Liposomes embedding cross-linking reagents and their use in in situ analysis of protein complexes in living organisms
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2024-11-28
- Publication Date
- 2026-05-29
Smart Images
Figure CN122109401A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a liposome containing a cross-linking agent and its application in in situ analysis of in vivo protein complexes, belonging to the field of bioanalytical technology. Background Technology
[0002] Liposomes are spherical, vesicle-like supramolecular structures formed by the self-assembly of amphiphilic components (phospholipids and cholesterol) dispersed in an aqueous solution. Liposomes possess a hydrophilic core and a hydrophobic outer bilayer, allowing hydrophilic substances to be encapsulated within the vesicle core while hydrophobic active substances are incorporated into the lipid bilayer. This property not only provides comprehensive advantages for obtaining a variety of effective drug or cosmetic carriers but also improves the bioavailability of active substances, prevents premature degradation, and ensures controlled delivery. Due to their high biocompatibility, significant efforts have been devoted to developing liposome-based drug delivery systems over the past few decades. To date, an increasing number of FDA-approved liposome-based therapies are available, and numerous liposomes are undergoing clinical trials.
[0003] Cancer is one of the leading causes of disease-related deaths worldwide. More than 52,900 people are diagnosed with cancer every day, and more than 27,000 die from it (Signal Transduction and Targeted Therapy, 2024, 175, 9). Protein-protein interactions are fundamental to the formation of protein complexes and are key to cellular activities required to regulate cell vitality and homeostasis. Therefore, abnormalities in protein-protein interactions are associated with a variety of human diseases, including cancer. The occurrence and development of cancer exhibit stages and individual variability; however, the lack of structural information about proteins and complexes present within tumors or other tissues limits the understanding of related disease pathways and the development of new therapies.
[0004] Chemical cross-linking mass spectrometry can covalently bond the proximal amino groups of interacting proteins. The unique ability of chemical cross-linking mass spectrometry (CCSS) to preserve natural interactions in living cells and simultaneously identify protein interaction sites allows for the identification of cross-links that can be used not only to validate and fine-tune protein structures but also to assist in modeling to elucidate the structure of protein complexes. However, most current CCSS experiments are limited to in vitro experiments, primarily due to the high complexity of tissue samples. The analysis of protein structures and interactions at the in vivo level is even more complex, and the high reactivity and easy hydrolysis of chemical cross-linking agents also limit in-situ cross-linking at the tissue level. Furthermore, traditional two-dimensional (2D) cell culture models cannot reproduce the cellular microenvironment, tissue structure, and complex multi-organ interactions of real cells and organs (Trends in Analytical Chemistry, 2024, 180, 117905). Therefore, there is an urgent need to develop methods for in-situ analysis of protein complexes in living tissues at the in vivo level to meet the demand for in-situ structural resolution of protein complexes in vivo.
[0005] In this patent, to address the difficulty of in-situ analysis of protein complexes in living tissues due to the inability of existing chemical cross-linking agents to be delivered in vivo, a method for in-situ analysis of protein complexes in vivo based on liposome cross-linking agent carrier is developed to achieve spatiotemporal dynamic analysis of protein complexes at the living level. Summary of the Invention
[0006] The purpose of this invention is to provide a liposome with an embedded cross-linking agent and its application in in situ analysis of in vivo protein complexes. The method of this invention solves the problem of the difficulty in in situ analysis of in vivo protein complexes.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] A liposome encapsulating a cross-linking agent and its application in in situ analysis of in vivo protein complexes, specifically including the following steps:
[0009] (1) Liposomes with embedded crosslinking agents, wherein the liposomes use phospholipids and cholesterol as the basic membrane material, wherein the phospholipids include natural phospholipids, synthetic phospholipids, synthetic phospholipids-polyethylene glycol, synthetic phospholipids bonded with targeting groups, or a mixture of the above. The targeting groups include one or more of the following: strongly positively charged compound groups, charge-reversed groups, and tissue or organ targeting groups.
[0010] (2) Crosslinking agents include succinimide groups, halogenated aromatic hydrocarbons, and imine esters whose two sides are succinimide groups, 2-mercaptopyridine, thiosulfonates, and halogenated acetyl groups that react with thiol groups on proteins; carbodiimide and isocyanates that react with carboxyl groups on proteins; acylhydrazine groups that react with sugar chains on proteins; or phenyl azide groups, diazidopyridine, and diphenyl ketones that are non-specific reactive groups that react with amino acid residues in proteins.
[0011] (3) The liposome preparation method is the thin film hydration method. The liposome membrane material, cholesterol and crosslinking agent are dissolved in a mixed solution of chloroform and methanol, and the membrane is formed by rotary evaporation at 37°C. Phosphate buffer solution is added for hydration. Then the liposome suspension is sonicated, centrifuged or ultrafiltered to obtain liposomes with embedded crosslinking agent.
[0012] (4) The animals used for the cross-linking agent transporter are normal mice / rats or constructed animal models. The constructed animal model is a primary tumor mouse / rats model, that is, a subcutaneous tumor mouse / rats constructed by injecting tumors into the liver of mice / rats or by injecting tumors into the subcutaneous tissue of mice / rats.
[0013] (5) The embedding cross-linking agent is injected into the animal body or added to the culture medium containing live cells or tissues.
[0014] The injection method is one of tail vein injection, intraperitoneal injection, intratumoral injection, subcutaneous injection, or portal vein injection. The tissue is one or more of the lung, liver, kidney, heart, spleen, and brain. A cross-linking agent is released via an endocytosis carrier, thereby enabling the cross-linking agent to cross-link protein complexes at the cellular and / or sub-organelle levels in situ. The cells are one or more of tumor cells, stem cells, hepatocytes, spleen cells, kidney cells, or tumor model cells. Proteins and their complexes are extracted from the collected cells using an ionic liquid or sodium dodecyl sulfate, followed by denaturation at 65°C-95°C using dithiothreitol. The denatured sample is then transferred to a 10K filter membrane and alkylated in the dark using iodoacetamide or N-ethylmaleimide. The ionic liquid or sodium dodecyl sulfate is repeatedly washed away using ammonium bicarbonate solution. The proteins and their complexes are then digested on the membrane using trypsin at a protein-to-enzyme ratio of 1:25-1:50, and peptides and their cross-linked peptides are collected by centrifugation.
[0015] The obtained peptides were analyzed using high-precision and high-resolution mass spectrometry, including one or more of the following: electrostatic orbital trap, time-of-flight tube, and Fourier transform mass spectrometry.
[0016] The collected mass spectrometry data were analyzed using one or more databases, including MaxQuant, pFind, and pLink.
[0017] This invention provides an application of an in situ analysis method for in vivo protein complexes, which is applied to: large-scale analysis of in vivo protein complexes, in situ target protein complex analysis, spatial structure resolution of proteins, and protein-protein interaction resolution.
[0018] In this method, a liposome carrier encapsulating a cross-linking agent is first designed and injected into a live in vivo model. The liposomes, after circulating through the bloodstream, reach specific tissues and are internalized, releasing the cross-linking agent in situ to achieve in-situ cross-linking of protein complexes. Subsequently, proteins from specific tissues are extracted using protein extraction methods such as ionic liquids, and mass spectrometry is used to analyze the protein complexes at the live in vivo level. This method is of great significance for elucidating protein interactions and dynamics in vivo, understanding disease mechanisms, and screening biomarkers.
[0019] The present invention has the following advantages:
[0020] (1) This invention solves the problem of easy hydrolysis of crosslinking agents by using liposomes as a carrier to encapsulate highly active crosslinking agents.
[0021] (2) This invention utilizes liposomes to deliver cross-linking agents, solving the problems of easy inactivation and lack of targeting of cross-linking agents in blood circulation.
[0022] (3) This invention solves the problem of difficult in situ protein analysis in living organisms. Attached Figure Description
[0023] Figure 1 Figure 1: Simulated cross-linking results of liposomes containing cross-linking agent and BSA Detailed Implementation
[0024] Example 1
[0025] Weigh 20 mg of liposome membrane material according to the following ratio: 1,2-bishexadecyl-sn-glycerol-3-phosphocholine (Xi'an Ruixi): distearate phosphatidylethanolamine-polyethylene glycol:cholesterol = 6.4:0.6:3. Add an equal mass of crosslinking agent bissuccinimide octanoate. Dissolve all raw materials in 10 ml of a 1:2 volume ratio methanol and chloroform mixture, sonicate to dissolve, and then rotary evaporate at 37°C. Add 20 ml of phosphate buffer solution, sonicate to dissolve the membrane, and further sonicate to break down the liposomes using cell sonication to obtain a mixed solution of liposomes and crosslinking agent. Centrifuge the sonicated suspension for 30 min to remove unencapsulated crosslinking agent; the supernatant is the prepared liposome solution.
[0026] At the cellular level, the vector and cells were co-incubated in DMEM medium at 37°C and 5% CO2, allowing the vector to enter the cells. After 6 hours of co-incubation, the cells were collected. Protein extraction was performed by adding 1-dodecyl-3-methylimidazolium chloride ionic liquid (300 μl of ionic liquid treated approximately 3e6 cells) to the collected cell pellet. The protein was then denatured by adding 50 mM dithiothreitol at 95°C for 5 min. The denatured protein sample was transferred to a 10K fasp membrane and subjected to reductive alkylation with 20 mM iodoacetyl. The ionic liquid was repeatedly washed away with ammonium bicarbonate solution. The protein and its complexes were digested on the membrane with trypsin at a protein-to-enzyme ratio of 1:25–1:50 (1:30 in this case). Peptides and their cross-linked peptides were collected by centrifugation. The obtained peptides were analyzed by high-precision, high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information on proteins and their cross-linked proteins. Seventeen cross-linked peptide pairs were identified, five loop-linked peptide pairs were identified, and 127 mono-linked peptide pairs were identified.
[0027] Example 2
[0028] Weigh 20 mg of liposome membrane material according to the following ratio: 1,2-dioleoyl-3-co-choline:distearatephosphatidylethanolamine-polyethylene glycol:choline = 6.4:0.6:3. Add an equal mass of crosslinking agent, bis(succinimide) octanoate. Dissolve all raw materials in 10 ml of a 1:2 methanol and chloroform mixture (v / v), sonicate to dissolve, and then rotary evaporate at 37°C. Add 20 ml of phosphate buffer solution, sonicate to dissolve the membrane, and further sonicate to break down the liposomes using cell sonication to obtain a mixture of liposomes and crosslinking agent. Centrifuge the sonicated suspension for 30 min to remove unencapsulated crosslinking agent; the supernatant is the prepared liposome solution. At the cellular level, co-incubate the carrier and cells in DMEM medium at 37°C and 5% CO2, and collect the cells. Add 300 μl of 1-dodecyl-3-methylimidazolium chloride ionic liquid to the collected cell pellet (approximately 3 eV / L). 6Proteins were extracted from individual cells and denatured at 95°C for 5 min with 50 mM dithiothreitol. The denatured protein samples were then transferred to a 10K fasp membrane and subjected to reductive alkylation with 20 mM iodoacetyl. The ionic liquid was repeatedly washed away with ammonium bicarbonate solution. The proteins and their complexes were digested on the membrane with trypsin at a protein-to-enzyme ratio of 1:25–1:50 (1:30 in this case). Peptides and their cross-linked peptides were collected by centrifugation. The obtained peptides were analyzed by high-precision and high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information on the proteins and their cross-linked proteins. Two cross-linked peptide pairs were identified, four loop-linked peptide pairs were identified, and 83 mono-linked peptide pairs were identified.
[0029] Example 3
[0030] Weigh 20 mg of liposome membrane material according to the following ratio: 1,2-bishexadecyl-sn-glycerol-3-phosphocholine ester: distearate phosphatidylethanolamine-disulfide bond-polyethylene glycol:cholesterol = 6.4:0.6:3. Add an equal mass of crosslinking agent bissuccinimide octanoate. Dissolve all raw materials in 10 ml of a 1:2 volume ratio methanol and chloroform mixture, sonicate to dissolve, and then rotary evaporate at 37°C. Add 20 ml of phosphate buffer solution, sonicate to dissolve the membrane, and further sonicate to disrupt the liposomes using cell sonication to obtain a mixed solution of liposomes and crosslinking agent. Centrifuge the sonicated suspension for 30 min to remove unencapsulated crosslinking agent; the supernatant is the prepared liposome solution.
[0031] At the cellular level, the vector and cells were co-incubated in DMEM medium at 37°C and 5% CO2 for 6 hours, and the cells were collected. 1-Dodecyl-3-methylimidazolium chloride ionic liquid (300 μl of ionic liquid treated approximately 3e6 cells) was added to the collected cell pellet to extract proteins. Dithiothreitol was added to a final concentration of 50 mM for protein denaturation at 95°C for 5 min. The denatured protein sample was transferred to a 10K fasp membrane and iodoacetyl was added to a final concentration of 20 mM for reductive alkylation. The ionic liquid was repeatedly washed away with ammonium bicarbonate solution. Trypsin was used to digest the proteins and their complexes on the membrane at a protein-to-enzyme mass ratio of 1:25–1:50 (1:30 in this case). Peptides and their cross-linked peptides were collected by centrifugation. The obtained peptides were analyzed by high-precision and high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information on the proteins and their cross-linked proteins. Twenty-two cross-linked peptide pairs were identified, ten loop-linked peptide pairs were identified, and 122 mono-linked peptide pairs were identified.
[0032] Example 4
[0033] A 1 mg / ml liposome solution was injected into the tumor of mice with small skin tumors. Six hours later, the mice were humanely sacrificed, and approximately 50 mg of subcutaneous tumor tissue was completely excised and extracted. The tissue was then cut into 1 cm³ pieces. 3 Small fragments were washed with phosphate buffer solution to remove blood, and then treated with 300 μl of 1-dodecyl-3-methylimidazolium chloride ionic liquid (approximately 3e). 6Proteins were extracted from individual cells and denatured at 95°C for 5 min with 50 mM dithiothreitol. The denatured protein samples were then transferred to a 10K fasp membrane and subjected to reductive alkylation with 20 mM iodoacetyl. The ionic liquid was repeatedly washed away with ammonium bicarbonate solution. The proteins and their complexes were digested on the membrane with trypsin at a protein-to-enzyme ratio of 1:25–1:50 (1:30 in this case). Peptides and their cross-linked peptides were collected by centrifugation. The obtained peptides were analyzed by high-precision, high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information about the proteins and their cross-linked proteins. 101 cross-linked peptide pairs were identified, 109 loop-linked peptide pairs were identified, and 1178 mono-linked peptide pairs were identified.
[0034] Example 5
[0035] In vivo, mice with in situ tumors were injected via the tail vein with a 1 mg / ml liposome solution. Six hours later, the mice were humanely euthanized, and approximately 50 mg of subcutaneous tumor tissue was completely excised and extracted. The tissue was then minced into 1 cm³ pieces. 3 Small fragments were washed with phosphate buffer solution to remove blood, and then treated with 300 μl of 1-dodecyl-3-methylimidazolium chloride ionic liquid (approximately 3e). 6 Proteins were extracted from individual cells and denatured at 95°C for 5 min with 50 mM dithiothreitol. The denatured protein samples were then transferred to a 10K fasp membrane and subjected to reductive alkylation with 20 mM iodoacetyl. The ionic liquid was repeatedly washed away with ammonium bicarbonate solution. The proteins and their complexes were digested on the membrane with trypsin at a protein-to-enzyme ratio of 1:25–1:50 (1:30 in this case). Peptides and their cross-linked peptides were collected by centrifugation. The obtained peptides were analyzed by high-precision, high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information on the proteins and their cross-linked proteins.
[0036] Example 6
[0037] In vivo, mice with orthotopic liver tumors were injected via the portal vein with a 1 mg / ml liposome solution. Six hours later, the mice were humanely euthanized, and approximately 50 mg of subcutaneous tumor tissue was completely excised and extracted. The tissue was then minced into 1 cm³ pieces. 3Small fragments were washed with phosphate buffer solution to remove blood, and then treated with 300 μl of 1-dodecyl-3-methylimidazolium chloride ionic liquid (approximately 3e). 6 Proteins were extracted from individual cells and denatured at 95°C for 5 min with 50 mM dithiothreitol. The denatured protein samples were then transferred to a 10K fasp membrane and subjected to reductive alkylation with 20 mM iodoacetyl. The ionic liquid was repeatedly washed away with ammonium bicarbonate solution. The proteins and their complexes were digested on the membrane with trypsin at a protein-to-enzyme ratio of 1:25–1:50 (1:30 in this case). Peptides and their cross-linked peptides were collected by centrifugation. The obtained peptides were analyzed by high-precision, high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information about the proteins and their cross-linked proteins.
Claims
1. A liposome encapsulating a crosslinking agent, characterized in that, The membrane includes a crosslinking agent and a liposome membrane material; the liposome membrane material includes phospholipids and cholesterol; the phospholipid content in the liposome membrane material ranges from 40% to 80%, preferably 60% to 70%, and more preferably 65% to 70% by mass; the crosslinking agent is a compound with reactive groups and crosslinking arms; the mass ratio of the crosslinking agent to the liposome membrane material ranges from 0.1:1 to 1:1, preferably 0.5 to 1:1, and more preferably 0.9 to 1:
1.
2. The liposomes according to claim 1, characterized in that: Phospholipids include one or more of the following: natural phospholipids, synthetic phospholipids, synthetic phospholipids-polyethylene glycol, and synthetic phospholipids bonded with targeting groups.
3. The liposomes according to claim 1, characterized in that: The active group includes one or more of the following groups: The protein contains one or more of the following functional groups: N-hydroxysuccinimide ester, halogenated aromatic hydrocarbons, and imine esters, which react with the amino group of proteins; or one or more of the following functional groups: diazonium, carbonyl imidazole, and carbodiimide, which react with the carboxyl group of proteins; or maleimide, 2-mercaptopyridine, thiosulfonate, and haloacetyl, which react with the thiol group of proteins.
4. A method for preparing liposomes with an embedded crosslinking agent as described in any one of claims 1-3, characterized in that: The preparation method of liposomes is thin film dispersion method. Specifically, phospholipids, cholesterol and cross-linking agent are dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1-3:
1. The solution is rotary evaporated in the temperature range of 37-45℃ to form a film. Phosphate buffer solution with an equal volume (mg mass of liposomes / ml volume of phosphate buffer solution) is added for hydration. The liposome suspension is then sonicated or shaken, centrifuged or filtered to obtain liposomes with embedded cross-linking agent.
5. The application of the liposomes according to any one of claims 1-3 in the in situ analysis of protein complexes in living cells.
6. The application according to claim 5, characterized in that: Liposomes are co-cultured with live cells, and cross-linking agents are released via endocytosis carriers, thereby enabling the cross-linking agents to cross-link protein complexes at the cellular and / or subcellular organelle levels in situ.
7. The application according to claim 6, characterized in that: The liposome-targeting groups include one or more of the following: strongly positively charged compound groups, charge-reversed groups, and tissue or organ-targeting groups; The cells are one or more of the following: tumor cells, stem cells, immune cells, liver cells, spleen cells, and kidney cells.
8. The application according to claim 5 or 6, characterized in that: Proteins and protein complexes were collected from cells. Proteins and their complexes were extracted using ionic liquids or sodium dodecyl sulfate surfactants. The concentration of collected proteins was determined. The proteins were then denatured at 56℃-95℃ using dithiothreitol at a final concentration of 10-100 mM. The denatured protein samples were transferred to a 10K filter membrane and alkylated at room temperature in the dark using iodoacetamide or ethyl maleimide at a final concentration of 20 mM-200 mM. Subsequently, trypsin was added to digest the proteins at a protein-to-trypsin mass ratio of 1:25-1:
50. Peptides were collected by centrifugation.
9. The application according to claim 5 or 6, characterized in that: The analysis process employs liquid chromatography-mass spectrometry (LC-MS), and the mass spectrometry includes one or more of the following: electrostatic orbital trap, time-of-flight tube, and Fourier transform mass spectrometry.
10. The cells according to claim 7 are derived from one or more of the lung, liver, kidney, heart, spleen, and brain. These cells are bound to the liposomes containing the embedding cross-linking agent by injection into the animal body or tissue section via tail vein injection, intraperitoneal injection, intratumoral injection, subcutaneous injection, or portal vein injection.
11. The application according to claim 5 or 6, characterized in that: The protein complex analysis was performed using one or more of the following search databases: MaxQuant, pFind, and pLink.