, and the deprotection is carried out using a 20% piperidine / DMF solution, with each deprotection for 2×5 minutes. Amino acids are coupled sequentially from the C-terminus to the N-terminus, and after each step of the reaction, it is rinsed 3 times with DMF and dried with nitrogen;

[0054] By combining computer simulation and empirical design, an amphiphilic α-helix polypeptide sequence is constructed, integrating the mitochondrial localization signal (MLS) and alternating hydrophobic / hydrophilic residues to form a transmembrane driving structure. Solid-phase synthesis uses the Fmoc chemical strategy to prepare a high-purity linear polypeptide with a resin as the carrier, ensuring accurate control of the sequence, laying a molecular foundation for subsequent crosslinking and functionalization, and avoiding non-specific side reactions.

[0055] Step S11, in the sequence design, the MLS sequence is specifically MLRVLLVLAAFSATAGSA;

[0056] Step S12, in the solid-phase synthesis, the particle size of Rink Amide MBHA resin is 100 - 200 mesh, and the substitution value is 0.5 mmol / g; the molar ratio of the activator HBTU to HOBt when mixed is 1:1; the preparation ratio of the 20% piperidine / DMF solution is by volume ratio;

[0057] Step S2, thiol-directed crosslinking:

[0058] Step S21, thiol group activation: Immerse the synthesized polypeptide-resin complex in a 50 mM TCEP (tris(2-carboxyethyl)phosphine) solution with a pH of 6.0, shake at room temperature for 1 hour to reduce the thiol groups on the Cys side chains;

[0059] Step S22, bifunctional crosslinker coupling: Use SMCC (sulfosuccinimidyl-4-N-maleimidomethyl) cyclohexane-1-carboxylate) as the crosslinker, dissolve it in PBS buffer with a pH of 7.4 until the final concentration is 5 mmol / L; mix according to the molar ratio of polypeptide to SMCC of 1:3, react at 4 °C in the dark for 12 hours to form a thioether bond crosslinked structure; add 10 volumes of pre-cooled ether to precipitate, centrifuge to collect the precipitate, with a centrifugation speed of 10000×g for 10 minutes;

[0060] By utilizing the thiol-maleimide click chemical reaction, introduce reversible crosslinking bonds (thioether bonds) at specific cysteine sites, enhance the stability of the polypeptide during intracellular transport, and achieve precise regulation of intra- or intermolecular crosslinking by controlling the concentration of the crosslinker (SMCC) and the reaction time, balance the structural rigidity and mitochondrial membrane potential responsiveness, and prevent premature degradation or aggregation of the polypeptide;

[0061] Step S3, cleavage and purification:

[0062] Step S31, resin cleavage: Prepare the cleavage solution TFA (trifluoroacetic acid): TIS (triisopropylsilane): H2O = 95:2.5:2.5, pre-cool in an ice bath; immerse the crosslinked polypeptide-resin in the cleavage solution, shake at room temperature for 3 hours, filter to remove the resin, and concentrate by nitrogen purging to 1 / 10 of the original volume;

[0063] Step S32, reverse-phase chromatography purification: Use a C18 preparative column and perform gradient elution with mobile phase A and mobile phase B;

[0064] During gradient elution, the concentration of mobile phase B was gradually increased from 5% to 60% over 30 minutes at a flow rate of 10 mL / min. The wavelength used for ultraviolet detection of the polypeptide was 220 nm. The main peak eluate with a retention time of 18 - 22 minutes was collected and freeze-dried to obtain a white powder;

[0065] The polypeptide chain was released from the resin using TFA cleavage solution, simultaneously removing the protecting groups and retaining the active functional groups. Reverse-phase chromatography purification separated impurities through hydrophobic interaction to ensure the purity of the final product, eliminating the interference of uncrosslinked fragments or truncated sequences, and ensuring the consistency of subsequent liposome drug-loading efficiency and biological function.

[0066] In step S31, the mixing ratio of the cleavage solution in resin cleavage is by volume ratio;

[0067] In step S32, the particle size of the C18 preparative column in reverse-phase chromatography purification is 5 μm, and the pore size is Mobile phase A is an aqueous solution containing 0.1% TFA, and mobile phase B is an acetonitrile solution containing 0.1% TFA. The elution time range of the target polypeptide in the chromatographic column;

[0068] Step S4, Liposome assembly:

[0069] Step S41, Lipid film preparation: Weigh DOPE (dioleoyl phosphatidylethanolamine): DOTAP (dioleoyl trimethylammonium propane): cholesterol in a molar ratio of 5:3:2 and dissolve it in a mixed solvent of chloroform and methanol; Rotate and evaporate to form a film, and purge with nitrogen for 30 minutes to remove residual solvents;

[0070] Step S42, Polypeptide loading: Dissolve the purified polypeptide in HEPES buffer with a final concentration of 2 mg / mL; Then mix the lipid and polypeptide in a mass ratio of 10:1, vortex for 2 minutes, perform ultrasonic treatment, and finally extrude through a 0.22 μm polycarbonate membrane 3 times to obtain homogeneous liposomes;

[0071] The polypeptide was encapsulated in cationic liposomes (DOPE / DOTAP / cholesterol) by the thin film hydration method, and the electrostatic interaction was used to enhance the binding efficiency between the polypeptide and the lipid membrane; Extrusion homogenization controlled the liposome particle size to 100 ± 20 nm, optimizing cell uptake and mitochondrial targeting, while reducing immunogenicity and enhancing in vivo delivery stability.

[0072] In step S41, the volume ratio of the mixed solvent of chloroform and methanol in lipid film preparation is 9:1; The temperature for rotating and evaporating to form a film is 40 °C, and the vacuum degree is -0.09 MPa;

[0073] Step S42: The concentration of HEPES buffer during polypeptide loading is 10 mmol / L, the pH is 7.4, and it contains 150 mmol / L sodium chloride; the frequency of sonication is 40 kHz, the power is 200 W, and it lasts for 5 minutes; the particle size range of the liposomes is 100 ± 20 nm, and PDI < 0.2;

[0074] Step S5. Freeze-dried preparation forming:

[0075] Step S51. Predrying treatment: Add a freeze-drying protectant to the liposome suspension and equilibrate it at 4°C for 2 hours;

[0076] Step S52. Freeze-drying: Quick-freeze the liposome suspension in an environment of -80°C for 4 hours; then transfer it to a freeze-dryer and conduct primary drying for 24 hours under vacuum conditions of -45°C and 0.1 mbar; finally, raise the temperature to 25°C and reduce the vacuum to 0.01 mbar, and continue secondary drying for 6 hours to finally obtain a porous and loose freeze-dried powder;

[0077] Add trehalose and mannitol as freeze-drying protectants, remove moisture through pre-freezing and gradient drying to form a porous freeze-dried powder; this process preserves the structural integrity of the liposomes, extends the storage stability of the preparation (> 12 months at 2 - 8°C), facilitates the rapid restoration of the nanoparticle dispersibility after reconstitution, and ensures the convenience of clinical use.

[0078] In step S51, the concentration of the freeze-drying protectant in the predrying treatment is expressed as weight / volume percentage, containing 5% trehalose and 1% mannitol.

[0079] A specific application of this example is:

[0080] Preparation of a novel mitochondrial-targeted MAPS polypeptide preparation based on an amphiphilic α-helical structure

[0081] 1. Materials and equipment:

[0082] Reagents: Fmoc-protected amino acids (GL Biochem), Rink Amide MBHA resin (substitution value 0.5 mmol / g, Sigma), HBTU / HOBt (TCI), TFA (Alfa Aesar), SMCC (Thermo Fisher), DOPE / DOTAP / cholesterol (Avanti Polar Lipids), TCEP (Sigma);

[0083] Equipment: Peptide synthesizer (CEM Liberty Pro), high performance liquid chromatograph (Agilent 1260), rotary evaporator (Buchi R-300), nano extruder (Avanti Mini Extruder), freeze dryer (Labconco FreeZone);

[0084] 2. Specific implementation steps:

[0085] Step 1. Solid-phase peptide synthesis:

[0086] Resin pretreatment:

[0087] Weigh Rink Amide MBHA resin (1.0 g, 0.5 mmol / g) and place it in the synthesis column. Wash it successively with DMF (10 mL × 3) and dichloromethane (10 mL × 2), and dry it with nitrogen;

[0088] Sequence coupling:

[0089] Couple successively according to the designed sequence KLAKLAK-Lys-Leu-Arg-Ile-Lys-Ile-Arg-Leu-Cys (C→N direction):

[0090] Deprotection: Treat it with 20% piperidine / DMF solution (5 mL) for 2 × 5 minutes, and rinse it with DMF 3 times;

[0091] Coupling: Add a DMF solution (5 mL) of Fmoc-amino acid (4 eq), HBTU / HOBt (4 eq / 4 eq), and DIEA (8 eq) to each step, bubble with nitrogen and react for 30 minutes, and rinse with DMF 3 times;

[0092] Key control point: After each coupling step, use Kaiser reagent to detect whether the amino group is free (blue indicates complete coupling);

[0093] Terminal modification:

[0094] N-terminal acetylation: Treat it with acetic anhydride / pyridine / DMF (1:1:8, v / v) for 30 minutes, and rinse with DMF;

[0095] Step 2. Thiol cross-linking reaction:

[0096] Reduction of thiol groups:

[0097] Transfer the resin to a centrifuge tube, add TCEP solution (50 mM, pH 6.0, 10 mL), shake at room temperature for 1 hour, centrifuge (3000 × g, 5 minutes), discard the supernatant, and wash it with DMF 3 times;

[0098] SMCC cross-linking:

[0099] Prepare the SMCC cross-linking solution: Dissolve 5 mM SMCC in PBS (pH 7.4, containing 1 mM EDTA), pre-cool in the dark;

[0100] Mix the resin with the cross-linking solution at a molar ratio of 1:3, react at 4 °C in the dark for 12 hours, and collect the crude cross-linked polypeptide by centrifugation (10000×g, 10 minutes);

[0101] Step 3. Polypeptide purification:

[0102] Resin cleavage:

[0103] Cleavage solution formula: TFA / TIS / H2O = 95:2.5:2.5 (v / v, 10 mL), pre-cool in an ice bath;

[0104] Soak the resin in the cleavage solution, shake at room temperature for 3 hours, filter to remove the resin, and concentrate the filtrate by nitrogen blowing to 1 mL;

[0105] HPLC purification:

[0106] Chromatographic conditions: Agilent Zorbax C18 column (4.6×250 mm, 5 μm), mobile phase A (0.1% aqueous TFA solution), B (0.1% TFA in acetonitrile);

[0107] Gradient program: 0 - 5 minutes, 5% B → 5 - 30 minutes, 5% - 60% B → 30 - 35 minutes, 60% - 95% B;

[0108] Collect the main peak with a retention time of 18 - 22 minutes, and obtain a white powder after lyophilization (purity > 95%, molecular weight confirmed by mass spectrometry: calculated value 2456.8 Da, measured value 2456.5 Da);

[0109] Step 4. Preparation of liposome carriers:

[0110] Lipid membrane formation:

[0111] Weigh DOPE (50 mg), DOTAP (30 mg), cholesterol (20 mg), dissolve in chloroform-methanol (9:1, 10 mL), and rotary evaporate at 40 °C, -0.09 MPa to form a uniform lipid membrane;

[0112] Polypeptide loading and extrusion:

[0113] Dissolve the purified polypeptide (10 mg) in HEPES buffer (10 mM, pH 7.4, containing 150 mM NaCl, 5 mL), mix with the lipid membrane, and vortex for 2 minutes;

[0114] Ultrasonic treatment: 40 kHz, 200 W, ultrasonic in an ice bath for 5 minutes (pulse mode: on for 2 seconds / off for 3 seconds);

[0115] Extruded three times through a 0.22 μm polycarbonate membrane (Avanti) to obtain a homogeneous liposome suspension (detected by dynamic light scattering: particle size 102 ± 5 nm, PDI 0.18, Zeta potential +35 mV);

[0116] Step 5, Preparation of freeze-dried preparation:

[0117] Addition of cryoprotectant:

[0118] Add trehalose (final concentration 5%, w / v) and mannitol (final concentration 1%, w / v) to the liposome suspension, and let it stand at 4 °C for 2 hours;

[0119] Freeze-drying:

[0120] Pre-freezing: Rapidly frozen at -80 °C for 4 hours;

[0121] Primary drying: Maintained under vacuum at -45 °C and 0.1 mbar for 24 hours;

[0122] Secondary drying: Maintained at 25 °C and 0.01 mbar for 6 hours to obtain a loose white freeze-dried powder (particle size after reconstitution 105 ± 8 nm, encapsulation efficiency 87.3%);

[0123] 3. Effects of the example:

[0124] Transmembrane efficiency: Confocal microscopy imaging showed that the mitochondrial localization efficiency of this preparation in HeLa cells was 3.2 times higher than that of the traditional TPP carrier (quantified by fluorescence labeling);

[0125] Stability: After incubation in serum at 37 °C for 12 hours, the proportion of remaining intact polypeptide > 80% (HPLC area normalization method).

[0126] In the description of this specification, the description referring to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0127] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for preparing a highly active mitochondrial-targeted MAPS polypeptide preparation, characterized in that: The preparation method comprises the following steps: Step S1, peptide design and synthesis: design a peptide containing an MLS targeting sequence, an amphipathic α-helical structure and a cysteine ​​cross-linking site, and sequentially couple amino acids through solid phase chemistry to construct a complete skeleton; Step S2, thiol-directed cross-linking: using SMCC cross-linking agent to react with the reduced polypeptide thiol group to form a reversible covalent cross-linking structure; Step S3, cleavage and purification: cleaving the resin and separating and purifying the polypeptide using reverse phase chromatography to ensure high purity and correct folding conformation; Step S4, liposome assembly: loading the polypeptide into DOPE / DOTAP / cholesterol liposomes, and forming nanocarriers with uniform particle size by ultrasonic extrusion; Step S5, freeze-dried preparation molding: After adding a freeze-dried protective agent, drying is performed in stages to obtain a solid preparation that can be stored for a long time, and the targeting and transmembrane activities are retained after reconstitution.

2. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 1, characterized in that: The step S1, polypeptide design and synthesis, specifically comprises the following steps: Step S11, sequence design: using KLAKLAK-NH2 as the mitochondrial targeting core, connecting the MLS sequence at the N-terminus, and inserting cysteine ​​at the C-terminus as a cross-linking site; The middle segment is inserted with alternating hydrophobic and hydrophilic residues to form an amphipathic α-helical template; Step S12, solid phase synthesis: using Fmoc chemical strategy, Rink Amide MBHA resin as carrier; HBTU / HOBt as activator, coupling time 30 minutes, deprotection using 20% ​​piperidine / DMF solution, each deprotection time 2×5 minutes, amino acids are coupled successively from C-terminus to N-terminus, rinsed with DMF 3 times after each reaction, and blown dry with nitrogen.

3. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 2, characterized in that: In the step S11, the MLS sequence in the sequence design is specifically MLRVLLVLAAFSATAGSA; In the step S12, the particle size of Rink Amide MBHA resin in solid phase synthesis is 100-200 mesh, and the substitution value is 0.5 mmol / g; the molar ratio of the activator HBTU and HOBt when mixed is 1:1; and the preparation ratio of 20% piperidine / DMF solution is a volume ratio.

4. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 1, characterized in that: The step S2, thiol-oriented cross-linking, specifically comprises the following steps: Step S21, thiol group activation: soak the synthesized polypeptide-resin complex in a 50 mM TCEP solution at pH 6.0, shake at room temperature for 1 hour to reduce the thiol group of the Cys side chain; Step S22, bifunctional cross-linker coupling: SMCC is used as a cross-linker, dissolved in a PBS buffer solution with a pH of 7.4 until the final concentration is 5 mmol / L; the polypeptide and SMCC are mixed at a molar ratio of 1:3, and reacted at 4°C in the dark for 12 hours to form a thioether bond cross-linking structure; 10 times the volume of pre-cooled ether precipitation is added, and the precipitate is collected by centrifugation at a speed of 10,000×g for 10 minutes.

5. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 1, characterized in that: The step S3, cutting and purification specifically comprises the following steps: Step S31, resin cutting: prepare a cutting solution of TFA:TIS:H2O=95:2.5:2.5, and pre-cool in an ice bath; immerse the cross-linked polypeptide-resin in the cutting solution, shake at room temperature for 3 hours, filter to remove the resin, and purge with nitrogen to concentrate to 1 / 10 of the original volume; Step S32, reverse phase chromatography purification: using a C18 preparative column, and gradient elution with mobile phase A and mobile phase B; During the gradient elution process, the concentration of mobile phase B was gradually increased from 5% to 60%, which took 30 minutes at a flow rate of 10 mL / min. The wavelength used for UV detection of polypeptides was 220 nm. The main peak effluent with a retention time of 18 to 22 minutes was collected and freeze-dried to obtain a white powder.

6. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 5, characterized in that: In step S31, the cutting liquid is prepared in a volume ratio in resin cutting; In step S32, the particle size of the C18 preparative column in the reverse phase chromatography purification is 5 μm, and the pore size is Mobile phase A is an aqueous solution containing 0.1% TFA, mobile phase B is an acetonitrile solution containing 0.1% TFA, and the time range of the peak of the target polypeptide in the chromatographic column.

7. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 1, characterized in that: The step S4, liposome assembly specifically comprises the following steps: Step S41, lipid film preparation: weigh DOPE:DOTAP:cholesterol with a molar ratio of 5:3:2, dissolve in a mixed solvent of chloroform and methanol; rotary evaporation to form a film, and nitrogen purge for 30 minutes to remove the residual solvent; Step S42, polypeptide loading: the purified polypeptide was dissolved in HEPES buffer, and the final concentration of the polypeptide was 2 mg / mL; the lipid and polypeptide were then mixed at a mass ratio of 10:1, vortexed for 2 minutes, ultrasonically treated, and finally extruded through a 0.22 μm polycarbonate membrane for 3 times to obtain homogeneous liposomes.

8. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 7, characterized in that: In step S41, the volume ratio of the mixed solvent of chloroform and methanol in the lipid film preparation is 9:1; the temperature of the rotary evaporation film is 40° C., and the vacuum degree is -0.09 MPa; In step S42, the concentration of the HEPES buffer in which the polypeptide is loaded is 10 mmol / L, the pH is 7.4, and it contains 150 mmol / L sodium chloride; the frequency of the ultrasonic treatment is 40 kHz, the power is 200 watts, and it lasts for 5 minutes; the particle size range of the liposome is 100±20 nm, and the PDI is <0.

2.

9. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 1, characterized in that: The step S5, freeze-dried preparation molding specifically comprises the following steps: Step S51, pre-lyophilization treatment: adding a lyophilization protective agent to the liposome suspension and equilibrate at 4° C. for 2 hours; Step S52, freeze drying: the liposome suspension is placed in a -80°C environment for quick freezing for 4 hours; then transferred to a freeze dryer, and primary drying is performed for 24 hours at -45°C and 0.1 mbar vacuum conditions; finally, the temperature is raised to 25°C, the vacuum degree is reduced to 0.01 mbar, and secondary drying is continued for 6 hours to finally obtain a loose porous freeze-dried powder.

10. The method for preparing a highly active mitochondrial targeted MAPS polypeptide preparation according to claim 9, characterized in that: In step S51, the concentration of the lyoprotectant in the pre-lyophilization treatment is expressed as a weight volume percentage, and contains 5% trehalose and 1% mannitol.

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