CN115260304A - Lipid-based protein degradation tool, application and preparation method thereof - Google Patents

Abstract

The invention provides a lipid-based protein degradation tool, application and a preparation method thereof. Wherein the lipid-based protein degradation means comprises: a POI recognition group, and a lipid hybrid attached to the POI recognition group. The POI recognition groups are modified on the surface of the lipid hybrid substance, and the POI recognition groups are exposed outside the lipid hybrid substance after being assembled, so that the degradation of target protein based on the lipid hybrid substance is realized, the synthesis difficulty for constructing a protein degradation tool is greatly reduced, and mass compounds, polypeptides, antibodies, nucleic acid aptamers and the like with binding force with the target protein can be upgraded into protein degradation drugs through a plug-and-play mode, so that new functions are exerted in the related fields of traditional liposomes and LNP, and the development of combined therapy is realized in scientific research and industrial application.

Description

Lipid-based protein degradation tool, application and preparation method thereof

Technical Field

The invention belongs to the technical field of targeted drugs, and particularly relates to a lipid-based protein degradation tool, application and a preparation method thereof.

Background

A targeted Protein degradation Tool (TPD) is a tool that specifically hijacks proteins of interest (POI) to an intracellular Protein recycling site to achieve targeted Protein degradation. TPD has become a powerful tool for biomedical research and the pharmaceutical industry.

Compared with the traditional enzyme inhibition/antagonism small molecule drugs, the proteolytic targeting chimera (PROTAC) as a representative member of the most representative first generation TPD can target and degrade the target spots which are traditionally difficult to form drugs, but the target spots are only limited by intracellular proteins. Extracellular and membrane proteins play an important role in the development of disease, with approximately 40% of the total protein encoded by a gene being non-intracellular. To extend the range of target protein degradation beyond the cytoplasm, previous research groups developed lysosome-targeting chimeras (LYTAC) linked through a cationic phosphooligomannose tail to antibodies/polypeptides that were endocytosed into the lysosome for degradation via the mannose-6-phosphate receptor. Subsequently, the group utilizes the characteristic that hepatic cells are rich in asialoglycoprotein receptor (ASGPR) to replace the lyTAC phosphomannose with a trigalactose structure, and further upgrades the lyTAC into liver-specific lyTAC, and although the LYTAC method has application potential, the synthetic method is complex. LYTAC is receptor dependent, requires special design on the LYTAC structure to help protein complexes enter cells, and also targets different cells, requires tail replacement, and is therefore tedious and difficult to design and synthesize.

The successful development of TPD, currently recognized, requires the design of (1) appropriate target protein recognition groups, (2) receptor-ligand matched pairs to hijack protein entry and intracellular trafficking, (3) initiation of appropriate protein degradation mechanisms, (4) design of targeting and biological barrier penetration specific to cell type from a pharmaceutical perspective. However, the mainstream TPD tools, including PROTAC, LYTAC, and the like, require laborious case design in developing tools for any new POI, where a de novo synthesis of compounds and extensive screening procedures are not rare for different diseases and cell types.

In conclusion, the existing targeted protein degradation tool has the defects of complex design and synthesis method, need of independent design aiming at each case of disease and cell type, poor flexibility, inconvenience in modification, inconvenience in-vivo targeted delivery, no crossing capability of biological barriers such as blood brain barrier and the like, and brings great inconvenience to a targeted treatment mode aiming at a patient.

Disclosure of Invention

To solve the above problems, the present invention provides a lipid-based protein degradation tool comprising:

a POI recognition group, and a lipid hybrid attached to the POI recognition group;

wherein the POI recognition group comprises an antibody, protein, polypeptide, aptamer or small molecule capable of specifically binding to the POI;

the lipid hybrid substance comprises liposome, exosome, cell membrane and LNP.

Preferably, when the POI recognition group is coupled to the lipid hybrid, the lipid-based protein degradation tool is a nanoparticle for protein degradation composed of the lipid hybrid in the core with the POI recognition group at the periphery.

Preferably, the lipid-based protein degradation means further comprises the lipid-based protein degradation means provided with a linking member between the POI recognition group and the lipid hybrid;

preferably, the linking member has a molecular weight of 0-1000kDa;

the connecting component is one of a polymer connecting arm and a lipid connecting arm;

preferably, the polymer linker arms comprise a hydrophilic polymer, a hydrophobic polymer, and an amphiphilic polymer;

preferably, the lipid linker arm is an amphiphilic lipid linker arm.

Preferably, when the POI recognition group is coupled to the lipid hybrid substance through the linking member, the POI recognition group and the linking member constitute a set of linking units; the lipid-based protein degradation tool is a lipid-based protein degradation tool having a multi-layered structure in which the lipid-hybridized substance is provided at the core, the plurality of sets of the linking members to which the lipid-hybridized substance is linked are provided at the intermediate layer, and the plurality of sets of linking units to which the POI recognition groups linked to the linking members are provided at the periphery.

Preferably, the lipid linking arm at least comprises two ends, one end is a lipophilic end which can be linked with the lipid hybrid substance, and the other end is a hydrophilic end;

preferably, the lipophilic terminus is a lipid molecule.

Preferably, the amphiphilic polymer is a polymer of a chain/branched molecular structure, which has at least one molecular end having hydrophilicity and one molecular end having hydrophobicity;

preferably, the amphiphilic polymer has a linear molecular structure, one end of which is a hydrophilic molecular end and the other end of which is a hydrophobic molecular end.

Preferably, further comprising a nanoparticle, wherein the nanoparticle is coated by the lipid hybrid substance at the core of the lipid-based protein degradation means;

the nanoparticles comprise hydrophilic particles, hydrophobic particles and inorganic nanoparticles;

preferably, the nanoparticles have a particle size of 5-1000nm.

In addition, in order to solve the above problems, the present invention also provides a lipid-based protein degradation tool as described above, and its use in the preparation of a medicament and a delivery system for treating and preventing diseases caused by abnormal accumulation of harmful proteins; wherein the diseases caused by abnormal accumulation of harmful proteins comprise tumors, immune system diseases, neurodegenerative diseases, blood system diseases and metabolic diseases.

In addition, in order to solve the above problems, the present invention also provides a lipid-based protein degradation tool as described above, and its application in the preparation of a detection product/kit for diseases caused by abnormal accumulation of harmful proteins; wherein the diseases caused by abnormal accumulation of harmful proteins comprise tumors, immune system diseases, neurodegenerative diseases, blood system diseases and metabolic diseases.

In addition, in order to solve the above problems, the present invention also provides a method for preparing the lipid-based protein degradation tool, wherein when the lipid-based protein degradation tool is a protein degradation tool in which the POI recognition group is coupled to the lipid hybrid, the method comprises: non-covalently bonding the POI recognition group to the lipid hybrid substance; alternatively, the POI recognition group is covalently bonded to the lipid hybrid based on a coupling group, thereby constituting the lipid-based protein degradation means.

Preferably, the lipid-based proteolytic means further comprises a proteolytic means in which the POI recognition group is linked to the lipid hybrid via a linking member;

wherein, when the lipid-based protein degradation tool is a protein degradation tool in which the POI recognition group is connected with the lipid hybrid through the connecting member, the preparation method comprises the following steps:

firstly, coupling the POI recognition group with the connecting member to form a coupling intermediate; then linking the coupling intermediate to the lipid hybrid substance to form the lipid-based protein degradation means;

or, constructing a nanocomposite structure with the connecting member as an outer layer and the lipid hybrid substance as a core; and coupling the POI recognition group with the nano-composite structure to form the lipid-based protein degradation tool.

The invention provides a lipid-based protein degradation tool, application and a preparation method thereof, wherein the lipid-based protein degradation tool comprises: a POI recognition group, and a lipid hybrid substance linked to the POI recognition group; wherein the POI recognition group comprises an antibody, protein, polypeptide, aptamer or small molecule capable of specifically binding to the POI; the lipid hybrid includes liposomes, exosomes, cell membranes and LNPs.

According to the invention, the POI recognition group is modified on the surface of the lipid hybrid substance, and the POI recognition group is exposed outside the lipid hybrid substance after being assembled, so that the degradation of the target protein based on the lipid hybrid substance is realized, the synthesis difficulty of constructing a protein degradation tool is greatly reduced, and through a plug-and-play mode, a large amount of compounds, polypeptides, antibodies, nucleic acid aptamers and the like which have binding force with the target protein can be upgraded into protein degradation drugs, so that new functions are exerted in the related fields of traditional liposomes (Liposome) and Lipid Nanoparticles (LNP), such as mRNA vaccines, nucleic acid delivery carriers and drug delivery, and the development of combined therapy in scientific research and industrial application is realized.

In addition, the application of ligand-targeted lipid nanoparticles (lipid hybrid substances) as protein degradation tools and the exploration of the mechanism thereof are still blank, the invention greatly expands the application range of the current lipid nanoparticles, provides basic knowledge for the TPD and nano delivery fields, and can degrade various human disease-related extracellular/membrane-related/intracellular proteins in vivo in principle.

Drawings

FIG. 1 is a schematic diagram of the structure of the POI recognition group of the present invention coupled to a lipid hybrid;

FIG. 2 is a schematic diagram of the structure of the present invention in which a POI recognition group containing a linking member is coupled to a lipid hybrid;

FIG. 3 is a schematic diagram of the structure of the present invention containing the POI recognition group of the linking member-lipid linking arm linked to the lipid hybrid substance;

FIG. 4 is a schematic diagram of the synthetic structure of NTZ-PEGlipo in example 1 of the present invention;

FIG. 5 shows the results of protein electrophoresis of the effect of NTZ-PEGlipo on the degradation of EGFR, a target protein, in M231 cells, in example 1 of the present invention;

FIG. 6 is a schematic view showing the synthesis of NTZ-lipo1, NTZ-lipo2, INE-lipo, palb-lipo, AV45-lipo in example 2 of the present invention;

FIG. 7 shows the results of protein electrophoresis of the effect of NTZ-lipo1 of different concentrations on the degradation of EGFR in M231 cells in example 2 of the present invention;

FIG. 8 shows the results of protein electrophoresis of the effect of NTZ-lipo1 of different lipid hybrid composition ratios on the degradation of the target protein EGFR in M231 cells in example 2 of the present invention;

FIG. 9 shows the results of protein electrophoresis of the effect of NTZ-lipo2 on the degradation of EGFR, a target protein, in M231 cells, according to example 2 of the present invention;

FIG. 10 shows the results of protein electrophoresis showing the effect of INE-lipo on the degradation of the target protein HER2 in M231 cells in example 2 of the present invention;

FIG. 11 shows the NMR results of the coupling of the lipid linker arm used in Palb-lipo with the POI recognition group in example 2 of the present invention;

FIG. 12 shows the NMR results of hydrogen spectra of the lipid linker arm used in AV45-lipo of example 2 coupled with the recognition group of POI;

FIG. 13 shows the results of protein electrophoresis of the effect of varying concentrations of Palb-lipo on the degradation of the target protein CDK4 in M231 cells in example 2 of the present invention;

FIG. 14 is a schematic view of the structure of AV45-lipo in embodiment 2 of the present invention;

FIG. 15 is a graph showing the results of confocal microscopy for detecting autofluorescence of AV45-lipo and beta-amyloid (A beta) oligomers labeled with FITC (fluorescent dye) for cells in a human glial cell culture medium after incubation of AV45-lipo with beta-amyloid (A beta) oligomers in example 2 of the present invention followed by PBS washing;

fig. 16 is a schematic structural diagram of NTZ-LNP1, NTZ-LNP1s, and NTZ-LNP2 in embodiment 3 of the present invention;

FIG. 17 is a graph showing the particle size distribution of the NTZ-LNP1, NTZ-LNP1s, and NTZ-LNP2 Dynamic Light Scattering (DLS) in example 3 of the present invention;

FIG. 18 shows the results of protein electrophoresis of the effect of NTZ-LNP1 in example 3 and NTZ-lipo1 in example 2 on the degradation of EGFR, a target protein, in M231 cells;

FIG. 19 shows the results of protein electrophoresis of the effect of NTZ-LNP1s on the degradation of the target protein EGFR in M231 cells in example 3 of the present invention;

FIG. 20 shows the results of protein electrophoresis of the effect of NTZ-LNP2 on the degradation of the target protein EGFR in M231 cells in example 3 of the present invention;

FIG. 21 is a graph of the DLS particle size distribution and the average dispersion coefficient PDI of NTZ-exo in example 4 of the present invention;

FIG. 22 shows the results of protein electrophoresis of the effect of NTZ-exo on the degradation of EGFR, a target protein, in M231 cells, in example 4 of the present invention;

FIG. 23 is a schematic view of the structure of NTZ-lipoP in example 5 of the present invention;

FIG. 24 shows the results of protein electrophoresis of the effect of NTZ-lipoP of different concentrations on the degradation of the target protein EGFR in M231 cells in example 5 of the present invention;

FIG. 25 is a schematic structural diagram of a CTX-RBCmD according to embodiment 5 of the present invention;

FIG. 26 is a graph showing the DLS particle size distribution results of CTX-RBCmD in example 5 of the present invention;

FIG. 27 shows the results of protein electrophoresis of the effect of CTX-RBCmD on the degradation of EGFR, a target protein, in M231 cells, according to example 5 of the present invention.

Reference numerals:

100, a lipid-based protein degradation means; 1,poi recognition group; 2, liposomes, exosomes or cell membranes in lipid hybrids; LNP in lipid hybrids; 4, a lipid linker arm; 5, a polymer linker arm; 6, a coupling group; 7, nano particles.

The implementation, functional features and advantages of the present invention will be further described with reference to the accompanying drawings.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of 823030a" is considered a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The term "about" in the present invention denotes an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following is provided merely to aid in understanding the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

The technical solution of the present invention is further described in detail by referring to the specific embodiments, but the present invention is not limited thereto, and any limited number of modifications made by anyone within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

Referring to fig. 1, the present example provides a lipid-based protein degradation tool comprising:

a POI recognition group, and a lipid hybrid substance linked to the POI recognition group;

wherein the POI recognition group comprises an antibody, protein, polypeptide, aptamer or small molecule capable of specifically binding to the POI;

the lipid hybrid includes liposomes, exosomes, cell membranes and LNPs.

The POI recognition group is an antibody, protein, polypeptide or small molecule capable of specifically binding to POI.

The lipid hybrid substance includes Liposome (Liposome), exosome, cell membrane and LNP. Wherein LNP is lipid Nanoparticle (lipid Nanoparticle).

It should be noted that lipid nanoparticles are mainly used for in vivo drug delivery, and have been widely used in medical treatment in the fields of small molecule drug delivery, nucleic acid drug delivery, nano-vaccines and the like, from traditional liposomes (liposomes) to Lipid Nanoparticles (LNPs). The mRNA vaccine of COVID-19 mostly consists of LNP.

Lipid nanoparticles have the advantages of low cost and convenient preparation, the composition rule of the lipid nanoparticles is relatively clear, and the lipid nanoparticles for medical treatment and drug development have a relatively regular composition, and are mainly membrane skeletons and cholesterol auxiliary materials. The lipid nanoparticles for clinical use can be added with other components according to needs, and can include but are not limited to the following components:

1. the membrane skeleton, which constitutes the main component of lipid bilayer membrane.

2. Auxiliary materials: (1) Cholesterol (cholestrol) regulates membrane fluidity and improves particle stability, and (2) auxiliary phospholipid maintains liposome microscopic morphology to make lysosome membrane unstable.

3. The PEG lipid can reduce the combination of the particles and plasma protein in vivo and prolong the systemic circulation time.

4. Cationic lipids, highly efficient entrapment of nucleic acid drugs, positive charge, in vivo transfection, pH sensitivity (ionizable).

5. The stabilizer has the freeze-drying protection effect and maintains the stable structure of the liposome in the freeze-drying process.

The lipid hybrid substance comprises liposome, exosome, cell membrane, LNP, LPP and lipid nanoemulsion. Among them, liposomes (lipopome), also known as classical liposomes, are vacuolated and are mainly used to carry hydrophobic drugs. As the liposome is used as an amphiphilic membrane, hydrophobic drugs can be loaded in the hydrophobic vacuoles, and hydrophilic drugs can be loaded in the hydrophilic vacuoles. The LNP is a lipid nanoparticle, the interior of the LNP contains cationic lipid, the existing mRNA vaccine and some nucleic acid vaccines are the LNP used for the existing mRNA vaccine, the nucleic acid is negatively charged, the cationic lipid is positively charged, the entrapment efficiency can be effectively improved, and the LNP is also used for delivering CRISPR gene editing elements. The traditional liposome lipo has low efficiency of carrying nucleic acid, and generally carries hydrophobic and hydrophilic micromolecular drugs. The LPP has the characteristics of lipid and polymer due to the inclusion of the polymer core, and has better drug-loading compatibility and stability.

In addition, LNP and LPP have the advantage of being easily modified, liposomes, LNP are easily modified by amphiphilic pegylated lipids and similar structures, which can be used to bind to receptors via PEG-terminally linked ligands (ligands), possibly facilitating drug delivery to target organs, also known as actively targeted lipid nanocarriers.

The lipid hybrid substance is convenient for carrying drugs, and comprises hydrophilic and hydrophobic micromolecular drugs, nucleic acid drugs, protein drugs, gene editing vectors and other biotechnology drugs which can be effectively loaded and delivered. However, the application of the drug delivery system still focuses on drug delivery, the physiological and pathological properties of the structure of the drug delivery system still need to be further explored, and more therapeutic potential and possibility need to be explored.

In summary, in the embodiment, the POI identifying group is modified on the surface of the lipid hybrid substance, and the POI identifying group is exposed outside the lipid hybrid substance after being assembled, so that the degradation of the target protein based on the lipid hybrid substance is realized, the synthesis difficulty of constructing a protein degradation tool is greatly reduced, and a large amount of compounds, polypeptides, antibodies, aptamers and the like having binding force with the target protein can be upgraded into protein degradation drugs through a plug and play mode, and further, new functions are exerted in the related fields of traditional liposomes (Liposome) and Lipid Nanoparticles (LNP), such as mRNA vaccines, nucleic acid delivery vectors and drug delivery, so that the development of combination therapy in scientific research and industrial applications is realized.

In addition, the application of ligand-targeted lipid nanoparticles (lipid hybrid substances) as protein degradation tools and the exploration of the mechanism thereof are still blank, the invention greatly expands the application range of the current lipid nanoparticles, provides basic knowledge for the TPD and nano delivery fields, and can degrade various human disease-related extracellular/membrane-related/intracellular proteins in vivo in principle.

Further, when the POI recognition group is coupled to the lipid hybrid, the lipid-based protein degradation tool is a nanoparticle for protein degradation composed of the lipid hybrid in the core and the POI recognition group in the periphery.

The above shows the structure of the lipid-based protein degradation tool, wherein the whole body can be a granular structure, including a core part and a periphery, the core part can be a lipid hybrid substance, and the periphery is a plurality of POI recognition groups connected with the lipid hybrid substance, i.e. a layer of POI recognition groups connected with the lipid hybrid substance is arranged on the outer surface of the lipid hybrid substance, thereby forming the whole lipid-based protein degradation tool.

Furthermore, depending on the form of the lipid hybrid, in principle, if it is a classical liposome, its form is vacuolated.

Further, referring to fig. 2, the lipid-based protein degradation means may further include a lipid-based protein degradation means having a connection member between the POI recognition group and the lipid hybrid.

Preferably, the linking member has a molecular weight of 0-1000kDa;

the connecting component is one of a polymer connecting arm and a lipid connecting arm; wherein, the structure of the lipid-based protein degradation tool containing the lipid linker arm is shown with reference to fig. 3.

Preferably, the polymer linker arms comprise hydrophilic polymers, hydrophobic polymers and amphiphilic polymers.

Preferably, the lipid linker arm is an amphiphilic lipid linker arm.

In the above, another lipid-based protein degradation tool is provided, similar to the basic structure, with a linking member between the lipid hybrid and the POI recognition group. The sequential connection combination of the lipid hybrid substance, the connecting component and the POI recognition group is formed, wherein the connecting component is provided with a coupling group which can be connected with the POI recognition group, thereby realizing the combination of the two.

Further, when the POI recognition group is coupled to the lipid hybrid through the linking member, the POI recognition group and the linking member constitute a group of linking units; the lipid-based protein degradation tool is a lipid-based protein degradation tool having a multi-layered structure in which the lipid-hybridized substance is provided at the core, the plurality of sets of the linking members to which the lipid-hybridized substance is linked are provided at the intermediate layer, and the plurality of sets of linking units to which the POI recognition groups linked to the linking members are provided at the periphery.

The above shows that the lipid-based protein degradation tool has a spatial structure, the core of the lipid-based protein degradation tool is a lipid hybrid material (core), the surface layer of the lipid-based protein degradation tool is connected with a plurality of connecting members (middle layers), and each connecting member is connected with a lipid hybrid material (periphery), so that an inner layer and an outer layer of granular structures are formed, and the whole lipid-based protein degradation tool can be spherical in theory or in other shapes.

Further, the molecular weight of the linking member is 0-1000kDa.

The linking member is one of a polymer linking arm and a lipid linking arm.

Preferably, the polymer linker arms comprise hydrophilic polymers, hydrophobic polymers, and amphiphilic polymers.

TABLE 1 three structural forms of Polymer linker arms

Preferably, the lipid linker arm is an amphiphilic lipid linker arm.

Above, the characteristics of the connecting member are defined, which are divided into two broad categories, including: the polymer connecting arm and the lipid connecting arm can be any one according to the synthesis requirement and the water solubility requirement of a substance, and the lipid connecting arm is amphiphilic.

The hydrophilic polymer, as described above, may include, but is not limited to: polyethylene glycol (PEG), polyethylene oxide (PEO), poly (ethylene glycol) methacrylate (POEG), poly (2-methacryloyloxyethyl phosphoryl) (PMPC), polycarboxylic betaine (PCB), dextran, hyaluronic acid, chitosan, beta-cyclodextrin, hyperbranched polyglycidyl ether (HPG), poly N- (2-hydroxypropyl) methacrylamide (PHPMA), polyhydroxyethylmethacrylate (PHEMA), polyacrylamide (PAM), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polymaleic anhydride (HPMA), polyquaternary ammonium salts and pharmaceutically acceptable polymer salts thereof, polyethyleneimine (PEI), N-dimethylaminoethyl methacrylate (PDMAEMA), polylysine (PLL), polyglutamic acid (PGu), polyaspartic acid (PASp) and other hydrophilic polyaminoacids, and derivatives of the above polymers and pharmaceutically acceptable salts thereof.

The hydrophobic polymer may include: polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polycarbonate (PMC) and derivatives thereof, glycolide/lactide/caprolactone/carbonate various combinations and copolymers of components, polyurethane (PU), polyether ether ketone (PEEK), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyethylene (PE), poly (phenylalanine) and other various hydrophobic polyamino acids, derivatives of the polymers and pharmaceutically acceptable salts thereof.

Amphiphilic polymers include: PEG-PLGA, PEG-PCL, PEG-PLA, PEG-PMC, and various amphiphilic block polymers and derivatives of the combination of the hydrophilic polymer and the hydrophobic polymer, and various amphiphilic polymers and derivatives of the combination of the hydrophilic polymer and the hydrophobic molecule (such as lipid).

Furthermore, the lipid connecting arm at least comprises two ends, one end of the lipid connecting arm is a lipophilic end which can be connected with the lipid hybrid substance, and the other end of the lipid connecting arm is a hydrophilic end;

preferably, the lipophilic terminus is a lipid molecule.

When the linking member is a lipid linking arm, it may structurally comprise a multi-terminal structure, but at least two terminals, a lipophilic terminal (linked to the lipid hybrid) and a hydrophilic terminal, and the lipophilic terminal is a lipid molecule.

The lipid molecules include: fatty acids (fatty acids), glycerolipids (glycerolipids), glycerophospholipids (glycerophospholipids), sphingolipids (sphingolipids), sterol lipids (sterol lipids), pregnenolone lipids (prenol lipids), glycolipids (saccharolipids), polyvinyls (polyketides), cationic lipids (cationic lipids) and ionizable lipids (ionizable lipids).

Further, the lipid molecule may be DSPE (distearoylphosphatidylethanolamine), distearoylphosphatidylcholine (DSPC), 1,2-Dimyristoyl-SN-GLYCEROL (DMG, 1, 2-Dimyristoyl-SN-GLYCEROL), 1,2-DIPALMITOYL-SN-GLYCEROL (DPG, 1, 2-DIPALMITOYL-SN-GLYCEROL), 1,2-DIPALMITOYL-SN-GLYCEROL (DPyG, 1, 2-diphytoyl-SN-GLYCEROL), diacylglycerol (DAG); triglycerides (TAG), 1, 2-Dipalmitin (DPG), 1' - [ (1R) -1- (hydroxymethyl) -1, 2-ethanediyl]Octadecanoic acid ester (D)SG), dianhydroxanoyl phosphatidylcholine (DAPC), 1-palmitoyl-2-lauroyl-sn-glycero-3-phosphocholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1, 2-dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), 1, 2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI); ceramide (Cer), sphingomyelin (SM), cholesterol (Cho), cholesteryl Ester (CE), 1, 2-dimyristoyl-SN-glycero-3-phosphate (DMPA), dilauroylphosphatidic acid (DLPA), phosphocholine (Phosphocholine), trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP), ionizable lipids including DLin-MC3-DMA, A6, OF-02, A18-ISO5-2DC18, 98_N12-5、9A1P9、C12-200、cKK-E12、7c1、G0-C14、L319、304O₁₃、OF-Deg-Lin、306-OB12、306-O_i10And FTT5, and derivatives of the foregoing lipid molecules and pharmaceutically acceptable salts thereof. The PEG lipid can be PEG and conjugates of PEG derivatives and the aforementioned lipids.

Preferably, the lipid molecule may be a pegylated lipid, wherein PEG has a relative molecular weight of 2000 and the pegylated lipid may comprise: PEG-DSPE (polyethylene glycol-distearoylphosphatidylethanolamine), PEG-DMG (polyethylene glycol-dimyristoyl glyceride), conjugates of the aforementioned lipid molecules and PEG, and pharmaceutically acceptable salts thereof. The PEG comprises PEG and PEG with methoxy or other groups at the tail end of the PEG.

Further, the amphiphilic polymer is a polymer with a chain/branch molecular structure, and at least one molecular end with hydrophilicity and one molecular end with hydrophobicity exist in the polymer;

the structure of the amphiphilic polymer is defined as a chain/branched molecular structure as described above, and may have a plurality of branches and a plurality of chains because of the characteristics of the chain/branched molecular structure, but it has at least two ends, one being a molecular end having hydrophilicity and one being a molecular end having hydrophobicity.

Preferably, the amphiphilic polymer has a linear molecular structure, one end of which is a hydrophilic molecular end and the other end of which is a hydrophobic molecular end.

The amphiphilic polymer is further defined as having a linear molecular structure.

Preferably, the lipid-based protein degradation tool further comprises a nanoparticle, wherein the nanoparticle is coated at the core of the lipid-based protein degradation tool by the lipid hybrid substance;

the nanoparticles comprise hydrophilic particles, hydrophobic particles and inorganic nanoparticles;

preferably, the nanoparticles have a particle size of 5-1000nm.

In another embodiment, a special structure is provided, that is, the nanoparticle is at the core, and the lipid hybrid material wraps the nanoparticle to form a composite structure.

Thus, the lipid-based protein degradation means may comprise several morphological structures as follows:

TABLE 2 morphological Structure of lipid-based protein degradation tools

In addition, in another embodiment, a special structure is provided, namely, the nano-particles are at the core, the lipid hybrid substance is coated on the nano-particles to form a composite structure,

wherein the nanoparticle is coated by the lipid-hybrid substance at the core of the lipid-based protein degradation means;

the nanoparticle (nanoparticle coated at the core in the lipid hybrid) includes:

(1) The hydrophilic particles may be a variety of nanogels composed of a dendrimer, a hyperbranched polymer, and the hydrophilic polymer and derivatives thereof as claimed in claim 4, and a nano albumin.

(2) The hydrophobic particles may be nanoparticles prepared from various hydrophobic polyamino acids and derivatives thereof, such as polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polycarbonate (PMC) and derivatives thereof, various combinations and copolymers of glycolide/lactide/caprolactone/carbonate, polyurethane (PU), polyetheretherketone (PEEK), polymethylmethacrylate (PMMA), polyvinyl alcohol (PVA), polyethylene (PE), and poly (phenylalanine).

(3) Inorganic nanoparticles, which can be gold nanoparticles, carbon nanoparticles, silicon nanoparticles, iron oxide nanoparticles, calcium phosphate nanoparticles, barium sulfate and iodide contrast agents, aluminum nitride nanoparticles, aluminum oxide nanoparticles, titanium oxide nanoparticles, aluminum iron alloy particles, and titanium iron alloy particles, are all materials with stable and low biological toxicity.

(4) And mixed nanoparticles composed of the aforementioned nanoparticles.

Among them, when the nanoparticle is one of a hydrophilic particle and a hydrophobic particle, and is a polymer, after being coated with a lipid hybrid substance, it can also be called lipid polymer (LPP), i.e., a lipid film is coated on the surface of the nanoparticle of the polymer having hydrophilic or hydrophobic properties, and Cationic nanoemulsion (Cationic lipid) is also widely studied and applied in vaccine and drug delivery.

Further, in the POI recognition group, the antibody is a therapeutic monoclonal antibody, a multispecific antibody, a nano antibody, an antibody derivative or an antibody coupling drug;

the polypeptide is a polypeptide with POI specific binding capacity;

the small molecule is a compound with POI specific binding capacity;

preferably, the small molecule comprises a derivative of the CDK4/6 protein inhibitor Palbociclib (Palbociclib), a derivative of the BRD4 protein inhibitor JQ1, a derivative of the beta-Amyloid protein probe AV-45, a derivative of the Pittsburgh compound PiB, and a derivative of the Tau protein probes GTP1 and PBB 3.

Preferably, the polypeptide comprises a binding polypeptide of ACE2, CD13, beta-Amyloid;

preferably, the antibody comprises: CTX, NTZ, PTZ, CRLZ, INE, ATZ, and aducanumab, miltuximab.

In addition, the invention also provides a lipid-based protein degradation tool, and application of the lipid-based protein degradation tool in preparation of drugs, vaccines and delivery systems for treating and preventing diseases related to abnormal accumulation of proteins; wherein the diseases caused by abnormal accumulation of the protein comprise tumors, immune system diseases, inflammation and pathogen infection, neurodegenerative diseases, blood system diseases and metabolic diseases.

In addition, the invention also provides a protein degradation tool based on lipid, and the application of the protein degradation tool in preparing a detection product/kit aiming at the research of abnormal protein accumulation related diseases and protein interaction; wherein the diseases caused by abnormal accumulation of the protein comprise tumors, immune system diseases, inflammation and pathogen infection, neurodegenerative diseases, blood system diseases and metabolic diseases.

In addition, the invention also provides a preparation method of the lipid-based protein degradation tool, wherein when the lipid-based protein degradation tool is a protein degradation tool of which the POI recognition group is coupled with the lipid hybrid substance, the preparation method comprises two steps:

(1) Non-covalently bonding the POI recognition group to the lipid hybrid;

(2) Alternatively, the POI recognition group is covalently bonded to the lipid hybrid substance based on a coupling group, thereby constituting the lipid-based proteolytic tool.

Further, the lipid-based proteolytic means further comprises a proteolytic means in which the POI recognition group is linked to the lipid hybrid via a linking member;

wherein, when the lipid-based protein degradation tool is a protein degradation tool in which the POI recognition group is connected with the lipid hybrid through the connecting member, the preparation method comprises the following steps:

(1) Firstly, coupling the POI recognition group with the connecting member to form a coupling intermediate; then linking the coupling intermediate to the lipid hybrid substance to form the lipid-based protein degradation means;

(2) Or, constructing a nanocomposite structure with the connecting member as an outer layer and the lipid hybrid substance as a core; and coupling the POI recognition group with the nano-composite structure to form the lipid-based protein degradation tool.

The invention is further illustrated by the following specific examples, but it should be understood that these examples are included merely for purposes of illustration in more detail and are not intended to limit the invention in any way.

TABLE 3 structural morphology of the Nanoprotein degradation tools prepared in examples 1-5

Example 1

Referring to table 3, in this example, the degradation tool of example 1 was prepared by coupling the POI recognition group to a lipid hybrid, wherein the POI recognition group is: monoclonal antibody drug Nitol (NTZ); the lipid hybrid substance is PEG (polyethylene glycol) liposome, namely the liposome (liposome) contains PEG exposed on the surface of the lipid hybrid substance, and the tail end of the PEG is provided with an active reaction site NHS (N-hydroxysuccinimide) for coupling with the amino group of the POI recognition group NTZ antibody. The structure obtained was NTZ-PEGlipo.

The preparation method comprises the following steps:

(1) Pretreatment: the buffer of the original antibody was replaced with PBS. In this example, EGFR monoclonal beads (NTZ, nimotuzumab) that have been used for clinical treatment were used.

Taking a nimotuzumab solution, centrifuging the nimotuzumab solution for 2 minutes at 4000 Xg by using a 3kDa ultrafiltration tube, further concentrating the antibody, and then diluting the antibody by using PBS (phosphate buffer solution); after repeated concentration and dilution, the buffer was replaced with PBS, and after dilution, the protein concentration was measured by IgG mode a280 of NanoDrop one C instrument, and the result was verified by BCA protein quantification kit.

(2) Preparation of lipid hybrid: the lipid hybrid substance used is liposome, which is composed of hydrogenated soybean lecithin (HSPC), cholesterol (CHO, cholestrol), DSPE (distearoylphosphatidylethanolamine) -PEG (polyethylene glycol) (polyethylene glycol molecular weight is 2 kDa) by a classical film hydration method, wherein the mass ratio HSPC: DSPE-PEG = 56. HSPC, CHO was dissolved in chloroform and then evaporated to concentrate and dried to form a film. After the chloroform was completely evaporated, 1mL of DSPE-PEG-NHS PBS solution was added, followed by sonication at 100W for 3min at room temperature. The resulting solution was then passed through 800nm, 400nm and 200nm filters to allow insertion of DSPE-PEG-NHS onto the lipid membrane, which should be controlled to be completed within 10 min.

(3) Coupling of POI recognition group NTZ antibody to PEG lipid hybrid:

the coupling reaction was initiated in an ice-water mixture and protected with nitrogen. The lipid hybrid solution was then added to the antibody PBS solution stirred at 800rpm (revolutions per minute); the reaction was then stirred and incubated at 4 ℃ on a 20rpm rotator for 24 hours.

(4) And (3) purification: the reaction mixture was then concentrated by ultrafiltration 3 times using a 50kDa centrifugal ultrafiltration tube and subsequently made up to 100. Mu.L using PBS. Protein concentration was determined by a NanoDrop one C instrument by a280 absorbance of IgG and verified by BCA method.

The experimental results are as follows: referring to FIG. 4, a diagram of the NTZ-PEGlipo synthesis process is shown. FIG. 5 shows the result of protein electrophoresis detection of EGFR protein expression by collecting protein after MDA-MB-231 (M231) human breast cancer tumor cells are treated with 500nM NTZ-PEGlipo for 24 hours, which shows that NTZ-PEGlipo can effectively degrade target protein EGFR.

The cell culture medium volumes were the same in all examples, and the molar concentrations of the treatments were in units of μ M (μ Mol/L) and nM (nMol/L). In the experiment, the cell culture and treatment were carried out in 12-well plate cell culture dishes in 500. Mu.L of the medium. Cell culture was performed according to standard cell culture specifications. All control groups were used at the same molar concentration as the degradation tool group, for example, the NTZ group and NTZ-lipo group had the same molar concentration and volume of antibody NTZ, while the lipo group without antibody and NTZ-lipo group had the same molar concentration and volume of lipid-hybrid. Wherein the lipid-hybrid control group in each example refers to the lipid-hybrid in the example to which the POI recognition group is not attached. In all examples involving multi-concentration testing, the control POI recognition group or the control lipid hybrid group was treated at an equal molar concentration to the highest concentration treatment in the concentration test group. GAPDH or VIN (vinculin) of all protein electrophorograms are internal controls.

Example 2

Referring to table 3, in this example, the degradation tool of example 2 was prepared by coupling the POI recognition group to the amphiphilic lipid linker arm followed by self-assembly with the lipid hybrid species. Wherein the POI recognition groups in example 2a and example 2b are: monoclonal antibody drug Nitol (NTZ); the linker arm is NHS-PEG-DSPE with NHS group. The lipid hybrid substance comprises a membrane skeleton and cholesterol (CHO, cholestrol), the membrane skeleton is hydrogenated soybean lecithin (HSPC) in example 2a, and the degradation tool of the membrane skeleton is NTZ-lipo1, the membrane skeleton is distearoyl phosphatidylcholine (DSPC) in example 2b, and the degradation tool of the membrane skeleton is NTZ-lipo2. The POI recognition groups in example 2c are: therapeutic monoclonal antibody of HER2 Inetetamab (INE, inetetamab), HER2 is tumor proliferation signal receptor and marker, the connecting arm is NHS-PEG-DSPE with NHS group, the lipid hybrid substance is composed of HSPC membrane skeleton and CHO, and the degradation tool composed of the HSPC membrane skeleton and CHO is INE-lipo.

The POI recognition groups in example 2d are: the small molecular CDK4 inhibitor Palbociclib (Palb) is characterized in that a connecting arm is NHS-PEG-DSPE with NHS groups, palbociclib is coupled with NHS through amino reaction, a lipid hybrid substance consists of an HSPC membrane skeleton and CHO, and a degradation tool formed by Palb-lipo is Palb-lipo.

The preparation method comprises the following steps:

experiment 1: the POI recognition group is an antibody. Wherein the POI recognition groups in example 2a and example 2b are: the mab drug Nitol (NTZ). The POI recognition groups in example 2c are: monoclonal antibody drug Inunitol (INE).

(1) Pretreatment: the buffer of the original antibody was replaced with phosphate buffered saline PBS. In this example, the NTZ and INE monoclonal antibody solutions were centrifuged at 4000 xg for 2 minutes using a 3kDa ultrafiltration tube, and then the antibodies were concentrated and diluted with PBS; after repeated concentration and dilution 3 times, the buffer main component was replaced with PBS, and after dilution with an antibody solution, the protein concentration was measured by IgG mode a280 of NanoDrop one C instrument, and the result was verified by BCA protein quantification kit.

(2) Coupling reaction of the POI recognition group and the amphiphilic lipid connecting arm: the antibody was reacted with NHS-PEG (2 kDa) -DSPE by reacting the amino group on the antibody with NHS (N-Hydroxysuccinimide) group at the end of DSPE (distearoylphosphatidylethanolamine) -PEG. The coupling reaction was initiated in an ice water mixture and protected with nitrogen. To improve dispersibility, DSPE-PEG was optionally sonicated in PBS buffer at 40kHz for 3 minutes, followed by addition of equimolar concentrations and volumes (molar ratio is not limited to 1, reference antibody conjugated drug) of NHS-PEG-DSPE PBS solution to the antibody PBS solution stirred at 800rpm (revolutions per minute); the reaction was then incubated at 4 ℃ for 24 hours on a 20rpm rotator (incubation time and conditions were not specifically required, with lower rpm and 4 degrees being preferred to maintain antibody activity). Obtaining NTZ-PEG-DSPE reaction mixture or INE-PEG-DSPE reaction mixture.

The coupling method has no special requirement, and can be coupling methods of general antibody coupling drugs, including but not limited to non-site coupling and site-specific coupling, such as amino coupling, carboxyl coupling and bridging sulfhydryl coupling. Site-directed conjugation may be click chemistry, selenium bond conjugation, serine conjugation, cysteine conjugation, unnatural amino acid conjugation, coupling after enzymatic catalysis, and sugar site conjugation.

(3) And (3) purification: the reaction mixture containing NTZ-PEG-DSPE or INE-PEG-DSPE was concentrated by ultrafiltration 3 times using a 50kDa centrifugal ultrafiltration tube, and then made up to 100. Mu.L with PBS. Protein concentration was determined by a NanoDrop one C instrument by a280 absorbance of IgG and verified by BCA method.

(4) Preparation of lipid hybrid: the lipid hybrid used was a liposome consisting of hydrogenated soy lecithin (HSPC) or distearoyl phosphatidylcholine (DSPC), cholesterol (CHO, cholestrol), DSPE-PEG (2 kDa) by classical membrane hydration, where the mass ratio [ HSPC or DSPC ]: CHO: DSPE-PEG (without POI recognition group) = 56. HSPC or DSPC, CHO were dissolved in chloroform and then concentrated by evaporation to form a film.

Preparation of NTZ-lipo1, NTZ-lipo2, INE-lipo: after the solvent chloroform of the lipid hybrid substance solution was completely evaporated, 1mL of DSPE-PEG-antibody in PBS was added, followed by sonication at 100W for 3min at room temperature. The resulting solution was then passed through 800nm, 400nm and 200nm filters. Following further purification, the reaction mixture was concentrated by ultrafiltration 3 times using a 50kDa centrifugal ultrafiltration tube and then made up to 100. Mu.L using PBS. Protein concentration was determined by a NanoDrop one C instrument by a280 absorbance of IgG and verified by BCA method.

Cell experiments were then performed depending on antibody concentration.

Experiment 2: POI recognition groups are small molecules. In example 2d, the POI recognition group is Palbociclib small molecule drug or beta-amyloid probe AV-45.

Palbociclib is a clinical commercial CDK4 inhibitor, and the conjugate of the lipid hybrid substance is Palb-lipo in the embodiment. CDK4 is a cyclin-dependent kinase that is widely involved in cell senescence tumorigenesis.

Palbociclib was reacted with NHS-PEG (2 kDa) -DSPE via the amino group on Palbociclib with the NHS group at the end of DSPE-PEG (molar ratio 1.2. Stirring and reacting for 24 hours at room temperature under the protection of nitrogen to obtain a Palb-PEG-DSPE reaction mixture. Palb-PEG-DSPE was dialyzed for 24 hours and lyophilized, and the concentration was determined by a standard curve of ultraviolet absorption peaks.

Lipid hybrids were prepared as in experiment 1 of this example.

And (3) preparing Palb-lipo, adding 1mL of PBS solution of Palb-PEG-DSPE after the solvent chloroform of the lipid hybrid substance solution is completely evaporated, and then carrying out ultrasonic treatment at room temperature for 3min at 100W. The resulting solution was then passed through 800nm, 400nm and 200nm filters. The concentration was determined by a standard curve of the UV absorption peak and cell experiments were performed according to the Palb molarity.

AV-45 is a beta-amyloid probe of a classical marker of Alzheimer's disease, and the lipid hybrid substance conjugate is AV45-lipo.

Beta-amyloid is a potential pathogenic protein of Alzheimer's disease, which is an extracellular protein, a precursor of the beta-amyloid is expressed on a cell membrane, and the precursor is secreted to form an oligomer (beta-amyloid 1-42 oligomer) and fibers outside the cell after shearing, wherein the oligomer is considered to have the greatest toxicity, the accumulation of the extracellular protein beta-amyloid oligomer brings pathological risks such as neuroinflammation, and the isotope labeled AV-45 is a clinical commercial beta-amyloid probe. The isotope-free AV-45 derivative selected in this example is AV-45-SH, AV-45 has blue autofluorescence, and is convenient for cell tracking, and its sulfhydryl SH is used for coupling with Mal (maleimide) -PEG-DSPE.

The preparation method of AV45-PEG-DSPE comprises the following steps: combining the AV45-SH with the sulfydryl with Mal-PEG (molecular weight is 2 kDa) -DSPE, namely dissolving the AV45-SH and the Mal-PEG-DSPE in DMSO at a molar ratio of 1.2, and protecting water bath at 37 ℃ for 24 hours in a dark nitrogen atmosphere. Followed by lyophilization by dialysis. The concentration was determined by a standard curve through the uv absorption peak.

Lipid hybrids were prepared as in experiment 1 of this example. Preparation of AV 45-lipo: after the solvent chloroform of the lipid hybrid solution was completely evaporated, 1mL of PBS solution of AV45-PEG-DSPE was added, followed by sonication at 100W for 3min at room temperature. The resulting solution was then passed through 800nm, 400nm and 200nm filters. The concentration was determined by a standard curve of uv absorbance peaks and cell experiments were performed according to AV45 molarity.

The experimental results are as follows: referring to fig. 6, a schematic diagram of the synthesis process is shown. FIG. 7 shows that the protein expression result of EGFR protein expression detection by protein electrophoresis of protein harvested after M231 human breast cancer tumor cells are treated by NTZ-lipo1 with different concentrations for 24 hours shows that NTZ-lipo1 can effectively degrade the target protein EGFR. FIG. 8 shows the results of protein electrophoresis of the degradation effect of the target protein EGFR by treating M231 cells with NTZ-lipo1 whose lipid hybrid substances are composed of different HSPC and CHO ratios for 24 hours, showing that different compositions have the protein degradation effect, and the ratios of the membrane skeleton HSPC and the helper lipid cholesterol have an influence on the protein degradation effect. FIG. 9 shows that the protein expression result of EGFR protein detection by protein electrophoresis is obtained after M231 human breast cancer tumor cells are treated by 500nM NTZ-lipo2 for 24 hours, and the result shows that the NTZ-lipo2 can effectively degrade the target protein EGFR. FIG. 10 shows the results of protein electrophoresis detection of HER2 protein expression from protein harvest after M231 human breast cancer tumor cells were treated with 500nM INE-lipo for 24 hours, showing that INE-lipo can effectively degrade the target protein.

FIG. 11 shows the successful ligation of Palb-PEG-DSPE by hydrogen nuclear magnetic resonance of the lipid linker arm used in Palb-lipo coupled to the POI recognition group. FIG. 12 shows the successful ligation as a result of hydrogen nuclear magnetic resonance of AV45-PEG-DSPE after the lipid linker arm used in AV45-lipo is coupled with the POI recognition group. FIG. 13 shows the result of protein electrophoresis detection of CDK4 protein expression from protein collected from M231 human breast cancer tumor cells after being treated with Palb-lipo for 24 hours at different concentrations, which shows that Palb-lipo can effectively degrade target proteins. FIG. 14 shows a schematic diagram of the structure of AV45-lipo and the structure of AV 45-SH. FIG. 15 shows confocal microscopy imaging of human glial cells after 24 hours of treatment with different concentrations of AV45-lipo, i.e., detection of β -amyloid (Abeta) being hijacked into cells. mu.M of oligomers prepared from beta-amyloid 1-42 polypeptide labeled with FITC green dye was incubated with 10 mu.M of AV45-lipo for 24 hours, followed by washing away the non-cellular processed material with PBS and confocal microscopy imaging to detect the green fluorescence of beta-amyloid 1-42 oligomers, which indicates greater amount of hijacking into cells, theoretically greater hijacking and phagocytosis and better degradation of lysosomal translocation energy, to reduce the toxicity of extracellular beta-amyloid oligomers accumulated, and the lysosomal dye LySoTracker was used to label lysosomes at a scale of 10 μ M.

Example 3

Referring to table 3, in this example, the degradation tool of example 3 was prepared by coupling the POI recognition group to the amphiphilic lipid linker arm followed by self-assembly with the lipid hybrid. Wherein the POI recognition group is: monoclonal antibody drug nitol bead (NTZ); the connecting arm is NHS-PEG-DSPE or NHS-PEG-DMG (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, polyethylene glycol end-modified N-hydroxysuccinimide) with NHS group. The lipid hybrid material consists of a membrane backbone, HSPC, cholesterol, and the cationic lipid, DOTAP (trimethyl-2, 3-dioleoyloxypropylammonium bromide). Cationic Lipid DOTAP is a representative cationic Lipid that facilitates Lipid hybridization of substances to form Lipid Nanoparticles (LNPs) for the encapsulation of negatively charged nucleic acid drugs. When the composition of the lipid hybrid substance is PEG-DSPE/cholesterol/HSPC, the degradation tool of the composition is NTZ-LNP1, and when the NTZ-LNP1 carries nonsense sequence no-load small interfering RNA (siRNA), the degradation tool of the composition is NTZ-LNP1s; when the composition of the lipid hybrid substance is PEG-DMG/cholesterol/HSPC, the degradation tool of the composition is NTZ-LNP2.

The preparation method comprises the following steps:

pretreatment, attachment of POI recognition group and linker arm, purification, using the method used in example 2.

Preparation of lipid hybrid: the lipid hybrid used was lipid nanoparticles, consisting of hydrogenated soy lecithin (HSPC) or Distearoylphosphatidylcholine (DSPC), cholesterol (CHO, cholestrol), DSPE-PEG (2 kDa) by classical thin film hydration, where the mass ratio [ HSPC or DSPC ]: CHO: DSPE-PEG (without POI recognition group) = 56. HSPC, CHO was dissolved in chloroform and then evaporated to concentrate and dried to form a film.

After synthesis of the lipid hybrid described above, 1mL of DSPE-PEG-antibody in PBS solution was added while DOTAP solution (molar ratio HSPC: DOTAP = 1) after complete evaporation of chloroform and then sonicated at 100W for 3min at room temperature. The resulting solution was then passed through 800nm, 400nm and 200nm filters.

NTZ-LNP1s: after the synthesis of the lipid hybrid described above, 1mL of DSPE-PEG-antibody in PBS was added after the chloroform had evaporated completely, and the amount of siRNA was calculated from the nitrogen to phosphorus ratio to DOTAP (N/P = 3. Simultaneously adding a DOTAP solution (molar ratio HSPC: DOTAP = 1) and an siRNA solution. Then sonicated at 100W for 3min at room temperature. The resulting solution was then passed through 800nm, 400nm and 200nm filters.

And (3) purification: the reaction mixture was then concentrated by ultrafiltration 3 times using a 50kDa centrifugal ultrafiltration tube and subsequently made up to 100. Mu.L using PBS. Protein concentration was determined by a NanoDrop one C instrument by a280 absorbance of IgG and verified by BCA method.

The experimental results are as follows: referring to FIG. 16, it is a schematic structural diagram of NTZ-LNP1, NTZ-LNP1s, and NTZ-LNP2. Fig. 17, particle size distribution for Dynamic Light Scattering (DLS) detection of the lipid hybrid. FIG. 18 shows the results of protein electrophoresis of protein harvest after 24 hours of 500nM NTZ-LNP1 treatment of M231 human breast cancer tumor cells for detecting EGFR protein expression, in which lipo1 in example 2 is also used as a control. The results show that NTZ-LNP1 can effectively degrade the target protein EGFR. FIG. 19 shows that protein electrophoresis detection of EGFR protein expression by protein harvest after M231 human breast cancer tumor cells are treated with NTZ-LNP1s for 24 hours shows that NTZ-LNP1s can effectively degrade target protein EGFR. FIG. 20 shows that protein electrophoresis detection of EGFR protein expression by protein harvest after M231 human breast cancer tumor cells are treated with NTZ-LNP2 for 24 hours shows that NTZ-LNP2 can effectively degrade target protein EGFR.

Example 4

Referring to table 3, in this example, the degradation tool of example 4 was prepared by coupling the POI recognition group to the amphiphilic lipid linker arm followed by self-assembly with the lipid hybrid. Wherein the POI recognition group is: monoclonal antibody drug Nitol (NTZ); the linking arm is NHS-PEG-DSPE with NHS group. Lipid hybrids consist of exosomes (exosomes). The exosome is an extracellular vesicle secreted by eukaryotic organisms such as animal or plant cells, is spontaneously formed, can perform intercellular communication, generally contains protein and a small amount of nucleic acid, has high biocompatibility and drug-loading capacity, and has a phospholipid membrane structure on the surface.

The preparation method comprises the following steps:

pretreatment, attachment of POI recognition group and linker arm, purification, using the method used in example 2.

Preparation of lipid hybrid: the exosome is separated, purified and identified by DC2.4 cells according to standard steps, and the exosome is coupled with a connecting arm through two modes of ultrasound and membrane extrusion. Namely, 1mL of PBS solution of DSPE-PEG-antibody is added into the PBS solution of exosome,

the combination of the connecting arm and the lipid hybrid substance adopts a membrane extrusion method or an ultrasonic method. Sonication consists of adding a PBS solution of DSPE-PEG-antibody to a PBS solution of exosomes (e.g. mass ratio exosomes: DSPE-PEG = 25 μ g in a 1mL system, i.e. 40. The extrusion method comprises the following steps: to the exosomes were added 1mL of a PBS solution of DSPE-PEG-antibody (mass ratio exosomes: DSPE-PEG =1mg, i.e. 40. The resulting solution was then passed through 800nm, 400nm and 200nm filters.

And (3) purification: the reaction mixture was then concentrated by ultrafiltration 3 times using a 50kDa centrifugal ultrafiltration tube and subsequently made up to 100. Mu.L using PBS. Protein concentration was determined by a NanoDrop one C instrument by a280 absorbance of IgG and verified by BCA method.

The experimental results are as follows: referring to fig. 21, DLS-detected particle size and average dispersion coefficient PDI (Polymer dispersion index) of the lipid hybrid material, the smaller the PDI, the more uniform the particle size, and the result indicates that the particle size is uniform. FIG. 22 shows that the protein collected from M231 human breast cancer tumor cells treated with NTZ-exo of different concentrations for 24 hours is subjected to protein electrophoresis to detect EGFR protein expression, and the result shows that NTZ-exo can effectively degrade target protein EGFR.

Example 5

Referring to table 3, in this example, the degradation tool of example 4 was prepared by coating the surface of the nanoparticle with the lipid hybrid, coupling the POI recognition group to the amphiphilic lipid linker arm, and then self-assembling with the lipid hybrid, with the POI recognition group exposed to the outside. Wherein the POI recognition group is: EGFR-targeting mab drug Nitol (NTZ) or Cetuximab (CTX); the linking arm is NHS-PEG-DSPE with NHS group. When the lipid hybrid substance consists of HSPC/cholesterol and wraps PLGA nano-particles, the degradation tool is NTZ-lipoP. Polylactic-co-glycolic acid (PLGA) is a representative polymer nanoparticle, has hydrophobicity, high biocompatibility, stable properties, and is biodegradable, and is widely used in medical treatment. The lipid hybrid is composed of mouse red cell membrane (RBCm) and encapsulates acetalized Dextran (Dextran), and when the POI recognition group is composed of CTX, the degradation tool is CTX-RBCmd. Dextran is representative of hydrophilic biodegradable nanoparticles. The erythrocyte membrane is a representative of cell membrane and organelle membrane, is a lipid hybrid substance, and is convenient to separate and extract.

The preparation method comprises the following steps:

pretreatment, attachment of POI recognition group and linker arm, purification, using the method used in example 2.

Preparation of lipid hybrid:

preparation of NTZ-lipoP: the composition is prepared from HSPC and CHO by a classical film rehydration method, wherein the mass ratio of the HSPC to the CHO: PEG-DSPE (without POI recognition group) = 56. HSPCs and CHO were dissolved in chloroform and subsequently concentrated and evaporated to form a lipid film. After the chloroform was completely evaporated, 1mL of antibody-PEG-DSPE dissolved in PBS was added for resuspension, followed by sonication at 100W for 3min at room temperature. The resulting solution was then passed through 800nm, 400nm and 200nm filters to insert the DSPE-PEG-antibody onto the lipid membrane.

And (3) coating PLGA: PLGA (15 kDa) was dissolved in DMF (10. Mu.g/. Mu.L), 2. Mu.L was added to 1mL of PBS solution every 10 seconds, the mixture was stirred at 700rpm for 3 hours, after DMF was evaporated, 1mg of LIPO (referred to as 1mg total of HSPC + CHO) was coated on each 0.01. Mu. Mol PLGA, and appropriate amounts of HSPC and CHO were dissolved in chloroform, and after concentration by evaporation, the mixture was resuspended in DSPE-PEG-NTZ PBS solution and sonicated at 100W for 3min. Incubating for 30min in a shaking table at 37 ℃, performing ultrasonic treatment for 5min at room temperature of 100W, and passing through a membrane of 800nm, 400nm and 200 nm. Mixing the two mixtures with PLGA PBS solution, performing 100W ice bath ultrasonic treatment for 2min, passing through 800, 400 and 200nm membranes, and performing ultrafiltration concentration. IgG concentration was measured.

Preparation of CTX-RBCmD: the synthesis of CTX-PEG-DSPE is as in example 2.

1mg of acetalized dextran was dissolved in 200. Mu.L of tetrahydrofuran, 10. Mu.L of the solution was dropped every 10s one drop into 1mL of alkaline water with pH =8 under stirring, and the solution was stirred at 700rpm for 3 hours. 1mg of mouse erythrocyte membrane was taken and resuspended in 1mL of PBS. PBS solution of red cell membrane was mixed with PEG solution of CTX-PEG-DSPE (calculated from membrane: acetalized dextran =1mg, molar ratio of acetalized dextran core to DSPE-PEG is 10 1) and incubated at 200rpm at 37 ℃ for 30min in shaker, followed by 100W at room temperature for 5min, and then the resulting solution was filtered through 400nm and 200nm filter membranes in order to obtain uniform size. Then mixed with the acetalized dextran solution, sonicated in a 100W ice bath for 2min, and the resulting solution was filtered through 400nm and 200nm filters to obtain the desired product. Then, ultrafiltration concentration was carried out. Protein concentration was measured in IgG format by NanoDrop one C (thermoldissher) and verified by BCA protein assay kit.

TABLE 4 DLS particle size statistics and PDI statistics for NTZ-lipoP and lipo-PLGA not linked by NTZ-PEG-DSPE

The experimental results are as follows: referring to FIG. 23, it is a schematic diagram of NTZ-lipoP structure. The results of DLS particle size statistics and PDI statistics for NTZ-lipoP and lipo-PLGA not linked by NTZ-PEG-DSPE show that the particle size is uniform and the hydrated particle size is slightly increased after the NTZ-PEG-DSPE is linked. FIG. 24 shows that the protein obtained from M231 human breast cancer tumor cells treated with NTZ-lipoP of different concentrations for 24 hours is subjected to protein electrophoresis to detect EGFR protein expression, and the result shows that the EGFR protein expression can effectively degrade the target protein EGFR. FIG. 25 is a schematic diagram of the CTX-RBCmD structure. Fig. 26 is a graph showing the result of DLS particle size distribution. FIG. 27 shows that the EGFR protein expression is detected by protein electrophoresis of protein harvested after M231 human breast cancer tumor cells are treated by 500nM CTX-RBCmD for 24 hours, and the result shows that the EGFR protein expression can be effectively degraded.

While the preferred embodiment and the corresponding examples of the present invention have been described, it should be understood that various changes and modifications, including but not limited to, adjustments of proportions, flows and amounts, which are within the scope of the invention, may be made by those skilled in the art without departing from the inventive concept thereof. While the preferred embodiment and the corresponding examples have been described, it should be understood that various changes and modifications, including but not limited to, adjustments of proportions, flows and amounts, which are within the scope of this invention, may be made by those skilled in the art without departing from the inventive concept.

Claims (11)

1.A lipid-based protein degradation tool, comprising:

a POI recognition group, and a lipid hybrid substance linked to the POI recognition group;

wherein the POI recognition group comprises an antibody, protein, polypeptide, aptamer or small molecule capable of specifically binding to the POI;

the lipid hybrid substance comprises liposome, exosome, cell membrane and LNP.

2. The lipid-based protein degradation means of claim 1,

when the POI recognition group is coupled to the lipid hybrid, the lipid-based protein degradation tool is a nanoparticle for protein degradation composed of the lipid hybrid in the core and the POI recognition group in the periphery.

3. The lipid-based protein degradation means of claim 1,

the lipid-based protein degradation means further comprises the lipid-based protein degradation means provided with a linking member between the POI recognition group and the lipid hybrid;

preferably, the linking member has a molecular weight of 0-1000kDa;

the connecting component is one of a polymer connecting arm and a lipid connecting arm;

preferably, the polymer linker arms comprise hydrophilic polymers, hydrophobic polymers, and amphiphilic polymers;

preferably, the lipid linker arm is an amphiphilic lipid linker arm.

4. The lipid-based proteolytic tool of claim 3, wherein the POI recognition group and the linking member form a set of linking units when the POI recognition group is coupled to the lipid hybrid through the linking member; the lipid-based protein degradation tool is a lipid-based protein degradation tool having a multi-layered structure in which the lipid hybrid is disposed at the core, the plurality of sets of the connection members connected to the lipid hybrid are disposed at the intermediate layer, and the plurality of sets of the connection units connected to the connection members are disposed at the periphery.

5. The lipid-based protein degradation kit according to claim 3, wherein the lipid linker arm comprises at least two ends, one end being a lipophilic end capable of linking to the lipid hybrid substance, and the other end being a hydrophilic end;

preferably, the lipophilic terminus is a lipid molecule.

6. The lipid-based protein degradation means of claim 3,

the amphiphilic polymer is a polymer with a chain/branched molecular structure, and at least one hydrophilic molecular end and one hydrophobic molecular end exist in the amphiphilic polymer;

preferably, the amphiphilic polymer has a linear molecular structure, one end of which is a hydrophilic molecular end and the other end of which is a hydrophobic molecular end.

7. The lipid-based protein degradation tool of claim 1, further comprising a nanoparticle, wherein the nanoparticle is coated at the core of the lipid-based protein degradation tool by the lipid hybrid;

the nanoparticles comprise hydrophilic particles, hydrophobic particles and inorganic nanoparticles;

preferably, the nanoparticles have a particle size of 5-1000nm.

8. Use of a lipid-based protein degradation means according to any of claims 1 to 7 for the preparation of a medicament, vaccine and delivery system for the treatment and prevention of diseases associated with abnormal accumulation of proteins; wherein, the diseases caused by abnormal accumulation of the protein comprise tumors, immune system diseases, inflammation and pathogen infection, neurodegenerative diseases, blood system diseases and metabolic diseases.

9. Use of a lipid-based protein degradation means according to any of claims 1 to 7 for the preparation of a test product/kit for the study of diseases associated with abnormal accumulation of proteins and protein interactions; wherein, the diseases caused by abnormal accumulation of the protein comprise tumors, immune system diseases, inflammation and pathogen infection, neurodegenerative diseases, blood system diseases and metabolic diseases.

10. A method for preparing a lipid-based protein degradation means according to any of claims 1 to 7,

when the lipid-based protein degradation tool is a protein degradation tool in which the POI recognition group is coupled to the lipid hybrid, the preparation method thereof is: non-covalently bonding the POI recognition group to the lipid hybrid; alternatively, the POI recognition group is covalently bonded to the lipid hybrid substance based on a coupling group, thereby constituting the lipid-based proteolytic tool.

11. The method of claim 10, wherein the lipid-based protein degradation means further comprises a protein degradation means wherein the POI recognition group is linked to the lipid hybrid via a linking member;

wherein, when the lipid-based protein degradation tool is a protein degradation tool in which the POI recognition group is connected with the lipid hybrid through the connecting member, the preparation method comprises the following steps:

firstly, coupling the POI recognition group with the connecting member to form a coupling intermediate; then linking the coupling intermediate to the lipid hybrid substance to form the lipid-based protein degradation means;

or, constructing a nanocomposite structure with the connecting member as an outer layer and the lipid hybrid substance as a core; and coupling the POI recognition group with the nano-composite structure to form the lipid-based protein degradation tool.

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