A nanoliposome-based transport carrier for delivering a crosslinking agent to mitochondria in cells and preparation and use thereof

Abstract

The present application relates to a kind of nanoliposome class transport carrier for delivering crosslinking agent to mitochondrion in cell and its preparation and application.In the method, nanoliposome class transport carrier with intracellular targeting transport function is designed and prepared, which is incubated with cells, endocytosed in cells, reaches mitochondrion, and releases chemical crosslinking agent in situ, captures protein complex information of mitochondrion or other membrane structure layer in situ.Subsequently, protein extraction method based on ionic liquid or sodium dodecyl sulfate (SDS) surfactant protein extraction method is used to extract protein and its complex, and after sample pretreatment, protein complex of mitochondrion or protein in membrane structure of cell membrane and mitochondrion membrane is in situ analyzed on the level of whole cell proteome.This method has great significance for analyzing protein complex of mitochondrion and membrane structure passed through to mitochondrion, tracking mitochondrion involved in cell function, understanding life process, revealing disease mechanism, screening biomarker and finding drug target.

Description

Technical Field

[0001] This invention relates to a nanoliposome-like transporter for delivering cross-linking agents to intracellular mitochondria, its preparation and application, and belongs to the field of bioanalytical technology. Background Technology

[0002] Proteins are the basic building blocks of cells and one of the most important executors of cellular biological functions. Proteins work in conjunction with other proteins or biomolecules to achieve specific biological functions. Therefore, a deep understanding of protein-protein interactions is essential for accurately understanding the underlying molecular biological processes. Furthermore, the cellular native environment, such as pH, subcellular organelles, and buffer salt concentrations, is also necessary for proteins to perform their functions. Therefore, studying the structure of proteins in their native environment or within the body is a crucial approach to fully understanding how proteins perform their functions.

[0003] Currently, numerous methods exist for characterizing protein-protein interactions and protein conformation. Among these, chemical crosslinking mass spectrometry (XL-MS) is an emerging method for identifying covalently crosslinked residues through crosslinking reactions and mass spectrometric analysis. It provides information on the internal and intermolecular crosslinking of spatially compact amino acid residues, primarily related to the linker arms of the crosslinking agent. (For example, through molecular dynamics simulations, the maximum constraint distance between the Cα (alpha carbon of the protein backbone) atoms of lysine residues crosslinked by bis(succinimide) octanoate (DSS) is...) XL-MS methods have been able to capture functionalized protein conformations in complex biological samples, including freshly isolated mitochondria, intact cells, and tissues, thereby enabling protein conformations to cross-link in their native environment. Combined with a bottom-up, peptide-level identification mass spectromics workflow and high-throughput and sensitive mass spectrometry detection, XL-MS studies have been successfully conducted to study mitochondrial protein complexes on a large scale.

[0004] Mitochondria, acting as the cell's fuel station, provide energy. Studying mitochondrial protein complexes and analyzing their spatiotemporal dynamics is crucial for understanding how these complexes function in biological pathways. However, due to limitations such as the lack of targeting and high reactivity of cross-linking agents, chemical cross-linking techniques for analyzing protein interactions in mitochondria still require cross-linking in vitro after mitochondrial separation. The use of non-biocompatible solvents and the organelle separation process can irritate the cell's natural state, making it impossible to obtain information on protein complex structure and interactions under natural cellular conditions. Therefore, there is an urgent need to develop methods for in-situ analysis of mitochondrial protein complexes at the living cell level to meet the demand for in-situ structural resolution of mitochondrial protein complexes.

[0005] Liposomes possess unique and excellent properties: 1) Good biocompatibility: The carrier consists of phospholipid-like bilayers encapsulating aqueous vesicles; 2) Broad adaptability to loaded drugs: Water-soluble drugs can be loaded into the aqueous phase, lipid-soluble drugs can dissolve within the phospholipid membrane, and amphiphilic drugs can be intercalated onto the lipid membrane, achieving simultaneous loading of hydrophilic and hydrophobic drugs; 3) High bioavailability: Phospholipids themselves are cell membrane components, are non-toxic after injection into the body, and do not induce immune responses; 4) Protects the loaded drug, preventing dilution by body fluids and degradation by enzymes in the body. Due to their good biocompatibility, stability, and rapid cellular entry, liposome-based drug carriers have entered clinical applications. Therefore, combining the advantages of liposome drug delivery, they can be applied to solve the problem of in-situ analysis of mitochondrial protein complexes in living cells.

[0006] In this patent, in order to address the difficulties in in-situ analysis of mitochondrial protein complexes in living cells caused by the need for organic solvents to aid dissolution and the inability to target delivery of existing chemical cross-linking agents, a nanoliposome-like transporter is developed to deliver the cross-linking agent to intracellular mitochondria, thereby enabling in-situ analysis of intracellular mitochondrial protein complexes and achieving spatiotemporal dynamic analysis of protein complexes on subcellular organelles. Summary of the Invention

[0007] The purpose of this invention is to provide a nanoliposome-like transporter for delivering cross-linking agents to intracellular mitochondria, as well as its preparation and application. The method of this invention solves the problem of difficult in-situ analysis of protein complexes at the organelle level, including mitochondria, in living cells.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A nanoliposome-like transporter for delivering cross-linking agents to intracellular mitochondria, its preparation and application, specifically including the following steps:

[0010] (1) Prepare mitochondrial-targeting nanoliposomes with cross-linking agent encapsulated. The carrier is synthetic phospholipid as liposome carrier, cholesterol as stabilizer, and functionalized phospholipid as the targeting driving force of liposomes, and the cross-linking agent is encapsulated.

[0011] (2) The characteristics of natural phospholipids and synthetic phospholipids include: natural phospholipids include high-purity egg yolk lecithin PC-98T, egg yolk lecithin, hydrogenated soybean lecithin, and hydrogenated soybean lecithin; synthetic phospholipids include, or PE-type synthetic phospholipids: dimyristoyl phosphatidylethanolamine DMPE, distearyl phosphatidylethanolamine DSPE, dipalmitoyl phosphatidylethanolamine DPPE, dioleoyl phosphatidylethanolamine DOPE, cationic liposome-assisted phospholipids, etc.; or PC-type synthetic phospholipids: dilauryl lecithin DLPC, disqualyl phosphatidylcholine DEPC, dioleoyl lecithin DOPC, dimyristoyl lecithin DMPC, 1-palmitoyl-2-oleoyl lecithin POPC, di... Stearoyl phosphatidylcholine (DSPC), dipalmitoyl lecithin (DPPC); or PS-type synthetic phospholipids: dipalmitoyl phosphatidylserine (DPPS), dioleoyl phosphatidylserine (DOPS); or PG-type synthetic phospholipids: dioleoyl phosphatidylglycerol (DOPG), egg yolk phosphatidylglycerol (EPG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG-Na), 1,2-palmitoyl phosphatidylglycerol (DPPG-NA), distearyl phosphatidylglycerol (DSPG-Na), dimyristoyl phosphatidylglycerol (DMPG-Na); or PA-type synthetic phospholipids: distearyl phosphatidyl acid (DSPA), dipalmitoyl phosphatidyl acid (DPPA); one or more of the above natural or synthetic phospholipids.

[0012] The nanoliposome transporter is modified with mitochondrial-targeting functionalized phospholipids, characterized in that: DSPE-PEG modifies phospholipids targeting mitochondrial groups or DPPE-PEG modifies phospholipids targeting groups or cationic liposomes; the modified mitochondrial-targeting groups include: peptides targeting mitochondria with amino acid residues that have both positive charge and lipophilicity, wherein the amino acid residues providing positive charge are arginine (R) and / or lysine (L), and the amino acid residues providing lipophilicity are phenylalanine (F) and / or cyclohexylalanine (Fx), and the number of amino acid residues in the peptide ranges from 2 to 100. Specifically, it is an SS-31 peptide (D-Arg-Dmt-Lys-Phe-NH2), or a delocalized lipophilic cation, specifically triphenylphosphine (TPP) and dequalinium (DQA), with a hydrophilic charged group; or a strongly positively charged compound, specifically bis(dodecyldimethylammonium bromide) (DDAB) and hexadecyltrimethylammonium bromide (CTAB). The cationic liposome is characterized by: (2,3-dioleopropyl)trimethylammonium chloride (DOTAP), DOP-DEDA, dioleoylpropyltrimethylamine chloride (DOTMA), one of the above functionalized phospholipids or cationic liposomes.

[0013] (3) The encapsulating crosslinking agent is characterized in that: the encapsulating crosslinking agent comprises: succinimide groups, halogenated aromatic hydrocarbons, and imine esters that react with amino groups on the protein; or maleimide groups, 2-mercaptopyridine, thiosulfonate, halogenated acetyl groups, and pyridinium disulfide that react with thiol groups on the protein; or carbodiimide and isocyanate that react with carboxyl groups on the protein; or acylhydrazine groups that react with glycan chains on the protein; or phenyl azide groups and diazidopyridine that are non-specific reactive groups that react with amino acid residues in the protein; or the crosslinking agent comprises any of the above-mentioned reactive groups on both sides.

[0014] (4) The carrier preparation method includes thin film ultrasonication, which is characterized by: dissolving phospholipids, cholesterol, functional phospholipids and crosslinking agent raw materials in a mixture of chloroform and methanol that are immiscible with water to form a raw material dissolved oil phase, and then rotating and evaporating the raw material dissolved oil phase in a round bottom flask to dry it into a film, wherein the ratio of chloroform to methanol is 0-100%, and then adding an aqueous phase in a volume ratio with the raw material mass to the aqueous phase for ultrasonication, wherein the mass-volume ratio of the raw material mass to the aqueous phase is 0.1 mg / ml-100 mg / ml, and then using it directly after ultrasonication or after extruding a membrane with a determined pore size, wherein the membrane pore size ranges from 10-1000 nm.

[0015] (5) The nanoliposome transporter with the modified targeting group of the cross-linking agent is incubated with the cell delivery for 1-48 hours. The cells are then digested with trypsin or collected by scraping off the cells. Alternatively, specific sites within the cells can be collected through further separation.

[0016] When the carrier encapsulating the cross-linking agent is delivered through an animal model, the drug is administered for 1-48 hours. After delivery, mouse tissue is collected, and cells are collected by tissue grinding. Alternatively, specific intracellular sites can be collected through further separation processing.

[0017] The mitochondria and the protein complexes that pass through the cell membrane layer before reaching the mitochondria are one or more of the following: cell membrane proteins, Golgi apparatus proteins, endoplasmic reticulum proteins, intracytoplasmic corpuscle proteins, outer mitochondrial membrane proteins, and inner mitochondrial membrane proteins.

[0018] (6) Add ionic liquid or 0.5-4 wt% SDS to the collected cells and extract proteins and / or their complexes. Then add dithiothreitol or tris(2-carboxyethyl)phosphine and denature at high temperature of 56℃-95℃. Transfer the denatured sample to a filter membrane or precipitate it with acetone and then reconstitute the protein in 5-10M urea. Alkylate the protein on the filter membrane or in the solution using iodoacetamide or N-ethylmaleimide. Digest the protein and / or their complexes on the filter membrane or dilute 5-10M urea with 10-50mM ammonium bicarbonate to 1M using trypsin. Collect the peptides and / or their cross-linked peptides by centrifugation.

[0019] (7) The collected peptides and their cross-linked peptides are analyzed by high-resolution mass spectrometry, including: qualitative and / or quantitative analysis of proteins and their protein complexes using liquid chromatography-mass spectrometry (LC-MS), wherein the mass analyzer used in the LC-MS is one or more of the following mass spectrometers: orbital trap, time-of-flight (TOF), and Fourier transform mass spectrometry (FT-ICR).

[0020] (8) Mass spectrometry data were analyzed using Mascot, pFind, and pLink to obtain information on proteins and their crosslinked proteins. Protein-protein interaction information and protein PDB site matching information were analyzed using Cytoscape and Proxl-MS.

[0021] (9) A nanoliposome-like transporter for delivering a crosslinking agent to intracellular mitochondria, and its preparation and application, including large-scale analysis of intracellular mitochondrial or cell membrane-associated protein complexes, spatial structure determination of proteins, protein-protein interaction determination, and spatiotemporal dynamic analysis of mitochondrial proteins.

[0022] The present invention has the following advantages:

[0023] (1) The present invention solves the problem of the difficulty of in-situ cross-linking of cross-linking agents in living cells.

[0024] (2) This invention solves the problem of the difficulty in realizing the interaction and structural information analysis of in situ subcellular organelle proteins and membrane chimeric proteins by cross-linking agents.

[0025] (3) The present invention solves the problem of targeted delivery of cross-linking agents to mitochondria via liposome-based transporters. Detailed Implementation

[0026] Example 1 (Preparation and application of thin-film ultrasonication of DSS-embedded mitochondrial liposome-based transporter)

[0027] A liposome transporter was prepared using distearate phosphatidylcholine (DSPC), a PC-type phospholipid from natural phospholipids, as the liposome transporter, cholesterol as the stabilizer, and functionalized phospholipid DSPE-PEG-TPP (manufacturer: Xi'an Ruixi) as the targeting driving force for the liposome transporter. The cross-linking agent (DSS, Chinese: bis(succinimide) octanoate) was encapsulated and delivered.

[0028] Eight parts by weight of DSPC-synthesized phospholipids, four parts by weight of cholesterol, four parts by weight of DSPE-PEG-TPP, and two parts by weight of crosslinking agent DSS1 were dissolved in a mixed organic phase of nine parts by weight of chloroform and five parts by weight of methanol. The mixed organic phase was placed in a round-bottom flask and vacuum-dried to form a film. Phosphate buffer was then added as the aqueous phase to achieve a film-to-volume ratio of 1.5 mg / ml. The film was then sonicated in a water bath for 5 min to prepare liposomes. The surface charge and particle size of the prepared crosslinking agent-loaded nanoliposomes are shown in Table 1.

[0029] Table 1: Particle size and surface potential characterization of liposome transporters (DSPC:cholesterol:DSPE-PEG-TPP:DSS) with embedded crosslinking agents.

[0030] DSPC: cholesterol: DSPE-PEG-TPP: DSS 149.6±2.779 16.1±1.71

[0031] Under air conditions of 37°C and 5% CO2 by volume, 10 mg of a liposome nanotransporter (DSPC:cholesterol:DSPE-PEG-TPP:DSS) loaded with the cross-linking agent DSS was subjected to cross-linking with 1*10 7HepG2 cells were co-incubated in DMEM medium to allow liposomes carrying cross-linking agents to enter the cells for targeted delivery of organelles (mitochondria). After 6 hours of incubation with cells, cells were collected, and 1 ml of ionic liquid (1-dodecyl-3-methylimidazolium chloride (C12Im-Cl)) was added to extract proteins. Proteins were then denatured and reduced with 100 mM DTT (dithiothreitol) at 95°C for 5 min, transferred to a FASP membrane, and reduced with IAA (iodoacetamide). Excess ionic liquid was then washed away with 50 mM ABC, and trypsin was added to digest the peptides (protein:enzyme, 50:1) overnight. After desalting, the peptides were dissolved in 0.1% formic acid for mass spectrometry analysis. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the information captured by the collected peptides and their cross-linked peptides. A high-precision, high-resolution electrostatic orbital trap (Oribitrap) was selected for the mass spectrometry analysis. Mass spectrometry analysis was performed using Mascot and pLink to analyze the mass spectrometry data. 89 cross-linked peptide pairs were identified, 76 loop-linked peptide pairs were identified, and 1024 mono-linked peptide pairs were identified.

[0032] Experimental results demonstrate that the nanoliposome-like transporter prepared by thin-film sonication achieves the goal of delivering it to mitochondria at the cellular level for in-situ resolution of mitochondrial protein complex information.

[0033] Example 2 (Preparation and application of thin film of DSS-embedded mitochondrial liposome-based transporter by ultrasound)

[0034] A liposome transporter was prepared using egg yolk lecithin, a natural phospholipid, as the transporter, cholesterol as the stabilizer, and cationic liposome DOTAP (manufacturer: Avitol) as the carrier, encapsulating and delivering a cross-linking agent (DSS). 10 parts by weight of natural phospholipid from egg yolk lecithin, 16 parts by weight of cholesterol, 4 parts by weight of DOTAP, and 20 parts by weight of the cross-linking agent DSS were dissolved in a mixed organic phase of 20 parts by weight of chloroform and 5 parts by weight of methanol (Vchloroform:Vmethanol = 3:1). The mixed organic phase dissolved in chloroform and methanol was placed in a round-bottom flask and vacuum-dried. Then, 15 parts by volume of phosphate buffer was added as the aqueous phase to achieve a mass-to-volume ratio of 1 mg / ml. The mixture was sonicated in water for 3 min, and then liposomes were extruded through a 200 nm pore size membrane to prepare liposomes. The surface charge and particle size of the prepared cross-linking agent-loaded nanoliposomes are shown in Table 2.

[0035] Table 2: Particle size and surface potential characterization of liposome transporters (PE:cholesterol:DOTAP:DSS) with embedded crosslinking agents.

[0036] Egg yolk phosphatidylcholine: cholesterol: DOTAP: DSS 227.3±3.995 31.2±1.44

[0037] Under conditions of 37°C and 5% CO2, 70 mg of the DSS nanoliposome transporter (PE:cholesterol:DOTAP:DSS) loaded with cross-linking agent was mixed with 1e 8 HepG2 cells were co-incubated in DMEM medium to allow liposomes carrying cross-linking agents to enter the cells for targeted delivery of organelles (mitochondria). After 6 hours of liposome-cell incubation, cells were harvested, and proteins were extracted using 4% SDS (sodium dodecyl sulfate). The proteins were then denatured and reduced with TCEP (tris(2-chloroethyl) phosphate) in a 56°C aquatic environment for 1 hour, followed by IAA reduction, and then enzymatically digested with trypsin to obtain peptides. After desalting, the peptides were dissolved in 0.1% formic acid for mass spectrometry analysis. A liquid chromatography-mass spectrometry (LC-MS) system was used to analyze the information captured by the collected peptides and their cross-linked peptides. A high-precision, high-resolution mass spectrometer, the Oribitrap, was selected. Mass spectrometry analysis was performed using Mascot and pLink to analyze the mass spectrometry data. Ten cross-linked peptide pairs were identified, six loop-linked peptide pairs were identified, and 70 mono-linked peptide pairs were identified.

[0038] Experimental results demonstrate that the nanoliposome-like transporter prepared by the thin-film ultrasonic method, after extrusion, can be delivered to mitochondria at the cellular level to resolve mitochondrial protein complex information in situ.

[0039] Example 3 (Preparation and application of thin film of BSPNO-embedded mitochondrial liposome-based transporter by ultrasound)

[0040] A liposome transporter was prepared using distearate phospholipid (DSPA) from the PA-type phospholipids of synthetic phospholipids, cholesterol as a stabilizer, and functionalized phospholipid DPPE-PEG-TPP. The functionalized phospholipid was generated by modifying the DPPE-PEG targeting group triphenylphosphine (TPP) into DPPE-PEG phospholipid. The cross-linking agent BSPNO (full name: bis(succinimide) propargyl nitro cross-linking agent, where the cross-linking arm is 7 carbons long and grafted with the enrichment group biotin) was embedded. Five parts by weight of synthetic phospholipid containing DSA, four parts by weight of cholesterol, one part by weight of functionalized phospholipid DPPE-PEG-TPP, and five parts by weight of cross-linking agent BSPNO were dissolved in a mixed organic phase of nine parts by weight of chloroform and six parts by weight of methanol (Vchloroform:Vmethanol = 3:1). The above-dissolved organic phase in chloroform and methanol was placed in a round-bottom flask and vacuum evaporated. Then, 10 volumes of phosphate buffer were added as the aqueous phase to make the mass-volume ratio 1.5 mg / ml. The liposomes were prepared by sonication in water for 5 minutes and used directly after sonication.

[0041] Specific pathogen-free (SPF) mice were selected, and the mouse model was established as a primary liver cancer tumor mouse model. 100 μl of liposomes (DSPA:cholesterol:DPPE-PEG-TPP:BSPNO) were subcutaneously injected at 2, 4, 9, and 12 hours after administration. Following delivery, mouse tissues (heart, brain, liver, skeletal muscle (legs), tumor sites, lymph nodes, spleen, etc.) were collected. Tissues at each time point were ground and cells were collected. Each collected cell sample underwent sample pretreatment, including extraction of proteins using ionic liquids. After extraction, a click reagent was added to click biotin onto cross-linked peptides. The clicked proteins were precipitated with acetone to remove excess biotin molecules, then reconstituted in 8M urea. The proteins underwent denaturation, reduction, and alkylation, followed by enzymatic digestion to produce peptides. Peptide enrichment was performed, followed by desalting and fractionation. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the collected peptides and their cross-linked peptides. A high-precision, high-resolution electrostatic field orbital trap (Oribitrap) mass spectrometer was selected. Mass spectrometry data were analyzed using Mascot and pLink. One set of cross-linking identification results showed 5 cross-linked peptide pairs, 4 loop-linked peptide pairs, and 12 mono-linked peptide pairs, as shown in Table 3.

[0042] Table 3 shows the identification of protein crosslinking information after in situ crosslinking of liposomes containing crosslinking agents in mice. Plink was used to retrieve crosslinking information (FDR = 1%).

[0043] Identification of Peptides(Pairs)Under 1% FDR Control.

[0044]

[0045] Experimental results demonstrate that the targeted encapsulation of cross-linking agents by liposomes achieves the goal of delivering cross-linking agents to mitochondria at the in vivo level to resolve mitochondrial protein complex information in situ.

Claims

1. A nanoliposome-like transporter, characterized in that: The nanoliposome-like transporter uses phospholipids as liposome carriers and cholesterol as stabilizers. It uses one or more of the following: functional phospholipids or cationic liposomes with mitochondrial targeting as the driving force for mitochondrial targeting. It is encapsulated with a cross-linking agent and constructed by thin film-ultrasound method. The mitochondrial-targeting functional phospholipids are phospholipids with mitochondrial targeting groups modified on DSPE-PEG phospholipids or DPPE-PEG phospholipids. The cationic liposomes include one or more of the following: (2,3-dioleopropyl)trimethylammonium chloride (DOTAP), DOP-DEDA, and dioleopropyltrimethylamine chloride (DOTMA).

2. The nanoliposome-based transport carrier as set forth in claim 1, wherein, The liposome carrier is one or more of natural phospholipids and synthetic phospholipids, including: natural phospholipids such as high-purity egg yolk lecithin PC-98T, egg yolk lecithin, and hydrogenated soybean phospholipids; synthetic phospholipids such as PE-type synthetic phospholipids: dimyristoyl phosphatidylethanolamine DMPE, distearate phosphatidylethanolamine DSPE, dipalmitoyl phosphatidylethanolamine DPPE, dioleoyl phosphatidylethanolamine DOPE, and cationic liposome-assisted phospholipids; or PC-type synthetic phospholipids: dilauroyl lecithin DLPC and disqualyl phosphatidylcholine DEPC. Dioleoyl lecithin (DOPC), dimyristoyl lecithin (DMPC), 1-palmitoyl-2-oleoyl lecithin (POPC), distearyl phosphatidylcholine (DSPC), dipalmitoyl lecithin (DPPC); or PS-type synthetic phospholipids: dipalmitoyl phosphatidylserine (DPPS), dioleoyl phosphatidylserine (DOPS); or PG-type synthetic phospholipids: dioleoyl phosphatidylglycerol (DOPG), egg yolk phosphatidylglycerol (EPG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG-Na), 1,2-palmitoyl phosphatidylglycerol (DPPG-NA), distearyl phosphatidylglycerol (DSPG-Na), dimyristoyl phosphatidylglycerol (DMPG-Na); or PA-type synthetic phospholipids: distearyl phosphatidyl acid (DSPA), dipalmitoyl phosphatidyl acid (DPPA) or one or more of these.

3. The nanoliposome-like transporter as described in claim 1, characterized in that, Preparation of thin film by ultrasonic method: Phospholipids, cholesterol, functional phospholipids, and crosslinking agent raw materials are dissolved in a mixture of chloroform and methanol, which are immiscible with water, to form a raw material dissolved oil phase. The raw material dissolved oil phase is then evaporated by rotary evaporation in a round-bottom flask to form a film. The ratio of chloroform to methanol is in the range of 0-100%. Then, an aqueous phase is added in a volume ratio with the raw material, and ultrasonication is performed. The mass-volume ratio of the raw material to the aqueous phase is in the range of 0.1 mg / ml-100 mg / ml. The film can be used directly after ultrasonication or after extrusion through a membrane with a defined pore size, in the range of 10-1000 nm.

4. The nanoliposome-like transporter as described in claim 1, characterized in that: The modified mitochondrial-targeting groups include: mitochondrial-targeting peptides containing both positively charged and lipophilic amino acid residues, wherein the positively charged amino acid residues are arginine (R) and / or lysine (L), and the lipophilic amino acid residues are phenylalanine (F) and / or cyclohexylalanine (Fx), wherein the number of amino acid residues in the peptide ranges from 2 to 100, and the peptide is SS-31 peptide (D-Arg-Dmt-Lys-Phe-NH2); or delocalized lipophilic cations, wherein the delocalized lipophilic cations are triphenylphosphine (TPP) and / or dequalinium (DQA); or hydrophilic charged groups; or strongly positively charged compounds, wherein the strongly positively charged compounds are one or more of bis(dodecyl dimethyl ammonium bromide) (DDAB) and / or hexadecyl trimethyl ammonium bromide (CTAB).

5. The nanoliposome-based transport carrier as set forth in claim 1, wherein the polyethylene glycol is linked to the surface of the nanoliposome-based transport carrier through a carbodiimide coupling reaction. The encapsulating crosslinking agent includes a crosslinking agent with any of the following reactive groups on both sides: succinimide groups, halogenated aromatic hydrocarbons, or imine esters that react with amino groups on proteins; maleimide groups, 2-mercaptopyridine, thiosulfonates, halogenated acetyl groups, or pyridinium disulfides that react with thiol groups on proteins; carbodiimide or isocyanates that react with carboxyl groups on proteins; acylhydrazine groups that react with glycan chains on proteins; or phenyl azide groups or biacrylic acid groups that are non-specific reactive groups that react with amino acid residues in proteins.

6. The nanoliposome-based transport carrier as claimed in claim 5, wherein: The crosslinking agent has crosslinking arms that connect the reactive groups on both sides. These arms have a carbon number range of 1-100 hydrocarbon chains. The arm length of the crosslinking agent provides a distance constraint, enabling crosslinking of protein amino acid sites within the distance range covered by the crosslinking arm. The crosslinking arm is grafted with enrichment groups, such as biotin. The crosslinking arm contains cleavable sites, and under certain conditions, the two ends of the crosslinking arm break apart. These certain conditions are cleavable sites under mass spectrometry conditions.

7. The nanoliposome-type transporter according to any one of claims 1-6, characterized in that: The mass ratio of one or more of the following—liposome carrier, stabilizer, functional phospholipid or cationic liposome—and crosslinking agent is 1-100:1-50:1:1-200.

8. The nanoliposome-like transporter as described in claim 7, characterized in that: The mass ratio of one or more of the following components—liposome carrier, stabilizer, functional phospholipid or cationic liposome—and crosslinking agent is 2:1:1:

3.

9. The nanoliposome-like transporter as described in claim 8, characterized in that: The mass ratio of one or more of the following components—liposome carrier, stabilizer, functional phospholipid or cationic liposome—and crosslinking agent is 5:4:1:

5.

10. A method for preparing the nanoliposome-based transport carrier according to any one of claims 1 to 9, characterized by: One or more of the following raw materials—liposome carrier, cholesterol, functional phospholipids, or cationic liposomes—and a crosslinking agent are dissolved in a mixture of chloroform and methanol to form a raw material dissolved oil phase. The raw material dissolved oil phase is then rotary evaporated and dried to form a membrane, wherein the mass ratio of the membrane to the oil phase ranges from 1 to 100:1, and the mass concentration of chloroform in the mixture ranges from 1 to 99%. Subsequently, an aqueous phase is added and ultrasonically sonicated, wherein the mass-volume ratio of the membrane to the aqueous phase ranges from 0.1 mg / ml to 100 mg / ml. The membrane can be used directly after ultrasonication or after membrane extrusion, wherein the membrane pore size ranges from 10 to 1000 nm.

11. The method for preparing the nanoliposome-like transporter as described in claim 10, characterized in that: The mass ratio of the membrane to the oil phase is 2:

1. The raw material is then dissolved in the oil phase and dried by rotary evaporation to form a membrane. The mass concentration of chloroform in the mixture is 66%.

12. The use of the nanoliposome-based transport carrier according to any one of claims 1 to 9, characterized in that: Nanoliposome-based transporters can be used to deliver cross-linking agents to mitochondria targeted by intracellular targeting groups, or to cell membrane proteins along the pathway.

13. The application as described in claim 12, characterized in that: In solution, nanoliposomes containing cross-linking agents are co-incubated with cells or injected into animal models. Following endocytosis, the nanoliposomes, through the action of targeting groups, deliver the cross-linking agent to the mitochondria or the cell membrane via the pathway within the cells or animal models, and release the cross-linking agent. This allows the cross-linking agent to cross-link mitochondria and / or other protein complexes at the cell membrane level via pathways other than mitochondria in situ. The cells are one or both of tumor cells and normal cells; The mitochondria and the protein complexes that pass through the cell membrane layer before reaching the mitochondria are one or more of the following: cell membrane proteins, Golgi apparatus proteins, endoplasmic reticulum proteins, intracytoplasmic corpuscle proteins, outer mitochondrial membrane proteins, and inner mitochondrial membrane proteins.

14. Use according to claim 13, wherein: The cells are immune cells or tumor model cells.

15. The use of claim 13, wherein: The cells in question are stem cells.

16. An application of the nanoliposome-like transporter according to any one of claims 1-9, which can be used for in-situ analysis of proteins and / or intracellular protein complexes; specifically: 1) The vector and cells are co-incubated in cell culture medium for 1-12 hours, and the cells are collected; or the vector is injected into an animal model and the tissue is collected and cells are extracted 1-48 hours after delivery. 2) The collected cells are used to extract proteins and / or protein complexes from the cells by means of ionic liquid or by means of 0.5-4% SDS. The extracted proteins and / or protein complexes are pretreated by means of denaturation, membrane reduction or direct reduction or alkylation. 3) In-situ analysis of cross-linked proteins and / or intracellular protein complexes delivered intracellularly by nanoliposome-based transporters using high-resolution mass spectrometry.

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